Semiautomated preparation of 3,5,6-trichloro-2-pyridinol in human urine using a Zymate XP laboratory robot with quantitative determination by gas chromatography-negative-ion chemical ionization mass spectrometry

A rapid and sensitive semiautomated method was developed for quantitation of the chlorpyrifos metabolite 3,5,6-trichloro-2-pyridinol (TCP) in human urine. A Zymark Zymate XP laboratory robotics system was used to mix urine samples, transfer aliquots, add the stable-isotope-labeled TCP internal standard (13C2- or 13C2,15N-), and liberate conjugates of TCP from urine via acid hydrolysis. Samples were manually extracted into toluene, derivatized, and analyzed by gas chromatography-negative-ion chemical ionization mass spectrometry. Determination of the metabolic TCP was performed by selected ion monitoring of the dichloropyridinol fragment ions: m/z 161 for TCP and m/z 165 for 13C2-TCP or m/z 168 for 13C2,15N-TCP. Interday precision and accuracy were demonstrated over 3 years of analyses using the 13C2-TCP internal standard, with an average recovery from fortified urine samples of 93+/-12% (N = 54, concentration range 1-140 ng/mL). The method was found to be linear over the range of 0.5 to 200 ng/mL, and the limit of detection for TCP in urine was estimated to be 0.2 ng/mL with a limit of quantitation of 1 ng/mL. The effect of solids distribution on the concentration of TCP in the thawed urine samples was examined, and the results indicated that homogeneous distribution is critical for quantitation. The precision and accuracy of the automated method with respect to the transfer of homgeneous urine aliquots and delivery of internal standard yielded equivalent or improved results over the manual techniques. Overall, this method is more simple than existing methodologies, and it yields results with improved precision, accuracy, and sensitivity over previously developed methods.     

Decreased β2-adrenoceptor density and decreased isoproterenol induced c-AMP increase in juvenile type I diabetes mellitus: An additional cause of severe hypoglycaemia in childhood diabetes?

Little is known about the receptor and post receptor mechanisms of sympathoadrenal signal transmission in type I diabetes mellitus. Therefore, we examined the maximum binding of granulocyte ₂-adrenoceptors and the in vitro c-AMP accumulation in lymphocytes of 24 children and adolescents with diabetes mellitus and 14 similarly aged healthy subjects. The number of high affinity ₂-adrenoceptors on granulocytes correlated significantly with unstimulated (r=0.6,P<0.004) and with isoproterenol stimulated c-AMP values in lymphocytes (r=0.68,P<0.0007) showing the proportional changes of ₂-adrenoceptors and c-AMP in two different cells. The number of ₂-adrenoceptors on granulocytes was significantly reduced in diabetic as compared to healthy children (median 1397, range 599–3405 vs. 2205, 825–3200 ₂-adrenoceptors per granulocyte,P=0.014). Moreover, the percentage in vitro stimulation of c-AMP by isoproterenol in lymphocytes was significantly reduced in diabetic children as compared to healthy individuals (120%, 39%–278% vs. 225%, 66%–500%,P=0.012). These results indicate a decreased sympathoadrenergic signal transmission in peripheral blood cells as a model for the liver probably contributing to severe hypoglycaemia in diabetic children.     

Differential inhibition and potentiation by cell-permeant analogues of cyclic AMP and cyclic GMP and NO-containing compounds of exocytosis in human neutrophils

Summary The chemoattractants, N-formyl-L-methio-nyl-L-leucyl-L-phenylalanine (fMet-Leu-Phe), complement C5a and platelet-activating factor (PAF), induce ß-glucuronidase release and aggregation and an increase in cytosolic Ca²⁺ [Ca²⁺]_i in human neutrophils. We studied the roles of cAMP and cGMP in neutrophil avtivation, using their cell-permeant analogues, N⁶,2-O-dibutyryl adenosine 3:5-cyclic monophosphate (Bt2cAMP) and N² ,2-O-dibutyryl guanosine 3:5-cyclic monophosphate (Bt₂cGMP) and the NO-containing compounds, sodium nitroprusside (SNP), 3-morpholino-sydnonimine (SIN-1) and its prodrug, molsidomine (SIN-10). Bt₂cAMP, Bt₂cGMP, SIN-1 and SIN-10 but not SNP inhibited exocytosis induced by fMet-Leu-Phe. Superoxide dismutase potentiated the inhibitory effect of SIN-1. Bt₂cGMP and SNP potentiated C5a-induced ß-glucuronidase release, Bt₂cAMP, KCN, SIN-1 and SIN-10 being ineffective. KCN partially reversed the stimulatory effect of SNP, and in the presence of superoxide dismutase, SIN-1 potentiated C5a-induced exocytosis. PAF-induced ß-glucuronidase release was not affected by Bt₂cAMP, Bt₂cGMP, SNP and SIN-1. Bt₂cGMP was more effective than Bt2cAMP to inhibit aggregation and the increase in [Ca²⁺]_i induced by fet-Leu-Phe at submaximally effective concentrations. C5a-induced rises in [Ca²⁺]_i were not affected by Bt₂cAMP and Bt₂cGMP. Bt₂cAMP but not Bt₂cGMP inhibited the effect of PAF at submaximally effective concentrations on [Ca²⁺]_i. Our data suggest (I) that Bt2cGMP and Bt2cAMP differentially modulate neutrophil activation, that (II) NO-containing compounds partially mimick the effects of Bt₂cGMP on exocytosis and that (III) cGMP plays an inhibitory role in fMet-Leu-Phe- and a stimulatory role in C5a-induced ß-glucuronidase release. Send offprint requests to R. Seifert at the above address     

The therapeutic results of programmed relaparotomy in diffuse peritonitis]

The therapeutical concept of programmed relaparotomy was performed in 184 patients with diffuse peritonitis from 4/1984 to 4/1991. Clinical results were prospectively documented and a total of 46 variables (e.g. risk factors, clinical parameters, laboratory tests, microbiological screenings, score systems) both univariate and multivariate were tested for prognostic significance. Total lethality rate was 26% (48/184 patients). If complete eradication of the source of infection was surgically achieved (150 patients/82%) lethality rate was only 9%. In contrast, lethality rate was 100% in patients with unsuccessful surgical focus eradication. Eradication of the source of infection during the first operation (104 patients/56%) resulted in a lethality rate of 6%, compared to 17% for patients who needed two or even more operations (46 patients/25%). Eradication of the source of infection during the first laparotomy ("focus eradication on time") was the most important prognostic parameters. Of further prognostic significance but with declining importance where serum-creatinine at the beginning of the treatment, patient's age (greater than less than 70 years) and preexistent hepatic disease.     

Patterns of structural deterioration due to ischemia in Purkinje fibres and different layers of the working myocardium

Cellular and mitochondrial swelling are regarded as typical intra-ischemic alterations ("IIA"), contraction band lesions (CBL), in contrast, as products of post-ischemic reperfusion. The occurrence of both types of structural deterioration was investigated in Purkinje fibres and subendocardial and intramural working myocardium: initially after St. Thomas- or HTK cardioplegia, then during ensuing global ischemia up to the "practical limit of resuscitability", and following post-ischemic reperfusion. Generally, Purkinje fibres are not better preserved than neighbouring working myocardium. Comparing St. Thomas- and HTK cardioplegia, considerable quantitative, but not qualitative differences in the reaction patterns of different cell types or layers arise. Immediately after cardioplegia, CBL are completely lacking in both cell types. During ischemia, CBL occur occasionally in Purkinje fibres and seldom in subendocardial working myocardium, "IIA" predominate. During post-ischemic reperfusion "IIA" tend to reverse in all layers, whereas CBL are found to remain in the subendocardial cell types. In intramural layers, CBL occur only during reperfusion. Thus, we deduce that cardioplegia only modulates the severity of "IIA" and the frequency of CBL, but cannot abolish the particular sensitivity of subendocardial Purkinje fibres to global ischemia. Prerequisites for the development of irreversible CBL are on the one hand ischemic metabolic alterations and corresponding energy deficits, and, on the other hand, a supply of oxygen. The oxygen may be inadequately supplied via diffusion during ischemia or may be subsequently provided by reperfusion.     

Neurodevelopmental disorders in males related to the gene causing Rett syndrome in females (MECP2).

Mutations in the MECP2 (methyl-CpG-binding protein 2) gene are known to cause Rett syndrome, a well-known and clinically defined neurodevelopmental disorder. Rett syndrome occurs almost exclusively in females and for a long time was thought to be an X-linked dominant condition lethal in hemizygous males. Since the discovery of the MECP2 gene as the cause of Rett syndrome in 1999, MECP2 mutations have, however, also been reported in males. These males phenotypically have classical Rett syndrome when the mutation arises as somatic mosaicism or when they have an extra X chromosome. In all other cases, males with MECP2 mutations show diverse phenotypes different from classical Rett syndrome. The spectrum ranges from severe congenital encephalopathy, mental retardation with various neurological symptoms, occasionally in association with psychiatric illness, to mild mental retardation only. We present a 21-year-old male with severe mental retardation, spastic tetraplegia, dystonia, apraxia and neurogenic scoliosis. A history of early hypotonia evolving into severe spasticity, slowing of head growth, breathing irregularities and good visual interactive behaviour were highly suggestive of Rett syndrome. He has a de novo missense mutation in exon 3 of the MECP2 gene (P225L). The clinical spectrum and molecular findings in males with MECP2 mutations are reviewed.     

Computed tomography (CT) evaluation of breast cancer patients with osteolytic bone metastases undergoing palliative radiotherapy—a feasibility study

Twenty-five patients with osteolytic metastases had computed tomography (CT) scans before and 3 months after palliative radiotherapy. The median % density change following single 8 Gy, 20 Gy/5#, 30 Gy/10# were: 128 (range 98–255), 141 (79–342), and 145 (65–235), respectively. It is feasible to evaluate remineralization of osteolytic lesions with palliative radiotherapy.     

Effects of whole body hyperthermia (41.8 degrees C) on the frequency of tumor cells in the peripheral blood of patients with advanced malignancies.

PURPOSE: Combining heat with antineoplastic drugs has produced evidence of antitumor synergism. An increasing number of trials are investigating whole body hyperthermia (WBH) in combination with chemotherapy in patients with advanced malignancies. Here we investigated whether the hyperdynamic state of the circulation as a consequence of WBH may stimulate dissemination of malignant cells. EXPERIMENTAL DESIGN: WBH in combination with chemotherapy was administered by a radiant heat device to 20 consecutive patients with advanced epithelial malignancies. One WBH session lasted for approximately 4 h (90 min heating time, 60 min plateau at 41.8 degrees C, and 60-80 min cooling). Peripheral blood was drawn before WBH treatment (baseline), at the end of the plateau (1 h), and 24 h and 48 h thereafter. After removal of leukocytes using anti-CD45 magnetic beads, circulating tumor cells were detected immunocytochemically using the monoclonal antibody A45-B/B3, which binds to a common epitope present on various cytokeratins. RESULTS: The method used to detect tumor cells in the peripheral blood proved to be specific and very sensitive (detection limit 1 tumor cell per 1.7 x 10(5) peripheral blood mononuclear cell). Before WBH, 6 of 20 patients had cyto-keratin-positive cells in their blood. A treatment-induced increase in the number of circulating tumor cells became statistically significant at 24 h after WBH (P = 0.043) and was detected in a total of 9 patients, 5 of whom had no detectable malignant cells at baseline. There was no evidence of a correlation between an increase in the number of circulating tumor cells and increased metastasis frequency. CONCLUSIONS: Our findings suggest that WBH might induce a temporary release of tumor cells into the circulation, but this spread appears to be clinically not significant in patients with advanced malignancies.