Heinz Breuer

Uptake of thyroid hormone by isolated rat liver cells.

L-Triiodothyronine is taken up by isolated rat liver cells by a process which is saturable and exhibits sigmoidity. Two uptake systems make themselves evident: A system with high affinity with an apparent Kt value of 52±22 nM and a system with low affinity with an apparent Kt value of 1446±764 nM. Cells heated at 60°C or after freezing do not show saturability of uptake. KCN inhibits the uptake by the low affinity system. In the presence of L-thyroxine and L-tyrosine the uptake of L-triiodothyronine is increased. The results suggest transport of L-triiodothyronine by proteins in the plasma membrane of the liver cell.

An improved assay for steroid glucuronyltransferase in rat liver microsomes.

Abstract A simple and economical method of assaying rat liver microsomal estrone and testosterone glucuronyltransferase activity has been developed. Liver microsomes were activated by pretreatment with Lubrol WX. The incubation was carried out at 37°C for 30 min and contained 30–600 μ m steroid, 1–2 m m UDP-glucuronic acid, 10 m m MgCl 2 , and 80–150 μg of microsomal protein. Enzyme activities showed a maximum at pH 8.8 with Tris-HCl buffer. After incubation the unreacted substrate was quantitatively removed by a single extraction with dichloromethane. The glucuronide was estimated by counting an aliquot of the aqueous phase in a liquid scintillation counter. The variation coefficients with estrone and testosterone as substrates were 6.0 and 4.0%, respectively.

A highly sensitive method for the demonstration of carcinoembryonic antigen in normal and neoplastic colonic tissue

SummaryA highly sensitive method for the immuno-histochemical localisation of carcinoembryonic antigen (CEA) is described. This method is based on the binding of a peroxidase-antiperoxidase complex (PAP) to anti-CEA antibodies by means of an anti-gamma-globulin which reacts with both the anti-CEA and the antiperoxidase antibodies. Using the technique described here, CEA was localised in conventionally processed normal and cancerous colonic tissue. In normal as well as in neoplastic tissues, a CEA-specific staining of cell membranes and cytoplasm was demonstrated. In frozen sections of normal colonic tissue CEA was found even at high dilutions of the first antibody; this indicates the high sensitivity of the method. The applicability of the method to conventionally processed and thereby well preserved tissue specimens opens the possibility to identify CEA by light microscopy even at very low concentrations.

Zur gas-chromatographisch-massenspektrometrischen Bestimmung von Steroidhormonen in Körperflüssigkeiten unter Verwendung eines Multiple Ion Detectors (Fragmentographie)

The quantitative determination of small amounts of steroids in body fluids by physical-chemical methods (gas chromatography, double isotope derivative dilution) requires a considerable amount of time and effort. The use of the combination gas chromatography-mass spectrometry provides a simple method which is highly specific and sensitive. The mass spectrometer can be used as a specific detector for gas chromatography; this is achieved by adjusting to a suitablem/e value. By means of the multiple ion detector, it is possible to record several masses simultaneously. The applicability of this method is demonstrated by the determination of the following steroid hormones: testosterone in plasma, aldosterone in urine, oestradiol-17β and oestrone in plasma. For the determination of oestrogens, the use of the corresponding dideutero compounds as standards offers a special advantage. Preliminary experiments showed that the lower limits of detection are 1 ng for aldosterone and testosterone, and 0.05 ng for oestradiol-17β and oestrone.ZusammenfassungDie quantitative Bestimmung geringer Steroidkonzentrationen in Körperflüssigkeiten erfordert mit den heute bekannten physikalisch-chemischen Verfahren (Gas-Chromatographie, Doppelisotopenderivatverdünnungsmethoden) einen erheblichen methodischen und zeitlichen Aufwand. Eine wesentliche Vereinfachung bei hoher Empfindlichkeit und Spezifität bietet der Einsatz des Gas-Chromatographen-Massenspektrometers. Hierbei wird das Massenspektrometer als hochspezifischer Detektor für den Gas-Chromatographen benutzt; dies erfolgt durch Einstellung eines geeignetenm/e-Wertes. Mit Hilfe des Multiple Ion Detectors ist es auch möglich, mehrere Massen gleichzeitig aufzuzeichnen. Die Brauchbarkeit dieses Verfahrens wurde am Beispiel folgender Methoden zur Bestimmung von Steroidhormonen demonstriert: Testosteron im Plasma, Aldosteron im Urin, Oestradiol-17β und Oestron im Plasma. Als besonders vorteilhaft erwies sich bei der quantitativen Bestimmung der Oestrogene die Verwendung der entsprechenden Dideuteroverbindungen als Standardsubstanzen. Die vorläufigen Nachweisgrenzen der hier angegebenen Verfahren liegen für Aldosteron und Testosteron bei 1 ng, für Oestron und Oestradiol-17β bei 0,05 ng.

Mass fragmentography as reference method in clinical steroid assay

Abstract Mass fragmentography may serve as a powerful tool in the fields of steroid biochemistry and clinical chemistry; it provides a simple and highly specific method for the determination of steroids in body fluids. Isotopically labelled steroids, corresponding to the natural occurring steroids, may be used as internal standards in order to correct for procedural losses as well as adsorption effects on the gas Chromatographie columns. Since the mass spectrometer is capable of separating isotopes, quantitative determinations may be performed by comparing the peak areas of the unlabelled and the isotopically labelled steroids; the peaks are recorded simultaneously by monitoring two m/e- values (multiple ion detection). With respect to sensitivity and specificity, the method may be considerably improved by the use of special derivatives, e.g. heptafluorobutyrates. Because of their high specificity, determinations by mass fragmentography will be increasingly used as reference methods in the field of steroid hormones. In the present paper the procedure has been applied to the specific determination of oestrogens, testosterone, 5α-dihydrotestosterone, cortisol and aldosterone in human plasma.

Kinetics of steroid transport through cell membranes: Comparison of the uptake of cortisol by isolated rat liver cells with binding of cortisol to rat liver cytosol

Abstract Viable rat liver cells suspended in a medium containing cortisol take up the steroid rapidly; the uptake is temperature dependent. After incubation for 1 min at 27°C the uptake is 20 times higher than that observed at 5°C. The activation energy calculated for the uptake of cortisol was 18 kcal between 5° to 20°C and 6 kcal from 20° to 32°C. With increasing concentrations of cortisol two saturable systems were observed, one with an apparent K M value of 0.2 μM and the other with 2.0 μM. The uptake after 1 min of incubation at 27°C was 9 pmol per mg cell protein. Diffusion of cortisol into the cell was also observed at the steroid concentrations tested. Metabolic inhibitor KCN inhibited the saturable uptake system; pCMB decreased the uptake of cortisol. Binding of cortisol to liver cytosol, carried out under conditions of uptake of cortisol by rat liver cells was more rapid; however, the binding after 1 min of incubation at 27°C was twice that observed at 5°C. The Arrhenius plot was linear from 5° to 32°C; the activation energy was calculated to be 1.2 kcal. The binding was linear from 5 to 10,000 nM cortisol. After incubation for 1 min at 27°C the binding of cortisol to cytosol proteins was 5% of the uptake by liver cells. KCN did not inhibit the binding, whereas pCMB decreased the binding. The results suggest that the uptake of cortisol by liver cells is mediated by carrier proteins in the plasma membrane and that cytosol proteins are not directly involved in this initial uptake process.

Specific interaction of corticosteroids with components of the cell membrane which are involved in the translocation of the hormone into the intravesicular space of purified rat liver plasma membrane vesicles

Abstract The uptake of [ 3 H]-corticosterone was studied by a highly purified rat liver plasma membrane vesicle fraction. At 23°C, [ 3 H]-corticosterone is taken up very rapidly; equilibrium is reached as early as 5 s. At high concentrations of corticosterone (30–8100 nM) uptake occurs predominantly by a non-saturable process, but the presence of a saturable process is also detectable. When the intravesicular space is successively decreased by increasing the osmolarity of the external medium, uptake of [ 3 H]-corticosterone decreased. About 45% of the total steroid taken up is translocated into the lumen of the vesicle. In the presence of a 200-fold molar excess of non-labeled corticosterone about 40% of the total uptake of [ 3 H]-corticosterone is displaceable. At low concentrations of corticosterone (2—230 nM) and considering only the specific portion of uptake the presence of a high and a low affinity uptake system is observable. The K D of the two systems were 7.2 ± 2.0 nM and 234 ± 67 nM, respectively. The K D of the high affinity system corresponds to the concentration of free corticosterone (about 8 nM) in the plasma of the female rat. The maximal binding capacity was about 180 fmol/(mg membrane protein). Structural analogues of corticosterone reduce the displaceable portion of the uptake of [ 3 H]corticosterone; cortisol, progesterone. dexamethasone and 5α-dihydrocorticosterone are strong inhibitors. Among the sex hormones 5α-dihydrotestosterone and diethylstilbestrol are the most efficient inhibitors. These results suggest that corticosterone reacts in a specific manner with components of the plasma membrane which may function to translocate the steroid into the cell.

Bestimmung von Aldosteron im Plasma des Menschen unter Verwendung der Kombination Gas-Chromatographie/Massenspektrometrie

The method is based on the principle of isotope dilution: a known amount of isotopically labelled standard ([4-14C]-aldosterone) is added to the plasma sample. After purification of the aldosterone fraction and formation of a suitable derivative, the ratio of isotopes (12C/14C) is measured by mass spectrometry. The analytical procedure consists of the following steps: 1. Chromatography of the plasma sample (10 ml) on Amberlite XAD-2, 2. formation of the acetal of aldosterone, 3. purification of the acetal by a single thin-layer chromatography, 4. formation of the heptafhuorobutyrate of the aldosterone acetal, 5. combined gas chromatography/mass spectrometry. The lower limit of detection is at 100 pg/10 ml plasma. Because of its high specificity, the present method is recommended as a reference method. 10 determinations can be carried out per working day.ZusammenfassungEs wird eine gas-chromatographisch-massenspektrometrische Methode zur Bestimmung von Aldosteron in Plasma des Menschen beschrieben. Bei diesem Verfahren wird das Prinzip der Isotopenverdünnung verwendet: Eine bekannte Menge isotopenmarkierter Standardsubstanz ([4-14C]Aldosteron) wird der zu untersuchenden Plasmaprobe zugesetzt. Nach Anreicherung der Aldosteronfraktion und Bildung eines geeigneten Derivats wird mit dem Massenspektrometer das Isotopenverhältnis (12C/14C) bestimmt. Die Analyse umfaßt folgende Schritte: 1. Chromatographie der Plasmaprobe (10 ml) an Amberlite XAD-2,2. Bildung des Aldosteron-Vollacetals, 3. Reinigung des Vollacetals durch einmalige Dünnschicht-Chromatographie, 4. Bildung des Heptafluorbutyrats des Vollacetals, 5. Kombinierte Gas-Chromatographie/Massenspektrometrie. Die untere Nachweisgrenze liegt bei 100 pg/10 ml Plasma. Auf Grund seiner hohen Spezifität ist das Verfahren als Referenzmethode geeignet. Pro Arbeitstag können 10 Bestimmungen durchgeführt werden.

Untersuchungen über den Stoffwechsel von Steroidhormonen bei Vertebraten: VI. Reduktiver Stoffwechsel von Testosteron und verwandter C19-Steroide in leberpräparationen der Forelle, des Tritons, des Frosches und des Huhnes☆

Zusammenfassung Es wurde der reduktive Stoffwechsel von Testosteron und einiger verwandter C 19 -Steroide in Schnitten sowie in den Mikrosomen- und Cytoplasma-Fraktionen der Leber der Forelle, des Tritons, des Frosches und des Huhnes untersucht. Die Identifizierung der Metaboliten erfolgte auf Grund ihrer R F -Werte und Wanderungs-strecken in verschiedenen dunnschichtchromatographischen Systemen sowie durch ihr Verhalten bei der Anisaldehyd-Schwefelsaure-Farbreaktion. Bei allen untersuchten Tieren konnten folgende Enzyme des Steroidstoffwechsels nachgewiesen werden: 5α- und 5β-Reduktasen sowie 3α-, 3β- und 17β-Hydroxysteroid-Oxydoreduktasen. Der Nachweis einer 17α-Hydroxysteroid-Oxydoreduktase gelang mit Sicherheit nur bei der Forelle, beim Frosch und beim Huhn. Alle Enzyme reagierten mit NAD und/oder NADP als Cofaktoren. Die 5α-Reduktasen sind ausschliesslich in den Mikrosomen-Fraktionen, die 5β-Reduktasen ausschliesslich in den Cytoplasma-Fraktionen lokalisiert. Aus den hier dargestellten Ergebnissen geht hervor, dass der reduktive Stoffwechsel von C 19 -Steroiden in der Leber von niederen Vertebraten und Vogeln in ahnlicher Weise verlauft wie bei Saugetieren.

Factors involved in the uptake of corticosterone by rat liver cells.

Isolated rat liver cells take up corticosterone rapidly; the initial rates increase with increasing temperature. A plot of the initial rates against the concentration of corticosterone indicated the presence of saturable and nonsaturable uptake systems. The Eadie-Hofstee plot showed the presence of two saturable and one nonsaturable uptake components. The apparent Kt values of the saturable systems were 64 +/- 40 nM (n = 3) and 1085 +/- 313 nM (n = 12). The nonsaturable system, probably diffusion, contributed 12% to the total uptake between 15 and 72 nM corticosterone, the physiological concentration of the free corticosterone in rat serum. Metabolic inhibitors did not influence the uptake of corticosterone. N-Ethylmaleimide, 1-fluoro-2,4-dinitrobenzene and sodium ethyl mercurithiosalicylate (1 mM each) decreased the uptake by 40%. Iodoacetate did not have any influence. Treatment of cells with phospholipase A inhibited the uptake 35--45%. In the presence of cortisone, cortisol, dexamethasone, aldosterone, testosterone, estradiol-17beta and estrone (2 muM each) the uptake decreased 30--50%. The presence of serum proteins in the external medium inhibits the uptake of corticosterone. These results suggest that corticosterone is transported into the cell and is accumulated. Only the free hormone is available for uptake which in turn may be regulated by protein and lipid components in the plasma membrane of the liver cell.