Effect of fasting on blood lipid peroxidation parameters of sheep

Research in Veterinary Science 1993, 55, 104-107

Effect of fasting on blood lipid peroxidation parameters of sheep T. GAAL, Department of Medicine, University of Veterinary Science, Istvdn Street 2, H-1078, Budapest, Hungary, M. MEZES, Department of Nutrition, University of Agricultural Sciences, GOdgll6, Hungary, O. MISKUCZA, Department of Medicine, P. RIBICZEY-SZABO, Central Laboratory, University of Veterinary Science, Istvdn Street 2, H-1078, Budapest, Hungary

The effects of three-day fasting and one-day

refeeding on some blood metabolites and parameters of lipid peroxidation were studied in eight non-pregnant merino ewes, Fasting produced an immediate decrease in blood glucose accompanied by an increase of free fatty acid, total lipid, total cholesterol and urea in the plasma. Starvation increased the concentration of thiobarbituric acid-reactive substances (malondialdehyde), with a slower but more sustained increase in the plasma than in the red blood cell haemolysate. Changes in glutathione peroxidase activity were the reverse of those in malondialdehyde concentration. Catalase activity was not measurable in plasma but was consistently increased in the haemolysate on fasting. Superoxide dismutase activity in the whole blood haemolysate significantly increased only on the first day of food deprivation. The vitamin E content of plasma showed no significant changes. The results indicate that energy deficiency, a well-known phenomenon in ruminants, affects not only the metabolic parameters of the blood but its lipid peroxidative status as well. THE metabolic consequences of nutritional deficiencies on carbohydrate, nitrogen and lipid metabolism are largely understood. During the past few years evidence has emerged indicating an association between food deprivation and effects upon tissue antioxidant systems (Lammi-Keefe et al 1984, Wohaieb and Godin 1987). The present knowledge in this area has recently been the subject of a review (Godin and Wohaieb 1988). As a result it was postulated that the effect of dietary restriction occurred either via a specific cofactor deficiency or as a result of complex but interrelat-

ed metabolic alterations arising in response to starvation. In the review there was virtually no reference to the consequences of food deprivation or starvation on antioxidative processes in ruminant species. The absence of such data is somewhat surprising as nutritional (energy) deficiency is frequently a feature in the metabolism of ruminant species; indeed it is an important factor in the development of such well-known metabolic disorders as ketosis of dairy cows and pregnant ewes (pregnancy toxaemia) or fatty liver (fat cow, fat mobilisation) syndrome (Schultz 1968, Morrow 1976, Roberts et al 1981, Reid and Roberts 1982). Ketosis can even be modelled through the starvation of cows (Brumby et al 1975). During the development of these well known disorders there is extensive mobilisation of body reserves, in particular tissue fat, to maintain the energy balance. Consequential large scale effects on aspects of lipid metabolism and composition within the tissues, plasma and milk have been noted (Brumby et al 1975, Husvdth et al 1983). Although it is to be expected that ruminant animals display similar alterations in their antioxidant status during periods of starvation or energy deficiency to that of other animals, such data are not available. Accordingly an in vivo experiment to study the effect of a three-day starvation period in sheep on a selection of blood parameters associated with lipid peroxidation has been conducted.

Materials and methods

Animals and sampling Eight non-pregnant merino ewes, three to four years old, were housed individually during early 104

Blood lipid peroxidation parameters spring. The animals were habituated to being handled for several weeks before the experiment while blood samples were sometimes also taken. During this time the diet of the sheep consisted of a commercial pelleted food received twice daily (08.00 and 17.00) and an ad libitum supply of grass hay. On the day before the period of starvation, heparinised blood samples were collected from a jugular vein at 07.00 to provide basal values. Further blood samples were then collected during the three-day starvation period at 24, 48 and 72 hours, following which the standard dietary regime was resumed. A final blood sample was taken one day after feeding was resumed,

Analyses Immediately following sampling an aliquot of whole blood was removed for the determination of superoxide dismutase (SOD). Plasma and red blood cells (RBC) were separated by centrifugation (2500 g, 15 minutes at 40C) and the plasma was stored frozen (-20°C) until further determination. Packed cells were then washed three times with 0.9 per cent sodium chloride and then haemolysed by exposure to nine parts of redistilled water followed by freezing (-20°C, 18 h) and thawing before analysis. SOD activity was measured by the production of formazan through the action of superoxide ion on nitrotetrazolium blue (Fried et al 1970, Nishikimi et al 1972) and was expressed in a widely used arbitrary unit g-1 protein of the whole blood haemolysate. One unit means 50 per cent inhibition of formazan production min -1 at 25 °C. Plasma metabolites of energy, lipid and nitrogen metabolism, that is, glucose, free fatty acids, total lipid, total cholesterol and urea were quantified using standard commercial kits (Boehringer Mannheim, Germany). Thiobarbituric acid-reactive Substances and the activity of glutathione peroxidase (6SH-Px) were measured in both the plasma and the RBC haemolysate. Thiobarbituric acid-reacting compounds were assayed according to Placer et al (1966) and expressed as malondialdehyde (MDA) equivalents in gmol litre-1 plasma and gmol g-1 haemoglobin in RBChaemolysate. GSH-Px was determined by using cumene hydroperoxide and reduced glutathione (GSH) as substrates and the loss of a s h following enzymic reaction was measured with Ellman reagent (Lawrence and Burk

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1976). The enzyme activity was expressed in iu (gmol a s h oxidised rain -1 at 25°C). Catalase activity was measured in the RBC haemolysate only using a direct ultraviolet detection system for the determination of hydrogen peroxide concentration (Beers and Sizer 1952). The enzyme activity was expressed in iu (1 gmol hydrogen peroxide loss min -1 at 25°C). The vitamin E content of the plasma was measured spectrophotometrically using the Emmerie-Engel reaction (Bieri 1964).

Statistical analysis Mean values of the determinations were compared to the basal values using the paired Student's t test. Results

As can be seen from Table 1 a significant decrease of blood glucose occurred immediately TABLE 1: The effects of three-day fasting and one-day refeeding on some blood parameters in sheep (n=8; means [SEMI) Basal value Glucose, 3.03 mmol litre -1 (0.12) FFA, 0'36 mmoI litre -1 (0.05) TCh, 1.51 mmol litre -1 (0-09) TL, 1"98 g litre -1 (0.07) Urea, 4.52 mmol litre -1 (0.63)

1

Days of fasting 2 3

2-55* (0.12) 0"57** (0.05) 1.89" (0.15) 1-91 (0'07) 6.79** (0.34)

2-68** (0.16) 0'85*** (0.08) 1.96"** (0.05) 2"56*** (0"07) 6.85** (0.66)

2-66** (0.17) 1"28"** (0-12) 2.46*** (0.16) 3"95*** (0'16) 5-49 (0.69)

Refeeding value 2.83 (0.06) 0"72** (0.12) 2-16" (0-10) 3'33** (0.21) 5.52 (0.89)

Probability of difference from the basal value *P<0.05; **P<0.01; ***P<0-001 FFA Free fatty acids TL Total lipid TCh Total cholesterol

after food deprivation and was accompanied by consistent increases over the three-day fasting period in the concentrations of free fatty acids, total lipid and total cholesterol in the plasma. When feeding was resumed the concentrations of all three lipid parameters decreased but still remained significantly higher than the basal values. Urea concentration also increased but significantly only for the first two days of starvation after which time the concentration was substantially reduced.

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T. Gadl, M. Mdzes, O. Miskucza, P. Ribiczey-Szab6

TABLE 2: The effects of three-day fasting and one-day refeeding on blood lipid peroxidation parameters in sheep (n=8; means [SEM]) Basal value Plasma MDA, ~mollitre -1 RBC haemolysate MDA, ~mo[ litre-1 Plasma GSH-Px iu g-1 protein RBC haemolysate GSH-Px, iu g-1 Hb RBC haemolysate CAT, iu g-1 Hb Whole blood haemolysate SOD, U g-1 protein Plasma vitamin E, mmol litre-1

1

Days of fasting 2 3

Refeeding value

1'49 1'66 2'09 (0'11) (0"14) (0'27) 4.64 6-36 6.00 (0'32) (0"57) (0"69) 1.74 1.77 1'18 (0"26) (0"21) (0"30) 15.44 15.23 17.12 (0'94) (0'56) (1"15) 68"15 74"04* 89'46** (4"09) (3.74) (6.36) 2.06 4.02* 2.93 (0.24) (0.64) (0.65)

3'10"** (0"19) 4.86 (0'66) 0.85* (0'15) 17.28 (1"47) 95"84** (9'76) 2.31 (0.39)

2'88*** (0'16) 4.70 (0"65) 0.96* (0"16) 18.36 (1'87) 93'99** (4.59) 2.95 (0.47)

0'133 0"127 0'130 (0'002) (0"005) (0"004)

0"133 (0'005)

0"126 (0"005)

CAT Catalase Probability of difference from the basal value *P<0.05; **P<0.01; ***P<0.001

The blood parameters of lipid peroxidation are shown in Table 2. MDA concentration in the blood plasma and that of the RBC haemolysate behaved differently during the starvation period. In plasma the steady increase in MDA concentration reached statistical significance on the third day of fasting; although after feeding was resumed a slight decrease was observed, the concentration remained significantly higher than the basal value. In the RBC haemolysate the concentration of MDA did not change significantly during the experiment. In general terms, GSH-Px activity showed the reverse of the changes in MDA concentration. The plasma activity of the enzyme showed little change following one-day of fasting but thereafter decreased until by the end of the starvation period when its activity was only some 50 per cent of the basal value. The activity increased only slightly after feeding was resumed but still remained significantly lower than the basal value. The red cell haemolysate GSH-Px activity did not significantly change during the period of the experiment. Catalase activity was not measurable in the plasma. Its activity in the RBC haemolysate showed a constant increase during starvation reaching significance after the first day of fasting. Following refeeding, catalase values underwent a slight but not significant reduction. The SOD activity of the whole blood haemolysate showed a significant increase only on the first day of food deprivation.

The vitamin E content of plasma did not change during the experimental period. Discussion The study investigated the effect of three days food deprivation on some parameters of lipid peroxidation in the blood of healthy sheep. Deficiences of several important nutritional factors, such as selenium, copper, manganese, vitamin E, can markedly alter tissue antioxidant status (I-Iafeman et al 1974, Hausewirth and Nair 1975, Williams et al 1975, deRosa et al 1980). The effect of these particular dietary factors is well understood, because they are well-defined components of tissue antioxidant systems. By contrast, general undernutrition is involved in a series of complex effects on the free radical defence mechanisms. Food deprivation causes alterations in the activities of tissue antioxidant enzymes, the extent of alteration varying considerably depending upon the particular tissue involved and the enzyme system in question (Godin and Wohaieb 1988). One of the most important effects of starvation is a decreased aSH concentration in the liver and other tissues (Igarashi et al 1983). As a result of a lack of substrate (reduced GSH) the effect may consequently give rise to a decrease of GSI-I-Pxactivity in the blood plasma. Another consequence of fasting is an acceleration in skeletal muscle proteolysis and a decrease in 6stt:oxidised glutathione ratio (Tischler et al 1985). Such results have been observed most notably during investigations using laboratory animals and in certain instances from human clinical observations. By contrast, data concerning alterations of the antioxidant status of fasted ruminants are not available. Similarly, there are very few clinical studies that point out the importance of alterations in the antioxidant status of energy-deficient ruminants, such as ketotic dairy cows and pregnant ewes or cows with fatty liver. Nevertheless, clinical studies on energy-deficient ruminants have documented changes in some blood parameters associated with lipid peroxidation. Hidiroglou and Hartin (1982) found lower vitamin E and selenium concentrations in the blood of cows with fatty liver than in controls. In a field survey it has been found that in energy-deficient dairy cows displaying fatty liver, MDA values were some two to two and a half times higher in blood plasma and haemolysate than in controls. GSH-Px activity

Blood lipid peroxidation parameters either in the plasma or in the RBC haemolysate and catalase activity of RBC haemolysate were significantly lower in energy-deficient cows (Ga~l et al 1990). Metabolic disorders such as hypoglycaemia, increased levels of free fatty acids, ketone bodies and urea in the blood plasma and accumulation of fat in the liver are common laboratory findings (Schultz 1968, Reid and Roberts 1982, Oltner and Wiktorsson 1983). In the present experiment with starved sheep, such changes of blood composition reflected the energy deficiency. The interpretation and comparison of the changes of lipid peroxidation in the blood during the acute fasting of the sheep in the present experiment with those of other clinical reports is rather difficult. Thus during the course of a field survey on dairy cows (Gafil et al 1990), changes in MDA and ~SH-Px but not catalase values were found similar to those observed in this study during starvation. It may be concluded, therefore, that the effects of total and immediate food deprivation differ from those of partial and long-term undernutrition. However, taken together it would appear that either starvation or undernutrion of ruminants causes significant alterations to the lipid peroxidative processes in the blood. The permanent increase of catalase activity within the RBC-haemolysate observed here would fully support such a hypothesis about the lipid peroxidative changes of blood during starvation. The temporary change in SOD activity only on the first day of the experiment would suggest that the primary defence of abe against oxygen-free radicals is catalase rather than SOD. References BEERS, R. F. & SIZER, 1. W. (1952) A spectrophotometric method for measuring the breakdown of hydrogen peroxide by catalase. Journal of Biological Chemistry 195, 133-140 BIERI, J. G. (1964) Serum vitamin E levels in a normal adult population in the Washington DC area. Proceedings of the Society for Experimental Biology and Medicine 117, 131-133 BRUMBY, P. E., ANDERSON, M., TUCKLEY, B. & STORRY, J. E. (1975) Lipid metabolism in the cow during starvation-induced ketosis. Biochemical Journal 146, 609-615 DEROSA, G., KEEN, C. L., LEACH, R. M. & HURLEY, L. S. (1980) Regulation of superoxide dismutase activity by dietary manganese. Journal of Nutrition 110, 795-804 FRIED, R., FRIED, L. W. & BABIN, D. (1970) The inhibition of reduc-

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Received September 7, 1992 Accepted December 2, 1992