Bengt Borgström

Mode of action of tetrahydrolipstatin: a derivative of the naturally occurring lipase inhibitor lipstatin

Tetrahydrolipstatin is a specific lipase inhibitor derived from lipstatin, a lipid produced by Streptomyces toxytricini. In addition to pancreatic lipase, it is shown in the present study that tetrahydrolipstatin also inhibits human gastric lipase, carboxyl ester lipase (cholesterol esterase) of pancreatic origin and the closely related bile-salt-stimulated lipase of human milk. It does not inhibit the exocellular lipase from Rhizopus arrhizus or a lipase recently isolated from Staphylococcus aureus. In the presence of a water-insoluble substrate, such as tributyrin, the inhibition has the characteristics of an irreversible inactivation of the uncompetitive type, thus indicating that an enzyme.substrate.inhibitor complex is formed, which cannot undergo further reaction to yield the normal product. This reaction probably takes place at the aqueous/oil interface of the substrate. In aqueous solution, in the absence of substrate, the inhibition of carboxyl ester lipase by tetrahydrolipstatin has the characteristics of being reversible, and finally becomes of a temporary nature analogues to the trypsin-trypsin inhibitor system. It is suggested that an enzyme-inhibitor complex of an acyl-enzyme type is formed that is slowly hydrolysed, with water as the final acceptor, leaving an intact enzyme and an inactive form of the inhibitor. The enzyme thus consumes the inhibitor, which undergoes a chemical conversion, as indicated by a change in mobility in an appropriate thin-layer chromatographic system, indicating an increase in hydrophilicity. Evidence is presented that the reaction product is an acid and that the functional group of tetrahydrolipstatin is the beta-lactone reacting with the active site of the enzyme.

Aqueous lipid phases of relevance to intestinal fat digestion and absorption.

The phase behavior of monoglyceride/water systems, with oleic and linoleic acid as the dominating fatty acid residues, was investigated. Increased solubilization of triglycerides (oil) or oleic acid in the cubic liquid-crystalline phase formed by monoglyceride and water resulted in the formation of a reversed hexagonal liquid-crystalline phase followed by an L2-phase. The liquid-crystalline phases have different dispersion properties compared to each other in dilute micellar bile salt solutions. The cubic phase is found to be easily dispersed. The relevance of aqueous lipid phases other thah micellar is discussed in relation to intestinal lipid digestion and absorption.

On the mechanism of pancreatic lipolysis of glycerides.

Abstract During hydrolysis of triglycerides of long chain fatty acids by pancreatic juice from rat an exchange occurs between glyceride fatty acids and liberated free fatty acids. In thi] exchange only fatty acids in the 1- and 3-position of the glyceride take part and these fatty acids rapidly come into equilibrium with the free fatty acids. The exchange between the glyceride fatty acids and the free fatty acids is at least partly due to a resynthesis of glyceride ester bonds occuring simultaneously with the hydrolysis. This resynthesis has been evidenced for the step 1.2-diglyceride to triglyceride. Under the conditions of the experiments, resembling those prevailing during digestion in the lumen of the small intestine, no resynthesis takes place from glycerol and fatty acids by the action of pancreatic juice and the overall hydrolysis of glycerides accordingly proceeds to completion in vitro . The results of this investigation indicate that the hydrolysis of glycerides by pancreatic juice enzyme proceeds via the 1.2-diglyceride to both 1- and 2- monoglycerides, the hydrolysis to the 2-monoglyceride prevailing. The pH/activity curves for the hydrolysis and resynthesis of glyceride ester bonds by pancreatic juice enzyme with olive oil as substrate in the absence of bile acids are parallel and show a maximum at pH about 8. Addition of Ca ions accelerates the rate of hydrolysis of triglycerides by pancreatic juice both in acid and alkaline media. The increased rate of hydrolysis brought about by Ca ions is parallel to a decreased rate of resynthesis of glyceride ester bonds. Different fatty acids are built into glycerides during hydrolysis with pancreatic enzyme at different rates.

Purification and characterization of two proteins with Co-lipase activity from porcine pancreas

Abstract Two co-lipases, named co-lipases I and II, have been isolated from extracts of porcine pancreatic gland. The two proteins can be separated by ion-exchange chromatography and disc electrophoresis. They probably have identical amino acid compositions and have the same sequence of nine N-terminal amino acids. They contain the same number of acidic and basic amino acids and therefore most probably only differ in the extent of amidation of their glutamic and aspartic acid residues. Two co-lipases also exist in porcine pancreatic juice. A co-lipase previously purified from the porcine pancreatic gland and reported to have an N-terminal isoleucine had lost six N-terminal amino acids most probably during the purification procedure. The biological activity of this co-lipase and those described here is, however, the same.

Intestinal digestion and absorption of cholesterol and lecithin in the human. Intubation studies with a fat-soluble reference substance.

Cholesterol and lecithin absorption in man was studied by intestinal intubation. β-3H-sitosterol lipid-soluble marker. Cholesterol absorption was limited and progressed over the length of the small intestine; most extensive in the upper part. Lecithin was hydrolyzed to L-α-lysolecithin and free fatty acids, which were absorbed in parallel in the upper small intestine. 3.5–6.5 g lecithin and 0.2–0.4 g cholesterol were delivered via the bile during passage of a test meal. Phospholipids partitioned in a glyceride bile salt mixture in favour of the bile salt micellar phase.

Evidence for a pancreatic pro-colipase and its activation by trypsin

Pancreatic colipase is a polypeptide secreted into the pancreatic juice, necessary for the binding of lipase to its triglyceride substrate in the bile-containing content of the small intestine [ 11. Colipase has been purified from the pancreatic glands of several species [I] and obtained with a varying number of amino acids. Colipases from porcine sources have been described with 68-70,83-84 and 94-95 amino acid residues and N-terminal glycine [2]. The complete sequence of colipase, has been determined [3]. Colipases with 102-l 07 amino acids and N-terminal valine was first isolated from the porcine gland [4] and later from bovine and horse pancreas [5,6]. The N-terminal sequence of these colipases is Val-P:o-Asp-Pro1 3 4 Arg-Gly-Ile-Ile-IF[4,5,6] (in the horse IF is 5 6 7 8 replaced by Val [5] ). It appears that these later colipases are the forms produced by the glands and that the shorter ones have been formed by limited proteolysis before or during the purification. As the -

Fat Digestion and Absorption

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Studies in choline deficiency. Fate of injected 1-C14-palmitic acid and fatty acid spectra in fasting and refed rats.

The field of fat digestion and absorption has interested physiological chemists, physical chemists and gastroenterologists since Claude Bernard [1] in 1856 so convincingly demonstrated the importance of bile and pancreatic juice for these processes.

Hydrolysis of long-chain monoglycerides in micellar solution by pancreatic lipase.

Discussion and summary The hypothesis of an impaired transport of neutral fat from the liver of fasting choline deficient animals is sustained by the isotopic data showing a persistently high activity in the liver neutral fat and low plasma triglyceride activity. However, in accord with the in vitro findings of Artom( 8 ), a decreased oxidation of the labeled fatty acids in the liver in choline deficiency might also be a factor of importance for the accumulation of fatty acids. In the refed animals in which oxidation is suppressed to a minimum, there is little difference in rate of disappearance of labeled fatty acids from the livers in both types of experiment. These curves also are rather similar to the liver activity curve in the fasting choline deficient rats. This can also be so expressed that the liver disappearance curves are rather similar for the choline deficient rats whether they have been starved or refed. The specific activity relationship between liver and plasma triglycerides in the choline deficient animals indicates that only a small fraction of the total liver triglycerides is in rapid equilibrium with the plasma triglycerides and that this “actively metabolizing pool”( 9 ) of liver triglycerides is not increased in proportion to the total triglyceride content of the liver in the choline deficient animals. Most of the liver triglycerides which pile up in the livers of choline deficient rats therefore are not easily available for metabolism. The rapid disappearance of neutral fat from the livers of the choline deficient animals on refeeding is not consistent with known data on lipid turnover rates in liver and blood plasma of the rat. The rapid changes in lipid composition and distribution caused by overnight refeeding of control animals maintained on the synthetic diet supplemented with choline has been reproduced in animals reared on ordinary pellet diet.

Quantitative study of the pathways of triglyceride synthesis by hamster intestinal mucosa.

Abstract 1. 1. Certain long-chain monoglycerides have a high solubility in bile-salt solutions forming mixed monoglyceride-bile-salt micelles. Monoglycerides in such an optically clear, micellar solution are readily hydrolyzed by pancreatic lipase (glycerol ester hydrolase, EC 3.1.1.3). Using a rat-pancreatic-juice preparation containing negligible esterase activity, the hydrolysis of a number of pure monoglycerides in micellar solution has been studied. 2. 2. The rate of hydrolysis was essentially linear with time and enzyme concentration up to a level of around 60–70% hydrolysis of the substrate. The extent of hydrolysis fell when the concentration of micelles of identical composition was increased. There was a broad pH optimum 5.5–7.5. Bile salts greatly enhanced the rate of hydrolysis at low concentrations, but this effect was possibly chiefly due to their dispersing effect; at higher concentrations, they caused a marked inhibition of hydrolysis. Typical anionic detergents influenced hydrolysis in the same manner as bile salts. With a cationic detergent the enzyme concentration had to be considerably increased to obtain the same rate of hydrolysis. 3. 3. 2-Monoolein was not attacked to any appreciable extent by pancreatic lipase; thus the specificity of lipase for the 1-ester position could be directly shown. Glycol monooleate was readily hydrolyzed; 1-α,α-dimethyl-monodecanoin was not split. 4. 4. Under experimental conditions where 1-monoolein was extensively hydrolyzed, middle-chain saturated 1-monoglycerides were hydrolized to a very limited extent. Extensive hydrolysis could be obtained by increasing the micellar amphiphile to micellar bile-salt ratio, by adding other amphiphiles such as fatty acid, or by using a two-phase heptane-buffer system where the monoglyceride was concentrated at the interface. The results of these and other experiments were interpreted as showing the tightness of packing of the micelle to be a critical factor influencing the extent of hydrolysis. 5. 5. Titration of the fatty acid of a mixed bile salt-monoglyceride-fatty acid micelle indicated a pK a of 6.9 with the experimental conditions present.