Cancer Letters, 72 (1993) 11-16

Elsevier Scientific Publisllers Ireland Ltd.

71

Tumour necrosis factor-a alters the blood compartmentation of amino acids in the rat

Marta Llovera, Francisco J. Lbpez-Soriano and Josep M. Argilks

Departament de Bioquimira i Fisiologia. Universitat de Barcelona. Barcelona (Spain)

(Received 30 March 1993)

(Revision received 12 May 1993)

(Accepted 14 May 1993)

Summary

The work presented focuses on the importance of studying the distribution of blood amino acids in both the cellular and plasmatic fraction when performing studies concerning the effects of cytokines on amino acid metabolism. Tumour necrosis factor treatment resulted in important changes in blood amino acid compartmentation between plasma and red blood cells in rats. The animals showed a change in compartmentation with an increase in the concentration of most amino acids in the cellular fraction with the excep- tion of phenylalanine, glutamate, aspartate and tyrosine.

Keywords: tumour necrosis factor-o; blood; amino acids; rats

Introduction

Red blood cells have been recognized as having an important role in the interorganic transport of several metabolites [ 1,2] including amino acids [3,4]. Although plasma amino acids are generally used as an index of the amino acid-transporting

Correspondence to: Josep M. Argilis, Unitat de Bioquimica i

Biologia Molecular B, Dcpartament de Bioquimica i Fisiologia,

Facultat de Biologia, Universitat de Barcelona, 08071-

Barcelona, Spain.

ability of blood, the erythrocyte content is not always dependent on the blood levels [5]. On the other hand, the quantitative importance of red blood cells in amino acid transport has not thus far being completely established. In addition, blood amino acid compartmentation may depend on the physiological conditions of the animal and the measurements of plasma levels underestimate the actual blood capacity for amino acid transport.

Tumour necrosis factor-o (TNF) is a molecule belonging to a polypeptide network made up of several cytokines and growth factors that have wide and varied effects on the growth, differentia- tion, and functions of immune system and normal cells [6,7]. It is produced primarily by activated macrophages in response to invasive stimuli. The main observations relating TNF to amino acid me- tabolism indicate that the cytokine stimulates hepatic amino acid uptake in vivo [g-10] and that it promotes amino acid release from skeletal mus- cle, these two observations are possibly related to the increased protein synthesis in liver and enhanc- ed protein degradation in muscle following the ad- ministration of the cytokine. Although there are some reports concerning changes in blood amino acid concentrations after TNF treatment [lo], not a single study has considered the effects of the cytokine on the cellular and plasmatic distribution of these compounds.

For the above reasons, it was our aim to esti- mate blood amino acid content and distribution in the plasmatic and cellular compartments in rats treated with recombinant TNF.

0304-3835/93/$06.00 0 1993 Elsevier Scientific Publishers Ireland Ltd

Printed and Published in Ireland

Experimental Procedures

Animals Female Wistar rats from our own colony at the

Faculty of Biology, University of Barcelona weighing loo-150 g were used. They were housed in animal quarters with a daily photoperiod of 12 h light between 08:OO and 20:00 h and were in- dividually caged in polypropylene cages, maintain- ed at 22 f 2°C and fed standard laboratory chow (Panlab, S.A., Barcelona).

Biochemicals They were all reagent grade and obtained either

from Boehringer Mannheim, S.A. (Barcelona, Spain) or from Sigma Chemical Co. (St. Louis, U.S.A). Recombinant-derived TNF was generous-

60

50

40

30

20

ly given by BASF/Knoll, A.G.. Ludwigshafen, Germany.

Cytokine administration TNF was given intraperitoneally for 8 days at a

dose of 100 pglkg per day (two administrations at 08:OO and 20:00 h). Control animals received 0.5 ml of vehicle (physiological saline).

Amino acid analysis Following pentobarbital anaesthesia (60 mg/kg),

blood samples were obtained from the aorta, using heparinized syringes on day 0, 2 and 4 after cytokine treatment. A blood aliquot was sonicated to attain cellular lysis. In another aliquot, plasma was separated by means of a refrigerated cen- trifuge. Aliquots of plasma and sonicated blood

0 Control

0 TNF-a

0 2 4 6 Days after treatment

a

Fig. 1. Hematocrit values in TNF-treated rats. The results are mean values f S.E.M. for live different animals. Statistical slgnifi-

cance of the results: *P < 0.05, **P < 0.01, ***P < 0.001

were deproteinized with 10% (w/v) trifluoroacetic acid and after centrifugation the clear supernatant was used for amino iacid determination by means of an amino acid ana.lyzer equipped with an spec- trophotometric detector and using ninhydrine as a reagent. Norleucine was used as an internal stan- dard. Blood amino acid compartmentation was calculated correcting plasma and blood levels by hematocrit values [5] as E = [WP-(1-H) x PI/H; free amino acid concentrations in erythrocytes (E); free amino acid concentrations in whole blood (WP); free amino acid concentrations in plasma (P); hematocrit (H) values are expressed as a frac- tion 11 I]. Statistical differences were calculated using the Student’s I-test.

Results and Discussion

The loss of body weight and development of cachexia are common signs associated with neo- plastic diseases. Cachexia is a poorly understood syndrome characterized by anorexia, weight loss,

73

profound metabolic abnormalities and progressive host wasting which may result in death [12-141. Tissue wasting involves mainly adipose tissue and skeletal muscle. Numerous reports attribute the cachectic state of the host to cytokines released as a result of invasive stimuli; see [15] for review. However, while the role of cytokines, particularly, tumour necrosis factor-a (TNF), on lipid meta- bolism seems to be clear [ 16- 181, the effects of the cytokine on amino acid metabolism lead to more confusing results; see [19] for review, In addition, very few data concerning how cytokines alter the circulating amino acid concentrations have been reported. It was for this reasons that we were in- terested in assessing how a prolonged TNF treat- ment could affect blood amino acid levels and compartmentation.

In vitro experiments have shown that the equi- libration ratio between blood fractions is very slow [20] having postulated that there is no rapid amino acid exchange. However, despite these low in vitro rates of interchange between both blood pools,

Table I. Whole blood amino acid concentration and plasma/cell ratios in TNF-treated rats.

Amino acid Control TNF (2) TNF (4) - Total P/C Total PIG Total P/C

Val 142 zt 9 1.21 164 f I4 1.19 163 f I4 0.61

Ile 64*4 1.19 90 f 12* 0.45 II f II 0.79

Leu 129 f 6 0.70 148 f IO 0.49 145 f 25 0.42

Met 27 zt 2 1.91 46 f 19 0.34 42 f 6** 0.76

cilu 356 zt 25 0.24 468 f 43* 0.22 330 f 24 0.20

Gln 430 f 26 21.2 256 zt 9** <O.lO 1278 f 89*** 0.59

Asp 34 f 2 0.26 47 f I1 0.21 68 f 19** 0.22

Asn 82 iz 7 1.04 107 f 6 0.77 150 f II*** 0.38

Pro 207 zt I8 1.73 319 f 32** 0.62 247 f 64 1.09

Ala 515 l 39 1.12 701 f 19* 0.72 544 f 46 0.82

GlY 320 f 23 0.80 477 f 30** 0.46 446 zt 44* 0.60

Ser 328 zt 16 0.76 345 f 24 0.53 321 *6 0.59

Thr 336 f 24 1.20 395 f 35 I.18 251 + 13 0.74

Tyr 61 zt 8 0.67 75 l 12 0.68 67 f 7 0.75

Phe 18 f 15 0.38 79 l 5 0.31 117 f I6 0.31

His 52 l 3 1.32 70 i 5* 0.59 73 f 6*+ 0.52

LYS 481 + 30 0.86 606 f 61 0.47 463 zrz 23 0.65

Arg 213 f 16 0.55 285 f 43 0.32 226 f 33 1 .oo

Om 50 f 4 1.13 73 f 4** 0.64 52 f 6 1.12

The results are mean values f S.E.M. for five different animals. P/C. plasma/cell ratios. Amino acid concentrations are given in nmoles/ml. Statistical significance of the results: *P < 0.05, **P < 0.01, ***P < 0.001. TNF (2). 2 days after treatment; TNF (4).

4 days after treatment.

f u ii

considerable changes are found in blood amino acid compartmentation in mammals under dif- ferent physiological c~oncentrations. In addition, it was demonstrated, however, that in vivo the equi- libration between blood fractions occurs very rapidly (2 I].

TNF treatment resulted in important changes in hematocrit values at ‘days 2 and 4 after treatment (Fig. 1). It was precisely for this reason that the amino acid compartmentation was studied at these time-points. The hematocrit changes reported here agree with the worlk [22], who found a 40% decrease in total red b’lood count following admin- istration of sublethal TNF doses to rats. The decrease in red blood. cells seems to be related to reduced synthesis and a decreased half-life of the erythrocytes [23]. Table I shows the whole blood amino acid concentration found in control and TNF-treated rats. Two days of cytokine treatment resulted in increases in the concentration of isoleucine (41%), hiaidine (35%), alanine (36%). proline (54%), glycine (49%) and ornithine (46%) and decreases in the concentration of glutamine (40%) (Table I). After four days of treatment, the concentrations of methionine (36%), histidine (40%), glutamine (197%), aspartate (100%) and asparagine (83%) were significantiy elevated versus the control animals. The blood amino acid com- partmentation is shown in Fig. 2. In the control animals, most of the amino acids are more concen- trated in the plasmatic compartment with the exceptions of leucine, phenylalanine, lysine, gluta- mate, aspartate, glycine, serine, tyrosine and argin- ine. TNF treatment promoted an increase in the concentration of total amino acids in both the cellular and plasmatic compartments. The total concentrations of amino acids (including, those presented in Table I, phosphoserine, taurine and citrulline) in each fraction were 4579 + 634 (con- trols), 6704 + 1186 (TNF-2 days), 7829 f 1014 * (TNF-4 days) for the cellular fraction and 3484 f 171 (control,s), 4208 f 134 * (TNF-2 days), 42 19 + 6 13 (TNF-4 days) for the plasmatic fraction. Table I also shows the plasma/cell ratio for the different amino acids studied. TNF-treated animals showed decreased ratios for most amino acids with the exceptions of phenylalanine, tyrosine, aspartate and glutamate (Table I).

Although erythrocyte amino acid levels as com- pared to plasma levels have rarely been used in metabolic disorders in patients [24], the present study is the first one to consider blood amino acid compartmentation in experimental models with high circulating cytokine levels, an interesting approach that may lead to future promising research in amino acid in different pathological situations such as sepsis, trauma, tumour growth.

Acknowledgements

This work was supported by grants from the Fondo de Investigaciones Sanitarias de la Segur- idad Social (F.I.S.) (90/663) of the Spanish Health Ministry and from the DGICYT (PB90-0497) from the Spanish Ministry of Education and Science. M.L. is recipient from a pre-doctoral scholarship from the Generalitat de Catalunya. We are very grateful to BASF/Knoll A.G. (Lud- wigshafen, Germany) for the generous supply of recombinant-derived TNF.

References

Prats, E., Monfar, M., Argiles. J.M. and Alemany. M.

(1987) Effects of starvation and a high energy diet on rat

blood compartmentation of injected radioactive alanine

and glucose. Biochem. Int., 14, 95-101.

Watts, R.P., Brendel, K., Luthra. M.G. and Kim, H.D.

(1979) Inosine from liver as a possible energy source for

pig red blood cells. Life Sci., 25, 1577-1582.

Felig, P., Wahren, J. and Raf, L. (1973) Evidence of inter-

organ amino acid transport by blood cells in humans.

Proc. Natl. Acad. Sci. USA, 70, 1775-1779.

Haitman, R.N. and Bergman, E.N. (1980) Transport of

amino acids in whole blood and plasma of sheep. Am. J.

Physiol., 239, 242-247.

Hagenfeldt, L. and Arvidsson. A. (1980) The distribution

of amino acids between plasma and erythrocytes. Clin.

Chim. Acta., 100, 133-141.

Beutler. B. and Cerami, A. (1986) Cachectin and tumor

necrosis factor as two sides of the same biological coin.

Nature, 320, 584-588.

Old, L.J. (1987) Polypeptide mediated network. Nature,

326, 330-33 I.

Roh, MS., Moldawer. L.L., Elkman. L.C., Dinarello.

C.A., Bistrian, B.R., Jeevanandam, M. and Brennan. M.F. (1986) Stimulatory effect of interleukin-I upon

hepatic metabolism. Metabolism, 35. 419-424.

Warren. W.R., Starnes, F.H. Jr., Alcock, N.. Calvano, S.

and Brennan. M.F. (1988) Hormonal and metabolic re-

10

11

12

13

14

15

16

17

18

sponse to recombinant human tumor necrosis factor in

rat: in vitro and in vivo. Am. J. Physiol., 255, E206-E212.

Argilts, J.M.. Lopez-Soriano, F.J., Wiggins, D. and

Williamson, D.H. (1989) Comparative effects of tumour

necrosis factor-o (cachectin), interleukin 1-p and tumour

growth on amino acid metabolism in the rat. Biochem. J.,

261, 355-362

Rivera, S., Lopez-Soriano, F.J., Azcon-Bieto, J. and

Argilts, J.M. (1987) Blood amino acid compartmentation

in mice bearing the Lewis lung carcinoma. Cancer Res.,

41, 5644-5646.

Strain, A.J. (1979) Cancer cachexia in man: a review. In-

vest. Cell. Pathol., 2, 181-193.

Theologides, A. (1979) Cancer cachexia. Cancer, 43.

2004-2012.

Lawson, D.H., Richmond, A., Nixon, D.W. and Rud-

man, D. (1982) Metabolic approaches to cancer cachexia.

Annu. Rev. Nutr., 2, 227-301.

Evans, R.D., Argiles, J.M. and Williamson, D.H. (1989)

Metabolic effects of tumour necrosis factor-a (cachectin)

and interleukin-I. Clin. Sci., 77, 357-364.

Price, S.R.. Olivecrona, T. and Pekala, P.H. (1986) Regu-

lation of lipoprotein lipase synthesis by recombinant

tumor necrosis factor - the primary regulatory role of the

hormone in 3T3-LI adipocytes. Arch. Biochem. Biophys,

251, 738-746.

Semb, H., Peterson, J., Tavernier, J. and Olivecrona. T.

(1987) Multiple effects of tumor necrosis factor on lipo-

protein lipase in vivo. J. Biol. Chem., 262, 8390-8394.

Evans, R.D. and Williamson, D.H. (1988) Tumour

necrosis factor-o (cachecin) mimics some of the effects of

tumour growth on the disposal of a [‘4C]lipid load in

virgin, lactating and litter-removed rats. Biochem. J., 256,

1055-1058.

I9 Argiles, J.M., Garcia-Martinez, C., Llovera, M. and

Lopez-Soriano, F.J. (1992) The role of cytokines in mus-

cle wasting: its relation with cancer cachexia. Med. Res.

Rev., 12, 637-652.

20 Elwyn, D.H. (1972) Distribution of amino acids between

the plasma and red blood cells in the dog. Fed. Proc.. 25,

8S4-861.

21 Elwyn, D.H., Launder, W.J., Parikh, H.C. and Wyse,

E.M. (1972) Role of plasma and erythrocytes in inter-

organ transport of amino acids in dogs. Am. J. Physiol.,

222, 1333-1342.

22 Tracey, K.J., Wei, H., Manogue. K.R.. Fong. Y ., Hesse,

D.C., Nguyen, H.T.. Kuo, G.C., Beutler. B., Cotran.

R.S., Cerami, A. and Lowry, S.F. (1988) Cachectinitumor

necrosis factor induces cachexia, anemia and inflamma-

tion. J. Exp. Med., 167, 1211-1227.

23 Moldawer. L.L., Marano, M.A., Wei. H.. Fong, Y., Silen.

M.L., Kuo, G., Manogue, K.R., Vlassara, H., Cohen, H..

Cerami, A. and Lowry, S.F. (1989) Cachectin/tumor

necrosis factor-o alters red blood cell kinetics and induces

anemia in vivo. FASEB J., 3, 1637-1643.

24 Levy, H.L. and Barkin, E. (1971) Comparison of amino

acid concentrations between the plasma and erythrocytes.

Studies in normal subjects and those with metabolic dis-

orders. J. Lab. Clin. Med., 78, 517-523.