Variation in induction of human placental CYP2E1: possible role in susceptibility to fetal alcohol syndrome?

Fetal alcohol syndrome occurs in less than 10% of women who drink heavily during pregnancy. One potential mechanism for this intersubject variation is differences in placental alcohol metabolism. Alcohol dehydrogenase is present at low concentrations in the placenta and is not inducible. CYP2E1 has not been found in human placentas at early gestation time points or in random term placentas. Hepatic CYP2E1 is induced by alcohol and other environmental agents, but induction varies among heavy drinkers and may be genetically controlled. To test whether CYP2E1 is induced in placenta by heavy drinking during pregnancy, we performed a Western blot analysis on placental microsomes from women (n = 8) whose periconceptional average daily absolute alcohol intake was greater than 1 ounce. Using anti-human CYP2E1, bands consistent with CYP2E1 were identified in six samples, although considerable variation among individuals was observed. Among drinking mothers, offspring head size was smaller among those with placental CYP2E1 (p = 0.04). The association between the presence of the protein and smaller birth weight and birth length was equivocal (p = 0.09). Our data are consistent with placental CYP2E1 being inducible by drinking, but with induction being variable among heavy drinking women. We speculate intersubject variation in induction may have a genetic basis and may play a role in susceptibility to alcohol related defects.

An accurate, automated, simultaneous gas chromatographic headspace measurement of whole blood ethanol and acetaldehyde for human in vivo studies.

Simultaneous assessment of ethanol and acetaldehyde concentrations is necessary to address hypotheses in alcohol research. Accurate measurement of in vivo acetaldehyde following ethanol exposure is problematic because acetaldehyde is present in blank blood, is volatile, and is formed enzymatically and nonenzymatically in blood containing ethanol. Because acetaldehyde is carried in red blood cells, previously reported plasma methods may not reflect total body acetaldehyde. We developed an accurate, sensitive, automated gas chromatographic whole blood method using headspace injection and flame ionization detection. Sensitivity was 5.4 mumol/L and 1.13 mumol/L for ethanol and acetaldehyde, respectively. Linearity (r2 > 0.99, both) and reproducibility (coefficients of variation = 1.6-7.7%) were acceptable. Because a whole blood method completely inhibiting in vitro oxidation of ethanol has not been reported, we evaluated multiple reported sample processing methods. The optimum method, which uses saturated sodium nitrite as the inhibitor, resulted in a 30% in vitro increase in acetaldehyde in blood containing 21.7 mmol/L (0.1 g/dL) ethanol, in contrast to the 9-40-fold increase observed with other inhibitors (p = 0.001). Using the described technique, the median acetaldehyde and ethanol peak concentrations in six African-American women following a 0.5 g/kg oral ethanol dose were 6.1 microM and 17.1 mM, respectively.

Maternal assessment of infant development: associations with alcohol and drug use in pregnancy.

Surveillance by parental concern has been advocated to assess whether formal child developmental testing is needed. To determine whether alcohol intake or illicit drug use in pregnancy is associated with differences in maternal perception of infant development, mothers with acknowledge alcohol and drug habits during pregnancy (N = 120) were interviewed at 11 months' postpartum, within 1 month before infant testing by use of the Bayley Scales of Infant Development. Women with heavy alcohol intake during pregnancy (> 3.5 oz absolute alcohol per week) were 15-fold more likely to overestimate their infant's mental development (P < 0.05), whereas mothers using illicit drugs were 4-fold more likely to overestimate their infant's physical development (P = 0.02). Given the frequent denial of substance abuse, we suggest that health care providers be cautious in accepting a lack of parental concern about a child's development and rely more heavily on formal testing, particularly in high-risk populations.

Human hepatic alcohol dehydrogenase and human erythrocyte catalase do not metabolize the cytochrome P-4502E1 substrate, chlorzoxazone.

Studies of cytochrome P-4502E1 (CYP2E1)-mediated oxidation of ethanol have been hampered by the lack of a suitable probe for in vivo human studies. Chlorzoxazone, a prescribed skeletal muscle relaxant, is metabolized to 6-hydroxychlorzoxazone by CYP2E1 and has been advocated as a specific probe of this enzyme on the basis of microsomal studies. The applications of this probe may include delineating the contribution of CYP2E1 to in vivo human ethanol metabolism. However, the activity of nonmicrosomal enzymes in metabolizing chlorzoxazone is unknown. Alcohol dehydrogenase (ADH), predominantly a hepatic cytosolic enzyme, may be more important than CYP2E1 in the oxidation of ethanol to acetaldehyde. The contribution of catalase in the in vivo oxidation of ethanol to acetaldehyde is controversial. To determine if either of these enzymes metabolizes chlorzoxazone and whether ethanol oxidation by either enzyme is inhibited by chlorzoxazone or its metabolite, multiple in vitro studies were performed. ADH enzyme kinetics were performed with human recombinant beta 1 beta 1 and beta 3 beta 3 ADH with ethanol and chlorzoxazone (0.5 to 2.5 mM). Neither ADH isoenzyme exhibited NAD(+) -dependent oxidation of chlorzoxazone, but displayed Michaelis-Menten kinetics for ethanol with K(m) values of 89 microM and 34 mM, for beta 1 beta 1 and beta 3 beta 3, respectively. Typical in vivo concentrations of chlorzoxazone and its metabolite, 6-hydroxychlorzoxazone, did not alter beta 1 beta 1 or beta 3 beta 3 ADH-mediated oxidation of ethanol to acetaldehyde. Studies of human hepatic nonmicrosomal enzyme activity were expanded to include all nonmicrosomal NAD(+) -dependent hepatic enzymes by starch gel electrophoresis assessment. Human hepatic enzymatic activity in the presence of chlorzoxazone was similar to that observed in the control sample (no added substrate), suggesting a lack of metabolism by NAD(+)-dependent enzymes. Similarly, human erythrocyte catalase, in the presence of a hydrogen peroxide generating system, did not metabolize chlorzoxazone. Furthermore, neither chlorzoxazone nor 6-hydroxychlorzoxazone altered the catalase-induced formation of acetaldehyde from ethanol. These data are consistent with chlorzoxazone as a specific probe of CYP2E1 that may be useful to alcohol researchers.

Comparison of chloral hydrate and midazolam for sedation of neonates for neuroimaging studies.

In a crossover study of seven term neonates who had neuroimaging studies, chloral hydrate (75 mg/kg administered orally) was more efficacious (p<0.05) but similar with regard to toxic effects than midazolam (0.2 mg/kg administered intravenously). Thus newer drugs are not necessarily better, and monitoring is essential even after a single oral sedative dose.