Puromycin-sensitive alanyl aminopeptidase from human liver cytosol: purification and characterization

A cytosolic alanyl aminopeptidase (AAP-S) was purified to homogeneity from human liver cytosol. The molecular weight of the purified enzyme was calculated to be approximately 98,000 on TOF-MS and 90,000 on SDS-PAGE in the presence of beta-ME. These findings suggest that the enzyme exists as a monomeric form in human liver cytosol. The enzyme rapidly hydrolyzed the substrates Ala-, Lys- and Phe-MCAs, and moderately hydrolyzed Met-, Leu-, Tyr- and Lys-Ala-MCAs at pH ranging from 7.5 to 8.0. The order of the K(cat)/K(m) values of AAP-S at the optimal pH was Arg->Arg-Arg->Met->Leu->Lys->Phe->Lys-Ala->Tyr->Ala-MCAs. It was strongly inhibited by bestatin, leuhistin, actinonin, amastatin, 1, 10-phenanthroline, DFP, PCMBS, Zn(2+), Cd(2+), Co(2+), Cu((2+)), Hg(2+) and puromycin. AAP-S was approximately 80 times more sensitive than human seminal plasma AAP (aminopeptidase N, membrane type). The amino acid sequence of the first 60 residues of AAP-S was highly homologous with the N-terminal amino acid sequence of the rat liver puromycin-sensitive enkephalin-degrading aminopeptidase. These physicochemical properties and findings indicate that AAP-S from human liver cytosol is identical to those of other puromycin-sensitive aminopeptidase(s). Furthermore, with immunohistochemistry the enzyme was strongly stained in the cytoplasm of liver cells and renal tubules, and was ubiquitously localized in various human tissues.     

Comparison of the rates of hydrolysis of lorazepam-glucuronide, oxazepam-glucuronide and tamazepam-glucuronide catalyzed by E. coli beta-D-glucuronidase using the on-line benzodiazepine screening immunoassay on the Roche/Hitachi 917 analyzer

The catalytic rates of hydrolysis of lorazepam-glucuronide, oxazepam-glucuronide, and temazepam-glucuronide when catalyzed by E. Coli. beta-glucuronidase both in phosphate buffer and buffered drug-free urine were compared as well as the pH dependence of enzyme activity. In 50 mM phosphate buffer pH 6.4, lorazepam-glucuronide has the highest turnover rate of 3.7 s(-1) with an associated Km of about 100 microM, followed by oxazepam-glucuronide, which has a turnover rate of 2.4 s(-1) with an associated Km of 60 microM. Temazepam-glucuronide has the lowest rate of 0.94 s(-1) with an associated Km of 34 microM. In buffered drug-free urine, a similar trend was observed. In addition, an optimal pH for beta-glucuronidase was determined to be between 6 and 7 when the enzyme hydrolyzes the benzodiazepine conjugates in buffered drug-free urine. Effects of temperature and incubation time were also examined. It can be concluded that the electron donating or withdrawing of the individual benzodiazepine structure may play an important role in the reactivity of the lorazepam-glucuronide, oxazepam-glucuronide and temazepam-glucuronide catalyzed by beta-glucuronidase. This is consistent with other observations made for monosubstituted phenyl-beta-glucuronides by Wang et al. (1).     

血中安定及其代谢物的酶水解研究

目的考察木瓜蛋白酶水解血中安定及其代谢物蛋白结合物的酶解条件,提高安定的提取率。方法采用正交试验确定酶解的最优条件,检材经蛋白酶水解,固相萃取后,应用LC-MS／MS方法进行检测,运用保留时间和MRM（多离子反应监测）方式来对血中安定及其代谢物进行定性定量分析。结果安定、去甲安定、去甲异安定、羟基安定和去甲羟基安定的最佳酶解条件分别为55℃,2．5h,pH7．0,8000U;50℃,1h,pH7．5,8000U;50℃,1h,pH7．5,8000U;50℃,1．5h,pH7．5,8000U;50℃,1．5h,pH7．5,8000U。结论酶水解后的血液中安定及其代谢物检出量明显增加。     

闭合性颅脑损伤后大鼠BAEP及脑损伤的动态变化

目的观察大鼠闭合性弥漫性颅脑损伤后脑干听觉诱发电位(BAEP)的动态改变及脑组织病理学变化,探讨BAEP在颅脑损伤后评估听觉功能障碍的价值。方法使用自制弹簧式小型生物打击机打击大鼠颅顶部,制造闭合性弥漫性轻型颅脑损伤模型。观察对照组以及伤后15min和1,3,6,12h及1,2,4,7,10,14,21d等时间点大鼠脑组织病理学改变,干/湿法检测脑组织含水量。分别于伤前、伤后各时间点以50Hz刺激率记录大鼠BAEP,对其结果进行比较。结果损伤后15min,BAEP的Ⅰ～Ⅴ、Ⅲ～ⅤIPL即较损伤前延长(P<0.05),至6～12h,Ⅲ、ⅤPL较损伤前延长。损伤后1～2d,Ⅲ、ⅤPL和Ⅰ～Ⅲ、Ⅰ～Ⅴ、Ⅲ～ⅤIPL均较损伤前显著延长(P<0.001),14d后BAEP逐渐恢复正常。伤后15min脑组织含水量开始升高,伤后1d达高峰,持续至4d后逐渐下降,至10d后降至正常水平。结论BAEP可作为闭合性弥漫性轻型颅脑损伤后听觉功能障碍的客观评价指标。     

Postmortem changes in cytochrome c oxidase activity in various organs of the rat and in human heart

Cytochrome c oxidase (COX), a mitochondrial enzyme, is inactivated by cyanide or carbon monoxide (CO) intoxication. To test whether cytochrome c has potential as an indicator of these toxins in cadavers, we measured COX activity in the main organs of the rat, and in the human heart, at various times after death. Each tissue sample or organ was homogenized and the COX activity in the mitochondrial fraction was measured using ferrous cytochrome c as the substrate. COX activity was significantly higher in rat brain, heart and kidney than in lung and liver from 0 to 4 days after death. The loss of COX activity was significantly slower in the brain and heart than in the lung, liver and kidney. Most importantly, COX activity correlated with the time-since-death for each of the rat organs we tested (r2=0.70-0.95), but for the human heart (r2=0.47). It may be possible that COX activity is likely to be a useful indicator of the time-since-death, and is worth pursuing as an indicator of the tissue cyanide and CO content.     

High performance liquid chromatography-immunoassay of delta 9-tetrahydrocannabinol and its metabolites in urine

High performance liquid chromatographic-immunoassay (HPLC-IA) profiles of cannabinoid metabolites in urine samples were obtained using four different antisera. The urines were chromatographed on a reverse phase system using a gradient of acetonitrile in water (pH 3.3) and fractions collected every 30 s. Some urine samples were hydrolyzed with methanolic sodium hydroxide before fractionation. Peaks of immunoreactivity were detected at a fraction corresponding to 11-nor-9-carboxy-delta 9-tetrahydrocannabinol (COOH-THC) and at an early eluting fraction; however, the profiles depended upon the specificity of the antisera used.     

Cleavage of synthetic pentazocine glucuronide with hydrochloric acid

It has been reported before that pentazocine (I) and pentazocine-glucuronide (II) form an artifact (III) by the addition of water to the double bond in the presence of HCl. This reaction leads to different results concerning the investigation of the rate of hydrolysis of II and the recovery of I. The glucuronide was quantitatively hydrolyzed by 20% HCl, but yielded only 15% of I (about 64% was detected as III). With 5% HCl the rate of hydrolysis only amounted to 40%-43%, whereas I yielded 31% (only 9% was recovered as III). The best III yield was obtained with a HCl concentration of 17.5%.     

In vitro loss of potassium from erythrocytes during the 0-108 h postmortem period in rats: relationship between potassium loss and postmortem interval.

A simple technique was employed for measurement of potassium loss (K(los)s) from normal and from postmortem rat erythrocytes during controlled exposure to physiologically isosmotic NaCl solution. Potassium loss from the cells decreased in a (non-linear) time-related manner during the 0-108 h postmortem period; expression of the data in the form of a double logarithmic plot (log K(loss) versus log postmortem interval (PMI) linearized the relationship between 18 and 108 h post mortem (r = -0.86; P less than 0.001 (n = 24)). Experimental data revealed that the observed postmortem changes in K(loss) were associated with and probably resulted from, the postmortem decrease in magnitude of the potassium concentration gradient across the erythrocyte membrane. Attention is drawn to the possibility of utilizing measurements of in vitro loss of potassium from erythrocytes as a means of estimating time elapsed since death.     

Disobedience to Law – Debbie Purdy's Case

This case note examines the implications of the House of Lords decision to order the DPP to issue offence specific guidelines allowing those contemplating assisting terminally ill persons to commit suicide to know the risk they face of prosecution under section 2(1) of the Suicide Act 1961. On the assumption that these guidelines will be law, and binding upon the DPP as well as the CPS, does this represent a change in the law, or a situation in which it may be unlawful to enforce the law, or even generate a legal right of disobedience to law?     

The chemical interconversion of GHB and GBL: forensic issues and implications.

In this work, the interconversion of GHB and GBL in a variety of aqueous media was studied. The effects of solution pH and time were determined by spiking GHB or GBL into pure water and buffered aqueous solutions, and determining the GHB and GBL contents at various time intervals. The degree of GBL hydrolysis to GHB was determined for several commercial aqueous-based GBL products, and further studied as a function of time. The effects of temperature and time were also determined for five commercial beverages spiked with GHB or GBL. GHB and GBL contents were determined using high performance liquid chromatography (HPLC). GHB and/or GBL confirmations were made using gas chromatography-mass spectrometry (GC-MS) and/or infrared spectroscopy (IR). Solution pH, time, and storage temperature were determined to be important factors affecting the rate and extent of GBL hydrolysis to GHB. Under strongly alkaline conditions (pH 12.0), GBL was completely converted to GHB within minutes. In pure water, GBL reacted to form an equilibrium mixture comprising ca. 2:1 GBL:GHB over a period of months. This same equilibrium mixture was established from either GHB or GBL in strongly acidic solution (pH 2.0) within days. A substantial portion of GBL (ca. 1/3) was hydrolyzed to GHB in aqueous-based GBL products, and in spiked commercial beverages, after ambient storage for a period ranging from several weeks to several months. Heat increased and refrigeration decreased the rate of GBL hydrolysis relative to ambient conditions. These studies show that hydrolysis of GBL to GHB does occur in aqueous-based solutions, with samples and time frames that are relevant to forensic testing. Implications for forensic testing and recommendations are discussed.     

大鼠撞击性肝挫伤后MMP-2和MMP-9蛋白时序性表达

目的观察大鼠撞击性肝挫伤后不同时间肝组织中基质金属蛋白酶(matrix metalloproteinase,MMP)-2和MMP-9的蛋白表达规律。方法选取健康成年雄性SD大鼠50只,随机平均分入对照组和伤后1 h、3 h、6 h、12 h、18 h、24 h、3 d、5 d、7 d组。采用自由落体撞击法建立大鼠肝挫伤模型,在相应时间点处死大鼠,提取挫伤的肝叶,运用免疫组织化学染色法(SP法)和Western印迹法观察各组大鼠肝组织内MMP-2和MMP-9的蛋白表达情况。结果肝挫伤后,阳性细胞表达和蛋白半定量结果显示:MMP-2在伤后6 h表达开始增强,24 h达到峰值,3~5 d逐渐下降,7 d时接近正常水平,损伤6 h后各组(除18 h组)与相邻上组比较,差异均有统计学意义(P0.05);MMP-9在伤后1 h即明显上升,18 h达到高峰,3~7 d时逐渐下调,但仍高于对照组,伤后各组(除12 h、24 h组)与相邻上组比较,差异均有统计学意义(P0.05)。结论 MMP-2和MMP-9的蛋白表达在撞击性肝挫伤组织中有较好的时序性,有望作为肝挫伤的时间推断的参考指标。     

A sensitive and simple assay of saliva on stamps

Identification of saliva on stamps or envelope flaps remains yet a not widely studied problem. In most forensic laboratories it is seldom carried out, but this fact does not reduce the importance of the assay. Most authors consider amylase a sufficiently specific marker of the presence of saliva; really, the only other human body fluid that contains high amounts of this enzyme is the pancreatic juice (and therefore feces). Here we present a simple and sensitive assay for the determination of alpha-amylase that uses a commercially available and well-known substrate. It is hydrolyzed by amylase with the production of soluble blue fragments, that can be measured by photometry, obtaining objective results. The presented assay identifies 1 X 10(-6) diluted saliva or that present on 0.5 mg of a stamp; 16-year-old samples can also be identified. Intra-assay and day-to-day CV resulted in 10.8% and 13.7%, respectively. Owing to the high sensitivity of the test, handling samples or reagents can introduce contamination with saliva traces, giving false-positive results. Addition of EDTA 0.1 mol/l to the incubation mixture, lowering the sensitivity to 1 X 10(-3) diluted saliva, overcomes this problem.     

体内痕量乌头碱的高相液相色谱测定

<正> 乌头碱是乌头属植物中所含的二萜双酯型生物碱类中的一种,成人口服纯品致死量仅约5mg,由乌头属植物引起的中毒与死亡屡见报导但一直缺乏较好的检测方法,使中毒后体内乌头碱分布与分解规律的研究难以进行,也给这类中毒案件的法医学鉴定带来一定困难。近年来应用HPLC分离检测生药中乌头碱类生物碱的方法已有报导。本文应用HPLC     

尿中吗啡的氮磷检测——气相色谱分析法

目的建立尿中吗啡的简便快速、灵敏可靠的GC/NPD分析方法。方法样品尿加内标烯丙吗啡,酶或酸催化水解,氯仿-异丙醇(9:1)液液提取或GDX403树脂固相提取,BSA衍生化,HP-5柱和氮磷检测器进行分析。结果 提取率62％～85％,检出限1.2～3.1ng/ml,线性范围20～2000ng/ml,回收率(97％～99％)±(6％~9％)(Mean±cv,N=5)。结论 方法适合于实际案件中尿样的检验。     

Simultaneous electrophoretic determination of phosphoglucomutase subtypes, adenosine deaminase, erythrocyte acid phosphatase, and adenylate kinase enzyme phenotypes

Many of the conventional agarose phosphoglucomutase (PGM) subtyping systems presently in use fail to provide a good separation between the 1 + and 2- bands as well as the 2+ band and the more anodic moving bands. Use of a 1-mm-thick gel composed of 1% ISO GEL (FMC Corp.) and phosphate-citric acid gel and tank buffers with a pH of 5.3 provided exceptionally good separation between all four of the major subtyping bands. The additional criteria for this procedure is a voltage of 21 V/cm and a run time of 4 h. Utilization of this procedure using case samples of varied ages proved the reliability of the procedure. Also examined were the effects of several reducing agents on the enzyme band patterns and the use of this system for the simultaneous determinations of the adenosine deaminase (ADA), erythrocyte acid phosphatase (EAP), and adenylate kinase (AK) enzyme phenotypes.     

A sandwich enzyme immunoassay for human pancreatic elastase III

To develop a method for the determination of pancreas injuries using a pancreas-specific antigen as a marker, human elastase III was purified from the pancreas by chromatographic methods. A rabbit anti-human elastase III antibody was prepared, and this antibody was confirmed using immunoblotting to react only with elastase III among proteins from the pancreas. A sensitive sandwich enzyme immunoassay for human elastase III was developed. The detection limit for human elastase III was 0.3 pg (10 amol) per assay. Proteins extracted from the pancreas showed the strongest response, whereas reactions of the other organs were less than the detection limit. These results suggest that a sandwich enzyme immunoassay for human elastase III is useful for the determination of pancreas injury.     

A highly specific and sensitive sandwich enzyme immunoassay for human hemoglobin A

A highly specific and sensitive sandwich enzyme immunoassay for human hemoglobin A (Hb A) is described. A rabbit anti-human Hb A IgG-coated polystyrene ball was incubated with human Hb A and subsequently with affinity-purified anti-human Hb A Fab'-horseradish peroxidase conjugates, which had been prepared before and after absorption with Japanese monkey Hb-Sepharose 4B and dog Hb-Sepharose 4B. Bound peroxidase activity was measured by fluorimetry using 3-(4-hydroxyphenyl)propionic acid as a substrate. The detection limits of human Hb A using the conjugate before and after the absorption were 0.65 pg/tube (3 X 10(10)-fold dilution of whole blood) and 2.0 pg/tube (1 X 10(10)-fold dilution of whole blood), respectively. Human Hb A could be discriminated from Hb of animals such as Japanese monkey, dog, cat, pig, horse, sheep, chicken and cow by measuring bound peroxidase activity in the presence and absence of the conjugates prepared before and after the absorption. Human Hb A in bloodstains on cotton gauze could be discriminated from Hb of animals described above even after seven washings. Human Hb A in 220,000-fold diluted bloodstains on cotton gauze could also be discriminated from Hb of animals described above.     

Detection of drugs using XAD-2 resin. II:Analysis of liver in medical examiner's cases

Liver tends to concentrate drugs in quantities generally higher than those found in blood or other body compartments. This fact as well as the general availability of liver in postmortem cases makes it an important specimen for comprehensive toxicologic investigation. A scheme for the analysis of liver for drugs with tissue hydrolysis, XAD-2 resin extraction, and TLC has been developed and the parameters affecting recovery have been studied. The hydrolysis of liver specimens at various pH conditions resulted in an improved recovery for morphine by using pH 2 (2N hydrochloric acid). Recoveries of barbiturates, codeine, and meperidine were essentially the same at pH 2 and pH 3. A considerable loss (22 to 55%) was observed for four drugs (pentobarbital, morphine, codeine, and meperidine) as a result of drug binding to the tissue pellets during the process of centrifuging the liver homogenates. This method is recommended as a comprehensive screening procedure for drugs in liver tissue. For quantitative purposes, however, it is necessary to determine a correction factor for all the losses occurring at the various steps of the procedure. This procedure compared favorably with other procedures for liver analysis reported in literature.     

HPLC-MS法测定鲜鱼中河豚毒素

目的建立用液相色谱-质谱（HPLC-MS）法测定鲜鱼中河豚毒素含量的方法。方法样品经粉碎、酸性水溶液提取、蛋白酶解、石油醚脱脂、有机溶剂甲醇沉淀、固相萃取柱净化后,以液相色谱-质谱法测定,外标法定量。结果鱼肉和鱼肝中河豚毒素在0.10～10 mg/kg范围内呈良好线性关系,相关系数分别为0.9998和0.9991,平均回收率为94.6%~102%,相对标准偏差RSD小于6.6%,方法的最低检出限为0.03 mg/kg。结论该方法操作简便、快速,为饮用水及水产品中河豚毒素的检测、相关的司法鉴定等提供了准确有效的科学依据。     

Postmortem disposition of morphine in rats

The antemortem and postmortem distribution of morphine was studied in rats for the purpose of establishing whether drug distribution is altered after death. Samples were examined for free and total morphine concentration, pH and water content at 0-96 h after death. Morphine was administered antemortem at various intervals. All groups of rats studied showed a significant (P less than 0.05) increase in postmortem cardiac blood morphine concentrations. These changes, which are detectable within 5 min after death are likely to be related to an observed, rapid decrease in cardiac blood pH from 7.34 +/- 0.02 to 6.74 +/- 0.05. Significant increases in free morphine levels were, also, observed 24 and 96 h after death in liver, heart and forebrain while urine morphine levels decreased. The liver showed the greatest increase (20-fold) in free morphine levels 96 h after death, while hindbrain levels did not significantly change. Bacterial hydrolysis of morphine glucuronides accounted only in part for the observed increase in free morphine concentration. Postmortem fluid movement and pH-dependent drug partitioning was detected. It would appear that several mechanisms are responsible for postmortem drug distribution. Understanding the mechanisms and patterns responsible may eventually lead to better choices of postmortem tissue which may better represent antemortem drug levels.