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The sulfhydration of cysteine residues in proteins is an important mechanism involved in diverse biological processes. We have developed a proteomics approach to quantitatively profile the changes of sulfhydrated cysteines in biological systems. Bioinformatics analysis revealed that sulfhydrated cysteines are part of a wide range of biological functions. In pancreatic β cells exposed to endoplasmic reticulum (ER) stress, elevated H2S promotes the sulfhydration of enzymes in energy metabolism and stimulates glycolytic flux. We propose that transcriptional and translational reprogramming by the Integrated Stress Response (ISR) in pancreatic β cells is coupled to metabolic alternations triggered by sulfhydration of key enzymes in intermediary metabolism.
Posttranslational modification is a fundamental mechanism in the regulation of structure and function of proteins. The covalent modification of specific amino acid residues influences diverse biological processes and cell physiology across species. Reactive cysteine residues in proteins have high nucleophilicity and low pKa values and serve as a major target for oxidative modifications, which can vary depending on the subcellular environment, including the type and intensity of intracellular or environmental cues. Oxidative environments cause different post-translational cysteine modifications, including disulfide bond formation (-S-S-), sulfenylation (-S-OH), nitrosylation (-S-NO), glutathionylation (-S-SG), and sulfhydration (-S-SH) (also called persulfidation) (Finkel, 2012; Mishanina et al., 2015). In the latter, an oxidized cysteine residue included glutathionylated, 60 sulfenylated and nitrosylated on a protein reacts with the sulfide anion to form a cysteine persulfide. The reversible nature of this modification provides a mechanism to fine tune biological processes in different cellular redox states. Sulfhydration coordinates with other post-translational protein modifications such as phosphorylation and nitrosylation to regulate cellular functions (Altaany et al., 2014; Sen et al., 2012). Despite great progress in bioinformatics and advanced mass spectroscopic techniques (MS), identification of different cysteine-based protein modifications has been slow compared to other post-translational modifications. In the case of sulfhydration, a small number of proteins have been identified, among them the glycolytic enzyme glyceraldehyde phosphate dehydrogenase, GAPDH (Mustafa et al., 2009). Sulfhydrated GAPDH at Cys150 exhibits an increase in its catalytic activity, in contrast to the inhibitory effects of nitrosylation or glutathionylation of the same cysteine residue (Mustafa et al., 2009; Paul and Snyder, 2012). The biological significance of the Cys150 modification by H2S is not well-studied, but H2S could serve as a biological switch for protein function acting via oxidative modification of specific cysteine residues in response to redox homeostasis (Paul and Snyder, 2012). Understanding the physiological significance of protein sulfhydration requires the development of genome-wide innovative experimental approaches. Current methodologies based on the modified biotin switch technique do not allow detection of a broad spectrum of sulfhydrated proteins (Finkel, 2012). Guided by a previously reported strategy (Sen et al., 2012), we developed an experimental approach that allowed us to quantitatively evaluate the sulfhydrated proteome and the physiological consequences of H2S synthesis during chronic ER stress. The new methodology allows a quantitative, close-up view of the integrated cellular response to environmental and intracellular cues, and is pertinent to our understanding of human disease development.
The ER is an organelle involved in synthesis of proteins followed by various modifications. Disruption of this process results in the accumulation of misfolded proteins, causing ER stress (Tabas and Ron, 2011; Walter and Ron, 2011), which is associated with development of many diseases ranging from metabolic dysfunction to neurodegeneration (Hetz, 2012). ER stress induces transcriptional, translational, and metabolic reprogramming, all of which are interconnected through the transcription factor Atf4. Atf4 increases expression of genes promoting adaptation to stress via their protein products. One such gene is the H2S-producing enzyme, γ-cystathionase (CTH), previously shown to be involved in the signaling pathway that negatively regulates the activity of the protein tyrosine phosphatase 1B (PTP1B) via sulfhydration (Krishnan et al., 2011). We therefore hypothesized that low or even modest levels of reactive oxygen species (ROS) during ER stress may reprogram cellular metabolism via H2S-mediated protein sulfhydration (Figure 1A).
In summary, sulfhydration of specific cysteines in proteins is a key function of H2S (Kabil and Banerjee, 2010; Paul and Snyder, 2012; Szabo et al., 2013). Thus, the development of tools that can quantitatively measure genome-wide protein sulfhydration in physiological or pathological conditions is of central importance. However, a significant challenge in studies of the biological significance of protein sulfhydration is the lack of an approach to selectively detect sulfhydrated cysteines from other modifications (disulfide bonds, glutathionylated thiols and sulfienic acids) in complex biological samples. In this study, we introduced the BTA approach that allowed the quantitative assessment of changes in the sulfhydration of specific cysteines in the proteome and in individual proteins. BTA is superior to other reported methodologies that aimed to profile cysteine modifications, such as the most commonly used, a modified biotin switch technique (BST). BST was originally designed to study protein nitrosylation and postulated to differentiate free thiols and persulfides (Mustafa et al., 2009). A key advantage of BTA over the existing methodologies, is that the experimental approach has steps to avoid false-positive and negative results, as target proteins for sulfhydration. BST is commonly generating such false targets for cysteine modifications (Forrester et al., 2009; Sen et al., 2012). Using mutiple validations, our data support the specificity and reliability of the BTA assay for analysis of protein sulfhydration both in vitro and in vivo. With this approach, we found that ATF4 is the master regulator of protein sulfhydration in pancreatic β cells during ER stress, by means of its function as a transcription factor. A large number of protein targets have been discovered to undergo sulfhydration in β cells by the BTA approach. Almost 1,000 sulfhydrated cysteine- containing peptides were present in the cells under the chronic ER stress condition of treatment with Tg for 18 h. Combined with the isotopic-labeling strategy, almost 820 peptides on more than 500 proteins were quantified in the 405 cells overexpressing ATF4. These data show the potential of the BTA method for further systematic studies of biological events. To our knowledge, the current dataset encompasses most known sulfhydrated cysteine residues in proteins in any organism. Our bioinformatics analyses revealed sulfhydrated cysteine residues located on a variety of structure-function domains, suggesting the possibility of regulatory mechanism(s) mediated by protein sulfhydration. Structure and sequence analysis revealed consensus motifs that favor sulfhydration; an arginine residue and alpha-helix dipoles are both contributing to stabilize sulfhydrated cysteine thiolates in the local environment.
Pathway analyses showed that H2S-mediated sulfhydration of cysteine residues is that part of the ISR with the highest enrichment in proteins involved in energy metabolism. The metabolic flux revealed that H2S promotes aerobic glycolysis associated with decreased oxidative phosphorylation in mitochondria during ER stress in β cells. The TCA cycle revolves by the action of the respiratory chain that requires oxygen to operate. In response to ER stress, mitochondrial function and cellular respiration are down-regulated to limit oxygen demand and to sustain mitochondria. When ATP production from the TCA cycle becomes limited and glycolytic flux increases, there is a risk of accumulation of lactate from pyruvate. One way to escape accumulation of lactate is the mitochondrial conversion of pyruvate to oxalacetic acid (OAA) by pyruvate carboxylase. This latter enzyme was found to be sulfhydrated, consistent with the notion that sulfhydration is linked to metabolic reprogramming towards glycolysis.
The switch of energy production from mitochondria to glycolysis is known as a signature of hypoxic conditions. This metabolic switch has also been observed in many cancer cells characterized as the Warburg effect, which contributes to tumor growth. The Warburg effect provides advantages to cancer cell survival via the rapid ATP production through glycolysis, as well as the increased conversion of glucose into anabolic biomolecules (amino acid, nucleic acid and lipid biosynthesis) and reducing power (NADPH) for regeneration of antioxidants. This metabolic response of tumor cells contributes to tumor growth and metastasis (Vander Heiden et al., 2009). By analogy, the aerobic glycolysis trigged by increased H2S production could give β cells the capability to acquire ATP and nutrients to adapt their cellular metabolism towards maintaining ATP levels in the ER (Vishnu et al., 2014), increasing synthesis of glycerolphospholipids, glycoproteins and protein (Krokowski et al., 2013b), all important components of the ISR. Similar to hypoxic conditions, a phenotype associated with most tumors, the decreased mitochondria function in β cells during ER stress, can also be viewed as an adaptive response by limiting mitochondria ROS and mitochondria-mediated apoptosis. We therefore view that the H2S-mediated increase in glycolysis is an adaptive mechanism for survival of β cells to chronic ER stress, along with the improved ER function and insulin production and folding, both critical factors controlling hyperglycemia in diabetes. Future work should determine which are the key proteins targeted by H2S and thus contributing to metabolic reprogramming of β cells, and if and how insulin synthesis and secretion is affected by sulfhydration of these proteins during ER stress.
Abnormal H2S metabolism has been reported to occur in various diseases, mostly through the deregulation of gene expression encoding for H2S-generating enzymes (Wallace and Wang, 2015). An increase of their levels by stimulants is expected to have similar effects on sulfhydration of proteins like the ATF4- induced CTH under conditions of ER stress. It is the levels of H2S under oxidative conditions that influence cellular functions. In the present study, ER stress in β cells induced elevated Cth levels, whereas CBS was unaffected. The deregulated oxidative modification at cysteine residues by H2S may be a major contributing factor to disease development. In this case, it would provide a rationale for the design of therapeutic agents that would modulate the activity of the involved enzymes.
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 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.
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.
Regulation
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.
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.
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.
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.
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.
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.
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.
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
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.
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.
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:
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.
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).
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.
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.
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).
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
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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Extracellular deposits of Amyloid-beta (Aβ) peptides (amyloidogenic pathway) and
Intracellular deposits of phosphorylated protein TAU (fibrillogenic pathway)
For many years, both these two pathways (amyloidogenic and fibrillogenic) contended the role of “responsible” for AD onset in the researchers’ debates, even originating respectively the two groups of “BAptists” and “TAUists” scientists. In the recent years, however, these absolutist hypotheses were confuted by the emerging data evidencing that late onset AD (LOAD) has the characteristics to be considered a multifactorial disease and by scientific reports demonstrating possible interconnection between (but not limited to) the two above-mentioned “pathogenic” pathways.
For example, it was demonstrated that
GSK-3β (glycogen synthase kinase 3-beta), a phosphorylase involved in tau phosphorylation, is also responsible for APP (Amyloid Precursor Protein) phosphorylation and that
Aβ peptides are able to induce GSK-3β.
Among the several possible cocauses and interconnected pathways involved in LOAD onset and progression, a very rapidly emerging topic is related to the role of epigenetics. Moreover, it was hypothesized that methylation impairment could be a common promoter and/or a connection between amyloid and tau pathogenic pathways involving not only DNA methylation but also protein methylation mechanisms. This observation rises from studies on PP2A (protein phosphatase 2A) protein methylation showing that downregulation of neuronal PP2A methylation occurs in affected brain regions from AD patients, causing the accumulation of both phosphorylated tau and APP isoforms and increased secretion of Aβ peptides.
Altered methylation metabolism could represent the connection between B vitamins and LOAD. B vitamins are essential cofactors of homocysteine (HCY) metabolism, also called 1-carbon metabolism. One-carbon metabolism is a complex biochemical pathway regulated by the presence of folate, vitamin B12 and B6 (among other metabolites), and leading to the production of methyl donor molecule S-adenosylmethionine (SAM). High HCY and low B vitamin levels are associated to LOAD, even if a cause-effect relationship is still far to be ascertained; moreover, a clear correlation between HCY and Aβ levels has been found.
In addition, SAM, the principal metabolite in the HCY cycle and the main methyl donor in eukaryotes, appears to be altered in some neurological disorders, including AD. HCY, a thiol containing amino acid produced during the methionine metabolism via the adenosylated compound SAM, once formed is either converted to cysteine by transsulfuration or remethylated to form methionine. In the remethylation pathway HCY is remethylated by the vitamin B12-dependent enzyme methionine synthase (MS) using 5-methyltetrahydrofolate as cosubstrate. Alternatively, mainly in liver, betaine can donate a methyl group in a vitamin B12-independent reaction, catalyzed by betaine-homocysteine methyltransferase (BHMT). In the transsulfuration pathway, HCY can condense with serine to form cystathionine in a reaction catalyzed by the cystathionine beta synthase (CBS), a vitamin B6-dependent enzyme, and the cystathionine is hydrolyzed to cysteine (Cys). Cysteine is used for protein synthesis, metabolized to sulfate, or used for glutathione (GSH) synthesis. The tripeptide GSH is the most abundant intracellular nonprotein thiol, and it is a versatile reductant, serving multiple biological functions, acting, among others, as a quencher of free radicals and a cosubstrate in the enzymatic reduction of peroxides. HCY accumulation causes the accumulation of S-adenosylhomocysteine (SAH) because of the reversibility of the reaction converting SAH to HCY and adenosine (Ado); the equilibrium dynamic favors SAH synthesis. The reaction proceeds in the hydrolytic direction only if HCY and adenosine are efficiently removed. SAH is a strong DNA methyltransferases inhibitor, which reinforces DNA hypomethylation (Chiang et al., 1996). Thus, an alteration of the metabolism through either remethylation or transsulfuration pathways can lead to hyperhomocysteinemia, decrease of SAM/SAH ratio (methylation potential; MP), and alteration of GSH levels, suggesting that hypomethylation is a mechanism through which HCY is involved in vascular disease and AD, together with the oxidative damage. To add insult to injury, oxidative stress also promotes the formation of oxidized derivatives of HCY, like homocysteic acid and homocysteine sulfinic acid. These compounds, through the interaction with glutamate receptors, generate intracellular free radicals.
The first observations about B vitamins or HCY deficiency in neurological disorders were hypothesized in the 80 seconds. Despite this recent acknowledgement, alterations of HCY levels and related compounds were only recently widely recognized as risk factors for LOAD and other forms of dementia. Few mechanisms are suggested as possible protagonists in the toxic pathway of HCY in LOAD onset:
oxidative stress and neurotoxicity,
vascular damage,
alteration of cholesterol and lipids,
alteration of protein function by methylation and
deregulation of gene expression by DNA methylation.
These results were obtained by using both transgenic and dietary models of hyperhomocysteinemia or altered 1-carbon metabolism. On the one hand, this variety of experimental models allowed to investigate multiple aspects of the biochemical alterations and their consequences; on the other, the lacking of common methods or goals generated a large body of literature in part overlapping for some aspects but fragmentary or incomplete for others. This aspect represents, together with the scarce interplay between clinical/epidemiological and biomolecular research, one of the reasons for the poor relevance given by the scientific community to the role of 1-carbon metabolism in certain diseases like dementia.
A causal connection between 1-carbon alterations:
hyperhomocysteinemia,
low B vitamins,
low SAM, or
high SAH
and biological alterations responsible for LOAD onset and progression is still missing. So, it was previously demonstrated that 1-carbon metabolism was related to AD-like hallmarks (increased Aβ production) via PSEN1 (presenilin 1) and BACE (beta-site APP cleaving enzyme 1) upregulation in cellular and animal models. More recently, it was added to the rising literature body dealing with 1-carbon metabolism and GSK-3β and PP2A modulation; it was also demonstrated that PSEN1 promoter is regulated by site-specific DNA methylation in cell cultures and mice and that this modulation of methylation is dependent on the regulation of the DNA methylation machinery. Although all the proposed pathways of HCY toxicity are possibly involved and nonmutually exclusive, as suggested by the multifactorial origin of LOAD, the recent advances in the connection between epigenetics and LOAD (as discussed above) stress a primary role for methylation dishomeostasis dependent on 1-carbon metabolism alterations.
Positioning a Therapeutic Concept for Endogenous Augmentation of cEPCs — Therapeutic Indications for Macrovascular Disease: Coronary, Cerebrovascular and Peripheral
Introduction: The following document is a seminal article concerning the relationship between hyoerhomocysteinemia and cardiovascular and other diseases. It provides a new insight based on the metabolism of S8 and geographic factors affecting the distribution, the differences of plant and animal sources of dietary intake,
and the great impact on methylation reactions. The result is the finding that hyperhomocysteine is a “signal”, just as CRP is a measure of IL-6, IL-1, TNFa -mediated inflammatory response. A deficiency of S8 due to the unavailability of S8, leads to CVD, and is seen in sulfur deficient regions with inadequate soil content and with veganism. Hyperhomocysteinemia is also an indicator of CVD risk in the well fed populations, and that gives us a good reason to ASK WHY?
I have trimmed the content to make the necessary points that would be sufficient for this content. The article can be viewed at OCCJ online.
The Oxidative Stress of Hyperhomocysteinemia Results from Reduced Bioavailability of Sulfur-Containing Reductants
Yves Ingenbleek*
Laboratory of Nutrition, Faculty of Pharmacy, University Louis Pasteur Strasbourg, France
Abstract
A combination of subclinical malnutrition and S8-deficiency
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
Impairment of CbS activity in protein malnutrition, entails
supranormal accumulation of Hcy in body fluids,
stimulation of (5) 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 is a limiting factor for
its non-enzymatic reduction to **H2S which contributes to
downsizing a common body pool (dotted circle).(Fig 1)
Combined protein- and S-deficiencies work in concert
to deplete Cys, GSH and H2S from their body reserves,
impeding these reducing molecules from countering
the oxidative stress imposed by hyperhomocysteinemia.
Hyperhomocysteinemia
Hyperhomocysteinemia (HHcy) is an acquired metabolic anomaly first identified by McCully [1]
The current consensus is that dietary deficiency in any of three water soluble vitamins may operate as causal factor of HHcy.
PLP–deficiency may trigger the upstream accumulation of Hcy in biological fluids [2] whereas
the shortage of vitamins B9 or B12 is held responsible for its downstream sequestration [3,4].
HHcy is regarded as a major causal determinant of CVD
working as an independent and graded risk factor
unrelated to the classical Framingham criteria such as
hypercholesterolemia,
dyslipidemia,
sedentary lifestyle,
diabetes and
smoking.
Hcy may invade the intracellular space of many tissues and locally generate [5]
endothelial dysfunction working as early harbinger of blood vessel injuries and atherosclerosis.
Most investigators contend
that production of harmful reactive oxygen and nitrogen species (ROS, NOS), notably
hydrogen peroxide (H2O2), superoxide anion (O2 .-) and peroxinitrite (ONOO.-),
constitutes a major culprit in the development of HHcy-induced vascular damages [7-10].
Accumulation of ROS associated with increased risk for
cardiovascular diseases [11]
stroke [12],
arterial hypertension [6],
kidney dysfunction [13],
Alzheimer’s disease [14],
cognitive deterioration [15],
inflammatory bowel disease [16] and
bone remodeling [17].
These effects overlook the protective roles played by
extra- and intracellular reductants such as cysteine (Cys) and glutathione (GSH)
in the sequence of events leading from HHcy to tissue damage.
Hydrogen Sulfide (H2S)
After the discovery of nitric oxide (NO) and carbon oxide (CO), hydrogen sulfide (H2S) is the
third gaseous signaling messenger found in mammalian tissues [18].
H2S is a reducing molecule displaying strong scavenging properties
as the gasotransmitter significantly attenuates [19, 20] or
even abolishes [21,22] the oxidative injury imposed by HHcy burden.
The endogenous production of the naturally occurring H2S reductant depends on
Cys bioavailability through
the mediation of TS enzymes [23,24].
H2S may also be produced in human tissues starting from elemental sulfur,
by a non-enzymatic reaction requiring the presence of Cys, GSH, and glucose [25,26].
It would be worth disentangling the respective roles played by
Cys,
GSH
H2S
for the prevention and restoration of HHcy-induced oxidative lesions.
but the plasma concentration of Cys and GSH is severely depressed in
subclinically malnourished HHcy patients [27],
impeding appropriate biogenesis of H2S molecules.
The present paper reviews the biological consequences
resulting from the complex interplay existing between the 3 reducing molecules,
to gain insight into the pathophysiologic mechanisms associated with HHcy states.
CLINICAL BACKGROUND
Numerous surveys have conclusively shown that the water soluble vitamin deficiency concept,
provides only partial causal account of the HHcy metabolic anomaly.
The components of body composition, mainly
the size of lean body mass (LBM),
constitutes a critical determinant of HHcy status [28,29].
Because nitrogen (N) and sulfur (S) concentrations
maintain tightly correlated ratios in tissues, we hypothesize
defective N intake and accretion rate would cause concomitant and
proportionate depletion of total body N (TBN) and total body S (TBS) stores [30].
Our clinical investigation undertaken in Central Africa in apparently healthy but
nevertheless subclinically malnourished vegetarian subjects has
documented that reduced size of LBM could lead to HHcy states [27].
The field study conducted in the Republic of Chad, populated by the Sara ethnic group [27], is a semi-arid region and
the staple food consists mainly of cassava, sweet potatoes, beans, millets and groundnuts.
Participants were invited to fill in a detailed dietary questionnaire whose results were compared with values reported in food composition tables [32-34] [27].
The dietary inquiry indicates that participants
consumed a significantly lower mean SAA intake (10.4 mg.kg-1.d-1)[27]
than the Recommended Dietary Allowances (RDAs) (13 mg.kg-1.d-1)[33,34].
Blood Analytes
The blood lipid profiles of rural subjects were confined within normal ranges
ruling out this class of parameters as causal risk factors for CVD disorders.
The normal levels measured for pyridoxine, folates, and cobalamins
precluded these vitamins from playing any significant role in the rise of Hcy
plasma concentrations [27]. Analysis of plasma SAAs revealed
unmodified methioninemia, significantly
elevated Hcy values (18.6 umol/L)
contrasting with significantly decreased plasma Cys and GSH values [27].
The significant lowering of classical
anthropometric parameters
(body weight, BW;
body mass index, BMI)
together with that of the main plasma and urinary biomarkers of
metabolic (visceral) and
structural (muscular) compartments point to
an estimated 10 % shrinking of LBM [27].
Transthyretin (TTR) and Lean Body Mass (LBM)
We have attached peculiar importance to the measurement of plasma transthyretin (TTR)
this indicator integrates the evolutionary trends outlined by body protein reserves [35],
providing from birth until death an overall and balanced estimate of LBM fluctuations [29].
In the absence of any superimposed inflammatory condition,
LBM and TTR profiles indeed reveal striking similarities [29].
Scientists belonging to the Foundation for Blood Research (Scarborough, Maine, 04074, USA) have recently published a large number of TTR results recorded
in 68,720 healthy US citizens aged 0-100 yr which constitute a comprehensive reference material to follow the shape of LBM fluctuations in relation with sex and age [29].
TTR concentrations plotted against Hcy values reveal a strongly negative correlation (r = –0.71) [29,30], confirming that
the depletion of TBN and TBS stores plays a predominant role in the development of HHcy states.
The body of a reference man weighing 70 kg contains 64 M of N (1,800 g) and 4,400 mM of S (140 g) [36]. Our vegetarian subjects consume diets providing
low fat and high fiber content conferring a large spectrum of well described health benefits notably for the prevention of several chronic disorders such as
cancer and diabetes, together with an effective protection against the risk of hypercholesterolemia-induced CVD [37,38].
Plant-based regimens, however, do not supply appropriate amounts of
nitrogenous substrates of good biological value which are required to adequately fulfill mammalian tissue needs [30].
vegetable items contain suboptimal concentrations of both SAAs [33,34,39] below the customary RDA guidelines.
This dietary handicap may be further deteriorated by
unsuitable food processing [40] and by
the presence in plant products of naturally occurring anti-nutritional factors
such as tannins in cereal grains and
anti-trypsin or anti-chymotrypsin inhibitors in soybeans and kidney beans [41].
LBM loss
LBM shrinking may be the result of either
dysmaturation of body protein tissues as an effect of protracted dietary SAA deprivation
or of cytokine-induced depletion of body stores.
Although causally unrelated and evolving along dissimilar adaptive processes,
both physiopathologic entities lead to comparable LBM downsizing best
identified by declining plasma TTR ( measured alone or within combined formulas )
and subsequently rising Hcy values.
All parameters are downregulated with the sole exception of RM flux rates, indicating that
maintenance of Met homeostasis remains a high metabolic priority in protein-depleted states.
Stressful disorders are characterized by
overstimulation of all
TM
RM
TS flux rates.
The severity and duration of initial impact determine the magnitude of protein tissue breakdown,
rendering an account of N : S urinary losses,
fluctuations of albuminuria and of
insulin resistance striving to contain LBM integrity.
Both physiopathologic entities are compromized in reducing the oxidative burden imposed by HHcy states owing to
defective synthesis and/or
enhanced overconsumption of Cys-GSH-H2S reducing molecules,
a condition still worsened by its co-existence with elemental S-deficiency.
IMPAIRMENT OF THE TRANSSULFURATION PATHWAY
The hypothesis that subclinical protein malnutrition might be involved in the occurrence of HHcy states via inhibition of cystathionine-b-synthase (CbS) activity
first arose in Senegal in 1986 [42] and was later corroborated in Central Africa [43]. The concept was clearly counterintuitive in that it was unexpected that
high Hcy plasma values might result from low intake of its precursor Met molecule.
Despite the low SAA intake of our vegetarian patients [27], plasma Met concentrations disclosed noticeable stability permitting
maintenance of the synthesis and functioning of myriads of Met dependent molecular, structural and metabolic compounds
These clinical investigations have received strong support from recent mouse [45] and rat [46] experiments submitted to Met-restricted regimens.
At the end of the Met-deprivation period, both animal species did manifest meaningful HHcy states (p<0.001) contrasting with
significantly lower BW (p<0.001) reduced by 33 % [45] and 44 % [46] of control, respectively.
the uniqueness of Met behavior stands in accordance with balance studies performed on large mammalian species showing
that the complete withdrawal of Met from otherwise normal diets causes the greatest rate of body loss,
nearly equal to that generated by protein-free regimens [47,48].
This efficient Met homeostatic mechanism is classically ascribed to a PLP-like inhibition of CbS activity exerted through
allosteric binding of S-adenosylmethionine (SAM) to the C-terminal regulatory domain of the enzyme [49,50].
The loss of CbS activity may develop via a (post)translational defect
independently from intrahepatic SAM concentrations [45].
We have postulated the existence of an independent sensor mechanism set in motion by TBS pool shrinkage and
reduced bioavailability of Met – its main building block – working as an inhibitory feedback loop of CbS activity [30].
Such Met-bodystat, likely to be centrally mediated, is to maintain unaltered Met disposal in conditions of
decreased dietary provision implies the fulfillment
of high metabolic priorities of survival value [30,44].
Whereas HHcy may be regarded as the dark side of a beneficial adaptive machinery [43],
impairment of the TS pathway also depresses the production of compounds situated downstream to the CbS blockade level,
notably Cys and GSH, keeping in mind that Cys may undergo reversible GSH conversion (Fig. 1).
The plasma concentration of both Cys and GSH reductants is indeed significantly decreased in our vegetarian subjects
by 33 % and 67 % of control, displaying negative correlations (r = –0.67 and –0.37, respectively) with HHcy values [27].
Reduced dietary intake of the preformed Cys molecule [27] and diminished Cys release from protein breakdown in malnourished states [51]
may contribute to the lowering effect.
The significantly decreased GSH blood levels may similarly be attributed to dietary composition since the tripeptide is mainly found in meat products
but is virtually absent from cereals, roots, milk and dairy items [52] and
because regimens lacking SAAs may lessen the production of blood GSH and its intrahepatic sequestration [53].
BIOGENESIS OF HYDROGEN SULFIDE
The TS degradation pathway schematically proceeds along two main PLP-dependent enzymatic reactions working in succession (Fig. 1).
The first is catalyzed by CbS (EC 4.2.1.22) governing the replacement of the hydroxyl group of serine with Hcy to generate Cysta plus H2O.
Cys may however substitute for serine and the replacement of its sulfhydryl group with Hcy releases Cysta and H2S instead of water [54].
The second is regulated by cystathionine-g-lyase (CgL, EC 4.4.1.1.) hydrolyzing Cysta to release Cys and alpha-ketobutyrate plus ammonia as side-products [55].
Cys may also undergo nonoxidative desulfuration pathways leading to H2S or sulfanesulfur production [56] under the control of CbS or CgL enzymes.
Cys may otherwise undergo oxidative conversion regulated by cysteine-dioxygenase (CDO, EC 1.13.11.20) which
catalyzes the replacement of the SH- group of Cys by SO3 – to yield cysteine-sulfinate [56].
This last compound may be further decarboxylated to hypotaurine that is finally oxidized to Tau (67 %) and SO4 2- oxyanions (33 %) [56]. CbS and CgL, both cytosolic enzymes,
their relative contribution to the generation of H2S may vary according to
animal strains,
tissue specificities and
nutritional or physiopathological circumstances [23,24].
CbS and CgL are expressed in most organs such as liver, kidneys, brain, heart, large vessels, ileum and pancreas [57,58] potentially
subjected to HHcy-induced ROS injury while keeping the capacity to desulfurate Cys and to
locally produce H2S as cytoprotectant signaling agent.
CbS is the principal TS enzyme found in
cerebral glial cells and astrocytes [59].
CgL predominates in the
vascular system [60] whereas
both enzymes are present in the renal proximal tubules [61].
H2S is the third gaseous substrate found in the biosphere [18] after NO and CO. All three gases are characterized by
severe toxicity when inhaled at high concentrations.
In particular, H2S produced by anaerobic fermentation is
capable of causing respiratory death by
inhibition of mitochondrial cytochrome C oxidase [62].
NO, CO and H2S are synthesized from arginine, glycine and Cys, respectively, exerting at low concentrations major biological functions in living organisms.
Most of our knowledge on these atypical signal messengers [63] are derived from animal experiments and tissue cultures. These transmitter molecules may
share some properties in common such as penetration of cellular membranes independently from specific receptors [64].
They are also manifesting dissimilar activities: whereas NO and CO activate guanylyl cyclase to generate biological responses via cGMP-dependent kinases,
H2S induces Ca2+-dependent effects through ATP-sensitive K+ channels [65].
Some of these potentialities may work in concert while others operate antagonistically. For instance,
NO and H2S express vasorelaxant tone on endogenous smooth muscle [66]
but reveal different effects on large artery vessels [67].
These gaseous substances maintain whole body homeostasis through complex interactions and multifaceted crosstalks between signaling pathways.
Elemental S (32.064 as atomic mass) is a primordial constituent of lava flows in areas of volcanic or sedimental origin usually presenting as crown-shaped
stable octamolecules – hence its S8 symbolic denomination – which may conglomerate to form brimstone rocks. The vegetable kingdom is
unable to assimilate S8 and requires as prior step its natural or bacterial oxidation to SO4 2- derivatives before launching
the synthesis of SAA molecules along narrowly regulated metabolic pathways [30,44].
Distinct anabolic processes are identified in mammalian tissues which lack the enzymatic equipment required to organize sulfate oxyanions
but possess the capacity of direct S8 conversion into H2S.
S8 is poorly soluble in tap waters [68] may be taken up and transported to mammalian tissues loosely fastened to serum albumin (SA) [69].
S may also be covalently bound to intracellular S-atoms taking the form of sulfane-sulfur compounds [70] either
firmly attached to cytosolic organelles or in
untied form to mitochondria [57,58,71,72] to undergo
later release in response to specific endogenous requirements [71].
Sulfane-sulfur compounds are somewhat unstable and may decompose in the presence of reducing agents allowing the restitution of S [70,71].
S may either endorse the role of stimulatory factor of several mammalian apoenzyme activities as shown for
succinic dehydrogenase [73] and NADH dehydrogenase [74] or
operate as inhibitory agent of other mammalian apoenzymes such as
adenylate kinase [75] and liver tyrosine aminotransferase [76].
Elemental S resulting from dietary supply or from sulfane-sulfur decay may be subjected to
non-enzymatic reduction in the presence of Cys and GSH [25,26] and/or reducing equivalents obtained from
glucose oxidation [25], hence yielding at physiological pH additional provision of H2S.
The gaseous mediator is a weakly acidic molecule endowed with strong lipophilic affinities. In experimental models, the blockade of the TS cascade
at CbS or CgL levels significantly depresses or even
abolishes the vitally required production of Cys
operating at the crossroad of multiple converting processes (Fig. 1).
Addition of Cys to the incubation milieu
resumes the generation of H2S [19] in a Cys concentration-dependent manner [77].
The compounds situated downstream both cystathionases in the context of SAA deprivation
keep their functional potentialities
but are unable to express their converting Cys – H2S capacities
in the absence of precursor substrate.
Summing up
inhibition of CbS activity contributes to
promote efficient RM processes and
maintenance of Met homeostasis
but entails as side-effects
upstream sequestration of Hcy molecules in biological fluids
while decreasing the bioavailability of Cys and GSH
working as limiting factors for H2S production.
These last adverse effects thus constitute the Achilles heel of a remarkable adaptive machinery.
ROLES PLAYED BY HYDROGEN SULFIDE
The first demonstration that human tissues may reduce S to H2S was incidentally provided in 1924 when a man given colloid sulfur
for the treatment of polyarthritis did rapidly exhale the typical rotten egg malodor [78].
H2S may be produced by the intestinal flora [79] and serves as a metabolic fuel for colonocytes [80].
Prevention of endogenous poisoning by excessive enteral production is insured by the detoxifying activities of mucosal cells [81],
hindering any systemic effect of the gaseous substrate.
The normal H2S concentration measured in mammalian plasmas usually ranges from 10 to 100 μM with a mean average turning around 40-50 μM [19,21,82,83].
This H2S plasma level, appearing as the net product of organs possessing CbS and CgL enzymes and supplemented by the non-enzymatic conversion of S,
flows transiently into the vasculature and freely penetrates into all body cells.
Supposing that the gaseous reductant is evenly distributed in total body water (45 L in a 70 kg reference man) allows an estimate of
bioavailable H2S pool turning around 2 mM which represents, in terms of S participation, largely less than 1 / 1,000 of TBS.
The peculiar adaptive physiology of vegetarian subjects renders very unlikely that their TBS pool might be solicited to release
S-substrates prone to undergo conversion to nascent H2S molecules since
they adapt to declining energy and nutrient intakes
by switching overall body economy toward downregulated steady state activities.
The release from TBS of substantial amounts of S-compounds occurs
only during the onset of hypercatabolic states as documented in trauma patients [31]
and in infectious diseases [84], exacting as preliminary step
cytokine-induced breakdown of tissue proteins, a selective hallmark of stressful disorders [85].
H2S in fulfilling ROS Scavenger Tasks
The limited disposal of H2S endogenously produced might be readily exhausted in fulfilling ROS scavenging tasks at the site of oxidative lesions.
All body organs generating H2S from TS enzymes are
simultaneously producers and consumers of the gaseous substrate whose actual concentration
reflects the balance between synthetic and catabolic rates [86].
Clinical investigations show that H2S concentrations found in cerebral homogenates from Alzheimer’s disease (AD) patients are
very much lower than expected from values measured in healthy brains [87], suggesting that
the gaseous messenger is locally submitted to enhanced consumption rates reflecting disease severity.
The concept is strongly supported by studies pointing to the
negative correlation linking the severity of AD to H2S plasma values [88].
in pediatric [89] and elderly [90] hypertensive patients as well
more severe HHcy-dependent oxidative burden is
associated with more intense H2S uptake rates.
These H2S cleansing properties are mainly exerted by mitochondrial organelles
known to be centrally involved in oxidative disorders [20,91].
Malnourished subjects deprived of Cys and GSH disposal thus incur the risk of H2S-deficiency
rendering them unable to properly overcome HHcy-imposed oxidative lesions.
The rapid exhaustion of H2S stores have detrimental consequences as shown disclosing
the beneficial effects of exogenous administration of commonly used sulfide salt donors (Na2S and NaHS)
generating H2S gas once in solution.
Such supply significantly augments
H2S plasma concentrations allowing to counteract ROS damages.
H2S was primarily recognized as a physiological substrate working as
neuromodulator [92] and soon later as
vasorelaxant factor [65].
H2S is now regarded as endowed with a broader spectrum of biological properties [18],
operating as a general protective mediator
against most degenerative organ injuries,
being capable of neutralizing or
abolishing most ROS harmful effects.
Table 1 collects findings displaying that H2S may promote the synthesis and activity of several
anti-oxidative enzymes (catalases, Cu- and Mn-superoxide dismutases, GSH-peroxidases) and
stimulate the production of anti-inflammatory reactants (interleukin-10) or
conversely downregulate
pro-oxidative enzymes (collagenases, elastases),
pro-inflammatory cytokines (interleukine-1b, tumor-necrosis factor a) and
It has been calculated that 81.5% of H2S undergoes catabolic disintegration in the form of hydrosulfide anion (HS-) or sulfide anion (S2-) [117].
Since S is the main element in the diprotonated H2S molecule (34.08 as molecular mass), it may be considered that
partial or complete repair of HHcy-induced lesions constitutes the therapeutic proof that
S-deficiency is causally involved in the development of ROS damages.
The concept is sustained by the observation that all synthetic drugs (diclofenac, indomethacine, sildenafil) utilized as surrogate providers of H2S [64,118] are
characterized by a large diversity of molecular conformations but
share in common the presence of Satom(s) mimicking, once released,
H2S-like pharmacological properties.
It remains to be clarified whether the beneficial effects of S-fortification to S-deficient subjects are mediated, among other possible mechanisms, via
stimulation [73,74] of anti-oxidative enzymes or inhibition [75,76] of pro-oxidative enzymes.
It is only very recently that the essentiality of S has been recognized, causing Hcy elevation in deficient individuals [119]. It is worth reminding that the
gaseous NO substrate may work in concert or antagonistically [66,83] to fine-tuning the helpful properties exerted by H2S on body tissues.
Preliminary studies suggest for instance that NO operates, in combination with H2S, as a potential modulator of endothelial remodeling since
NO-synthase isoforms contribute to the activation of metalloproteinases involved in the regulation of the collagen/elastin balance defining vascular elastance [83,120].
SUBCLINICAL MALNUTRITION AS WORLDWIDE SCOURGE
A growing body of data collected along the last decades indicates that
large proportions of mankind still suffer varying degrees of protein and energy deficiency that is associated with
increased morbidity and mortality rates.
The determinants of malnutrition are complex and interrelated, comprising
socioeconomic and political conditions,
insufficient dietary intakes,
inadequate caring practices and
superimposed inflammatory burden.
Children living in developing countries are paying a heavy toll to chronic malnutrition [121,122] whereas adult populations are handicapped by
feeble physical and working capacities,
increased vulnerability to infectious complications and
reduced life expectancy [123,124].
Cross-sectional studies collected in the eighties indicate that chronic malnutrition remains a worldwide scourge with
top prevalence recorded in Asia, whereas
sub-Saharan Africa endures medium nutritional distress and
Latin America appears as the least affected [125,126].
Along the last decades, significant progresses have been achieved in some countries such as Vietnam [127] and Bangladesh [128]
owing to appropriate education programs and improved economic development.
Inequalities however persist between middle class population groups mainly located in affluent urban areas and
underprivileged rural communities remaining stagnant on the sidelines of household income growth.
Representative models of these socio-economic disparities in global nutrition and health are illustrated in the two most populated countries in the world, China and India.
Large surveys undertaken in 105 counties of China and recently published have concluded that the rural communities haven’t yet reached the stage of overall welfare [129].
In India, similar investigations have documented that extreme poverty still prevails in the northern mountainous states of the subcontinent [130]. Taken together, southern
Asian countries fail to overcome malnutrition burden [131]. In some African countries, there exists even upward trends suggesting nutritional
deterioration over the years [132] still aggravated by a severe drought. The assessment of malnutrition in children usually rely on anthropometric criteria such as height-for-age, weight for-height, mid upper arm circumference and skinfold thickness allowing to draw the degree of stunting and wasting from these estimates. In adult subjects, BW and BMI are currently selected parameters to which some biochemical measurements are frequently added, notably SA, classical marker of protein nutritional status, and creatininuria (u-Cr), held as indicator of sarcopenia. The former biometric approaches are very useful in that they correctly provide a static picture of the declared stages of malnutrition but fail to recognize the dynamic mechanisms occurring during the preceding months and the adaptive alterations running behind.
Table 1. Reversal of HHcy-Induced Oxidative Damages by Administration of Exogenous H2S
BRAIN EFFECTS
H2S is overproduced in response to neuronal excitation [93], and
increases the sensitivity of N-methyl-D-aspartate (NMDA) reactions to glutamate in hippocampal neurons [23,94].
improves long-term potentiation, a synaptic model of memory [92,93]
stimulates the inhibitory effects of catalase and superoxide dismutase (SOD) in oxidative stress of endothelial cells [95].
regulates Ca 2+ homeostasis in microglial cells [96]and it inhibits TNFa expression in microglial cultures [97].
protects brain cells from neurotoxicity by preventing the rise of ROS in mitochondria [98].
CARDIOVASCULAR EFFECTS
H2S releases vascular smooth muscle,
inhibits platelet aggregation and
reduces the force output of the left ventricule of the heart [18].
maintains vascular smooth muscle tone [66] and
insures protection against arterial hypertension [99].
modifies leucocyte-vascular epithelium interactions in vivo by
modulating leucocyte adhesion and
diapedesis at the site of inflammation [100].
attenuates myocardial ischemia-reperfusion injury by
depressing IL-1b and mitochondrial function [20].
upregulates the expression of depressed anti-oxidative enzymes in heart infarction and
inhibits myocardial injury [21].
alleviates smooth muscle pain by
stimulating K+ ATP channels [101].
prevents apoptosis of human neutrophil cells
by inhibiting p38 MAP kinase and caspase 3 [102].
potentiates angiogenesis and wound healing [103].
RENAL EFFECTS
H2S downregulates the increased activity of metalloproteinases 2 and 9 involved in extracellular matrix degradation (elastases, collagenases) [19].
Prevents apoptotic cell death in renal cortical tissues [19].
Improves the expression of desmin (marker of podocyte injury) and
restores the drop of nephrin (component of normal slit diaphragm) in the cortical tissues
resulting in reduced proteinuria [19].
Induces hypometabolism revealing protective effects on renal function and survival [104].
Normalizes GSH status and production of ROS in renal diseases [19].
Controls renal ischemia-reperfusion injury and dysfunction [105].
Depresses the expression of inflammatory molecules involved in glomerulosclerosis [106].
Prevents lung oxidative stress in hypoxic pulmonary hypertension caused by low GSH content [113].
Promotes SOD and catalase activities and reduces the production of malondialdehyde in oxidative lung injury [114].
Reduces lung inflammation and remodeling in asthmatic animals [115] and in pulmonary hypertension [116]. ..(see OCCJ 2011;4:34-44)
Assessing Protein-Depleted States
SA is an insensitive marker of protein-depleted states compared to TTR [134]
SA is an indicator of population than of individual protein status in subclinical PEM.
u-Cr is likewise a meagerly informative tool as 10 % loss of muscle mass is required before it reaches significantly decreased urinary concentrations [135].
The data imply that the magnitude of subclinical malnutrition is largely
underscored when classical biometric and laboratory investigations are performed.
Moreover, ruling out the protein component involved in HHcy epidemiology and confining solely attention to the B-vitamin triad led to unachieved conclusions.
surveys undertaken in Taiwan [136] and in India [137] established HHcy variance turning around 30 %, indicating that
a sizeable percentage of subjects do not come within the vitamin shortage concept.
only one recent review recommending the use of TTR in vegetarian subjects [138].
The main reason for making the choice of TTR is grounded on the striking similar plasma profile disclosed by this marker with both LBM and Hcy [29].
Under healthy conditions, the 3 parameters –
TTR,
LBM,
Hcy –
indeed show low concentrations at birth,
linear increase without sexual difference in preadolescent children,
gender dimorphism in teenagers with higher values recorded in adolescent male subjects
thereafter maintenance of distinct plateau levels during adulthood [29,139,140].
Under morbid circumstances, the plasma concentrations of
Hcy manifest gradual elevation
negatively correlated with LBM downsizing and
TTR decline.
In vegetarian subjects and subclinically malnourished patients,
rising Hcy and
diminished TTR plasma concentrations look as mirror image of each other,
revealing divergent distortion from normal and
allowing early detection of preclinical steps
at the very same time both SA and u-Cr markers still remain silent.
Any disease process characterized by quantitative or qualitative dietary protein restriction or intestinal malabsorption
may cause LBM shrinking,
downregulation of TTR concentrations and
subsequent HHcy upsurge.
These conditions are documented in frank kwashiorkor [141], subclinical protein restriction [27,43] and anorexia nervosa [142].
In patients submitted to weight-reducing programs,
LBM was found the sole independent variable
negatively correlated with rising Hcy values [143].
Morbid obesity may be alleviated by medical treatment [143] or surgical gastroplasty [144,145],
conditions frequently associated with secondary malabsorptive syndromes and malnutrition [146],
How does this account play out in the typical patient with excessive body fat, lipoprotein disoreder, and perhaps diabetes and disordered sleep – an account of acquired HHcy?
Have the studies been done? Would you expect to see a clear benefit from reduced HHcy_emia based on a 30 min daily walk, and
eating of well fat trimmed meats, fruits and vegetables, and fish, flax seed, or krill oil?
In westernized countries, subclinical protein-depleted states are illustrated in immigrants originating from
developing regions but keeping alive their traditional feeding practices [147] or
by communities having adopted, for socio-cultural reasons, strict vegan dietary lifestyles [148].
THE ADDITIONAL BURDEN OF S-DEFICIENCY
After N, K and P, elemental S is recognized as the fourth most important macronutrient required for plant development. The essentiality of S in the vegetable kingdom
arose from observations made many decades ago by pedologists and agronomists [149,150] revealing that the withdrawal of sulfate salts from nutrient sources produces
rapid growth retardation,
depressed chlorophyllous synthesis,
yellowing of leaves and
reduction in fertility and crop yields.
A large number of field studies, mainly initiated for economical reasons, has provided continuing gain in fundamental and applied knowledge and led to the overall consensus
that SO4 2- -deficiency is a major wordwide problem [151,152].
Field investigations have shown that the concentration of SO4 2- oxyanions in soils and drinking waters
may reveal considerable variations ranging from less than 2 mg/L to more than 1 g/L,
meaning a ratio exceeding 1 / 500 under extreme circumstances [30].
The main causal factors responsible for unequal distribution of SO4 2- oxyanions are geographical distance from eruptive sites and
intensity of soil weathering in rainy countries.
SO4 2- -dependent nutritional deficiencies entail detrimental effects to most African and Latin American crops [151]
reaching nevertheless top incidence in southeastern Asia [151,153].
and the Indo-Gangetic plain extending from Pakistan to Bangladesh and covering the North of India and Nepal [154].
Intensive agricultural production,
lack of animal manure and
use of fertilizers providing N, K and P substrates
but devoid of sulfate salts may further aggravate that imbalanced situation.
As global population increases steadily and the production of staple plants predicted to escalate considerably,
SO4 2- deficient disorders are expected to become more pregnant along the coming years [155] with significant harmful impact for mankind.
Nevertheless, effective preventive efforts are developed in some countries aiming at fortification of soils mainly
by ammonium sulfate or calcium sulfate (gypsum) salts,
resulting in meaningful improvements in crop yield,
SAAs content and biological value and
opening more optimistic perspectives for livestock and human consumption [152,155-158].
Contrasting with the tremendously high amounts of data accumulated over decades by pedologists and agronomists on sulfate requirements and metabolism,
the available knowledge on elemental sulfur in human nutrition looks like a black hole. Despite the fact that S8 follows H, C, O, N, Ca and P as the seven most
abundant element in mammalian tissues, it appears as a forgotten item. Not the slightest attention is dedicated to S8 in the authoritative “Present Knowledge
in Nutrition” series of monographs even though they go over most oligo- and trace-elements in minute detail.
The geographical distribution of S8 throughout the earth’s crust is not well-known
as extreme paucity of measurements in soils and tap waters prevents reaching a comprehensive overview.
Nevertheless, and because S8 is the obligatory precursor substrate for the oxidative production of sulfate salts,
a decremental dispersion pattern paralleling those of SO4 2- oxyanions is likely to occur with
highest values recorded in the vicinity of volcano sources
and lowest values found in remote and washed-out areas.
Obviously, a great deal of research on elemental S remains to be completed by clinical biochemists before rejoining the status of plant agronomy.
Taken together, these data imply that subclinically malnourished subjects living in areas recognized as
SO4 2- -deficient for the vegetable kingdom also
incur increased risks to become S8-depleted.
This clinical entity most probably prevails in all regions, notably Northern India, where protein malnutrition [130] and sulfur-deficiency [154] coexist.
Combination of both nutritional deprivations explains why the bulk of local dwellers, including young subjects [159,160], may develop HHcy states and CVD disorders
characterized by strong refractoriness to vitamin-B supplementation [160] or
high incidence of stroke [161] unrelated to the classical Framingham criteria.
The current consensus is that “the problem of CVD in South Asia is different in etiology and magnitude from other parts of the world” [162]. These disquieting findings are
confirmed in several Asian countries [163] and have prompted local cardiologists to exhort their governments to focus more attention on CVD epidemiology [164].
CONCLUDING REMARKS
vegetarian subjects are not protected against the risk of CVD and stroke which should no longer be regarded as solely affecting populations living in westernized societies
whose morbidity and mortality risks are stratified by classical Framingham criteria.
Likewise hypercholesterolemia, hyperhomocysteinemia should be incriminated as
emblematic risk factor for a panoply of CVD and related disorders.
Whereas the causality of cholesterol and lipid fractions largely prevails in affluent societies consuming high amounts of animal-based items,
that of homocysteine predominates in population groups whose dietary lifestyle gives more importance to plant products.
MAIN PHYSICO-CHEMICAL AND METABOLIC CHARACTERISTICS* OF 3 CARRIER-PROTEINS INVOLVED IN THE STRESS RESPONSE
CBG
TTR
RBP
Molecular mass (Da.)
42,650
54,980
21,200
Conformation
monomeric
tetrameric
monomeric
Amino acid sequence
383
4 x 127
182
Carbohydrate load
18 % glycosylated
unglycosylated
unglycosylated
Hormonal binding sites
one for cortisol
two for TH
one for retinol
Association constant (M-1)
3 x 107
7 x 107 (T4)
1.9 x 107
Normal plasma concentration
30 mg/L.
300 mg/L.
50 mg/L.
Biological half-life
5 days
2 days
14 hrs
Bound ligand concentration
120 µg/L.
80 µg TT4/L.
500 µg/L.
Free ligand concentration
5 µg/L.
20 ng FT4/L.
1 µg/L.
Ratio free : bound ligands
4 %
0.034 %
0.14 %
Distribution volume of free moieties
18 L.
12 L.
18 L.
STIMULATORY AND INHIBITORY EFFECTS MODULATED
BY GLUCOCORTICOIDS
TARGET SYSTEMS
INDUCED EFFECTS
REF.
Thymidine kinase
_
transcription of induced DNA into RNA
112
Alkaline phosphodiesterase I
_
cleavage of phosphodiester bonds
113
Tyrosine transaminase
_
transfer of tyrosine amino group
114
Tryptophane oxygenase
_
formylkynurenine and Trp catabolites
115
Alkaline phosphatase
_
release of P from phosphoric esters
116
Phosphoenolpyruvate carboxykinase (liver)
_
glycolysis from pyruvate and ATP production
117
Mannolsyltransferases
_
dolichol-linked glycosylation of APRs
118
Haptoglobin
_
APR combining with hemoglobin
119
α1-Anti (chymo) trypsin (α1 AT, α1 ACT)
_
serpin molecules allowing N-sparing effects
120
α1-Acid glycoprotein (AGP)
_
glycosylated APR with antibody-like actions
121
Serum amyloid protein (SAA)
_
defense systems against oxidative burst
122
γ-Fibrinogen
_
clotting processes and tissue repair
123
C-Reactive Protein (CRP)
_
complement processes and opsonization
124
Corticosteroid-binding globulin (CBG)
_
CBG levels, favoring free hypercortisolemia
100
Phosphoenolpyruvate carboxykinase (adipocytes)
_
ATP turnover and glycolysis
113
THE DUAL MORBID ENTITIES CAUSING LBM DOWNSIZING AND SUBSEQUENT Hcy UPSURGE
TTR coupled with CRP or other inflammatory indices (31,177,178,284,285)
Insulin resistance status
Normal or low (286)
Increased in proportion of tissue breakdown (177,178,181-183)
status of Cys-GSH-H2S reducing molecules
Decreased enzymatic and non-enzymatic production (39,161,162,287)
Increased production cancelled out by tissue overconsumption (78,171)
Urinary SO42- and S-compounds
Decreased kidney output (76,78,79)
Variable depending on exogenous SAA supply and
extent of tissue breakdown (78,163,168,173)
Transmethylation pathway
Depressed (48,93)
Overstimulated (169)
Remethylation pathway
Stimulated (76,83,153)
Overstimulated (169)
Transsulfuration pathway
Inhibited (49,76,83)
Overstimulated (170,173)
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anamikasarkar
apreconasia
aviralvatsa
David Orchard-Webb, PhD
danutdaagmailcom
Demet Sag, Ph.D., CRA, GCP
Dror Nir
dsmolyar
Ethan Coomber
evelinacohn
Gail S Thornton
Irina Robu
jayzmit48
jdpmd
jshertok
kellyperlman
Ed Kislauskis
larryhbern
Madison Davis
marzankhan
megbaker58
ofermar2020
Dr. Pati
pkandala
ritusaxena
Rick Mandahl
sjwilliamspa
Srinivas Sriram
stuartlpbi
Dr. Sudipta Saha
tildabarliya
vaishnavee24
zraviv06
zs22
