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Nuclear Magnetic Resonance Analysis of Tumor Necrosis Factor-induced Alterations of Phospholipid Metabolites and pH in Friend Leukemia Cell Tumors and Fibrosarcomas in Mice¹

Laboratory of Cell Biology [F. P., G. C., M. D. V., M. G.] and Virology [E. P., F. B.], Istituto Superiore di Sanita, Viale Regina Elena, 299, 00161 Rome, Italy; Laboratory of Molecular Biology, State University of Ghent, Ledeganckstraat 35, B-9000 Ghent, Belgium [W.F.]; and Institut de Recherches Scientifiques sur le Cancer, BP 8 94802 Villejuif Cedex, France [I.G.]

The alterations induced on the pool sizes of five phospholipid metabolites, glycerol 3-phosphorylcholine, glycerol 3-phosphorylethanolamine, phosphorylcholine, sn-glycerol 3-phosphate, and choline were studied by nuclear magnetic resonance (NMR) spectroscopy in murine tumors injected with recombinant murine tumor necrosis factor (TNF).

Solid tumors were obtained by s.c. injection of either Friend leukemia cells (clones 3C1-8 and 745) in DBA/2 mice or murine fibrosarcoma cells (HeN4) in C3H/HeN mice. After tumor nodules had developed, TNF or bovine serum albumin was injected intratumorally. Treatment of both tumors with TNF resulted in a marked inhibition of tumor growth.

³¹P-NMR analyses of Friend leukemia cell tumors (and tissue extracts), 6 h after injection of TNF, showed: (a) a 1.5- to 3.5-fold decrease in the pool sizes of glycerol 3-phosphorylcholine and glycerol 3-phosphorylethanolamine; (b) a 7- to 8-fold increase of sn-glycerol 3-phosphate; (c) a 2- to 3.5-fold decrease of phosphorylcholine; (d) an alkaline shift (0.2 units) in intratumoral pH. Similar metabolic alterations occurred in TNF-treated HeN4 fibrosarcoma. ¹H-NMR analyses of Friend leukemia cell tumor extracts also indicated, 6 h after tumor injection with TNF: (a) elevated choline levels (9x); (b) a 19-fold increase in the ratio [choline]/[phosporylcholine]; (c) elevated (1.4x) levels of lactic acid; and (d) a 1.6-fold decrease in the [taurine]/[glycine] ratio.

The results are interpreted in the light of possible alterations in the activity of enzymes controlling the de novo biosynthesis and catabolism of phospholipids.

We conclude that NMR spectroscopy can be a useful means to monitor the level of some phospholipid precursors and/or derivatives as early markers of therapeutic efficacy in intact neoplastic tissues.

¹ Supported in part by CNR Special Grants on Oncology (Grants 85.02560.44 and 85.02025.44), Biomedical and Clinical Engineering (Grant 85.01516.57), and "Controllo delle Malattie da Infezione" (Grant 85.02797.52). Research in W. F.'s laboratory was supported by Biogent.

² To whom requests for reprints should be addressed.

Received 4/24/87. Revised 7/27/87. Accepted 8/27/87.