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Abstract

An elisa was developed to evaluate the concentration of apolipoprotein A-I, a major apoprotein in high-density lipoprotein, in the serum of cattle. Serum apolipoprotein A-I was purified electrophoretically, and antibodies to this protein were raised in rabbits. The specificity of the antiserum was assessed by use of several immunologic techniques including western blotting. The elisa was sensitive (detection limit was 70 ng of apolipoprotein A-I/ml) and reliable (coefficients of variance were in the range of 3.5 to 8.2%). By use of this method, the serum apolipoprotein A-I concentration was higher in 2- to 6-year-old Holstein cows (mean ± SD, 0.580 ± 0.304 mg/ml) than in 7- to 15-month-old heifers (0.339 ± 0.237 mg/ml), 6-month-old heifers (0.238 ± 0.188 mg/ml), and 6-month-old steers (0.173 ± 0.146 mg/ml). The concentration, however, is not largely different in cows in early, middle, and late lactation and in nonlactating stages. Results also indicated that apolipoprotein A-I concentration was decreased in cows with hepatic lipidosis (fatty liver) induced by administration of ethionine, suggesting that this method is a useful tool for the pathophysiologic study of lipid metabolism and its impairment in cattle.

Free access
in American Journal of Veterinary Research

Abstract

Objective

To determine: whether apolipoprotein A-I (apoA-I) is secreted by calf liver parenchymal cells; the isoprotein pattern and association with lipids of secreted apoA-I; and effects of steroid hormones on apoA-I secretion.

Sample Population

6 male Holstein calves (1 to 2 weeks old) as a cell culture source.

Procedure

apoA-I in culture medium was detected by immunoblot analysis, and its concentration was measured by use of an ELISA. The isoprotein pattern was analyzed by use of two-dimensional polyacrylamide gel electrophoresis. Associations of apoA-I with cholesterol and phospholipids were examined by ultracentrifugal analysis of the culture medium.

Results

Concentration of apoA-I in culture medium increased linearly up to 24 hours. The protein synthesis inhibitors, actinomycin D and cycloheximide, suppressed apoA-I accumulation in the medium in dose-dependent manner. Molecular mass of this protein in culture medium was 28 kd, and was indistinguishable from apoA-I of plasma. Four isoproteins with different isoelectric points (1 = 5.75; 2 = 5.67; 3 = 5.58; and 4 = 5.46) were detected. Of these, isoprotein 2 was the major species. By comparison, isoprotein 4 was the predominant species in plasma, and isoprotein 5 (isoelectric point = 5.38) was newly detected instead of isoprotein 1. Approximately 33% of apoA-I in culture medium was found in lipid-rich fractions, whereas the rest was found in nonlipoprotein fractions. Dexamethasone (10−8 to 10−6 M) significantly (P < 0.001) increased apoA-I concentration in the medium, and the stimulatory effect was significantly (P < 0.001) suppressed by the simultaneous addition of 10−5 M progesterone. Progesterone itself (10−6 to 10−5 M) had little effect on apoA-I secretion; estradiol (10−14 to 10−6 M) also had no significant effect.

Conclusions

apoA-I is synthesized by calf liver parenchymal cells, and the secreted protein is modified during circulation. Moreover, apoA-I synthesis and secretion by the cells appear not to be largely influenced by hormones, except for dexamethasone. (Am J Vet Res 1997;58:811–815)

Free access
in American Journal of Veterinary Research

Abstract

Objective

To determine serum apolipoprotein C-III (apoC-III) concentration in cows in various stages of lactation by use of an ELISA.

Sample Population

Sera obtained from 29 Holstein cows during early lactation, 65 cows during midlactation, 42 cows during late lactation, and 23 cows during the nonlactating stage.

Procedure

A 7.3-kd bovine apoC-III antiserum raised in rabbits was purified by affinity chromatography, and an ELISA was developed.

Results

In the immunoblot analysis, the antiserum reacted with the 7.3-kd apoC-III and moreover with another 8.2-kd apoC-III isoform. The 2 isoforms of apoC-III were also indistinguishable in the developed ELISA, the 2 proteins being measured as total apoC-III. In the ELISA for serum apoC-III concentration, addition of 2-mercaptoethanol to the coating buffer (50 mM sodium carbonate buffer, pH 9.6) was required. Mean ± SEM bovine serum apoC-III concentration (μg/ml of serum) was 71.6 ± 12.1 for the early lactating stage, 115 ± 14.0 for the midlactating stage, 104 ± 18.8 for the late lactating stage, and 55.3 ± 8.4 for the nonlactating stage. Concentration of apoC-III was significantly (P < 0.05) higher in cows during midlactation than in cows during the nonlactating stage and was correlated negatively with serum triglyceride concentration (r = −0.479; P< 0.01) and positively with total cholesterol (r = 0.421; P < 0.05) and phospholipids (r = 0.415; P < 0.05) concentrations.

Conclusions

Changes of apoC-III concentration in various stages of lactation suggest that this apolipoprotein is involved in a function related to lactation. (Am J Vet Res 1998;59:1358–1363)

Free access
in American Journal of Veterinary Research

SUMMARY

An elisa was developed to determine serum concentration of apolipoprotein B-100, a major triglyceride-binding protein in very low-density lipoproteins and a putative maker for hepatic lipidosis of dairy cows. Serum apolipoprotein B-100 was prepared electrophoretically, and antibodies to this protein were raised in rabbits. The antiserum prepared was further purified by affinity chromatography, using bovine serum albumin-Sepharose 4B, to remove antibodies to albumin. For the elisa, addition of 2-mercaptoethanol to the coating buffer (50 mM sodium carbonate, pH 9.6) was required to evaluate apolipoprotein B-100 concentration in serum. The elisa developed was sensitive (detection limit was 300 to 400 ng/ml of serum) and reliable (coefficients of variance were in the range of 3.3 to 7.6%). By use of the established elisa, the serum apolipoprotein B-100 concentration was found to be significantly (P < 0.01) lower during the early lactating stage than during other stages of lactation. Reduced hepatic synthesis or secretion of apolipoprotein B-100 during the early lactating stage, together with the excess uptake by the liver of serum nonesterified fatty acids, is suggested to be relevant in the accelerated accumulation of triglycerides in the liver of dairy cows during the periparturient period.

Free access
in American Journal of Veterinary Research

Summary

Ethionine, an analogue of methionine, induces fatty liver in rats by inhibiting protein synthesis, including that of apolipoproteins in liver. Ethionine was administered to cows to elucidate the participation in fatty liver development of impaired triglyceride secretion from liver attributable to decreased apolipoprotein synthesis. The administration resulted in a significant increase of liver triglyceride contents. Several apolipoproteins were found to have decreased concentrations. In particular, apolipoprotein B-100 in very low-density (0.95 to 1.006 g/ml) lipoprotein and in low-density (1.006 to 1.063 g/ml) lipoprotein fractions was greatly reduced. The decreases of apolipoprotein B-100 concentrations in the 2 lipoprotein fractions were at least partly correlated to the decreased triglyceride concentrations in the respective fractions. Decreased concentrations of apolipoprotein A-I in high-density (1.063 to 1.210 g/ml) lipoprotein were also observed, although not as distinctly as with apolipoprotein B-100. Total cholesterol and phospholipid concentrations in low- and high-density lipoprotein fractions were decreased. The decrease in cholesterol was attributed to reduced concentrations of cholesteryl esters. It was suggested that the impaired lipid secretion from liver attributable to the decreased apolipoprotein concentrations has a role in ethionine-induced fatty liver of cows.

Free access
in American Journal of Veterinary Research

Abstract

Objective

To determine whether annexins or haptoglobin could be detected in bronchoalveolar lavage (BAL) fluid specimens obtained from calves experimentally inoculated with Pasteurella haemolytica.

Animals

Twelve 2- to 3-month-old male Holstein calves.

Procedure

Pasteurella haemolytica was inoculated into the right lung lobes of each of 6 calves. Six other calves received vehicle alone and were used as control calves. Specimens of BAL fluid were obtained from 3 control and 3 inoculated calves 1 day after inoculation and from the other calves 2 days after inoculation. The amount of annexins I, II, IV, and VI, and haptoglobin in BAL fluid specimens was examined by use of immunoblot analysis.

Results

Annexins I and IV were detected in BAL fluid specimens obtained from the right lung lobes of each of the inoculated calves, but annexins II and VI were not. Annexin I also was found in BAL fluid specimens obtained from the left lung lobes of each inoculated calf and from left and right lung lobes of the control calves. By comparison, detection of annexin IV was essentially limited to the right lung lobes of inoculated calves. Haptoglobin was detected in some, but not all, BAL fluid specimens from the right lung lobes of inoculated calves, and its detection in BAL fluid was associated with serum proteins such as albumin.

Conclusions and Clinical Relevance

Annexin IV was detected most specifically in response to inoculation of P haemolytica. This protein could be used as a marker for inflammatory pulmonary disease caused by P haemolytica. (Am J Vet Res 1999;60:1390–1395)

Free access
in American Journal of Veterinary Research

Abstract

Objective

To identify, purify, and analyze distribution of apolipoprotein C-III in lipoprotein fractions and to evaluate its concentration in serum from calves, heifers, and cows during various stages of lactation.

Sample Population

Sera from 3 female calves, 3 heifers, and 12 cows during early, middle, late, and nonlactation stages.

Procedure

Apolipoprotein C-III was identified by use of amino-terminal amino acid sequence analysis of bands separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Purification was performed by extraction with acetone, delipidation with 2-propanol, and 2 chromatographic steps. The apolipoprotein C-III concentration in the total lipoprotein fraction was evaluated by densitometric scanning of the bands for apolipoprotein C-III separated by electrophoresis, using purified apolipoprotein C-III as an internal standard.

Results

Apolipoprotein C-III was identified as 8.2- and 7.3-kd proteins with identical amino acid sequences. The 2 proteins were mainly found in the high-density lipoprotein fraction, then were purified separately. Serum apolipoprotein C-III concentration was significantly (P< 0.05) higher in cows during lactation than in nonlactating cows and was negatively correlated with serum triglyceride concentration (r = −0.620, P < 0.01).

Conclusions

The role of apolipoprotein C-III in cows may involve a function related to lactation. (Am J Vet Res 1998;59:667-672)

Free access
in American Journal of Veterinary Research

Abstract

Objective

To assess the relevance of hepatic lipidosis (fatty liver) in the development of ketosis and left displacement of the abomasum (LDA).

Sample Population

Sera from 22 healthy cows in early lactation. 21 cows with ketosis, and 19 cows with LDA, and serum and liver specimens from 35 slaughtered cows with or without fatty liver, ketosis, and/or LDA.

Procedure

Apolipoprotein B-100 and A-I concentrations were measured in sera of healthy farm cows and of farm cows with ketosis and LDA. Serum apolipoprotein concentration, together with liver triglyceride content, also were surveyed in a subset of slaughtered cows.

Results

Compared with those in healthy cows or controls, apolipoprotein B-100 and A-I concentrations were decreased in cows with ketosis and LDA.

Conclusions

Decreases in apolipoprotein B-100 and A-I concentrations in cows with ketosis and LDA indicate that the 2 disorders may be intimately associated with fatty liver.

Clinical Relevance

Monitoring of the apolipoprotein B-100 and A-I concentrations during the stages of nonlactation and early lactation is helpful for detecting cows susceptible to ketosis and LDA. (Am J Vet Res 1997;58:121–125)

Free access
in American Journal of Veterinary Research

Summary

Haptoglobin (Hp), an acute-phase protein, is detected in serum of cows with hepatic lipidosis (fatty liver). To assess the relevance of Hp in fatty liver, induction of Hp was examined, using conditions similar to those involving development of fatty liver in cows. Induction of Hp was achieved by a combination of dexamethasone administration (0.1 mg/kg of body weight) and 2 days’ starvation. Haptoglobin appearance in serum was not associated with the increase of α1-acid glycoprotein (a marker for inflammation). This treatment increased serum nonesterified fatty acids concentration and decreased serum triglycerides concentration. Protein kinase C activity was decreased in the cytosolic fractions of liver and mononuclear cells. Reduction of protein kinase C-catalyzed endogenous protein phosphorylation also was observed, particularly in the cytosolic fractions of the tissue and cells. Detection of Hp in serum of cows with fatty liver appears to be explained by the fact that Hp is induced by dexamethasone administration and starvation, which are similar to the condition responsible for fatty liver development. The change of protein kinase C-catalyzed phosphorylation was suggested to be involved in the induction of Hp in cows.

Free access
in American Journal of Veterinary Research

Summary

Estradiol was administered to 3 steers (0.12 mg/kg of body weight/d for 14 consecutive days), followed by 2 days of nonfeeding (starvation). During estradiol administration, liver nuclear estrogen receptor and serum apolipoprotein B-100 (apoB-100), as well as serum triglycerides concentrations were increased, compared with values before administration. Starvation, together with interruption of estradiol administration, resulted in rapid decreases of the receptor, serum apoB-100, and serum triglycerides concentrations, and increase of nonesterified fatty acids concentration. Of the 3 steers, 2 had higher liver triglyceride content, compared with values before treatment. In the control group (3 steers that received vehicle alone, then starved similarly), these concentrations, except for serum nonesterified fatty acids and triglycerides concentrations after starvation, were not changed. In another experiment, serum apoB-100 concentration in dairy cows was significantly (P< 0.05) lower at parturition than values before and after parturition. These results indicate that estradiol may be involved in development of fatty liver in cattle.

Free access
in American Journal of Veterinary Research