Isotope kinetics of human skin cholesterol secretion.

Specific radioactivity (SA) time curves of plasma and skin surface cholesterol collected at several skin areas were recorded in 10 patients on formula diets after single intravenous injections of radioactive cholesterol. Earliest detectable radioactivity on skin surface was found in 4-6 days; depending on the skin site, SA's peaked in 13-75 days. SA's of free cholesterol were almost always higher than those of esterified cholesterol. The general forms of the SA time curves were in keeping with the idea that plasma cholesterol is carried to the skin surface in association with the epidermal and sebaceous cells, whereby (a) cholesterol synthesized de novo is mixed with derived from plasma and (b) appearances of plasma cholesterol on the skin surface is delayed by a time factor that corresponds to the movement of epidermal and sebaceous cells from the basal layer to the skin surface. The shorter mean transit times of plasma cholesterol on skin areas rich in sebaceous glands (22-24 days on the head) than on those poor in these glands (38 days on forearms and 72 days on feet) suggest that cholesterol passes faster through the sebaceous glands than through the epidermis, and faster through thin than thick epidermis. The fraction of skin surface cholesterol (f) that is derived from plasma cholesterol was estimated by three independent methods, and comparable results were obtained. Values of f were lower on skin areas rich in sebaceous glands (0.29-0.46 on forehead) than on areas poor in these glands (0.41-0.70 for forearms; 0.60 on feet) and lower for esterified (0.27-0.33) than for free (0.39-0.48) cholesterol. These data suggest that higher proportions of sebaceous gland and of esterified cholesterol, respectively, are synthesized de novo than epidermal and of free cholesterol. In two patients it was possible to calculate that f of total skin surface cholesterol was 0.49 and 0.37, respectively, and that the maximum amount of plasma cholesterol lost through the skin was 29 and 22 mg/day, respectively. Knowing the total daily excretion of total neutral and acidic steroids in feces in these patients, and assuming a total daily urinary steroid excretion 50 mg, we estimated that no more than 3.2% of total steroid excretion occurred via the skin.

Human adipocyte cholesterol. Concentration, localization, synthesis, and turnover.

By analysis of 124 specimens in 16 different patients, isolated human adipocyte cholesterol concentration is highly correlated with fat cell size but not with plasma cholesterol concentration. Less than 6 percent of total cholesterol is esterified; after subcellular fractionation, 88 percent of the cholesterol is recovered in the triglyceride-rich supernatant oil. This latter finding supports the observation that fat cell cholesterol is determined by triglyceride content, and hence by fat cell size. After intravenous administrtion of radioactive cholesterol, the sum of a three-exponential equation was fit simultaneously to both the plasma and adipocyte specific activity time curves in six patients. In five of the six, a slowly turning over pool (pool 3) closely fit the adipocyte data. Two model structures, mammillary and catenary, were fitted to the data. There was no synthesis in pool 3 using a mammillary model but a mean 5.3 percent of the total body production rate was found in compartment 3 if a catenary model was assumed. Although a catenary model is biologically unlikely, it could not be excluded. Obesity is associated with an increased cholesterol synthetic rate equal to 20 mg/day for each kilogram of body fat. To test (by an independent method) if this synthesis might be occurring in adipose tissue, human fat cells were obtained under a wide variety of dietary conditions and incubated in vitro with radioactive glucose or acetate. Incorportation of these precursors into sterol could account for no more than 1 mg cholesterol synthesis/kg fat per day. These in vitro data taken together with the in vivo mammillary compartmental analysis data are compatible with the possiblity that the excess cholesterol synthesis of obesity occurs in pool 1, most likely from hepatic or intestinal sites.

Cholesterol metabolism in human obesity.

An experiment was undertaken to test whether in severe obesity cholesterol production rates obtained by isotope kinetic analysis (two-pool compartmental analysis) are comparable to those measured by chemical sterol balance techniques. Eight severely obese but normocholesterolemic patients were studied by the balance method, and five of these eight were studied by compartmental analysis. Cholesterol turnover was 10% higher by compartmental analysis. In the entire group of eight patients cholesterol turnover was greater than twice that found previously in nonobese patients studied under similar conditions with bile acids and neutral sterols both participating in the increase. This increment was directly related to excess body fat and to adipose cellularity, with correlation co-efficients of 0.66 and 0.72, respectively. The amount of cholesterol in the slowly turning over pool B was related to degree of adiposity, but that in plasma and in pool A did not differ from values in nonobese patients.

Abnormal lipoprotein lipase in familial exogenous hypertriglyceridemia.

A 5-yr old male proband and his sister have had hypertriglyceridemia and hepatosplenomegaly since birth. When studied on a metabolic ward, they demonstrated rapid decreases in serum triglycerides on 3 g fat/day diets. Oral glucose tolerance tests were normal. Postheparin lipolytic activity (PHLA) against chylomicrons was virtually absent in both children whereas the mother and a normolipemic sister had levels approximately 50% normal. However, all four had a normal PHLA against commercial triglyceride emulsion (Intralipid). Two unrelated children from different kindreds of typical type 1 hyperlipoproteinemia and two patients with acquired type V hyperlipoproteinemia had deficient PHLA against both substrates. No inhibitors of PHLA could be demonstrated in the proband's plasma, and his own PHLA could not be enhanced by either normal concentrated plasma or pooled d > 1.063 lipoprotein fraction. The proband's postheparin plasma required almost 20 times the normal chylomicron-triglyceride concentration to reach one-half maximal lipase velocity. Both affected siblings showed heavy pre-beta lipoprotein electrophoretic bands plus chylomicrons in their fasting plasmas while ingesting a 33% carbohydrate, 30% fat diet. Incubation of their postheparin plasma with S(f) > 400 chylomicrons in vitro produced a smaller S(f) 20-400 "remnant" with pre-beta electrophoretic mobility that was not seen under the same conditions when normal postheparin plasma was used. Postheparin monoglyceridase and phospholipase activities were either normal or only moderately decreased when determined with appropriate artificial substrates. These data are consistent with either (a) a mutant gene producing a lipoprotein lipase with unusual substrate specificities or (b) an absolute deficiency of normal lipoprotein lipase with a compensatory increase in some other postheparin triglyceridase.

Mobilization of triglyceride but not cholesterol or tocopherol from human adipocytes during weight reduction.

Adipocyte triglyceride, cholesterol, and tocopherol contents were examined in three human obese subjects, all over 150% of ideal weight, who were placed on weight reduction formula diets for 11, 21, and 25 wk, respectively. Body weight decreased from 77 to 63 kg, 188 to 147 kg, and 147 to 99 kg, respectively. All subjects had normal serum cholesterol and triglyceride levels, and these did not vary greatly during weight reduction. Subcutaneous fat was obtained at frequent intervals by needle aspiration from the buttocks before and during weight reduction. Adipocyte size was measured by osmium tetroxide fixation technique, tissue cholesterol content by GLC, tissue triglyceride content by lipid extraction, and tissue tocopherol by thin-layer chromatography. Initial adipocyte size was 0.54, 1.06, and 0.96 micrograms of lipid per cell, respectively (normal 0.60), and the mean cell size decrease during weight reduction was 40%, all due to triglyceride mobilization. Adipocyte cholesterol and tocopherol content did not change significantly during weight reduction. These data are consistent with the concepts that triglycerides but not cholesterol or tocopherol are mobilized from the fat cell during up to 6 months of weight reduction and that independent mechanisms control the efflux of these adipocyte constituents.

In vivo studies of sterol and squalene secretion by human skin.

This work was aimed at studying the quantity and composition of sterols and squalene secreted by the human skin. Lipids secreted by the entire skin were recovered by Soxhlet extraction of the clothing worn by a patient for 24 hr with a chloroform-methanol azeotrope and by extracting the water of a shower taken by the patient at the end of the 24-hr period. Squalene and sterols were quantified by gas-liquid chromatography. Plant sterols were separated from total sterols by thin-layer chromatography. Free and esterified cholesterol were separated by digitonin precipitation. In eight adults, seven of them with hyperlipoproteinemia, the total skin secretion of cholesterol ranged from 59 to 108 mg/day, with a mean of 88 +/- 17 (SD) mg/day. There was no difference in cholesterol secretion between the normocholesterolemic individual and the hypercholesterolemic ones, nor were there any differences according to type of hyperlipoproteinemia. Free cholesterol amounted to 54 +/- 5% of the total cholesterol. The secretion of squalene ranged from 125 to 475 mg/day in five patients. The secretion of both squalene and cholesterol was quite constant for any individual on a given diet. Cholesterol constituted 95.6 +/- 0.5% of the digitonin-precipitable total body surface sterols of eight patients, and lathosterol, the next largest fraction, 3.4 +/- 0.4%. Total plant sterols formed only 0.65 +/- 0.38% and beta-sitosterol 0.35 +/- 0.23% of the skin surface sterols in six patients whose dietary beta-sitosterol intake ranged from 230 to 3400 mg/day.

Mechanisms of action of clofibrate on cholesterol metabolism in patients with hyperlipidemia.

The influence of clofibrate on cholesterol metabolism in patients with hyperlipidemia was studied by means of sterol balance and isotope kinetic techniques and by measurements of flow rates of cholesterol through the biliary tract. Long-term balance studies were carried out on a metabolic ward in 24 patients with all currently recognized types of hyperlipidemia; in five other patients with hypercholesterolemia, pool sizes and turnover rates of cholesterol were defined by compartmental analysis before and after three years' daily administration of the drug. Except in fat-induced hypertriglyceridemia (two patients), clofibrate caused reduced plasma levels of triglycerides and cholesterol in all categories of hyperlipidemia. As a general rule, excretion of cholesterol into bile and feces was significantly increased and fecal bile acid excretion was decreased, regardless of the type of lipoprotein abnormality. Despite a net increase in steroid excretion in most patients with hyperlipidemia, cholesterol synthesis was not increased; indeed, in many patients synthesis appeared to be decreased. While the data obtained in 29 patients were not always consistent, the bulk of the evidence suggests that, in all forms of hyperlipidemia except fat-induced hyperglyceridemia, the drug causes an increased output of cholesterol while simultaneously inhibiting any compensatory increase in cholesterol synthesis. Therefore, it appeared that the increased excretion of steroids was most likely derived from cholesterol stored in tissues. This conclusion was strengthened by finding that long-term administration of the drug can cause marked reduction in body pools of cholesterol. These findings are reflected clinically by resolution of skin and tendon xanthomatosis. However, it is not yet known whether the accumulation of cholesterol in arterial walls that is part of the process of atherogenesis can be inhibited or reversed by the drug.

HYPERLIPIDEMIA IN AN ADULT DIABETIC POPULATION.

Of 98 adult-onset diabetic patients selected at random from an ambulatory clinic, 37.8 per cent were found to have hyperlipidemia. In follow-up examination of the hyperlipidemic patients, 23 per cent had hyper-ß-lipoproteinemia (Type II), 58 per cent had hyper-pre-ß-lipoproteinemia (Type IV), 13 per cent had increased plasma ß-and pre-ß-lipoproteins (combined type) and 6 per cent had reverted to normal values. Type IV patients, as a group, were distinguished by greater hyperglycemia and relative body weight. Chylomicronemia was not observed in this study group. Hyperlipoproteinemia was frequent in this adult-onset diabetic population and differed in several respects from similar data in untreated juvenile-onset diabetes reported by others.

Measurement of squalene in human tissues and plasma: validation and application.

A method is described for accurate and reproducible measurement of squalene in plasma, feces, urine, bile, and tissue that depends on isolation by alumina column chromatography after mild saponification and on measurement by gas-liquid chromatography. Recoveries from all tissues exceeded 80% and from plasma 96%; losses were accurately corrected by appropriate additions of squalene as an overall recovery standard.

Measurement of cholesterol synthesis in man by isotope kinetics of squalene.

A method for measuring the rate of total daily cholesterol synthesis in man has been developed through isotope kinetic studies of squalene biosynthesis after intravenous administration of [14C]mevalonic acid. Plasma squalene becomes rapidly labeled, reaching maximal specific activity approximately 100 min after mevalonate administration and then decays exponentially to reach undetectable levels in 12 hr. The rate of daily squalene synthesis equals the percent dose of mevalonate converted to cholesterol divided by the area under the specific activity curve of squalene; the fraction of the dose of mevalonate converted to cholesterol is calculated by the simultaneous injection of [3H]- and [14C] cholesterol in plasma. The premise that squalene and cholesterol synthetic rates are equivalent was tested. In seven patients it was found that the mean daily cholesterol synthesis rates estimated simultaneously by sterol balance and by sqyalene kinetic methods agreed within 8%. In addition, fractional conversions of mevalonic acid to cholesterol were highly correlated with cholesterol synthesis rates. Maximum estimates of the pool sizes and half-lives of metabolically "active" squalene also were obtained. This measurement of daily cholesterol synthesis by squalene kinetics minimizes patient inconvenience, is suitable for outpatient studies, and yields results in 4 weeks or less. Because of the rapidity of the rate of squalene synthesis, the results obtained reflect cholesterol synthesis over a period of less than 10 hr and are therefore uniquely applicable to unsteady state situations.