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home - Biliary - Miscellaneous - Bilirubin Determination Methods Written by Dr Sebastian Zeki

Bilirubin Determination Methods

Written by Dr Sebastian Zeki Several other methods that use dry reagent chemistry have been reported.One method, which is used in many clinical chemistry laboratories, is based upon photographic film technology.Can be automated and detects delta bilirubin. Bilirubin diazo compound creates two relatively stable dipyrryl azopigments. detected spectrophoto-metrically (their maximal absorption occurs at 540 nm). The indirect and direct fractions can be distinguished based upon their rate of production in the presence or absence of alcohol. Direct Indirect Alcohol The fraction produced within one minute in the absence of alcohol represents the concentration of direct bilirubin; the total serum bilirubin is that amount that reacts in 30 minutes after the addition of alcohol; and the indirect fraction is the difference between the total and the direct bilirubin.The fast reaction of direct (conjugated bilirubin) is due to the absence of internal hydrogen bonding and the fact that it is water soluble.Total serum bilirubin concentrations using this technique are 0.2-0.9 mg/dL (2 -15.4 ul/L) in 95 % of the general population, and < 1 mg/dL (18 ul/L) in 99 %. Direct bilirubin represents up to 30 %, or 0.3 mg/dL (5.1ul/L), of the total bilirubin concentration. van den Bergh method Can give false results as endogenous substances can contaminateAlso can overestimate conjugated fraction Measurement of serum bilirubin —Reaction of bilirubin with diazo reagents causes breakdown of the tetrapyrrole to two azodipyrroles (the van den Bergh reaction) which can be readily measured spectrophotometrically.Diazo assays are most commonly used for quantification of bile pigments for clinical purposes via the following sequence.Because the central carbon bridge of bilirubin is buried within the molecule by hydrogen bonds, unconjugated bilirubin reacts slowly with the diazo reagent.In comparison, conjugated bilirubin, which lacks hydrogen bonds, reacts rapidly even in the absence of accelerators ("direct" van den Bergh reaction).On disruption of hydrogen bonds by addition of "accelerators" such as methanol or caffeine, the reaction is completed rapidly (total bilirubin).Unconjugated bilirubin ("indirect" bilirubin) concentration is calculated by subtracting the direct-reacting fraction from total bilirubin.Direct reacting bilirubin slightly overestimates the conjugated bilirubin concentration because a fraction of unconjugated bilirubin (about 10 to 15 %) also gives a direct van den Bergh reaction.There are several other potential sources of error.As an example, endogenous substances, such as plasma lipids, and drugs, such as propranolol, interfere with the diazo assay.This can produce unreliable results, but only when the bilirubin concentration is normal or slightly elevated.Albumin-bilirubin complexes (delta-bilirubin) also may give a direct reaction.More accurate and sensitive quantification of bilirubin requires chromatographic analysis, such as high performance liquid chromatography and reflectance fluorimetry. Bilirubin alkaline methanoly-sis bilirubin methyl esters. chloroform extraction Separation of these esters is performed using high-performance liquid chromatography (HPLC), and spectrophotometry. Alkaline methanolysis — Dry reagent chemistry — Bilirubin Determination Methods