Fusaric acid as a novel proton-affinitive derivatizing reagent for highly sensitive quantification of hydroxysteroids by LC-ESI-MS/MS.

A highly sensitive derivatization method for liquid chromatography (LC)-electrospray ionization (ESI) tandem mass spectrometry of dehydroepiandrosterone (DHEA), testosterone (T), pregnenolone (P5), and 17alpha-OH-pregnenolone (17-OHP5) was developed based on the use of fusaric acid as a reagent. DHEA, P5, and 17-OHP5 were rapidly and quantitatively converted to the 3-fusarate esters by treatment with fusaric acid and 2-methyl-6-nitrobenzoic anhydride. The positive ESI-mass spectra of the fusarate esters of each steroid were dominated by the appearance of [M + H](+) as base peaks. The fusarate derivatization of these steroids showed 17.6-fold (DHEA), 11.9-fold (P5), 3.3-fold (17-OHP5), and 1.8-fold (T) higher sensitivity to those of the corresponding picolinate derivatives in LC-selected reaction monitoring.

Development and application of electrospray-active derivatization reagents for hydroxysteroids.

New derivatization reagents, 1-(2,4-dinitro-5-fluorophenyl)-4-methylpiperazine (PPZ) and 4-(4-methyl-1-piperazyl)-3-nitrobenzoyl azide (APZ), were developed for the liquid chromatography-electrospray ionization-mass spectrometric (LC-ESI-MS) determination of steroids having a hydroxy group. PPZ reacted with a phenolic hydroxy group in estrogens. After quaternarization of the PPZ-estrogens with methyl iodide, the resulting derivatives provided more than a 2000-fold higher sensitivity compared to the intact estrogens. After derivatization of steroids having an alcoholic hydroxy group (5-ene-steroids or 5alpha-reduced steroids) with APZ followed by methylation, their detection responses increased more than 500 times. These derivatization procedures coupled with LC-ESI-MS/MS were successfully used for the determination of estrogens in the serum and prostatic 5alpha-dihydrotestosterone.

Synthesis of pyridine-carboxylate derivatives of hydroxysteroids for liquid chromatography-electrospray ionization-mass spectrometry.

Synthesis and liquid chromatography-electrospray ionization-mass spectrometric (LC-ESI-MS) behaviors of the picolinoyl, 6-methylpicolinoyl, nicotinoyl, 2-methoxynicotinoyl and isonicotinoyl derivatives of the hydroxysteroids estrone, estradiol, 3beta-hydroxyandrost-5-en-17-one (dehydroepiandrosterone) and testosterone in positive mode were investigated. Each steroid was converted to the corresponding pyridine-carboxylate derivative by the acyl chloride method or the mixed anhydride method using the corresponding free acids and 2-methyl-6-nitrobenzoic anhydride; in each case, the latter method principally gave a better yield. The pyridine-carboxylate derivative of each steroid exhibited a clear single peak in liquid chromatography with a reversed phase column and CH(3)CN-0.1% CH(3)COOH as a mobile phase. The positive-ESI-mass spectra of the picolinoyl, 6-methylpicolinoyl and 2-methoxynicotinoyl derivatives showed a predominance of [M+H](+), whereas [M+H+CH(3)CN](+) was observed with high intensity in the nicotinoyl and isonicotinoyl derivatives. Even in the case of estradiol, with its two hydroxyl groups, a single charged ion of [M+H](+) or [M+H+CH(3)CN](+) was observed in the positive-ESI-mass spectrum of each derivative. The results revealed that picolinoyl derivatization is a simple and versatile method suitable for the sensitive and specific determination of hydroxysteroids by LC-ESI-MS (selected reaction monitoring).

Novel polyhydroxysterols from the Red Sea marine sponge Lamellodysidea herbacea.

Chemical investigation of the dichloromethane extract of the Red Sea marine sponge Lamellodysidea herbacea led to the isolation of four novel polyhydroxysteroids: cholesta-8-en-3beta,5alpha,6alpha,25-tetrol (1), cholesta-8(14)-en-3beta,5alpha,6alpha,25-tetrol (2), cholesta-8,24-dien-3beta,5alpha,6alpha-triol (3), and cholesta-8(14),24-dien-3beta,5alpha,6alpha-triol (4). Their structures were identified through 1D and 2D NMR studies. Relative stereochemistries were established by analysis of chemical shifts, coupling constants, and NOESY correlations. Compounds 3-4 showed antifungal activity against Candida tropicalis, with an inhibition diameter of 13 and 11 mm at 10 microg/disc, respectively.

Seasonal variations in the levels of polyhydroxysteroids and related glycosides in the digestive tissues of the starfish Patiria (Asterina) pectinifera.

Seasonal variations in the levels of polar steroids including polyhydroxylated steroids and related glycosides in digestive organs of the starfish Patiria (=Asterina) pectinifera have been studied. The concentration of polar steroids is related to the annual reproductive cycle of the starfish and periods of active feeding. Two peaks in concentrations of polar steroids in pyloric caeca and stomach were found, the first in winter during reorganization and the second in summer during intensive gametogenesis before spawning. Probable biological functions of polyhydroxysteroids and related glycosides are discussed. The data support the hypothesis these compounds are involved in digestion in the starfish.

Immunoassay of 7-hydroxysteroids: 2. Radioimmunoassay of 7alpha-hydroxy-dehydroepiandrosterone.

High sensitivity radioimmunoassay of 3beta, 7alpha-dihydroxy-5-androsten-17-one (7alpha-OH-DHEA) has been developed and evaluated. The method is based on polyclonal rabbit antisera raised against 19-O-(carboxymethyl)oxime bovine serum albumin conjugate and bridge- and position homologous [(125)I]iodotyrosine methyl ester as a tracer. Sensitivity of the assay amounted to 3.12 fmol (0.95 pg)/tube, precision as a mean intra- and interassay coefficient of variation was 7.1 and 10.6%, respectively, and the average recovery of the analyte added to steroid-free serum was 110%. Out of the steroids occurring in human serum which may interfere with the assay, the only important cross-reactants were dehydroepiandrosterone and 3beta, 7beta-dihydroxy-5-androsten-17-one (7beta-OH-DHEA) with cross-reactivities of 1.95 and 1.16%, respectively. The levels of free (unconjugated) 7alpha-OH-DHEA have been determined in 29 sera from healthy volunteers (23 females and 6 males), and from 48 patients (43 females and 5 males) in which dehydroepiandrosterone and its sulfate (DHEA/S) had been measured for various endocrinopathies. The levels in healthy subjects ranged from 0.21 to 6.57 (mean 2.33+/-1.50) nM, while those of the patients from 0 to 5. 99 (mean 1.46+/-1.52) nM. The levels of 7alpha-OH-DHEA in patients significantly correlated with those of DHEA and its sulfate.

Sensitive assay system for bile acids and steroids having hydroxyl groups utilizing high-performance liquid chromatography with peroxyoxalate chemiluminescence detection.

3 alpha- or 3 beta-hydroxysteroids, such as bile acids (free and glycine and taurine conjugates), 3 beta-hydoxy-5-cholenic acid, pregnanediol, 5-pregnene-3 beta, 20 beta-diol and 5-pregnene-3-beta,20 alpha-diol, were converted to 3-oxosteroids by enzymatic reaction using immobilized hydroxysteroid dehydrogenase, derivatized with dansylhydrazine to the corresponding dansyl hydrazones and purified by gel permeation chromatography. The dansyl hydrazones were chromatographed on a C18 column with a tetrahydrofuran-containing eluent and detected at the level of a few femtomoles by a peroxyoxalate chemiluminescence post-column reaction using bis[4-nitro-2-(3,6,9-trioxadecyloxycarbonyl)phenyl] oxalate as a chemilumigenic reagent. The dansyl hydrazones of chenodeoxycholic acid and deoxycholic acid (free and glycine and taurine conjugates) in particular, which coeluted under the chromatographic conditions above, were separated using an eluent including acetonitrile and 2,6-di-O-methyl-beta-cyclodextrin and detected in the same way.

Liquid chromatographic and spectral analysis of the 17-hydroxy anabolic steroids.

The liquid chromatographic properties of various 17-hydroxy anabolic steroids are examined under reversed-phase conditions. These anabolic steroids are now listed as controlled drugs in many states due to their abuse potential in athletics, body building, and other areas. These nonesterified steroids are separated on a C18 stationary phase with a 70% methanol in water mobile phase. In a few cases, two compounds display very similar retention properties. However, dual-wavelength detection at 254 and 280 nm allows for their differentiation. Reversed-phase retention parallels steroid lipophilicity based on hydroxyl and methyl group substituents. Also, those steroids containing a dienone substructure are more polar than steroids containing an enone moiety.

Selective gas chromatographic analysis of monohydroxysteroids as their cyanosyl derivatives.

A new silylating reagent that forms cyanosyl (cyanoethyldimethylsilyl) derivatives with monohydroxysteroids is introduced and its usefulness for the gas chromatographic analysis of these compounds evaluated. The reactivity of the new reagent, N-methyl-N-cyanoethyldimethylsilyltrifluoroacetamide, has been studied using a series of monohydroxysteroids and compared to that of other reagents forming cyanosyl or alkylsilyl derivatives. The results obtained indicate that at room temperature, in polar solvents, the reagent reacts rapidly and selectively with most hydroxyl groups found in steroids. The cyanosyl derivatives formed have retention properties that are comparable to those of the corresponding alkylsilyl derivatives. However, the former offer advantageous properties for detection since they can be analyzed with a nitrogen-phosphorus detector, and they exhibit mass spectral features that are suitable for identification and quantitation at low levels using selected ion monitoring mass spectrometry. The detection limits for all the monohydroxysteroids studied in this work, as cyanosyl derivatives, are typically on the order of 30-50 pg and the relative response factors are unity within a range of 25 percent.

Metabolic fate of [3H]deoxycorticosterone and [14C]progesterone--I. Early tissue metabolites and analysis of 21-hydroxysteroids by high pressure liquid chromatography.

Rabbits injected with mixtures of [3H]deoxycorticosterone and [14C]progesterone had significant levels of both 3H and 14C in several tissues and fluids extracted 10-45 min later. The distribution of radioactivity between 21-deoxysteroid, 21-hydroxysteroid, steroid acid and steroid glucuronide fractions was determined by alumina adsorption chromatography. Steroid acids derived from both steroids accumulated in the liver, kidney and urine, but were quantitatively less significant in the bile, duodenum, uterus, spleen and lung and were detected in the blood for the first time. Different 21-hydroxysteroid profiles were detected in the tissue and fluid extracts by reverse and straight phase high pressure liquid chromatography. [3H]Deoxycorticosterone accumulated in the kidney, lung, spleen and uterus, whereas tetra and hexahydro reduced metabolites predominated in the liver, bile and duodenum. By contrast, [14C]progesterone was metabolised to more polar 21-hydroxylated metabolites which were detected in the liver, kidney and urine. These results show the influence of a steroid 21-hydroxyl function, when administered, as opposed to being formed in in vivo, on the metabolic fate and excretory pathways of 21-hydroxysteroids by the rabbit.

Bile acid sulfotransferase I from rat liver sulfates bile acids and 3-hydroxy steroids: purification, N-terminal amino acid sequence, and kinetic properties.

A bile acid:3'phosphoadenosine-5'phosphosulfate:sulfotransferase (BAST I) from adult female rat liver cytosol has been purified 157-fold by a two-step isolation procedure. The N-terminal amino acid sequence of the 30,000 subunit has been determined for the first 35 residues. The Vmax of purified BAST I is 18.7 nmol/min per mg protein with N-(3-hydroxy-5 beta-cholanoyl)glycine (glycolithocholic acid) as substrate, comparable to that of the corresponding purified human BAST (Chen, L-J., and I. H. Segel, 1985. Arch. Biochem. Biophys. 241: 371-379). BAST I activity has a broad pH optimum from 5.5-7.5. Although maximum activity occurs with 5 mM MgCl2, Mg2+ is not essential for BAST I activity. The greatest sulfotransferase activity and the highest substrate affinity is observed with bile acids or steroids that have a steroid nucleus containing a 3 beta-hydroxy group and a 5-6 double bond or a trans A-B ring junction. These substrates have normal hyperbolic initial velocity curves with substrate inhibition occurring above 5 microM. Of the saturated 5 beta-bile acids, those with a single 3-hydroxy group are the most active. The addition of a second hydroxy group at the 6- or 7-position eliminates more than 99% of the activity. In contrast, 3 alpha,12 alpha-dihydroxy-5 beta-cholan-24-oic acid (deoxycholic acid) is an excellent substrate. The initial velocity curves for glycolithocholic and deoxycholic acid conjugates are sigmoidal rather than hyperbolic, suggestive of an allosteric effect. Maximum activity is observed at 80 microM for glycolithocholic acid. All substrates, bile acids and steroids, are inhibited by the 5 beta-bile acid, 3-keto-5 beta-cholanoic acid. The data suggest that BAST I is the same protein as hydrosteroid sulfotransferase 2 (Marcus, C. J., et al. 1980. Anal. Biochem. 107: 296-304).

High-performance liquid chromatography of hydroxysteroids detected with post-column immobilized enzyme reactors.

Assaying the low concentrations of steroid hormones in extracts of body fluids requires detectors that are both highly sensitive to the steroid and relatively insensitive to interfering compounds usually present in much higher concentrations. To explore the use of moderately specific enzymes in post-column reactors, we immobilized 3 alpha- and beta-hydroxysteroid dehydrogenase on controlled pore glass beads, 37 microns in diameter, and constructed 4.6-mm diameter reactor columns, 3-cm long, packed with one of the two kinds of these beads. Hydroxysteroids eluted from the analytical column were mixed with the coenzyme, nicotinamide adenine dinucleotide (NAD), before passing through the reactor. The effluent from the reactor was passed through the 70-microliters flow cell of a fluorometer in which the fluorescence of the NADH produced in the enzyme-catalyzed oxidation of the hydroxysteroid was monitored. At the conventional high-performance liquid chromatography flow-rates used, oxidation of the steroids was almost complete. The yield depended on both the residence time of steroids in the reactor column and the concentration of organic modifier in the reaction mixture. Maximal yield was obtained with buffer having a low organic solvent concentration and passing through the reactor slowly. In assays of mixtures of epimeric hydroxysteroids, the 3 alpha-hydroxysteroids were detected with the 3 alpha-hydroxysteroid dehydrogenase reactor; the beta-hydroxysteroids were not, confirming the specificity of the enzymatic detection. With the fluorometer used, picomole quantities of steroids could easily be distinguished from noise.

Chronobiologic dynamics of delta 5-3 beta-hydroxysteroids and glucocorticoids in rat brain and plasma and human plasma.

While a different timing of circadian rhythms does not necessarily demonstrate the operation of independent mechanisms, it is one step in the isolation of separate interacting rhythmic factors underlying the dynamics of all life. With this step in mind, circadian rhythms are quantified in rat plasma and brain for corticosterone (B), pregnenolone (P), and dehydroepiandrosterone (D) by the rejection of the zero-amplitude assumption with the single cosinor method, from data obtained every 3 hr for 24 hr on groups of three male Sprague-Dawley rats, 11-12 weeks of age. The rats were killed; steroids were extracted from brain and plasma and were radioimmunoassayed. In relation to the acrophase (phi) of B, the phi of P in brain (P = 0.035) and of D in plasma (P = 0.0012) preceded the phi of B. By contrast, in clinically healthy women, the phi of plasma dehydroepiandrosterone sulfate (DHEA-S) lags behind that of cortisol (F), on the average by 6 hr 28 min. Such a lag is also seen in men. A species difference in the time relations of delta 5-3 beta-hydroxysteroids vs. glucocorticoids in plasma is obvious (P less than 0.01). A difference in time relations of human circulating D, DHEA-S, and F between schizophrenic and clinically healthy men renders the time relations of glucocorticosteroids and delta 5-3 beta-hydroxysteroids in brain particularly interesting, as do relations of an egocentric and expansive personality to the rhythm characteristics of DHEA-S. The fact that the acrophases for some of the steroids investigated in brain and plasma, respectively, are differently timed is in keeping with the assumption that they involve partly different mechanisms, even if differences in the metabolic handling of different steroids also remain to be investigated.

A one-step enzymatic assay for the measurement of 17 beta-hydroxy- and 17-oxo-steroid profiles in biological samples.

We describe a simple enzymatic method for the sensitive and specific detection and quantitation of families of hydroxy- and oxo-steroids in biological mixtures. Analysis of the profiles of individual steroids may be achieved following their chromatographic separation. The objectives of this analytical system are, therefore, different from conventional methods which are designed to measure single steroids with a high degree of specificity. The method employs highly purified and active bacterial hydroxysteroid dehydrogenases (HSD) which promote stereospecific, nicotinamide nucleotide-dependent oxidations and reductions at specified positions of steroids. In the presence of catalytic quantities of steroids these enzymes promote the transfer of hydrogen (transhydrogenation) between NADH and NAD analogues. A recently purified 17 beta-HSD from an Alcaligenes species (D. W. Payne and P. Talalay, J. biol. Chem., 260, 13468-13655, 1985) shows almost complete specificity for the 17 beta-hydroxy- and 17-oxo-groups of both C18 and C19 steroids. This enzyme catalyzes steroid-dependent transhydrogenation between NADH and the thionicotinamide analogue of NAD (S-NAD). When these components are incubated at pH 8.5 in the presence of minute quantities of steroid substrates, S-NADH (measured at 398 nm where NADH does not absorb) accumulates at a constant rate which is proportional to the concentrations of steroid and enzyme. The linear increase in absorbance with time is a measure of the total concentration of 17 beta-hydroxy- and 17-oxo-steroids, and can be used to detect subpicomol quantities of steroids. The method is illustrated by the detection and identification of free and conjugated androgens in human serum following their separation by high pressure liquid chromatography. The specificity of the transhydrogenase assay is completely dependent on the specificity of the enzyme and is thus applicable to the detection of other hydroxy- and oxo-steroids by making use of HSDs with appropriate specificities (e.g. 3 alpha-HSD for the measurement of 3 alpha-hydroxy- and 3-oxo-steroids). The simple one-step reaction lends itself to automation, needs no auxiliary detection systems, and requires only an inexpensive colorimeter.

Sex steroids and 5-en-3 beta-hydroxysteroids in specific regions of the human brain and cranial nerves.

Sex steroids and 5-en-3 beta-hydroxysteroids were determined by radioimmunoassay in specific regions of the human brain, in the anterior and posterior pituitary, in one sensory organ, the retina and in the cranial nerves. Progesterone, androstenedione, testosterone and estrone were found in all areas of the brain and in all the cranial nerves but not in all cases. There was no sex difference except in the case of androstenedione where values were higher in women in some brain areas. Estrone values were always higher than those of estradiol in both men and women. No 5 alpha-dihydrotestosterone was detected in any of the samples studied. The values for pregnenolone, dehydroepiandrosterone and their sulfates were much higher than those of the sex steroids in all areas of the brain and in all the cranial nerves. Values for pregnenolone were greater than those of its sulfate while those of dehydroepiandrosterone were in general equal to or higher than those of its sulfate. The values for pregnenolone were greater than those of dehydroepiandrosterone. There were no obvious regional differences in the concentrations of the 5-en-3 beta-hydroxysteroids either in specific areas of the brain or in the cranial nerves. But there was a definite trend for the free dehydroepiandrosterone values to be higher in women. The possible significance of these observations is discussed.

Bis-trifluoromethylbenzoyl derivatives for steroid analysis by gas chromatography electron capture negative ion chemical ionisation mass spectrometry.

3,5-Bis-trifluoromethylbenzoyl derivatives of eight monohydroxy steroids and eight dihydroxy steroids were synthesised. Under the reaction conditions described, the monohydroxy steroids each gave a single derivative while the dihydroxy steroids, with the exception of corticosterone, showed multiple product formation. The reactivity of hydroxyl groups relative to their stereochemistry is discussed. The electron capture negative ion chemical ionisation mass spectra of these derivatives were recorded. When the derivatised hydroxyl group was alicyclic or aromatic, a molecular ion was normally the base peak in the mass spectrum. Selected ion monitoring of molecular ions indicated that, in certain cases, as little as 1 pg of the parent steroid could be detected. The potential use of this derivative in the quantitative analysis of steroids by gas chromatography-mass spectrometry is discussed.

Determination of serum delta 5-3 beta-hydroxysteroid sulphates by combined high-performance liquid chromatography and immobilized 3 beta,17 beta-hydroxysteroid dehydrogenase in column form.

We studied the use of an immobilized enzyme, covalently bound to aminopropyl-CPG, in the analysis of individual delta 5-3 beta-hydroxysteroid sulphates. A microcolumn with immobilized 3 beta,17 beta-hydroxysteroid dehydrogenase was prepared and used together with high-performance liquid chromatography (HPLC). The reduced nicotinamide-adenine dinucleotide produced from delta 5-3 beta-hydroxysteroids by this enzyme was fluorimetrically determined. The immobilized enzyme was sufficiently stable for at least one month or for 180 tests when used repeatedly. A clinical trial demonstrated that this HPLC-immobilized enzyme method is superior to the soluble enzyme method, giving reliable and reproducible results at a low cost.

[Evaluation of liver function in chronic liver diseases using the bile acid spectrum]. / Die Beurteilung der Leberfunktion bei chronischen Hepatopathien mit Hilfe des Gallensäurenspektrums.

In 24 persons with healthy liver and 44 patients with without exception morphologically ascertained chronic hepatopathies of different degree of severity the diagnostic valency of the bile acids was tested. In these cases the bile acids estimated in the C-bile proved as sensitive indicators of a chronic lesion of the liver parenchyma. Quantitative deviations of the bile acids, the relation from tri- to dihydroxycholan acids, qualitative alterations in the spectre of the free bile acids as well as changes of the form of conjugation were of importance concerning the functional diagnosis. The concentration of bile acids decreased the more expressed was the chronic lesion of the liver parenchyma. Trihydroxycholan acids and dihydroxycholan acids variably participated in the reduction of the bile acids in different forms of chronic hepatopathies. From the change of the quotient tri- to dihydroxycholan acids causal connections between the degree of severity of the chronic hepatopathy and the dominance of the chenodesoxycholic acid could be derived. Moreover, the conjugation of the bile acids with glycine prevailing in chronic hepatopathies underwent a degradated change. From the examinations results that quantitative and qualitative deviations of the bile acid spectre under defined morphological prerequisites render possible a differentiated judgment of the liver function in chronic hepatopathies.

Reversed- and normal-phase high-performance liquid chromatography of 18-hydroxylated steroids and their derivatives. Comparison of selectivity, efficiency and recovery from biological samples.

The chromatographic behaviour of adrenal 18-hydroxysteroids, and a series of derivatives thereof, has been studied in reversed-phase and normal-phase high-performance liquid chromatography (HPLC). Both 18-hydroxycorticosterone and 18-hydroxy-11-deoxycorticosterone exhibited a marked loss of chromatographic efficiency when separated on incompletely-covered C18 reversed-phase supports (as determined by methyl red adsorption) showed no such effect, nor was it seen when aprotic solvents, such as dioxane, were used. This phenomenon is unique among the wide range of adrenal and testicular steroids that we have studied and affords a useful test, applicable under aqueous conditions, of coverage by the alkylsilane reversed phase, a factor of considerable importance in the successful resolution of complex mixtures of adrenal steroids by reversed-phase HPLC. The retention times of the 20-methoxy, 20-ethoxy, 21-acetoxy, etiolactone, 11 beta, 18-ether and dimeric derivatives of the naturally-occurring 18-hydroxysteroids have been determined in relation to major adrenal steroids. Procedures for extraction and reversed-phase HPLC of 18-hydroxysteroids from tissues without the formation of these less polar forms, which can complicate their separation by other chromatographic techniques, are illustrated with respect to a human malignant adrenocortical tumour which caused hypermineralocorticism.