Sirolimus quantification by high-performance liquid chromatography with ultraviolet detection

The need to adapt optimal conditions of sirolimus blood level monitoring in laboratories led us to optimize an high-performance liquid chromatography-ultraviolet method and compare the elution performances using the mobile phase A, 68% MeOH/2% acetonitrile (ACN)/30% H(2)O and mobile phase B, 30% MeOH/42% ACN/28% H(2)O. Samples were assayed with 1-chlorobutane, redissolved in MeOH/water and injected onto a C-18 column at 50 degrees C. The assay achieved sensitivity of 2.5-150 ng/ml (CV = 10.6%) and recovery of 92-103.6%. The intra- and interassay precisions ranged from 3.3% to 13% and from 5.9% to 15% for quality controls of 7.5, 60 and 120 ng/ml. The mobile phase A was unable to elute and recover sirolimus and internal standard in the expected retention time and concentration. Under our working conditions, the assay was precise, accurate and sensible, stressing the importance of establishing for the best working conditions according to the staff and demands of the laboratory.     

超高效液相色谱法测定男性精浆中生物胺含量的研究

目的:建立一种超高效液相色谱测定人精浆中腐胺、亚精胺、精胺3种生物胺含量的方法. 方法:精浆样品用5％三氯乙酸提取,丹磺酰氯柱前衍生化,采用C18(2.1×50mm,1.7 μm)色谱柱,以水-乙腈为流动相梯度洗脱,流速为0.3 ml/min,检测波长为245 nm,柱温:35℃;进样量3.0μl.测定了捐精志愿者52...     

Determination of urinary oxalate by high-performance liquid chromatography monitoring with an ultraviolet detector

Summary High-performance liquid chromatography (HPLC) monitoring with an ultraviolet detector was carried out to measure urinary oxalate levels in urolithiasis. Interfering substances in urine were removed by anion exchange prior to chromatography. This procedure was found excellent with respect to sensitivity, reproducibility, and analytical recovery. The findings were in agreement with colorimetric date. The mean oxalate level in 24-hour urine was 30.5±15.1 mg in patients with a single episode and 36.3±9.8 mg in recurrent stone formers. The latter values was significantly higher than the normal control level (27.4±3.8 mg).     

Analysis of dentine glycosaminoglycans using high-performance liquid chromatography

Puppy dentine was prepared using ultracentrifugation of tooth powder in organic density gradients. The glycosaminoglycans of the obtained tissue fraction were prepared after papain digestion and beta-elimination, using preparative chromatography on DEAE-cellulose and CPC-cellulose. These polysaccharide fractions were analyzed using highly sensitive HPLC procedures. One such HPLC procedure allowed hyaluronic acid to be determined in less than microgram amounts. The glycosaminoglycans thus prepared consisted only of chondroitin-4-sulfate, chondroitin-6-sulfate, and small amounts of highly hybridized dermatan sulfate, while the experiments failed to demonstrate even trace amounts of keratan sulfate, hyaluronic acid or heparan sulfate.     

Significance of malondialdehyde concentration in seminal plasma of infertile men

Objective: To determine the concentration of malondialdehyde (MDA), product of lipid peroxidation, in seminal plasma and evalu ate its significance. Methods: Ninety-three cases of infertile pa tients were divided into the obstructive azoospermic (12 cases), the non-obstructive azoospermic (15 cases), the oligozoospermic (21 cases), the asthenozoospermic (19 cases), the oligoastheno zoospermic (16 cases) and the oligoasthenoteratozoospermic (10) groups. Eighteen fertile males served as the controls. MDA con centration was assessed by high-performance liquid chromatogra phy (HPLC). Results: With the exception of the obstructive azoospermic group, the MDA concentration in the seminal plasma was significantly different between the control group and the infer tile groups (P<0.01) as well as between the different infertile groups. Conclusion: Determination of MDA concentration in the seminal plasma is helpful to the diagnosis of male infertility induced by overproduction of reactive oxygen species.     

Cerebrospinal fluid concentrations of atracurium,laudanosine and vecuronium following clinical subarachnoid hemorrhage

BACKGROUND: Neuromuscular blocking agents may exert central nervous system effects when they reach the brain. This study assessed the concentrations and the time course of passage of vecuronium, atracurium, and its metabolite laudanosine in the cerebrospinal fluid (CSF) of patients undergoing intracranial aneurysm clipping. METHODS: Twenty-five patients with subarachnoid hemorrhage were randomly allocated to receive an intravenous infusion of vecuronium (n=13) or atracurium (n=12). Arterial blood and lumbar CSF were sampled before and 1, 2, 3, 4 and 8 h after the start of the relaxant infusion. The samples were analyzed by liquid chromatography-electrospray ionization mass spectrometry (vecuronium) and high-pressure liquid chromatography (atracurium and laudanosine). RESULTS: The data of 20 patients (10 in both groups) were analyzed. In 11 CSF samples from five patients atracurium was detected in concentrations from 10 to 50 ng/ml. Laudanosine was retrieved in all CSF samples at 1, 2, 3, 4 and 8 h; the highest CSF concentration of laudanosine occurred at 3 h [38 (18-63) ng/ml: median (range)]. Vecuronium was not found in any CSF sample. CONCLUSION: Significant concentrations of atracurium and laudanosine but not of vecuronium were detected in the CSF of patients during and immediately after intracranial aneurysm surgery.     

阿屈库铵对离体原代培养大鼠肝细胞的毒性研究

目的：研究阿屈库铵对离体鼠肝细胞的毒性作用。方法：采用离体鼠肝细胞培养方法，以培养基中乳酸脱氢酶（ＬＤＨ）释放量及肝细胞内Ｋ＋含量的变化为定量指标。结果：阿屈库按（５～５００μｇ／ｍｌ）使离体培养肝细胞ＬＤＨ净释放量增加，细胞内Ｋ＋含量明显减少（Ｐ＜０．０１）。结论：阿屈库铵对离体鼠肝细胞有毒性，并随浓度的增加而加重；用ＴＯＴＰ（ｔｒｉｏｒｔｈｏｔｏｌｙｌｐｈｏｓｐｈａｔｅ）抑制阿屈库铵水解降解途径的同时抑制了ａｃｒｙｌａｔｅｓ的灭活，加重其细胞毒性，表明阿屈库铵毒性的根源可能为ａｃｒｙｌａｔｅｓ。     

Significance of simultaneous determination of serum and seminal plasma alpha-tocopherol and retinol in infertile men by high-performance liquid chromatography

High-performance liquid chromatography was used for the simultaneous determination of alpha-tocopherol and retinol in serum and semen of 40 subfertile men. The serum levels of the two vitamins were significantly higher in serum than in semen (3- to 10-fold) (P < 0.001). The mean alpha-tocopherol levels were higher in the serum and semen of men with normal sperm parameters (20 +/- 5 and 5 +/- 4 mumol L-1) than those with oligozoospermia (10 +/- 4 and 3 +/- 2 mumol L-1), azoospermia (8 +/- 3 and 3 +/- 1 mumol L-1) and asthenozoospermia (9 +/- 6 and 3 +/- 2 mumol L-1) (P < 0.002). Mean retinol levels in serum were similar in men with normal sperm parameters (2.4 +/- 2 mumol L-1) as in those with defective sperm parameters such as oligozoospermia (2.5 +/- 2 mumol L-) and asthenozoospermia (2.1 +/- 1.0 mumol L-) (P = 0.15), but significantly lower in men with azoospermia (1.3 +/- 0.3 mumol L-1) (P < 0.05). The alpha-tocopherol:retinol ratio was significantly higher in semen than in serum of men with normal sperm parameters (11.5) and those with asthenozoospermia (10.3) compared with ratios in those with oligozoospermia (8.3) and azoospermia (6.3). This may be related to reduced antioxidant activity in sperm dysfunction as a result of lipid peroxidation, from increased generation of reactive oxygen species.     

Inactivation of atracurium in human and rat plasma

The contribution of enzyme-catalyzed hydrolysis to inactivation of atracurium in human and rat plasma was determined in vitro by inhibiting the enzyme carboxylesterase with triorthotolyl phosphate. The inhibitor was removed by centrifugation and aspiration. Atracurium was then added to both the control and the inhibited plasma samples, and all samples were incubated at 37 degrees C for 45 min. The amount of atracurium present in aliquots of plasma was determined using a bioassay technique in anesthetized rats. Inactivation of atracurium proceeded rapidly in control rat plasma but was markedly slowed in samples treated with the inhibitor of carboxylesterase. In contrast, the inactivation was slow in control human plasma, and inhibition of carboxylesterase produced only marginal, if any, effects. We conclude that the inactivation of atracurium proceeds, in part, by enzyme-catalyzed ester hydrolysis. In species with high enzyme activity in plasma, e.g., the rat, enzyme-catalyzed hydrolysis is clearly evident. In humans, the level of enzyme activity is low and the contribution of enzyme-catalyzed inactivation is less manifest. By exclusion, Hofmann elimination or other reactions probably represent the major inactivation pathway in humans.     

阿曲库铵对兔心内传导系统的影响

目的 观察阿曲库铵对心内传导系统的影响。方法 选择家兔10只，阿曲库铵0.20mg/kg静脉推注，约30秒推完。测定用药前，后心率（HR），窦房结传导时间（SACT），最在窦房结恢复时间9SNRTMAX），矫正窦房结恢复时间（CSNRT），窦房结恢复时间指数（SNRTI），文氏A-V传导频率（WB），P-R间期和QRS波。结果 用药前后上述测定结果均无显著性差异。结论 阿曲库铵0.20mg/kg静脉注射对兔心内传导系统无显著影响。     

Resolution of proteins in the kidney stone matrix using high-performance liquid chromatography

The organic matrix accounts for 2-3% of the total stone weight and has been considered to play a role in stone formation and growth. Thus far, fractionation of the matrix proteins has been insufficient due to low resolution and reproducibility. In this report the matrix proteins of 22 stones were resolved by means of high-performance liquid chromatography. Following pulverization, the organic matrix was obtained by dialysis against EDTA. The average content of nondialyzable extractable proteins was 1.6% of the total stone weight. Analysis of the matrix proteins with high-performance gel permeation liquid chromatography and high-performance ion-exchange liquid chromatography has indicated that the protein composition of the stone matrix is identical regardless of the mineral composition. The major component of the matrix proteins was identified as glycoprotein and/or proteoglycan from their absorption to a concanavalin A Sepharose column. Higher molecular weight matrix proteins seem to be polymers or condensation products, since they have been degraded into lower molecular weight subfractions by sodium dodecyl sulfate treatment.     

Analysis of lipid peroxidative levels in seminal plasma of infertile men by high-performance liquid chromatography

For the purpose to evaluate the significance of lipid peroxidative products on male infertility, the levels of malondialdehyde (MDA), which is one of the final products of lipid peroxidation in seminal plasma, were determined. Ninety-three male infertile patients were divided into obstructive azoospermic group (12 cases), non-obstructive azoospermic group (15 cases), oligozoospermic group (21 cases), asthenozoospermic group (19 cases), oligoasthenozoospermic group (16 cases) and oligoasthenoteratozoospermic group (10 cases). Eighteen fertile males were included in the control group. MDA concentrations of seminal plasma in the fertile and infertile men were detected by high-performance liquid chromatography (HPLC). The results showed that the concentration of MDA in seminal plasma differed significantly between the control group and all the infertile groups (P < 0.01) except the obstructive azoospermic group, between the oligoasthenozoospermic group and the oligozoospermic and asthenozoospermic groups (P < 0.01), and between the oligoasthenoteratozoospermic group and the oligozoospermic and asthenozoospermic groups (P < 0.01). MDA concentration of seminal plasma in the oligoasthenoteratozoospermic group differed significantly from that in the oligoasthenozoospermic group (P <0.05). The results suggested that detection of MDA concentrations in seminal plasma by HPLC has an indicative value on the diagnosis of male infertility induced by overproduction of reactive oxygen species in male reproductive system.     

中国人遗传性非息肉病性结直肠癌家系错配修复基因突变的研究

目的探讨中国人遗传性非息肉病性结直肠癌(HNPCC)家系中hMLH1、hMSH2基因遗传性突变情况。方法取14个符合中国人HNPCC标准的HNPCC家系肿瘤先证者外周血DNA样本,聚合酶链反应扩增hMLH1、hMSH2基因共35个外显子,应用变性高效液相色谱技术(DHPLc)结合DNA测序法检测突变。结果14个家系中共发生41个大肠癌和19个肠外恶性肿瘤,其中胃癌是最常见的肠外恶性肿瘤类型。14个患者中检测到分属于9个家系的12个遗传性单个碱基改变,其中8％为无义突变,25％为错义突变,其余42％为单核苷酸多态,17％为内含子区的单碱基改变,8％为同义突变。结论(1)应用DHPLc成功检测到hMLH1、hMSH2基因杂合性突变。(2)符合中国人HNPCC标准家系约有1／3可检出hMLH1、hMSH2基因遗传杂合性突变,其中错义突变较多见。     

Selective isolation of acrosome-reacted human spermatozoa with progressive motility by using cell affinity chromatography on Concanavalin A Sepharose

Summary. In order to ensure fertility, mammalian spermatozoa have to undergo acrosome reaction, the most obvious morphological change during this being the exposure of the inner acrosomal membrane. In the present study, the acrosome-reacted human spermatozoa were successfully separated without loss of viability by using cell affinity chromatography on Concanavalin A (Con A) Sepharose. Con A demonstrated affinity for both the intact and the acrosome-reacted spermatozoa regardless of their viability; the latter, however, gave higher affinity than the former against Con A. Prior to the column chromatography, the immotile spermatozoa and the seminal plasma were excluded by means of a modified swim-down procedure and the resulting spermatozoa were subsequently immobilized by slow rate cooling in ice-cold water. Cell affinity chromatography was performed at 4 °C. To prevent mechanical trapping of the spermatozoa among the packed gel beads, the column was interconnected with a reservoir, the vertical drive of which was allowed to lose the gel bed and thereby release the trapped spermatozoa. Stepwise competitive elution with 5.0 μM mannose and 25% heat-inactivated human serum was capable of separating the intact spermatozoa and the acrosome-reacted spermatozoa from each other. The acrosome reaction rate of sperm fraction which was adsorbed to Con A Sepharose and eluted with 25% serum was found to be 83±2.3%, and motility and viability of these fractions were measured to be 80±6.3% and 83±7.6%, respectively ( n = 8, mean±SD). The status of the acrosome in a final preparation (motility 92%, acrosome reaction rate 88%) was observed by scanning electron microscopy, and 81% spermatozoa lost their acrosome cap.
Acrosome-reacted spermatozoa—     

Method for simultaneous determination of creatine phosphate and adenine nucleotides in the intestinal smooth muscle of guinea-pig taenia caeci using high-performance liquid chromatography

A method for simultaneous determination of adenosine 5'-triphosphate (ATP), adenosine 5'-diphosphate (ADP) and creatine phosphate (PCr) by high-performance liquid chromatography is described. This method was applied to the isolated intestinal smooth muscle tissue of guinea pig taenia caeci weighing approximately 30 mg. It was found that one g of the muscle tissue contained 3.55 mumol PCr, 2.40 mumol ATP and 0.477 mumol ADP.     

HPLC-ELSD法测定尿液中甘露醇和乳果糖浓度

目的:建立甘露醇和乳果糖的高效液相色谱检测方法.方法:采用高效液相色谱-蒸发光散射检测器分析法(HPLC-ELSD),以对照品为外标,检测6例正常人和6例腹水患者尿液标本中甘露醇和乳果糖浓度.结果:该方法甘露醇和乳果糖能得到良好分离.甘露醇和乳果糖的线性范围分别为20～960 mg/L(r=0.997 3)和25～800 mg/L(r=0.996 2);甘露醇和乳果糖日内精密度(RSD)分别为1.5%和1.3%(n=6);甘露醇的加样回收率在93.0%～98.8%之间,乳果糖的加样回收率在93.3%～98.9%之间.本方法测定甘露醇的最大检出限为200 ng,乳果糖为250 ng.腹水患者的肠道通透性(乳果糖排泄率/甘露醇排泄率:0.400±0.340)较正常人(乳果糖排泄率/甘露醇排泄率:0.025±0.005)显著增高.结论:HPLC-ELSD检测肠道通透性的准确度高、重复性好,样品前处理简单,是临床评价小肠黏膜屏障功能的实用方法.     

Effects of etomidate on free intracellular amino acid concentrations in polymorphonuclear leucocytes in vitro

BACKGROUND: Previous studies have shown the inhibitory effects of etomidate on polymorphonuclear leucocyte (PMN) function. No reports exist, however, regarding free intracellular amino acid metabolism, although physiological cell metabolism and basic cell functions rely upon a balanced intracellular amino acid content and the cell membrane-mediated separation of cellular amino acids from the extracellular plasma amino acid pool. Thus, in the current study, we evaluated the effects of etomidate on free intracellular amino acid metabolism in PMN. METHODS: With ethics committee approval, blood was withdrawn from 35 healthy volunteers and incubated (1 h) either with 0 microg/ml, 0.0156 microg/ml, 0.0625 microg/ml or 0.5 microg/ml of etomidate as well as with its additives (propylene glycol and Lipofundin MCT 10%). The PMN were separated using standardized Percoll-gradient and centrifugation procedure before deep-freezing and lyophilization techniques were employed. All PMN samples were dissolved in methanol/H2O, and the concentrations of free intracellular amino acids were monitored using both novel advanced PMN-separation and high-performance liquid chromatography techniques. RESULTS: Etomidate influenced important free amino acid profiles in PMN in a dose-dependent manner, indicating complex changes of cellular amino acid turnover. Neither propylene glycol nor Lipofundin MCT 10% changed free amino acid concentrations in PMN. CONCLUSIONS: For the first time, the effects of etomidate on free intracellular amino acid metabolism in PMN have been investigated. Our results draw attention to the biochemical pathways which may be involved in etomidate-induced alterations in PMN function and cellular immunocompetence.