An update on maternal cardiac hemodynamics in fetal growth restriction and pre-eclampsia

Pre-eclampsia and fetal growth restriction (FGR) have been long related to primary placental dysfunction, caused by abnormal trophoblast invasion. Nevertheless, emerging evidence has led to a new hypothesis for the origin of pre-eclampsia and FGR. Suboptimal maternal cardiovascular adaptation has been shown to result in uteroplacental hypoperfusion, ultimately leading to placental hypoxic damage with secondary dysfunction. In this review, we summarize current evidence on maternal cardiac hemodynamics in FGR and pre-eclampsia. We also discuss the different approaches for antihypertensive treatment according to the hemodynamic phenotype observed in pre-eclampsia and FGR.     

Stimulatory effect of cigarette smoking on the 15 alpha-hydroxylation of estradiol by human term placenta

OBJECTIVE: Our objective was to characterize the oxidative metabolism of estradiol by human term placenta and its modulation by cigarette smoking. METHODS: Placental microsomes were prepared from term placentas obtained from 13 cigarette smokers (20 to 30 cigarettes per day until the time of delivery) and 13 control subjects who were nonsmokers. Estrogen metabolism was studied by incubation of 250 nmol/L [(3)H]estradiol with placental microsomes and NADPH, and the estrogen metabolites were determined by HPLC and gas chromatography-mass spectrometry. RESULTS: 2-Hydroxyestradiol was the major hydroxyestrogen detected, followed by 6alpha-hydroxyestradiol. Small amounts of several other hydroxyestrogen metabolites (4-hydroxyestradiol, 6beta-hydroxyestradiol, 7alpha-hydroxyestradiol, and 16alpha-hydroxyestradiol) were also detected. Large amounts of estrone plus small amounts of 2-hydroxyestrone and unidentified nonpolar metabolites were formed. Cigarette smoking stimulated the placental hydroxylation of benzo[a ]pyrene by about 16-fold. Cigarette smoking had little or no effect on the overall rate of placental estradiol metabolism or on the formation of estrone, 2-hydroxyestradiol, 2-hydroxyestrone, or 16alpha-hydroxyestradiol. However, placental formation of 4-hydroxyestradiol and 7alpha-hydroxyestradiol was increased 38% (P =.08) and 150% (P =.05), respectively, in cigarette smokers. The formation of 6alpha-hydroxyestradiol was decreased 33% (P =.04). Metabolic formation of 15alpha-hydroxyestradiol was observed during incubations of estradiol with placental microsomes from 11 of the 13 cigarette smokers, but this metabolite was not detected during incubations with placental microsomes from any of the 13 nonsmokers. Analysis of data from all 26 placentas showed that the 15alpha-hydroxylation of estradiol was highly correlated with benzo[a ]pyrene hydroxylation (r = 0.93; P <.001). CONCLUSIONS: Many hydroxylated estradiol metabolites were formed by placental microsomes from cigarette smokers and nonsmokers. 15alpha-Hydroxylation of estradiol was markedly stimulated in the placentas of cigarette smokers.     

Influence of smoking habits on Down’s syndrome risk evaluation at mid-trimester through biochemical screening

Expectant mothers who smoke have higher levels of maternal serum alpha-fetoprotein and lower levels of unconjugated estriol and total human chorionic gonadotrophin than non-smoking mothers. This significantly affects performance of screening for Down’s syndrome. This study includes 22,169 pregnant women: 18,876 non-smokers, 2,660 smoking ≤10 cigarettes/day, and 633 smoking >10 cigarettes/day. Mean maternal age (32.6 years), maternal weight (60.5 kg), and gestational age (114.7 days) were similar or only slightly different between the three groups. To verify the effects of smoking on screening, we studied retrospectively 130 sequential Down’s syndrome cases (47 from the screening program, 83 from the prenatal diagnosis program). The proportion of smokers in the Down’s syndrome and unaffected pregnancies was similar, whilst the false-positive rate and detection rate, based on fetal outcome, differed: false-positive rates were 5.63% in smokers and 9.42% in non-smokers, and detection rate 55.6% in smokers and 83.0% in non-smokers. Since the prevalence of Down’s syndrome pregnancies was the same at mid-trimester in smokers and non-smokers and the proportion of smokers was not related to maternal age, we propose an adjustment of the Down’s syndrome risk evaluation algorithm according to smoking habits.     

IMMUNOHISTOLOGICAL AND ELUTION STUDIES OF THE HUMAN PLACENTA

An immunohistological survey of 28 full-term human placentas has demonstrated deposits of IgG, β1C, β1E, and fibrinogen/fibrin in areas of fibrinoid necrosis and on the trophoblast basement membrane in approximately 35% of placental villi. Traces of IgM were detected at similar sites in 18 of 28 full-term placentas. In 11 specimens of immature placentas (10–18 wk gestation) traces of IgG and β1C and deposits of fibrinogen/fibrin were also present, but IgM was not detected in this material. IgG was recovered in acidic eluates from an homogenized placenta which behaved as an antibody reactive with unidentified material present in fibrinoid deposits and on the thickened trophoblast basement membrane of some villi. It could not be determined whether this IgG was derived from the maternal or fetal circulation.     

Reduced maternal expression of adrenomedullin disrupts fertility, placentation, and fetal growth in mice

Adrenomedullin (AM) is a multifunctional peptide vasodilator that is essential for life. Plasma AM expression dramatically increases during pregnancy, and alterations in its levels are associated with complications of pregnancy including fetal growth restriction (FGR) and preeclampsia. Using AM+/- female mice with genetically reduced AM expression, we demonstrate that fetal growth and placental development are seriously compromised by this modest decrease in expression. AM+/- female mice had reduced fertility characterized by FGR. The incidence of FGR was also influenced by the genotype of the embryo, since AM-/- embryos were more often affected than either AM+/- or AM+/+ embryos. We demonstrate that fetal trophoblast cells and the maternal uterine wall have coordinated and localized increases in AM gene expression at the time of implantation. Placentas from growth-restricted embryos showed defects in trophoblast cell invasion, similar to defects that underlie human preeclampsia and placenta accreta. Our data provide a genetic in vivo model to implicate both maternal and, to a lesser extent, embryonic levels of AM in the processes of implantation, placentation, and subsequent fetal growth. This study provides the first genetic evidence to our knowledge to suggest that a modest reduction in human AM expression during pregnancy may have an unfavorable impact on reproduction.     

Accuracy of Prenatal Ultrasound in Evaluating Placental Pathology Using Superb Microvascular Imaging: A Prospective Observation Study

Superb microvascular imaging (SMI) is a new Doppler method that enables the visualization of low-velocity blood flow. The aim of the study described here was to clarify whether SMI can detect prenatal pathological findings in pathologic placentas. In this prospective diagnostic observational study, pregnant women who were admitted to our center for perinatal management were enrolled. Ultrasound examinations to identify placental pathologies using SMI were performed before delivery. After delivery, the placental tissue was clipped for microscopic examination at locations determined under ultrasound guidance. The accuracy of prenatal ultrasound detection of placental pathologies was compared between women who were admitted because of fetal growth restriction (FGR), pre-eclampsia and other indications. The highest accuracy was observed with placental infarction in FGR (positive predictive value = 100%, sensitivity = 89%, area under the curve = 0.945). As a result, it became clear that SMI can accurately detect placental pathologic findings, such as placental infarction and avascular villi. This modality may improve perinatal management in cases of placental dysfunction.     

Protective effect of metformin on a rat model of lipopolysaccharide‐induced preeclampsia

Recent in vitro and clinical studies have found that metformin (MET) may play a preventive or therapeutic role in preeclampsia (PE) and may be a candidate drug for the prevention and/or treatment of PE. In this study, we used lipopolysaccharide (LPS) to induce a PE‐like rat model and investigated the intervention effect of MET from the perspectives of clinical manifestations, placental morphology, serum marker for placental injury, systemic inflammatory response and oxidative/nitrative stress, and placental nuclear factor‐κB (NF‐κB) signaling. The results showed that MET improved LPS‐induced hypertension, proteinuria, fetal growth restriction (FGR) and stillbirth, alleviated placental injury and decreased maternal serum marker alpha‐fetoprotein (MS‐AFP) level; MET suppressed LPS‐induced TNF‐α and IL‐6 productions, reduced oxidative/nitrative stress as evidenced by increased superoxide dismutase (SOD) activity, decreased inducible nitric oxide synthase (iNOS) activity, and decreased levels of malondialdehyde (MDA) and nitric oxide (NO); MET inhibited LPS‐induced NF‐κB activation in placentas. Based on these findings, it can be concluded that MET is beneficial to the PE‐like rat model by protecting placentas from injury, suppressing systemic inflammatory response and oxidative/nitrative stress, and inhibiting placental NF‐κB signaling pathway. MET is a promising drug for prevention and/or treatment of PE.     

胎儿生长受限孕妇血及脐血内脂质过氧化物和一氧化氮水平

目的探讨母血及脐血中脂质过氧化物(LPO)和一氧化氮(NO)水平与胎儿生长受限(FGR)发生的关系及其临床应用价值。方法取38例妊娠合并FGR(FGR组)及50名正常孕妇(对照组)肘静脉血及其新生儿脐血,应用改良的硫代巴比妥酸(TBA)法测定血清LPO水平;应用硝酸盐还原酶法测定血清NO水平。结果FGR组患者血LPO和NO水平与对照组相比均显著升高(P<0.01),其新生儿脐血LPO和NO水平也显著升高(P<0.001)。两组孕妇母血、脐血中LPO和NO水平均呈正相关(P均<0.01)。结论LPO和NO均可能参与了FGR的发病,检测母血及脐血中LPO和NO水平可作为FGR的辅助诊断指标之一。     

Experimental congenital toxoplasmosis. II. Transmission of toxoplasmosis to the placenta and fetus following vaginal infection in the pregnant mouse

Pregnant mice infected with Toxoplasma by the vaginal route have been found to transmit toxoplasmosis to the placentas and fetuses in utero. The microorganism entered the blood stream of the mother from primary foci of infection in the vaginal wall and produced disseminated lesions in the labyrinth of the allantoic placenta at the same time as other peripheral maternal tissues were involved. Placental lesions were observed in mice infected with Toxoplasma by vagina between the 3rd and the 9th day of pregnancy. They consisted of microscopic foci of degeneration, without inflammation, in the syncytial trophoblast, and parasites undergoing multiplication were readily identified in them. Here Toxoplasma gained access to the fetal circulation. Following the vaginal instillation of Toxoplasma on the 8th day of pregnancy, subinoculation of test animals revealed the parasites in the maternal peripheral and placental blood on the 13th day and later, while the first histopathologic changes in the placenta were found on the 17th day. Toxoplasma could be demonstrated in suspensions of fetal tissues on and after the 17th day by the injection of normal test animals. However, no lesions of toxoplasmosis, or Toxoplasma, were found in histologic sections of fetuses 11 to 21 days old removed at autopsy from vaginally infected mothers. It is concluded that before birth the parasites were confined to the fetal blood. The experiments provide the first direct histological demonstration of placental toxoplasmosis. The possible bearing of the experimental disease on human placental and fetal toxoplasmosis is briefly considered. It is probable that a maternal parasitemia during the latter part of pregnancy, whatever the portal of entry may be, is an essential factor in the pathogenesis of human congenital toxoplasmosis and that this occurs shortly after exposure to Toxoplasma rather than in a later chronic stage of the infection. The suggestion is offered that some instances of spontaneous abortion or fetal death in man, as in the mouse, may be due to inapparent toxoplasmosis.     

影响临床胎儿生长受限的多因素分析及预防措施

目的分析影响临床胎儿前3个月生长受限的因素及相应的预防措施。方法选择2013年1月—2014年12月本院56例住院分娩FGR孕妇为研究对象(FGR组),同时选择同期56例正常足月妊娠的孕妇为对照组,对孕妇一般资料进行问卷调查,并测量孕妇的身高和体质量,计算体质量指数,测量血压,检测血细胞比容和血红素水平。记录头三个月胎儿的生长。同时,对孕早期因素对顶臀长度的影响进行多变量分析,评估早期胎儿生长受限对出生胎儿的不良影响。结果 FGR组和对照组的孕妇在年龄、血细胞比容、血红素水平、文化水平、吸烟、饮酒、叶酸补充、初次分娩、舒张压等单因素方面差异均具有统计学意义(P0.05);FGR组和对照组胎儿在前3个月股骨长度、出生头围、出生身长、出生体质量、出生孕龄等方面差异均具有统计学意义(P0.05)。产妇的年龄、血细胞比容、血红素水平、吸烟(≥10支/d)、叶酸补充等因素对胎儿顶臀长度的影响差异显著(P0.05)。早期胎儿顶臀长度的生长受限会增加胎儿早产、出生体质量轻、胎龄小等风险。结论产妇高龄、文化程度低、高血细胞比容、血红素水平、吸烟、饮酒、叶酸补充不足、高舒张压均是胎儿生长受限发生的高危因素,孕早期胎儿的生长受限对出生胎儿会产生不利的影响。因此,在孕期应针对以上高危因素积极采取及时、有效、综合的防治措施。     

Placental diffusing capacities at varied carbon monoxide tensions.

To test the hypothesis that carbon monoxide transfer across the placenta is, in part, a facilitated process, we have looked for evidence of saturation kinetics for carbon monoxide. In eight pregnant ewes, fetal to maternal carbon monoxide transfer was examined in a preparation in which the fetal side of the placenta was perfused with blood. The carboxyhemoglobin concentrations on the fetal side of the placenta were varied from 4.8 to 70% in 23 measurements. At increased carbon monoxide tensions, the transfer from fetus to mother always decreased. The slope of log rate of carbon monoxide transfer vs. log partial pressure gradient across the placenta was significantly different from 1. Placental membrane diffusing capacity was calculated separately from total placental diffusing capacity which includes hemoglobin reaction rates and erythrocyte membrane diffusion. Placental membrane diffusing capacity decreased at increased carbon monoxide tensions. Placental permeability for urea did not change with increasing carbon monoxide tensions. These results are consistent with the hypothesis that carbon monoxide diffusion in the placenta is, in part, carrier mediated.     

Inflammation in rat pregnancy inhibits spiral artery remodeling leading to fetal growth restriction and features of preeclampsia

Fetal growth restriction (FGR) and preeclampsia (PE) are often associated with abnormal maternal inflammation, deficient spiral artery (SA) remodeling, and altered uteroplacental perfusion. Here, we provide evidence of a novel mechanistic link between abnormal maternal inflammation and the development of FGR with features of PE. Using a model in which pregnant rats are administered low-dose lipopolysaccharide (LPS) on gestational days 13.5–16.5, we show that abnormal inflammation resulted in FGR mediated by tumor necrosis factor-α (TNF). Inflammation was also associated with deficient trophoblast invasion and SA remodeling, as well as with altered uteroplacental hemodynamics and placental nitrosative stress. Moreover, inflammation increased maternal mean arterial pressure (MAP) and was associated with renal structural alterations and proteinuria characteristic of PE. Finally, transdermal administration of the nitric oxide (NO) mimetic glyceryl trinitrate prevented altered uteroplacental perfusion, LPS-induced inflammation, placental nitrosative stress, renal structural and functional alterations, increase in MAP, and FGR. These findings demonstrate that maternal inflammation can lead to severe pregnancy complications via a mechanism that involves increased maternal levels of TNF. Our study provides a rationale for the use of antiinflammatory agents or NO-mimetics in the treatment and/or prevention of inflammation-associated pregnancy complications.Preeclampsia (PE) is a serious condition that affects 3–10% of all pregnancies (Lyall and Belfort, 2007), and is characterized by the development of maternal hypertension (>140/90 mm Hg) and proteinuria (≥300 mg/24 h; Redman and Jefferies, 1988). PE usually develops after 20 wk of gestation, and is difficult to treat except by early delivery, which can result in neonatal complications. In addition to increasing the maternal and fetal risk of morbidity and mortality, the pathological processes associated with PE can restrict fetal growth and impair development. Fetal growth restriction (FGR) occurs when the fetus fails to achieve its genetically predetermined growth potential (Gardosi et al., 1992). It is the second leading cause of fetal death and is often associated with PE (Peleg et al., 1998; Vatten and Skjaerven, 2004).Although the precise mechanisms leading to the development of FGR/PE remain unknown, there is evidence that these complications are associated with an aberrant maternal inflammatory response. Women afflicted by FGR/PE exhibit a heightened inflammatory state; proinflammatory cytokines and chemokines, such as TNF, IL-6, and MCP-1, are elevated systemically and locally in the placenta (Redman et al., 1999; Borzychowski et al., 2006; Jain et al., 2007; LaMarca et al., 2007; Szarka et al., 2010). This abnormal inflammatory response leads to oxidative and nitrosative stress associated with decreased nitric oxide (NO) bioavailability (Roggensack et al., 1999; Lowe, 2000; Borzychowski et al., 2006).It is widely recognized that the placenta plays an important role in the pathophysiology of FGR/PE (Page, 1939; Koga et al., 2010). In the two-stage model of PE initially proposed by Redman (1991), deficient placental perfusion (stage one) leads to the release of vasoactive factors that precipitate the onset of the maternal syndrome (stage two) (Roberts and Gammill, 2005). Placental perfusion is dependent on adequate remodeling of the uterine spiral arteries, a process whereby endovascular trophoblast cells invade and replace the endothelium and vascular smooth muscle of these vessels (Boyd and Hamilton, 1970). Hence, a deficiency in this remodeling process is thought to account for the poor placental perfusion associated with FGR/PE.Despite the strong association between abnormal maternal inflammation and FGR/PE, it is not known whether inflammation is causally linked to the deficient spiral artery (SA) remodeling that characterizes these pregnancy complications. Reister et al. (1999) examined vessels from preeclamptic pregnancies and found that an increase in the distribution of macrophages around the spiral arteries was associated with impaired trophoblast invasion. Moreover, our in vitro studies revealed that activated macrophages inhibit trophoblast invasion by secreting TNF at subapoptotic levels (Renaud et al., 2005). Other studies have additionally described an inhibitory role of TNF in the regulation of trophoblast motility and migration (Todt et al., 1996; Renaud et al., 2007; Venegas-Pont et al., 2010). Using a rat model, we describe a novel mechanism by which abnormal maternal inflammation results in deficient SA remodeling and altered uteroplacental perfusion. Our study also reveals that this inflammation-induced deficiency in vascular adaptation is associated with FGR and features of PE.     

Pregnancy outcome and fibrinolytic, endothelial and coagulation markers in women undergoing uterine artery Doppler screening at 23 weeks

Summary.  Background:  Pre-eclampsia (PET) and/or fetal growth restriction (FGR) remain a major cause of maternal and fetal morbidity and mortality. In pregnancy, fibrinolysis is controlled by the maternal endothelium and placenta, both of which are central to the pathogenesis of PET/FGR. Clinically, uterine artery Doppler screening at 23 weeks is used to predict PET/FGR. An abnormal uterine artery Doppler finding is defined as early diastolic bilateral uterine artery notching (BN) in the waveform. However, about 50% of mothers with BN do not develop PET/FGR. Objectives:  We investigated fibrinolytic changes and uterine artery Doppler findings in the second trimester, and related them to pregnancy outcome; in particular assessing whether fibrinolytic markers could discriminate between normal and abnormal outcome in mothers with BN. Patients/methods:  Plasma levels of tissue-type plasminogen activator (t-PA), plasminogen activator inhibitor-1 (PAI-1), plasminogen activator inhibitor-2 (PAI-2), plasmin-α₂ antiplasmin (PAP), D-dimers and markers of endothelial dysfunction were measured with Doppler ultrasound at 23 weeks. Results:  Those with BN had decreased PAP and D-dimer levels, and raised PAI-1 and thrombomodulin levels. Mothers with BN and PET/FGR had significantly increased t-PA levels and reduced PAI-2 levels. Conclusions:  BN at 23 weeks of gestation is associated with increased PAI-1 levels. Within the BN group, mothers who developed PET/FGR had increased t-PA levels and decreased PAI-2 levels, although there was no net change in fibrinolysis as measured by D-dimer levels. No single fibrinolytic marker is helpful in determining pregnancy outcome in those with BN, but t-PA and PAI-2 are worthy of study in a multifactorial algorithm.     

The placental transfer and materno-fetal disposition of methadone in monkeys.

A method is described for the quantitation of levo-methadone[3H] in biological samples which involves sample extraction and thin-layer chromatographic separation. Four pregnant Macaca mulata monkeys, two in early gestation and two in late gestation, were given single i.m. injections of levo-methadone[3H]. Twenty-nine fetal and maternal tissues and fluids were assayed to provide quantities of unchanged methadone and methadone plus metabolites. Little placental transfer of methadone or its metabolites occurred during early gestation, but equivalent concentrations of unchanged methadone were found in maternal and fetal tissues during late gestation (maternal brain, 172 ng/g; fetal brain, 123 ng/g). With few exceptions, tissues or fluids from the late gestation mothers showed higher levels of unchanged methadone than those from early gestation mothers at both 1 hour (P less than .001) and 6 hours (P less than .010) after administration. The late gestation mother had a 40.2% greater concentration of unchanged methadone at 6 hours and a 50.1% greater concentration at 1 hour than the early gestation mothers. These data suggest a slowing of metabolism during advanced pregnancy. At 6 hours after administration the eyes of both early and late gestation mothers and late gestation fetuses showed the highest concentrations of unchanged methadone of any maternal or fetal tissue. This localization of methadone appears to be associated with pigmented epithelium.     

Altered placental development and intrauterine growth restriction in IGF binding protein-1 transgenic mice

IGF binding protein-1 (IGFBP-1) is a secretory product of decidualized endometrium and a major constituent of amniotic fluid. It is thought to modulate the actions of the IGFs on trophoblast cells and is therefore potentially important in regulating placental development and fetal growth. To investigate this hypothesis, we have studied the effects of decidual IGFBP-1 excess on fetoplacental growth in transgenic mice overexpressing human IGFBP-1. Endogenous fetal IGFBP-1 overexpression is associated with a transient impairment of fetal growth in midgestation. Maternal decidual IGFBP-1 excess is also associated with impaired fetal growth in midgestation independent of fetal genotype, indicating placental insufficiency. Our data also demonstrate that amniotic fluid IGFBP-1 is derived almost exclusively from maternal sources. Decidual IGFBP-1 overexpression has a marked effect on placental development. Placental morphology is abnormal in transgenic females due to altered trophoblast invasion and differentiation. These changes result in an increase in placental mass throughout pregnancy. This study provides the first compelling in vivo evidence that IGFBP-1 plays a role in placentation and suggests that IGFBP-1 has a pathological role in preeclampsia, a disorder characterized by shallow uterine invasion and altered placental development.     

Alterations of human placental epidermal growth factor receptor in intrauterine growth retardation.

We studied human placental microvillous EGF receptor (EGFR) and its relationship with maternal and placental features in 14 cases of intrauterine growth retardation. Placental EGFR phosphorylation was significantly decreased or absent in 12 cases of small for gestational age neonates, as shown by SDS-PAGE, autoradiography, and scanning analysis. Specific [125I]EGF binding and Scatchard plots of the binding data showed a decreased number of EGFR in 6 of the 12 cases, with a mean maximal binding capacity of 1.09 +/- 0.32 pmol/mg for high affinity sites (mean control value = 2.30 +/- 0.23 pmol/mg). Most of the hypertensive women and smokers belonged to this subgroup. In three of the remaining six cases of small gestational age placentas with low EGFR phosphorylation, there was no maternal pathology or significant parenchymatous placental lesions. Five showed a 175-kD EGFR species when probed by [125I]EGF cross-linking and Western blotting with RK2 and C-Term, two polyclonal anti-EGFR antibodies, suggesting abnormal transduction of the EGF-induced signal. The sixth placenta yielded a single 145-kD EGFR band consistent with an abnormal EGFR structure; Western blot analysis showed no immunoreactive band. In conclusion, maternal and placental pathologies in intrauterine growth retardation are associated with various alterations of placental EGFR, pointing out the importance of EGFR ligands in the regulatory pathway of placental and fetal growth.     

Effects of cigarette smoking and carbon monoxide on nicotine and cotinine metabolism

OBJECTIVES: To examine the effects of cigarette smoking on the disposition kinetics of nicotine and cotinine, to determine the effects of cigarette smoking on pathways of nicotine and cotinine metabolism, and to test the hypothesis that carbon monoxide inhibits the metabolism of nicotine. STUDY DESIGN: Twelve cigarette smokers were studied in three treatment conditions, each lasting 7 days, during which they smoked cigarettes, breathed carbon monoxide to achieve carboxyhemoglobin levels similar to cigarette smoking, or breathed air. In each treatment condition, subjects received a combined infusion of deuterium-labeled nicotine (d2) and cotinine (d4), with measurement of disposition kinetics and urine metabolite profile. RESULTS: Cigarette smoking significantly inhibited the metabolism of nicotine but had no effect on cotinine metabolism. Cigarette smoking markedly induced the O-glucuronidation of trans-3'-hydroxycotinine but had no effect on the N-glucuronidation of nicotine or cotinine. Carbon monoxide had no effect on nicotine or cotinine kinetics or metabolic profile. CONCLUSIONS: This study confirms previous observations that cigarette smoking inhibits nicotine metabolism but disproves the hypothesis that this effect is due to carbon monoxide. Induction of glucuronidation must be considered in understanding the effects of cigarette smoking on drug metabolism.