Correlation between Glutathione Peroxidase Activity and the Quantity of Selenium in the Whole Blood of Beef Calves

Harapin I . , M. Bauer , L. Bedrica, D. Potoãnjak: Correlation Between Glutathione Peroxidase Activity and The Quantity of Selenium in The Whole Blood of Beef Calves. Acta Vet. Brno 2000, 69: 87–92. The aim of the study was to establish concentration of selenium and the pertinent activity of glutathione peroxidase (GSH-Px ) in whole blood that could be used as reference values in future research. The first part of study was carried out on clinical healthy beef calves (n = 35), fed a basal diet and a ready-made fodder mix that contained 0.1 mg.kg-1 selenium. In the second part of our research, we investigated the GSH-Px activity in a group of calves (n = 47) that had not received supplements added to the basal diet. The research was carried out in the north-west Croatia, in the region known to be poorer in selenium. We determined GSH-Px activity in whole blood by the “Ransel” method adapted to a Technicon RA-1000 at 37 °C. Determination of Se concentration was done with a modification of the method given by Perkin-Elmer HGGS. The mean value of the Se concentration in the whole blood of the first group of calves was 200.22 ± 45.2 μg.l-1 and pertinent average GSH-Px activity was 764.6 ± 197.8 μkat.l-1. From the results obtained, a correlation between Se and GSH-Px was calculated (r = 0.82; P < 0.001). The mean values of the GSH-Px activity in herd that did not receive a supplement were 435.3 ± 155.76 μkat.l-1. Out of 47 animals of the second herd, four animals (8.51%) had lower values than those recommended as sufficient. This study confirmed that after the calculation of the correlation between Se and GSHPx, glutathione peroxidase activity determination can be used as a rapid and simple proxy for the determination of selenium concentration in whole blood. Glutathione-peroxidase, selenium, white muscle disease, beef cattle The discovery that glutathione peroxidase (GSH-Px) protects haemoglobin from oxidative denaturalisation by hydrogen peroxide (Mi l l s 1957) and that the erythrocytes of rats with selenium deficiency are subject to haemolysis brought about by hydrogen peroxide (Rotruck et al. 1972) led to the realisation that glutathione peroxidase is an enzyme which contains the micro-element selenium (Rot ruck et al. 1973; F lohe et al. 1973). This knowledge enabled the construction of a biochemical model through which it is possible to monitor the relation between selenium, vitamin E, glutathione peroxidase, polyunsaturated fatty acids and sulphur containing amino acids (Chow and Tappe l 1974; Hoeks t ra 1974). Vitamin E and glutathione peroxidase protect biological membranes from the harmful effect of free radicals. Glutathione peroxidase works in the cytosol of the cell, and vitamin E is incorporated into the lipid membranes (Smi th et al. 1988). Soon after the discovery that glutathione peroxidase is a potent antioxidant, containing four selenium atoms in its molecule, it was determined that there is also a glutathione peroxidase that does not contain selenium (Lawrence and Burk 1978). Scho lz et al. (1981) studied the distribution of Se-glutathione peroxidase and non-Se-glutathione peroxidase in calf tissues and blood. It was established that the spleen, cardiac muscle, erythrocytes, brain, thymus, fatty tissue and striated muscles contain only Se-glutathione peroxidase. The liver, lungs, adrenaline, testes and kidneys ACTA VET. BRNO 2000, 69: 87–92 Address for correspondence: Dr. Sc. Ivica Harapin Clinic for Internal Diseases of Domestic Animals Veterinary Faculty Heinzelova 55, 10000 Zagreb, Croatia Phone: +385 1 2390 342 Fax: +3851 214 697 E-mail: [email protected] http://www.vfu.cz/acta-vet/actavet.htm contain both isozymes. Of the internal organs, the liver had the most non-selenium glutathione peroxidase activity. This statement was confirmed by Pehrson (1985). Scho lz and Hutch inson (1979) investigated the distribution of glutathione peroxidase activity and selenium concentration in the body fluids of dairy cows and determined that 98.6% of glutathione peroxidase activity, when the activity of the enzyme is expressed in mU.ml-1 of whole blood, is related to erythrocytes in the peripheral blood. In the cellular elements of the blood, there is also a larger proportion of selenium (73%) than in the plasma. The research of Thompson et al. (1981) shows that GSH-Px activity in the plasma is a more sensitive indicator than whole blood activity, but since the values in the plasma are very low, and the results are accordingly less reliable. Diagnostics are based on detailed data concerning the composition of the soil and feedstuffs, and on laboratory tests that can show selenium deficiency (the element being an indispensable component of the enzyme glutathione peroxidase) and/or vitamin E, as well as on pathohistological evidence of muscles that show muscular degeneration. However, post-mortem findings only confirm the damage, and do not reduce it. Determination of the quantity of selenium in food or in the serum of animals at threat is relatively expensive because it requires a complex equipment. At present, the enzyme glutathione peroxidase has been used as an indicator of the extent to which the organism is supplied with selenium, for glutathione peroxidase has four Se atoms and a very high and significant positive correlation with quantities of selenium in the blood. Although glutathione peroxidase research goes back thirty years, the actual process has only recently been standardised for biochemical analyses. Because of the diversity of methods and ways of presenting the results (authors have not shown the enzyme activities obtained only in terms of IU.l-1 of whole blood (or in μkat.l-1), but linked to haemoglobin content (IU.gHb-1) or packed cell volume value (IU.ml PCV-1), many data in the literature have not been comparable. Lack of standardisation in the determination and presentation of the results of glutathione peroxidase activity led us to study this problem. Our research (Harapin 1996) aimed at establishing our own values for glutathione peroxidase activity and the pertinent concentration of selenium in whole blood, which would be usable as reference values in future research. Materials and Methods The experiment was carried out on 35 crossbreed six-month-old Simmental calves. The animals were clinically healthy, and according to anamnesis had not had any kind of sickness for at least one month before the blood samples were taken. They had been fed maize grits silage, whole maize plant silage, hay and a ready-made fodder mix that contained 0.1 mg.kg-1 selenium. In the second part of the study, we investigated the GSH-Px activity in a group of beef calves that had not received supplements added to the basal diet. The animals were chosen from individual breeds after clinical test. The main criterion was weaker growth and stunted animals. A total of 47 fattened calves, 5-9-month-old were included in the study. The calves had been fed with the usual fattening feed ground maize corn mixed with cereal crops, maize silage and hay ad libitum. The study was carried out in the north-western Croatia, in the region known to be poorer in selenium, as indicated by previous investigations (Vulinec et al. 1985). The blood was taken from v. jugularis by the Vacutainer system into test tubes with 143 IU sodium heparinate/7 ml. We determined GSH-Px activity in whole blood by the commercial “Ransel” method from Randox Laboratories Ltd. adapted to a Technicon RA-1000 biochemical analyser at 37 °C. The method was based on the work of Pagl ia and Valent ine (1967), which described a direct spectrophotometric method for determining the glutathione peroxidase of blood cells. Determination of selenium concentration was done with a modification of the method by Perkin-Elmer HGGS. For a reading of the selenium in the samples, an atomic absorption spectrophotometer had a non-electrode lamp for selenium connected to it as well as apparatus for selenium hydride generation. The experimental data were statistically evaluated by standard descriptive methods and regression analysis (Chase and Bown 1997) made by SAS Software Release 6.12. 88