2.2. Laboratory Analyses

Biological samples were collected from peripheral blood of enrolled patients and kept refrigerated at 4°C–8°C for immediate routine biochemical processing or stored frozen at −80°C for further analysis for the present study.

The samples were processed for clinical biochemistry following protocols, quality control, and routine laboratory analytical technical instructions. The glycemic, lipid, enzymatic, metabolic, and inflammatory profiles of each participant were analyzed. Levels of glucose, creatinine, cholesterol, triglycerides, HDL-C, AST (GOT), ALT (GPT), gamma-GT, total proteins, and albumin were measured by enzymatic, kinetic, or colorimetric tests, with reagents compatible with a HumanStar 600 (InVitro®) equipment. The same automation was used to determine ultrasensitive C-reactive protein levels by turbidimetry. Glomerular filtration rate was estimated by the Cockcroft–Gault formula [23], while LDL-C and VLDL-C fractions were estimated by the Friedewald formula [24].

Hematological analyses were performed by automation using ABBOTT® Cell Dyn 3700 equipment, operated according to the manufacturer's recommendations. Glycated hemoglobin was measured by high-performance liquid chromatography (HPLC) technique, while total PSA, free PSA, insulin, vitamin D, and TSH levels were obtained by electrochemiluminescence using the Roche® Cobas e411 system. The HOMA beta cell function index and HOMA-IR index were calculated, as well as the free PSA/PSA ratio.

Total circulating MMP-1 was assayed by enzyme-linked immunosorbent assay (ELISA) (R&D Systems—DuoSet, lot 339081), using serum as a sample, and processed according to the manufacturer's guidelines.