Determination of Hexosamines in Coniunction with Electrophoresis on Starch OTTO W. NEUHAUS and MARCIA LETZRING Departments of Anatomy and Physiological Chemistry, Wayne State University College of Medicine, Detroit, Mich.
b A modification of the Elson and Morgan procedure for glucosamine is readily applicable to establishing a “glycoprotein” pattern with zone electrophoresis on starch. The compound(s) formed by glucosamine and acetylacetone is extracted with isoamyl alcohol and an aliquot of this extract is then treated with Ehrlich’s This procedure minimizes reagent. interferences from hydrolyzate color and nonglucosamine substances that frequently react with Ehrlich’s reagent. Each of the steps involved in this procedure has been re-evaluated to establish optimum conditions, A typical glycoprotein curve obtained by the electrophoretic separation of human serum on starch is presented. In addition to its applicability to starch electrophoresis, the extraction procedure may be used to determine total hexosamines with 0.0 1 to 0.02-mi. samples of serum. With pooled human sera a value of 94.5 mg. % was obtained.
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separations of the glycoproteins in biological fluids are commonly performed on paper and visualized by an appropriate staining technique (3, 14, 16, 17, 20). Also used for this purpose are measurements of the hexosamine content in the a-, 8-, y-globulin, and albumin bands of a paper electropherogram ( 6 ) . An electrophoretic pattern of glycoproteins comparable with the total protein curve should be easily obtained by zone electrophoresis on starch, if hexosamine determinations could be performed on the individual eluates. Such a method for hexosamines would have to combine sensitivity with ease of operation, since as many as 20 to 30 determinations are required for a single electrophoretic separation. Two methods mere considered-those of Elson and Morgan (8, 21) and Dische ( 7 ) . Preliminary attempts to use these methods indicated the former to be the more applicable. Of the many modifications available (4,21, 26, 26) that of Blix (4)was chosen because of its sensitivity. However, considerable difficulty with hydrolyzate color and a reaction of nonglucosamine substances in the procedure was observed, especially with the eluates low in glucosamine. Spurious peaks and shoulders were obtained on the “glycoprotein” pattern and all values were LECTROPHORETIC
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ANALYTICAL CHEMISTRY
subject to question. Thus it became necessary to find a modification of the Elson and Morgan procedure in which the hexosamine is in some way separated from interfering materials. The methods employing ion exchange columns were considered too involved for this application ( 5 ) . The studies of Schloss (22) and Prodi (19) suggested that the compound(s) formed by the reaction of glucosamine with acetylacetone might be extracted with a water-immiscible alcohol. The final chromogen would then be produced by simply adding Ehrlich’s reagent to an aliquot of the alcoholic extract. Isoamyl alcohol was found suitable for this purpose. The extraction method described here combines the advantages of sensitivity and ease of operation with a minimization of interfering substances. REAGENTS AND APPARATUS
Solutions of glucosamine. A stock solution of glucosamine hydrochloride (dried over phosphorus pentoxide in vacuo) was prepared fresh weekly and stored a t 5” C. (9). Dilutions were made to cover the range of 3 to 40 y per ml. Acetylacetone. Acetylacetone (Eastman Kodak reagent), 0.3 ml., was added to 10 ml. of a carbonate buffer of pH 10 Ilt0.l (8 grams of sodium carbonate and 2.1 grams of sodium bicarbonate in 100 ml.). This solution was prepared fresh daily. Ehrlich’s reagent. The Ehrlich’s reagent was prepared by dissolving 0.4 gram of p-dimethylaminobenzaldehyde in 1.5 ml. of concentrated hydrochloric acid and 13.5 ml. of isoamyl alcohol. This solution was also prepared fresh daily. Isoamyl alcohol. The isoamyl alcohol used, “Baker’s analyzed,” gave no trace of a pink color with Ehrlich’s reagent on standing for 30 minutes. Apparatus. Glass-stoppered centrifuge tubes were used for all steps except in the final color formation for which Klett selected colorimeter cells were employed. Readings of absorptivity were taken with a Bausch and Lomb Spectronic 20 colorimeter. PROCEDURE
Hydrolyzates. Suitable aliquots, usually 1-ml., of the electrophoretic eluates were concentrated to dryness in glass-stoppered centrifuge tubes. This process was hastened by heating in a boiling water bath and by directing a gentle stream of air onto the surface of the sample. Then 1 ml. of 3N hy-
drochloric acid was added, and the tubes were stoppered and placed in a boiling water bath for 4 hours. The hydrolyzates were concentrated to dryness in vacuo over solid sodium hydroxide (13). To these concentrates were added 0.5 ml. of 0.1N sodium hydroxide (1 drop of phenolphthalein) and sufficient 1N sodium hydroxide to make the mixture distinctly alkaline. The pink color was then just dispelled by dropwise addition of 0.1N hydrochloric acid and the final volume was adjusted to 1 ml. To this neutralized hydrolyzate was added 1 ml. of the acetylacetone solution (final pH between 9.5 and 9.7) and after thorough mixing the tubes were closed. The mixtures were heated for 20 minutes in a boiling water bath. After cooling, 5 ml. of isoamyl alcohol were added and the tubes were shaken vigorously for 2 minutes. The layers were separated by brief centrifugation. To 4 ml. of the alcoholic extract in the Klett colorimeter tubes was added 1 ml. of Ehrlich’s reagent. After thorough mixing, the color was allowed to develop for 15 minutes before measuring the absorbances a t 530 mp. All measurements were made against a reagent blank prepared by substituting 1 ml. of water for the sample. The absorbances were converted t o micrograms of glucosamine by means of a standard curve and were then corrected for the aliquot of alcohol used--e.g., 5/4. Calibration Curve. The calibration curve was prepared by using 1-ml. aliquots of the diluted standard solutions (3 to 40 y per ml.). A straight line was obtained a t least between the limits of 8 and 32 y of glucosamine (per 4 ml. of isoamyl alcohol), although with some lots of isoamyl alcohol the straight line could be extended to zero. Thus it is necessary to re-establish the curve for new lots of isoamyl alcohol. With pure solution of glucosamine the standard deviation from the mean was 1.3% within the limits of 8 and 32 y. Samples of galactosamine hydrochloride, 10 and 30 y of galactosamine, gave identical absorptivities and absorption curves as glucosamine. Therefore, as with the Elson and Morgan procedure, the isoamyl alcohol extraction method does not distinguish between the tlTo amino sugars and the results are best expressed as hexosamines. RESULTS AND DISCUSSION
I n preliminary studies using the modification of Blix (4), efforts were made to increase the sensitivity by reducing
the final dilution with ethyl alcohol (l/6 recommended volume). This reduced the alcohol concentration to 50% by volume which, according to some workers (2, 28), should not create difficulties. However, a n unstable color resulted with Ehrlich's reagent and accurate timing of the colorimeter readings was essential. The use of this procedure (the direct method), was also hampered by hydrolyzate color and the formation of color by nonglucosamine substances (2, 6, 10-12, 23). These factors are critical with samples low in glucosamine. The extent of interferences by humin of a hydrolyzed eluate is illustrated by curve B of Figure 1. The hydrolyzed and dried eluate was heated with a sodium carbonate solution, in the absence of acetylacetone, and then mixed with Ehrlich's reagent and ethyl alcohol. I n this way only the initial hydrolyzate color and those substances reacting directly with Ehrlich's reagent were recorded. However, when the isoamyl alcohol extraction method was used, there was no significant interference, as shown by curve C of Figure 1. When starch is used as a supporting medium for electrophoresis, there is always danger of contaminating the eluates with starch. Thus glucose is also a potential interfering material. Curve D of Figure 1 illustrates the absorption spectrum of 300 y of glucose in the direct method. This potential
interference is eliminated in the isoamyl alcohol procedure, as shown by Table I. Because mixtures of amino acids and glucose are potentially reactants in the "browning" reaction, it was necessary to determine the degree of interference, if any. Table I clearly shows the lack of significant interference on the part of glucose and some amino acids when using the isoamyl alcohol procedure. Increasing amounts of crystalline, bovine, serum albumin (Armour and Co.), when hydrolyzed for 4 hours with 3N hydrochloric acid, show a significant color production above the 5-mg. level (Table 11). The addition of 150 y of glucose did not increase the color produced by 10 mg. of protein. Sometimes a yellow color was exTable 1. Effect of Nonglucosamine Substances on Determination of Glucosamine
Glucosamine, Recovery, Test Substance" Added Found % Glucose 0 0 ... Glucose 20 18.8 94 0 0 ... Serine Serine 20 19.1 95.5 Glucose Serine 0 0 Lysine.HC1 0 0 20 20 100 Lysine.HC1 Glucose lysine. HC1 0 0 ... Glucose, 3N HClb ... 0
