- Email: [email protected]
Idiopathic recurrent rhabdomyolysis
Idiopathic
Recurrent
Rhabdomyolysis*
A Clinical, Chemical and Morphological Skd_y DANTE G.
SCARPELLI, M.D., PH.D.,~
and WALTER
Columbus, N 1910 Meyer-Bet2 [ 71 first described a disease entity characterized by paroxysmal voluntary muscle pain, weakness and myoglobinuria. Three years later a report of a similar condition in draft horses was published by Carlstrom [ZJ. Since then, despite numerous reports [&6] and several review articles [7-91 which have appeared in both human and veterinary medical literature, data concerning the basic nature of this illness are few. In our study of this disorder various technics including electron microscopy were used. Biochemical data on the patient and two members of her family are reported, and an alteration of muscle fine structure which, to our knowledge, has not been hitherto observed in this malady.
H.
GREIDER,
PH.D.
PH.D.
Ohio blood pressure 120/60 mm. Hg. The lungs were clear to percussion; there were coarse inspiratory rales at the left apex anteriorly and posteriorly. No abnormalities of heart size, sounds or rhythm were present. Examination of the abdomen revealed no enlarged organs or abnormal masses. The flexor and extensor muscles of the forearms and legs were weak. These muscles, especially the gastrocnemius, quadriceps and biceps femoris muscles, were markedly tender, board-like in consistency and swollen. The neurologic examination was within normal limits. Roentgenographic studies of the chest showed small, well defined, partially calcified nodular densities in the apex of the left lung. Roentgenograms of the abdomen showed no abnormalities. Electrocardiograms showed a prolonged electrical systole and borderline low voltage. Laboratory examination of the blood revealed a white blood cell count of 13,930 per cu. mm. with 77 per cent neutrophils. The red blood cell count was 5,000,OOO per cu. mm., the hematocrit 47.5 per cent and the hemoglobin 15.5 gm. per cent. A lupus erythematosus preparation from the peripheral blood was negative. The blood urea nitrogen was 9 mg. per cent. Chemical studies of the blood serum revealed creatinine 2 mg. per cent, total protein 5.9 gm. per cent with 3.6 albumin and 2.3 globulin, potassium 4.8 mEq. per L., sodium 146 mEq. per L., chloride 97 mEq. per L., calcium 5.0 mEq. per L., inorganic phosphorus 4.2 mEq. per L., creatine 4.3 mg. per cent and an alkaline phosphatase activity of 6.4 Shinowara-Jones-Rinehart units. The serum total bilirubin was 0.5 mg. per cent with a direct bilirubin fraction of 0.1 mg. per cent. The urine was mahogany brown, with a specific gravity of 1.025, a pH of 6.0 and a protein concentration of 1.97 gm. per cent. Microscopic examination of the sediment revealed 4 to 7 white blood cells per high powered field, moderate numbers of epithelial cells and no red blood cells. Results of tests for urine bile and urobilinogen were negative. Twenty-four-hour excretion studies of the urine revealed normal concenof delta-amino-levulinic trations acid, porpho-
I
CASE
MARIE
J. FRAJOLA,
REPORT
A forty-three year old white woman was admitted to the hospital because of cramping pain in both lower extremities and the passage of dark brown urine. She had enjoyed good health until five years before, when she experienced similar but less severe symptoms. Three days prior to admission the patient worked continuously for fourteen hours at her occupation as a barmaid. Two days later she awakened with severe cramping pain in the muscles of both lower extremities, which confined her to bed. Later that evening she noticed that her urine had become dark brown, and the muscles of her upper extremities were painful. Upon attempting to walk, she became alarmed at the weakness and loss of control that she experienced in her lower limbs. The muscles of both calves were tender, swollen and hard. She did not have any nausea, fever, vomiting or abdominal pain. Physical examination revealed a well developed, well nourished, alert white woman who complained of considerable pain in both the upper and lower extremities. The oral temperature was 102.4’%., pulse 124 beats per minute, respirations 18 per minute and
* From the Laboratory of Cellular Pathology, Department of Pathology, and the Herman A. Hester Research Laboratory, The Ohio State University, Columbus, Ohio. Manuscript received February 12, 1962. t Senior Research Fellow in Pathology, National Institutes of Health Grant GM-K3-15, 104-C4. 426
AMERICAN
JOURNAL
OF
MEDICINE
idiopathic
Recurrent
Khabdornyolysis--&al-pelli
bilinogen, uroporphyrins and coproporphyrins. The reaction to a benzidine test on the urine was strongly positive. Electrophoretic study of the urine showed the mahogany colored pigment to be myoglobin. (Fig. 1.) Serum enzyme studies were begun on the day of admission and continued serially for the subsequent eight days. Levels of glutamic oxaloacetic @GOT) and glutamic pyruvic transaminases (SGPT), lactic (LDH), malic (MDH), isocitric (ICD) and glutamic dehydrogenases (GDH), aldolase (ALD), phosphohexose isomerase (PHI) and leucine aminopeptidase (LAP) activities were markedly elevated but decreased by the eighth day, paralleling the patient’s clinical improvement. On the day of admission, 10 units of succinic dehydrogenase (SD) activity was detected in the serum. Subsequent serial studies showed lower values as her condition improved. Except for a low level of activity demonstrated on the day of admission, serum acetylcholine esterase (ACE) activity remained slightly below the lower limit of the normal range throughout her period of hospitalization. (Table I.) The patient was placed at absolute bed rest. She continued to experience muscle pain in the upper and lower extremities and passed small amounts of dark brown urine. Her oliguria persisted for five days following admission. During this period her urine gradually became lighter in color and the total twenty-four-hour output increased from 270 to 1,240 cc. During the next three days her muscular
FIG. 1. Paper electrophoresis patterns. ing hemoglobin. B, urine containing
is identical
Day
~
SGOT
I
11-1
SGPT
/
I
OF SERUM
306 525 400 200 162 138 86
3 4 6 8 9
MD
LDH
ENZYMES
ICD
~
1:
ALD
GDH
PHI
LAP __
14,400 4,800 3,420 1,920 1,600 1,600 1,075
430 100 200 100 69 78 50
1
*
SD
68 ...
23,750 6,400 5,200 2,700 2,800 2,600 5,600
0
10 ... 4 0
;I
:0
Serum Enzymes of Siblings A. M. (sister) J. L. (brother)
with myoglobin.
pain decreased and the affected muscle masses lost their board-like rigidity. On the ninth hospital day an erythematous, mildly pruritic rash developed on her cheeks and nose. The rash disappeared within the next two days. By the twelfth hospital day she was free of symptoms and her urine devoid of protein, although she still exhibited a slight degree of residual
Enzyme Hospital
A, urine containhemoglobin and
myoglobin. C, patient’s urine. D, control solution of myoglobin. The patient’s urine contains a protein which has approximately half the mobility of hemoglobin and
TABLE ACTIVITY
el al.
z
1
TZ
j
‘E
1
1:
I
0”
149 113 141 111 120 100 84
ii
ACE
IL
~ 1
520 440 380 400 360 390 350
50 130 130 155 130 105
’
z::
:I:
_______ 1
1:
)
1::
,
_______ ,-O-30 * For explanation VOL.
34,
MARCH
1 O-30 ~,I,,,,~:::::~~~ of abbreviations, 1963
see text.
0
~50-100~
lo-20
~100-310~130-260
Idiopathic
428
Recurrent
Rhabdonlyolysis
FIG. 2. Gastrocnemius muscle biopsy specimen obtained during the acute phase of muscle pain and weakness with myoglobinuria. A, coagulation necrosis of muscle fibers. Original magnification X 92. B, an area of muscle necrosis with sarcolemmal nuclear proliferation. Note the segmental distribution of the necrosis. Original magnification X 125. C, liquefaction necrosis of sarcous substance immediately adjacent to normal appearing muscle fibers. Note the absence of an inflammatory reaction. Original magnification X 205. muscle weakness in both legs. The patient charged on the sixteenth hospital day. SPECIAL
STUDIES
0~
was dis-
MUSCLE
Electromyography. Electromyographic studies* of both weakened and grossly pathologic muscles * Performed by Dr. J. D. Guyton, Department of Physical Medicine.
Scurpelli et al.
exhibited increased insertion potentials and complex motor unit potentials which were reduced both in amplitude and duration. In addition, considerable fibrillation and occasional fasciculation were noted. These findings were interpreted as indicative of a diffuse myopath! with extensive random destruction of muscle fibers. Histopathology. A biopsy of the left gastrocnemius muscle revealed a pale grayish pink muscle tissue. It was fixed immediately in 10 per cent neutral formalin for light microscopy and histochemistry, and in buffered 1 per cent osmium tetroxide for electron microscopy. Paraffin sections showed extensive coagulation necrosis of muscle fibers characterized by masses of structureless, densely clumped, eosinophilic sarcoplasm. A few fibers showed liquefaction of sarcous substance. The necrosis involved either complete muscle fibers immediately adjacent to normal appearing ones or segments of muscle fibers with zones of necrosis interspersed between areas of undamaged sarcoplasm. Many muscle fibers exhibited lesser degrees of damage, such as myofibrillar swelling and obscuration of sarcoplasmic structure. In certain areas there was evidence of sarcolemmal nuclear proliferation. Despite the widespread necrosis there was a paucity of inflammatory cells. (Fig. 2.) The periodic acid-Schiff reaction showed occasional granules of glycogen scattered in the sarcoplasm which were removed by diastase during previous digestion. None of the muscle fibers showed the intrasarcoplasmic cleft formation characteristic of familial periodic paralysis. Electron Microscopy. Under the electron microscope areas of muscle fiber necrosis showed marked disruption of sarcoplasmic structure with absence of organized bundles of myofibrils. Isolated myofibrils were present in which occasional Z bands were still visible; the H bands were absent. The interfibrillar sarcoplasm consisted of numerous vesicles of varying size, occasional swollen mitochondria (sarcosomes) and an electron-translucent amorphous material. (Fig. 3.) Although the sarcolemmal membrane of damaged muscle segments appeared less osmiophilic and less intact than that of undamaged ones, the fine structure was surprisingly well preserved despite widespread damage of the muscle fibers. (Fig. 4.) The sarcolemmal nuclei of necrotic muscle fibers appeared undamaged. (Fig. 5.) Less damaged AMERICAN
JOURNAL
OF
MEDICINE
Idiopathic
Recurrent
Rhabdornyolysis-Scar-eZZi
et al.
FIG. 3. A longitudinal section of a necrotic muscle fiber adjacent to an undamaged one. Within the necrotic fiber isolated remnants of myofibrils (MF) are present in which the Z bands are still visible. The necrotic sarcoplasm contains several swollen sarcosomes (S), numerous vesicles of varying size, and amorphous material. An arrow indicates the sarcolemma (SL) of the necrotic cell. An undamaged sarcolemmal membrane (SL) and muscle fiber are shown in the lower portion of the micrograph. Original magnification X 12,000.
fFIo. 4. The sarcolemmal membrane (SL) of a normal muscle fiber is shown at the left. The sarcolemma of a necrotic muscle cell appears at the right. The laminae, especially the inner one, show marked thinning and numerous interruptions of continuity. Note the absence of myofibrils in the necrotic sarcoplasm (NS). Original magnification X 28,000. 34,
MARCY
1963
429
Idiopathic
Recurrent
Rhabdomyolysis-Scarpelli
et al.
FIG. 5. A sarcolemmal cell with associated perinuclear cytoplasm is shown immediately adjacent to necrotic sarcoplasm and separated from it by a distinct plasma membrane (PM). A portion of the nucleus (N) appears in the upper right of the micrograph. Note the rod-shaped dense sarcosome (S) in the sarcolemmal cell cvtoulasm and the swollen sarcosome (S) in the necrotic sarcoplasm. Original magnification X 28,500.’ A
muscle fibers with intact myofibrils exhibiting both Z and H bands had swollen sarcosomes which were notable in that they were often contiguous to morphologically normal-appearing sarcosomes. (Fig. 6.) The swollen sarcosomes showed remnants of cristae which were indistinct and in most instances had lost their characteristic double membraned structure. (Fig. 7.) The sarcoplasmic reticulum appeared undamaged. In several areas of muscle the fine structure of the myofibrils showed a curious change, having become granular and more electron-dense. Only a few bundles of fibrils had remnants of the 2 bands. (Fig. 8.) SERUM
ENZYME
STUDIES
OF TWO
SIBLINGS
Enzyme studies were performed on the serums of a sister and brother of the patient, both of whom were historically and clinically free of stigmata of the disease. The serum of the sister
had elevated malic and succinic dehydrogenase activity. The enzyme activities of the brother’s serum showed no abnormalities. (Table I.) COMMENTS
The classic communication by Meyer-Betz describing idiopathic rhabdomyolysis does not mention whether the attack was preceded by physical exercise. It has become apparent now that although in many cases of idiopathic rhabdomyolysis the attacks appear to be precipitated by physical exercise, a considerable number are not. In the most recent review of the literature [S] twenty-one of the forty-six cases reported were of the exertional type. Bowden et al. [7l state that the exertional type has been observed exclusively in males. The case reported here was definitely of the exertional type and occurred in a female, a relationship which has not been reported previously. AMERICAN
JOURNAL
OF
MEDICINE
idiopathic Recurrent Khabdornyolysis -Scar~elli et (11.
FIG. 6. A longitudinal section of non-necrotic muscle showing focal sarcosomal (S) swelling and membrane alteration. The myofibrils are undamaged, exhibiting intact sarcomeres in which both the Z and H bands are clearly visible. Original magnification X 9,600.
FIG. 7. A higher magnification of an area similar to Figure 6, showing the focal sarcosomal akerations in greater detail. Note the swollen sarcosomes contiguous to normal appearing ones. The arrow points to the remnant of a double outer membrane of a sarcosome in which the cristae have lost their characteristic double-membraned structure. Original magnification X 37,600. VOL.
34,
MARCH
1963
432
Idiopathic Recurrent Rhabdolnyolysis--~Scar~elli el al.
FIG. 8. A section showing a curious alteration of the myofibrils (MF); these appear granular and more electron-dense than their adjacent normal counterparts. The arrow points to remnants of the Z bands; the H bands are absent. Original magnification X 9,600.
In view of the high concentration of potassium [70,77] and creatine within the muscle cell, the muscular necrosis, and the clinical and chemical signs of impaired renal function in this patient, one would expect the serum levels of creatine and potassium to be elevated. The normal serum and potassium levels encountered creatine during the acute phase of the disease are similar to those reported for creatine by Hipp and Shukers [72], and for potassium by Bowden et al. [7]. The absence of a high serum creatine level may result from the depletion of muscular phosphocreatine prior to necrosis. Increased activity of the various enzymes in the serum is most probably the result of leakage of soluble enzymes from damaged muscle cells. This is in accord with the findings of Sibley and Fleisher [ 731, Wroblewski and La Due [ 741, and Dreyfus, Schapira and Schapira [75]. The sarcolemma of the damaged muscle ceils in this case showed marked alterations of ultrastructure which were, no doubt, reflected in gross disturbances of selective permeability. Essentially
similar morphologic changes have been observed by Mauro and Adams [76] in the sarcolemmal membrane of traumatically damaged frog skeletal muscle. Alteration of membrane permeability may be a sufficient basis on which to explain the diffusion of soluble enzymes from a damaged cell [ 771. However, in the light of the work of Watson and Siekevitz [78], the appearance of succinic dehydrogenase activity in the serum suggested a more severe degree of damage. Watson and Siekevitz were able to show in cornminuted liver mitochondria that the fraction which exhibited the majority of succinoxidase activity consisted largely of mitochondrial membranes. Thus it is considered that succinic dehydrogenase is intimately bound to the structural proteins of the mitochondrial membranes, so that its appearance in the serum could result only from considerable alteration in sarcosomal fine structure as demonstrated in this case. This is in contrast to the more soluble glutamic dehydrogenase which would leak from mitochondria across damaged membranes. That the membrane alteration in this patient was severe is supported by the fact that the damaged sarcosomal cristae lost their characteristic doublemembraned structure. The selective nature of cellular damage in this disease was emphasized under the electron microscope by the presence of sarcolemmal nuclei and perinuclear cytoplasm in intimate contact with necrotic sarcoplasm. The origin of these cells has been the subject of many [ 7$.22]. Currently two concepts investigations concerning their origin are held: (1) sarcolemma1 nuclei and cytoplasm originating from muscle cells, and (2) emigration of extramuscular “free cells” which gain access into the sarcolemmal lined myotubes and assume a subsarcolemmal position. The activation of sarcolemmal nuclei constitutes an important part of the reaction of muscle to injury. Reconstruction of muscle begins with the proliferation of sarcolemmal nuclei and an increase in the amount of perinuclear cytoplasm. The presence of a distinct plasma membrane between these cells and the necrotic sarcoplasm (Fig. 5) suggests to us that they may represent two separate cellular entities. Although the sarcolemmal nuclei are distinctly beneath the sarcolemmal membrane, our data shed no light on whether these nuclei originate in situ or migrate from without the myotube. AMERICAN
JOURNAL
OF
MEDICINE
Idiopathic
Recurrent
Rhabdornyolysis-
The possible genetic basis of recurrent idiopathic rhabdomyolysis was first pointed out by Hittmair [23]. Such a relationship has been suggested subsequently by Hed [Z], Schaar, La Bree and Gleason [25], and Bowden et al. 171. The abnormal serum enzyme activities resembling those of the patient in a female sibling who, so far as we know, is free of any disease suggests that she may possess a similar metabolic disturbance. The absence of stigmata of the disease in this sibling may be attributed to subclinical attacks due to either incomplete penetrance of the genetic defect, if one indeed exists, or exercise levels of insufficient severity and duration to precipitate the attacks. Subclinical attacks of rhabdomyolysis during which the patient was free of overt symptoms have been observed and documented by a study of serial muscle biopsies in a patient reported by Reiner et al. [26]. A variety of etiologic factors has been implicated in acute recurrent rhabdomyolysis ranging infection and alcohol to an from exercise, enzymatic defect of muscle glycogenolysis [27]. The electron microscopic data obtained in the present study suggest still another possible etiologic factor-that of impaired mitochondrial function. A few statements concerning current knowledge of the mechanism of muscular contraction and relaxation should precede a discussion of this possibility. Muscular contraction results from an interaction of the contractile proteins, actin and give rise to myosin, which upon combination the actomyosin complex. This is followed by a shortening of the myofilaments and thus contraction [28,29]. Since actin and myosin combine very readily with a high free energy of interaction [30], muscle possesses mechanisms bywhich the interaction and dissociation of these proteins are controlled. Contraction and relaxation of muscle may be controlled by the following mechanisms: (1) Adenosine triphosphate (ATP) in sufficiently high concentration around myosin molecules can inhibit their spontaneous interaction with actin [28]. (2) ATP in high concentrations can dissociate the actomyosin complex [28,37]. (3) A water-soluble muscle fraction called relaxing factor [32] which consists of creatine phosphate and a protein [33] may prevent the interaction of actin and myosin by the inhibition of myofibrillar ATP-ase (myosin), thus maintaining a high concentration of ATP in muscle fibers, and by the rephosVOL.
34.
MARCH
1963
Scqbelli
et al.
433
phorylation of adenosine diphosphate (ADP) to ATP [34]. The morphologic evidence of sarcosomal damage may be the result of, or give rise to, a defect in the synthesis of ATP. This could lead to the following sequence of events, which presently is purely speculative although based on established biochemical and physiological facts: A deficiency of ATP would lead to a loss of intrasarcosomal calcium. ATP binds calcium within the sarcosomes by chelation in a manner similar to EDTA [35,36]. Loss of calcium impairs seriously oxidative phosphorylation and results in sarcosomal swelling [37]. In addition, depletion of ATP around the myofibrils and inhibition of the phosphorylative and relaxing [38] functions of relaxing factor by free calcium ions would result in a spontaneous combination of actin and myosin. This would lead to a continuous state of uncontrolled contraction which would eventually damage the muscle cell. Failure of the relaxing factor system is certainly suggested by the clinical finding of board-like tonic muscles during the acute phase of the disease. SUMMARY
The results of clinical, chemical and morphologic studies of a woman suffering from paroxysmal idiopathic rhabdomyolysis are presented. Increased serum enzyme activities observed during the acute phase of the disease returned toward normal levels as the patient’s clinical state improved. Of exceptional interest was the of succinic dehydrogenase in the presence patient’s serum. A biopsy specimen of the gastrocnemius muscle revealed extensive focal coagulation necrosis of muscle fibers. Some of the fibers exhibited lesser degrees of damage, such as myofibrillar and sarcoplasmic swelling. Under the electron microscope focal alteration of sarcosomal fine structure was observed in mildly damaged myofibrils. This consisted of swollen sarcosomes and extensive obscuration of the double membraned cristae. Damaged sarcosomes were often contiguous to completely normal-appearing ones. The possible significance and relationship of these findings to the etiology and pathogenesis of this disease are discussed. Acknowledgment: We gratefully acknowledge the cooperation of Dr. W. F. Ashe for allowing us to study his patient, and for his encourage-
434
Idiopathic
Recurrent
Khabdorllyolysis-S~~~~~~
ment and advice; and the technical assistance of Ruth Austad, Modestine Brown, Devinia Jefferson, Lynn Shawhan, Dr. Nicanor Atillo, C. V. Sittler and G. R. Millard. REFERENCES
1. MEYER-BETZ, F. Beobachtungen an einem eigenartigen mit MuskellHhmungen verbundenen Fall van Hzmoglobinurie. Da&he Arch. klin. Med., 101: 85,191l. 2. CARLSTRGM, B. eber die Aetiologie und Pathogenese der Kreuzlihme des Pferdes (Haemoglobinaemia paralytica). Skandinav. Arch. Physiol., 61: 161, 1931. 3. BYWATERS,E. G. L. and DIBLE, J. H. Acute paralytic myohaemoglobinuria in man. J. Path. & Bat., 55: 7, 1943. 4. ACHESON,D. and MCALPINE, D. Muscular dystrophy associated with paroxysmal myoglobinuria and excessive excretion of ketosteroids. Lancet, 2: 372, 1953. 5. SCHAAR, F. E. Paroxysmal myoglobinuria. Am. J. Dis. Child., 89: 23, 1955. 6. FARMER, T. A., JR., HAMMACK, W. J. and FROMMEYER, W. B. Idiopathic recurrent rhabdomyolysis associated with myoglobinuria. New Engkmd J. Med., 60: 66, 1961. 7. BOWDEN, D. H., FRASER, D., JACKSON,S. H. and WALKER, N. F. Acute recurrent rhabdomyolysis aroxysmal myohaemoglobinuria). Medicine, 35: $5 1956. 8. COM&, D. A. and ROSENFELD, H. Idiopathic paroxysmal myoglobinuria. Ann. Int. Med., 55: 647, 1961. 9. MINETT, F. C. The haemoglobinuria and myoglobinurias of animals. PYOC. Roy. Sot. Med., 28: 672, 1935. 10. RITCHIE, A. D. The Comparative Physiology of Muscular Tissue. Cambridge, England, 1928. Cambridge University Press. 11. STEINBACH,H. B. Intracellular inorganic ions and muscle action. Ann. New York Acad. SC., 47: 849, 1947. 12. HIPP, H. R. and SHUKERS, C. R. Spontaneous myoglobinuria: report of a case with symptoms of myotonia. Ann. Znt. Med., 42: 197, 1955. 13. SIBLEY, J. A. and FLEISHER, G. A. The clinical significance of serum aldolase. Proc. Stofl Meet. Mayo Clin., 29: 591, 1954. 14. WROBLEWSKI,F. and LA DUE, J. S. Lactic dehydrogenase activity in blood. Proc. Sot. E@er. Biol. &T Med., 90: 210, 1955. 15. DREYFUS, J. C., SCHAPIRA, G. and SCHAPIRA, F. Serum enzymes in the physiology of muscle. Ann. New York Acod. SC., 75: 235, 1958. 16. MAURO, A. and ADAMS, W. R. The structure of the sarcolemma of the frog skeletal muscle fiber. J. Biophys. & B&hem. Cytol., (supp.), 10: 177,196l. 17. ZIERLER, K. Factors which influence movement of aldolase from excised rat diaphragm. Am. J. Physiol., 183: 675, 1955. 18. WATSON, M. L. and SIEKEVITZ,P. The isolation and analysis of a mitochondrial membrane fraction. J. Biophys. & Biochem. Cytol. (supp.), 2: 379,1956.
et (II.
19. WALDEYER, W. Ueber die Versnderungen der quergestieiften Muskeln bei Entziinduig und dem Typhusprozess, sowei iiber die Regeneration derselben nach Substanzdefecten. V&chows Arch. path. Anat., 34: 473, 1865. 20 CLARK, W. E. L. An experimental study of the regeneration of mammalian striped muscle. J. Anat., 80: 24, 1946. 21. GODMAN, G. C. On the regeneration and redifferentiation of mammalian striated muscle. J. Morphol., 100: 27, 1957. 22. LASH, J. W., HOLTZER, H. and SWIFT, H. Regeneration of mature skeletal muscle. Anat. Rec., 128: 679,1957. 23. HITTMAIR, A. Haemoglobinuria paroxysmalis paralytica. Wien. klin. Wchnschr., 38: 431, 1925. 24. HED, R. En familjar form av paroxysmal myoglobinuri. Nerd. med., 35: 1586, 1947. 25. SCHAAR, F. E., LA BREE, J. W. and GLEASON,D. F. Paroxysmal myohemoglobinuria with fatal renal tubular injury. J. Lab. &? Clin. Med., 34: 1744, 1949. 26. REINER, L., KONIKOFF, N., ALTSCHULE, M. D.. DAMMIN, G. J. and MERRILL, J. P. Idiopathic paroxysmal myoglobinuria. Arch. Int. Med., 97: 537, 1956. 27. SCHMID, R. and MAHLER, R. Chronic progressive myopathy with myoglobinuria: demonstration of glycogenolytic defect in muscle. J. Clin. Znvesf., 38: 2044,1959. 28. SZENT-GY~RGYI, A. Chemistry of Muscular Contraction. New York, 1951. Academic Press, Inc. 29. HUXLEY, H. E. Muscle cells. In: The Cell. Biochemistry, Physiology, Morphology, vol. 4, p. 366. Edited by Brachet, J. and Mirsky, A. E. New York, 1960. Academic Press, Inc. 30. GERGELY, J. and KHOLER, H. Light scattering studies on the stepwise formation and dissociation of actomyosin. In: Proceedings of the Conference on Muscle Contraction, p. 14. Tokyo, 1957. Igaku Shoin, Ltd. 31. STRAUB, F. B. The specificity of the adenosine triphosphoric acid effect. Studies Inst. M. Chem. Univ. Szeged., 3: 38, 1943. 32. MARSH, B. B. A factor modifying muscle synaeresis. Nature, London, 167: 1065, 1951. 33. GOODALL, M. C. and SZENT-GYBRGYI, A. G. Relaxing factor in muscle. Nature, London, 172: 84, 1953. 34. LORAND, L. Adenosine triphosphate-creatine transphosphorylase as relaxing factor of muscle. Nature, London, 172: 1181, 1953. 35. ERNSTER,L. and LB,, H. Reconstruction of oxidative phosphorylation in aged mitochondrial systems. Exper. Cell Res., (supp.), 3: 133, 1955. 36. RAAFLAUB, J. Die Metallpufferfunktion der Adenosinphosphate. Helvet. phvsiol. et pharmacol. acta, 14: 304,1956. 37. LEHNINGER,A. L. Physiology of mitochondria. In: Enzymes: Units of Biological Structure and Function, p. 217. Edited by Gaebler, 0. H. New York, 1956. Academic Press, Inc. 38. BOZLER, E. and PRINCE,J. T. The control of energy release in extracted muscle fibers. J. Gen. Phyriof., 37: 53,1953. AMERICAN
JOURNAL
OF
MEDICINE
Recurrent
Rhabdomyolysis*
A Clinical, Chemical and Morphological Skd_y DANTE G.
SCARPELLI, M.D., PH.D.,~
and WALTER
Columbus, N 1910 Meyer-Bet2 [ 71 first described a disease entity characterized by paroxysmal voluntary muscle pain, weakness and myoglobinuria. Three years later a report of a similar condition in draft horses was published by Carlstrom [ZJ. Since then, despite numerous reports [&6] and several review articles [7-91 which have appeared in both human and veterinary medical literature, data concerning the basic nature of this illness are few. In our study of this disorder various technics including electron microscopy were used. Biochemical data on the patient and two members of her family are reported, and an alteration of muscle fine structure which, to our knowledge, has not been hitherto observed in this malady.
H.
GREIDER,
PH.D.
PH.D.
Ohio blood pressure 120/60 mm. Hg. The lungs were clear to percussion; there were coarse inspiratory rales at the left apex anteriorly and posteriorly. No abnormalities of heart size, sounds or rhythm were present. Examination of the abdomen revealed no enlarged organs or abnormal masses. The flexor and extensor muscles of the forearms and legs were weak. These muscles, especially the gastrocnemius, quadriceps and biceps femoris muscles, were markedly tender, board-like in consistency and swollen. The neurologic examination was within normal limits. Roentgenographic studies of the chest showed small, well defined, partially calcified nodular densities in the apex of the left lung. Roentgenograms of the abdomen showed no abnormalities. Electrocardiograms showed a prolonged electrical systole and borderline low voltage. Laboratory examination of the blood revealed a white blood cell count of 13,930 per cu. mm. with 77 per cent neutrophils. The red blood cell count was 5,000,OOO per cu. mm., the hematocrit 47.5 per cent and the hemoglobin 15.5 gm. per cent. A lupus erythematosus preparation from the peripheral blood was negative. The blood urea nitrogen was 9 mg. per cent. Chemical studies of the blood serum revealed creatinine 2 mg. per cent, total protein 5.9 gm. per cent with 3.6 albumin and 2.3 globulin, potassium 4.8 mEq. per L., sodium 146 mEq. per L., chloride 97 mEq. per L., calcium 5.0 mEq. per L., inorganic phosphorus 4.2 mEq. per L., creatine 4.3 mg. per cent and an alkaline phosphatase activity of 6.4 Shinowara-Jones-Rinehart units. The serum total bilirubin was 0.5 mg. per cent with a direct bilirubin fraction of 0.1 mg. per cent. The urine was mahogany brown, with a specific gravity of 1.025, a pH of 6.0 and a protein concentration of 1.97 gm. per cent. Microscopic examination of the sediment revealed 4 to 7 white blood cells per high powered field, moderate numbers of epithelial cells and no red blood cells. Results of tests for urine bile and urobilinogen were negative. Twenty-four-hour excretion studies of the urine revealed normal concenof delta-amino-levulinic trations acid, porpho-
I
CASE
MARIE
J. FRAJOLA,
REPORT
A forty-three year old white woman was admitted to the hospital because of cramping pain in both lower extremities and the passage of dark brown urine. She had enjoyed good health until five years before, when she experienced similar but less severe symptoms. Three days prior to admission the patient worked continuously for fourteen hours at her occupation as a barmaid. Two days later she awakened with severe cramping pain in the muscles of both lower extremities, which confined her to bed. Later that evening she noticed that her urine had become dark brown, and the muscles of her upper extremities were painful. Upon attempting to walk, she became alarmed at the weakness and loss of control that she experienced in her lower limbs. The muscles of both calves were tender, swollen and hard. She did not have any nausea, fever, vomiting or abdominal pain. Physical examination revealed a well developed, well nourished, alert white woman who complained of considerable pain in both the upper and lower extremities. The oral temperature was 102.4’%., pulse 124 beats per minute, respirations 18 per minute and
* From the Laboratory of Cellular Pathology, Department of Pathology, and the Herman A. Hester Research Laboratory, The Ohio State University, Columbus, Ohio. Manuscript received February 12, 1962. t Senior Research Fellow in Pathology, National Institutes of Health Grant GM-K3-15, 104-C4. 426
AMERICAN
JOURNAL
OF
MEDICINE
idiopathic
Recurrent
Khabdornyolysis--&al-pelli
bilinogen, uroporphyrins and coproporphyrins. The reaction to a benzidine test on the urine was strongly positive. Electrophoretic study of the urine showed the mahogany colored pigment to be myoglobin. (Fig. 1.) Serum enzyme studies were begun on the day of admission and continued serially for the subsequent eight days. Levels of glutamic oxaloacetic @GOT) and glutamic pyruvic transaminases (SGPT), lactic (LDH), malic (MDH), isocitric (ICD) and glutamic dehydrogenases (GDH), aldolase (ALD), phosphohexose isomerase (PHI) and leucine aminopeptidase (LAP) activities were markedly elevated but decreased by the eighth day, paralleling the patient’s clinical improvement. On the day of admission, 10 units of succinic dehydrogenase (SD) activity was detected in the serum. Subsequent serial studies showed lower values as her condition improved. Except for a low level of activity demonstrated on the day of admission, serum acetylcholine esterase (ACE) activity remained slightly below the lower limit of the normal range throughout her period of hospitalization. (Table I.) The patient was placed at absolute bed rest. She continued to experience muscle pain in the upper and lower extremities and passed small amounts of dark brown urine. Her oliguria persisted for five days following admission. During this period her urine gradually became lighter in color and the total twenty-four-hour output increased from 270 to 1,240 cc. During the next three days her muscular
FIG. 1. Paper electrophoresis patterns. ing hemoglobin. B, urine containing
is identical
Day
~
SGOT
I
11-1
SGPT
/
I
OF SERUM
306 525 400 200 162 138 86
3 4 6 8 9
MD
LDH
ENZYMES
ICD
~
1:
ALD
GDH
PHI
LAP __
14,400 4,800 3,420 1,920 1,600 1,600 1,075
430 100 200 100 69 78 50
1
*
SD
68 ...
23,750 6,400 5,200 2,700 2,800 2,600 5,600
0
10 ... 4 0
;I
:0
Serum Enzymes of Siblings A. M. (sister) J. L. (brother)
with myoglobin.
pain decreased and the affected muscle masses lost their board-like rigidity. On the ninth hospital day an erythematous, mildly pruritic rash developed on her cheeks and nose. The rash disappeared within the next two days. By the twelfth hospital day she was free of symptoms and her urine devoid of protein, although she still exhibited a slight degree of residual
Enzyme Hospital
A, urine containhemoglobin and
myoglobin. C, patient’s urine. D, control solution of myoglobin. The patient’s urine contains a protein which has approximately half the mobility of hemoglobin and
TABLE ACTIVITY
el al.
z
1
TZ
j
‘E
1
1:
I
0”
149 113 141 111 120 100 84
ii
ACE
IL
~ 1
520 440 380 400 360 390 350
50 130 130 155 130 105
’
z::
:I:
_______ 1
1:
)
1::
,
_______ ,-O-30 * For explanation VOL.
34,
MARCH
1 O-30 ~,I,,,,~:::::~~~ of abbreviations, 1963
see text.
0
~50-100~
lo-20
~100-310~130-260
Idiopathic
428
Recurrent
Rhabdonlyolysis
FIG. 2. Gastrocnemius muscle biopsy specimen obtained during the acute phase of muscle pain and weakness with myoglobinuria. A, coagulation necrosis of muscle fibers. Original magnification X 92. B, an area of muscle necrosis with sarcolemmal nuclear proliferation. Note the segmental distribution of the necrosis. Original magnification X 125. C, liquefaction necrosis of sarcous substance immediately adjacent to normal appearing muscle fibers. Note the absence of an inflammatory reaction. Original magnification X 205. muscle weakness in both legs. The patient charged on the sixteenth hospital day. SPECIAL
STUDIES
0~
was dis-
MUSCLE
Electromyography. Electromyographic studies* of both weakened and grossly pathologic muscles * Performed by Dr. J. D. Guyton, Department of Physical Medicine.
Scurpelli et al.
exhibited increased insertion potentials and complex motor unit potentials which were reduced both in amplitude and duration. In addition, considerable fibrillation and occasional fasciculation were noted. These findings were interpreted as indicative of a diffuse myopath! with extensive random destruction of muscle fibers. Histopathology. A biopsy of the left gastrocnemius muscle revealed a pale grayish pink muscle tissue. It was fixed immediately in 10 per cent neutral formalin for light microscopy and histochemistry, and in buffered 1 per cent osmium tetroxide for electron microscopy. Paraffin sections showed extensive coagulation necrosis of muscle fibers characterized by masses of structureless, densely clumped, eosinophilic sarcoplasm. A few fibers showed liquefaction of sarcous substance. The necrosis involved either complete muscle fibers immediately adjacent to normal appearing ones or segments of muscle fibers with zones of necrosis interspersed between areas of undamaged sarcoplasm. Many muscle fibers exhibited lesser degrees of damage, such as myofibrillar swelling and obscuration of sarcoplasmic structure. In certain areas there was evidence of sarcolemmal nuclear proliferation. Despite the widespread necrosis there was a paucity of inflammatory cells. (Fig. 2.) The periodic acid-Schiff reaction showed occasional granules of glycogen scattered in the sarcoplasm which were removed by diastase during previous digestion. None of the muscle fibers showed the intrasarcoplasmic cleft formation characteristic of familial periodic paralysis. Electron Microscopy. Under the electron microscope areas of muscle fiber necrosis showed marked disruption of sarcoplasmic structure with absence of organized bundles of myofibrils. Isolated myofibrils were present in which occasional Z bands were still visible; the H bands were absent. The interfibrillar sarcoplasm consisted of numerous vesicles of varying size, occasional swollen mitochondria (sarcosomes) and an electron-translucent amorphous material. (Fig. 3.) Although the sarcolemmal membrane of damaged muscle segments appeared less osmiophilic and less intact than that of undamaged ones, the fine structure was surprisingly well preserved despite widespread damage of the muscle fibers. (Fig. 4.) The sarcolemmal nuclei of necrotic muscle fibers appeared undamaged. (Fig. 5.) Less damaged AMERICAN
JOURNAL
OF
MEDICINE
Idiopathic
Recurrent
Rhabdornyolysis-Scar-eZZi
et al.
FIG. 3. A longitudinal section of a necrotic muscle fiber adjacent to an undamaged one. Within the necrotic fiber isolated remnants of myofibrils (MF) are present in which the Z bands are still visible. The necrotic sarcoplasm contains several swollen sarcosomes (S), numerous vesicles of varying size, and amorphous material. An arrow indicates the sarcolemma (SL) of the necrotic cell. An undamaged sarcolemmal membrane (SL) and muscle fiber are shown in the lower portion of the micrograph. Original magnification X 12,000.
fFIo. 4. The sarcolemmal membrane (SL) of a normal muscle fiber is shown at the left. The sarcolemma of a necrotic muscle cell appears at the right. The laminae, especially the inner one, show marked thinning and numerous interruptions of continuity. Note the absence of myofibrils in the necrotic sarcoplasm (NS). Original magnification X 28,000. 34,
MARCY
1963
429
Idiopathic
Recurrent
Rhabdomyolysis-Scarpelli
et al.
FIG. 5. A sarcolemmal cell with associated perinuclear cytoplasm is shown immediately adjacent to necrotic sarcoplasm and separated from it by a distinct plasma membrane (PM). A portion of the nucleus (N) appears in the upper right of the micrograph. Note the rod-shaped dense sarcosome (S) in the sarcolemmal cell cvtoulasm and the swollen sarcosome (S) in the necrotic sarcoplasm. Original magnification X 28,500.’ A
muscle fibers with intact myofibrils exhibiting both Z and H bands had swollen sarcosomes which were notable in that they were often contiguous to morphologically normal-appearing sarcosomes. (Fig. 6.) The swollen sarcosomes showed remnants of cristae which were indistinct and in most instances had lost their characteristic double membraned structure. (Fig. 7.) The sarcoplasmic reticulum appeared undamaged. In several areas of muscle the fine structure of the myofibrils showed a curious change, having become granular and more electron-dense. Only a few bundles of fibrils had remnants of the 2 bands. (Fig. 8.) SERUM
ENZYME
STUDIES
OF TWO
SIBLINGS
Enzyme studies were performed on the serums of a sister and brother of the patient, both of whom were historically and clinically free of stigmata of the disease. The serum of the sister
had elevated malic and succinic dehydrogenase activity. The enzyme activities of the brother’s serum showed no abnormalities. (Table I.) COMMENTS
The classic communication by Meyer-Betz describing idiopathic rhabdomyolysis does not mention whether the attack was preceded by physical exercise. It has become apparent now that although in many cases of idiopathic rhabdomyolysis the attacks appear to be precipitated by physical exercise, a considerable number are not. In the most recent review of the literature [S] twenty-one of the forty-six cases reported were of the exertional type. Bowden et al. [7l state that the exertional type has been observed exclusively in males. The case reported here was definitely of the exertional type and occurred in a female, a relationship which has not been reported previously. AMERICAN
JOURNAL
OF
MEDICINE
idiopathic Recurrent Khabdornyolysis -Scar~elli et (11.
FIG. 6. A longitudinal section of non-necrotic muscle showing focal sarcosomal (S) swelling and membrane alteration. The myofibrils are undamaged, exhibiting intact sarcomeres in which both the Z and H bands are clearly visible. Original magnification X 9,600.
FIG. 7. A higher magnification of an area similar to Figure 6, showing the focal sarcosomal akerations in greater detail. Note the swollen sarcosomes contiguous to normal appearing ones. The arrow points to the remnant of a double outer membrane of a sarcosome in which the cristae have lost their characteristic double-membraned structure. Original magnification X 37,600. VOL.
34,
MARCH
1963
432
Idiopathic Recurrent Rhabdolnyolysis--~Scar~elli el al.
FIG. 8. A section showing a curious alteration of the myofibrils (MF); these appear granular and more electron-dense than their adjacent normal counterparts. The arrow points to remnants of the Z bands; the H bands are absent. Original magnification X 9,600.
In view of the high concentration of potassium [70,77] and creatine within the muscle cell, the muscular necrosis, and the clinical and chemical signs of impaired renal function in this patient, one would expect the serum levels of creatine and potassium to be elevated. The normal serum and potassium levels encountered creatine during the acute phase of the disease are similar to those reported for creatine by Hipp and Shukers [72], and for potassium by Bowden et al. [7]. The absence of a high serum creatine level may result from the depletion of muscular phosphocreatine prior to necrosis. Increased activity of the various enzymes in the serum is most probably the result of leakage of soluble enzymes from damaged muscle cells. This is in accord with the findings of Sibley and Fleisher [ 731, Wroblewski and La Due [ 741, and Dreyfus, Schapira and Schapira [75]. The sarcolemma of the damaged muscle ceils in this case showed marked alterations of ultrastructure which were, no doubt, reflected in gross disturbances of selective permeability. Essentially
similar morphologic changes have been observed by Mauro and Adams [76] in the sarcolemmal membrane of traumatically damaged frog skeletal muscle. Alteration of membrane permeability may be a sufficient basis on which to explain the diffusion of soluble enzymes from a damaged cell [ 771. However, in the light of the work of Watson and Siekevitz [78], the appearance of succinic dehydrogenase activity in the serum suggested a more severe degree of damage. Watson and Siekevitz were able to show in cornminuted liver mitochondria that the fraction which exhibited the majority of succinoxidase activity consisted largely of mitochondrial membranes. Thus it is considered that succinic dehydrogenase is intimately bound to the structural proteins of the mitochondrial membranes, so that its appearance in the serum could result only from considerable alteration in sarcosomal fine structure as demonstrated in this case. This is in contrast to the more soluble glutamic dehydrogenase which would leak from mitochondria across damaged membranes. That the membrane alteration in this patient was severe is supported by the fact that the damaged sarcosomal cristae lost their characteristic doublemembraned structure. The selective nature of cellular damage in this disease was emphasized under the electron microscope by the presence of sarcolemmal nuclei and perinuclear cytoplasm in intimate contact with necrotic sarcoplasm. The origin of these cells has been the subject of many [ 7$.22]. Currently two concepts investigations concerning their origin are held: (1) sarcolemma1 nuclei and cytoplasm originating from muscle cells, and (2) emigration of extramuscular “free cells” which gain access into the sarcolemmal lined myotubes and assume a subsarcolemmal position. The activation of sarcolemmal nuclei constitutes an important part of the reaction of muscle to injury. Reconstruction of muscle begins with the proliferation of sarcolemmal nuclei and an increase in the amount of perinuclear cytoplasm. The presence of a distinct plasma membrane between these cells and the necrotic sarcoplasm (Fig. 5) suggests to us that they may represent two separate cellular entities. Although the sarcolemmal nuclei are distinctly beneath the sarcolemmal membrane, our data shed no light on whether these nuclei originate in situ or migrate from without the myotube. AMERICAN
JOURNAL
OF
MEDICINE
Idiopathic
Recurrent
Rhabdornyolysis-
The possible genetic basis of recurrent idiopathic rhabdomyolysis was first pointed out by Hittmair [23]. Such a relationship has been suggested subsequently by Hed [Z], Schaar, La Bree and Gleason [25], and Bowden et al. 171. The abnormal serum enzyme activities resembling those of the patient in a female sibling who, so far as we know, is free of any disease suggests that she may possess a similar metabolic disturbance. The absence of stigmata of the disease in this sibling may be attributed to subclinical attacks due to either incomplete penetrance of the genetic defect, if one indeed exists, or exercise levels of insufficient severity and duration to precipitate the attacks. Subclinical attacks of rhabdomyolysis during which the patient was free of overt symptoms have been observed and documented by a study of serial muscle biopsies in a patient reported by Reiner et al. [26]. A variety of etiologic factors has been implicated in acute recurrent rhabdomyolysis ranging infection and alcohol to an from exercise, enzymatic defect of muscle glycogenolysis [27]. The electron microscopic data obtained in the present study suggest still another possible etiologic factor-that of impaired mitochondrial function. A few statements concerning current knowledge of the mechanism of muscular contraction and relaxation should precede a discussion of this possibility. Muscular contraction results from an interaction of the contractile proteins, actin and give rise to myosin, which upon combination the actomyosin complex. This is followed by a shortening of the myofilaments and thus contraction [28,29]. Since actin and myosin combine very readily with a high free energy of interaction [30], muscle possesses mechanisms bywhich the interaction and dissociation of these proteins are controlled. Contraction and relaxation of muscle may be controlled by the following mechanisms: (1) Adenosine triphosphate (ATP) in sufficiently high concentration around myosin molecules can inhibit their spontaneous interaction with actin [28]. (2) ATP in high concentrations can dissociate the actomyosin complex [28,37]. (3) A water-soluble muscle fraction called relaxing factor [32] which consists of creatine phosphate and a protein [33] may prevent the interaction of actin and myosin by the inhibition of myofibrillar ATP-ase (myosin), thus maintaining a high concentration of ATP in muscle fibers, and by the rephosVOL.
34.
MARCH
1963
Scqbelli
et al.
433
phorylation of adenosine diphosphate (ADP) to ATP [34]. The morphologic evidence of sarcosomal damage may be the result of, or give rise to, a defect in the synthesis of ATP. This could lead to the following sequence of events, which presently is purely speculative although based on established biochemical and physiological facts: A deficiency of ATP would lead to a loss of intrasarcosomal calcium. ATP binds calcium within the sarcosomes by chelation in a manner similar to EDTA [35,36]. Loss of calcium impairs seriously oxidative phosphorylation and results in sarcosomal swelling [37]. In addition, depletion of ATP around the myofibrils and inhibition of the phosphorylative and relaxing [38] functions of relaxing factor by free calcium ions would result in a spontaneous combination of actin and myosin. This would lead to a continuous state of uncontrolled contraction which would eventually damage the muscle cell. Failure of the relaxing factor system is certainly suggested by the clinical finding of board-like tonic muscles during the acute phase of the disease. SUMMARY
The results of clinical, chemical and morphologic studies of a woman suffering from paroxysmal idiopathic rhabdomyolysis are presented. Increased serum enzyme activities observed during the acute phase of the disease returned toward normal levels as the patient’s clinical state improved. Of exceptional interest was the of succinic dehydrogenase in the presence patient’s serum. A biopsy specimen of the gastrocnemius muscle revealed extensive focal coagulation necrosis of muscle fibers. Some of the fibers exhibited lesser degrees of damage, such as myofibrillar and sarcoplasmic swelling. Under the electron microscope focal alteration of sarcosomal fine structure was observed in mildly damaged myofibrils. This consisted of swollen sarcosomes and extensive obscuration of the double membraned cristae. Damaged sarcosomes were often contiguous to completely normal-appearing ones. The possible significance and relationship of these findings to the etiology and pathogenesis of this disease are discussed. Acknowledgment: We gratefully acknowledge the cooperation of Dr. W. F. Ashe for allowing us to study his patient, and for his encourage-
434
Idiopathic
Recurrent
Khabdorllyolysis-S~~~~~~
ment and advice; and the technical assistance of Ruth Austad, Modestine Brown, Devinia Jefferson, Lynn Shawhan, Dr. Nicanor Atillo, C. V. Sittler and G. R. Millard. REFERENCES
1. MEYER-BETZ, F. Beobachtungen an einem eigenartigen mit MuskellHhmungen verbundenen Fall van Hzmoglobinurie. Da&he Arch. klin. Med., 101: 85,191l. 2. CARLSTRGM, B. eber die Aetiologie und Pathogenese der Kreuzlihme des Pferdes (Haemoglobinaemia paralytica). Skandinav. Arch. Physiol., 61: 161, 1931. 3. BYWATERS,E. G. L. and DIBLE, J. H. Acute paralytic myohaemoglobinuria in man. J. Path. & Bat., 55: 7, 1943. 4. ACHESON,D. and MCALPINE, D. Muscular dystrophy associated with paroxysmal myoglobinuria and excessive excretion of ketosteroids. Lancet, 2: 372, 1953. 5. SCHAAR, F. E. Paroxysmal myoglobinuria. Am. J. Dis. Child., 89: 23, 1955. 6. FARMER, T. A., JR., HAMMACK, W. J. and FROMMEYER, W. B. Idiopathic recurrent rhabdomyolysis associated with myoglobinuria. New Engkmd J. Med., 60: 66, 1961. 7. BOWDEN, D. H., FRASER, D., JACKSON,S. H. and WALKER, N. F. Acute recurrent rhabdomyolysis aroxysmal myohaemoglobinuria). Medicine, 35: $5 1956. 8. COM&, D. A. and ROSENFELD, H. Idiopathic paroxysmal myoglobinuria. Ann. Int. Med., 55: 647, 1961. 9. MINETT, F. C. The haemoglobinuria and myoglobinurias of animals. PYOC. Roy. Sot. Med., 28: 672, 1935. 10. RITCHIE, A. D. The Comparative Physiology of Muscular Tissue. Cambridge, England, 1928. Cambridge University Press. 11. STEINBACH,H. B. Intracellular inorganic ions and muscle action. Ann. New York Acad. SC., 47: 849, 1947. 12. HIPP, H. R. and SHUKERS, C. R. Spontaneous myoglobinuria: report of a case with symptoms of myotonia. Ann. Znt. Med., 42: 197, 1955. 13. SIBLEY, J. A. and FLEISHER, G. A. The clinical significance of serum aldolase. Proc. Stofl Meet. Mayo Clin., 29: 591, 1954. 14. WROBLEWSKI,F. and LA DUE, J. S. Lactic dehydrogenase activity in blood. Proc. Sot. E@er. Biol. &T Med., 90: 210, 1955. 15. DREYFUS, J. C., SCHAPIRA, G. and SCHAPIRA, F. Serum enzymes in the physiology of muscle. Ann. New York Acod. SC., 75: 235, 1958. 16. MAURO, A. and ADAMS, W. R. The structure of the sarcolemma of the frog skeletal muscle fiber. J. Biophys. & B&hem. Cytol., (supp.), 10: 177,196l. 17. ZIERLER, K. Factors which influence movement of aldolase from excised rat diaphragm. Am. J. Physiol., 183: 675, 1955. 18. WATSON, M. L. and SIEKEVITZ,P. The isolation and analysis of a mitochondrial membrane fraction. J. Biophys. & Biochem. Cytol. (supp.), 2: 379,1956.
et (II.
19. WALDEYER, W. Ueber die Versnderungen der quergestieiften Muskeln bei Entziinduig und dem Typhusprozess, sowei iiber die Regeneration derselben nach Substanzdefecten. V&chows Arch. path. Anat., 34: 473, 1865. 20 CLARK, W. E. L. An experimental study of the regeneration of mammalian striped muscle. J. Anat., 80: 24, 1946. 21. GODMAN, G. C. On the regeneration and redifferentiation of mammalian striated muscle. J. Morphol., 100: 27, 1957. 22. LASH, J. W., HOLTZER, H. and SWIFT, H. Regeneration of mature skeletal muscle. Anat. Rec., 128: 679,1957. 23. HITTMAIR, A. Haemoglobinuria paroxysmalis paralytica. Wien. klin. Wchnschr., 38: 431, 1925. 24. HED, R. En familjar form av paroxysmal myoglobinuri. Nerd. med., 35: 1586, 1947. 25. SCHAAR, F. E., LA BREE, J. W. and GLEASON,D. F. Paroxysmal myohemoglobinuria with fatal renal tubular injury. J. Lab. &? Clin. Med., 34: 1744, 1949. 26. REINER, L., KONIKOFF, N., ALTSCHULE, M. D.. DAMMIN, G. J. and MERRILL, J. P. Idiopathic paroxysmal myoglobinuria. Arch. Int. Med., 97: 537, 1956. 27. SCHMID, R. and MAHLER, R. Chronic progressive myopathy with myoglobinuria: demonstration of glycogenolytic defect in muscle. J. Clin. Znvesf., 38: 2044,1959. 28. SZENT-GY~RGYI, A. Chemistry of Muscular Contraction. New York, 1951. Academic Press, Inc. 29. HUXLEY, H. E. Muscle cells. In: The Cell. Biochemistry, Physiology, Morphology, vol. 4, p. 366. Edited by Brachet, J. and Mirsky, A. E. New York, 1960. Academic Press, Inc. 30. GERGELY, J. and KHOLER, H. Light scattering studies on the stepwise formation and dissociation of actomyosin. In: Proceedings of the Conference on Muscle Contraction, p. 14. Tokyo, 1957. Igaku Shoin, Ltd. 31. STRAUB, F. B. The specificity of the adenosine triphosphoric acid effect. Studies Inst. M. Chem. Univ. Szeged., 3: 38, 1943. 32. MARSH, B. B. A factor modifying muscle synaeresis. Nature, London, 167: 1065, 1951. 33. GOODALL, M. C. and SZENT-GYBRGYI, A. G. Relaxing factor in muscle. Nature, London, 172: 84, 1953. 34. LORAND, L. Adenosine triphosphate-creatine transphosphorylase as relaxing factor of muscle. Nature, London, 172: 1181, 1953. 35. ERNSTER,L. and LB,, H. Reconstruction of oxidative phosphorylation in aged mitochondrial systems. Exper. Cell Res., (supp.), 3: 133, 1955. 36. RAAFLAUB, J. Die Metallpufferfunktion der Adenosinphosphate. Helvet. phvsiol. et pharmacol. acta, 14: 304,1956. 37. LEHNINGER,A. L. Physiology of mitochondria. In: Enzymes: Units of Biological Structure and Function, p. 217. Edited by Gaebler, 0. H. New York, 1956. Academic Press, Inc. 38. BOZLER, E. and PRINCE,J. T. The control of energy release in extracted muscle fibers. J. Gen. Phyriof., 37: 53,1953. AMERICAN
JOURNAL
OF
MEDICINE