Abstract
Background/Aim: The liver of pregnant women undergoes physiological and pathological changes and the changes in liver enzyme activity and release reflect changes in serum enzymatic activity. We aimed to assess the activity of alcohol dehydrogenase (ADH) isoenzymes, and aldehyde dehydrogenase (ALDH) in the sera of women with intrahepatic cholestasis of pregnancy (ICP), the most common pregnancy-related liver disease. Patients and Methods: Serum samples were taken from 40 women with ICP in the second or third trimester of pregnancy. Serum samples were also obtained from 40 healthy pregnant women at the same time of pregnancy and 40 healthy non-pregnant women. Class I and II of ADH and ALDH activity was measured by a spectrofluorometric method. Class III, IV ADH and total ADH activity was measured by photometric methods. Results: The total ADH activity was significantly higher in women with ICP than in healthy pregnant and non-pregnant women (about 42%). The median total activity of ADH was 1067 mU/l in women with ICP, 628 mU/l in healthy pregnant and 605 mU/l in non-pregnant women. A statistically significant increase in class I ADH isoenzymes was found in the sera of pregnant women with ICP. The median activity of this class in the ICP group increased about 62% and 80% in comparison to the healthy pregnant women and non-pregnant women, respectively. Conclusion: The activity of class I ADH isoenzymes in the sera of women with ICP is statistically significantly increased and may have a diagnostic significance.
Intrahepatic cholestasis of pregnancy (ICP) is a liver disease unique to pregnant women. It usually occurs in the second or third trimester and affects 0.1–2.0% of pregnant women in Europe (1). The cause of disease remains unknown. Currently, research on the pathogenesis of ICP focuses on genetic and hormonal factors (2). Pruritus and abnormal liver function are the classical symptoms of ICP. Diagnosis is based on elevated serum bile acid and/or transaminases activity. These enzymes are important diagnostic parameters but they may be normal in up to 30% of cases (3). An accurate diagnostic test is needed to diagnose ICP and prevent stillbirth. Many authors have noted that alterations in enzyme activity in hepatocytes in the course of liver diseases are reflected by the change of their activity in the blood (4-6). The cellular damage of hepatocytes leads to the release of cytoplasmic enzymes. Some studies have shown that the activity of alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) in the sera is an indicator of liver cell injury (7, 8). ADH and ALDH, which are most abundant in the liver, play a significant role in the metabolism of many important biological substances. The best characterized function of alcohol dehydrogenases is protection against excess of endogenous alcohols, products of lipid peroxidation and some exogenous xenobiotics (9). Human ALDH is responsible for the oxidation of aldehydes and participates in polyamine catabolism and oxidation of retinal (10).
In the present study, we investigated the effect of liver cell damage in the course of ICP on the activity of serum alcohol dehydrogenase isoenzymes and aldehyde dehydrogenase. We hypothesized that changes in the activities of these enzymes in hepatocytes are reflected in the sera and could be helpful for diagnosing ICP.
Materials and Methods
Materials. The research protocol was approved by the Human Care Committee of the Medical University in Bialystok, Poland (Approval Nr R-I-002/434/2017). All patients gave their informed consent for the examination.
Serum samples were taken for routine biochemical examinations from 40 pregnant women (age range=20-37 years) with ICP in the second or third trimester of pregnancy, who were hospitalized in the 2nd Department of Obstetrics and Gynecology, Medical University of Warsaw (Poland). Diagnosis was performed on the basis of clinical and laboratory examinations (total bile acid concentration, transaminases activities). Inclusion criteria were: skin pruritus starting on the second or third trimester of pregnancy and elevation of serum total bile acids (>10 μmol/). Exclusion criteria were: chronic liver disease and hepatic viral infection (HAV, HBV or HCV), pre-eclampsia, hemolysis elevated liver enzymes low platelets (HELLP), acute fatty liver of pregnancy (AFLP). None of the pregnant women had received rifampicin at least 1 year before serum collection.
The tested group was compared with 40 healthy pregnant women (age range=19-39 years) in the second or third trimester of pregnancy and 40 healthy non-pregnant women (age range=20-42 years). The healthy controls were recruited from the same geographical location and ethnic population as the patients. Control subjects were volunteers and were defined as those with normal results in all physical and blood examinations (LTFs), and computerized tomography (CT). None of the women consumed any alcohol.
Methods
Determination of class I and II ADH isoenzymes. Class I and II alcohol dehydrogenase isoenzyme activity were measured using fluorogenic substrates (4-methoxy-1-naphthaldehyde for class I and 6-methoxy-2-naphthaldehyde for class II) in a reduction reaction according to Wierzchowski et al. (11). The assays were performed in a reaction mixture containing the 4-methoxy-1-naphthaldehyde or 6-methoxy-2-naphthaldehyde (150 ml of 300 mM), NADH (100 ml of 1 mM) and sodium phosphate buffer (2.69 ml of 0.1 M pH 7.6), and serum (60 ml) (12). The measurements were performed on a Shimadzu RF-5301 spectrofluorophotometer (Shimadzu Europa GmbH, Duisburg, Germany) at excitation wavelength 316 nm for both substrates and emission of 370 nm for class I and 360 nm for class II isoenzymes.
Determination of class III ADH isoenzyme. The assay mixture for class III of alcohol dehydrogenase activity contained n–octanol as a substrate (31 ml of 1 mM), NAD (240 ml of 1.2 mM) in 0.1 M NaOH-glycine buffer (pH 9,6) and serum (100 ml) (13). The reduction of NAD was monitored at 340 nm and 25°C on a Shimadzu UV/VIS 1202 spectrophotometer.
Determination of class IV ADH isoenzyme. The assay mixture for class IV of alcohol dehydrogenase activity contained m–nitrobenzaldehyde as a substrate (132 ml of 80 mM), NADH (172 ml of 86 mM) in 0.1 M sodium phosphate buffer (pH 7.5) and serum (50 ml) (14). The oxidation of NADH was monitored at 340 nm and 25°C on a Shimadzu UV/VIS 1202 spectrophotometer.
Determination of total ADH activity. Total alcohol dehydrogenase activity was estimated by the photometric method with p-nitrosodimethylaniline as a substrate (15). The reaction mixture (2 ml) contained 1.8 ml of a 26 mM solution of substrate in 0.1 M of sodium phosphate buffer, (pH 8.5) and 0.1 ml of a mixture containing 5 mM NAD and 0.25 M n-butanol and 0.1 ml of serum. The reduction of the substrate was monitored at 440 nm on a Shimadzu UV/VIS 1202 spectrophotometer.
Determination of total ALDH activity. Aldehyde dehydrogenase activity was measured using the fluorogenic method based on the oxidation of 6-methoxy-2-naphtaldehyde to the fluorescent 6-methoxy-2 naphtoate (16). The reaction mixture contained 60 ml of substrate, 20 ml of 11.4 mM NAD and 2.8 ml of 50 mM of sodium phosphate buffer (pH 8.5) and 60 ml of serum. The measurements were performed on a Shimadzu RF-5301 spectrofluorophotometer (Shimadzu Europa GmbH, Duisburg, Germany) at excitation wavelength 310 nm and emission of 360 nm.
Statistical analysis. Statistical analyses were carried out using the STATISTICA12.1.PL program (Statsoft, Cracow Poland). A preliminary statistical analysis (Chi-square test) revealed that the distribution of ADH and ALDH activities did not follow a normal distribution. Consequently, the Mann-Whitney test for non parametric data was used for comparison between groups. Statistically significant differences were defined as p<0.05. Results are presented as median, range and mean.
Results
Table I presents the median, range and mean for ADH, ALDH and isoenzymes of alcohol dehydrogenase activities in the sera of women with ICP, healthy pregnant women and non-pregnant women (control group). The total activity of ADH was significantly higher in women with ICP than in healthy pregnant and non-pregnant women (about 42%). The median total activity of ADH was 1067 mU/l in patients with ICP, 628 mU/l in pregnant women and 605 mU/l in non-pregnant women. The analysis of ALDH activity did not show significant difference between tested group and healthy women. The comparison of alcohol dehydrogenase isoenzymes activities showed that the highest difference was exhibited for ADH I. The median activity of this class in the ICP group (2.68 mU/l) increased respectively about 62% and 80% in the comparison to the pregnant healthy women (1.65 mU/l) and non-pregnant women (1.49 mU/l). This increase was statistically significant (p<0.05). The analysis of the other tested classes of ADH isoenzymes activity did not indicate any significant differences.
The analysis of alcohol dehydrogenase, aldehyde dehydrogenase and ADH isoenzymes activities in the serum did not show significant differences between healthy pregnant women and non-pregnant women.
Discussion
During pregnancy the body of the mother undergoes physiological changes. Every organ system in the organism needs to adapt in order to support a healthy pregnancy. For example, the normal ranges for many biochemical indices change, and there is an increased requirement for folate, vitamin B12 or iron and bile acid. While in most women the increase in serum bile acid level is moderate, a subset of women develop ICP.
It is a reversible form of cholestasis, which affects 0.1-2.0% of pregnant women in Europe (1). The etiology of ICP is not fully understood and seems to be multifactorial. Its pathogenesis is related to increased sex hormone synthesis, genetic predisposition and environmental factors (17). Clinical symptoms resolve quickly after delivery, however, recurrence in subsequent pregnancies has to be expected. Women with ICP have a higher incidence of liver disease, bile ducts, including hepatitis C virus infection, non-alcoholic cirrhosis, cholangitis, cholecystitis and cholelithiasis cholangitis. Moreover, ICP is associated with increased risk of severe fetal complication, including preterm delivery. Therefore, it is very important to find disease markers that detect the disorder as early as possible. Currently no single reliable test unequivocally identifies all cases of intrahepatic cholestasis of pregnancy or other liver disease. The biochemical parameter commonly used to diagnose ICP is determination of the concentration of serum bile acids. Many studies have shown that there is a relationship between bile acid level and preterm delivery (18). However total bile acid levels can fluctuate depending on the fasting state or gestational age (19, 20). Additional clinical features comprise raised serum activities of alanine transaminase (ALT), and to a lesser extent of aspartate transaminase (AST). Transaminase activities increased over the course of pregnancy with peak values of 86 and 110 U/l for AST and ALT, respectively, at 35 weeks of gestation (21). It is difficult to diagnose ICP only by routine tests, however, regular fetal monitoring using cardiotocography and/or ultrasound in women with intrahepatic cholestasis of pregnancy has not been shown to reduce the risk of perinatal complications. While various markers have been studied for ICP, the diagnostic significance of ADH isoenzymes and ALDH activities have not been reported. In the present study, we showed that the serum total ADH activity changes in the course of ICP. The increased enzyme activity was positively correlated with ADH I. Therefore, the cause of the increase in total ADH in the course of ICP is an elevation of class I isoenzymes. More than 95% of ADH I is present in the liver. Thus, the elevated activity of ADH I in the serum of women with ICP seems to be caused by isoenzymes released from altered hepatocytes. The changes in the activities of other ADH isoenzymes in the sera of patients with ICP were not significant. Class II in humans is found only in the liver whereas class III is present in all examined tissues. Physiologically, the activity of these classes in the liver is significantly lower compared to ADH I. Class IV alcohol dehydrogenase exists in the digestive tract organs while its activity in the liver is negligible (5). ALDH is also present in the liver cells although its activity seems to be disproportionally low compared to the ADH activity. The serum levels of ALDH were not significantly higher in women with ICP in comparison to healthy pregnant and non-pregnant women. The activity of ADH isoenzymes did not increase during pregnancy, and did not show significant differences in comparison with non-pregnant women.
Serum activity of ADH and ALDH in ICP women and healthy pregnant and non-pregnant women.
The results of this study are similar to those of other studies performed in various liver diseases. In our previous study, we have found that the activity of ADH I and ADH II were significantly higher in the sera of patients with hepatitis C (22). Chrostek et al. have found much higher ADH I and II isoenzymes activity in the sera of patients with acute viral hepatitis B (23). We have also observed that ADH I activity was elevated in the serum of patients with metastatic liver cancer (8). Furthermore, we have reported that the activity of class I alcohol dehydrogenase was significantly higher in the serum of patients with autoimmune hepatitis or nonalcoholic fatty liver disease than in healthy controls (24, 25). In our study, we excluded women with different hepatobiliary diseases.
In conclusion, the increase in the activity of total ADH and ADH I isoenzyme in the sera of women with ICP seems to be caused by the release of this isoenzyme from damaged liver cells to the blood.
Footnotes
Authors' Contributions
Wojciech Jelski- general concept, compiling results and editing the thesis; Joanna Piechota - general concept, editing the thesis and collecting material for research; Karolina Orywal- compiling results and collecting material for research; Barbara Mroczko- general concept and discussion of results.
Conflicts of Interest
The Authors have no conflicts of interest to declare regarding this study.
- Received February 14, 2020.
- Revision received February 26, 2020.
- Accepted February 27, 2020.
- Copyright© 2020, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved
