Studies on Determination of Hydroxyproline SIR: The procedure of Neuman and Logan (S), which utilizes hydrogen peroside in the oxidation of hydroxyproline to form A*-pyrroline+hydroxy2-carbosylic acid, is commonly used today. A recent modification (6) recommends chloramino-T dissolved in aqueous methyl Cellosolve and acetatecitrate buffer as the oxidant, with p dimethylaminobenzaldehyde dissolved in HC104 to form the red dye. In the current study, sodium peroxide has been used as the oxidant. The intermediate product is rapidly converted to pyrrole2-carboxylic acid in acid solution (4). The color reagent p-dimethylaminobenzaldehyde (p-DAB) is mixed with sulfuric acid prior to use. These two modifications result in a more rapid analysis and ease of removal of excess hydrogen peroxide. Experimental. I n the first of three colorimetric tubes, 1.8 cm. in diameter, place 1 ml. of standard (1 to 20 pg. of hydroxyproline per ml.), in the second 1 ml. of water (the blank), and in the third the unknown containing 33 to 112 pg. of hydrolyzed collagen. To each tube add 1.0 ml. of 0.01M copper sulfate. Then add 2.0 ml. of 0.625M sodium peroxide solution. Let stand for 5 minutes a t room temperature, shaking occasionally. Heat for 5 minutes in 80" C. water bath. At this time mix 5% solution of p-DAB in propanol with 3.0N sulfuric acid in 1 to 2 ratio, respectively. Add 6.0 ml. of p-DAB-sulfuric acid mixture to each tube, and shake well. Place
in a 70" C. water bath for 20 minutes. Cool to room temperature and read a t 550 mp in either a Coleman Junior spectrophotometer or a Beckman DU spectrophotometer, using a 1.0-cm. cuvette for the latter. Run a standard curve each time with 5, 10, and 15 pg. of hydroxyproline.
recovery of hydroxyproline and absorbance of color at different levels. Discussions of the interferences in the determination of hydroxyproline and methods of eliminating them, given by Mitoma et a2. (2) and Fels ( I ) , apply equally well to this procedure.
RESULTS
Effect of Sodium Peroxide.
The maximum absorbance is obtained at the level of 0.625M sodium peroxide. Any change from this in sodium peroxide concentration results in a decrease in absorbance. The hydrolysis of 0.625M sodium peroxide produces 1.25N sodium hydroxide and 2% hydrogen peroxide solution. The use of 2 ml. of this solution yields sufficient sodium hydroxide and hydrogen peroxide for complete oxidation. This solution is stable for 30 minutes. Effect of Sulfuric Acid. Color intensity is maximum when a 5Oj, solution of p-DAB in propanol is mixed in 1 to 2 ratio with 3.ON sulfuric acid. Heating for 20 minutes results in a most stable color complex. This complex is the condensation of pyrrole-2-carboxylic acid with p-DAB. Its stability, in this method, remains unchanged for 1 hour. However, i t loses 9% of its absorbancy by the end of 2 hours. Beer's law is well obeyed up to 20 pg. of hydroxyproline. At 25 pg. it is slightly deviated. Table I shows both
Table 1. Recovery of Hydroxyproline from a Gelatin Hydrolyzate
Hydroxyproline, pg. Added to Drotein hydrolFound yzate 5.00 7.50 10.00 15.00
5.00 f 0.05 7.50 f 0 . 0 5 10.00 f 0.10 15.00 f 0 . 1 5
%
A Error 0.100 1 . 0 0.150 0 . 7 0.200 1.0 0.300 1 . 0
LITERATURE CITED
( 1 ) Fels, G., Clin. Chem. 4 , 62 (1958). (2) Mitoma, C., Smith, T. E., Davidson, J. D., Udenfriend, S., Dacosta, F. M., Sjoerdsma, A., J. Lab. Clin. Med. 53, 970 (1959). (3) Neuman, R. E., Logan, M. A., J. Biol. Chem. 184, 299 (1950). (4) Radhakrishnan, A. N., Meister, A., Zbad., 226,559 (1957).
(5) Stegemann, H., 2. physiol. Chem. Jll,41(1958).
ISAAC J. BEEHOR LUCIENA. B A V E ~ A
School of Dentistry University of Southern California Los Angeles 7, Calif. INVESTIQATION supported by U.S.P.H.S. grants D-626 and D-1021.
Carrier-Free Separation of Praseodymium-144 from Cerium-1 4 4 by Anion Exchange Douglas MacDonald, Atomic Power Division, Westinghouse Electric Corp., Idaho Falls, Idaho
separation of carrier-free Pr144 A from CeI4' may be effected on an anion exchange resin. Ce144-Prl4' tracer, RAPID
oxidized to the Ce+' state with NaBrOs in 9N "03, is absorbed on a Dowex 1 column in the nitrate form. The column, 5 X 70 mm., is prepared from 100- to 200-mesh resin which has been boiled for several minutes in a solution 6N in HNOs and 0.5M in NaBrOB. The Pr144,which is not absorbed, may be cluted from the column in a few minutes by passing 10 ml. of 9 N HXO3 through the column under pressure. Less than 0.2% CeI44activity is present in the eluate. The separated Pr144
then may be carried on a LaF3 precipitate to provide a counting sample. Total time from separation to counting is on the order of 20 minutes. Separation of microcurie amounts of carrier-free Pr144 in the above manner provided large amounts of short-lived (17.5-minute) beta activity for the direct determination of coincidence counting losses a t high counting rates for beta counting equipment. The adsorption of ceric ion on an anion resin as described above has been incorporated into the procedure in use a t this laboratory for the radiochemical determination of Ce144 as follows.
After the adsorption of ceric carrier and activity on the resin, the column is washed with 10 ml. of 3N "0s. Cerium may then be eluted a t a flow rate of about 0.5 ml. per minute with 15 ml. of 3N HNOI to which 5 drops of hydrazine hydrate have been added. A fluoride precipitation and final determination of cerium as the oxide yield sufficient decontamination for most purposes. Yields are on the order of 50 to 60%.
WORKsu ported by the United States
Atomic %ergy Commission under Contract AT-11-1-Gen-14 with Westinghouse Electric Corp. VOL. 33, NO. 12, NOVEMBER 1961
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