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Original Research Communications |
1 From the Division for Medical Statistics, Department of Public Health and Primary Health Care and the Department of Pharmacology, University of Bergen, Norway.
2 Supported by the Norwegian Research Council.
3 Address reprint requests to L El-Khairy, Division for Medical Statistics, Department of Public Health and Primary Health Care, University of Bergen, Armauer Hansens Hus, N-5021 Bergen, Norway. E-mail: Lina.El-Khairy{at}smis.uib.no.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Objective: Our objective was to search for the determinants of plasma total cysteine (tCys) and compare them with those of tHcy.
Design: In this cross-sectional study, we studied 7591 healthy men and 8585 healthy women aged 40–67 y with no history of hypertension, diabetes mellitus, coronary heart disease, or cerebrovascular disease.
Results: In the group aged 40–42 y, tCys was significantly higher in men (
: 273 µmol/L; 2.5–97.5 percentile: 219–338 µmol/L) than in women (253 µmol/L; 202–317 µmol/L) (P < 0.001). In the group aged 65–67 y, there was no significant sex difference in tCys: men (296 µmol/L; 233–362 µmol/L) and women (296 µmol/L; 234–361 µmol/L). As with tHcy, tCys was positively associated with age, total cholesterol concentration, diastolic blood pressure, and coffee consumption. Body mass index was a strong determinant of tCys but was not related to tHcy. Several factors known to influence tHcy, including smoking status, folate and vitamin intake, heart rate, and physical activity, were not associated or were only weakly associated with tCys.
Conclusion: Plasma tCys is strongly related to several factors that constitute the cardiovascular disease risk profile. This should be an incentive to determine the role of tCys in cardiovascular disease.
Key Words: Plasma total cysteine • homocysteine • body mass index • total cholesterol concentration • coffee consumption • blood pressure • humans • cardiovascular disease risk factors • lifestyle • Hordaland Homocysteine Study • Norway
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Homocysteine is formed from the essential amino acid methionine as a product of numerous S-adenosylmethionine–dependent transmethylation reactions (6). Homocysteine may be remethylated to methionine. This reaction is in most tissues catalyzed by the ubiquitous enzyme 5-methyltetrahydrofalate–homocysteine S-methyltransferase (methionine synthase), which requires vitamin B-12 as a cofactor and methyltetrahydrofolate as a cosubstrate (6). Alternatively, homocysteine is degraded to cysteine via the transsulfuration pathway in 2 sequential vitamin B-6–dependent reactions (6). Inhibition of homocysteine metabolism as a result of enzymatic defects or vitamin deficiencies causes homocysteine export into extracellular compartments and thereby causes elevated plasma tHcy (7).
Despite the strong evidence that tHcy is an independent risk factor for cardiovascular disease (5), the question of causality and the mechanism or mechanisms by which tHcy exerts its pathogenicity are still not clear. Experimental evidence suggests that the mechanism may be related to the reactivity or redox properties of the sulfhydryl group (8, 9). If this is the case, other thiols might also be expected to confer increased cardiovascular disease risk. Total cysteine (tCys), like homocysteine, is an aminothiol, and its concentration in plasma is 
20-fold higher than tHcy (250 µmol/L) (10). However, there are only 2 reports showing higher tCys concentrations in vascular patients than in healthy control subjects (11, 12). In vascular disease (11) and in conditions characterized by transient (13, 14) or long-term (15, 16) elevation of tHcy, hyperhomocysteinemia was associated with complex changes in tCys and overall aminothiol redox status. Thus, hyperhomocysteinemia should not be considered an isolated event, but rather as a component of an interactive redox thiol system (10), the impairment of which may cause vascular lesions.
In this study we investigated the possible relations between tCys and several demographic and lifestyle factors associated with increased cardiovascular disease risk. We used baseline tCys data from the Hordaland Homocysteine Study of 16176 healthy women and men (17). In this population, we showed that tHcy increases with age, is higher in men than in women, and is associated with risk factors such as smoking, high blood pressure, high blood cholesterol concentrations, and low physical activity. In addition, plasma tHcy is increased by high coffee consumption and is reduced by intake of folate and vitamins (18). A similar investigation of tCys may shed light on the possible role of tCys and tHcy as mediators of cardiovascular disease risk.
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SUBJECTS AND METHODS |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To allow for any effect on tCys of disease, treatment, or change in lifestyle, 1866 participants who reported a previous diagnosis of coronary heart disease, cerebrovascular disease, hypertension, or diabetes mellitus were excluded from the analyses. One patient with homocystinuria was also excluded. Thus, 16176 subjects were included in this study. Because 96.2% of the participants belonged to the youngest and oldest age groups, most of the analyses were confined to these 2 groups. The study protocol was approved by the Regional Ethical Committee of western Norway, whose directives are based on the Helsinki Declaration.
Data collection
Data were collected via questionnaires, examinations, and blood tests. The questionnaires provided information about age; smoking habits; physical activity; type of work; personal history of cardiovascular diseases, hypertension, and diabetes; family history of disease; recent food intake; lifestyle; medical history; dietary habits; use of alcohol; and frequency of intake of various food items and vitamin supplements. Details on the collection and categorization of data were published previously (17).
Physical activity was categorized as 1) sedentary or no activity, 2) moderate activity (walking, cycling, or other type of moderate activity for 
4 h/wk), 3) active exercise (exercise, gardening with physical exertion, or a similar degree of activity for 4 h/wk, or 4) heavy training (regular heavy training or participation in competitive sports several times a week). Subjects were classified into 5 categories: never smokers, former smokers, light smokers (1–9 cigarettes/d), moderate smokers (10–19 cigarettes/d), and heavy smokers ( 20 cigarettes/d). Coffee consumption was divided into 5 categories according to the number of cups consumed per day: 0, < 1, 1–4, 5–8, and 9.
A vitamin-supplement score was created on the basis of use during the year and frequency of intake during the week of any type of vitamin supplement and was divided into 5 categories. The lowest category consisted of subjects who never used vitamin supplements and the highest category consisted of those who took vitamins 6–7 d/wk during the whole year (18). A folate score was computed from food-frequency information and data on the use of vitamin supplements. The calculation of the folate score and its validation against plasma folate and tHcy concentrations in 329 healthy subjects were reported previously (18).
Clinical examinations, blood sample collection, and biochemical analysis
The examinations included measurements of height, weight, and blood pressure; details of the procedures were reported previously (17). Blood was drawn from nonfasting subjects. Procedures for blood sample collection, processing, transport, and storage were described previously (17). Plasma tCys and tHcy were determined by HPLC and fluorescence detection (19, 20). The precision (between-day CV) of the assay is <3%. Serum total cholesterol and triacylglycerol concentrations were measured with enzymatic methods at the Department of Clinical Chemistry, Ullevål Hospital, Oslo.
Statistical methods
Because the distribution of tCys was symmetrical, no transformations were performed; arithmetic means are given for tCys. The distribution of tHcy was skewed with a long tail toward high values, and therefore the analyses were done by using log10 tHcy values.
Multiple linear regression models were used to assess the simultaneous relation among the various predictors of tCys. Plasma tCys was the dependent variable, whereas the independent variables were represented in the models as indicator variables denoting membership to 1 of 4 categories for cholesterol, heart rate, diastolic blood pressure, body mass index (BMI), physical activity, triacylglycerols, and folate intake and membership to 1 of 5 categories for smoking status, coffee consumption, and vitamin intake. Thus, the regression coefficient was used to estimate the difference in mean tCys between the reference category and the other categories for each factor. tCys concentrations across categories of each risk factor were tested jointly for homogeneity of means and for linear trend.
The multiple regression technique determines the relation between a set of independent variables and the mean of a normally distributed dependent variable (tCys). Thus, this model implies that the independent variables have the same effect on high and low values of the dependent variable. Biologically, however, it is possible that a factor influences extreme high or low values of the dependent variable with only moderate effects on the overall mean (21). Such effects can be overlooked with linear regression but may be studied with a set of logistic regression analyses, each at a different cutoff for the dichotomous dependent variable. Therefore, a series of logistic regressions were performed to assess the effect of the different factors on high and low tCys concentrations. The 5th (tCys 
217 µmol/L) and 95th (tCys 330 µmol/L) percentiles of the total study sample were chosen as cutoffs. The results, presented as odds ratios, represented an estimate of the risk that an individual with a specific risk profile was hypo- or hypercysteinemic relative to subjects with a baseline risk profile.
Furthermore, Pearson correlation coefficients were computed to provide a simpler summary of the linear relations between tCys and the various factors. The analyses were performed by using the statistical package BMDP (22). In addition, S-PLUS software (23) was used to construct Lowess' plots (24) of the smoothed relations between 2 variables and to estimate the density distribution of tCys. All tests were two-tailed, and a P value <0.05 was considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Mean tCys values and the 2.5 and 97.5 percentiles in the 3 age groups and in both men and women are shown in Table 1. Mean tCys values were higher in the older groups than in the youngest group. In the oldest age group there was no sex difference, whereas in the younger age groups tCys concentrations were higher in men than in women.
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. For all variables, we estimated the difference in mean tCys concentrations between categories of each risk factor and its reference category. The results, adjusted for sex, age, or both, showed that the largest overall differences in tCys concentrations were observed across different categories of age, sex, BMI, and cholesterol concentration and, to a lesser extent, coffee consumption and diastolic blood pressure. Small but significant differences in tCys concentrations were found across different categories of smoking status, but there was no marked linear trend. Nonsignificant differences in tCys were observed for physical activity, heart rate, triacylglycerol concentration, and folate intake (data not shown). Additional adjustment for all factors in Table 2, in addition to folate score, heart rate, and physical activity, showed that the relations with age, BMI, cholesterol concentration, sex, coffee consumption, and diastolic blood pressure remained strong, whereas smoking status no longer contributed to the prediction of tCys concentrations.
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). Age, sex, BMI, cholesterol concentration, diastolic blood pressure, and coffee consumption were associated with a shift of the tCys distribution to higher values. Although the associations with age and sex were somewhat stronger at low concentrations and stronger with BMI at high concentrations, the analyses of tail effects provided essentially the same information as the analyses of mean plasma tCys concentrations.
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). In all age and sex groups, plasma tCys concentrations correlated strongly with BMI (Figure 2) and also correlated with cholesterol concentration and diastolic blood pressure. The relation with BMI was attributed mainly to weight and not to height (data not shown). The association of tCys with coffee intake was strongest in the youngest age group. In contrast, smoking was negatively associated with tCys in the elderly. Weaker or no correlations were found between tCys and triacylglycerols, heart rate, physical activity, and vitamin intake (data not shown).
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). To exclude the possibility that the associations between tCys and the various factors investigated were secondary to changes in tHcy, we repeated the correlation analyses after dividing the subjects into those with tHcy concentrations >12 µmol/L and those with tHcy concentrations 12 µmol/L. The association between tCys and the various factors (except for tHcy) remained essentially unaltered (Table 4
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). Group 1 were factors associated with both tCys and tHcy: age, cholesterol concentration, diastolic blood pressure, and coffee consumption. BMI was strongly related to tCys but not to tHcy (group 2), whereas smoking status, folate and vitamin intakes, heart rate, and physical activity were mainly associated with tHcy and not with tCys (group 3). Finally, triacylglycerol concentrations were weakly associated with both aminothiols (group 4).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Such classification may point to possible mechanisms behind the associations and to a possible role of tCys as a cardiovascular disease risk factor.
Age, sex, cholesterol concentration, diastolic blood pressure, and coffee consumption were strong determinants of both tHcy and tCys and therefore complied with the criteria of group 1. Like tHcy (17, 26), tCys (Table 1) increased with age in both men and women. As with tHcy (27), the effect was most pronounced at the lower part of the tCys distribution (Table 3). Possible mechanisms are an age-dependent decrease in enzymatic activities involved in both cysteine and homocysteine metabolism (28) and impaired renal function. The latter possibility is supported by consistent observations that tCys and tHcy increase considerably in renal insufficiency (29). Notably, the decline in glomerular filtration rate may explain the age-related increase in tCys and tHcy (30).
Some (31), but not all (32), previous studies showed higher tCys concentrations in men than in women; such sex differences have been consistently shown for tHcy (17, 26). In the present study, plasma tCys concentrations were higher in men than in women in the youngest but not in the older age groups. The sex difference may have been due to hormonal effects, which vanish at advanced ages. Higher creatine-creatinine synthesis (a function of muscle mass and the major source of Hcy formation) in men than in women may contribute to the sex difference in tHcy concentration (26) but may have less of an effect on cysteine homeostasis.
Both tCys and tHcy were related to cholesterol concentration and diastolic blood pressure, and the relations remained strong after adjustment for other risk factors (Table 2). We are aware of no experimental or clinical observations that explain the relation between plasma aminothiol and cholesterol concentrations. The association of both tCys and tHcy with blood pressure may be related to the fact that both compounds react with nitric oxide to form vasoactive nitrosothiol adducts (8). In addition, cysteine may exert endothelium-dependent contraction by generating O2–, which rapidly inactivates the endothelium-derived relaxing factor (33).
A moderately strong relation between tCys and coffee consumption was observed. A similar relation was seen with tHcy and was attributed to the possible influence of caffeine (27). Notably, coffee consumption was associated with a complete shift of the tCys distribution (Table 3), but it was related to tHcy only at low concentrations (27).
BMI was a strong determinant of tCys in both men and women and in both age groups and was particularly predictive of high tCys concentrations. BMI has only rarely been associated with tHcy concentrations (34); in the present study we found no association and, therefore, BMI was classified as a group 2 factor. The mechanism for the association between BMI and tCys is unclear, but the association may reflect a possible role of plasma cysteine availability in stabilizing body cell mass (35).
Smoking status, vitamin and folate intakes, physical activity, and heart rate are determinants of plasma tHcy concentrations (17) but were essentially unrelated to tCys (Figure 4). Accordingly, these factors were classified as group 3 factors. However, the differential effects were not absolute because tCys was weakly related to heart rate and weakly and inversely related to smoking in elderly men and women. In young men and women, tCys was unrelated to smoking. In line with these findings, a recent study showed no effect of smoking on various cysteine forms in plasma (32). On the contrary, plasma tHcy showed a significant relation with smoking status that may have been attributable to changes in the thiol redox status or to the lower concentrations of plasma folate and vitamin B-12 in smokers (36).
As expected, tCys was unrelated to the intake of vitamin supplements or to the folate score, both of which are strong determinants of tHcy (Figure 4) (17, 18). Vitamin B-6, which is a cofactor in the 2 sequential enzymes that convert homocysteine into cysteine (6), was not assessed in the present study but should be considered as a possible factor for explaining some of the relations between tCys and several lifestyle factors. For instance, the strong relation between tHcy and coffee consumption (27) may have been due to impaired vitamin B-6 function, especially if caffeine functions as a vitamin B-6 antagonist, as shown recently for another xanthine derivative, theophylline (37). However, inhibition of the transsulfuration pathway is expected to decrease tCys, whereas we found that tCys increased in coffee drinkers. Smoking may also impair vitamin B-6 status, but elevated tHcy (38) and low tCys (Table 4) concentrations were observed only in the elderly smokers. Thus, our data do not support the hypothesis that coffee and smoking affect tCys by interfering with vitamin B-6 status. However, caffeine was shown recently to reduce the glomerular filtration rate (39), an effect that may partly have accounted for the increase in concentrations of both tCys and tHcy.
The present study has no clinical endpoints but addresses several questions of clinical relevance. Despite the relation between tCys and tHcy (Figure 3), the unique associations observed for each aminothiol (Figure 4) suggest that tCys and tHcy may confer independent cardiovascular disease risk. The possibility of an effect of tCys independent of tHcy is supported by similar relations between tCys and BMI, coffee consumption, serum cholesterol concentration, and blood pressure in subgroups with a high or low tHcy concentration (Table 4). Several in vitro studies on vascular or atherogenic effects of aminothiols showed similar effects for cysteine and homocysteine (5, 40). Such findings have been interpreted as a lack of specificity for homocysteine (5, 40) but could suggest that high concentrations of tCys and tHcy have a synergistic effect. Such synergy may explain the high cardiovascular mortality and morbidity predicted by hyperhomocysteinemia in patients with renal failure (41, 42), which, in contrast with vitamin deficiency, elevates both tHcy and tCys concentrations (43).
In conclusion, both tHcy and tCys are interactive components that undergo disulfide exchange and redox reactions (plasma redox thiol status) (10). Although tHcy has been established as an independent cardiovascular disease risk factor (5), only sparse data link tCys to human health and disease. Our study showed that tCys is strongly related to several biochemical, physical, and lifestyle factors, some of which also correlate with tHcy. Thus, these plasma aminothiols have distinct correlation profiles with established cardiovascular disease risk factors. Our data may suggest interactive effects in relation to the pathogenesis of vascular disease and should motivate clinical studies of these aminothiols as risk factors.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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