J. Sci. FoodAgric. 1981,32, 1033-1036

Creatine as an Indicator of Net Muscle Proteins

ZdenGk DvoPak

Meat Industry Research Institute, 61200 Brno, Czechoslovakia

(Manuscript received 15 October 1980)

Creatine and creatinine levels in different cuts of pork, beef, veal and lamb have been estimated and expressed in relation to the content of total proteins (N x 6.25) and net muscle proteins (N x 6.25 minus hydroxyproline x 8). The relationship between creatine and total proteins was not linear. Creatine values were linear with those for net muscle protein, giving a mean value of 23 mg creatine g-1 net muscle protein in these meats. Creatinine values averaged 1 mg g-I net muscle protein. The results are discussed in connection with using creatine as an index of meat quality and with using urinary creatinine as an index of muscle mass of the animal.

1. Introduction

The physiological function of creatine is to serve, in its phosphorylated form, as a reservoir of high energy phosphate which is then used for adenosine triphosphate regeneration during muscle contraction. This explains the relatively high content of creatine in muscle in comparison with other tissues.

D a h P 3 confirmed the opinion of Hunter4 that the level of creatine in lean meat is relatively constant. He found1 0.74-3.28 g creatine 100 g-l of crude protein in skeletal muscles of adult animals, but a narrower range (2.15-2.50) in meat cuts without any visible connective tissue. Since smaller amounts of creatine were found in meat samples with higher contents of connective tissue, Dahl3 suggested that the creatine content as a percentage of the crude protein could be used as an aid in evaluating comminuted meat products.

From the indirect relationship between the levels of hydroxyproline and creatine in meat which was found by Dahl,3 it may be concluded that the amount of creatine in muscle tissue is related to the amount of contractile muscle proteins, i.e. the total protein minus the connective tissue protein (N x 6.25 minus hydroxyproline x 8). These will be called ‘net muscle proteins’. Here the ratio between creatine and net muscle protein in pork, beef, veal and lamb has been studied.

2. Experimental 2.1. Materials Different meat cuts with variable amounts of fat and connective tissue were used for the analyses. From pork, 38 samples of head, neck, jowl, shoulder, round, shank, loin and belly meat from different animals of various quality groups were used. Samples of cuts were taken at random so that they did not represent the whole meat. Two samples of meat from two stunted pigs were bulked.

From beef, 29 samples of neck, shoulder, round, hind shin, loin, top rib, thin flank and diaphragm (pars costalis diaphragmatis) were taken at random.

Samples of veal represented muscles of animals before birth. Calves were taken by Caesarean section; ten samples from round, shoulder, fillet (M. psoas maior), thin rib and hind shin were taken from two animals.

From lambs, ten samples from round, shoulder, top rib, thin flank and hind shin were taken from two animals.

0022-5142/81/1000-1033 $02.00 0 1981 Society of Chemical Industry 1033

1034 2. Dvoiik

Fresh meat samples were homogenised in a Moulinette mill-this guaranteed that any vari- ability between portions of the same cut was very small. In some meat cuts, e.g. beef belly, the variability in hydroxyproline between two portions reached 6 %. Different contents of fat did not influence the results. Accuracy and reproducibility of results were insured by taking duplicate samples for each of the analyses (about 10, 4 and 2 g accurately for creatine, hydroxyproline, and nitrogen, respectively).

2.2. Methods Creatine (CR) was determined by the a-naphthol-diacetyl reaction,5 but with some modifications: 10 g of the sample was homogenised with 200 ml of 3 % trichloroacetic acid (the acidity of the solution hydrolyses any creatine phosphate present), and after 1 h the mixture was filtered using Whatman no. 3 filter paper. Standard solutions of creatine (creatine monohydrate, Merck) were used and analyses completed within 1 day to avoid any cyclisation of the creatine in solution.

Creatinine was determined in the same filtrates by the Jaffe reaction6. The procedure consisted of diluting neutralised filtrates with sodium picrate solution (made from 10 vols of 0.5% picric acid and 1 vol. of 10% NaOH) and then measuring the colour intensity at 525 nm. To obtain a value for creatine the amount of creatinine was multiplied by a factor of 1.159 (for such values the symbol CR' is used).

Total protein (N x 6.25) was determined by reaction with sodium hypochlorite and phenolate after dige~tion.~ Such values are equivalent to those obtained using a semi-micro-Kjeldahl deter- mination.

Connective tissue protein was determined as hydroxyproline multiplied by a factor of 8 using the IS0 method.8

3. Results

The relationship of creatine to total protein in beef, veal, lamb and pork is illustrated in Figure 1 ; it increases with increasing amounts of protein but the relationship is not linear and variability is

/ O 0 I

Figure 1. The relationship between creatine and total protein in different cuts of: 0, pork; 0, beef; A, veal: and a , lamb.

Total proteins (g 100 g-' tissue)

Creatine as an indicator of net muscle protein 1035

considerable. When the same creatine values are plotted against net muscle proteins, the relation- ship is linear and the variability decreases (Figure 2). The regression equation is:

Y = -0.12f23.18 X sh= k0.51; r = 0.98

The constant of the equation may be neglected. The slope then indicates a value of 23 mg creatine g-l net muscIe protein.

600

500

- a, 2 400 '0

- +

0 300

7 - W

+ G f3 200 P 0

I00 Figure 2. The relationship between creatine and net muscle protein in different cuts of: 0, pork; 0 , beef; A, veal; and A, lamb.

The creatinine contents of meat cuts were found to vary from 0 to 24.5 mg 100 g-1 fresh tissue; depending on net muscle proteins but with greater variability. Because creatinine is formed by a non-enzymic cyclisation of creatine, the values were recalculated to creatine (CR'), and expressed as mg 8-1 net muscle proteins. Values for the various types of meat are presented in Table 1. The average value for creatinine is about 4 % of CR + CR'.

4. Discussion

The results obtained agree well with those of Dahl,3 who found 2.5% of creatine in meat with a minimum collagen content. The present results establish more certainly a constant ratio of creatine

Table 1. The amount of creatine (CR) and creatine from creatinine (CR') (mg g-l net muscle protein) in the different types of meat (mean+ standard

deviation)

n CR CR' CR + CR' Pork 38 23.3 k 1.4 1.0k0.5 24.3 + 1.4 Beef 29 23.3 + 1 . 5 0 . 9 k 0 . 3 24.2 + 1 .6 Veal 10 22.7f I .O 1 . 3 k 0 . 2 23.9 k 1 .O Lamb 10 22.7 + O .9 0 . 7 + 0 . 2 23.2 + 0 .9 Average 87 23.2 f I . 4 1.0f0.4 24.1 f 1 . 4

1036 Z. Dvoihk

to net muscle proteins for meat cuts with a wide range of collagen contents. The creatine values vary little from 23 mg 8-l net muscle protein. All values illustrated in Figure 2 are in the range of a reliability interval defined as twice the standard deviation of the slope of the equation:

B=byktX2Sb= i2.69; P=O.O1

i.e. 20.5 and 25.9 mg creatine 8-l net muscle protein. This relationship was obtained throughout life and included the as yet inactive muscles taken from calves before birth. It could be suggested that this value is universal for all mammals, since Khan and Cowen5 and Palansk89 found similar values (22.3 and 22.7) in beef and rabbit meat, respectively.

This constant ratio of creatine to net muscle protein could be of practical importance as an index of meat quality. The amount of net muscle protein is used in West Germany as an indicator of meat quality under the name BEFFElO ('Bindegewebeeiweissfreies Fleischeiweiss'), and is described on the basis of total nitrogen and hydroxyproline analyses. The creatine content could be a simpler indicator of this index of quality, at least for raw meat.

Similarly, it might help in the understanding of urinary creatinine levels as an index of lean body mass.ll Assuming that creatinine excretion depends upon muscle mass, the creatinine excretion (kg-l body weight) is much less in infants and young children than in adults. Since excreted creati- nine is derived from muscle creatine, its level will depend upon the net muscle protein rather than the muscle mass which, in infants, is lower because infantile muscle contains more collagen than adult.

References 1. 2.

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4. 5.

6. 7. 8.

9.

10. 11.

Dahl, 0. Creatine content as an index of the quality of meat products. J. Agric. Food Chem. 1963, 11, 351-355. Dahl, 0. Possibility of using creatine content as an index of the quality of meat products. 8th Europ. Congress Meat Res. Inst. 1962, No. 10, Moscow. Dahl, 0. Estimating protein quality of meat products from the content of typical amino acids and creatine. J . Sci. Food Agric. 1965, 16, 619-621. Hunter, A. Creatine and Creatinine Longmans, Green and Co., London, 1928. Khan, A. W.; Cowen, D. C. Rapid estimation of muscle proteins in beef-vegetable protein mixtures. J. Agric. Food Chem. 1977,25,236-238. Dvoiak, Z. Kreatin a kreatinin v mase a masnfch v);rohcich. Primyslpotravin 1961,12, 381-384. Dvoiak, Z. ZavdreEna zprava. Zpravy Vjhkumne'ho Bstavu masne'ho prdmyslu v Brnd 1976, No. 14. ISO, Meat and meat products-Determination of hydroxyproline content (reference method). Draft Znter- national Standard 1977, ISO/DIS, 3496.2. Palanskl, 0. Studium fysikAlnich, chemickgch a biochemickfch charakteristik v kostrovem svalstve kralika domlceho v priebehu postnatllnbho vfvoja. Kandiddtskd prace Slovenske' vysoke' Skoly technicke' Bratislava, 1977. Neufassung der Fleischverordnung von 28 Marz 1973, Bonn, 1973. Waterlow, J. C. The assessment of protein nutrition and metabolism in the whole animal, with special reference to man. In Mammalian Protein Metabolism Vol. 3 (Munro, H. N., Ed.), Academic Press, New York, 1969, pp. 325-390.