Robert E. Pitas

Synthesis of Some Acylglycerols and Phosphoglycerides

Publisher Summary This chapter presents explicit directions for the synthesis of some acylglycerols and phosphoglycerides. Gas-liquid chromatography is the method of choice for separation and identification of fatty acids. Thin layer chromatography (TLC) is unequaled for the quick separation and tentative identification of lipids. All acylglycerols are checked for polar contaminants with TLC systems. Appropriate standards must be used to locate the separated materials. The well-known specificity of pancreatic lipase for primary fatty acid esters has been utilized to determine the positional structure of synthetic triacylglycerols. This chapter also discusses isopropylidene glycerols. These compounds, which are the immediate precursors of 1-monoacylglycerols, are prepared by condensing acetone with glycerol, using p-toluenesulfonic acid as a catalyst. The racemic compound rarely needs to be isolated after being made as the synthesis of the 1-monoacylglycerol is done in the same flask. However, the enantiomeric isopropylidene glycerols, obtained in several steps from D- or L-mannitol, are used in the synthesis of several enantiomeric lipids.

Digestion of butyrate glycerides by pancreatic lipase.

The racemic triglycerides, glyceryl-1-palmitate-2,3-dibutyrate (PBB), glyceryl-1-butyrate-2,3-dipalmitate (PPB), glyceryl-2-butyrate-1,3-dipalmitate (PBP), and the diglyceride, racemic glyceryl-1-palmitate-3-butyrate (P-B) were synthesized and digested with pancreatic lipase. Each triglyceride was mixed with equimolar amounts of triolein (OOO) prior to incubation.The following order of digestion rates was observed: PBB>PPB>PBP>P-B. There was no evidence for short-chain fatty acid specificity; however the triglycerides containing butyric acid were hydrolyzed more rapidly than OOO. Based upon the fatty acid composition of partial glycerides, digestion of butyrate glycerides was not a simple phenomenon. For example, in the digestion of PBB, butyric acid accumulated faster than palmitic acid in the diglycerides, and monobutyrin was found to accumulate when the diglyceride, P-B, was digested. As evidenced by the fatty acid composition of the monoglycerides, positional specificity of pancreatic lipase was always maintained.

Effects of diet and type IIa hyperlipoproteinemia upon structure of triacylglycerols and phosphatidyl cholines from human plasma lipoproteins.

Four normal and two individuals with Type IIa hyperlipoproteinemia were placed on the National Heart and Lung Institute Type IIa diet (low cholesterol, <300 mg/day, high polyunsaturated, low saturated fat diet) for 1 week and on a normal diet the following week. Plasma samples were obtained and the triacylglycerols, phospholipids, and cholesterol contents of plasma and of very low density lipoproteins, low density lipoproteins, and high density lipoproteins determined. Triacylglycerol fatty acid composition was determined and stereospecific analyses of triaclglycerols and phosphatidyl cholines performed. Structural determinations were limited to one normal and one Type IIa individual. In normal and Type IIa individuals, chylomicrons contained twice the amount of 18∶0 as did the very low density lipoproteins, low density lipoproteins, or high density lipoproteins. The structure of the triacylglycerols from the very low density lipoproteins and low density lipoproteins was asymmetric with at least 50M% 16∶0 in thesn-1 position and mostly 18∶1 in positionssn-2 and 3. There was a marked difference in the distribution of 18∶2 in low density lipoproteins of the normal and Type IIa individuals. The control contained equal amounts of 18∶2 in thesn-1 andsn-3 positions, whereas IIa low density lipoprotein was asymmetric with 26% of the 18∶2 in positionsn-1 and 3% in thesn-3 position. Very low density lipoprotein was asymmetric with regard to 18∶2 in control and IIa samples with an average of 5% of the 18∶2 in positionsn-1 and 40% in positionsn-3. The phosphatidyl cholines contained predominantly 16∶0 and 18∶0 in positionsn-1, whereas the acids in positionsn-2 were unsaturated with very little difference between lipoprotein classes. Neither the short dietary periods nor source of plasma affected the structure of the phosphatidyl cholines.

Pancreatic lipolysis of enantiomeric triglycerides

Pancreatic lipase hydrolyzed fatty acids in equimolar quantities from thesn-1- and 3-positions of three synthetic enantiomeric triglycerides, two of which could make a racemic pair. The monoglycerides from digestions of five enantiomeric triglycerides were at least 99% representative of the 2-position. The data confirm that pancreatic lipase did not distinguish between thesn-1- and 3-positions and that with these triglycerides pancreatic lipolysis can be used to help establish structure.

Convenient method for concentration of esters prior to gas liquid chromatographic analysis

Sir: In the preparation of lipid samples for gas liquid chromatographic analysis, a recurring problem is the extraction and concentration of esters from the esterification mixture prior to injection. Often the amount of reagent is limited so as to permit injection of the reaction mixture directly; however, when working with unknown quantities of lipid extracts, this can lead to samples too dilute to analyze properly or with overload of the reagents with lipid. For years we have used a convenient procedure which is applicable to a wide variety of esterification methods and sample concentrations. When water soluble reagents, for example 2N sodium methoxide or HCI in methanol (S.W. Christopherson, and R.L. Glass, J. Dairy Sci. 52:1289 [1969]) , are used, the amount of reagent does not have to be limited. The sample is routinely evaporated under reduced pressure prior to the addition of reagents. When the reaction is complete, the mixture is transferred to a Babcock milk fat test bottle with a Pasteur pipette. The reaction vessel then is washed several times with small portions of hexane which are also transferred to the Babcock bottle. The bottle is then half filled with water, shaken vigorously, filled with water well into the narrow neck, and then centrifuged for I0 rain. The esters are now either injected immediately or the hexane layer is removed with a Pasteur pipette for storage or for evaporation under a stream of nitrogen prior to injection. Babcock test bottles are available at low cost from most laboratory supply firms.

Effect of chronic ingestion of DDT on physiological and biochemical aspects of essential fatty acid deficiency

Male weanling rats were fed semipurified diets with and without essential fatty acid (EFA) and DDT (150 ppm) for 14 weeks to determine the effects of the pesticide on physiological and biochemical aspects of EFA deficiency (EFAD). DDT did not affect EFAD-induced reduction in growth rate or final body weight, nor did the pesticide affect EFAD-induced changes in feed efficiency or skin dermatitis. The pesticide did increase liver/body mass ratios, but did not interact with EFAD, which also increased this ratio. The pesticide produced complex changes in total fatty acid composition of liver and tail skin: liver levels of 18∶0, 18∶2 and 20∶3ω9 were increased, whereas levels of 12∶0, 14∶0 and 16∶0 were decreased. In both tissues, DDT interacted with EFA to increase 18∶2 levels. DDT did not change the total fatty acid 20∶3ω9/20∶4ω6 ratio in either tissue. In this study, although DDT did not exacerbate the physiological aspects of EFAD, DDT-induced changes in fatty acid composition of liver and tail skin indicated that 150 ppm DDT in the diets did alter lipid metabolism of the rats in an unexplained manner.

Transport of diacylalkylglycerols in chylomicrons and very low density lipoproteins of rat intestinal lymph following intragastric administration of 1,3-dioctadecenoyl-2-hexadecylglycerol.

The triacylglycerol (TG) analog 1,3-dioctadecenoyl-2-hexadecyl glycerol was used in the study of the transport of dietary lipids by lipoprotein fractions of rat intestinal lymph. 1,3-Diacyl-2-alkyl glycerols (DAG) are hydrolyzed by pancreatic lipase to form 2-alkyl glycerols and free fatty acids. These hydrolysis products are then absorbed, and DAG are resynthesized within the intestinal mucosa. Intestinal lymph of rats was collected following intragastric administration of 1,3-dioctadecenoyl-2-hexadecyl glycerol. The DAG to TG ratios in very low density lipoprotein (VLDL) and chylomicron fractions were determined as a measure of the incorporation of lipid of dietary origin. The ratio of DAG to TG in the VLDL-2 (Sf 12–100) fraction ranged from0.06 to 0.56 indicating a significant amount of DAG transported relative to TG. The glyceryl ether to TG ratio increased with mean lipoprotein volume from the VLDL-2 fraction to the chylomicron (Sf>400) fraction. The correlation between glyceryl ether to TG ratio and average volume and between the amount of DAG per ml of original lymph and average volume within the chylomicron fraction was 0.99. Thus, the amount of dietary fat transported was correlated with the size of the chylomicrons produced. The glyceryl ether to TG ratio was positively correlated with the average volume of the lipoprotein fractions isolated (chylomicrons, chylomicron rich (Sf>100), VLDL-1 (Sf 100–400) and VLDL-2) (r=0.87). These results suggest that the size of the lipoproteins produced by the intestine is determined by the amount of fat available for transport and that particles of larger diameter are formed by the addition of lipid of dietary origin to existing VLDL.

Effect of diet on fatty acids in the lipoprotein cholesteryl esters of type IIa and normal individuals.

Four normal and two individuals with type IIa hyperlipoproteinemia were placed on the National Heart and Lung Institute Type IIa Diet (<300 mg of cholesterol per day, high polyunsaturated, low saturated fat diet) for 1 week and on a normal diet the following week. Plasma samples were obtained and the cholesterol contents of plasma and of very low density, low density and high density lipoproteins determined. The cholesteryl esters in one type IIa and two normal individuals were identified.The cholesteryl esters in type IIa very low density lipoproteins from blood drawn 45 min after the last meal in each dietary period, contained less 18∶2 than from the normal. After the first dietary period, the very low density lipoprotein cholesteryl ester 18∶2 content for the type IIa was 37.2M% and for the normals, 54.7M%. After the second dietary period, the corresponding values were 49.7M% and 56.7M%. Fasting samples had lower 18∶2 contents in the low density lipoproteins from the Type IIa subject following both dietary periods and in the high density lipoproteins following the Type II diet.

Glycerol ester hydrolase activity in the pulp of unerupted calves’ teeth

The pulp from unerupted calves’ teeth was found to contain low levels of glycerol ester hydrolase activity. Solutions in Tris buffer cleared tributyrylglycerol agar and hydrolyzed emulsified olive oil. The average quantity of free fatty acids released in five assays with olive oil was: 2.8, S.D.±1.4. Specific activities were: 0.25, S.D.±0.13, mkatals/kg protein.