Disposition of 14C-labelled ENDOTELON in humans.

In order to study the disposition of ENDOTELON in humans, this compound was labelled with 14C by photosynthesis. ENDOTELON consists of a complex of procyanidolic oligomers extracted from the seeds of a variety of vine cultivated in the Bordeaux wine-growing region, and is prescribed for the treatment of chronic venous insufficiency and retinal lesions. Considering the difficulty in labelling the various constituents of the product, the labelling procedure was based on providing radioactive CO2 to the plant. After isolation and purification, 150 mg of active material (50 microCi) was administered orally to six healthy volunteers. Radioactivity was measured in the blood over time until 72 and 120 hours in the same subjects after drug administration. Urinary and faecal elimination was measured for a period of 167 hours. Urinary elimination of the radioactive compounds represented 12 to 27% of the administered dose and faecal elimination represented 47 to 75% depending on the subject. The radioactivity of the 14CO2 eliminated in the breath was also measured, and represented around 8% of the total radioactivity for the 72-hour period after administration. Although the disposition of ENDOTELON is based on the total radioactivity measured over time, this technique allows the evaluation of the elimination rate of the product and its metabolites from the human body.

The bioaccessibility of low level radionuclides from two Savannah river site soils.

Risk assessments for inorganic contaminated soils are often based on total concentration of a contaminant. However, strong binding of metals in soil can reduce the oral bioavailability. Since oral bioavailability of inorganics is generally less than 100% and partially dissolution-limited, human gastrointestinal dissolution models that measure bioaccessibility instead of the total extractable mass should be used to develop radionuclide source terms. For the reported study, a published bioaccessibility method was modified to allow measurements of bioaccessible radionuclides. The technique can be used to model human exposure and radionuclide dose from soil ingestion pathways. A step that included the addition of organic acids to the gastrointestinal fluids did not considerably affect the bioaccessibility of 90Sr and 137Cs. The bioaccessibility of 137Cs in the soils was significantly correlated to soil physicochemical characteristics, with a negative correlation with clay content, while 90Sr was significantly correlated to calcium bioaccessibility. These relationships can be used to prioritize remediation according to soil type.

Allele-specific receptor-ligand interactions in Brassica self-incompatibility.

Genetic self-incompatibility in Brassica is determined by alleles of the transmembrane serine-threonine kinase SRK, which functions in the stigma epidermis, and of the cysteine-rich peptide SCR, which functions in pollen. Using tagged versions of SRK and SCR as well as endogenous stigma and pollen proteins, we show that SCR binds the SRK ectodomain and that this binding is allele specific. Thus, SRK and SCR function as a receptor-ligand pair in the recognition of self pollen. Specificity in the self-incompatibility response derives from allele-specific formation of SRK-SCR complexes at the pollen-stigma interface.

Flavonoid 6-hydroxylase from soybean (Glycine max L.), a novel plant P-450 monooxygenase.

Cytochrome P-450-dependent hydroxylases are typical enzymes for the modification of basic flavonoid skeletons. We show in this study that CYP71D9 cDNA, previously isolated from elicitor-induced soybean (Glycine max L.) cells, codes for a protein with a novel hydroxylase activity. When heterologously expressed in yeast, this protein bound various flavonoids with high affinity (1.6 to 52 microm) and showed typical type I absorption spectra. These flavonoids were hydroxylated at position 6 of both resorcinol- and phloroglucinol-based A-rings. Flavonoid 6-hydroxylase (CYP71D9) catalyzed the conversion of flavanones more efficiently than flavones. Isoflavones were hardly hydroxylated. As soybean produces isoflavonoid constituents possessing 6,7-dihydroxy substitution patterns on ring A, the biosynthetic relationship of flavonoid 6-hydroxylase to isoflavonoid biosynthesis was investigated. Recombinant 2-hydroxyisoflavanone synthase (CYP93C1v2) efficiently used 6,7,4'-trihydroxyflavanone as substrate. For its structural identification, the chemically labile reaction product was converted to 6,7,4'-trihydroxyisoflavone by acid treatment. The structures of the final reaction products for both enzymes were confirmed by NMR and mass spectrometry. Our results strongly support the conclusion that, in soybean, the 6-hydroxylation of the A-ring occurs before the 1,2-aryl migration of the flavonoid B-ring during isoflavanone formation. This is the first identification of a flavonoid 6-hydroxylase cDNA from any plant species.

The male determinant of self-incompatibility in Brassica.

In the S locus-controlled self-incompatibility system of Brassica, recognition of self-related pollen at the surface of stigma epidermal cells leads to inhibition of pollen tube development. The female (stigmatic) determinant of this recognition reaction is a polymorphic transmembrane receptor protein kinase encoded at the S locus. Another highly polymorphic, anther-expressed gene, SCR, also encoded at the S locus, fulfills the requirements for the hypothesized pollen determinant. Loss-of-function and gain-of-function studies prove that the SCR gene product is necessary and sufficient for determining pollen self-incompatibility specificity, possibly by acting as a ligand for the stigmatic receptor.

Molecular characterization and functional expression of dihydroxypterocarpan 6a-hydroxylase, an enzyme specific for pterocarpanoid phytoalexin biosynthesis in soybean (Glycine max L.).

Four cytochrome P450-dependent enzymes, among them dihydroxypterocarpan 6a-hydroxylase (D6aH), are specifically involved in the elicitor-inducible biosynthesis of glyceollins, the phytoalexins of soybean. Here we report that CYP93A1 cDNA, which we isolated previously from elicitor-induced soybean cells, codes for a protein with D6aH activity. Analysis of the catalytic properties of recombinant CYP93A1 expressed in yeast, its NADPH dependency, stereoselectivity and high substrate affinity confirmed that D6aH is the physiological function of CYP93A1. It thus represents the first isoflavonoid-specific CYP to be characterized at the molecular level. In elicitor-treated soybean cells producing phytoalexins, increases in D6aH activity were correlated with elevated transcript levels which indicates that expression of the enzyme is regulated at the level of transcription. Therefore, CYP93A1 cDNA can be used as a specific molecular marker for the inducible defense response against pathogen attack.

Identification of elicitor-induced cytochrome P450s of soybean (Glycine max L.) using differential display of mRNA.

Elicitor-inducible glyceollin biosynthesis in soybean depends on five presumably transcriptionally regulated cytochrome P450-dependent enzymes (P450s). In order to isolate corresponding cDNA clones, we devised a novel polymerase chain reaction (PCR)-based approach targeting P450s that are transcriptionally activated under glyceollin-inducing conditions. The differential display of mRNA (DD-RT-PCR) technique was performed with upstream primers based on the conserved heme-binding region of P450s, and ten different 3'-terminal partial P450 sequences were isolated. They were subsequently used to isolate nine different full-length cDNA clones from a cDNA library. As shown by Northern blot analysis, eight of the clones represented P450s, which were activated under glyceollin-inducing conditions similar to two enzymes of the glyceollin biosynthesis pathway, CHS and IFR. Therefore, these eight clones are candidate cDNAs for the glyceollin-related P450s. Functional expression in yeast identified one cDNA clone coding for cinnamate 4-hydroxylase. Thus, at least one of the isolated clones definitively encodes a P450 of the glyceollin pathway. Consequently, this approach offers a straightforward alternative to classical P450 isolation strategies via protein purification and should prove especially useful for isolating P450s that are expressed at a low level.

Pharmacokinetics of pipecuronium in infants, children and adults.

In order to explain the reported shorter clinical duration of action of cumulative ED95 of pipecuronium in infants as compared to children or adults, the pharmacokinetic profiles of pipecuronium were compared in infants (n = 6; mean age 6.8 months; mean weight 7.3 kg) in children (n = 6; mean age 4.6 years; mean weight 19.2 kg) and in adults (n = 7; mean age 42 years; mean weight 58.2 kg). Equipotent doses (2 x ED95) of pipecuronium were injected i.v. as single bolus and arterial blood was sampled for 4-5 h. Pipecuronium was quantified by complex formation with [125I]-labelled rose bengal. Pharmacokinetic parameters were calculated using a two-compartment open model. The median for the distribution half-life of pipecuronium was 2.54 min (interquartile range: 1.0-2.5 min) in infants and 2.04 min (0.26-2.04 min) in children; both were significantly shorter than in adults (5.75 [3.7-9.7] min). The plasma clearance of pipecuronium was significantly decreased in infants (1.50 [0.6-1.5] ml.min-1.kg-1; P < 0.05) as compared to children and adults (2.27 [0.88-2.27] and 2.45 [1.7-3.2] ml.min-1.kg-1, respectively). The total volume of distribution was similar in all three groups. We conclude that the pharmacokinetic features of pipecuronium are age-dependent: differences as compared to adults consisted of a faster distribution in both infants and children and a slower elimination in infants. The pharmacokinetic profile of pipecuronium does not explain the faster recovery from neuromuscular blockade in infants as compared to children. Because of the low total plasma clearance in infants, pipecuronium dosage should be carefully monitored to avoid accumulation and prolonged paralysis.

Vecuronium neuromuscular blockade reflects liver function during hepatic autotransplantation in pigs.

BACKGROUND: Rapid assessment of hepatic function early after reperfusion of the liver graft is of great importance, because it may allow for prompt detection of incipient hepatic graft failure. The current study was undertaken to determine whether the continuous recording of neuromuscular transmission could be used as an on-line assessment of hepatic function during liver transplantation when a muscle relaxant with high hepatic uptake is used. METHODS: We quantified and compared the effect of liver exclusion and graft reperfusion on the level of vecuronium-induced neuromuscular blockade in nine pigs studied twice within 3 days. During the 1st day (control session), an intravenous infusion of vecuronium was administered to maintain a constant 90-95% twitch depression during 180 min. The twitch response was then allowed to recover spontaneously to 75% of its prerelaxant value. Neuromuscular transmission was continuously measured on the right anterior leg using an acceleration transducer. During the same time period, the metabolic rate of 14C-labeled aminopyrine (a well-established quantitative test of the liver microsomal function) was determined by measuring the excretion of 14CO2 in expired air after administration of an intravenous bolus of 14C-labeled aminopyrine. Two days later, the pigs underwent a hepatic autotransplantation, during which vecuronium was administered to maintain a constant 90-95% twitch depression. After reperfusion of the liver graft, the vecuronium infusion rate was maintained at its anhepatic level, and the recovery index of the neuromuscular blockade (the time from 25% to 75% recovery of twitch height) was calculated. The aminopyrine breath test was performed during the last 30 min of the anhepatic phase, and during 3 h after reperfusion of the liver graft. RESULTS: During control studies, the mean infusion rate of vecuronium was 1.30 +/- 0.33 mg.kg-1.h-1 and the recovery index was 3.4 +/- 0.5 min. During liver dissection, the infusion rate of vecuronium was similar to the control value (1.18 +/- 0.16 mg.kg-1.h-1), then considerably decreased to 0.05 +/- 0.03 mg.kg-1.h-1 during the anhepatic phase. After reperfusion of the liver graft, the recovery index was markedly prolonged to 35.5 +/- 15.8 min, indicating a prolongation of the recovery of neuromuscular blockade by a factor of 10.4. Excretion of 14CO2 was equal to zero during the anhepatic phase and then increased to 0.19 +/- 0.11% during the 1st h after reperfusion of the liver graft, an excretion rate corresponding to 11.2% of control conditions. The relationship between individual changes in the recovery index of the neuromuscular blockade and 14CO2 excretion in expired air after reperfusion of the liver graft showed a strong significant correlation (r2 = 0.71). CONCLUSIONS: These results indicate that, compared with the control studies, there is a similar decrease in the recovery rate of vecuronium-induced neuromuscular blockade and in the metabolic rate of 14C-labeled aminopyrine during the progressive recovery of hepatic function immediately after unclamping of the liver vessels. Metabolism of 14C-labeled aminopyrine increased progressively during the reperfusion phase. Therefore, recording of neuromuscular transmission during liver transplantation could serve as a continuous and easy to perform assessment of liver graft function provided that a muscle relaxant with a high hepatic uptake is used for neuromuscular blockade.

Hepatic function during general hypothermia in the pig: assessment by aminopyrine breath test.

We investigated the influence of pre-harvesting general hypothermia on liver metabolic activity by means of Aminopyrine Breath Test (ABT). This study was conducted in pigs which were anesthetized, curarised and cooled on an ice bed. Each animal received labelled aminopyrine and 14CO2 in expired air was measured between 37.5 and 25.5 degrees C. The liver metabolic activity at 31.5 degrees C represents 53.3% of the normothermic value. At 25.5 degrees C, this activity is reduced by 75.1%. The mean decreasing rate is 6.2%/degrees C for a fall in temperature of 12 degrees C. A change of slope occurred at 31.5 degrees C. The first decreasing rate is 7.47 +/- 1.62%/degrees C and the second one is 4.48 +/- 2.27%/degrees C. Thus, use of general hypothermia during liver harvesting should improve the quality of organ preservation: the important reduction of metabolism should decrease the oxygen debt resulting from anaerobic cold perfusion.

[Aminopyrine breath test in hepatic auto-transplantation in pigs]. / L'aminopyrine breath test (ABT) au cours de l'autotransplantation hépatique chez le porc.

The aminopyrine breath test (ABT) was performed during hepatic autotransplantation in the pig. The test is reproducible in this animal and only hepatic cytochromes perform the demethylation of aminopyrine. Ischemia, varying from 90 to 200 minutes, generated a major decrease in the elimination of the 14CO2 and a marked change in the elimination curve. In this specific model it was not possible to assess survival of the animal by the ABT.

Influence of hepatic dysfunction on cyclosporine metabolism in the pig.

Cyclosporine (CyA) is eliminated from the body via biliary excretion at a rate directly proportional to bile production and the functional status of the liver. Previous reports demonstrated that disturbances in the hepatic excretory function with a rise in the plasma bilirubin level are positively correlated with high blood concentrations of CyA and CyA plus metabolites (CyA + M). Less information is available about the blood concentrations of the CyA parental substance or CyA metabolites in the case of liver dysfunction when there was no elevation of serum bilirubin content. To answer this question, we compared the pharmacokinetic profile of CyA in a cholestatic and in a ischemic model in pigs. Our results show that in pigs receiving a single dose of CyA after liver ischemia, the blood concentrations of CyA and CyA + M are significantly increased independently of the serum bilirubin concentration, probably through a slow down of CyA metabolism by impairment of cytochrome P450 III A.

Pharmacokinetics of arteether in dog.

A pharmacokinetic study has been conducted in six beagle dogs after i.m. administration of 25 mg/kg of arteether, a qinghaosu (artemisinin) derivative of high anti-malarial activity. Arteether plasma concentrations were measured during a 24 h period using HPLC with an electrochemical detector in the reductive mode. The pharmacokinetic parameters were established using an open two-compartment model. Results showed a relatively rapid absorption phase: T1/2ka was 0.300 +/- 0.096 h and a mean elimination half-life of 27.95 +/- 11.93 h. Cmax was 110 +/- 16 ng/ml, Cltot/F was 1.69 +/- 0.34 ml/min and AUC was 2797 +/- 476 ng/ml/h.

Dose requirements and plasma concentrations of pipecuronium during bilateral renal exclusion and orthotopic liver transplantation in pigs.

We have studied five pigs undergoing bilateral clamping of the renal pedicles, seven pigs undergoing orthotopic liver transplantation and three control animals without surgery in order to examine the roles of the kidney and liver in the plasma clearance of pipecuronium. An i.v. infusion of pipecuronium was controlled to maintain a constant 90-95% twitch depression throughout the investigation. The right sciatic nerve was stimulated continuously with supramaximal stimuli at 0.1 Hz and the force of the corresponding evoked isometric muscle contraction was recorded continuously. Control pigs needed an infusion rate of pipecuronium 8-10.7 micrograms kg-1 min-1. In the renal group, it was necessary to reduce the infusion rate of pipe-curonium by about 25% after clamping both renal vascular pedicles (P less than 0.05 compared with controls); in pigs undergoing liver transplantation, it was necessary to reduce the rate by approximately 80% after clamping hepatic vessels (P less than 0.05 compared with controls and from the period after clamping of renal vessels). After hepatic recirculation, the infusion rate of pipecuronium was increased progressively to a rate which corresponded to 50% of baseline values (P less than 0.05 compared with the anhepatic phase and from controls). Plasma concentrations of pipecuronium were comparable in the three animal groups and did not change significantly during the study. These data suggest that the liver plays a more important role than the kidney in the plasma clearance of pipecuronium in pigs.

Plasma concentrations of laudanosine, but not of atracurium, are increased during the anhepatic phase of orthotopic liver transplantation in pigs.

To quantify the changes in plasma concentrations of atracurium and laudanosine induced by the lack of hepatic function and circulation, the authors studied nine domestic pigs (22-25 kg) undergoing an orthotopic liver transplantation, and three control animals without surgery, using atracurium as the muscle relaxant. After intubation facilitated by isoflurane 2-3%, anesthesia was maintained with isoflurane (0.5% in oxygen) and fentanyl (4 micrograms.kg-1.hr-1). Ventilation was controlled to keep end-tidal CO2 at 35-40 mmHg, body temperature maintained at 35.5-37.5 degrees C, and arterial pH at 7.35-7.50. The right sciatic nerve was stimulated with a nerve stimulator delivering a single twitch at 0.1 Hz with 0.2-ms duration, at supramaximal stimulation. The force of the corresponding evoked isometric muscle contraction was continuously measured by a force-displacement transducer. A single iv bolus of atracurium (2 mg/kg) was given to obtain a 90-95% twitch depression, followed 5 min later by a constant-rate iv infusion of atracurium at 120 micrograms.kg-1.min-1 maintained during the entire investigation. Blood samples for plasma atracurium and laudanosine concentrations were drawn every 15 min. In the control group, plasma concentrations of atracurium remained stable between 6.5-8.0 micrograms/ml following initial bolus injection; plasma concentrations of laudanosine increased during the first 60 min, then remained stable between 0.69-0.74 micrograms/ml up to the end of the study. In animals undergoing transplantation, plasma concentrations of atracurium remained stable between 10-12 micrograms/ml, despite a 90-min duration of liver exclusion.(ABSTRACT TRUNCATED AT 250 WORDS)

Pharmacokinetics of atracurium besylate in the pig after a single i.v. injection.

A pharmacokinetic study of atracurium besylate was performed in the pig after a single i.v. bolus injection of 2 mg/kg, the dose needed to produce surgical neuromuscular blockade. The plasma concentration values were obtained by high performance liquid chromatography. Using a two-compartment pharmacokinetic model, the elimination half life was found to be 28.6 +/- 6.3 min (mean +/- SEM), the total volume of distribution 341 +/- 56 ml/kg and the plasma clearance 8.7 +/- 1.1 ml/min/kg. Although the doses required to obtain a satisfactory neuromuscular blockade as well as the plasma level, volume of distribution and plasma clearance values were higher in the pig than in man, the distribution and elimination half-lives were similar to those recently reported.

[Prevalence of allergies in children and adolescents]. / Prévalence de l'allergie chez les enfants et les adolescents.

As for other chronic diseases, surveys of allergic conditions can give useful data about their prevalence and natural history. A study of the frequency of major allergic manifestations--asthma, allergic rhinitis, hay fever, atopic dermatitis and urticaria--could be performed twice at 13 years interval (1968/1981) among kindergarten children (4-6 years old) and 9th grade students (15 years old) attending the Geneva public schools. These samples represent about 90% of the corresponding age groups of the total population. Both surveys were conducted with individual interviews and physical examinations by trained physicians and nurses. The total prevalence of allergy was 5.4% in 1968 and 7.0% in 1981 for the children, and 10.3% and 11.5% for the adolescents. Asthma prevalence was 1.7% in 1968 vs 2.0% in 1981 among children, and 1.9% vs 2.8% among adolescents. For the other diseases the figures are: allergic rhinitis 0.6% vs 0.2% and 1.0% vs 0.6%; hay fever 0.5% vs 1.1% and 4.4% vs 6.1%; atopic dermatitis 2.2% vs 2.8% and 2.3% vs 1.5%; urticaria 0.4% vs 0.9% and 0.7% vs 0.5%. The rate increases over the years concern mainly the documented respiratory manifestations of atopy. Variables like sex, family atopy, ethnic origin and socio-economic status seem to be important factors influencing prevalence. Environmental factors may explain the increase of allergy in childhood and adolescence.