Gut, 1986, 27, SI, 25-39

Muscle function and nutritionK N JEEJEEBHOY

From the Department of Medicine, University of Toronto, Toronto General Hospital, Toronto, Ontario,Canada

Wasting of muscle and a negative nitrogen balanceare obvious effects of malnutrition and have led tothe use of anthropometric measurements and nitro-gen balance for assessing the extent of malnutrition.A positive nitrogen balance and an increase in limbmuscle circumference are believed to be indices ofthe beneficial effects of nutritional support. Inexperiments with growing rats and young childrennitrogen retention and growth are recognised to bethe desirable effects of optimal nutritional intake.This concept has been applied to malnourished adulthumans (non-growing) who have been consideredto be potentially able to "regrow" the lost tissue.While it is true that patients receiving long term

(more than six months) home total parenteralnutrition gain body weight and nitrogen over manymonths and years of observation, this process is notseen during shorter (less than 40 days) nutritionalintervention given in hospital.1 Despite adequateintakes of nitrogen and calories little or no increasein total body nitrogen is seen in a variety of patientsreceiving total parenteral nutrition in hospital overseveral weeks.2-7 Despite the absent or very modestgain in nitrogen nutritional support does seem toimprove outcome by reducing complications andmortality after a period of support so short that bodycomposition is hardly changed. Young et al showedthat although amino acids and amino acids pluscalories both resulted in equivalent sparing of bodynitrogen, amino acids plus calories was associatedwith ruicker wound healing and fewer complica-tions.-Thus the outcome and body composition data

suggest that the reversal of the adverse effects ofmalnutrition is not based on improvement of thetraditional variables of nutrition, such as gain inbody nitrogen, or a demonstrable increase in musclemass or plasma proteins.8 This discrepancy is furthersupported by the observation that global clinicalassessment is at least equivalent to, and in somerespects better than, individual objective traditionalmeasurements of nutritional state in predictingoutcome.9Correspondence to: Professor K N Jeejeebhoy, Room 6352, Medical SciencesBuilding, University of Toronto, Toronto, Ontario, Canada M5S 1A8.

On the basis of the foregoing evidence, there aregrounds for suspecting that functional abnormalitiesin adults may not be the result of simple loss of leantissue and may recover before such lean tissue isregained.One of the major organ systems of the human

body is the musculoskeletal system, and it istherefore important to determine the effect ofmalnutrition on the musculoskeletal system. Pre-vious studies of muscle function have been largelyrelated to the examination of fatigue, myopathy,and endocrine-metabolic abnormalities.1t 11 Thisreview will discuss the effect of nutrition on skeletalmuscle function in relation to the effects of feedingand fasting in normal subjects, in patients withcritical illness, and also in a rat model of malnutri-tion.

Techniques for investigating the effect of nutrition onmuscle function

MUSCLE FUNCTION TESTSThe contraction-relaxation characteristics and en-durance properties of the adductor pollicis muscle inman and the gastrocnemius muscle in rats have beenstudied.12 13

Human studiesSupramaximal ulnar nerve stimulation was performedwith square wave pulses for 60-70 microseconds(and surface EMG recordings made), at frequenciesincreasing from 10 Hz to 100 Hz, for one to twoseconds at a time, and the force of contractionrecorded. Then the maximal rate of muscle relaxationwas noted after stimulation at 30 Hz for two to threeseconds. Finally, the adductor pollicis muscle wasstimulated continuously at 20 Hz for 30 seconds, andthe degree of fatigue, judged by a fall in force ofcontraction with time, was noted over this period.Recently, the clinical protocol has been shortened toa sequence of 10, 20, and 50 Hz stimuli only, and therelaxation rate is observed during a one to twoseconds 20 Hz stimulation. The F1(:F20 and Fl(:F50ratios were found to give the same sorts of results asthe F1(:F11() ratio.

25

26

Anim1al stlidiesAnimals anaesthetised with barbiturates had thegastrocnemius and soleus muscles freed, keepingtheir blood supply intact and isolating their commonnerve supply, the sciatic nerve. The body and hindlimbs of the animal were immersed in modifiedLiley's solution kept at 37°C. The same measure-

ments were made as those made in the humanstudies but at frequencies from 0-5 to 200 Hz. Theeffects of stimulating the sciatic nerve on thecontraction characteristics of the gastrocnemius andsoleus muscles were noted.

MUSCILE BIOPSY STUDIES

Hiuman studiesMuscle biopsy specimens were obtained from thegastrocnemius in morbidly obese subjects, firstlyfrom those on a weight maintaining diet and thenafter two weeks from those on a 400 kcal/day diet. 14These were taken immediately after the musclefunction tests reported here had been performed.

Animal studiesThe biopsy specimens were taken from the contra-lateral gastrocnemius at the time of the musclefunction tests.

BIOPSY MEASUREMENTSBiopsy specimens from animals and humans were

analysed for total water content; total sodium,potassium, chloride, calcium, phosphate and mag-

nesium content (and the intracellular concentrationsof these chemicals were also calculated); activitiesof phosphofructokinase (PFK), succinate dehy-drogenase (SDH), and hydroxyacyl Co-A dehy-drogenase (ACDH); concentrations of adenosinetriphosphate (ATP), adenosine diphosphate (ADP),adenosine monophosphate (AMP), creatinephosphate (CP); and pyruvate and lactate values.Histochemical treatment for myosin ATPase andsodium and potassium ATPase and electron micro-scopic examination were also performed to look forchanges in fibre type. Details of these studies havebeen published elsewhere.'3 14

The muscle electrolytes were measured using a

modification of the methods described by Grahamet all-5 for human muscle. Muscle chloride contentwas determined by a modification of the methodof Schales and Schales.'6 The sodium, potassium,calcium and magnesium contents of the musclebiopsy specimen were determined by atomic absorp-tion spectrophotometry. Muscle phosphate was

determined by a colorimetric method.'7The determination of extracellular and intracellu-

lar water was based on the chloride method. '8

Chloride is freely diffusible across the skeletalmuscle fibre membrane at rest and is distributed

Jeejeebhoy

according to the Nernst equation." 2" Assuming aconstant membrane potential of 85 mV, the Cll.:Cl1ratio calculated from the Nernst equation is 24:1. Ifthe total water and chloride of the muscle tissue andthe extracellular concentration of the chloride(obtained by correcting the plasma chloride concen-tration for a Donnan factor and a factor for plasmawater21) are known extracellular and intracellularelectrolyte concentrations can be calculated.As the validity of this calculation depends on the

assumption that the muscle membrane potential was85 mV, we also measured the membrane potentialdirectly using intracellular electrodes in control rats,those fasted for five days, and those fed a hypo-caloric diet for 21 days. The measurements weremade in the muscles of livinp anaesthetised rats, asdescribed by Charlton et a12- in 1(-20 muscle fibresin the first and second layers of the muscle, using amicroelectrode filled with potassium chloride of5-10 megohms resistance and low (<5 mV) tippotential. The exposed muscles were immersed inmodified Liley's solution at 37°C during measure-ment. The results showed that hypocaloric feedingresulted in only minimal changes to the membranepotential, such that there was no appreciable effecton the calculated value of intracellular electrolytes.The details of this process have been publishedelsewhere. '3

OBJECTIVE MEASUREMENTS OF NUTRITIONAISTATENutritional state is a term used to denote lean bodymass, as determined by anthropometric measure-ments, determination of serum protein (albuminand transferrin), measurement of total body nitrogen,and total body potassium, creatinine-height index,and observations of delayed cutaneous hyper-sensitivity (DCH) to recall antigens.

Studies of muscle function in models of puremalnutrition in manDEFINITION OF MALNUTRITIONIntake of a diet sufficient to meet or exceedindividual needs will keep the composition andfunction of otherwise healthy subjects within thenormal range. This equilibrium is disturbed by threeprocesses: decreased intake; increased require-ments; and changed use, all of which preventnutrients from being used for tissue repair. Whenthe above mentioned states of disequilibrium occurthen loss of body tissue ensues. Not all bodyconstituents, however, are lost at the same rate.Proteins are the least dispensable and most import-ant of the body components; hence the adverseeffects of insufficient food intake are reduced bycompensatory mechanisms that protect body pro-

Muscle fuinction and nutrition

teins at the expense of body fat. Even thoughstarvation or undernutrition result in a loss of bothfat and protein, the loss of protein is minimised byreducing the need to use it as a source of energy.23This is effected by mobilising fat and enhancing fatoxidation as the principal source of energy. Unfortu-nately, protein wasting continues and rapidlyaccelerates after fat stores have been consumed.

Losses of body fat and protein result in a fall inbody weight, reduced fat fold thickness, andreduced muscle bulk. Reduced muscle bulk will beseen as thin arms and legs and a reduced excretionof creatinine in the urine. Weight loss may bemasked by concomitant retention of fluid.24 25 Lackof nutrients will ultimately reduce weight, skin foldthickness, and arm muscle circumference, all ofwhich have been advocated for assessing the nutri-tional state - and that goes for everybody.

Unfortunately, body wasting occurs as the resultof several factors not related to the nutritionaldisequilibrium described above - for example,inactivity will reduce muscle protein synthesis andcause wasting.2" Trauma and cancer likewise inducewasting and loss of lean tissue. Thus malnutritiondefined on the basis of lean body mass or factorsbased on it (such as body potassium and nitrogen),will not distinguish those instances caused by a truelack of nutrients from those caused by other formsof injury and illness. Furthermore, such a definitiondoes not make allowance for body and especiallymuscle function being reduced independently ofwasting and change in body composition.

Because of these factors, we have defined musclemalnutrition as the presence of abnormalities thatare seen when nutrients are withdrawn and whichare correctable by refeeding. Such changes occurindependently of body composition. More import-antly, after hypocaloric feeding the ultrastructure ofmuscle has been observed to change, showing fibreatrophy at a time when total body composition wasstill normal. 14 27

In clinical terms such a definition is relevantbecause it alerts the clinician to an abnormality thatcan be positively and acutely influenced by refeed-ing rather than being seen as an untreatable one.

EFFECT OF FASTING AND REFEEDING OR REDUCEDNUTRIENT INTAKE ON MUSCI E FUNCTIONFasting in the morbidly obeseThe object of this study was to observe the effect ofpure fasting in obese but otherwise normal subjects.In six morbidly obese subjects27 muscle function wasmeasured before and after two weeks of a 400kcal/day diet, and again after an additional twoweeks of fasting. After two weeks of refeeding thesemeasurements were repeated.

27

Refeeding patients with severe malnutrition caused byanorexia nervosaSix severely depleted patients with primary anorexianervosa, with a mean loss of 23% of body weight,were studied before and after four and eight weeksof refeeding.28 Muscle function tests and objectivemeasurements of nutritional state were performedat these intervals.

MUSCLE FUNCTION TESTSThere were important changes in three variables ofmuscle function in malnourished humans andanimals compared with those of normal controls(Fig. 1). In both humans and animals hypocaloricdieting and fasting and the malnutrition associatedwith anorexia nervosa had a uniform effect,irrespective of species or clinical setting.

Change in force-frequency curveIn normal humans and unstarved obese patientsthere was a rise in the force of muscle contraction asthe frequency of stimulation increased from 10 Hz to100 Hz. The maximum force was attained at 50 Hz.Expressed as a percentage of this maximumattained, the force developed at 10 Hz was 29%(Fig. Ia).

Hypocalorically fed and fasted humans and un-treated patients with anorexia had a decrease in theincrement of the force at higher stimulation frequen-cies, with maintenance of force at lower frequencies.Thus the ratio or percentage of the latter, in relationto the maximum attainable (which was less), in-creased in these instances. The force at 10 Hz wasabout 48% of the maximum in these malnourishedstates. This value was significantly higher than thatof controls (Fig. la).

Change in muscle relaxation rateIn normal human subjects the muscle relaxation rateat 30 Hz was 9-6% force loss/10 milliseconds.The relaxation rate was significantly slower with

fasting (766% force loss/10 milliseconds and inuntreated anorectic patients (6.6% force loss/10milliseconds).

FatigueIn controls the muscle scarcely lost power whenstimulated continuously for 30 seconds (3-5(Yo forceloss/30 seconds).

In contrast, there was a significant increase inmuscle fatiguability after fasting (13.7%Yo force loss/30 seconds) and in the untreated anorectic patient(18.6% force loss/30 seconds).

Refeeding the starved obese patient and theanorectic resulted in the disappearance of musclefunction abnormalities within two and four weeks,respectively. 14 28

Jeejeebhlov

Hunan Rat Human Rat Human Rat(i) F10 /Fmax (1.) ® MRR (1.foreloss/10ms) ( Fatigue (Mforceloss)

/ 30seconds / 5secondsFig. 1 Contraction characteristics ofhuman adductorpollicis and rat gastrocnemius muscles under conditions ofnutritional deprivation. Open bars= reported normals. Wide diagonal hatched=obese patients after two weeks offasting.Shaded=anoretic patients at baseline. Close diagonal hatched=rats after 21 days ofhypocaloric feeding. MRR=-maximalrelaxation rate. *p<005, **p<0.025, ***p<0.01, ****p<0.001. (AfterJPEN 1985; 9: 415-21.)

CORREILATION OF OBJECTIVE MEASUREMENTS OFBODY COMPOSITION WITH MUSCLE FUNCTIONThe fasted obese human did not lose significantamounts of total body potassium or total bodynitrogen, nor was there a significant change increatinine-height index at a time when abnormalitiesof muscle function were obvious (Table 1). Con-

Table 1 Changes in standard variables

versely, during refeeding these subjects rapidly lostmuscle fatiguability and regained normalcontraction-relaxation characteristics at a time whenthere was no significant increase in lean bodycomponents or in body weight.

In the anorectic patient gross muscle fatigue andabnormal function were present when first seen. At

of nutritional assessment during period of abnormal muscle function

Total body Total body Creatininie-nitrogen (kg) potassiumn (g) height intdex (%)

Albumin(gIdl)

7fotal iro,t biti(lidigcapac-ity (Igdldl)

Obesity study:Normal (predicted7")Baseline

Fasted

1-52 (0-()5)1 56 (0-02)(n =4)1-38 (0()5)

.11So%o

96 (14)129 (8)(n =4)III (11)1-14 0%1

10)135 (7)(n=6)115 (8)1- 14-81

None of these changes are significant.

Anorexia study:Predicted7'

Baseline

Four weeks refed

Eight weeks refed

1 70 (0(08)(n=5)1 21 (0(11)(n=5)1-33 (0110))[+9 9%11 37 (0.10)*[+13-2%]

61 (7)(n=5)73 (8)*1+19-7%I81 (7)*1+32-8%]

49.7 (7.4)(n=5)64-9 (3.4)1+30.61670 (7.7)1+34.71

*p<O)0)5 compared with hascline valuc. None of the other changes arc significant.(From JPEN 1985; 9: 415-21.)

3. 5-5()3-9 (0-2)(n =6)3-9 (0-1)

25(1-4(X)268 (21)(n =6)245 (8)

4.0 ((()1)(n =6)4-4 (0(3)

4*4 (0.3)

283 (49)(n =6)333 (63)

384 (76)

28

Muscle function and nutrition

that time these patients had normal plasma proteins.Initially they had pronounced loss of lean body massand of total body nitrogen and potassium. Whenrefed, muscle fatigue disappeared within fourweeks, and all functional abnormalities were re-stored by eight weeks of refeeding, at which time thecreatinine-height index was still very low at 67% andtotal body nitrogen had risen by only 13%. Interest-ingly, the total body potassium had risen at fourweeks by only 19-6% and at eight weeks by 32-7% inpatients who, based on their creatinine-height in-dices, had lost 50% of their muscle mass. Thus thetotal body potassium was still well below the normalexpected for their height (Table 1). Body fat, whichis regarded as "average" for a woman at 24%,29 rosefrom 14.0 (SE 0-5%) at baseline to 17 1 (SE 1.0%)at eight weeks.

Despite an incomplete return to normal bodycomposition clinically these patients had restoredtheir ability to exercise with normal muscle function.

Studies of muscle function in models of malnutritionin the rat

To determine whether these abnormalities could bereproduced in animals by hypocaloric feeding andreversed by refeeding a rat model was studied. Theeffects of short and long term changes were alsomeasured, and the muscle under study was biopsied.Muscle function tests and muscle biopsies were

performed in rats initially weighing about 250 g(eight weeks old). They were studied as follows:

(i) Nine control rats eating purina laboratorychow unrestrainedly, eight rats fasted for twodays, and five rats fasted for five days.

(ii) Six control rats eating purina laboratory chowunrestrainedly and six hypocalorically fedanimals given only 25% of the food eaten byits pair fed control.

MUSCLE FUNCTION TESTSThere were significant changes in three variables ofmuscle function in experimental animals comparedwith those of normal controls (Fig. 1). Hypocaloricdieting and fasting had the same effect as in thehuman.

Change in force-frequency curveIn normal rats there was a rise in the force of musclecontraction as the frequency of stimulation in-creased from 10 Hz to 100 Hz. The maximum forcewas attained at 100 Hz. Expressed as a percentage ofthis maximum attained, the force developed at 10Hz was 29% (Fig. la).

Hypocaloric dieting and fasting in rats resulted ina decrease in the increment of force being exerted at

higher stimulation frequencies, with maintenance offorce at lower stimulation frequencies. Thus theratio or percentage of the latter, in relation to themaximum attainable, which was less, increased inthese instances. In malnourished rats it was 40% ofthe maximum and was significantly higher than thatof controls (Fig. la).

Change in muscle relaxation rateIn normal rats the muscle relaxation rate at 100 Hzwas 36-3% force loss/10 milliseconds (Fig. lb).

In rats five days of fasting slowed the relaxation to19-3% force loss/10 milliseconds and 21 days ofhypocaloric dieting to 108% force loss/10 milli-seconds (Fig. lb).

FatigueWhen stimulated continuously for 30 seconds thecontrol rat lost 20-8% force/five seconds (Fig. 1c).

Fasted rats (five days) and hypocalorically fedanimals showed double the loss of force on sustainedstimulus (45% force loss/five seconds) (Fig. 1c).

EFFECT OF REFEEDING ON MUSCLE FUNCTION OF

THE RAT

Rats weighing 250 g were allocated to three groups,one control and two pair, fed 25% of the controlintake. After three weeks the control and onehypocaloric group were studied, as described above.The third hypocaloric group was refed freely for twoweeks and studied. As indicated above the force at10 Hz expressed as a percentage of that at 100 Hzwas higher in the hypocaloric group and the relaxa-tion rate slower. On refeeding, these changes werereversed and the refed group was not different fromthe control group. 30

CHANGES IN THE MUSCLE BIOPSY ON FASTING ORHYPOCALORIC FEEDINGFibre typeIn obese patients fasting resulted in type II fibreatrophy. In animals two to five days' fasting resultedin an increase of slow twitch oxidative fibres (typeI), but prolonged hypocaloric dieting resulted in theappearance of fibres depleted in both myosin andsodium and potassium ATPase, a fibre type notnormally seen (Atwood, Russell, and Jeejeebhoy,unpublished data). Despite these very different fibretype patterns the muscle function abnormalities andfatiguability were similar to those seen in nutritionaldeprivation. In human studies specific Z banddegeneration was also noted, with preservation of Aand I bands (Fig. 2).

Electrolyte abnormalitiesThe most striking finding during hypocaloric dieting

29)

Jeejeebhoy

I V

Human Ra model

LI Baseline. control Fd two

Hypocloric diet FastedL cl~~~~~ays

Fig. 2 Biopsy specimen ofhuman gastrocnemius muscleafter two weeks ofhypocaloric dieting showing atrophicfibres with degeneration ofZ bands but preservation ofand especially A bands. (From Am J Clin Nutr 1984; 39:503-15.)

and fasting in both humans and rats was an increasein the total water content, mainly extracellular, andan increase in the intracellular concentration ofmuscle calcium in both human and rat studies(Fig. 3). In contrast, total potassium, magnesium,chloride and phosphate concentrations remainednormal.

Metabolite abnormalitiesIn both rats and humans the phosphocreatine:ATPratio fell while the ATP value remained normal.Muscle lactate rose in hypocalorically fed rats. Asmuscle pH is closely linked to lactate activity31 a fallin pH is likely. The formula of Sahlin et al3lpredicted that the pH had fallen from 7-19 to 6-97.Supporting our findings are studies by Jacobs et al(Fig. 4).3

Muscle enzyme contentPFK fell with short term fasting while SDH and

Fig. 3 Concentrations of intracellular calcium in humanand rat gastrocnemii after nutritional deprivation. *p<o005,**p<0 001. (From JPEN 1985; 9: 415-21.)

ACDH remained normal or rose, suggesting achange to more oxidative fibres with early starva-tion. Prolonged hypocaloric feeding, however, wasfollowed by a reduction in PFK and SDH in humansand rats, and ACDH in the rat (Fig. 5).

Effect of non-nutritional factors on muscle function

AGE AND SEXThere is a weak (r2=0-09) but significant (p<0l001)correlation between age and the ratio of the force at10 Hz with that at 50 Hz (Fio:F50). The maximalrelaxation rate did not correlate with age.33

EFFECT OF RENAL FAILURE, CHRONICOBSTRUCTIVE LUNG DISEASE, STEROIDADMINISTRATION AND MAJOR SURGICALPROCEDURESNeither renal failure per se, nor peritoneal dialysis,nor haemodialysis changed the F10:F50 ratio or themaximal relaxation rate.34 The same was true ofchronic obstructive lung disease.35 Steroid adminis-tration for Crohn's disease in doses of 20 mg/day forat least three weeks also did not change the F10:F50ratio or the maximal relaxation rate from the control

-

.-

Bl@sSEE

3()

.t

Mluscle function1 (It(l militritioti

Adenosine triphosphate/Adenosine diphosphate

[I

rT80

70

60

50

40

30

UP

I .**

IControt Fasted two

E Fasted five Hypocaloricdas2ldy

Fig. 4 Ratios of A TP:ADP and lactate:pyruvate in ratmodel of nutritional deprivation. *p<0.05, * *p<O0O1.(Frot JPEN1985; 9: 415-21.)

Phosphofnjolkinae Succxtte l-ldtcycyl CoA- -

Rot Human Rot Hrnon Ra Hurnc

10 Ctol * HypocolDric diet

Fig. 5 Enzyme concentrations ofhuman and ratgastrocnemius muscles after hypocaloric diets. *p<0Q05,**p<0.02, ***p<0.001. (From JPEN 1985; 9: 415-21.)

value.33 Patients studied in the recovery roomimmediately after a laparotomy or aortic surgeryalso had normal Fl(:F50, Fl(:F20, ratios and maximalrelaxation rate.33

EFFECT OF MAJOR TRAUMA WITHOUT SEPSIS

Major trauma was associated with a slight fall inmaximal relaxation rate when studied in theemergency room. Next day and a week later theF1(:F50 and F1(:F21( ratios and maximal relaxationrate were normal despite a severely negative nitro-gen balance not compensated for b' an increasednutrient intake or special support.3

EFFECT OF SEPSIS

Severe sepsis in patients who were eating wellbefore the episode or those receiving parenteralnutrition when studied (Stoner index >1036) wasassociated with a noticeable but slight rise in F1(:F50ratio but a normal maximal relaxation rate. Thechanges were considerably less than those seen inpatients with a caloric intake less than 90% of theirmeasured resting metabolic rate. These subjects alsohad an appreciably slower maximal relaxationrate.

EFFECT OF REDUCED CALORIE INTAKE AND

NUTRITIONAL SUPPORT IN PATIENTS WITH SEPSISAND GASTROINTESTINAL DISEASEProtocol of studyPatients admitted to a general surgical service werestudied before and sequentially at weekly intervalsduring their treatment with parenteral nutrition.They were assessed for sepsis score,36 musclefunction, dietary and parenteral nutrient intake,metabolic rate by indirect calorimetry, and objectivenutritional assessment, as defined above.

SENSITIVITY AND SPECIFICITY OF MUSCLE

FUNCTION IN DETECTING REDUCED CAL ORIE INTAKE

Patients, irrespective of their clinical state, were

divided at the time of admission into those notmalabsorbing and taking calories >=90% of theirresting metabolic rate, and those with less than thatintake and with malabsorption. A receiver operatorcharacteristic (ROC) curve was drawn to determinethe best sensitivity and specificity of the Fl(:F5( ratio

FI(:F20 ratio, maximal relaxation rate, and maximalforce at 50 Hz in separating these two states ofnutrition. The F10:F501 separated these two states at a

an ratio of 0-4 (Flo, above 40% of F50,) with a sensitivityof 0-83 and a specificity of 0 98. The maximalrelaxation rate likewise separated these groups at a

value of 10% of maximum force lost/10 milli-seconds with a sensitivity of 0 80 and a specificity of0 93%. During this study several healthy women fed

14

12'

10'

8'

4

2

0

31

32

hypocaloric diets were also studied. They had beenon a self administered diet, which was not accuratelyadhered to for variable periods of time. Whencontrols were added to the patient data the sensitiv-ity and specificity remained unchanged, but whendata from such dieters were added the sensitivity fellto 0-6 for both the Fl0:F5o ratio and maximalrelaxation rate without affecting specificity. Inshort, periodic dieting does not affect musclefunction to the same degree as poor intake withmalabsorption. Even under these circumstances,however, the specificity remains high.33

EFFECT OF PARENTERAL NUTRITION

Parenteral nutrition appreciably reduced the F1(:F50and F1o:F20 ratios and increased maximal relaxationrate within two to three weeks. There was also an

appreciable rise in the force of muscular contractionwith nutritional support without an increase in thearm muscle circumference, triceps skin fold thick-ness, or the serum albumin concentration. Thusnutrition increased the force of contraction withoutincreasing the muscle mass or albumin concentra-tions.There was a linear fall in F1o:F5( ratio and a rise in

maximal relaxation rate and force of contractionwith increased duration of parenteral nutrition.Thus there seems to be a progressive effect ofnutritional support with time.

CHANGES IN BODY COMPOSITION AND MUSCLEFUNCTION BY REDUCING NUTRIENT INTAKEThese studies indicate that there is a lack of relationbetween changes in total body nitrogen or potassium(measures of lean body mass) and muscle function.It was possible to improve muscle force and othervariables in patients without affecting muscle mass,

fat stores, and albumin concentration. It is clear thatthe loss or restoration of lean body mass is notessential for the occurrence of correspondingchanges in muscle contraction-relaxation character-istics, force, and endurance properties. Our findingsare consistent with the somewhat similar dataconcerning morbidity and mortality mentioned ear-lier. Thus there is a need for questioning whetherthe proof of malnutrition should depend on changesin lean body mass, and conversely, whether therestoration of lean body mass and improved nitrogenbalance constitute the gold standards for goodnutritional support. Our findings indicate that thefailure to restore body nitrogen, observed in earlierstudies, does not negate the fact that such supportmay have restored function.The findings of the changes in the muscle biopsy

specimens in patients and animals suggest thatskeletal muscle function may be changed because of

Jeejeebhov

factors affecting the energy state of the muscle,which affects both the contraction-relaxation charac-teristics of muscle and inhibits calcium efflux acrossthe sarcolemma. Calcium efflux may promotemuscle proteolysis and injury.

Clinical importance of changed muscle function

FATIGUE AT 30 HZ STIMULATION (HUMAN) AND100 HZ (ANIMALS)

There is an inability to sustain tetanic force whencompared with the control for 30 seconds (human)and five seconds (animal) at a tension which is closeto 86% of the maximum in the human14 and 100% inanimals. 13 This fatigue in the human studies is at lowfrequency stimulation and has been shown, insimilar studies with the adductor pollicis by Bigland-Ritchie,7 to be caused by muscular factors and notby a failure of nerve conduction.Even though the animal muscles fatigued at 100

Hz, at which frequency of stimulation nerve conduc-tion may also be affected, it has been shown that anidentical pattern of fatigue occurs in curarised ratextensor digitorum longus,38 which is comparable(type II fibres) with the gastrocnemius seen in ourdata. 13 Studies by Luttgau39 have shown that unlessthe muscle contracts, continued stimulation does notaffect the action potential during tetanus. Hence itwould be reasonable to conclude that in human andanimal studies there is enhanced muscular fatigue inthe underfed human and rat, which can be reversedby refeeding. 12 27 28

SLOW RELAXATION RATEOf greater interest is that in the above mentionedstudies the relaxation rate is slowed when measuredafter brief tetanus of only one to two seconds. Onepossibility is that the type II fibre atrophy that hasbeen seen'4 could reduce the relaxation rate becausethe remaining type I fibres have a slower relaxationrate. Contradicting this explanation, however, arethe preliminary observations by Whittaker et al thatslow relaxation was seen in the malnourished soleusmuscle which is mainly composed of type I fibres.30There are other mechanisms causing a slow

relaxation rate. From as early as 1915 a slowrelaxation rate has been shown to be associated withfatigue.4'(48 Indeed Esau et a146 have proposed asimple clinical test of diaphragmatic fatigue basedon measurement of relaxation rate. As relaxationrate depends on the rate at which myosin crossbridges dissolve following binding with ATP47 it islikely to be affected by factors related to the ATPhydrolysis or binding to actomyosin. The exactmechanisms that slow relaxation are debatable, andthe proposed causes are given below:

Musclefunction and nutrition

1 Reduced energy release on ATP binding isrelated to a fall in the free energy change for ATPhydrolysis.

2 Raised ADP values reduce detachment rateand inhibit the calcium ATPase activity for pumpingcalcium into the sarcoplasmic reticulum (lateralcisternae).49

3 ATP production is reduced.50

RATIO OF LOW FREQUENCY TO HIGH FREQUENCYFORCEThe low:high frequency force ratio rises in themalnourished muscle mainly because, while itmaintains the level of force developed at lowfrequency stimulation, there is a reduction ofthe increment in the force produced at high fre-quencies.13 27 Previous studies compared the forcedeveloped at 10 Hz with that at 100 Hz, and itmay be argued that the loss of force at highfrequencies could be due to failure of nerve or endplate conduction. Our recent studies in humans,however, show that the ratio of the force at 10 Hz(F10) to that at 20 Hz (F20), F10:F20, is alsoappreciably increased in malnourished subjects andrestored by refeeding.33 What is the importance ofthis shift? Previous studies have shown that withfatigue the relaxation rate slows, and thus themuscle tetanises to an almost maximal extent at alower frequency, and also that increasing the fre-quency does not result in a further rise in tension. Inother studies this change has been shown to beassociated with a reduced heat production in theadductor pollicis and reduced ATP turnover inmouse soleus.42 51 Furthermore, during isometriccontraction the energy cost of generating anisometric force increases linearly with the force-timeintegral.52 53 Thus the lower total maximal forcegenerated and reduced thermogenesis can be inter-preted as an inability to receive or use the neces-sarily greater rate of energy required for thispurpose.Thus all the electrophysiological findings, particu-

larly the slow relaxation, which is independent ofthe mechanical factors of the experiment,54 suggesta reduction of the ability of the muscle to sustain amaximal change in the enthalopy (heat+work done)of the system. Why does this happen in themalnourished patient or animal?To understand the possible ways in which the

muscle in a malnourished creature may not be ableto use energy at the same rate as in a normallynourished one it is necessary to look at the followingfactors: (i) availability of substrate; (ii) activity ofenzyme pathways; (iii) changes in the pH; (iv)changes in free energy change due to ATP hydro-lysis; (v) changes in calcium kinetics.

SUBSTRATE AVAILABILITYGlycogen stores were not decreased in the obesepatients fed a 400 kcal diet, even though therelaxation rate was slowed and the F10:Fmax (ratioof the force at 10 Hz stimulation to that at 100 Hzstimulation) had significantly increased.27 Whileglycogen stores were reduced by 50% in the ratstudies, there was sufficient glycogen to provideenergy for the duration of the applied stimulus - twoseconds. Fatty acids are the other source of energy.Muscle contracting above 30% of maximal, how-ever, is ischaemic.55 Hence fatty acid oxidationcannot supply energy at the time of a stimulus of20 Hz or more.

ENZYME ACTIVITY RELATED TO ENERGYMETABOLISMThe muscle fibres derive energy from glycolysis oroxidative metabolism, depending on the fibre type.For a short duration of stimulation, however, theimmediate source of energy is creatinine phosphateand then glycolysis. Later oxidative recovery occurs,but it is not a factor when the muscle is contractingat more than 30% of its maximal force because of itsischaemia.55

In our experimental system limitation of glycolysiscould account partially for the fatigue after a fivesecond stimulus and perhaps even for relaxationslowing after a one to two second stimulus for thefollowing reasons. While data for the gastrocnemiusitself are not available we can use data derived froma muscle of similar fibre composition (mainly typeII) in the rat. It has been shown the maximum heatproduction during isometric contraction of theextensor digitorum longus is 43-9 mcal/g at 270C.56Furthermore, the heat rate increased in a linearfashion with increase in force. The amount of ATPrequired for hydrolysis to meet this energy need persecond depends on the free energy change for ATPhydrolysis (delta G):

delta G=delta GO+2-8 ln[ADP]/[ATP]X[P,]

At 37°C and pH 7 0 the delta Go=-37-4.57

A pH of 7-0 was chosen, because the malnourishedmuscle was shown to have this pH by NMR.32 Thenext part of the equation requires the measurementof the free ADP:ATP ratio, which cannot be donedirectly from experimental data. As the creatinekinase reaction is in equilibrium, however, the freeADP can be calculated from the creatine phosphateand creatine measurements as follows:

CrP+ADP+H+=ATP+Cr. . . . . . .(1)

or, as this is in equilibrium,

33

34

-delta G0=RT ln [ATP][Cr]/[CrP][ADP][H+]

As delta G0=-RT ln K, therefore,RT In K=RT ln [ATP][Cr]/[CrP][ADP][H+][ADP]=[ATP][Cr]/[CrP][H+] K.......(2)

Free energy change for ATP hydrolysis=delta G0+2-58 ln [ADP][Pi]/[ATP]

=delta G0+2*58 ln [Cr][Pi]/[CrP][H+]K

With pH=7-0, [H+]=10-7, and as creatine phos-phate concentration in malnourished muscle was0-009 M/1, the free creatine was 0-0016 M/,[Pi]=0-01103, and k=2x 109, (unpublished results).'3

Therefore:

delta G=-37.4+5.9 lo ,(O0-0016xO>00103/0-009x2x10 x10-7)

=-66-95 Kj/mol=0-06695 mj/micromol (mj=millijoules)

As the maximum heat production of extensordigitorum longus is 43-9 mcal/g/second=43-9x4 18=183-5 mj/g/second, then the rate of ATP hydro-lysis required for this heat production is 0-184/0-0669=2-7 micromol/second. With a possible Qlo of1.85 the ATP hydrolysis at 37°C would be 5 0micromol/second.The creatine phosphate content of malnourished

muscle was at its lowest in the 21 day hypocaloricallyfed rat - 45.8 micromol/g dry weight or 9-1 micromol/gwet weight (recalculated from reference13). Hencethis rate of ATP hydrolysis can be maintained forless than two seconds before creatine phosphatestores run out. Thus it would be necessary for ATPto be formed from glycolysis during a contractionslasting in excess of two seconds. In the hypocaloricrat'3 the maximum activity of 6-phosphofructo-kinase (PFK), which is a non-equilibrium enzymelimiting the rate of glycolysis, was 58-6 micromol/minute/g, or about 1 micromol/g/second. This willenable the synthesis of 3 micromol of ATP/g/second,which is about 3/4-9 or 61% of the maximal needs,and thus it would be calculated that after the firstone to two seconds, the rate of energy derived fromglycolysis would result in a fall in force of 100-61 =39% of the maximum force. Interestingly, the loss offorce in hypocaloric rats was 44% of the maximumattained, which is within 5% of the calculated lossexpected for the enzyme activity.

Similarly, the predicted glycolysis rate in controlrats should result in the regeneration of 3-85micromol/second/g muscle or enough to meet 3 85/4-9=78% of requirements for maximal force. This inturn would be expected to result in a fall of force ofabout 22%. This calculated value is 1% lower thanthe measured fatigue of 20*8% . Finally, when total

Jeejeebhoy

force at 200 Hz is plotted against the PFK activitythere is a linear correlation (p<001). Similarly,fatigue and the F1O:F100 ratio are negatively cor-related in a highly significant manner (p<001)(Fig. 6). Thus it seems that reduced PFK activitymay be related to the reduced force. Despite thisrelation between expected and observed "fatigue" itis unclear how the lower glycolytic activity would berecognised by the contractile machinery and trans-lated into a fall in force. In our studies the ATPvalue was normal in malnourished rats, but themeasurements were made in unstimulated muscle.Even if the concentration fell a little after stimula-tion it is unlikely that it would fall below thatrequired to saturate myofibrillar ATPase (km0.1 mM). This problem is not unique to fatigue inmalnutrition, and the same observations and ques-tions have arisen in connection with fatigue inducedby exercise.59 Hence other factors to be discussedbelow may contribute to the slow relaxation andfatigue seen in malnutrition.The above calculations are for a muscle with type

II fibres. The heat production for a comparable slowtwitch muscle, however, such as the soleus in therat, is only 4 mcal/g/second at maximum tension,which would result in only about 0.5 micromol ofATP being hydrolysed, per second/g.56 The differ-ence between fast and slow twitch muscle is not dueto a difference in the maximum force generated,because heat production, corrected for force, was17-9 and 3.0 mcal/g/kg force/cm2 for extensor digi-

.2'0

aU-

S220a

50 60 70 80Phosphofructokinase (,umol/g/minute)

Fig. 6 Correlation of (i) ratio offorce at 10 Hz to that at100 Hz as % (FJO:F100) (0) and (ii) fatigue (% force lossperfive seconds) (0) with PFK activity (Wnollglminute).Both negative correlations are significant (p<OOI).

0

01.1

Muscle function and nutrition

torum longus and the soleus of the rat, respectively,even when corrected for fibre length.56 Under thesecircumstances there is enough creatine phosphateactivity for a six second stimulus.

In preliminary studies, however, Whittaker et at30showed that the relaxation of even the soleus musclewas still slow in malnourished rats and recoveredwith refeeding. Clearly, the slower relaxation of thesoleus cannot be due to limitation of glycolyticenergy and must therefore be due to other factors tobe discussed below.

CHANGES IN MUSCLE pHThe lactate activities were higher in the hypocaloric-ally fed rats. As there is a good correlation betweenthe lactate activities and pH in muscle,3t thesefindings suggest that hypocalorically fed animalsmay have a low muscle pH. A lower pH may in-fluence the glZcolytic pathway through the inhibi-tion of PFK. 9 Furthermore. a fall in pH maydecrease the release of calcium by the sarcoplasmicreticulum and thus influence contractility,(( or evenhave a direct effect on muscle.6' Nevertheless, lowmuscle pH could not be the only factor influencingmuscle relaxation, as significant slowing of therelaxation was seen even after two and five day fastswhen lactate activities were not significantly raisedin the fasted animals.'3

FREE ENERGY FOR ATP HYDROLYSIS

This was calculated for the control, two day andfive day fasted animals from datat3 and unpublishedobservations (Table 2).The data show good correlation between the

relaxation rate and delta G, whereas there is no suchcorrelation with fatigue or the F10:F100 ratio. Thecorrelation coefficient is 0O968. Dawson et at48 founda linear correlation between the relaxation rate anddelta G expressed in the same way as ours. Theybelieved that the slow relaxation rate was due to afall in the free energy change for ATP hydrolysisduring fatigue. On the other hand, Hultman et al didnot find this correlation.50 Their measurement of

35

the delta G was based on total ADP measured bio-chemically. As the total ADP is not necessarilyrepresentative of the free ADP their results are dif-ficult to interpret. Our results, while consistent withthose of Dawson et al,48 are nevertheless incon-clusive as the calculations were done by amalgamat-ing data from two different studies.What is the importance of the relation of relax-

ation rate to free energy change for ATP hydrolysis?Muscle relaxation is caused by reuptake of calciuminto the lateral cisternae of the sarcoplasmic reticu-lum.The total force available to drive Ca2+ into the

endoplasmic reticulum during relaxation is:-(dG/dE)tota1= -(dG/dE)ATp -n (dG/dE)Ca

.()....48 62

The work required to pump a mole of calcium intothe sarcoplasmic reticulum is:(dG/dE)Ca=2-58 ln [free Ca2+] in/[free Ca2+] out

Kj mol-F. . (2)As the hydrolysis of 1 mol of ATP results in the

uptake of 2 mol of Ca2+, therefore n=2.Substituting (2) in (1),

when -(dG/dE)totai=0 and no calcium is pumpedthen:(dG/dE)ATp=5-98 ln [free Ca2+] in/[free Ca2+] outRearranging the equation,

[free Ca2+jIn/[free Ca2+] out= e(dG/dE)atp/5 9... (3)

Hence small changes in dG/dE of ATP (change inthe free energy change of ATP hydrolysis) may haveprofound effects on the regulation of free cytosoliccalcium.50 64

CHANGES IN CALCIUM KINETICSIn both human and the two animal studies13 14 30 wenoticed that the malnourished muscle has anappreciably higher intracellular calcium concentra-tion, compared with that of controls. The rise in cellcalcium concentration means that there is a netpositive balance of calcium across the sarcolemma.It is likely that this increase is largely due to an

Table 2 Free energy change for A TP hydrolysis (delta G)

FJO:F1OO Fatigue (% force Relaxation* Delta GStatus ratio loss in 5 seconds) (lIt ms ')* Kj/M

Control 26-6 20 8 5 0 -71-30Two day fast 35 8 35 0 3-16 -68-30Five day fast 38-3 41-7 2-67 -68-8021 day hypocaloric 39.5 44.5 1-49 -66-90

*69-3/t /2

36

increase in the mitochondrial calcium for the follow-ing reasons:

1 From the above equation a reduction in the freeenergy change for ATP hydrolysis will only result ina fall in the sarcoplasmic free calcium. Assumingthat the free calcium is in equilibrium with thebound calcium, this will result in a fall in sarcoplas-mic calcium concentration. Hence the increase is

unlikely to be in the sarcoplasmic reticulum (lateralcisternae).

2 The reduced free energy change for ATPhydrolysis means that the cytosolic uptake will bereduced, resulting in a rise in free cytosolic calcium.This will result in an increase of mitochondrialcalcium influx.63

3 The steep electrochemical gradient for calciumacross the sarcolemma makes the entry of calciuminto the cell a passive process.64 Hence calciumefflux has to occur to maintain homeostasis. Themechanisms for efflux are the CaMgATPase656system 5 and sodium exchange.66 More relevant to

our observations is that Jones et a167 showed thatmuscle fatigue, induced by stimulating in a hypoxicor normoxic medium, was followed by release oflactate dehydrogenase. If not stimulated, the hy-poxic muscle did not release lactate dehydrogenaseto any appreciable extent. Thus muscle fatigue was

shown to result in muscle enzyme release. In a

subsequent study68 using the hypoxia model theyshowed that there was Z band degeneration associ-ated with the enzyme release and that both could beprevented by excluding external calcium from themedium. Finally, they found that fatigued musclehad twice the amount of intracellular calcium whenincubated in medium containing calcium.69 It isinteresting to note that malnourished muscle alsoshowed Z band degeneration with an increase inintracellular calcium.'4 In the studies cited above itwas shown that, unlike cardiac muscle, inhibitingenergy metabolism by cyanide or iodoacetate alsoacts synergistically with fatigue to enhance lactatedehydrogenase release. Thus there is evidence tosuggest that fatigue associated with a reduction inavailable energy may prevent calcium efflux andinduce a positive calcium balance in the muscle cell.The postulated mechanism would be as follows:

the calcium entering the cell has to be removed by a

process of efflux. There are two processes of effluxfrom muscle - sodium and calcium exchange andCa2+ +Mg2+ATPase Ca2+ transporter. The first,while not linked to ATP hydrolysis, is stimulated byit. The second is linked to ATP hydrolysis, much as

it is in the sarcoplasmic reticulum. Thus it is possiblethat the CaMgATPase system may be inhibited bythe fall in free energy change for ATP hydrolysis.Furthermore, this process is activated by calmodulin

Jeejeebhoy

calcium and magnesium,70 and malnutrition maychange calmodulin concentrations.

Proposed central hypothesis

Based on the above analysis, we propose thefollowing central hypothesis:

1 Reduction in food intake depresses muscleglycolytic enzyme activity, thus reducing the avail-ability of energy from glycolysis during contraction.The limited rate of glycolysis reduces the total forceand thus the high frequency response of muscle.

2 When there is an imbalance between the abilityto generate ATP via the glycolytic pathway andenergy needs for muscle contraction, then creatinephosphate activity is used and the ratio of creatinephosphate:creatine falls. As the creatine kinasereaction is in equilibrium this is associated with alower ADP:ATP ratio and a rise in free phosphorus.Hence as the CR and Pi rise and CrP falls, the log ofthe right side of this equation will become lessnegative or more positive, and thus the overall freeenergy change will become less negative, andbecause it is a negative quantity, will be reduced.This reduction may in turn slow the relaxation rate.

3 The fall in force and the slow relaxation rate aresimilar to the changes seen in fatigue induced byexercise. Based on the observations of Jones,Jackson, and Edwards67-69 on experimental fatigueassociated with calcium accumulation and Z banddegeneration, together with the fact that both thesephenomena are seen in malnourished muscle, it ispostulated that similar mechanisms may apply tomalnourished muscle and further injure muscle.Accumulation of calcium could explain the lag inrecovery of muscle function noted for some timeafter the start of nutritional support, when malnutri-tion is severe and prolonged.

In conclusion, while many interesting avenuesneed to be confirmed and explored, there is suffi-cient evidence to suggest that in the adult human theadverse functional effects of malnutrition on musclefunction cannot be equated to, nor quantitated by, asimple loss of lean body mass. Furthermore, restora-tion of muscle function cannot be equated to"regrowth" of lean body mass assessed by a gain inbody nitrogen or the attainment of a positivenitrogen balance. The functional effects of malnutri-tion and changes in cellular electrolytes at a time offunctional impairment, and especially the role ofcalcium in modulating nutritional effects and pro-tecting muscle from "outstripping" its energy sup-plies need further study.

Muscle function and nutrition 37

This work was supported in part by the OntarioMinistry of Health (Grant PR 228) and the MedicalResearch Council of Canada (MT-3204).

References

1 Jeejeebhoy KN, Baker JP, Wolman SL, et al. Criticalevaluation of the role of clinical assessment and bodycomposition studies in patients with malnutrition andafter total parenteral nutrition. Am J Clin Nutr 1982;35: 1117-27.

2 Yeung CK, Smith RC, Hill GL. Effect of an elementaldiet on body composition. A comparison with in-travenous nutrition. Gastroenterology 1979; 77: 652-7.

3 Young GA, Hill GL. A controlled study of protein-sparing therapy after excision of the rectum: effects ofintravenous amino acids and hyperalimentation onbody composition and plasma amino acids. Ann Surg1980; 192: 183-91.

4 Elwyn DH, Gump FE, Munro HN, et al. Changes innitrogen balance of depleted patients with increasinginfusions of glucose. Am J Clin Nutr 1979; 32:1597-611.

5 Hill GL, King RFGJ, Smith RC, et al. Multi-elementanalysis of the living body by neutron activationanalysis - application to critically ill patients receivingintravenous nutrition. Br J Surg 1979; 66: 868-72.

6 Cohn SH, Gartenhaus W, Sawitsky A, et al. Compart-mental body composition of cancer patients bymeasurement of total body nitrogen, potassium andwater. Metabolism 1981; 30: 222-9.

7 Greenberg GR, Jeejeebhoy KN. Intravenous protein-sparing therapy in patients with gastrointestinal dis-ease. JPEN 1979; 3: 427-32.

8 Starker PM, Gump FE, Askanazi J, et al. Serumalbumin levels as an index of nutritional support.Surgery 1982; 91: 194-9.

9 Baker JP, Detsky AS, Wesson DE, et al. Nutritionalassessment: a comparison of clinical judgment andobjective measurements. N Engl J Med 1982; 306:969-72.

10 Edwards RHT. Human muscle function and fatigue.In: Porter R, Whelan J, eds. Human muscle fatigue:physiological mechanisms. London: Pitman Medical,1981: 1-18.

11 Wiles CM, Young A, Jones DA, Edwards RHT.Muscular relaxation rate, fibre-type composition andenergy turnover in hyper- and hypo-thyroid patients.Clin Sci 1977; 57: 375-84.

12 Lopes JM, Russell DMcR, Whitwell J, et al. Skeletalmuscle function in malnutrition. Am J Clin Nutr 1982;36: 602-10.

13 Russell DMcR, Whittaker JS, Atwood HL, et al. Theeffect of fasting and hypocaloric diets on the functionaland metabolic characteristics of rat gastrocnemiusmuscle. Clin Sci 1984; 67: 185-95.

14 Russell DMcR, Walker PM, Leiter LA, et al. Metabolicand structural changes in skeletal muscle during hypo-caloric dieting. Am J Clin Nutr 1984; 39: 503-13.

15 Graham JA, Lamb JF, Linton AL. Measurement of

body water and intracellular electrolytes by means ofmuscle biopsy. Lancet 1967; ii: 1172-6.

16 Tietz NW. Methods for determination of chloride inbody fluids. Mercurimetric titration. (Schales andSchales modified). In: Tietz NW, ed. Fundamentals ofclinical chemistry. 2nd ed. Philadelphia: WB SaundersCo, 1976: 880-2.

17 Fiske CH, Subbarow Y. The colorimetric determina-tion of phosphorus. J Biol Chem 1925; 66: 375-400.

18 Bergstrom J, Hultman E. Water, electrolyte andglycogen content of muscle tissue in patients under-going regular dialysis therapy. Clin Nephrol 1974; 2:24-34.

19 Wilde WS. The chloride equilibrium in muscle. Am JPhysiol 1945; 143: 666-76.

20 Conway EJ. Nature and significance of concentrationrelations of potassium and sodium ions in skeletalmuscle. Physiol Rev 1957; 37: 84-132.

21 Bergstrom J. Muscle electrolytes in man: determinedby neutron activation analysis on needle biopsy speci-mens; a study on normal subjects, kidney patients andpatients with chronic diarrhoea. Scand J Clin LabInvest 1962; 14 (Suppl 68): 1-110.

22 Charlton MP, Silverman H, Atwood HL. Intracellularpotassium activities in muscles of normal and dys-trophic mice: in in vivo eletrometric study. Exp Neurol1981; 71: 203-19.

23 Cahil GF. Starvation in man. N Engl J Med 1970; 282:668-75.

24 Keys A, Brozek J, Hanschel A, Michelson 0, TaylorHL. The biology of human starvation. Minneapolis:University of Minnesota Press, 1950.

25 Garrow JS. Is there a body protein reserve? Proc NutrSoc 1982; 41: 373-9.

26 Shonheyder F, Heilskov NCS, Oleson K. Isotopestudies on the mechanism of negative nitrogen balanceproduced by immobilization. Scand J Clin Lab Invest1954; 6: 178-88.

27 Russell DMcR, Leiter LA, Whitwell J, Marliss EB,Jeejeebhoy KN. Skeletal muscle function during hypo-caloric diets and fasting: a comparison with standardnutritional assessment parameters. Am J Clin Nutr1983; 37: 133-8.

28 Russell DMcR, Prendergast PJ, Darby PL, et al. Acomparison between muscle function and body com-position in anorexia nervosa: the effect of refeeding.Am J Clin Nutr 1983; 38: 229-37.

29 Sinning WE. Body composition assessment. In: WilsonPK, ed. Adult fitness and cardiac rehabilitation. Balti-more: University Park Press, 1975: 362-77.

30 Whittaker JS, Desai M, Atwood HL, Walker PM,Jeejeebhoy KN. Effect of hypocaloric feeding andrefeeding on rat soleus muscle function and composi-tion. Clin Res 1984; 32: (2), 474A.

31 Sahlin K, Alvestrand A, Brandt R, Hultman E.Intracellular pH and bicarbonate concentration inhuman muscle during recovery from exercise. JournalofApplied Physiology: Respiratory, Environmental andExercise Physiology 1978; 45: 474-80.

32 Jacobs DO, Whitman G, Maris J, et al. In vivo P31nuclear magnetic resonance spectroscopy of rat skeletalmuscle during starvation. JPEN 1985; 9: 107.

38 Jeejeebhoy

33 Brough WA, Horne G, Irving MH, Jeejeebhoy KN. Astudy of malnutrition, sepsis, trauma, steroid adminis-tration and surgery on muscle function. Br Med J(in press).

34 Berkelhammer CH, Leiter LA, Jeejeebhoy KN, et al.Skeletal muscle function in chronic renal failure: anindex of nutritional status. Am J Clin Nutr 1985; 42:845-54.

35 Fraser IM, Russell DMcR, Whittaker JS, et al. Skeletaland diaphragmatic muscle function in malnourishedchronic obstructive lung disease patients. Am RevRespir Dis 1984; 129 (4): A269.

36 Elebute EA, Stoner HB. The grading of sepsis. Br JSurg 1983; 70: 29-31.

37 Bigland-Ritchie EMG. Fatigue of human voluntary andstimulated contractions. In: Porter R, Whelan J, eds.Human muscle fatigue: physiological mechanisms. Lon-don: Pitman Medical, 1981: 130-56.

38 Jones DA. Muscular fatigue due to changes beyond theneuromuscular junction. In: Porter R, Whelan J, eds.Human muscle fatigue: physiological mechanisms. Lon-don: Pitman Medical, 1981: 178-96.

39 Luttgau HC. The effect of metabolic inhibitors on thefatigue of the action potential in single muscle fibres.J Physiol (London) 1965; 178: 45-67.

40 Mosso A. Fatigue. London: Allen and Unwin, 1915:334.

41 Edwards RHT, Hill DK, Jones DA. Effect of fatigueon the time course of relaxation from isometriccontractions of skeletal muscle in man. J Physiol(Lond) 1972; 227: 26-27P.

42 Edwards RHT, Hill DK, Jones DA. Metabolic changesassociated with the slowing of relaxation in fatiguedmouse muscle. J Physiol (Lond) 1975; 251: 287-301.

43 Wiles CM, Young A, Jones DA, Edwards RHT.Relaxation rate of constituent muscle fibre types inhuman quadriceps. Clin Sci 1979; 56: 47-52.

44 Viitasalo JT, Komi PV. Effects of fatigue on isometricforce and relaxation time characteristics in humanmuscle. Acta Physiol Scand 1981; 111: 87-95.

45 Esau SA, Bellamare F, Grassino A, Permutt S,Roussos C, Pardy RL. Rate of the relaxation of thediaphragm during fatigue. Journal of Applied Physio-logy: Respiratory, Environmental and ExercisePhysiology 1983; 54: 1353-60.

46 Esau SA, Bye PTP, Pardy RL. Changes in rate ofrelaxation of sniffs with diaphragmatic fatigue inhumans. J Appl Physiol 1983; 55: 731-5.

47 Lymn RW, Taylor EW. Mechanism of adenosinetriphosphate hydrolysis by actomyosin. Biochemistry1971; 10: 4617-24.

48 Dawson MJ, Gadian DG, Wilkie DR. Mechanicalrelaxation rate and metabolism studied in fatiguingmuscle by phosphorus nuclear magnetic resonance.J Physiol (Lond) 1980; 299: 465-84.

49 Blinks JR, Rudel R, Taylor SR. Calcium transients inisolated amphibian skeletal muscle fibres: Detection byaequorin. J Physiol 1978; 277: 291-323.

50 Hultman E, Sjoholm H, Sahlin K, Edstrom L. Glycoly-tic and oxidative energy metabolism and contractioncharacteristics of intact muscle. In: Porter R, Whelan J,

eds. Human muscle fatigue: physiological mechanisms.London: Pitman Medical, 1981: 19-40.

51 Wiles CM, Edwards RHT. Metabolic heat productionin isometric ischaemic contractions of human adductorpollicis. Clin Physiol 1982; 2: 499-512.

52 Rome LC, Kushmeric MJ. Energetics of isometriccontractions as a function of muscle temperature. Am JPhysiol 1983; 244: (Cell Physiol 13): C0I-C109.

53 Homsher E, Kean CJ. Skeletal muscle energetics andmetabolism. Ann Rev Physiol 1978; 40: 93-131.

54 Jewell BR, Wilkie DR. The mechanical properties ofrelaxing muscle. J Physiol 1960; 152: 30-47.

55 Edwards RHT, Harris RC, Hultman E, et al. Effect oftemperature on muscle energy metabolism and endur-ance during successive isometric contractions, sus-tained to fatigue, of the quadriceps in man. J Physiol(Lond) 1972; 220: 335-52.

56 Wendt IR, Gibbs CL. Energy production of ratextensor digitorum longus muscle. Am J Physiol 1973;224: 1081-6.

57 Alberthy RA. Calculation of the Gibbs free energy,enthalopy and entropy changes for the hydrolysis ofATP at 00, 250, 370, and 75°. In: San Pietro A, Gest H,eds. Horizons of bioenergetics. New York: AcademicPress, 1972: 135-47.

58 Newsholme E, Leech AR. Metabolism in exercise. In:Biochemistry for the medical sciences. Chichester: JWiley and Sons, 1983: 366.

59 Danforth WH. Activation of glycolytic pathway inmuscle. In: Chance B, Eastbrook RW, eds. Control ofenergy metabolism. New York: Academic Press, 1965:287-97.

60 Nakamura Y, Schwartz Z. The influence of hydrogenion concentrations on calcium binding and release byskeletal muscle sarcoplasmic reticulum. J Gen Physiol1972; 59: 22-32.

61 Donaldson SBK, Kerrick W, Hermansen L. Differen-tial direct effects of H4 on Ca'2 -activated form ofskinned fibres from soleus, cardiac and adductormagnus muscles of rabbits. Pflugers Arch 1978; 376:55-65.

62 Kodama T. Thermodynamic analysis of muscle ATPasemechanisms. Physiol Rev 1985; 65: 468-551.

63 Nicholls D. Some recent advances in mitochondrialcalcium transport. Trends in Biochemical Sciences1981; 36-8.

64 Li CL, Shy GM, Wells J. Some properties of mamma-lian skeletal muscle fibres with particular reference tofibrillation potentials. J Physiol (Lond) 1957; 135:522-35.

65 Sulakhe PV, Drummond GI, Ng DC. Calcium bindingby skeletal muscle sarcolemma. J Biol Chem 1973; 248:4150-7.

66 Caputo C, Balanos P. Effect of external sodium andcalcium on calcium efflux in frog striated muscle.J Membr Biol 1978; 41: 1-14.

67 Jones DA, Jackson MJ, Edwards RHT. Release ofintracellular enzymes from an isolated mammalianskeletal muscle preparation. Clin Sci 1983; 65: 193-201.

68 Jones DA, Jackson MJ, McPhail G, Edwards RHT.Experimental mouse muscle damage: the importanceof external calcium. Clin Sci 1984; 66: 317-22.

Muscle function and nutrition 39

69 Jackson MJ, Jones DA, Edwards RHT. Experimentalskeletal muscle damage: the nature of calcium-activated degenerative process. Eur J Clin Invest 1984;14: 369-74.

70 Ritz E. The role of intracellular calcium and calmodu-lin in cellular metabolism: possible implications for

renal failure. Kidney Int 1983; 24: (Suppl 16):S161-S166.

71 Harrison JE, McNeill KG, Strauss AL. A nitrogenindex-total body protein normalized for body size - fordiagnosis of protein status in health and disease. NutrRes 1984; 4: 209-24.