- Email: [email protected]
Chronic maternal smoking and cord blood amino acid and enzyme levels at term
Chronic Maternal Smoking and Cord Blood Amino Acid and Enzyme Levels at Term ERIC JAUNIAUX, MD, PhD, VINCIANE BIERNAUX, MD, ERIK GERLO, PhD, AND BEATRICE GULBIS, MD, PhD Objective: To assess the influence of chronic active maternal smoking on cord blood amino acid and enzyme levels at term. Methods: The concentrations of 24 free amino acids, total protein, and five enzymes were measured in samples of maternal and fetal cord venous plasma from 24 nonsmokers who were not exposed to tobacco smoke and 24 chronic smokers. Cotinine levels were also measured in maternal plasma to evaluate fetal tobacco exposure. The pregnancies were between 37 and 40 weeks’ gestation, were uncomplicated, and were delivered vaginally. Results: Fetal weight was significantly (P < .01) lower in the smokers than in controls. A positive significant (P < .001) correlation was found between maternal and umbilical venous cotinine concentrations. Significantly lower concentrations of aspartic acid (P < .01), hydroxyproline (P < .05), threonine (P < .005), alanine (P < .05), ␣-aminobutyric acid (P < .001), methionine (P < .05), tyrosine (P < .001), phenylalanine (P < .01), and lysine (P < .05) were found in the venous cord plasma of the smokers compared with nonsmokers. The fetomaternal ratios were similar in both groups. The umbilical plasma alkaline phosphatase activity was significantly (P < .01) lower in the smokers than in the controls. Conclusion: Chronic maternal smoking is associated with alterations of protein metabolism and enzyme activity in fetal cord blood. These may be secondary to irreversible changes in the cellular functions of the trophoblast and may contribute to fetal growth restriction. (Obstet Gynecol 2001; 97:57– 61. © 2001 by The American College of Obstetricians and Gynecologists.)
From the Academic Departments of Obstetrics and Gynaecology, University College London, London, United Kingdom; the Departments of Clinical Chemistry and Obstetrics and Gynecology, Academic Hospital Erasme, Universite´ Libre de Bruxelles, Brussels, Belgium; and the Department of Clinical Chemistry, Academisch Ziekenhuis, Vrije Universiteit van Brussels, Brussels, Belgium. Supported by a grant from the David & Alice Van Buuren Foundation (Universite´ Libre de Bruxelles, Brussels, Belgium) and a grant from the Fonds National de la Recherche Scientifique (no. 9.4523.97).
VOL. 97, NO. 1, JANUARY 2001
Tobacco smoke compounds such as nicotine and carbon monoxide have been shown to act indirectly on the fetus through uteroplacental vasoconstriction.1 The placental structure seems to be extremely vulnerable to smoke toxins such as cadmium, which damage its vascular system2 and inhibit some of its enzyme activities.3 Nicotine, its metabolites, and most tobacco carcinogens and teratogens have low molecular weights and high water solubility and therefore readily cross the placenta. Cotinine has been found to accumulate in the fetal compartment as early as 7 weeks’ gestation in both active and passive smokers.4 Recently, a tobaccospecific carcinogen and its metabolite have been demonstrated in amniotic fluid samples of smokers at 16 –21 weeks.5 Commonly reported adverse perinatal outcomes of maternal cigarette smoking during pregnancy include an increased miscarriage rate6 and a two-fold increase in the risk of delivering a low birth weight infant because of prematurity and/or fetal growth restriction (FGR).1,7 These effects are linked to alterations in placental structure and function induced by some of the many tobacco smoke compounds, in particular DNA adduct.8 –11 The findings that these carcinogens induce gene mutations in utero12 and that modified DNA can be transmitted to embryos by spermatozoa13 have recently received widespread interest. Active smoking during pregnancy is associated in all trimesters with placental ultrastructural lesions, including a decrease in syncytiotrophoblast microvilli and pinocytotic activity, focal syncytial necrosis, and degeneration of cytoplasmic organelles.8,14,15 These morphologic alterations are irreversible and are associated with a direct effect on the placental metabolic and transfer functions.16 We have recently reported that the fetal plasma levels of some amino acids and amylase at 12–17 weeks are modified by maternal smoking.17 In the present study, we further evaluated the effect of chronic maternal smoking throughout pregnancy on the levels
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of free amino acids and enzyme activity in umbilical cord plasma collected at term.
Materials and Methods Samples of fetal cord and maternal venous blood were obtained from 48 women at 37– 40 weeks’ gestation. The study included a consecutive series of 24 chronic smokers, who smoked three to 20 cigarettes per day (median 11.5 cigarettes per day), and a series of 24 nonsmokers prospectively collected over a 6-month period in the same population living in the same area. All women were white, 21– 40 years of age (median 27.5 years), had no medical history, and attended the antenatal clinic at Erasme Academic Hospital. Gestational age was determined from the date of the last menstrual period and confirmed by ultrasound measurement of the crownrump length between 9 and 14 weeks’ gestation. Exposure to cigarette smoke is currently determined in our clinic by a questionnaire that is distributed at the first visit to all women attending the antenatal clinic. All women are encouraged to avoid cigarette smoke and are provided with the same counseling on nutrition. The groups included only pregnancies after 36 weeks that had been uncomplicated. Each study case was matched prospectively with a control, who was the next woman to deliver. Forty pairs were initially enrolled in this study. Passive smokers (n ⫽ 10) were excluded as controls before delivery when their urine cotinine level did not corroborate the self-reported exposure level.4 Six pairs were excluded at the time of delivery because either the study case or the corresponding control had a complicated delivery. Collection of umbilical cord blood for research has been approved by the Erasme Academic Hospital and University College Hospitals Committees on the Ethics of Human Research. Both departments are conducting ongoing research projects on cord blood. After written informed consent, 3.0 –5.0 mL of cord blood was aspirated from the vein into preheparinized syringes. Simultaneously, maternal blood samples were collected from an antecubital vein and then centrifuged. All samples were stored at ⫺70C without preservative until assayed. The concentration of free amino acids was measured as described previously.17,18 The lower limit of detection of the method was 5 mol/L for all amino acids except for arginine and tryptophan, for which it was 10 mol/L. Total protein concentration was measured by a Biuret method (Roche Diagnostics, Brussels, Belgium). Activities of ␥-glutamyl transferase (Enzyme Commission [EC] 2.3.2.2), alkaline phosphatase (EC 3.1.3.1), aspartate aminotransferase (EC 2.6.1.1), amylase (EC
58 Jauniaux et al
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3.2.1.1), and lipase (EC 3.1.1.3) were measured at 30C with commercially available kits (Roche Diagnostics). Cotinine was analyzed by a double-antibody liquidphase radioimmunoassay, as described previously.4 Intra- and interassay coefficients of variation were less than 10%, and the lower limit of detection of the assay was 25 ng/mL. Data were analyzed with a biomedical processing statistical package (Statgraphics; Manugistics, Rockville, MD). Because some distributions were skewed, the data are presented as medians and interquartile ranges. Differences in the medians of plasma amino acids between the nonsmokers and the smokers were tested by the Mann-Whitney U rank test at the 95% confidence interval. Individual correlations between the concentration of cotinine in the different compartments and the number of cigarettes smoked per day were calculated by the least-squares method, and their slopes were tested for significance by the F ratio test. Results were considered statistically significant at P ⬍ .05.
Results The median gestational age of the pregnancies in both groups was 39 weeks (interquartile range 37– 41). The female to male ratios of the fetuses were similar (11 of 13 in smokers compared with 12 of 12 in controls). The fetal weight was significantly (P ⬍ .01) lower in the smokers (3240 g; range 3000 –3600) than in the controls (3570 g; range 3300 –3940). Cotinine was detected in all maternal and fetal plasma samples from the smokers. The corresponding medians (interquartile range) were 207 ng/mL (136 – 279) in maternal venous plasma and 231 ng/mL (114 – 267) in umbilical venous plasma. The cotinine level was below the limit of detection of the assay in all samples from the nonsmokers. In smokers, positive linear correlations were found between maternal venous and umbilical venous cotinine concentrations (r ⫽ .93; F ⫽ 141; P ⬍ .001). There was no significant correlation between the maternal venous cotinine concentration and the number of cigarettes smoked per day (r ⫽ .36; F ⫽ 3.6; P ⫽ .07). Table 1 presents the median amino acid concentrations in matched samples of umbilical cord venous plasma. Compared with the nonsmokers, the smokers had significantly lower concentrations of aspartic acid, hydroxyproline, threonine, alanine, ␣-aminobutyric acid, methionine, tyrosine, phenylalanine, and lysine in the umbilical venous plasma. Serine and phenylalanine were the only amino acids in significantly lower concentration (P ⬍ .01 and P ⬍ .05, respectively) in maternal venous plasma of the smokers compared with controls. Comparison of fetal to maternal venous con-
Obstetrics & Gynecology
Table 1. Umbilical Cord Venous Plasma Amino Acid Concentrations (mol/L) in Nonsmokers and Smokers Amino acid
Nonsmokers
Smokers
Mann-Whitney rank test* P
Taurine Aspartic acid Hydroxyproline Threonine Serine Asparagine Glutamic acid Glutamine Proline Glycine Alanine Citrulline ␣-Aminobutyric acid Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Ornithine Lysine Histidine Arginine Tryptophan
166 (132–222) 29 (23–38) 22 (20 –26) 286 (254 –319) 162 (143–191) 46 (33–59) 285 (158 –365) 339 (266 – 417) 198 (170 –221) 329 (292–399) 566 (529 –769) 12 (10 –16) 18 (15–21)
144 (131–192) 22 (17–27) 18 (16 –22) 232 (213–266) 148 (127–182) 43 (35– 47) 215 (149 –270) 324 (282–376) 184 (161–201) 303 (271–328) 509 (473–572) 12 (10 –16) 11 (10 –14)
.320 ⬍.01 ⬍.05 ⬍.005 .169 .056 .113 .699 .156 .080 ⬍.05 .742 ⬍.001
207 (180 –221) 31 (28 –36) 61 (50 –71) 99 (87–126) 71 (63–75) 70 (59 –76) 177 (151–195) 386 (347– 429) 119 (108 –127) 24 (11–26) 48 (39 –55)
189 (162–205) 28 (26 –30) 61 (56 – 67) 107 (89 –117) 51 (46 – 63) 58 (53– 65) 157 (143–184) 342 (328 –382) 112 (98 –120) 24 (20 –28) 47 (40 –57)
.080 ⬍.05 .833 .981 ⬍.001 ⬍.01 .085 ⬍.05 .082 .318 .992
Data are presented as median (interquartile range). * P value for the two-sided test (U).
centration ratios of both groups (Table 2) showed no significant difference. There were no differences in maternal and umbilical vein total protein plasma concentrations or ␥-glutamyl transferase, aspartate aminotransferase, amylase, and lipase activities in smokers compared with nonsmokers (Table 3). The umbilical plasma alkaline phosphatase activity was significantly (P ⬍ .01) lower in the smokers than in the controls.
Discussion The present data confirm and expand those of previous reports showing a relation between active maternal smoking and impairment of fetoplacental development. There is a relation between fetal and placental side effects of cigarette smoke and the maternal urinary or blood level of cotinine, the main nicotine metabolite.7,19 The maternal cotinine level reflects smoking habits and thus fetal exposure more accurately than self-reported number of cigarettes smoked per day.4,20 In a previous study4 on women requesting termination of pregnancy, we reported a positive correlation between maternal blood cotinine level at 7–17 weeks and cigarette con-
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Table 2. Fetal-Maternal Venous Concentration Ratios in Nonsmokers and Smokers Amino acid
Nonsmokers
Smokers
Mann-Whitney rank test* P
Taurine Aspartic acid Hydroxyproline Threonine Serine Asparagine Glutamic acid Glutamine Proline Glycine Alanine Citrulline ␣-Aminobutyric acid Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Ornithine Lysine Histidine Arginine Tryptophan
1.9 (1.5–3.1) 1.3 (0.9 –1.6) 1.9 (1.2–2.5) 1.4 (1.3–1.5) 1.4 (1.2–1.6) 1.1 (0.9 –1.2) 2.1 (1.5–2.8) 0.9 (0.8 –1.2) 1.2 (0.9 –1.4) 1.8 (1.5–2.0) 1.5 (1.2–1.7) 0.8 (0.6 – 0.9) 1.9 (1.5–2.1)
1.8 (1.4 –2.3) 1.3 (1.1–1.7) 1.9 (1.2–2.7) 1.3 (1.1–1.4) 1.5 (1.3–1.8) 1.0 (0.9 –1.1) 2.0 (1.5–3.0) 0.9 (0.8 –1.1) 1.2 (1.1–1.3) 1.7 (1.5–2.0) 1.5 (1.2–1.6) 0.7 (0.6 – 0.9) 1.8 (1.5–1.9)
.451 .821 .687 .119 .138 .145 .979 .633 .725 .615 .546 .624 .352
1.3 (1.1–1.5) 1.7 (1.5–2.1) 1.2 (0.9 –1.5) 1.1 (0.9 –1.4) 1.6 (1.4 –1.8) 1.3 (1.1–1.6) 2.3 (2.0 –2.9) 2.3 (2.0 –2.4) 1.2 (1.1–1.3) 0.6 (0.4 – 0.8) 1.7 (1.6 –1.8)
1.2 (1.1–1.4) 1.7 (1.5–1.9) 1.2 (1.1–1.4) 1.1 (1.0 –1.3) 1.5 (1.3–1.6) 1.4 (1.2–1.6) 2.5 (2.0 –3.0) 2.3 (2.0 –2.6) 1.1 (1.0 –1.2) 0.8 (0.6 –1.2) 1.8 (1.5–2.0)
.632 .669 .881 .821 .352 .529 .546 .880 .563 .056 .743
Data are presented as median (interquartile range). * P value for the two-sided test (U).
sumption. In the present study, there was no similar relation at term. Furthermore, although the women who we investigated at 7–17 weeks4 reported that they
Table 3. Maternal and Umbilical Venous Plasma Concentrations of Total Protein and Enzyme Activities in Nonsmokers and Smokers Variable
Nonsmokers
Smokers
Maternal Total protein (g/L) 71 (68 –74) 72 (69 –73) GGT (U/L) 9 (7–13) 10 (9 –14) ALP (U/L) 342 (274 – 439) 375 (310 – 462) AST (U/L) 19 (14 –24) 18 (14 –23) Amylase (U/L) 150 (117–175) 131 (110 –162) Lipase (U/L) 30 (25– 44) 32 (25– 46) Umbilical vein Total protein (g/L) 64 (62– 68) 63 (60 – 65) GGT (U/L) 90 (49 –78) 85 (63–114) ALP (U/L) 357 (291–388) 260 (227–307) AST (U/L) 34 (30 – 41) 35 (29 – 44) Amylase (U/L) 11 (10 –17) 10 (10 –16) Lipase (U/L) 15 (12–15) 15 (13–16)
Mann-Whitney rank test P* .532 .171 .179 .978 .128 .826 .284 .854 ⬍.01 .838 .813 .227
GGT ⫽ ␥-glutamyl transferase; ALP ⫽ alkaline phosphatase; AST ⫽ aspartate aminotransferase. Data are presented as median (interquartile range). * P value for the two-sided test (U).
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Maternal Smoking and Cord Blood 59
smoked more cigarettes per day, their cotinine levels and those of their fetuses were lower than those found in this study. This can be explained by the fact that women with planned pregnancies underestimate their consumption and change their manner of smoking with advancing gestation (eg, length and depth of inhalation), especially during the third trimester, because of increased anxiety about the birth and increased immobility.20 The developing fetus is totally dependent on the supply of amino acids from the maternal blood for protein synthesis. Higher amino acid concentrations in the umbilical vein compared with maternal venous blood have been interpreted to indicate active transport systems within the placenta.21,22 These systems are functional as early as 5 weeks’ gestation.18 Using the same methodology, we have previously demonstrated that at 12–17 weeks’ gestation, the levels of serine, proline, ␣-aminobutyric acid, leucine, and arginine are lower in the fetal plasma of smokers than in nonsmokers.17 The levels of nine amino acids were found to be decreased in the current study (Table 1), confirming that tobacco exposure has a marked effect on the fetoplacental metabolism. The level of ␣-aminobutyric acid in fetal blood was decreased in similar proportions in smokers at 12–17 weeks and at term. This finding suggests that the metabolism of some amino acids is specifically altered by tobacco compounds and that this will be associated with a chronic amino acid deficit for protein synthesis by the fetus. Women who smoke have smaller infants, and their placentas demonstrate morphologic changes from as early as 9 weeks’ gestation.14 These changes occur without any alteration in placental weight,15 and thus FGR in smokers results from the combined effect of reduction in the villous capillary network and alteration of the placental cellular function. At the time of fetal blood sampling, the umbilical venous blood of growthrestricted fetuses contains lower concentrations of many amino acids.21–23 In contrast, the maternal arterial concentrations of most essential amino acids are increased in cases of FGR compared with appropriate for gestational age pregnancies.23 This results in reduced fetomaternal amino acid concentration gradients, particularly for alanine. Economides et al21 reported lower fetal venous plasma concentrations of valine, threonine, arginine, glycine, serine, taurine, proline, and asparagine in FGR. They also found a correlation between the decrease in essential amino acids and fetal hypoxemia. Cetin et al22,23 found a similar decrease in venous plasma amino acid levels in pregnancies complicated by FGR. However, they found no correlation between changes in fetomaternal ratios and the severity of FGR as evaluated by Doppler velocimetry and fetal oxygen-
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ation and acid-base balance.23 Different study designs and definitions of FGR can explain these variations. Interestingly, none of these studies reported the smoking status of the mother. In our study, we found no difference in fetomaternal venous amino acid ratios between smokers and controls (Table 2). In vitro, cigarette smoke induces the formation of new trophoblastic carriers for the uptake of ␣-aminobutyric acid, suggesting that part of the fetal amino acid deficit induced by maternal smoking may be compensated for by the induction of new amino acid transport systems.24 Our data suggest that this is a temporary phenomenon and indicate that chronic maternal smoking induces major alterations in the placental metabolism of some amino acids from as early as the first month of pregnancy. Several authors have demonstrated that tobacco compounds such as cadmium or DNA adducts have an effect in vitro on placental enzymes involved in xenobiotic metabolism.25,26 In first- and third-trimester placental explants exposed to tobacco xenobiotics, enzymes such as quinone reductase and catecholamine-omethyltransferase demonstrated increased activity. This suggests that placental tissue can inactivate these carcinogens or mutagens, thus limiting their transfer to the fetus. In adults, smoking enhances the secretion of amylase by the exocrine pancreas.27 At 12–17 weeks’ gestation, amylase activity is increased in the fetal plasma of mothers who smoke as compared with nonsmokers.17 At the same stage of gestation, fetal plasma alkaline phosphatase activity is also increased in smokers, but not significantly. At term, we found no increase in fetal blood amylase activity, but alkaline phosphatase activity was significantly decreased in chronic smokers compared with controls (Table 3). These data suggest that early in pregnancy, maternal smoking induces an increase in enzymatic activity in the fetoplacental unit, whereas chronic exposure throughout pregnancy impairs placental enzymatic activity. This also suggests that chronic maternal smoking progressively diminishes placental defense mechanisms. Thus, as pregnancy advances, the placenta may lose its potential to inactivate carcinogens locally and to regulate the transfer of metabolized toxic agents into the fetal compartment. Our biochemical investigations have confirmed disturbances in placental function associated with chronic maternal cigarette smoking, the most important of which are reductions in the metabolism of ␣-aminobutyric acid and alkaline phosphatase activity. This may contribute to FGR observed in mothers who smoke, and it also indicates that the overall placental metabolic and xenobiotic capability in response to chronic tobacco smoke exposure is limited.
Obstetrics & Gynecology
References 1. Lambers DS, Clark KE. The maternal and fetal physiologic effects of nicotine. Semin Perinatol 1996;20:115–26. 2. Copius Peereboom-Stegeman JHJ, van der Velde WJ, Dessing JWM. Influence of cadmium on placental structure. Ecotoxicol Environ Saf 1983;7:79 – 86. 3. Boadi WY, Urbach J, Brandes JM, Yannai S. Effect of cadmium on some enzyme activities in first-trimester human placenta. Toxicol Lett 1992;60:155– 64. 4. Jauniaux E, Gulbis B, Acharya G, Thiry P, Rodeck C. Maternal tobacco exposure and cotinine levels in fetal fluids in the first half of pregnancy. Obstet Gynecol 1999;93:25–9. 5. Milunsky A, Carmella SG, Ye M, Hecht SS. A tobacco-specific carcinogen in the fetus. Prenat Diagn 2000;20:307–10. 6. Armstrong BG, McDonald AD, Sloan M. Cigarette, alcohol and coffee consumption and spontaneous abortion. Am J Public Health 1992;82:85–7. 7. Shah NR, Bracken MB. A systematic review and meta-analysis of prospective studies on the association between maternal cigarette smoking and preterm delivery. Am J Obstet Gynecol 2000;182:465– 72. 8. Burton GJ. The effects of maternal cigarette smoking on placental structure and function in mid- to late gestation. In: Poswillo D, Alberman E, eds. Effects of smoking on the fetus, neonate and child. Oxford: Oxford Medical Publications, 1992:60 – 80. 9. Hansen C, Sorensen LD, Asmussen I, Autrup H. Transplacental exposure to tobacco smoke in human-adduct formation in placenta and umbilical cord vessels. Teratog Carcinog Mutagen 1992;12:51– 60. 10. Genbacev O, Bass KE, Joslin RJ, Fisher SJ. Maternal smoking inhibits early human cytotrophoblast differentiation. Reprod Toxicol 1995;9:245–55. 11. Daube H, Sherer G, Riedel K, Ruppert T, Tricker AR, Rosenbaum P, et al. DNA adducts in human placenta in relation to tobacco smoke exposure and plasma antioxidant status. J Cancer Res Clin Oncol 1997;123:141–51. 12. Finette BA, O’Neill JP, Vacek PM, Albertini RJ. Gene mutations with characteristic deletions in cord blood T lymphocytes associated with passive maternal exposure to tobacco smoke. Nat Med 1998;10:1144 –51. 13. Zenzes MT, Puy LA, Bielecki R, Reed TE. Detection of benzo[␣]pyrene diol epoxide-DNA adducts in embryos from smoking couples: Evidence for transmission by spermatozoa. Mol Hum Reprod 1999;5:125–31. 14. Jauniaux E, Burton GJ. The effect of smoking in pregnancy on early placental morphology. Obstet Gynecol 1992;79:645– 8. 15. Bush PG, Mayhew TM, Abramovich DR, Aggett PJ, Burke MD, Page KR. A quantitative study on the effects of maternal smoking on placental morphology and cadmium concentration. Placenta 2000;21:247–56. 16. Sastry BV. Placental toxicology: Tobacco smoke, abused drugs, multiple chemical interactions and placental function. Reprod Fertil Dev 1991;3:355–72. 17. Jauniaux E, Gulbis B, Acharya G, Gerlo E. Fetal amino acid and
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18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
enzyme levels with maternal smoking. Obstet Gynecol 1999;93: 680 –3. Jauniaux E, Gulbis B, Gerlo E, Rodeck C. Free amino acid distribution inside the first trimester human gestational sac. Early Hum Dev 1998;51:159 – 69. Ellard GA, Johnstone FD, Prescott RJ, Ji-Xian W, Jian-Hua M. Smoking during pregnancy: The dose dependence of birthweight deficits. Br J Obstet Gynaecol 1996;103:806 –13. Cope G, Nayyar P, Holder R, Gibbons J, Bunce R. A simple near-patient test for nicotine and its metabolite in urine to assess smoking habit. Clin Chim Acta 1996;256:135– 49. Economides DL, Nicolaides KH, Gahl WA, Bernardi I, Evans MI. Plasma amino acids in appropriate- and small-for-gestational age fetuses. Am J Obstet Gynecol 1989;161:1219 –27. Cetin I, Corbetta C, Serini L, Marconi A, Bozzetti P, Pardi G, et al. Umbilical amino acid concentrations in normal and growth retarded fetuses sampled in utero by cordocentesis. Am J Obstet Gynecol 1990;162:253– 61. Cetin I, Ronzoni S, Marconi AM, Perugino G, Corbetta C, Battaglia FC, et al. Maternal concentrations and fetal-maternal concentration differences of plasma amino acids in normal and intrauterine growth-restricted pregnancies. Am J Obstet Gynecol 1996;174: 1575– 83. Sastry BV, Horst MA, Naukam RJ. Maternal tobacco smoking and changes in amino acid uptake by human placental villi: Induction of uptake systems, gammaglutamyltranspeptidase and membrane fluidity. Placenta 1989;10:345–58. Barnea ER. Modulatory effect of maternal serum on xenobiotic metabolizing activity of placental explants: Modification by cigarette smoking. Hum Reprod 1994;9:1017–21. Sanyal MK, Li YL, Blanger K. Metabolism of polynuclear aromatic hydrocarbon in human placenta influenced by cigarette smoke exposure. Reprod Toxicol 1994;5:411– 8. Dubick MA, Conteas CN, Billy HT, Majumdar AP, Geokas MC. Raised serum concentrations of pancreatic enzymes in cigarette smokers. Gut 1987;28:330 –5.
Address reprint requests to:
Eric Jauniaux, MD, PhD Academic Department of Obstetrics and Gynaecology University College London Medical School 86-96 Chenies Mews London WC1E 6HX United Kingdom E-mail: [email protected]
Received May 15, 2000. Received in revised form July 20, 2000. Accepted August 10, 2000. Copyright © 2001 by The American College of Obstetricians and Gynecologists. Published by Elsevier Science Inc.
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Maternal Smoking and Cord Blood 61
From the Academic Departments of Obstetrics and Gynaecology, University College London, London, United Kingdom; the Departments of Clinical Chemistry and Obstetrics and Gynecology, Academic Hospital Erasme, Universite´ Libre de Bruxelles, Brussels, Belgium; and the Department of Clinical Chemistry, Academisch Ziekenhuis, Vrije Universiteit van Brussels, Brussels, Belgium. Supported by a grant from the David & Alice Van Buuren Foundation (Universite´ Libre de Bruxelles, Brussels, Belgium) and a grant from the Fonds National de la Recherche Scientifique (no. 9.4523.97).
VOL. 97, NO. 1, JANUARY 2001
Tobacco smoke compounds such as nicotine and carbon monoxide have been shown to act indirectly on the fetus through uteroplacental vasoconstriction.1 The placental structure seems to be extremely vulnerable to smoke toxins such as cadmium, which damage its vascular system2 and inhibit some of its enzyme activities.3 Nicotine, its metabolites, and most tobacco carcinogens and teratogens have low molecular weights and high water solubility and therefore readily cross the placenta. Cotinine has been found to accumulate in the fetal compartment as early as 7 weeks’ gestation in both active and passive smokers.4 Recently, a tobaccospecific carcinogen and its metabolite have been demonstrated in amniotic fluid samples of smokers at 16 –21 weeks.5 Commonly reported adverse perinatal outcomes of maternal cigarette smoking during pregnancy include an increased miscarriage rate6 and a two-fold increase in the risk of delivering a low birth weight infant because of prematurity and/or fetal growth restriction (FGR).1,7 These effects are linked to alterations in placental structure and function induced by some of the many tobacco smoke compounds, in particular DNA adduct.8 –11 The findings that these carcinogens induce gene mutations in utero12 and that modified DNA can be transmitted to embryos by spermatozoa13 have recently received widespread interest. Active smoking during pregnancy is associated in all trimesters with placental ultrastructural lesions, including a decrease in syncytiotrophoblast microvilli and pinocytotic activity, focal syncytial necrosis, and degeneration of cytoplasmic organelles.8,14,15 These morphologic alterations are irreversible and are associated with a direct effect on the placental metabolic and transfer functions.16 We have recently reported that the fetal plasma levels of some amino acids and amylase at 12–17 weeks are modified by maternal smoking.17 In the present study, we further evaluated the effect of chronic maternal smoking throughout pregnancy on the levels
0029-7844/01/$20.00 PII S0029-7844(00)01108-X
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of free amino acids and enzyme activity in umbilical cord plasma collected at term.
Materials and Methods Samples of fetal cord and maternal venous blood were obtained from 48 women at 37– 40 weeks’ gestation. The study included a consecutive series of 24 chronic smokers, who smoked three to 20 cigarettes per day (median 11.5 cigarettes per day), and a series of 24 nonsmokers prospectively collected over a 6-month period in the same population living in the same area. All women were white, 21– 40 years of age (median 27.5 years), had no medical history, and attended the antenatal clinic at Erasme Academic Hospital. Gestational age was determined from the date of the last menstrual period and confirmed by ultrasound measurement of the crownrump length between 9 and 14 weeks’ gestation. Exposure to cigarette smoke is currently determined in our clinic by a questionnaire that is distributed at the first visit to all women attending the antenatal clinic. All women are encouraged to avoid cigarette smoke and are provided with the same counseling on nutrition. The groups included only pregnancies after 36 weeks that had been uncomplicated. Each study case was matched prospectively with a control, who was the next woman to deliver. Forty pairs were initially enrolled in this study. Passive smokers (n ⫽ 10) were excluded as controls before delivery when their urine cotinine level did not corroborate the self-reported exposure level.4 Six pairs were excluded at the time of delivery because either the study case or the corresponding control had a complicated delivery. Collection of umbilical cord blood for research has been approved by the Erasme Academic Hospital and University College Hospitals Committees on the Ethics of Human Research. Both departments are conducting ongoing research projects on cord blood. After written informed consent, 3.0 –5.0 mL of cord blood was aspirated from the vein into preheparinized syringes. Simultaneously, maternal blood samples were collected from an antecubital vein and then centrifuged. All samples were stored at ⫺70C without preservative until assayed. The concentration of free amino acids was measured as described previously.17,18 The lower limit of detection of the method was 5 mol/L for all amino acids except for arginine and tryptophan, for which it was 10 mol/L. Total protein concentration was measured by a Biuret method (Roche Diagnostics, Brussels, Belgium). Activities of ␥-glutamyl transferase (Enzyme Commission [EC] 2.3.2.2), alkaline phosphatase (EC 3.1.3.1), aspartate aminotransferase (EC 2.6.1.1), amylase (EC
58 Jauniaux et al
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3.2.1.1), and lipase (EC 3.1.1.3) were measured at 30C with commercially available kits (Roche Diagnostics). Cotinine was analyzed by a double-antibody liquidphase radioimmunoassay, as described previously.4 Intra- and interassay coefficients of variation were less than 10%, and the lower limit of detection of the assay was 25 ng/mL. Data were analyzed with a biomedical processing statistical package (Statgraphics; Manugistics, Rockville, MD). Because some distributions were skewed, the data are presented as medians and interquartile ranges. Differences in the medians of plasma amino acids between the nonsmokers and the smokers were tested by the Mann-Whitney U rank test at the 95% confidence interval. Individual correlations between the concentration of cotinine in the different compartments and the number of cigarettes smoked per day were calculated by the least-squares method, and their slopes were tested for significance by the F ratio test. Results were considered statistically significant at P ⬍ .05.
Results The median gestational age of the pregnancies in both groups was 39 weeks (interquartile range 37– 41). The female to male ratios of the fetuses were similar (11 of 13 in smokers compared with 12 of 12 in controls). The fetal weight was significantly (P ⬍ .01) lower in the smokers (3240 g; range 3000 –3600) than in the controls (3570 g; range 3300 –3940). Cotinine was detected in all maternal and fetal plasma samples from the smokers. The corresponding medians (interquartile range) were 207 ng/mL (136 – 279) in maternal venous plasma and 231 ng/mL (114 – 267) in umbilical venous plasma. The cotinine level was below the limit of detection of the assay in all samples from the nonsmokers. In smokers, positive linear correlations were found between maternal venous and umbilical venous cotinine concentrations (r ⫽ .93; F ⫽ 141; P ⬍ .001). There was no significant correlation between the maternal venous cotinine concentration and the number of cigarettes smoked per day (r ⫽ .36; F ⫽ 3.6; P ⫽ .07). Table 1 presents the median amino acid concentrations in matched samples of umbilical cord venous plasma. Compared with the nonsmokers, the smokers had significantly lower concentrations of aspartic acid, hydroxyproline, threonine, alanine, ␣-aminobutyric acid, methionine, tyrosine, phenylalanine, and lysine in the umbilical venous plasma. Serine and phenylalanine were the only amino acids in significantly lower concentration (P ⬍ .01 and P ⬍ .05, respectively) in maternal venous plasma of the smokers compared with controls. Comparison of fetal to maternal venous con-
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Table 1. Umbilical Cord Venous Plasma Amino Acid Concentrations (mol/L) in Nonsmokers and Smokers Amino acid
Nonsmokers
Smokers
Mann-Whitney rank test* P
Taurine Aspartic acid Hydroxyproline Threonine Serine Asparagine Glutamic acid Glutamine Proline Glycine Alanine Citrulline ␣-Aminobutyric acid Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Ornithine Lysine Histidine Arginine Tryptophan
166 (132–222) 29 (23–38) 22 (20 –26) 286 (254 –319) 162 (143–191) 46 (33–59) 285 (158 –365) 339 (266 – 417) 198 (170 –221) 329 (292–399) 566 (529 –769) 12 (10 –16) 18 (15–21)
144 (131–192) 22 (17–27) 18 (16 –22) 232 (213–266) 148 (127–182) 43 (35– 47) 215 (149 –270) 324 (282–376) 184 (161–201) 303 (271–328) 509 (473–572) 12 (10 –16) 11 (10 –14)
.320 ⬍.01 ⬍.05 ⬍.005 .169 .056 .113 .699 .156 .080 ⬍.05 .742 ⬍.001
207 (180 –221) 31 (28 –36) 61 (50 –71) 99 (87–126) 71 (63–75) 70 (59 –76) 177 (151–195) 386 (347– 429) 119 (108 –127) 24 (11–26) 48 (39 –55)
189 (162–205) 28 (26 –30) 61 (56 – 67) 107 (89 –117) 51 (46 – 63) 58 (53– 65) 157 (143–184) 342 (328 –382) 112 (98 –120) 24 (20 –28) 47 (40 –57)
.080 ⬍.05 .833 .981 ⬍.001 ⬍.01 .085 ⬍.05 .082 .318 .992
Data are presented as median (interquartile range). * P value for the two-sided test (U).
centration ratios of both groups (Table 2) showed no significant difference. There were no differences in maternal and umbilical vein total protein plasma concentrations or ␥-glutamyl transferase, aspartate aminotransferase, amylase, and lipase activities in smokers compared with nonsmokers (Table 3). The umbilical plasma alkaline phosphatase activity was significantly (P ⬍ .01) lower in the smokers than in the controls.
Discussion The present data confirm and expand those of previous reports showing a relation between active maternal smoking and impairment of fetoplacental development. There is a relation between fetal and placental side effects of cigarette smoke and the maternal urinary or blood level of cotinine, the main nicotine metabolite.7,19 The maternal cotinine level reflects smoking habits and thus fetal exposure more accurately than self-reported number of cigarettes smoked per day.4,20 In a previous study4 on women requesting termination of pregnancy, we reported a positive correlation between maternal blood cotinine level at 7–17 weeks and cigarette con-
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Table 2. Fetal-Maternal Venous Concentration Ratios in Nonsmokers and Smokers Amino acid
Nonsmokers
Smokers
Mann-Whitney rank test* P
Taurine Aspartic acid Hydroxyproline Threonine Serine Asparagine Glutamic acid Glutamine Proline Glycine Alanine Citrulline ␣-Aminobutyric acid Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Ornithine Lysine Histidine Arginine Tryptophan
1.9 (1.5–3.1) 1.3 (0.9 –1.6) 1.9 (1.2–2.5) 1.4 (1.3–1.5) 1.4 (1.2–1.6) 1.1 (0.9 –1.2) 2.1 (1.5–2.8) 0.9 (0.8 –1.2) 1.2 (0.9 –1.4) 1.8 (1.5–2.0) 1.5 (1.2–1.7) 0.8 (0.6 – 0.9) 1.9 (1.5–2.1)
1.8 (1.4 –2.3) 1.3 (1.1–1.7) 1.9 (1.2–2.7) 1.3 (1.1–1.4) 1.5 (1.3–1.8) 1.0 (0.9 –1.1) 2.0 (1.5–3.0) 0.9 (0.8 –1.1) 1.2 (1.1–1.3) 1.7 (1.5–2.0) 1.5 (1.2–1.6) 0.7 (0.6 – 0.9) 1.8 (1.5–1.9)
.451 .821 .687 .119 .138 .145 .979 .633 .725 .615 .546 .624 .352
1.3 (1.1–1.5) 1.7 (1.5–2.1) 1.2 (0.9 –1.5) 1.1 (0.9 –1.4) 1.6 (1.4 –1.8) 1.3 (1.1–1.6) 2.3 (2.0 –2.9) 2.3 (2.0 –2.4) 1.2 (1.1–1.3) 0.6 (0.4 – 0.8) 1.7 (1.6 –1.8)
1.2 (1.1–1.4) 1.7 (1.5–1.9) 1.2 (1.1–1.4) 1.1 (1.0 –1.3) 1.5 (1.3–1.6) 1.4 (1.2–1.6) 2.5 (2.0 –3.0) 2.3 (2.0 –2.6) 1.1 (1.0 –1.2) 0.8 (0.6 –1.2) 1.8 (1.5–2.0)
.632 .669 .881 .821 .352 .529 .546 .880 .563 .056 .743
Data are presented as median (interquartile range). * P value for the two-sided test (U).
sumption. In the present study, there was no similar relation at term. Furthermore, although the women who we investigated at 7–17 weeks4 reported that they
Table 3. Maternal and Umbilical Venous Plasma Concentrations of Total Protein and Enzyme Activities in Nonsmokers and Smokers Variable
Nonsmokers
Smokers
Maternal Total protein (g/L) 71 (68 –74) 72 (69 –73) GGT (U/L) 9 (7–13) 10 (9 –14) ALP (U/L) 342 (274 – 439) 375 (310 – 462) AST (U/L) 19 (14 –24) 18 (14 –23) Amylase (U/L) 150 (117–175) 131 (110 –162) Lipase (U/L) 30 (25– 44) 32 (25– 46) Umbilical vein Total protein (g/L) 64 (62– 68) 63 (60 – 65) GGT (U/L) 90 (49 –78) 85 (63–114) ALP (U/L) 357 (291–388) 260 (227–307) AST (U/L) 34 (30 – 41) 35 (29 – 44) Amylase (U/L) 11 (10 –17) 10 (10 –16) Lipase (U/L) 15 (12–15) 15 (13–16)
Mann-Whitney rank test P* .532 .171 .179 .978 .128 .826 .284 .854 ⬍.01 .838 .813 .227
GGT ⫽ ␥-glutamyl transferase; ALP ⫽ alkaline phosphatase; AST ⫽ aspartate aminotransferase. Data are presented as median (interquartile range). * P value for the two-sided test (U).
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Maternal Smoking and Cord Blood 59
smoked more cigarettes per day, their cotinine levels and those of their fetuses were lower than those found in this study. This can be explained by the fact that women with planned pregnancies underestimate their consumption and change their manner of smoking with advancing gestation (eg, length and depth of inhalation), especially during the third trimester, because of increased anxiety about the birth and increased immobility.20 The developing fetus is totally dependent on the supply of amino acids from the maternal blood for protein synthesis. Higher amino acid concentrations in the umbilical vein compared with maternal venous blood have been interpreted to indicate active transport systems within the placenta.21,22 These systems are functional as early as 5 weeks’ gestation.18 Using the same methodology, we have previously demonstrated that at 12–17 weeks’ gestation, the levels of serine, proline, ␣-aminobutyric acid, leucine, and arginine are lower in the fetal plasma of smokers than in nonsmokers.17 The levels of nine amino acids were found to be decreased in the current study (Table 1), confirming that tobacco exposure has a marked effect on the fetoplacental metabolism. The level of ␣-aminobutyric acid in fetal blood was decreased in similar proportions in smokers at 12–17 weeks and at term. This finding suggests that the metabolism of some amino acids is specifically altered by tobacco compounds and that this will be associated with a chronic amino acid deficit for protein synthesis by the fetus. Women who smoke have smaller infants, and their placentas demonstrate morphologic changes from as early as 9 weeks’ gestation.14 These changes occur without any alteration in placental weight,15 and thus FGR in smokers results from the combined effect of reduction in the villous capillary network and alteration of the placental cellular function. At the time of fetal blood sampling, the umbilical venous blood of growthrestricted fetuses contains lower concentrations of many amino acids.21–23 In contrast, the maternal arterial concentrations of most essential amino acids are increased in cases of FGR compared with appropriate for gestational age pregnancies.23 This results in reduced fetomaternal amino acid concentration gradients, particularly for alanine. Economides et al21 reported lower fetal venous plasma concentrations of valine, threonine, arginine, glycine, serine, taurine, proline, and asparagine in FGR. They also found a correlation between the decrease in essential amino acids and fetal hypoxemia. Cetin et al22,23 found a similar decrease in venous plasma amino acid levels in pregnancies complicated by FGR. However, they found no correlation between changes in fetomaternal ratios and the severity of FGR as evaluated by Doppler velocimetry and fetal oxygen-
60 Jauniaux et al
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ation and acid-base balance.23 Different study designs and definitions of FGR can explain these variations. Interestingly, none of these studies reported the smoking status of the mother. In our study, we found no difference in fetomaternal venous amino acid ratios between smokers and controls (Table 2). In vitro, cigarette smoke induces the formation of new trophoblastic carriers for the uptake of ␣-aminobutyric acid, suggesting that part of the fetal amino acid deficit induced by maternal smoking may be compensated for by the induction of new amino acid transport systems.24 Our data suggest that this is a temporary phenomenon and indicate that chronic maternal smoking induces major alterations in the placental metabolism of some amino acids from as early as the first month of pregnancy. Several authors have demonstrated that tobacco compounds such as cadmium or DNA adducts have an effect in vitro on placental enzymes involved in xenobiotic metabolism.25,26 In first- and third-trimester placental explants exposed to tobacco xenobiotics, enzymes such as quinone reductase and catecholamine-omethyltransferase demonstrated increased activity. This suggests that placental tissue can inactivate these carcinogens or mutagens, thus limiting their transfer to the fetus. In adults, smoking enhances the secretion of amylase by the exocrine pancreas.27 At 12–17 weeks’ gestation, amylase activity is increased in the fetal plasma of mothers who smoke as compared with nonsmokers.17 At the same stage of gestation, fetal plasma alkaline phosphatase activity is also increased in smokers, but not significantly. At term, we found no increase in fetal blood amylase activity, but alkaline phosphatase activity was significantly decreased in chronic smokers compared with controls (Table 3). These data suggest that early in pregnancy, maternal smoking induces an increase in enzymatic activity in the fetoplacental unit, whereas chronic exposure throughout pregnancy impairs placental enzymatic activity. This also suggests that chronic maternal smoking progressively diminishes placental defense mechanisms. Thus, as pregnancy advances, the placenta may lose its potential to inactivate carcinogens locally and to regulate the transfer of metabolized toxic agents into the fetal compartment. Our biochemical investigations have confirmed disturbances in placental function associated with chronic maternal cigarette smoking, the most important of which are reductions in the metabolism of ␣-aminobutyric acid and alkaline phosphatase activity. This may contribute to FGR observed in mothers who smoke, and it also indicates that the overall placental metabolic and xenobiotic capability in response to chronic tobacco smoke exposure is limited.
Obstetrics & Gynecology
References 1. Lambers DS, Clark KE. The maternal and fetal physiologic effects of nicotine. Semin Perinatol 1996;20:115–26. 2. Copius Peereboom-Stegeman JHJ, van der Velde WJ, Dessing JWM. Influence of cadmium on placental structure. Ecotoxicol Environ Saf 1983;7:79 – 86. 3. Boadi WY, Urbach J, Brandes JM, Yannai S. Effect of cadmium on some enzyme activities in first-trimester human placenta. Toxicol Lett 1992;60:155– 64. 4. Jauniaux E, Gulbis B, Acharya G, Thiry P, Rodeck C. Maternal tobacco exposure and cotinine levels in fetal fluids in the first half of pregnancy. Obstet Gynecol 1999;93:25–9. 5. Milunsky A, Carmella SG, Ye M, Hecht SS. A tobacco-specific carcinogen in the fetus. Prenat Diagn 2000;20:307–10. 6. Armstrong BG, McDonald AD, Sloan M. Cigarette, alcohol and coffee consumption and spontaneous abortion. Am J Public Health 1992;82:85–7. 7. Shah NR, Bracken MB. A systematic review and meta-analysis of prospective studies on the association between maternal cigarette smoking and preterm delivery. Am J Obstet Gynecol 2000;182:465– 72. 8. Burton GJ. The effects of maternal cigarette smoking on placental structure and function in mid- to late gestation. In: Poswillo D, Alberman E, eds. Effects of smoking on the fetus, neonate and child. Oxford: Oxford Medical Publications, 1992:60 – 80. 9. Hansen C, Sorensen LD, Asmussen I, Autrup H. Transplacental exposure to tobacco smoke in human-adduct formation in placenta and umbilical cord vessels. Teratog Carcinog Mutagen 1992;12:51– 60. 10. Genbacev O, Bass KE, Joslin RJ, Fisher SJ. Maternal smoking inhibits early human cytotrophoblast differentiation. Reprod Toxicol 1995;9:245–55. 11. Daube H, Sherer G, Riedel K, Ruppert T, Tricker AR, Rosenbaum P, et al. DNA adducts in human placenta in relation to tobacco smoke exposure and plasma antioxidant status. J Cancer Res Clin Oncol 1997;123:141–51. 12. Finette BA, O’Neill JP, Vacek PM, Albertini RJ. Gene mutations with characteristic deletions in cord blood T lymphocytes associated with passive maternal exposure to tobacco smoke. Nat Med 1998;10:1144 –51. 13. Zenzes MT, Puy LA, Bielecki R, Reed TE. Detection of benzo[␣]pyrene diol epoxide-DNA adducts in embryos from smoking couples: Evidence for transmission by spermatozoa. Mol Hum Reprod 1999;5:125–31. 14. Jauniaux E, Burton GJ. The effect of smoking in pregnancy on early placental morphology. Obstet Gynecol 1992;79:645– 8. 15. Bush PG, Mayhew TM, Abramovich DR, Aggett PJ, Burke MD, Page KR. A quantitative study on the effects of maternal smoking on placental morphology and cadmium concentration. Placenta 2000;21:247–56. 16. Sastry BV. Placental toxicology: Tobacco smoke, abused drugs, multiple chemical interactions and placental function. Reprod Fertil Dev 1991;3:355–72. 17. Jauniaux E, Gulbis B, Acharya G, Gerlo E. Fetal amino acid and
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18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
enzyme levels with maternal smoking. Obstet Gynecol 1999;93: 680 –3. Jauniaux E, Gulbis B, Gerlo E, Rodeck C. Free amino acid distribution inside the first trimester human gestational sac. Early Hum Dev 1998;51:159 – 69. Ellard GA, Johnstone FD, Prescott RJ, Ji-Xian W, Jian-Hua M. Smoking during pregnancy: The dose dependence of birthweight deficits. Br J Obstet Gynaecol 1996;103:806 –13. Cope G, Nayyar P, Holder R, Gibbons J, Bunce R. A simple near-patient test for nicotine and its metabolite in urine to assess smoking habit. Clin Chim Acta 1996;256:135– 49. Economides DL, Nicolaides KH, Gahl WA, Bernardi I, Evans MI. Plasma amino acids in appropriate- and small-for-gestational age fetuses. Am J Obstet Gynecol 1989;161:1219 –27. Cetin I, Corbetta C, Serini L, Marconi A, Bozzetti P, Pardi G, et al. Umbilical amino acid concentrations in normal and growth retarded fetuses sampled in utero by cordocentesis. Am J Obstet Gynecol 1990;162:253– 61. Cetin I, Ronzoni S, Marconi AM, Perugino G, Corbetta C, Battaglia FC, et al. Maternal concentrations and fetal-maternal concentration differences of plasma amino acids in normal and intrauterine growth-restricted pregnancies. Am J Obstet Gynecol 1996;174: 1575– 83. Sastry BV, Horst MA, Naukam RJ. Maternal tobacco smoking and changes in amino acid uptake by human placental villi: Induction of uptake systems, gammaglutamyltranspeptidase and membrane fluidity. Placenta 1989;10:345–58. Barnea ER. Modulatory effect of maternal serum on xenobiotic metabolizing activity of placental explants: Modification by cigarette smoking. Hum Reprod 1994;9:1017–21. Sanyal MK, Li YL, Blanger K. Metabolism of polynuclear aromatic hydrocarbon in human placenta influenced by cigarette smoke exposure. Reprod Toxicol 1994;5:411– 8. Dubick MA, Conteas CN, Billy HT, Majumdar AP, Geokas MC. Raised serum concentrations of pancreatic enzymes in cigarette smokers. Gut 1987;28:330 –5.
Address reprint requests to:
Eric Jauniaux, MD, PhD Academic Department of Obstetrics and Gynaecology University College London Medical School 86-96 Chenies Mews London WC1E 6HX United Kingdom E-mail: [email protected]
Received May 15, 2000. Received in revised form July 20, 2000. Accepted August 10, 2000. Copyright © 2001 by The American College of Obstetricians and Gynecologists. Published by Elsevier Science Inc.
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