Immunonephelometric Assay

Demographic and clinical characteristics, including age, sex, race, income, education, smoking status, and alcohol use, were determined by self-report. Height and weight were measured during the in-person visit to estimate the body mass index. Hypertension was defined when an average of two seated BP readings had a systolic BP of ≥140 mm Hg and/or diastolic BP of ≥90 mm Hg or when the person reported use of antihypertensive medications. Diabetes was defined as self-reported use of insulin or oral hypoglycemic agents, fasting glucose ≥126 mg/dl, or random glucose ≥200 mg/dl. History of coronary heart disease (CHD) was defined as having any of the following: evidence of myocardial infarction on the baseline electrocardiograph, self-report of a history of a cardiac procedure (coronary artery bypass surgery or percutaneous coronary intervention), or self-reported history of myocardial infarction. History of stroke was determined by self-report as well. A suspected history of heart failure at baseline was determined by current use of heart failure–related medications at the baseline visit. The lipid profile, including high-density lipoprotein and triglycerides, was measured from fasting samples. High-sensitivity C-reactive protein was measured using a high-sensitivity particle–enhanced immunonephelometric assay. Serum creatinine was calibrated to an international isotope dilution mass spectroscopic-traceable standard, measured by colorimetric reflectance spectrophotometry, and eGFR was calculated using the 2009 Chronic Kidney Disease Epidemiology Collaboration equation.^{20 (link)} Albumin and creatinine were measured using the random spot urine specimen by nephelometry (BN ProSpec Nephelometer, Dade Behring, Marburg, Germany) and Modular-P chemistry analyzer (Roche/Hitachi, Indianapolis, IN), respectively.

Study participants provided a medical history and a physical examination. Participants self‐reported their demographics and smoking status. Weight was measured using a balance beam scale and height was measured using a Harpenden stadiometer (Holtain Ltd). Body mass index (BMI) was calculated in kg/m². Baseline prevalent diabetes was defined using self‐reported histories, use of antidiabetic agents, fasting plasma glucose concentration >125 mg/dL, or a 2‐h oral glucose tolerance test result >199 mg/dL. Systolic and diastolic blood pressures were measured twice using a conventional mercury sphygmomanometer and averaged. Medications were brought into study visits by participants and categorized using the Iowa Drug Information System.
Serum cystatin C and urine albumin and urine creatinine measurements were available only at year 1 (baseline), so we carried forward these measurements to year 2 when vitamin D metabolites and all other data were collected, consistent with prior studies.^[6, ^11] Urine albumin was measured using a particle‐enhanced turbidimetric inhibition immunoassay allowing for direct albumin quantification (Siemens). Measurement of urine creatinine was done by a modified Jaffé method on a clinical chemistry analyzer (Siemens). Cystatin C was measured at the Health ABC core laboratory (University of Vermont, Burlington, VT, USA) with a BNII nephelometer (Dade Behring Inc.) that used a particle‐enhanced immunonephelometric assay (N Latex Cystatin C). Estimated glomerular filtration rate (eGFR) was calculated using the 2012 CKD Epidemiology Collaboration (CKD‐EPI) cystatin C equation.^[22] Serum calcium and phosphate were measured using direct quantitative colorimetric determination (Stanbio Laboratory). Intact parathyroid hormone (iPTH) was measured in EDTA plasma using a two‐site immunoradiometric assay kit (N‐tact PTHSP; DiaSorin). Fibroblast growth factor‐23 (FGF‐23) was measured using an intact assay (Kainos Laboratories). Serum albumin was measured using the same assay for VDBP with the addition of internal standards for three albumin‐specific peptides, as described previously.^[11]