Determination of Exogenous Galactose

in Blood using “Specially Purifled”

Glucose Oxidase

Sheldon S. Waldstein, Alvin Dubin, Albert Newcomer,

and Charles H. McKenna

A method is described for the determination of exogenous galactose in blood by amodification of the Nelson-Somogyi hexose procedure in which glucose oxidase is

used to eliminate interfering glucose. The use of “specially purified” glucoseoxidase (SPGO) achieves low, regular blanks and satisfactory replicates, and

enhances accuracy, in contrast to the results obtained with crude enzyme prepara-tions. The ready availability of SPGO makes the method satisfactory and practical

for the clinical laboratory.

rFBE ACC�TRATE DETERMINATION of exogenous galactose in blood by a

method which is rapid and easily adapted to routine blood sugar ineth-

ods is desirable, particularly for the study of liver function and dis-

ease. (ilucose oxidase has been useful for elimination of glucose from

biologic fluids ill order to measure other sugars by methods in which

glucose would be an interfering substance (1). For the past 5 years,

we have used glucose oxidase for this purpose in a colorimetric method

for the determination of exogenous galactose in blood. Until recently,

most enzyme preparations that have been available commercially have

been crude, and when used in this manner have had the important dis-

advantage of producing high, irregular, and unpredictable blanks (1).

In the past 2 years, we have tested and used a preparation, designated

“speciallY purified” glucose oxidase (SPGO) by the manufacturer,

that has proved to he satisfactory for colorimetry.

From the Department of Medicine, Northwestern University Medical School, and the

Hektoen Institute for Medical Research of the Cook County Hospital, Chicago, Ill.

This study was supported by Research Grant H-2187 from the National Heart Tiistitute,

U.S. Public Health Service.

Received for publication Apr. 6, 1962.

381

382 WALDSTEIN fT AL. Clinical Chemistry

It is the purpose of this report to present a simple modification of

the Nelson-Somogyi hexose method suitable for the measurement of

exogenous galactose in blood, and in which the use of SPGO has elim-

inated the irregular color development which occurs when crude glu-

cose oxidase is used.

Materials and MethodsReagents

SPGO* Stock enzyme SPGO is supplied as a lyophilized powder

in vials containing 4400 U. of glucose oxidase.t In addition, catalase

is present in an unquantitated amount. When protracted storage is

anticipated, store the vials in the refrigerator in the dark. To prepare

a working enzyme solution containing 400 U./ml. dissolve the lyophi-

]ized powder in distilled water. Ordinarily, no more than a weeks’

supply of working enzyme solution should be kept on hand.

Zinc sulfate (ZnSO4 . 7H,O), reagent-grade, w/v 5%

Barium hydroxide, reagent-grade, O.3N Upon titration, 5 ml. of

zinc sulfate solution diluted to 25 ml. with distilled water should re-

quire 4.7-4.8 ml. of the barium hydroxide solution to produce a pink

color with phenolphthalein. Adjust the solutions as needed.

Caprylic alcohol, reagent-grade

Somogyi copper reagent (2) Before use, add 1 ml. 15% copper

sulfate (3) to each 24 ml. of this reagent.

Nelson arsenomolybd ate solution (2)

Galactose standard solutions Prepare solutions of galactose in

varying concentrations between 10 and 300 mg./100 ml. with saturated

benzoic acid solution. Galactose, C.P.,1 or 50% galactose injectable

solutionsl are satisfactory. Store in the refrigerator for no more than

2 weeks.

Apparatus

Oxygen train The reaction is carried out in 175 X 22-mm. bac-

teriologic culture tubes. The reaction tubes are closed with rubber

stoppers through which pass two hollow glass tubes, one of which ex-

*Fer,neozynle Type 785, Fermco Laboratories, Inc., Chicago, Ill., distributed by Pfanstiehl

Laboratories, Inc., Waukegan, Ill.

tA unit of glucose oxidase is defined by the manufacturer of SPGO as that amount ofenzyme which will cause up take of 10 ml. oxygen per minute, using 3.3% glucose substrate,

at 300 and pH 5.9.

Available from Pfanstiehl Laboratories, Inc.

Vol. 10, No. 5, 1964 DETERMINATION OF GALACTOSE 383

tends to within 5 mm. of the bottom of the reaction tube and the other

of which extends to a distance well above the surface of the fluid. Con-

nect these to a source of oxygen, whose flow should be adjusted care-

fully to provide equal oxygenation by noting the bubbling in all tubes.

A suitable rack to support the tubes may be constructed from a 2 X 4-

iii. board.

Heparinized containers Add 3 drops of heparin solution, 1000 TJ./

ml., to a number of 25-ml. containers and dry at 60#{176}.These contain

sufficient heparin to provide adequate anticoagulation for as much as

25 ml. of blood.

Preparation of Samples

Reagent blank without enzyme (RB) Add 16 ml. of distilled water

to a reaction tube.

Reagent blank with enzyme (EB) Add 15 ml. of distilled water

and 1 ml. of working enzyme solution to a reaction tube.

Control blood sample Before the introduction of galactose into

the subject, collect (without stasis) 10-25 ml. whole blood for the

measurement of fasting blood sugar and residual reducing substances,

and for the construction of the standard curve for galactose. When

desired, plasma may be substituted for whole blood.

Galact’ose standard curve For each standard, add 13 ml. of dis-

tilled water, 1 ml. of control blood, 1 ml. of the appropriate galactose

standard solution, and 1 ml. of working enzyme solution to a reaction

tube. The galactose standard curve should be constructed to include

the anticipated range of galactose concentration in the unknown

samples.

Residual reducing substances (RB) Add 14 ml. of distilled water,

1 ml. of control blood, and 1 ml. of working enzyme solution to a

reaction tube.

Blood glucose Add 15 ml. of distilled water and 1 ml. of control

blood to a reaction tube.

Unknown samples Collect, without stasis, 3-4 ml. whole blood per

sample at various times after the introduction of galactose into the

subject. Immediately upon collection, add each sample to a heparinized

container, mix, and pipet 1 ml. into a reaction tube containing 14 ml.

of distilled water. Add 1 ml. of working enzyme solution.

Where applicable in all samples described above, add the blood as

soon after shedding as practical, but add the working enzyme solution

just before reaction.

384 WALDSTEIN fT AL. Clinical Chemistry

Procedure

Reaction of Blood with Glucose Oxidase

Add a fine drop of caprylic alcohol to each reaction tube. Connect

the oxygen train and oxygenate the mixtures at room temperature for

30 mm. (Care should be taken that each oxygen delivery tube is placed

to the same depth in each reaction tube.) After 30 mm., disconnect the

oxygen train carefully to avoid aspiration of the sample into the

delivery tubes. From this point on, follow the usual hexose method.

Precipitation

Add 2 ml. of barium hydroxide to each reaction tube, mix, and allow

to stand until all color change has been completed. Add 2 ml. of the

zinc sulfate solution, shake thoroughly, and filter through medium

filter paper. The mixture is now a 1 :20 dilution of the original sample,

and should yield at least 14 ml. of a clear and colorless filtrate which

contains no enzyme activity. Should the filtrate be distinctly colored

or turl)id, discard the sample.

Color Development

Add 1 ml. of the filtrate to a Folin tube. Add I ml. of the modified

copper reagent. Place the Folin tubes in a boiling-water bath for 20

mm. Although glucose develops maximum color in 10-iS mm., galac-

tose does not reach its maximum in less than 20 mm. (3). Cool the

tubes rapidly in an ice bath. Add 1 ml. of the arsenomolybdate reagent

rapidly. Mix thoroughly, and after all foaming has ceased, dilute with

distilled water to 25 ml.

Colorimetry

Galactose develops approximately 83% of the absorbance as glucose

under identical conditions (3). Readings may be made in an Evelyn

colorimeter, using the macrocuvet (10-mm. slit) at 515 mp�, or in other

appropriate instruments. Set the colorimeter to 100% transmittance

using the reagent blank without enzyme (RB). Read the reagent

blank containing enzyme (EB) against the RB. Satisfactory enzyme

solutions produce an EB which gives no more color than 95% T

against the RB. Reset the colorimeter to 100% T, using the EB. Read

all unknown samples and galactose standards against the EB. Con-

struct the standard curve. It sho#{252}ldcross the ordinate at the RS value.

Read the values for the unknown samples from the standard curve for

galactose constructed for each run.

Vol. 10, No. 5, 1964 DETERMINATION OF GALACTOSE 385

ResultsShelf Life

Under usual laboratory conditions, SPGO appeared stable for pro-

tracted periods. Seven lots of SPGO solution were frozen for up to

9 months and tested at regular monthly intervals for activity against

glucose. No loss of activity was noted. Lyophilized preparations of

SPGO stored in the refrigerator or at room temperature in the dark

were likewise found to maintain full activity for several months. When

such precautions were not taken, partial loss of activity occurred.

Recalibratiori of the unitage of a batch of lyophilized SPGO after 10

months of storage at room temperatures under ambient 1i�-ht showed

that 10% of the original glucose oxidase activity and 30% of the

original catalase activity was lost (4). Thus it can be expected that no

appreciable loss of glucose oxidase activity will occur for protracted

periods if lyophilized SPGO is stored in darkness in the refrigerator.

For the present method, the enzyme solution must produce a low

and regular blank. Storage of the working solution was found to re-

sult in an increasing blank with time, so that eventually the color of

the blank exceeded the color produced by low concentrations of galac-

tose. This occurred at varying intervals, depending upon the mode

of storage. Working enzyme solutions stored in the refrigerator ex-

cept when used remained satisfactory for 10-14 days. Solutions

frozen immediately after preparation were stored for periods up to

1 year, and were as satisfactory when thawed as were freshly prepared

solutions. Daily thawing and freezing, however, appeared to cause

more rapid deterioration than storage at refrigerator temperatures.

Therefore it has been our practice to divide large volumes of working

enzyme solution into aliquots (each of a volume sufficient for the num-

ber of weekly determinations) and to freeze them. At the beginning

of each week, a new aliquot is thawed, stored in the refrigerator, and

discarded at the end of the week. A careful record of each EB is kept,

and the remaining frozen aliquots of the original batch discarded if

the EB begins to show consistently excessive color. Six- to 12-month

supplies of working enzyme solution have been stored and used satis-

factorily in this way. With the present availability of lvophilized

SPGO in convenient quantities, however, such extremes of storage

time for working solutions of the enzyme need not be encountered.

Enzyme Characteristics which Affect the Method

Glucose oxidase exhibits significant activity over a pH range of 4-8(s). Gluconic acid is generated rapidly, and in unbuffered aqueous

386 WALDSTEIN FT AL. Clinical Chemistry

solutions, a prompt drop of pH below 4.0 occurs, which limits the re-

action. Buffering is therefore necessary in aqueous media. A phos-

phate buffer at pH 7.0 has proved satisfactory for the present method,

since it is near the optimum pH of the enzyme and does not interfere

with the protein precipitants used. In blood, the buffers contained

within a 1-ml. sample have been found to be sufficient to maintain the

pH of the reaction mixture well within the range of optimum activity.

The enzyme is active over a wide range of temperature. Impairment

in activity is noted at 0#{176},but at room temperatures and at 37#{176},destruc-

tion of glucose in the medium is virtually complete within the specified

reaction time. Accordingly, the method is carried out at room tem-

perature.

Oxygen, required for the oxidation of glucose, must be supplied to

the reaction mixture. Although it has been possible to secure oxygena-

tion by placing the 16-mi. reaction mixture in a large, flat-bottomed

flask, thereby creating a thin layer of fluid into which oxygen can pass

by diffusion, optimum oxygenation has been achieved by bubbling

oxygen through the mixture.

Oxidation of glucose in the presence of glucose oxidase is a first-

order reaction. Under the conditions of the method, the reaction ap-

proaches completion within 15 mm. but does not reach equilibrium for

60-120 mm. Since it has been shown previously that glucose oxidase

attacks galactose to a limited extent (3), a variety of reaction times

were studied to select one resulting in the most glucose removal with

the least galactose loss. A minimum of 15 mm. and a maximum of 60

mm. met these objectives when the recommended concentration of

enzyme was used. Therefore, 30 mm. of reaction time was selected

as most convenient.

The effect of enzyme concentration was studied in buffered aqueous

solution and in blood (Fig. 1). In buffer, approximately 40 U. SPGO

pci’ tube was sufficient to eliminate glucose at an initial concentrationgreater than 2000 mg./i00 ml. In blood, however, 400 U. of SPGO per

tube was necessary to effect elimination of glucose at a typical fasting

concentration, and 800 U. SPGO per tube did not effect complete re-

moval of glucose when an initial concentration over 500 mg./100 ml.

was present. The reason for inhibition of enzyme activity in blood is

unknown. Because blood may contain peroxidase inhibitors, an at-

tempt was made to overcome the inhibition by addition of catalase in

excess of that already contained in SPGO. No effect upon the inhibi-

tion was found. When blood is reacted with the enzyme, the value for

residual reducing substances (RS) represents nonglucose-reducing

Initial Glucose Concentration (mgI 100 nil.)

�--‘ 2250 (in buffer)I 05 (in blood)

#{176} 520 (inblood)

0 100 200 300Units SPGO added

400 800

Vol. 10, No. 5, 1964 DETERMINATION OF GALACTOSE 387

substances (including endogenous galactose) and residual glucose,

if any. Blood samples obtained during the course of oral glucose

tolerance tests were treated with SPGO (400 U.) and the value for RS

compared to that obtained for the corresponding fasting sample.

Fig. 1. Effect of vary-

jug amounts of SPGO on

glucose removal under

standard conditions of

method.

Although the RS value for the fasting bloods varied among different

subjects, there was no measurable difference in each subject between

the RS values for nonfasting and for fasting blood samples, even

though the glucose concentration had increased by as much as 150 mg./

100 ml. during the course of the test. This large a variation in glucose

concentration has not been encountered in galactose tolerance tests in

fasting subjects. Therefore, 400 U. SPGO per tube was selected for the

method because this concentration has minimal effect upon galactose,

is effective for complete removal of normal fasting concentrations of

blood glucose, and permits a moderate increase in glucose concentra-

tion above fasting without change in the RS value. Regular measure-

ment of both the RS value and the fasting glucose concentration is

recommended. If either is unusually high, the possibility of incom-

plete glucose removal should be considered. It has been our practice

to defer galactose tolerance testing if the blood glucose concentration

is above 250 mg/100 ml.

Accuracy

Although destruction of galactose by glucose oxidase may be con-

siderable upon protracted incubation (3), under the conditions select-

ed for the present method, an average of 5% (1-9%) of galactose in

buffered aqueous solution was lost at concentrations between 10 and

250 mg./100 ml. When the enzyme was first inactivated by boiling,

N represents Number of pairs; D., means difference between pairs; S.D., Standard Devia-

tion of difference between pairs.

388 WALDSTEIN FT AL. Clinical Chemistry

complete recovery of all concentrations of galactose was obtained. In

blood, less galactose is probably lost because of the inhibition of

enzyme activity. Nevertheless, it is recommended that, in each blood

studied, a standard curve for galactose be constructed encompassing

the anticipated range of blood concentrations.

An estimate of the accuracy of the present method was obtained by

analysis of the difference between duplicate measurements of known

concentrations of galactose between 10 and 250 mg./100 ml., both in

buffered aqueous solution and in whole blood (Table 1). These differ-

ences include procedural error as well as error due to variation iii

enzyme activity. Concentration had no apparent effect upon accuracy.

In aqueous solution, the difference between duplicates was well within

1% T. This is of the same order of accuracy obtained for the Nelson-

Somogyi hexose method alone using the same instrumentation, and

suggests that the enzyme, SPGO, contributes little sampling error

per se to the method. In blood, greater sampling variation was found,

although the mean difference between duplicates was 1% T or less.

The reason for the variation being greater in blood than in aqueous

solution is not known, but may be related to the partial inhibition of

enzyme activity in blood, noted above. Because of the shallow slope

of the galactose standard curve, the mean difference between dupli-

cates is equivalent to approximately 2 mg./100 ml. galactose in aqueous

solution, and to 4 mg./100 ml. galactose in blood; differences as great

as 10 mg./100 ml. may be encountered in blood.

On the other hand, when ordinary commercial crude glucose oxidase

was employed under the conditions of the present method, duplication

was unsatisfactory (Fig. 2). The differences between duplicates using

a crude preparation were equivalent to 30-40 mg./100 ml. galactose.

Table 1. DIFFERENCES BErWEEN DUPLICATES OF GALACPOSE STANDARDS WITH USE OF SPGO

Concentration gaiaetose

added (mg/joe mi)

Galactos e standards in buffer G&actose standards in blood

N D.±S.D. (% T) N D.±S.D. (% T)

10 41 0.22 ± 0.28 43 0.71 ± 0.72

25 72 0.39 ± 0.50 66 0.84 ± 0.90

40 30 0.20 ± 0.23 27 0.93 ± 0.72

50 76 0.28 ± 0.30 70 0.78 ± 0.83

100 64 0.37 ± 0.30 60 0.82 ± 0.78

150 49 0.50 ± 0.55 46 1.17 ± 0.89

200 55 0.37 ± 0.30 49 1.09 ± 0.97

250 22 0.44 ± 0.44 19 1.05 ± 0.84

All concentrations (ToTAL) 409 0.41 ± 0.32 380 0.90 ± 0.82

#{149}

#{149}

S

S

S

Vol. 10, No. 5, 1964 DETERMINATION OF GALACTOSE 389

Variation of such magnitude is largely inherent in crude preparations

of the enzyme and does not appear only in the presence of glucose or

galactose. The color produced by duplicate blanks containing no en-

zyme or of duplicate blanks containing SPGO has been consistently

#{149}Crude glucose o.s’idose.#{149} I O�pe�ilol/ypurif�jd

Fig. 2. Variation of final color developed glucose oxidose

under conditions of method from replicate - 1 :“

samples of blood (with and without added

galactose) reacted with crude glucose oxidase

or SPGO. Dotted lines indicate 1% trans. -

mittance difference. -

GLUCOSE GALACTOSE GALACTOSE9Omg./IOOml. 25 mg/lOOmI. l5OmG/I00 ml.

within 1% T, but the color produced by duplicate blanks containing

crude glucose oxidase varies irregularly and has been as great as

6% T. For these reasons, crude glucose oxidase is unsatisfactory for

the present method and the use of SPGO is recommended.

Discussion

In 1928, Muller (6) obtained an enzyme from fungi of Aspergillus

and Penicillium capable of catalyzing the oxidation of glucose. He

named this enzyme “glucose oxidase,” and although it has since been

shown to function as a dehydrogenase (7), the original and familiar

designation has been used in this report.

In this country, glucose oxidase is commonly derived from the mold

mycelia of Aspergillus niger grown on liquid media containing glucose.

Most preparations available here are crude, if highly concentrated.

However, preparations of greater purity have been made and used in

reported methods (1,8-10). One such preparation, now available corn-

mercially, contains “specially purified” glucose oxidase (SPGO);its coenzyme, FAD; and amounts of catalase adequate for the removal

of 11202 as it is formed.

Glucose oxidase catalyzes the following reaction:

glucoSeC,;Hi.,0,i + 1120 + Oi . � C611,207 + H20�

oxidase

Alternative approaches for the use of the enzyme in the determina-

tion of glucose have been developed. In one, the hydrogen peroxide

produced is reacted in the presence of peroxidase with a chromogenic

oxygen acceptor such as o-tolidine or o-dianisidine. The intensity of

the resulting color is proportional to the amount of glucose (11). This

390 WALDSTEIN ET AL. Clinical Chemistry

approach is the basis for the commonly used test strips for estimating

urine glucose (12). In another method, oxygen uptake is measured

manometrically, there being a stoichiometric relation between glucose

oxidized and oxygen consumption (13). In a third approach, total

hexose is measured before and after incubation of the sample with the

enzyme, the difference representing glucose (14).

Of more than 50 sugars (other than glucose) tested, glucose oxidase

has been found to catalyze the oxidation of galactose (3), mannose,

xylose, 6-methylglucose, 4-6-dimethylglucose (5), and 2-deoxy-d-glu-

cose (is). Since it attacks these sugars to a very limited extent it is

possible to use the enzyme to eliminate glucose from biologic fluids

and to determine these “foreign” sugars by appropriate means with-

out interferelice. In this manner, Tygstrup et al. (9) used “notatin”

to measure galactose, and Froesch et al. (1) used a commercially un-

available, highly purified glucose oxidase preparation to measure

inulin. Because the Nelson-Somogyi method is used widely for the

measurement of blood glucose, it seemed to us appropriate to adapt it

in like manner for the measurement of galactose. The procedure, as

developed in this laboratory, adds but 35-40 mm. to the usual glucose

method and requires little additional equipment. The use of SPGO,

a potent, stable, and uniform preparation of glucose oxidase, has

eliminated the irregularities produced by crude glucose oxidase and

has greatly enhanced the accuracy of galactose determination. The

commercial availability of SPGO should facilitate measurement of

galactose and permit greater use of galactose tolerance tests in clin-

ical medicine.

References

1. Froesch, E. H., Reardon, J. B., and Renold, A. E., J. Lab. 4� Gun. Med. 50, 918 (1957).

2. Nelson, N., J. Biol. Chem. 153, 375 (1944).

3. w�ld�t�th, S. S., Areilla, H. A., and Dubin, A., Quart. Bull. I’Torthwe,itern Univ. Mcd.

School 31, 323 (1957).

4. Se�tt, D., Personal communication, April, 1961.

5. Keilin, D., and Hartree, E. F., Bioche’m. J. 42, 221 (1948).

6. Miller, D., Bioehem. Ztschr. 199, 136 (1928).

7. Theorell, H., in The Enz2,mes (vol. 2). J. Summer, and K. Myrback, Eds. Academic,

New York, 1951, p. 348.

8. Coulthard, C. E., Miehaelis, R., Short, W. F., Sykes, G., Skrimshire, 0., Standfast, A.,

Birkinshaw, J., and Raistriek, H., Biochem. J. 39, 24 (1945).

9. Tygstrup, N., Winkler, K., Lund, E., and Engell, H. C., Scand. J. Clin. 4� Lab. Invest.

6,43 (1954).

10. Marks, \T�, Clin. Chim. Acta 4, 395 (1959).11. Saifer, A., and Gerstenfeld, S., J. Lab. 4�Ctin. Med. 51,448 (1958).

12. Comer, J. P., Anal. (Them. 28, 1748 (1956).

13. Keilin, D., and Hartree, E. F., Biochein. J. 42, 230 (1948).

14. Froeseh, E. B., and Renold, A. E., Diabetes 5, 1 (1956).

15. MeComb, B. B., and Yushok, W. D., J. Franklin Inst. 265, 417 (1958).