[CANCER RESEARCH 38, 3663-3667, November 1978] 0008-5472/78/0038-0000$02.00 Increased Tyrosine Phenol-Lyase Activity in Mice following Pyridoxal Phosphate Administration1 Gary W. Elmer,2 Leonard Minor, Gary G. Meadows,3 Darrell H. Spackman, and Vernon Riley Department ot Pharmaceutical Sciences, School of Pharmacy, University of Washington, Seattle [G. W. £., L. U., G. G. U.), and the Pacific Northwest Research Foundation, and The Fred Hutchinson Cancer Research Center, Seattle, Washington 98104 [D. H. S., V. Ft.] ABSTRACT was determined both with and without adding the usual optimal levels of PLP (10 /ng/ml) to the assay mixture. One A limiting factor in the depletion of plasma tyrosine IU of TPL is the amount of enzyme that will catalyze the following tyrosine phenol-lyase injection into normal mice degradation of tyrosine to produce 1 /¿molof pyruvate per was found to be the availability of an essential cofactor, min at 37°. pyridoxal phosphate. Because of the extremely short half- Mice. Female C57BL/6 x DBA/2 F, (hereafter called life of this cofactor, adequate elevation of circulating BD2F,) mice, 17to 21 g, were obtained from the Charles River cofactor levels for prolonged periods by injection of a Breeding Laboratory, Wilmington, Mass. All mice were pyridoxal phosphate solution was not practical. Similarly, housed in groups of 5 in metal cages with solid floors long-term diets enriched with pyridoxine and pyridoxal covered with hardwood shavings for bedding. The mice phosphate did not significantly improve the efficiency of were equilibrated for at least 7 days prior to use and were the injected holoenzyme. A repository dosage form was given food and water ad libitum except that access to food devised that consisted of an s.c. implant of pyridoxal was denied during the 5 hr prior to obtaining blood sam phosphate suspended in a spermaceti and peanut oil ples. Since the ubiquitous LDH virus, which is present in mixture. Under these conditions a sustained increase in many transplantable tumors (29), can cause significant holoenzyme activity levels and a significant resulting immunological and other alterations in the host (26, 27), all decrease in plasma tyrosine levels were obtained. mice were given injections of the virus 2 to 3 days prior to experimentation as previously described (18), in order to INTRODUCTION assure experimental uniformity. The LDH virus, by itself, does not significantly affect the growth rate of the pig- Interest in extending the application of highly purified mented B-16 melanoma (28). enzymes that selectively degrade specific amino acids in vivo has been stimulated by the utilization of L-asparaginase Analyses. Blood samplesfor the determination of plasma as a chemotherapeutic agent in humans (1, 5-7,10, 22, 41). enzyme and amino acid levels were obtained by orbital Certain glutaminase preparations may also have potential bleeding (25). As tubes of blood were collected, they were for cancer therapy (31, 32, 39). Two PLP"-dependent en immediately chilled in ice water and centrifuged at 0°.The zymes, L-methioninase and TPL, have been reported to ex plasma samples were pooled in cups packed in ice; aliquots hibit antineoplastic activity in vivo (14, 18). Both enzymes were then removed for subsequent enzyme assay. The actively degraded their respective amino acid substrates in remainder of the plasma, to be used for amino analysis, was added to a preweighed tube containing 0.6 M sulfo- vivo, but total depletion of these amino acids in the plasma of treated animals has not been achieved. salicylic acid, to precipitate all proteins immediately, thus Dissociation in vivo of the essential PLP cofactor from the inactivating any enzyme activity and stabilizing the amino holoenzymes following injection is an important theoretical acid content. After further preparation, the plasma samples factor in regulating enzyme activity and thus in establishing were analyzed for tyrosine and all other free amino acids the extent and duration of amino acid depletion and, with a modified Beckman Model 120B amino acid analyzer, by the methods of Spackman (35-38). This preparative subsequently, the antineoplastic activity of such enzymes. This report describes the relative instability of holotyrosine procedure has been found to be effective in stopping phenol-lyase in vivo and the enhanced activity of the in vivo plasma enzyme activity in other studies when plasma en zymes were present such as asparaginase (26-30) and enzyme that results when plasma levels of PLP are in arginase (D. H. Spackman, unpublished studies). creased by injections or repository implants. Tumors. Pigmented B-16 melanomas were implanted and measured as described by Meadows era/. (18). MATERIALS AND METHODS PLP. Free plasma PLP levelswere determined by measur Enzyme. Purified TPL was prepared and assayed as ing the relative activation of apo-TPL (19). described previously (18). Where indicated, TPL activity RESULTS 1 These studies were supported in part by Institutional Cancer Grant IN-26 and Grant PDT-73 from the American Cancer Society. 2 To whom requests for reprints should be addressed. Effect of PLP on TPL Activity. In the absence of the 3 Present address: College of Pharmacy, Washington State University, added PLP cofactor, holo-TPL was rapidly converted to the Pullman, Wash. 99163. ' The abbreviations used are: PLP, pyridoxal 5'-phosphate; TPL, tyrosine apoenzyme when injected into normal mice (Table 1). At 5 hr after holoenzyme administration, the plasma was as phenol-lyase; LDH virus, láclate dehydrogenase-elevating virus. sayed for the "effective" TLP activity with no PLP added to Received April 6, 1978: accepted July 31, 1978. NOVEMBER 1978 3663 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1978 American Association for Cancer Research. G. W. Elmer et al. Table 1 In vivo activity of TPL as a function of PLP administration Groups of 5 mice were given injections i.p. of either TPL (200 ID/kg), 10 mw potassium phosphate buffer (pH 7.4), or PLP (50 mg/kg). PLP was reinjected in Groups 2 and 4 again at 2, 3, and 4 hr after the initial treatment. All mice were bled 5 hr subsequent to the initial injections. Plasma samples from all mice within each group were pooled prior to performing the analyses. Single amino acid analyses were performed on each plasma pool. With regular calibration of the amino acid analyzer with calibration mixtures, the results are accurate to better than ±2%over the range of 10 to 200 nmol/ml (35-38). Plasma TPL activity was determined 5 hr following holoenzyme administration, both without and with PLP (10 /*g/ml) added to the assay mixture in order to compare the effective TPL activity in the host with its potential activity in the presence of optimal cofactor. Replicate assays were performed. TPLGroup1 Plasma tyrosine level activity activity treatmentBuffer (nmol/ml)74.3 (mlU/ml)0 (mlU/ml)0 ±1.5" (100)* 2 PLP only 47.5 ±1.0 (64) 0 0 3 TPL only 31.2 ±0.6 (42) 40 ± 1.6 (7) 570 ±23(100) 4MouseTPL + PLPPlasma10.1 ±0.2 (14)Effective280 ±11. (37)Potential760 ±30(100) " Mean ±S.E. '' Numbers in parentheses, percentage. the assay mixture. These values were then compared with or pyridoxine were not significantly reduced as compared the "potential" TPL activity in a replicate plasma sample, to to the normal levels of 70 to 80 nmol of plasma tyrosine per which PLP had been added to the optimal level. These ml found in control groups on normal diets. comparative data showed that the effective activity was only Long-Acting PLP Implants. With the goal of substantially 7% of the potential enzyme activity. In comparing Groups 1 increasing plasma PLP levels, a time release repository and 3 in Table 1, it may be seen that the nonsupplemented implant of PLP was devised. A mixture of spermaceti in enzyme was capable of effecting a reduction in plasma peanut oil (30% v/v) was found to be a convenient and tyrosine to 42% of normal. When large amounts of the PLP effective vehicle. This mixture melted at 40-43°and could cofactor (50 mg/kg) were injected together with TPL (Group thus be implanted as a liquid. After cooling in situ, it 4), effective enzyme activity levels were substantially ele hardened to a semisolid gel. Release of the PLP suspended vated, to 37% of potential activity. This increased enzyme in the vehicle appeared to be dependent, in part, on the activity was reflected by a corresponding decrease in cir host metabolizing the spermaceti and peanut oil. Surgical culating tyrosine, to 10.1 nmol/ml. PLP administration examinations at 7 days revealed that very little of the alone also resulted in a significant reduction in plasma implant material remained from the i.m. implantation in the tyrosine. This is possibly due to an activation of endoge right hip. nous apotyrosine aminotransferase which also requires Effect of Elevated PLP. Following i.p. administration of this cofactor (43, 44). TPL to PLP-implanted mice, effective plasma enzyme activ PLP Diets. It was previously shown that exogenously ities were increased to 37% of maximal potential activity administered PLP is rapidly cleared from plasma, with a and were maintained at an increased level starting at half-life for free PLP of about 15 min (19). It therefore approximately 24 hr and persisting for at least 100 hr after seemed impractical to elevate plasma PLP levels substan PLP implantation (see Chart 1). The extent of TPL-mediated tially for sustained periods of time by the repeated paren- plasma tyrosine depletion in the mice with PLP implants teral injection of PLP solutions. Studies in humans have was greater (to 8% of normal) than was achieved in mice shown that a daily p.o.