Homocysteine induces expressions of adhesive molecules on leukocytes in whole blood.

OBJECTIVE: To observe whether homocysteine can directly alter the expressions of CD11b, CD18, CD14 and L-selectin on neutrophils, monocytes, and lymphocytes in whole blood from healthy human subjects. METHODS: Leukocyte surface adhesive molecule expressions were analyzed by immunofluorescence flow cytometry. RESULTS: Homocysteine at the lowest concentration (20 mumol/L) significantly increased surface adhesive molecule expressions of CD11b and CD18 on each cell type and CD14 on monocytes and neutrophils in whole blood. These effects were increased at homocysteine concentrations of 200 and 400 mumol/L, but at concentrations > or = 1 mmol/L, CD11b/CD18 and CD14 expressions on all types of leukocytes were decreased. L-selectin expression was slightly decreased on all cell types in whole blood by homocysteine. CONCLUSION: Homocysteine alters leukocyte expressions of CD11b/CD18, CD14 and L-selectin on leukocytes, which may be involved into homocysteine-induced leukocyte adhesion and migration.

Interrelations between plasma homocysteine and intracellular S-adenosylhomocysteine.

S-Adenosylhomocysteine, a potent intracellular methylation inhibitor, is suggested as a potential mediator for hyperhomocysteinemia-related vascular changes. We investigated the effect of acute and chronic hyperhomocysteinemia on intracellular S-adenosylhomocysteine and S-adenosylmethionine in rats and humans. Elevated plasma homocysteine in rats infused with homocysteine produced an increase in S-adenosylhomocysteine (P < 0.001) but not S-adenosylmethionine levels (P > 0.05) in various rat tissues. However intraerythrocyte S-adenosylhomocysteine and S-adenosylmethionine levels were not changed in homocysteine-infused rats and human subjects with experimentally acute hyperhomocysteinemia by methionine loading test. In contrast, erythrocyte S-adenosylhomocysteine levels were significantly higher in chronic renal failure patients, who had chronically elevated plasma homocysteine levels, than in either vascular disease patients or healthy controls (P < 0.05). In conclusion, acute hyperhomocysteinemia can increase intracellular S-adenosylhomocysteine levels in tissues actively involved in homocysteine metabolism. The findings are relevant to homocysteine-related endothelial dysfunction since S-adenosylhomocysteine modulates endothelial cell apoptosis.

Physical proximity and functional association of glycoprotein 1balpha and protein-disulfide isomerase on the platelet plasma membrane.

Platelet function is influenced by the platelet thiol-disulfide balance. Platelet activation resulted in 440% increase in surface protein thiol groups. Two proteins that presented free thiol(s) on the activated platelet surface were protein-disulfide isomerase (PDI) and glycoprotein 1balpha (GP1balpha). PDI contains two active site dithiols/disulfides. The active sites of 26% of the PDI on resting platelets was in the dithiol form, compared with 81% in the dithiol form on activated platelets. Similarly, GP1balpha presented one or more free thiols on the activated platelet surface but not on resting platelets. Anti-PDI antibodies increased the dissociation constant for binding of vWF to platelets by approximately 50% and PDI and GP1balpha were sufficiently close on the platelet surface to allow fluorescence resonance energy transfer between chromophores attached to PDI and GP1balpha. Incubation of resting platelets with anti-PDI antibodies followed by activation with thrombin enhanced labeling and binding of monoclonal antibodies to the N-terminal region of GP1balpha on the activated platelet surface. These observations indicated that platelet activation triggered reduction of the active site disulfides of PDI and a conformational change in GP1balpha that resulted in exposure of a free thiol(s).

Identification and characterization of a cystathionine beta/gamma-lyase from Lactococcus lactis ssp. cremoris MG1363.

This paper describes the nucleotide sequence of a gene encoding cystathionine beta/gamma-lyase from Lactococcus lactis ssp. cremoris MG1363, its overexpression in Escherichia coli and some functional characteristics of the purified recombinant protein.

The effect of diet on plasma homocysteine concentrations in healthy male subjects.

OBJECTIVE: To determine the effect of habitual omnivorous and vegetarian diets on folate and vitamin B12 status and the subsequent effect on homocysteine concentration. DESIGN: Cross-sectional comparison of free-living habitual meat-eaters and habitual vegetarians. SETTING: The study was conducted at RMIT University, Melbourne. SUBJECTS: One hundred and thirty-nine healthy male subjects (vegans n=18, ovolacto vegetarians n=43, moderate meat-eaters n=60 and high meat-eaters n=18) aged 20-55 y who were recruited in Melbourne. OUTCOME MEASURES: Fasting plasma or serum from each subject was analysed for folate, vitamin B12 and homocysteine concentration. A semi-quantitative Food Frequency Questionnaire was completed by a subset of subjects from each group to determine methionine intake. RESULTS: The two meat eating groups consumed significantly greater levels of methionine (P<0.001). There was no clear trend in plasma folate status between groups, however the plasma vitamin B12 concentration decreased progressively from the high-meat-eating group to vegans (P<0.05). An inverse trend was observed with plasma homocysteine concentration, with vegans showing the highest levels and high meat eaters the lowest (P<0.05). CONCLUSIONS: Dietary methionine intake has no observable effect on plasma homocysteine concentration. In habitual diets, where folate intake is adequate, lowered vitamin B12 intake from animal foods leads to depleted plasma vitamin B12 concentration with a concomitant increase in homocysteine concentration. The suggested mechanism is the failure to transfer a methyl group from methyl tetrahydrofolate by vitamin B12 in the remethylation of homocysteine to methionine.

Homocysteine enhances neutrophil-endothelial interactions in both cultured human cells and rats In vivo.

Despite intense investigation, mechanisms linking the development of occlusive vascular disease with elevated levels of homocysteine (HCY) are still unclear. The vascular endothelium plays a key role in regulating thrombogenesis and thrombolysis. We hypothesized that vascular lesions in individuals with elevated plasma HCY may be related to a dysfunction of the endothelium triggered by HCY. We investigated the effect of HCY on human neutrophil adhesion to and migration through endothelial monolayers. We also examined the effect of HCY on leukocyte adhesion and migration in mesenteric venules of anesthetized rats. We found that pathophysiological concentrations of HCY in vitro induce increased adhesion between neutrophils and endothelial cells. This contact results in neutrophil migration across the endothelial layer, with concurrent damage and detachment of endothelial cells. In vivo, HCY infused in anesthetized rats caused parallel effects, increasing leukocyte adhesion to and extravasation from mesenteric venules. Our results suggest that extracellular H2O2, generated by adherent neutrophils and/or endothelial cells, is involved in the in vitro endothelial cell damage. The possibility exists that leukocyte-mediated changes in endothelial integrity and function may lead to the vascular disease seen in individuals with elevated plasma HCY.

Mutational analysis of the cystathionine beta-synthase gene: a splicing mutation, two missense mutations and an insertion in patients with homocystinuria. Mutations in brief no. 120. Online.

RT-PCR and direct sequence analyses were used to define mutations in the cystathionine beta-synthase (CBS) gene in two unrelated male patients with vitamin B6 nonresponsive homocystinuria. Both patients were compound heterozygotes for CBS alleles containing point mutations. One patient had a maternally derived G->A transition in the splice-donor site of intron 1, resulting in aberrant splicing of CBS mRNA. The other allele contained a missense mutation resulting in the previously reported E144K mutant CBS protein. The second patient had a maternally derived 4 bp insertion in exon 17, predicted to cause a CBS peptide of altered amino acid sequence. A 494G->A transition was found in exon 4 of the other allele, predicting a C165Y substitution. Expression of recombinant CBS protein, containing the C165Y mutation, had no detectable catalytic activity. Each mutation was confirmed in genomic DNA.

Gender differences and other determinants of the rise in plasma homocysteine after L-methionine loading.

The methionine load test is widely used to identify subjects with mild homocysteinemia. We studied factors influencing the rise in plasma homocysteine (tHcy) levels following oral methionine loading, after taking into account determinants of the basal level. The study population comprised 522 persons (mean age 53, range 17 to 95; 63% male; 60% with coronary disease). tHcy was measured fasting and 4 h after administration of L-methionine (dose 4 g/m2). The percentage rise in tHcy was greater in females, at younger ages, and with lower serum creatinine. Basal tHcy was lower in those with high levels of serum folate, red cell folate and serum B12; they also showed a greater percentage rise with loading. After adjustment for age, creatinine and serum vitamins the percent rise was greater in females (mean 230%, S.D. 92) than in males (mean 172%, S.D. 71, P < 0.0001). About 1/4 of this effect was explained by the methionine dose, but important independent gender effects remained. When the results of the loading test were compared with an overall reference range, females were systematically labelled as 'high' but comparison with age and gender-specific reference ranges normalised the sex distribution. Our data show that a single L-methionine loading protocol for both sexes is not appropriate. Gender-specific reference ranges should be used to define post-load homocysteinemia and previous studies which have identified a high proportion of females with this trait probably reflect the need for gender specific reference ranges.

Fasting and post-methionine homocyst(e)ine levels in a healthy Australian population.

BACKGROUND: Plasma homocyst(e)ine (H[e]) is frequently measured in patients with occlusive vascular disease, but levels vary between populations and between laboratories. AIMS: We sought to derive reference values for an Australian population over a wide age range. METHODS: We measured plasma H(e) in the fasting state and after methionine loading in 116 volunteers selected at random from the Hunter districts of the New South Wales electoral roll and in 49 apparently healthy, active subjects recruited from the region's lawn bowling clubs. Reference ranges were derived for both sexes, in three age strata. We collaborated with two international laboratories in standardising our results. RESULTS: Mean fasting H(e) levels were approximately 2.7 mumol/L higher in men than women, at all ages (95% CI for the difference, 1.2 to 4.1 mumol/L). Levels increased with age (approximately 1.0 mumol/L/decade, 95% CI 0.5 to 1.5) and were correlated with serum creatine (r = 0.48), serum folate (r = -0.30), red cell folate (r = -0.25) and serum B12 (r = -0.31) (all p < 0.001). These results are similar to those reported in other populations. CONCLUSIONS: We have defined reference ranges for a typical Australian population, following careful laboratory standardisation. H(e) levels must be interpreted with regard to age, sex, renal function and vitamin B12 and folate status.

Characterisation of five missense mutations in the cystathionine beta-synthase gene from three patients with B6-nonresponsive homocystinuria.

Homocystinuria, due to a deficiency of the enzyme cystathionine beta-synthase (CBS), is an inborn error of sulphur-amino acid metabolism. This is an autosomal recessive disease which results in hyperhomocysteinaemia and a wide range of clinical features, including optic lens dislocation, mental retardation, skeletal abnormalities and premature thrombotic events. We report the identification of 5 missense mutations in the protein-coding region of the CBS gene from 3 patients with pyridoxine-nonresponsive homocystinuria. Reverse-transcription PCR was used to amplify CBS cDNA from each patient and the coding region was analysed by direct sequencing. The mutations detected included 3 novel (1058C-->T, 992C-->A and 1316G-->A) and 2 previously identified (430G-->A and 833C-->T) base alterations in the CBS cDNA. Each of these mutations predicts a single amino acid substitution in the CBS polypeptide. Appropriate cassettes of patient CBS cDNA, containing each of the above defined mutations, were used to replace the corresponding cassettes of normal CBS cDNA sequence within the bacterial expression vector pT7-7. These recombinant mutant and normal CBS constructs were expressed in Escherichia coli cells and the catalytic activities of the mutant proteins were compared with normal. All of the mutant proteins exhibited decreased catalytic activity in vitro, which confirmed the association between the individual mutation and CBS dysfunction in each patient.

Assay of plasma homocysteine: light sensitivity of the fluorescent 7-benzo-2-oxa-1, 3-diazole-4-sulfonic acid derivative, and use of appropriate calibrators.

The recognition of homocysteine as a vascular risk factor has led to increased clinical interest in assaying plasma homocysteine concentrations. Our aim was to improve the reliability of a widely used assay based on HPLC of the fluorescent 7-benzo-2-oxa-1, 3-diazole-4-sulfonic acid (SBD) derivative. We found that SBD derivatives of homocysteine, cysteine, and N-acetylhomocysteine were highly unstable in light but essentially stable in the dark for several hours at either 0 degree C or 25 degrees C. As our primary calibrator, we chose homocystine added to human serum for more consistent results than homocysteine or homocystine in an aqueous buffer. N-acetylcysteine was effective as an internal recovery standard. We observed a previously unreported peak with a prolonged elution time in some plasma samples from subjects who had ingested methionine. Our findings suggest improvements in this and other assay procedures for plasma homocysteine.

Variable hyperhomocysteinaemia phenotype in heterozygotes for the Gly307Ser mutation in cystathionine beta-synthase.

BACKGROUND: A deficiency of cystathionine beta-synthase (CBS) activity is the most frequent cause of homocystinuria, an autosomal recessive disease with multiple clinical manifestations. Mutations in the CBS gene have been reported in several patients with homocystinuria. AIMS: To establish the molecular basis of CBS deficiency in a female patient with pyridoxine non-responsive homocystinuria, and to apply the findings to genetic screening of her family members. METHODS: The entire coding region of the CBS cDNA was amplified by PCR and used for direct sequence analysis. Mutant alleles were confirmed by direct sequence analysis of PCR-amplified genomic DNA, and by a combination of single strand conformation polymorphism and temperature gradient gel electrophoresis analysis. RESULTS: The proband was homozygous for a G919A base transition which predicts the substitution of serine for glycine at codon 307 in the CBS protein (G307S). The parents (both of Irish background) were heterozygotes for the G307S allele, while an asymptomatic sibling had normal CBS sequence, Plasma homocysteine, assessed after an oral methionine load, indicated the mother clearly had moderate hyperhomocysteinaemia, whereas the father had normal concentrations of homocysteine. This is the first report of a normal methionine load test in a proven heterozygote for a CBS mutation which causes severe homocystinuria in the homozygote. Other factor(s) may have contributed to hyperhomocysteinaemia in the mother. The G307S allele has been reported in other patients and appears to be a common allele among families of Celtic origin.

Serial measures of plasma homocyst(e)ine after acute myocardial infarction.

To determine whether plasma levels of homocyst(e)ine are affected by the acute phase response, we studied 10 subjects serially after acute myocardial infarction. Our data indicate that measurement of homocyst(e)ine in patients with myocardial infarction should ideally be deferred for 7 days if spuriously low levels are to be averted.

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

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.

1H NMR determination of urinary betaine in patients with premature vascular disease and mild homocysteinemia.

Urinary N,N,N-trimethylglycine (betaine) and N,N-dimethylglycine (DMG) have been identified and quantified for clinical purposes by proton nuclear magnetic resonance (1H NMR) measurement in previous studies. We have assessed these procedures by using both one-dimensional (1-D) and 2-D NMR spectroscopy, together with pH titration of urinary extracts to help assign 1H NMR spectral peaks. The betaine calibration curve linearity was excellent (r = 0.997, P = 0.0001) over the concentration range 0.2-1.2 mmol/L, and CVs for replicate betaine analyses ranged from 7% (n = 10) at the lowest concentration to 1% (n = 9) at the highest. The detection limit for betaine was < 15 mumol/L. Urinary DMG concentrations were substantially lower than those of betaine. Urinary betaine and DMG concentrations measured by 1H NMR spectroscopy from 13 patients with premature vascular disease and 17 normal controls provided clinically pertinent data. We conclude that 1H NMR provides unique advantages as a research tool for determination of urinary betaine and DMG concentrations.

Effect of serum lipoprotein(a) on estimation of low-density lipoprotein cholesterol by the Friedewald formula.

The calculation of serum low-density lipoprotein cholesterol (LDL-C) by the Friedewald formula does not account for the cholesterol associated with lipoprotein(a) [Lp(a)]. To quantify the contribution of Lp(a) cholesterol to total serum cholesterol, we measured concentrations of serum Lp(a) by an ELISA and concentrations of other serum lipids and lipoproteins by standard assays in 23 normolipemic women, ages 50-60 years. In measuring serum high-density lipoprotein we found that polyethylene glycol 6000 precipitated > 99.8% of all Lp(a). When serum Lp(a) concentrations were < or = 300 mg/L, 301-600 mg/L, and > 600 mg/L, the uncorrected serum LDL-C was overestimated, respectively, by a mean of 4.1% (n = 7), 8.5% (n = 8), and 21.4% (n = 8). Serum Lp(a) concentrations were positively correlated with percentage overestimation (P < 0.001), but were not correlated with either corrected or uncorrected serum LDL-C. We conclude that the Friedewald formula should be modified to take into account the contribution of Lp(a) cholesterol to total serum cholesterol.

The relationship between infant and parent Lp(a) levels.

Since atherogenesis may begin in childhood, and elevated serum lipoprotein(a) (Lp(a)) concentrations increase cardiovascular risk, we explored the early expression of the apolipoprotein(a) (apo(a)) gene and relationships between infants' and parents' serum levels. In a consecutive series of 1032 babies aged 3-5 days the distribution of apo(a) levels was positively skewed as in adults but with lower levels: 50th and 95th percentiles were the equivalent of 30 mg/l and 130 mg/l of Lp(a) in serum. Concentrations were re-measured in 51 infants when aged 8.5 +/- 2 months together with parental values. Levels at 3-5 days and 8.5 months were highly correlated (r = 0.73, P < 0.0001, n = 51) with a twofold increase at 8.5 months. Regression coefficients between 8.5 months concentrations and those of fathers, of mothers and the average level of both parents were 0.439, 0.521 and 0.93, respectively (P < 0.0001 for each), and infant and parental levels were then not different. The positive and negative predictive values of first post-natal week capillary blood apo(a) measurements detecting a parent with serum Lp(a) above 300 mg/l were 95% and 70%. We conclude that the apo(a) gene is virtually fully expressed before 1 year during which apo(a) levels track closely and are predictive of parental values. Childhood Lp(a) measurements may identify families at enhanced cardiovascular risk and facilitate targeted prevention.

Effects of homocysteine and related compounds on prostacyclin production by cultured human vascular endothelial cells.

Elevated plasma homocysteine is associated with an increased risk of intravascular thrombosis. Platelet aggregation and thrombosis are inhibited by prostacyclin produced by the vascular endothelium. Our aim was to investigate whether homocysteine and related metabolites inhibit endothelial prostacyclin production. We used a radioimmunoassay for 6-ketoprostaglandin-F1 alpha to assay medium which had been in contact with confluent cultured endothelial cells. In medium containing 20% human serum, endothelial prostacyclin production was not specifically inhibited by homocysteine, S-adenosylhomocysteine or protein-bound homocysteine. Further, there was no consistent difference in prostacyclin production by cells cultured in medium containing sera from homocystinuria patients, compared with medium containing normal healthy sera. We conclude that vascular disorder in homocystinuria is unlikely to result from effects of homocysteine or related metabolites on endothelial prostacyclin production. By contrast, S-adenosylhomocysteine and protein-bound homocysteine specifically inhibited prostacyclin production by cells cultured in medium containing 20% fetal calf serum.