The Potential Use of Xylitol in Glucose-6-

Phosphate Dehydrogenase Deficiency Anemia

Y. M. WANG,J. H. PATrERSON, and J. vAN EYsFrom the Departments of Biochemistry and Pediatrics, Vanderbilt UniversitySchool of Medicine, Nashville, Tennessee 37203

A B S T R A C T NADP-linked xylitol dehydrogenase hasbeen found to be present in human red blood cells. Thisenzyme activity is normal in most glucose-6-phosphatedehydrogenase (G6PD) -deficient red cells. Xylitol wasexplored as a potential agent for treatment of hemolysisin patients with G6PD-deficiency. Intracellular GSH(glutathione, reduced) was first converted to its oxi-dized form by incubation of the erythrocytes with acetyl-phenylhydrazine or by pretreatment with methyl phenyl-diazenecarboxylate. The addition of 0.15 M xylitol wasshown to be more effective than 0.15 M glucose in main-taining the levels of GSH in G6PD-deficient red cellsduring such oxidative challenge. Rabbit erythrocytescontain less activity of G6PDand glutathione reductasecompared with the normal human adult values, but havean active xylitol dehydrogenase. The rabbit erythrocyteis sensitive to acetylphenylhydrazine and primaquinephosphate. In both in vivo and in vitro experiments,xylitol was found to partially prevent acetylphenylhy-drazine induced acute hemolysis of the rabbit red celland GSH content was found to be preserved. The in-travenous injection of xylitol (0.5 g/kg body weightper 6 hr) for 6 days, seemed to be nontoxic to the ani-mal. The results suggest that xylitol should be furtherinvestigated as an agent for the treatment of G6PD-deficient patients during acute hemolytic episodes.

INTRODUCTIONRed blood cell glucose-6-phosphate dehydrogenase(G6PD)1 deficiency is a widespread genetic disorder.About 3% of the world population is estimated to beaffected. 10% of American Negroes have been foundto carry such genetic defect. Hemolysis, resulting

Received for publication 30 November 1970 and in revisedform 1 March 1971.

1 Abbreviations used in this paper: APH, acetylphenyl-hydrazine; G6PD, glucose-6-phosphate dehydrogenase;GSSG-R, glutathione reductase; PVC, packed cell volume;XDH, xylitol dehydrogenase.

from G6PD-deficiency can be expressed in five waysdepending on the clinical setting; (a) drug induced he-molytic anemia; (b) hemolytic anemia in infectious andother febrile illness; (c) chronic nonspherocytic hemo-lytic anemia; (d) favism; and (e) neonatal jaundice.Drug induced hemolysis is usually a self-limited disease,but among enzyme-deficient patients, 2% have a chronichemolytic anemia in the absence of drug challenge (1).The disease is characterized biochemically by a lowor almost absent G6PD-activity. Secondarily, the GSHis unstable on in vitro incubation of such red cells inthe presence of oxidizing agents. There are many en-zyme variants, and clinical expression correlates bothwith enzyme activity, and, directly or indirectly, withthe type of enzyme. The mechanism of hemolysis is stillfar from clear. It has been presumed that a normal GSHconcentration plays an important role in the maintenanceof the integrity of the red cell. Treatment with eitherACTH (adrenocorticotropin) (2) or cortisone (3, 4),appears to be of no value for the congenitial nonsphero-cytic hemolytic anemia secondary to G6PD-deficiency.Splenectomy is much less effective than in hereditaryspherocytosis. In fact, no adequate treatment of suchdisease has thus far been developed. Transfusion is theonly method known to maintain the patient duringacute hemolytic crises.

Recently, we were able to demonstrate the presenceof a triphosphopyridine nucleotide (NADP)-linked xyli-tol dehydrogenase (XDH) in the human red blood cell.The red cell hemolysate can utilize xylitol and produceL-xylulose and NADPH(reduced form of triphospho-pyridine nucleotide) (5). It thus seems possible thatxylitol might be able to replace glucose-6-phosphate forthe maintenance of NADPH in the G6PD-deficientstate. In such a way, the intracellular GSHconcentra-tion could be restored in the deficient cells, thereby pro-tecting red cells from oxidative injury as shown in theScheme 1. The purpose of this report is to present evi-dence from both in vitro and animal experiments which

The Journal of Clinical Investigation Volume 50 1971 1421

|Glucose-6- Phosphate]

6-Phospho- NADPgluconate

[Xylitol NIkNADPH

L-xylu lose

Scher

suggest that administration of xitients with G6PD-deficient anemi.lytic crises.

METHODSBlood samples and enzyme determ

G6PD-deficiency were diagnosed -

Hospital Patient population by a s(diagnosis was confirmed by spectraall samples from deficient patientstrophoresis using a standard buffeusing cellulose acetate in place of speared to have the A- variant. AlNormal blood was obtained from hefaculty and students. G6PD was mmethod by Zinkham, Lenhard, and (reductase (GSSG-R) was determinedand Carson (9). XDH was measutNADPHat pH 8.0 as described FGSSG-R, and XDH activities werecell hemolysates by the same method

In vitro restoration of GSH. Expemaintenance and regeneration of intrmal and G6PD-deficient red cells wfollowing two ways. First, a modifiestest was used (10): an equal volumeglycine buffer (0.1 M glycylglycine,0.55 M NaCl at pH 7.2) was added tcells. Xylitol or glucose were added acrit of these suspensions varied fromincubation volume was 2 ml. Exp4hydrazine (APH) was continued thrperiod. A concentration of 5 mg AXtion mixture was used. Control expeand/or xylitol or glucose were include

When methyl phenyldiazeneca:NCOOCH8) was used as oxidant,dissolved in the isotonic glycylglycinecentration 1.6 jsg/ml as described byand London (11). The cell suspensitemperature for 10 min and washedwithout azoester until the supernataally, the GSH-content was reduced toof the original value. Such pretreaterbated at 370C with glycylglycine bufcose as additives. A 0.4 ml sampletube for GSH-determination. GSHman's reagent (12), but the freezingomitted. The same experiments werebit erythrocytes. In addition to experthe azoester, primaquine phosphatemixture) was used.

Animal experiments. New Zealand-white male rabbitswere used throughout the study. For a long-term xylitol ad-

GSH H202 ministration experiment, 12 rabbits were divided into fourgroups for evaluation of the effectiveness of xylitol in vivo.The optimum dose of APH to induce a reproducible de-gree of acute hemolysis in the rabbit was evaluated. The

GSSG H. O toxicity for rabbits of a single intravenous injection ofxylitol was checked. The details of each experiment aredescribed in the legend of the appropriate figures.

Chemical determinations. Xylitol was measured as for-meI maldehyde after periodate oxidation (13). Glucose concen-

tration was determined using a commercial version of theglucose oxidase method (Worthington Kit) (14). Xylulose

ylitol might help pa- was estimated as ketopentose by the cysteine carbozolea during acute hemo- method (15) using 6% perchloric acid as deproteinizing

solution. Total and conjugated serum bilirubin were deter-mined by a manual method (16).

Materials. Methyl phenyldiazenecarboxylate was synthe-sized by a method described by Huang and Kosower (17).The compound was identified by infrared, ultraviolet, and

inations. Patients with nuclear magnetic-resonance spectra analysis. The use ofamong the Vanderbilt the azoester and its mechanism of reactions have beencreening test (6). The discussed in detail by the same group (18).photometric assay and Xylitol was kindly supplied by the Eisai, Co., Ltd. Tokyo,were checked by elec- Japan. No inhibition of growth of Escherichia coli couldor and stain (7) but be detected. No contaminant could be detected in the xylitoltarch. All patients ap- infusion solution as judged by the ultraviolet absorption

11 patients were black. spectrum.althy volunteers among All other reagents were purchased from standard Ameri-easured by a modified can commercial sources.Tihilds (8). Glutathioneby the method of Long RESULTS

red by the increase ofpreviously (5). G6PD, To evaluate the usefulness of xylitol as a potential formmeasured in rabbit red of rational therapy, the following questions needed to beIs. answered: (a) is XDHpresent in normal and G6PD-ritments to evaluate the deficient red cells in equal activity; (b) is xylitol perme--acellular GSH in nor-tere carried out in the able to the red cell and is it effective in restoration of

d glutathione instability intracellular GSH in oxidatively challenged red bloode of an isotonic glycyl- cell in vitro; (c) is it effective in animal models in0.03 M Na2HPO4, and vivo, and (d) is it justified for use in human subjectsto saline-washed packed on basis of the known toxicity of the compound and itsis needed. The hemato- onboites.35 to 40%o. The final metabolites.osure to acetylphenyl- XDH in G6PD-deficient patients. G6PD and XDHroughout the incubation were measured in groups of normal and G6PD-deficient'H/ml of final incuba-eriments without APH subjects. Table I reports the activities of the two en-

d. zymes in the red cell hemolysates. It is clear that therboxylate (CeH5N = G6PD-deficient patients have a normal XDH activitythe azoester was first averaging 7.3 ±1.2 nm NADPH/min per g Hb.

e buffer in a final con-Kosower, Vanderhoff,

ion was kept at room TABLE Iwith the same buffer Glucose-6-Phosphate Dehydrogenase (G6PD)- and NADP-

nt became clear. Usu- Linked Xylitol Dehydrogenase (XDH) Activities inapproximately 10-20%o Normal and G6PD-Deficient Erythrocytes

d cells were then incu-fer and xylitol or glu-was taken from each

was measured by Eli-and thawing step wascarried out with rab-

-iments with APH and(2.5 mg/ml incubation

G6PD XDH

IU/100 ml nMNADPH/RBC min per g Hb

Normal erythrocytes* 153.5 1=15.3 7.0 4=0.8G6PD-deficient erythrocytes* 12.9 4:18.0 7.3 4=11.2* Average of eight subjects 1= SD.

1422 Y. M. Wang, J. H. Patterson, and J. pan Fys

Maintenance and restoration of intracellular GSHbyxylitol. Xylitol is actively metabolized by red cells andhas been shown to stimulate lactate formation (19) andmethemoglobin reduction (20, 21). Xylitol can restoreGSH in sodium nitrite-treated normal erythrocytes(22). The permeability of xylitol to the red cell wasevaluated by Asakura et al. (22), and a simple diffusionmechanism tas been suggested. Xylitol passes throughthe red cell membrane easily and equilibrates in less thanI min. Long-term storage with xylitol as an additive tored cells has been shown in our laboratory to help themaintenance of the 2, 3 DPG-level.2 A direct demonstra-tion of the effectiveness of xylitol in restoring GSH inboth normal and G6PD-deficient cells has been made byusing APH and methyl phenyldiazenecarboxylate.

Fig. 1 gives the results of a representative experimentwith APH. Fig. 1A shows that glucose could maintainthe GSH level better than xylitol in normal red cells.However, Figure 1B demonstrates that xylitol preservedmore intracellular GSH in the G6PD-deficient cells. Inthe normal cells, glucose maintains 90% GSHand xylitol55% after 4 hr incubation. In the deficient cell, glucosepreserved only 26% and xylitol 35%. This result wasreproducible on multiple experiments and the differenceis significant (P < 0.05). Fig. 2 shows similar resultsof a representative experiment using the azoester. Fig.2A illustrates that glucose is far better than xylitol inGSH-recovery in the normal blood cells, in which, glu-cose restores 85% GSHand xylitol 56%. In the G6PD-deficient cell xylitol shows better ability to regenerateGSH as shown in Fig. 2B. Xylitol restores 65% ofGSH and glucose only 48%. The difference is repro-ducible on multiple trials and is significant (P < 0.05).A 100% recovery is not likely to be reached, since theGSSGhas been reported to leak out of the red cells(23). Even without an oxidant challenge, xylitol main-tained GSHbetter than glucose in the G6PD-deficientcells. This is shown in the top curve of Figs. 1B and2B. In red cells from persons heterozygous for G6PD-deficiency (around 55 IU per 100 ml RBC) the benefitof xylitol was much less marked.

Glucose and xylitol at final concentrations of 0.015 M,which is 10 times lower than that used in the experi-ments described, gave similar results to glucose andxylitol at 0.15 M.

The effect of xylitol on rabbit erythrocytes in vitro.There is no suitable animal model for G6PD-deficientstudies. The rabbit was chosen because it is known tobe drug sensitive. In a preliminary experiment the lev-els of the pertinent enzymes were measured. Table IIgives the G6PD, GSSG-R, and XDH activities in therabbit red cell hemolysates. With the average of 4.3%

2 Chan, S. H., and J. van Eys. The possible use of xylitolin preservation of stored red cells. To be published.

E 6 ~

a \~~\ \b

3L

0 2 4 0 2 4TIME

FIGURE 1 The maintenance of intracellular GSHin humanerythrocytes during APH stress. Fig. (A) refers to normalcells and (B) to G6PD-deficient cells. The following sym-bols are used: solid lines indicate control, i.e. withoutAPH, and dotted lines represent experimental samples towhich APH is added; (0) control, i.e. no xylitol or glu-close added; (II) 0.15 M xylitol, and (A) 0.15 M glucose.

reticulocytes, GSSG-R activity was found to be halfthat of the normal human level; G6PD was also lowerwhen considered relative to the per cent reticulocytes.Rabbit XDH-activity has been found the same as hu-man values. The average activity in five samples wasaround 8.6 ±1.3 nM NADPH/min per g Hb. comparedto 7.0 ±0.8 nM/min per g Hb. in humans. TheMichaelis-Menten constant of xylitol for the rabbitXDH is estimated to be 5.4 X 10' M. In the GSH in-stability test, rabbit red cells were more drug sensitivethan normal human red cells under the same experi-mental conditions. Xylitol helped in maintenance of in-tracellular GSH, though glucose was shown to be better.In experiments in which primaquine phosphate was

TIME hr

FIGURE 2 The regeneration of intracellular GSHin humanerythrocytes after azoester stress. (A) Normal cells, (B)G6PD-deficient cells. The same symbols are used as de-scribed in Fig. 1 except azoester instead of APH was usedas described in the text.

Xylitol and G6PD-Deficiency 1423

TABLE I IComparison of G6PD-, Glutathione Reductase (GSSG-R)-,

and XDH-Activities and Reticulocyte Percentage inHumanand Rabbit Erythrocytes

Human Rabbit

G6PDIU/100 mlRBC 153.5 415.3 (8)*t 166.0 4:15.2 (5)

GSSG-RIU/100 mlRBC 95.8 4:15.4 (5) 54.9 :1:9.1 (5)

XDHnM NADPH/min per Hb 7.0 4-0.8 (8) 8.6 411.3 (5)

Average reticulo-cytes, % 1 4.3

* Mean :4:1 SD.I Figure in parenthesis indicates the number of subjects.

added, the same results were obtained. Fig. 3 shows arepresentative experiment.

Optimal dose of APH-induction of in vivo hemolysis.Fig. 4 shows the percentage hemolysis after differentdoses of APH were injected intraperitoneally. 40% he-molysis was observed consistently after 72 hr whenmore than 20 mg APH per kg body weight was in-jected into rabbits. Some animals died with a dosageexceeding 25 mg APH per kg body weight. There wasa drastic decrease of the GSH content along with asevere drop of PCVand hemoglobin concentration. Thereticulocyte count rose proportionately. A 70% reticu-locyte count was observed in one rabbit before death.Serum hemoglobin was found increased occasionally.10 mg APH per kg body weight was chosen to be areasonable dose and is therefore, the one used in thefollowing long-term animal experiments.

i0c I I I

A B C

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FIGURE 3 The maintenance and regeneration of intracellu-lar GSH in rabbit erythrocytes. A) During APH-stress,B) After azoester challenge, C) During primaquine phos-phate treatment. The same symbols are used as shown inFig. 1.

Administration of xylitol to rabbits with APH-inducedhemolysis. Fig. 5 gives the result in a long-term xylitoltreatment of animals anemic from an APH-injection.PCV, hemoglobin concentration, reticulocyte counts, andGSH content of single animals are shown. Rabbits ineach group followed a similar pattern in their hemato-logical values. While there are no observable changesin control animals, animals to which only xylitol wasgiven intracellular GSH content increased slightly.The value returned to normal at the end of experi-mental period. In animals to which only APH was ad-ministered, the PCV dropped from 36 to 24%, the he-moglobin concentration fell from 12.8 to 8.6 g per 100ml in 120 hr. GSH content showed an 18% decrease.With the injection of xylitol and the administration ofAPH, the animal showed only an absolute drop in PCVof 6%, while hemoglobin fell only from 12.2 to 10.3 gper 100 ml blood. GSHmeasured within no-rmal range.An increase of reticulocyte count was seen after the 5thday. In the animals of the control and xylitol treatedgroup, the reticulocyte counts increase, slightly, whichis likely due to the continued withdrawal of 2-3 ml bloodfrom the animals daily.

Fig. 6 summarizes the average differences of PCV-and GSH-content in per cent of initial value among thefour groups. The baseline value is set at 100%. Fig. 6Aindicates the differences in PCV among groups. Fig.6B demonstrates that GSHhas been conserved by thetreatment of xylitol in the APH-treated animals. Itseems clear that the production of NADPHby xylitolmaintained the intracellular GSHcontent.

Toxicity of xylitol to the rabbit. Xylitol and glucoselevels in three animals after a single xylitol intravenousinjection (0.5 g xylitol per kg body weight in a 20%solution) are shown in Fig. 7. The values are expressedin mg per 100 ml. The plasma xylitol declines rapidlyfive min after intravenous infusion. The mean half disap-

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FIGURE 4 APH-dose response in rabbit. The oxidant wasgiven intraperitoneally. The percentage hemolysis was thenmeasured at 72 hr by the decrease of packed cell volumefrom the baseline.

1424 Y. M. Wang, J. H. Patterson, and J. van Eys

pearance rate, ti, of xylitol is estimated to be about 20min. The decay constant of xylitol in plasma is com-puted to be 3.3% per min. The results are in agreementwith the results of the elimination study in human blood(24).

The xylitol content in the red cell has been found ele-vated after xylitol injection. Fig. 7A points out thatxylitol is higher in whole blood than plasma after 6 hr.Red cell xylitol reached about 13 mg per 100 ml. Theglucose concentration in both whole blood or plasma wasnot significantly altered by the injection of xylitol. Theparallel curve between plasma and whole blood glucoseindicates that red cell glucose was maintained unchanged.The fall of glucose at 60 min might be caused by an

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group, (-) APH-group, (A) experimental group towhich both APH and xylitol were given. APH at dose of10 mg per kg body weight was injected intraperitoneally atzero time. Xylitol, in a 20%o solution, was injected fourtimes a day for 6 days. 0.5 g xylitol per kg body weight wasinjected over 4 min each time through an ear vein.

tL

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r. . . . .. . . . . . . . . . . . -

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FIGURE 6 Hematological changes in rabbits expressed asper cent of initial values. The same symbols are used asdescribed in Fig. 5. Baseline values were used as 100%.Each point represents an average of three animals ± 1 SD.

increase of insulin in the plasma (25). Fig. 8 shows thetime course of the plasma xylulose after an intravenousxylitol injection. The ketopentose formation reached apeak around 5 min after the injection. The total con-version to xylulose was estimated to be only 4% of thexylitol injected. It is likely that the ketopentose is L-xylu-lose. However, the plasma ketopentose was not char-acterized.

Serum bilirubin has been determined in two animalswho received xylitol only. There was no elevation ofserum bilirubin in 24 or 48 hr during chronic xylitoladministration.

No deterioration has been observed in the group whichreceived only xylitol and all animals survived through-out the study.

TIME min

FIGURE 7 A) Xylitol; B) glucose concentration in wholeblood and plasma after a single xylitol injection (a 20%osolution at 0.5 g xylitol per kg body wt). (0) refers towhole blood; and (A) to plasma. Each point indicatesan average of three animals +1 SD.

Xylitol and G6PD-Deficiency 1425

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90

801

701

601

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0 60 120 180 240 360TIME min

FIGURE 8 Time course of xylitol disappearance and keto-pentose formation in whole blood after a single xylitol in-jection. (0) refers to xylitol concentration and (A) toketopentose concentration. Each point indicates an averagevalue of three animals.

DISCUSSION

The results show that xylitol might be useful to patientswith severe hemolytic episode. The in vitro data ob-tained on human red cells indicate the usefulness ofxylitol to restore or maintain the GSH-content in theG6PD-deficient state either when challenged or inchronic nonspherocytic hemolytic anemia. The long-term animal experiments prove that xylitol infusion couldprotect rabbits from acute hemolysis induced by acetyl-phenylhydrazine. The GSH is also conserved by con-

tinuing treatment with xylitol. It is, of course, possiblethat the comparatively small drop of GSH-concentra-tion in APH-treated animals might not be the majorcause for the acute hemolysis. In addition to being theintermediate in maintenance of a normal level of GSH,NADPHmay be directly involved in the maintenance ofhemoglobin and other functional or structural proteinsin a reduced active form after oxidative injury. How-ever, the state of hemoglobin as well as the total sulf-hydryl concentration was not measured. Methemoglobinreductase has been shown to use both NADH andNADPH. Xylitol is uniquely suited to maintain fer-rous hemoglobin because it can generate both reducedcoenzymes readily (5). Since APH may not be solelyan oxidative challenge to the red cell (26), the action

of xylitol may be a general one in other cells besides theerythrocyte. In patients with G6PD-deficiency this en-zyme may also be decreased in liver (27), platelets (28),leucocytes (29), skin (30), and in lens tissue (31, 32).The NADP-linked xylitol dehydrogenase has been foundin almost all the organs of mammals (33). Therefore,the potential usefulness of xylitol may not be limited tothe erythrocyte.

Xylitol has been introduced for clinical application10 yr ago as an adjuvant in parenteral alimentation. Ithas been reported that xylitol is independent of insulinfor transport (34). It is also an adequate calorie source(35). Xylitol has been used in the human in Germany(36), Japan (37), Russia (38, 39), Italy (40), andSouth Africa (25). Besides normal subjects, it has beenused to treat patients with diabetes (41), bile duct andliver disease (38), renal disease (25), ketonemia (42),pulmonary tuberculosis (43), and for parenteral nu-trition during and after surgery (36). No harmful effecthas been observed in these published series. Recently,however, Donahoe and Powers (44) reported that thehyperuricemia, hyperuricosuria and hyperbilirubinemiahave occasionally been observed in normal subject withxylitol doses of 1.22-3.13 g per kg body weight. The in-fusion time was not indicated in this note. Similar ad-verse effects were also reported by an Australian group(45). Once again, the xylitol dose and the infusion timewere not mentioned. At a lower dose, 1.5 g xylitol perkg body weight Forster, Meyer, Ziege (46) reportedtransient rise in serum uric acid and serum bilirubinthough remaining within the normal range, in normalsubjects after rapid xylitol infusion. SGOT- and SGPT-levels were not influenced by the xylitol administration.Our experience with xylitol in rabbits showed no clini-cal toxicity, and no serum bilirubin elevation, thoughthe literature did mention xylitol toxicity in rabbits(47).

The plasma and whole blood glucose was found un-changed for 6 hr after a single xylitol injection. Similarresults were found in dogs (48), rabbits (49), andhumans (50) with different xylitol dosage. Therefore, themaintenance of the hematocrit was not due to increasedserum glucose, but likely from the effect of xylitol per se.The xylitol concentration was estimated to be 13 mgper 100 ml red blood cell after a single injection ofxylitol. The NADP-linked xylitol dehydrogenase has alow Km for xylitol (5). Although the xylitol content ofthe red cell increased after repeated injection, it is un-likely that the intracellular xylitol concentration reachedsaturating levels for the enzyme. Even so, saturationis not necessary if the NADPHformed is rapidly uti-lized to reduce GSSG.

The liver is the major organ which metabolizes xyli-tol. The major products, except L-xylulose are similar

1426 Y. M. Wang, 1. H. Patterson, and 1. van Eys

0

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to that of glucose. The excess L-xylulose will be ex-creted through the urine, and is likewise a harmlesssubstance, as shown by pentosurics who are asympto-matic in spite of the excretion of gram quantities of thatsugar (51). A mild increase of lactate was reported(25, 42, 50) in the blood after intravenous xylitol ad-ministration. The serum concentration of xylulose wasvery low in our experiments.

For the time being human experiments are excluded.The Australian groups reported multiple cases of deathfrom xylitol infusion although the presence of a con-taminant in the xylitol solution was strongly suspected(52). Since these observations are at present not pub-lished but only informally reported, a decision mustawait more details.

ACKNOWLEDGMENTSThe authors are grateful to Dr. D. E. Pearson, Dr. K. N.Subbaswami, and Mr. D. J. Thoennes, for their advice insynthesizing and analyzing methyl phenyldiazenecarboxylate.

This investigation was supported by a grant from theJohn A. Hartford Foundation, Inc.

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