Parathyroid hormone reference ranges in healthy individuals classified by vitamin D status.

PURPOSE: Parathyroid hormone (PTH) concentrations are routinely measured in the diagnosis and management of bone and kidney diseases, but reference ranges can be overestimated if determined in otherwise healthy individuals for whom vitamin D deficiency was not evaluated. We establish PTH reference ranges in apparently healthy, normocalcemic, normophosphatemic individuals categorized by 25-hydroxyvitamin D (25(OH)D) status using the Elecsys® PTH (cobas e 601) and Elecsys® Vitamin D total II electrochemiluminescence immunoassays (cobas e 411). METHODS: This prospective, non-interventional study measured PTH in serum from 653 apparently healthy adults [56.7% female; 68.2% white/Caucasian; 28.6% African American; median age 44 years (range 21-83)] from three diverse geographic sites across the USA during summer and winter months. Subjects were classified by concomitant vitamin D sufficiency (≥ 30 ng/mL), insufficiency (> 20 to < 30 ng/mL) or deficiency (≤ 20 ng/mL). RESULTS: In vitamin D sufficiency, median PTH was 31.9 pg/mL [range (2.5th-97.5th percentile) 17.9-58.6] compared with 35.5 pg/mL (17.0-60.4) for insufficiency, and 39.8 pg/mL (19.5-86.4) for deficiency. A significant inverse relationship was found between PTH and 25(OH)D (P < 0.001). After accounting for vitamin D, potential effects of race or season as covariates were relatively small or absent. CONCLUSIONS: Upper reference limits (URL) for PTH in vitamin D sufficiency/insufficiency were similar and lower than current values. Clinically important PTH elevations were observed in vitamin D deficiency, where revised reference ranges with a higher URL may be appropriate. These data may help to distinguish vitamin D-related PTH elevations from other causes [e.g., primary (normocalcemic) or secondary hyperparathyroidism].

The effects of phytosterols present in natural food matrices on cholesterol metabolism and LDL-cholesterol: a controlled feeding trial.

BACKGROUND/OBJECTIVES: Extrinsic phytosterols supplemented to the diet reduce intestinal cholesterol absorption and plasma low-density lipoprotein (LDL)-cholesterol. However, little is known about their effects on cholesterol metabolism when given in native, unpurified form and in amounts achievable in the diet. The objective of this investigation was to test the hypothesis that intrinsic phytosterols present in unmodified foods alter whole-body cholesterol metabolism. SUBJECTS/METHODS: In all, 20 out of 24 subjects completed a randomized, crossover feeding trial wherein all meals were provided by a metabolic kitchen. Each subject consumed two diets for 4 weeks each. The diets differed in phytosterol content (phytosterol-poor diet, 126 mg phytosterols/2000 kcal; phytosterol-abundant diet, 449 mg phytosterols/2000 kcal), but were otherwise matched for nutrient content. Cholesterol absorption and excretion were determined by gas chromatography/mass spectrometry after oral administration of stable isotopic tracers. RESULTS: The phytosterol-abundant diet resulted in lower cholesterol absorption (54.2±2.2% (95% confidence interval 50.5%, 57.9%) vs 73.2±1.3% (69.5%, 76.9%), P<0.0001) and 79% higher fecal cholesterol excretion (1322±112 (1083.2, 1483.3) vs 739±97 mg/day (530.1, 930.2), P<0.0001) relative to the phytosterol-poor diet. Plasma lathosterol/cholesterol ratio rose by 82% (from 0.71±0.11 (0.41, 0.96) to 1.29±0.14 µg/mg (0.98, 1.53), P<0.0001). LDL-cholesterol was similar between diets. CONCLUSIONS: Intrinsic phytosterols at levels present in a healthy diet are biologically active and have large effects on whole-body cholesterol metabolism not reflected in circulating LDL. More work is needed to assess the effects of phytosterol-mediated fecal cholesterol excretion on coronary heart disease risk in humans.

Enantiospecificity of cholesterol function in vivo.

The importance of the absolute configuration of cholesterol for its function in vivo is unknown. To directly test this question in vivo, we synthesized the enantiomer of cholesterol (ent-cholesterol) and tested its ability to substitute for natural cholesterol (nat-cholesterol) in the growth, viability, and behavior of Caenorhabditis elegans, a cholesterol auxotroph. First-generation animals grown on ent-cholesterol were viable with only mild behavioral defects. However, ent-cholesterol produced 100% lethality/arrest of their second generation progeny. Isotopically labeled ent-cholesterol incorporated into animals, indicating that its lethality was not secondary to cholesterol starvation. When mixed with nat-cholesterol, ent-cholesterol was not inert; rather, it antagonized the activity of nat-cholesterol. These results demonstrate for the first time that the absolute configuration of cholesterol, not just its physical properties, is essential for its functions in vivo.

Orlistat inhibits dietary cholesterol absorption.

OBJECTIVE: Orlistat decreases the absorption of dietary triglycerides by inhibiting intestinal lipases. Orlistat therapy is associated with a greater decline in plasma low-density lipoprotein-cholesterol concentrations than that expected from weight loss alone. Therefore, we evaluated the effect of orlistat treatment on dietary cholesterol absorption as a possible mechanism for the independent effect of orlistat on plasma cholesterol concentration. RESEARCH METHODS AND PROCEDURES: Cholesterol absorption from a standardized meal, containing 72 mg of cholesterol, was determined in 18 subjects with class II abdominal obesity (BMI, 35.0 to 39.9 kg/m(2)) by simultaneous administration of intravenous ([(2)H(6)] cholesterol) and oral ([(2)H(5)] cholesterol) cholesterol tracers. In protocol 1 (n = 9), cholesterol absorption was determined on two different occasions, 10 to 20 days apart, to assess the reproducibility of the tracer method. In protocol 2 (n = 9), cholesterol absorption was determined with and without orlistat therapy in a prospective, randomized, crossover design to assess the effect of orlistat on cholesterol absorption. RESULTS: In protocol 1, cholesterol absorption from the test meal was the same on both occasions (53 +/- 5% and 51 +/- 5%). In protocol 2, orlistat treatment caused a 25% reduction in cholesterol absorption, from 59 +/- 6% to 44 +/- 5% (p < 0.01). DISCUSSION: These data demonstrate that orlistat inhibits dietary cholesterol absorption, which may have beneficial effects on lipoprotein metabolism in obese subjects that are independent of weight loss itself.

Decreased hepatic accumulation and enhanced esterification of cholesterol in mice deficient in mdr1a and mdr1b P-glycoproteins.

Class I P-glycoproteins [Pgp; MDR1 (ABCB1) in humans, mdr1a and mdr1b in mice] confer resistance to structurally diverse chemotherapeutic drugs in cultured cells and intact animals, but the function of these proteins in normal physiology remains poorly characterized. Based on studies in cell culture, a putative role for class I Pgp in absorption and intracellular trafficking of sterols has been proposed. We examined wild-type and mdr1a(-/)-/1b(-/)- mice to determine whether class I Pgp affects cholesterol absorption and esterification in vivo. Using a dual-isotope protocol, absorption of orally administered radiolabeled cholesterol into serum did not differ between wild-type and mdr1a(-/)-/1b(-/)- mice, demonstrating that class I Pgp is not essential for overall absorption of cholesterol through the intestine. However, the ratio of oral to intravenous labeled cholesterol in liver was decreased significantly in mdr1a(-/)-/1b(-/)- mice. In the liver, but not other tested organs, deletion of class I Pgp enhanced kinetics of esterification of an oral bolus of radiolabeled cholesterol without affecting esterification of cholesterol administered intravenously. Steady-state hepatic content of cholesterol and esterified cholesterol also were unaffected by absence of mdr1a and mdr1b.Thus, in normal animals, class I Pgp functions to kinetically increase hepatic accumulation and decrease esterification of orally administered cholesterol in vivo.

Relationships of plasma C-peptide and gender to the urinary excretion of inositols in older people.

PURPOSE: The urinary excretions of myo-inositol and D-chiro-inositol are elevated in diabetes, and have been suggested as possible markers or effectors of insulin action. The aim of the present study was to measure the urinary excretion of these compounds, and to assess possible relationships with the metabolic control of glucose, in older, non-diabetic men and women. SUBJECTS: 32 older (age range 54-71 yrs), moderately overweight (body mass index 29.1 +/- 0.4 kg/m2, mean +/- SEM), non-diabetic men (n = 17) and women (n = 15). METHODS: 75 g oral glucose tolerance testing was done the day after all subjects had consumed nutrient-defined menus for five days. Plasma samples were analyzed for the concentrations of glucose, insulin, and C-peptide, and the 180-minute area under the curve (AUC) for each of these compounds was calculated. Samples from 24-hour urine collections were analyzed for the concentrations of myo-inositol, D-chiro-inositol, L-chiro-inositol, and pinitol. RESULTS: The fasting glucose, insulin, and C-peptide, and the AUC for glucose and insulin, were not different between men and women. C-peptide AUC was greater in the men versus the women (p < 0.001). The median urinary excretions (micromol/g creatinine) of myo-inositol (p < 0.001), D-chiro-inositol (p < 0.001), L-chiro-inositol (p < 0.05), and pinitol (p < 0.001) were higher, and the myo-inositol:D-chiro-inositol ratio was lower (p < 0.001), in the men versus women. For all subjects combined, C-peptide AUC was positively correlated with the urinary excretion of each of the measured inositols, as well as the myo-inositol:D-chiro-inositol ratio. The correlations between C-peptide AUC and these inositols were strongly influenced by the co-linear relationship between C-peptide AUC and gender. CONCLUSIONS: Collectively, these data show that older, moderately overweight, non-diabetic men and women with gender-related differences in glucose-stimulated C-peptide AUC, an indirect indicator of insulin secretion, also display differences in the urinary excretion of myo-inositol, D-chiro-inositol, L-chiro-inositol, and pinitol. The gender-related difference in the myo-inositol:D-chiro-inositol ratio suggests that, while the urinary excretion of all of the inositols measured were higher in the men than the women, the difference was more pronounced for D-chiro-inositol.

Effect of pinitol treatment on insulin action in subjects with insulin resistance.

OBJECTIVE: Endogenous low-molecular-weight glycans containing pinitol (3-O-methyl-D-chiro-inositol) and D-chiro-inositol are thought to mediate certain actions of insulin. We tested the hypothesis that oral administration of soybean-derived pinitol would improve insulin sensitivity in obese subjects (BMI = 36.6 kg/m2) with diet-treated type 2 diabetes or glucose intolerance (HbA1c = 6.8%). RESEARCH DESIGN AND METHODS: There were 22 subjects randomized to receive either pinitol 20 mg x kg(-1) x day(-1) (n = 12) or placebo (n = 10) in a 28-day double-blinded trial. RESULTS: No toxicity due to the pinitol was observed during the study The sensitivity of glucose and lipid metabolism to insulin were assessed by measurement of whole-body glucose, palmitate, and glycerol kinetics during basal conditions and a hyperinsulinemic-euglycemic clamp. Metabolic measurements were made at baseline and again at the end of the study Final plasma levels of pinitol were 48-fold (1.06 +/- 0.15 vs. 0.02 +/- 0.01 micromol/l, P < 0.0001) and D-chiro-inositol levels 14-fold (0.56 +/- 0.08 vs. 0.04 +/- 0.02 micromol/l, P < 0.0001) greater in the pinitol group compared with placebo. CONCLUSIONS: Four weeks of pinitol treatment did not alter baseline glucose production, insulin-mediated glucose disposal, or rates of appearance of free fatty acids and glycerol in plasma. We conclude that plasma levels of both pinitol and D-chiro-inositol are very responsive to pinitol ingestion, but insulin sensitivity does not increase after pinitol treatment in individuals with obesity and mild type 2 diabetes.

Lack of effect of cholesterol esterase inhibitor CVT-1 on cholesterol absorption and LDL cholesterol in humans.

Two clinical trials were performed to test the hypothesis that CVT-1, a potent inhibitor of pancreatic cholesterol esterase, reduces percent cholesterol absorption and LDL cholesterol in humans. Measurements of cholesterol absorption were made with deuterated cholesterol tracers given orally and intravenously and detected in plasma by a new technique using negative ion mass spectrometry. Study 1 was a randomized, double-blind parallel study of CVT-1 treatment at doses of 0, 300, 1500, and 3000 mg/day in 19 subjects. Percent cholesterol absorption measured at baseline and again after 2 and 6 weeks showed no treatment effect and LDL cholesterol was unchanged. Study II was a randomized open-label crossover comparison between CVT-1 given as 1000 mg three times daily for 2 weeks and 187.5 mg hourly 16 hours/day for 2 weeks. Percent cholesterol absorption and plasma LDL cholesterol were not different between periods. We conclude that cholesterol esterase is not required for unesterified cholesterol absorption in human subjects.

Sitostanol administered in lecithin micelles potently reduces cholesterol absorption in humans.

BACKGROUND: Phytosterol feeding in human clinical trials has had generally small and inconsistent effects on serum cholesterol concentrations, raising doubts about the importance of phytosterols in natural diets and supplements. OBJECTIVE: The hypothesis tested was that the low intestinal bioavailability of purified phytosterols can be increased by formulation with lecithin. DESIGN: The ability of sitostanol to reduce cholesterol absorption was measured directly by including hexadeuterated cholesterol tracer in a standard test breakfast and measuring plasma tracer concentration 4 and 5 d later by gas chromatography-negative ion mass spectrometry. The tracer amount after a test meal containing sitostanol was compared with that after an identical meal containing placebo. Each subject served as his or her own control and the order of testing was random. Sitostanol was formulated either as a powder or as a sonicated micellar solution with lecithin. A total of 38 single-meal tests were performed in 6 healthy subjects. RESULTS: Sitostanol powder (1 g) reduced cholesterol absorption by only 11.3 +/- 7.4% (P = 0.2), confirming in vitro data showing poor solubility of sitostanol powder in artificial bile. In contrast, sitostanol in lecithin micelles reduced cholesterol absorption by 36.7 +/- 4.2% (P = 0.003) at a dose of 700 mg and by 34.4 +/- 5.8% (P = 0.01) at a dose of 300 mg. CONCLUSIONS: Sitostanol reduced cholesterol absorption at doses lower than reported previously, but only if presented in lecithin micelles. Properly formulated sitostanol as well as naturally occurring complexes of phytosterol and phospholipid might be therapeutically useful for cholesterol lowering.

Cholesterol absorption efficiency declines at moderate dietary doses in normal human subjects.

While unphysiologically large cholesterol doses are known to reduce percent cholesterol absorption, smaller amounts are reported to have no effect in human subjects. To determine the dose;-response relation between dietary cholesterol consumed and the efficiency of intestinal cholesterol absorption, we fed 18 normal subjects two test meals containing different amounts of natural cholesterol. In each test pentadeuterated cholesterol tracer was given orally, hexadeuterated cholesterol tracer was given intravenously, and the tracer ratio was measured in plasma 4 days later by gas chromatography/negative ion mass spectrometry. Baseline cholesterol absorption in the presence of 26 mg cholesterol tracer was 40.7 +/- 2.3%. This decreased by 4.9 percentage points (P = 0.05) when a total of 188 mg cholesterol was included in the meal and by 15.6 percentage points (P = 0.006) when 421 mg cholesterol was given, showing that the efficiency of cholesterol absorption declines appreciably even with modest increases in cholesterol dose. Considerable variation was noted in the response of different subjects and, on the higher cholesterol dose, dietary cholesterol absorption varied 5-fold from 40 mg to 212 mg. Fasting plasma insulin was correlated with the ability to absorb higher cholesterol doses without loss of efficiency (r(s) = 0.700, P = 0.036). Percent cholesterol absorption in a single meal is significantly influenced by the amount of cholesterol in that meal, suggesting that acute dietary factors influencing cholesterol absorption need further study.

The effects of metoprolol and captopril on heart rate variability in patients with idiopathic dilated cardiomyopathy.

BACKGROUND: The effects of treatment with captopril or metoprolol on heart rate variability (HRV) were investigated in 38 patients (29 men and 9 women) with mild to moderate symptoms of heart failure due to idiopathic dilated cardiomyopathy (DCM). HYPOTHESIS: The aim of the study was to investigate and compare the effects of the angiotensin-converting enzyme inhibitor captopril with those of the selective beta-adrenergic receptor blocker metoprolol on HRV in patients with idiopathic DCM. METHODS: Heart rate variability was analyzed in the time and frequency domains from 18th of Holter monitoring before randomized treatment was started, after 6 months of therapy, and 1 month after therapy was stopped. RESULTS: Captopril treatment increased HRV expressed as total power and low-frequency power in the frequency domain. There was no change in the time domain. In the metoprolol group, there was a pronounced increase in both time- and frequency-domain indices of HRV. The increase in total power was partly maintained 1 month after therapy was stopped in both treatment groups. CONCLUSION: Treatment with captopril and metoprolol increases HRV in patients with DCM. This effect seems to be maintained for at least 1 month after therapy is stopped. The increase in HRV seems to be more pronounced with metoprolol, and the two different pharmacologic approaches may have additive effects that are of prognostic importance in patients with heart failure.

Effects of alcohol and cholesterol feeding on lipoprotein metabolism and cholesterol absorption in rabbits.

Alcohol fed to rabbits in a liquid formula at 30% of calories increased plasma cholesterol by 36% in the absence of dietary cholesterol and by 40% in the presence of a 0.5% cholesterol diet. The increase was caused almost entirely by VLDL, IDL, and LDL. Cholesterol feeding decreased the fractional catabolic rate for VLDL and LDL apoprotein by 80% and 57%, respectively, and increased the production rate of VLDL and LDL apoprotein by 75% and 15%, respectively. Alcohol feeding had no effect on VLDL apoprotein production but increased LDL production rate by 55%. The efficiency of intestinal cholesterol absorption was increased by alcohol. In the presence of dietary cholesterol, percent cholesterol absorption rose from 34.4+/-2.6% to 44.9+/-2.5% and in the absence of dietary cholesterol, from 84.3+/-1.4% to 88.9+/-1.0%. Increased cholesterol absorption and increased LDL production rate may be important mechanisms for exacerbation by alcohol of hypercholesterolemia in the cholesterol-fed rabbit model.

Percent cholesterol absorption in normal women and men quantified with dual stable isotopic tracers and negative ion mass spectrometry.

Percent cholesterol absorption was measured in 94 normal subjects aged 17- 80 years while consuming diets generally low in cholesterol (mean intake = 226 +/- 126 mg/day). A new dual stable isotope method was used where a cholesterol tracer containing 6 extra mass units was given intravenously and another tracer with 5 extra mass units was given orally during a standard test meal. The ratio of tracers in plasma was determined by negative ion mass spectrometry of pentafluorobenzoyl sterol esters. Absorption values ranged widely from 29.0% to 80.1% with mean 56.2 +/- 12.1 (SD) %. Cholesterol absorption was significantly increased in African-Americans (63.4 +/- 11.8% vs. 55.1 +/- 11.9%, P = 0.027) but was similar for women (53.3 +/- 11.9%) and men (57.6 +/- 12.1%). It was not related to plasma lipoproteins, age, apoE3/E3 or E3/E4 genotype, or chronic dietary intake of energy, fat, or cholesterol quantitated from 7- day food records. However, dietary cholesterol intake was positively related to plasma cholesterol (P = 0.036) and triglycerides (P = 0.026). The milligram amount of dietary cholesterol absorbed (but not percent absorption) was positively correlated with fasting plasma insulin (r = 0.525, P < 0.0001), C-peptide (r = 0.367, P = 0.0003) and glucagon (r = 0.421, P < 0.0001) independent of gender, body fat percent and age.The efficiency of intestinal cholesterol absorption and the milligram amount of dietary cholesterol absorbed were not related to plasma cholesterol or LDL cholesterol in individuals consuming a low-cholesterol low-fat diet. The dominant factor determining dietary cholesterol absorption was intake rather than absorption efficiency. Dietary cholesterol and fat were strongly and independently related to hormonal measures of insulin resistance.-Bosner, M. S., L. G. Lange, W. F. Stenson, and R. E. Ostlund, Jr. Percent cholesterol absorption in normal women and men quantified with dual stable isotopic tracers and negative ion mass spectrometry.

Normal intestinal dietary fat and cholesterol absorption, intestinal apolipoprotein B (ApoB) mRNA levels, and ApoB-48 synthesis in a hypobetalipoproteinemic kindred without any ApoB truncation.

The purpose of this study was to characterize intestinal apolipoprotein B (apoB) metabolism in subjects with familial hypobetalipoproteinemia (FHBL), where segregation analysis supports linkage to the apoB gene but no apoB truncations are present. We investigated cholesterol and fat absorption, intestinal apoB mRNA synthesis and editing, as well as apoB-48 synthesis. Plasma triglycerides (TG) and retinyl palmitate in the chylomicron fractions were analyzed after 12 hours of fasting and then repeatedly for 14 hours after ingestion of a vitamin A-containing high-fat meal. Cholesterol absorption was assessed using a dual stable-isotope method. Mean peak times and concentrations and areas under the curve (AUCs) for fat absorption and mean percentages of cholesterol absorption were comparable in affected and nonaffected family members. Intestinal biopsies were extracted for total RNA and also incubated with 35S-methionine for measurements of apoB synthesis. Similar quantities of apoB mRNA were found to be expressed in the intestine in affected and control subjects by RNase protection assay. ApoB mRNA editing assay showed that the majority of apoB-100 mRNA was edited to the apoB-48 form to a similar extent in both groups. Virtually no apoB-100 protein was synthesized by the intestine in any subject, and apoB-48 protein synthesis was not significantly different in the affected individuals. These data are consistent with in vivo metabolism data that show normal production rates for liver-derived apoB-100 but increased apoB-100 fractional catabolic rates in affected members of this family. Thus, the molecular defect probably does not affect transcription, translation, or secretion of apoB-containing lipoproteins, but may instead affect their clearance.

The effect of oxidizing cholesterol on gastrointestinal absorption, plasma clearance, tissue distribution, and processing by endothelial cells.

Little is known about the absorption or metabolism of oxysterols. Toward better appreciating the metabolic consequences of oxidizing cholesterol, we compared labeled cholesterol with the labeled oxysterols 7 alpha-hydroxycholesterol, 7 beta-hydroxycholesterol, and 7-ketocholesterol prepared from [4-14C]cholesterol, [26,26,26,27,27,27-2H6]cholesterol, and [23,24,25,26,27-13C5] cholesterol. Gastrointestinal absorption of oxysterols in rats was 91.5 +/- 0.3% compared with 75 +/- 1.1% for cholesterol, determined by fecal collection (P < .001). When injected intravenously and followed by gas chromatography/mass spectrometry, 7 alpha-hydroxycholesterol was cleared at 23 times the rate of cholesterol. After 5 minutes, only 1.2 +/- 0.2% of 7 alpha-hydroxycholesterol remained in the plasma, whereas 28.0 +/- 1.7% of cholesterol and 40.0 +/- 2.5% of a triglyceride emulsion injected simultaneously were still present. [14C]7 alpha-Hydroxycholesterol injected intravenously was also rapidly cleared from plasma, was widely distributed in tissues and organs, and showed evidence of extensive metabolism at 5 minutes. The fractional rate of uptake of radiolabeled oxysterols by cultured endothelial cells was 15.7 times that of cholesterol (P < .001), and the fractional rate of efflux was 3.4 times that of cholesterol (P < .001). Oxysterols passed through endothelial cells grown on transwell membranes at a rate 4.3 times that of cholesterol (P < .001). Fractional oxysterol transport across the endothelial cell monolayer was increased 62 +/- 17% when HDL was added to the medium in the lower chamber (P = .003). Oxysterols were extensively metabolized to even more polar metabolites during endothelial cell transit. These properties of oxysterols potentially provide a mechanism for enhancing transport of cholesterol through tissues and preventing accumulation of cholesterol in those cells that can oxidize it.

Quantification of cholesterol tracers by gas chromatography--negative ion chemical ionization mass spectrometry.

Because of its high sensitivity, gas chromatography negative ion chemical ionization mass spectrometry (GC-NCI-MS) is a potentially valuable analytical tool for the study of cholesterol metabolism. Of several derivatives prepared for potential use in tracer studies pentafluorobenzoyl cholesterol was selected because it formed rapidly at ambient temperature and was stable for long periods, could be detected at a level of 1 fmol, and yielded a mass spectrum in which the molecular ion was the principal component. Hexadeuterated cholesterol tracer ([26,26,26,27,27,27-2H6]cholesterol) could be detected in dilutions up to 2700 in unlabeled cholesterol by selected ion monitoring with a coefficient of variation averaging 3.2%. In seven normal subjects tracer cholesterol was infused intravenously and plasma cholesterol enrichment was determined after 4 h. The measured rapidly miscible cholesterol pool was 391.0 +/- 38.6 mg cholesterol/kg. Negative ion mass spectrometry of pentafluorobenzyol cholesterol will facilitate analysis of both small amounts of natural cholesterol and labeled cholesterol in applications where sensitivity is critical.

Relation between plasma leptin concentration and body fat, gender, diet, age, and metabolic covariates.

We measured plasma leptin concentrations by RIA in 204 normal weight and obese subjects, aged 18-80 yr, using full-length recombinant human leptin as a standard. Fasting levels between 1.2-97.9 ng/mL were observed. The plasma leptin concentration was highly correlated with percent body fat (r = 0.710; P < 0.0001) and was 3 times as high in women as in men (17.1 vs. 5.8 ng/mL; P < 0.0001). Circulating leptin was inversely related to age and was reduced 53% in subjects over age 60 yr. A statistical model containing percent body fat, gender, and age accounted for 65% of the variance in plasma leptin levels. Leptin was not independently related to abdominal fat distribution, plasma lipids and lipoproteins, chronic energy intake, diet composition, plasma insulin, or maximum oxygen consumption. However, plasma leptin was reduced by 26% in 5 obese subjects who consumed a 1000-Cal diet for 10 days (P = 0.004). We conclude that circulating leptin rises continuously with increasing adiposity. Gender, age, and short term caloric restriction may be important secondary regulators of plasma leptin.

Microscale preparation of natural and labeled oxysterols.

The preparation of cholesterol and radiocholesterol oxidation products on a microscale is difficult. Cholesterol generally resists oxidation unless it is well dispersed under controlled conditions. A method was developed to reliably produce 7 alpha- and 7 beta-hydroxycholesterol and 7-ketocholesterol. Small changes in pH, metal ions present, or in the colloidal dispersion, resulted in production of completely different oxysterols. Attempts to oxidize aged radiocholesterol were not successful even after purification by several thin-layer chromatographic steps, and this appeared to be due to a time-related change in the radioactive material. Fresh radiocholesterol oxidized readily.

A stereospecific myo-inositol/D-chiro-inositol transporter in HepG2 liver cells. Identification with D-chiro-[3-3H]inositol.

D-chiro-Inositol is an epimer of myo-inositol that is found in certain mammalian glycosylphosphatidylinositol protein anchors and inositol phosphoglycans possessing insulin-like bioactivity. In order to generate a probe for metabolic studies, D-chiro-[3-3H]inositol was synthesized by selective reduction of D-chiro-3-inosose at pH 6.5 with sodium borotritide. D-chiro-[3-3H]Inositol was taken up by HepG2 human liver cells through a saturable and stereospecific pathway in which D-chiro-inositol and myo-inositol competed equally but L-chiro-inositol was not recognized. Dd-Glucose, but not L-glucose, competed for D-chiro-[3-3H]inositol uptake over glucose concentrations of 4-28 mM. Maximum transport capacity was 717 pmol/mg cell protein/3 h with a Km value of 348 microM. Uptake was reduced by 76% when sodium was eliminated from the medium and by 94% when the experiment was performed at 0 degrees C. The new myo/D-chiro-inositol transporter is distinct from the sodium-myo-inositol co-transporter found in many tissues and accounts for all of the saturable D-chiro-inositol uptake and for a portion of the saturable low affinity myo-inositol uptake in HepG2 cells. It may allow D-chiro-inositol to be used by cells in the presence of a relatively large amount of competing myo-inositol.