The quantitative determination of galactose—An enzymic method using galactose oxidase, with applications to blood and other biological fluids

ANALYTICAL

BIOCHEMISTRY

10,

32-52 (1965)

The Quantitative Determination of G,alactose-An Enzymic Method Using Galactose Oxidase, with Applications to Blood and Qther Biological Fluids HENRY From

the Metabolic

ROTH, Clinical

STANTON

Endocrinology Diseases, National

SEGAL, Branch, Institutes

AND

DOLORES

BERTOLI

National Institute of Arthritis of Health, Bethesda, Maryland

Received April

and

1, 1964

INTRODUCTION

Work in numerous areas of biological investigation has been handicapped by the lack of a convenient and reliable technique for determining the galactose content of biological solutions. Such techniques as have been available have been seriously limited in usefulness and applicability by complexity, by lack of specificity, or by a range of sensitivity inadequate to detect the small concentrations frequently encountered. A major problem encountered in the analysis of biological solutions has been the presence of other sugars, especially glucose, which react along with gala&se. Most early methods for assaying galactose specifically depended upon the ability of certain yeasts to metabolize glucose and galactose at different rates, and involved the determination of total sugar before and after fermentation with yeast (l-3) ; the nonfermentable reducing material present was presumed to be galactose. The fastidious and delicate nature of the microorganisms, the high incidence of adaptation, the necessity of performing two separate sugar analyses on each specimen, and the introduction of numerous “correcting factors” to adjust for differences in the rates at which the various detecting agents reacted with glucose, galactose, and other material present, rendered these techniques inconvenient and occasionally capricious (4, 5). Other methods for the quantitative determination of galactose have been suggested, which involve its prior purification by means of paper chromatography or electrophoresis and elution (6-9). More recently, most of these methods have been replaced (at least in blood analyses) by the enzymic elimination of glucose, the major interfering material, through incubation of the sample with glucose oxidase (10, 11). This technique, although possessing certain advantages over the yeast fermentation 32

ENZYMIC

DETERMINATION

OF

GALACTOSE

33

method, shares most of the difficulties, especially with respect to specificity, the identical procedure being used for the determination of several different sugars in biological solutions (12, 13). The isolation of the enzyme galactose oxidase (14, 15) ) which catalyzes the oxidation of galactose at the sixth carbon atom:

0 H

+02-gJ+Hs* Ctl

H

OH

offered promise of a simple and specific technique for the determination of galactose, exploiting the properties of the H,O, produced stoichiometrically in the reaction, in a manner analogous to the determination of glucose with glucose oxidase (16). A number of specialized applications of this principle have already been reported (17-21). Among these is the commercially available “Galactostat” kit, which is useful for galactose determinations at certain concentrations, but fails to yield a linear response over a large segment of its range, and can detect only relatively large amounts of galactose. It was our purpose to define the conditions which permit adaptation of the galactose oxidase reaction to a generally useful method for the determination of galactose in a variety of situations, but particularly in blood, incubation media, and similar fluids. MATERIALS

Authentic D(+)-galactose, C.P., for the preparation of standard solutions, was purchased from Pfanstiehl Chemicals, Waukegan, Illinois. Paper chromatographic analysis of this material revealed the presence of trace quantities of at least four other materials capable of reacting with the color reagent used (aniline hydrogen phthalate) , but since these appeared to amount to less than 1% of the total galactose present their significance was felt to be minimal with respect to the use of this preparation as a reference standard in the present assay. Two preparations of galactose oxidase were used. The first was obtained from Worthington Biochemical Corporation, Freehold, New Jersey, through the courtesy of Mr. J. D. Teller, and is the basic component of the “Galactostat” kit marketed by this firm. It was supplied as a powder with approximately 6000 units of enzymic activity per gram, the unitage being that defined by Avigad et al. (15). A second galactose oxidase preparation was obtained from Ames Research Laboratory, Elkhart, Indiana, through the courtesy of Dr. C. 0. Rupe. The

34

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material was supplied as a dry powder, containing 35,000 to 43,000 units per gram, unitage being expressed in terms of equivalent glucose oxidase units as defined by Scott (22). Both preparations were stored at -15°C. The horse-radish peroxidase employed was the type II marketed by Sigma Chemical Co. of St. Louis, Missouri. It was stored at -15”. Benzidine base, reagent-grade, was purchased from Hartman-Leddon Co., Philadelphia, Pennsylvania. Prior to use, it was recrystallized from hot water after treatment with activated charcoal and filtration. The recrystallized reagent was stored in the dark. Spectrophotometric measurements were made in a Beckman DU spectrophotometer, using quartz cells of l-ml capacity and l-cm light path. METHODS

General Assay

Procedure

(Method

I)

Stock Solutions. (A) Buffer: 0.05 M glycine buffer, pH 8.3, is stable for at least 1 month if stored at 4°C. It is prepared by making a 1: 10 dilution of 0.5 M glycine buffer. The concentrated buffer is prepared by dissolving 7.5 gm of glycine in 200 ml of water and adding 5 N NaOH dropwise until the pH reaches 8.4. The concentrated buffer is stored at 4°C. (B) Galactose oxidase: an aqueous solution, containing 35 unitsl/ml is prepared and divided into individual l-ml portions which are stored at -15” until used. (No detectable change in activity has been noted after 12 months of such storage.) (C) Horse-radish peroxidase: an aqueous solution, containing 1 mg/ml is prepared and stored at 4°C. The use of solutions more than 30 days old is not recommended. (D) Ben&dine base: 100 mg of the recrystallized powder is dissolved in 10 ml of absolute ethanol, and the solution is stored at -15°C in the dark. The use of solutions more than 30 days old is not recommended. [The absorption of this compound, which may occur through the intact skin or by inhalation of the powder, is associated with grave toxicity (23) and should be carefully avoided.] Reagent Solution. Prepared within 15 min before use, by mixing together 10 ml of solution A, 1 ml of solution B, 0.5 ml of solution C, and 0.25 ml of solution D. This provides enough reagent solution to react with approximately 21 unknown solutions and standards. Chilling of the ‘Units are those defined by Scott (22) or those of Avigad et al., (151, depending on the source of the enzyme preparation. One unit of either preparation yields approximately the same amount of enzymic activity under the conditions described herein.

ENZYMIC

DETERMINATION

OF

35

GALACTOSE

reagent solution does not appreciably retard deterioration and may cause precipitation of the benzidine (which, however, redissolves readily when the mixture is brought back to room temperature). Procedure. Unknown solutions and standards containing from 0 to 150 pg of galactose are brought to 0.5 ml with water in 5-ml test tubes and warmed to 37°C. To each sample, 0.5 ml of reagent solution is added, with brief mixing. After incubating for exactly 30 min at 37”, the reactions are stopped by the addition of 1 drop of 5 N hydrochloric acid to each test tube. Samples are transferred to l-ml Beckman cuvettes and the optical density of the solutions is measured at 310 rnp, using water as a blank. The ultraviolet absorption at this wavelength is stable for about 30 min after acidification (if samples are protected from temperatures exceeding 50”), but then begins to deteriorate slowly. Samples with optical densities up to 0.700 may be read directly on the spectrophotometer ; samples with high extinctions should be diluted with water before 2.600

I

I

I

I

I

I

I

I

I

1

Fro. 1. Standard curve for galactose assay (Method I). Optical density at 310 mp as a function of galactose concentration in the general assay procedure described in the text. Experimental points represent duplicate determinations. All samples with optical densities exceeding 0.700 were diluted with waster before reading.

36

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reading. Optical densities higher than 2000, however, must be interpreted with caution since they indicate galactose concentrations above the linear range of the assay. A standard curve, typical of some 50 such curves run under these conditions, is illustrated in Fig. 1. A linear response is obtained with galactose concentrations ranging from 0 to 150 pg (per 0.5-ml sample), with an optical density (OD) change of 0.0128 unit per microgram of galactose present. Micro

Procedure

(Method

ZZ)

Where the measurement of smaller concentrations of galactose (<20 pg) with higher sensitivity is desired, and a wide range of permissible concentrations is not required, the following procedural variation is useful : The reagent mixture is made up to contain 10 ml of solution A, 1 ml of solution B, 0.5 ml of solution C, and 0.080 ml of solution D. Standard and unknown samples of 0.5 ml are brought to 37”C, mixed with 0.5 ml of reagent mixt,ure, and allowed to incubate at 37”. The reaction is terminated at the end of 120 min by the addition of 1 drop of 5 N HCI. I

I

I

I

I

I

I

I

I

.6X’-

I 2

I

4

I

6 MICROGRAMS

FIG. 2. Standard (Method II). Optical the assay procedure Experimental points

I

I

6

IO OF

I

I

I

I

12

14

16

I8

I

20

GALACTOSE

curve for galactose assay at low concentrations of galactose density at 295 rnp as a function of galactose concentration in as modified for measurement of small quantities of galactose. represent duplicate determinations.

ENZYMIC

DETERMINATION

OF

37

GALACTOSE

Solutions are transferred to l-ml cuvettes and the absorbance at 295 rnp is recorded, A standard curve is illustrated in Fig. 2, and it can be seen that this procedure affords linear measurement of quantities of galactose ranging from 0 to 20 pg, with a sensitivity of 0.0236 OD units per pg of galactose. This variation may be applied to “micro” samples of blood, the preparation of which is described below. Preparation

of Samples

for Galactose

Assay

A. General. Samples should be at approximately neutral pH, and free of the interfering substances enumerated below. Protein precipitants and other substances capable of injuring enzymes in general (e.g., large amounts of ethanol) should, obviously, be eliminated. One further condition which has been found to affect the galactose oxidase reaction adversely appears to be high ionic strength, or osmolarity. Thus high concentrations (exceeding approximately 0.5 M) of simple salts (chlorides, sulfates, and phosphates of sodium or potassium) and organic compounds (glucose, glycine, Tris, succinate, sucrose) have been found to inhibit the enzyme. B. Blood samples. The analysis of blood specimens for galactose content requires deproteinization of the blood and the removal of a number of potentially interfering substances present. Good results have been obtained with blood extracts prepared by the method of Nelson (24) : One-half milliliter of heparinized blood (the use of fluoridated or oxalated anticoagulant mixt’ures is discouraged because of potential inhibition of the enzyme system by these ions) is added to 2.5 ml of water, in a 25ml flask or beaker. When the erythrocytes have hemolyzed (after 2 to 5 min), 1 ml of a 0.3 N solution of Ba(OH) 2 is added, with agitation, and this is followed by the addition of 1 ml of a 5% solution of &SO,. [The Ba(OH)* and ZnSO, solutions should be matched against one another so as to be perfectly isoequivalent. Directions for their preparation have been published elsewhere (24) 1. The mixture is agitated and allowed to stand for 10 min, after which it is centrifuged at 2000 rpm-approximately 1800 X g (25)-for 15 min. The clear supernatant fluid is aspirated off and provides sufficient volume for as many as three repeat analyses for gala&se, each analysis requiring the use of 0.5 ml of the centrifugate. From the assay procedure as described, the galactose concentration will be expressed in terms of micrograms per 0.5 ml of centrifugate; since the centrifugate represents a ten-fold dilution of the

38

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original blood, this value is multiplied by the factor 20 to obtain the galactose concentration in pg/ml of original blood sample. C. Micro Blood Samples. A conical centrifuge tube (12-ml capacity or less) containing 0.18 ml of water is prepared and brought to the bedside. A standard (Sahli-type) hemoglobin pipet (capacity = 0.020 ml) is filled to the mark with blood from finger or ear-lobe puncture, and then this blood is pipetted into the water in the centrifuge tube (the pipet being rinsed 3 or 4 times with the water). The blood is allowed to hemolyze, and then 0.4 ml of 0.03 N Ba (OH), is added with mixing, followed by 0.4 ml of 0.5% ZnSO,. The mixture is allowed to stand for 10 min and is then centrifuged at 2000 rpm (approximately 1800 X g) for 10-15 min. Sufficient clear supernatant fluid is aspirated off to allow pipetting of 0.5 ml for the assay. The centrifugate represents a 50: 1 dilution of the original blood. The galactose content in 0.5 ml would therefore have to be multiplied by 100 to obtain the concentration in terms of &ml of blood. D. Tissue Incubation Media. Somogyi-Nelson extracts, similar to those described above for blood, are prepared at dilutions of 1:5 by adding 1 ml of incubation medium (or tissue homogenate) to 2 ml of water, and then adding 1 ml of 0.3 M Ba (OH), and 1 ml of 5% ZnSO,. Samples are centrifuged after standing for 10 min or longer and the clear centrifugate may be analyzed for galactose content as above. E. Urine and Other Fluids. Specimens of urine, bacterial culture media, hydrolyzates of complex macromolecules, and similar essentially nonproteinaceous solutions containing excessive concentrations of inorganic ions, compounds absorbing ultraviolet light, and other miscellaneous interfering constituents, are best treated in a different manner. In order to prepare such samples for galactose analysis using galactose oxidase, lo-ml aliquots of such samples (or 10 ml of appropriate dilutions thereof) containing no more than 0.5 mg of galactose per milliliter (50 mg%) are treated with 1 gm of hydrated aluminum trisilicate (Lloyd reagent) and 300 mg of activated charcoal. (We do not generally weigh out these materials each time, but simply add them from spatulas known to contain the correct volume of the dry reagents when filled.) The samples are allowed to stand for 30 min at room temperature, with gentle agitation, and are then filtered through Whatman No. 1 (or equivalent grade) filter paper. The UV absorption of 2: 1 dilutions of these filtrates at 310 rnp is recorded (for later subtraction from final readings, and also to verify that the majority of UV-absorbing materials has been removed; if substantial amounts remain, it is best to repeat the decolorizing procedure on fresh aliquots of higher dilution). The filtrates are then analyzed for galactose by the procedure outlined above, using

ENZYMIC

DETERMINATION

OF

GALACTOSE

39

0.5 ml of filtrate for each determination. Experiments with urine specimens to which known amounts of galactose had been added, and with standard aqueous solutions of galactose, have shown (see below) that the decolorizing step wit,h Lloyd reagent and charcoal, when carried out under the conditions just described, also removes 50% of the galactose contained in these solutions, at galactose concentrations ranging from 0 to 500 pg (0.5 mg) per milliliter. Therefore, the concentration of galactose indicated by the assay (in &0.5 ml) should be doubled to obtain the true galactose content of the solution before decolorization, and mult.iplied by 4 to obtain the true galactose concentration in pg/ml. RESULTS

Blood Snmples. When blood specimens are prepared as described above, concentrations of galactose ranging from 5 to 300 mg% can be measured linearly (since the effective linear range of the assay extends from 2.5 to 150 pg of galactose per 0.5 ml of fluid analyzed and the blood is diluted 1: 10). The measurement of blood concentrations exceeding 300 mg% would require further dilution of the blood extracts (although it is difficult to visualize situations in which such concentrations could be anticipated). Upon analyzing blood samples from 25 individuals (including 17 patients with various endocrine diseases and 8 normal volunteers), it was found that the blood of all persons tested gave a reaction equivalent to approximately 6.4 mg% of galactose. Paper chromatographic techniques sensitive to one-tenth this concentration of true galactose failed to demonstrate any galactose in the blood of these persons. Incubation of blood extracts in control tubes containing no galactose oxidase failed to give this reaction. It must therefore be concluded that some constituent of normal blood, (which is neither galactose nor glucose) is capable of interacting with galactose oxidase (or some contaminant thereof) in such a way as to produce H,O, in an amount equivalent to that produced by the interaction of 3.2 pg of galactose with the enzyme. The identity of the material yielding this adventitious reaction has not been established. Possibly it will become less important as purer preparations of galactose oxidase become available. Its concentration varied little from one person to the next, and was equivalent to an average of 6.4 2 0.8 mg% of galactose, the highest reading being 8.7 mg%, and the lowest, 5.2 mg%. A typical standard curve representing the values obtained from human blood samples to which known amounts of galactose were added is shown in Fig. 3, where the linearity of response is demonstrated over a range of blood concentrations from 25 to 227 mg%. From the linearity of curve A in Fig. 3, and from the parallel nature of curves A and B, it

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1.600^i 1.400E ”E! 1.2002 E

LOOOl.OOO-

0 I 25

I 50

I 75

EQUIVALENT CONCENTRATION OF GALACTOSE ASSAY ALIPUOTS AND WATER STANDARDS

I loo

!5

IN DILUTED (pg/k ml)

FIG. 3. Standard curves: (A) Determination of galactose content (using Method I, described in the text) of blood samples to which known amounts of galactose had previously been added. Optical density developed after application of the procedure described in the text is plotted as a function of galactose concentration in mg/lOO ml original blood specimen (upper row of figures along abscissa) and of galactose content in pg/O.5 ml of blood extract analyzed (lower row of figures along abscissa). (B) Simultaneous determinations of galactose content of standard solutions known to contain the same amounts of galactose as theoretically present in the blood extracts.

can be deduced that the recovery of galactose in the procedure resulting in curve A was complete and that the effect of the adventitious (i.e., not originating from the oxidation of galactose) optical density increment contributed by the blood is simply additive, over the entire extent of the curve, to the true galactose present. ,In the measurement of 25 blood samples (from 20 different patients) to each of which 100 mg% of known galactose had been added, the mean galactose concentration measured was 106 & 5 mg% with the highest reading, 113 mg%, and the lowest, 82 mgO/o. On the basis of results such as these, confidence in the assay technique seems warranted, at least for purposes of routine clinical and investigational measurements of blood galactose. The prior construction of standard reference curves for each specimen to be analyzed (to com-

ENZYMIC

DETERMINATION

OF

41

GALACTOSE

pensate for any individual variations in the interaction of blood extracts with the assay system) is unnecessary; the blood concentration can simply be computed from the absolute concentration of galactose measured in the blood extract, subtracting 6.4 mg% from the apparent final blood concentration. Figure 4 illustrates the application of the above technique in the performance of intravenous gala&se tolerance tests on a normal individual and a patient with congenital galactosemia. 5073 46 -

I

40

60

80 MINUTES

*

100

,

1

120

,

,

140

Fra. 4. Galactose tolerance tests in a normal individual and in a patient with congenital galactosemia. The normal subject was given 5 gm (92.4 mg/kg), and the galactosemic patient was given 6 gm (78.2 mg/kg) of galactose intravenously at “zero” time. Blood specimens were drawn at frequent intervals and analyzed as described in the text.

Micro Blood Samples. In this case, the blood extract represents a 5O:l dilution of the original specimen. Again, correction must be made for the presence in blood of some substance giving an adventitious reaction equivalent to approximately 5 mg% of galactose. The results again indicated the apparent presence of more galactose than the extracts were known to contain, pointing to the presence of some material in blood

42

which yields reaction.

ROTH,

an adventitious

SEGAL,

AND

BERTOLI

(but predictable

and simply

additive)

Tissue Incubation Media. The technique has been applied to the analysis of tissue incubation media and liver homogenates for galactose content. Figure 5 shows the amount of galactose remaining in the medium

I

30

INCUBATION

I

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60

90 TIME

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120

(Minutes)

FIQ. 5. Disappearance of galactose from tissue incubation media. Ten rat liver slices, each weighing 215 + 15 mg were suspended in individual flasks containing 2 ml Krebs-Ringer bicarbonate buffer to which 500 mg/ml of galactose had been added, and were incubated at 37°C. At each time point, two flasks were removed from the incubator and the media therein analyzed for galactose content by the method described in the text. (Independent duplicate values were therefore obtamed for each time point.) By means of further calculations, the rate of galactose consumption per mg tissue can be accurately derived.

(i.e., the disappearance rate) when 215 k 15 mg (wet weight) of adult rat liver tissue is incubated for varying lengths of time at 37°C in 2 ml of Krebs-Ringer bicarbonate buffer to which 1 gm (500 mg/ml) of galactose had been added initially. A set of experiments using this method to study the metabolism of gala&se in liver tissue in vitro has been reported elsewhere (21). The identical procedure has also been found useful for measuring the free galactose content of whole tissue homogenates.

ENZYYIIC

DETERMINATION

OF

TABLE

43

GALACTOSE

1

ANaLYSIS OF URINE SPECIMENS CONTAINING KNOWN AMOUNTS OF GALXTOSE (procedure

for preparation

of samples

and galactose

assay

described

in text)

(9

Th?oreticnl (iii) galactose concn. Optical (WI ‘p%tl$l density Optical density ‘zfo2yf ) authentic (ii) (310 mP) galactose Dilutioll dilution of sample (u) added to before de- of decolor- a;;;er&- Net change specimen), colorieing ized film opticnl step trate assay density mx% 20 30 40 50 50 50 50 50 50 60 0

1O:l 1O:l 1O:l 1O:l 1O:l 1O:l 1O:l 1O:l 1O:l 1O:l

0.060 0.080 0.075 0.100 0.070 0.078 0.070 0.053 0.100 0.088

C&lactose recovered in

mg%

0.175 0.230 0.260 0.315 0.295 0.303 0.285 0.263 0.328 0.340 = (column

0.115 0.150 0.185 0.215 0.225 0.225 0.210 0.210 0.228 0.252 vi)

(4

Gala&xc present iu aliquot. assayed(from standard CUPW,as in Fig. 11, fig

x (column

4.5 7.5 10.1 12.4 13.0 13.0 12.0 12.0 13.1 15.2

(vii) Galsetose recovered (uili) in o@ginal Per cant spec*men.” recover> 9.0 15.0 20.2 24.4 26.0 26.0 24.0 24.0 26.2 30.4

ii)

45.0 50.0 50.5 49.6 52.0 52.0 48.0 48.0 52.4 51.7

x 2 x 0.1.

Urine. Table I illustrates the application of the set of procedures recommended in what is essentially a group of recovery trials, where urine specimens, to which known amounts of galactose had been added, were then analyzed for galactose content. The same kind of table (without columns i and viii, and with the calculation formula modified to account for the 50% recovery) can be used as a protocol outline for calculating galactose concentrations in unknown specimens. Although, as shown, only 50% of the gaIactose is recovered in the preparation of samples, the fact that the recovery rate remains constant over a range of galactose concentrations extending from 0 to 500 &ml (0 to 0.5 gm/lOO ml) has rendered this procedure useful for measuring the galactose content of urine and similar fluids. In cases of clinical galactosemia, or in experimental studies involving the infusion of galactose, where the urinary galactose concentrations generally encountered are in the range of 0.5 to 2.0 gmJlO0 ml, 20-fold or higher dilutions (made before the addition of the decolorizing agents) will often be necessary.

PROPERTIES

OF THE REACTION

SYSTEM

Use of Benxidine for Determination of Hydrogen Peroxide. The oxida-

tion of galactose oxidase proceeds by the reaction outlined above, with the evolution of stoichiometric quantities of hydrogen peroxide. The reaction of hydrogen peroxide with any of a number of reducing agents

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and dyes, catalyzed by peroxidase, results in the production of a variety of compounds which absorb light in the visible or ultraviolet regions, and the conditions can be adjusted so as to ensure that the production of these follows in stoichiometric proportions. A large number of reducing agents is available and those that have been used in photometric assay procedures depending upon the oxidizing properties of hydrogen peroxide include pyrogallol (26)) o-phenylenediamine (27)) o-anisidine (20, 28), and o-tolidine (19). The results obtained after testing a large series of such compounds indicated that only benzidine (among the compounds tested) afforded both linearity of color response and appreciable sensitivity over a range of H,Oz concentrations useful for the determination of galactose in the concentrations likely to be encountered, as well as relative stability of color. Curve (a) of Fig. 6 is a plot of the ultraviolet absorption spectrum of the reaction product obtained in the galactose assay procedure de-

.060.020260

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300

320

I 340

I 360

WAVELENGTH

I 360

I 400

I 420

I 440

I 460

I 460

500

(mp)

FIQ. 6. Ultraviolet absorption spectra: (a) Spectrum of combined reaction product(s) after incubation of 20 gg of galactose with reagent mixture for 30 min; optical densities were measured using the reagent blank as the reference solution in order to subtract out the absorbances of unreacted materials and thereby yield as close an approximation as possible of the absorption spectrum of the reaction product(s). (b) Spectrum of reagent (i.e., the same mixture as curve a, but with the reaction terminated at zero time), measured using distilled water as reference solution.

ENZYMIC

DETERMINATION

OF

GALACTOSE

45

scribed above. The peak absorption occurs at 295 mp. Curve (b) is a plot of the absorption spectrum of the unreacted benzidine under the same conditions. The absorption of this material at 295 my is appreciable and can (and does) interfere with the linearity of the standard curve for galactose at this wavelength, since the concentration of unreacted benzidine remaining (and, therefore, its contribution to the absorption at 295 mp) varies inversely with the amount of benzidine oxidized. However, the extinction of the unreacted benzidine-as seen from curve (b) -falls off rapidly with increasing wavelength, and at 310 rnp is no longer sufficiently intense to interfere with the linearity of the assay response. It is evident from the curves that 310 rnp is the wavelength yielding the maximum absorption from the oxidation of galactoee with the minimum contribution from the unreacted materials. Even at this wavelength, however, some absorption does occur, and this accounts for t’he major part of the optical density reading obtained at “zero” galactose concentrations, and, therefore, for the failure of the standard curve to intersect the origin of the graph. This initial absorption would be much higher at 295 rnp and would cause the standard curve to intersect the vertical axis at about 0.250 OD unit. Since the extinction of the oxidized benzidine is maximal at 295 rnp., the slope of the standard curve would be st,eeper at this wavelength, and the sensitivity of the assay would, therefore, be greater (in terms of OD units developed per pg of galactose) than at 310 rnp. Clearly, the choice of 310 rnp represents a compromise designed to achieve linearity of response, minimal “zero” readings, and maximal range, at the expense of a certain amount of sensitivity. The spectrum shown in curve (b) does not differ substantially from the spectrum of a control solution containing water instead of galactose and incubated for 30 min and then acidified. The use of a lower concentration of benzidine in the basic reagent mixture would reduce the range of the assay by limiting the amount of benzidine available for oxidation, but would also avoid the problems caused by significant basal absorption at 295 mp and would thereby make it possible to operate the assay at this wavelength and achieve maximal sensitivity. This property of the system is exploited in the variation of the procedure described for the determination of small amounts of galactose (1 to 20 pg) with greater sensitivity. On carrying out the assay procedure, it will become evident that a pale pink color is also produced; this color is in the visible region of the spectrum, and has a wavelength quite different from that used in the photometric assay. The visible color produced is, however, useful as a rough guide for deciding whether or not to dilute samples before

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reading the optical density at 310 mp: in general, any sample with enough visible pink color to be readily identified by t’he unaided eye will have an optical density in excess of 0.400 at 310 mp. A number of reaction products, differing from one another in charge, electron configuration, oxidation state, degree of molecular association, and combination with other ions, is possible when benzidine is oxidized with H,O, (29). An analysis of the identity of the product (or products) whose optical properties permitted the construction of the assay procedure described above was not carried out, and a detailed discussion of the various possibilities is beyond the scope of this report. The relevance of the foregoing lies in the fact that variations in such conditions as pH, reaction rate, or temperature can result in the appearance of oxidation products of benzidine exhibiting entirely different optical properties, absorbing light maximally in the visible spectrum (510 or 410 rnp) or not at all. Some of these other products, obtained with slight variations in procedure, are potentially useful in this type of assay by virtue of their linear response and very high extinction coefficients, but present difficulties because they are relatively unstable. In addition to the free base, benzidine is available as the hydrochloride, a form which is generally more stable, more easily soluble, and supplied in purer form. However, the acidity of the hydrochloride is so great that even the small amount added exceeds the capacity of the buffer system, and the use of the free base is therefore recommended. Effect of PH. Figure 7 shows initial rate curves for the reaction of 50 pg of galactose per milliliter in glycine buffer at various pH levels, and a plot of the reaction rate as a function of the pH. The maximal reaction rate is achieved at pH 8.3. (Both of the enzyme preparations used yielded identical curves.) The variation of the reaction rate (and therefore the color yield at the end of 30 min) with varying pH is small in this range, and, therefore, errors introduced as a result of small pH shifts (which may be caused by buffer systems present in the samples to be analyzed) will generally be negligible. The pH response which has been described elsewhere (14) for galactose oxidase may provide a truer characterization of the properties of the enzyme itself; the pH optimum described here is valid only with respect to the development of ultraviolet absorbance in the mixture containing galactose oxidase, peroxidase, and benzidine. The galactose oxidase reaction proceeds satisfactorily in a number of buffer systems, including phosphate and Tris, but the solubility characteristics of the color reagent (benzidine) are such that the best results, for the purpose of this assay, are obtained with glycine buffer. Rate of Reaction. Rate curves for two concentrations of galactose are

ENZYMIC

.700(

PH

DETERMINATION

7;5

I 5

7;

I IO

7.9

I I 15 20 INCUBATION

OF

8;

9.;

I 25 TIME

a;5

I 30 (Minute.)

47

GALACTOSE

8;

I 35

9;

I 40

1

I 45

PIQ. 7. Effect of pH on rate of galactose oxidation by galactose oxidase. Three rate curves are shown describing the development of absorbance at 310 mp under the conditions of Method I as a function of time (refer to abscissa at bottom of graph) at pH 7.5, 8.3, and 9.0. A pH optimum curve is also shown, describing the absorbance at 310 rnp developed in 30 mm as a function of pH (refer to abscissa at top of graph).

presented in Fig. 8. Approximately 60% of the final ultraviolet absorption is developed in the 30 min allowed for the reaction in the assay procedure. Greater sensitivity can thus be achieved, when necessary, by prolonging the incubation time (although at the expense of a reduction in the range of linearity), and this property is exploited in the procedural variation described for determining smaller amounts of galactose with greater sensitivity. Standard Curves. Sensitivity, Precision, and Range. On inspection of the standard curves in Figs. 1 and 2, it will be noted that at LLzero” galactose concentration, a definite reading is always obtained, and this fa,ilure of the standard curve to intersect at the origin is one of the chief

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90040,ug

GALACTOSE

.aoo-

0 GALACTOSE I IO

I 20

I 30

I 40

I 50

INCUBATION

I 60

I 70

TIME

(Minutes)

I 00

I 90

I 100

I 110

120

FIG. 8. Rate of galactose oxidation by galactose oxidase: generation of ultraviolet absorption as a function of time, under the conditions of Method I. Oxidation of 40 pg and 20 pg of galactose was allowed to proceed for varying intervals, instead of the standard 30 min used in the assay procedure. The lowest curve describes the %pontaneous” increase in the absorbance of the reaction mixture (i.e., rate of oxidation of bensidine by components other than the H20, produced in the oxidation of galactose) ; if galactose oxidase is omitted from the mixture, this increase is not observed.

shortcomings of the method. The magnitude of the zero reading is a function of the amount of benzidine present in the original reagent mixture, the wavelength at which absorption is measured, and the time of incubation. Since these three factors, as discussed above, also affect the sensitivity of the method (i.e., the optical density developed per microgram of galactose), it was necessary to find the optimal combination which would yield maximum sensitivity without producing a prohibitively high “background” absorption. As indicated by the slope of the standard curve (Fig. l), the ultraviolet absorption (or extinction) generated per microgram of galactose in the general assay procedure amounts to 0.0128 optical density unit. The slopes of many standard curves run over the course of several months tended to vary somewhat (between 0.0100 and 0.0135 OD unit per

ENZYMIC

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microgram of galactose), depending on the batch of galactose okkxe used, since no attempt was made to quantitate precisely the enzyme unitage of the galactose oxidase preparations received from the suppliers. However, strict internal linearity was always observed. This means that the inclusion of at least 3 standards (a “zero” blank and 2 different concentrations of galactose) to define the slope of the curve is advisable when this technique is to be applied to precise quantitative measurements. In practice, we have found that these 3 extra standards never have varied by more than 5% from a previously prepared complete standard curve, so long as the same batch of galactose oxidase was used. Within any single standard curve, no points were ever found to deviate from the rectilinear plot by more than 2%. The response of the general assay procedure as described above, is linear for quantities of galactose ranging from 0 to 150 pg (per 0.5 ml of fluid assayed), i.e., for concentrations between 0 and 300 pg/ml. The smallest amount of galactose which can be measured quantitatively with reliability is in the neighborhood of 2.5 pg, and therefore the effective range of the assay is from 5 to 300 pg/ml. Since the upper limit of the linear range is determined by the solubility characteristics of the beneidine oxidation product, it is possible to increase the range (at the expense of sensitivity) through the use of a shorter incubation time. (Alternatively, as with most such methods, the range can also be extended upward indefinitely by appropriate dilution of the original samples, although this always involves a corresponding loss of sensitivity.) In any case, the development of an optical density greater than 2.000 is an indication that the linear range of the assay procedure has been exceeded. Interfering Substances. In the course of attempts to apply the technique reported here, a number of potentially interfering substances have been identified. Others are evident from consideration of the mechanism of the assay procedure. The following is a list of the substances so far identified whose presence will interfere with the accuracy or sensitivity of the assay: (a) Hydrogen peroxide, or sources thereof (as might be represented by other biological reactions proceeding concurrently with the evolution of H,O.>). (6) Other peroxides, as Na,O, or ethanol peroxide. (c) Significant amounts of catalase or other agents (e.g., catalytic metals) which will absorb, consume, or destroy H,O,. (Catalase activity is almost ubiquitous in biological systems and has even been detected in the galactose oxidase preparations used in the assay procedure. However,

50

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the affinity of the peroxidase, which is present in great excess, for H,O, is so high that the effect of these small amounts of catalase activity is easily overcome.) (d) Cyanide ions, fluoride ions, and peroxidase inhibitors in general. Traces of cyanide will inhibit both peroxidase and galactose oxidase; the latter is not affected by fluoride ions. (e) Oxidizing agents or systems capable of oxidizing benzidine under these conditions (as for instance perchlorates, silicates, and certain metals of low electromotive force at high oxidation states). (f) Reducing agents which will compete with benzidine for the peroxide, as ascorbic acid. the benzidine present by (g) Sulfate ions, which will precipitate reacting with it to form the insoluble benzidine sulfate. (h) Large quantities of compounds with significant ultraviolet absorption, as mates, purines in general, proteins, certain amino acids, etc. (i) Benzoic acid, which will inhibit galactose oxidase when present in small concentrations. (Benzoic acid is often used to retard the growth of microorganisms in stock solutions of various sugars; obviously, its use must be avoided in the galactose standard solutions. We have found no other suitable preservative, and have had to resort to storing the galactose solutions in the frozen state at -15°C or else to making up fresh solutions at 2- to S-week intervals.) (j) Other sugars capable of reacting with galactose oxidase (15). All of the potentially interfering agents enumerated above either do not occur in ordinarily encountered biological materials or else are readily removed by one or more of the techniques described above for the preparation of samples. Additional data concerning the effect of various inhibitors on the galactose oxidase reaction have recently been reported by Amaral et al. (30). Comparison

of Galactose Oxidase Preparations. Although it was possible to obtain satisfactory curves using various batches of galactose oxidase purchased from Worthington Biochemical Corporation, the galactose oxidase preparations obtained from Ames Research Laboratories yielded superior results under the conditions of the assay procedure as described here. The usefulness of the Worthington enzyme was limited by several factors: when dissolved, it formed a turbid suspension which imparted an appreciable absorption of light throughout the ultraviolet range to the reagent mixture; it contained sulfate ions in concentrations sufficient to precipitate the benzidine in the reagent solution at room temperature; even in the absence of galactose, sufficient oxidizing activity was present to produce considerable color (amounting to 0.145 OD

ENZYMIC

DETERMINATION

OF

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51

unit) with benzidine during the 3%min incubation; catalase activity was high enough to completely destroy the H,O, in 1 ml of a 0.03% solution in 5 min. The enzyme obtained from Ames Research Laborntories was supplied as a dry powder containing 35,006 to 43,000 units/gm, which dissolved readily in water to produce a clear colorless solution of 1 mg/ml, with negligible ultraviolet absorption; no materials incompatible with benzidine were present; color production with benzidine after 30 min of incubation in the absence of galactose, although definitely present, amounted to only 0.010 OD unit (cf. Fig. 8) ; the catalase activity present was such that 50% of the H,O, in a 0.0006% solution was destroyed in 15 min. DISCUSSION

The properties of the galactose oxidase reaction have been studied, and adapted to a convenient, procedurally simple photometric method for galactose determinations. The technique seems well suited for application to a variety of situations, some of which have been illustrated in this report. As evident from Fig. 1, the color response to increasing concentrations of galactose obeys a strictly rectilinear relationship over the range between 0 and 150 pg of galactose, yielding an optical density increment of 0.0128 unit per microgram. The method possesses a high, although not absolute degree of specificity. The complete absence of any reaction with glucose, as reported by Avigad et al. (15) was confirmed in this laboratory. The reactivity of galactose oxidase with substrates other than galactose has been examined and described by Avigad et al. (15)) who found that certain other sugars-notably derivatives of galactose such as galactosamine, as well as galactosides such as stachyose-may react appreciably. Certain of these sugars react at rates sufficient to permit adaptation of this procedure to their determination, but, in general, the likelihood of their presence in the routinely encountered biological solutions is small. The section describing potential interfering substances is intended for the guidance of investigators who may wish to extend the method to more complex situations. In actual practice, the system has been found to be quite ‘hardy” and entirely reproducible (except as influenced by variations in the potency of the enzyme preparations furnished) when samples are prepared in the manner described. SUMMARY

Techniques are described for the quantitative analysis of biological solutions for galactose content, involving incubation of unknown samples with the enzyme galactose oxidase in the presence of peroxidase and

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benzidine. Some characteristics of the reaction are discussed and conditions are defined for two procedural variations, depending upon the sensitivity and range desired. Directions are stated for the application of the method to blood, urine, and other biological fluids, and illustrations of a number of these applications are presented. REFERENCES 1. HARDING, V. J., AND GRANT, G., J. Biol. Chem. 94, 529 (1931). 2. RAYMOND, L., AND BLANCO, G., J. Biol. Chem. 79, 649 (1928). 3. STENSTAM, T., Acta Med. Stand., suppl. CLXXVII, 125, 1-114 (1946). 4. LARRSON, P. A., AND SVEINSSON, S. L., Slcand. Arch. Physiol. 83, 58 (1939). 5. KUTSCHERA, W., AND RETTENBACHER, R., Wien. Klin. Wochschr. 68, 528 (1956). 6. ALBERS, P., AND FREISKORN, R., Ann. Chem. 622, 150 (1959). 7. ATKINSON, M. R., BURTON, R. M., AND MORAN, R. K., Biochem. J. 78, 813 (1961). 8. LEOPOLD, B., Anal. Chem. 34, 170 (1962). 9. DATE, J. W., Stand. J. Clin. Lab. Znz;est. 10, 155 (1958). 10. TYGSTRUP, N., WINKLER, K., LUND, H., AND ENGELL, H. C., Scund. J. Clin. Lnh. Invest. 6, 43 (1954). 11. SONDERGAARD, G., &and. J. CZin. Lab. Invest. lb, 203 (1958). 12. GROGER, W. K. L., C&n. Chim. Acta 6, 866 (1961). 13. WYNGAARDEN, J. F., SEGAL, S., AND FOLEY, J., J. CZin. Invest. 36, 1395 (1957). 14. COOPER, J. A. D., SMITH, W., BACILA, M., AND MEDINA, H., J. Biol. Chem. 234, 445 ( 1959). 15. AVIGAD, G., AMARAL, D., ASENSIO, C., AND HORECKER, B. L., J. BioZ. Chem. 237, 2736 (1962). 16. KESTON, A. S., Abstmcts, 129th meeting American Chemical Society, April, 1965, p. 31c. 17. SEGAL, S., AND BERNSTEIN, H., J. Pediut. 62, 363 (1963). 18. AGRANOFF, B. W., RADIN, N., AND SUOMI, W., Biochim. Biophys. Actu 57, 194 (1962). 19. ROREM, E. S., AND LEWIS, J. C., Anal. Biochem. 3, 230 (1962). 20. DE VERDIER, C. H., AND HJELM, M., CZin. Chim. Actu 7, 742 (1962). 21. SEGAL, S., ROTH, H., AND BERTOLI, D., Science 142, 1311 (1963). 22. SCOTT, D., J. Agr. Food Chem. 1, 727 (1953). 23. SCOTT, T. S., Brit. J. Znd. Med. 9, 127 (1952). 24. NELSON, N., J. Biol. Chem. 155, 374 (1944). 25. DOLE, V. P., AND COTZIAS, G. C., Science 113, 552 (1951). 26. BIELEFELDT, J., 2. Vituminforsch. 13, 286 (1943). 27. WALLERSTEIN, J. S., ALVA, R. T., HALE, M. G., AND LEVY, H., Biochim. Biophys. Actu 1, 327 (1947). 28. COMER, J. P., AND BRICKLEY, H. F., Anal. Chem. 31, 109 (1959). 29. WELCHER, F. J., “Organic Analytic Reagents,” Vol. II, p. 275 ff. Van Nostrand, Princeton, N. J. 1947. 30. AMARAL, D., BERNSTEIN, L., MORSE, D., AND HORECKER, B. L., J. Biol. Chcm. 238, 2281 (1963).