THE CREATININE LEVEL OF BLOOD SERUM AS AN INDICATOR OF CARCASS COMPOSITION 1 PAUL R. WUTHIER AND P. O. STRATTON Wyoming Agricultural Experiment Station URRENT trends in consumer demand emphasize the importance of C the relative proportions of fat and lean in the meat-animal carcass. Major differences in carcass meatiness have been shown among cattle considered uniform by the most critical visual appraisal. The development of meat-type cattle would be greatly aided by a practical and accurate in vivo determination of carcass composition. Evidence has been accumulated by Brody (1945) showing that, in general, urinary creatinine reflects the mass of supporting muscle in the body, In a study of the body composition of humans, Miller and Blyth (1952) found that the lean body mass of individuals predicted from creatinine excretion agreed within 13.1% of the determined specific gravity of body fat from body weight in 90% of the cases studied. Creatinine excretion as related to the percentage of separable lean in yearling steers was studied by Lofgreen and Garrett (1954). The percentage of lean was estimated by a densimetric determination of the lean in the 9, 10, llth rib cut. The correlation between creatinine excretion and percentage of lean was highly significant (r-~.67). Creatinine in blood serum was considered as a source of information, since total urine samples are difficult to obtain. Brody (1945) has shown that the quantity of blood varies linearly with body weight. Since creatinine is not re-absorbed in the kidney, the level of creatinine in the blood should reflect the quantity being produced. Hankins and Howe (1946) have shown that the percentage of separable lean in the carcass can be accurately estimated by the lean in the 9, 10 and llth rib cut (r----.90). A recent study by Cahill et al (1956) has shown a highly significant relationship (r~---.85) between the area of rib eye and the edible portion of the carcass. Experimental The data reported in this study are from cattle of the University of Wyoming beef herd. Included were 15 bulls and 12 heifers of the 1955 progeny-test cattle as well as 4 fat steers, 3 fat heifers, and 1 cow. Ages ranged from 12 to 14 months on the progeny-test cattle and 19 to 23 on the fat cattle. The cow was 34 months old. Blood samples were collected one to ten days before slaughter. Blood 1 In cooperation with the Animal and Poultry Husbandry Research Branch, A.R.S., U.S.D.A. under Western Regional Project W-l, Beef Cattle Breeding Research. Published with approval of the Director, Wyoming Agricultural Experiment Station as Journal Paper No. 94. 961
962 WUTHIER AND STRATTON creatinine was determined with modifications of the method of Folin and Wu (1919) where its reaction in a protein free filtrate with alkaline picrate forms a red color (the Jaffe reaction). The Jaffe reaction is not specific for creatinine. Chromogenic material in red blood cells contribute to the formation of red color (Hawk et al., 1951). Preliminary analyses were by the procedure of Hawk et al., using a protein-free filtrate prepared from blood serum in 1/10 dilution and alkaline pi~rate prepared from saturated picric acid. A Bausch and Lomb monochromatic colorimeter using a 505 m/~ filter was used to measure color intensity in terms of percentage of light transmission. The repeatability of early determinations using this method indicated limited precision. The use of a 1/5 acidic dilution in preparation of protein free filtrate (Mandel and Jones, 1953) and alkaline picrate prepared from 0.26% picric acid (Bausch and Lomb manual, 1950) were found to give more precise measurement with the Bausch and Lomb coiorimeter. The technique of measurement is described below. A standard curve was prepared by obtaining percent transmission readings from known quantities of creatinine. A standard creatinine solution containing 1 rag. creatinine per ml. was used as the source of creatinine. The first dilution (S~) was made by diluting 2.5 ml. of the standard 25 ml. in a volumetric flask. The second dilution ($2) was prepared by diluting 5 ml. of $1 to 100 ml. in a volumetric flask. All dilutions were made with distilled water. The $2 solution in 0, 1, 2, 3, 4, and 5 ml. quantities was placed in transmission curvettes and filled to 5 ml. with distilled water. The resultant dilutions were 0, 0.1, 0.2, 0.3, 0.4 and 0.5 mg% creatinine (rag. per 100 ml.) respectively. The preparations were run in triplicate on different days. Alkaline picrate, prepared within 30 minutes of the time of use, consisted of 1 part of 10% sodium hydroxide solution to 5 parts of 0.26% picric acid solution. Two cc. of the alkaline picrate were added to each curvette and allowed to stand for 20 minutes at room temperature. The 0% level or blank was used as the base or 100% transmission level. The percent transmission for each concentration of creatinine was determined from this base, and plotted on semi-logarithmic graph paper for a standard curve. Protein-free filtrate was prepared according to the technique of Mandel and Jones (1953). Five ml. distilled water were added to 2 ml. of the blood serum to be tested in a 20 ml. centrifuge tube. Two ml. of 2/3 N. sulfuric acid were added slowly and shaken by mechanical shaker for five minutes. After this, 1 ml. of 10% sodium tungstate solution was added and mixed to a homogeneous state by inverting the tube to avoid foam production. Contents were again shaken for five minutes and then centrifuged at 1800 rpm. for 15 minutes. Five ml. of the resulting clear filtrate were drawn from each tube and placed in curvettes for creatinine reading. Blanks, consisting of 5 ml. dis-
CREATININE AND CARCASS COMPOSITION 963 tilled water plus 2 ml. of fresh alkaline picrate, were run in duplicate with each group determination to establish the 100% transmission level. The percent transmission was determined to the nearest 0.2%, 20 minutes after the addition of the alkaline picrate. Creatinine in mg. per 100 ml. was read from the standard curve. The fat-lean ratio of the 9, 10 and llth rib cut was measured densimetrically with modifications of the method of Lofgreen and Garrett (1954). The cut was separated from the left side after 10 to 14 days aging by the method of Hankins and Howe (1945). The cut was boned to eliminate errors due to difference in bone development or improper splitting. The weight in air of the boned cut was recorded to the nearest gram. The weight in water was measured by attaching the boned cut to the scale with a piece of fine wire so as to allow it to hang free when submerged in water. Water temperature of 20 ~ C. was used for all determinations. A standard weight of 100 gin. was attached to the wire and weighed under water. This weight, including wire and the submerged standard, was subtracted from each underwater weight as tare. In this manner cuts or samples such as fatty tissue could be weighed under water even though their specific gravities were less than one. To standardize specific gravity measurements, density determinations were made of tap water at 20 ~ C. Eight replications were made with different volumetric flasks from 10 to 100 cc. The averagedensity (weight in grams/volume in ml.) at 20 ~ C was 0.9957 Specific gravity measurements were made of fat and lean samples of rib cuts. Samples were taken at the same time as boned rib cut densities, 10-14 days after slaughter, and were measured shortly after removal from the cooler at 34 ~ F. (approximately 1 ~ C.). Average specific gravity values of separable fat and lean and of the boned rib cut from which the samples were taken are listed in table 1. The specific gravity of the boned 9, 10 and 1 lth rib cut was determined for each carcass. Since the same correction for water density is applicable for the numerator and denominator of the prediction formula below, specific gravity values were used to determine the proportion of fat and lean in the carcass from the formulas: s.g. of lean minus s.g. of whole cut proportion of fat~ s.g. of lean minus s.g. of fat proportion of leanz I minus proportion of fat. Carcass composition was also estimated by rib eye area measured on the left front quarter separated between the 12th and 13th rib from color photographs by the procedure of Schoonover et al (1957). The area of external fat was determined by plotting two perpendicular lines at either end of a straight line drawn through the long axis of the longissimus dorsi muscle. The external fat within the boundry of these two perpendicular
964 WUTH~ER AND STRATTON TABLE 1. SPECIFIC GRAVITY OF FAT, LEAN AND BONED RIB CUT No. Av. specific Standard Av. specific gravity Standard samples gravity error of boned rib cut error Fat 23 0.9229 0.0039 1.028.000055 Lean 16 1. 0659 0. 0013 1. 033.000072 lines was measured for an estimate of area of fat. The proportion of lean in the rib cut was estimated by the formula: rib eye area proportion of lean=ri b eye area@area of external fat Results Creatinine levels ranged from 1.28 to 2.24 mg. per 100 ml. of blood serum. The average level for all cattle was 1.58 mg. Average levels of 1.73, 1.47, and 1.48 were determined for the progeny-test bulls, progeny-test heifers, and older cattle, respectively. The percentage of lean in the boned 9, 10, and 11th rib cut ranged from 41.7 to 99.4. In both age groups males had a higher percentage of lean than females. The difference is shown by average values of 84.7 and 61.5% lean for bulls and heifers in the progeny-test cattle, and 58.6 and 49.6% lean for steers and heifers in the older cattle. Correlations between the percentage of lean and serum creatinine are presented in table 2. Difference in fatness within age groups, line and sex was less than the difference between these divisions; thus the highest correlation existed where all variation was considered. Variation in rib-cut composition in females was small, so that the higher correlation existed in males. Additional carcass data were obtained from photographic measure of rib-eye area on the progeny-test cattle. Rib-eye areas ranged from 4.19 to 9.70 sq. in. A portion of the wide range in rib-eye area can be attributed to variations in carcass weight as shown in table 3. Regression analysis showed significant increases of 1.27 and 0.40 sq. in. TABLE 2. CORRELATION BETWEEN LEAN PERCENTAGE AND SERUM CREATININE Within age, Classification No. Total Within age group line and sex All cattle 35.55**.54**.23 Males 19.55*.40*.16 Females 16.22.35.30 * Significant at the 5% level. ** Significant at the 1% level.
CREATININE AND CARCASS COMPOSITION 965 TABLE 3, AVERAGE LIVE WEIGHT, CARCASS WEIGHT AND RIB EYE AREA OF PROGENY-TEST CATTLE BY SEX Sex No. Av. age Av. live wt. Av. carcass wt. Av. rib eye area mo. lb. lb. sq. in. Bulls 15 13 652 127.9 370.3--+82.7 7.70-+1.20 Heifers 12 13 532 59.1 307.5-+43.2 5.16-+0.65 of rib-eye area per 100 lb. of carcass weight for bulls and heifers~ respectively. The difference in regression values was highly significant. These regression values were used to correct rib-eye area for deviations from average carcass weight in each sex. The correlations between serum creatinine and the corrected and uncorrected rib-eye areas are reported in table 4. The highest correlation existed between serum creatinine and rib-eye area in heifers. In bulls, the correlation between uncorrected rib-eye area and serum creatinine was negative. However, when the carcass-weight correction was applied and line differences removed, the correlation coefficient was positive and approached significance. Carcass-weight correction and line difference had a lesser effect on the correlation between serum creatinine and rib-eye area in females. This is explained in part by a rather uniform carcass weight and degree of condition in females. A wide range in fatness and carcass weight (table 3) existed in bulls. The largest rib-eye areas were associated with the greatest percentage of fat. A negative correlation between rib-eye area and percentage of lean (r~--.53) was found for the 15 bulls on progeny test. The total relationships between the two estimates of carcass composition studied are of interest. The correlation between the proportion of lean in the 9, 10, and llth rib cut as determined by specific gravity and the proportion of lean in the rib-eye cut as determined by photographic measurement was highly significant (r----.94). These data indicate that the ratio of area of rib eye to total area of rib cut as measured by photographic means may give an accurate estimate of the lean tissue in the carcass. TABLE 4. CORRELATION BETWEEN RIB EYE AREA AND SERUM CREATININE Uncorrected Corrected Corr. rib eye area Classification No. rib eye area rib eye area within sex and line Progeny test cattle 27.22.52**.45* Bulls 15 --. 16.00.44 Heifers 12.65".63".51 Significant at the 5% level. ~ Significant at the 1% level.
966 WUTHIER AND STRATTON Summary Serum levels of creatinine were found to l~e significantly related to percentage of lean in the boned 9, 10, and llth rib cut @=.55, n=35) and rib-eye area corrected for carcass weight (r=.52, n~27). Although the above correlations were highly significant, the predictions by serum creatinine were low, r2=.27, and r2=.31. Thus only 27% of the variation of lean of the rib eye as measured by area and 31 ~ of the variation of lean in the boned 9, 10, and llth rib cut as measured by specific gravity could be accounted for by differences in serum creatinine levels. The relationships between the estimates of carcass composition were highly significant. The correlation between corrected rib-eye area and percentage of rib-cut lean was.79. The area of external fat and the area of rib-eye measured from colored photographs were used to estimate the proportion of lean. The correlation between this estimate and the proportion of lean in the boned 9, 10, and llth rib cut determined by specific gravity was.94. The data indicate that this photographic estimate could be used as an accurate estimate of carcass composition. References Bausch and Lomb, Monochromatic Colorimeter Manual. 1950. Determination of blood creatinine. Bausch and Lomb Optical Co., Rochester, N. Y. Brody, S. 1945. Bienergetics and Growth. Reinhold Publishing Corp., New York. Cahill, V. R., L. E. Kunkle, E. W. KIosterman, F. E. Deatherage and E. Wierbicki. 1956. Effect of diethystilbestrol implantation on carcass composition and the weight of certain endocrine glands of steers and bulls. J. Animal Sci. 15:701. Folin, O. and H. Wu. 1919. A system of blood analysis. J. Biol. Chem. 38:81. Hankins, O. G. and P. E. Howe. 1946. Estimation of the composition of beef carcasses and cuts. U.S.D.A. Tech. Bul. 926. Hawk, P. B., B. L. Oser and W. H. Summerson. 1951. Practical Physiological Chemistry. 12th Ed. The BIakiston Co. New York, Philadelphia and Toronto. Lofgreen, G. P. and W. N. Garrett. 1954. Creatinine excretion and specific gravity as related to the composition of the 9, 10, and llth rib cut to Hereford steers. J. Animal Sci. 13:496. Mandel, E. E. and F. L. Jones. 1953. Studies in non-protein nitrogen III. Evaluation of methods measuring creatinine. J. Lab. Clin. Med. 41:323. Miller, A T. and C. S. Blyth. 1952. Estimation of lean body mass and body fat from basal oxygen consumption and creatinine excretion. J, Applied Physiol. 5:73. Schoonover, C. O. and P. O. Stratton. 1957. A photographic grid used to measure rib eye areas. J. Animal Sci. 16:957.