Van den Bergh reaction

Van den Bergh reaction Solubility in aqueous solution at acid Indirect Direct... 

The constants for these second order reactions have been determined and they indicate that the first reaction proceeds at a considerably faster rate than the second reaction. It is possible that the van den Bergh reaction, as used in clinical chemistry, takes place in the same way but, as it is not taking place in a homogeneous medium, it is probable that the kinetics will be more complicated. 

Bilirubin is a lipid-soluble substance and for it to give a direct van den Bergh reaction it must be converted into a water-soluble state. This can be achieved in alkaline solution or by the use of solvents, such as ethanol, which are soluble in both water and chloroform (Cl). Conjugation with glucuronic acid also causes the solubilization of bilirubin and in view of the well-known versatility of the liver s detoxifying systems, the possibility that bilirubin may be conjugated with other substances has been considered. 

K6. Klatskin, G., and Bungards, L., Bilirubin-protein linkages in serum and their relationship to the van den Bergh reaction. J. Clin. Invest. 36, 537-551 (1956). 

Conjugated and unconjugated bilirubin concentrations in serum may be measured via the van den Bergh reaction. The conjugated bilirubin reacts directly with diazotized sulfanylic acid and is thus termed "direct bilirubin." Unconjugated (albumin-bound) bilirubin does not react with the reagent unless the serum is treated with alcohol, and its concentration is thus termed "indirect bilirubin." The determination of direct and indirect serum bilirubin, as well as... 

After an incubation time of 75 seconds the reflectance of the dye that has formed is measured at 560 nm (modified van den Bergh reaction). Calculation is effected via a calibration curve that is obtained with the help of two calibrators (low/high). 

Billing BH, Cole PG, Lathe GH. The excretion of bihrubm as a glucuronide giving the direct van den Bergh reaction. Biochemical J 1957 65 774-83.. 

P5. Paulson, M., and Wyler, C. I., Multiple tests of hepatic function with gastroenteric malignancy the value of bromsulphthalein, hippuric acid and the van den Bergh reaction in detecting hepatic metastases, with an evaluation of normality of hippuric acid test. Ann. Internal Med. [N. S.] 16, 872-878 (1942). 

Rotor, A. B., Manahan, L., and Florentin, A., Familial non-hemolytic jaundice with direct van den Bergh reaction. Acta Med. Philippina 6, 37-47 (1966). Rowntree, L. G., Hurvitz, S. H., and Bloomfield, A. L., An experimental and clinical study of the value of phenol tetrachloro-phthalein as a test for hepatic function. Bull. Johns Hopkins Hosp. 24, 327—342 (1913). 

UDP-glucose is necessary for the conjugation of bilirubin to form bilirubin glucuronide. Consequently, reduction in the levels of UDP-glucose could explain the increased indirect van den Bergh reaction and the jaundice observed in galactosemia. 

Bilirubin is not used as such but is excreted by the liver cell. Bilirubin excretion starts with the conversion of bilirubin to bilirubin glucuronide, which was discovered rather recently. It was first observed that bile bilirubin hydrolysis yields two molecules of glucuronic acid per molecule of bilirubin. Bilirubin glucuronide was found in blood serum and was demonstrated to be the compound responsible for the classical van den Bergh reaction. 

Diazo methods Many laboratories estimate bilirubin by its reaction with diazotized sulphanilic acid to form azobilirubin (the Van den Bergh reaction). Conjugated bilirubin reacts rapidly and is therefore sometimes referred to as direct-acting bilirubin. On the other hand, unconjugated bilirubin will only react if an accelerating agent is present and this is referred to as the indirect reaction. 

Many modifications of the Van den Bergh reaction have been tried, using different accelerators. Among these accelerators are methanol (Malloy and Evelyn), ethanol (King and Coxon), benzoate and urea (Powell), diphylline (Michaelson) and caffeine/benzoate (Jendrassik and Grof). This latter method is one of the most widely used. In this technique the diazotization reaction is terminated by the addition of ascorbic acid which destroys the excess diazo reagent. Alkali is added at the end of the reaction to give a more stable colour and also to dissolve any proteins that may have been precipitated. 

A synonym for conjugated bilirubin, i.e. water-soluble bilirubin which reacts directly in the Van den Bergh reaction without the need for an accelerator. 

Fio. 27. The van den Bergh reaction of bilirubin with diasotiaed solfanilie acid to form 2 moles of azobilirubin (SS4). 

The reaction with diazotized sulfanilic acid (van den Bergh reaction) yields almost quantitatively 2 moles of isomeric purple azobUirubin [(II) and (IV), Fig. 27] per mole of bilirubin (S 4)- The reaction mechanism undoubtedly proceeds by attack of the diazonium ion on the pyrrole carbon adjacent to the methylene bridge of bilirubin [(I).d, B). The bilirubin reacts faster than the resulting hydrox3rpyrromethene carbinol (III). In ethanol-chloroform containing 0.006 M HCl the Bmu for azobUirubin at 530 m t is 29,800 for bilirubin at 450 m/i it is 55,200 and for the hydroxy-pyrromethene carbinol at 425 m it is 32,700. 

The quantitative determination in serum of bilirubin and bilirubin diglucuronide, distinguished on the basis of a fast or slow van den Bergh reaction, serves as a valuable clinical diagnostic test (SUS) (Table IX). 

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