Homocysteine Catabolism Research Articles

Engineered Carbon Nanocatalysts for Homocysteine Catabolism

Cystathionine-β-synthase (CBS) deficiency or homocystinuria (HCU) is a rare inherited disorder with main clinical features of osteoporosis, dislocation of the optic lenses, learning difficulties and thromboembolism. CBS is a key enzyme in methionine catabolism pathway. CBS deficiency impairs the conversion of homocysteine (Hcy) to cystathionine, which leads to extracellular buildup of Hcy in toxic concentrations. Patients with CBS deficiency have elevated plasma Hcy and methionine.Currently, there is no FDA approved drug for CBS deficiency. Clinical treatment options are low-protein diet and betaine for severely affected patients, and vitamin supplementation for mildly affected adults. Artificial protein-based enzymes for Hcy treatment are in clinical trials. However, protein-based artificial enzymes can be immunogenic, prone to intracellular modifications, and the treatment may not be viable after few months.We propose a novel nanotechnology-based approach for the treatment of CBS deficiency. Carbon nanomaterials with multiple unsaturated carbons provide an excellent scaffold for engineering enzyme mimicking catalysts. We have designed carbon nanomaterial based catalytic artificial enzymes (CAEs) by surface engineering to mimic metabolic enzymes that catabolize Hcy under physiological conditions. Using a combination of paper chromatography and H1-NMR, our lab has shown that CAE can breakdown homocysteine and produce hydrogen sulfide similar to CGL enzyme. The degree of surface functionalization as well as surface curvature has profound effect on specificity and catalysis. We have shown that CAE can enter cells and lower homocysteine levels in cellular models. In preliminary studies, CAE was able to lower serum homocysteine levels in a murine model. Further studies will involve safety assessments of CAE as a treatment for HCU.

Exogenous administration of hydrogen sulfide alleviates homocysteine induced inflammation in ARPE-19 cells

Plasma homocysteine (Hcy) is an independent risk factor for Age related macular degeneration (AMD) and an inducer of inflammation. Homocysteine catabolism releases hydrogen sulfide (H2S). H2S has controversial effects on inflammation. In this study we have analysed the endogenous and exogenous H2S in modulating inflammation using adult retinal pigment epithelial (ARPE-19) cells as an in vitro model for AMD. ARPE-19 cells were treated with various concentrations of Hcy (15, 30 and 50 μM) for 3 h. Expression of Hcy transulfuration genes (CBS, CSE) by qPCR and western blot. H2S levels were measured using Free Radical Analyzer System (WPI, USA). The inflammatory markers (IL-6 and IL-8) were evaluated using real-time PCR and ELISA. Hcy exposure increased CBS protein expression, hydrogen sulfide levels and pro-inflammatory cytokines, modulating CBS by silencing did not alter H2S levels, but inhibition of CSE with PAG inhibited H2S production and decreased cytokine (IL-6 and IL-8) levels. On the contrary exogenous supply of hydrogen sulfide with NaHS and by compound 1c showed anti-inflammatory effects even in the presence of Hcy. This study shows that exogenous delivery of H2S decreases inflammation in retinal pigment epithelial cells on exposure to Hcy in ARPE-19 cells.

Chronoeffects of the Herbal Medicines Puerariae radix and Coptidis rhizoma in Mice: A Potential Role of REV-ERBα.

Identifying drugs with dosing time-dependent effects (chronoeffects) and understanding the underlying mechanisms would help to improve drug treatment outcome. Here, we aimed to determine chronoeffects of the herbal medicines Puerariae radix (PR) and Coptidis rhizoma (CR), and investigate a potential role of REV-ERBα as a drug target in generating chronoeffects. The pharmacological effect of PR on hyperhomocysteinemia in mice was evaluated by measuring total homocysteine, triglyceride levels and lipid accumulation. PR dosed at ZT10 generated a stronger effect on hyperhomocysteinemia than drug dosed at ZT2. Furthermore, PR increased the expression levels of REV-ERBα target genes Bhmt, Cbs and Cth (encoding three key enzymes responsible for homocysteine catabolism), thereby alleviating hyperhomocysteinemia in mice. Moreover, CR attenuated chronic colitis in mice in a dosing time-dependent manner based on measurements of disease activity index, colon length, malondialdehyde/myeloperoxidase activities and IL-1β/IL-6 levels. ZT10 dosing generated a stronger anti-colitis effect as compared to ZT2 dosing. This was accompanied by lower production of colonic inflammatory cytokines (i.e., Nlrp3, IL-1β, IL-6, Tnf-α and Ccl2, REV-ERBα target genes) in colitis mice dosed at ZT10. The diurnal patterns of PR and CR effects were respectively consistent with those of puerarin (a main active constituent of PR, a REV-ERBα antagonist) and berberine (a main active constituent of CR, a REV-ERBα agonist). In addition, loss of Rev-erbα in mice abolished the dosing time-dependency in PR and CR effects. In conclusion, the therapeutic effects of PR and CR depend on dosing time in mice, which are probably attributed to diurnal expression of REV-ERBα as the drug target. Our findings have implications for improving therapeutic outcomes of herbal medicines with a chronotherapeutic approach.

Toxicoproteomic Profiling of hPXR Transgenic Mice Treated with Rifampicin and Isoniazid.

Tuberculosis is a global health threat that affects millions of people every year, and treatment-limiting toxicity remains a considerable source of treatment failure. Recent reports have characterized the nature of hPXR-mediated hepatotoxicity and the systemic toxicity of antitubercular drugs. The antitubercular drug isoniazid plays a role in such pathologic states as acute intermittent porphyria, anemia, hepatotoxicity, hypercoagulable states (deep vein thrombosis, pulmonary embolism, or ischemic stroke), pellagra (vitamin B3 deficiency), peripheral neuropathy, and vitamin B6 deficiency. However, the mechanisms by which isoniazid administration leads to these states are unclear. To elucidate the mechanism of rifampicin- and isoniazid-induced liver and systemic injury, we performed tandem mass tag mass spectrometry-based proteomic screening of mPxr−/− and hPXR mice treated with combinations of rifampicin and isoniazid. Proteomic profiling analysis suggested that the hPXR liver proteome is affected by antitubercular therapy to disrupt [Fe–S] cluster assembly machinery, [2Fe–2S] cluster-containing proteins, cytochrome P450 enzymes, heme biosynthesis, homocysteine catabolism, oxidative stress responses, vitamin B3 metabolism, and vitamin B6 metabolism. These novel findings provide insight into the etiology of some of these processes and potential targets for subsequent investigations. Data are available via ProteomeXchange with identifier PXD019505.

Chronopharmacological targeting of Rev-erbα by puerarin alleviates hyperhomocysteinemia in mice

Hyperhomocysteinemia is associated with poor health, including cardiovascular and brain diseases. Puerarin, initially isolated from Puerariae radix, has been shown to possess anti-hyperhomocysteinemia effect. However, the mechanism of puerarin action remains unknown. Here, we uncovered that puerarin targeted the circadian clock protein Rev-erbα to alleviate hyperhomocysteinemia in mice in a circadian time-dependent manner. We first identified puerarin as an antagonist of Rev-erbα based on luciferase reporter, Gal4 co-transfection and target gene expression assays. Consistent with an antagonistic effect, puerarin induced mRNA and protein expressions of Bhmt, Cbs and Cth (three enzymes involved in homocysteine catabolism and known targets of Rev-erbα) in Hepa-1c1c7 cells. These induction effects of puerarin were lost in Rev-erbα-deficient cells. Furthermore, puerarin dose-dependently alleviated methionine-induced hyperhomocysteinemia in mice as evidenced by decreased levels of total homocysteine and triglyceride. This was accompanied by increased expressions of Bhmt, Cbs and Cth in the liver. Moreover, puerarin dosed at ZT10 generated stronger pharmacological effects than drug dosed at ZT22 consistent with diurnally rhythmic expression of Rev-erbα (a high expression at ZT10 and a low expression at ZT22). In conclusion, puerarin targets Rev-erbα to alleviate hyperhomocysteinemia in mice in a circadian time-dependent manner. The finding of a circadian gene as drug target encourages chronotherapeutic practices on puerarin and related medications for optimized efficacy.

Decreased Homocysteine Trans-Sulfuration in Hypertension With Hyperhomocysteinemia: Relationship With Insulin Resistance.

Homocysteine is an independent cardiovascular risk factor and is elevated in essential hypertension. Insulin stimulates homocysteine catabolism in healthy individuals. However, the mechanisms of hyperhomocysteinemia and its relationship with insulin resistance in essential hypertension are unknown. To investigate whole body methionine and homocysteine kinetics and the effects of insulin in essential hypertension. Eight hypertensive male subjects and six male normotensive controls were infused with l-[methyl-2H3,1-13C]methionine for 6 hours. In the last 3 hours a euglycemic, hyperinsulinemic clamp was performed. Steady-state methionine and homocysteine kinetics were determined in postabsorptive and hyperinsulinemic conditions. Postabsorptive hypertensive subjects had elevated homocysteine concentrations (+30%, P = 0.035) and slightly (by 15% to 20%) but insignificantly lower methionine rates of appearance (Ras) (P = 0.07 to P = 0.05) and utilization for protein synthesis (P = 0.06) than postabsorptive normotensive controls. Hyperinsulinemia suppressed methionine Ra and protein synthesis, whereas it increased homocysteine trans-sulfuration, clearance, and methionine transmethylation (the latter only in the normotensive subjects). However, in the hypertensive subjects trans-sulfuration was significantly lower (P < 0.05) and increased ~50% less [by +1.59 ± 0.34 vs +3.45 ± 0.52 µmol/kg lean body mass (LBM) per hour, P < 0.005] than in normotensive controls. Homocysteine clearance through trans-sulfuration was ~50% lower in hypertensive than in normotensive subjects (P < 0.005). In the hypertensive subjects, insulin-mediated glucose disposal was ~45% lower (460 ± 44 vs 792 ± 67 mg/kg LBM per hour, P < 0.0005) than in normotensive controls and was positively correlated with the increase of trans-sulfuration (P < 0.0015). In subjects with essential hypertension, hyperhomocysteinemia is associated with decreased homocysteine trans-sulfuration and probably represents a feature of insulin resistance.

Ratios of One-Carbon Metabolites Are Functional Markers of B-Vitamin Status in a Norwegian Coronary Angiography Screening Cohort

Background: Functional (metabolic) markers of B-vitamin status, including plasma total homocysteine (tHcy) for folate and plasma methylmalonic acid (MMA) for vitamin B-12, suffer from moderate sensitivity and poor specificity. Ratios of metabolites belonging to the same pathway may have better performance characteristics.Objective: We evaluated the ratios of tHcy to total cysteine (tCys; Hcy:Cys), tHcy to creatinine (Hcy:Cre), and tHcy to tCys to creatinine (Hcy:Cys:Cre) as functional markers of B-vitamin status represented by a summary score composed of folate, cobalamin, betaine, pyridoxal 5'-phosphate (PLP), and riboflavin concentrations measured in plasma.Methods: Cross-sectional data were obtained from a cohort of patients with stable angina pectoris (2994 men and 1167 women) aged 21-88 y. The relative contribution of the B-vitamin score, age, sex, smoking, body mass index, and markers of renal function and inflammation to the variance of the functional B-vitamin markers was calculated by using multiple linear regression.Results: Compared with tHcy alone, Hcy:Cys, Hcy:Cre, and Hcy:Cys:Cre all showed improved sensitivity and specificity for detecting plasma B-vitamin status. Improvements in overall performance ranged from 4-fold for Hcy:Cys to ∼8-fold for Hcy:Cys:Cre and were particularly strong in subjects with the common 5,10-methylenetetrahydrofolate reductase (MTHFR) 677CC genotype.Conclusions: Ratios of tHcy to tCys and/or creatinine showed a severalfold improvement over tHcy alone as functional markers of B-vitamin status in Norwegian coronary angiography screenees. The biological rationale for these ratios is discussed in terms of known properties of enzymes involved in the catabolism of homocysteine and synthesis of creatine and creatinine.

Targeted metabolomics and mathematical modeling demonstrate that vitamin B-6 restriction alters one-carbon metabolism in cultured HepG2 cells.

Low vitamin B-6 nutritional status is associated with increased risk for cardiovascular disease and certain cancers. Pyridoxal 5'-phosphate (PLP) serves as a coenzyme in many cellular processes, including several reactions in one-carbon (1C) metabolism and the transsulfuration pathway of homocysteine catabolism. To assess the effect of vitamin B-6 deficiency on these processes and associated pathways, we conducted quantitative analysis of 1C metabolites including tetrahydrofolate species in HepG2 cells cultured in various concentrations of pyridoxal. These results were compared with predictions of a mathematical model of 1C metabolism simulating effects of vitamin B-6 deficiency. In cells cultured in vitamin B-6-deficient medium (25 or 35 nmol/l pyridoxal), we observed >200% higher concentrations of betaine (P < 0.05) and creatinine (P < 0.05) and >60% lower concentrations of creatine (P < 0.05) and 5,10-methenyltetrahydrofolate (P < 0.05) compared with cells cultured in medium containing intermediate (65 nmol/l) or the supraphysiological 2,015 nmol/l pyridoxal. Cystathionine, cysteine, glutathione, and cysteinylglycine, which are components of the transsulfuration pathway and subsequent reactions, exhibited greater concentrations at the two lower vitamin B-6 concentrations. Partial least squares discriminant analysis showed differences in overall profiles between cells cultured in 25 and 35 nmol/l pyridoxal vs. those in 65 and 2,015 nmol/l pyridoxal. Mathematical model predictions aligned with analytically derived results. These data reveal pronounced effects of vitamin B-6 deficiency on 1C-related metabolites, including previously unexpected secondary effects on creatine. These results complement metabolomic studies in humans demonstrating extended metabolic effects of vitamin B-6 insufficiency.

Effects of vitamin B6 metabolism on oncogenesis, tumor progression and therapeutic responses

Pyridoxal-5'-phosphate (PLP), the bioactive form of vitamin B6, reportedly functions as a prosthetic group for >4% of classified enzymatic activities of the cell. It is therefore not surprising that alterations of vitamin B6 metabolism have been associated with multiple human diseases. As a striking example, mutations in the gene coding for antiquitin, an evolutionary old aldehyde dehydrogenase, result in pyridoxine-dependent seizures, owing to the accumulation of a metabolic intermediate that inactivates PLP. In addition, PLP is required for the catabolism of homocysteine by transsulfuration. Hence, reduced circulating levels of B6 vitamers (including PLP as well as its major precursor pyridoxine) are frequently paralleled by hyperhomocysteinemia, a condition that has been associated with an increased risk for multiple cardiovascular diseases. During the past 30 years, an intense wave of clinical investigation has attempted to dissect the putative links between vitamin B6 and cancer. Thus, high circulating levels of vitamin B6, as such or as they reflected reduced amounts of circulating homocysteine, have been associated with improved disease outcome in patients bearing a wide range of hematological and solid neoplasms. More recently, the proficiency of vitamin B6 metabolism has been shown to modulate the adaptive response of tumor cells to a plethora of physical and chemical stress conditions. Moreover, elevated levels of pyridoxal kinase (PDXK), the enzyme that converts pyridoxine and other vitamin B6 precursors into PLP, have been shown to constitute a good, therapy-independent prognostic marker in patients affected by non-small cell lung carcinoma (NSCLC). Here, we will discuss the clinical relevance of vitamin B6 metabolism as a prognostic factor in cancer patients.

Plasma homocysteine levels in patients with liver cirrhosis

Homocysteine (2-amino-4-mercaptobutyric acid) is an amino acid that may be found in small quantities in all cells, and is quantitatively the major methionine metabolite. The most prevalent form is protein-bound homocysteine (about 80%), mostly to albumins. If catabolism of homocysteine is impaired either due to enzyme defect or deficiency of required intracellular cofactors, homocysteine accumulates in cells and reaches the circulation. The aim of our study was to determine homocysteine values and factors affecting homocysteine metabolism in patients with liver cirrhosis. The prospective study included 35 patients with liver cirrhosis and 30 age and sex matched healthy controls. All the examinations were based on: medical history, physical examination, laboratory tests including serum homocysteine levels and liver biopsy. The degree of liver failure was assessed according to the Child-Pugh classification. The mean plasma homocysteine levels were much higher in the patients with liver cirrhosis than in the healthy controls (t-test, p < 0.001). There was no significant difference between the plasma homocysteine concentration and etiology of liver cirrhosis (ANOVA, p > 0.05). Correlation analysis showed a positive correlation between the homocysteine and creatinine concentrations and between the serum albumin and homocysteine values, (Pearson's correlation, p < 0.01, and p < 0.05 respectively). In liver cirrhosis, the genesis of homocysteinemia is multifactorial, influenced significantly by impaired catabolic liver function, renal failure and hypoalbuminemia.

Content of Total Homocysteine and Major Aminothiols in Rats with Experimental Renal Ischemia

The contents of total homocysteine, cysteine, and glutathione in blood plasma and tissue of rats with renal ischemia were measured by HPLC. Our study was performed on the "two-kidney, one-clip (0.13 mm)" model. The concentrations of homocysteine and cysteine in blood plasma from treated rats were higher than in sham-operated animals (control; by 36 and 14%, respectively). Homocysteine level in the intact and clipped kidneys of treated rats was 40% higher than in the control. However, no differences were found in homocysteine level in the ischemic and intact kidneys of treated animals. Cysteine concentration in the clipped kidney was lower than in the kidneys of intact and sham-operated animals (by 1.6 and 1.5 times, respectively). Glutathione concentration in the ischemic kidney did not differ from the control. No differences were revealed in the content of aminothiols in liver samples from rats of the treatment and control groups. Our results suggest that functional inactivation of one kidney is accompanied by impairment of homocysteine catabolism (trans-sulfonation).

Foreword to special issue on homocysteine disorders

Foreword to special issue on homocysteine disorders

Plasma homocysteine levels in patients with β‐thalassaemia major

Objective. We investigated the level of homocysteine (HCY) and its relation with vitamin B12, folate and oxidative stress in patients with β‐thalassaemia major. Material and methods. Plasma HCY, methionine, advanced oxidation protein products (AOPP) and serum vitamin B12, folate, ferritin and total antioxidant capacity (TAC) were determined in 32 thalassaemic patients and 27 control subjects. Results. HCY (6.44±0.44 versus 8.71±0.57 µmol/L), methionine (12.57±1.8 versus 22.2±3.8 µmol/L), folate (9.14±0.48 versus 15.38±0.71 nmol/L) and TAC (0.34±0.03 versus 0.56±0.03 mmol/L) significantly decreased in thalassaemic patients, whereas AOPP (20.26±1.8 versus 11.30±0.2 μmol/L) and ferritin (3481.0±512 versus 46.9±4.6 ng/mL) significantly increased. Vitamin B12 levels were similar in both groups (259.1±16.6 versus 228.9±7.4 pmol/L). Conclusions. These findings suggest that increased and uncompensated oxidative stress may lead to an increment in HCY catabolism in thalassaemic patients.

Homocysteine and Parkinson's disease: A dangerous liaison?

Homocysteine, a sulphur-containing amino acid formed by demethylation of methionine, is involved in numerous processes of methyl group transfer, all playing pivotal roles in the biochemistry of the human body. Increased levels of plasma homocysteine (hyperhomocysteinemia) – which may result from a deficiency of folate, vitamin B6 or B12 or mutations in enzymes regulating the catabolism of homocysteine – are associated with a wide range of clinical manifestations, mostly affecting the central nervous system ( e.g., mental retardation, cerebral atrophy and epileptic seizures). Recent evidence suggests that changes in the metabolic fate of homocysteine, leading to hyperhomocysteinemia, may also play a role in the pathophysiology of neurodegenerative disorders, particularly Parkinson's disease (PD). The nervous system might be particularly sensitive to homocysteine, due to the excitotoxic-like properties of the amino acid. However, experimental findings have shown that homocysteine does not seem to posses direct, cytotoxic activity, while the amino acid has proven able to synergize with more specific neurotoxic insults. Hyperhomocysteinemia has been repeatedly reported in PD patients; the increase, however, seems mostly related to the methylated catabolism of l-Dopa, the main pharmacological treatment of PD. Therefore, hyperhomocysteinemia may not be specific to movement disorders or other neurological diseases, the condition being, in fact, rather the result of the combinations of different factors, mainly metabolic, but also genetic and pharmacological, intervening in the neurodegenerative process.

Effects of catechin on homocysteine metabolism in hyperhomocysteinemic mice

We have recently focused on the interaction between hyperhomocysteinemia, defined by high plasma homocysteine levels, and paraoxonase-1 expression and found a reduced activity of paraoxonase-1 associated with a reduced gene expression in the liver of cystathionine beta synthase (CBS) deficient mice, a murine model of hyperhomocysteinemia. As it has been demonstrated that polyphenolic compounds could modulate the expression level of the paraoxonase-1 gene in vitro, we have investigated the possible effect of flavonoid supplementation on the impaired paraoxonase-1 gene expression and activity induced by hyperhomocysteinemia and have evaluated the link with homocysteine metabolism. High-methionine diet significantly increased serum homocysteine levels, decreased hepatic CBS activity, and down-regulated paraoxonase-1 mRNA and its activity. However, chronic administration of catechin but not quercetin significantly reduced plasma homocysteine levels, attenuated the reduction of the hepatic CBS activity, and restored the decreased paraoxonase-1 gene expression and activity induced by chronic hyperhomocysteinemia. These data suggest that catechin could act on the homocysteine levels by increasing the rate of catabolism of homocysteine.

New insights into the regulation of methyl group and homocysteine metabolism.

Hepatic folate, methyl group, and homocysteine metabolism are interrelated pathways that when disrupted are associated with numerous pathologies. Maintenance of normal methyl group and homocysteine homeostasis is dependent on the balance between: S-adenosylmethionine (SAM)-dependent transmethylation, which utilizes methyl groups and produces homocysteine; remethylation of homocysteine back to methionine by folate-dependent and -independent mechanisms; and homocysteine catabolism via the transsulfuration pathway. Recent studies have demonstrated that hormonal imbalance is a factor in the control of key proteins that regulate these pathways. A diabetic state is characterized by increased expression of specific methyltransferases that utilize SAM-derived methyl groups and produce homocysteine. Although the supply of methyl groups from the folate-dependent 1-carbon pool appears to be diminished under diabetic conditions, the increased production of homocysteine is compensated for by stimulation of folate-independent remethylation and catabolism by transsulfuration, resulting in hypohomocysteinemia. Similar changes have been observed with glucocorticoid administration and in a growth hormone-deficient model, which can be prevented by insulin and growth hormone treatment, respectively. Taken together, these reports clearly indicate that hormonal regulation is a major factor in the metabolic control of folate, methyl groups, and homocysteine, thereby providing a potential link between the pathologies associated with these pathways and hormonal imbalance.

Effects of Insulin Deprivation and Treatment on Homocysteine Metabolism in People with Type 1 Diabetes

Abnormal homocysteine metabolism may contribute to increased cardiovascular death in type 1 diabetes (T1DM). Amino acid metabolism is altered in T1DM. In vitro, insulin reduces hepatic catabolism of homocysteine by inhibiting liver transsulfuration. It remains to be determined whether methionine-homocysteine metabolism is altered in T1DM. We sought to determine whether insulin deficiency during insulin deprivation or high plasma insulin concentration after insulin treatment alters homocysteine metabolism in T1DM. This was an acute interventional study with paired and comparative controls. The study was conducted at a general clinical research center. We used stable isotope tracers to measure methionine-homocysteine kinetics in six patients with T1DM during insulin deprivation (I-) and also during insulin treatment (I+) and compared them with nondiabetic controls (n = 6). Homocysteine kinetics (transmethylation, transsulfuration, and remethylation) were from plasma isotopic enrichment of methionine and homocysteine and (13)CO(2). T1DM (I-) had lower rates of homocysteine-methionine remethylation (P < 0.05 vs. control and I+). In contrast, transsulfuration rates were higher in I- than controls and I+ (P < 0.05). Insulin treatment normalized transsulfuration and remethylation (P < 0.05 vs. I- and P > 0.8 vs. control). Plasma homocysteine concentrations were lower in T1DM (P < 0.05 vs. control during both I- and I+), which may be explained by increased homocysteine transsulfuration. Thus, significant alterations of methionine-homocysteine metabolism occur during insulin deprivation in humans with T1DM. Insulin plays a key role in the regulation of methionine-homocysteine metabolism in humans, and altered homocysteine may occur during insulin deficiency in type 1 diabetic patients.

Expression of mutant human cystathionine β-synthase rescues neonatal lethality but not homocystinuria in a mouse model

Cystathionine beta-synthase (CBS) deficiency is a recessive genetic disorder in humans characterized by elevated levels of total plasma homocysteine (tHcy) and frequent thrombosis in humans. The I278T mutation is the most common mutation found in human CBS-deficient patients. The T424N mutation was identified as a mutation in human CBS that could restore function to I278T in Saccharomyces cerevisiae. In this report, we have engineered mice that express human I278T and I278T/T424N proteins from a metallotheinein-driven transgene. These transgene-containing mice were then bred to CBS knockout animals (Cbs-) to generate mice that express only human I278T or I278T/T424N protein. Both the I278T and the I278T/T424N transgenes are able to entirely rescue the previously described neonatal mortality phenotype despite the animals having a mean tHcy of 250 microm. The transgenic Cbs-/- animals exhibit facial alopecia, have moderate liver steatosis and are slightly smaller than heterozygous littermates. In contrast to human CBS deficiency, these mice do not exhibit extreme methioninemia. The mutant proteins are stable in the liver, kidney and colon, and liver extracts have only 2-3% of the CBS enzyme activity found in wild-type mice. Surprisingly, the I278T/T424N enzyme had exactly the same activity as the I278T enzyme indicating that T424N is unable to suppress I278T in mice. Our results show that elevated tHcy per se is not responsible for the neonatal lethality observed in Cbs-/- animals and suggests that CBS protein may have a function in addition to its role in homocysteine catabolism. These transgenic animals should be useful in the study of homocysteine related human disease.

Alteration of homocysteine catabolism in pre-eclampsia, HELLP syndrome and placental insufficiency

Hyperhomocysteinemia is a risk factor in obstetrical complications such as pre-eclampsia, 'hemolysis, elevated liver enzymes, low platelet' (HELLP)-syndrome and placental insufficiency. The aim of our study was to investigate the alterations of homocysteine catabolism in these patients in relation to serum B-vitamins and renal function. Maternal fasting serum from pre-eclampsia (n=24), HELLP (n=20) and placental insufficiency (n=25) patients at the time of diagnosis and pregnant controls (n=34) was analyzed for homocysteine and its metabolites cystathionine and methylmalonic acid, the vitamins B6, B12 and folate, renal and additional parameters. Cystathionine, a parameter of homocysteine catabolism, was significantly increased in pre-eclampsia and HELLP compared with controls and placental insufficiency patients (mean concentrations: 343, 324, 248, 227 nmol/l; p=0.001). Homocysteine, folic acid, vitamin B6 and methylmalonic acid, however, did not differ significantly between groups. The main determinants of cystathionine are cystatin C and vitamin B6, whereas the main determinants of homocysteine are folate and uric acid. The strongest dependency of cystathionine on vitamin B6 was observed in pre-eclampsia and HELLP patients. The results suggest that the vitamin B6-dependent trans-sulfuration pathway is activated in pre-eclampsia and HELLP syndrome, probably by oxidative stress. Therefore, the demand for vitamin B6 is increased in these patients. Furthermore, renal dysfunction and low vitamin B6 levels contribute to the increase of cystathionine in pre-eclampsia and HELLP patients.

Altered Gene Expression in Liver from a Murine Model of Hyperhomocysteinemia

Cystathionine beta-synthase (CBS) deficiency causes severe hyperhomocysteinemia and other signs of homocystinuria syndrome, in particular a premature atherosclerosis with multiple thrombosis. However, the molecular mechanisms by which homocysteine could interfere with normal cell function are poorly understood in a whole organ like the liver, which is central to the catabolism of homocysteine. We used a combination of differential display and cDNA arrays to analyze differential gene expression in association with elevated hepatic homocysteine levels in CBS-deficient mice, a murine model of hyperhomocysteinemia. Expression of several genes was found to be reproducibly abnormal in the livers of heterozygous and homozygous CBS-deficient mice. We report altered expression of genes encoding ribosomal protein S3a and methylthioadenosine phosphorylase, suggesting such cellular growth and proliferation perturbations may occur in homozygous CBS-deficient mice liver. Many up- or down-regulated genes encoded cytochromes P450, evidence of perturbations of the redox potential in heterozygous and homozygous CBS-deficient mice liver. The expression of various genes involved in severe oxidative processes was also abnormal in homozygous CBS-deficient mice liver. Among them, the expression of heme oxygenase 1 gene was increased, concomitant with overexpression of heme oxygenase 1 at the protein level. Commensurate with the difference in hepatic mRNA paraoxonase 1 abundance, the mean hepatic activity of paraoxonase 1, an enzyme that protects low density lipoprotein from oxidation, was 3-fold lower in homozygous CBS-deficient mice. Heterozygous CBS-deficient mice, when fed a hyperhomocysteinemic diet, have also reduced PON1 activity, which demonstrates the effect of hyperhomocysteinemia in the paraoxonase 1 activity.