Evaluation of specific high-performance liquid-chromatographic determinations of urinary metanephrine and normetanephrine by comparison with isotope dilution mass spectrometry.

A method for the determination of metanephrine (MN; also known as metadrenaline), normetanephrine (NMN; also known as normetadrenaline) and 3-methoxytyramine (3-MT) in human urine using high-performance liquid chromatography followed by electrochemical detection (ECD) was validated primarily by comparing the results with those obtained by a gas chromatography mass spectrometry (GC-MS) reference method. Correlation coefficients of 0.93, 0.94 and 0.91 were obtained for MN, NMN and 3-MT, respectively, in a group of healthy controls consisting of 30 women and 30 men. A systematic difference was detected only for 3-MT (-16%). Further tests of accuracy (linearity and recovery) and precision demonstrated that the described method must be considered to be a reliable approach to assess urinary metanephrines in the diagnosis of phaeochromocytoma. At lower concentrations (MN, 248 nmol/L; NMN, 434 nmol/L; 3-MT, 402 nmol/L), within-assay coefficients of variation were close to 5% or less (5.3, 4.6 and 2.2%, respectively) and between-assay coefficients of variation were 8.9, 11.2 and 12.3%, respectively, for the same low levels. This raises the possibility that this method can also be applied to assess urinary free, unconjugated metanephrines. Sex differences were detected for MN and NMN excretion when expressed in nmol per 24h and nmol/mmol creatinine, respectively, by both ECD and GC-MS methods.

Evaluation of specific high-performance liquid-chromatographic determinations of urinary adrenaline and noradrenaline by comparison with isotope dilution mass spectrometry.

A method for the determination of metadrenaline (MA), normetadrenaline (NMA) and 3-methoxytyramine (3-MT) in human urine using high-performance liquid chromatography followed by electrochemical detection (ECD) was validated primarily by comparing the results with those obtained by a gas chromatography-mass spectrometry (GC-MS) reference method. Correlation coefficients of 0.93, 0.94 and 0.91 were obtained for MA, NMA and 3-MT, respectively, in a group of healthy controls consisting of 30 women and 30 men. A systematic difference was detected only for 3-MT (-16%). Further tests of accuracy (linearity and recovery) and precision demonstrated that the described method must be considered to be a reliable approach to assess urinary metadrenalines in the diagnosis of phaeochromocytoma. At lower concentrations (MA, 248 nmol/L; NMA, 434 nmol/L; 3-MT, 402 nmol/L), within-assay coefficients of variation were close to 5% or less (5.3, 4.6 and 2.2%, respectively) and between-assay coefficients of variation were 8.9, 11.2 and 12.3%, respectively, for the same low levels. This raises the possibility that this method can also be applied to assess urinary free, unconjugated metanephrines. Sex differences were detected for MA and NMA excretion when expressed in nmol per 24 h and nmol/mmol creatinine, respectively, by both ECD and GC-MS methods.

Cerebrotendinous xanthomatosis.

Cerebrotendinous xanthomatosis is a rare autosomal recessive lipid-storage disease caused by mutations in the sterol 27-hydroxylase gene. The accumulation of cholestanol in various tissues characterizes this disease. Diagnosis is based on determination of urinary bile alcohols. Therapy with chenodeoxycholic acid may arrest the progression of the disease. A 55-year-old woman presented with a slowly progressive paraparesia and two firm subcutaneous tumors over the knees. Her medical history revealed difficulty in standing and walking since infancy, bilateral juvenile cataracts, and mental retardation. Histopathologic examination of one subcutaneous tumor was consistent with tendinous xanthoma. Substantial elevation of urinary bile alcohols confirmed the diagnosis. Treatment with oral chenodeoxycholic acid was started, with only mild improvement of spasticity. Recognition of tendon xanthomas in a young patient with neurologic symptoms or cataracts (or both) is crucial to start early treatment and to avoid irreversible neurologic sequelae.

Investigation of applicability of a mid-infrared spectroscopic method using an attenuated total reflection accessory and a new near-infrared transmission method for determination of faecal fat.

In many laboratories, the titrimetric method of Van de Kamer is used for the analysis of faecal fat content of patients suspected of steatorrhoea. We investigated the applicability of a mid-infrared (MIR) spectroscopic method, using an attenuated total reflection (ATR) accessory, and a new near-infrared (NIR) spectroscopic method. For the NIR method, sealed plastic bags containing the stool samples were used as transmission cells. Standardization was obtained using a previously described MIR method, with a NaCl flow-cell, as reference method. Partial least-squares regression was used for the calibration of each method. Full cross-validation of the calibration set was used for the internal validation of each method. Fifteen per cent of the stool samples could not be estimated with the ATR method within reasonable accuracy limits compared with the reference. The standard error of prediction of the NIR method was 1.1 g/dL. We conclude that the new NIR method is a promising technique for routine use. However, further experiments need to be done with triplicate measurements of each sample and the use of an external validation set.

New method for faecal fat determination by mid-infrared spectroscopy, using a transmission cell: an improvement in standardization.

Current techniques used in clinical laboratories for faecal fat determination, such as the Van de Kamer method, are not very accurate or precise. This became apparent when results obtained by different laboratories were compared, and could explain the disappointing performance of near-infrared and mid-infrared spectroscopy since the accuracy of these techniques depends upon the accuracy of the calibration used (i.e. inaccurate wet chemical analysis). In order to improve standardization, we developed and tested a new quantitative method in three laboratories, based on Fourier transform infrared (FT-IR) spectroscopy. Fatty acids were extracted from faecal samples with acidified petroleum ether-ethanol and the extracts were dried and dissolved in chloroform. An infrared spectrum of the extracts was recorded in the range 4000-650 cm(-1), using an infrared transmission cell. Standard mixtures of stearic and palmitic acids (65:35) were used for calibration. Quantification was based on the absorbance band of the CH2 group (2855 cm(-1)) of free fatty acids and fatty acid glycerol esters. The calibration curve showed excellent linearity. The correlation coefficient between the titrimetric Van de Kamer and FT-IR methods was 0.96 (y = 1.12x-0.02, standard error of prediction = 0.89 g% fat). No significant difference was found when the FT-IR results of 28 faecal samples from patients were compared between two different university hospital laboratories. The new FT-IR method, using primary standards, is simple and rapid, and provides satisfactory intra- and inter-laboratory precision for the diagnosis and monitoring of steatorrhoea.

Lack of relationship between 11beta-hydroxysteroid dehydrogenase setpoint and insulin sensitivity in the basal state and after 24h of insulin infusion in healthy subjects and type 2 diabetic patients.

OBJECTIVES: To test whether insulin resistance in type 2 diabetes mellitus is associated with an altered overall setpoint of the 11beta-hydroxysteroid dehydrogenase (11betaHSD) mediated cortisol to cortisone interconversion towards cortisol, and to evaluate whether changes in insulin sensitivity induced by antecedent hyperinsulinaemia are related to changes in the 11betaHSD setpoint. PATIENTS AND MEASUREMENTS: The urinary ratio of (tetrahydrocortisol + allo-tetrahydrocortisol)/tetrahydrocortisone ((THF + allo-THF)/THE) and of free cortisol/free cortisone (UFF/UFE), as well as the plasma cortisol/cortisone ratio were measured in 8 male type 2 diabetic patients and 8 healthy male subjects without and after 24 h of insulin infusion. Insulin was infused at a rate of 30 mU/kg/h with blood glucose being clamped at euglycaemic levels in healthy subjects and at isoglycaemic levels in diabetic patients. Insulin sensitivity was assessed by measurement of whole body glucose uptake (M-value) during a 3-4 h euglycaemic clamp, directly after the 24 h insulin infusion and compared to the M-value on a control day, at least 1 week apart from the 24 h insulin infusion. RESULTS: Despite impaired insulin sensitivity (M-value, 11.6 +/- 7.7 vs. 28.5 +/- 11.6 micromol/kg/minutes, in type 2 diabetic and healthy subjects, respectively, P < 0.05), urinary (THF + allo-THF)/THE ratio and baseline plasma cortisol/cortisone ratio at 0800 h were similar in type 2 diabetic patients (0.82 +/- 0.07 and 3. 77 +/- 0.70, respectively) and healthy subjects (0.76 +/- 0.14 and 3. 81 +/- 0.88, respectively, ns). Insulin sensitivity was not correlated with urinary (THF + allo-THF)/THE ratio nor with baseline plasma cortisol/ cortisone. In type 2 diabetic patients, insulin sensitivity was further impaired by antecedent hyperinsulinaemia (P < 0.05), but the urinary (THF + allo-THF)/THE ratio (0.80 +/- 0.14, ns) and the plasma cortisol/cortisone at 0800 h (3.66 +/- 0.72, ns) did not change. In healthy subjects, insulin sensitivity did not change significantly (M-value, 22.5 +/- 9.7 micromol/kg/minutes, ns), although the urinary (THF + allo-THF)/THE ratio (0.92 +/- 0.25, P < 0.05) and the plasma cortisol/cortisone (4.59 +/- 0.63, P < 0.05) increased. Insulin did not affect the UFF/UFE ratio in either group. CONCLUSION: The present study does not support the hypothesis that insulin resistance in type 2 diabetes mellitus is associated with an overall change in the 11betaHSD set point towards cortisol. In view of the stimulatory effects of insulin and cortisol on adipogenesis, long-term stimulation of 11betaHSD reductase activity by insulin could aggravate visceral obesity.

Determination of urinary 18 beta-glycyrrhetinic acid by gas chromatography and its clinical application in man.

A sensitive and quantitative gas chromatographic assay for the determination of 18 beta-glycyrrhetinic acid (18 beta-GA), the main metabolite of glycyrrhizin after oral licorice consumption in human urine, has been developed and validated. For the extraction of 18 beta-GA from urine two Sep-Pak C18 extractions, hydrolysis with Helix pomatia and three liquid-liquid extractions were performed, using 18 alpha-glycyrrhetinic acid (18 alpha-GA) as internal standard. Both 18 beta-GA and internal standard were converted into their pentafluorobenzyl-ester/trimethylsilyl-ether derivatives and detected by flame ionization detection using a WCOT-fused-silica capillary column. Good quality control data were obtained in precision and accuracy tests. The detection limit of the gas chromatographic method was 10 micrograms/l with a urine volume of 10 ml. A detection limit of 3 micrograms/l was obtained by performing GC-MS. The GC method was used to monitor the urinary excretion of 18 beta-GA after licorice consumption by two healthy volunteers and a patient suspected of licorice abuse. Furthermore, it was shown that this GC assay enables to detect other metabolites related to licorice consumption.

Biliary fibrosis associated with altered bile composition in a mouse model of erythropoietic protoporphyria.

BACKGROUND & AIMS: Reduced activity of ferrochelatase in erythropoietic protoporphyria (EPP) results in protoporphyrin (PP) accumulation in erythrocytes and liver. Liver disease may occur in patients with EPP, some of whom develop progressive liver failure that necessitates transplantation. We investigated the mechanisms underlying EPP-associated liver disease in a mouse model of EPP. METHODS: Liver histology, indicators of lipid peroxidation, plasma parameters of liver function, and bile composition were studied in mice homozygous (fch/fch) for a point mutation in the ferrochelatase gene and in heterozygous (fch/+) and wild-type (+/+) mice. RESULTS: Microscopic examination showed bile duct proliferation and biliary fibrosis with portoportal bridging in fch/fch mice. PP content was 130-fold increased, and thiobarbituric acid-reactive substances (+30%) and conjugated dienes (+75%) were slightly higher in fch/fch than in fch/+ and +/+ livers. Levels of hepatic thiols (-12%) and iron (-52%) were reduced in fch/fch livers. Liver enzymes and plasma bilirubin were markedly increased in the homozygotes. Plasma bile salt levels were 80 times higher in fch/fch than in fch/+ and +/+ mice, probably related to the absence of the Na(+)-taurocholate cotransporting protein (Ntcp) in fch/fch liver. Paradoxically, bile flow was not impaired and biliary bile salt secretion was 4 times higher in fch/fch mice than in controls. Up-regulation of the intestinal Na(+)-dependent bile salt transport system in fch/fch mice may enhance efficiency of bile salt reabsorption. The bile salt/lipid ratio and PP content of fch/fch bile were increased 2-fold and 85-fold, respectively, compared with +/+, whereas biliary glutathione was reduced by 90%. Similar effects on bile formation were caused by griseofulvin-induced inhibition of ferrochelatase activity in control mice. CONCLUSIONS: Bile formation is strongly affected in mice with impaired ferrochelatase activity. Rather than peroxidative processes, formation of cytotoxic bile with high concentrations of bile salts and PP may cause biliary fibrosis in fch/fch mice by damaging bile duct epithelium.

Clinical applications of gas chromatography and gas chromatography-mass spectrometry of steroids.

This review article underlines the importance of gas chromatography-mass spectrometry (GC-MS) for determination of steroids in man. The use of steroids labelled with stable isotopes as internal standard and subsequent analysis by GC-MS yields up to now the only reliable measurement of steroids in serum. Isotope dilution GC-MS is the reference method for evaluation of routine analysis of serum steroid hormones. GC-MS is an important tool for detection of steroid hormone doping and combined with a combustion furnace and an isotope ratio mass spectrometer the misuse of testosterone by athletes can be discovered. Finally the so called urinary steroid profile by GC and GC-MS is the method of choice for detection of steroid metabolites in health and disease.

Effect of simvastatin in addition to chenodeoxycholic acid in patients with cerebrotendinous xanthomatosis.

The effects of combination therapy with chenodeoxycholic acid (CDCA) and simvastatin on serum cholestanol, low-density lipoprotein (LDL) cholesterol, and lathosterol levels were investigated in seven adult patients with cerebrotendinous xanthomatosis (CTX) who were on long-term treatment with CDCA. The patients were treated with a combination of CDCA 750 mg daily and an increasing dose of simvastatin from 10 mg to 40 mg daily for a period of 6 months. We found a significant effect of this combination therapy compared with CDCA alone in terms of decreasing the serum cholestanol and LDL cholesterol levels, particularly with a daily dose of 40 mg simvastatin. The mean cholestanol level decreased from 9.27 micromol/L (baseline) to 6.69 micromol/L (40 mg simvastatin), while the mean LDL cholesterol level decreased from 5.08 mmol/L (baseline) to 3.04 mmol/L (40 mg simvastatin). No side effects were reported, and there were no effects on the clinical condition, cerebral magnetic resonance imaging (MRI), visual evoked potentials, and electroencephalographic features. We conclude that a combination of 750 mg CDCA and 40 mg simvastatin daily is effective to further reduce serum cholestanol, LDL cholesterol, and lathosterol in adult CTX patients treated with long-term CDCA. Whether this combination treatment will be effective for the long-term prevention of neurological deterioration and atherosclerosis remains to be established.

Gas chromatographic-mass spectrometric analysis of urinary glycyrrhetinic acid: an aid in diagnosing liquorice abuse.

OBJECTIVES: Liquorice abuse can lead to severe clinical complications, caused by its active compound 18beta-glycyrrhetinic acid (18betaGA). 18betaGA inhibits dehydrogenase activity of 11beta-hydroxysteroid dehydrogenase (11betaHSD). This enzyme catalyses the interconversion between cortisol and cortisone and normally protects the mineralocorticoid receptor from being activated by cortisol. Diagnosing liquorice abuse can be notoriously difficult. The aim of our study was to develop an accurate and clinically applicable 18betaGA urinary assay. DESIGN: We developed a urinary 18betaGA assay based on gas chromatography and mass spectrometry (GCMS) with sufficient sensitivity to detect 18betaGA at low concentrations. The assay was validated in four volunteers consuming different amounts of liquorice. We applied its use in two patients with hypokalaemic hypertension and suppressed plasma renin activity and serum aldosterone, who were suspected of liquorice abuse. RESULTS: The detection limit for 18betaGA of the GC assay was 10 microg L-1, which was lowered to 3 microg L-1 by subsequent application of MS. In all volunteers, urinary 18betaGA was detected during liquorice intake. Urinary 18betaGA remained detectable until 5 days after stopping continued liquorice intake and until at least 51 h after ingestion of a single large amount. Urinary 18betaGA was demonstrated in both patients, establishing a diagnosis of liquorice abuse. One patient showed changes in urinary cortisol metabolites, consistent with 11betaHSD inhibition. Changes in cortisol metabolites were less pronounced in the other patient. CONCLUSION: Liquorice abuse can result in hypokalaemic hypertension with prolonged suppression of plasma renin activity and aldosterone concentration. This is caused by 18betaGA-mediated inhibition of 11betaHSD, resulting in activation of the renal mineralocorticoid receptor by cortisol. Urinary 18betaGA measurement by GCMS is a useful aid in establishing liquorice abuse.

The daily cortisol production reinvestigated in healthy men. The serum and urinary cortisol production rates are not significantly different.

We have measured the urinary cortisol production rate (uCPR) simultaneously with the serum cortisol production rate (sCPR) in four healthy men within a period of 3 days. uCPR, determined by isotope dilution of 11-oxoetiocholanolone was compared with sCPR, which was measured in three different ways (a, b, c). Blood was sampled at 10-min intervals for 24 h, and deconvolution analysis was applied to the cortisol concentrations. The daily serum cortisol production per liter, multiplied by the distribution volume yielded sCPR. The measurement methods are characterized as follows: a) the secretion and elimination terms were free; b) like method a, but with the input of the rate constants alpha and beta into the elimination function; c) the average 24-h cortisol concentration was multiplied by the metabolic clearance rate. uCPR was 25.4 +/- 4.7 [range: 21.3-31.4] micromol/(m2 x day), sCPR (method a) was 28.8 +/- 4.5 [range: 23.5-34.3] micromol/(m2 x day), sCPR (method b) was 27.9 +/- 8.1 [range: 18.5-37.7] micromol/(m2 x day), and sCPR (method c) was 29.3 +/- 4.8 [range: 22.7-33.2] micromol/(m2 x day). uCPR did not significantly differ from each of the 3 sCPR values (P > 0.30; > 0.46; and > 0.06, respectively). The patterns of the cortisol secretory rates in the present and previous studies do not necessarily represent the physiological process of the secretory bursts. We conclude that the estimated CPR, being 25-30 micromol/(m2 x day) [9-11 mg/(m2 x day)], can serve as a guideline for glucocorticoid replacement dose and that the urinary route to measure CPR is preferred because of its relative ease.

Exon skipping in the sterol 27-hydroxylase gene leads to cerebrotendinous xanthomatosis.

We report a new mutation in the sterol 27-hydroxylase (CYP 27) gene in a Dutch family with cerebrotendinous xanthomatosis: a G-->A transition in the splice donor site in intron 4. This mutation leads to skipping of exon 4, resulting in a loss of 66 amino acids in the CYP 27 enzyme molecule.

Determination of urea kinetics by isotope dilution with [13C]urea and gas chromatography-isotope ratio mass spectrometry (GC-IRMS) analysis.

1. Stable urea isotopes can be used to study urea kinetics in humans. The use of stable urea isotopes for studying urea kinetic parameters in humans on a large scale is hampered by the high costs of the labelled material. We devised a urea dilution for measurement of the distribution volume, production rate and clearance of urea in healthy subjects and renal failure patients using the inexpensive single labelled [13C]urea isotope with subsequent analysis by headspace chromatography-isotope ratio MS (GC-IRMS) of the [13C]urea enrichment. 2. The method involves measurement of the molar percentage excess of [13C]urea in plasma samples taken over a 4 h period after an intravenous bolus injection of [13C]urea. During the sample processing procedure, the plasma samples together with calibration samples containing a known molar percentage excess of [13C]urea are acidified with phosphoric acid to remove endogenous CO2, and are subsequently incubated with urease to convert the urea present in the plasma samples into CO2. The 13C enrichment of the generated CO2 is analysed by means of GC-IRMS. This method allows measurement of the molar percentage excess of [13C]urea to an accuracy of 0.02%. 3. Reproducibility studies showed that the sample processing procedure [within-run coefficient of variation (CV) < 2.8% and between-run CV < 8.8%] and the GC-IRMS analysis (within-day CV < 1.3% and between-day CV < 1.3%) could be repeated with good reproducibility. 4. In clinical urea kinetic studies in a healthy subject and in a renal failure patient without residual renal function, reproducible values of the distribution volume, production rate and clearance of urea were determined using minimal amounts of [13C]urea (25-50 mg). 5. Because only low [13C]urea enrichments are needed in this urea dilution method using GC-IRMS analysis, the costs of urea kinetic studies are reduced considerably, especially in patients with renal failure.

A patient with adrenocortical carcinoma: characterization of its biological activity and drug resistance profile.

We describe a patient with a metastasized adrenocortical cancer who exhibited excessive production of both glucocorticoids and mineralocorticoids combined with suppressed androgen production. Unusual steroid metabolites found in the patient's urine have not been described previously in association with this tumor type. Investigation of the multidrug resistance phenotype in single-cell suspensions of the tumor revealed low expression of multidrug resistance protein but high expression of P-glycoprotein (Pgp) and lung resistance-related protein. Functional Pgp in these tumor cells was shown by the modulatory effect of PSC833 on daunorubicin accumulation. Mitotane, at a concentration achieved in this patient's plasma, completely reversed the Pgp-related resistance both in the Pgp-overexpressing KB8-5 cell line and in the patient's tumor cells. On the basis of these in vitro results, the patient was treated with a combination of multidrug resistance drugs (doxorubicin, vincristine, and etoposide) plus mitotane as a Pgp modulator. This treatment was ineffective, however. A chemosensitivity assay demonstrated that the tumor cells were highly resistant to the drugs used. The adrenocortical cancer cells expressed mutant p53, and no evidence for induction of apoptosis by these drugs was found.

Evaluation of urinary metanephrine and normetanephrine enzyme immunoassay (ELISA) kits by comparison with isotope dilution mass spectrometry.

Determination of urinary 3-O-methylated catecholamines (metanephrines) is generally considered a principal test for the clinical chemical diagnosis of pheochromocytoma and is currently performed predominantly with chromatographic techniques such as gas-liquid chromatography and HPLC. Enzyme immunoassays based on microtiter plate technology have recently been developed for the quantitative determination of urinary metanephrine (M) and normetanephrine (NM). We compared the results for urinary M and NM determined by these ELISA methods with those obtained by a recently developed isotope dilution mass spectrometric method. From this comparative study we can conclude that the investigated ELISA methods are applicable in the quantification of urinary M and thus can be successfully used to establish the diagnosis of pheochromocytoma. These relatively simple methods can be executed in any clinical laboratory and in time may replace the present, more complicated, chromatographic techniques.

Kinetics of intravenously dosed cortisol in four men. Consequences for calculation of the plasma cortisol production rate.

The kinetics of cortisol in the serum of 4 healthy men were studied following single i.v. doses of 2 and 0.8 mg of cortisol. The disappearance of cortisol was determined by blood sampling frequently over 2.5 h and analysing the apparently biexponential cortisol decay. The main results, shown as the mean (+/-SD), were: (a) the average distribution volume of cortisol at steady state (Vd,ss), which was 7.1 l/m2 body surface area. The extrapolated distribution volume (Vd,ext) was 8.4 l/m2, being 18% higher than the corresponding Vd,ss. (b) It was confirmed that plasma cortisol disappears biexponentially. Since the rapid phase remains unnoticed if cortisol is measured at an interval of 10 or more minutes, the obscured rapid-phase parameters can be found only if the known ratio of the two rate constants is used. (c) The fraction of cortisol, which during this fast phase irreversibly disappeared according to the two-compartment open model, was 5 to 8% larger than that found using the monocompartment model. (d) The half-life of the slow or beta phase was equal for the 2 and 0.8 mg experiments, namely t1/2(beta) = 66 +/- 18 min. The kinetics of cortisol in the same 4 men were also measured after an i.v. dose of radioactive cortisol (82 +/- 7 kBq 3H/m2). All urine was collected in 15 portions during the next 3 days, followed by measuring the cumulative radioactivity and analysing the triexponential increase of urinary radioactivity [1]. The main results with the urinary model were: (a) the half-life of cortisol elimination from the circulation was 40 +/- 11 min, (b) the maximal radioactivity (69 +/- 7% of the dose) in the first pool (liver) was found at 2 +/- 0.3 h, (c) the half-life of the cortisol metabolites in the body was 6.8 +/- 0.7 h. Forcing the measured cortisol concentrations in plasma to fit a monoexponential function, allowed us to compare the half-life of cortisol decay with that from the urinary model. It was found that these half-lives were similar with values between 30 and 40 min. Finally, the distribution volume has to be measured individually if a 24 h plasma cortisol profile is used for the calculation of the cortisol production rate.

Optimization of a classical aromatase activity assay and application in normal, adenomatous and malignant breast parenchyma.

The tritium water release assay, originally described for the analysis of aromatase activity in placental tissue, was used to estimate aromatase activity in breast tissue samples. The lower activity in this tissue necessitates longer incubation times and thus optimization of the assay conditions. To prevent oxidative and proteolytic inactivation of aromatase, dithiothreitol and albumin were added to the incubation mixture. Extra NADPH, cofactor in the aromatase reaction, also improved reaction rate in placental incubations, but after approximately 120 min activity rapidly decreased. Inhibitors gradually produced during the incubation could explain this phenomenon. Quantitative gas chromatography-mass spectrometry (GC-MS) analyses of testosterone, oestradiol, oestrone and androstenedione after incubation with non-labelled androstenedione proved that a substantial amount of the substrate is converted into testosterone. Qualitative GC-MS steroid profiling of the incubation mixture demonstrated the presence of hydroxylated oestradiol and hydroxylated testosterone, produced during incubation, which could have caused partial aromatase inhibition. The adjusted assay was used to analyse 84 breast tissue samples, histologically classified as normal, adenoma or carcinoma. Aromatase activity was found in 56% of all samples and ranged from 0.6 to 26 pmol oestrogen/g protein per hour. Aromatase positivity was found in 80% of the normal samples, 56% of the adenoma samples and 48% of the carcinoma samples. Although carcinoma samples were less often aromatase positive than normal tissue samples (chi2 = 4.80; P < 0.050) there was no difference in absolute aromatase activity. Because no less than approximately 50% of the carcinomas contained aromatase activity and because of the non-routine character of the assay we conclude that it is justified to start aromatase inhibition therapy without previous knowledge of the aromatase status.

Pharmacodynamic effects of depot-medroxyprogesterone acetate (DMPA) administered to lactating women on their male infants.

Normal postpartum women, who had a spontaneous vaginal delivery of one full-term male infant, free of congenital abnormalities and other diseases, were recruited for this study. Thirteen women received 150 mg depot-medroxy-progesterone acetate (DMPA), intramuscularly on days 42 + 1 and 126 + 1 postpartum. Infants of nine mothers, who did not receive DMPA, served as controls. Blood samples were collected from treated mothers on days 44, 47, 74, 124, 128, and 130 postpartum for medroxyprogesterone acetate (MPA) measurements. Four-hour urine collections were obtained from all 22 infants in the morning on days 38, 40, 42, 44, 46, 53, 60, 67, 74, 88, 102, 116, 122, 124, 126, 128, 130, and 137. Urinary follicle stimulating hormone (FSH), luteinizing hormone (LH), unconjugated testosterone, and unconjugated cortisol were measured by radioimmunoassay, and serum MPA and urinary MPA metabolites were measured by gas chromatography-mass spectrometry (GC-MS). No MPA metabolites could be detected in the urine of the infants from the DMPA-receiving mothers. Hormonal profiles in the urine samples were not suppressed in comparison with those of the control infants. The present study demonstrates that DMPA, administered to the mother, does not influence the hormonal regulation of the breast-fed normal male infant.

Relationship between biliary lipid and protoporphyrin secretion; potential role of mdr2 P-glycoprotein in hepatobiliary organic anion transport.

BACKGROUND/AIMS: Erythropoietic protoporphyria, caused by ferrochelatase deficiency, leads to protoporphyrin accumulation in the liver. Therapeutic attempts to increase the secretion of this hydrophobic organic anion into bile are hampered by a lack of understanding of the secretory mechanism(s) involved. We have investigated biliary secretion of protoporphyrin in rats and mice, primarily targeted on the role of biliary lipids in this process. METHODS: Gel permeation chromatography was applied to investigate the association of porphyrins with lipid fractions in bile. Secretion of endogenous porphyrins was studied in (GY mutant) rats and mdr2 P-glycoprotein deficient mice, under conditions of widely varying biliary lipid secretion rates. RESULTS: Gel permeation chromatography revealed that, in native human and rat bile, protoporphyrin associated with cholesterol/phospholipid vesicles upon elution with bile salt-free buffer. In contrast, the more hydrophilic coproporphyrin isomers I and III were found only in bile salt/organic anion hybrid particles and smaller aggregates. Interruption of the enterohepatic circulation in normal Wistar rat resulted in parallel decrease of endogenous protoporphyrin-, lipid-, and bile salt secretion, but did not alter the secretion of coproporphyrin I and III. Uncoupling of lipid- from bile salt secretion by sulfated taurolithocholate resulted in impaired secretion into bile of protoporphyrin only. Conversely, secretion of coproporphyrin I and III, but not that of protoporphyrin, was impaired in mutant Groningen Yellow rats with defective ATP-dependent hepatobiliary organic anion transport. In mice homozygous for a disruption of the mdr2 P-glycoprotein gene, resulting in complete absence of phospholipids in bile and strongly reduced cholesterol output, secretion of protoporphyrin was reduced by 90%, whereas that of coproporphyrin I and III was affected to a much lesser extent. CONCLUSIONS: Our data demonstrate a close association between protoporphyrin and lipid secretion into bile, indicating that these processes are, at least functioning coupled. This finding implicates a role of mdr2 P-glycoprotein activity in hepatobiliary removal of the hydrophobic organic anion protoporphyrin. Hence, it may be speculated that protoporphyrin secretion can be influenced by drugs, diet or other means that affect biliary lipid secretion.