Conjugation of androsterone by the guinea-pig kidney in vitro

Radioactive androsterone was incubated with kidney slices from guinea pigs in a Ringer bicarbonate buffer. For analysis, the radioactivity was partitioned between chloroform and water for the separation of non-polar and polar steroids. After multiple chromatography procedures, the non-polar fraction was shown to contain androsterone and at least four other similar steroids, not identified in this study. The polar fraction yielded numerous conjugates from which androsterone glucuronide and androsterone sulphate in approximately equal quantities were isolated and characterized.     

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.     

Use of monoclonal antibodies to separate the enantiomers of abscisic acid

The resolution of racemates often requires difficult and time consuming purification procedures. McAb technology allows the production of specific antibodies in quantities suitable for the preparation of matrices for large scale affinity purification. Here we report the rapid separation of abscisic acid (ABA) enantiomers by affinity chromatography using McAb. This method appears to be far superior to previously published separations based on crystallization, chromatography, and affinity purification with conventional antisera. The approach here described will be particularly attractive in a wide variety of similar situations.     

Nutrition and somatomedin--XII. Fractionation of somatomedins and somatomedin inhibitors in normal and diabetic rats

Bioassayable somatomedins and somatomedin inhibitors were examined after chromatographic separation, using serum from normal rats (enriched in somatomedins) and diabetic rats (enriched in somatomedin inhibitors). At neutral pH, gel filtration on Sephacryl S-300 revealed somatomedins at mol. wt approximately 140,000 (presumably carrier-bound) and inhibitors at mol. wts approximately 250,000, approximately 24,000 and approximately 1,000. At acid pH, gel filtration on Sephadex G-50 revealed somatomedins at mol. wt approximately 8,000 (presumably carrier-free) and a single inhibitor at mol. wt approximately 21,000. Ion exchange chromatography revealed that the inhibitor(s) may be more acidic than the somatomedins, but only low quantities of somatomedins were recovered. Sephadex G-50 fractionation was applied to pathophysiologic models in rats: 3 days of fasting were associated with a 62% fall in somatomedins and a 159% rise in inhibitors; 2 days of diabetes were associated with a 60% fall in somatomedins and a 344% rise in inhibitors. Since chromatography on Sephadex G-50 at pH 2.4 appears to provide adequate separation of somatomedins and somatomedin inhibitors with good estimated recovery of biological activity, this simple approach may be a probe useful in examining the regulation of somatomedins and somatomedin inhibitors in vivo.     

Sample purification using a C18-bonded reversed-phase cartridge for the quantitative analysis of corticosteroids in adrenal cell cultures by high-performance liquid chromatography or gas chromatography—mass spectrometry

Quantitative extraction and subsequent purification of small biological samples often involve cumbersome procedures. We have devised a short and efficient method for the quantitative extraction of the corticosteroid and the 20α reduced steroid series from culture medium containing 20% sera in a single, pure fraction with separation from cholesterol. Passage through a C₁₈-bonded reversed-phase Sep-Pak^® cartridge of the acidified culture medium and subsequent extraction of the steroid fraction with methanol yields a single fraction containing all steroids in 90% recovery and reduced quantities of cholesterol down to 30%. The extract can then be used without further purification for quantitative analysis by high-performance liquid chromatography or derivatized and analyzed by gas chromatography and gas chromatography—mass spectrometry.     

Steroids and hematopoiesis II. The effect of steroids on in vitro erythroid colony growth: Evidence for different target cells for different classes of steroids

Androgenic steroids and their non-androgenic 5p-H metabolites enhance the number of colonies of hemoglobin synthesizing cells grown from rat bone marrow in response to a standard (0.25 unitlml) concentration of erythropoietin. The target cells for two steroids were found to be different. Cells influenced by the androgen, fluoxymesterone (fluoxy), resembled cells responding to erythropoietin in their cycle characteristics, as measured by tritiated thymidine suicide, and in their physical characteristics, as determined by velocity sedimentation gradient separation. Cells responding to etiocholanolone (etio) had a much lower tritiated thymidine suicide rate and different sedimentation velocities. Reincubation of marrow cells with etio for two hours was sufficient to enhance erythroid colony growth by 84%, whereas a similar incubation with fluoxy produced no increment. These studies demonstrate that different classes of steroids may influence in vitro erythropoiesis by acting on distinct populations of marrow cells. Fluoxymesterone appears to act through cells already committed to respond to erythropoietin, while etiocholanolone appears to act on a separate, perhaps more primitive population of marrow cells.     

Gas chromatographic determination of unconjugated dehydroepiandrosterone, androstenedione, testosterone, pregnenolone and progesterone in human ovarian tissue.

A gas chromatographic method has been presented for the determination of unconjugated dehydroepiandrosterone, androstenedione, testosterone, pregnenolone and progesterone in human ovarian tissue. The procedure utilized radioactive tracers added to homogenate for correcting methodological loss, preliminary separation of steroids by thinlayer chromatography, acetylation, rechromatography on chromatoplate and gas chromatography on 3% SE-30 or 1% XE-60 columns with flame ionisation detection of steroids by using internal standards. Results of control experiments and representative clinical findings on normal and polycystic ovaries are reported.     

Affinity chromatography of endoglucanase of Trichoderma viride by concanavalin A-agarose.

Endoglucanase (C kappa cellulase) and cellobiase are often cross-contaminated in separation procedures by ion-exchange chromatography such as DEAE-cellulose. By using concanavalian A (Con A)-agarose chromatography, C kappa cellulase and cellobiase from Trichoderma virde can be separated. C kappa cellulase showed affinity toward Con A. indicating a glycoprotein containing alpha-D-mannopyransyl and alpha-D-glucopyranosyl end groups or internal 2-O-D-mannopyranosyl residues in sugar moieties. This method provides a way to estimate the quantities of C kappa enzyme produced by T. viride and possibly by other organisms.     

Fractionation of fecal neutral steroids by high performance liquid chromatography

Fecal neutral steroids were fractionated by high performance liquid chromatography (HPLC) into three major fractions: 5 beta-H, 3-keto steroids; 5 beta-H, 3 beta-hydroxy steroids; and 5 alpha-H and delta 5-3 beta-hydroxy steroids. This separation was achieved in about 10 minutes, with greater than 97% recovery of standards in each fraction. Gas-liquid chromatographic quantitation of fecal steroids fractionated by either HPLC or thin-layer chromatography gave nearly identical results. A method using both C18 reverse phase and silica HPLC to purify radiolabeled sterols is also described.     

Fluorometric labeling of tetrahydroprogesterones.

7-Diethylaminocourmarin-3-carbohydrazide was used to label the ketone group of the tetrahydroprogesterones to form fluorescent derivatives with high sensitivity. The four isomeric 3-hydroxypregnanes separated readily on high-performance, thin-layer chromatography after derivatization. This separation was not possible with the underivatized isomers. Standards and steroids from biologic mixtures were separated and showed similar characteristics. The methods used are described.     

High performance liquid chromatographic separation of steroids from ovarian follicles of fresh water perch Anabas testudineus: identification and characterization of the maturation-inducing hormone

Accurate separation and identification of steroids from the postvitellogenic ovarian follicles of Indian climbing perch Anabas testudineus was performed by high-performance liquid chromatography (HPLC) and specific radioimmunoassay (RIA). The steroids from such follicles, incubated in Cortland's saline with or without homologous fish pituitary extract (FPE), were extracted with dichloromethane and separated on a micro Bondapak C(18) column. Identification of the HPLC fractions was further confirmed by thin layer chromatography. As HPLC peaks for 17 alpha, 20 beta-dihydroxy-4-pregnen-3-one (DHP) and testosterone (T) were close, clear separation of these steroids and accurate measurement of their quantities was achieved by RIA of HPLC fractions using specific antibodies. Altogether, nine eluted fractions in the FPE-untreated and ten in FPE-treated samples were obtained. Of these, six were identified as: 5 beta-pregnan-3 alpha,17 alpha,20 beta-triol (5 beta-3 alpha,17 alpha,20 beta-P); DHP; T; 17 alpha-hydroxyprogesterone (17 alpha-P(4)); progesterone (P(4)); and androstenedione (AD). Three fractions from untreated and four from FPE-treated samples, however, remained unidentified. Of all the HPLC fractions examined for their relative maturational inducing (MI) potency on full grown (postvitellogenic) ovarian follicles of perch, the fraction identified as DHP was found to be the most effective inducer of germinal vesicle breakdown (GVBD) both at low and high concentrations. Fractions identified as 5 beta-3 alpha, 17 alpha, 20 beta-P and 17alpha-P(4) could induce only 32% and 20% GVBD at their highest concentration, while none of the unidentified fractions showed any MI activity. FPE caused increased production of DHP, testosterone, and 5 beta-3 alpha, 17 alpha, 20 beta-P. The qualitative differences between the fractions obtained from FPE-treated samples and those from FPE-untreated samples were only the appearance of a new polar metabolite of unknown function. The present study showed that, as a single steroid, DHP was the most potent MIH for A. testudineus.     

Isolation of secondary metabolites from roots of Plumbago auriculata Lam. by countercurrent chromatography

Centrifugal liquid-liquid partition chromatography presents significant advantages for the separation and purification of plant metabolites owing to the short operational time of the process and the elimination of possible irreversible adsorption of compounds. The crude chloroform extract from roots of Plumbago auriculata was analysed by countercurrent chromatography using hexane:ethyl acetate:methanol:water (40:10:10:2, v/v) as solvent system. The isolation of the naphthoquinones plumbagin and epi-isoshinanolone, the steroids sitosterol and 3-O-glucosylsitosterol, plumbagic and palmitic acids was easily achieved. Naphthoquinones are typical components of Plumbago species and they show interesting biological activities.     

Preparation of enterochelin from Escherichia coli.

A convenient method has been developed for the preparation of enterochelin, the natural iron carrier produced by Escherichia coli. The method employs a mutant strain which is unable to transport the ferric-enterochelin complex into the cell and which excretes large quantities of enterochelin into the culture medium. The addition of excess iron to the medium allows the enterochelin to accumulate as the ferric-enterochelin complex which is purified by ion-exchange chromatography and then dissociated and the free enterochelin further purified by differential extraction and crystallization. The enterochelin is isolated in good yield and appears to be of high purity as judged by a number of criteria.     

Sensitive detection of specific repair endonucleases: radial diffusion assay utilizing differential alkaline denaturation of supercoiled and nicked PM2 DNA in agarose gels

An improved method for separation and quantitation of sulfated neutral and acidic steroids in human feces was developed. The procedure consists of separation of sulfated steroids on Sephadex LH-20 and hydrolysis by cholylglycine hydrolase followed by quantitation and identification of the trimethylsilylether derivatives by gas-liquid chromatography and gas-liquid chromatography-mass spectroscopy. Using this procedure, we detected no sulfated bile acids in human feces. However, sulfated cholesterol was detected in the sulfated bile acid fraction obtained from human fecal extracts. Analysis showed that cholesterol sulfate comprised 12.3, 11.2, and 31.0% of the total neutral sterol fraction in the three fecal samples. Using our procedures, cholesterol sulfate and bile acid sulfates in a biological mixture can be quantitated and identified when they are present.     

Bioconversion of 2 alpha-hydroxyprogesterone to 2-hydroxylated corticosteroids by newborn rat adrenal cells in primary culture

The bioconversion of 2 alpha-hydroxyprogesterone into 2-hydroxylated steroids was accomplished using newborn rat adrenal cells in primary culture. The products were purified using column and thin-layer chromatography, and identified by GC-MS. They resulted principally from the enzymatic reactions of 21-hydroxylation, 11 beta-hydroxylation, reduction of 20-oxo and 3-oxo groups, and epimerization of the substrate. In addition, minor metabolites resulted from 18-hydroxylation, 6 beta-hydroxylation and reduction of the 3-oxo-4-ene group. The identification of these compounds allowed us to conclude that the metabolism of 2 alpha-hydroxyprogesterone is similar to that of progesterone in this cellular system. Assuming that the 2 beta-epimers of the different metabolites arose principally from the transformation of 2 beta-hydroxyprogesterone, the specificity of the various enzyme systems seems to be similar for both epimers except in the case of the 11 beta-hydroxylation where the reaction appears stereospecific for the 2 beta-epimer. The 2 alpha-hydroxyl group on ring A seems to favor the reduction of the 3-oxo group and it does this stereospecifically to the 3 beta-structure. The epimerization of the substrate, which is most likely enzymatically induced, is the first example of steroid epimerization reported in the adrenal. This is a practical preparative method for synthesizing a variety of steroids hydroxylated at C-2 from a single substrate and could be adjusted to the production of important quantities of 2-hydroxylated metabolites of corticosteroids.     

Protein/enzyme inactivation during different chromatographic methods of separation.

Protein denaturations encountered during the different types of chromatographic separations are presented. The analysis of different protein denaturations presented along with the causes of such denaturations provides a judicious framework to compare protein denaturations encountered by such separation techniques. Especially of interest are those studies which compare the mass recovery of proteins and the retention of activity by different chromatographic techniques. Reversed-phase chromatography is presented even though it is utilized nowadays only for specialized cases such as separation of small peptides. It appears that relatively mild interactions that are encountered generally in hydrocarbon-interaction chromatography are favorable to the preservation of the native (active) protein state. The few available mechanistic studies presented provide judicious physical insights into protein conformational behavior on chromatographic columns.     

Bioconversion of 2α-hydroxyprogesterone to 2-hydroxylated corticosteroids by newborn rat adrenal cells in primary culture

The bioconversion of 2α-hydroxyprogesterone into 2-hydroxylated steroids was accomplished using newborn rat adrenal cells in primary culture. The products were purified using column and thin-layer chromatography, and identified by GC-MS. They resulted principally from the enzymatic reactions of 21-hydroxylation, 11β-hydroxylation, reduction of 20-oxo and 3-oxo groups, and epimerization of the substrate. In addition, minor metabolites resulted from 18-hydroxylation, 6β-hydroxylation and reduction of the 3-oxo-4-ene group. The identification of these compounds allowed us to conclude that the metabolism of 2α-hydroxyprogesterone is similar to that of progesterone in this cellular system. Assuming that the 2β-epimers of the different metabolites arose principally from the transformation of 2β-hydroxyprogesterone, the specificity of the various enzyme systems seems to be similar for both epimers except in the case of the 11β-hydroxylation where the reaction appears stereospecific for the 2β-epimer. The 2α-hydroxyl group on ring A seems to favor the reduction of the 3-oxo group and it does this stereospecifically to the 3β-structure. The epimerization of the substrate, which is most likely enzymatically induced, is the first example of steroid epimerization reported in the adrenal. This is a practical preparative method for synthesizing a variety of steroids hydroxylated at C-2 from a single substrate and could be adjusted to the production of important quantities of 2-hydroxylated metabolites of corticosteroids.     

Method development for corticosteroids and anabolic steroids by micellar liquid chromatography

A systematic optimization of the HPLC separation of a complex mixture containing urinary steroids (anabolics and corticoids), boldenone and bolasterone (synthetic anabolics) by micellar liquid chromatography has been carried out. The isocratic micellar mobile phases (from binary to quaternary) consisted of sodium dodecyl sulphate and organic modifiers such as acetonitrile, tetrahydrofuran, propanol, butanol or pentanol. The effect of the organic modifiers, surfactant concentration, temperature, ionic strength and flow-rate on the separation has been studied. A micellar mobile phase made of 5% propanol and 40 mM surfactant allowed the separation of 13 steroids in about 23 min. A bivariant optimization method for the micellar mobile phase surfactant-propanol corroborated the above results. The separations obtained show good perspectives for future developments.     

Errata

Trichloroacetic acid extracts of red cell often produce an iron-ATP complex after ion exchange chromatography of the extract amounting to about $\text{1}\text{3}$ of the total ATP. In the present work the presence of 14–50% of iron-ATP in such extracts from human and Rhesus monkey blood has been shown.Experiments designed to clarify the possible role and origin of iron-ATP revealed that non-acid treatment of human blood or red cells, as in the freeze-thaw process, followed by separation on a Sephadex column did not produce an iron-ATP fraction. In addition, purified hemoglobin and ATP were combined and incubated at pH 7.4. After Sephadex chromatography, there was no evidence of an iron-ATP fraction. However, similar combinations of incubated hemoglobin and ATP treated with trichloroacetic and separated by ion exchange chromatography did produce an iron-ATP fraction similar to that obtained from acid-extracted blood.It appears that iron-ATP in quantities found in acid-extracted blood is the result of iron release from hemoglobin and the subsequent complexing of such iron with available ATP.     

Separation of drugs by packed-column supercritical fluid chromatography

Packed-column supercritical fluid chromatography (pSFC) has been expected to analyze various kinds of compounds. Many researchers have expected a new chromatographic technique that overcomes the limitations of other techniques, HPLC and GC. In pharmaceutical development, chromatography plays an important role in the evaluation of safety and efficacy of a new compound. This article provides an overview of the separation of drugs by pSFC. The effects of the chromatographic parameters were studied for the separation of steroids. In chiral separation, the successful results were shown and compared with HPLC.