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Biomedical subjects

W Kalow

Publications and source records attributed to W Kalow.

At least 73 records · Page 4Linked to original sources
PubMed1991

Caffeine as a metabolic probe: exploration of the enzyme-inducing effect of cigarette smoking.

It has been realized recently that the primary metabolism of caffeine in humans is catalyzed by P-450IA2 and that the rate of caffeine metabolism can be estimated from a metabolic ratio in a single urine sample. A population of 178 students including 19 smokers were subjected to this caffeine test to establish their P-450IA2 index. Both stated numbers of cigarettes smoked per day and urinary cotinine levels as a confirmatory measure correlated significantly with enzyme activity showing dose-effect relationships (r = 0.62 and 0.89, respectively). Nevertheless, more nonsmokers than smokers had the highest enzyme indexes, suggesting that dietary elements or other factors may determine P-450IA2 activities in populations. Because P-450IA2 is a monooxygenase that may be confined to the liver, caffeine reveals directly the Ah-receptor-dependent enzyme induction only in the liver, but it may also be a signal of induction elsewhere.

Caffeine↗
PubMed1991

Caffeine as a metabolic probe: validation of its use for acetylator phenotyping.

The use of two caffeine metabolite ratios for acetylator phenotyping was validated by demonstrating concordance with two sulfamethazine tests in 178 unrelated healthy subjects. The caffeine metabolites used for this purpose were 5-acetylamino-6-amino-3-methyluracil (AAMU), 1-methylxanthine (1X), and 1-methylurate (1U). The ratio AAMU/(AAMU + 1X + 1U), referred to as molar ratio or N-acetyltransferase, was compared with the ratio AAMU/1X. The results indicated that, for screening purposes, the acetylator phenotype can be determined by analysis of a 6-hour urine sample after a cup of coffee or strong tea or a can of caffeine-containing soft drink. The ratio AAMU/1X is the ratio of choice for the study of subjects in whom variability of xanthine oxidase can be neglected; use of the ratio AAMU/(AAMU + 1X + 1U) appears appropriate for special purposes. Gender, ethnic origin, habitual or moderate consumption of coffee, tea, soft drinks, or ethanol, or cigarette smoking have little if any effect on the caffeine tests for acetylator phenotyping.

Acetylation↗
PubMed1991

Identification of a new variant CYP2D6 allele lacking the codon encoding Lys-281: possible association with the poor metabolizer phenotype.

A variant CYP2D6(C) P450 protein was found in a liver characterized by deficient microsomal metabolism of bufuralol and sparteine, prototypical substrates for the debrisoquine-sparteine drug oxidation polymorphism. This protein was present at decreased levels in liver and had a slightly different relative mobility on SDS-polyacrylamide gels. The cDNA cloning and sequencing of the variant, designated CYP2D6(C), revealed that its mRNA lacked a single codon resulting in deletion of Lys281. This was the result of a three base pair deletion at the 3' end of CYP2D6 exon 5. The CYP2D6(C) P450, produced in HepG2 cells using vaccinia virus mediated cDNA expression displayed Km values toward bufuralol, debrisoquine and sparteine that were not significantly different from wild type CYP2D6. These data suggest that the poor metabolizer phenotype in livers expressing CYP2D6(C) is not due to a catalytically defective enzyme but perhaps due to decreased levels of the P450 protein in microsomal membranes. Low microsomal CYP2D6(C) contents could result from deficient membrane insertion or decreased stability of the P450 protein. A polymerase chain reaction-based procedure, developed to detect CYP2D6(C) alleles, indicates that this variant probably represents less than 1.5% of all CYP2D6 alleles.

Alleles↗
PubMed1991

Pharmacogenetics of caffeine and caffeine-halothane contractures in biopsies of human skeletal muscle.

In vitro pharmacological responses of fresh biopsy specimens of human skeletal muscle were used as indicators of some intrinsic muscle properties. The measured parameters that were utilized for the current study were contractures induced in vitro by caffeine or by caffeine plus halothane. The opportunity to study such specimens arose from clinical testing for diagnosing the genetic predisposition to malignant hyperthermia, a potentially fatal complication of anaesthesia. The current analysis covers data from over 1,000 subjects, most of whom were clinical suspects and relatives of these. Responsiveness of the muscle specimens varied over two orders of magnitude. The frequency distribution curves suggest that the variation does not represent a continuum but that there are three or more clusters of functional variants. Muscle specimens from males were on average more responsive to caffeine than were those from females. Correlations within father-son and brother-brother pairs indicated complete heritability of responsiveness; this might have been expected but the surprise was a lack of correlation within mother-daughter pairs. There was an intermediate correlation in father-daughter pairs. The sex difference in heritability could be due to gender-related modifying genes or due to secondary modification of the muscle response in females by sex-related, perhaps hormonal factors. Among the effects of age appeared to be poor development in early childhood of the potentiation of the caffeine contracture by halothane.

Adolescent↗
PubMed1991

Oxidation of reduced haloperidol to haloperidol: involvement of human P450IID6 (sparteine/debrisoquine monooxygenase).

1. The conversion of haloperidol (HAL) to reduced haloperidol (RHAL) and then back to HAL has been established in vivo and observed in psychiatric patients. The reduction of HAL to RHAL is known to be catalysed by a ketone reductase, while the nature of oxidation back to HAL is the subject of the present study. 2. We examined the in vitro oxidation of RHAL to HAL in human livers. The activity was microsomal and evidence is presented to suggest that the sparteine/debrisoquine metabolizing isoenzyme P450IID6 contributes to this oxidation. 3. Reciprocal inhibition studies between RHAL and sparteine, a specific substrate for cytochrome P450IID6, indicated that both compounds compete for the same binding site. Quinidine, the most specific inhibitor for this cytochrome P450 potently inhibited the oxidative conversion of reduced haloperidol to haloperidol. A significant correlation (rs = 0.62, P less than 0.01) was found between RHAL oxidation and sparteine oxidation in a study involving 17 human liver samples.

Cytochrome P-450 CYP2D6↗
PubMed1991

Neuronal cytochrome P450IID1 (debrisoquine/sparteine-type): potent inhibition of activity by (-)-cocaine and nucleotide sequence identity to human hepatic P450 gene CYP2D6.

Catalytic, pharmacological, and molecular criteria have been used to identify cytochrome P450IID1 in mammalian brain (enzyme, P450IID; gene, CYP2D). Sparteine metabolism in canine striatal membranes was shown to be inhibited in a concentration-dependent and stereoselective manner by quinidine (Ki, approximately 51 nM), quinine (Ki, approximately 5.9 microM), and various other known substrates and inhibitors of hepatic P450IID1 activity. In addition, canine striatal P450IID1 was inhibited with high affinity by dopamine uptake blockers, such as (-)-cocaine (Ki, approximately 74 nM), d-amphetamine (Ki, approximately 4.5 microM), and methylphenidate (Ki, approximately 15 microM). Inhibitory constants (Ki) of numerous compounds for inhibition of sparteine metabolism in canine striatal membranes correlated well with (a) Ki values observed in human liver microsomes (r = 0.95), (b) [3H]GBR-12935 binding to P450IID1 in canine striatal membranes (r = 0.85), and (c) the inhibition (IC50) of sparteine metabolism in HepG2 cells expressing human CYP2D6 cDNA (r = 0.93). Moreover, antibodies raised against rat hepatic enzyme inhibited, in a concentration-dependent manner, sparteine metabolism in canine striatal membranes. Enzymatic activity was unevenly distributed throughout the canine brain and ranged from 0.5 to 21 pmol/mg of protein/hr in cerebellum and supraorbital cortex, respectively, with the striatum displaying moderate levels of activity (8 pmol/mg of protein/hr). The polymerase chain reaction was used to amplify cDNA from a human caudate lambda gt11 library encoding exons 6-9 of the human CYP2D6 gene, which revealed, upon sequencing, 100% nucleic acid sequence identity. These data indicate that P450IID1 is expressed centrally and is similar, at the functional and molecular levels, to the human hepatic P450IID1 enzyme. Because the debrisoquine/sparteine mono-oxygenase is a polymorphic enzyme, in which 5-10% of caucasians are deficient in metabolism of various drugs, a genetic difference in human brain metabolism of P450IID1 substrates may possibly lead to differences in drug response and toxicity.

Animals↗
PubMed1990

The dopamine transporter and cytochrome P45OIID1 (debrisoquine 4-hydroxylase) in brain: resolution and identification of two distinct [3H]GBR-12935 binding proteins.

Two [3H]GBR-12935 binding proteins, identified as the dopamine transporter and cytochrome P45OIID1, were solubilized in digitonin from canine striatal membranes, and were resolved following wheat germ agglutinin (WGA)-lectin column chromatography. Protein adsorbed to and specifically eluted from WGA-lectin with N-acetylglucosamine displayed saturable, high affinity (KD approximately 3 nM), and sodium-dependent binding of [3H]GBR-12935, which was inhibited in a concentration-dependent and stereoselective manner by dopamine uptake blockers and substrates with a pharmacological profile indicative of the dopamine uptake site. Protein not adsorbed to WGA-lectin also bound [3H]-GBR-12935 with high affinity (approximately 7 nM), in a sodium-independent manner, and was insensitive to classical dopamine uptake blockers and substrates such as mazindol or dopamine, corresponding to the so-called "piperazine acceptor" site seen in native membranes. [3H]GBR-12935 binding to this latter protein was, however, inhibited by various compounds with a pharmacological profile indicative of a form of cytochrome P450 designated P45OIID1 (debrisoquine/sparteine monooxygenase) with the following rank order of inhibitory potency: GBR-12909 greater than budipine greater than alpha-lobeline greater than quinidine greater than alpha flupenthixol greater than SKF-525A greater than sparteine greater than quinine. Ki values obtained for inhibition of [3H]-GBR-12935 binding to neuronal WGA passthrough fractions by these drugs correlate well with their respective Ki values for liver P45OIID1 activity. Western blotting and immunoprecipitation analysis with rabbit anti-rat P45OIID1 antibody also supported the identity of the mazindol-insensitive [3H]GBR-12935 binding site (or piperazine acceptor site) as P45OIID1. Furthermore, a [3H]GBR-12935 binding protein with pharmacological and immunological characteristics similar to those of P45OIID1 was solubilized from both bovine and human liver membranes, and GBR-12909 was found to be a potent competitive inhibitor (Ki approximately 100 nM) of sparteine monooxygenase activity in human liver microsomes. These data clearly indicate that [3H]GBR-12935 and its analogs display similar affinities for both the dopamine transporter and neuronal P45OIID1, and that this radioligand may be a useful probe of P45OIID1 activity in brain and liver. The exact molecular and functional association (if any) between these two distinct binding protein populations remains to be established; however, it is tempting to speculate that P45OIID1 is involved in the catabolism and processing of neurotransmitters subsequent to their reuptake into target cells.

Animals↗
PubMed1990

Sparteine metabolism capacity in human liver: structural variants of human P450IID6 as assessed by immunochemistry.

An antibody raised against rat P450dbl was used to examine the heterogeneity of the human enzyme involved in the sparteine/debrisoquine polymorphism. The extent to which the antibody was able to inhibit sparteine metabolism varied in different human livers (10-80%, n = 9) and reflected the amount of sparteine metabolism carried out by the polymorphic P450IID6 in individual liver specimens. The individual sample variation in inhibition by the antibody correlated with the inhibition caused by quinidine, a prototype competitive inhibitor of the P450IID6 enzyme active site. Western immunoblots of the liver microsomes confirmed that the variation in the inhibition of sparteine metabolism by this antibody reflected the amount of P450IID6 protein. In addition, a detailed study of one of the livers (K19) which demonstrated a lack of inhibition by the antibody was performed which confirmed the lack of P450IID6 in this liver specimen and suggested that the nascent sparteine metabolism activity was due to other forms of P450.

Animals↗
PubMed1990

The caffeine breath test and caffeine urinary metabolite ratios in the Michigan cohort exposed to polybrominated biphenyls: a preliminary study.

A field biochemical epidemiology study was conducted using the Michigan cohort consisting of 51 rural residents exposed to polybrominated biphenyls (PBB). The study had three major objectives: a) to determine the serum half-life of the major PBB congener, hexabromobiphenyl (HBB), in the human, b) to determine if the PBB-exposed subjects had elevated cytochrome P-450I function as determined by the caffeine breath test (CBT) and the caffeine urinary metabolite ratio (CMR), and c) to determine the applicability of the CBT and CMR in field studies. PBB serum levels were detected in 36 of the 51 PBB-exposed subjects. The serum half-life of HBB was determined by comparing the current serum HBB values to the subject's previous serum values obtained 5 to 8 years earlier. The median HBB half-life was 12 years (range 4-97 years). The CBT and CMR were elevated in the subjects exposed to PBBs as compared to the values obtained from urban nonsmokers and were similar to those found in adults who smoke. A gender effect was seen in the PBB-exposed subjects, the median CBT and CMR values of the females being lower than the values of males. There was a correlation between the CBT and the HBB serum values (r2 = 0.2, p = 0.01) but not between CMR and HBB serum values. The CBT and CMR were easily conducted in the field and appear to be useful metabolic probes of cytochrome P-450I activity in human environmental toxicology.

Adult↗
PubMed1989

cDNA cloning and sequence and cDNA-directed expression of human P450 IIB1: identification of a normal and two variant cDNAs derived from the CYP2B locus on chromosome 19 and differential expression of the IIB mRNAs in human liver.

A cDNA designated hIIB1, representing the entire coding sequence of a P450 in the IIB gene family, was isolated from a human liver lambda gt11 library by using the rat IIB1 cDNA as a probe. The hIIB1 protein, deduced from the cDNA sequence, contained 491 amino acids, had a calculated molecular weight of 56,286, and displayed 76% amino acid similarity with the rat IIB1 protein. Expression of this cDNA, using the vaccinia virus system, yielded a P450 that had a reduced CO-binding spectrum with an absorption maximum of 452 nm. The expressed human enzyme was able to catalyze the deethylation of 7-ethoxy-coumarin. Total RNA from 13 livers was probed for levels of hIIB mRNA. Two livers had high levels, four contained moderate levels, and eight contained very low, or no detectable, mRNA. These data suggest either that defective hIIB1 genes exist in humans or that the hIIB1 gene is regulated and variably induced in our liver specimens. To search for mutant mRNA transcripts, libraries were constructed from livers expressing low levels of hIIB1 mRNA. A cDNA, designated hIIB2, was isolated that was identical with the hIIB1 cDNA except for the presence of an unusual alteration of the DNA near its 5' end corresponding to the putative exon 4. This alteration was caused by a deletion of 29 bp and an insertion of 44 bp of nonhomologous DNA. This sequence replacement occurs at the junction of the third and fourth exons as predicted from the structure of the rat IIB1 gene, suggesting that a faulty splice might have given rise to the variant hIIb2 transcript.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1989

Cytochrome P-450 hPCN3, a novel cytochrome P-450 IIIA gene product that is differentially expressed in adult human liver. cDNA and deduced amino acid sequence and distinct specificities of cDNA-expressed hPCN1 and hPCN3 for the metabolism of steroid hormones and cyclosporine.

Immunoblotting analysis of human liver microsome preparations revealed that human cytochrome P-450 PCN1 (hPCN1, Mr approximately 52,000) was expressed in each of 40 individual specimens examined. In about 10-20% of the livers, an immunologically related protein having a lower electrophoretic mobility (Mr approximately 52,500) was also detected. A single liver was found that expressed only the lower mobility protein, designated hPCN3, and RNA isolated from this liver was used to construct a lambda gt11 library. The library was screened with an hPCN1 cDNA probe resulting in the isolation of a unique full-length cDNA that was sequenced and shown to encode hPCN3. The deduced amino acid sequence of this cDNA contained 502 residues, a calculated molecular mass of 57,115 daltons, and displayed 84% similarity with hPCN1. The deduced amino-terminal sequence of hPCN3 was identical to that of HFLa, a major cytochrome P-450 expressed in human fetal liver that is immunologically cross-reactive with several family III cytochrome P-450s. hPCN1 and hPCN3 cDNAs were expressed in Hep G2 cells using a vaccinia virus expression system and shown to encode active enzymes, both characterized by reduced CO-binding spectra with lambda max at 450 nm. Enzymatic analysis revealed that both cytochrome P-450s were similarly active in catalyzing oxidation of the calcium channel blocking drug nifedipine. Both enzymes also catalyzed 6 beta-hydroxylation of the steroid hormones testosterone, progesterone, and androstenedione, although hPCN1 exhibited several-fold higher expressed activity than hPCN3. Several minor oxidation products of these steroids (e.g. 15 beta-hydroxytestosterone), comprising up to approximately 20% of the total metabolites, were formed by hPCN1 but not hPCN3, indicating that hPCN3 is a more highly regiospecific monooxygenase catalyst with steroid substrates. Clear differences were also detected in their catalytic activities toward the immunosuppressive drug cyclosporine, with two hydroxylated metabolites (M1 and M17) and one demethylated metabolite (M21) formed by hPCN1 but only one metabolite (M1) formed by hPCN3. These studies establish that hPCN3 is a newly described cytochrome P-450 that is differentially expressed in the adult human population and that has overlapping substrate specificity compared to hPCN1 for metabolism of steroid and drug substrates.

Amino Acid Sequence↗
PubMed1989

Genetic polymorphism in drug oxidation.

Of the two clearly established drug oxidation polymorphisms, only the one referred to as debrisoquine polymorphism affects many drugs. The only known polymorphic substrates of mephenytoin hydroxylase are mephenytoin and mephobarbital. Relatively recently discovered drug substrates of debrisoquine hydroxylase are propafenone, diltiazem, and codeine. The list of substrates contains 28 items. The fate of slightly less than half of these is clinically affected in poor metabolizers, and several of the latter drugs are no longer marketed. There are many reasons why a failure of metabolism may not alter the fate of a drug sufficiently to affect its clinical use. Of interest and clinical importance is the inhibition of debrisoquine hydroxylase by inhibitors such as quinidine and by some neuroleptics; also the simultaneous use of two substrates has led to serious toxicity by mutual metabolic inhibition. The study of these oxidation polymorphisms has been instructive not only for formal pharmacogenetics but also for the understanding of problems of therapy in patients without genetic defects.

Animals↗
PubMed1989

Haloperidol reduction can be assayed in human red blood cells.

One metabolite of haloperidol present in plasma is "reduced haloperidol." This study demonstrates that human red blood cells are capable of converting haloperidol to reduced haloperidol in vitro. The reductase involved requires NADPH, as does haloperidol (ketone) reductase in human liver cytosol.

Adult↗
PubMed1988

High-performance liquid chromatographic analysis of desferrioxamine. Pharmacokinetic and metabolic studies.

A high-performance liquid chromatographic method has been developed that permits determination and quantitation of desferrioxamine and metabolites as their iron (III) complexes in small samples of mammalian plasma at levels encountered with ion-specific chelation treatments. The technique permits measurement of desferrioxamine and metabolite concentrations which can be used in pharmacokinetic studies. A human study is presented as an example.

Animals↗
PubMed1988

Method to determine the enantiomers of ibuprofen from human urine by high-performance liquid chromatography.

By means of ethyl chloroformate, ibuprofen enantiomers were coupled to 4-methoxyaniline. The resulting amides were resolved from each other and from urinary constituents on a Pirkle column using an isocratic mobile phase with ultraviolet detection at 254 nm. Applicability of this method for the determination of inter-individual differences in urinary metabolic profiles of ibuprofen enantiomers is demonstrated. The chromatographic behavior of the corresponding amide derivatives of two ibuprofen metabolites is also described.

Biotransformation↗