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[CANCER RESEARCH 41, 3192-3199. August 1981] 0008-5472/81 /0041-OOOOS02.00 of Xylitol and in Bearing Hepatocellular Carcinomas1

Junko Sato,2 Yeu-Ming Wang, and Jan van Eys

Department of Pediatrics, The University of Texas System Cancer Center, M. D. Anderson Hospital and Tumor Institute, Houston, Texas 77030

ABSTRACT glycolysis, were also revealed in the catabolism and anabolism of nucleic acids, lipids, and proteins (33-35). The variation in metabolism of glucose and xylitol by diverse Consequently, although the metabolism by tumors was not hepatocellular carcinomas and partially hepatectomized rat always qualitatively different from normal cells, it is significantly livers was studied. The AS-30D and FB56 tumors demon different quantitatively. The characteristic differences and re strated a significantly different degree of utilization of glucose quirements that distinguish cancer cells from normal cells could and xylitol in vitro. This correlated partially with the low activity be used to manipulate and control tumor metabolism and of dehydrogenase when xylitol was used as a substrate. growth. The different rates and pathways of energy consump The activity of a nicotinamide adenine dinucleotide-depend- tion in host and tumor tissues could also be an important clue ent polyol dehydrogenase in various hepatomas ranged from to the exact nature of the responses of a host to a growing nondetectable to 30 nmol/min/mg protein, with the lower tumor. The differential metabolism of xylitol from glucose may activities in FB56 and AS-30D tumors at 0 and 0.22 nmol/ exert selective block or disadvantage to the tumor of this min/mg, respectively; while nicotinamide adenine dinucleotide nutrient. To assess the significance of nutritional factors in phosphate-dependent xylitol dehydrogenase activities ranged cancer, biochemical alterations following nutrient intake must from 0 in FB56 to 3.31 nmol/min/mg protein in liver regener be carefully observed. Qualitative and/or quantitative differ ated for 1 week. The activities of nicotinamide adenine dinu ences in metabolism between tumor and host may reveal clues cleotide phosphate-dependent enzyme for normal liver and AS- as to the complexity of the host-tumor relationship. 30D tumors measured 2.2 and 0.14 nmol/min/mg protein, Xylitol has been increasingly used in several countries as an respectively. Although only the 311C tumor had an activity effective substance for nutrition and for the provision of energy equivalent to that of normal liver, the range of the nicotinamide in clinical practices (31). The differential utilization between adenine dinucleotide phosphate-dependent polyol dehydro xylitol and glucose has been well documented in dental plaque, genase activities among the cell lines studied is narrow. diabetic animals, and humans, particularly in liver. In addition, The ratios of metabolites of ['"CJglucose or [14C]xylitol were our recent finding has suggested that xylitol can produce a determined in rats bearing AS-30D tumors. Animals were given cancerostatic substance, methylglyoxal, in normal rat liver (22). i.v. injections of a 10% solution of [14C]glucose or [14C]xylitol, These observations have prompted us to explore the metabo 2 g/kg body weight. Assays of neutral metabolites from lism of polyalcohol xylitol in rats bearing hepatoma. each substrate in the acid-soluble fraction of liver or AS-30D Accordingly, a comparative study of xylitol and glucose tumor showed that xylitol in the liver was converted primarily metabolism in solid and ascites rat hepatocellular carcinomas into glucose while in the tumor 80 to 90% of the xylitol remained was undertaken to determine whether normal and cancerous unchanged. This hepatocellular carcinoma is also markedly liver tissue differs in utilization both in vitro and deficient in the ability to synthesize acid-insoluble glycogen in vivo. Ultimately, we attempt to answer whether metabolic and glycoprotein from xylitol as compared to the liver. alteration effects retardation of tumor cell growth without caus ing nutritional depletion or disturbing the function of the host INTRODUCTION liver. In this paper, we report the results of the comparative study Changes in metabolism or in structural constitution of living of the metabolism of glucose and the pentitol xylitol in several materials may cause an overall alteration of normal cells, recently developed rat hepatocellular carcinomas and in the leading to the development of neoplastic cells. One striking host. difference found to exist between normal and tumor cells reported by Warburg (32) is that tumor cells exhibit a higher rate of aerobic and anaerobic glycolysis than do normal cells. MATERIALS AND METHODS This is not a characteristic of all types of tumors (1 ); correlation Animals and Chemicals. Xylitol was donated by the J. P. has been found to exist between the growth rate of different Pfrimmer Co., Erlangen, West Germany. Radioactive xylitol tumor cells and the degree of glycolysis (35); glycolysis par (87.5 mCi/mmol) and glucose (248 mCi/mmol) were obtained allels the rate of growth. Similar correlations, as clear as in from Amersham/Searle Co., Arlington Heights, III. Glucose and boric acid were purchased from the Fisher Scientific Co., ' The project was supported in part by a generous donation from Mr. and Mrs. Pittsburgh, Pennsylvania. 0-NAD+ and /J-NADP" came from Leland Anderson. Presented at the Annual Meeting of the Federation of American Boehringer/Mannheim, Mannheim, West Germany. The anion- Societies for Experimental Biology, April 1 to 10, 1979, Dallas, Texas (21). 2 Recipient of a Predoctoral Fellowship from the Clayton Foundation for exchange resin (AG1-X4 CD came from Bio-Rad Laboratories, Research, Houston. Texas, in partial support of this project. To whom requests Richmond, Calif. Aquasol (xylene-based) scintillation solution for reprints should be addressed. Received July 18, 1980; accepted May 11. 1981 was purchased from New England Nuclear, Boston, Mass. The

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remaining chemicals were of analytical reagent grade and were ice for 10 min and then centrifuged at 16,000 x g for 30 min from various commercial sources. at 4°.The resulting supernatant was dialyzed overnight against Male Sprague-Dawley rats weighing 125 to 150 g were 0.01 M sodium phosphate buffer (pH 7.0). obtained from the Timco Breeding Laboratory, Houston, Texas. For the hepatocellular carcinomas, 2 to 3 g solid tumors The transplantable hepatocellular carcinomas 252, 311C, and minced in a small amount of cold 0.9% NaCI solution and 2 to FB56 were all originated by exposure of male inbred ACI rats 3 ml packed cells from the ascitic tumors prepared after to the carcinogen W-2-fluorenylacetamide. All received the removal of contaminating rat erythrocytes by centrifuging at 7 standard 4-cycle diet. The generation of the tumors were x g for 10 min were successively processed in the manner approximately: 252, generation; 311C, generation 78; and previously described. The final supernatant was adjusted to a FB56, generation 49. protein concentration of 2 mg/ml. The polyol dehydrogenase Carcinomas 252 and 311C are well differentiated and take activity assay mixture contained 0.1 mmol Tris-HCI (pH 9.0), approximately 1 month to grow between transplants. FB56 is 1.5 jumol MgCI2, 0.5 mg ß-NAD+or /?-NADP+, 1 to 100 /imol more aneuploid and grows in approximately 2.5 weeks. Rat xylitol, and homogenate containing 0.1 to 0.2 mg protein in a hepatocellular carcinoma lines, 252, 311C, and FB56 solid total volume of 1 ml. Control sample contained all components tumors were generously furnished by Dr. F. F. Becker. The save the substrate. The enzyme activity was determined at 340 Novikoff ascites tumor was kindly provided by Dr. R. B. Hurl- nm for 5 to 10 min at room temperature in a Beckman spectro- bert, and AS-30D ascites tumor was obtained from Dr. E. F. photometer. Walborg, Jr. (27, 28). All 3 are colleagues from the Depart Preparation of Tissue Homogenates from Rats Infused with [14CJXylitol or [14C]Glucose. Each rat had a 15-ml air ments of Pathology or Biochemistry, M. D. Anderson Hospital and Tumor Institute. Subsequently, the AS-30D and Novikoff pouch injected s.c. interscapularly with a 20-ml syringe and an ascites tumors were maintained in our laboratory by injecting 18-gauge needle (5). One million cells of AS-30D ascites tumor 106 cells i.p. into male Sprague-Dawley rats weekly. as single-cell suspensions were then inoculated into each air Preparation of Tissue and Tumor Homogenates. Two to 3 pouch. In 10 to 14 days, a 15- to 20-g AS-30D solid tumor g of solid hepatocellular carcinoma lines, 311C, 252, and FB56 developed as sheets of tumor cells adhering to the wall of the freshly removed from rats were finely minced with a scalpel air pouch. Fluid containing tumor cells accumulated in the air blade in a small amount of cold 0.9% NaCI solution. The minced pouch was discarded. These rats were fasted for 48 hr and tumors were placed in a 50-ml Teflon Potter-Elvehjem homog- given injections of 2 /iCi [L/-14C]glucose or [U-14C]xylitol, 2 g/ enizer and disrupted in 5 volumes of 0.023 M potassium kg body weight in a 10% solution. Labeled were phosphate buffer (pH 7.4) containing 0.133 M KCI with 15 administered at a rate of 1 ml/min into the tail vein, and the strokes of a pestle at a speed of 500 rpm (Fisher Stedi-Speed rats were killed 15 to 45 min after infusion. The liver, kidneys, Motor). The homogenates were dialyzed against the same spleen, and tumor of each animal were carefully removed and buffer without 0.133 M KCI for 5 hr and then diluted with 0.023 weighed. These organs were then finely minced separately in M potassium phosphate buffer with 0.133 M KCI to a protein 5 to 20 ml of cold 0.9% NaCI solution, the amount depending concentration of 100 mg/ml. To prepare the homogenates of on the weight of the organs. One-mi aliquots were removed for the AS-30D and Novikoff ascites tumors, 30 ml of fluids were nucleic acids assay. The minced tissues were then added to withdrawn from abdominal cavities of rats bearing these tu 0.5 volume of cold 12% PCA3 and homogenized with 15 mors. Twenty ml of cold 0.9% NaCI solution were added to the strokes in a Teflon Potter-Elvehjem homogenizer at a maximum fluid, and the mixture was subjected to centrifugation (7 x g) speed of 500 rpm. Aliquots of the homogenates were removed for 5 min. for assays of acid-insoluble fractions. After 15 min of centrifu This was repeated 5 times to minimize the contamination by gation at 400 x g, the remaining cold PCA extracts were rat erythrocytes. The ascites tumors thus prepared were sub neutralized with 6 N K2CO3, and the KCIO4 precipitates were jected to homogenization and dialysis identical to the solid removed. tumors. All steps were performed at 4°.All surgical procedures Radioactivity of Total Nucleic Acids, Acid-insoluble Gly- for obtaining regenerating liver from male Sprague-Dawley rats cogen (19), and Glycoprotein in Tissues. One ml of the (125 to 150 g) were carried out under ether anesthesia ac remaining 0.9% NaCI suspension of the tumors and normal cording to the method of Higgins and Anderson (10). Five g of livers was added to 10 volumes of 2.5% PCA, stirred, and then the regenerating livers (12 hr and 1 week) and freshly removed allowed to precipitate for about 5 min in an ice bath. The normal Sprague-Dawley rat liver without tumor were perfused sample was centrifuged at 400 x g for 5 min. To the sediment with 50 ml Hanks' medium without glucose and calcium at 4° were added 5 ml of 95% to remove lipoidal compound. and minced separately in a Retri dish filled with cold 0.9% NaCI Then, the final precipitate was dissolved in 1.0 ml of distilled solution. The tissues then were centrifuged at 7 x g for 2 min, water and 1.0 ml of 5% PCA and placed in a boiling water bath homogenized, and dialyzed as above. for 15 min. Subsequently, the sample tube was allowed to Determination of Polyol Dehydrogenase Activity. The nor stand in ice for 5 min and centrifuged at 400 x g for 5 min. mal and regenerated livers perfused with Hanks' medium with The extraction was repeated with 1 ml hot PCA. The 2 extracts out Ca++ and glucose were homogenized in an equal volume were combined, and 0.5 ml-aliquots were counted for nucleic of 0.01 M sodium phosphate buffer (pH 7.0) as described acids. An aliquot was also assayed for DNA (37) and RNA (23). previously. The homogenates were then sonicated 4 times for The remaining acid-insoluble fractions of the tumors and 15 sec each (output set on 5; Sonicator; Ultrasonic, Inc.). The normal liver were lyophilized. One g of each dried sample sonicate was centrifuged at 4500 x g for 15 min. To the (Fraction A) (2 to 2.5 g) was added to 12 ml of 30% KOH, and supernatant was added 10% protamine sulfate at one-tenth of the volume of the supernatant. The mixture was left standing in 3 The abbreviation used is: PCA, perchloric acid.

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Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1981 American Association for Cancer Research. J. Sato et al. the mixture was boiled for 20 min and then precipitated with second with 0.6 N boric acid at pH 10.0. 15 ml of 95% ethanol for 10 min. The tube was allowed to Assay Systems. Glucose was analyzed enzymatically (9). stand at room temperature for about 2 hr prior to the centrifu- Xylitol was assayed colorimetrically by the method of Bailey gation for 15 min at 1000 x g to separate the tissue protein (3). Protein content was determined by the method of Lowry et hydrolysates (Fraction B) from glycogen and glycoprotein (24, al. (18). The remaining experimental procedures are delineated 36). The precipitate containing the latter materials was redis- in the legends of the appropriate tables and charts. solved in 1 ml of distilled water and twice precipitated with 95% ethanol. The washed precipitate was dissolved in 5 ml of RESULTS distilled water (Fraction C1), 4.3 ml of which were added to 5 ml of 40% (w/v) Dowex 50W-HT (50-X8-400, 200 to 400 Metabolism of Xylitol and Glucose in Various Rat Hepato- mesh) in 0.02 N HCI, followed by acid for 40 hr at cellular Carcinomas. Glucose is known to be metabolized by 100°. The acid hydrolysate (Fraction C2) was separated from a variety of hepatocellular carcinomas (34, 35), resulting in an the Dowex resin by centrifuging at 500 rpm fo 5 min. Aliquots accumulation of lactate. The utilization of xylitol and glucose of Fractions B, C1, and C2 (0.2 to 0.5-ml) were counted in 10 by relatively newly developed hepatocellular carcinoma lines ml of scintillation counting fluid. Ten to 20 mg of the Fraction was compared. Regenerating and normal young adult livers A were oxidized in a small oxidizer (Packard Tri-Carb) and served as controls. Utilization of glucose ranged from 21.3 to counted in 7 ml of carbon dioxide absorber and 11 ml of 29.1% in the transplantable tumors studied (Table 1). These Permfluor V (Packard). These samples were counted in a figures were comparable to those found for the 12-hr and 1- Beckman LS7500 scintillation counter. All the counts were week regenerating and normal livers. In contrast, xylitol utili automatically corrected to dpm depending upon the counting zation was significantly different among the cell lines. The AS- efficiency. 30D and FB56 tumors used only 2.7 and 1.3% of the xylitol, Borate Column Chromatography of Neutral in Acid- respectively. Xylitol metabolism in the AS-30D tumor was stud soluble Fractions (16). Ten to 15 ml of neutralized acid ex ied further relative to the differences in energy expenditure tracts from the tumor and liver tissues were passed through a between tumor and host tissue. In general, the conversion of mixed-bed Amberlite MB-3 column (2 x 20 cm) and eluted xylitol to D- in the presence of NAD* is accomplished with 100 to 150 ml distilled water. The eluate was lyophilized. through polyol dehydrogenase (2, 29). The NADP+-dependent The lyophilisâtes were dissolved in 2 ml of 0.1 N boric acid (pH xylitol dehydrogenase oxidizes xylitol to L-xylulose (12, 29). 7.0). Maximum Polyol Dehydrogenase Activity. To investigate A strong-base anión exchanger (AG1-X4, C1 ~, 200 to 400 the correlation between the degree of xylitol metabolism and mesh) was washed free of fines by décantation, slurried into polyol dehydrogenase activity in these tumors, the soluble the column (1.5 x 30 cm), and washed with 0.15 N boric acid enzyme activity of the dehydrogenase was assayed in the (pH 7.0) until the C1 " ion was completely removed. The column cytosol fractions. Table 2 summarizes the data on the maximum was washed with water to remove excess boric acid and then polyol dehydrogenase activities when xylitol is used as the with 30 ml of 0.01 N boric acid (pH 7.0). A 2-ml sample was substrate. The NAD*-dependent polyol dehydrogenase activi applied to the anión exchange column, and the column was ties in the fast-growing tumors, Novikoff and AS-30D ascites, washed with 2 ml of the same eluting agent. It was then were 0.56 and 0.22 nmol/min/mg protein, respectively. Activ successively eluted with a linear gradient in concentration and ities for the other solid tumors, 311C and 252, were 12.11 and pH at a flow rate of 1 ml/min. The contents of both chambers 5.90 nmol/min/mg protein, respectively. The enzyme activity were 500 ml, the first with 0.1 N boric acid at pH 7.0 and the for FB56 was not detectable under the conditions used. The

Table 1 Percentage of total metabolism of xylitol and glucose in various rat hepatocellular carcinomas and regenerating livers Each incubation mixture (2 ml) contained 4 (imol NAD, SOfimol substrate, 280/imol KCI. 400 nmol ATP, 12 /imol sodium phosphate buffer (pH 7.4), and 50 to 60 mg protein of each homogenate. Incubation time was 30 min at 37°. After incubation, each mixture was immediately added to 2 ml of cold 12% PCA and cooled in an ice bath for 10 min. Aliquots (0.1 to 2 ml) were assayed for glucose and xylitol. % of total metabolism

Hepatocellular carcinomasControlRegenerating

liverSubstrateGlucose0

1C29.1* wk38.8 liver32.9

Xylitol31 9.025221.3 12.8FB5625.71.3AS-30D23.52.7Novikoff25.77.612hr44.534.51 24.0Normal23.7 a Glucose present in the tissues at 0 time was measured. The percentages for glucose were shown after correction. Percentages of total metabolism were calculated as follows:

remaining glucose after incubation 100 x glucose at 0 time + administered glucose

Each value is an average of 2 experiments. The difference between 2 experiments ranged within 5 to 15% of each value.

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NADP+-dependent xylitol dehydrogenase activities were within tion of xylitol metabolites in the tumor was slow. Rapid phos- a close range among the livers and tumors studied except for phorylation of glucose metabolite in the tumor can be inferred the FB56 and AS-30D hepatomas. The highest activity was 3.3 from the large ratio of the specific activity between xylitol and nmol/min/mg protein in the 1-week regenerating rat liver. glucose in the same sample. The liver can metabolize xylitol as Acid-soluble and Acid-insoluble Fractions in Various Tis well as glucose. sues Obtained from Rats Bearing AS-30D Tumor Treated The ratios of the specific activity obtained from acid-insoluble with [lA14C]Xylitol or [U-14C]Glucose. Under normal circum fractions of the tumor to the liver are about 0.16 in the xylitol- stances, xylitol is readily converted to glucose in the liver. To treated rat and 0.48 for the glucose-treated rat, suggesting a investigate the extent to which xylitol was transformed to glu rapid transformation of radioactive precursors to macromole- cose in the AS-30D tumor, uniformly labeled [14C]xylitol or cules of the catalytic or structural cellular components with 3- [14C]glucose were given i.v. through a tail vein of the tumor- fold more in the tumor treated with glucose. Furthermore, to bearing rats. Table 3 shows the results of the distribution of determine whether quantitative changes to the acid-insoluble radioactivity in acid-soluble and acid-insoluble fractions in dif fractions take place, major components such as a mixture of ferent organs. In the rats receiving [14C]xylitol, the total radio protein-bound glycogen, glycoprotein (19), and nucleic acids activity from [14C]xylitol was about 12% in the tumor and 39% were separated as described in the "Materials and Methods." in the liver. The rats given injections of [14C]glucose, by the A protein-bound glycogen and glycoprotein cannot be disso same procedure, do not demonstrate this divergence between ciated under our experimental conditions used. The result is tumor and host; both retained about 30% radioactivity. shown in Table 4. The radioactivity recovered from the tumor acid-soluble frac The percentages of radioactivity in the mixture of acid-insol tion was 0.76% (1681 dpm/g tissue) at 15 min and 0.73% uble glycogen and glycoprotein from xylitol and glucose in the (1618 dpm/g tissue) at 45 min of the total [14C]xylitol injected. tumor were strikingly different; radioactivities from xylitol at 15 These figures became 0.68% (1503 dpm/g tissue) at 15 min and 45 min in the tumor were 1.2 and 1.4%, respectively, of and 0.56% (1238 dpm/g tissue) at 45 min after Amberlite MB- the total dose administered, and radioactivities from glucose 3 column chromatography. This suggested that phosphoryla- were 9.8 and 11.5% in the same time periods. In the liver, the percentages of conversion to the acid-insoluble material were Table 2 virtually identical for both sugars. The total radioactivity from Maximum polyol dehydrogenase activity neutral sugar moieties after acid hydrolysis in the presence of protein)NAD*-dependent30.21(nmol/min/mg Dowex SOW resin offers only the incorporation of '4C in both Rat hepatocellular carcino masNormal glycogen and glycoprotein. There was no difference between ±3.28a (3)b rat liver ±0.46(3) Fractions C1 and C2 in the amount of radioactivity, indicating Novikoff (ascites) 0.55 ±0.11 (4) 1.06 ±0.39(4) that no radioactivity from peptide was trapped by Dowex 50W. AS-30D (ascites) 0.22 ±0.15 (3) 0.14 ±0.06(3) 252 (solid) 12.11 ±1.69 (3) 0.99 ±0.07 (3) Therefore, the gross determination of 95% ethanol precipitate 31 1C (solid) 5.90 ±0.002 (3) 2.28 ±0.07 (3) (Fraction CD revealed an absence of the peptide moieties for FB56 (solid) Not detectable (3) Not detectable (3) incorporation of [14C]xylitol or[14C]glucose. As a consequence, 12-hr regenerating rat liver 27.50 ±0.65 (3) 3.16 ±0.01 (3) 1-wklivera regenerating rat (3)number36.90 ±0.57 3.31 ±0.26(3) the radioactivity obtained after removal of protein hydrolysate Mean ±S.D. appears to be derived solely from sugar moieties. The tumor 6 Numbers in parentheses,Activity of assays.NADP*-dependent2.22 acid-insoluble glycogen and glycoprotein from xylitol was only

Table 3 Distribution of total acid-soluble and acid-insoluble fractions in various tissues in rats bearing AS-30D hepatocellular carcinoma treated with ["Clxylitol or ["CJ- glucose at varying intervals Total acid-soluble traction Total acid-insoluble fraction

Before Amberlite MB-3 After Amberlite MB-3

[14C]XylitolTissue3Tumor15

re re re re re (x102dpm/g re (x102 covered/tissue60.760.731.081.120.030.160.060.061.922.07Specificactivity(dpm/gtissue)1681161446264797594317040454075['"C]Glucose%covered/tissue1.100.741.070.930.210.070.090.032.471.77Specificactivity(dpm/gtissue)24331637458339834160139661462037[14C]Xylitol%covered/tissue0.680.560.510.510.230.040.030.051.451.16Specificactivity(dpm/gtissue)1503123821842186456679220713429[14C]Glucose%covered/tissue0.120.060.400.540.100.040.040.010.660.65Specificactivity(dpm/gtissue)2651321713231519917922716697[MC]Xylitol%covered/tissue11.411.736.937.816.814.69.828.4374.9072.50Specificactivitycovered/tissue29.430.731.422.49.018.536.836.5176.3068.10Specificactivitydpm/g tissue)252259158014603328289266705726['"CJGIucose%tissue)6501678913459591785169046394422

min45 minLiver1

5min45 minKidney15

min45 minSpleen1

5min45 minTotal15

min45 min% Average weight of the tissues: tumor, 21.5 g; liver, 11.1 g; kidneys, 2.4 g; spleen, 0.7 g. ' Each value is an average of 2 experiments. The difference between 2 experiments ranged within 2 to 15% of each value.

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Table 4 Components of total acid-insoluble fractions in tumors and livers of rats bearing AS-30D hepatocellular carcinoma treated with ¡"dxylitol or ["Cfalucose in vivo at varying intervals

XylitolTumorFraction3Acid-insoluble

of ra of ra of ra of ra dioac 102dpm/g)1,2801,4901.1701,3802,3822,498213214Liver%dioac 102dpm/g)45,98043,62045,58039,6909,89610.125852969GlucoseTumor%dioac 102dpm/g)8,53010.0108,62010,3603.5632,990299346Liver%dioac 102dpm/g)41,71030,91039,48029,0507,4154,2451,3641,243 tivity61.21.41.11.310.310.8<0.1<0.1Specificactivity(X tivity11.711.111.610.125.926.5<0.2<0.2Specificactivity(Xtivity9.811.59.911.919.916.7<0.1<0.1Specificactivity(Xtivity11.28.310.67.825.514.6<0.2<0.2Specificactivity(X glyco-gen andglyco-protein (C1)c15 min45 minTotal

neutralsugarsrecovered fromC1 (C2)"15 min45 minProtein (B)815 min45 minTotal

nucleicacids15 min45 min% a Content of nucleic acids, 7 to 10 mg/g liver, 10 to 12 mg/g tumor; contents of protein, 112 to 147 mg/g liver, 96 to 124 mg/g tumor; contents of acid-insoluble glycogen and glycoprotein, 10 to 12 mg/g liver, 21 to 25 mg/g tumor. 6 Each value is an average of 2 experiments. The difference between 2 experiments ranged from 5 to 20% of each value. c The 95% ethanol precipitates. Acid hydrolysates obtained after 40 hr incubatioin at 100°in the presence of Dowex 50W resin. Specific activities for Fraction C2 are expressed as the ratios of dpm of Fraction C2 to unit weight of acid-insoluble glycogen and glycoprotein. e Protein hydrolysates obtained after alkaline hydrolysis of acid-insoluble fractions.

3% that of the liver, whereas the same components from Table 5 glucose consisted of 20 to 32%. Therefore, the tumor appears Percentage of neutral sugars and neutral metabolites recovered after borate column Chromatography to incorporate the carbon from xylitol into the above substance. At the end of the elution in borate column Chromatography (details of the However, the tumor sustains an ability to form the acid-insolu procedure were provided in "Materials and Methods"), the eluatesthat contained ble glycogen and glycoprotein from glucose. more than 500 cpm/tube associating with the peaks of xylitol and glucose were The labeled carbon of the acid-insoluble fraction from xylitol pooled and recounted. recovered3TissueLiver in the tumor was predominantly confined to the tissue protein Experi moiety. ment1 Borate Column Chromatography of Neutral Sugar Metab olites. In an attempt to estimate the carbon flow of neutral 2 Liver Xylitol 6.6 54.0 39.4 carbohydrates more specifically, the residual radioactivities in 3 AS-30D Xylitol 81.4 5.3 13.3 AS-30D the acid-soluble fractions were further analyzed. The distribu 4 93.4xylitolXylitol 0.0 6.6 5 Liver Glucose 69.4 30.6 tion of the neutral metabolites following borate column chro- 6 Liver Glucose 65.7 34.3 matography in the tumor and host tissue is summarized in 7 AS-30D Glucose 44.5 55.5 AS-30D38% Glucose["CJXylitol8.4 43.7x 46.3 Table 5. Only xylitol and glucose were identified. The amount Calculated as: of [14C]xylitol converted to [14C]glucose by the host liver tissue

cpm of""C-SubstrateXylitolor glucose recovered[14C]Glucose60.9 was 50 to 60%, indicating that the liver was a primary site for moOthers30.7 the metabolism of xylitol. The Chromatographie profile of the Total cpm of neutral sugar metabolites recovered after Amberllte MB-3 column Chromatography neutral sugar metabolites in host liver tissue showed 8 distinc tive peaks with radioactivities from xylitol. One of the peaks was the major metabolite glucose (Chart 1). Some of the (Table 2). Further, the percentage of radioactivity recovered labeled peaks are associated with periodic acid-positive sugars after removal of ionized sugar metabolites also suggests a [xylitol was used as a standard (3) to indicate the intensity of reduced metabolism of xylitol by the solid AS-30D tumor (Table unknown periodic acid-positive compounds]. The neutral sugar 3). metabolites from the AS-30D tumor treated by injection of The Chromatographie profile of the same fraction taken from [14C]xylitol indicated primarily intact [14C]xylitol, with glucose the liver after [14C]glucose injection showed a much simpler as a minor metabolic product (Chart 2). The results obtained in pattern; the radioactivity was incorporated into a few remaining xylitol-injected tumor-bearing rats are consistent with the low neutral metabolites due to the rapid metabolism of glucose NAD+-dependent polyol dehydrogenase activity of the tumor (Chart 3). The peaks of radioactivity were widely scattered in

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(pH7-10) c 160 Glucose E340inSMaterial

..g/TuO000V1 O)O

I £ 100 S 160£S o.u- - 600 s <120ui 1(_- < 60 400

800.l

«0.10'3)/Tube*- 20

400 600 200 400 600 800 Volum«(ml) Volume (ml) Chart 4. Borate column chromatography of neutral metabolites from the total Chart 1. Borate column chromatography of neutral sugar metabolites from acid extract in AS-30D tumor treated with [U-'4C]glucose in vivo. For chromatog the total acid extract in host liver treated with [U-MC]xylitol in vivo. Exchanger, AG1-X4 (borate form), 200 to 400 mesh; column size, 1.5 x 30 cm; eluting agent, raphy conditions, see Chart 1. pH, and concentration gradients, 500 ml each (0.1 N boric acid at pH 7.0 to 0.6 N boric acid at pH 10.0); volume collected per tube, 5 ml. the tumor (Chart 4). Glucose was significantly metabolized to a variety of glycolytic metabolites, as indicated by the results

800 of Table 5. a 700 - DISCUSSION à i •5600 " 3 - Numerous studies regarding metabolic comparison of xylitol • with glucose and other sugars in rat liver and human have been s soo a. reported (7,13,17, 31 ). None of the data, however, have dealt u with metabolism of this polyol in tumors. Differences in carbo 400 hydrate metabolism between host and tumor have been ob

300 served in the present study. To find sugars that tumors might M not be able to utilize, the polyol xylitol was compared with •g200 glucose as substrate for tumor cells. All hepatocellular carci O. nomas and normal rat liver showed similar rates of utilizing 1 100 glucose, while xylitol was poorly metabolized in all tumors studied. The tumors of FB56 and AS-30D demonstrated a particularly low utilization of xylitol. Since NAD+-dependent 200 400 600 800 polyol dehydrogenase and NADP^-dependent xylitol dehydro- Volume (ml) genase activities in cells of AS-30D hepatocarcinoma were Chart 2. Borate column chromatography of neutral sugar metabolites from the total acid extract in AS-30D tumor treated with [l/-'4C]xylitol in vivo. For markedly low, this poor utilization reflects as either a qualitative chromatography conditions, see Chart 1. or a quantitative metabolic deficiency by the tumor. It is known 1800 that increased glycolysis parallels an increased growth rate of 260 p 4 r tumors (34). If this biochemical phenomenon implies large 240 energy requirements for tumor proliferation, the faster-growing

è tumors may be sensitive to unfavorable substrates which do i not participate in energy production. Rats bearing FB56 and AS-30D tumors, therefore, are good models to further study the tumor-host relationship in energy metabolism. 100 Further evaluation of metabolism in the AS-30D tumor model 80 - 600 demonstrated distinctively different patterns for glucose and xylitol. As shown in Table 4, most of the radioactivity in the acid-insoluble fraction from the tumor treated with xylitol was recovered in protein. The conversion of [14C]xylitol into protein 40 by the tumor (90 to 92%) was relatively higher than in the liver (70%). The conversion of [14C]glucose, on the other hand, was at a similar order of magnitude in both tumor (53 to 68%) and liver (65 to 81%), when conversion of 14C into protein was 200 400 600 MM Volume (ml) expressed as percentage of the total acid-insoluble fraction. Chart 3. Borate column chromatography of neutral sugar metabolites from the total acid extract in host liver treated with [U-MC]glucose in vivo. For The biological diversity between acid-insoluble glycoprotein, chromatography conditions, see Chart 1. glycogen, and protein synthesis in xylitol metabolism in the

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Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1981 American Association for Cancer Research. J. Sato et al. tumor is an interesting phenomenon. At this time, there is no growth also seems to fluctuate to the degree to which overall knowledge regarding biological significance of the reduced metabolism of the cellular components shifts towards a direc uptake of xylitol to acid-insoluble glycogen and glycoprotein. tion unfavorable for the energy production when xylitol is Amino acid biosynthesis from carbohydrates via the tricarbox- administered (30). ylic acid cycle followed by the formation of protein is generally Furthermore, this experimental observation that there is im accepted accordingly. It seems reasonable to hypothesize that paired metabolism of xylitol due to lack of polyol dehydrogen xylitol is metabolized through the same pathway to form tumor ase in AS-30D tumor justifies a detailed study of the effect of protein. However, the conversion of [14C]xylitol into protein by continuous infusion of xylitol in rat hepatomas. the tumor could result in part from a conversion to methyl- glyoxal and methylglyoxal-protein complex formation. Kato et REFERENCES al. (15) and Fodor ef al. (6) imply that methylglyoxal may bind 1. Aisenberg, A.C. The anaerobic and aerobic glycolysis of normal and tissues. to structural proteins. Furthermore, we showed that xylitol was In: The Glycolysis and Respiration of Tumors, pp. 1-53. New York: Aca demic Press. Inc.. 1961. transformed to methylglyoxal at a rate 10 times greater than 2. Arsenis, C., and Touster, O. Nicotinamide adenine dinucleotide phosphate- that of glucose in rat liver (22). These observations support the linked xylitol dehydrogenase in guinea pig liver cytosol. J. Biol. Chem.. 244: above hypothesis. 3895-3899, 1969. 3. Bailey. J. M. A microcolorimetric method for the determination of . The metabolic path of xylitol to tissue glycogen via glucose and in biologic fluids. J. Lab. Clin. Med.. 54: 158-162, is based on the rapid conversion of xylitol to glucose in the 1959. normal rat liver (13). The findings at present argue against a 4. Bloch-Frankenthal, L.. Langan, J.. Morris, H. P., and Weinhouse, S. Fatty acid oxidation and ketogenesis in transplantable liver tumors. Cancer Res.. rapid conversion to glucose, since the radioactivity retained in 25. 732-736, 1965. the tumor was predominantly from xylitol and not via the 5. Chang, J. P.. Gibley, C. W.. Jr., and Kihyoe, I. Establishment of transplant- glucose moiety (Table 5). Apparently, a slow transformation of able hepatomas induced by 3'-methyl-4-methylaminoazobenzene with spe cial reference to the histologie natures of the transplants of early passages. xylitol into glucose did occur in the tumor, regulated by the low Cancer Res., 27. 2065-2071, 1967. activity of the NAD+-dependent polyol dehydrogenase. This 6. Fodor, G., Mujumdar, R., and Szent-Gyorgi, A. Isolation of methylglyoxal accounts for low incorporation of radioactivity of [14C]xylitol from liver. Proc. Nati. Acad. Sei. U. S. A. 75. 4317-4319, 1978. 7. Froesch, E. R., and Jakob, A. The metabolism of xylitol. In: H. C. Sipple and into glycogen in the tumor. K. W. McNutt (eds.) Sugars in Nutrition, pp. 241 -253. New York: Academic Low 14C recovery in total nucleic acid suggests that ribose Press, Inc.. 1974. 5-phosphate, a nucleic acid precursor, is not a major carbo 8. Cullino, P. M., Grantham, F. H., and Courtney, A. H. Glucose consumption by transplanted tumors in vivo. Cancer Res.. 27. 1031-1040, 1967. hydrate metabolite of glucose or xylitol by the AS-30D tumor 9. Gutmann, I., and Wahlefeld. A. W. D-Glucose. Methods Enzymatic Anal. 3: in this experimental setting. During a prolonged starvation of 1186-1201, 1974. 10. Higgins, G. M., and Anderson, R. M. Experimental pathology of the liver. rat (over 48 hr), the amount of RNA is drastically reduced in Arch. Pathol., 12: 186-203, 1931. the liver due to degradation of the rough endoplasmic reticulum 11. Hirsh. C. A., and Hiatt, H. H. Turnover of liver ribosomes in fed and fasted (11, 20). This may well explain the results obtained. rats. J. Biol. Chem. 241: 5936-5940. 1966. 12. Hollman, S.. and Touster, O. The L-xylulose-xylitol enzyme and other polyol Borate column chromatography showed 4 distinctive profiles dehydrogenase of guinea pig liver mitochondria. J. Biol. Chem., 225: 87- when the metabolic pattern of xylitol was compared to that of 102, 1957. glucose in the host tissue and in AS-30D tumor. The low activity 13. Jakob, A., Williamson, J. R., and Asakura, T. Xylitol metabolism in perfused rat liver. J. Biol. Chem., 246. 7623-7631. 1971. of the AS-30D tumor in metabolizing xylitol could be beneficial 14. Kaneki, T., Oka, H., Oda, T., and Toshitoshi. Y. 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Determination of nucleic acids in tissues by pentose xylitol may slow tumor growth. Xylitol certainly may not be the analysis. Methods Enzymol., 3: 680-684, 1957. major regulatory factor of the tumor growth. The level of 24. Seifer, S., Dayton, S.. Novic, B.. and Muntwyler, E. The estimation of glycogen with the anthrone reagent. Arch. Biochem., 25. 191-200. 1950. adenine nucleotides, NAD and NADP, required for an energy 25. Sestoft, L., and Gammeltoft, A. The effect of intravenous xylitol on the metabolism is known to be changed by xylitol (14, 25). The concentration of adenine nucleotides in human liver. Biochem. Pharmacol.. sulfhydryl group believed to be a potential agent regulating cell 25. 2619-2621, 1976.

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26. Shonk, C. E., Morris, H. P.. and Boxer, G. E. Patterns of glycolytic enzymes 31. Wang, Y. M., and van Eys, J. Nutritional significance of fructose and sugar in rat liver and hepatoma. Cancer Res., 25: 671-676, 1965. . Annu. Rev. Nutr., T: 437-475, 1981. 27. Smith, D. F., and Walborg, E. F., Jr. Isolation and chemical characterization 32. Warburg, O. The Metabolism of Tumors. London: Arnold Constable, 1930. of cell surface sialo-glycopeptide fraction during progression of rat ascites 33. Weber, G. Enzymology of Cancer Cells. N. Engl. J. Med.. 296. 486-493, hepatoma AS-30D. Cancer Res., 32. 543-549, 1972. 1977. 28. Smith, D. F., Walborg, E. F., Jr., and Chang, J. P. Establishment of a 34. Weber, G., and Lea, M. A. The molecular correlation concept. An experi transplantable ascites variant of a rat hepatoma induced by 3'-methyl-4- mental and conceptual method in cancer research, Methods Cancer Res., dimethyl aminoazobenzene. Cancer Res., 30: 2306-2309. 1970. 2. 523-578, 1967. 29. Touster, O., and Shaw, D. R. D. Biochemistry of the acyclic . Physiol. 35. Weber. G., and Morris, H. P. Comparative biochemistry of hepatomas. Res., 42 181-225, 1962. Cancer Res., 23: 987-994. 1963. 30. van Eys, J., Wang, Y. M., Chan, S., Tanphaichitr, V. S., and King, S. M. 36. Wood, N., and Fillios, L. C. Bound sugars in hepatic glycoproteins from male Xylitol as a therapeutic agent in glucose-6-phosphate dehydrogenase defi rats during early protein depletion. J. Nutr., ) 70. 324-329, 1980. ciency. In: H. C. Sipple and K. W. McNutt (eds.). Sugars in Nutrition, pp. 37. Zamenhof, S. Preparation and assay of deoxyribonucleic acid from animal 613-651. New York: Academic Press, Inc., 1974. tissue. Methods Enzymol. 3: 696-704, 1957.

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Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1981 American Association for Cancer Research. Metabolism of Xylitol and Glucose in Rats Bearing Hepatocellular Carcinomas

Junko Sato, Yeu-Ming Wang and Jan van Eys

Cancer Res 1981;41:3192-3199.

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