609
Biochem. J. (1975) 146, 609-615 Printed in Great Britain
Lipid Metabolism in the Cow during Starvation-Induced Ketosis By PHILIP E. BRUMBY, MALCOLM ANDERSON, BRIAN TUCKLEY and JOHN E. STORRY National Institute for Research in Dairying, Shinfield, Reading RG2 9AT, U.K. and KENNETH G. HIBBIIT Agricultural Research Council Institute for Research on Animal Diseases, Compton, Berks. RG16 ONN, U.K. (Received 16 September 1974)
1. Concentrations and compositions of liver, serum and milk lipids of cows were measured during 6 days' starvation and serum lipids during 60 days' re-feeding. 2. The concentration of free fatty acid in serum increased fivefold during starvation. 3. The content of total lipid in liver (g/100g of liver dry matter) doubled owing to a 20-fold increase in triglyceride, an eightfold increase in cholesterol ester, a threefold increase in free fatty acid and a 20 % increase in cholesterol. There were no changes in the content or composition of liver phospholipids. 4. Starvation lowered the concentrations of total lipid, phospholipid and cholesterol ester of dextran sulphate-precipitable serum lipoproteins. Total lipid and cholesterol ester concentrations in lipoproteins of d> 1.055 and in lipoproteins not precipitable by dextran sulphate decreased from day 4 ofthe starvation period and during the first 20 days' re-feeding. 5. During starvation there were decreases in percentages of stearic acid and increases in oleic acid in serum free fatty acids and triglycerides and in liver neutral lipid. 6. Throughout starvation total milk lipid yield decreased, yields and percentages of C4_14 fatty acids decreased and percentages of C18 fatty acids increased. 7. It is suggested that accumulation of triglyceride in liver may be caused by increased uptake of plasma free fatty acids without corresponding increase in lipoprotein secretion. Spontaneous bovine ketosis is characteristically found in high-producing animals in early lactation when the energy demands for production exceed the dietary supply. In addition to the well-documented ketonaemia and hypoglycaemia the condition is also marked by heavy fatty infiltration of the liver (Schultz, 1968, 1971; Bergman, 1971) and changes in the lipid and fatty acid composition of milk (Luick & Smith, 1963; Waterman & Schultz, 1972) and blood plasma (McCarthy et al., 1968a,b; Yamdagni & Schultz, 1970; Waterman & Schultz, 1972; Waterman et al., 1972). Responses are similar during decreased energy intake or complete starvation in sheep (Ford, 1962; Patterson, 1966; Leat & Ford, 1966; Jackson & Winkler, 1970; Mann, 1972), cows (Luick & Smith, 1963; Kinsella & Butler, 1970; Fisher et al., 1971) and goats (Annison et al., 1968; Yamdagni & Schultz, 1969). The accumulation of lipid in the liver and the changes in composition of blood and milk lipids suggested some aberration in lipid transport. To provide further evidence on this suggestion the simultaneous measurement of changes in milk, serum and liver lipids was undertaken in cows in which ketosis was induced by completely withholding food for 6 days. Vol. 146
Studies on carbohydrate metabolism (Baird et al., 1972) and ultrastructure of the liver (Reid & Isenor, 1972) were carried out on the same animals.
Experimental Experimental animals Three lactating cows were starved for 6 days and sampled for blood and liver tissue, as described by Baird et al. (1972). Further samples of blood were taken approx. 20, 40 and 60 days after liver biopsies had been performed and when the animals were again receiving their normal food intake. Samples of milk were also taken from two of the cows before and during the period of starvation. In addition, liver samples were taken at slaughter from three fed lactating cows for the purposes of lipid and fatty acid comparison. Methods of analysis
Milk. Total lipid was determined by the Gerber method (British Standards Institution, 1969). Fatty acid composition was determined as described by Storry et al. (1973). 20
610
P. E. BRUMBY, M. ANDERSON, B. TUCKLEY, J. E. STORRY AND K. G. HIBBITT
Blood. Blood samples were allowed to clot at room temperature for 2h and the serum was decanted. Lipoproteins were separated ultracentrifugally into fractions of d> 1.055 and d< 1.055 and by dextran sulphate into precipitable and non-precipitable fractions (Brumby & Welch, 1970). Lipids were extracted from whole serum and from lipoprotein fractions and their weights determined as described by Brumby et al. (1972). Lipids were separated by t.l.c. (Tuckley & Storry, 1974). Heptane extracts of cholesterol and cholesterol esters were determined by the method of Brown (1959) after adaptation to the Technicon Autoanalyser. The extracts were reacted with colour reagent [1 ml of 72% (w/w) HC104+100ml of acetyl chloride+400ml of 1,2-dichloroethane] in the ratio 1: 10 (v/v) for 10min at 50°C. HC104 digests of phospholipids were analysed by the method of Chen et al. (1956) after adaptation to the Autoanalyser. The digest was treated with molybdate reagent [2vol. of 0.3% (w/v) ammonium molybdate in 1.1 M-HC104 + 1 vol. of 2% (w/v) ascorbic acid] in the ratio 1: 3 (v/v) for 5min at 90°C. Fatty acids were released from triglycerides by heating at 70°C for 30min with 0.4% (w/v) KOH in 95 % (v/v) ethanol, converted into acid form with an excess ofHCI and extracted into chloroform. Triglyceride fatty acids and serum free fatty acids were determined by the method of Antonis (1965), in which the bead-filled column was replaced by a coiled polythene tube (Sm x I mm bore), cupric sulphate was used in the buffered copper reagent and 0.3 % (w/v) zinc dibenzyldithiocarbamate in chloroform was used to detect the copper soaps. The fatty acid compositions of the free fatty acids and triglycerides of serum were analysed by g.l.c. (Brumby et al., 1972) on a Hewlett-Packard model 7620 A chromatograph (Hewlett-Packard Ltd., Wokingham Berks., U.K.) linked to an on-line Data Processor (Digital Equipment Co. Ltd., Reading, Berks., U.K.).
Liver. Total lipid was extracted from liver tissue in chloroform-methanol (2: 1, v/v) as described by Folch et al. (1957). The various component lipids were determined as described for blood. Phospholipids were separated by t.l.c. (Parsons & Patton, 1967) and individual components were identified by reference to authentic standards. Phospholipids were determined as described above. Fatty acid compositions of neutral lipids were determined by g.l.c. after their separation from polar lipids by t.l.c. (Anderson, 1974). Statistical analysis. Differences between fed and starved cows were analysed as described in the legends to the Tables and Figures. Results Milk secretion During starvation there was a significant decrease in yield of milk from 17.6 to 4.3kg/day per cow (Baird et al., 1972) and a significant increase in milk lipid percentage (Table 1). Changes in the proportions and yields of individual acids in milk differed according to their origin (see Storry, 1970, 1972). The proportions and yields of C414 acids, synthesized within the mammary gland, were progressively decreased throughout lactation (Tables 1 and 2). The proportions and yields of C16 acids, derived from both intramammary synthesis and pre-formed plasma C16 acids, were also decreased, but the regression coefficients were not statistically significant. There was no significant change in the yield of C18 acids, which are considered to be derived entirely from blood plasma C18 fatty acids (see Storry, 1970), but their proportion in milk increased progressively. The decrease in yield of total milk fatty acids was not statistically
significant (Table 2).
Table 1. Effect Ofstarvation on thepercentage andfatty acidcomposition ofcows' milk lipid
Milk samples were taken at approx. 12h intervals from two cows for 2 days before and during 6 days' starvation. For each cow, sample from 2-day periods were combined in proportion to milk yield and analysed for lipid percentage (g/lOOg of milk) and fatty acid percentage (g/lO0g of fatty acid) as described under 'Methods of analysis'. Fatty acid percentages for acids with 4-14,16 and 18 carbon atoms which changed similarly during starvation were combined. For total lipid and each group of fatty acids the s.E. of a 'day' mean and the S.E. of the linear effect of time (regression coefficient) was derived from the error variance 'days x cows'. The significance of the regression coefficient from zero was determined by the t test (t = regression coefficient-s.E. of regression coefficient). Significance of regression coefficients from zero: * P
Biochem. J. (1975) 146, 609-615 Printed in Great Britain
Lipid Metabolism in the Cow during Starvation-Induced Ketosis By PHILIP E. BRUMBY, MALCOLM ANDERSON, BRIAN TUCKLEY and JOHN E. STORRY National Institute for Research in Dairying, Shinfield, Reading RG2 9AT, U.K. and KENNETH G. HIBBIIT Agricultural Research Council Institute for Research on Animal Diseases, Compton, Berks. RG16 ONN, U.K. (Received 16 September 1974)
1. Concentrations and compositions of liver, serum and milk lipids of cows were measured during 6 days' starvation and serum lipids during 60 days' re-feeding. 2. The concentration of free fatty acid in serum increased fivefold during starvation. 3. The content of total lipid in liver (g/100g of liver dry matter) doubled owing to a 20-fold increase in triglyceride, an eightfold increase in cholesterol ester, a threefold increase in free fatty acid and a 20 % increase in cholesterol. There were no changes in the content or composition of liver phospholipids. 4. Starvation lowered the concentrations of total lipid, phospholipid and cholesterol ester of dextran sulphate-precipitable serum lipoproteins. Total lipid and cholesterol ester concentrations in lipoproteins of d> 1.055 and in lipoproteins not precipitable by dextran sulphate decreased from day 4 ofthe starvation period and during the first 20 days' re-feeding. 5. During starvation there were decreases in percentages of stearic acid and increases in oleic acid in serum free fatty acids and triglycerides and in liver neutral lipid. 6. Throughout starvation total milk lipid yield decreased, yields and percentages of C4_14 fatty acids decreased and percentages of C18 fatty acids increased. 7. It is suggested that accumulation of triglyceride in liver may be caused by increased uptake of plasma free fatty acids without corresponding increase in lipoprotein secretion. Spontaneous bovine ketosis is characteristically found in high-producing animals in early lactation when the energy demands for production exceed the dietary supply. In addition to the well-documented ketonaemia and hypoglycaemia the condition is also marked by heavy fatty infiltration of the liver (Schultz, 1968, 1971; Bergman, 1971) and changes in the lipid and fatty acid composition of milk (Luick & Smith, 1963; Waterman & Schultz, 1972) and blood plasma (McCarthy et al., 1968a,b; Yamdagni & Schultz, 1970; Waterman & Schultz, 1972; Waterman et al., 1972). Responses are similar during decreased energy intake or complete starvation in sheep (Ford, 1962; Patterson, 1966; Leat & Ford, 1966; Jackson & Winkler, 1970; Mann, 1972), cows (Luick & Smith, 1963; Kinsella & Butler, 1970; Fisher et al., 1971) and goats (Annison et al., 1968; Yamdagni & Schultz, 1969). The accumulation of lipid in the liver and the changes in composition of blood and milk lipids suggested some aberration in lipid transport. To provide further evidence on this suggestion the simultaneous measurement of changes in milk, serum and liver lipids was undertaken in cows in which ketosis was induced by completely withholding food for 6 days. Vol. 146
Studies on carbohydrate metabolism (Baird et al., 1972) and ultrastructure of the liver (Reid & Isenor, 1972) were carried out on the same animals.
Experimental Experimental animals Three lactating cows were starved for 6 days and sampled for blood and liver tissue, as described by Baird et al. (1972). Further samples of blood were taken approx. 20, 40 and 60 days after liver biopsies had been performed and when the animals were again receiving their normal food intake. Samples of milk were also taken from two of the cows before and during the period of starvation. In addition, liver samples were taken at slaughter from three fed lactating cows for the purposes of lipid and fatty acid comparison. Methods of analysis
Milk. Total lipid was determined by the Gerber method (British Standards Institution, 1969). Fatty acid composition was determined as described by Storry et al. (1973). 20
610
P. E. BRUMBY, M. ANDERSON, B. TUCKLEY, J. E. STORRY AND K. G. HIBBITT
Blood. Blood samples were allowed to clot at room temperature for 2h and the serum was decanted. Lipoproteins were separated ultracentrifugally into fractions of d> 1.055 and d< 1.055 and by dextran sulphate into precipitable and non-precipitable fractions (Brumby & Welch, 1970). Lipids were extracted from whole serum and from lipoprotein fractions and their weights determined as described by Brumby et al. (1972). Lipids were separated by t.l.c. (Tuckley & Storry, 1974). Heptane extracts of cholesterol and cholesterol esters were determined by the method of Brown (1959) after adaptation to the Technicon Autoanalyser. The extracts were reacted with colour reagent [1 ml of 72% (w/w) HC104+100ml of acetyl chloride+400ml of 1,2-dichloroethane] in the ratio 1: 10 (v/v) for 10min at 50°C. HC104 digests of phospholipids were analysed by the method of Chen et al. (1956) after adaptation to the Autoanalyser. The digest was treated with molybdate reagent [2vol. of 0.3% (w/v) ammonium molybdate in 1.1 M-HC104 + 1 vol. of 2% (w/v) ascorbic acid] in the ratio 1: 3 (v/v) for 5min at 90°C. Fatty acids were released from triglycerides by heating at 70°C for 30min with 0.4% (w/v) KOH in 95 % (v/v) ethanol, converted into acid form with an excess ofHCI and extracted into chloroform. Triglyceride fatty acids and serum free fatty acids were determined by the method of Antonis (1965), in which the bead-filled column was replaced by a coiled polythene tube (Sm x I mm bore), cupric sulphate was used in the buffered copper reagent and 0.3 % (w/v) zinc dibenzyldithiocarbamate in chloroform was used to detect the copper soaps. The fatty acid compositions of the free fatty acids and triglycerides of serum were analysed by g.l.c. (Brumby et al., 1972) on a Hewlett-Packard model 7620 A chromatograph (Hewlett-Packard Ltd., Wokingham Berks., U.K.) linked to an on-line Data Processor (Digital Equipment Co. Ltd., Reading, Berks., U.K.).
Liver. Total lipid was extracted from liver tissue in chloroform-methanol (2: 1, v/v) as described by Folch et al. (1957). The various component lipids were determined as described for blood. Phospholipids were separated by t.l.c. (Parsons & Patton, 1967) and individual components were identified by reference to authentic standards. Phospholipids were determined as described above. Fatty acid compositions of neutral lipids were determined by g.l.c. after their separation from polar lipids by t.l.c. (Anderson, 1974). Statistical analysis. Differences between fed and starved cows were analysed as described in the legends to the Tables and Figures. Results Milk secretion During starvation there was a significant decrease in yield of milk from 17.6 to 4.3kg/day per cow (Baird et al., 1972) and a significant increase in milk lipid percentage (Table 1). Changes in the proportions and yields of individual acids in milk differed according to their origin (see Storry, 1970, 1972). The proportions and yields of C414 acids, synthesized within the mammary gland, were progressively decreased throughout lactation (Tables 1 and 2). The proportions and yields of C16 acids, derived from both intramammary synthesis and pre-formed plasma C16 acids, were also decreased, but the regression coefficients were not statistically significant. There was no significant change in the yield of C18 acids, which are considered to be derived entirely from blood plasma C18 fatty acids (see Storry, 1970), but their proportion in milk increased progressively. The decrease in yield of total milk fatty acids was not statistically
significant (Table 2).
Table 1. Effect Ofstarvation on thepercentage andfatty acidcomposition ofcows' milk lipid
Milk samples were taken at approx. 12h intervals from two cows for 2 days before and during 6 days' starvation. For each cow, sample from 2-day periods were combined in proportion to milk yield and analysed for lipid percentage (g/lOOg of milk) and fatty acid percentage (g/lO0g of fatty acid) as described under 'Methods of analysis'. Fatty acid percentages for acids with 4-14,16 and 18 carbon atoms which changed similarly during starvation were combined. For total lipid and each group of fatty acids the s.E. of a 'day' mean and the S.E. of the linear effect of time (regression coefficient) was derived from the error variance 'days x cows'. The significance of the regression coefficient from zero was determined by the t test (t = regression coefficient-s.E. of regression coefficient). Significance of regression coefficients from zero: * P
