Objective: To test the hypothesis that endogenous synthesis of taurine from methionine is impaired in people with coronary heart disease (CHD).
Design: Nested case–control.
Subjects: Indian Asian and white European males aged 35–60 y. Both racial group included patients with CHD and healthy controls. Samples from 20 subjects in each of the four groups were selected at random.
Interventions: Fasting blood samples were taken before and 6 h after consumption of methionine (100 mg/kg body weight)
Measurements: Plasma concentrations of taurine, cysteine, pyridoxal-5-phosphate and 4-pyridoxic acid.
Results: Fasting plasma taurine values were higher in Indian Asian cases than controls, but not significantly different between European cases and controls. Postload taurine values were higher in cases than controls in both racial groups (P=0.002). Fasting plasma cysteine was higher in cases than controls (P=0.002) and higher in Indian Asians than Europeans (0.007), but there were no significant differences between any of the groups in postload cysteine values, nor in plasma pyridoxal-5-phosphate or 4-pyridoxic acid.
Conclusions: Taurine production from methionine was not impaired in patients with CHD, but fasting plasma cysteine was higher in CHD cases than controls.
Epidemiological evidence has shown an inverse relationship between urinary taurine excretion and mortality from coronary heart disease (CHD) in different populations (Yamori et al, 1996, 2001). This may reflect a direct effect of taurine, since supplementation with pharmacological doses of taurine has been shown to improve a number of processes including endothelial-dependent arterial dilatation (Fennessy et al, 1998) and neutrophil activation and endothelial adhesion (McCarty, 1999), and to have a potential role in the control of hypertension (Fujita et al, 1987) and hypercholesterolaemia (Mizushima et al, 1996). On the other hand, it may be an artefact arising from a relationship between vitamin B6 status and CHD, since taurine can be produced endogenously by a series of reactions, several of which involve vitamin B6 (pyridoxal phosphate, PLP) as a cofactor (see Figure 1). Several reports have shown a strong association between decreased plasma PLP concentration and the incidence of CHD, and vitamin B6 has been proposed to be an independent risk factor for CHD (Robinson et al, 1995; Folsom et al, 1998).
Taurine is synthesised endogenously from cysteine, which in turn can be synthesised from methionine. The pathway from methionine to cysteine begins with the conversion of methionine to homocysteine (Hcy) (see Figure 1). Although much of the Hcy is normally remethylated to methionine, some is converted to cystathionine and then to cysteine in what is known as the transulphuration pathway. If this pathway did not function optimally, the result might be decreased production of cysteine and taurine accompanied by increased levels of Hcy. A high circulating Hcy concentration has consistently been identified as a risk factor for CHD, although again the mechanism is not clear (McCully, 1996; Hankey & Eikelboom, 1999).
It is thus a plausible hypothesis that production of taurine from methionine is impaired in people who are at high risk of CHD. One way of testing this hypothesis would be to measure circulating levels of taurine following the ingestion of a loading dose of methionine. The methionine load causes a considerable increase in the concentration of S-adenosylmethionine (Miller et al, 1993), and this tends to suppress the remethylation of Hcy by inhibiting methylenetetrahydrofolate reductase and inactivating betaine-homocysteine methyltransferase (Finkelstein, 1990). Thus, the stress of the methionine load should reveal any defect in the transulphuration pathway for disposal of Hcy to cysteine and taurine. The methionine loading protocol has been used in a recent case–control study of plasma Hcy levels in CHD in Indian Asian and European men living in the same area of the UK (Chambers et al, 2000). In the present study, we measured plasma taurine and PLP concentrations in a representative subgroup drawn from this case–control study, in order to begin to test the above hypothesis.
Plasma samples were selected from the case–control study, which is fully described by Chambers et al (2000). In brief, subjects were males aged between 35 and 60 y of age. Indian Asians had been resident in the UK for a mean of 27 y, and had all four grandparents of north Indian descent, while Europeans were white and had been born in the UK. CHD patients (cases) were identified from three west London hospitals and controls were selected by age from the registers of local general practitioners. Criteria for CHD were: myocardial infarction (chest pain associated with electrocardiographic (ECG) evidence of myocardial infarction or raised cardiac enzymes or both); unstable angina (cardiac pain associated with dynamic ECG abnormalities); angiographically proven coronary artery disease (>50% stenosis in one or more major epicardial vessel in multiple projections). Exclusion criteria for both patients and controls included cardiomyopathy, serious organ disease, systemic illness, chronic alcohol abuse, serious psychiatric illness, anticonvulsant therapy, and, for controls, the presence of pathological Q waves on the ECG. The study was approved by the local research ethics committee and all individuals gave written, informed consent.
Venous blood samples were collected after an overnight fast, and subjects were given an oral dose of methionine (100 mg/kg body weight) in orange squash. A second venous blood sample was taken after 6 h, during which time the subjects were allowed to consume water but no food. Blood samples were placed on ice and centrifuged within 1 h and plasma samples were stored at −70°C till analysis. Total plasma Hcy and cysteine were measured in all samples by high-performance liquid chromatography (HPLC) (Fiskerstrand et al, 1993).
For the present study, 20 CHD cases and 20 controls from each of the two racial groups were selected at random. For each subject, both plasma samples were analysed for free amino acid concentrations by HPLC (Mann et al, 1988). The taurine peak was identified and quantified by reference to an internal standard (homoserine). Additional fasting blood samples which had been taken at the same time were analysed for plasma PLP and its major metabolite, 4-pyridoxic acid, by HPLC (Bates et al, 1999).
The data were analysed by two-way analysis of variance using Minitab (version 12.21, Minitab Inc., State College, PA, USA). Probabilities of less than 0.05 were considered significant.
The data for plasma taurine, cysteine, PLP and 4-pyridoxic acid concentrations are summarised in Table 1. Fasting plasma taurine concentration appeared to be greater in the CHD cases than the controls, but the significant interaction term indicates that this was mainly due to a large case–control difference in the Indian Asian subjects, with no significant effect in the European subjects. Following the methionine load, there was a clear difference between cases and controls in both racial groups, with no significant interaction. The same result was obtained when the postload taurine values were reanalysed using preload taurine as a covariate (data not shown). Thus, it is clear that the postload taurine concentration was higher in CHD cases than controls, regardless of the preload value.
Both race and disease status had independent effects on fasting plasma cysteine concentrations, with CHD cases having higher values than controls and Indian Asian subjects having higher values than European subjects. However, after the methionine load there were no significant differences between any of the groups. Again this was confirmed when the postload cysteine values were reanalysed using preload cysteine as a covariate (data not shown), with no significant effects of either race or disease status being found.
There were no significant differences between any of the groups in either plasma PLP or plasma 4-pyridoxic acid concentrations.
This study was designed to test the hypothesis that endogenous synthesis of taurine from methionine is impaired in people with CHD. The results show clearly that this is not the case, since postload taurine values were actually higher in CHD cases than controls. The fasting plasma taurine values were also not lower in CHD cases than controls, suggesting that basal taurine production was also unimpaired, and in fact it was enhanced in the Indian Asian cases. One possible explanation for the higher plasma taurine concentrations is that renal clearance of taurine may have been impaired in subjects with CHD. However, adjusting the data for plasma creatinine concentration made no difference to the results (data not shown). Taurine appears to have many possible physiological roles, and has been hypothesised to be protective against hypertension and CHD (Yamori et al, 1996). The present data do not appear to support this hypothesis.
Fasting plasma cysteine concentrations were higher in CHD cases than controls, in line with the findings of El-Khairy et al (2001), and in Indian Asian subjects than Europeans. Fasting plasma cysteine levels have previously been shown to correlate with fasting Hcy (Brattstrom et al, 1994), and in the present study there was also a significant correlation between these two variables (r=0.304, P=0.007). Hence, the present findings are in line with the data from the large case–control study from which these samples were selected (Chambers et al, 2000), which showed greater fasting Hcy concentrations in cases than controls and in Indian Asians than in Europeans. There was also a significant correlation between postload cysteine and Hcy concentrations (r=0.246, P=0.031). Again the present finding of no significant difference in postload cysteine concentrations is in line with the finding from the larger study of no significant case–control difference in postload Hcy concentration (Chambers et al, 2000). The mechanism by which Hcy affects risk of CHD, which may involve effects on lipoprotein metabolism, atherosclerosis, endothelial function and thrombogenesis, is not yet clear (McCully, 1996). However, cysteine has very similar chemical properties to Hcy and is present in the plasma at 20 times greater concentrations, so it may also play a role in the pathogenesis of CHD.
Perhaps the most striking feature of these results is that plasma levels of cysteine and taurine did not rise 6 h after the ingestion of 100 mg/kg methionine, a dose that corresponds to almost five times as much methionine as an average person consumes in a whole day. In fact, for the group as whole the plasma concentrations of both taurine and cysteine decreased significantly between 0 and 6 h (P<0.001, paired t-test). A fall in plasma cysteine concentration after a methionine load has been reported previously (Mansoor et al, 1992; Ubbink et al, 1996; Suliman et al, 2001). A period of 6 h was chosen as the time for postmethionine load sampling because plasma Hcy values are normally maximal at this time. Plasma methionine was elevated by more than 10-fold at this time (570 vs 44 μM), so it is unlikely that the measurements were made too early or too late to detect a significant rise in plasma taurine and cysteine. Moreover, we have recently found that plasma levels of cysteine and taurine were not significantly elevated 1 or 3 h after a similar methionine load in healthy volunteers, while plasma methionine had peaked at around 3 h (A Shakouri, O Obeid and P Emery, unpublished data). Mansoor et al (1992) also noted that plasma cysteine concentrations were lower 2 and 4 h after the methionine load.
It is possible that any cysteine and taurine produced from the methionine load was retained in the cells or rapidly cleared into other tissues. Suliman et al (2001) observed an increase in the concentration of taurine, but not cysteine, in erythrocytes following a similar methionine load. Unfortunately, we do not have any data on intracellular amino acid concentrations.
Another possibility is that cysteine and taurine were rapidly cleared into the urine. However, our recent observations on healthy volunteers given the same methionine load showed no significant rise in urinary cysteine excretion and only a small rise in taurine excretion, amounting to no more than 1% of the methionine load, up to 24 h after the methionine load (A Shakouri, K Tsang, O Obeid and P Emery, unpublished data). Previous studies with a slightly smaller methionine load showed no significant rise in taurine excretion (Park & Linkswiler, 1970).
Thus, the most likely explanation for these data is that the methionine load actually had very little effect on the production of cysteine and taurine. The transulphuration pathway has the characteristics of a biosynthetic rather than a catabolic pathway, with the rate-limiting enzyme cystathionine-β-synthase being inhibited by the product of the pathway, cysteine (Marks et al, 1996). Hence flux through this pathway responds to changes in the requirement for cysteine rather than the availability of methionine (Bender, 1985). For people on a normal protein intake, the exogenous supply of cysteine would be more than adequate, so the transulphuration pathway would not be induced.
The results of this study do not support the hypothesis that endogenous synthesis of taurine from methionine is impaired in people with CHD.
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We are grateful to Dr John Chambers and Dr Jaspal Kooner (Imperial College School of Medicine) for access to the blood samples and for constructive criticism of the manuscript. We thank Mr Peter Milligan (King's College London) for expert statistical advice.
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