Effects of an exercise and hypocaloric healthy eating program on biomarkers associated with long-term prognosis after early-stage breast cancer: a randomized controlled trial.

Excess body weight at diagnosis and weight gain after breast cancer are associated with poorer long-term prognosis. This study investigated the effects of a lifestyle intervention on body weight and other health outcomes influencing long-term prognosis in overweight women (BMI > 25.0 kg/m(2)) recovering from early-stage (stage I-III) breast cancer. A total of 90 women treated 3-18 months previously were randomly allocated to a 6-month exercise and hypocaloric healthy eating program (n = 47, aged 55.6 ± 10.2 year) or control group (n = 43, aged 55.9 ± 8.9 year). Women in the intervention group received three supervised exercise sessions per week and individualized dietary advice, supplemented by weekly nutrition seminars. Body weight, waist circumference, waist/hip ratio [WHR], cardiorespiratory fitness, blood biomarkers associated with breast cancer recurrence and cardiovascular disease risk, and quality of life (FACT-B) were assessed at baseline and 6 months. Three-day diet diaries were used to assess macronutrient and energy intakes. A moderate reduction in body weight in the intervention group (median difference from baseline of -1.09 kg; IQR -0.15 to -2.90 kg; p = 0.07) was accompanied by significant reductions in waist circumference (p < 0.001), WHR (p = 0.005), total (p = 0.021) and saturated fat (p = 0.006) intakes, leptin (p = 0.005), total cholesterol (p = 0.046), and resting diastolic blood pressure (p = 0.03). Cardiopulmonary fitness (p < 0.001) and FACT-B quality of life (p = 0.004) also showed significant improvements in the intervention group. These findings suggest that an individualized exercise and a hypocaloric healthy eating program can positively impact upon health outcomes influencing long-term prognosis in overweight women recovering from early-stage breast cancer.

Sensitivity of markers of DNA stability and DNA repair activity to folate supplementation in healthy volunteers.

We have previously reported that supplementation with folic acid (1.2 mg day(-1) for 12 week) elicited a significant improvement in the folate status of 61 healthy volunteers. We have examined effects of this supplement on markers of genomic stability. Little is known about the effect of folate supplementation on DNA stability in a cohort, which is not folate deficient. Preintervention, there was a significant inverse association between uracil misincorporation in lymphocyte DNA and red cell folate (P < 0.05). In contrast, there were no associations between folate status and DNA strand breakage, global DNA methylation or DNA base excision repair (measured as the capacity of the lymphocyte extract to repair 8-oxoGua ex vivo). Folate supplementation elicited a significant reduction in uracil misincorporation (P < 0.05), while DNA strand breakage and global DNA methylation remained unchanged. Increasing folate status significantly decreased the base excision repair capacity in those volunteers with the lowest preintervention folate status (P < 0.05). Uracil misincorporation was more sensitive to changes in folate status than other measures of DNA stability and therefore could be considered a specific and functional marker of folate status, which may also be relevant to cancer risk in healthy people.

Research goals for folate and related B vitamin in Europe.

In the past decade, the understanding of folate bioavailability, metabolism and related health issues has increased, but several problems remain, including the difficulty of delivering the available knowledge to the populations at risk. Owing to the low compliance of taking folic acid supplements, for example, among women of child-bearing age who could lower the risk of having a baby with a neural tube defect, food-based strategies aimed at increasing the intake of folate and other B-group vitamins should be a priority for future research. These should include the development of a combined strategy of supplemental folate (possibly with vitamin B(12)), biofortification using engineered plant-derived foods and micro-organisms and food fortification for increasing folate intakes in the general population. Currently, the most effective population-based strategy to reduce NTDs remains folic acid fortification. However, the possible adverse effect of high intakes of folic acid on neurologic functioning among elderly persons with vitamin B(12) deficiency needs urgent investigation. The results of ongoing randomized controlled studies aimed at reducing the prevalence of hyperhomocysteinemia and related morbidity must be available before food-based total population approaches for treatment of hyperhomocysteinemia can be recommended. Further research is required on quantitative assessment of folate intake and bioavailability, along with a more thorough understanding of physiological, biochemical and genetic processes involved in folate absorption and metabolism.

Antioxidant supplementation enhances antioxidant capacity and mitigates oxidative damage following acute ischaemic stroke.

OBJECTIVE: To test whether supplementary antioxidants immediately following acute ischaemic stroke will enhance antioxidant capacity and mitigate oxidative damage. DESIGN: A randomised controlled trial. SETTING: A university teaching hospital. SUBJECTS: A total of 48 acute ischaemic stroke patients within 12 h of symptom onset. INTERVENTION: Daily oral 800 IU (727 mg) of alpha-tocopherol and 500 mg of vitamin C (n = 24), or no treatment (n = 24) for 14 days. Treatment group and controls were matched for stroke subtype and age. MAIN OUTCOME MEASURES: alpha-Tocopherol, ascorbic acid, total antioxidant capacity (TAOC), plasma malondialdehyde (MDA) and C-reactive protein (CRP) before treatment, at day 7 and day 14 following recruitment. RESULTS: In all, 14 days of vitamin supplementation significantly improved plasma alpha-tocopherol and ascorbic concentrations in the treatment group compared with the decrease seen in the control group (P < 0.005 for difference in cumulative changes). TAOC increased significantly in the treatment group compared with controls (P < 0.003). There was a significant reduction in plasma MDA concentration in the treatment group, in contrast to the increase seen in the control group (P < 0.002). After adjusting for clinical complications CRP concentrations within 90 days postinfarct were significantly lower in the treatment group compared with controls. CONCLUSION: Supplementation with antioxidant vitamins within 12 h of onset of acute ischaemic stroke increased antioxidant capacity, reduced lipid peroxidation products and may have an anti-inflammatory effect. SPONSORSHIP: Sheffield Teaching Hospital NHS Trust.

Downstream effects on human low density lipoprotein of homocysteine exported from endothelial cells in an in vitro system.

A model system is presented using human umbilical vein endothelial cells (HUVECs) to investigate the role of homocysteine (Hcy) in atherosclerosis. HUVECs are shown to export Hcy at a rate determined by the flux through the methionine/Hcy pathway. Additional methionine increases intracellular methionine, decreases intracellular folate, and increases Hcy export, whereas additional folate inhibits export. An inverse relationship exists between intracellular folate and Hcy export. Hcy export may be regulated by intracellular S-adenosyl methionine rather than by Hcy. Human LDLs exposed to HUVECs exporting Hcy undergo time-related lipid oxidation, a process inhibited by the thiol trap dithionitrobenzoate. This is likely to be related to the generation of hydroxyl radicals, which we show are associated with Hcy export. Although Hcy is the major oxidant, cysteine also contributes, as shown by the effect of glutamate. Finally, the LDL oxidized in this system showed a time-dependent increase in uptake by human macrophages, implying an upregulation of the scavenger receptor. These results suggest that continuous export of Hcy from endothelial cells contributes to the generation of extracellular hydroxyl radicals, with associated oxidative modification of LDL and incorporation into macrophages, a key step in atherosclerosis. Factors that regulate intracellular Hcy metabolism modulate these effects.

Absence of luminal riboflavin disturbs early postnatal development of the gastrointestinal tract.

An increase in the size and cellularity of duodenal crypts and a decreased incidence of bifurcating crypts is observed in response to very short-term feeding of a riboflavin-deficient diet to weanling rats. A study was conducted to determine whether the absence of riboflavin in the lumen of the small intestine impairs gastrointestinal development. Forty-eight female weanling Wistar rats were allocated to one of two treatment regimens, to receive either a riboflavin-deficient diet and a daily intraperitoneal injection of flavin mononucleotide (luminally deficient group) or a complete diet and a daily intraperitoneal injection of saline (control group). Animals were killed at 93, 141, or 165 hr from feeding. The flavin injection regimen maintained normal systemic riboflavin status in the luminally deficient group. In this group, however, crypt hypertrophy and reduced crypt bifurcation were evident by 141 hr of luminal riboflavin deprivation. The absence of riboflavin in the duodenal lumen impairs normal development, suggesting that a crypt sensing mechanism may be involved in the response to riboflavin deficiency.

Reduction in plasma total homocysteine through increasing folate intake in healthy individuals is not associated with changes in measures of antioxidant activity or oxidant damage.

BACKGROUND: Various mechanisms have been proposed to explain the association between plasma total homocysteine (tHcy) and risk of cardiovascular disease, including oxidative activity of homocysteine. OBJECTIVE: To explore the putative role of reactive oxygen species in the association between plasma tHcy and risk of cardiovascular disease in healthy individuals. DESIGN: A double-blind, placebo-controlled crossover intervention to increase folate intake through diet (increased consumption of folate-rich foods) and supplement (400 micro g folic acid) was carried out in 126 healthy men and women. Measurements were made of antioxidant activity in red blood cells and plasma, and products of oxidant damage in plasma. RESULTS: Diet and supplement-based interventions led to an increase in measures of folate status and a reduction in plasma tHcy. This was not associated with any significant change in measures of antioxidant activity (plasma and red blood cell glutathione peroxidase activity and red blood cell superoxide dismutase activity) or oxidant damage (plasma malondialdehyde), although an improvement in plasma total antioxidant capacity just failed to reach significance. CONCLUSIONS: In healthy individuals lowering plasma tHcy does not have any functional implications regarding oxidative damage.

Vitamin C therapy ameliorates vascular endothelial dysfunction in treated patients with homocystinuria.

OBJECTIVES: We sought to investigate the effects of short- and long-term vitamin C therapy on endothelial dysfunction in patients with homocystinuria. BACKGROUND: Untreated homocystinuria due to cystathionine beta-synthase deficiency is associated with premature atherothrombotic disease; 25% of untreated patients suffer a vascular event by the age of 16 years and 50% by 29 years. Treatment directed at reducing homocysteine accumulation significantly reduces this risk. However, despite 'optimal' treatment and compliance, hyperhomocysteinaemia usually persists and individuals exhibit endothelial dysfunction indicative of an adverse cardiovascular prognosis. Additional intervention is therefore required to further reduce cardiovascular risk. METHODS: We investigated the endothelial effects of acute (2 g single dose) and chronic (1 g/day for 6 months) administration of oral vitamin C in 5 patients with homocystinuria (mean age 26 years, 1 male) and 5 age- and sex-matched controls. Brachial artery endothelium-dependent flow-mediated dilatation (FMD) and endothelium-independent responses to nitroglycerin (NTG) were measured using high-resolution ultrasonic vessel wall-tracking. RESULTS: Baseline: Plasma total homocysteine was 100.8 +/- 61.6 and 9.2 +/- 1.9 micromol/L in the patient and control groups, respectively (p < 0.001). FMD responses were impaired in the patient group (20 +/- 40 microm) compared with the controls (116 +/- 30 microm) (p < 0.001). Vitamin C administration: FMD responses in the patient group improved both acutely, 160 +/- 65 microm at 4 h (p < 0.001), and chronically, 170 +/- 70 microm at 2 weeks (p < 0.001) and 170 +/- 40 microm at 6 months (p < 0.001). FMD responses in the control group were unaltered (p = 0.526). Within both groups, neither the vascular response to NTG nor plasma homocysteine was altered (p > 0.4). CONCLUSIONS: Vitamin C ameliorates endothelial dysfunction in patients with homocystinuria, independent of changes in homocysteine concentration and should therefore be considered as an additional adjunct to therapy to reduce the potential long-term risk of atherothrombotic disease.

Folate protects against oxidative modification of human LDL.

Elevated plasma total homocysteine is considered to be a graded risk factor for cardiovascular disease. Folate, through its homocysteine-lowering potential, may therefore be protective. Folate, however, may have protective effects independent of homocysteine-lowering. We have measured the effects of folate on Cu-catalysed oxidative damage to the unsaturated lipids in human LDL. Experiments were carried out in the presence of citrate, and followed increases in absorption at 234 nm, which measures the amount of conjugated diene produced. There is a lag time during which endogenous antioxidants are oxidised, followed by rapid oxidation of lipid. Addition of 0-6 microm - 5-methyltetrahydrofolate produced a dose-dependent increase in the lag time, suggesting that folate may have a direct anti-oxidant role in vivo, which is independent of any indirect effects through lowering of homocysteine levels.

Riboflavin deficiency: early effects on post-weaning development of the duodenum in rats.

The aim of this present study was to identify the earliest point at which riboflavin deficiency affects post-weaning bowel development in rats. After weaning, eighty Wistar rats were weight-matched as pairs, one animal being fed a normal synthetic diet and the other being fed the same diet but deficient in riboflavin. Body weight, feeding and rates of growth were monitored and eight pairs of animals were taken for analysis at 45, 69, 93, 117 and 141 h. Riboflavin status was monitored by determining the erythrocyte glutathione reductase activation coefficient (EGRAC), and hepatic flavins were measured by a fluorescence assay. Changes to the number and dimensions of villi and crypts in the duodenum were determined, as well as crypt division (bifurcation) and the DNA synthesis index of the crypt epithelium by bromodeoxyuridine (BrdU) labelling. Riboflavin deficiency was established in the experimental rats, as demonstrated by a significant increase in EGRAC after 45 h (P<0.001) and decreased liver flavins after 96 h (P<0.001). After 96 h a significant increase in the size and cellularity of the crypts (P<0.001 in both cases) was seen in these riboflavin-deficient animals, with a decreased incidence of bifurcating crypts and of BrdU-labelled cells. No changes to villus number or size were observed. The present study has demonstrated that developmental changes to the duodenal crypt arise shortly after circulating riboflavin measurements show evidence of deficiency. These changes primarily affect cell proliferation and crypt bifurcation, and precede long-term changes such as the reduction of villus number.

Optimization of dietary folate or low-dose folic acid supplements lower homocysteine but do not enhance endothelial function in healthy adults, irrespective of the methylenetetrahydrofolate reductase (C677T) genotype.

OBJECTIVES: We sought to study the effect of low-dose folic acid supplementation or optimization of dietary folate intake on plasma homocysteine and endothelial function in healthy adults. BACKGROUND: Elevated homocysteine is associated with cardiovascular disease, but it is not known whether this relationship is causal. Individuals homozygous (TT) for the C677T mutation in the methylenetetrahydrofolate reductase (MTHFR) gene ( approximately 12% of the population) have increased homocysteine levels, particularly in association with suboptimal folate intake. METHODS: Healthy subjects (n = 126; 42 of each MTHFR genotype) were included in this cross-over study of three interventions of four months each: 1) placebo plus natural diet; 2) daily 400-microg folic acid supplement plus natural diet; and 3) increased dietary folate intake to 400 microg/day. RESULTS: At baseline, homocysteine was inversely related to plasma folate and was higher in TT homozygotes. For the whole group, plasma folate increased by 46% after dietary folate and by 79% after supplementation, with reductions of homocysteine of 14% and 16%, respectively. Within the genotype, TT homozygotes exhibited the most marked changes in these variables. Brachial artery endothelial function, as determined by a change in end-diastolic diameter in response to increased flow, was not changed by increased folate intake (98 +/- 73 microm at baseline, 110 +/- 69 microm after a high-folate diet, 114 +/- 59 microm after supplementation and 118 +/- 68 microm after placebo). Plasma von Willebrand factor antigen was unaltered. CONCLUSIONS: Optimization of dietary folate or low-dose folic acid supplementation reduces plasma homocysteine but does not enhance endothelial function, irrespective of the MTHFR (C667T) genotype.

Antioxidant vitamin status and carotid atherosclerosis in the elderly.

BACKGROUND: The oxidative modification of LDL is thought to play a crucial role in the initiation of atherosclerosis. Antioxidant vitamins can protect LDL from oxidation, and high intakes or blood concentrations of these vitamins have been linked with a reduced risk of cardiovascular disease. Few data are available on the importance of antioxidant vitamins in earlier stages of atherogenesis. OBJECTIVE: We investigated the cross-sectional relation between antioxidant vitamin status and carotid atherosclerosis in a group of elderly persons. DESIGN: The study sample comprised 468 men and women aged 66-75 y living in Sheffield, United Kingdom. Duplex ultrasonography was used to measure intima-media thickness and the degree of stenosis in the extracranial carotid arteries. Antioxidant vitamin status was assessed by measuring fasting plasma concentrations of vitamin C, vitamin E, and beta-carotene. RESULTS: In the men, after adjustment for age and cardiovascular disease risk factors, a 20% higher plasma vitamin C concentration was associated with a 0.004-mm smaller intima-media thickness; a 20% higher beta-carotene concentration was associated with a 0.005-mm smaller intima-media thickness. Compared with men with high blood concentrations of beta-carotene or cholesterol-adjusted vitamin E, those with low blood concentrations of these vitamins were 2.5 times as likely to have carotid stenosis of >30%. We found no significant trends between plasma concentrations of antioxidant vitamins and either measure of carotid atherosclerosis in the women. CONCLUSION: A high antioxidant vitamin status may help to prevent the initiation and progression of early atherosclerotic lesions in men.

Role of aminothiols as a component of the plasma antioxidant system and relevance to homocysteine-mediated vascular disease.

Hyperhomocysteinaemia is considered to be an independent risk factor for vascular disease. Elevated plasma homocysteine may pose an oxidative stress, leading to the development of vascular damage. A component of this effect may be a disturbance of the extracellular aminothiol redox state. The relative contributions of plasma total homocysteine (tHcy) and plasma total cysteine (tCys) to the total antioxidant capacity (TAOC) of plasma was established in subjects with normal and elevated plasma tHcy. A total of 10 subjects with severe hyperhomocysteinaemia (due to inherited metabolic defects), 13 of their heterozygous parents and 72 normal healthy subjects were recruited to the study. The mean plasma tHcy in the patients was 91.8 micromol/l, compared with 13.2 micromol/l in the parents and 14.7 micromol/l in healthy control subjects. Plasma tCys and plasma TAOC were significantly lower in the subjects with severe hyperhomocysteinaemia compared with the parents and healthy control subjects (P<0.05). In blood samples from subjects with a normal tHcy, a positive correlation was observed between tCys and tHcy (P=0.0001). In contrast, in blood samples with tHcy >or=20 micromol/l, plasma tCys was negatively correlated with tHcy (P=0.0001). In samples with tHcy >or=20 micromol/l, tHcy was inversely correlated with TAOC (P=0.0001), whereas tCys was positively associated with TAOC (P=0.0001). Multiple regression analysis revealed that tCys was the most important independent determinant of TAOC in the patient and control groups when the effects of tHcy and several factors known to influence TAOC, such as urate, were taken into account. Thus hyperhomocysteinaemia may pose an oxidative stress not only through the direct cytotoxicity of homocysteine, but also from an associated fall in plasma cysteine.

Elevated plasma homocysteine elicits an increase in antioxidant enzyme activity.

Elevated plasma homocysteine is considered to be a risk factor for cardiovascular disease. The mechanisms for this effect are not fully understood but there is some evidence for a role for reactive oxygen species (ROS). This study was conducted to explore the effects of elevated plasma total homocysteine (tHcy) concentration on activity of antioxidant enzymes in the circulation. The study group consisted of 10 patients with inherited defects of homocysteine metabolism, from whom 41 blood samples were collected over a period of six months. Blood samples were also collected from 13 of their obligate heterozygous parents. For data analysis samples were classified as those with plasma tHcy < 20 microM or > 20 microM. The activity of erythrocyte superoxide dismutase (SOD) and plasma glutathione peroxidase (GSHPx) was elevated in samples with plasma tHcy > 20 microM. Moreover, a significant correlation was demonstrated between plasma GSHPx activity, plasma glutathione peroxidase protein and plasma tHcy. III vitro studies confirmed that this observation was not due to a simple chemical enhancement of enzyme activity. Homocysteine protected GSHPx from loss of activity following incubation at 37 degrees C. A similar effect was seen with another thiol-containing amino acid, cysteine. Results suggest that elevated plasma tHcy represents an oxidative stress, resulting in an adaptive increase in activity of antioxidant enzymes in the circulation.

Homocysteine-induced inhibition of endothelium-dependent relaxation in rabbit aorta: role for superoxide anions.

Hyperhomocysteinemia is associated with endothelial dysfunction, although its mechanism is unknown. Isometric tension recordings and lucigenin chemiluminescence were used to assess the effects of homocysteine exposure on endothelium-dependent and -independent relaxation in isolated rabbit aortic rings and superoxide anion (O(2)(-)) production by cultured porcine aortic endothelial cells, respectively. Homocysteine (0.1 to 10 mmol/L) produced a significant (P<0.001) concentration- and time-dependent inhibition of endothelium-dependent relaxation in response to both acetylcholine and the calcium ionophore A23187. Only the intracellular O(2)(-) scavenger 4,5-dihydroxy-1,3-benzene disulfonic acid (Tiron, 10 mmol/L) significantly (P<0.001) inhibited the effect of homocysteine on acetylcholine- and A23187-induced relaxation. Incubation of porcine aortic endothelial cells with homocysteine (0.03 to 1 mmol/L for up to 72 hours) caused a significant (P<0.001) time-dependent increase in the O(2)(-) released by these cells on the addition of Triton X-100 (1% [vol/vol]), with levels returning to values comparable to those of control cells at the 72-hour time point. These changes in O(2)(-) levels were associated with a time-dependent increase in endothelial cell superoxide dismutase activity, becoming significant (P<0.001) after 72 hours. Furthermore, the homocysteine-induced increase in endothelial cell O(2)(-) levels was completely inhibited (P<0.001) by the concomitant incubation with either Tiron (10 mmol/L), vitamin C (10 micromol/L), or vitamin E (10 micromol/L). These data suggest that the inhibitory effect of homocysteine on endothelium-dependent relaxation is due to an increase in the endothelial cell intracellular levels of O(2)(-) and provide a possible mechanism for the endothelial dysfunction associated with hyperhomocysteinemia.

Developments in the measurement of plasma total homocysteine.

Elevated plasma homocysteine is considered to be a graded risk factor for cardiovascular disease, and for this reason there is great interest in high-performance analytical techniques. Methods have evolved from ion-exchange chromatography to embrace high-performance liquid chromatography with fluorescence or electrochemical detection, immunoassays, gas chromatography-mass spectroscopy and liquid chromatography with tandem mass spectrometry. Immunoassays and high-performance liquid chromatography methods are currently available in kit form, fluorescence polarization immunoassay showing the best performance.

Nutritional status of rural elderly Malays: dietary and biochemical findings.

A cross-sectional nutritional survey was carried out on 350 elderly Malays aged 60 and above from 11 randomly selected villages in a rural area on the East Coast of Malaysia. The findings indicated that the mean intakes of energy and all of the nutrients investigated were below the Malaysian Recommended Dietary Allowances, excepts for protein and vitamin C. Nutrients most likely to be inadequate were vitamin A, thiamine, riboflavin, niacin and calcium, with more than 50% of the subjects having estimated intakes of below 2/3 of the recommendations. However, vitamin A status was adequate, with only 2 subjects being biochemically deficient (plasma retinol < or = 0.7 mmol/l). Approximately a third of the subjects had hypoalbuminaemia (plasma albumin < 3.3 g/dl) and anaemia (Haemoglobin < 12 g/dl for men; < 13 g/dl for women). Riboflavin deficiency, as assessed by an erythrocyte glutathione reductase activation coefficient (EGRAC) of more than 1.35 was identified in 77% of the subjects. The prevalence of vitamin E deficiency (plasma a-tocopherol < or = 12 mmol/l) was 27%, with men being at a greater risk. In conclusion, the dietary intakes of these rural elderly Malays was inadequate. Over three quarters of the sample were biochemically deficient in riboflavin, the functional consequences of which need to be further investigated.

Current knowledge concerning optimum nutritional status of riboflavin, niacin and pyridoxine.

The term 'optimum nutrition' has evolved from a perceived need to base recommendations for nutrient intakes firmly in the context of function. It follows that 'optimum nutritional status' for individual nutrients should be defined in terms of biochemical or physiological markers having some functional value but also showing an appropriate relationship to nutrient intake. The present short review considers the current position regarding such markers for riboflavin, pyridoxine and niacin. It is concluded that whilst there are several biochemical measures which respond to changes in intake of each of these vitamins, no single measure is wholly satisfactory as a marker of optimum status.

Recommended approaches for the laboratory measurement of homocysteine in the diagnosis and monitoring of patients with hyperhomocysteinaemia.

Several recent studies have indicated that an increased concentration of plasma homocysteine is an independent risk factor for the premature development of vascular disease. These important findings emphasize the need for careful selection of an appropriate analytical approach to diagnose and treat individuals who may be at risk. We compared the results obtained from the measurement of plasma total homocysteine (free + protein-bound fractions) by high-performance liquid chromatography (HPLC) with the measurement of plasma free homocystine (free fraction) by conventional ion-exchange chromatography in 10 patients with inherited defects of homocysteine metabolism and 13 obligate heterozygote individuals. This study can be used to formulate recommendations on the appropriate use of these assays in different clinical circumstances. Our results show that the concentration of total plasma homocysteine must exceed 60 mumol/L before plasma free homocystine becomes detectable by conventional ion-exchange chromatography. Similarly, assessment of the urinary excretion of homocysteine in these patients indicates that it may not become consistently detectable by conventional ion-exchange chromatography or HPLC until plasma total homocysteine exceeds 150 mumol/L. On this basis, while most patients with classical homocystinuria would be detected by analysis of plasma using conventional ion-exchange chromatography or by measurement of of the urinary homocysteine excretion, occasional patients would be missed. When monitoring patients receiving treatment for classical homocystinuria, in whom metabolic control is good, and when investigating individuals with a suspected inherited defect of cobalamin or folate metabolism, a method which measures plasma total homocysteine should be used. The identification of moderate hyperhomocysteinaemia of undefined cause investigated in relation to a history of early vacsular disease can only be identified by this approach.