Merle L. Blank

Antihypertensive activity of an alkyl ether analog of phosphatidylcholine.

Summary An alkyl ether analog of phosphatidylcholine was prepared from choline plasmalogen of fresh beef heart by reducing the alk-1-enyl moiety to an alkyl group and by hydrolyzing the sn -2 acyl moiety and replacing it with an acetoyl group. The basis for preparing and testing this compound resulted from experience with antihypertensive lipids derived from the renal medulla and from experience with ether lipid biochemistry. The alkyl lipid analog was found to possess powerful antihypertensive properties in the one-kidney, one-clip hypertensive rats when given either intravenously or orally. This activity induced acute and prolonged depressor effects. These actions were similar to those of a glycerophosphate obtained from fresh renal medulla and designated as APRL (antihypertensive polar renomedullary lipid).

Novel quantitative method for determination of molecular species of phospholipids and diglycerides

A novel method is described for the quantitative analysis of subclasses (alk-1-enylacyl, alkylacyl, and diacyl types) and molecular species within each subclass of glycerophosphatides. Diradylglycerols from phospholipase C hydrolysis of the phospholipids are converted to benzoate derivatives, the benzoates are separated into their respective subclasses by thin-layer chromatography, and quantitated by measuring absorbance at 230 nm. Molecular species within individual subclasses are separated using a combination of argentation thin-layer chromatography and reversed-phase high-performance liquid chromatography with direct, on-line quantitation at 230 nm. We applied the method to the analysis of ethanolamine phosphatides from beef brain and were able to quantitate the three diradylglycerol subclasses (alk-1-enylacyl, alkylacyl, and diacyl types) as well as ca. 29 molecular species within each of these subclasses. This new quantitative approach for the analysis of specific molecular species of glycerolipids should be applicable to studies involving a variety of biologically important lipids, such as phosphatidylcholine, phosphatidylinositol, platelet activating factor, plasmalogens, and neutral type glycerolipids including diacylglycerols.

Inactivation of 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine by a plasma acetylhydrolase: Higher activities in hypertensive rats

We have partially characterized the properties of a specific acetylhydrolase in plasma from spontaneous hypertensive rats. This enzyme inactivates 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine (a lipid involved in platelet aggregating, hypotensive, and allergic responses) by removal of the acetate group. The extent of acetate hydrolysis was linear with both time and protein concentration, and the enzyme had an apparent Km of 2.5 microM and a Vmax of 2.6 nmol/min/mg protein. As with an intracellular acetylhydrolase previously characterized by us, the plasma activity was not affected by addition of phosphatidylcholine, EDTA, or Ca2+. However, in contrast to the acetylhydrolase activity in the rat kidney soluble fraction, the plasma activity was associated with a higher molecular weight protein resolved on a Sepharose 6B column and the plasma acetylhydrolase was not inhibited by treatment with trypsin, pronase, or subtilisin. We also compared the acetylhydrolase activity in plasma of age-matched spontaneous hypertensive rats and their normotensive controls, and found approximately 20% higher levels of activity in plasma from the hypertensive animals (P less than 0.01).

1-alkyl-2-acetyl-sn-glycero-3-phosphocholine (platelet-activating factor) stimulates calcium influx in rabbit platelets

Summary 1-Alkyl-2-acetyl- sn -glycero-3-phosphocholine (platelet-activating factor) induces an influx of Ca 2+ in rabbit platelets that is time-, temperature-, and concentration-dependent. The ionophoretic activity of 1-alkyl-2-acetyl- sn -glycero-3-phosphocholine is much higher (>2-fold at 10 −10 M) than 3-hexadecyl-2-acetyl- sn -glycero-l-phosphocholine, 1-hexadecyl-2-lyso- sn -glycero-3-phosphocholine, 1-acyl-2-acetyl- sn -glycero-3-phosphocholine, or 1-hexadecanoyl-2-lyso- sn -glycero-3-phosphocholine.

Occurrence and nature of O-alkyl and O-alk-1-enyl moieties of glycerol in lipids of morris transplanted hepatomas and normal rat liver

Abstract 1. 1. The lipid class composition and nature of the O-alkyl, O-alk-1-enyl, and acyl chains of major lipid classes were investigated in normal rat liver and 7794A and 7777 Morris hepatomas. Although both hepatomas contained less phospholipid than the normal liver, the relative distribution of the classes within the phospholipid fraction was similar in the three tissues. Almost half the phosphoglycerides consisted of phosphatidyl choline and the remainder was mostly phosphatidyl ethanolamine and phosphatidyl serine-phosphatidyl inositol. The phosphatidyl choline and phosphatidyl ethanolamine fractions of normal rat liver did not contain any significant quantities of ether-containing lipids, but both the 7794A and the 7777 hepatomas contained up to 7% O-alk-1-enyl acyl phosphatidyl ethanolamine. The phosphatidyl choline fraction isolated from the 7777 hepatoma contained more alkyl ethers than the phosphatidyl choline in the 7794A hepatoma or normal rat liver. 2. 2. The neutral lipids in all three tissues were mainly sterol esters, triglycerides, free fatty acids, and cholesterol. The 7777 hepatoma also contained significant quantities of glyceryl ether diesters, and accumulated sterol esters and cholesterol at the expense of triglycerides and phosphoglycerides compared to normal rat liver or the 7794A hepatoma. The 7794A hepatoma did not contain any glyceryl ether diesters, but compared to normal rat liver this tumor also contained increased quantities of sterol esters, free fatty acids, and cholesterol. 3. 3. In the 7777 hepatoma, the O-alkyl moieties in the glyceryl ether diesters and phosphatidyl choline and the O-alk-1-enyl moieties in the phosphatidyl ethanolamine were similar and consisted mainly of 16:0, 18:0, and 18:1 hydrocarbon chains. The proportion of 18:0 to 18:1 side chain was higher in the alk-1-enyl ethers of the phosphatidyl ethanolamines than in those of the alkyl ethers of the phosphatidyl chorines. 4. 4. In all tissues studied, the fatty acid side chains differed completely from the alkyl and alk-1-enyl ether-linked side chains. Both tumors contained about twice as much 18:1 fatty acids in the sterol esters as did normal rat liver, primarily at the expense of 16:0 fatty acids. In contrast, the phosphatidyl ethanolamine and phosphatidyl choline fractions from the hepatomas contained higher quantities of linoleic acid and lower quantities of arachidonic acid than these fractions from normal liver. 5. 5. Our data demonstrate that glyceryl ether diesters and alkyl-acyl cholinecontaining lipids tend to accumulate in hepatoma 7777 which is a relatively fastgrowing tumor. In addition, the relative proportion of 18:2 and 20:4 fatty acids in the phosphoglycerides indicates that essential fatty acids are poorly utilized by hepatomas.

In vivo metabolism of a new class of biologically active phospholipids: 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine, a platelet activating-hypotensive phospholipid

Abstract This report describes the in vivo metabolism of a new class of naturally occurring biologically active phospholipids (1-alkyl-2-acetyl- sn -glycero-3-phosphocholines) that can cause hypotension and platelet aggregation. After intravenous injection in male rats, the acetylated ether phospholipid (1-[1′,2′-3H]alkyl) is rapidly cleared ( T 1 2 ⋍30 s ) from blood and its metabolites are found in a variety of tissues. The tissues containing the highest levels of radioactivity are lung, liver, spleen, and kidney. Chromatographic results showed that a considerable portion of the active lipid is not readily catabolized in some of the major tissues examined; however, inactive metabolites were also found, mainly 1-alkyl-2-lyso- sn -glycero-3-phosphocholine and 1-alkyl-2-acyl- sn -glycero-3-phosphocholine; the latter has a long chain fatty acid at the sn -2 position instead of the acetate. The findings are consistent with our earlier data that show these same tissues have the most active enzyme systems for metabolizing 1-alkyl-2-acetyl- sn -glycero-3-phosphocholine.

Relationships of chain lengths and double bond locations in O-alkyl, O-alk-1-enyl, acyl, and fatty alcohol moieties in preputial glands of mice☆

Abstract Wax esters, glyceryl ether diesters, triglycerides, and sterol esters are the major lipid classes present in normal preputial glands of mice. Small amounts of phosphatidyl choline (PC) and phosphatidyl ethanolamine (PE) were also found. Three-fourths of the PE fraction was comprised of alk-1-enyl ethers, whereas most of the PC fraction (⋍90%) was present in the diacyl form. The ether linkage was found to be at the 1-position of glycerol. The chain lengths of the alkyl and alk-1-enyl hydrocarbon moieties of glyceryl ether diesters, phosphatidyl choline, and phosphatidyl ethanolamine were similar to one another and to the alcohol moieties of the waxes. Principal hydrocarbon chains in ether linkage with glycerol were 14:0, 14:1, 16:0, and 16:1. The location of double bonds in the acyl, alkyl, and alk-1-enyl hydrocarbon moieties of the various lipid classes was determined by gas-liquid chromatography of the aldehyde fragments produced by reductive-ozonolysis. The 18:1 acyl moieties from the triglycerides and waxes consisted primarily of the Δ-9 isomer but small amounts of the Δ-11 isomer were also found. The 16:1 acyl moieties of triglycerides were also mainly the Δ-9 isomer, whereas in waxes the 16:1 acyl moieties were equally divided between Δ-6 and Δ-9 isomers. Location of double bonds in the 16:1 and 14:1 alcohol and acyl moieties of waxes and the alkyl moieties of glyceryl ether diesters was similar. The 16:1 fraction was comprised mainly of the Δ-6 isomer and smaller quantities of the Δ-7 and Δ-9 isomers. The Δ-5 and Δ-6 isomers accounted for more than 90% of the 14:1 chains in the fatty alcohols of waxes and in the alkyl moieties of glyceryl ether diesters; the acyl moieties of waxes contained approximately 70% of these isomers. The data indicate that in preputial glands of mice the intermediary pathways or enzymatic sites responsible for the biosynthesis of fatty alcohol, and to some extent fatty acids, in the waxes must be identical to those pathways or sites responsible for the biosynthesis of the alkyl chains of glyceryl ether diesters. The similarities of alkyl and alk-1-enyl ether-linked hydrocarbon moieties suggest that these two classes of lipids can be interconverted enzymatically.

The retention of arachidonic acid in ethanolamine plasmalogens of rat testes during essential fatty acid deficiency

To assess the possible role of plasmalogens in essential fatty acid deficiency, the metabolism of [6,8,9,11,12,14,15-3H7]arachidonic acid was examined in testicular tissue of rats maintained on essential fatty acid-deficient and control diets. The findings demonstrated that this essential fatty acid is metabolized more rapidly in the diacyl phospholipids and is retained to a greater extent in the plasmalogens of testicular lipids of the animals fed the essential fatty acid-deficient diet. It would appear that the build-up of labeled arachidonic acid in the plasmalogens from rats fed the fat-free diet is explained by an acyl exchange reaction, since the quantity of ethanolamine plasmalogens in the testes remained constant.

Long chain fatty alcohols in normal and neoplastic tissues.

Small quantities of long chain fatty alcohols (esterified or free or both) were found in four normal tissues (about 0.01% of total neutral lipids) and three neoplasms (about 0.3% of total neutral lipids). The major chain lengths (16∶0, 18∶0 and 18∶1) of the fatty alcohols in both normal and neoplastic cells qualitatively resemble the O-alkyl chain lengths of glyceryl ethers. Our data showing that long chain fatty alcohols occur in vivo support the biological significance of the metabolic pathway that uses fatty alcohols as a substrate for the alkyl chain in glyceryl ether biosynthesis.

Substrate specificity in the biocleavage of the O-alkyl bond: 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine (a hypotensive and platelet-activating lipid) and its metabolites☆

Abstract 1-Alkyl-2-acetyl- sn -glycero-3-phosphocholine possesses both hypotensive and platelet-activating properties. Our data show that after removal of the acetyl group at the sn -2 position by an acetyl hydrolase, the product, 1-alkyl-2-lyso- sn -glycero-3-phosphocholine, can be cleaved by a microsomal tetrahydropteridine-dependent alkyl monooxygenase in the liver and spleen of rats. Results obtained for the tetrahydropteridine requirement, tissue distribution, responses to thermal inactivation and catalase, and substrate inhibition of the enzyme indicate that the ether linkage of 1-alkyl-2-lyso- sn -glycero-3-phosphocholine is hydrolyzed by a monooxygenase that appears to be identical to the one responsible for cleavage of the O -alkyl moiety of alkylglycerols.