Direct Spectrometric Determination of the Concentrations of (Unconjugated) Bilirubin and Conjugated Bilirubin in Serum

1974 ◽  
Vol 34 (3) ◽  
pp. 265-273 ◽  
Author(s):  
H. Hertz ◽  
R. Dybkaer ◽  
M. Lauritzen
Keyword(s):  
Unconjugated Bilirubin ◽  
Conjugated Bilirubin

Comparison of the Weber-Schalm Method with the Ducci-Watson Modification of the Malloy-Evelyn Method for Serum Bilirubin Determination

1970 ◽  
Vol 16 (3) ◽  
pp. 239-246 ◽  
Author(s):  
W Vincent Perrelli ◽  
C J Watson
Keyword(s):  
Human Serum ◽  
Total Bilirubin ◽  
Serum Bilirubin ◽  
Unconjugated Bilirubin ◽  
Human Sera ◽  
Conjugated Bilirubin

Abstract Unconjugated bilirubin, conjugated bilirubin, and mixtures of various proportions of each were dissolved in depigmented human serum. These, together with normal and pathologic native human sera, were analyzed for conjugated, unconjugated, and total bilirubin by the Weber-Schalm method. Results were compared with the determination of "prompt direct reacting bilirubin" and of total bilirubin by the Ducci-Watson modification of the Malloy-Evelyn method. Comparisons show that the Weber-Schalm method more accurately measures conjugated bilirubin concentrations in serum in which the bilirubin is mostly conjugated, but that it gives falsely high values for unconjugated bilirubin in serum in which more of the bilirubin is in the unconjugated form. The two methods gave statistically identical total bilirubin values for the native serum tested. A method is described for correcting the unduly low values for prompt, direct reacting bilirubin in terms of percentage of conjugated bilirubin.

scholarly journals Inaccuracies in measurement of conjugated and unconjugated bilirubin in bile with ethyl anthranilate diazo and solvent-partition methods

1978 ◽  
Vol 173 (1) ◽  
pp. 263-267 ◽  
Author(s):  
J D Ostrow ◽  
S T Boonyapisit
Keyword(s):  
Unconjugated Bilirubin ◽  
Lower Phase ◽  
Rat Bile ◽  
Conjugated Bilirubin

A criticial evaluation was made of the ethyl anthranilate diazo and two solvent-partition methods for the determination of conjugated and unconjugated bilirubin in human and rat bile. The ethyl anthranilate diazo reagent, which reacts only with conjugated bilirubin in serum, also diazotized a variable proportion of unconjugated bilirubin in bile and thus overestimated the concentration of monoconjugates. With the Weber-Schalm and modified Folsch solvent-partition methods applied to human or rat bile, 4–9% of added 14C-labelled unconjugated bilirubin partitioned with the conjugated bilirubin in the upper phase, and 4–9% of added 14C-labelled conjugated bilirubin partitioned into the lower phase. With dog bile, the spill-over of 14C-labelled bilirubin into the lower phase was 9–11%. Analysis of azopigments from the Weber-Schalm partition confirmed that over two-thirds of the bilirubin in the lower phase represents monoconjugates, principally the less-polar monoxylosides and monoglucosides. These solvent-partition methods thus overestimate the concentration of unconjugated bilirubin in bile.

Jaundice

2015 ◽  
Author(s):  
Hugo Farne ◽  
Edward Norris-Cervetto ◽  
James Warbrick-Smith
Keyword(s):  
Blood Cells ◽  
Sclerosing Cholangitis ◽  
Bile Ducts ◽  
Water Soluble ◽  
Red Cells ◽  
Unconjugated Bilirubin ◽  
Bile Canaliculi ◽  
Complete Obstruction ◽  
Metabolism Of Bilirubin ◽  
Conjugated Bilirubin

The metabolism of bilirubin in humans is summarized in Figure 14.1 and can be divided into three sequential steps: 1 Production of unconjugated bilirubin. Red blood cells are broken down by macrophages (mainly in the spleen), which degrade haemoglobin into iron and unconjugated (water insoluble) bilirubin. The iron is stored inside transferrin proteins. Unconjugated bilirubin travels to the liver bound to albumin. In disease, unconjugated bilirubin can be produced by haemolysis of red cells intravascularly, rather than in the spleen. 2 Conjugation of bilirubin. Liver hepatocytes uptake unconjugated bilirubin and conjugate it to glucuronate, thus making water soluble, conjugated bilirubin. 3 Excretion of bilirubin. Once conjugated, bilirubin is secreted into the bile canaliculi. Conjugated bilirubin flows with bile down the bile ducts and into the duodenum. Inside the bowel, conjugated bilirubin is metabolized by bacteria into colourless products (urobilinogen, stercobilinogen). Some of these can be reabsorbed by the gut and excreted via the kidneys, but the vast majority are oxidized in the gut into coloured pigments (urobilin, stercobilin) which give faeces their brown colour. Consequently, if there is complete obstruction of the bile ducts there will be no flow of conjugated bilirubin into the gut, no conversion into urobilinogen, and therefore not even a trace of urobilinogen in the urine. The terminology is confusing because different people mean different things. If you are going to use this terminology, make sure that you and your colleagues agree on the definitions. Nonetheless, this is what people usually mean: • Prehepatic jaundice: this refers to jaundice caused by an excessive production of bilirubin. Remember that bilirubin is produced by the breakdown of haemoglobin in the blood vessels or the spleen, hence the term prehepatic. • Hepatic jaundice: for some people, this means any jaundice due to pathology in the liver (anatomically), such as points 3, 4, and 5 in Figure 14.1, and can thus include problems with hepatocytes (e.g. hepatitis) or with the bile canaliculi (e.g. primary sclerosing cholangitis, PSC).

Characterization of sulfasalazine's interference in the measurement of conjugated bilirubin by the Ektachem slide method.

1989 ◽  
Vol 35 (8) ◽  
pp. 1760-1762 ◽  
Author(s):  
D W Franquemont ◽  
J L Sutphen ◽  
D A Herold ◽  
D E Bruns
Keyword(s):  
Inflammatory Bowel Disease ◽  
Human Serum ◽  
Bowel Disease ◽  
Multilayer Film ◽  
Total Bilirubin ◽  
Inflammatory Bowel ◽  
Film Analyzer ◽  
Conjugated Bilirubin

Abstract We describe the cases of four patients who were taking sulfasalazine for inflammatory bowel disease, whose conjugated bilirubin concentrations in serum exceeded their corresponding total bilirubin concentrations as measured with a multilayer film analyzer, the Ektachem 400. Sulfasalazine added to pooled human serum at therapeutic concentrations increased the apparent conjugated bilirubin, as measured with the Ektachem, in a linear and dose-related fashion. Measured unconjugated bilirubin was simultaneously decreased to values less than -3 mg/L. The same interference occurred on the Ektachem 700, but an algorithm prevented the instrument from reporting the results. The major metabolites of sulfasalazine in blood did not interfere with analysis for those fractions of bilirubin. Sulfasalazine's strong absorbance at 400 nm explains its interference with determination of conjugated bilirubin in this instrument.

Methotrexate interferes with determinations of conjugated bilirubin with the Kodak Ektachem 400.

1986 ◽  
Vol 32 (5) ◽  
pp. 863-864 ◽  
Author(s):  
S Swanson ◽  
T E Mifflin ◽  
J C Boyd
Keyword(s):  
Absorption Spectrum ◽  
Intravenous Infusion ◽  
Total Bilirubin ◽  
Unconjugated Bilirubin ◽  
High Dose ◽  
Wide Range ◽  
Dose Concentration ◽  
Interference Study ◽  
Conjugated Bilirubin

Abstract Immediately after intravenous infusion of a high dose (concentration in serum greater than 1000 mumol/L) of methotrexate, the apparent conjugated bilirubin (Bc) concentrations in serum of two osteosarcoma patients, as measured by the Kodak Ektachem 400 analyzer, were greater than the corresponding total bilirubin concentrations, but decreased as the concentrations of methotrexate in serum decreased. In an interference study we found that methotrexate added to sera containing a wide range of basal Bc concentrations increased the measured Bc concentration in a linear and dose-related fashion. Methotrexate also interfered negatively with measurements of unconjugated bilirubin (Bu). The source of the interference appears to be an overlap in the absorption spectrum of methotrexate with Bc and Bu at 400 nm.

Measurement of direct bilirubin by use of bilirubin oxidase.

1987 ◽  
Vol 33 (8) ◽  
pp. 1349-1353 ◽  
Author(s):  
B T Doumas ◽  
B Perry ◽  
B Jendrzejczak ◽  
L Davis
Keyword(s):  
Direct Bilirubin ◽  
Unconjugated Bilirubin ◽  
Enzymatic Method ◽  
Mean Values ◽  
Bilirubin Oxidase ◽  
Box Method ◽  
Conjugated Bilirubin

Abstract We developed an enzymatic method for measuring direct-reacting bilirubin (DBIL) in serum. At pH 4.5, bilirubin oxidase (BOX) oxidizes mono-conjugated bilirubin, di-conjugated bilirubin, and most of the delta-bilirubin to biliverdin. The resulting decrease in absorbance at 460 nm is linearly related to the concentration of DBIL in serum. Mean DBIL values in the 51 patients' sera examined by the BOX method and a diazo procedure (Clin Chem 1982;28:2305) were 45.4 and 42.8 mg/L, respectively. For the same samples, mean values for DBIL and conjugated bilirubin by the Kodak "Ektachem" methods were 50.2 and 24.8 mg/L, respectively. Hemoglobin, up to 1.5 g/L, does not interfere. Unconjugated bilirubin reacts negligibly. Day-to-day CVs were 2.2% and 2.4% at DBIL concentrations of 37 and 74 mg/L, respectively.

scholarly journals Isolation and properties of conjugated bilirubin from bile

1970 ◽  
Vol 120 (2) ◽  
pp. 311-327 ◽  
Author(s):  
J. Donald Ostrow ◽  
Nancy H. Murphy
Keyword(s):  
Bile Salts ◽  
Absorption Maximum ◽  
Water Soluble ◽  
Unconjugated Bilirubin ◽  
Human Bile ◽  
Gunn Rats ◽  
Partition Method ◽  
Reaction In Water ◽  
Rat Bile ◽  
Conjugated Bilirubin

1. A simple, rapid solvent partition method is described for isolation of conjugated bilirubin, free of unconjugated bilirubin, bile salts, phospholipids and cholesterol, from rat bile. Yields are 40–58%. The product is a phosphate-buffered solution containing approx. 0.4mg of bilirubin/ml, principally as mono- and di-glucuronide conjugates. The method may be modified for isolation of conjugates from human bile with 15–22% yield, and for preparation of unconjugated bilirubin from rat or human bile with yields of 55–62%. 2. The conjugated pigment has red–brown fluorescence and an absorption maximum at 450nm with ∈mM 59.8cm−1. Diazotization by the Malloy–Evelyn method gives a direct Van den Bergh reaction (in water) 12% greater than the total reaction (in methanol), with ∈total 28.4×103lmol−1cm−1 at 550nm. After desalting by elution from Sephadex LH-20 in 50% (v/v) ethanol, the product gave water-soluble mustard-yellow crystalline needles. Such desalted conjugates were precipitated by Pb2+ but not by Ba2+, Ca2+ or Zn2+. 3. At pH7.0 and 37°C the conjugated bilirubin was oxidized at a rate of 1%/h without hydrolysis, whereas 84% was hydrolysed by β-glucuronidase or aqueous alkali. 4. Mono- and di-glucuronides were separated by elution from Sephadex LH-20 in 95% (v/v) ethanol or by extraction with chloroform at pH3.2–3.4. The monoconjugated bilirubin did not become labelled during incubation with unconjugated [14C]bilirubin, and chromatographed as a single spot without dissociating into unconjugated bilirubin and diglucuronide as would be expected of a complex. 5. After intravenous injection of mono- or di-conjugated [14C]bilirubin into normal or Gunn rats, 79–91% was excreted in bile and 2–7% in urine over 2h. In these experiments injected diglucuronide was not hydrolysed whereas 30–41% of injected monoglucuronide was converted into diglucuronide by the normal but not by the Gunn rats. The evidence favours the existence of a true bilirubin mono-glucuronide that is not a complex.

SEPSIS AND JAUNDICE IN EARLY INFANCY

PEDIATRICS ◽  
1962 ◽  
Vol 29 (6) ◽  
pp. 873-882
Author(s):  
Jay Bernstein ◽  
Audrey K. Brown
Keyword(s):  
Giant Cell ◽  
Acute Pyelonephritis ◽  
Cell Transformation ◽  
Early Infancy ◽  
Unconjugated Bilirubin ◽  
Intracellular Accumulation ◽  
Bile Stasis ◽  
Hepatocellular Damage ◽  
Conjugated Bilirubin ◽  
Cellular Alterations

In jaundice that developed in association with sepsis in early infancy, elevation of bilirubin levels in serum occurred in both the direct- and indirect-reacting fractions. The regurgitative nature of the jaundice, with retention of conjugated bilirubin, served to distinguish this condition from physiologic jaundice. Jaundice in the nine cases under study was most often associated with coliform sepsis, particularly in the presence of acute pyelonephritis, but other bacteria and other infectious processes were also implicated. The most consistent histopathologic observation in the liver was bile stasis, indicating an inability on the part of the cell to excrete conjugated bilirubin. This was accompanied by the intracellular accumulation of unconjugated bilirubin and by "toxic" cellular alterations, such as giant-cell transformation. These findings have been interpreted as evidence of primary hepatocellular damage.

Sign in / Sign up

Export Citation Format

Share Document