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).     

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.     

Estrone sulfate, estrone and estradiol concentrations in normal and cirrhotic postmenopausal women

Circulating levels (mean +/- SD) of estrone sulfate (E1S), estrone (E1) and estradiol-17 beta (E2) were measured in normal and cirrhotic postmenopausal women matched for body weight and age. In cirrhotic postmenopausal women, the E1S concentrations (201 +/- 46 pg/ml), while both E1 and E2 levels showed an increase (46 +/- 7 and 30 +/- 8 pg/ml) compared to control subjects (32 +/- 6 and 18 +/- 7 pg/ml). These data suggest that the liver plays an important role on the control of estrogen sulfation.     

Estrone sulfate and dehydroepiandrosterone sulfate concentrations in normal subjects and men with cirrhosis.

Circulation levels of estrone sulfate (E1S) and dehydroepiandrosterone sulfate (DHAS) have been measured in plasma using a radioimmunoassay for estrone and dehydroepiandrosterone following extraction and hydrolysis of the sulfate. The mean +/- SE concentrations of E1S and DHAS in normal men were 458 +/- 25 pg/ml and 1.45 +/- 0.19 micrograms/ml, respectively. In normal women the values for days 5-7 of the cycle were 880 +/- 117 pg/ml and 1.25 +/- 0.12 micrograms/ml which were not different than the values for days 20-22 of 1195 +/- 176 pg/ml and 1.58 +/- 0.29 micrograms/ml. The mean values in post-menopausal women were 250 +/- 33 pg/ml and 0.47 +/- 0.07 micrograms/ml, both lower than the values in young women. In a group of cirrhotic men the mean values were 325 +/- 55 pg/ml and 0.38 +/- 0.12 micrograms/ml, both significantly lower than the normal values. This suggests a defect in sulfurylation in men with hepatic cirrhosis.     

A non-chromatographic radioimmunoassay of prugesterone in serum

Convenient methodology based on separation of progesterone from alcoholic neutral steroids by means of a sulfation-procedure has been developed for the radioimmunoassay (RIA) of progesterone in male and female serum. When coordinated with our previously published nonchromatographic procedure for the RIA of estrone and estradiol in serum, all 3 seteroids can be determined in the same specimen. Validation of the procedure was based on: 1. Agreement between results obtained using TLC and sultation to fractionate progesterone (r=0.98; b=0.86), 2. accurate recovery of different quantities of progesterone added to serum, 3. independence of the concentration of progesterone and volume of serum used for assay, 4. low procedural blanks (3.6 ± 1.3 pg), 5. low intraassay (9.7 – 10.3%) and interassay (11.0 – 11.6%) variability and 6. correspondence of observed values for progesterone in male serum (108 ± 20 pg/ml) and in female serum (follicular, 285 ± 149 pg/ml; luteal, 3.46±1.45 ng/ml) with those reported previously by others.     

A non-chromatographic radioimmunoassay of estrone and estradiol-17beta serum

A sensitive and efficient non-chromatographic procedure employing the Girard reagent and solvent-partitioning has been developed for the accurate radioimmunoassay (RIA) of estrone (E1) and estradiol-17β(E2) in a single 1.0 ml specimen of male or female serum. Using standard curves which permitted the discrimination of zero from 0.75–1.5 pg (p=0.05), the following mean procedural blanks (pg ± S.D.) were determined (1.0 ml water, n= 24): estrone, 2. 1 ± 1.1 (range 0–4.1); estradiol 1.0± 1.1 (range 0–3.6).A comparison of RIA of estrogens (1) in serum after separation by the Girard procedure and by TLC yielded correlation coefficients of 0.99 and 0.98 for E1 and E2 respectively. The following results (pg/ml ± S.D.) were obtained on RIA of E1 and E2 in 12 different 1.0 ml specimens of male and female serum using the Girard procedure: male. E1 (32.0 ± 9.2), E2 (24.1 ± 10.9); female, E1 (108.5 ± 60.8), E2 (126.4 ± 63.2).The intra-assay variability (c.v.) was found to be 12.6% for E1 and 9.4% for E2. The interassay variability was 14.2% for both estrogens.Twenty-four assays of E1 and E2 can be completed by one person in 2 working days.     

Use of pregnancy-specific protein-B and estrone sulfate for determination of pregnancy on Day 49 in fallow deer (Dama dama )

The objective of this study was to determine if pregnancy specific protein-B (PSPB) and estrone sulfate (E(1)SO(4)) could be used to determine pregnancy status in fallow deer (Dama dama ). Forty mature does were synchronized for estrus with an intravaginal progesterone-releasing device (CIDR) and then artificially inseminated via laparoscopy with frozen semen on one day. Ultrasound examination and jugular blood sampling were done 49 days later. Transrectal ultrasonography was done to presumptively determine the pregnancy status at the time of blood sampling. Serum estrone sulfate concentrations were significantly (P < 0.05) greater in pregnant (n=31) than nonpregnant (n=9) females at 49 days of gestation (166.7 +/- 25.9 pg/ml vs 36.3 +/- 11.1 pg/ml, respectively). The percentage of [(125)I]PSPB bound was significantly (P < 0.01) lower when sera of pregnant (n=29) versus nonpregnant (n=9) females was added to RIA tubes (63.7 +/- 1.6% vs 98.1 +/- 1.6%, respectively). There were 30 fawns born from the group of females that were diagnosed pregnant based on ultrasound. We conclude that estrone sulfate and PSPB can be used to determine pregnancy status in fallow deer at 49 days of gestation.     

A new method for hydrolyzing sulfate and glucuronyl conjugates of steroids

A new method for hydrolyzing steroid conjugates (both sulfates and glucuronides conjugates) that is efficient, effective, and inexpensive is described. This method comprises incubation of the conjugates--after salting-out into ethyl acetate or elution from a C18 cartridge--with anhydrous methanolic hydrogen chloride (methanolysis) for 10 min. It has been successfully applied to our routine radioimmunoassay screening and GC/MS confirmation studies of steroids in prerace and postrace equine urine samples. Comparative GC/MS studies on entire (male horse) urine samples showed that methanolysis gave amounts of free steroids (estrone, estradiols, testosterone, estrenediols, nandrolone, androstanediols) at least as large as those obtained by solvolysis. Similar studies on urine samples from a gelding that had been administered nandrolone phenylpropionate showed that methanolysis gave larger amounts of free steroids (nandrolone, estranediols) than Helix pomatia enzymatic hydrolysis or solvolysis. Also, TLC studies on methanolysis of corticosteroid conjugates such as hydrocortisone 21-sulfate and hydrocortisone 21-phosphate showed that free corticosteroid was released in 5 min.     

Biosynthesis, characterization and inhibition of leukotriene B4 in human whole blood

We have evaluated the biosynthesis, characterization and inhibition of Leukotriene (LT) B4 in unstimulated and in A23187-stimulated human whole blood. LTB4 was assayed by radioimmunoassay (RIA) both in unextracted serum and after extraction and thin-layer chromatography (TLC). Unstimulated human whole blood allowed to clot at 37 degrees C for 60 min produced only trace amounts of LTB4 (0.16 +/- 0.05 ng/ml, mean +/- SD, n = 3). LTB4-like immunoreactivity (ir-LTB4) detectable in unstimulated serum samples was largely overestimated by direct RIA, most likely because of interfering substance(s) unrelated to cyclooxygenase or lipoxygenase activity. Incubation of human whole blood with A23187 (2-10 microM) resulted in a concentration-dependent stimulation of LTB4 production. At 10 microM A23187, ir-LTB4 was 18 +/- 2.4 ng/ml (mean +/- SEM, n = 28). In A23187-stimulated serum samples, LTB4 concentrations measured by direct RIA correlated in a statistically significant fashion with those measured after extraction and TLC. Nafazatrom added in vitro caused a dose-dependent inhibition of A23187-stimulated ir-LTB4 production with an IC50 of 17 microM.     

Superiority of gas chromatography/tandem mass spectrometry assay (GC/MS/MS) for estradiol for monitoring of aromatase inhibitor therapy

Currently available radioimmunoassay methods for estradiol in serum lack sufficient sensitivity and precision to monitor estradiol levels in patients placed on third generation aromatase inhibitors. We recently validated a gas chromatography/tandem mass spectrometry assay (GC/MS/MS) for estradiol and determined estrogen levels in normal post-menopausal women and in women with breast cancer before and during administration of aromatase inhibitors. Validation of the GC/MS/MS assay in human plasma and human serum included determination of assay sensitivity (<0.63 pg/ml), precision (all CVs less than 17.8%), recovery (98-103%), and linearity of recovery (R=0.998). Levels of estradiol were lower when assayed by GC/MS/MS compared to RIA under all conditions (7.26+/-4.82 pg/ml versus 11.9+12.0 pg/ml in normal post-menopausal women; 5.88+/-3.43 pg/ml versus 13.8+/-7.5 pg/ml in breast cancer patients prior to treatment; and<0.63 pg/ml versus 5.8+/-4.1 pg/ml during aromatase inhibitor therapy). Fifty-five women treated either with atamestane/toremiphene or letrozole/placebo were monitored for estradiol levels at 4, 8 and 12 weeks of therapy. The mean levels of estradiol during aromatase inhibitor therapy was 5.8+/-4.1 pg/ml as measured by RIA and <0.63 pg/ml by GC/MS/MS. The degree of suppression with the aromatase inhibitors as detected by RIA was 58% versus >89% by GC/MS. These results suggest that most RIA methods detect cross-reacting estrogen metabolites and yield higher measured levels than GC/MS/MS. Several pharmacological and clinical considerations suggest that GC/MS/MS should become the preferred method for monitoring aromatase inhibitor therapy.     

Radioimmunoassay of arginine vasopressin in the plasma and urine.

We introduced the radioimmunoassay (RIA) of arginine vasopressin (AVP) with standard AVP and antiserum to AVP (both Calibiochem). The sensitivity of the system was increased from the declared 4pg to 1 pg per tube by preparing AVP-125I of high specific activity (about 1,500 mCi/mg) and by modifying the reaction conditions. The sensitivity of the method was adequate for measuring AVP in urine and in concentrated plasma extracts, even under physiological conditions. Reliability of the results depended upon maintenance of approximately the same osmolarity in all the RIA samples. The mean plasma AVP level, uncorrected for AVP extraction losses, was 1.52 +/- 0.20 pg/ml for an ad libitum fluid intake; in fluid deprivation it rose in proportion to the osmolarity of the plasma to 5.83 +/- 0.42 pg/ml at 12 hours and to 19.09 +/- 4.51 pg/ml at 36 hours. Extraction recovery of added AVP was about 63%. The urinary AVP concentration varied according to the patients' state of hydratation from undetectable values at UOsm less than 200 mOsm/1 to a mean 16.5 +/- 7.9 pg/ml in the presence of an ad libitum fluid intake and to 29.1 +/- 7.5 pg/ml after 12 hours' and 117.2 +/- 13.7 pg/ml after 36 hours' deprivation of fluids.     

A simple, accurate, and sensitive assay method of dehydroepiandrosterone sulfate: application for quantitative determination in human breast cyst and duct fluids

Radioimmunoassay (RIA) is the most prevalent method for measuring small amounts of hormones, peptides, and other compounds in human body fluids. The method, however, has several problems, such as cross reactions or non-specific reactions of the antibody used. In order to establish an improved method for assaying dehydroepiandrosterone sulfate (DHEAS) and cholesterol, which are the largest components of human breast cyst and duct fluids, we describe a simple, accurate, and sensitive method using high-performance liquid chromatography (HPLC). The samples were treated with cholesterol oxidase for quantitation of dehydroepiandrosterone (DHEA) and free cholesterol, and the respective oxidized substances, 4-androstene-3,17-dione and 4-cholesten-3-one, were extracted with n-hexane. The extracts were analyzed by straight phase HPLC. Effluents were monitored by measuring absorption at 240 nm, where a newly introduced chromophoric group, an alpha,beta-unsaturated ketone, showed intense absorption (epsilon = 16,000). When the total amount of DHEA (DHEAS plus DHEA) was measured, the sample had been solvolyzed by sulfatase beforehand. The amounts of DHEAS were quantified by comparing the amounts of DHEA before and after solvolysis. Levels of free cholesterol, DHEAS, and DHEA in human breast cyst fluids (n = 30) were 1.77 +/- 1.12 mmol/dl, 8.27 +/- 10.24 micromol/dl, and 0.02 +/- 0.02 micromol/dl (means +/- SD), respectively. The levels of sterol and steroid measured in breast duct fluids that were turbid, brown, dark green, or milky in color (n = 9) (mean levels, 3.20 +/- 2.97 mmol/dl for free cholesterol and 14.77 +/- 13.75 micromol/dl for DHEAS) were significantly (P < 0.01) higher than the levels in clear or serous breast fluids (n = 21) (mean levels, 0.14 +/- 0.13 mmol/dl for free cholesterol and 0.04 +/- 0.07 micromol/dl for DHEAS).     

Plasma estrone sulfate levels in postmenopausal women

Estrone sulfate levels were measured in the plasma of 63 postmenopausal women. The assay method involved prior extraction of the free estrogens, enzyme hydrolysis of the estrone sulfate with sulfatase and radioimmunoassay of the estrone liberated. The plasma levels ranged from 37 to 320 pg/ml (expressed as free estrone) with a mean value of 178 ± 79 pg/ml. As observed in premenopause, estrone sulfate is quantitatively the most important circulating estrogen in postmenopausal women.     

Pregnenolone, dehydroepiandrosterone, and their sulfate and fatty acid esters in the rat brain

The rat brain contains large amounts of pregnenolone (P) and dehydroepiandrosterone (D) arising from local biosynthetic pathways. We have devised a procedure for the measurement of both "neurosteroids" either unconjugated or released from their sulfate (S) or fatty acid (L) esters. The measurements were performed at the acrophase of the circadian variation of neurosteroids, and confirmed the large accumulation of P (25 +/- 8 ng/g, mean +/- SD) and of PS (19 +/- 6 ng/g) and DS (2.1 +/- 0.5 ng/g) in the brain of adult male rats. We found that fatty acid esters constitute the major species of neurosteroids in brain (PL 46 +/- 14, and DL 36 +/- 7 ng/g, in adult males). The levels of P and DS were increased by daily injection of vehicle to intact males, whereas castration, without or with testosterone or estradiol supplementation (2 mg daily for 7 days), did not produce a significant change of neurosteroids concentrations. Measurements of neurosteroids had not been previously reported in cyclic females. The levels of P, PL, and DS were identical in proestrous females and in intact males, whereas PS (26 +/- 6 ng/g) and DL (50 +/- 16 ng/g) were increased in females. Compared to proestrous females, diestrous females had lower levels of PS (19 +/- 6 ng/g), DS (1.7 +/- 0.4 ng/g), and PL (43 +/- 19 ng/g). These differences suggested a modulatory role of ovarian secretions on the metabolism of neurosteroids.     

Establishment of radioimmunoassay for human islet amyloid polypeptide and its tissue content and plasma concentration

Using a synthetic C- terminal tetradecapeptide of human islet amyloid polypeptide (IAPP), we prepared an antiserum for human IAPP [24-37] and established a highly sensitive radioimmunoassay (RIA) for human IAPP. Analyses of human pancreatic extract using reverse-phase high performance liquid chromatography coupled with the RIA revealed that the antiserum specifically detects human IAPP. The content of IAPP in the pancreas of two non-diabetic patients was 604.0 and 1447.7 pg/mg wet weight, and a small amount of IAPP-immunoreactivity was detected in the stomach, duodenum, and jejunum. The mean plasma concentration of IAPP in 10 normal individuals was 13.5 +/- 4.8 (SD) pg/ml. The RIA established in this study provides a useful tool to elucidate the physiological function of IAPP and its pathophysiological significance in non-insulin-dependent diabetes mellitus (NIDDM).     

A radioimmunoassay for rat ghrelin: evaluation of method and effects of nonylphenol on ghrelin secretion in force-fed young rats

Antiserum YJC 13-31 against the rat ghrelin conjugated to bovine serum albumin (BSA) was produced in the rabbit and a double antibody radioimmunoassay (RIA) for ghrelin has been developed. Characterization results of this antiserum revealed no cross-reaction with human growth hormone and somatostatin. Weak cross-reactions with insulin (0.1%), rat growth hormone (0.1%) and glucagon (0.3%) were observed, which scarcely interfered the assay system. The sensitivity of this RIA was 5 pg per assay tube. With the rat serum samples, the within-assay precision was 7.1% and the between-assay precision was 12.3%. The RIA was also available to detect the ghrelin in rat tissue extracts with good parallelism to the rat ghrelin standard. In application, the serum ghrelin and corticosterone levels in weaned rats were measured by RIA. Gavage of saline was sufficient to raise serum ghrelin from 2.6 +/- 0.18 to 6.7 +/- 0.7 ng/ml (P < 0.01). Gavage with nonylphenol (NP) suppressed the elevation of serum ghrelin levels in a dose-dependent manner. Besides, gavages of saline elevated the serum levels of corticosterone from 108.8 +/- 13.5 to 188.7 +/- 23.5 ng/ml (P < 0.01) but the elevation effects of corticosterone from gavages were overcome by NP in the low dose of 50 mg/kg. It can be speculated that ingestion of NP is harmful to young animals during growth and environmental adaptation.     

Determination of plasma melatonin levels by enzyme-linked immunosorbent assay (EIA) in turbot (Scophtalmus maximus L.) and tench (Tinca tinca L.)

An enzymoimmunoassay (EIA) kit for plasma melatonin (MLT) measurements was employed in tench (Tinca tinca) and in turbot (Scophtalmus maximus). Tench and turbot plasma samples were purified with a C18 reversed phase extraction columns because this kit is designed for human serum measurements. The lowest detection limit of the technique was 11.48 pg/well with a sensitivity at 50% binding of 100 pg/well. Intra-assay and inter-assay CV (%) were always less than 5% (n=8), and 9% (n=6) in tench plasma samples, and less than 5% (n=8) and 13% (n=5) in turbot plasma samples, respectively. Correlation coefficients between EIA and RIA measurements in tench and turbot plasma samples were 0.93 and 0.89 (p<0.001) respectively. Diurnal and nocturnal plasma melatonin mean levels were 14.7+/-2.1 pg/ml and 87.4+/-11 pg/ml in tench (n=15), and 3.5+/-0.4 pg/ml and 28.1+/-2.1 pg/ml in turbot (n=15). These species showed a melatonin circadian rhythm as in other animals studied. The results suggest that the commercial kit used in this experiment could be a suitable and alternative method to RIA for plasma MLT determinations in tench and turbot although it is necessary to increase volumes (1ml) and concentrate daytime samples.     

A bile alcohol sulfate as a major component in the bile of the small skate (Raja erinacea)

The nature of bile alcohols and bile acids in gall-bladder and hepatic bile from perfused livers of the small skate (Raja erinacea) has been investigated. The main bile alcohol sulfate was isolated by thin-layer chromatography and analyzed by fast atom bombardment mass spectrometry and 13C NMR. Following solvolysis and purification on Lipidex-DEAP, the bile alcohol profile was measured by capillary gas-liquid chromatography-electron impact mass spectrometry. Based on these studies and on comparison with authentic scymmnol sulfate and scymnol, the main bile alcohol was identified as 5 beta-cholestane-3 alpha,7 alpha,12 alpha,24 xi,26,27-hexol sulfate. The mean +/- SD concentration in gallbladder bile from five different skates was 24.6 +/- 8.7 mmol/l. Only 0.1 mmol/l of cholic acid and its conjugates was found in a pool of skate bile. The main bile alcohol sulfate in the bile of the small skate seems to be a metabolic end product, present in a concentration comparable to that of bile salts in mammals.     

Characteristics of the response of dispersed guinea-pig adrenal cells to low doses (1-50 pg/ml) of alpha 1-24 adrenocorticotrophin

Isolated adrenal cells prepared by tryptic digestion of the guinea-pig adrenal gland are sensitive to low concentrations (less than 25 pg/ml) of adrenocorticotrophin (ACTH). Cell which have been pre-incubated for 2 h. centrifuged and resuspended in fresh culture medium prior to the introduction of 10 pg/ml ACTH for 60 min show a marked increase (328 +/- 109 nmol/l; mean +/- SD) in cortisol secretion over the control compared to freshly dispersed cells (75 +/- 45 nmol/l). Further potentiation of the ACTH effect was seen with the pre-incubated cells by suplementing the medium with calcium (8 mM) and ascorbate (2 mM) but not with theophylline (1 mM). Basal cortisol secretion was not affected by any of the additives. In the presence of 8 mM calcium and after 60 min incubation 10 pg/ml ACTH stimulated cortisol secretion from 328 nmol/l over the control to 839 +/- 382 nmol/l. The effect of ascorbate (2 mM) was to further increase the effect of ACTH at all dose levels tested (1-25 pg/ml). The concentration of ACTH required to provoke half maximal cortisol secretion decreased from 95 pg/ml with normal medium to 12 pg/ml with calcium -ascorbate supplemented medium. Using this supplemented medium the cells were sensitive to 1 pg/ml and cortisol secretion was stimulated 10-fold over the control with 50 pg/ml, a dose which saturated the system.     

Atrial natriuretic factor in human plasma

A reproducible and sensitive radioimmunoassay (RIA) was developed to measure ANF in human plasma. Immunoreactive ANF was extracted from plasma with Sep-Pak cartridges, using 0.2% ammonium acetate (pH 4) with acetonitrile. The sensitivity of the assay was 3.9 pg/ml. The coefficient of variance for inter-assay and intra-assay was 16.8% and 6.8%, respectively. In normal healthy subjects (n = 67), ANF content was 11.9 +/- 1.3 pg/ml (mean +/- SEM). Significantly-higher ANF concentrations were found in proximal coronary sinus blood, being 6 to 37 times greater than in the peripheral circulation. Comparison of the prior extraction method with direct RIA revealed a good correlation (r = 91) in samples containing higher than 100 pg/ml ANF. No correlation was observed with lower values. The elution profiles of reverse-phase HPLC of peripheral and coronary sinus plasma extracts were similar but somewhat complex, with the main immunoreactive peak corresponding to a low-molecular-weight peptide.