Posts Tagged ‘Sulfur’

The biochemistry of S amino acids

Larry H. Bernstein, MD, FCAP, Curator


Amino Acid and Sulfur Metabolism

Dr. Rainer Höfgen

 Sulfur is together with nitrogen, phosphorous and potassium a plant macronutrient and a crucial element affecting plant growth, plant performance and yield. The group of Dr. Rainer Hoefgen focuses on characterising the regulation of cysteine and methionine as a result of sulfate uptake and assimilation in the model plant Arabidopsis thaliana.

Cysteine and methionine are two essential amino acids which contain sulfur. We are also looking at interconnections between sulfur metabolism and other plant nutrients. Further, we are investigating means of improving the nutritional quality of crops, with a current focus on rice (Oryza sativa) with respect to a balanced amino acid composition.

In our studies of plant sulfur metabolism, we use two mutually supporting approaches as the basis of our research portfolio. The first is a targeted, pathway-oriented approach aimed at understanding pathway architecture and coordination, and the regulation of the sulfur-containing metabolites as such. The second is a non-biased approach in which functional genomics is used to work out how sulfur metabolism is embedded and controlled within the whole plant system.

sulfur uptake and assimilation

sulfur uptake and assimilation

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Sulfur is a required macronutrient, sulfur uptake and assimilation are crucial determinants in how quickly plants grow and cope with various stresses, and therefore, in how well crops yield.

Inorganic sulfate is taken up through plant roots and, via cysteine biosynthesis, incorporated as organic sulfur. Our investigations focus on fundamental questions about cysteine (cys) and methionine (met) biosynthesis and on the possibility of engineering crop plants enriched in these sulfur-containing amino acids. Methionine is essential for non-ruminant mammals (including man) and uptake of cysteine reduces the methionine requirement. We have used transgenic strategies to generate many plant lines affected in cysteine and methionine biosynthesis, and subjected them to detailed molecular and biochemical analyses. Recently, we embarked on a course to study sulfur metabolism in a holistic way, rather than focusing on single pathways as such. By applying functional genomic tools like transcript, metabolite, and protein profiling in our analysis of transgenic potato (Solanum tuberosum) and of the model plant Arabidopsis thaliana, we are heading for a better understanding of the sulfur metabolism network in plants.

To learn about the control mechanisms involved in sulfur-containing amino acid biosynthesis, we are isolating and studying the involved genes and their promoters. The model plant systems of our investigations are potato and Arabidopsis, although a limited amount of work is also dedicated to rice (Oryza sativa), cucumber (Cucumis sativus), and tomato (Lycopersicon esculentum). Various transgenic plants exhibiting reduced or increased expression of relevant genes in the pathway have been produced and analysed. Fundamental knowledge of pathway regulation has been obtained as well as an improvement of the nutritional quality of a crop plant: Nutritional quality is largely determined by methionine, which is often the most limited of the essential amino acids.

The main thrust of our research recently shifted to analysing sulfur metabolism networks. In a systems biology approach, we investigate the response of Arabidopsis to different periods or degrees of sulfur starvation by applying non-biased, multiparallel tools including transcript, protein, and metabolite profiling. Our results are integrated to form working models for further detailed investigations with a focus on regulatory aspects of metabolism. This work entails the detailed analysis of Arabidopsis mutants and pulls many of our earlier results together into biological context (eg. the increased thiol levels seen during SAT over-expression, glutathione involvement in stress response mechanisms towards active oxygen species, etc.). Our long-term goal is to imbed sulfur metabolism in a broader context such as carbohydrate and nitrogen metabolic networks, which will occur through close collaborations with external and in house research groups.


metabolite profiling

metabolite profiling


Plants are sessile organisms; if they are to survive and reproduce, they must adapt to the growth conditions in which they find themselves. We use variations in sulfur levels as a stimulus and analyse the complex response using diverse multiparallel techniques, particularly transcript and metabolite profiling, trying to piece together the total system response. The plant of choice here is, obviously, Arabidopsis thaliana, although results obtained in this model system are likely to be transferable to other plant species and crop plants. The goal is to provide a consistent and holistic description of plant sulfur metabolism and its regulation.

H Hesse and R Höfgen (2001) Application of Genomics in Agriculture. In: Molecular analysis of plant adapatation to the environment. Eds: Malcolm J. Hawkesford, Peter Buchner. Kluwer AP, Dordrecht, The Netherlands, 61-79

V Nikiforova, J Freitag, S Kempa, M Adamik, H Hesse, R Hoefgen (2003) Transcriptome analysis of sulfur depletion in Arabidopsis thaliana: Interlacing of biosynthetic pathways provides response specificity. The Plant Journal, 33, 633-650.



Plants adapt to available sulfur soil levels by regulating gene expression and protein activity to maintain homeostasis. Sulfur availability in the environment is not static, nor is the plant’s dependence on sulfur at various developmental stages. Thus, one can assume not only that the activities of regulatory proteins are dynamic, but also that changes in the expression of transcription factors involved in triggering downstream gene expression change temporally. Sulfur deprivation triggers a slow adaptive process that resets the level of sulfur homeostasis. Using transcript profiling, we have been able to identify a number of transcription factors involved in this process, which are now the target of further investigations.


Metabolome analysis and bioinformatics

system response

system response

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Gene expression, metabolite spectrum and enzyme activities change under sulfur-limiting conditions.

The response of steady state transcription levels to the sulfur stimulus is but the first chapter of the story. To understand the system response, we have to turn the page and look at protein profiles – levels and activities – and before closing the book, at metabolite profiles, which adjust rapidly in response to changes in protein expression. We are now focusing on metabolome analysis: The same samples used for transcriptome analysis are examined using element analysis (ICP-AES) and metabolite analyses (HPLC, CE, GC/MS, GC/TOF, LC/MS), either in house or in collaboration with outside research groups.

Malcolm J. Hawkesford, Rothamsted Research, UK

As these analyses are refined and data accumulates, it will become more and more important to overlay and compare transcript and metabolite profiles in order to try to generate an in silico representation of the plant sulfur regulatory complement. Various approaches are and will be followed here: bioinformatic tools have to be developed and/or adapted to fully mine the data. Otherwise, it will not be possible to fully describe the system: by looking only at the most highly expressed genes in isolation, we would simply be scratching at the surface.


Transcriptome Analysis

gene expression

gene expression

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Scatterplots of gene expression of the ratio -/+ S

Plants and some photoautotrophic bacteria assimilate inorganic sulfur from sulfates into cysteine, the first sulfur-containing organic compound and, effectively, the sole molecular doorway for reduced sulfur in all living beings. This essential process has been as finely tuned through millennia of evolution as photosynthesis. Cysteine is subsequently converted to methionine, and then into a variety of other sulfur-containing organic compounds. Sulfur assimilation is even more spendy in terms of reduction equivalents than nitrogen assimilation. Obviously, such a costly enterprise is highly controlled in juxtaposition with the rest of metabolism.

To elucidate this network of interactions, we stimulate Arabidopsis with sulfur (i.e. sulfate) at its rhizosphere with various concentrations and at different developmental stages to institute periods of starvation and replenishment. The plant tissue samples (roots, shoots) are then subjected to array hybridisation/transcript profiling after RNA extraction using either macro-arrays of 7,200 non-redundant genes on nylon filters and now full genome chips. The expression profiles are processed to select differentially expressed genes. Depending on the duration of treatment, anything between a handful and thousands of genes exhibit altered expression mirroring the gradual response of the system to conditions of altered sulfur availability. Among these responsive genes we expect to find sulfur-regulated genes; genes involved in perception, signalling, and immediate responses; and genes further down the line involved in more pleiotropic mechanisms like general stress responses. Since they arise in response to sulfur stimulation, the latter are still regarded as sulfur-responsive genes.

Sulfur-responsive genes are grouped by functional category or biosynthetic pathway. As expected, genes of the sulfur assimilation pathway are altered in expression. Furthermore, genes involved in the flavonoid, auxin, and jasmonate biosynthesis pathways are up regulated when sulfur is limiting. We focus most of our attention, however, on the regulatory elements, transcription factors.

V Nikiforova, J Freitag, S Kempa, M Adamik, H Hesse, R Hoefgen (2003) Transcriptome analysis of sulfur depletion in Arabidopsis thaliana: Interlacing of biosynthetic pathways provides response specificity. The Plant Journal, 33, 633-650

Further reading

MY Hirai, T Fujiwara, M Awazuhara, T Kimura, M Noji, K Saito (2003) Global expression profiling of sulfur-starved Arabidopsis by DNA macroarray reveals the role of O-acetyl-L-serine as a general regulator of gene expression in response to sulfur nutrition. Plant Journal. 33(4)651-663

A Maruyama-Nakashita, E Inoue, A Watanabe-Takahashi, T Yarnaya, and H Takahashi (2003) Transcriptome profiling of sulfur-responsive genes in Arabidopsis reveals global effects of sulfur nutrition on multiple metabolic pathways. Plant Physiology. 132(2)597-605

Sulfur and Other Plant Nutrients

The plant sulfur assimilation pathway is intricately interconnected with various other pathways and regulatory circuits.

Systems Analysis of Plant Sulfur Metabolism

Every organism is a complex, multi-elemental, multi-functional system living in an ever-changing environment. The viability of the system is provided by, and likewise dependent upon, flexible, effective control circuits of multiple informational fluxes, which interconnect in a dense network of metabolic physiological responses.



L-cysteine L-Met

L-cysteine L-Met

Methionine is synthesised from cysteine and phosphohomoserine

Methionine is synthesised from cysteine and phosphohomoserine


Pathway Analysis of Sulfur Containing Amino Acids

To learn about the control mechanisms involved in the biosynthesis of sulfur-containing amino acids, we are isolating and studying genes involved and their promoters. Methionine is synthesised from cysteine and phosphohomoserine via the enzymes cystathionine gamma-synthase (CgS), cystathionine beta-lyase (CbL), and methionine synthase (MS); we have cloned and characterised these three genes in potato.

Biosynthesis of Sulfur-Containing Amino Acids

Biosynthesis of Sulfur-Containing Amino Acids

Genes from Arabidopsis and potato and, when appropriate, E. coli involved in cysteine and methionine biosynthesis have also been cloned, including various isoforms of O-acetylserine (thiol)-lyase, the enzyme that converts O-acetylserine to cys; ATP-sulfurylase, the enzyme activating the inert sulfate through binding to ATP; and serine acetyltransferase (SAT), the enzyme catalysing the activation of serine to O-acetylserine. We manipulated the expression of these genes in an attempt to create conditions in which flux to either cysteine or methionine is increased.

For example, the over-expression of SAT using an E. coli gene targeted to plastids resulted in cysteine and glutathione (a tripeptide containing glutamic acid, cysteine, and glycine) levels almost twice as high as usual. By blocking the competing pathway to threonine using the partial antisense inhibition of threonine synthase in Arabidopsis and potato, we were able to increase leaf and tuber methionine levels significantly. Moreover, analysis of these transformants made it clear that there are species-specific differences in the regulation of methionine biosynthesis.

Our results in Nicotiana plumbaginifolia and potato have established the essential, but not rate-limiting, role of CbL in plant methionine biosynthesis. Furthermore, we found that regulation at the level of CgS differs between the plant species Arabidopsis and potato. Our objective now is to deepen our understanding of the regulation of methionine biosynthesis and to exploit what we learn in order to improve the nutritional quality of crop plants, which is largely determined by methionine content.

Cysteine Biosynthesis

Cysteine biosynthesis represents the essential step in the incorporation of inorganic sulfide to organic sulfur in plants. In order to gain insight into the control mechanisms involved in cysteine biosynthesis, we are isolating and studying the involved genes and their promoters, including genes coding for O-acetylserine(thiol)-lyase (OAS-TL), the enzyme which converts O-acetylserine to cysteine, and serine acetyltransferase (SAT), the enzyme catalysing the activation of serine to O-acetylserine.

Serine acetyltransferase

Serine acetyltransferase

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Serine acetyltransferase

In addition, spatial and developmental aspects of regulation are investigated with respect to gene expression and enzyme activity. We are manipulating the expression of various genes in transgenic potato plants in an attempt to create conditions in which flux to either cysteine or methionine is increased. For example, the heterologous over-expression of an E. coli SAT gene targeted to plastids resulted in a doubling of both cysteine and glutathione (a tripeptide containing glutamic acid, cysteine, and glycine that is involved in stress tolerance) levels. However, these alterations had no effect on outward plant appearance or on the expression and enzymatic activity of OAS-TL. This example demonstrates the importance of SAT in plant cysteine biosynthesis and shows that the accumulation of cysteine and related sulfur-containing compounds is limited by the supply of activated carbon backbones derived from serine. We are currently investigating this and other transgenic plants affected in cysteine and methionine biosynthesis in respect to sulfur assimilation and glutathione-mediated stress tolerance.

Despite the increase of reduced organic sulfur in our potato SAT over-producers, we did not observe an increase in methionine, although other groups reported methionine increases when using a similar approach in maize (Tsakraklides et al., 2002). Again, species specific differences, probably as a result of adaptation to specific environmental or physiological conditions, have to be taken into account, especially when generalising and transferring these data to plant breeding.

V Nikiforova, S Kempa, M Zeh, S Maimann, O Kreft, A P Casazza, K Riedel, E Tauberger, R Hoefgen, H Hesse. (2002) Engineering of cysteine and methionine biosynthesis in potato. Amino Acids 22(259-278).

K Harms, P von Ballmoos, C Brunold, R Höfgen, and H Hesse (2000) Expression of a bacterial serine acetyltransferase in transgenic potato plants leads to increased levels of cysteine and glutathione. Plant J. 22, 335-343

Further reading

MJ Hawkesford (2003) Transporter gene families in plants: the sulphate transporter gene family – redundancy or specialization? Physiologia Plantarum, 117,155-163

G Tsakraklides, M Martin, R Chalam,, MC Tarczynski, A Schmidt, and T Leustek (2002) Sulfate reduction is increased in transgenic Arabidopsis thaliana expressing 5′-adenylylsulfate reductase from Pseudomonas aeruginosa. Plant J. 32, 879

Annu Rev Nutr. 1986;6:179-209.
Metabolism of sulfur-containing amino acids.

Met metabolism occurs primarily by activation of Met to AdoMet and further metabolism of AdoMet by either the transmethylation-transsulfuration pathway or the polyamine biosynthetic pathway. The catabolism of the methyl group and sulfur atom of Met ultimately appears to be dependent upon the transmethylation-transsulfuration pathway because the MTA formed as the co-product of polyamine synthesis is efficiently recycled to Met. On the other hand, the fate of the four-carbon chain of Met appears to depend upon the initial fate of the Met molecule. During transsulfuration, the carbon chain is released as alpha-ketobutyrate, which is further metabolized to CO2. In the polyamine pathway, the carboxyl carbon of Met is lost in the formation of dAdoMet, whereas the other three carbons are ultimately excreted as polyamine derivatives and degradation products. The role of the transamination pathway of Met metabolism is not firmly established. Cys (which may be formed from the sulfur of Met and the carbons of serine via the transsulfuration pathway) appears to be converted to taurine and CO2 primarily by the cysteinesulfinate pathway, and to sulfate and pyruvate primarily by desulfuration pathways in which a reduced form of sulfur with a relatively long biological half-life appears to be an intermediate. With the exception of the nitrogen of Met that is incorporated into polyamines, the nitrogen of Met or Cys is incorporated into urea after it is released as ammonium [in the reactions catalyzed by cystathionase with either cystathionine (from Met) or cystine (from Cys) as substrate] or it is transferred to a keto acid (in Cys or Met transamination). Many areas of sulfur-containing amino acid metabolism need further study. The magnitude of AdoMet flux through the polyamine pathway in the intact animal as well as details about the reactions involved in this pathway remain to be determined. Both the pathways and the possible physiological role of alternate (AdoMet-independent) Met metabolism, including the transamination pathway, must be elucidated. Despite the growing interest in taurine, investigation of Cys metabolism has been a relatively inactive area during the past two decades. Apparent discrepancies in the reported data on Cys metabolism need to be resolved. Future work should consider the role of extrahepatic tissues in amino acid metabolism as well as species differences in the relative roles of various pathways in the metabolism of Met and Cys.

The Sulfur-Containing Amino Acids: An Overview1,2

John T. Brosnan3 and Margaret E. Brosnan

J. Nutr. June 2006; 136(6): 1636S-1640S

Methionine and cysteine may be considered to be the principal sulfur-containing amino acids because they are 2 of the canonical 20 amino acids that are incorporated into proteins. However, homocysteine and taurine also play important physiological roles (Fig. 1). Why does nature employ sulfur in her repertoire of amino acids? The other canonical amino acids are comprised only of carbon, hydrogen, oxygen, and nitrogen atoms. Because both sulfur and oxygen belong to the same group (Group 6) of the Periodic Table and, therefore, are capable of making similar covalent linkages, the question may be restated: why would methionine and cysteine analogs, in which the sulfur atom is replaced by oxygen, not serve the same functions? One of the critical differences between oxygen and sulfur is sulfur’s lower electronegativity. Indeed, oxygen is the second most electronegative element in the periodic table. This accounts for the use of sulfur in methionine; replacement of the sulfur with oxygen would result in a much less hydrophobic amino acid. Cysteine readily forms disulfide linkages because of the ease with which it dissociates to form a thiolate anion. Serine, on the other hand, which differs from cysteine only in the substitution of an oxygen for the sulfur, does not readily make dioxide linkages. The difference results from the fact that thiols are much stronger acids than are alcohols, so that the alcohol group in serine does not dissociate at physiological pH. Substitution of oxygen for sulfur inS-adenosylmethionine would produce so powerful a methylating agent that it would promiscuously methylate cellular nucleophiles without the need for an enzyme.


Structures of the sulfur-containing amino acids.

Methionine and cysteine in proteins.

Although both methionine and cysteine play critical roles in cell metabolism, we suggest that, in general, the 20 canonical amino acids were selected for the roles they play in proteins, not their roles in metabolism. It is important, therefore, to review the role played by these amino acids in proteins. Methionine is among the most hydrophobic of the amino acids. This means that most of the methionine residues in globular proteins are found in the interior hydrophobic core; in membrane-spanning protein domains, methionine is often found to interact with the lipid bilayer. In some proteins a fraction of the methionine residues are somewhat surface exposed. These are susceptible to oxidation to methionine sulfoxide residues. Levine et al. (1) regard these methionine residues as endogenous antioxidants in proteins. In E. coli glutamine synthetase, they tend to be arrayed around the active site and may guard access to this site by reactive oxygen species. Oxidation of these methionine residues has little effect on the catalytic activity of the enzyme. These residues may be reduced to methionine by means of the enzyme methionine sulfoxide reductase (2). Thus, an oxidation–reduction cycle occurs in which exposed methionine residues are oxidized (e.g., by H2O2) to methionine sulfoxide residues, which are subsequently reduced:FormulaFormula

It is considered that the impaired activity of methionine sulfoxide reductase and the subsequent accumulation of methionine sulfoxide residues are associated with age-related diseases, neurodegeneration, and shorter lifespan (2).

Methionine is the initiating amino acid in the synthesis of eukaryotic proteins; N-formyl methionine serves the same function in prokaryotes. Because most of these methionine residues are subsequently removed, it is apparent that their role lies in the initiation of translation, not in protein structure. In eukaryotes, translation initiation involves the association of the initiator tRNA (met-tRNAimet) with eIF-2 and the 40S ribosomal subunit together with a molecule of mRNA. Drabkin and Rajbhandary (3) suggest that the hydrophobic nature of methionine is key to the binding of the initiator tRNA to eIF-2. Using appropriate double mutations (in codon and anticodon), they were able to show that the hydrophobic valine could be used for initiation in mammalian cells but that the polar glutamine was very poor.

Cysteine plays a critical role in protein structure by virtue of its ability to form inter- and intrachain disulfide bonds with other cysteine residues. Most disulfide linkages are found in proteins destined for export or residence on the plasma membrane. These disulfide bonds can be formed nonenzymatically; protein disulfide isomerase, an endoplasmic reticulum protein, can reshuffle any mismatched disulfides to ensure the correct protein folding (4).


S-Adenosylmethionine (SAM)4 is a key intermediate in methionine metabolism. Discovered in 1953 by Cantoni (5) as the “active methionine” required for the methylation of guanidioacetate to creatine, it is now evident that SAM is a coenzyme of remarkable versatility (Fig. 2). In addition to its role as a methyl donor, SAM serves as a source of methylene groups (for the synthesis of cyclopropyl fatty acids), amino groups (in biotin synthesis), aminoisopropyl groups (in the synthesis of polyamines and, also, in the synthesis of ethylene, used by plants to promote plant ripening), and 5′-deoxyadenosyl radicals. SAM also serves as a source of sulfur atoms in the synthesis of biotin and lipoic acid (6). In mammals, however, the great bulk of SAM is used in methyltransferase reactions. The key to SAM’s utility as a methyl donor lies in the sulfonium ion and in the electrophilic nature of the carbon atoms that are adjacent to the sulfur atom. The essence of these methyltransferase reactions is that the positively charged sulfonium renders the adjoining methyl group electron-poor, which facilitates its attack on electron-rich acceptors (nucleophiles).

Metabolic versatility of S-adenosylmethionine.

Metabolic versatility of S-adenosylmethionine.


Metabolic versatility of S-adenosylmethionine.

SAM can donate its methyl group to a wide variety of acceptors, including amino acid residues in proteins, DNA, RNA, small molecules, and even to a metal, the methylation of arsenite (7,8). At present, about 60 methyltransferases have been identified in mammals. However, the number is almost certainly much larger. A bioinformatic analysis of a number of genomes, including the human genome, by Katz et al. (9) has suggested that Class-1 SAM-dependent methyltransferases account for 0.6–1.6% of open reading frames in these genomes. This would indicate about 300 Class 1 methyltransferases in humans, in addition to a smaller number of Class 2 and 3 enzymes. In humans, it appears that guanidinoacetate N-methyltransferase (responsible for creatine synthesis) and phosphatidylethanolamine N-methyltransferase (synthesis of phosphatidylcholine) are the major users of SAM (10). In addition, there is substantial flux through the glycine N-methyltransferase (GNMT) when methionine intakes are high (11). An important property of all known SAM-dependent methyltransferases is that they are inhibited by their product, S-adenosylhomocysteine (SAH).

Methionine metabolism.

Methionine metabolism begins with its activation to SAM (Fig. 3) by methionine adenosyltransferase (MAT). The reaction is unusual in that all 3 phosphates are removed from ATP, an indication of the “high-energy” nature of this sulfonium ion. SAM then donates its methyl group to an acceptor to produce SAH. SAH is hydrolyzed to homocysteine and adenosine by SAH hydrolase. This sequence of reactions is referred to as transmethylation and is ubiquitously present in cells. Homocysteine may be methylated back to methionine by the ubiquitously distributed methionine synthase (MS) and, also, in the liver as well as the kidney of some species, by betaine:homocysteine methyltransferase (BHMT). MS employs 5-methyl-THF as its methyl donor, whereas BHMT employs betaine, which is produced during choline oxidation as well as being provided by the diet (10). Both MS and BHMT effect remethylation, and the combination of transmethylation andremethylation comprise the methionine cycle, which occurs in most, if not all, cells.

Major pathways of sulfur-containing amino acid metabolism.

Major pathways of sulfur-containing amino acid metabolism.

Major pathways of sulfur-containing amino acid metabolism.

The methionine cycle does not result in the catabolism of methionine. This is brought about by the transsulfuration pathway, which converts homocysteine to cysteine by the combined actions of cystathionine β-synthase (CBS) and cystathionine γ-lyase (CGL). The transsulfuration pathway has a very limited tissue distribution; it is restricted to the liver, kidney, intestine, and pancreas. The conversion of methionine to cysteine is an irreversible process, which accounts for the well-known nutritional principle that cysteine is not a dietary essential amino acid provided that adequate methionine is available, but methionine is a dietary essential amino acid, regardless of cysteine availability. This pathway for methionine catabolism suggests a paradox: is methionine catabolism constrained by the need for methylation reactions? If this were so, the methionine in a methionine-rich diet might exceed the methylation demand so that full catabolism could not occur via this pathway. GNMT methylates glycine to sarcosine, which may, in turn, be metabolized by sarcosine dehydrogenase to regenerate the glycine and oxidize the methyl group to 5,10-methylene-THF.

Application of sophisticated stable isotope tracer methodology to methionine metabolism in humans has yielded estimates of transmethylation, remethylation, and transsulfuration. Such studies reveal important points of regulation. For example, the sparing effect of cysteine on methionine requirements is evident as an increase in the fraction of the homocysteine pool that is remethylated and a decrease in the fraction that undergoes transsulfuration (12). In young adults ingesting a diet containing 1–1.5 g protein·kg−1·d−1, about 43% of the homocysteine pool was remethylated, and 57% was metabolized through the transsulfuration pathway (transmethylation = 9.7, transulfuration = 5.4, remethylation = 4.4 μmol·kg−1·h−1) (13).

Methionine metabolism affords a remarkable example of the role of vitamins in cell chemistry. MS utilizes methylcobalamin as a prosthetic group, 1 of only 2 mammalian enzymes that are known to require Vitamin B-12. The methyl group utilized by MS is provided from the folic acid 1-carbon pool. Methylenetetrahydrofolate reductase (MTHFR), which reduces 5,10-methylene-THF to 5-methyl-THF, contains FAD as a prosthetic group. Both of the enzymes in the transsulfuration pathway (CBS and CGL) contain pyridoxal phosphate. It is hardly surprising, therefore, that deficiencies of each of these vitamins (Vitamin B-12, folic acid, riboflavin, and pyridoxine) are associated with elevated plasma homocysteine levels. The oxidative decarboxylation of the α-ketobutyrate produced by CGL is brought about by pyruvate dehydrogenase so that niacin (NAD), thiamine (thiamine pyrophosphate), and pantothenic acid (coenzyme A) may also be regarded as being required for methionine metabolism.

Not only are vitamins required for methionine metabolism, but methionine metabolism plays a crucial role in the cellular assimilation of folate. MS has 2 principal functions. In addition to its role in methionine conservation, MS converts 5-methyl-THF to THF, thereby making it available to support DNA synthesis and other functions. Because 5-methyl-THF is the dominant circulating form that is taken into cells, MS is essential for cellular folate assimilation. Impaired MS activity (e.g., brought about by cobalamin deficiency) results in the accumulation of the folate coenzymes as 5-methyl-THF, the so-called methyl trap (14). This hypothesis explains the fact that Vitamin B-12 deficiency causes a functional cellular folate deficiency.

The combined transmethylation and transsulfuration pathways are responsible for the catabolism of the great bulk of methionine. However, there is also evidence for the occurrence of a SAM-independent catabolic pathway that begins with a transamination reaction (15). This is a very minor pathway under normal circumstances, but it becomes more significant at very high methionine concentrations. Because it produces powerful toxins such as methane thiol, it has been considered to be responsible for methionine toxicity. The identity of the initiating transaminase is uncertain; the glutamine transaminase can act on methionine, but it is thought to be unlikely to do so under physiological conditions (15). In view of the likelihood that this pathway plays a role in methionine toxicity, more work is warranted on its components, tissue distribution, and physiological function.

Regulation of methionine metabolism.

The major means by which methionine metabolism is regulated are 1) allosteric regulation by SAM and 2) regulation of the expression of key enzymes. In the liver, SAM exerts powerful effects at a variety of loci. The liver-specific MAT has a highKm for methionine and, therefore, is well fitted to remove excess dietary methionine. It exhibits the unusual property of feedback activation; it is activated by its product, SAM (16). This property has been incorporated into a computer model of hepatic methionine metabolism, and it is clear that it renders methionine disposal exquisitely sensitive to the methionine concentration (17). SAM is also an allosteric activator of CBS and an allosteric inhibitor of MTHFR (18). Therefore, elevated SAM promotes transsulfuration (methionine oxidation) and inhibits remethylation (methionine conservation). Many of the enzymes involved in methionine catabolism (MAT 1, GNMT, CBS) are increased in activity on ingestion of a high-protein diet (18).

In addition to its function in methionine catabolism, the transsulfuration pathway also provides cysteine for glutathione synthesis. Cysteine availability is often limiting for glutathione synthesis, and it appears that in a number of cells (e.g., hepatocytes), at least half of the cysteine required is provided by transsulfuration, even in the presence of physiological concentrations of cysteine (19). Transsulfuration is sensitive to the balance of prooxidants and antioxidants; peroxides increase the transsulfuration flux, whereas antioxidants decrease it (20). It is thought that redox regulation of the transsulfuration pathway occurs at the level of CBS, which contains a heme that may serve as a sensor of the oxidative environment (21).


Taurine is remarkable, both for its high concentrations in animal tissues and because of the variety of functions that have been ascribed to it. Taurine is the most abundant free amino acid in animal tissues. Table 1 shows that, although taurine accounts for only 3% of the free amino acid pool in plasma, it accounts for 25%, 50%, 53%, and 19%, respectively, of this pool in liver, kidney, muscle, and brain. The magnitude of the intracellular taurine pool deserves comment. For example, skeletal muscle contains 15.6 μmol of taurine per gram of tissue, which amounts to an intracellular concentration of about 25 mM. In addition to its role in the synthesis of the bile salt taurocholate, taurine has been proposed, inter alia, to act as an antioxidant, an intracellular osmolyte, a membrane stabilizer, and a neurotransmitter. It is an essential nutrient for cats; kittens born to mothers fed taurine-deficient diets exhibit retinal degeneration (24). Taurine is found in mother’s milk, may be conditionally essential for human infants, and is routinely added to most infant formulas. Recent work has begun to reveal taurine’s action in the retina. It appears that taurine, via an effect on a glycine receptor, promotes the generation of rod photoreceptor cells from retinal progenitor cells (25).

View this table:


Taurine concentrations in rat tissues (22,23)


The sulfur-containing amino acids present a fascinating subject to the protein chemist, the nutritionist, and the metabolic scientist, alike. They play critical roles in protein synthesis, structure, and function. Their metabolism is vital for many critical functions. SAM, a remarkably versatile molecule, is said to be second, only to ATP, in the number of enzymes that require it. Vitamins play a crucial role in the metabolism of these amino acids, which, in turn, play a role in folic acid assimilation. Despite the great advances in our knowledge of the sulfur-containing amino acids, there are important areas where further work is required. These include methionine transamination and the molecular basis for the many functions of taurine.

Disorders of Sulfur Amino Acid Metabolism

  • Generoso Andria,  Brian Fowler,  Gianfranco Sebastio

Chapter  Inborn Metabolic Diseases  pp 224-231


Several defects can exist in the conversion of the sulfur-containing amino acid methionine to cysteine and the ultimate oxidation of cysteine to inorganic sulfate (Fig. 18.1). Cystathionine-β-synthase (CBS) deficiency is the most important. It is associated with severe abnormalities of four organs or organ systems: the eye (dislocation of the lens), the skeleton (dolichostenomelia and arachnodactyly), the vascular system (thromboembolism), and the central nervous system (mental retardation, cerebrovascular accidents). A low-methionine, highcystine diet, pyridoxine, folate, and betaine in various combinations, and antithrombotic treatment may halt the otherwise unfavorable course of the disease. Methionine adenosyltransferase deficiency and γ-cystathionase deficiency usually do not require treatment. Isolated sulfite oxidase deficiency leads (in its severe form) to refractory convulsions, lens dislocation, and early death. No effective treatment exists.

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    Mudd SH, Skovby F, Levy HL, Pettigrew KD, Wilcken B, Pyeritz RE, Andria G, Boers GHJ, Bromberg IL, Cerone R, Fowler B, Grobe H, Schmidt H, Schweitzer L (1985) The natural history of homocystinuria due to cystathionine (3-synthase deficiency. Am J Hum Genet 37: 1–31 PubMed

  5. 5.

    de Franchis R, Sperandeo MP, Sebastio G, Andria G. The Italian Collaborative Study Group on Homocystinuria (1998) Clinical aspects of cystathionine ß-synthase deficiency: how wide is the spectrum? Eur J Pediatr 157: S67–7o

  6. 6.

    Kraus JP (1994) Molecular basis of phenotype expression in homocystinuria. J Inherited Metab Dis 17: 383–390 PubMedCrossRef

  7. more…

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The good, the bad and the ugly of sulfur and volcanic activity

Larry H Bernstein, MD, FCAP, Curator



Climate change deniers have promulgated much ignorance about the planet and our life on earth.  Nevertheless, I shall deal with geophysical and geochemical issues and indirectly, climate change in this portion of the discussion.  The good, the bad, and the ugly has everything to due with the elements and to life on earth.  This is the case, regardless of claims propagated by the tobacco and the carbon fuels interests.  I shall proceed as I have done in the previous discussions.

Is a Lack of Water to Blame for the Conflict in Syria?

A 2006 drought pushed Syrian farmers to migrate to urban centers, setting the stage for massive uprisings

By Joshua Hammer



An Iraqi girl stands on former marshland, drained in the 1990s because of politically motivated water policies. (Essam Al-Sudani / AFP / Getty Images)

The world’s earliest documented water war happened 4,500 years ago, when the armies of Lagash and Umma, city-states near the junction of the Tigris and Euphrates rivers, battled with spears and chariots after Umma’s king drained an irrigation canal leading from the Tigris. “Enannatum, ruler of Lagash, went into battle,” reads an account carved into an ancient stone cylinder, and “left behind 60 soldiers [dead] on the bank of the canal.”


Water loss documented by the Gravity Recovery and Climate Experiment (GRACE), a pair of satellites operated by NASA and Germany’s aerospace center, suggests water-related conflict could be brewing on the riverbank again. GRACE measured groundwater usage between 2003 and 2009 and found that the Tigris-Euphrates Basin—comprising Turkey, Syria, Iraq and western Iran—is losing water faster than any other place in the world except northern India . During those six years, 117 million acre-feet of stored freshwater vanished from the region as a result of dwindling rainfall and poor water management policies. That’s equal to all the water in the Dead Sea. GRACE’s director, Jay Famiglietti, a hydrologist at the University of California, Irvine, calls the data “alarming.”

While the scientists captured dropping water levels, political experts have observed rising tensions. In Iraq, the absence of a strong government since 2003, drought and shrinking aquifers have led to a recent spate of assassinations of irrigation department officials and clashes between rural clans. Some experts say that these local feuds could escalate into full-scale armed conflicts .

In Syria, a devastating drought beginning in 2006 forced many farmers to abandon their fields and migrate to urban centers. There’s some evidence that the migration fueled the civil war there, in which 80,000 people have died. “You had a lot of angry, unemployed men helping to trigger a revolution,” says Aaron Wolf, a water management expert at Oregon State University, who frequently visits the Middle East.

Tensions between nations are also high. Since 1975, Turkey’s dam and hydro­power construction has cut water flow to Iraq by 80 percent and to Syria by 40 percent. Syria and Iraq have accused Turkey of hoarding water.

Hydrologists say that the countries need to find alternatives to sucking the aquifers dry—perhaps recycling wastewater or introducing desalination—and develop equitable ways of sharing their rivers. “Water doesn’t know political boundaries. People have to get together and work,” Famiglietti says. One example lies nearby, in an area not known for cross-border cooperation. Israeli and Jordanian officials met last year for the first time in two decades to discuss rehabilitating the nearly dry Jordan River, and Israel has agreed to release freshwater down the river.

“It could be a model” for the Tigris-Euphrates region, says Gidon Bromberg, a co-director of Friends of the Earth Middle East, who helped get the countries together. Wolf, too, remains optimistic, noting that stress can encourage compromise.

History might suggest a way: The world’s first international water treaty, a cuneiform tablet now hanging in the Louvre, ended the war between Lagash and Umma.

“Rebel forces are targeting water installations to cut off supplies to the largely Shia south of Iraq,” says Matthew Machowski, a Middle East security researcher at the UK houses of parliament and Queen Mary University of London.

“It is already being used as an instrument of war by all sides. One could claim that controlling water resources in Iraq is even more important than controlling the oil refineries, especially in summer. Control of the water supply is fundamentally important. Cut it off and you create great sanitation and health crises,” he said

Isis now controls the Samarra barrage west of Baghdad on the River Tigris and areas around the giant Mosul Dam, higher up on the same river. Because much of Kurdistan depends on the dam, it is strongly defended by Kurdish peshmerga forces and is unlikely to fall without a fierce fight, says Machowski.

Iraqi troops were rushed to defend the massive 8km-long Haditha Dam and its hydroelectrical works on the Euphrates to stop it falling into the hands of Isis forces. Were the dam to fall, say analysts, Isis would control much of Iraq’s electricity and the rebels might fatally tighten their grip on Baghdad.

Isis fighters in Fallujah captured the smaller Nuaimiyah Dam on the Euphrates and deliberately diverted its water to “drown” government forces in the surrounding area. Millions of people in the cities of Karbala, Najaf, Babylon and Nasiriyah had their water cut off but the town of Abu Ghraib was catastrophically flooded along with farms and villages over 200 square miles. According to the UN, around 12,000 families lost their homes.

Earlier, Kurdish forces reportedly diverted water supplies from the Mosul Dam. Equally, Turkey has been accused of reducing flows to the giant Lake Assad, Syria’s largest body of fresh water, to cut off supplies to Aleppo, and Isis forces have reportedly targeted water supplies in the refugee camps set up for internally displaced people.

Iraqis fled from Mosul after Isis cut off power and water and only returned when they were restored, says Machowski. “When they restored water supplies to Mosul, the Sunnis saw it as liberation. Control of water resources in the Mosul area is one reason why people returned,” said Machowski.

Both Isis forces and President Assad’s army are said to have used water tactics to control the city of Aleppo. The Tishrin Dam on the Euphrates, 60 miles east of the city, was captured by Isis in November 2012.

“The deliberate targeting of water supply networks … is now a daily occurrence in the conflict. The water pumping station in Al-Khafsah, Aleppo, stopped working on 10 May, cutting off water supply to half of the city.

A satellite view showing the two main rivers running from Turkey through Syria and Iraq. Credits: MODIS/NASA

The Euphrates River, the Middle East’s second longest river, and the Tigris, have historically been at the centre of conflict. In the 1980s, Saddam Hussein drained 90% of the vast Mesopotamian marshes that were fed by the two rivers to punish the Shias who rose up against his regime. Since 1975, Turkey’s dam and hydropower constructions on the two rivers have cut water flow to Iraq by 80% and to Syria by 40%. Both Syria and Iraq have accused Turkey of hoarding water and threatening their water supply.

The Barada River, shown here in Damascus, is the only notable river flowing entirely within Syrian territory. The city’s water supplies are under huge strain

DAMASCUS, 25 March 2010 (IRIN) – Poor planning and management, wasteful irrigation systems, intensive wheat and cotton farming and a rapidly growing population are straining water resources in Syria in a year which has seen unprecedented internal displacement as a result of drought in eastern and northeastern parts of the country.

In 2007 Syria consumed 19.2 billion cubic metres of water – 3.5 billion more than the amount of water replenished naturally, with the deficit coming from groundwater and reservoirs, according to the Ministry of Irrigation.

Agriculture accounts for almost 90 percent of the country’s water consumption, according to government and private sector.

Agricultural policies encourage water-hungry wheat and cotton cultivation, and inefficient irrigation methods mean much water is wasted.


South Asia is a desperately water-insecure region, and India’s shortages are part of a wider continental crisis. According to a recent report authored by UN climate scientists, coastal areas in Asia will be among the worst affected by climate change. Hundreds of millions of people across East, Southeast and South Asia, the report concluded, will be affected by flooding, droughts, famine, increases in the costs of food and energy, and rising sea levels.

Groundwater serves as a vital buffer against the volatility of monsoon rains, and India’s falling water table therefore threatens catastrophe. 60 percent of north India’s irrigated agriculture is dependent on ground water, as is 85 percent of the region’s drinking water. The World Bank predicts that India only has 20 years before its aquifers will reach “critical condition” – when demand for water will outstrip supply – an eventuality that will devastate the region’s food security, economic growth and livelihoods.

Analysts fear that growing competition for rapidly dwindling natural resources will trigger inter-state or intra-state conflict. China and India continue to draw on water sources that supply the wider region, and a particularly concerning flashpoint is the Indus River Valley basin that spans India and Pakistan. The river’s waters are vital to the economies of areas on both sides of the border and a long-standing treaty, agreed by Pakistan and India in 1960, governs rights of access. But during the “dry season,” between October and March, water levels fall to less than half of those seen during the remainder of the year. The fear is that cooperation over access to the Indus River will fray as shortages become more desperate.

Farm worker heading for the paddy fields at Gubinder Singh’s farm

The Indo-Gangetic Basin, which lies at the foothills of the Himalayas, is one of the areas in the world facing a huge water crisis.  The Basin spans from Pakistan, across Northern India into Bangladesh. Apart from runoff from mountainous streams and glaciers, it also holds one of the largest underground bodies of water in the world. But it’s also in one of the most populous regions of the world, with more than a billion people living on the subcontinent.  Still, parts of the region are well-resourced when it comes to water supplies – like the Indian state of Punjab, which has three rivers running through it and a network of canals in some parts.

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NASA Satellites Unlock Secret to Northern India’s Vanishing Water



NASA Hydrologist Matt Rodell discusses vanishing groundwater in India. Credit:NASA
› Watch Video


soil moisture belt

soil moisture belt


Groundwater resides beneath the soil surface in permeable rock, clay and sand as illustrated in this conceptual image. Many aquifers extend hundreds of feet underground and in some instances have filled with water over the course of thousands of years. Credit: NASA

groundwater withdrawals as a percentage of groundwater recharge

groundwater withdrawals as a percentage of groundwater recharge



The map, showing groundwater withdrawals as a percentage of groundwater recharge, is based on state-level estimates of annual withdrawals and recharge reported by India’s Ministry of Water Resources. The three states included in this study are labeled. Credit:NASA/Matt Rodell

The averaging function (spatial weighting) used to estimate terrestrial water storage changes from GRACE data is mapped. Warmer colors indicate greater sensitivity to terrestrial water storage changes. Credit: NASA/Matt Rodell

Beneath northern India’s irrigated fields of wheat, rice, and barley … beneath its densely populated cities of Jaiphur and New Delhi, the groundwater has been disappearing. Halfway around the world, hydrologists, including Matt Rodell of NASA, have been hunting for it.

Where is northern India’s underground water supply going? According to Rodell and colleagues, it is being pumped and consumed by human activities — principally to irrigate cropland — faster than the aquifers can be replenished by natural processes. They based their conclusions — published in the August 20 issue of Nature — on observations from NASA’s Gravity Recovery and Climate Experiment (GRACE).

“If measures are not taken to ensure sustainable groundwater usage, consequences for the 114 million residents of the region may include a collapse of agricultural output and severe shortages of potable water,” said Rodell, who is based at NASA’s Goddard Space Flight Center in Greenbelt, Md.

The Himalayas are representative of a modern and active mountain-building event, called anorogeny in geologic parlance. Both the Himalayas and the Cascade Range are the result of plate-to-plate collision in the Theory of Plate Tectonics.
The difference between the Himalayas and the Cascade Range volcanoes is based on density of the lithospheric plates. Yes. The Cascade Range is caused by subduction of more dense ocean crust into and underneath lighter, lower density continental crust. As the oceanic plate dives deeper and deeper, the ocean crust warms, melts, and rises upward through the overriding continental crust “inland” from the plate collision boundary. As that molten rock punches through the continental crust, a curvilinear series of volcanoes, generally parallel to the plate collision boundary, begins to form.

Cascade Range Subduction

Cascade Range Subduction


Cascade Range Subduction from J. Wiley & Sons – 2010
In the case of the Cascade Range, the name of this type of volcanic formation is unique in process, as well as geochemistry, and has been referred to as an Andesitic-type after the Andes Mountains. Regardless, the Cascade Range is comprised of intermediate igneous rocks, with a fairly high silica content. High silica makes for high siliceous acid. That creates “sticky” igneous extrusions that often have quite dramatic eruptions [May 1980 Mt. St. Helens eruption].


Igneous Rock Classification

Igneous Rock Classification

Igneous Rock Classification Chart – Public Domain

The Himalayas are also a plate-to-plate collision tectonic boundary. In this case, the Indian Plate [of the Indian Subcontinent] is colliding head-on with the Eurasian Plate. Both plates are comprised of continental lithospheric crust, so there is no appreciable distinction in density. Both have a density of approximately 2.7 g/cm³. This as opposed to ocean crust with a mean density of 3.3 g/cm³. The plates try to compete in the plate-to-plate collision but the equal densities of the two plates cannot push one under the other very deep like that in a subduction zone.  The result is large-scale thickening of the continental crust in the region at and surrounding the collision boundary. Other processes occurring in the Himalayas region associated with the orogeny are metamorphism, thrust [compression] faulting, and plateau uplift.

Depiction of Himalayan Collision

Depiction of Himalayan Collision

Generalized Depiction of Himalayan Collision from FHSU – 2010
A perfect analogy is two trucks of the same make and model colliding head-on. The Himalayan Orogeny is the oft mentioned “crumple zone”. Metal does not deform in a brittle sense like competent rock does, so don’t confuse that too much.

With all that being said, there are tremendous temperatures attained at a continental plate-to-plate collision boundary. However, the crust is simply too thick, and too “squashed together” to allow anything to squeeze up and break through to the surface as volcanic eruptions.

FHSU,  2010.  Image of Himalayan Collision.  Fort Hays State University.  Hays, Kansas.  2010.
Wiley & Sons, J.,  2010.  Image of Cascade Range Subduction Zone.  J. Wiley & Sons.  Hoboken, New Jersey.  2010.


Mt. Everest was formed (is forming) by two tectonic plates colliding–the Indo-Australian Plate and the Eurasian plate.

Sometimes, when two tectonic plates collide, volcanoes form (such as the Juan de Fuca plate and the North American Plate forming the Cascades). However, this has to do with one plate–in this case the Juan de Fuca Plate sliding or subducting beneath another–the North American Plate. This happens because the oceanic plate (the Juan de Fuca Plate) is more dense than than the continental plate (the NA Plate). For reasons I won’t get into here, magma forms between the two plates as one subducts beneath the other and volcanoes are formed.

Mt. Everest is formed by two continental plates colliding. Continental plates are generally too buoyant to subduct beneath each other. While some subduction occurred during this collision, most of what happened was crustal shortening. Think about what happens when you have a rug on a wood floor and push two ends toward each other. It buckles and folds up in itself. This is a simplified version of what happened in the Himalaya.

Because little to no subduction is occurring, no magma is forming and Mt. Everest will not become a volcano.

The Himalayas were created by two continental plates colliding. What happens when two masses of rocks with some similarities, like in density, collide? Both of them rise. There is a lot of heat produced. However, there isn’t enough heat to melt rocks completely. For there to be a volcano, there has to be a source of molten rock.

This material can occur if the two masses of rocks have vastly different densities. In this case, the heavier mass will slide above the other. The mass on the bottom will melt. This molten rock material will rise and create a volcano. or two or more. This, however can not happen in the HImalayas. The two masses in action are the Indian Plate and the Eurasian Plate, which have similar rock density.


Volcanic Eruptions and the Role of Sulfur Dioxide in Climate Change

In March and April of this year, a series of severe volcanic eruptions shook Alaska’s Mount Redoubt.1  To date, the largest of the eruptions produced an ash plume that reached 50,000 feet above sea level and released a significant amount of sulfur dioxide (CAS Registry Number® 7446-09-5) into the earth’s atmosphere.  According to the Alaska Volcano Observatory, “The main concerns for human health in volcanic haze consist of ash, sulfur dioxide gas (SO2), and sulfuric acid droplets (H2SO4), which forms when volcanic SO2 oxidizes in the atmosphere.”1

While there is obvious reason for alarm among local populations, sulfur dioxide from the Mount Redoubt eruption could also have more widespread impacts, particularly on the climate.  According to a 1997 article published in the Journal of Geology, “The mechanism by which large eruptions affect climate is generally accepted: injection of sulfur into the stratosphere and conversion to sulfate aerosol, which in turn reduces the solar energy reaching the earth’s surface.”2

In the years following a volcanic eruption, sulfate aerosol that remains in the atmosphere is thought to cause surface cooling by reflecting the sun’s energy back into space.  In fact, sulfate aerosol from the massive eruption of Indonesia’s Mount Tambora in 1815 is blamed, at least in part, for the “year without a summer” reported in Europe and North America in 1816:

  • “Daily temperatures (especially the daily minimums) were in many cases abnormally low from late spring through early fall; frequent northwest winds brought snow and frost to northern New England and Canada, and heavy rains fell in western Europe.  Many crops failed to ripen, and the poor harvests led to famine, disease, and social distress…”3

Supporting this claim, sulfate aerosol-related climate changes were also reported after the 1991 eruption of Mount Pinatubo in the Philippines.4  An article published inScience in 2002 summarizes a decade’s worth of research on Pinatubo’s effects on the global climate, highlighting impacts far more widespread and complex than previously thought:

You can use SciFinder® or STN® to search the CAS databases for additional information about sulfur dioxide from volcanic eruptions.  If your organization is enabled to use the web version of SciFinder, you can click the links in this article to directly access details of the substances and references.


Volcanic ash vs sulfur aerosols

The primary role of volcanic sulfur aerosols in causing short-term changes in the world’s climate following some eruptions, instead of volcanic ash, was hypothesized by scientists in the early 1980’s. They based their hypothesis on the effects of several explosive eruptions in Indonesia and the world’s largest historical effusive eruption in Iceland.

Scientists studied three historical explosive eruptions of different sizes in Indonesia–Tambora (1815), Krakatau (1883), and Agung (1963). They noted that decreases in surface temperatures after the eruptions were of similar magnitude (0.18-1.3 °C). The amount of material injected into the stratosphere, however, differed greatly. By comparing the estimated amount of ash vs. sulfur injected into the stratosphere by each eruption, it was suggested that the longer residence time of sulfate aerosols, not the ash particles which fall out within a few months of an eruption, was the paramount controlling factor (Rampino and Self, 1982).

In contrast to these explosive eruptions, one of the most severe volcano-related climate effects in historical times was associated with a largely nonexplosive eruption that produced very little ash–the 1783 eruption of Laki crater-row in Iceland. The eruption lasted 8-9 months and extruded about 12.3 km3 of basaltic lava over an area of 565 km2. A bluish haze of sulfur aerosols all over Iceland destroyed most summer crops in the country; the crop failure led to the loss of 75% of all livestock and the deaths of 24% of the population (H. Sigurdsson, 1982). The bluish haze drifted east across Europe during the 1783-1784 winter, which was unusually severe.

Clearly, these examples suggested that the explosivity of an eruption and the amount of ash injected into the stratosphere are not the main factors in causing a change in Earth’s climate. Instead, scientists concluded that it must be the amount of sulfur in the erupting magma.

The eruption of El Chichon, Mexico, in 1982 conclusively demonstrated this idea was correct. The explosive eruption injected at least 8 Mt of sulfur aerosols into the atmosphere, and it was followed by a measureable cooling of parts of the Earth’s surface and a warming of the upper atmosphere. A similar-sized eruption at Mount St. Helens in 1980, however, injected only about 1 Mt of sulfur aerosols into the stratosphere. The eruption of Mount St. Helens injected much less sulfur into the atmosphere–it did not result in a noticeable cooling of the Earth’s surface. The newly launched TOMS satellite (in 1978) made it possible to measure these differences in the eruption clouds. Such direct measurements of the eruption clouds combined with surface temperatures make it possible to study the corrleation between volcanic sulfur aerosols (instead of ash) and temporary changes in the world’s climate after some volcanic eruptions.


Hazards Of Volcanic Ash

A multitude of dangerous particals and gases, such as aerosols, are carried in volcanic ash. Some of these include;

  • Carbon dioxide
  • Sulfates (sulfur dioxide)
  • Hydrochloric acid
  • Hydroflouric acid

These each have different but serious effects on human health if exposed, which will be discussed later.

In addition, volcanic ash can cause reduced visibility, and it is recommended that precautions are taken when driving.

Sources: Where Does It Come From?

Figure 1

volcanoes found all over the Earth, particularly at plate boundaries

volcanoes found all over the Earth, particularly at plate boundaries

There are volcanoes found all over the Earth, particularly at plate boundaries (see figure 1). This is due to the collision of plates, which causes uplift in the overlying crust. This uplift results in the formation of mountainous landforms; melting of the crust due to frictional heating is what creates magma, which can erupt out of these mountains when pressure gets too high.

Some of the most notable volcanic eruptions are:

  • the 1783 eruption of Mt. Laki in Iceland
    • released clouds of poisonous flourine and sulfur dioxide which killed off about 50% of the livestock population
    • that summer in Great Britain was known as “sand-summer” due to ash carried over the Atlantic
    • poisonous clouds spread over Europe, and a buildup of aerosols caused a cooling effect in the entire Northern Hemisphere
  • the 1815 eruption of Mt. Tambora in Indonesia
    • gas releases caused the Stratosphere to change drastically
    • noxious ash and poisoned rain clouds killed off vegetation
  • the 1902 eruption of Mt. Pelee in Martinique
    • spewed toxic clouds traveling at speeds of 600mph
    • largest eruption in the 20th century

For further information on volcanoes around the world, visit


  • EEA-33 emissions of sulphur oxides (SOX) have decreased by 74% between 1990 and 2011. In 2011, the most significant sectoral source of SOX emissions was ‘Energy production and distribution’ (58% of total emissions), followed by emissions occurring from ‘Energy use in industry’ (20%) and in the ‘Commercial, institutional and households’ (15%) sector.
  • The reduction in emissions since 1990 has been achieved as a result of a combination of measures, including fuel-switching in energy-related sectors away from high-sulphur solid and liquid fuels to low-sulphur fuels such as natural gas, the fitting of flue gas desulphurisation abatement technology in industrial facilities and the impact of European Union directives relating to the sulphur content of certain liquid fuels.
  • All of the EU-28 Member States have reduced their national SOX emissions below the level of the 2010 emission ceilings set in the National Emission Ceilings Directive (NECD)[1]. Emissions in 2011 for the three EEA countries having emission ceilings set under the UNECE/CLRTAP Gothenburg protocol (Liechtenstein, Norway and Switzerland) were also below the level of their respective 2010 ceilings.
  • Environmental context: Typically, sulphur dioxide is emitted when fuels or other materials containing sulphur are combusted or oxidised. It is a pollutant that contributes to acid deposition, which, in turn, can lead to changes in soil and water quality. The subsequent impacts of acid deposition can be significant, including adverse effects on aquatic ecosystems in rivers and lakes and damage to forests, crops and other vegetation. SO2 emissions also aggravate asthma conditions and can reduce lung function and inflame the respiratory tract. They also contribute, as a secondary particulate pollutant, to the formation of particulate matter in the atmosphere, an important air pollutant in terms of its adverse impact on human health. Furthermore, the formation of sulphate particles in the atmosphere following the release of SO2 results in reflection of solar radiation, which leads to net cooling of the atmosphere.
faults  sn-seafloor

faults sn-seafloor


Glacier - Helheim

Glacier – Helheim


Making North America

Making North America





What caused the Nepal earthquake

What caused the Nepal earthquake

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Pyrroloquinoline quinone (PQQ) – an unproved supplement

Larry H. Bernstein, MD, FCAP, Curator


Pyrroloquinoline quinone (PQQ) 

Pyrroloquinoline quinone (henceforth PQQ) is a small quinone molecule which has the ability to be a REDOX agent, capable of reducing oxidants (an antioxidant effect) and then being recycled by glutathione back into an active form. It appears to be quite stable as it can undergo several thousand cycles before being used up, and it is novel since it associates with protein structures inside the cell (some antioxidants, mostly notably carotenoids like β-carotene and Astaxanthin, are located at specific areas of a cell where they exert proportionally more antioxidant effects due to proximity; PQQ seems to do this near proteins like carotenoids do so at the cell membrane).

The aforementioned REDOX functions can alter protein function and signaling pathways, and while there is a lot of promising in vitro (outside of a living model) research on what it could do there are only a few promising results of PQQ supplementation, mostly related to either altering some signaling pathways or via its benefits to mitochondria (producing more of them and increasing their efficiency).

It is a coenzyme in bacteria (so, to bacteria, this would be something like a B-vitamin) but this role does not appear to extend to humans. Since this does not extend to humans, the designation of PQQ as a vitamin compound has fallen through and it is only considered ‘vitamin-like’ at best.

PQQ seems to modify oxidation in a cell after binding to some proteins, and this modulatory role it plays can alter the signalling processes that go on in a cell. Due to PQQ being a REDOX agent (capable of both reducing and oxidizing) it is not a pure antioxidant, but it is involved in a cyclical antioxidative cycle with an antioxidant enzyme known as glutathione

For human evidence, the limited evidence we have right now suggests a possible neuroprotective role in the aged (no research in clinical situations of neurodegeneration nor in youth) and it may have an antiinflammatory role. This limited evidence also suggests that the main claim of PQQ, an enhancement of mitochondrial function, occurs in otherwise healthy humans given PQQ supplementation.

The animal evidence that might apply to humans (using oral supplementation at doses similar to what humans use) include a radioprotective effect, possible benefits to insulin resistance, and being a growth factor when PQQ is added to the diet over a long period of time. Higher than normal oral doses in rodents seem to also enhance peripheral neurogenesis (nerve growth outside of the brain) but not necessarily in the brain.

A large amount of the evidence for a direct antioxidant role or the neurological actions related to NMDA signalling of PQQ seem to use very high concentrations in cells, due to possible transportation issues to the brain and low concentrations of PQQ found in the blood following oral ingestion.

It holds a potential to modify signalling in humans, and although the oxidation in the blood (easiest thing to measure) in mostly unaffected it also retains the potential to act as an intracellular antioxidant. The enhancement of mitochondrial function may also occur, but beyond some alterations in signalling and the mitochondrial biogenesis most other properties of PQQ are unlikely to extend to humans.

  1. Sources and Structure

1.1. Sources

Pyrroloquinoline quinone (PQQ) is a quinone molecule that was first identified as an enzymatic cofactor in bacteria, acting as a prosthetic group similar to how B-vitamins work in humans.[1] It is doubtful that PQQ is an enzymatic cofactor in humans, although it still appears to have affinity to proteins in the human body and can bind to them to confer biolgical effects. The proteins that seem to bind to PQQ are called quinoproteins,[2] and via modifying their actions in the body PQQ can exert biological activity.

PQQ was once thought to be a novel vitamin compound, although this view has since had doubts cast upon it and is no longer seen as accurate. Despite the lack of a vitamin role in mammals, it does appear to have growth promoting properties in rodents and may be active in humans following supplementation

PQQ naturally occurs in most foods (in miniscule amounts) although the highest levels can be found in:

  • Fermented Soybeans products such as Nattō (highest estimate of 61+/-31 ng/g wet weight,[3] lower estimates in the range of 1.42 +/- 0.32ng/g[4])
  • Green Soybeans (9.26+/-3.82ng/g wet weight)[3]
  • Spinach (7.02 +/- 2.17ng/g fresh weight)[4]
  • Rape blossoms (blossoms of the brassica napus plant at 5.44 +/- 0.8ng/g fresh weight)[4]
  • Field Mustard (5.54 +/-1.50ng/g fresh weight)[4]
  • Tofu (24.4+/-12.5ng/g wet weight)[3]
  • Teas from Camellia Sinensis, aka Green Tea (around 30ng/g dry weight of leaves)[3] with the lower range of estimates at 0.16 +/- 0.05[4]
  • Green peppers, Parsely, and Kiwi fruits (around 30ng/g wet weight or so)[3] although some estimates are lower (2.12 +/- 0.40ng/g for green peppers)[4]
  • Human Breast milk at 140-180ng/mL (total PQQ and IPQ)[5]

Overall content of PQQ in foods seems to range from 0.19-7.02ng/g fresh weight in one study[4] up to 3.7-61ng/g in another,[3] low numbers may not adequately reflect total content in foods due to excluding IPQ in the measurements whereas higher levels tend to include both PQQ and IPQ.[5]

PQQ is present in a wide variety of foods, but currently the estimates of its contents are quite variable. This may be due to confusion as to whether solely PQQ should be counted or PQQ conjugates (it is not known if these confer dietary benefit). In general, the PQQ content of food products listed above is substantially lower than the content of supplemented PQQ (10-20mg) and food ingestion is unlikely to replicate the effects of supplementation due to the magnitude of difference

It should be noted that due to an affinity of PQQ to bind to amino acids and form imidazolopyrroloquinoline derivatives that the PQQ content of foods may not be the same as the total bioactive amounts of PQQ,[6] probably due to rapid association with proteins forming amino acid conjugates (Imidazolopyrroloquinoline, or IPQ).[7] Human milk, for example, contained 15% PQQ and 85% IPQ derivatives. That being said, no direct studies have been undertaken to see whether PQQ and IPQ have similar or different properties in vivo.

PQQ may form conjugates with dietary protein similar to how it is known to react with proteins in the body, but it is not known if this potential interaction with dietary protein is beneficial or negatively influences bioavailability

1.2. Structure and Properties

Pyrroloquinoline quinone is heat-stable and water soluble,[1] and appears to be stable at ambient temperatures in the form of PQQ disodium salt either as trihydrate (12.7% water[8]) or pentahydrate (22.9% water[9]). It is thought to be a relatively stable REDOX factor in vivo, and is able to carry generally around 20,000 REDOX reactions before degradation,[10][11] and when it carries out REDOX reactions by itself it gets converted into its reduced form known as pyrroloquinoline dihydroquinone (PQQH2)[12] and is replenished (back to the PQQ form) by glutathione.[12]

PQQ binds to proteins via forming a schiff base, which is a spontaneous (no enzyme required) reaction to amino acids found in the protein structures such as lysine.[13] The binding of PQQ to proteins uses the carbonyl groups (C=O),[14]including the three carboxylic groups opposite of the two ketones used in REDOX reactions.

Pyrroloquinline quinone (PQQ) is a quinone structure with three carboxylic acid groups which are used to bind to proteins, and two ketone groups which are involved in the REDOX capacities of the molecule

In some in vitro studies, combining PQQ with reducing agents (SIN-1, sodium borohydride) can form a green precipitate[15] and the reddish coloration of PQQ turns increasing brown when water content is removed.[8]

PQQ (as a powder) appears to be able to change color depending on its hydration status and oxidation status

1.3. Biological Significance

PQQ was initially thought to be synthesized via the α-amino adipic acid-Δ-semialdehyde (AASDH; also known as U26) enzyme[16] although this seems to be incorrect[17][18] since despite this protein having many PQQ binding sites[19] its mRNA levels are not negatively regulated by PQQ levels[20] which would most likely occur if the enzyme synthesized PQQ. It is known to be synthesized (in bacteria) from the amino acids L-Tyrosine and glutamate[21][22] in a process requiring a series of enzymes labelled PqqA-F where PqqA formed the peptide precursor and the other enzymes structurally modify it into active PQQ.[23]

Although mammalian synthesis is not certain, PQQ does occur normally in the mammalian body[24] and approxiamtely 100-400 nanograms of PQQ are thought to be made in humans each day;[3][25] leading some authors to claim an estimated tissue concentration of approximately 0.8−5.9ng/g in humans.[3]

Since complete deprivation from the diet of animals has been shown to hinder growth and reproductive performance,[26][7] it was initially thought that this (paired with the initial guess of endogenous synthesis via AASDH) indicated a vitamin deficiency. However, due to the definition of vitamins being one that requires a disease state to occur during deficiency[16] and no apparent dysfunction aside from impaired growth seen with PQQ deficeincy it was not classified as an essential vitamin;[17] this claim of no vitamin-like property being supported by the idea that AASDH is not actually used for PQQ sythesis in humans.[17][18]

Pyrroloquinoline quinone (PQQ) is known to occur in both the diet and in mammalian tissue, and appears to have biological activity in the body. It was initially thought to be a new vitamin, but this conclusion seems unlikely and it is more likely a bioactive non-vitamin compound.

PQQ has been investigated for being a growth factor in youth (since deprivation in rats impairs growth[26][7]), secondary to its effects at improving mitochondrial biogenesis (making more mitochondria) at seemingly effective doses of 0.2-0.3mg/kg foodstuff (in mice),[27] which is surprisingly close to the levels found in human breast milk.[5] Preliminary evidence for mitochondrial efficacy has also been noted in adult humans given 0.075-0.3mg/kg daily,[28] with the latter dose being close to the recommended 20mg serving for a 150lb adult.

PQQ is thought to be a non-vitamin growth factor, in part due to its naturally high levels in breast milk and reduced growth in rats without dietary PQQ. It may do so via beneficially influencing mitochondrial function

It is seen as a novel REDOX catalyzing agent due to its stability, which prevents most self-oxidation (seen in catechins) and polymerization (tannins).[10] A case has been made that PQQs effects are constant between species and bacteria, which aims to validate extrapolation from one species to humans.[10] The potency of PQQ and its quinoproteins in REDOX cycling appears to be approximately 100-fold greater than Vitamin C or other polyphenolic compounds, when in alkaline conditions.[25][29][30]

PQQ, after associating with proteins (not in the role of a cofactor) appears to be capable of REDOX cycling suggesting that it can have conditional prooxidant and antioxidant roles. The association with proteins suggests that it can modify their structures either directly or via modifying the levels of oxidation at the level of the protein (similar to how carotenoids such as Astaxanthin are located at the cellular membrane which localizes their effects)

  1. Molecular Targets

2.1. Enzymatic Cofactor

Pyrroloquinoline quinone (PQQ) was discovered in 1979 as an enymatic cofactor in bacteria;[31] preliminary evidence in pig kidneys and adrenal glands suggested a similar role in mammals.[32][33][34][35] Doubts were later cast upon the role of PQQ as a mammalian enzymatic cofactor,[36][37][38] and currently the consensus is that PQQ is unlikely to be an enzymatic cofactor in humans as it is in bacteria and plants.

Pyrroloquinoline quinone (PQQ) was first discovered as a bacterial enzymatic cofactor (being required by bacterial enzymes to function properly) and preliminary evidence suggested it could play the same role in mammals, which would make PQQ a vitamin. But further study found no quality evidence supporting this role in mammals; it is currently believed that PQQ does not act as an enzymatic cofactor in humans

2.2. REDOX Signalling

REDOX (REDuction OXidation) signalling refers to stimulation or inhibition of cellular signalling systems by molecules that can switch from an oxidized state to a reduced state, such as the well-known REDOX-acting supplements Vitamin C andAlpha-Lipoic Acid.[39] Pyrroloquinoline quinone (PQQ) may have this property as well, although its primary mode of action seems to be acting on known REDOX proteins in the cell; this is in line with its high binding affinity for some proteins, despite not acting as their coenzyme.[40][21] For example, PQQ may function as a mammalian growth factor via signal transduction modification by both oxidation and redox cycling[41], and has been shown to improve insulin signalling in mice by redox cycling.[42]

PQQ may have an indirect influence on REDOX signalling in a cell by modifying the actions of proteins, which may underlie some antioxidative (and prooxidative) changes in a cell similar to any other REDOX agent

2.3. Thioredoxin Reductase 1

PQQ has been noted to partially inhibit thioredoxin reductase 1 (TrxR1), which is an enzyme in the cytosol that reducesthioredoxin.[43] PQQ has low potency yet high affinity in binding to TrxR1 and seems to outcompete thioredoxin binding.[44] When PQQ binds to TrxR1, the enzyme’s activity is modified so it acts more on an alternate substrate known as juglone.[45] Overall, NADPH oxidase activity of TrxR1 (a measure of the activity of this enzyme) is increased in the presence of 10-50µM PQQ due to increased activity of the TrxR1-Juglone interaction.[45]

Pyrroloquinoline quinone (PQQ) binds to an antioxidant enzyme (TrxR1) and alters its function, reducing its affinity towards its normal substrate and increasing its affinity towards an alternate substrate. Overall activity of this enzyme appears to be enhanced at high concentrations of PQQ, but the effect of more physiologically realistic (nanomolar) concentrations are not known

Inhibition of TrxR1 activity is known to cause an increase in the activity of the Nrf2 protein, which acts on the nucleus (via the antioxidant response element or ARE) to increase antioxidant gene expression.[46][47][48] Since oral supplementation of PQQ appears to influence a large amount of genes under control of TrxR1-related transcripts[49] it is thought that TrxR1 inhibition by PQQ occurs in vivo.[49]

It is thought that PQQ inhibits thioredoxin reductase (TrxR1) when ingested orally, since genes that would normally be activated when TrxR1 is inhibited do seem to be activated with PQQ in rats

2.4. Glutathione Reductase

PQQ has also been shown to inhibit glutathione reductase, but despite a decreased KM towards juglone (which would increase NAPDH oxidation and enzyme activity) the Kcat was also reduced and enzyme activity remains similar with or without PQQ.[45] However, GSSG reduction with 5µM PQQ was reduced approximately 2-fold relative to control.[45]

An inhibitory effect has been noted in regards to glutathione reductase as well, although the practical significance of this particular enzyme interaction is not known

2.5. Mitochondrial Biogenesis

In rats, PQQ depletion is known to influence genetic expression (238 out of 10,000 tested genes) and dietary repletion is known to influence 847 transcripts;[49] of these, the major pathways affected include Thioredoxin and MAPK signalling but also PGC-1α, a positive regulator of mitochondrial biogenesis[50]).[49] PQQ activates PGC-1α via CREB phosphorylation[51]and appears to positively regulate mitochondrial biogenesis in vivo. It also has other possible roles in blood pressure regulation, cellular cholesterol homeostasis, energy production, and protection of mitochondrial activity, all of which are beneficially associated with increased PGC-1α activity[10][50]).

When studies are undertaken in rats comparing a PQQ deficient diet, in which the rats must rely solely on de novobiogenesis of PQQ) against PQQ sufficient diets, the PQQ supplemented diets tend to promote up to 20-30% more mitochondria in the liver (on a mass basis, as assessed by mtDNA) over the rats’ lifetime.[27][26][10][52][7][49][51] Decreased permeability of the mitochondrial membrane has also been noted without alterations in functional capacity or mitochondrial size,[26] along with the mitochondrial count per cell increasing 60% from 56.8+/-7.8 to 91+/-6.6 with 2mg/kg PQQ fed by gavage starting from 2 weeks of age in rats on a PQQ deficient diet.[26]

Pyrroloquinoline quinone (PQQ) appears to be capable to increasing the activity of PGC-1α, which then promotes mitochondrial proliferation and membrane stabilization. This occurs in rats using oral doses similar to those in humans, and occurs secondary to CREB phosphoylation; this may suggest bioenergetic benefits of supplementation, but human evidence does not yet exist

When humans supplement PQQ (0.075-0.3mg/kg for one week at a time for each dose), urinary lactate decreased by 15% along with a reduction in urinary pyruvic acid.[28] A minor reduction of fumarate was noted, but other Kreb’s cycle intermediates (Isoaconitate, Citric acid, 2-oxoglutarate, and succinate) were not altered in the urine.[28] It was hypothesized, on the assumption that urinary metabolites reflect cellular energy status, that this indicated an increase in mitochondrial efficiency.[53][54]

A nonsignificant decreasing trend in urinary 4-hydroxyphenylacetate was noted with PQQ;[28] decreases in this and other urinary metabolites tend to suggest increased β-oxidation rates.[55]

The currently lone human study using doses of PQQ commonly found in supplements suggest that supplementation may increase mitochondrial efficiency

2.6. PTP1B

Pyrroloquinoline quinone (PQQ) is known to enhance signalling of some MAPK proteins, most notably ERK1/2, to significant extents, rivalling its effects on thioredoxin and PGC-1α.[49][56] This may be secondary to oxidative changes on the PTP1B protein; the changes occur when PQQ facilitates the production of hydrogen peroxide by associating with other proteins[57]) within a cell via direct REDOX cycling.[41] Hydrogen peroxide then modifies PTP1B on Cys-215.[58] The change of Cys-215 from a sulfenic acid moiety (-SOH) into a more oxidized sulfinic acid (–SO2H) or sulfonic acid (–SO3H) causes reversible inhibition of PTP1B.[59][60]

PTP1B is a negative regulator of the insulin receptor,[61] and is also a negative regulator of the epidermal growth factor receptor (EGFR).[58] By alleviating a negative inhibition, PQQ (via H2O2) can enhance signalling through the EGFR resulting in more ERK1/2 activation.

By acting as a direct REDOX couple, PQQ can inhibit PTP1B activity via hydrogen peroxide production within a cell. This inhibition of PTP1B enhances growth factor signalling (via EGFR signalling) and can enhance insulin sensitivity in a cell (by enhancing insulin receptor signalling)

  1. Pharmacology

3.1. Absorption

PQQ is absorbed well in the intestines, but its absorption is highly variable; 62% of PQQ is absorbed on average in rats in a fed state, with a range from 19-89%.[62]

3.2. Serum

A single dose (0.2mg/kg) of PQQ ingested by humans in a fruit-flavored drink has a tmax of about two hours and a Cmax of approximately 9nM.[28] Doubling PQQ dose from 0.075mg/kg PQQ daily for one week to 0.15mg/kg and then 0.3mg/kg in healthy subjects increased plasma PQQ levels in a linear manner. Fasting blood levels of PQQ ranged from 2 to 14nM when measurements were taken on day four of supplementation.[28] These levels may be similar to the steady state values as they were measured after the fourth day of dosing on the morning after PQQ was ingested.[28]

Daily supplementation of pyrroloquinoline quinone (PQQ) appears to increase plasma PQQ concentrations to a steady state level of around 10nM in humans

3.3. Distribution

PQQ appears to be eliminated from mice 24 hours after ingestion except in the skin and kidneys, which retain detectable levels of PQQ following oral ingestion.[62] In the skin, it was noted that 0.3% of the ingested dose was detectable six hours following a dose and 1.3% of the oral dose was detected after 24 hours. Greater than 95% of the PQQ in the blood seems to be associated with the blood cell fraction, with less than 5% remaining in the plasma fraction.[62]

3.4. Elimination

86% of an ingested dose of PQQ in mice appears to be eliminated via the kidneys within 24 hours of oral ingestion[62] and is excreted in a manner directly correlated with serum levels in humans;[28] in humans, less than 0.1% of the ingested dose is detected as unmodified PQQ, suggesting that PQQ is highly metabolized prior to elimination.[28]

3.5. Mineral Bioaccumulation

Pyrroloquinoline quinone has been noted to bind directly to metals such as uranium. This explains the toxicity of uranium to bacteria, which depend on PQQ as a cofactor for enzymes;[63] uranium displaces a calcium ion which is required to bind PQQ to certain enzymes in bacteria.[64][65]

Pyrroloquinoline quinone has a known affinity for some minerals, but the role of PQQ in the human body in regards to minerals is not known. It is unlikely to play a role in heavy mineral elimination due to the very low serum concentrations of PQQ

  1. Interactions with Neurology

4.1. Glutaminergic Neurotransmission

The NMDA receptor possesses a sulfhydryl REDOX modulatory site that is susceptible to oxidation[66] where oxidation suppresses NMDA signalling and reduction enhances NMDA signalling.[67][68] PQQ (50µM) does not affect basal currents through the receptor, but it can block reducing agents from enhancing signalling[69][70] in the 5-200µM range. The reduction of signalling is thought to be due to acting on the REDOX site, since PQQ can reduce excitotoxicity but fails to protect from H2O2 (which causes toxicity independent of the NMDA receptor).[71]

This mechanism is thought to underlie protective benefits of PQQ supplementation[70] seen at low concentrations of 5µM (other mechanisms require PQQ concentrations of up to 50µM in order to become appreciable).[71]

PQQ appears to have a regulatory effect on the glutamate receptor known as NMDA, by causing some oxidation of the REDOX site and preventing excess reduction from occurring it can suppress abnormal spikes in NMDA signalling; since an excess of NMDA signalling can be toxic, the result is a neuroprotective effect. This is thought to be applicable to oral supplementation due to a low concentration being required

4.2. Neuroprotection

100µM PQQ has been noted to protect cells from glutamate-induced cytotoxicity[72][73] associated with an increase in antioxidant enzyme activity, as assessed by Nrf2 and HO-1.[72] This is thought to be downstream of Akt/PI3K and GSK-3β activation,[74] of which the former is known to occur with PQQ in the 50-100µM range in vitro.[74]

PQQ also appears to prevent an increase in JNK signalling seen with NMDA-mediated toxicity, but it is not related to the protective effects on cellular survival[74] and PI3K activation cannot fully predict the protective effects of PQQ.[72]

PQQ appears to be related to an activation of PI3K/Akt signalling, which is known to cause an induction in antioxidant enzymes via Nrf2. This is thought to underlie some of the protective benefits of PQQ on cellular structure seen in vitro, but its significance to oral supplementation is not known

Protective effects against glutamate have been noted when PQQ is directly injected into the brain in a manner that is associated with the aforementioned antioxidant effects (PI3K activation and Nrf2/HO-1 induction).[73]

Injections of PQQ into the brain are known to be neuroprotective, but it is not known if this applies to oral ingestion as well

4.3. Neurogenesis

In fibroblastic cells (L-M), incubation of PQQ disodium salt (approximately 100µg/mL) for 24 hours has resulted in a peak 40-fold increase in Nerve Growth Factor (NGF) synthesis, with minor (around 5 to 10-fold) increases at 10-20µg/mL[75][76]in a manner dependent on COX2 induction[77] and PI3K/Akt.[78] Prostaglandins D2 and E2 (from Arachidonic acid) have been reported in vitro,[77] and while they were not tested as a mandatory intermediate the former (and its metabolite prostaglandin J2) are known to promote NGF synthesis in the 6.3-25µg/mL range[77] via CHRT2[79] extending to a variety of cell lines.[80][81][82]

This increase in NGF synthesis has also been noted in isolated mouse astrocytes exceeding Alpha-Lipoic Acid (ALA) in potency, but less than ALA in c/3T3 (embyotic fibroblast) cells.[83]

When tested in vitro, PQQ appears to concentration-dependently increase NGF synthesis up to a peak efficacy at 100µg/mL. The increase noted in isolated cells appears to be quite large. Eicosanoid signalling appears to be involved in this phenomena, suggesting that PQQ works via manipulating the actions of eicosanoids

When fat-soluble derivatives were tested (PQQ trimethyl esters) at injections of 0.1-1mg/kg every other day, it was noted that peripheral sciatic nerves had enhanced regeneration;[75] injections into the periphery failed to cause an increase in NGF in the neocortex, thought to be due to poor diffusion of PQQ across the blood brain barrier due to complexation with proteins in serum.[75] A pharmaceutical modification of the PQQ enzyme (oxapyrroloquinoline; OPQ) was able to enhance brain NGF concentrations,[75] and since OPQ is known to be metabolized into PQQ in bacteria (hypothesized to occur in rodents) and is fat soluble it was thought to act as a prodrug.

When tested later, PQQ added to silicon tubes confirmed an increase in the rate of physical recovery in a mouse model of physical nerve injury with benefits seen after four weeks extending to twelve weeks.[84] This improvement was associated with an increase in well-myelinated neurons.[84]

In a spinal cord injury model, 5mg/kg PQQ injected into the spine daily for a week after injury was able to suppress the expression of iNOS after one day (a biomarker for inflammation[85][86]) and improved both locomotor performance and neuronal health (axonal density) in the area relative to control.[87] Benefits to peripheral nerve function (in a rat model of sciatic nerve injury) have been noted orally; a low dose (20mg/kg) prevented hyperalgesia from the nerve injury while only the higher dose (40mg/kg) prevented muscular atrophy and lipid peroxidation.[88]

The enhancement of neurogenesis has been noted in the periphery (tissue excluding the brain) with injections of low doses of PQQ, but an increase in neurogenesis in the brain has failed to be noted which is thought to be due to transportation issues to the brain. While there are no oral studies in rodents yet, PQQ has been noted to enhance peripheral neurogenesis following nerve injuries

4.4. Neurooxidation

As mentioned in the glutaminergic section, the oxidative effects of PQQ on the NMDA modulatory site[69][70] can ultimately cause a reduction in NMDA-induced superoxide formation in the neuron[71] at concentrations (5uM) that do not affect oxidation per se (no effect against hydrogen peroxide which circumvents the receptor).[71]

The anti-glutaminergic effects that occur at lower concentrations may also ultimately cause anti-oxidative effects by suppressing NMDA signalling, despite this mechanism being reliant on the pro-oxidant effects of PQQ

PQQ does not appear to influence the toxicity of peroxynitrate (a combination of nitric oxide and the superoxide radical), despite inhibiting its formation.[89] When using SIN-1 as a way to produce peroxynitrate and induce cell death in vitro, PQQ at 100uM abolished cell death prior to peroxynitrate formation with an EC50 of 15+/-8.4uM, yet actually potentiated pre-existing peroxynitrate toxicity (also seen with superoxide dismutase, an anti-oxidant enzyme, when catalase was not present).[15] The mechanism appears to be through sequestering the superoxide radical without significantly influencing nitric oxide, as PQQ does not appear to modify many parameters of nitric oxide or peroxynitrate per se yet potentiated a SIN-1 induction of cGMP and production of nitrate, theoretically caused by a backlog of nitric oxide that could not convert to peroxynitrate due to less free superoxide radicals.[15] Interactions with PQQ and superoxide radicals has been noted previously.[90][91]

Can prevent superoxide radical induced cell death, but does not significantly influence nitric oxide cell death per se

4.5. Epilepsy and Convulsions

NMDA receptors are involved in the pathology of seizures (as seizures are involved with excessive NMDA signalling[92][93]) and the REDOX modulatory site that PQQ is known to interact with (suppressing high levels of activity) is further implicated[94] since seizures are associated with a high level of reducing agents in the brain[95][96] which can act upon that site to promote increased NDMA signalling;[94] it is thought that PQQ could have a therapeutic role (seen with pharmaceutical NMDA antagonist[97][98]) since by its oxidative role it hinders this particular site on NMDA receptors[69][70]and PQQ is thought to not associated with side-effects from excess suppression due to only suppressing high levels of NMDA signalling but not basal levels.

When seizures occur, they are potentiated by excessive signalling through the NMDA receptors and due to this NMDA receptor antagonists (or anything that can suppress excess signalling) are thought to be therapeutic. Since PQQ has been implicated in suppressing excess NMDA signalling, it is being investigated for anti-epileptic effects

Application of 200µM PQQ to isolated neurons undergoing epileptic activity can fully abolish such activity if induced by reducing agents (no effect on epileptic activity induced by other means),[94] supporting the role PQQ plays in epilepsy via NMDA antagonism which may occurs to limited levels at concentrations as low as 5µM.[71]

In vitro evidence support a role for PQQ, but due to quite high concentrations being used (relative to what is seen in the blood) and a hypothesized low transportation to the brain it is not sure if this will occur in a living organism following oral ingestion

4.6. Hypoxia and Stroke

Pyrroloquinoline quinone (PQQ) appears to have protective effects against ischemia (assessed by infart size) when 10mg/kg is injected either 30 minutes prior to ischemia (reducing the infarct size from a 95+/-3.6% increase to 68.8+/-10.4%)[99] and is slightly less effective when injected immediately after rather than preloaded (37.6% reduction seen previously reduced to 18.5%).[99] This has been replicated elsewhere with 3-10mg/kg (70-81% protection) but not 1mg/kg was given an hour after MCAO injury.[100]

Injections of PQQ have been noted to have protective effects in rats subject to stroke, but due to high injection doses being used and the low dose being ineffective preliminary evidence does not appear to look promising for oral supplementation of PQQ in this role; oral testing, however, has not yet been conducted

4.7. Brain Injury

Injections (intraperitoneal) of PQQ in the range of 5-10mg/kg to rats for three days prior to tramautic brain injury was able to dose-dependently protect the brain from injury with the highest dose appearing to confer absolute protection (assessed by histology and cognitive behaviour post-injury).[101]

4.8. Memory and Learning

When injected into rats at 10mg/kg bodyweight, PQQ does not appear to cause overt behavioural changes in regards to sedation, activty, or heart rate[99] with no alterations in EEG readings being observed.[99]

Several morphological changes are associated with PQQ that may confer pro-cognitive effects, such as proliferation of Schwann cells secondary to PI3K/Akt activation,[78] PQQ is also able to induce production of Nerve Growth Factor (NGF)[76] secondary to COX induction;[77] increases in NGF have been observed in vivo when using trimethylesters (for permeability into the brain) with a maximal increase of 1.7-fold over baseline associated with a PQQ metabolite named oxazopyrroloquinoline.[75]

PQQ supplementation has also been associated with preventing stress-associated (oxidative stress mediated) declines in memory[102] reducing damage done by methylmercury toxicity,[103][104] and reducing memory impairment induced by a lack of oxygen;[105] at 20mg/kg bodyweight PQQ has a potency nonsignificantly different than 200mg/kg Vitamin E (as R-R-R-Alpha tocopherol) in reversing age-related memory decline in rats.[105] which, together with its neuroprotective status, assure it a position as a rehabilitative Nootropic.

Currently, one study has been conducted in humans using PQQ at 20mg daily or using PQQ at 20mg paired with 300mgCoQ10.[106] This study used the supplements once-daily at breakfast for 12 weeks in persons aged 51.7-52.3yrs with the three tests being a Verbal Memory test (seven words read aloud and then asked to recite), the Stroop Test, and the CogHealth test. The results suggested a tendency towards improvement in the Verbal memory test (nonsignificant) a significant increase in performance in the Stroop test with PQQ+CoQ10 but not PQQ in isolation, and the choice reaction and simple reactions subsets of the CogHealth test showed statistically significant improvements with PQQ and PQQ+CoQ10 but the degree of improvement was not recorded.[106]

General nootropic benefit for those with impaired cognitive function (due to age, neural damage, etc.) but does not have ample evidence to be claimed a cognition promoting nootropic in otherwise healthy. The one study conducted in humans does not claim a 50% or doubling of memory, and was not suited to answer this question

4.9. Sedation

One open-label human study conducted with 20mg PQQ for 8 weeks in 17 persons with fatigue or sleep impairing disorder noted that PQQ was able to significantly improve sleep quality, with improvements in sleep duration and quality appearing at the first testing period 4 weeks after usage while a decrease in sleep latency required 8 weeks to reach significance.[107] This study also noted improved appetite, obsession, and pain ratings that may have been secondary to improved sleep; contentness with life trended toward significance over 8 weeks but did not reach.[107]

  1. Cardiovascular Health

5.1. Cardiac Tissue

Protective effects have been noted in cardiac myocytes subject to ischemia, secondary to scavenging of peroxynitrate radicals, at injectible doses of 15mg/kg bodyweight 30 minutes prior to ischemia.[108][109] PQQ was studied alongside metprolol as a combiantion anti-oxidant/beta-blocker therapy, and 3mg/kg PQQ and 1mg/kg metprolol were both insignificantly different in reducing mortality (40% of control passed, 8% of PQQ and 14% of metprolol) while no deaths were recorded in combination therapy.[110] Combination was also more effective in reducing infarct size relative to either therapy in isolation, and both groups using PQQ had a reduction of creatine kinase release that was insignificantly different between groups.[110]

The combination therapy study noted increased cardiac mitochondrial respiration with PQQ but neither metprolol nor PQQ+metprolol, and respiration was further increased even in the contrl groups with no ischemia/reperfusion done.[110]

Secondary to the pro-mitochondrial effects and anti-oxidative effects during ischemia/reperfusion, PQQ appears to be cardioprotective under certain contexts

5.2. Atherosclerosis

In otherwise healthy humans supplementing PQQ at 0.075-0.3mg/kg for three weeks (increasing the dose each week), supplementation was associated with a decrease in C-reactive protein concentrations in serum (45%).[28] This study also noted that urinary trimethylamine-N-oxide (TMAO) was reduced[28] and since both C-reactive protein (CRP)[111] and TMAO[112] are thought to be biomarkers for atherosclerosis PQQ is thought to have a role.

5.3. Triglycerides

In rats fed a PQQ deficient diet relative to the same diet fed with 2mg/kg PQQ, plasma diglycerides and triglycerides (DAG and TAG) were elevated 20-50% (higher value related to triglycerides) in the PQQ deficient diet relative to 2mg/kg with no significant difference in free fatty acids,[27] which is similar to levels previously seen with this experimental protocol.[26] The elevation of triglycerides in the deficient mice does not influence the n3/n6 omega fatty acid ratios.[27]

The increase seen in triglycerides may be due to this study being conducted for a long period of time, where previous research has demonstrated that PQQ deficient diets reduce mitochondrial density by 20-30%[26] and levels of mRNA for PPAR, Fatty Acid binding protein, and Acyl CoA oxidase being significantly reduced with PQQ deficiency.[27] Additionally, higher levels of beta-hydroxybutryic acid (indicative of less beta-oxidation) were seen in PQQ deficient rats. Inducing PQQ deficiency from a sufficient state can also elevate triglyceride levels to almost two-fold the previous levels, with the trend being reversed upon acute administration of PQQ in pharmacological amounts (2mg/kg bodyweight).[49]

Appears to reduce triglycerides very potently (to a greater extent than Fish Oil, empirically) in research animals relative to a PQQ deficient diet, and this is thought to be due to increased mitochondrial β-oxidation of fatty acids

The one human study to use supplemental PQQ (0.075-0.3mg/kg for three weeks in escalating doses) failed to find any significant influence on triglyceride concentrations in serum of otherwise healthy adults consuming a standard (but uncontrolled) diet.[28] This study also noted alterations in urinary metabolites (4-Hydroxyphenylacetate and 4-Hydroxyphenylactate) suggestive of an increase in mitochondrial β-oxidation despite no apparent changes in triglycerides.[28]

First study to assess the effects of PQQ on triglycerides has failed to find an influence in otherwise healthy humans

  1. Interactions with Glucose Metabolism

6.1. Glucose Deposition

PQQ (500nM) has been noted to inhibit protein tyrosine phosphatase 1B (PTP1B) secondary to producing H2O2[41] (H2O2is known to inactivate PTP1B in a reversible manner[58]), and aside from PTP1B being a negative regulator of a growth factor receptor (EGFR[58]) it also negatively influences insulin receptor signalling;[61] inhibition of PTP1B, seen also withBerberine and Ursolic Acid (albeit by different mechanisms), tends to increase the activity of the insulin receptor.

Sequestering the hydrogen peroxide made from PQQ appears to block its inhibition on PTP1B.[41]

Via prooxidative changes within a cell, PQQ can produce hydrogen peroxide which then impairs PTP1B function. Since PTP1B normally suppresses signalling via the insulin receptor, the result is a compensatory increase in insulin signalling

6.2. Serum Glucose

In young rats (before sexual maturation), PQQ either at 3mg/kg in the diet or having a PQQ deficient diet does not seem to significantly affect blood glucose or insulin levels.[27] An increased glucose AUC was seen when PQQ deficient mice were subject to an oral glucose tolerance test, but no single time point was significnatly different.[27] Injections of PQQ at 4.5mg/kg bodyweight also did not significantly influence blood sugar or insulin levels in healthy rats, but was able to significantly reduce glucose AUC (by 7%) and glucose disposition in diabetic rats fed glucose and injected with PQQ, with no effect of PQQ on fasting glucose levels in rats.[27]

6.3. Insulin resistance

It has potential for alleviating fat-induced insulin resistance (characterized by a dysregulation in beta-oxidation of the TCA cycle) by increasing mitochondrial biogenesis in muscle cells, similar to exercise.[113]

At this moment in time, nothing remarkable about PQQ and glucose metabolism

  1. Interactions with Obesity

7.1. Metabolic Rate

When comparing a rat diet deemed sufficient in dietary pyrroloquinoline quinone (PQQ; 2mg/kg) to a diet deficient in one, the deficient diet appeared to have a decreased metabolic rate (reaching only 90% of the control rats)[27] with the difference being more prominent during the fed rather than fasted state;[27] it appears that this decreased metabolic rate did not influence the rats of lipolysis nor glycolysis as assessed by the respiratory quotient.[27]

Depleting the rat diet of PQQ appears to reduce their metabolic rates relative to a diet with adequate levels of PQQ, but no studies have investigated whether an increase in metabolic rate occurs with extra supplemental PQQ

  1. Bone and Joint Health

8.1. Osteoclasts

Pyrroloquinoline quinone (PQQ) has been noted to inhibit RANKL-induced osteclast formation in RAW 264.7 macrophage-like cells at a concentration of 10µM, which occurred at all stages of cell maturation.[114]

RANKL normally signals through the transcription factor NFATc1[115][116] via a particular AP-1 signalling protein that contains c-Fos and c-Jun.[117][118] PQQ inhibited c-Fos induction from RANKL,[114] but other RANKL-induced proteins (NF-kB and MAPKs) were unaffected suggesting that RANKL signalling overall was unaffected.[114]

There is a negative regulatory pathway from RANKL, where RANKL increases IFN-β production which signals via its receptor (IFNAR[119]) to activate STAT1 and JAK1 to suppress the actions of RANKL.[120][121] IFN-β was not affected by PQQ, but the receptor expression (and its targets) appeared to be increased which were thought to underlie the observed inhibitory effects seen with PQQ.[114]

PQQ appears to enhance the negative feedback mechanism controlling osteoclastogenesis (production of osteoclasts, which are negative regulators of bone mass) and via this enhancement overall osteoclast activity is hindered somewhat and this is thought to promote bone mass over time. Due to a higher than normal concentration being used, it is not sure if this occurs following oral supplementation

  1. Skeletal Muscle and Physical Performance

9.1. Mechanisms

One study using 0.075-0.3mg/kg PQQ supplementation daily for three weeks (increasing with dose each week) in otherwise healthy adults has noted a decrease in overall urinary amino acid levels by approximately 15%,[28] with the decrease in some (serine, asparangine, aspartic acid) being biomarkers for skeletal muscle consumption of nitrogen (via being converted into Glutamine and alanine[28][122]).

Preliminary evidence suggests that oral PQQ supplementation can influence skeletal muscle metabolism in otherwise healthy humans with standard supplemental doses, but the practical significance of this is not yet known

  1. Immunology and Inflammation

10.1. Mechanisms

PQQ appears to have some interactions with the immune system, as deprivation of PPQ from the diet (relative to a PQQ sufficient diet) appears to cause abnormal immune function in mice, with altered immune response after stressors.[52][7]

A study on parental (intravenous) nutrition found that the addition of 3mcg PQQ to the parental nutrition in mice was able to increase the count of CD8+ cells and lymphocytes in intestinal Peyer’s Patches, although not to the level of oral control.[123]

10.2. Macrophages

Application of PQQ to macrophages in vitro was able to prevent osteoclast differentiation at doses as low as 0.1uM (but more potency at 10uM) secondary to increasing IFN-β secretion; IFNβ is a negative regulator of osteoclast differentiation normally released after inflammation, and PQQ increases its release (and subsequent suppression), which is also demonstrated by increased levels of proteins induced by IFN-β (iNOS, STAT1, JAK1).[114] PQQ was found to phosphorylate NF-kB, p38, and IKKβ in these cells which is a pro-inflammatory response in macrophages.[114]

Practical relevance unknown

  1. Interactions with Oxidation

11.1. Singlet Oxygen

The reduced form of pyrroloquinoline quinone (PQQ), known as pyrroloquinoline equinol or dihydroquinone pyrroloquinoline (PQQH2) appears to be able to sequester singlet oxygen (1O2) with a potency 6.4-fold less than β-carotene as reference yet higher than that of Vitamin E (2.2-fold) and Vitamin C (6.3-fold).[12]

PQQH2 appears to be produced (via reduction) from PQQ when in a buffer in the presence of glutathione[12] and this process is known to use the semiquinone (PQQH) as an intermediate;[57] exposure to oxygen either by ambient atmosphere or by singlet oxygen readily oxidizes PQQH2 back into PQQ.[12] This suggests that glutathione is capable of recycling PQQ as an antioxidant.

PQQ and its reduced form PQQH2 appear to form a cyclical relationship where PQQH2 sequesters oxygen radicals, and glutathione reduces it back into PQQ so it may sequester more radicals; the potency of this reaction, on a molecular level, seems intermediate to β-carotene (PQQ is lesser) and Vitamin C/E (greater)

11.2. Reactive Nitrogen Species

One study assessing whether PQQ could directly sequester peroxynitrate (ONOO) failed to find such a property of PQQ, as despite protecting cells form the toxic effects of SIN-1 (produces nitric oxide and superoxide radicals,[124] of which PQQ scavenged the superoxide radicals[15]) the toxicity of peroxynitrate directly was not protected against (in fact, it appeared to be augmented at 100-300µM PQQ).[15]

Pyrroloquinoline quinone (PQQ), even at impractically high concentrations, does not appear to direct sequester reactive nitrogen species (nitrogen based pro-oxidants) such as peroxynitrate

11.3. Lipid Peroxidation

One human study using supplemental pyrroloquinoline quinone (PQQ) and measuring serum antioxidant capacity via TBARS and TRAP values failed to find any significant influence on TRAP values but noted a decrease in TBARS (indicative of lipid peroxidation) to the degree of 0.2% when measured at peak serum PQQ values (6-12nM) seen with up to 300µg/kg supplementation;[28] this decrease in TBARS was noted to be significantly less than other dietary supplements such as procyanidins from Cocoa Extract which (560mg) can reduce TBARS by 25-35%[125] or sources of anthocyanins such as Aronia melanocarpa or Blueberry.

The decrease in serum biomarkers of lipid peroxidation that is known with PQQ supplementation is probably much too low to be indicative of anything significant

11.4. Radiation

Oral ingestion of 4mg/kg PQQ to mice (more effective than both 2mg/kg and 8mg/kg, as well as the reference drug of 10mg/kg nilestriol[126]) appears to reduce death from gamma irradiation when given an hour before and again seven days after irradiation; damage to select cells tested (white blood cells, reticulocytes, bone marrow cells) was also reduced with 4mg/kg PQQ supplementation to mice.[126]

Oral ingestion of PQQ (estimated human equivalent of 0.32mg/kg) appears to be able to protect mice from gamma irradiation to a respectable degree

  1. Peripheral Organ Systems

12.1. Liver

An intraperitoneal injection of pyrroloquinoline quinone (PQQ) to rats at 5mg/kg twice before CCl4 liver toxicity appeared to exert protective effects;[127] when tested in vitro, PQQ showed protective effects in isolated liver cells with most potency at 3µM.[127]

12.2. Intestines

Due to the involvement of pyrroloquinoline quinone (PQQ) in bacteria (from where it was discovered in 1979[31]) and the involvement of quinoproteins in the fermentation process [128] (which PQQ associates with) and the above higher count of PQQ recorded in fermented foods; it is hypothesized that fermentation may increase PQQ content. Interestingly, common strains of bacteria in the human intestinal tract do not appear to synthesis much PQQ[129][130] and in antibiotic fed mice (lacking intestinal microflora) it seems that dietary intake is the major determinent of bodily PQQ levels.[130]

Pyrroloquinoline quinone was thought to be synthesized by intestinal bacteria due to its discovery being that of a bacterial cofactor, but preliminary evidence does not support the intestinal microflora as a major producer of PQQ in the body

12.3. Kidney

Pyrroloquinoline quinone (PQQ) was once implicated in being an enzymatic cofacter for diamine oxidase (pig kidney)[32][33]and DOPA decarboxylase (pig kidney)[34] (as well as dopamine β-hydroxylase, albeit in the renal medulla[35]), although it is generally accepted to not be a significant component of eukaryotic enzymes in vivo (in the role of a cofactor) like it is in bacterial and plant enzymes.[36][37][38] Still, it is detectable in the kidney after oral ingestion in the rat[62] and elimination of PQQ is primarily via the urine[62] suggesting it may still play a role independent of being an enzymatic cofactor.

PQQ is not thought to play a role as a cofactor of enzymes in the kidneys like initially thought, but due to being eliminated by the kidneys and accumulating in them following oral ingestion in the rat it is still thought to play a role (perhaps as a REDOX couplet, like other mechanisms)

  1. Interactions with Cancer

13.1. Leukemia

PQQ has been shown to be cytotoxic to U937 leukemia cells, but not NIH3T3 nor L929 cells, in a dose-dependent manner.[131] Catalase treatment neutralized these effects, as they appear to be secondary to hydrogen peroxide production in cells which PQQ has been repeatedly shown to induce.[132] Superoxide dismutase had no effect on PQQ cytotoxicity, while glutathione or N-AcetylCysteine increased cytotoxicity 2-5fold without affecting the cells on their own (and thus working via PQQ by increasing H2O2 production form PQQ 1.5-2fold).[131] PQQ by itself decreased intracellular glutathione levels, and when glutathione was depleted (via BSO, an inhibitor of γ-glutamylcysteine synthetase) the apoptosis of cells morphed into necrosis, and this necrosis was still mediated by H2O2 due to being inhibited by catalase.[131]

Induces cell death via H2O2, and uses glutathoine to produce even more H2O2 to augment its efficacy. A depletion of glutathione induces necrosis

13.2. Melanoma

PQQ has been implicated in reducing melanogenic (melanin producing) protein expression in cultured B16 cells, where it can inhibit tyrosinase expression and reduce gene activity[133] and can prevent stimulation of tryosinase mRNA by alpha-melanocyte stimulating hormone.[134]

  1. Interactions with Medical Conditions

14.1. Parkinson’s Disease

Parkinson’s disease is known to be associated with what are known as Lewy Bodies (irregular cytoplasmic inclusions[135][136]) which are comprised of a molecule known as α-synuclein[137] which is known to damange dopaminergic neurons and is involved in the pathology of Parkinson’s disease when it aggregates.[138][139] It is involved in normal physiological function (as a chaperone) when unaggregated,[140] so the process of α-synuclein aggregation itself is seen as pathological.

Pyrroloquinline quinone (PQQ) is known to bind to some of these α-synuclein peptides directly via forming a schiff basewith the lysine amino acids in the peptides[13] similar to both EGCG (Green Tea Catechins) and baicalein (skullcap)[13]although baicalein seems relatively more potent.[141] This direct binding also reduces formation of truncated α-synuclein[142] (which accelerate the formation of larger aggregates[143]) and the larger protein aggregates themselves[13] by around 14.8-50% at 280µM.[142] This may indirectly reduce the cytotoxicity that is seen with large aggregates,[13] although PQQ seems to be capable of reducing cytotoxicity from pre-formed aggregates independent of the aforementioned binding.[142]

Protein aggregates tend to occur normally in the brain, and their aggregation is accelerated and seem to be central to the development of Parkinson’s Disease. PQQ appears to physically bind to these proteins in vitro to prevent the aggregation, but it occurs at a very high concentration and it does not seem likely to occur with respectable potency following oral supplementation

6-hydroxydopamine (6-OHDA), a metabolite of dopamine which is known to cause oxidative damage to dopaminergic neurons and detected at higher levels in persons with Parkinson’s,[144] may have its toxicity attenuated with coincubation of PQQ.[145] Oxidative neurotoxicity and DNA fragmentation induced by 6-hydroxydopamine was reduced in a concentration dependent manner with concentrations of 300nM showing efficacy, yet this protective effect was not seen with Vitamin C or Vitamin E, two other anti-oxidants tested at concentrations up to 100µM.[145]

Elsewhere in isolated neurons, the protein DJ-1 (plays roles in oxidative protection[146][147] and mutations in it underly some genetic cases of early onset Parkinson’s Disease[148]) does not have its expression altered by PQQ[149] but 15µM PQQ appeared to preserve cell survival in the presence of oxidants by preserving the actions of DJ-1;[149] excessive oxidation of DJ-1 at C106 ablates its antioxidant potential[150] and PQQ appears to prevent this from occurring despite no direct binding.[149]

There may be some protective effects at the level of dopaminergic neurons with PQQ that is not related to preventing the formation of protein aggregation, and although this happens at a much more respectable (lower) concentration it is still uncertain if this applies to oral supplementation of PQQ

14.2. Alzheimer’s Disease

Pyrroloquinline quinone appears to inhibit the formation of amyloid fibrils (Aβ1-42; full inhibition at 70μM PQQ[151]), and although it can also bind to α-synuclein this binding does not indirectly inhibit Aβ1-42 aggregation.[13]

and to reduce the cytotoxicity of these fibrils on neuronal cells.[152]

  1. Nutrient-Nutrient Interactions

15.1. Glutathione

PQQ has been shown to be cytotoxic to U937 leukemia cells, but not NIH3T3 nor L929 cells (but was observed in EL-4), in a dose-dependent manner with most significance at 20-50uM.[131] Catalase treatment neutralized these effects, as they appear to be secondary to hydrogen peroxide production in cells which PQQ has been repeatedly shown to induce.[132]Superoxide dismutase had no effect on PQQ cytotoxicity, while glutathione or N-AcetylCysteine increased cytotoxicity 2-5fold without affecting the cells on their own (and thus working via PQQ by increasing H2O2 production form PQQ 1.5-2fold).[131] PQQ by itself decreased intracellular glutathione levels, and when glutathione was depleted (via BSO, an inhibitor of γ-glutamylcysteine synthetase) the apoptosis of cells morphed into necrosis, and this necrosis was still mediated by H2O2 due to being inhibited by catalase.[131]

Glutathione can be increased by cysteine containing supplements including N-AcetylCysteine or Whey Protein

In cancer cells susceptible to PQQ’s induction of H2O2, adding glutathione to the cell by consuming Cysteine-containing supplements can augment the efficacy of PQQ

  1. Safety and Toxicology

16.1. General

PQQ has been associated with renal tubule inflammation at the dose of 11-12mg/kg bodyweight in rats after injections, and some symptoms of both renal and hepatic toxicity are seen with injections of 20mg/kg in rats.[110][153] Acute death from PQQ injections between doses of 500-1000mg/kg bodyweight has been recorded in rats.[10][153]

11-12mg/kg bodyweight, based on rudimentary body surface area conversions, is approximately 120-131mg/PQQ daily (although injections) if extrapolated to humans.

One human study using 20mg PQQ alone or in combination with 300mg CoQ10 noted that there were no toxicological signs or symptoms associated with treatment over a 12 week period,[106] and consumption of up to 0.3mg/kg PQQ (around 20mg for a 150lb person) for one week has been noted to be safe.[28]

Chronic toxicity to the kidneys and liver may be achieved at a relatively low dose, although acute death requires a very high and unpractical dose. Until more evidence surfaces, it would be prudent to avoid superloading

16.2. Genotoxicity

In an Ames test (TA1535, TA1537, TA98, and TA100 strains), 10-5000μg PQQ per plate (without metabolic activation) and 156-5000μg per plate (with activation) has failed to show appreciable genotoxic effects.[154]

In lung fibroblasts derived from chinese hamsters, 12.5-400μg/mL (no metabolic activation) and 117.2-3750μg/mL (with activation; highest concentration being 10mM) and the latter concentration in isolated lymphocytes failed to exert appreciable genotoxic effects as assessed by structural abberations and polyploidy.[154]

The aforementioned disodium salt of PQQ has failed to acutely exert genotoxic effects in mice (up to 2,000mg/kg) as assessed by a micronucleus assay and in bone marrow erythrocytes.[154]

No genotoxiticity has been noted with the disodium salt of PQQ

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(Users who contributed to this page include shrillthrillGregoryLopezKurtisFrankKamalPatel )

Pyrroloquinoline quinone

Pyrroloquinoline quinone (PQQ) was discovered by J.G. Hauge as the third redox cofactor after nicotinamide and flavin in bacteria (although he hypothesised that it was naphthoquinone).[1] Anthony and Zatman also found the unknown redox cofactor in alcohol dehydrogenase and named it methoxatin.[2] In 1979, Salisbury and colleagues[3] as well as Duine and colleagues[4] extracted this prosthetic group from methanol dehydrogenase of methylotrophs and identified its molecular structure. Adachi and colleagues identified that PQQ was also found in Acetobacter.[5]

These enzymes containing PQQ are called quinoproteins. Glucose dehydrogenase, one of the quinoproteins, is used as a glucose sensor. Subsequently, PQQ was found to stimulate growth in bacteria.[6] In addition, antioxidant and neuroprotective effects were also found.[7]

Research in animals[edit]

Mitochondrial biogenesis in mice[edit]

In 2010, researchers at the University of California at Davis released a peer-reviewed publication showing that PQQ’s critical role in growth and development stems from its unique ability to activate cell signaling pathways directly involved in cellular energy metabolism, development, and function. The study demonstrated that PQQ not only protects mouse hepatocyte mitochondria from oxidative stress—it promotes the spontaneous generation of new mitochondria within aging cells, a process known asmitochondrial biogenesis.[8]

The team of researchers at the University of California analyzed PQQ’s influence over cell signaling pathways involved in the generation of new mitochondria and found that there are three mouse proteins activated by PQQ that cause cells to undergo spontaneous mitochondrial biogenesis: peroxisome proliferator-activated receptor gamma coactivator 1-alpha, cAMP response element-binding protein, and the DJ-1 protein.[8]

Cardioprotection in rat models[edit]

Damage from a heart attack, like a stroke, is inflicted via ischemic reperfusion injury. PQQ administration reduces the size of damaged areas in animal models of acute heart attack (myocardial infarction). Significantly, this occurs irrespective of whether the chemical is given before or after the ischemic event itself, suggesting that administration within the first hours of medical response may offer benefits to heart attack victims.[9]

Researchers at the University of California at San Francisco investigated this potential, comparing PQQ with the beta blocker metoprolol—a standard post-MI clinical treatment. Independently, both treatments reduced the size of the damaged areas and protected against heart muscle dysfunction. When given together, the left ventricle’s pumping pressure was enhanced. The combination of PQQ with metoprolol also increased mitochondrial energy-producing functions—but the effect was modest compared with PQQ alone. Only PQQ favorably reduced lipid peroxidation. These results led the researchers to conclude that “PQQ is superior to metoprolol in protecting mitochondria from ischemia/reperfusion oxidative damage.” [10]

Subsequent research has also demonstrated that PQQ helps heart muscle cells resist acute oxidative stress by preserving and enhancing mitochondrial function.[11]

Radiation poisoning in mice[edit]

In a study of gamma radiation poisoning in mice, 4mg/kg of PQQ improved 30-day survival from 2/20 to 12/20 at an 8 Gy dose.[12]


PQQ is a neuroprotective compound that has been shown in a small number of preliminary studies to protect memory and cognition in aging animals and humans.[13][14] It has been shown to reverse cognitive impairment caused by chronic oxidative stress in animal models and improve performance on memory tests.[15] PQQ supplementation stimulates the production and release of nerve growth factors in cells that support neurons in the brain,[16] a possible mechanism for the improvement of memory function it appears to produce in aging humans and rats.

PQQ has also been shown to safeguard against the self-oxidation of the DJ-1 protein, an early step in the onset of some forms of Parkinson’s disease.[17]

PQQ protects brain cells against oxidative damage following ischemia-reperfusion injury—the inflammation and oxidative damage that result from the sudden return of blood and nutrients to tissues deprived of them by stroke.[18] Reactive nitrogen species (RNS) arise spontaneously following stroke and spinal cord injuries and impose severe stresses on damaged neurons, contributing to subsequent long-term neurological damage.[19] PQQ suppresses RNS in experimentally induced strokes,[20] and provides additional protection following spinal cord injury by blocking inducible nitric oxide synthase (iNOS), a major source of RNS.[21]

In animal models, administration of PQQ immediately prior to induction of stroke significantly reduces the size of the damaged brain area.[22] These observations have been compounded by the observation in vivo that PQQ protects against the likelihood of severe stroke in an experimental animal model for stroke and brain hypoxia.[18]

PQQ also affects some of the brain’s neurotransmitter systems. It protects neurons by modulating the properties of the N-methyl-D-aspartate (NMDA) receptor,[23][24] and so reducing excitotoxicity—the damaging consequence of long-term overstimulation of neurons that is associated with many neurodegenerative diseases and seizures.[25][26][27][28]

PQQ also protects the brain against neurotoxicity induced by other powerful toxins, including mercury[29](a suspected factor in the development of Alzheimer’s disease[30]) and oxidopamine[31] (a potent neurotoxin used by scientists to induce Parkinsonism in laboratory animals by destroying dopaminergic and noradrenergic neurons.[32])

PQQ prevents aggregation of alpha-synuclein, a protein associated with Parkinson’s disease.[33] PQQ also protects nerve cells from the toxic effects of the amyloid-beta protein linked with Alzheimer’s disease,[34]and reduces the formation of new amyloid beta aggregates.[35]


Although Nature Magazine published the 2003 paper by Kasahara and Kato which essentially stated that PQQ was a new vitamin, they also subsequently published, in 2005, an article by Chris Anthony and his colleague L.M. Fenton of the University of Southhampton which states that the 2003 Kasahara and Kato paper drew incorrect and unsubstantiated conclusions.[36] On his website,[37] Anthony discusses the Nature Magazine publications:

When I pointed out to the journal Nature that their high reputation was being used to justify investments of millions of dollars in the development of PQQ as a vitamin, they investigated the original paper, agreed with our objections and published our argument against it (Felton & Anthony, Nature Vol. 433, 2005). They also published (alongside ours) a paper by Rucker disagreeing with the conclusions of Kasahara and Kato on nutritional grounds, concluding “that insufficient information is available so far to state that PQQ uniquely performs an essential vitamin function in animals”.

Anthony further states on his website that “No mammalian PQQ-containing enzyme (quinoprotein) has been described” and that PQQ therefore cannot be called a “vitamin”. The latter statement is an exaggeration, since there is one mammalian enzyme which appears to use PQQ as a cofactor:[38]


    1. Jump up^ Hauge JG (1964). “Glucose dehydrogenase of bacterium anitratum: an enzyme with a novel prosthetic group”. J Biol Chem 239: 3630–9. PMID 14257587.
    2. Jump up^ Anthony C, Zatman LJ (1967). “The microbial oxidation of methanol. The prosthetic group of the alcohol dehydrogenase of Pseudomonas sp. M27: a new oxidoreductase prosthetic group”. Biochem J 104 (3): 960–9. PMC 1271238PMID 6049934.
    3. Jump up^ Salisbury SA, Forrest HS, Cruse WB, Kennard O (1979). “A novel coenzyme from bacterial primary alcohol dehydrogenases”. Nature 280 (5725): 843–4. doi:10.1038/280843a0PMID 471057.
    4. Jump up^ Westerling J, Frank J, Duine JA (1979). “The prosthetic group of methanol dehydrogenase from Hyphomicrobium X: electron spin resonance evidence for a quinone structure”. Biochem Biophys Res Commun87 (3): 719–24. doi:10.1016/0006-291X(79)92018-7PMID 222269.
    5. Jump up^ Ameyama M, Matsushita K, Ohno Y, Shinagawa E, Adachi O (1981). “Existence of a novel prosthetic group, PQQ, in membrane-bound, electron transport chain-linked, primary dehydrogenases of oxidative bacteria”.FEBS Lett 130 (2): 179–83. doi:10.1016/0014-5793(81)81114-3PMID 6793395.
    6. Jump up^ Ameyama M, Matsushita K, Shinagawa E, Hayashi M, Adachi O (1988). “Pyrroloquinoline quinone: excretion by methylotrophs and growth stimulation for microorganisms”. BioFactors 1 (1): 51–3. PMID 2855583.
    7. Jump up^ Rucker R, Chowanadisai W, Nakano M. (2009). “Potential physiological importance of pyrroloquinoline quinone”. Altern Med Rev. 14 (3): 179–83.
    8. Jump up to:a b Chowanadisai, W.; Bauerly, K. A.; Tchaparian, E.; Wong, A.; Cortopassi, G. A.; Rucker, R. B. (January 2010). “Pyrroloquinoline quinone stimulates mitochondrial biogenesis through cAMP response element-binding protein phosphorylation and increased PGC-1alpha expression”Journal of Biological Chemistry 285 (1): 142–152. doi:10.1074/jbc.M109.030130PMC 2804159PMID 19861415.
    9. Jump up^ Zhu, B. Q.; Zhou, H. Z.; Teerlink, J. R.; Karliner, J. S. (November 2004). “Pyrroloquinoline quinone (PQQ) decreases myocardial infarct size and improves cardiac function in rat models of ischemia and ischemia/reperfusion”. Cardiovascular Drugs and Therapy 18 (6): 421–431. doi:10.1007/s10557-004-6219-x.PMID 15770429.
    10. Jump up^ Zhu, B. -Q.; Simonis, U.; Cecchini, G.; Zhou, H. -Z.; Li, L.; Teerlink, J. R.; Karliner, J. S. (June 2006). “Comparison of pyrroloquinoline quinone and/or metoprolol on myocardial infarct size and mitochondrial damage in a rat model of ischemia/reperfusion injury”. Journal of Cardiovascular Pharmacology and Therapeutics 11 (2): 119–128. doi:10.1177/1074248406288757PMID 16891289.
    11. Jump up^ Tao, R; Karliner, J; Simonis, U; Zheng, J; Zhang, J; Honbo, N; Alano, C (2007). “Pyrroloquinoline quinone preserves mitochondrial function and prevents oxidative injury in adult rat cardiac myocytes”Biochemical and Biophysical Research Communications 363 (2): 257–62. doi:10.1016/j.bbrc.2007.08.041PMC 2844438.PMID 17880922.
    12. Jump up^ Xiong, X. H.; Zhao, Y; Ge, X; Yuan, S. J.; Wang, J. H.; Zhi, J. J.; Yang, Y. X.; Du, B. H.; Guo, W. J.; Wang, S. S.; Yang, D. X.; Zhang, W. C. (2011). “Production and radioprotective effects of pyrroloquinoline quinone”. International Journal of Molecular Sciences 12 (12): 8913–23. doi:10.3390/ijms12128913.PMC 3257108PMID 22272111.
    13. Jump up^ Takatsu, H; Owada, K; Abe, K; Nakano, M; Urano, S (2009). “Effect of vitamin E on learning and memory deficit in aged rats”. Journal of nutritional science and vitaminology 55 (5): 389–93. doi:10.3177/jnsv.55.389.PMID 19926923.
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    15. Jump up^ Ohwada, K.; Takeda, H.; Yamazaki, M.; Isogai, H.; Nakano, M.; Shimomura, M.; Fukui, K.; Urano, S. (January 2008). “Pyrroloquinoline quinone (PQQ) prevents cognitive deficit caused by oxidative stress in rats”. Journal of Clinical Biochemistry and Nutrition 42 (1): 29–34. doi:10.3164/jcbn.2008005.PMC 2212345PMID 18231627.
    16. Jump up^ Murase, K; Hattori, A; Kohno, M; Hayashi, K (1993). “Stimulation of nerve growth factor synthesis/secretion in mouse astroglial cells by coenzymes”. Biochemistry and molecular biology international 30 (4): 615–21.PMID 8401318.
    17. Jump up^ Nunome, K; Miyazaki, S; Nakano, M; Iguchi-Ariga, S; Ariga, H (2008). “Pyrroloquinoline quinone prevents oxidative stress-induced neuronal death probably through changes in oxidative status of DJ-1”. Biological & Pharmaceutical Bulletin 31 (7): 1321–6. doi:10.1248/bpb.31.1321PMID 18591768.
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PQQ and Statin Damage
By Dr. Duane Graveline MD, MPH

Those of you who have been following my research during the past two years will know that I consider mitochondrial DNA damage as the ultimate result for some of statin drug intake.

Through mevalonate blockade, statins directly inhibit CoQ10 synthesis making mitochondrial damage and mutation all but inevitable. Furthermore, the inhibitory effect of statins on dolichol synthesis makes repair of DNA damage all the more difficult because of dolichol’s vital role in glycoprotein (glycohydrolase) synthesis.

Recently I have learned of another biochemical substance that also is implicated in this process of mitochondrial maintenance. The name of this biochemical is pyrroloquinoline quinone with the shorthand version being PQQ.

This substance has been discovered only in the past decade with its vital role in mitochondrial support having been documented only in the past several years. From what I have read of this substance, trying to get beyond the hype, it is worth considering for those of us who have been damaged by statins, whether by cognitive dysfunction, permanent myopathy, ALS like symptoms, or peripheral neuropathy.

Dietary sources of PQQ include many fruits and vegetables and egg yolk. Natto ( fermented soybeans ) has the highest concentration but parsley, green peppers, papaya, kiwi fruit and spinach are all good sources. PQQ is also available as a dietary supplement. Human trials and studies will need to be performed to support any claims for the benefits of PQQ supplementation.

One promotion for PQQ begins with, “The more functional mitochondria you have in your cells, the greater your overall health and durability,” which is the premise of my new e-book, The Dark Side of Statins, so my interest in this substance is obvious.

The problem is that as we age, our mitochondria degrade and become dysfunctional. Compared with nuclear DNA, mitochondrial DNA is left almost entirely exposed to the ravages of free radicals. It attaches directly to the inner membrane where the mitochondria’s furnace rages continuously.

Statin drugs directly hasten this process of mitochondrial DNA degradation by direct inhibition of CoQ10 and dolichol synthesis. The ultimate cause of statin associated adverse reactions is this progressive deterioration of mitochondrial DNA.  PQQ is being touted not only for its extra anti-oxidant protection in the fight against free radicals but also for its potential use for mitochondrial genesis

This is part 1 (of nine parts) of the Preventing and Reversing Alzheimer’s Disease presentation, an earlier version of which was presented to the San Francisco bay area Smart Life Forum in January of 2009. This part covers the verbal introduction and the falling-dominoes illustration of the Alzheimer’s cascade

This is part three of the Prevention and Reversal of Alzheimer’s Disease presentation. This part covers the Alzheimer’s Map (schematic), mitochondria, and creatine kinase (the first domino in the Alzheimer’s disease cascade).

This is part six of the Prevention and Reversal of Alzheimer’s Disease presentation. This part covers the antioxidant defense system, glutathione (the “star of the movie”), and the brain’s phosphorylation cycle (the brains “biorhythm).

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Transthyretin and Lean Body Mass in Stable and Stressed State

Curator: Larry H Bernstein, MD, FCAP

Chapter 20
Plasma Transthyretin Reflects the Fluctuations
of Lean Body Mass in Health and Disease
Yves Ingenbleek

Transthyretin (TTR) is a 55-kDa protein secreted mainly by the choroid plexus and the liver. Whereas its intracerebral production appears as a stable secretory process allowing even distribution of intrathecal thyroid hormones, its hepatic synthesis is influenced by nutritional and inflammatory circumstances working concomitantly. Both morbid conditions are governed by distinct pathogenic mechanisms leading to the reduction in size of lean body mass (LBM). The liver production of TTR integrates the dietary and stressful components of any disease spectrum, explaining why it is the sole plasma protein whose evolutionary patterns closely follow the shape outlined by LBM fluctuations. Serial measurement of TTR therefore provides unequalled information on the alterations affecting overall protein nutritional status. Recent advances in TTR physiopathology emphasize the detecting power and preventive role played by the protein in hyperhomocysteinemic states, acquired metabolic disorders currently ascribed to dietary restriction in water-soluble vitamins. Sulfur (S)-deficiency is proposed as an additional causal factor in the sizeable proportion of hyperhomocysteinemic patients characterized by adequate vitamin intake but experiencing varying degrees of nitrogen (N)-depletion. Owing to the fact that N and S coexist in plant and animal tissues within tightly related concentrations, decreasing LBM as an effect of dietary shortage and/or excessive hypercatabolic losses induces proportionate S-losses. Regardless of water-soluble vitamin status, elevation of homocysteine plasma levels is negatively correlated with LBM reduction and declining TTR plasma levels. These findings occur as the result of impaired cystathionine-b-synthase activity, an enzyme initiating the transsulfuration pathway and whose suppression promotes the upstream accumulation and remethylation of homocysteine molecules. Under conditions of N- and S-deficiencies,the maintenance of methionine homeostasis indicates high metabolic priority.
Y. Ingenbleek
Laboratory of Nutrition, University Louis Pasteur Strasbourg
S.J. Richardson and V. Cody (eds.), Recent Advances in Transthyretin Evolution, 329
Structure and Biological Functions,
DOI: 10.1007/978‐3‐642‐00646‐3_20, # Springer‐Verlag Berlin Heidelberg 2009

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Vegan Diet is Sulfur Deficient and Heart Unhealthy

Larry H. Bernstein, MD, FCAP, Curator


The following is a reblog of “Heart of the Matter: Plant-Based Diets Lead to High Homocysteine, Low Sulfur and Marginal B12 Status”
Posted on September 26, 2011 by Dr Kaayla Daniel in WAPF Blog and tagged B12, Forks over Knives, Kaayla T. Daniel, Kilmer S. McCully, Yves Ingenbleek

It is a report of a scientific study carried out by Kilmer S. Cully and Yves Ingenbleek, Harvard Pathology and Univ Louis Pasteur.  I have previously written about the conundrum of transthyretin as an accurate marker of malnutrition, but also being lowered by the septic state.  This is accounted for by the catabolic state that sets off autocannabalization of skeletal muscle and lean body mass to provide gluconeogenic precursors to sustain life.  While serum albumin and transthyretin both decline, the former has a half-life of 20 days, while the latter is 48 hours.  Much work has been done to gain a better understand this rapid turnover protein that transports thyroxine, and the immediate result of the decline in concentration is a shift the the hormone protein binding equilibrium increasing the free thyroxine, a euthyroid hyperthyroid effect.  However, much work by Prof. Inglenbleek, some ion collaboration with Vernon Young, at MIT, showed that transthyretin reflects the sulfur stores of animals.  The sulfur to nitrogen ratio of plants is 1:20, but it is 1:12 in man, so the dietary intake would affect an omnivorous animal.  Recall that S is carried on amino acids that take part in disulfide linkage.  A deficiency in S containing amino acids would have a negative health effect.  The story is presented here.

The World Health Organization (WHO) reports that 16.7 million deaths occur worldwide each year due to cardiovascular disease, and more than half of those deaths occur in developing countries where plant-based diets high in legumes and starches are eaten by the vast majority of the people.

Yet “everyone knows” plant-based diets prevent heart disease.  Indeed this myth  is repeated so often that massive numbers of educated, health-conscious individuals in first world countries are consciously adopting third world style diets in the hope of preventing disease, optimizing health and maximizing longevity.   But if the WHO statistics are correct, plant-based diets might not be protective at all.   And today’s fashionable experiment in veganism could end very badly indeed.

A study out August 26 in the journal Nutrition makes a strong case against plant-based diets for prevention of heart disease.  The title alone  –  “Vegetarianism produces subclinical malnutrition, hyperhomocysteinemia and atherogenesis” — sounds a significant warning.   The article establishes  why subjects who eat mostly vegetarian diets develop morbidity and mortality from cardiovascular disease unrelated to vitamin B status and Framingham criteria.

Co-author Kilmer S. McCully, MD, “Father of the Homocysteine Theory of Heart Disease,” is familiar to WAPF members as winner of the Linus Pauling Award, WAPF’s Integrity in Science Award, and author of numerous articles published in peer-reviewed journals as well as the popular books The Homocysteine Revolution and The Heart Revolution.   In 2009 Dr. McCully was one of the signers of the Weston A. Price Foundation’s petition to the FDA in which we asked the agency to retract its unwarranted 1999 soy/heart disease health claim.  (

Dr. McCully teamed up with Yves Ingenbleek, MD, of the University Louis Pasteur in Strasbourg, France, which funded the research.   Dr. Ingenbleek is well known for his work on malnutrition, the essential role of sulfur to nitrogen, and sulfur deficiency as a cause of  hyperhomocysteinemia.

The study took place in Chad, and involved 24 rural male subjects age 18 to 30, and 15 urban male controls, age 18-29.   (Women in this region of Chad could not be studied because of their animistic beliefs and proscriptions against collecting their urine.)

The rural men were apparently healthy, physically active farmers with good lipid profiles.  Their staple foods included cassava, sweet potatoes, beans, millet and ground nuts.   Cassava leaves, cabbages and carrots provided good levels of carotenes, folates and pyridoxine (B6).  The diet is plant-based there because of a shortage of grazing lands and livestock, but subjects occasionally consume  some B12-containing foods, mostly poultry and eggs, though very little dairy or meat.   Their diet could be described as high carb, high fiber,  low in both protein and fat, and low in the sulfur containing amino acids.    In brief, the very diet recommended by many of today’s nutritional “experts” for overall good health and heart disease prevention.

The urban controls were likewise healthy and ate a similar diet, but with beef, smoked fish and canned or powdered milk regularly on the menus.  Their diet was thus higher in protein, fat and the sulfur-containing amino acids though roughly equivalent in calories.

Dr. McCully’s research over the past 40 years on the pathogenesis of atherosclerosis has shown the role of homocysteine in free radical damage and the protective effect of  vitamins B6, B12 and folate.   Indeed, many doctors today recommend taking this trio of B vitamins as an inexpensive heart disease “insurance policy.”

In Chad, both groups showed adequate levels of B6 and folate.  The B12 levels of the vegetarian group were lower, but the difference was only of “borderline significance.”   However, as the researchers point out, ”A previous study undertaken in the same Chadian area in a larger group of 60 rural participants did demonstrate a weak inverse correlation between B12 and homocysteine concentrations in the 20 subjects most severely protein depleted .  .  .  It is therefore likely that the hyperhomocysteinemia status of some of our rural subjects in the present survey might have resulted from combined B12 and protein deficiencies.   The correlation of B12 deficiency with hyperhomocysteinemia could well reach statistical significance if a larger groups of subjects were studied.”

Clearly it’s wise for people on plant-based diets to supplement their diets with B12, but protein malnutrition must also be addressed.   And the issue is not just getting enough protein to eat, but the right kind.   Quality, not just quantity.   The bottom line is we must eat  protein rich in bioavailable, sulfur-containing amino acids — and that means animal products.   (Vegans at this point will surely claim the issue is insufficient protein and trot out soy as the solution.   Soy is indeed a  complete plant based protein, but notoriously low in methionine.  It does contain decent levels of cysteine, but the cysteine is bound up in protease inhibitors, making it largely  biounavailable. (For more information, read  my book The Whole Soy Story: The Dark Side of America’s Favorite Health Food, endorsed by Dr. McCully, as well as our petition to the FDA noted above.)

So what did  Drs. Ingenbleek and  McCully find among the study group of protein-deficient people?   Higher levels of homocysteine, of course.  Also significant alterations in body composition,  lean body mass, body mass index and plasma transthyretin levels.  In plain English, the near-vegetarian subjects were thinner, with poorer muscle tone and showed subclinical signs of protein malnutrition.   (So much for popular ideas of extreme thinness being healthy. )

The plant-based diet of the study group was low in all of the sulfur-containing amino acids.   As would be expected, labwork on these men showed lower plasma cysteine and glutathione levels compared to the controls.  Methionine levels, however,  tested comparably.   The explanation for this is  “adaptive response.”   In brief, mammals trying to function with insufficient sulfur-containing amino acids will do whatever’s necessary to survive.   Given the essential role of methionine in metabolic processes, that means deregulating the transsulfuration pathway, increasing homocysteine levels, and methylating homocysteine to make methionine.

Ultimately, it all boils down to our need for sulfur.   As Stephanie Seneff, PhD, and many others have written in Wise Traditions and on this website, sulfur is vital for disease prevention and maintenance of good health.   In terms of heart disease, Drs. Ingenbleek and McCully have shown sulfur deficiency not only leads to high homocysteine levels, but is the likeliest reason some clinical trials using B6, B12 and folate interventions have proved ineffective for the prevention of cardiovascular and cerebrovascular diseases.    Over the past few years, headlines from such studies have led to widespread dismissal of Dr. McCully’s  “Homocysteine Theory of Heart Disease” and renewed media focus on cholesterol, c-reactive protein and other possible culprits that can be treated by statins and other profitable drugs.   In contrast, Drs. McCully and Ingenbleek research suggests we can better prevent heart disease with three inexpensive B vitamins and traditional diets rich in the sulfur-containing amino acids found in animal foods.

In the blaze of publicity surrounding Forks Over Knives and other blasts of vegan propaganda, few people are likely to hear about this study.   That’s sad, for it provides an important missing piece in our knowledge of heart disease development, a strong argument against the plant-based fad, and a bright new chapter in what the New York Times has called “The Fall and Rise of Kilmer McCully.”

*  *  *  *  *

Thanks to Sylvia Onusic PhD who was able to access a full text copy of this article to share with  me.

This entry was posted in WAPF Blog and tagged B12, Forks over Knives, Kaayla T. Daniel, Kilmer S. McCully, Naughty Nutritionist, soy, sulfur, Yves Ingenbleek. Bookmark the permalink.

Sylvia says:

September 26, 2011 at 5:32 pm

Kaayla, I found the article but you brought it to life- what a great explanation backed by high levels of knowledge and analysis. We are grateful for your numerous contributions to the field of health!
Thanks so much.

Sylvia Onusic



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A Second Look at the Transthyretin Nutrition Inflammatory Conundrum

Subtitle: Transthyretin and the Systemic Inflammatory Response


Author and Curator: Larry H. Bernstein, MD, FACP, Clinical Pathologist, Biochemist, and Transfusion Physician


Brief introduction

Transthyretin  (also known as prealbumin) has been widely used as a biomarker for identifying protein-energy malnutrition (PEM) and for monitoring the improvement of nutritional status after implementing a nutritional intervention by enteral feeding or by parenteral infusion. This has occurred because transthyretin (TTR) has a rapid removal from the circulation in 48 hours and it is readily measured by immunometric assay. Nevertheless, concerns have been raised about the use of TTR in the ICU setting, which prompted a review of the  benefit of using this test in acute and chronic care. TTR is easily followed in the underweight and the high risk populations in an ambulatory setting, which has a significant background risk of chronic diseases. It is sensitive to the systemic inflammatory response syndrome (SIRS), and needs to be understood in the context of acute illness to be used effectively. There are a number of physiologic changes associated with SIRS and the injury/repair process that affect TTR. The most important point is that in the context of an ICU setting, the contribution of TTR is significant in a complex milieu.  A much better understanding of the significance of this program has emerged from studies of nitrogen and sulfur in health and disease.

Transthyretin protein structure

Transthyretin protein structure (Photo credit: Wikipedia)

Age-standardised disability-adjusted life year...

Age-standardised disability-adjusted life year (DALY) rates from Protein-energy malnutrition by country (per 100,000 inhabitants). (Photo credit: Wikipedia)


The systemic inflammatory response syndrome C-reactive protein and transthyretin conundrum.
Larry H Bernstein
Clin Chem Lab Med 2007; 45(11):0
ICID: 939932
Article type: Editorial

The Transthyretin Inflammatory State Conundrum
Larry H. Bernstein
Current Nutrition & Food Science, 2012, 8, 00-00

Keywords: Tranthyretin (TTR), systemic inflammatory response syndrome (SIRS), protein-energy malnutrition (PEM), C- reactive protein, cytokines, hypermetabolism, catabolism, repair.

Transthyretin has been widely used as a biomarker for identifying protein-energy malnutrition (PEM) and for monitoring the improvement of nutritional status after implementing a nutritional intervention by enteral feeding or by parenteral infusion. This has occurred because transthyretin (TTR) has a rapid removal from the circulation in 48 hours and it is readily measured by immunometric assay. Nevertheless, concerns have been raised about the use of TTR in the ICU setting, which prompts a review of the actual benefit of using this test in a number of settings. TTR is easily followed in the underweight and the high risk populations in an ambulatory setting, which has a significant background risk of chronic diseases. It is sensitive to the systemic inflammatory response syndrome (SIRS), and needs to be understood in the context of acute illness to be used effectively.

There are a number of physiologic changes associated with SIRS and the injury/repair process that affect TTR and  in the context of an ICU setting, the contribution of TTR is essential.  The only consideration is the timing of initiation since the metabolic burden is sufficiently high that a substantial elevation is expected in the first 3 days post admission, although the level of this biomarker is related to the severity of injury. Despite the complexity of the situation, TTR is not to be considered a test “for all seasons”. In the context of age, prolonged poor meal intake, chronic or acute illness, TTR needs to be viewed in a multivariable lens, along with estimated lean body mass, C-reactive protein, the absolute lymphocyte count, presence of neutrophilia, and perhaps procalcitonin if there is remaining uncertainty. Furthermore, the reduction of risk of associated complication requires a systematized approach to timely identification, communication, and implementation of a suitable treatment plan.

The most important point is that in the context of an ICU setting, the contribution of TTR is significant in a complex milieu.


Title: The Automated Malnutrition Assessment
Accepted 29 April 2012. Nutrition (2012), doi:10.1016/j.nut.2012.04.017.
Authors: Gil David, PhD; Larry Howard Bernstein, MD; Ronald R Coifman, PhD
Article Type: Original Article

Keywords: Network Algorithm; unsupervised classification; malnutrition screening; protein energy malnutrition (PEM); malnutrition risk; characteristic metric; characteristic profile; data characterization; non-linear differential diagnosis

We have proposed an automated nutritional assessment (ANA) algorithm that provides a method for malnutrition risk prediction with high accuracy and reliability.  The problem of rapidly identifying risk and severity of malnutrition is crucial for minimizing medical and surgical complications. These are not easily performed or adequately expedited. We characterized for each patient a unique profile and mapped similar patients into a classification. We also found that the laboratory parameters were sufficient for the automated risk prediction.


Title: The Increasing Role for the Laboratory in Nutritional Assessment
Article Type: Editorial
Section/Category: Clinical Investigation
Accepted 22 May 2012.
Clin Biochem (2012), doi:10.1016/j.clinbiochem.2012.05.024
Keywords: Protein Energy Malnutrition; Nutritional Screening; Laboratory Testing
Author: Dr. Larry Howard Bernstein, MD

The laboratory role in nutritional management of the patient has seen remarkable growth while there have been dramatic changes in technology over the last 25 years, and it is bound to be transformative in the near term. This editorial is an overview of the importance of the laboratory as an active participant in nutritional care.

The discipline emerged divergently along separate paths with unrelated knowledge domains in physiological chemistry, pathology, microbiology, immunology and blood cell recognition, and then cross-linked emerging into clinical biochemistry, hematology-oncology, infectious diseases, toxicology and therapeutics, genetics, pharmacogenomics, translational genomics and clinical diagnostics.

In reality, the more we learn about nutrition, the more we uncover of metabolic diversity of individuals, the family, and societies in adapting and living in many unique environments and the basic reactions, controls, and responses to illness. This course links metabolism to genomics and individual diversity through metabolomics, which will be enlightened by chemical and bioenergetic insights into biology and translated into laboratory profiling.

Vitamin deficiencies were discovered as clinical entities with observed features as a result of industrialization (rickets and vitamin D deficiency) and mercantile trade (scurvy and vitamin C)[2].  Advances in chemistry led to the isolation of each deficient “substance”.  In some cases, a deficiency of a vitamin and what is later known as an “endocrine hormone” later have confusing distinctions (vitamin D, and islet cell insulin).

The accurate measurement and roles of trace elements, enzymes, and pharmacologic agents was to follow within the next two decades with introduction of atomic absorption, kinetic spectrophotometers, column chromatography and gel electrophoresis.  We had fully automated laboratories by the late 1960s, and over the next ten years basic organ panels became routine.   This was a game changer.

Today child malnutrition prevalence is 7 percent of children under the age of 5 in China, 28 percent in sub-Saharan African, and 43 percent in India, while under-nutrition is found mostly in rural areas with 10 percent of villages and districts accounting for 27-28 percent of all Indian underweight children. This may not be surprising, but it is associated with stunting and wasting, and it has not receded with India’s economic growth. It might go unnoticed viewed alongside a growing concurrent problem of worldwide obesity.

The post WWII images of holocaust survivors awakened sensitivity to nutritional deprivation.

In the medical literature, Studley [HO Studley.  Percentage of weight loss. Basic Indicator of surgical risk in patients with chronic peptic ulcer.  JAMA 1936; 106(6):458-460.  doi:10.1001/jama.1936.02770060032009] reported the association between weight loss and poor surgical outcomes in 1936.  Ingenbleek et al [Y Ingenbleek, M De Vissher, PH De Nayer. Measurement of prealbumin as index of protein-calorie malnutrition. Lancet 1972; 300[7768]: 106-109] first reported that prealbumin (transthyretin, TTR) is a biomarker for malnutrition after finding very low TTR levels in African children with Kwashiorkor in 1972, which went unnoticed for years.  This coincided with the demonstration by Stanley Dudrick  [JA Sanchez, JM Daly. Stanley Dudrick, MD. A Paradigm ShiftArch Surg. 2010; 145(6):512-514] that beagle puppies fed totally through a catheter inserted into the superior vena cava grew, which method was then extended to feeding children with short gut.  Soon after Bistrian and Blackburn [BR Bistrian, GL Blackburn, E Hallowell, et al. Protein status of general surgical patients. JAMA 1974; 230:858; BR Bistrian, GL Blackburn, J Vitale, et al. Prevalence of malnutrition in general medicine patients, JAMA, 1976, 235:1567] showed that malnourished hospitalized medical and surgical patients have increased length of stay, increased morbidity, such as wound dehiscence and wound infection, and increased postoperative mortality, later supported by many studies.

Michael Meguid,MD, PhD, founding editor of Nutrition [Elsevier] held a nutrition conference “Skeleton in the Closet – 20 years later” in Los Angeles in 1995, at which a Beckman Prealbumin Roundtable was held, with Thomas Baumgartner and Michael M Meguid as key participants.  A key finding was that to realize the expected benefits of a nutritional screening and monitoring program requires laboratory support. A Ross Roundtable, chaired by Dr. Lawrence Kaplan, resulted in the first Standard of Laboratory Practice Document of the National Academy of Clinical Biochemists on the use of the clinical laboratory in nutritional support and monitoring. Mears then showed a real benefit to a laboratory interactive program in nutrition screening based on TTR [E Mears. Outcomes of continuous process improvement of a nutritional care program incorporating serum prealbumin measurements. Nutrition 1996; 12 (7/8): 479-484].

A later Ross Roundtable on Quality in Nutritional Care included a study of nutrition screening and time to dietitian intervention organized by Brugler and Di Prinzio that showed a decreased length of hospital stay with $1 million savings in the first year (which repeated), which included reduced cost for dietitian evaluations and lower complication rates.

Presentations were made at the 1st International Transthyretin Congress in Strasbourg, France by Mears [E Mears.  The role of visceral protein markers in protein calorie malnutrition. Clin Chem Lab Med 2002; 40:1360-1369] on the impact of TTR in screening for PEM in a public hospital in Louisiana, and by Potter [MA Potter, G Luxton. Prealbumin measurement as a screening tool for patients with protein calorie malnutrition in emergency hospital admissions: a pilot study.  Clin Invest Med. 1999; 22(2):44-52] that indicated a 17% in-hospital mortality rate in a Canadian hospital for patients with PCM compared with 4% without PCM (p < 0.02), while only 42% of patients with PCM received nutritional supplementation. Cost analysis of screening with prealbumin level projected a saving of $414 per patient screened.  Ingenbleek and Young [Y Ingenbleek, VR Young.  Significance of transthyretin in protein metabolism.  Clin Chem Lab Med. 2002; 40(12):1281–1291.  ISSN (Print) 1434-6621, DOI: 10.1515/ CCLM.2002.222, December 2002. published online: 01/06/2005] tied the TTR to basic effects reflected in protein metabolism.


Transthyretin as a marker to predict outcome in critically ill patients.
Arun Devakonda, Liziamma George, Suhail Raoof, Adebayo Esan, Anthony Saleh, Larry H Bernstein
Clin Biochem 2008; 41(14-15):1126-1130
ICID: 939927
Article type: Original article

TTR levels correlate with patient outcomes and are an accurate predictor of patient recovery in non-critically ill patients, but it is uncertain whether or not TTR level correlates with level of nutrition support and outcome in critically ill patients. This issue has been addressed only in critically ill patients on total parenteral nutrition and there was no association reported with standard outcome measures. We revisit this in all patients admitted to a medical intensive care unit.

Serum TTR was measured on the day of admission, day 3 and day 7 of their ICU stay. APACHE II and SOFA score was assessed on the day of admission. A registered dietician for their entire ICU stay assessed the nutritional status and nutritional requirement. Patients were divided into three groups based on initial TTR level and the outcome analysis was performed for APACHE II score, SOFA score, ICU length of stay, hospital length of stay, and mortality.

TTR showed excellent concordance with the univariate or multivariate classification of patients with PEM or at high malnutrition risk, and followed for seven days in the ICU, it is a measure of the metabolic burden.  TTR levels decline from day 1 to day 7 in spite of providing nutritional support. Twenty-five patients had an initial TTR serum concentration more than 17 mg/dL (group 1), forty-eight patients had mild malnutrition with a concentration between 10 and 17 mg/dL (group 2), Forty-five patients had severe malnutrition with a concentration less than 10 mg/dL (group 3).  Initial TTR level had inverse correlation with ICU length of stay, hospital length of stay, and APACHE II score, SOFA score; and predicted mortality, especially in group 3.


A simplified nutrition screen for hospitalized patients using readily available laboratory and patient
Linda Brugler, Ana K Stankovic, Madeleine Schlefer, Larry Bernstein
Nutrition 2005; 21(6):650-658
ICID: 825623
Article type: Review article
The role of visceral protein markers in protein calorie malnutrition.
Linda Brugler, Ana Stankovic, Larry Bernstein, Frederick Scott, Julie O’Sullivan-Maillet
Clin Chem Lab Med 2002; 40(12):1360-1369
ICID: 636207
Article type: Original article

The Automated Nutrition Score is a data-driven extension of continuous quality improvement.

Larry H Bernstein
Nutrition 2009; 25(3):316-317
ICID: 939934

Transthyretin: its response to malnutrition and stress injury. clinical usefulness and economic implications.
LH Bernstein, Y Ingenbleek
Clin Chem Lab Med 2002; 40(12):1344-1348
ICID: 636205
Article type: Original article


Yves Ingenbleek  MD  PhD  and  Larry Bernstein MD
J CLIN LIGAND ASSAY  (out of print)

The acute reaction to stress is characterized by major metabolic, endocrine and immune alterations. According to classical descriptions, these changes clinically present as a succession of 3 adaptive steps – ebb phase, catabolic flow phase, and anabolic flow phase. The ebb phase, shock and resuscitation, is immediate, lasts several hours, and is characterized by hypokinesis, hypothermia, hemodynamic instability and reduced basal metabolic rate. The catabolic flow phase, beginning within 24 hours and lasting several days, is characterized by catabolism with the flow of gluconeogenic substrates and ketone bodies in response to the acute injury. The magnitude of the response depends on the acuity and the severity of the stress. The last, a reparative anabolic flow phase, lasts weeks and is characterized by the accretion of amino acids (AAs) to rebuilding lean body mass.

The current opinion is that the body economy is reset during the course of stress at novel thresholds of metabolic priorities. This is exemplified mainly by proteolysis of muscle, by an effect on proliferating gut mucosa and lymphoid tissue as substrates are channeled to support wound healing, by altered syntheses of liver proteins with preferential production of acute phase proteins (APPs) and local repair in inflamed tissues (3). The first two stages demonstrate body protein breakdown exceeding the rate of protein synthesis, resulting in a negative nitrogen (N) balance, muscle wasting and weight loss. In contrast, the last stage displays reversed patterns, implying progressive recovery of endogenous N pools and body weight.

These adaptive alterations undergo continuing elucidation. The identification of cytokines, secreted by activated macrophages/monocytes or other reacting cells, has provided further insights into the molecular mechanisms controlling energy expenditure, redistribution of protein pools, reprioritization of syntheses and secretory processes.

The free fraction of hormones bound to specific binding-protein(s) [BP(s)] manifests biological activities, and any change in the BP blood level modifies the effect of the hormone on the end target organ.  The efficacy of these adaptive responses may be severely impaired in protein-energy malnourished (PEM) patients. This is especially critical with respect to changes of the circulating levels of transthyretin (TTR), retinol-binding protein (RBP) and corticosteroid-binding globulin (CBG) conveying thyroid hormones (TH), retinol and cortisol, respectively.  This reaction is characterized by cytokine mediated autocrine, paracrine and endocrine changes. Among the many inducing molecules identified, interleukins 1 and 6 (Il-1, Il-6) and tumor necrosis factor a (TNF) are associated with enhanced production of 3 counterregulatory hormonal families (cortisol, catecholamines and glucagon). Growth hormone (GH) and TH also have roles in these metabolic adjustments.

There is overproduction of cortisol mediated by several cytokines acting on both the adrenal cortex (10) and on the pituitary through hypothalamic CRH with loss of feedback regulation of ACTH production (11). Hypercortisolemia is a major finding observed after surgery (12), sepsis (13), and medical insults, usually correlated with severity of insult and of complications. Rising cortisol values parallel hyperglycemic trends, as an effect of both gluconeogenesis and insulin resistance. Working in concert with TNF, glucocorticoids govern the breakdown of muscle mass, which is regarded as the main factor responsible for the negative N balance.

Under normal conditions, GH exerts both lipolytic and anabolic influences in the whole body economy under the dual control of the hypothalamic hormones somatocrinin (GHRH) and somatostatin (SRIH). GH secretion is usually depressed by rising blood concentrations of glucose and free fatty acids (FFAs) but is paradoxicaly elevated despite hyperglycemia in stressed patients.

The oversecretion of counterregulatory hormones working in concert generates subtle equilibria between glycogenolytic/glycolytic/gluconeogenic adaptive processes. The net result is the neutralization of the main hypoglycemic and anabolic activities of insulin and the development of a persisting and controlled hyperglycemic tone in the stressed body. The molecular mechanisms whereby insulin resistance occurs in the course of stress refer to
cytokine-  and  hormone-induced  phosphorylation abnormalities affecting receptor signaling. The insulin-like anabolic processes of GH are mediated by IGF1 working as relay agent. The expected high IGF1 surge associated with GH oversecretion is not observed in severe stress as plasma values are usually found at the lower limit of normal or even in the subnormal range.  The end result of this dissociation between high GH and low IGF1 levels is to favor the proteolysis of muscle mass to release AAs for gluconeogenesis and the breakdown of adipose tissue to provide ketogenic substrates.

The acute stage of stress is associated with the onset of a low T3 syndrome typically delineated by the drop of both total (TT3) and free (FT3) triiodothyronine plasma levels in the subnormal range. In contrast, both total (TT4) and free (FT4) thyroxine values usually remain within normal ranges with declining trends observed for TT4 and rising tendencies for FT4 (44). This last free compound is regarded as the sensor reflecting the actual thyroid status and governing the release of TSH whereas FT3 works as the active hormonal mediator at nuclear receptor level. The maintenance of an euthyroid sick syndrome is compatible with the down-regulation of most metabolic and energetic processes in healthy tissues. These inhibitory effects , negatively affecting all functional steps of the hypothalamo-pituitary-thyroid axis concern TSH production, iodide uptake, transport and organification into iodotyrosyl residues, peroxidase coupling activity as well as thyroglobulin synthesis and TH leakage. Taken together, the above-mentioned data indicate that the development of hyperglycemia and of insulin-resistance in healthy tissues – mainly in the muscle mass – are hallmarks resulting from the coordinated activities of the counterregulatory hormones.

A growing body of recent data suggest that the stressed territory, whatever the causal agent – bacterial or viral sepsis, auto-immune disorder, traumatic or toxic shock, burns, cancer – manifest differentiated metabolic and immune reactions. The amplitude, duration and efficacy of these responses are reportedly impaired along several ways in PEM patients. These last detrimental effects are accompanied by a number of medical, social and economical consequences, such as extended length of hospital stay and increased complication / mortality rates. It is therefore mandatory to correctly identify and follow up the nutritional status of hospitalized patients. Such approaches are prerequisite to timely and scientifically grounded nutritional and pharmacological mediated interventions.

Contrary to the rest of the body, energy requirements of the inflamed territory are primarily fulfilled by anaerobic glycolysis, an effect triggered by the inhibition of key-enzymes of carbohydrate metabolism, notably pyruvate-dehydrogenase. This non-oxidative combustion of glucose reveals low conversion efficiency but offers the major advantage to maintain, in the context of hyperglycemia, fuel provision to poorly irrigated and/or edematous tissues. The depression of the 5’-monodeiodinating activity (5’-DA) plays a pivotal role in these adaptive changes, yielding inactive reverse T3 (rT3) as index of impaired T4 to T3 conversion rates, but at the same time there is an augmented supply of bioactive T3 molecules and local overstimulation of thyro-dependent processes characterized by thyroid down-regulation.  The same differentiated evolutionary pattern applies to IGF1. In spite of lowered plasma total concentrations, the proportion of IGF1 released in free form may be substantially increased owing to the proteolytic degradation of IGFBP-3 in the intravascular compartment. The digestion of  BP-3 results from the surge of several proteases occurring the course of stress, yielding biologically active IGF1 molecules available for the repair of damaged tissues. In contrast, healthy receptors oppose a strong resistance to IGF1 ligands freed in the general circulation, likely induced by an acquired phosphorylation defect very similar in nature to that for the insulin transduction pathway.

PEM is the generic denomination of a broad spectrum of nutritional disorders, commonly found in hospital settings, and whose extreme poles are identified as marasmus and kwashiorkor. The former condition is usually regarded as the result of long-lasting starvation leading to the loss of lean body mass and fat reserves but relatively well-preserved liver function and immune capacities. The latter condition is typically the consequence of (sub)acute deprivation predominantly affecting the protein content of staplefood, an imbalance causing hepatic steatosis, fall of visceral proteins, edema and increased vulnerability to most stressful factors. PEM may be hypometabolic or hypermetabolic, usually coexists with other diseased states and is frequently associated with complications. Identification of PEM calls upon a large set of clinical and analytical disciplines comprising anthropometry, immunology, hematology and biochemistry.

CBG, TTR and RBP share in common the transport of specific ligands exerting their metabolic effects at nuclear receptor level. Released from their specific BPs in free form, cortisol, FT4 and retinol immediately participe to the strenghtening of the positive and negative responses to stressful stimuli. CBG is a relatively weak responder to short-term nutritional influences (73)  although long-lasting PEM is reportedly capable of causing its significant diminution (74). The dramatic drop of CBG in the course of stress appears as the combined effect of Il-6-induced posttranscriptional blockade of its liver synthesis (75) and peripheral overconsumption by activated neutrophils (61). The divergent alterations outlined by CBG and total cortisolemia result in an increased disposal of free ligand reaching proportions considerably higher than the 4 % recorded under physiological conditions.

The appellation of negative APPs that was once given to the visceral group of carrier-proteins. The NDAD concept takes the opposite view, defending the opinion that their suppressed synthesis releases free ligands which positively contribute to strengthen all aspects of the stress reaction, justifying the ABR denomination. This implies that the role played by ABRs should no longer be interpreted in terms of concentrations but in terms of functionality.


Yves Ingenbleek. The Open Clinical Chemistry Journal, 2011, 4, 34-44.

Vegetarian subjects consuming subnormal amounts of methionine (Met) are characterized by subclinical protein malnutrition causing reduction in size of their lean body mass (LBM) best identified by the serial measurement of plasma transthyretin (TTR). As a result, the transsulfuration pathway is depressed at cystathionine-β-synthase (CβS) level triggering the upstream sequestration of homocysteine (Hcy) in biological fluids and promoting its conversion to Met. Maintenance of beneficial Met homeostasis is counterpoised by the drop of cysteine (Cys) and glutathione (GSH) values downstream to CβS causing in turn declining generation of hydrogen sulfide (H2S) from enzymatic sources. The biogenesis of H2S via non-enzymatic reduction is further inhibited in areas where earth’s crust is depleted in elemental sulfur (S8) and sulfate oxyanions. Combination of subclinical malnutrition and S8-deficiency thus maximizes the defective production of Cys, GSH and H2S reductants, explaining persistence of unabated oxidative burden. The clinical entity increases the risk of developing cardiovascular diseases (CVD) and stroke in underprivileged plant-eating populations regardless of Framingham criteria and vitamin-B status. Although unrecognized up to now, the nutritional disorder is one of the commonest worldwide, reaching top prevalence in populated regions of Southeastern Asia. Increased risk of hyperhomocysteinemia and oxidative stress may also affect individuals suffering from intestinal malabsorption or westernized communities having adopted vegan dietary lifestyles.

Metabolic pathways: Met molecules supplied by dietary proteins are submitted to TM processes allowing to release Hcy which may in turn either undergo Hcy – Met RM pathways or be irreversibly committed into TS decay. Impairment of CbS activity, as described in protein malnutrition, entails supranormal accumulation of Hcy in body fluids, stimulation of activity and maintenance of Met homeostasis. This last beneficial effect is counteracted by decreased concentration of most components generated downstream to CbS, explaining the depressed CbS- and CbL-mediated enzymatic production of H2S along the TS cascade. The restricted dietary intake of elemental S further operates as a limiting factor for its non-enzymatic reduction to H2S which contributes to downsizing a common body pool. Combined protein- and S-deficiencies work in concert to deplete Cys, GSH and H2S from their body reserves, hence impeding these reducing molecules to properly face the oxidative stress imposed by hyperhomocysteinemia.

see also …

McCully, K.S. Vascular pathology of homocysteinemia: implications for the pathogenesis of arteriosclerosis. Am. J. Pathol., 1996, 56, 111-128.

Cheng, Z.; Yang, X.; Wang, H. Hyperhomocysteinemia and endothelial dysfunction. Curr. Hypertens. Rev., 2009, 5,158-165.

Loscalzo, J. The oxidant stress of hyperhomocyst(e)inemia. J. Clin.Invest., 1996, 98, 5-7.

Ingenbleek, Y.; Hardillier, E.; Jung, L. Subclinical protein malnutrition is a determinant of hyperhomocysteinemia. Nutrition, 2002, 18, 40-46.

Ingenbleek, Y.; Young, V.R. The essentiality of sulfur is closely related to nitrogen metabolism: a clue to hyperhomocysteinemia. Nutr. Res. Rev., 2004, 17, 135-153.

Hosoki, R.; Matsuki, N.; Kimura, H. The possible role of hydrogen sulfide as an endogenous smooth muscle relaxant in synergy with nitric oxide. Biochem. Biophys. Res. Commun., 1997, 237, 527-531.

Tang, B.; Mustafa, A.; Gupta, S.; Melnyk, S.; James S.J.; Kruger, W.D. Methionine-deficient diet induces post-transcriptional downregulation of cystathionine-􀀁-synthase. Nutrition, 2010, 26, 1170-1175.

Elshorbagy, A.K.; Valdivia-Garcia, M.; Refsum, H.; Smith, A.D.; Mattocks, D.A.; Perrone, C.E. Sulfur amino acids in methioninerestricted rats: Hyperhomocysteinemia. Nutrition, 2010, 26, 1201- 1204.


Yves Ingenbleek. Plasma Transthyretin Reflects the Fluctuations of Lean Body Mass in Health and Disease. Chapter 20. In S.J. Richardson and V. Cody (eds.), Recent Advances in Transthyretin Evolution, Structure and Biological Functions, DOI: 10.1007/978‐3‐642‐00646‐3_20, # Springer‐Verlag Berlin Heidelberg 2009.

Transthyretin (TTR) is a 55-kDa protein secreted mainly by the choroid plexus and the liver. Whereas its intracerebral production appears as a stable secretory process allowing even distribution of intrathecal thyroid hormones, its hepatic synthesis is influenced by nutritional and inflammatory circumstances working concomitantly. Both morbid conditions are governed by distinct pathogenic mechanisms leading to the reduction in size of lean body mass (LBM). The liver production of TTR integrates the dietary and stressful components of any disease spectrum, explaining why it is the sole plasma protein whose evolutionary patterns closely follow the shape outlined by LBM fluctuations. Serial measurement of TTR therefore provides unequalled information on the alterations affecting overall protein nutritional status. Recent advances in TTR physiopathology emphasize the detecting power and preventive role played by the protein in hyperhomocysteinemic states, acquired metabolic disorders currently ascribed to dietary restriction in water-soluble vitamins. Sulfur (S)-deficiency is proposed as an additional causal factor in the sizeable proportion of hyperhomocysteinemic patients characterized by adequate vitamin intake but experiencing varying degrees of nitrogen (N)-depletion. Owing to the fact that N and S coexist in plant and animal tissues within tightly related concentrations, decreasing LBM as an effect of dietary shortage and/or excessive hypercatabolic losses induces proportionate S-losses. Regardless of water-soluble vitamin status, elevation of homocysteine plasma levels is negatively correlated with LBM reduction and declining TTR plasma levels. These findings occur as the result of impaired cystathionine-b-synthase activity, an enzyme initiating the transsulfuration pathway and whose suppression promotes the upstream accumulation and remethylation of homocysteine molecules. Under conditions of N- and S-deficiencies, the maintenance of methionine homeostasis indicates high metabolic priority.

Schematically, the human body may be divided into two major compartments, namely fat mass (FM) and FFM that is obtained by substracting
FM from body weight (BW). The fat cell mass sequesters about 80% of the total body lipids, is poorly hydrated and contains only small quantities of lean tissues and nonfat constituents. FFM comprises the sizeable part of lean tissues and minor mineral compounds among which are Ca, P, Na, and Cl pools totaling about 1.7 kg or 2.5% of BW in a healthy man weighing 70 kg. Subtraction of mineral mass from FFM provides LBM, a composite aggregation of organs and tissues with specific functional properties. LBM is thus nearly but not strictly equivalent to FFM. With extracellular mineral content subtracted, LBM accounts for most of total body proteins (TBP) and of TBN assuming a mean 6.25 ratio between protein and N content.

SM accounts for 45% of TBN whereas the remaining 55% is in nonmuscle lean tissues. The LBM of the reference man contains 98% of total
body potassium (TBK) and the bulk of total body sulfur (TBS). TBK and TBS reach equal intracellular amounts (140 g each) and share distribution patterns (half in SM and half in the rest of cell mass).  The body content of K and S largely exceeds that of magnesium (19 g), iron (4.2 g) and zinc (2.3 g). The average hydration level of LBM in healthy subjects of all age is 73% with the proportion of the intracellular/extracellular fluid spaces being 4:3. SM is of particular relevance in nutritional studies due to its capacity to serve as a major reservoir of amino acids (AAs) and as a dispenser of gluconeogenic substrates. An indirect estimate of SM size consists in the measurement of urinary creatinine, end-product of the nonenzymatic hydrolysis of phosphocreatine which is limited to muscle cells.

During ageing, all the protein components of the human body decrease regularly. This shrinking tendency is especially well documented for SM  whose absolute amount is preserved until the end of the fifth decade, consistent with studies showing unmodified muscle structure, intracellular K content and working capacit. TBN and TBK are highly correlated in healthy subjects and both parameters manifest an age-dependent curvilinear decline
with an accelerated decrease after 65 years.  The trend toward sarcopenia is more marked and rapid in elderly men than in elderly women decreasing strength and functional capacity. The downward SM slope may be somewhat prevented by physical training or accelerated by supranormal cytokine status as reported in apparently healthy aged persons suffering low-grade inflammation. 2002) or in critically ill patients whose muscle mass undergoes proteolysis and contractile dysfunction.

The serial measurement of plasma TTR in healthy children shows that BP values are low in the neonatal period and rise linearly with superimposable concentrations in both sexes during infant growth consistent with superimposable N accretion and protein synthesis rates. Starting from the sixties, TTR values progressively decline showing steeper slopes in elderly males. The lowering trend seems to be initiated by the attenuation of androgen influences and trophic stimuli with increasing age. The normal human TTR trajectory from birth to death has been well documented by scientists belonging to the Foundation for Blood Research. TTR is the first plasma protein to decline in response to marginal protein restricion, thus working as an early signal warning that adaptive mechanisms maintaining homeostasis are undergoing decompensation.

TTR was proposed as a marker of protein nutritional status following a clinical investigation undertaken in 1972 on protein-energy malnourished (PEM) Senegalese children (Ingenbleek et al. 1972). By comparison with ALB and transferrin (TF) plasma values, TTR revealed a much higher degree of sensitivity to changes in protein status that has been attributed to its shorter biological half-life (2 days) and to its unusual Trp richness (Ingenbleek et al. 1972, 1975a). Transcription of the TTR gene in the liver is directed by CCAAT/enhancer binding protein (C/EBP) bound to hepatocyte nuclear factor 1 (HNF1) under the control of several other HNFs. The mechanism responsible for the suppressed TTR synthesis in PEM-states is a restricted AA and energy supply working as limiting factors (Ingenbleek and Young 2002). The rapidly turning over TTR protein is highly responsive to any change in protein flux and energy supply, being clearly situated on the cutting edge of the equipoise.

LBM shrinking may be the consequence of either dietary restriction reducing protein syntheses to levels compatible with survival or that of cytokine-induced tissue proteolysis exceeding protein synthesis and resulting in a net body negative N balance. The size of LBM in turn determines plasma TTR concentrations whose liver production similarly depends on both dietary provision and inflammatory conditions. In animal cancer models, reduced TBN pools were correlated with decreasing plasma TTR values and provided the same predictive ability. In kidney patients, LBM is proposed as an excellent predictor of outcome working in the same direction as TTR plasma levels.  High N intake, supposed to preserve LBM reserves, reduces significantly the mortality rate of kidney patients and is positively correlated with the alterations of TTR plasma concentrations appearing as the sole predictor of final outcome. It is noteworthy that most SELDI or MALDI workers interested in defining protein nutritional status have chosen TTR as a biomarker, showing that there exists a large consensus considering the BP as the most reliable indicator of protein depletion in most morbid circumstances.

Total homocysteine (tHcy) is a S-containing AA not found in customary diets but endogenously produced in the body of mammals by the enzymatic transmethylation of methionine (Met), one of the eight IAAs supplied by staplefoods. tHcy may either serve as precursor substrate for the synthesis of new Met molecules along the remethylation (RM) pathway or undergo irreversible kidney leakage through a cascade of derivatives defining the transsulfuration (TS) pathway. Hcy is thus situated at the crossroad of RM and TS pathways that are regulated by three water-soluble vitamins (pyridoxine, B6; folates, B9; cobalamins, B12).

Significant positive correlations are found between tHcy and plasma urea and plasma creatinine, indicating that both visceral and muscular tissues undergo proteolytic degradation throughout the course of rampant inflammatory burden. In healthy individuals, tHcy plasma concentrations maintain positive correlations with LBM and TTR from birth until the end of adulthood. Starting from the onset of normal old age, tHcy values become disconnected from LBM control and reveal diverging trends with TTR values. Of utmost importance is the finding that, contrary to all protein
components which are downregulated in protein-depleted states, tHcy values are upregulated.  Hyperhomocysteinemia is an acquired clinical entity characterized by mild or moderate elevation in tHcy blood values found in apparently healthy individuals (McCully 1969). This distinct morbid condition appears as a public health problem of increasing importance in the general population, being regarded as an independent and graded risk factor for vascular pathogenesis unrelated to hypercholesterolemia, arterial hypertension, diabetes and smoking.

Studies grounded on stepwise multiple regression analysis have concluded that the two main watersoluble vitamins account for only 28% of tHcy variance whereas vitamins B6, B9, and B12, taken together, did not account for more than 30–40% of variance. Moreover, a number of hyperhomocysteinemic conditions are not responsive to folate and pyridoxine supplementation. This situation prompted us to search for other causal factors which might fill the gap between the public health data and the vitamin triad deficiencies currently incriminated. We suggest that S – the forgotten element – plays central roles in nutritional epidemiology (Ingenbleek and Young 2004).

Aminoacidemia studies performed in PEM children, adult patients and elderly subjects have reported that the concentrations of plasma IAAs invariably display lowering trends as the morbid condition worsens. The depressed tendency is especially pronounced in the case of tryptophan and for the so-called branched-chain AAs (BCAAs, isoleucine, leucine, valine) the decreases in which are regarded as a salient PEM feature following the direction outlined by TTR (Ingenbleek et al. 1986). Met constitutes a notable exception to the above described evolutionary profiles, showing unusual stability in chronically protein depleted states.

Maintenance of normal methioninemia is associated with supranormal tHcy blood values in PEMadults (Ingenbleek et al. 1986) and increased tHcy leakage in the urinary output of PEM children. In contrast, most plasma and urinary S-containing compounds produced along the TS pathway downstream to CbSconverting step (Fig. 20.1) display significantly diminished values. This is notably the case for cystathionine (Ingenbleek et al. 1986), glutathione, taurine, and sulfaturia. Such distorted patterns are reminiscent of abnormalities defining homocystinuria, an inborn disease of Met metabolism characterized by CbS refractoriness to pyridoxine stimuli, thereby promoting the upstream retention of tHcy in biological fluids. It
was hypothesized more than 20 years ago (Ingenbleek et al. 1986) that PEM is apparently able to similarly depress CbS activity, suggesting that the enzyme is a N-status sensitive step working as a bidirectional lockgate, overstimulated by high Met intake (Finkelstein and Martin 1986) and downregulated under N-deprivation conditions (Ingenbleek et al. 2002). Confirmation that N dietary deprivation may inhibit CbS activity has recently provided. The tHcy precursor pool is enlarged in biological fluids, boosting Met remethylation processes along the RM pathway, consistent with studies showing overstimulation of Met-synthase activity in conditions of protein restriction. In other words, high tHcy plasma concentrations observed in PEM states are the dark side of adaptive mechanisms for maintaining Met homeostasis. This is consistent with the unique role played by Met in the preservation of N body stores.

The classical interpretation that strict vegans, who consume plenty of folates in their diet and manifest nevertheless higher tHcy plasma concentrations than omnivorous counterparts, needs to be revisited. On the basis of hematological and biochemical criteria, cobalamin deficiency is one of the most prevalent vitamin-deficiencies wordwide, being often incriminated as deficient in vegan subjects. It seems, however, likely that its true causal impact on rising tHcy values is substantially overestimated in most studies owing to the modest contribution played by cobalamins on tHcy
variance analyses. In contrast, there exists a growing body of converging data indicating that the role played by the protein component is largely underscored in vegan studies. It is worth recalling that S is the main intracellular anion coexisting with N within a constant mean S:N ratio (1:14.5) in animal tissues and dietary products of animal origin (Ingenbleek 2006). The mean S:N ratio found in plant items ranges from 1:20 to 1:35, a proportion that does not optimally meet human tissue requirements (Ingenbleek 2006), paving the way for borderline S and N deficiencies.

A recent Taiwanese investigation on hyperhomocysteinemic nuns consuming traditional vegetarian regimens consisting of mainly rice, soy products,
vegetables and fruits with few or no dairy items illustrates such clinical misinterpretation (Hung et al. 2002). The authors reported that folates and cobalamins, taken together, accounted for only 28.6% of tHcy variance in the vegetarian cohort whereas pyridoxine was inoperative (Hung et al. 2002). The daily vegetable N and Met intakes were situated highly significantly (p < 0.001) below the recommended allowances for humans (FAO/WHO/United Nations University 1985), causing a stage of unrecognized PEM documented by significantly depressed BCAA plasma
concentrations. Met levels escaped the overall decline in IAAs levels, emphasizing that efficient homeostatic mechanisms operate at the expense of an acquired hyperhomocysteinemic state. The diagnosis of subclinical PEM was missed because the authors ignored the exquisitely sensitive TTR detecting power. A proper PEM identification would have allowed the authors to confirm the previously described TTR–tHcy relationship that was established in Western Africa from comparable field studies involving country dwellers living on plant products.

The concept that acute or chronic stressful conditions may exert similar inhibitory effects on CbS activity and thereby promote hyperhomocysteinemic states is founded on previous studies showing that hypercatabolic states are characterized by increased urinary N and S losses maintaining tightly correlated depletion rates (Cuthbertson 1931; Ingenbleek and Young 2004; Sherman and Hawk 1900) which reflect the S:N ratio found in tissues undergoing cytokine induced proteolysis. This has been documented in coronary infarction and in acute pancreatitis where tHcy elevation evolves too rapidly to allow for a nutritional vitamin B-deficit explanation.  tHcy is considered stable in plasma and the two investigations report unaltered folate and cobalamin plasma concentrations.

The clinical usefulness of TTR as a nutritional biomarker, described in the early seventies (Ingenbleek et al. 1972) has been substantially disregarded by the scientific community for nearly four decades. This long-lasting reluctance expressed by many investigators is largely due to the fact that protein malnutrition and stressful disorders of various causes have combined inhibitory effects on hepatic TTR synthesis. Declining TTR plasma concentrations may result from either dietary protein and energy restrictions or from cytokine-induced transcriptional blockade (Murakami et al. 1988) of its hepatic synthesis. The proposed marker was therefore seen as having high sensitivity but poor specificity. Recent advances in protein metabolism settle the controversy by throwing further light on the relationships between TTR and the N-components of body composition.

The developmental patterns of LBM and TTR exhibit striking similarities. Both parameters rise from birth to puberty, manifest gender dimorphism during full sexual maturity then decrease during ageing. Uncomplicated PEM primarily affects both visceral and structural pools of LBM with distinct kinetics, reducing protein synthesis to levels compatible with prolonged survival. In acute or chronic stressful disorders, LBM undergoes muscle proteolysis exceeding the upregulation of protein syntheses in liver and injured areas, yielding a net body negative N balance. These adaptive responses are well identified by the measurement of TTR plasma concentrations which therefore appear as a plasma marker for LBM fluctuations.
Attenuation of stress and/or introduction of nutritional rehabilitation restores both LBM and TTR to normal values following parallel slopes. TTR fulfills, therefore, a unique position in assessing actual protein nutritional status, monitoring the efficacy of dietetic support and predicting the patient’s outcome (Bernstein and Pleban 1996).

see also…

Acosta PB, Yannicelli S, Ryan AS, Arnold G, Marriage BJ, Plewinska M, Bernstein L, Fox J, Lewis V, Miller M, Velazquez A (2005) Nutritional therapy improves growth and protein status of children with a urea cycle enzyme defect. Mol Genet Metab 86:448–455.

Arroyave G, Wilson D, Be´har M, Scrimshaw NS (1961) Serum and urinary creatinine in children with severe protein malnutrition. Am J Clin Nutr 9:176–179.

Bates CJ, Mansoor MA, van der Pols J, Prentice A, Cole TJ, Finch S (1997) Plasma total homocysteine in a representative sample of 972 British men and women aged 65 and over. Eur J Clin Nutr 51:691–697.

Battezzatti A, Bertoli S, San Romerio A, Testolin G (2007) Body composition: An important determinant of homocysteine and methionine concentrations in healthy individuals. Nutr Metab Cardiovasc Dis 17:525–534.

Bernstein LH, Bachman TE, Meguid M, Ament M, Baumgartner T, Kinosian B, Martindale R, Spiekerman M (1995) Prealbumin in nutritional care Consensus Group. Measurement of visceral protein status in assessing protein and energy malnutrition: Standard of care. Nutrition 11:169–171

Bernstein LH, Ingenbleek Y (2002) Transthyretin: Its response to malnutrition and stress injury. Clinical usefulness and economical implications. Clin Chem Lab Med 40:1344–1348.

Boorsook H, Dubnoff JW (1947) The hydrolysis of phosphocreatine and the origin of creatinine. J Biol Chem 168:493–510.

Briend A, Garenne M, Maire B, Fontaine O, Dieng F (1989) Nutritional status, age and survival: The muscle mass hypothesis. Eur J Clin Nutr 43:715–726

Brouillette J, Quirion R (2007) Transthyretin: A key gene involved in the maintenance of memory capacities during aging. Neurobiol Aging 29:1721–1732

Chertow GM, Goldstein-Fuchs DJ, Lazarus JM, Kaysen GA (2005) Prealbumin, mortality, and cause-specific hospitalization in hemodialysis patients. Kidney Int 68:2794–2800

Cohn SH, Gartenhaus W, Sawitsky A, Rai K, Zanzi I, Vaswani A, Ellis KJ, Yasumura S, Cortes E, Vartsky D (1981) Compartmental body composition of cancer patients by measurement of total body nitrogen, potassium and water. Metabolism 30:222–229

Cuthbertson DP (1931) The distribution of nitrogen and sulphur in the urine during conditions of increased catabolism. Biochem J 25:236–244

Devakonda A, George L, Raoof S, Esan A, Saleh A, Bernstein LH (2008) Transthyretin as a marker to predict outcome in critically ill patients. Clin Biochem 41:1126–1130

Ellis KJ, Yasumura S, Vartsky D, Vaswani AN, Cohn SH (1982) Total body nitrogen in health and disease: Effects of age, weight, height, and sex. J Lab Clin Med 99:917–926

Etchamendy N, Enderlin V, Marighetto A, Vouimba RM, Pallet V, Jaffard R, Higueret P (2001) Alleviation of a selective age-related relational memory deficit in mice by pharmacologically induced normalization of brain retinoid signaling. J Neurosci 21:6423–6429

Evans WJ (1991) Reversing sarcopenia: How weight training can build strength and vitality. Geriatrics 51:46–53

Evans WJ, Campbell WW (1993) Sarcopenia and age-related changes in body composition and functional capacity. J Nutr 123:465–468

Finkelstein JD, Martin JJ (1984) Methionine metabolism in mammals. Distribution of methionine between competing pathways. J Biol Chem 259:9508–9513

Garg UC, Zheng ZJ, Folsom AR, Moyer YS, Tsai MY, McGovern P, Eckfeldt JH (1997) Short-term and long-term variability of plasma homocysteine measurement. Clin Chem 43:141–145

Goodman AB, Pardee AB (2003) Evidence for defective retinoid transport and function in late onset Alzheimer’s disease. Proc Natl Acad Sci USA 100:2901–2905

Gray GE, Landel AM, Meguid MM (1994) Taurine-supplemented total parenteral nutrition and taurine status of malnourished cancer patients. Nutrition 10:11–15

Heymsfield SB, McManus C, Stevens V, Smith J (1982) Muscle mass: Reliable indicator of protein-energy malnutrition and outcome. Am J Clin Nutr 35:1192–1199

Ingenbleek Y (2006) The nutritional relationship linking sulfur to nitrogen in living organisms. J Nutr 136:S1641–S1651
Ingenbleek Y (2008) Plasma transthyretin indicates the direction of both nitrogen balance and retinoid status in health and disease. Open Clin Chem J 1:1–12
Ingenbleek Y, Bernstein LH (1999a) The stressful condition as a nutritionally dependent adaptive dichotomy. Nutrition 15:305–320
Ingenbleek Y, Bernstein LH (1999b) The nutritionally dependent adaptive dichotomy (NDAD) and stress hypermetabolism. J Clin Ligand Assay 22:259–267
Ingenbleek Y, Carpentier YA (1985) A prognostic inflammatory and nutritional index scoring critically ill patients. Internat J Vitam Nutr Res 55:91–101

Ingenbleek Y, Young VR (1994) Transthyretin (prealbumin) in health and disease: Nutritional implications. Annu Rev Nutr 14:495–533
Ingenbleek Y, Young VR (2002) Significance of transthyretin in protein metabolism. Clin Chem Lab Med 40:1281–1291
Ingenbleek Y, Young VR (2004) The essentiality of sulfur is closely related to nitrogen metabolism. Nutr Res Rev 17:135–151

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Mitochondrial Damage and Repair under Oxidative Stress

Curator: Larry H Bernstein, MD, FCAP


Keywords: Mitochondria, mitochondrial dysfunction, electron transport chain, mtDNA, oxidative stress, oxidation-reduction, NO, DNA repair, lipid peroxidation, thiols, ROS, RNS, sulfur,base excision repair, ferredoxin.
Summary: The mitochondrion is the energy source for aerobic activity of the cell, but it also has regulatory functions that will be discussed. The mitochondrion has been discussed in other posts at this site. It has origins from organisms that emerged from an anaerobic environment, such as the bogs and marshes, and may be related to the chloroplast. The aerobic cell was an advance in evolutionary development, but despite the energetic advantage of using oxygen, the associated toxicity of oxygen abundance required adaptive changes. Most bacteria that reduce nitrate (producing nitrite, nitrous oxide or nitrogen) are called facultative anaerobes use electron acceptors such as ferric ions, sulfate or carbon dioxide which become reduced to ferrous ions, hydrogen sulfide and methane, respectively, during the oxidation of NADH (reduced nicotinamide adenine dinucleotide is a major electron carrier in the oxidation of fuel molecules).

The underlying problem we are left with is oxidation-reduction reactions that are necessary for catabolic and synthetic reactions, and that cumulatively damage the organism associated with cancer, cardiovascular disease, neurodegerative disease, and inflammatory overload. Aerobic organisms tolerate have evolved mechanisms to repair or remove damaged molecules or to prevent or deactivate the formationof toxic species that lead to oxidative stress and disease. However, the normal balance between production of pro-oxidant species and destruction by the antioxidant defenses is upset in favor of overproduction of the toxic species, which leads to oxidative stress and disease. How this all comes together is the topic of choice.

Schematic diagram of the mitochondrial .

The transformation of energy is central to mitochondrial function. The system of energetics includes:

  • the enzymes of the Kreb’s citric acid or TCA cycle,
  • some of the enzymes involved in fatty acid catabolism (β-oxidation), and
  • the proteins needed to help regulate these systems,

central to mitochondrial physiology through the production of reducing equivalents. Reducing equivalents are also used for anabolic reactions.
Electron Transport Chain
It also houses the protein complexes involved in the electron transport component of oxidative phosphorylation and proteins involved in substrate and ion transport. The chemical energy contained in both fats and amino acids can also be converted into NADH and FADH2 through mitochondrial pathways. The major mechanism for harvesting energy from fats is β-oxidation; the major mechanism for harvesting energy from amino acids and pyruvate is the TCA cycle. Once the chemical energy has been transformed into NADH and FADH2, these compounds are fed into the mitochondrial respiratory chain.
Under physiological conditions, electrons generally enter either through complex I (NADH-mediated, examined in vitro using substrates such as glutamate/malate) or complex II (FADH2-mediated, examined in vitro using succinate).

Electrons are then sequentially passed through a series of electron carriers.

The progressive transfer of electrons (and resultant proton pumping) converts the chemical energy stored in carbohydrates, lipids, and amino acids into potential energy in the form of the proton gradient. The potential energy stored in this gradient is used to phosphorylate ADP forming ATP.

In redox cycling the reductant is continuously regenerated, thereby providing substrate for the “auto-oxidation” reaction.

When partially oxidized compounds are enzymatically reduced, the auto-oxidative generation of superoxide and other ROS to start again. Several enzymes

  •  NADPH-cytochrome P450 reductase,
  • NADPH-cytochrome b5 reductase [EC]
  • NADPH-ubiquinone oxidoreductase [EC], and
  • xanthine oxidase [EC]),

can reduce quinones into semiquinones in a single electron process.

The semiquinone can then reduce dioxygen to superoxide during its oxidation to a quinone.

Redox cycling is thought to play a role in carcinogenesis. The naturally occurring estrogen metabolites (the catecholestrogens) have been implicated in hormone-induced cancer, possibly as a result of their redox cycling and production of ROS. It is thought that diethylstilbestrol causes the production of the mutagenic lesion 8-hydroxy-2’deoxyguanosine. It can also cause DNA strand breakage.

Another oxidative reaction that is associated with H2O2 is a significant problem for living organisms as a consequence of the reaction between hydrogen peroxide and oxidizable metals, the Fenton reaction [originally described in the oxidation of an α-hydroxy acid to an α-keto acid in the presence of hydrogen peroxide (or hypochlorite) and low levels of iron salts (Fenton (1876, 1894)).
Chemical Reactions and Biological Significance

The hydroxyl free radical is so aggressive that it will react within 5 (or so) molecular diameters from its site of production. The damage caused by it, therefore, is very site specific. Biological defenses have evolved that reduce the chance that the hydroxyl free radical will be produced to repair damage. An antioxidant would have to occur at the site of hydroxyl free radical production and be at sufficient concentration to be effective.

Some endogenous markers have been proposed as a useful measures of total “oxidative stress” e.g., 8-hydroxy-2’deoxyguanosine in urine. The ideal scavenger

  • must be non-toxic,
  • have limited or no biological activity,
  • readily reach the site of hydroxyl free radical production,
  • react rapidly with the free radical, be specific for this radical, and
  • neither the scavenger nor its product(s) should undergo further metabolism.

Unlike oxygen, nitrogen does not possess unpaired electrons and is therefore considered diamagnetic. Nitrogen does not possess available d orbitals so it is limited to a valency of 3. In the presence of oxygen, nitrogen can produce Nitric oxide which occurs physiologically with the immune system which, when activated, can produce large quantities of nitric oxide.

Nitric oxide is produced by stepwise oxidation of L-arginine catalyzed by nitric oxide synthase (NOS). Nitric oxide is formed from the guanidino nitrogen of the L-arginine in a reaction that

  • consumes five electrons and
  • requires flavin adenine dinucleotide (FAD),
  • flavin mononucleotide (FMN) tetrahydrobiopterin (BH4), and
  • iron protoporphyrin IX as cofactors.

The primary product of NOS activity may be the nitroxyl anion that is then converted to nitric oxide by electron acceptors.

NOS cDNAs show homology with the cytochrome P450 reductase family. Based on molecular genetics there appears to be at least three distinct forms of NOS:

  • A Ca2+/calmodulin-requiring constitutive enzyme (c-NOS; ncNOS or type I)
  • A calcium-independent inducible enzyme (i-NOS; type II), which is primarily involved in the mediation of the cellular immune response; and
  • A second Ca2+/calmodulin-requiring constitutive enzyme found in aortic and umbilical endothelia (ec-NOS or type III)

This has been discussed extensively in this series of posts. Recently, a mitochondrial form of the enzyme, which appears to be similar to the endothelial form, has been found in brain and liver tissue. Although the exact role of nitric oxide in the mitochondrion remains elusive, it may play a role in the regulation of cytochrome oxidase.
Nitric Oxide
Nitric oxide appears to regulate its own production through a negative feedback loop. The binding of nitric oxide to the heme prosthetic group of NOS inhibits this enzyme, and c-NOS and ec-NOS are much more sensitive to this regulation than i-NOS. It appears that in the brain, NO can regulate its own synthesis and therefore the neurotransmission process.

  • On the one hand, inhibition of ec-NOS will prevent the cytotoxicity associated with excessive nitric oxide production.
  • On the other, the insensitivity of i-NOS to nitric oxide will enable high levels of nitric oxide to be produced for cytotoxic effects.

Endogenous inhibitors of NOS (guanidino-substituted derivatives of arginine) occur in vivo as a result of post-translational modification of protein contained arginine residues by S-adenosylmethionine. The dimethylarginines (NG,NG-dimethyl-L-arginine and NG,N’G-dimethyl-L-arginine) occurs in tissue proteins, plasma, and urine of humans and they are thought to act as both regulators of NOS activity and reservoirs of arginine for the synthesis of nitric oxide.
It has been calculated that even though membrane makes up about 3% of the total tissue volume, 90% of the reaction of nitric oxide with oxygen occurs within this compartment. Thus the membrane is an important site for nitric oxide chemistry.
There are two major aspects to nitric oxide chemistry.

  • It can undergo single electron oxidation and reduction reactions producing nitrosonium and nitroxyl
  • Having a single unpaired electron in its π*2p molecular orbital it will react readily with other molecules that also have unpaired electrons, such as free radicals and transition metals.

Examples of the reaction of nitric oxide with radical species include:

  • Nitric oxide will react with oxygen to form the peroxynitrite (nitrosyldioxyl) radical (ONO2)
  • and with superoxide to form the powerful oxidizing and nitrating agent, peroxynitrite anion (ONO2-). Peroxynitrite causes damage to many important biomolecules


  • nitrosothiols that are important in the regulation of blood pressure terminates lipid peroxidation
  • 3-nitrosotyrosine and/or 4-O-nitrosotyrosine can affect the activity of enzymes that utilize tyrosyl radicals
  • rapidly reacts with oxyhemoglobin, the primary route of its destruction in vivo
  • the reaction between nitric oxide and transition metal complexes

During the last reaction a “ligand” bond is formed (the unpaired electron of nitric oxide is partially transferred to the metal cation),

 resulting in a nitrosated (nitrosylated) complex.

For example, such complexes can be formed with free iron ions,

iron bound to heme or iron located in iron-sulfur clusters.

Ligand formation allows nitric oxide to act as a signal, activating some enzymes while inhibiting others. Thus, the binding of nitric oxide to the Fe (II)-heme of guanylate (guanalyl) cyclase [GTP-pyrophosphate lyase: cyclizing] is the signal transduction mechanism. Guanylate cyclase exists as cytosolic and membrane-bound isozymes.
Thiol-Didulfide Redox Couple

The thiol-disulfide redox couple is very important to oxidative metabolism. For example, GSH is a reducing cofactor for glutathione peroxidase, an antioxidant enzyme responsible for the destruction of hydrogen peroxide.

The importance of the antioxidant role of the thiol-disulfide redox couple:

Thiols and disulfides can readily undergo exchange reactions, forming mixed disulfides. Thiol-disulfide exchange is biologically very important. For example,

  • GSH can react with protein cystine groups and influence the correct folding of proteins.
  • GSH may also play a direct role in cellular signaling through thiol-disulfide exchange reactions with membrane bound receptor proteins
  •                        the insulin receptor complex)
  •                        transcription factors (e.g., nuclear factor κB)
  •                        and regulatory proteins in cells

Conditions that alter the redox status of the cell can have important consequences on cellular function.

The generation of ROS by redox cycling is only one possible explanation for the action of many drugs. Rifamycin not only owes its activity to ROS generation but also to its ability to block bacterial RNA synthesis as well. Quinones (and/or semiquinones) can also form adducts with nucleophiles, especially thiols. These adducts may act as toxins directly or indirectly through the inhibition of key enzymes (e.g., by reacting with essential cysteinyl residues) or the depletion of GSH.
DNA Adduct Formation

By far the most intense research in this field has been directed towards the chemistry and biology of DNA adduct formation. Attack of the free bases and nucleosides by pro-oxidants can yield a wide variety of adducts and DNA-protein cross-links. Such attack usually occurs

  • at the C-4 and C-8 position of purines and
  • C-5 and C-6 of pyrimidines.

Hydroxyl free radical-induced damage to purine bases and nucleosides can proceed through a C-8-hydroxy N-7 radical intermediate, and then either undergo oxidation with the production of an 8-hydroxy purine, or reduction, probably by cellular thiols, followed by ring opening and the formation of FAPy (formamido-pyrimidine) metabolites (hydroxyl free radical-induced damage to guanosine). Although most research has focused on 8-hydroxy-purine adducts a growing number of publications are attempting to measure the FAPy derivative.

Nitrosation of the Amines of the Nucleic Acid Bases.

Primary aromatic amines produce deaminated products, while secondary amines form N-nitroso compounds.
Formation of Peroxynitrite from Nitric Oxide.

Peroxynitrite shows complex reactivity

  • with DNA initiating DNA strand breakage, oxidation (e.g., formation of 8-hydroxyguanine, 8-OH2’dG, (5-hydroxymethyl)-uracil, and FAPyGua),
  • nitration (e.g., 8-nitroguanine), and
  • deamination of bases.

Peroxynitrite can also promote the production of lipid peroxidation related active carbonyls and cause the activation of NAD+ ADP-ribosyltransferase.

Modification of Guanine
Although all DNA bases can be oxidatively damaged, it is the modification of guanine that is the most frequent. 8OH2’dG is the most abundant DNA adduct. This can affect its hydrogen bonding between base-pairs. These base-pair substitutions are usually found clustered into areas called “hot spots”. Guanine normally binds to cytosine.

8OH2’dG, however, can form hydrogen bonds with adenine. The formation of 8OH2’dG in DNA can therefore result in a G→T transversion.

8-Hydroxyguanine was also shown to induce codon 12 activation of c-Ha-ras and K-ras in mammalian systems. G→T transversions are also the most frequent hot spot mutations found in the p53 supressor gene which is associated with human tumors.

Other mechanisms by which ROS/RNS can lead to mutations have been
proposed. Direct mechanisms include:

  • conformational changes in the DNA template that reduces the accuracy of replication by DNA polymerases
  • altered methylation of cytosine that affects gene control

Indirect mechanisms include:

  • Oxidative damage to proteins, including DNA polymerases and repair enzymes.
  • Damage to lipids causes the production of mutagenic carbonyl compounds
  • Misalignment mutagenesis (“slippery DNA”)
DNA Mismatch Repair 5

DNA Mismatch Repair 5 (Photo credit: Allen Gathman)

Repair of ROS/RNS-induced DNA Damage
The repair of damaged DNA is an ongoing and continuous process involving a
number of repair enzymes. Damaged DNA appears to be mended by two major mechanisms:

  1. base excision repair (BER) and
  2. nucleotide excision repair (NER)

Isolated DNA is found to contain low levels of damaged bases, so it appears that these repair processes are not completely effective.
Base Excision Repair

BER is first started by DNA glycosylases which recognize specific base
modifications (e.g., 8OH2’dG). For example,

  • Formamido-pyrimidine-DNA glycosylase (Fpg protein) recognizes damaged purines such as 8-oxoguanine and FAPyGua.
  • Damaged pyrimidines are recognized by endonuclease III, which acts as both a glycosylase and AP endonuclease.
  • Glycosylases cleave the N-glycosylic bond between the damaged base and the sugar

Following the glycosylase step, AP endonucleases then remove the 3′-deoxyribose moiety by cleavage of the phosphodiester bonds thereby generating a 3’-hydroxyl group that can then be extended by DNA polymerase.

The final step in mending damaged DNA is the rejoining of the free ends of DNA by a DNA ligase. It also appears that the presence of 8-oxoguanine modified bases in DNA is not only a result of ROS attack on this macromolecule. Oxidized nucleosides and nucleotides from free cellular pools can also be incorporated into DNA by polymerases and cause AT to CG base substitution mutations.

Mitochondrial DNA Repair

The mitochondrion genome encodes the various complexes of the electron transport chain, but contains no genetic information for DNA repair enzymes. These enzymes must be obtained from the nucleus. As mitochondria are continuously producing DNA damaging pro-oxidant species, effective DNA repair mechanisms must exist within the mitochondrial matrix in order for these organelles to function. Mitochondria have a short existence, and excessively damaged mitochondria will be quickly removed. Mitochondria contain many BER enzymes and are proficient at repair, but they do not appear to repair damaged DNA by NER mechanisms.

Single Strand DNA Damage and PARP Activation

Single strand DNA breakage activates NAD+ ADP-ribosyltransferase (PARP). PARP is a protein-modifying, nucleotide-polymerizing enzyme and is found at high levels in the nucleus. Activated PARP

  1. cleaves NAD+ into ADP-ribose and nicotinamide
  2. then attaches the ADP-ribose units to a variety of nuclear proteins (including histones and its own automodification domain).
  3. then polymerizes the initial ADP-ribose modification with other ADP-ribose units to form the nucleic acid-like polymer, poly (ADP) ribose.

PARP only appears to be involved with BER and not NER. In BER PARP does not appear to play a direct role but rather it probably helps by keeping the chromatin in a conformation that enables other repair enzymes to be effective. It may also provide temporary protection to DNA molecules while it is being repaired. Conflicting evidence suggests that PARP may not be an important DNA repair enzyme as cells from a PARP knockout mouse model have normal repair characteristics.

Activation of PARP can be dangerous to the cell. For each mole of ADP-ribose transferred, one mole of NAD+ is consumed, and through the regeneration of NAD+ four ATP molecules are wasted. Thus the activation of PARP can rapidly deplete a cell’s energy store and even lead to cell death. Some researchers suggest that this may be one mechanism whereby cells with excessive DNA damage are effectively removed. However, a variety of diseases may involve PARP overactivation including

  • circulatory shock,
  • CNS injury,
  • diabetes,
  • drug-induced cytotoxicity, and
  • inflammation.

The Indirect Pathway.
This (mutation) pathway does not involve oxidative damage to the protein per se. This process involves oxidative damage to the DNA molecule encoding the protein. Thus pro-oxidants can cause changes in the base sequence of the DNA molecule. If such base modification is in a coding region of DNA (exon) and not corrected, the DNA molecule may be transcribed incorrectly. Translation of the mutant mRNA can result in a mutant protein containing a wrong amino acid in its primary sequence. If this modified amino acid occurs in an essential part of the protein (e.g., the active site of an enzyme or a portion that alters folding), the function of that protein may be impaired. Fortunately, unlike modified DNA
that can pass from cell to cell during mitosis thereby continuing the production of mutant protein, damage to a protein is non-replicating and stops with its destruction.

The Direct Pathway

This (post-translational) pathway involves the action of a pro-oxidant on a protein resulting in

  • modification of amino acid residues,
  • the formation of carbonyl adducts,
  • cross-linking and
  • polypeptide chain fragmentation.

Such changes often result in altered protein conformation and/or activity. Proteins will produce a variety of carbonyl products when exposed to metal-based systems (metal/ascorbate and metal/hydrogen peroxide) in vitro. For example, histidine yields aspartate, asparagine and 2-oxoimidazoline, while proline produces glutamate, pyroglutamate, 4-hydroxyproline isomers, 2-pyrrolidone and γ-aminobutyric acid. Metal-based systems and other pro-oxidant conditions can oxidize methionine to its sulfoxide.

This portion of the presentation is endebted to THE HANDBOOK OF REDOX
BIOCHEMISTRY, Ian N. Acworth, August 2003, esa. (
We shall now identify more recent work related to this presentation.

Oxygen and Oxidative Stress

The reduction of oxygen to water proceeds via one electron at a time. In the mitochondrial respiratory chain, Complex IV (cytochrome oxidase) retains all partially reduced intermediates until full reduction is achieved. Other redox centres in the electron transport chain, however, may leak electrons to oxygen, partially reducing this molecule to superoxide anion (O2_•). Even though O2_• is not a strong oxidant, it is a precursor of most other reactive oxygen species, and it also becomes involved in the propagation of oxidative chain reactions. Despite the presence of various antioxidant defences, the mitochondrion appears to be the main intracellular source of these oxidants. This review describes the main mitochondrial sources of reactive species and the antioxidant defences that evolved to prevent oxidative damage in all the mitochondrial compartments.

Reactive oxygen species (ROS) is a phrase used to describe a variety of molecules and free radicals (chemical species with one unpaired electron) derived from molecular oxygen. Molecular oxygen in the ground state is a bi-radical, containing two unpaired electrons in the outer shell (also known as a triplet state).

Since the two single electrons have the same spin, oxygen can only react with one electron at a time and therefore it is not very reactive with the two electrons in a chemical bond.

On the other hand, if one of the two unpaired electrons is excited and changes its spin, the resulting species (known as singlet oxygen) becomes a powerful oxidant as the two electrons with opposing spins can quickly react with other pairs of electrons, especially double bonds.

The formation of OH• is catalysed by reduced transition metals, which in turn may be re-reduced by O2 -•, propagating this process. In addition, O2-• may react with other radicals including nitric oxide (NO•) in a reaction controlled by the rate of diffusion of both radicals. The product, peroxynitrite, is also a very powerful oxidant. The oxidants derived from NO• have been recently called reactive nitrogen species (RNS).

‘Oxidative stress’ is an expression used to describe various deleterious processes resulting from an imbalance between the excessive formation of ROS and/or RNS and limited antioxidant defences.

  • Whilst small fluctuations in the steady-state concentration of these oxidants may actually play a role in intracellular signalling,
  • uncontrolled increases in the steady-state concentrations of these oxidants lead to free radical mediated chain reactions

which indiscriminately target

  • proteins,
  • lipids,
  • polysaccharides.

In vivo, O2-• is produced both enzymatically and nonenzymatically.

Enzymatic sources include

  • NADPH oxidases located on the cell membrane of
  • polymorphonuclear cells,
  • macrophages and
  • endothelial cells and
  • cytochrome P450-dependent oxygenases.

The proteolytic conversion of xanthine dehydrogenase to xanthine oxidase provides another enzymatic source of both O2 -• and H2O2 (and therefore constitutes a source of OH•) and has been proposed to mediate deleterious processes in vivo.

Given the highly reducing intramitochondrial environment, various respiratory components, including flavoproteins, iron–sulfur clusters and ubisemiquinone, are thermodynamically capable of transferring one electron to oxygen. Moreover, most steps in the respiratory chain involve single-electron reactions, further favouring the monovalent reduction of oxygen. On the other hand, the mitochondrion possesses various antioxidant defences designed to eliminate both O2- • and H2O2.

The rate of O2 -• formation by the respiratory chain is controlled primarily by mass action, increasing both when electron flow slows down (increasing the concentration of electron donors, R•) and when the concentration of oxygen increases (eqn (1); Turrens et al. 1982).

d[O2]/dt = k [O2] [R•].

The energy released as electrons flow through the respiratory chain is converted into a H+ gradient through the inner mitochondrial membrane (Mitchell, 1977). This gradient, in turn, dissipates through the ATP synthase complex (Complex V) and is responsible for the turning of a rotor-like protein complex required for ATP synthesis. In the absence of ADP,

  • the movement of H+ through ATP synthase ceases and
  • the H+ gradient builds up
  • causing electron flow to slow down and
  • the respiratory chain to become more reduced (State IV respiration).

Mitochondrial Antioxidant Defences

The deleterious effects resulting from the formation of ROS in the mitochondrion are, to a large extent, prevented by various antioxidant systems. Superoxide is enzymatically converted to H2O2 by a family of metalloenzymes called superoxide dismutases (SOD). Since O2-• may either reduce transition metals, which in turn can react with H2O2 producing OH• or spontaneously react with NO• to produce peroxynitrite, it is important to maintain the steady-state concentration of O2-• at the lowest possible level. Thus, although the dismutation of O2-• to H2O2 and O2 can also occur spontaneously, the role of SODs is to increase the rate of the reaction to that of a diffusion-controlled process.

The mitochondrial matrix contains a specific form of SOD, with manganese in the active site, which eliminates the O2 -• formed in the matrix or on the inner side of the inner membrane. The expression of MnSOD is further induced by agents that cause oxidative stress, including radiation and hyperoxia, in a process mediated by the oxidative activation of the nuclear transcription factor NFkB .

Turrens JF. Mitochondrial formation of reactive oxygen species. J Physiol 2003; 552(2): 335–344. DOI: 10.1113/jphysiol.2003.049478.

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Reactive Oxygen Species and Control of Apoptosis

Reactive oxygen species (ROS) are products of normal metabolism and xenobiotic exposure, and depending on their concentration, ROS can be beneficial or harmful to cells and tissues.

  • At physiological low levels, ROS function as “redox messengers” in intracellular signaling and regulation, whereas
  • excess ROS induce oxidative modification of cellular macromolecules, inhibit protein function, and promote cell death.

Additionally, various redox systems, such as

  • the glutathione,
  • thioredoxin, and
  • pyridine nucleotide redox couples,
  • NADPH and antioxidant defense
  • NAD+ and the function of sirtuin proteins

participate in cell signaling and modulation of cell function, including apoptotic cell death. Cell apoptosis is initiated by extracellular and intracellular signals via two main pathways,

  1. the death receptor and
  2. the mitochondria-mediated pathways.

ROS and JNK-mediated apoptotic signaling

              GSH redox status and apoptotic signaling

Various pathologies can result from oxidative stress-induced apoptotic signaling that is consequent to

  • ROS increases and/or antioxidant decreases,
  • disruption of intracellular redox homeostasis, and
  • irreversible oxidative modifications of lipid, protein, or DNA.

We focus on several key aspects of ROS and redox mechanisms in apoptotic signaling and highlight the gaps in knowledge and potential avenues for further investigation. A full understanding of the redox control of apoptotic initiation and execution could underpin the development of therapeutic interventions targeted at oxidative stress-associated disorders.

Circu, M. L.; Aw, T. Y., Reactive oxygen species, cellular redox systems, and apoptosis, Free Radic. Biol. Med. 2010. FRB-10057; pp 14. doi:10.1016/j.freeradbiomed.2009.12.022

Assembly of Iron-sulfur (FeyS) Clusters

Iron-sulfur (FeyS) cluster-containing proteins catalyze a number of electron transfer and metabolic reactions. The components and molecular mechanisms involved in the assembly of the FeyS clusters have been identified only partially. In eukaryotes, mitochondria have been proposed to execute a crucial task in the generation of intramitochondrial and extramitochondrial FeyS proteins. Herein, we identify the essential ferredoxin Yah1p of Saccharomyces cerevisiae mitochondria as a central component of the FeyS protein biosynthesis machinery. Depletion of Yah1p by regulated gene expression resulted in a

30-fold accumulation of iron within mitochondria,

similar to what has been reported for other components involved in FeyS protein biogenesis. Yah1p was shown to be required for the assembly of FeyS proteins both inside mitochondria and in the cytosol. Apparently, at least one of the steps of FeyS cluster biogenesis within mitochondria requires reduction by ferredoxin. Our findings lend support to the idea of a primary function of mitochondria in the biosynthesis of FeyS proteins outside the organelle. To our knowledge, Yah1p is the first member of the ferredoxin family for which a function in FeyS cluster formation has been established. A similar role may be predicted for the bacterial homologs that are encoded within iron-sulfur cluster assembly (isc) operons of prokaryotes.
H Lange, A Kaut, G Kispal, and R Lill. A mitochondrial ferredoxin is essential for biogenesis of cellular iron-sulfur proteins. PNAS 2000; 97(3): 1050–1055.

DNA Charge Transport

Damaged bases in DNA are known to lead to errors in replication and transcription, compromising the integrity of the genome. The authors proposed a model where repair proteins containing redoxactive [4Fe-4S] clusters utilize DNA charge transport (CT) as a first step in finding lesions. In this model, the population of sites to search is reduced by a localization of protein in the vicinity of lesions. Here, we examine this model using single-molecule atomic force microscopy (AFM). XPD, a 5′-3′ helicase involved in nucleotide
excision repair, contains a [4Fe-4S] cluster and exhibits a DNA bound redox potential that is physiologically relevant.

In AFM studies, they observe the redistribution of XPD onto kilobase DNA strands containing a single base mismatch, which is not a specific substrate for XPD but, like a lesion, inhibits CT. They also provide evidence for DNA-mediated signaling between XPD and Endonuclease III (EndoIII), a base excision repair glycosylase that also contains a [4Fe-4S] cluster.

  • When XPD and EndoIII are mixed together, they coordinate in relocalizing onto the mismatched strand.
  • However, when a CT-deficient mutant of either repair protein is combined with the CT-proficient repair partner, no relocalization occurs.

The data presented here indicate that XPD, an archaeal protein from the NER pathway, may cooperate with other proteins that are proficient at DNA CT to localize in the vicinity of damage. XPD, a superfamily 2 DNA helicase with 5′-3′ polarity, is a component of TFIIH that is essential for repair of bulky lesions generated by exogenous sources such as UV light and chemical carcinogens. XPD contains a conserved [4Fe-4S] cluster suggested to be conformationally controlled by ATP binding and hydrolysis.

Mutations in the iron-sulfur domain of XPD can lead to diseases including TTD and XP, yet the function of the [4Fe-4S] cluster appears to be unknown.

Electrochemical studies have shown that when BER proteins MutY and EndoIII bind to DNA, their [4Fe-4S] clusters are activated toward one electron oxidation. XPD exhibits a DNA-bound midpoint potential similar to that of EndoIII and MutY when bound to DNA (approximately 80 mV vs. NHE), indicative of a possible role for the [4Fe-4S] cluster in DNA-mediated CT.

For EndoIII we have also already determined a direct correlation between the ability of proteins to redistribute in the vicinity of mismatches as measured by AFM, and the CT proficiency of the proteins measured electrochemically. Thus, we may utilize single-molecule AFM as a tool to probe the redistribution of proteins in the vicinity of base lesions and in so doing, the proficiency of the protein to carry out DNA CT.

Here we show that, like the BER protein EndoIII, XPD, involved both in transcription and NER, redistributes in the vicinity of a lesion. Importantly, this ability to relocalize is associated with the ability of XPD to carry out DNA CT. The mutant L325V is defective in its ability to carry out DNA CTand this XPD mutant also does not redistribute effectively onto the mismatched strand.

These data not only indicate a general link between the ability of a repair protein to carry out DNA CT and its ability to redistribute onto DNA strands near lesions but also provide evidence for coordinated DNA CT between different repair proteins in their search for damage in the genome. These data also provide evidence that two different repair proteins, each containing a [4Fe-4S] cluster at similar DNA bound potential, can communicate with one another through DNA-mediated CT.

Sontz PA, Mui TP, Fuss JO, Tainer JA, and Barton JK. DNA charge transport as a first step in coordinating the detection of lesions by repair proteins. PNAS 2012; 109(6):1856–1861. doi:10.1073/pnas.1120063109/-/ DCSupplemental.

Janus Bifron 

The signaling function of mitochondria is considered with a special emphasis on their role in the regulation of redox status of the cell, possibly determining a number of pathologies including cancer and aging. The review summarizes the transport role of mitochondria in energy supply to all cellular compartments (mitochondria as an electric cable in the cell), the role of mitochondria in plastic metabolism of the cell including synthesis of

  • heme,
  • steroids,
  • iron-sulfur clusters, and
  • reactive oxygen and nitrogen species.

Mitochondria also play an important role in the Ca2+-signaling and the regulation of apoptotic cell death. Knowledge of mechanisms responsible for apoptotic cell death is important for the strategy for prevention of unwanted degradation of postmitotic cells such as cardiomyocytes and neurons.

In accordance with P. Mitchell’s chemiosmotic concept, vectorial transmembrane transfer of electrons and protons is accompanied by generation of electrochemical difference of proton electrochemical potential on the inner mitochondrial membrane; its utilization by ATP synthase induces conformational rearrangements resulting in ATP synthesis from ADP and inorganic phosphate. Details of the mechanism responsible for ATP synthesis are given elsewhere.

Membrane potential (DY) generated across the inner mitochondrial membrane is the component of the transmembrane electrochemical potential of H+ ions (DμH+), which provides ATP synthesis together with the concentration component (DpH). Maintenance of constant membrane potential is a vitally important precondition for functioning of mitochondria and the cell. Under conditions of limited supply of the cell with oxygen (hypoxia) and inability to carry out aerobic ATP synthesis, mitochondria become ATP consumers (rather than generators) and ATP is hydrolyzed by mitochondrial ATPase, and this is accompanied by generation of membrane potential.

Redox homeostasis, i.e. the sum of redox components (including proteins, low molecular weight redox components such as NAD/NADH, flavins, coenzymes Q, oxidized and reduced substrates, etc.) is one of important preconditions for normal cell functioning.

Single-strand and double-strand DNA damage

Single-strand and double-strand DNA damage (Photo credit: Wikipedia)

Mitochondria generate such potent regulators of redox potential as

  • superoxide anion,
  • hydrogen peroxide,
  • nitric oxide,
  • peroxynitrite, etc.

They are actively involved in regulation of cell redox potential and consequently

  • control proteolysis,
  • activation of transcription,
  • changes in mitochondrial DNA (mDNA),
  • cell metabolism, and
  • cell differentiation.

Zorov DB, Isaev NK, Plotnikov EY, Zorova LD, et al. The Mitochondrion as Janus Bifrons. Biochemistry (Moscow) 2007; 72(10): 1115-1126. ISSN 0006-2979.
DOI: 10.1134/S0006297907100094

Structure of the human mitochondrial genome.

Structure of the human mitochondrial genome. (Photo credit: Wikipedia)

Gene Expression Associated with Oxidoreduction and Mitochondria
The naked mole-rat (Heterocephalus glaber) is a long-lived, cancer resistant rodent and there is a great interest in identifying the adaptations responsible for these and other of its unique traits. We employed RNA sequencing to compare liver gene expression profiles between naked mole-rats and wild-derived mice. Our results indicate that genes associated with oxidoreduction and mitochondria were expressed at higher relative levels in naked mole-rats. The largest effect is nearly

300-fold higher expression of epithelial cell adhesion molecule (Epcam), a tumour-associated protein.

Also of interest are the

  • protease inhibitor, alpha2-macroglobulin (A2m), and the
  • mitochondrial complex II subunit Sdhc,

both ageing-related genes found strongly over-expressed in the naked mole-rat.

These results hint at possible candidates for specifying species differences in ageing and cancer, and in particular suggest complex alterations in mitochondrial and oxidation reduction pathways in the naked mole-rat. Our differential gene expression analysis obviated the need for a reference naked mole-rat genome by employing a combination of Illumina/Solexa and 454 platforms for transcriptome sequencing and assembling transcriptome contigs of the non-sequenced species. Overall, our work provides new research foci and methods for studying the naked mole-rat’s fascinating characteristics.

C Yu, Y Li, A Holmes, K Szafranski, CG Faulkes, et al. RNA Sequencing Reveals Differential Expression of Mitochondrial and Oxidation reduction Genes in the Long-Lived Naked Mole-Rat When Compared to Mice. PLoS ONE 2011; 6(11): 1-9. e26729.

The complete set of viable deletion strains in Saccharomyces cerevisiae was screened for sensitivity of mutants to five oxidants to identify cell functions involved in resistance to oxidative stress. This screen identified a unique set of mainly constitutive functions providing the first line of defense against a particular oxidant; these functions are very dependent on the nature of the oxidant. Most of these functions are distinct from those involved in repair and recovery from damage, which are generally induced in response to stress, because there was little correlation between mutant sensitivity and
the reported transcriptional response to oxidants of the relevant gene. The screen identified 456 mutants sensitive to at least one of five different types of oxidant, and these were ranked in order of sensitivity. Many genes identified were not previously known to have a role in resistance to reactive oxygen species. These encode functions including

  • protein sorting,
  • ergosterol metabolism,
  • autophagy, and
  • vacuolar acidification.

two mutants were sensitive to all oxidants examined,
12 were sensitive to at least four,

Different oxidants had very different spectra of deletants that were sensitive. These findings highlight the specificity of cellular responses to different oxidants:

  • No single oxidant is representative of general oxidative stress.
  • Mitochondrial respiratory functions were overrepresented in mutants sensitive to H2O2, and
  • vacuolar protein-sorting mutants were enriched in mutants sensitive to diamide.

Core functions required for a broad range of oxidative-stress resistance include

  • transcription,
  • protein trafficking, and
  • vacuolar function.

GW Thorpe, CS Fong, N Alic, VJ Higgins, and IW Dawes. Cells have distinct mechanisms to maintain protection against different reactive oxygen species: Oxidative-stress-response genes. PNAS 2004;101: 6564–6569. cgi doi 10.1073 pnas.0305888101
Subcellular Thiol Redox State in Complex I Deficiency

Isolated complex I deficiency is the most common enzymatic defect of the oxidative phosphorylation (OXPHOS) system, causing a wide range of clinical phenotypes. Th authers reported before that the rates at which reactive oxygen species (ROS)-sensitive dyes are converted into their fluorescent oxidation products are markedly increased in cultured skin fibroblasts of patients with nuclear-inherited isolated complex I deficiency.

Using videoimaging microscopy we show here that these cells also display a marked increase in NAD(P)H autofluorescence. Linear regression analysis revealed a negative correlation with the residual complex I activity and a positive correlation with the oxidation rates of the ROS sensitive dyes (5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein and hydroethidine for a large cohort of 10 patient cell lines.

On the other hand, video-imaging microscopy of cells selectively expressing reduction-oxidation sensitive GFP1 in either the mitochondrial matrix or cytosol showed the absence of any detectable change in thiol redox state. In agreement with this result, neither the glutathione nor the glutathione disulfide content differed significantly between patient and healthy fibroblasts.

Finally, video-rate confocal microscopy of cells loaded with C11-BODIPY581/591 demonstrated that the extent of lipid peroxidation, which is regarded as a measure of oxidative damage, was not altered in patient fibroblasts. Our results indicate that fibroblasts of patients with isolated complex I deficiency maintain their thiol redox state despite marked increases in ROS production.

S Verkaart, WJH Koopman, J Cheek, SE van Emst-de Vries. Mitochondrial and cytosolic thiol redox state are not detectably altered in isolated human NADH:ubiquinone oxidoreductase deficiency. Biochimica et Biophysica Acta (BBA) – Molecular Basis of Disease 2007; 1772(9): 1041. DOI : 10.1016/j.bbadis.2007.05.004

  • Mitochodrial mtDNA and Cancer
  • Mitochondrial research has recently been driven by the

identification of mitochondria-associated diseases and 
the role of mitochondria in apoptosis.

Moreover, mitochondria have been implicated in the process of carcinogenesis because of their vital role in

  • energy production,
  • nuclear-cytoplasmic signal integration and
  • control of metabolic pathways.

At some point during neoplastic transformation, there is an increase in reactive oxygen species (ROS), which damage the mitochondrial genome. This accelerates the somatic mutation rate of mitochondrial DNA.

Mitochondrial characteristics

There are several biological characteristics which cast mitochondria and, in particular, the mitochondrial genome, as a biological tool for early detection and monitoring of neoplasia and its potential progression. These vital characteristics are important in cancer research, as not all neoplasias become malignant. Mitochondria are archived in the cytoplasm of the ovum and as such do not recombine.

This genome has an accelerated mutation rate, by comparison with the nucleus, and accrues somatic mutations in tumour tissue. Moreover, mitochondrial DNA (mtDNA) has a high copy number in comparison with the nuclear archive of DNA. There are potentially thousands of mitochondrial genomes per cell, which enables detection of important biomarkers, even at low levels. In addition, mtDNA can be heteroplasmic, which means that disease-associated mutations occur in a subset of the genomes.

The presence of heteroplasmy is an indication of disease and is found in many human tumours. Identification of low levels of heteroplasmy may allow unprecedented early identification and monitoring of neoplastic progression to malignancy.

Coding for just 13 enzyme complex subunits, 22 transfer RNAs and two ribosomal RNAs, the mitochondrial genome is packaged in a compact 16,569 base pair (bp) circular molecule. These products participate in the critical electron transport process of ATP production. Collectively, mitochondria generate 80 per cent of the chemical fuel which fires cellular metabolism.

As a result, nuclear investment in the mitochondria is high — that is, several thousand nuclear genes control this organelle in order to accomplish the complex interactions required to maintain a network of pathways, which coordinate energy demand and supply.

It has been proposed that these mutations may serve as an early indication of potential cancer development and may represent a means for tracking tumour progression.

Does this provide a potential utility in that these mutations may be used for the identification and monitoring of neoplasia and malignant transformation where appropriate body fluids or non-invasive tissue access is available for mtDNA recovery? Specifically discussed are:

  • prostate,
  • breast,
  • colorectal,
  • skin and
  • lung cancers

There are many important questions yet to be addressed: such as

  • the relationship between mtDNA and the actual disease;
  • are mutations causative or merely a reflection of nuclear instability?
  • And, are these processes independent events?

Alterations in the non-coding D-loop suggest genome instability;
however, as studies focus more on the coding regions of the
mitochondrial genome,

Particularly in the case of nonsynonymous mutations in the genes
contributing products to the electron transport process, metabolic
implications are evident. Moreover, mutations in mitochondrial
transfer RNAs indicate the possibility of a global mitochondrial
translational shut down.

RL Parr, GD Dakubo, RE Thayer, K McKenney, MA Birch-Machin. Mitochondrial DNA as a potential tool for early cancer detection. HUMAN GENOMICS 2006; 2(4). 252–257.
Mitochondrial DNA (mtDNA) is particularly prone to oxidation due to the lack of histones and a deficient mismatch repair system. This explains an increased mutation rate of mtDNA that results in heteroplasmy, e.g., the coexistence of the mutant and wild-type mtDNA molecules within the same mitochondrion. Hyperglycemia is a key risk factor not only for diabetes-related disease, but also for cardiovascular and all-cause mortality. One can assume an increase in the risk of cardiovascular disease by 18% for each unit (%) glycated hemoglobin HbA1c. In the Glucose Tolerance in Acute Myocardial Infarction study of patients with acute coronary syndrome, abnormal glucose tolerance was the strongest independent predictor of subsequent cardiovascular complications and death. In the Asian Pacific Study, fasting plasma glucose was shown to be an independent predictor of cardiovascular events up to a level of 5.2 mmol/L.

Glucose level fluctuations and hyperglycemia are triggers for inflammatory responses via increased mitochondrial superoxide production and endoplasmic reticulum stress. Inflammation leads to insulin resistance and β-cell dysfunction, which further aggravates hyperglycemia. The molecular pathways that integrate hyperglycemia, oxidative stress, and diabetic vascular complications have been most clearly described in the pathogenesis of endothelial dysfunction, which is considered as the first step in atherogenesis according to the response to injury hypothesis.

  • In diabetes mellitus,
  • glycotoxicity,
  • advanced oxidative stress,
  • collagen cross-linking, and
  • accumulation of lipid peroxides

in foam macrophage cells and arterial wall cells may significantly

  • decrease the mutation threshold,
  • endothelial dysfunction,
  • promoting atherosclerosis.

Alterations in mitochondrial DNA (mtDNA), known as homoplasmic and heteroplasmic mutations, may influence mitochondrial OXPHOS capacity, and in turn contribute to the magnitude of oxidative stress in micro- and macrovascular networks in diabetic patients.
The authors critically consider the impact of mtDNA mutations on the pathogenesis of cardiovascular diabetic complications.

Mutation Threshhold

Although cells may harbor mutant mtDNA, the expression of disease is dependent on the percent of alleles bearing mutations. Modeling confirms that an upper threshold level might exist for mutations beyond which the mitochondrial population collapses, with a subsequent decrease in ATP. This decrease in ATP results in the phenotypic expression of disease. It is estimated that in many patients with clinical manifestations of mitochondrial disorders, the proportion of mutant DNA exceeds 50%.

For the MELAS (mitochondrial encephalopathy, lactic acidosis and stroke-like syndrome)-causing mutation m.3243 A>G in the mitochondrial gene encoding tRNALeu, which is also associated with diabetes plus deafness, a strong correlation between the level of mutational heteroplasmy and documented disease has been found. Increased percentages of mutant mtDNA in muscle cells (up to 71%) can lead to mitochondrial myopathy. Levels of heteroplasmy of over 80% may lead to recurrent stroke and mutation levels of 95% have been associated with MELAS.

Regardless of the type of mutation or the level of heteroplasmy in affected mitochondria, unrepaired damage leads to a decrease in ATP, which in turn causes the phenotypic manifestation of disease. The manifestation of disease not only depends on the ATP level but also on the tissue affected. Various tissues have differing levels of demand on OXPHOS capacity. To evaluate a tissue threshold, Leber’s hereditary optic neuropathy can be used as a model for mitochondrial neurodegenerative disease. For neural and skeletal muscle tissues, the tissue threshold should be as high as or higher than 90% of
damaged (mutated) mtDNA. To induce mitochondrial malfunctions, the tissue threshold of the cardiac muscle is estimated to be significantly lower (approximately 64%-67%). In chronic vascular disease such as atherosclerosis, a mutation threshold in the affected vessel wall (e.g., in the postmortem aortic atherosclerotic plaques) was observed to be significantly lower. For example, for mutations m.3256 C>T, m.12315 G>A, m.15059 G>A, and m.15315 G>A, the heteroplasmy range of 18%-66% in the atherosclerotic lesions was 2-3.5-fold that in normal vascular tissue.

Mitochondrial stress and insulin resistance

  • Mitochondrial damage precedes the development of atherosclerosis and tracks the extent of the lesion in apoE-null mice, and
  • mitochondrial dysfunction caused by heterozygous deficiency of a superoxide dismutase increases atherosclerosis and vascular mitochondrial damage in the same model.

Blood vessels destined to develop atherosclerosis may be characterized by inefficient ATP production due to the uncoupling of respiration and OXPHOS. Blood vessels have regions of hypoxia, which lower the ratio of state 3 (phosphorylating) to state 4 (nonphosphorylating) respiration. Human atherosclerotic lesions have been known for decades to be deficient in essential fatty acids, a condition that causes respiratory uncoupling and atherosclerosis.

The finding by Kokaze et al.  helps to explain, at least in part, the anti-atherogenic effect of the allele m. 5178A due to its relation with the favorable lipid profile. The nucleotide change causes leucine-to-methionine substitution at codon 237 (Leu-237Met) of the NADH dehydrogenase subunit 2 located in the loop between 7th and 8th transmembrane domains of the mitochondrial protein. Given that this methionine residue is exposed at the surface of respiratory Complex I, this residue may be available as an efficient oxidant scavenger. Complex I

  • accepts electrons from NADH,
  • transfers them to ubiquinone, and
  • uses the energy released to pump protons across the mitochondrial inner membrane.

Thus, the Leu237Met replacement in the ND2 subunit might have a protective effect against oxidative damage to mitochondria.

Most fatty acid oxidation, which is promoted by peroxisome proliferator-activated receptor α (PPARα) activation, occurs in the mitochondria. Mitochondrial effects could explain why PPARα- deficient mice are protected from diet-induced insulin resistance and atherosclerosis as well as glucocorticoid induced insulin resistance and hypertension. Caloric restriction,

  • improves features of insulin resistance,
  • increases mitochondrial biogenesis and, surprisingly,
  • enhances the efficiency of ATP production.

Dysfunctional mitochondria in cultured cells can be rescued by transfer of mitochondria from adult stem cells, raising the possibility of restoration of normal bioenergetics in the vasculature to treat atherosclerosis associated with insulin resistance.
Chistiakov DA, Sobenin IA, Bobryshev YV, Orekhov AN. Mitochondrial dysfunction and mitochondrial DNA mutations in atherosclerotic complications in diabetes. World J Cardiol 2012; 4(5): 148-156. ISSN 1949-8462 (online). doi:10.4330/wjc.v4.i5.148.

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