Mutarotation of D-Glucose in Body Fluids and Perfused Rat Liver

4332 Vol. 35 (1987)

Chem. Pharm, Bull. 350014332-4337(19871

JUN OKUDA," TADAO TAGUCHI,a and AIKO TOMIMURAb

Departmentof Clinical Biochemistry,Faculty of Pharmacy, Meijo University," Tempaku-ku,Nagoya 468, Japan and ClinicalLaboratory, Holy Spirit Hospital,bShowa-ku, Nagoya 466, Japan

(Received May 11, 1987)

Mutardtation of n-glucose ƒ¿- and ƒÀ-anomers in some human body fluids, physiological salt solutions, and perfused rat liver was measured by the fl-D-glucose oxidase-mutarotase method or by

polarimetry. The mutarotational half-lives of a-n-glucose at 20 •Ž (37 •Ž) were as follows: whole blood, 9.0 min (2.3 min); serum, 10.6 min (2.5 min); saliva, 22.4 min (4.5 min); urine, 17.9 min

(4.3 min); and distilled water, 41.0 min (9.8 min). It seems that the rapid mutarotation of D-glucose in blood may be due to the presence of mutarotase, phosphate ions, and bicarbonate ions. The

mutarotation of a-n-glucose infused into isolated rat liver, warmed at 37 •Ž, was rapid (t1/2 = 7.2 mm), probably due to the presence of mutarotase in the liver, compared with that in the control system without liver (t112 = 50.3 min). The anomeric composition of n-glucose released from the perfused rat liver was almost at equilibrium.

Keywords ƒ¿-D-glucose; fl-D-glucose; mutarotase; D-glucose anomer mutarotation; blood; saliva; serum; urine; perfused liver

Introduction

D-Glucose in the crystalline state is well known to exist in either the a or ƒÀ anomeric form. After either of these anomers is dissolved in aqueous solution, there is a mutarotation into an equilibrium state in which 63.5% is in the )6 form, 36.5% is in the a form, and 0.003% is in the so-called aldehyde form.1,2) Many reports have recently appeared on the anomeric specificity of D-glucose metabolism3,4) and D-glucose anomeric recognition4-6)by tissues of higher animals. The mutarotation of D-glucose anomers in pure water1 ,7) and some salt solutions 8,9) has been studied by polarimetry. In body fluids, the mutarotation of the two anomers of D-glucose was supposed to be accelerated by the presence of mutarotase (aldose l-epimerase, EC 5.1.3.3), phosphate ions, etc. However, no report has heretofore been published on the mutarotation of D-glucose anomers in serum and blood. Thus we attempted to determine the mutarotational half-lives of D-glucose in normal human body fluids, such as blood, serum, saliva, and urine by our enzymatic method. We also determined by polarimetry the mutarotation of a-D-glucose in several physiological salt solutions which are frequently used in the physiological study of D-glucose anomers. On the other hand, the mutarotation of D-glucose anomers in the organs has been studied by a few investigators. Hill et al. reported that among the organs rich in mutarotase activity, the liver of the anesthetized dog caused great changes in the relative D-glucose anomer content of the blood flowing through it.101 Keston et al. reported that when ƒ¿- or )ƒÀ-D-glucose was infused into the renal artery of the dog, the D-glucose appearing in urine due to the renal threshold being exceeded was not substantially mutarotated, but the D-glucose in the renal vein blood plasma had been subjected to catalyzed mutarotation.11 ) However, their in- vestigations were done under conditions influenced by blood, in which D-glucose anomers might have mutarotated very rapidly. Thus, we reevaluated the mutarotation of D-glucose No. 10 4333 anomers in perfused rat liver by using a system with modified Ringer's solution to eliminate blood-related variables. The results are described in this paper.

Experimental

Materials Normal human blood, serum, saliva, and urine samples were obtained from healthy adults. To prepare high-and low-molecular-weight fractions of human serum, normal human serum was fractionated by cen- trifugation at 600 x g for 15 min using ultrafiltration membrane cones (Centriflo CF 25, Amicon Corporation, Lexington, U.S.A). The ultrafiltrate of serum contained low-molecular-weight substances (MW below 25000) including ions. The unfiltered substances (MW above 25000) were diluted with Tris–HCI buffer (20 mm, pH 7.4) to the initial volume of the serum, and were used as the high-molecular-weight fraction. Water used in this experiment was distilled before use. The compositions of physiological buffers used in the experiment were as follows: Gamble's solution (119 mm NaCl, 2.5 mm CaCl2, 0.8 mm MgSO4, 50 mm KHCO3, 21.0 mm Na2HPO4, pH 7.4); Ringer's solution (147 mm NaCl, 4.0 mm KC1, 2.3 mm CaCl2, pH 7.5); Krebs–Ringer bicarbonate solution (118 mm NaC1, 4.7 mm KC1, 1.2 mm CaCl2, 1.2 mm KH2PO4, 1.2 mm MgSO4, 24 mm NaHCO3, pH 7.4); Krebs–Ringer phosphate solution (118 mm NaCl, 4.7 mm KC1, 1.2 mm CaCl2, 1.2 mm KH2PO4, 1.2 mm MgSO4, 16 mm Na2HPO4, pH 7.4). Improved Recrystallization Method of a- and P-D-Glucose12) a-D-Glucose: a-, fl-, or equilibrated D-glucose (100 mg) was dissolved in 80% acetic acid (170 pl) and heated in a water bath at 90 °C until the crystals had dissolved completely. The solution was cooled to room temperature (20 ± 2 °C) for 2-3 d. The crystals obtained were washed with cold ethyl alcohol (6 ml) and dried at 80 °C until the odor of acetic acid had disappeared. Yield, 63-70 mg, purity, over 98%. fl-D-Glucose: a-, 13-,or equilibrated D-glucose (100 mg) was dissolved in anhydrous pyridine (9041) and heated in an oil bath at 100 °C until the crystals had dissolved completely. The solution was evaporated under reduced pressure and the resulting crystals were kept at room temperature (20 ± 2 °C) for 2-3 d. The crystals were first washed with cold ethyl alcohol (2 ml), and then with cold ethyl ether (4 ml), and dried at 50 °C under reduced pressure until the odor of pyridine had disappeared. Yield, 80-90 mg, purity, over 98%. The anomeric purities of a- and fl-D-glucose were checked with a digital polarimeter (DIP 181, JASCO, Tokyo). Determination of Mutarotational Half-Lives of a-D-Glucose in Buffers and Salt Solutions The changes in mutarotation of a-D-glucose in various buffers and salt solutions were measured with a polarimeter. The half-lives of mutarotation of a-D-glucose in these solutions were estimated from semilogarithmic plots of the rotation by the method of Nelson and Beegle.7) We also measured the half-lives of mutarotation of /3-D-glucose in some buffers, as 'well as of a-D-glucose, and similar data were obtained. Therefore, we measured the half-lives in body fluids and physiological salt solutions by using mainly a-D-glucose. Determination of Mutarotational Half-Lives of a-D-Glucose in Body Fluids For determination of mutarotational half-lives of a-D-glucose in blood, serum, saliva, and urine, the anomeric compositions of D-glucose in these body fluids were determined by our enzymatic method") with fl-D-glucose oxidase (/3-D-glucose:oxygen oxidoreduc- tase, EC 1.1.3.4), mutarotase, and an oxygen analyzer (model 777, Beckman Instruments Inc., Fullerton, U.S.A.). a- D-Glucose (2 mg) was dissolved in the sample fluids (1 ml) and each mixture was incubated at 20 or 37 °C. Then a 15 pl aliquot was taken for analysis of the anomeric compositions of D-glucose at 5 min intervals until the anomers were almost fully equilibrated. In the case of blood, we used potassium ferricyanide to release oxygen from oxyhemoglobin in blood samples, and the anomers of D-glucose were determined by another of our enzymatic methods") with /3-D- glucose oxidase and mutarotase. After measurements of the anomeric compositions, the half-lives of mutarotation of a-D-glucose in these body fluids were calculated from semilogarithmic plots of the decrease of a-D-glucose. Perfusion of Rat Liver The liver perfusion methods reported by Sugano et al.") and us° were modified and used in this experiment. The isolated rat liver was perfused from a Teflon cannula inserted into the portal vein; the perfusion medium was a modified Ringer's solution: 154 mm NaC1, 1.8 mm CaCl2, 2.7 mm MgCl2, 2.0 mm N-2- hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), pH 7.2, saturated with 95% 02+ 5% CO2. The solutions of D-glucose anomers were cooled in the reservoir with ice in order to prevent spontaneous mutarotation as much as possible. The perfusion medium and D-glucose solution were warmed at 37 °C by passage through a stainless steel coil immersed in warm water just before their infusion into the liver. A peristaltic pump was used for perfusion, and the rate of flow was kept at about 37 ml/min. The mean oxygen consumption by a perfused rat liver was 0.93 mol 02/min/g of wet liver. The bile production by the perfused rat liver was also measured and the mean flow rate was 16 pl/h/g of wet liver. For the determination of anomeric composition of D-glucose released from the perfused liver, perfusion was carried out in the flow-through mode. For study of the mutarotation of D-glucose anomers in the perfused liver, the recycle mode was used, in which the perfusate from the liver was returned to the initial reservoir cooled at about 4 °C with ice. 4334 Vol. 35 (1987)

The liver was first perfused for 20 min with perfusion medium containing equilibrated D-glucose solution

(120 mg/100 ml). Then, fresh a-D-glucose solution, fresh fl-D-glucose solution (500 mg/100 ml) or the D-glucose-free perfusion medium, was introduced into the perfused liver by changing a three-way cock. Aliquots of the perfusate from the liver were collected at 2-min intervals for 8 min, and immediately frozen in a dry ice•@ cetone mixture. These samples were stocked at •@ 70 °C for the determinations of anomeric composition and amount of D-glucose in the perfusate.

Results and Discussion

Mutarotation of a-D-Glucose in Body Fluids and Physiological Solutions

The mutarotational half-life of a-D-glucose in purified water at 20 or 37 °C was quite slow

(41.0 or 9.8 min, respectively), and these values agree fairly well with those of previous reports

(45 min at 20 °C," 10 min at 37 °C7)). The half-life of a-D-glucose in normal whole blood was 2.3 min at 37°C. This value in blood is very low in comparison with the value (9.8 min) in water, as shown in Table I.

Bailey et al.16) reported that the mutarotational half-life of a-D-glucose under physiological conditions was 7.1 min at 37 °C, although the conditions used for measurement of mutarotation were not described in detail. However, our results indicate that the mutarotational half-lives of a-D-glucose in normal human whole blood and serum are very small, 2.3 and 2.5 min respectively, at 37 °C, as shown in Table I.

The mutarotation of a-D-glucose in normal human saliva progressed slowly, and the half- lives at 20 and 37 °C were 22.4 and 4.5 min, respectively (Table I). Those in urine at 20 and

TABLEI. Mutarotational Half-Lives (t112)of a-D-Glucose in Normal Human Body Fluids

TABLEII. Mutarotational Half-Lives (t,/2) of a-D-Glucose in Various Buffers and Physiological Salt Solutions No. 10 4335

37 °C were 17.9 and 4.3 min. We also investigated the mutarotation of a-D-glucose in 20 mm Tris—HC1 buffer (pH 7.4) containing human serum albumin (4.2 g/100 ml) or human serum y-globulin (4.7 g/100 ml), and these mutarotational half-lives were determined to be 7.0 min at 37 °C. The half-lives of mutarotation of a-D-glucose in these protein solutions were larger than those in blood and serum. The half-life of mutarotation of a-D-glucose in the low-molecular-weight fraction of serum (2.4 min) was almost the same as those in blood and serum, and that in the high- molecular fraction of serum (MW above 25000) was 3.7 min. These results indicate that the rapid spontaneous mutarotation of a-D-glucose in blood and serum is mainly caused by the low-molecular-weight compounds (MW below 25000), including ions, in it. Table II shows the mutarotational half-lives of a-D-glucose in various buffers which are frequently used in biochemical studies. As shown in Table II, phosphate buffer (pH 6.0, 7.4) caused rapid mutarotation of a-D-glucose as compared with Tris—HC1 buffer (pH 7.4) at the same molar concentration, and the half-life (6.0 min) of the mutarotation in phosphate buffer

(pH 7.4) was consistent with that given in a previous report.8) We also investigated the mutarotation of a-D-glucose in various salt solutions (10 mM) at 37 °C. The chlorides of K , Na , and Mg' dissolved in 20 mm Tris—HC1 buffer (pH 7.4) shortened the mutarotational half-life of a-D-glucose in comparison with that (8.0 min) in only 20mm Tris—HC1 buffer (pH 7.4) as follows: KC1, 6.5 min; NaCl, 7.0 min; and MgCl2, 6.0 min. Calcium chloride, however, did not alter the half-life of the mutarotation. To compare the effects of anions on the mutarotation, we also measured the half-lives of mutarotation in the presence of potassium salts (20 mM) of HPO42- , HCO3 - , and SO,' . The values were calculated to be 3.0, 6.0, and 8.0 min, respectively. The half-life of mutarotation of D-glucose in Gamble's solution at 37 °C was calculated as 4.0 min, as shown in Table II. In Krebs—Ringer bicarbonate and Krebs•@ inger phosphate solutions, the a-D-glucose mutarotation was as rapid as that in the serum. However, the mutarotational half-life (10.0 min) of a-D-glucose in Ringer's solution at 37 °C was almost the same as that in distilled water. Since human serum contains 1.35 mm phosphate ion and 25 mm bicarbonate ion, which accelerate the mutarotation of D-glucose," the rapid mutarotation of a-D-glucose observed in blood and serum is undoubtedly due to the presence of phosphate and bicarbonate ions in them.

Mutarotation of a, fl-D-Glucose in Perfused Rat Liver Prior to the perfusion experiments with isolated rat liver, the mutarotation of D-glucose anomers in the perfusion medium at •@ 70 °C and at 37 °C was measured. There was no mutarotation of a- or /3-D-glucose at •@ 70 °C over at least a 10 d period. Thus, the perfusates from the liver were frozen in a dry ice—acetone mixture and stocked at •@ 70 °C prior to measurement. The mutarotational half-life of a-D-glucose in the perfusion medium at 37 °C

TABLE III. Mutarotational Half-Lives of D-Glucose Anomers in the Perfusion System at 37°C 4336 Vol. 35 (1987)

Fig. 1. The Concentration and the Anomeric Composition of D-Glucose Released from Perfused Rat Liver

Isolated rat liver (12.5 + 0.9 g, mean weight + S.D.) was perfused with modified Ringer's solution containing 120 mg/100 ml of D-glucose for about 20 min. Then the perfusion medium was replaced by the same medium without D-glucose. An arrow indicates the time of the medium change. The concentration (0) and the percent of n-glucose as /-D-glucose (0) in the perfusate were measured as described in Materials and Methods. Results are given as the means (•} S .D.) of five experiments.

was calculated to be 9.2 + 0.7 min (mean + S.D., n = 5). In the perfusion system in which the liver was replaced by a silicone tube (0.96 ml, mean volume of the vascular system of rat liver as determined by the method of Marklund17)), a slow mutarotation of ƒ¿- or fl-D-glucose was observed at 37 °C: the half-lives of mutarotation of ƒ¿- and fl-D-glucose were calculated to be 50.3 min (a-D-glucose) and 51.4 min (fl-D-glucose). In contrast with the above results, the mutarotation of infused ƒ¿- and ƒÀ-D-glucose during perfusion with rat liver was considerably more rapid, as shown in Table III, with the half-lives being 7.2 min for a-D-glucose and 7.6 min for fl-D-glucose. These values were about 7 times lower than those in the perfusion system using the silicone tube as described above. Since rat liver contains a large amount of mutarotase,18) both ƒ¿- and ƒÀ-anomers of D-glucose entering the liver cells might be rapidly mutarotated by this enzyme. The liver was first perfused with the medium containing D-glucose for 20 min; then perfusion was continued with medium lacking D-glucose. Within 8 min of the medium change, the concentration of D-glucose in the perfusate decreased from 135 mg/100 ml to 12 mg/100 ml

(Fig. 1). The release of D-glucose from the perfused liver continued at least for 12 min. The anomeric composition of ƒÀ-D-glucose in the perfusates from 4 to 12 min was about 63%, the equilibrium state. These phenomena can be explained by the presence of mutarotase in the liver and (or) the rapid spontaneous mutarotation of ƒ¿- and ƒÀ-D-glucose 6-phosphate as reported previously.19) The result suggests that D-glucose released from the liver, probably by glycogenolysis and gluconeogenesis, is almost fully equilibrated. Recently, the physiological roles of ƒ¿- and fl-D-glucose and the aldehyde form have become more clearly understood; e.g., ƒ¿-D-glucose is used as the signal for D-glucose sensing cells (Langerhans ƒ¿-, fl-cells and taste bud cells for sweetness of D-glucose), while ƒÀ-D-glucose is used for energy production in many cells and organs in higher animals. Hence, the study of mutarotation of D-glucose anomers in body fluids, physiological salt solutions, and in organs is quite important for elucidation of the biochemical roles4) of D-glucose anomers and the aldehyde form in higher animals.

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