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thesis entitled
CHEMICAL AND HISTOCHEMICAL OBSERVATIONS‘kON L. W
MUSCLE FROM BEEF AND PORK
presented by

Clarence Eugene Bodwell

has been accepted towards fulfillment
of the requirements for

Ph.D. degree in Food Science

' Majgr professor

Date Se t. 25 1964

 

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ABSTRACT
CHEMICAL AND HISTOCHEMICAL OBSERVATIONS ON L, DORSI‘
iMUSCLE FROM BEEF AND PORK

by Clarence Eugene Bodwell

Chemical changes in.l,.gg£§i_muscle of five beef carcasses were fol-
lowed from less than 10 minutes after death through 20 days post-mortem.

In addition, the activity of 17 specific enzymes was followed by histochemi-
cal procedures.

Initial levels of pH, glycogen and creatine phosPhate were similar
to previously reported values. ATP levels were more accurately estimated
by an enzymic method than by acid hydrolysis procedures. Results suggest
that the onset of rigor was initiated at about 12-15 hrs post-mortem. Ap-
proximately ZOZ and 3% of the initial glycogen remained at 96 and 480 hrs
post-mortem, resPectively. Glycogen was stoichiometrically degraded to
lactic acid and reducing sugars under post-mortem conditions.

The histochemical activity of lactate dehydrogenase, alpha-glycero-
phosphate dehydrogenase, succinate dehydrogenase, isocitrate dehydrogenase,
TPN diaphorase and DPN diaphorase showed a steady decrease with increasing
times post-mortem. Reactions for both alcohol dehydrogenase and glutamate
dehydrogenase were very weak or absent at 48 hrs post-mortem and all sub-
sequent sampling periods.

Positive reactions for glucose-6-ph03phate dehydrogenase and beta-
hydroxybutyrate dehydrogenase were observed in the initial samples only.
Acid phoSphatase, leucine amino peptidase, and 6-phosphogluconate dehydro-

genase were not detected in any of the samples.

‘ .

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Clarence Eugene Bodwell

Chemical and histochemical procedures similar to those described for
beef were used to follow the changes from O to 24 and/or 48 hrs post-mortem
in the l, ggrgi muscle of eighteen pork carcasses. One side from each of
thirteen pork carcasses was placed at -29°C, while the other side from
each carcass was subjected to 37°C for the first 4 1/2-5 hrs post~mortem
in an attempt to induce soft, watery pork.

The muscles of the pork carcasses studied exhibited marked differences
in initial levels of pH, glycogen, glucose and lactic acid. Post-mortem
accumulation of glucose and lactic acid was similar to that noted in beef
-muscle. A similar stoichiometric relation to that found in beef muscle
between post-mortem decrease in glycogen and the correSponding accumulation
of lactic acid and glucose existed in the carcasses from Poland China pigs.
The equivalent relationship for the carcasses from Hampshire pigs was less
apparent.

The 37°C treatment rarely induced soft, watery muscle as was expected
from reports of previous workers. It was concluded that a low pH at a
high muscle temperature pg; se was not a causal factor in the developement
of the soft, watery condition. The loss in fibrillar water-binding capacity
as the result of low pH values at high muscle temperatures reported by pre-
vious workers was confirmed. In contrast to previous studies, decreases
in fibrillar water binding capacity induced by the low pH-high temperature
phenomenon did not usually result in soft, watery muscle. Although the
loss in fibrillar water-binding capacity is a characteristic encountered
in soft, watery muscle, this characteristic by itself is not the only causal

factor in the developement of this condition.

     

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Clarence Eugene Bodwell

The activity of lactate dehydrogenase, alpha-glycerophosphate dehy-
drogenase, succinate dehydrogenase, isocitrate dehydrogenase and DPN diar
phorase was slightly weaker at both 0 and 24 hrs than similar activity in
beef muscle. Alcohol dehydrogenase activity was weaker than that observed
for the above enzymes, while the activity of glutamate dehydrogenase was
of intermediate intensity. Glucose—6-phosphate dehydrogenase and 6-phos-
phogluconate dehydrogenase showed traces of activity in only a portion of
the initial samples. Similarly, weak reactions for beta-hydroxybutyrate
dehydrogenase were observed only at 0 hrs post-mortem. None or only weak
activity was observed for malic dehydrogenase in all samples studied.
Likewise, TPN diaphorase activity was unexplainably absent from all sam-
ples. Moderate levels of cytochrome oxidase activity were observed at
both 0 and 24 hrs post-mortem.

UDPG-glycogen transferase was completely inactivated by the 37°C
treatment, but the treatment had a less marked effect upon phosphorylase
and branching enzyme. The presence of acid and alkaline phosphatase acti—
vity in muscles of 7 pork carcasses suggested that a degenerative condition

existed in these muscles.

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Clarence Eugene Bodwell

The activity of lactate dehydrogenase, alpha-glycerophosphate dehy—
drogenase, succinate dehydrogenase, isocitrate dehydrogenase and DPN dia-
phorase was slightly weaker at both 0 and 24 hrs than shnilar activity in
beef muscle. Alcohol dehydrogenase activity was weaker than that observed
for the above enzymes, while the activity of glutamate dehydrogenase was
of intermediate intensity. Glucose-6-phosphate dehydrogenase and 6-phos-
phogluconate dehydrogenase showed traces of activity in only a portion of
the initial samples. Similarly, weak reactions for beta—hydroxybutyrate
dehydrogenase were observed only at 0 hrs post-mortem. None or only weak
activity was observed for malic dehydrogenase in all samples studied.
Likewise, TPN diaphorase activity was unexplainably absent from all sam-
ples. Moderate levels of cytochrome oxidase activity were observed at
both 0 and 24 hrs post-mortem.

UDPG-glycogen transferase was completely inactivated by the 37°C
treatment, but the treatment had a less marked effect upon phosphorylase
and branching enzyme. The presence of acid and alkaline phosphatase acti-
vity in muscles of 7 pork carcasses suggested that a degenerative condition

existed in these muscles.

 

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CHEMICAL AND HISTOCHEMICAL OBSERVATIONS ON L. DORSI

'MUSCLE FROM BEEF AND PORK

By

CLARENCE EUGENE BODWELL

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Food Science

1964

HUI (HM H.335! In'Jflf-I S-DETR‘.’

 

 

ACKNOWLEDGMENTS

The author expresses his most sincere appreciation and gratitude to
his major professor, Dr. A. M. Pearson, for his willing counsel, continé
uous encouragement, personal understanding, and patience throughout the
course of his Ph.D. studies. The author also expresses his appreciation
to Professor L. J. Bratzler for his advice, interest, and general esprit.

The general counsel of Dr. J. Wismer-Pedersen, and the advice of
Dr. R. A. Fennell in the histochemical portions of the author's research
studies are gratefully acknowledged.

The author also wishes to thank the other members of his guidance
committee, Dr. L. E. Dawson, Dr. E. J. Benne, and Dr. W. A. WOods.

Appreciation is expressed to Dr. B. S. Schweigert, Chairman of the
Department of Food Science, for his interest and stimulative encourage-
ment.

Special thanks are due to Mrs. Dora Spooner and Miss Anne Sanderson
for technical assistance with the chemical determinations.

Lastly, the author expresses his appreciation to his parents for
their encouragement, faith and assistance during the years of graduate

study.

ii

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TABLE OF CONTENTS

INTRODUCTION . o o . o . . . . . . o o . .

REVIEW OF LITERATURE . . . . . . . . . . .
Classification of Muscle . . . . . .
Chemical Composition of Muscle . . .
Structure and Muscle Contraction . .

General Structure . . . . . . .
Myofibrils and Filaments . . . .
The Sarc0plasmic Reticulum . . .
‘Muscle Contraction . . . . . . .
The Mechanism of Contraction . .
RigorMortiS............
Studies on Rabbit Muscle . . . .
The State of the Myofilaments in
The ”Resolution" of Rigor . . .
Rigor Mortis in Beef Muscle . . . . .
Rigor Mortis in Pork.Musc1e . . . . .
DaniSh StUdieS o o o o o o o o 0
British Studies . . . . . . . .
American Studies . . . . . . . .

Irish Studies . . . . . . . . .

Histochemistry of Striated Muscle . .

Mortis

0

‘Methods for Detecting Dehydrogenase Activity . . .

Mbthods for Detecting Respiratory Enzyme Activity
Methods for Detecting the Activity of Other Enzymes

Distribution of Enzymic Activity . . . . . . . . . .

EXPERIMENTAL PROCEDURES 0 o . . . o 0

iii

Page

19
22

22
26
27
29

3O
3O
31

31
32

37

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Page

Beef carcasses o o o o o o o o o o o o o o o o o o o o o o 37

Experimental Animals and Sampling Procedures . . . . . 37
Temperature Measurements . . . . . . . . . . . . . . . 38
:Measurement 0f PH 0 o o o o o o o o o o o o o o o o o 38
Chemical Analyses . . . . . . . . . . . . . . . . . . 39
StatiStical Analyses . o o o o o o o o o o o o o o o o 39
Histochemical Procedures . . . . . . . . . . . . . . . 40

Pork Carcasses

Experimental Animals 0 o a o o o o o o o o o o o o o o 45
Slaughtering and Sampling Procedures . . . . . . . . . 46
Temperature Treatments . . . . . . . . . . . . . . . . 48
Determination of Fibrillar water-Binding Capacity . . 48
Temperature, pH and Chemical Determinations . . . . . 50
HiStOChemiCal PrOCEdureS o o o o o o o o o o o k 0 o O 51
Statistical Analyses . . . . . . . . . . . . . . . . . 55
Panel Evaluation 0 o o o o o o o o o o o o o o o o o o 55

RESULTS AND DISCUSSION . O O O O I O O O O O I O O C O . 0 O C O 5 6

PosteMortem,Levels and/or Interrelationships of Chemical
Constituents, pH and Temperature in Beef Muscle . . . . . . 56

LeVElS Of PH 0 o o o o o o o o o o o o o o o o o o o o 56
Creatine Phosphate o o o o o o o o o o o o o o o o o o 56
ATP 0 O O O O O O O O O O O O O O O O O O O O O O O O 58
TSP and ortho P 0 O O O O O 0 0 O O O O O I O O O O O 59
GlyCOgen . . . . . . . . . . . . . . . . . . . . . . . 60
Glucose .. . . . . . . . . . . . . . . . . . . . . . . 61
Lactic Acid . . . . . . . . . . . . . . . . . . . . . 62
Temperature . . . . . . . . . . . . . . . . . . . . . 62
ATP, CP, and pH . . . . . . . . . . . . . . . . . . . 63
Lactic Acid and pH . . . . . . . . . . . . . . .
Glycogen, Lactic Acid and Glucose . . . . . . . . .

Chemical Constituents, Interrelationships and Properties of
Pork MUSCI-e O O O O O O O O O O O O O 0 O O O O O O O O O O 68

Carcasses 1 through 5 . . . . . . . . . . .
Muscle Temperatures . . . . . . . . . . . . . . . . 69
PrOperties of Fibrillar Protein . . . . . . . . . . . 75
Creatine Phosphate . . . . . . . . . . . . . . . . . . 78
ATP . . . . . . . . . . . . . . . . . . . . . . . . . 79
Glycogen, Glucose and Lactic Acid . . . . . . . . . . 81
Effect of Temperature on Glycogen, Glucose and Lactic

Acid Levels . . . . . . . . . . . . . . . . . . . . 85
Degradation of Glycogen . . . . . . . . . . . . . . . 87

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Histochemical Observations in Beef Muscle . . . . . . . . . 88

Glycolytic Enzymes . . . . . . . . . . . . . . . . . . 90
Enzymes of the TCA Cycle . . . . . . . . . . . . . . . 90
Other Enzymes . . . . . . . . . . . . . . . . . . . . 93
General Observations . . . . . . . . . . . . . . . . . 95

Histochemical Observations in Pork Muscle . . . . . . . . . 95

TCA Cycle Enzymes . . . . . . . . . . . . . . . . . . 97
Glycolytic Enzymes . . . . . . . . . . . . . . . . . 97
Electron Transport Enzymes . . . . . . . . . . . . . . 97
Other Metabolic Enzymes . . . . . . . . . . . . . . 100
Acid and Alkaline Phosphatases . . . . . . . . . . . 100
UDPG— —Glycogen Transferase, PhosphorylaSe and Branching
Enzyme Activity 1 . . . . . . . . . . . . . . . . . 102
General Observations . . . . . . . . . . . . . . . . . 105

SUMMARY

Studies on Beef . . . . . . . . . . . . . . . . . . . . . . 106

Pork Studies . . . . . . . . . . . . . . . . . .

BIBLIOGRAPHY . . . . . . . . . . . . . . . . . .

APPENDIX . . . . . . . . . . . . . . . . . . . . . . . .

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LIST OF TABLES
Page

Approximate sampling times (mins post-mortem) in pork
carcasses . . . . . . . . . . . . . . . . . . . . . . . . 47

Temperature treatments (Pork carcasses) . . . . . . . . . 49

Values for chemical constituents and pH of beef muscle at
various periods post-mortem . . . . . . . . . . . . . . . 57

Average post-mortem temperature of the l. dorsi muscle of
five beef carcasses . . . . . . . . . . . . . . . . . . . 63

 

Levels of ATP, CP, and pH during the initial 48 hrs post-
mortem . . . . . . . . . . . . . . . . . . . . . . . . . 64

Post-mortem pH and ultimate muscle condition as affected
by temperature treatments of carcasses 6 through 10 and
llY I O O I I O I O 0 a O Q l O O O U 0 O O I O l O O O I 69

Fibrillar protein content and water binding of washed
fibrils from 24 hr post-mortem samples of carcasses 6
through 10 and llY . . . . . . . . . . . . . . . . . . . 76

Post-mortem levels of ATP and pH . . . . . . . . . . . . 80
Means and standard deviations of pH, glycogen, glucose and
lactic acid at various times post-mortem as related to
temperature treatments for carcasses of groups 1 and 2 . 82
Means and standard deviations of sums of glycogen, glucose
and lactic acid and estimates of measuring error for each
constituent . . . . . . . . . . . . . . . . . . . . . . . 87

Relative enzyme activity in l, dorsi muscle from beef . . 89

Relative enzyme activity in l. dorsi muscle of carcasses
1 through 10 and llY . . . . . . . . . . . . . . . . . . 96

Levels of UDPG-glycogen transferase, phosPhorylase, and

branching enzyme in carcasses A through G at 3 hrs post-
mortem . . . . . . . . . . . . . . . . . . . . . . . . . 104

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LIST OF FIGURES AND PLATES
Page

The relationship between pH and lactic acid in beef
1. dorsi muscles . . . . . . . . . . . . . . . . . . . . 66

The relationship between post-mortem levels of glycogen,
lactic acid and glucose in beef muscle (glucose, which
includes total reducing sugarg and glycogen is expressed

in glucose equivalents) . . . . . . . . . . . . . . . . . 67

Post—mortem temperature of pork l. dorsi muscle of repre-
sentative sides subjected to —29°C or 37°C temperatures
immediately after death . . . . . . . . . . . . . . . . . 70

Relation between pH, temperature treatment and ultimate
muscle condition . . . . . . . . . . . . . . . . . . 74

Effect of temperature treatment on levels of glycogen,
glucose and lactic acid in carcasses A through G (Group 2) 86

L, dorsi muscle from beef. Lactate (I), alpha-glycero-

phosphate (II), and alcohol (III) dehydrogenase activity
at O (A), 48 (B) and 480 (C) hrs post-mortem (x 225). . . 91

 

L. dorsi muscle of beef. Isocitrate (I) succinate (II)

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and glutamate (III) dehydrogenase activity at O (A), 48 (B)
and 480 (C) hrs post—mortem (X 225). . . . . . . . . . 92

 

L. dorsi muscle from beef. TPN diaphorase (I) and DPN
diaphorase (II) activity at 0 (A), 48 (B) and 480 (C)

hrs post—mortem: glucose— 6- -phosphate (III, A) and beta-
hydroxybutyrate (111,3) dehydrogenase activity at 0 hrs

post-mortem. Magnificantion: 225X. . . . . . . . . . 94

 

L. dorsi muscle from pork. Isocitrate dehydrogenase (I),
succinate dehydrogenase (II) and DPN diaphorase (III)
activity at 0 (A) and 24 hrs post- mortem from carcasses
subjected to the —29°C (B) and 37°C (C) treatments (x 225) 98

L dorsi muscle of pork. Alpha- glycerophOSphate (I) and
lactate (II) dehydrogenase activity at O (A) and 24 hrs post—
mortem in -29° C (B) and 37° C (C) treatments (x 225); cytochrome
oxidase activity (III) A) at 0 hrs (A) post-mortem (x 860);

and Aniline Blue- -0range C stained sections from 24 hrs post-
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(x 225) . . . . . . . . . . . . . . . . . . . . . . . . 99

 

 

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L, dorsi muscle of pork: varied levels of acid phosphatase
activity in isolated fibers (I, A,B, and C; X 225); strong
acid phosphatase activity in degenerating fiber (II, A;

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ment of cross-linkages between fibers at points of high
activity (III, B; X 860).

viii

 

  
   

  
        
       
   

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Appendix A
Table

Table

Appendix B
Table

Table

Table

LIST OF APPENDIX TABLES
Page
Sources of chemicals for histochemical procedures 125
Post-mortem levels of chemical constituents, and

pH for individual beef carcasses at various periods
Post—mortem . . . . . . . . . . . . . . . . . . . 126

Sampling locations on pork carcasses . . . . . . 128

Post-mortem levels of pH, glycogen, glucose, lactic
acid, ATP, fibrillar protein content, and water-
binding capacity for each side of pork carcasses

1 through 10 and llY. . . . . . . . . . . . . . . 129

Post-mortem levels of glucose, glycogen, lactic

acid, ATP and pH for each side of carcasses A through
G . . . . . . . o . . . . . . . . . . . . . . . . 136

ix

 

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INTRODUCTION

The phenomenon of rigor mortis is extremely important in the trans-
formation of living muscle into meat. In beef muscle, the chemical and
physical changes associated with rigor mortis and the subsequent period
of post—rigor aging or ripening are believed to have a strong influence
on various qualitative attributes, i.e. color, tenderness, flavor, etc.
(Whitaker, 1959). How these changes are related to each of the various
quality factors has not been fully established. However, departures from
the norm in certain characteristics, which are related to the chemical
events involved in rigor, are known to result in correSponding undesirable
traits (Lawrie, 1962; Bendall, 1960).

Distinct variations in the rate and extent of certain chemical changes
during the post-mortem pre—rigor and rigor stages in pork muscle have been
shown to have considerable influence on the quality of the resulting meat
(Lawrie, 1962; Bendall, 1960). In particular, the occurrence of a low pH
while muscle temperature is high has been reported to produce soft, watery
pork (Bendall e£_g;, 1963; Wismer-Pedersen, 1959).

Although data are available on various post-mortem chemical and phy-
sical changes in both beef and pork muscle, the causal factors remain
largely unexplored. The post—mortem activity of oxidative and respiratory
enzymes in muscle would appear to be potentially relevant, but this has
not been studied to any extent. Given these considerations, the experi-

mental objectives of this study were:

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1. To determine the levels of certain chemical substances in beef
muscle from shortly after death to approximately three weeks post-mortem.

2. To observe the activity of various enzymes in beef muscle during
the same post-mortem period using histochemical methods.

3. To investigate similar chemical and histochemical characterise
tics during the first 48 hrs post-mortem in pork muscle using carcasses
subjected to controlled post-mortem temperature environments, which were
intended to either induce, accentuate, or retard the developement of

soft, watery muscle.

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REVIEW OF LITERATURE

CLASSIFICATION OF MUSCLE

Muscle is generally classified into three types--smooth, cardiac,
and voluntary or striated. Cardiac muscle is restricted to the heart,
while smooth muscle is found in the digestive, vascular, respiratory, and
other involuntary systems. The predominating musculature of the animal
body is striated muscle, and likewise, this type of muscle is the major
constituent of meat. For convenience, the term muscle as used herein

refers to striated or voluntary muscle.

CHEMICAL COMPOSITION OF MUSCLE

According to Bendall (1962), muscle is composed of approxfinately 1%
ash, 3-10% fat, 21—22% non-lipid organic matter, and 70—75% water. The
predominate inorganic constituents are P, K, Na, Mg, and Ca. Protein
makes up about 95% of the non—lipid organic matter and constitutes about
20% of the muscle mass. Contractile proteins contribute approximately 52%
of the total protein, while sarcoplasmic proteins constitute about 32%,
and the stroma (connective tissue) proteins about 5-6%.

About 11% of the total nitrogen in muscle is derived from non-protein
sources (Bendall, 1962). ATP (adenosinetriphosphate) and CP (creatine
phosphate) account for over half of the NPN (non-protein nitrogen). Di-
peptides, particularly anserine and carnosine, and free amino acids pro-
vide a small amount of NPN, but their concentration is highly variable
even within muscles of a given animal (Bendall, 1962; Bate Smith, 1938;

Davey, 1957, l960a,b).

3.152;». In no: 1.

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The approximate values given herein for various constituents of
'muscle are subject to variation from species to species and within muscles
of the same and/or different animals within a Species. However, such
values are in general representative of mammalian muscle (Bendall, 1962).
These broad groupings obviously ignore some biologically important sub-
stances, such as vitamins and hormones. Likewise, some relatively unex-
plored substances such as some proteins of presumed contractile origin

(Szent-Gygrgyi, 1960) are excluded.

STRUCTURE AND MUSCLE CONTRACTION

Several comprehensive discussions on various asPects of the structure
and function of muscle have been published CMommaerts, 1950, 1954; Szent-
Gygrgyi, 1951, 1953, 1960; weber and Portzehl, 1952; Dubuisson, 1954;
Bailey, 1954; Hamoir, 1955; Morales and Botts, 1956; weber, 1957; Huxley
and Hanson, 1960; Bennett, 1960; Needham, 1960; Bendall, 1962; 1963;
Venable, 1963). Accordingly, only a brief summary of muscle structure and

function has been included.

General Structure

 

On a gross level, muscle is composed of a mass of tissue subdivided
into increasingly smaller units by limiting interstitial and enveloping
connective tissue (Walls, 1960; Bloom and Fawcett, 1963). A muscle is
contained within the collagenous fascia or spimysium, which penetrates
into the muscle to form the perimysium. The perimysial stroma consists
of collagenous, elastic, and reticular fibers and a variety of connective

tissue cells. The perimysium separates the muscle into tertiary, secondary,

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and primary muscle bundles. The primary bundles are composed of varying
numbers of muscle cells or fibers.

In most mammalian muscles, the nuclei are located in the sarcoplasm
immediately beneath the sarcolemna. In addition to other entities, the
sarcoplasm also contains the mitrochondria, the glycolytic enzymes, myo-
globin, and sometimes glycogen granules and lipid droplets. The mitochon-
dria also contain the oxidative enzymes. The sarcoplasmic fluid surrounds
and fills the spaces between the highly organized contractile structures

of the muscle fiber--the fibrils or myofibrils (walls, 1960).

The Myofibrils and Filaments

When viewed in longitudinal section with a light microscope, the
fibrils appear as long, parallel threads (Walls, 1960; Bloom and Fawcett,
1963). A cross-banded or striated appearance results from the presence
of cylindrical segments of two main types, which alternate in repeating
units along the fibril. These two segments appear as a dark band or A
band, and a lighter band or I band. In the center of the 1 band is a
narrow dark line, the Z disc. The lighter areas of the I band, on either
side of the Z disc, are called the N bands. That portion of a fibril
located between adjacent Z discs represents the structural unit of the
fibril and is known as a sarcomere. 0n proceeding from one Z disc to an
adjacent Z disc, three bands are apparent, an N band, the A band, and a
second N band.

In electronmicrographs (H. E. Huxley, 1953,1957; Hodge £5 31., 1954;

Huxley and Hanson, 1957b, 1960), a lighter area centrally located in the

  

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A band and called the H band is apparent. The A and I bands are found
to be of almost equal length in a resting fibril.

Electron microsc0pe studies, together with data from.x-ray diffrac-
tion and phase contrast or interference microsc0py (Huxley and Hanson,
1954, 1957a; Hanson and Huxley, 1953, 1955; A. F. Huxley, 1957), proved
that the fibril consisted of an array of distinctly organized myofila-
ments or filaments. These filaments were of two general types, one being

about twice as thick as the other and shorter in length. These filaments,

both thick and thin, overlapped along part of their lengths in ordered

array within a sarcomere. This resulted in the banded or striated appear-
ance. Within the I band, only thin filaments, bisected by the Z disc
were present and within the H band only thick filaments. The portions of
the A band lying on either side of the H band contained overlapping, inter-
digited filaments of both types.

The thick filaments are composed of myosin and the thin filaments
of actin (Huxley and Hanson, 1960). A cross-section within the areas of
overlap in the A band shows that each actin filament is surrounded by
three filaments of myosin, while each myosin filament is surrounded by
six filaments of actin. When treated with tryosin, myosin is degraded to
two primary products--L or light meromyosin and H or heavy meromyosin
(Mihalyi and Szent-Gygrgyi, 1953). The H-meromyosin contains the actin-
combining and the ATPase prOperties of myosin. H-meromyosin has been
shown to be located in the H band, while bound ATP in resting fibrils is
found only in the L-meromyosin, which is located towards either end of

the A band (Marshall §£_§l., 1959). The myofilaments thus described

  

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provide the mechanical basis for muscle contraction.

The Sarcoplasmic Reticulum

Bennett (1960) reviewed descriptions of the sarcoplasmic reticulum,
which were published between 1888 and 1902. He remarked, "It is aston-
ishing that a structure once described as accurately and as beautifully
as the reticulum was ----- should have so quickly become almost lost to
man's knowledge". Textbooks, reviews and other papers were essentially
devoid of any reference to the sarcoplasmic reticulum when it was first
encountered in electromicrographs by Bennett and Porter (1953). Many
studies on the reticulum have since been published (Bennett, 1955, 1956;
Porter, 1956, 1961; Sjostrand, 1956; Porter and Palade, 1957). The reti-
culum consists of a network of membrane-limited tubules, which surround
each myofibril in a continuous system. The system is composed of trans-
verse and longitudinal tubules, which are inter-connected by thinner
tubules to form ”triads”. In mammalian muscle, two triads are found with-
in each sarcomere, each encircling the A band near an A band-I band junc-
tion. The triads of adjacent fibrils are inter-connected, while those of
fibrils adjacent to the sarcolemna come into close contact with the sarco-

lemna.

Muscle Contraction
The explanation of contraction has been wrought with much controversy
and opposing interpretations (Needham, 1960), but the primary questions

concerning the mechanical events appear to be largely resolved (Bendall,

    

 

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1963). In a sarcomere undergoing contraction, first the H band disappears
and then the N bands shorten (Huxley and Hanson, 1960). The length of
neither actin nor myosin filaments changG except upon extreme shortening,
when they are "obliged to do so for steric reasons occurring as a result
of shortening" (Huxley and Hanson, 1960). Thus, upon contraction the
thin actin filaments are pulled into the A band alongside or into the
thick myosin filaments. The degree ofinterdigitalp overlapping of the
two types of filaments increases as contraction progresses.

The primary contributions to an understanding of the chemical events
occurring in muscle contraction are summarized below:

In the presence of ATP, a solution of actomyosin decreases in vis-
cosity and turgidity due to dissociation of the protein into actin and
myosin. Upon depletion of the ATP, actomyosin is reformed (Engelhardt
and Ljubimova, 1939; weber, 1956; Gergely, 1956). In 1941, Szent-Gygrgyi
reported that actomyosin threads contract upon the addition of ATP (Need-
ham, 1960). Conversely, upon suppression of ATPase activity, the addition
of ATP induces extensibility of the threads. Pyrophosphate likewise
induces extensibility, but not contraction, and dissociates an actomyosin
solution into actin and myosin (Mommaerts, 1947). Hence, ATP was shown
to have a passive "plasticizing" effect on actomyosin in the absence of
active ATPase hydrolysis, while a reverse effect equivalent to contraction
occurred during hydrolysis of ATP.

The plasticizing influence was found to be effected only when Mg was
present in a defined concentration together with ATP in the form of a

Mg-ATP complex (Bendall, 1963; Engelhardt, 1946). Thus, in the absence

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of Mg and/or ATP, the actin and myosin filaments of the muscle become
rigidly attached forming an actomyosin complex. Conversely, the Mg-ATP
complex was shown to be necessary for contraction (Weber and WEber, 1951;
Hasselbach and Weber, 1953; Bendall, 1953, 1963). Mg-ATP was thereby
shown to perform two functions in the fibri1---a plasticizing function
essential for extensibility and to provide the active energy for con-
traction.

Marsh (1952) reported the presence of a relaxing factor in muscle,
which completely inhibits contraction if added to washed fibrils in the
presence of ATP and Mg° In the absence of the relaxing factor, contrac-
tion occurs. Ca ions suppress the effect of the relaxing factor (Bendall,
1953).

The relaxing factor is formed from granules of the triads of the
sarcoplasmic reticulum (Porter and Palade, 1957; Nagai E£.El-: 1962;
Porter, 1961; Ebashi, 1961b) and acts as an ATP-driven Ca-pump (Ebashi,
1961a,b; Hasselbach and Makinose, 1962). The reticulum also serves as
the conduction system for transferring impulses from the fiber membrane
into the individual fibrils (Huxley and Taylor, 1955).

Bendall (1963) has succinctly summarized the phenomenon of muscle

relaxation and contraction. He states, "

....the Marsh relaxing factor
organized in the muscle as the triads of the sarcoplasmic reticulum, ex-
ercises its control over the contractile filaments by pumping out all
traces of calcium from the fibrils. The opposite process, contraction,

can also be visualized as initiated by an ion-exchange process, origin—

ating at the fibre membrane as a wave of depolarization and travelling

 

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inwards, often at the level of the Z-discs at which the triads of the
reticulum are located in many muscles, as a wave of calcium ions, removed
from their exchange-sites by the ionic changes accompanying the depolari-
zation wave. The calcium ions then immediately stimulate the explosive
splitting of ATP by the actin and myosin filaments, and contraction ensues.
This process is reversed as the fibre-membrane is repolarized, the triads
of the reticulum recapture the Ca ions, ATP floods back into the system
and the conditions for relaxation are once more established.”

In View of more recent data (weber g£_21., 1963; Wéber and Herz, 1963),
this scheme requires clarification on two points. Firstly, the relaxing
factor achieves its relaxing effect by reducing the concentration of Ca
ions and not by removing these ions completely. Secondly, for contraction
to occur Ca ions must be present. In the presence of Mg, the ATPase acti-
vity of actomyosin results from the effect of the actin on myosin, which
greatly increases the relatively low rate of ATPase hydrolysis by myosin
alone. Myosin ATPase is unaltered by either the relaxing factor or by
the removal of Ca, while the opposite is true for actomyosin ATPase (Weber
gt a1., 1963). Actomyosin and myofibrils contract only when complexed
with Ca, and the complex is formed only when a defined concentration of
Ca ions are present in the surrounding medium (Weber and Herz, 1963).

The relaxing factor was thus shown to exert its effect through lowering
the concentration of ionized Ca below a critical level beyond which the
Ca-actomyosin complex dissociates. Since 0.1 micromole of Ca per gram
of muscle is sufficient to saturate the actomyosin, many of the earlier
Mg-ATP studies were unknowingly contaminated with sufficient Ca ions to

facilitate contraction.

  

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Earlier research on muscle contraction failed to prove the breakdown
of ATP during contraction (Bowen, 1951, 1954; Bowen and Martin, 1958;
Mommaerts, 1955; Hoeve gt_§g,, 1963). However, Cain and Davies (1962)
and Cain g; al., (1962) demonstrated ATP breakdown during a single con-
traction.

A somewhat shnilar but still unresolved problem is the identity of
the relaxing factor in chemical terms. Tentative identifications have
been made (Lorand st 31., 1957; Marsh, 1960; E113 and Faulkner, 1961;
Uchida and Mommaerts, 1963), but have been refuted (Briggs §£_al,, 1959;
Bendall, 1960; Parker, 1961; Lorand and Molver, 1962; Mommaerts 32 al.,

1963; Takauji and Nagai, 1963).

The Mechanism of Contraction

Theories have been advanced for explaining the interaction of actin
and myosin during contraction by Astbury (1947), Riseman and Kirkwood
(1948), Pauling and Corey (1951), Hill (1953), Huxley and Hanson (1954),
Huxley and Niedergerke (1954), Flory (1956a,b; 1957), and Davies (1963).
The sliding filament model of Huxley has been one of the more intricately
developed theories (A. F. Huxley, 1957; Huxley and Hanson, 1960; Needham,
1960). However, studies by Hoeve and Willis (1963) and Hoeve g£_§l.,
(1963) indicated that the only theory fully compatible with their data
on phase transitions of fibrils was that detailed by Flory (1956a,b, 1957).
Flory had suggested that a phase transition from an oriented, crystalline
condition to a random, amorphous state is reSponsible for contraction.

A more plausible and complete theory of contraction has been recently

advanced by Davies (1963). This theory satisfies the major postulates of

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the Huxley model as well as the requirement for a change of phase. Davies'
theory, which involves both sliding and folding, can be summarized as
follows:

Upon stimulation, Ca ions move from the sarcoplasmic reticulum to
polypeptides on the H-meromyosin units of the myosin filaments. The Ca
ions form chelate links between bound ATP on the end of the H-meromyosin
polypeptides and the bound ADP of F—actin filaments. The Ca ions neutra-
lize the electric charge on the bound ATP of the polypeptides, which
then Spontaneously contract to form an alpha-helix with the energy derived
from hydrogen bond and hydrophobic bond formation. The contraction drags
the actin filament along the myosin filament, which brings the ATP into
the range of action of the H-meromyosin ATPase. The ATPase cleaves off
the terminal phOSphate and breaks the link. Upon rephOSphorylation of
the ADP, the helix is pulled out to an extended chain by the repulsion of
the negative charge on the ATP and a fixed charge on the H—meromyosin.
Thus, the peptide extends towards and along the actin filament. The cycle
is repeated many times during a single contraction and steps when the
calcium is pumped back into the sarcoplasmic reticulum.

In a subsequent paper from the same laboratory (Iyengar EE.E£-: 1964),
an acto-H—meromyosin system was studied in which a polypeptide in the
meromyosin was observed to undergo the postulated change in conformation
under the conditions set forth in Davies' theory, i.e. an alpha-helix to
a mixture of random coil and extended beta-form type of conformational

change.

   

 

 

 

 

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RIGOR MORTIS

After death muscle eventually becomes stiff and inflexible, a condi-
tion known as rigor mortis. Rigor mortis, or rigor, can be more fully
defined as the collective chemical and physical events within the muscle
which result in a rigid, inextensible post-mortem condition.

Observations and investigations of rigor prior to the 1930's were
largely limited to those of forensic medicine. These were detailed in
various editions of Taylor's ”Manuel of Medical Jurisprodence" between
1844 and 1928 (Bendall, 1960). These early observations expounded the
belief that the stiffening process was due to a coagulation of the water-
soluble proteins of muscle. Coagulation was assumed to be caused by the
accumulation of lactic acid, which resulted from the post-mortem break-
down of glycogen. Although rigor had been observed to occasionally occur
at a low level of acidity (high pH or "alkaline" rigor), this was a rela-
tively unexplained phenomenon.

In 1877, Claude Bernard observed that ”alkaline” rigor developed
more rapidly than acid rigor (Bendall, 1960). In 1926, Hoet and Marks
described a similar phenomenon and suggested that a third change was in-

volved, which was common to both "alkaline” and acid rigor (Bendall, 1960).

Studies on Rabbit Muscle

From 1939 to 1951, four studies on rabbit muscle established a scien-
tific basis for the interpretation of the events occurring during rigor
mortis (Bate Smith, 1939; Bate-Smith and Bendall, 1947, 1949; Bendall,

1951). Bate Smith (1939) reported a study on the changes in extensibility

     
   

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of rabbit muscle undergoing rigor. This was the first attempt to relate
chemical changes to extensibility changes during the onset and develope-
ment of rigor. He concluded that as the onset of rigor occurred, a ten-
fold increase in elastic modulus was observed. Connective tissue in
muscle did not contribute to the stiffening during rigor. He found that
in normal muscle with not less than 0.7% glycogen, the onset of rigor
closely paralleled the production of lactic acid. No correlation was
found between these factors in muscles which contained insufficient gly—
cogen to bring the pH to 6.3 by production of lactic acid. The rapidity
of pH decrease and the rate of onset of rigor could be influenced by ante-
mortem exercise and/or nutritional state prior to death. He concluded
almost prophetically, that the onset of rigor "is related to the loss of
elasticity of the fibril, which must be associated with changes in con-
figuration of myosin”. The "myosin" of which Bate Smith (1939) spoke
included actomyosin. It was further postulated that these changes were

a result of the breakdown of the fiber membrane and the accompanying free
exchange of ions throughout the muscle.

Later, Erdos in Szent-GySrgyi's laboratory in Hungary demonstrated
that ATP destruction closely paralled the developement of rigor in the
rabbit (Bate-Smith and Bendall, 1947; Szent-Gygrgyi, 1947). Bate-Smith
and Bendall (1947) showed that the onset and developement of rigor mortis
was closely correlated with a decrease in ATP in both acid and "alkaline”
rigor. In acid rigor, pH at death also influenced the lag period before
onset of rigor, and the developement of rigor was closely paralleled by
a decrease in pH. Shortening occurred only if the pH was greater than

6.2 at the onset of rigor.

 

-15-

In a subsequent paper Bate-Smith and Bendall (1949) concluded that
ATP was necessary to prevent the muscle from passing into rigor regardless
of the pH immediately post-mortem. The ATP of post-mortem muscle had a
high rate of turnover and during the lag phase before onset of rigor, its
breakdown was balanced by resynthesis from the glycolytic cycle. The on—
set of rigor began when this balance was not maintained due to insuffi-
cient glycogen. The pre-rigor lag period was long, short, or very short
according to whether the muscles came from well—fed, starved, or exhausted
animals, respectively. The initial pH after death was dependent upon the
extent of struggling while the ultimate or final pH was determined by the
level of feeding or degree of fatigue of the annual before death. The
pH at onset of rigor was linearly related to the ultimate pH. Both the
lag period and the time required for the onset of rigor were markedly
increased by lowering the muscle temperature from 37°C to 17°C.

Bendall (1951) showed that although CP was the first chemical com-
pound to be broken down after death, it had no effect on the onset of
rigor except as a replenisher of ATP. ATP breakdown began only after 70%
or more of the CP had disappeared and proceeded relatively quickly there-
after, regardless of the rate of glycolysis. The overall rate of ATP
turnover depended on pH in a manner similar to that of myosin ATPase.

In animals killed by stunning and decapitation, little or no CP was pre-
sent immediately after death and the ATP level at the onset of rigor was
approximately 50% of the level immediately after death. The decline of
the ratio of resynthesis to rate of breakdown of ATP had a closer rela-

tionship to the shortening and extensibility changes than the fall in

 

-16-

ATP level pg; fig. The latter explained the phenomenon reported by Bate-
Smith and Bendall (1949) indicating that the ultimate pH and pH at the
onset of rigor were linearily related. Data from calculations of various
rate-ratios of the resynthesis and breakdown of ATP at different pH levels
was shown to account for the variations encountered in critical ATP level
with decreasing pH. The importance of the ATP rate-ratio was surmised

by Bendall (1951) to be due to the requirement of small but constant
levels of ATP at sites in the fibril to ensure a pre-rigor state. The
requirement was lower with lower pH levels. Although the theoretical
explanation was correct, Bendall (1960) concluded that the use of the

ATP rate-ratio concept was unnecessary in view of subsequent knowledge

on the plasticizing effects of the Mg—ATP complex. In the presence of
the Marsh factor, the ATP level required for a plasticizing effect had
been found to decrease with lower pH (Briggs and Portzehl, 1957). Ben-
dall (1960) concluded that onset of rigor occurs when the ATP level is
approximately 2 micromoles/g if the final pH falls below 6.3, but is about
4 micromoles/g if the pH fall is restricted.

Changes occurring during rigor mortis of other Species have been
found to generally adhere to the principles evolved in these studies.
Research on other species includes that on the draught horse (Lawrie 1953,
1955), whale (Marsh, 1952b), pork (Wismer-Pedersen, 1959), beef (Marsh,
1954; Howard and Lawrie, 1956, 1957), chicken (deFremery and Pool, 1960,
1963) and fish (Partmann, 1963).

However, two German researchers (Hamm, 1960; Partmann, 1963) have

disagreed with Bate-Smith and Bendall (1949) and Bendall (1951). They

 

 

-17-

have emphasized the role of the relaxing factor in delaying the onset of
rigor as opposed to Bendall's (1951, 1960) emphasis on the decrease in
ATP level. Hamm (1960) stated that the breakdown of ATP immediately after
death is inhibited by the presence of the relaxing factor. During the
first few hours post-mortem, the activity of calcium ions increases, and
the relaxing factor is then inhibited. The myofibrillar ATPase is fully
activated and the onset of rigor occurs. Partmann (1963) agreed and con-
cluded that the rapid phase of ATP breakdown commences only after the
relaxing factor is more or less inactivated by the calcium ions. He
suggests that the release of Ca ions and the consequent inactivation of
the relaxing factor appears to be correlated with membrane changes of the
fiber.

However, Bendall (1960) concluded that less than 3% of the ATPase
activity after death was due to actomyosin or myofibrillar ATPase. He
presented evidence indicating that the sarcoplasmic ATPase of Kielley
and Meyerhoff (1948) is the major, if not the only, active ATPase in
normal post-mortem rigor. Bendall (1960) further concluded that the myo-
fibrillar ATPase is activated only if the muscle is depolarized in some
way, such as in thaw rigor, and then only to a small extent. Bendall
(1963) gave further theoretical and experimental evidence for the passive

role of myofibrillar ATPase during normal rigor.

The State of the Myofilaments in Rigor Mortis

 

Bate-Smith (1948) suggested a theory on the state of the muscle

fibrils extant upon the completion of rigor and discussed a similar theory

-18-

advanced by Szent-GySrgyi (1947). Bate-Smith emphasized that the two
theories were complementary rather than competitive. Szent—Gygrgyi (1947)
had suggested that actin and myosin existed in an extended form in a non-
contractile interaction of the actomyosin complex. In essence, Bate-
Smith (1948) was in agreement, but carried the theory further by suggest-
ing a mechanism for the non—contractile interaction of actin and myosin.
He postulated that the mechanizm consisted of interfilament cross linkages
between actin and myosin.

Bendall (1960) concluded that "as rigor ensues, the actin and myosin
chains combine, and the extensibility falls to that residing in the newly
formed actomyosin of the A—band and the residual extensibility of the
free chains of the I band", and that "rigor will result in nearly the
same proportional decrease in extensibility whether the length of the
muscle at the time is greater or smaller than the rest length." Using a
glycerinated fiber-bundle, Bendall (1963) subsequently demonstrated that
the conditions of rigor could be imposed in either the relaxed or contracted
state of the muscle fiber bundle. The contraction-type filament inter-
action was shown to be of consequence only during thaw rigor as opposed
to normal rigor.

Davies (1963) has considered the filament condition in rigor in his
theory of muscle contraction. Using his model, he demonstrated how stiff-
ness or rigidity would occur during rigor. As a result of a low ATP level,
a strong attraction would deve10pe between the positive (in the absence
of ATP) binding sites on the H-meromyosin polypeptide and both the actin

ADP and the fixed negative charge on the H-meromyosin. The latter would

 

 

-19-

induce contraction of the extended polypeptide. Actomyosin would be

:brmed and a stiff, inextensible state would result.

The ”Resolution" of Rigor‘
Marsh (1954), Bendall (1960) and Lawrie (1956) have stressed the

lack of any extensibility increase after the completion of rigor mortis,
and consequently the absence of any ”resolution" of rigor or dissociation
of actomyosin. In agreement with this, Wierbicki ££_g1., (1956) found no
evidence for actomyosin dissociation during the aging of beef. Bendall
(1960) has suggested that any softening of the muscle is probably due to
putrefaction. However, Zender £3 31., (1959) presented evidence of post-
rigor softening under aseptic conditions. Lawrie (1956), Hamm (1960) and
Whitaker (1959) do not preclude a possible role of ion-shifts or the action
of catheptic enzymes in "post-rigor softening”.
Partmann (1963) has suggested that the actomyosin may dissociate

post-rigor. The interpretation by Partmann (1963) of certain experimen-
tal phenomenon as evidence for such dissociation is refuted, however, by

the work of Bendall (1960) and Perry and Grey (1956).

RIGOR MORTIS IN BEEF MUSCLE
Marsh (1954) studied the relationship between extensibility and

changes in ATP, CP and pH during the onset of rigor in the 1. dorsi muscle

 

of beef. In general, his results were compatible with the earlier studies
on rabbit muscle (Bendall, 1951; Bate-Smith and Bendall, 1947, 1949;
Bate Smith, 1939). In muscle strips held at 37°C, CP decreased to 5%

or less of the TSP (total acid-soluble phosphate) before the rapid phase

 

-20-

of ATP disappearance and the first detectable change in extensibility
occurred. Marsh (1954) also confirmed the earlier work showing that the
ATP level was the primary factor controlling the onset of rigor. He
demonstrated that the ATP level could be approximated from pH values of
any muscle which eventually attained a pH level below 5.70.

Howard and Lawrie (1956) examined the effects of pre-slaughter treat-
ments on the development of rigor mortis and the associated biochemical
changes occurring in the muscles of Australian beef. Muscle samples,
removed at 1 hr post-mortem, were analyzed for ATP, CP, TSP, glycogen and
total Ca and Mg. Measurements of pH were taken at l and 24 hrs post-
mortem. Residual glycogen was determined after the carcasses had been
held for 20 weeks at -10°C. Muscle strips were also removed at 1 hr post-
mortem and held at 37°C and extensibility measurements were taken to ob-
serve the onset of rigor. At the onset of rigor, samples were removed
from the strips for determination of ATP levels and pH. Howard and Lawrie
(1956) concluded that the general biochemical characteristics of beef
muscle were similar to those of rabbit. However, neither fasting nor
enforced exercise alone depleted muscle glycogen reserves sufficiently to
raise the ultimate pH above normal values. This was in contrast to the
effect of such treatments observed with rabbits (Bendall, 1951; Bate-Smith
and Bendall, 1947, 1949; Bate Smith, 1939). On the other hand, a combin-
ation of fasting and exercise appeared to have a limited influence in
raising the ultimate pH. The authors suggested that the ruminant was
capable of utilizing fatty acids to a greater extent than monogastric

animals, and that consequently, the glycogen reserves could be more easily

 

 

-21-

maintained. The authors concluded that of the various treatments used,
the most striking was the "relaxation associated with severe hypermagne-
saemia and hypercalcaemia", which respectively slowed down and accelerated
the onset of rigor. Insulin-induced convulsions were found to virtually
deplete muscle glycogen reserves.

In a further study (Howard and Lawrie, 1957), the ante-mortem injec-
tion of pyrophosphate appeared to accelerate the onset of rigor, which
was in contrast to the plasticizing effect of 22.22239 studies. Although
the authors were again unable to deplete glycogen stores by exercise or
starvation, ante-mortem injection of neopyithiamin resulted in depletion
of glycogen reserves. Howard and Lawrie (1957) considered this as support
for the postulated role of fatty acid oxidation in beef muscle. However,
they did not preclude the possibility that beef muscle contained a more
efficient process for glycogen synthesis than muscles from monogastric
animals.

Swift g£_§1., (1960) studied the levels of glycogen, protein, ulti-
mate pH, and buffering capacity of eight beef muscles. Glycogen was
determined at 3,10, and 28 hrs post-mortem. All muscles contained resi-
dual glycogen, even after 28 hrs post-mortem. The content was found to
be largest in those muscles attaining the lowest ultimate pH values.

Fredholm (1963) reported that muscle from beef stored under refrig-
eration still contained 50% and 23% of their original ATP, respectively,
after 3 and 5 days storage. These results were not expected in View of

the work of Marsh (1954) and Howard and Lawrie (1956, 1957).

 

-22-

Andrews Eggng., (1952) determined ATPase, succinic dehydrogenase,
aldolase and total glycolytic activity in beef muscle at 48 hrs, 2 weeks
and 4 weeks post-mortem. The amount of glycogen at each storage period
was also determined. The authors found that ATPase, succinic dehydrogen-
ase and total glycolytic activity did not decrease. However, aldolase
activity was shown to decrease to about two-thirds its 48 hrs value after
2 weeks and to one-half after 4 weeks. It was concluded that the lack
of substrates in intact muscle was the limiting factor in post-mortem

muscle metabolism.

RIGOR.MORTIS IN PORK MUSCLE

Early studies related to rigor mortis in pork muscle are largely
limited to those of Callow (1937, 1938) who conducted investigations on
pork muscle from pigs that had been subjected to various ante—mortem
treatments. The general effect of pH was studied and he concluded that
pork muscle could be classified into two groups according to pH-structure
relationships. An open structure was characterized by a moist appearance
and a lower final pH, while a closed structure exhibited a dry out sur-

face and a higher pH.

Danish Studies

Studies on rigor mortis BE£.§E in pork muscle were not carried out
until after Ludvigsen (1953) reported a condition in Danish Landrace pigs
which he called "muscular degeneration" disease. He described the condi-
tion as "alterations of the musculature, appearing macroscopically as a

discoloration, the altered musculature having a pale or greyish colour"

 

 

 

  
 

   

     
 

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and as being "very juicy". Ludvigsen noted that the condition was espec-
ially prevalent in the l. dorsi, but that on occasion it was found in all
muscles of a given animal. The pH of normal muscle was 6.8-7.0 at approxi-
mately 45 mins post-mortem, while values as low as 5.3-5.5 were found when
"muscular degeneration" was present.

Wismer-Pedersen (1959) observed that the incidence of the condition
described by Ludvigsen (1953) was very rare in Danish Landrace pigs and
called the condition usually encountered "pale and watery pork". Wismer—
Pedersen (1959) studied pH, glycogen, reducing sugars, and ATP levels at
45 mins and 20 hrs post-mortem and water-holding capacity at 20 hrs post-
mortem. He concluded that the soft, watery condition was accompanied by
a pH of less than 6.2 at 45 mins post-mortem. The low pH was due to a
high level of lactic acid and a low level of glycogen 45 mins after
slaughter, and thus was associated with very rapid post-mortem glycolysis.
No differences were found in levels of ATP 45 mins post-mortem, but the
soft, watery muscles showed a general decrease in water-holding capacity
at 20 hrs post—mortem. The frequency of the soft, watery condition was
found to be higher in summer than in winter and appeared to be influenced
by the amount of stress and excitability immediately ante-mortem.

Wismer-Pedersen and Briskey (1961a) and Briskey and Wismer-Pedersen
(1961a) reported four distinct types of muscle according to their post-
mortem pH patterns. They reported that types 1, 2, and 3 were more or
less normal while type 4 was soft and watery. The type 4 muscle exhibited
a Sharp, significant decrease in pH to approximately 5.1 at 1 1/2 hrs

post-mortem with a subsequent elevation to 5.3-5.6. The authors further

 

 

-24-

stated that sections of l. dorsi muscle removed immediately after death
and held at 37°C for 1 1/2 hrs displayed the characteristics of the type
4 muscle. Briskey and Wismer-Pedersen (1961b) concluded that the pH-time
sequence and the consequent development of watery or normal tissue was
dependent on a number of factors. However, only three factors were em—
phasized which were as follows: 1) the extent of glycogen reserves in
the muscle tissue at the time of slaughter, 2) the phosphorylase activity
and the state of glycogen in the tissue, and 3) the methylene-blue reduc-
tion activity of the tissue.

Wismer-Pedersen and Briskey (1961b) studied the effects of various
combinations of temperature and acidity in relation to muscle structure.
The authors suggested that pale, exudative pork could be produced by re-
taining body temperature for an extended post-mortem period. Conversely,
they pointed out that rapid chilling of muscle samples, which had a low
pH within 45 mins of slaughter prevented the developement of the soft,
watery condition.

Bendall and Wismer-Pedersen (1962) showed that washed fibrils obtained
from soft, watery pork had a lower water retention at low ionic strength
and much lower extractability at high ionic strength than the fibrils
from normal pork. Washed fibrils from soft, watery pork showed a greater
protein content than similar fibrils from normal pork. The authors con-
cluded that the greater fibrillar protein content in the watery fibrils
was probably caused by a layer of denatured sarcoplasmic protein, which
was firmly bound to the surface of the filaments. Histological evidence

supported this conclusion. According to these authors, all of the changes

 

 

1|
1-

-25-

reported, including the characteristic gain of protein, could be artifi-
cially induced in normal muscle by allowing it to pass into rigor at 37°C.
It was concluded that the immediate cause of wateriness was the combined
effect of high temperature and low pH on the muscle proteins, which was
in agreement with the suggestion of Wismer-Pedersen and Briskey (1961b).
Bendall and Wismer-Pedersen (1962) also concluded that the depression

and elevation of pH values which was reported by Briskey and Wismer-
Pedersen (1961a) was a reversible phenomenon due to the effect of temper-
ature on the pK values of charged groups on the fibrillar and sarcoplasmic
proteins. They consequently refuted the suggestion of Wismer-Pedersen
and Briskey (1961a) that the depression and elevation of pH was a factor
in the developement of soft, watery pork.

Bendall g£_a1., (1963) reported a further study on the rates of ATP
turnover in relation to pH and onset of rigor mortis in muscle samples
removed hmnediately after slaughter and held at 37°C. Muscle which was
eventually soft and watery showed a mrch faster rate of ATP turnover and
a shorter lag period prior to the onset of rigor. The authors were unable
to explain the differences in rates, which could not be altered by various
pre-mortem treatments. The authors noted that muscle which was allowed
to go into rigor at a constant temperature of 37°C became watery and pale.
They concluded that the probable reason for soft, watery pork was a com-

bination of high temperature and low pH and suggested that the condition

could be prevented by cooling rapidly to 30°C or below.

-26-

British Studies

Lawrie et al., (1958) reported that the ultimate pH was abnormally
low in l. dorsi of exudative muscles. An ultimate pH of 5.1 or below was
frequently found, with the lowest pH observed being 4.78. It was further
reported that the exudative muscle had a considerably lower buffering
capacity than normal muscle.

Lawrie (1960) reported a study on English pigs, mostly of the breed
of Large White X Middle White. ATP, CP and pH levels and egtensibility
were determined on samples removed from six pigs held at 37°C under nitro-
gen. Glycogen and pH determinations were made on samples removed at l
and 24 hrs post-mortem. Lawrie (1960) defined a normal 1. dorsi muscle
as having a pH of 6.7 at 1 hr post-mortem and an ultimate pH of about 5.5.
He observed in normal muscle that the time taken for the muscles to pass
from an identical pH at 1 hr post—mortem to the same ultimate pH varied
3-fold when held at 37°C. An apparently inverse relationship existed
between the rate of pH decrease and the exudation of fluid. Rate of pH
fall was emphasized as the major factor resulting in low water-holding
capacity as Opposed to the level of pH at 24 hrs post-mortem. Measure-
ments of pH at 24 hrs in two groups of British Landrace pigs showed that
they had considerably lower pH values relative to those observed for the
other English pigs. Again, however, no relationship was evident between
24 hr pH and the soft exudative condition, which was in agreement with
Briskey and Wismer-Pedersen (l96la,b). Lawrie observed distinct histolo-
gical differences between the normal and exudative muscles in the Landrace
pigs. However, these differences appeared to be primarily related to the

24 hr post-mortem pH level rather than the soft, watery condition.

-27-

American Studies

Briskey gg‘gl., (1959a,b,c; 1960) reported a decrease in glycogen
in ham muscles from pigs subjected to exhaustive ante-mortem exercise.
An inverse relationship was reported between initial glycogen level and
24 hr pH, water-binding capacity, and color.

Sayre g; 31., (1961) subjected pigs to a 0-5°C environment for 30-40
mins prior to slaughter. They reported that the treatment resulted in a
decrease in initial muscle glycogen and post-mortem accumulation of lactic
acid. Although color intensity of the chilled muscle was increased, the
water-binding capacity was not consistently affected. The authors con—
cluded that factors other than initial glycogen content, 24 hr pH and
rate of glycolysis were important in determining water-binding capacity.

Meyer §£_§1., (1962), using 20 Poland China and Chester White pigs,
concluded that glucose tolerance was not an absolute indicator of post-
mortem changes in muscle characteristics, but that there was a definite
trend for those animals with a high glucose tolerance to have higher
initial muscle glycogen levels and an ultimately inferior muscle quality.
Sayre and Briskey (1963) reported that with the onset of rigor mortis at
a pH below 5.7 and a temperature above 35°C, the muscle became extremely
pale, soft and exudative and exhibited a marked loss in sarcoplasmic and
fibrillar protein solubility. The latter was in agreement with the re-
sults of Bendall and Wismer-Pedersen (1962).

Sayre ggigl., (1963a) reported that short-term excitement and exer-
cise immediately prior to slaughter resulted in muscle with inferior

water-binding properties. Long- and short-term ante—mortem sucrose feeding

 

 

-23-

elevated initial glycogen levels and resulted in muscle that was slightly
soft and pale. These authors further concluded that total phosphorylase
activity was not affected by pre-slaughter treatment and did not appear
to be associated with the rate of post~mortem glycolysis or with the ulti-
mate muscle characteristics.

Sayre ggugl., (1963b) subjected Hampshire, Poland China and Chester
White pigs to elevated environmental temperatures immediately antedmortem.
They reported that the treatment caused an increase in muscle temperature
at slaughter and induced a rapid rate of pH decline and a decrease in
ultimate color intensity. According to these authors, phosphofructokin-
ase activity was not affected by ante~mortem.treatments and did not appear
to be related to glycolytic rate or to the condition of the muscle 24 hrs
post-mortem. In contrast to the previous study (Sayre gt EL'J 1963a),
the results of ante-mortem feeding of sucrose were not uniform.

Kastenschmidt £2 31., (1964) studied the effects of four ante~mortem
environmental temperature treatments, which were warm (42-45°C for 30-60
mins), cold (l~3°C for 30 mins), the warm followed by the cold treatment,
and the cold followed by the warm treatment. The warm treatment alone
was reported to induce the deve10pement of extremely pale, soft and exu-
dative muscle. The authors concluded that warm treatment followed by
cold treatment resulted in dark, dry firm muscle. The other two treat-
ments resulted in less marked changes.

Borchert and Briskey (1964) removed wholesale cuts immediately post-
mortem and immersed them in liquid nitrogen (-195°C) for various periods

of time. They concluded that immersion in liquid nitrogen with subsequent

 

 

-29_

equilibration and thawing at either -l8°C or 4°C prevented the develope—
ment of pale, soft exudative muscle.

Sayre gt g1., (1964) concluded that when the onset of rigor mortis
occurred at pH values below 5.9 with temperatures above 35°C, the 1. dorsi
muscle became pale and exudative. Conversely, the authors reported that
if the onset of rigor mortis occurred when pH values remained above pH

6.0, the muscle was dark and firm.

Irish Studies

McLoughlin and GoldSpink (1963a) reported that pale, exudative post-
rigor muscle resulted if a rapid fall in pH to less than 6.0 in the l;
dgrgi occurred within 45 mins post-mortem. Extremely low (below 5.3) 24
hr pH values were found in many, but not all, specimens of pale, exudative
muscle. The authors did not observe any histological changes character-
istic of muscle degeneration in the soft, watery muscles.

McLoughlin (1963) concluded that the solubility of the sarcoplasmic
and fibrillar proteins from exudative muscle was reduced at low and high
ionic strengths, which is in agreement with Bendall and Wismer—Pedersen
(1963). McLoughlin and GoldSpink (1963b) and Goldspink and McLoughlin
(1964) observed the effects of temperature and pH on the solubility of
the sarcoplasmic protein. They concluded that the color of post-rigor
muscle could be maintained if the temperature of the muscle was reduced
to about 30°C before the pH approached 6.0. This conclusion was in agree-

ment with the suggestion of Bendall st 31., (1962).

 

 

 

_30-

HISTOCHEMISTRY 0F STRIATED MUSCLE

Several comprehensive discussions on the developement and use of
histochemical techniques have been published (Burstone, 1962; Pearse,
1960; Lillie, 1954; Danelli, 1963; Gomori, 1952; Click, 1949). These
references include detailed discussions of methods utilized for the de-
tection of various cellular constituents and the activity of numerous
enzymes in muscle. Accordingly, the present discussion is restricted to
a brief description of current methods for demonstrating certain enzymic
reactions which are pertinent. Furthermore, a brief account of the dis-
tribution of these enzymes in different muscle tissues is included, for

example, the distribution between white and red ribers.

Methods for Detecting Dehydrogenase Activity
Nachlos g£.al., (l958a,b), Pearse (1957) and Hess g; 31., (1958)

described methods for detecting the activities of the various DPN (diphos-
ph0pyridine nucleotide) and TPN (triphosphopyridine nucleotide) linked
dehydrogenases. Nachlos §£_§1., (l958a,b) utilized Nitro BT (nitro blue
tetrazolium) as the final electron acceptor, while Pearse and co—workers
used MTT (3- 4,5-dimethyl-thiazoyl-2 -2-5—diphenyl tetrazolium bromide).

Hess g2 g1., (1958) described how the activity of two enzymes of the
pentose shunt (glucose-6-ph05phate dehydrogenase and 6-phosphogluconate
dehydrogenase) and one enzyme of the beta-fatty acid oxidation cycle (beta-
hydroxybutyrate dehydrogenase) could be demonstrated using MIT as the
electron acceptor.

Nachlos g£_al,, (l958a,b) detected lactate, alpha-glycerophosPhate,

and ethanol dehydrogenase reactions, which are indicative of glycolytic

-31-

activity utilizing Nitro ET. The same authors likewise detected the

 

activities of three TCA (tricarboxylic acid) cycle enzymes, i.e. malate,

 

isocitrate, and alpha-keto glutarate dehydrogenases. Since alpha-keto
glutarate dehydrogenase is also a catalyst for the conversion of L-glutamic
acid to alpha-keot glutarate, the reaction obtained was not regarded to
be indicative of the TCA cycle activity pg; gs.

Pearse (1960) has described the use of either Nitro BT or MIT in
detecting succinic dehydrogenase activity. He has also discussed the
role of this enzyme as a link between the TCA cycle and the electron

transport system in addition to its involvement in the TCA cycle per fig.

Methods for Detecting Respiratory Enzyme Activity

Nachlos E£.i£-: (l958a) used Nitro BT while Scarpelli ggig1., (1958)
utilized MIT for the detection of TPN- or DPN- linked diaphorase (TPN/
DPN-cytochrome c reductase) activity. Pearse (1960) has discussed the
use of DPNH and TPNH as substrates, and alternatively, exogenous enzyme-
substrate systems for conversion of TPN or DPN to the reduced substrate
form. Moog (1943), Nachlos 35 $1,, (1958) and Burstone (1959, 1961a)
demonstrated cytochrome oxidase activity by utilizing n—phenyl—p-phenylene
diamine, dimethyl-p—phenylene diamine or 4-amino-1,N,N-dimethylnaphthy—

lamine as substrate, respectively.

Methods for Detecting the Activity of Other Enzymes

A method for determining adenosine trithSphatase activity was re-
ported by Padykula and Herman (l955a,b) while Wachstein and Meisel (1957)

reported a different method. According to Padykula and Gauthier (1963)

-32-

these two methods detect myofibrillar and sarcoplasmic ATPases, respect-
ively.

Takeuchi (1956, 1958) reported detection of the synthesis of alpha-
1,4 linked glycogen from glucose-l-phosphate. The activity of the two
enzymes involved, i.e. phosphorylase and branching enzyme (anylo—1,4
1,6-transglycosidase) could be individually demonstrated. Takeuchi and
Glennar (1960, 1961) investigated the syntehsis of glycogen by a second
pathway. This pathway involved the synthesis of an amylose-type glycogen
from UDPG (uridine diphosphoglucose) by UDPG-glycogen transferase.

Procedures for the detection of LAP (leucine amino peptidase) acti-
vity were reported by Burstone and Folk (1956) and Nachlos et al., (1957,
1962) utilizing either L-leucyl—beta naphthylamide or L-leucy1—4-methoxy-
2-naphthylamide as substrate. Pearse (1960) has discussed the demonstra-
tion of thiamine pyrophosphatase activity. Gomori (1952), and Burstone
(1961b) have reported different methods for detecting alkaline phosphatase
activity. Likewise, Gomori (1950), Grogg and Pearse (1952), and Burstone

(1961b) have deve10ped methods for demonstrating acid phosPhatase reactions.

Distribution of Enzymic Activity

The concept of red and white muscles which ontain a predominance of
either red or white fibers, respectively, has been recognized for many
years (Needham, 1926). However, the development of histochemical methods
for detecting enzymic activity has greatly facilitated elucidation of the
characteristics of the two types of fibers (Dubowitz and Pearse, 1961).

wachstein and Meisel (1955) studied the distribution of succinic

dehydrogenase activity and mitochondria in various rat and rabbit muscles.

_33-

They reported considerable muscle to muscle variation in the amount of
activity and in the apparent number of mitochondria present. Nachimias
and Padykula (1958) found no positive correlation between fiber size in
rat muscle and glycogen content, although the latter tended to be more
abundant in the large white fibers. Dubowitz and Pearse (1960, 1961)
reported low concentrations of oxidative enzymes and high concentrations
of phosphorylase in the large white fibers of striated muscle from the
human, rat, and pigeon. A reverse realtionship was observed for the
small, red fibers.

Stein and Padykula (1962) observed three types of fibers in rat
muscle. Type A were rich in glycogen and low in succinic dehydrogenase
activity. Types B and C were high in enzyme activity and variable in
their content of glycogen. According to the authors, the type A fibers
were equivalent to large white fibers while types B and C represented two
kinds of small red fibers.

Ogata and Mori (1963, 1964) suggested that there were three types
of fibers present in mammalian muscle. Small fibers with a weak or nega-
tive phOSphorylase reaction showed a high concentration of oxidative
enzymes. However, large fibers showed a reverse relationship. The third
type of fiber was intermediate for both phosphorylase and oxidative en-
zyme activity.

Pearse (1961) found a high concentration of alpha-glycerophosphate
dehydrogenase in large fibers and a low concentration or absence of acti-
vity in small fibers. Thus, alpha-glycerophosPhate dehydrogenase, which

is not linked to a co-enzyme, paralleled phOSphorylase activity.

 

 

-34-

Blancher and Van Wijhe (1962), when using a biochemical assay, found
a higher level of diaphorase activity in red than in white muscle, but an
equal lactate dehydrogenase activity in these muscles. Accordingly,
these authors suggested that the differences between red and white fibers
were probably due to inadequate diaphorase activity in the large, white
fibers. However, these same workers (Van Wijhe g£_§l,, 1963; Blancher
et al., 1963) later concluded that glycolytic dehydrogenases are more
active in white than in red fibers. They further reported that white
muscles of the guinea pig had two to three times greater alpha-glycero-
phosPhate and lactic dehydrogenase activity than red muscles.

Hess and Pearse (1961) suggested that glycogen may be synthesized
in large white fibers, via phosphorylase and branching enzyme, and in
small red fibers by UDPG-glycogen transferase. However, Sasaski and
Takeuchi (1963) found intense phosphorylase reaction in small fibers and
a weak phosphorylase plus branching enzyme activity in fibers of large
diameter.

Pearse (1961) and Blanchaer g£_§l., (1963) have alluded to a possible
relationship between the high levels of alpha-glycerophosphate dehydro-
genase in the large white fibers and the postulated alpha—glycerOphosphate
"shuttle" (Esterbrook and Sackton, 1958). They suggested that this was
possibly a preferred metabolic pathway in these fibers. Recent chemical
studies (Blanchaer, 1964) on the respiration of mitochondria isolated from
red and white muscles support this view.

Goldspink (1962) suggested that large fibers are merely a different

phase of the small fibers. He concluded that various histochemical ob-

 

 

-35-

servations can be explained as being due to a decrease in the concentra—
tion of enzyme in the large fibers, but not the amount of total enzymic
activity. However, Dubowitz and Pearse (1961) have indicated the discre-
pancies involved in interpreting the occurrence of large and small fibers
as phases in activity of the same fiber type.

The results reported previously on the activities of various enzymes
in normal muscle have been confirmed and expanded by Ogata and Mori (1963,
1964). These authors reported that red fibers of mouse muscle contained
moderate to high concentrations of succinate, lactate, malate and alpha-
glycerOphosphate dehydrogenases, while large white fibers contained only
traces. Traces of glutamate dehydrogenase activity were observed in red
fibers, but no activity was observed in the small fibers. Beta-hydroxy-
butyrate dehydrogenase and DPN-diaphorase were moderately active in red
fibers but only slightly active in large, white fibers. TPN-diaphorase
activity was absent in the large fflaers and present in moderate amounts
in the small red fibers. TPN—isocitrate dehydrogenase and Glucose-6-
phosphate dehydrogenase were not detected in either type of fiber. Simi-
lar results were generally obtained for these enzymes in cat and human
muscle. Succinate, lactate, malate, alpha-glycerophosphate, and glutamate
dehydrogenases were studied in dog, pig and ox muscles. Results were
similar to those reported for muscles from the mouse. However, according
to the authors, the white fibers of these Species had higher levels of
lactate, malate and glutamate dehydrogenase activity than is found in
mouse muscle. Amino peptidase activity was absent in all muscles studied.
Results reported for phosphorylase activity were in agreement with those

of Dubowitz and Pearse (1961, 1961).

 

 

—36-

In the same studies (Ogata and Mori, 1963, 1964), all muscles observed

were devoid of alkaline phosphatase activity. Acid phosphatase activity
vvas not found using diazo dye coupling method. The latter result was in
contrast to the observations of Beckett and Bourne (1958), who used a
modification of Gomori's lead nitrate method (Gomori, 1952). The latter
workers reported the presence in normal muscle of acid phosphatase acti-
vity in the peripheral nerves and the Golgi apparatus in both muscle
fibers and connective tissue. Ogata and Mori (1963) suggested that such
results were diffusion artifacts. However, Beckett and Bourne (1960)
believe the two methods are each Specific for a different enzyme or en-
zymes.

Cytochrome oxidase and both sarcoplasmic and myofibrillar ATPase
have been reported to occur in normal striated muscle without any consis-

tent pattern of distribution (Beckett and Bourne, 1960).

  
   
 

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EXPERIMENTAL PROCEDURES

BEEF CARCASSES

This investigation was carried out to follow the chemical and histo-
chemical changes in the 1. dorsi muscle under post—mortem conditions
shnilar to the usual commercial procedures. Previous studies (Marsh,
1954; Howard and Lawrie, 1956, 1957) on beef have primarily involved ob—

servations on muscle strips held at 37°C post-mortem.

Experimental Animals and Sampling Procedures

Five Hereford heifers weighing approximately 850 lbs were obtained
from the University Farm. The animals were fasted 12 hrs prior to
slaughtering at one-half hr intervals. Immediately after death, the
animals were partially hoisted and an incision was made through the hide
in the vicinity of the last rib. Three cores were removed from the 1;

dorsi muscle of each carcass using a l-inch core borer. All initial sam-

 

ples were removed within 10 mins after death. Each carcass was then
dressed in the conventional manner and moved into a 3—4°C cooler. Further
samples were removed at 6, 12, 24, 48, 126, 288 and 480 hrs for pH, chem-
ical and histochemical observations. Additional samples were removed for
pH measurements at 72, 96, 102, 173 and 198 hrs. The holes resulting
from the removal of a sample were packed with adsorbent cotton. Consecu-
tive sampling locations were separated by 1 1/2 — 2 inches.

Immediately after removal from the carcass, samples for histochemi-
cal analysis were frozen in isopentane cooled to approximately -158°C
with liquid nitrogen. Samples for glucose and glycogen analyses were

_37_

-38-

 

frozen in liquid nitrogen. All frozen samples were wrapped in Dow Handi-
Wrap and stored at -29°C until analyzed.

Samples for lactic acid, creatine phosphate, ATP, TSP, and ortho
phosphate analyses were weighed on an analytical balance and homogenized
in a waring blendor with 50 m1 of cold (4°C) 7% TCA (trichloroacetic acid)
solution for one min. The homogenate was filtered through Whatman No. 1
paper in the cold (4°C). The homogenizing cup was rinsed with an addi-
tional 25 m1 of 7% TCA which was added to the original homogenate before
filtering. The filtrate was adjusted to pH 7.1-7.3 with NaOH, made up
to 100 ml with cold (4°C) distilled water and stored at -29°C until ana-
lyzed. After thawing for analyses, one-half m1 chloroform was added to

each sample.

Temperature Measurements

A thermocouple, attached to a Minneapolis—Honeywell temperature re-
corder, was inserted into the center of the l. dorsi muscle of each car-
cass at the approximate level of the 4th lumbar vertebra immediately
after the carcass had been placed in the cooler. Temperature recordings

were taken continuously throughout the first 72 hrs post-mortem.

Measurement of pH

A sample weighing approximately 2 gms was homogenized in 25 ml of
0.005 M sodium iodoacetate for one—half min in a Waring blendor. Dupli-

cate pH measurements were made with a Beckman, Model G, pH meter.

_39-

Chemical Analyses

Creatine phosphate was determined according to the procedure of
Ennor and Rosenberg (1952) and lactic acid according to the method of
Barker and Summerson (1941). ATP, TSP, and inorganic phosphate analyses
were made using the method of Allen (1940) as modified by Marsh (1954).
ATP was also determined using a Turner fluoromenter by the bioluminescent
enzymic method of Strehler and Totter (1952) and Strehler (1953).

For glucose or glycogen determinations, one-half to 1 gm of tissue
was chipped from a frozen tissue block in the cold (~10°C). The tissue
was introduced into a previously weighed digestion solution as outlined
by Folin and Wu (1920) or an extraction solution as given by Hansen 25
55L., (1952) for glucose and glycogen, respectively. After warming to
room temperature, each solution was reweighed to obtain the sample weight.
The analyses were then carried out according to the methods reported

earlier herein.

Statistical Analyses

Means and standard deviations were calculated for the chemical values
for each sampling period according to Snedecor (1959). An estimate of
measuring error was calculated for glucose, glycogen and lactic acid de—
terminations according to Magee (1964). In calculating this estimate,
all values recorded for a given sample including all repeated analyses
Were utilized, even if some values appeared to be in obvious error.
Accordinigly, the estimates probably approach the largest expected error

Values.

-40-

Histochemical Procedures

Blocks of tissue which had been frozen as previously described, were
trimmed and mounted on chucks with distilled water in the cold (-29°C).
Sections 10-12 microns in thickness were cut on a Slee—Pearse cryostat at
-l6°C within one week of sampling. The sections were mounted on cover-
slips, and subjected to the procedures outlined by Pearse (1960) which
are given below.

a) TPN diaphorase: Sections were incubated for 15-20 mins at 20-22°C
in 0.1-0.3 ml of a reaction mixture which included 0.6 ml 1.1 M Na DL-
isocitrate solution (pH 7.4), 0.5 ml 2.5 M Na-L-malate solution (pH 7.4),
0.3 ml 5 mg/ml solution of Nitro BT, 0.2 ml 5 mg/ml solution of TPN, 0.3
ml 0.005 M MnClZ, and 1.1 ml 0.05 M veronal acetate buffer (pH 7.4). The
sections were then rinsed in saline, fixed in formal-saline for 10 mins
and mounted in glycerin jelly.

b) DPN diaphorase: Sections were incubated at 22°C for 15—20 mins
in 0.1—0.3 ml of a reaction mixture consisting of 0.6 ml of pH 7.4 sodium
lactate (0.5 M), 0.2 ml of 5 mg/ml nitro BT solution (pH 7.4), 0.2 ml of
a 1.5% aqueous solution of lactic dehydrogenase, 0.3 ml of a 5 mg/ml solu-
tion of DPN, 1.0 ml of pH 7.4 phosphate buffer (0.2 M), and 0.6 ml dis-
tilled water. The sections were then rinsed briefly in 0.85% saline solu-
tion, fixed in 10% formol-saline for 10 mins, and mounted on slides in
glycerin jelly.

c) Isocitrate dehydrogenase: Sections were incubated for 15-30 mins
in air at 37°C in 0.1-0.3 m1 of a reaction mixture consisting of 0.1 m1

0f SOdILum DL-isocitrate adjusted to pH 7.0 with Tris buffer, 0.1 ml of

 

.

. . . .

. . .

. . .

. . . 5

. .
.
.
. .

 

-41-

0.1 M sodium cyanide solution (pH 7.2), 0.25 ml of 0.06 M phosphate buffer
(pH 7.0), 0.25 ml of a 1 mg/ml Nitro BT solution, 0.1 ml of 0.05 M MgClz,
0.3 ml of distilled water and 75 mg of PVP (polyvinylpyrrolidone). The
sections were then fixed in 10% formol-saline or 10% formol-calcium for

10 mins, rinsed with 10-15% ethanol, and mounted in glycerin jelly.

d) Glutamate dehydrogenase: Procedures were identical to those used
for isocitrate dehydrogenase except that 1.0 M sodium-L-glutamate (mono-
hydrate) solution buffered at pH 7.0 with phosphate buffer was utilized
as substrate.

e) G1ucose-6-phosphate dehydrogenase: Sections were incubated for
15 mins at 37°C in 0.1-0.3 ml of a reaction mixture, which included 0.1
ml of a 1.0 M di Na-glucose-é-phosphate solution buffered at pH 7.0 with
Tris buffer, 0.1 m1 of 0.1 M sodium ozide (pH 7.0), 0.25 ml of 0.2 M Tris
buffer (pH 6.8-7.0), 0.25 ml of a 1 mg/ml MTT solution (pH 7.0), 0.05 ml
0.01 M Na F (pH 7.0), 0.1 ml 0.1 M TPN, 0.1 ml of 0.5 M CoClz, 0.1 ml
distilled water and 75 mg PVP. If cobalt-phosphate precipitates were
present, the sections were rinsed briefly (30 secs) in 1% HCl, but usually
this step was not necessary. The sections were then fixed in 10% formol-
saline for 10 mins and mounted in glycerin jelly containing 0.5 M cobalt-
ous acetate.

f) Beta-hydroxybutyrate dehydrogenase: Sections were incubated for
15 mins in a reaction mixture containing 0.3 m1 of a filtered 1:10 mix—
ture of 0.5 M CoClz and 0.06 M phOSphate buffer (pH 6.8-7.0), 0.1 ml DL-
beta-hydroxybutyric acid (Na salt), 0.1 m1 of 0.1 M sodium ozide or anytal

)

0.25 ml of MTT (1 mg/ml, pH 7.0), 0.1 ml 0.1 M DPN, 0.3 ml distilled water,

 

-42-

and 75 mg PVP. The sections were then fixed in 10% formol-saline for 10
mins and mounted in glycerin jelly containing 0.5 M cobaltous acetate or
in plain jelly.

g) Succinate dehydrogenase: Sections were incubated for 15 mins at
37°C in a reaction mixture consisting of 2 m1 of a 1:1 solution of 0.2 M
phosphate buffer (pH 7.6) and 0.2 M sodium succinate, and 2 m1 of 1 mg/ml
Nitro BT solution (pH 7.6). The sections were then washed in saline,
fixed in 10% formol-saline for 10 mins, rinsed in 15% ethanol for 5 mins
and mounted in glycerin jelly.

h) 6—phosphogluconate dehydrogenase: Sections were incubated in
0.1-0.2 ml of a reaction mixture consisting of 0.1 m1 of barium 6-phos-
phogluconate (pH 7.0), 0.1 m1 of amytal or sodium ozide (0.1 M, pH 7.0),
0.25 ml of 0.2 M Tris buffer (pH 6.8-7.0), 0.25 ml of a 1 mg/ml MTT solu-
tion, 0.1 ml of 0.1 M TPN, 0.05 ml 0.5 M 00012, 0.3 ml distilled water,
and 75 mg PVP. The sections were fixed in 10% formol—saline or 10% formol-
calcium for 10 mins and mounted in glycerin jelly.

i) Alpha-glycerophosphate dehydrogenase: Sections were incubated for
15 mins at 37°C in 0.1-0.3 ml of a reaction mixture consisting of 0.1 ml
0.1 M sodium-DL-alpha—glycerophOSphate (pH 7.0), 0.1 ml 0.1 M sodium
cyanide (pH 7.2), 0.25 ml 0.2 M Tris buffer (pH 6.8-7.0), 0.25 ml of a
1 mg/ml Nitro BT solution (pH 7.0), 0.1 ml 0.1 M DPN, 0.1 ml 0.05 M MgClZ,
0.3 ml distilled water, and 75 mg PVP. The sections were then fixed in
10% formol-saline or a 10% formol-calcium solution for 10 mins and mounted

in glycerin jelly.

-43_

j) Lactate and alcohol dehydrogenase: The proceduresused were iden-
tical to that for alpha-glycerOphosphate dehydrogenase except for the use
of 0.06 M phosphate as the buffer, and 1.0 M sodium-DL-lactate or 1.0 M
ethanol as substrate.

k) Acid phosphatase: Sections were post-fixed in cold (4°C) acetone
for 2 hrs and incubated for 40 mins to 2 hrs in 0.1-0.3 ml of a reaction
mixture containing 2 vols. of 12% sodium-beta-glycerophosphate, 1 vol.
0.1 M acetate buffer (pH 5.0-6.0), 1 vol. 2% lead acetate, and 0.3 vol.
l—5% MgClz. The sections were then rinsed in 5% sodium thiosulfate for
5 mins, and mounted in glycerin jelly.

1) Alkaline phosphatase: After drying for 1-2 hrs at room tempera-
ture, sections were incubated for 30 mins to 2 hrs in 0.1-0.3 m1 of a
reaction mixture consisting of 10 ml 3% sodium-beta-glycerophosphate,

10 ml 2% sodium diethyl barbiturate, 5 ml distilled water, 20 m1 2% CaC12
and 1 ml 5% magnesium sulfate. The sections were then washed in distilled
water, treated with a 2% cobalt solution, washed in distilled water,
treated with dilute ammonium sulfide solution, and mounted in glycerine
with or without counterstaining in 1% aqueous eosin.

m) ATPase (fibrillar): Sections were incubated for 15 mins at 37°C
in a freshly prepared reaction mixture adjusted to pH 9.4 with 0.1 M
NaOH and containing 20 ml 0.1 M sodium barbiturate, 10 ml 0.18 M CaClZ,
30 ml of distilled water and 152 mg of di-Na-ATP. The sections were then
washed in three changes of 1% CaC12, transferred to 2% CoClz for 3 mins,
washed in distilled water, developed in 0.1% ammonium sulfide for l min,
washed in distilled water, dehydrated, cleared and mounted in Permount

(Fisher Chemical Company).

-44-

n) ATPase (sarcoplasmic): Sections were incubated for 15-20 mins

at 37°C in a pH 7.2 reaction mixture containing 20 m1 of 125 mg % ATP

 

(di Na) solution, 20 ml 0.2 M Tris-HCl buffer (pH 7.2), 3 m1 2% Pb (NO3)2,
5 ml 0.1 M MgSO4, and 4 ml distilled water. The sections were then rinsed
in distilled water, developed in dilute ammonium sulfide, rinsed again in
water, and mounted in glycerin jelly.

o) Thiamine pyrophosphatase: Sections were incubated at 42°C for 1
hr or at 37°C for 24 hrs in a freshly filtered reaction mixture containing
60 mg Pb (N03)2, 45 m1 0.005 M acetate buffer (pH 5.0) and 5 m1 5% thia-
mine pyrophosphate. The sections were rinsed in 1% acetic acid, blotted,
dried at 60°C for 5 mins, covered with a thin film of celloidin, developed
in dilute ammonium sulfide, rinsed in water and mounted in glycerin jelly.

p) Leucine aminopeptidase: Sections were incubated for 2 hrs at 37°C
in 0.1-0.3 ml of a reaction mixture containing 1 m1 of an 8 mg/ml L-leucyl-
beta-naphthylamine solution, 10 ml of sodium acetate buffer (0.1 M, pH
6.5), 8 m1 of 0.85% saline solution, 1 m1 of 0.02 M KCN solution, and 10
mg of Fast Blue B salt. The sections were then rinsed in 0.85% saline
for 2 mins, immersed in 0.1 M cupric sulphate for 2 mins, rinsed again in
saline and mounted on slides in glycerin jelly.

The activity of glucose-6-phOSphate dehydrogenase was also observed
in the initial samples with a Nitro BT procedure as adapted from Pearse
(1960). In this procedure, sections were incubated for 30 mins at 37°C
in a reaction mixture containing 0.1 ml di—Na-glucose-6-phosphate (1.0 kg
pH 7.0), 0.1 ml 0.1 M amytal (pH 7.0), 0.25 ml Tris-buffer (0.2 M, pH 6.8-

7.0), 0.05 ml of a 1 mg/ml Nitro BT solution (pH 7.0), 0.05 ml 0.01 M NaF

 

 

-45-

(pH 7.0), 0.1 ml 0.1 M TPN, 0.1 ml 0.05 M Ng012, and 75 mg PVP. The sec-
tions were fixed in 10% formol-saline or formol-calcium for 10 mins, and
mounted in glycerin jelly.

In all cases, two or more sections were treated as outlined above.
Additional sections were utilized for substrate blanks, i.e., reaction
mixture containing no substrate. The commercial sources from which the
various substrates, co-enzymes, etc. were obtained are listed in Appendix
A, Table 1.

Over 1200 mounted sections resulted from application of the above
histochemical procedures. The intensity of the reactions was evaluated
by microscopic observation and rated as — (none), i (traces), + (weak),
++ (moderate), +++ (moderately strong), and ++++ (strong).

Photomicrographs were made of representative sections using a Leitz
Dialux microscope equipped with a Kodak Colorsnap 35 camera and a Vickers
Instruments, Ltd. Photo-Multiplier and Automatic Integrating Timer Unit.

Ansco Veraspan 35 (135 mm, A.S.A. #125) film was used.

PORK CARCASSES

Experimental Animals

Ten Hampshire gilts acquired from a private breeder were kept for
two to three weeks at the Michigan State University Farm prior to slaugh—
tering. These gilts and one Yorkshire gilt from the University herd were
slaughtered in groups of 2 or 3 upon reaching a weight of 190 to 220 lbs.
The animals were brought to the Meat Laboratory 2 1/2 days prior to slaugh-

ter and provided with feed and water until slaughter. The carcasses from

 

. . . K

 

-46- w

the ten Hampshire gilts and one Yorkshire gilt were designated as 1 through
10, and llY, respectively.

Six Poland China gilts were obtained from a private breeder and
housed at the Meat Laboratory for 3-6 days. They were allowed free access
to feed and water up to the time of slaughter. Carcasses from these gilts

were designated as A through G.

Slaughtering and Sampling Procedures

The pigs were electrically stunned, shackled, hoisted, bled and pro-
cessed in the usual manner. Initial samples were removed from the unevis-
cerated carcasses at an average time of approximately 11 1/2 mins after
death. The maximum time interval between death and initial sampling was
16 mins.

Carcasses 1 through 5 were dressed in the usual manner and the car-
casses split down the backbone with the fatback left intact. On all other
carcasses a similar procedure was followed except that on the side to be
placed at —29°C the backfat over the 1. gpgpi muscle was trimmed to less
than one-fourth inch thickness.

Samples were removed and treated as previously described for beef,
except that chloroform was added to the TCA extracts prior to storage.

In addition, samples were removed for determination of fibrillar water-
binding capacity in carcasses 1 through 10 and llY. The sampling locations
and sequence of removal of samples are listed in Appendix B, Table 1. The
times post-mortem at which samples were removed for chemical analysis and
pH determinations, and where applicable, histochemical determinations and

fibrillar water-binding capacity are listed in Table 1.

  
   

 
  
       

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-47-

Table 1. Approximate sampling times (mins post-mortem) in pork carcasses*

 

 

Carcass Right side Left side
1 10 28,73,139, (193), 253, 33, 80, 133, 193, 253, 491, 1440,
493, 1440, 2280 2880
2 10 83,144,236, 324, 564, 80, 146, 235, 327, 564, 1440,
1440, 2880 2880
3 47, 65, 121, 187, 251, 325, 12, 48, 63, 123, 190, 269, 320,
566, 1440, 288 80 554, 1440, 2880
4 207, 308, 503, 668, 1440, lib 173, 312, 503, 668, 1440,
2880 2880
5 11, 143, 301, 438, 633, 1040, 166, 294, 438, 633, 1440, 2880
2880
6 169, 288, 518, 1440, 2880 14, 167, 291, 516, 1440, 2880
7 16, 177, 287, 524, 739, 1440, 176, 289, 526, 1440, 2880
2880
8 182, 294, 532, 787, 1440, 15, 187, 302, 532, 787, 1440,
28802880
9 fig 173, 298,525, 720, 1440, 175, 305, 525, 720, 1440, 2880
288 80
10 133, 253, 493, 733, 1440, 13, 120, 253, 493, 733, 1440,
880 2880
11 12, 266, 1440, 2880 266, 1440, 2880
A $2, 187, 329, 1440, 2880 175, 322, 1440
B 186, 333, 1440, 2880 1;, 172, 322, 1440
C $9, (70), 180, 340, 1440, (73), 175, 343, 1440
D (70), 181, 346, 1440, 2880 19, (72), 169, 348, 1440
E 1&5 (89), 160, 312, 1440, (85), 170, 303, 1440
F (75), 172, 298, 1440, 2880 13, (74), 164, 290, 1440
G 13, (74), 175, 302, 1440, (73), 166, 293, 1440
2880

 

*Underlined values are initial sampling times, and values in parenthesis
are time periods at which only samples for pH were removed.

-43-

.Tgpperature Treatpents

The various temperature treatments to which individual sides were
subjected are listed in Table 2.

All sides were placed at a given temperature within 13 to 27 mins
after death. The treatments utilized in carcasses 1 through 5 were in-
tended to result in a difference in ultimate muscle condition between the
two sides of a given carcass. One side of each of these five carcasses
was subjected to so-called "normal cooling conditions" (3-4°C), while the
opposite side was subjected to one or more of three different temperature
treatments as specified in Table 2. When necessary to prevent freezing,
the sides held at -29°C were removed and held at 4°C for 5 to 10 min and
then placed back at -29°C°

Since the procedures used for carcasses 1 through 5 did not result
in marked differences in the ultimate muscle condition of the two sides
of a given carcass as was expected from the observations of previous work-
ers (Wismer-Pedersen and Briskey, 1961a; Briskey and Wismer-Pedersen,
l961a,b; Bendall and Wismer-Pedersen, 1962; Bendall et al., 1963), the
two extremes in temperature (+37°C, ~29°C) were subsequently employed for

the remaining carcasses (6 through 10, llY, and A through G).

Determination of Fibrillar water-Binding Capacity

The water-binding capacity of the fibrils was determined according
to the general procedure of Bendall and Wismer-Pedersen (1962). A sample
weighing approximately 2 gms was homogenized in 25 m1 of a 0.04 M KH2P04
buffer (pH 7.0; approximate ionic strength = 0.09) with a VirTis homogen-

izer, which was taken to full speed in 3 bursts of 10 seconds duration so

 

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-49-

Table 2. Temperature treatments.

 

 

 

Treatment
Carcass
HQ) Right side Hours* Left side Hours
1 4°C - 37°C 4 1/2-5
2 4°C - -29°C** 1
+25°c 1 1/2
3 37C 4 1/2—5 4°C -
4 4°C - -29°C 4-4 1/2
5 4°C — -29°C 4-4 1/2
6 through 10, llY ~29°C 4-4 1/2 +-3TC 4 1/2-5
A through G -29°c 4—4 1/2 +:3_7*’c 4 1/2-5

 

* Number of hours a side was subjected to a given treatment prior to
being placed in the 4°C cooler.

**The left side of carcass 2 was first placed in the -29°C treatment for
1 hr then moved to room temperature for 1 1/2 hrs prior to being placed
in the 4°C cooler.

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as to avoid excessive heating of the homogenate. Furthermore, the homogen-
izing flask was placed in an ice—bath during homogenization. The homogen-
ate was centrifuged at 2000 x g for 15 mins and the supernatant discarded.
The precipitated fibrils were re-mixed with 25 m1 of buffer and centrifuged
as before. The supernatant was discarded. The concentration of nitrogen
in the washed fibrils was determined using a micro-Kjeldahl method des-
cribed by the American Instrument Company (1961). Samples removed at 24
hrs post-mortem were weighed on an analytical balance before homogenization.
The weight of the precipitated fibrils was also determined prior to nitro-
gen measurements. Grams of water retained per gm of fibrillar protein was
calculated for all samples. Fibrillar protein precipitated per gm of
sample was also calculated for the 24 hr samples. Calculations were made

according to Bendall and Wismer-Pedersen (1962).

Temperature, pH and Chemical Determinations

Temperature was recorded and pH determinations were made according
to the procedures described for beef. However, temperature measurements,
were limited to the initial 6-8 hrs post-mortem.

The levels of CP, glucose, glycogen and lactic acid were determined
according to the methods described for beef. The initial levels of ATP
for carcasses 6 through 10 and llY were determined using the two methods
described for beef muscle. On all other samples, ATP analyses were made

by utilizing only the enzymic method.

     

 
 

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Histochemical Procedures

The methods described for beef were utilized to observe TPN and DPN
diaphorase, as well as isocitrate, glutamate, beta-hydroxybutyrate, suc-
cinate, alpha-glycerophosphate, alcohol, and lactate dehydrogenase activity
in the initial and 24 hrs post-mortem samples of carcasses 1 to 10 and llY.
G1ucose-6-phosphate dehydrogenase activity was observed using the Nitro BT
method described for beef muscle.

A Nitro BT method was also used to observe the activity of 6-phospho-
gluconate dehydrogenase as adapted from Pearse (1960). In the same samples
the activity of malate dehydrogenase and cytochrome oxidase were observed
according to Pearse (1960). Methods described by Pearse (1960) were also
used to observe alkaline and acid phosphatase activity in carcasses 1
through 5. These five procedures are listed below.

a) Acid phosphatase: Sections were incubated for 5 hrs at 37°C in a
filtered reaction mixture containing 5 ng Na-AS-BI—phosphate, 0.10 ml
dimethyl sulfoxide, 2.5 ml 0.2 M acetate buffer (pH 5.2-5.6), 35 mg Red
Violet LB salt and 2 drops of 10% MnClz. Sections were washed in running
water and mounted in glycerin jelly.

b) Alkaline phosPhatase: Sections were air-dried for l to 3 hrs and
incubated for 30 mins at room temperature in a reaction mixture filtered
onto the sections and containing 20 mg sodium-alpha-maphthyl phosphate,

20 ml of 0.1 M Tris-buffer (pH 10.0), and 20 mg of diazote 5—chloro—o-
toluidine. The sections were then washed in running water for 1-3 mins,
counterstained in 0.05% anilin blue for 3 to 6 mins, washed in running

water for 40 mins and mounted in glycerin jelly.

 

-52-

c) Cytochrome oxidase: Sections were incubated for 60 mins at room
temperature in a filtered reaction mixture containing 10 mg of l-hydroxy-
2 naphthoic acid, 10 mg of N—phenyl-p-phenylene diamine, 0.5 m1 ethanol,
35 ml distilled water and 15 ml Tris-buffer (0.2 M; pH 7.4). The sections
were then transferred directly to 1% cobalt acetate in 10% formalin for
1 hr, washed in distilled water, and mounted in glycerin jelly.

d) 6-ph03phog1uconate dehydrogenase: Each section was incubated
for 30 mins at 37°C in 0.1 to 0.3 ml of a reaction mixture containing 0.1
ml 0.01 M barium—6-phosphogluconate (pH 7.0), 0.1 ml sodium ozide (0.1 M,
pH 7.0), 0.25 ml Tris-buffer (0.2 M; pH 6.8—7.0), 0.25 ml of a 1 mg/ml
solution of Nitro BT, 0.1 ml 0.1 M TPN, 0.3 ml distilled water and 75 mg
PVP. The sections were then fixed in 10% formol-calcium or formol-saline
for 10 mins and mounted in glycerin jelly.

e) Malate dehydrogenase: Sections were incubated for 15-20 mins at
37°C in a reaction mixture including 0.1 ml 1.0 M L-malic acid solution
(pH 7.0), 0.1 ml 0.1 M sodium cyanide solution (pH 7.2), 0.25 ml 0.06 M
phosphate buffer (pH 7.2), 0.25 ml of a 1 mg/ml solution of Nitro BT (pH
7.0), 0.1 ml 0.1 M DPN solution, 0.1 ml 0.05 M MgClz, 0.1 ml distilled
water and 75 mg PVP. The sections were then fixed in 10% formol-calcium
and mounted in glycerin jelly.

The activity of phOSphorylase, branching enzyme, and UDPG-glycogen
transferase was observed on some of the initial samples of carcasses 6
through 10. Activity was detected according to the methods ofTakeuchi
(1958) and Takeuchi and Glenner (1961) as described below.

a) Phosphorylase and branching enzyme: Different sections were in-

cubated in three different reaction mixtures (A, B, C) for 1 hr at 37°C.

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Reaction mixture A contained 50 mg potassium-glucose-1-phosphate, 20 mg
AMP, 10 mg glycogen, 10 ml distilled water, and 10 ml 0.2 M acetate buffer
(pH 5.7-5.9). Reaction mixtures B and C were identical to the above except
for the addition of 5 ml ethanol or 5 ml of 0.0006 HgClz, respectively.
The same three reaction mixtures with the pH adjusted to 5.0 or 6.0 were
also used in some cases. Likewise, AMP and/or glycogen were omitted in
similar reaction mixtures. After incubation the sections were stained in
Gram's iodine and mounted in iodine-glycerin jelly or in plain glycerin
jelly. Both ethanol and HgClz inhibit branching enzyme activity. The in-
hibition by alcohol is strong while that of HgClz is relatively complete.
Con ersely, the inhibition of phosphorylase by either of these substances
is slight. The sections stained blue to dark blue when phosphorylase acti-
vity is predominant and violet red to brownish purple when the branching
enzyme predominates.

b) UDPG-glycogen transferase: Sections were incubated for 1 hr at
37°C or 1—3 hrs at 25°C in a reaction mixture containing 50 mg UDPG, 10
mg glycogen, 20 mg Versene, 10 mg glucose-6-phosphate, 14 ml distilled
water, 10 ml 0.2 M Tris-buffer (pH 7.4), and 1 ml ethanol. UDPG was
omitted in substrate-blank reaction mixtures. Following incubation, the
sections were treated as described in the above procedures for phosPhory-
lase and branching enzyme. A reddish purple to reddish brown color indi-
cated UDPG-glycogen transferase activity.

With carcasses A through G, only four, and in some cases five, en-
zymes were studied. These included phosphorylase, branching enzyme, and
UDPG-glycogen transferase, which were observed by the procedures given

above. Additionally, succinic dehydrogenase and/or glutamate dehydrogenase

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-54_

was/were detected using procedures previously described for beef muscle.
The activity of these enzymes was observed on all initial and 2 1/2-3
hrs post-mortem samples and on a limited number of samples removed at 24
hrs after death.

Sections from all the 2 l/2—3 hr samples and on a limited number of
24 hr samples of carcasses A through C were subjected to an HCl-Orange
B-Anilin Blue staining procedure as modified from Guyer (1953). Sections
were fixed in 10% formalin for 10 mins, stained 6 mins in acid iron-
hematoxylin, washed in distilled water, rinsed in 1% acetic acid, stained
in anilin blue-orange G-HCl solution, washed in 3 changes of acetic acid,
dehydrated, and cleared by an acetone-xylene sequence and mounted in Per-
mount.

In all cases, two or more sections were treated as outlined above.
Substrate blanks were also utilized as described for beef muscle. Further-
more, where feasible sections were also incubated in complete reaction
mixtures containing an inhibitor. Three-tenths ml of a saturated solution
of PCMB (Na-p-chloromercurobenzoate) was included for each 1.1 ml of re-
action mixture as an inhibitor when observing DPN and TPN diaphorase, and
isocitrate, beta—hydroxybutyrate, alpha-glycerophosphate, lactate and al-
cohol dehydrogenase activity. As an inhibitor for succinate dehydrogenase,
0.6 ml of a saturated solution of PCMB was included in 2 ml of reaction
mixture. A concentration of 0.001 M NaN3 was used to inhibit cytochrome
oxidase activity. The commercial sources from which these substrates

used in the above procedures are included in Appendix B, Table 1.

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- ..' "..x. -. II- '.Z. .'"- . I. '5 . .. . 5 . .. -1 .. . i 3' .-.-_" .3 35': _
. - . ' ' '. I
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-55-

Over 1350 mounted sections resulted from application of the above
histochemical procedures. The intensity of the reactions was evaluated

and photomicrographs were made as described for beef muscle.

Statistical Analyses

Calculations were made as previously described for beef. However,
means for sampling periods were calculated only where such periods were

at approximately the same time post—mortem.

Panel Evaluation

The extent of the soft, watery and pale condition of the cut loin
surface of carcasses 6 through 10, llY, and carcasses A through G was
subjectively evaluated at 48 hrs post-mortem by a four-member panel. The
panel rated each loin as being either extremely, moderately, slightly, or

not at all soft and watery.

 

 

RESULTS AND DISCUSSION
POST—MORTEM LEVELS AND/0R INTERRELATIONSHIPS OF CHEMICAL CONSTITUENTS,
pH AND TEMPERATURE IN BEEF MUSCLE
The means and standard deviations of the values for chemical consti-
tuents and pH at various periods post-mortem are listed in Table 3. The
individual values for each carcass at each time period are tabulated in

Appendix A, Table 2.

Levels of pH

The average initial pH value was 6.99. The lowest initial pH value
was 6.90, while the highest was 7.07. At 48 hrs post-mortem, the mean
pH was 5.46 and at 480 hrs it was 5.57.

The initial pH values are in agreement with Marsh (1954), who calcu—
lated the approximate pH at death for beef muscle and found the values
varied from about 6.65 to above 7.40. Similarly, the initial pH values
are also in agreement with Howard and Lawrie (1956), who obtained an
average beef l. dorsi muscle pH value of 6.74 at 1 hr post-mortem.

The values for 48 and 480 hrs post—mortem are in agreement with the
final pH value of 5.50 determined under similar conditions by Howard and

Lawrie (1956).

Creatine Phosphate

Creatine phosphate decreased to about 22% and 16% of the initial
level (9.1 micromoles/g of muscle) at 6 and 12 hrs post-mortem, respect—
ively. The initial CP value is in general agreement with Howard and

Lawrie (1956), who reported an average value equivalent to about 7.3

-56-

 

 

 

 

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~58-

micromoles/g in the l. dorsi muscle of 13 steers at 1 hr post-mortem.

ATP

The initial ATP(1) (acid hydrolyses) and ATP(2) (enzymic assay)
values were 6.4 and 10.9 micromoles, respectively. The ATP(2) value had
decreased to nil within 24 hrs post-mortem, while the ATP(1) value was
still at a level of 1.7 micromoles/g. The ATP(1) value is in good agree-
ment with that of Howard and Lawrie (1956), who reported a comparable ATP
value equivalent to approximately 5.3 micromoles/g of tissue at 1 hr post-
mortem, and with that of 5.6 micromoles/g at 60-80 mins post-mortem as
reported by Marsh (1954).

However, the ATP(2) values are considerably higher than the ATP(1)
values (Table 3). The acid—molybdate reagent of Allen (1940) used for
color developement in the ATP(1) analysis has been shown to hydrolyze the
two acid labile phosphates of ATP, and especially the terminal phosPhate
in varying degrees. The amount of hydrolysis is primarily dependent on
temperature and time of exposure to the acid—molybdate reagent (Whil-
Malherbe and Green, 1951). Since Allen's (1940) method allows the time
of exposure to the acid-molybdate reagent to vary from 5 to 25 mins,
values for ortho phosphate estimated by this method would be expected to
include some ATP phOSphate. Consequently, the initial ATP(1) values in
the current study as well as similar values of Howard and Lawrie (1956)
would be expected to be less than the true ATP value.

Conversely, when using acid hydrolysis followed by determination of

free phosphate by the acid-molybdate procedure, residual ATP and ADP are

..miosvflaoq m 1 3:. usage :1 in 91mm .1 1 i,

 

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present at a combined level, which is less than 2 micromoles/g (ATP(1);
Table 3). Such a residual quantity is not found when assayed with the
enzymic method (ATP(2); Table 3). Bendall (1951) suggested that the re-
sidual ATP and/or ADP obtained by the acid hydrolysis method was due to
ATP and/or ADP, which is tightly bound to the muscle protein. However,
Bendall and Davey (1957) concluded that acid labile-P content was a re-
liable guide to true ATP and/or ADP content only in the early stages of
rigor, when CP and glycolysis resynthesis maintained ATP at a fairly
constant level. They further concluded that when the rate of resynthesis
begins to decrease, side-reactions invalidated any simple equation for
labile-P to ATP.

Furthermore, in support of the absence of detectable ATP at 24

(2)
hrs post-mortem in the current study (Table 3), it can be calculated
from data presented by Davies (1963) that less than 0.2 micromole/g of
tissue of ATP and/or ADP would be bound to the muscle protein in full
rigor for rabbit muscle.

Fredholm (1963) reported that 50 and 23% of the initial ATP was pre-
sent in beef ”gracilis-muscle” after storage at cooler temperatures for
3 and 5 days, respectively. This is not in agreement with results of

the current study, where the ATP(2) level was negligible at 24 hrs (Table

3).

TSP and Ortho P
In the current study, the overall mean for TSP was 54.4 microatoms/g

of muscle. Marsh (1954) and Lawrie (1959) reported values equivalent to

 

-60..

55.5 and 57.1 microatoms/g for beef muscle, respectively. Values reported
for rabbit muscle have varied from 61 to over 70 microatoms/g (Bate-Smith
and Bendall, 1947; Bendall and Davey (1957).

The initial level of ortho P was 22.1 microatoms/g of tissue. This
level increased to 35.5 microatoms/g at 480 hrs post-mortem. Comparable
values are not available for beef. However, Bate-Smith and Bendall (1947)
reported an initial level equivalent to 22.9 microatoms/g and a 444 min
post-mortem level (at 37°C) of 31 microatoms/g in rabbit muscle. Hence,
the concentration of ortho phosphate would appear to increase 1 l/2 times

after death in muscle from both beef and rabbit.

Glycogen

The initial glycogen level was 56.7 micromoles/g. This value de-
creased to approximately 54% at 12 hrs and to less than 20% at 24 hrs
post-mortem. At 288 hrs, the glycogen was still 17.5% of the initial
value, but decreased thereafter to less than 3% at 480 hrs. It seems
likely that the breakdown of residual glycogen occurred as a result of
the combined activity of alpha-amylase, amylo-lz6-glycosidase, and mal-
tase, which has been suggested by Sharp (1962). Although comparable
V alues for all the various post-mortem time periods are not available,
the values found in the present study are in good agreement with the
reports of previous workers at specified times post-mortem. Initial
levels of glycogen calculated from the data of Howard and Lawrie (1956,
1957) are 51.9 and 51.7 micromoles/g of l, dgggi muscle from 13 and 5
steers, respectively. Swift_gt.§;., (1960) reported glycogen levels

equivalent to 40.3 micromoles/g at 2-3 hrs post-mortem.

 

 

 

  

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-61-

In the current study, the 24 hr value for glycogen was 10.1 micro-
moles/g. This is in agreement with the results of Lawrie and Howard (1956),
who reported residual glycogen levels equivalent to as much as 8.3 micro-
moles/g in beef muscle after being frozen at 24 hrs post-mortem and stored

at -10°C for twenty weeks.

Glucose

The average glucose (total reducing sugar) level increased from 7.9
to 17.9 micromoles/g between the initial and 480 hr post-mortem sampling
periods. The marked variation observed in glucose levels at intervening
periods (Table 3) suggests that the amount of glucose may vary consider-
ably from location to location, and/or it may be converted to a form
other than a reducing sugar at certain times post-mortem.

The values for total reducing sugars found in the current study
(Table 3) are in good agreement with those reported by Sharp and Rolfe
(1958). These workers observed that free glucose and glucose-6-phosphate
were present in mammalian muscle immediately post-mortem at levels equi-
valent to 2.8 and 2.4 micromoles of glucose per g of muscle, reSpectively.
Only relatively small concentrations of other reducing substances were
observed. Glucose—6-phosphate increased during post-mortem glycolysis
to a constant level, which approached about 14 micromoles (glucose equi-
valents)/g of tissue. They reported an accumulation of free glucose
equivalent to 5.6 and 31.6 micromoles/g in beef and pork muscle, respect-
ively, which was held at l8-20°C for 24 hrs post-mortem. Hence, the
combined value of free glucose and glucose-6-ph05phate in beef muscle

approached 19.6 micromoles/g. If it is assumed that a maximum of 14

 

 

-62-

micromoles of glucose—6-ph03phate accumulates post-mortem per g of beef
muscle, then in the current study approximately 4-5 micromoles of free
sugar or other reducing substances were present at 480 hours post-mortem.
Such a value is in good agreement with the observations of Sharp and

Rolfe (1958).

Lactic Acid

Lactic acid, which was present at an initial concentration of 13.1
micromoles/g,had increased almost 3 1/2 times at 6 hrs post-mortem (Table
3). A further slower, but steady increase was observed up to 48 hrs to
approxhnately 6 1/2 times the initial level. Thereafter, little or no
increase was evident. Results support Marsh's (1954) suggestion that

active glycolysis is complete in beef 1. dorsi muscle within 36 hrs post-

 

mortem.
Although comparable lactic acid values are not available, Howard
and Lawrie (1956) suggested that a ”minimum quantity” of 700 mg of gly-

cogen per 100 g of beef 1, dorsi muscle was necessary to reach an ultimate

 

pH of 5.44. If, as these workers assumed, glycogen were completely con-
verted to lactic acid, the value for lactic acid would be 77.8 micromoles/
g of muscle at pH 5.44. Such a value is in good agreement with the results

of the current study (Table 3).

Temperature
The average temperatures of the 1. dorsi muscle of the five beef
carcasses are given in Table 4. It would appear that the cooling process

was completed for this muscle prior to 20 hrs post-mortem.

flood-19 g '16! Manhunt; angina-mar. saw

99:1 3:0 aslmm‘toin li—I- 1.79:; ant-rams

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-63-

Table 4. Average post-mortem temperature of the l. dorsi muscle of five
beef carcasses.

 

 

Time post-mortem Temperature

(hrs)

Initial 38.9

3 , 27.2

6 20.0

10 13.3

12 10.0

16 6.7

20 2.8

24 2.8

48 2.8

 

ATPz CP and pH
CP and ATP levels as a percent of their initial levels and their

relation to pH are shown in Table 5. CP decreased to approximately 20%
of its initial value within 6 hrs post-mortem. During the same period,
ATP2 had decreased by less than 10%. At 12 hrs post-mortem, ATPZ values
had decreased to about 50% of the initial value, with a concurrent aver-
age pH level of 5.96. As previously discussed, the mean temperature of
the l. dorsi muscle was 20.0, 13.3, and 10.0°C at 6, 10, and 12 hrs post-

mortem, reSpectively.

 

   

-64-

Table 5. Levels of ATP, CP and pH during the initial 48 hrs post-mortem

 

 

Time ATP level CP as a % of
post-mortem as a % of the the initial
(hrs) pH initial level level

ATP1a ATP9E CP

Initial 6.99 100.0 100.0 100.0

6 6.57 78.1 91.7 22.0

12 5.96 60.9 48.6 16.5
24 5.74 26.6 0.0 -
48 5.57 17.2 0.0 -

 

aBased on acid hydrolysis values.
bBased on enzymic assay values.

The rapid depletion of CP prior to any significant decrease in ATP
level is in agreement with the observations of Marsh (1954).

Howard and Lawrie (1956) noted that the onset of rigor in beef muscle
held at 37°C occurred when 1/2 to 3/4 of the initial ATP had disappeared
with a corresponding pH level of 6.02. Bendall and Davey (1957) reported
that the onset of rigor in rabbit muscle occurred when 1/2 of the initial
ATP was depleted in muscle held at 37°C. If held at room temperature,
however, onset of rigor occurred when 3/4 of the initial level was depleted.

In the current study, it is suggested that the average time post-
mortem the onset of rigor in the l, d2£§i_muscle was initiated was about
12-15 hrs post-mortem. Results also indicated that the developement of
rigor was virtually complete prior to 24 hrs post-mortem. This observa-

tion is supported by the data on lactic acid (Table 3).

 

-65—

Lactic Acid and pH

The general relationship between post-mortem levels of lactic acid
and pH is shown in figure 1. Increasing levels of lactic acid resulted
in lower levels of pH. This is in agreement with the observations of
Bate Smith (1939), and Bate-Smith and Bendall (1947) on rabbit muscle.

In the current study, 46.5 micromoles of lactic acid/g of muscle
were produced for every unit decrease in pH during the initial to 24 hr
period. During the same period, pH decreased from 6.99 to 5.74. This
value is in good agreement with a similar increase of 44 micromoles of
lactic acid/g of tissue for every unit pH decrease as calculated from the
data of Howard and Lawrie (1956) in beef 1. gg$§i_muscle. However, an
equivalent value for approximately the same pH interval of 63.3 micromoles
of lactic acid per g of tissue for each unit pH decrease in rabbit muscle
was reported by Bate-Smith and Bendall (1949). Both the observations of
Howard and Lawrie (1956) and those in the current study would suggest
that the buffering capacity in beef 1, dorsi muscle is considerably lower

than that found in rabbit muscle within the same pH range.

Glycogen, Lactic Acid and Glucose

 

The relationship between post-mortem levels of glycogen, glucose (total
reducing sugars) and lactic acid is shown in figure 2. The sum in micro-
moles of glucose, glycogen and lactic acid (expressed as glucose equiva-
lents) is approximately constant at all times post-mortem. Deviations
from a completely constant relationship are within the lhnits of the errors
of measurements which were i 2.2, i 1.2, and i 6.6 micromoles/g for gly-

cogen, glucose, and lactic acid, reSpectively.

 

-66-

 

901'

80"

70-

60-

50"

4o

30'

,UM LACTIC ACID /9 FRESH TISSUE

20-

I

l0

 

 

1 1 1 .1 1
O

7.5 7.0 6.5 6.0 5.5
pH

Figure l. The relationship between pH and lactic acid in beef _1. dorsi
muscle.

 

-57-

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-68

It is concluded that glycogen in beef muscle is degraded to glucose
and lactic acid during post-mortem glycolysis in an approximately stoi4
chiometric manner. This is in agreement with the work of Sharp and Rolfe
(1958) on the accumulation of free glucose and glucose-6-phosphate.
Furthenmore, this conclusion supports the observation of Howard and Lawrie
(1956) that in beef muscle, ultimate pH level could not be fully exPlained

by assuming a stoichiometric degradation of glycogen to lactic acid.

CHEMICAL CONSTITUENTS, INTERRELATIONSHIPS, AND PROPERTIES OF PORK MUSCLE
Individual values for post-mortem levels of pH5 glycogen, glucose,
lactic acid, ATP(1) and ATP(2) for each side of carcasses 1 through 10
and llY are given in Appendix B, Table 2. Corresponding values for the
fibrillar protein content/g of precipitate, the amount of water retained/g
of fibrillar protein and the content of fibrillar protein/g of muscle at
24 hrs post-mortem.are also included in Appendix B, Table 2. Likewise,
individual values for post-mortem levels of pH, glycogen, glucose, lactic
acid and ATP2 for each side of carcasses A through G are tabulated in

Appendix B, Table 3.

Carcasses 1 Through 5

The various temperature treatments utilized for carcasses 1 through
5 did not result in any appreciable differences between the two sides of
a given carcass. The l, dg£§i_muscle surface of all five carcasses was

considered normal at 48 hrs post-mortem.

 

 

 

 

 

4694

IMuscle Temperatures

In figure 3 are shown two typical temperature curves for two sides
of a carcass. One side was subjected to -29°C immediately after death
and the other to a temperature greater than 37°C. The initial temperature
of the 1, ggggibmuscle in both sides was approximately 41°C prior to being
subjected to the two different temperatures. The temperature of the side
held at -29°C decreased to about 30°C after approximately 65 mins post-
mortem and at 2 hrs had fallen to l9-20°C. The muscle in the side sub-
jected to the higher temperature was still more than 35°C at 3 1/2 hrs
post-mortem.

The effect of the two temperature treatments on pH value and ultimate
muscle condition are given in Table 6 for the group 1 carcasses (carcasses
6 through 10) and carcass llY.

Table 6. Post-mortem pH and ultimate muscle condition as affected by
temperature treatments of carcasses 6 through 10 and llY

 

 

 

 

 

 

Time post- pH Degree of soft,

mortem: Initial 3 hrs 48 hrs watery conditiona:b
Treatment: -29°C >37°C -29°C >37°C ~29°C >37°C

Carcass 6 5.6 5.4 5.3 5.3 5.3 S S to M
7 6.7 6.5 6.2 5.6 5.3 N S to M

8 6.1 5.3 5.3 5.3 5.3 N M

9 6.4 5.7 5.4 5.3 5.5 N N

10 6.3 6.3C 5.5c 5.5 5.5 N N

11y 6.6 6.3d 5.4d 5.7 5.5 N N

 

a48 hrs post-mortem

bRated on a scale of N = none, S = slight, M = moderate and E = extreme.
cApproximately 2 hrs post-mortem.

dApproximately 4 hrs post-mortem.

From the reports of previous workers (Wismer-Pedersen, 1959; Briskey

and Wismer-Pedersen, 1961b; Bendall gt al., 1963), the extremely low iniital

 

 

 

     

TEMPERATURE ’C

     
  

l8.’3 (-29’C)
l5. 6 -

l2.8-

I0.0

   

l l l l l l
30 80 90 l20 ISO I80 2l0
TIME POST- MORTEM (M INS.)

 

Figure 3. Post-mortem temperature of pork 1. dorsi muscle of representative
sides subjected to -29°C or 37°C temperatures immediately after
death.

 

 

-71-

pH values for carcass 6 were expected to result in a soft, watery muscle
condition Observed in both sides of this carcass. Rapid cooling would
not have been expected to greatly alter the muscle condition of the side

placed in the ~29°C treatment since the prevailing conditions believed
)

 

to have a causal relationship to the developement of the soft, watery
condition were already present within 16 mins post-mortem. These condi-

tions were the occurrence of a low pH level (less than pH 6.0) while

 

'muscle temperature was still high, i.e., above 30-35°C (Wismer-Pedersen,
1959, Briskey and Wismer-Pedersen, l96la,b; Bendall 25.31,, 1963).

The muscle condition of the two sides of carcass 7 were evaluated
as normal, and slightly to moderately soft and watery for the -29°C and
37°C treatments, respectively. The rate of glycolysis immediately after
death was not greatly different in the two sides as evidenced by the 3
hr pH levels, but differences in ultimate pH were obtained (Table 6).
The high 3 hr pH value of 6.2 for the sides in the 37°C treanment was re-
flected in the high levels for ATPZ, and glycogen, which were much greater
than the correSponding values from all other carcasses of group 1 at 3
hr post-mortem (Appendix B, Table 2). The relatively slow rate of glyco-
lysis was expected to result in a normal muscle condition (Briskey and
Wismer-Pedersen, 1961b; Bendall 33.21., 1963). However, the deve10pement
of the soft, watery condition in the 37°C treatment supported the obser-
vations of previous workers as to the causal role of the post-mortem.pH-
temperature relationship. Several workers (Briskey and'Wismer-Pedersen,
1961b; Wismer-Pedersen and Briskey, 1961b; Bendall £5 31., 1963; Sayre

‘g£‘§1., 1961), have suggested that rapid cooling to below 30°C before

 

-72-

the pH level has decreased to 5.9-6.0 or less would be expected to pre-
vent the developement of the soft, watery condition.

Sim_lar differences (Table 6) in ultimate muscle condition were
apparent in the two sides of carcass 8. The identical pH levels in the
two sides of this carcass at 3 hrs post-mortem would not necessarily
preclude a faster rate of glycolysis in the 37°C side. However, the
initial pH of 6.1 suggested that the pH in the side at -29°C would have
declined to below 5.9-6.0 prior to any significant drop in muscle temper-
ature.

In the two sides of carcasses 9, 10 and llY, marked differences were
observed in 3 hr pH levels between the two treatments. The results (Table
6) on these three carcasses strongly suggested that the occurrence of a
low pH at a high muscle temperature can not be equated with the routine
developement of the soft and watery condition. Furthermore, these results
are in disagreement with the observations of several workers (Wismer—
Pedersen, 1959; Bendall and Wismer-Pedersen, 1962; Bendall gt al., 1963).
These investigators reported that the soft, watery condition could be
induced by holding a sample of muscle removed from the carcass immediately
after death at 37-41°C until it passed into rigor, and/or by merely heating
a minced sample of normal muscle at 37°C for 1 1/2 hrs. A wi-e discre-
pancy would appear to exist between the observations made on small isolated
samples of muscles and the results of the current study in which intact
carcasses were utilized.

The failure to substantiate the causal effect of the low pH-high

temperature relationship was further supported by the data obtained with

 

 

 

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-73-

the group 2 carcasses (carcasses A through G). In these carcasses, the
relation between 3 hr pH, temperature treatment and ultimate muscle con-
dition at 48 hrs post-mortem is Shown in Figure 4. All of these carcasses
had an initial pH of 6.6 or higher. The ultimate 48 hr pH levels varied
from 5.5 to 5.8 for muscles held at -29°C, while those in muscles at 37°C
varied from 5.2 to 5.7 (Appendix B, Table 3). In general, the 37°C

treatment greatly lowered the 3 hr pH values. However, alteration of the

 

ultimate condition of the muscles was less apparent.

The results for carcass A followed the pattern that would be expected
from the literature. The initial pH value was 6.7 (Figure 4). At 3 hrs
post-mortem, the muscle at ~29°C had a pH of 6.4, w-ile the side subjected
to the 37°C treatment had a pH of 5.4. The 48 hr pH values were 5.5 and
5.3 for the muscle from the low and high temperature treatments, respect-
ively. No evidence of the soft, watery condition was found in the muscle
held at -29°C while a slight to moderate degree was observed at 37°C.
Similar results were obtained with carcass E (Figure 4).

In the remaining five carcasses of group 2, significant changes in
ultimate muscle condition were not apparent. The data for carcass F were
of particular interest. The initial pH value was 6.8. The three hr pH
values were 6.7 and 5.3 for the -29°C and 37°C treatments. The eventual
condition of the muscles from both treatments was assessed as being normal
(Figure 4).

From the results on the two groups of carcasses, it was concluded
that the occurrence of a low pH at a high temperature in pork muscle was

not a causal factor in producing soft, watery muscle. However, occurrence

 

 

 

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-75-

of the low pH-high temperature phenomenon immediately post-mortem.as a
normally encountered characteristic of muscle eventually termed soft and

watery is not precluded.

PrOperties of Fibrillar Protein
The fibrillar protein content/g of muscle and the water retained/g
of fibrillarprotein from 24 hr post-mortem samples of carcasses 6 through

10 and llY are given in Table 7. The means and standard deviation for

 

the grams of fibrillar protein/g of muscle were 0.0635i0.007 and 0.0970
i0.045 grams for the -29°C and 37°C treatments, respectively. Correspond-
ing values for grams of water retained/g of fibrillar protein were 34.77
ill.98 and 13.25i2.9l g. The data for individual sides parallelled the
observations on alterations in pH (Table 6). The -29°C treatment was
ineffective in significantly altering either the fibrillar protein con-
tent or the water retention of the washed fibrils in carcass 6. The
muscle in this carcass from both sides was ultimately soft and watery as
previously discussed. This was expected because of the low initial pH
(5.6, Table 6).

A pronounced response to the temperature treatments was observed in
carcass 7, while an intermediate effect was obtained for carcass 8. The
grams of water retained/g of fibrillar protein in the 37°C treatment was
only 39.7 and 46.7% of that retained in the -29°C treatment for carcasses
7 and 8, resPectively. A similar effect was observed in an increased
content of fibrillar protein from the ~29°C treatment (Table 7).

However, in carcasses 9, 10 and llY, the effect of the two treat-

Inents was equally as marked as that observed in carcasses 7 and 8. This

 

-76..

Table 7. Fibrillar protein content and water binding of washed fibrils
from 24 hr post-mortem samples of carcasses 6 through 10 and
llY.

Grams of
fibrillar protein
per gram of

Grams of H20
retained per gram

Ultimate degree
of soft, watery

 

 

 

muscle of fibrillar protein conditiona
Treatment: -29°C >37°C -29°C >67°C -29°C >37°C
Carcass 6 0.0745 0.0763 14.1 10.1 S S to‘M
7 0.0579 0.1135 36.5 14.5 N S to'M
8 0.0642 0.0901 29.8 13.8 N 'M
9 0.0525 0.0888 45.6 18.2 N N
10 0.0621 0.1059 35.8 11.7 N N
llY 0.0698 0.1072 46.8 10.9 N N

 

 

3N = none; S = slightly; M = moderately; and E = extremely.

was expecially apparent in carcass llY, in which the water bound/g of
fibrillar protein in the muscle from the 37°C treatment was only 23.3%
of that for the corresponding muscle from the -29°C treatment. In con-
trast to carcasses 6 and 7, both sides of carcasses 9, 10, and llY were
assessed as being normal (Table 7).

In both carcasses 7 and 8, the 37°C treatment appeared to induce
the soft, watery muscle condition. Results on these two carcasses would
support the observations of Bendall and Wismer-Pedersen (1962). These
workers found that the washed fibrils from muscle exhibiting the soft,
watery condition had a lower water retention and that the fibrils showed
a gain in protein content as compared to fibrils from normal muscle.

The authors suggested that the fibrils from soft watery muscle were
covered with a layer of denatured sarc0plasmic protein, which was firmly
bound to the surface of the myofilaments. Bendall and Wismer-Pedersen

(1962) also theorized that this was caused by the occurrence of a low pH

 

 

 

 

-77-

while muscle temperature was still high. Furthermore, they observed a
similar phenomenon when muscle samples were allowed to pass into rigor
at 37°C.

The effect of the 37°C treatment in increasing fibrillar protein con-
tent and in decreasing fibrillar water binding capacity in carcasses 9,
10 and llY is in agreement with Bendall and'Wismer-Pedersen's (1962) ob-
servation that this phenomenon pg£“§g_resulted from the occurrence of a
low pH at a high muscle temperature. However, results from.these three
carcasses do not support the suggestion of Bendall and Wismer-Pedersen
(1962) that the decrease in water binding capacity resulting from a low
pH at a high temperature is a primary causal factor in the deve10pement
of the soft, watery condition. These results do not preclude the occuré
rence of decreased fibrillar water binding capacity and the concurrent
gain in protein by the fibrils in soft, watery muscle.

It is suggested that factors other than the low pH-high temperature
phenomenon may also have contributed to the amount of protein gained by
the fibrils, and thus, the corresponding decrease in fibrillar water re-
tention in the muscles of the pigs studied by Bendall and Wismer-Pedersen
(1962). The values for grams of water retained/g of fibrillar protein
by these workers were 11.22 and 6.47 grams for normal and soft, watery
muscles, resPectively. Such values are even lower than the values found
for the muscles from the 37°C treatment in the current study (Table 7).
It might be suggested that the muscles studied by Bendall and Wismer-
Pedersen (1952) underwent a much sharper decline in pH immediately after

death than the carcasses in the present study with a resultant accentuated

 

 

 

 

 

-73-

decrease in fibrillar water-binding capacity. However, observations in
the current study do not support such a suggestion. For example, carcass
6 had a very high glycolytic rate immediately after slaughter which re-
sulted in an initial pH value of 5.6. The side of this carcass which was
subjected to the 37°C treatment, had a 24 hr water—binding capacity of
10.1 grams of water/g of fibrillar protein (Table 7). This value corres-
ponds to similar values for normal muscle from the carcasses from Danish
Landrace pigs studied by Bendall and Wismer-Pedersen (1962). Thus, it is
suggested that muscles from Danish Landrace pigs have a generally lower
fibrillar water-binding capacity than the pigs utilized in the current
study. It is further suggested that another factor(s) besides the occur-
rence of a low pH at a high temperature and/or a sharp decline in post-
mortem pH in the muscles from Danish Landrace pigs may be contributing

to their greatly decreased fibrillar water binding capacity.

Creatine Phosphate

Creatine phosphate was not present in detectable amounts (>0.3 micro-
moles/g of tissue) in any of the initial samples of the l8 carcasses. The
absence of CP may have resulted from electrical stunning, which was used
prior to slaughter. Electrical stunning has been shown to accelerate
the rate of post-mortem pH decline (McLoughlin, l964a,b), which may be
due in part to a rapid depletion of all CP within a few mins after death.

The absence of CP in the present study is in contrast to the results
reported by Bendall gt al., (1963), who found average CP concentrations

of 8.0 and 5.5 micromoles/g of l. dorsi muscle for two groups of Danish

-79-

Landrace pigs. Likewise, Lawrie (1959) reported 1 hr post-mor-em levels ,
of CP equivalent to about 7.2 micromoles/g 0f.l- dgrgi muscle in Large
White X Middle White pigs. The methods of stunning were not given by
Bendall 25 al., (1963) or Lawrie (1959). Kastenschmidt 35.31,, (1964)
reported "low creatine phosphate levels" in the l, dgg§i_muscle from P0-
land China pigs immediately after death, but did not give any values.
Bendall gt al., (1963) and Lawrie (1959) used an acid-molybdate procedure
to determine CP, which has been shown to overestimate CP compared to

other methods (Ennor and Rosenberg, 1952).

ATP

The averages and standard deviations of ATP2 and the corresponding
pH levels for various times post-mortem are given in Table 8. for the
two groups of carcasses, i.e. group 1 (carcasses 6 through 10) and group
2 (carcasses A through G). The average initial pH values were 6.2 and
6.7 for groups 1 and 2, respectively. The initial ATP2 value for both
carcass groups was 5.6 micromoles/g of tissue. In the -29°C treatment
more than 50 and 25% of the initial ATPZ was present in both groups at
approximately 3 and 4 1/2-5 hrs post—mortem, respectively. No ATPZ was
detected at approximately 3 hrs post-mortem in the sides held at >37°C,
except in the case of carcass 7. The levels of ATP for this carcass were
8.7 and 8.9 micromoles/g of tissue for the >37°C and ~29°C treatments,
respectively. In support of these observations, the corresponding pH
values were 6.22 and 6.51.

The average initial ATP(1) value (determined by acid hydrolysis) of

6 micromoles/g of tissue for the carcasses in group 1 was over 23% higher

 

 

 

-80-

ble 8. Post-mortem levels of ATP and pH.

 

Average time

 

 

post-mortem ATP lia ATP(2)B
gasses: Treatment (mins) EH (micrgmg es/g) (micrgmgles/g)
sup 1
arcasses
through - 13 6.2i0.4 6.9i0.9 5.6i4.1
10)
“29°C 166 5.8i006 " 3.2i3.7
286 5.8i0.5 — 2.6:2.4
518 5.7i0.4 - 0.5i0.7
745 5o4i003 - 0.4i-OQ9
37°C 166 5.5t0.4 - * -
285 5.4i001 - 0.0
)up 2
lrcasses
:hrough - 11 6.7i0.l - **5.6i2.2
G)
-29°C 177 6.5i0.2 - 3.7i2.8
323 6.4i0.l - 1.6i2.l
1440 5.7i0.1 _ ase _
37°C 170 5.4i001 - 0.0
317 504i0.1 - 0.0

 

P(1) determined by acid hydrolysis.

P(2) determined by enzymic assay.

Value for carcass 7 was 8.7 micromoles/g; all others = 0.0.

This value based on 6 carcasses.

l carcass contained 0.3 micromole/g; all others = 0.0.

1 the corresponding ATPZ value (determined by enzymic assay; Table 8).

of the carcasses had ATP(2) levels of less than 2 micromoles/g of tissue
= 1.6 micromoles) while the average corresponding ATP(1) value for the

: carcasses was 6.3 micromoles/g. Hence, these ATP(1) values probably

.ect levels of acid hydrolyzable phosphate compounds other than ATP

:uggested by Bendall and Davey (1957).

 

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—81-

However, the initial ATP(1) values (Table 8) are in general agree-
ant with the values of 7.1 and 6.8 micromoles/g of l. dorsi muscle ob-
iined by a similar method of analysis as reported by Bendall g£_§l,,
L963) for two groups of Danish Landrace pigs. The corresponding pH
llues were 6.78 and 6.66 (Bendall E£.EL°: 1963).

An ATP value equivalent to less than 3 micromoles/g of Poland China

dgrsi muscle with a pH level of about 6.5 at "0 hrs" was calculated
Tom the data of Kastenschmidt et al., (1964). This is in good agreement

th a similar value of 3.7 micromoles/g of Hampshire 1. dorsi muscle

 

th a similar pH level in the current study (group 2 carcasses, Table 8),
t is at variance with the results obtained for the Poland China car?
sses (group 1, Table 8).

In disagreement with results of the current study (Table 7), Lawrie
960) reported labile phosphate levels equivalent to about 6.9 micro-

Les/g of l. dorsi muscle from Large White x Middle White pigs at 110

 

IS post-mortem. The corresponding pH level was 6.5. Lawrie (1960)

; commented on the high level of ATP found in these pigs.

'cogen, Glucose and Lactic Acid
The means and standard deviations of pH, glycogen, glucose, and lac-
acid at various times post-mortem for the sides of the two groups of
casses are given in Table 9. The initial levels of glycogen were 28.3
75.8 micromoles/g of tissue in groups 1 and 2, respectively. Wismer-
ersen (1959) reported a comparable value equivalent to 31.1 micromoles/g

L. dorsi muscle from Danish Landrace pigs at 45 mins post-mortem. The

-82..

 

 

 

 

 

 

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-84-

rresponding pH level was 6.24. This is in good agreement with the values
r group 1 carcasses (pH 6.2) in the current study, but considerably

wer than comparable values in group 2 (Table 9). Also, in disagreement
th the results of the current study, Lawrie (1959) reported a value
uivalent to about 34.9 micromoles/g of l. dgggi muscle from Large White
Hiddle White pigs. The corresponding pH value was 6.7 at 1 hr post-mortem.
vever, the average initial glycogen level for the group 1 carcasses

)le 9) are in good agreement with an equivalent level of 33.9 micromoles
of 1, ggrsi muscle from Danish Landrace pigs with a correSponding pH

6.2 at 15 mins post-mortem (Briskey and Wismer-Pedersen, 1961b). These
kers classified this type of muscle as having a "sharp" post-mortem pH
.line.

The much lower initial levels of glycogen in the Group 1 carcasses
land China pigs) than in the group 2 carcasses (Hampshire pigs) is in
eement with the results of Sayre st al., (1963c). These workers found
imes more glycogen in the l. ggggi muscle of Hampshire pigs than in
and China pigs.

The initial glucose levels (total reducing sugars) were 12.6 and 11.6
:mnoles/g of tissue in groups 1 and 2, respectively. These values are
:iderably lower than the equivalent values of 28.3 and 15.5 reported

Vismer-Pedersen (1959) for l. dorsi muscle from Danish Landrace car-

 

es, with pH levels of 6.24 and 6.76, respectively, at 3/4 hr post-
an.

However, the initial glucose values (Table 9) are higher than the

L of 7.4 micromoles/g of l. dgrsi muscle immediately after death cal-

.ed .from the data of Sharp (1962).

The 24 and 48 hr glucose values varied from 24.4 to 35.4 micromoles/g
muscle (Table 7). In general agreement with these values, Sharp (1962)
ported glucose levels equivalent to 32.2 micromoles/g of 1. 5193.11; muscle
ter being held at 18°C for 26 hrs and a further 144 hrs at 1°C.

The initial lactic acid levels were 54.8 and 31.4 micromoles/g of
ssue for groups 1 and 2, resPectively, with corresponding pH levels of
2 and 6.7. These results are in general agreement with those of Wismer-
iersen (1959), who reported values equivalent to 67.8 and 37.8 micromoles
of _1. Q3123; muscle from Danish Landrace pigs when the reSpective pH
rels were 6.2 and 6.7 at 45 mins post-mortem.

However, the initial lactic acid values (Table 9) were lower than
a values of 68.9, 68.9, and 97.8 micromoles/g of Danish Landrace 1. £95.53;
:cle as calculated from the data of Briskey and Wismer-Pedersen (1961b).

: corresponding pH levels were 6.4, 6.6 and 6.2 at 15 mins post-mortem.

The 48 hr lactic acid values in the current study varied from 75.9
88.7 micromoles/g of 1. 5133s]; muscle. These levels are much lower than
values of 124.4 to 128.9 micromoles/g of Danish Landrace _l. _d_9_1_:_s_i_._ mus-

at 24 hrs after death calculated from the data of Briskey and Wismer-

arsen (1961b).

act of Temperature on Glycogen, Glucose and Lactic Acid Levels.

The effect of the two temperature treatments on the average levels
;lucose, glycogen and lactic acid for the group 2 carcasses are shown ,.
'igure 5. As expected, in the carcasses held at 37°C, the glycogen

1 decreased rapidly. At 3 hrs post-mortem, the accumulation of glu-

was evident, while the lactic acid concentration had reached a

-86..

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astant level. In the muscles from the ~29°C treatment, over 90% of
awinitial glycogen was present at 3 hrs postemortem.and over 70% was
asent at 5 hrs. For the same group, appreciable glucose accumulation
1 not occur until after 3 hrs. Lactic acid had attained a level of

>roximately 50% of that in the 37°C treatment at 5 hrs post~mortem.

gradation of Glycogen

The means and standard deviations of the sums of glycogen, glucose
)tal reducing sugars) and lactic acid eXpressed as micromoles of glu-
;e equivalents, and the estimates of the errors in measurement for each
lstituent are given in Table 10.
ple 10. .Means and standard deviations of sums of glycogen, glucose and

lactic acid and estimates of measuring error for each consti-
tuent.

 

I:

casses: Group 1 Group 2

 

L.

COGEN + GLUCOSE +~LACTIC ACIDa:

Initial 68.3f7.3 103.2i33.9
-29°C treatment 67.5i2.9 98.3i35.8
37°C treatment 68.5i4.2 87.3i27.3

[MATED ERROR OF MEASUREMENT:

Glycogen il.9 i4.6
Glucose il.4 i2.0
Lactic acid i4.8 $4.3

 

ues expressed in micromoles of glucose equivalents/g of tissue.
'ns are based on sums calculated for each individual sample in all
438'within treatments. The values for glucose used included total
ucing sugars.

-33-

The values for the muscles of the carcasses in group 1 are very
nilar to those obtained for beef muscle. The overall mean for beef
5 approximately 69 micromoles/g of muscle (Figure 2, p. 67) for these
ne chemical constituents. Hence, on the basis of the data for the
yup 1 carcasses (Table 10), there is nothing to suggest that glycogen
3 not broken down to glucose and lactic acid in a stoichiometric manner
nilar to that observed in beef muscle.

However, the values from the group 2 carcasses do not completely
>port the above conclusion. In these carcasses, there was a general
Idency for the sums of the three components to decrease from the initial
.ue, to the -29°C value, and to the 37°C value, in that order (Table
. This may suggest that some undetected glycolytic intermediate(s)
/were accumulating, and that the leve1(s) was/were influenced by the
.perature treatments used. Alternatively, the trend for decreasing
ues for the post-mortem carbohydrate pool from the group 2 carcasses

‘merely reflect the higher errors of measurements in contrast to those
group 1 (Table 10). The higher errors of measurement were probably
to the high dilutions that were necessary in the glycogen determina-

n for the samples from these carcasses.

EOCHEMICAL OBSERVATIONS IN BEEF MUSCLE

The relative activity of the 17 enzymes studied at 0 and 480 hrs are
11ated in Table 11. The differences in intensity of enzyme activity
:rved between small red, large white and intermediate fibers are in

ement with established patterns of distribution (Pearse, 1960).

3:24! :1.-.:'. 1' car"

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-89-

able 11. Relative enzyme activity in.l, dorsiymuscle from.beef.

 

 

 

Activitya .
L Enzyme 0 hrs 480 hrs
LYCOLYSIS
Lactate dehydrogenase + +’to + +~+ + +.+
a-glycer0phosphate dehydrogenase + + to + + + + to +|+ +
Alcohol dehydrogenase + to + +-+ + +'to +

CA CYCLE

Isocitrate dehydrogenase
Succinate dehydrogenase
Glutamate dehydrogenase

LECTRON TRANSPORT

DPN diaphorase
TPN diaphorase

EXOSE MDNOPHOSPHATE CYCLE

Glucose-6-ph03phate dehydrogenase
6-ph03phog1uconate dehydrogenase

ATTY ACID OXIDATION
-hydroxybutyrate dehydrogenase
EHERS

Acid phOSphatase
Alkaline phosphatase
ATPase (Ca++)
ATPase (Mg++)
TPPase
Leucine amino peptidase

+ to +~+ + +
+ + to + +-+
+ to + +

>+++
>+++

++

 

+ to +-+’+'+
+ to + +
- to

l+

-+ to-+-+-+

+++

++++

++++
++

 

Ihe following code was used for expressing the amount of activity:
- none, r traces, + weak, ++ moderate, +++ moderately strong,-++++ strong.

 

.nhulrrlll

 

 

-90..

lycolyt ic Enzymes

Photomicrographs of representative reactions at 0, 48 and 480 hrs
astdmortem for lactate, dehydrogenase,alpha-glycerOphosphate dehydrogen-
se and alcohol dehydrogenase are shown in Plate 1. Of these three gly-
>1ytic enzymes, lactate dehydrogenase and alpha-glucogen phosphate
:hydrogenase were more uniformly reactive at all times postdmortem than
.cohol dehydrogenase. Alcohol dehydrogenase activity was highly variable
rom carcass to carcass, especially with increasing periods of time post?
»rtem. Furthermore, beyond 24 hrs post-mortem, reactions for alcohol
hydrogenase were usually either very weak or completely absent (Plate

III B and C).

zymes of the TCA Cycle

Typical isocitrate dehydrogenase, succinate dehydrogenase, and glu-
nate dehydrogenase reactions observed in 0, 48 and 480 hr post-mortem
nples are shown in Plate 2. Under post-mortem conditions, isocitrate
lydrogenase appeared to be the most active enzyme of this group as well
the most stable (Table 11). Succinate dehydrogenase activity was de-
:ted at all times post-mortem but was highly variable in intensity.
: lower level of glutamate dehydrogenase activity detected at 0 hrs as
qpared to that observed for succinate dehydrogenase is in agreement
11 the observations on beef muscle of Ogata and Mori (1964). Reactions
gglutamate dehydrogenase were very weak or absent at 48 hrs post-mortem

sill subsequent sampling times (Plate 2, III B and C).

!

 

 

 

I . .'. J:
r
. _ _
. .
.
r
.

 

 

  

‘51:"
, ’97

1. _I_._. dorsi muscle from beef. Lactate (I), alpha-glycerophosPhate
(II), and alcohol (III) dehydrogenase activity at 0 (A), 48 (B)
and 480 (C) hrs post-mortem (x 225).

 

 

 

         

2. L. dorsi muscle of beef. Isocitrate (I), succinate (II), and glutamate
(III) dehydrogenase activity at 0 (A), 48 (B) and 480 (C) hrs post-
mortem (x 225).

 

 

 

 

-93-

Other Enzymes

Plate 3 shows representative reactions for TPN and DPN diaphorase
at 0, 48 and 480 hrs post-mortem, beta-hydroxybutyrate dehydrogenase at
0 hrs, and glucose-6-phosphate dehydrogenase at 0 hrs. The two diaphor-
ases were both generally quite active throughout 480 hrs post-mortem.
However, the DPN diaphorase reactions were uniformly stronger than the
corresponding TPN reactions (Table 11). 1
Muscle from two of the five carcasses studied showed a weak glucose—
6—phosphate dehydrogenase activity at 0 hrs (Plate 3, III A). Very weak
to moderate reactions were observed for beta—hydroxybutyrate dehydrogenase
at 0 hrs (Plate 3, III, B). How-ver, no activity was detected for these
enzymes in either the 24 hr post-mortem or subsequent samples. This
would suggest that these enzymes are more labile under post-mortem condi-
tions than the majority of the enzymes in the current study (Table 11).
The presence of detectable glucose-6—phosphate dehydrogenase activity
was unexpected in View of the work of Ogata and Mori (1964). These in-
vestigators did not detect any activity in normal mouse, cat or human
muscle. However, the activity detected for this hexase monophosphate
shunt enzyme in the current study was confirmed by the Nitro BT procedure
previously described. It is of interest that no activity was observed
for another hexase monophosphate cycle enzyme, 6-phosphogluconate dehydro-
genase (Table 11). The latter may be less stable immediately after death
than glucose-6-phosphate dehydrogenase. The presence of beta-hydroxy-
butyrate dehydrogenase activity supports the suggestion of Howard and
Lawrie (1956) that fatty acid oxidation may play an important role in the

metabolism of beef muscle.

 

Plate 3.

 

L. dorsi muscle from beef.

TPN diaphorase (I) and DPN diaphorase
(II) activity at 0 (A), 48 (B) and 480 (C) hrs post-mortem; glucose-

6-phosphate (III, A) and beta-hydroxybutyrate (III,‘B) dehydrogenase
activity at 0 hrs post-mortem. Magnification:' 225x.

 

 

-95-

Acid phosphatase and leucine amino peptidase activity was not de-
tected in any of the muscles from the five carcasses (Table 11). These
results are in agreement with the results of Ogata and Mori_(l964) who
found no acid phosphatase or leucine amino peptidase activity in normal
human, cow, pig, cat or mouse muscle.

The methods used to detect alkaline phosphatase, the two ATPases
and TPPase resulted in moderate to strong reactions for all samples stu-

died. Activities were similar throughout the post-mortem period (Table
11). The Gomori (1952) method used for alkaline phosphatase resulted in

a very diffuse general reaction and consequently' had no significance.

General Observations

In general, the activity of the enzymes of the glycolytic and TCA
cycles and of the electron transport system, which were observed in the
current study exhibited a steady decrease in activity with increasing time
post-mortem. Results would support the observation of Andrews £5 31,,
(1952) that the lack of substrates is the limiting factor in post—mortem

metabolism in beef muscle.

HISTOCHEMICAL OBSERVATIONS IN PORK MUSCLE

The relative activity at 0 and 24 hrs post-mortem of 15 of the enzymes
studied in carcasses 6 through 10 and llY are shown in Table 12. In gen-
eral, the variation observed in activity at 24 hrs post-mortem did not

appear to be related to the temperature treatment or to the pH level of

a given muscle at the time of removal of a given sample.

-96-

Table 12. Relative enzyme activity in l. dorsi muscle of carcasses 1
through 10 and llY.

 

 

 

Activitya
Enzyme 0 hrs 24 hrs
GLYCOLYSIS
Lactate dehydrogenase + to + + + i to + +
a—glyceroPhOSphate dehydrogenase i to + + + i to + + +
Alcohol dehydrogenase - to + + - to i
TCA CYCLE
Succinate dehydrogenase + to + + + i to + +
Isocitrate dehydrogenase i to + + + —'to + +
Glutamate dehydrogenase i to + + + - to + +
Malate dehydrogenase - to i -
ELECTRON TRANSPORT
DPN diaphorase + to + + + + to + +
TPN diaphorase - _
HEXOSE MONOPHOSPHATE CYCLE
Glucose-6—phosphate dehydrogenase - to i —
6-phosphog1uconate dehydrogenase i to + -
FATTY ACID OXIDATION
-hydroxybutyrate dehydrogenase - to + -
OTHERS
Cytochrome oxidase i to + + i to + +
Acid phosphataseb positive positive
Alkaline phosphataseb positive positive

 

aThe following code was used for expressing the amount of activity:

- no activity, i very weak, + weak, ++ moderate, +++-moderately strong,
++++ strong.
bObserved on carcasses 1 through 7 only.

 

 

 

 

-97_

TCA Cycle Enzymes

Succinate dehydrogenase, isocitrate dehydrogenase and glutamate de-
hydrogenase exhibited similar decreases in activity between 0 and 24 hrs
postdmortem (Table 12). Photomicrographs of typical reactions obtained
for isocitrate dehydrogenase and succinate dehydrogenase at 0 hrs and
24 hrs post-mortem from carcasses subjected to the -29°C and 37°C treat-
ments are shown in Plate 4. However, malate dehydrogenase was observed
to be either weakly active or completely inactive at 0 hrs post-mortem.
In all cases no activity for this enzyme was detected at 24 hrs post-

mortem.

Glycolytic Enzymes

Lactate dehydrogenase and alpha-glycerophosphate dehydrogenase acti-
vity were generally similar at both 0 and 24 hrs post-mortem (Table 12).
However, individual muscles showed higher 24 hr levels of alpha-glycero-
phosphate dehydrogenase activity than that found for lactate dehydrogen-
ase in any of the muscles studied. Photomicrographs representative of
the reactions observed for these two enzymes are shown in Plate 5. Alco-
hol dehydrogenase reactions were weaker than the reactions observed for

either of the other two glycolytic enzymes studied (Table 12).

Electron Transport Enzymes

Weak to moderately strong reactions were obtained when observing DPN
diaphorase activity (Table 12). Typical DPN reactions are shown in Plate
4. However, TPN diaphorase activity was not detected in any of the mus-

cles studied.

 

 

Plate 4.

L. dorsi muscle from pork.
dehydrogenase (II) and DPN diap
24 hrs post-mortem from carcasses subjected t

(C) treatments (X 225).

 

 

Isocitrate dehydrogenase (I), succinate
horase (III) activity at 0 (A) and
o the -29°C (B) and 37°C

 

 

 

 

     

\

- 'rgf-i ewe. ' h ’0
. , v c - ‘ 4
.-‘ a _ ‘ . ’ A... ._.‘I.
Plate 5. L. dorsi muscle of pork. Alpha-glycerophosphate (I) and lactate (II)
dehydrogenase activity at 0 (A) and 24 hrs post—mortem in -29°C (B)
and 37°C (C) treatments (x 225); cytochrome oxidase activity (III, A)

at 0 hrs (A) post-mortem (x 860); and Aniline blue—orange C stained
sections from 24 hrs post—mortem muscle in —29°C (III B) and 37°C

 

(III C) treatments (x 225).

 

 

 

 

-100-

Other Metabolic Enzymes

Glucose-6-phosphate dehydrogenase activity was detected in traces
in some of the muscles studied. Reactions for 6-phosphogluconate dehy—
drogenase and beta-hydroxybutyrate dehydrogenase were slightly more
positive (Table 12). Cytochrome oxidase activity was detected at levels
which varied in intensity from very weak to moderate at both 0 and 24

hrs post-mortem. A typical 0 hrs reaction is shown in Plate 5.

Acid and Alkaline Phosphatases

Positive areas were observed for acid and alkaline phosphatase
activity in all muscles in which these enzymes were studied (carcasses 1
through 7). In all cases, the activity was not effected by any of the
temperature treatments. The incidence of positive areas did not appear
to be related to the appearance of the muscles at 48 hrs post-mortem.

Various levels of acid phosphatase activity are shown in Plate 6
(I A, B, and C); II A). It was concluded that the fiber showing an in-
tense level of acid phosphatase activity (II A) was in an active state of
degeneration. This conclusion was supported by the results of other
workers. Fennell and West (1963) concluded that a striking feature of
dystrophic muscle in mice was the strong acid phosphatase reactions of
atroPhic fibers and the interfibrillar connective tissues. They further
suggested that acid phosphatase may function in catabolic processes. This
suggestion was supported by the results of Klamer and Fennell (1962), who
observed increased acid phosphatase activity in specimens of Tetrahymena
during the declining phase of growth. Furthermore, Beckett and Bourne
(1960) observed that groups of inflammatory cells invading degenerating
fibers often showed a moderate to strong histochemical reaction for acid

phosphatase.

 

 

II
III
Plate 6.

 

a

L, dorsi muscle of pork: varied levels of acid phosphatase activity
in isolated fibers (I, A, B, and C; x 225); strong acid phosphatase
activity in degenerating fiber (II, A; x 860); alkaline phosphatase
activity (11, B, C; x 225); alkaline phosphatase activity in "frag-
mented” fiber (III A; x 860); and alkaline phOSphatase activity show-
ing developement of cross—linkages between fibers at points of high
activity (III, B; x 860).

 

 

 

 

-102-

The distribution of alkaline phsophatase activity is shown in Plate
6 (photomicrographs II B, C; III A, B). The general distribution of
activity is shown in the two photomicrographs at the lower magnification
(II, B, C). The photomicrographs III A and B are higher magnifications
of the section shown in II B (Plate 6). In photomicrograph III A, alka-
line phosphatase activity is shown in a fragmented or possibly degenerating
fiber. The positive area in the upper part of photomicrograph III B
shows alkaline phosphatase in adjacent areas of two separate fibers. In
the lower part of the same photomicrograph, a similar area is observed,
but in this case the fibers appear to be joined together at the point of
phosphatase activity.

It is suggested that the alkaline phosphatase activity observed is
indicative of an abnormal condition in these muscles. Such a conclusion
is supported by the observations of Beckett and Bourne (1960) who have
noted that alkaline phosphatase activity is limited to the walls of the
capillaries and the endothelial lining of the larger blood vessels in
normal muscle. These workers have observed positive alkaline phosphatase
activity in fibers from human muscle taken from patients with various
muscular or neuromuscular disorders. However, the occurrence of the alka-
line phosphatase positive fibers observed by Beckett and Bourne (1960)
was not correlated with the general histological state of the muscle or

with the diagnosis.

UDPG—Glycogen Transferasez Phosphorylasez and Branching Enzyme Activity

Histochemical procedures for observing phosphorylase and BE (branch-

ing enzyme or amylo-l, 4-—>1,6-transglucosidase) activity were carried

 

. ulna-L

 

 

 

 

 

 

 

 

.'-.....

-lO3-

out on some of the samples of carcasses 6 through 10. Some differences
appeared to be present, but these differences were highly variable.
Furthermore, the tissue blocks had been stored at -29°C for 2 1/2-3 wks.
Consequently, the generally weak reactions as well as excessive ice
crystal growth precluded any conclusions concerning the differences ob-
served.

Phosphorylase, BE, and UDPG-glycogen transferase activity was ob-
served in the initial sample, at approximately 3 hrs, and at 24 hrs
post-mortem for carcasses A through G. Results of these observations
are given in Table 13.

In the muscles from the -29°C treatment, considerable variation
existed in amount of activity observed between different carcasses (Table
13). However, any relationship between activity, pH and ultimate muscle
condition was not apparent. An exception to this observation was the low
UDPG-glycogen transferase activity observed in carcass 6. This carcass
had a pH value of 6.1 at 3 hrs post-mortem, which may suggest that the
low pH level may have inactivated this enzyme.

There was no detectable UDPG-glycogen transferase activity present
at 3 hrs post-mortem in any of the muscles of the sides subjected to the
37°C treatment. However, it is impossible to distinguish whether complete
inactivation resulted from low pH values alone or from the combination of
a low pH value and high muscle temperatures.

The variable effect of the 37°C treatment on phosphorylase and phos-
phorylase + BE activity (Table 13) could not be explained on the basis

of muscle pH or ultimate 48 hr muscle condition.

 

 

 

 

 

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~105-

The reactions observed in the initial samples were generally similar
to those found for the -29?C treatment at 3 hrs post-mortem. The reactions
observed for all of the muscles at 24 hrs post-mortem generally reflected
the activity at 3 hrs post-mortem. However, all reactions at 24 hrs were
considerably weaker than those observed in the -29°C treatment at 3 hr
post-mortem. Photomicrographs showing an extreme effect of the heat treat?
ment on the histological condition of carcass A have been included in

Plate 5 (III B, c).

General Observations

Generally, weaker activities were observed for pork muscle in the
initial and the 24 hrs post-mortem samples than was observed at corres-
ponding times for beef muscle. This observation may merely reflect a
much faster pace of post-mortem chemical changes in pork muscle. The
apparent absence of detectable malic dehydrogenase or TPN diaphorase
activity in pork muscle may suggest that these two enzymes are quite
labile under the conditions extant immediately after death in pork muscle.
However, it is suggested that this observation requires further substan-
tiation.

Although the degree of acid and alkaline phosphatase activity did
not appear to be related to the ultimate muscle condition, the detection
of such activity, together with the implications previously discussed,

would seem to warrant further elucidation.

 

 

 

 

 

SUMMARY

Studies on Beef

Chemical changes in l. dorsi muscle of five beef carcasses were fol-

 

lowed from less than 10 minutes after death through 20 days post-mortem.
In addition, the activity of 17 specific enzymes was followed by histo-
chemical procedures.

Initial levels of pH, creatine phosphate and glycogen were in agree~
ment with values reported by previous workers. Initial ATP levels aver-
aged 10.9 micromoles/g of tissue when determined enzymically, but were
considerably lower (6.4 micromoles/g) when measured by an acid hydrolyses
procedure. It is believed that the latter underestimates the true ATP
value found in beef muscle immediately post-mortem.

Results suggest that the onset of rigor was initiated at about 12—15
hrs post-mortem. A fairly linear breakdown of glycogen occurred during
the first 24 hrs post—mortem, after which breakdown occurred at a much
slower rate. When the minimum pH of 5.4 was reached at about 96 hrs after
death, approximately 20% of the total initial glycogen remained. However,
at 480 hrs post-mortem, less than 3% of the glycogen remained.

There was an increase in reducing sugars during the first 24 hrs
post-mortem, and thereafter, little change. Lactic acid increased rapidly
during the first 36-48 hrs post-mortem, with only slight further increases
up to 480 hrs. It was concluded that glycogen was stoichiometrically
degraded to lactic acid and reducing sugars. Of the two products, lactic

acid predominated and increased some 6 1/2 times from within 10 mins after

~106-

 

 

 

 

-107-

death to 480 hrs post-mortem. During the same period of time, glucose
only slightly more than doubled.

The histochemical activity of lactate dehydrogenase, alpha glycero-
phosphate dehydrogenase, succinate dehydrogenase, isocitrate dehydrogenase,
TPN diaphorase and DPN diaphorase showed a steady decrease with increasing
times post-mortem. Reactions for both alcohol dehydrogenase and glutamate
dehydrogenase were very weak or absent at 48 hrs post-mortem and all sub-
sequent sampling periods.

Positive reactions for g1ucose-6-phosphate dehydrogenase and beta-
hydroxybutyrate dehydrogenase were observed in the initial samples only.
Acid phosphatase, leucine amino peptidase, and 6-phosphogluconate dehydro~

genase were not detected in any of the samples.

Pork Studies

Chemical and histochemical procedures similar to those described for
beef were used to follow the changes from 0 to 24 and/or 48 hrs post-
mortem in the l. g2;§;_muscle of eighteen pork carcasses. One side from
each of thirteen pork carcasses was placed at —29°C, while the other side
from each carcass was subjected to 37°C for the first 4 1/2-5 hrs post-
mortem in an attempt to induce soft, watery pork.

The muscles of the pork carcasses studied exhibited marked differences
in initial levels of pH, glycogen, glucose and lactic acid. Post-mortem
accumulation of glucose and lactic acid was similar to that noted in beef
muscle. A similar stoichiometric relation to that found in beef muscle

between post—mortem decrease in glycogen and a corresponding accumulation

 

 

 

~108-

of lactic acid and glucose existed in the carcasses from Poland China
pigs. An equivalent relationship for the carcasses from Hampshire pigs
was less apparent.

The 37°C treatment did not consistently induce soft, watery muscle
as was expected from reports of previOus workers. It was concluded that
a low pH at a high muscle temperature Egg gm was not a causal factor in
the developement of the soft, watery condition. The loss in fibrillar
water-binding capacity as the result of low pH values at high muscle temp-
eratures as reported by previous workers was confirmed. In contrast to
previous studies, decreases in fibrillar water binding capacity induced
by the low pH-high temperature phenomenon did not usually result in soft,
watery muscle. Although the loss in fibrillar water-binding capacity as
a result of the low pH value-high temperature relationship is a character-
istic encountered in soft, watery muscle, this characteristic by itself
is not a primary causal factor in the developement of this condition.

The activity of lactate dehydrogenase, alpha-glycerophosphate dehy-
drogenase, succinate dehydrogenase, isocitrate dehydrogenase and DPN dia-
phorase was slightly weaker at both 0 and 24 hrs than shuilar activity in
beef muscle. Alcohol dehydrogenase activity was weaker than that observed
for the above enzymes, while the activity of glutamate dehydrogenase was
of intermediate intensity. Glucose-6-ph03phate dehydrogenase and 6-phos-
phogluconate dehydrogenase showed traces of activity only in some of the
initial samples. Similarly, weak reactions for beta-hydroxybutyrate
dehydrogenase were observed only at 0 hrs post-mortem. None or only weak

activity was observed for malic dehydrogenase in all samples studied.

 

 

 

 

 

 

-109-

Likewise, TPN diaphorase activity was unexplainably absent from all sam-
ples. Moderate levels of cytochrome oxidase activity were observed at
both 0 and 24 hrs post-mortem.

UDPG-glycogen transferase was completely inactivated by the 37°C
treatment, but the treatment had a less marked effect upon phosphorylase
and branching enzyme. The presence of acid and alkaline phosphatase
activity in muscles of 7 pork carcasses suggested that a degenerative

condition existed in these muscles.

 

 

 

 

 

 

BIBLIOGRAPHY

Allen, R. J. L. 1940. The estimation of phosPhorous. Biochem. J. 24, 858.

Andrews, M. M., Guthneck, B. T., MtBride, B. H., and Schweigert, B. S.
1952. Stability of certain respiratory and glycolytic enzyme systems
in animal tissues. J. Biol. Chem._g, 715.

Astbury,'W. T. 1947. On the structure of biological fibres and the pro-
blem of muscle. Proc. Roy. Soc. (London), SE 134, 304.

Bailey, K. 1942. 'Myosin and adenosine triphOSphatase. Biochem. J. 39,
121.

Bailey, K. 1954. Structure Proteins, II. 'Muscle. In The Proteins, Vol.
II, p. 951. Ed. H. Neurath and K. Bailey. Academic Press. N. Y.

 

Barker, S. B. and Summerson, W. H. 1941. The colorimetric determination
of lactic acid in biological material. J. Biol. Chem. 138, 535.

Bate Smith, E. C. 1938. The buffering of muscle in rigor; protein,
phosPhate and carnosine. J. Physiol. 22, 336.

Bate Smith, E. C. 1939. Changes in elasticity of mammalian muscle under-
going rigor mortis. J. Physiol. 26, 176.

Bate-Smith, E. C. 1948. The physiology and chemistry of rigor mortis
with special reference to the aging of beef. Adv. in Food Res. 1, 1.

Bate-Smith, E. C. and Bendall, J. R. 1947. Rigor mortis and adenosine-
triphosphate. J. Physiol. 106, 177.

Bate~Smith, E. C. and Bendall, J. R. 1949. Factors determining the time
course of rigor mortis. J. Physiol. 110, 47.

Beckett, E. B. and Bourne, G. H. 1958. Acid and alkaline phosphatases
in normal and diseased human muscle. Acta Anat. 32, 326.

Beckett, E. B. and Bourne, G. H. 1960. Histochemistry of skeletal muscle
and changes in some muscle diseases. In Structure and Function of
‘Muscle, Vol. III, 276. Ed. G. H. Bourne. Academic Press, N.Y.

Bendall, J. R. 1951. The shortening of rabbit muscles during rigor mor-
tis: its relation to the breakdown of adenosine triphOSphate and
creatine phOSphate and to muscular contraction. J. Physiol. 114, 71.

Bendall, J. R. 1953. Further observations on the factor (the "Marsh"
factor) effecting relaxation of ATP-shortened muscle-fibre models,

and the effect of Ca andng ions upon it. J. Physiol. (London) 121,
232.

~110-

 

 

 

 

 

 

 

 

 

-lll-

Bendall, J. R. l960a. Relaxing factors in muscle. Biochem. Biophys.
Research Comm. 3, 641.

Bendall, J. R. 1960b. Post mortem changes in muscle. In The Structure
and Function of Muscle, Vol. III, p. 227. Ed. G. H. Bourne, Academic
Press, N. Y.

Bendall, J. R. 1962. The structure and composition of muscle. In Recent
Advances in Food Science. Eds. J. Hawthorn and J. M. Leitch. Vol. I,
p. 58.

Bendall, J. R. 1963. Physiology and chemistry of muscle. Proc. Meat
Tenderness Symposium, p. 33. Campbell Soup Co., Camden, N. J.

Bendall, J. R. and Davey, C. L. 1957. Ammonia liberation during rigor
mortis and its relation to changes in the adenine and inosine nucleo-
tides of rabbit muscle. Biochim. Biophys Acta 26, 93.

Bendall, J. R., Hallund, O. and Wismer-Pedersen, J. 1963. Post-mortem
changes in the muscles of Landrace pigs. J. Food Sci. 28, 156.

Bendall, J. R. and Wismer-Pedersen, J. 1962. Some properties of the
fibrillar proteins of normal and watery pork muscle. J. Food Sci.
2_7_, 144.

Bennett, H. S. 1955. Modern concepts of structure of striated muscle.
Am. J. Phys. Mad. 333 46.

Bennett, H. S. 1956. The concepts of membrane flow and membrane vesicu-
lation as mechanisms for active transport and ion pumping. J. Biophys.
Biochem. Cytol. g Suppli., 99.

Bennett, H. S. 1960. The structure of striated muscle as seen by the
electron microscope. In The Structure and Function of Muscle, Vol. I,
137. Ed. G. H. Bourne. Academic Press, N.Y.

Bennett,.H. S. and Porter, K. R. 1953. An electron microscope study of
sectioned breast muscle of the domestic fowl. Am. J. Anat. 23, 61.

Blanchaer, M. and VanWijhe, M. 1962. Distribution of lactic dehydrogen-
ase in skeletal muscle. Nature 193, 887.

Blanchaer, M. C., VanWijhe, M., and Mozersky, D. 1963. The oxidation of
lactate and a-glycerophosphate by red and white skeletal muscle: I.
Quantitative studies. J. Histochem. Cytochem. 11, 500.

Blanchaer, M. C. 1964. Respiration of mitochondria of red and white
skeletal muscle. Am. J. Physiol. 206, 1015.

 

 

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.
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-112-

Bloom, W. and Fawcett, D. W. 1963. A textbook of Histology. W. B. Saun-
ders, Philadelphia.

Borchert, L. L. and Briskey, E. J. 1964. Prevention of pale, soft,
exudative porcine muscle through partial freezing with liquid nitro-
gen post-mortem. J. Food Sci. 22, 203.

Bosanquet, F. D., Daniel, P. M. and Parry, H. B. 1960. Myopathy: the
pathological changes in intrinsic diseases of muscles. In The Struc-
ture and Function of Muscle, Vol. III, 336. Ed. G. H. Bourne.
Academic Press, N. Y.

Bowen, W. J. 1951. Phosphorylysis of adenosine triphosphate and rate of
contraction of Myosin B threads. Am. J. Physiol. 165, 10.

Bowen, W. J. 1954. Relation of maximum shortening of glycerine-treated
muscle and the concentration of ATP. Am. J. Physiol. 179, 620.

Bowen, W. J. and Martin, H. L. 1958. Rate of dephosphorylation of adeno-
sinetriphosphate and the isotonic and isometric contraction of glycerol-
treated muscle. Am. J. Physiol. 195, 311.

Briggs, F. N., Kaldor, G. and Gergely, J. 1959. Participation of a dia-
lyzable cofactor in the relaxing system of muscle. III. Substitution
of pyrophosphate for the cofactor. Biochim. Biophys. Acta. 24, 224.

Briggs, F. N. and Portzehl, H. 1957. The influence of relaxing factor
on the pH dependence of the contraction of muscle models. Biochim.
Biophys. Acta 24, 482.

Briskey, E. J., Bray, R. W., Hoekstra, W. C., Grummer, R. H., and Phillips,
P. H. 1959a. The chemical and physical characteristics of various
pork ham muscle classes. J. Anim. Sci. lfi, 146.

Briskey, E. J., Bray, R. W., Hoekstra, W. G., Grummer, R. H., and Phillips,
P. H. 1959b. The effect of various levels of exercise in altering
the chemical and physical characteristics of certain pork ham muscles.
J. Anim. Sci. 2§, 153.

Briskey, E. J., Bray, R. W., Hoekstra, W. G., Grummer, R. H., and Phillips,
P. H. 1959c. The effect of exhaustive exercise and high sucrose
regimen on certain chemical and physical pork ham muscle characteris-
tics. J. Anim. Sci. 2§, 173.

Briskey, E. J., Bray, R. W., Hoekstra, W. C., Phillips, P. H. and Grummer,
R. H. 1960. Effect of high protein, high fat and high sucrose rations
on the water-binding and associated properties of pork muscle. J.
Anim. Sci. 12, 404.

-113-

Briskey, E. J. and Wismer-Pedersen, J. 1961a. Biochemistry of pork muscle
structure. 1. Rate of anaerobic glycolysis and temperature change
versus the apparent structure of muscle tissue. J. Food Sci. 26, 297.

Briskey, E. J. and Wismer—Pedersen, J. 1961b. Biochemistry of pork muscle
structure. II. Preliminary observations of bi0psy samples versus
ultimate muscle structure. J. Food Sci. 26, 306.

Burstone, M. S. 1958. The relationship between fixation and techniques
for the histochemical localization of hydrolytic enzymes. J. Histo-
chem. Cytochem. 9, 322.

Burstone, M. S. 1959. New histochemical techniques for the demonstration
of tissue oxidase (cytochrome oxidase). J. Histochem. Cytochem. 2, 112.

Burstone, M. S. 1961a. Modifications of histochemical techniques for the
demonstration of cytochrome oxidase. J. Histochem. Cytochem. 2, 59.

Burstone, M. S. 1961b. Histochemical demonstration of phosphatases in
frozen sections with naphthol AS-phosphates. J. Histochem. Cytochem.
9 146
_.’ O

Burstone, M. S. and Folk, J. E. 1956. Histochemical demonstration of
aminopeptidase. J. Histochem. Cytochem. 4, 217.

Cain, D. F. and Davies, R. E. 1962. Breakdown of adenosine triphosphate
during a single contraction of working muscle. Biochem. Biophys.
Res. Comm. §, 361.

Cain, D. F., Infante, A. A. and Davies, R. E. 1962. Chemistry of muscle
contraction. Nature 196, 214.

Callow, E. H. 1937. The electrical resistance and micro-structure of
muscular tissue. Ann. Rept. Food Invest. Bd., p. 46. (London).

Callow, E. H. 1938. The structure of muscular tissue. Ann. Rept. Food
Invest. Bd., p. 55 (London).

Danielli, J. F. 1953. Cytochemistgy. Wiley and Sons, N. Y.

Davey, C. L. 1957. An ion exchange method of determining carnosine,
anserine and their precursors in animal tissues. Nature 179, 209.

Davey, C. L. l960a. The effects of carnosine and anserine on glycolytic
reactions in skeletal muscle. Arch. Biochem. Biophys. §2, 296.

Davey, C. L. 1960b. The significance of carnosine and anserine in striated
skeletal muscle. Arch. Biochem. Biophys. §2, 303.

 

 

 

 

-114-

Davies, R. E. 1963. A molecular theory of muscular contraction: Calcium
dependent contractions with hydrogen bond formation plus ATP-dependent
extensions of part of the myosin-actin cross-bridges. Nature 199,
1068.

deFremery, D. and Pool, F. 1960. Biochemistry of chicken muscle as
related to rigor mortis and tenderization. Food Res. 25, 73.

deFremery, D. and Pool, M. F. 1963. The influence of post-mortem glyco-
lysis on poultry tenderness. J. Food Sci. 2§, 173.

Dubowitz, V. and Pearse, A. G. E. 1960. A comparative histochemical
study of oxidative enzyme and phosphorylase activity in skeletal
muscle. Histochemie 2, 105.

Dubowitz, V. and Pearse, A. G. E. 1961. Enzymic activity of normal and
dystrophic human muscle: a histochemical study. J. Path. Bact. 82,
365.

Dubuisson, M. 1954. Muscular Contraction, C. C. Thomas, Springfield, Ill.

Ebashi, S. 1961a. Calcium binding activity of vesicular relaxing factor.
J. Biochem. 22, 236.

Ebashi, S. 1961b. The role of "relaxing factor” in contraction-relaxation
cycle of muscle. Progr. Theoret. Phys. 22, 35.

E113, H. A. and Faulkner, P. 1961. Inhibition of myofibrillar adenosine-
triphosphatase and syneresis by 1,3-diphosphog1yceric acid. Biochem.
Biophys. Research Comm. 2, 255.

Engelhardt, W. A° 1946. Adenosine triphosphatase prOperties of myosin.
Adv. in Enzymol. 6, 147.

Engelhardt, W. A. and Ljubimova, M. N. 1939. Myosin and adenosinetri-
phosphatase. Nature 144, 668.

Ennor, A. H. and Rosenberg, H. 1952. The determination and distribution
of phosphocreatine in animal tissues. Biochem. J. 52, 606.

Esterbrook, R. W. and Sackton, B. 1958. a-glycerophosphate oxidase of
flight muscle mitochondria. J. Biol. Chem. 233:1014.

Fennell, R. A. and west, W. T. 1963. Oxidative and hydrolytic enzymes
of homozygous dystrophic and heterozygous muscle of the house mouse.
J. Histochem. Cytochem. 22, 374.

Flory, P. J. 1956a. Role of crystallization in polymers and proteins.
Science 124, 53.

Flory, P. J. 1956b. Theory of elastic mechanisms in fibrous proteins.
J. Am. Chem. Soc. 18, 5222.

 

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n
. n a u n .
. u p
. n n n
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-115-

Flory, P. J. 1957. Crystallinity and dimensional changes in fibrous
proteins. J. Cellular Comp. Physiol. 42 (Suppl. 1), 175.

Folin, A. and VH5 H. 1920. A simplified and improved method for deter—
mination of sugar. J. Biol. Chem. 42, 367.

Fredholm, H. 1963. 5'- mono-, di- and triphosphates of adenosine in beef
and pork kept under refrigeration. Acta Agri. Scand. 22, 352.

Gergely, J. 1956. The interaction between actomyosin and adenosine tri-
phosphate. Light scattering studies. J. Biol. Chem. 220, 917.

Glick, D. 1949. Techniques of Histo- and Cyto-Chemistry. Interscience,

O Y.

Goldspink, G. 1962. Biochemical and physiological changes associated
with the postnatal developement of the biceps brachii. Comp. Biochem.
Physiol. 2; 157.

Goldspink, G. and McLoughlin, J. V. 1964. Studies on pig muscle. 3.

The effect of temperature on the solubility of the sarcoplasmic pro—
teins in relation to colour changes in post-rigor muscle. Irish J.
Agri. Res. 2, 9.

Gonnori, G. 1952. Microscopic Histochemistry. Chicago Univ. Press.

Grogg, E. and Pearse, A. G. E. 1952. A critical study of the histochemi—
cal techniques for acid phosphatase, with a description of an azo-
dye method. J. Path. Bact. 64, 627.

Guyer, M. F. 1953. Animal Micrology. Univ. of Chicago Press.

Hamm, R. 1960. Biochemistry of meat hydration. Adv. Food Res. 29, 355.

Hamoir, G. 1955. Fish Proteins. Advances in Protein Chem. 29, 227.

Hanson, J. and Huxley, H. E. 1953. The structural basis of the cross-
striations in muscle. Nature 172, 530.

Hanson, J. and Huxley, H. E. 1955. The structural basis of contraction
in striated muscle. Symposia Soc. Exptl. Biol. 2, 228.

Hansen, R. C., Rutter, W. G. and Crowe, E. M. 1952. A nephelometric
method for determination of glycogen. J. Biol. Chem. 195, 127.

Hasselbach, W. and Makinose, M. 1962. ATP and active transport. Biochem.
Biophys. Res. Comm. 2, 132.

Hasselbach, W. and Weber, H. H. 1953. The influence of the MB factor on
the contraction of fiber models. Biochim. Biophys. Acta 22, 160.

-116-

Hess, R. and Pearse, A. G. E. 1961. Dissociation of uridine diphosphate
glucose-glycogen trans-glucosylase from phOSphorylase activity in
individual muscle fibers. Proc. Soc. Exp. Biol. Med. 107, 569.

Hess, R., Scarpelli, D. G. and Pearse, A. G. E. 1958. The cytochemical
localization of oxidative enzymes. II. Pyridine nucleotide-linked
dehydrogenases. J. BioPhys. Biochem. Cytol. 4, 753.

Hill, T. L. 1953. Energetics and molecular mechanisms in muscle action.
Part II. Statistical thermodynamical treatment of contractile systems.
Disc. Faraday Soc. 22, 132.

Hodge, A. J., Huxley, H. E. and Spiro, D. 1954. Electron microscope stu-
dies on ultrathin sections of muscle. J. Exptl. Med. 22, 201.

Hoeve, C. A. J. and Willis, V.A. 1963. Elasticity of the fibrous muscle
proteins. Biochem. 2, 279.

Hoeve, C. A. J., Willis, V. A., and Martin, D. J. 1963. Evidence for a
phase transition in muscle contraction. Biochem. 2, 282.

Howard, A. and Lawrie, R. A. 1956. Studies on beef quality. 11. Physio-
logical and biological effects of various pre-slaughter treatments.
Tech. Paper, No. 2, p. 18; Div. Food Pres. Transp. C.S.I.R.O., Mel-
bourne.

Howard, A. and Lawrie, R. A. 1957. Studies on beef quality V. Further
observations on biochemical and physiological responses to pre-
slaughter treatments. Tech. Paper, No. 4, Div. Food Pres. Transp.
C.S.I.R.O., Melbourne.

Huxley, A. F. 1957. Muscle structure and theories of contraction. Progr.
in Biophys. and Biophys. Chem. 2, 257.

Huxley, H. E. 1953. Electron-microscope studies on the organization of
the filaments in striated muscle. Biochim. Biophys. Acta 22, 387.

Huxley, H. E. 1957. The double array of filaments in cross-striated
muscle. J. Biophys. Biochem. Cytol. 2, 631.

Huxley, H. E. and Hanson, J. 1954. Changes in the cross-striation of
muscle during contraction and stretch and their structural interpre-
tation. Nature 173, 973.

Huxley, H. E. and Hanson, J. 1957a. Quantitative studies on the structure
of cross-striated myofibrils. I. Investigations by interference
microscopy. Biochim. Biophys. Acta 22, 229.

Huxley, H. E. and Hanson, J. 1957b. Preliminary observations on the
structure of inset flight muscle. Proc. Stockholm Cont. Electron
Microscopy 1956, p. 202.

 

 

 

 

-117-

Huxley, H. E. and Hanson, J. 1960. The molecular basis of contraction

in cross-striated muscles. In The Structure and Function of Musclez
Vol. I, 183. Ed. G. H. Bourne. Academic Press, N.Y.

Huxley, A. F. and Niedergerke, R. 1954. Structural changes in muscle
during contraction. Nature 173, 971.

Huxley, A. F. and Taylor, R. E. 1955. Function of Krause's membrane.
Nature 176, 1068.

Iyengar, M. R., Glauser, S. C., and Davies, R. 1964. An ATP-induced
conformational change in the acto-H—meromyosin system. Biochem.
Biophys. Res. Comm. 2Q, 379.

Kastenschmidt, L. L., Briskey, E. J. and Hoekstra, W. G. 1964. Preven-
tion of pale, soft, exudative porcine muscle through regulation of
ante-mortem environmental temperature. J. Food Sci. 22, 210.

Kielley, W. W. and Meyerhof, O. 1948. Studies on adenosinetriphosphate
of muscle. II. A new magnesium—activated adenosinetriphosphatase.
J. Biol. Chem. 176, 591.

Klamer, B. and Fennell, R. A. 1963. Acid phosphatase activity during
growth and synchronous division of Tetrahymena pyriformis. Exp.
Cell. Res. 22, 166.

Lawrie, R. A. 1953. The onset of rigor mortis in various muscles of the
draught horse muscle. J. Physiol. 121, 275.

Lawrie, R. A. 1955. Residual glycogen at high ultimate pH in horse muscle.
Biochim. Biophys. Acta 21, 282.

Lawrie, R. A. 1958. Physiological stress in relation to dark-cutting beef.
J. Sci. Food Agr. 2, 721.

Lawrie, R. A. 1960. Post mortem glycolysis in normal and exudative long-
issimus dorsi muscles of the pig in relation to so-called white muscle
disease. J. Comp. Path. 12, 273.

Lawrie, R. A. 1962. The conversion of muscle to meat. In Recent Advances

in Food Science, Vol. I, p. 68. Eds. J. Hawthorn and J. M. Leitch.
Butterworths, London.

Lawrie, R. A., Gatherum, D. P. and Hale, H. P. 1958. Abnormally low ulti-
mate pH in pig muscle. Nature 182, 807.

Lillie, R. D. 1954. Histopathologie Technic and Practical Histochemistgy.

Blakiston Co., N. Y.

 

 

. u n u I u I . .
n
. .. n . u . u u u v
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-1184

Lorand, L., Molner, J. and Moos, C. 1957. Conference on the chemistry
of muscular contraction, Tokyo, 1957, p. 85. Igaku Shoin Ltd., Tokyo.

Lorand, L. and Molner, J. 1962. Muscle as a tissue, p. 126. McGraw-Hill,
N. Y.

Ludvigsen, J. 1953. "Muscular degeneration" in hogs (preliminary report).
Proc. XVth Intern. Veterin. Cong. Part 1, Vol. 1, 602. Boktryckeri,
Stockholm.

Magee, W. T. 1964. Personal communications.

Marsh, B. B. 1952a. The effects of ATP on the fibre-volume of a muscle
homogenate. Biochim. Biophys. Acta 2, 247.

Marsh, B. B. 1952b. Observations on rigor mortis in whale muscle. Bio-
chim. Biophys. Acta. 2, 127.

Marsh, B. B. 1954. Rigor mortis in beef. J. Sci. Food Agr. 2, 70.

Marsh, B. B. 1960. Identification of the muscle relaxing factor. Bio-
chem. Biophys. Research Comm. 3, 233.

Marshall, J. M., Holtzer, H., Frick, H. and Pepe, F. 1959. The distribu-
tion of protein antigens in striated myofibrils. Exptl. Cell. Res.
2, 219 (Suppl.).

McLoughlin, J. V. 1963. Studies on pig muscle. 2. The effect of rapid
post-mortem pH fall on the extraction of the sarcoplasmic and myo-
fibrillar proteins of post-rigor muscle. Irish J. Agri. Res. 2, 117.

McLoughlin, J. V. 1964a. Observations on pale, soft exudative musculature.
Proc. 17th Ann. Reciproc. Meats Conf., Madison, Wisc.

McLoughlin, J. B. 1964b. Personal communcation.

McLoughlin, J. V. and GoldSpink, G. 1963a. Studies on Pig Muscle. I.
Exudative pig muscle. Irish J. Agri. Res. 2, 27.

McLoughlin, J. V. and Goldspink, G. 1963b. Post—mortem changes in the
colour of pig longissimus dorsi muscle. Nature 198, 584.

Meyer, J. A., Briskey, E. J., Hoekstra, W. G. and Bray, R. W. 1962. Glu-
cose tolerance in swine as related to post-mortem muscle characteristics.
J. Anim. Sci. 21, 543.

Mihalyi, E. and Szent-Gygrgyi, A. G. 1953. Trypsin digestion of muscle
proteins. I. Ultracentrifugal analysis of the process. J. Biol.
Chem. 201, 189.

 

 

 

 

 

 

 

-ll9—

Mommaerts, W. F. H. M. 1947. The reaction between actomyosin and adeno-
sine triphosphate. J. Gen. Physiol. 31, 361.

Mommaerts, W. F. H. PL 1950. Muscular Contraction. Interscience, N. Y.

Mommaerts, W. F. H. M. 1954. The biochemistry of muscle. Ann. Rev.
Biochem. 23, 381.

Mommaerts, W. F. H. M. 1955. Investigation of the presumed breakdown of
adenosine-triphosphate and phosphocreatine during a single muscle
twitch. Am. J. Physiol. 182, 585.

Mommaerts, W. F. H. M., Seraydarian, K. and Uchida, K. 1963. On the re-
laxing substance of muscle. Biochem. Biophys. Res. Comm. 1;, 58.

Moog, F. 1943. Cytochrome oxidase in early chick embryo. J. Cell Comp.
Physiol. 22, 223.

Morales, M. F. and Botts, J. 1956. A theory of the primary event in
muscle action. In Currents in Biochemical Research, p. 609, Ed. D.
E. Green, Interscience, N. Y.

Morales, M. F., Botts, J., Blum, J. J. and Hill, T. L. 1955. Elementary
processes in muscle action: an examination of current concepts.
Physiol. Revs. 35, 475.

Nachlas, M. M., Crawford, D. T. and Seligman, A. M. 1957. The histochemi-
cal demonstration of leucine amin0peptidase. J. Histochem. Cytochem.
5 264
_J O

Nachlas, M. M., Crawford, D. T., Goldstein, T. P. and Seligman, A. M.
1958. The histochemical demonstration of cytochrome oxidase with a
new reagent for the Nadi reaction. J. Histochem. Cytochem. 6, 445.

Nachlas, M. M., Friedman, M. M. and Seligman, A. M. 1962. New observa-
tions on discrepancies in the histochemical localization of leucine
amin0peptidase. J. Histochem. Cytochem. 19, 315.

Nachlas, M. M., Monis, B., Rosenblatt, D. and Seligman, A. M. 1960.
Improvement in the histochemical localization of leucine aminopepti—
dase with a new substrate, L-leucyl-4-methoxy-Z-naphthylamide. J.
Biophys. Biochem. Cytol. Z, 261.

Nachlas, M. M., Walker, D. G., and Seligman, A. M. 1958a. The histochemi-
cal localization of triphosphoPyridine nucleotide diaphorase. J.
Biophys. Biochem. Cytol. 4, 467.

Nachlas, M. M., walker, D. G. and Seligman, A. M. 1958b. A histochemical
method for the demonstration of diphosphopyridine nucleotide diaphor-
ase. J. Biophys, Biochem. Cytol. 4, 29.

 

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~120-

Nachmias, V. T. and Padykula, H. A. 1958. A histochemical study of normal
and denervated red and white muscles of the rat. J. Biophys. Biochem.
Cytol. 4, 47.

Nagai, T., Uchida, K. and Yasuda, M. 1962. Some further properties of
the muscle relaxing-factor system and the separation of the effective
substance. Biochim. Biophys. Acta 56, 205.

 

Needham, D. M. 1926. Red and white muscle. Physiol. Rev. £5 1.

Needham, D. M. 1960. Biochemistry of Muscular Action. In The Structure
and Function of Muscle, Vol. II, 55. Ed. G. H. Bourne. Academic
Press, N. Y.

Ogata, T. and Mori, M. 1963. A histochemical study of hydrolytic enzymes
in muscle fibers of various animals. J. Histochem. Cytochem. 2;, 645.

 

Ogata, T. and Mori, M. 1964. Histochemical study of oxidative enzymes
in vertebrate muscles. J. Histochem. Cytochem. 22, 171.

Padykula, H. A. and Gauthier, G. F. 1963. Cytochemical studies of adeno-
sine triphosphatases in skeletal muscle fibers. J. Cell Biol. 22, 87.

Padykula, H. A. and Herman, E. 1955a. Factors affecting the activity of
adenosine triphosphatase and other phosphatases as measured by histo—
chemical techniques. J. Histochem. Cytochem. 2, 161.

Padykula, H. A. and Herman, E. 1955b. The specificity of the histochemi-
cal method for adenosine triphosphatase. J. Histochem. Cytochem° 2,
170.

 

 

Parker, C. J. 1961. The role of a-glycerol phosphate in muscle relaxation.
Biochem. Biophys. Research Comm. 4, 309.

Partmann, W. 1963. Post-mortem changes in chilled and frozen muscle. J.
Food Sci. 22, l.

Pauling, L. and Corey, R. 1951. Configuration of polypeptide chains.
Nature 168, 550.

Perry, S. V. and Grey, T. C. 1956° A study of the effects of substrate
concentration and certain relaxing factors on the magnesium-activated
myofibrillar adenosine triphosphatase. Biochem. J. 64, 184.

Pearse, A. G. E. 1957° Intracellular localization of dehydrogenase systems
using monotetrazolium salts and metal chelation of their formayons.
J. Histochem. Cytochem. 5, 515.

Pearse, A. G. E. 1960. Histochemistry. Theoretical and Applied. Little,

Brown and Co. Boston.

 

 

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-121-

Pearse, A. G. E. 1961. Direct relationship of phosphorylase and mite?
chondrial a—glycerophosphate dehydrogenase activity in skeletal muscle.
Nature 191, 504.

Porter, K. R. 1956. The sarcoplasmic reticulum in muscle cells of Amblye
stoma larvae. J. BiOphys. Biochem. Cytol. 2 Suppli., 163. '

Porter, K. R. 1961. The sarcoplasmic reticulum. Its recent history and
present status. J. Bi0phys. Biochem. Cytol. 2&5 Suppli., 219.

Porter, K. R. and Palade, G. E. 1957. Studies on the endoplasmic reti¥
culum III. Its form and distribition in striated muscle cells. J.
Biophys. Biochem. Cytol. 8, 269.

Riseman, J. and Kirkwood, J. G. 1948. Remarks on the physico chemical
mechanisms of muscular contraction and relaxation. J. Am. Chem. Soc.
255 2820.

Sayre, R. N. and Briskey, E. J. 1963. Protein solubility as influenced
by physiological conditions in the muscle. J. Food Sci. 28, 675.

Sayre, R. N., Briskey, E. J., Hoekstra, W. G. and Bray, R. W. 1961.
Effect of preslaughter change to a cold environment on characteris-
tics of pork muscle. J. Anim. Sci. 28, 487.

Sayre, R. N., Briskey, E. J. and Hoekstra, W. G. 1963a. Effect of excite-
ment, fasting and sucrose feeding on porcine muscle phosphorylase and
post-mortem glycolysis. J. Food Sci. 28, 472.

Sayre, R. N., Briskey, E. J. and Hoekstra, W. G. 1963b. Alteration of
post-mortem changes in porcine muscle by preslaughter heat treatment
and diet modification. J. Food Sci. 28, 292.

Sayre, R. N., Briskey, E. J. and Hoekstra, W. G. 1963c. Comparison of
muscle characteristics in three breeds of swine. J. Anim. Sci. 22,
1012.

Sayre, R. N., Kiernat, B., and Briskey, E. J. 1964. Processing character-
istics of porcine muscle related to pH and temperature during rigor
mortis developement and to gross morphology 24 hr post-mortem. J.
Food Sci. 22, 175.

Sasaski, M. and Takeuchi, T. 1963. Histochemical and electron microscopic
observations of glycogen synthesized from glucose-l-phOSphate by phos-
phorylase and branching enzyme in human muscle. J. Histochem. Cytochem.
22, 342.

Scarpelli, D. G., Hess, R. and Pearse, A. G. E. 1958. Cytochemical locali-
zation of oxidative enzymes. I. Diphosphopyridine nucleotide diaphor-
ase and triphosphopyridine nucleotide diaphorase. J. Biophys. Biochem.
Cytol. 4, 747.

 

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-122-

Sharpe, J. G. 1962. Non—enzymic browning deterioration in dehydrated meat.
In Recent Advances in Food Science, Vol. 2, p. 65. Eds. J. Hawthorne
and J. M. Leitch, Butterworths, London.

Sharpe, J. G., and Rolfe, E. J. 1958. Deterioration of dehydrated meat
during storage. In Fundamental Aspects of the Dehydration of Food-

stuffs, p. 197. Soc. Chem. Ind., London.

Sjgstrand, F. S. 1956. The ultrastructure of cells as revealed by the
electron microscope. Intern. Rev. Cytol. 8, 455.

Snedecor, G. W. 1959. Statistical Methods. 5th Ed. Iowa State Press.

Stein, J. M. and Padykula, H. A. 1962. Histochemical classification of
individual skeletal muscle fibers of the rat. Am. J. Anat. 110, 103.

Strehler, B. L. 1953. Firefly liminescence in the study of energy trans-
fer mechanisms. II. Adenosine triphOSphate and photosynthesis. Arch.
Biochem. Biophys. 48, 67.

Strehler, B. L. and Totter, J. R. 1952. Firefly luminescence in the study
of energy transfer mechanisms. I. Substrate and enzyme determination.
Arch. Biochem. Biophys. 49, 28.

Swift, C. E., Berman, M. D., and Lockett, C. 1960. Factors affecting the
water retention of beef. II. Variation in some pH determinants among
eight muscles. Food Tech. 24, 74.

Szent-Gygrgyi, A. 1947. The Chemistgy of Muscular Contraction. Academic

Press, N. Y.

Szent-Gygrgyi, A. 1951. Chemistry of Muscular Contraction, 2nd ed.

Academic Press, N. Y.

Szent-Gygrgyi, A. 1953. Chemical Physiology of Contraction in Body and

Heart Muscle. Academic Press, N. Y.

H
Szent-Gyorgyi, A. G. 1960. Proteins of the Myofibril. In the Structure
and Function of Muscle, Vol. II, p. 1, Ed. G. H. Bourne. Academic
Press, N. Y. .

Takeuchi, T. 1956. Histochemical demonstration of phosphorylase. J.
Histochem. Cytochem. 4, 84.

Takeuchi, T. 1958. Histochemical demonstration of branching enzyme (anylo-
l,4-->l,6-transglucosidase) in animal tissues. J. Histochem. Cytochem.
6 208
_, O

Takeuchi, T. and Glenner, G. G. 1960. Histochemical demonstration of a
pathway for polysaccharide synthesis from uridine diphosphoglucose.
J. Histochem. Cytochem. 8, 227.

 

 

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-123-

Takeuchi, T. and Glenner, G. G. 1961. Histochemical demonstration of
uridine diphosphate glucose-glycogen transferase in animal tissues.
J. Histochem. Cytochem. 2, 304.

Takauji, M. and Nagai, T. 1963. Is adenosine-3', 5'-phosphate identical
with the relaxing substance? Biochem. Biophys. Res. Comm. 13, 54.

Uchida, K. and Mommaerts, W. F. H. M. 1963. Modification of the contractile
responses of actomyosin by cyclic adenosine 3', 5' phosphate. Biochem.
Biophys. Research Comm. 28, 1.

VanWijhe, M., Blanchaer, M. C. and Jacyk, W. R. 1963. The oxidation of
lactate and a—glycerophosphate by red and white skeletal muscle: II.
Histochemical studies. J. Histochem. Cytochem. 2}, 505.

Venable, J. H. 1963. The histology of muscle. Proc. Meat Tenderness
Symposium, p. 7. Campbell Soup Co., Camden, N. J.

Wachstein, M. and Meisel, E. 1955. The distribution of demonstrable
succinic dehydrogenase and of mitochondria in tongue and skeletal
muscle. J. Biophys. Biochem. Cytol. l, 483.

Wachstein, M. and Meisel, E. 1957. Histochemistry of hepatic phosphatases
at a physiologic pH. With special reference to the demonstration of
bile canaliculi. Am. J. Clin. Path. 21, 13.

Walls, E. W. 1960. The microanatomy of muscle. In The Structure and
Function of Muscle, Vol. I, 21. Ed. G. H. Bourne. Academic Press,
N. Y.

Weber, H. H. 1957. The biochemistry of muscle. Ann. Rev. Biochem. gfb

Weber, A. 1956. The ultracentrifugal separation or L—my051n and actin
in an actomyosin sol under the influence of ATP. Biochim. Biophys.
Acta 22, 345.

weber, A. and Herz, R. 1963. The binding of calcium to actomyosin systems
in relation to their biological activity. J. Biol. Chem. 238, 581.

Weber, A., Herz, R. and Reiss, I. 1963. On the mechanism of the relaxing
effect of fragmented sarcoplasmic reticulum. J. Gen. Physiol. 48, 679.

Weber, H. H. and Portzehl, H. 1952. Muscle contraction and fibrous muscle
proteins. Adv. in Protein Chem. Z, 161.

Weber, A. and Weber, H. H. 1951. Zur Thermodynamik der Kontraktion des
Fasermodells. (English Summary). Biochim. Biophys. Acta 1, 339.

-124—

weil-Malherbe, H. and Green, R. H. 1951. The catalytic effect of molyb?
date on the hydrolysis of organic phosphate bonds. Biochem. J. 42, 286.

Whitaker, J. R. 1959. Chemical changes with aging of meat with emphasis
on the proteins. Adv. in Food Res. 2, l.

Wismer-Pedersen, J. 1959. Quality of pork in relation to rate of pH
change post mortem. Food Res. 24, 711.

Wismer-Pedersen, J., and Briskey, E. J. 1961a. Rate of anaerobic glyco-
lysis versus structure in pork muscle. Nature 189, 318.

Wismer—Pedersen, J., and Briskey, E. J. 1961b. Relationship of post-
mortem acidity and temperature. Food Tech. 18, 232.

Zender, R., Lataste-Dorolle, C., Collet, R. A., Rowinski, P. and Mouton,
R. F. 1958. Aseptic autolysis of muscle: biochemical and micro-
scopic modifications in rabbit and lamb muscle during aseptic and
anaerobic storage. Food Res. 28, 305 (1958).

I

 

 

 

 

 

 

APPENDIX

 

 

 

 

 

-125-
i
1

Appendix A. Table 1. Sources of chemicals for histochemical procedures.

 

 

 

Chemical Source*
AMP (adenosine-5'-phosphate; Na-salt, crystalline;

from muscle Sigma
ATP (adenosine—5'-triphosphate; di Na, 3 H20,

crystalline) Sigma
Diazote 5-chloro-o-toluidine Dajac
DPN (beta—diphosphopyridine nucleotide) Sigma
Fast Blue B salt (O-dianisidine, tetrazotized) Sigma
Fast red violet LB salt Sigma
G1ucose—6-phosphate (di Na salt) Sigma
Glucose—l-phosphate (di-K salt) Sigma 1
Glucose-l-phOSphate (di-Na salt) Sigma
L-Glutamate (Na) Sigma
DL-alpha-Glycerophosphate Sigma
beta-Glycerophosphate (di-Na) Sigma
Glycogen (from rabbit) Mann
DL—beta-Hydroxybutyrate (Na salt) Sigma
DL-isocitrate (tri-Na) Sigma
Lactic dehydrogenase Sigma
DL-Lactate (Na salt, 60% syrup) Sigma
L—Malic acid (Na) Sigma
MTT (MTT tetrazolium) Sigma
l-Hydroxy-Z-Naphthoic acid Kodak
Nitro BT (Nitro blue tetrazolium) Sigma
N-Phenyl-p-phenylene diamine Kodak
6-Phosphogluconate (Ba) Sigma
Sodium-AS—BI-phosphate Sigma
Succinate (Na) Sigma
Thiamine pyrophosphate (Cl) Mann
TPN (triphosphopyridine nucleotide, Na salt) Sigma
UDPG (uridine-5'—diphosphoglucose, Na salt) Sigma
*Sigma: Sigma Chemical Co., 3500 DeKalb St., St. Louis 18, Mo.
Dajac: Borden Co. Chemical Division, 5000 Langdon St., Philadelphia 24, Pa.
Kodak: Eastman Kodak Co., Rochester 3, N. Y.
Mann:

Mann Research Laboratories, 136 Liberty St., New York 6, N.Y.

 

 

 

 

 

 

 

-126-

Appendix A. Table 2. Post-mortem levels of chemical constituents, and pH
for individual beef carcasses at various periods
post-mortem

 

 

 

Time
post-mortem CaroaqS
(hrs) 1 2 3 4 5
pH:
Initial 6.90 6.95 7.07 6.98 7.03
6 6.41 6.50 6.73 6.60 6.60
12 5.80 5.92 6.07 5.90 6.13
24 5.60 5.80 6.02 5.53 5.73
48 5.47 5.50 5.67 5.50 5.72
72 5.43 5.60 5.40 5.40 5.48
96 5.35 5.38 5.40 5.37 5.32
102 5.42 5.58 5.40 5.35 5.33
126 5.45 5.52 5.45 5.52 5.55
151 5.47 5.48 5.49 5.67 5.52
173 5.46 5.50 5.48 5.68 5.58
198 5.55 5.59 5 60 5.72 5.69
288 5.63 5.65 5 50 5.58 5.58
480 5.47 5.40 5 50 5.50 5.42
Glycogen:
(mg/g) Initial 10.20 9.30 9.81 7.44 9.29
6 6.58 4.34 9.47 7.32 6.06
12 3.66 4.79 7.22 3.44 5.53
24 1.45 2.18 2.39 0.65 1.54
48 1.68 1.70 1.84 1.40 1.50
102 2.32 3.52 3.03 2.85 1.56
288 1.56 1.06 3.06 1.62 0.69
480 0.40 0.28 0.28 0.12 0.23
Glucose:
(mg/g) Initial 1.52 1.49 1.18 1.63 1.32
6 1.30 0.95 1.76 0.87 0.80
12 2.12 3.18 1.45 2.74 1.47
24 3.66 3.98 2.26 3.25 3.14
48 3.52 3.51 2.81 2.35 2.18
102 3.02 2.31 1.85 1.83 1.90
288 3.89 4.35 2.60 2.26 2.00
480 3.79 3.20 3.66 3.21 2.26
Lactic acid:
(mg/g) Initial 1.93 0.76 0.60 1.46 1.13
6 4.50 5.13 2.66 5.48 2.39
12 7.34 6.57 4.30 4.57 3.33
24 7.05 7.75 6.02 6.09 5.14
48 6.96 8.57 7.02 7.46 7.11
102 6.88 8.04 7.06 7.30 7.14
288 7.80 6.97 7.46 7.47 7.52
480 7.35 7.49 7.97 7.96 7.44

 

 

 

 

 

 

 

-127-

Appendix A. Table 2. Post-mortem levels of chemical constituents, and pH
for individual beef carcasses at various periods
post-mortem (continued)

 

 

 

 

Time
post-mortem Carraqq
(hrs) 1 2 3 4 5
ATPl
(mg P/g) Initial .328 .425 .517 .388 .309
6 .330 .283 .383 .228 .270
12 .138 .207 .368 .267 .222
24 .080 .051 .125 .104 .156
48 .050 .040 .189 .055 .007
ATPz
(micromoles Initial 10.1 12.6 10.7 9.2 11.8
/g) 9.9 8.5 11.0 7.3 13.2
12 0.0 2.5 6.7 7.9 9.4
24 0 0 0.0 1.4 0.5 1.3
48 0 0 0.0 0.0 0.0 0.0
TSP
(mg P/g) Initial 1.703 1.879 1.547 1.770 1.600
6 1.837 1.776 1.556 1.688 1.676
12 1.743 1.886 1.642 1.673 1.569
24 1.584 1.914 1.620 1.689 1 483
48 1.573 1.868 1.640 1.686 1.629
102 1.637 1.866 1.561 1.671 1.585
288 1.736 1.880 1.590 1.662 1.631
480 1.479 1.878 1.701 1.680 1.655
Ortho P
(mg P/g) Initial 0.6274 0.6570 0.6090 0.6693 0.8497
6 0.6487 0.6044 0.6640 0.7494 0.9329
12 0.8440 0.7234 0.7490 0.8156 0.8789
24 0.6782 0.7047 0.6930 0.6796 0.5923
48 0.8044 0.8505 0.8190 0.9134 0.8833
102 0.7576 0.7713 1.0530 1.1052 1.0124
288 1.0336 0.8772 1.0500 1.0617 1.1902

480 1.0095 1.0215 1.1580 1.1407 1.1867

 

-128-

Appendix B. Table 1° Sampling locations* on pork carcasses

 

 

 

Carcass Right side Left side
1 1L, 9I, 6L, I3I, 3L, 4/5L, 2T, 3L, III, 6L, 13T, 9T
11T** 7T, 1L, 4/5L
2 7T, 9T, 6L, 13T, IL, 11T, 8T 5L, I3I, 4/5L, 3/4L,
llT, IL, 7T
3 6L, 9T, 11T, 4/5L, 7T, 3L, I3I, IL, 3L, III, 4/5L, 6L,
IL, 5L 13T, 7T, 9T, 7T
4 4/5L, 2/3L, IL, 6L, 7T, 9T 2/3L, 4/5L, IL, 6L,
I, 9
5 4/5L, III, IL, 9T, 2/3L, I4I 2/3L, III, IL, 9T,
4/5L, I4I
6 2/3L, III, IL, 9T, 4/5L, 14T Same as in right side
7 II II
8 4/5L, 7T, IL, 6L, 2/3L, 9T "
9 H II
10 6L, 4/5L, 2/3L, IL, I3/I4I, 9T "
11 n n

A through G 2/3L, III, IL, 9T, 4/5L, 14T "

 

* L = lumbar vertebra; T = thoracic vertebra. Numbers refer to vertebra
numbers, i.e., 1 = lst, 2 = 2nd, etc.
**Listed in order of sampling.

 

 

 

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I 00.50 00N0. H.H 00.0 00.N No.0 50.0 000
I 0H.0N 0000. H.0 00.5 0N.N 00.H 5N.0 00N
I 00.0N 5000. 0.0 05.0 00.N 00.0 H0.0 0NH 060+
I 0N.H0 HH00. 0.0 00.0 00.N 00.0 00.0 0H I 0H
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-136-

Appendix B. Table 3. Post-mortem levels of glucose, glycogen, lactic
acid, ATP and pH for each side of carcasses A

 

 

through G1
Time Lactic ATP4
post-mortem Glycogen2 Glucose3 'acid (micro-
Carcass Treatment mins H m m m / moles/ )
A - 10 6.69 14.08 2.22 4.21 —
-29°C 187 6.43 14.26 0.98 4.77 0.0
329 6.35 9.31 2.70 '3.90 0.0
1440 5.72 8.36 3.22 7.33 -
2880 5.52 — - - -
>37°C 165 5.42 4.79 3.56 7.93 0.0
312 5.34 4.36 5.72 8.08 0.0
1440 5.33 00.75 9.30 7.73 —
B — ll 6 61 2 86 2 09 3 84 3 6
-29°C 186 6.10 1.17 2.54 5.84 0.0
333 6.29 0.90 3.36 5.14 0 0
1440 5.80 0.00 2.41 6.22 -
2880 5.67 — - - -
>37°C 172 5.70 0.10 1.52 7.48 0.0
322 5.64 O 28 1.80 6.60 0.0
1440 5.67 0.12 1.77 7.64 —
C - 10 6.86 20.13 1.65 1.71 3.8
-29°C 70 6.79 - - _ _
180 6.45 21.89 0.78 1.66 4.0
330 6.42 17.82 2.96 4.22 1.6
1440 5.68 13.80 3.93 6.81 0.0
2880 5.52 - - - _
>37°C 63 6.40 ~ - _ 0:0
170 5.38 9.34 4.24 7.04 0.0
343 5.31 8.72 6.30 6.79 -
1440 5.34 5.89 8.58 6.67 -

 

1A11 chemical values expressed on a per gram of fresh tissue basis.
2Expressed in glucose-equivalents.

Total reducing sugars expressed in glucose equivalents.
4Determined enzymatically.

 

 

 

 

-137-

Appendix B. Table 3. Post-mortem levels of glucose, glycogen, lactic
acid, ATP and pH for each side of carcasses A
through Gl (continued)

 

 

 

Time Lactic ATPZ
post-mortem Glycogen2 Glucose3 acid (micro-
Garcass Treatment (mins) pH gmg/g! {mg/g) (mg/g) moles/g)
D - '10 6.79 9.39 2.29 2.53 7.2
-29°C 70 6.84 - - - -
181 6.56 9.32 1.42 3.43 4.0
346 6.35 5.35 2.68 4.26 1.0
1440 5.67 2.15 2.25 6.34 0.0
2880 5.49 - - - -
>37°C 72 6.52 - - - -
169 5.49 1.16 3.80 7.52 0.0
348 5.36 0.22 3.90 7.42 -
1440 5.40 0.00 4.08 7.38 -
E 10 6 69 10.33 2.26 2 45 3.5
~29°C 89 6.54 - - - -
160 6.57 8.66 2.06 3.72 4.8
312 6.45 4.57 3.73 3.96 2.8
1440 5.61 2.37 2.26 6.44 0.0
2880 5.47 - - - -
>37°C 85 6.43 - - - -
170 5.37 0.49 3.64 6.48 0.0
289 5.37 0 39 4.30 6.51 -
1440 5.33 0.14 4.70 6.77 -
F - 14 6.78 17.59 1.83 2.63 8.3
-29°C 75 6.62 - - - -
172 6.72 16.77 2.76 2.07 7.2
298 6.59 17.13 1.76 3.49 5.7
1440 5.66 10.15 2.44 6.81 0.3
2880 5.70 - - - -
>37°C 74 6.40 - - _ -
164 5.32 8.81 3.55 6.76 0.0
290 5.25 6.43 5.44 7.80 -
1440 5.23 2.40 7.74 7.13 -

 

1'rAll chemical values expressed on a per gram of fresh tissue basis.
Expressed in glucose-equivalents.

3Total reducing sugars expressed in glucose equivalents.
4Determined enzymatically.

 

 

-138-

Appendix B. Table 3. Post-mortem levels of glucose, glycogen, lactic
acid, ATP and pH for each side of carcasses A
through G1 (continued)

 

 

 

Time Lactic ATP4
post-mortem Glycogen2 Glucose3 acid (micro-
Carcass Treatment (mins) H m m m / moles/
G - 13 6.78 21.22 2.40 2.42 7.4
-29°C 74 6.84 - - - -
175 6.66 20.93 3.00 3.40 5.9
302 6.45 17.75 3.14 4.23 0.0
1440 5.62 14.21 2.48 7.88 -
2880 5.60 - - - -
>37°C 60 6.35 - - - -
166 5.38 10.18 4.14 7.34 0.0
293 5.28 5.60 6.56 7.40 -
1440 5.30 4.62 8.51 7.60 -

 

 

1All chemical values expressed on a per gram of fresh tissue basis.
2Expressed in glucose-equivalents.

3Total reducing Sugars expressed in glucose equivalents.
4Determined enzymatically.

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

    

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