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The influence of fluorocarbon FC 43 on the metabolism of oestrogens in the isolated perfused rat liver was investigated. In comparative once-through perfusions with FC 43 emulsion or albumin solution as perfusion medium, the clearance rates of oestrone and its metabolites were determined. In perfusions with FC 43, the clearance of oestrone was lower, and the metabolites formed in the liver were more concentrated in the outflowing medium than in perfusions without fluorocarbon. This can be explained by the high affinity, even of conjugated oestrogens, to FC 43, which is established by equilibrium dialysis and partition coefficient. The results presented here show that the fluorocarbon has a strong influence of its own on the metabolism of steroids in the isolated perfused liver. Therefore, this solvent should be avoided as medium when the metabolism of steroids is studied in perfusion experiments.

A specific and sensitive method is described for the separation and determination of metabolites of radioactively labelled noradrenaline in very small amounts of brain tissue. This method gives reproducible results, it allows the direct determination of all major metabolites of noradrenaline from the same extract, and is suitable for quantitating a large number of samples in relatively short time. This method was used to investigate how the half-life (turnover) and the metabolism of noradrenaline in rat brain may be influenced by anaesthesia with nembutal. It was found that both the half-life time as well as the metabolism of noradrenaline are reduced during nembutal anaesthesia; in addition, the release of metabolites of noradrenaline into the blood is delayed. When studying the metabolism of neurotransmitters, it has to be considered that nembutal-narcosis influences the metabolic pattern of noradrenaline as well as its temporal changes.

In patients with liver cirrhosis the concentrations of amino acids were measured by ion exchange chromatography in the serum of blood samples taken from various vessels during and after the performance of a porto-caval anastomosis. Statistical evaluation was carried out with a nonparametric test. In three female and eight male patients, amino acids were determined intra operationem in blood samples of the following vessels (n = number of blood samples): arm vein (n = 8), arteria femoralis (n =2), vena cava (n = 7), vena portae (n = p) and aorta abdominalis (n = 5). With exception of ornithine (aorta abdominalis versus vena cava), no statistically significant differences in the concentrations of amino acids were observed in the various blood vessels. One to three years after the introduction of the porto-caval shunt, amino acid concentrations were measured in blood from the arm vein and arteria femoralis in four female and five male patients. The concentrations of glutamic acid, phenylalanine and lysine were significantly higher in blood from the arm vein than in blood of the arteria femoralis. The concentrations of valine, leucine and isoleucine were markedly lower in patients with liver cirrhosis than in normal persons. On the basis of the present findings and of the results obtained with normal subjects, it may be concluded that porto-caval anastomosis does not exert a noticeable effect on the metabolism of amino acids in patients with liver cirrhosis.

The effect of spirolactones on the urinary excretion of individual 17-oxosteroids and of pregnanediol in man was investigated under various conditions. After purification by thin-layer chromatography, the 17-oxosteroids and pregnanediol were determined by gas chromatography. Eleven male subjects (age 21-34 years) received a daily dose of 200 mg spironolactone (Aldactone) orally on seven consecutive days. Five female subjects (age 20-35 years) and ten pregnant women (age 20-35 years; 28th.-39th. week of gestation) were given a daily intravenous injection of 600 mg potassium canrenoate (Aldactone pro injectione) on three consecutive days. In the male subjects, the excretion of individual 17-oxosteroids and of pregnanediol was significantly reduced during administration of spironolactone. In the nonpregnant female subjects, the excretion of etiocholanolone and dehydroepiandrosterone was diminished, whereas in the pregnant women, the excretion of pregnanediol and dehydroepiandrosterone was decreased during treatment with potassium canrenoate. These results suggest that the reduced urinary excretion of steroids may be due to an inhibition of steroid metabolising enzymes by spirolactones. It seems likely that spirolactones affect the enzymatic conversion fo cholesterol to pregnenolone.

It has been shown previously that the biosynthesis and metabolism of oestrogens is affected by rifampicin. The present study was undertaken to investigate if rifampicin may also influence the plasma concentrations of testosterone and cortisol. Five male patients suffering from tuberculosis received rifampicin or streptomycin. During treatment with rifampicin testosterone concentrations were significantly elevated as compared to control conditions; this increase was found in the morning (8.00 h) as well as in the afternoon (16.30 h). When the same patients were treated with streptomycin, only irregular changes of testosterone concentrations were observed. The blood level of cortisol was increased in two patients, but unchanged in three patients who were treated with rifampicin. No changes at all were detected in patients under treatment with streptomycin. The results presented here show that rifampicin -- in contrast to streptomycin -- influences the biosynthesis of testosterone and possibly also that of cortisol.

Diuresis, the excretion of aldosterone-18-glucuronide and the excretion of sodium and potassium were measured before and during two periods of immersion in four male athletes in training and four untrained male subjects (age 20-27 years). The increase in diuresis during immersion was accompained by a concomitant decrease of aldosterone excretion and increase of sodium excretion in urine. These changes were more pronounced in untrained subjects than in trained subjects. From these results it may be concluded that trained subjects react more sensitive to slight changes in aldosterone secretion than untrained subjects.

A definitive method is described for the determination of cortisol in human plasma. The method is based on the principle of isotope dilution-mass spectrometry. The analytical procedure comprises the following steps: (1) Addition of [4-14C]cortisol to the plasma sample; (2) extraction of the 14C-labelled and of the non-labelled cortisol by dichloromethane; (3) purification of the cortisol fraction by column chromatography on Sephadex LH-20; (4) formation of the trimethylsilyl ether of cortisol methoxime; (5) selected ion monitoring at m/z-values 605, 607, 636 and 638 during gas liquid chromatography. The accuracy of the method is based on the high specificity of mass spectrometry and on the exact control of recovery employing the principle of isotope dilution. The precision of the method was evaluated by calculation of the coefficient of variation from day to day; this varied from 0.7-2.3% in the range of 256-845 nmol/l. The lower limit of detection (ratio of signal to noise 3:1) is 200 pg cortisol per sample.

Studies on the individual components of the renin-angiotensin-aldosterone system are of crucial importance for the investigation of hypertension. The purpose of the workship conference was to give occasion for discussion amongst German speaking research groups on the determination of the individual components of this system, and for the consideration of new results and their pathophysiological significance, as well as possibilities for the evaluation of radioimmunological data with the aid of electronic data processing. The following report is a summary of the contributions to the discussion by those taking part in the workshop conference.

The uptake of cortisol by isolated rat liver cells was studied. Cortisol was taken up rapidly; the uptake increased with increasing temperature and reached a plateau after 45 s at 37 degrees, C, after 60 s at 27 degrees C, and after 90 s at 22 degrees C; at 5 degrees C the uptake increased linearly with time. The uptake was linear up to 1.5 mg of cell protein. Analysis of uptake as a function of increasing concentration of cortisol in the external medium indicated the presence of two saturable systems: a high-affinity system with an apparent Km value of 190 +/- 25 nM and a low-affinity system with an apparent Km value of 2200 +/- 180 nM. Above 600 nM, the rate of uptake of cortisol increased almost linearly with increasing cortisol concentration. Treatment of cells with KCN or 2,4-dinitrophenol inhibited the two saturable components, leaving the nonsaturable system unaffected. The affinity constants, Ka, were 6 X 10(6) M-1 and 0.6 X 10(6) M-1 for the high and low affinity components, respectively. These values increased approximately two-fold when uptake rates were corrected for diffusion. Cortisone and corticosterone inhibited the uptake of cortisol by liver cells competitively; dexamethasone inhibited cortisol uptake noncompetitively. Similarly, oestrone, oestradiol and testosterone decreased the uptake of cortisol, at a concentration of 2000 nM in the external medium, by 20, 49 and 35 percent, respectively; the inhibition was noncompetitive. p-Chloromercuribenzoate, N-ethylmaleimide and 1-fluoro-2,4-dinitrobenzene decreased the uptake of cortisol. Ouabain did not influence the uptake of cortisol; varying the external sodium concentration also did not affect uptake of cortisol. Cyclic-3',5'-adenosine monophosphate had a stimulatory effect. The results show that the first step, before cortisol is bound to intracellular binding proteins, is the uptake of cortisol by proteins in the plasma membrane. At lower concentrations of cortisol, uptake takes place by saturable processes; at higher concentrations saturation is not achieved, indicating that simple diffusion becomes the major route of transport into the cell. The proteins in the plasma membrane probably function as carriers to transport the glucocorticoid into the cell.

In 1980 and 1981, four quality control surveys for urinary calculus analyses were carried out, within the framework of external quality controls of the Deutsche Gesellschaft für Klinische Chemie. During these quality control surveys the participating laboratories received a total of 16 samples (eight pure substances and eight mixtures of two components), to be analyzed qualitatively and quantitatively. For the qualitative analyses, a laboratory could provide simplified descriptions of the analytes - for example, without stating the number of molecules of the water of crystallization; or, to the extent permitted by the mode of analysis, the exact chemical composition could be presented. Of 1283 analyses, 818 (64%) attained the goals of the simplified analysis. The most frequent errors in chemical analyses were false negative and false positive results for calcium oxalate, and false negative results for calcium phosphate. Laboratories using analysis by infrared spectroscopy also submitted the biggest proportion of incorrect analyses for these components. The most reliable results were produced by X-ray-diffraction: 86% of the results in this category represented correct differentiated analyses. The generally accurate results achieved by some participants using chemical analysis suggest, however, that - in addition to the principle of analysis used - the qualification of the operator is an important determinant in the reliability of the results.

The binding capacities of SHBG and CBG were measured by agar gel electrophoresis in 63 men with cirrhosis of the liver and in 42 healthy male subjects. The normal range (X- +/- 2s) for SHBG was 8.3-17.1 microgram/l, for CBG 46.4-82.8 micrograms/l. SHBG binding capacity was significantly higher in men with liver cirrhosis (mean 18;1 microgram/l; p less than 0.001) but CBG binding capacity was significantly lower (mean 49.7 microgram/l; p less than 0.001). Although SHBG was lower in patients with decreased CBG binding capacity, a correlation between both steroid binding proteins did not exist. Moreover, there was no correlation between SHBG or CBG on one hand and other parameters of hepatic protein synthesis such as serum protein concentration, cholinesterase activity and the coagulation factors V and VII on the other hand. In contrast to liver cirrhosis, 12 patients with fatty liver and 11 patients with toxic fibrosis of the liver did not reveal changes in SHBG or CBG. Treatment with spironolactone (200 mg daily for one week in 9 subjects) did not change the steroid binding capacity of human serum.

An improved method for the simultaneous gas-chromatographic determination of individual 17-oxosteroids and of pregnanediol in the same sample of urine is described. The method involves the following steps. The urinary sample is chromatographed on Amberlite XAD-2. The fraction containing the steroid conjugates is evaporated; the residue is dissolved in n-heptane and the organic phase extracted with water. The steroid glucuronides are hydrolysed by a bacterial beta-glucuronidase; subsequently, the steroid sulphates are solvolysed in ethyl acetate. Free 17-oxosteroids and pregnanediol are separated by two-dimensional thin-layer chromatography. The zone containing the steroids to be investigated is eluted; after evaporation, the residue is treat with N-methyl N-trimethyl silyl trifluoroacetamide. The reaction mixture is injected into a gas chromatograph; the trimethyl silyl ethers of the individual 17-oxosteroids and of pregnanediol are quantitated by measuring the peak heights.

It is known that all forms of life possess biological rhythms. The structure of this biological daily periodicity is caused by endogenous and exogenous factors. The reasons of circadian periodicity are endogenous processes, which can be modified by exogenous factors. This review describes the daily fluctuations of concentrations or excretions of haematologic, clinical chemical and endocrinological parameters. In blood and serum the largest daily variations are found for the concentrations of haemoglobin, potassium, iron, cortisol, testosterone, somatotropin, prolactin, adrenaline and noradrenaline. In contrast to the serum concentrations of the electrolytes sodium, potassium, calcium and phosphate, their urinary excretions demonstrate pronounced daily rhythms with amplitudes being twice as high as the daily means. There is a remarkable phase shift of 180 degrees between phosphate and the cations both in the serum concentrations and urinary excretions. A daily rhythm comparable to the urinary excretion of electrolytes, has been measured for the urinary excretion of adrenaline, noradrenaline and cortisol. A constant, well defined timing of the sampling is important for those constituents with a marked daily variation. For numerous parameters very contradictory results have been reported in the literature concerning their daily variations, especially in cases of less pronounced rhythms. For some parameters the reasons for the differences in the observed daily variations are discussed.

A Workshop Conference on UDP-glucuronyltransferase was held in order to bring together different investigators specialising in a particular aspect of the enzyme. The intention was to find a basis for a closer cooperation between the different groups and to exchange ideas and methods making it possible to cross the hurdles facing those who work in the field of glucuronidation of endogenous and exogenous substances. The topics which were chosen for the meeting covered the following aspects: methods of determination of UDP-glucuronyltransferase activity; problems relating to the origin of the enzyme and the nature of the substrate; application of assays of UDP-glucuronyltransferase in clinical chemistry; enzyme-substrate interactions and factors influencing the enzymic reaction; the role of UDP-glucuronyltransferase in the entero-hepatic circulation of glucuronides, regulation and induction of UDP-glucuronyltransferase, with a subsection on solubilisation and purification.

In the years 1977 to 1981, 14 quality-control surveys for the determination of steroid hormones were performed in cooperation with the Deutsche Gesellschaft für Klinische Chemie. Hereby the laboratories participating could in each case analyze the following steroids: aldosterone, cortisol, oestradiol-17 beta, oestriol, progesterone, and testosterone. In the light of the results an investigation was made as to whether, in the course of time, an improvement in the accuracy or in the precision of the determinations had been attained, and to what extent the determinations depend on the qualities of the test material. A clear improvement in the accuracy of the results of the analyses could only be ascertained for oestradiol-17 beta. For aldosterone and cortisol, values were found in pool-plasma whose medians were significantly above the definitive values. An improvement in precision could be noted especially with oestradiol-17 beta and to lesser degrees with cortisol and oestriol. The kind of test material--plasma which contained only the hormones to be analyzed on the one hand, and, on the other hand, pool-plasma, which also contained all endogenous hormones--had no influence on the precision of the results from various laboratories. Low concentrations of the individual steroids led--on the basis of the methodological principle of radioimmunoassays--in almost all cases to a reduced interlaboratory precision in regard to values. The accuracy of the analysis values was considerably impaired only with aldosterone and oestradiol-17 beta by low concentrations: the medians here were in part twice as high as the definitive values.

After incubation of [4-14C]oestrone (E1) with kidney cortex slices of minipigs, [4-14C]oestradiol-17 beta (E2) and small amounts of a polar metabolite were detected in the ether-soluble fraction. E1, E2 and polar metabolites were found in the protein-bound fraction. The water-soluble fraction contained E1-3-glucuronide (80% of total glucuronides), E2-3-glucuronide and trace amounts of the 3-monoglucuronide of oestriol (E3). When E2 was used as substrate, the main product formed was E1; it was detected in both the ether-soluble and protein-bound fractions. E1-3-Glucuronide was the main metabolite in the water-soluble fraction, which also contained some E2-3-Glucuronide. In male minipigs, the mast, the rate of conversion of E1 and E2 as well as the formation of glucuronides were significantly greater in fertile females than in infertile females. Whereas no sex difference was observed in the metabolism of oestrogens in kidneys of infertile minipigs, the rate of oxidoreduction and glucuronidation was more pronounced in fertile female animals than in the corresponding males. The present results suggest that, in the kidneys of minipigs, the ratio of E1 to E2 is shifted towards the former; furthermore, by a comparatively rapid metabolism of the oestrogenic hormone, the renal tissue contributes to the maintenance of hormonal equilibrium.

Human carcinoembryonic antigen (CEA) was measured in perchloric acid extracts from 9 malignant gastrointestinal tumours, using two different radioimmunological methods (Hoffmann-La Roche and CEA-IRE-SORIN). When standard curves were set up, quantitative differences in the degree of inhibition were found between the standards from the two manufacturers. In the Hoffmann-La Roche assay the CEA binding curves of all tumour extracts ran parallel with the standard. However, when the CEA-IRE-SORIN assay was applied two of the nine tumours exhibited different slopes. In one case, using the CEA-IRE-SORIN method, various parts of the tumour exhibited binding curves with non-parallel slopes. The present results suggest that radioimmunologically heterogeneous CEA occurs in different gastrointestinal tumours as well as in various parts of the same tumour.

After incubation of [4-14C]oestrone (E1) with liver slices from minipigs, the ether-soluble fraction contained [4-14C]oestradiol-17 beta (E2). In the protein-bound fractions, only polar metabolites were found, whereas in the water-soluble fraction the 3-monoglucuronide of oestriol (E1) was the preferred conjugate. When E2 was used as a substrate, E1 was present as main metabolite in the ether-soluble fraction. The radioactive metabolites in the protein-bound and water-soluble fractions were similar to those in the experiments with E1. The metabolism of E1 and E2 was dependent on age. Thus, the rate of conversion of oestrogens was greater in liver tissue of infertile male animals than in fertile males. In contrast, the two steroids were metabolised more rapidly in liver of fertile female minipigs than in infertile female animals. In fertile animals, the metabolic pattern of oestrogens in the ether-soluble, the protein-bound and the water-soluble fractions showed sex dependence: In females, E1 and E2 were metabolised to a greater extent by liver slices than in males. On the other hand, in experiments with male minipigs, E3-3-monoglucuronide was the only metabolite in the water-soluble fraction, whereas liver slices of female animals not only form E3-3-monoglucuronide, but also the 3-glucuronides of E1 and E2. The results described here show that, in liver tissue of minipigs, the oxidoreduction of E2 and E1 is the predominant reaction; in contrast to human liver, hydroxylation reactions play only a minor role. It may be concluded that there are differences in the metabolism of steroid hormones in man and minipig.

The binding of [125I]CEA to an anti-CEA Sepharose-4B immunosorbent has been investigated in the presence of detergents. Non-ionic detergents (Triton-X-100, Lubrol PX and Tørgitol NPX) at concentrations of 5-40 milligrams did not inhibit the binding of [125I]CEA to the immunosorbent. Sodium deoxycholate decreased the binding from 98% (concentration of detergent 0.6 milligram) to 41% (concentration of detergent 20 milligrams). Sodium dodecyl sulfate inhibited the binding of [125I]CEA to the immunosorbent considerably, even at low concentrations.

The isolated rat liver was perfused with a haemoglobin-free, albumin containing salt solution at 28 degrees and 37 degrees C, respectively, with [4-14C]oestrone as substrate. During perfusion, the functional state of the liver was checked by continuously measuring the oxygen pressure and hydrogen ion concentration in the perfusion medium, flow rate, oxygen tension at various areas of the liver surface, and oxygen consumption; in addition, the following biochemical parameters were determined: ATP, ADP, lactate and pyruvate in liver tissue, and lactate and pyruvate in the perfusion medium. After 30 min of perfusion, free steroids, steroid glucuronides, steroid sulphates and the remaining water-soluble metabolites, present in liver tissue, perfusion medium and bile, were separated from each other and characterised by thin-layer and paper chromatography. It was found that, during perfusion at 37 degrees C, less hydroxylated metabolites were formed than at 28 degrees C. In contrast, metabolites whose formation is not directly oxygen-dependent, such as glucuronides and sulphates, arose in higher amounts at 37 degrees C than at 28 degrees C. It may be concluded that the relative shortage of oxygen at 37 degrees C leads to a selective impairment of metabolic pathways requiring a sufficient supply of molecular oxygen. Since oxidative processes play an important role in the biogenesis and metabolism of steroid hormones, it is evident that results, obtained in perfusion experiments with haemoglobin-free media at 37 degrees C, must be treated with reserve.