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tive shunt, and that E represents the fraction of the. CO, formed glycolytically. If nz moles of COz are formed from glucose, mE moles will be formed ...
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April 20, 1954

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COMMUNICATIONS TO THE EDITOR

Cl4O2from l-C1*-gIucoseexceeded that from eyenly labeled and from 6-C14-glucose. They derived equations relating the C1402 formed from the evenly and the 1-labeled glucose t o that from 1-, 2- and 3-C14-lactates, and on the basis of these equations concluded that glucose catabolism in rat 0 liver does not proceed mainly v i a the EmbdenCHzCH=CHz Meyerhof scheme but involves the direct oxidation and decarboxylation of carbon 1 of glucose (hexose CHzC=CHz monophosphate shunt). They stated that not CH2=IZLz/@ ( A ) CHa more than 20-25% (and possibly none) of the CO? arising from glucose was formed glycolytically. in turn, undergoes similar internal rearrangement We have confirmed the observations of Bloom, to give the phenols (111) and (IV). et aL2 I t is shown here, however, that the derivaThe possibility remains that a species such as tion of their equations is questionable. When the (.I)is not in the direct path between the ether (I) experimental data of Bloom, et al.,, are applied and the pnvn-rearrangement product (111) but that to revised equations presented here, we find that a t (:I rearranges ) to (111) via (I). The modified least 80% of the COz derived from glucose in liver Dewar mechanism for the para-rearrangement is, could have arisen v i a glycolysis. therefore, not excluded. Let us designate, as in ref. 2, the C1402 yields To settle this point, recovered starting material from evenly labeled and l-C14-glucoseas a and b, and products were analyzed after incomplete respectively, and the ratio a l b as U. I t is assumed, rearrangement of (I). Indeed, after 70% comple- as in ref. 2 , that glucose is oxidized to CO,by two tion, infrared analysis of 10% solutions of the ethers pathways, namely, glycolysis and the direct oxidain carbon tetrachloride using the maxima a t 1050 tive shunt, and that E represents the fraction of the and 923 cm.-l showed the recovered ether to con- CO, formed glycolytically. tain only 85% of (I) and 15 i 2% of the rearranged If nz moles of COz are formed from glucose, mE ether (11) and when (I) was heated without solvent, moles will be formed via glycolysis, and m(1 - E ) analysis of the recovered ether after 5Oy0reaction cia the shunt. The radioactive yield of each fracshowed that it had rearranged to (11) to the extent tion can be represented by mE and m(1 - E ) of 48%. It is likely that the (I) rearranges to (X) multiplied by their respective molar specific which can undergo reversion to (I), or rearrange- activities. ment to (11). When the ether fraction recovered If the molar specific activity of glucose is taken after 30% rearrangement of (I) was examined, as 1, the molar specific activity of the Cl4O2derived however, i t contained not more than 5% of the from evenly labeled glucose will be 1/G, irrespective isomeric ether (11) and the composition of the of its mode of formation. The molar specific phenolic products was the same as that after activity of the Cl4O2formed from glucose-1-C" complete reaction. by the direct oxidative path will be 1, and that I t seems clear that the interconversion of the formed glycolytically will be e ' 2 ( c d e), ethers (I) and (11) cannot provide the explanation where c, d and e represent the extent of conversion of the formation of the large amount of the p- to Cl4O2of the carboxyl, alpha and beta carbons of methallylphenol (IV) found in the rearrangement pyruvate (or lactate) formed from glucose. Let of the allyl ether (I). The present results strongly R = (c d e ) /3e (Ris equivalent to the exsupport, therefore, the Hurd and Pollack mech- pression fi = ( N T 1) '3 in ref. 21, then the anism for the para-Claisen rearrangement and ap- molar specific activity of the CI4O2formed glycolytpear to exclude the possibility that the Dewar mech- ically from glucose-l-C14 will be 1 'GR. Thus, anism plays an important role in this reaction. the ratio, a/b, ;.e. The rearrangement of dienone (A) to the methmE -~ + ( I - E)WI allyl ether (I) which is suggested by the work above, glucose - 6 6- ._ . - seems to be an example of a rearrangement of a C"02 from evenly labeled - u '2'402 from l C L 4 labeled glucose mE new type and we hope to investigate it further (1 - E h and 1-methallyloxy-2-propyl-6-isobutylbenzene (b. p. 67-68' a t 0.05 mm., n Z 0 D 1.4915). The results are consistent with the mechanism proposed by Hurd and Pollack' in which (I), for example, rearranges to the intermediate (A) which,

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Noms CHEMICAL LABORATORY DAVIDY CURTIN UNIVERSITY OF ILLINOIS HARRYW. JOHNSON, JR. ~TRBANA,

ILLIVOIS RECE~VED JANUARY 22, 1954

IMPORTANCE OF GLYCOLYTIC AND "OXIDATIVE" PATHWAYS IN GLUCOSE UTILIZATION BY LIVER1

Sir: Bloom and Stetten2p*studied the formation of C1402from C14-labeledglucose and lactate in tissue slices, and observed that in rat liver the yield of ( 1 ) Supported hy contract with the U. S. Atomic Energy Commission. (2) B. Bloom, M . R Stetten and D Stetten, Jr., J . Biol. Chcm , 204, 681 (1953). (3) B. Bloom and D. Stetten, Jr., T H r s JOURNAL, 75, 5446 (1953).

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R(6U - 1) whence E = U ( 6 R 1)

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By using the experimental values of Bloom, et a1.,2for R and U , namely, 2 and 0.6, respectively, in our equation ( l ) , we find that E = 0.79, or that 79% of the COZ derived from glucose was formed v i a the classical glycolytic scheme.

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(4) Compare with equation (4) of ref, ( 2 ) , U = [BRE il - E ) ] / [BE 6(1 E)],whence E = (6U - 1)/(6R - I ) E in this equation actually represents the fraction of the COP formed from the first carbon of glucose w'a glycolysis, and does not have the meaning designated by Bloom, et o2.1 I t should be pointed out that, while referring to the same numerical values in a subsequent paper.' these authors have defined E as the fraction contributed by glycolysis "to the over-all conversion of glucose to carbon dioxide," which has a different meaning and different numerical value from the E used in ref. 2 and here.

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COMMUNICATIONS TO TIIE EDITOR

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In a similar manner we have derived the following equation for the ratio (Cl4O2from 6-C14-glucose,/ C1402from l-C14-glucose),which we shall designate W

electrophoresis in 0.01 ;1.f Na2HP04,pH 8.8, a t I .-I3. Moving boundary electrophoresis in cacodylate buffer of ionic strength 0.1 and p H 6.5 a t 1.4' showed a component with the mobility of hemoglobin F and a component with a mobility greater (E'6R) E ) , whence = ( E / 6 R ) (1 than that of hemoglobin A but slightly less than 6RW that of sickle cell (S) hemoglobin. The mobilities E = 1 + W ( 6 R - 1) of hemoglobins A, S and C in this buffer are, reset.-' Again, using the experimental values of Bloom, spectively, 2.4, 2.9 and 3.2 X 10-5 et a1.,2.afor R and W , namely, 2 and 0.3, respec- volt-'.' The component having the mobility of tively, we calculate that 84YGof the COZ derived from hemoglobin F comprised 41% of the total by elecglucose was formed glycolytically, a value in good trophoretic analysis. This component was isolated agreement with that obtained from our equation (1). from the ascending limb of a moving boundary In our experiments with rat liver slices, the ob- experiment in 0.01 M Na2HP04; its ultraviolet served ratio U = a / b was higher than that reported absorption spectrum was found to be that of hemoby Bloom, et a1.,2 and varied between 1 and 1.8. globin F. The ultraviolet spectrum of the original However, these workers added lactate, acetate specimen did not differ significantly from that of a and gluconate, in high concentrations, to their comparable mixture of hemoglobins A and F." media. Under such conditions, as shown in Table When the original specimen was partially deI, Cl4OZformation from glucose is depressed, and natured with dilute sodium hydroxide, the amount the relative importance of the hexose monophos- of alkali-resistant hemoglobin recovered corresponded to the amount of the fetal electrophoretic phate shunt is increased. component in the specimen. The alkali-resistant TABLE I component was found to have the ultraviolet 250-mg. rat liver slices incubated with 2.5 ml. of Krehsspectrum of hemoglobin F. The non-fetal cornRinger bicarbonate buffer containing 55 pmoles of labeled ponent, isolated from the ascending limb of the glucose. The addition was 50 mm. each of lactate, acetate 5% Cor; incubated p H 6.5 moving boundary experiment, had the color and gluconate. Gas phase 95% 0 2 and visible spectrum of hemoglobin. The solufor 3 hours a t 37". bility of the original specimen as amorphous ferroLabel in yo of C" Ea X glucose Addition in C'4O2 L: = a / b 100mo hemoglobin in 2.58 M phosphate buffer of pH 6.8 even-C'd None a 2.8 a t 25' was 1.94 g. per liter, a value similar to those 1.2 94 of mixtures of hemoglobins A and F under the same 1-ci4 None b 2.8 condition^.^ We conclude from these results that the hemoglobin specimen examined consisted of a a 0.8 e ~ e n - C ~ ~+ mixture of hemoglobin F and a hitherto undescribed 0.6 79 abnormal human hemoglobin, which we shall call b 1.4 1-cl4 These values of E are calculated from our equation (1) hemoglobin E. It differs from all of the previously using the value of R = 2 determined experimentally hy described forms in its electrophoretic behavior. Bloom, et ~ 1 . ~ Its absorption spectrum, solubility and lability to Thus, in rat liver slices, over 90YG of the COZ alkali denaturation are similar to those of normal is derived from glucose via glycolysis, and even adult hemoglobin. A detailed account of this under the special conditions of Bloom, et aZ.,213 work will be published later. about SOY0 of the CO, is formed glycolytically. (3) G. H. Beaven, H. Hoch a n d E . R. Holiday, Biochem. J . , 49, 374

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DEPARTMENT OF PHYSIOLOGY J. KATZ (4) H. A . Itano, Arch. Biochem. B i n p h y s . , 47, 148 (1953). UNIVERSITY OF CALIFORNIA S. ABRAHAM OF CHEMISTRY SCHOOL OF MEDICINE R. HILL GATESAND CRELLINLABORATORIES No. 1889) BERKELEY, CALIFORNIA I. L. CHAIKOFF (COSTRIBUTION INSTITUTE OF TECHNOLOGY CALIFORNIA RECEIVED JANUARY 25, 1954 PASADEXA 4, CALIFORNIA -_ AND THE HARVEY A . ITAKO CHILDREN'S HOSPITAL, OF BIOCHEMISTRY AND OF PEDIATRICS DEPARTMENTS IDENTIFICATION OF A FOURTH ABNORMAL HUMAN USIVERSITY OF SOUTHERN CALIFORXIA IT.R . BERGREN HEMOGLOBIN LOSASGELES,CALIFORNIA PHILLIPSTURGEON Sir: RECEIVED MARCH22, 1951

I n addition to normal adult (A) and fetal (F) hemoglobins three abnormal forms (S, C and D) of human hemoglobin have been described. 1 , 2 We wish to report the identification of a fourth abnormal form in the erythrocytes of a child (M. &with I.)an atypical anemia. Filter paper electrophoresis of hemoglobin from this individual in 0.01 k?sodium barbital, PH 9.2, a t room temperature revealed two components, one with the mobility of hemoglobin F and the other with a mobility very nearly that of hemoglobin C. The same result was obtained by moving boundary II. A . Itano, S c i e n r e , 117,89 (1953). ( 2 ) J. V. Neel, et at., i b i d . , 118, 116 (1853). (1)

REACTION OF BIS-(CYCLOPENTAD1ENYL)-TITANIUM DICHLORIDE WITH ARYLLITHIUM COMPOUNDS'

Sir: Ris-(cyclopentadieny1)-iron was first described? in 1051. Since then, analogous complexes of n number of other transition elements have been ~ r e p a r e d . ~The structure of the iron complex has ( 1 ) This work was carried o i i t under Contract Nonr-R82(00) with t h e Office of Naval Research. ( 2 ) T. J. Kealy a n d P. L. Paiison. .\'aluve, 168, 1039 (19.51). (3) (a) G . Wilkinson, THISJ O U R N A L , 7 4 , 6146, 6148 (1952); 76, 209 (1954); (b) G. Wilkinson, P. L. Pauson, J . hl. Birmingham and F . A. Cotton, i b i d . , 76, 1011 (1953).