Association between serum 25-hydroxyvitamin D levels and carotid atherosclerosis in chronic kidney disease patients

This was a cross sectional study conducted between September 2015 and March 2016. It has adhered to the STROBE guidelines for observational studies. A consecutive sampling methodology was used for participant recruitment. The flow diagram is shown in Fig. 1.

A total of 100 pre-dialysis CKD stage 3–4 patients who attended nephrology, diabetic, and outpatient clinics at the University of Malaya Medical Centre (UMMC) were included. Patients who were 70 years old and above, had underlying coronary artery disease, heart failure, malignancy, active infection, or were on immunosuppressive drugs or vitamin D supplements were excluded.

Ethics approval (MECID 201412-878) was obtained from the UMMC Medical Ethics Committee which governs all research involving humans. Participants provided written informed consent prior to being enrolled in the study.

Socio-demographic data, medical history, alcohol and smoking history, and medication history were obtained from all subjects. Body mass index (BMI) was calculated as weight in kilograms divided by height in meters squared (kg/m²). During the enrolment phase, previous biochemical tests were reviewed through the laboratory information system (TD-Web version 11.61B, Technidata SAS, France) and results within 2 weeks were exempted from repeat analysis. The biochemical analysis of blood samples was conducted in the same laboratory. The Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) formula was used to calculate the estimated glomerular filtration rate (eGFR). Participants were categorized into three CKD stages: stage 3A (45–59 mL/min per 1.73 m²), stage 3B (30–44 mL/min per 1.73 m²) and stage 4 (15–29 mL/min per 1.73 m²) according to Kidney Disease Outcome Quality Initiative (KDOQI) criteria.

Serum 25(OH)D and intact parathyroid hormone (PTH) levels were determined using direct chemiluminesent immunoassay (ADVIA Centaur XP; Siemens Healthcare Diagnostics, Tarrytown, New York, USA). The detection limit of the device was <10 nmol/L, with the intra- and inter-assay coefficient of variations of 4.2 and 11.9 % respectively. This assay demonstrated comparable performance with the reference standard, liquid chromatography tandem mass spectrometry (LC-MS/MS) [14‐16]. A mean of 5 nmol/L was assumed for results reported as <10 nmol/L. Vitamin D status was defined as deficiency (< 25 nmol/L) [17] and non-deficiency (≥ 25 nmol/L). Serum calcium, inorganic phosphate, renal profiles and lipid profiles were measured using colorimetric methods (ADVIA 2400 Chemistry System; Siemens Healthcare Diagnostics, Tarrytown, New York, USA). Complete blood counts were analysed using fluorescent flow cytometry (XN-10; Sysmex, Kobe, Japan).

An ultrasound system equipped with high-resolution, linear-array transducers in the frequency of 4–9 MHz (Philips iU22; Philips, Seattle, Washington, USA) was used. Carotid arteries were imaged with L9-3 and L8-4 transducers. It was performed by a single certified sonographer and images were verified by an experienced stroke neurologist. All participants underwent a carotid ultrasound within 2 weeks from 25(OH)D assay to measure intima-media thickness (IMT) and to assess the presence of carotid plaque or stenosis at the common carotid arteries (CCA), internal carotid arteries (ICA), and external carotid arteries (ECA). Three IMT measurements were obtained at the far walls of the distal, 2 cm from the common carotid artery. The mean of these three measurements was recorded as the IMT for that side (left and right). CCA-IMT was defined as the average of left and right IMT. The images were obtained at a 30° angle and the length of the segments measured was 1 cm. The image analysis was done using QLAB Vascular Ultrasound Quantification software, version 7.1. The analysis was done offline. The presence of carotid plaque or significant stenosis (≥ 50 %) in the CCA, bifurcation, and ICA bilaterally were documented. Abnormal CCA-IMT was defined as CCA-IMT ≥ 0.8 mm which is beyond the 75th percentile as per the American Society of Echography Task Force recommendations [18, 19]. Plaque was defined as a focal structure that encroaches into the arterial lumen of ≥ 0.5 mm or 50 % of the surrounding IMT value [20]. Significant stenosis was defined as peak-systolic velocities ≥ 125 cm/s and end-diastolic velocities ≥ 40 cm/s as recommended by the Society of Radiologists in Ultrasound [21].

A statistical analysis was performed using the Statistical Package for Social Sciences for Windows, version 23.0 (IBM Corp., Armonk, New York, USA). Clinical characteristics data was expressed as means ± standard deviation (SD). Differences between groups were analysed using independent t-test and one-way analysis of variance (ANOVA) when the dependent variable was normally distributed, otherwise by Mann-Whitney U and Kruskal-Wallis H tests. Appropriate transformations were made when necessary to ensure normality or linearity assumptions were met. The correlation between serum 25(OH)D levels and CCA-IMT was determined using Pearson’s product-moment correlation coefficients. Linear regression analysis was performed to determine the predictors of serum 25(OH)D levels. Multiple linear regression models were used to identify the predictors of CCA-IMT, abnormal CCA-IMT, and plaque presence. Age, gender, race, BMI, smoking habits, diabetes status, hypertension status, eGFR, serum triglyceride, LDL cholesterol, HDL cholesterol, calcium, phosphate, PTH, and 25(OH)D levels were taken as independent variables for modelling. Backward elimination was applied to remove insignificant variables. All P-values were calculated two-sided and a value of < 0.05 was considered as significant.