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Percutaneous needle-biopsy of skeletal muscle in diagnosis and research
THE LANCET, SRPTllMnlm
11, 1971
593 51yl I
Occasional Survey PERCUTANEOUS NEEDLE-BIOPSY OF SKELETAL MUSCLE IN DIAGNOSIS AND RESEARCH
R. II. T.
(])~======
EDWARDS
Department of Medicille" Royal Postgraduate Medical School, Hammersmith Hospital, London W.12
,
Percutaneous needle-biopsy of skeletal muscle has been successfully used [tH' several YCU1'S in other countries, but only rarely, if at all, in Great Britain. Complicntions-e-hrematoma Iormution or infection-e-are exceedingly uncommon. The technique is simple and relatively atraumatic and biopsy specimens can be taken in the ward or outpatient clinic for puthological or biochemical studies. Such biopsies will contribute information about muscle as a tissue and on the distribution of electrolytes between the cellular and extracellular spaces of the body. Since samplea can be obtained very rapidly, and repeatedly, the technique also makes it possible for muscle energy metabolism to be studied during exercise. The biopsy technique offers u practical means of studying the cellular basis of the symptoms of muscle weakness, excessive fatigue, and effort intolerance. .summClry
INTIl<>UUCTION
we in Great Britain are neglecting a potentially valuable diagnostic and research technique, which is widely used elsewhere. Recent reviews in British journals of current practice in soft-tissue biopsy techniques 1,2 omit any reference to percutaneous needle-biopsy of skeletal muscle. Yet it is over a century since Duchenne described the use of his needle (or" harpoon ") to obtain" living pathological anatomy" in patients with pseudohypertrophic muscular dystrophy.s.! Further, it is notable that he describes his biopsy needle as an instrument inoffensif (fig. 1) as its use was associated with little discomfort (in days before the introduction of adequate local antesthesia) or harm to the patient. With such an auspicious start it is perhaps surprising that this technique for musele biopsy was so little used subsequently. A percutaneous needle-biopsy technique was recently introduced by Bergstrom, G who modified an existing synovial biopsy needle G for the purpose. An estimated 3000-4000 biopsies of skeletal muscle have been taken with this needle (fig. 2) in Scandinavia during the past ten years. The present account will be limited to the technique of obtaining samples of muscle with this needle, together with a review of EVrDHN'l'LY
C ··'r"I:,:-_......
L - -_ _..:.:..:.:.. ..~
FiR'. l-Duchenne's biopsy needle.'
w
i
~===============~ - -
- - - 1JO mm
Fill, 2-·Del'gstrlim's biopsy needl".'
current methods of studying the biopsy sample and the diagnostic and research opportunities offered by the technique. Biopsy with Bel'IJstl'om Needle Local anesthetic (2-4 mi. 1 % lignocaine) is infiltrated into the skin and subcutaneous tissues down to the deep fascia, after first shaving and disinfecting the skin, The biopsy needle is introduced through a 4 mm, incision in the skin and subcutaneous tissue made with a Swan Morton blade (no. 11). The needle is advanced until the tip is about 3-5 em. below the skin surface and a sample of tissue is guillotined with the hollow cylinder and secured within tile barrel of the needle (fig. 2), which is then rapidly withdrawn from the muscle. Wh.en several biopsy specimens are to be taken over a short period of time as in some physiological studies, the skin incisions are made beforehand; these are covered with pads of sterile gauze which are removed in turn as specimens are required. The edges of the skin incisions are later opposed with a sterile adhesive material (' Steristrip '). They heal well and are generally invisible after a few months. With this "no-touch" technique there is no need for extensive towelling, "scrubbing up ", or the use of sterile gloves. This means that the technique can be used as a ward or clinic procedure, thus obviating the need for surgical expertise and operating-theatre facilities. The technique is remarkably atraumatic, Generally the most painful part of the procedure is the infiltration of the local aneesthetic into the skin and subcutaneous tissues. Though the biopsy is taken from muscle which has not been ansesthetised the sensation (described from personal experience of nine such biopsies) is of deep pressure together with a " visceral ,. feeling (as when the ulnar nerve at the elbow is accidentally knocked). The residual sensation is one of moderate muscle stiffness, such as would follow unaccustomed, strenuous exercise. This clears in a few days. Muscle function is little impaired by the procedure, as shown by the fact that subjects have been able to continue competing in strenuous cross-country ski races after several such biopsiea.t The simplicity of the operation, which takes less than three seconds, allows a number of specimens to be taken in quick succession, and it is possible to study changes occurring as the result of muscular contraction. The principal disadvantage of this technique is that it is "blind" and therefore applicable only to large
594
l'HH f.ANCln', SIlP'rBMOnR
II, 1971
muscles, but biopsy specimens have been taken without complication from the posterior part of the deltoid muscle." The vastus Iateralis of the quadriceps femoris muscle is preferred, as this muscle is free of important blood-vessels and nerves. Virtually all specimens have in the past been taken from the quadriceps, and the technique is likely to be of regular diagnostic use only when a disorder of this muscle is present. But proximal-muscle weakness is a common feature of many muscle diseases so that limitation to this anatomical site does not seriously affect the usefulness of the technique. Possible complications are htemorrhage and infection. In my experience, hrematcma formation occurred twice in a series of 100 biopsy specimens taken from normal subjects participating in research studies in Sweden. One of these was in a medical colleague, who later admitted that he was liable to heavy bleeding following dental extractions. He experienced severe muscle swelling and stiffness and complete recovery took four weeks. In another subject hsernatoma caused some swelling and muscle stiffness which persisted for two weeks. Both these subjects had four biopsies in quick succession. Infection of the skin incision was seen only once in the whole series. Infection of the deep tissues did not occur. Damage to nerves is of course a possibility, though this has never been seen in practice. The danger of heemorrhage into the muscle is slight but, should symptoms or signs appear) hematoma formation can be greatly reduced by immediately applying firm pressure to the biopsy site with a pad of sterile gauze. It is recommended that biopsies should not be performed on persons with a bleeding tendency or those on anticoagulant therapy. In the rare event of a diagnostic biopsy being indicated in a patient on anticoagulants, the biopsy should be delayed until the whole-blood clotting-time has been temporarily restored to normal following a period of intravenous heparin therapy. The Muscle Sample The biopsy technique yields a sample which generally has a wet-weight of 20-40 mg. For morphological or histochemical studies there is no need to weigh the sample, but for all other analyses accurate
weighing is essential. WIlen fresh muscle is needed (as for measurement of enzyme activity) the sample is placed on an elecrrobalance. Weight is plotted against time for a short period to allow for the effect of water evaporation by extrapolation to zero-time." If metabolites are to be measured it is essential that metabolism be arrested as quickly us possible, and this is done by freezing the sample in liquid • Freon' at --I50°c or liquid nitrogen. Connective tissue and blood are removed by dissecting the frozen sample in a cryostat 8 or, more conveniently, by dissecting the freeze-dried tissue at room temperature, The sample is weighed accurately after removal of this extraneous material.
,Fig. 3-Needle-biopsy sample showing normal differentiation of fibre types by staining for myosin A.T.P.-ase (see text).
Fig. 4-Electron microphotograph of a needle-biopsy sample of muscle from a patient with polymyositis, showing atrophy of myofibrils.
MORPHOT,OGICAI, AND HISTOClll1MIc:AL Sl'Ulllf(S
The biopsy technique provides samples which nrc suitable for light microscopy (after stainlng with lucmatoxylin and eosin) and for hiarochemicnl lnvestigations using cryostat sectlona.!" Several muscleenzymes have been studied using conventlonal histochemical methods. It is possible in this way to identify different types of muscle-fibre, Stainlng nil' myosin A:r.l'.-llse (fig. 3) allows a distinction to be made between fast (white) fibres, which pOSHCSS a high activity of this enzyme, from slow (red) fibres with a low activity. This differentiadon is of Interest in view of recent accounts of selective degeneration of nne fibre type (e.g., fast fibres in parkinaonism 11 and slow fibres in disuse atrophy due to joint injury). 11 Samples for electron microscopy arc fixed immediately in 3%) glutaraldehyde and subsequently treated by conventional techniques. Structure is well preserved and abnormalities can be explored in detail. Considerable enlargement and alteration of the form of the cristee of mitochondria have been found in human skeletal muscle biopsied immediately after severe exercise. 13 A recent finding of considerable physiological interest is that improved physical fitness is not associated with increased capillary density in skeletal muscle. H , 18 This is contrary to what had previously been supposed. CHEMICAL ANALYSIS OF
NEEDLE~BIOPSY
SAMPLES
Electrolytes The needle-biopsy technique was developed by
595
n IH I,ANCHT, SIlPTEMnnu 11) 1971
Bergstrom for measurement of muscle-electrolytes. Skeletal muscle represents about 40 e/(, of the bodyweight and it is the largest and most easily accessible fra ction of th e in traccllulur-Iluid compartment. As u seq ucl to the interest in water and electrolyte balance in the extracellular fluid following the introdu ction of flame photometry in the 1950s) techniques were developed by Bergstrom • for the study of the much larger intra cellular- fluid compartment. These were used for investig ation s on patients with disturbances of fluid and electrolyte balance. Neutronactivation analysis was employed for assaying certain ions. Atomic absorbtion flume photometry has also been used to measure concentrations of sodium; potassium, chloride, and rnagncaium.!" lilll.'I'J{Y Stores and lvIccabolircs The largest local energy store in muscle is provided by glycogen, which conulhures 1-·l ·5 % of the muaclc's we t-wei gh t." There have been several biops y studies of the influence of dlet und heavy exercise 011 glycogenolysis lind rcsynchcsia. !" There nrc changes in various types of diseuse-for instance, unt reated juvenile diabetics have a low mus cle-glycogen which is res tored to normal by insulin therapy. 10 Muscle-glycogen is low 1I180 during acu te oliguric renal Iailu rc. : " S nullea have also been made of the con centration s in muscle of the energy-rich compounds 1\.'1'.1'. and phos~h()rylcrelltinc (r.c.) .• . 0, 0 I Even in fatiguing ClCcr<.:ISC depiction of A/ r .t'. is limited because it is mai ntained lit the expe nse of 1'.<:., the level of which ca n fall almost to zel'o.B.ll n L ittle is known as to h ow disease atlcc ts these hi gh-energy compounds in mu scle, but recent eviden ce suggests tha t the ir amounts can be increased significa ntly by physical training. 2 ' Several glycolytic intermediates and co-factors have been studied in human muscle obtained by the needlebiopsy technique.B.D,2 ~,25 The aim has been to elucidate the factors controlling the energy-supplying mechanisms in working muscle. 3',5' cyclic A.M.P. has been measured in a needle-biopsy sample by Dr. B. Tulloch (Royal Postgraduate Medical School) by a recently developed ana lytical method. 2 6 Enzymes Succinic dehydrogenase has been measured to indicate the oxidati ve activity of the mitochondria. 2. The activity of this enzyme was found to be increased followin g physical tr aining. S& No reduction in activit y was found in patients with dystrophia myotonica.w The activity of muscle lactate-dehydrogenase has been found to be increased following prolonged exercise in normal subjects,"! and to be present with an ab norma l isoenzyme str ucture in patients with chronic alcoholism. v' Information can also be gained hy studying the metabolite balance for reactions catalyscd by particular enzymes. F or example, changes in the proportions of fr uctose 1: (i-dlpho sphatc lind fructose 6-phospilate in isometric exercise sugg est that .the activity of phosphofr uctokinase may be imp aired by mu scular fatigue/ particularly when mu scle temperature is increased. 33 Lipids Recent biochemical analyses have shown a significant decrease in musc1e-triglycerides as a result of pro-
longed severe exercise. "1 Electron micrograph s have not been sufficiently "quantitative" to show a corresponding reduction in the Dumber 01' size of fat droplets in muscle-cells even when the glycog en granules are much depleted. w O ther Analyses The influence of arninoacid solutions on the catabolic nitrogen metabolism after major surgery 30 and in ul'remia' 7 have been st u d ied by meas uring the incorporation of the isotope toN into blood and muscle proteins following the infusion of 15Nlabelled glycine. Mass spectrometry was used to assay the lGN in needle-biopsy samples. The effect of intermittent dialysis on the arsenic content of m uscle has been studied by neutron-activation analysis of biops y samples. aH FUTUR ll O\'l'OR'l'UNt1'I[lS
Wider usc of the needle- biopsy t echnique cou ld be of value for explo iting histological and hi stochemical methods of di agno sis in p at ients with muscle weak ness. Further, the technique provides an opportunity to remedy the present luck of direct information, largely attributable to difficulty in obtaining suitable sam p les. w concerning the biochemical basis of impaired muscle function in disease. Skeletal muscle, like many specialised tissu es in th e body, r esponds to the many form s of dise ase on ly in a limited number of ways, and consequently muscle biopsies ar c more likely in the Immediate future to provide information about basic mechani sms than to indicate the retiology of particular diseases. However, there is reason to hope that wid er use of the muscle-biopsy techn iqu e will ultimately provide a cl ue as to why these basic mechanisms are disturbed and thereby elucidate the common sym ptoms of muscular weakness and fatigue. I thank Mrs. Caroline Maunder of the Department of Histochemistry, Royal Postgraduate Medical School, for the Itistochemi cal and electron rnicroacopical studies illustrated here; and the Wellcome Trust and the Muscular Dystrophy Group of G reat Britain for supp ort . F ig. 2 is .reproduce d by permission. REFERENCES 1- Turner Warwick, R., T urner W arwi ck, M . 8 ,·. .... d. J. 19 70, Hi, 149. 2 . G ear, M. W. L . Br.]. H Ofp. M,d. 19 69,2, 17013. Duchen ne,·G . B. A rcll. ·g.". M.a. 1868, 11, l7 9. 4. Nntrass, F. J. in: Resea rch in Muscu lar D ystrophy (edit ed by th e M usculor D yst rophy Group); p .5. Lon don, 196 B. 5. Bergatrbm, J. S eal/d.]. eli". Lu b.l"uoll . 1962,14, su p pl, 68. 6. Polley, H , P., Bickel, W . H. Proc , May o Olin . 1951,26,273. 7. Hultman, E. S carul . J. clin, La b. I nuest, 19 67,19 , suppl, 94. 8. Kurlason, J. A cm pnYJ,·ol. rcemd. 1971, supp l, 35B. 9. Bergst r om, T., Harris, H. C ., Hultm an , E ., No r clesjti, 1.-0. Adu. ."p. M.d. 13;0[. 1971, It, 311. 10. D ubowi tz, V. ill D lsor de rs of Vo lunta r y M uscle (edited b y I. N . Wolto n); p, 239. L ondon, 1969. 1 1. I!dslrllm, L . J. muroto Sc i. 1970, 11, 537. 12. Bdstrom , L . ibid. p. 55!. l3 . Gollnic k, P. D . , I anuzzo , C. n., K ing, D . \'Q. Ad". expo Mod. Bioi. 1971, 11, 69 . 14. Sal rln, n., lllom qvist , G ., Mitch ell, J. H ., JohnslIn, R. L ., Ir. , Wildenthal, 1<., Chap ma n, C. II , Circulation, 1968, 37,38, s uppl, 7. 15. H ermansen, L., Waeh tlova, M. Ac!u physi"l. scan d, 1970, 79,16", 16. llerionod e, V ., Bergstr om , J., Hultm an, E. Scand, y, clin, Lab, Inu est, (in the press). 17. Hultm on, E. ibid. 1967, 19,209. l B. See Hultrnsn, E., Bergstrom, I ., R och-Norlund, A. E. Ad'll. expo Med. Blol. 1971,11, 273. 19. Ro ch-Norlund, A. E., Bergstrom, J., Castenfors, H., Hultman, E. A cta wed. scand, 1970, 187, 445.
References continu ed at foot ·of ne xt column
11, 1971
593 51yl I
Occasional Survey PERCUTANEOUS NEEDLE-BIOPSY OF SKELETAL MUSCLE IN DIAGNOSIS AND RESEARCH
R. II. T.
(])~======
EDWARDS
Department of Medicille" Royal Postgraduate Medical School, Hammersmith Hospital, London W.12
,
Percutaneous needle-biopsy of skeletal muscle has been successfully used [tH' several YCU1'S in other countries, but only rarely, if at all, in Great Britain. Complicntions-e-hrematoma Iormution or infection-e-are exceedingly uncommon. The technique is simple and relatively atraumatic and biopsy specimens can be taken in the ward or outpatient clinic for puthological or biochemical studies. Such biopsies will contribute information about muscle as a tissue and on the distribution of electrolytes between the cellular and extracellular spaces of the body. Since samplea can be obtained very rapidly, and repeatedly, the technique also makes it possible for muscle energy metabolism to be studied during exercise. The biopsy technique offers u practical means of studying the cellular basis of the symptoms of muscle weakness, excessive fatigue, and effort intolerance. .summClry
INTIl<>UUCTION
we in Great Britain are neglecting a potentially valuable diagnostic and research technique, which is widely used elsewhere. Recent reviews in British journals of current practice in soft-tissue biopsy techniques 1,2 omit any reference to percutaneous needle-biopsy of skeletal muscle. Yet it is over a century since Duchenne described the use of his needle (or" harpoon ") to obtain" living pathological anatomy" in patients with pseudohypertrophic muscular dystrophy.s.! Further, it is notable that he describes his biopsy needle as an instrument inoffensif (fig. 1) as its use was associated with little discomfort (in days before the introduction of adequate local antesthesia) or harm to the patient. With such an auspicious start it is perhaps surprising that this technique for musele biopsy was so little used subsequently. A percutaneous needle-biopsy technique was recently introduced by Bergstrom, G who modified an existing synovial biopsy needle G for the purpose. An estimated 3000-4000 biopsies of skeletal muscle have been taken with this needle (fig. 2) in Scandinavia during the past ten years. The present account will be limited to the technique of obtaining samples of muscle with this needle, together with a review of EVrDHN'l'LY
C ··'r"I:,:-_......
L - -_ _..:.:..:.:.. ..~
FiR'. l-Duchenne's biopsy needle.'
w
i
~===============~ - -
- - - 1JO mm
Fill, 2-·Del'gstrlim's biopsy needl".'
current methods of studying the biopsy sample and the diagnostic and research opportunities offered by the technique. Biopsy with Bel'IJstl'om Needle Local anesthetic (2-4 mi. 1 % lignocaine) is infiltrated into the skin and subcutaneous tissues down to the deep fascia, after first shaving and disinfecting the skin, The biopsy needle is introduced through a 4 mm, incision in the skin and subcutaneous tissue made with a Swan Morton blade (no. 11). The needle is advanced until the tip is about 3-5 em. below the skin surface and a sample of tissue is guillotined with the hollow cylinder and secured within tile barrel of the needle (fig. 2), which is then rapidly withdrawn from the muscle. Wh.en several biopsy specimens are to be taken over a short period of time as in some physiological studies, the skin incisions are made beforehand; these are covered with pads of sterile gauze which are removed in turn as specimens are required. The edges of the skin incisions are later opposed with a sterile adhesive material (' Steristrip '). They heal well and are generally invisible after a few months. With this "no-touch" technique there is no need for extensive towelling, "scrubbing up ", or the use of sterile gloves. This means that the technique can be used as a ward or clinic procedure, thus obviating the need for surgical expertise and operating-theatre facilities. The technique is remarkably atraumatic, Generally the most painful part of the procedure is the infiltration of the local aneesthetic into the skin and subcutaneous tissues. Though the biopsy is taken from muscle which has not been ansesthetised the sensation (described from personal experience of nine such biopsies) is of deep pressure together with a " visceral ,. feeling (as when the ulnar nerve at the elbow is accidentally knocked). The residual sensation is one of moderate muscle stiffness, such as would follow unaccustomed, strenuous exercise. This clears in a few days. Muscle function is little impaired by the procedure, as shown by the fact that subjects have been able to continue competing in strenuous cross-country ski races after several such biopsiea.t The simplicity of the operation, which takes less than three seconds, allows a number of specimens to be taken in quick succession, and it is possible to study changes occurring as the result of muscular contraction. The principal disadvantage of this technique is that it is "blind" and therefore applicable only to large
594
l'HH f.ANCln', SIlP'rBMOnR
II, 1971
muscles, but biopsy specimens have been taken without complication from the posterior part of the deltoid muscle." The vastus Iateralis of the quadriceps femoris muscle is preferred, as this muscle is free of important blood-vessels and nerves. Virtually all specimens have in the past been taken from the quadriceps, and the technique is likely to be of regular diagnostic use only when a disorder of this muscle is present. But proximal-muscle weakness is a common feature of many muscle diseases so that limitation to this anatomical site does not seriously affect the usefulness of the technique. Possible complications are htemorrhage and infection. In my experience, hrematcma formation occurred twice in a series of 100 biopsy specimens taken from normal subjects participating in research studies in Sweden. One of these was in a medical colleague, who later admitted that he was liable to heavy bleeding following dental extractions. He experienced severe muscle swelling and stiffness and complete recovery took four weeks. In another subject hsernatoma caused some swelling and muscle stiffness which persisted for two weeks. Both these subjects had four biopsies in quick succession. Infection of the skin incision was seen only once in the whole series. Infection of the deep tissues did not occur. Damage to nerves is of course a possibility, though this has never been seen in practice. The danger of heemorrhage into the muscle is slight but, should symptoms or signs appear) hematoma formation can be greatly reduced by immediately applying firm pressure to the biopsy site with a pad of sterile gauze. It is recommended that biopsies should not be performed on persons with a bleeding tendency or those on anticoagulant therapy. In the rare event of a diagnostic biopsy being indicated in a patient on anticoagulants, the biopsy should be delayed until the whole-blood clotting-time has been temporarily restored to normal following a period of intravenous heparin therapy. The Muscle Sample The biopsy technique yields a sample which generally has a wet-weight of 20-40 mg. For morphological or histochemical studies there is no need to weigh the sample, but for all other analyses accurate
weighing is essential. WIlen fresh muscle is needed (as for measurement of enzyme activity) the sample is placed on an elecrrobalance. Weight is plotted against time for a short period to allow for the effect of water evaporation by extrapolation to zero-time." If metabolites are to be measured it is essential that metabolism be arrested as quickly us possible, and this is done by freezing the sample in liquid • Freon' at --I50°c or liquid nitrogen. Connective tissue and blood are removed by dissecting the frozen sample in a cryostat 8 or, more conveniently, by dissecting the freeze-dried tissue at room temperature, The sample is weighed accurately after removal of this extraneous material.
,Fig. 3-Needle-biopsy sample showing normal differentiation of fibre types by staining for myosin A.T.P.-ase (see text).
Fig. 4-Electron microphotograph of a needle-biopsy sample of muscle from a patient with polymyositis, showing atrophy of myofibrils.
MORPHOT,OGICAI, AND HISTOClll1MIc:AL Sl'Ulllf(S
The biopsy technique provides samples which nrc suitable for light microscopy (after stainlng with lucmatoxylin and eosin) and for hiarochemicnl lnvestigations using cryostat sectlona.!" Several muscleenzymes have been studied using conventlonal histochemical methods. It is possible in this way to identify different types of muscle-fibre, Stainlng nil' myosin A:r.l'.-llse (fig. 3) allows a distinction to be made between fast (white) fibres, which pOSHCSS a high activity of this enzyme, from slow (red) fibres with a low activity. This differentiadon is of Interest in view of recent accounts of selective degeneration of nne fibre type (e.g., fast fibres in parkinaonism 11 and slow fibres in disuse atrophy due to joint injury). 11 Samples for electron microscopy arc fixed immediately in 3%) glutaraldehyde and subsequently treated by conventional techniques. Structure is well preserved and abnormalities can be explored in detail. Considerable enlargement and alteration of the form of the cristee of mitochondria have been found in human skeletal muscle biopsied immediately after severe exercise. 13 A recent finding of considerable physiological interest is that improved physical fitness is not associated with increased capillary density in skeletal muscle. H , 18 This is contrary to what had previously been supposed. CHEMICAL ANALYSIS OF
NEEDLE~BIOPSY
SAMPLES
Electrolytes The needle-biopsy technique was developed by
595
n IH I,ANCHT, SIlPTEMnnu 11) 1971
Bergstrom for measurement of muscle-electrolytes. Skeletal muscle represents about 40 e/(, of the bodyweight and it is the largest and most easily accessible fra ction of th e in traccllulur-Iluid compartment. As u seq ucl to the interest in water and electrolyte balance in the extracellular fluid following the introdu ction of flame photometry in the 1950s) techniques were developed by Bergstrom • for the study of the much larger intra cellular- fluid compartment. These were used for investig ation s on patients with disturbances of fluid and electrolyte balance. Neutronactivation analysis was employed for assaying certain ions. Atomic absorbtion flume photometry has also been used to measure concentrations of sodium; potassium, chloride, and rnagncaium.!" lilll.'I'J{Y Stores and lvIccabolircs The largest local energy store in muscle is provided by glycogen, which conulhures 1-·l ·5 % of the muaclc's we t-wei gh t." There have been several biops y studies of the influence of dlet und heavy exercise 011 glycogenolysis lind rcsynchcsia. !" There nrc changes in various types of diseuse-for instance, unt reated juvenile diabetics have a low mus cle-glycogen which is res tored to normal by insulin therapy. 10 Muscle-glycogen is low 1I180 during acu te oliguric renal Iailu rc. : " S nullea have also been made of the con centration s in muscle of the energy-rich compounds 1\.'1'.1'. and phos~h()rylcrelltinc (r.c.) .• . 0, 0 I Even in fatiguing ClCcr<.:ISC depiction of A/ r .t'. is limited because it is mai ntained lit the expe nse of 1'.<:., the level of which ca n fall almost to zel'o.B.ll n L ittle is known as to h ow disease atlcc ts these hi gh-energy compounds in mu scle, but recent eviden ce suggests tha t the ir amounts can be increased significa ntly by physical training. 2 ' Several glycolytic intermediates and co-factors have been studied in human muscle obtained by the needlebiopsy technique.B.D,2 ~,25 The aim has been to elucidate the factors controlling the energy-supplying mechanisms in working muscle. 3',5' cyclic A.M.P. has been measured in a needle-biopsy sample by Dr. B. Tulloch (Royal Postgraduate Medical School) by a recently developed ana lytical method. 2 6 Enzymes Succinic dehydrogenase has been measured to indicate the oxidati ve activity of the mitochondria. 2. The activity of this enzyme was found to be increased followin g physical tr aining. S& No reduction in activit y was found in patients with dystrophia myotonica.w The activity of muscle lactate-dehydrogenase has been found to be increased following prolonged exercise in normal subjects,"! and to be present with an ab norma l isoenzyme str ucture in patients with chronic alcoholism. v' Information can also be gained hy studying the metabolite balance for reactions catalyscd by particular enzymes. F or example, changes in the proportions of fr uctose 1: (i-dlpho sphatc lind fructose 6-phospilate in isometric exercise sugg est that .the activity of phosphofr uctokinase may be imp aired by mu scular fatigue/ particularly when mu scle temperature is increased. 33 Lipids Recent biochemical analyses have shown a significant decrease in musc1e-triglycerides as a result of pro-
longed severe exercise. "1 Electron micrograph s have not been sufficiently "quantitative" to show a corresponding reduction in the Dumber 01' size of fat droplets in muscle-cells even when the glycog en granules are much depleted. w O ther Analyses The influence of arninoacid solutions on the catabolic nitrogen metabolism after major surgery 30 and in ul'remia' 7 have been st u d ied by meas uring the incorporation of the isotope toN into blood and muscle proteins following the infusion of 15Nlabelled glycine. Mass spectrometry was used to assay the lGN in needle-biopsy samples. The effect of intermittent dialysis on the arsenic content of m uscle has been studied by neutron-activation analysis of biops y samples. aH FUTUR ll O\'l'OR'l'UNt1'I[lS
Wider usc of the needle- biopsy t echnique cou ld be of value for explo iting histological and hi stochemical methods of di agno sis in p at ients with muscle weak ness. Further, the technique provides an opportunity to remedy the present luck of direct information, largely attributable to difficulty in obtaining suitable sam p les. w concerning the biochemical basis of impaired muscle function in disease. Skeletal muscle, like many specialised tissu es in th e body, r esponds to the many form s of dise ase on ly in a limited number of ways, and consequently muscle biopsies ar c more likely in the Immediate future to provide information about basic mechani sms than to indicate the retiology of particular diseases. However, there is reason to hope that wid er use of the muscle-biopsy techn iqu e will ultimately provide a cl ue as to why these basic mechanisms are disturbed and thereby elucidate the common sym ptoms of muscular weakness and fatigue. I thank Mrs. Caroline Maunder of the Department of Histochemistry, Royal Postgraduate Medical School, for the Itistochemi cal and electron rnicroacopical studies illustrated here; and the Wellcome Trust and the Muscular Dystrophy Group of G reat Britain for supp ort . F ig. 2 is .reproduce d by permission. REFERENCES 1- Turner Warwick, R., T urner W arwi ck, M . 8 ,·. .... d. J. 19 70, Hi, 149. 2 . G ear, M. W. L . Br.]. H Ofp. M,d. 19 69,2, 17013. Duchen ne,·G . B. A rcll. ·g.". M.a. 1868, 11, l7 9. 4. Nntrass, F. J. in: Resea rch in Muscu lar D ystrophy (edit ed by th e M usculor D yst rophy Group); p .5. Lon don, 196 B. 5. Bergatrbm, J. S eal/d.]. eli". Lu b.l"uoll . 1962,14, su p pl, 68. 6. Polley, H , P., Bickel, W . H. Proc , May o Olin . 1951,26,273. 7. Hultman, E. S carul . J. clin, La b. I nuest, 19 67,19 , suppl, 94. 8. Kurlason, J. A cm pnYJ,·ol. rcemd. 1971, supp l, 35B. 9. Bergst r om, T., Harris, H. C ., Hultm an , E ., No r clesjti, 1.-0. Adu. ."p. M.d. 13;0[. 1971, It, 311. 10. D ubowi tz, V. ill D lsor de rs of Vo lunta r y M uscle (edited b y I. N . Wolto n); p, 239. L ondon, 1969. 1 1. I!dslrllm, L . J. muroto Sc i. 1970, 11, 537. 12. Bdstrom , L . ibid. p. 55!. l3 . Gollnic k, P. D . , I anuzzo , C. n., K ing, D . \'Q. Ad". expo Mod. Bioi. 1971, 11, 69 . 14. Sal rln, n., lllom qvist , G ., Mitch ell, J. H ., JohnslIn, R. L ., Ir. , Wildenthal, 1<., Chap ma n, C. II , Circulation, 1968, 37,38, s uppl, 7. 15. H ermansen, L., Waeh tlova, M. Ac!u physi"l. scan d, 1970, 79,16", 16. llerionod e, V ., Bergstr om , J., Hultm an, E. Scand, y, clin, Lab, Inu est, (in the press). 17. Hultm on, E. ibid. 1967, 19,209. l B. See Hultrnsn, E., Bergstrom, I ., R och-Norlund, A. E. Ad'll. expo Med. Blol. 1971,11, 273. 19. Ro ch-Norlund, A. E., Bergstrom, J., Castenfors, H., Hultman, E. A cta wed. scand, 1970, 187, 445.
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