Observations on rigor mortis in whale muscle

VOL. 9 ti952)

OBSERVATIONS

BIOCHIMICA ET BIOPHYSICA ACTA

ON RIGOR

MORTIS

IN WHALE

127

MUSCLE

by B. B. MARSH*

Low Temperature Station/or Research in Biochemistry and Biophysics, University o~ Cambridge and Department o/Scientific and Industrial Research, Cambridge (England)

Various physical a n d biochemical changes in fresh whale muscle have been studied, as p a r t of a n i n v e s t i g a t i o n of the lean m e a t from A n t a r c t i c whales. A l t h o u g h for the most p a r t this work, which was carried out on b o a r d a w h a l i n g factory ship, has merely e x t e n d e d to whales o b s e r v a t i o n s p r e v i o u s l y m a d e on other m a m m a l s , it has at the same time revealed some i n t e r e s t i n g differences b e t w e e n the muscles of the whale a n d those of the species more generally used in l a b o r a t o r y experiments. F r e s h whale muscle is u s u a l l y either dry, glistening a n d firm, or wet a n d dull, with a d i s r u p t e d a p p e a r a n c e ; i n a third, a n d less u s u a l state, it is dry, h a r d a n d r u b b e r y . The i n v e s t i g a t i o n s described here i n d i c a t e t h a t these c o n d i t i o n s are m a n i f e s t a t i o n s of differe n t stages of the onset of rigor mortis. T h e appreciable decrease i n fluid r e t e n t i o n which accompanies these changes has since been e x a m i n e d in r e l a t i o n to synaeresis i n muscle fibres, a n d forms the s u b j e c t of a later c o m m u n i c a t i o n . EXPERIMENTAL

Materials. Blue (Balaenoptera musculus) a n d F i n (B. physalus) whales provided most of the e x p e r i m e n t a l material, a l t h o u g h a few o b s e r v a t i o n s were m a d e on the muscles of S p e r m whales (Physeter Catadon). The longissimus dorsi a n d psoas muscles were used, a n d a l t h o u g h samples were t a k e n as soon as the muscles were exposed, d u r i n g the flensing o p e r a t i o n on the deck of the factory vessel, great v a r i a t i o n s were e n c o u n t e r e d because of the v a r i a b l e d u r a t i o n of the chase a n d d e a t h struggle, a n d the differences in times p o s t - m o r t e m . Thus, while some whales were killed w i t h i n a few seconds of the b e g i n n i n g of the chase, others s u c c u m b e d only after a struggle of several hours; in addition, the times p o s t - m o r t e m before the muscles b e c a m e accessible v a r i e d from one to over t w e n t y hours. D u r i n g these periods the b o d y t e m p e r a t u r e of a b o u t 35 ° C was m a i n tained, because of tile great b u l k a n d the protective layer of b l u b b e r . 3/Iethods. pI-I was determined with the glass electrode after grinding 1-2 g muscle in io ml o.oo 5 M iodoacetate, and buffering capacity by successive pH measurements on the brei after addition of o.5 ml aliquots of o.o5 N NaOH or HCI over the pH range 5-8. Lactic acid was estimated on the same brei, after deproteinization with lO% (w/v) trichloroacetic acid (TCA), by the method of FRIEDEMANN, COTONIOAND SHAFFER2. On another TCA extract prepared directly from the whole muscle, phosphorus was determined by the method of ALLEN3; colour development in the perchloricmolybdate reagent was permitted to continue for 20-30 minutes to complete the hydrolysis of " Present Address: Dominion Laboratory, Department of Scientific and Industrial Research, Wellington, New Zealand.

Re]erences p. I32.

128

B.B. MARSH

VOL. 9 (I952)

creatine phosphate. The acid-labile phosphate of adenosine triphosphate (ATP) was determined approximately, with inorganic and creatine phosphates, after hydrolysis in N HC1 for 7 rain at lOO% Nitlogen was estimated by the macro-iKjeldahl method. In order to follow the physical changes occurring in fresh muscle, a "penetrometer" was made from a io kg spring balance, to the pan support of which was attached vertically a blunt steel rod 15 cm × i cm. When the rod was pressed on the surface of a large block of muscle in a direction perpendicular to the axis of the fibres, the force applied was continuously recorded by the instrument until the rod, under increasing pressure, pierced the fibres, The maximum reading just prior to penetration thus provided a measure of the "hardness" of the muscle. RESULTS

I n view of t h e a b i l i t y of some w h a l e species to m a i n t a i n s t r e n u o u s s u b m a r i n e a c t i v i t y for c o n s i d e r a b l e p e r i o d s 4, 5 i t first s e e m e d d e s i r a b l e to a s c e r t a i n t h a t in whale muscle t h e p r o d u c t i o n of lactic acid b y a n a e r o b i c glycolysis resembles t h a t in o t h e r m a m m a l i a n muscle. This was done b y d e t e r m i n a t i o n of t h e " b a s a l " p H (BATE-SMITHS), a m e a s u r e of t h e p H in c o m p l e t e absence of l a c t i c acid which n o t only gives t h e l i m i t i n g p H of t h e r e s t i n g muscle b u t also, f r o m e x t e n t of its v a r i a t i o n , i n d i c a t e s if t h e p r o d u c t i o n of lactic acid is the p r i n c i p l e cause of p H change. F r o m e s t i m a t i o n s of lactic acid c o n t e n t , buffering c a p a c i t y a n d p H on a n u m b e r of s a m p l e s t h e results, s u m m a r i z e d in T a b l e I, were o b t a i n e d . T h e y i n d i c a t e t h a t t h e " b a s a l " p H of w h a l e muscle does n o t differ from t h a t of r a t muscle, in which BATE-SMITHs o b s e r v e d v a l u e s of 7.4-7.7, a n d t h a t lactic acid is t h e o n l y a c i d a c c u m u l a t i n g in q u a n t i t y , b e i n g sufficient to a c c o u n t for all o b s e r v e d p H values. A f t e r m a k i n g an a p p r o x i m a t e n e t c o r r e c t i o n of o.3 p H u n i t for loss of CO s a n d for d e c o m p o s i t i o n of A T P a n d c r e a t i n e p h o s p h a t e 7, t h e p r o b a b l e resting p H of w h a l e muscle is f o u n d to b e 7.2-7.4. Sufficient l a c t i c a c i d was p r o d u c e d to give a p H in full rigor (the " u l t i m a t e p H " ) of 5.7 + 0.2.

THE " B A S A L "

Species

Sperm Blue Fin

TABLE I pH oF WHALE

NO.

MUSCLE

o/ analyses

Mean "basal pH"

4 9 7

7.68 ± o-o7 7.6o ± o.o8 7-44 ± °-13

I n c i d e n t a l to these d e t e r m i n a t i o n s of b a s a l p H , a high buffering c a p a c i t y was obs e r v e d in t h e muscles of b a l e e n (Blue a n d Fin) whales. To p e r m i t c o m p a r i s o n w i t h p u b l i s h e d d a t a for o t h e r species, buffering c a p a c i t y has been c a l c u l a t e d for t h e p H r a n g e 6 - 7 (Table II). W h i l e s p e r m whale muscle has a v a l u e c o m p a r a b l e w i t h t h a t of o t h e r m a m m a l i a n muscle, in which BATE-SMITHs r e p o r t e d a range of 5.3-5.8 m i l l i - e q u i v a l e n t s b a s e / I o o g, t h e m u s c u l a r tissue of b a l e e n whales buffers a b o u t 25 % m o r e effectively. This high v a l u e is a s s o c i a t e d w i t h an i n c r e a s e d n o n - p r o t e i n n i t r o g e n c o n t e n t , which was f o u n d to r e p r e s e n t 18 ~ 2 % of t h e t o t a l nitrogen, or 0.65 % of t h e whole muscle, c o m p a r e d w i t h a b o u t 0.4% for r a t a n d r a b b i t muscle s . As w i t h o t h e r species, m a x i m u m buffering p o w e r was e x e r t e d a t a p H of a b o u t 6. 9. A l t h o u g h no t r a n s i t i o n r e s e m b l i n g t h e onset of rigor m o r r i s h a d been o b s e r v e d prev i o u s l y in w h a l e muscle, an i n d i c a t i o n t h a t such a c h a n g e s h o u l d occur was p r o v i d e d b y Re[erences p. x32.

VOL. 9 (1952)

t~IGOR MORTIS IN WHALE MUSCLE

129

TABLE II THE

BUFFERING

POWER

No. Species

OF W H A L E

MUSCLE

o/analyses

Mean buUering power (milli-equivalents base/Ioo g muscle)

4 5 7

5.2 i 0.5 6.8 4- 0.7 6.7 ± 0.5

Sperm Blue Fin

phosphate ester analysis. Table I I I , illustrating the relation found between pH and ATP, shows that a high ATP content is maintained until a pH below 6.2 is reached, after which ATP decomposition is rapid relative to pH decrease. Since the ultimate pH did not differ greatly from 5.7, this result provides an interesting extension of the experiments of BATE-SMITH AND BENI)ALL ~, who showed that, for rabbit muscle having this ultimate pH, the phase of rapid ATP decomposition (and onset of rigor mortis) coincides with the attainment of a p H of about 6.o. TABLE III TIlE

pH

AND

ATP

CONTENT

OF 13ALEEN W H A L E

No. o! analyses

A TP (rag acid-labile P/g muscle)

7.4-7.0 7.0-6.6 6.6-6.2

2 8 7

6.2-5.8

IO

5'8--5'4

3

0.37 0.35 4- 0.08 0.32 ± 0.07 O.IO ~ O.IO O'03

p H range .

MUSCLE

Attention was directed at this point of the investigation towards the dry and wet states in which whale muscle was usually observed, and complete correlation between the physical state and pH was found. Those muscles with a pH greater than 6.3 were invariably of the dry type, while only wet muscle was observed when pH values below 6.1 were recorded. In the pH range 6.1-6. 3 both wet and dry muscles were encountered. Thus while a borderline pH of about 6.2 separated wet and dry muscle, a similar pH coincided with the commencement of the rapid phase of ATP decomposition. The results strongly suggested that the dry and wet states correspond respectively to muscle before and after the onset of rigor mortis, which is determined by the ATP content% To confirm this view, experiments were conducted to detect the transition to wetness with onset of rigor. One series of observations is shown in Table IV, while the results of another experiment are illustrated in Fig. I, in which the graphical representation employed by BATESMITH AND BENDALL9 is followed ; in each case a 2o kg block of dry muscle, initially at a temperature of 35 °, was held at a laboratory temperature of 22 °, and samples for analysis were taken from a depth of several cms. It is clear that the increase in resistance to penetration parallelled the dephosphorylation of ATP, the physical appearance of the muscle changing at the same time to that described as "rubbery". ReJerenees p. ~32.

B. B. MARSIt

13o

VOL. 9 (1952)

TABLE IV RIGOR

Time post-mortem

IN

XVHALE

MUSCLE

A TP

Hardness

pH

Hours

9.5 lO.2 lO.8 11. 7 14.1

MORTIS

mg laWe

%

P/g muscle

initial

g

maximum

0.45 o.43 o.33 0.33 o.io

ioo 95 73 73 22

750 875 15oo 2125 325 °

23 27 46 65 ioo

6.72 6.39 6.28 6.18 5.84

%

Visual o b s e r v a t i o n s were m a d e for a t o t a l of 72 hours in t h e e x p e r i m e n t r e p r e s e n t e d in Fig. I. T h e " r u b b e r y " c o n d i t i o n was s u p e r s e d e d b y a slow r e t u r n to low (56%) penet r o m e t e r readings and, b y t h e 63rd h o u r ~ loc p o s t - m o r t e m , t h e muscle was quite m o i s t on freshly-cut surfaces, b u t was X • n o t so wet as m a n y muscles o b s e r v e d pH on fresh carcasses. The o b s e r v a t i o n s of r, oc 5.8 ROBINSON 1, who f o u n d t h a t t h e a m o u n t of e x u d e d fluid increased w i t h increas5.0 ing d e p t h into the muscle, suggest t h a t t h e degree of wetness of whale muscle 5.2 6C is l a r g e l y d e t e r m i n e d b y t h e t e m p e r a 5.4 t u r e a t t h e onset of rigor, an effect comparable with the increased shorten5.6 ing in rigor w i t h increase of t e m p e r a 40 t u r e of r a b b i t muscle 7. A t t e m p t s to 5.8 c a r r y o u t rigor e x p e r i m e n t s on whale muscle at 37 ° were, however, d e f e a t e d o z0 b y r a p i d bacterial putrefaction. 2~ I n c i d e n t a l to t h e m a i n i n v e s t i g a z2 tion, t h e e x p e r i m e n t i l l u s t r a t e d in Fig. ® o PH , i r e v e a l e d an effect which a p p e a r s to be p e c u l i a r to whale muscle. T h e p H lb 2b 3b Hours posf-mortern r e m a i n e d a t 7.20 from 2 ~ - I 8 ~ hours p o s t m o r t e m , a n d t h e n d e c r e a s e d at a Fig. i. The onset of rigor morris in whale muscle r a t e c o m p a r a b l e w i t h t h a t o b s e r v e d in r a b b i t muscle in p o s t - m o r t e m glycolysis 7. I n a n o t h e r series of o b s e r v a t i o n s a t l a b o r a t o r y t e m p e r a t u r e t h e p H d e c r e a s e d from 6.67 to 6.50 from t h e second to t h e n i n e t e e n t h h o u r p o s t - m o r t e m , a n d rigor m o r t i s was n o t d e t e c t a b l e till 5 ° hours p o s t - m o r t e m . Single p H d e t e r m i n a t i o n s on muscles r e c e n t l y r e m o v e d from t h e carcass also suggested a " s t a t i o n a r y p h a s e " of p H , since values of 7.0-7.3 were s o m e t i m e s r e c o r d e d after a postm o r t e m i n t e r v a l of 6 hours, d u r i n g which t i m e a t e m p e r a t u r e of a b o u t 35 ° h a d been m a i n t a i n e d . Because a q u i t e a p p r e c i a b l e r a t e of decrease of p H was o b s e r v e d in some cases (for instance, t h a t q u o t e d in T a b l e IV), it a p p e a r s t h a t lactic acid p r o d u c t i o n m a y be g r e a t l y suppressed, a n d a t t i m e s c o m p l e t e l y arrested, in pre-rigor whale muscle.

/

i

Re]erences p. 132.

VOL. 9 (1952)

RIGOR gORTIS IN WHALE MUSCLE

131

DISCUSSION

For the most part the results of this investigation indicate that in most respects the post-mortem behaviour of whale muscle is very similar to that of the muscle of other mammalian species. This conclusion is of some interest since the physiological processes of respiration and locomotion in the whale have appeared sufficiently unusual to merit considerable research, despite the difficulties of studying a m a m m a l of such great bulk and unusual habitat. The striking transition of whale muscle during the onset of rigor mortis from a dry state to one of extreme wetness is a change which, in other species, is scarcely detectable. T h a t such a transition does occur in other muscle, however, has been shown in a study 1° which the present work suggested; the dephosphorylation of ATP now appears to be a factor of some importance in determining the extent of fluid release by muscle, the diminution in fluid-retaining ability due to ATP decomposition being of a magnitude similar to that caused b y normal post-mortem acid production 11. The unusually high buffering capacity of baleen whale muscle, probably owing to an exceptionally high content of carnosine and anserine 12, m a y be related to the extended periods of anaerobic activity demanded by the diving habits of these animals. In whales capable of submarine movement for fifteen minutes or more s an increase in muscle buffering power of about 25 % would permit, for the same fall in pH, a prolongation of several minutes in underwater activity. Since the Sperm whale can remain submerged for even longer periods 4, its muscles might be expected to exert an even higher buffering power, but it appears, instead, to prolong its submarine movement by a great increase in myoglobin. CASE1 has found that Sperm whale muscle contains almost 5 % of myoglobin, compared with only 0.6 % in the muscles of baleen whales. In addition the Sperm whale does not attain the speeds of which the Blue and Fin species are capable, owing perhaps to an inability to support the necessary rate of anaerobic expenditure of energy. Evidence has been presented to show that the production of lactic acid in fresh whale muscle m a y be completely arrested for a long period during which, despite the absence of ATP resynthesis by anaerobic glycolysis, the muscle remains in its dry prerigor condition with a correspondingly high ATP content. No comparable p H arrest has been detected in other whole m a m m a l i a n muscle, although a t e m p o r a r y cessation of acid formation, and sometimes a slight rise in pH, has been observed in minced horse muscle in oxygen xS, the fine state of sub-division ensuring a ready diffusion of oxygen throughout the material. The samples of whale muscle, however, were taken from a depth of several centimetres where oxygen diffusing inward from the surface could not penetrate 1~, and since the stationary p H effect was observed in baleen whale muscle of myoglobin content about 0.6 % - - a value frequently exceeded in horse muscle 15 - - the oxygen retained by that protein cannot explain the observations. A fuller investigation of the effect has been prevented by the difficulty of obtaining fresh whale muscle. The author is grateful to United Whalers L t d for the facilities provided aboard their whaling factory ship " B a l a e n a " ; to Dr J. G. SHARP, of this laboratory, who was largely responsible for organising the research programme; and to Mr G. S. BEATTIE, M.R.C.V.S., D.V.S.M., of the Ministry of Agriculture and Fisheries, for his interest and advice during the investigation. The work described in this paper was carried out as part of the programme of the Food Investigation Organisation of the United Kingdom, Department of Scientific and Re/erences p.

I32.

I32 Industrial

I~. B. lVfARSH Research,

during the author's

of Scientific and Industrial

secondment

VOL. 9 (1952) from the New Zealand Department

Research.

SUMMARY T h e o n s e t of rigor m o r r i s in baleen w h a l e m u s c l e h a s been i n v e s t i g a t e d . A t e m p o r a r y , t h o u g h s o m e t i m e s prolonged, a r r e s t of lactic acid f o r m a t i o n m a y precede t h e p h y s i c a l change. T h e l a t t e r is a c c o m p a n i e d b y a considerable decrease in fluid r e t e n t i o n a n d a r e l a t i v e l y r a p i d t r a n s i t i o n to a s t a t e of wetness. R~SUMI~ O n a 6tudi6 le c o m m e n c e m e n t de "rigor m o r t i s " d a n s le m u s c l e de la baleine. U n e i n t e r r u p t i o n t e m p o r a i r e m a i s parfois prolong6e p e u t se p r o d u i r e d a n s la p r o d u c t i o n de l'acide l a c t i q u e a v a n t q u e la m o d i f i c a t i o n p h y s i q u e c o m m e n c e . Cette dernibre e s t a c c o m p a g n 6 e d ' u n d 6 c r o i s s e m e n t consid6rable de la r 6 t e n t i o n de fluide et d ' u n e t r a n s i t i o n r e l a t i v e m e n t r a p i d e ~ u n 6 t a t d ' h u m i d i t ~ . ZUSAMMENFASSUNG D a s E i n s e t z e n der T o t e n s t a r r e i m W a l f i s c h m u s k e l w u r d e u n t e r s u c h t . B i n voriibergehender, doch m a n c h m a l l a n g e d a u e r n d e r S t i l l s t a n d der M i l c h s / i u r e b i l d u n g k a n n der p h y s i k a l i s c h e n A n d e r u n g v o r a n g e h e n . L e t z t e r e ist y o n einer b e d e u t e n d e n A b n a h m e der F l i i s s i g k e i t s - R e t e n t i o n u n d e i n e m verh~Lltnism~ssig r a s c h e n ~ b e r g a n g in den f e u c h t e n Z u s t a n d begleitet. REFERENCES 1 D . S . I . R . Special R e p o r t . I n p r e p a r a t i o n . 2 T. E. FRIEDEMANN, M. COTONIO, AND P. A. SHAFFER, J. Biol. Chem., 73 (1927) 335. 3 R. J. L. ALLEN, Biochem. J., 34 (194 °) 858. 4 p. V. SCHOLANDER,Hvalradets Skri/ter (Oslo), No. 22. 5 E. R. GUNTHER, Discovery Reports, 25 (1949) 113. (Cambridge U n i v e r s i t y Press). G E . C. BATE-SMITH, J. Soc. Chem. Ind., 67 (1948) 83. 7 E. C. BATE-SMITH AND J. R. BENDALL, dr. Physiol., IiO (1949) 47. 8 E. C. BATE-SMITH, J. Physiol., 92 (1938) 336. 9 E . C. BATE-SMITH AND J. R. BENDALL, J. Physiol., 106 (1947) 177. 10 B. B. MARSH, Nature, 167 (1951) lO65. 11 W. A. E~PEY, J. Soc. Chem. Ind., 52 (1933) 23oT. 1~ E. C. BATE-SMITH AND J. G. SHARP, Food Manu[acture, 21 (1946) 371. la R. A. LAWRIE, P r i v a t e C o m m u n i c a t i o n . 14 A. V. HILL, Proc. Roy. Soc., B, lO 4 (I929) 39. 16 R. A. LAWRIE, dr. dgric. Sci., 4 ° (195 o) 356. Received

November

22nd, 1951