Familial dup(8)(p12p21.1): mild phenotypic effect and review of partial 8p duplications.

We describe a family with direct transmission of a duplication of 8p12-->8p21.1. The phenotype of affected relatives included mild mental retardation but no minor anomalies. The duplication was identified by means of GTG-banding and fluorescence in situ hybridization with a probe specific for 8p12 generated by microdissection and degenerate oligonucleotide primed-polymerase chain reaction. Assay of glutathione reductase, which has been localised to 8p21.1, was significantly increased when compared with controls with normal chromosomal constitution. To the best of our knowledge, a proximal direct duplication of 8p restricted to subbands p12-->p21.1 has not been reported so far. The reported aberration is compared with other partial duplications of 8p, in particular to inversion duplications 8p and to small direct distal duplications involving 8p23.1. Am. J. Med. Genet. 94:306-310, 2000.

Erythrocyte glutathione S transferase as a marker of oxidative stress at birth.

AIMS: To determine the level of oxidative stress and cell damage as a result of exposure to O(2) at birth. METHODS: Using glutathione S transferase (GST) as an indicator of oxidative stress, GST activity in cord blood was compared with that in samples taken three hours after birth. Twenty four prematurely born infants and eight full term infants were studied. To test whether stronger effects occur under less favourable conditions, the neonates were divided in three groups: healthy premature; sick premature; and healthy full term infants. RESULTS: GST activity three hours after birth was significantly decreased compared with that at birth in all three groups tested. There were no significant differences in the magnitude of this effect among the three groups. CONCLUSIONS: These results indicate that a sudden increase in oxygenation exposes the neonate to oxidative stress. Measurement of GST activity might be useful for the evaluation of protective treatment in trials considering antioxidant strategies.

Two mechanisms for toxic effects of hydroxylamines in human erythrocytes: involvement of free radicals and risk of potentiation.

The toxic potency of three industrially used hydroxylamines was studied in human blood cells in vitro. The parent compound hydroxylamine and the O-ethyl derivative gave very similar results. Both compounds induced a high degree of methemoglobin formation and glutathione depletion. Cytotoxicity was visible as Heinz body formation and hemolysis. High levels of lipid peroxidation occurred, in this respect O-ethyl hydroxylamine was more active than hydroxylamine. In contrast H2O2 induced lipid peroxidation was lowered after O-ethyl hydroxylamine or hydroxylamine treatment, this is explained by the ferrohemoglobin dependence of H2O2 induced radical species formation. Glutathione S-transferase (GST) and NADPH methemoglobin reductase (NADPH-HbR) activities were also impaired, probably as a result of the radical stress occurring. The riboflavin availability was decreased. Other enzyme activities glutathione reductase (GR), glucose 6-phosphate dehydrogenase (G6PDH), glucose phosphate isomerase and NADH methemoglobin reductase, were not or only slightly impaired by hydroxylamine or O-ethyl hydroxylamine treatment. A different scheme of reactivity was found for N,O-dimethyl hydroxylamine. This compound gave much less methemoglobin formation and no hemolysis or Heinz body formation at concentrations up to and including 7 mM. Lipid peroxidase induction was not detectable, but could be induced by subsequent H2O2 treatment. GST and NADPH-HbR activities and riboflavin availability were not decreased. On the other hand GR and G6PDH activities were inhibited. These results combined with literature data indicate the existence of two different routes of hematotoxicity induced by hydroxylamines. Hydroxylamine as well as O-alkylated derivatives primarily induce methemoglobin, a process involving radical formation. The radical stress occurring is probably responsible for most other effects. N-alkylated species like N,O-dimethyl hydroxylamine primarily lead to inhibition of the protective enzymes G6PDH and GR. Since these enzymes play a key role in the protection of erythrocytes against oxidative stress a risk of potentiation during mixed exposure does exist.

Tissue distribution of selective warfarin binding sites in the rat.

Liver microsomes contain a specific warfarin binding site that is related to the target enzyme vitamin KO reductase [Thijssen HHW and Baars LGM, Biochem Pharmacol 38: 1115-1120, 1989]. In this study the distribution of the warfarin binder in the rat was investigated. Rats were given tracer doses of [14C]warfarin and tissue distribution was estimated after a time period. The selectivity of the distribution was verified by the ability of unlabeled warfarin to displace in vivo the tissue accumulated [14C]warfarin. The relation to the target enzyme vitamin KO reductase was verified by comparing the results with distribution behavior in the Scottish warfarin-resistant rat strain. The results show that in addition to liver various non-hepatic tissues accumulate warfarin. Among the tissues having a high accumulation ratio and a high rate of exchange by unlabeled warfarin are liver, pancreas, kidney, and salivary gland. Also arteria (aorta), bone, lung and spleen show exchangable [14C]warfarin accumulation. In HS rats the [14C]warfarin distribution was affected similarly for all tissues; lower levels of accumulation and higher rates of exchange by unlabeled warfarin. The tissue-bound warfarin was recovered predominantly in the microsomal fraction. Its release could only be accomplished in the presence of dithiothreitol and appeared to be stereoselective. The in vivo distribution pattern correlated with the number of warfarin binding sites in the tissue microsomes. The microsomal vitamin KO reductase activity did not always correlate to the binding capacity. The distribution was not affected by vitamin K deficiency. Warfarin-treated rats showed vitamin K epoxide accumulation in most of the organs having the warfarin binder.

Enantioselective structure-pharmacokinetic relationships of ring substituted warfarin analogues in the rat.

The enantiomer specific pharmacokinetics of ring substituted warfarin analogues have been studied in the rat after the administration of 2 mg kg-1 of the racemates. The stereoselective differences observed were due to stereoselective plasma protein binding and stereoselective intrinsic hepatic clearance. Greater binding was observed for the S-enantiomers except for 2'-substituted analogues where the R-enantiomers were more tightly bound. The stereoselectivity in the binding ranged up to a factor of about 4. All substituted warfarins showed a higher intrinsic clearance than warfarin. Enantiomer selectivity depended on the position of the substituent; warfarin and 3'-substituted analogues showed R greater than S; 4'- and 2' substituted warfarins showed S greater than R stereoselectivity. Exceptions to this generality were seen for 4'- methoxy- and 4'-methylwarfarin which did not show stereoselective hepatic clearance.

Microsomal warfarin binding and vitamin K 2,3-epoxide reductase.

Rat liver microsomal 4-hydroxycoumarin binding was studied by assaying specific [14C]warfarin binding. Microsomes of warfarin-sensitive rats contained about 40 pmole of specific binding sites per mg of microsomal protein. There was no difference for R- or S-[14C]warfarin. Neither was there any difference between the enantiomers of acenocoumarol and phenprocoumon to prevent the in vitro racemic [14C]warfarin binding. Pretreatment of the microsomes with dithiothreitol, the in vitro reductor for microsomal vitamin K epoxide reductase activity, reduced the warfarin binding. Vitamin K epoxide nor vitamin K affected the warfarin binding. Microsomes of the Welsh warfarin resistant genotype showed weak warfarin binding properties. The Scottish resistant variant, on the other hand, did not differ from sensitive microsomes. Warfarin binding was reduced in microsomes of rats to which S-warfarin was administered. The reduction in warfarin binding was linear with the inhibition of microsomal vitamin K epoxide reductase activity and was linear with the amount of S-warfarin present in the microsomes. The results show the microsomal 4-hydroxycoumarin binding to be related to the target enzyme vitamin K epoxide reductase.

Vitamin K 2,3-epoxide reductase: the basis for stereoselectivity of 4-hydroxycoumarin anticoagulant activity.

1. The administration of S-warfarin (1 mg kg-1 i.v.) to rats that were pre-loaded 48 h before with tracer doses (6 micrograms) of 14C-labelled R- or S-warfarin caused the plasma levels of these compounds to increase. This is due to the substitution of the microsomal (vitamin K 2,3-epoxide (K0) reductase) bound R- or S-[14C]-warfarin by the unlabelled 4-hydroxycoumarin administered. The rate of reappearance was 3-4 fold higher for R- than for S-warfarin; t1/2 of release: 1.2 +/- 0.04 and 3.7 +/- 0.6 h, respectively. 2. Liver microsomes prepared from rats pretreated with R- or S-[14C]-warfarin, released these compounds only in the presence of dithiothreitol (DTT; 10 mM). The rate of release was higher for R- than for S-warfarin-treated microsomes. 3. Liver microsomes treated in vitro with R- or S-acenocoumarol could be reactivated by DTT (10 mM). Reactivation was higher for the R- than for the S-acenocoumarol-treated microsomes. 4. The microsomal vitamin K0 reductase activity under 'normal' assay conditions ([DTT] = 2 mM) was as sensitive for R- as for S-4-hydroxycoumarins. At elevated DTT concentrations (= 42 mM) the rate of vitamin K0 conversion was about 1.5 fold higher in the presence of the R-isomers than in the presence of the S-isomers. For instance, at 2 mM DDT the reductase activities in the presence of 2.6 microM R- and S-warfarin were about 15% of control. At 42 mM DTT the activities were 90 and 65% of control, respectively. 5. In the in vitro experiments acenocoumarol appeared to be more potent than warfarin and phenprocoumon. 6. The following mechanism is proposed: vitamin K0 reductase becomes oxidized during substrate reduction. The oxidized (i.e. inactive) form binds equally to the R- and S-enantiomers of 4- hydroxycoumarins. The attached (covalently bound?) coumarin is released by the reactivation (i.e. reduction) of the enzyme. However, the rate of reactivation is strongly attenuated by the attached coumarin. This effect is more pronounced for the S-configuration of the 4-hydroxycoumarin anticoagulants.

Phenylbutazone-hydroxycoumarol interactions. Effects on steady state disposition, hepatocellular distribution, and biliary excretion of (S)-acenocoumarol in rats.

The effect of phenylbutazone on the disposition of (S)-acenocoumarol in the rat was studied at steady state conditions of distribution and elimination. (S)-Acenocoumarol was administered by constant rate infusions (1 microgram/min). The biliary excretion of 6- and 7-hydroxylated acenocoumarol was followed and the intrahepatic distribution was investigated. Phenylbutazone (50 mg/kg) increased the plasma unbound fraction about 4-fold. (S)-Acenocoumarol plasma clearance was enhanced (2.8 +/- 0.15 vs. 1.54 +/- 0.14 ml/min) but the unbound plasma clearance was reduced by 50% (67 +/- 9 vs. 140 +/- 27 ml/min). Phenylbutazone caused an intrahepatic redistribution of (S)-acenocoumarol, i.e. the drug shifted from the cytosol to the 10,000g pellet. The cytosolic unbound concentration, however, was increased. The (S)-acenocoumarol content in the microsomal fraction was not affected. The biliary excretion rate of total metabolite (free plus conjugated) comprised 50% of the (S)-acenocoumarol infusion rate in controls and was slightly stimulated (+20%) by phenylbutazone. The biliary excretion of free metabolites, however, was greatly increased (62 +/- 7 vs. 22 +/- 6 ng/min for 6-hydroxy-acenocoumarol; 337 +/- 38 vs. 141 +/- 32 ng/min for 7-hydroxy-acenocoumarol). This effect is probably due to stimulation of a hepatic biliary transport system; the rate constant for transport of 7-hydroxy-acenocoumarol was enhanced 5-fold (0.107 +/- 0.03 vs. 0.021 +/- 0.007 min-1).

Hepatic uptake and storage of warfarin. The relation with the target enzyme vitamin K 2,3-epoxide reductase.

The mechanisms of the reported dose-dependent warfarin pharmacokinetics were investigated using [14C]warfarin. When administered in microdoses (9 micrograms i.v.) to rats (male Wistars, 270-300 g), a steep distribution phase (T1/2 = 0.25 hr) was followed by a relatively slow beta-phase (T1/2 = 40 hr). The observed volume of distribution was 390 ml. This pharmacokinetic behavior contrasted highly with the one seen for higher (greater than 0.2 mg/kg) doses (unlabeled) warfarin; volume of distribution = 45 ml, T1/2 = 12.5 hr. If a "macrodose" (0.2 mg/kg) preceded (16 hr) the "microdose," "normal" pharmacokinetics were observed for the latter, suggesting a saturable "deep compartment." The administration of 4-hydroxycoumarins (i.e., acenocoumarol, phenprocoumon and warfarin) after the microdose of [14C]warfarin was in its beta-phase caused a rapid rise of plasma [14C]warfarin indicating [14C]warfarin to be displaced from the "deep compartment." The rate of appearance of [14C]warfarin was 0.3 hr-1 irrespective the 4-hydroxycoumarin used. The hepatic distribution of [14C]warfarin was investigated and the effect of a displacer thereupon. Fifty-three hours after the [14C]warfarin administration, the liver contained about 40% of the dose; 45% of it was bound to microsomes. The administration of acenocoumarol (0.2 mg/kg) at 48 hr, halved the liver content. [14C]warfarin was redistributed from microsomes (-65%) and from the 10,000 X g pellet (-65%) into the cytosol (+260%) and the plasma (+320%). Microsomal bound [14C]warfarin in vitro could not be washed out or be displaced unless dithiothreitol (50 mM) was included in the washing buffers.(ABSTRACT TRUNCATED AT 250 WORDS)

The biliary excretion of acenocoumarol in the rat: stereochemical aspects.

Within 24 h, 50% of a single dose of the acenocoumarol enantiomers was recovered in bile and 20% in urine of Wistar rats. The elimination products were mainly (greater than 90%) the 6- and 7-hydroxyacenocoumarol as conjugates in the bile but free in the urine. Only R-acenocoumarol, free and conjugated, was excreted in bile. There were no gross differences between the enantiomers in metabolic pattern or in the amount of metabolites formed. A significant difference was observed for the biliary excretion of the 7-hydroxy metabolite; the ratio of free and conjugated 7-hydroxyacenocoumarol was three times higher for the S- than for the R-isomer. An unknown third metabolite was recovered in bile in higher amounts with the S- than with the R-acenocoumarol. Only traces of this metabolite were recovered from urine. The data show an extensive biliary excretion of acenocoumarol and demonstrate stereoselective mechanisms in the excretion processes.

Lack of effect of cimetidine on pharmacodynamics and kinetics of single oral doses of R- and S-acenocoumarol.

The kinetics and dynamics of single doses (5 mg p.o.) of the optical isomers of acenocoumarol (R-AC and S-AC) were followed in healthy subjects and the effect on them of cimetidine 800 mg/day was also investigated. The AC enantiomers differed greatly in their pharmacokinetics. The mean residence time (MRT) of R-AC was about 10 times longer than that of S-AC, 15 h vs 1.2 h. There was no difference in the volume of distribution. Depression of blood clotting activity (Thrombotest) was observed only after administration of R-AC. The inactivity of S-AC as a vitamin K antagonist must be ascribed to its short MRT. Cimetidine did not affect the acute oral kinetics of R- and S-AC, nor did it affect the anticlotting activity of R-AC. The urinary excretion pattern of the 6- and 7-hydroxylated AC metabolites was not altered during cimetidine treatment. Although the present studies showed no effect of cimetidine on the pharmacokinetics and dynamics of acenocoumarol, the findings of Serlin et al. suggest that cimetidine should not be administered during acenocoumarol therapy.

Stereoselective aspects in the pharmacokinetics and pharmacodynamics of acenocoumarol and its amino and acetamido derivatives in the rat.

The stereoselectivity of the pharmacokinetics and of the pharmacodynamics of the oral anticoagulant acenocoumarol (AC) and of two of its potential metabolites, the amino (AM) and the acetamido (AA) derivatives, were investigated in the rat. The pharmacokinetics and pharmacodynamics were investigated following the acute subcutaneous (1 mg/kg) administration of the pure enantiomers. For AC and AA, the S(-)-enantiomer was preferentially eliminated, whereas for AM the R(+)-enantiomer showed the shortest half-life. The differences in elimination between the AC enantiomers were entirely due to differences in total clearance, 183 +/- 14 and 714 +/- 148 ml X h-1 X kg-1 (+/- SD) for R(+)- and S(-)-AC. Also the differences in elimination between the AM enantiomers were mainly due to differences in body clearance, 50 +/- 13 and 18 +/- 4 ml X h-1 X kg-1 for R(+)- and S(-)-AM. For S(-)-AA the higher total clearance as well as the smaller volume of distribution accounted for its 2-fold higher rate of elimination. Acetylation of AM, i.e. the conversion to AA, which accounted for about 50% of its total clearance was stereoselective for R(+)-AM. The renal clearance of AA which accounted for 50-60% of the AA clearance was not selective for one of the AA enantiomers. Stereoselectivity in plasma protein binding was observed, the differences, however, were small. Thus, stereoselectivity in plasma protein binding did not account for the observed differences in the pharmacokinetics. The differences in anticlotting activity between the enantiomers of AC and AM were determined mainly by their pharmacokinetics.(ABSTRACT TRUNCATED AT 250 WORDS)

The role of the intestinal microflora in the reductive metabolism of acenocoumarol in man.

Although the oral anticoagulant acenocoumarol (AC) is very effectively metabolized by the intestinal microflora to its amino metabolite, under clinical conditions this route of AC-disposition is of no importance because the compound is rapidly absorbed from its pharmaceutical application form. Only when the gastro-intestinal absorption is retarded, for instance by using a capsule as vehiculum, are appreciable amounts of reduced metabolites recovered in urine.

Acenocoumarol and its amino and acetamido metabolites. Comparative pharmacokinetics and pharmacodynamics in the rat.

The elimination, distribution and anticoagulant activity of racemic acenocoumarol, its amino (AM) and acetamido (AA) derivative were determined in male Wistar rats after subcutaneous injection of a single dose of 2 mg kg-1. The effect of daily administration of acenocoumarol on the prothrombin complex activity (PCA) was also investigated. A rapid onset of the hypothrombogenic effect was observed for all three derivatives with the half-life of decline of PCA = 3.6 h. The duration of the hypothrombogenic effect was short for the drug and its AA derivative and long for the AM compound (normalization at about 24 and 70 h, respectively), parallelling the half-life of elimination of the compounds of, 3.3, 4, and 8 h respectively. Daily administration of acenocoumarol for 8 days showed no change in the kinetics of the anticoagulant effect. Elimination of the drug is solely by metabolism. Reduction of the 4'-nitro group was not a metabolic route of importance; the amount of its two derivatives cumulatively excreted in urine over 5 days accounted for 2-3% of the dose only. Elimination of AM derivative is mainly by acetylation to AA, the compound which itself is eliminated by renal excretion. The distribution of acenocoumarol between liver and plasma was determined. The liver to plasma ratio was higher than 1 beyond 10 min after administration. The elimination rate of the drug from liver was slower than from plasma giving an increase in liver to plasma ratio in time. Plasma protein binding was extensive, being the highest for the drug; 1.81, 2.84 and 3.89% free for drug, AM, and AA derivative, respectively.

Active metabolites of acenocoumarol: do they contribute to the therapeutic effect?

The pharmacokinetics and pharmacodynamics of racemic acenocoumarol (AC), the amino (AM) and acetamido (AA) derivative were investigated in healthy volunteers after administration of a single oral (10 mg) dose. All three coumarins were rapidly absorbed from the gastrointestinal tract. The elimination half-lives were 10.9, 10.4, and 4.1 h, for AC, AM and AA, respectively. After the oral administration of AC, no AM and AA was detected in the plasma, and less than 1% of the dose was recovered in the urine (0-24 h) as AM. Renal clearance was the main route of elimination of AM and AA; the former by glomerular filtration, the latter by active excretion. Binding to serum proteins was the highest for AC (1.52% free). AM and AA were less tightly bound (about 3% free). AM or AA did not interfere with the AC plasma protein binding. The oral administration of AC resulted in a rise in prothrombin time with a maximum effect at 24 to 30 h. No anticoagulant activity was observed upon the administration of AM and AA. The results indicate that the compounds AM and AA, if they are formed out of AC at all, do not contribute to the anticoagulant activity of AC.

Analysis of acenocoumarin and its amino and acetamido metabolites in body fluids by high-performance liquid chromatography.

Acenocoumarin and its acetamido metabolite, after extraction at pH 4.4, were analysed by isocratic reversed-phase high-performance liquid chromatography using aqueous acetonitrile (pH 4.90) as eluent. Warfarin was used as internal standard. The amino metabolite, after back-extraction into 0.5 N HCl, was derivatized by diazotization and heat treatment. The resulting product was analysed by the same reversed-phase system. The sensitivity of the method for acenocoumarin and its amino metabolite was in the range of 20 ng/ml. To achieve this sensitivity for the analysis of the acetamido compound, the acetonitrile content of the eluent had to be lowered. The assay was applied to the analysis of plasma samples of patients under acenocoumarin therapy. The disposition of the amino compound in rats was investigated.