A novel spectrophotometric assay for lipase activity utilizing cis-parinaric acid.

A new spectrophotometric assay for determining the activity of acylglycerol hydrolases (lipases, E.C. 3.1.1.3) was developed and optimized for yeast lipase (Candida cylindracea). Studies with porcine pancreatic lipase were also conducted and the influence of various detergents and divalent cations on the assay was evaluated. The assay uses cis-parinaric acid (PnA), a naturally occurring fatty acid that has unique spectroscopic properties, and takes advantage of the reversible binding of fatty acids to bovine serum albumin (BSA). Free PnA has an ultraviolet absorption peak at 321.2 nm. When PnA is bound to BSA, however, the peak shifts to 324.2 nm. The assay mixture contains 6 microM PnA, 1 microM BSA, 75 microM triolein, and 0.3 mM taurocholate in a 50 mM tris-HCl buffer with 1 microM EDTA. The release of oleic acid from triolein is monitored over time by measuring the ratio of optical densities (OD) at 319.0 and 329.0 nm. Initially, there is maximum binding of PnA to BSA, and the OD ratio is approximately 1.0. Upon addition of lipase, PnA is displaced from the BSA by oleic acid released from triolein, and the OD ratio increases to a maximum of about 1.8. However, when calcium is present in the reaction mixture an insoluble calcium-PnA complex forms, resulting in a progressive decrease in OD at both 319.0 and 329.0 nm. The kinetic assay described here is simple, rapid, sensitive, reproducible, inexpensive, and it can be adapted to measure the activity of a variety of calcium-independent lipases. Under similar assay conditions, activities for Candida cylindracea lipase obtained with this assay are similar to those obtained with 14C-labelled triolein.

Liver subcellular fatty acid profiles of chicks fed diets containing hydrogenated fats and varying linoleate levels.

Day-old male broiler chickens were fed semipurified diets containing 5% lipid from one of four different lipid sources: corn oil (CO), partially hydrogenated soybean oil (HSBO), a spent restaurant grease (SRG) and a purified mixture of triolein, tripalmitin and tristearin (OPS). Diets CO and HSBO contained adequate amounts of linoleic acid, but diets SRG and OPS were deficient in linoleate. In addition, SRG and HSBO contained trans isomers of 16:1 and 18:1. The diets were fed for 3 wk to determine the effects of low linoleate levels and trans isomers on fatty acid profiles in liver microsomes, mitochondria and cytosol. Chicks fed HSBO had the highest body weights, while those fed SRG and OPS had the lowest. The incidence and severity of dermatitis were similar for all treatments. The proportions of linoleate and arachidonate in lipids from liver subcellular fractions were reduced significantly in chicks fed OPS and SRG; however, levels of 20:3 omega 9 were not increased. Feeding HSBO, which is high in both linoleate and linolenate, resulted in higher levels of 18:3 omega 3 and 20:5 omega 3 in liver subcellular fractions and lower levels of 20:4 omega 6 than those seen in chicks fed CO. The isomeric forms of 18:1 present in the partially hydrogenated fats (HSBO and SRG) appeared to be incorporated into the lipids of liver fractions. The results of this study show that dietary lipids influence fatty acid profiles of chick liver microsomes, mitochondria and cytosol. Decreases in linoleate and arachidonate in these organelles occur before overt essential fatty acid (EFA), deficiency signs in chicks fed EFA-deficient diets.

Alteration of lipid metabolism in Japanese quail by feeding oat hulls and brans.

Five varieties of oat brans fed for 4 weeks at 2.27% and oat hulls fed at 11.36% in a semi-purified diet containing .5% cholesterol were ineffective in lowering cholesterol levels in the serum of 5-week-old Japanese quail. Oat brans and hulls reduced the level of fat in livers of both males and females. No differences in cholesterol levels were observed between sexes, but hypertriglyceridemia and high liver fat contents were more evident in females. This may be the result of sexual maturation stimulated by a 14L:10D photoperiod.

Hypocholesterolemia and growth-depression in chicks fed guar gum and konjac mannan.

Two mannans (guar gum and konjac mannan) were fed to growing chicks at a level of 2% in a semipurified diet containing 0.5% cholesterol for 4 weeks. The mannans were nearly identical in producing growth depression, pancreatic hypertrophy and reduction in plasma and hepatic cholesterol when compared to controls fed corn starch or the sugars D-galactose or D-mannose. Hepatic triglyceride levels were higher for chicks fed konjac mannan, but no significant differences in fasting plasma glucose were observed. Apparent metabolizable energy levels for the mannan diets were significantly less than those of the other diets. The effects of the mannans in this study were not correlated with viscosity measurements made in vitro.