published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

The reactions of aldehydes with proteins are of nutritional and physiological significance. The nutritive value of food materials can be impaired by a reaction necessitating additional dietary protein, and enzymes have been shown in vitro to be inhibited by reactions with aldehydes. Aldehydes present may arise from the autoxidation of lipid materials. A diversity of carbonyl products can occur. Several autoxidizing food lipid systems have been analyzed by other investigators, and the carbonyls present isolated and identified. In most cases the short chain aldehydes predominated. Reactions between aldehydes and proteins have been studied mainly with formaldehyde. Reactions between the aldehyde and many protein functional groups have been suggested including amino, amide, guanidyl, sulfhydryl and imidazole. The amino groups of the protein have received the most attention. The reactions proposed have included: a. methylolamine formation followed by condensation forming a cross link, or b. Schiff's base formation. The reactions have been found to be pH specific with the pH of maximum reaction depending upon the particular protein used. Possible reactions of dialdehydes with proteins have been little explored. Of interest are the possible reactions of malonaldehyde, one of the dialdehydes occurring in autoxidizing lipid systems. Herein, the action of malonaldehyde has been compared to that of formaldehyde, considered a reactive aldehyde, and to propionaldehyde, its monoaldehyde counterpart. The enzyme lipase was used as both the protein and the system of analysis for following the course of the reaction. Lipase activity was determined by potentiometrically titrating the fatty acids liberated from an olive oil emulsion. The commiercial bovine pancreatic lipase preparation used was found to have an activity optimum near pH 9.0. The relationship of the amount of enzyme present to the amount of fatty acids liberated was determined and found to be linear. Maximum stability of the control enzyme appeared between pH 6.0 and 6.5, while maximum lability in the presence of MA-Na (the sodium salt of the enolic form of malonaldehyde) was also near pH 6.0. Formaldehyde and propionaldehyde, however, both exhibited a non-pH-specific inhibition of lipase. Of the three aldehydes reacted with lipase at pH 6.0 and analyzed at pH 9.0, MA-Na was the most inhibitory. The reaction appeared to be two stage, the first being rapid and a function of the MA-Na concentration. The second stage was slower, and the rate was related linearly to the MA-Na concentration. Methanol-free formaldehyde did not inhibit lipase as effectively as MA-Na. Low concentrations (0.01 M) of formaldehyde had no apparent effect on the enzyme while concentrations above 0.05 M produced only slight changes in the degree of inhibition. Propionaldehyde showed only slight inhibition of lipase activity. Its reaction was two stage, with the second stage paralleling the inhibition seen in the control due to factors other than the aldehyde present. Reactions such as these, especially for the more reactive aldehydes such as malonaldehyde, may occur in vitro and in vivo with other proteins and could be of important nutritional or physiological significance. / Graduation date: 1964

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The suckling rat obtains about 70% of its energy from the catabolism of lipid, compared to only about 25% in the adult rat. In addition, lipids are in great demand for membrane synthesis in the rapidly growing tissues. Thus the suckling rat represents an important system in which to study lipid metabolism. In this dissertation research the following experiments were performed on rats during the suckling period: (1) Determination of the concentration and fatty acid composition of triacylglycerols, cholesteryl esters, phospholipids, and free fatty acids in dam's milk, and in suckling rat lymph, portal plasma, vena cava plasma and liver; (2) Determination of positional distribution of fatty acids among the three different positions in triacylglycerols of milk, lymph, plasma and liver; (3) Isolation of plasma and mesenteric lymph lipoproteins by density gradient centrifugation, and identification of the individual classes of lipoproteins by measurement of their density and by polyacrylamide gel electrophoresis; (4) Determination of the concentration of each lipoprotein by refractometry and determination of the concentration and fatty acid composition of different lipid components in each lipoprotein fraction; (5) Studies of physical properties of the lipoproteins using analytical ultracentrifugation; (6) Determination of apoprotein composition and concentration in each lipoprotein fraction. The results show that the suckling rat consumes a diet which is rich in medium chain fatty acids (35% of total milk fatty acids). Mesenteric lymph triacylglycerols carry a significant amount of these medium chain fatty acids, unlike the adult in which they would be transported as free fatty acids, unlike the adult in which they would be transported as free fatty acids via the portal vein. Medium chain fatty acids are enriched at the sn-3 position in all triacylglycerols. Lymph contains chylomicrons, very low density lipoproteins, and high density lipoproteins, but no low density lipoproteins. Plasma contains all classes of lipoproteins but at concentrations higher than found in the adult, especially low density lipoproteins which are present at levels six times that found in the adult. The plasma very low density lipoproteins have a lipid composition which resembles that of chylomicron remnants. Apoprotein, lipid composition, and electrophoresis patterns show that the plasma lipoproteins in the suckling rat are heterogeneous.

In this study, synthetic routes to certain short acyl chain phospholipids were developed. Their inhibitory or substrate properties for phospholipase A₂ were then examined. An improved method for the acylation of glycerophosphocholine is described using a mixed fatty acid - trifluoroacetic acid anhydride. This partial synthetic route is particularly suitable for obtaining short acyl chain phosphatidylcholines. A new pathway for constructing the unnatural sn-1-phosphatidylcholines is also described, starting from L-arabinose. This is converted first to a key intermediate, 2,3-O-isopropylidene-sn-glycerol, which is then phosphorylated and transformed into sn-glycero-1-phosphocholine. This can be acylated as above to give sn-1-phosphatidylcholines. Routes to the short chain phosphatidylethanolamines were investigated and discussed. A procedure, using phospholipase D, was used to convert L-diC₆ PC into L-diC₆ PE in a transphosphatidylation reaction. Failed attempts to obtain shorter chain homologs by this and other methods are also detailed. The kinetics of inhibition of the phospholipase A₂ hydrolysis of L-diC₆ PC by the D-isomer are also reported for the monomeric concentration range. It was found that the D- enantiomer did not behave as a pure competitive inhibitor, and that an enzyme-substrate-inhibitor complex can exist. The implications of these results with regard to PLA₂ hydrolysis of mixed micelles is discussed. The PLA₂ substrate properties of both the anionic and zwitterionic diC₆ PE's were also studied. It was established that the anionic diC₆ PE is either a very poor substrate relative to the zwitterionic diC₆ PE, or acts as a competitive inhibitor towards its hydrolysis. Similarly, the rates of base-catalyzed acyl ester hydrolysis are about 18 times greater for the zwitterionic than for the anionic diC₆ PE. The importance of a protonated amino group in both these hydrolyses studies is noted. In addition, certain physical properties of diC₆ PE, such as its critical micelle concentration and carbon-13 NMR spectrum, are also given.

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