Micro-HPLC and standard-size HPLC for the separation of peptide stereoisomers employing an ion-exchange principle

Standard-size (4 mm ID) and micro-HPLC columns (0.5 mm ID) packed with a quinine-based ion-exchange type chiral stationary phase are comparatively evaluated for the separation of peptide enantiomers with up to six amino acid residues. The results show that downscaling the separation system in order to gain the advantages of miniaturized HPLC is possible without sacrificing separation power. Further, five different N-terminal protections (3,5-dinitrobenzoyl, 2,4-dinitrophenyl, 3,5-dinitrobenzyloxycarbonyl, carbazole-9-carbonyl, and 9-fluorenylmethoxycarbonyl) of the analytes are investigated regarding their effect on enantioselectivity. A comparison between a hydro-organic and a polar-organic mobile phase is also reported. The enantiomers of the peptides containing one to four amino acid residues were baseline resolved, while for the penta- and hexamers only partial separations were possible. In addition, all four stereoisomers of alanylalanine could be baseline separated.     

Application of the CFU-GM assay to predict acute drug-induced neutropenia: an international blind trial to validate a prediction model for the maximum tolerated dose (MTD) of myelosuppressive xenobiotics.

In a previous study of prevalidation, a standard operating procedure (SOP) for two independent in vitro tests (human and mouse) had been developed, to evaluate the potential hematotoxicity of xenobiotics from their direct and the adverse effects on granulocyte-macrophages (CFU-GM). A predictive model to calculate the human maximum tolerated dose (MTD) was set up, by adjusting a mouse-derived MTD for the differential interspecies sensitivity. In this paper, we describe an international blind trial designed to apply this model to the clinical neutropenia, by testing 20 drugs, including 14 antineoplastics (Cytosar-U, 5-Fluorouracil, Myleran, Thioguanine, Fludarabine, Bleomycin, Methotrexate, Gemcitabine, Carmustine, Etoposide, Teniposide, Cytoxan, Taxol, Adriamycin); two antivirals (Retrovir, Zovirax,); three drugs for other therapeutic indications (Cyclosporin, Thorazine, Indocin); and one pesticide (Lindane). The results confirmed that the SOP developed generates reproducible IC90 values with both human and murine GM-CFU. For 10 drugs (Adriamycin, Bleomycin, Etoposide, Fludarabine, 5-Fluorouracil, Myleran, Taxol, Teniposide, Thioguanine, and Thorazine), IC90 values were found within the range of the actual drug doses tested (defined as the actual IC90). For the other 10 drugs (Carmustine, Cyclosporin, Cytosar-U, Cytoxan, Gemcitabine, Indocin, Lindane, Methotrexate, Retrovir, and Zovirax) extrapolation on the regression curve out of the range of the actual doses tested was required to derive IC90 values (extrapolated IC90). The model correctly predicted the human MTD for 10 drugs out of 10 that had "actual IC90 values" and 7 drugs out of 10 for those having only an extrapolated IC90. Two of the incorrect predictions (Gemcitabine and Zovirax) were within 6-fold of the correct MTD, instead of the 4-fold range required by the model, whereas the prediction with Cytosar-U was approximately 10-fold in error. A possible explanation for the failure in the prediction of these three drugs, which are pyrimidine analogs, is discussed. We concluded that our model correctly predicted the human MTD for 20 drugs out of 23, since the other three drugs (Topotecan, PZA, and Flavopiridol) were tested in the prevalidation study. The high percentage of predicitivity (87%), as well as the reproducibility of the SOP testing, confirm that the model can be considered scientifically validated in this study, suggesting promising applications to other areas of research in developing validated hematotoxicological in vitro methods.     

The need for phase-encoding flow compensation in high-resolution intracranial magnetic resonance angiography

PURPOSE: To demonstrate that the time delay between phase and frequency encoding and the presence of pulsatile blood flow in high-resolution time-of-flight (TOF) imaging of the intracranial arteries (especially near the circle of Willis) can distort the appearance of blood vessels and result in a cross-hatch-appearing artifact in surrounding tissue. MATERIALS AND METHODS: Two techniques to reduce the artifact, tri-directional flow compensation (3DFC) and elliptical-centric (EC) phase-encoding order, are investigated in five volunteer studies. RESULTS: 3DFC eliminates the pulsation-related artifacts and the vessel distortion. A residual amplitude variation artifact is observed. EC phase encoding nearly eliminates the pulsatile motion-related artifact, but it does not eliminate vessel distortion. CONCLUSION: The combination of 3DFC and EC phase encoding appears to provide the greatest artifact reduction in the five volunteer studies performed.     

A multicentre phase II trial of bryostatin-1 in patients with advanced renal cancer

Protein kinase C (PKC) has a critical role in several signal transduction pathways, and is involved in renal cancer pathogenesis. Bryostatin-1 modulates PKC activity and has antitumour effects in preclinical studies. We conducted a multicentre phase II clinical trial in patients with advanced renal cancer to determine the response rate, immunomodulatory activity and toxicity of bryostatin-1 given as a continuous 24 h infusion weekly for 3 out of 4 weeks at a dose of 25 mug m(-2). In all, 16 patients were recruited (11 males and five females). The median age was 59 years (range 44-68). Patients had been treated previously with nephrectomy (8) and/or interferon therapy (9) and/or hormone therapy (4) and/or radiotherapy (6). Eight, five and three patients had performance statuses of 0, 1 and 2, respectively. A total of 181 infusions were administered with a median of 12 infusions per patient (range 1-29). Disease response was evaluable in 13 patients. Three patients achieved stable disease lasting for 10.5, 8 and 5.5 months, respectively. No complete responses or partial responses were seen. Myalgia, fatigue, nausea, headache, vomiting, anorexia, anaemia and lymphopenia were the commonly reported side effects. Assessment of biological activity of bryostatin-1 was carried out using the whole-blood cytokine release assay in six patients, two of whom had a rise in IL-6 levels 24 h after initiating bryostatin-1 therapy compared to pretreatment values. However, the IL-6 level was found to be significantly lower at day 28 compared to the pretreatment level in all six patients analysed.     

鼻咽癌患者5-FU血药浓度与毒性及疗效的关系

背景与目的:5-氟尿嘧啶(5-fluorouracil,5-FU)对人体的作用有几条途径,不同个体对5-FU的代谢有很大的差异,故很难预测5-FU对不同个体的治疗效果和毒副作用。本研究拟探讨5-FU的稳态血药浓度在接受相同初始剂量(按体表面积计算)的患者之间的差异,以及其与不良反应、治疗反应之间的关系。方法:在接受顺铂(cisplatin,DDP)联合5-FU连续静脉灌注治疗的20例鼻咽癌患者中,开始5-FU治疗后24h采集血样,用高效液相色谱仪(HPLC)测定5-FU的血药浓度。HPLC条件:1mmol/L磷酸盐缓冲液作为流动相,流速1.3ml/min,波长260nm,柱温25℃。结果:5-FU血药浓度在鼻咽癌患者中呈正态分布,个体间有明显差异。5-FU血药浓度低于600μg/L时,患者均没有不良反应,而高于1000μg/L时,患者均出现较严重的不良反应。不同级别不良反应及不同的治疗反应所对应的5-FU血药浓度之间存在明显的统计学差异(P<0.05)。结论:5-FU血药浓度在接受相同初始剂量(按体表面积计算)的患者之间存在明显的差异。5-FU血药浓度与其毒副作用、治疗反应有关。     

Polymorphisms of the glutathione S-transferase P1 gene and head and neck cancer susceptibility

BACKGROUND: Factors determining the individual susceptibility to head and neck squamous cell carcinoma (HNSCC) are still largely unknown. An imbalance between enzymes involved in the toxification and detoxification of (pre)-carcinogens closely related to HNSCC, which may appear during smoking and alcohol consumption, may play a role. Genetic polymorphisms in glutathione S-transferases (GSTs) often result in altered detoxification, which may contribute to individual susceptibility to HNSCC. METHODS: We studied the frequencies of polymorphic variants in the GSTP1 gene in 235 patients with HNSCC and 285 healthy controls. In addition, data on exposure to alcohol and tobacco consumption were recorded. DNA was extracted from whole blood, and polymerase chain reaction-based methods were used to detect genetic polymorphisms. RESULTS: In patients with HNSCC and control groups, the homozygous GSTP1 BB genotype was observed in 12.3% and 13.6%, respectively. No statistical differences were found for the GSTP1 AA and GSTP1 AB/GSTP1BB genotypes. CONCLUSIONS: Our study showed that genetic polymorphisms of GSTP1 are not associated with altered susceptibility to HNSCC.     

Gabaergic signaling mediates the morphological organization of astrocytes in the adult rat forebrain

Previous studies have provided evidence that the morphological organization of immature astrocytes is influenced by the inhibitory neuronal transmitter gamma amino-butyric acid (GABA). The present study was designed to determine whether the occurrence of differential organization of mature astrocytes throughout various regions of the adult brain is related to differential GABAergic signaling. For this we first used Western blotting and high-performance liquid chromatography to quantify the levels of the astrocytic protein glial fibrillary acidic protein (GFAP) and GABA, respectively, within the same tissue punches taken from different forebrain regions of the adult rat, as well as immunocytochemistry for GFAP, GABA, or glutamate decarboxylase to visualize the morphological organization of astrocytes and of GABAergic axons in these regions. These data indicate that GFAP and GABA contents are correlated throughout the different forebrain regions analyzed, and that the regions containing the highest densities in GABAergic terminals are those that contain astrocytes exhibiting the highest degree of stellation. Secondly, we chronically increased GABAergic signaling in vivo by the systemic administration of an inhibitor of GABA transaminase or by the intracerebroventricular infusion of muscimol, a potent agonist of GABA(A) receptors. Our data show that in both cases, the GFAP content of the different forebrain regions is significantly augmented, in close association with a marked increase in the number of astrocytic processes and with their degree of branching. Taken together, these data strongly suggest that GABAergic signaling mediates the morphological organization of astrocytes and their expression of GFAP in the adult brain.     

Neurochemical development of the brainstem catecholaminergic cell groups in rat

The postnatal development of tyrosine hydroxylase activity has been studied in the brainstem catecholaminergic cell groups (A1C1, A2C2, A5, A6, A7), involved in cardiorespiratory control. In rat, at birth and at postnatal days P3, P7, P14, P21 ant P68, we used a microdissection technique followed by in vivo measurement of the tyrosine hydroxylase (TH) activity, the rate-limiting enzyme in catecholamine synthesis. There is two successive marked increases in TH activity: at P3 in every catecholaminergic cell groups (A1C1, +225%; A2C2, +300%; A5, +190%; A6, +205% compared to birth) and during the third postnatal week with a peak of TH activity at P14 (A6, +90% above the P7 level) or at P21 (A1C1, +715%; caudal A2C2, +585%; rostral A2C2, +15%; A5, +445%; A7, +180% compared to P7). The data suggest the existence of two temporal windows during the neurochemical development of the catecholaminergic cell groups, which correspond to two metabolic transitions. The first one could be related to the intra-, extrauterine transition and the second one, to a deep energetic phase of maturation in the rat brain, closely related to the maturation of cardiorespiratory processes.     

Human SP-A and a pharmacy-grade porcine lung surfactant extract can be reconstituted into tubular myelin--a comparative structural study of alveolar surfactants using cryo-transmission electron microscopy

Cryo-transmission electron microscopy (cryo-TEM) is a rather artefact-free method, well suited to study the alveolar surfactant system. A pharmacy grade porcine lung surfactant extract (HL-10) was mixed with human SP-A and Ringer's solution (for calcium ions), and it was shown by cryo-TEM that the tubular myelin (TM) type of structure was reconstituted. These aggregates were associated to liposomal aggregates, and resulted in macroscopic phase-separation. This phase showed a weak birefringence in the polarising microscope, which is characteristic for a liquid-crystalline type of structure. TM from rabbit lung lavage was also examined, and showed the same periodic arrangement of bilayers as alveolar surface layer from freshly cut rabbit lungs deposited directly on the cryo-TEM grids. The distance between the bilayers of TM was 40-50 nm, and an electron dense material, assumed to be SP-A, was sometimes seen to occur periodically along the bilayers, oriented perpendicularly to the tubuli. The results are consistent with the surface-phase model of the alveolar lining.     

Total testing process: appropriateness in laboratory medicine

BACKGROUND: The need to reduce costs in Laboratory Medicine is often related to the possibility of reducing test requests without taking into account patients' outcomes. Therefore, the term "appropriateness" in Laboratory Medicine as referred to the specific steps (pre-analytical, analytical, post-analytical) and related to the clinical process could allow the improvement of clinical effectiveness and economic efficiency. METHODS: Our experience has shown an improvement in analytical appropriateness (reorganization and re-engineering by Laboratory automation) and pre-analytical appropriateness (critical revision of the panel for cardiac markers) by evaluating the workload and errors rate in the pre-analytical phase. RESULTS: We obtained an economic saving (119,580 euro/year) in cardiac markers request (analytical appropriateness: 60%, pre-analytical appropriateness: 40%) and also an improvement in clinical appropriateness (diagnosis and therapy). CONCLUSIONS: Our data confirm the need to improve communications between physicians and Laboratory Medicine as regards the pre-analytical step and to implement educational programs for defining criteria and procedures. Appropriateness in analytical and post-analytical steps contribute to achieve economic saving (Core lab, POCT) and improvement of the turn-around time (TAT).