Pathways of glucose metabolism in human placental tissue

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leukemia L-i2IO. Utilization of intermediates of the citric acid cycle and of glutamate was essentially the same in both cases. In contrast, the rate of oxidation of ~-glycerophosphate by the leukemic spleen was less than 5o % that of normal. A similar decrease in the mitochondriai oxidation of ~-glycerophosphate was found in spleen infiltrated with leukemia B82T. Mitochondria from the solid B82T tumor also had low levels of ~-glycerophosphate oxidase activity; the rate was only one-tenth that for succinate. These findings indicate that the mitochondrial oxidation of ~-glycerophosphate, as well as the formation of ~-glycerophosphate by the cytoplasmic dehydrogenase, is reduced in leukemic tissues and further support the view that the ~-glycerophosphate shuttle system is inoperative in these malignant cells.

Experimental Zoology Branch, Physiology Division, Direaorate of Medical Research, U.S. Army Chemical Research and Development Laboratories, Edgewood Arsenal, Md.

BERTRAM SACKTOR

1 B. SACKTOR AND A. lc{. DICK, Cancer Res., 2o (196o) 14o8. I TH. Bf2CHER AND M. KLINGENBERG, A~gew. Chem., 7 ° (I958) 552.

a R. 4 B. 5 p. 6 p. A. s G.

W. ESTABROOK AND B. SACKTOR, J. Biol. Chem., 233 (1958) IOI4. SACKTOR AND A. R. DICK, J. Biol. Chem., 237 (1962) 3259. BOEST, Bloc.him. Biophys. Aaa, 57 (1962) 27o. HOLZER, P. G'LOGNER AND G. SEDLMAYR, Biochem. Z., 330 (1958) 59. DELER0CK, H. SCHIMASSEK, K. BARTSCH AND TH. B0CHER, Bioc.hem. Z., 331 (1959) 297. E. BOXER AND C. E. SHONK, Canc.er Res., 2o (196o) 85.

9 E. I. CIACClO, D. L. KELLER AND G. E. BOXER, Bioc.him. Biophys. Ac.ta, 37 (196o) 191.

x0 p. EMMELOT AND C. J. B o s , Bloc.him. Biophys. Aaa, 59 (I962) 495. 11 C. E. WENNER AND R. CEREIJO-SANTALO, J. Biol. Chem., 238 (I963) 1584. 1~ W . C. SCaNEIVER, J. Biol. Chem., 176 (1948) 259. 13 B. HAGIHARA, Bloc.him. Biophys. Ac.ta, 46 (1961) 134. 14 D. E. GREEN, Comp. Bloc.hem. Physiol., 4 (1962) 81. 15 B. CHANCE, ill S. P. COLOWICK AND N. O. KAPLAN, Methods in En,ymology, Vol. 4, A c a d e m i c

Press, N e w York, 1957, P. 273.

lB B. SACKTOR AND L. PACKER, J. Neuroc.hem., 9 (1962) 371. 17 B. CHANCE AND G. l~. WILLIAMS, J . Biol. Chem., 217 (1955) 295. 18 E. MAXWELL AND G. ASHWELL, Arc.h. Bloc.hem. Biophys., 43 (1953) 389.

19 D. W. VAN BEKKUM, Bloc.him. Biophvs. Ac.ta, 16 (1955) 437. so T. L. BENJAMIN AND H. T. YOST, JR., Radiation Res., 12 (196o) 6I 3. 21 t{. W. ESTABROOK AND B. SACKTOR, Arch. Bloc, hem. Biophys., 76 (1958) 532. 13 M. KLINGENBERG AND TH. B0CHER, Biochem. Z., 334 (1961) 1.

Received March 24th, 1964

Bioc.him. Biophys. Ac.ta, 9o (1964) 163-166

sc 23029

Pathways of glucose metabolism in human placental tissue Studies of the relative importance of the glycolytic and hexose-monophosphate pathways for glucose degradation in human foetal tissues have shown that, early in gestatiop, each tissue has established its own characteristic pattern of carbohydrate metabolism resembling that of the corresponding adult tissue and which remains unchanged during the first 25 weeks of gestation1. There is however no comparable information on the routes of glucose metabolism in the placenta. The relative utilisation of the glycolytic and direct oxidative pathways for glucose metabolism was therefore investigated in the human placenta at different stages of gestation. Bloc.him. Biophys. Ac.ta, 90 (1964) 166-168

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pathway to placental glucose metabolism in the first trimester of pregnancy. However the stage in gestation when the direct oxidative pathway is no longer measurable and the factors, whether of maternal or foetal origin, which determine this altered placental metabolic activity, must be of profound importance in foetal development. This research was supported by the Dan Mason Foundation and the Clinical Research Fund of Chafing Cross Hospital.

Department of Obstetrics, Chafing Cross Hospital Medical School, London (Great Britain)

J E A N GINSBURG MARJORIE K . JEACOCK

z C. A. "VILLEE AND J. M. LORING, Biochem. J., 8I (1961) 488. l B. BLOOM AND D. STETTEN, J. Am. Chem. Sot., 75 (1953) 5446. 8 p. BEACONSFIELD, Expevientia, 19 (1963) 437. 4 p. BEACONSFIELD AND A. CARPI, Nature, 2oi (1964) 825. 5 C. A. VILLEE, Biochem. J., 83 (1962) 191. 6 G. E. GLOCK AND P. McLEAN, Biochem. J., 56 (1954) 171. ? M. K. JEACOCK, N. F. MORRIS AND J. A. PLESTER, J. Obstet. Gynaecol. Brit. Commonwealth, 7 ° (1963) 267. 8 p. BEACONSFIELD AND A. LIUZZI, Life Sciences. 7 (1963) 459. 9 R. V. COXON AND R. J. ROBINSON, Proc. Roy. Soc. London, Set. B, 145 (1956) 232. 10 j . KATZ AND H. G. WOOD, J. Biol. Chem., 235 (196o) 2165. 11 j . KATZ AND H. G. WOOD, J. Biol. Chem., 238 (I963) 517 •

Received April I6th, 1964 Biochim. Biophys. Acta, 9o (1964) 166-168

sc 23 o17

Multiple-reflectance filter-paper spectrophotometry of microvolumes of turbid cytochrome preparations Since the pioneering efforts of KEILIN AND HARTREEz, 2, determination of cytochrome spectra in turbid systems has presented difficulties. CHANCE8, LUNDEG,~RDH 4, DUYSENS s, FRENCHe and SHIBATA~,8 have contributed notably to our ability to understand, and cope with, these difficulties. In this report we present a simple method for obtaining excellent absorption spectra from microvolumes of turbid cytochrome preparations. Its developement stems especially from the work of CHANCE8 and LUNDEGARDH4. We have found (initially by accident) that ordinary laboratory filterpaper can be used instead of glass or silica cuvettes, to carry as little as 15 ~1 of turbid cytochrome-containing preparations in a spectrophotometer light beam, and yet yield sharp and clear absorption curves. The phenomenon of multiple reflectance, as discussed by SHIBATA ?, appears predominantly responsible for the effectiveness of the method, and for this reason we have called it "multiple-reflectance filter-paper spectrophotometry". Methods, Instruments: The Cary Model 14, the Beckman Models DK2 and DU, and the Process and Instruments Co., Model RS3, spectrophotometers were used. On the latter instrument, a 1.5 V pen-light battery operated voltage-bucking device was an indispensable aid (on per cent absorption outputs) in shifting recorder-pen zero-position full scale in either direction, to accommodate variations in base-line absorbancies of different preparations. Filter paper: Schleicher and Schfill papers, No. 47o, No. 576 and No. 598 were Biochim. Biophys. Acla, 90 (I964) 168-17o

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I~7

H a c e n t a e were o b t a i n e d a t o p e r a t i o n or after s p o n t a n e o u s v a g i n a l d e l i v e r y a n d cere i n c u b a t e d w i t h i n half an h o u r of r e m o v a l from t h e p a t i e n t . The a m o u n t s of tC02 p r o d u c e d r e s p e c t i v e l y from [I-l*C]glucose a n d [5-1*C]glucose were d e t e r m i n e d .ccording to t h e m e t h o d of BLOOM AND STETTEN 2. T h e results are shown in Table I. The C-I/C-6 ratio was t a k e n as a n i n d i c a t i o n of he relative utilisation of the g l y c o l y t i c a n d h e x o s e - m o n o p h o s p h a t e p a t h w a y s . R a t i o s ,Teater t h a n one were t a k e n t o indicate the utilisation of b o t h p a t h w a y s ; a r a t i o of one mplied t h a t t h e E m b d e n - M e y e r h o f p a t h w a y was the o n l y one m e a s u r a b l e . TABLE I PRODUCTION OF 14COlt FROM [I-14C]GLUCOSE AND [6-14C]GLUCOS]~ IN HUMAN PLACENTAL TISSUE

;onditions of incubation: tissue slices were incubated in Krebs--Ringer bicarbonate buffer conaining o.x ~/o glucose for 90 rain at 37 ° in 95 ~o oxygen and 5 % COs. The radioactive material w a s diluted with inert substrate to give a specific activity of about 2~C[mg. Mean liCOt produaion

Placenta

Stage of

Mode of

in countalminlzoo rag

C-zlC-6

number

gesto21on in reeel'.s

delivery

wet wt. tissue

Ratio

6 8 xo Io II 12 12 13 14 20 Term 37-4 ° Term 37-4 ° Term 37-4 o Term37-4 o

D. & C. D. & C. H. H. H. D. & C. H. H. H. H. S.V.D. S.V.D. S.V.D. C.S.

I 2 3 4 5 6 7 8 9 IO 11 12 13 14

[ z.t qYJ Gluoose

[ 6-t~ ]Cdat,ose

2128 3368 4376 5o l l 2764 4600 4347 565 4875 3971 8o 7 531 IO47 803

637 873 911 lO73 611 948 I°o4 81

981

13oo 9oi 640 981 793

3.3 3.9 4.8 4.7 4.5 4.9 4-3 7.0 5.O 3.I o.9 0.8 1.I i.o

). & C., dilatation and curretage; H., hysterotomy; S.V.D., spontaneous vaginal delivery; C.S., ;aesarian section. The relative utilisation in p l a c e n t a l tissue of t h e direct o x i d a t i v e p a t h w a y for ~lucose d e g r a d a t i o n t h u s v a r i e d w i t h t h e stage of gestation. The g r e a t e r utilisation of :he p e n t o s e - p h o s p h a t e p a t h w a y in t h e e a r l y weeks of g e s t a t i o n t h a n a t t e r m parallels ~lacental growth, a n d it is r e l e v a n t t h a t BEACONSFIELD8,4 has c o r r e l a t e d increased t c t i v i t y of t h e p e n t o s e s h u n t w i t h i n j u r y a n d repair. The a p p a r e n t absence of the p e n t o s e - p h o s p h a t e p a t h w a y a t t e r m corresponds to :he low levels of N A D P (ref. 5) a n d glucose-6-phosphate d e h y d r o g e n a s e (D-glucose-6~hosphate: N A D P oxidoreductase, EC 1.1.1.49 ) (ref. 6) f o u n d in p l a c e n t a l tissue. ~Iowever, levels of co-enzyme a n d e n z y m e a c t i v i t y do n o t necessarily reflect the p o :ential a c t i v i t y of a p a r t i c u l a r p a t h w a y *-8 a n d i n d e e d t h e a d d i t i o n of m e t h y l e n e blue, m electron acceptor, to t h e i n c u b a t i o n m e d i u m resulted in a small b u t m e a s u r a b l e ltilisation of the pentose p a t h w a y in t e r m p l a c e n t a l tissue. I n view of t h e difficulty of d e t e r m i n i n g the q u a n t i t a t i v e i m p o r t a n c e of the phos~hogluconate p a t h w a y from t h e 14C02 yields from [I-14C]glucose a n d [6-1~C]glucose [ref. 9-11) it has n o t y e t been possible t o e v a l u a t e the precise c o n t r i b u t i o n of e i t h e r Biochim. Biophys. Acta,

90 (I964) 166-168