Lipase

LIPASE (from Greek lipos- fat), a lipolytic enzyme belonging to the group of esterases, i.e., enzymes that break down complex esters according to the equation R.CO-OR' + H₂O → RCOOH + R'OH into alcohol and acid. The action of esterases extends to esters of both monohydric alcohols and lower fatty acids, as well as polyhydric alcohols (e.g., glycerin) and higher fatty acids. By L. in the strict sense are meant those enzymes that predominantly break down esters of the latter type, primarily triglycerides of higher fatty acids, to which fats also belong. However, it is not yet possible to draw a strict boundary around the group of true L.s, since on the one hand, enzymes that vigorously break down ordinary fats can also hydrolyze other esters, and on the other hand, esterases that predominantly break down lower esters can, although weaker than true L.s, also saponify triglycerides. - According to origin, L.s can be divided into plant lipases (phytolipases) and animal lipases (zoolipases). The former are found in bacteria; they are especially abundant in oily seeds, e.g., in castor seeds. In the animal organism, we encounter L.s throughout the digestive tract; the most active enzyme is secreted in the pancreatic juice (steapsin, steaptase); intestinal and gastric juices also contain L.s; according to Scheer and Koldaev and Pikul, L.s are also present in saliva. In addition to these enzymes secreted into the digestive tract, we encounter L.s in all tissues and organs: in the liver, brain, kidneys, blood, etc. The properties of L.s have been largely elucidated through the work of Willstätter's laboratory from recent years. The long-held view of the insolubility of L.s in water has not been confirmed, since after proper purification it is possible to obtain preparations that are easily and completely soluble, although unstable in aqueous solution. The purest preparations give no reactions for proteins or carbohydrates, contain about 10% nitrogen, and quite a lot of ash. When studying digestive juices in their natural state, the L.s contained in them show various pH optima: for pancreatic L.s about 7.5, blood 8.0, liver 7.8-8.5, for gastric juice L.s about 5.0. However, according to Willstätter, this difference is due not to different natures of the enzymes but only to the presence of various impurities, "accompanying substances" that have either activating or inhibiting effects depending on the pH. These accompanying substances to a large extent account for the different relationship of L.s of various origins to various poisons discovered by Rona (see table).

According to Willstätter, the pancreatic and gastric juice L.s are identical or at any rate very close; the liver L.s are undoubtedly different from them. With respect to fat, pancreatic L.s are almost 10,000 times more active than liver L.s, with respect to tributyrin only 100 times, and with respect to methyl butyrate liver L.s are 2.5 times more active than pancreatic ones. Thus, the pancreatic gland enzyme is a true lipase capable of breaking down even simple esters, while the liver enzyme is an esterase that only weakly hydrolyzes ordinary fats. Like carbohydrases, L.s exhibit stereospecificity, acting predominantly on one or the other of stereoisomeric substrates. On lipases, the reversibility of enzyme action was first definitely discovered: in a mixture of alcohol and acid, over time the same state of equilibrium is established as is achieved in the hydrolysis of the corresponding complex ester. The main methods for quantitative determination of L.s are based either on determining the amount of fatty acids formed or on measuring the surface tension, which changes as capillary-active substrates such as mono- or tributyrin are broken down. For accurate accounting, it is necessary to eliminate the influence of activating or inhibiting accidental impurities. For this, Willstätter proposed the method of "equalizing activation or inhibition." For alkalimetric determination in an alkaline medium, activators are albumin and calcium chloride, in the stalagmometric method—albumin and calcium oleate. The activating effect of these substances is explained by the fact that they form adsorption compounds of the type: albumin^xlipase or complex adsorbates of the type: calcium oleate-albumin fat. The long-known activating effect of bile is also explained not by the conversion of the L. zymogen into an active form, but precisely by the formation of such adsorption compounds. - Alkalimetric determination according to Willstätter: the material under investigation (juice, solution of dry preparation) is brought to 10 cm³ with water, 2.5 g of olive oil, 2 cm³ of buffer solution (n-solution NH₃ + n-solution NH₄Cl in the ratio 1:2, pH=8.9), 0.5 cm³ of 2% CaCl₂ and 0.5 cm³ of 3% albumin solution are added, shaken for 3 minutes and placed in a thermostat at 30°. Before titration, alcohol is added to 125 cm³ and 20 cm³ of ether, and titrated with n/10 or n/1 alkali with thymolphthalein as indicator. Conditions are chosen so that no more than 24% of the available fat is broken down; the pH at the end shifts to 5.5. - Rona and Laznitsky proposed a gasometric method: as substrate an emulsion of tributyrin in Ringer's solution is used. The splitting off of butyric acid displaces carbon dioxide from the Ringer's solution bicarbonate, causing an increase in pressure, which is read on a manometer. - The method of Rona and Michaelis based on the stalagmometer has now become most widespread; it is based on the fact that tributyrin strongly lowers the surface tension of water, while its breakdown products (glycerin and butyric acid) have this property only to a very small degree. Thus, from the change in surface tension (it is determined by counting the number of drops from a capillary pipette of the stalagmometer) one can judge the breakdown of tributyrin.