Thiamine diphosphate in growing tissues

THIAMINE

DIPHOSPHATE

1. THIAMINE

DIPHOSPHATE JIALIGNANT

K.-H. Institute

IN GROWING

of Zoophysiology,

IN

NORMAL

TISSUES ANI)

TISSITES

KIESSLIKG University

of C’ppsula, Sweden

Received December 15, 1960

‘r111;. \\ell-established fact that thiamine cliphosphatel plays an important role in the metabolism of such a key metabolite as p,vru\-iv acid, means that a great many catabolic and anaholic processes may Ix tlisturhcd by thiamine dcfivicncy. The thiamine content of a large number of normal anti tumor tissues has been studied by M’illiams et trl. [20, 22]. Their data sholv no general cliffcrencc~ in the thiamine content of normal and tumor tissues. ;\Iassayama anti Yokoyama [lx ’ found only minor differences between the thiamine contthnt of’ normal rat liver and hepatoma. Hriggs [l ~, on the other hand, \vhfAn comparing hepatomas induced by butter-yellow \vith normal liver, found a decrease of thiamine greater than 50 per cent. The thiamine level, however, is not necessarily proportional in the tissues to the level of ‘I’DI’. Certainly a low thiamine level may affccbt the synthesis of’ ‘I’l)l’, but can also result from an active thiamino kinasc. Inversely, :t high thiamine level may reflect either an impaired synthesis or an incrcascti tl~~l-‘hosphorvlation of ‘TDP. 13esides, the distribution of ‘I’DI’ within the cells, i.e., lwt\\-een the ‘I‘l)l’dependent reactions in the mitochondria and those in the hesosc monophosl)hatv shunt in the soluble fraction, might be of the samr importance as the total amount of ‘I’l)P in the cell. In the present xvork the content of 7’111’ in dilt’crent 1~onna1 and malignant tissues has het‘n studied, as \\cll as the distribution of ‘TI)I’ bet\vecn thr mitochondrial anti the supernatant fraction in the tlill’crcnt cell types. An attempt has hccn made to correlate the ‘I’l)l’ content of the tissues with thrb a(*tivitics of phosphatases decomposing thiamine phosphntcs. L Abbreviations: TDI’, thiamine diphosphate, TMP, thiamine monophosphate, ‘I‘l)Pasc, mine tliphosphatasc. TMPasr, thiamine monophosphatase, tri\. (‘2-atrlino-‘_‘-hvdro~~r~lethyl-l pi-opandiol).

thia.:3-

312

K.-H.

Kiessling

EXPERIMENTAL

Malignant and normal tissues used.-The tumors used have been spontaneous C3H mammary carcinoma, transplanted methylcholantrene fibrosarcoma and hyperdiploid (ELD), and stable hypertetraploid (ELT) Ehrlich ascites tumors, all from inbred mouse strains (cf. [ll]). Tissue homogenization and preparation of cell fractions.-Disintegration of the tissues was mainly performed in a Potter-Elvehjem homogenizer for two minutes with an interruption of one minute for the prevention of a rise of temperature. Ehrlich ascites cells, however, were very resistant, and required four minutes of homogenization to give separable amounts of mitochondria. The temperature was not allowed to rise above 3°C. With these cells also ultrasonic disintegration with an MSE-Mullard Ultrasonic disintegrator was tried, resulting in a richer yield of mitochondria. All tissues were homogenized in cold 0.25 M sucrose. Besides, skeletal muscle, heart, and the malignant tissues were disintegrated in tris-potassium chloride buffer as described for muscle by Chappell and Perry [3]. The homogenates were centrifuged at 800 g for ten minutes at O-2% in an International refrigerated centrifuge, model PR-2, the precipitate discarded, and the remaining solution recentrifuged at 10,000 g for 20 minutes. The two fractions thus obtained are called the mitochondrial and the supernatant fraction. The latter still contains microsomes and lyzosomes. The mitochondrial fraction was resuspended in the medium, and recentrifuged for 15 minutes as above. In the case of brain mitochondria a somewhat different technique was used, described by Brody and Bain

[21.

Estimation of TDP in homogenates and cell fractions.-The mitochondrial and supernatant fractions, as well as samples taken from the homogenates before centrifugation, were treated according to Westenbrink and Steyn Parve [27], and TDP was estimated as described by them. The mitochondria were always suspended in the same medium as used in the corresponding supernatant. Remaining determinations.-Oxygen consumption was measured at 37°C in Warburg vessels containing 100 mg tissue homogenate in Krebs’ phosphate Ringer solution. Glucose, 0.3 ml of 0.3 M solution and 0.1 ml MgCl, 0.1 IC’Iwere added. The final volume was made up to 2 ml with Ringer solution. The center well contained 0.3 ml 2 M KOH. The activities of phosphatases hydrolyzing TDP to TMP and TMP to thiamine, which are localized mainly in the supernatant fractions, were determined as described earlier [23]. Homogenates from malignant mouse tissues were compared with those from the livers of the same animals. Protein was estimated with the Biuret reaction at 550 rnp according to Cleland and Slater [4]. RESULTS

TDP

and normal tissues.-The contents of TDP in different in the present investigation are shown in Table I together with

in malignant

tissues found Experimental

Cell Research 24

Thiamine &phosphate in growing tissues. 1

3 13

‘There are great differences in thc~ cwmplementary data from the literature. content of ‘I’DI’ between different normal tissues. High TI)I’ levels are found, e.g., in liyer and heart from rat and mouse, and lo~v levels in skeletal muscle. In the tumors the ‘TDI’ content xyas X-25 per cent of that in liver and ahout the same as in skeletal muscle, i.e., the normal tissue from rat ancl mouw with the lowest content of Tl)P known up to no\v. Also the clistrihution of ‘TI)P between mitocl~ondria anti siipernatani differed \videly lx%\-ten different types of tissues (‘I’ahle II). The ratio 1x4\\-wn jig ‘I’l)l’ in supernatant and mitochondria from ont 1 gram \\-et tissue \\-a~ IO\\ in brain, liver, kidney, and skeletal muscle treated in tris-potassium hul’fer, to Ihe sho\ving that a large part of the ‘I’I)P of these cells \vas ~onccnlratccl

The t’igurw

without

A-om7ul

refercnccs

arc those obtained in the present values of at least four experinlenty.

investigation,

and arc n1cai1

/i.SSllP.S

I.ivcr, I,iver, I.ivcr,

muse, adult n~ouse, ,juveniles wt. adult

I Aver, liitinrv I.ivcr, Ilrain. Ilrain, Jluscle, Musclr, Elead, nhsck.

Homo 3 rat Hon~o u10use rat leg of mouse teg of rat rat t i0~~~0

I’ccthologicd

1.0-1.2 X5-8.1 1.X 3.2 2.6-3.9 2.5 0.5 -1.5 X8-7.3 3.0

28. x. 28, 2x. ‘2x 5 2x.

21, 1 I. Iti li 17 21

21. lki. 19

2x 2x, 19 ‘Lx

contlilions

I;atty liver (CCI,), rat I.ivcr from alcholic rat ‘I’hiamillc-tleficient rat liver 1’rlmor.s

1-II.l) and I
7.2-12.2 7.0 1.0

13, ti 10 x

314

K.-H.

Kiessling

mitochondria. For the preparation of muscle mitochondria tris-potassium buffer is preferable to 0.25 M sucrose, as has already been shown by others [3, 71. Also in the case of TDP the medium used seemed to be of significant importance. In tris-potassium buffer (Table II) the ratio was two, whereas in sucrose it was more than seven. The total amount of TDP in muscle, however, was found to be the same, irrespective of the medium used. This indicated a leakage in sucrose of TDP from the mitochondria to the supernatant. Heart muscle from normal rat, and tumors, whether treated in sucrose or in TABLE

II.

The ratio between TDP in the supernatant and the mitochondrial fractions of normal and malignant tissues.

The figures without references are those obtained in the present investigation, and are mean values of at least four experiments. The ratios are based on pug TDP in supernatant and mitochondria from one gram wet tissue. Tissue

Sormal

rat

Pathological

Jlagtignant

2.6 2.7 1.5-1.9 2.0 1.2 1.6 t” 2.0 t” 7.7 sa 8.1 ta 10.1 sa

16 14, 8, 6

conditions

Fatty liver (CCI,). rat Liver from alcoholic rat (old) Thiamine-deficient rat liver

ELD

Reference

tissue

Liver, mouse Liver, rat (old) Liver, rat (young) Kidney, mouse Brain, rat Muscle, leg of mouse Muscle, leg of rat Heart,

Ratio

2.5-3.3 1.8 1.7

14, 6 16 8

tissues

and EL?

Mammary carcinoma Cholantrene sarcoma

5.2 P-Eb 6.1 us” 4.1 4.4

a t, homogenized in tris-potassium buffer; s, prepared in 0.25 M sucrose. b P-E, homogenized in 0.25 M sucrose in a Potter-Elvehjem homogenizer; in 0.25 M sucrose by ultrasonic disintegration. Experimental

Cell Research 24

US, homogenized

Thiamine

&phosphate

in growing

fis.suus. 1

315

t&potassium buffer or disintegrated in a I’otter-~lvel~.i(~m homogenizer or with ultrasonic vibration, showed only a minor part of the T1)P to be situated in the mitochondria. No appreciable dilferences were found between 1:1,1) and EL’I’, either in the total amount of ‘I’DI’ or in the ratio of’ ‘1’1)l’ between the supernatant and the mitochondrial fra&)n.

‘I’.\I~I.I:

III.

7’I)P in mitochondrinl trnd supernrritrnt /krctions mciligncrnt tisrut3 from mouse. The figures represent

T-issue

I .ivel Kidney xusc1c, leg 1X1) and ELT \Iammai-y carcinoma (lholantrene sarcoma

pg TDP/lOO Mitochondria

4.5 5.0 5.0 1.5 1.x 0.9

front normrrl rrntl

mg protcin. supernatant

.1.x S.6 l.li

1..I 1.3 1.0

The high ratio in tumors between the TI)l’ content in the supernatant and that in the mitochondria may depend on a low content of 7’111’ per mitochondrion or on a low number of mitochondria per cell. It is known thal certain tumors may contain less mitochondria than corresponding normal tissues. In Table III the amounts of TDP per 100 mg protein are given for mitochondria and supernatant in tumors and in mouse liver. The figures sho\v that the first of the above alternatives may be predominant. Assuming that the homogenization was equally efficient in the case 01’ tumors and li\rr, a comparison of the amounts of proteins in the mitochondrial fractions indicates that the concentration of mitochondria in mammary carcinoma and in cholantrenc sarcoma is about 40-N per cent of’ that in liver. Thus, also the differences in the content of mitochondria should, although to a 1cssr1 cbstcnt, be responsible for the high ratios in tumors as compared \\-ith most normal tissues (Table II j. Pho.s~~lirrtrrs~s in normrrl crnd nirrlignfrnt lissurs fiydrolyziny fliirxniinr phosphtrtrs---As \vas pointed out in the introduction, a high phosphatasc activit) might be one reason for a low TI)P content in a tissue. ‘I’hc ‘I’I)P content of mouse and rat liver was higher than in any other tissue examind front these animals. The phosphatasrs from tumors, the

K.-H. Kiessling TDP content of which is rather low, has therefore been compared with those from the liver of the same individual. The figures of the phosphatases from tumor and liver are seen in Table IV. TABLE IV. Dephosphorylation of TDP and TMP by phosphatases malignant tissues from mouse.

in liver and

The figures are given as percent ages of TDP and TMP hydrolyzed in 30 min at 30°C and are mean values of five experiments. No significant difference was found between livers from mice bearing different tumors. They are therefore treated as one single group. TMPase

TDPase l-issue

Liver ELD and ELT Mammary carcinoma Cholantrene sarcoma

TABLE V. Oxygen

consumption

PH 7

PH 9

PH 6

PH 7

22 16 14 6

56 35 31 23

37 70 29 It)

18 55 14 11

of liver and tumor homogenates

from mouse.

The figures represent oxygen consumption as mm3130 min/lOO mg wet tissue. Temp. 37°C. ,Medium: Krebs phosphate Ringer solution. Mean values of four experiments. Tissue

Liver ELD and ELT Mammary carcinoma Cholantrene sarcoma

Glucose

112 76 19 25

No substrate

75 61 18 15

No connection seems to exist between the TDP content of a tissue and its activity of phosphatases decomposing TDP and TMP. In tumors with a low TDP content the TDPase and TMPase activities were rather low as compared with liver, with the exception of the TMPase of ELD and ELT. In kidney, on the contrary, with a lower TDP content than liver, the activities of the phosphatases have been shown to be about twice those of liver [13]. Oxygen consumption.-In Table V the oxygen consumption of tumor and mouse liver homogenates is shown. The substrate added only slightly increased the respiration. The oxygen consumption of mammary carcinoma and cholantrene sarcoma was slow compared with that of ascites tumor cells and liver. Experimental

Cell Research 24

Thiamine diphosphate in growing tissues. I

31;

DISCUSSION

‘The distribution of TDP in rat liver fractions has been studied by Goethart [8 , Dianzani and Dianzani Mor [G], and Kiessling and Tilander [l J--l6 , The data agree that about 60 per cent is localized in the soluble fraction, X-40 per cent in the mitochondria, and only traces in the nuclei and microsomcs. In thiamine-deficient animals, the total content of ‘IWP in liver was decreased to 30 per cent, the dec,rease being about the same in the mitochondrial and the soluble fraction [8]. Wright and Scott [29j found that in thiamine deficiency, the rate of oxidation of pyruvate in homogenates of liver, kidney, and heart from rat was lowered, whereas that of a-ketoglutarate was less influenced; they could demonstrate stimulation of the respiration by addition of ‘I’DI’. ‘Thus, normal tissues may reveal specific, metabolic defects which are directly caused by the low content of the cofactor. Great variations in the content of TDP are found among normal tissues (Table I). In the three types of tumors investigated the TDl- level was 01 about the same. Although this level is rather low, there are normal tissues \vith about the same ‘TDE content, as, for example, skeletal muscle. In this case, how-ever, the distribution of TDP within the cells is such that a proportionately high percentage of the total amount is situated in the mitochondria (‘l’ablc 1I). In heart muscle the distribution is unfavourable for the mitochontlria, but the total amount of TDP, on the other hand, is rather high. In the thrrr lyprs of tumors investigated the TDP is mainly recovered from the supernatant fraction. This fact, together with the low total content of ‘1‘111’ Found in these tissues, seems to mean that only a minor amount of the total TDI) of these cells is localized in the mitochondria. \\‘arburg’s investigations [24, 2.51 have revealed a high aerobic and anaerobic glycolysis, and a formation of relatively large quantities of lactate, as characteristics of cancer cells. In the light of this abnormal metabolism the IO\\- mitochondrial ‘1’1)1-’ content of the tumors investigated may be ot certain interest. In compilations by Greenstein [Y, lo], a comprehensive revie\v is given of’ concentrations and activities of the components and cnzyrncs involved in thr oxidation of pyruvate. From a qualitative point of vie\v it is evident that tumors contain all the knolvn enzymes involved in the Krebs cycle. Quantitatively there are \vide ranges in the activities of these enzymes in various normal tissues. The activities of these enzymes in tumors generally approach Ihcl lo\vrst normal values, the tumors showing in this respect a uniform be-

K.-H. Kiessling havior. Wenner and Weinhouse [26] and Kielley [ 121, however, she\\-ctl that the oxidation of pyruvate and of the intermediates of the Krehs cycle 1,~ tumor homogenates can be enhanced by addition of DPN. Moreover, Ernster et al. [7] give evidence of a rapid oxidation of pyruvate in rat muscle mitochondria, a normal tissue with perhaps the lowest mitochondrial TDP content of those investigated, although not as low as in the tumors. Taking these findings into consideration the low TDP content of tumors may be of no significant importance in the attempt to explain their abnormal metabolism. SUMMARY A study of the thiamine diphosphate content and its distribution within the cells of normal and malignant tissues is reported and a comparison is made between the activities of phosphatases decomposing thiamine phosphates. Quantitatively there are wide ranges in the thiamine diphosphate content of various normal tissues. In the tumors examined the amounts are rather small, and are comparable with the lowest normal values. In normal tissues about 30 per cent of the thiamine diphosphate occurs in the mitochondrial fraction and the rest mainly in the supernatant fraction. Only in heart tissue a very low thiamine diphosphate content of the mitochondria, as compared with the supernatant, was found. Also in tumors the main part of thiamine diphosphate was refound in the supernatant fraction. This fact, combined with the low total content of thiamine diphosphate in tumors, means that tumors, as compared with normal tissues, have an extremely low mitochondrial level of thiamine diphosphate. The significance of this phenomenon is discussed. So correlation between thiamine diphosphate content and activity of phosphatases decomposing thiamine phosphates was found. This work is part of investigations supported by the Swedish Natural Science Research Council. I am very much indebted to Prof. G. Klein and Dr. K. E. HellstrGm, Karolinska Institutet, Stockholm, for supplying the mice bearing mammary carcinoma and cholantrene-induced sarcoma, to Prof. P. E. Lindahl, Institute of Zoophysiology, Uppsala, for making available the ascites tumor mice and to Miss Margareta Schiiild for technical assistance. REFERENCES 1. BRIGGS, M. H., Nature 187, 249 (1960). 2. BRODY, T. M. and BAIN, ,J. A., J. Bid. Chem. 195, 685 (1952). 3. CHAPPELL, J. B. and PERRY, S. V., Nature 173, 1094 (1954).

Experimental

Cell Research 24

Thiamine diphosphate in growing tissues. 1

3 19

4. CLELANU, I<. W. and SLATER, E. C., Biochem. J. 53, 547 (1953). G., Experientia 13, 165 (1957). 5. & CARO, L., RISDI, G., PERRI, V. and FERRARI, 6. L)I~\SZASI, ikl. I’. and DIANZANI MOR, M. A., Biochim. et Hiophys. Acfu 24, 564 (1957). 7. ERSSTEK, I,., IKK~S, D. and LUFT, R., Nature 184, 1851 (1959). X. GOETHART, G., Jjiocftim. et Biophys. Acta 8, 479 (1952). 9. GHEIENS.FISIN,.I. I’., Cancer Research 16, 641 (1956). 10. -.~Biochemistry of Cancer, 2nd ed. Academic Press, Sew York, 19.54. 11. H\r.scrr~,\, T. S., GRtNNEL, S. T., RBvBsz, L. and KLEIN. (+., .J. Strff. (Zuncer Inst. 19. 1X

(1957). IL I;., L’trncer Reseurch 12, 124 (1952). 13. I
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17. 18. 19. 20.

--- --~ L!npublishrd results. MusAY,~~, T. and YOKOY~SI~, T., (iann 33 (1939). OCHOA, S. and PETERS, R. A., Biochem. J. 32, 1501 (1938). POLLACK, RI. A., TAYLOR, A. and WILLIAMS, R. J., Cniu. Texas Pub. No. 4237, 56 (1942). 21. HINDI, G. and t)E GIUSEPPE, L., Experienfia 16, 447 (1960). 22. Thy~on, A., POI.LACK, M. A. and %‘ILLIAMS, R. J., Univ. II’excrs I’uhl. No. 4237, 41 (1932). 23. TtL.\St>ER, K. and KIESSLINU, K.-H., Acfa Chem. &and. In press. 24. \V:\RBYR(;, O., The Metabolism of Tumors. London, 1930.

2.5.

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26. 27. 28.

\VXSNER, C. I:. and \I\;EINH~USE, S., Cancer Research 13, 21 (1953). \\7wt%XBRtNK. H. G. K. and STEW-PARVE, E. P., Inf. Reu. Vitamin Research 21, 461 (1950). WESTENBRISK, H. G. K.. STEYN-P,\Rw%, E: P. and THOX~SS~S, H. .J., %. l’ifamirtfors~h. 13.

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\Ytwtm,

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Experimental

(lrlf Rcseurch 24