Biochemical determinations

The enzymatic activity was assayed according to the method of Sassa, (1982[45]), by measuring the rate of product (porphobilinogen - PBG) formation, as previously described (Peixoto et al., 2003[36]). Incubation was started by adding 200 µL of S1 or hemolyzed blood and carried out for 30, 60 and 120 min for the kidney, liver and blood, respectively, at 39 °C. The reaction was stopped by the addition of trichloroacetic acid (TCA) 10 % containing HgCl₂ 0.05 M; PBG was measured with Ehrlich's reagent, using the molar absorption coefficient of 6.1 x 10⁴ for Ehrlich-PBG salt. The specific enzymatic activity was expressed as nmol of PBG formed per hour per mg protein.

Thiol levels from the kidney and liver were determined as previously described by Ellman (1959[11]). For non-protein thiol (NPSH) determination, the protein fraction of 200 μL S1 was precipitated with 200 μL of 4 % TCA (v/v) followed by centrifugation (1,050 g, 10 min) and the supernatant used for analysis. The colorimetric test was carried out in a 1 M phosphate buffer, pH 7.4. A curve using glutathione as standard was constructed in order to calculate the SH in the tissue samples. TSH and NPSH levels were expressed as mmol SH per g tissue.

Ascorbic acid (AA) determination was performed as described Roe (1954[40]) with some modifications. S1 from the kidney and liver were precipitated in 10 volumes of cold 4 % TCA solution and centrifuged (1,050 g, 10 min). An aliquot of the sample in a final volume of 500 µL of the solution was incubated for 3 h at 37 °C; afterwards, 500 µL of H₂SO₄ 65 % (v/v) was added into the medium. The reaction product was determined using color reagent containing 4.5 mg/mL dinitrophenyl hydrazine and CuSO₄ (0.075 mg/mL). Ascorbic acid levels were expressed as µg of AA per g tissue.

Enzymatic activity was determined by the Thomas (1998[51]) method, using a commercial kit in a medium containing Tris-HCl buffer 55.8 mM pH 7.15, L-alanine 500 mM, 2-Oxoglutarate 15 mM and NADH 0.18 mM, with 50 µL of serum or tissue. The specific enzymatic activity was expressed in serum as U per L and in the liver as U per mg protein.

The estimation of serum creatinine levels was performed by measuring the formed product, creatinine picrate. Creatinine was used as standard, utilizing a Labtest commercial kit. The reaction was conducted in a medium containing picric acid 20.2 mM and NaOH 145.4 mM at 37 °C with 50 µL of serum. Levels were expressed as mg of creatinine per dL of serum.

Incubation at 37 °C for 5 min was started by adding 10 µL of serum sample to a medium containing phosphate buffer 19.34 mM pH 6.9, sodium salicylate 58.84 mM, sodium nitroprusside 3.17 mM, and urease (≥ 12.63 UK/L), using a Labtest commercial kit. The reaction was stopped by adding oxidant solution (final concentrations: NaOH 0.07 M and sodium hypochlorite 3.01 mM), and the mixture was incubated for 5 min to achieve color development. Levels were expressed as mg of urea per dL of serum.