Use of a soft-agar colony-forming assay to determine the photosensitizing effects of several components of hematoporphyrin derivative.

The photosensitizing/cytotoxic effects of four porphyrin compounds derived from hematoporphyrin were compared using two human tumor cell lines with a soft-agar colony-forming assay. Reproducible dose- and time-dependent increases in reproductive cell death were observed for each porphyrin tested. Two fractions derived from hematoporphyrin derivative (HPD) were found to be better photosensitizers than HPD itself, indicating the potential value of this in vitro assay for detecting the most active component from a heterogeneous mixture.

Synthesis of mono- and diglucosiduronates of metabolites of deoxycorticosterone and corticosterone and analysis by a new mass spectrometric technique.

By condensing 3 alpha,21-dihydroxy-5 beta-pregnan-20-one, or its appropriate monoacetate, with methyl 2,3,4-tri-O-acetyl-1-bromo-1-deoxy-alpha-D-glucuronate in the Koenigs-Knorr reaction beta-D-glucosiduronates 10, 4, and 7 were obtained as polyacetate methyl esters. Alkaline hydrolysis of these substances cleaved the ester groups and gave the corresponding steroidal glucosiduronic acids 12, 6 and 8. Upon treatment with diazomethane, these acids produced the equivalent methyl esters. The C-3, the C-21 and the C-3,21 glucosiduronates of 3 alpha,21-dihydroxy-5 beta-pregnan-11,20-dione were prepared by previously reported methods and converted into the corresponding C-20 semicarbazones (14, 20 and 26). With C-20 stabilized by the semicarbazone group against reduction, it was possible to reduce the 11-oxo function in these substances to an 11 beta-hydroxyl group; after removal of the semi-carbazone moiety from these products at pH 2.0, glucosiduronic acids 18, 22 and 28 were obtained. The mass spectra of a representative group of the mono- and diglucosiduronic acids and esters were determined by utilizing fast atom bombardment and monitoring ions in both positive and negative modes of operation.

Analysis of intact steroid conjugates by secondary ion mass spectrometry (including FABMS) and by gas chromatography.

The analysis of intact steroid conjugates by two different methods is described. One method employed secondary ion mass spectrometry (SIMS) using a Cs+ beam for ionisation, although comparable data were obtained by fast atom bombardment (FAB) using a Xe0 beam. In both of these mass spectrometric techniques the samples were analysed in a liquid matrix (glycerol). Positive and negative ion spectra have been obtained, the latter being most useful for steroid sulphate and glucuronide analysis. The negative ion spectrum of each steroid is dominated by a pseudomolecular ion at m/z [M - H]- (M of free acid) and a lack of marked fragmentation. Mixtures of steroids can be resolved in a single spectrum, providing the individual steroids differ in mass. The second method was gas chromatography. The carboxylic acid moieties of the steroid glucuronides were derivatised with diazomethane and the remaining functional groups in the steroids were thermally protected by methyloxime formation (for carbonyls) and trimethylsilylation (for all steroidal and glucuronic acid hydroxyls). Satisfactory analysis of steroid glucuronides was achieved through the use of glass or fused silica columns stable at high temperature (330 degrees C). Conveniently, trimethylsilylation resulted in exchange of the sulphate in 3 beta-hydroxy-5-ene steroid sulphates for a trimethylsilyl group so these could effectively be analysed as "free" steroids.

Chemical synthesis of glucuronidated metabolites of cortisol.

During in vivo metabolism the addition of six atoms of hydrogen to cortisone at the appropriate location and configuration can lead to formation of either 3 alpha,17,20 alpha,21-tetrahydroxy 5 beta-pregnan-11-one (cortolone) or 3 alpha,17,20 beta,21-tetrahydroxy-5 beta-pregnan-11-one (beta-cortolone). Likewise, metabolic reduction of cortisol can lead to formation of either 5 alpha-pregnane-3 alpha,11 beta,17,20 alpha,21-pentol (cortol) or the 20 beta isomer (beta-cortol). This paper describes the chemical syntheses of the C-3 beta-D-glucosiduronates of cortolone, beta-cortolone, cortol and beta-cortol-conjugates which are normal excretory products of man. The foregoing conjugates are characterized as free acids (or salts), as methyl esters and as polyacetate methyl esters.

C-3 glucosiduronates of metabolites of adrenal steroids.

On treatment with methyl 2,3,4-tri-O-acetyl-1-bromo-1-deoxy-alpha-D-glucuronate and silver carbonate, tetrahydrocortisone 21-acetate gave the corresponding 3-glucosiduronate triacetyl methyl ester. This product was converted into the 20-semicarbazone which, by treatment with alkali to hydrolyze the ester functions and acid to hydrolyze the semicarbazone moiety, gave tetrahydrocortisone 3-glucosiduronic acid. The acid was converted into the crystalline barium salt and into the methyl ester. An analogous series of reactions was carried out on tetrahydrocortexolone 21-acetate. Treatment of the 20-semicarbazone of tetrahydrocortisone 3-glucosiduronic acid with potassium borohydride reduced the 11-oxo function to an 11 beta hydroxyl group; acid-catalyzed removal of the semicarbazone group produced tetrahydrocortisol 3-glucosiduronic acid which also was obtained as the barium salt and the methyl ester.

Evidence for a monoglucuronide of 1,25-dihydroxyvitamin D3 in rat bile.

We have isolated and characterized a monoglucuronide fraction of 9,10-secocholesta-5,7,10(19)triene-1 alpha, 3 beta, 25-triol, 5,6-cis isomer (1,25-dihydroxyvitamin D3) from rat bile. Polar radioactive metabolites of 1,25-dihydroxyvitamin D3 were purified by a sequence of chromatographic procedures which utilized Amberlite XAD-2, diethylaminohydroxypropyl Sephadex LH-20, liquid-liquid partition on paper, and reverse phase chromatography on C-18 microparticulate columns. A purified radioactive substance showed maximal absorbance at 264 nm, indicating the presence of a triene in the 5,6-cis configuration. Mass spectrometry by fast atom bombardment of the product demonstrated an ion at m/z 637 atomic mass units that is consistent with a natriated sodium salt of a monoglucuronide of 1,25-dihydroxyvitamin D3 ([MNa]Na+). Following methylation of the carboxylic acid group and formation of trimethylsilyl ethers of the hydroxyl groups, the fragmentation pattern of the product was compatible with that of a monoglucuronide of 1,25-dihydroxyvitamin D3. The intact metabolite was treated with beta-glucuronidase and the aglycon was isolated by chromatography on microparticulate silica. The aglycon co-migrated with authentic 1,25-dihydroxyvitamin D3 during chromatography and it gave a mass fragmentation pattern consistent with 1,25-dihydroxyvitamin D3. The aglycon was bound by an intestinal cytosol receptor with essentially the same affinity as 1,25-dihydroxyvitamin D3. These findings indicate that bile contains a monoglucuronide of 1,25-dihydroxyvitamin D3.

Enterohepatic physiology of 1,25-dihydroxyvitamin D3.

After intravenous administration of radiolabeled 1,25-dihydroxyvitamin D3 to rats, approximately 25% of the administered radioactivity appeared in the bile within 24 h. Instillation of the biliary radioactivity into the duodena of other rats was followed by recovery of 15% of the radioactivity in newly secreted bile within 24 h. The process by which products of 1,25-dihydroxyvitamin D3 were excreted in bile was not saturable in the dose range tested (0.275-650 ng). The metabolites of 1,25-dihydroxyvitamin D3 present in bile were found to be much more polar than 1,25-dihydroxyvitamin D3 and were resolved into three fractions on high performance liquid chromatography. 60% of the radioactivity present in bile was retained selectively by DEAE-cellulose; the radioactive material could be eluted from the gel at a low pH or at high salt concentrations. When bile containing the radiolabeled metabolites was incubated at 37 degrees C and pH 5 with beta-glucuronidase, there was an increase in the amount of radioactivity comigrating with 1,25-dihydroxyvitamin D3. Treatment of the products of radiolabeled 1,25-dihydroxyvitamin D3 in bile with diazomethane, an agent which converts acids into methyl esters, transformed one of the metabolites into a less polar compound. These results demonstrate that there is a quantitatively important enterophepatic circulation of the products of 1,25-dihydroxyvitamin D3 in the rat.

An evaluation of systems for paper chromatography of C21 steroids.

A group of moderately polar C21 steroids (3-5 oxygen functions) has been chromatographed in 9 solvent systems. Using the concept that standard deviation of the mean RF is an index of chromatographic resolution, and that coefficients of correlation between sets of RF data can be used to quantify the similarities of chromatographic systems, we have evaluated the resolving properties of the systems when used individually, and also when used in combinations of two, three and four. The discriminating powers of some of the most effective individual systems, and some of the sequences of systems which are most efficient, are shown graphically as chromatography trees. The relationship between the total effective standard deviation of a group of systems which are used in sequence and the probability that a pair of compounds will be separated by more than 0.10 RF is discussed.

Isolation, determination of structure and synthesis of the acid-labile conjugate of aldosterone.

1. After administration of 600mg of 3H-labelled aldosterone to human volunteers, 57 mg of homogeneous acid-labile conjugate was isolated from the urine and identified as aldosterone 18 beta-D-glucosiduronic acid. 2. Esterification and acetylation of the conjugate gave a tetra-acetate methyl ester, which, by measurement of the optical rotation and nuclear-magnetic-resonance spectrum, was shown to be a beta-glucosiduronate. This tetra-acetate methyl ester was synthesized in approx. 10% yield by the Koenigs-Knorr procedure. 3. Removal of the acetyl and methyl ester groups from the tetra-acetate methyl ester with alkali was accompanied by almost complete isomerization at C-17 to give 17-isoaldosterone 18 beta-D-glucosiduronic acid. 4. To prevent inversion at C-17 during removal of the acetate and ester groups of beta-glucosiduronate (a) the 3,20-disemicarbazone was prepared, (b) the acetate and ester groups were removed from the disemicarbazone by treatment with alkali, and (c) the semicarbazone groups were removed from the product at pH 2.0, and aldosterone 18 beta-D-glucosiduronic acid was obtained in 47% overall yield. 5. In the presence of components used to synthesize beta-glucosiduronate by the Koenigs-Knorr reaction this substance is converted slowly into the alpha-glucosiduronate; this conversion is responsible, in part, for the low yield of beta-glucosiduronate. 6. Two additional conjugates were obtained in the Koenigs-Knorr reaction; a provisional structure was assigned to one substrate. The other substance is a C-18 alpha-glucosiduronate. Removal of the acetyl and ester groups from C-18 alpha-glucosiduronate gave the alpha-glucosiduronic acid in 84% yield and the 17-isoaldosterone alpha-glucosiduronic acid in 12% yield. 7. The rate at which several types of beta-glucuronidase hydrolyse the foregoing steroidal alpha- and beta-glucosiduronic acids is given.

Preparation of 3beta, 16beta-dihydroxyandrost-5-en-17-one: stabilization of its alpha-ketolic group toward alkali by formation of a semicarbazone.

Alkaline hydrolysis of a 16beta-acetoxy-17-oxo steroid is accompanied by almost complete rearrangement of the product to a 16-oxo-17beta-hydroxy steroid. Hydrolysis can be achieved without rearrangement by 1) formation of a C-17 semicarbazone, 2) alkaline removal of the acetate group, and 3) removal of the semicarbazone group in the presence of pyruvic acid-acetic acid. By employing this technique, the title compound was obtained from its diacetate in a yield of 65%.

Liquid ion exchangers in reversed-phase systems for chromatography of steroidal glucosiduronic acids.

Steroidal glucosiduronic acids were chromatographed on paper by the reversed-phase technique using five different liquid ion exchangers as stationary phase and aqueous KCl as mobile phase. The relationship of mobility of the acids (Rm) to both the amount of exchanger on the paper and the concentration of KCl in the mobile phase is discussed: the relationships may be expressed as Rm=n.log [exchanger] + const. and RM=-N.LOG [KCl] + const., respectively. Migration of the acids in the presence of different exchangers is correlated by use of the equation Rm (exchanger Y)=a.Rm (exchanger X) + b. The lack of appreciable correlation between migration of the acids in a reversed-phase system and a corresponding straight-phase system is discussed and expressed by means of regression equations. The correlation coefficients and standard errors of estimate from these equations provide useful indices for selecting two solvent systems that are to be used sequentially to obtain maximal resolution of a group of compounds. deltaRm values obtained for various functional groups with reversed-phase and straight-phase techniques are compared.

Liquid ion exchangers in paper chromatography of steoidal glucosiduronic acids. Influence of different exchangers on the mobility in chloroform-formamide and correlation of chromatographic data.

A group of 25 steroidal glucosiduronic acids was chromatographed on paper chloroform-formamide in the presence of several different liquid ion exchangers. Chromatograms were run also in three Bush-type systems. RF values were converted into RM values and the data were correlated by use of a series of regression equations of the type RM(Y) = a-RM(X) + b, in which X designates a standard system to which each other system (Y) is compared. The ratio of the slope a to the correlation coefficient r (i.e., a/r) is a measure of the resolving power of system Y relative to the standard system; intercept b, in association with slope a, is an indication of the polarity of system Y relative to X. The correlation coefficient r and the standard error of estimate sy-x are indications of whether solvent systems Y and X have very similar or relatively different resolving properties for a group of solutes. The regression equations are useful for correlating chromatographic data obtained from a group of compounds in several solvent systems. Properties of the chromatography systems are discussed and the relative importance of ion exchange and hydrogen bonding with the various solvent systems is pointed out. Delta RMg and delta RMr values are given for functional groups at several locations in the conjugates for ten of the chromatography systems.

Liquid ion exchangers in paper chromatography of steroidal glucosiduronic acids, glucosiduronic esters and free steroids. Influence of concentration of exchanger and counterion.

The chromatographic mobility of steroidal glucosiduronic acids on paper in chloroform-formamide increases as the concentration of ion exchanger in the chloroform phase increases; mobility decreases as the concentration of counterion in formamide increases. Mobility of glucosiduronic esters and of hydroxylated free steroids increases with an increase in concentration of exchanger; small changes in concentration of counterion in the stationary phase do not alter the migration of these nonionizable compounds. Data are presented which suggest that partition of the glucosiduronic acids between the two phases occurs predominantly by an ion-exchange process and that hydrogen bonding plays a secondary role. Partition of the glucosiduronic esters and hydroxylated free steroids appears to occur primarily by a hydrogen-bonding process.