Enzymatic Fluorimetric Method Research Articles

Determination of polyphenolic content in beverages using laccase, gold nanoparticles and long wavelength fluorimetry

An enzymatic fluorimetric method for the determination of polyphenol compounds in beverages is described, which is based on the temporal inhibition caused by these compounds on the oxidation of the long wavelength fluorophor indocyanine green ( λ ex 764 nm, λ em 806 nm), in the presence of the enzyme laccase and positively charged gold nanoparticles (AuNPs). The oxidation of the dye gives rise to a fast decrease in its fluorescence, but it is delayed by the polyphenol, obtaining a time period directly proportional to its concentration, which has been used as the analytical parameter. The behaviour of several benzenediols and benzenetriols in the system and the modification of the activity of the enzyme by its interaction with AuNPs have been studied. The system has been optimized using gallic acid as a polyphenol model, but the dynamic ranges of the calibration graphs and the detection limits for several of the polyphenols assayed were obtained (μmol L −1): gallic acid (0.13–5, 0.04), catechol (0.08–5, 0.01), hydroquinone (0.05–2, 0.01), hydroxyhydroquinone (0.09–5, 0.03), pyrogallol (0.17–5, 0.04). Most of the values of the regression coefficients were 0.999 and the precision of the method, expressed as RSD% and checked at two concentration levels of each analyte, ranged between 1.8 and 5.6%. The method has been applied to the determination of polyphenol content in several foodstuff samples and the results compared with those obtained with the standard Folin–Ciocalteu method.

An enzymatic-fluorimetric method for monitoring of ethanol in ambient air

A method is described for the continuous monitoring of ethanol in ambient air. The system consists of a scrubber coil for enrichment of the analyte from air in an aqueous solution and a directly connected fluorescence detector. Because of using a reagent solution containing alcohol dehydrogenase (ADH) and nicotinamide adenine dinucleotide (NAD+) for absorption, ethanol can react directly with ADH and NAD+ during air sampling, producing NADH, which can be measured by fluorescence detection. The influence of reagent concentrations, gas flow rate and scrubber solution flow rate on the performance of the instrument was tested. Possible ozone interferences can be avoided by placing a KI coated filter in front of the scrubber inlet. The response time of the system was found to be 2.3 min and the detection limit about 1 ppbV. The applicability of the developed method was demonstrated during a field campaign in Brazil.

DISTRIBUTION AND INHIBITION OF NEUTRAL METALLOENDOPEPTIDASE (NEP) (EC 3.4.24.11), THE MAJOR DEGRADATIVE ENZYME FOR ATRIAL NATRIURETIC PEPTIDE, IN THE RAT KIDNEY

1. Atrial natriuretic peptide (ANP) is degraded by neutral metalloendopeptidase (NEP) (EC 3.4.24.11) and the kidney is a major site of ANP clearance. 2. The regional distribution of NEP in the rat kidney was investigated. 3. The activity of NEP, measured with an enzymatic fluorimetric method employing N-dansyl-D-alanyl-glycyl-L-4-nitrophenylalanyl-glycine as a synthetic substrate, was 18 times and eight times higher in the outer stripe of the medulla and inner cortex than in the outer cortex (OC). 4. Low concentrations of NEP were found in the OC, inner stripe and inner medulla. 5. NEP activity in the rat kidney was inhibited by the specific NEP inhibitors (SCH39370, phosphoramidon and thiorphan) at micromolar concentrations. 6. The present result suggests that degradation of ANP by NEP occurs mainly in the proximal tubules of the juxtamedullary nephrons, rather than cortical nephrons, and that the convoluted tubule in the OC is not a major site of location of NEP. 7. The relationship between NEP activities in the kidney in vitro and plasma clearance of ANP in vivo remains to be clarified.

Enzymatic Fluorimetric Determination of Ursodeoxycholic Acid in Urine Using Clostridium Absonum 7β-Hydroxysteroid Dehydrogenase

Abstract A simple and rapid enzymatic fluorimetric method for the determination of ursodeoxycholic acid (UDCA) and its glycine (GUDCA) and taurine (TUDCA) conjugates in urine has been developed. Octadecylsilane-bonded silica cartridges (Sep-Pak C18) are used for the solid-phase extraction of bile acids (BA) from urine samples. the method is based on the fluorimetric monitoring of NADPH formed via the reaction of 7β hydroxylated BA (7β-BA) with β-nicotinamide adenine dinucleotide phosphate (β-NADP+) catalysed by 7β hydroxysteroid dehydrogenase (7β-HSD). the 7β-HSD, which is not yet commercially available, was isolated from Clostridium absonum cultures (ATCC # 27555) and purified by affinity chromatography. The method has a limit of detection of 2 μmol/L (initial sample concentration), within-run precision varied from 8.3% to 5.3% and between-run precision varied from 12% to 1.8% for low and high concentrations respectively. the recovery of ursodeoxycholic acid added to urine samples was about 98% (range 88...

A simple enzymatic fluorimetric method for the determination of branched-chain l-amino acids in microlitre volumes of plasma

Existing methods for the estimation of the branched-chain amino acids (BCAA) normally require sophisticated, expensive instrumentation. An alternative is an enzymatic spectrophotometric method but this requires relatively large volumes of blood and is rather time-consuming. A simple enzymatic fluorimetric method for the measurement of the BCAA in microlitre samples of plasma is described here. The method is based on the oxidative deamination of l-leucine, l-isoleucine and l-valine by leucine dehydrogenase from Bacillus species. The NADH which is formed in stoichiometric quantities is estimated fluorimetrically. In the presence of the ketone-trapping agent hydrazine the reaction goes to completion in an alkaline incubation medium at 37°C. By this method the combined BCAA can be measured routinely in a 20 μl sample of plasma. The test exhibits acceptable precision and reproducibility.

A simple enzymatic fluorimetric method for the determination of triglycerides in 10 μl of serum

An assay for the determination of triglyceride (acyl glycerol) in microlitre volumes of serum or plasma is described. The method is based on enzymatic hydrolysis of triglyceride by lipase from Rhizopus arrhizus followed by enzymatic fluorimetric assay of the glycerol so released. Under the conditions of the assay the enzymatic hydrolysis is complete for concentrations up to 5 mmol/l in less than 15 min at room temperature. The determination of free glycerol and glycerol released is based on the formation of dihydroxyacetone in the presence of NAD and glycerol dehydrogenase. The NADH which is formed in stoichiometric quantities is estimated fluorimetrically. In the presence of the ketone-trapping agent hydrazine the reaction goes to completion above pH 9.0. By this method acyl glycerol can be measured routinely in a 10 μ1 sample of serum or plasma. The test is extremely sensitive compared to previously described methods and exhibits acceptable precision and reproducibility.

Semiautomatic bioluminescence determination of glycerol using a computer controlled luminescence analyser (Berthold LB 950 T).

A semiautomatic determination of glycerol is described, in which luminescence produced by bacterial NADH-linked luciferase is measured by an automatic luminescence analyser (Berthold LB 950 T). The glycerol determination is based on the enzymatic conversion of glycerol to 3-phosphoglycerate, made irreversible by the presence of arsenate. NADH, formed in the glycerol-3-phosphate and glyceraldehyde-3-phosphate dehydrogenase reactions, is subsequently determined by the bacterial luciferase system. Stable kinetics of light emission were obtained by reducing the catalytic concentration of NAD(P)H: FMN oxidoreductase from 85 U/1 to 8.5 U/1. This method was applied to serum samples and validated by comparison with an enzymatic fluorimetric method. The new method is approximately 10 times more sensitive than the fluorimetric one. Moreover, it is simpler, more convenient, less time consuming and also less expensive than spectrophotometric, fluorimetric or radiochemical methods used for glycerol determination.

Enzymatic fluorimetric determination of the individual bile acids in blood serum

An enzymatic fluorimetric method is described for the determination of total bile acids (cholic acid and deoxycholic acid), primary bile acids (cholic and chen acids and individual bile acids in serum without prior separation of the acids. Total and primary bile acids are determined by equilibrium procedures by conver of the 3α- and 7α-hydroxy bile acids to 3-oxo and 7-oxo bile acids by α-NAD +, in the presence of 3α- and 7α-hydroxysteroid dehydrogenase (HSD), respectively, and measurement of the generated NADH fluorimetrically. Chenodeoxycholic acid is determined with 7α-HSD in the presence of cholic and deoxycholic acids by a differential kinetic procedure, and cholic and deoxycholic acids are calculated by difference. Interferents are removed by treatment of serum with Sachrom rein. Only 1.00 ml of serum is required. Low cost, simplicity and reliability are the main features of the method. The recovery of bile acids added to serum averaged 103% (range 83–122%). The method is suitable for routine use in small clinical laboratories.

Enzymatic fluorimetric determination of cholic acid and chenodeoxycholic acid in aqueous solutions and blood serum using a differential kinetic method

An enzymatic fluorimetric method is described for the determination of chenodeoxycholic acid and its conjugates and of cholic acid and its conjugates in aqueous solutions and serum. The method is based on the oxidation of 7 α-hydroxy bile acids by β-NAD + in the presence of 7 α-hydroxysteroid dehydrogenase; the NADH produced is monitored fluorimetrically. Chenodeoxycholic acid is determined in the presence of cholic acid by a differential kinetic procedure; the sum of the two acids (primary bile acids) is determined by an equilibrium procedure, and cholic acid is calculated by difference. The r.s.d. was ca. 3% and 10% for aqueous solutions and sera, respectively. Recoveries of chenodeoxycholic acid, cholic acid and primary bile acids added to serum samples averaged 100.5, 105.1, and 102.9%, respectively. Ten samples can be analyzed per working day.

Determination of total bile acids in serum. A comparison of a radioimmunoassay with an enzymatic-fluorimetric method

A solid phase radioimmunoassay kit method for total conjugated bile acids has been compared to an enzymatic fluorimetric method for total serum bile acids. The methods were compared with respect to: precision, cross-reactivity (molar equivalence) of different bile salts, recovery of different bile salts from serum, the reference range for a healthy population, linearity, coefficient of correlation, diagnostic effectiveness, cost and ease of assay. Both assays seemed equally capable of predicting the presence or absence of liver disease. Radioimmunoassay had little advantage over the enzymatic-fluorimetric method. Its relative ease was far outweighed by its greater cost and poorer analytical performance.

Serum Bile Acid Concentrations in Patients with Liver Disease

An enzymatic-fluorimetric method using a highly purified 3alpha-hydroxysteroid dehydrogenase (Sterognost-3alpha, Nyco) was used to determine fasting serum bile acid concentrations on 49 occasions in 43 patients with various liver diseases. A two-hour postprandial bile acid determination was carried out on 29 occasions in 27 of the patients. Fasting bile acid concentration correlated significantly both in cholestatic hepatobiliary and in parenchymatous liver disease to serum bilirubin and aspartate aminotransferase (ASAT) but not to alanine aminotransferase (ALAT), alkaline phosphatase, or albumin. The two-hour postprandial bile acid concentration was above normal in all patients with biochemical and/or histological signs of hepatobiliary disease, also when fasting concentration was within normal limits. In parenchymatous liver disease correlations existed between the two-hour postprandial bile acid concentration and bilirubin, ASAT, and ALAT. The sensitivity of serum bile acid estimation was compared to other liver function tests. Both the fasting and the postprandial serum bile acid concentrations tended to be more sensitive tests of hepatobiliary disease than bilirubin, ASAT and ALAT.