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  • Author or Editor: Shin Oikawa x
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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.

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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

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

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

Abstract

Objective

To detect localization of α1-acid glycoprotein (α1,-AG) antigens in the liver tissue of cattle by use of immunoperoxidase technique.

Sample Population

Liver specimens from 6 bovine fetuses, 2 healthy bovine neonates, 2 healthy adult cattle, 3 cattle with experimentally induced hepatic abscesses, and 2 cattle with enzootic bovine leukosis (EBL).

Procedure

3 cattle (with hepatic abscesses) were inoculated with a suspension of Fusobacterium necrophorum in the ruminal vein. Serum α1-AG concentration was determined by use of the single radial immunodiffusion method. Livers from fetuses, newborn calves, and adult or sick cattle were fixed in buffered 10% formalin, dehydrated in alcohol, embedded in paraffin, sectioned, and stained by use of the avidin-biotin complex/immunoperoxidase technique.

Results

Sites of localization of the α1-AG antigen positive reaction (AGPR) in the liver obtained from bovine fetuses, neonates, or sick cattle were different. In fetal and newborn calves, the AGPR was detected in the cytoplasm of hepatocytes. Intensity of the reaction varied in direct proportion to α1-AG serum concentration. In adult cattle, the AGPR was particularly intense in hepatocytes adjacent to abscesses or EBL-induced tumors.

Conclusions

The pattern of distribution of cells with AGPR in the liver varied, depending on severity of inflammation. In the cattle with EBL, whether the AGPR was attributable to inflammation could not be clarified, although suppression of immunologic response to tumors may have been a cause of the observed reaction. This association suggests that the glycoprotein may be synthesized, mainly in hepatocytes. (Am J Vet Res 1997;58:725–728)

Free access
in American Journal of Veterinary Research