Mechanism of interference by hemoglobin in the determination of total bilirubin. I. Method of Malloy-Evelyn.

Abstract Oxyhemoglobin is the species of hemoglobin in erythrocyte hemolysates that inhibits the diazo reaction. Ferric hemoglobin derivatives and species with relatively low molecular mass do not interfere. Conversion of oxyhemoglobin to acid hematin under assay reaction conditions is associated with rapid destruction of bilirubin, which accounts for the diazo reaction error. The most probable mechanism for this destruction of bilirubin is an oxidative reaction involving H2O2, formed in the oxidation of hemoglobin, and acid hematin acting as a pseudoperoxidase. We could find no evidence for other mechanisms of interference such as spectral error or azobilirubin destruction. Addition of potassium iodide, 4.0 mmol/L final concentration in the reaction mixture, eliminates interference from hemoglobin added to give concentrations as great as 10 g/L. It also eliminated the effects of hemolysis in the method of Ertingshausen et al. (Clin. Chem. 19: 1366, 1973), in which ethylene glycol is used as the accelerator.

Download Full-text

Mechanism of interference by hemoglobin in the determination of total bilirubin. II. Method of Jendrassik-Grof.

Clinical Chemistry ◽

10.1093/clinchem/26.1.26 ◽

1980 ◽

Vol 26 (1) ◽

pp. 26-29 ◽

Cited By ~ 17

Author(s):

B C Shull ◽

H Lees ◽

P K Li

Keyword(s):

Hydrogen Peroxide ◽

Related Species ◽

Total Bilirubin ◽

Probable Mechanism ◽

Spectral Interference ◽

Nitrite Ion ◽

Reaction Conditions ◽

Hemoglobin Oxidation ◽

Rapid Formation

Abstract Oxyhemoglobin in erythrocyte hemolysates interferes with the Jendrassik-Grof assay. Destruction of azobilirubin occurs when oxyhemoglobin is oxidized to methemoglobin during diazotization or to alkaline hematin with addition of alkaline tartrate. The most probable mechanism is by oxidation with an agent such as hydrogen peroxide or a related species resulting from hemoglobin oxidation. Methemoglobin also appears to cause some destruction of azobilirubin during diazotization. Methemoglobin forms during diazotization because of reactions of oxyhemoglobin with both diazo reagent and nitrite ion. Formation of methemoglobin is, therefore, more rapid in the test than in the blank mixture and, under reaction conditions, its absorbance is less than that of oxyhemoglobin. This results in spectral interference when neutral azobilirubin is assayed. Alkaline tartrate abolishes this spectral error by causing rapid formation of alkaline hematin in both test and blank.

Download Full-text

Mechanism of interference by hemoglobin in the determination of total bilirubin. II. Method of Jendrassik-Grof.

Clinical Chemistry ◽

10.1093/clinchem/26.1.0026 ◽

1980 ◽

Vol 26 (1) ◽

pp. 26-29 ◽

Cited By ~ 1

Author(s):

B C Shull ◽

H Lees ◽

P K Li

Keyword(s):

Hydrogen Peroxide ◽

Related Species ◽

Total Bilirubin ◽

Probable Mechanism ◽

Spectral Interference ◽

Nitrite Ion ◽

Reaction Conditions ◽

Hemoglobin Oxidation ◽

Rapid Formation

Abstract Oxyhemoglobin in erythrocyte hemolysates interferes with the Jendrassik-Grof assay. Destruction of azobilirubin occurs when oxyhemoglobin is oxidized to methemoglobin during diazotization or to alkaline hematin with addition of alkaline tartrate. The most probable mechanism is by oxidation with an agent such as hydrogen peroxide or a related species resulting from hemoglobin oxidation. Methemoglobin also appears to cause some destruction of azobilirubin during diazotization. Methemoglobin forms during diazotization because of reactions of oxyhemoglobin with both diazo reagent and nitrite ion. Formation of methemoglobin is, therefore, more rapid in the test than in the blank mixture and, under reaction conditions, its absorbance is less than that of oxyhemoglobin. This results in spectral interference when neutral azobilirubin is assayed. Alkaline tartrate abolishes this spectral error by causing rapid formation of alkaline hematin in both test and blank.

Download Full-text

Single-Reagent Method for Rapid Determination of Total Bilirubin with the "CentrifiChem" Analyzer

Clinical Chemistry ◽

10.1093/clinchem/19.12.1366 ◽

1973 ◽

Vol 19 (12) ◽

pp. 1366-1369 ◽

Cited By ~ 7

Author(s):

Gerhard Ertingshausen ◽

Diane L Fabiny Byrd ◽

Thomas O Tiffany ◽

Sandra J Casey

Keyword(s):

Ethylene Glycol ◽

Total Bilirubin ◽

Rapid Determination ◽

Precision Data ◽

Union Carbide

Abstract We report a rapid single-reagent method for determining total bilirubin with the " CentrifiChem Analyzer" (Union Carbide Corp.). The reagent is the stable 1,5-disulfonate of diazosulfanilic acid, and ethylene glycol is used as an accelerator. The reaction requires less than 5 min. Only 25 µl of serum is needed. We present precision data (x = 2.62 mg/dl, SD = .082 mg/dl, CV = 3.13%) and results (r = .998) as compared with those obtained by an automated Jendrassik—Grof method.

Download Full-text

Plastics. Determination of average molecular mass and mixture ratio of poly(ethylene glycol) and its derivatives by MALDI-TOF-MS

10.3403/30321524u ◽

2017 ◽

Keyword(s):

Ethylene Glycol ◽

Molecular Mass ◽

Poly Ethylene Glycol ◽

Average Molecular Mass ◽

Mixture Ratio ◽

Maldi Tof Ms ◽

Maldi Tof ◽

Poly Ethylene ◽

Tof Ms

Download Full-text

Plastics. Determination of average molecular mass and mixture ratio of poly(ethylene glycol) and its derivatives by MALDI-TOF-MS

10.3403/30321524 ◽

2017 ◽

Keyword(s):

Ethylene Glycol ◽

Molecular Mass ◽

Poly Ethylene Glycol ◽

Average Molecular Mass ◽

Mixture Ratio ◽

Maldi Tof Ms ◽

Maldi Tof ◽

Poly Ethylene ◽

Tof Ms

Download Full-text

Determination of Total and Direct Bilirubin in Plasma by Means of a Bichromatic Method on a Centrifugal Analyser

Annals of Clinical Biochemistry International Journal of Laboratory Medicine ◽

10.1177/000456328201900304 ◽

1982 ◽

Vol 19 (3) ◽

pp. 151-156 ◽

Cited By ~ 2

Author(s):

A Westwood

Keyword(s):

Sodium Dodecyl Sulphate ◽

Total Bilirubin ◽

Sodium Dodecyl ◽

Direct Bilirubin ◽

Alternative Methods ◽

Binding Agent ◽

Reaction Conditions ◽

Sulphanilic Acid

A bichromatic method is described for the determination of both total and direct-reacting bilirubin using a miniature centrifugal analyser. Bilirubin was coupled with diazotised sulphanilic acid in strongly acidic reaction conditions, and sodium dodecyl sulphate was used as accelerator for the measurement of total bilirubin. The inclusion of dyphylline as a haem-binding agent greatly reduced interference, even in the presence of extreme haemolysis. The method was shown to be precise, to be more sensitive and less affected by haemolysis than alternative methods, and to allow the estimation of total and direct bilirubin within the same run.

Download Full-text

Studies on the Determination of Bile Pigments

Clinical Chemistry ◽

10.1093/clinchem/10.2.95 ◽

1964 ◽

Vol 10 (2) ◽

pp. 95-102 ◽

Cited By ~ 11

Author(s):

G W Stevenson ◽

S L Jacobs ◽

R J Henry

Keyword(s):

Ethylene Glycol ◽

Extraction Method ◽

Total Bilirubin ◽

Rapid Determination ◽

Bile Pigments ◽

Single Tube ◽

Chloroform Layer ◽

The Difference ◽

Conjugated Bilirubin

Abstract The absorbance at 450 mµ of serum diluted with acidified ethylene glycol is used as a measure of the serum's total bilirubin content in a single-tube, single-extraction method for rapid determination of free and total bilirubin in 0.1 ml. of serum. With the addition of chloroform, free bilirubin is extracted into the chloroform but conjugated bilirubin remains in the ethylene glycol. Free bilirubin is determined from the absorbance at 450 mµ in the chloroform layer. Conjugated bilirubin is calculated from the difference. Absorbance readings at 520 mµ compensate for the absorbance at 450 mµ, due to hemoglobin in the serum. Comparison with the Malloy-Evelyn procedure showed equivalent levels of total but higher levels of conjugated bilirubin.

Download Full-text