Urinary metabolites of cannabidiol in dog, rat and man and their identification by gas chromatography—mass spectrometry

Urinary metabolites of cannabidiol (CBD), a non-psychoactive cannabinoid of potential therapeutic interest, were extracted from dog, rat and human urine, concentrated by chromatography on Sephadex LH-20 and examined by gas chromatography—mass spectrometry as trimethylsilyl (TMS), [²H₉]TMS, methyl ester—TMS and methyloxime—TMS derivatives. Fragmentation of the metabolites under electron-impact gave structurally informative fragment ions; computer-generated single-ion plots of these diagnostic ions were used extensively to aid metabolite identification. Over fifty metabolites were identified with considerable species variation. CBD was excreted in substantial concentration in human urine, both in the free state and as its glucuronide. In dog, unusual glucoside conjugates of three metabolites (4″- and 5″-hydroxy- and 6-oxo-CBD), not excreted in the unconjugated state, were found as the major metabolites at early times after drug administration. Other metabolites in all three species were mainly acids. Side-chain hydroxylated derivatives of CBD-7-oic acid were particularly abundant in human urine but much less so in dog. In the latter species the major oxidized metabolites were the products of β-oxidation with further hydroxylation at C-6. A related, but undefined pathway resulted in loss of three carbon atoms from the side-chain of CBD in man with production of 2″-hydroxy-tris,nor-CBD-7-oic acid. Metabolism by the epoxide-diol pathway, resulting in dihydro-diol formation from the Δ-8 double bond, gave metabolites in both dog and human urine. It was concluded that CBD could be used as a probe of the mechanism of several types of biotransformation; particularly those related to carboxylic acid metabolism as intermediates of the type not usually seen with endogenous compounds were excreted in substantial concentration.     

Chromosome preparations from microplate cultures of man,dog, rat,and mouse

Summary A simple method for making chromosomal preparations from 10⁵ leukocytes from man, dog, mouse, and rat and from 0.01 ml total human and dog blood is developed. The leukocytes and the peripheral blood are cultured in Cooke microtiter plates in a culture volume of 0.1 ml. The culture medium is R.P.M.I. 1640 supplemented with serum and phytohemagglutinin. The culture time is 72 or 96 h.     

Significance of the peri-insular extracellular matrix for islet isolation from the pancreas of rat,dog, pig,and man

Summary The presence and distribution in the peri-insular region of extracellular matrix, and in particular basement membrane, was investigated in a comparative study comprising pancreata of rat, dog, pig, and man. Basement membrane markers, collagen type-IV and laminin, were determined immunohistochemically. Additional information pertaining to the structural relationships between endocrine and exocrine pancreas, in particular cell-to-cell and cell-to-matrix contacts, was obtained by electron microscopy. In pig, very little periinsular capsule is present, and the structural integration of the porcine islet in the exocrine pancreas almost exclusively depends on cell-to-cell adhesion. In the canine pancreas, the islets are almost completely encapsulated with very little direct exocrine-to-endocrine cell-to-cell contact. In rat and man, the situation is intermediate with a tendency towards predominance of cell-to-matrix adhesion. The intra-insular adhesion mechanisms depend largely on cell-to-cell adhesion in all four species. The ultrastructural results suggest that collagenase preparations employed in islet isolation procedures should be of high purity as to preserve the protease-sensitive intra-islet cell-to-cell adhesion. Under these conditions, however, the endocrine-to-exocrine cell-to-cell contacts will be conserved also, resulting in an exocrine-tissue contamination of the islets of Langerhans. Consequently, additional steps for the effective removal of exocrine tissue and the purification of islets are required.     

Biotransformation of terodiline I. Identification of metabolites in dog urine by mass spectrometry

Nine metabolites of terodiline (N-tert-butyl-4,4-diphenyl-2-butylamine) have been identified in dog urine by various chromatographic techniques and mass spectrometry. The main metabolic pathway is aromatic hydroxylation, leading to the quantitatively most important metabolite, N-tert-butyl-4-(4-hydroxyphenyl)-4-phenyl-2-butylamine, and to two dihydroxylated metabolites, one mono substituted in both rings (N-tert-butyl-4,4'-bis(4-hydroxyphenyl)-2-butylamine), and one disubstituted in one ring (N-tert-butyl-4-(3,4-dihydroxyphenyl)-4-phenyl-2-butylamine). The latter is further metabolized by methylation, forming N-tert-butyl-4-(4-hydroxy-3-methoxyphenyl)-4-phenyl-2-butylamine, the second most abundant metabolite. Still another metabolite is formed by hydroxylation in the tert-butyl group to N-(2-hydroxymethyl-2-propyl)-4,4-diphenyl-2-butylamine. A very minor dihydroxylated metabolite results from oxidation both in an aromatic ring and in the tert-butyl group, giving N-(2-hydroxymethyl-2-propyl)-4-(4-hydroxyphenyl)-4-phenyl-2-butylamine. Oxidation of the carbon adjacent to the nitrogen and subsequent deamination gives the two ketones 4-(4-hydroxyphenyl)-4-phenyl-2-butanone and 4-(4-hydroxy-3-methoxyphenyl)-4-phenyl-2-butanone. Reduction of the carbonyl function in the former yields the corresponding alcohol, 4-(4-hydroxyphenyl)-4-phenyl-2-butanol. Some unchanged terodiline is also present. All metabolites formed by functionalization appear to be extensively conjugated, presumably with glucuronic acid.     

Metabolism of metandienone in man: identification and synthesis of conjugated excreted urinary metabolites, determination of excretion rates and gas chromatographic-mass spectrometric identification of bis-hydroxylated metabolites

After oral administration of metandienone (17 alpha-methyl-androsta-1,4-dien-17 beta-ol-3-one) to male volunteers conjugated metabolites are isolated from urine via XAD-2-adsorption, enzymatic hydrolysis and preparative high-performance liquid chromatography (HPLC). Four conjugated metabolites are identified by gas chromatography-mass spectrometry (GC/MS) with electron impact (EI)-ionization after derivatization with N-methyl-N-trimethyl-silyl-trifluoroacetamide/trimethylsilyl-imidazole (MSTFA/TMS-Imi) and comparison with synthesized reference compounds: 17 alpha-methyl-5 beta-androst-1-en-17 beta-ol-3-one (II), 17 alpha-methyl-5 beta-androst-1-ene-3 alpha,17 beta-diol (III), 17 beta-methyl-5 beta-androst-1-ene-3 alpha,17 alpha-diol (IV) and 17 alpha-methyl-5 beta-androstane-3 alpha,17 beta-diol (V). After administration of 40 mg of metandienone four bis-hydroxy-metabolites--6 beta,12-dihydroxy-metandienone (IX), 6 beta,16 beta-dihydroxy-metandienone (X), 6 beta,16 alpha-dihydroxy-metandienone (XI) and 6 beta,16 beta-dihydroxy-17-epimetandienone (XII)--were detected in the unconjugated fraction. The metabolites III, IV and V are excreted in a comparable amount to the unconjugated excreted metabolites 17-epimetandienone (VI), 6 beta-hydroxy-metandienone (VII) and 6 beta-hydroxy-17-epimetandienone (VIII). Whereas the unconjugated excreted metabolites show maximum excretion rates between 4 and 12 h after administration the conjugated metabolites III, IV and V are excreted with maximum rates between 12 and 34 h.     

Antinociceptive activity of clonidine in the mouse, rat and dog

The antinociceptive activities of clonidine have been determined against several qualitatively different noxious stimuli in the mouse, rat and dog. In these tests clonidine given subcutaneously was 6 to 7 times more potent than morphine. Both clonidine and morphine were more potent against responses to heat induced nociceptive stimuli than against responses to heat induced nociception or that induced by electrical tail stimulation. However, unlike morphine the effects of clonidine in these latter tests were only seen at doses that also caused sedation and so these animals were less able to respond to the nociceptive stimuli. In contrast in pressure, chemical and tooth pulp stimulation tests clonidine produced increases in nociceptive thresholds at doses which caused no overt signs of behavioural depression. Comparisons of the relative potencies of clonidine and the less lipophilic analogue 4-hydroxyclonidine given subcutaneously and intracerebroventricularly indicate that clonidine induced antinociception is predominantly centrally mediated. However, a peripheral component may also be present in the inhibition of acetylcholine-induced abdominal constriction in the mouse.     

Methandrostenolone metabolism in humans: potential problems associated with isolation and identification of metabolites

Methandrostenolone dose (amount and duration) and methods of isolation from urine can influence the identification and quantitation of methandrostenolone metabolites. Long-term use of methandrostenolone at high dosages led to the appearance of unmetabolized drug in the urine and contributed to the identification of a previously unreported metabolite, 3 beta, 6 section, 17 beta-trihydroxy-17 alpha-methyl-5 section-1-androstene. Exposure of methandrostenolone in vitro to acid conditions induced a retropinacol rearrangement in the D-ring of the methandrostenolone molecule, causing the formation of 18-nor-17,17-dimethyl-1,4,13(14)-androstatrien-3-one in large amounts. The same acidic conditions led to the addition of a hydroxyl at the 6 position of the B-ring of either the retropinacol rearrangement products or native methandrostenolone resulting in the formation of 6 beta-hydroxy-18-nor-17,17-dimethyl-1,4,13(14)-androstatrien-3-one, 6 alpha- hydroxy-18-nor-17,17-dimethyl-1,4,13(14)-androstatrien, 6 beta-17 alpha-methyl-1,4-androstadien-3-one and 6 alpha,17 beta-dihydroxy-17 alpha-methyl-1,4-androstadien-3-one. Hydroxylation of native methandrostenolone at the 6 position also occurs endogenously. However, no evidence of an endogenous retropinacol rearrangement was found. Silylating agents alone can induce the formation of small amounts of 6 beta-17 beta-dihydroxy-17 alpha-methyl-1,4-androstadien-3-one. Discrepancies between previously published reports on methandrostenolone metabolism in man are discussed and compared with an animal model.     

Cell types in rat liver cultures: their identification and isolation

This paper reviews the various types of cells in the liver in vivo and in hepatic cellular suspensions produced by perfusion of the liver with collagenase solutions. Methods to identify and isolate different types of hepatic cells are discussed. In vitro culture of various types of liver cells is reviewed and the identification of cultured cells is considered.     

Nuclear proteoglycans from mouse liver cells: isolation and identification]

Proteoglycans (PG) have been isolated from mouse liver nuclei and identified. Nuclear PG are represented by various classes: i) PG containing dermatan sulfate (DS) chains; ii) PG containing heparan sulfate (HS) chains and, apparently, iii) mixed PG whose protein core contains both DS and HS chains.     

Testosterone secretion is severely impaired after hepatectomy in rat, dog and man

In 90%-hepatectomized rats, the plasma testosterone level (0.34 +/- 0.07 ng.ml, mean +/- SEM) is significantly lower (P less than 0.001) than in sham operated male rats (1.7 +/- 0.26 ng.ml, mean +/- SEM). In dogs, after 90% hepatectomy, the mean plasma testosterone concentration fell to 1/10 of the plasma testosterone level measured in sham operated animals either 24 or 72 h after surgery. In hepatectomized men, plasma testosterone is markedly decreased in contrast to what is observed after duodeno-pancreatectomy performed under the same conditions of anesthesia. These results suggest that 90% hepatectomy severely alters the Leydig cell function.     

Identification of the major urinary metabolite of alprenolol in man, dog and rat

After oral administration of ³H-alprenolol to man, dog and rat, urinary metabolites of the drug have been separated by ion-exchange chromatography on Bio-Rex 70, a carboxylic acrylate resin. The major metabolite has been identified by GC-MS as 4-hydroxyalprenolol. Occurring in the urine largely in a conjugated form, it represents about 40 % of the excreted amount in man and dog and about 30 % in rat. Including alprenolol, which also appears largely as a conjugate, about 80 % of the amount of radioactivity excreted in human urine can be accounted for.     

Biotransformation of aflatoxin B1 and its conjugated metabolites by rat gastrointestinal microfloras

Rat cecal microflora from high- and low-fiber-fed animals hydrolyzed aflatoxin conjugates to metabolites indistinguishable from aflatoxin B1 and aflatoxin P1, but aflatoxicol was not a transformation product.     

Separation and identification of phase I and phase II [14C]antipyrine metabolites in rat and dog urine

A simple and accurate HPLC procedure was developed to quantify, in a single run, all phase I and phase II [¹⁴C]antipyrine metabolites that occur in rat and dog urine. All metabolites were subjected to thermospray-LC-MS and EI-MS in order to establish their structure. The rat metabolizes antipyrine to eight major metabolites, six of which are conjugated; 1.4% of the dose was excreted unchanged, 18.9% in a free form, 30.6% as sulfates and 21.1% as glucoronides. The dog metabolizes antipyrine to four metabolites, all as sulfate (61.0% of the dose) or glucoronide conjugates (16.2% of the dose).     

Biotransformation of 17-alkylsteroids in the equine: gas chromatographic-mass spectral identification of ten intermediate metabolites of methyltestosterone

The metabolism of the orally active anabolic steroid methyltestosterone in the equine was investigated by administration of the drug along with a tritiated traces. In this study some of the metabolites were identified and a radio immunoassay screen and immunoaffinity chromatography gel for methyltestosterone were also evaluated. Pathway intermediates, in particular the 17-methylandrostanediols, were studied to gain an insight into the most likely stereochemistry of the major metabolites. The predominant phase I biotransformations involve reduction of the A ring 3-oxo and 4-ene groups to yield predominantly 3β-hydroxy-5-androstane products and hydroxylation of the steroid nucleus at several positions. Epimerisation of the 17-methyl group also occurred. Ten steroids could be positively identified by comparison with authentic references materials and many other triol, tetrol and pentols were also observed. Phase II metabolites and sulphate conjugates in particular, were common.     

Biotransformation of isoeugenol to vanillin by a newly isolated Bacillus pumilus strain: identification of major metabolites

A bacterial strain S-1 capable of transforming isoeugenol to vanillin was isolated. The strain was identified as Bacillus pumilus based on biochemical tests, cellular fatty acid composition, riboprint pattern and 16S rRNA gene sequence analyses. In the biotransformation of isoeugenol, vanillin was the main product. With the growing culture of B. pumilus S-1, 10 g l⁻¹ isoeugenol was converted to 3.75 g l⁻¹ vanillin in 150 h, with a molar yield of 40.5% that is the highest up to now. Dehydrodiisoeugenol, a dimer of isoeugenol, was separated by preparative thin layer chromatography and identified by gas chromatography–mass spectrometry. Based on the accurate masses obtained from gas chromatography–high resolution mass spectrometry, two key intermediates, isoeugenol-epoxide (IE) and isoeugenol-diol (ID), were identified by mass spectra interpretations. The biotransformation with resting cells showed that vanillin was oxidized to vanillic acid and then to protocatechuic acid before the aromatic ring was broken. These findings suggest that isoeugenol is degraded through an epoxide-diol pathway.     

Biotransformation of aflatoxin B1 and its conjugated metabolites by rat gastrointestinal microfloras.

Rat cecal microflora from high- and low-fiber-fed animals hydrolyzed aflatoxin conjugates to metabolites indistinguishable from aflatoxin B1 and aflatoxin P1, but aflatoxicol was not a transformation product.     

The isolation, identification and synthesis of two metabolites of guanethidine formed in pig and rabbit liver homogenates

1. Two metabolites of radioactively labelled guanethidine were isolated from rabbit and pig liver homogenates by ion-exchange chromatography on a sulphonic acid resin. 2. One of the metabolites was eluted from the column with ammonia and identified as 2-(6-carboxyhexylamino)ethylguanidine on the basis of the elemental analysis, i.r. spectrum and pH titration curve of the pure compound, and the observed partial loss of tritium for ring-labelled guanethidine during the formation of this metabolite. 3. This identification was confirmed by synthesis. 4. 2-(6-Carboxyhexylamino)ethylguanidine underwent ring-closure in hot alkaline solution to 1-(6-carboxyhexyl)-2-iminoimidazolidine. 5. The other metabolite of guanethidine was eluted from the ion-exchange column with 6m-hydrochloric acid along with the unchanged drug. It was purified by countercurrent distribution and shown to be identical with synthetic guanethidine N-oxide. 6. The two metabolites and the product of ring-closure had less than one-tenth of the antihypertensive activity of guanethidine in the renal-hypertensive rat and are unlikely to contribute to the pharmacological properties of the drug.