Posts Tagged ‘homocysteine’

Serum Folate and Homocysteine, Mood Disorders, and Aging

Larry H. Bernstein, MD, FCAP, Curator



Dietary Folate and the Risk of Depression in Finnish MiddleAged Men

Tolmunen T, et al.


Serum Folate, Vitamin B-12, and Homocysteine and Their Association With Depressive Symptoms Among U.S. Adults

PSYCHOSOM MED · NOV 2010;             DOI:

Objective: To examine, in a nationally representative sample of U.S. adults, the associations of serum folate, vitamin B-12, and total homocysteine (tHcy) levels with depressive symptoms. Several nutritional and physiological factors have been linked to depression in adults, including low folate and vitamin B-12 and elevated tHcy levels.
Methods: Data on U.S. adults (age, 20–85 years; n 2524) from the National Health and Nutrition Examination Survey during the period 2005 to 2006 were used. Depressive symptoms were measured with the Patient Health Questionnaire (PHQ), and elevated symptoms were defined as a PHQ total score of 10. Serum folate, vitamin B-12, and tHcy were mainly expressed as tertiles. Multiple ordinary least square (OLS), logistic, and zero-inflated Poisson regression models were conducted in the main analysis.
Results: Overall, mean PHQ score was significantly higher among women compared with men. Elevated depressive symptoms (PHQ score of 10) were inversely associated with folate status, particularly among women (fully adjusted odds ratio [tertiles T3 versus T1] 0.37; 95% confidence interval, 0.17–0.86), but not significantly related to tHcy or vitamin B-12. No interaction was noted between the three exposures in affecting depressive symptoms. In older adults (50 years) and both sexes combined, tHcy was positively associated with elevated depressive symptoms (fully adjusted odds ratio [tertiles T2 versus T1] 3.01; 95% confidence interval, 1.01–9.03), although no significant dose-response relationship was found. Conclusions: Future interventions to improve mental health outcomes among U.S. adults should take into account dietary and other factors that would increase levels of serum folate.
Key words: depression, folate, vitamin B-12, homocysteine, adults.


Relationship of homocysteine, folic acid and vitamin B12 depression in a middle-aged community sample

P.S. SACHDEV, et al.   PSYCHOL MED · MAY 2005;   35, 529–538 /10.1017/S0033291704003721 

Background. Case control studies have supported a relationship between low folic acid and vitamin B12 and high homocysteine levels as possible predictors of depression. The results from epidemiological studies are mixed and largely from elderly populations.
Method. A random subsample of 412 persons aged 60–64 years from a larger community sample underwent psychiatric and physical assessments, and brain MRI scans. Subjects were assessed using the PRIME-MD Patient Health Questionnaire for syndromal depression and severity of depressive symptoms. Blood measures included serum folic acid, vitamin B12, homocysteine and creatinine levels, and total antioxidant capacity. MRI scans were quantified for brain atrophy, subcortical atrophy, and periventricular and deep white-matter hyperintensity on T2-weighted imaging.
Results. Being in the lowest quartile of homocysteine was associated with fewer depressive symptoms, after adjusting for sex, physical health, smoking, creatinine, folic acid and B12 levels. Being in the lowest quartile of folic acid was associated with increased depressive symptoms, after adjusting for confounding factors, but adjustment for homocysteine reduced the incidence rate ratio for folic acid to a marginal level. Vitamin B12 levels did not have a significant association with depressive symptoms. While white-matter hyperintensities had significant correlations with both homocysteine and depressive symptoms, the brain measures and total antioxidant capacity did not emerge as significant mediating variables. Conclusions. Low folic acid and high homocysteine, but not low vitamin B12 levels, are correlates of depressive symptoms in community-dwelling middle-aged individuals. The effects of folic acid and homocysteine are overlapping but distinct.


Association of folate intake with the occurrence of depressive episodes in middle-aged French men and women

P. Astorg, et al.    BRIT J  NUTR · AUG 2008; 100, 183–18

A low folate intake or a low folate status have been found to be associated with a higher frequency of depression in populations, but the existence and the direction of a causal link between folate intake or status and depression is still uncertain. The aim of this study was to seek the relation between the habitual folate intake in middle-aged men and women and the occurrence of depressive episodes. In a subsample of 1864 subjects (809 men and 1055 women) from the French SU.VI.MAX cohort, dietary habits have been measured at the beginning of the follow-up (six 24 h records) and declarations of antidepressant prescription, taken as markers of depressive episodes, have been recorded during the 8-year follow-up. No significant association was observed between folate intake and the risk of any depressive episode or of a single depressive episode during the follow-up, in both men and women. In contrast, the risk of experiencing recurrent depressive episodes (two or more) during the follow-up was strongly reduced in men with high folate intake (OR 0·25 (95% CI 0·06, 0·98) for the highest tertile v. the lowest, P for trend 0·046). This association was not observed in women. These results suggest that a low folate intake may increase the risk of recurrent depression in men.   Folate: Depression: Cohort studies


Homocysteine, vitamin B12, and folic acid levels in Alzheimer’s disease, mild cognitive impairment, and healthy elderly: baseline characteristics in subjects of the Australian Imaging Biomarker Lifestyle study.

Faux NG1, Ellis KA, Porter L, Fowler CJ,…, Ames D, Masters CL, Bush AI.
J Alzheimers Dis. 2011; 27(4):909-22.

There is some debate regarding the differing levels of plasma homocysteine, vitamin B12 and serum folate between healthy controls (HC), mild cognitive impairment (MCI), and Alzheimer’s disease (AD). As part of the Australian Imaging Biomarker Lifestyle (AIBL) study of aging cohort, consisting of 1,112 participants (768 HC, 133 MCI patients, and 211 AD patients), plasma homocysteine, vitamin B12, and serum and red cell folate were measured at baseline to investigate their levels, their inter-associations, and their relationships with cognition. The results of this cross-sectional study showed that homocysteine levels were increased in female AD patients compared to female HC subjects (+16%, p-value < 0.001), but not in males. Red cell folate, but not serum folate, was decreased in AD patients compared to HC (-10%, p-value = 0.004). Composite z-scores of short- and long-term episodic memory, total episodic memory, and global cognition all showed significant negative correlations with homocysteine, in all clinical categories. Increasing red cell folate had a U-shaped association with homocysteine, so that high red cell folate levels were associated with worse long-term episodic memory, total episodic memory, and global cognition. These findings underscore the association of plasma homocysteine with cognitive deterioration, although not unique to AD, and identified an unexpected abnormality of red cell folate.


Homocysteine and folate as risk factors for dementia and Alzheimer disease1,2,3

Giovanni RavagliaPaola FortiFabiola Maioli, …., Nicoletta BrunettiElisa Porcellini, and Federico Licastro
Am J Clin Nutr Sept 2005; 82(3): 636-643


Background: In cross-sectional studies, elevated plasma total homocysteine (tHcy) concentrations have been associated with cognitive impairment and dementia. Incidence studies of this issue are few and have produced conflicting results.

Objective: We investigated the relation between high plasma tHcy concentrations and risk of dementia and Alzheimer disease (AD) in an elderly population.

Design: A dementia-free cohort of 816 subjects (434 women and 382 men; mean age: 74 y) from an Italian population-based study constituted our study sample. The relation of baseline plasma tHcy to the risk of newly diagnosed dementia and AD on follow-up was examined. A proportional hazards regression model was used to adjust for age, sex, education, apolipoprotein E genotype, vascular risk factors, and serum concentrations of folate and vitamin B-12.

Results: Over an average follow-up of 4 y, dementia developed in 112 subjects, including 70 who received a diagnosis of AD. In the subjects with hyperhomocysteinemia (plasma tHcy > 15 μmol/L), the hazard ratio for dementia was 2.08 (95% CI: 1.31, 3.30; P = 0.002). The corresponding hazard ratio for AD was 2.11 (95% CI: 1.19, 3.76; P = 0.011). Independently of hyperhomocysteinemia and other confounders, low folate concentrations (≤11.8 nmol/L) were also associated with an increased risk of both dementia (1.87; 95% CI: 1.21, 2.89; P = 0.005) and AD (1.98; 95% CI: 1.15, 3.40; P = 0.014), whereas the association was not significant for vitamin B-12.

Conclusions: Elevated plasma tHcy concentrations and low serum folate concentrations are independent predictors of the development of dementia and AD.


In Western societies, the prevalence and economic costs of Alzheimer disease (AD) are soaring in step with the increased number of elders in the population (1). Therefore, it is important to identify modifiable risk factors for this disease. The sulfur amino acid homocysteine is a unique candidate for this role because of its direct neurotoxicity (24) and its association with cerebrovascular disease (5), which is currently believed to play a significant role in AD etiology (6). Moreover, elevated concentrations of plasma total homocysteine (tHcy) are an indicator of inadequate folate and vitamin B-12 status (7) and can directly affect brain function via altered methylation reactions (8).

An association between AD and elevated tHcy concentrations has been reported in case-control (9, 10) and cross-sectional (11, 12) studies. Moreover, in nondemented elderly populations, plasma tHcy is inversely associated with poor performance at simultaneously performed tests of global cognitive function (1315) and specific cognitive skills (13, 16). However, cross-sectional studies cannot determine causality. Only 2 longitudinal studies investigated the relation between hyperhomocysteinemia and risk of incident AD, but their results were inconsistent; the Framingham Study reported a strong association (17), and the Washington Heights–Inwood Columbia Ageing Project (WHICAP) reported no association (18). Clarification of this issue is important because consistent evidence of a prospective association between homocysteine and AD would more strongly support the need for intervention trials testing the effectiveness of homocysteine-lowering vitamin therapy in preventing dementia.

Therefore, we examined baseline plasma tHcy in relation to risk of incident dementia and AD in the Conselice Study of Brain Aging (CSBA), an Italian population-based study of older persons.

Study population

The CSBA is a population-based survey, already described in detail elsewhere (19,20), the principal aim of which is to provide data about epidemiology and risk factors for dementia in the elderly. Its design includes both cross-sectional and longitudinal components. The study was approved by the Institutional Review Board of the Department of Internal Medicine, Cardioangiology, and Hepatology, University of Bologna, and written informed consent was obtained from all participants.

Briefly, in 1999–2000, 1016 (75%) of the 1353 individuals aged ≥65 y residing in the Italian municipality of Conselice (province of Ravenna, Emilia Romagna region) participated in the prevalence study. Data on cognitive status at the follow-up examination in 2003–2004 were collected for 861 of the 937 participants free of dementia at baseline. A flow chart detailing the derivation of the incidence sample used in this study is reported in Figure 1.

This prospective population-based study was the first to replicate previous findings from the Framingham Study (17), indicating that hyperhomocysteinemia doubles the risk of developing dementia and AD independently of several major confounders. Our results disagree with the negative findings recently reported in the WHICAP study (18). Possible explanations for this difference are the acknowledged insufficient statistical power of the WHICAP study, the rather homogeneously high tHcy concentrations of its sample—which did not permit enough variability to detect an association—and methodologic issues related to the prolonged time between blood sample collection and processing, which could have affected tHcy measurements.

Inconsistent results were also given by the only 2 studies that examined the association between homocysteine and cognitive decline at follow-up as measured with the MMSE (30, 31). These studies, however, differed in sample size and in which confounders were taken into account. Moreover, MMSE is a reliable global screening measure of cognitive function but was not developed to estimate changes in cognitive function or to diagnose dementia (32).

The substantial evidence that tHcy is an independent vascular risk factor (5) supports the role of hyperhomocysteinemia in AD. Subjects with vascular risk factors and cerebrovascular disease have an increased risk of AD (6), and hyperhomocysteinemia has been related to cerebral macro- and microangiopathy, endothelial dysfunction, impaired nitric oxide activity, and increased oxidative stress (3335). Moreover, as shown in cell cultures, homocysteine can directly cause brain damage through several mechanisms: increased glutamate excitoxicity via activation of N-methyl-D-aspartate receptors (2), enhancement of β-amyloid peptide generation (4), impairment of DNA repair, and sensitization of neurons to amyloid toxicity (3).

On the basis of cross-sectional observations, some authors have suggested that elevated plasma tHcy concentrations are not a causative factor in dementia and AD but are only a marker for concomitant vascular disease, independently of cognitive status (36, 37). Results from other cross-sectional investigations (9, 12, 38), as well as those from the present investigation and the Framingham Study (17), argue against this interpretation, but only intervention trials can give the ultimate proof of a causal relation between hyperhomocysteinemia and AD.

In contrast with both the Framingham (17) and WHICAP (18) studies, we also found that, independent of homocysteine and other confounders (including vitamin B-12), low serum folate is associated with an increased risk of incident dementia and AD. Mandatory folate fortification of food might partially explain the negative results of the US studies, whereas in Italy, where folate fortification is not practiced, relative folate deficiency may be endemic among the elderly population. Nondemented patients with poor cognitive performance and AD patients often exhibit poor folate status (reviewed in 8), but only one study specifically examined B vitamins in relation to incident dementia. In a selected sample of nondemented Swedish elderly participants in the Kungsholmen Study, low serum folate and vitamin B-12 were predictive of AD at 3 y of follow-up (39). The sample, however, was small (370 subjects), and a clear association was detected only when both vitamins were taken into account.

Biologic explanatory mechanisms relating folate deficiency to dementia include impaired methylation reactions in the central nervous system, with a consequent insufficient supply of methyl groups, which are required for the synthesis of myelin, neurotransmitters, membrane phospholipids, and DNA (8). However, because of the study design and the relatively short follow-up time, we cannot definitely establish whether the independent association between low folate and dementia risk indicates an actual effect of folate status on cognitive function or, on the contrary, that subtle functional alterations may affect the dietary intake of folate in the early preclinical stages of dementia.


Neurotoxicity associated with dual actions of homocysteine at the N-methyl-D-aspartate receptor

Stuart A. Lipton*Won-Ki KimYun-Beom Choi*,…, Derrick R. Arnelle§, and Jonathan S. Stamler
NAS 1997; 94(11):5923–5928

Severely elevated levels of total homocysteine (approximately millimolar) in the blood typify the childhood disease homocystinuria, whereas modest levels (tens of micromolar) are commonly found in adults who are at increased risk for vascular disease and stroke. Activation of the coagulation system and adverse effects of homocysteine on the endothelium and vessel wall are believed to underlie disease pathogenesis. Here we show that homocysteine acts as an agonist at the glutamate binding site of the N-methyl-D-aspartate receptor, but also as a partial antagonist of the glycine coagonist site. With physiological levels of glycine, neurotoxic concentrations of homocysteine are on the order of millimolar. However, under pathological conditions in which glycine levels in the nervous system are elevated, such as stroke and head trauma, homocysteine’s neurotoxic (agonist) attributes at 10–100 μM levels outweigh its neuroprotective (antagonist) activity. Under these conditions neuronal damage derives from excessive Ca2+ influx and reactive oxygen generation. Accordingly, homocysteine neurotoxicity through overstimulation of N-methyl-D-aspartate receptors may contribute to the pathogenesis of both homocystinuria and modest hyperhomocysteinemia.


Vitamin B12 and folate in relation to the development of Alzheimer’s disease

H-X. Wang, Å. WahlinH. Basun, …, B. Winblad, and L. Fratiglioni
Neurology May 8, 2001; 56(9):1188-1194    http:/​/​dx.​doi.​org/​10.​1212/​WNL.​56.​9.​1188

Objective: To explore the associations of low serum levels of vitamin B12 and folate with AD occurrence.

Methods: A population-based longitudinal study in Sweden, the Kungsholmen Project. A random sample of 370 nondemented persons, aged 75 years and older and not treated with B12 and folate, was followed for 3 years to detect incident AD cases. Two cut-off points were used to define low levels of vitamin B12 (≤150 and ≤250 pmol/L) and folate (≤10 and ≤12 nmol/L), and all analyses were performed using both definitions. AD and other types of dementia were diagnosed by specialists according to DSM-III-R criteria.

Results: When using B12 ≤150pmol/L and folate ≤10 nmol/L to define low levels, compared with people with normal levels of both vitamins, subjects with low levels of B12or folate had twice higher risks of developing AD (relative risk [RR] = 2.1, 95% CI = 1.2 to 3.5). These associations were even stronger in subjects with good baseline cognition (RR = 3.1, 95% CI = 1.1 to 8.4). Similar relative risks of AD were found in subjects with low levels of B12or folate and among those with both vitamins at low levels. A comparable pattern was detected when low vitamin levels were defined as B12 ≤250 pmol/L and folate ≤12 nmol/L.

Conclusions: This study suggests that vitamin B12 and folate may be involved in the development of AD. A clear association was detected only when both vitamins were taken into account, especially among the cognitively intact subjects. No interaction was found between the two vitamins. Monitoring serum B12 and folate concentration in the elderly may be relevant for prevention of AD.


Assessing the association between homocysteine and cognition: reflections on Bradford Hill, meta-analyses, and causality

Hyperhomocysteinemia is a recognized risk factor for cognitive decline and incident dementia in older adults. Two recent reports addressed the cumulative epidemiological evidence for this association but expressed conflicting opinions. Here, the evidence is reviewed in relation to Sir Austin Bradford Hill’s criteria for assessing “causality,” and the latest meta-analysis of the effects of homocysteine-lowering on cognitive function is critically examined. The meta-analysis included 11 trials, collectively assessing 22 000 individuals, that examined the effects of B vitamin supplements (folic acid, vitamin B12, vitamin B6) on global or domain-specific cognitive decline. It concluded that homocysteine-lowering with B vitamin supplements has no significant effect on cognitive function. However, careful examination of the trials in the meta-analysis indicates that no conclusion can be made regarding the effects of homocysteine-lowering on cognitive decline, since the trials typically did not include individuals who were experiencing such decline. Further definitive trials in older adults experiencing cognitive decline are still urgently needed.
Mouse model for deficiency of methionine synthase reductase exhibits short-term memory impairment and disturbances in brain choline metabolism
, , , , , , ,
Biochem. J. 2014 461: 205212
Hyperhomocysteinaemia can contribute to cognitive impairment and brain atrophy. MTRR (methionine synthase reductase) activates methionine synthase, which catalyses homocysteine remethylation to methionine. Severe MTRR deficiency results in homocystinuria with cognitive and motor impairments. An MTRR polymorphism may influence homocysteine levels and reproductive outcomes. The goal of the present study was to determine whether mild hyperhomocysteinaemia affects neurological function in a mouse model with Mtrr deficiency. Mtrr+/+, Mtrr+/gt and Mtrrgt/gtmice (3 months old) were assessed for short-term memory, brain volumes and hippocampal morphology. We also measured DNA methylation, apoptosis, neurogenesis, choline metabolites and expression of ChAT (choline acetyltransferase) and AChE (acetylcholinesterase) in the hippocampus. Mtrrgt/gt mice exhibited short-term memory impairment on two tasks. They had global DNA hypomethylation and decreased choline, betaine and acetylcholine levels. Expression of ChAT and AChE was increased and decreased respectively. At 3 weeks of age, they showed increased neurogenesis. In the cerebellum, mutant mice had DNA hypomethylation, decreased choline and increased expression of ChAT. Our work demonstrates that mild hyperhomocysteinaemia is associated with memory impairment. We propose a mechanism whereby a deficiency in methionine synthesis leads to hypomethylation and compensatory disturbances in choline metabolism in the hippocampus. This disturbance affects the levels of acetylcholine, a critical neurotransmitter in learning and memory.

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Adenosine Receptor Agonist Increases Plasma Homocysteine

Larry H. Bernstein, MD, FCAP, Curator



The Adenosine Receptor Agonist 5’-N-Ethylcarboxamide-Adenosine Increases Mouse Serum Total Homocysteine Levels, Which Is a Risk Factor for Cardiovascular Diseases

Spring Zhou Editor at Scientific Research Publishing

I would like to share this paper with you. Any comments on this article are welcome.


An increase in total homocysteine (Hcy) levels (protein-bound and free Hcy in the serum) has been identified as a risk factor for vascular diseases. Hcy is a product of the methionine cycle and is a precursor of glutathione in the transsulfuration pathway. The methionine cycle mainly occurs in the liver, with Hcy being exported out of the liver and subsequently bound to serum proteins. When the non-specific adenosine receptor agonist 5’-N-ethylcarboxamide-adenosine (NECA; 0.1 or 0.3 mg/kg body weight) was intraperitoneally administered to mice that had been fasted for 16 h, total Hcy levels in the serum significantly increased 1 h after its administration. The NECA treatment may have inhibited transsulfuration because glutathione levels were significantly decreased in the liver. After the intraperitoneal administration of a high dose of NECA (0.3 mg/kg body weight), elevations in total Hcy levels in the serum continued for up to 10 h. The mRNA expression of methionine metabolic enzymes in the liver was significantly reduced 6 h after the administration of NECA. NECA-induced elevations in total serum Hcy levels may be maintained in the long term through the attenuated expression of methionine metabolic enzymes.



  1.  Is level of protein consumption a factor?
  2. Is reliance on plant food products a factor?
  3. What are the levels of transthyretin?
  4. Is there a concomitant decrease in vitamin A or vitamin D?



The Adenosine Receptor Agonist 5’-N-Ethylcarboxamide-Adenosine Increases Mouse Serum Total Homocysteine Levels, Which Is a Risk Factor for Cardiovascular Diseases

Shigeko Fujimoto Sakata*, Koichi Matsuda, Yoko Horikawa, Yasuto Sasaki     Faculty of Nutrition, Kobe Gakuin University, Kobe, Japan.    DOI: 10.4236/pp.2015.610048

Cite this paper

Sakata, S. , Matsuda, K. , Horikawa, Y. and Sasaki, Y. (2015) The Adenosine Receptor Agonist 5’-N-Ethylcarboxamide-Adenosine Increases Mouse Serum Total Homocysteine Levels, Which Is a Risk Factor for Cardiovascular Diseases. Pharmacology & Pharmacy, 6, 461-470. doi: 10.4236/pp.2015.610048.
An increase in total serum homocysteine levels (total Hcy: serum protein-bound and free Hcy) has been identified as a risk factor for cardiovascular disease [1] [2] and liver fibrosis [3]. The normal range of total Hcy in adults is typically 5 – 15 μM, with the mean level being approximately 10 μM [2]. Plasma Hcy concentrations were previously found to be strongly associated with the presence and number of small infarctions, or infarction of the putamen in elderly diabetic patients [4]. High levels of Hcy have been shown to induce endoplasmic reticulum (ER) stress and increase the production of reactive oxygen species (ROS) [5]. Hcy has strong reducibility and modifies disulfide bonds in proteins. Only 1% to 2% of Hcy occurs as thiol homocysteine in the serum; 75% of Hcy has been suggested to bind to proteins through disulfide bonds with protein cysteines [6]. Hcy is formed as an intermediary in methionine metabolism [7] [8]. Methionine metabolism mainly occurs in the livers of mammals. Methionine receives an adenosine group from ATP to become S-adenosylmethionine (AdoMet) in the methionine cycle. This reaction is catalyzed in the liver by liver-specific methionine adenosyltransferase I/III (MAT I/III), which is encoded by the methionine adenosyltransferase 1A (MAT1A) gene [9]. AdoMet then transfers its methyl group to a large number of compounds, a process that is catalyzed by various methyltransferases (e.g., glycine N-methyltransferase: GNMT; DNA methyltransferase; phosphatidylethanolamine N-methyl- transferase), to produce S-adenosylhomocysteine (AdoHcy). Hcy is formed from AdoHcy by AdoHcy hydrolase (SAHH). The reaction that generates Hcy from AdoHcy is reversible, and AdoHcy from Hcy is shown to be thermodynamically favored over the synthesis of Hcy [10]. A previous study reported that Hcy levels were very low in the liver [11]. This reaction then proceeds toward the synthesis of Hcy when the products (Hcy and adenosine) are removed by further metabolism [12]. Three enzymes metabolize Hcy, with the betaine-homocysteine S-methyltransferase (BHMT) and methionine synthase (MS) reactions both yielding methionine. A large proportion of Hcy in the liver is remethylated by BHMT [3]. The third enzyme, cystathionine β-synthase (CBS) catalyzes Hcy to cystathionine in the transsulfuration pathway. Previous studies of whole body methionine kinetics demonstrated that 62% of Hcy was converted to cystathionine during each cycle in males fed a basal diet, resulting in the production of glutathione (GSH), while 38% of Hcy was remethylated to methionine [13]. Hcy is located at an important regulatory branch point: remethylation to methionine; conversion to cystathionine; export from the cells.
A decrease in intracellular ATP levels, accompanied by the accumulation of 5’-AMP and subsequently adenosine, is known to follow ischemia. Adenosine levels in interstitial fluids were shown to increase 100 – 1000- fold from basal levels (10 – 300 nM) with ischemia [14]. Furthermore, adenosine levels in hepatocytes were increased by a hypoxic challenge, with excess amounts of adenosine being exported out of cells [14]. Adenosine levels were also found to increase 10-fold due to hypoxia, stress, and inflammation [15]. Adenosine has been shown to activate A1, A2a, and A3 receptors with EC50 values in the range of 0.2 – 0.7 μM, and also A2b receptors with an EC50 of 24 μM [16]. A1 and A3 receptors have been classified as adenylate cyclase inhibitory receptors, and A2a and A2b receptors as adenylate cyclase-activating receptors [17]. The activation of adenosine receptors accompanied by ischemia may increase total Hcy levels in the serum because hepatic ischemia is known to decrease the content of GSH and activity of MAT [18].
We previously reported that the non-specific adenosine receptor agonist 5’-N-ethylcarboxamide-adenosine (NECA) increased serum glucose levels and the expression of a glucogenic enzyme (glucose 6-phosphatase) in the liver [19] [20]. Based on the dose of NECA administered in these studies and plasma concentrations after the administration of other adenosine agonists [21], it was inferred that the serum NECA concentration was in the μM range and also that NECA activated adenosine A2b receptors. In the present study, we measured methionine metabolites, including Hcy, in NECA-treated mice in order to determine whether the activation of adenosine receptors increased total Hcy levels in the serum. The results obtained clearly demonstrated that NECA increased total Hcy levels in the serum.
Measurement of Methionine Metabolites AdoMet and AdoHcy levels in the liver were measured using an HPLC method [25] and total GSH in the liver was measured using a microtiter plate assay [26], as described previously [23]. Total Hcy and total cysteine levels (total Cys: free and protein-bound cysteine) in the serum were measured using an HPLC method [27]. Briefly, a mixture of 50 μL of serum, 25 μL of an internal standard, and 25 μL of phosphate-buffered saline (PBS, pH 7.4) was incubated with 10 μL of 100 mg/mL TCEP for 30 min at room temperature in order to reduce and release protein-bound thiols. After this incubation, 90 μL of 100 mg/mL trichloroacetic acid containing 1 mmol/L EDTA was added for deproteinization, centrifuged at 15,000 ×g for 10 min, and 50 μL of the supernatant was added to a tube containing 10 μL of 1.55 mol/L NaOH; 125 μL of 0.125 mol/L borate buffer containing 4 mmol/L EDTA, pH 9.5; and 50 μL of 1 mg/mL SBD-F in the borate buffer. The sample was then incubated for 60 min at 60˚C. HPLC was performed on a Waters M-600 pump equipped with a Waters 2475 Multi λ Fluorescence Detector (385 nm excitation, 515 nm emission). The separation of SBD-derivatized thiols was performed on a μ-BONDASPHERE C18 column (Waters, 5 μm, 100 A, 150 × 3.9 mm) with a 20-μL injection volume and 0.1 mol/L acetate buffer, pH 5.5, containing 30 ml/L methanol as the mobile phase at a flow rate of 1.0 mL/min and column temperature of 29˚C.
3.1. Effects of NECA on Total Hcy and Total Cys Levels in the Serum As shown in Table 1, serum total Hcy and total Cys levels significantly increased after 16 h of fasting. The administration of a low dose of NECA (NECA0.1 group) to mice fasted for 16 h resulted in higher serum total Hcy levels than those in the control group at 1 h (Experiment 1). Serum total Hcy levels were also significantly elevated at 3 h (Experiment 2), but were not significantly different from those in the control group at 6 h (Experiment 3). The administration of a high dose of NECA (NECA0.3 group) resulted in significantly higher serum total Hcy levels than those in the control group at 1 h, 3 h, 6 h, and 10 h (Experiments 4, 5, 6, and 7), gradually increasing Hcy levels to 19.7 μM. The effects of NECA on serum total Cys levels were the same as those on total Hcy levels.
Table 1. Effects of NECA on the content of total homocysteine and total cysteine in the serum.

3.2. Effects of NECA on Other Methionine Metabolite Levels in the Liver We previously reported that fasting for 16 h decreased AdoMet and GSH levels, and increased AdoHcy levels in the livers of mice [23]. In the present study, as shown in Table 2, the administration of a low dose of NECA (NECA0.1 group) to mice fasted for 16 h resulted in lower liver GSH levels than those in the control group at 1 h (Experiment 1). Liver GSH levels were also significantly lower at 3 h (Experiment 2), while GSH levels were not significantly different from those in the control group at 6 h (Experiment 3). The administration of a high dose of NECA (NECA0.3 group) resulted in liver GSH levels that were significantly lower than those in the control group at 1 h, 6 h, and 10 h (Experiments 4, 6, and 7). The effects of NECA on total Hcy levels in the serum and GSH levels in the liver were similar at each dose and time. Furthermore, the low and high doses of NECA both led to significantly higher AdoMet levels than those in the control group at 1 h (Experiments 1 and 4). AdoMet levels at 3 h, 6 h, and 10 h were not significantly different from those in the control group (Experiments 2, 3, 5, 6, and 7). AdoHcy levels were significantly lower in the NECA0.3 group than in the control group 6 h and 10 h after the administration of NECA (Experiments 6 and 7), while the administration of a low dose of NECA had less of an impact on AdoHcy levels.

Table 2. Effects of NECA on the content of methionine metabolites in the liver.

3.3. Effects of NECA on mRNA Expression of Methionine Cycle Enzymes in the Liver Figure 1 shows changes in the mRNA expression of methionine cycle enzymes in Experiments 4, 5, and 6. The expression of methionine cycle enzymes did not significantly change 1 h after the administration of NECA. The expression of MAT1A mRNA was significantly decreased in the liver 6 h after the NECA treatment, while that of MAT2A was increased. The changes observed in the expression of MAT in the present study were consistent with previous findings obtained in ischemic livers [18] or with liver regeneration [28]. The expression of GNMT, which eliminates excess AdoMet, was significantly decreased 6 h after the NECA treatment. The expression of CBS, which converts Hcy to cystathionine through the transsulfuration pathway, and BHMT, which converts Hcy to methionine, was also decreased at 6 h.

Figure 1 shows changes in the mRNA expression of methionine cycle enzymes in Experiments 4, 5, and 6. The expression of methionine cycle enzymes did not significantly change 1 h after the administration of NECA. The expression of MAT1A mRNA was significantly decreased in the liver 6 h after the NECA treatment, while that of MAT2A was increased. The changes observed in the expression of MAT in the present study were consistent with previous findings obtained in ischemic livers [18] or with liver regeneration [28]. The expression of GNMT, which eliminates excess AdoMet, was significantly decreased 6 h after the NECA treatment. The expression of CBS, which converts Hcy to cystathionine through the transsulfuration pathway, and BHMT, which converts Hcy to methionine, was also decreased at 6 h.
Figure 1. Effects of NECA on the mRNA expression of methionine cycle enzymes in the mouse liver. Northern hybridization was performed on the liver RNA of mice in experiments 4, 5, and 6. The mean ± SEM of the ratio of each enzyme mRNA to the level of the 18S rRNA signal is shown as an arbitrary unit. Unpaired Student’s t-tests were used to compare NECA- treated groups with the control groups. *p < 0.05, **p < 0.01: significantly different from each control.
4. Discussion In the present study, an increase in total Hcy levels and AdoMet levels, and decrease in GSH levels occurred 1 h after the NECA treatment. These results were not due to changes in the expression of methionine metabolic enzymes, which remained unchanged 1 h after the NECA treatment (Figure 1). The effects of NECA on methionine metabolism are summarized in Figure 2. No previous study has demonstrated that adenosine has the ability to directly affect CBS; however, the overproduction of carbon monoxide (CO), which is generated by heme oxygenase (HO), is found to inhibit transsulfuration [11]. CO has been shown to inhibit CBS activity and increase AdoMet concentrations [11]. Adenosine and NECA were previously reported to markedly induce HO in macrophages [29]. Hcy, which is a substrate of CBS, may be increased by NECA via the CO-induced inhibition of CBS, and GSH may be decreased by the CO-induced inhibition of transsulfuration. However, the mechanism by which NECA affects transsulfuration in the short term has not yet been elucidated.
Figure 2. Effects of NECA on the methionine metabolic pathway. MAT: methionine adenosyltransferase, GNMT: glycine N-methyltransferase, CBS: cystathionine β-synthase, BHMT: betaine-homocysteine S-methyltransferase, MS: methionine synthase (Map is based on Sakata SF 2005).
GSH was maintained at a low level for up to 10 h by the NECA0.3 treatment and transsulfuration may have been continuously inhibited by the NECA0.3 treatment. Total Hcy levels were also continuously increased for up to 10 h by the NECA0.3 treatment, and decreased AdoHcy levels were observed 6 h and 10 h after the NECA0.3 treatment. Long-term elevations in serum total Hcy levels by NECA may be maintained by attenuating the expression of methionine metabolic enzymes via the following mechanisms: The expression of methionine metabolic enzymes in the liver was reduced 6 h after the NECA0.3 treatment (Figure 1); the flow of the methionine cycle may have been decreased by changes in the expression of MAT (decreased liver-specific MAT1A expression and increased non-liver type MAT2A expression) because MATIII (Km for methionine: 215 μM – 7 mM) is the true liver-specific isoform responsible for methionine metabolism [30] and the generation rate of AdoMet by MATII (non-liver type enzyme) was modest with a low Km (80 μM for methionine) [31]; inhibition of the methyltransferases, BHMT [32] and GNMT [33], induces hyperhomocysteinemia; decreases in AdoHcy levels may be caused by reductions in methyltransferase levels. However, the mechanisms by which NECA continuously increased total Hcy levels have not yet been elucidated in detail. 5. Conclusion The present study confirmed that the non-specific adenosine receptor agonist NECA continuously increased total Hcy levels in the serum. The inhibition of adenosine receptors may decrease the risk of cardiovascular diseases because an increase in serum total Hcy levels is a known risk factor.


[1] Antoniades, C., Antonopoulos, A.S., Tousoulis, D., Marinou, K. and Stefanadis, C. (2009) Homocysteine and Coronary Atherosclerosis: from Folate Fortification to the Recent Clinical Trials. European Heart Journal, 30, 6-15.
[2] Refsum, H., Ueland, P.M., Nygard, O. and Vollset, S.E. (1998) Homocysteine and Cardiovascular Disease. Annual Review of Medicine, 49, 31-62.
[3] Garcia-Tevijano, E.R., Berasain, C., Rodriguez, J.A., Corrales, F.J., Arias, R., Martin-Duce, A., Caballeria, J., Mato, J.M. and Avila, M.A. (2001) Hyperhomocysteinemia in Liver Cirrhosis: Mechanisms and Role in Vascular and Hepatic Fibrosis. Hypertension, 38, 1217-1221.
[4] Araki, A., Ito, H., Majima, Y., Hosoi, T. and Orimo, H. (2003) Association between Plasma Homocysteine Concentrations and Asymptomatic Cerebral Infarction or Leukoaraiosis in Elderly Diabetic Patients. Geriatrics & Gerontology International, 3, 15-23.
[5] Elanchezhian, R., Palsamy, P., Madson, C.J., Lynch, D.W. and Shinohara, T. (2012) Age-Related Cataracts: Homocysteine Coupled Endoplasmic Reticulum Stress and Suppression of Nrf2-Dependent Antioxidant Protection. Chemico-Biological Interactions, 200, 1-10.
[6] Mudd, S.H., Finkelstein, J.D., Refsum, H., Ueland, P.M., Malinow, M.R., Lentz, S.R., Jacobsen, D.W., Brattstrom, L., Wilcken, B., Wilcken, D.E., Blom, H.J., Stabler, S.P., Allen, R.H., Selhub, J. and Rosenberg, I.H. (2000) Homocysteine and Its Disulfide Derivatives: A Suggested Consensus Terminology. Arteriosclerosis Thrombosis and Vascular Biology, 20, 1704-1706.
[7] Finkelstein, J.D. (1990) Methionine Metabolism in Mammals. The Journal of Nutritional Biochemistry, 1, 228-237.
[8] Stipanuk, M.H. (2004) Sulfur Amino Acid Metabolism: Pathways for Production and Removal of Homocysteine and Cysteine. Annual Review of Nutrition, 24, 539-577.
[9] Chou, J.Y. (2000) Molecular Genetics of Hepatic Methionine Adenosyltransferase Deficiency. Pharmacology & Therapeutics, 85, 1-9.
[10] De La Haba, G. and Cantoni, G.L. (1959) The Enzymatic Synthesis of S-Adenosyl-L-Homocysteine from Adenosine and Homocysteine. The Journal of Biological Chemistry, 234, 603-608.

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The biochemistry of S amino acids

Larry H. Bernstein, MD, FCAP, Curator


Amino Acid and Sulfur Metabolism

Dr. Rainer Höfgen

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

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

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

sulfur uptake and assimilation

sulfur uptake and assimilation

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

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

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

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


metabolite profiling

metabolite profiling


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

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

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



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


Metabolome analysis and bioinformatics

system response

system response

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

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

Malcolm J. Hawkesford, Rothamsted Research, UK

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


Transcriptome Analysis

gene expression

gene expression

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Scatterplots of gene expression of the ratio -/+ S

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

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

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

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

Further reading

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

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

Sulfur and Other Plant Nutrients

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

Systems Analysis of Plant Sulfur Metabolism

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



L-cysteine L-Met

L-cysteine L-Met

Methionine is synthesised from cysteine and phosphohomoserine

Methionine is synthesised from cysteine and phosphohomoserine


Pathway Analysis of Sulfur Containing Amino Acids

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

Biosynthesis of Sulfur-Containing Amino Acids

Biosynthesis of Sulfur-Containing Amino Acids

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

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

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

Cysteine Biosynthesis

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

Serine acetyltransferase

Serine acetyltransferase

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Serine acetyltransferase

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

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

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

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

Further reading

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

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

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

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

The Sulfur-Containing Amino Acids: An Overview1,2

John T. Brosnan3 and Margaret E. Brosnan

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

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


Structures of the sulfur-containing amino acids.

Methionine and cysteine in proteins.

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

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

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

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


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

Metabolic versatility of S-adenosylmethionine.

Metabolic versatility of S-adenosylmethionine.


Metabolic versatility of S-adenosylmethionine.

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

Methionine metabolism.

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

Major pathways of sulfur-containing amino acid metabolism.

Major pathways of sulfur-containing amino acid metabolism.

Major pathways of sulfur-containing amino acid metabolism.

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

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

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

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

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

Regulation of methionine metabolism.

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

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


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

View this table:


Taurine concentrations in rat tissues (22,23)


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

Disorders of Sulfur Amino Acid Metabolism

  • Generoso Andria,  Brian Fowler,  Gianfranco Sebastio

Chapter  Inborn Metabolic Diseases  pp 224-231


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

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    Rubba P, Faccenda F, Pauciullo P, Carbone L, Mancini M, Strisciuglio P, Carrozzo R, Sartorio R, Del Giudice E, Andria G (1990) Early signs of vascular disease in homocystinuria: a noninvasive study by ultrasound methods in eight families with cystathionine ß-synthase deficiency. Metabolism 39: 1191–1195 PubMedCrossRef

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    Kang S-S, Wong PWK, Malinow MR (1992) Hyperhomocyst(e)inemia as a risk factor for occlusive vascular disease. Annu Rev Nutr 12: 279–288 PubMedCrossRef

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    Boushey CJ, Beresford SA, Omenn GS, Motulsky AG (1995) A quantitative assessment of plasma homocysteine as a risk factor for vascular disease. Probable benefits of increasing folic acid intakes. JAMA 274: 1049–1057

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

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

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

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Summary – Volume 4, Part 2: Translational Medicine in Cardiovascular Diseases

Summary – Volume 4, Part 2:  Translational Medicine in Cardiovascular Diseases

Author and Curator: Larry H Bernstein, MD, FCAP


We have covered a large amount of material that involves

  • the development,
  • application, and
  • validation of outcomes of medical and surgical procedures

that are based on translation of science from the laboratory to the bedside, improving the standards of medical practice at an accelerated pace in the last quarter century, and in the last decade.  Encouraging enabling developments have been:

1. The establishment of national and international outcomes databases for procedures by specialist medical societies

Stent Design and Thrombosis: Bifurcation Intervention, Drug Eluting Stents (DES) and Biodegrable Stents
Curator: Aviva Lev-Ari, PhD, RN

On Devices and On Algorithms: Prediction of Arrhythmia after Cardiac Surgery and ECG Prediction of an Onset of Paroxysmal Atrial Fibrillation
Author, and Content Consultant to e-SERIES A: Cardiovascular Diseases: Justin Pearlman, MD, PhD, FACC

Mitral Valve Repair: Who is a Patient Candidate for a Non-Ablative Fully Non-Invasive Procedure?
Author, and Content Consultant to e-SERIES A: Cardiovascular Diseases: Justin Pearlman, MD, PhD, FACC and Article Curator: Aviva Lev-Ari, PhD, RN

Cardiovascular Complications: Death from Reoperative Sternotomy after prior CABG, MVR, AVR, or Radiation; Complications of PCI; Sepsis from Cardiovascular Interventions
Author, Introduction and Summary: Justin D Pearlman, MD, PhD, FACC and Article Curator: Aviva Lev-Ari, PhD, RN

Survivals Comparison of Coronary Artery Bypass Graft (CABG) and Percutaneous Coronary Intervention (PCI) /Coronary Angioplasty
Larry H. Bernstein, MD, Writer And Aviva Lev-Ari, PhD, RN, Curator

Revascularization: PCI, Prior History of PCI vs CABG
Curator: Aviva Lev-Ari, PhD, RN

Outcomes in High Cardiovascular Risk Patients: Prasugrel (Effient) vs. Clopidogrel (Plavix); Aliskiren (Tekturna) added to ACE or added to ARB
Reporter and Curator: Aviva Lev-Ari, PhD, RN

Endovascular Lower-extremity Revascularization Effectiveness: Vascular Surgeons (VSs), Interventional Cardiologists (ICs) and Interventional Radiologists (IRs)
Curator: Aviva Lev-Ari, PhD, RN

and more

2. The identification of problem areas, particularly in activation of the prothrombotic pathways, infection control to an extent, and targeting of pathways leading to progression or to arrythmogenic complications.

Cardiovascular Complications: Death from Reoperative Sternotomy after prior CABG, MVR, AVR, or Radiation; Complications of PCI; Sepsis from Cardiovascular Interventions Author, Introduction and Summary: Justin D Pearlman, MD, PhD, FACC and Article Curator: Aviva Lev-Ari, PhD, RN

Anticoagulation genotype guided dosing
Larry H. Bernstein, MD, FCAP, Author and Curator

Stent Design and Thrombosis: Bifurcation Intervention, Drug Eluting Stents (DES) and Biodegrable Stents
Curator: Aviva Lev-Ari, PhD, RN

The Effects of Aprotinin on Endothelial Cell Coagulant Biology
Co-Author (Kamran Baig, MBBS, James Jaggers, MD, Jeffrey H. Lawson, MD, PhD) and Curator

Outcomes in High Cardiovascular Risk Patients: Prasugrel (Effient) vs. Clopidogrel (Plavix); Aliskiren (Tekturna) added to ACE or added to ARB
Reporter and Curator: Aviva Lev-Ari, PhD, RN

Pharmacogenomics – A New Method for Druggability  Author and Curator: Demet Sag, PhD

Advanced Topics in Sepsis and the Cardiovascular System at its End Stage    Author: Larry H Bernstein, MD, FCAP

3. Development of procedures that use a safer materials in vascular management.

Stent Design and Thrombosis: Bifurcation Intervention, Drug Eluting Stents (DES) and Biodegrable Stents
Curator: Aviva Lev-Ari, PhD, RN

Biomaterials Technology: Models of Tissue Engineering for Reperfusion and Implantable Devices for Revascularization
Author and Curator: Larry H Bernstein, MD, FACP and Curator: Aviva Lev-Ari, PhD, RN

Vascular Repair: Stents and Biologically Active Implants
Author and Curator: Larry H Bernstein, MD, FACP and Curator: Aviva Lev-Ari, RN, PhD

Drug Eluting Stents: On MIT’s Edelman Lab’s Contributions to Vascular Biology and its Pioneering Research on DES
Author: Larry H Bernstein, MD, FACP and Curator: Aviva Lev-Ari, PhD, RN

MedTech & Medical Devices for Cardiovascular Repair – Curations by Aviva Lev-Ari, PhD, RN

4. Discrimination of cases presenting for treatment based on qualifications for medical versus surgical intervention.

Treatment Options for Left Ventricular Failure – Temporary Circulatory Support: Intra-aortic balloon pump (IABP) – Impella Recover LD/LP 5.0 and 2.5, Pump Catheters (Non-surgical) vs Bridge Therapy: Percutaneous Left Ventricular Assist Devices (pLVADs) and LVADs (Surgical)
Author: Larry H Bernstein, MD, FCAP And Curator: Justin D Pearlman, MD, PhD, FACC

Coronary Reperfusion Therapies: CABG vs PCI – Mayo Clinic preprocedure Risk Score (MCRS) for Prediction of in-Hospital Mortality after CABG or PCI
Writer and Curator: Larry H. Bernstein, MD, FCAP and Curator: Aviva Lev-Ari, PhD, RN

ACC/AHA Guidelines for Coronary Artery Bypass Graft Surgery Reporter: Aviva Lev-Ari, PhD, RN

Mitral Valve Repair: Who is a Patient Candidate for a Non-Ablative Fully Non-Invasive Procedure?
Author, and Content Consultant to e-SERIES A: Cardiovascular Diseases: Justin Pearlman, MD, PhD, FACC and Article Curator: Aviva Lev-Ari, PhD, RN 

5.  This has become possible because of the advances in our knowledge of key related pathogenetic mechanisms involving gene expression and cellular regulation of complex mechanisms.

What is the key method to harness Inflammation to close the doors for many complex diseases?
Author and Curator: Larry H Bernstein, MD, FCAP

CVD Prevention and Evaluation of Cardiovascular Imaging Modalities: Coronary Calcium Score by CT Scan Screening to justify or not the Use of Statin
Curator: Aviva Lev-Ari, PhD, RN

Richard Lifton, MD, PhD of Yale University and Howard Hughes Medical Institute: Recipient of 2014 Breakthrough Prizes Awarded in Life Sciences for the Discovery of Genes and Biochemical Mechanisms that cause Hypertension
Curator: Aviva Lev-Ari, PhD, RN

Pathophysiological Effects of Diabetes on Ischemic-Cardiovascular Disease and on Chronic Obstructive Pulmonary Disease (COPD)
Curator:  Larry H. Bernstein, MD, FCAP

Atherosclerosis Independence: Genetic Polymorphisms of Ion Channels Role in the Pathogenesis of Coronary Microvascular Dysfunction and Myocardial Ischemia (Coronary Artery Disease (CAD))
Reviewer and Co-Curator: Larry H Bernstein, MD, CAP and Curator: Aviva Lev-Ari, PhD, RN

Notable Contributions to Regenerative Cardiology  Author and Curator: Larry H Bernstein, MD, FCAP and Article Commissioner: Aviva Lev-Ari, PhD, RD

As noted in the introduction, any of the material can be found and reviewed by content, and the eTOC is identified in attached:


This completes what has been presented in Part 2, Vol 4 , and supporting references for the main points that are found in the Leaders in Pharmaceutical Intelligence Cardiovascular book.  Part 1 was concerned with Posttranslational Modification of Proteins, vital for understanding cellular regulation and dysregulation.  Part 2 was concerned with Translational Medical Therapeutics, the efficacy of medical and surgical decisions based on bringing the knowledge gained from the laboratory, and from clinical trials into the realm opf best practice.  The time for this to occur in practice in the past has been through roughly a generation of physicians.  That was in part related to the busy workload of physicians, and inability to easily access specialty literature as the volume and complexity increased.  This had an effect of making access of a family to a primary care provider through a lifetime less likely than the period post WWII into the 1980s.

However, the growth of knowledge has accelerated in the specialties since the 1980’s so that the use of physician referral in time became a concern about the cost of medical care.  This is not the place for or a matter for discussion here.  It is also true that the scientific advances and improvements in available technology have had a great impact on medical outcomes.  The only unrelated issue is that of healthcare delivery, which is not up to the standard set by serial advances in therapeutics, accompanied by high cost due to development costs, marketing costs, and development of drug resistance.

I shall identify continuing developments in cardiovascular diagnostics, therapeutics, and bioengineering that is and has been emerging.

1. Mechanisms of disease

REPORT: Mapping the Cellular Response to Small Molecules Using Chemogenomic Fitness Signatures 

Science 11 April 2014:
Vol. 344 no. 6180 pp. 208-211

Abstract: Genome-wide characterization of the in vivo cellular response to perturbation is fundamental to understanding how cells survive stress. Identifying the proteins and pathways perturbed by small molecules affects biology and medicine by revealing the mechanisms of drug action. We used a yeast chemogenomics platform that quantifies the requirement for each gene for resistance to a compound in vivo to profile 3250 small molecules in a systematic and unbiased manner. We identified 317 compounds that specifically perturb the function of 121 genes and characterized the mechanism of specific compounds. Global analysis revealed that the cellular response to small molecules is limited and described by a network of 45 major chemogenomic signatures. Our results provide a resource for the discovery of functional interactions among genes, chemicals, and biological processes.


Laura Zahn
Sci. Signal. 15 April 2014; 7(321): ec103.

In order to identify how chemical compounds target genes and affect the physiology of the cell, tests of the perturbations that occur when treated with a range of pharmacological chemicals are required. By examining the haploinsufficiency profiling (HIP) and homozygous profiling (HOP) chemogenomic platforms, Lee et al.(p. 208) analyzed the response of yeast to thousands of different small molecules, with genetic, proteomic, and bioinformatic analyses. Over 300 compounds were identified that targeted 121 genes within 45 cellular response signature networks. These networks were used to extrapolate the likely effects of related chemicals, their impact upon genetic pathways, and to identify putative gene functions

Key Heart Failure Culprit Discovered

A team of cardiovascular researchers from the Cardiovascular Research Center at Icahn School of Medicine at Mount Sinai, Sanford-Burnham Medical Research Institute, and University of California, San Diego have identified a small, but powerful, new player in thIe onset and progression of heart failure. Their findings, published in the journal Nature  on March 12, also show how they successfully blocked the newly discovered culprit.
Investigators identified a tiny piece of RNA called miR-25 that blocks a gene known as SERCA2a, which regulates the flow of calcium within heart muscle cells. Decreased SERCA2a activity is one of the main causes of poor contraction of the heart and enlargement of heart muscle cells leading to heart failure.

Using a functional screening system developed by researchers at Sanford-Burnham, the research team discovered miR-25 acts pathologically in patients suffering from heart failure, delaying proper calcium uptake in heart muscle cells. According to co-lead study authors Christine Wahlquist and Dr. Agustin Rojas Muñoz, developers of the approach and researchers in Mercola’s lab at Sanford-Burnham, they used high-throughput robotics to sift through the entire genome for microRNAs involved in heart muscle dysfunction.

Subsequently, the researchers at the Cardiovascular Research Center at Icahn School of Medicine at Mount Sinai found that injecting a small piece of RNA to inhibit the effects of miR-25 dramatically halted heart failure progression in mice. In addition, it also improved their cardiac function and survival.

“In this study, we have not only identified one of the key cellular processes leading to heart failure, but have also demonstrated the therapeutic potential of blocking this process,” says co-lead study author Dr. Dongtak Jeong, a post-doctoral fellow at the Cardiovascular Research Center at Icahn School of  Medicine at Mount Sinai in the laboratory of the study’s co-senior author Dr. Roger J. Hajjar.

Publication: Inhibition of miR-25 improves cardiac contractility in the failing heart.Christine Wahlquist, Dongtak Jeong, Agustin Rojas-Muñoz, Changwon Kho, Ahyoung Lee, Shinichi Mitsuyama, Alain Van Mil, Woo Jin Park, Joost P. G. Sluijter, Pieter A. F. Doevendans, Roger J. :  Hajjar & Mark Mercola.     Nature (March 2014)


“Junk” DNA Tied to Heart Failure

Deep RNA Sequencing Reveals Dynamic Regulation of Myocardial Noncoding RNAs in Failing Human Heart and Remodeling With Mechanical Circulatory Support

Yang KC, Yamada KA, Patel AY, Topkara VK, George I, et al.
Circulation 2014;  129(9):1009-21.    …/CIRCULATIONAHA.113.003863.full

The myocardial transcriptome is dynamically regulated in advanced heart failure and after LVAD support. The expression profiles of lncRNAs, but not mRNAs or miRNAs, can discriminate failing hearts of different pathologies and are markedly altered in response to LVAD support. These results suggest an important role for lncRNAs in the pathogenesis of heart failure and in reverse remodeling observed with mechanical support.

Junk DNA was long thought to have no important role in heredity or disease because it doesn’t code for proteins. But emerging research in recent years has revealed that many of these sections of the genome produce noncoding RNA molecules that still have important functions in the body. They come in a variety of forms, some more widely studied than others. Of these, about 90% are called long noncoding RNAs (lncRNAs), and exploration of their roles in health and disease is just beginning.

The Washington University group performed a comprehensive analysis of all RNA molecules expressed in the human heart. The researchers studied nonfailing hearts and failing hearts before and after patients received pump support from left ventricular assist devices (LVAD). The LVADs increased each heart’s pumping capacity while patients waited for heart transplants.

In their study, the researchers found that unlike other RNA molecules, expression patterns of long noncoding RNAs could distinguish between two major types of heart failure and between failing hearts before and after they received LVAD support.

“The myocardial transcriptome is dynamically regulated in advanced heart failure and after LVAD support. The expression profiles of lncRNAs, but not mRNAs or miRNAs, can discriminate failing hearts of different pathologies and are markedly altered in response to LVAD support,” wrote the researchers. “These results suggest an important role for lncRNAs in the pathogenesis of heart failure and in reverse remodeling observed with mechanical support.”

‘Junk’ Genome Regions Linked to Heart Failure

In a recent issue of the journal Circulation, Washington University investigators report results from the first comprehensive analysis of all RNA molecules expressed in the human heart. The researchers studied nonfailing hearts and failing hearts before and after patients received pump support from left ventricular assist devices (LVAD). The LVADs increased each heart’s pumping capacity while patients waited for heart transplants.

“We took an unbiased approach to investigating which types of RNA might be linked to heart failure,” said senior author Jeanne Nerbonne, the Alumni Endowed Professor of Molecular Biology and Pharmacology. “We were surprised to find that long noncoding RNAs stood out.

In the new study, the investigators found that unlike other RNA molecules, expression patterns of long noncoding RNAs could distinguish between two major types of heart failure and between failing hearts before and after they received LVAD support.

“We don’t know whether these changes in long noncoding RNAs are a cause or an effect of heart failure,” Nerbonne said. “But it seems likely they play some role in coordinating the regulation of multiple genes involved in heart function.”

Nerbonne pointed out that all types of RNA molecules they examined could make the obvious distinction: telling the difference between failing and nonfailing hearts. But only expression of the long noncoding RNAs was measurably different between heart failure associated with a heart attack (ischemic) and heart failure without the obvious trigger of blocked arteries (nonischemic). Similarly, only long noncoding RNAs significantly changed expression patterns after implantation of left ventricular assist devices.


Decoding the noncoding transcripts in human heart failure

Xiao XG, Touma M, Wang Y
Circulation. 2014; 129(9): 958960, 

Heart failure is a complex disease with a broad spectrum of pathological features. Despite significant advancement in clinical diagnosis through improved imaging modalities and hemodynamic approaches, reliable molecular signatures for better differential diagnosis and better monitoring of heart failure progression remain elusive. The few known clinical biomarkers for heart failure, such as plasma brain natriuretic peptide and troponin, have been shown to have limited use in defining the cause or prognosis of the disease.1,2 Consequently, current clinical identification and classification of heart failure remain descriptive, mostly based on functional and morphological parameters. Therefore, defining the pathogenic mechanisms for hypertrophic versus dilated or ischemic versus nonischemic cardiomyopathies in the failing heart remain a major challenge to both basic science and clinic researchers. In recent years, mechanical circulatory support using left ventricular assist devices (LVADs) has assumed a growing role in the care of patients with end-stage heart failure.3 During the earlier years of LVAD application as a bridge to transplant, it became evident that some patients exhibit substantial recovery of ventricular function, structure, and electric properties.4 This led to the recognition that reverse remodeling is potentially an achievable therapeutic goal using LVADs. However, the underlying mechanism for the reverse remodeling in the LVAD-treated hearts is unclear, and its discovery would likely hold great promise to halt or even reverse the progression of heart failure.


Efficacy and Safety of Dabigatran Compared With Warfarin in Relation to Baseline Renal Function in Patients With Atrial Fibrillation: A RE-LY (Randomized Evaluation of Long-term Anticoagulation Therapy) Trial Analysis

Circulation. 2014; 129: 951-952​CIR.0000000000000022

In patients with atrial fibrillation, impaired renal function is associated with a higher risk of thromboembolic events and major bleeding. Oral anticoagulation with vitamin K antagonists reduces thromboembolic events but raises the risk of bleeding. The new oral anticoagulant dabigatran has 80% renal elimination, and its efficacy and safety might, therefore, be related to renal function. In this prespecified analysis from the Randomized Evaluation of Long-Term Anticoagulant Therapy (RELY) trial, outcomes with dabigatran versus warfarin were evaluated in relation to 4 estimates of renal function, that is, equations based on creatinine levels (Cockcroft-Gault, Modification of Diet in Renal Disease (MDRD), Chronic Kidney Disease Epidemiology Collaboration [CKD-EPI]) and cystatin C. The rates of stroke or systemic embolism were lower with dabigatran 150 mg and similar with 110 mg twice daily irrespective of renal function. Rates of major bleeding were lower with dabigatran 110 mg and similar with 150 mg twice daily across the entire range of renal function. However, when the CKD-EPI or MDRD equations were used, there was a significantly greater relative reduction in major bleeding with both doses of dabigatran than with warfarin in patients with estimated glomerular filtration rate ≥80 mL/min. These findings show that dabigatran can be used with the same efficacy and adequate safety in patients with a wide range of renal function and that a more accurate estimate of renal function might be useful for improved tailoring of anticoagulant treatment in patients with atrial fibrillation and an increased risk of stroke.

Aldosterone Regulates MicroRNAs in the Cortical Collecting Duct to Alter Sodium Transport.

Robert S Edinger, Claudia Coronnello, Andrew J Bodnar, William A Laframboise, Panayiotis V Benos, Jacqueline Ho, John P Johnson, Michael B Butterworth

Journal of the American Society of Nephrology (Impact Factor: 8.99). 04/2014;     http://dx.

Source: PubMed

ABSTRACT A role for microRNAs (miRs) in the physiologic regulation of sodium transport in the kidney has not been established. In this study, we investigated the potential of aldosterone to alter miR expression in mouse cortical collecting duct (mCCD) epithelial cells. Microarray studies demonstrated the regulation of miR expression by aldosterone in both cultured mCCD and isolated primary distal nephron principal cells.

Aldosterone regulation of the most significantly downregulated miRs, mmu-miR-335-3p, mmu-miR-290-5p, and mmu-miR-1983 was confirmed by quantitative RT-PCR. Reducing the expression of these miRs separately or in combination increased epithelial sodium channel (ENaC)-mediated sodium transport in mCCD cells, without mineralocorticoid supplementation. Artificially increasing the expression of these miRs by transfection with plasmid precursors or miR mimic constructs blunted aldosterone stimulation of ENaC transport.

Using a newly developed computational approach, termed ComiR, we predicted potential gene targets for the aldosterone-regulated miRs and confirmed ankyrin 3 (Ank3) as a novel aldosterone and miR-regulated protein.

A dual-luciferase assay demonstrated direct binding of the miRs with the Ank3-3′ untranslated region. Overexpression of Ank3 increased and depletion of Ank3 decreased ENaC-mediated sodium transport in mCCD cells. These findings implicate miRs as intermediaries in aldosterone signaling in principal cells of the distal kidney nephron.


2. Diagnostic Biomarker Status

A prospective study of the impact of serial troponin measurements on the diagnosis of myocardial infarction and hospital and 6-month mortality in patients admitted to ICU with non-cardiac diagnoses.

Marlies Ostermann, Jessica Lo, Michael Toolan, Emma Tuddenham, Barnaby Sanderson, Katie Lei, John Smith, Anna Griffiths, Ian Webb, James Coutts, John hambers, Paul Collinson, Janet Peacock, David Bennett, David Treacher

Critical care (London, England) (Impact Factor: 4.72). 04/2014; 18(2):R62.

Source: PubMed

ABSTRACT Troponin T (cTnT) elevation is common in patients in the Intensive Care Unit (ICU) and associated with morbidity and mortality. Our aim was to determine the epidemiology of raised cTnT levels and contemporaneous electrocardiogram (ECG) changes suggesting myocardial infarction (MI) in ICU patients admitted for non-cardiac reasons.
cTnT and ECGs were recorded daily during week 1 and on alternate days during week 2 until discharge from ICU or death. ECGs were interpreted independently for the presence of ischaemic changes. Patients were classified into 4 groups: (i) definite MI (cTnT >=15 ng/L and contemporaneous changes of MI on ECG), (ii) possible MI (cTnT >=15 ng/L and contemporaneous ischaemic changes on ECG), (iii) troponin rise alone (cTnT >=15 ng/L), or (iv) normal. Medical notes were screened independently by two ICU clinicians for evidence that the clinical teams had considered a cardiac event.
Data from 144 patients were analysed [42% female; mean age 61.9 (SD 16.9)]. 121 patients (84%) had at least one cTnT level >=15 ng/L. A total of 20 patients (14%) had a definite MI, 27% had a possible MI, 43% had a cTNT rise without contemporaneous ECG changes, and 16% had no cTNT rise. ICU, hospital and 180 day mortality were significantly higher in patients with a definite or possible MI.Only 20% of definite MIs were recognised by the clinical team. There was no significant difference in mortality between recognised and non-recognised events.At time of cTNT rise, 100 patients (70%) were septic and 58% were on vasopressors. Patients who were septic when cTNT was elevated had an ICU mortality of 28% compared to 9% in patients without sepsis. ICU mortality of patients who were on vasopressors at time of cTNT elevation was 37% compared to 1.7% in patients not on vasopressors.
The majority of critically ill patients (84%) had a cTnT rise and 41% met criteria for a possible or definite MI of whom only 20% were recognised clinically. Mortality up to 180 days was higher in patients with a cTnT rise.


Prognostic performance of high-sensitivity cardiac troponin T kinetic changes adjusted for elevated admission values and the GRACE score in an unselected emergency department population.

Moritz BienerMatthias MuellerMehrshad VafaieAllan S JaffeHugo A Katus,Evangelos Giannitsis

Clinica chimica acta; international journal of clinical chemistry (Impact Factor: 2.54). 04/2014;

Source: PubMed

ABSTRACT To test the prognostic performance of rising and falling kinetic changes of high-sensitivity cardiac troponin T (hs-cTnT) and the GRACE score.
Rising and falling hs-cTnT changes in an unselected emergency department population were compared.
635 patients with a hs-cTnT >99th percentile admission value were enrolled. Of these, 572 patients qualified for evaluation with rising patterns (n=254, 44.4%), falling patterns (n=224, 39.2%), or falling patterns following an initial rise (n=94, 16.4%). During 407days of follow-up, we observed 74 deaths, 17 recurrent AMI, and 79 subjects with a composite of death/AMI. Admission values >14ng/L were associated with a higher rate of adverse outcomes (OR, 95%CI:death:12.6, 1.8-92.1, p=0.01, death/AMI:6.7, 1.6-27.9, p=0.01). Neither rising nor falling changes increased the AUC of baseline values (AUC: rising 0.562 vs 0.561, p=ns, falling: 0.533 vs 0.575, p=ns). A GRACE score ≥140 points indicated a higher risk of death (OR, 95%CI: 3.14, 1.84-5.36), AMI (OR,95%CI: 1.56, 0.59-4.17), or death/AMI (OR, 95%CI: 2.49, 1.51-4.11). Hs-cTnT changes did not improve prognostic performance of a GRACE score ≥140 points (AUC, 95%CI: death: 0.635, 0.570-0.701 vs. 0.560, 0.470-0.649 p=ns, AMI: 0.555, 0.418-0.693 vs. 0.603, 0.424-0.782, p=ns, death/AMI: 0.610, 0.545-0.676 vs. 0.538, 0.454-0.622, p=ns). Coronary angiography was performed earlier in patients with rising than with falling kinetics (median, IQR [hours]:13.7, 5.5-28.0 vs. 20.8, 6.3-59.0, p=0.01).
Neither rising nor falling hs-cTnT changes improve prognostic performance of elevated hs-cTnT admission values or the GRACE score. However, rising values are more likely associated with the decision for earlier invasive strategy.


Troponin assays for the diagnosis of myocardial infarction and acute coronary syndrome: where do we stand?

Arie Eisenman

ABSTRACT: Under normal circumstances, most intracellular troponin is part of the muscle contractile apparatus, and only a small percentage (< 2-8%) is free in the cytoplasm. The presence of a cardiac-specific troponin in the circulation at levels above normal is good evidence of damage to cardiac muscle cells, such as myocardial infarction, myocarditis, trauma, unstable angina, cardiac surgery or other cardiac procedures. Troponins are released as complexes leading to various cut-off values depending on the assay used. This makes them very sensitive and specific indicators of cardiac injury. As with other cardiac markers, observation of a rise and fall in troponin levels in the appropriate time-frame increases the diagnostic specificity for acute myocardial infarction. They start to rise approximately 4-6 h after the onset of acute myocardial infarction and peak at approximately 24 h, as is the case with creatine kinase-MB. They remain elevated for 7-10 days giving a longer diagnostic window than creatine kinase. Although the diagnosis of various types of acute coronary syndrome remains a clinical-based diagnosis, the use of troponin levels contributes to their classification. This Editorial elaborates on the nature of troponin, its classification, clinical use and importance, as well as comparing it with other currently available cardiac markers.

Expert Review of Cardiovascular Therapy 07/2006; 4(4):509-14. 


Impact of redefining acute myocardial infarction on incidence, management and reimbursement rate of acute coronary syndromes.

Carísi A Polanczyk, Samir Schneid, Betina V Imhof, Mariana Furtado, Carolina Pithan, Luis E Rohde, Jorge P Ribeiro

ABSTRACT: Although redefinition for acute myocardial infarction (AMI) has been proposed few years ago, to date it has not been universally adopted by many institutions. The purpose of this study is to evaluate the diagnostic, prognostic and economical impact of the new diagnostic criteria for AMI. Patients consecutively admitted to the emergency department with suspected acute coronary syndromes were enrolled in this study. Troponin T (cTnT) was measured in samples collected for routine CK-MB analyses and results were not available to physicians. Patients without AMI by traditional criteria and cTnT > or = 0.035 ng/mL were coded as redefined AMI. Clinical outcomes were hospital death, major cardiac events and revascularization procedures. In-hospital management and reimbursement rates were also analyzed. Among 363 patients, 59 (16%) patients had AMI by conventional criteria, whereas additional 75 (21%) had redefined AMI, an increase of 127% in the incidence. Patients with redefined AMI were significantly older, more frequently male, with atypical chest pain and more risk factors. In multivariate analysis, redefined AMI was associated with 3.1 fold higher hospital death (95% CI: 0.6-14) and a 5.6 fold more cardiac events (95% CI: 2.1-15) compared to those without AMI. From hospital perspective, based on DRGs payment system, adoption of AMI redefinition would increase 12% the reimbursement rate [3552 Int dollars per 100 patients evaluated]. The redefined criteria result in a substantial increase in AMI cases, and allow identification of high-risk patients. Efforts should be made to reinforce the adoption of AMI redefinition, which may result in more qualified and efficient management of ACS.

International Journal of Cardiology 03/2006; 107(2):180-7. · 5.51 Impact Factor


3. Biomedical Engineerin3g

Safety and Efficacy of an Injectable Extracellular Matrix Hydrogel for Treating Myocardial Infarction 

Sonya B. Seif-Naraghi, Jennifer M. Singelyn, Michael A. Salvatore,  et al.
Sci Transl Med 20 February 2013 5:173ra25

Acellular biomaterials can stimulate the local environment to repair tissues without the regulatory and scientific challenges of cell-based therapies. A greater understanding of the mechanisms of such endogenous tissue repair is furthering the design and application of these biomaterials. We discuss recent progress in acellular materials for tissue repair, using cartilage and cardiac tissues as examples of application with substantial intrinsic hurdles, but where human translation is now occurring.

 Acellular Biomaterials: An Evolving Alternative to Cell-Based Therapies

J. A. Burdick, R. L. Mauck, J. H. Gorman, R. C. Gorman,
Sci. Transl. Med. 2013; 5, (176): 176 ps4

Acellular biomaterials can stimulate the local environment to repair tissues without the regulatory and scientific challenges of cell-based therapies. A greater understanding of the mechanisms of such endogenous tissue repair is furthering the design and application of these biomaterials. We discuss recent progress in acellular materials for tissue repair, using cartilage and cardiac tissues as examples of applications with substantial intrinsic hurdles, but where human translation is now occurring.

Instructive Nanofiber Scaffolds with VEGF Create a Microenvironment for Arteriogenesis and Cardiac Repair

Yi-Dong Lin, Chwan-Yau Luo, Yu-Ning Hu, Ming-Long Yeh, Ying-Chang Hsueh, Min-Yao Chang, et al.
Sci Transl Med 8 August 2012; 4(146):ra109. 10.1126/scitranslmed.3003841

Angiogenic therapy is a promising approach for tissue repair and regeneration. However, recent clinical trials with protein delivery or gene therapy to promote angiogenesis have failed to provide therapeutic effects. A key factor for achieving effective revascularization is the durability of the microvasculature and the formation of new arterial vessels. Accordingly, we carried out experiments to test whether intramyocardial injection of self-assembling peptide nanofibers (NFs) combined with vascular endothelial growth factor (VEGF) could create an intramyocardial microenvironment with prolonged VEGF release to improve post-infarct neovascularization in rats. Our data showed that when injected with NF, VEGF delivery was sustained within the myocardium for up to 14 days, and the side effects of systemic edema and proteinuria were significantly reduced to the same level as that of control. NF/VEGF injection significantly improved angiogenesis, arteriogenesis, and cardiac performance 28 days after myocardial infarction. NF/VEGF injection not only allowed controlled local delivery but also transformed the injected site into a favorable microenvironment that recruited endogenous myofibroblasts and helped achieve effective revascularization. The engineered vascular niche further attracted a new population of cardiomyocyte-like cells to home to the injected sites, suggesting cardiomyocyte regeneration. Follow-up studies in pigs also revealed healing benefits consistent with observations in rats. In summary, this study demonstrates a new strategy for cardiovascular repair with potential for future clinical translation.

Manufacturing Challenges in Regenerative Medicine

I. Martin, P. J. Simmons, D. F. Williams.
Sci. Transl. Med. 2014; 6(232): fs16.

Along with scientific and regulatory issues, the translation of cell and tissue therapies in the routine clinical practice needs to address standardization and cost-effectiveness through the definition of suitable manufacturing paradigms.




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Hyperhomocysteinemia interaction with Protein C and Increased Thrombotic Risk

Reporter and Curator: Larry H Bernstein, MD, FCAP


This document explores the relationship between thromboembolic risk related to hyperhomocysteinemia related to the HHcy interaction with and blocking the protective effect of APC.

Previous Venous Thromboembolism Relationships With Plasma Homocysteine Levels

Marco Cattaneo, Franca Franchi, Maddalena L. Zighetti, Ida Martinelli, Daniela Asti, P. Mannuccio Mannucci
Arterioscler Thromb Vasc Biol. 1998;18:1371-1375.
Received January 28, 1998; revision accepted March 16, 1998. From the Angelo Bianchi Bonomi Hemophilia and Thrombosis Center, Institute of Internal Medicine, IRCCS Ospedale Maggiore, University of Milano, Italy.
Correspondence to Marco Cattaneo, MD, Hemophilia and Thrombosis Center, Via Pace 9, 20122 Milano, Italy. E-mail © 1998 American Heart Association, Inc. 1371 Original Contributions


The proteolytic enzyme activated protein C (APC) is a normal plasma component, indicating that protein C (PC) is continuously activated in vivo. High concentrations of homocysteine (Hcy) inhibit the activation of PC in vitro;

  • this effect may account for the high risk for thrombosis in patients with hyperhomocysteinemia (HyperHcy).

We measured the plasma levels of APC in 128 patients with previous venous thromboembolism (VTE) and in 98 age- and sex-matched healthy controls and

  • correlated them with the plasma levels of total Hcy (tHcy) measured before and after an oral methionine loading (PML).

Forty- eight patients had HyperHcy and 80 had normal levels of tHcy. No subject was known to have any of the congenital or acquired thrombophilic states at the time of the study.  Because the plasma levels of APC and PC were correlated in healthy controls,  the APC/PC ratios were also analyzed.

Plasma APC levels and APC/PC ratios were significantly higher in VTE patients than in controls (P < 0.03 and 0.0004, respectively).

  • Most of the increase in APC levels and APC/PC ratios were attributable to patients with HyperHcy.

Patients with normal tHcy had intermediate values, which did not differ significantly from those of healthy controls.

  • There was no correlation between the plasma levels of tHcy or its PML increments and APC or APC/PC ratios in controls.
  • The fasting plasma levels of APC and APC/PC ratios of 10 controls did not increase 4 hours PML, despite a 2-fold increase in tHcy.

This study indicates that

  • APC plasma levels are sensitive markers of activation of the hemostatic system in vivo and
  • that Hcy does not interfere with the activation of PC in vivo.

Key Words: homocysteine, protein C, thromboembolism, activated protein C, hypercoagulability,  T mechanism.

The zymogen protein C is converted to the active protease, activated protein C (APC),

  • through proteolytic cleavage by thrombin bound to its endothelial membrane receptor thrombomodulin.1

The demonstration that APC is a normal plasma component,2,3whose enzymatic activity can be detected with specific and sensitive methods,4,5indicates that

  • the protein C anticoagulant pathway is continuously activated in vivo.

Measurement of APC plasma levels might therefore be helpful in determining the in vivo integrity of the protein C anticoagulant pathway. More generally,

  • APC levels might mirror the in vivo activation of the coagulation system and
  • serve as a marker of thrombin activity in the circulation.4

The mechanism(s) by which a moderate elevation of plasma levels of homocysteine (Hcy) increases the risk for arterial and venous thrombotic disease is still unclear.6,7 In vitro studies showed that

  • Hcy inhibits the thrombomodulin- dependent protein C activation to APC and
  • interferes with the expression of thrombomodulin on human umbilical vein endothelial cells.8–10

These findings may be relevant to unravel the thrombogenic mechanism of Hcy, because

the protein C anticoagulant system is of major physiological importance in the regulation of the hemostatic  congenital or acquired disorders

  • characterized by impaired production or function of APC are associated with a high risk for venous thromboembolism (VTE).11

It must be noted, how ever, that these in vitro findings have been obtained by using very high concentrations of Hcy,

  • at least 1 order of magnitude higher than the plasma concentrations found in patients with homozygous homocystinuria.12,13

Their clinical relevance is therefore uncertain and awaits confirmation from ex vivo and/or in vivo studies in humans. In this study, we compared the plasma levels of APC with those of the prothrombin fragment F1,2, a marker of thrombin generation,14in healthy subjects and patients with previous episodes of VTE and

  • tested whether the levels are affected by plasma Hcy concentrations.



L-Methionine, tri-n-butylphosphine, 7-fluoro-2,1,3-benzoxadiazole- 4-sulfonamide (ABDF), L-cystine, Tween 20, Tween 80, benzamidine, and HEPES were from Sigma. (4-Amidinophenyl)-methanesulfonylfluoride (APMSF) was from Boehringer, BSA from Calbiochem, and the chromogenic substrate L-homocystine, ovalbumin, S-2366 from Chromogenics. The monoclonal antibody directed against the light chain of protein C (C3-Mab) was a kind gift of Dr H.P. Schwarz (Immuno, Vienna, Austria). All other chemicals were of reagent grade. Subjects We studied 128 patients with previous VTE and 98 healthy controls. All diagnoses of thrombotic episodes, excluding those of superficial veins, had been confirmed by objective methods: compression ultrasonography or venography for deep vein thrombosis; and ventilation/perfusion scintigraphy for pulmonary embolism. The contemporary presence of deep vein thrombosis in patients with superficial vein thrombosis had not been excluded by objective methods. Table 1 shows the characteristics of the patients studied.

They belonged to a cohort of 315 patients who had been screened for thrombophilic states at our Center between December 1993 and July 1995 and were selected on the basis of the following characteristics:
(1) absence of congenital or acquired thrombophilic states except hyperhomocysteinemia (HyperHcy) (see below);
(2) oral anticoagu- lant therapy discontinued at least 1 month before screening;
(3) at least 4 months elapsed since the last thrombotic episode; and
(4) willingness to participate in the study.

The screening for thrombophilia included the following tests:

  • prothrombin time;
  • activated partial thromboplastin time;
  • thrombin time;
  • plasma levels of fibrinogen,
  • protein C,
  • protein S, and
  • antithrombin;
  • APC resistance; and
  • screening for antiphospholipid syndrome15 and
  • plasma levels of total homocysteine (tHcy)

before and 4 hours after an oral methionine load. Patients with abnormal APC resistance were also screened for factor V Leiden.16

The study was designed and completed before the demonstration that the mutation G20210A of the prothrombin gene is a risk factor for deep vein thrombosis.17 This mutation therefore was looked for retrospectively only in those subjects whose DNA was still available for analysis (all controls and 50 patients): 5 patients (10%) and 2 controls (2.1%) were heterozygous for the mutation. Of the 128 patients enrolled in the study,

  • 48 had hyperhomocysteinemia (VTE-HyperHcy) according to the diagnostic criteria outlined below, and
  • 80 had normal Hcy levels (VTE-NormoHcy).
    • The healthy controls, who were age and sex matched with the patients (male/female, 50/45; median age, 40 years [range, 20 to 73 years]), had been chosen from the same geographical area and with the same socioeconomic background as the patients.
  1. Previous episodes of thrombosis had been ruled out by a validated structured questionnaire.18
  2. No subject had abnormal liver or renal function, or overt autoimmune or neoplastic disease.
  3. Informed consent to participate in the study was obtained from all subjects.
  4. The study was approved by the ethics committee of the University of Milano.

Study Protocol

After an overnight fast, blood samples were drawn between 8:30 and 9:30 AM in K3-EDTA for measurement of total Hcy (tHcy), in 0.013 mol/L trisodium citrate for measurement of F1?2 and protein C, and in citrate plus 0.03 mol/L benzamidine (a reversible inhibitor of APC) for measurement of APC. L-Methionine (3.8 g/m2body surface area) was then administered orally in approximately 200 mL of orange juice. Four hours later, a second blood sample was collected in EDTA for tHcy measurement from all subjects and in citrate plus benzamidine for measurement of APC plasma levels from 10 controls. All subjects remained in the fasting state until the second blood sample had been taken. Plasma Hcy Assay Blood samples in K3-EDTA were immediately placed on ice and centrifuged at 2000xG, 4°C, for 15 minutes. The supernatant was stored in aliquots at < 70°C until assay.
The plasma levels of tHcy (free and protein bound) were determined by high-performance liquid chromatography (Waters Millipore 6000A pump, Millipore) and fluorescence detection (Waters 474) by the method of Ubbink et al,19with slight modifications.20 Briefly, 100 uL of plasma was incubated with 10 uL of 10% tri-n-butylphosphine in dimethylfor- mamide at 4°C for 30 minutes to reduce homocystine and mixed disulfide and deconjugate Hcy from plasma proteins. Then, 100 uL of 10% trichloroacetic acid was added, and the mixture was centrifuged in an Eppendorf microcentrifuge at 13 000 rpm for 10 minutes.
After centrifugation, the mixture was incubated with 1 mg/mL ABDF in borate buffer to derivatize the thiols. The mobile phase, pumped at 1 mL/min, consisted of 0.1 mol/L potassium dihydrogenophosphate, 0.06 mmol/L EDTA, and 12% acetonitrile (pH = 2.1).

Criteria for Diagnosis of HyperHcy  HyperHcy was diagnosed when
  1. fasting plasma levels of tHcy or its postmethionine load absolute increments above fasting levels exceeded the 95th percentiles of distribution of values obtained in 388 healthy controls.
Measurement of Plasma APC  Plasma APC levels were measured with < enzyme capture assay, essentially as described by Gruber and Griffin.4 Blood samples were

Patients With Previous VTE-NormoHcy

Demographic Characteristics of Patients With Previous VTE-HyperHcy
VTE-HyperHcyVTE-NormoHcy                                                                                                        4880
No. Males/females                                                                                                                                                23/25
Median age, y (range)                                                                                                                                     36 (19–69)
Median age at the first thrombotic episode, y (range)                                                                     32 (17–62)
Time elapsed since last episode, mo (range)                                                                                        14 (4–70)
Time elapsed since discontinuation of oral anticoagulant therapy, mo (range)                   11 (1–64)Type of first thrombotic episode
Deep vein thrombosis                                                                                                                                       31/49
Pulmonary embolism                                                                                                                                    36 (14–62)
Superficial vein thrombosis                                                                                                                       31 (13–60)
Venous thrombosis of other sites                                                                                                           14 (4–90)                                                                                                                                                                          
With 1 or more episodes                                                                                                                              11 (1–70)
2233                                                                                                                                                                    26 (54.2%)
With circumstantial risk factors* at first episode                                                                             44 (55%)
*The following circumstantial risk factors were considered: surgery (26), trauma (50), immobilization (47), pregnancy/puerperium (16,21), and oral contraceptives (22).


Activated Protein C, Thrombosis, and Homocysteine

centrifuged within 60 minutes from collection at 1200xG, 4°C, for 30 minutes to obtain platelet-poor plasma, which was frozen in aliquots at < 70°C. A plasma pool from 30 healthy individuals (15 men, 15 women) was obtained in the same way and used to prepare the standards.
(removed)…  The chromogenic substrate for APC S-2366 (0.46 mmol/L in Tris-buffered saline, pH 7.4) was then added to the wells. After incubation of the sealed plates at 4°C in wet chambers for 3 weeks, hydrolysis of the substrate was monitored at a dual wavelength setting of 405/655 nm.
The concentration of APC in the unknown samples was calculated from the absorbance of each sample with the standard curve as a reference. Results were expressed as percentage of pooled normal plasma. Measurement of Plasma F1?2 F1?2 was assayed by a commercial ELISA (Behringwerke), as previously described.21

Statistical Analysis

The two-tailed t test was used to compare VTE patients and healthy controls. ANOVA was used to compare VTE-HyperHcy, VTE controls, and healthy controls, followed by the Dunnett’s test for internal contrasts. The Pearson r value was calculated for correla- tions between the variables studied.


The results obtained in all VTE patients and controls are presented, including those with the heterozygous G20210A mutation of the prothrombin gene. A subanalysis of the results obtained in the 40 patients and 98 controls, in whom the mutation was looked for, revealed that

  • exclusion of the subjects heterozygous for the mutation did not significantly affect the results.

Plasma tHcy Levels

The mean (SD) fasting levels of plasma tHcy were significantly higher in VTE-HyperHcy (28.8?19.5 ?mol/L) than in VTE-NormoHcy (12.0+5.2, P<0.001) and healthy con- trols (11.0+5.3, P<0.001). The mean postmethionine load increments of tHcy above fasting levels were also higher in VTE-HyperHcy (32.9+13.5 umol/L) than in VTE- NormoHcy (19.8+7.5, P<0.001) and healthy controls (16.1+7.6, P<0.001). Differences between VTE-NormoHcy and healthy controls were not statistically significant. Six healthy controls (6.3%) had HyperHcy, according to the diagnostic criteria previously outlined. Plasma Levels of APC Healthy Controls The mean plasma level of APC in healthy controls was 116(20%). There was a statistically significant correlation between the plasma levels of APC and those of protein C (r?0.48, P?0.001) (Figure 1). Therefore, because APC levels are influenced by the concentration of their zymogen, both the absolute APC levels and the activated protein C/protein C (APC/PC) ratios were used for subsequent analysis. The mean value of the APC/PC ratio in healthy controls was 1.01?0.2.

There was no correlation between the plasma levels of APC (not shown) or the APC/PC ratios and the fasting plasma levels of tHcy (Figure 2) or its postmethionine load increments above fasting levels (not shown). The mean APC plasma levels and APC/PC ratios were similar in healthy controls whose tHcy plasma levels fell within the first (115 and 1.0), second (118 and 0.96), or third (115 and 1.01) tertiles of distribution. The mean fasting plasma levels of APC and the APC/PC ratios of 10 healthy controls

– did not significantly differ from those measured in the same subjects 4 hours after an oral methionine load,
– which increased the concentration of tHcy by more than 2-fold (Table 2).

VTE Patients

The mean plasma levels of APC and APC/PC ratios were higher in VTE patients than in healthy controls (124?32 versus 116?20, P?0.03 and 1.12?0.32 versus 0.99?0.19, P?0.0004). This difference was mostly due to VTE- HyperHcy patients whose plasma APC levels and APC/PC ratios were significantly higher than those of healthy controls (Table 3). In contrast, differences between VTE-NormoHcy and healthy controls and between VTE-HyperHcy and VTE- NormoHcy did not reach statistical significance (Table 3). Results did not change substantially when we excluded patients with thrombosis of the superficial veins (APC levels, 124+26 in VTE-HyperHcy and 121?31 in VTE-NormoHcy; APC/PC ratio, 1.17?0.25 in VTE-HyperHcy and 1.09?0.3 Figure 1. Correlation between the plasma levels of protein C and APC in 98 healthy volunteers. Values are expressed as per- centage of the concentrations measured in pooled normal plasma from 30 healthy blood donors. Figure 2. Correlation between the fasting plasma levels of tHcy and APC/PC ratios of 98 healthy volunteers. Cattaneo et al September 1998 1373 in VTE-NormoHcy) or women taking oral contraceptives (APC levels, 115?19 in controls, 130?29 in VTE- HyperHcy, and 121+33 in VTE-NormoHcy; APC/PC ratio, 0.98?0.23 in controls, 1.13?0.4 in VTE-HyperHcy, and 1.08?0.3 in VTE-NormoHcy). The prevalence of high APC/PC ratios was significantly higher in VTE patients than in controls, independent of the tHcy levels in their plasma (Table 4),

-whereas that of high plasma APC levels was significantly increased in VTE- HyperHcy patients only (Table 4).

Plasma Levels of F1?2

The mean plasma level of F1?2 in VTE patients (1.6?0.5 nmol/L) did not significantly differ from that measured in healthy controls (1.5?0.6 nmol/L). There was no statistically significant difference between plasma levels of F1?2 in VTE-HyperHcy (1.6?0.6 nmol/L), VTE-NormoHcy (1.6?0.6 nmol/L), and healthy controls. The mean F1?2 plasma levels were similar in healthy controls whose plasma levels of tHcy fell within the first, second, or third tertiles of distribution (not shown). F1?2 levels and APC/PC ratios were significantly correlated in controls (r?0.28, P?0.005) but not in VTE-HyperHcy (r? ?0.03, P?0.05) or VTE- NormoHcy (r?0.08, P?0.05).


This study shows that

–  patients with previous episodes of VTE have higher circulating plasma levels of APC than healthy controls, particularly if they have HyperHcy.

The patients studied had none of the known congenital or acquired thrombophilic states, in which

–  the circulating levels of markers of activation of the coagulation system may be increased.21–24Even
– though the recently described G20210A mutation of the prothrombin gene17could be looked for retrospectively in only approximately one third of the pa- tients, also those patients in whom the prothrombin mutation was ruled out had high APC levels,
– excluding that they were mainly due to the presence of the mutation.

APC is generated from its plasma precursor, protein C, on activation by thrombin-thrombomodulin complex on the endothelial cell surface, probably acting in concert with the endothelial cell protein C receptor.1Subcoagulant amounts of thrombin in the circulation may increase the plasma levels of endogenous APC, which can therefore be considered markers of a hypercoagulable state.4Accordingly, the high APC plasma levels that we measured in patients with previous episodes of VTE may be interpreted as an index of ongoing thrombin formation,
despite the fact that at least 4 months (and a median of 14 months) elapsed since their last thrombotic episode. However,

–  the plasma concentrations of F1?2, a marker of thrombin generation, were not increased signifi cantly in the same VTE patients and were not correlated with APC levels or APC/PC ratios.

In contrast to VTE patients, a statistically significant correlation between APC and F1?2 plasma levels was found in healthy controls. On the basis of these data, we hypothesize that

–  the increased plasma levels of APC found in patients with previous episodes of VTE are not caused by heightened thrombin generation but by alternative mechanisms. Although we did not measure markers of activation of the fibrinolytic system,

– the possibility that high plasma levels of plasmin could be responsible for protein C activation25in these patients should be considered.

The greatest increase of APC plasma levels in VTE patients was observed in subjects with fasting and/or postmethionine-loading HyperHcy. VTE patients with nor mal plasma levels of tHcy had lower concentrations of APC than patients with HyperHcy, but this

–  difference could be due to chance alone, because it was not statistically significant. These results contrast with the alleged inhibitory effect of Hcy on protein C activation that was shown in in vitro studies.8–10

Our data obtained in healthy individuals

– support the view that Hcy does not affect protein C activation in vivo, because the
– mean plasma levels of APC of subjects in the highest tertile of distribution of tHcy levels were not different from those of subjects in the lowest tertile. Moreover,
– the rapid increase in plasma tHcy brought about by an oral methionine load did not affect the concentration of circulating APC

TABLE 2. Healthy Controls Before and 4 Hours After Methionine Loading (PML) Plasma Levels tHcy, APC, and APC/PC Ratios in 10 tHcy, ?mol/LAPC, %APC/PC Ratio Baseline 4 h PML* P† 10.5?3.8 29.5?7.6 0.0001 118?43 113?32 0.57 0.98?0.2 0.95?0.1 0.7 Data are mean?SD. *Methionine was given orally at a dose of 3.8 g/m2body surface area. †t test for paired samples. TABLE 4. APC/PC Ratios in Healthy Controls, Patients With Previous VTE-HyperHcy, and Patients With Previous VTE-NormoHcy Prevalences of High Plasma Levels of APC and Subjectsn With High APC LevelsWith High APC/PC Ratio n (%)OR (95% CI) n (%)OR (95% CI) Healthy controls 98 10 (10.2) 1.0 (reference) 10 (10.2) 1.0 (reference) VTE-HyperHcy48 12 (25.0) 2.9 (1.1–8.3) VTE-NormoHcy80 16 (20.0) 2.2 (0.9–5.7) 16 (33.3) 4.4 (1.7–11.4) 22 (27.5) 3.3 (1.4–8.1) CI indicates confidence interval. The cutoff points, which corresponded to the 90th percentiles of distribution among healthy controls, were 143.1% for APC levels and 1.22 for APC/PC ratios.

TABLE 3. Controls, Patients With Previous VTE-HyperHcy, and Patients With Previous VTE-NormoHcy

Plasma Levels of APC and APC/PC Ratios in Healthy Subjects nAPC,* %APC/PC Ratio† Healthy controls VTE-HyperHcy VTE-NormoHcy P (ANOVA) 98 48 80 116?20 128?29 121?33 0.03 0.99?0.19 1.15?0.33 1.10?0.31 0.002 Data are mean?SD. *VTE-HyperHcy versus VTE-NormoHcy (Dunnett’s test), P?NS; VTE- HyperHcy versus healthy controls, P?0.01; VTE-NormoHcy versus healthy controls, P?NS. †VTE-HyperHcy versus VTE-NormoHcy (Dunnett’s test), P?NS; VTE- yperHcy versus healthy controls, P?0.001; VTE-NormoHcy versus healthy controls, P?0.01. 1374

Activated Protein C, Thrombosis, and Homocysteine

Therefore, the results of our study suggest that Hcy does not negatively influence the plasma APC levels and argue against the hypothesis that

– it inhibits the activation of protein C in vivo by interfering with the activity of thrombomodulin.

Recently, Lentz et al,26in an experimental study of mon- keys with diet-induced moderate HyperHcy, showed that

– the thrombin-stimulated endothelium of aortas from hyperhomocysteinemic animals activated protein C in vitro less effectively than that of control animals.

This study, which supports the hypothesis that Hcy interferes with protein C activation, is in apparent contradiction with our results. At least two possible explanations for their different results can be proposed.

First, Hcy would not affect protein C activation that is ongoing in vivo under physiological conditions, whereas it would interfere with its activation at sites at which athero- genic or thrombogenic stimuli injured the endothelium and increased the local concentration of thrombin.
Second, due to the different relative densities of endothelial cell protein C receptor and thrombomodulin on the endothelium of large vessels and capillaries,1the regulation of protein C activation may differ in the two vascular districts. Although Lentz et al26 measured protein C activation by the endothelium of the aorta, we measured circulating APC, which mostly reflects protein C activation occurring in the microcirculation.

On the basis of the considerations above, we speculate that
– Hcy does not interfere with protein C activation ongoing in the micro- circulation under physiological conditions, whereas
– it could inhibit protein C activation on large, injured vessels.

In conclusion, our study shows that APC plasma levels are high in patients with previous episodes of VTE in whom the plasma levels of F1?2 are normal. Therefore, APC plasma levels represent a sensitive marker of activation of the hemostatic system. In addition, the study showed that high Hcy levels are not associated with heightened thrombin generation and do not interfere with the activation of protein C under physiological conditions in vivo. Further studies are needed to unravel the mechanism(s) by which HyperHcy increases the risks for atherosclerosis and thrombosis.


1. Esmon CT, Ding W, Yasuhiro K, Gu J-M, Ferrel G, Regan LM, Stearns- Kurosawa DJ, Kurosawa S, Mather T, Laszik Z, Esmon NL. The protein C pathway: new insights. Thromb Haemost. 1997;78:70–74.
2. Bauer KA, Kass BL, Beeler DL, Rosenberg RD. Detection of protein C activation in humans. J Clin Invest. 1984;74:2033–2041.
3. Heeb MJ, Mosher D, Griffin JH. Inhibition and complexation of activated protein C by two major inhibitors in plasma. Blood. 1989;73:446–454.
4. Gruber A, Griffin JH. Direct detection of activated protein C in blood from human subjects. Blood. 1992;79:2340–2348.
5. Espan ˜a F, Zuazu I, Vicente V, Estelle ´s A, Marco P, Aznar J. Quantifi- cation of circulating activated protein C in human plasma by immuno- assays: enzyme levels are proportional to total protein C levels. Thromb Haemost. 1996;75:56–61.
6. Cattaneo M. Hyperhomocysteinemia: a risk factor for arterial and venous thrombotic disease. Int J Clin Lab Res. 1997;27:139–144.
7. Harpel PC, Zhang X, Borth W. Homocysteine and hemostasis: patho- genetic mechanisms predisposing to thrombosis. J Nutr. 1996;126: 1285S–1289S.
8. Rodgers GM, Conn MT. Homocysteine, an atherogenic stimulus, reduces protein C activation by arterial and venous endothelial cells. Blood. 1990;75:895–901.
9. Lentz SR, Sadler JE. Inhibition of thrombomodulin surface expression and protein C activation by the thrombogenic agent homocysteine. J Clin Invest. 1991;88:1906–1914.
10. Hayashi T, Honda G, Suzuki K. An atherogenic stimulus homocysteine inhibits cofactor activity of thrombomodulin and enhances thrombo- modulin expression in human umbilical vein endothelial cells. Blood. 1992;79:2930–2936.
saee original manuscript for further referencesz  and for figures (not shown)

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Late Onset of Alzheimer’s Disease and One-carbon Metabolism

Reporter and Curator: Dr. Sudipta Saha, Ph.D.


AD (Alzheimer’s disease)

amyloid-beta ()

late onset AD (LOAD)

GSK-3β (glycogen synthase kinase 3-beta)

PP2A (protein phosphatase 2A)

homocysteine (HCY)

S-adenosylmethionine (SAM)

methionine synthase (MS)

betaine-homocysteine methyltransferase (BHMT)

cystathionine beta synthase (CBS)

cysteine (Cys)

glutathione (GSH)

S-adenosylhomocysteine (SAH)

adenosine (Ado)

presenilin 1 (PSEN1)

beta-site APP cleaving enzyme 1 (BACE)

The two main molecular signs of AD are:

  • 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.

Source References:;jsessionid=FE6A683C10230B201295DDF1388DAC68.d02t01

Other articles related to this topic were published on this Open Access Online Scientific Journal, including the following:

Introduction to Nanotechnology and Alzheimer disease

Tilda Barliya PhD, RN 03/14/2013

Alzheimer’s disease conundrum – Are we near the end of the puzzle?

Larry H Bernstein, MD, FCAP, RN 03/09/2013

Ustekinumab New Drug Therapy for Cognitive Decline resulting from Neuroinflammatory Cytokine Signaling and Alzheimer’s Disease

Aviva Lev-Ari, PhD, RN 02/27/2013

The Alzheimer Scene around the Web

Larry H Bernstein, MD, FCAP, Reporter, RN 11/02/2012

Alzheimer’s before Symptoms show: Imaging Techniques for Detection and Pre-Clinical Diagnosis

Aviva Lev-Ari, PhD, RN 09/29/2012

Blood markers for Alzheimer’s disease

Dr. Venkat S Karra, Ph.D., RN 09/05/2012

THREE new drugs for Alzheimer’s Disease: Two Antibodies against AMYLOID and one IV Immune Globulin

Aviva Lev-Ari, PhD, RN 07/17/2012

New ADNI Project to Perform Whole-genome Sequencing of Alzheimer’s Patients,

Aviva Lev-Ari, PhD, RN 07/03/2012

New Bio-markers in Alzheimer’s & Stress Induced Changes in the Brains of Alzheimer’s Patients

Dr. Venkat S Karra, Ph.D., RN 06/26/2012


How Methionine Imbalance with Sulfur-Insufficiency Leads to Hyperhomocysteinemia

Larry H Bernstein, MD, FACP, RN 04/04/2013


Problems of vegetarianism

Dr. Sudipta Saha, Ph.D., RN 04/22/2013


Amyloidosis with Cardiomyopathy

Larry H Bernstein, MD, FACP, RN 03/31/2013


Liver endoplasmic reticulum stress and hepatosteatosis

Larry H Bernstein, MD, FACP, RN 03/10/2013


Assessing Cardiovascular Disease with Biomarkers

Larry H Bernstein, MD, FACP, RN 12/25/2012


Telling NO to Cardiac Risk

Stephen J. Williams, PhD, RN 12/10/2012


A Second Look at the Transthyretin Nutrition Inflammatory Conundrum

Larry H Bernstein, MD, FACP, RN 12/03/2012


Special Considerations in Blood Lipoproteins, Viscosity, Assessment and Treatment

Larry H Bernstein, MD, FACP, RN 11/28/2012


The Molecular Biology of Renal Disorders: Nitric Oxide – Part III

Larry H Bernstein, MD, FACP, RN 11/26/2012


Nitric Oxide Function in Coagulation

Larry H Bernstein, MD, FACP, RN 11/26/2012


The Potential for Nitric Oxide Donors in Renal Function Disorders

Larry H Bernstein, MD, FACP, RN 11/20/2012


Nitric Oxide, Platelets, Endothelium and Hemostasis

Larry H Bernstein, MD, FACP, RN 11/08/2012


Expanding the Genetic Alphabet and linking the genome to the metabolome

Larry H Bernstein, MD, FACP, RN 09/24/2012


Interaction of Nitric Oxide and Prostacyclin in Vascular Endothelium

Larry H Bernstein, MD, FACP, RN 09/14/2012


Positioning a Therapeutic Concept for Endogenous Augmentation of cEPCs — Therapeutic Indications for Macrovascular Disease: Coronary, Cerebrovascular and Peripheral

Aviva Lev-Ari, PhD, RN 08/29/2012


Drug Eluting Stents: On MIT’s Edelman Lab’s Contributions to Vascular Biology and its Pioneering Research on DES

Larry H Bernstein, MD, FACP, RN 04/25/2013


Personalized Medicine in NSCLC

Larry H Bernstein, MD, FACP, RN 03/03/2013


Nitric Oxide and Immune Responses: Part 2

Aviral Vatsa PhD, MBBS, RN 10/28/2012


Mitochondrial Damage and Repair under Oxidative Stress

Larry H Bernstein, MD, FACP, RN 10/28/2012


Is the Warburg Effect the cause or the effect of cancer: A 21st Century View?

Larry H Bernstein, MD, FACP, RN 10/17/2012


Ubiquitin-Proteosome pathway, Autophagy, the Mitochondrion, Proteolysis and Cell Apoptosis: Part III

Larry H Bernstein, MD, FACP, RN 02/14/2012

Special Considerations in Blood Lipoproteins, Viscosity, Assessment and Treatment

Larry H Bernstein, MD, FACP, RN 11/28/2012

Nitric Oxide and iNOS have Key Roles in Kidney Diseases – Part II

Larry H Bernstein, MD, FACP, RN 11/26/2012

New Insights on Nitric Oxide donors – Part IV

Larry H Bernstein, MD, FACP, RN 11/26/2012

The Essential Role of Nitric Oxide and Therapeutic NO Donor Targets in Renal Pharmacotherapy

Larry H Bernstein, MD, FACP, RN 11/26/2012

Paclitaxel vs Abraxane (albumin-bound paclitaxel)

Tilda Barliya PhD, RN 11/17/2012

Ubiquinin-Proteosome pathway, autophagy, the mitochondrion, proteolysis and cell apoptosis

Larry H Bernstein, MD, FACP, RN 10/30/2012

Advances in Separations Technology for the “OMICs” and Clarification of Therapeutic Targets

Larry H Bernstein, MD, FACP, RN 10/22/2012

Nitric Oxide and Immune Responses: Part 1

Aviral Vatsa PhD, MBBS, RN 10/18/2012

Crucial role of Nitric Oxide in Cancer

Ritu Saxena, Ph.D., RN 10/16/2012

Nitric Oxide Covalent Modifications: A Putative Therapeutic Target?

Stephen J. Williams, PhD, RN 09/24/2012

Nitric Oxide Signalling Pathways

Aviral Vatsa, PhD, MBBS, RN 08/22/2012

Proteomics and Biomarker Discovery

Larry H Bernstein, MD, FACP, RN 08/21/2012

The rationale and use of inhaled NO in Pulmonary Artery Hypertension and Right Sided Heart Failure

Larry H Bernstein, MD, FACP, RN 08/20/2012

Bystolic’s generic Nebivolol – positive effect on circulating Endothelial Progenitor Cells endogenous augmentation

Larry H Bernstein, MD, FACP, RN 07/16/2012

The mechanism of action of the drug ‘Acthar’ for Systemic Lupus Erythematosus (SLE)

 Dr. Venkat S. Karra, Ph.D., RN 07/08/2012

Arthritis, Cancer: New Screening Technique Yields Elusive Compounds to Block Immune-Regulating Enzyme

Prabodh Kandala, PhD, RN 05/11/2012

In Focus: Targeting of Cancer Stem Cells

Ritu Saxena, Ph.D, RN 03/27/2013

Novel Cancer Hypothesis Suggests Antioxidants Are Harmful

Ritu Saxena, Ph.D, RN 01/27/2013

What can we expect of tumor therapeutic response?

Larry H Bernstein, MD, FACP, RN 12/05/2012

Nitric Oxide has a ubiquitous role in the regulation of glycolysis -with a concomitant influence on mitochondrial function

Larry H Bernstein, MD, FACP, RN 09/16/2012

Targeting Mitochondrial-bound Hexokinase for Cancer Therapy

Ziv Raviv, PhD, RN 04/06/2013

Genomics-based cure for diabetes on-the-way

Ritu Saxena, Ph.D, RN 03/04/2013

PLATO Trial on ACS: BRILINTA (ticagrelor) better than Plavix® (clopidogrel bisulfate): Lowering chances of having another heart attack

Aviva Lev-Ari, PhD, RN 12/28/2012

Biochemistry of the Coagulation Cascade and Platelet Aggregation – Part I

Larry H Bernstein, MD, FACP, RN 11/26/2012

Mitochondria: Origin from oxygen free environment, role in aerobic glycolysis, metabolic adaptation

Larry H Bernstein, MD, FACP, RN 09/26/2012

Mitochondrial Mechanisms of Disease in Diabetes Mellitus

Aviva Lev-Ari, PhD, RN 08/01/2012

Cardiovascular Disease (CVD) and the Role of Agent Alternatives in endothelial Nitric Oxide Synthase (eNOS) Activation and Nitric Oxide Production

Aviva Lev-Ari, PhD, RN 07/19/2012

Mitochondria: More than just the “powerhouse of the cell”

Ritu Saxena, Ph.D, RN 07/09/2012

Ovarian Cancer and fluorescence-guided surgery: A report

Tilda Barliya PhD, RN 01/19/2013

NO Nutritional remedies for hypertension and atherosclerosis. It’s 12 am: do you know where your electrons are?

Meg Baker, Ph.D., Registered Patent Agent, RN 10/07/2012

High Doses of Certain Dietary Supplements Increase Cancer Risk

Prabodh Kandala, PhD, RN 05/17/2012

Read Full Post »

How Methionine Imbalance with Sulfur-Insufficiency Leads to Hyperhomocysteinemia

Curator: Larry H Bernstein, MD, FACP

The Open Clinical Chemistry Journal, 2011; 4: 34-44 2011/
Bentham Open   Open Access

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


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
    • in underprivileged plant-eating populations
    • regardless of Framingham criteria and vitamin-B status.

Although unrecognized up to now,

  • the nutritional disorder is one of the commonest worldwide,
  • reaching top prevalence in populated regions of Southeastern Asia.

Increased risk of hyperhomocysteinemia and oxidative stress may also affect

  • individuals suffering from intestinal malabsorption or
  • westernized communities having adopted vegan dietary lifestyles.

Vegetarian subjects

  • consuming subnormal amounts of methionine (Met) are characterized by
  • subclinical protein malnutrition causing reduction in size of their lean body mass (LBM) best
  • identified by the serial measurement of plasma transthyretin (TTR).

As a result, the transsulfuration pathway is depressed at cystathionine-beta-synthase (CbS) level

  • triggering the upstream sequestration of homocysteine (Hcy) in biological fluids and
  • promoting its conversion to Met.

Maintenance of beneficial Met homeostasis is

  • counterpoised by the drop of cysteine (Cys) and glutathione (GSH) values downstream to
  • CbS causing in turn declining generation of hydrogen sulfide (H2S) from enzymatic sources.

The biogenesis of H2S via non-enzymatic reduction is further inhibited in areas where

  • earth’s crust is depleted in elemental sulfur (S8) and sulfate oxyanions.

Keywords: Vegetarianism, malnutrition, sulfur-deficiency, hyperhomocysteinemia, oxidative stress, hydrogen sulfide, cardiovascular diseases, developing countries, Asia.

Homocysteine (Hcy) Generated by Transmethylation Pathway and Degraded via Transsulfuration Pathway

Homocysteine (Hcy) is a nonproteogenic sulfur containing amino acid (SAA)

  • generated by the intrahepatic transmethylation (TM) of dietary Met.
  • It may either be recycled to Met following remethylation (RM) pathways or
  • catabolized along the transsulfuration (TS) cascade.

Under normal circumstances, the Met-Hcy cycle stands under the regulatory control of three water soluble B-vitamins:

  • folates (5-methyl-tetrahydrofolates, B9) are regarded as the main factor working as donor of the CH3 group involved in the remethylation process,
  • pyridoxine (pyridoxal-5’-phosphate, PLP, B6) plays the role of co-factor of both
  • cystathionase enzymes belonging to the TS pathway and cobalamins (B12) ensure that of methionine-synthase.

Met-Hcy-Met Cycle

The main steps of the Met _ Hcy _Met cycle are summarized in Fig. (1).


Fig. (1). Schematic representation of the methionine cycle and homocysteine degradation pathways.

Compounds: ATP, adenosyltriphosphate; THF, tetrahydrofolate; SAM, S- adenosylmethionine; SAH, adenosylhomocysteine; Cysta, cystathionine; Cys, cysteine;
GSH, glutathione; H2S, hydrogen sulfide; Tau, taurine; SO4-2 , sulfate oxyanions.
Enzymes: (1) Met-adenosyltransferase; (2) SAM-methyltransferases; (3) adenosyl-homocysteinase; (4) methylene-THF reductase; (5) Metsynthase; (6) cystathionine
, CbS;  (7) cystathionine-b-lyase, CbL; (8) g-glutamyl-synthase; (9) g-glutamyl-transpeptidase; (10)oxidase; (11) reductase; (12) cysteine-dioxygenase, CDO.

Metabolic pathways

Met molecules supplied by dietary proteins are

  • submitted to TM processes
  • releasing Hcy which may in turn either
    • undergo Hcy_Met RM pathways or be
    • irreversibly committed into TS decay.

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 (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

  1. working as an independent and graded risk factor
  2. 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

  1. Cys,
  2. GSH
  3. 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.


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[27]
  • than the Recommended Dietary Allowances (RDAs) (13[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)

  1. this indicator integrates the evolutionary trends outlined by body protein reserves [35],
  2. 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
  1. TM
  2. RM
  3. 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.


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].


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 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 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 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

  1. upstream sequestration of Hcy molecules in biological fluids
  2. 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.


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
    • immune reactions (hyperleukocytosis, diapedesis, phagocytosis).

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].


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


H2S is overproduced in response to neuronal excitation [93], and

  1. increases the sensitivity of N-methyl-D-aspartate (NMDA) reactions to glutamate in hippocampal neurons [23,94].
  2.  improves long-term potentiation, a synaptic model of memory [92,93]
  3. stimulates the inhibitory effects of catalase and superoxide dismutase (SOD) in oxidative stress of endothelial cells [95].
  4.  regulates Ca 2+ homeostasis in microglial cells [96]and it inhibits TNFa expression in microglial cultures [97].
  5.  protects brain cells from neurotoxicity by preventing the rise of ROS in mitochondria [98].


  1. H2S releases vascular smooth muscle,
  2. inhibits platelet aggregation and
  3. reduces the force output of the left ventricule of the heart [18].
  4. maintains vascular smooth muscle tone [66] and
  5. insures protection against arterial hypertension [99].
  6. modifies leucocyte-vascular epithelium interactions in vivo  by
    1. modulating leucocyte adhesion and
    2. diapedesis at the site of inflammation [100].
  7. attenuates myocardial ischemia-reperfusion injury by
    1. depressing IL-1b and mitochondrial function [20].
  8. upregulates the expression of depressed anti-oxidative enzymes in heart infarction and
    1. inhibits myocardial injury [21].
  9. alleviates smooth muscle pain by
    1. stimulating K+ ATP channels [101].
  10. prevents apoptosis of human neutrophil cells
    1. by inhibiting p38 MAP kinase and caspase 3 [102].
  11. potentiates angiogenesis and wound healing [103].


  1. H2S downregulates the increased activity of metalloproteinases 2 and 9 involved in extracellular matrix degradation (elastases, collagenases) [19].
  2. Prevents apoptotic cell death in renal cortical tissues [19].
  3. Improves the expression of desmin (marker of podocyte injury) and
  4. restores the drop of nephrin (component of normal slit diaphragm) in the cortical tissues
    1. resulting in reduced proteinuria [19].
  5. Induces hypometabolism revealing protective effects on renal function and survival [104].
  6. Normalizes GSH status and production of ROS in renal diseases [19].
  7. Controls renal ischemia-reperfusion injury and dysfunction [105].
  8. Depresses the expression of inflammatory molecules involved in glomerulosclerosis [106].
  9. Increases renal blood flow, glomerular filtration and urinary Na+ excretion [77].


  1. H2S insures protection against ROS stress in gastric mucosal epithelia [22].
  2. Accelerates gastric ulcer healing [107].
  3. Reduces gastric injury caused by nonsteroidal anti-inflammatory drugs [108].
  4. Relaxes ileal smooth muscle tone and increases colonic secretions [79].
  5. Attenuates intestinal ischemia-reperfusion injury by increasing SOD and GSH peroxidase status [109].
  6. Stimulates insulin secretion [110] and controls inflammatory events associated with acute pancreatitis [111].
  7. Alleviates hepatic ischemia-reperfusion injury [112].


  1. Prevents lung oxidative stress in hypoxic pulmonary hypertension caused by low GSH content [113].
  2. Promotes SOD and catalase activities and reduces the production of malondialdehyde in oxidative lung injury [114].
  3. Reduces lung inflammation and remodeling in asthmatic animals [115] and in pulmonary hypertension [116].  ..(see OCCJ 2011;4:34-44)

Assessing Protein-Depleted States

  1.  SA is an insensitive marker of protein-depleted states compared to TTR [134]
  2. SA is an indicator of population than of individual protein status in subclinical PEM.
  3. 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].


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].
  1. Intensive agricultural production,
  2. lack of animal manure and
  3. use of fertilizers providing N, K and P substrates
  4. 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].


  1.  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.





Molecular mass (Da.)








Amino acid sequence


4 x 127


Carbohydrate load

18 % glycosylated



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.







Thymidine kinase


transcription of induced DNA into RNA


Alkaline phosphodiesterase I


cleavage of phosphodiester bonds


Tyrosine transaminase


transfer of tyrosine amino group


Tryptophane oxygenase


formylkynurenine and Trp catabolites


Alkaline phosphatase


release of P from phosphoric esters


Phosphoenolpyruvate carboxykinase (liver)


glycolysis from pyruvate and ATP production




dolichol-linked glycosylation of APRs




APR combining with hemoglobin


α1-Anti (chymo) trypsin (α1 AT, α1 ACT)


serpin molecules allowing N-sparing effects


α1-Acid glycoprotein (AGP)


glycosylated APR with antibody-like actions


Serum amyloid protein (SAA)


defense systems against oxidative burst




clotting processes and tissue repair


C-Reactive Protein (CRP)


complement processes and opsonization


Corticosteroid-binding globulin (CBG)


CBG levels, favoring free hypercortisolemia


Phosphoenolpyruvate carboxykinase (adipocytes)


ATP turnover and glycolysis



Primary causal factor

  1. Reduced dietary intake of methionine (39,151,152)
  2. Cytokine-induced tissue breakdown (164,165)

Main clinical conditions

  1. Protein malnutrition,
  2. veganism,
  3. intestinal malabsorption (139,155,156,158-160,281)
  4. Trauma,
  5. sepsis,
  6. burns,
  7. Inflammatory & neoplastic disorders (163,166,170,176,179,180)

Physiopathologic mechanisms

  1. Unachieved LBM replenishment (30,33)
  2. Excessive LBM losses (33,167,179)

Overall protein metabolic status

  1. Downregulated
  2. Upregulated

Plasma biomarker(s) of protein status

  1. Transthyretin (TTR) (144,145)
  2. TTR coupled with CRP or other inflammatory indices (31,177,178,284,285)

Insulin resistance status

  1. Normal or low (286)
  2. Increased in proportion of tissue breakdown (177,178,181-183)

status of Cys-GSH-H2S reducing molecules

  1. Decreased enzymatic and non-enzymatic production (39,161,162,287)
  2. Increased production cancelled out by tissue overconsumption (78,171)

Urinary SO42- and S-compounds

  1. Decreased kidney output (76,78,79)
  2. Variable depending on exogenous SAA supply and
  • extent of tissue breakdown (78,163,168,173)

Transmethylation pathway

  1. Depressed (48,93)
  2. Overstimulated (169)

Remethylation pathway

  1. Stimulated (76,83,153)
  2. Overstimulated (169)

Transsulfuration pathway

  1. Inhibited (49,76,83)
  2. Overstimulated (170,173)


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Received: September 30, 2011 Revised: October 12, 2011 Accepted: October 12, 2011
© Yves Ingenbleek; Licensee Bentham Open.
This is an open access article licensed under the terms of the Creative Commons Attribution Non-Commercial License (
by-nc/3.0/) which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited.

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