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Homocysteine and coronary artery disease

Robinson K.; Mayer E.; Jacobsen D.W. Department of Cardiology, Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195 USA CLEVEL. CLIN. J. MED. (USA) , 1994, 61/6 (438-450)

Background: Homocystinuria is a rare autosomal recessive disease complicated by early and aggressive occlusive arterial disease. This may be related to the grossly increased homocysteine concentrations seen in this disease. More recently, milder hyperhomocysteinemia has been proposed as an independent risk factor for coronary artery disease.

Summary: Many patients with homozygous homocystinuria develop severe premature atherosclerosis and thromboembolism probably caused by abnormally high concentrations of homocysteine. Homocysteine undergoes metabolism either by remethylation or transsulfuration, and deficiency or dysfunction of any of the substances that regulate these reactions may lead to hyperhomocysteinemia. Homocysteine may have adverse effects on platelets, clotting factors, and endothelial cells. Studies have demonstrated significantly higher plasma homocysteine levels in patients with occlusive arterial disease than in controls. The causes are not clearly understood but may include deficiency of vitamin G6, vitamin B12, and folic acid and heterozygosity for cystathionine synthase deficiency. Vitamin supplementation can lower plasma homocysteine levels.

Conclusions: Whether measuring plasma homocysteine levels in patients with coronary artery disease should be routine and whether treating hyperhomocysteinemia in these patients may reduce the risk of coronary events remains to be determined.

Importance of elevated plasma homocysteine levels as a risk factor for atherosclerosis

Masser P.A.; Taylor L.M. Jr.; Porter J.M. Division of Vascular Surgery, Oregon Health Sciences University, 3181 SW Sam Jackson Park Road, Portland, OR 97201-3098 USA ANN. THORAC. SURG. (USA) , 1994, 58/4 (1240-1246)

Atherosclerosis is a leading cause of death and disability in the Western world, and an important risk factor for it may be an elevated level of the plasma amino acid homocysteine. The biochemical characteristics of homocysteine, along with historical laboratory, and clinical evidence for its pathologic role in atherosclerosis, are reviewed. Possible therapies for reducing elevated homocysteine levels and the possible impact of therapy in atherosclerosis are examined.

Relationship between homocysteine and cardiovascular risk factors in obesity

Suter P.M.; Locher R.; Lehmann E.; Vetter W. Ramistrasse 100, CH-8091 Zurich Switzerland NIEREN- HOCHDRUCKKR. (Germany) , 1994, 23/10 (511-512)

Increased homocystein levels may be a risk factor for the development of cardiovascular disease. In 22 overweight subjects (mean plus or minus SD, 39 plus or minus 14 years, body mass index 33 plus or minus 4 kg/m2) a positive correlation between the plasma homocysteine levels and the systolic, diastolic blood pressure, plasma triglyceride levels and the waist/hip ratio was found. A negative correlation was found between the plasma homocysteine levels and the HDL-cholesterol, HDL2-cholesterol, HDL3-cholesterol. Our data show, that the presence of certain cardiovascular risk factors (high blood pressure hypercholesterolemia, increased abdominal fat) are associated with higher plasma homocysteine levels. This supports the concept, that homocysteine may play an important role in the pathogenesis of atherosclerosis.

A prospective study of plasma homocyst(e)ine and risk of ischemic stroke

Verhoef P.; Hennekens C.H.; Malinow M.R.; Kok F.J.; Willett W.C.; Stampfer M.J. Channing Laboratory, 180 Longwood Ave, Boston, MA 02115 USA STROKE (USA) , 1994, 25/10 (1924-1930)

Background and Purpose: Several studies have reported elevated circulating homocyst(e)ine levels in subjects with cerebral atherosclerosis. We assessed prospectively whether high plasma levels of homocyst(e)ine affect risk of ischemic stroke and evaluated whether high blood pressure modifies any such effect.

Methods: The study sample was drawn from the Physicians' Health Study, a randomized, double-blind, placebo-controlled trial of aspirin and beta-carotene in 22 071 US male physicians. A total of 14 916 subjects 40 to 84 years old with no prior history of stroke, transient ischemic attack, or myocardial infarction provided blood samples at baseline and were followed for 5 years, with 99.7% morbidity and 100% mortality follow-up. Using a nested case-control design, we assayed homocyst(e)ine in samples from 109 subjects who subsequently developed ischemic stroke and 427 control subjects.

Results: The mean plasma concentration of homocyst(e)ine was slightly higher in subjects with stroke (11.1plus or minus4.0 (plus or minusSD) nmol/mL) than in control subjects (10.6plus or minus3.4 nmol/mL), but the difference was not statistically significant (P=.12). The crude odds ratio of ischemic stroke for subjects in the upper 20% (>12.7 nmol/mL) compared with those in the bottom 80% of homocyst(e)ine levels was 1.4 (95% confidence interval, 0.8 to 2.2). The odds ratio was 1.2 (95% confidence interval, 0.7 to 2.0) after controlling for several risk factors and other potential confounders. In subgroup analyses, elevated homocyst(e)ine levels appeared to be more strongly predictive of ischemic stroke in normotensive subjects and in men 60 years or younger. Although not statistically significant, in these subgroups increases in risks of 100% and 70%, respectively, were observed for men in the upper 20% of homocyst(e)ine values.

Conclusions: In this study, the data were compatible with a small but nonsignificant association between elevated plasma homocyst(e)ine and risk of ischemic stroke. However, since the sample size is small and the confidence intervals are wide, either no association or a moderate increase in risk cannot be excluded, particularly in subgroups otherwise at low risk, eg, younger men and those with normal blood pressure.

Arterial disease in hyperhomocysteinaemia and the effect of treatment: A pilot study

Van Den Berg M.; Stehouwer C.D.A.; Boers G.H.J.; Rauwerda J.A.; Kluft C. Dept. of Surgery, Institute for Cardiovasc. Research, Free University Hospital, Amsterdam Netherlands FIBRINOLYSIS (United Kingdom) , 1994, 8/SUPPL. 2 (88-90)

Elevated plasma levels of homocysteine, which are associated with endothelial dysfunction and atherosclerosis, can be reduced by treatment with vitamin B6 and folic acid. We studied the effects of one year of such treatment in 18 hyperhomocysteinaemic patients with vascular disease. Treatment was associated with decreases in the plasma levels of von Willebrand factor, thrombomodulin and endothelin (all elevated at baseline) but not of tissue-type plasminogen activator and sE-selectin (normal at baseline). These preliminary results suggest amelioration of endothelial dysfunction through treatment of hyperhomocysteinaemia.

Impaired homocysteine metabolism: A risk factor in young adults with atherosclerotic arterial occlusive disease of the leg

Aronson D.C.; Onkenhout W.; Raben A.M.T.J.; Oudenhoven L.F.I.J.; Brommer E.J.P.; Van Bockel J.H. Department of Surgery, University Hospital, PO Box 9600, 2300 RC Leiden Netherlands BR. J. SURG. (United Kingdom) , 1994, 81/8 (1114-1118)

To assess the prevalence of impaired homocysteine metabolism in young adults with arterial occlusive disease, 80 consecutive patients under 45 years old were screened. Various laboratory blood investigations and a standardized methionine loading test were performed. In the first 52 patients plasma levels of free homocysteine were determined; thereafter the levels of total homocysteine (a more sensitive measure of impaired homocysteine metabolism) were measured. The methionine loading test was abnormal in 15 patients (19 per cent) who did not differ from the other 65 with respect to prevalence of other risk factors, clinical characteristics, and electrocardiographic and angiographic findings. Blood levels of glucose, vitamins B6 and B12, folate, protein C and protein S, fibrinogen and low-density lipoprotein cholesterol did not differ significantly between the two groups. The prevalence of impaired homocysteine metabolism in young patients with arterial occlusive disease is greater than the 1-2 per cent found in the normal population.

Hyperhomocysteinaemia: A significant risk factor for cardiovascular disease in renal transplant recipients

Massy Z.A.; Chadefaux-Vekemans B.; Chevalier A.; Bader C.A.; Drueke T.B.; Legendre C.; Lacour B.; Kamoun P.; Kreis H. Division of Nephrology, Department of Medicine, Hennepim County Medical Center, 914 South 8th Street, Minneapolis, MN 55404 USA NEPHROL. DIAL. TRANSPLANT. (United Kingdom) , 1994, 9/8 (1103-1108)

Moderate hyperhomocysteinaemia has been shown to constitute an independent risk factor for cardiovascular disease (CVD), a frequent cause of morbidity and mortality in renal transplant recipients (RTR). In these patients few data regarding both total homocysteine levels and their influence on cardiovascular risk have been reported. We therefore studied serum homocysteine levels in deep-frozen sera from 42 kidney transplant recipients with a follow-up of 11 plus or minus 4.5 years (mean plus or minus SD) after transplantation. Eighteen patients had one or more ischaemic events (CVD(+)) and 24 patients had none (CVD(-)). Serum samples had been drawn 1-6 months prior to the first vascular event in CVD(+) patients and serum storage time was comparable in both CVD(-) and CVD(+) patients. Serum homocysteine levels were measured using a radioenzymatic method. Mean homocysteine level was significantly higher in 42 RTR males and females (15.5 plus or minus 6.3, 13.5 plus or minus 5.5 microM respectively) compared with 35 control subjects matched for age and sex (8.7 plus or minus 1.9, 7.5 plus or minus 1.9 microM, P < 0.001). The difference in serum homocysteine levels between CVD(+) and CVD(-) RTR nearly reached statistical significance in male patients (18.6 plus or minus 7.8 versus 13.1 plus or minus 3.4 microM, P < 0.06) but not in female patients (P = NS). In the CVD(+) group 11/18 patients had homocysteine levels > 14 microM (the upper limit in healthy control) versus 7/24 in the CVD(-) group (P = 0.04). In these patients we simultaneously measured in the same serum samples, serum triglycerides, and total and HDL cholesterol, and calculated LDL cholesterol. By stepwise discriminant analysis and by logistic regression analysis in this relatively small patient population, only serum triglycerides and homocysteine were selected as risk factors associated with CVD. We conclude that significant hyperhomocysteinaemia is present in renal transplant recipients and represents a potential risk factor for cadiovascular disease in these patients.

Promotion of vascular smooth muscle cell growth by homocysteine: A link to atherosclerosis

Tsai J.-C.; Perrella M.A.; Yoshizumi M.; Hsieh C.-M.; Haber E.; Schlegel R.; Lee M.-E. Harvard School of Public Health, 677 Huntington Avenue, Boston, MA 02115 USA PROC. NATL. ACAD. SCI. U. S. A. (USA) , 1994, 91/14 (6369-6373)

Plasma homocysteine levels are elevated in 20-30% of all patients with premature atherosclerosis. Although elevated homocysteine levels have been recognized as an independent risk factor for myocardial infarction and stroke, the mechanism by which these elevated levels cause atherosclerosis is unknown. To understand the role of homocysteine in the pathogenesis of atherosclerosis, we examined the effect of homocysteine on the growth of both vascular smooth muscle cells and endothelial cells at concentrations similar to those observed in clinical studies. As little as 0.1 mM homocysteine caused a 25% increase in DNA synthesis, and homocysteine at 1 mM increased DNA synthesis by 4.5-fold in rat aortic smooth muscle cells (RASMC). In contrast, homocysteine caused a dose-dependent decrease in DNA synthesis in human umbilical vein endothelial cells. Homocysteine increased mRNA levels of cyclin D1 and cyclin A in RASMC by 3- and 15-fold, respectively, indicating that homocysteine induced the mRNA of cyclins important for the reentry of quiescent RASMC into the cell cycle. Furthermore, homocysteine promoted proliferation of quiescent RASMC, an effect markedly amplified by 2% serum. The growth-promoting effect of homocysteine on vascular smooth muscle cells, together with its inhibitory effect on endothelial cell growth, represents an important mechanism to explain homocysteine-induced atherosclerosis.

Homocysteine, atherosclerosis and arterial thrombosis

Quere I.; Tobelem G. Hopital Lariboisiere, Service Hematologie Clinique, 2, Rue Ambroise-Pare, 75010 Paris France SANG THROMB. VAISS. (France) , 1993, 5/9 (599-606)

Homocysteine is a thiol-containing amino acid resulting from demethylation of methionine. Clinical features of all forms of homocystinuria due to homozygote deficiency include thromboembolism and arterioscleriosis. The incidence of these thromboembolic events is about 25% by the age of 16 years and 50% by the age of 30 years and is the major cause of high mortality (75% in 30 years non-treated patients). The recent development of a simple and automated method of measuring total homocysteine in plasma has enabled the investigation of the possible relation between mild homocysteinemia, heterozygote cystathionine-beta synthetase deficiency and premature arteriosclerosis and thromboembolism. The present stage of knowledge suggests a role of mild homocysteinemia in the development of arteriosclerotic lesions both in cerebral, coronary and peripheral vessels. It is important to confirm this relation. This will require large prospective studies following asymptomatic heterozygotes for cystathionine beta-synthase deficiency. Overall, because of the great overlap between cases and controls, molecular methods should be preferred to actually detecting cystathionine-beta synthase deficiency (methionine loading test, enzymatic activity on fibroblast cultures).

Oxidation of low density lipoprotein by thiols: Superoxide-dependent and - independent mechanisms

Heinecke J.W.; Kawamura M.; Suzuki L.; Chait A. Division of Atherosclerosis, Nutrition and Lipid Research, Box 8046, 660 South Euclid, St. Louis, MO 63110 USA J. LIPID RES. (USA) , 1993, 34/12 (2051-2061)

Oxidatively damaged low density lipoprotein (LDL) may cause macrophages to accumulate cholesterol in an unregulated manner, initiating the development of atherosclerotic lesions. Cultured smooth muscle cells oxidize LDL by a superoxide (O2-)-dependent mechanism that requires L-cystine and redox- active transition metal ions in the incubation medium. To test the hypothesis that cellular reduction of L-cystine to a thiol might be involved, we exposed LDL to L-cysteine, glutathione, and D,L-homocysteine. In a cell-free system each thiol modified LDL by a pathway that required either Cu2+ or Fe3+. Thiol- and Cu2+-modified LDL underwent lipid peroxidation and exhibited a number of properties of cell-modified LDL, including increased mobility on agarose gel electrophoresis and fragmentation of apolipoprotein B-100. Superoxide dismutase inhibited modification of LDL by L-cysteine/Cu2+, whereas catalase and mannitol were without effect. In striking contrast, superoxide dismutase had little effect on oxidation of LDL by Cu2+ and either homocysteine or glutathione. Moreover, only L-cysteine/Cu2+- modified 125I-labeled LDL was degraded more rapidly than 125I-labeled LDL by human monocyte-derived macrophages: superoxide dismutase in the reaction mixture blocked the facilitated uptake of L-cysteine/Cu2+-modified 125I-labeled LDL, suggesting involvement of O2-. These results indicate that LDL oxidation by L-cysteine and Cu2+ requires O2- but not H2O2 or hydroxyl radical. The reaction may involve the metal ion-dependent formation of L-cystine radical anion which is oxidized by oxygen, yielding O2- and the disulfide. LDL modified by L-cysteine and smooth muscle cells exhibit similar physical and biological properties, indicating that thiol-dependent generation of O2- may be the oxidative mechanism in both systems. Thiols also promote lipid peroxidation by O2--independent reactions but human macrophages fail to rapidly degrade these oxidized LDLs.

Chemical pathology of homocysteine I. Atherogenesis

McCully K.S. Pathology and Lab. Medicine Service, VA Medical Center, Providence, RI 02908-4799 USA ANN. CLIN. LAB. SCI. (USA) , 1993, 23/6 (477-493) :

The atherogenic properties of homocysteine were discovered by observation of arteriosclerosis in children with homocystinuria caused by inherited deficiency of three different enzymes. Hyperhomocysteinemia is generally recognized as an independent risk factor for coronary, cerebral, and peripheral atherosclerosis. Hyperhomocysteinemia is caused by heterozygosity for homocystinuria, micronutrient deficiency from dietary imbalance, toxins, drugs, hormones, and other factors, explaining many key observations concerning the epidemiology of atherosclerosis. The etiological factors for atherosclerosis are believed to increase conversion of methionine to homocysteine thiolactone, the reactive cyclic internal lactone of homocysteine. The free amino groups of low density lipoprotein (LDL) are thiolated by homocysteine thiolactone, causing aggregation and increased luptake of LDL by macrophages, explaining lipid deposition in atheromas. Homocysteine thiolactone, released from homocysteinylated LDL within vascular wall, promotes intimal injury, oxidation of cholesterol and unsaturated lipids, platelet aggregation, thrombogenic factors, myointimal hyperplasia, deposition of sulfated glycosaminoglycans, fibrosis and calcification of atherosclerotic plaques.

Hyperhomocysteinemia in patients operated for lower extremity ischaemia below the age of 50 - Effect of smoking and extent of disease

Bergmark C.; Mansoor M.A.; Swedenborg J.; de Faire U.; Svardal A.M.; Ueland P.M. Department of Surgery, Karolinska Hospital, S-104 01 Stockholm Sweden EUR. J. VASC. SURG. (United Kingdom) , 1993, 7/4 (391-396)

Moderate hyperhomocystinemia may be a risk factor for atherosclerotic peripheral vascular disease (PVD). In order to develop PVD at an early age risk factors are more strongly expressed and hyperhomocysteinemia may be one such factor. Homocysteine is derived from methionine and is metabolised by cystathionine-synthase to cystathionine or remethylated to methionine. Cystathionine-synthase activity is dependent on vitamin B6 while the remethylation of homocysteine is dependent on vitamin B12 and folate. The present study analyses homocysteine in patients operated on for lower extremity ischaemia before the age of 50. Homocysteine before and after loading with methionine, vitamin B6, B12 and folate were measured at follow-up. The patients were compared to age- and sex-matched controls. Significantly more patients than controls had hyperhomocysteinemia, 16/58 vs. 4/65, defined as fasting total homocysteine above 18.6 micromol/l. Loading with methionine did not further discriminate between patients and controls. Smoking patients had higher levels of homocysteine than non-smoking patients or smoking and non-smoking controls. Smoking patients also had lower levels of vitamin B6. When comparing patients with suprainguinal, infrainguinal and multilevel disease the highest homocysteine levels were seen in the latter group. Also, in this group smoking patients had higher homocysteine levels. Multivariate analysis revealed that homocysteine was associated with low levels of vitamin B12, folate and smoking. Smoking therefore seems to be connected to increased homocysteine levels in patients with early development of atherosclerosis, partly explained by decreased levels of B6, B12 and folate.

Hyperhomocysteinemia, a risk factor for atherosclerosis in chronic uremic patients

Chauveau P.; Chadefaux B.; Coude M.; Aupetit J.; Hannedouche T.; Kamoun P.; Jungers P. Departement de Nephrologie, Hopital Necker, 161 Rue de Sevres, 75743 Paris Cedex 15 France KIDNEY INT. SUPPL. (USA) , 1993, -/41 (S-72-S-77)

Hyperhomocysteinemia has been shown to constitute an independent risk factor for premature occlusive arterial disease (N Engl J Med 324: 1149), a frequent complication in chronic uremic patients in whom homocysteine (Hcy) accumulation has been reported to occur. We prospectively determined fasting plasma level of total, protein-bound Hcy in 118 adult chronic uremic patients, either dialyzed or not. In 79 non-dialyzed patients (47 male) with various degrees of chronic renal failure (RF) assessed by creatinine clearance (C(Cr)), none receiving folate, B6 or B12 vitamin supplementation, mean (plus or minus 1 sD) plasma Hcy level was 16.2 plus or minus 8.1 micromol/liter in 28 patients with mild RF (C(Cr) 30 to 75 ml/min), 23.3 plus or minus 14.7 in 29 patients with moderate RF (C(Cr) 10 to 29.9), and 29.5 plus or minus 14.4 in 22 patients with advanced RF (C(Cr) < 10), a significant difference (P < 0.01 for all groups) compared to 45 healthy controls (8.2 plus or minus 2.2 micromol/liter). Linear regression analysis showed a significant correlation between plasma creatinine and Hcy concentrations (r = 0.49, P < 0.0001) Hcy was significantly higher in 20 patients (16 males) who had past histories of occlusive arterial disease than in the 59 (31 males) who did not (30.9 plus or minus 19.1 vs. 19.6 plus or minus 9.7 micromol/liter, P < 0.001) and all of the former had Hcy level > 14.1 micromol/liter (the upper limit in healthy controls) versus 35 of 59 in the latter. Predialysis Hcy concentration in 39 dialyzed patients who received folate supplementation (23.5 plus or minus 10.7 micromol/liter, P < 0.001) was significantly higher than in controls despite a significant (nearly 50%) decrease postdialysis (11.5 plus or minus 6.3 micromol/liter, P < 0.01). We conclude that significant hyperhomocysteinemia is present from the early stage of chronic renal failure and increases in parallel with progression of renal failure, with only partial correction by chronic hemodialysis. Such long-lasting hyperhomocysteinemia may constitute a risk factor for premature atherosclerosis in uremic patients.

Betaine effects on hepatic methionine metabolism elicited by short-term ethanol feeding

Barak A.J.; Beckenhauer H.C.; Tuma D.J. Liver Study Unit, VA Medical Center, 4101 Woolworth Avenue, Omaha, NE 68105 USA Alcohol (USA) , 1996, 13/5 (483-486)

Previous studies in this laboratory have shown that feeding of ethanol to rats produces prompt inhibition of methionine synthetase (MS) as well as a subsequent increase in activity of betaine homocysteine methyltransferase (BHMT). Further studies have shown that supplemental dietary betaine enhanced methionine metabolism and S-adenosylmethionine (SAM) generation in control and ethanol-fed rats. Because MS and BHMT are both involved in the formation of SAM. This study was conducted to determine early of effects of ethanol on hepatic SAM levels and the influence of betaine supplementation on parameters of methionine metabolism during the early periods of MS inhibition and enhanced BHMT activity. Results showed that ethanol feeding produced a significant loss in SAM in the first week with a return to normal SAM levels in the second week. Betaine feeding enhanced hepatic betaine pools in control as well as ethanol-fed animals. This feeding attenuated the early loss of SAM in ethanol fed animals, produced an early increase in BHMT activity, and generated increased levels of SAM in both control and ethanol-fed groups. Furthermore, betaine lowered significantly the accumulation of hepatic triglyceride produced by ethanol after 2 weeks of ingestion.

Hepatic betaine-homocysteine methyltransferase activity in the chicken is influenced by dietary intake of sulfur amino acids, choline and betaine

Emmert J.L.; Garrow T.A.; Baker D.H. 290 ASL, 1207 West Gregory Drive, Urbana, IL 61801 USA Journal of Nutrition (USA) , 1996, 126/8 (2050-2058)

There is much interest in the metabolism of homocysteine, because elevated plasma homocysteine (hyperhomocyst(e)inemia) is an independent risk factor for the development of cardiovascular disease. Four chick assays were conducted to determine the effects of varying dietary sulfur amino acids, choline and betaine on the activity of hepatic betaine-homocysteine methyltransferase (BHMT), an enzyme likely to be important in modulating plasma homocysteine. In Experiment 1, chicks were fed a purified crystalline amino acid diet containing adequate sulfur amino acids and choline. Excess dietary methionine, or the combination of excess cystine with choline or betaine, caused a small increase (P < 0.05) in BHMT activity. In Experiment 2, use of a methionine-deficient purified diet resulted in a threefold increase (P < 0.05) in BHMT activity, and addition of choline or betaine further increased (P < 0.05) BHMT activity. In Experiment 3, use of a methionine-deficient corn-peanut meal diet increased BHMT (P < 0.05) relative to that of chicks supplemented with adequate methionine, and addition of surfeit choline to the methionine-deficient basal diet caused a further increase (P < 0.05). In Experiment 4, addition of both surfeit choline and surfeit betaine to the methionine-deficient corn-peanut meal diet caused an increase (P < 0.05) in BHMT activity relative to that observed in chicks fed the methionine-deficient basal diet. These assays show that large increases in BHMT activity can be produced under methionine-deficient conditions, especially in the presence of excess choline or betaine.

Homocystinuria: What about mild hyperhomocysteinaemia?

Van den Berg M.; Boers G.H.J. Institute Cardiovascular Research, Department of Vascular Surgery, Free University Hospital, PO Box 7057, 1007 MB Amsterdam Netherlands Postgraduate Medical Journal (United Kingdom) , 1996, 72/851 (513-518)

Hyperhomocysteinaemia is associated with an increased risk of atherosclerotic vascular disease and thromboembolism, in both men and women. A variety of conditions can lead to elevated homocysteine levels, but the relation between high levels and vascular disease is present regardless of the underlying cause. Pooled data from a large number of studies demonstrate that mild hyperhomocysteinaemia after a standard methionine load is present in 21% of young patients with coronary artery disease, in 24% of patients with cerebrovascular disease, and in 32% of patients with peripheral vascular disease. From such data an odds ratio of 13.0 (95% confidence interval 5.9 to 28.1), as an estimate of the relative risk of vascular disease at a young age, can be calculated in subjects with an abnormal response to methionine loading. Furthermore, mild hyperhomo-cysteinaemia can lead to a two- or three-fold increase in the risk of recurrent venous thrombosis. Elevated homocysteine levels can be reduced to normal in virtually all cases by simple and safe treatment with vitamin B6, folic acid, and betaine, each of which is involved in methionine metabolism. A clinically beneficial effect of such an intervention, currently under investigation, would make large-scale screening for this risk factor mandatory.

Purification, kinetic properties, and cDNA cloning of mammalian betaine- homocysteine methyltransferase

Garrow T.A. 445 Bevier Hall, University of Illinois, 905 S. Goodwin Ave., Urbana, IL 61801 USA Journal of Biological Chemistry (USA) , 1996, 271/37 (22831-22838)

Porcine liver betaine-homocysteine methyltransforuse (BHMT; EC was purified to homogeneity, and the Michaelis constants for betaine, dimethylacetothetin, and L-homocysteine are 23, 155, and 32 microM, respectively. The maximum rate of catalysis is 47-fold greater using dimethylacetothetin as a methyl donor compared with betaine. Partial amino acid sequence of porcine BHMT was obtained, and inosine-containing redundant oligonucleotide primers were used to amplify an 815-base pair sequence of the porcine cDNA by polymerase chain reaction (PCR). Nondegenerate oligonucleotide primers based on the porcine cDNA were synthesized and used to isolate a 463-base pair fragment of the human cDNA by PCR. The human PCR DNA product was then used to screen a cDNA library by plaque hybridization, and cDNAs encoding human BHMT were isolated. The primary structure of the human cDNA is reported here, and the open reading frame encodes a 406- residue protein of M(r) 44,969. The deduced amino acid sequence of human BHMT shows limited homology to bacterial vitamin B12-dependent methionine synthases (EC A plasmid containing the human BHMT cDNA fused in frame to the N terminus of beta-galactosidase was transformed into Escherichia coli, and transformants expressed BHMT activity, an activity that is absent from wild type E. coli.

Betaine, ethanol, and the liver: A review

Barak A.J.; Beckenhauer H.C.; Tuma D.J. Liver Study Unit (151), VA Medical Center, 4101 Woolworth Avenue, Omaha, NE 68105 USA Alcohol (USA) , 1996, 13/4 (395-398)

Two of the most important biochemical hepatic pathways in the liver are those that synthesize methionine and S adenosylmethionine (SAM) through the methylation of homocysteine. This article reviews some recent findings in this laboratory, which demonstrate that ethanol feeding to rats impairs one of these pathways involving the enzyme methionine synthetase (MS), but by way of compensation increases the activity of the enzyme betaine:homocysteine methyl transferase (BHMT), which catalyzes the second pathway in methionine and SAM biosynthesis. It has been shown that despite the inhibition of MS, the enhanced BHMT pathway utilizes hepatic betaine pools to maintain levels of SAM. Subsequent to the above findings, it has been shown that minimal supplemental dietary betaine at the 0.5% level generates SAM twofold in control animals and fivefold in ethanol-fed rats. Concomitant with the betaine-generated SAM, ethanol-induced hepatic fatty infiltration was ameliorated. In view of the fact that SAM has already been used successfully in the treatment of human maladies, including liver dysfunction, betaine, shown to protect against the early stages of alcoholic liver injury as well as being a SAM generator, may become a promising therapeutic agent and a possible alternative to expensive SAM in the treatment of liver disease and other human maladies.

Defective cystathionine beta-synthase regulation by S-adenosylmethionine in a partially pyridoxine responsive homocystinuria patient

Kluijtmans L.A.J.; Boers G.H.J.; Stevens E.M.B.; Renier W.O.; Kraus J.P.; Trijbels F.J.M.; Van den Heuvel L.P.W.J.; Blom H.J. Department of Pediatrics, University Hospital Nijmegen, P.O. Box 9101, 6500 HB Nijmegen Netherlands Journal of Clinical Investigation (USA) , 1996, 98/2 (285-289)

We determined the molecular basis of cystathionine beta-synthase (CBS) deficiency in a partially pyridoxine-responsive homocystinuria patient. Direct sequencing of the entire CBS cDNA revealed the presence of a homozygous G1330A transition. This mutation causes an amino acid change from aspartic acid to asparagine (D444N) in the regulatory domain of the protein and abolishes a TaqI restriction site at DNA level. Despite the homozygous mutation, CBS activities in extracts of cultured fibroblasts of this patient were not in the homozygous but in the heterozygous range. Furthermore, we observed no stimulation of CBS activity by S- adenosylmethionine, contrary to a threefold stimulation in control fibroblast extract. The mutation was introduced in an E. coli expression system and CBS activities were measured after addition of different S-adenosylmethionine concentrations (0-200 microM). Again, we observed a defective stimulation of CBS activity by S-adenosylmethionine in the mutated construct, whereas the normal construct showed a threefold stimulation in activity. These data suggest that this D444N mutation interferes in S-adenosylmethionine regulation of CBS. Furthermore, it indicates the importance of S-adenosylmethionine regulation of the transsulfuration pathway in homocysteine homeostasis in humans.

Human homocysteine catabolism: Three major pathways and their relevance to development of arterial occlusive disease

Dudman N.P.B.; Guo X.-W.; Gordon R.B.; Dawson P.A.; Wilcken D.E.L. Clinical Sciences Building, Prince Henry Hospital, Little Bay, NSW 2036 Australia Journal of Nutrition (USA) , 1996, 126/4 SUPPL. (1295S-1300S)

Two separate metabolic pathways that methylate homocysteine to methionine are known in humans, utilizing, respectively, 5-methyltetrahydrofolate and betaine as methyl donors. Deficiency of the folate-dependent methylation system is linked to hyperhomocysteinemia. Our data suggest that this deficiency leads to concurrent metabolic down-regulation of homocysteine transsulfuration that may contribute to hyperhomocysteinemia. By contrast, no instances have been reported of hyperhomocysteinemia resulting from deficiencies of betaine-dependent homocysteine methylation. Long-term betaine supplementation of 10 patients, who had pyridoxine-resistant homocystinuria and gross hyperhomocysteinemia due to deficiency of cystathionine beta- synthase activity, caused a substantial lowering of plasma homocysteine, which has now been maintained for periods of up to 13 years. Betaine had to be taken regularly because the effect soon disappeared when treatment was stopped. In conclusion, depressed activity of the transsulfuration pathway may contribute to hyperhomocysteinemia because of primary deficiencies of enzymes of either the transsulfuration or of the folate-dependent methylation pathways. Stimulation of betaine-dependent homocysteine remethylation causes a commensurate decrease in plasma homocysteine that can be maintained as long as betaine is taken.

Treatment of hyperhomocyst(e)inemia: Physiological basis

Kang S.-S. Rush-Presbyterian SLMC, 1750 W. Harrison Street, Chicago, IL 60612 USA Journal of Nutrition (USA) , 1996, 126/4 SUPPL. (1273S-1275S)

Hyperhomocyst(e)inemia (HCY) is caused either by genetic or nongenetic defect(s), and the clinical severity of HCY is correlated with the biochemical abnormality. Treatment of HCY is approached on the basis of its etiology and severity of defect. The preferred method of treatment for genetic HCY is activation of mutant enzyme activity with the cofactor or the precursor of cofactor. If HCY does not respond to this treatment, pharmacological doses of betaine or folic acid should be used to enhance the alternative pathway of homocysteine turnover. Phenotypic expression of minor genetic defects, such as heterozygous cystathionine synthase deficiency and thermolabile methylenetetrahydrofolate reductase (MTHFR) can be amplified or masked by nongenetic (nutritional) factor(s). Hence, supplementation of folic acid, vitamin B-12, pyridoxine and choline to maintain their serum concentrations above low normal range may satisfactorily prevent the development of moderate HCY due to a minor genetic defect.

Plasma homocyst(e)ine: A risk factor for arterial occlusive diseases

Malinow M.R. Oregon Regional Primate Res. Center, 505 NW 185th Avenue, Beaverton, OR 97006 USA Journal of Nutrition (USA) , 1996, 126/4 SUPPL. (1238S-1243S)

Results of basal plasma homocyst(e)ine concentrations in patients reported in the literature are reviewed, with emphasis on the series of subjects analyzed by the author. Findings support the hypothesis that plasma homocyst(e)ine is a risk factor for coronary, cerebral and peripheral arterial occlusive diseases, as well as for carotid thickening. Results of four studies show that heritability influences plasma homocyst(e)ine. Moreover, data suggest that a graded risk for atherothrombotic disease is distributed across the entire distribution of plasma homocyst(e)ine levels. Elevated levels of homocyst(e)ine can be decreased effectively by supplementary folate, occasionally requiting the addition of vitamin B-12, vitamin B-6, choline or betaine. Consequently, it is important that placebo- controlled clinical trials be conducted to determine whether the clinical evolution of arterial occlusive diseases is influenced by those supplements.

Long term treatment with betaine in methylenetetrahydrofolate reductase deficiency

Ronge E.; Kjellman B. Department of Paediatrics, Central Hospital, S-541 85 Skovde Sweden Archives of Disease in Childhood (United Kingdom) , 1996, 74/3 (239-241)

A girl aged 7.5 years with deficiency of 5,10 -methylenetetrahydrofolate reductase was treated from early infancy with betaine, 3-6 g daily. She has slight microcephaly, moderate developmental delay, and impaired vision but there have been no obvious signs of folate deficiency. From 4 years of age, she developed an unexplained extreme increase in appetite and weight. Recent magnetic resonance imaging of her brain was normal. The plasma methionine levels have been normal but in the lower range, and the total plasma homocysteine concentrations have been moderately increased (54 to 85 micromol/l) without obvious correlation with the different betaine doses given. Folic acid has sometimes been added.

Atypical hemolytic uremic syndrome due to an intracellular abnormality in vitamin B12 metabolism

Loirat C.; Ogier De Baulny H.; Baudouin V.; Peuchmaur M. Service de Nephrologie, Hopital Robert Debre, 48, Boulevard Serurier, 75019 Paris France Annales de Pediatrie (France) , 1995, 42/10 (625-634)

Hemolytic uremic syndrome is exceedingly rare during the first few weeks after birth. Five cases in neonates with an inherited group Cb1C intracellular vitamin B12 abnormality comparable to that in four previously reported cases are described herein. All five patients developed feeding difficulties, inadequate weight gain, hypotonia and lethargy within two weeks of birth. Hemolytic uremic syndrome occurred between 28 and 90 days of age, with severe hemolytic anemia, schizocytosis, thrombocytopenia (7/9), proteinuria, hematuria, renal failure (8) and arterial hypertension (4/9). All patients had unusual clinical and laboratory test findings including severe acidosis (8 documented cases), leukoneutropenia (6/9), liver failure (7/8), gastrointestinal bleeding and diarrhea (6/8), respiratory failure due to interstitial pneumonia (6/8), heart failure (3/5), hypotonia and lethargy (7/7), coma (n = 2), seizures (n = 1), and hydrocephalus (n = 2). Pigmentary retinitis was found in three of the five patients who underwent ophthalmologic evaluation. The diagnosis of group Cb1C vitamin B12 metabolic deficiency was established based on very high plasma levels of homocysteine and methylmalonic acid, very low plasma levels of methionine, and very high urinary excretions of homocysteine and methylmalonic acid. There were eight deaths, of which six were due to multiorgan failure and two to permanent neurologic damage. The most recently diagnosed patient was doing well with no sequellae at age 2 years 2 months; this is the only patient who was treated with hydroxycobalamine, folinic acid, and betaine within 24 hours of onset of the hemolytic uremic syndrome. Histologic studies disclosed glomerular and arteriolar thrombi and/or thrombotic glomerular microangiopathy in the renal parenchyma; thrombi in pulmonary arterioles and small arteries, with severe interstitial pneumonia; complete atrophy of the gastric mucosa and cystic dilations of the gastric glands; and fatty infiltration with hemosiderosis of the liver. The fact that the only survivor was treated early emphasizes the need for very early diagnosis and treatment which is probably the only means of avoiding death or permanent neurologic damage.

Homocysteine and vascular disease

Berwanger C.S.; Jeremy J.Y.; Stansby G. Academic Surgical Unit, QEQM, St Mary's Hospital Medical School, Praed Street, London W2 1NY United Kingdom British Journal of Surgery (United Kingdom) , 1995, 82/6 (726-731)

There is increasing evidence that a raised blood level of homocysteine (HC) is a risk factor for premature atherosclerosis. With a gene frequency between one in 70 and one in 200 this condition may be more common than previously thought. It should be suspected especially in young patients in whom other risk factors are absent. The diagnosis may be made by demonstrating raised plasma HC levels, either basally or after methionine loading. Studies have shown significantly increased levels of HC inpatients with premature coronary artery, peripheral vascular and cerebrovascular disease. The mechanisms by which HC produces vascular damage are, as yet, not completely understood but endothelial injury is probably a central factor. The principle of treatment is to lower HC levels in the blood by administration of vitamin B6, vitamin B12, folate or betaine. How effective this strategy will be in preventing complications is not yet known.

Tyrosinase inhibition due to interaction of homocyst(e)ine with copper: The mechanism for reversible hypopigmentation in homocystinuria due to cystathionine beta-synthase deficiency

Reish O.; Townsend D.; Berry S.A.; Tsai M.Y.; King R.A. Department of Pediatrics, Institute of Human Genetics, University of Minnesota, 420 Delaware Street S.E., Minneapolis, MN 55455 USA American Journal of Human Genetics (USA) , 1995, 57/1 (127-132)

Deficiency of cystathionine beta-synthase (CBS) is a genetic disorder of transsulfuration resulting in elevated plasma homocyst(e)ine and methionine and decreased cysteine. Affected patients have multisystem involvement, which may include light skin and hair. Reversible hypopigmentation in treated homocystinuric patients has been infrequently reported, and the mechanism is undefined. Two CBS-deficient homocystinuric patients manifested darkening of their hypopigmented hair following treatment that decreased plasma homocyst(e)ine. We hypothesized that homocyst(e)ine inhibits tyrosinase, the major pigment enzyme. The activity of tyrosinase extracted from pigmented human melanoma cells (MNT-1) that were grown in the presence of homocysteine was reduced in comparison to that extracted from calls grown without homocysteine. Copper sulfate restored homocyst(e)ine-inhibited tyrosinase activity when added to the culture cell media at a proportion of 1.25 mol of copper sulfate per 1 mol of DL-homocysteine. Holo-tyrosinase activity was inhibited by adding DL-homocysteine to the assay reaction mixture, and the addition of copper sulfate to the reaction mixture prevented this inhibition. Other tested compounds, L-cystine and betaine did not affect tyrosinase activity. Our data suggest that reversible hypopigmentation in homocystinuria is the result of tyrosinase inhibition by homocyst(e)ine and that the probable mechanism of this inhibition is the interaction of homocyst(e)ine with copper at the active site of tyrosinase.

Measurement of homocyst(e)ine in the prediction of arteriosclerosis

Fortin J.-J.; Genest J. Jr. Cardiovascular Genetics Laboratory, Clinical Research Inst. Montreal, Montreal, Que. Canada Clinical Biochemistry (USA) , 1995, 28/2 (155-162)

Homocyst(e)ine (H(e)), the sum of homocysteine, homocystine, and the homocysteine-cysteine mixed disulfide, free and protein-bound, has been shown to be associated in retrospective case control studies, and in one prospective study, with vascular disease, including coronary artery disease (CAD), cerebrovascular disease, and peripheral vascular disease. Elevated levels of homocyst(e)ine severe enough to cause homocystinuria are seen in severe nutritional deficiencies of vitamin B12, folic acid and vitamin B6. Rare genetic disorders of vitamin B12 synthesis of 5'-10'-methylene tetrahydrofolate reductase, or the pyridoxal phosphate-dependent enzyme cystathionine beta-synthase may cause severe hyperhomocyst(e)inemia and homocystinuria. The clinical manifestation of these disorders are mental retardation, neurological disorders, and widespread thromboembolic phenomena. The measurement of H(e) is currently performed using high-pressure liquid chromatography with fluorescence detection. Other methods, especially mass spectroscopy, are also used. Internal standards using increasing concentrations of hemocystine and acetylcysteine and several external standards are used to ensure accuracy of the assay. Milder elevations of H(e) have recently been associated with vascular disease, in both men and women. The strength of this association appears to be stronger for peripheral and cerebrovascular disease than for CAD. Nevertheless, several case control studies in Europe, Canada, and the United States have shown that H(e) levels are elevated in CAD patients compared with controls, and H(e) levels are independent of the conventional cardiovascular risk factors (age, gender, lipid and lipoprotein cholesterol levels, hypertension, or cigarette smoking). One prospective study, the Physicians' Health Study, has shown that H(e) levels are slightly but significantly higher in CAD cases vs controls in a population of US physicians. Because CAD is a major public health issue, new biochemical markers for the disease are of potential interest. Low-dose folic acid, vitamin B6 or both are used to reduce elevated H(e) levels in affected individuals. Resistant cases may be treated with betaine. it remained to be determined in large, prospective studies, whether H(e) is an important cardiovascular risk factor and whether treatment of an elevated H(e) level will lead to a decrease in cardiovascular events and mortality.

Combined vitamin B6 plus folic acid therapy in young patients with arteriosclerosis and hyperhomocysteinemia

Van den Berg M.; Franken D.G.; Boers G.H.J.; Blom H.J.; Jakobs C.; Stehouwer C.D.A.; Rauwerda J.A. Division of Vascular Surgery, Department of Surgery, Free University Hospital, De Boelelaan 1117, 1081 HV Amsterdam Netherlands J. VASC. SURG. (USA) , 1994, 20/6 (933-940)

Purpose: Hyperhomocysteinemia is associated with arteriosclerotic and thromboembolic events. The homocysteine-lowering effect of combined treatment with vitamin B6 plus folic acid has never been explored in a large group of patients with vascular disease. Therefore we studied the effects of at least 6 weeks treatment with these vitamins in 72 patients with cardiovascular disease and mild hyperhomocysteinemia (defined as an increase of the plasma homocysteine level after methionine loading greater than 97.5 percentile of age-matched control subjects but less than 200 micromol/L).

Methods: The existence of mild hyperhomocysteinemia was investigated in 309 consecutive patients under 50 years of age with peripheral arterial occlusive disease, cerebral arterial occlusive disease, or coronary artery occlusive disease. All patients with an abnormal loading test result were treated with vitamin B6, 250 mg daily, plus folic acid, 5 mg daily. After 6 weeks of treatment a second methionine loading test was performed to assess the homocysteine- lowering effect. Results: Mild hyperhomocysteinemia was detected in 72 patients (23%), 33 (46%) of whom also had hyperhomocysteinemia when fasting. Treatment with vitamin B6 plus folic acid normalized the postload plasma homocysteine concentration in 66 of the 72 patients (92%), whereas fasting hyperhomocysteinemia was normalized in 30 of 33 (91%) patients. In six patients therapy failed to achieve normalization of the postload homocysteine levels. In three of these patients, the same treatment was continued for an additional 6 weeks, and in the remaining three patients betaine was added to the treatment regimen. After 6 weeks of additional treatment all six patients had normal postload plasma homocysteine concentrations.

Conclusion: The prevalence of mild hyperhomocysteinemia in young patients with arterial occlusive disease is high. Simple and inexpensive therapy with vitamin B6 plus folic acid will normalize homocysteine metabolism, as assessed by the homocysteine plasma level after methionine loading, in virtually all these patients.

Plasma homocyst(e)ine and arterial occlusive diseases: A mini-review

Malinow M.R. Oregon Regional Primate Res. Center, 505 NW 185th Ave., Beaverton, OR 97006 USA CLIN. CHEM. (USA) , 1995, 41/1 (173-176)

Homocysteine (HCY), which is derived from the intracellular metabolism of methionine, is exported into plasma, where it circulates mostly in oxidized forms (i.e., homocystine and cysteine-HCY disulfide) and mainly bound to proteins. Concentrations of total HCY, or homocyst(e)ine (H(e)), are increased in 15-40% of patients with coronary, cerebral, or peripheral arterial diseases. Such association of H(e) with arterial occlusive diseases has been documented in retrospective, cross-sectional, and prospective studies. Concentrations of H(e) are also increased in subjects having thickened carotid arteries, as determined by ultrasonography, and who are asymptomatic for atherosclerosis. Statistical analyses of data from several series of patients demonstrate that H(e) concentrations are associated with coronary artery disease, independently from most other risk factors for atherosclerosis. The increased concentrations of H(e) are readily corrected by folic acid, occasionally supplemented with pyridoxine, vitamin B12, choline, or betaine. Whether these supplements affect the evolution of atherosclerotic disease needs to be established by prospective, placebo- controlled clinical trials.

Hyperhomocysteinaemia: A newly recognized risk factor for vascular disease

Boers G.H.J. Department of Medicine, Division of Endocrinology, University Hospital, P.O. Box 9101, 6500 HB Nijmegen Netherlands NETH. J. MED. (Netherlands) , 1994, 45/1 (34-41)

In the past decade significant p}ogress has been made in understanding of hyperhomocysteinaemia and its association with the proneness to premature development of vascular disease. Pooled data from a large number of studies demonstrate that mild hyperhomocysteinaemia after standardized methionine loading is present in 21% of patients with coronary artery disease, in 24% of patients with cerebrovascular disease, and in 32% of patients with peripheral vascular disease. A relative risk of 13.0 (95% confidence interval 5.9-28.1) of vascular disease at relatively young age can be calculated in subjects with such abnormal response to methionine loading. Pathological homocysteine levels are affected by genetic defects in homocysteine metabolism which have still not been completely clarified and which are more complex than originally supposed. Furthermore, a variety of non-genetic determinants such as deficiency of folate or vitamine B12 has to be taken into account. Mild hyperhomocysteinaemia can be reduced to normal in virtually all cases by simple and safe treatment with vitamin B6, folic acid, and betaine, each of which is involved in methionine metabolism. A clinically beneficial effect of such an intervention, which is currently under investigation, could make large-scale screening mandatory for this risk factor.

Therapeutical control of homocystinuria by means of the determination of total homocysteine in plasma

Vilaseca Busca M.A.; Campistol Plana J.; Vernet Bori A.; Poo Arguelles P.; Monso G.; Ramon Bauza F. Servicio de Bioquimica, Hospital 'Sant Joan de Deu', Carretera d'Esplugues, s/n, 08034 Barcelona Spain AN. ESP. PEDIATR. (Spain) , 1994, 40/6 (411-416)

Homocysteinc exists in human plasma as various mixed disulfides: homocystine, cystein-homocystein disulfide and, mainly, protein-bound. The determination of total homocysteine or its different fractions appears to be indispensable for the reliable therapeutical control of homocystinuric patients. Methods for total and protein-bound homocysteine determinations have been adapted so that they can be rapidly and effectively performed in a hospital laboratory for the routine control of treatment. We compared the results of free, total and protein-bound homocysteine during one year of treatment of four homocystinuric patients, 2 with classical forms and 2 in combination with methylmalonic aciduria (Cb1C mutations). We discuss the advantages of total homocysteine determinations (simple, rapid, reproducible and informative of the plasma levels) for the control of an effective therapy.

Treatment of mild hyperhomocysteinemia in vascular disease patients

Franken D.G.; Boers G.H.J.; Blom H.J.; Trijbels F.J.M.; Kloppenborg P.W.C. Department of Medicine, University Hospital Nijmegen, Post Box 9101, 6500 HB Nijmegen Netherlands ARTERIOSCLER. THROMB. (USA) , 1994, 14/3 (465-470)

Mild hyperhomocysteinemia is recognized as a risk factor for premature arteriosclerotic disease. A few vitamins and other substances have been reported to reduce blood homocysteine levels, but normalization of elevated blood homocysteine concentrations with any of these substances has not been reported. Therefore, we screened 421 patients suffering from premature peripheral or cerebral occlusive arterial disease by oral methionine loading tests for the presence of mild hyperhomocysteinemia. Thirty-three percent of patients with peripheral and 20% of patients with cerebral occlusive arterial disease were identified with mild hyperhomocysteinemia (14% of the men, 34% of the premenopausal women, and 26% of the postmenopausal women). Mildly hyperhomocysteinemia patients were administered vitamin B6 250 mg daily. After 6 weeks methionine loading tests were again assessed to evaluate the effect of treatment. Patients with nonnormalized homocysteine concentrations were further treated with vitamin B6 250 mg daily and/or folic acid 5 mg daily and/or betaine 6 g daily, solely or in any combination. Vitamin B6 treatment normalized the afterload homocysteine concentration in 56% of the treated patients (71% of the men, 45% of the premenopausal women, and 88% of the postmenopausal women). Further treatment resulted in a normalization of homocysteine levels in 95% of the remaining cases. Thus, mild hyperhomocysteinemia, which is frequently encountered in patients with premature arteriosclerotic disease, can be reduced to normal in virtually all cases by safe and simple treatment with vitamin B6, folic acid, and betaine, each of which is involved in methionine metabolism.

Methylenetetrahydrofolate reductase deficiency: Prenatal diagnosis and family studies

Marquet J.; Chadefaux B.; Bonnefont J.P.; Saudubray J.M.; Zittoun J. Laboratoire d'Hematologie, Faculte de Medecine, 8 Rue du General Sarrail, 94010 Creteil France PRENATAL DIAGN. (United Kingdom) , 1994, 14/1 (29-33)

Prenatal diagnosis of methylenetetrahydrofolate reductase (MTHFR) deficiency and family studies were performed because of a severely affected first child in this family. The fetus at risk was found to be heterozygous was found to be heterozygous as confirmed by the enzymatic activity assay performed several times after birth. In the father, MTHFR activity was normal in lymphocytes and decreased in fibroblasts, whereas in the asymptomatic mother, the activity was not detectable in fibroblasts and was very low in lymphocytes. The absence of any clinical symptoms in the mother despite a clear MTHFR deficiency and hyperhomocystinemia emphasizes the heterogeneity of this disease.

Serum betaine, N,N-dimethylglycine and N-methylglycine levels in patients with cobalamin and folate deficiency and related inborn errors of metabolism

Allen R.H.; Stabler S.P.; Lindenbaum J. Division of Hematology, Colorado Univ. Health Sciences Ctr., Campus Box B170, 4200 E Ninth Ave, Denver, CO 80262 USA METAB. CLIN. EXP. (USA) , 1993, 42/11 (1448-1460)

Homocysteine and 5-CH3-tetrahydrofolate (5-CH3-THF) are converted to methionine and THF by the CH3-cobalamin (CH3-Cbl)-dependent enzyme methionine synthase. Serum homocysteine levels are elevated in more than 95% of patients with Cbl or folate deficiency and in patients with inborn errors involving the synthesis of 5-CH3-THF or CH3-Cbl. Homocysteine and betaine are converted to methionine and N,N-dimethylglycine by betaine-homocysteine methyltransferase. It requires neither Cbl nor folate, although N,N- dimethylglycine is converted to N-methylglycine and then to glycine in reactions that both involve the formation of 5,10-CH2-THF from THF. Large amounts of betaine are often given orally to patients with inborn errors, even though little is known about its metabolism in normal subjects or these patients. Thus we developed new gas chromatographic-mass spectrometric assays for serum betaine, N,N-dimethylglycine, and N-methylglycine. In 60 blood donors, we found ranges for normal serum of 17.6 to 73.3, 1.42 to 5.27, and 0.60 to 2.67 micromol/L for the three metabolites, respectively, which were normal in the majority of 50 patients with Cbl deficiency, none of whom had increased levels of N-methylglycine. In 25 patients with folate deficiency, serum betaine level was normal in most, but 76% and 60% had elevations of N,N-dimethylglycine and N-methylglycine levels that ranged as high as 343 and 43.2 micromol/L, respectively. All of seven patients on betaine therapy for inborn errors had high values for betaine (167 to 3,900 micromol/L), N,N- dimethylglycine (15.1 to 250 micromol/L), and N-methylglycine (2.93 to 49.3 micromol/L). Serum total homocysteine levels remained very high at 47.2 to 156 micromol/L (normal, 5.4 to 16.2). In patients with cbl C and cbl D mutations, methionine levels remained low or low-normal at 8.3 to 15.6 micromol/L (normal, 13.3 to 42.7) despite betaine treatment. We conclude that (1) betaine levels are maintained in most patients with Cbl and folate deficiency; (2) levels of N,N-dimethylglycine and N-methylglycine are increased in most patients with folate deficiency; and (3) betaine therapy is relatively ineffective in patients with defective synthesis of CH3-Cbl.

Glycerophosphocholine and phosphocholine are the major choline metabolites in rat milk

Rohlfs E.M.; Garner S.C.; Mar M.-H.; Zeisel S.H. Department of Nutrition, School of Medicine, University of North Carolina, Chapel Hill, NC 27599-7400 USA J. NUTR. (USA) , 1993, 123/10 (1762-1768)

Choline is a constituent of cell membranes, surfactant and acetylcholine and is also a major source of methyl groups for the regeneration of methionine from homocysteine. Previous analyses of rat, human and bovine milk measured only choline, phosphatidylcholine and sphingomyelin. Choline- containing compounds in milk from rats lactating for 15 d were measured by HPLC and gas chromatograph-mass spectrometry. In addition to the previously reported choline metabolites, substantial concentrations of glycerophosphocholine (3.7 mmol/L) and phosphocholine (653 micromol/L) were also detected. At 1 h after oral administration of (methyl-14C)choline to lactating rats, the major labeled metabolites were phosphocholine (91% of label in milk) and betaine (9%). Twenty-four hours after the dose, glycerophosphocholine was the major labeled metabolite (69% of label in milk). Rat mammary epithelial cells, in primary culture, synthesized and secreted phosphatidylcholine, phosphocholine, glycerophosphocholine and betaine. Thus, the mammary gland was able to synthesize the choline metabolites found in milk, but these metabolites may not be derived exclusively from uptake from maternal blood. We have established that the total choline concentration in rat milk is sevenfold higher than previously reported, with >80% present as glycerophosphocholine and phosphocholine.