A new reversed phase, paired ion thin-layer chromatographic method for steroid sulfate separations.

The sulfoconjugated steroids estrone sulfate (ES) and dehydroepiandrosterone sulfate (DS) were separated in the reversed phase mode on polyamide-coated TLC plates. Baseline resolution was obtained between tritiated ES and DS standards when run with a mobile phase of 20% acetonitrile in 5mM aqueous triethylamine, triethanolamine, tris-hydroxymethylaminomethane, tributylamine or ammonia. ES and DS showed no mobility in the absence of an ion-pair reagent. The radioactive peaks were detected and integrated non-destructively by scanning. Quantitation was confirmed by elution of cut-out peak areas and liquid scintillation counting. Similar results were obtained with washed ethanol extracts of serum labeled with tritiated ES and DS. The extracts were defatted on the plate with hexane: ethyl acetate (1:1) prior to the reversed phase development.

Radioimmunoassay of estrone sulfate in the serum of normal men after a chromatographic procedure that eliminates dehydroepiandrosterone sulfate interference.

Fifty fresh-frozen normal male sera containing tritiated estrone sulfate (ES) and dehydroepiandrosterone sulfate (DS) were extracted with ethanol after ether extraction of unconjugated steroids. Washed extracts were defatted and chromatographed on polyamide-coated plates by reversed phase paired ion TLC. Plates were scanned for radioactivity, and ES peaks were cut, eluted and assayed by direct RIA with a commercially available antiserum. Mean ES values were 445 +/- 209 pg/mL (SD), in agreement with the three lowest of the seven laboratories which had previously reported normal male ES values. No differences were observed in ES values when samples were rechromatographed prior to assay, or when up to 4 micrograms/mL unlabeled DS was added to serum before extraction. These data confirm the absence of interference by DS in the current study and suggest that previously reported high (716-1194 pg/mL) mean normal male ES values reflect DS interference. The present study also demonstrates the the stability of ES in sera stored frozen at -40 C for an average of 17 years (mean: 406 +/- 258 pg/mL; [SD]; n = 41).

Radioimmunoassay of estrone sulfate in the serum of normal men after a non-chromatographic procedure that eliminates interference from dehydroepiandrosterone sulfate.

Duplicate aliquots of 20 fresh-frozen normal human male sera were prepared for estrone sulfate (ES) radioimmunoassay (RIA) by each of three different methods: the thin-layer chromatography (TLC) procedure we previously reported, a new procedure including overnight heating (100 C) of an ethanol extract reconstituted in dilute acetate buffer, and the new procedure with the hot incubation omitted. The purpose of the 100 C incubation was the selective thermal solvolysis of dehydroepiandrosterone sulfate (DS), the only steroid conjugate present in serum in high enough concentrations to interfere with a high-specificity ES RIA. Dehydroepiandrosterone released by solvolysis and endogenous unconjugated steroids were extracted from the samples with ether before RIA. Estrone sulfate values obtained after the thermal solvolysis preparation averaged 854 +/- 501 pg/ml (SD) versus 826 +/- 474 pg/ml (SD) after the TLC method, with excellent correlation between the two (r = 0.97). Samples prepared by the new method but with thermal solvolysis omitted averaged a 33.8% elevation of measured ES level, an elevation significantly correlated (P less than 0.02) with DS levels obtained from the same specimens. In addition, a single specimen showed no elevation after preparation by the thermal solvolysis method when up to 8 micrograms/ml authentic DS as added before extraction. Compared with the TLC method, the new method also provides substantial savings in specimen volume requirements and sample processing time.

A simple, differential extraction method for the simultaneous direct radioimmunoassay of androgens and androgen glucuronides in human serum.

A method is described for the differential extraction of unconjugated androgens (testosterone and dihydrotestosterone) and glucuronidated androgens (androstane-3 alpha,17 beta-diol glucuronide and androsterone glucuronide) from human serum using solid-phase, gravity-flow extraction columns. In this method, 100-microL aliquots of serum are loaded onto the normal-phase columns, unconjugated androgens are eluted with ethyl ether, and glucuronides are eluted with ethyl ether containing 2% acetic acid. Glucuronide eluates are washed with 1% aqueous acetic acid to remove cross-reacting steroid sulfates. Assays of sera for the four steroids were performed using standard radioimmunoassay methodology, except for androsterone glucuronide. This steroid was assayed with a novel radioimmunoassay method that employees a tritiated, unconjugated androsterone tracer and an anti-dehydroepiandrosterone sulfate antiserum. The new method is well suited for the assay of conjugated and unconjugated steroids in large numbers of specimens, particularly where the sample volume is limited.

A new partition thin-layer chromatographic method for steroid separations.

TLC plates with a 25 mu thick polyamide stationary phase were modified for the separation of neutral steroids by impregnation with propylene glycol. A mixture of tritiated 5 alpha-androstan-3 alpha,17 beta-diol, testosterone, 17 beta-hydroxy-5 alpha-androstan-3-one and 4-androstene-3,17-dione was applied to the plate and developed in a toluene mobile phase to a height of 13.6 cm. This resulted in complete resolution of the 4 compounds as detected by a gas flow scanner or imaging analyzer. Cutting and elution of peak areas with methanol resulted in quantitative recovery of all four steroids. The thinness of the layer also permitted a 3-5% counting efficiency on scanning, resulting in good quantitation of recovery without liquid scintillation counting. The high sorptive capacity of the polyamide layer also enabled extracts of normal human serum to be defatted on the TLC plate by development with pure hexane prior to the toluene step. The new method thus offers several advantages over existing methods for steroid separations and should be adaptable to separations of other relatively non-polar compounds.

Increased circulating levels of plasma ATP in cystic fibrosis patients.

Recent studies have shown that the cystic fibrosis transmembrane conductance regulator (CFTR), an ATP-binding cassette (ABC) transporter whose mutations are responsible for cystic fibrosis (CF), permeates ATP. However, little information is available concerning extracellular ATP concentrations in CF patients. Thus, the goal of this preliminary study was to determine the circulating levels of plasma ATP in CF patients. Circulating levels of plasma ATP were determined by the luciferin-luciferase assay in both CF patients and healthy volunteer control subjects. The two groups were compared using an analysis of variance. CF genotype and age, which ranged from 7 to 56 years, were also used to compare data by single-blind analysis. With comparable sample numbers, CF patients had statistically higher levels of circulating ATP (34%, P<0.01) when compared by analysis of covariance with the age of the subjects as the cofactor. The CF patients bearing the DeltaF508 genotype had a 54% (n=33, P<0.01) higher plasma ATP concentration compared to controls, while patients bearing other CF genotypes were similar to controls (n=10, P<0.4). We conclude that CF patients have higher circulating levels of ATP when compared to controls. Increased levels of plasma ATP, which is an important autocrine/paracrine hormone in many cell types, may be associated with chronic manifestations of the disease.