STUDIES OF RIBOSE METABOLISM. III. THE PATHWAY OF RIBOSE CARBON CONVERSION TO GLUCOSE IN MAN Howard H. Hiatt J Clin Invest. 1958;37(5):651-654. https://doi.org/10.1172/JCI103649. Research Article Find the latest version: https://jci.me/103649/pdf STUDIES OF RIBOSE METABOLISM. III. THE PATHWAY OF RIBOSE CARBON CONVERSION TO GLUCOSE IN MAN' BY HOWARD H. HIATT (From the Departments of Medicine, Harvard Medical School and Beth Israel Hospital, Boston, Mass.) (Submitted for publication November 22, 1957; accepted January 2, 1958) The conversion of ribose carbon to hexose via protein. Following centrifugation the clear supernatant the reactions of the pentose phosphate pathway solution was reduced to a volume of approximately 30 ml. has been observed in studies with rat liver ho- by lyophilization and then deionized by passage through a mixed bed resin (Amberlite MB-3, Fisher Scientific mogenates (1), rat liver slices (2), and in the Co.). Since the effluent contained radioactive material intact mouse (3). Since most of these reactions other than glucose, further purification was carried out are reversible (4), they provide mechanisms not by phosphorylation of the glucose, adsorption of the sugar only for ribose conversion to hexose, but for ri- phosphate on an ion-exchange resin, washing the phos- bose synthesis as well. Indeed, has phate ester free of contaminants, elution of the ester, and, recent work finally, dephosphorylation. One-half of the deionized demonstrated that a human carcinoma cell in solution, containing approximately 200 micromoles of tissue culture (5) as well as the intact human glucose, was brought to pH 7.6 with 0.1 N KOH and subject (6) synthesize ribose via this pathway. treated with 400 micromoles of adenosine triphosphate This report provides further evidence that in man (ATP), first brought to pH 7.6, 100 micromoles of the reactions of this pathway MgCl2, and 10 mg. of crude yeast hexokinase (8). The mediate the inter- mixture was stirred magnetically at 250 C. in a beaker conversion of ribose and glucose. D-Ribose spe- in which external electrodes from a Beckman pH meter cifically labeled with radioactive carbon was given were suspended. The reaction was followed by meas- to a patient with inoperable carcinoma. Isotope uring the quantity of 0.1 N NaOH required to maintain distribution in glucose subsequently isolated from the pH, since for every mole of glucose phosphorylated, this patient's blood is consistent with the con- one mole of acid is produced (8). Following phosphory- lation of 82 per cent of the glucose, the mixture was ad- version of ribose carbon to hexose via the pen- sorbed on Dowex-1-acetate (the chloride form of the tose phosphate pathway. resin is equally satisfactory). The resin was washed with several volumes of water until the effluent was free METHODS of radioactivity, and the glucose-6-phosphate was eluted Sixty microcuries of D-ribose-l-C14 (24 mg.) in 10 with 0.1 N HCl. The eluate was neutralized with 1.0 N ml. of isotonic saline was sterilized by passage through a KOH, and the volume reduced to 5.0 ml. by lyophiliza- sintered glass bacteriological filter. This solution was tion. The pH was adjusted to 5.0, and the ester was de- administered intravenously to a 75 year old female phosphorylated by treatment with 10 mg. of acid phos- three hours after breakfast. The patient was suffering phatase at 370 C. for three hours. The solution was from carcinoma of the head of the pancreas, demon- then deproteinized with perchloric acid and deionized as strated to be inoperable on abdominal laparotomy, but described above. An aliquot of this solution was chro- her fasting blood sugar level, renal function, and he- matographed in butanol-acetic acid-water (9), and was patic parenchymal function (serum thymol turbidity, found to contain a single radioactive spot on radio- cephalin flocculation, albumin and globulin, and cho- autography. lesterol, total and esterified) were normal. Fifteen min- Glucose was degraded by incubation with Leuconostoc utes and two hours following the ribose injection, 50 ml. mesenteroides (10). The fermentation products were of heparinized blood was removed from an antecubital converted to BaCO, by techniques which have been sum- vein of the opposite arm. The blood was treated with 3 marized elsewhere (11), and which permit the isolation ml. of 70 per cent perchloric acid, and the protein precipi- of each carbon atom of the glucose molecule free of the tate removed by centrifugation and washed with 10 ml. of others. In order to determine whether added glucose-6- water. The supernatant solution and wash were brought phosphate could be degraded by L. mesenteroides the to pH 7.0 with 5.0 N KOH, and the insoluble potassium sugar phosphate was incubated with a lyophilized prepa- perchlorate removed by centrifugation. The super- ration of the organism. The lyophilized bacteria fer- natant solution was treated with barium hydroxide and mented glucose actively, but had no discernible effect on zinc sulfate (7) to remove a small amount of residual the phosphate ester. Thus, the dephosphorylation step was required prior to further degradation of the isolated 1 This investigation has been aided by grants from sugar. the Jane Coffin Childs Memorial Fund for Medical Re- Chemical and enzymatic procedures. Ribose was ana- search and from the National Cancer Institute (C-3151), lyzed by the orcinol procedure (12) and glucose by the United States Public Health Service. method of Nelson (13) and Somogyi (7). Glucose-6- 651 652 HOWARD H. HIATT phosphate was assayed spectrophotometrically with glu- unlabeled glucose and ribose-1-C14 was degraded cose-6-phosphate dehydrogenase and triphosphopyridine nucleotide (TPN) (14). by L. mesenteroides. No radioactivity was found Materials. Ribose-l-C"' was obtained from the Na- in carbons 1 or 3, the sites of major isotope con- tional Bureau of Standards through the courtesy of Dr. centration in the blood glucose of the patient given H. S. Isbell. Crude yeast hexokinase, glucose-6-phos- ribose-1-C14. phate dehydrogenase, TPN, and ATP were purchased DISCUSSION from Sigma Chemical Co. Glucose-6-phosphate was purchased from Schwarz Biochemical Co. The fraction of ribose carbon which was con- Radioactivity determinations were carried out with a verted to blood glucose within the 15 minutes fol- "micromil" end-window counter with a counting effi- lowing ribose administration may be calculated ciency of approximately 20 per cent. Barium carbonate from the was counted at "infinite" thickness and all other samples product of the specific activity of the at "infinite" thinness. blood glucose [395 cpm per mg.], the blood glu- cose concentration [90 mg. per 100 ml.], and the RESULTS glucose space [24 per cent (16) of the patient's Blood glucose samples, obtained 15 minutes and body weight, 50 kilograms]. This figure, 4.80 x 2 hours following the injection of ribose-C14, had 106 cpm, which represents 16 per cent of the ad- specific activities of 395 and 133 counts per min- ministered radioactive carbon, is a minimal one, ute (cpm) per mg., respectively. for it does not include the glucose catabolized dur- The degradation data are presented in Table I. ing the 15 minutes following ribose administration. Radioactivity was found predominantly in carbons Six to fifteen per cent of the administered radio- 1 and 3 of the glucose molecule, with the latter activity was found in liver glycogen of mice given more active than the former. The isotope pattern ribose-1-C14 (3). in the sample isolated at 2 hours was virtually Studies with plants, bacteria and animals sug- identical with that observed in the 15 minute gest a uniformity in the pathway of conversion of specimen, and very similar to the distribution of ribose to hexose (4). The pentose must first be radioactivity in mouse (3) and in rat (15) liver phosphorylated, and a specific kinase for this reac- glycogen isolated following ribose-1-C14 adminis- tion has been isolated from mammalian liver (17). tration (Table I). Ribose ATP -* + ADP The unlikely possibility that trace quantities of 1) + ribose-5-phosphate radioactive ribose remaining in the circulation (adenosine diphosphate) could have contributed to the isotope pattern can This pentose phosphate may then be converted be ruled out as a result of a previously reported to hexose phosphate via the nonoxidative series investigation (3). In this study a mixture of of reactions of the pentose phosphate pathway: HC*O H2C*OH H2C*OH H&OH (=0 IC= Isomerase Epimerase 2) HCOHI I~~ IHOHHCOH HOCH HCOH HCOH H$OH H2C-OP03- H2C-0P03- H2C-0P03 Ribose-5-P Ribulose-5-P Xylulose-5-P H2C*OHI H2C*OH HC*O C=O HCOH HO*H HCO ___ _ Transketolase 3) HOCH + H1COH HCOH + HCOH I~~~~~~ HCOH HCOH HCOH H2C-0PO3- H2C-0P03- H2CO-P3Oa HCOH H2C-OP03- Xylulose P Ribose P Sedoheptulose P Triose P RIBOSE CARBON CONVERSION TO GLUCOSE IN MAN 653 H2C*OH c=o H2C*OH HOC*H --I-- Transaldolase 4) HCOH HOb*H HCO HCOH + HCO HhOH + HLOH HCOH HCOH HbOH HCOH H2C-0P03- H2C-OP03- H2S-OPO3 H2C-0P03- Sedoheptulose P Triose P Fructose P Erythrose P H2C*OH H2C*OH C==o c=o HCO Transketolase HOCH 5) - - - HOCH + HCOH I~~~~~~~ HLOH + HCO HCOH HCOH HCOH HCOH H2C-OP03= H2C-OP03 H2C-OPO3- H2Ch-OPO3- Xylulose P Erythrose P Fructose P Triose P To summarize these reactions, three molecules jected with ribose-1-C4 (Table I); this glucose of ribose phosphate would give rise to two mole- was labeled almost exclusively in carbons 1 and 3. cules of fructose phosphate and one of triose phos- The striking similarity of isotope distribution in phate.