Homocysteine - from disease biomarker to disease prevention.

We have reviewed the literature and have identified more than 100 diseases or conditions that are associated with raised concentrations of plasma total homocysteine. The commonest associations are with cardiovascular diseases and diseases of the central nervous system, but a large number of developmental and age-related conditions are also associated. Few other disease biomarkers have so many associations. The clinical importance of these associations becomes especially relevant if lowering plasma total homocysteine by B vitamin treatment can prevent disease and so improve health. Five diseases can at least in part be prevented by lowering total homocysteine: neural tube defects, impaired childhood cognition, macular degeneration, primary stroke, and cognitive impairment in the elderly. We conclude from our review that total homocysteine values in adults of 10 µmol/L or below are probably safe, but that values of 11 µmol/L or above may justify intervention. Homocysteine is more than a disease biomarker: it is a guide for the prevention of disease.

Impact of age, gender and CYP2C9/2C19 genotypes on dose-adjusted steady-state serum concentrations of valproic acid-a large-scale study based on naturalistic therapeutic drug monitoring data.

PURPOSE: Valproic acid (VPA) has an extensive interindividual pharmacokinetic variability. Published data regarding the impact of gender, age, and CYP2C9/2C19 genetics on VPA variability are conflicting, and the purpose of present study is to clarify the effect of these factors on dose-adjusted steady-state serum VPA concentration (C:D ratio) in a large, naturalistic patient material. METHODS: In patients who had been subjected to cytochrome P450 (CYP) genotyping and therapeutic drug monitoring of VPA, information about serum concentrations, dose, gender, age, and CYP2C9/2C19 genotypes was retrospectively collected from a routine TDM database during the period 2008-2012. The effects of age, gender, and CYP2C9/CYP2C19 genotypes on C:D ratios of VPA were investigated by multivariate analyses (mixed model) including sampling time as covariate. RESULTS: In total, 857 serum concentrations from 252 patients were included. A significant gender effect was observed with a 1.3-fold higher estimated C:D ratio in females than in males, i.e., geometric means 0.34 vs. 0.27 µM/mg/day, respectively (p < 0.001). A similar and significant difference in estimated geometric means was found between patients >65 vs. ≤65 years, i.e., 0.36 vs. 0.26 µM/mg/day (p < 0.001), respectively. Finally, no association between the various CYP2C9/2C19 variant genotypes and C:D ratio of VPA was observed (p > 0.1). CONCLUSION: The present study shows that age and gender significantly influence VPA serum concentration. In order to obtain similar drug exposure, our findings suggest that older female patients would generally require 30-50 % lower dosing of VPA compared to younger males. Moreover, we conclude that CYP2C9/2C19 genotype is not relevant for variability in VPA exposure.

Association between serum lipids and membrane fatty acids and clinical characteristics in patients with schizophrenia.

OBJECTIVE: Earlier reports indicate that patients with schizophrenia have altered lipid levels in serum and cell membranes. The purpose of this study was to determine the relationship between clinical characteristics and serum and membrane lipids. METHOD: Fifty-five patients with schizophrenia and 51 healthy controls were included. The patients were characterized with Positive and Negative Syndrome Scale (PANSS) and Global Assessment of Functioning (GAF). Serum lipids [high- and low-density lipoprotein cholesterol (HDL, LDL) and triglyceride (TG)] and erythrocyte polyunsaturated fatty acids (PUFA) were measured. RESULTS: Among the participants with schizophrenia, there was a significant correlation between serum triglyceride levels and PANSS-positive symptoms (r = 0.28, P = 0.04), GAF-S (r = -0.48, P = 0.001) and GAF-F (r = -0.32, P = 0.01), and between HDL level and GAF-S (r = 0.37, P = 0.008) and GAF-F (r = 0.28, P = 0.04). Long-chain PUFA were significantly associated with PANSS-negative symptoms (r = 0.52, P < 0.001), GAF-S (r = -0.32, P = 0.02), and GAF-F (r = -0.29, P = 0.04). The patients with schizophrenia had significantly higher TG (P < 0.001) and lower HDL (P < 0.001) levels than healthy controls. HDL was also lower in the subgroup (n = 11) not receiving antipsychotic medication (P = 0.02). CONCLUSION: The results suggest associations between lipid profile and clinical characteristics. This may indicate a role for lipid biology in schizophrenia pathophysiology.

Changes in cognitive function during psychogeriatric treatment in relation to benzodiazepine cessation.

INTRODUCTION: The aim of this study was to investigate cognitive functions after admission to a geriatric psychiatric hospital, and to study the short-term effects of cessation of benzodiazepine use on cognitive functions. METHODS: Details of benzodiazepine use and serum concentration measurements were recorded on admission. The Hopkins verbal learning test, the Stroop test, Digit Vigilance Test and the Mini Mental Status Examination were performed on admission, and after 4 weeks of hospitalization. Test results were compared for the total group of patients, as well as for benzodiazepine "continuers" and the "quitters". RESULTS: For all patients (n=224), improved performances were observed in 10 out of 12 cognitive tests. Significant improvements were seen in 4 out of 12 tests. Benzodiazepine "quitters" improved significantly more than the "continuers" (p=0.027) only on the Hopkins verbal learning test, delayed recall performance. DISCUSSION: Among elderly psychiatric patients, cognitive function improved slightly during the 4 weeks of hospital treatment, but only for one of the memory tests, the improvement was related to the cessation of benzodiazepine treatment.

A clinical study of the cognitive effects of benzodiazepines in psychogeriatric patients.

Previous studies have shown cognitive impairment in long-term benzodiazepine users compared to non-users. However, little is known about such effects in a population of geriatric psychiatry patients. The aim of this study was to identify differences between benzodiazepine users and non-users on standardized tests of the cognitive fields of learning and memory, executive functions and vigilance, at admittance to a department of geriatric psychiatry.Hopkins verbal learning test, Stroop test and digit vigilance test were performed in all patients. Test performances were compared between benzodiazepine users (n=168) and non-users (n=73). A multiple linear regression model was used, adjusting for different baseline characteristics (years of education, dementia and depression).No significant differences in test results were found between benzodiazepine users and non-users on 11 out of 12 cognitive tests results. On one of the 12 test results (time used on the digit vigilance test), benzodiazepine users showed better performance compared to non-users (ß=-0.20, p=0.032). This finding was not statistically significant after Bonferroni correction for multiple testing.This study of geriatric psychiatry benzodiazepine users did not reveal cognitive impairment compared to non-users on the cognitive areas tested. Other possible negative consequences of benzodiazepine use should, however, also be considered when prescribing drugs to older patients.

The association of fasting plasma sulfur-containing compounds with BMI, serum lipids and apolipoproteins.

BACKGROUND AND AIM: Sulfur amino acids are recognized as potent modulators of lipid metabolism. Plasma total cysteine (tCys) is associated with fat mass, obesity and serum LDL-cholesterol and apolipoprotein (Apo)-B in large population studies. It is not known how fasting plasma concentrations of cysteine precursors and products relate to these associations in humans, given that sulfur-containing compounds (SCC) influence rodent weight gain and serum lipids. METHODS AND RESULTS: We investigated the cross-sectional associations of fasting plasma SCC (methionine, total homocysteine, cystathionine, tCys, taurine and total glutathione) with BMI and fasting serum lipids and apolipoproteins in 854 men and women with and without cardiovascular disease (CVD). In multiple linear regression analysis adjusted for age, gender, CVD and other SCC, neither methionine, taurine, nor total glutathione was associated with BMI. Plasma taurine was, however, inversely related to HDL-cholesterol (partial r = -0.12, p = 0.004) and its associated apoA1 (partial r = -0.18, p < 0.001). Plasma cystathionine correlated positively with triglycerides and BMI, while tCys positively correlated with total cholesterol, LDL-cholesterol (partial r = 0.20, p < 0.001) and its associated apoB. The associations of SCC with serum lipids were independent of BMI. tCys was also independently associated with BMI (partial r = 0.20, p < 0.001) after adjustment for other SCC, glucose, lipids and apolipoproteins. CONCLUSIONS: Fasting tCys is associated with BMI independently of metabolically related SCC. Elevation of plasma SCC is generally associated with an unfavorable lipid profile. The negative relations of plasma taurine with HDL-C and apoA1 deserve further investigation.

Effectiveness of B Vitamins and Their Interactions with Aspirin in Improving Cognitive Functioning in Older People with Mild Cognitive Impairment: Pooled Post-Hoc Analyses of Two Randomized Trials.

BACKGROUND AND OBJECTIVES: A randomized placebo-controlled trial found a significant negative interaction between aspirin and B vitamins in cognitive functioning in older people with mild cognitive impairment (MCI). To validate this finding, we pooled data of this trial with that of a similar B-vitamin trial (VITACOG) to examine the effectiveness of B vitamins and their interactions with aspirin in improving global cognitive functioning and slowing brain atrophy in older people with MCI. DESIGN: Pooled post-hoc analyses of two randomized placebo-controlled trials. PARTICIPANTS: In total, 545 older people with MCI were included in the study. INTERVENTION: Placebo or B-vitamin supplements (vitamin B12, folic acid with or without vitamin B6) for 24 months. MEASUREMENTS: The primary outcome was the Clinical Dementia Rating scale-global score (CDR-global). The secondary outcomes were CDR-sum of box score (CDR-SOB), memory Z-score, executive function Z-score, and whole brain atrophy rate. RESULTS: 71 (26.2%) and 83 (30.3%) subjects in the active and placebo group respectively were aspirin users. Overall, B vitamins reduced whole brain atrophy rate significantly (P = 0.003), but did not have significant effect on CDR-global, CDR-SOB, memory and executive function. Aspirin use had significant negative interaction effects on B vitamins in CDR-global and CDR-SOB (Beta = 0.993, P = 0.038, and Beta = 0.583, P = 0.009, respectively), but not in memory or executive function Z-scores. Among aspirin non-users, B-vitamin group subjects had more favourable changes in CDR-global and CDR-SOB (P = 0.019 and 0.057, respectively). B vitamins significantly slowed brain atrophy in aspirin non-users (P = 0.001), but not in aspirin users, though the interaction term was not significant (Beta = 0.192, P = 0.276). CONCLUSION: In older people with MCI, B vitamins had significantly favourable effects on global cognitive functioning and whole brain atrophy rate in those who were not taking aspirin, but not in aspirin users.

Combined analyses and extended follow-up of two randomized controlled homocysteine-lowering B-vitamin trials.

OBJECTIVES: In the Norwegian Vitamin Trial and the Western Norway B Vitamin Intervention Trial, patients were randomly assigned to homocysteine-lowering B-vitamins or no such treatment. We investigated their effects on cardiovascular outcomes in the trial populations combined, during the trials and during an extended follow-up, and performed exploratory analyses to determine the usefulness of homocysteine as a predictor of cardiovascular outcomes. DESIGN: Pooling of data from two randomized controlled trials (1998-2005) with extended post-trial observational follow-up until 1 January 2008. SETTING: Thirty-six hospitals in Norway. SUBJECTS: 6837 patients with ischaemic heart disease. INTERVENTIONS: One capsule per day containing folic acid (0.8 mg) plus vitamin B12 (0.4 mg) and vitamin B6 (40 mg), or folic acid plus vitamin B12, or vitamin B6 alone or placebo. MAIN OUTCOME MEASURES: Major adverse cardiovascular events (MACEs; cardiovascular death, acute myocardial infarction or stroke) during the trials and cardiovascular mortality during the extended follow-up. RESULTS: Folic acid plus vitamin B12 treatment lowered homocysteine levels by 25% but did not influence MACE incidence (hazard ratio, 1.07; 95% CI, 0.95-1.21) during 39 months of follow-up, or cardiovascular mortality (hazard ratio, 1.12; 95% CI, 0.95-1.31) during 78 months of follow-up, when compared to no such treatment. Baseline homocysteine level was not independently associated with study outcomes. However, homocysteine concentration measured after 1-2 months of folic acid plus vitamin B12 treatment was a strong predictor of MACEs. CONCLUSION: We found no short- or long-term benefit of folic acid plus vitamin B12 on cardiovascular outcomes in patients with ischaemic heart disease. Our data suggest that cardiovascular risk prediction by plasma total homocysteine concentration may be confined to the homocysteine fraction that does not respond to B-vitamins.

Serum concentrations of citalopram--dose-dependent variation in R- and S-enantiomer ratios.

INTRODUCTION: The antidepressive effect of racemic citalopram (CIT) is exerted by S-CIT, while R-CIT is a partial antagonist to S-CIT. Since R-and S-CIT are metabolized by different pathways, we investigated whether the ratio of S- and R-CIT may differ between individuals on the same dose of racemic CIT, and if a possible variability in the R/S-ratio could be dose-dependent. METHODS: A chiral analysis of R- and S-CIT in serum samples taken from 88 female patients receiving treatment with racemic CIT was performed using high-pressure liquid chromatography. RESULTS: The mean levels of R-CIT were significantly higher than those of S-CIT in all dose groups. The R/S-CIT ratio increased from 1.99 to 2.45 with an increase in the dose (p<0.05), and the interindividual variance in the R/S-CIT ratio was up to four-fold on the same dosage. DISCUSSION: Our findings show that the stereoselective metabolism of citalopram IN VIVO has pharmacokinetic consequences reflected by dose dependent variations of enantiomeric drug concentrations, as well as substantial interindividual variabilities in the ratios of the concentrations. The clinical consequences, however, are unclear and should be further explored.

Identification of a novel CYP2C19-mediated metabolic pathway of S-citalopram in vitro.

Systemic exposure of the antidepressant S-citalopram (escitalopram, SCIT) differs several-fold according to variable cytochrome P450 2C19 activity, demonstrating the importance of this enzyme for the metabolic clearance of SCIT in vivo. However, previous studies have indicated that the involvement of CYP2C19 in formation of the metabolite N-desmethyl S-citalopram (SDCIT) is limited. Therefore, the purpose of the present in vitro study was to investigate to what extent the CYP2C19-mediated clearance of SCIT was due to a metabolic pathway different from N-desmethylation and to identify the product(s) of this possible alternative metabolic reaction. CYP2C19-mediated metabolism of SCIT was investigated using recombinant Supersomes expressing human CYP2C19. Initial experiments showed that approximately half of the CYP2C19-mediated clearance of SCIT was accounted for by the N-desmethylation pathway. Subsequent experiments identified that, in addition to SDCIT, the propionic acid metabolite of SCIT (SCIT PROP) was formed by CYP2C19 in vitro. Formation of SCIT PROP accounted for 35% of total CYP2C19-mediated clearance of SCIT (calculated as the ratio between metabolite formation rate and substrate concentration at low substrate concentration). Moreover, analysis of samples from six CYP2C19-genotyped patients treated with SCIT indicated that differences in serum concentrations of SCIT between CYP2C19 genotypes may be due to a combined effect on SCIT PROP and SDCIT formation. Identification of SCIT PROP as a metabolic pathway catalyzed by CYP2C19 might explain why impaired CYP2C19 activity has a substantially larger effect on SCIT exposure than estimated from in vitro data based solely on formation of SDCIT.

Plasma vitamin B12 status and cerebral white-matter lesions.

BACKGROUND AND OBJECTIVE: Elevated homocysteine has been associated with a higher prevalence of cerebral white-matter lesions and infarcts, and worse cognitive performance. This raises the question whether factors involved in homocysteine metabolism, such as vitamin B(12), are also related to these outcomes. This study examined the association of several markers of vitamin B(12) status with cerebral white-matter lesions, infarcts and cognition. METHODS: The study evaluated the association of plasma concentrations of vitamin B(12), methylmalonic acid, holotranscobalamin and transcobalamin saturation with cerebral white-matter lesions and infarcts at baseline and cognition at baseline and during follow-up among 1019 non-demented elderly participants of the population-based Rotterdam Scan Study. Analyses were adjusted for several potential confounders, including homocysteine and folate concentration. RESULTS: Poorer vitamin B(12) status was significantly associated with greater severity of white-matter lesions, in particular periventricular white-matter lesions, in a concentration-related manner. Adjustment for common vascular risk factors (including blood pressure, smoking, diabetes and intima media thickness) did not alter the associations. Adjustment for homocysteine and folate modestly weakened the associations. No association was observed for any of the studied markers of vitamin B(12) status with presence of brain infarcts and baseline cognition or cognitive decline during follow-up. CONCLUSIONS: These results indicate that vitamin B(12) status in the normal range is associated with severity of white-matter lesions, especially periventricular lesions. Given the absence of an association with cerebral infarcts, it is hypothesised that this association is explained by effects on myelin integrity in the brain rather than through vascular mechanisms.

Serum concentrations of sertraline and N-desmethyl sertraline in relation to CYP2C19 genotype in psychiatric patients.

OBJECTIVE: To investigate the impact of CYP2C19 genotype on serum concentrations of sertraline and N-desmethyl sertraline in psychiatric patients. METHODS: Patients treated with sertraline (n = 121) were divided into six subgroups according to CYP2C19 genotype: CYP2C19*17/*17, CYP2C19*1/*17, CYP2C19*1/*1, CYP2C19*17/def, CYP2C19*1/def and CYP2C19def/def (def = allele encoding defective CYP2C19 metabolism, i.e. *2 and *3). Dose-adjusted serum concentrations were compared by linear mixed model analyses using the CYP2C19*1/*1 subgroup as reference. RESULTS: Subgroups carrying one or two alleles encoding defective CYP2C19 metabolism achieved significantly higher mean dose-adjusted serum concentrations of sertraline and N-desmethyl sertraline compared to the CYP2C19*1/*1 subgroup (P < 0.05). The effect of CYP2C19 genotype was expressed as 3.2-fold (sertraline) and 4.5-fold (N-desmethyl sertraline) higher dose-adjusted serum concentrations in the CYP2C19def/def subgroup compared to the CYP2C19*1/*1 subgroup (P < 0.01). The CYP2C19*17 allele had no influence on the dose-adjusted serum concentrations of sertraline and N-desmethyl sertraline. CONCLUSION: The significantly higher serum concentrations associated with alleles encoding defective CYP2C19 metabolism might be of relevance for the clinical outcome of sertraline treatment.

Serum concentrations of venlafaxine and its metabolites O-desmethylvenlafaxine and N-desmethylvenlafaxine in heterozygous carriers of the CYP2D6*3, *4 or *5 allele.

OBJECTIVE: Increased systemic exposure of the antidepressant venlafaxine and increased risk of side effects has previously been observed in patients with defective CYP2D6 function [poor metabolisers (PMs)]. The aim of this study was to evaluate venlafaxine pharmacokinetics in carriers of one functional and one defective CYP2D6 allele [heterozygous extensive metabolisers (HEMs)]. METHODS: Data was collected retrospectively from a therapeutic drug-monitoring database. All CYP-genotyped patients with steady-state serum concentration measurements of venlafaxine and metabolites were included in the study. Patients were divided in groups: *1/*1 [homozygous extensive metabolisers (EMs)], *1/*3, *4 or *5 (HEMs) and *4/*4 (PMs). Dose-adjusted serum concentrations of venlafaxine, O-desmethylvenlafaxine, N-desmethylvenlafaxine, and the metabolic ratio (O-desmethylvenlafaxine/venlafaxine) were compared between the different genotype groups. RESULTS: The sum of venlafaxine and O-desmethylvenlafaxine serum concentrations was not significantly different between genotype groups. Metabolic ratio was 50% lower in HEMs (n = 18) than in EMs (n = 20) (p < 0.05). Serum concentration of N-desmethylvenlafaxine was 5.5-fold higher in HEMs (p < 0.01) and 22-fold higher in PMs (p < 0.001) than in EMs. CONCLUSION: The study showed a shift in the metabolic pathway resulting in substantially higher levels of N-desmethylvenlafaxine in HEMs than in EMs. The metabolic pattern of venlafaxine in HEMs was similar to previous observations in PMs and possibly represents an increased risk of venlafaxine-related side effects in HEM patients.

Determinants and vitamin responsiveness of intermediate hyperhomocysteinemia (> or = 40 micromol/liter). The Hordaland Homocysteine Study.

From 1992-93, we screened 18,043 subjects, aged 40-67 yr, and found 67 cases (0.4%) with total plasma homocysteine (tHcy) > or = 40 micromol/liter. Compared to 329 controls, the cases had lower plasma folate and cobalamin levels, lower intake of vitamin supplements, consumed more coffee, and were more frequently smokers. Homozygosity for the C677T mutation in the methylenetetrahydrofolate reductase gene was observed in 73.1% of the cases and 10.2% of the controls. Only seven cases with cobalamin deficiency and one with homocystinuria received specific therapeutic instructions. 2 yr after the screening, 58 subjects were reinvestigated. 41 still had tHcy > 20 micromol/liter, and in 37 of these, intervention with low dose folic acid (0.2 mg/d) was started. Notably, 34 of 37 (92%) had homozygosity for the C677T mutation. Plasma tHcy was reduced in all but two after 7 wk, and became normal within 7 mo in 21 of 37 subjects. Most of the remaining subjects obtained a normal tHcy level with 5 mg/d of folic acid. We conclude that most subjects with hyperhomocysteinemia > or = 40 micromol/liter in the general population have the C677T mutation combined with low folate status. Daily supplement of low dose folic acid will reduce and often normalize their tHcy level.

Apolipoprotein E epsilon4 and impaired episodic memory in community-dwelling elderly people: a marked sex difference. The Hordaland Health Study.

BACKGROUND: Among elderly people without dementia, the apolipoprotein E epsilon4 allele (APOE4) has been associated with cognitive deficit, particularly in episodic memory, but few reports are available on whether this association differs by sex. METHODS: In a community-dwelling Norwegian cohort of 2181 elderly people (55% women), aged 70-74 years, episodic memory was examined in relation to sex and APOE4 zygosity, with the Kendrick Object Learning Test (KOLT). RESULTS: Possession of at least one APOE4 allele had a modest, detrimental effect on episodic memory in women, whereas in men, heterozygotes were unaffected and homozygotes had markedly lower scores across the distribution of KOLT scores. This sex difference was found consistently in all analyses: on comparing means and medians, examining trends across quintiles, and studying the distribution of scores and the risk of cognitive impairment. Results were broadly similar when adjusted for known determinants of cognition and also when severely impaired participants were excluded. The adjusted odds ratio (OR) of cognitive impairment in women was shown to be 1.8 (95% confidence interval (CI): 1.1 to 2.8) for heterozygotes and 1.1 (0.3 to 3.7) for homozygotes; the adjusted OR in men was observed to be 1.1 (0.6 to 2.1) for heterozygotes and 10.7 (4.7 to 24) for homozygotes. CONCLUSIONS: Although the harmful effect of APOE4 on episodic memory was modest in women, the risk was found to occur in about 30%. APOE4 was observed to have a dramatic effect on episodic memory in men, but only in homozygotes, who comprised about 3% of men: the whole male homozygous group showed a marked shift to lower memory scores.

Factor V Leiden, pregnancy complications and adverse outcomes: the Hordaland Homocysteine Study.

BACKGROUND: The factor V Leiden (FVL) mutation is the most common cause of inherited thrombophilia in Caucasian populations, and women with this variant allele are at increased risk for pregnancy complications. AIM: To examine whether the FVL allele is associated with pregnancy complications and adverse outcomes in a population-based study, and to identify potential factors that interact with the FVL genotype. DESIGN: Retrospective cohort study in a geographically-defined area. METHODS: Polymorphisms of factor V 1691G-->A, methylenetetrahydrofolate reductase (MTHFR) 677C --> T and 1298A --> C and plasma levels of total homocysteine, folate and vitamin B(12) were determined in blood samples collected in 1992-1993 from 5874 women aged 40-42 years, and linked with 14 474 pregnancies in the same women, recorded in the Medical Birth Registry of Norway, 1967-1996. RESULTS: The allelic frequency of FVL was 3.7% (6.9% heterozygotes, 0.3% homozygotes). Maternal FVL mutation was associated with significantly higher risks of pre-eclampsia (OR 1.63, 95%CI 1.15-2.30), pre-eclampsia at <37 weeks (OR 2.76, 1.34-5.70), low birth weight (OR 1.34, 95%CI 1.03-1.74) and stillbirth (OR 2.20, 95%CI 1.45-3.36). The presence of a variant allele for the 677C --> T MTHFR polymorphism strengthened the association between FVL and stillbirth (OR 3.34, 95%CI 1.95-5.73) (p(interaction) = 0.034). DISCUSSION: FVL mutation is a significant risk factor for pregnancy complications and adverse outcomes, and MTHFR 677CT/TT genotype can further enhance the risk of stillbirth.

Investigation of relationship between reduced, oxidized, and protein-bound homocysteine and vascular endothelial function in healthy human subjects.

Previous studies investigating homocysteine and vascular disease have relied on total plasma homocysteine as the sole index of homocysteine status. We examined the dynamic relationship between vascular endothelial function and concentrations of total, protein-bound oxidized, free oxidized, and reduced homocysteine to identify the homocysteine form associated with endothelial dysfunction in humans. We investigated 14 healthy volunteers (10 men, 4 women). Brachial artery flow-mediated dilatation was measured at baseline and at 30, 60, 120, 240, and 360 minutes after oral (1) L-methionine (50 mg/kg), (2) L-homocysteine (5 mg/kg), and (3) placebo. Plasma concentrations of total, protein-bound oxidized, free oxidized, and reduced homocysteine were measured at each time point, and nitroglycerin-induced dilatation at was assessed at 0, 120, and 360 minutes. Flow-mediated dilatation fell, and concentrations of total, protein-bound oxidized, free oxidized, and reduced homocysteine increased after oral homocysteine and oral methionine (all P<0.05 for difference in time course compared with placebo). Flow-mediated dilatation showed a reciprocal relationship with reduced homocysteine during both homocysteine and methionine loading. In both loading studies, peak reduction in flow-mediated dilatation coincided with maximal reduced homocysteine concentrations. In contrast, there was no consistent relationship between flow-mediated dilatation and free oxidized homocysteine, protein-bound oxidized homocysteine, or related species. Nitroglycerin-induced dilatation was unchanged by oral homocysteine and oral methionine (P>0.10 compared with placebo). Reduced homocysteine is closely associated with endothelial dysfunction during oral methionine and oral homocysteine loading. Our observations support the hypothesis that reduced homocysteine is the deleterious form of homocysteine for vascular function in vivo and suggest a less important role for other homocysteine species.

Disposition of endogenous homocysteine by mouse fibroblast C3H/10T1/2 Cl 8 and the chemically transformed C3H/10T1/2 MCA Cl 16 cells following methotrexate exposure.

The tumorigenic cell line termed "MCA Cl 16" was derived from C3H/10T1/2 clone (Cl) 8 cells by chemical transformation in the presence of 3-methylcholanthrene [(MCA) CAS: 56-49-5]. Transformed (Cl 16) cells were more sensitive toward the cytotoxic effect of methotrexate (MTX) than their normal counterpart Cl 8 cells. The disposition of endogenous L-homocysteine (Hcy) was investigated in these two cell lines after MTX exposure. Both nonmalignant and transformed cells exported Hcy into the extracellular medium, and only small amounts were retained within the cells. The Hcy efflux from the malignant cells was markedly increased after MTX exposure (0.5-10 microM), and this effect was almost completely prevented by 5-formyl-tetrahydrofolate (THF), whereas treatment with thymidine plus hypoxanthine did not inhibit the MTX-dependent Hcy efflux. Cytotoxic concentration of MCA reduced rather than increased the Hcy efflux from these cells. High concentrations of MTX (greater than 10 microM) were required to increase the release of Hcy from nonmalignant cells. The enhancement of Hcy export from the malignant cells in the presence of MTX was not associated with cellular build-up of S-adenosyl-L-homocysteine (AdoHcy), indicating that the amount of intracellular Hcy was kept below the level required for inhibition or reversion of the AdoHcy hydrolase reaction. MTX-dependent Hcy efflux probably reflects cellular deficiency of 5-methyl-THF required for the salvage of Hcy to methionine and may therefore be a measure of lack of this reduced folate relative to the metabolic demand.

Acute and long-term effects of high-dose methotrexate treatment on homocysteine in plasma and urine.

The amino acid, homocysteine, is not supplied by food but is a product formed by cleavage of S-adenosylhomocysteine; a product of transmethylation. Homocysteine is further salvaged to methionine. Since this reaction is in most tissues dependent on 5-methyltetrahydrofolate, we investigated the effect of the antifolate drug, methotrexate (MTX), on homocysteine in patients treated with this drug against cancer. Free and protein-bound homocysteine in plasma and urinary excretion of this amino acid were monitored in seven patients before, during, and after infusion with MTX (1-13.6 g). Each patient was investigated during one to five consecutive MTX treatments, which were separated by intervals of 1 to 4 weeks. Three components of the homocysteine response could be distinguished. An acute effect appeared after a lag period of about 6 h, lasted for about 24 h, and was characterized by a transient increase in free and protein-bound homocysteine and a concomitant increase in urinary excretion of homocysteine. Some patients showed a marked plasma response, whereas in others, enhancement of urinary excretion predominated. A long-term effect developed within 48-72 h after each infusion and was characterized by a progressive decrease in both plasma homocysteine and urinary excretion of homocysteine to amounts below those observed prior to the infusion. This effect lasted for at least 4 weeks. In this way the amount of homocysteine in plasma and urine decreased as a function of the number of MTX infusions. This long-term effect was associated with a decrease in acute homocysteine response in plasma and/or urine. Notably, MTX induced no acute or long-term effect on plasma methionine, suggesting that the homocysteine response is not caused by an imbalance in methionine metabolism due to malignant disease or chemotherapy. The cause and possible consequences of altered homocysteine metabolism during MTX therapy are discussed.

Plasma homocysteine in children with acute lymphoblastic leukemia: changes during a chemotherapeutic regimen including methotrexate.

Plasma homocysteine was determined in 12 children with acute lymphoblastic leukemia. The patients were investigated prior to chemotherapy (stage I), during seven weeks of induction chemotherapy (stage II), and thereafter during intermittent high-dose methotrexate (HD-MTX) therapy (stage III). The patients were followed for a period of three to 15 months, and the study included a total of 80 HD-MTX courses. Before start of chemotherapy (stage I), the average plasma homocysteine level in the children with leukemia was 13.18 +/- 6.23 (SD) mumol/liter, which is significantly (P less than 0.001) higher than the level in control children (6.52 +/- 1.21 mumol/liter). The plasma homocysteine level in the patients was positively correlated with the peripheral white blood cell count (P less than 0.01) and negatively correlated with serum folate (P less than 0.02). The serum folate was normal or subnormal in these patients. During induction therapy with cytotoxic drugs such as vincristine, asparaginase, and intrathecal MTX (stage II), there was a drastic change in plasma homocysteine as a function of time. A reciprocal alteration in serum folate was observed, suggesting fluctuating intracellular folate status at this stage of therapy. At the end of stage II (about seven weeks), there was a significant (P less than 0.01) reduction in total homocysteine (to 7.08 +/- 3.84 mumol/liter). HD-MTX (8 g/m2) therapy with 5-formyltetrahydrofolate "rescue" (stage III) was usually begun about seven weeks after start of chemotherapy, and the patients were followed for two to eight courses separated by three to eight weeks. Plasma homocysteine showed a transient increase (26-64%) following each MTX infusion. After three MTX infusions, basal total plasma homocysteine was reduced to 5.56 +/- 1.12 mumol/liter. During most MTX infusions, there was a variable reduction (17-56%) in plasma methionine followed by a rebound increase. It is concluded that plasma homocysteine in children with acute lymphoblastic leukemia is elevated prior to therapy, probably because of occasional folate deficiency and increased burden of proliferating cells. During induction therapy, monitoring plasma homocysteine and serum folate both suggest a labile folate homeostasis, usually a deficiency state. HD-MTX induced a temporary intracellular folate depletion before 5-formyl-tetrahydrofolate was administered, as judged by a transient homocysteinemia. The methionine depletion may interfere with the antileukemic effect of MTX.