Identification of cytochrome p450 enzymes involved in the metabolism of 4'-methyl-alpha-pyrrolidinopropiophenone, a novel scheduled designer drug, in human liver microsomes.

4'-Methyl-alpha-pyrrolidinopropiophenone (MPPP) is a new drug of abuse. It is believed to have an abuse potential similar to that of amphetamines. Previous studies with Wistar rats had shown that MPPP was metabolized mainly by hydroxylation in position 4' followed by dehydrogenation to the corresponding carboxylic acid. The aim of the study presented here was to identify the human hepatic cytochrome p450 (p450) enzymes involved in the biotransformation of MPPP to 4'-hydroxymethyl-pyrrolidinopropiophenone. Baculovirus-infected insect cell microsomes and human liver microsomes were used for this purpose. Only CYP2C19 and CYP2D6 catalyzed this hydroxylation. The apparent Km and Vmax values for the latter were 9.8 +/- 2.5 microM and 13.6 +/- 0.7 pmol/min/pmol p450, respectively. CYP2C19 was not saturable over the tested substrate range (2-1000 microM) and interestingly showed a biphasic kinetic profile with apparent Km,1 and Vmax,1 values of 47.2 +/- 12.5 microM and 8.1 +/- 1.4 pmol/min/pmol p450, respectively. Experiments with pooled human liver microsomes also revealed biphasic nonsaturable kinetics with apparent Km,1 and Vmax,1 values of 57.0 +/- 20.9 microM and 199.7 +/- 59.7 pmol/min/mg of protein for the high affinity enzyme, respectively. Incubation of 2 microM MPPP with 3 microM of the CYP2D6-specific inhibitor quinidine resulted in significant (p < 0.01) turnover inhibition (11.8 +/- 1.6% of control). Based on kinetic data corrected for the relative activity factors, CYP2D6 is the enzyme mainly responsible for MPPP hydroxylation, confirmed by CYP2D6 inhibition studies.     

Behandlungsergebnisse nach primärer und sekundärer Radiusköpfchenresektion

Apart from comminuted fractures of the radial head the indication for resection of the radial head is discussed controversially. To evaluate our own results, the hospital notes of 113 patients treated between 1.1.1984-31.12.1994 in our clinic by posttraumatic primary or secondary radial head resection were examined retrospectively. 79 patients were controlled by clinical and radiological examination with an average follow-up of 37.8 months. We examined at 46 patients the influence of additional lesions of the elbow joint on the functional outcome. In 33 patients follow up was done with special regard to the time of resection. Poor results due to the score from Radian and Riseborough especially followed secondary radial head resection (> 14 days after trauma) and in case of additional elbow lesions. We could not observe problems of the wrist joint after radial head resection as described by other authors. According to our own experience primary resection is recommended in case of doubtful reconstruction of the radial head. In these fracture types radial head resection should not be seen as an alternative treatment because of the worse results following secondary resection.     

Electron microscopic localization of nitric oxide I synthase in the organ of Corti of the guinea pig

Nitric oxide synthase (NOS) activity has been detected previously in the mammalian cochlea at a light microscopic level. Here we present results of electron microscopic analysis for post-embedding immunoreactivity of neural-type NOS I in the cochlea of the guinea pig. Strong enzyme immunoreactivity was identified in the cytoplasm of inner and outer hair cells. Gold-labeled NOS I antibodies were mainly located in electron-dense areas of the cytoplasm, whereas electron-lucent regions of the receptor cells were nearly free from any immunoreactivity. In both types of hair cells anti-NOS I antibodies were also visible in the cuticular plates, hair bundles and nuclei. Further ultrastructural analysis revealed that the submembranous cisternae of the outer hair cells were nearly free from any reaction product, demonstrating that the whole cytoplasm of this hair cell was not immunoreactive. Other NOS I immunoreactivity was identified in the cuticular plates of the inner and outer pillar cells and in the cytoskeletal elements located in the apical parts of Deiter cells, forming the lamina reticularis or in cytoskeletal-containing regions in basal Deiter cells. Anti-NOS antibodies were visible in the nuclei of various cell types. Our findings suggest that nitric oxide produced by NO I synthase in the organ of Corti may act as a modulator of hair cell physiology during the processes of signal transduction with frequence selectivity.     

Alteration of loosely bound calcium in the guinea pig organ of Corti after treatment with diltiazem as calcium channel blocker

After oral administration of the organic calcium channel blocker diltiazem to guinea pigs for 7 days, calcium ions were precipitated with potassium antimonate in the cochleae. The spatial distribution of the precipitates was studied by energy-filtering transmission electron microscopy and the amount of the ultrastructural reaction products formed was determined semiquantitatively by an image processing system. Compared with untreated control ears, the number of the formed precipitates was reduced drastically in the inner hair cells after diltiazem treatment. In addition, electron microscopic analysis revealed that the number of calcium precipitates attached at the basolateral membrane of the outer hair cells was clearly reduced when compared with untreated control specimens. A large number of histochemical reaction products could be identified in the basilar membrane and were also observed in the untreated control specimens. The spatial distribution of the calcium precipitates in the lamina reticularis was not affected by diltiazem treatment and calcium precipitates could be identified within different cell membranes. The technique used was considered to be helpful for identifying calcium channels ultrastructurally in intact undissected tissues and to support light microscopic analyses and patch-clamp electrophysiological measurements.     

Respiratory syncytial virus genotypes and disease severity among children in hospital

OBJECTIVES: To determine the spectrum of N and G genotypes of respiratory syncytial virus (RSV) causing respiratory tract infection and whether particular genotypes are associated with severity of infection. PATIENTS AND METHODS: Nasopharyngeal aspirates (NPAs) were obtained from 114 infants with acute respiratory tract infection due to RSV over two seasons. Viral mRNA was extracted from NPAs or cultured virus, reverse transcribed, and the cDNA amplified by the polymerase chain reaction using primers directed to parts of the N and G gene respectively. Amplicons were separately digested with four different restriction endonucleases for each gene. The fragments were separated by agarose gel, electrophoresis, and the electrophoretic patterns used to assign the various genotypes. Disease severity was assessed as very mild (upper respiratory tract signs only), mild (coryza and signs of lower respiratory tract infection), moderate (requiring nasogastric or intravenous fluids), and severe (requiring oxygen or ventilation). RESULTS: Five of the six known N genotypes were detected, but NP4 and NP2 were found most frequently. There was no association between N genotype and disease severity. Six G (SHL) genotypes were detected. Significantly (p = 0.04) more of the infants infected with the SHL2 genotype had severe or moderate disease. CONCLUSIONS: During the seasonal peaks of RSV respiratory tract infection at least 10 different RSV genotypes cocirculated. While there is no association between N genotypes and disease severity, infection with the SHL2 G genotype appears to result in moderate to severe disease.     

Use of electron spectroscopic imaging to determine element composition of the melanin granules in the stria vascularis of the guinea pig

Electron spectroscopic imaging (ESI) was used to analyze the element content of melanin granules in the stria vascularis seen in ultrathin sections of Spurr-embedded cochleae of the guinea pig. To determine element composition, ESI images were taken at different ionization edges, and non-specific background signals were subtracted digitally by an image processing system. The presence of calcium and nitrogen in the melanin granules could be demonstrated clearly. The calcium identified in the melanin granules was then compared with the spatial distributions of calcium binding sites after the application of an antimonate precipitation method, which was used to localize loosely bound calcium. Despite a high calcium concentration within the granules, only very small single scattered calcium precipitates could be detected between these structures as compared with the amount of calcium precipitates attached to the plasma membrane or located within the cell nuclei. The nearly complete absence of precipitates within the melanin granules after the application of antimonate suggests differences in calcium binding and mobility involved in various physiological processes of ion balance regulation within the stria vascularis. Received: 14 October 1997 / Accepted: 11 February 1998     

Total but not resting energy expenditure is increased in infants with ventricular septal defects

OBJECTIVE: The purpose of this study was to determine the effect of left-to-right shunting on the resting energy expenditure (REE), total energy expenditure (TEE), and energy intake in a group of 3- to 5-month-old infants with moderate to large unrepaired ventricular septal defects (VSDs) compared with age-matched, healthy infants. METHODS: Eight infants with VSDs and 10 healthy controls between 3 to 5 months of age participated in the study. Indirect calorimetry was used to measure REE and the doubly-labeled water method was used to measure TEE and energy intake. An echocardiogram and anthropometric measurements were performed on all study participants. Daily urine samples were collected at home for 7 days. Samples were analyzed by isotope ratio mass spectrometry. Data were compared using analysis of variance. RESULTS: No significant differences were found in REE (VSD, 42.2 +/- 8.7 kcal/kg/d; control, 43.9 +/- 14.1 kcal/kg/d) or energy intake (VSD, 90.8 +/- 19.9 kcal/kg/d; control, 87.1 +/- 11.7 kcal/kg/d) between the groups. The percent total body water was significantly higher in the VSD infants and the percent fat mass was significantly lower. TEE was 40% higher in the VSD group (VSD, 87.6 +/- 10.8 kcal/kg/d; control, 61.9 +/- 10.3 kcal/kg/d). The difference between TEE and REE, reflecting the energy of activity, was 2.5 times greater in the VSD group. CONCLUSIONS: REE and energy intake are virtually identical between the two groups. Despite this, infants with VSDs have substantially higher TEE than age-matched healthy infants. The large difference between TEE and REE in VSD infants suggests a substantially elevated energy cost of physical activity in these infants. These results demonstrate that, although infants with VSDs may match the energy intake of healthy infants, they are unable to meet their increased energy demands, resulting in growth retardation.