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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 post partum 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.
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 post partum female. Using an extensive maternal questionnaire, post partum women where queried regarding their alcohol consumption during pregnancy. Any drinking, the occurrence of drinking > 3 drinks/occasion, and excessive drinking of >5 drinks/occasion during pregnancy were noted. Using HPLC, maternal plasma samples were analyzed for GSH, oxidized GSSH and the redox potential of the GSH/GSSG antioxidant pair calculated.
Maternal alcohol use occurred in 25% (83/321) of our study sample. Two in ten women reported consuming > 3 drinks/occasion during pregnancy, while one in ten women reported consuming excessive alcohol at > 5 drinks/occasion. Any alcohol use during pregnancy significantly decreased plasma GSH (p<.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).
Alcohol use during pregnancy, particularly at levels of more than 3 drinks/occasion, caused significant oxidation of the systemic GSH system in the post partum 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.
Increased systemic oxidant stress contributes to a variety of maternal complications during pregnancy (Orhan et al., 2003). In women who develop preeclampsia during pregnancy, markers of increased systemic oxidant stress are characteristic (Kaur et al., 2008; Nemeth et al., 2001; Patil et al., 2007) although oxidant stress’ contribution to the initiation of the disease remains under interrogation (Llurba et al., 2004). Furthermore, pregnancies complicated by maternal diabetes (Djordjevic et al., 2004) or intra uterine growth retardation (Biri et al., 2007) also demonstrate increased systemic oxidative stress in the pregnant female.
The maternal use of alcohol during pregnancy continues to be a significant problem in our society despite extensive public education noting alcohol’s dangers for the developing fetus (Albertsen et al., 2004; Ebrahim and Gfroerer, 2003; Lester et al., 2001). In utero alcohol exposure causes increased oxidant stress in the exposed offspring in multiple organs in a variety of animal models (Devi et al., 1993; Gauthier et al., 2005; Henderson et al., 1999; Rathinam et al., 2006). However, for the alcohol-consuming pregnant female, controversy remains whether alcohol-induced maternal systemic oxidative stress contributes to the adverse outcomes of the alcohol-exposed newborn (Cohen-Kerem and Koren, 2003; Signore et al., 2008).
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., 2006; Brown et al., 2004; 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 post partum female.
Data for these analyses were abstracted from a larger parent study of 351 women and their newborn infants who were enrolled in a two year longitudinal study of infant language development at two 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 post partum. 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.
Mothers completed an informed consent procedure approved by the Institutional Review Boards of the Emory University School of Medicine and the birth hospitals. 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 three 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.
After enrollment, the mothers were asked to provide a blood sample, which was collected by the nursing staff of the hospital. Plasma samples for GSH and GSSG analyses were collected in specially prepared tubes allowing immediate acidification with perchloric acid (5% final) and 5µM (final) γ-glutamyl-glutamate (an internal standard). 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 controling 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.
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 excessive drinking of >5 drinks/occasion during pregnancy. 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% versus 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).
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).
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 more than 3 drinks/occasion or Yes to more than 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.
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% (Figure 1A: * p<0.05). There was no significant difference in the percent of oxidized GSSG between women who reported no alcohol versus any alcohol (Figure 1B: p=NS). Furthermore, there was no difference in the GSH redox potential for the GSH/GSSG pair between these groups (Figure 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).
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 (Figure 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 17mV (Figure 4: No alcohol: −110.8 ± 29.7 versus >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 post-partum women.
To determine if excessive 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 more than 5 drinks/occasion during pregnancy resulted in significantly decreased plasma GSH by 40% and increased percentage of GSSG by over 75% (Figure 5A and 3B, 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 (Figure 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 excessive consumption of alcohol during pregnancy. In this otherwise healthy sample of post-partum women, alcohol consumption occurred in approximately one in four women, with one out of every ten admitting to excessive alcohol of more than five 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 the stereotypical views that alcohol use is a maternal problem limited to the underclass, poor population. 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 post-partum women demonstrated that pregnancy alone increased systemic oxidant stress in the female. The normal GSH redox potential in the plasma of healthy non-pregnant adults has been reported as −137 ± 9 mV (Jones et al., 2000). In our study population, pregnant non-drinkers demonstrated an oxidized GSH redox potential of 109.28 ± 29.9 mV, significantly higher than the reported normal adult values (p<0.001). 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).
However, with maternal consumption of alcohol, particularly an excess of 3 drinks/occasion, the current study demonstrated significant systemic oxidation in the post-partum female, characterized by decreased systemic GSH, increased percentage of oxidized GSSG and a near 20mV oxidation of the plasma GSH redox potential. Despite tobacco use, significant independent alterations in the GSH redox system remained. This level of oxidation is striking, in an otherwise clinically healthy post-partum 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 post-partum women, has the potential to significantly contribute to maternal disease states during or after pregnancy.
Is 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 C/S or length of labor between the groups (data not shown), since maternal fasting has been postulated to effect the systemic GSH status in the post partum 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, 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, particularly 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 post partum 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).