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Biomedical subjects

H Nau

Publications and source records attributed to H Nau.

At least 253 records · Page 14Linked to original sources
PubMed1975

Gas chromatography-mass spectrometry for probing the structure and mechanism of action of enzyme active sites. The role of Glu-270 in carboxypeptidase A.

A new technique for the study of the mechanism of enzymes has been developed. An enzyme, modified by an active-site directed reagent, is digested by one or more proteases. The resulting mixture of oligopeptides is then analyzed directly by gas chromatography-mass spectrometry without the use of separation or isolation procedures. A comparison with unmodified enzyme identifies the modified residue as well as quantifies the reaction. This approach has been applied to the identification of Glu-270 in the active site of carboxypeptidase A using a carbodiimide as modification reagent. Studies on the possible incorporation of 18O (from 18O-enriched water) into Glu-270 or other acidic residues near the active site of carboxypeptidase A show that the oxygens of the carboxyl groups of these residues are not exchangeable.

Amino Acid Sequence↗
PubMed1975

The amino acid sequence of a carboxypeptidase inhibitor from potatoes.

The carboxypeptidase inhibitor from Russet Burbank potatoes (C. A. Ryan et al. (1974b), J. Biol. Chem 249, 5495) is a mixture of approximately equal amounts of two polypeptide chains containing 38 and 39 amino acid residues, respectively. The chains differ in their amino terminal sequence only, one beginning with smaller than Glu-His-Ala ... and the other with smaller than Glu-Gln-His-Ala ..... Specific cleavage procedures utilized in determining the complete amino acid sequence of the inhibitor included acid cleavage of the aspartyl-proline bond and tryptic and chymotryptic digestion. Mass spectrometry, automatic Edman degradation, and subtractive Edman degradation were employed in sequencing the resulting peptide fragments.

Amino Acid Sequence↗
PubMedDate not supplied

Pharmacokinetics of valproic acid and metabolites in mouse plasma and brain following constant-rate application of the drug and its unsaturated metabolite with an osmotic delivery system.

Human therapeutic valproic acid (VPA) levels could be maintained in the mouse for a period of 1 week by constant rate application via subcutaneously implanted osmotic minipumps. Also, the concentrations of VPA metabolites observed in mouse plasma were similar to those seen in human plasma. The drug application could be prolonged by replacing exhausted pumps with freshly-filled devices. Removal of the implanted pumps and measurement of the decay of the drug levels revealed that the half-life of the main plasma metabolite 2-en(2-propyl-2-pentenoic acid) exceeded that of VPA. This result was confirmed by constant-rate application of this metabolite; the plasma clearance of 2-en (as calculated from the steady-state levels observed) was found to be lower than that of VPA. Brain levels of VPA and 2-en during steady-state were 3-10 per cent of corresponding plasma levels. The blood-brain kinetics of 2-en following administration of VPA were similar to those observed following application of 2-en itself. VPA was cleared faster from the brain than from the plasma, while 2-en was more persistent in the brain than in the plasma. Our results indicate that controlled, constant-rate application of drugs such as VPA, via implantable osmotic minipumps, may be a valuable procedure for a number of pharmacological and toxicological studies, particularly where persistent drug levels must be maintained for extended time periods.

Animals↗
PubMedDate not supplied

Valproic acid pharmacokinetics in the mouse following controlled-release of pharmacologic and toxic doses via novel implantable and refillable drug reservoirs.

Novel drug reservoirs are described which, after their implantation under mouse skin, continuously released organic liquids such as the anti-epileptic drug valproic acid at preselected rates for periods up to several weeks. The liquids, which were filled into the reservoirs, diffused through silastic membranes. The area and thickness of these membranes determined the administered dose. Administration of the anti-epileptic drug valproic acid at various selected doses resulted in persistent drug concentrations spanning from subtherapeutic to toxic levels. The drug reservoirs were easily refillable in situ which greatly extended the duration of the experiment. The dose administered could be determined within 5-15 per cent (rel. S.D.). It is suggested that the maintenance of persistent drug levels in small laboratory animals may be an appropriate model for the pharmacological and toxicological study of those compounds with short half-lives and high clearance rates in these species.

Animals↗
PubMedDate not supplied

Valproic acid and active unsaturated metabolite (2-en): transfer to mouse liver following human therapeutic doses.

The transfer of valproic acid (VPA, 2-propylpentanoic acid) and its unsaturated active metabolite (2-en, 2-propyl-2-pentenoic acid) from plasma to liver has been studied in the mouse between 2 min and 6 h following oral administration of 50 mg of the sodium salts per kg body weight. Transfer of both compounds was extremely rapid. Liver concentrations of VPA were higher than those in plasma, while liver concentrations of 2-en were lower than those in plasma. The low hepatic levels of 2-en may be explained by extensive plasma protein binding of this metabolite. The liver/plasma concentration ratios were concentration-dependent, indicating the presence of active transport mechanisms and/or saturation of plasma protein binding. Our results indicate that 2-en should be further studied in regard to its potential for the induction of liver toxicity. The desirable low level of 2-en reached in the liver, seen together with previously observed favourable-anticonvulsant profile and low teratogenicity, would indicate that this compound may be a valuable alternative antiepileptic agent.

Animals↗
PubMedDate not supplied

Quantitative determination of valproic acid and 14 metabolites in serum and urine by gas chromatography/mass spectrometry.

A method for the determination of the antiepileptic drug valproic acid and 14 of its metabolites in serum and urine by gas chromatography/mass spectrometry with selected ion monitoring of the trimethylsilylated derivatives has been developed. Sample preparation, including hydrolysis of VPA-conjugates and removal of urea in urine is carried out at pH 5.0 and is rapid and simple. The samples are extracted with ethyl acetate and the concentrated extracts are trimethylsilylated. Analysis with adequate separation of metabolites is achieved with a DB 1701 fused silica (Megabore) capillary column. The method exhibits high recovery and reproducibility and is sufficiently sensitive and selective for analysis of small sample volumes. Application of the method for screening patient serum and urine samples for unusual metabolite patterns, with possible predictive value for early detection of liver injury, is presented.

Chromatography, Liquid↗
PubMedDate not supplied

Embryotoxicity of stable isotopes and use of stable isotopes in studies of teratogenetic mechanisms.

Experiments on teratogenic effects of stable isotopes from our own and other laboratories are evaluated. In the first series of investigations, the enrichment of the stable isotope 13C derived from U-13C-glucose was studied in mouse embryos at various stages of development, including limb buds in organ culture. Preimplantation mouse embryos incubated in vitro in 13C-enriched medium for 48 hours showed normal development during subsequent differentiation in vitro and also in vivo after embryo transfer to faster mothers. These embryos were 15% to 20% enriched in 13C. Administration of U-13-C-glucose to pregnant mice during organogenesis led to an increase of the absolute 13C content of the embryo for several days after the end of isotope administration, whereas the enrichment in maternal tissue decreased. No alterations of embryonic development were detected due to stable isotope enrichment. Development of cultured mouse limb buds was unaffected by incubation with 82 mol% U-13C-glucose as judged from morphologic and biochemical criteria. The second part of the article describes the value of deuterium-labeled drugs as probes into the mechanism of activation of teratogenic metabolites. A comparison of the pharmacokinetics as well as the teratogenicity between cyclophosphamide and some specific deuterium-labeled analogues showed that the isotope effect observed can be related to a particular metabolic pathway crucial for teratogenic activation by this drug.

Animals↗
PubMedDate not supplied

Trimethoprim potentiates valproic acid-induced neural tube defects (NTDs) in mice.

The antiepileptic drug valproic acid (VPA) may produce NTDs because of interference with folate metabolism. Therefore, the possible interactions of VPA with the dihydrofolate reductase inhibitor trimethoprim (TM) was investigated. The combination of TM with sulfamethoxazol is used for treatment of urinary infections, the most common complications of pregnancy. TM (80 and 160 mg/kg) was given i.p. and orally, 0.5 and 1 h, respectively, prior to valproic acid (VPA, 300 and 400 mg/kg, s.c.) in day 8 pregnant NMRI mice. Fetuses were examined for exencephaly, resorptions, and fetal weight retardation on day 18 of gestation. TM (160 mg/kg, i.p.) produced no exencephaly or embryolethality, but increased fetal weight. Administration of TM (80 mg/kg, i.p.) increased VPA-induced exencephaly and fetal weight retardation but not embryolethality. Exencephaly rates induced by VPA (300 and 400 mg/kg) were 4% and 12.9% and were increased by coadministration of TM to 22.7% and 42.5%, respectively (P < 0.01). Oral TM also increased VPA-induced exencephaly and fetal weight retardation but with lower potency than i.p. injection. The observed effects were not due to altered VPA pharmacokinetics. These results support the view that VPA-induced neural tube defects may be mediated via an interaction with folate metabolism, and advise against TM-use in VPA-treated epileptics during pregnancy.

Animals↗
PubMedDate not supplied

Ethanol potentiates valproic acid-induced neural tube defects (NTDs) in mice due to toxicokinetic interactions.

Both the antiepileptic drug valproic acid (VPA) and ethanol interfere with fetal folate metabolism, which may contribute to their mechanism of teratogenesis. Therefore, the possible interaction between VPA and ethanol was investigated in mice. Ethanol (2 x 2.5 g/kg) was given orally 4 and 1 h prior to VPA (300 and 400 mg/kg, SC) in day 8.25 pregnant NMRI mice. Fetuses were examined for exencephaly, embryolethality, and fetal weight retardation on day 18 of gestation. Higher doses of ethanol (2 x 5 g/kg, orally) at day 7.5 and 8 of gestation resulted in 22% embryolethality and 1.7% exencephaly with no effect on fetal weight. Ethanol, however, increased VPA (400 mg/kg, SC)-induced exencephaly, embryolethality, and fetal weight retardation. It also increased VPA (300 mg/kg, SC)-induced exencephaly without affecting embryotoxicity. A minimum of two oral doses of 2.5 g/kg ethanol, 1 and 4 h, or 1 and 6 h prior to VPA administration were needed to produce maximum potentiation of the effects observed. These ethanol doses increased plasma VPA levels of day 8.25 pregnant mice given 400 mg/kg VPA to values comparable to the levels of mice given only VPA at a higher dose level (500 mg/kg). The incidence of exencephaly was increased from 35% for VPA (400 mg/kg) to 59% when VPA was given with ethanol. This incidence was similar to that of 60% for the high dose of VPA (500 mg/kg) administered without ethanol. Maternal plasma ethanol concentration peaked at 193, 196, and 183 mg/dL 15, 30, and 60 min, respectively, after oral ethanol administration (2.5 g/kg), and fell to 110 mg/dL by 2 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Abnormalities, Drug-Induced↗
PubMedDate not supplied

Valproate-induced limb malformations in mice associated with reduction of intracellular pH.

Valproic acid (VPA) is a commonly used antiepileptic agent that recently has been found useful in the treatment of affective disorders and prophylaxis of migraine. VPA induces congenital malformations, especially spina bifida, in the offspring of women treated with this agent during early pregnancy. The mechanism by which VPA induces abnormal development remains unknown despite many studies in experimental animals in which VPA causes malformations similar to those seen in human infants. Because of its chemical structure as a weak organic acid and its capability to induce postaxial forelimb ectrodactyly in C57BL/6 mice, we postulated that VPA acts to perturb limb morphogenesis by reducing embryonic intracellular pH (pHi). We administered VPA, 200 to 400 mg/kg, to C57BL/6 mice on day 9 of gestation. A dose-dependent incidence of postaxial forelimb ectrodactyly was observed. Forelimb bud pHi was estimated by computer-assisted image analysis from the transplacental distribution of 14C-DMO. At the highest doses, 300 and 400 mg/kg, a decrease of pHi of 0.2 to 0.3 pH units was observed uniformly throughout the limb bud 1 h after VPA treatment. None of these changes were seen after treatment with 2-en VPA, a nonteratogenic analog of VPA. Furthermore, the capability of VPA to induce postaxial forelimb ectrodactyly was greatly enhanced by coadministration of agents that inhibit pHi regulatory processes. These data support the hypothesis that VPA-induced postaxial ectrodactyly in murine fetuses can be attributed to reduction in limb bud pHi.

Abnormalities, Drug-Induced↗
PubMedDate not supplied

Further branching of valproate-related carboxylic acids reduces the teratogenic activity, but not the anticonvulsant effect.

In the present study, compounds derived from the anticonvulsant drug valproic acid (VPA, 2-n-propylpentanoic acid) and analogues known to be teratogenic were synthesized with an additional carbon-branching in one of the side chains. The substances were tested for their ability to induce anticonvulsant activity and sedation in adult mice, and neural tube defects (exencephaly) in the offspring of pregnant animals (Han:NMRI mice). In all cases, the rates of exencephaly, embryolethality, and fetal weight retardation induced by the methyl-branched derivatives were very low when compared to those of the parent compounds. These novel compounds exhibited anticonvulsant activity which was not significantly different from that of VPA. Neurotoxicity was considerably lower for some compounds as compared to VPA. Anticonvulsant activity and neurotoxicity of branched short chain fatty acids are far less structure-dependent and not related to teratogenic potency. Within this series of compounds, (+/-)-4-methyl-2-n-propyl-4-pentenoic acid and (+/-)2-isobutyl-4-pentenoic acid exhibited the most favorable profile in regard to high anticonvulsant effect, low sedation, and teratogenicity. Valproic acid analogues with additional methyl branching may be valuable antiepileptic agents with low teratogenic potential.

Animals↗
PubMedDate not supplied

Weak acids may act as teratogens by accumulating in the basic milieu of the early mammalian embryo.

Among the eleven drugs or chemicals which are well-documented human teratogens, eight (or their main metabolites) are weak acids whereas none is a weak base. Moreover, 23 out of 32 acids tested have been found to be teratogenic in at least one animal species. The acidic property of drugs may therefore be an important determinant of teratogenicity. We demonstrate here that the intracellular pH (pHi) of the mouse and rat embryo is higher than that of maternal plasma, as determined by the relative accumulation of dimethadione. The antiepileptic drug valproic acid and its pharmacologically active unsaturated metabolite accumulate in embryonic tissue to higher concentrations than in maternal plasma, whereas the essentially neutral amide of valproic acid (valpromide) or ethosuximide do not accumulate in the embryo; we further demonstrate in the rat that the pHi of the embryo decreases with advancing gestation; in general agreement with the pH partition hypothesis, the exposure of the embryo to valproic acid also decreases significantly during that period. Furthermore, the amides of two weak acid teratogens, valpromide and methoxyacetamide, and the imide ethosuximide, are much less teratogenic than their acid counterparts. Our results suggest that weakly acidic drugs, by virtue of their physico-chemical nature, accumulate in the early embryo with its relatively high pHi.

Acids↗