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Biomedical subjects

N P Dudman

Publications and source records attributed to N P Dudman.

At least 19 recordsLinked to original sources
PubMed2001

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.

Adult↗
PubMed2000

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.

Adult↗
PubMed2000

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).

Antibodies↗
PubMed1999

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.

Animals↗
PubMed1999

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.

Adult↗
PubMed1998

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.

Alternative Splicing↗
PubMed1996

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.

Arterial Occlusive Diseases↗
PubMed1996

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.

Adolescent↗
PubMed1996

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.

Calibration↗
PubMed1995

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.

Adult↗
PubMed1994

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.

Adult↗
PubMed1994

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.

Chemical Precipitation↗
PubMed1993

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.

6-Ketoprostaglandin F1 alpha↗
PubMed1993

Circulating lipid hydroperoxide levels in human hyperhomocysteinemia. Relevance to development of arteriosclerosis.

Elevated circulating homocyst(e)ine is a risk factor for occlusive vascular disease. We explored whether elevated plasma homocyst(e)ine is associated with increased plasma lipid hydroperoxides that might trigger vascular disease. We obtained plasma containing high levels of homocyst(e)ine from four patients with a homozygous deficiency of cystathionine beta-synthase activity and also from four heterozygotes with a deficiency of this enzyme after an oral methionine load. The mean plasma non-protein-bound homocyst(e)ine level in all subjects was more than 11-fold higher than the mean normal fasting value. Levels of high density lipoprotein (HDL) cholesteryl ester hydroperoxides (CEOOH), normalized against the concentration of free cholesterol in HDL, were not elevated in our subjects (mean +/- SD, 0.0091 +/- 0.0061) compared with values for 14 fasting healthy donors (0.0164 +/- 0.0086). An inverse dependency was observed between plasma total homocyst(e)ine and HDL CEOOH (r = -0.78, p = 0.023). Also, the ubiquinol-10/ubiquinone-10 ratio in HDL, which is expected to fall during oxidative stress, increased with plasma homocyst(e)ine. Since HDL contains the majority of detectable plasma lipid hydroperoxides, of which CEOOHs are the most abundant, our data suggest that an elevated plasma homocyst(e)ine level does not enhance oxidative stress, increase the levels of lipid hydroperoxides in plasma, or generate vascular damage by this mechanism.

Adult↗
PubMed1993

Disordered methionine/homocysteine metabolism in premature vascular disease. Its occurrence, cofactor therapy, and enzymology.

Mild homocysteinemia occurs surprisingly often in patients with premature vascular disease. We studied the possible enzymatic sources of this mild hyperhomocysteinemia and the control of homocysteine levels in plasma by treatment of patients with the cofactors and cosubstrates of homocysteine catabolism. We assessed homocysteine metabolism in 131 patients who had premature disease in their coronary, peripheral, or cerebrovascular circulation by using a standard oral methionine-load test. Impaired homocysteine metabolism occurred in 28 patients. We assayed levels of the primary enzymes of homocysteine catabolism in cultured skin fibroblast extracts from 15 of these 28 patients. The patients' cystathionine beta-synthase levels (3.68 +/- 2.52 nmol/h per milligram of cell protein, mean +/- SD) were markedly depressed compared with those from 31 healthy adult control subjects (7.61 +/- 4.49, P < .001). The patients' levels of 5-methyltetrahydrofolate: homocysteine methyltransferase were normal. While betaine: homocysteine methyltransferase was not expressed in skin fibroblasts, 24-hour urinary betaine and N,N-dimethylglycine measurements were consistent with normal or enhanced remethylation of homocysteine by betaine: homocysteine methyltransferase in the 13 patients tested. When treated daily with choline and betaine, pyridoxine, or folic acid, there was a normalization of the postmethionine plasma homocysteine level in 16 of 19 patients. Our results indicate that mild homocysteinemia in premature vascular disease may be caused by either a folate deficiency or deficiencies in cystathionine beta-synthase activity. It does not necessarily involve deficiencies of either 5-methyltetrahydrofolate:homocysteine methyltransferase or betaine:homocysteine methyltransferase. Effective treatment regimens are also defined.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗