Urobilin

Urobilin, a pigment discovered in urine by Jaffe; formed from urobilinogen chromogen upon standing of urine. In fresh urine, U. is normally absent or present in very small quantities; in pathological cases, it may be found even in fresh urine. U. is a normal component of feces (stercobilin) and is also present in bile. U. apparently represents a mixture of pigments formed by oxidation of urobilinogen, as indicated by the difficulty of isolating it in pure form from urine and differences in empirical formulas established by various authors: e.g., Garrod and Hopkins give C-63.6%, H-7.8%, N-4.1%; H. Fischer: C-65.9%, H-7.8%, N-5.1%. Based on recent work by H. Fischer, urobilin can be considered a tetrapyrrothrien. U. is an amorphous substance, depending on the conditions of isolation, red, reddish-brown or reddish-yellow in color; poorly soluble in water and ether, easily soluble in alcohol, amyl alcohol, chloroform, alkalis and ammonia. From solutions of U., it precipitates upon saturation with (NH₄)₂SO₄ in an acidic medium, upon addition of phosphotungstic acid and neutral or basic lead acetates, and partially upon addition of ZnCl₂ or ZnSO₄ and ammonia. A characteristic property of U. is that its neutral alcoholic solutions, as well as alkaline and ammoniacal solutions, give green fluorescence, intensified upon addition of zinc salts, and absorption spectra: in transmitted light, very dilute acidic solutions (non-fluorescent) give absorption bands between lines D and F, closer to the latter at λ=480-501 μμ, with higher concentration extending beyond D; in alkaline solutions the band is much weaker and shifts toward D, λ=500-510 μμ. U. gives the biuret test, and the resulting mixture also has its absorption spectrum. For detection of U. in urine, the Nencki reaction is used: to 10-20 cm³ of urine (alkaline urine is slightly acidified with diluted HCl), 5-10 cm³ of amyl alcohol is added to extract U. Carefully decanting the alcoholic extract, it is examined in a spectroscope. Upon addition of several drops of 1% alcoholic solution of ZnCl₂ (strongly alkalized with ammonia and filtered), green fluorescence appears: in transmitted light the liquid is pink, in reflected light-green. ZnCl₂ can be replaced by an ammoniacal solution of Zn(C₂H₃O₂)₂ (Jaffe reaction). This same reaction can be used for detection of U. in feces, after extracting them with ligroin (Schlesinger).- Urobilinogen, mesobilirubinogen C₃₃H₄₂N₄O₆, the chromogen of U., a constant component of normal urine, is a very unstable substance, oxidizing in air, especially in light, turning into U.; it is also easily oxidized by the action of HNO₃, H₂O₂, KMnO₄ and other oxidants. Urobilinogen has been isolated in pure form by Jaffe, crystallizing well in colorless prisms of the monoclinic system; melting point 202°; very difficultly soluble in ether, benzene, ligroin and water, easily soluble in alkalis and ammonia, chloroform and naphthalene. Unlike U., urobilinogen has no absorption spectra and does not fluoresce. For its detection, the Ehrlich reaction is used: to 10 cm³ of urine, 1 cm³ of Ehrlich's reagent is added, consisting of 2 g of p-dimethylaminobenzaldehyde dissolved in 50 cm³ of concentrated fuming HCl and diluted with water to 100 cm³; the solution of urobilinogen after shaking, depending on concentration, takes on a color from pink to intense red and gives an absorption band between D and E. Urobilin and urobilinogen have a close relationship to bile pigments; urobilinogen is identical with mesobilirubinogen (H. Fischer), obtained upon reduction of bile pigments, and possibly also with Maly's hydrobilirubin, a product of reduction of bilirubin subjected to oxidation. In the organism, the site of formation of urobilinogen is the intestine, where it is formed from bile pigments under the action of putrefactive bacteria. Whether U. is formed in other places has not yet been established. The formed U. is partially excreted with feces, while the larger part returns to the liver, from which it passes into bile and in some amount through the kidneys into urine. In newborns, U. appears in urine only on the third day after birth; it is absent in meconium. In some animals, e.g., in adult horses and cattle, in rabbits (Fromhold), U. is not contained in urine; however, the data regarding rabbits are disputed. In calves, U. is contained in urine only up to the 10-14th day; later its appearance is pathological. The daily content of U. in human urine varies depending on physiological conditions: according to Saillet-30-130 mg; Hoppe-Seyler-80-140 mg; Adler-21-25 mg. The content of U. varies with food: with protein and fatty foods it is greater, with carbohydrate foods less. The amount of U. is different at different times of day. The content of U. in urine sharply increases (urobilinuria) in diseases of the liver (congested liver) and bile ducts (it can serve as one of the diagnostic signs of cancer of the liver and bile ducts), in disorders of blood circulation, in various infectious diseases and poisonings causing breakdown of red blood cells, in fasting; in some cases U. is found in serous fluids and lymph, very rarely in blood. U. is absent in urine with obstruction of the bile ducts, as well as in chronic nephritis. Methods for quantitative determination of U. and urobilinogen in urine and feces: 1) the Terwen method is based on the reduction of urobilin to urobilinogen and condensation of the latter with p-dimethylamido-benzaldehyde into a compound, the hydrochloric acid solution of which is colored red with a blue tint. Reagents: 1) saturated aqueous solution of salicylic acid; 2) 16% solution (freshly prepared) of Mohr's salt FeSO₄(NH₄)₂SO₄·6H₂O; 3) 12% solution of NaOH; 4) ether (purified by shaking with 30% NaOH and washing three times with water); 5) tartaric acid (20% solution); 6) p-dimethylamidobenzaldehyde-saturated cold ether solution; 7) concentrated HCl; 8) saturated cold solution of sodium acetate; 9) phenolphthalein-0.05% alcoholic solution; 10) soda solution, saturated cold. Urine for 24 hours is collected in a dark bottle; to prevent putrefaction, a solution of salicylic acid is added. Into a wide glass or mortar, 80 cm³ of well-shaken urine (urates capture U.) is measured, 20 cm³ of iron alum solution is added, and while shaking, 20 cm³ of NaOH is added. The entire mixture is poured into a dark glass vessel with a well-fitting stopper of such a size that no air remains. It is left to stand in a dark place overnight. The completeness of reduction of U. is checked by the disappearance of absorption bands corresponding to the alkaline solution of U. (if there is much U. in urine, all reagents are taken in double amount). After a day, the urine is filtered through a dry filter into a dark bottle. 20 cm³ of the filtrate, acidified with 5 cm³ of tartaric acid, is extracted with 40 cm³ of purified ether in a separatory funnel, well shaken (up to 60 times). After draining the lower layer, the extract is shaken 4 times with 3 cm³ of water, after which 30 cm³ of the extract is measured into a clean, dry separatory funnel, 3 cm³ of p-dimethylamidobenzaldehyde solution and 10 drops of concentrated HCl are added and shaken vigorously for 1/2 hour to extract the condensation product of U. with aldehyde. Then, pouring several cm³ of water, it is shaken again and 3 cm³ of sodium acetate solution is added. It is shaken once more and the colored liquid is poured into a cylinder. The ether solution is washed several times with water, which is combined with the main portion. If U. is abundant, the ether extraction is repeated several times, taking only 5 drops of HCl and 1.5 cm₃ of NaC₂H₃O₂ solution. If U. is small, the reagents should be taken such that the final volume is as small as possible-5 cm³ (take 5 drops of HCl and 1.5 cm³ of NaC₂H₃O₂ solution). The resulting liquid is colored reddish-blue; in the spectrum there is one absorption band at λ=555-570 μμ. The liquid is diluted with water until the intensity of the color becomes weaker than that of the standard solution. It is colorimetrically measured in an Autenrite colorimeter. The wedge is prepared each time before determination: 5 cm³ of solution No. 10 + 94 cm³ of water + 1 cm³ (exactly) of solution No. 9. Calculation: as a unit of urobilinogen, the concentration of phenolphthalein solution=0.0005% is taken. The number obtained upon colorimetry is multiplied by the degree of dilution of urine (10 cm³ of the resulting solution correspond to 10 cm³ of the original urine). One unit of urobilinogen = 0.4 mg of U. in 100 cm³. All processing must be carried out very quickly.

Adler's method. Principle: to urine or liquid is added zinc acetate and then diluted with water until "the fluorescence disappears. It is compared in a comparator with a solution of U. of known concentration. Urobilinogen is converted to U. before determination by oxidation. 0.085 mg of U. in 100 cm³ of liquid gives under the experimental conditions barely noticeable fluorescence. Reagents: 1) powdered zinc acetate; 2) absolute alcohol; 3) 3% alcoholic solution of iodine; 4) dilution fluid: 20 g of zinc acetate are dissolved in water in a 100 cm³ flask to the mark and diluted with 100 cm³ of alcohol. Procedure: 10 cm³ of urine, which is acid-reacting or acidified with acetic acid, are diluted with 10 cm³ of absolute alcohol; add 1 g of powdered zinc acetate and 3 drops of iodine solution. After thorough shaking, filter (the filtrate should be clear). In a preliminary test, approximately determine the degree of dilution to set the scale to zero. Then, with a pipette, measure different amounts of urine, bring the volume to 2 cm³ with the dilution fluid (No. 4) and determine the moment of fluorescence disappearance. When determining in feces, 10 g of freshly weighed and well-mixed in a mortar excrement are mixed with 20 cm³ of petroleum ether or ligroin for 2-3 minutes. Extraction continues until a negative test with zinc acetate. The residue is triturated with 10 cm³ of absolute alcohol, 1 g of zinc acetate and 3 drops of iodine solution. Filter and proceed further as with urine. Table for determining urobilin by Adler's method. Filtrate of substance (cm³) for dilution (in cm³) U. (mg in 100 cm³ of substance) Filtrate of substance diluted 1:10 for dilution (in cm³) U. (mg in 100 cm³ of substance) 2.0 0.17 0.8 1.2 4.25 0.5 0.31 0.63 1.34 5.10 1.0 1.00 0.34 0.57 1.43 5.95 0.8 1.2 0.43 0.5 1.5 6.80 0.5 1.6 0.68 0.4 1.6 8.50 0.4 1.6 0.85 0.3 1.7 11.00 0.3 1.7 1.10 0.26 1.74 12.75 0.26 1.74 1.28 0.2 1.80 17.05 0.2 1.8 1.70 0.16 1.84 21.25 0.16 1.84 2.13 0.13 1.87 25.50 0.13 1.87 2.55 0.1 1.9 34.00 0.1 1.9 3.40 filtrate of substance diluted 1:100 - - 0.80 1.2 42.5 - - 0.66 1.34 61.0 - - 0.57 1.43 59.50 - - 0.50 1.5 68.0 - - 0.4 1.6 77.0 - 0.3 1.7 85.0 Lit.: Burovaya E., Comparison of methods for determining urobilin in urine and its clinical significance, Scientific Notes of the Smolensk Institute, vol. VI, No. 2, 1928; Mirsky I., Urobilin formation, Klin. med., 1921, No. 21; Fromgold G., Studies on urobilin, diss., M., 1811; Bang O., Clinical Urobilin Studies, Oslo, 1929; Brule M., Origin and value of urobilinuria, Presse. méd., v. XXVII, p. 714, 1919; Fischer H., Constitution of the protein-free dye components and their derivatives (Handb. d. norm. u. path. Physiol., hrsg. y. A. Betlie u. G. Bergman, B. VI, T. 1, B., 1928); Meyer-Betz F., The doctrine of Urobilin, Erg. d. Inn. Med. u. Kinderh., B. XII, 1913 (lit.); Pashkis K., Blood destruction and urobilin metabolism, Erg. inn. Med., B. XLV, 1933 (lit.); Couvet M., L'urobiline à l'état normal et pathologique, P., 1930; Budert H. u. Heilmeyer L., Spectrophotometric studies on Urobilin, Biochem. Zeitschr., B. CCLXI, 1933. See also lit. to the article Urobilinuria.