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Mainly skatole and androstenone have so far been considered causative for boar taint. Using a mixed methods approach it is shown herein that 2-aminoacetophenone (AAP) affects human perception of pork, too. We explored the importance of AAP in four trials: (1) chemical analyses of 221 fat samples from boar carcasses revealed that AAP occurs, on average, in similar quantities as skatole while the levels of androstenone being four-fold. (2) ranking tests with mixtures of androstenone and/or skatole with AAP presented on smell strips to trained sensory assessors showed that AAP amplifies boar odour. In order to study AAP's importance in meat products, four experimental variants of Lyon type sausage were then produced: a control, a product with added skatole (0.075 μg/g fat tissue), with added AAP (0.075 μg/g fat tissue), and with addition of both compounds. (3) results of a consumer discrimination test panel (n = 71) showed that, when added to a sausage system, APP causes a sensory difference of similar size as skatole while the methodology chosen affects the effect size: tetrad tests proved to be more sensitive than duo trio difference tests, in the tetrad test a sensory difference expressed as d' (d-prime) of 1.0 was reached. (4) a hedonic consumer test (n = 121) finally revealed that APP decreased consumer liking of the APP-spiked sausage - even to a stronger extent than skatole. APP caused significant drops in smell, taste, mouth-feel, after-taste and overall liking in Lyoner. Overall the findings suggest that, in the context of pork meat, AAP is of similar olfactory importance as skatole., Competing Interests: Declaration of competing interest None., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Urinary sulfate fraction of the anabolic androgenic steroids is not analyzed routinely in anti-doping analyses but has demonstrated in the last years an increasing interest among the anti-doping community. Sulfate conjugates are linked to plasma proteins increasing the residence time in the body compared to glucuro-conjugated metabolites, and then their analyses may allow improving the detection time window of specific metabolites. Hydrolysis of sulfates can be made enzymatically or chemically and can be challenging, depending on the strategy selected., Methods: Hydrolysis by solvolysis was validated for metabolic studies, focusing on setting a quality control able to assess the hydrolytic step. To the internal standards mixture, androsterone-D4 and etiocholanolone-D5 sulfate were added. The proposed protocol was applied over samples collected after dehydroepiandrosterone (DHEA) administrations., Results: The stability of the structures showed good results, and no evident formation of degradation products was observed. Internal standard to monitor the efficiency of hydrolysis, recovery, and retention time was successfully introduced. Additional analytes (4β-hydroxy-DHEA, 5-androstene-3β,17β-diol and 5α-androstane-3β,17β-diol) were found to be affected besides of DHEA and epiandrosterone (epiA) as previously described., Conclusions: Results in terms of linearity, precision, and accuracy, showed that the method is suitable to quantify seven analytes in urine in the sulfated fraction. The validated method was successfully applied to urine samples after administration of DHEA to detect this compound in the sulfate fraction and preliminarily to negative samples from athletes of both sexes, to determine Q1 and Q3 inter-quartiles. A quality control assessment for the hydrolysis efficiency was established for every individual sample., (Copyright © 2020 Elsevier Inc. All rights reserved.)

While forming mixtures is a widely used approach for other raw materials in food industry, it has not yet been systematically analyzed for boar tainted meat. That is why we simultaneously studied four factors relevant for the production of emulsion-type sausages: percentage boar meat (skatole concentrations up to 0.3 μg/g, androstenone up to 3.8 μg/g in melted backfat), duration of traditional smoke and concentration levels of two spices. 16 variants of Frankfurters were produced in two independent studies and evaluated by in total 211 consumers. A linear mixed effects model revealed that increased levels of boar tainted meat significantly reduced consumer acceptance which could not be compensated by increased smoke or spice levels. We propose a non-inferiority test to identify the mixture which is similarly accepted as the reference made without boar tainted meat. Up to 33% tainted boar meat is proposed, assuming a liking drop of 0.5 on a 9 point liking scale as benchmark for an inferior product., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

This study investigated the effects of ageing period (2 or 7days), endpoint temperature (70 or 75°C), cut type (loin (M. longissimus thoracis et lumborum), silverside (M. biceps femoris), blade (M. triceps brachii) and chuck tender (M. supraspinatus)), cooking method (roast and stir fry (all cuts) and steak (loin only)) on eating quality attributes of pork from entire and immunocastrated male pigs. Higher intramuscular fat levels were found in all cuts from immunocastrated males compared with entire males. Of the seven cut type×cooking method combinations evaluated, shoulder cuts had the lowest fail rate (P<0.001) and loin steak and silverside cuts scored lowest (P<0.001) for tenderness, juiciness, overall liking, quality grade and re-purchase intention. Although no differences in sensory scores due to gender were observed, boar taint was identified in 10% of entire males. Minimising adverse risks in eating quality due to boar taint in males remains a key industry issue to ensure consistent delivery of high quality pork., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

3α-Hydroxysteroid dehydrogenase/carbonyl reductase (3α-HSD/CR) catalyzes the oxidation of androsterone with NAD + to form androstanedione and NADH with the rate limiting step being the release of NADH. In this study, we elucidate the role of remote substrate binding interactions contributing to the rate enhancement by 3α-HSD/CR through steady-state kinetic studies with the truncated substrate analogs. No enzyme activity was detected for methanol, ethanol, and 2-propanol, which lack the steroid scaffold of androsterone, implying that the steroid scaffold plays an important role in enzyme catalytic specificity. As compared to cyclohexanol, the activity for 2-decalol, androstenol, and androsterone increases by 0.9-, 90-, and 200-fold in k cat , and 37-, 1.9 × 10 6 -, and 1.8 × 10 6 -fold in k cat /K B , respectively. The rate limiting step is hydride transfer for 3α-HSD/CR catalyzing the reaction of cyclohexanol with NAD + based on the observed rapid equilibrium ordered mechanism and equal deuterium isotope effects of 3.9 on V and V/K for cyclohexanol. The k cat /K B value results in ΔG ‡ of 14.7, 12.6, 6.2, and 6.2 kcal/mol for the 3α-HSD/CR catalyzed reaction of cyclohexanol, 2-decalol, androstenol, and androsterone, respectively. Thus, the uniform binding energy from the B-ring of steroids with the active site of 3α-HSD/CR equally contributes 2.1 kcal/mol to stabilize both the transition state and ground state of the ternary complex, leading to the similarity in k cat for 2-decalol and cyclohexanol. Differential binding interactions of the remote BCD-ring and CD-ring of androsterone with the active site of 3α-HSD/CR contribute 8.5 and 6.4 kcal/mol to the stabilization of the transition state, respectively. The removal of the carbonyl group at C17 of androsterone has small effects on catalysis. Both uniform and differential binding energies from the remote sites of androsterone compared to cyclohexanol contribute to the 3α-HSD/CR catalysis, resulting in the increases in k cat and k cat /K B ., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Boar taint is a contemporary off-odor present in meat of uncastrated male pigs. As European Member States intend to abandon surgical castration of pigs by 2018, this off-odor has gained a lot of research interest. In this study, rapid evaporative ionization mass spectrometry (REIMS) was explored for the rapid detection of boar taint in neck fat. Untargeted screening of samples (n=150) enabled discrimination between sow, tainted and untainted boars. The obtained OPLS-DA models showed excellent classification accuracy, i.e. 99% and 100% for sow and boar samples or solely boar samples, respectively. Furthermore, the obtained models demonstrated excellent validation characteristics (R 2 (Y)=0.872-0.969; Q 2 (Y)=0.756-0.917), which were confirmed by CV-ANOVA (p<0.001) and permutation testing. In conclusion, in this work for the first time highly accurate and high-throughput (<10s) classification of tainted and untainted boar samples was achieved, rendering REIMS a promising technique for predictive modelling in food safety and quality applications., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Yeast inoculation of dry fermented sausages manufactured with entire male fat was evaluated as a strategy to improve sausage quality. Four different formulations with entire male/gilt back fat and inoculated/non-inoculated with Debaryomyces hansenii were manufactured. The use of entire male back fat produced the highest weight losses, hardness and chewiness in dry sausages. Consumers clearly distinguished samples according to drying time and D. hansenii inoculation while the use of entire/gilt back fat was not highly perceived. The presence of androstenone and skatole was close to their sensory thresholds. Androstenone was not degraded during the process but skatole was affected by yeast inoculation. D. hansenii growth on the surface regulated water release during ripening, reduced hardness and chewiness in entire male sausages and resulted with similar texture to gilt sausages. Yeast inoculation inhibited lipid oxidation providing fruity odours and less oxidized fatty sausages in the sensory analysis. The effectiveness of yeast to mask boar taint was demonstrated by sensory analysis., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Direct electrochemistry and bioelectrocatalysis of a newly discovered C-19 steroid 1α-hydroxylase (CYP260A1) from the myxobacterium Sorangium cellulosum So ce56 were investigated. CYP260A1 was immobilized on screen-printed graphite electrodes (SPE) modified with gold nanoparticles, stabilized by didodecyldimethylammonium bromide (SPE/DDAB/Au). Cyclic voltammograms in argon-saturated substrate free 0.1 M potassium phosphate buffer, pH 7.4, and in enzyme-substrate complex with androstenedione demonstrated a redox processes with a single redox couple of E(0') of -299 ± 16 mV and -297.5 ± 21 mV (vs. Ag/AgCl), respectively. CYP260A1 exhibited an electrocatalytic activity detected by an increase of the reduction current in the presence of dissolved oxygen and upon addition of the substrate (androstenedione) in the air-saturated buffer. The catalytic current of the enzyme correlated with substrate concentration in the electrochemical system and this dependence can be described by electrochemical Michaelis-Menten model. The products of CYP260A1-depended electrolysis at controlled working electrode potential of androstenedione were analyzed by mass-spectrometry. MS analysis revealed a mono-hydroxylated product of CYP260A1-dependent electrocatalytic reaction towards androstenedione., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Characteristic off-flavours may occur in uncastrated male pigs depending on the accumulation of androstenone and skatole. Feasible processing of strongly tainted carcasses is challenging but gains in importance due to the European ban on piglet castration in 2018. This paper investigates consumers' acceptability of two sausage types: (a) emulsion-type (BOILED) and (b) smoked raw-fermented (FERM). Liking (9 point scales) and flavour perception (check-all-that-apply with both, typical and negatively connoted sensory terms) were evaluated by 120 consumers (within-subject design). Proportion of tainted boar meat (0, 50, 100%) affected overall liking of BOILED, F (2, 238)=23.22, P<.001, but not of FERM sausages, F (2, 238)=0.89, P=.414. Consumers described the flavour of BOILED-100 as strong and sweaty. In conclusion, FERM products seem promising for processing of tainted carcasses whereas formulations must be optimized for BOILED in order to eliminate perceptible off-flavours. Boar taint rejection thresholds may be higher for processed than those suggested for unprocessed meat cuts., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Consumer liking was assessed for streaky bacon and pork belly roll from entire male pigs with an androstenone (AND) content of up to 9.4 ppm and a skatole (SKA) content of up to 0.92 ppm in the back fat and castrates. No clear effect of either AND or SKA was seen in consumer liking, although an insignificant tendency was seen for SKA. A sensory profile analysis showed that AND increased the boar taint of bacon, while both AND and SKA increased the boar taint of the pork belly roll. Consumer sensitivity towards AND and SKA did not affect liking of the meat products. The lack of effect of AND and SKA on consumer liking could be due to a masking effect of the spices and smoke. Three consecutive weeks' exposure to bacon did not change the liking score, irrespective of the AND and SKA content. This indicates that the consumers did not become more sensitive towards boar taint., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The aim of this study was to evaluate the performance of various boar taint detection methods, measure the relationship between them and identify possible points of improvement for boar taint detection. The methods used to evaluate boar taint in the carcasses of 448 entire male pigs and 17 barrows were the hot iron method (n = 442), a standardised (n = 323) and home (n = 58) consumer meat-evaluation panel, an expert panel assessment of meat and fat (n = 464) and laboratory analysis of skatole, androstenone and indole in fat (n = 464). The axillary odour of a number of slaughtered entire male pigs was also investigated (n = 231). As correlation coefficients were generally weak, a positive result for one of these detection methods did not per se result in a positive result for all other methods. Results of one detection method could not be generalised. The choice to use one or more detection methods deserves consideration depending on the aim of the study. In this paper, we suggest some possible improvements for evaluating boar taint with a consumer panel based on our results and experience. The home consumer evaluation was correlated with the concentration of indole (r = 0.27) but not with skatole or androstenone. We therefore recommend that lab analyses include indole testing. The hot iron method seems to be an easy and fast detection method, which yields comparable or better correlation coefficients with the other detection methods than an expert panel evaluating fat samples. However, the reliability of the hot iron method depends on the training and reliability of one or two assessors. Efforts should be made to further optimise this method by evaluating the effect of testing conditions. The axillary odour score was moderately correlated with the other detection methods (up to 0.32). More research is needed to evaluate the possibilities of axillary odour as a boar taint detection method.

A 3α-hydrosteroid biosensor for androsterone determination has been prepared by immobilizing the enzyme 3α-hydroxysteroid dehydrogenase (3α-HSD) in a composite electrode platform constituted of a mixture of multi-walled carbon nanotubes (MWCNTs), octylpyridinium hexafluorophosphate (OPPF(6)) ionic liquid and NAD(+) cofactor. This configuration allowed the fast, sensitive and stable electrochemical detection of the NADH generated in the enzyme reaction. All the experimental variables involved in the preparation and performance of the enzyme biosensor were optimized. Amperometry in stirred solutions at +400 mV provided a linear calibration plot for androsterone in the 0.5-10 μM concentration range with a slope value more than 200-times higher than that previously reported. The detection limit achieved was 0.15 μM and a low value of the apparent Michaelis-Menten constant (K(app)(M)), 36.0 μM, similar to that reported for the enzyme in solution, was calculated. The 3α-HSD/MWCNTs/OPPF(6)/NAD(+) biosensor provided good results in the determination of androsterone in spiked human serum samples., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Consumer acceptability of entire male pork at eating was assessed in three experiments. The 140 consumers involved in each experiment were classified as insensitive (INSENS) to the odor of pure androstenone or sensitive perceiving it as pleasant (SENS-PLEA) or unpleasant (SENS-UNPL). Entire male pork with very low skatole and androstenone levels (LS-LA) was as well accepted as gilt pork, whatever the consumer category. Entire male pork with elevated levels in both skatole and androstenone (HS-HA) was clearly differentiated from LS-LA pork by SENS-UNPL, but not by SENS-PLEA or INSENS consumers. Whatever the consumer category, entire male pork with elevated levels of androstenone and very low levels of skatole (LS-HA and LS-HHA) were not significantly differentiated from LS-LA pork. The results suggest that, in the conditions of the present experiment, androstenone and skatole totally explain boar taint at eating and that the acceptability threshold for androstenone, in the absence of skatole, is in the range of 2-3 μg/g liquid fat., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

This comprehensive review describes the analytical methods developed for quantification of the boar taint compounds skatole and androstenone in porcine adipose tissue. The following parts are considered; sampling, sample preparation, calibration and instrumentation. Additionally, method performance characteristics and level of validation of the existing methodology are discussed. It is concluded that there is a need for further validation of existing methods and need for standardisation of methodology to quantify boar taint compounds. Facing a possible near future ban of castration of male piglets would enforce further method harmonisation in this field., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Due to the impact in the media and the requirements of sensitivity and robustness, the detection of the misuse of forbidden substances in sports is a really challenging area for analytical chemistry, where any study focused on enhancing the performance of the analytical methods will be of great interest. The aim of the present study was to evaluate the usefulness of using hydrogen instead of helium as a carrier gas for the analysis of anabolic steroids by gas chromatography-mass spectrometry with electron ionization. There are several drawbacks related with the use of helium as a carrier gas: it is expensive, is a non-renewable resource, and has limited availability in many parts of the world. In contrast, hydrogen is readily available using a hydrogen generator or high-pressure bottled gas, and allows a faster analysis without loss of efficiency; nevertheless it should not be forgotten that due to its explosiveness hydrogen must be handled with caution. Throughout the study the impact of the change of the carrier gas will be evaluated in terms of: performance of the chromatographic system, saving of time and money, impact on the high vacuum in the analyzer, changes in the fragmentation behaviour of the analytes, and finally consequences for the limits of detection achieved with the method., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The objective of this study was to determine the effect of using a gonadotropin-releasing factor (GnRF) vaccine on follicle-stimulating hormone (FSH) and luteinizing hormone (LH) concentrations in plasma, the size of testicles, and the expression of boar taint in male pigs. Vaccinated pigs were compared with surgically castrated pigs and entire males. Pigs were randomly assigned to three treatment groups: surgically castrated during the first week of life (T01, n=274), immunized twice during the fattening period with a GnRF vaccine, the first when 13 to 14 wk of age and the second when 20 to 21 wk of age (T02, n=280), and entire males (T03, n=56). From a subgroup of both T01 and T02 and from all pigs of group T03, blood samples were collected immediately before second vaccination (T02) and again before slaughter at either 24 to 25 or 26 to 27 wk of life to determine the plasma concentrations of LH and FSH. Testicles were removed after slaughter and their size was determined. Meat and fat samples from all pigs of T02 and T03 as well as 25% of the pigs of T01 were examined with the cold cooking and fat melting test. Immediately before the second vaccination (T02 only), LH and FSH concentrations were not significantly different between T02 and T03. However, LH and FSH concentrations were significantly higher in T01 compared with T02 and T03. Before the first slaughter date, LH and FSH concentrations were significantly lower in T02 than in T03. Testicle size was significantly lower in T02 compared with that in T03. In T02, 98% (235 of 239) of the samples were rated negative for boar taint by the cooking test, whereas in T03, 94% (48 of 51) were rated positive. In the fat melting test, 97% of T02 were rated negative and 3% (7 pigs) were rated positive, including the pigs tested positive in the cold cooking test. In T03, 94% were rated positive. All pigs (7 of 239) in T02 that were positive for boar taint in the cooking or melting test and that were tested had androstenone and skatole concentrations in backfat below threshold levels of 1 microg/g and 0.2 microg/g, respectively.

Together with steroid glucuronides, sulfoconjugates may be used as markers of steroid administration as well as endogenous steroid production. A fast and sensitive analytical procedure has been developed for the simultaneous separation, determination and quantification of sulfate and glucuronide derivatives of testosterone (T), epitestosterone (E), androsterone (A), etiocholanolone (Etio) and dehydroepiandrosterone (DHEA) in human urine. First, a weak anion-exchange solid-phase extraction support (SPE Oasis WAX) was used for complete and rapid separation of sulfates and glucuronides in two extracts after loading of urine sample (2 mL). Then sulfates were analyzed directly by high-performance liquid chromatography-ion-trap mass spectrometry (LC-MS/MS) with electrospray ionization in negative mode. Chromatographic separation of the targeted sulfoconjugates was achieved using a Waters XBridge C18 column (150 mm x 4.6 mm I.D., 5 microm) with gradient elution. Assay validation demonstrated good performance for instance for T sulfate (TS) and E sulfate (ES) in terms of trueness (89-107%), repeatability (3.4-22%) and intermediate precision (5.8-22%) over the range of 2-200 ng/mL (corresponding to 1.5-147 ng/mL as free steroids). Results obtained on biological samples demonstrated the suitability of this analytical strategy for direct measurement of androgen sulfoconjugates and glucuroconjugates in human urine.

A short and efficient synthesis of pentadeuterated 2,2,3,4,4-d5-19-nor-5alpha-androsterone 7 starting from 19-norandrost-4-ene-3,17-dione 1 by a d1-L-Selectride mediated stereo- and regioselective reduction of the 3-keto group is presented. The use of compound 7 as internal standard for the detection of anabolic steroids via mass spectrometric techniques such as gas chromatography-mass spectrometry (GC-MS) is discussed.

Androstenone (5 alpha-androst-16-en-3-one) is a steroid pheromone produced in the testis. Excessive accumulation of androstenone together with skatole (3-methyl-indole) in the adipose tissue of some male pigs leads to "boar taint". In isolated pig hepatocytes androstenone represses the expression of cytochrome P450IIE1 (CYP2E1), the enzyme principally responsible for skatole metabolism. Androstenone can be metabolised in liver microsomes but the pathway has not been established. We have investigated androstenone metabolism in liver microsomes from two breeds of pigs exhibiting low and high levels of androstenone in adipose tissue-Large White (LW) and Meishan (M), respectively. Androstenone was reduced in isolated liver microsomes mainly to beta-androstenol using NADH as a co-factor. The rate of beta-androstenol formation in the presence of NADPH was very low. In microsomes from LW pigs the rate of beta-androstenol formation from androstenone was six times higher than in M pigs. 3beta-hydroxysteroid dehydrogenase (3beta-HSD) was investigated as a likely candidate for the enzyme catalysing androstenone reduction in pig liver. RT-PCR analysis showed that there was no sequence difference in the cDNA encoding 3beta-hydroxysteroid dehydrogenase from LW and M pigs. However, competitive RT-PCR analysis showed that the expression of 3beta-hydroxysteroid dehydrogenase mRNA was about 12 times higher in the case of LW compared to M pigs. It is concluded that the rate of androstenone metabolism in pig liver microsomes is determined by the level of expression of hepatic 3beta-hydroxysteroid dehydrogenase. The differential expression of this enzyme could be a factor affecting the rate of hepatic androstenone metabolism which in turn may influence the level of hepatic CYP2E1 expression and hence the rate of hepatic skatole metabolism.

The 5alpha-reduced androgens have been implicated as antagonists of follicular development. In this experiment, we examined the effect of active immunization against 5alpha-reduced androgen on follicular development in ewes. During the breeding season, cyclic Merino ewes were either actively immunized three times against 5alpha-androstane-3alpha,17beta-diol (3alpha-diol) or served as controls. Six to nine weeks after the last immunization, they were treated with PGF(2alpha) analog (PG, 125mg cloprostenol i.m.) and luteolysis was induced. Fourteen days after the PG treatment, the ewes were either killed (mid-luteal phase) or treated a second time with PG and killed 24h later (early follicular phase). At slaughter, blood samples were collected and ovaries recovered. All CL and follicles larger than 2mm were dissected and their size and appearance were recorded. Follicular fluid was collected and concentrations of estradiol-17beta (E(2)), progesterone (P), androstenedione (A(4)), testosterone (T), dihydrotestosterone (DHT), 5alpha-androstane-3alpha-ol,17beta-one (androsterone: 3alpha-ol) and 3alpha-diol were determined by RIA. Immunization induced antibodies primarily to DHT and its 5alpha-reduced substrates 3alpha-diol and 3alpha-ol but not to E(2), P, A(4) or T. Immunization increased ovulation rate, size of ovulatory follicles and weight of CL. Immunization appeared to increase ovulation rate by decreasing the incidence of atresia in large preovulatory follicles. Regardless of their physiological status follicles contained only low levels of DHT; 3alpha-ol and 3alpha-diol were not detected in most follicles. Immunization did not appear to affect levels of DHT or other steroids in the follicular fluid. In conclusion, the induction of antibodies to 5alpha-reduced androgens increases ovulation rate by enhancing follicular viability during the preovulatory period in ewes. However, this effect is not brought about by the direct immune-neutralization of DHT or its 5alpha-reduced substrates 3alpha-ol and 3alpha-diol at the ovarian level.

In order to elucidate the mechanism of action of androgen in human hair follicles, we studied testosterone metabolism by cultured beard outer root sheath cells in comparison with that of epidermal keratinocytes. When cells were incubated as a monolayer in serum-free keratinocyte growth medium with physiological concentrations (25 nM) of [3H]testosterone, the major metabolite was androstenedione in either type of cells. Small amounts of androstanedione, dihydrotestosterone and androsterone were also formed. The ratio of apparent 5 alpha-reductase to 17 beta-hydroxy-steroid dehydrogenase activities ranged from 0.11 to 0.81 and did not differ between these two kinds of cells. After these cells were cultured in the presence of 10% fetal calf serum for 10 days, the activities of both metabolic pathways markedly increased without significant changes of the ratio of activities of these two enzymes. Thus, testosterone was rather converted to weak androgens even in beard outer root sheath cells under the experimental conditions.

Androgen metabolism is one of the major keys for a better understanding of conditions such as androchronogenetic alopecia, acne, and other androgen-related skin disorders. This paper addresses the process by which testosterone metabolism leads to the following major androgens: androstenedione, dihydrotestosterone, 3 alpha- and 3 beta-androstanediol, androsterone, epiandrosterone, and androstanedione. Our report describes a methodology developed for the direct quantitative measurement from the silica gel plate of these metabolites. After detailing the chromatographic procedures to achieve the complete separation of the seven testosterone metabolites on a single plate, specifications are given for obtaining accurate measurements by 1) calibration of the radiodetectors and 2) internal and external standardization of samples and plates. Analytical criteria are discussed in terms of comparison of the level of sensitivity, reproducibility, and practicability obtained by both the linear analyzer and the direct digital autoradiograph. Signals as weak as 25 dpm were easily detected and calibration curves were obtained for the range of 50-500 dpm. For biological measurements the coefficients of variation do not exceed 10%. Given the difficulty of obtaining large amounts of microdissected subfractions of the pilosebaceous unit and the necessity of evaluating the complete pattern of testosterone transformed into its 3 alpha,3 beta-, 5 alpha-reduced and 17 beta-dehydrogenated secondary derivatives, digital autoradiography appears to be a powerful yet simple tool for studying androgen metabolism. In addition, this methodology offers an important environmental advantage: the high sensitivity of the detectors makes it possible to minimize the quantities of radioactive materials that must be handled or discarded.

In 28 breast cyst fluids obtained from 20 patients (age 29-65 years) sodium, potassium and the sulfates (S) of estrone (E1), estradiol (E2), dehydroepiandrosterone (DHEA) and androsterone (A) were determined. The radioimmunoassays (RIA) used were validated for this particular biological fluid. According to electrolyte ratio (Na+/K+) the cyst fluids were subdivided into two groups: the first with low (less than 3) (n = 16) and the other with high (greater than 3) (n = 12) values. Markedly higher steroid sulfate levels were observed in the first group, the mean levels being: 147.7 nmol/l, 54.6 nmol/l, 108.1 mumol/l and 158.0 mumol/l for E1S, E2S, DHEAS and AS respectively. The mean levels in the second group were: 13.6 nmol/l, 6.7 nmol/l, 68.8 mumol/l and 33.6 mumol/l for E1S, E2S, DHEAS and AS, respectively. In the first group only E1S and E2S levels were significantly correlated (r = 0.51; P less than 0.05). Conversely, the steroid sulfate levels were significantly correlated with each other in the group with high electrolyte ratio. These data have confirmed preceding results and have clearly shown that breast cyst fluids with low electrolyte ratio contain more E2S than the other group. This finding might be correlated with the fact that patients with these breast cysts lined by with apocrine epithelium may be at a greater risk of breast cancer than those with the other type.

A digital autoradiographic-thin-layer chromatographic method involving simple steps is described for thorough separation of eight major androgens (testosterone, androstenedione, dihydrotestosterone, androsterone, epiandrosterone, androstanedione, 3 alpha-androstanediol and 3 beta-androstanediol) derived from testosterone metabolism. Their direct quantification is performed by radioscanning, which avoids tedious plate-scraping and liquid scintillation counting. Satisfactory accuracy is obtained by both external standardization of plates by calibrated amounts of radiolabelled standard steroids and internal standardization of bioassays by radiolabelled squalane. Coefficients of variation are below 8.5% in the range 100-500 dpm. Some analytical criteria related to chromatographic conditions and quantification parameters depending on position-sensitive proportional counter are discussed.

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.

Steroids were derivatized with dimethylthiophosphinic chloride to produce steroidal thiophosphinic esters. A catalyst, 4-dimethylaminopyridine, was used to promote quantitative and reproducible thiophosphinic ester formation at low reaction temperatures. The derivatives were analyzed by capillary supercritical fluid chromatography (SFC) with phosphorus thermionic detection. The phosphorus thermionic detector exhibited linearity over 3-4 orders of magnitude. A sensitivity of 120 fg P/s was obtained for the dithiophosphinic ester of pregnanediol at a signal-to-noise ratio of 3. The efficiency of capillary SFC and the excellent sensitivity of the phosphorus thermionic detector were demonstrated by the analysis of steroids isolated from both human urine and plasma.

Androsterone (3alpha-hydroxy-5alpha-androstan-17-one), 5alpha-androstane-3alpha, 17beta-diol and 5alpha-androstane-3beta, 17beta-diol were conjugated at C-16 through sulfur to bovine and human serum albumin. Rabbits injected with these conjugates produced antibodies suitable for radioimmunoassays of these hormone metabolites. Samples were purified on Sephadex LH-20 columns. Levels of these steroids were measured in a rat blood serum pool and in ovarian tissue extract pools.

High-pressure liquid chromatography (HPLC) should be used more frequently in biomedical research because heavy molecules can be analysed at ambient temperature without derivatisation to volatile compounds. In this paper the separation of four free 17-ketosteroids, their ester sulfates and glucuronides by reversed-phase HPLC on a Micropak CH column is described. In order to improve the separation several parameters have been studied. Retention data allow comparison of the hydrophilic behaviour of free and conjugated steroids.

Analysis of the secretion of the human apocrine gland has shown the presence of dehydroepiandrosterone and androsterone sulfates, two androgen steroids previously identified in axillary sweat. These steroid sulfates were characterized by the gas chromatographic/mass spectrometric analysis of the odorous steroids formed on direct injection of the apocrine secretion into the host gas chromatographic injector. No spectral evidence was found for the presence of the delta16-androgen steroids which have axillary-like odors and have also been reported in axillary sweat. Cholesterol was the major steroid component of the secretion.

A gas chromatographic-mass spectrometric screening procedure is described for the identification and differentiation of the following alkylamine antihistamines and their metabolites in urine: azatadine, benzquinamide, brompheniramine, chlorphenamine, (clofedanol), cyproheptadine, dimetindene, ketotifen, mebhydroline, phenindamine, pheniramine, pyrrobutamine, terfenadine and tolpropamine. After acid hydrolysis of the conjugates, extraction and acetylation, the urine samples were analysed by computerized gas chromatography-mass spectrometry. Using ion chromatography with the selective ions m/z 58, 169, 203, 205, 230, 233, 262 and 337, the presence of alkylamine antihistamines and/or their metabolites was indicated. The identity of positive signals in the reconstructed ion chromatograms was confirmed by a visual or computerized comparison of the stored full mass spectra with the reference spectra. The ion chromatograms, reference mass spectra and gas chromatographic retention indices (OV-101) are documented. The procedure presented is integrated in a general screening procedure (general unknown analysis) for several groups of drugs.

Sensitive methods for quantifying androgens were lacking. Therefore, a relatively simple procedure for separating steroids was combined with highly specific assay methods so that eight androgens could be measured with high accuracy, precision and sensitivity. Semi-automated separations on Sephadex LH-20 columns used heptane:methylene chloride:ethanol:water (50:50:1:0.12) and a flow rate of 17.0 min/ml. The six peaks eluted contained androstenedine; androsterone, epiandrosterone and dihydrotestosterone; testosterone and dehydroepiandrosterone; 3alpha-androstanediol; 3beta-androstanediol; and androstenediol. Androstenedione, dehydroepiandrosterone and androstenediol were quantified using specific antisera (sensitivity less than or equal to 75 pg). Testosterone and dihydrotestosterone were measured by competitive protein-binding assays using rabbit TeBG (sensitivity less than or equal to 150 pg). 3alpha- and 3beta-androstanediol were similarly assayed using human TeBG (sensitivity approximately 150 pg). Androsterone was reduced with NaBH4 and the resulting 3alpha-androstanediol was assayed using human TeBG (sensitivity approximately 200 pg). Inter- and intra-assay variations were less than 10% for radioimmunoassays and less than 16% for competitive protein-binding assays over the entire dose response curve.