Reprints requests: Theresa W. Gauthier, MD, Division of Neonatal-Perinatal Medicine, Department of Pediatrics, Emory University, 2015 Uppergate Drive NE, Atlanta, GA 30322; Fax: 404-727-3236; E-mail: email@example.com
Background: Increased systemic oxidant stress contributes to a variety of maternal complications of pregnancy. Although the antioxidant glutathione (GSH) and its oxidized component glutathione disulfide (GSSG) have been demonstrated to be significantly altered in the adult alcoholic, the effects of maternal alcohol use during pregnancy on oxidant stress in the postpartum female remain under investigation. We hypothesized that maternal alcohol use would increase systemic oxidant stress in the pregnant female, evidenced by an oxidized systemic GSH redox potential.
Methods: As a subset analysis of a larger maternal language study, we evaluated the effects of alcohol consumption during pregnancy on the systemic GSH redox status of the postpartum female. Using an extensive maternal questionnaire, postpartum women where queried regarding their alcohol consumption during pregnancy. Any drinking, the occurrence of drinking >3 drinks/occasion, and heavy drinking of >5 drinks/occasion during pregnancy were noted. Using HPLC, maternal plasma samples were analyzed for GSH, oxidized GSSG and the redox potential of the GSH/GSSG antioxidant pair calculated.
Results: Maternal alcohol use occurred in 25% (83/321) of our study sample. Two in ten women reported consuming >3 drinks/occasion during pregnancy, while 1 in 10 women reported consuming alcohol at >5 drinks/occasion. Any alcohol use during pregnancy significantly decreased plasma GSH (p < 0.05), while alcohol at >3 drinks/occasion or >5 drinks/occasion significantly decreased plasma GSH concentration (p < 0.05), increased the percent of oxidized GSSG (p < 0.05), and substantially oxidized the plasma GSH redox potential (p < 0.05).
Conclusions: Alcohol use during pregnancy, particularly at levels >3 drinks/occasion, caused significant oxidation of the systemic GSH system in the postpartum women. The clinical ramifications of the observed alcohol-induced oxidation of the GSH redox system on high risk pregnancies or on the exposed offspring require more accurate identification and further investigation.
Alcohol-induced oxidant stress contributes to the pathophysiology of end-organ damage in the adult in multiple organ systems including the lung, liver and pancreas (Apte et al., 2006; Brown et al., 2004, 2006; Kono et al., 2000; Yeh et al., 2007). The antioxidant glutathione (GSH, γ-glutamyl-cysteinylglycine) and its oxidized component glutathione disulfide (GSSG) are altered in the adult alcoholic. A decrease in GSH and an increase in GSSG result in significant oxidation of the GSH redox potential in the plasma of otherwise healthy alcoholics (Yeh et al., 2007). Altered systemic GSH redox plays a pathophysiologic role in the progression of a variety of disease states in the adult (Ballatori et al., 2009; Jones, 2006; Jones et al., 2000). However, the effects of maternal alcohol consumption on systemic GSH redox potential in the context of pregnancy have not been described.
We hypothesized that maternal alcohol use would increase systemic oxidant stress in the pregnant female. The purpose of this analysis was to determine the effects of alcohol consumption during pregnancy on the systemic GSH redox status of the postpartum female.
Materials and Methods
Data for these analyses were abstracted from a larger parent study of 351 women and their newborn infants who were enrolled in a 2-year longitudinal study of infant language development at 2 large hospitals in the Atlanta, GA, metropolitan area (Kable et al., 2009). Women were approached for enrollment in the hospital when they were at least 24 hours postpartum. Eligibility criteria for enrollment into the parent study included a maternal age of at least 18 years, the primary language within the home of English, and delivery of a singleton infant at least 34 weeks gestational age with no known medical conditions such as genetic disorders, severe complications of prematurity, perinatal trauma, visual or hearing impairments.
Questionnaire of Substance Exposure
Mothers completed an informed consent procedure approved by the Institutional Review Boards of the Emory University School of Medicine and the birth hospitals. This procedure was consistent with the provisions of the Health Insurance Portability and Accountability Act of 2001. During the same visit, a research interviewer performed an extensive maternal interview noting prenatal care, maternal education, family income, and tobacco, alcohol, and other drug use in the 3 months prior to conception and during the pregnancy. For cigarette use, mothers were asked how many cigarettes per day they smoked during the pregnancy. Participants were asked to describe the pattern and quantity of drinking alcohol prior to conception and during the pregnancy. Maternal medical information and information concerning tobacco and other drug use was also obtained through abstraction of the medical record.
GSH and GSSG Measurements
After enrollment, the mothers were asked to provide a blood sample, which was collected by the nursing staff of the hospital. Plasma samples were available from 60% of the cohort (213 women). These samples were collected in specially prepared tubes allowing immediate acidification with perchloric acid (5% final) and 5 μM (final) γ-glutamyl-glutamate (an internal standard) for GSH and GSSG analyses. The collected samples were transported for analyses to the Brown Laboratory and stored at −80°C until batch analysis. GSH and GSSG in the maternal plasma were determined by high performance liquid chromatography (HPLC) analysis as previously described by this laboratory (Brown et al., 2004; Gauthier et al., 2005; Yeh et al., 2007). Briefly, after derivitization with iodoacetic acid and dansyl chloride, the GSH and GSSG fractions were separated by HPLC using an amino μBondaPak column (Waters). Fluorescent detection was used to separate and quantitate the dansyl derivatives relative to the fluorescence of the γ-glutamyl-glutamate standard. The redox potential (Eh) of the GSH/GSSG redox pair in the plasma was calculated with the Nernst equation: Eh = Eo + RT/nF 1n [disulfide]/([thiol1] × [thiol2]), where Eo is the standard potential for the redox couple, R is the gas constant, T is the absolute temperature, n is 2 for the number of electrons transferred, and F is Faraday’s constant. The standard potential Eo for 2 GSH/GSSG couple is −264 mV at pH 7.4 (Brown et al., 2006). The Eo was adjusted by a factor of a 5.9-mV change in Eo with every 0.1 decrease in pH.
SPSS 15.0 for Windows (SPSS, Inc., Chicago, IL) was used for all analyses. Group median comparisons were made with the nonparametric Mann–Whitney test.
ANCOVA analysis was also used to determine the independent effects of alcohol controlling for maternal tobacco use. Data were presented either as mean ± SEM or as medians with the first and third quartiles noted. A p value of ≤0.05 was considered statistically significant.
Characteristics of the Study Group
In the maternal interview, 25.5% of the study population (83/325) reported alcohol use during their pregnancy. A maximum alcohol use of greater than 3 drinks/occasion was reported in approximately 21% (67/325) of the population, while 9% of the mothers (30/325) reported heavy alcohol intake of drinking >5 drinks/occasion during pregnancy (National Institute on Alcohol Abuse and Alcoholism of the NIH, 2000). Other substances reported included tobacco use in 65%, cocaine use in 2% and marijuana use in 7% of the group. Complications of pregnancy included reported hypertension in 4% of the sample, gestational diabetes in 4%, and preterm labor in approximately 2%. Medical illnesses noted prior to the current pregnancy were infrequent and included hypertension in 4% of the sample, diabetes mellitus in 3%, cardiac disease in 1%, and asthma in 7%.
Table 1 describes the demographics of those women who reported no alcohol use during pregnancy (n = 238 available) compared to those who reported alcohol use during pregnancy (n = 83). There was no difference in maternal age, maternal race, or prenatal care during this pregnancy between those who did not drink alcohol and those who did report alcohol use in the maternal interview (p = NS). Similarly, there were no differences between the groups in terms of pregnancy history, number of premature deliveries, abortions or living children (p = NS). There was no difference in the development of pregnancy induced hypertension or gestational diabetes between the groups (p = NS, respectively). Significantly more women who reported drinking alcohol during pregnancy smoked cigarettes compared to those who did not drink alcohol during pregnancy (57.8% vs. 45.8%, p < 0.05). Among the smokers in both groups, there was no significant difference in the average number of cigarettes smoked/day (p = NS).
Table 1. Maternal Demographics
Alcohol use – NO (n = 238)
Alcohol use – YES (n = 83)
aOther to include Pacific Islander, Asian, or mixed race.
Maternal age (years)Mean ± SD [range]
25.3 ± 5 [18–41]
26.9 ± 5 [18–40]
Maternal race (%)
Received prenatal care (%)
Gravida (mean ± SD)
2.5 ± 1.5
2.8 ± 1.7
Full term (mean ± SD)
0.8 ± 1
0.8 ± 1
Preterm (mean ± SD)
0.08 ± 0.4
0.1 ± 0.3
Abortions (mean ± SD)
0.6 ± 0.9
0.8 ± 1
Living (mean ± SD)
0.9 ± 1
0.9 ± 1
Gestational diabetes (%)
Number of cigarettes/dMean ± SD [range]
3.6 ± 6.5 [0–40]
3.8 ± 5.9 [0–30]
Maternal Education and Family Income
We then contrasted mothers who reported alcohol use to those who denied alcohol use in terms of reported maternal education and family income (Table 2). Interestingly, although there was no difference in the highest education obtained by the mother between the groups, the distribution of total yearly family income of mothers who reported alcohol use was significantly higher compared to those who did not use alcohol (p < 0.05 Chi square). This suggested that lower education or poorer socioeconomic status did not contribute to the use of alcohol during pregnancy in this sample population. We did not find, however any differences in maternal education or total family income when comparing those who drank >3 to those who drank an excess of 5 drinks/occasion (p = NS).
Table 2. Maternal Education and Income
Alcohol use – NO (n = 231)
Alcohol use – YES (n = 81)
Maternal education (%)
Some high school
High school diploma
Total family income (%)
Newborn Infant Characteristics
The gestational age, birth weight, and head circumferences of the newborns born to this study population are presented in Table 3. The newborns were approximately 39 weeks of gestation. There were no significant differences in gestational age, birth weight, or head circumferences between pregnancies where mothers reported, “No” to Alcohol Use, “Yes” to Alcohol Use, “Yes” to >3 drinks/occasion, or “Yes” to >5 drinks/occasion (p = NS, respectively). There were also no significant differences in the growth distribution of babies characterized as appropriate for gestational age (AGA), small for gestational age (SGA), or large for gestational age (LGA) in the medical record between the alcohol use groups (p = NS, respectively; Table 3).
Table 3. Newborn Infant Characteristics
Alcohol use – NO
Alcohol use – YES
YES >3 drinks/ occasion
YES >5 drinks/ occasion
Gestational age (weeks; mean ± SD)
38.7 ± 1.4
38.8 ± 1.4
38.9 ± 1.1
39.2 ± 1.3
Birth weight (kg; mean ± SD)
3.3 ± 0.5
3.2 ± 0.5
3.2 ± 0.5
3.2 ± 0.5
Head circumference (cm; mean ± SD)
33.5 ± 1.7
33.8 ± 1.8
33.8 ± 1.7
33.7 ± 2.0
Systemic GSH Availability Is Decreased With Any Reported Alcohol Use
To address our hypothesis that maternal alcohol would increase systemic oxidative stress, maternal plasma was evaluated via HPLC for GSH, the percent of its oxidized component GSSG, and the GSH redox potential of this antioxidant couple. Any alcohol use during pregnancy significantly decreased systemic GSH concentration by ∼38% (Fig. 1A: *p < 0.05). There was no significant difference in the percent of oxidized GSSG between women who reported no alcohol versus any alcohol (Fig. 1B: p = NS). Furthermore, there was no difference in the GSH redox potential for the GSH/GSSG pair between these groups (Fig. 2: p = NS). When controlling for maternal tobacco use in the analysis, the independent decrease in GSH due to any alcohol use was lost and did not reach statistical significance (p = 0.15).
Oxidized Systemic GSH Redox Potential With >3 Drinks Per Occasion
Because of the substantial occurrence of reported alcohol use in excess of 3 drinks/occasion during pregnancy (21% of the population), we similarly compared this group of women to those who reported no alcohol use, hypothesizing that increased amounts of alcohol would cause further systemic oxidant stress in the mother. With reported alcohol consumption of >3 drinks/occasion, maternal systemic GSH was significantly reduced by ∼40% and the percent of oxidized GSSG significantly increased by ∼80% compared to moms who denied alcohol use (Fig. 3A and 3B, respectively: *p < 0.05). This decrease in GSH and increase in GSSG resulted in significant oxidization of the GSH redox potential by over 17 mV (Fig. 4: No alcohol: −110.8 ± 29.7 vs. >3 drinks/occasion: −93.1 ± 5.8 mV, p < 0.05). In the ANCOVA analysis controlling for maternal tobacco use, the significant effects of alcohol use at >3 drinks/occasion remained. Alcohol independently decreased GSH (p = 0.018) and oxidized the GSH redox potential (p = 0.03). Therefore, maternal alcohol consumption of >3 drinks/occasion significantly altered the systemic GSH redox system in these otherwise healthy postpartum women.
Alcohol Intake of >5 Drinks Per Occasion Caused Systemic Oxidant Stress
To determine if heavy amounts of alcohol exposure altered systemic GSH redox status, we similarly compared the 9% of the population who reported consuming excessive alcohol at a level of >5 drinks/occasion to those who denied alcohol use. Maternal report of >5 drinks/occasion during pregnancy resulted in significantly decreased plasma GSH by 40% and increased percentage of GSSG by over 75% (Figs.5A and 5B, respectively: *p < 0.05). Finally, systemic oxidation was evident in those with alcohol consumption of >5 drinks/occasion as demonstrated by a significant increase of 19 mV in the GSH redox potential (Fig. 6, p < 0.05). When controlling for maternal tobacco use, alcohol at excessive amounts of >5 drinks/occasion independently decreased systemic GSH (p = 0.034) and increased the percent of oxidized GSSG (p = 0.04).
The current analysis demonstrated the continued frequent and in some cases heavy consumption of alcohol during pregnancy. In this otherwise healthy sample of postpartum women, alcohol consumption occurred in approximately 1 in 4 women, with 1 of every 10 admitting to heavy alcohol of >5 drinks/occasion. Maternal use of alcohol was more common in women with higher yearly income, as has been suggested by others (Alvik et al., 2006; Caetano et al., 2006). This contrasts with the stereotypical view that maternal alcohol use is a problem limited to women of lower socioeconomic status or who are ethnic minorities. The offspring of these women were essentially healthy babies, with similar gestational ages, birth weights, and head circumferences, suggesting that alcohol exposure, if based on infant growth characteristics, would have gone undetected in the normal newborn nursery.
Our analysis of the plasma GSH/GSSG redox pair in postpartum women demonstrated that pregnancy alone increased systemic oxidant stress in the female. The normal GSH redox potential in the plasma of healthy nonpregnant adults has been reported as −137 ± 9 mV (Jones et al., 2000). In our study population, pregnant nondrinkers demonstrated an oxidized GSH redox potential of 109.28 ± 29.9 mV, significantly higher than the reported normal adult values (p < 0.001). We cannot exclude that the increased oxidation demonstrated in the nondrinkers was in part due alcohol consumption during pregnancy that was denied in the maternal interview. However, similar to other reported markers of systemic oxidant stress due to pregnancy, our data suggested that pregnancy alone caused systemic oxidation of the GSH redox pair (Orhan et al., 2003).
With maternal consumption of alcohol, particularly in excess of 3 drinks/occasion, the current study demonstrated significant systemic oxidation in the postpartum female, characterized by decreased systemic GSH, increased percentage of oxidized GSSG and a near 20 mV oxidation of the plasma GSH redox potential. Even when effects of tobacco use are controlled, significant independent alterations in the GSH redox system remained. This level of oxidation is striking, in an otherwise clinically healthy postpartum population with a low incidence of maternal illnesses, either prior to or during pregnancy. This extent of oxidation has been demonstrated to significantly alter the structure and function of proteins and lipids, influencing the pathophysiology of a variety of disease processes (Fruhwirth et al., 2007; Jones et al., 2002; Oettl and Stauber, 2007). Our data suggested that alcohol-induced systemic oxidant stress, although clinically “unseen” in these otherwise healthy postpartum women, has the potential to significantly contribute to maternal disease states during or after pregnancy.
It is important to note that was no difference in the age of the women who reported drinking compared to those who did not consume alcohol since the systemic GSH redox can be affected by age, with progressive oxidation with advancing years (Jones et al., 2002). Similarly, there were no differences in the rates of cesarean section or length of labor between the groups (data not shown), since maternal fasting has been postulated to effect the systemic GSH status in the postpartum women (Roes et al., 2005). In our sample population, complications of pregnancy known to increase oxidant stress, such as pregnancy induced hypertension or gestational diabetes, were infrequent. There was no difference in the development of these complications of pregnancy between the alcohol groups.
As a subset analysis of a larger study with subgroup analyses of maternal drinking, the results of our study remain limited. However, the strengths of the current study are its extensive questionnaire and detailed interview to ascertain alcohol use during pregnancy. Multiple standardized questionnaires have attempted to accurately define alcohol exposure during pregnancy, but the reliability of these methods remains a continual question (Chang et al., 1999; Russell et al., 1996). Other studies have assessed maternal blood markers such as carbohydrate-deficient transferring and γ-glutamyl transferase, but specificity and sensitivity of these biological tests are lacking (Sarkola et al., 2000; Stoler et al., 1998). Thus, the maternal interview remains the mainstay strategy for identifying the alcohol-exposed pregnancy and likely underestimates the real impact of maternal alcohol use during pregnancy.
We speculate that the observed state of increased oxidation of the systemic GSH redox potential may increase other oxidant-mediated complications in the pregnant female as well as in her offspring. The current study evaluated otherwise healthy term infants with minimal to no adverse outcomes of pregnancy. These infants did not demonstrate any outward effects of alcohol exposure, such as low birth weight or decreased head circumference. Furthermore, the current study did not address the at-risk newborns particularly if born prematurely. Because of gestationally-dependant maturation of antioxidant systems such as GSH, prematurity itself is a state of increased risk of oxidant-induced injury to the newborn (Boda et al., 1998; Grigg et al., 1993; Jain et al., 1995). The current study suggested that alcohol exposure during pregnancy compounds the risk of oxidant-induced injury for the alcohol-exposed newborn. We speculate that this would be important for newborns at risk, especially if born at premature gestations. The clinical ramifications of the observed alcohol-induced oxidation of the GSH redox system on high risk pregnancies or the GSH redox status of newborns born prematurely require more accurate identification and warrant further investigation.
In summary, the current study described the novel finding of alcohol-induced oxidation of the systemic GSH redox potential in the otherwise healthy postpartum woman. The implications of these findings on the potential for maternal complications during and after pregnancy as well as neonatal complications which are modulated by oxidant stress remain under investigation.
This research was funded by the National Institute of Child Health and Human Development grant R01 HD041203-01A2 (JAK and CDC) and the Emory Alcohol and Lung Biology Center P50 AA 135757 (TWG and LAB).