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Letters |
1
Department of Biochemistry, University Hospital, "Virgen de la Arrixaca", Murcia, Spain, ,
2
Department of Physiology, University of Murcia School of Medicine, Murcia, Spain, ,
3
Department of Anesthesiology, University Hospital, "Virgen de la Arrixaca", Murcia, Spain
a Address correspondence to this author at: C/José Maria Mortes Lerma, 32 Dpl, Pta 14. 46014-Valencia, Spain. Fax 34-926-431668.
To the Editor:
A variety of free radicals (FRs) are produced as a consequence of normal cell metabolism. In healthy tissue, these potentially toxic by-products are removed by endogenous scavengers, but under pathological conditions, the rate of FR release is increased, with consequent lipid peroxidation (1)(2). FRs and lipid peroxidation are assumed to play an essential and causative role in the pathogenesis of cellular damage induced by different afflictions (1)(2)(3). Nitric oxide (NO) is a pluripotential molecule that acts as both an autocrine and paracrine mediator of homeostasis, and derangement of its metabolism can be linked with many pathophysiological events (4). NO is a lipid-soluble gas that, in solution, has a half-life <30 s, undergoing oxidation to nitrate and nitrite. In addition, it can be inactivated by superoxide anions and binds to heme-containing proteins; it has also been shown to interact with FRs. However, NO can form a potent oxidant, peroxynitrite, with a half-life of 1 s (4). Peroxynitrite decomposes to form a reactive hydroxyl radical, and its degradation products have been linked to lipid peroxidation. NO will combine with protein-bound thiol groups (GSHs) to form S-nitrosyl compounds. Finally, each of these interactions can contribute to cell injury, and the effect of NO during oxidative stress injury in human diseases still remains controversial.
However, because FRs are very reactive and short-lived, their direct detection is difficult (2)(3)(4). Determination of lipoperoxides (LPOs), total antioxidant status (TAS), GSHs, and NO end products is therefore of practical importance in determining the effects of free radicals in biological systems. In recent years, several methods have been proposed for measuring these analyte concentrations in biological samples (3)(5)(6)(7)(8)(9). Many literature reports are for individual analytes and are based on small numbers and unspecified statistical evaluation, and reported reference intervals for plasma vary widely. This may be because of methodological variations, problems related to the sampling, time required for analysis, and limitations corresponding to each method, which make it difficult to choose the best method. Nonetheless, measuring the degree of oxidative stress is not in wide clinical use, although no standardized method has been accepted as measuring the oxidative stress status and lipid peroxidation in humans. Because these analyte measurements are important and the need to investigate relationships between clinical outcome and laboratory tests requires reliable reference values, we undertook the current study to provide reliable reference intervals for human plasma, including the establishment of possible sex-related differences.
With the consent of our hospital's Clinical Research Committee we measured LPOs, GSHs, TAS, and NO end products in the plasma of 200 nonhospitalized individuals. Adult subjects were selected for absence of known organic disease and were carefully screened for infectious, malignant, and other serious disorders. The mean age was 42 years (range, 18–65 years), and all persons enrolled in the study had similar lifestyles and dietary habits. To further check their state of health, they were subjected to a conventional biochemical screening and hematological analysis (10). Subjects were classified in two groups according to sex, group A (men) and group B (women). Blood was collected from overnight fasted subjects by venipuncture into 5-mL evacuated tubes containing EDTA/K3 solution as anticoagulant (Becton Dickinson Vacutainer Tube Systems). After centrifugation (2500g) for 10 min in a centrifuge cooled to 4 °C, plasma was removed carefully within 30 min after sample collection. During the assay period, samples were stored at -80 °C until analyzed (usually within 30 days) in trace-element-free tubes to maintain the stability of the plasma samples and inhibit in vitro lipid peroxidation.
To determine interassay precision, we froze aliquots of plasma from a control subject at -80 °C, thawing these only before analysis. Interassay precision was calculated from 20 assays performed over a 30-day period.
LPOs were analyzed using LPO-586 assay (Bioxytech®, OXIS International S.A.; interassay CV, 4.7%). This assay is based on the reaction of a chromogenic reagent (N-methyl-2-phenylindole in acetonitrile), with malondialdehyde (MDA) and 4-hydroxyalkenals (4-HNEs) at 45 °C. One molecule of either MDA or a 4-HNE reacts with two molecules of N-methyl-2-phenylindole in acid medium (methanesulfonic acid), to yield a stable chromophore with a maximum absorbance at 586 nm. Calibration solutions consisted of 1,1,3,3,-tetraethoxypropane in Tris-HCl buffer, pH 7.4, and a 4-HNE as the diethylacetal in acetonitrile (6). When the sample is mixed with reagents, most of the proteins precipitate, and the MDA and the 4-HNEs are extracted simultaneously. After the sample was homogenized and centrifuged, MDA and the 4-HNEs were measured as described above (6).
The TAS was measured using a kit supplied by Randox Laboratories® (interassay CV, 5.0%). The assay principle is that metmyoglobin reacts with H2O2 to form the radical species, ferrylmyoglobin. A chromogen (2,2'-azino-di-[ethylbenzthiazoline sulfonate]; ABTS®) is incubated with the ferrylmyoglobin to produce the radical cation species ABTS. This has a relatively stable blue-green color, which is measured at 600 nm. Antioxidants in the added sample cause suppression of this color production to a degree that is proportional to their concentration (7). Although plasma is not a simple chemical system in regard to antioxidant activity (the different antioxidants may be active at specific sites and hence perform special functions), it has been suggested that one can extrapolate from the above a ranking of antioxidants in the plasma water (7). The GSHs (protein-bound and free content in plasma) were estimated spectrophotometrically by the method of Sedlak and Lindsay(9), which uses Ellman's reagent [5,5'-dithio-bis(2-nitrobenzoic acid); DTNB]; the interassay CV was 4.0%. DTNB and reduced glutathione were obtained from Sigma Chemical Co.
The NO end products in vivo are nitrite and nitrate, which were analyzed by Nitric Oxide Colorimetric Assay® (Boehringer Mannheim; interassay CV, 4.9%). The assay principle is that the nitrate present in the sample is reduced to nitrite by reduced NADPH in the presence of the enzyme nitrate reductase; the nitrite is quantified colorimetrically after the reaction with the Griess reagent (8). Specimens were analyzed in duplicate, and absorbance was measured on a microplate reader (BioWhittaker Microplate Reader model 2001). Statistics (mean, SD, 0.90 confidence interval, unpaired Student t-test, and Kolmogorov–Smirnov test) were determined with the SPSS statistical package (SPSS Inc.).
For each group, there was a representative sample of the reference
population, according to the IFCC guidelines
(11)(12). From the initial 100 individuals in
each group, we discarded the results of those in whom some disease was
found or more than one abnormal biochemical or hematological
measurement was present. For both groups, the distribution of LPO, TAS,
GSH, and NO end product concentrations in human plasma were not
significantly different from a gaussian frequency distribution
(Kolmogorov–Smirnov test). Aberrant values were excluded, according to
the IFCC guidelines (11)(12). Between groups,
the composite distributions were not significantly different from the
distribution for either sex separately. Accordingly, we calculated
parametric (mean ± 2 SD) reference intervals (Table 1
). In conclusion, these analytes can be adapted for screening
patients who may be subjected to oxidative stress, and can be used for
the routine monitoring of lipid peroxidation in human disorders.
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Acknowledgments
This work was supported in part by Fondo de Investigaciones Sanitarias (Madrid, Spain; grants FIS 96/1631, FIS 97/5249, and 98/0606), and a grant from Fundacion para el Desarrollo del Trasplante Hepatico and Novartis Farmaceutica S.A. (Madrid, Spain).
References
The following articles in journals at HighWire Press have cited this article:
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  M. G. Salom, B. Arregui, L. F. Carbonell, F. Ruiz, J. L. Gonzalez-Mora, and F. J. Fenoy Renal ischemia induces an increase in nitric oxide levels from tissue stores Am J Physiol Regulatory Integrative Comp Physiol, November 1, 2005; 289(5): R1459 - R1466. [Abstract] [Full Text] [PDF]
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 J. F. Bentzon, E. Skovenborg, C. Hansen, J. Moller, N. S.-C. de Gaulejac, J. Proch, and E. Falk Red Wine Does Not Reduce Mature Atherosclerosis in Apolipoprotein E-Deficient Mice Circulation, March 27, 2001; 103(12): 1681 - 1687. [Abstract] [Full Text] [PDF]
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 M. LEAL, J. DIAZ, E. SERRANO, J. ABELLAN, and L. F. CARBONELL Hormone Replacement Therapy for Oxidative Stress in Postmenopausal Women With Hot Flushes Obstet. Gynecol., June 1, 2000; 95(6): 804 - 809. [Abstract] [Full Text] [PDF]
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