Determination of Protein-Bound Carbohydrate in Serum by a Modified Anthrone Method SIR: The accurate determination of serum protein-bound carbohydrate by the anthrone method (3) is hindered by the simultaneous release of tryptophan from the protein (5-7). Dissatisfaction with existing modifications (1, 2, 6, 7 ) prompted further study of the behavior of carbohydrate and tryptophan with anthrone. Confirming the work of Tuller and Keiding ( 7 ) for a given concentration of carbohydrate, all the absorption curves converged a t 585 nip despite varying concentrations of tryptophan. This observation offers an effective solution to the tryptophan interference problem. This communication establishec the validity of a modified anthrone method, which is compared with existing ninclifications. REAGENTS
Anthrone Solution. Anthrone, 0.2y0 w./v. (British Drug Houses) in 96% sulfuric acid (British Drug Houses, AR). The solution mas made up a t least 4 hours before use (8) and prepared fresh each day. Carbohydrate Solution. An equimolar mixture, 100 mg. 7, w./v., of Dgalactose and D-mannose. The sugars (100 mg. of each) are dissolved in 200 ml. of distilled water. Working solution was prepared daily by 1 in 10 dilution of this stock solution, which was refrigerated when not in use. Tryptophan Solution. Aqueous solution, 40 mg. yo,of L-tryptophan (Light
2
0
A
0
0
& Co.). Concentrations of sugar and tryptophan are reported as milligram per cent in the solution (made up to 4 ml. with distilled water) prior to the addition of the anthrone. PROCEDURE
Portions (8 ml.) of anthrone solution were added dropwise to 4 ml. of the carbohydrate-containing solution, previously cooled to under 4" C. by immersion in an ice bath for 45 minutes. After mixing the contents, the firmly stoppered tubes were immersed in a water bath a t 92' C. for 8 minutes. On withdrawal, the tubes B-ere reimmersed in the ice bath t o stop the reaction. The solutions were transferred to cuvettes and their absorbancies read after 30 minutes in an Unicam SP 350 spectrophotometer. All estimations were done in duplicate. The importance of temperature in the development of the color reaction has been reviewed by Helbert and Brown (4). I n these experiments, the optimal conditions were found to be heating a t 92" C. for 8 minutes, identical to those of Goa ( 2 ) . EXPERIMENTAL
The behavior of varying concentrations of carbohydrate and tryptophan with anthrone was studied. Portions (2.5, 10.0, and 20.0 mg. %) of tryptophan solution were added to con-
4 m q . O l ~ qalactose -mannose
0 mg. Oi0 tryptophan 2 0 mg.
O/O
s
A 0
tryptophan
6
+ A
c
A
IO m g . %
tryptophan
c
D
0
2 . 5 mg.%
tryptophan
D
Reed of 6 2 0 ~
6
&ad at 585 m p
Figure 1. Effect of tryptophan on absorbance of galactosemannose solutions
centrations of carbohydrate, increasing by 1-mg. yo amounts from 2 to 8 mg. 7,. For each concentration of carbohydrate the spectral curves crossed a t 585 mp, regardless of the amount of tryptophan added. The absorbances of a given concentration of carbohydrate, read a t 585 nip, were not affected by the addition of tryptophan (Figure 1). On the other hand, the absorbancies of the carbohydrate solution read a t 620 nip were lowered linearly as tryptophan was added. The Beer-Lambert law was obeyed a t 585 mp. Readings at 585 mp were only slightly less sensitive than those a t 620 mp. Recovery of Carbohydrate from Carbohydrate-Tryptophan Mixtures. Recoveries of carbohydrate were determined from solutions containing 4 , 5 , 6, and 7 mg. Yo galactosemannose, alone and with the addition of 2.5, 5.0, i . 5 , and 10.0 mg. % tryptophan. Recoveries were low, 7 5 to 81% n i t h Graff's and Goa's methods; lowest when 10 mg. % of tryptophan were added; in the absence of tryptophan there was almost complete recovery. Tuller and Keiding's method gave 106 to 118% recovery. With Bjornesjo's and the present modification, recovery \vas 100% in almost every case, despite varying concentrations of tryptophan. Recovery of Added Carbohydrate from Alcohol-Precipitated Serum Protein. Galactose-mannose vias added t o alcohol-precipitated serum protein so t h a t the final concentrations of added carbohydrate, before the addition of anthrone, were 100, 150, and 200 mg. %, respectively. The total carbohydrate present was then determined by the various methods; from this value was subtracted the value for the serum protein-bound carbohydrate, obtained a t the same time by the same method. The recovery of the added carbohydrate was then calculated. Because duplicate determinations were made on 20 sera with the addition of three concentrations of carbohydrate to each, 120 individual recoveries n-ere done for each method. The results are summarized in Table I. The methods of Graff et al., Goa, and Tuller and Keiding are biased-ie., only occasionally and by chance can they give 1007, recovery. Bjornesjo's and the present modification nil1 frequently give lOOyo recovery and are thus unbiased. Protein-Bound Carbohydrate in Serum. By the five methods, the VOL. 32, NO. 7, JUNE 1960
885
Table I.
Concentration of Protein-Bound Carbohydrate in 20 Sera, and Recovery of Added Carbohydrate from These Sera
Method Graff et al. Goa Tuller and Keiding Bjornesjo Revised method
Concentration Mean, S.E. of mg. 70 mean 137 3.183 140 3.183 160 3.176 156 3.321 159 3.131
Recovery S.E. of mean 0.63 1.4 1.9 3.2 0.88
Mean,
%
95 93.4 89.8 95.4 98.9
work and James Sylvie, Department of Mathematics, University of Glasgow, for his help with the statistical analyses.
Range,
%
93-97 90-96 86-94 88-102 96-101
LITERATURE CITED
(1) Bjornesjo, I
