Enzymic ethanol assay: a new colorimetric method based on measurement of hydrogen peroxide.

1987 ◽  
Vol 33 (4) ◽  
pp. 486-489 ◽  
Author(s):  
L Prencipe ◽  
E Iaccheri ◽  
C Manzati
Keyword(s):  
Hydrogen Peroxide ◽  
Gas Chromatography ◽  
Human Plasma ◽  
Colorimetric Method ◽  
Alcohol Oxidase ◽  
Blue Dye ◽  
Standard Curve ◽  
Analytical Recovery ◽  
Sample Dilution ◽  
Coupled Assay

Abstract We describe a new colorimetric method for measuring ethanol in plasma by use of a peroxidase-coupled assay system and alcohol oxidase (EC 1.1.3.13) from Pichia species. Absorptivity is low enough to give useful results without sample dilution. The procedure is also applicable to saliva samples and utilizes only one working reagent. The absorbance of the blue dye that is formed is measured at 600 nm. The standard curve for the method is linear for ethanol concentrations up to 4 g/L. Average analytical recovery of ethanol in human plasma exceeded 99%. Within-run and between-run CVs were less than 2.45% and less than 1.92%, respectively. Results correlate very well with those by gas chromatography (r = 0.9977). The method is adaptable to automation.

Enzymic creatinine assay: a new colorimetric method based on hydrogen peroxide measurement.

1983 ◽  
Vol 29 (8) ◽  
pp. 1494-1496 ◽  
Author(s):  
P Fossati ◽  
L Prencipe ◽  
G Berti
Keyword(s):  
Hydrogen Peroxide ◽  
Room Temperature ◽  
Colorimetric Method ◽  
Creatinine Concentration ◽  
Standard Curve ◽  
Urine Creatinine ◽  
Analytical Recovery ◽  
Human Sera ◽  
Fuller’S Earth ◽  
Sample Measurement

Abstract We describe a new colorimetric method for measuring creatinine in serum and urine. Creatinine hydrolysis is catalyzed by creatinine amidohydrolase, and the creatine so produced is assayed in reactions catalyzed sequentially by creatine amidinohydrolase and sarcosine oxidase in a system that generates hydrogen peroxide. The hydrogen peroxide is measured at 510 nm in a reaction catalyzed by horseradish peroxidase, with 3,5-dichloro-2-hydroxybenzenesulfonic acid/4-aminophenazone as the chromogen. This series of reactions is complete in 30 min at room temperature. A blank sample measurement corrects for endogenous creatine. The standard curve is linear for creatinine concentrations as great as 2.21 mmol/L. Analytical recovery of creatinine in human sera and urine averaged 99.8%. Within-run and between-run precision studies gave CVs of less than or equal to 3.3 and less than or equal to 4.3% for a serum with a creatinine concentration of 69 mumol/L. Results by this method agree well (r greater than 0.99) with those by both the enzymic ultraviolet method of Wahlefeld and the fuller's earth/Jaffé method.

Evaluation of a modified alcohol dehydrogenase assay for the determination of ethanol in blood.

1982 ◽  
Vol 28 (10) ◽  
pp. 2125-2127 ◽  
Author(s):  
A Poklis ◽  
M A Mackell
Keyword(s):  
Gas Chromatography ◽  
Reaction Time ◽  
Alcohol Dehydrogenase ◽  
Correlation Coefficients ◽  
Least Square ◽  
Standard Curve ◽  
Analytical Recovery ◽  
Sigma Chemical ◽  
Enzymic Assay

Abstract We evaluated a new alcohol dehydrogenase (EC 1.1.1.1) enzymic assay (ADH-glycine, Sigma Chemical Co.) for the determination of ethanol in blood. This assay differs from the manufacturer's previous assay (ADH-pyrophosphate) in that glycine replaces pyrophosphate as the buffer and hydrazine replaces semicarbazide as the trapping agent. The standard curve for the assay was linear over blood ethanol concentrations of 0.50-5.00 g/L. The reaction time of the assay was 10 min. At 1.00 g/L within-run and between-run CVs were 3.96% (n = 20) and 4.01% (n = 20), respectively. Mean analytical recovery of ethanol added to whole blood at 0.50-5.00 g/L was 99.7% (SD 2.6%). We performed 100 consecutive clinical and forensic determinations by the ADH-glycine assay, the ADH-pyrophosphate assay, and gas chromatography. Correlation coefficients of the results by least-square linear regression were 0.995 for ADH-pyrophosphate vs ADH-glycine, and 0.990 for gas chromatography vs ADH-glycine. The major advantage of the ADH-glycine assay over the ADH-pyrophosphate assay is the shorter reaction time, 10 min vs 30 min.

Improved radioimmunoassay of urinary estriol.

1979 ◽  
Vol 25 (1) ◽  
pp. 99-102 ◽  
Author(s):  
M J Jawad ◽  
E A Wilson ◽  
H L Kincaid
Keyword(s):  
Incubation Time ◽  
Colorimetric Method ◽  
Detection Range ◽  
Standard Curve ◽  
Double Antibody ◽  
Analytical Recovery ◽  
Urinary Glucose

Abstract We report a rapid double-antibody radioimmunoassay for urinary estriol. Advantages over other current methods include: (a) 30-min hydrolysis; (b) total incubation time, 55 min; (c) assay unaffected by urinary glucose; (d) no degradation of estriol evident during hydrolysis; (e) superior (85%) analytical recovery of estriol conjugates; (f) linear standard curve by logit-log extrapolation; (g) good correlation (r = 0.83) with total estrogen determination by a generally accepted colorimetric method; (h) only 20 muL of urine required; and (i) the detection range is 1.9 to 100.5 mg/24-h urine.

Enzymic urea assay: a new colorimetric method based on hydrogen peroxide measurement.

1989 ◽  
Vol 35 (4) ◽  
pp. 654-658 ◽  
Author(s):  
F Lespinas ◽  
G Dupuy ◽  
F Revol ◽  
C Aubry
Keyword(s):  
Hydrogen Peroxide ◽  
Horseradish Peroxidase ◽  
Glutamine Synthetase ◽  
Pyruvate Kinase ◽  
Present Method ◽  
Colorimetric Method ◽  
Reference Method ◽  
Urea Hydrolysis ◽  
Pyruvate Oxidase ◽  
Standard Curve

Abstract We describe a new enzymic colorimetric method in which urea is measured in serum by use of a single reagent mixture. Ammonia produced by urea hydrolysis, catalyzed by urease, reacts with glutamate and ATP in the presence of glutamine synthetase. The ADP so produced is assayed in reactions catalyzed sequentially by pyruvate kinase and pyruvate oxidase in a system that generates hydrogen peroxide. The hydrogen peroxide is measured at 500 or 550 nm in a reaction catalyzed by horseradish peroxidase, with phenol/4-aminophenazone as the chromogen. The reaction is complete in 15 min at 37 degrees C. The standard curve is linear up to a urea concentration of 40 mmol/L. Precision is good; CVs ranged from 2.5% to 3.1%. Results by the present method compared well with those by a candidate Reference Method and are not subject to interferences from commonly used drugs and anticoagulants.

Serum triglycerides determined colorimetrically with an enzyme that produces hydrogen peroxide.

1982 ◽  
Vol 28 (10) ◽  
pp. 2077-2080 ◽  
Author(s):  
P Fossati ◽  
L Prencipe
Keyword(s):  
Hydrogen Peroxide ◽  
Room Temperature ◽  
Volume Ratio ◽  
Glycerol Phosphate ◽  
Serum Triglycerides ◽  
Standard Curve ◽  
Analytical Recovery ◽  
Human Sera ◽  
Sample Measurement ◽  
Colorimetric Procedure

Abstract In this direct colorimetric procedure, serum triglycerides are hydrolyzed by lipase, and the released glycerol is assayed in a reaction catalyzed by glycerol kinase and L-alpha-glycerol-phosphate oxidase in a system that generates hydrogen peroxide. The hydrogen peroxide is monitored in the presence of horseradish peroxidase with 3,5-dichloro-2-hydroxybenzenesulfonic acid/4-aminophenazone as the chromogenic system. The high absorbance of this chromogen system at 510 nm affords useful results with a sample/reagent volume ratio as low as 1:150, and a blank sample measurement is not needed. A single, stable working reagent is used; the reaction is complete in 15 min at room temperature. The standard curve is linear for triglyceride concentrations as great as 13.6 mmol/L. Average analytical recovery of triglycerides in human sera is 100.1%, and within-run and between-run precision studies showed CVs of less than or equal to 1.6 and less than or equal to 3.0%, respectively. The method is suitable for automation.

Sensitivity of the direct oxalate oxidase assay of urinary oxalate improved.

1987 ◽  
Vol 33 (10) ◽  
pp. 1931-1933 ◽  
Author(s):  
J E Buttery ◽  
P R Pannall
Keyword(s):  
Gas Chromatography ◽  
Calcium Chloride ◽  
Colorimetric Method ◽  
Comparison Method ◽  
Oxalate Oxidase ◽  
Urinary Oxalate ◽  
Modified Method ◽  
Analytical Recovery ◽  
And Control

Abstract The direct colorimetric method for urinary oxalate has been modified to improve its sensitivity. Oxalate is precipitated overnight with calcium chloride and ethanol, the precipitate is redissolved, and the oxalate is measured by use of oxalate oxidase (EC 1.2.3.4), methylbenzothiazolinone hydrazone, and dimethylaniline. The color developed is more intense, analytical recovery averages 102%, and the overall imprecision is less than 5%. To assess the accuracy of the method, we used a gas-chromatography comparison method and control sera. Interference from ascorbate is negligible. The modified method retains its simplicity and is less expensive.

Turbidimetric immunoassay of serum C-reactive protein.

1982 ◽  
Vol 28 (10) ◽  
pp. 2121-2124 ◽  
Author(s):  
S Otsuji ◽  
H Shibata ◽  
M Umeda
Keyword(s):  
Radial Immunodiffusion ◽  
Tween 20 ◽  
C Reactive Protein ◽  
Standard Curve ◽  
Reactive Protein ◽  
Analytical Recovery ◽  
Polyethylene Glycol 6000 ◽  
Sample Dilution ◽  
Immunoprecipitation Reaction ◽  
Turbidimetric Immunoassay

Abstract This rapid, reliable equilibrium turbidimetric immunoassay for serum C-reactive protein involves a potent monospecific antibody. Polyethylene glycol-6000 to accelerate and enhance the immunoprecipitation reaction, and Tween-20 surfactant to lower and stabilize the sample blank values. Grossly lipemic, icteric, or hemolyzed sera can be assayed. Values up to about 220 mg/L, for which the standard curve is linear, can be measured without sample dilution. Results by the proposed method and by radial immunodiffusion (r 0.989) or laser nephelometry (r = 0.957) correlated well. Analytical recovery averaged 101.3%. Within-, between-, and day-to-day CVs ranged from 0.9% to 3.5%, 0.8% to 5.5%, and 1.9% to 4.8%, respectively. The method is demonstrably superior to radial immunodiffusion or nephelometry. Any spectrophotometer that can measure turbidimetrically at 340 nm can be used.

Simple, optimized liquid-chromatographic method for measuring total hydroxyproline in urine evaluated

1991 ◽  
Vol 37 (2) ◽  
pp. 285-290 ◽  
Author(s):  
P Reed ◽  
I B Holbrook ◽  
M L G Gardner ◽  
J R McMurray
Keyword(s):  
Solid Phase ◽  
Colorimetric Method ◽  
Internal Standard ◽  
Ion Exchange Resin ◽  
Reversed Phase ◽  
Standard Curve ◽  
Liquid Chromatographic Method ◽  
Analytical Recovery ◽  
Assay Time ◽  
Liquid Chromatographic

Abstract We have optimized a method for measuring total hydroxyproline (HYP) in urine by HPLC after release from urinary peptides by solid-phase hydrolysis on Dowex 50W x 8 ion-exchange resin. The HYP was derivatized with 4-chlor-7-nitrobenzo-2-oxa-1,3-diazole, and excess reagent was removed with the use of a 100-mg C18 Bond-Elut cartridge. The HYP derivative was separated isocratically at 30 degrees C on a 250 x 4.6 mm reversed-phase column containing 5-microns particles of Spherisorb S5 ODS-2, with S-carboxymethylcysteine as internal standard. Total assay time was 14 min. The standard curve for the method was linear from the detection limit for HYP, 3.6 mumol/L, to 10 mmol/L. The between-batch CV was less than 5.1% and the mean analytical recovery of HYP was 95% +/- 1.4%. Comparison with a commercially available colorimetric method showed good correlation: y = 1.158x + 19.76 mumol/L (Syx = 74, n = 120), but HPLC results were 15% higher, probably from incomplete hydrolysis with the colorimetric method. This method offers a considerable improvement in assay time, specificity, sensitivity, precision, and cost compared with the colorimetric method.

Determination of β2-Microglobulin in Serum by a Microparticle-Enhanced Nephelometric Immunoassay

1992 ◽  
Vol 38 (12) ◽  
pp. 2464-2468 ◽  
Author(s):  
J A Viedma ◽  
S Pacheco ◽  
M D Albaladejo
Keyword(s):  
Linear Regression Analysis ◽  
Reference Interval ◽  
Serum Samples ◽  
Standard Curve ◽  
Analytical Recovery ◽  
Beta 2 ◽  
Beta 2 Microglobulin ◽  
Scattering Signal ◽  
Sample Dilution

Abstract This fully automated nephelometric immunoassay to quantify beta 2-microglobulin in human serum measures the light-scattering signal produced by agglutination of commercially available latex microparticles (diameter 0.1 micron) coated with specific F(ab')2 against beta 2-microglobulin. The calibration curve, generated by serial dilutions of a beta 2-microglobulin standard of known concentration, is used to calculate beta 2-microglobulin concentrations in serum samples by the logit-log function and linear-regression analysis. The assay range (sample dilution 400-fold) extends from 0.3 to 40.0 mg/L. No antigen excess appears at beta 2-microglobulin concentrations up to 320 mg/L. Within-run CVs ranged from 1.0% to 3.4%, and between-days from 1.2% to 2.8%. Total imprecision (CV) was < 5%. Analytical recovery averaged 99.5% +/- 2.8%. Rheumatoid factor, complement, bilirubin (up to 340 mumol/L), and hemoglobin (up to 2.0 g/L) do not interfere. Strongly turbid lipemic samples must be cleared before analysis. Standard curve linearity was very good in samples covering the clinical useful range of concentrations. Results of the method correlated well with those of radioimmunoassay and microparticle enzyme-linked immunoassay (r = 0.979 and 0.975, respectively). The reference interval (nonparametric estimation) in apparently healthy adults (n = 303) was 0.87 (0.80-0.94) to 2.42 (2.28-2.45) mg/L; the median value was 1.54 mg/L.

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