Dihydroxy Metabolites Research Articles

Identification of In Vitro (Rat Liver Postmitochondrial S9 Fraction) Metabolites of the Antiprotozoal Agent 3a,4,5,6,7,7a-Hexahydro-3-(l-methyl-5-nitro-1H-imidazol-2-yl)-l,2-benzisoxazole

Twelve in vitro oxygenated metabolites of 3a,4,5,6,7,7a- hexahydro-3-(l-methyl-5-nitro-lH-imidazol-2-yl) -1,2-benzisoxazole (MK-0436) were produced by incubation of this antiprotozoal agent with the postmitochondriai supernatant (S9) fraction isolated from the livers of rats treated with phenobarbital. Metabolite structure elucidation was achieved using NMR and mass spectrometry. Seven monohydroxy and two dihydroxy metabolites were fully characterized; two other metabolites were partially characterized as dihydroxy derivatives of the drug. The major in vitro metabolite is the 5 axial hydroxy compound, and a minor metabolite is the corresponding ketone. In all cases metabolite formation involved biotransformation on the hexahydrobenzisoxazole ring.

Serum dihydroxyvitamin d metabolite concentrations in patients on chronic anticonvulsant drug therapy: Response to pharmacologic doses of vitamin D 2

We examined the effects of chronic anticonvulsant drug administration on the serum concentrations of dihydroxy-vitamin D metabolites and the response of these metabolite levels to short-term treatment with pharmacologic doses of vitamin D 2. Twelve patients maintained on chronic combined diphenylhydantoin and phenobarbital therapy were studied before and after the administration of vitamin D 2, 75,000 units/week for 6 weeks. Prior to vitamin D 2 administration, the patient group demonstrated decreased serum calcium ( P < 0.01) and increased serum iPTH ( P < 0.02) concentrations relative to 18 matched controls. Serum 250HD and 24,25(OH) 2D concentrations in the patient group were reduced by 38% and 75% ( P < 0.001), while serum 1,25(OH) 2D concentration was increased by 27% ( P < 0.05) relative to control values. After vitamin D administration serum calcium and iPTH concentrations in the patient group were restored to values that were not significantly different from control levels. Serum 250HD and 24,25(OH) 2D concentrations were increased by 4.6-and 6.3-fold, respectively, to supranormal levels. Serum 1,25(OH) 2D concentration exhibited an unexpected further 30% increase over pretreatment values, resulting in a return of the serum 1,25(OH) 2D/24,25(OH) 2D concentration ratio to a normal value. These data indicate that in patients treated chronically with anticonvulsant drugs, serum 250HD concentration responds appropriately to vitamin D2 administration, but regulation of renal dihydroxy metabolite formation may be altered.

Dopaminergic properties of nomifensine

Nomifensine and a proposed dihydroxy metabolite produced stimulation of motor behavior in mice with nomifensine being more potent. Weak cage-climbing behavior (stereotypy) was also produced. The stimulatory effects were greater in mice in which dopamine receptor sensitivity was increased by long-term haloperidol. Both of the analogs were potent inhibitors of dopamine and norepinephrine uptake in vitro with nomifensine approximately 3 times more potent than the metabolite. In contrast, the two analogs had weak affinity for the post-synaptic dopamine receptor (as measured by displacement of ligand binding in vitro) with dihydroxynomifensine approximately 6 times more potent than nomifensine. These results suggest that the behavioral effects of nomifensine are largely dependent on presynaptic catecholamine mechanisms but that weak direct dopamine agonist properties do exist, particularly in vivo where the drug may be metabolized to a more active form.

A simple and sensitive assay for 25-hydroxyvitamin D, 24,25-dihydroxyvitamin D and 1,25-dihydroxyvitamin D in human serum

An improved method is described which permits the simultaneous determination of 25-hydroxyvitamin D [25-(OH)D], 24,25-dihydroxyvitamin D [24,25-(OH) 2D] and 1,25-dihydroxyvitamin D [1,25-(OH) 2D] in 2 milliliters of human serum. Methodological improvements enabled a rapid and almost complete extraction of the three metabolites from serum and omission of adding labeled internal standards to each serum sample for the calculation of individual recoveries. Commercially available stable chick embryo intestinal mucosa cytosol preparation made the troublesome preparation of cytosol receptor for 1,25-(OH) 2D unnecessary. The procedure involves saturation of serum with ammonium carbonate and extraction with methanol/ethyl acetate, followed by separation of 25-(OH)D from the dihydroxy metabolites of vitamin D by Sephadex LH-20 column chromatography and further separation of the dihydroxy metabolites into 24,25-(OH) 2D and 1,25-(OH) 2D by high-pressure liquid chromatography. This is followed by individual determination of each metabolite by competitive protein-binding assay or radioreceptor assay.

Evidence for extrarenal production of 1 alpha ,25-dihydroxyvitamin D in man.

Recent studies provide evidence for extrarenal production of 1 alpha ,25-dihydroxyvitamin D [1 alpha ,25(OH)2D]. To investigate this possibility, serum vitamin D, 25-hydroxyvitamin D (25-OHD), 24,25-dihydroxyvitamin D [24,25(OH)2D], and 1 alpha ,25(OH)2D were measured in eight adult anephric subjects. All were undergoing hemodialysis and three of them were receiving vitamin D, 50,000 or 100,000 U/d. Serum vitamin D was elevated in two of the patients given vitamin D and was abnormally low in the others. Mean serum 25-OHD was increased in patients given vitamin D (94.0 +/- 7.6 ng/ml) and was normal in the others (16.4 +/- 0.9 ng/ml, P less than 0.001). Mean serum 24,25(OH)2D was normal in patients given vitamin D (1.38 +/- 0.27 ng/ml) and was low in the others (0.25 +/- 0.08 ng/ml, P less than 0.001). Serum 24,25(OH)2D correlated significantly with serum 25-OHD (r = 0.848, P less than 0.01). Mean serum 1 alpha ,25(OH)2D determined by receptor assay was 5.8 +/- 1.9 pg/ml in patients who were not given vitamin D and was 14.1 +/- 0.6 in those who were given vitamin D (P less than 0.001). Serum 1 alpha ,25(OH)2D correlated significantly with serum 25-OHD (r = 0.911, P less than 0.01). Mean serum 1 alpha ,25(OH)2D, measured by bioassay, was 8.3 +/- 1.9 pg/ml in patients who were given vitamin D and was 15.9 +/- 2.4 pg/ml in those who were given vitamin D (P less than 0.05). There was a significant correlation between the values for serum 1 alpha ,25(OH)2D obtained with the two methods (r = 0.728, P less than 0.01). The results (a) provide evidence in man for extrarenal production of both 24,25(OH)2D and, by two independent assays, of 1 alpha , 25(OH)2D, and (b) indicate that serum values of the two dihydroxy metabolites of vitamin D in anephric subjects vary with the serum concentration of the precursor 25-OHD.

Metabolism of dichlorobiphenyls by highly purified isozymes of rat liver cytochrome P-450.

Hepatic mixed-function oxidase metabolism of the ubiquitous pollutant polychlorinated biphenyls (PCBs) is implicated in their toxification and detoxification. We used dichlorobiphenyls (DCBs) as models to investigate the effect of the chloro substituent sites on this metabolism experimentally and by molecular orbital calculations. Reconstituted, purified cytochrome P-450 PB-B and BNF-B, the major terminal oxidase isozymes of this system, from phenobarbital (PB)- and beta-naphthoflavone (BNF)-induced rats were used to investigate this metabolism. Both isozymes are also induced by PCBs. High-performance liquid chromatography (HPLC) was used to detect, quantify, and isolate metabolites. Metabolite structures were identified by mass spectrometry, dechlorination to identifiable hydroxybiphenyls, and HPLC retention times. All DCBs yielded 3- and 4- but no 2-monohydroxylated metabolites (3,3'-DCB also yielded a dihydroxy metabolite). Di-o-chloro-substituted DCBs were metabolized primarily by cytochrome P-450 PB-B, mono-o-chloro substituted DCBs by both isozymes approximately equivalently, and DCBs without o-chloro substituents by BNF-B primarily. Thus PB-B preferentially metabolizes noncoplanar DCBs and BNF-B coplanar DCBs. The cytochrome isozymes exhibited differing regioselectivities for DCB metabolism - PB-B hydroxylated unchlorinated phenyl rings and BNF-B chlorinated rings. Incorporation of epoxide hydrolase yielded DCB dihydrodiols, and hydroxy metabolite patterns were consistent with those calculated from ring-opened arene oxide intermediates. Thus the rates and regioselectivities of metabolism and thus possibly the toxicity and carcinogenicity of DCBs are dependent on the cytochrome P-450 isozymes induced.

A gas-liquid chromatography assay for phencyclidine and its metabolites.

A GLC assay for phencyclidine (PCP) is described, which also simultaneously measures three primary hydroxylated metabolites formed from incubating PCP in tissue homogenates. Using the FID detector, the limits of reliable detection of PCP and both monohydroxy metabolites, 4-phenyl-4-piperidino-cyclohexanol, 2, and 1-(1-phenylcyclohexyl)-4-hydroxypiperidine, 3, are 0.02 mumol per injection and 0.05 mumol for the dihydroxy metabolite, 4-(4'-hydroxypiperidino)-4-phenylcyclohexanol, 2A. Baseline separation of an compounds was achieved and coefficients of variation (between-run) was 3-6% for PCP, and both monohydroxy metabolites, and 12% for the dihydroxy metabolite. A GCMS assay is also reported herein for the analysis of PCP at low levels, and can detect 5 pmol per injection of PCP, with a linear standard curve from 50 to 2000 pmol.

Determination of circulating 25,26-dihydroxycholecalciferol in man by radioimmunoassay.

1. A radioimmunoassay of 25,26-dihydroxycholecalciferol has been developed with an antiserum raised in a sheep, tritiated 1,25-dihydroxycholecalciferol as tracer and synthetic 25,26-dihydroxycholecalciferol as standard. The metabolite was purified from serum extracts by Sephadex LH 20 and high-pressure liquid chromatography; recovery was monitored with biologically generated, tritiated 25,26-dihydroxycholecalciferol. 2. The mean +/- SEM concentration of 25,26-dihydroxycholecalciferol in serum from 18 healthy subjects was 587 +/- 65 pmol/l. Seven Asian patients with osteomalacia due to vitamin D deficiency had very low or undetectable (< 96--231 pmol/l) circulating 25,26-dihydroxycholecalciferol concentrations. 3. The metabolite was detectable in the sera from seven anephric patients (mean 262 +/- 43 pmol/l), indicating that extrarenal sites for the 26-hydroxylation of 25-hydroxycholecalciferol exist in man. 4. A strong positive correlation between the concentrations of 25-hydroxycholecalciferol and those of 25,26-dihydroxycholecalciferol in serum was obtained. Thus it appears that in man the production of this dihydroxy metabolite of vitamin D depends on the concentration of its precursor, 25-hydroxycholecalciferol.

Nuclear translocation of the 1,25-dihydroxycholecalciferol receptor in mouse kidney.

We have recently demonstrated that cytosol from mouse kidney contains a receptor-like binding protein for 1,25-dihydroxycholecalciferol (1,25-(OH)2D3). The present studies were undertaken to investigate whether this component undergoes nuclear translocation, thus adding support to the contention that this binding moiety represents a receptor for 1,25-(OH)2D3. Kidney slices from vitamin D-deficient mice were incubated with 1,25-(OH)2[3H]D3 and specific binding was assessed in cytosol and 0.3 M KCl extracts of isolated nuclei. The appearance of specific nuclear-bound 1,25-(OH)2[3H]D3 exhibited a saturation time course and the increase in nuclear binding was accompanied by a concomitant decrease in cytoplasmic binding such that, by 120 min, approximately 80% of specifically bound 1,25-(OH)2[3H]D3 was associated with the nuclear fraction and approximately 20% remained in the cytoplasmic compartment. The nuclear transfer was found to be temperature-dependent and specific for the dihydroxy metabolite. Both the cytoplasmic and nuclear receptors sedimented at 3.2 S in 0.3 M KCl sucrose density gradients. However, under low salt conditions, the cytoplasmic receptor was transformed to yield an approximately 5.4 S peak while the nuclear receptor sedimented unchanged t 3.2 S. In summary, the cytoplasmic 1,25-(OH)2D3 receptor in mammalian kidney undergoes temperature-dependent nuclear translocation and the cytoplasmic and nuclear forms of the receptor can be distinguished by their sedimentation properties.

Metabolism of monochlorobiphenyls by hepatic microsomal cytochrome P-450

In vitro rat hepatic microsomal metabolism of the monochlorobiphenyls (MCBs) 2-, 3- and 4-chlorobiphenyl, has been investigated as a model for the metabolism of polychlorinated biphenyl pollutants. MCB metabolism was catalyzed by cytochrome P-450, as indicated by a dependence on NADPH and O 2, inhibition by 2-diethylaminoethyl-2,2-diphenylpropylacetate (SKF 525-A), metyrapone and CO, and the formation of type I difference spectra, on the addition of MCBs to microsomes. All MCBs yielded a 4'-monohydroxy MCB as the major metabolite, as determined by mass and nuclear magnetic resonance spectroscopy, dechlorination to 4-hydroxybiphenyl, and high-pressure liquid chromatography retention times. Minor monohydroxy and dihydroxy metabolites were also produced from the MCBs. The regioselectivity of control cytochrome P-450 for metabolism of MCBs at the 4' position was not altered by preinduction of cytochrome P-450 with 2,4,2',4'-tetrachlorobiphenyl (TCB) or cytochrome P-448 with 3,4,3', 4'-TCB. 2-Chlorobiphenyl was metabolized only by control and induced cytochrome P-450; 3- and 4-chlorobiphenyl were metabolized by control and by induced cytochrome P-450 and P-448. Thus, the regioselectivity of metabolism of MCBs is independent of the chlorine position or the form of the induced cytochrome involved, but the extent of metabolism of polychlorinated biphenyls (PCBs) is determined by induction of the hepatic cytochromes P-450.

Leukotriene C: a slow-reacting substance from murine mastocytoma cells.

Murine mastocytoma cells treated with calcium ionophore A23187 produced a slow-reacting substance (SRS) that caused guinea pig ileum to contract. The response was reversed by the SRS antagonist FPL 55712. On the basis of isotope incorporation experiments, spectroscopy, and chemical degradations, the SRS was identified as a cysteine-containing derivative of 5-hydroxy-7,9,11,14-icosatetraenoic acid. This amino acid was attached in thioether linkage at C-6. The SRS is structurally related to previously identified epoxy and dihydroxy metabolites of arachidonic acid in leukocytes. A common feature is the presence of a conjugated triene, and the name "leukotriene" has been introduced to designate these compounds. Leukotriene A (5,6-epoxy-7,9,11,14-icosatetraenoic acid) is an intermediate in the formation of leukotriene B (5,12-dihydroxy-6,8,10,14-icosatetraenoic acid) and is proposed to be a precursor also of leukotriene C, which is the SRS identified here.

The metabolism of biphenyl. V. Phenolic metabolites in some marine organisms.

The phenolic metabolites of biphenyl in the crustacean Cirolana borealis LILJEBORG (ISOPODA), the gastropod Buccinum undatum L and the ophiuroid Ophiocomina nigra ABILDGAARD have been analysed qualitatively and semi-quantitatively as trimethylsilyl (TMS) ethers by combined gas chromatography/mass spectrometry and gas chromatography, respectively. The findings demonstrate the in vivo metabolic capacity of these organisms to convert biphenyl into its hydroxylated derivatives. Thus, 2-hydroxybiphenyl is the most prominent metabolite detected in all these experiments, which also showed that minor amounts of the 4-hydroxy and 4,4'-dihydroxy metabolites are formed. Metabolites of biphenyl origin are found both in the sea water from the aquaria and in the tissue of the respective marine organisms. The experiments, moreover, indicate a slow metabolism of biphenyl in C. borealis, B. undatum and O. nigra, between which no qualitative metabolic differences are found.

PCB metabolism in rats following prolonged exposure to Aroclor 1242 and aroclor 1016.

Several mono- and dihydroxy metabolites of di-, tri, and tetrachlorobiphenyl have been identified in the urine of rats fed prolonged diets of Aroclor 1016 or Aroclor 1242. Combined gas chromatography-mass spectrometry was used for characterization of the metabolic products.

A rapid assay for 25-OH-vitamin D3 without preparative chromatography.

ABSTRACT A technique for determination of the 25-OH-D content of serum directly without preparative chromatograpy has been developed and validated by comparison with reference assays of the 25-OH-D fraction separated by silicic acid chromatography. The direct assay technique selectively measures the 25-OH-D content of sample extracts even in the presence of a 3-5-fold excess of vitamin D as in the serum of hypervitaminotic D animals and subjects and without apparent interference from the dihydroxy metabolites of vitamin D. Meaningful clinical information can be obtained with this technique, allowing differentiation of vitamin D deficiency from other causes of hypocalcemia and hypervitaminosis D from other causes of hypercalcemia.

Metabolism and distribution of cannabinoids in rats after different methods of administration

Lungs of rats given 14C-Δ 9-tetrahydrocannabinol (Δ 9-THC) by smoke inhalation showed a retention and metabolism of the cannabinoid, with further retention and metabolism by the liver. Only the liver showed retention and metabolism when 14C-Δ 9-THC was given by i.v. injection. The brain showed a greater penetration or retention of unmetabolized cannabinoid after smoke inhalation than after i.v. injection. 11-Hydroxy-Δ 9-THC was observed soon after smoke inhalation of 14C-Δ 9-THC in all of the organs and tissues except brain, and concentrations of a dihydroxy metabolite increased with time. A still unidentified metabolite was retained in the liver and spleen 15 days after an i.v. or chronic i.p. injection of 14C-Δ 8-THC or 14C-Δ 9-THC.

The Biological Activity and Metabolism of 24,25-Dihydroxyvitamin D3

Abstract 24,25-Dihydroxyvitamin D3 (24,25-(OH)2D3) gives a marked increase in intestinal calcium transport and serum calcium concentration of vitamin D-deficient rats fed a diet containing adequate calcium and phosphorus. These responses occur after a considerable time lag following administration of the metabolite. The 24,25-(OH)2D3 on a long term basis and at low doses has striking activity in the stimulation of intestinal calcium transport but not bone calcium mobilization. Radioactive 24,25-(OH)2D3 is metabolized to a more polar compound which increases in concentration in serum, intestinal mucosa, and bone over a 48-hour period. Nephrectomy or the feeding of a high calcium diet abolishes the stimulation of intestinal calcium transport by 24,25-(OH)2D3 and the in vivo genesis of the more polar compound from tritiated 24,25-(OH)2D3. It is concluded that 24,25-(OH)2D3, the usual dihydroxy metabolite of vitamin D3 in normal rats, has potential physiological importance through its conversion to a more polar metabolite. This metabolite possibly acts to facilitate intestinal calcium absorption without stimulating bone calcium mobilization.

Embryonic chick intestine in organ culture: response to vitamin D 3 and its metabolites.

Embryonic chick intestine maintained in organ culture responded to vitamin D(3) and its metabolites 25-hydroxycholecalciferol and 1,25-dihydroxycholecalciferol by synthesis of calcium-binding protein and enchanced calcium-45 uptake. The dihydroxy metabolite was by far the most potent inducer of the protein and also acted more rapidly than vitamin D(3) to stimulate isotope uptake. Despite its lower potency, vitamin D(3) itself was effective.

Renal Osteodystrophy

Renal osteodystrophy (or the osseous manifestations of chronic renal failure) consists mainly of a varying combination of two main abnormalities—impaired mineralization producing rickets or osteomalacia, and secondary hyperparathyroidism producing osteitis fibrosa. Impaired mineralization is due chiefly to deficiency of the dihydroxy metabolites of vitamin D normally made in the kidney; this deficiency also results in hypocalcemia and in impaired calcium absorption. All patients go through a phase of defective mineralization, but this is more commonly asymptomatic in the United States than in Great Britain because of sociological and geographic differences in vitamin D status. Secondary hyperparathyroidism is due to a combination of hypocalcemia and phosphate retention and serves to minimize disruptions of both calcium and phosphate homeostasis. Although parathyroid hormone levels rise very early in chronic renal failure, osteitis fibrosa is a relatively late manifestation because the level required for this is higher than that required to promote phosphate excretion. Occasionally the secretion of parathyroid hormone exceeds homeostatic demands because of a defect in the synthesis or release of hormone (secretory autonomy) or, more commonly, a defect in the control of cell division and proliferation (mitotic autonomy). The clinical features of renal osteodystrophy depend on the age of onset and rate of progression of the renal disease. Before epiphyseal closure occurs there may be retarded growth and late rickets; genu valgum and slipped femoral capital epiphyses are common deformities, especially in adolescence. Mild degrees result mainly from rickets alone, but severe degrees result from metaphyseal collapse due to the subperiosteal erosion of osteitis fibrosa. After epiphyseal closure, typical symptomatic osteomalacia may occur. In patients without Looser zones, mild osteitis fibrosa may be the only radiographic abnormality. Osteitis fibrosa may be asymptomatic unless a giant cell cyst causes pressure on surrounding structures, or unless a pathologic fracture occurs. Osteosclerosis is common, especially in the axial skeleton, but rarely causes symptoms. Soft tissue calcification may cause lesions resembling calcific periarthritis or tumoral calcinosis. Maintenance hemodialysis is often complicated by worsening osteodystrophy, but with two exceptions this can be prevented by adequate dialysis and maintenance of normal levels of both calcium and phosphate in the blood. If there is parathyroid autonomy, these measures may be unavailing, and some patients develop a new type of osteodystrophy with severe osteoporosis and multiple fractures, which can be neither prevented nor treated except by renal transplantation. After transplantation, hypercalcemia may occur mainly because of delayed parathyroid involution, but this usually resolves spontaneously. A more intractable complication is aseptic necrosis of bone, most commonly affecting the femoral heads; this is due mainly to corticosteroid therapy. Treatment of renal osteodystrophy may be medical (chiefly administration of vitamin D to correct impaired mineralization), surgical (parathyroidectomy for autonomous hyperparathyroidism), or orthopedic (treatment of fractures or correction of deformity). Advances in the treatment of renal failure have greatly increased the possible scope for orthopedic intervention, but timely diagnosis and optimal medical treatment of osteodystrophy should make this unnecessary in most patients.

Factors affecting the enzymatic formation of o-methylated dihydroxy derivatives

The effect of 3,4-benzpyrene and cold stress on the enzymatic formation of O-methylated dihydroxy compounds from monohydroxy compounds in rats was investigated. Enzyme activity was increased in treated rats only when monohydroxy N-acetyl derivatives (N-acetylserotonin, N-acetyltyramine, and N-acetyl- p-amino-phenol) were used as subtrates. The effect of cold stress and 3,4-benzpyrene was shown to be on the hydroxylating enzyme in the microsomes. Pretreatment with phenobarbital increased, while serotonin decreased, the formation of the O-methylated dihydroxy metabolite of N-acetylserotonin. An enzyme was found in liver microsomes that could O-methylate catechols and that differed from soluble catechol O-methyltransferase in its response to 3,4-benzpyrene and cold stress treatment as well as in pH optimum.

Role of impaired transmethylation of epinephrine in ethionine-induced hepatic fat accumulation

1. (1)|Ethionine interieres in vitro with the reaction sequence leading to the transmethylation of epinephrine by competing with methionine for ATP and for the activating enzyme, forming S-adenosylethionine. Catechol- O-methyltransferase does not use S-adenosylethionine as an ethyl donor to epinephrine. 2. (2)|Ethionine treatment blocks the epinephrine-metanephrine pathway in vivo, resulting in a prolonged half-life of epinephrine and in an increase in the proportion of unmethylated 3,4-dihydroxy dihydroxy metabolites in the urine. 3. (3)|Epinephrine stimulates hepatic fat accumulation in adrenalectomized cortisone- and ethionine-treated rats, while metanephrine is ineffective. This indicates that the transmethylation is a true inactivation of epinephrine, and blocking of this inactivation prolongs the action of epinephrine on fat mobilization.