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Nondenaturing gradient gel electrophoresis continues to be used widely for resolution and characterization of lipoprotein subclasses. Methods for making such gels in the laboratory have been published, but occasionally samples do not display uniform mobilities for all lanes in a laboratory-made gel. To help overcome this limitation, we recommend a modification

We fed 634 baboons three diets to assess the separate effects of increasing dietary fat and cholesterol intakes on three independent measures of HDL phenotype: concentrations of HDL cholesterol and apoAI, and size distributions of HDL cholesterol. Increasing dietary fat significantly increased concentrations of HDL cholesterol and apoAI (both, P<0.0001), but did not affect HDL particle sizes, whereas increasing dietary cholesterol increased HDL cholesterol (P<0.0001) concentrations and HDL particle sizes (P=0.08), but did not affect apoAI concentrations. A substantial proportion of variation in each of the HDL traits was influenced by genes (heritabilities ranged from 25 to 61%) and a common set of genes influenced HDL variation on each of the diets (genetic correlations ranged from 0.64 to 1.0). However, genes exerted a smaller effect on HDL response to changes of dietary fat and of dietary cholesterol. Therefore, dietary fat and cholesterol alter HDL levels and characteristics, but the dietary responses are not strongly mediated by additive genetic effects.

Syntheses of the 6 alpha-O-carboxymethyl ether derivatives of estrone and estradiol-17 beta and the preparation of their bovine serum albumin conjugates are described. The generation and evaluation of antisera produced from these conjugates is discussed.

We describe a protocol for making a new type of gradient gel, the Composite gradient gel, that was designed to resolve plasma lipoproteins using nondenaturing gradient gel electrophoresis. The new gel format allows analysis both of high density lipoproteins (HDLs) and low density lipoproteins (LDLs) on the same gel. The gel gave highly repeatable (r2 = 0.999) size estimates. We compared lipoprotein phenotypes determined from the new gradient gel with those obtained using specialized HDL and LDL gradient gels. The comparisons indicated that the Composite gel gave lipoprotein particle size estimates for HDLs and LDLs that were virtually identical to those obtained, respectively, from the specialized HDL and LDL gradient gels. We measured median diameters, which reflect the distributions of absorbance, for LDLs and for HDLs and found that the Composite gel gave lipoprotein size distributions that were virtually identical to those measured using the specialized LDL and HDL gels. Finally, comparison of fractional absorbance for six lipoprotein size intervals obtained from the Composite and specialized gels revealed a close correlation (r2 = 0.828). Thus, it appears that both LDL and HDL size phenotypes may be evaluated simultaneously using a single gradient gel format.

We conducted genetic analyses to determine the components of variation for size distributions of apolipoprotein (apo) A-I among human plasma lipoproteins resolved on the basis of size. Analyses used data for 717 individuals in 26 pedigrees. Apo A-I distributions among lipoprotein size classes were measured by nondenaturing gradient gel electrophoresis (GGE) and immunoblotting procedures. Curves were fitted to apo A-I absorbance profiles to estimate fractional absorbance in each of five high-density lipoprotein (HDL) subclasses. Multivariate regression analyses revealed several covariates (sex, age, diabetes, and apo A-I concentrations) that were significantly associated with variation in one or more HDL subclasses. Female gender and elevated apo A-I concentrations were associated with increases in proportion of apo A-I in larger HDLs, while increasing age and diabetes were associated with decreases. The analyses showed significant heritabilities. h2, for each variable representing the different HDL subclasses. Both genetic and nongenetic effects on apo A-I size distributions were generally exerted across the range of lipoprotein sizes, as suggested by high genetic and environmental correlations between HDL subclass variables. Decomposition of total overall variance showed that unidentified environmental factors accounted for 48% of variation in apo A-I size distribution, while genetic factors explained about 36% and the identified covariates explained the remaining 16%. When considered separately, apo A-I concentration explained only 5% of the total variation in apo A-I size distribution, indicating that apo A-I concentration is a poor predictor of apo A-I size distribution. In summary, the data suggest that there are significant genetic and environmental effects on apo A-I size distribution in humans, and that they are general metabolic effects rather than effects on specific HDL subclasses.

The effects of diabetes on lipoprotein particle sizes were assessed using samples from 94 subjects with non-insulin-dependent diabetes mellitus. From a larger population of nondiabetic subjects who showed normal glucose tolerance, we selected an exact match in terms of age, sex, and menopausal status. We designed a protocol to make nondenaturing gradient gels for the resolution of LDL subfractions and generated two measures of LDL size: diameter of the predominant LDL species and proportion of LDL cholesterol (LDL-C) in particles larger than 25.5 nm (large LDL-C). Similarly, we made two measures of HDL size, large HDL cholesterol (HDL-C) and large HDL-apoAI, which represents the proportion of HDL-C and apoAI, respectively, occurring on particles larger than HDL-3. In pairwise comparisons, diabetes was associated with significantly (P < .004) smaller lipoprotein particles for all measures except large HDL-C. Each of the size measures was significantly and positively correlated with each of the others, suggesting that common metabolic mechanisms influence lipoprotein particle sizes across classes of lipoproteins. In addition, each of the size measures was correlated with a variety of measures of HDL and beta-lipoprotein concentrations, which included HDL-C, LDL-C, triglycerides, and apoAI, apoB, and apoE. We used stepwise regression analyses to select from the measures of lipoprotein concentrations those independently correlated with each of the lipoprotein size measures. After adjusting for these metabolic correlates of lipoprotein size measures, we found the effect of diabetes on lipoprotein size measures was no longer significant except for a modest effect (P = .027) on large HDL-apoAI.

The levels of 3 alpha-hydroxy-5 alpha-pregnan-20-one (allopregnanolone) and the epimeric 3 alpha-hydroxy-5 beta-pregnan-20-one (pregnanolone) were studied in women with prospectively confirmed premenstrual syndrome (n = 15) and in a group of asymptomatic control women (n = 12) during the luteal phase of the menstrual cycle. Single late luteal phase plasma samples were selected to make comparisons of plasma hormone levels between patients and controls in the following measures: allopregnanolone, pregnanolone, the ratio of allopregnanolone to pregnanolone, the ratio for each of these anxiolytic steroids to the parent compound progesterone, and the ratio of the sum of allopregnanolone and pregnanolone to progesterone. Differences in these measures were compared by analysis of variance. Additionally, correlations were performed among the various hormone measures and between the hormone measures and the symptom self-ratings. Analysis of variance showed no significant between group differences in the plasma levels of allopregnanolone, pregnanolone, and progesterone. Plasma levels of both allopregnanolone and pregnanolone were correlated with plasma progesterone levels. However, there were no significant correlations between the severity of mood and behavioral symptoms and plasma levels of progesterone, allopregnanolone, and pregnanolone. These data suggest that symptoms of premenstrual syndrome are not associated with a simple deficiency state of either progesterone or its anxiolytic steroid metabolites.

Synthesis of the 11 alpha-hemiglutaryl derivative of 5 alpha-androstane-3 alpha,17 beta-diol 17-glucuronide (androstane-diol-17G) starting from androsta-4,9(11)-diene-3,17-dione through a 10-step sequence and the preparation of its bovine serum albumin conjugate is described. By using this conjugate, antiserum was raised in rabbits which proved to be very specific for androstanediol-17G. A direct radioimmunoassay using a double antibody procedure is described for the measurement of androstanediol-17G from plasma without prior chromatography.

A 3 alpha-hydroxy A-ring-reduced metabolite of progesterone, 3 alpha-hydroxy-5 alpha-pregnan-20-one (allopregnanolone), and one of deoxycorticosterone (DOC), 3 alpha,21-dihydroxy-5 alpha-pregnan-20- one (allotetrahydroDOC), are among the most potent known ligands of gamma-aminobutyric acid (GABA) receptors designated GABAA in the central nervous system. With specific radioimmunoassays, rapid (less than 5 min) and robust (4- to 20-fold) increases of allopregnanolone and allotetrahydroDOC were detected in the brain (cerebral cortex and hypothalamus) and in plasma of rats after exposure to ambient temperature swin stress. Neither steroid was detectable in the plasma of adrenalectomized rats either before or after swim stress. However, allopregnanolone, but not allotetrahydroDOC, was still present in the cerebral cortex (greater than 3 ng/g) after adrenalectomy. These data demonstrate the presence of allopregnanolone and allotetrahydroDOC in brain and show that acute stress results in a rapid increase of these neuroactive steroids to levels known to modulate GABAA receptor function.

5 alpha-Androstane-3 alpha, 17 beta-diol glucuronide (androstanediol-G) is a dihydrotestosterone metabolite whose serum levels are elevated in hirsute women. Current assay methods do not distinguish between the two androstanediol-G isomers, androstanediol 3-G and androstanediol 17-G. Since the production of these isomers may be influenced by different factors, we have developed a specific radioimmunoassay for androstanediol 17G. The antibody was raised against 5 alpha-androstane-3 alpha, 17 beta-diol 17-G conjugated to bovine serum albumin (BSA). [9,11(-3H]5 alpha-Androstane-3 alpha, 17 beta-diol 17-G was used for determination of procedural losses and as the labeled ligand in the assay. Unlabeled androstanediol 17-G was used as assay standard. Serum levels of total androstanediol-G, androstanediol 3-G and androstanediol 17-G were measured in 8 normal men. Total androstanediol-G levels were 16.5 +/- 5.2 nmol/l, androstanediol 17-G levels were 12.9 +/- 5 nmol/l, and androstanediol 3-G levels were 3.3 +/- 1.8 nmol/l. 77 +/- 13% of total androstanediol-G was androstanediol 17-G. These results confirm our previous findings that androstanediol 17-G is the predominant androstanediol-G isomer in human serum and suggests that 5 alpha-dihydrotestosterone (DHT) is preferentially metabolized to androstanediol 17-G.

A radioimmunoassay for measuring 3 alpha-hydroxy-5 alpha-pregnan-20-one in plasma has been developed. Polyclonal antibodies were raised in rabbits against 3 alpha-hydroxy-20-oxo-5 alpha-pregnan-11 alpha-yl carboxymethyl ether coupled to bovine serum albumin. 3 alpha-Hydroxy-5 alpha-pregnan-20-one was purified from either extracts of plasma by high-performance liquid chromatography. These antibodies were then used for the radioimmunoassay of this centrally active progesterone metabolite in rat and human plasma. 3 alpha-Hydroxy-5 alpha-pregnan-20-one was detected in plasma from female rats on the day of estrus (2.0 to 9.3 ng/ml) and in the plasma of women during the luteal phase of the menstrual cycle at levels ranging from 0.25 to 2.5 ng/ml. The latter was highly correlated with plasma progesterone levels.

The syntheses of 3-O-carboxymethyl ether derivatives of estrone, estradiol-17beta, and estriol and the preparation of their bovine serum albumin (BSA) conjugates are described. These conjugates were employed for the generation of specific antisera suitable for radioimmunoassay (RIA) of estrone, estradiol-17beta, and estriol. The previous concept that specific antisera for estrogens cannot be obtained by employing estrogens derivatized at the 3-position is unfounded.

A radioimmunoassay (RIA) procedure has been developed for measurement of testosterone in male plasma after ether:chloroform (4:1) extraction of the plasma sample without resorting to chromatography. The highly specific anti-testosterone serum was generated from both rabbits and sheep immunized with 15beta-carboxyethylmercapto-testosterone-BSA conjugate. The synthesis of 15beta-carboxyethylmercaptotestosterone and the preparation of its BSA conjugate are described. The high affinity (Ka=2.38 X 10(9) liters/mole) antiserum binds 50% of 50 picograms of tritiated testosterone at working dilutions of 1:100,000 to 1:200,000. Both 5alpha and 5beta-dihydrotestosterone compounds exhibited less than 2% cross-reaction. The only other steroids that showed minor cross-reaction were 11beta-hydroxytestosterone (3.8%), progesterone (2.1%), corticosterone (1.6%), and deoxycorticosterone (7.7%).

Ethinyl estradiol sulfates are major circulating metabolites of ethinyl estradiol (EE2); this is a relationship analogous to that of endogenous estrone and estrone sulfate. Because of the wide use of contraceptives containing EE2, the pharmacokinetics of its sulfate conjugates are of some importance. In previous studies of the intermediate metabolism of ethinyl estrogens we have shown that the baboon is an appropriate animal model. Accordingly, oral and/or intravenous doses of EE2 or each of its three sulfates were administered to castrate female baboons, and plasma levels of EE2 and its sulfates were studied by specific radioimmunoassay or radioisotope counting. After intravenous administration of EE2, the 3-sulfate and the 3,17-disulfate are the major circulating metabolites. After oral dosage administration, the 3-glucuronide and, in some cases, the 3,17-diglucuronide also become important. After intravenous administration, about twice as much of the drug exists in the sulfate as in the free form, as reflected by the areas under the plasma level curves. The bioavailability of orally administered EE2 was about 60%, confirming the presence of a substantial first-pass effect. Hydrolysis at the 17-position occurs when EE2-17-sulfate is administered orally but appears not to occur with intravenous administration. EE2 and the three sulfates, given intravenously, exhibited two-compartment open-model kinetics. The elimination phase half-lives of all four compounds were similar, ranging from 8.8 to 11.2 hours. The area under the plasma level curve of EE2 resulting from the intravenous administration of the 3-sulfate was approximately 8% of the total area under the plasma level curve of both EE2 and EE2 sulfates. The ratio of the area under the plasma level curve of sulfates resulting from 3-sulfate administration compared to the other two sulfates was approximately 0.3, reflecting the existence of other metabolic pathways for its disposition.

The increasing use of radionuclides in medical diagnosis raises the possibility of accidental spills and skin contamination. This study was performed to evaluate the effectiveness of several decontaminating agents. Most nuclides were easily removed to levels of less than 5% of their original activity. Sodium pertechnetate (Tc-99m) was the most difficult compound to remove. Little difference was found between the effectiveness of tap water, soap and water, and two commercially available decontaminating agents.

A case of Rhizopus osteomyelitis is described. The radionuclide and radiographic findings differ from those of osteomyelitis secondary to common pathogens: low-level radionuclide activity is observed, while soft-tissue swelling, periosteal reaction, and loss of fascial margins are absent.

[3H]Dihydrotestosterone (50 microCi) was infused into normal men and women for 8 h. It was previously shown that this was sufficient time for this material to reach a steady state. Venous plasma was obtained at 6 and 8 h, pooled, and the unconjugated steroids removed by ether extraction. The remaining plasma was adjusted to pH 4.9 and the steroid conjugate was extracted first with ethyl acetate and then with an ether-ethanol mixture. The extracts were combined and taken to dryness. Steroid sulfates were solvolyzed using dioxane, and the mixture partitioned between ether and 1% NaOH. The aqueous phase was acidified and added to an XAD-2 column, washed with water, and the glucuronide fraction eluted with methanol. The solvent was concentrated and the methanol extract was passed through a C18 Sep-Pak, filtered through an Acrodisc CR and then subjected to gradient high performance liquid chromatography [HPLC] (Nova-Pak C18, KH2PO4, pH 3, and methanol). The fractions containing steroid glucuronides were collected and esterified with diazomethane and then acetylated with acetic anhydride in pyridine. The glucuronide triacetyl methyl ester (GAME) derivatives were then run in a second HPLC system (3 Lichrosorb 5 mu columns, 4 mm x 25 cm) using a gradient of ethanol-heptane and heptane. We clearly established that this system separates 3 alpha-diol GAME conjugated at the 17 and 3 positions (44 vs 50 min) with authentic samples previously synthesized in our laboratory. We concluded that the pooled plasma contained only the 17-GAME conjugate. No significant activity of the 3-glucuronide was detected. The natural compound in circulation, therefore, is 5 alpha-androstane-3 alpha, 17 beta-diol 17-glucuronide.

Antiserum for radioimmunoassay (RIA) of dehydroepiandrosterone (DHA) was raised in rabbits with the conjugate obtained by coupling 15beta-carboxyethylmercapto-dehydroepiandrosterone with bovine serum albumin. The antiserum proved to have high affinity and specificity for DHA and showed only minor cross-reaction to androst-5-ene-3beta, 17beta-diol (3.5%). The Rivanol-treated antiserum was covalently coupled to Enzacryl Polyacetal to evaluate its utility in solid-phase RIA.

The syntheses of 15beta-carboxyethylmercapto-5alpha-dihydrotestosterone, 15beta-carbosyethylmercapto-5alpha-androstane-3beta, 17beta-diol and 15beta-carboxyethylmercapto-5alpha-androstane-3alpha, 17beta-diol and the preparation of their bovine serum albumin (BSA) conjugates are described. These conjugates were employed for the generation of specific antisera suitable for radioimmunoassay (RIA) of 5alpha-dihydrotestosterone (5alpha-DHT), 5alpha-androstane-3beta, 17beta-diol (3beta-diol) and 5alpha-androstane-3alpha, 17beta-diol (3alpha-diol).