Spectrophotometric Study of Some Oxidative Products of Bilirubin BENNIE ZAK, NORMAN MOSS', ALBERT 1. BOYLE, and ALBERT ZLATKISZ DepartmeBt o f Pathology, W a y n e University College o f Medicine, Detroit Receiving Hospital,
and
Department o f Chemistry and Medicine, W a y n e University, Detroit, M i c h .
T
HE determination of bilirubin in various media is important in clinical chemistry and several methods (8, la-14) with their many modifications are available for use in the quantitative analysis of this open chsin tetrapyrrolic system. The simplest procedure is its determination as a serum constituent by icteric index ( 5 , 1 0 ) in which the yellow color of the diluted jaundiced serum is compared to a n empirically determined standard of dilute potassium dichromate of the proper concentration. A modification of the latter requires acetone extraction of bilirubin before measuring against the standard (11). This step remcrvrs the possibility of interference by carotenoids or dilute hemoglobin and eliminates the turbidity which occurs in some pathologicd sera by the first procedure. I n other quantitative analysrs, bilirubin is oxidized to biliverdin in serum ( 9 ) , urine and bile ( d ) , a n d the final measurement is made on the intense green color of the bilatriene formed. The most acceptable approach require -some modifications (6, 16), of the van den Bergh adaptation ( I ) , of the Ehrlich diazo reaction ( 9 ) , in which bilirubin is coupled with a diazonium salt by a splitting of the bilirubin molecule at the central methylene group before adding on the diazo aryl sulfonic acid ( 7 ) .
Table I.
Sample Blank A
B C D
E
Preparationof Xonprotein and Protein Solutions for Spectral Analysis Medium
MI,
...
Water Gelatin" Albumina NormalserumC Jaundicedserumd
1.0 1.0 1.0
1.0
Bilirubin, RI1. Water, 20mg. per 85.4% 1\11. 100 ml. H,POa 4.0 0.0 3.0 3.0 1.0 3.0 2.0 1.0 3.0 2.0 1.0 3.0 2.0 1.0 3.0 3.0 0.0 3.0
Sulfuric Acid Color Reagent, RI1. 2.0 2.0 2.0 2.0 2.0 2.0
hydroxide dissolved in it. Transfer to a 100-ml. volumetric flask and dilute to the mark with distilled water and mix thoroughly. Refrigerate and use fresh. Albumin Solution. Pipet 25 ml. of 25 grams per 100 ml. of saltpure albumin (Armour & Co., Chicago, Ill.) into a 100-ml. volumetric flask and dilute to the mark with distilled water, mixing thoroughly. This corresponds to 6.25 grams of protein per 100 ml. of solution. Normal Serum. Icteric Serum. PROCEDURE
Reaction with Sulfuric Acid-Glacial Acetic Color Reagent. To 1 mg. of dry bilirubin in a IO-ml. volumetric flask is added 10 ml. of color reagent. The solution is mixed until all of the bilirubin has reacted to form a clear green color. A spectral curve is obtained over the visible range and is shown in Figure 1, A . ]\-hen an equal volume of distilled water is added to an aliquot of this solution with mixing, a deep blue color forms immediately and its spectral curve over the visible range is shown in Figure 1, B. Reaction with Perchloric Acid Color Reagent. To 1.0 mg. of dry bilirubin in a 10-ml. volumetric flask is added 10 ml. of the perchloric acid color reagent. The solution is mixed to form a clear reddish color. The addition of an equal volume of water to an aliquot causes a change in color to a deep purple. Both spectral curves were obtained for the visible region to get curves .4and B shown in Figure 2 . Reaction with Ferric Chloride-Sulfuric Acid in a Phosphoric Acid-Water Medium. The addition of water and phosphoric acid to bilirubin, either alone or in the presence of proteins, resulted in deep green solutions containing an oxidized form of bilirubin when the sulfuric acid color reagent is added. The peaks of the curves appeared a t several wave lengths depending on the protein or proteins used. The solutions were propared according to Table I and the various curves for the visible range are shown in Figure 3. Curve A represents bilirubin run with no
a 6.0 g. per 100 ml. b 6.25 g. per 100 ml. C
Contains 0.4 mg. per 100 ml. of serum total bilirubin.
d Contains 7.0 mg. per 100 ml. of Seruiii total bilirubin.
The purpose of the investigation is to show some spectrophotometric evidence of several oxidative products of bilirubin-Le., biladienes and bilatrienes-which lead to the estimationof bilirubin by oxidation with an iron reagent. I n previous work on the analysis of total serum cholesterol ( 2 6 ) the effect of the interference of bilirubin was determined. The quantitative aspects of the chromophore found from bilirubin and the added effect of water on the stability of the color also were studied. REAGENTS USED
Reagents are analytical grade unless indicated otherwise. Ferric Chloride Solution. Dissolve 1.0 gram of ferric chloride hexahydrate, reagent grade, in 10 ml. of glacial acetic acid. Sulfuric Acid Color Reagent. Dilute 1.0 ml. of the ferric chloride solution to 100 ml. with C.P. concentrated sulfuric acid. Sulfuric Acid-Glacial Acetic Color Reagent. Dilute 40 ml. of the sulfuric acid color reagent to 100 ml. with glacial apetic acid. Perchloric Acid Color Reagent. Dilute 1.0 ml. of the ferric chloride solution to 100 ml. with C.P. 7 2 y 0 perchloric acid. Gelatin Solution. Xeigh out 6 grams of C.P. gelatin and dissolve in 100 ml. of distilled water with warming. Bilirubin Solution. Teigh out 20 mg. of bilirubin and dissolve in 20 ml. of distilled water containing one pellet of C.P. sodium
I
400
500
I
600
701
Wave lengih in millimicrons 1 Present address, Medical Student, Unil ersity of Michigan, Ann Arbor, AIich. 3 Present address, Shell Research, Houston, Tex.
Figure 1. Spectra of Bilirubin with Sulfuric Acid-Glacial Acetic Acid Color Reagent 1220
1221
V O L U M E 2 6 , N O . 7, J U L Y 1 9 5 4
going from one state to the other. The shift in band from 660 to 640 mp may be due then to a small decrease in this large number of resonance states of the green chromophore caused by the addition of distilled water to form the blue one, although it is only a rough picture. Both colors are extremely stable showing no change after several hours and are entirely reproducible, obeying Beer's law betv-een 0 and 50 p.p.m.
O"-
13
;0 4 12 03
I 1
10
09
0,L Figure 2.
1
I
600 Wove Iewfh in rntllirnicrons
70
500
06
Spectra of Bilirubin with Perchloric Acid Color Reagent
g 07 7.
Table 11. Recoveries of Bilirubin Added to Serum (A phosphoric acid-water medium) Serum Sample
xb
06
Bilirubin, lhIp./lOO 311.
Piesent"
Added
Found
05
04
I J K L
11
N 0
a
10.4 7.8 3.9 1.8 11.6 5,s 4.7 0.0
6 0 6 0 6 0 6 0
8 0 8 0 8 0
P 80 Each is based on a n average of four sarnplrs.
16.0 13.5 9 8 7 7 19 1 14 3 12 6 8 1
0.3 Bilirubin : A. Inwater
02
8. In gelatin C. In humon albumin
01
D. Added to normal serum E. Present in jaundiced serum (7mg /100ml.)
proteins present, B in the presence of 6 gram % gelatin, C in the presence of 6.25 gram 70albumin, D in the presence of normal serum containing 0.4 mg. per 100 ml. of bilirubin by the diazo reaction, whereas E represents jaundiced serum to which no bilirubin was added prior to carrying out the reaction.
9
I
0
1
500 600 Wave length in millimicrons
L
70
Figure 3. Spectra of Bilirubin with Water and Phosphoric Acid Aledium, Alone and in Presence of Proteins
DISCUSSION
Curve A of Figure 1 shows the spectral curve for the stable green chromophore nith a peak at 660 mp. Curve B, made after the addition of an equal volume of distilled water to a n aliquot of green solution. shom the hypsochromic effect obtained as the absorption band shifts toa-ard a higher frequency. The color changes to blue with an absorption peak a t 640 mp. The acidiron reagent reacts 1%-ithbilirubin to form bilatriene. The reaction involves dehydrogenation, where the increase in conjugation from the pair of five conjugated bonds of bilirubin to 10 conjugated and one cross-conjugated bonds of biliverdin causes the color to change from orange to green in the absence of water. The sulfuric acid may also cause dehydration of the hilatriene, formed by the dehydrogenation. The change in number of bonds in long conjugation, going from bilirubin to biliverdin, accounts for the deepening of color for this leads to the formation of a large number of resonance states (5). According to Lemberg and Legge ( 7 ) this may result in a smaller energy requirement in
The reaction of the perchloric acid solution of ferric chloride on bilirubin is interesting because the products isolated by spectrum analysis appear to be biladienes. The purple color is characteristic of the violins, of which mesobiliviolin is an example and has eight conjugated-double bonds. Red is a characterietic color of the erythrins (rhodins), of 1%-hichmesobilierythrin (mesobilirhodin) with seven double bonds in conjugation is an example. The addition of water to an aliquot of the erythrin formed from bilirubin causes a bathochromic shift to a longer wave length and the formation of a violin. These colors are very stable, showing no change in spectra after several hours and obey Beer's law over the range measured from 0 to 50 p.p.m. Because of the interference of cholesterol in a physiological medium such as serum, it is impossible under the conditions of the experiment and under almost anhydrous conditions to determine bilirubin by the sulfuric acid color reagent. In a like manner,
1222
A N A L Y T I C A L CHEMISTRY
the complexity of the serum system, u-hich contains substances like tryptophan in proteins with which perchloric acid reacts to give a greenish fluorescence, makes i t impossible t o carry out the analysis by the perchloric acid color reagent. When sulfuric acid color reagent is added to a mixture of water and phosphoric acid, deep green solutions result and have different absorption peaks depending on the presence of proteins. The curves obtained for water and gelatin are similar with a peak a t 670 mp, although the intensity of the gelatin solution is a little greater than that of the water solution These are shown in curves A and B of Figure 3. C, D,and E in the same figure were obtained with human albumin alone and human albumin in which bilirubin was added in normal serum (bilirubin content of 0.4 mg. per 100 ml.), and jaundiced serum, respectively. The hyperchromic effect of the proteins in C and D is very marked, for the intensity of color is much greater than the n-ater or gelatin solutions. All of the green chromophores are stable and obey Beer’s law over the range of 0 to 20 mg. per 100 ml. of serum. Additions of bilirubin t o several sera show good recovery when compared to standards prepared by the addition of bilirubin to normal serum which s h o w no bilirubin by diazo reaction (Table 11). Measurements were made a t 600 mp where both sample and standards had similar abmrption peaks.
LITERATURE CITED
Bergh, A. -4.H. van den, and Muller, P., “Handbuch der biologeschen Arbeitsmethoden,” Abt. IV, T1. I, p. 901 Urban & Schww-aenburg, Berlin, 1927. Ehrlich, P., Zentr. Kliniken, 45,721 (1883); 2. anal. Chew?.,22, 301 (1883). Hausser, K. W., et a/., 2. physik. Chem., 29B,363 (1935). Hersfeld, E., Biochem. Z., 251, 394 (1932). Hosley, R. J., A m . J . Clin. Pathol., 19,884 (1949). Jendrassik. L., and Grof, P., Biochem. Z., 297,81 (1938). Lernberg, R., and Legge, J. W., “Hematin Compounds and Bile Pigments,” Sew York, Interscience Publishers, 1939. hIalloy, H. T., and Evelyn, K. A , J . B i d . Chem., 119, 481 (1937). Ibid., 122,597 (1937). Meulengracht, E., Deut. Arch. klin. Med., 132,285 (1920). Newberger, R. A , , J . Lab. Clin. Med., 22, 1192 (1937). Rabinowitch, I. XI.,J . Biol. Chem., 97,163 (1932). Scott, L. D., Brit.J . Ezptl. Pathol., 22,17 (1941). Sheard, C., Baldes, E. J., hlann, F. C., and Bollnian, J. L.. Am. J . Physiol., 76,577 (1926). With, T. K., 2 . physiol. Chem., 278, 120 (1943). Zlatkis, .1.,Zak, B., and Boyle, A. J., J . Lab. Clin. M e d . , 41. 486 (1953). RECEIVED for review October 27, 1953. Accepted -4pril 3, 1954. Presented before the Division of Riological Chemistry a t t h e 123rd M w t i n g of the A I I E R I C W CHEMICAL SOCIETY, Lou Angeler, Calif.
Determination of Tetraethyllead in Gasoline By Titration with Ethylenediamine Tetraacetate 0.I . MILNER and G. F. SHIPMAN Research and Development Department, Socony- Vacuum Laboratories, faulsboro,
S
OME widely used methods for the determination of
tetraethyllead in gasoline are the ASTM method ( I ) , and polarographic ( 2 , 1 4 ) or x-ray (6, 1 1 ) procedures. I n the ASTM procedure, the lead is extracted with concentrated hydrochloric acid and determined gravimetrically as lead chromate. This method is precise and accurate, but is rather long Tlie polarographic procedure, in which lead is first extracted as in the gravimetric method, is equal t o the gravimetric in precision and accuracy, and is far more rapid, but is somewhat specialized for many small control laboratories. The x-ray method eliminates the preliminary extraction and is extremely rapid, but is quite specialized and requires expensive instrumentation; furthermore, the sulfur content of the sample must be knonn, 80 that an appropriate rorrection can be applied. This paper evaluates a simple rapid titration as compared n i t h the lengthy gravimetric method for the determination of the extracted lead Disodium ethylenediaminetetraacetic acid (Versene, E D T d , Sequestrene) is a powerful complexing agent for many metals. I t a a s first used by Schwarxenbach and coworkers and was applied to the titration of magnesium n i t h Eriochrome Black T as an indicator (3, 4,7 , 8, 12, 15). Manns et al. have modified the titration to permit the determination of barium ( I S ) . This proposed procedure parallels earlier work with respect t o the titration. It is based on a technique devised by Flaschka (9) in which the lead is titrated a t p H 10 in ammoniacal tartrate medium. The indistinct purple-to-blue end point, obtained by Flaschka, is improved by adding a known amount of standard magnesium solution. The stability constants of the numerous complexes in the system are such that the color change a t the end point is the same as in the titration of magnesium alone-namely, pink to blue. After the work described in this paper was completed, Grun-
N. 1.
nald ( 1 0 ) published a method in which the same titrant is used; however, the two methods are quite different in other respects. A single determination (including the extraction) can be completed in 1 hour by the method described below. By making several determinations simultaneously the working time per determination can be reduced to considerably lese than l hour. RECOMMENDED METHOD
Preparation of Indicator and Standard Solutions. Eriochrome Black T Indicator. Prepare by grinding 0.2 gram of the dye (Eastman Kodak Co., Rochester, X. Y., P6361) with 100 grams of ammonium chloride to a fineness of 40 to 50 mesh. Store in a tightly stoppered bottle. Standard Lead Solution. Prepare a 0.05.t’ solution from reagent-grade lead nitrate crystals which were crushed and dried a t 105” C. before weighing. Standard Magnesium Chloride Solution. Prepare an approximatelv 0.05N solution from the hexahvdrated salt. To a meas;red portion of the solution add 0.3- gram of ammonium chloride, 3.0 ml. of concentrated ammonium hydroxide, and 75 mg. of prepared indicator. Titrate with 0.055 disodium ethylenediamine tetraacetate solution, and express the strength in terms of volume of tetraacetate equivalent t o 1 ml. of magnesium solution. Standard Disodium Ethylenediamine Tetraacetate Solution. Prepare a 0.055 solution from the dihydrated salt and standardize against the standard lead solution as follows: To a measured volume of standard lead solution, add 2 grams of tartaric acid, 0.3 gram of ammonium chloride, 7 ml. of concentrated ammonium hydroxide, and 75 mg. of prepared indicator. Pipet exactly 1.00 ml. of standard magnesium solution and titrate with the tetraacetate solution. Calculate the normality after subtracting the volume of tetraacetate solution equivalent t o the magnesium added. (The reagent, disodium Versenate analytical reagent, is available from Bersworth Chemical Co., Framingham, Mass.) Procedure. Proceed as in the ASTM method ( I ) , obtaining the combined acid extract and washings in a 300-ml. Erlenmeyer.
