Hematological and biochemical parameters.

ABO and Rhesus (Rh) blood groups were determined by a hemagglutination technique using specific antibodies.

Hemoglobin (Hb) was measured by a HemoCue analyzer (HemoCueHb 201, Sweden). WHO references with different ranges related to the altitude were used. In Puna, anemia was considered mild at 14.5–14.9 g/dL; moderate 11.5–14.4 g/dL, or severe < 11.5 g/dL according to published levels for healthy residents in altitude. In the Yungas, mild anemia was defined as 11–11.4 g/dL, moderate 8–10.9 g/dL, and severe < 8 g/dL.

After mixing heparinized blood samples, capillary tubes were centrifuged for 5 minutes at 2,500 g. Microhaematocrit tubes and a reader (Hawksley & Sons Ltd., Sussex, United Kingdom) were used to calculate the final percentage.

Levels of 25(OH)-vitamin D (vitD) in serum were measured using a commercial enzyme-linked immunosorbent assay kit (Immundiagnostik AG, Bensheim, Germany). Subjects were classified as vitD sufficient if > 75 nmol/L; insufficient 30–75 nmol/L; and deficient < 30 nM.

Albumin and zinc were measured in samples using a Cobas 8000 modular analyser (Roche). Tested normal range used for albumin was 3.6–4.8 g/dL and for zinc 11–18 μM.¹⁰