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Fetal Amino Acid and Enzyme Levels With Maternal Smoking

From the Academic Department of Obstetrics and Gynaecology, University College London, London, United Kingdom; Department of Clinical Chemistry, Academic Hospital Erasme, Université Libre de Bruxelles; and Department of Clinical Chemistry, Academisch Ziekenhuis, Vrije Universiteit Brussel, Brussels, Belgium.

Address reprint requests to: Eric Jauniaux, MD, PhD Academic Department of Obstetrics and Gynecology University College of 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 active maternal smoking on fetal amino acid and enzyme levels in early pregnancy.

Methods: The concentrations of 23 free amino acids and total protein, and the activity levels of four enzymes were measured in samples of maternal and fetal plasma from nine nonsmokers who were not exposed to tobacco smoke and nine long-term, heavy smokers matched for gestational age. To determine fetal exposure to smoking, cotinine levels were measured in maternal and fetal plasma and fetal liver samples from both groups. The pregnancies were between 12 and 17 weeks’ gestation.

Results: In women who smoke, the median cotinine concentrations were 156 mg/mL in maternal plasma and 89 ng/mL in fetal plasma, but only one fetal liver sample contained detectable cotinine. Significantly lower concentrations of serine, proline, -aminobutyric acid, leucine, and arginine were found in smokers compared with nonsmokers, with the lowest in arginine. Fetal plasma amylase activity was significantly higher in smokers than controls. There were no differences in concentrations of other amino acids or activity levels of other enzymes in the two groups.

Conclusion: Maternal smoking affected placental and fetal protein metabolism and enzyme activity from at least 12 weeks’ gestation. That finding indicates that high levels of tobacco exposure in the first trimester might cause irreversible changes in the cellular functions of the villous trophoblastic barrier.

Cigarette smoking during pregnancy is associated with fetal side effects in every trimester. The perinatal outcomes of women who smoke during pregnancy are poorer than those of nonsmokers, in a dose-dependent manner.¹ In the first trimester, maternal smoking increases the risk of spontaneous abortion by 33% over that of nonsmoking controls. In the third trimester, women who smoke have a twofold higher risk of delivering a low birth weight infant, due to prematurity or poor fetal growth, with a resultant increase of 33% in perinatal mortality.¹

It is not known which tobacco components contribute to reduction in birth weight. In humans and experimental animals, tobacco-smoke compounds, such as nicotine and carbon monoxide, were shown to act indirectly on the fetus through uteroplacental vasoconstriction and directly by crossing the placenta and entering fetal tissues.^2,3 However, there is little evidence of decreased uterine blood flow in response to nicotine in humans.³ Nicotine and its main metabolite, cotinine, cross the placenta easily and accumulate in fetal fluid and tissues from the first month of pregnancy but do not have a distinctive effect on fetal cells.^2–6 Carbon monoxide has a strong affinity for fetal hemoglobin, resulting in higher fetal levels of carboxyhemoglobin than maternal levels,³ which shifts the oxygen-hemoglobin saturation to the right, reducing oxygen delivery and release into fetal tissues.

The toxicity of tobacco carcinogenic and teratogenic agents on placental and fetal cells varies considerably.^5,6 Placental structure is vulnerable to smoke toxins such as cadmium, which damage its vascular system⁷ and inhibit some of its enzyme activities,⁸ but little is known about the effect of those toxins on fetal cellular metabolism. In the present study, we evaluated the effect of active maternal smoking on fetal plasma levels of free amino acids and enzymes in early pregnancy.

	Material and Methods

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Samples of fetal blood, liver tissue, and maternal venous blood were collected from 18 healthy women at 12–17 weeks’ gestation during surgical termination (dilation and evacuation) of pregnancy for psychologic reasons under general anesthesia. At University College London, women were enrolled in an ongoing research program on placental drug transfer during pregnancy, approved by the University College London Hospitals Committee on the Ethics of Human Research. Demographic characteristics of the study population were reported previously.⁴ The study groups included a consecutive series of nine long-term, heavy smokers (15–25 cigarettes per day) and a concurrent series of nine nonsmokers. Exposure to cigarette smoke was determined by a questionnaire distributed to all women at the hospital. The groups included only pregnancies that had been uncomplicated and were retrospectively matched for gestational age after those exposed to passive smoke had been excluded, ie, when urine cotinine level did not corroborate self-reported exposure level.⁴ Gestational age was determined from date of last menstrual period and confirmed by ultrasound measurement of biparietal diameter.

After written informed consent was obtained, 2.0–3.0 mL of pure fetal blood was aspirated by ultrasound-guided transabdominal puncture of the fetal heart using a 22-gauge needle. Simultaneously, maternal blood samples were collected from an antecubital vein and centrifuged. All procedures were done between 10:00 and 12:00 AM, after an overnight fast of 12 hours, and all samples were stored at -70C without preservative until assayed.

The concentration of free amino acids was measured as previously reported.⁹ 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. A dyebinding method (Sopachem cat. 003. 0309. 02, Sopar Biochem, Brussels, Belgium) was used to measure total protein concentrations by spectrophotometry. Gamma-glutamyl transferase, alkaline phosphatase, aspartate amniotransferase, and amylase activity were measured at 30C, with commercially available kits (Boehringer Mannheim, Mannheim, Germany). All measurements were done on the Hitachi 717 automatic analyzer (Hitachi, Tokyo, Japan). The lower limit of detection of the enzyme assays was 5 U/L. Cotinine was analyzed by a double-antibody, liquid-phase radio-immunoassay as reported.⁴ Intraassay and interassay coefficients of variation for that method 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 Inc, Rockville, MD). Because some distributions were skewed, the data are presented as medians and interquartile ranges. Differences in median fetal-plasma amino acid levels, total protein concentrations, and enzyme activity levels between nonsmokers and smokers were tested by the Mann-Whitney test at the 95% confidence level. Results were considered statistically significant at P < .05.

	Results

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The median (interquartile range) gestational age in both groups was 14 weeks (13–16 weeks). Cotinine was detected in all maternal and fetal plasma samples from smokers, with medians of 156 ng/mL (109–208 ng/mL) and 89 ng/mL (45–123 ng/mL), respectively. A cotinine level of 65 ng/mL was also found in one sample of fetal liver tissue homogenate, corresponding to the mother and fetus with the highest plasma cotinine levels (545 and 232 ng/mL, respectively). The cotinine level was below the limit of detection of the assay in the remaining fetal liver samples from smokers and in all samples from nonsmokers.

Table 1 presents the median amino acid concentrations in matched samples of fetal plasma. Compared with nonsmokers, the fetal plasma of smokers contained significantly lower concentrations of serine, proline, -aminobutyric acid, leucine, and arginine. No differences were found in the concentrations of amino acids in the maternal plasma of both groups.

	Discussion

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The placenta has a high metabolic rate throughout pregnancy and until delivery is an active synthesizer of glycogen, lactate, fatty acids, cholesterol, proteins, and enzymes.¹⁰ Nicotine, the main alkaloid in tobacco, concentrates in placental tissue,³ and active smoking during pregnancy is associated in all trimesters with placental ultrastructural lesions, including decreases in syncytiotrophoblast microvilli and pinocytotic activity, focal syncytial necrosis, and degenerated cytoplasmic organelles.^11–15 Identical changes were observed at term in the placentas of women who gave up smoking early in pregnancy, implying that trophoblastic cellular alterations were irreversible.¹³ Those findings suggest that tobacco smoking affects progressively placental development, starting with the trophoblast that is in direct contact with maternal circulation.

In vitro, nicotine was shown to block the trophoblast acetylcholine-facilitated amino acid transport¹¹ and to inhibit the trophoblast differentiation pathways that lead to column formation and uterine invasion.¹² Other studies in vitro indicated that maternal smoking decreased the active uptake of amino acids by placental villi from the maternal circulation and their transfer into the fetal circulation.¹¹ Present data showing decreases in the levels of five amino acids in the fetal plasma of smokers suggest a direct effect of maternal tobacco smoking on trophoblastic functions of transfer and synthesis.

Placental tissue exposure in vitro to cigarette smoke induces the formation of new trophoblastic carriers for the uptake of aminobutyric acid,¹⁶ which suggests that part of the fetal amino acid deficit induced by maternal smoking might be compensated for by the induction of new amino acid transport systems. The present finding, showing lower levels of most amino acids, in particular aminobutyric acid, in the fetal plasma of mothers who smoke, does not support that concept in vivo.

In a recent study we found that cotinine accumulates in fetal fluids and serum early in gestation in active and passive smokers and that fetal and maternal concentrations were related.⁴ Investigations of distribution of nicotine in adult rat tissues demonstrated that the highest concentrations were found in the kidneys and that concentrations were low in the liver.¹⁷ In the present study, only one sample of fetal liver tissue contained detectable cotinine in the corresponding assay. Although nicotine is metabolized by the liver,³ it does not accumulate there and is rapidly eliminated by the kidneys in adults and fetuses.

Retention of nicotine after continuous intravenous infusion was higher in esophagus, stomach, spleen, caecum, pancreas, testes, and heart than in other adult rat organs, indicating their predisposition to pathologic manifestation.¹⁷ Nicotine induced cytoplasmic vacuolation and cellular edema in adult rat pancreas and was implicated in the etiology of pancreatitis and pancreatic carcinoma.¹⁸ In humans, smoking enhances the secretion of amylase by the exocrine pancreas¹⁹ but not by the salivary glands.²⁰ Our data showed significantly higher fetal plasma amylase activity in mothers who smoked compared with nonsmokers, indicating that nicotine or its metabolites might affect the fetal pancreas as early as 12 weeks’ gestation.

Tobacco toxins such as cadmium accumulate mainly in villous tissue, and only when the placenta is saturated does cadmium leak through to the fetus.⁷ The placenta has the potential to inactivate carcinogens locally and to regulate the transfer of metabolized toxic agents into the fetal compartments.^5,6 Those mechanisms might protect the fetus to some extent, until the trophoblast detoxification function is overwhelmed by long-term exposure to the same toxins.

	Footnotes

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

PII S0029-7844(98)00548-1

Received August 21, 1998. Received in revised form October 27, 1998. Accepted November 12, 1998.

	References

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