Measurement of high-energy phosphates in tiny fragments of human muscle fibres

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This research method was  developed in the group headed by Professor Anthony J Sargeant in Amsterdam. Arnold de Haan had developed the basis of the technique in the 1980s as part of his own PhD work. This was subsequently refined to enable very small fragments of human muscle fibre obtained by needle biopsy to be analysed. The present research paper describes that refined techniques and its sensitivity. The work formed part of the PhD thesis the outstanding Greek PhD student, Christina Karatzaferi, who was supervised by Tony Sargeant and Arnold de Haan.

Improved high-performance liquid chromatographic assay for the determination of “high-energy” phosphates in mammalian skeletal muscle. Application to a single-fibre study in man

Christina Karatzaferi, Arnold de Haan, Carla Offringa, Anthony J Sargeant.

Journal of Chromotography

J Chromatogr B Biomed Sci Appl. 1999 Jul 9;730(2):183-91
Abstract
A sensitive and reproducible method for the determination of adenine nucleotides (ATP, IMP) and creatine compounds [creatine (Cr), phosphocreatine (PCr)] in freeze-dried single human muscle fibre fragments is presented. The method uses isocratic reversed-phase high-performance liquid chromatography of methanol extracts. Average retention times (min) of ATP, IMP and PCr, Cr from standard solutions were 10.6+/-0.42, 2.11+/-0.06 (n=6) and 10.5+/-0.31 and 1.19+/-0.02 (n=9), respectively. Detection limits in extracts from muscle fibre fragments were 2.0, 1.0, 3.0 and 2.0 mmol/kg dm, respectively. The assay was found successful for analysis of fibre-fragments weighing > or = 1 microg.
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Functional and structural changes after disuse of human muscle – first study to quantify disuse muscle atrophy at fibre level in humans

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Clinical Science and Molecular Medicine (1977) 52, 337-342. Functional and structural changes after disuse of human muscle – Authors: ANTHONY J SARGEANT,* C. T. M. DAVIES,* R. H. T. EDWARDS, C. MAUNDER AND A. YOUNG *Medical Research Council Environmental Physiology Unit, London School of Hygiene and Tropical Medicine, University of London, and Jerry Lewis Muscle Research Centre, Royal Postgraduate Medical School, Hammersmith Hospital, London

Summary

1. Seven patients who had suffered unilateral leg fracture were studied after removal of immobilizing plaster casts.

2. Leg volume measured anthropometrically was reduced by 12% in the injured leg (5.68 f 1.05 litres) compared with the uninjured (6.43 f 0.87 litres). Associated with this loss was a similar reduction in the net maximum oxygen uptake achieved in one-leg cycling, from 1.89 k 0.21 l/min in the uninjured leg to 1.57+0.18 l/min in the injured.

3. Measured by a percutaneous needle biopsy technique, a reduction of 42% was found in the cross-sectional area of the muscle fibres sampled from the vastus lateralis of the injured compared with the uninjured leg.

4. Staining for myosin adenosine triphosphatase activity showed that both type I and I1 fibres were affected, being reduced respectively from 3410 to 1840 pm2 and from 3810 to 2390 pm2 cross-sectional area.

5. Possible reasons and implications are discussed for the discrepancy between the magnitude of the difference observed in the gross measurement of leg function (maximum oxygen uptake) and structure (leg volume) as compared with the cellular level (cross-sectional fibre area).

 

Correspondence: Dr A. J. Sargeant, MRC Environmental Physiology Unit, London School of Hygiene and Tropical Medicine, University of London, Keppel Street (Gower Street), London WClE 7HT.

Introduction

Atrophy of the affected limb and loss of muscle power follows bone fracture and subsequent immobilization. Years of experience have enabled the rehabilitation professions to develop empirical programmes to reverse these changes. However, the efficacy of such programmes may be further improved if we can increase our understanding of the atrophic response to disuse in human muscle. Recent studies showed that 15 weeks immobilization in a long-leg plaster cast after fracture reduced the fat-free volume of the affected leg by 12%, which was accompanied by a similar fall in the maximum oxygen uptake ( ~oz,,,,=.) achieved with oneleg pedalling (Davies & Sargeant, 1975a,b). However, it was not known how far these changes in gross structure and function were reflected at a cellular level within the affected muscles. Since the work of pedalling is performed mainly by the leg extensors (A. J. Sargeant & C. T. M. Davies, unpublished work) needle biopsy was used (Edwards, Maunder, Lewis & Pearse, 1973) to study fibre atrophy in the quadriceps femoris muscle and to compare this with measurements of the gross leg volume and maximal oxygen uptake of patients recovering from unilateral leg fracture.

http://www.clinsci.org/content/ppclinsci/52/4/337.full.pdf

Human Muscle Power

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The ability of generating muscle power power is important whether you are an Olympic athlete, a ballet dancer, or an elderly person wanting to climb the stairs to go to bed. In this comprehensive review of his research Anthony Sargeant points out the importance of different types of muscle fibres that make up the human skeletal muscles that produce power in legs and arms. Tony also points out that in research seeking to measure human muscle power it is essential to measure or control the speed at which the power is generated (this is because power is the product of work and velocity).

Structural and functional determinants of human muscle power
by Anthony J Sargeant

Experimental Physiology
Exp Physiol. 2007 Mar;92(2):323-31

Measurements of human power need to be interpreted in relation to the movement frequency, since that will determine the velocity of contraction of the active muscle and hence the power available according to the power-velocity relationship. Techniques are described which enable movement frequency to be kept constant during human exercise under different conditions. Combined with microdissection and analysis of muscle fibre fragments from needle biopsies obtained pre- and postexercise we have been able ‘to take the muscle apart’, having measured the power output, including the effect of fatigue, under conditions of constant movement frequency. We have shown that fatigue may be the consequence of a metabolic challenge to a relatively small population of fast fatigue-sensitive fibres, as indicated by [ATP] depletion to approximately 30% of resting values in those fibres expressing myosin heavy chain isoform IIX after just 10 s of maximal dynamic exercise. Since these same fibres will have a high maximal velocity of contraction, they also make a disproportionate contribution to power output in relation to their number, especially at faster movement rates. The microdissection technique can also be used to measure phosphocreatine concentration ([PCr]), which is an exquisitely sensitive indicator of muscle fibre activity; thus, in just seven brief maximal contractions [PCr] is depleted to levels < 50% of rest in all muscle fibre types. The technique has been applied to study exercise at different intensities, and to compare recruitment in lengthening, shortening and isometric contractions, thus yielding new information on patterns of recruitment, energy turnover and efficiency.

Anthony J Sargeant