Estimation of Nicotinic Acid in Tissues by the Cyanogen Bromide Reaction HAROLD C. GOLDTHORPE AND DORIS TIPPIT University of Utah, Salt Lake C i t y , U t a h A number of procedures for estimating nicotinic acid by the use of cyanogen bromide were found to suffer from interfering colors, instability, and insensitiveness. A study of the factors influencing the cyanogen bromide procedures developed various w n ditions that improved the estimation. The reagent is best prepared from crystalline cyanogen bromide. The temperature of the reaction must not go above 40" C. for 5 minutes or 25" C. for 30 minutes of reaction time. The pH of the medium is very critical, as color development falls off very rapidly on either side of pH 4.9 to 5.3. The reaction is best conducted in the dark. When p-methylaminophenol is used as the amine, the chromogen produced is very stable over 4 hours. The procedure described, if carefully followed, is more selective and precise than other cyanogen bromide methods which have been applied to animal tissues for estimation of nicotinic acid.
I
(3). However, cyanogen bromide reagents prepared this way must be of slightly different compositions and therefore more difficult to buffer to the same pH. Roggen's procedure (fa)of making the reagent was also tried, but constant readings could not be obtained. In agreement with Bandier and Hald (f?),the preferred reagent was found to be a solution of crystalline cyanogen bromide, Eastman's No. 919, in water. EFFECT OF TEMPERATURE. The effect of temperature on the cyanogen bromide reaction was found to be important (Pigure 1). Most methods ( 2 , 6, 6) allow this reaction to take place a t a temperature of 75" to 80" C. The color which quickly developed a t 75 C. decreased rapidly thereafter, and only when the temperature was not allowed to go above 40" C. was the greatest color obtained. Continuing the study, it was found that the maximum color developed if the reactants were allowed to react a t 25" C. for 30 minutes. A great part of the inconsistency obtained by the published
N PREPARING for a study of the effect of individual amino
acid deficiencies, intake of all other dietary constituents being maintained a t normal levels by forced feeding, chemical methods of estimation of nicotinic acid Kere sought which could be applied to the analysis of tissues. It was found that the published methods of colorimetric analysis suffered from interfering colors, instability, or insensitiveness. An improved modification of the cyanogen bromide method has now been developed, which largely overcomes these di, wltieb EXPERIMENTAL
Bandier m d Hald's modification of the cyanogen bromide reaction ( I , 2) was used as a basis for this study, primarily because they (>]aimgreater stability of the chromogen produced with p methylaminophenol sulfate than when other anlines are used, and also because the color development is completed in an aqueous medium, However, it was hard to obtain consistent results. Using a Coleman Universal spectrophotometer, concentration curve8 were prepared by the original method. The galvanometer readings obtained within any one set of determinations checked fairly well, but the values obtained in different SPL- using the same nicotinic acid concentrations did not check. In other words, the K values obtained were not constant. The normal greenish-yellow color which should have developed was often masked by a reddish-brown color. Because the procedure did not give duplicable results on known nicotinic acid solutions, a study of the factors involved in this reaction was undertaken. Cyanogen Bromide Reaction. EFFECTOF PURITY OF CYANOGEK BROMIDE.The cyanogen bromide reagent as ge'nerally used is made by adding cold 10% sodium cyanide to a cold saturated bromine solution until the solution is just colorless. The authors found that this procedure does not give a reproducible reagent. Apparently the eye is not senpitive enough t o detect when all the bromine has been combined. Some "colorless" reagents developed an amethyst color on addition of the amine. This was responsible for off-color in the final reading and was thought to be due to a trace of free bromine which oxidizes the amine to a colored derivative. Adding a slight excess of sodium cyanide when preparing the reagent is claimed to overcome this defect 484
1I
2I
3(
4I
rime Figure 1.
51
6 I
7 I
8 I
I9
d
j , it.flji.rrtes
Effect of Temperature on Reaction
In each case from a series of tubes one was removed every minute, the color developed, and per cent transmittance determined
I 4
1
1
4.5
Figure 2.
72,
5.5
6.,
Effect of pH on Reaction
Duplicate sets of tubes were prepared over the pH range' one tube at each pH was used for pH measurement and the otder for color development. Transmittance of one tube was plotted against pH of the other
V O L U M E 23, NO. 3, M A R C H 1 9 5 1
Figure 3.
Effect of Cyanogen Bromide Concentration
Color developed according to procedure that did not give intensity finally achieved by method described i n text
3 ml. of buffer, 4 d.of cyanogen bromide reagent, and 14 ml. of pmethylaminophenol sulfate in 0.1 N sulfuric acid. Chromogen developed in darkness for 80 minutes and then left in light
methods appears to be due to the use of high temperatures, which cause loss of color. The tubes are not always in the water bath for the same time, and as this reaction is very sensitive to temperature, different values for the same nicotinic acid concentration are obtained. This can be avoided by using a temperature of 25' C. and allowing longer time for the reaction. EFFECTOF PH. The p H of the medium in which the cyanogen bromide reacts is very critical. In various procedures this reaction is carried out a t p H from 4.3 to 7.5 ( 6 , 8 , 9 ) . In these studies it was found that the p H range must be kept between 4.9 and 5.3 for maximum color development. On either side of these values color development rapidly declines (Figure 2). Because the reaction of the p-aminophenol sulfate with the cyanogen bromidenicotinic acid complex requires a low pH, it was found impossible to combine the two reactions as recommended by Melnick and Field (9) in the case of aniline. To control p H a t this point phosphate-citric acid buffers were used ('7): one of p H 5.25 for solutions containing protein-split products and one of p H 4.9 for pure aqueous solutions. The buffer must be so chosen that the p H is about 5.1 when the buffered solution of the sample and the cyanogen bromide reagent are added together. The control of p H is important for sensitivity and intensity of color development. EFFECTOF CYANOGEN BROMIDE CONCENTRATION. The sensitivity of the reaction is also dependent to some extent on the concentration of cyanogen bromide used in the reaction. This con-
485
centration varies from 0.4 to 2.4% in different procedures ( 2 , 9). The best color development was found when 4 ml. of a 4% cyanogen bromide reagent were made to a total volume of 10 ml. with the buffered solution of the unknown. This corresponds t o a 1.6% concentration (Figure 3). EFFECTOF LIGHT. When the reaction of cyanogen bromide was allowed to proceed in indirect daylight, the color was only 94% of that produced in the dark. Reaction with Aromatic Amine. The chromogens produced by different amines vary in their stability to light. Martinek et al. (8)state that the color produced using aniline reaches a maximum in 5 minutes and then rapidly fades, whereas orthoform (methylrn-amino-p-oxybenzoate) also reaches a maximum development of color in 5 minutes but is stable for about 15 minutes. Harris and Raymond (6) and Kodicek (6) used p-aminoacetophenone as the amine. Their spectrophotometric readings had to be made within 15 minutes and their chromogens were not exposed to light. The authors substantiated Bandier's claim that the chromogeii produced using p-methylaminophenol sulfate is stable to light ( 1 , 9). They left the tubes out in diffuse daylight for 24 hours with only very slight fading (Figure 4). The development of full color is relatively slow with this amine, taking about 80 minutes to reach full value. There was a slight increase in color intensity when the reaction proceeded in the dark. Allowing both the cyanogen bromide and amine reactions to proceed in the dark increased the color intensity 5.2%. There was also a slight increase in color intensity when the amine reaction proceeded a t 15' C. Above p H 2 an off-color developed, but this wm prevented when the amine reacted in a solution of p H 1.0. The amine reagent was therefore dissolved in 0.1 N sulfuric acid. The p-methylaminophenol sulfate obtained on the market sometimes has a slight pink color in solution or develops one on standing 2 to 3 hours a t room temperature. The impurity responsible was removed from the amine by washing with alcohol. Preparation of Tissue Extracts for Analysis. I n view of the effect of the factors mentioned above, a procedure has been developed for analysis of biological tissues and fluids. Pepsin was used m a means of hydrolysis in preference to Bandier and Hald's ( 1 , a )alkali treatment or Dann and Handler's ( 3 )acid treatment. This was done because the pepsin hydrolyzate could be used for the determination of other vitamins. Highly colored digests were avoided, so that color interference in the hydrolyzate was a t a minimum. A volatile solvent such as acetone waa not used, for extraction of the nicotinic acid from the hydrolyzate, aa large errors could result from evaporation of acetone. One to 5 grams of minced tissue were placed in a 250-ml. Erlenmeyer flask. To this were added 0.5 gram of pepsin and 50 ml. of 0.1 N hydrochloric acid and the mixture was allowed to digest a t a temperature of 37" C. in an incubator for 24 hours. This acid-pepsin digest was adjusted with 5 ml. of 1 N sodium hydroxide to a pH of approximately 5.2, quantitatively transferred to a 100-ml. glass-stoppered volumetric flask, made up to volume, and well mixed. Ten-ndliliter aliquots were used for the determinations. Of the reagents investigated for precipitation of the larger protein-split products, only zinc sulfate was satisfactory, as clear centrifugates were obtained ( 4 ) and no loss of nicotinic acid resulted from its use. The zinc sulfate is added to the hydrolyzate after alkaline hydrolysis of the nicotinic acid derivatives and subsequent partial neutralization. METHOD
Reagents. Hydrochloric acid, 0.1 ,V and 1.0 N . Sodium hydroside, 1.0 S. Zinc sulfate, 10%. McIlvaine's buffer was prepared by making 15.2 grams of disodium hydrogen phosphate and 9.75 grams of citric acid (HaceH ~ O T . H ~to O )1000 ml. with water, checking on pH meter, and adjusting to the correct pH of 5.25. Cyanogen bromide, 4.Oyo. I t is best to use crystalline cyanogen
486
ANALYTICAL CHEMISTRY
Table I.
Nicotinic Acid Recoveries from Egg Albumin Digests
Nicotinic Acid Added Found 7
Y
Y
7
250 250 500 500 625 825 750 750 1000
245.8 245.8 493.7 495.8 626.7 626.7 766.7 747.5 995.9 1000.0 1262.5 1241.7 1275.0
3 3 6
2.95 2.95 5.90 5.95 7.52 7.52 9.2 8.97 11.95 12.00 15.15 14.9 15.3
1000 8250 1250 1250
Table 11.
Tissue
Amide Added X 1 . 00S4 7
E g g albumin
Rat kidney Rat heart Rat liver Beef liver Rat liveJ Kiacin 250 Amide 630 a
Nicotinic Acid in Aliquot Found Recovery
1260 1260 1260 1260 630 630 630 630 945 37.8 880
6
7.5 7.5 9 9 12 12 1.3 1.5 15
%
98.3 98.3 98.7 99.2 100.2 100.2 102.2 99.7 99.6 100.0 101.0 99.3 102,o .", = 99.9 Standard deviation 1 . 2
Nicotinamide Recoveries Amide Recovered a8 Nicotinic Acid
Amide as Kicotinic Aoid in Aliquot . R Found of Amide
Y
Y
7
%
1241.7 1245.9 1262.5 1233.4 615 642 634.7 629.2 945.9 37.8 893
13.12 15.12 15.12 15.12 7.56 7.56 7.56 7.56 11.35 0.45 10.58
14 9 14.95 15.15 14.8 7.30 7.7 7.62 7.55 11.36 0.45 10.72
98.5 98.9 100.2 97.9 97.6 101 .8 100.8 100.0 100.1 100.0 101.4
bromide is added to tubes 5 and 6. If the pH is higher than 5.1 in tube 7 as determined by a glass electrode pH meter, a drop or two of 10% citric acid solution is added to tubes 5 and 6; if below 5.1, a drop or two of 10% dibasic sodium phosphate solution is added. After pH correction, the cyanogen bromide is added to the tubes and is allowed to react in the dark for 30 minutes a t 25" C. The tubes are then cooled to 10" to 15" C. and 14 ml. of cooled 5% p-methylaminophenol sulfate reagent are added. The samples are made up to volume and the tubes are allowed to stand in the dark a t 10" to 15' C. for 80 minutes. Density readings are taken, a Coleman Universal spectrophotometer being used and set a t a wave length of 410 mp. Distilled water is used as a reference sample in setting the machine. For calculation, the average density reading of tubes 1 and 2 is added to that of tubes 3 and 4 and the sum is subtracted from the average density reading of assay tubes 5 and 6. The values obtained are converted into nicotinic acid values either by use of a concentration reference curve or by calculation from a K value determined from known amounte of the acid carried through the procedure. As nicotinic acid may be present in the pepsin preparations used, an analyeis should be carried out on the material and a proper correction ~ ~ made~ if necessary. ~ ~S o n e was ~ found ~ in the Merck's pepsin preparations used in these studies.
Table 111.
Nicotinic Acid Values in Male Rat Tissues
(Comparison with values obtained b y other chemical and microbiological procedures) Dann and Handler Age of Precient" Authorsa Originalb SingalC Tissue Rats Method analvsis reoort et ol. Daw Y/g. Y/B. Y/& Y/I. Liver 24 ... 159 ... (148-176) 45-55 172 143 ... ... (159-183) (123-171) 60-72 146 137 ... ... (138-152) (132-144) ,,, ... 91-94 ... ... 157 (130-173) 135 1+8 ... ... ... (147-156) .., 270 144 ... 175 ... (127-157) (151-191) ... ... 115 ... Kidney cortex 24 (98-122) Kidney whole 60-72 144 95 ... ... (98-123) (79-106) 91-94 ... ... ... 87 (80-92)
...
M = 99.75 Standard deviation 1.31 Fnctor for conversion of nicotinamide t o nicotinic acid.
...
bromide. The solution is stable for months when kept in a refrigerator. p-Methylaminophenol sulfate, 5% solution in 0.1 W sulfuric acid, is purified by washing solid amine with ethyl alcohol. Dibasic sodium phosphate, 10%. Citric acid solution, 10%. Pepsin, U.S.P Procedure. Two 10-ml. aliquots from the acid-pepsin digest &e transferred t o two 25-ml volumetric flasks, and 6 ml. of N sodium hydroxide are added. This makes the digest solution 0.38 N alkali in strength, with a pH of about 12. The flask and its contents are now heated in a boiling water bath a t 96 O C. (spproximately the boiling point a t this elevation) for 1 hour. This hydrolyzes the nicotinamide diphosphopyridine nucleotide and triphosphopyridine nucleotide into nicotinic acid. The values thus obtained are total nicotinic acid in the oxidized form. reduced nicotinic acid is not determined by cyanogen brom'ide method ( 1 2 ) . After heating, excess alkalinity is partially neutralized by adding 5 ml. of 1 N hydrochloric acid. The remaining alkali is now utilized together with zinc sulfate for the precipitation of the protein-split products produced by pepsin action. To the partially neutralized alkaline digest 2 ml. of 10% zinc sulfate are added, the volume ie made up to 25 ml., mixed well, allowed to stand 10 minutes, and centrifuged, and the supernatant liquid is decanted. This is used for the analysis and must have a pH of 7.0 to 7.2 for a successful precipitation with zinc sulfate. For color development 25 X 200 mm. test tubes are used, graduated a t 25 ml. From each of the centrifugates sets of tubes are set up as shown: Tube
Aliquot, MI.
Buffer, M1.
CSBr
Reagent, MI.
Amine Rea ent,
MY.
270
...
Heart
90
...
Leg
24
...
...
Abdominal
60-72
62 (55-73)
52 (39-62)
Kidney cortex
Leg
270
Abdominal
270
a
* C
...
...
...
...
132 (112-151)
...
77 (63-87)
...
... 114
... ...
86 (81-92) ... .. 54 (40-62) Sprague-Dawley strain of rats. Dann and Handler CKBr procedure (3) Vanderbilt strain of rata. Sinpal el al. microbiological values (Ib),'Wistar strain of rats.
...
Hi0, MI. RECOVERY EXPERIMENTS
Tubes 1 and 2 are controls on the reagents; tubes 3 and 4 are controls on the color in the aliquot. The authors have not found any color to develop between unknown substances in the digest and the amine reagent (6). Tubes 5 and 6 are for the analysis, while tube 7 is set up to control the pH before the cyanogen
Recoveries of nicotinic acid'added to egg albumin are shown in Table I, and added nicotinamide recoveries from egg albumin and tissue digests are shown in Table 11. The range of nicotinic acid recovery was from 98.3 to 102.2'% with a mean of 99.9% and a standard deviation of 1.2Q/,. The range of nicotinamide recoveries was from 97.6 to 101.870with a mean of 99.75% andastandard deviation of 1.31%. As shown in the last line in Table 11,
V O L U M E 2 3 , NO. 3, M A R C H 1 9 5 1 recoveries were good where hoth nic.otinamide and iiicotinic acid were added. In Table I11 a comparison is made of values for aliquots of the same rat tissues, using the present procedure and that of Dann and Handler (3). For additional comparison the values originally reported by Dann and Handler (3)are given, as well as those obtained by Singal et al. ( 1 2 ) using a microbiological procedure (13). I t is the finding of this laboratory that many factors enter into the amounts of nicotinic acid and its derivatives which are found in tissues. 4ge, the amount of tryptophan in the diet, and the strain of rat must be considered when comparing values reported from different laboratories. When the Dann and Handler method and the present procedure were used on the same tissues, higher values were obtained by the latter. The microbiological procedure would be expected t o give higher values, as the values obtained by the cyanogen bromide procedures do not include any reduced diphosphopyridine nucleotide present in tissues. The cyanogen bromide reaction does not take place where the acarbon in nicotinic acid is substituted (14). In spite of this, the values of Singal et al. fall in the same range. Excellent recoveries of nicotinic acid in egg albumin digest were obtained when only 3 micrograms were present (Table I ) . In other recovery experiments values on the order of 1 microgram of nicotinic acid per h a 1 aliquot were determined quantitatively with an accuracy of 98%. Table I1 shon-s that 0.45 microgram of nicotinamide added per aliquot was quantitatively recovered. These recoveries show the applicability of the procedure for the accurate determination of small amounts of nicotinic acid and its derivatives. TIigonelline was not converted into nicotinic acid by this procedure (11). Xicotinuric acid was not tried. Kicotinic acid has been estimated in various ingredients of a rat's diet Dextrin gave no nicotinic acid, zein contained 791 microgram< per
487 100 grams, while celluflour contained 825 micrograms per 100 grams. Using a diphosphopyridine nucleotide preparation obtained from the Schwarz Laboratories, Xew York, which wae rated a t a 60% diphosphopyridine nucleotide content, the authors found values of 58.2 and 59.1%. The procedure as described appears applicable, therefore, to the accurate determination of nicotinic acid plus diphosphopyridine n icleotidr in a \vide variety of plant and animal products. ACKNOWLEDGMENT
The authors wish t o express their appreciation for the grants obtained from the University Research Committee of the Cnivcrsity of Utah, and the Sugar Research Foundation. LITER4TURE CITED
Bandiei, E., Btochenz. J . , 33, 1130 (1939). Bandier, E., and Hald, Jens, Ibid., 33, 264 (1939). Dann, W.J., and Handler, P., J . Biol. Chem., 140, 201 (1941). Friedemann, T. E., and Barborka, C. J., Ibid., 138, 786 (1941). Harris, L. J., and Raymond, IV. D., Beochem. J., 33, 2037 (1939).
Kodicek, E., Ibid., 34, i 1 2 (1940). McIlvaine, J. C., J . Biol. Chem., 49, 183 11921). Martinek, R. G., Kirch, E. R., and JTebster, G. L., Ibzd.,
149,
245 (1943).
Melnick, D., and Field, I%.,Ibid., 134, 1 (1940). Roggen, J. C., .l'edrrland. Tijdschr. G e n e e s h n d e .
85, 4603 -8
(7941); Chein. Z e n t r . , 1942, I, 1917.
Sarett, H. P., Perlzweig, W. A., and Levy, E. D., J . Btol. Chem., 135,483 (1940).
Singal, S. A , , Sydenstricker, V. P., and Littlejohn, J. h l . , I b t d . , 176, 1069 (1948).
Snell, E., and Wright, L. D., Ibid., 139, 675 (1941). Waisnian, H. A,, and Elvehjem, C. -I., IND. ENG.CHEM.,ANAL. ED.,13, 221 (1941). RECEIVED March 20, 1950.
Stable Isotope Dilution Method for Nicotinic Acid Determination N. R. TRENNER, R. W. WALKER, BYRON ARISON, AND CAROL T R U M B i C ' E R .yerck & Co., Znc., Rahway, .V. J . The application of the isotope dilution technique to the determination of nicotinic acid was developed because it became necessary to assay accurately for the nicotinic acid content of crude products known to contain substantial amounts of other compounds containing pyridine rings, and because a review of the literature revealed the existence of no analytical method which would not be seriously affected by the presence of such impurities. A stable isotope dilu-
E
VER since the discovery ( 4 ) of the vitamin activity of nico-
tinic acid in 1937, there has been considerable interest in methods for its determination, especially in complex mixtures. As a result there has appeared in the literature a relatively large number of papers describing such assay methods. K O attempt is made here to review these methods, as adequate bil liographies are available in a number of places ( 1 , s ) . In general, these methods of determination may be divided into two types, microbiological and chemical. Microbiological methods are characterized by relatively good specificity, but are most difficult to carry out with any high degree of precision. A great many papers have been written describing chemical methods of assay, but in every case the fundamental reaction involved is the
tion method is described in which deuteronicotinic acid is used as the tracer and its dilution is determined by infrared spectrophotometry. The method provides an absolute assay for nicotinic acid in complex mixtures, and has the general significance for analytical chemistry of further illustrating the importance of the isotope dilution principle in the development of specific (absolute) assay methods, a field which has been neglected in the past.
same-that is, the Konig reaction (?',8)in which the pyridine ring is opened by reaction with cyanogen bromide to give a product which is capable of further reaction with a primary or secondary amine to give a yellow colored product, the amount of which may be determined spectrophotometrically. Because many pyridine ring-containing substances will give this same reaction, such a method cannot be very specific and this fact is amply borne out by experience ( 2 ) . Under these circumstances and because the materials for which assays were required were known to contain closely related pyridine-ring compounds (Bmethyl nicotinic acid), the authors again (11)turned to the method of isotope dilution as one which would not be subject to any of the above disadvantages.
