Marion Barclay

Separation of lipid classes by thin-layer chromatography.

Abstract 1. 1. A one-dimensional two-step thin-layer Chromatographie system for separation of classes of neutral lipids on silica gel without CaSO4 binder is described. In the first step of development of chromatograms, the solvent isopropyl ether-acetic acid (96:4, v/v) is used and in the second step, light petroleum-ethyl ether-acetic acid (90:10 : 1, v/v). The main lipid classes separated are: hydrocarbons, cholesterol esters, methyl esters of fatty acids, triglycerides, fatty acids, 1,3-diglycerides, 1,2-diglycerides, cholesterol, monoglycerides and phospholipids together with cerebrosides, which remain at the origin. 2. 2. Application of this system for the separation of neutral lipid classes in rat liver and in human serum is demonstrated .

Quantitative analysis of simple lipid classes by thin-layer chromatography.

Abstract 1. 1. A quantitative thin-layer Chromatographie procedure for the analysis of simple lipid classes (‘neutral lipids’) is described. They were separated by two-step development on silica gel thin-layer chromatoplates. In the first step the solvent diisopropyl ether-acetic acid (96:4, v/v) was used and in the second step, petroleum ether-diethyl ether-acetic acid (90:10:1, v/v/v). 2. 2. After separation, lipid bands were detected by Rhodamine 6G spray, the silica gel with adhered lipids was scraped off and the lipids were eluted. Triglycerides, diglycerides, free fatty acids and hydrocarbons were eluted with diethyl ether, whereas monoglycerides, free cholesterol and cholesteryl esters were eluted by chloroform-methanol (4:1, v/v). The eluates were filtered through sintered glass (‘fine’ porosity) funnels. 3. 3. The eluted lipids were analyzed in the following ways: quantity of triglycerides, diglycerides, monoglycerides and hydrocarbons were determined by quantitative infrared spectrophotometry; free fatty acids by titration; and cholesteryl esters and cholesterol by a standard chemical procedure. 4. 4. The average recovery of individual lipid classes was in the range 98.9–102.0%. 5. 5. Applicability of the procedure for quantitative analyses of tissue lipids is demonstrated on lipid extracted from rat livers.

Separation of acidic phospholipids by one-dimensional thin-layer chromatography

1. 1. Three one-dimensional thin-layer Chromatographie systems for separation of acidic phospholipids such as cardiolipin, phosphatidic acid and phosphatidyl-glycerol, are described. System I: adsorbent is silica gel without CaSO4 binder. Two step developing system is used. The first solvent is acetone-light petroleum (1:3, by vol.); the second solvent is a mixture of chloroform-methanol-acetic acid-water (80:13:8:0.3, by vol.). The separated compounds from the top of the chromatogram are: monoglyceride, cardiolipin, phosphatidic acid, ceramidemonohexosides (cerebrosides), phosphatidylglycerol and phosphatidylethanolamine. System II: silica gel thin-layer plates are prepared with 0.1 M Na2CO3. The first solvent is pyridine-light petroleum (3:1, by vol.); the second solvent is chloroform-methanol-pyridine-2 M NH4OH (35:12:65:1, by vol.). The following compounds are separated by the two-step developing system (from the top of chromatogram): ceramidemonohexosides, sulfatides and ceramidedihexosides (overlapping), phosphatidic acid, phosphatidylglycerol and cardiolipin. System III: one-step development modification of System II. This system is suitable only for quantitative determination of phospholipids if the presence of glycolipids is not deemed detrimental. 2. 2. Application of the Systems I and II for separation of acidic phospholipids along with some glycolipids is demonstrated on total lipid extracts from animal tissues. 3. 3. The systems described for the separation of acidic phospholipids are complementary to the previously reported system for separation of phospholipids.

SERUM LIPOPROTEINS AND HUMAN NEOPLASTIC DISEASE.

Abstract Serum lipoproteins have been measured m 20 normal subjects. Young women from a convent were studied weekly for a 12-week period, and women from the Institute staff in single experiments. The subjects were divided on the basis of family history for cancer. The same studies were done on 9 young women with advanced cancer of the breast. Lipoproteins with densities less than 1.006 g/ml, e.g., Sf20–100 and Sf0–20, and Sf10–20 class floating in solution density 1.0635 g/ml, are elevated in serum from subjects with advanced cancer of the breast. Of these, Sf0–20 (1.006, unaltered serum) was significantly higher in subjects with cancer. These elevations were not present in the normal subjects who had a negative family history for cancer. The high-density lipoprotein Sf0–4 (1.125, NaCl) which is the major component floating at solution density 1.125 g/ml, was very low in subjects with cancer. It was low consistently in only the normal subjects with cancer in their immediate blood relatives. These differences were highly significant. Although this high-density lipoprotein fluctuates during the menstrual cycle, and each serial subject had a stable range of values, it is possible that a pronounced family history for cancer may influence the serum levels of Sf0–4 or HDL2.

Disclosure and characterization of new high-density lipoproteins in human serum

Abstract Sequential removal, with solutions of increasing density, of the lower-density lipoproteins has made possible the unmasking of previously undescribed high-density (D = 1.0635−1.125 g/ml) lipoproteins. These new high-density lipoproteins were revealed as one or two discrete boundaries in the Spinco Model E ultracentrifuge. They are in the density range between HDL 1 and HDL 2 , and have been classified as S f 12–20 (1.125, NaCl) and S f 4–12 (1.125, NaCl), while the heavier boundary is termed S f 0–4 (1.125, NaCl). This heavier boundary has usually been named HDL 2 .

Properties of lipoprotein lipase extracted from livers of normal rats and livers and tumors of rats bearing Walker carcinosarcoma 256

Abstract Extracts of acetone powders made from normal rat livers, and livers and tumor tissues from male rats bearing the Walker carcinosarcoma 256 have been made. No heparin was given to the animals at any time. The properties of the lipases studied indicate that they are lipoprotein lipases, even though they will hydrolyze well a 1.5% coconut oil emulsion. There is some indication that these enzymes preferentially hydrolyze the lower-density lipoproteins i.e., S f 100–400 and then proceed to the S f 20–100 and 0–20 classes, in in vitro experiments. The enzyme from the liver tissue of rats bearing the Walker carcinoma 256 is especially active in reducing the quantity of all the low-density lipoproteins measured in these experiments. The enzyme from tumor tissue is quite dependent upon heparin. The enzymes from all the tissues examined have characteristics which are like the lipoprotein lipase extracted from normal rat heart, and it may be suggested that the tissue extracts tested in these experiments do have lipoprotein lipase, or clearing factor, activities.

Low-density lipoproteins and lipoprotein lipase activity in tissues from rats bearing Walker carcinosarcoma 256

Abstract It has been observed that the low-density lipoproteins, especially the triglyceride-carrying S f 20–400, are present in large quantities in sera of rats with the Walker carcinosarcoma 256. At the same time, although the lipoprotein lipase activity is maintained at high levels in the sera to the 21st day after implantation, the hyperlipemia remains. Large quantities of similar lower density lipoproteins were observed also in tumor tissue between the 14th and 21st day, suggestive of transport to and penetration of triglyceride hydrolysis products into the tumor tissue. By the 28th day, the S f 20–400 lipoproteins had accumulated greatly in the blood along with diminished lipoprotein lipase activity, while virtually no lipoproteins nor enzyme activity could be measured in the tumor tissue.

ADDITIONAL EVIDENCE FOR THE EXISTENCE OF "NEW" HIGH-DENSITY LIPOPROTEINS IN HUMAN SERUM.

Abstract Two recently described high-density lipoproteins are characterized further. The hydrated densities for these components floating in solution density 1.125 g/ml are: S f 20−12, 1.071 g/ml; S f 12−4, 1.079 g/ml. When they occur in human sera, S f 12−4 especially, is quite consistently present. They have not been observed in rat sera. Experimental measurements of densities during separation procedures are reported.