The measurement and distribution of carnosine in the rat.

Preparation of Tissue Extra&+-Adult male rats of the SpragueDawley strain, fed Purina chow ad libitum, were killed with ether. The thigh muscles, or other tissues, were sliced finely, obvious fat and connective tissues having been discarded, and a 1 g portion was transferred to a large test tube. The tissue was extracted twice with 10 ml of water by placing the tube in a water bath at 100” for 10 minutes. The extracts were decanted through a filter into a 25-ml volumetric flask, and the solution was made to volume. Chromatographic Procedure-Descending paper chromatography on Whatman No. 4 paper with a solution of butanol, propanel, and 20% ammonium hydroxide (2:2 : 1) for 60 hours resulted in a clear-cut separation of carnosine, histidine, anserine, and methylhistidine. These compounds were detected by dipping the paper in butanol containing 0.4% ninhydrin, followed by heating it (at 100-110”) for 10 minutes. Sodium and potassium ions decreased the rate of movement of these substances. Carnosine and histidine were detected on paper by spraying the paper with diazotized sulfanilic acid, prepared by adding 5 ml of 5% (weight per volume) sodium nitrite to 5 ml of 0.9% (weight per volume) sulfanilic acid (in 0.1 N HCl), and after mixing and 15 minutes of refrigeration, adding 10 ml of 10% sodium carbonate. The solution was used immediately since deterioration occurs on standing. Extracts of muscle containing 10 to 30 pg of carnosine were chromatographed on paper, and the papers then allowed to stand at least 16 hours to allow the solvents to evaporate. The area containing carnosine (determined on other strips run simultaneously) was cut out and placed in a tube containing the color reagent. After 10 minutes on a mechanical shaker, the tubes were centrifuged, the extract was decanted into a cuvette, and its color was read immediately. Predicted values were obtained by the calorimetric procedure on the crude extract. The recoveries in 12 tests ranged from 81 to 112% of the predicted value with a mean of 97.5%. Identi,fication of Imidazole-reacting Materials in MuscleMuscle extracts were applied to paper and chromatographed. Development with the imidazole reagent showed the presence of one major chromogenic constituent, carnosine. The material in the corresponding area on an unstained paper was eluted and hydrolyzed by autoclaving for 2 hours with 2 N HCl. Chromatography of this solution indicated the presence of only histidine and /3-alanine. No free histidine was detected in muscle extracts either by staining the paper chromatogram or by carrying out a colorimetric determination on an eluate from the corresponding area on a duplicate chromatogram. This observation suggests that there are less than 5 pg of free histidine per g of muscle. A minor constituent was also found that gave a positive imidazole reaction and that moved 0.3 as far as carnosine. By visual estimation and comparison with standard carnosine and histidine spots, there appeared to be about 5 pg of this unknown substance per g of muscle. It is possible to conclude that 2% or less of the total material measured in a crude extract of skeletal muscle by the imidazole reaction is not carnosine. Intracellular Distribution of Carnosine in Skeletal MuscleSamples of rat thigh muscle were homogenized with an ElvejhemPotter blender in 4 ml of 0.88 M sucrose per g of tissue and centrifuged at 650 x g in the cold for 10 minutes. The supernatant fluid was decanted and centrifuged at 0” in a high speed centrifuge at 15,000 x g for 20 minutes to obtain the mitochondria (6). The supernatant fluid, the mitochondria, and the debris were analyzed for earnosine content. Paper chromatographic identification was made in each case. In six analyses, the debris contained 43.1’% (s.d. 1.84), the supernatant 49.4% (s.d. 2.90), and the mitochondria 7.3% (s.d. 2.80) of the total carnosine. With the nitrogen content of each fraction as the basis for comparison, the debris, supernatant, and mitochondria contained 23.9 (s.d. 3.22), 71.5 (s.d. 6.34), and 15.3 (s.d. 1.86) pg of carnosine per mg of nitrogen, respectively. It has been noted previously (6) that the particulate matter in skeletal muscle varies in size; therefore, the supernatant fluid obtained from the initial centrifugation was treated at various rates from 12,000 to 20,000 x g for 10 to 20 minutes to obtain the mitochondria. The carnosine content of the mitochondrial fractions thus obtained did not change when the speed or the time was varied. The mitochondrial carnosine was not washed off by resuspending the particles in either 0.88 M or 0.25 M sucrose and centrifuging, suggesting that the carnosine is tightly bound to or located within the mitochondria. Carnosine is soluble in water, and its presence in the debris even after the resuspension in 0.88 M sucrose and centrifugation indicates that it is probably bound. A suspension of the debris in distilled water, 50% ethanol-water, or 50% glycerol-water for 24 hours at 04” ex-