Analysis by Enzyme-Linked Immunosorbent Assay and 2-Dimensional Electrophoresis of Haptoglobin in the High-Density Lipoprotein Fraction in Cows.

Abstract

ABSTRACT. Haptoglobin (Hp) is a hemoglobin (Hb)-binding acute-phase protein. Besides its relevance in inflammation, Hp is involved in the regulation of lipid metabolism. In cattle, in addition to the lipoprotein-deficient fraction, Hp is distributed in high-density lipoprotein (HDL) and very high-density lipoprotein (VHDL) fractions. The purpose of this study was to determine Hp concentrations in the lipoprotein fractions using an enzyme-linked immunosorbent assay (ELISA) based on the affinity with Hb, and also to detect structural differences of HDL Hp from that in the lipoprotein-deficient fraction using 2-dimensional electrophoresis. When purified Hp was used as the antigen for the ELISA, the detection limit was 7.4 ng/ml and linearity was obtained from 14.8 to 475 ng/ml. The correlation coefficient between the ELISA and single radial immunodiffusion was 0.884. The ELISA was shown to be applicable to evaluate Hp concentrations in the lipoprotein fractions. Hp concentrations in the lipoprotein fractions were in the range of 0.94 to 8.77 µg of Hp/ml (n=4), and concentration ratios were 0.2 to 0.3% of whole serum Hp. Of the lipoprotein fractions, Hp was most abundant in HDL, moderate in VHDL and faint in chylomicrons, the very low-density lipoprotein fraction and low-density lipoprotein fraction. By 2-dimensional electrophoresis, α-and β-chains of serum Hp were each separated into 5 spots, and their isoelectric point (pI) values were from 5.05 to 6.28 in the α-chain and from 5.92 to 6.95 in the β-chain. The pI values of HDL Hp were indistinguishable from those of serum Hp. These results indicate that the ELISA based on the affinity with Hb is useful for evaluating Hp concentrations in lipoprotein fractions, and also suggest that HDL Hp is structurally similar to that in the lipoprotein-deficient fraction. KEY WORDS: bovine, enzyme-linked immunosorbent assay, haptoglobin, high-density lipoprotein, 2-dimensional electrophoresis.

The effect of the season on some blood metabolites and haptoglobin in dairy cows during postpartum period

“…The effect of climatic conditions on haptoglobin in dairy cows is controversial: some authors did not find a season influence on its concentrations (Chan et al 2004), while other authors found higher concentrations in summer than in winter (Wenz et al 2010). It is believed that serum haptoglobin in cows could be involved in the regulation of lipid metabolism (Kanno & Katoh 2001). In our study the patterns of haptoglobin and NEFA were similar among periods.…”

Detection of SNPs in Porcine Haptoglobin and Apolipoprotein Genes

“…Isoelectric point values of Hp in cattle range from 5.05 to 6.28 (Kanno and Katoh 2001), which concurs with the findings of this study. A pI for APOA4 has not been reported, however, and the estimated pI value was 5.51.…”

Relevance of Apolipoproteins in the Development of Fatty Liver and Fatty Liver-Releted Peripartum Diseases in Dairy Cows.

“…In sera from cows with fatty liver, Hp and Hb are detected, in addition to the lipoproteindeficient fraction (d>1.25), in HDL and VHDL fractions [76]. The association of Hp in HDL and VHDL fractions is also found in sera from cows with mastitis [66]. In a calf with hyperlipidemia, Hp, together with apoA-I, was detected in both CM and HDL fractions [193].…”