Vanadium in Late-onset Preeclampsia

This observational case-control study was conducted at the Department of Obstetrics and Gynecology, Cengiz Gokcek Public Hospital, Gaziantep, Turkey, between March 2018 and June 2019. The protocol was approved by the Ethics Committee for Clinical Research of Gaziantep University (reference no: 2019/36). The study strictly adhered to the principles of the Declaration of Helsinki. All subjects included in the study gave oral and written informed consent. Ninety-two women were enrolled in the study in two groups.

Data collection and Study intervention:

The diagnosis of L-PrE, as defined by the Committee on Terminology of the American College of Obstetricians and Gynecologists (ACOG), is established based on the presence of proteinuria (urinary excretion of protein ≥300 mg in a 24-h urine specimen, or proteinüria ≥1+ in dipstick) and a blood pressure level of ≥90/140 mmHg (two blood pressure measurements 6 h apart) that occurs after 34 weeks of gestation in a previously normotensive woman. The diastolic and/or systolic blood pressure <110/160 mm Hg, it was accepted as mild; and in case these values exceeded this level, it was accepted as severe. Every women in the study population underwent obstetric ultrasound examination and fetal-maternal assesment was carried out by one of the authors. The obstetric anamnesis were obtained from all subjects. The dempograhic data like age, gravidity, parity, body mass index (BMI) and gestational age were recorded. The gestational age was determined by calculation from the last menstrual period and supported by the ultrasonography measurements at first trimester of gestation. Maternal venous plasma samples were taken for measurement of cadmium, lead and vanadium levels after the diagnosis of L-PrE in outpatient clinic. These samples quickly centrifuged at 1,500 g for 10 min, plasma samples were seperated and stored at -80 C until the day of measurement. All patients with L-PrE were also hospitalized and their pregnancies were terminated. The samples of the control groups were obtained during the routine obstetrical care examination in the third trimester of pregnancy. Then, these pregnant women were followed-up until the delivery. Both groups were compared in terms of maternal age, BMI, week of gestation, gravida, parity, live born, systolic/diastolic blood pressure, total protein in spot urine sample, hemoglobin, hematocrit, platelet count, white blood cell count, urea, uric acid, albümin, blood urea nitrogen, creatinine, liver function tests (AST, ALT), lactic acid dehydrogenase, cadmium, lead and vanadium concentrions and infant weight at delivery. Small for gestational age (SGA) neonate was defined as birth weight <10th percentile for gestational age with Turkey's national nomogram as the reference for fetal growth. The samples were transferred within boxes in ice molds to Yozgat Bozok University Science and Technology Application and Research Center (Occupational and Environmental Toxicology Laboratory) for measuring heavy metal and trace element levels. The frequency of seafood consumption was divided into four categories: 2-3 per week, 1-2 per week, 1-2 per month, and rare. Smoking was classified into 3 groups (none, formerly smoker, and active smoking). Passive smoking was also categorized into 2 groups (no and yes). The education levels was classified into 4 groups (illiterate, primary education, high school, and university). The residences of women was classified as rural and urban. Furthermore, the residences of women have divided either living near a busy street (around 100 metres) or not.Small for gestational age (SGA) neonate was defined as birth weight <10th percentile for gestational age with Turkey's national nomogram as the reference for fetal growth.

The method developed by Aliyev et al. was used for preparing the samples for analysis One milliliter of plasma samples was placed into high-temperature-resistant Teflon tubes in a microwave oven, and 5 mL Suprapur® (Merck, Darmstadt, Germany) nitric acid (HNO3) and 5 mL ultra-pure water was added. All plasma samples were digested using a microwave digestion system (Start D; Milestone, MD, USA). After digestion, each sample was made to a total volume of 20 mL with 9 mL ultra-pure water in a 50 mL polypropylene tube. Nitric acid (Suprapur®, 65%) was used for digestion of samples and standard reference material. Ultra-pure water (Direct-Q®; Millipore, Darmstadt, Germany) was used for dilution of the standard (multi-element standard Chem-Lab, Zedelgem, Belgium) and sample preparations.

The three metals (cadmium, lead and vanadium) were measured using inductively coupled plasma-mass spectrometry (Thermo Scientific ICAPQc, USA). The operating parameters were set as follows: radiofrequency power 1550 W, nebulizer gas 0.96 L/min, plasma gas 0.88 L/min, nebulizer pressure 3.01 bar, dwell time 0.01 milliseconds, and spray chamber temperature 3.7°C. The sampler probe was washed between injections by rinsing with ultrapure water for 30 s, followed by washing with 2% HNO3 for 45 s, and finally rinsing with ultrapure water for 45 s. After the washing steps, the instrument automatically ran the next sample. An 11-point calibration curve (0.5-500 µg/L) was used to measure the level of each element. The r2 values of the calibration curves of all metals were calculated, and the minimum value was 0.9994. To ensure the accuracy of the results, each measurement of the samples and standards was repeated three times. The results of these measurements showed that the relative standard deviation (RSD) did not exceed 5%. Certified Reference Material (CRM-Seronorm™ Trace Elements Whole Blood L-2, Sero AS, Billingstad, Norway) was used for the validation method. To check the stability and sensitivity of the instrument, a mixture of internal standards (Hf) was used, and the mean and RSD values of metals were also calculated. The variations of each measurement of the quality controls was <15%. The relative percentage differences in replicate analyses were <5% in the samples and standards.