The lipase and esterase activities of the pancreas and small intestine of the chick

Abstract

Apart from the work of Hewitt & Schelkopf (1955), little in the nature of a general study of the digestive enzymes of the chick has appeared since the papers of Shaw (1912-13) and Plimmer & Rosedale (1922). The findings presented in these three papers are somewhat limited since the techniques employed were only qualitative or, at the most, semi-quantitative. Some of the enzymes concerned with the digestive processes in the chick were therefore investigated. The work here reported is concerned with a comparison of the activities of pancreatic and intestinal lipase and esterase in chicks of different ages. During this investigation it became clear that lipase activity (as defined by Desnuelle, 1961) with an emulsified triglyceride as substrate was confined almost entirely to the pancreatic extracts, whereas esterase activity with a water-soluble substrate was confined almnost entirely to extracts of the small intestine. This partition of the enzymes between pancreas and small intestine allowed certain properties of the two enzymes to be studied without previous attempts at purification. EXPERIMENTAL Measurement of lipase and esterase activity of the pancreas and small intestine of chicks of different ages Female chicks (1 day old) of a broiler strain were obtained from a local breeder. On arrival at the Laboratory, before they had any access to food, 24 of them were killed. The remainder were given a commercial chick starter diet and drinking water ad lib. Groups of 12, 6 and 6 chicks chosen at random were killed at 10, 20 and 30 days of age respectively. So that each bird should be in a similar post-absorptive state the food trays were removed 12 hr. before the birds were killed. Immediately after death the pancreas and small intestine were removed from each bird as rapidly as possible and the contents of the intestine quantitatively removed by washing with water. The small intestines of the 10-, 20-and 30-day-old chicks were divided into three sections: the duodenum (from the gizzard to the pancreatic and bile ducts), the upper and lower small intestine (ob-tained by dividing the intestine from the pancreatic and bile ducts to the ileocaecal junction into two sections of equal length). The small intestines of the 1-day-old chicks were not subdivided and were treated subsequently as a whole. With minimum delay all tissues were transferred to a deep-freeze cabinet, where they were stored for further use at-20°. Before acetone-dried powders of these tissues were prepared certain of them were bulked, owing to the limited amount of material available from the younger birds. For the 1-day-old chicks four samples of each tissue (pancreas and whole small intestine) were obtained by randomly pooling the tissues from groups of six birds. For the 10-day-old chicks four samples of each tissue (pancreas, duodenum, upper and lower small intestine) were obtained by pooling the tissues obtained from groups of three birds. The tissues from the 20-and 30-day-old chicks were all treated individually. Acetone-dried powders were prepared by a method similar to that of Morton (1955). The frozen tissues were chopped into small sections and homogenized with 10 vol. of acetone at 00 either in a Potter-Elvehjem glass homo-genizer or, according to the quantity of tissue available, a stainless-steel Waring Blendor. The homogenates were centrifuged and the solid residues washed and re-centrifuged with three successive portions of ice-cold acetone. They were then dried under reduced pressure and passed through a sieve (B.S. size 50) to remove any remaining connective tissue. The acetone-dried powders were weighed and stored in a desiccator at 00. Enzyme solutions were prepared by extracting known weights of the acetone-dried powders with 015M-sodium chloride (Morton, 1955) and portions of the extracts were taken for enzyme assay. All enzyme assays were performed at pH 7-0 in the sodium acetate buffer described by Boissonnas (1948), since despite the liberation of acid during incubation the pH of the mixture remains under these conditions relatively constant (Boissonnas, 1948). In view of the accumulating evidence that water-soluble substrates are unsuitable for the measurement of lipase activity and the concept that there are two distinct groups of hydrolytic enzymes, one acting exclusively on emulsified esters and the other on esters in true solution (Desnuelle, 1961), the activities of the pancreatic and small-intestinal extracts were each determined with a water-soluble sub-strate and an emulsified triglyceride. The water-soluble ester used was Tween 20 (polyoxyethylene sorbitan mono-laurate) obtained from Honeywill and Stein Ltd., London; it was purified by the method of Bier (1955). The substrate consisted of a 20 % (v/v) solution of the purified Tween 20 in 0 2M-sodium acetate adjusted to pH 7-0. So that enzyme extracts could be assayed for activity against an emulsified triglyceride, a 5 % (w/v) emulsion of triolein was 632