Plasma lipoproteins and fatty liver in dairy cows

Research in Veterinary Science 1988, 45, 389-393

Plasma lipoproteins and fatty liver in dairy cows Y. RAYSSIGUIER, A. MAZUR, E. GUEUX, Laboratoire des Maladies Metaboliques, INRA, Theix, 63122 Ceyrat, France, I. M. REID, C. J. ROBERTS, AFRC Institute/or Animal Health, Compton, Newbury, Berkshire RG16 ONN . .

Dairy cows were classified on the basis of a histological study after a hepatic biopsy conducted in the second week post partum (mild fatty liver, moderate fatty liver, severe fatty liver). Plasma lipoproteins were separated into various density classes by repeated ultracentrifugation. The results indicate that the beginning of lactation is associated with a low concentration of the t- 006 to I- 063 g ml- I lipoprotein fraction. The lowest concentrations occurred in cows with severe steatosis or during the evolution of moderate steatosis. SOME degree of hepatic lipid infiltration is frequently observed in high producing dairy cows which are unable to make the necessary metabolic adjustments at the time of parturition (Reid and Roberts 1983). The increased fat mobilisation of early lactation results in increased free fatty acid uptake by the liver. Free fatty acids can form ketone bodies or can be completely oxidised in the Krebs' cycle. Alternatively free fatty acids can be re-esterified ~nd the resulting triglycerides can be secreted as lipoproteins. When the uptake of fatty acids exceeds that necessary to sustain a maximal rate of secretion of lipoproteins, triglyceridesaccumulate in the liver (Bell 1981). Liver biopsy samples taken from a large number of dairy cows at various times after calving have indicated the pattern of development of fatty infiltration of the liver. Normally, the maximal lipid infiltration occurs one to two weeks after calving and then disappears progressively. This liver fat accumulation has been associated with an increased incidence of metabolic, infectious and reproductive disorders (Reid and Roberts 1983). Cows with less than 20 per cent fat in the liver are classified as normal on the basis of histological analysis of liver biopsy samples taken one to two weeks after calving. Those with 20 to 40 per cent fat were considered moderately fatty, while those with over 40 per cent fat were considered severely fatty (Gall et al 1983). In the absence of hepatic biopsy, the diagnosis of fatty liver is difficult even though different authors have proposed various combinations of blood parameters to assess the risk of steatosis (Roberts et al 1981, Grohn et al1983, Reid et alI983). Abnormal

lipid and lipoprotein concentrations are often associated with liver disorders (Novikoff 1982) and the determination of plasma lipoprotein in dairy cows in early lactation seemsof interest both in understanding the pathogenesis of fatty liver syndrome and for diagnostic purposes. In the present study, blood samples were taken from dairy cows showing different degrees of lipid infiltration in the liver in order to determine the relationship between lipoproteins and the degree of steatosis. Materials and methods Animals

British Friesian cows from the AFRC Compton Laboratory's dairy herd were housed in yards and fed ad libitum a mixture of lucerne and maize silage supplemented on occasion with alkali treated straw, molasses, brewers' grains and grass silage. A concentrate containing 18per cent crude protein was individually fed according to appetite. The average milk yield of the cows" in the previous lactation was 6480 kg. Animals were classified on the basis of a histological study, after a hepatic biopsy conducted in the second week post partum (mild fatty liver six animals, moderate fatty liver eight, severe fatty liver six.) Blood sampling

Blood samples were taken from the jugular vein immediately before the liver biopsy, seven to 13 days following calving (two weekssample), and again 28 to . 34 days after calving (four weeks sample). Two animals with moderate fatty liver and animals with severe fatty liver were not sampled during the fourth week because different treatments given would have interfered with the parameters studied. Collected plasma was stored at - 20°C before analysis. Liver biopsy and histological analysis

Liver samples were taken from each cow by percutaneous needle biopsy (Davies and Jebbett 1981)

389

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Y. Rayssiguier, A. Mazur, E. Gueux, I. M. Reid, C. J. Roberts 200

-.

stained with oil red 0 (ORO). The percentage of visible fat in parenchymal liver cellswas estimated by stereological point-counting methods (Reid I980a). Ten fields from each ORO section of the biopsy sample were examined at a final magnification of 110x with a light microscope that had a 100 point eyepiece graticule. The points overlyingextracellular space and hepatocytic fat were counted. From these figures, the fractional volume (v/v) of the liver cell occupied by fat droplets was calculated. Cows with 20 per cent fat in the liver sample taken one week after calving were considered normal (mild fatty liver, control animals). Those with 20 to 40 per cent fat were considered moderate, and cows with 40 per cent fat were considered severely fatty (Gall et al1983).

2nd week

E

~ '" '.5 150

50

Plasma analysis

4th week

150

100

50

o

im"IIII!!IIl!=:I..--_ VlDl

I

FIG 1: Lipid content of various plasma lipoprotein fractions in cows. Cows with less than 20 per cent fat in the liver are classified as normal (II!I mild fatty Iiverl. Those with 20 to 40 per cent fat were considered moderately fatty (~) while those with over 40 per cent fat were considered severely fatty (1ZIl. Lipoproteins tvun, lDl, HDl) were isolated by ultracentrifugation. Values are means ± SEM (six to eight animals per group). Significant difference from cows with mild fatty liver. *P
between seven to 13 days after calving. A portion of the liver sample was put in 12 per cent (v/v) neutralbuffered formalin and fixed for at least 24 hours. A suitable length of this sample (5 mm) was sectioned on a Leitz Kryomat at 15 um thickness, mounted on a clean glass slide, air dried for several hours, and

Plasma lipoproteins were separated into various density classes by repeated ultracentrifugation as previously described (Havel et al 1955). Ultracentrifugation was performed at 15°C in a Beckman L-5 model ultracentrifuge (Beckman Instruments) with a type 50 titanium rotor. Before lipoprotein separation, sodium azide (0,02 per cent), merthiolate (0,005 per cent) and EDTA (0,04 per cent) were added to prevent lipid oxidation and microbial contamination. Very low density lipoproteins (VLDL) were isolated at plasma density d<1·oo6 g ml- 1 by centrifugation at 100,000 g for 18 hours. Other lipoprotein fractions were isolated by sequentially raising the plasma density to d = 1.063 g ml ' ! low density lipoproteins (LDL), and l' 21 g ml- I high density lipoproteins (HDL) by adding crystalline potassium bromide and centrifugation at 100,000 g for 20 and 24 hours, respectively. Free fatty acids (Nefa C; Wako Chemicals, Biolyon, France), triglycerides (Triglenzyme Color; Biotrol, France), cholesterol (Cholesterol enzymatique PAP; BiOMl:rieux, France) and phospholipids (Phospholipides enzymatiques PAP; BioMl:rieux, France) in plasma and lipoprotein fractions were determined by sensitiveenzymatic procedures. Statistics

Results are given as mean ± test was used to analyse data.

SEM.

The Student's t

Results The degrees of lipid infiltration were, respectively, 6·8 ± 2'0; 32'5 ± 0'5 and 54 ± 3·0 per cent for the control group and animals with moderate and severe fatty liver. Fig 1 and Table 1show the value of plasma lipoproteins and the distribution of triglycerides, cholesterol and phospholipids, two weeks after calv-

Lipoproteins in bovine fatty livers TABLE 1: Distribution of triglycerldes. cholesterol and phospholipids among the various plasma lipoprotein fractions of cows with mild. moderate or severe fatty liver two weeks after calving Lipoprotein fractions

VLDL

(d< Hl06)

LDL

(d=HX16 to 1·063)

HDL

(d=H163 to 1·211

Triglycerides (mg 100ml- 1) Mild fatty liver Moderate fatty liver Severefatty liver

2·1 ±0'1 2·0±0·1 1·9±0·2

6·1 ±0·1 5·5± 0·1 5·4±0·2

3·6±0·1 3·3±0·1 3·4± 0·1

Cholesterol (mg 100ml- 1) Mild fatty liver Moderate fatty liver Severefatty liver

0·4±0·1 0·5±0·1 0·4± 0·1

12·9± 1·2 11·7± 1·3 7·9 ± 0'8**

86·9 ± 1-6 76·5 ± 3·7 86·7 ± 5·7

Phospholipids (mg l00ml- 1) Mild fatty liver Moderate fatty liver Severefatty liver

0·9±0·1 0·8±0·2 0·8±0·1

10·7± 1·3 11-1 ± 1·6 5·4 ± 0'7'*

93·9± 3·1 66·4±8·1* 00·1 ± 5,0*

Valuesare means± SEM (six to eight animalsper group). Significant difference from cows with mild fatty liver *P<0·05. **P<0'01

ing. The concentrations of VLDL and LDL were extremely low, particularly VLDL. A high proportion of the plasma lipids is transported in HDL. Cholesterol and phospholipids are the principal components of lipoproteins and are accompanied by much smaller quantities of triglycerides. Triglyceride distribution into the different lipoprotein fractions was not modified by the levelof lipid infiltration of the liver whereas significant chllrnges occurred in the cholesterol and phospholipidsvdistribution. The animals with moderate steatosis had significantly lower values of HDL than controls as shown by the decrease in HDL cholesterol and HDL phospholipid concentrations, whereas the LDL fraction was not modified. Severesteatosis was associated with a large decrease in LDL concentration and a smaller change in HDL concentration. HDL phospholipid concentrations slightly decreased in cows with severe steatosis when compared to control animals, whereas HDL cholesterol concentrations were not modified. The LDL cholesterol and phospholipid concentrations for the animals with severe fatty liver were significantly lower than those of control animals, LDL cholesterol and . phospholipids concentrations for these animals were 61 and 51 per cent of the control values, respectively. Fig 1 and Table 2 indicate the values of different lipoprotein fractions and the distribution of triglycerides, cholesterol and phospholipids four weeks after calving. LDL and HDL levels were significantly higher than one week after calving, the major difference being in the LDL levels. The animals with moderate steatosis at the beginning of lactation had

391

TABLE2: Distribution of triglycerldes. cholesterol and phospholipids among the various plasma lipoprotein fractions four weeks post partum in cows which were classified In mild or moderate fatty liver group two weeks after calving Lipoprotein fractions

VLDL

LDL

HDL

(d
(d=HXl6 to 1·063)

(d=H163 to 1·21)

Triglycerides (mg 100ml- 1) Mild fatty liver Moderate fatty liver

2·6± 0·2 1'9±0'1**

8·5±0·5 4·1 ± 0,1***

3·3 ± 0·2 2·8±0·1

Cholesterol (mg 100ml- 1) Mild fatty liver Moderate fatty liver

0·6±0·1 0·4±0·1

50·9 ± 8·9 25·4± 4,6*

132·7±8·4 124·1 ± 7·1

Phospholipids (mg 100ml- 1) Mild fatty liver Moderate fatty liver

0·9±0·1 0·8±0·1

73·8 ± 16·1 35·8±3·1*

144·0± 8·2 107·3± 5,7*

Values are means ± SEM (six animals per group). Significant difference from cows with mild fatty liver *P<0·05. **P<0·01. ***P<0'001

significantly lower values of LDL and HDL fraction than control animals with mild fatty liver four weeks post calving. HDL phospholipid concentrations were significantly decreased when compared to control animals. The differences were more pronounced in the LDL fractions. The triglyceride, cholesterol and phospholipid concentrations of this fraction were around 50 per cent of the values of control animals. Finally, the results indicate that the beginning of lactation is associated with a low concentration of the 1'006 to 1'063 g ml- I lipoprotein fraction. The lowest concentrations occurred in cows with severe steatosis or during the evolution of moderate steatosiS. Discussion Several studies indicate that most of the lipid infiltration of the liver in dairy cows after calving is in the form of triglycerides. Estimates of the amount of fat in the liver by stereological point counting methods provide an acceptable alternative to biochemical estimates of the triglyceride content (Gall et aI1983). Cows were divided into experimental groups according to the extent that the liver was fatty as determined stereologically. In the present study, the fat concentration of the liver of cows with moderate or severe fatty liver was five and eight times greater, respectively, than in controls. This classification was rather arbitrary but the rationale was that the fat cow syndrome occurs with. moderate or severe fatty infiltration of the liver along with an increased incidence of metabolic reproductive and infectious disorders (Reid and Roberts 1983). The role of lipoprotein formation and release in

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Y. Rayssiguier, A. Mazur, E. Gueux, I. M. Reid, C. J. Roberts

this syndrome is not well known (Bell 1981). Plasma lipoproteins are complex molecules that are heterogeneous in composition, size and biological activity. This heterogeneity is further complicated by movements of both the lipids and the apoproteins between the different particles (Eisenberg 1980). In the present experiments, the authors separated lipoproteins under the same conditions used to separate the lipoproteins of human serum (Havel et al 1955), since there are no standard conditions for isolating ruminant lipoproteins from plasma (Mazur and Rayssiguier 1988). The most significant result obtained two weeks post partum in cows with severe fatty liver was the dramatic decrease in plasma LDL. The fact that LDL originate from VLDL is well established (Eisenberg 1980). The rate of turnover of plasma VLDL in the lactating cow is rapid relative to that of other lipoproteins (Palmquist and Mattos 1978) and LDL determination provides information on VLDL metabolism. Decreased LDL concentration in the plasma of cows with severe fatty liver could result from several mechanisms including decreased VLDL secretion, decreased conversion of VLDL to LDL or increased LDL uptake. Factors involved in the VLDL formation and secretion in early lactation are not well known. To be secreted by the liver as VLDL, triglycerides must be associated with other lipoprotein constituents: cholesterol, phospholipids and apoproteins. Availability of one or more of these components may have limited lipoprotein synthesis. In the present experiments, triglyceridecontent of LDL in cows with severe fatty liver was not reduced, whereas cholesterol and phospholipid concentration decreased. Another hypothesis is that lipoprotein synthesis may have been limited by the amount of available apoproteins. In fatty liver, a marked decrease in the proportion of hepatic endoplasmic reticulum has been reported (Reid and Collins 1980). A decreased hepatic protein synthesis could have important implications for the ability of the liver to secrete lipoproteins. Another explanation for the LDL decrease would be that VLDL transformation into LDL was reduced or that LDL catabolism was increased. Decreased plasma insulin levelshave been reported in high yielding cows (Reid et al 1983) but the hypothesis of a decreased lipoprotein lipase activity as a primary mechanism in reduced LDL concentration seems unlikely in the absence of VLDL elevation. Alternatively, no evidence that LDL catabolism increase would cause plasma LDL decrease has been reported in cows with fatty liver. In the case of bovine lipoproteins, no entirely satisfactory density for isolation of LDL has been identified and the fraction qualified as LDL is heterogeneous and contains some particles with HDL-like properties (Ferreri and Gleockler 1979,Grummer et al

1983, Mazur and Rayssiguier 1988). A reduced concentration of this fraction could result from a decrease of light HDL. A reduction in the content of light HDL could either result from a decreased secretion of HDL by the liver or from a lowered conversion-of HDL into light HDL. Since this process involves an enrichment of HDL with constituents of VLDL (Eisenberg' 1980), a lower synthesis and secretion of the VLDL by the liver would be also implied. Finally, the decrease in the 1,006 to 1'063 g ml- I fraction in cows with severe steatosis appears to be the consequence of the reduced synthesis or secretion of VLDL by the liver. This agrees with previous observations. A marked reduction in hepatic triglyceride output occurs in response to fasting in dairy cows (Reid et aI1979). However, differences in serum and liver lipids exist between naturally occurring and fast induced bovine steatosis and fasted cows are not suitable models of severe bovine steatosis (Herdt et al 1983). Hepatic steatosis which occurs in association with abomasal displacement is also accompanied by a reduction of the dextran sulphate precipitable serum lipid fraction (Herdt et al 1983). However, the use of cows with displaced abomasum also seems questionable for studying naturally occurring steatosis. Thus the present investigation provides what is believed to be the first direct evidence of modification in the profile of lipoproteins isolated by ultracentrifugation in cows with spontaneous lipid infiltration of the liver at the beginning of lactation. All the cows, even those in the control group, showed a reduction of the I, 006 to I· 063 g ml- I fraction following calving compared to a later stage of lactation. This modification indicates a defect in the hepatic triglyceride-rich lipoprotein release and may explain the increase in hepatic lipids in all dairy cows following calving, even in healthy animals (Reid 1980b). The decrease of this fractltm was especially pronounced in the cows with severe steatosis. During this same period, the animals in the moderately steatosic group were no different from the controls as regards the LDL levels. In contrast, by the fourth week, the moderately steatosic animals had an LDL fraction that was significantly lower than control animals. In normal dairy cows, with low lipid infiltration of the liver post partum, different workers have shown that lipid infiltration reached a maximum one to two weeks post partum, and regressed rapidly. Thus, lipid infiltration practically disappeared during the fourth week post partum (Reid and Roberts 1983). Whether the decrease in the LDL fraction in the group of cows that had moderate steatosis in the second week was related to the persistence of fatty liver in this group deserves investigation. In conclusion, the origin of the decrease of the lipoprotein fraction isolated at I' 006 to I, 063 g ml- I in

Lipoproteins in bovine fatty livers cows with severe steatosis or during the evolution of moderate steatosis should be clarified; and the determination of LDL cholesterol, LDL phospholipids might be useful for diagnostic purposes.

References BELL, A. W. (1981) Lipid Metabolism in Ruminant Animals. Ed W. W. Christie. Oxford, Pergamon Press. pp 363-410 DAVIES, D. C. & JEBBETT, I. H. (1981) Veterinary Record Supplement In Practice 3, 14-16 EISENBERG, S. (1980) Annals of the New York Academy of Sciences 348, 30-47 FERRERI, L. F. & GLEOCKLER, D. H. (1979) Journal of Dairy Science 62,1577-1582 GALL, T., REID, I. M., COLLINS, R. A., ROBERTS, C. J. & PIKE, B. v. (1983) Research in Veterinary Science 34, 245-249 GROHN, Y., LINDBERG, L. A., BRUSS, M. L. & FARVER, T. B. (1983) Journal of Dairy Science 66, 2320-2328 GRUMMER, R. R., DAVIS, C. L. & HEGARTY, H. M. (1983) Lipids 18, 795-802 HAVEL, R. J., EDER, A. H. & BRAGDON, J. M. (1955) Journal Of Clinical Investigation 34, 1345-1363 HERDT, T. D., LIESMAN, J. S., GERLOFF, B. J. & EMERY, R. S. (1983) American Journal of Veterinary Research 44, 293-296

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MAZUR, A. & RAYSSIGUlER, Y. (1988) Annales de Recherches vetennatres 19, 53-58 NOVIKOFF, A. B. (1982) The Liver Biology and Pathology. Eds J. Arias, H. Popper, D. Schachter and D. A. Schafritz. New York, Raven Press. pp 9-25 PALMQUIST, D. L. & MATTOS, W. (1978) Journal of Dairy Science 61, 561-565 REID, I. M. (1980a) Veterinary Pathology 17, 522-543 REID, I. M. (1980b) Veterinary Record 107, 281-284 REID, I. M., COLLINS, R. A., BAIRD, G. D., ROBERTS, C. J. & SYMONDS, H. W. (1979) Journal of Agricultural Science, Cambridge 93, 253-256 REID, I. M. & COLLINS, R. A. (1980) Investigative and Cellular Pathology 3, 237-249 REID, I. M. & ROBERTS, C. J. (1983) Irish Veterinary Journal 37, 104-110 REID, I. M., ROWLANDS, G. J., DEW, A. M., COLLINS, R. A., ROBERTS, C. J. &MANSTON, R. (1983)Journal ofAgricultural Science, Cambridge 101, 473-480 ROBERTS, C. J., REID, I. M., ROWLANDS, G. J. & PATTERSON, A. (1981) Veterinary Record 108, 7-9

Received September 15, 1987 Accepted January 27, 1988

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