Evaluation of serum uric acid and liver function tests among pregnant women with and without preeclampsia at the University of Gondar Comprehensive Specialized Hospital, Northwest Ethiopia.

BACKGROUND: Pre-eclampsia can be described as new-onset hypertension (blood pressure ≥140/90 mmHg) together with proteinuria (24-hr urinary protein ≥ 0.3 g) or any indication of end-organ damage after 20 weeks of gestation. Liver and kidney dysfunction, thrombocytopenia, pulmonary edema, and neurologic dysfunction are common manifestations of end-organ damage due to pre-eclampsia. Pre-eclampsia is the most common cause of liver and kidney dysfunction due to hypoxia and endothelial dysfunction. Hyperuricemia indicates kidney dysfunction and is considered a predictor of the severity of preeclampsia. Therefore, the objective of this study is to evaluate the utility of the levels of serum uric acid and liver function tests [alanine aminotransferase (ALT) and aspartate aminotransferase (AST)] as biomarkers of preeclampsia-related organ damage. METHODS AND MATERIALS: An institutional-based comparative cross-sectional study design was conducted, and a total of 102 subjects (51 patients with preeclampsia and 51 normotensive pregnant women) were recruited. The parameters measured were levels of serum uric acid and liver function tests. RESULTS AND DISCUSSION: There were statistically significant differences in the mean serum uric acid, ALT, and AST levels between preeclamptic pregnant women and normotensive pregnant women (p<0.05). There were no statistically significant differences in the mean total and direct bilirubin levels. There was also a significant difference in mean serum uric acid, alanine transaminase, and aspartate transaminase levels across different gestational age categories.
CONCLUSION: Our study revealed that serum uric acid, ALT, and AST levels were higher in pre-eclamptic pregnant women compared to those of normotensive pregnant women, and the differences were statistically significant. As such, serum uric acid and liver function tests may be considered biomarkers of pre-eclampsia-related end-organ damage.

Introduction

Pregnancy-induced hypertension is a major cause of maternal and fetal morbidity and mortality. During pregnancy, four classes of hypertensive disorders exist. This includes chronic hypertension, gestational hypertension, preeclampsia-eclampsia, and chronic hypertension with superimposed preeclampsia [1].

Gestational hypertension develops after 20 weeks of gestation and has no diagnostic criteria for preeclampsia, and it can change to chronic hypertension if the blood pressure remains increased more than 12 weeks postpartum. Almost 15–46% of women will develop preeclampsia among those initially diagnosed with gestational hypertension [2]. This represents the highest degree of feto-maternal complications [3].

Pre-eclampsia is defined as new-onset hypertension (blood pressure ≥140/90 mmHg) in combination with proteinuria (24-hr urinary protein ≥ 0.3 g) or any sign of end-organ damage after 20 weeks of gestation. The presence of 300 mg or more of protein in a 24-hour urine collection or a urine dipstick protein of +1 is termed proteinuria [4]. Proteinuria, however, is not a requirement anymore to make a diagnosis of pre-eclampsia [5].

Disease severity varies, and it is considered severe if it is associated with one or more of the following; elevated blood pressure ≥160/110 mmHg on two circumstances at least 6 hours apart, a 24-hour protein of ≥5g, and/or other symptoms, such as headache, blurring of vision, vomiting. The main underlying disease mechanism is endothelial dysfunction affecting multiple organs, including the brain, liver, kidneys, and placenta [5, 6] in conjunction with other symptoms such as impaired liver and kidney function, oliguria, headache, hyperreflexia, or right upper quadrant and epigastric pain, and thrombocytopenia (hemolysis elevated liver enzymes and low platelets syndrome (HELLP)) [6, 7].

Approximately 50,000 maternal deaths worldwide are caused annually by preeclampsia, of which 25% are due to intrauterine growth restrictions (IUGR) and 15% are due to preterm birth in developed countries [8].

Its global prevalence is extremely variable and ranges from 2–10% of pregnancies. The World Health Organization reports that the incidence in developing countries (2.8% of live births) is sevenfold greater than that in developed countries (0.4%) [9]. In Africa, it occurs in 10% of pregnancies, which is higher than the global average. Around 4 in 100 women experience problems with high blood pressure and kidney dysfunction [10]. Ethiopian National Emergency Obstetric and Newborn Care found that approximately 5 percent of all pregnancies and 1 percent of all deliveries result in complications from preeclampsia. The UN 2010 study found that Ethiopia is one of the five countries that accounts for 50 percent of the world’s maternal deaths, most of which are due to preeclampsia [11].

Liver and kidney dysfunctions are common manifestations of end-organ damage due to preeclampsia [4].

Hyperuricemia indicates kidney dysfunction because of decreased glomerular filtration, decreased tubular secretion, and/or increased proximal tubular reabsorption [12]. It is also an independent risk factor for cardiovascular diseases as it has been suggested that it alters vascular function and mediates vascular inflammation. As such, hyperuricemia can perhaps predict the severity of pre-eclampsia [13].

Pre-eclampsia is also the most common cause of liver dysfunction in 3% of pregnancies because of microvesicular fat deposition and reduced blood flow to the liver potentially causing ischemia and periportal hemorrhage [14, 15]. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels are usually normal, but when they become elevated and are accompanied by abdominal pain, it almost always suggests the severe end of the disease spectrum [16-18].

As such, this study was undertaken to evaluate the utility of levels of serum uric acid and liver function tests as biomarkers of pre-eclampsia-related end-organ damage among pregnant women with and without preeclampsia.

Methods and materials

Study design and patients

An institutional-based comparative cross-sectional study design was implemented. We enrolled 102 participants (51 preeclamptic pregnant women as confirmed by physicians and 51 normotensive pregnant women). All patients had antenatal care follow-up at the University of Gondar Comprehensive Specialized Hospital. We excluded pregnant women who had chronic hypertension, liver disease, kidney disease, gout, cardiac disease, diabetes mellitus, infections, a history of medication use (e.g., aspirin, phenytoin, tetracycline, sulphonamides, etc.), and those with substance abuse (eg smoking and alcohol consumption) as these factors may affect the outcome variables.

Study area and period

The study was conducted from February to October 2020 at the University of Gondar Comprehensive Specialized Hospital, Northwest Ethiopia, to evaluate the levels of serum uric acid and liver function tests among pregnant women with and without preeclampsia.

Sample size determination

To determine the sample size, G-Power Version 3.1.9.2 was used as a tool. G-Power is one of the software packages that perform sample size calculations such as Minitab and Epi-info, covering a wider range of study designs. As an input, G-Power requires selecting an appropriate test family (t-test in this case) and statistical test within the test family (linear regression in this case) and specifying alpha error probability, power (1-β error probability), and effect size. By considering alpha = 0.05, power (1- β) = 0.8 (80%), and effect size = 0.5, the total sample size was 102.

The number of participants enrolled in the study was 102 (51 preeclamptic pregnant women as confirmed by physicians and 51 normotensive pregnant women).

Data collection methods, blood sample collection, and processing

Trained nurses collected sociodemographic data after careful examination of the patients’ histories. Four laboratory technicians were oriented on blood sample collection and storage. Questionnaires were filled out through face-to-face interviews with participants. Pre-pregnancy BMIs were calculated from prepregnancy body weight (kg) and height (meter) as follows: BMI = Weight (in kg)/(Height in m) 2. Using the WHO (2008), classification of five categories of BMI can be identified as follows: underweight, <18.5 kg/m2; normal, (18.5–24.9 kg/m2); overweight, (25.0–29.9 kg/m2); and obesity, 30.0–34.9 or 35.0–39.9 kg/m2, extreme obesity > 40.0 kg/m2 [19]. Two consecutive blood pressure readings were also measured.

Approximately 5 ml of venous blood was collected in a sterile plain vacutainer tube and properly labeled with a specific code of the patient. Blood collected in SST tubes was allowed to stand for 30 minutes at room temperature to allow complete clotting and clot retraction. It was then centrifuged (MEGAFUGE R 1.0 HERAEUS) at 3500 rpm for 15–30 min to extract serum. The serum was kept at -80°C refrigerators until biochemical analysis was carried out. The extracted serum was used to determine the biochemical levels (uric acid, aspartate aminotransferase (AST), and alanine aminotransferase (ALT)) and analyzed using Biosystem kits on a Mindray BS200E Chemistry Analyser at the UoG main lab.

Data quality control and management

To assure data quality, orientation was given to data collectors on the objective of the study, data collection process, and relevance of the study before data collection. The validity of the questionnaire was maintained by a pretested questionnaire. Quantitative data were double entered to check if there is any data inconsistency and to avoid any problem with data entry processes. The data collectors were supervised daily throughout the data collection. The data were checked for completeness on-site, and before data entry, the incomplete data were discarded. All the laboratory procedures were handled by professional laboratory technicians. All the laboratory instruments were automated and standardized.

Data processing and analysis

All data were checked for cleaning and completeness. Data processing and analysis obtained from laboratory analyses of the blood samples and questionnaires were performed by coding and entering the data into Epi-Data version 4.6.0 statistical software. The data were exported to STATA 14.0 software for analysis. Continuous variables are expressed as the mean ± standard deviation (SD), while categorical variables are presented as frequencies and percentages. Tables and graphs were also used to present the data. Levene’s test of homogeneity of variances, Shapiro–Wilk test of normality, multicollinearity diagnosis, and other assumptions was checked before performing any statistical analysis and were fulfilled. Descriptive analyses, independent sample t-tests, and ANOVA were performed. Bivariable and multivariable linear regression analyses were used to assess the association of different variables. With bivariable linear regression analysis, variables with p values <0.2 were selected for multivariable analysis while with multivariable linear regression analysis, variables with p values<0.05 at 95% confidence intervals were considered statistically significant in all analyses.

Ethics approval and consent to participate

Ethical clearance was obtained from the University of Gondar College of Medicine and Health Sciences School of Medicine with protocol number 1995/05/20. The objectives of the study were briefly explained to the participants in a local language (Amharic), and clarification was given. Verbal permission and written consent were also obtained from each study participant before starting data collection.

Results

Sociodemographic characteristics of study participants

The present study recruited 102 female study participants, 51 of whom were pre-eclamptic pregnant women while 51 of who were normotensive pregnant women with gestational age above 20 weeks. The mean ages of preeclamptic patients and controls were 32.9 and 29.5 years with a minimum age of 21 and 23 years and a maximum age of 42 and 35 years, respectively. The mean gestational ages of pre-eclamptic patients and normotensive pregnant women were 33.6 weeks (range: 22–39) and 29.3 weeks (range: 21–38) respectively. The majority (98%) of the study participants were married. Among the preeclamptic patients, 31 (60.8%) were living in rural areas. Among the normotensive pregnant women, 41 (80.4%) were living in urban areas. Most of the 44 (86.3%) and 41 (80.4%) patients were Orthodox Christians, and the rest were Muslims in the case and control groups, respectively. Most of the study participants were housewives (76.5%) (Table 1).

Table 1

Baseline characteristics of study participants, University of Gondar Comprehensive Specialized Hospital, Northwest Ethiopia, 2020.

Variables	Preeclampsia (51)		Normotensive pregnancy (51)
	Frequency (count)	%	Frequency(count)	%	P-value
Age (years)
19–25	11	21.7	18	35.3	0.851
26–34	12	23.5	24	47.06
≥35	28	54.9	9	17.7
Marital status
Single	1	1.96	1	1.96	0.095
Married	50	98.1	50	98.04
Residence
Urban	20	39.2	41	80.4	0.302
Rural	31	60.8	10	19.6
Educational status
Illiterate	21	41.2	11	21.6
Only read and write	9	17.6	5	9.8	0.41
Elementary	14	27.5	27	52.9
school	5	9.8	7	13.7
High school College and above	2	3.9	1	1.96
Occupational status
Housewife	39	76.5	39	76.5	0.235
Gov. employee	2	.92	1	1.96
Self-employed	10	19.6	10	19.6
Unemployed	0	0	1	1.96
Gestational age at diagnosis (in weeks)
20–30	10	19.6	28	54.9	0.061
31–35	12	23.5	13	25.5
≥36	29	56.7	10	19.6
Previous history of preeclampsia
Yes	8	15.7	4	7.8	0.062
No	43	84.3	47	92.2
Family history of preeclampsia
Yes	1	1.196	1	1.96	0.04
No	50	98.04	50	98.04
Parity
Nullipara	31	60.8	4	11.8	0.213
≥1 parity	20	39.2	47	88.2
Fetus of the current pregnancy
Singleton	41	80.4	49	96.1	0.14
Twin	10	19.6	2	3.9
BMI
<18.5Kg/m2	0	0	0	0	0.714
18.5–24.9 Kg/m2	41	80.4	48	94.1
25–29.9 kg/m2	8	15.7	3	5.9
≥30 kg/m2	2	3.9	0	0
SBP
≤ 140 mmHg	14	27.4	1	1.96
≥ 140 mmHg	37	72.6	50	98.04
DBP
≤ 90 mmHg	14	27.4	1	1.96
≥90 mmHg	37	72.6	50	98.04

Anthropometric and clinical characteristics of study participants

Prepregnancy BMI was calculated using their prepregnancy weight and height. Based on the calculation, the majority of the patients (41, 80.4%) had a normal BMI, 8 (15.7%) were overweight, and 2 (3.9%) were obese. The majority of the normotensive pregnant women (94.1%) had normal BMI while the rest (5.9%) were overweight. None in the control group was underweight. Blood pressures of the study participants were recorded and found to be high in preeclampsia patients, with a mean SBP of 142.8±6.34 and a mean DBP of 92.8±5.22.

Descriptive characteristics of serum uric acid and liver function tests between preeclampsia patients and normotensive pregnancy

The normal concentrations of serum uric acid for females were 2.4–5.7 mg/dl, serum ALT 0–31 IU/L, serum AST 0–32 IU/L, serum total bilirubin 0–1.2 mg/dl, and serum direct bilirubin 0–0.25 mg/dl. The majority of the preeclamptic patients (70.6%, 80.4%, and 90.2%) had above normal serum uric acid (SUA), ALT, and AST levels, respectively but had normal total and direct bilirubin levels (60.8% and 62.7% respectively). Most of the normotensive pregnant women had normal SUA, ALT, AST, and total and direct bilirubin levels.

An independent sample t-test showed that there was a statistically significant difference in the mean serum uric acid level between preeclamptic pregnant women and normotensive pregnant women (p-value = 0.001). Similarly, there was also a statistically significant difference in the mean serum ALT (p = 0.002) and mean serum AST levels (p = 0.001) between preeclamptic women and normotensive pregnant women. There was, however, no significant difference in the total and direct bilirubin levels in preeclamptic pregnant women were not significantly different from those in normotensive pregnant women (p = 0.135 and p = 0.181, respectively). The age and gestational age of the study participants were also not significantly different between the case and control groups (p = 0.059 and p<0.061). Pre-pregnancy BMI was not significantly higher in preeclamptic patients compared to that of normotensive pregnant women (p = 0.580) (Table 2).

Table 2

Independent t-test for mean comparison of serum uric acid and liver function tests between preeclamptic and normotensive pregnant women, University of Gondar Comprehensive Specialized Hospital, Northwest Ethiopia.

	Preeclampsia (n = 51)	Normotensive pregnancy (n = 51)
	Mean (95% CI)	Mean (95% CI)	p value
SUA (mg/dl)	6.17 ±1.04	3.65± 1.19	0.001*
ALT(IU/L)	35.9± 7.28	20.2 ± 7.23	0.002*
AST(IU/L)	39.7± 6.73	20.5±7.65	0.001*
Bilirubin total(mg/dl)	0.95± 0.45	0.8±0.27	0.135
Bilirubin direct(mg/dl)	0.31±0.12	0.276±0.2	0.181
Age (years)	32.9±6.3	29.5±3.3	0.059
Gestational age (in Weeks)	33 .5±4.3	29.4±4.9	0.061
Prepregnancy BMI	23.4±0.51	21.5±0.23	0.580

*p<0.05 is statistically significant

Comparison of mean SUA, ALT, and AST levels across different age groups of study participants

A post hoc (Bonferroni) test showed that there was a significant difference in mean serum ALT between the age group of 26–34 years and age ≥ 35 years (p = 0.005). However, there was no significant difference in the mean ALT levels between 19-25-year-old and 26-34-year-old (p = 0.72). There was also no significant mean ALT difference between the age groups 19–25 years and ≥ 35 (p = 0.19).

A post hoc (Bonferroni) test also showed that there was a significant difference in mean AST level between the age group of 26–34 years and age ≥ 35 years (p = 0.006). There was also a significant difference in mean AST level between the age group 19–25 years and age ≥ 35 years (p = 0.025). However, there was no significant mean AST level difference between the 19-25-year-old and 26-34-year-old age groups (p = 1.000).

Comparison of mean SUA, ALT, and AST levels at different gestational age categories of study participants

The post hoc (Bonferroni) test showed that there was a significant difference in mean ALT between the gestational age groups of 20–30 weeks and ≥ 36 weeks (p<0.001). However, there was no significant mean ALT level difference between the gestational ages of 20–30 weeks and 31–35 weeks (p = 0.23). Similarly, there was also no significant difference in mean ALT between the age groups 31–35 weeks and ≥ 36 weeks (p = 0.255).

A post hoc (Bonferroni) test also showed that there was a significant difference in mean AST level between the gestational age groups of 20–30 weeks and ≥ 36 weeks (p<0.001). However, there was no significant mean AST level difference between the gestational ages of 20–30 weeks and 31–35 weeks (p = 0.144). Similarly, there was also no significant difference in mean AST between the 31-35-week and ≥ 36-week gestational age groups (p = 0.344).

Multiple linear regression analysis of SUA with different predictor variables of study participants

Variables with a p-value less than 0.2 in the bivariable linear regression analysis were selected for multivariable linear regression analysis. Bivariable linear regression analysis of all predictor variables with SUA level was performed separately, and only age, gestational age, BMI, systolic blood pressure, diastolic blood pressure, and preeclamptic status had a p-value of less than 0.2. The remaining variables were not eligible for multiple linear regressions.

In the multivariable linear regression analysis, age, SBP, DBP, gestational age at diagnosis, and being preeclamptic were significantly associated with SUA level (p = 0.041, p = 0.013, p = 0.041, p = 0.034 p = 0.01, respectively). BMI (P = 0.191) did not show a significant association with SUA. Keeping other variables constant, a one-year increase in age increases SUA by a factor of 0.81, a one-unit increase in SBP increases SUA by a factor of 0.4 and a one-unit increase in DBP increases SUA level by a factor of 0.2. A one-week change in gestational age increased/decreased SUA level by a factor of 0.18. Being preeclamptic increased SUA levels by a factor of 2.37.

Multiple linear regression analysis of ALT with different predictor variables of study participants

Bivariable linear regression analysis of all predictor variables with ALT was performed separately, and only age, gestational age at diagnosis, prepregnancy BMI, systolic blood pressure, diastolic blood pressure, parity, and preeclamptic status had a p-value less than 0.2. The remaining variables were not eligible for multiple linear regressions.

In the multivariable linear regression analysis, age, SBP parity, and preeclamptic status were significantly associated with ALT (p = 0.045, p = 0.046, p = 0.016, p = 0.001, respectively). Keeping other variables constant, a one-year increase in age increases the level of ALT by a factor of 0.51, a one-unit increase in SBP of respondents increases ALT by a factor of 0.2, and being nullipara increases ALT by a factor of 1.2. Being preeclamptic increases serum ALT by a factor of 2 (Table 3).

Table 3

Multiple linear regression analysis of SUA, ALT, AST, and bilirubin levels with different predictor variables of preeclamptic women, University of Gondar Comprehensive Specialized Hospital, Northwest Ethiopia, 2020.

Variables	SUA	ALT	AST	Total bilirubin	Direct bilirubin
	β(95%CI, p value)	β(95%CIp value)	β(9%CI, p value)	β(95%CI,p- value)	β (95%CI, p value)
Age	0.81,0.04	0.51,0.4	1.56,0.031	0.07,0.153	1.9,0.89
Gestational age	0.18,0.03	0.88,0.3	1.51,0.53	0.052,0.89	0.0073,0.9
SBP	0.4,0.013	0.2,0.05	0.42,0.002	0.054,0.962	0.04,0.72
DBP	0.2,0.013	0.1,0.17	0.1,0.037	0.06,0.44	0.05,0.62
BMI	1.82,0.19	0.87,0.6	2.43,0.26	1.4,0.72	0.52,0.63
Nulliparity	0.4,0.2	0.12,0.02	0.78,0.48	0.3,0.07	0.04,0.09
Being preeclamptic	2.37,0.01	2.02,0.001	1.9,0.015	0.8,0.021	0.65,0.02
Previous history of PE	1.3,0.7	3.3,0.15	2.7,0.24	1.4,0.07	2.9,0.61
Family history of PE	0.74,0.34	3.9,0.09	1.62,0.8	2.3,0.87	0.58,0.45
No of fetus of pregnancy	0.9,0.056	1.7,0.14	0.36,0.08	2.7,0.65	0.22,0.76

* P values are considered significant when <0.05.

Multiple linear regression analysis of AST level with different predictor variables of study participants

Bivariable linear regression analysis of all predictor variables with AST was performed separately, and age, gestational age at diagnosis, prepregnancy BMI, systolic blood pressure, diastolic blood pressure, parity, and preeclampsia status had a p-value <0.2.

In the multivariable linear regression analysis, age, SBP, DBP, and being preeclamptic were significantly associated with AST level (p = 0.031, p = 0.002, p = 0.037, p = 0.015, respectively). Keeping other variables constant, a one-year increase in age increases AST level by a factor of 1.56, a one-unit increase in SBP increases AST level by a factor of 0.42 and a one-unit increase in DBP increases AST level by a factor of 0.06. Being preeclamptic increases serum AST by a factor of 1.9 (Table 3).

Multiple linear regression analysis of total bilirubin with different predictor variables of study participants

Bivariable linear regression analysis of all predictor variables with total bilirubin was performed, and age, gestational age at diagnosis, systolic blood pressure, diastolic blood pressure, and preeclamptic status had a p-value less than 0.2. However, only preeclamptic status showed a significant association with total bilirubin in multiple linear regressions (p = 0.025). Being preeclamptic increased the total bilirubin level by a factor of 0.8 (Table 3).

Multiple linear regression analysis of direct bilirubin with different predictor variables of study participants

Bivariable linear regression analysis of all predictor variables with direct bilirubin was performed, and gestational age at diagnosis, systolic blood pressure, diastolic blood pressure, and preeclamptic status had a p-value less than 0.2. Only being preeclamptic showed a significant association with direct bilirubin in multiple linear regressions (p = 0.02) (Table 3).

Discussion

In our study, the majority of the pre-eclamptic patients were found ≥35 years old which is in keeping with the results of a study conducted by Tessema et al., and Jasovic-Siveska, et al., showing that advanced maternal age is associated with the development of preeclampsia [20, 21]. Other studies conducted in Finland and Japan also demonstrated that women of advanced maternal age were 1.5 times more likely to have preeclampsia than women under the age of 35 years. This may be related to the aging of the uterine blood vessels [22, 23].

Our study also showed that the mean ± SD of prepregnancy BMI was found to be 32.4± 0.51 kg/m2 and 21.5 ± 0.23 kg/m2 for the case and control groups, respectively, and there was no statistically significant difference between the two groups. This finding is in contrast with the findings of other studies [24, 25], and this difference may be due to nutritional variation, sample size variation, and/or lifestyle differences.

In our study, the mean serum uric acid level was significantly higher in cases compared to controls (p = 0.001) which is again consistent with the findings of other studies [15, 26].

In addition, the mean serum ALT and AST levels were also significantly higher. Other researchers also noticed an increase in serum ALT levels in preeclampsia [15, 17]. This elevation may be due to placental ischemia followed by a systemic inflammatory response, resulting in endothelial dysfunction causing vasoconstriction and eventual liver and kidney dysfunctions. Other similar studies conducted by Ekun et al. also concluded that there was an increase in transaminases in preeclamptic patients compared to controls [27].

On the other hand, our study showed that there was no significant difference in the total and direct bilirubin levels in preeclamptic pregnant women and those in normotensive pregnant women. Our findings are in contrast with the findings of other studies wherein there were significant increases in serum bilirubin [28], alanine transaminase, and aspartate aminotransferase levels in preeclamptic women compared to normotensive pregnant women [29, 30]. This discrepancy may be due to variations in the extent of hemolysis and/or reference value of the chemistry analyzer. In pre-eclampsia, the main cause of hyperbilirubinemia is thought to be disseminated intravascular coagulation caused by hemolysis and liver cell necrosis [16].

In our study, there were significant differences in the mean SUA, ALT, and AST levels across the different age groups (p = 0.0023, p = 0.0063, p = 0.0037, respectively). The mean SUA level in the age group 26–34 years was 4.43 mg/dL, which then increased to 5.66 mg/dL in the age group >35 years (p = 0.002). The mean ALT level in the 26-34-year-old age group was 24.41 U/L and increased to 32.18 U/L in the age group ≥35 years (p = 0.005). The mean AST level among the 26–34 years age group was 26.17 U/L and increased to 34.93 U/L in the age group ≥35 years (p = 0.006). These findings were consistent with those of other studies, which showed that serum uric acid and liver function tests increased with advancing maternal age [31, 32].

Similarly, there were also significant differences in the mean SUA, ALT, and AST levels across the different gestational age groups (p = 0.005, p = 0.007, p = 0.0005, respectively). The mean SUA levels among the gestational age group of 20–30 weeks was 4.35 mg/dl and increased to 5.69 mg/dl in the gestational age group of ≥ 36 weeks (p<0.001). The mean ALT in the gestational age group of 20–30 weeks was 23.49 U/L and increased to 32.55 U/L in the gestational age group of ≥36 weeks (p<0.001). The mean AST level among the gestational age group of 20–30 weeks was 24.02 U/L and increased to 34.75 U/L in the gestational age group of ≥36 weeks (p<0.001). This was in line with previous studies reporting that higher gestational age was associated with preeclampsia and resulted in high serum uric acid and liver function tests [15].

Lastly, we performed a multivariable linear regression analysis and found a statistically significant positive association between SUA and age, SBP, DBP, gestational age, and preeclamptic status (p = 0.041, p = 0.013, p = 0.041, p = 0.034, p = 0.01, respectively). A study conducted in Ukraine and Europe found that SUA levels increased with increasing age and gestational age[33, 34].

In the present study, preeclamptic patients were positively and significantly associated with SUA, ALT, and AST levels. Preeclampsia was associated with a marked increase in SUA, AST, and ALT levels compared to controls. This was in line with other studies [29, 30].

Conclusion

Our study revealed that serum uric acid, ALT, and AST levels were higher in pre-eclamptic pregnant women compared to those of normotensive pregnant women, and the differences were statistically significant. The study also shows that a significant difference was observed in mean SUA, ALT, and AST levels across different age and gestational age categories among preeclamptic patients.

Transfer Alert

This paper was transferred from another journal. As a result, its full editorial history (including decision letters, peer reviews and author responses) may not be present.

12 Apr 2022

Please address reviewers comments

Please submit your revised manuscript by May 27 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Maria Lourdes Gonzalez Suarez, MD, PhD

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. Please update your submission to use the PLOS LaTeX template. The template and more information on our requirements for LaTeX submissions can be found at http://journals.plos.org/plosone/s/latex.

3. Thank you for stating the following financial disclosure:

NO

At this time, please address the following queries:

a) Please clarify the sources of funding (financial or material support) for your study. List the grants or organizations that supported your study, including funding received from your institution.

b) State what role the funders took in the study. If the funders had no role in your study, please state: “The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.”

c) If any authors received a salary from any of your funders, please state which authors and which funders.

d) If you did not receive any funding for this study, please state: “The authors received no specific funding for this work.”

Please include your amended statements within your cover letter; we will change the online submission form on your behalf.

4. Thank you for stating the following in the Acknowledgments Section of your manuscript:

We would like to express our gratitude to …the University of Gondar

College of Medicine and Health Sciences for the financial, material, and equipment support

for this research work.

We note that you have provided additional information within the Acknowledgements Section that is not currently declared in your Funding Statement. Please note that funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form.

Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows:

NO

Please include your amended statements within your cover letter; we will change the online submission form on your behalf.

5. Thank you for stating the following in your Competing Interests section:

NO

Please complete your Competing Interests on the online submission form to state any Competing Interests. If you have no competing interests, please state "The authors have declared that no competing interests exist.", as detailed online in our guide for authors at http://journals.plos.org/plosone/s/submit-now

This information should be included in your cover letter; we will change the online submission form on your behalf.

6. In your Data Availability statement, you have not specified where the minimal data set underlying the results described in your manuscript can be found. PLOS defines a study's minimal data set as the underlying data used to reach the conclusions drawn in the manuscript and any additional data required to replicate the reported study findings in their entirety. All PLOS journals require that the minimal data set be made fully available. For more information about our data policy, please see http://journals.plos.org/plosone/s/data-availability.

Upon re-submitting your revised manuscript, please upload your study’s minimal underlying data set as either Supporting Information files or to a stable, public repository and include the relevant URLs, DOIs, or accession numbers within your revised cover letter. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories. Any potentially identifying patient information must be fully anonymized.

Important: If there are ethical or legal restrictions to sharing your data publicly, please explain these restrictions in detail. Please see our guidelines for more information on what we consider unacceptable restrictions to publicly sharing data: http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions. Note that it is not acceptable for the authors to be the sole named individuals responsible for ensuring data access.

We will update your Data Availability statement to reflect the information you provide in your cover letter.

Additional Editor Comments:

Thank you for submitting your manuscript. I apologize for the delay during the peer review process. Please address the comments made by our reviewers, especially your suggestions regarding liver function panel to be considered as biomarker of early stages of preeclampsia, please provide data on when the samples where obtained regarding gestational age.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: No

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: I Don't Know

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: No

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The manuscript by Dejenie et al. explores common biochemical biomarkers such as uremic acid and liver function enzymes, ALT and AST, in pregnant women with and without preeclampsia. While the manuscript provides valuable insight into the biochemical profile of women with preeclampsia, there is some major insufficiency.

Minor comments:

1. Table 4: it is not clear which column is "Cases" and which is "Controls"

2. t-test was applied on several spots where it was obvious from presented data that CV was above 30%

3. The authors did not address early vs. late preeclampsia differences.

4. Comparison studies of biochemical markers across gestational age and different age groups would look better it it were presented in tables rather than textually.

Major comments:

1. Based on the results we can only conclude that there are differences among PE vs normotensive pregnancies. However the authors make a conclusion of SUA, ALT and AST as early biomarkers of preeclampsia. This is incorrect, as they did not report when were the samples collected. Based on their study design, these women have already been diagnosed with PE so increase of these biochemical parameters could be the consequence of the disease, not early marker of it.

2. The authors report that women with preeclampsia were older compared to the control group. In the same time they observe positive association between age and biochemical markers such as ALT and AST. It is possible that age is actually pulling the difference so authors should have performed adequate statistical analysis to check this.

3. I am confused that the authors did not observe the difference in bilirubin concentrations between cases and controls, but report "being preeclamptic shows significant association with total bilirubin in multiple linear regressions"

Reviewer #2: Review:

In this cross-sectional study, Dr. Hassen et al aimed to evaluate the utility of levels of serum uric acid and liver function tests/transaminases as early biomarkers of pre-eclampsia-related end organ damage. Both elevated serum uric acid and liver function tests/transaminases are potential laboratory findings in pre-eclampsia. Although the findings are not necessarily novel, the text is clear and easy to read, but I do have some suggestions as outlined below. There are also inconsistencies in some of the authors’ statements, which need to be corrected/modified.

Abstract:

Line 11: Pre-eclampsia can be described as new-onset hypertension…

Line 12: …or ANY indication…

Line 13: Liver and kidney dysfunction…

Line 14: …common MANIFESTATIONS of end-organ damage…

Line 15: …most common cause of LIVER and KIDNEY dysfunction…(Please change all “renal” terms to “kidney” instead.)

Line 15: Pre-eclampsia is the most common cause of liver and kidney dysfunction – in what patient population? – due to hypoxia and endothelial dysfunction.

Line 17: Hyperuricemia indicates kidney dysfunction…

Line 18: …severity of pre-eclampsia; therefore, the objective of this study is to evaluate the utility of the levels of serum uric acid and liver function tests [alanine aminotransferase (ALT) and aspartate aminotransferase (AST)] as early biomarkers…

Line 20: An institution-based comparative cross-sectional study design…

Line 21: …conducted, …

Line 22: The parameters measured were levels of serum uric acid and liver function tests.

Line 24: There were statistically significant differences in the mean serum uric acid, ALT, and AST levels …

Line 26: There were no statistically significant differences in the mean total and direct bilirubin levels…

Line 27: There were also statistically significant differences in the mean serum uric acid, ALT, and AST levels across different gestational age categories.

Line 30: Our study revealed that serum uric acid, ALT, and AST levels were HIGHER in pre-eclamptic pregnant women compared to THOSE of healthy pregnant women, and the differences were statistically significant. As such, serum uric acid and liver function tests may be considered as early biomarkers of pre-eclampsia-related end organ damage.

Background:

Line 38: Pregnancy-induced hypertension is a major cause of maternal and fetal morbidity and mortality.

Line 40: …hypertensive disorders exist, including…

Lines 43-53: I’m not sure if these definitions should be included as they make the background longer than it should be.

Line 54: Pre-eclampsia is defined as new-onset hypertension…

Line 55: …or ANY sign of end-organ damage…

Line 56: Please add – “Proteinuria, however, is not a requirement anymore to make a diagnosis of pre-eclampsia.” (Reference: PMID: 32063058)

Line 58: Disease severity varies, and it is considered severe if it is associated with one or more of the following: …

Line 59: … at least 6 hours apart, a 24-hour protein of >5 g, and/or other symptoms, such as…

Line 62: Please add – “The main underlying disease mechanism is endothelial dysfunction affecting multiple organs, including brain, liver, kidneys, and placenta.” (Reference: PMID: 32063058)

Line 66: Its global prevalence is extremely variable…

Line 69: In Africa, it occurs in 10% of pregnancies…

Line 70: Around 4 in 100 women experience PROBLEMS with high blood pressure and kidney dysfunction…

Line 75: …maternal deaths, most of which are…

Line 76: Liver and kidney dysfunction…common manifestations of end-organ damage…

Line 78: Hyperuricemia indicates kidney dysfunction because of decreased glomerular filtration, decreased tubular secretion, and/or increased proximal tubular reabsorption. (Reference: PMID: 15660336) It is also an independent risk factor for cardiovascular diseases as it has been suggested that it alters vascular function and mediates vascular inflammation. As such, hyperuricemia can perhaps predict the severity of pre-eclampsia.

Lines 81-85: Pre-eclampsia is also the most common cause of liver dysfunction in 3% of pregnancies because of microvesicular fat deposition and reduced blood flow to the liver potentially causing ischemia and periportal hemorrhage. (References: PMID 3189435 and PMID 8607495). ALT and AST are usually normal, but when they become elevated and are accompanied by abdominal pain, it almost always suggests the severe end of the disease spectrum.

Line 86: As such, this study was undertaken to evaluate the utility of levels of serum uric acid and liver function tests as early biomarkers of pre-eclampsia-related end organ damage…

Methods and Materials:

Line 91: An institution-based comparative cross-sectional study design…

Line 92: All patient had antenatal care follow-up…

Line 94: We excluded pregnant women who had…, those with a history of medication USE (eg…), and those with substance abuse (eg smoking and alcohol consumption) as these factors may affect the outcome variables.

Line 101: …to evaluate the levels of serum uric acid and liver function tests…

Line 104: …sample size calculations, like Minitab and Epi-info, …

Line 105: As an input, G-Power requires selecting appropriate test family (t-test in this case) and statistical test within test family (linear regression in this case) and specifying alpha error probability, …

Line 108: …(80%), and effect size…

Line 112: Trained nurses…

Line 113: Four laboratory technicians were oriented about blood sample collection and storage. Questionnaires were filled by face-to-face interviews with participants. Pre-pregnancy BMIs were calculated from…

Line 118: New sentence – “Two consecutive blood pressure readings were also measured.”

Line 122: It was then centrifuged…

Line 124: …refrigerator UNTIL…

Line 132: …to check IF there is any data inconsistency and to avoid any problem with data entry processes…

Line 138: All data were checked for cleaning and completeness. Data processing and analysis obtained from…

Line 144: …and other assumptions WERE checked before doing any statistical analysis and WERE fulfilled.

Line 147: WITH bivariable linear regression analysis, variables with…WHILE WITH multivariable linear regression analysis, variables with…

Line 152: Ethical clearance was obtained from…

Line 159: The present study recruited 102 female study participants, 51 of whom were pre-eclamptic pregnant women while 51 of whom were healthy pregnant women with gestational age above 20 weeks.

Line 161: The mean ages of pre-eclamptic patients and healthy controls were 32.9 years (range: 21-42) and 29.5 years (range: 23-35) RESPECTIVELY.

Line 162: The mean gestational ages of pre-eclamptic patients and healthy controls were 33.6 weeks (range: 22-39) and 29.3 weeks (range: 21-38) RESPECTIVELY.

Line 164: Majority of the study participants were married. I will delete the statement - “and the rest were single in both case and control groups.”

Line 166: Among the healthy controls, 41 (80.4%) were living in urban areas.

Line 176: Based on the calculation, …

Line 178: Majority of the healthy controls (94.1%) had normal BMI while the rest (5.9%) were overweight.

Line 179: None in the control group WAS underweight. Blood pressures of the study participants were recorded…

Line 186: I think it would be better to include the reference ranges for the different laboratory parameters in Table 3.

Line 188: Majority of the pre-eclamptic patients…but had normal total and direct bilirubin levels (60.8% and 62.7% respectively).

***Under paragraphs 3.3 and 3.4, the abbreviation SUA was used instead of serum uric acid. For consistency, the authors can start using the abbreviations at the start of the manuscript and make sure to spell them out on first usage and then use the abbreviations afterwards. Aside from SUA, the rule also applies to ALT and AST.

Lines 196-199: …statistically significant difference…

Line 200: There was, however, no significant difference in the total and direct bilirubin levels…

Line 202: The age and the gestational age of the study participants were also not significantly different…

Line 204: …compared to that of healthy controls…

Line 215: …; however, there was no significant difference in the mean SUA levels between the age groups 19-25 years and >35 years…

Line 219: …in mean ALT levels across different age groups…

Line 221: …; however, there was no significant difference in the mean ALT levels between the age groups…

***General comments for the results sections:

1. The word “level” should be inserted after all these phrases “mean SUA, ALT, and AST,” i.e. mean SUA level, mean ALT level, and mean AST level.

2. The preposition“of” can be deleted in all sentences containing the phrase “between the age groups,” i.e. between the age groups 19-25 years and 26-34 years. There is no need for the preposition “of” in this case.

3. To be grammatically correct, there should be a semicolon and a comma before and after the word “however” - …; however,…

Discussion:

Lines 303-306: I think that these statements need not be included in the discussion. I would consider starting the discussion by summarizing your key findings.

Line 307: In our study, majority of the pre-eclamptic patients were found…, which is in keeping with the results of a study conducted by…showing that advanced maternal age…

Line 309: Other studies conducted in Finland and Japan also demonstrated that…

Line 312: Our study also showed that the…were found to be…

Line 313: …, and there was no statistically significant difference between the two groups. This finding is in contrast in with the findings of other studies, and this difference may be due to…

Line 317: In our study, the mean serum uric acid level was significantly higher in cases compared to controls…, which is again consistent with the findings of other studies. I would elaborate more on these findings and explain the mechanism/s behind these findings. In addition, the mean serum ALT and AST levels were also significantly higher…

Line 323: …inflammatory response, resulting in endothelial dysfunction causing vasoconstriction and eventual liver and kidney dysfunction…

Lines 325-331: These statements are somewhat a repetition of the statements in lines 320-324. I would combine these 2 paragraphs to be more cohesive.

Line 332: On the other hand, our study showed that there was no significant difference in the total and direct bilirubin levels…

Line 334: Our findings are in contrast with the findings of other studies wherein there were significant increases in…

Line 336: …due to variations in the extent of…

Line 337: In pre-eclampsia, the main cause of hyperbilirubinemia is thought to be disseminated…

Line 340: In our study, there were significant differences in the mean SUA, ALT, and AST levels across the different age groups…The mean SUA level in the age group 26-34 years was 4.43 mg/dL, which then increased to 5.66 mg/dL in the age group >35 years. Please modify the sentences after this one to the same format.

Line 346: These findings were consistent with those of other studies, which showed that…

Line 348: Similarly, there were also significant differences in the mean SUA, ALT, and AST levels across the different gestational age groups…Please also modify the sentences after this one to the same format as mentioned above.

Line 357: Lastly, we performed a multivariable linear regression analysis and found a statistically significant positive association between…

Lines 362-365: If I am not mistaken, these statements are inaccurate as your study did not show any significant difference in the total and direct bilirubin levels between pre-eclamptic and healthy pregnant women.

Conclusion:

Our study revealed that serum uric acid, ALT, and AST levels were HIGHER in pre-eclamptic pregnant women compared to THOSE of healthy pregnant women, and the differences were statistically significant. As such, serum uric acid and liver function tests may be considered as early biomarkers of pre-eclampsia-related end organ damage.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

Submitted filename: PLOS ONE_Review.docx

Click here for additional data file.

27 Apr 2022

April 27, 2022

Rebuttal letter

Pone-d-21-30928

Evaluation of serum uric acid and liver function tests among pregnant women with and without preeclampsia at the University of Gondar comprehensive specialized hospital, northwest Ethiopia

Dear Editors and Reviewers,

We appreciate the constructive comments and suggestions for improving our manuscript. Based on the comments and suggestions, we have made corrections and modifications and provided point-by-point responses to the comments and suggestions. Please find our responses in green under the comments and suggestions presented by academic editors and reviewers in yellow. Below are our responses to each point raised by the academic editor and reviewers.

Best regards!

Tadesse Asmamaw Dejenie, on behave of all authors

Response to academic editor’s comment

Comment 1: Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming

Author response: Thank you very much for sending the link to the PLOS ONE style templates; we have taken your advice and updated our manuscript to meet the style requirements of PLOS ONE. Please check it in the revised manuscript.

Comment 2a: Please clarify the sources of funding (financial or material support) for your study. List the grants or organizations that supported your study, including funding received from your institution.

Author response: We'd like to thank you for your insightful remarks and suggestions. We've considered the suggestions and made the necessary revisions. For this work, we did not receive any funds.

Comment 2b: State what role the funders took in the study. If the funders had no role in your study, please state: “The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.”

Author response: We stated that we did not receive any funding for this study and that the University of Gondar was the study setting.

Comment 2c: If any authors received a salary from any of your funders, please state which authors and which funders.

Author response: No author received a salary since there is no funder for this work.

Comment 2d: If you did not receive any funding for this study, please state: “The authors received no specific funding for this work.

Author response: Many thanks for your suggestion; in the revised manuscript, we stated that "the authors received no specific funding for this work." Please check it in the revised manuscript.

Comment 3: We note that you have provided additional information within the Acknowledgements Section that is not currently declared in your Funding Statement. Please note that funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form.

Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows:

Author response: We'd like to thank you for your insight. This section has been removed entirely. For further information, kindly check the revised manuscript.

Comment 4: Please complete your Competing Interests on the online submission form to state any Competing Interests. If you have no competing interests, please state "The authors have declared that no competing interests exist."

Author response: We are grateful for your suggestions and stated that the authors have declared that no competing interests exist." Please find it in the revised manuscript.

Comment 5: In your Data Availability statement, you have not specified where the minimal data set underlying the results described in your manuscript can be found. Upon re-submitting your revised manuscript, please upload your study’s minimal underlying data set as either Supporting Information files or to a stable, public repository and include the relevant URLs, DOIs, or accession numbers within your revised cover letter.

Author response: We'd want to express our gratitude once more for bringing this to our attention. We stated that the authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the manuscript in the revised manuscript. Please check it.

Response to Reviewers’ comment

Reviewer #1:

Minor comments:

Comment 1: Table 4: it is not clear which column is "Cases" and which is "Controls"

Author response: Thank you so much; we've made the necessary modifications. Please check it in the revised manuscript.

Comment 2: t-test was applied on several spots where it was obvious from presented data that CV was above 30%

Author response: Thank you very much for taking the time to share your insights. We used an independent t-test because we thought it would be a reasonable statistical model test for comparing measurable parameters across case and control groups in the case of this study.

Comment 3: The authors did not address early vs. late preeclampsia differences.

Author response: We really appreciate your insights. We considered that it is obvious that the complication is more common in late preeclamptic women than in early preeclamptic women.

Comment 4: Comparison studies of biochemical markers across gestational age and different age groups would look better it were presented in tables rather than textually.

Author response: We sincerely appreciate your insight. Since we were afraid of having a bulky table in our manuscript, we removed those suggested tables, but we have now inserted these tables as per your suggestion. Please find tables 5 and 6 of the revised manuscript.

Major comments:

Comment 1: Based on the results we can only conclude that there are differences among PE vs normotensive pregnancies. However the authors make a conclusion of SUA, ALT and AST as early biomarkers of preeclampsia. This is incorrect, as they did not report when the samples were collected. Based on their study design, these women have already been diagnosed with PE so increase of these biochemical parameters could be the consequence of the disease, not early marker of it.

Author response: Thank you so much for everything. We've taken your concern into account and made the necessary modifications. Please check it in the revised manuscript.

Comment 2: The authors report that women with preeclampsia were older compared to the control group. In the same time they observe positive association between age and biochemical markers such as ALT and AST. It is possible that age is actually pulling the difference so authors should have performed adequate statistical analysis to check this.

Author response: Thanks, Even though we have stated that women with preeclampsia are older than the control groups, their age difference is insignificant. Please see table 4 of the manuscript.

Comment 3: I am confused that the authors did not observe the difference in bilirubin concentrations between cases and controls, but report "being preeclampsia shows significant association with total bilirubin in multiple linear regressions"

Author response: Please accept our apology for the confusion; we have already done that in table 4 of the manuscript. I think this is because of Table 4; it was not clear which column was "cases" and which was "controls" for you. Now we separate the case and control columns in this table. Please find it in the revised manuscript.

Reviewer #2:

Abstract:

Suggestion 1: Line 11: Pre-eclampsia can be described as new-onset hypertension…

Author response: Indeed, thank you very much for your suggestion to improve the clarity of our manuscript. We have modified it as per your suggestion. Please check it in line 11 of the revised manuscript.

Suggestion 2: Line 12: …or ANY indication…

Author response: Indeed, thank you very much for your suggestion to improve the clarity of our manuscript. We have modified it as per your suggestion. Please check it in line 12 of the revised manuscript.

Suggestion 3: Line 13: Liver and kidney dysfunction…

Author response: Indeed, thank you very much for your suggestion to improve the clarity of our manuscript. We have modified it as per your suggestion. Please check it in line 13 of the revised manuscript.

Suggestion 4: Line 14: …common MANIFESTATIONS of end-organ damage…

Author response: Indeed, thank you very much for your suggestion to improve the clarity of our manuscript. We have modified it as per your suggestion. Please check it in line 14 of the revised manuscript.

Suggestion 5: Line 15: …most common cause of LIVER and KIDNEY dysfunction… (Please change all “renal” terms to “kidney” instead.)

Author response: Indeed, thank you very much for your suggestion to improve the clarity of our manuscript. We have modified it as per your suggestion. Please check it in line 15 & 16 of abstract section of the manuscript and throughout the revised manuscript.

Suggestion 6: Line 15: Pre-eclampsia is the most common cause of liver and kidney dysfunction – in what patient population? – Due to hypoxia and endothelial dysfunction.

Author response: In a population of pregnant women, ischemia caused by hypoperfusion of the placenta leads to the release of many mediators, causing endothelial dysfunction and a cascade of systemic disorders including hypertension, proteinuria, edema, and platelet aggregation (Szpera-Gozdziewicz et al., 2014).

Suggestion 7: Line 17: Hyperuricemia indicates kidney dysfunction…

Author response: Author response: Indeed, thank you very much for your suggestion to improve the clarity of our manuscript. We have modified it as per your suggestion. Please check it in line 17 of the revised manuscript.

Suggestion 8: Line 18: …severity of pre-eclampsia; therefore, the objective of this study is to evaluate the utility of the levels of serum uric acid and liver function tests [alanine aminotransferase (ALT) and aspartate aminotransferase (AST)] as early biomarkers…

Author response: Indeed, thank you very much for your suggestion to improve the clarity of our manuscript. We have modified it as per your suggestion. Please check it in lines 18-20 of the revised manuscript.

Suggestion 9: Line 20: An institution-based comparative cross-sectional study design…

Author response: Author response: Indeed, thank you very much for your suggestion to improve the clarity of our manuscript. We have modified it as per your suggestion. Please check it in line 21 of the revised manuscript.

Suggestion 10: Line 21: …conducted,

Author response: Author response: Indeed, thank you very much for your suggestion to improve the clarity of our manuscript. We have modified it as per your suggestion. Please check it in line 21 of the revised manuscript.

Suggestion 11: Line 22: The parameters measured were levels of serum uric acid and liver function tests.

Author response: Indeed, thank you very much for your suggestion to improve the clarity of our manuscript. We have modified it as per your suggestion. Please check it in line 23 of the revised manuscript.

Suggestion 12: Line 24: There were statistically significant differences in the mean serum uric acid, ALT, and AST levels …

Author response: Indeed, thank you very much for your suggestion to improve the clarity of our manuscript. We have modified it as per your suggestion. Please check it in line 24-26 of the revised manuscript.

Suggestion 13: Line 26: There were no statistically significant differences in the mean total and direct bilirubin levels…

Author response: Indeed, thank you very much for your suggestion to improve the clarity of our manuscript. We have modified it as per your suggestion. Please check it in line 27 of the revised manuscript.

Suggestion 14: Line 27: There were also statistically significant differences in the mean serum uric acid, ALT, and AST levels across different gestational age categories.

Author response: Indeed, thank you very much for your suggestion to improve the clarity of our manuscript. We have modified it as per your suggestion. Please check it in lines 27 & 28 of the revised manuscript.

Suggestion 15: Line 30: Our study revealed that serum uric acid, ALT, and AST levels were HIGHER in pre-eclamptic pregnant women compared to THOSE of healthy pregnant women, and the differences were statistically significant. As such, serum uric acid and liver function tests may be considered as early biomarkers of pre-eclampsia-related end organ damage.

Author response: Indeed, thank you very much for your suggestion to improve the clarity of our manuscript. We have modified the conclusion section of this manuscript as per your and reviewer #1 suggestions. Please check it in the conclusion and abstraction section of the revised manuscript.

Introduction:

Suggestion 16: Line 38: Pregnancy-induced hypertension is a major cause of maternal and fetal morbidity and mortality.

Author response: Indeed, thank you very much for your suggestion to improve the clarity of our manuscript. We have modified it as per your suggestion. Please check it in lines 36 & 37 of the revised manuscript.

Suggestion 17: Line 40: …hypertensive disorders exist, including…

Author response: Indeed, thank you very much for your suggestion to improve the clarity of our manuscript. We have modified it as per your suggestion. Please check it in line 37 of the revised manuscript.

Suggestion 18: Lines 43-53: I’m not sure if these definitions should be included as they make the background longer than it should be.

Author response: Indeed, thank you very much for your suggestion to improve the clarity of our manuscript. We have accepted and deleted it as per your suggestion. Please check it in the revised manuscript.

Suggestion 19: Line 54: Pre-eclampsia is defined as new-onset hypertension…

Author response: Indeed, thank you very much for your suggestion to improve the clarity of our manuscript. We have modified it as per your suggestion. Please check it in line 45 of the revised manuscript.

Suggestion 20: Line 55: …or ANY sign of end-organ damage…

Author response: Indeed, thank you very much for your suggestion to improve the clarity of our manuscript. We have modified it as per your suggestion. Please check it in line 46 of the revised manuscript.

Suggestion 21: Line 56: Please add – “Proteinuria, however, is not a requirement anymore to make a diagnosis of pre-eclampsia.” (Reference: PMID: 32063058)

Author response: Thank you very much for your suggestion to improve the clarity of our manuscript. We have added and cited the above-mentioned sentences as per your suggestion. Please check it in lines 48-49 of the revised manuscript.

Suggestion 22: Line 58: Disease severity varies, and it is considered severe if it is associated with one or more of the following: …

Author response: Thank you so much for your suggestion to improve the clarity of our manuscript. We have modified it as per your suggestion. Please check it in line 50 of the revised manuscript

Suggestion 23: Line 59: … at least 6 hours apart, a 24-hour protein of >5g, and/or other symptoms, such as…

Author response: Indeed, thank you very much for your suggestion to improve the clarity of our manuscript. We have modified it as per your suggestion. Please check it in line 52 of the revised manuscript.

Suggestion 24: Line 62: Please add – “The main underlying disease mechanism is endothelial dysfunction affecting multiple organs, including brain, liver, kidneys, and placenta.” (Reference: PMID: 32063058)

Author response: Thank you very much for your suggestion to improve the clarity of our manuscript. We have added and cited the above-mentioned sentences as per your suggestion. Please check it in lines 53-54 of the revised manuscript.

Suggestion 25: Line 66: Its global prevalence is extremely variable…

Author response: Thank you so much for your advice on how to make our manuscript clear. We've made the changes you suggested. Please double-check it in the revised manuscript on line 61.

Suggestion 26: Line 69: In Africa, it occurs in 10% of pregnancies…

Author response: Thank you so much for your suggestions on how to improve the clarity of our manuscript. We've made the modifications you recommended. Please check it on lines 63-64 of the revised manuscript.

Suggestion 27: Line 70: Around 4 in 100 women experience PROBLEMS with high blood pressure and kidney dysfunction…

Author response: Thank you for your suggestions for improving the clarity of our manuscript. Your suggestions have been accepted. In the revised manuscript, on lines 64-65, please double-check it.

Suggestion 28: Line 75: …maternal deaths, most of which are…

Indeed, thank you very much for your suggestion to improve the clarity of our manuscript. We have modified it as per your suggestion. Please check it in lines 68-69 of the revised manuscript

Suggestion 29: Line 76: Liver and kidney dysfunction…common manifestations of end-organ damage…

Author response: Thank you for your suggestions for improving the clarity of our manuscript. Your suggestions have been accepted. Please double-check lines 70-71 in the revised manuscript.

Suggestion 30: Line 78: Hyperuricemia indicates kidney dysfunction because of decreased glomerular filtration, decreased tubular secretion, and/or increased proximal tubular reabsorption. (Reference: PMID: 15660336) It is also an independent risk factor for cardiovascular diseases as it has been suggested that it alters vascular function and mediates vascular inflammation. As such, hyperuricemia can perhaps predict the severity of pre-eclampsia.

Author response: Thank you very much for your suggestion to improve the clarity of our manuscript. We have added and cited the above-mentioned sentences as per your suggestion. Please check it in lines 72-73 of the revised manuscript.

Suggestion 31: Lines 81-85: Pre-eclampsia is also the most common cause of liver dysfunction in 3% of pregnancies because of micro vesicular fat deposition and reduced blood flow to the liver potentially causing ischemia and periportal hemorrhage. (References: PMID 3189435 and PMID 8607495). ALT and AST are usually normal, but when they become elevated and are accompanied by abdominal pain, it almost always suggests the severe end of the disease spectrum.

Author response: Thank you very much for your suggestion to improve the clarity of our manuscript. We have added and cited the above-mentioned sentences as per your suggestion. Please check it in lines 77-82 of the revised manuscript

Suggestion 32: Line 86: As such, this study was undertaken to evaluate the utility of levels of serum uric acid and liver function tests as early biomarkers of pre-eclampsia-related end organ damage…

Author response: Thank you for your suggestions on making our manuscript more clear. Your recommendations have been taken into consideration. Please double-check lines 83–85 in the revised manuscript.

Methods and Materials:

Suggestion 33: Line 91: An institution-based comparative cross-sectional study design…

Author response: Thank you for your suggestions on making our manuscript more clear. Your recommendation has been accepted, and we have modified it. Please see line 88 of the revised manuscript.

Suggestion 34: Line 92: All patients had antenatal care follow-up…

Author response: Thank you for your suggestions on making our manuscript more clear. Your recommendation has been accepted, and we have modified it. Please see lines 89-90 of the revised manuscript.

Suggestion 35: Line 94: We excluded pregnant women who had…, those with a history of medication USE (eg…), and those with substance abuse (eg smoking and alcohol consumption) as these factors may affect the outcome variables.

Author response: you for your suggestions on making our manuscript more clear. Your recommendation has been accepted, and we have modified it. Please see lines 91-94 of the revised manuscript.

Suggestion 36: Line 101: …to evaluate the levels of serum uric acid and liver function tests…

Author response: Thank you for your suggestions on making our manuscript more clear. Your recommendation has been accepted, and we have modified it. Please see line 88 of the revised manuscript.

Suggestion 37: Line 104: …sample size calculations, like Minitab and Epi-info, …

Author response: Thank you for your suggestions on making our manuscript more clear. Your recommendation has been accepted, and we have modified it. Please see line 101 of the revised manuscript.

Suggestion 38: Line 105: As an input, G-Power requires selecting appropriate test family (t-test in this case) and statistical test within test family (linear regression in this case) and specifying alpha error probability, …

Author response: Thank you for your suggestions on making our manuscript more clear. Your recommendation has been accepted, and we have modified it. Please see lines 102-104 of the revised manuscript.

Suggestion 39: Line 108: …(80%), and effect size…

Author response: Thank you for your suggestions on making our manuscript more clear. We've taken your suggestion and made modifications to it. Please check line 105 of the revised manuscript.

Suggestion 40: Line 112: Trained nurses…

Author response: Thank you for your suggestions on making our manuscript more clear. We've taken your suggestion and made modifications to it. Please check line 109 of the revised manuscript.

Suggestion 41: Line 113: Four laboratory technicians were oriented on blood sample collection and storage. Questionnaires were filled out through face-to-face interviews with participants. Pre-pregnancy BMIs were calculated from…

Author response: Thank you for your suggestions on making our manuscript more clear. We've taken your suggestion and made modifications to it. Please check lines 110-112 of the revised manuscript.

Suggestion 42: Line 118: New sentence – “Two consecutive blood pressure readings were also measured.”

Author response: Thank you for your suggestions on making our manuscript more clear. We've added this new sentence to our manuscript to make it more clear. Please line 116 of the revised manuscript.

Suggestion 43: Line 122: It was then centrifuged…

Author response: Thank you for your suggestions on making our manuscript more clear. We've taken your suggestion and made modifications to it. Please check line 19 of the revised manuscript.

Suggestion 44: Line 124: …refrigerator UNTIL…

Author response: Thank you for your suggestions on improving the clarity of our manuscript. We've taken your advice and modified it. Please check line 121 of the revised manuscript.

Suggestion 45: Line 132: …to check IF there is any data inconsistency and to avoid any problem with data entry processes…

Author response: Thank you for your suggestions on improving the clarity of our manuscript. We've taken your advice and modified it. Please check line 129 of the revised manuscript.

Suggestion 46: Line 138: All data were checked for cleaning and completeness. Data processing and analysis obtained from…

Author response: Thank you for your suggestions on improving the clarity of our manuscript. We've taken your advice and modified it. Please check line 135 of the revised manuscript.

Suggestion 47: Line 144: …and other assumptions WERE checked before doing any statistical analysis and WERE fulfilled.

Author response: Thank you for your suggestions on improving the clarity of our manuscript. We've taken your advice and modified it. Please check lines 141 & 142 of the revised manuscript.

Suggestion 48: Line 147: WITH bivariable linear regression analysis, variables with…WHILE WITH multivariable linear regression analysis, variables with…

Author response: Thank you for your suggestions on improving the clarity of our manuscript. We've taken your advice and modified it. Please check lines 145 & 146 of the revised manuscript.

Suggestion 49: Line 152: Ethical clearance was obtained from…

Author response: Thank you for your suggestions on improving the clarity of our manuscript. We've taken your advice and modified it. Please check line 402 of the revised manuscript.

Suggestion 50: Line 159: The present study recruited 102 female study participants, 51 of whom were pre-eclamptic pregnant women while 51 of whom were healthy pregnant women with gestational age above 20 weeks.

Author response: Thank you for your suggestions on improving the clarity of our manuscript. We've taken your advice and modified it. Please check lines 150-152 of the revised manuscript.

Suggestion 51: Line 161: The mean ages of pre-eclamptic patients and healthy controls were 32.9 years (range: 21-42) and 29.5 years (range: 23-35) RESPECTIVELY. Line 162: The mean gestational ages of pre-eclamptic patients and healthy controls were 33.6 weeks (range: 22-39) and 29.3 weeks (range: 21-38) RESPECTIVELY.

Author response: Thank you for your suggestions on improving the clarity of our manuscript. We've taken your advice and modified it. Please check lines 155-159 of the revised manuscript.

Suggestion 52: Line 164: Majority of the study participants were married. I will delete the statement - “and the rest were single in both case and control groups.”

Author response: Thank you for your suggestions on improving the clarity of our manuscript. We've accepted and modified it. Please check line 155 of the revised manuscript.

Suggestion 53: Line 166: Among the healthy controls, 41 (80.4%) were living in urban areas.

Author response: Thank you for your suggestions on improving the clarity of our manuscript. We've taken your advice and modified it. Please check lines 156-159 of the revised manuscript.

Suggestion 54: Line 176: Based on the calculation, …

Author response: Thank you for your suggestions on improving the clarity of our manuscript. We've taken your advice and modified it. Please check line 171 of the revised manuscript.

Suggestion 55: Line 178: Majority of the healthy controls (94.1%) had normal BMI while the rest (5.9%) were overweight. None in the control group WAS underweight. Blood pressures of the study participants were recorded…

Author response: Thank you for your suggestions on improving the clarity of our manuscript. We've taken your advice and modified it. Please check lines 173-175 of the revised manuscript.

Suggestion 56: Line 186: I think it would be better to include the reference ranges for the different laboratory parameters in Table 3.

Author response: Thanks to your suggestion, we've included the reference ranges for the different laboratory parameters in Table 3. Please check in table 3 of the revised manuscript.

Suggestion 57: Line 188: Majority of the pre-eclamptic patients…but had normal total and direct bilirubin levels (60.8% and 62.7% respectively).

Author response: Thank you for your suggestions on improving the clarity of our manuscript. We've taken your advice and modified it. Please check lines 183-185 of the revised manuscript.

Suggestion 58: ***Under paragraphs 3.3 and 3.4, the abbreviation SUA was used instead of serum uric acid. For consistency, the authors can start using the abbreviations at the start of the manuscript and make sure to spell them out on first usage and then use the abbreviations afterwards. Aside from SUA, the rule also applies to ALT and AST.

Author response: Thank you for your suggestions. We've corrected it. Please check it throughout the revised manuscript.

Suggestion 59: Lines 196-199: …statistically significant difference…

Author response: Thank you for your suggestions on improving the clarity of our manuscript. We've taken your advice and modified it. Please check lines 190-193 of the revised manuscript.

Suggestion 60: Line 200: There was, however, no significant difference in the total and direct bilirubin levels…

Author response: Thank you for your suggestions on improving the clarity of our manuscript. We've taken your advice and modified it. Please check line 194 of the revised manuscript.

Suggestion 61: Line 202: The age and the gestational age of the study participants were also not significantly different…

Author response: Thank you for your suggestions on improving the clarity of our manuscript. We've taken your advice and modified it. Please check line 197 of the revised manuscript.

Suggestion 62: Line 204: …compared to that of healthy controls…

Author response: Thank you for your suggestions on improving the clarity of our manuscript. We've taken your advice and modified it. Please check line 199 of the revised manuscript.

Suggestion 63: Line 215: …; however, there was no significant difference in the mean SUA levels between the age groups 19-25 years and >35 years…

Author response: Thank you for your suggestions on making our manuscript more clear. We've taken your suggestion and modified it. Kindly check line 208 of the revised manuscript.

Suggestion 64: Line 219: …in mean ALT levels across different age groups…

Author response: Thank you for your suggestions on making our manuscript more clear. We've taken your suggestion and modified it. Kindly check lines 213-215 of the revised manuscript.

Suggestion 65: Line 221: …; however, there was no significant difference in the mean ALT levels between the age groups…

Author response: Thank you for your suggestions on making our manuscript more clear. We've taken your suggestion and modified it. Kindly check lines 213-215 of the revised manuscript.

Suggestion 66: ***General comments for the results sections:

1. The word “level” should be inserted after all these phrases “mean SUA, ALT, and AST,” i.e. mean SUA level, mean ALT level, and mean AST level.

2. The preposition“of” can be deleted in all sentences containing the phrase “between the age groups,” i.e. between the age groups 19-25 years and 26-34 years. There is no need for the preposition “of” in this case.

3. To be grammatically correct, there should be a semicolon and a comma before and after the word “however” - …; however,…

Author response: Thank you for your suggestions on how to improve the clarity of our manuscript. We've made significant changes based on your suggestions. Please check throughout the result section of our revised manuscript.

Discussion:

Suggestion 67: Lines 303-306: I think that these statements need not be included in the discussion. I would consider starting the discussion by summarizing your key findings.

Author response: As per your suggestions, we have deleted and made significant changes. Please find it in the first paragraph of the discussion section of the revised manuscript.

Suggestion 68: Line 307: In our study, majority of the pre-eclamptic patients were found…, which is in keeping with the results of a study conducted by…showing that advanced maternal age…

Author response: Thank you for your suggestions on improving the clarity of our manuscript. We took your suggestion and modified it. Please take a look at lines 306-308 of the revised manuscript.

Suggestion 69: Line 309: Other studies conducted in Finland and Japan also demonstrated that…

Author response: Thank you for your suggestions on making our manuscript more clear. We acknowledged your suggestion and modified it to make it better. Please see line 309 of the revised manuscript for more information.

Suggestion 70: Line 312: Our study also showed that the…were found to be…

Author response: Thank you for your suggestions on making our manuscript more clear. We acknowledged your suggestion and modified it to make it better. Please check it line 312 of the revised manuscript for more information.

Suggestion 72: Line 313: …, and there was no statistically significant difference between the two groups. This finding is in contrast in with the findings of other studies, and this difference may be due to…

Author response: Thank you for your suggestions on improving the clarity of our manuscript. We accepted your suggestion into consideration and improved it. More information can be found on lines 313-115 of the revised manuscript.

Suggestion 73: Line 317: In our study, the mean serum uric acid level was significantly higher in cases compared to controls…, which is again consistent with the findings of other studies. I would elaborate more on these findings and explain the mechanism/s behind these findings. In addition, the mean serum ALT and AST levels were also significantly higher…

Author response: Thank you for your suggestions. We've corrected it. Please check lines 317–319 of the revised manuscript.

Suggestion 74: Line 323: …inflammatory response, resulting in endothelial dysfunction causing vasoconstriction and eventual liver and kidney dysfunction…

Author response: Thank you so much for your suggestions. It has now been addressed. Please check lines 321–322 of the revised manuscript.

Suggestion 75: Lines 325-331: These statements are somewhat a repetition of the statements in lines 320-324. I would combine these 2 paragraphs to be more cohesive.

Author response: Thank you so much for your advice. We gratefully accepted and modified the recommendation. Please check the revised manuscript.

Suggestion 76: Line 332: On the other hand, our study showed that there was no significant difference in the total and direct bilirubin levels…

Author response: Thank you for your suggestions on making our manuscript more clear. Your recommendation has been accepted, and we have modified it. Please see line 325 of the revised manuscript.

Suggestion 77: Line 334: Our findings are in contrast with the findings of other studies wherein there were significant increases in…

Author response: Thank you for your suggestions on making our manuscript more clear. Your recommendation has been accepted, and we have modified it. Please see line 327 of the revised manuscript.

Suggestion 78: Line 336: …due to variations in the extent of…

Author response: Thank you for your suggestions on making our manuscript more clear. Your recommendation has been accepted, and we have modified it. Please see line 330 of the revised manuscript.

Suggestion 79: Line 337: In pre-eclampsia, the main cause of hyperbilirubinemia is thought to be disseminated…

Author response: Thank you for your suggestions on making our manuscript more clear. Your recommendation has been accepted, and we have modified it. Please see line 331 of the revised manuscript.

Suggestion 80: Line 340: In our study, there were significant differences in the mean SUA, ALT, and AST levels across the different age groups…The mean SUA level in the age group 26-34 years was 4.43 mg/dL, which then increased to 5.66 mg/dL in the age group >35 years. Please modify the sentences after this one to the same format.

Author response: Thank you for your suggestions on making our manuscript more clear. Your recommendation has been accepted, and we have modified it. Please see lines 333-336 of the revised manuscript.

Suggestion 81: Line 346: These findings were consistent with those of other studies, which showed that…

Author response: Thank you for your suggestions on making our manuscript more clear. Your recommendation has been accepted, and we have modified it. Please see line 339 of the revised manuscript.

Suggestion 82: Line 348: Similarly, there were also significant differences in the mean SUA, ALT, and AST levels across the different gestational age groups…Please also modify the sentences after this one to the same format as mentioned above.

Author response: Thank you for your suggestions on improving the clarity of our manuscript. Your suggestion has been accepted, and it has been modified. Please find lines 341-342 of the revised manuscript.

Suggestion 83: Line 357: Lastly, we performed a multivariable linear regression analysis and found a statistically significant positive association between…

Author response: Thank you for your suggestions on improving the clarity of our manuscript. Your suggestion has been accepted, and it has been modified. Please find line 350 of the revised manuscript.

Suggestion 84: Lines 362-365: If I am not mistaken, these statements are inaccurate as your study did not show any significant difference in the total and direct bilirubin levels between pre-eclamptic and healthy pregnant women.

Author response: We appreciate your insight. We accepted it and made significant changes.

Conclusion:

Suggestion 80: Our study revealed that serum uric acid, ALT, and AST levels were HIGHER in pre-eclamptic pregnant women compared to THOSE of healthy pregnant women, and the differences were statistically significant. As such, serum uric acid and liver function tests may be considered as early biomarkers of pre-eclampsia-related end organ damage.

Author response: Indeed, thank you very much for your suggestion to improve the clarity of our manuscript. We have modified the conclusion section of the revised manuscript as per your and other reviewers' suggestions. Please check the revised manuscript.

Submitted filename: Response to Reviewers.rtf

Click here for additional data file.

24 Jun 2022

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Maria Lourdes Gonzalez Suarez, MD, PhD

Academic Editor

PLOS ONE

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

1 Jul 2022

June 30, 2022

Rebuttal letter

Pone-d-21-30928

Evaluation of serum uric acid and liver function tests among pregnant women with and without preeclampsia at the University of Gondar comprehensive specialized hospital, northwest Ethiopia

Dear Editors and Reviewers,

We appreciate the constructive comments and suggestions for improving our manuscript. Based on the comments and suggestions, we have made corrections and modifications and provided point-by-point responses to the comments and suggestions. Please find our responses in green under the comments and suggestions presented by the academic editors in yellow. Below are our responses to each point raised by the academic editor.

Best regards!

Tadesse Asmamaw Dejenie, on behave of all authors

Response to academic editor’s comment

Comment 1: Describe the definition that was used to diagnose preeclampsia in this study, were the controls having normal BP? If so please refer to controls as normotensive pregnancy.

Author response: We'd like to thank you for your insightful remarks and suggestions. We have selected those preeclampsia women confirmed by physicians (high blood pressure ≥ 140/90 mmHg after 20 weeks of pregnancy and having proteinuria)

Comment 2: Combine the information in table 1 and table 2 into one larger table with "baseline characteristics". Consider adding p value

Author response: Indeed, thank you very much for your suggestion to improve the clarity of our manuscript. We have combined table1 and table 2 as per your suggestion. Please check it in the revised manuscript.

Comment 3: Please change the column names in tables 1, 2 and 4 from "Case" and "Control" to "Preeclampsia" and "Normotensive Pregnancy"

Author response: Thank you so much; we've accepted your suggestions and made the necessary modifications. Please check it in the revised manuscript.

Comment 4: In table 2, rather than using the words: "below normal", "normal", "above normal"; provide values of BMI, SBP, and DBP. Also add proteinuria values for "cases" and "Controls", add platelet cell counts for both groups

Author response: Indeed, thank you very much for your suggestion to improve the clarity of our manuscript. We have modified the words: "below normal", "normal", and "above normal". We have provided values of BMI, SBP, and DBP as per your suggestion but we did not do proteinuria and platelet cell count for this study. Please find it in table 1 of the revised manuscript.

Comment 5: Consider deleting table 3, it does not provide extra information and becomes redundant with the information provided in table 4

Author response: We want to express our gratitude for your expertise. This table has been deleted. For more information, kindly check out the revised manuscript

Comment 6: Please review the format of the columns and the name of the columns of tables 5 and 6; it is not clear what it is being compared, and it is important for these tables to be as clear as possible, since it is the main evidence of your discussion.

Author response: We sincerely appreciate your insight. We removed those tables (5 and 6) since the information is already described textually. We did a post hoc (Bonferroni) test to see the SUA, ALT, and AST levels across different age and gestational age of study participants.

Comment 7: In table 7, consider deleting the following rows: marital status, religion, education, occupation and residence. They should not be important in order to address the difference in the lab values you are measuring.

Author response: Indeed, thank you very much for your suggestion to improve the clarity of our manuscript. We have accepted your suggestion and deleted the rows as per your suggestion. Please check it in the revised manuscript.

Comment 8: Please review your conclusion, while I agree that your "study revealed that serum uric acid, ALT, and AST levels were higher in pre-eclamptic pregnant women compared to those of healthy pregnant women, and the differences were

statistically significant."

Author response: Thanks, We've taken your concern into account and made the necessary modifications. Please check the conclusion section of the revised manuscript.

Comment 9: Please clarify if any of the patients with preeclampsia in this study were diagnosed with HELLP syndrome

Author response: Thank you so much for your insight. During the study period, none of the pregnant women were diagnosed with HELLP syndrome.

Comment 10: Please provide information on the maternal and fetal outcomes of those with preeclampsia in this study when compared to normal pregnancy, including maternal and fetal survival, preterm delivery, birth weight, etc.

Author response: We really appreciate your thoughtful suggestions. A longitudinal study is required to provide information on maternal and fetal outcomes such as maternal and fetal survival, preterm delivery, and birth weight, but this is a cross-sectional study. At the time of data collection, those normotensive pregnant women came to the hospital for antenatal care follow up and those preeclampsia women were admitted to the University of Gondar hospital’s labor ward for the treatment of preeclampsia.

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

14 Jul 2022

EVALUATION OF SERUM URIC ACID AND LIVER FUNCTION TESTS AMONG PREGNANT WOMEN WITH AND WITHOUT PREECLAMPSIA AT UNIVERSITY OF GONDAR COMPREHENSIVE SPECIALIZED HOSPITAL, NORTHWEST ETHIOPIA

PONE-D-21-30928R2

Dear Dr. Dejenie,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Maria Lourdes Gonzalez Suarez, MD, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Thank you for addressing our comments and concerns.

Reviewers' comments:

21 Jul 2022

PONE-D-21-30928R2

Evaluation of serum uric acid and liver function tests among pregnant women with and without preeclampsia at the University of Gondar comprehensive specialized hospital, Northwest Ethiopia

Dear Dr. Dejenie:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Maria Lourdes Gonzalez Suarez

Academic Editor

PLOS ONE