化学发光法测定葡萄糖

葡萄糖在葡萄糖氧化酶作用下氧化生成葡萄糖酸和过氧化氢,用化学发光法可以测定生成的过氧化氢,从而知道葡萄糖的含量。本法所需样品量少,快速方便,灵敏度较比色法高,适用于大量样品的分析,如血糖,也可用于测定葡萄糖氧化酶、过氧化物酶、过氧化氢以及金属离子等。测定方法包括葡萄糖的氧化和发光强度的测量两个步骤。     

环糊精交联固定酶的生物传感器及临床应用

通过交联方式将辣根过氧化物酶固定在Eastman-AQ-N-甲基吩嗪修饰电极上,制备成过氧化氢生物传感器.通过循环伏安法和计时电流法证明固定在Eastman-AQ阳离子交换树脂中的N-甲基吩嗪有效地在辣根过氧化物酶和玻碳电极之间传递电子.由于该生物传感器对过氧化氢具有良好的生物电催化还原的功能,所以将它与葡萄糖氧化酶和半乳糖苷酶结合,制备成双酶和三酶体系的生物传感器,用于葡萄糖和乳糖的测定.该生物传感器具有灵敏度高、响应快、响应范围宽及选择性好等优点.对糖尿病患者的血糖测定结果与采用葡萄糖氧化酶和辣根过氧化物酶的分光光度法的结果一致.     

葡萄糖传感器及其应用

着重阐述了二种固定化酶膜的制备方法:一种是明胶包埋的固定化葡萄糖氧化酶膜;一种是白蛋白共交联的固定化葡萄糖氧化酶膜。介绍了葡萄糖传感器在临床血糖测定、发酵工业中葡萄糖含量测定、油菜籽中硫代葡萄糖甙测定上的应用。     

醋酸纤维素膜为基础的葡萄糖生物传感器的研制

用共价法将酶固定在醋酸纤维素膜上,方法简便易行,制造的酶膜稳定,比活力高。同时采用该方法制备了葡萄糖氧化酶酶膜,与氧电极组装成测定葡萄糖的生物传感器,线性范围为50~800mg／dl,仪器工作的最适pH为6．0,最适温度为40℃。将该膜与过氧化氢电极组装得到的传感器具有以下特性：线性范围为10~200mg／dl,最适pH为6．0,测定结果与酶试制盒有良好相关性。     

蔗糖—葡萄糖双功能酶传感器的研究

结合蔗糖转化酶酶管与葡萄糖氧化酶－葡萄糖变旋酶双酶电极构成一种新的蔗糖传感器。该传感器可以分别用于蔗糖及葡萄糖的测定。蔗糖经酶管作用产生α－Ｄ－葡萄糖,再用ＧＯＤ－ＭＵＴ双酶电极定糖。若是样品中蔗糖和葡萄糖共存,比较样品流经不同路径时传感器的响应值,可以排除葡萄糖对蔗糖测定的干扰。传感器的最适ｐＨ和温度范围分别为：５．０－６．５和３０－４０℃,在稳态法实验中,传感器的线性范围为：２．５×１０＾－４     

二茂铁－交联剂修饰的葡萄糖传感器

用年清白蛋白－戊二醛交联剂把葡萄糖氧化酶固定在Ｎａｆｉｏｎ－二茂铁修饰电极上,最后在电极上修饰一层Ｎａｆｉｏｎ膜,制备成葡萄糖传感器,电活物质如抗坏血酸,尿酸等对葡萄糖的测定无干扰。该传感器的线性范围为５．０×１０＾－＾４－１．３×１０＾－＾２ｍｏｌ／Ｌ,响应时间小于６０ｓ。     

磁化水治疗糖尿病揭谜

磁化水能预防和治疗各种结石和血栓、动脉硬化、高血压、小儿蛔虫病、糖尿病等。所有这些都是在一些临床观察下取得的结果,许多结论都缺乏必要的理论依据。例如对于治疗糖尿病的生理生化机制,目前仍不清楚。针对这些问题,我们对磁化水进行了生物化学方面的研究,发现磁化水对人体内许多生物活性酶的活性有激活作用,如葡萄糖氧化酶等。患有糖尿病的人表现为血糖含量过高,而催化葡萄糖分解的正是与葡萄糖有关的各种活性酶,磁化水对酶的激活作用,能大大加快血液中葡萄糖的代谢,降低血糖含量,达到缓解、治疗糖尿病的效果。为了模仿人体…     

苯醌修饰葡萄糖氧化酶电极

本文用苯醌作为电子传递介体,石墨电极为基础电极。葡萄糖氧化酶和苯醌吸附在石墨电极表面,再用戊二醛交联固定。制成的酶电极以电流法测定底物葡萄糖浓度,其线性响应范围为(0－15mmol/L)。本工作测定了介体改良酶电极对葡萄糖的响应值,酶电极的pH范围,介体的循环伏安图谱,以及温度对酶电极的影响。     

PVA－葡萄糖氧化酶膜制备的研究

合成了新的光交联载体PVA—sbQ—GA,通过物理包理和化学键合相结合的办法固定了葡萄糖氧化酶。对SbQ和GA在载体中量及光照时间进行了优化试验,酶膜活力回收达40％。与氧电极结合制成葡萄糖传感器,对其性能进行了测定。     

尿素氮-葡萄糖双功能分析仪的研究

由固定化脲酶、谷氨酸脱氢酶、谷氨酸氧化酶、葡萄糖氧化酶的复合酶膜组成的双电极系统,可以同时测定尿素氮和葡萄糖的含量,每次进样量为25μl,20s即可测定出尿素氮和葡萄糖的含量。在0～60mg/dl尿素氮、0～500mg/dl葡萄糖范围内具有良好的线性关系。连续测定20次的变异系数分别为1.02％和1.05％。酶膜使用寿命为两星期以上。此仪器可广泛应用于临床检验和体育训练中。     

Influence of Temperature on Glucose Utilization by Pseudomonas fluorescens

The influence of temperature on the conversion of glucose into cell material and into energy for maintenance was determined for Pseudomonas fluorescens by a steady-state turbidity method and by a substrate utilization method. Conversion of glucose into cell material was measured as yield; conversion of glucose into energy for maintenance was measured as specific maintenance, the minimum dilution rate in continuous culture below which a steady state is not possible. The values obtained by the two methods were nearly identical; with both, the yield and specific maintenance decreased with decreasing temperature. The specific maintenance consumption rate (milligrams of glucose taken up per milligram of cell dry weight per hour at zero growth) was also calculated by the substrate utilization method and found to decrease with decreasing temperature. However, the amount of glucose consumed per generation for maintenance increased with decreasing temperature. This increased glucose consumption for maintenance may provide a partial explanation for the decrease in yield at low temperatures. Small amounts of glucose were also converted into pigment at all temperatures tested, with the greatest amount formed at 20 C.     

Glucose determination using immobilized polyphosphate glucokinase.

Polyphosphate glucokinase (EC 2.7.1.63, polyphosphate:glucose phosphotransferase) was covalently coupled to collagen-coated silica gel beads. The immobilized enzyme, as a packed-bed reactor, was used to determine glucose in serum and other samples. The method was based on a spectrophotometric measurement of NADPH produced by two consecutive reactions, similar to the hexokinase method. The described approach takes advantage of the greater stability of polyphosphate compared to that of ATP, the greater specificity of polyphosphate glucokinase versus that of hexokinase, and the reusability of the immobilized enzyme. Linearity, precision, and accuracy of the method were tested and found to be very good. The results were linear between 10 and 50 nmol of glucose in a 50-microliter sample and the coefficient of variation was less than 4% in five successive determinations. The recovery of glucose was about 100% after calibration of the method. The results of the measurements correlated well with those obtained with soluble polyphosphate glucokinase (r = 0.997, y = 1.036x - 0.016). The immobilized-enzyme reactor showed good operational stability during a month of use, losing about 12% of its initial activity.     

Continuous Monitoring of Extracellular Glucose Concentrations in the Striatum of Freely Moving Rats with an Implanted Glucose Biosensor

Abstract: We have used a glucose oxidase-based sensor implanted in the striatum of freely moving rats to determine the concentration of extracellular glucose in two distinct ways. With a modification of the zero net flux method, in which different concentrations of glucose are infused through a dialysis probe glued to the biosensor, we calculated the concentration at which there was no change in glucose current by regression analysis; this gave a concentration of 0.351 ± 0.016 m M . Calculating the concentration from the basal current and the in vitro calibration of the biosensor was not significantly different from this. The basal extracellular glucose concentration determined by either method remained constant over a period of several days. Infusion of 50 µ M veratridine through the adjacent dialysis probe caused a steep decrease in glucose current as soon as the drug reached the brain in contrast to the delayed fall (7.5 min) seen with microdialysis in previous experiments from this laboratory. These results demonstrate that this biosensor provides a direct, real-time measure of the extracellular concentration of glucose.     

Comparative Glucose Catabolism of Xanthomonas Species

Glucose catabolism in eight Xanthomonas species has been comparatively examined by means of the radiorespirometric method. The basic mechanisms for the respective xanthomonads closely resembled each other. The Entner-Doudoroff pathway, in conjunction with the tricarboxylic acid cycle pathway, was the predominant mechanism for glucose catabolism. A small portion (8 to 16%) of substrate glucose was routed into the pentose phosphate pathway. The hexose cycle pathway did not appear to play any significant role in glucose catabolism of these xanthomonads. The results are also consistent with the well-recognized close phylogenic relationship between xanthomonads and pseudomonads.     

Effects of Hyperprolactinemia on Plasma Prolactin and Glucose and on Local Cerebral Glucose Utilization

Elevated blood levels of prolactin increase the synthesis, turnover, and release of 3,4-dihydroxyphenylethylamine (dopamine) from the tuberoinfundibular dopaminergic neurons, which project to the median eminence. The present study examined whether hyperprolactinemia also increases local cerebral glucose utilization, as determined by the 2-deoxy-D-[1-14C]glucose method, in the median eminence and other brain structures. Adult male rats were given ovine prolactin (4 mg/kg) subcutaneously every 8 h for 48 h. This treatment exerted an autoregulatory feedback effect on endogenous rat prolactin secretion, as evidenced by decreased circulating levels of rat prolactin. Ovine prolactin treatment also decreased plasma glucose concentrations. However, in both partially immobilized and free-ranging rats, glucose utilization in brain structures containing tuberoinfundibular dopaminergic cell bodies (the arcuate nucleus) and terminals (the median eminence) was not affected by ovine prolactin treatment. Hyperprolactinemia was, however, associated with decreased glucose utilization in the medial forebrain bundle and the CA subfield of the dorsal hippocampus. The lack of a significant effect of prolactin treatment on glucose utilization in the median eminence indicates that the resolution of the deoxyglucose technique, as used here, is not adequate to detect the ovine prolactin-induced increase in tuberoinfundibular dopaminergic neuronal activity, that the median eminence does not utilize glucose as its primary energy substrate, or that ovine prolactin treatment causes a counterbalancing decrease in the activity of other neurons projecting to the median eminence.     

Effect of Portal Glucose Sensing on Systemic Glucose Levels in SD and ZDF Rats

BackgroundThe global epidemic of Type-2-Diabetes (T2D) highlights the need for novel therapeutic targets and agents. Roux-en-Y-Gastric-Bypass (RYGB) is the most effective treatment. Studies investigating the mechanisms of RYGB suggest a role for post-operative changes in portal glucose levels. We investigate the impact of stimulating portal glucose sensors on systemic glucose levels in health and T2D, and evaluated the role of sodium-glucose-cotransporter-3 (SGLT3) as the possible sensor.MethodsSystemic glucose and hormone responses to portal stimulation were measured. In Sprague-Dawley (SD) rats, post-prandial state was simulated by infusing glucose into the portal vein. The SGLT3 agonist, alpha-methyl-glucopyranoside (αMG), was then added to further stimulate the portal sensor. To elucidate the neural pathway, vagotomy or portal denervation was followed by αMG+glucose co-infusion. The therapeutic potential of portal glucose sensor stimulation was investigated by αMG-only infusion (vs. saline) in SD and Zucker-Diabetic-Fatty (ZDF) rats. Hepatic mRNA expression was also measured.ResultsαMG+glucose co-infusion reduced peak systemic glucose (vs. glucose alone), and lowered hepatic G6Pase expression. Portal denervation, but not vagotomy, abolished this effect. αMG-only infusion lowered systemic glucose levels. This glucose-lowering effect was more pronounced in ZDF rats, where portal αMG infusion increased insulin, C-peptide and GIP levels compared to saline infusions.ConclusionsThe portal vein is capable of sensing its glucose levels, and responds by altering hepatic glucose handling. The enhanced effect in T2D, mediated through increased GIP and insulin, highlights a therapeutic target that could be amenable to pharmacological modulation or minimally-invasive surgery.     

Mathematical Modeling of Renal Tubular Glucose Absorption after Glucose Load

A partial differential Progressive Tubular Reabsorption (PTR) model, describing renal tubular glucose reabsorption and urinary glucose excretion following a glucose load perturbation, is proposed and fitted to experimental data from five subjects. For each subject the Glomerular Filtration Rate was estimated and both blood and urine glucose were sampled following an Intra-Venous glucose bolus. The PTR model was compared with a model representing the conventional Renal Threshold Hypothesis (RTH). A delay bladder compartment was introduced in both formulations. For the RTH model, the average threshold for glycosuria varied between 9.90±4.50 mmol/L and 10.63±3.64 mmol/L (mean ± Standard Deviation) under different hypotheses; the corresponding average maximal transport rates varied between 0.48±0.45 mmol/min (86.29±81.22 mg/min) and 0.50±0.42 mmol/min (90.62±76.15 mg/min). For the PTR Model, the average maximal transports rates varied between 0.61±0.52 mmol/min (109.57±93.77 mg/min) and 0.83±0.95 mmol/min (150.13±171.85 mg/min). The time spent by glucose inside the tubules before entering the bladder compartment varied between 1.66±0.73 min and 2.45±1.01 min.The PTR model proved much better than RTH at fitting observations, by correctly reproducing the delay of variations of glycosuria with respect to the driving glycemia, and by predicting non-zero urinary glucose elimination at low glycemias. This model is useful when studying both transients and steady-state glucose elimination as well as in assessing drug-related changes in renal glucose excretion.     

Glucose Metabolism in Neisseria gonorrhoeae

The metabolism of glucose was examined in several clinical isolates of Neisseria gonorrhoeae. Radiorespirometric studies revealed that growing cells metabolized glucose by a combination on the Entner-Doudoroff and pentose phosphate pathways. A portion of the glyceraldehyde-3-phosphate formed via the Entner-Doudoroff pathway was recycled by conversion to glucose-6-phosphate. Subsequent catabolism of this glucose-6-phosphate by either the Entner-Doudoroff or pentose phosphate pathways yielded CO(2) from the original C6 of glucose. Enzyme analyses confirmed the presence of all enzymes of the Entner-Doudoroff, pentose phosphate, and Embden-Meyerhof-Parnas pathways. There was always a high specific activity of glucose-6-phosphate dehydrogenase (EC 1.1.1.49) relative to that of 6-phosphogluconate dehydrogenase (EC 1.1.1.44). The glucose-6-phosphate dehydrogenase utilized either nicotinamide adenine dinucleotide phosphate or nicotinamide adenine dinucleotide as electron acceptor. Acetate was the only detectable nongaseous end product of glucose metabolism. Following the disappearance of glucose, acetate was metabolized by the tricarboxylic acid cycle as evidenced by the preferential oxidation of [1-(14)C]acetate over that of [2-(14)C]acetate. When an aerobically grown log-phase culture was subjected to anaerobic conditions, lactate and acetate were formed from glucose. Radiorespirometric studies showed that under these conditions, glucose was dissimilated entirely by the Entner-Doudoroff pathway. Further studies determined that this anaerobic dissimilation of glucose was not growth dependent.     

Contribution of Glucose Phosphorylation to Glucose Metabolism in Brevibacterium fuscum

When the ‘dihydroxyacetone-fermentation’ was carried out in a steady state by the cells of Br. fuscum, it was suggested that the consumption rate of glucose in the medium might be regulated at the initial stages of glucose degradation such as; (a) glucose isomerization, (b) glucose dehydrogenation, and (c) glucose phosphorylation. Of these three enzymatic reactions, the isomerization and the dehydrogenation were proved to be unable to occur or negligible in vivo. So, in consideration of the pool sizes of Mg^{+ +}, Pi, H⁺, glucose, G6P*, ATP, ADP, etc., the intracellular glucokinase** activity was calculated. Results indicate that glucokinase reaction may be the limiting factor for direct glucose metabolism in Br. fuscum.     

Microcalorimetric Study of Glucose Permeation in Microbial Cells

A microcalorimetric method for measuring the influence of extracellular glucose concentration on the rate of catabolism is described. This method has been applied to anaerobically growing cultures of Zymomonas mobilis and of a respiratory-deficient ("petite") mutant of Saccharomyces cerevisiae (strain YFa). The Michaelian kinetics recorded with both organisms were apparently related to glucose transport. With Z. mobilis, it was found that, in the range of glucose concentrations at which this organism was growing exponentially, cell activity was limited by the maximal rate of the catabolic enzymes; at lower concentrations, glucose transport was the rate controlling step. The metabolic activity of yeast always depended on external glucose concentration; when this was lowered under a threshold, a change of kinetics took place. The microcalorimetric method described seems to be widely applicable to kinetic studies of the permeation of metabolizable substrates in microorganisms.