The inhibition of fluorescence resonance energy transfer between quantum dots for glucose assay

Fluorescence resonance energy transfer (FRET) between two quantum dots of different sizes causes fluorescence quenching. Hereby a binding site pre-blocking approach is proposed to avoid this effect. Pre-binding of glucose on the donor occupies the binding sites and thus blocks resonance energy transfer between the two quantum dots, protecting the fluorescence from being quenched. A glucose assay is developed based on this approach. The glucose content is correlated with the fluorescence difference in the absence and in the presence of glucose. In practice, Green QDs-Con A conjugates are used as donors and Red QDs-NH(2)-glu conjugates as acceptors to form FRET system. The inhibition of fluorescence quenching is then measured in the presence of glucose. A linear calibration graph is achieved within 0.1-2.0 mmolL(-1), along with a detection limit of 0.03 mmolL(-1) and a RSD of 2.1% (1.0 mmolL(-1)). 91-105% of glucose in serum and urine samples is recovered. It is worth mentioning that the present glucose assay approach also generates a fluorescence chromatic difference imaging, and the color display clearly identifies the glucose contents by visual detection with a distinguishing ability of ca. 0.5 mmolL(-1). The present approach can potentially be used for the clinical determination of glucose in biological samples which can be further developed into a glucose sensor.     

Estimation of total and direct serum bilirubin using modified micro assay method

Estimation of total and direct bilirubin in serum plays an important role in differential diagnosis of hyperbilirubinemia. Several direct spectrophotometric methods are commercially available for total and direct bilirubin estimation in which the amount of the sample (serum) varies from 200 ml to 800 ml. It is difficult to collect such amount of serum from infants, as neonatal jaundice is the most common problem in this age group. To overcome this problem modified micro assay method was developed using dimethylsulfoxide (DMSO). The amount of the serum sample is reduced from 100 ml to 20 ml per test for both total and direct bilirubin. A method comparison study was performed using 100 consecutive serum samples, by modified micro assay method and a reference Jendrassik-Grof method. Total bilirubin in these human serum samples ranged from 0.4-15.0 mg/dl and direct bilirubin ranged from 0.05-12.0 mg/dl. The results conclude that modified micro assay method had significant correlation with r-value of 0.99989 for total serum bilirubin and with r-value of 0.99971 for direct serum bilirubin. Linearity of the method is 20 mg/dl and 15 mg/dl for total and direct bilirubin, respectively. Monoreagent used during the assay is stable for 24 hours at 2-8 degrees C while the kit is stable for one year at 2-8 degrees C. In conclusion this micro assay method is rapid, reliable, simple and accurate for the estimation of total and direct bilirubin with small serum quantities. It is equally reliable for manual; semi automated and automated chemistry analyzers.     

Spectrophotometric determination of serum nitrite and nitrate by copper-cadmium alloy

A macro and micro assay for the spectrophotometric determination of serum nitrite and nitrate was developed. Nitrite/nitrate in biological samples can be estimated in a single step by this method. The principle of the assay is the reduction of nitrate by copper-cadmium alloy, followed by color development with Griess reagent (sulfanilamide and N-naphthylethylenediamine) in acidic medium. This assay is sensitive to 1 microM nitrate and is suitable for different biological fluids, including sera with a high lipid concentration. The copper-cadmium alloy used in the present method is easy to prepare and can completely reduce nitrate to nitrite in an hour. The present method provides a simple, cost-effective assay for the estimation of stable oxidation products of nitric oxide in biological samples.     

Glycosylated hemoglobin as a stable alternative to serum glucose in white-tailed deer

We compared serum glucose concentration and percent glycosylated hemoglobin (GH) in captive and wild white-tailed deer (Odocoileus virginianus) to determine stability of glucose relative to GH. Temporal changes in levels of serum glucose and GH were ascertained from serial blood samples collected from three captive deer over a 2-week period. State of glycemia also was determined for 17 wild deer that were collected from three populations in southeastern Oklahoma and southwestern Arkansas (USA). Concentration of serum glucose of captive deer decreased (P = 0.04) from 190.4 to 155.8 mg/dl over the 2 weeks; percent GH did not differ temporally (P = 0.30). Percent GH of wild deer did not differ (P = 0.23) when deer were separated into 2 groups (high and low state of glycemia) based on the median serum glucose concentration. We found a significant difference (P = 0.04) in percent GH among wild deer populations; serum glucose concentration did not differ (P = 0.72) among populations. Our results indicate that percent GH is more stable than serum glucose concentration and may be useful in population comparisons of nutritional condition.     

Radiometric assays for glycerol, glucose, and glycogen

We have developed radiometric assays for small quantities of glycerol, glucose and glycogen, based on a technique described by Thorner and Paulus (1971, J. Biol. Chem. 246, 3885-3894) for the measurement of glycerokinase activity. In the glycerol assay, glycerol is phosphorylated with [32P]ATP and glycerokinase, residual [32P]ATP is hydrolyzed by heating in acid, and free [32P]phosphate is removed by precipitation with ammonium molybdate and triethylamine. Standard dose-response curves were linear from 50 to 3000 pmol glycerol with less than 3% SD in triplicate measurements. Of the substances tested for interference, only dihydroxyacetone gave a slight false positive signal at high concentration. When used to measure glycerol concentrations in serum and in media from incubated adipose tissue, the radiometric glycerol assay correlated well with a commonly used spectrophotometric assay. The radiometric glucose assay is similar to the glycerol assay, except that glucokinase is used instead of glycerokinase. Dose response was linear from 5 to 3000 pmol glucose with less than 3% SD in triplicate measurements. Glucosamine and N-acetylglucosamine gave false positive signals when equimolar to glucose. When glucose concentrations in serum were measured, the radiometric glucose assay agreed well with hexokinase/glucose-6-phosphate dehydrogenase (H/GDH)-based and glucose oxidase/H2O2-based glucose assays. The radiometric method for glycogen measurement incorporates previously described isolation and digestion techniques, followed by the radiometric assay of free glucose. When used to measure glycogen in mouse epididymal fat pads, the radiometric glycogen assay correlated well with the H/GDH-based glycogen assay. All three radiometric assays offer several practical advantages over spectral assays.     

Enzymatic microtiter plate-based nitrate detection in environmental and medical analysis

Our microtiter plate assay is based on the enzymatic reduction of nitrate by dissimilatory nitrate reductase from Pseudomonas stutzeri [EC 1.7.99.4]. Exogenous redox mediators like methyl viologen, methylene blue, and cibachron blue were applied to reduce nitrate reductase. Concentrations of 0.02-0.9 mM nitrate can be detected with +/-6% standard deviation, by using a photometric Griess reaction for the formed nitrite. Nitrate reductase is stable in the pH range 6.5-9.0 and works in the temperature range 4-76 degrees C. The assay shows no interferences with salt content up to 1 M chloride or 11 mM chlorate, and serum albumin content up to 50 mg/ml. The time demand of our two-step procedure is 20 min/100 samples. Nitrate reductase could be conserved on site of the wells of microtiter plates for at least 6 months at room temperature. The nitrate assay was applied in environmental and consumer goods analysis, and for medical diagnostics in human plasma samples.     

Long-term stability of parameters of antioxidant status in human serum

Abstract

The antioxidant status of serum or plasma can be determined using several commercially available assays. Here, four different assays, total antioxidant status (TAS), its second-generation assay (TAS2), biological antioxidant potential (BAP), and enzymatic assay using horseradish peroxidase (EAOC), were applied on human serum samples to test the temperature stability of antioxidants, upon storage of serum for 12 months. The two or three most commonly used temperatures for storage, that is, ? 20, ? 70 (or ? 80), and ? 196°C, were selected. The general conclusion is that all assays were stable at the temperatures tested. In addition, there were almost no statistically significant differences between the samples stored at different temperatures. Only the rank order of the EAOC assay was not very good in samples stored at ? 20°C. Also three components contributing to the total antioxidant capacity, uric acid, creatinine and bilirubin, showed no statistically significant differences between the temperatures. Therefore, storage at ? 20°C is sufficient to maintain a proper assay outcome of most of the total antioxidant assays, although storage at ? 70/80°C is to be preferred for longer storage times.     

Thalidomide enantiomers: determination in biological samples by HPLC and vancomycin-CSP

Thalidomide is a racemate with potentially different pharmacokinetics and pharmacodynamics of the component (+)-(R)- and (-)-(S)-thalidomide enantiomers. As part of a project on the adjunctive effects of thalidomide and cytotoxic agents, a method for the chiral separation and quantitation of thalidomide was developed and validated. Thalidomide in relevant serum and tissue homogenate samples was stabilized by buffering with an equal volume of citrate-phosphate buffer (pH 2, 0.2M), and stored at -80 degrees C pending assay. The thalidomide enantiomers, extracted from the samples with diethyl ether, were well separated on a chiral HPLC column of vancomycin stationary phase and a mobile phase of 14% acetonitrile in 20 mM ammonium formate adjusted to pH 5.4; their concentrations were determined with phenacetin as internal standard at 220 nm detection. Over a thalidomide concentration range of 0.1-20 microg/ml, assay precision was 1-5% (CV) for both enantiomers, and calibration curves were linear with all correlation coefficients being >0.99. The estimated limit of quantification for both enantiomers was 0.05 microg/ml with 0.2-0.6 ml serum samples. Thalidomide in rat and human serum, acidified and stored as described above, was found to be chemically and chirally stable over 1 year. The method has been successfully applied to serum samples from human patients undergoing thalidomide treatment for mesothelioma, and to serum, blood and tissue samples from a laboratory rodent model using transplanted 9l gliosarcoma. Enantioselectivity in thalidomide pharmacokinetics has been found, thereby reinforcing the need for considering the relevance of chirality in thalidomide pharmacology.     

Antiamyloidogenic Effects of Ellagic Acid on Human Serum Albumin Fibril Formation Induced by Potassium Sorbate and Glucose

Oxidative stress has the main role in protein conformational changes and consequent direct involvement in different kind of diseases. Potassium sorbate as a widespread industrial preservative and glucose are two important oxidants that can be involved in oxidative stress. In this study the effect of ellagic acid as a phenolic antioxidant on amyloid fibril formation of human serum albumin upon incubation of potassium sorbate and glucose was studied using thioflavin T assay, surface tension, atomic force microscopy, Amadori product, and carbonyl content assays. The thioflavin T assay and atomic force microscopy micrographs demonstrated the antiamyloidogenic effect of ellagic acid on the human serum albumin fibril formation. This antioxidant also had the repair effect on surface tension of the modified human serum albumin (amyloid intermediates), which was destructed, caused by potassium sorbate and glucose. This mechanism takes place because of potent carbonyl stress suppression effect of ellagic acid, which was strengthening by potassium sorbate in the presence and absence of glucose.     

Effect of D,L-carnitine, acetyl-D,L-beta-methylcholine chloride and glycine betaine on some processes of carbohydrate metabolism of humans and goats.

Oral administration of carnitine in normal and diabetic subjects showed a marked decrease in the level of blood glucose during the oral glucose tolerance test (OGTT) except for the three hour samples in diabetic subjects, while a decrease in the level of subsequent blood pyruvate samples was observed during the OGTT in normal and diabetic subjects after the administration of carnitine. During the OGTT, the peak of blood glucose and blood pyruvate level was generally delayed in the diabetic subjects. Furthermore, the mean blood pyruvate levels were elevated above those of normal subjects during the late stages of the test. The mean levels of blood glucose and blood pyruvate of all samples after the administration of carnitine were significantly higher in diabetics than the corresponding values in noramls. Carnitine administration decreased the total blood amino acid nitrogen level only in diabetic subjects. Carnitine caused a highly significant increase in the activity of serum alanine aminotransferase in normal and diabetic subjects, while it had no effect on the activity of serum aspartate aminotransferase. In goats, the level of blood glucose during the intravenous glucose tolerance test (IVGTT) was not affected by carnitine (1,3 or 6 mg/kg body weight). Carnitine in all doses used had no effect on the total blood amino acid nitrogen during the IVGTT, or on the activity of serum alanine aminotransferase and serum aspartate aminotransferase in the fasting samples. Acetyl-D,L-beta-methylcholine had no effect on the level of blood glucose, total blood amino acid nitrogen, the activity of serum alanine aminotransferase or serum aspartate aminotransferase in normal and diabetic subjects. The level of blood pyruvate decreased both in normal and diabetic subjects, in the samples that represented the peak of the curve. Glycine betaine had no effect on blood glucose, pyruvate, total blood amino acid nitrogen and the activity of serum alanine aminotransferase or serum aspartate amino transferase in normal and diabetic subjects or in goats.     

Determination of biocytin

Biocytin (epsilon-N-[d-biotinyl]-L-lysine) is generally undetected in serum and other body fluids of normal healthy individuals in view of the ubiquitous distribution of biotinidase. It has been suggested that biocytin may be present in serum and urine of patients with inherited biotinidase deficiency. We have developed a noncompetitive assay for biocytin based on its interaction with avidin. Biocytin can be determined in biological samples containing both biotin and biocytin. Biotin from such samples is removed by an anion-exchange resin and the biocytin is determined by the avidin-binding assay. The effect of above-normal levels of biotin in the sample on the assay of biocytin is discussed.     

Development and validation of a method to determine the unbound paclitaxel fraction in human plasma

Paclitaxel is pharmaceutically formulated in a mixture of Cremophor EL and ethanol (1:1, v/v). The unbound fraction of the anticancer drug paclitaxel in plasma is dependent on both plasma protein binding and entrapment in Cremophor EL micelles. We have developed a simple and reproducible method for the quantification of the unbound paclitaxel fraction in human plasma. Human plasma was spiked with [3H]paclitaxel and [14C]glucose (unbound reference) and incubated at 37 degrees C for 30 min. Plasma ultrafiltrate was prepared by a micropartition system (MPS-1) and collected in a sample cup containing 100 microl of plasma to prevent the loss of paclitaxel due to adsorption. The radionuclides were separated after combustion of the biological samples using a sample oxidizer and the radioactivity was determined by liquid scintillation counting. The unbound fraction of paclitaxel was calculated by dividing the ratios of 3H and 14C in plasma ultrafiltrate and in plasma. The method was thoroughly validated using human plasma spiked with pharmacologically relevant concentrations of paclitaxel (10-1000 ng/ml) and Cremophor EL (0.25-2.0%). The method was precise, with a within-day precision ranging from 3.9 to 11.0% and a between-day precision ranging from 5.8 to 13.1%. In patient plasma with low serum albumin values containing 1% of Cremophor EL, the unbound fraction appeared to be significantly higher than that in plasma with normal albumin values. The determination of the unbound fraction of paclitaxel proved to be stable during a 10-week storage at -20 degrees C. Furthermore, the assay was applicable in patient samples. This assay can be used to determine the unbound fraction of paclitaxel in plasma. Moreover, its design should allow the determination of the unbound concentrations of other hydrophobic drugs.     

A colorimetric assay for the measurement of D-glucose consumption by cultured cells

A colorimetric method is described for measuring glucose consumption by tissue culture cells. This procedure, which utilizes the coupled activities of glucose oxidase and horseradish peroxidase, is insensitive to the spectral interferences caused by the phenol red and sera present in most tissue culture media. The spectral properties (absorbance maxima and apparent absorption coefficients) and stability of a large number of chromogenic horseradish peroxidase substrates were surveyed for their ability to perform in an assay for glucose in the presence of phenol red and sera components. One of these chromophores, the product of an oxidative couple between 4-aminoantipyrine and N-ethyl-N-sulfopropyl-m-toluidine, was subsequently used to develop a fixed time assay for glucose in media samples. The assay required only 10 microliters of media in a 1-ml assay volume; reproducibility studies showed variabilities of less than 5% in the assay of a single sample, and values obtained in glucose analyses correlated well with those obtained using commercially available test kits. The assay was used to study the rate of glucose consumption in two different cell types, bovine corneal endothelial cells and human diploid fibroblasts.     

A stable, radioactive substrate emulsion for assay of lipoprotein lipase.

A method is described for the assay of lipoprotein lipase, using a stable, radioactive substrate emulsion. Fatty acid-labeled trioleoylglycerol was emulsified by homogenization in glycerol with lecithin as detergent. This anhydrous emulsion was stable for at least six weeks. Substrate solutions for enzyme assay were prepared by diluting the emulsion with buffer containing serum and albumin. The fatty acid produced on hydrolysis was isolated in a one-step liquid-liquid partition system. Incubations with extracts of acetone powders from adipose tissue displayed characteristics of lipoprotein lipase activity, i.e., serum dependence and inhibition by NaCl and protamine. The method is rapid (less than 1 hour), sensitive and reproducible, and suitable for routine use.     

Diagnosis of active tuberculosis using MPB64, a specific antigen of Mycobacterium bovis

Because the incidence of tuberculosis (TB) is still high in developing countries, an inexpensive and rapid diagnostic test for this infection is needed. To develop a screening test for TB, MPB64 antigen was produced by recombinant technology and purified with a polyhistidine tag. Next, serum and urine samples from patients with TB and uninfected individuals were examined by the dot‐blot assay method using this purified antigen. Serum samples from patients with TB reacted more strongly with MPB64 antigen than did those from uninfected individuals. In addition, serum samples from TB patients with active infection reacted more strongly with the antigen than did samples from patients with inactive TB. When urine samples were assessed using this assay, similar results were obtained. Correlations between the data obtained from serum and urine samples were analyzed for all subjects, including uninfected individuals, and a strong positive correlation between the results of serum and urine tests (n = 36, r = 0.672) was found. The sensitivity and specificity of this assay for serum samples was 85.7 % and 85.0 %, and for urine samples 75.0 % and 85.0 %, respectively. These results suggest that dot‐blot assay with MPB64 antigen could be a useful screening test for active TB. Because urine samples can be obtained more easily than serum samples and because urine is less contagious, urine testing should probably be employed for screening purposes.     

Glucose determination in samples taken by microdialysis by peroxidase-catalyzed luminol chemiluminescence

An automatic, luminometric assay of glucose in samples of the extracellular water space obtained by microdialysis is described. The assay involves oxidation by glucose oxidase (EC 1.1.3.4) and mutarotation of glucose by aldose mutarotase (EC 5.1.3.3.). The H2O2 formed is subsequently determined in a reaction catalyzed by horseradish peroxidase (EC 1.11.1.7) using luminol as electron donor. The assay is linear between 0.01 and 1 nmol in the cuvette. The detection limit, defined as 3 standard deviations of the reagent blank, was 0.008 mumol/liter in the cuvette. A complete oxidation of glucose is obtained within 4 min and 25 samples are automatically assayed within 75 min. Addition of microdialysate sample obtained from human adipose tissue in vivo did not interfere with the standard curves. Glucose added to microdialysate resulted in a complete recovery compared to a H2O2 standard. Analytical interference from different factors was investigated. No interference was observed up to the following concentrations: 5 mumol/liter epinephrine, 1 mumol/liter norepinephrine, 100 mumol/liter insulin, 500 mumol/liter pyruvate, 50 mmol/liter lactate, and 1 mumol/liter ascorbate. The glucose values with the present method correlated strongly (r = 0.984) with values obtained using a routine method involving glucose oxidase and peroxidase.     

Hyperinsulinism after removal of a pheochromocytoma.

The finding of hypoglycemia after the surgical removal of a pheochromocytoma in two patients in a previous study led to monitoring of the serum glucose and plasma C-peptide levels in two other patients with a pheochromocytoma and one with unilateral adrenocortical hyperplasia. In the two patients with a pheochromocytoma endogenous insulin secretion, as measured by a C-peptide assay, was suppressed before removal of the tumours and resumed immediately after removal. The serum glucose levels decreased in these patients, but sufficient intravenous administration of glucose prevented postoperative hypoglycemia. In the patient with adrenocortical hyperplasia the plasma C-peptide level was not decreased before tumour removal, nor did it increase abruptly following removal. It therefore seems likely that the rapid fall in the serum glucose level following removal of a pheochromocytoma is caused by prompt resumption of beta-cell activity, with rebound hyperinsulinism.     

A colorimetric method for the estimation of serum glycated proteins based on differential reduction of free and bound glucose by sodium borohydride

A new colorimetric method based on the phenol-sulfuric acid reaction is described for the estimation of serum glycated proteins by the differential reduction of free glucose and hexose bound nonenzymatically with 2.0 and 20 mg of NaBH4 in 0.02 ml of serum, respectively, at room temperature for 15 min. The values (microgram hexose/mg protein) in control subjects (n = 60) and diabetics (n = 90) were estimated to be 5.60 +/- 0.85 and 10.8 +/- 1.6, respectively. The increase was highly significant (P less than 0.001) in diabetics. The serum glycated protein levels correlate well with fasting blood sugar values (r = 0.77, P less than 0.001, n = 25). There was also a highly significant correlation between glycated protein level and glycated albumin value in individual serum samples (r = 0.85, P less than 0.001, n = 25). Values of borohydride reducible glyco-groups bound to serum proteins also correlated well with serum glycated protein levels (r = 0.96, p less than 0.001, n = 20) determined by the thiobarbituric acid assay method. The method is found to be simple and rapid, with a coefficient of variations of +/- 3.8%.     

A homologous radioimmunoassay for guinea pig corticosteroid-binding globulin

A rapid, specific, and sensitive (requiring only 20 fmole of antigen equivalent to 0.007 microliter of serum) radioimmunoassay (RIA) was developed for the measurement of guinea pig corticosteroid-binding globulin (CBG). CBG was purified to homogeneity from guinea pig serum by affinity chromatography and used for immunization, as the standard and as the radiolabeled trace in the RIA. The antiserum to CBG was raised in rabbits. It was judged specific by immunoelectrophoresis and by comparison of RIA values with steroid-binding assay profiles obtained on serum separated on the basis of size and ion-exchange properties. The results of the radioimmunoassays agree with those of a steroid-binding assay run on identical samples. The sensitivity of the assay allows detection of CBG in serial serum samples, other biologic fluids such as milk, and cell culture supernatants.