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Technical Briefs |
1
Dept. of Surg., Karolinska Hosp., Stockholm, Sweden.;
2
Dept. of Clin. Chem., Central Hosp. in Rogaland, Stavanger, Norway;
3
Dept. of Clin. Biol., Div. of Pharmacol., Univ. of Bergen, Haukeland Hosp., Bergen, Norway;
4
Dept. of Epidemiol., Inst. of Environ. Med., Karolinska Inst., and Div. of Cardiovasc. Med., Dept. of Med., Karolinska Hosp., Stockholm, Sweden;
a corresponding author: fax 619-534-2005, e-mail cbergmark{at}ucsd.edu
Cigarette smoking is a dominant risk factor for atherosclerotic vascular disease. Moderate increase of plasma homocysteine (Hcy) is also associated with various forms of vascular disease (1). Several genetic and nutritional factors, which interact in a complex manner, determine the concentration of plasma Hcy. On theoretical grounds one would suspect an indirect effect of smoking on Hcy metabolism, mediated by the effects on the cofactors for Hcy metabolism, vitamin B12, B6, and folate (2)(3). In a previous report we found current smoking to be associated with moderately increased Hcy in patients with premature peripheral atherosclerosis but not in control subjects (4).
Several mechanisms involving pro-oxidant properties, e.g., redox changes in glutathione, can explain why smoking contributes to atherosclerotic vascular disease (5)(6). Because of a previously found correlation with redox status of thiols related to glutathione (8), we hypothesized that smoking could interfere with the redox status of these thiols.
In the present pilot study we used a newly developed method (9) to investigate the redox status of plasma Hcy and related aminothiols in healthy young subjects, differing in current smoking habits. Possible interactions between smoking habits and cofactors for Hcy metabolism were also analyzed.
All 41 subjects—19 women and 22 men, mean age 26 years (range 20–33)—had completed a health declaration, and those with signs of cardiovascular disease had been excluded. Nineteen were nonsmokers and 22 current smokers. A nonsmoker was defined as a person who had never smoked or who had not smoked for at least 1 year. Current smokers consumed at least 10 cigarettes/day and had done so for at least 2 years; they were told not to change their daily smoking habits during the study. The following factors were recorded: smoking habits, length, weight, history of cardiovascular disease, medication, last menstruation, and previous medical admissions.
Blood samples were taken in the morning after an overnight fast and analyzed with the following laboratory tests: hemoglobin, erythrocyte count, platelet count, leukocyte count, total cholesterol, triglycerides, HDL-cholesterol, and apolipoproteins A-I and B. LDL values were derived from the Friedewald formula. Serum cobalamin and serum folate were measured by the Simultrac-SNB Radioassay Kit (Becton Dickinson). Vitamin B6 was measured as its first active metabolite, pyridoxal 5-phosphate, by an enzymatic method (10).
For thiol analysis, we analyzed blood collected into 3 evacuated
tubes containing either monobromobimane or N-ethylmaleimide
as thiol-derivatizing reagents or no derivatizing additive. The blood
was centrifuged at 10 000g for 1 min at room temperature.
Analysis of the plasma from blood collected in monobromobimane solution
yielded the amounts of reduced thiols; analysis of plasma from blood
collected in N-ethylmaleimide gave the amounts of the
oxidized forms; and the total amount of thiol components was assayed in
the untreated plasma. The protein-bound fraction was calculated by
subtracting the reduced and free oxidized species from the total
amount. The total, protein-bound, reduced, and oxidized forms of Hcy,
cysteine, and cysteinylglycine (a degradation product of glutathione)
are reported in Table 1
. A more extensive description of the method is given by Mansoor
et al. (9).
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Differences between groups were tested with Wilcoxon's two-sample tests. Multiple linear regression analysis and multiple logistic regression analysis were performed with JMP software (SAS Institute). Mean values were determined for each sex and for smokers and nonsmokers. Univariate analysis for differences between smokers and nonsmokers were made within each sex. We found no significant differences in basic and hematological characteristics between the groups except that male smokers had a lower weight and higher concentrations of apo A-I.
As shown in Table 1
, no fraction other than reduced Hcy differed
between smokers and nonsmokers; also, the young men had higher total
Hcy concentrations than the young women. The distribution of reduced
Hcy among male and female smokers and nonsmokers is shown in Fig. 1
. The concentrations of the various cofactors for Hcy metabolism
showed no significant differences between smokers and nonsmokers.
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Multiple regression analysis with the reduced fraction of Hcy as responder revealed that no other factors significantly influenced the association between current smoking and the concentration of reduced Hcy, but vitamin B6 was noted to have an effect on reduced Hcy among nonsmokers, both in univariate and multiple regression analysis. A substantial part of this finding, however, depended on one outlier, which was from the only nonsmoker with a high concentration of reduced Hcy. Notably, she had a very low value for pyridoxal phosphate (vitamin B6), the next to lowest value for total Hcy, and the highest serum folate content. Multiple logistic regression analysis with smoking habit as responder suggested that only reduced Hcy and triglycerides were significant discriminators.
A recent study on Hcy and other risk factors for vascular disease in 3000 healthy individuals demonstrated a positive correlation between current smoking and total Hcy (15). This association is thought to be caused by the effects of smoking on the cofactors for Hcy metabolism: vitamins B6, B12, and folate (2)(3). The present study, however, does not demonstrate a difference in total Hcy between smokers and nonsmokers—possibly because of the relatively small number of subjects or their youth. Nonetheless, the difference in reduced Hcy is clear and independent of gender or registered covariates of smoking, except for a negative correlation between vitamin B6 and reduced Hcy in nonsmokers.
Two major different mechanisms could be of importance for the association of smoking and increased concentrations of reduced Hcy. One is displacement of disulfide-bound homocyst(e)ine in plasma by compounds in the cigarette smoke. The other is extracellular export of reduced Hcy as a defense reaction against oxidative stress by cigarette smoke. If displacement is important, it should probably influence the redox status of other thiols as well.
Similar results, with even higher concentrations of reduced Hcy, but not increased total Hcy, are found in AIDS patients (16), indicating a role of the immune system. In patients with homocystinuria, whose concentrations of Hcy are very high, the reduced fraction is relatively greater than in subjects with moderate hyperhomocysteinemia (7). Previously, we demonstrated that the ratio between reduced and total Hcy correlates with the same ratio for cysteine in patients with premature peripheral atherosclerotic disease (8). This correlation was not seen in the present study (data not shown). An explanation for this discrepancy may be that smoking in some way might shift the equilibrium among thiols/disufides/mixed disulfides in plasma (6) and that the present study involved only healthy subjects. The reduced fraction of Hcy is highly labile and prone to autoxidation of its sulfhydryl group. For this reason the reduced fraction of Hcy, representing only 1.3% and 2.9% of the total amount of Hcy in nonsmokers and smokers, respectively, is thought to be the metabolically most active fraction, whereas the stable oxidized fractions are more inert.
Hultberg and colleagues (11) found that reduced Hcy was increased and total Hcy was decreased after administration of N-acetylcysteine (NAC), indicating a displacement effect of NAC on plasma fractions of Hcy. Other possible causes for the plasma compartment of reduced Hcy to be increased are related to intracellular free radical defense, e.g., cellular import of disulfide-Hcy or intracellular reduction of the disulfide bond. The latter mechanism involves scavenging of free radical-generated electrons and subsequent export from the intracellular compartment of the reduced Hcy (12). Several explanations for the metabolic activity of reduced Hcy have been proposed. Reduced Hcy may cause redox cycling and disulfide interchange with other thiols, preferably cyst(e)ine, thus interfering with disulfide bonds. This mechanism may be important in protein folding and enzyme activation (8).
The reasons why smoking causes a more than twofold increase of reduced Hcy in plasma cannot be settled from the present study. The many complex actions of cigarette smoking may be related to oxidizing components, e.g., oxygen free radicals, aldehydes, or NO2. Sulfhydryl groups in thiols have an important role in scavenging carbon radicals (13), and sulfur radicals could also be formed by NO2 from cigarette smoke and plasma thiols (14).
The marked increase of the small fraction of reduced Hcy we found in young, healthy smoking subjects as compared with nonsmoking controls might be what contributes to the damaging effects of smoking.
References
The following articles in journals at HighWire Press have cited this article:
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  A. De Bree, W. M. M. Verschuren, D. Kromhout, L. A. J. Kluijtmans, and H. J. Blom Homocysteine Determinants and the Evidence to What Extent Homocysteine Determines the Risk of Coronary Heart Disease Pharmacol. Rev., December 1, 2002; 54(4): 599 - 618. [Abstract] [Full Text] [PDF]
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 A. d. Bree, W. M. M. Verschuren, H. J. Blom, and D. Kromhout Lifestyle Factors and Plasma Homocysteine Concentrations in a General Population Sample Am. J. Epidemiol., July 15, 2001; 154(2): 150 - 154. [Abstract] [Full Text] [PDF]
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 L. El-Khairy, P. M Ueland, O. Nygard, H. Refsum, and S. E Vollset Lifestyle and cardiovascular disease risk factors as determinants of total cysteine in plasma: the Hordaland Homocysteine Study Am. J. Clinical Nutrition, December 1, 1999; 70(6): 1016 - 1024. [Abstract] [Full Text] [PDF]
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