High performance liquid chromatography system

Baseline information was obtained during clinic visits or at home using standardized questionnaires including: demographics (age, sex), occupation (management/professional versus not), level of education (≥Bachelor’s degree versus 2) [17 (link)]. EGFR was included as a covariate due to a known inverse association with aldosterone [17 (link)]. Sodium, potassium, creatinine, and chloride were measured on a Vitros 950 or 250, Ortho-Clinical Diagnostics analyzer (Raritan, NJ, USA) at the University of Mississippi Medical Center [13 ,18 (link)]. A1c concentrations were measured using a high-performance liquid chromatography system (Tosoh Corp, Tokyo, Japan) [18 (link)].

Diabetes was defined as HbA_1c of 6.5% or greater (to convert to proportion of total hemoglobin, multiply by 0.01), fasting blood glucose of 126 mg/dL or greater (to convert to millimoles per liter, multiply by 0.0555), using diabetes medications, or a self-reported physician diagnosis.^{12 (link)} Fasting glucose was measured on a Vitros 950 or 250, Ortho-Clinical Diagnostics analyzer using standard procedures that met the College of American Pathologists accreditation requirement.^{13 (link)} A high-performance liquid chromatography system (Tosoh Corporation) was used to measure HbA_1c concentrations. History of CHD was defined as evidence of a previous myocardial infarction by electrocardiogram (ECG) based on Minnesota Code criteria (codes 1.1 and 1.2 plus 4.1-4.2, or 5.1-5.2) or history of physician-diagnosed myocardial infarction, percutaneous coronary intervention, or coronary bypass surgery. The definition of stroke was based on the history of stroke (by personal history, stroke signs, and symptoms ascertained by standardized questionnaires), transient ischemic attack, or carotid endarterectomy and/or angioplasty. Details of these procedures have been described previously.^{14 (link)}

Urine samples (100 µL) were treated with 100 µL of 10% (w/v) trichloroacetic acid (TCA), and centrifuged at 15,000× g for 10 min. The supernatant (50 µL) was used to measure polyamines, which were measured as described previously [16 (link)], using Hitachi high-performance liquid chromatography system on which a TSKgel Polyaminepak column (4.6 × 50 mm) (Tosoh, Tokyo, Japan) was heated to 50 °C. The flow rate of buffer (0.35 M citric acid buffer, pH 5.1, 2 M NaCl and 20% methanol) was 0.42 mL/min. Retention times for putrescine, spermidine and spermine were 6, 12 and 24 min, respectively. Detection of polyamines was by fluorescence intensity with an o-phthalaldehyde solution containing 0.06% o-phthalaldehyde, 0.4 M boric buffer (pH 10.4), 0.1 M Brij-35, and 37 mM 2-mercaptoethanol. The flow rate of the o-phthalaldehyde solution was 0.4 mL/min, and fluorescence was measured at an excitation wavelength of 340 nm and an emission wavelength of 455 nm.