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Technical Briefs |
1
Dept. of Clin. Chem.,
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Blood Bank;
3
Dept. of Cardiol., Central Hosp. in Rogaland (SiR), 4003 Stavanger;
4
Hydro Aluminium AS Karmøy;
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Dept. of Clin. Chem., Haugesund Hosp., Rogaland, Norway);
a address for correspondence: fax 47-51519907
Recent data suggest that hyperhomocysteinemia is associated with an increased risk for premature vascular disease. Total plasma homocysteine (tHcy) may be increased by impaired activity of enzymes or suboptimal availability of B vitamins (1).
Homocysteine transsulfuration reactions are catalyzed by the enzymes cystathionine ß-synthase and cystathionine lyase in the presence of coenzyme vitamin B6, and remethylation of homocysteine is performed by the enzymes methionine synthase and betaine-homocysteine methyltransferase with vitamin B12 as a coenzyme for the former enzyme. 5-Methyltetrahydrofolate (5-methylTHF) or betaine are methyl donors during remethylation. The enzyme 5,10-methylenetetrahydrofolate reductase (MTHFR) with coenzyme FADH2 synthesizes 5-methyl-THF from 5,10-methylenetetrahydrofolate (5,10-MTHF) (2). The postmethionine load (PML) abnormal tHcy increments may reflect suboptimal plasma vitamin B6 or deficiency of cystathionine ß-synthase (3). The present report describes the effects of methionine loading on serum and erythrocyte folate, tHcy, and related thiols in smokers and nonsmokers.
We recruited 63 apparently healthy smokers [ages 37.8 ± 0.8
years; body mass index (BMI), 25.4 ± 0.5; male/female, 39/24;
cigarettes/day, 16.2 ± 0.8; smoking period (years), 19.7 ±
0.7], 44 nonsmokers (ages 37.8 ± 1.0 years; BMI, 24.5 ±
0.5; male/female, 26/18), and 23 former smokers who refrained from
smoking for 
3 months (ages 39.5 ± 1.0 years; BMI, 27.7 ±
0.5; male/female, 18/5) in the study. BMI and age of the participants
were not statistically different. Informed consent was obtained from
the participants, and the study was approved by the regional ethics
committee.
Blood sampling and the methionine loading test were performed as described previously (4). Participants were asked to eat a light breakfast or lunch with low contents of fats and proteins. Serum and erythrocyte folate and vitamin B12 were measured by a Dualcount, solid-phase, no-boil RIA developed by Diagnostic Products. tHcy, cysteine, and cysteinylglycine were measured according to the method described previously (5).
The Mann–Whitney U-test was applied to investigate the differences between the groups, and the Wilcoxon signed rank test was used to explore changes within the groups during methionine loading. Significance of difference between more than two groups was estimated by analysis of variance with the use of the Bonferroni/Dunn procedure. StatView for the Macintosh developed by Abacus Concepts was used for statistical calculations.
The concentrations of serum and erythrocyte folate of smokers were
significantly lower than those of nonsmokers (P <0.05)
(Table 1
). Lower intake of fruits and vegetables, free radicals in
cigarette smoke, and increased excretion of folates may have
contributed to a gradual decline in the concentration of serum and
erythrocyte folate (6)(7)(8)(9). An 11–16% reduction in the
concentrations of serum folate was detected during methionine loading
in smokers as well as in nonsmokers 2 h (P <0.01 and
<0.001, respectively) and 6 h afterwards (P <0.01 and
<0.001, respectively) (Fig. 1
, A). A significant decrease in serum folate of former smokers
was observed after 6 h (P <0.01) (not shown in the
figure). A drop in the concentration of serum folate may have occurred
because of the reduced activity of enzyme MTHFR. Methionine loading
increases the concentration of S-adenosylmethionine, and
increased concentrations of S-adenosylmethionine inhibit the
reductase activity of MTHFR (10).
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Women had higher concentrations of serum folate than men (P <0.05) (data not shown). Nonsmokers had higher concentrations of serum folate than former smokers (P <0.01), but the difference in erythrocyte folate was not statistically different. Former smokers may not exhibit a significant change in serum folate until erythrocytes gradually accumulate folate to an optimal amount during the period of smoking cessation.
The concentrations of erythrocyte folate in smokers and nonsmokers
remained unaffected during methionine loading (Fig. 1
, B), but a
significant reduction in erythrocyte folate was detected in former
smokers after 2 h (P <0.05) (not shown in the figure).
Because 5-methyl-THF makes up ~40–50% of total folate
polyglutamates in erythrocytes, minor changes in the concentration of
5-methyl-THF may not influence substantially total erythrocyte folate.
Mean tHcy concentrations were significantly lower in women than in men (P <0.01); the significance disappeared when data were analyzed for male smokers vs female smokers (P = 0.07) and male nonsmokers vs female nonsmokers (P = 0.09). Smokers had slightly higher concentrations of tHcy than nonsmokers, but the difference was not significant even if data were analyzed separately for gender (data not shown). Previously, it has been reported that heavy smokers have significantly higher concentrations of tHcy than nonsmokers (11).
The mean concentration of tHcy increased, and plasma concentrations of
cysteine and cysteinylglycine decreased during methionine loading, as
shown previously (Fig. 1
, C, D, and E) (5). Large doses of
methionine given to mice and rats seem to perturb activity of enzymes
and concentrations of amino acids involved in methionine metabolism
(12)(13).
The 2-h PML response was abnormal (
tHcy concentration >90th
percentile) in 10% of smokers, 9% of former smokers, and 7% of
nonsmokers. The 6-h PML response was abnormal in 10% of smokers, 9%
of former smokers, and 9% of nonsmokers. The concentrations of serum
vitamin B12 in smokers, nonsmokers, and former smokers
(338, 353, and 356 pmol/L, respectively) were not significantly
different, and the PML concentrations did not change significantly in
the groups (data not shown).
We conclude that smokers have lower concentrations of serum and erythrocyte folate than nonsmokers and that methionine loading decreases serum folate in all groups.
Acknowledgments
We thank the following persons for their cooperation and technical assistance: Anne Marie Aarstad, Kari Skibeli, Gunn Walmestad, Kirstin Gard, Ingjerd Rasmussen, and Otlo Osvik (Central Hospital in Rogaland), Helga Lie (Hydro Aluminium), and Kirsten Gundersen (Haugesund Hospital).
References
-glutamyl cycle functions in vivo using intracellular glutathione: effects of amino acids and selective inhibition of enzymes. Proc Natl Acad Sci U S A 1978;75:5405-5408.
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,
nonsmokers. A, serum folate; B, erythrocyte folate;
C, plasma homocysteine; D, cysteine;
E, cysteinylglycine, ns, nonsignificant.