HPLC method for quantitation of cholesterol and four of its major oxidation products in muscle and liver tissues.

A fast, sensitive, high performance liquid chromatographic method was developed for the quantitation of cholesterol and four of its major oxidation products: 3 beta-hydroxycholest-5-en-7-one (7-ketocholesterol), cholest-5-ene-3 beta, 7 alpha-diol (7 alpha-hydroxycholesterol), cholest-5-ene-3 beta, 7 beta-diol (7 beta-hydroxycholesterol), and cholest-5-ene-3 beta,25-diol (25-hydroxycholesterol). In this procedure 2:1 chloroform:methanol (v/v) extracts of tissue homogenate were combined, dried over anhydrous Na2SO4, filtered, evaporated to dryness under N2 and dissolved with a mobile phase of either 97:3 or 93:7 hexane:isopropanol (v/v). After membrane filtration and without further purification, aliquots were directly injected onto a 10-microns pore size, 30 X 0.39 cm mu-Porasil normal phase column. The separation of cholesterol and its oxidation products was monitored by a UV detector at 206 and 233 nm. This method was successfully applied to pork muscle as well as mouse liver tissues and was able to detect cholesterol oxidation products (COP) in the ppm range. The identity of the COP was confirmed by mass spectroscopy.

Lysophosphatidic acids. II. Interaction of the effects of adenosine diphosphate and lysophosphatidic acids in dog, rabbit, and human platelets.

In order to explore a possible relationship between platelet aggregation induced by lysophosphatidic acid (LPA) and that induced by adenosine diphosphate (ADP), we have studied the influence of palmitoyl-LPA (P-LPA) on platelets from dogs and rabbits and on human platelets made refractory to LPA. Dog platelets did not aggregate with P-LPA alone, but P-LPA enhanced ADP aggregation, and after a small dose of ADP, P-LPA was itself effective in causing aggregation and internal contraction in dog platelets. Rabbit platelets showed no response to P-LPA alone, but, as with dog platelets, P-LPA enhanced ADP aggregation. In addition, when P-LPA was added during or immediately after ADP aggregation, it caused a contraction within the platelets and a small wave of aggregation by itself. P-LPA added to human platelets caused aggregation without the need for ADP. However, when a small dose of P-LPA was added to human platelets and the wave of aggregation was allowed to reverse, these platelets subsequently were unresponsive to P-LPA, although they showed an enhanced response to ADP. The addition of a small dose of ADP to the P-LPA refractory platelets partially reversed the refractory state, and the platelets then showed aggregation with P-LPA. The results demonstrate that ADP and P-LPA have significant interactions in their effects on platelets. These interactions are discussed in terms of a two-component mechanism for the ADP-induced intracellular calcium flux, LPA, or possibly phosphatidic acid, being one component.

Lysophosphatidic acids. Influence on platelet aggregation and intracellular calcium flux.

Decanoyl-, palmitoyl-, and oleoyl-lysophosphatidic acid (LPA) were studied for their effects on platelet aggregation and intracellular calcium flux. Palmitoyl-LPA and oleoyl-LPA both caused a concentration-dependent aggregation of human blood platelets at concentrations of 12--300 microM. Aggregation by adenosine diphosphate (ADP) was enhanced at slightly lower concentrations. First-wave aggregation induced by these LPAs was not blocked by aspirin, indomethacin, or heparin, suggesting similarities to ADP aggregation. However, in washed platelets with a high calcium concentration, no serotonin secretion was observed, even though full aggregation occurred, suggesting that aggregation was not due to released ADP. This concept was supported by studies of platelets deficient in the storage pool of ADP and serotonin, which had a normal first-wave aggregation response to palmitoyl-LPA. Aggregation induced by palmitoyl LPA was inhibited by prostaglandin E1 (PGE1), theophylline, and ethylenediaminotetraacetate (EDTA), though in the presence of EDTA shape change occurred. Aggregation stimulated by palmitoyl-LPA or oleoyl-LPA was characterized by changes in the shape of the platelets with development of pseudopods and centralization of granules closely surrounded by contractile microfilaments and supporting microtubules. The addition of palmitoyl-LPA and oleoyl-LPA, but not decanoyl-LPA, caused the release of calcium from a platelet membrane fraction that contains elements of the intracellular calcium storage system and actively concentrates this cation in the presence of adenosine triphosphate (ATP) and magnesium. It is suggested that LPAs cause aggregation by stimulating the release of calcium intracellularly.