Measurements

Blood samples were obtained by venipuncture of an antecubital vein, collected in vacutainers after overnight fasting. Urine samples were collected by the patients. Hs-CRP, glucose, insulin, cholesterol, low-density lipoprotein (LDL), triglycerides, and creatinine, and albuminuria and creatininuria were analyzed in the central laboratory at OUHSC. Samples for G3 were obtained in an ethylenediamine tetraacetic acid (EDTA) tube, centrifuged, aliquoted and stored at −80°C and were analyzed in batches.

G3 was measured using a commercially available enzyme linked immunosorbent assay (ELISA): the G3 assay (BG Medicine, Waltham, MA, USA). The test uses a rat monoclonal antibody against mouse galectin-3. At the epitope for the assay there is 100% homology between mouse and human G3. To avoid cross reactivity, patients receiving any other murine antibody within 6 months were excluded

Samples, standardization dilutions and kit controls were run in duplicates (e.g. each sample, standard or control was tested in two wells in each plate). Participant samples were allocated in the plates following a randomly generated order, and the operator performing the ELISAs was blind to the ABI and clinical variables.

The plates were read with the Synergy 2 multi-detection microplate reader (BioTek Instruments Inc. Winooski, VT) and standardization curves were generated by the built-in software using a 4-parameter logistic method.

This ELISA has a low within-run, run-to-run and day-to-day variability (less than 10% in all cases) and G3 levels demonstrated linearity between observed and expected values in the range between 0.96ng/mL to 130ng/mL.^18,19

Arterial elasticity was determined by diastolic pulse contour analysis (DPCA) with the HDI/Pulse wave CR-2000 Research Cardiovascular Profiling System (Hypertension Diagnostics, Eagan, MN). A blood pressure cuff appropriately sized was placed on the left upper arm of the participant. The right wrist was immobilized with a rigid wrist stabilizer in neutral supine. An Arterial Pulsewave™ Sensor was placed on the skin directly over the radial artery at the point of the strongest pulse by palpation, overlaying a bony prominence.²⁰ The non-invasive acoustic sensor was adjusted to the highest relative signal strength. Then the blood pressure was measured on the left with an oscillometric blood pressure cuff and the arterial waveform was recorded on the right wrist for 30 seconds at a rate of 200 per second, and subsequently analyzed with the software algorithm based on a modified Windkessel model.²¹ Measurements were averaged over three consecutive trials. To convert values to whole numbers, the units for large arterial elasticity index (LAEI) (ml × mmHg⁻¹) were multiplied by 10 and the units for small arterial elasticity index (SAEI) (ml × mmHg⁻¹) were multiplied by 100. The test-retest intra-class reliability coefficient is r=0.87 for LAEI and r=0.83 for SAEI.²²

The LAEI, represents the compliance of the aorta and main branches, and the SAEI represents the compliance of the smaller arteries.