The causes of cancer in France.

BACKGROUND: While external factors are responsible for many human cancers, precise estimates of the contribution of known carcinogens to the cancer burden in a given population have been scarce. METHODS: We estimated the proportion of cancer deaths which occurred in France in 2000 attributable to known risk factors, based on data on frequency of exposure around 1985. RESULTS: In 2000, tobacco smoking was responsible for 23.9% of cancer deaths (33.4% in men and 9.6% in women), alcohol drinking for 6.9% (9.4% in men and 3.0% in women) and chronic infections for 3.7%. Occupation is responsible for 3.7% of cancer deaths in men; lack of physical activity, overweight/obesity and use of exogenous hormones are responsible for 2%-3% of cancer deaths in women. Other risk factors, including pollutants, are responsible for <1% of cancer deaths. Thus, known risk factors explain 35.0% of cancer deaths, and 15.0% among never smokers. CONCLUSIONS: While cancer mortality is decreasing in France, known risk factors of cancer explain only a minority of cancers, with a predominant role of tobacco smoking.

Cloning and expression of genes involved in 4-chlorobiphenyl transformation by Pseudomonas testosteroni: homology to polychlorobiphenyl-degrading genes in other bacteria.

The genes of Pseudomonas testosteroni strain B-356, specifying the transformation of 4-chlorobiphenyl (4-CB) into 4-chlorobenzoic acid (4-CBA) were cloned into Pseudomonas putida KT2440 using a broad-host-range cosmid, pPSA842. Of 10,000 clones tested, four were able to transform 4-CB. Gas chromatographic and mass spectrometric analysis of the catabolic products from two of the 4-CB-transforming clones carrying the hybrid plasmids, pDA1 and pDA2, demonstrated that pDA1 carried a complete set of structural genes involved in the transformation of 4-CB into 4-CBA, while pDA2 contained part of the pathway genes leading up to the meta-cleavage compound. Restriction endonuclease mapping and subcloning of pDA1 and pDA2 showed that the clones contained a common stretch of DNA of about 9.1 kb and that pDA2 carried gene(s) involved in regulation. Probing blots of genomic DNA from 13 different polychlorinated biphenyl(PCB)-degrading bacteria with radio-labelled pDA1 and pDA2, suggested that many PCB-degrading pathways have a common phylogenetic origin.

Quantitative magnetic resonance and isotopic imaging: early evaluation of radiation injury to the brain.

PURPOSE: Using magnetic resonance (MR) and isotopic imaging to investigate the cerebral alterations after highdose single-fraction irradiation on a pig model. We assessed the nuclear magnetic resonance (NMR) relaxation times as early markers of radiation injury to the healthy brain. METHODS AND MATERIALS: A total of 17 animals was studied; 15 irradiated and 2 unirradiated controls. Pigs were irradiated with a 12 MeV electron beam at a rate of 2 Gy/min. Ten animals received 40 Gy at the 90% isodose, five animals received 60 Gy, and two animals were unirradiated. The follow-up intervals ranged from 2 days to 6 months. T1-weighted scans, T2-weighted scans, and scintigrams were performed on all animals to study neurological abnormalities, cerebral blood flow, and blood-brain barrier (BBB) integrity. T1 and T2 relaxation times were measured in selected regions of interest (ROIs) within the irradiated and contralateral hemispheres. A ratio T1 after irradiation/T1 before irradiation, and a ratio T2 after irradiation/T2 before irradiation, were calculated, pooled for each dose group, and followed as a function of time after irradiation. RESULTS: Scintigraphy visualized the brain perfusion defect and BBB disruption in all irradiated brains. The ratio T2 after irradiation/T2 before irradiation was proportional to the effective dose received. The T2 ratio kinetics could be analyzed in three phases:an immediate and transient phase, two long-lasting phases, which preceded compression of the irradiated lateral ventricle, and edema and necrosis at later stages of radiation injury, respectively. The magnetic resonance imaging (MRI) observations correlated well with histological analysis. CONCLUSION: The results show that quantitative imaging is a sensitive in vivo method for early detection of cerebral radiation injury. The reliability and dose dependence of T2 relaxation time may offer new opportunities to detect and understand brain pathophysiology after high-dose single-fraction irradiation.

Overview of pulmonary alveolar macrophage renewal in normal rats and during different pathological processes.

We report experimental results on pulmonary alveolar macrophage (PAM) renewal in healthy rats and in rats treated with particles introduced in the lungs. Morphometric studies showed that the lungs of normal rats of the strain used in our study contain 20 x 10(6) PAM, 50 x 10(6) monocytes in alveolar capillaries, and about 3 x 10(5) interstitial macrophages. Pulse labeling with a tritiated thymidine (3HT) gave a labeling index of 0.4% for the monocytes, of which a few could be observed in mitosis within alveolar capillaries. These monocytes are likely to be the PAM precursors. The daily input (greater than 4%) by PAM proliferation exceeds PAM loss by migration to the upper respiratory tract (2.5%). The life span of PAM was measured by sequential counting of lavaged cells after labeling with [125I]iododeoxyuridine instilled intratracheally. The pulmonary lavage procedure used allowed us to recover at least 80% of the whole PAM population. A daily loss of PAM of 8-9% was measured, of which loss by death in the endoalveolar compartment was estimated at 5-6%. During the pathological processes studied, several parameters of PAM renewal were shown to be modified. PAM migration to the upper respiratory tract was frequently inhibited, PAM cytotoxicity was observed, and PAM proliferation increased in some cases and decreased in others. Under most of the pathological conditions investigated, the renewal of endoalveolar macrophages appeared quite different from that in normal rats, and direct blood monocyte migration to the endoalveolar compartment became a major component of PAM renewal.

Degradation pathways of PCB upon gamma irradiation.

In order to understand the modifications of the chromatographic profile of Aroclor 1260 upon gamma irradiation, a total of 14 pure polychlorinated biphenyl (PCB) congeners were separately irradiated in solution. Dechlorination was observed, and the generated products were investigated by gas chromatography and mass spectrometry. Degradation proceeds more rapidly in methanol/water mixture than in petroleum ether, but the relative amount of ortho-dechlorinated congeners formed upon irradiation was smaller in the former solvent. Ortho chlorines are preferentially lost in petroleum ether except when they are involved in a 2.5 (or 3.6) substitution pattern, in which case para dechlorination becomes predominant. The precursors of some toxicologically important congeners such as congeners 77, 118, 167, and 189 have been identified. These data are useful to rationalize the modifications of the chromatographic profile of PCB complex mixture upon gamma irradiation.

In vitro dissolution of uranium oxide by baboon alveolar macrophages.

In vitro cellular dissolution tests for insoluble forms of uranium oxide are technically difficult with conventional methodology using adherent alveolar macrophages. The limited number of cells per flask and the slow dissolution rate in a large volume of nutritive medium are obvious restricting factors. Macrophages in suspension cannot be substituted because they represent different and poorly reproducible functional subtypes with regard to activation and enzyme secretion. Preliminary results on the dissolution of uranium oxide using immobilized alveolar macrophages are promising because large numbers of highly functional macrophages can be cultured in a limited volume. Cells were obtained by bronchoalveolar lavages performed on baboons (Papio papio) and then immobilized after the phagocytosis of uranium octoxide (U3O8) particles in alginate beads linked with Ca2+. The dissolution rate expressed as percentage of initial uranium content in cells was 0.039 +/- 0.016%/day for particles with a count median geometric diameter of 3.84 microns(sigma g = 1.84). A 2-fold increase in the dissolution rate was observed when the same number of particles was immobilized without macrophages. These results, obtained in vitro, suggest that the U3O8 preparation investigated should be assigned to inhalation class Y as recommended by the International Commission on Radiological Protection. Future experiments are intended to clarify this preliminary work and to examine the dissolution characteristics of other particles such as uranium dioxide. It is recommended that the dissolution rate should be measured over an interval of 3 weeks, which is compatible with the survival time of immobilized cells in culture and may reveal transformation states occurring with aging of the particles.

Most ten-year-old children with negative or unknown histories of chickenpox are immune.

To evaluate the proportion of children to vaccinate against varicella in a catch-up program targeting 9- to 10-year-old children, a study was conducted among children age 10 years to assess the age-specific incidence of varicella and document the immunity against varicella in those with negative or unknown chickenpox history. Of the latter 62% were seropositive for varicella.

Effectiveness of a whole cell pertussis vaccine in child-care centers and schools.

BACKGROUND: Pertussis has substantially increased in Quebec, Canada, since 1990. We estimated pertussis vaccine effectiveness and vaccine coverage in child-care centers and elementary schools. METHODS: Two retrospective cohort studies were simultaneously conducted. One included 4482 children attending 88 public child-care centers and the other included 3429 pupils in 14 elementary schools. Cough and pertussis symptoms were assessed through a questionnaire and medical records; immunization status was ascertained by examination of written records. RESULTS: In child-care centers 95% of children had received at least three vaccine doses at the beginning of the follow-up; in schools more than 98% of pupils had received at least 4 doses. With > or = 4 doses of vaccine and a standard case definition used for surveillance (cough > or = 2 weeks, > or = 1 pertussis symptom and no other apparent cause for cough), vaccine effectiveness was estimated at 61% (95% confidence interval, 44 to 72%) in child-care centers and at 60% (95% confidence interval, 10 to 82%) in schools. With the same number of doses but a case definition requiring a cough > or = 5 weeks, vaccine effectiveness increased to 71% (95% confidence interval, 49 to 83) in child-care centers and to 86% (95% confidence interval, 66 to 94%) in schools. CONCLUSIONS: The increase in pertussis in Quebec is not caused by a low vaccine coverage. A low vaccine effectiveness may contribute to the resurgence of pertussis in the past decade.

Comparison of the induction of pulmonary neoplasms in Sprague-Dawley rats by fission neutrons and radon daughters.

Pulmonary carcinomas were recorded in a life-span experiment of male Sprague-Dawley rats exposed to fission neutrons. Mortality-corrected prevalences are obtained by the method of isotonic regression. In a second part of the paper a comparison is made with data obtained earlier for radon-daughter inhalations in the same strain of rats. A simultaneous maximum likelihood analysis is applied jointly to all experimental groups from the radon inhalation and the fission neutron study. The dependence of the resulting coefficients for the different groups on absorbed dose or inhalation dose permits a derivation of equivalence ratios. At low doses the equivalence ratio is 3 WLM (working level months) of radon-daughter exposure to 1 mGy of fission neutrons. At higher doses the equivalence ratio decreases. The neutron data are also utilized to derive mortality-corrected lifetime incidences of pulmonary carcinomas in the exposed animals. At low doses the relation is consistent with linearity, but sublinearity (dose exponent less than 1) cannot be excluded.

Gastrointestinal absorption of neptunium in primates: effect of ingested mass, diet, and fasting.

Absorption and retention of neptunium were determined in baboons after intragastric administration of neptunium nitrate solutions at pH 1. The effects of mass, diet, and fasting on absorption were studied. At higher mass levels (400-800 micrograms Np/kg), absorption was about 1%; at lower mass intakes (0.0009-0.005 micrograms Np/kg), absorption was reduced by 10- to 20-fold. The addition of an oxidizing agent (Fe3+) increased gastrointestinal absorption and supported the hypothesis of a reduction of Np (V) when loss masses were ingested. Diets depleted of or enriched with hydroxy acids did not modify retention of neptunium but increased urinary excretion with increasing hydroxy acid content. The diet enriched with milk components reduced absorption by a factor of 5. Potatoes increased absorption and retention by a factor 5, not necessarily due to the effect of phytate. Fasting for 12 or 24 h increased retention and absorption by factors of about 3 and 10, respectively. Data obtained in baboons when low masses of neptunium were administered suggest that the f1 factor used by ICRP should be decreased. However, fasting as encountered in certain nutritional habits is a factor to be taken into consideration.

Neutron RBE for induction of tumors with high lethality in Sprague-Dawley rats.

The effectiveness of fission neutrons is compared to that of gamma rays and X rays with regard to the induction of malignancies in male Sprague-Dawley rats. The analysis is based on autopsy results. It is focused on tumors that tend to be present in animals dying early, which is indicative of a high degree of lethality. The relative biological effectiveness (RBE) is deduced from a comparison of the cumulative hazard functions. Different nonparametric models-the constant relative risk model, a time shift model, and an acceleration model-are employed in the comparison, and the resulting values of RBE are seen to be substantially independent of the choice of model. The results are in good agreement with earlier studies of nonlethal lung tumors in the same series of experiments. At neutron doses of 20 to 60 mGy, the RBE of fission neutrons is about 50.

Lung carcinomas in Sprague-Dawley rats after exposure to low doses of radon daughters, fission neutrons, or gamma rays.

The effectiveness of radon-daughter inhalation and irradiation with fission neutrons and gamma rays in the induction of lung carcinomas in Sprague-Dawley rats at low doses is compared. Earlier reports which compared radon-daughter inhalations and neutron irradiations over a wider range of doses were based on dosimetry for the radon-daughter inhalations which has recently been found to be faulty. In the present analysis, low-dose experiments were designed to derive revised equivalence ratios between radon-daughter exposures, and fission neutron or gamma irradiations. The equivalence is approximately 15 working level months (WLM) of radon daughters to 10 mGy of neutrons (the earlier value was 30 WLM to 10 mGy). The relative biological effectiveness (RBE) of neutrons is 50 or more at a gamma-ray dose of 1 Gy. In these experiments with low doses and exposures, the lifetime incidences can be estimated from the raw incidences, while the derivation of the time dependence of the prevalence is essential for the estimation of RBE values and equivalence ratios.

Subcellular and intranuclear localization of neptunium-237 (V) in rat liver.

The present investigation was aimed at establishing the distribution of 237Np within the different structures of hepatocytes. Rats were contaminated experimentally by intravenous injection of 237Np (V) and the subcellular structures of the liver were separated by ultracentrifugation. Twenty-four hours after contamination, the nuclear and cytosolic fractions bound 54 and 32%, respectively, of the total radionuclide. Purification of the nuclei followed by dissociation of the protein components in medium of increasing ionic strength showed a specific binding of neptunium to the structural proteins of the nuclear matrix.

Use of unstable chromosome aberrations for biological dosimetry after the first postirradiation mitosis.

The loss of unstable chromosome aberrations after the first postirradiation mitosis makes their use difficult in radiation dosimetry. We describe here a method which, in a cell population observed at this stage, allows retrospective estimation of the frequencies of the unstable aberrations induced at the time of irradiation, and their use as a dosimeter. The laws controlling the behavior of unstable aberrations during mitosis were defined from a large-scale experiment on irradiated human lymphocytes. For cells undergoing the first, second, or third mitosis after irradiation, relationships were determined between the frequency, at irradiation time, of acentric fragments not arising from formation of dicentrics or rings, and the ratio of dicentrics and centric rings appearing without acentric fragments to the total number of dicentrics plus rings. On the basis of this ratio, the method described here provides an assessment of the postirradiation mitotic activity in a cell population. This assessment permitted estimation of the cell distribution and frequency of dicentrics plus centric rings, and of the frequency of acentric fragments at the time of irradiation. The use of this method for retrospective dosimetry after whole-body irradiation under various conditions of exposure is illustrated.

Studies on anabolic steroids--4. Identification of new urinary metabolites of methenolone acetate (Primobolan) in human by gas chromatography/mass spectrometry.

The metabolism of methenolone acetate (17 beta-acetoxy-1-methyl-5 alpha-androst-1-en-3-one), a synthetic anabolic steroid, has been investigated in man. After oral administration of a 50 mg dose of the steroid to two male volunteers, twelve metabolites were detected in urine either in the glucuronide, sulfate or free steroid fractions. Methenolone, the parent steroid was detected in urine until 90 h after administration. Its cumulative urinary excretion accounted for 1.63% of the ingested dose. With the exception of 3 alpha-hydroxy-1-methylen-5 alpha-androstan-17-one, the major biotransformation product of methonolone acetate, metabolites were excreted in urine at lower levels, through minor metabolic routes. Most of methenolone acetate metabolites were isolated from the glucuronic acid fraction, namely methenolone, 3 alpha-hydroxy-1-methylen-5 alpha-androstan-17-one, 3 alpha-hydroxy-1 alpha-methyl-5 alpha-androstan-17-one, 17-epimethenolone, 3 alpha,6 beta-dihydroxy-1-methylen-5 alpha-androstan-17-one, 2 xi-hydroxy-1-methylen-5 alpha-androstan-3,17-dione, 6 beta-hydroxy-1-methyl-5 alpha-androst-1-en-3,17-dione, 16 alpha-hydroxy-1-methyl-5 alpha-androst-1-en-3,17-dione and 3 alpha,16 alpha-dihydroxy-1-methyl-5 alpha-androst-1-en-17-one. Interestingly, the metabolites detected in the sulfate fraction were isomeric steroids bearing a 16 alpha- or a 16 beta-hydroxyl group, whereas 1-methyl-5 alpha-androst-1-en-3,17-dione was the sole metabolite isolated from the free steroid fraction. Steroids identity was assigned on the basis of the mass spectral features of their TMS ether, TMS enol-TMS ether, MO-TMS, and d9-TMS ether derivatives and by comparison with reference and structurally related steroids. The data indicated that methenolone acetate was metabolized into several compounds resulting from oxidation of the 17-hydroxyl group and reduction of A-ring substituents, with or without concomitant hydroxylation at the C6 and C16 positions.

Studies on anabolic steroids--11. 18-hydroxylated metabolites of mesterolone, methenolone and stenbolone: new steroids isolated from human urine.

New metabolites of mesterolone, methenolone and stenbolone bearing a C18 hydroxyl group were isolated from the steroid glucuronide fraction of urine specimens collected after administration of single 50 mg doses of these steroids to human subjects. Mesterolone gave rise to four metabolites which were identified by gas chromatography/mass spectrometry as 18-hydroxy-1 alpha-methyl-5 alpha-androstan-3,17-dione 1, 3 alpha,18-dihydroxy-1 alpha-methyl-5 alpha-androstan-17-one 2, 3 beta,18-dihydroxy-1-alpha-methyl-5 alpha-androstan-17-one 3 and 3 alpha,6 xi,18-trihydroxy-1 alpha-methyl-5 alpha-androstan-17-one 4. These data suggest that mesterolone itself was not hydroxylated at C18, but rather 1 alpha-methyl-5 alpha-androstan-3,17-dione, an intermediate metabolite which results from oxidation of mesterolone 17-hydroxyl group. In addition to hydroxylation at C18, reduction of the 3-keto group and further hydroxylation at C6 were other reactions that led to the formation of these metabolites. It is of interest to note that in the case of both methenolone and stenbolone, only one 18-hydroxylated urinary metabolite namely 18-hydroxy-1-methyl-5 alpha-androst-1-ene-3,17-dione 5 and 18-hydroxy-1-methyl-5 alpha-androst-1-ene-3,17-dione 6 were both detected in post-administration urine specimens. These data indicate that the presence of a methyl group at the C1 or C2 positions in the steroids studied is a structural feature that seems to favor interaction of hepatic 18-hydroxylases with these steroids. These data provide further evidence that 18-hydroxylation of endogenous steroids can also occur in extra-adrenal sites in man.

Studies on anabolic steroids--12. Epimerization and degradation of anabolic 17 beta-sulfate-17 alpha-methyl steroids in human: qualitative and quantitative GC/MS analysis.

The epimerization and dehydration reactions of the 17 beta-hydroxy group of anabolic 17 beta-hydroxy-17 alpha-methyl steroids have been investigated using the pyridinium salts of 17 beta-sulfate derivatives of methandienone 1, methyltestosterone 4, oxandrolone 7, mestanolone 10 and stanozolol 11 as model compounds. Rearrangement of the sulfate conjugates in buffered urine (pH 5.2) afforded the corresponding 17-epimers and 18-nor-17,17-dimethyl-13(14)-enes in a ratio of 0.8:1. These data indicated that both epimerization and dehydration of the 17 beta-sulfate derivatives were not dependent upon the respective chemical features of the steroids studied, but were instead inherent to the chemistry of the tertiary 17 beta-hydroxy group of these steroids. Interestingly, in vivo studies carried out with human male volunteers showed that only methandienone 1, methyltestosterone 4 and oxandrolone 7 yielded the corresponding 17-epimers 2, 5 and 8 and the 18-nor-17,17-dimethyl-13(14)-enes 3, 6 and 9 in ratios of 0.5:1, 2:1 and 2.7:1, respectively. No trace of the corresponding 17-epimers and 18-nor-17,17-dimethyl-13(14)-enes derivatives of mestanolone 10 and stanozolol 11 was detected in urine samples collected after administration of these steroids. These data suggested that the in vivo formation of the 17-epimers and 18-nor-17,17-dimethyl-13(14)-enes derivatives of 17 beta-hydroxy-17 alpha-methyl steroids is also dependent upon phase I and phase II metabolic reactions other than sulfation of the tertiary 17 beta-hydroxyl group, which are probably modulated by the respective chemical features of the steroidal substrates. The data reported in this study demonstrate that the 17-epimers and 18-nor-17,17-dimethyl-13(14)-enes are not artifacts resulting from the acidic or microbial degradation of the parent steroids in the gut as previously suggested by other authors, but arise from the rearrangement of their 17 beta-sulfate derivatives. Unchanged oxandrolone 7 was solely detected in the unconjugated steroid fraction whereas unchanged steroids 1, 4 and 11 were recovered from the glucuronide fraction. These data are indirect evidences suggesting that the glucuronide conjugates of compounds 1 and 4 are probably enol glucuronides and that of compound 11 is excreted in urine as a N-glucuronide involving its pyrazole moiety. The urinary excretion profiles of the epimeric and 18-nor-17,17-dimethyl-13(14)-ene steroids are presented and discussed on the basis of their structural features.

Studies on anabolic steroids--6. Identification of urinary metabolites of stenbolone acetate (17 beta-acetoxy-2-methyl-5 alpha-androst-1-en-3-one) in human by gas chromatography/mass spectrometry.

The metabolism of stenbolone acetate (17 beta-acetoxy-2-methyl-5 alpha-androst-1-en-3-one), a synthetic anabolic steroid, has been investigated in man. Nine metabolites were detected in urine either as glucuronic or sulfuric acid aglycones after oral administration of a single 50 mg dose to a male volunteer. Stenbolone, the parent compound, was detected for more than 120 h after administration and its cumulative excretion accounted for 6.6% of the ingested dose. Most of the stenbolone acetate metabolites were isolated from the glucuronic acid fraction, namely: stenbolone, 3 alpha-hydroxy-2-methyl-5 alpha-androst-1-en- 17-one, 3 alpha-hydroxy-2 xi-methyl-5 alpha-androst-17-one; 3 isomers of 3 xi, 16 xi-dihydroxy-2-methyl-5 alpha-androst-1-en-17-one; 16 alpha and 16 beta-hydroxy-2-methyl-5 alpha-androst-1-ene-3, 17-dione; and 16 xi, 17 beta-dihydroxy-2-methyl-5 alpha-androst-1-en-3-one. Only isomeric metabolites bearing a 16 alpha or a 16 beta-hydroxyl group were detected in the sulfate fraction. Interestingly, no metabolite was detected in the unconjugated steroid fraction. The steroids identities were assigned on the basis of their TMS ether, TMS enol-TMS ether, MO-TMS and d9-TMS ether derivatives and by comparison with reference and structurally related steroids. Data indicated that stenbolone acetate was metabolized into several compounds resulting from oxidation of the 17 beta-hydroxyl group and/or reduction of A-ring delta-1 and/or 3-keto functions with or without hydroxylation at the C16 position. Finally, comparison of stenbolone acetate urinary metabolites with that of methenolone acetate shows similar biotransformation pathways for both delta-1-3-keto anabolic steroids. This indicates that the position of the methyl group at the C1 or C2 position in these steroids has little effect on their major biotransformation routes in human, to the exception that stenbolone cannot give rise to metabolites bearing a 2-methylene group since its 2-methyl group cannot isomerize into a 2-methylene function through enolization of the 3-keto group as previously observed for methenolone.

Studies on anabolic steroids--8. GC/MS characterization of unusual seco acidic metabolites of oxymetholone in human urine.

One of the biotransformation routes of oxymetholone (17 beta-hydroxy-2-hydroxymethylene-17 alpha-methyl-5 alpha-androstan-3-one) in man leads to the formation of 17 beta-hydroxy-17 alpha-methyl-5 alpha-androstan-3-one (mestanolone). To demonstrate that this latter steroid may be formed by decarboxylation of an intermediate metabolite of oxymetholone bearing a 2-carboxylic group, we studied the urinary excretion of oxymetholone acidic metabolites. Five new acidic metabolites are reported here for the first time, among which four are unusual seco steroids resulting from the oxidative cleavage of the A-ring. The most abundant compound is 17 beta-hydroxy-17 alpha-methyl-2,3-seco-5 alpha-androstane-2,3-dioic acid 1, the cumulative excretion of which accounted for 1.52% of the dose. Three other seco diacids were produced in smaller amounts, namely 17 beta-hydroxy-17 alpha-methyl-2,3-seco-5 alpha-androstane-2,4- dicarboxylic acid 3, 17 beta-hydroxy-17 alpha-methyl-1,3-seco-5 alpha-androstane-1,3-dioic acid 4 and 17 beta-hydroxy-17 alpha-methyl-2,4-seco-5 alpha-androstane-2,4-dioic acid 5. The fifth acidic metabolite was identified as 3 alpha, 17 beta-dihydroxy-17 alpha-methyl-5 alpha-androstane-2 beta-carboxylic acid 2. The excretion in urine of these acidic metabolites suggests that the 2-hydroxymethylene group in oxymetholone is readily oxidized to yield the corresponding beta-keto acid which can be (1) decarboxylated to form mestanolone; (2) reduced at C-3 to give compound 2; and (3) further oxidized to afford the unexpected seco diacids 1, 3, 4 and 5. The identity of compounds 1 and 2 was ascertained by GC/MS and 1H and 13C-NMR analysis of reference compounds. The other metabolites were characterized by GC/MS analysis.

Proposed definitive methods for measurement of plasma testosterone and 17 alpha-hydroxyprogesterone.

OBJECTIVES: This report provides the results of the development and evaluation of definitive isotope dilution/mass spectrometry (ID/MS) methods for the determination of testosterone and 17 alpha-hydroxyprogesterone in human plasma at concentrations ranging from 1.4 to 37.9 nmol/L and 1.5 to 45.4 nmol/L, respectively. The internal standards were 16, 16, 17-2H-testosterone and 21,21,21, -2H-17 alpha-hydroxyprogesterone. The development of optimum extraction and derivatization procedures, and studies of storage time, temperature effects, accuracy, and precision are presented. RESULTS: The results indicate that the methods employing the TBDMS derivative of testosterone and MO-TMS derivative of 17 alpha-hydroxyprogesterone are capable of generating accurate and precise data at the inherently low concentrations given, with recovery greater than 95%. Accuracy of testosterone in the fortified steroid-free and pooled plasma by ID/MS measurement was good because the relative error ranged from +3.1% to -0.7, with a mean of 0.9% over the concentration levels of 1.4 to 37.9 nmol/L testosterone, and the imprecision ranged from 4.2% to 0.7% CV, with a mean of 1.9%. Accuracy of 17 alpha-hydroxyprogesterone in the fortified steroid-free and pooled plasma was also good, considering the inherently low concentrations. The relative error ranged from -2.1% to +1.5%, with a mean of 1.1%, and the imprecision ranged from 3.8% to 0.8% CV with a mean of 1.3% over concentration levels of 1.5 to 15.1 nmol/L. The high precision and accuracy and absence of statistically significant bias qualifies these methods as candidate definitive methods for plasma testosterone and 17 alpha-hydroxyprogesterone.