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Chronic Maternal Smoking and Cord Blood Amino Acid and Enzyme Levels at Term

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, Université Libre de Bruxelles, Brussels, Belgium; and the Department of Clinical Chemistry, Academisch Ziekenhuis, Vrije Universiteit van Brussels, Brussels, Belgium.

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: e.jauniaux{at}ucl.ac.uk

	Abstract

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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.

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 tobacco-specific 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 of free amino acids and enzyme activity in umbilical cord plasma collected at term.

	Materials and Methods

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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 µmol/L for all amino acids except for arginine and tryptophan, for which it was 10 µmol/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 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 liquid-phase 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

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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 concentration ratios of both groups (Table 2) showed no significant difference.

	Discussion

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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 consumption. 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 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 growth-restricted 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 al^22,23 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 oxygenation 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-o-methyltransferase 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 non-smokers.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.

	Footnotes

 
Supported by a grant from the David & Alice Van Buuren Foundation (Université Libre de Bruxelles, Brussels, Belgium) and a grant from the Fonds National de la Recherche Scientifique (no. 9.4523.97).

PII S0029-7844(00)01108-X

Received May 15, 2000. Received in revised form July 20, 2000. Accepted August 10, 2000.

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