Clinical Biochemistry 

Abstract: Objectives: To develop a new, colorimetric and automated method for measuring total oxidation status (TOS). Design and methods: The assay is based on the oxidation of ferrous ion to ferric ion in the presence of various oxidant species in acidic medium and the measurement of the ferric ion by xylenol orange. The oxidation reaction of the assay was enhanced and precipitation of proteins was prevented. In addition, autoxidation of ferrous ion present in the reagent was prevented during storage. The method was applied to an automated analyzer, which was calibrated with hydrogen peroxide and the analytical performance characteristics of the assay were determined. Results: There were important correlations with hydrogen peroxide, tert-butyl hydroperoxide and cumene hydroperoxide solutions (r = 0.99, P b 0.001 for all). In addition, the new assay presented a typical sigmoidal reaction pattern in copper-induced lipoprotein autoxidation. The novel assay is linear up to 200 μmol H2O2 Equiv./L and its precision value is lower than 3%. The lower detection limit is 1.13 μmol H2O2 Equiv./L. The reagents are stable for at least 6 months on the automated analyzer. Serum TOS level was significantly higher in patients with osteoarthritis (21.23 ± 3.11 μmol H2O2 Equiv./L) than in healthy subjects (14.19 ± 3.16 μmol H2O2 Equiv./L, P b 0.001) and the results showed a significant negative correlation with total antioxidant capacity (TAC) (r = �0.66 P b 0.01). Conclusions: This easy, stable, reliable, sensitive, inexpensive and fully automated method that is described can be used to measure total oxidant status.

Abstract: Objectives: To develop a novel colorimetric and automated direct measurement method for total antioxidant capacity (TAC). Design and Methods: A new generation, more stable, colored 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid radical cation (ABTS *+ ) was employed. The ABTS *+ is decolorized by antioxidants according to their concentrations and antioxidant capacities. This change in color is measured as a change in absorbance at 660 nm. This process is applied to an automated analyzer and the assay is calibrated with Trolox. Results: The novel assay is linear up to 6 mmol Trolox equivalent/l, its precision values are lower than 3%, and there is no interference from hemoglobin, bilirubin, EDTA, or citrate. The method developed is significantly correlated with the Randox- total antioxidant status (TAS) assay ( r = 0.897, P n = 91) and with the ferric reducing ability of plasma (FRAP) assay ( r = 0.863, P n = 110). Serum TAC level was lower in patients with major depression (1.69 ± 0.11 mmol Trolox equivalent/l) than in healthy subjects (1.75 ± 0.08 mmol Trolox equivalent/l, P = 0.041). Conclusions: This easy, stable, reliable, sensitive, inexpensive, and fully automated method described can be used to measure total antioxidant capacity.

Abstract: Objectives To describe the importance of the antioxidant enzymes superoxide dismutase, glutathione peroxidase, and catalase working together in human cells against toxic reactive oxygen species, their relationship with several pathophysiologic processes and their possible therapeutic implications. Conclusions Reactive oxygen species (ROS) are involved in the cell growth, differentiation, progression, and death. Low concentrations of ROS may be beneficial or even indispensable in processes such as intracellular signaling and defense against micro-organisms. Nevertheless, higher amounts of ROS play a role in the aging process as well as in a number of human disease states, including cancer, ischemia, and failures in immunity and endocrine functions. As a safeguard against the accumulation of ROS, several nonenzymatic and enzymatic antioxidant activities exist. Therefore, when oxidative stress arises as a consequence of a pathologic event, a defense system promotes the regulation and expression of these enzymes.

Abstract: Objectives: Oxidative damage of biomolecules occurs as a result of potent free radical reactions. In this study, a novel, colorimetric and fully automated method for measuring total antioxidant response (TAR) against potent free radical reactions is described. Design and methods: Potent free radical reactions were initiated with the production of hydroxyl radical (OH) via Fenton reaction, and the rate of the reactions was monitored by following the absorbance of colored dianisidyl radicals. Ortho-dianisidine (10 mM) and ferrous ammonium sulfate (45 μM) were dissolved in KCl/HCl solution (75 mM, pH 1.8). This mixture was named as Reagent 1 and hydrogen peroxide solution (7.5 mM) as Reagent 2. The OH, produced by mixing of R1 and R2, oxidized o-dianisidine molecules into dianisidyl radicals, leading to a bright yellow-brown color development within seconds. Antioxidants, present in the sample, suppressed the color formation to a degree that is proportional to their concentrations. The method was applied to an automated analyzer and analytical performance characteristics of the assay were determined. Results: Vitamin C and Trolox, reduced glutathione, bilirubin, uric acid and (±)-catechin solutions suppressed the color formation depending on their concentrations. Serum TAR against potent free radical reactions was lower in patients with chronic renal failure (1.13 ± 0.21 mmol Trolox equiv./l) and was higher in the individuals with neonatal icterus (2.82 ± 1.18 mmol Trolox equiv./l) than in healthy subjects (1.54 ± 0.15 mmol Trolox equiv./l). Conclusions: The easy, inexpensive and fully automated method described can be used to measure TAR of samples against potent free radical reactions.

Abstract: A critical study of the different steps involved in previous procedure for hydroxyproline assay allows the direct measurement of collagen content in tissue homogenates without losing the advantages of the method. The procedure is based on alkaline hydrolysis of the tissue homogenate and subsequent determination of the free hydroxyproline in hydrolyzates. Chloramine-T was used to oxidize the free hydroxyproline for the production of a pyrrole. The addition of Ehrlich's reagent resulted in the formation of a chromophore that can be measured at 550 nm. Optimal assay conditions were determined using tissue homogenate and purified acid soluble collagen along with standard hydroxyproline. Critical parameters such as the amount of chloramine-T, sodium hydroxide, p-dimethylaminobenzaidehyde, pH of the reaction buffer, and length of oxidation time were examined to obtain satisfactory results. The method has been applied to samples of tissue homogenate and purified acid soluble collagen, with recovery of added hydroxyproline of 101 ± 6.5 and 104 ± 6.0 (SD) percent, respectively. The method is highly sensitive and reproducible when used to measure the imino acid in tissue homogenates. The modified hydroxyproline assay presented in this communication will be useful for routine measurement of collagen content in extracts of various tissue specimens. In addition, the modified method can be used for batch processing of column fractions to monitor the collagen concentrations during purification.