Effect of smoking on serum concentrations of total homocysteine and B vitamins in mid-pregnancy

Effect of smoking on serum concentrations of total homocysteine and B vitamins in mid-pregnancy

Clinica Chimica Acta 306 Ž2001. 103–109 www.elsevier.comrlocaterclinchim Effect of smoking on serum concentrations of total homocysteine and B vitami...

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Clinica Chimica Acta 306 Ž2001. 103–109 www.elsevier.comrlocaterclinchim

Effect of smoking on serum concentrations of total homocysteine and B vitamins in mid-pregnancy Karla Pagan ´ a, Jinrong Hou b, Robert L. Goldenberg b, Suzanne P. Cliver b, Tsunenobu Tamura a,) a

Department of Nutrition Sciences, 218 Webb Building., UniÕersity of Alabama at Birmingham, Birmingham, AL 35294-3360, USA b Department of Obstetrics and Gynecology, UniÕersity of Alabama at Birmingham, Birmingham, AL 35294, USA Received 26 October 2000; received in revised form 19 January 2001; accepted 30 January 2001

Abstract There are conflicting findings in the literature on the effect of smoking on total homocysteine ŽtHcy. concentrations in non-pregnant subjects. We evaluated the effect of smoking on serum concentrations of tHcy, folate, vitamin B-12 pyridoxal 5X-phosphate ŽPLP, a coenzyme form of vitamin B-6. in 196 women at 18 and 30 weeks’ gestation. The smokers were defined as those who self-reported cigarette smoking and had serum concentrations of thiocyanate, a biomaker of smoking, in the highest quartiles of the population. Mid-pregnancy serum tHcy concentrations were not significantly different between smokers and non-smokers. Folate, vitamin B-12 and PLP concentrations were generally lower in smokers than non-smokers. In smokers, tHcy concentrations had significant negative correlations with folate at both time points. The multiple regression analyses indicated that serum folate concentration was the most significant factor associated with tHcy concentrations among smokers, whereas thiocyanate concentrations showed no such effect. We conclude that serum tHcy concentrations were most strongly associated with the nutritional status of folate among the B vitamins tested during mid-pregnancy in our subjects. We suggest that it is essential to consider the nutritional status of folate, vitamin B-12 and vitamin B-6 in evaluating the effect of smoking on homocysteine metabolism. q 2001 Published by Elsevier Science B.V. Keywords: Homocysteine; Folate; Smoking; Thiocyanate; Human; Pregnancy

1. Introduction Moderately increased serumrplasma total homocysteine ŽtHcy. concentrations Žhyperhomocysteinemia. have been implicated as a risk factor for occlusive vascular disease w1x. Many researchers in the field of obstetrics have associated hyperhomo) Corresponding author. Tel.: q1-205-934-7478; fax: q1-205934-7049. E-mail address: [email protected] ŽT. Tamura..

cysteinemia with various pregnancy complications including fetal-growth restriction w2,3x, although serum tHcy concentrations in pregnant women are generally low w4,5x. The hypotheses associated with hyperhomocysteinemia and an adverse outcome of pregnancy include: Ž1. the atherogenic effect of hyperhomocysteinemia leading to the circulatory disturbances in the placenta; Ž2. the deficiencies of B-vitamins Žfolate, vitamin B-12 and vitamin B-6. resulting in elevated tHcy; and Ž3. the direct adverse effect of these deficiencies w2,3x.

0009-8981r01r$ - see front matter q 2001 Published by Elsevier Science B.V. PII: S 0 0 0 9 - 8 9 8 1 Ž 0 1 . 0 0 4 0 2 - 8

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K. Pagan ´ et al.r Clinica Chimica Acta 306 (2001) 103–109

Smoking is known to be one of the most significant factors adversely affecting pregnancy outcome including fetal growth w6x. Various investigators have suggested that smoking leads to elevated serum tHcy concentration in non-pregnant adults, although the findings are controversial w7–20x. To our knowledge, however, the association between smoking and homocysteine metabolism in pregnancy has not been evaluated. Therefore, we undertook this investigation to evaluate the relationship between smoking Ždefined by admission of smoking and elevated serum concentrations of thiocyanate, a biomarker of smoking w21x. and serum tHcy concentrations in women at 18 and 30 week’s gestation. Furthermore, we studied the association between smoking and the nutritional status of folate, vitamin B-12 and vitamin B-6, each of which regulates homocysteine metabolism w1x.

2. Materials and methods 2.1. Subject and samples A total of 285 pregnant women were selected using a random-computerized program from more than 1500 women from a low-socioeconomic background, who participated in a study to identify risk factors for the development of fetal-growth restriction. This original study took place in Birmingham, Alabama, USA between 1986 and 1988 w22x. All women delivered a term-singleton infant, and their mean age was 25.5 years old. They were 46% African American and 54% Caucasian. These women were offered daily oral supplements of folic acid Žpteroylglutamic acid, 1.0 mg. and iron Ž65 mg. during pregnancy with estimated routine usage of the supplements of about 50%, which was based on serum folate concentrations being over 46 nmolrl as previously reported w22x. Non-fasting blood samples were collected at 18 and 30 weeks’ gestation using plastic syringes ŽMonovette, Sarstedt, Princeton, NJ, USA. and were centrifuged within 40 min of drawing to separate the serum, which was kept at y708C until assayed w22x. Of 285 women, 196 subjects were selected for the evaluation presented here, because the information on tobacco smoking Žadmission of smoking and

serum thiocyanate concentrations. and the concentrations of serum tHcy, folate, vitamin B-12 and pyridoxal 5X-phosphate ŽPLP, an active coenzyme of vitamin B-6. concentrations were available wPagan ´ et al., unpublished datax. The study was approved by the Institutional Review Board for Human Use of the University of Alabama at Birmingham. 2.2. Definition of smokers and non-smokers Smokers were defined based on their positive admission of smoking and serum thiocyanate concentrations at or above the lowest value of the highest quartile of this population at 18 and 30 week’s gestation. The cutoff values of thiocyanate concentrations were different between the time points, and these were 122 mmolrl at 18 weeks and 113 mmolrl at 30 weeks’ gestation. The women in the highest quartile of serum thiocyanate noted that they smoked an average of 19.5 Ž"14.2, SD. and 9.9 Ž"9.4. cigarettesrday at 18 and 30 weeks’ gestation, respectively. The women who denied smoking and had their serum thiocyanate concentrations below the lowest value of the highest quartile were defined as non-smokers. Based on the criteria noted above, at 18 weeks’ gestation, 65 and 131 women were identified as smokers and non-smokers, respectively, and at 30 weeks’ gestation, 57 and 122 women were identified as smokers and non-smokers, respectively. Of these, 42 women were identified as smokers at both 18 and 30 weeks’ gestation, and 118 women did not smoke at both time points. 2.3. Determination of total homocysteine, Õitamin B-12, PLP and thiocyanate Serum tHcy concentrations were measured using high-performance-liquid chromatography with fluorescence detection using homocysteine thiolactone as a standard w23x. Serum vitamin B-12 concentrations were determined by a radioassay kit ŽCiba Corning, Palo Alto, CA, USA., and serum PLP concentrations were measured by tyrosine-decarboxylase-assay method using w3 Hx-tyrosine Žspecific activity, 53.4 Cirmmol; Moravek Biochemicals, Brea, CA, USA. as a substrate w24,25x. Serum thiocyanate concentrations were measured colorimetrically w26x. Serum

K. Pagan ´ et al.r Clinica Chimica Acta 306 (2001) 103–109

folate concentrations were taken from an investigation published in 1992 w22x, where they were assayed in 1989 by a Lactobacillus casei microbiological method w27x. The inter-assay coefficients of variation for tHcy, folate, vitamin B-12 and PLP were approximately 8%, 10%, 8% and 11%, respectively, in our laboratory w25x. 2.4. Statistical analyses Data are presented as mean " S.D., where appropriate. A two-tailed Student t-test was used to determine the significance of the differences in the means of serum thiocyanate, tHcy and the vitamins between the smokers and non-smokers. A Pearson correlation coefficient was used to evaluate the relationships between the various serum indices. A multiple regression analysis was employed to estimate a change in serum tHcy concentrations as a result of a change in serum concentrations of thiocyanate and B vitamins by controlling maternal race. The level of significance was set at p - 0.05 in this study.

3. Results As shown in Table 1, there was no significant difference in serum tHcy concentrations between smokers and non-smokers at both 18 and 30 weeks’ gestation. Serum folate concentrations were significantly lower in smokers than in non-smokers at 30 weeks’ gestation, whereas no such difference was observed at 18 weeks’ gestation. Furthermore, the percentages of women who smoked and likely took folic acid supplementation assessed based on serum folate concentrations Ž) 46 nmolrl w22x. were 42% and 35% of all smokers and those were 57% and 53% at 18 and 30 weeks’ gestation, respectively. The differences between the two groups were significant at 18 and 30 weeks’ gestation Ž p s 0.05 and p 0.05, respectively.. Serum vitamin B-12 concentrations were significantly lower in smokers than in non-smokers at both time points. Overall, PLP concentrations were lower than generally accepted values w25x, and smokers had significantly lower values at 18 weeks’ gestation, whereas such a difference was not found at 30 weeks’ gestation. Serum vitamin B-12 concentrations in African Americans were sig-

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Table 1 Serum indices of smokers and non-smokers at 18 and 30 weeks’ gestation Smoker

Non-smoker

p

18 weeks’ gestation Thiocyanate Žmmolrl. Total homocysteine Žmmolrl. Folate Žnmolrl. Vitamin B-12 Žpmolrl. X Pyridoxal 5 -phosphate Žnmolrl.

n s65 170"39 5.2"2.4

ns131 42"25 5.0"1.6

0.0001 NS

47"31 319"99 15"10

54"38 379"139 24"23

NS - 0.005 - 0.0005

30 weeks’ gestation Thiocyanate Žmmolrl. Total homocysteine Žmmolrl. Folate Žnmolrl. Vitamin B-12 Žpmolrl. X Pyridoxal 5 -phosphate Žnmolrl.

ns 57 166"42 5.7"3.4

ns122 46"24 4.9"1.6

0.0001 NS

38"30 254"60 11"15

54"39 309"102 15"16

- 0.005 0.0001 NS

Mean"S.D.

nificantly higher than those in Caucasians at both time points Ž p - 0.0001., whereas there were no racial differences for serum tHcy, folate, and PLP concentrations. In smokers, there were significant negative correlations between serum tHcy and folate concentrations at 18 and 30 weeks’ gestation Ž r s y0.33, p s 0.05 and r s y0.38, p s 0.01, respectively.. The scattergram of the association at 30 weeks’ gestation is presented in Fig. 1. Similar relationships between serum tHcy and folate concentrations were also observed in non-smokers at both time points. Significant negative correlations were found between serum tHcy and vitamin B-12 concentrations at 18 weeks’ gestation in both smokers and non-smokers Ž r s y0.37, p s 0.005 and r s y0.28, p - 0.005, respectively., whereas those were not significant at 30 weeks’ gestation Ž r s y0.21, p s NS, and r s y0.10, p s NS, respectively.. There were significant positive correlations between serum tHcy and thiocyanate concentrations at both 18 and 30 weeks’ gestation Ž r s 0.26, p - 0.05 and r s 0.29, p - 0.05, respectively.. This association at 30 weeks’ gestation is depicted in Fig. 2. However, this relationship was significant only among smokers. The correlations between serum

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K. Pagan ´ et al.r Clinica Chimica Acta 306 (2001) 103–109

Fig. 1. Relationship between serum total homocysteine and folate concentrations in smokers at 30 weeks’ gestation Ž r s y0.38, p - 0.01..

thiocyanate and either folate, vitamin B-12 or PLP concentrations were not significant at both 18 and 30 weeks’ gestation Ž p s NS. among smokers, indicating no dose–response relationships.

Similar analyses were performed in 42 and 118 women who were identified as smokers and nonsmokers, respectively, at both 18 and 30 weeks’ gestation. Overall findings of these analyses were

Fig. 2. Relationship between serum total homocysteine and thiocyanate concentrations in smokers at 30 weeks’ gestation Ž r s 0.29, p - 0.05..

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Table 2 Effect of serum indices on plasma total homocysteine concentrations at 18 and 30 weeks’ gestation using multiple regression analyses Regressors

Thiocyanate Folate Vitamin B-12 X Pyridoxal 5 -phosphate a

18 weeks’ gestation

30 weeks’ gestation

Effect size Žmmolrl. a

p

Effect size Žmmolrl. a

p

0 y0.0120 y0.0037 y0.0219

NS NS NS NS

q0.0181 y0.0416 y0.0078 y0.0227

NS - 0.05 NS NS

Changes equivalent of the increases in one unit of regressors.

essentially similar to those observed in a total of 196 women. To further evaluate the relationship between the concentrations of serum tHcy and thiocyanate or B vitamins in smokers, multiple regression analyses were performed ŽTable 2.. The analyses indicated that serum folate concentration was the only significant factor associated with serum tHcy concentrations. A decrease of 10 nmolrl in serum folate concentrations corresponded to the increase of 0.42 mmolrl in serum tHcy concentrations at 30 weeks’ gestation Ž p - 0.05.. However, no such association was found at 18 weeks’ gestation as shown in Table 2.

4. Discussion We found that mid-pregnancy serum tHcy concentrations were not significantly different between smokers and non-smokers. The concentrations of folate, vitamin B-12 and PLP were generally lower in smokers than non-smokers in mid-pregnancy, and these results are consistent with those found in the literature w28–31x. However, in our pregnant population, the difference in serum folate concentrations between smokers and non-smokers at 30 weeks’ gestation may be due to the fact that a higher percentage of non-smokers took folic acid supplementation. Our observation of the difference in vitamin B-12 concentrations between pregnant African Americans and Caucasians is similar to that reported in a group of elderly subjects by Carmel et al. w32x; however, the mechanism of such a difference is unknown at present.

Serum tHcy concentrations negatively correlated with folate concentrations at both time points among smokers. Based on the multiple regression analyses, serum folate concentration was the most significant factor associated with tHcy concentrations among smokers, whereas serum thiocyanate concentrations showed no significant relationship to tHcy concentrations. Therefore, we conclude that serum tHcy concentrations in mid-pregnancy were most strongly regulated by the nutritional status of folate among the B vitamins; however, they were not directly affected by smoking. To our knowledge, 11 groups of investigators have evaluated the effect of smoking on serum tHcy concentrations in a total of 12 independent populations of non-pregnant subjects w7–20x. The results of these studies are summarized in Table 3. Of these 12 studies, five indicated that smoking was associated with elevated serumrplasma tHcy concentrations w8,10,12,13,15,16,19x. However, as shown in Table 3, most of the increases in tHcy concentrations secondary to smoking were small with the range of increases between 1.1 and 1.5 mmolrl. The only exception was a study by Bergmark et al. w8x who reported an increase of 4.7 mmolrl. In contrast to these five studies, seven other groups did not report a significant effect w7,9,11,14,17,18,20x. We also found that the mean tHcy concentrations in smokers were only 0.2 and 0.8 mmolrl higher than non-smokers and this difference was not significant. Therefore, if there is a relationship between smoking and plasma tHcy concentrations, the associated increase in the concentrations tends to be small and may have little physiological significance. It should be pointed out that a limited number of these investigations involved the nutritional assess-

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Table 3 Investigations on the effect of smoking on serumrplasma total homocysteine concentrations in non-pregnant subjects Investigators wReferencex

Location

Number of subjects

Change in total homocysteine Žmmolrla .

Berg et al. w7x Bergmark et al. w8x Brattstrom ¨ et al. w9x Nygard et al. w10,13,15x Monsoor et al. w11x Shaw et al. w12x Woodside et al. w14x Kark et al. w16x Bostom et al. w17x Walmsley et al. w18x de Jong et al. w19x Al-Obaidi et al. w20x

Norway Norway Sweden Norway Norway Australia Northern Ireland Israel USA England Netherlands England

155 58 243 ) 16,000 107 365 509 1,788 1,947 1,191 510 117

q0.48 q4.74 b y0.40 q1.21b q0.70 q1.50 b 0 q1.10 c y0.07 q1.20 increased but data not available y0.40

a

The values represent the maximum differences presented in the articles. The effect of smoking was significant. c This was significant among males only. b

ment of the B vitamins w9,11,14,17x. The study by Bostom et al. w17x is the only one in which concentrations of all three vitamins were determined. It has been well recognized that smoking affects the nutritional status of folate, vitamin B-12 and vitamin B-6 w28–31x, each of which regulates homocysteine metabolism w1x. Therefore, it is important to take the nutritional status of the B vitamins into account when evaluating the effect of smoking on tHcy concentrations. Based on our data, it is apparent that the elevated tHcy concentration is a surrogate for compromised status of B vitamins, particularly of folate, secondary to smoking. It seems unlikely that smoking directly affects homocysteine metabolism. Kark et al. w16x reported a positive correlation between the concentrations of serum tHcy and thiocyanate in elderly men in Israel. Their data are similar to our observation of a weak but significant positive correlation between serum thiocyanate and tHcy concentrations in pregnant subjects. These two independent findings suggest that there is a dose–response relationship between smoking and the metabolism of the vitamins that leads to an alteration in homocysteine metabolism. In conclusion, smoking does not appear to significantly affect serum tHcy concentrations in our pregnant population. Our multiple regression analyses indicated that serum folate concentration is the major factor affecting serum tHcy concentrations. We sug-

gest that it is essential to consider the nutritional status of folate, vitamin B-12 and vitamin B-6 in evaluating the effect of smoking on homocysteine metabolism.

Acknowledgements This study was supported in part by National Institutes of Health contract N01-4-2811.

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