DIRECT SPECTROMETRIC DETERMINATION OF SERUM BILE-ACIDS IN THE DOG AND CAT

  • 1 January 1984
    • journal article
    • research article
    • Vol. 45  (10) , 2043-2050
Abstract
Serum bile acid concentrations were shown to be a predictive indicator of hepatobiliary disease in persons. There has been only limited use of bile acid values in the clinical diagnosis of hepatobiliary disease in the dog and cat because of technical difficulties associated with many bile acid assays. A rapid enzymatic method previously developed for the quantitation of 3-hydroxy bile acids in persons was adapted for use in the dog and cat. Nonsulfated 3-hydroxy bile acids are converted to 3-oxo bile acids by 3.alpha.-hydroxysteroid dehydrogenase and reduction of NAD+ to NADH. In a coupled diaphorase catalyzed reaction, H+ is transferred to nitrotetrazolium blue to produce a diformazan dye, which is measured spectrophotometrically at 540 nm. Nonspecific interfering dehydrogenase activities present in the dog and the cat serums were inhibited by heating the serum to 60.degree. C for 30 min or by the addition of sodium pyruvate. Standard curves prepared from various serum sodium taurocholate concentrations in dogs and cats are linear to 250 .mu.mol/L. The assay is sensitive for the detection of bile acid concentrations as low as 2.5 .mu.mol/l in sera from dogs and cats. In validation studies quantitative recovery of known concentrations of 7 primary and secondary, conjugated and unconjugated, 3-hydroxy bile acids from pooled canine serum was 95.3 .+-. 7.9% (mean .+-. SEM [standard error of the mean]) and that from pooled feline serum was 101.4 .+-. 8.2%. Intra-assay precision on 10 replicate samples of pooled serums resulted in a mean value of 3.2 .+-. 0.2 .mu.mol/l, with a 6.6% coefficient of variation (CV) for the dog, and a mean value of 2.7 .+-. 0.3 .mu.mol/l with a 12.5% CV for the cat. Interassay precision on 11 separate assays of a known bile acid standard containing 29.0 .mu.mol/l gave a mean value of 29.0 .+-. 3.5 .mu.mol/l, with a 12.2% CV. Serum bile acid concentrations were determined in 20 healthy mature male and female dogs after they were fasted and after they were fed. The dogs were alloted to 2 groups of 10 dogs each and those in the 2 groups were fed diets having different protein and fat content. Group 1 dogs were fed a diet containing 11.4% protein and 6.9% fat, and group 2 dogs were fed a diet containing 8.9% protein and 1.1% fat. Serum bile acid concentrations were determined after 12 h fasting at 2, 4, 6, and 8 h after the dogs were fed. The mean value .+-. SE of the mean (.hivin.X .+-. SEM) of serum bile acids in fasting animals for both groups was 2.3 .+-. 0.4 .mu.mol/l. A significant (P < 0.05) increase in mean serum bile acid concentrations occurred in each group at the 2 and 4 h postprandial times, but not at the 6 and 8 h times. The maximal increases in mean serum bile acid value occurred in group 1 at 2 h after feeding with a mean of 8.3 .+-. 2.2 .mu.mol/l, and in group 2 at 4 h after feeding with a mean of 4.8 .+-. 1.0 .mu.mol/l. Serum bile acid concentrations were determined in 20 healthy mature male and female cats fasted for 12 h and then fed a diet containing 13.6% protein and 8.4% fat. The mean serum bile acid concentration after fasting was 1.7 .+-. 0.3 .mu.mol/l. The mean serum bile acid concentration at 2 h after cats were fed was significantly (P < 0.05) elevated to 8.3 .+-. 0.8 .mu.mol/l. There were no significant differences between the dog and cat in the serum bile acid values after a 12 h fast or at 2 h after feeding.