Lipidex chromatography in the radioimmunoassay of serum and urinary cortisol.

A highly specific method for the determination of cortisol in human serum and urine is described. The sample is first extracted with diethyl ether/ethyl acetate (1 : 1, by vol.), then chromatographed on a highly lipophilic derivative of Sephadex (hydroxyalkoxypropyl Sephadex, Lipidex) in light petroleum/chloroform (1 : 1, by vol.), and finally cortisol is measured by radioimmunoassay using a cortisol-21-BSA antiserum. Bound and unbound radioactivities are separated using dextran-coated charcoal technique. The 8 a.m. values (mean +/- S.D.) of cortisol among 11 young females and 16 young males were 152 +/- 32 ng/ml (range 111-235) and 185 +/- 21 ng/ml (103-232), respectively. The respective values at 4 p.m. were 84 +/- 29 ng/ml (26-117) and 84 +/- 36 ng/ml (32-172). The importance of Lipidex chromatography was demonstrated with assays of serum samples from children with congenital adrenal hyperplasia; without chromatography cortisol values were 6 times those with chromatography. Specific cortisol assays from pregnancy serum also necessitated Lipidex chromatography. Among 11 young men and 9 young women the mean daily urinary cortisol excretion was 56 +/- 26 mug (32-109) and 60 +/- 24 mug (39-109), respectively. Specific urinary cortisol determination could not be achieved without Lipidex chromatography.     

Congenital adrenal hyperplasia. Plasma and urinary steroid conjugates in seven children with steroid 21-hydroxylase deficiency

Plasma neutral steroid sulphates and urinary neutral steroid sulphates and glucuronides from seven children (0.17–10.4 years) with steroid 21-hydroxylase deficiency were determined using gas-liquid chromatography and gas chromatography -mass spectrometry. Three of the patients were salt-losers. Large inter-individual variation in plasma concentration and urinary excretion of these compounds was observed. However, the group did have certain characteristic features. In plasma, the main compounds present were 3β-hydroxy-5-ene steroids. A progesterone metabolite, 5α-pregnane-3β,20α-diol, and a 17α-hydroxyprogesterone metabolite, 5β-pregnane-3α, 17α, 20α-triol, were present as sulphate conjugates and 3α, 17α-dihydroxy-5β-pregnan-20-one sulphate was identified for the first time in human peripheral plasma. In the urine, metabolites of 17α-hydroxyprogesterone were predominant and 5β-pregnane-3α, 17α,20α-triol, excreted mainly as a glucuronide, alone comprised about 50% of the neutral steroid excretion in these subjects. The next most abundant steroid was 3α,17α,20α-trihydroxy-5β-pregnan-11-one. The following compounds have not previously been found in the urine of patients with steroid 21-hydroxylase deficiency but were found in the present subjects: 5-androstene-3β, 17β-diol, 5α-pregnane-3β, 20α-diol and 3β,17α-dihydroxy-5β-pregnan-20-one. The pattern of the plasma and urinary steroids determined clearly differentiates the subjects with a steroid 21-hydroxylase defect from normal subjects and from patients with a 3β-hydroxysteroid dehydrogenase deficiency.     

The early recognition of the 21-hydroxylase deficiency variety of congenital adrenal hyperplasia

The urinary steroids excreted by three newborn infants with 21-hydroxylase deficiency and by 15 healthy newborns aged two days have been compared after analysis by gas liquid chromatography (GLC). The identity of each steroid was carefully checked by gas chromatography/mass spectroscopy (GC-MS). The enzyme deficiency leads to the elevated excretion of urinary precursor metabolites, mainly 3α,17α,20α-trihydroxy-5β-pregnan, 3α,17α,20α-trihydroxy-5β-pregnan-11-one and 3α,17α-dihydroxy-5β-pregnan-20-one.In the search for a quick and firm confirmation of suspected 21-hydroxylase deficiency in a newborn baby by means of a GLC-profile of urinary steroids, most attention has up to now been paid to 3α,17α,20α-trihydroxy-5β-pregnan. However, 3α,17α-dihydroxy-5β-pregnan-20-one is a better indicator, as it enables one to confirm the existence of this disease soon after birth directly from the GLC-profile without further analyses by GC-MS.     

A semiautomated method for the determination of 11-deoxy-17-oxo-steroids in urine

A semiautomated method is described for the determination of total 11-deoxy-17-oxo-steroids (11-DOOS: androsterone, etiocholanolone plus dehydroepiandrosterone) in urine.

Urinary conjugates are manually extracted on a XAD-2 resin, hydrolysed by (β-glucuronidase and “solvolysed” in acid ethyl-acetate according to Burnstein-Lieberman. The free 11-DOOS are extracted automatically with iso-octane and estimated colorimetrically by the Zimmerman reaction in an Auto-Analyzer II system.

The method was evaluated by investigation of its precision, accuracy, sensitivity and specificity. It was found to be satisfactory for the rapid and reliable screening of large numbers of urine samples.     

The effect of ibuprofen on the excretion of steroid metabolites

An inexpensive gas Chromatographic method is described that allows simultaneous measurement in urine of androsterone (A), aetiocholanolone (E), 11-hydroxyandrosterone (11-OA), 11-hydroxyaetiocholanolone (11-OE), pregnanediol (PD), pregnanetriol (PT), tetrahydrocortisone (THE) and tetrahydrocortisol (THF). Dehydroepiandrosterone was also resolved by the column.Ibuprofen was administered to five healthy normal males at a dose used therapeutically in rheumatoid arthritis (RA). The above urinary steroids were measured weekly during a control period, during a four week period of drug treatment and for four weeks after drug treatment had ceased.The excretion of A fell to a mean of 63% of the control value (P < 0.02) and returned to the control value within two weeks. 11-OA, which showed a greater variability than A, fell to the same extent (P < 0.1). No other steroid measured showed a change that could be related to the drug.     

Interferences in the radioimmunological determination of urinary free cortisol

The magnitude of interferences arising in the radioimmunological assessment of urinary free cortisol is studied (a) by comparing cortisol immunoreactivities from crude urine, after organic solvent extraction of different selectivity and after additional chromatography by high pressure liquid chromatography (HPLC) and (b) by evaluation of the profile of immunoreactivity resulting from the fractions eluted by HPLC. Three antisera from different sources have been investigated. Values of cortisol-immunoreactivity in crude urine were about six times and values of a simple dichloromethane extract about three times higher than values obtained after HPLC. The main part of the interfering compounds arising in organic extracts have a polarity similar to cortisol, which cannot be easily eliminated by simple solvent extraction procedures. Specific estimation of urinary cortisol by radioimmunoassay requires a preceding Chromatographic technique of high efficiency, such as HPLC, which represents an adequate tool for the routine laboratory.     

An assay for human erythrocyte catechol-O-methyltransferase activity using a catechol estrogen as the substrate.

A radiometric assay for catechol-O-methyltransferase (COMT) activity in human erythrocytes is described that employs 2-hydroxy[3H]estrone, and non-radiolabeled S-adenosylmethionine (SAM) as the cosubstrates. The ease of separation of the product of the reaction, 2-methoxy[3H]estrone from 2-hydroxy[3H]estrone makes it possible to achieve low reaction blanks. The assay is very sensitive, and only 200 microliter of whole blood are used per determination. The assay is highly reproducible. The interassay variability (coefficient of variation) was 6.5% for 24 assays of COMT activity in red blood cells in blood obtained daily for 24 days from one person. In incubations conducted at 37 degrees C for 30 min, the catechol-O-methyltransferase activity was a linear function of enzyme concentration (equivalent to 11 to 180 microliter of packed red blood cells). Employing this assay, we evaluated the catalytic conversion of 2-hydroxyestrone to 2-methoxyestrone by catechol-O-methyltransferase from human red blood cells and found that the apparent Michaelis constant and the apparent maximal rate of reaction were 3 x 10(-7) M and 6.7 x 10(-9) mol . ml-1 erythrocytes . h-1, respectively. The catechol-O-methyltransferase activity measured in erythrocytes obtained from 100 healthy subjects (men and nonpregnant women) was 8.2 +/- 0.17 (mean +/- S.E.) nmol 2-methoxyestrone . ml-1 erythrocytes . h-1.     

The effect of glycosaminoglycans on the in vitro activity of human skin fibroblast glycosphingolipid beta-galactosidases and neuraminidases.

The apparent low level of synthetic substrate beta-D-galactosidase activity in liver from patients with the Hurler-Hunter syndrome is caused by the inhibitory effect of accumulated glycosaminoglycans. We have demonstrated complete inhibition of GM1 ganglioside beta-galactosidase activity in vitro by both heparan sulfate and dermatan sulfate, but the effect on lactosylceramide and galactosylceramide hydrolysis was less marked. In contrast, lysosomal neuraminidase activity in vitro was enhanced by the addition of glycosaminoglycans. These observations are discussed in relationship to the observed storage pattern of glycosphingolipids in liver from patients with mucopolysaccharidoses.     

Relative competition of corticosterone, cortisol, cortisone, 11-dexycortisol and prednisolone with (1,2-3H)-cortisol in various protein-binding radioassay systems.

The relative influence of some endogenous corticosteroids and of synthetic prednisolone on competitive protein-binding radioassay was compared with that of cortisol, using as a source of transcortin pooled plasmas from various species and at different dilutions. Human (different clinical situations), cow, dog, sheep, hog, rabbit and chicken plasma were examined. The ability of corticosterone, cortisone, 11-deoxycortisol and prednisolone to displace [1,2-3-H] cortisol from corticosteroid-binding globulin (CBG) was measured: (1) by assessing the amounts (ng/tube) which produce a displacement equal to 10 ng/tube of cortisol, and (2) by calculating the integrated areas of displacement defined by the binding curves and by expressing them as a percentage of the cortisol curve area. While corticosterone, 11-deoxycortisol and prednisolone have a binding potency not very different from that of cortisol in almost the entire set of competitive protein-binding assays tested, the binding ability of cortisone was found to be particularly dependent upon the species of diluted plasma. Differences in the relative specificity of binding are apparent also within the human species, depending on the source of diluted plasma, whereas the concentration of endogenous steroids does not seem to significantly affect binding curves under the conditions examined. It is suggested that binding proteins in various conditions differ from those of healthy adults not only quantitatively but also qualitatively.     

浙江省衢州市应用聚合酶链反应检测钩端螺旋体效果评价

目的 建立聚合酶链反应(PCR)检测钩端螺旋体的快速方法,应用于宿主动物钩端螺旋体的快速检测,同时比较PCR方法和常规检测的效果.方法 根据中国疾病预防控制中心提供的引物,以致病性大肠杆菌、产毒性大肠杆菌、溶藻弧菌、沙门菌、霍乱弧菌、痢疾志贺菌、非O1、O139霍乱弧菌、副溶血性弧菌、肠出血性大肠杆菌O157:H7为对照,建立PCR检测钩端螺旋体的快速方法,用于钩端螺旋体的快速检测.结果 衢州市2006年分离钩体菌株进行PCR鉴定结果与血清学鉴定结果相符.100只鼠和100只蛙肝、脾、肾标本进行PCR检测,PCR检测阳性率10%,钩体菌株培养分离率仅为1%,二者差异有统计学意义(2=16.05,P0.005).结论 PCR检测钩端螺旋体灵敏度高、特异性强,可用于钩端螺旋体的快速检测.     

BG、CD4T淋巴细胞联合检测在早期诊断艾滋病患者肺部侵袭性真菌感染的临床意义

目的：分别测定（1-3）-β-D-葡聚糖（BG）、CD4T 淋巴细胞的水平,以早期诊断艾滋患者肺部侵袭性真菌感染（IPFI）。方法根据合格的下呼吸道标本真菌培养结果选择153例有肺部感染症状并已确诊的艾滋病患者,分为肺部侵袭性真菌感染阳性组63例和肺部侵袭性真菌感染阴性组90例;然后检测肺部侵袭性真菌感染阳性组63例、肺部侵袭性真菌感染阴性组90例和50例健康对照组血浆中的 BG,同时检测各组的 CD4T 淋巴细胞数量,各组同一项目两两比较,进行统计学分析。结果肺部侵袭性真菌感染阳性组 BG 均值为（56.30±15.38）pg／mL,CD4T 淋巴细胞均值为（56±41）／μL;肺部侵袭性真菌感染阴性组 BG 均值为（21.32±14.26）pg／mL,CD4T 淋巴细胞均值为（200±53）／μL;健康对照者组 BG 均值为（2.89±1.55）pg／mL,CD4淋巴细胞均值为（480±89）／μL。3组中 BG 水平与 CD4T 淋巴细胞两两比较,两者进行 t 检验,差异均有统计学意义（P ＜0.01）。结论联合检测 BG、CD4淋巴细胞对艾滋病患者 IPFI 感染具有明显的早期诊断价值。     

肺结核合并感染患者3项指标联合检测的临床应用

目的：探讨肺结核合并细菌、真菌感染患者血浆内毒素、降钙素原（PCT）及（1,3）-β-D 葡聚糖（BG）水平检测的临床意义。方法回顾性分析该院2013年3月至2013年12月结核病防治所收治的肺结核确诊病例240例临床资料,根据细菌培养结果将患者分为革兰阴性（G－）菌生长组39例、革兰阳性（G＋）菌（包括真菌）生长组45例、无菌生长组156例,另选该院健康体检中心体检健康者48例纳入健康对照组。比较各组患者的血浆内毒素、PCT 及 BG 水平变化。结果G－菌生长组内毒素水平为（0．682±0．418）EU／mL、PCT 水平为（2．93±0．87）μg／L,明显高于 G＋菌（包括真菌）生长组的（0．063±0．034）EU／mL 及（0．85±0．52）μg／L,比较差异有统计学意义（P ＜0．05）。G－菌生长组内毒素、PCT 水平明显高于无菌生长组（P ＜0．05）。健康对照组与无菌生长组内毒素、PCT、BG 水平比较差异无统计学意义（P ＞0．05）,而与其他组比较差异有统计学意义（P ＜0．05）。结论血浆内毒素、PCT 及 BG 水平检测具有快速、敏感的特点,联合检测有助于快速判断肺结核患者是否合并 G－菌、真菌感染,对指导临床合理用药、及时对症治疗具有重要意义。     

结直肠癌Septin9基因甲基化检测质控品制备及其应用

目的制备可模拟临床标本的血浆Septin9基因甲基化(mSEPT9)质控品并用于室间质量评价。方法选择Septin9区域已发生/未发生甲基化的HeLa/Jurkat细胞进行培养,收集并抽提细胞基因组DNA,经mSEPT9试剂盒检测,依据检测Ct值用阴性血浆稀释分装成含有不同浓度的质控品,对质控品均匀性和稳定性进行评价后,将5个样品随机编号后发送至参评实验室,分析评价回报结果。结果抽提得到的细胞基因组DNA纯度和浓度均可满足使用需求,且可用作mSEPT9检测的质控品。均匀性和稳定性均符合中国合格评定国家认可委员会对能力验证样品要求。部分参评实验室出现假阳性和假阴性情况,仅有约55.6%实验室的检测结果(Ct值)呈良好线性相关。9家参评实验室中,7家实验室(77.8%)成绩优秀,1家实验室(11.1%)成绩合格,1家实验室(11.1%)成绩不合格。结论成功研制血浆mSEPT9检测的质控品并应用于室间质评,有利于评估并提升临床实验室检测能力。     

Use of a sensitive enzyme immunoassay for human thyrotropin in the diagnosis of hyperthyroidism

A sensitive, simultaneous sandwich enzyme immunoassay for TSH was evaluated especially for its ability to distinguish hyperthyroid patients from the euthyroid population. A total of 140 patient samples was analzyed by this assay as well as with a two-step sandwich radioimmunoassay. The diagnostic sensitivity of the thyrotropin assay was 92.5% and the specificity was 88%. False negatives by thyrotropin assay included two patients with Graves' disease who were being treated with propranolol at the time of testing and one patient who was considered hyperthyroid while receiving synthroid. Twelve patients with elevated free thyroxine index levels were considered euthyroid and 50% of these had thyrotropin values that were undetectable; most were elderly patients with nonthyroidal illnesses. Although the thyrotropin enzyme immunoassay had good sensitivity and precision for the detection of hyperthyroidism, our data suggest the limitation of a single thyrotropin determination in establishing the euthyroid state, especially in elderly patients with associated nonthyroidal illnesses and hyperthyroxinemia.     

A new biodegradable pasty-type copolymer of l-lactic acid and δ-valerolactone with relatively low molecular weight for application in drug delivery systems

Copolymers of l-lactic acid (LA) and δ-valerolactone (VL) with a number-average molecular weight of 1500–2600 were developed as biodegradable carriers for drug delivery. Copolymers were synthesized by direct copolycondensation in the absence of a catalyst. Depending on the mol% of l-lactic acid residues, three different morphologies are obtained: solid state (100-80 mol%), pasty state (70-30 mol%), and waxy state (15-0 mol%). The degradation of the copolymer was markedly influenced by Rhizopus delemer lipase. The rate of degradation of the pasty copolymer in the presence of the lipase is much faster than that for the solid or the waxy Copolymers. A nitrogen mustard derivative of estradiol-17β, Estramustine, was incorporated into a pasty-type copoly(LA/VL, 70/30 mol%) with . The system was completely degraded in vivo (rat) 5 weeks after insertion. A pharmacological effect on the prostates of rats was observed during a period of 5 weeks.     

320排CT在百草枯中毒致兔早期肾损伤中的诊断价值

目的 通过320排CT灌注扫描观察兔百草枯中毒早期肾脏血流动力学变化,根据其灌注参数指标评估肾损伤的程度.方法 24只新西兰白兔,随机分为百草枯组12只,对照组12只.百草枯组一次性腹腔注射35 mg/kg剂量百草枯溶液,对照组腹腔注射等体积生理盐水.两组均于注射后2h、12 h及24h行320排CT灌注扫描以获取灌注图像、肾脏血流量(renal blood flow,RBF)与肾脏血容量(renal blood volume,RBV)的灌注参数值;各时间点耳缘静脉采血2mL检测血清肌酐(sCr)与尿素氮(BUN).结果 百草枯组:sCr、BUN于注射百草枯后2h较对照组无明显变化、12 h略有升高,但差异无统计学意义(P＞0.05);而百草枯组24h时检测sCr、BUN升高明显,差异有统计学意义(P＜0.05或P＜0.01);RBF、RBV于注射百草枯后2h较对照组无明显变化,12 h及24h均明显降低,差异均有统计学意义(P＜0.01).RBF、RBV较sCr、BUN能更早地反映肾脏的损伤程度.光镜下,百草枯组病理改变有肾小球毛细血管内皮细胞充血水肿、空泡变性,近端小管上皮细胞肿胀、坏死等;对照组肾小球及肾小管结构清晰,未见细胞充血水肿、变性等病理改变.结论 320排CT可早期评估百草枯中毒肾损伤的程度.     

Development of a multiple-run high-resolution melting assay for Salmonella spp. genotyping HRM application for Salmonella spp. subtyping

A quick and robust Salmonella spp. differentiation method based on high-resolution DNA melting (HRM) was developed. DNA samples from 134 Salmonella spp. strains and 20 serotypes were tested. Each serotype was represented by at least 2 strains. All raw data were derived on the Rotor-Gene 65H0-100 system using the designed 8 primer pairs. The reference samples for HRM error evaluation between runs were applied. Raw data error minimization and fluorescence normalization between runs were carried out by application of the proposed calculations. The data analysis showed that repetitive sequence targets are much more informative than the nonrepetitive ones. The method possesses a high potential and can be adopted for further subtyping analyses.     

Assessment of the EMIT-(TM) technique as a screening test for opiates and methadone for a methadone maintenance clinic and its calibration by Bayesian statistics.

1. The performance of the semi-quantitative enzyme multiplied immunoassay technique (EMIT, Syva Corp.) for opiates and methadone has been examined as a screening procedure of urine samples from a methadone maintenance programme. The predictive value model that is based upon Bayesian statistics was used to determine screening levels for the EMIT assays. 2. With a predictive value of a negative result of 100%, the EMIT opiate assay can be used to show the absence of the indicated drugs, while positive results can be confirmed by a non-immunological technique. A screening level of 1.0 micrograms/ml for the opiate assay, conforms to this model. 3. The EMIT methadone assay was shown to have a predictive value of a negative result of 28% with respect to thin-layer chromatographic (TLC) results. This discrepancy between EMIT and TLC can not be explained by sensitivity alone. The manufacturer's recommended 0.5 micrograms/ml cutoff has been used therefore for the methadone assay.     

高仿真模拟人在大学生心肺复苏术培训中的应用效果研究

[目的]评价高仿真模拟人在大学生心肺复苏术培训中的应用效果。[方法]对113名非医学专业大一本科生进行心肺复苏教学,随机分为实验组(高仿真模拟人)56名和对照组(传统模拟人)57名,培训结束即刻和1个月后进行理论和操作测试,并用视觉模拟评分(VAS)评估急救自信心和教学满意度。[结果]培训结束即刻,两组理论成绩相近,但是实验组胸外按压(t=30.49,P0.01)和人工呼吸(t=14.97,P0.01)成绩明显优于对照组,此外,实验组学生表现出更强的急救信心(t=30.49,P0.01),且对教学效果更为满意(t=13.23,P0.01),1个月后实验组理论知识和人工呼吸技能保留较好,而对照组理论和操作成绩均下降显著。[结论]采用高仿真模拟人进行大学生心肺复苏术教学培训可有效提高学生操作能力。