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Characterization of homocysteine metabolism in the rat liver
Abstract
Recent evidence suggests that an increased plasma concentration of the sulphur amino acid homocysteine is a risk factor for the development of vascular disease. The tissue(s) responsible for homocysteine production and export to the plasma are not well known. However, given the central role of the liver in amino acid metabolism, we developed a rat primary hepatocyte model in which homocysteine (and cysteine) production and export were examined. The dependence of homocysteine export from incubated hepatocytes on methionine concentration fitted well to a rectangular hyperbola, with half-maximal homocysteine export achieved at methionine concentrations of approx. 0.44 mM. Hepatocytes incubated with 1 mM methionine and 1 mM serine (a substrate for the transulphuration pathway of homocysteine removal) produced and exported significantly less homocysteine (25-40%) compared with cells incubated with 1 mM methionine alone. The effects of dietary protein on homocysteine metabolism were also examined. Rats fed a 60% protein diet had a significantly increased total plasma homocysteine level compared with rats fed a 20% protein diet. In vitro effects of dietary protein were examined using hepatocytes isolated from animals maintained on these diets. When incubated with 1 mM methionine, hepatocytes from rats fed the high protein diet exported significantly more homocysteine compared with hepatocytes from rats fed the normal protein diet. Inclusion of serine significantly lowered homocysteine export in the normal protein group, but the effect was more marked in the high protein group. In vivo effects of serine were also examined. Rats fed a high protein diet enriched with serine had significantly lower total plasma homocysteine (25-30%) compared with controls. These data indicate a significant role for the liver in the regulation of plasma homocysteine levels.
... Homocysteine (Hcys) is produced from S-adenosylhomocysteine (SAH) through the catalysis of (SAH) hydrolase, (Li et al., 2006). Hcys is derived from the essential amino acid methionine, during normal condition, excess Hcys is converted back to methionine or broken down be excreted (Stead et al., 2000). While the remethylation process of Hcys was 5-methyltetrahydrofolate and the activity of betaine homocystein S-methyltransferase (BHMT) as the methyl donor (Finkelstein et al., 1971;Castro et al., 2002 andFinkelstein, 2007). ...
... al, 1997B & 1999. According to Stead et al., 2000, the liver is a key organ of Hcys metabolism and potentially control the plasma Hcys level. Betaine was involved in Hcys metabolism as an alternative methyl donor, it is used in the treatment of homocystineuria in human as revealed by Schwab et al., 2002. ...
... On the other hand, some studies reported that the levels of homocysteine were either reduced or normal under diabetic conditions [11,12]. These inconsistent results seem to be attributable to varying extents of hepatic and renal dysfunction [11,13], the methionine content of the diet [14,15], and the clinical status of diabetic disease [12,16,17]. Here, we focused on the fact that a high-fat diet affects the metabolism of both methionine and homocysteine in a diabetic rat model [14]. ...
... The K SH value of ZDF rats was higher than that of controls, which meant that upregulation of the insulin-signaling pathway induced the increased SAHH activity [23]. In addition, our model showed that elevated levels of enzymes catalyzing homocysteine production and recycling to methionine, corresponding with previous studies conducted using diabetic in vivo rat models [13,24]. Among all catalyzing enzymes, BHMT played a significant role in catalyzing remethylation of homocysteine to methionine. ...
Background
Although alterations in the methionine metabolism cycle (MMC) have been associated with vascular complications of diabetes, there have not been consistent results about the levels of methionine and homocysteine in type 2 diabetes mellitus (T2DM). The aim of the current study was to predict changes in plasma methionine and homocysteine concentrations after simulated consumption of methionine-rich foods, following the development of a mathematical model for MMC in Zucker Diabetic Fatty (ZDF) rats, as a representative T2DM animal model.
Method
The model building and simulation were performed using NONMEM® (ver. 7.3.0) assisted by Perl-Speaks-NONMEM (PsN, ver. 4.3.0). Model parameters were derived using first-order conditional estimation method with interactions permitted among the parameters (FOCE-INTER). NCA was conducted using Phoenix (ver. 6.4.0). For all tests, we considered a P-value < 0.05 to reflect statistical significance.
Results
Our model featured seven compartments that considered all parts of the cycle by applying non-linear mixed effects model. Conversion of S-adenosyl-L-homocysteine (SAH) to homocysteine increased and the metabolism of homocysteine was reduced under diabetic conditions, and consequently homocysteine accumulated in the elimination phase.
Using our model, we performed simulations to compare the changes in plasma methionine and homocysteine concentrations between ZDF and normal rats, by multiple administrations of the methionine-rich diet of 1 mmol/kg, daily for 60 days. The levels of methionine and homocysteine were elevated approximately two- and three-fold, respectively, in ZDF rats, while there were no changes observed in the normal control rats.
Conclusion
These results can be interpreted to mean that both methionine and homocysteine will accumulate in patients with T2DM, who regularly consume high-methionine foods.
... For instance, three amino acids (glycine, L-threonine, and serine) that are involved in one-carbon metabolism-the central pathway that facilitates arsenic methylation and elimination-were inversely related to water or urinary arsenic levels. Glycine and serine participate in the metabolism of methionine as a methyl-group acceptor and as a substrate for cystathionine synthesis, respectively (Benevenga and Harper 1970;Stead et al. 2000). Both animal and human studies (Benevenga and Harper 1970;Fukada et al. 2006;Girard-Globa et al. 1972;Stead et al. 2000;Verhoef et al. 2004) have shown that serine can lower homocysteine-a risk factor for CVD. ...
... Glycine and serine participate in the metabolism of methionine as a methyl-group acceptor and as a substrate for cystathionine synthesis, respectively (Benevenga and Harper 1970;Stead et al. 2000). Both animal and human studies (Benevenga and Harper 1970;Fukada et al. 2006;Girard-Globa et al. 1972;Stead et al. 2000;Verhoef et al. 2004) have shown that serine can lower homocysteine-a risk factor for CVD. Glycine is synthesized endogenously from serine, threonine, choline, or glyoxylate in the liver and kidney (Wang et al. 2013). ...
Background:
Chronic exposure to inorganic arsenic from drinking water has been associated with a host of cancer and noncancer diseases. The application of metabolomics in epidemiologic studies may allow researchers to identify biomarkers associated with arsenic exposure and its health effects.
Objective:
Our goal was to evaluate the long-term reproducibility of urinary metabolites and associations between reproducible metabolites and arsenic exposure.
Methods:
We studied samples and data from 112 nonsmoking participants (58 men and 54 women) who were free of any major chronic diseases and who were enrolled in the Health Effects of Arsenic Longitudinal Study (HEALS), a large prospective cohort study in Bangladesh. Using a global gas chromatography-mass spectrometry platform, we measured metabolites in their urine samples, which were collected at baseline and again 2 y apart, and estimated intraclass correlation coefficients (ICCs). Linear regression was used to assess the association between arsenic exposure at baseline and metabolite levels in baseline urine samples.
Results:
We identified 2,519 molecular features that were present in all 224 urine samples from the 112 participants, of which 301 had an ICC of ≥0.60. Of the 301 molecular features, water arsenic was significantly related to 31 molecular features and urinary arsenic was significantly related to 74 molecular features after adjusting for multiple comparisons. Six metabolites with a confirmed identity were identified from the 82 molecular features that were significantly associated with either water arsenic or urinary arsenic after adjustment for multiple comparisons.
Conclusions:
Our study identified urinary metabolites with long-term reproducibility that were associated with arsenic exposure. The data established the feasibility of using metabolomics in future larger studies. https://doi.org/10.1289/EHP1992.
... In human 9 omnivores, one-half of Cr required is provided in the diet and the remainder is endogenously 10 synthesized [6]. Recent studies have shown Cr synthesis and Hcy formation are metabolically 11 connected ( Figure 1) [7,8]. Cr synthesis involves the reversible transfer of the amidino group 12 of arginine to glycine to form guanidinoacetic acid (GAA) and ornithine in a reaction 13 catalyzed by the enzyme arginine: glycine amidinotransferase (AGAT), which is very active 14 in kidneys. ...
... In Studies have demonstrated that Cr synthesis is responsible for considerable SAM 6 consumption in the liver and Hcy formation [6]. Stead et al. [8] based on studies using stable 7 isotopes to determine the methylation flux, demonstrated that the synthesis of 8 phosphatidylcholine and Cr are both the major consumers of SAM in the liver; and Cr is 9 responsible for at least 40% of the methylation demand and consequently for Hcy formation. were modulated by Cr and GAA intake, using rats receiving either 0.4% Cr, 0.36% GAA or a 17 control diet. ...
Background: Increasing evidence has shown that an elevated a level of homocysteine (Hcy) in the blood is related to several diseases. Over the last few years, studies have demonstrated creatine (Cr) synthesis and Hcy formation are metabolically connected; and Cr supplementation can decrease Hcy blood levels in different situations. This data however is inconsistent and still controversial.
Objective: The aim of this critical review with meta-analysis was to discuss and ascertain the effects of Cr supplementation on blood Hcy levels.
Method: A review was conducted according to the PRISMA guidelines using PubMed Discuss and Scielo online databases to identify relevant studies through November 2015. RevMan was used to calculate the effect size of the change in Hcy plasma/serum concentration from baseline to post-supplementation with Cr vs. placebo groups. Weighted mean differences were calculated using random effect models.
Results: Cr supplemented trials were divided into two subgroups according to whether the experimental design included animals or humans participants. Overall, 14 studies were included in the meta-analysis. The six rodent included studies reported decreased plasma Hcy concentration after Cr supplementation with a mean effect size equal to −2.43 μmol/l (95% CI: 3.60, −1.26, P
... Também a menor ingestão protéica parece causar o aumento de homocisteína sérica no plasma de ratos, como demonstrado em um estudo de Okawa et al. (2006) 5 , o qual discorda de outros dados reportados por Stead et al. (2000) 6 , que verificaram que o aumento de homocisteína está ligado a uma maior ingestão protéica, já Smolin et al. (1984) 7 reportaram que a concentração de homocisteína não apresentou diferenças entre animais alimentados com 10% ou 60% de caseína. A suplementação de creatina isoladamente demonstrou ser outro fator que pode elevar a homocisteína sérica 8 . ...
... Também a menor ingestão protéica parece causar o aumento de homocisteína sérica no plasma de ratos, como demonstrado em um estudo de Okawa et al. (2006) 5 , o qual discorda de outros dados reportados por Stead et al. (2000) 6 , que verificaram que o aumento de homocisteína está ligado a uma maior ingestão protéica, já Smolin et al. (1984) 7 reportaram que a concentração de homocisteína não apresentou diferenças entre animais alimentados com 10% ou 60% de caseína. A suplementação de creatina isoladamente demonstrou ser outro fator que pode elevar a homocisteína sérica 8 . ...
Model of study: Experimental study. Objectives: this study had as objective evaluate and characterize a low protein diet as an experimental model for nonalcoholic fatty liver disease (NAFLD). Methods: male Wistar rats were divided in two groups with isocaloric diets: control (GC) in which the used diet followed praised for the AIN-93 and the low protein (GH), with reduced amount of protein of 20% for 10%. The diets and water had been offered ad libitum over four weeks. After this period the animals were sacrificed and analyzed: glycemia; urinary nitrogen; serum protein; liver total lipids; cholesterol; weight variation and food consumed. Results: the glycemia and the urinary nitrogen had not presented significant differences between GC and GH (p>0,05), The change of weight in the last day of the experiment was significant (p<0,02). The percentage of total liver lipids was higher in the GH, when compared with the GC (p<0,04). There was lower levels of cholesterol (p<0,01) and serum protein (0,005) in GH. The food consumed was not different between the groups. Conclusions: in this paper the low protein diet constitutes a model of NAFLD induction that can be characterized for serum protein and plasmatic cholesterol and increased fat in the liver, however not alterations in the glycemia suggest no changes in insulin sensitivity, thus constituting a defective model to study one of the main factors of risk for the establishment of the NAFLD, the resistance to insulin.
... Earlier studies demon- strating the combined effects of folic acid and riboflavin were performed using lymphocytes [8,9]. The liver is reportedly the major site of methionine catabolism [10], and hepatocytes can make a major contribution to the plasma Hcy level [11]. Though folate and riboflavin presumably interact in hepatocytes, no prior study has yet used hepatocytes to examine this interaction. ...
... To our knowledge, this is the first study to examine the effects of riboflavin alone and combined with 5-MTHF or THF on changes in the levels of Hcy and folate de- rivatives in rat primary hepatocytes. In prior studies us- ing rat hepatocytes, significant amounts of methionine were added to EMEM so that Hcy could be recovered [14] and the dependence of Hcy export on methionine levels in rat hepatocytes in Krebs-Henseleit medium [11] has been reported. ...
... While we did not quantify hepatic homocysteine in our study, other studies show that it does not change in rats on a SAAR diet (Tamanna et al., 2018). On the other hand, homocysteine export was reported to be sensitive to Ser availability and the ratio of S-adenosylmethionine to S-adenosylhomocysteine (Melnyk et al., 2000;Stead et al., 2000). Overall, due to a lack of data on the hepatic levels of these compounds, it is difficult to ascertain any definitive role for increased Ser in homocysteine regulation. ...
Decreasing the dietary intake of methionine exerts robust anti-adiposity effects in rodents but modest effects in humans. Since cysteine can be synthesized from methionine, animal diets are formulated by decreasing methionine and eliminating cysteine. Such diets exert both methionine restriction (MR) and cysteine restriction (CR), that is, sulfur amino acid restriction (SAAR). Contrarily, SAAR diets formulated for human consumption included cysteine, and thus might have exerted only MR. Epidemiological studies positively correlate body adiposity with plasma cysteine but not methionine, suggesting that CR, but not MR, is responsible for the anti-adiposity effects of SAAR. Whether this is true, and, if so, the underlying mechanisms are unknown. Using methionine-and cysteine-titrated diets, we demonstrate that the anti-adiposity effects of SAAR are due to CR. Data indicate that CR increases serinogenesis (serine biosynthesis from non-glucose substrates) by diverting substrates from glyc-eroneogenesis, which is essential for fatty acid reesterification and triglyceride synthesis. Molecular data suggest that CR depletes hepatic glutathione and induces Nrf2 and its downstream targets Phgdh (the serine biosynthetic enzyme) and Pepck-M. In mice, the magnitude of SAAR-induced changes in molecular markers depended on dietary fat concentration (60% fat >10% fat), sex (males > females), and age-at-onset (young > adult). Our findings are translationally relevant as we found negative and positive correlations of plasma serine and cysteine, respectively, with triglycerides and metabolic syndrome criteria in a cross-sectional epidemiological study. Controlled feeding of low-SAA, high-polyunsaturated fatty acid diets increased plasma serine in humans. Serinogenesis might be a target for treating hypertriglyceridemia.
... It is assumed that supplying L-serine will enhance fluxes of homocysteine through the methionine cycle and transsulfuration pathway (Sections 5.2 and 5.3), which may subsequently reduce homocysteine levels and have therapeutic utility. L-serine decreased homocysteine synthesis in rat hepatocytes incubated with 1 mM methionine [83], reduced plasma homocysteine levels in hyperhomocysteinemia induced by high methionine diet in both rats and humans [84][85][86], and decreased homocysteine levels in mice with alcoholic fatty liver [87]. ...
L-serine plays an essential role in a broad range of cellular functions including protein synthesis, neurotransmission, and folate and methionine cycles and synthesis of sphingolipids, phospholipids, and sulphur containing amino acids. A hydroxyl side-chain of L-serine contributes to polarity of proteins, and serves as a primary site for binding a phosphate group to regulate protein function. D-serine, its D-isoform, has a unique role. Recent studies indicate increased requirements for L-serine and its potential therapeutic use in some diseases. L-serine deficiency is associated with impaired function of the nervous system, primarily due to abnormal metabolism of phospholipids and sphingolipids, particularly increased synthesis of deoxysphingolipids. Therapeutic benefits of L-serine have been reported in primary disorders of serine metabolism, diabetic neuropathy, hyperhomocysteinemia, and amyotrophic lateral sclerosis. Use of L-serine and its metabolic products, specifically D-serine and phosphatidylserine, has been investigated for the therapy of renal diseases, central nervous system injury, and in a wide range of neurological and psychiatric disorders. It is concluded that there are disorders in which humans cannot synthesize L-serine in sufficient quantities, that L-serine is effective in therapy of disorders associated with its deficiency, and that L-serine should be classified as a “conditionally essential” amino acid.
... Печень играет ключевую роль в метаболизме метионина и гомоцистеина [15,18]. Изменение режима питания (потребление продуктов, богатых метионином), прием ряда лекарственных препаратов (например, в спорте, бодибилдинге) приводят к изменению состояния печеночной ткани, следствием которых является повышение уровня гомоцистеина в крови (гипергомоцистеинемия, ГГЦ). ...
The aim of the study was to analyze the microvasculation of the liver in administration of tautomeric forms of orotic acid for a methionine-induced liver pathology to assess the efficacy of the modified drugs and the potentialof their clinical use.
Material and methods. The study included 30 white outbred rats, that were simulated methionine induced hyperhomocysteinemia by the administration of methionine, dosage 0.15 g/100 g of the body we ight, via food during 4 weeks. Animals with hyperhomocysteinemia were divided into 4 groups: animals of group 1 received orotic acid (OA) in the initial form (oxo-tautomer), animals of group 2 received hydroxy–tautomer OA after mechanomodification for 1 hour, animals of group 3 received dihydroxy-tautomer OA after mechanomodification for 6 hours. Histological preparations of the liver were used to evaluate the area of hepatocytes; diameters and areas of the central vein, the interlobular vein and artery, the bile duct, and the Visotto coefficient were calculated.
Results. Morphometric data of the hepatic microvascular bloodstream with simulated hyperhomocysteinemia evidenced a general increase in the diameter and area of blood vessels, changes affected the system of blood inflow and outflow at the organ level. Administration of OA had a normalizing effect on the liver bloodflow, but the effects were different: the most pronounced effect was detected in administration of the hydroxy-form of the preparation, this can be explained by a modificationin the dispersion of the preparation (without changing the crystal lattice), an increase in the rate of dissolution in water and aqueous solutions, an increasein the number of functionally active groups in the heterocycle of the hydroxy-form of OA. Thus, the hydroxy-tautomer of orotic acid had the greatest efficacy relating to vasodilation of microvessels of the liver bloodstream in hyperhomocysteinemia; the fact suggesting feasibility of its further study in the clinical environment.
... The liver is a major organ that maintains Hcy homeostasis in humans [112] and animals [113]. An impairment of the liver function (e.g., by alcohol) elevates Hcy and causes hepatic steatosis, colloquially called fatty liver [114,115]. ...
Introduction
Homocystinuria due to cystathionine β-synthase (CBS) deficiency, the most frequent inborn error of sulfur amino acid metabolism, is characterized biochemically by severely elevated homocysteine (Hcy) and related metabolites, such as Hcy-thiolactone and N-Hcy-protein. CBS deficiency reduces life span and causes pathological abnormalities affecting most organ systems in the human body, including the cardiovascular (thrombosis, stroke), skeletal/connective tissue (osteoporosis, thin/non-elastic skin, thin hair), and central nervous systems (mental retardation, seizures), as well as the liver (fatty changes), and the eye (ectopia lentis, myopia). Molecular basis of these abnormalities were largely unknown and available treatments remain ineffective.
Areas covered
Proteomic and transcriptomic studies over the past decade or so, have significantly contributed to our understanding of mechanisms by which the CBS enzyme deficiency leads to clinical manifestations associated with it.
Expert opinion
: Recent findings, discussed in this review, highlight the involvement of dysregulated proteostasis in pathologies associated with CBS deficiency, including thromboembolism, stroke, neurologic impairment, connective tissue/collagen abnormalities, hair defects, and hepatic toxicity. To ameliorate these pathologies, pharmacological, enzyme replacement, and gene transfer therapies are being developed.
... The molecular mechanisms through which the Met cycle is disrupted have been identified in animal experiments using a specific BHMT chemical inhibitor [126], leading to upstream sequestration of Hcy proportional to the duration and severity of the causal factor. Elevated Hcy levels in body fluids operate as precursor pools rapidly taken up by liver parenchymal cells to undergo intrahepatic RM of Hcy to novel Met molecules [127] which are exported back to the site of injury. The RM process is controlled by cystathionine-ß-synthase governing the transsulfuration pathway, which maintains Met homeostasis in LBM tissues [125] at the expense of increased Hcy-dependent toxicity [94]. ...
Skeletal muscle (SM) mass, the chief component of the structural compartment belonging to lean body mass (LBM), undergoes sarcopenia with increasing age. Decreased SM in elderly persons is a naturally occurring process that may be accelerated by acute or chronic nutritional deficiencies and/or inflammatory disorders, declining processes associated with harmful complications. A recently published position paper by European experts has provided an overall survey on the definition and diagnosis of sarcopenia in elderly persons. The present review describes the additional contributory role played by the noninvasive transthyretin (TTR) micromethod. The body mass index (BMI) formula is currently used in clinical studies as a criterion of good health to detect, prevent, and follow up on the downward trend of muscle mass. The recent upsurge of sarcopenic obesity with its multiple subclasses has led to a confused stratification of SM and fat stores, prompting workers to eliminate BMI from screening programs. As a result, investigators are now focusing on indices of protein status that participate in SM growth, maturation, and catabolism that might serve to identify sarcopenia trajectories. Plasma TTR is clearly superior to all other hepatic biomarkers, showing the same evolutionary patterns as those displayed in health and disease by both visceral and structural LBM compartments. As a result, this TTR parameter maintains positive correlations with muscle mass downsizing in elderly persons. The liver synthesis of TTR is downregulated in protein-depleted states and suppressed in cytokine-induced inflammatory disorders. TTR integrates the centrally-mediated regulatory mechanisms governing the balance between protein accretion and protein breakdown, emerging as the ultimate indicator of LBM resources. This review proposes the adoption of a gray zone defined by cut-off values ranging from 200 mg/L to 100 mg/L between which TTR plasma values may fluctuate and predict either the best or the worst outcome. The best outcome occurs when appropriate dietary, medicinal and surgical decisions are undertaken, resuming TTR synthesis which manifests rising trends towards pre-stress levels. The worst occurs when all therapeutic means fail to succeed, leading inevitably to complete exhaustion of LBM and SM metabolic resources with an ensuing fatal outcome. Some patients may remain unresponsive in the middle of the gray area, combining steady clinical states with persistent stagnant TTR values. Using the serial measurement of plasma TTR values, these last patients should be treated with the most aggressive and appropriate therapeutic strategies to ensure the best outcome.
... In addition, serine supplementation also increased the content of cysteine, which is one of the major determinants of GSH synthesis. These results were in lines with previous studies which found that serine supplementation increased expression of the gene encoding glutathione peroxidase [29], and serine also condensed with Hcy to form cysteine in vitro [30] and increased GSH content [24]. These effects suggested serine supplementation could prevent Hcy accumulation and protect the liver against HFD-induced dysfunction of the GSH antioxidant defense system, which often leads to severe oxidative stress and Cpt1a, carnitine palmitoyltransferase 1a; Acadm, medium-chain acyl-CoA dehydrogenase; DGAT, diacylglycerol O-acyltransferase; AMPKα, AMP-activated protein kinase, α subunit; pAMPKα, phosphor-Thr 172 -AMPKα; nrf2, nuclear factor-erythroid 2-related factor-2. ...
Serine deficiency has been observed in patients with nonalcoholic fatty liver disease (NAFLD). Whether serine supplementation has any beneficial effects on the prevention of NAFLD remains unknown. The present study was conducted to investigate the effects of serine supplementation on hepatic oxidative stress and steatosis and its related mechanisms. Forty male C57BL/6J mice (9week-old) were randomly assigned into four groups (n=10) and fed: i) a low-fat diet; ii) a low-fat diet supplemented with 1% (wt:vol) serine; iii) a high-fat (HF) diet; and iv) a HF diet supplemented with 1% serine, respectively. Palmitic acid (PA)-treated primary hepatocytes separated from adult mice were also used to study the effects of serine on oxidative stress. The results showed that serine supplementation increased glucose tolerance and insulin sensitivity, and protected mice from hepatic lipid accumulation, but did not significantly decreased HF diet-induced weight gain. In addition, serine supplementation protected glutathione (GSH) antioxidant system and prevented hypermethylation in the promoters of glutathione synthesis-related genes, while decreasing reactive oxygen species (ROS) in mice fed a HF diet. Moreover, we found that serine supplementation increased phosphorylation and S-glutathionylation of AMP-activated protein kinase α subunit (AMPKα), and decreased ROS, malondialdehyde and triglyceride contents in PA-treated primary hepatocytes. However, while AMPK activity or GSH synthesis was inhibited, the abovementioned effects of serine on PA-treated primary hepatocytes were not observed. Our results suggest that serine supplementation could prevent HF diet-induced oxidative stress and steatosis by epigenetically modulating the expression of glutathione synthesis-related genes and through AMPK activation.
... Based on these animal experiments, we postulate that each LBM component-with the sole exception of renal tissues endowed with normal BHMT activity [14]-may only locally trigger a sluggish inflammatory response due to poor MS reactivity and blunt RM aptitude to restore intracellular Met losses. The bulk of Hcy flooding into the extracellular space is eliminated in the kidneys [85], whereas the remaining fraction is removed by hepatocytes and redirected either toward RM processes to provide additional Met sources or channeled along the TS pathway to yield Cys [86] under the regulatory control of CBS and CGL. We postulate that quiescent BHMT behavior in LBM tissues followed by the substantial release of Hcy fluxes during inflammatory disorders supports the sensing mechanism concept. ...
Protein-depleted states generate allosteric inhibition of liver cystathionine β-synthase (CBS), which governs the first enzymatic step of the transsulfuration cascade, resulting in upstream accretion of homocysteine (Hcy) in body fluids. A similar Hcy increase may arise from normal hepatocytes undergoing experimentally-induced impairment of betaine-homocysteine methyltransferase (BHTM) activity or from components of lean body mass (LBM) submitted to any inflammatory disorder. LBM comprises a composite agglomeration of extrarenal tissues characterized by naturally occurring BHTM inactivity. As a result of cellular injury, LBM releases high concentrations of Hcy into the extracellular space, contrasting with the disruption of normal remethylation pathways. Hyperhomocysteinemia acts as a biomarker, reflecting the severity of insult and operating as an alarm signal. Elevated Hcy levels constitute a precursor pool recognized by a CBS coding region that reacts to meet increased methionine requirements in LBM tissues, using its enhanced production in hepatocytes. Preservation of methionine homeostasis benefits from its high metabolic priority and survival value.
... On the one hand, serine condenses with Hcy to form cysteine and this reaction also helps remove the accumulated Hcy by increasing transsulfuration pathway. These effects of serine have been clearly proved in previous studies [9,[35][36][37] and our results further confirmed it. On the other hand, the reaction catalyzed by SHMT1 not only provides glycine for GSH synthesis, but also donates a one-carbon unit to support the methionine cycle through 5,10-methylenetetrahy-drofolate (CH 3 -THF). ...
Scope:
serine lies at the central node linking biosynthetic flux from glycolysis to glutathione synthesis and one-carbon metabolic cycle which are closely related to antioxidant capacity. The present study was conducted to determine the effects of serine supplementation on oxidative stress and its relative mechanisms.
Methods and results:
diquat treatment was performed to induce oxidative stress in mice and primary hepatocytes. The results showed that hepatic glutathione anti-oxidant systems were impaired and reactive oxygen species and homocysteine were increased in diquat-induced mice and hepatocytes, while such disadvantageous changes were diminished by serine supplementation both in vivo and in vitro. However, when cystathionine β-synthase expression was inhibited by interference RNA in hepatocytes, the effects of serine supplementation on the improvement of glutathione synthesis and the alleviation of oxidative stress were diminished. Moreover, when hepatocytes were treated with cycloleucine, an inhibitor of methionine adenosyltransferase, the effects of serine supplementation on the improvement of methionine cycle and the alleviation of DNA hypomethylation and oxidative stress were also diminished.
Conclusion:
our results indicated that serine supplementation alleviated oxidative stress via supporting glutathione synthesis and methionine cycle, mostly by condensing with homocysteine to synthesize cysteine and providing one-carbon units for homocysteine remethylation. This article is protected by copyright. All rights reserved.
... Homocysteine (hcy) is a critical intermediate of methionine metabolism and has profound importance in health and diseases. Obviously, the metabolism of hcy mainly takes place in liver and growing evidence also indicates that chronic liver injury is closely associated with an impairment of hepatic sulfur-containing amino acid metabolism, which is attributed to abnormality in the critical enzymes involved in the metabolic reactions including cystathionine beta synthase (CBS), the first enzyme involved in the transsulfuration pathway [7][8][9]. We characterized hepatic lesions in hhcy mice due to CBS deficiency [10] and demonstrated that hhcy alters activities of phase I (paraoxonase 1, PON1) and phase II (NAD(P)H: quinone oxidoreductase, NQO1) xenobiotic metabolizing enzymes (XMEs). ...
Alcoholic liver diseases arise from complex phenotypes involving many genetic factors. It is quite common to find hyperhomocysteinemia in chronic alcoholic liver diseases, mainly due to deregulation of hepatic homocysteine metabolism. Dyrk1A, involved in homocysteine metabolism at different crossroads, is decreased in liver of hyperhomocysteinemic mice. Here, we hypothesized that Dyrk1A contributes to alcohol-induced hepatic impairment in mice. Control, hyperhomocysteinemic and mice overexpressing Dyrk1A were fed using a Lieber-DeCarli liquid diet with or without ethanol (5% v/v ethanol) for one month, and liver histological examination and liver biochemical function tests were performed. Plasma alanine aminotransferase and homocysteine levels were significantly decreased in mice overexpressing Dyrk1A compared to control mice with or without alcohol administration. On the contrary, the mean plasma alanine aminotransferase and homocysteine levels were significantly higher in hyperhomocysteinemic mice than that of control mice after alcohol administration. Paraoxonase 1 and CYP2E1, two phase I xenobiotic metabolizing enzymes, were found increased in the three groups of mice after alcohol administration. However, NQO1, a phase II enzyme, was only found increased in hyperhomocysteinemic mice after alcohol exposure, suggesting a greater effect of alcohol in liver of hyperhomocysteinemic mice. We observed positive correlations between hepatic alcohol dehydrogenase activity, Dyrk1A and ADH4 protein levels. Importantly, a deleterious effect of alcohol consumption on hepatic Dyrk1A protein level was found. Our study reveals on the one hand a role of Dyrk1A in ethanol metabolism and on the other hand a deleterious effect of alcohol administration on hepatic Dyrk1A level.
... A recent study performed on female rats investigated tissue homocysteine metabolism using stable-isotope approaches and tried to assess the contribution of individual tissues to plasma homocysteine by measuring the isotopic enrichment (55) . Interestingly, in contrast with previous studies (56,57) , they did not find a dominant role for the liver as a source of plasma homocysteine. Other tissues with higher intracellular homocysteine enrichments than in the plasma, including the small intestine, may also contribute to the homocysteinaemia (55) . ...
... 6,7) SAM is converted to decarboxylated SAM, which then releases an aminopropyl group used in polyamine synthesis. 8) On the other hand, the transsulfuration pathway connects SAM to cysteine biosynthesis and makes SAM an important precursor of glutathione (GSH); the transsulfuration pathway is especially active in the liver (Fig. 2). S-Adenosylhomocysteine (SAH), the byproduct of SAM in transmethylation reactions, is hydrolyzed into homocysteine and adenosine by S-adenosylhomocysteine hydrolase (SAHH). ...
Methionine is an essential sulfur-containing amino acid that is metabolized mainly in the liver, where it is converted to S-adenosylmethionine (SAM) by methionine adenosyltransferase. Importantly, SAM is a metabolically pleiotropic molecule that participates in three types of biochemical reactions; transmethylation, transsulfuration (which results in the transfer of sulfur from methionine to serine to form cysteine), and amino propylation (to synthesize polyamines). Critical roles of SAM in the liver have been extensively studied using transgenic animals with chronically reduced or increased hepatic SAM levels. Interestingly, both models with abnormal hepatic SAM concentrations develop liver disease suggesting that SAM homeostasis plays a pivotal role in liver disease. The transsulfuration pathway is connected to the production of glutathione (GSH), which has potent antioxidant capacity in the liver. Accumulating data show that GSH depletion renders the liver vulnerable to oxidative stress and prone to progression of liver disease. In this review, we highlight the importance of homeostasis in the metabolism of sulfur-containing amino acids with a particular focus on the transsulfuration pathway which could be a promising therapeutic target in liver injury.
... Numerous studies have linked high circulating Hcy levels and low concentrations of folate or vitamin B 12 with increased risk of low bone mineral density (BMD) in non-diabetic subjects [22,23]. ...
... Homocysteine is an intermediate in sulfur amino acid metabolism and is produced from hydrolysis of S-adenosylhomocysteine. The liver is the most important organ for homocysteine metabolism [1,2]. In the liver, homocysteine is utilized via the transmethylation pathway to regenerate methionine and via the transsulfuration pathway to produce cysteine [2]. ...
Non-alcoholic fatty liver disease (NAFLD) is associated with increased plasma homocysteine level, which is caused by down-regulation of hepatic cystathionine beta-synthase (CBS) activity. CBS catalyzes the first step in the transsulfuration of homocysteine to cysteine, which contributes ∼50% of the cysteine required for hepatic biosynthesis of glutathione (GSH), the most abundant antioxidant in cells. As the glucagon-like peptide-1 (GLP-1) receptor agonists (e.g. exendin-4) effectively reverse hepatic steatosis, the effect of exendin-4 on both homocysteine and redox status was investigated in the livers of rats fed with high-fat diet (HFD). It was found that HFD down-regulated CBS protein expression, which was probably due to induction of rno-miR-376c expression in the liver. The level of GSH was markedly reduced, whereas the level of malonydialdehyde, an indicator of lipid peroxidation, was significantly increased in the livers of rats fed with HFD. Exendin-4 treatment increased hepatic CBS protein and GSH levels, and reduced malonydialdehyde level in hyperlipidemic rats. Our findings suggest that GLP-1 receptor agonists have beneficial effects on redox homeostasis in NAFLD.
... Também a menor ingestão protéica parece causar o aumento de homocisteína sérica no plasma de ratos, como demonstrado em um estudo de Okawa et al. (2006) 5 , o qual discorda de outros dados reportados por Stead et al. (2000) 6 , que verificaram que o aumento de homocisteína está ligado a uma maior ingestão protéica, já Smolin et al. (1984) 7 reportaram que a concentração de homocisteína não apresentou diferenças entre animais alimentados com 10% ou 60% de caseína. A suplementação de creatina isoladamente demonstrou ser outro fator que pode elevar a homocisteína sérica 8 . ...
Modelo de estudo: Estudo experimental. Objetivos: este estudo teve como objetivo avaliar e caracterizar a dieta hipoproteica como um modelo experimental para estudo de EHNA. Metodos: foram utilizados ratos da linhagem Wistar divididos em dois grupos com dietas isocaloricas: controle (GC) no qual a dieta utilizada seguiu o preconizado pela AIN-93 e hipoproteico (GH) com quantidade de proteina reduzida de 20% para 10%. As dietas e agua foram ofertadas ad libitum por quatro semanas. Apos esse periodo, os animais foram sacrificados e analisados: glicemia; nitrogenio urinario; proteina serica; gordura hepatica; colesterol; variacao de peso e quantidade de racao consumida. Resultados: glicemia e nitrogenio urinario nao apresentaram diferencas significativas entre GC e GH (p>0,05), a variacao de peso no ultimo dia do experimento foi significativa (p<0,02). A porcentagem de gordura hepatica foi estatisticamente maior no GH, quando comparado ao GC (p<0,04). Foram menores o nivel de colesterol (p<0,01) e proteina serica (p<0,005) no GH. A quantidade de dieta consumida nao foi diferente entre os grupos, considerando-se as medias de ingestao semanal. Conclusoes: neste trabalho a dieta hipoproteica constitui um modelo de inducao de EHNA que pode ser caracterizada pela diminuicao da proteina serica e do colesterol plasmatico e aumento da gordura hepatica, entretanto nao ocorreram alteracoes na glicemia sugerindo que nao existiu mudanca na sensibilidade a insulina, constituindo assim um modelo falho para estudar um dos principais fatores de risco para o estabelecimento da EHNA, a resistencia a insulina.
... L-Serine reduces homocysteine concentrations in hepatocytes treated with methionine in rats fed a high-protein diet (9) and in humans who ingested meals fortified with methionine (10). However, it remains to be established whether the homocysteinelowering effects of L-serine can ameliorate fatty liver. ...
Hyperhomocysteinemia plays an important role in the development of hepatic steatosis, and studies indicate that homocysteine-lowering treatment inhibits the development of fatty liver.
We evaluated the effects of l-serine on alcoholic fatty liver and homocysteine metabolism.
In a binge ethanol study, male C57BL/6 mice were divided into 4 groups: control, ethanol + vehicle, and ethanol + 20 or 200 mg/kg l-serine. Mice were gavaged with ethanol (5 g/kg body weight) 3 times every 12 h with or without l-serine which was given twice 30 min before the last 2 ethanol doses. Control mice were fed isocaloric dextran-maltose. In a chronic ethanol study, male Wistar rats were divided into 3 groups: control, ethanol, and ethanol + l-serine. Rats were fed a standard Lieber-DeCarli ethanol diet (36% ethanol-derived calories) for 4 wk with or without dietary l-serine supplementation (1%; wt:vol) for the last 2 wk. In control rats, the ethanol-derived calories were replaced with dextran-maltose. The effects of l-serine were also tested in AML12 cells manipulated to have high homocysteine concentrations by silencing the genes involved in homocysteine metabolism.
Binge ethanol treatment increased serum homocysteine and hepatic triglyceride (TG) concentrations by >5-fold vs. controls, which were attenuated in the 200-mg/kg l-serine treatment group by 60.0% and 47.5%, respectively, compared with the ethanol group. In the chronic ethanol study, l-serine also decreased hepatic neutral lipid accumulation by 63.3% compared with the ethanol group. l-Serine increased glutathione and S-adenosylmethionine by 94.0% and 30.6%, respectively, compared with the ethanol group. Silencing betaine homocysteine methyltransferase, cystathionine β-synthase, or methionine increased intracellular homocysteine and TG concentrations by >2-fold, which was reversed by l-serine when l-serine-independent betaine homocysteine methyltransferase was knocked down.
These results demonstrate that l-serine ameliorates alcoholic fatty liver by accelerating l-serine-dependent homocysteine metabolism.
© 2015 American Society for Nutrition.
... Recentemente, Chen demonstrou que, em culturas de células do músculo liso da parede arterial de seres humanos e de carótidas de suínos, a homocisteína tem efeito prejudicial na proliferação das células do músculo liso e efeito citotóxico nas células endoteliais 7 . Stead verificou que, colocando metionina em contato com hepatócitos de ratos, estas células transportavam mais homocisteína, sugerindo que o fígado é o responsável pela regulação dos níveis plasmáticos da mesma 8 . O efeito da homocisteína elevada no plasma de ratos diminui a vasodilatação e contribui para inativar o óxido nítrico no endotélio, como foi demonstrado por Eberhardt 9 . ...
OBJECTIVE: To assess the effects of induced homocystinemia in the swine iliac artery. MATERIAL AND METHOD: A comparative experimental study with two homogeneous groups of seven pigs from the Macao specimen, weighed between 20 and 30 kg, which were assessed during 30 days. The pigs were divided into two groups. One of them was fed with a methionine-rich diet for a 4-week period. Blood samples were collected for analyses of total cholesterol, triglycerides, HDL and homocysteine concentrations. The animals were submitted to arteriography to evaluate the patency of iliac arteries and then sacrificed. The iliac artery segment was removed for histological analysis. RESULTS: All animals survived the procedure, and there were no significant changes in total cholesterol, triglycerides and HDL concentrations in both groups. Microscopic examinations of the control group did not show pathological changes and was similar in all analyses. In the group receiving the methionine diet, the plaques were formed by foamy macrophages, but smooth muscle cells, cholesterol crystals or inflammatory cells were not seen. The tunica media had the internal elastic lamina intact. In the control group, there was no change in homocysteine levels during the experiment. In the methionine group, there was an increase in plasma homocysteine levels, with an average value of 59.80 µmol/l after 30 days with a methionine-rich diet. CONCLUSION: Homocystinemia induced by methionine causes atherogenesis in the swine iliac artery.
... In rats, around 30 % of plasma Hcy is protein bound. In normal humans, however, 80 % of total Hcy bound to form mixed disulfide and the remaining is found in free form [29]. This is important because free Hcy is unstable, which makes the determination of accurate levels difficult [30]. ...
The purpose of the study is to evaluate the effects of creatine supplementation on homocysteine (Hcy) plasma levels after acute exercise in humans.
Twenty-three young (under-20) soccer players were divided into 2 groups: creatine (Cr)- and placebo (Pla)-supplemented groups. The supplementation was performed in double-blind controlled manner using creatine or placebo tablets with 0.3 g/kg during 7 days. Before and after 7 days of supplementation, the athletes performed an acute high-intensity sprint exercise (two consecutive running-based anaerobic sprint test protocol consisted in 6 × 35 m sprint with 10 s between them). Blood samples were collected before and after 7 days of supplementation as well as 0 and 1 h after exercise protocol.
Homocysteine concentration significant increased (P < 0.05) 1 h after acute exercise (18 %). Acute exercise also decreased red blood cell S-adenosylmethionine (SAM) 30 % with no changes in SAM/SAH ratio. Seven days of creatine supplementation were able to increase (P < 0.05) plasma creatine concentration (Pla 130.1 ± 21.7 vs Cr 1,557.2 ± 220.3 μmol/L) as well as decrease (P < 0.05) plasma guanidinoacetic acid (33 %). Controversially, creatine supplementation did not change Hcy plasma level after 7-day supplementation (Pla 6.9 ± 0.2 vs Cr 7.2 ± 0.2 μmol/L) or after acute exercise (Pla 8.2 ± 0.3 vs Cr 8.4 ± 0.3 μmol/L). No changes in plasma vitamin B12 and folate as well as cysteine and methionine were found.
Seven days of creatine supplementation does not avoid increased plasma Hcy induced by acute sprint exercise in humans.
... Remethylation patterns in the liver, the primary site for amino acid metabolism, did not coincide with the reduction in homocysteine concentrations observed in exercised mice for this study (179). Other mechanisms to reduce homocysteine may have been more prominent with exercise treatment, such as decreased methionine substrate availability for transmethylation. ...
Normal folate, homocysteine, and methyl group metabolism is critical for maintaining optimum health, and perturbations in these mechanisms may lead to a number of pathological conditions such as diabetes, cardiovascular disease, neural tube defects, and cancer. Hyperhomocysteinemia is a condition that occurs when blood plasma concentrations of homocysteine exceed 15μM and has been implicated as an independent risk factor in cardiovascular disease. Specifically, populations with genetic polymorphisms in folate-dependent remethylation or poor B-vitamin status are at risk for this condition. This study was conducted in order to determine the influence of exercise on homocysteine and methyl group metabolism in a folate-deficient and -sufficient mouse model. Elevated homocysteine concentrations observed in the folate-deficient sedentary group were reduced by more than 2-fold with the addition of both forced treadmill and free access wheel exercise (p < 0.001). Similar results were observed in the folate-sufficient control diet with treadmill and wheel exercised mice having about 50% lower homocysteine concentrations than their sedentary counterparts (p < 0.001). Increased renal homocysteine remethylation through the enzyme betaine-homocysteine S-methyltransferase (BHMT) was observed in wheel exercised mice suggesting an increasingly important role in kidney homocysteine metabolism with exercise (p < 0.05). To our knowledge, this is the first study showing the attenuation of hyperhomocysteinemia in a model known to experience perturbed homocysteine metabolism. Findings from this study will be useful in determining the influence exercise has in other populations of altered homocysteine metabolism such as diabetics, vegans, and individuals with genetic polymorphisms.
... Recentemente, Chen demonstrou que, em culturas de células do músculo liso da parede arterial de seres humanos e de carótidas de suínos, a homocisteína tem efeito prejudicial na proliferação das células do músculo liso e efeito citotóxico nas células endoteliais 7 . Stead verificou que, colocando metionina em contato com hepatócitos de ratos, estas células transportavam mais homocisteína, sugerindo que o fígado é o responsável pela regulação dos níveis plasmáticos da mesma 8 . O efeito da homocisteína elevada no plasma de ratos diminui a vasodilatação e contribui para inativar o óxido nítrico no endotélio, como foi demonstrado por Eberhardt 9 . ...
OBJECTIVE: To assess the effects of induced homocystinemia in the swine iliac artery. MATERIAL AND METHOD: A comparative experimental study with two homogeneous groups of seven pigs from the Macao specimen, weighed between 20 and 30 kg, which were assessed during 30 days. The pigs were divided into two groups. One of them was fed with a methionine-rich diet for a 4-week period. Blood samples were collected for analyses of total cholesterol, triglycerides, HDL and homocysteine concentrations. The animals were submitted to arteriography to evaluate the patency of iliac arteries and then sacrificed. The iliac artery segment was removed for histological analysis. RESULTS: All animals survived the procedure, and there were no significant changes in total cholesterol, triglycerides and HDL concentrations in both groups. Microscopic examinations of the control group did not show pathological changes and was similar in all analyses. In the group receiving the methionine diet, the plaques were formed by foamy macrophages, but smooth muscle cells, cholesterol crystals or inflammatory cells were not seen. The tunica media had the internal elastic lamina intact. In the control group, there was no change in homocysteine levels during the experiment. In the methionine group, there was an increase in plasma homocysteine levels, with an average value of 59.80 µmol/l after 30 days with a methionine-rich diet. CONCLUSION: Homocystinemia induced by methionine causes atherogenesis in the swine iliac artery.
... There are several possible explanations for the absence of intracellular Hcy lowering after FA. Plasma Hcy and its response to therapy may predominantly be a reflection of Hcy production/export by specific organs or tissues, such as the liver and kidney, rather than a reflection of Hcy concentrations in most cell types [ 23 ]. Alternatively, cellular Hcy may simply be too tightly regulated to be affected by folate administration, e.g., by export of out in isolated lymphoblasts, which may not fully represent what happens in vivo, either in PBMCs or in other cell types. ...
Background:
In randomized trails, folic acid (FA) lowered plasma homocysteine, but failed to reduce cardiovascular risk. We hypothesize this is due to a discrepancy between plasma and intracellular effects of FA.
Methods:
In a double-blind trial, 50 volunteers were randomized to received 500 µg FA daily for 8 weeks, or placebo. Plasma and peripheral blood mononuclear cell (PBMC) concentrations of homocysteine, S-adenosylmethionine (SAM), S-adenosylhomocysteine, methionine, cystathionine and 5-methyltetrahydrofolate (bioactive folate) were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS). PBMCs were used as a cellular model since they display the full spectrum of one-carbon (1C) enzymes and reactions.
Results:
At baseline, plasma concentrations were a poor reflection of intracellular concentrations for most 1C metabolites, except 5-methyltetrahydrofolate (R=0.33, p=0.02), homocysteine (Hcy) (R=0.35, p=0.01), and cystathionine (R=0.45, p=0.001). FA significantly lowered plasma homocysteine (p=0.00), but failed to lower intracellular homocysteine or change the concentrations of any of the other PBMC 1C metabolites. At baseline, PBMC homocysteine concentrations correlated to PBMC SAM. After FA supplementation, PBMC homocysteine no longer correlated with PBMC SAM, suggesting a loss of SAM's regulatory function. In vitro experiments in lymphoblasts confirmed that at higher folate substrate concentrations, physiological concentrations of SAM no longer effectively inhibit the key regulatory enzyme methylenetetrahydrofolate reductase (MTHFR).
Conclusions:
FA supplementation does not reduce intracellular concentrations of Hcy or any of its closely related substances. Rather, FA may disturb physiological regulation of intracellular 1C metabolism by interfering with SAM's inhibitory effect on MTHFR activity.
... Moreover, we recently found an increase in DYRK1A protein expression in brain of Cbs +/− mice [10]. Indeed, the liver constitutes the appropriate target for gene transfer since the liver plays a central role in the metabolism of methionine and contributes to much of the plasma Hcy [11,12]. ...
Hyperhomocysteinemia, characterized by high plasma homocysteine levels, is recognized as an independent risk factor for cardiovascular diseases. The increased synthesis of homocysteine, a product of methionine metabolism involving B vitamins, and its slower intracellular utilization cause increased flux into the blood. Plasma homocysteine level is an important reflection of hepatic methionine metabolism and the rate of processes modified by B vitamins as well as different enzyme activity. Lowering homocysteine might offer therapeutic benefits. However, approximately 50% of hyperhomocysteinemic patients due to cystathionine-beta-synthase deficiency are biochemically responsive to pharmacological doses of B vitamins. Therefore, effective treatments to reduce homocysteine levels are needed, and gene therapy could provide a novel approach. We recently showed that hepatic expression of DYRK1A, a serine/threonine kinase, is negatively correlated with plasma homocysteine levels in cystathionine-beta-synthase deficient mice, a mouse model of hyperhomocysteinemia. Therefore, Dyrk1a is a good candidate for gene therapy to normalize homocysteine levels. We then used an adenoviral construct designed to restrict expression of DYRK1A to hepatocytes, and found decreased plasma homocysteine levels after hepatocyte-specific Dyrk1a gene transfer in hyperhomocysteinemic mice. The elevation of pyridoxal phosphate was consistent with the increase in cystathionine-beta-synthase activity. Commensurate with the decreased plasma homocysteine levels, targeted hepatic expression of DYRK1A resulted in elevated plasma paraoxonase-1 activity and apolipoprotein A-I levels, and rescued the Akt/GSK3 signaling pathways in aorta of mice, which can prevent homocysteine-induced endothelial dysfunction. These results demonstrate that hepatocyte-restricted Dyrk1a gene transfer can offer a useful therapeutic targets for the development of new selective homocysteine lowering therapy.
... There are several possible explanations for the absence of intracellular Hcy lowering after FA. Plasma Hcy and its response to therapy may predominantly be a reflection of Hcy production/export by specific organs or tissues, such as the liver and kidney, rather than a reflection of Hcy concentrations in most cell types [ 23 ]. Alternatively, cellular Hcy may simply be too tightly regulated to be affected by folate administration, e.g., by export of [ 27 ]. ...
Disturbances in the levels of one-carbon (1C) metabolism metabolites have been associated with a wide variety of neuropsychiatric diseases. Cerebrospinal fluid (CSF) levels of homocysteine (Hcy) and the other 1C metabolites, nor their interrelatedness and putative determinants, have been studied extensively in a healthy population.
Plasma and CSF samples from 100 individuals free from neuropsychiatric diseases were analyzed (55 male, 45 female; age 50±17 years). In blood, we measured plasma Hcy, serum folate and serum vitamin B12. In CSF, we measured total Hcy, S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH) and 5-methyltetrahydrofolate (5-methylTHF). Highly selective analytical methods like liquid chromatography combined with either mass spectrometry or fluorescence detection were used.
CSF Hcy was inversely correlated with CSF 5-methylTHF and positively with plasma Hcy, independent of serum folate status. CSF SAH correlated with age, lower CSF 5-methylTHF and higher CSF Hcy. CSF 5-methylTHF showed independent negative correlations with age and positive correlations with serum folate. CSF SAM did not correlate with any of the 1C metabolites.
Aging is characterized by a reduction in CSF 5-methylTHF levels and increased CSF levels of the potentially neurotoxic transmethylation inhibitor SAH. CSF 5-methylTHF, which is itself determined in part by systemic folate status, is a powerful independent determinant of CSF levels of Hcy and SAH.
... Chistensen et al. [21] demonstrated that Hcy export from cultured hepatocytes was much greater than from other cell lines and that it was highly dependent on extracellular methionine. Finally, using a rat primary hepatocyte model, Stead et al. [22] recently confirmed a predominant role of the liver in regulating plasma Hcy levels. Therefore, the endothelial dysfunction observed in response to methionine administration in vivo is probably due to the production and export of large amounts of Hcy by the liver, leading to a plasma Hcy concentration above the pathological threshold of 15 mmol : L 21 . ...
The increased cardiovascular risk associated with hyperhomocysteinemia has been partly related to homocysteine (Hcy)-induced endothelial cell dysfunction. However, the intra or extracellular starting point of the interaction between Hcy and endothelial cells, leading to cellular dysfunction, has not yet been identified. We investigated the effects of both intracellular and extracellular Hcy accumulation on endothelin-1 (ET-1) synthesis by cultured human endothelial cells. Incubation of cultures with methionine (1.0 mmol·L−1) for 2 h induced a slight increase in cellular Hcy content but no change in ET-1 production. Incubation of cells with Hcy (0.2 mmol·L−1) led to a significant fall in ET-1 generation, accompanied by a significant increase in cellular Hcy content. Addition of the amino-acid transport system L substrate 2-amino-2-norbornane carboxylic acid had no effect on the Hcy-induced decrease in ET-1 production but significantly inhibited the Hcy-induced increase in the cellular Hcy content. Incubation of cells with a lower Hcy concentration (0.05 mmol·L−1) also reduced ET-1 production without increasing the cellular Hcy content. Co-incubation with extracellular free-radical inhibitors (superoxide dismutase, catalase and mannitol) markedly reduced the effect of Hcy on ET-1 production. Thus, it is extracellular Hcy accumulation that triggers the decrease in ET-1 production by endothelial cells through oxidative products.
... Methionine is condensed with ATP to form S-adenosylmethionine, the universal methyl donor in transmethylation reactions and the direct precursor of Hcys in vivo (Satta et al., 2006). During normal conditions excess Hcys is converted back to methionine or broken down for excretion (Stead et al., 2000). High Hcys appears to contribute to atherosclerosis in at least three ways: direct toxic effect that damages the cell lining of the arteries, interference with clotting factors and oxidation of low density lipoprotein (LDL) (Ridker et al., 1999). ...
he effects of homocysteine (Hcys) and/or the antioxidant Betaine (Bet) on the esterase activities of spleen and serum samples of rats were compared with control animals by using polyachrylamide gel electrophoresis by using two different substrates. Male albino rats were divided into four groups. Group (I) was considered as a control group, while groups (II), (III) and (IV) were orally administered with Bet (0.6 mg/kg b.wt.), Hcys (0.6 g/kg b.wt.) or both Hcys and Bet respectively for 8 weeks. Rats were sacrificed after 2, 4, 6 and 8 weeks and samples from spleen and sera were prepared for the determination of esterase activities. The electrophoretic pattern of α-esterase revealed 17 and 14 bands for the sera and spleen respectively. The number of common bands for sera of all the experimental groups was 3 with relative fragmentation (RF) of 0.14, 0.2 and 0.57, while the number of common bands for the spleen was 4 with RF of 0.085, 0.22, 0.36 and 0.66. Concerning β-esterase, the number of electrophoretic bands were 17 and 18 for serum and spleen respectively. Two common bands were recorded for each with RF 0.24 and 0.68 for sera and 0.055 and 0.14 for the spleen. The recorded changes in esterases pattern due to treatment with Hcys and/or Bet were associated with weight disturbance and aggressive behavior in rats treated with Hcys.
The modern understanding of the use and possibility of integrating artificial intelligence (AI) in medicine provides
for trends in the focus development and implementation of innovative approaches to the use of expert systems (ES)
and medical information systems (MIS), which are also considered in this systematic review. Development of new
clinical decision support systems (CDSS) using different AI models, in particular machine learning (ML), deep learning
(DL) and artificial neural network (ANN), based on the analysis of “big data”, datasets, followed by their integration
into the process of diagnosis and the formation of recommendations for the tactical management of patients with
a particular pathology. This conclusion follows from our analysis of more than 30 literature sources, which were
mostly meta-analyses, retrospective prospective studies in the field of development and the growing role of IT in
medicine over the past 20 years. As a demonstration of the growth in the development and application of innova�tive technologies, in this review we consider the determinations we have identified on the example of clinical and
morphological correlations and genotypic variability of prostate cancer, which most clearly reflects new diagnostic
and prognostic models of modern AI.
Keywords: prostate cancer, artificial intelligence, machine learning, risk stratification, artificial neural networks
Lean body mass (LBM) agglomerates the bulk of nitrogen (N)-containing molecules following well-identified age and sex evolutionary patterns best appraised in clinical practice using the serial measurement of plasma transthyretin (TTR). Methionine (Met), the sole essential amino acid bearing a sulfur (S) atom, presides at the initiation of protein synthesis while maintaining stable body tissue S:N molar ratios of approximately 1:14.5. In protein- depleted states, N- and Met-deficiencies operate as limiting factors for LBM protein synthesis and accretion, causing growth retardation and subnormal TTR plasma values. In inflammatory disorders, LBM is subjected to cytokine-induced tissue breakdown reflecting the S:N ratio found in healthy tissues whereas the liver secretion of TTR declines in proportion. Both malnutrition and inflammation are characterized by stepwise LBM downsizing and reduced bioavailability of Met body stores setting in motion molecular mechanisms safeguarding Met homeostasis at the expense of augmented homocysteine (Hcy) values in biological fluids. Divergent TTR and Hcy alterations indicate that rising Hcy values measured in plasma and cerebrospinal fluid should be regarded as the dark side of efficient compensatory processes. As a result, the neuroprotective activities normally exerted by TTR are weakened, whereas the oxidative burden generated by supranormal Hcy concentrations are strengthened. The combination of protein malnutrition and inflammatory disorders of any cause maximizes the risk of incurable neurodegenerative effects.
The incidence of cardiovascular diseases in vegetarian individuals is lower than that in the general population. Nevertheless, individuals who adhere to vegan diets have a higher prevalence of hyperhomocysteinemia with eventual adverse effects on vascular reactivity. Creatine supplementation (CrS) reduces plasma homocysteine levels and enhances vascular reactivity in the microcirculation. Thus, we investigated the effects of CrS on systemic microcirculation and homocysteine blood levels in strict vegan subjects. Forty‐nine strict vegan subjects were allocated to the oral CrS (5 g micronized creatine monohydrate daily for three weeks; n=31) and placebo (n=18) groups. Laser speckle contrast imaging coupled with acetylcholine skin iontophoresis was used to evaluate cutaneous microvascular reactivity, and intra‐vital video‐microscopy was used to evaluate skin capillary density and reactivity before and after CrS. We demonstrated that CrS reduces the plasma levels of homocysteine and increases those of folic acid. After the CrS period, the homocysteine levels of all of the vegan subjects normalized. CrS also induced increases in baseline skin functional capillary density and endothelium‐dependent capillary recruitment in both normo‐ (N‐Hcy) and hyperhomocysteinemic (H‐Hcy) individuals. CrS increased endothelium‐dependent skin microvascular vasodilation in the H‐Hcy vegan subjects but not in the N‐Hcy vegan subjects. In conclusion, three weeks of oral CrS was sufficient to increase skin capillary density and recruitment and endothelium‐dependent microvascular reactivity. CrS also resulted in plasma increases in folic acid levels and reductions in homocysteine levels among only the H‐Hcy individuals.
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Unlabelled:
Homocysteine, a sulfur-containing amino acid formed during the metabolism of methionine, is commonly slightly elevated in the plasma of the general population. Additionally, we previously found that cystathionine beta synthase-deficient mice, a murine model of hyperhomocysteinemia, exhibit altered activities of xenobiotic metabolizing enzymes (XME), which dispose of foreign chemicals, in the liver. Thus, hyperhomocysteinemia may result in susceptibility to xenobiotics like cadmium, a heavy-metal toxicant found in drinking water, atmospheric air, and food. Consequently, we exposed hyperhomocysteinemic mice to cadmium via their drinking water for one month to analyze the combined effects of hyperhomocysteinemia and cadmium exposure in liver. No difference in plasma homocysteine level was found after cadmium administration in control and hyperhomocysteinemic mice, but the glutathione level was significantly lower in exposed hyperhomocysteinemic mice compared to control mice, reflecting oxidative stress. We therefore analyzed the effect of Cd administration on hepatic XMEs known to be dysregulated in hyperhomocysteinemic mice: paraoxonase 1, a phase I XME, and
Nad(p)h:
quinone oxidoreductase, a phase II XME. Cadmium exposure negatively affected activity of paraoxonase 1, a calcium-dependent enzyme. Thus, we analyzed another calcium-dependent enzyme known to be dysregulated in liver of hyperhomocysteinemic mice, calpain, which was also significantly lower after cadmium administration. A comparison of the calculated affinities of cadmium docking versus calcium redocking suggested that cadmium ions may inhibit enzymatic activities by preventing the binding of calcium ions. Moreover, the increased
Nad(p)h:
quinone oxidoreductase activity observed after cadmium administration could indicate the presence of protective mechanisms in liver of mice. In conclusion, although cadmium administration had no effect on plasma homocysteine level, its effects on plasma glutathionine level suggest a susceptibility to cadmium in the condition of hyperhomocysteinemia, which could be countered by an increased
Nad(p)h:
quinone oxidoreductase activity.
Hyperhomocysteinemia is an independent risk factor for several cardiovascular diseases. The use of vitamins to modulate homocysteine metabolism substantially lowers the risk by reducing plasma homocysteine levels. In this study, we evaluated the effects of l-serine and related amino acids on homocysteine-induced endoplasmic reticulum (ER) stress and endothelial cell damage using EA.hy926 human endothelial cells. Homocysteine treatment decreased cell viability and increased apoptosis, which were reversed by cotreatment with l-serine. l-Serine inhibited homocysteine-induced ER stress as verified by decreased glucose-regulated protein 78kDa (GRP78) and C/EBP homologous protein (CHOP) expression as well as X-box binding protein 1 (xbp1) mRNA splicing. The effects of l-serine on homocysteine-induced ER stress are not attributed to intracellular homocysteine metabolism, but instead to decreased homocysteine uptake. Glycine exerted effects on homocysteine-induced ER stress, apoptosis, and cell viability that were comparable to those of l-serine. Although glycine did not affect homocysteine uptake or export, coincubation of homocysteine with glycine for 24h reduced the intracellular concentration of homocysteine. Taken together, l-serine and glycine cause homocysteine-induced endothelial cell damage by reducing the level of intracellular homocysteine. l-Serine acts by competitively inhibiting homocysteine uptake in the cells. However, the mechanism(s) by which glycine lowers homocysteine levels are unclear.
This chapter focuses on the metabolic processes involved in one-carbon (methyl) donor production and their impact on DNA methylation. It discusses the metabolic regulation of DNA methylation in mammals. More than 100 methyltransferase enzymes (including four DNA methyltransferases) have been described in mammals, involved in the transfer of methyl groups to a large array of proteins, phospholipids, and nucleotides. These reactions are fundamental to many different cellular functions, and it is therefore not surprising that insufficient methyl donor availability has the potential to disrupt a wide variety of biological processes, including DNA nucleotide synthesis and methylation, and gene expression (among others). The production of sufficient methyl-donors is therefore of critical importance for faithful cell division and development. The methylated maternal and paternal genomes are de-methylated at fertilization and specific patterns of methylation are then re-established progressively starting in the early post-conception period. The methyl groups required for establishment and maintenance of DNA methylation are derived solely from dietary methyl donors in association with specific enzymes and associated cofactors. The metabolic pathways that generate the primary methyl donors needed for the de novo establishment and maintenance of the DNA methylation profile are complicated and do not exist in isolation from other essential biochemical reactions necessary for cell survival, division, and differentiation.
The present study was aimed to investigate the metabolomics of sulfur amino acids in Zucker diabetic fatty (ZDF) rats, an obese type 2 diabetic animal model. Plasma levels of total cysteine, homocysteine and methionine, but not glutathione (GSH) were markedly decreased in ZDF rats. Hepatic methionine, homocysteine, cysteine, betaine, taurine, spermidine and spermine were also decreased. There are no significant difference in hepatic S-adenosylmethionine, S-adenosylhomocysteine, GSH, GSH disulfide, hypotaurine and putrescine between control and ZDF rats. Hepatic SAH hydrolase, betaine-homocysteine methyltransferase and methylene tetrahydrofolate reductase were up-regulated while activities of gamma-glutamylcysteine ligase and methionine synthase were decreased. The area under the curve (AUC) of methionine and methionine-d4 was not significantly different in control and ZDF rats treated with a mixture of methionine (60mg/kg) and methionine-d4 (20mg/kg). Moreover, the AUC of the increase in plasma total homocysteine was comparable between two groups, although the homocysteine concentration curve was shifted leftward in ZDF rats, suggesting that the plasma total homocysteine after the methionine loading was rapidly increased and normalized in ZDF rats. These results show that the AUC of plasma homocysteine is not responsive to the up-regulation of hepatic BHMT in ZDF rats. The present study suggests that the decrease in hepatic methionine may be responsible for the decreases in its metabolites, such as homocysteine, cysteine, and taurine in liver and consequently decreased plasma homocysteine levels.
Copyright © 2015. Published by Elsevier Inc.
Liver diseases lead to a complex syndrome characterized by neurological, neuro-psychiatric and motor complications, called hepatic encephalopathy, which is prevalent in patients and animal models of acute, sub-chronic and chronic liver failure. Although alterations in GABAergic, glutamatergic, cholinergic and serotonergic neuronal functions have been implicated in HE, the molecular mechanisms that lead to HE in chronic liver disease (CLD) is least illustrated. Due to hepatocellular failure, levels of ammonia and homocysteine (Hcy), in addition to others, are found to increase in the brain as well as plasma. Hcy, a non-protein forming amino acid and an excitotoxin, activates ionotropic glutamate (n-methyl-d-aspartate; NMDA) receptors, and thereby leads to influx of Ca(2+) into neurons, which in turn activates several pathways that trigger oxidative stress, inflammation and apoptosis, collectively called excitotoxicity. Elevated levels of Hcy in the plasma and brain, a condition called Hyperhomocysteinemia (HHcy), and the resultant NMDA receptor-mediated excitotoxicity has been implicated in several diseases, including Parkinson's disease and Alzheimer's disease. Although, hyperammonemia has been shown to cause excitotoxicity, the role of HHcy in the development of behavioral and neurochemical alterations that occur in HE has not been illustrated yet. It is hypothesized that CLD-induced HHcy plays a major role in the development of HE through activation of NMDA receptors. It is further hypothesized that HHcy synergizes with hyperammonemia to activate NMDA receptor in the brain, and thereby cause oxidative stress, inflammation and apoptosis, and neuronal loss that leads to HE.
Copyright © 2015. Published by Elsevier Ltd.
Non-alcoholic fatty liver disease (NAFLD) is a broad spectrum liver disorder diagnosed in patients without a history of alcohol abuse. NAFLD is growing at alarming rates worldwide. Its pathogenesis is complex and incompletely understood. The cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE) system regulates homocysteine and cysteine metabolism and contributes to endogenous hydrogen sulfide (H2S) biosynthesis. This review summarizes our current understanding of the hepatic CBS/CSE system, and for the first time, positions this system as a potential therapeutic target in NAFLD. As will be discussed, the CBS/CSE system is highly expressed and active in the liver. Its dysregulation, presenting as alterations in circulating homocysteine and (or) H2S levels, has been reported in NAFLD patients and in NAFLD-associated co-morbidities such as obesity and type 2 diabetes. Intricate links between the CBS/CSE system and a number of metabolic and stress related molecular mediators have also emerged. Various dysfunctions in the hepatic CBS/CSE system have been reported in animal models representative of each NAFLD spectrum. It is anticipated that a newfound appreciation for the hepatic CBS/CSE system will emerge that will improve our understanding of NAFLD pathogenesis, and give rise to new prospective targets for management of this disorder.
Hydrogen sulfide (H2S) has emerged as an important signaling molecule with beneficial effects on various cellular processes affecting, for example, cardiovascular and neurological functions. The physiological importance of H2S is motivating efforts to develop strategies for modulating its levels. However, advancement in the field of H2S-based therapeutics is hampered by fundamental gaps in our knowledge of how H2S is regulated, its mechanism of action, and its molecular targets. This review provides an overview of sulfur metabolism; describes recent progress that has shed light on the mechanism of H2S as a signaling molecule; and examines nutritional regulation of sulfur metabolism, which pertains to health and disease.
Cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE) catalyze homocysteine (Hcy) metabolism via the trans-sulfuration pathway. They are also responsible for hydrogen sulfide (H2S) production via desulfuration reactions. The liver contributes significantly to the regulation of Hcy and H2S homeostasis, which might participate in many physiological and pathological processes. The aim of this study was to investigate the effect of a high-fat diet (HFD) on hepatic CBS and CSE expression and its impact on Hcy and H2S metabolism. Mice (C57BL/6) fed a HFD (60% kcal fat) for 5 weeks developed fatty liver. The mRNA and protein levels of CBS and CSE in the liver were significantly elevated in mice fed a HFD. Subsequently the metabolism of Hcy by CBS and CSE was increased in the liver, and its level decreased in the circulation. Increased CBS and CSE expression also caused a significant elevation in H2S production in the liver. The level of lipid peroxides was elevated, indicating oxidative stress, while the level of total glutathione remained unchanged in the liver of HFD-fed mice. Upregulation of the trans-sulfuration pathway might play an adaptive role against oxidative stress by maintaining total glutathione levels in the liver.
A homocisteína, formada a partir da metionina hepática, é metabolizada nas vias de desmetilação e de transulfuração, sendo que seus valores plasmáticos e urinários refletem a síntese celular. Sua determinação, realizada em jejum e após sobrecarga de metionina, caracteriza as diferenças dessas vias metabólicas, principalmente quando de natureza genética. A hiper-homocisteinemia tem sido associada a maior risco de eventos aterotrombóticos, e a literatura sugere associação causal, independente de outros fatores de risco para doença arterial. Diminuição da homocisteína plasmática para valores normais é seguida de redução significante na incidência de doença aterotrombótica. A relação entre homocisteína e o fígado vem adquirindo importância nos dias atuais, uma vez que alterações das lipoproteínas e da depuração de metionina são comuns em pacientes com doença hepática crônica (hepatocelular e canalicular). O tratamento da hiper-homocisteinemia fundamenta-se na suplementação alimentar e medicamentosa de ácido fólico e vitaminas B6 e B12.
Objective: To assess the effects of induced homocystinemia in the swine iliac artery. Material and method: A comparative experimental study with two homogeneous groups of seven pigs from the Macao specimen, weighed between 20 and 30 kg, which were assessed during 30 days. The pigs were divided into two groups. One of them was fed with a methionine-rich diet for a 4-week period. Blood samples were collected for analyses of total cholesterol, triglycerides, HDL and homocysteine concentrations. The animals were submitted to arteriography to evaluate the patency of iliac arteries and then sacrificed. The iliac artery segment was removed for histological analysis. Results: All animals survived the procedure, and there were no significant changes in total cholesterol, triglycerides and HDL concentrations in both groups. Microscopic examinations of the control group did not show pathological changes and was similar in all analyses. In the group receiving the methionine diet, the plaques were formed by foamy macrophages, but smooth muscle cells, cholesterol crystals or inflammatory cells were not seen. The tunica media had the internal elastic lamina intact. In the control group, there was no change in homocysteine levels during the experiment. In the methionine group, there was an increase in plasma homocysteine levels, with an average value of 59.80 µmol/l after 30 days with a methionine-rich diet. Conclusion: Homocystinemia induced by methionine causes atherogenesis in the swine iliac artery.
The effects of dietary proteins on plasma lipids, on the process of oxidation and on homocysteinemia, platelet aggregation, atherosclerosis, arterial hypertension and thrombosis have been investigated in animal models and in humans. Although sometimes conflicting, the results of epidemiological surveys and experimental studies, show that, in contrast to vegetable proteins, animal proteins might be well promote the development of atherosclerosis in subjects genetically predisposed to atherosclerosis, particularly in hypercholesterolemic subjects. Their eventual effects on healthy subjects have not been still proven. For the analysis of the results, several factors have to be taken into account: genetic, dietary environment and fats in particular, cholesterol, dietary fibers and isoflavones. The atherogenic, hypertensive and thrombotic effects depend on the amino acid composition of proteins, on the nature of the peptides released during digestion and of the extent of their immunologic effects. The mechanisms behind these pathogenic effects are complex and still under discussion also new investigations are necessary.
AUTEUR(S)
Gérard DEBRY
MOTS-CLÉS
protéines alimentaires, lipides plasmatiques, athérosclérose, hypertension artérielle, thrombose.
KEYWORDS
dietary proteins, plasma lipids, atherosclerosis, arterial hypertension, thrombosis.
LANGUE DE L'ARTICLE
Français
Elevated plasma homocysteine has been identified as a risk factor for cardiovascular disease and non-alcoholic liver disease, which are major complications of diabetes. Hence, hepatic homocysteine metabolism has become a major focus of diabetes research. However, little information is available regarding plasma homocysteine levels in non-obese diabetic animals. Therefore, we investigated the hepatic metabolism of sulfur-amino acids in non-obese type-2 diabetic Goto-Kakizaki rats. The experiments were performed using 9-week-old Goto-Kakizaki rats and age-matched Wistar rats. The major finding of this study is that homocysteine levels in the liver and plasma are maintained by a balance between the up-regulation of betaine homocysteine methyltransferase and the inhibition of cystathionine β-synthase in non-obese type-2 diabetic rats. Hepatic levels of cysteine and its metabolites, such as hypotaurine, taurine, and glutathione, were increased despite inhibition of the transsulfuration of homocysteine to cysteine. The elevated hepatic taurine and glutathione levels may be attributed to the up-regulation of cysteine dioxygenase expression and increased cysteine availability for glutathione synthesis. Inhibition of hepatic methionine adenosyltransferase activity in Goto-Kakizaki rats was associated with a decrease in hepatic S-adenosylmethionine, which serves as an allosteric activator of cystathionine β-synthase. The non-obese type-2 diabetic condition results in profound changes in hepatic sulfur-amino acid metabolism and raises the possibility that sulfur-amino acid metabolism may be regulated by obesity- as well as diabetes-associated factors. Further study to elucidate the pathological significance of sulfur-amino acid metabolism in chronic liver disease in type-2 diabetic animals is underway in this laboratory.
High homocysteine (Hcy) concentrations are associated with the risk of cardiovascular disease. Despite the known high incidence of the use of food supplements by athletes, little is known about the possible effect of the use of protein supplements containing methionine (Met) on Hcy metabolism. The objective of the present study was to test the hypothesis that the consumption of protein supplements can increase the ingestion of Met and Hcy levels. Seventeen swimmers (8 males and 9 females) aged 20.11:26 years were evaluated in terms of biochemical profile and habitual food consumption. After the application of a questionnaire about the use of food supplements, the swimmers were divided into three groups: control (C), energy supplement consumption (ES), and protein-energy supplement consumption (PES). The results showed that 82% of the individuals used some type of supplement, with PES being consumed by 36% of them. The addition of a supplement did not significantly increase protein or methionine intake in the PES group compared to C and ES groups. No significant differences in plasma Hcy concentrations were detected between groups, but a positive correlation was observed between Met intake and serum Hcy (r = 0.65; p=0.01), and a negative correlation was also detected between serum Hcy and serum vitamin B12 (r = -0.52; p=0.03). In conclusion, it was observed that, even in the absence of protein intake modulation and changes in Hcy levels, the ingestion of methionine through food supplements is associated with plasma Hcy levels.
de creatina, homocisteína e estresse oxidativo. Medicina (Ribeirão Preto) 2007;40 (3): 368-77, jul./set. RESUMO: A creatina é uma substância popular entre atletas devido a sua possível proprie-dade ergogênica. Tal popularidade incentivou autores a estudar e explorar o possível potencial terapêutico desta substância. A síntese de creatina é responsável pela maioria das transferências de grupos metila no metabolismo hepático normal. Como a homocisteína é um aminoácido formado exclusivamente a partir da desmetilação da metionina, acredita-se que a creatina e homocisteína estejam metabolicamente conectadas. Estudos têm mostrado que a hiperhomo-cisteinemia está diretamente ligada à formação de espécies reativas de oxigênio pela auto-oxidação da homocisteína e/ou da cisteína e que tal auto-oxidação pode provocar danos celula-res. O objetivo desta revisão é discutir aspectos da suplementação com creatina relacionados aos níveis de homocisteína e o estresse oxidativo. Descritores: Creatina. Suplementação Alimentar. Homocisteína. Estresse Oxidativo.
To determine the effect of type-2 diabetes and obesity on the hepatic metabolism of sulfur amino acids, hepatic sulfur amino acid metabolism was determined in db/db mice. Hepatic methionine was markedly decreased in db/db mice, although the hepatic activity of betaine homocysteine methyltransferase was increased. The decrease in hepatic methionine was reflected by decreased sulfur-containing methionine metabolites, including S-adenosylmethionine, homocysteine, cysteine, and hypotaurine in liver and plasma. In contrast, S-adenosylhomocysteine, putrescine, and spermidine were increased in db/db mice. The hepatic level and activity of methionine adenosyltransferase I/III, an S-adenosylmethionine synthesizing enzyme, were significantly increased. These results suggest that increased polyamine synthesis, in conjunction with decreased hepatic methionine levels, is partly responsible for the reduction in hepatic S-adenosylmethionine. Decreased homocysteine in liver and plasma may be attributable to the decrease in hepatic methionine and upregulation of hepatic betaine homocysteine methyltransferase. Glutathione in liver and plasma did not change despite decreased -glutamylcysteine ligase activity. The decreased hepatic hypotaurine may be attributable to the downregulation of cysteine dioxygenase. The major finding of this study is that db/db mice exhibited decreases in hepatic methionine and its sulfurcontaining metabolites.
Diets rich in protein are often used for weight loss in obese patients, but their long-term effects are not fully understood. Homocysteine (Hcy) is considered to be a risk factor for cardiovascular diseases, and its levels are influenced by diet, particularly the protein and fat content. We studied the effect of diets with varying fat/protein content on body weight and composition, food intake, Hcy, B vitamins, leptin, and several pro-inflammatory cytokines.
For 2 mo, Long-Evans rats were fed either a low protein/high fat (LP), a standard control (C), or a high protein/low fat (HP) diet containing 5, 15, or 40% protein, respectively, and normal carbohydrate content (55% of total energy).
The HP rats ingested 12 to 15% fewer calories (P < 0.001), gained less weight (P < 0.04), and were less fatty (P < 0.01) than the other groups. Plasma Hcy was increased in HP rats compared to C (+23%) and LP (+29%) rats (P < 0.03). It was positively correlated with protein intake (r = 0.386; P < 0.01). No obvious signs of inflammation were observed in any of the groups. Hcy increase was related directly to decrease in plasma folate (r = -0.372; P < 0.02).
These data confirm the beneficial effects of HP diets on body weight but bring attention to the control of folate allowance to limit the adverse effects of elevated Hcy. Ingestion of folate-rich foods or even folate supplementation should be considered when using these HP diets over the long term for weight loss.
Nitrous oxide irreversibly inactivates cob(I)alamin, which serves as a cofactor of the enzyme methionine synthase catalyzing the remethylation of homocysteine to methionine. In patients exposed to nitrous oxide, increase in plasma homocysteine is a responsive indicator of cob(I)alamin inactivation. In the present work, we measured the inactivation of methionine synthase and the concurrent homocysteine export rate of two murine and four human cell lines during nitrous oxide exposure. When cultured in a standard medium with high content (2.3 microM) of folic acid, the methionine synthase of all cell types was inactivated at an initial rate of 0.05 to 0.14 h-1. The inactivation curves leveled off, and a residual activity of 15 to 45% was observed after 48 h of nitrous oxide exposure. The rate and extent of the nitrous oxide-induced inactivation were markedly reduced when the cells were transferred and cultured (greater than 10 days) in a medium containing low concentration (10 nM) of 5-methyltetrahydrofolate. The methionine synthase inactivation increased in a dose-dependent manner when the 5-methyltetrahydrofolate content of the medium was increased from 3 nM to 2.3 microM. The inactivation of methionine synthase was associated with a marked enhancement of homocysteine export rate of murine fibroblasts and a moderate increase in export from two human glioma cell lines. In contrast, in three leukemic cell lines (murine T-lymphoma R 1.1 cells, human promyelocytic leukemia HL-60 cells and human acute myelogenous leukemia KG-1a cells), the homocysteine export rates were not increased during nitrous oxide exposure. In the responsive murine fibroblasts and the glioma cells, the homocysteine export rate varied inversely to the changes in methionine synthase activity induced by nitrous oxide exposure at different concentrations of folate in the medium. The enhancement of homocysteine export rate of some cell types during nitrous oxide exposure probably reflects inhibition of homocysteine remethylation in intact cells, and highlights the utility of extracellular homocysteine as an indicator of metabolic flux through the methionine synthase pathway. No enhancement of homocysteine export despite inactivation of methionine synthase in three leukemic cell lines questions the functional state of the enzyme in these cells.
We used a newly developed procedure to determine reduced, oxidized, and protein-bound forms of homocysteine, cysteine, cysteinylglycine, and glutathione to measure the plasma concentrations of these species during methionine loading in six young healthy men with normal fasting concentrations of plasma homocysteine and cysteine. The methionine loading induced a transient increase in total homocysteine, which peaked after approximately 6-8 h. All six subjects showed a concurrent significant increase in reduced homocysteine and cysteine, which peaked 2 h after loading, and a rapid decrease in protein-bound cysteine and cysteinylglycine. The concentration of reduced cysteinylglycine was not altered. Plots of protein-bound cysteine and cysteinylglycine vs total homocysteine formed hysteretic loops, showing a time-dependent relation between these analytes. After the initial decrease, protein-bound cysteine and cysteinylglycine showed a slight, transient increase. From 12 to 24 h after loading, protein-bound cysteine approached preloading concentrations in two subjects and declined further in four subjects. The response pattern was similar for cysteine and cysteinylglycine in each subject. Simple displacement could not account for these effects, which suggests that plasma homocysteine may affect the disposition of other thiols through complex mechanisms. The presence of reduced homocysteine and the dynamic relation that exists between homocysteine, cysteine, and related compounds in plasma should be taken into account when evaluating plasma homocysteine as an indicator or causative agent of human disease.
We conducted a systematic evaluation of the effects of increasing levels of dietary methionine on the metabolites and enzymes of methionine metabolism in rat liver. Significant decreases in hepatic concentrations of betaine and serine occurred when the dietary methionine was raised from 0.3 to 1.0%. We observed increased concentrations of S-adenosylhomocysteine in livers of rats fed 1.5% methionine and of S-adenosylmethionine and methionine only when the diet contained 3.0% methionine. Methionine supplementation resulted in decreased hepatic levels of methyltetrahydrofolate-homocysteine methyltransferase and increased levels of methionine adenosyltransferase, betaine-homocysteine methyltransferase, and cystathionine synthase. We used these data to simulate the regulatory locus formed by the enzymes which metabolize homocysteine in livers of rats fed 0.3% methionine, 1.5% methionine, and 3.0% methionine. In comparison to the model for the 0.3% methionine diet group, the model for the 3.0% methionine animals demonstrates a 12-fold increase in the synthesis of cystathionine, a 150% increase in flow through the betaine reaction, and a 550% increase in total metabolism of homocysteine. The concentrations of substrates and other metabolites are significant determinants of this apparent adaptation.
Low concentrations (0.5-6 nmol/g) of homocysteine (Hcy) have recently been demonstrated in acid extracts of various tissues of the mouse and rat (Ueland, P.M., Helland, S., Broch, O.-J., and Schanche, J.-S. (1984) J. Biol. Chem. 259, 2360-2364). This is referred to as free Hcy in tissues. This paper describes a method for the determination of protein-bound Hcy, which involves precipitation and washing of tissue protein with ammonium sulfate, release of Hcy from native proteins in the presence of dithioerythritol, and determination of free Hcy by a sensitive radioenzymic assay. Both free and bound Hcy decreased markedly in rat tissues within a few seconds following death of the animal. The amount of protein-bound Hcy was highest in liver, somewhat lower in kidney, brain, heart, lung, and spleen. The ratio between free and bound Hcy was between 1 and 2 in most tissues, except in cerebellum, containing a large excess of free Hcy (free/bound ratio of 18). Free Hcy was almost exclusively localized to the soluble fraction of rat liver, whereas protein-bound Hcy was about equally distributed between this fraction and the microsomes. Isolated rat hepatocytes contained free and protein-bound Hcy in proportions observed in whole liver, but a large amount of Hcy was exported into the extracellular medium. The half-lives, as determined from pulse-chase experiments with [35S] methionine, were 53 s for S-adenosylmethionine, 2 s for S-adenosylhomocysteine and 3 s for Hcy (free and bound regarded as a single pool). Furthermore, isotope equilibrium between these metabolites and between free and bound Hcy throughout the rapid chase period suggests the turnover rates of S-adenosylhomocysteine and Hcy to be production rate limited, and the dissociation rate of the Hcy-protein complex may greatly exceed the turnover rate of Hcy. Thus, the half-lives of Hcy are such that participation of both free and bound Hcy in metabolic regulation is feasible.
Hyperhomocysteinemia is a common finding in dialysis-dependent end-stage renal disease (ESRD) patients, but its etiology and refractoriness to standard homocysteine-lowering B-vitamin therapy are poorly understood. In the absence of actual in vivo data, it has been hypothesized that loss of normal renal parenchymal uptake and metabolism of homocysteine is an important determinant of hyperhomocysteinemia in ESRD, given that urinary homocysteine excretion by healthy kidneys is trivial. We assessed net renal uptake and metabolism of homocysteine using an established rat model for measuring arteriovenous amino acid differences across the rat kidney, along with simultaneous determination of renal plasma flow, urine flow, and urinary homocysteine concentration. Substantial homocysteine uptake and metabolism by normal rat kidneys was demonstrated, and we also confirmed that urinary homocysteine excretion is minimal. These data suggest that loss of the sizable homocysteine metabolizing capacity of the intact kidneys may be an important determinant of the refractory, potentially atherothrombotic hyperhomocysteinemia frequently observed in ESRD.
We investigated total, free and protein-bound plasma homocysteine, cysteine and cysteinylglycine in 13 subjects aged 24-29 y after a breakfast at 0900 h containing 15-18 g of protein and a dinner at 1500 h containing approximately 50 g of protein. Twelve subjects had normal fasting homocysteine (mean +/- SD, 7.6 +/- 1.1 mumol/L) and methionine concentrations (22.7 +/- 3.5 mumol/L) and were included in the statistical analyses. Breakfast caused a small but significant increase in plasma methionine (22.2 +/- 20.6%) and a brief, nonsignificant increase followed by a significant decline in free homocysteine. However, changes in total and bound homocysteine were small. After dinner, there was a marked increase in plasma methionine by 16.7 +/- 8.9 mumol/L (87.9 +/- 49%), which was associated with a rapid and marked increase in free homocysteine (33.7 +/- 19.6%, 4 h after dinner) and a moderate and slow increase in total (13.5 +/- 7.5%, 8 h) and protein-bound (12.6 +/- 9.4%, 8 h) homocysteine. After both meals, cysteine and cysteinylglycine concentrations seemed related to changes in homocysteine, because there were parallel fluctuations in the free:bound ratios of all three thiols. Dietary changes in plasma homocysteine will probably not affect the evaluation of vitamin deficiency states associated with moderate to severe hyperhomocysteinemia but may be of concern in the risk assessment of cardiovascular disease in patients with mild hyperhomocysteinemia. Synchronous fluctuations in the free:bound ratio of the plasma aminothiol compounds indicate that biological effects of homocysteine may be difficult to separate from effects due to associated changes in other aminothiol compounds.
The plasma homocysteine response to methionine loading was assessed in vitamin B-6- and folate-deficient rats. Rats fed vitamin B-6- or folate-deficient diets for 4 wk were administered a gastric gavage of methionine (100 mg/kg body wt). Subsequent plasma analyses revealed a peak post-methionine load increase in plasma homocysteine concentration of > 300 mumol/L in the vitamin B-6-deficient rats. Folate-deficient rats exhibited no significant changes in plasma homocysteine after the load. These disparate responses can be explained by the observed increase in hepatic S-adenosylmethionine (SAM) concentration because of the load. In vitamin B-6 deficiency, increased SAM inhibits homocysteine remethylation, which, in conjunction with the impaired homocysteine catabolism due to the deficiency and the increased synthesis of homocysteine due to the methionine load, leads to a large elevation of homocysteine in the blood. In folate deficiency, increased SAM activates homocysteine catabolism, which compensates for the increased synthesis of homocysteine due to the load and thus no change in blood homocysteine is observed. These results have significant bearing on the interpretation of both positive and negative responses to methionine loading in humans.
Oxidative stress has been suggested to be an important factor in the immunopathogenesis of human immunodeficiency virus (HIV) infection. Reduced plasma thiols may lead to production of reactive oxygen species, thus contributing to the oxidative stress. We quantified the total, reduced, and protein-bound forms of the thiols homocysteine, cysteine, cysteinylglycine, and methionine in plasma from 21 HIV-infected patients and 15 healthy control subjects and compared the results with clinical and immunologic indexes. The HIV-infected patients had significantly higher concentrations of reduced homocysteine in plasma compared with control subjects. No significant differences in reduced homocysteine concentrations were noted when asymptomatic and symptomatic HIV-infected patients were compared, and we did not find any relation between reduced homocysteine concentrations and other markers of immunodeficiency. The HIV-infected patients had normal total homocysteine concentrations. The reduced cysteinylglycine concentration tended to be elevated in the patient group. No differences between HIV-infected patients and control subjects were found for reduced or total cysteine. Compared with control subjects, the HIV-infected patients had lower concentrations of methionine in plasma, and a significant correlation was found between low concentrations of methionine and low CD4+ lymphocyte counts in blood. Elevated concentrations of reduced homocysteine could possibly contribute to formation of reactive oxygen species, leading to accelerated immunologic deterioration and increased HIV replication.
Epidemiological studies have provided strong evidence that an elevated plasma homocysteine concentration is an important independent risk factor for cardiovascular disease. We have shown, in the rat, that the kidney is a major site for the removal and subsequent metabolism of plasma homocysteine [Bostom, Brosnan, Hall, Nadeau and Selhub (1995) Atherosclerosis 116, 59-62]. To characterize the role of the kidney in homocysteine metabolism further, we measured the disappearance of homocysteine in isolated renal cortical tubules of the rat. Renal tubules metabolized homocysteine primarily through the transulphuration pathway, producing cystathionine and cysteine (78% of homocysteine disappearance). Methionine production accounted for less than 2% of the disappearance of homocysteine. Cystathionine, and subsequently cysteine, production rates, as well as the rate of disappearance of homocysteine, were sensitive to the level of serine in the incubation medium, as increased serine concentrations permitted higher rates of cystathionine and cysteine production. On the basis of enrichment profiles of cystathionine beta-synthase and cystathionine gamma-lyase, in comparison with marker enzymes of known location, we concluded that cystathionine beta-synthase was enriched in the outer cortex, specifically in cells of the proximal convoluted tubule. Cystathionine gamma-lyase exhibited higher enrichment patterns in the inner cortex and outer medulla, with strong evidence of an enrichment in cells of the proximal straight tubule. These studies indicate that factors that influence the transulphuration of homocysteine may influence the renal clearance of this amino acid.
An elevated level of total homocysteine (tHcy) in blood, denoted hyperhomocysteinemia, is emerging as a prevalent and strong risk factor for atherosclerotic vascular disease in the coronary, cerebral, and peripheral vessels, and for arterial and venous thromboembolism. The basis for these conclusions is data from about 80 clinical and epidemiological studies including more than 10,000 patients. Elevated tHcy confers a graded risk with no threshold, is independent of but may enhance the effect of the conventional risk factors, and seems to be a particularly strong predictor of cardiovascular mortality. Hyperhomocysteinemia is attributed to commonly occurring genetic and acquired factors including deficiencies of folate and vitamin B12. Supplementation with B-vitamins, in particular with folic acid, is an efficient, safe, and inexpensive means to reduce an elevated tHcy level. Studies are now in progress to establish whether such therapy will reduce cardiovascular risk.
Mild hyperhomocysteinaemia is associated with increased risk for vascular disease. We studied homocysteine export from human umbilical vein endothelial cells (HUVECs) by measuring total homocysteine (tHcy) concentrations in the culture medium. Under standard culture conditions tHcy concentrations in the HUVEC culture medium increased by constant amounts after 24, 48 and 72h [mean=2.5 (SD ± 0.7) μmolL-1 homocysteine every 24 h]. As the cells are the only source of homocysteine increase in the culture medium, we designate this as homocysteine export from HUVEC. Folic acid supplementation to the culture medium lowered the homocysteine export in a dose-dependent manner. Methyl-tetrahydrofolate (MeTHF) and folinic acid (a stable precursor of MeTHF) were in this respect about 10 times more effective than folic acid. A 50% reduction in the homocysteine export was seen with 10-30 nmolL-1 MeTHF supplementation; reduction to almost zero was seen with 100-300 nmolL-1 MeTHF. Additions to the culture medium of the other vitamins involved in the homocysteine metabolism, such as vitamin B12, vitamin B6 and flavin adenine dinucleotide, did not show any effect on homocysteine export. Because homocysteine export reflects an imbalance in the homocysteine metabolism, our observations showed a susceptible dependency of this metabolism on folic acid in endothelial cells.
We studied two mammalian enzymes capable of remethylating homocysteine. Betaine-homocysteine methyltransferase was found only in rat liver while N5-methyltetrahydrofolate-homocysteine methyltransferase could be demonstrated in all tissues except small intestinal mucosa. Various hormones significantly affect the specific activity of the enzymes. These effects depend on hormone, enzyme, and tissue. The responses of the two transmethylases to dietary changes differed markedly. Betaine-homocysteine methyltransferase increased with protein and methionine feeding. N5-Methyltetrahydrofolate-homocysteine methyltransferase on the other hand increased under conditions that suggested the need for methionine synthesis. We conclude that N5-methyltetrahydrofolate-homocysteine methyltransferase activity contributes significantly to the regulation of methionine metabolism in mammals. High protein diets repress the synthesis of this enzyme in liver.
Article de synthese sur le metabolisme de la methionine, acide amine essentiel, chez les mammiferes. Etude du schema metabolique, de la regulation du metabolisme (mise en evidence et importance relative de chaque facteur). Etude de la transamination de la methionine, du role de la S-adenosyl-methionine, de l'oxydation de l'homocysteine et de la cystathionine. Etude du disfonctionnement du metabolisme et des consequences chez les hommes
1.1.|The organ distribution of the branched-chain aminotransferase ands α-ketoisocaproate (ketoleucine) dehydrogenase indicates that the muscle is the main site of deamination of branched-chain amino acids and the liver is the main site of oxidation of the carbon skeleton of leucine.2.2.|Rates of ketone body production from ketoleucine are high in the liver, and of the same order as from oleate in hepatocytes from fasted rats.3.3.|The procedures for the quantitative assay of the capacity of the ketoacid dehydrogenase are not satisfactory, as indicated by the fact that the rates of ketoleucine disappearance (corrected for amination) in hepatocytes are higher than the capacity of the enzyme in liver hemogenates supplemented optimally with co-factors.4.4.|Examples are given of other enzyem complexes where the in vivo flux shows gross discrepancies with the assayed enzyme capacity. Hepatic glutamine synthetase in the intact cell under optimal conditions exhibits only a fraction of the capacity in liver homogenates. On the other hand glutamate dehydrogenase may give unduly low rates in mitochondria incubated with glutamate because of end-product inhibition whereas in vivo the end-products are continuously removed.5.5.|Ketoleucine removal in the fasted laiver is accelerated by intermediates of the tricarboxylic acid cycle and glutamate (in homogeneates or isolated mitochondria) and by glutamate and asparagine (in hepatocytes), i.e., by ‘sparkers’ of fatty acid oxidation. The earlier hypothesis that sparkers provide ATP for the synthesis of fatty acyl CoA cannot explain their effect on ketoleucine metabolism because this substrate does not require ATP for the formation of the acyl CoA ester.6.6.|The mechanism of action of sparkers has been re-assessed. An alternative explanation is that they supply oxaloaccetate which promotes the citrate synthaase reaction and thereby regenerates CoA from the acetyl CoA arising from ketoleucine or fatty acids. In the absence of added sparkers, the supply of CoA may be rate-limiting for ketoleucine or fatty acid removal. However, there are certain sparker effects with glutamine and glutamate which cannot be explained on the basis.7.7.|The rates of disposal of the carbon skeletons of essential amino acids by reactions other than amination are of clinical interest in connection with the use of the ketoanalogues as therapeutic agents in the treatment of uremia and inborn errors of the urea cycle.
Normal young adult male and female subjects were maintained on fixed dietary regimens which were either essentially normal or were semisynthetic and curtailed in methionine and choline intakes and virtually free of cystine. The subjects maintained stable weights and remained in positive nitrogen balance or within the zone of sulfur equilibrium. Choline intakes were calculated, and urinary excretions of creatinine, creatine, and sacrosine were measured. Creatinine excretions of male subjects on essentially normal diets outweighed the total intakes of labile methyl groups. Taking into account the excretions of additional methylated compounds, as judged from published values, it appears that methyl neogenesis must normally play a role in both males and females. When labile methyl intake is curtailed, de novo formation of methyl groups is quantitatively more significant than ingestion of preformed methyl moieties. On the normal diets used in these experiments, the average homocysteinyl moiety in males cycled between methionine and homocysteine at least 1.9 times before being converted to cystathionine. For females, the average number of cycles was at least 1.5. When labile methyl intake was curtailed, the average number of cycles rose to 3.9 for males and 3.0 for females under the conditions employed.
Determination of homocysteine in plasma or serum for evaluation of cobalamin and folate deficiency is becoming an important diagnostic procedure. Accurate, rapid and low cost methods for measuring homocysteine are therefore required. We have improved an HPLC method and made it suitable for clinical application. The more important changes are the addition of an internal standard, mercaptopropionylglycine, and the use of a plasma/serum based calibration material. The method consists of the following steps: reduction of the sample with tri-n-butylphosphine, precipitation of proteins, derivatisation with ammonium 7-fluorobenzo-2-oxa-1,3-diazole-4-sulphonate, and HPLC separation followed by fluorescence detection. The linearity of the assays is established and the coefficient of variation is 3.0%. Stability studies show that blood samples must be cooled or centrifuged immediately after venipuncture. The method is useful for evaluation of cobalamin or folate deficiency, especially in patients with normal or moderately depressed cobalamin or folate concentrations in blood.
Determination of the transient increase in plasma homocysteine following administration of excess methionine is an established procedure for the diagnosis of defects in homocysteine metabolism in patients. This so-called methionine loading test has been used for 25 years, but the knowledge of the response of various cell types to excess methionine is limited. In the present paper we investigated homocysteine export from various cell types cultured in the presence of increasing concentrations (15-1,000 microM) of methionine. For comparison of homocysteine export, the export rates per million cells were plotted versus cell density for proliferating cells, and versus time for quiescent cells. The homocysteine export from growing cells was greatest during early to mid-exponential growth phase, and then decreased as a function of cell density. The export rate was higher from phytohemagglutinin-stimulated than non-stimulated lymphocytes, and higher from proliferating than from quiescent fibroblasts. The hepatocytes showed highest export rate among the cell types investigated. The enhancement of homocysteine export by excess methionine ranged from no stimulation to marked enhancement, depending on cell type investigated, and three different response patterns could be distinguished: 1) quiescent fibroblasts and growing murine lymphoma cell showed no significant increase in homocysteine export following methionine loading; export from human lymphocytes was only slightly enhanced in the presence of excess methionine; 2) the homocysteine export from proliferating hepatoma cells and benign and transformed fibroblasts was stimulated three to eightfold by increasing the methionine concentration in the medium from 15 to 1,000 microM; and 3) the response to methionine loading was particularly increased (about 15-fold) in non-transformed primary hepatocytes in stationary culture. The results outline a potentially useful procedure for the comparison of homocysteine export during cell growth in the presence of various concentrations of methionine. The results are discussed in relation to the special feature of homocysteine metabolism in various cell types and tissues including liver, and to the possible source of plasma homocysteine following methionine loading in vivo.
The aim of the present work was to evaluate in vivo the role of the transport step in hepatic amino acid metabolism. To vary hepatic utilization of amino acids, rats were adapted to diets containing various concentrations of casein (5, 15, and 60%). In rats fed 5 or 15% casein diets, Gln and Glu were released by the liver, and there was a significant uptake of Ala. Hepatic fluxes of amino acids increased considerably after adaptation to high-casein diet (up to 1.55 mumol.min-1.g liver-1 for Ala), because of the rise in afferent concentrations as well as enhanced uptake percentage (peaking at 60-75% for most glucogenic amino acids). Adaptation to a high-protein diet led to induction of not only system A but also of most of the other transport systems (Gly, anionic, T, y+, and to a lesser extent system N); only systems ASC and L were unchanged. The study of amino acid repartition between liver and plasma with different diets indicates that transport could modulate utilization of Ala, Ser, Thr, Gly, Gln, and Asp. For Arg and Asn, present in very low concentrations in liver under any condition, the transport step should be the major locus of control of their metabolism. For amino acids chiefly transported by nonconcentrative systems, such as aromatic amino acids, cellular metabolism could also be limited by the transport process. In conclusion, during adaptation to a high-protein diet, there is apparently a coordinated adaptation of amino acid transport and of their intracellular metabolism. For some amino acids, induction of catabolic enzymes seems greater than that of transport, so that the transport step may play an important role in control of metabolic fluxes. For example, concentration of amino acids such as Thr may be markedly depressed in rats adapted to a high-protein diet.
Tissue levels of S-adenosylmethionine (SAMe) were measured by a sensitive and specific isotope derivative, isotope dilution technique. The SAMe concentration in tissues can be altered by decreased production or increased utilization. Methionine administration increases SAMe concentration, while active methyl acceptors, such as pyrogallol, lower tissue levels. Tropolone, purpurogallin, pargyline, and imipramine also appear to lower tissue levels of this active methyl donor. Several environmental and pharmacological variables do not alter SAMe concentrations. Alteration of the activity of methionine-activating enzyme does not appear to be responsible for lowering SAMe levels with pyrogallol, tropolone, or pargyline. It is suggested that increased SAMe utilization is the important factor in the lowering of tissue SAMe by pyrogallol, purpurogallin, and pargyline.
There is considerable evidence that homocystine has a direct damaging effect on vascular endothelium and other tissues. The demonstration of the existence of protein-bound homocyst(e)ine has strengthened this hypothesis. In an attempt to remove bound homocyst(e)ine, D-penicillamine was given to three patients with pyridoxine-nonresponsive homocystinuria. Before the clinical trial, it had been demonstrated that 0.1 mumole per ml concentration of D-penicillamine or cysteamine released approximately 50% of the homocyst(e)ine bound to plasma proteins in vitro. Oral D-penicillamine effectively reduced both free and plasma protein-bound homocyst(e)ine in homocystinurics from the second day of treatment. The homocystine excreted in the urine was mainly in the form of homocysteine-penicillamine disulfide. No mixed disulfide was detectable in the plasma, indicating an extremely high renal clearance. These observations suggested that oral D-penicillamine removed a considerable quantity of the bound homocyst(e)ine accumulated in the tissue proteins.
Homocysteine is a thiol‐containing amino acid resulting from demethylation of methionine. The free and protein‐bound forms of the amino acid derived disulfides are called homocyst(e)ine [H(e)]. Multiple studies have shown elevated H(e) levels in patients with coronary, cerebrovascular, or peripheral arterial diseases; this association is frequent and independent of most other risk factors for atherosclerosis. In the 1993 Frontiers in Medicine Symposium* investigators discussed the genetic, physiological, nutritional, and pharmacological mechanisms involved in the regulation of plasma H(e), the association of H(e) with arterial occlusive diseases, and the relationships of H(e) with nitric oxide and haemostasis.
High plasma H(e) levels usually can be reversed with vitamin supplements. Whether vitamin supplements will affect the evolution of arterial occlusive diseases needs to be established in prospective, placebo‐controlled, randomized, clinical trials.
We investigated the nitrous oxide-induced inactivation of methionine synthase and the concurrent homocysteine (Hcy) export in mutant fibroblasts with defects in the homocysteine catabolizing enzyme, cystathionine beta-synthase, or in methionine synthase, which carries out homocysteine remethylation. The fibroblasts were incubated in various concentrations of methionine to create conditions favoring methionine conservation or catabolism. In cystathionine beta-synthase-deficient cells, high medium methionine partly protected the enzyme against inactivation, as previously found in normal fibroblasts. The Hcy export rate at low methionine levels was low (0.2-0.6 nmol/h/10(6) cells), and increased 2-3-fold at high methionine levels. Nitrous oxide enhanced Hcy export rate at low methionine, so that in the presence of nitrous oxide, the Hcy export became less dependent of methionine. In cb1G cells, the enzyme inactivation was moderate and independent of medium methionine. The Hcy export rate was intermediate (0.5-0.8 nmol/h/10(6) cells) at low methionine levels, and increased moderately (< 2-fold) at high methionine levels or following nitrous oxide exposure. In cb1E mutants, the enzyme activity was not affected by nitrous oxide, and the Hcy export was high (0.8-1.6 nmol/h/10(6) cells) and independent of methionine and nitrous oxide. These data suggest that Hcy remethylation and cystathionine beta-synthase activity are major determinants of Hcy export at low and high methionine, respectively. The low susceptibility of methionine synthase to nitrous oxide in the presence of high methionine or in cb1G or cb1E mutants is probably related to low catalytic turnover.
Mild hyperhomocysteinaemia is associated with increased risk for vascular disease. We studied homocysteine export from human umbilical vein endothelial cells (HUVECs) by measuring total homocysteine (tHcy) concentrations in the culture medium. Under standard culture conditions tHcy concentrations in the HUVEC culture medium increased by constant amounts after 24, 48 and 72 h [mean = 2.5 (SD +/- 0.7) mumol L-1 homocysteine every 24 h]. As the cells are the only source of homocysteine increase in the culture medium, we designate this as homocysteine export from HUVEC. Folic acid supplementation to the culture medium lowered the homocysteine export in a dose-dependent manner. Methyl-tetrahydrofolate (MeTHF) and folinic acid (a stable precursor of MeTHF) were in this respect about 10 times more effective than folic acid. A 50% reduction in the homocysteine export was seen with 10-30 nmol L-1 MeTHF supplementation; reduction to almost zero was seen with 100-300 nmol L-1 MeTHF. Additions to the culture medium of the other vitamins involved in the homocysteine metabolism, such as vitamin B12, vitamin B6 and flavin adenine dinucleotide, did not show any effect on homocysteine export. Because homocysteine export reflects an imbalance in the homocysteine metabolism, our observations showed a susceptible dependency of this metabolism on folic acid in endothelial cells.
Cystathionine beta-synthase (CBS) catalyzes the condensation of homocysteine and serine to cystathionine-an irreversible step in the eukaryotic transsulfuration pathway. The native enzyme is a homotetramer or multimer of 63-kDa (551 amino acids) subunits and is activated by S-adenosyl-l-methionine (AdoMet) or by partial cleavage with trypsin. Amino-terminal analysis of the early products of trypsinolysis demonstrated that the first cleavages occur at Lys 30, 36, and 39. The enzyme still retains the subunit organization as a tetramer or multimer composed of 58-kDa subunits. Analysis by electrospray ionization mass spectrometry showed that further trypsin treatment cleaves CBS in its COOH-terminal region at Arg 413 to yield 45-kDa subunits. This 45-kDa active core is the portion of CBS most conserved with the evolutionarily related enzymes isolated from plants, yeast, and bacteria. The active core of CBS forms a dimer of approximately 85 kDa. The dimer is about twice as active as the tetramer. It binds both pyridoxal 5'-phosphate and heme cofactors but is no longer activated by AdoMet. Further analysis suggests that the dissociation of CBS to dimers causes a decrease in enzyme thermostability and a threefold increase in affinity toward the sulfhydryl-containing substrate-homocysteine. We found that the COOH-terminal region, residues 414-551, is essential for maintaining the tetrameric structure and AdoMet activation of the enzyme. The inability of the active core to form multimeric aggregates has facilitated its crystallization and X-ray diffraction studies.
Elevated blood concentrations of total homocysteine (tHcy) have been implicated in the pathogenesis of atherosclerotic cardiovascular disease. Previous studies identified suboptimal nutritional status and dietary intake of folate, vitamin B-6, and vitamin B-12 as determinants of elevated tHcy.
We identified other nutritional factors associated with tHcy in 260 retired schoolteachers in the Baltimore metropolitan area.
We performed observational analyses of baseline and 2-4-mo follow-up data collected in a study designed to test the feasibility of conducting a large-scale clinical trial of vitamin supplements by mail. The study population consisted of 151 women and 109 men with a median age of 64 y. At baseline, each participant completed a food-frequency questionnaire. At follow-up, fasting serum tHcy was measured.
In multivariable linear regression and generalized linear models, there was an independent, inverse dose-response relation between dietary protein and In tHcy (P = 0.002) and a positive, significant dose-response relation between coffee consumption and In tHcy (P for trend = 0.01). Other significant predictors of In tHcy were creatinine (positive; P = 0.0001) and prestudy use of supplemental B vitamins (inverse; P = 0.03). In stratified analyses restricted to persons receiving standard multivitamin therapy, the association of 1n tHcy with dietary protein and coffee persisted.
These results support the hypothesis that increased protein intake and decreased coffee consumption may reduce tHcy and potentially prevent atherosclerotic cardiovascular disease and other disease outcomes.
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