2.7. Biochemical Analysis
BLOOD, PATHOLOGY
Blood samples (non-fasting) were collected from each participant with venipuncture by a skilled phlebotomist at the basic health unit. For plasma zinc assessment, the blood was collected in trace element-free tubes to avoid contamination. All samples were centrifuged to obtain plasma/serum in the pathology laboratory of DHQ hospital Parachinar Newly Merged District of Khyber Paktunkhawa (NMD-KPK) Pakistan. The separated plasma/serum was tightly packed in Eppendorf tubes and was transported in dry ice on a daily basis to the Biochemistry laboratory of Khyber Medical University for processing and storage at −80 °C until further biochemical analysis. The plasma/serum was used for the measurement of plasma albumin using a fully automated chemistry analyzer (Cobas 6000) (Roche Diagnostics, Switzerland, Rotkreuz). The 25-hydroxy vitamin D was analyzed with the ELISA technique (according to its available literature protocol) using BIOS Microwell Diagnostic System (Perkin Elmer, catalog No: 10501, USA) with a cut-off value of <30 ng/mL. The vitamin A (retinol) was analyzed through an ELISA kit (Elabscience, catalog No: E-EL-0135, Wuhan, China) with a cut-off value of <18.8 ng/mL. To measure the plasma zinc level, a proper wet acid digestion method was used to digest plasma and then followed by analysis through an atomic absorption spectrometer (model: AAS 700, make: Perkin Elmer, Waltham, MA, USA) with a cut-off value of <65 µg/dL [