Diagnosis of a case of acute chloroquine poisoning using 1H NMR spectroscopy: characterisation of drug metabolites in urine

The compromised optima for high intensity chemiluminescence (CL), using superoxide generators, were all above pH 9.0 for the CL probes luminol and lucigenin. With luminol the optima were at pH 9.0 and 9.4 for the generators KO2 and hypoxanthine/xanthine oxidase (HX/XO), respectively. Lucigenin, with the same generators, produced optima at pH 9.5 and 10.0, respectively. The probe methyl-Cypridina-luciferin analogue (MCLA) produced optima closer to neutral pH, which is preferred for physiological assessments. MCLA had optima at pH 6.0, 8.7 and 9.5 with KO2 and with HX/XO optima at pH 4.8, 6.0, 7.0 and 8.7. When CL was assessed at physiological pH, MCLA observed superoxide radicals with a sensitivity of 100- and 330-fold more than luminol or luicigenin respectively. For singlet oxygen, the sensitivity of MCLA at this pH was 45- and 5465-fold more than for the said probes respectively. H2O2 did not elicit CL between pH 4 and 9.5 with any of the probes and did not influence the production of superoxide or singlet oxygen when co-assessed. Therefore CL could only be obtained when enzymes were used as converters. The optima for the enzyme-conversion system horseradish peroxidase (HRP)/H2O2, and luminol, were at pH 8.0 and 9.2. Lucigenin and HRP/H2O2 also had a biphasic CL profile with optima at pH 7.4 and 9.6. MCLA and HRP/H2O2 had five optima, with the major ones at pH 6.1 and beyond 10. The optima for the myeloperoxidase/H2O system were at 8.6 and beyond 10.0 when luminol and 0.15 mol/L NaBr were used.     

Luminol-Based Enhanced Chemiluminescence Assay for Quantification of Peroxidase and Hydrogen Peroxide in Aqueous Solutions: Effect of Reagent pH and Ionic Strength

The effect of reagent pH and ionic strength was evaluated on the horseradish peroxidase (HRP)-luminol-p-iodophenol enhanced chemiluminescence assay. This assay was optimized for HRP and H2O2 quantification during enzyme-mediated remediation of groundwater or wastewater contaminated with phenolic chemicals. The maximum chemiluminescence intensity (ICLmax) and total area under the chemiluminescence intensity profiles were measured as a function of p-iodophenol concentration, reagent solution pH, and reagent ionic strength. ICLmax values were optimum at pH 8.5 and increased linearly with reagent ionic strength. Optimum chemiluminescence enhancement was produced at a p-iodophenol concentration of 0.3 mM under the reaction conditions. ICLmax values were linearly correlated with HRP and H2O2 concentrations within the ranges of 0.1–1 activity units/mL and 0.1–1 mM, respectively. Results indicate that the HRP-luminol-p-iodohenol enhanced chemiluminescence assay has a potential to be used for quantification of HRP activity and H2O2 concentration in aqueous solutions encountered in groundwater remediation or wastewater treatment scenarios.     

Evaluation of five imidazopyrazinone-type chemiluminescent superoxide probes and their application to the measurement of superoxide anion generated by Listeria monocytogenes

Superoxide-triggered chemiluminescence of five new imidazopyrazinone derivatives was investigated using the hypoxanthine-xanthine oxidase system as the source of superoxide anion. The results showed that they are highly sensitive and have favorable properties in measuring superoxide anion. With those new probes, the generation of superoxide anion from the bacteria Listeria monocytogenes was examined. The results confirmed the previous report that L. monocytogenes is an unusual organism that extracellularly and continuously generates a high level of superoxide anion in the presence of acetaldehyde. The data indicated that two of the probes, 3,7-dihydro-2-methyl-6-phenylethynylimidazo[1,2-a]pyrazin-3- one (4) and its methoxy derivative (5), are highly sensitive and useful in the measurements of superoxide anion and are clearly superior to 3,7-dihydro-2-methyl-6-(4-methoxyphenyl)imidazo[1,2-a]pyrazin-3-on e (MCLA), which-has been generally considered the most sensitive superoxide probe in the past. When tested at a probe concentration of 3.3 microM, the luminescence response and the signal-background ratio of compound 4 were 1.5 and 2.5 times those of MCLA, respectively, and the signal-background ratio of compound 5 was almost 15 times that of MCLA, though the luminescence response of this compound was slightly lower than that of MCLA. The low probe concentration used enhances the usefulness of probes in the measurements of superoxide in functioning biological systems.     

Biliary cystadenoma of the liver

The antioxidant effect of 2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo[1,2-alpha]pyraz in-3-one (MCLA), a Cypridina luciferin analog that acts as a chemiluminescence probe to detect O2.-, was investigated. MCLA produced a lag in oxygen consumption induced by cumene hydroperoxide in microsomes or by 2,2'-azobis (2-amidinopropane) dihydrochloride in liposomes and disappeared during the duration of the lag. MCLA profoundly inhibited the propagation reaction in Fe2+-dependent lipid peroxidation in liposomes, and MCLA disappearance accompanied by suppression of oxygen consumption markedly occurred in liposomes susceptible to peroxidation. Thiobarbituric acid-reactive substances in all systems used were also suppressed by MCLA dose dependently. These results indicate that MCLA has an antioxidant property through scavenging free radicals.     

New enhancers for the chemiluminescent peroxidase catalysed chemiluminescent oxidation of pyrogallol and purpurogallin

The effects of various boronate compounds, 4-biphenylboronic acid, 4-bromobenzene-boronic acid, trans-4-(3-propionic acid)phenylboronic acid and 4-iodophenylboronic acid, on the horseradish peroxidase (HRP) catalysed chemiluminescent oxidation of pyrogallol and purpurogallin by peroxide were investigated. trans-4-(3-Propionic acid)phenylboronic acid produced a 13.7-fold enhancement in the peak light emission from the chemiluminescent HRP catalysed pyrogallol reaction (detection limit for HRP < 1.25 fmol). At low enhancer concentration a single peak of light emission was observed and as the enhancer concentration increased the time to peak light emission became progressively longer. The chemiluminescence showed two peaks at higher concentrations (> 54.3 mumol/L) and the individual peak times depended upon the concentration of the enhancer. All of the boronates enhanced peak light emission in the chemiluminescent HRP catalysed purpurogallin reaction. 4-Biphenylboronic acid was the most effective and it enhanced peak light emission 314-fold. The practical detection limit for HRP (Type VIA) using this enhancer was 4.18 pmol (peak emission at 20 minutes). This compound also enhanced peak light emission 232-fold from a chemiluminescent HRP-purpurogallin reaction in which molecular oxygen replaced peroxide as the oxidant.     

Effects of diltiazem on myocardial perfusion abnormalities during exercise in patients with hypertrophic cardiomyopathy

We developed a novel chemiluminescent assay of beta-D-galactosidase (beta-gal) based on the chemiluminescence of indole. 5-Bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-gal) was used as a substrate for beta-gal and also as a light emitter. X-gal was hydrolysed by beta-gal to liberate free indoxyl, followed by oxidation to indigo dye, and simultaneously produces hydrogen peroxide (H2O2). H2O2 reacts with the residual X-gal in the presence of horseradish peroxidase (HRP) to emit light. The measurable range of beta-gal obtained by this method was 6 x 10(-14) mol/L to 6 x 10(-11) mol/L; the detection limit was 3 amol/assay. This chemiluminescent assay could be applied to an enzyme immunoassay of thyroxine using beta-gal as the enzyme label.     

Measurement of endogenous and TNF alpha-mediated H2O2 production in supernatants of SK-N-SH neuroblastoma cells with an enhanced chemiluminescence assay

A sensitive peroxidase-dependent luminol-enhanced chemiluminescence (ECL) assay for determination of hydrogen peroxide (H2O2) generation by tumour cells was established. This test system allows determination of H2O2 in concentrations as low as 25 pmol (50 nmol/L) and yields results which are comparable to those obtained using a less sensitive photometric method and a previously described scopoletin fluorescence assay. After 3 h incubation time 10(4) SK-N-SH neuroblastoma cells released 60 +/- 5 pmol H2O2 in the supernatant and this level was significantly (p < 0.025) increased by about 70% in the presence of 5 pmol (100 ng/mL) recombinant tumour necrosis factor alpha (TNF alpha). In contrast, H2O2 production was slightly reduced by TNF alpha at a very low concentration of 0.5 fmol (0.01 ng/mL).     

硒的酶催化荧光猝灭效应研究及其在土壤分析中的应用

在氨性缓冲介质中,痕量硒能阻抑辣根过氧化物酶(HRP)催化H₂O₂氧化L-酪氨酸(L-Tyr)产生荧光的反应。对该体系的荧光猝灭效应进行了探讨,并应用于土壤中痕量硒的测定。在pH 10.0的 NH₃·H₂O-NH₄Cl缓冲溶液中,控制L-Tyr、H₂O₂和HRP的浓度分别为7.50×10^-5 mol/L、1.00×10^-4 mol/L和5.00×10^-7 mol/L,在室温下反应20 min后,于激发波长为314 nm,发射波长为404 nm处测定体系的荧光强度猝灭值(ΔF)。结果表明,硒在质量浓度为0.1～10.0 μg/mL范围内与ΔF呈线性关系,线性回归方程为ΔF=83.123 ρ_{Se }(μg/mL)+0.068 5,相关系数r=0.999 5。方法中硒检出限为0.03 μg/mL。将体系应用于测定富硒土壤样品中痕量硒,测定值与原子吸收光谱法(AAS)相符,相对标准偏差(RSD,n=6)为1.6%～3.2%,加标回收率为98%～103%。     

恒电位电解流动注射化学发光分析法测定钢铁中微量钼

建立了钼的恒电位电解流动注射化学发光分析法。在0.02 mol/L的H2C2O4酸度下,使含不具发光活性钼（Ⅵ）的溶液,以1.7 mL/min的流速通过自制的流通式碳电解池时,在-0.60 V(vs∶Ag/AgCl)电位处,钼（Ⅵ）在线还原为钼（Ⅲ）,钼（Ⅲ）与鲁米诺在碱性条件下产生化学发光,且发光强度与钼的质量浓度在5.0×10-10～5.0×10-7 g/mL范围内呈线性关系,钼（Ⅵ）检出限为5×10-11 g/mL。大多数常见的阳离子和阴离子对钼的测定没有干扰,Fe3+和Fe2+的允许量较低,但试液通过测定流路中钠型离子交换柱后,Fe3+和Fe2+的允许量提高到1 000倍。方法已用于低合金钢和碳钢标准样品中微量钼的测定,测定值与认定值一致,相对标准偏差在0.68 %~1.3 %之间。     

Variation of nitric oxide concentration during inspiration

OBJECTIVE: To evaluate the pattern of inspiratory nitric oxide concentration in a simple, constant flow delivery system during the use of two phasic-flow ventilatory modes. DESIGN: Laboratory study in a lung model. SETTING: University experimental laboratory. SUBJECT: Nitric oxide (800 ppm in nitrogen) was administered continuously into the inspiratory circuit to deliver a nitric oxide concentration of 10 and 40 ppm to a test lung during volume-controlled (constant flow) and pressure-controlled (decelerating flow) ventilation, with an FIO2 of 1.0. INTERVENTIONS: In each mode, minute ventilation of 7, 14, and 21 L/min and installation of mixing chambers (none, 1-L, 2-L, and 3.2-L turbulence boxes) were studied, respectively. Nitric oxide and nitric dioxide were monitored by chemiluminescence. Since the nitric oxide/nitrogen gas is the only nitrogen source in the system during ventilation with an FIO2 of 1.0, we evaluated the fluctuation in the inspiratory nitric oxide (NOx) concentration by measuring nitrogen with a fast-response analyzer. To test the effect of the measurement site, we measured nitric oxide concentrations using chemiluminescence at different positions in the inspiratory and expiratory limbs, with and without the mixing chambers, with a minute ventilation of 14 L/min and a nitric oxide concentration of 40 ppm. MEASUREMENTS AND MAIN RESULTS: Nitrogen dioxide production was not influenced by the flow pattern. During a nitric oxide concentration of 10 ppm, nitrogen dioxide was always < 0.6 ppm. During a nitric oxide concentration of 40 ppm, the highest nitrogen dioxide (4.47 ppm) concentration was found at the lowest minute ventilation and the largest inspiratory circuit volume. Nitric oxide values displayed by chemiluminescence indicated stable concentrations at all settings. However, without mixing chambers, NOx concentration calculated from nitrogen measurements demonstrated marked inspiratory fluctuations and was highest with a minute ventilation of 21 L/min and higher during pressure-controlled ventilation compared with volume-controlled ventilation (nitric oxide concentration of 40 ppm, pressure-controlled ventilation: 14.5 to 130.5 ppm; volume-controlled ventilation: 21.6 to 104.7 ppm; nitric oxide concentration of 10 ppm, pressure-controlled ventilation: 3.2 to 30.9 ppm; volume-controlled ventilation: 4.5 to 27.1 ppm). NOx concentration fluctuation decreased with an increasing mixing chamber, and was negligible at all settings with the 3.2-L turbulence box. Nitric oxide concentration fluctuation influenced chemiluminescence measurements. The displayed nitric oxide values varied, depending on the sampling site, and did not accurately reflect mean inspiratory nitric oxide concentration. Incorporation of a mixing chamber eradicated this sampling site influence. CONCLUSIONS: Continuous flow delivery of nitric oxide into the circuit of a phasic-flow ventilator results in marked inspiratory nitric oxide concentration fluctuation that is not detected by a slow-response chemiluminescence analyzer. Moreover, nitric oxide concentration fluctuation can influence the accuracy of the chemiluminescence measurements. These effects can be diminished by using additional mixing chambers to facilitate a stable gas concentration. As these mixing volumes increase the contact time of nitric oxide with oxygen, an increase of nitrogen dioxide has to be taken into account.     

Investigation on possible antioxidative properties of the NMDA-receptor antagonists ketamine, memantine, and amantadine in comparison to nicanartine in vitro

Possible antioxidative properties of three N-methyl-D-aspartate (NMDA)-receptor antagonists, the anesthetic ketamine and the antiparkinson drugs memantine and amantadine were investigated in vitro on the microsomal cytochrome P450 (P450) system of rat livers and on rat whole blood chemiluminescence in comparison to nicanartine, a substance with known antiatherosclerotic, hypolipemic and antioxidative capacity. For this purpose, the effects on NADPH- and iron-stimulated lipid peroxidation (LPO), hydrogen peroxide (H2O2) production, and NADPH- and iron-stimulated lucigenin (LC) and luminol (LM) amplified chemiluminescence (CL) were examined using rat liver microsomes. Additionally, the influence on LM amplified whole blood chemiluminescence after zymosan activation of polymorphonuclear leukocytes (WB-CL) was investigated. Furthermore, binding to P450 and effects on P450 mediated monooxygenase function, as measured by the model reactions ethoxyresorufin O-deethylation (EROD), ethoxycoumarin O-deethylation (ECOD), and ethylmorphine N-demethylation (END), were assessed. Nicanartine concentration dependently reduced LPO and H2O2 production already at a concentration of 1 microM, whereas LC and LM amplified CL and WB-CL were not affected. EROD and END were concentration dependently diminished starting at 1 microM, and ECOD already at 0.1 microM. Ketamine decreased LPO, H2O2 production and LM and LC amplified CL, starting at 100 microM. WB-CL was significantly diminished already at 10 microM. EROD and ECOD were inhibited at 10 and 100 microM and END at 100 microM. With memantine a concentration dependent inhibition of LPO and WB-CL was seen at 100 and 1000 microM and a reduction of LC and LM amplified CL only at 1000 microM. H2O2 production was not affected. EROD and ECOD were significantly diminished by a concentration of 100 microM. No effect was observed on END. Amantadine significantly reduced LPO and WB-CL, but only at 1000 microM. H2O2 production and LC and LM amplified CL were not affected. EROD was significantly diminished at 100 microM, whereas no influence was seen on ECOD and END. Nicanartine displayed type II or reverse type I, ketamine, memantine and amantadine type I substrate binding to P450. The highest binding affinity to P450 was seen with nicanartine, followed by ketamine, memantine and then amantadine. These results demonstrate, that all four substances seem to act as radical scavengers and/or as inhibitors of the oxidative function of P450. All four substances seem to interfere with the monooxygenase function of P450. This may result in a possible influence on the biotransformation of endogenous as well as of foreign compounds. The effects of nicanartine were much more pronounced than those of ketamine, memantine, and amantadine.     

Nitric oxide scavenging by hemoglobin or nitric oxide synthase inhibition by N-nitro-L-arginine induces cortical spreading ischemia when K+ is increased in the subarachnoid space

We investigated the combined effect of increased brain topical K+ concentration and reduction of the nitric oxide (NO.) level caused by nitric oxide scavenging or nitric oxide synthase (NOS) inhibition on regional cerebral blood flow and subarachnoid direct current (DC) potential. Using thiopental-anesthetized male Wistar rats with a closed cranial window preparation, brain topical superfusion of a combination of the NO. scavenger hemoglobin (Hb; 2 mmol/L) and increased K+ concentration in the artificial cerebrospinal fluid ([K+]ACSF) at 35 mmol/L led to sudden spontaneous transient ischemic events with a decrease of CBF to 14+/-7% (n=4) compared with the baseline (100%). The ischemic events lasted for 53+/-17 minutes and were associated with a negative subarachnoid DC shift of -7.3+/-0.6 mV of 49+/-12 minutes' duration. The combination of the NOS inhibitor N-nitro-L-arginine (L-NA, 1 mmol/L) with [K+]ACSF at 35 mmol/L caused similar spontaneous transient ischemic events in 13 rats. When cortical spreading depression was induced by KCl at a 5-mm distance, a typical cortical spreading hyperemia (CSH) and negative DC shift were measured at the closed cranial window during brain topical superfusion with either physiologic artificial CSF (n=5), or artificial CSF containing increased [K+]ACSF at 20 mmol/L (n=4), [K+]ACSF at 3 mmol/L combined with L-NA (n=10), [K+]ACSF at 10 mmol/L combined with L-NA (five of six animals) or [K+]ACSF at 3 mmol/L combined with Hb (three of four animals). Cortical spreading depression induced longlasting transient ischemia instead of CSH, when brain was superfused with either [K+]ACSF at 20 mmol/L combined with Hb (CBF decrease to 20+/-20% duration 25+/-21 minutes, n=4), or [K+]ACSF at 20 mmol/L combined with L-NA (n=19). Transient ischemia induced by NOS inhibition and [K],ACSF at 20 mmol/L propagated at a speed of 3.4+/-0.6 mm/min, indicating cortical spreading ischemia (CSI). Although CSH did not change oxygen free radical production, as measured on-line by in vivo lucigenin-enhanced chemiluminescence, CSI resulted in the typical radical production pattern of ischemia and reperfusion suggestive of brain damage (n=4). Nimodipine (2 microg/kg body weight/min intravenously) transformed CSI back to CSH (n=4). Vehicle had no effect on CSI (n=4). Our data suggest that the combination of decreased NO. levels and increased subarachnoid K+ levels induces spreading depression with acute ischemic CBF response. Thus, a disturbed coupling of metabolism and CBF can cause ischemia. We speculate that CSI may be related to delayed ischemic deficits after subarachnoid hemorrhage, a clinical condition in which the release of Hb and K+ from erythrocytes creates a microenvironment similar to the one investigated here.     

The effect of norfloxacin on the luminol-dependent chemiluminescence and adhesion of leukocytes

The in vitro effect of norfloxacin on the luminol-dependent chemiluminescence and adherence of polymorphonuclear leukocytes of guinea pigs was studied with the use of a wide range of the drug concentrations from 0.1 to 100 micrograms/ml under two different conditions, i.e. during the cell incubation in the presence of norfloxacin various concentrations and after the cell washing to remove the drug. In a concentration of 1 microgram/ml norfloxacin stimulated the zymosan-induced chemiluminescence. When the drug was used in a concentration of 10 micrograms/ml, the stimulating effect was less pronounced. In a concentration of 100 mu/ml norfloxacin significantly inhibited the leukocyte chemiluminescence. In concentrations of 0.1 to 100 micrograms/ml norfloxacin had no effect on the leukocyte adherence to the plastic surface covered by albumin.     

鲁米诺-H2O2-Cu（Ⅱ）化学发光法测定镍（Ⅱ）

基于Ni（Ⅱ）能置换出Cu（Ⅱ）-EDTA络合物中的Cu（Ⅱ）,与鲁米诺-H2O2-Cu（Ⅱ）化学发光体系相偶合,建立了测定Ni（Ⅱ）的置换偶合反应流动注射化学发光法测定Ni（Ⅱ）的新方法。考察了Cu（Ⅱ）-EDTA络合比、反应介质与pH、氧化剂的选择及其浓度以及鲁米诺浓度对体系相对发光强度的影响,并采用酒石酸掩蔽剂消除了干扰组分对测定的影响。在最佳的实验条件下,该方法测定Ni（Ⅱ）的线性范围为5.0×10-8～5.0×10-6 g/mL,检出限为2.0×10-8 g/mL (3σ)。对环境水样中的镍（Ⅱ）进行测定,其结果同原子吸收光谱法的测定值相一致,相对标准偏差为3.7%～4.6%。     

锌(Ⅱ)-2-(5-溴-2-吡啶偶氮)-5-二乙氨基酚-亚硝酸钠三元络合物在碳糊电极上的电化学行为及应用

研究了锌(Ⅱ)-2-(5-溴-2-吡啶偶氮)-5-二乙氨基酚-亚硝酸钠[Zn(Ⅱ)-5-Br-PADAP-NaNO₂]三元络合物在碳糊电极(CPE)上的电化学行为,并建立了测定痕量锌的方波伏安法(SWV)。实验表明:在pH 5.4的六次甲基四胺-盐酸缓冲溶液中,当5-Br-PADAP浓度为3.0×10^-5 mol/L,NaNO₂浓度为5.0×10^-5 mol/L,振幅为0.025 V,频率60 Hz时, 于－0.40 V(vs.SCE)富集45 s后,以100 mV/s的扫速在-0.40～0.60 V 区间内用SWV进行扫描,Zn(Ⅱ)-5-Br-PADAP-NaNO₂络合物在0.292 V(vs.SCE)有一灵敏的吸附氧化峰。锌浓度在5.0×10^-9～5.0×10^-6 mol/L范围内与络合物的峰电流I_p呈现良好的线性关系,线性相关系数为0.998 9,检出限为1.7×10^-9 mol/L。方法用于环境水和管网水中痕量锌含量的测定,测得结果与双硫腙光度法测定值基本一致,相对标准偏差(RSD,n=6)为1.9%~2.5%,回收率在97%~105%之间。     

2-(5-氰基-2-吡啶偶氮)-5-二甲氨基苯胺双波长叠加分光光度法测定铜

以2-(5-氰基-2-吡啶偶氮)-5-二甲氨基苯胺(5-CN-PADMA)做显色剂,建立了双波长叠加分光光度法测定铜的新方法。研究发现,在pH 4.0~5.0的HAc-NaAc缓冲介质中,于室温下,5-CN-PADMA与铜即可形成稳定的2:1紫红色配合物,其吸收光谱呈现两个吸收峰,分别位于548 nm和583 nm处,且两个吸收峰处的吸光度具有良好的加合性,吸光度之和与铜质量浓度线性相关。铜质量浓度在0~0.8 μg/mL范围内服从比尔定律,双波长测量的表观摩尔吸光系数为1.06×10⁵ L·mol^-1·cm^-1。应用于铝合金中铜的测定,结果与火焰原子吸收光谱法一致,相对标准偏差(RSD)在0.86%~2.1%之间。     

Solvent extraction of heavy metals contained in phosphoric acid solutions by 7-(4-ethyl-1-methyloctyl)-8-hydroxyquinoline in kerosene diluent

The solvent extraction of zinc, cadmium and chromium contained in 5.5 mol/L phosphoric acid solutions (30% P₂O₅) was investigated using 7-(4-ethyl-1-methyloctyl)-8-hydroxyquinoline-Kelex 100® as extractant and treated kerosene as diluent. At organic-to-aqueous phase ratio (1/1) and at room temperature, 58% of zinc, 34% of chromium and 15% of cadmium were recovered in 240 min. In order to improve the kinetics of extraction, a modifier reagent was added to the organic phase. The addition of n-decanol (10 vol.%) increased the rate metals extraction by reducing the equilibrium time from 240 min to 30 min, along with 60% recovery of metals. Extraction of metal ions increased with increasing aqueous phase pH. The pH_0.5 values difference of 0.1 M with Kelex 100® indicates the possible separation of cadmium, zinc and chromium. Increasing the concentration of Kelex 100® increased the percentage extraction of metal ions. The loading capacity values were found to be 83%, 80% and 71% for zinc, chromium and cadmium, respectively, at 0.4 M Kelex 100® concentration, indicating that the extractant is highly selective for the metal ions considered.     

硫酸氢钾沉淀分离—2-(5-溴-2-吡啶偶氮)-5-二乙氨基苯酚光度法测定矿石中铅

2-(5-溴-2-吡啶偶氮)-5-二乙氨基苯酚(5-Br-PADAP)光度法应用于矿石中铅的测定时,Cu、Zn、Fe、Ni、Bi、Mn、Cd、Co、V、Ti、Nb和Ta等多种元素的干扰不容忽视。针对这一问题,实验提出了采用KHSO₄沉淀法对样品进行前处理,5-Br-PADAP光度法测定矿石中Pb的方法。实验表明,采用盐酸-硝酸体系溶解矿石后,加入10 mL 200 g/L KHSO₄溶液,放置20 min后即可见样品中的铅沉淀完全从而与上述离子分离;将沉淀过滤后,采用pH 5.6的HAc-NaAc缓冲溶液溶解沉淀,可使K₂SO₄·PbSO₄复盐沉淀溶解,从而与Ba、W、Sr和Ca等元素分离。在样品溶液中依次加入0.4 mL pH 9.3 H₃BO₃-KCl-NaOH缓冲溶液、0.4 mL饱和KI溶液、0.4 mL 10%(V∶V)Trion X-100溶液、1.0 mL 0.03 g/L 5-Br-PADAP乙醇溶液,于1 cm比色皿中,在波长558 nm处进行测定,Pb质量浓度在0.02～2 μg/mL范围内符合比尔定律,线性回归方程的相关系数为0.998。方法检出限为0.02 μg/mL。采用实验方法分别对铅矿、锌矿、铅锌矿标准物质中的Pb进行分析,测得结果与认定值基本一致,相对标准偏差(RSD,n=11)为2.3%～5.8%。     

Insulin-like growth factor binding protein production in bovine retinal endothelial cells

Retinopathy is the most frequent microangiopathic complication in diabetes. Many circulating hormones and locally produced mitogenic factors have been involved. Bovine retinal endothelial cells (BRECs) were cultured to investigate if insulin, insulin-like growth factors (IGFs), IGF binding proteins (IGFBPs), and a chronic high-glucose condition could control endothelial cell growth. Specific IGF-I receptors with two binding sites with high (Kd 0.03 nmol/L) and low (Kd 1.3 nmol/L) affinity were found when analyzing families of displacement curves between IGF-I versus IGF-I and IGF-I versus insulin. However, IGFs failed to be mitogenic factors in these cells. This could be explained by an inhibitory effect due to the presence of specific IGFBPs with a molecular weight between 24 and 43 kd. Using Western blot and immunoblot analysis, Northern blot study, and specific radioimmunoassay (RIA), these IGFBPs have been identified as IGFBP-3, -2, -5, and -4. Insulin, which does not bind to IGFBPs, was a potent mitogenic factor in these cells at a high concentration (10 nmol/L), suggesting a cross-reaction to IGF-I receptor. These IGFBPs, except the 24-kd form (IGFBP-4), were modulated by both IGF-I and IGF-II, with a maximum effect at 100 and 10 nmol/L, respectively. This regulation on IGFBPs was IGF-I receptor-independent. In fact, (1) IGFBP mRNA levels were not modified after stimulation with 100 nmol/L IGF-I, (2) 100 nmol/L IGF plus an equimolar concentration of alpha IR3 did not affect IGFBP production, (3) Des(1-3)IGF-I had no effect on IGFBP modulation, whereas at 10 nmol/L it enhanced BREC thymidine cell incorporation, and (4) 100 nmol/L insulin, which at this concentration can cross-react with the IGF-I receptor, did not modify the IGFBP pattern. Chronic exposure (4 weeks) of BRECs to 25 mmol/L glucose had no effect on cell growth. However, after 3 weeks, we observed a decreased IGFBP detection, and addition of 100 nmol/L IGF-I did not change IGFBP levels and did not modify cell growth. Conversely, BRECs grown in regular medium for 4 weeks showed increased IGFBP production. In conclusion, we showed that conditions mimicking hyperinsulinemia, rather than high levels of IGFs, could regulate BREC growth and that the IGF-I analog, Des(1-3), even with reduced affinity for IGFBPs but in part capable of binding to IGFBP-3, significantly stimulated BRECs growth only at 10 nmol/L. IGF actions are modulated by locally produced endothelial IGFBPs, and in turn, these endothelial IGFBPs are regulated, via in IGF-I receptor-independent mechanism, by the presence of IGFs. The autoregulatory IGF system together with the direct glucose modulation of IGFBPs could contribute in diabetic subjects to the retinal endothelial cell growth and metabolism through local changes in IGF bioavailability.     

Lactate and PO2 modulate superoxide anion production in bovine cardiac myocytes: potential role of NADH oxidase

BACKGROUND: Lactate increases lucigenin chemiluminescence (CL)-detectable superoxide anion (O2.-) generation in bovine vascular smooth muscle and endothelium, and a microsomal flavoprotein-containing NADH oxidase whose activity is regulated by PO2 and cytosolic NAD(H) redox appears to be the detected source of O2.- production. Little is known about the importance of this O2.(-)-producing system in cardiac myocytes. METHODS AND RESULTS: In isolated bovine cardiac myocytes, lactate (10 mmol/L) increased lucigenin-detectable O2.- levels to approximately 1.8 times baseline, whereas pyruvate (10 mmol/L) and mitochondrial probes did not increase the detection of O2.-. A nonmitochondrial NADH oxidase activity, found in microsomes containing a cytochrome b558, was a major source of O2.- production in the homogenate of myocytes, because NADH (0.1 mmol/L) increased basal lucigenin CL >100-fold. NADPH oxidases, mitochondria, and xanthine oxidase were minor sources of detectable O2.- production. However, mitochondria released H2O2 in the presence of 5 mmol/L succinate and 30 micromol/L antimycin, based on its detection as catalase-inhibitable luminol (+horseradish peroxidase)-elicited CL. Diphenyliodonium (DPI), an inhibitor of flavoprotein-containing oxidases, significantly attenuated basal, lactate, and NADH-elicited lucigenin CL. Hypoxia eliminated myocyte lucigenin CL, and posthypoxic reoxygenation caused an 8.6-fold increase in the detection of O2.- that was potentiated by lactate and inhibited by DPI. CONCLUSIONS: NADH oxidase activity linked to cytosolic NAD(H) redox appears to be a key source of O2.- production in cardiac myocytes that could contribute to oxidant signaling mechanisms and injury upon exposure to changes in PO2 and metabolites produced under hypoxia, such as lactate. These processes could contribute to the previously observed potentiation of injury caused by lactate in cardiac ischemia/reperfusion.