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					Beta Radioactivity of the Ash in Relation to the Composition of Ham R. Kulwich, L. Feinstein and C. Golumbic J Anim Sci 1960. 19:119-126.

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BETA R A D I O A C T I V I T Y OF T H E ASH I N R E L A T I O N TO T H E C O M P O S I T I O N OF HAM
R. K U L W l C H , L . F E I N S T E I N , A N D C . G O L U M B I C U. S. Department o/Agriculture 1, 2, 3

improved objective for estimating the T HE development ofanimals, carcasses andmethodscuts should increase composition of live meat the efficiency of the marketing operation. Techniques that would make it possible to estimate accurately the amount of lean, fat and bone in meat animals, carcasses and meat cuts would benefit the producer, the processor and the consumer. Some recent studies suggest that natural potassium-40 radioactivity of animals may furnish an index of the muscle mass. In 1956 Woodward et al. reported that the total body potassium of a group of human subjects was related to the body water content and, therefore, to the lean body weight and muscle mass. The total body potassium content was determined by the use of a sensitive gamma-ray detector (the Los Alamos "human counter") which was capable of measuring the naturally present radioistope, potassium=40. Anderson (1957a) reported on the basis of potassium-40 and tritium dilution measurements with human subjects that the potassium content was 0.28% on a "fat-free" weight basis and was independent of the sex, age or weight of the subject. The possibility of estimating the muscle mass of a sample by a nondestructive technique suggests many applications and warrants further investigation. In preliminary study Kulwich, Feinstein and Anderson (1958) investigated the relationship of the fat content and the potassium-40 gamma radioactivity of four hams as measured in the Los Alamos "human counter". Measurements were made on the intact hams, then again after removing the skin and external fat, and finally on the lean portion with most of the fat trimmings removed. In each case the value for disintegrations per second of gamma rays per pound of sample was proportional to the percent of fat-free lean present. The purpose of this study was to investigate further the relationship between' the radioactivity emitted and the composition of meat samples. Materials and M e t h o d s Potassium-40 has an isotopic abundance of 0.0119%, a half life of about 1.25X109 years, and emits 10 beta particles for every gamma ray (Suttle
1 Biological Sciences Branch, Marketing Research Division, Agricultural Marketing Service, Beltsville, Maryland. Acknowledgment is given to E. W. Kobernusz for technical assistance. 2 Appreciation is expressed to E. J. Koch of Biometrical Services, Agricultural Research Service for advice on statistical aspects of this study. s Appreciation is expressed to N. R. Ellis, R. L. Hiner and W. R. Kauffman of the Animal Husbandry Research Division, Agricultural Research Service, who made available the samples of ham lean and fat trimmings used in this study and supplied the ether extract protein, and moisture analyses. 119

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KULWICH, FEINSTEIN AND GOLUMBIC

and Libby, 1955). Potassium from different sources does not vary by more than -r in its potassium-40 content (Vinogradov, 1957), so that a determination of the potassium-40 content of a biological sample should provide an excellent index of the total amount of potassium present. Potassium-40 is the principal naturally radioactive isotope present in all organisms. The data of Vinogradov (1957) indicate that, in the case of humans, there is about 7 times as much radioacitvity emitted by potassium--40 as there is by the next most prevalent naturally radioactive isotope, carbon-14. Because the preparation of samples for beta-radioactivity counting used in this study includes ashing which removes carbonaceous materials, potassium-40 is by far the predominant naturally radioactive material remaining in soft tissue ash. Of the fission products resulting from atomic testing that may occur in meat, cesium-137 is probably of the greatest concern in connection with this study. Anderson etal. (1957b) reported that fresh hams analyzed had a cesium-137 to potassium-40 ratio of 0.30. The ashing procedure used in the present study may result in some volatilization of cesium. The ash samples were not analyzed for cesium content specifically. The total beta radioactivity of the ash was measured. In 1959 Laug and Wallace reported the results of a survey of radioactive residues in foods. There was no indication of significant amounts of beta radioactivity in the ash of meat-products samples above that which could be accounted for by the potassium content as determined by flame photometry. Since potassium and cesium are closely related chemically, belonging to the same group in the Periodic Table, cesium may follow potassium to some extent in its pattern of deposition. In this case, it might also tend to vary with the muscle mass. Anderson (1957a) reported that cesium followed to a rough approximation the potassium content in meat samples that were studied. The samples used came from the right hams of 34 different hogs. The animals involved were 3 Duroc gilts, 9 Duroc barrows, 12 Yorkshire gilts and 10 Yorkshire barrows. Although these animals had varied treatment histories, there was no indication of any pronounced effect of prior treatment or sex difference upon the distribution of potassium in the tissue of the hams. These animals were slaughtered at around 180 lb. live weight. The right hams were dissected to separate the bone, fat trimmings and separable lean. The separable lean and the fat trimmings portions of each ham were handled separately. They were run through a meat grinder twice with thorough mixing before carrying out ether extract, protein and moisture analyses on samples according to A.O.A.C. methods (1955). After chemical determinations were made, 36 samples were prepared for measurement of the beta radioactivity of the ash. These samples were dried and then ashed overnight at 550 ~ C. in a muffle furnace. A 10-ml. portion of 1 N sulfuric acid was added to each sample before drying and ashing to minimize volatilization of potassium halide (Blaxter and Rook,

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COMPOSITION

OF HAM

121

1956). Twelve of the samples consisted of 300 gm. of fat trimmings from 12 hams. The other 24 samples, two of which were from hams included among the 12 fat samples, consisted of various amounts of lean and fat trimmings from individual hams. The samples were dried and ashed in tared 250-ml. porcelain evaporating dishes. A preliminary charring treatment as described by Thiers (1957) was necessary to prevent the samples from foaming and spewing out of the dishes. Some of the excess fat was burned off by the use of low ash filter paper wicks in the case of fat-trimmings samples processed. After the samples were placed in the muffle furnace, the temperature was raised gradually and the furnace was left partly open. The samples were ignited at around 450 ~ C. After flaming ceased, the muffle furnace door was shut, and the temperature was set at 550 ~ C. for the night. There is a possibility of some potassium losses during the ashing operation. Carter and Greig (1957) reported the potassium content of rat tissues decreased when dry ashing was carried out in Pyrex glass at 550 ~ C., compared to results obtained when platinum crucibles were used. No report was made of results of ashing done in porcelain dishes. After ashing was completed, the sulfated ash was weighed. Then a portion of ash was weighed out and formed into a briquette 1-1/8 in. in diameter, weighing about 600 mg., by the use of 15,000 lb. per sq. in. pressure applied with a hydraulic press. Similar briquettes were prepared with analytical grade potassium chloride for use as standards. The briquettes were placed in cupped stainless steel pans that were 1-1/4 in. in diameter and 3/32 in. deep. The thickness of the briquetted ash samples was such that none of them protruded above the lip of the stainless steel pans. The beta radioactivity of the ash samples was measured by the use of a commercial gas-flow counter with a very thin plastic window having a density of less than 150 micrograms per cm 2. The efficiency of the counter was checked daily with the potassium chloride standards. Counting efficiency (averaging about 31% ) varied only slightly from day to day and corrections were made for these fluctuations. Each sample was counted for a total of at least 8000 counts, and the associated background for at least 200 counts, which resulted in a relative standard deviation of the sample net counting rate determination of less than 27o (Comar, 1955). Results and Discussion The relationship between the beta radioacivity of the ash expressed in terms of the equivalent percent of potassium in the fresh sample which would account for the amount of beta radioactivity measured, and the percent of ether extract in the fresh sample is illustrated in figure 1. The correlation between beta radioactivity and percent ether extract was highly significant (r value, --.99). The sample standard deviation from

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122

KULWlCH~FEINSTEIN

AND GOLUMBIC

regression (Snedecor, 1956) was 2.2% (ether extract). The 95% confidence interval for the predicted ether extract content from the beta radioactivity of the ash at the point of average beta radioactivity (0.213 units) was from 41.0 to 50.27o or 45.6+__4.6% ether extract. The range of ether extract content in the 36 samples studied was from 13.0 to 78.6%. The ranges of the protein and moisture contents of these samples were from 5.2 to 21.6~o and from 16.1 to 64.77o, respectively. The 9 5 ~ con-

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Figure 1. Regression of the percent ether extract (fresh weight basis) on the beta radioactivity of the ash ( e x p r e s s e d in terms of the equivalent percent potassium which would account for the amount of beta radioactivity measured). The regression line is indicated by the continuous line and its 95% confidence limits by the interrupted lines. fidence intervals of the predicted protein and moisture contents at the point of average beta radioactivity were 12.7+__1.3% and 40.8_3.5%, respectively. The average beta radioactivity of 0.213 units is that amount that would be emitted by a sample containing 0.213% potassium on a fresh basis with no other radioactive ash component. With the procedures used in this study, this corresponds to a measured beta radioactivity of 117 counts per minute per 100 gm. of fresh sample containing 45.6~o ether extract. The data relating to the correlations of protein and moisture with beta radioactivity are presented in figures 2 and 3. These results indicate a relationship between the beta radioactivity emitted and the fat-free lean content of the samples analyzed. More information is needed about the variability of the amount of radioactivity emitted by the fat-free lean of different muscles in a carcass as well as the overall differences among carcasses and among live animals. In the

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COMPOSITION OF HAM

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Figure 2. Regression of the percent protein (fresh weight basis) on the beta radioactivity of the ash (expressed in terms of the equivalent percent potassium which would account for the amount of beta radioactivity measured). The regression line is indicated by the continuous line and its 95% confidence limits by the interrupted lines.

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Figure 3. Regression of percent moisture (fresh weight basis) on the beta radioactivity of the ash (expressed in terms of the equivalent percent potassium which would account for the amount of beta radioactivity measured). The regression line is indicated by the continuous line and its 95% confidence limits by the interrupted lines.

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124

KULWICH, FEINSTEIN AND GOLUMBIC

present study, the average value for beta radioactivity of ash components on a fat-free fresh weight basis was 211 counts per minute per 100 gin. In making this calculation it is assumed that the fat contributes no ash components emitting beta radioactivity. On a fat-free fresh weight basis, 211 counts per minute per 100 gm. is equivalent to the beta radioactivity that would be emitted by a sample containing 0.40~o potassium and no other source of beta radioactivity in the ash. The coefficient of variation of the beta radioactivity emission rate on this basis was 7.67o. More information is needed about the gamma radioactivity emitted by meat cuts, carcasses, and live animals, since the gamma-ray measurement technique offers the possibility of making nondestructive measurements. This laboratory is planning to carry out further studies involving nondestructive gamma-ray measurements of meat samples. A review of the pertinent literature gives other indications of the possible usefulness of potassium measurements for providing an index of the muscle mass in addition to those previously cited. Muldowney et al. (1957) reported that total exchangeable potassium was closely correlated with the lean body mass in a study carried out with 16 normal human males and 14 normal females ( " r " z 0 . 9 0 ) . There is evidence that total exchangeable potassium may represent the bulk of total body potassium. Corsa et al. (1950), on the basis of a tracer study with human subjects, reported that the total exchangeable potassium content was probably 5% less than total body potassium. Rundo and Sagild (1955) reported that potassium --42 exchanges with about 85% of the body potassium --40 in humans. Cheek and West (1955) determined the total body potassium and the lean body mass of 30 rats of various weights and ages. They reported that there was a close correlation between total body potassium and lean body mass. Holliday et al. (1957) determined the potassium content of thigh and back muscle samples from albino rats of different ages. They reported a difference in the potassium content at the two sites when expressed on a fat-free dry solids basis, but not when potassium was expressed on the basis of its concentration in calculated intracellular water. Briskey et al. (1959a) studied the potassium content of pork ham muscles which ranged from watery and pale to dry and dark in appearance. These workers reported that there were no consistent differences in potassium concentrations (expressed on a fat-free, dry weight basis). Briskey et al. (1959b) reported that the differences in potassium concentrations (on a fat-free, dry tissue basis) of pork ham muscles associated with varying levels of forced exercise prior to slaughter were not significant. Huth and Elkinton (1959) studied the effect of acute fasting upon the electrolyte content of the muscle of male white rats. These workers noted that chloride and chloride to sodium ratio of fat-free skeletal muscle were increased after acute fasting for 3 to 6 days. Calculations based on the assumption that the extracellular phase is equivalent to the chloride space indicated that the intracellular water, sodium, and potassium concentrations in fat-free

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COMPOSITION OF HAM

125

skeletal muscle were essentially the same in the fasted rats as in the controls. The 13 fasted rats averaged 4 6 . 6 2 0 . 6 0 milliequivalents of potassium per 100 gm. of fat-free solids (mean ___ standard error), while the corresponding value for the 6 control rats was 4 6 . 8 2 1 . 1 5 . These data appear to indicate that under the experimental conditions which prevailed, potassium seemed to be a rather good index of the fat-free solids content of rat skeletal muscle. Summary The results of a study in which measurements were made of the beta radioactivity emitted by the ash of ham fat trimmings samples and samples with variable proportions of ham lean and fat trimmings from 34 hogs are presented. There is evidence of a relationship between the a m o u n t of beta radioactivity emitted, expressed on a fresh weight basis, and the percent of fat-free l e a n (fresh basis). D a t a pertaining to the correlation of beta radioactivity and the content of ether extract, protein and moisture in these samples are presented. Literature Cited Anderson, E. C. 1957a. Scintillation counters. The Los Alamos human counter. British J. Radiol., Suppl. No. 7:27. Anderson, E. C., R. L. Schuch, W. R. Fisher and W.-Langham. 1957b. Radioactivity of people and foods. Science 125:1273. Association of Official Agricultural Chemists. 1955. Official Methods of Analysis (8th ed.). Washington, D. C. Blaxter, K. L. and J. A. F. Rook. 1956. The indirect determination of energy retention in farm animals. I. The development of method. J. Agr. Sci. 48:194. Briskey, E. J., R. W. Bray, W. G. Hoekstra, P. H. Phillips and R. H. Grummer. 1959a. The chemical and physical characteristics of various pork ham classes. J. Animal Sci. 18:146. Briskey, E. J., R. W. Bray, W. G. Hoekstra, R. H. Grummer and P. H. Phillips. 1959b. The effect of various levels of exercise in altering the chemical and physical characteristics of certain pork ham muscles. J. Animal Sci. 18:153. Carter, M. K. and M. E. Greig. 1957. Erroneous values for sodium and potassium content of rat tissues dry-ashed in Pyrex. J. Am. Pharm. Assn. Scientific Ed. 46:624. Cheek, D. B. and C. D. West. 1955. An appraisal of methods of tissue chloride analysis: The total carcass chloride, exchangeable chloride, potassium and water of the rat. J. Clin. Invest. 34:1744. Comar, C. L. 1955. Radioisotopes in Biology and Agriculture. Principles and Practice. page 172. McGraw-Hill Book Company, Inc., New York. Corsa, L., Jr., J. M. Olney, Jr., R. W. Steenberg, M. R. Ball and F. D. Moore, 1950. The measurement of exchangeable potassium in man by isotope dilution. J. Clin. Invest. 29:1280. Holliday, M. A., W. E. Segar, A. Lukenbill, R. M. Valencia and A. M. Durell. 1957. Proc. Soc. Exp. Biol. Med. 95:786 . Huth~ E. J. and J. R. Elkinton. 1959. Effect of acute fasting in the rat on water and electrolyte content of serum and muscle and on total body composition. Am. J. Physiol. 196:299.

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Kulwich, R., L. Feinstein and E. C. Anderson. 1958. Correlation of potassium-40 and fat-free lean content of hams. Science 127:338. Laug, E. P. and W. C. Wallace. 1959. A survey of radioactive residues in foods before and after 1945: Evidence of possible fallout contamination. J. Assn. Official Agr. Chem. 42:431. Muldowney, F. P., J. Crooks and M. M. Bluhm. 1957. The relationship of total exchangeable potassium and chloride to lean body mass, red cell mass and creatinine excretion in man. J. Clin. Invest. 36:1375. Rundo, J. and U. Sagild. 1955. Total and "exchangeable" potassium in humans. Nature 175:774. Snedecor, G. W. 1956. Statistical Methods (Sth ed.). The Iowa State College Press, Ames, Iowa. Suttle, A. D., Jr. and W. F. Libby. 1955. Absolute assay of beta radioactivity in thick solids. Application to naturally radioactive potassium. Anal. Chem. 27:921. Theirs, R. E. 1957. Contamination in trace element analysis and its control. In Methods of Biochemical Analysis, Vol. V, edited by D. Glick, p. 273, Interscience Publishers, Inc., New York. Vinogradov, A. P. 1957. The isotope K 4~ and its role in biology. Biokhimiya 22:14. (English translation available from Consultant's Bureau, Inc., New York). Woodward, K. T., T. T. Trujillo, R. L. Schuch and E. C. Anderson. 1956. Correlation of total body potassium with body water. Nature 178:97.

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