Date of Award

1980

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Pathology and Laboratory Medicine

College

College of Graduate Studies

First Advisor

Russell A. Vincent, Jr.

Second Advisor

William Stillway

Third Advisor

ElsieTaber

Fourth Advisor

James M. Powers

Abstract

Fibroblasts obtained from a child with Wolman’s disease and maintained in culture demonstrated acid lipase deficiency, reached senescence prematurely and exhibited an abnormal #5 chromosome. When cytogenetic analysis was repeated on frozen and stored cells, the chromosomal defect could no longer be demonstrated, but other anomalies were present. The karyotypes of other Wolman’s disease cell cultures and the parents of the proband were normal. The mother’s fibroblasts had reduced acid lipase activity, consistent with a carrier-state, but the father’s fibroblasts had normal acid lipase activity. It was possible to classify a culture as Wolman’s disease, carrier or normal by the ability of medium from the culture to reduce the lipid stored in a Wolman’s disease cell culture. The culture of the proband stored more lipid than other Wolman’s disease cultures. Two enzymes present in fetal calf serum possessing paraoxonase activity could be differentiated by their sensitivity to heating at 56°C. The esterase inhibitor E600 was slightly more toxic to Wolman’s disease than to normal cells, and was also toxic to E. coli. Normal cells exhibited lipid storage when treated with 10(-4)M E600, but a more pronounced time-dependent accumulation of lipid occurred at 10(-3)M E600. p-Nitrophenol, the major metabolite of E600, had little effect on lipid storage. The amount of lipid stored varied directly with the serum concentration and was unaffected by heat inactivation of the serum. Histochemical stains and biochemical analyses were performed on cultured fibroblasts. Both Wolman’s disease and E600-treated cells showed storage of triglycerides and cholesteryl esters, although one Wolman’s disease culture had a normal level of triglyceride. The E600-treated cells also showed a large increase in phospholipid and small increases in free cholesterol and free fatty acid. Wolman’s disease cells treated with E600 showed increases comparable to the normal E600-treated cells, but no increase of free fatty acid was seen. Wolman’s disease heterozygotes appeared to have normal amounts of lipid. Lipid in Wolman’s disease and E600-treated cells fluoresced in ultraviolet light. Cholesteryl esters of untreated normal cells had more stearic (18:0) than oleic (18:1ω9) acid, whereas cholesteryl esters in Wolman’s disease, E600-treated cells, and fetal calf serum had three times more oleic than stearic acid, suggesting that serum lipid was taken up, but not degraded, in Wolman’s disease and E600-treated cells. Three Wolman’s disease cell cultures exhibited genetic deficiency of acid lipase. Normal fibroblasts had acid lipase activity, but possessed little neutral lipase activity. Acid lipase activity of normal cells was inhibited by E600. Wolman’s disease cells resembled normal cells by scanning electron microscopy. Except in a perinuclear zone which also had not stained with oil red O or neutral red, the cytoplasm of E600-treated cells had numerous bumps which appeared to form ridges along the path of actin stress filaments. Normal cells treated with free fatty acid or cholesterol, and Wolman’s disease cells treated with free fatty acid, exhibited lipid storage in large, peripheral lipid droplets. Lipid was generally stored in smaller granules closer to the nucleus in E600-treated and Wolman’s disease cells. E600-treated cells showed increased numbers of lysosomes by neutral red staining and had increased numbers of dense bodies ultrastructurally. The dense bodies of Wolman’s disease and E600-treated cells sometimes contained lipid clefts which probably consisted of cholesteryl ester. The dense bodies accumulated colloidal gold, suggesting their identity with secondary lysosomes. Although some differences were observed, E600-treated cells resembled Wolman’s disease cells histochemically, biochemically, and ultrastructurally.

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