Hans Brockerhoff

Positional distribution of fatty acids in triglycerides of animal depot fats

Abstract Several animal depot fats were subjected to stereospecific analysis, i.e., the fatty acid distribution in positions 1, 2, and 3 of the triglycerides were determined. The distribution of fatty acids among the positions 1, 2, and 3 in the triglycerides of animal depot fats is non-random. The distribution between position 2 and positions 1 and 3 seems to be governed by chain length and unsaturation. The shorter and the more unsaturated fatty acids show a greater tendency to occupy the position 2. This rule applies also to the apparent exceptions, the fats of pig and marine mammals, but in these the influence of chain length overrides that of unsaturation. On the other hand, in the three birds investigated unsaturation seems to be the only directing factor. We found fats with asymmetrical distribution of fatty acids as well as symmetrical fats. All mammalian fats were asymmetrical, as were the fats of one amphibian, one fish, and one arthropod, whereas the fats of one fish, one arthropod, and three birds were symmetrical. All asymmetrical fats have an excess of palmitic acid in position 1 and of oleic acid in position 3,

Substrate specificity of pancreatic lipase

Abstract 1. 1. The hydrolysis of esters of oleic acid by pancreatic lipase (glycerol-ester hydrolase, EC 3.1.1.3) depends on the nature of the alcohol. Two factors appear to influence the speed of the reaction: an inductive effect, and steric hindrance. 2. 2. Lipolysis is promoted by electrophilic substituents, as might be expected for a reaction involving nucleophilic attack on the carbonyl; e.g. oleyl oleate is split slowly, 2-fluoroethyl oleate or p-nitrobenzyl oleate rapidly. This inductive effect explains the following sequence: speed of lipolysis: triglyceride > 1,2-diglyceride > 1,3-diglyceride > 1-monoglyceride > 2-monoglyceride. It probably also regulates the attack of pancreatic lipase on different phospholipids. 3. 3. Bulkiness of the carbinol group inhibits lipolysis: vinyl oleate is hydrolysed, but isopropyl or phenyl oleate are not. This steric effect explains the specificity of the lipase for the α-chains of triglycerides. Electron-withdrawing substituents can counteract this hindrance; e.g. 1,3-difluoroisopropyl oleate and p-nitrophenyl oleate are slowly hydrolysed. 4. 4. The degree of hydration of the alcohol may be a third factor in lipolysis; e.g. trifluoroethyl ester reacts more slowly than monofluoroethyl ester, although the groups occupy similar space. This points to a change in the structure of the interphase. 5. 5. It is suggested that lipases differ from other esterases in being comparatively weak nucleophilic agents.

Positional distribution of fatty acids in depot triglycerides of aquatic animals

Stereospecific triglyceride analyses were performed on fats of the following animals: five aquatic invertebrates, five freshwater fish, six marine fish, three marine birds, two amphibia, two seals, a whale, and a marine turtle. The distribution of faty acids was asymmetrical in most cases. A formula is presented which describes the general tendencies of fatty acid distribution in many animal fats, and some special rules which modify this formula are stated.

Fatty acid distribution of triglycerides determined by deacylation with methyl magnesium bromide.

Summary Triglycerides can be degraded with CH3MgBr to yield partial glycerides. The α- and β-monoglycerides are partly isomerized, and the α,α-diglyceride contains a few percent isomerized material, but the α,β-diglyceride obtained has the required composition and can be used to calculate the α-β fatty acid distribution on the triglyceride. The method is demonstrated on lard. A representative α,β-diglyceride has been prepared from seal oil. A preliminary experiment with butter is discussed.

Substrate specificity of pancreatic lipase: Influence of the structure of fatty acids on the reactivity of esters

Abstract Various esters of monoenoic, polyenoic, methyl-branched, and ω-cyclohexyl or ω-phenyl substituted acids and also esters of aliphatic acids of various chain lengths were hydrolysed by porcine pancreatic lipase, and their maximal rates of lipolysis were compared to the rates of the corresponding oleates. 1. 1. Substituents or unsaturation on Carbon 2–5 led to a relative resistance of the esters against lipase. Cis and trans unsaturation and the one triple bond tested inhibited lipolysis by comparable degrees. Introduction of additional but more remote double bonds, as in marine polyenoic acids, introduced no additional resistance. Between unsaturation at Δ2,3 and Δ5,6, the resistance does not depend in a coherent manner on the actual position of the double bond. Δ2,3-acids or Δ4,5-acids may in fact be slightly better substrates than Δ5,6-acids. 2. 2. Compared to a monomethyl-branched acid, multiple branching introduced further resistance. Cyclohexylacetic acid was almost completely resistant. Inhibition by ω-phenyl groups disappeared gradually as the chain was lengthened to carbon 6. The ω-phenyl octanoates had an abnormally high rate of lipolysis, two to three times that of oleate. 3. 3. Lipolysis rates of esters of straight-chain saturated acids increased from C2 to a maximum at C4, decreased suddenly at C5 and then increased until around C9 the rates of the oleates were approached. 4. 4. Formates were attacked by lipase at rates comparable to those of oleates. 5. 5. It is concluded that the resistance of unsaturated or branched acids is due to steric hindrance during the formation of the activated complex. This hindrance disappears largely for structures after C5. The saturated C4-chain seems to be optimally adaptable to the enzyme. The adsorption of the enzyme to the interphase should be viewed as a process preceding and separate from the formation of the substrate-enzyme complex. In this complex the esters are fixed to the enzyme in a two-dimensional orientation, probably along the axis carbonyl carbon-ether oxygen of the carboxyl ester group. Three-point fixation is a possibility. It is suggested that fixation and activation of the substrate is achieved by hydrogen bonding to both carbonyl and ether oxygen, and that the leaving alkoxy group is received by a labile hydrogen of the enzyme.

Incorporation of fatty acids of marine origin into triglycerides and phospholipids of mammals.

Rats and mink were kept on a diet of mackerel, and the distribution of fatty acids over the different positions of triglycerides and phospholipids was analyzed. In the triglycerides of depot fat the monoenoic acids 20:1 and 22:1 accumulate in Positions 1 and 3∗, and the polyenoic acids in Position 3, in a pattern similar to that of marine mammals. The triglycerides of the livers incorporate less monoenoic acid than the depot fats. In the phosphatidyl-choline and phosphatidyl-ethanolamine of the livers (mink and rat) and of the adipose tissue (rat) the acids 20:5 and 22:6 are incorporated into the β-position, replacing linoleic and arachidonic acid. The fatty acid composition in the α-position remains essentially unchanged. There is little incorporation ot 20: 1 and no noticeable accumulation of 14:0, 16:1, 18:4 and 22:1 in the phospholipids. It would appear that the marine fatty acids can be accepted by depot fat for the purpose of energy storage, but that they do not serve other structural or metabolic purposes, with the exception of the long-chain polyenoic acids, especially 22:6.

Improved synthesis of choline phospholipids

Choline phospholipids (diether and dialkyl analogs of phosphatidyl choline, cholesteryl phosphocholine) were prepared, in yields of 72–83%, by condensation of the diglyceride analogs (or cholesterol) with phosphorusoxychloride and choline toluene-sulfonate.

Phosphatidylinositol-4,5-bisphosphate may antecede diacylglycerol as activator of protein kinase C.

Phosphatidylserine/calcium-dependent protein kinase C (PKC) from rat brain is activated fifty times more efficiently by phosphatidylinositol-4,5-bisphosphate (PIP2) (Kapp = 0.04 mole% in Triton-lipid micelles) than by diacylglycerol (DG) (Kapp = 2 mole%). Both effector lipids appear to bind to the same site but PIP2 may confer a narrower substrate specificity on the kinase. DG, which together with inositol trisphosphate (IP3) is generated by hydrolysis from PIP2 after cell stimulation, has been considered the natural activator of the kinase but it is likely to be anteceded in this function by PIP2; DG may perhaps retain the function of a back-up activator. The lack of PKC-activation by phosphatidylinositol (PI) or phosphatidylinositol-4-phosphate (PIP) opens the possibility that the Inositide Shuttle, PI reversible PIP reversible PIP2, has a role in controlling the activity of the kinase.

A model of pancreatic lipase and the orientation of enzymes at interfaces.

Abstract A model for the interfacial orientation and the mode of action of lipase is proposed. Lipase is oriented so that its active site is near the oil-water boundary. This orientation is achieved by oil-enzyme bonding at the “hydrophobic head” of the enzyme, a region free of electric charges and relatively resistant to unfolding. The measured KM is a complex constant including the dissociation constant of this oil-enzyme “complex”. The interfacial orientation of lipase is further aided by hydrophilic negative charges on the “back” of the enzyme and by a hydrophilic carbohydrate “tail”. It is suggested that similar hydrophobic heads and hydrophilic tails and asymmetric charge distributions establish the orientation of many enzymes which act at interfaces. Many phospholipases, for instance, appear to be charge-oriented, and the carbohydrate residues of ribonucleases and many other glycoproteins may be hydrophilic tails. Lipase is probably a serine enzyme with a catalytic center similar to that of chymotrypsin, but more hindered, perhaps owing to the presence of a leucine residue, and there is no binding of substrate lipid chains in the “active complex”. The substrate molecule is fixated on the enzyme in a two-dimensional orientation, because its leaving alkoxy group must be received by the serine hydroxyl hydrogen which is directed towards the imidazol ring of the reactive histidine through a hydrogen bond. The high turnover rate of lipolysis, 5 × 105/min, exceptional even for an enzyme, results from the extremely high substrate concentration near the active site, and from an almost complete extrusion of water because of the hydrophobicity of both the active site and the substrate. In addition, both substrate and enzyme, because of their polarity, are already so favorably positioned at the interface that the formation of the “active complex” requires only a proper two-dimensional alignment, perhaps with partial extraction of the substrate molecule from the lipid phase.

Can intact liposomes be absorbed in the gut

Abstract Inulin, EDTA, or maltose were given intragastrically to mice, either free or entrapped in mono-shell liposomes of egg phosphatidylcholine or the non-digestible diether and dialkyl analog of phosphatidylcholine. Results indicate that liposomes may protect a drug from premature digestion but cannot carry it through the intestinal wall.