In vitro apparatus for controlled release studies and intrinsic rate of permeation

The hydrodynamic characteristics of an in vitro apparatus for membrane moderated controlled drug release studies were investigated by measuring a dissolution rate from benzoic acid disk. An empirical correlation was developed for estimating the mass transfer coefficient. A simple method for correcting the effect of diffusion boundary layer on the rate of drug permeation was also described. The intrinsic rates of progesterone, testosterone and hydrocortisone through the silicone membrane were then evaluated by the correction method described.     

Serum dopamine beta-hydroxylase: assay and enzyme properties.

A method for the estimation of dopamine beta-hydroxylase activity in human serum is described, based on a thin layer chromatographic separation of the substrate ([14C]tyramine) from the reaction product ([14C]octopamine). The basic properties of the human serum enzyme, investigated by this method are described.     

A rapid gas-chromatographic method for the quantitative determination of clomethiazole in human serum

A rapid method without elaborate instrumentation for the determination of clomethiazole serum concentrations is described. This method is suitable for a routine clinical laboratory, and the results, from 37 patients investigated by this method which are presented, are comparable to results described by other authors. A relationship between dose ingested and serum concentration can be established. The determination of clomethiazole serum concentrations is useful as an adjunct to overall clinical assessment, and helpful in establishing a suitable dosage regimen of clomethiazole for the individual patient; it can also be used to verify suspected clomethiazole abuse or overdose.     

An ion-exchange assay for high molecular weight alkaline phosphatase.

An ion-exchange method for the measurement of a high molecular weight form of alkaline phosphatase in serum is described. The method is simple, rapid, precise and suitable for processing small batches of samples. Other forms of alkaline phosphatase commonly encountered in serum do not interfere. The correlation between results obtained by this method and those obtained by Sephadex 6B chromatography is discussed. Electrophoretic methods of measurement were also investigated but were found to be both imprecise and inaccurate.     

Serum water determination by means of microwave evaporation

A novel method is described for the determination of serum water using a microwave oven. The sources of experimental errors were analysed. Serum samples from two hundred patients were analysed for sodium, water and protein, and the data were used to calculate serum sodium molalities. A possible correlation was investigated between serum water content and protein concentration. The results were compared with those in the literature.     

Quantitative acylcarnitine profiling in fibroblasts using [U-13C] palmitic acid: an improved tool for the diagnosis of fatty acid oxidation defects

A method was developed for the investigation of mitochondrial fatty acid beta-oxidation in cultured fibroblasts. Monolayer cultures were incubated without foetal calf serum with commercially available [U-13C] palmitic acid and L-carnitine for 96 h. The acylcarnitines produced by the cells were extracted from the cell suspension and analysed either by quantitative stable isotope dilution gas chromatography chemical ionization mass spectrometry, or by fast atom bombardment mass spectrometry. Characteristic acylcarnitine profiles were obtained for all the different enzyme deficiencies investigated, with the exception of carnitine palmitoyltransferase II deficiency and carnitine/acylcarnitine carrier deficiency which showed similar patterns. Comparison between this method and the 3H-myristate and 3H-palmitate tritium release assays revealed that the method described here is superior, allowing unequivocal identification of patients.     

Determination of the rate of urea synthesis from serial measurements of plasma urea concentration after an alanine load: theoretical and methodological aspects

A method is described by which the rate of synthesis of urea can be calculated from the change of plasma concentration of urea after an alanine load. The results can be expressed in terms of f, the maximum increase in the rate of urea synthesis, and t, the time at which urea synthesis reaches its maximum. These parameters are calculated by an algebraic curve-fitting technique which is suitable for a desk computer. The method removes the need for isotopic analysis and urine collections. The effect of various errors and experimental conditions on the calculated synthesis parameters is investigated.     

AT1 receptor A1166C and AT2 receptor -1332A/G gene polymorphisms: efficient genotyping by single-tube PCR

Angiotensin II type 1 receptor (AT1) and angiotensin II type 2 receptor (AT2) genes have been investigated in recent years as potential etiologic candidates for cardiovascular and renal diseases. The pathogenic implications of AT1 A1166C and AT2 A-1332G gene polymorphisms have been shown. Here we describe a rapid and reliable method for detecting both AT1 and AT2 gene polymorphisms by a single-tube PCR, to reduce analysis time and simplify the genotyping procedure. In contrast to previously described methods, our method does not require hybridization, primer extension, or nested PCR for genotyping. In most previous studies concerning gene polymorphisms of RAS, both AT1 and AT2 receptor gene polymorphisms were investigated. The advantage of our method is that it makes it possible to detect both of these polymorphisms in a duplex PCR. The procedure described is convenient for routine laboratory use with manual sample processing, and offers the potential for further automation as well. Its simplicity makes it practical for large-scale screening of individuals and families at risk for cardiovascular or renal diseases.     

The quantitative determination of morphine in urine by gas-liquid chromatography and variations in excretion

A simple quantitative gas—liquid chromatography method for total morphine in urine is described. [¹⁴C] Morphine is used to correct for extraction losses. The precision and accuracy of the method have been investigated and the coefficient of variation over a period of six months was 5.6%. The percentage of the daily dose of diamorphine or morphine excreted in 37 terminal cancer patients varied from 20–112%. In one patient investigated for nine consecutive days the percentage varied from 40–124%. It is concluded that extreme caution should be exercised in drawing conclusions from results of morphine estimations on isolated urine specimens.     

ETHYLENE CHLOROHYDRIN DETERMINATION IN ETHYLENE OXIDE STERILIZED POLYVINYL CHLORIDE TUBING

A convenient rapid gas-chromatographic method is described for the quantitative determination of ethylene chlorohydrin. The method reported herein extracts the ethylene chlorohydrin with water. The method is simple and offers advantages since no elaborate and expensive gas extraction apparatus is required. The ethylene chlorohydrin levels in five different sterilized polyvinyl chloride samples were found to vary between 5 and 25 ppm. The effect of resterilization with ethylene oxide was investigated and was found to produce less ethylene chlorohydrin.     

Human peripheral blood and bone marrow cell separation using density gradient centrifugation on Lymphoprep and Percoll in haematological diseases

Density gradient centrifugation on Lymphoprep as described by Böyum [1] was used for mononuclear cell separation of peripheral blood (PB) and bone marrow (BM) samples in healthy subjects and in haematological diseases. The cells were analysed morphologically from cytocentrifuge preparations stained with May-Grünwald-Giemsa. At least 200 cells per preparation were counted manually. The separation results of PB samples in healthy subjects were comparable with those described by Böyum. This method was also suitable for the separation of lymphocytes and blast cells in chronic lymphoproliferative diseases and in acute leukaemias, respectively. In other haematological diseases, however, the mononuclear cell fractions of PB samples and especially of BM samples were contaminated with myeloid and erythroid cells. Percoll gradients were used with success for the enrichment of myeloid BM cells and PB reticulocytes. However, absolutely pure myeloid cell fractions from different maturation stages could not be separated. The results indicate that cells to be investigated should be morphologically examined, to avoid erroneous interpretation of biochemical and functional activity of the cells.     

Improved extraction procedure for determination of bile acids in faeces

A simplified extraction procedure for bile acids from wet faeces, using methanol/hydrochloric acid is described. Extracts were analyzed by gas-liquid chromatography, thin-layer chromatography and an enzymatic assay, with 3α-hydroxysteroid dehydrogenase.Recoveries of some stable bile acids, added to faeces, were studied; extraction efficiency was also investigated with a procedure using radioactive labelled bile acids given orally to patients.Resin treatment of faecal extracts, because of the sometimes dark colour of the extracts, resulted in a slightly lower recovery as determined by the enzymatic method.Recoveries were higher, using the proposed extraction procedure, than those obtained with extracts prepared by the standard procedure of Grundy et al [6].     

Human peripheral blood and bone marrow cell separation using density gradient centrifugation on Lymphoprep and Percoll in haematological diseases

Density gradient centrifugation on Lymphoprep as described by B?yum [1] was used for mononuclear cell separation of peripheral blood (PB) and bone marrow (BM) samples in healthy subjects and in haematological diseases. The cells were analysed morphologically from cytocentrifuge preparations stained with May-Grünwald-Giemsa. At least 200 cells per preparation were counted manually. The separation results of PB samples in healthy subjects were comparable with those described by B?yum. This method was also suitable for the separation of lymphocytes and blast cells in chronic lymphoproliferative diseases and in acute leukaemias, respectively. In other haematological diseases, however, the mononuclear cell fractions of PB samples and especially of BM samples were contaminated with myeloid and erythroid cells. Percoll gradients were used with success for the enrichment of myeloid BM cells and PB reticulocytes. However, absolutely pure myeloid cell fractions from different maturation stages could not be separated. The results indicate that cells to be investigated should be morphologically examined, to avoid erroneous interpretation of biochemical and functional activity of the cells.     

Enzymatic determination of acetate in serum or plasma using a centrifugal fast analyser

A simple enzymatic spectrophotometric micromethod is described for direct kinetic assay of acetate in serum or plasma using the Eni-Gemsaec centrifugal fast analyser. The method is based on the transformation of acetate and ATP into acetylphosphate and ADP by acetate kinase (EC 2.7.2.1). ADP is further measured by two coupling reactions involving pyruvate kinase (EC 2.7.1.40) and lactate dehydrogenase (EC 1.1.1.27) with measurement of NADH consumption at 340 nm.The method involves a reagent blank for compensation of reagent deterioration, a preincubation of 3 min without acetate kinase to eliminate any interference due to endogenous pyruvate, and a two-point kinetic protocol with measurements of absorbance at 95 s and 395 s. The analytical performances of the proposed method were investigated using an evaluation scheme proposed by the French Society of Clinical Biology.     

Determination of ascorbic acid in plasma and urine by high performance liquid chromatography with ultraviolet detection.

A reliable simple reversed-phase liquid chromatographic method for the routine determination of ascorbic acid in plasma and urine with ultraviolet detection is described. This method enables the complete separation of the ascorbic acid peak from others with a recovery of above 95% within 8 minutes. The method can be used for analysing multiple samples within a day. In addition, the storage conditions and stability of ascorbic acid in plasma and urine were investigated. Samples of plasma and urine can be stored on ice in darkness for at least 60 min without reduction of ascorbic acid concentration. Prepared samples can be stored in darkness at 4 degrees C for at least 120 min and in liquid nitrogen for 42 days.     

A micromethod for the estimation of free levels of anticonvulsant drugs in serum

A micromethod for estimating free levels of phenobarbitone, phenytoin and carbamazepine in patients' sera is described. Serum samples are subjected to a process of ultrafiltration, the filtrates treated with acetonitrile and the drug concentration quantified using high performance liquid chromatography. The stability of free levels in specimens before and after storage is investigated. The method is reproducible and mean recovery exceeds 98.5% showing that there is no significant absorption of drug onto the filters used. There is no interference from other substances normally present in patients' sera and there is a good correlation between results obtained by this method and a fluorescence polarisation immunoassay with correlation coefficient between 0.975 and 0.999. Serum samples can be stored for a lengthy period before ultrafiltration without adverse effects. The relevance of the method to patient care is discussed.     

Determination of Phenobarbital and Phenytoin in Serum by Ultraviolet Spectrophotometry

A modification of the method of Svensmark & Kristensen for the spectrophotometric determination of phenobarbital and phenytoin in serum has been described. By means of small alterations the method was made more suitable for routine use. It was shown that recovery of both drugs was 7–9 per cent higher when known amounts were added to water instead of serum. Some not previously described interfering drugs, ethoxybenzamid, trimethadione, sulthiame, sulphadimethoxine and sulphamethoxipyridazine, were investigated. It was shown that recording of the extinction curve in the range 220–300 nm offers a possibility to recognize interfering drugs, except other barbiturates. Phenytoin can only be determined by this method if interfering drugs or metabolites are not present, i.e. the intake of other drugs should be known not to interfere; otherwise the analysis must be carried out by a specific method (thin-layer chromatography).     

Sheep poxvirus identification from clinical specimens by PCR, cell culture, immunofluorescence and agar gel immunoprecipitation assay

Some 40 clinical specimens of skin lesions from sheep pox suspected cases were investigated by four different diagnostic assays: PCR, virus isolation in lamb testis cell cultures, direct immunofluorescent assay (DIFA) and antigen detecting agar gel immune precipitation test (AGIPT). All the specimens were positive by PCR and virus isolation, 29 were positive by DIFA and 16 by AGIPT. Using virus isolation on cell cultures as the gold standard, the PCR sensitivity was 100%, while that of DIFA and AGIPT was 73% and 40%, respectively. Skin samples with orf lesions or normal skin biopsies were PCR-negative. Cross-reactions with orf virus were observed in three samples only in the AGIPT assay. The PCR described combines high specificity and sensitivity with speed. PCR was therefore shown to be the method of choice for sheep poxvirus diagnosis directly from clinical specimens.     

Detection and typing of HSV-1, HSV-2, and VZV by a multiplex polymerase chain reaction

The development of a multiplex polymerase chain reaction method for the rapid and accurate detection and typing of HSV-1, HSV-2, and VZV from clinical specimens is described. A sensitive multiplex polymerase chain reaction was achieved by optimization of parameters such as the primers, magnesium, and dNTPs concentrations. False-negative results that sometimes arise due to inhibitors of DNA amplification or failure of DNA extraction procedure used may be avoided by assaying each specimen with alpha-tubulin primers. Multiplex PCR amplified viral sequences from all 55 specimens obtained from patients with clinical evidence of HSV or VZV infection indicated 100% sensitivity. From 55 patients who were investigated by multiplex PCR, HSV-1 was detected in 28, HSV-2 in 20, and VZV in 7 specimens. The reported results indicate that the present multiplex PCR assay has a potential application in clinical diagnosis when a rapid and accurate detection and typing of involved viruses HSV-1, HSV-2, or VZV is needed.     

细菌鉴定中鉴别试验的选择方法

目的 :选择最少的试验项目 ,获得最大的分离鉴定效果 ;方法 :根据 Gyllenberg的数学原理 ,编写计算机软件 ;结果 :从 2 2个试验中选择出 1 0个试验 ,即可鉴别 2 0种弧菌科细菌 ,达到最大的分离率 ;结论 :该方法是细菌鉴定中试验选择准确、快速而又实用的方法。