33*^ M. WOODS, K. WIGHT, J. HUNTER, D. BURK VOL. 12 (1953) 



obtained from Webster white mice of the N.I.H. strain; dba and C-57 black mice were used in some 

 experiments. No differences in brain metabohsm attributable to strain of mouse were noted in these 

 experiments. In a number of experiments, the tumor-bearing mice were exposed to controlled tempera- 

 tures for varying periods of time before the in vitro measurements. These are specifically described in 

 the text. Tissues were sectioned by hand with a sharp, single-edge, safety razor blade, rapidly weighed 

 on a torsion balance, and transferred to appropriate solutions in the respirometer vessels which were 

 immersed in ice and water until transferred onto the manometers. 



Anaerobic conditions were provided by gassing with a mixture of 95% N^ and 5% COg which 

 had been passed, together, with a small amount of Hj {ca. i %), over hot copper to remove traces of Og. 

 In aerobic experiments 95% O^ and 5% COg were employed in the gas phase. Two media, which gave 

 similar results, were used, HankSimms* and Krebs-Ringer-bicarbonate**. A few experiments were al- 

 so run in Warburg-Okamoto solution***. Dextrose and KHCO3 were added tothesemediaas required. 

 Unless otherwise stated all experiments were run at a level of 0.625% dextrose, a concentration found 

 to give maximal rates of glycolysis in both brain and melanoma slices without significantly inhibiting 

 respiration. Acid production was measured in terms of COj released from the bicarbonate-buffered 

 medium. Preliminary experiments in Hank-Simms medium with melanoma and brain slices showed 

 that practically all of the manometrically indicated acid could be accounted for as lactic. 0.03 M 

 KHCO3 was routinely employed with 5% CO2 in the gas phase to give an initial pH of ca. 7.6 at 38° C. 

 pH determinations were frequently made of the vessel contents at the end of each run. Aerobic de- 

 terminations were carried out with the Summerson differential manometer and modified Dixon- 

 Keilin flasks. 



Four types of insulin preparations were usedt, (i) Iletin (insulin, Lilly) containing crystalline 

 zinc-insulin and 0.2% phenol as a preservative; (2) zinc-insulin crystals (Lilly no. 535664); (3) zinc- 

 insulin crystals (Lilly no. 499667) which had been treated with trypsin to destroy all of the hyper- 

 glycemic factor (HGF) ; and (4) amorphous low-zinc (0.017%) insulin (Squibb no. 53 In-i). The sample 

 of purified hyperglycemic factor contained only 0.05 units of insulin per mg. Inorganic zinc was 

 supplied as ZnSO^- 7H2O, reported concentrations being for actual weight of zinc. Phenol (as separate 

 from commercial insulin preparations) was reagent grade, Merck. .\11 materials, except the commercial 

 insulin solution, were in most cases added in distilled water (0.25 ml per i.o ml of medium) directly 

 to the refrigerated respirometer vessels before placing them on the bath maintained at 38 °C. In certain 

 experiments the crystalline zinc-insulin was dissolved in 0.025 ^ NaOH and the pH immediately 

 adjusted to ca. 7.6. This method of preparation gave clear solutions which were useful in inaking high 

 dilutions of the hormone. The use of such preparations is specifically mentioned in the text. 



The following symbols have been used to designate the metabolic activities measured: Q02 = 



microliters of oxygen consumed per mg initial dry weight per hour; Q9i, = microliters of acid, in 



terms of COg, produced per mg initial dry weight per hour in the presence of oxygen ; Q^X , like the 

 preceding, but in the absence of oxygen. 



Comparative metabolism of melanoma and brain slices in Hank-Simnis medium 



The comparative behavior of brain and S-91 melanoma shces in Hank-Simms 

 medium is illustrated in Table L Values for embryonic brain slices (fetus ca. 10 days old) 

 and slices of total embryo (fetus S3veral days old) are given for comparison in the same 

 medium. Contrary to generality, <2c6 was greater then Q^^ in adult brain (9.4 vs 2.3), 

 which is all the more strking in view of the high Qq_^ (17.1) which would normally yield a 

 positive rather than a negative Pasteur effect. Monkey brain (young adult female 

 Rhesus)^\ although showing a somewhat different quantitative pattern, behaved in a 



* Hank-Sim7ns solution (values refer to molarities): Hank's solution-3 parts: NaCl 0.137, KCl 

 0.005, MgS04-7H20 0.004, MgCl2-6H20 0.005, CaClj o.ooi, Na^HPO,,- 12 HjO 0.00017, KHgPO, 

 0.00044; Simm's serum ultrafiltrate (prepared by Microbiological .\ssociatcs, Bethe.sda, Md., from 

 ox blood) I part NaHCOg was replaced by KHCO3 and dextrose varied as indicated in the text. 

 Phenol red (0.02 g/liter) was added as an indicator. 



** Krebs-Ringer-bicarbonate solution (values refer to molarities): NaCl 0.141, KCl 0.005, CaCI, 



0.003, KllgPOj 0.0014, MgSO, -71120 0.0014. KHCO3 and dextrose added as indicated in the text. 



Warburg-Okamoto solution (values refer to molarities): NaCl 0.148, CaClj 0.002, KCl 0.003. 



t We are indebted to Doctors H. A. Clowes and Otto Behrens of Eli Lilly and Co. for the 

 preparations of zinc-insulin crystals and the purified H.G.F. Dr. A. Borman of E. R. Squibb and Sons 

 kindly supplied the amorphous low-zinc insulin preparation. 



tt Data on comparative metabolism of various tissues of mouse and monkey were secured witli 

 Dr. Alexis Shelokov of the Laboratory of Infectious Diseases, National Microbiological Institute. 

 These will be reported elsewhere. 



References p. 346. 



