Rapid and successive microdeterminations of oxygen, d -glucose, and its anomers in blood with β-d -glucose oxidase and oxygen electrode

BIOCHEMICAL

MEDICINE

Rapid and D-Glucose,

7, 257-265

( 1973)

Successive Microdeterminations and Its Anomers in Blood Oxidase

and

JUN OKUDA Faculty

Oxygen AND

of Oxygen, with ,&D-Glucose

Electrode

GOU OKUDA

of Pharmaceutical Science, Meiio University, Showa-ku, Nagoya 468, and Clinical Laboratory, Daini Red Cross Hospital of Nagoya, Showa-ku, Nagoya 466, Japan

Received

May

24,

1972

In the previous papers (1,2), the authors reported a rapid polarographic microdetermination of n-glucose in biological fluid with P-Dglucose oxidase (EC 1.1.3.4, /3-n-glucose:oxygen oxidoreductase) and oxygen electrode. Recently, the authors reported polarographic determinations of n-glucose anomers with &n-glucose oxidase and mutarotase (EC 5.1.3.3, aldose-1-epimerase) (3, 4). Very recently, the authors also described the stoichiometric consumption of dissolved oxygen due to /3-n-glucose in water with /.!?-D-glucoseoxidase and oxygen electrode, and measured the amount of dissolved oxygen in water using a specified amount of n-glucose (5). These findings led us to study the rapid and successive determinations of oxygen, n-glucose and its anomers in blood with ,&n-glucose oxidase and oxygen electrode. In the present method, mutarotase was employed to measure the ratio of &n-glucose to the total n-glucose, and sodium azide ( catalase inhibitor ) was used to increase the sensitivity for determination of n-glucose ( l), and a specified amount of n-glucose was added in the reaction mixture as an internal standard for oxygen and n-glucose in the sample. Determinations of oxygen, D-glucose and its anomers in 15 ~1 of blood will be finished within only 3-4 min by the present method, The method is described in detail. APPARATUS

AND

REAGENTS

For measurement of oxygen consumption, a polarographic oxygen analyzer (Model 777, Beckman Instruments Inc., Fullerton, CA, diameter of the oxygen electrode is 1.2 cm) connected to a recorder (EPR-STC, Toa Electronics Ltd., Tokyo, Japan) was used. An acrylic plastics vial 257 Copyright All rights

@ 1973 by Academic Press, of reproduction in any form

Inc. reserved.

258

OKUDA

AND

OKUDA

(2.1 cm in height, 1.0 ml in volume) (5) was used, and the vial was placed on the magnetic stirrer. Microsyringes, 10 and 25 ~1 in volume (Jintan Terumo Co. Ltd., Tokyo, Japan) were used for addition of blood or reagents to the reaction mixture. Reagent grade n-glucose, usually a-n-glucose, was dissolved to a concentration of 5 mg/ml in water and allowed to stand overnight to reach mutarotational equilibrium prior to use. In an urgent case, n-glucose solution was heated at 90°C for 10 min to complete mutarotation. In water, n-glucose equilibrates among cr-anomer ( 36.5%), p-anomer ( 63.5%)) and so-called aldehyde form ( 0.003%). &D-Glucose oxidase used in this experiment was a preparation purified by Sephadex G-25 from crude enzyme preparation (Penicillium amagusakiense, Nagase & Co. Ltd., Osaka, Japan) with 0.1 M acetate buffer ( pH 5.6)) and dissolved in 0.5 M acetate buffer (pH 5.6) to a concentration of 15 mg/ml. Mutarotase was prepared from hog kidney cortex according to the purification procedures of Lapedes and Chase (6), and of Okuda and Miwa (7)) and dissolved in water to a concentration of 220 units/ml (8). For release of oxygen from hemoglobin and for inhibition of catalase in blood, 4.6% potassium ferricyanide-0.2% sodium azide mixture prepared, it can be used within 5 days when kept in a cold room. The arterial or venous blood was taken from aorta abdominalis or vena cava inferior of Wistar male rats (120-130 g) anesthesized with ether. PRINCIPLE

p-n-Glucose oxidase catalyses the following of catalase:

reaction in the absence

@-glucose+ 02 + Hz0 - D-gluconicacid + Hz02 If catalase is present in the reaction mixture, one molecule of hydrogen peroxide formed in the reaction mixture is decomposed to half a molecule of oxygen and one molecule of water. Oxygen thus formed can be used again for the oxidation of another molecule of /U-glucose by the above reaction. Therefore, the catalase was blocked by adding sodium azide as a strong inhibitor to the reaction mixture ( 1, 2, 7). So, the oxygen consumption in the presence of sodium azide is twice as much as that in the absence of sodium azide. The authors added mutarotase to the reaction mixture in order to mutarotate a-D-ghCOSe to /3-n-glucose and to oxidize the newly formed ,&n-glucose to n-gluconic acid in the presence of &n-glucose oxidase, so the addition of mutarotase makes the method possible to determine D-glucose anomers accurately and

OXYGEN

AND

GLUCOSE

IN

259

BLOOD

rapidly (4, 7). Oxygen in blood was released by addition of potassium ferricyanide in oxygen-depleted water, and was determined by the above reaction using a specified amount of n-glucose as an internal standard. PROCEDURES

Determinations

of Oxygen,

D-Glucose

and Its Anomers

in Arterial

Blood

Nitrogen gas was introduced into distilled water to deplete about half the amount of dissolved oxygen which was measured by the oxygen electrode (5), and 1.1 ml of the oxygen-depleted water containing ca. 4 ppm of dissolved oxygen was put into the vial with a wide-mouthed pipet. The oxygen electrode was immersed into the oxygen-depleted water as described in the previous paper (5). The meter reading of the oxygen electrode was set at about 40% on the recorder chart while stirring continuously with a stainless-steel stirring bar. In this case, the recorder was used at 50 mV full-scale. Then 5 ~1 of the potassium ferricyanide-sodium azide mixture was added; however, neither release nor consumption of oxygen was observed. A rapid release of oxygen bound to hemoglobin was recorded when 15 ,~l of arterial blood of rat was put into the vial (see Fig. la). Diffusion of oxygen from the atmosphere was not observed in the present method when the vial was used. Then, 5 ~1 of j?-D-ghCOSe oxidase solution was added to see the oxygen consumption (b) due to P-D-ghCOSC in blood, and finally 5 ~1 of standard

FIG. 1. Determinations K3Fe( CN),, p-D-glucose

of oxygen oxidase, and

and oxygen

D-ghCOSe

electrode.

in

arterial

blood

of

rat

with

260

OKUDA AND OKUDA

D-glucose solution (5 mglml) was added to record the oxygen consumption (c) due to /3-D-glucose of the standard D-glucose (25 ,ug) added (Fig. 1). As shown in Fig. 2, mutarotase was added to the reaction mixture after measurement of oxygen consumption (b) due to p-D-glucose in arterial blood with B-D-glucose oxidase. a-D-Glucose from blood remaining in the reaction mixture was rapidly changed to &D-glucose which was successively oxidized to D-gluconic acid with consumption of oxygen (b’), and the standard total ((Y + ~3) D-glucose (25 pg) was added to estimate the oxygen consumption (d).

FIG. 2. Determinations of rat with K3Fe( CN),,

of oxygen, P-n-glucose

D-ghCOSe,

oxidase,

and its anomers in arterial mutarotase, and oxygen electrode.

blood

If mutarotase was added with P-D-glucose oxidase in the reaction after release of oxygen from hemoglobin as shown in Fig. 3, total ( LY+ ,8) D-glucose in blood and standard total (01 + p) D-glucose (25 pg) were oxidized to D-gluconic acid with oxygen consumptions (e) and (d) successively as shown in Fig. 3. The value (e) should be the same with the value (b + b’) in Fig. 2.

mixture

Determinations

of Oxygen, D-Gkcose and Its Anomers in Venous Blood

Amounts of oxygen, D-glucose and its anomers in venous blood can be measured as in the case of arterial blood described above. However, when venous blood was added to the oxygen-depleted potassium ferricyanide-sodium azide mixture, sharp peak of oxygen consumption

OXYGEN

I 100

I

I

AND

GLUCOSE

IN

261

BLOOD

RELATIVEOXYGEN CONCENTRATION(%) 1 I I I I I I 50

0

-

t

FIG. 3. Determinations K,Fe ( CN )6, p-o-glucose

of oxygen and oxidase, mutarotase,

D-&COW

and

in oxygen

arterial blood electrode.

of

rat

with

(p) was followed by oxygen release (a’) from hemoglobin (see Fig. 4). A rapid oxygen consumption (a” ) was observed when venous blood was added to the oxygen-depleted water. Successive rapid oxygen release (a” + a’) was observed just after 5 ~1 of potassium ferricyanidesodium azide mixture was added to the reaction mixture. The deter-

I

I

+

I

FIG.

oxygen

4. Determinations electrode.

I

I

of oxygen

,

I

in venous

blood

I

of rat

with

K3Fe(

CN )8 and

262

OKUDA

AND

OKUDA

mination of n-glucose anomers in venous blood was carried out the case of arterial blood. The oxygen consumption (a”) was observed when venous blood added in the oxygen-depleted water as shown in Fig 4; however, oxygen consumption was not recorded when the arterial blood added in the oxygen-depleted water.

as in was such was

CALCULATIONS

The amounts of oxygen and n-glucose in blood estimated in the absence of mutarotase are calculated from the following expressions using a, b, and c of Fig. 1. Oxygen concentration (bmoles/ml blood) = “13’

c z f OA3’ = 0.0882 X a cxv

Oxygen concentration (pi/ml blood) = ‘*13’ ’ a cx:F

x 22*4 =-- 1.98 X a cxv

n-Glucose concentration (pmoles/ml) =

0.139 X b cx v

25 x b D-Glucose concentration (lg/ml blood) = -9 where 0.635 is the proportion of &n-glucose in the total n-glucose ((Yanomer + p-anomer ), and II ml is the volume of the blood used. The amounts of oxygen, o-glucose and its anomers in blood estimated in the presence of mutarotase are calculated from the following expressions using values of a, b, b’, and d of Fig. 2, or using values of a, e, and d of Fig. 3. Oxygen concentration (pmoles/ml blood) =

0.139 X a dx v

Oxygen concentration (pi/ml blood) = ‘*13’ 2;

f

22’4 = 3.11 X a dxv

D-Glucose concentration (pmoles/ml blood) =

0.139 X (b +bJ, dxv or 0.139 X e dXv

OXYGEN

D-Glucose

concentration

AND

GLUCOSE

IN

263

BLOOD

(pg/ml blood) =

25 X (b + b’) dXv ’ 25 X e Or dXv

The ratio of ,&n-glucose to total n-glucose in blood is calculated from two values b and b’ obtained from Fig. 2. Amounts of oxygen, n-glucose and its anomers in arterial and venous blood of rat were estimated by the above expressions. The results are summarized in Table 1. The amount of oxygen in venous blood was almost 60% as much as that in arterial blood, and the value (u” + a’) of oxygen in venous blood (Fig. 4) was almost equal to the value of that (a) in arterial blood (Figs. l-3) ; however, the amounts of n-glucose and its anomers in arterial blood were almost the same with those in venous blood. Ten estimations of a single venous blood sample for oxygen and n-glucose gave the same standard deviations of 3%, respectively. TABLE AMOUNTS

Arterial (Aort,a

OF OXYGEN,

D-GLUCOSE

blood abdominalis)

Venous blood (Vena cava inferior)

a All data

were

calculated

1

AND

ITS

ANOMERS

RAT

BLOODY

Oxygen

D-Glucose

8.99 pmoleslml 201.4 pi/ml

6 22 pmoles/ml 1119 fig/ml oc-anomer 35.0% p-snomer 65.0% 6.22 pmoles/ml 1119 pg/ml a-anomer 35.0% p-anomer 65.0%

5.14 pmoles/ml 115.1 pi/ml

from

IN

the

values

of Figs.

l-4.

DISCUSSION

An oxygen determination in blood with manometer had been established by Van Slyke (9) in 1925, and the method has been widely used in the physiological laboratories hitherto. In the last decade, the oxygen electrode has been developed, and the manometric technique is being replaced by oxygen electrode technique. Many reports on the determinations of oxygen in blood with oxygen electrode have appeared ( 10-13). In these oxygen-electrode methods, potassium ferricyanide aqueous solution of which total dissolved oxygen was depleted completely with nitrogen gas, was employed for release of oxygen from hemoglobin, and the same potassium ferricyanide aqueous solution

264

OKUDA AND OKUDA

aerated completely was used for calibration of the oxygen electrode, because the oxygen electrode is sensitive only to the partial pressure of oxygen, and not to the concentration of oxygen in the medium. Furthermore, a thermostatically controlled circulating system was required in these methods. On the other hand, Kadish and Hall (14) reported first the determination of D-glucose with a Clark-type oxygen electrode and P-Dglucose oxidase in 1965, then several reports on D-glucose determinations with similar technique appeared ( 15-18). As described above, the authors reported also a similar method for determination of D-glucose (1, 2) and developed the method for determination of D-glucose anomers using mutarotase (4), and also applied it for determination of dissolved oxygen in water (5). On the basis of these results, a new successive determination of oxygen, D-glucose and its anomers in blood with B-D-glucose oxidase, mutarotase, and oxygen electrode was devised in which a specified amount of D-glucose was used as the internal standard for oxygen and D-glucose. A thermostatically controlled circulating system is not necessary. Only the recorded value of oxygen release from hemoglobin, and those of oxygen consumption due to D-glucose and its anomers in blood and standard D-glucose, are required to know the absolute amounts of oxygen, D-glucose and its anomers in blood tested. Addition of both mutarotase and sodium azide to the reaction mixture increases the sensitivity of D-glucose determination three times larger than that without addition of these factors. The addition of mutarotase to the reaction mixture causes a rapid and complete oxidation of total D-glucose, not only P-D-glucose but also (U-D-glucose, and so the use of mutarotase makes the method more accurate and rapid, and the determination of two anomers of D-glucose became possible. The method without mutarotase can be used for determination of oxygen and Dglucose in blood; however, the oxygen consumption due to only P-Dglucose (corresponding to 63.5% of the total D-glucose) is observed in this case. Makin and Warren (18) pointed out the inhibition of &D-glucose oxidase by sodium azide which was recommended by the authors ( 1, 2) as a catalase inhibitor in P-D-glucose oxidase method for determination of blood glucose; however, no inhibition of ,&D-glucose oxidase by sodium azide at the indicated concentration (final concentration; 0.~1~) was observed as shown in the experimental part, and the oxygen consumption for P-D-glucose in the P-D-glucose oxidase method was confirmed to be twice larger than that without sodium azide as reported by the authors (I). It should be noticed that the molar ratios of oxygen to D-glucose in

OXYGEN

AND

GLUCOSE

IN BLOOD

265

venous or arterial blood exist within the range between 0.8 and 1.4 which makes possible the determinations of oxygen, n-glucose and its anomers in blood to record on the same chart. Determinations of oxygen, n-glucose and its anomers in human arterial and venous blood by the present method are being carried out in our laboratory. The results in detail will be published in the near future. SUMMARY

Amounts of oxygen, D-&COSe and its anomers in 15 ~1 of arterial or venous blood of rat are successively determined with /?-n-glucose oxidase and oxygen electrode in 3-4 min. The present method is based on the stoichiometric consumption of oxygen due to n-glucose with p-n-glucose oxidase and mutarotase. The procedure is composed of the addition of 5 ,~l of 4% K,Fe( CN),-O.2% NaN, mixture in 1 ml of distilled water of which about 60% of total dissolved oxygen is previously depleted with nitrogen gas, and measurement of release (a) of combined oxygen from hemoglobin by addition of 15 ~1 of blood, and that of successive oxygen consumption (b) due to @-glucose in blood with p-D-&CO%? oxidase, and that (c) due to a-n-glucose in blood with additional mutarotase, and that (d) d ue to standard total (a + p) n-glucose (25 fig) finally added to the reaction mixture. From the values of a, b, c, and d recorded on the chart, the amounts of oxygen, n-glucose and its anomers in blood are calculated. REFERENCES 1. OKUDA, J,, AND OKUDA, G., Chn. Chim. Actu 23, 365 ( 1969). OKUDA, J., OKUDA, G., AND MIWA, I., Chem. Phurm. Bull. 18, 1945 (1970). 3. OKIJDA, J., AND MIWA, I., Anal. Biochem. 39, 387 ( 1971). 4. OKUDA, J., AND MIWA, I., Anal. Biochem. 43, 312 ( 1971). 5. OKUDA, J., INOUE, T., AND MIWA, I., Analyst 96, 858 ( 1971). 6. LAPEDES, S. L., AND CHASE, A. M., Biochem. Biophys. Res. Commun. 31, 967 (1968). 7. OKUDA, J., AND MIWA, I., “Methods in Biochemical Analysis” ( D. Glick, Ed.), Interscience, New York, in press. 8. MIWA, I., Anal. Biochem. 45, 441 (1972). 9. VAN SLYKE, D. D., AND NEIL, J. M., J. BioZ. Chem. 61, 523 (1924). 10. MAYERS, L. B., AND FORSTER, R. E., J. Appl. Physiol. 21, 1393 (1966). 11. LAKER, M. B., MURPHY, A. J., SEIFEN, A., AND RADFORD, E. P., JR., J. AppZ. PhysioZ. 20, 1063 ( 1965). 12. HEDDEN, M., Brit. 1. Anuesthesiol. 42, 15 (1970). 13. SHIRAISHI, T., Jup. J. CZin. Puthol. 18, 636 (1970). 14. KADISH, A. H., AND HALL, D. A., Clin. Chem. 11, 869 (1965). 15. MAKINO, Y., AND KONNO, K., Jup. J. CZin. Puthol. 15, 391 (1967). 16. UPDIKE, S. J., AND HICKS, G. P., Nature London 214, 986 (1967). 17. KADISH, A. H., LITLE, R. L., AND STERNBERG, J. C., CZin. Chem. 14, 116 ( 1968). 18. MAKIX, H. L. J., AND WARREN, P. J., Clin. Chim. Actu 29, 493 (1970). 2.