ACTA FACULTATIS MEDICAE NAISSENSIS UDC: 616.61-008.6 DOI: 10.1515/afmnai-2015-0027 Original article Proteinuria as the Most Relevant Parameter Affecting Fetuin-A Levels in Preeclampsia Hussein Kadhem Al-Hakeim, Roaa Ali Muhammed Ali Department of Chemistry, College of Science, University of Kufa, Iraq SUMMARY The present study aimed to examine the factors affecting the possible changes in serum fetuin-A in patients with preeclampsia (PE). The examined factors included the parameters of insulin resistance (IR) [(insulin sensitivity (HOMA%S), insulin resistance (HOMA2IR), and beta-cell functions (HOMA%B)], which were calculated using the HOMA2 calculator, and total and ionized calcium and magnesium levels. Sixty PE patients and thirty healthy pregnant women, which comprised the study group and control group, respectively participated in the present study. Fetuin-A, estradiol, insulin, glucose, total and ionized calcium and magnesium, total protein, albumin, and globulins were measured in their sera. The results of the present study showed that serum total and ionized magnesium and the I.Ca/Mg ratio decreased in PE women. Although the fasting insulin level and HOMA2IR were higher and HOMA2%S was lower in PE compared with the control women, PE did not appear as an overt insulin-resistant state. Serum fetuin-A was low in PE patients compared with the control group because PE women had proteinuria. Fetuin-A levels were not correlated with the characteristics and IR parameters, cations, and estradiol levels, but it was correlated with the severity of proteinuria. These results confirmed the hypothesis that proteinuria results in the loss of fetuin-A because it has a low molecular weight. Key words: fetuin-A, insulin resistance, preeclampsia, magnesium, calcium Corresponding author: Hussein Kadhem Al-Hakeim e-mail: [email protected] Acta facultatis medicae Naissensis 2015;32(4):267-277 267 O riginal article INTRODUCTION SUBJECTS AND METHODS Preeclampsia (PE) is generally defined as new Subjects hypertension and substantial proteinuria at or after the second half of gestation (1). It is a dangerous A - Patients: Sixty PE female patients participated complication of pregnancy that affects 5–10% of in the study. Their age range was 28.72±6.96 years. pregnant women and leads to increased mortality of The samples were collected from the “Labor Hall and fetus and mothers (2). The exact mechanism of PE Operation Hall” in Al-Zahra Teaching Hospital in remains unknown. However, its pathogenesis is Najaf Governorate-Iraq from August, 2014 to thought to occur in two phases, namely, the placental December, 2014. PE was diagnosed by a gynecologist, phase and maternal phase, which is characterized by who treated patients during the pregnancy terms, an abnormal maternal endothelial response, resulting using ICD-10 code O14 (International Classification of in hypertension, proteinuria, and edema (3). Diseases, 9th and 10th revisions). PE is defined as Several risk factors have been identified with hypertension (systolic blood pressure ≥140 mmHg increased risk of PE, including insulin resistance, and/or diastolic blood pressure ≥90 mmHg measured diabetes mellitus, obesity, chronic hypertension, renal twice after 20 weeks of gestation in combination with diseases, preexisting thrombophilia, smoking, and proteinuria (≥1+ dipstick proteinuria). Self-voiding family history of PE (4). Fetuin-A is a carrier protein, was used for urine sampling. A single blood sample such as albumin, that forms soluble complexes with was taken from women with PE (n = 60) and control calcium and phosphate; thus, fetuin-A is a carrier of the pregnant women who were matched for age (+/−5 insoluble calcium phosphate (5). years) and gestational age of 34 weeks (+/−12 days). Fetuin-A, a negative acute phase protein reactant Blood samples were also obtained from 30 matched, (6), has been implicated in the pathophysiology of PE (7). healthy, pregnant women. The women were fasting. Fetuin-A functions as an important component of None was in active labor and none had ruptured diverse normal and pathological processes, including membranes. vascular calcification and bone metabolism regulation, These studies were approved by the Kufa insulin resistance, and protease activity control (8). University Research Ethics Committee, and written Furthermore, fetuin-A is an inhibitor of the insulin consent was obtained from each participant. receptor because of phosphorylation of tyrosine kinase B - Controls: Thirty apparently healthy women activity, and its serum level is correlated with insulin were selected as the control group. Their age range resistance (9). Estradiol (E2) might affect the synthesis was 25.17±5.55 years, which was comparable with of fetuin-A, as suggested by the alteration in its levels that of patients with PE. None of these subjects after estrogen therapy and menopause (10). suffered from hypertension. Low serum calcium and/or magnesium may C - Exclusion criteria: This study excluded patients cause high blood pressure by stimulating parathyroid with multifetal gestation, chronic hypertension, diabetes hormone and rennin release and also by inducing mellitus, autoimmune disease, renal disorder, maternal vasoconstriction by increasing its level in vascular or fetal infection, and fetal congenital anomaly. smooth muscle (11). Calcium might also have an indirect effect on smooth muscle function by increasing magnesium levels (12). Blood samples The present study aims to examine the factors affecting the possible changes in serum fetuin-A in PE Maternal blood samples were obtained from an patients, as well as the factors that may affect fetuin-A antecubital vein into plain tubes, allowed to clot for levels, including insulin resistance state parameters 15 min, and centrifuged at 3000 g for 5 min. The [insulin resistance (HOMA2IR), insulin sensitivity aliquots of serum were stored at -18 °C until further (HOMA%S), and beta-cell functions (HOMA%B)], analyses. which were calculated using the HOMA2 calculator, and total and ionized calcium and magnesium levels. 268 Acta facultatis medicae Naissensis 2015;32(4):267-277 Hussein Kadhem Al-Hakeim, Roaa Ali Muhammed Ali Measurements For the normally distributed variables, the results were expressed as the mean ± standard Serum fetuin-A and E2 were measured using deviation. Pooled t-test was used to compare data ELISA kits supplied by Monobind® (USA). Insulin between the patient and control groups and among levels in serum were measured using DRG® Insulin subdivided groups in the measured parameters. ELISA based on the sandwich principle. Pearson’s correlation coefficients (r) were calculated Total calcium in serum was measured using a to estimate the correlation between parameters. ready-to-use kit according to the Arsenazo III method. For nonparametric variables that were not Corrected calcium was calculated using the following normally distributed, the results were expressed as formula (13): medians, in addition to the mean ± standard Corrected calcium = total calcium (in mg/dl) / deviation. The Mann–Whitney U test was used to (0.6 + 0.05 × total protein) compare data between the patient and control groups Ionized calcium levels in serum were calculated and among subdivided groups in the measured according to the following formula (13): parameters. Spearman’s correlation coefficients (ρ, Ionized Ca=0.9 + (0.55 × T.Ca in mmol/l)–(0.3 × rho) were used to estimate the correlation between albumin) parameters. Total magnesium in serum was measured by The difference between groups was considered the calamite method using a kit supplied by statistically significant when p < 0.05. All statistical CypressCo. Serum ionized magnesium levels were analyses were performed using SPSS Statistics version calculated according to the following formula (14): 19.0.1 Multilingual program (2010, IBM, USA). Ionized magnesium in mmol/L = (0.66 × (T.Mg Figures were constructed using EXCEL (Microsoft in mmol/L)) + 0.039 Office 2010). Total protein, albumin, and glucose concentrations in serum were measured by colorimetric methods using ready-to-use kits supplied RESULTS AND DISCUSSION by Agapee® Company (Switzerland). Comparison between PE and control groups Estimation of HOMA2IR, HOMA%S, and HOMA%B Descriptive parameters The results of descriptive variables in PE and Insulin resistance parameters (HOMA2IR, control groups are presented in Table 1. The results HOMA%B, and HOMA%S) were calculated by a showed significant differences (p < 0.05) in weight, computer-based HOMA2 calculator (available at height, BMI, and the percentages of having children www.dtu.ox.ac.uk/homa) using fasting glucose and and abortion between PE and normal pregnant insulin. The model was recalibrated by the United women. Kingdom Prospective Diabetes Study (UKPDS) group at Oxford University to 100% in normal young adults Table 1: Descriptive parameters in PE patients and when using currently available assays for measuring control groups expressed as mean±standard deviation fasting blood glucose and insulin only. Ideal normal- (N.S. = not significant, p value is not cited if p > 0.05) weight individuals aged < 35 years should have HOMA2IR of 1 mol·µU/L2 and HOMA%B cell Parameter Patients Control p-value function of 100%. Age yr. 28.72±6.96 25.17±5.55 0.011 Wt kg 86.00±10.70 74.67±7.75 <0.001 Statistical analysis Height cm 1.57±0.21 1.56±0.14 0.002 BMI kg/m2 34.77±4.49 30.65±3.33 <0.001 The distribution types of the results were Wks of Preg. 37.18±1.75 37.63±0.67 N.S. examined using the Kolmogorov–Smirnov test. The 23% 10% results of the analysis divided the variables into two Abortion 0.033 (14 out of 60) (3 out of 30) types according to the statistical distribution: the 78.3% 70.0% Having child 0.037 normally distributed variables and nonparametric (47 out of 60) (21 out of 30) variables. Acta facultatis medicae Naissensis 2015;32(4):267-277 269 O riginal article These results indicated that BMI was associated with a significant reduction in the risk of significantly higher in the PE group than that in the PE (12).