Materials and Methods

Animals and serum specimens—Holstein cows with suspected ketosis that were brought to the Meh-met Akif Ersoy University Veterinary Clinical Center by their owners were used as principals in this experiment. Cows (n = 21) were of different ages and were in early lactation (2 to 37 days after parturition). Milk yield was approximately 8,000 kg/y/cow and was similar in healthy and diseased cows. Urine and milk samples were collected from each cow and the Rothera test³⁰ and test strips^a were applied to diagnose ketosis. Routine clinical examination was carried out, and rectal temperature and heart and respiratory rates were recorded for each animal before and after treatment. Clinical ketosis (diagnosed via positive results of the Rothera test in the milk and urine, high concentration of β-hydroxybutyrate in the serum, and clinical signs of disease, such as decrease of milk yield or loss of appetite) was diagnosed in 21 dairy cows that were allocated into 3 groups equally for treatment by use of 3 procedures.

Each cow in the first group received 750 mL of 30% serum dextrose IV and a dose of 4 mg of dexamethasone/100 kg of body weight IM for 2 consecutive days. Each cow in the second group received 750 mL of 30% serum dextrose IV plus 0.2 mg of prednisolone/kg IM daily for 2 days. Furthermore, 750 mL of 30% dextrose (IV, for 2 days) and 200 units of insulin (IV, twice, with 48 hours between treatments) were administered to each cow in the third group. Two control groups (7 healthy cows in the early lactation period and 7 healthy cows in the nonlactation period [> 300 days after parturition]) were used for comparison with cows with ketosis and between control groups. These healthy cows were obtained from a dairy farm in Burdur Province. Animals in control groups did not receive any treatment during the study.

Blood samples from each cow were collected in the morning (before feeding) and were allowed to clot at 15° to 25°C for 1 hour, and subsequently, the serum was separated by centrifugation at 500 X g at 20°C for 15 minutes. Lipoprotein precipitation procedures were carried out on the same day. The serum samples were stored at −20°C until total cholesterol and β-hydroxybutyrate analyses were performed.

Precipitation of serum β lipoproteins with DS-MgCl₂—A 20 g/L solution of dextran sulfate^b and a 1.0 mol/L solution of MgCl₂-6H₂O, adjusted to a pH of 7.0, were prepared. Both were stored at 4°C. The working reagent was prepared freshly each week by mixing equal volumes of the 2 solutions. Then, 75 μL of the working reagent was added to 1.0 mL of serum, and the solution was mixed thoroughly and incubated at 15° to 25°C for 15 minutes and subsequently at 4°C for 1 hour before centrifugation at 2,500 X g for 15 minutes.^31,32 The supernatant was separated, the precipitate was dissolved in 200 μL of saline (0.9% NaCl) solution, and magnesium ions were removed by the addition of 50 μL of a solution of 12.7% potassium oxalate and centrifugation at 500 X g for 5 minutes.³³ One part each of precipitate and supernatant was separated, and the other part was stored at −20°C for SDS-PAGE analysis.

Precipitation of serum α lipoproteins with DS-MgCl₂—Precipitation was made with modifications of methods developed by Burstein et al³⁴ and Bachorik and Albers.²¹ A 50 g/L solution of dextran sulfate and a 2.0 mol/L solution of MgCl₂-6H₂O were prepared and adjusted to a pH of 7.0. Both were stored at 4°C. The working reagent was prepared freshly each week by mixing equal volumes of the 2 solutions. Then, 200 μL of the working reagent was added to 1.0 mL of the supernatant, and the solution was mixed thoroughly and incubated at 15° to 25°C for 15 minutes and subsequently at 4°C for 1 hour before centrifugation at 2,500 X g for 15 minutes. The supernatant was removed by decantation, and the precipitate was dissolved in 400 μL of 0.5M potassium oxalate³⁵ and centrifuged at 500 X g for 5 minutes. One part each of precipitate and supernatant was separated, and the other part was stored at −20°C for SDS-PAGE analysis.

Separation of serum lipoproteins via agarose gel electrophoresis—Serum and the lipoproteins precipitated were separated and identified by use of an agarose gel electrophoretic system.^c Agarose was used at a concentration of 0.8%. Fifteen microliters of serum was applied directly to agarose gel. After mixing 200 μL of precipitate and supernatant with 50 μL of sample buffer (16 mL of barbital buffer at a pH of 8.6, 5 mL of glycerol, and 100 μL of 1.0% bromophenol blue were mixed and made up to 100 mL with distilled water), 18 μL of precipitate and supernatant was placed in the slot. Electrophoresis was performed in barbital buffer at a pH of 8.6 for 45 minutes at 83 V. After electrophoresis, the gels were fixed in a solution of ethanol-acetic acid-water (ratio of 60:10:30 vol/vol/vol) for 10 minutes, oven dried (60°C for 30 minutes), and stained overnight with a Fat Red 7B solution. Gels were destained in a solution of methanol-water (ratio of 1:4 vol/vol) until the lipoprotein bands were clear.^36,37 An image processing program^d was used for densitometric scanning of stained electrophoretograms with automatic integration of the peaks of optical density.

SDS-PAGE—Apolipoprotein composition was determined via discontinuous SDS-PAGE,^e as described by Laemmli.³⁸ Four percent stacking and 7.5% and 12% resolving gels (1.0 mm thick) were prepared. Apolipoproteins at high molecular weights were evaluated in the 7.5% gel, and apolipoproteins at low molecular weights were evaluated in the 12% gel. The precipitates were diluted 1:1 (vol/vol) with the sample buffer containing 5.0 mL of Tris-HCl buffer (pH, 6.8), 4.0 mL of glycerol, 8.0 mL of 10% SDS, 2.0 mL of 2-mercaptoethanol, 1.0 mL of 0.05% bromophenol blue, and 20 mL of distilled water. After adding the sample buffer, the precipitates were incubated at 100°C in a boiling water bath for 5 minutes. A 15-μL aliquot of precipitates (indicated as P₁ and P₂) and a 5- to 10-μL aliquot of standard protein markers^f,g were applied on gels. Electrophoresis was performed at a constant current of 20 mA/gel for 90 minutes. After electrophoresis, the gels were stained overnight with 0.025% Coomassie brilliant blue G-250 in methanol-acetic acid-water (ratio of 40:7:53 [vol/vol/vol]), destained with methanol-acetic acid-water (ratio of 40:7:53 [vol/vol/vol]), and stored in 5% acetic acid. An image processing program^d was used for estimation of molecular weight in gels.

Other methods—Serum concentration of β-hydroxybutyrate was determined by use of a commercially available test kit.^h Serum concentration of cholesterol was measured by use of an automatic analyzer.ⁱ

Statistical analysis—Data for cholesterol and β-hydroxybutyrate concentrations were analyzed by use of the Kruskal-Wallis and Mann-Whitney U tests. Values are expressed as mean ± SD.^j For all comparisons, values of P < 0.05 were considered significant.

Results

Agarose gel electrophoresis of serum β and α lipoproteins isolated via DS-MgCl₂ precipitation—Serum lipoprotein patterns in healthy cows during the lactation and nonlactation periods were compared with those in ketotic cows. In general, lipoproteins migrated into β (ie, LDL) and α (ie, HDL) regions. Pre-β lipoproteins (ie, VLDL) were not detectable in serum samples because of their small amounts. In agarose gel electrophoresis, lipoproteins with α₁ mobility were observed in some β regions during lactation but were absent during nonlactation. Compared with mean percentage areas for serum β and α lipoproteins in healthy cows during nonlactation, those in cows during lactation were higher, whereas those in cows with ketosis were lower (Table 1). Decreases were clearer for serum β lipoproteins than for α lipoproteins in cows with ketosis. In terms of ketotic cows, the serum lipoproteins migrated farther and in a much more widespread manner than those of nonketotic cows. In response to treatment, dextrose and insulin were more effective, regarding the recovery of serum β lipoproteins of ketotic cows, than were other treatment methods.

SDS-PAGE—Two protein bands, whose molecular weights were estimated to be 36 and 40 kDa, were found via analysis of bovine serum β lipoproteins. These proteins, identified as bovine apolipoprotein E, significantly increased during the lactation period, compared with the nonlactation period, whereas in the ketotic cows, the proteins decreased and even disappeared. After 3 days of treatment with prednisolone and dexamethasone, apolipoprotein E was increased in ketotic cows. Small amounts of the 19- and 24-kDa proteins, presumably apolipoprotein A-I, a major apolipoprotein of α lipoproteins, were detected in bovine serum β lipoproteins. Furthermore, in healthy cows during lactation, these dimeric proteins were in higher concentration than in cows during the nonlactation period and were in lowest concentration in ketotic cows. The same protein bands were also detected in bovine α lipoprotein in high concentration, and they were reduced in the ketotic cows (Table 2). However, decreases were not as pronounced for α lipoproteins as for β lipoproteins. The β lipoprotein fraction contained a decreased albumin band (66 kDa) in the ketotic cows and returned to basal concentration after treatment for 3 days. In contrast, in the α lipoprotein fraction of ketotic cows, increased concentrations of albumin were detected, compared with the lactation period, and the albumin band was slightly decreased after treatment with insulin for 3 days. A 10.2-kDa protein and a 8.8-kDa protein (near the 14.2-kDa molecular weight marker) were detected in β lipoproteins. These protein bands, which were considered to be similar to bovine apolipoprotein C-III, increased in cows during lactation, compared with the magnitude in cows during nonlactation. These protein bands decreased in ketotic cows and also after treatment. A 55-kDa protein band, probably apolipoprotein A-IV, was found in both β lipoprotein and α lipoprotein fractions. The band was decreased in the β lipoprotein fraction of ketotic cows but increased in the α lipoprotein fraction, compared with protein bands in cows during the lactation and nonlactation periods. After treatment, the protein band was restored. In the β lipoprotein fraction, a 222-kDa protein band was detected, which was absent in the α lipoprotein fraction. The band corresponding to apolipoprotein B was not detected in ketotic cows.

The cholesterol concentration was lower in the ketotic cows than in healthy cows during lactation and increased after treatment (Table 3). Concentrations of β-hydroxybutyrate in the ketotic cows were higher than those in healthy cows and returned to the values of healthy cows after treatment with insulin and dexamethasone.

Discussion

The electrophoretic distribution of bovine serum indicated that α lipoproteins were the major lipoprotein and that β lipoproteins were also present in lower amounts. Pre-β lipoproteins are associated with VLDL but were not detectable in serum samples because of the small amount.²² In cows, ultracentrifugally isolated LDL overlapped with a light form of HDL. These large α lipoproteins associated with hyperalphalipoproteinemia were detected in some LDL fractions during lactation and lactation-pregnancy but were absent during nonlactation.^3,22,25,39 As detected by use of agarose gel electrophoresis, α₁ (HDL₁) was associated with β mobility during nonlactation and with α and β mobility throughout lactation. The variety in HDL₁ mobility is related to lipoprotein lipase activity as blood triglycerides are hydrolyzed for milk fat synthesis.²² In bovine studies, dextran sulfate has been used to precipitate lipoproteins, but as Burstein et al³⁴ noted, this polysaccharide also can be used to precipitate human LDL and HDL. These authors note that the lipid-to-protein ratio is a determinant for the relative ease of formation of an insoluble lipoprotein-polyanion complex. As a result, α-LDL and HDL₁ in high concentrations in lactating cows, after interacting with dextran, coprecipitate with β lipoproteins. The increased concentration of α-LDL and HDL₁ in lactating cows then would be products of a primary particle secreted by the liver, rather than a remnant of VLDL catabolism.⁴⁰ In the present study, bovine lipoproteins migrated into the β and α region and pre-β lipoproteins were not detectable via agarose gel electrophoresis of precipitates with dextran sulfate. During the lactation period, β and α bands were stronger than those in cows during the nonlactation period. Lipoproteins with α₁ mobility were evident in the β region during lactation but were absent during nonlactation.

In cows with ketosis, LDL (β lipoproteins) and HDL (α lipoproteins) concentrations are reported to be lower.^16,18 Severe hepatic lipidosis has been associated with a large decrease in LDL concentration and a smaller change in HDL concentration.^6,8,16 Results of our study indicated that bovine serum β and α lipoproteins isolated via DS-MgCl₂ precipitation were decreased in ketotic cows. Via agarose gel electrophoresis, decreases in serum β lipoproteins of cows with ketosis were more distinct than in serum α lipoproteins. The dramatic decrease in serum β lipoproteins could result from several mechanisms, including decreased VLDL secretion, decreased conversion of VLDL to LDL, or increased LDL uptake.⁶ In the ketotic cows, the serum lipoproteins migrated farther than those in nonketotic cows. This suggested that NEFAs associated with serum albumin in α and β lipoprotein fractions of the ketotic cows increased as the severity of the disease increased. In humans, apolipoproteins have been recognized as obvious markers for identifying discrete lipoprotein particles. However, limited research has been conducted on ruminant apolipoproteins.⁴¹ The analysis of lipoproteins in ruminant animals is complicated because of an overlap of α lipoproteins (containing apolipoprotein A-I as the major apolipoprotein) and β lipoproteins (containing apolipoprotein B as the major apolipoprotein). Contrary to humans, in bovine serum, most of the circulating lipids are associated with apolipoprotein A-I–containing lipoproteins. In cattle, concentrations of β lipoproteins are about one-fifth to one-fourth those in healthy humans. This agrees with the low concentrations of plasma triglycerides in ruminants resulting from their nutritional and metabolic particularities, but the importance of these lipoproteins for milk fat formation has been recognized in dairy cows.⁴¹

In cows with hepatic lipidosis, notable decreases were determined in lipid and lipoprotein consentrations.⁸ Furthermore, in cows with fatty liver and ketosis, decreases of serum apolipoprotein B-100,¹² apolipoprotein A-I, apolipoprotein C-III, cholesterol esters, albumin concentrations, and LCAT activity are pronounced.^{3,4,7,10,11,13,15,36} It is possible to assume that β-hydroxybutyrate, hepatic triglycerides, and NEFAs reduce apolipoprotein synthesis or secretion.^10,15 Moreover, in a recent study,⁴² liver from ketotic cows was characterized by substantial downregulation of genes involved in protein synthesis, protein trafficking, protein ubiquitination, and molecular transport. The fact that the apolipoprotein B gene (APOB) was upregulated along with downregulation of genes associated with protein ubiquitination-degradation and triglyceride accumulation points at other mechanisms limiting VLDL synthesis and export (eg, lower de novo synthesis of cholesterol and oleic acid, rather than deficient apolipoprotein B synthesis). The functional consequences of impaired intracellular protein degradation in liver might include alterations in cellular turnover, effects on gene regulation, modulation of cell signaling, induction of apoptosis and necrosis, release of reactive oxygen species, and loss of gene-protein function. Such changes could be a primary factor involved in the impairment of hepatic function that contributes to the onset of clinical ketosis in dairy cows. Also, in the present study, the cholesterol concentration was significantly lower in the ketotic cows than in the lactating cows. The increased cholesterol in the lactating cows appeared to be associated with dynamic changes in and among the various lipoprotein classes, with the LCAT enzyme playing a pivotal role.⁴⁰

Marcos et al⁴ and Grummer¹³ reported that serum concentrations of albumin are decreased in cows with hepatic lipidosis, which may indicate a reduction in the ability of the liver to synthesize protein when triglyceride content is high. However, in other studies,^2,16 the albumin concentrations of ketotic cows were significantly higher than in the early lactation cows. In contrast, protein concentrations of ketotic cows were significantly lower, compared with the protein concentrations of the control cows.

The α lipoprotein fraction contains an increased concentration of NEFAs correlated with the severity of the ketosis. Free fatty acids associated with serum albumin are included in the extracts of the α lipoproteins. Increased concentrations of NEFAs in the serum of ketotic cows have been reported.² In severe cases of ketosis, the β lipoproteins also contained increased concentrations of NEFAs.

In our view, the β lipoprotein fraction contained a decreased concentration albumin band (66 kDa) in ketotic cows and returned to basal value after treatment. However, in α lipoproteins of ketotic cows, increased concentrations of albumin were detected, in comparison with lactating cows, and the albumin band was slightly decreased after treatment with insulin for 3 days.

Apolipoprotein B-100 and apolipoprotein A-I are essential for transportation of triglycerides from the liver to the mammary gland and of cholesterol to steroidogenic tissues (ovary and adrenal cortex).^10,12 Compared with values in cows during early lactation, apolipoprotein B-100 and apolipoprotein A-I concentration further decreased in cows with ketosis. However, the decreases in apolipoprotein B-100 concentration were more distinct than those of apolipoprotein A-I. The preferential decrease in apolipoprotein B-100 concentration indicated that apolipoprotein B-100 is synthesized by the liver, whereas apolipoprotein A-I is synthesized by the liver and the intestine. The intestinal synthesis appears not to be considerably affected in diseased cows.^5,10,11 In a previous study⁴³ in which bovine lipoproteins were isolated in plasma via gel filtration and apolipoprotein composition was determined via SDS-PAGE, the 22-kDa protein band was detected in bovine triglyceride-rich lipoproteins. This low–molecular-weight protein may be analogous to nonruminant apolipoprotein A-I. In the same study, the 220- and 290-kDa proteins were predominantly detected; however, > 25 proteins were detected between 110 and 370 kDa in triglyceride-rich lipoproteins. Also, bovine HDL contained proteins analogous to apolipoprotein A-I and apolipoprotein C. Differences in apolipoprotein profiles between nonlactating and lactating cows were not apparent. In another study, the apolipoprotein A-I band mainly distributed in HDL was detected in LDL and VLDL. The band appeared to be a mixture of apolipoprotein A-I (24 kDa) and γ-globulin light chain (23 kDa).⁴⁴ Because bovine HDL₁ (α₁) particles were detected in the LDL range, HDL₁ contained the major protein component of HDL (ie, apolipoprotein A-I).⁴⁵ However, the major apolipoprotein of bovine α-LDL and HDL₁ is apolipoprotein A-I.⁴⁰

In the β lipoprotein fraction, a 222-kDa protein band was detected, which was similar to apolipoprotein B, in cows in lactation and nonlactation; however, this was not detected in ketotic cows. The 19- and 24-kDa proteins (presumably apolipoprotein A-I) were reduced in β and α lipoprotein fractions of cows with ketosis. Moreover, in the α lipoprotein fraction of ketotic cows, apolipoprotein A-I had indistinct decreases.

One study⁴⁶ found that bovine apolipoprotein A-IV concentrations determined by use of ELISA during the lactation period in cows are significantly higher than during the nonlactation period. After 4 days of food withholding, the concentration of plasma apolipoprotein A-IV had decreased significantly at days 3 and 4 and was returned to the basal value by 3 days of feeding. In the same study, a minor band (42 kDa) was found in VLDL, LDL, and HDL fractions via SDS-PAGE. Another band (43 kDa) was found in the HDL fraction, and a 42-kDa protein band was identified as bovine apolipoprotein A-IV.⁴⁶ Puppione et al²⁵ suggested that the 46-kDa apolipoprotein in the HDL fraction might also reflect the conversion of triglyceride-rich lipoproteins into α lipoproteins if it corresponds to a bovine form of apolipoprotein A-IV. In the present study, a protein of approximately 55 kDa (probably apolipoprotein A-IV) in the α lipoprotein fraction was higher in concentration in cows during lactation than during nonlactation and was increased in ketotic cows. However, the same protein band was decreased in the β lipoprotein fraction during the lactation period, compared with the nonlactation period, and also was decreased in cows with ketosis. After treatment, the protein band was restored.

Apolipoprotein C-III is recognized as one of the most important serum triglyceride regulatory factors. Bovine apolipoprotein C-III is a low molecular mass protein mainly distributed in the HDL fraction, and it consists of at least 2 isoforms, 8.2 and 7.3 kDa; the latter is the major species.¹ Also, apolipoprotein C-III has been detected in chylomicrons and LDL and VLDL fractions.^24,44 Apolipoprotein C-III inhibits the activation of lipoprotein lipase by apolipoprotein C-II and further reduces hepatic uptake of the remnant lipoprotein. Lecithin cholesterol acyltransferase is also activated by apolipoprotein C-III. Bovine serum apolipoprotein C-III concentration has the lowest value in the nonlactation period, gradually increases around the early lactation period, and reaches the maximum concentration in the midlactation period. The serum apolipoprotein C-IIII concentration is decreased in cows with hepatic lipidosis, ketosis, left displacement of abomasum, hypocalcemia, and retained placenta, compared with healthy cows in early lactation.^1,9,11,14,44 In addition to LCAT and apolipoprotein B-100, determination of the apolipoprotein C-III concentration during the peripartum period appears to be helpful in diagnosing periparturient diseases.^1,9,11,14 It was also found that the amount of 10.2- and 8.8-kDa proteins, which are similar to bovine apolipoprotein C-III, increased during lactation, compared with that in cows during nonlactation, in the β lipoprotein fraction. However, a faint protein band was detected in ketotic cows and was not changed after treatment. Apolipoprotein C-III could not be detected in the α lipoprotein fraction because the β lipoprotein fraction used in this study was contaminated to some extent by HDL (probably HDL₁).

Apolipoprotein E is believed to have an important role in cholesterol metabolism, mediating hepatic uptake of HDL, which would be the final phase of the hypothesized reverse transport of cholesterol.⁴⁷ The existence of apolipoprotein E was reported in VLDL and LDL of lactating cows.⁴³ However, apolipoprotein E in lipoprotein fractions was not detected in calves and adult cows.^47,48 Takahashi et al⁴⁸ found an increase of a 38-kDa protein in the bovine VLDL fraction caused by withholding of food, and the lipoprotein was identified as bovine apolipoprotein E by determination of its N-terminal amino acid sequence. The concentration of bovine plasma apolipoprotein E was low, compared with that in other species. Apolipoprotein E was markedly increased in VLDL, LDL, and nonlipoprotein fractions by withholding of food. The concentration of apolipoprotein E in the HDL fraction was low, compared with those of VLDL or LDL, and was not changed by withholding of food. Increased concentrations of plasma apolipoprotein E in a cow from which food has been withheld imply the possible relevance of apolipoprotein E in the development of lipid-related disorders such as hepatic lipidosis in the peripartum period. Detection of an unusually low apolipoprotein E concentration in the peripartum period may be useful for early diagnosis of such disorders.

In another study,⁴⁷ it was found that the apolipoprotein E concentrations evaluated by use of ELISA during lactation were significantly higher than during nonlactation. Because most of the plasma apolipoprotein E is thought to be of liver origin, it was suggested that liver production had been upregulated during lactation. Via agarose gel electrophoresis, bovine apolipoprotein E was found in the β lipoprotein fraction, whereas it could not be found in the α lipoprotein fraction. The slow β particles (α₁) that had a high concentration of apolipoprotein E were probably deficient in neutral lipids.⁴⁷ The HDL₁ appears to be metabolically related to HDL, and the predominant apolipoprotein of HDL₁ is apolipoprotein E.⁴⁵

By use of SDS-PAGE, it was determined that in the β lipoprotein fraction, 2 protein bands, with molecular weights estimated to be 36 and 40 kDa (probably apolipoprotein E), had a higher concentration during the lactation period than during the nonlactation period, whereas in the ketotic cows, they decreased and even disappeared. Apolipoprotein E could not be detected in the α lipoprotein fraction because apolipoprotein E was the predominant apolipoprotein of HDL₁ (α₁). After ketotic cows were treated for 3 days with prednisolone and dexamethasone, apolipoprotein E concentration increased. Therefore, detection of apolipoprotein E in ketotic cows may indicate a better prognosis, and more studies on apolipoprotein E should be carried out.

In the present study, ketotic cows were treated with glucocorticoid (prednisolone and dexamethasone) plus dextrose and insulin plus dextrose. Insulin inhibits the release of NEFAs from adipose tissue, which is followed by a decrease in circulating plasma NEFAs. Insulin enhances lipogenesis and the synthesis of acylglycerol and increases oxidation of glucose to CO₂ via the pentose phosphate pathway. All these effects are dependent on glucose and can be explained, to a large extent, on the basis of the ability of insulin to enhance the uptake of glucose into adipose cells. A principal action of insulin in adipose tissue is to inhibit the activity of hormonesensitive lipase, reducing the release not only of NEFAs but of glycerol as well.⁴⁹ Reduction in glucose output by the liver could decrease blood glucose concentrations and insulin secretion, which, in turn, could lead to greater lipid mobilization from adipose tissue and increased rates of hepatic fatty acid uptake and ketogenesis. During ketosis, plasma NEFAs and liver triglyceride concentrations typically increase and liver glycogen content decreases.¹³

The glucocorticoids (11-oxysteroids) are secreted by the adrenal cortex and increase gluconeogenesis. This is a result of enhanced hepatic uptake of amino acids and increased activity of aminotransferases and key enzymes of gluconeogenesis. In addition, glucocorticoids inhibit the use of glucose in extrahepatic tissues. In all these actions, glucocorticoids act in a manner antagonistic to insulin.⁴⁹

Prednisone is the most widely prescribed of the glucocorticoids. Its short half-life, low cost, and negligible mineralocorticoid effect make it useful for most immunosuppressive and anti-inflammatory indications. Prednisolone is the active hepatic metabolite of prednisone and is useful for treatment of liver failure. Dexamethasone is the long-acting glucocorticoid of choice, being about 7 times as potent on a weight basis as prednisone and having a half-life of 24 hours. Prednisolone is preferred because of the low prevalence of adverse effects. A substantial decrease in milk production should be expected when administrating a corticosteroid to lactating cattle.

In an earlier study,⁷ cows with hepatic lipidosis were treated with insulin to suppress further adipose fat mobilization and to help increase lipoprotein output. Ketotic cows were treated with glucocorticoid plus insulin by McCarthy et al.² Glucocorticoids promote the breakdown or mobilization of tissue protein, whereas insulin suppresses the formation of gluconeogenic enzymes in the liver, and the dual hormonal action increases the availability of amino acids by promoting their mobilization but slowing their conversion to glucose. In the present study, it was observed that lipoprotein profiles and cholesterol and β-hydroxybutyrate concentrations of cows treated with insulin plus dextrose returned to reference range values more quickly than those of cows treated with dexamethasone and prednisolone plus dextrose.

From a practical aspect, our results suggested that precipitation of bovine serum lipoproteins with DS-MgCl₂ can be useful for diagnosis of ketosis and estimating a prognosis. In the same way, determination of bovine lipoprotein and apolipoprotein profiles might be useful for the study of other metabolic disorders. Further investigations are required for identification of apolipoproteins in ketotic cows.