Standardisation of 31phosphorus-nuclear magnetic resonance spectroscopy determinations of high energy phosphates in humans

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Eur J Appl Physiol (1994) 68:107-110

European

Applied

Journal of

Physiology

and Occupational Physiology

Standardisation of 31phosphorus-nuclear magnetic resonance

spectroscopy determinations of high energy phosphates in humans

L. Gariod ~, T. Binzoni ~, G. Ferretti 1, J. F. Le Bas 2, H. Reutenauer 2, P. Cerretelli ~

1 Ddpartement de Physiologie, Centre M6dical Universitaire, 9 Av. de Champel, CH-1211 Geneva 4, Switzerland z LMCEC, INSERM, Unit6 318, H6pital Albert Michallon, BP 217X, F-38043 Grenoble Cedex, France

Accepted: November 15, 1993

Abstract.

A procedure is described for standardising

the determination of adenosine 5'-triphosphate and

phosphocreatine concentration ([ATP] and [PC], re-

spectively, in absolute arbitrary units) in human muscle

by nuclear magnetic resonance (NMR) spectroscopy.

The individual 3~phosphorus (31p)-NMR spectra ob-

tained on equal hemispherical tissue volumes (muscle

plus skin and fat) were corrected for the thickness of

the skin and of the subcutaneous fat. The volumes in-

vestigated were standardised using an external refer-

ence. The procedure described made possible the com-

parison of high energy phosphate concentrations

among different subjects. It was applied to the assess-

ment of [ATP] and [PC] in four groups of sedentary

subjects (children, and adults aged 20-35, 35-50 and

over 50 years), and in a group of athletes (volleyball

players). The [ATP] and [PC] were not statistically dif-

ferent in the groups investigated.

Key words:

Adenosine 5'-triphosphate

-

Phosphocrea-

tine - Age - Training status - Method

Introduction

The determination of the absolute concentration of tis-

sue high energy phosphates (adenosine 5'-triphos-

phate [ATP] and phosphocreatine [PC]), and of inor-

ganic phosphate (P~) is of paramount importance in the

study of muscle energetics. Classical measurements

rely on chemical assays (extraction, freezing and spec-

trophotometric analysis) of biopsy samples. These

have been extensively employed for human studies

also (e.g. Bergstr6m 1967; Cheetham et al. 1986; Green

et al. 1989; Harris et al. 1977; Jones et al. 1985; Karls-

son and Saltin 1970; Karlsson et al. 1972; Larsson et al.

1978). The above techniques, however, only allow the

assessment of the composition of very small samples

Correspondence to: G. Ferretti

and, in addition, cannot provide repeated measure-

ments on the same muscle.

The 31phosphorus nuclear magnetic resonance spec-

troscopy (31p-NMRS) technique makes it possible to

perform multiple determinations of high energy phos-

phates in vivo in the same muscle sample. Although

31P-NMRS could theoretically provide absolute con-

centration (in millimoles per litre or millimoles per

gram) values, so far it has proved impossible to per-

form such measurements in intact animals, because of

the difficulty of detecting the precise size of the tissue

volume investigated. As a consequence, the phosphate

concentrations obtained by 31P-NMRS have been

mainly expressed either in relation to a known refer-

ence or as the ratio of two different molecules (mainly

PC to Pi; e.g. Arnold et al. 1984; Chance et al. 1981,

1985, 1986; Laurent et al. 1991; McCully et al. 1988;

Meyer et al. 1985; Taylor et al. 1983).

The aims of the present study were

1. To develop a 31P-NMRS procedure for tissue vol-

ume normalisation in vivo that would allow compari-

son of high energy phosphate concentration values (in

arbitrary units) among different subjects;

2. To determine in humans [ATP] and [PC] as a func-

tion of age and/or physical training.

Methods

Subjects. A group of 47 healthy male subjects aged 8-65 years participated in the study after giving their informed consent. Four age groups were selected among sedentary subjects. The fifth group consisted of 6 volley-ball players of international and na- tional standard aged 24 (SD 3) years. The physical characteristics of the subjects are summarised in Table 1. The [ATP] and [PC] values that appear in the Results section (Table 2), are the mean of four observations (two spectra on each leg).

Anthropometric measurements' of skin fold thickness. Skinfold thickness (skin plus subcutaneous fat) was measured by means of a skinfold caliper (Harpenden, Holtain Ltd, UK) on the posterior surface of both calves, above the triceps surae muscle at the site of contact of the coil with the limb surface. The skinfold thickness was then compared with the amount of fat detected from the 1H- NMR spectra (see next paragraph).

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108

Table 1. Physical characteristics of the subjects

Group Number of Age Height Mass

subjects (year) (cm) (kg)

mean SD mean SD mean

A 10 28.8 3.4 174 3 73.0 B 9 40.7 4.2 179 6 80.6 C 9 57.7 4.9 174 6 74.0 D 13 10.9 2.3 142 10 35.8 E 6 25.8 3.2 190 8 82.2 SD 6.6 5.6 9.7 7.7 8.2 INDUCTIVE

h .I:. COUPLING ex~er na I

~ ~ reference

c o u p l i n g c o i l s u r f a c e c o i l

Fig. 1. Diagram of the coil arrangement within the magnet, h.f,

High frequency

Table 2. Values for adenosine 5'-triphosphate [ATP] and phosphocreatine [PC] concentrations (in arbitrary units) ob- served in the groups of subjects

[ATP] Mean

SD

[PC] Mean

SD

Children Adults Adults Adults Athletes

20-35 years 35-50 years > 50 years

13.56 11.89 12.26 11.50 11.87

2.95 1.48 2.43 1.47 0.95

47.13 44.66 46.73 45.18 45.61

10.42 5.97 7.82 5.63 3.51

sip_ and 1H-NMR Spectroscopy. The measurements were made with a Brucker 2.35 T, 35-cm bore spectrometer. A double-tuned (1H-31P) 6-cm diameter radio-frequency coil (Decorps et al. 1985) was placed on the calf of the subject (at approximately one- third of the distance between the knee joint and the heel) in the homogeneous region of the magnetic field. The tissue mass investigated (about 70 cm 3) comprised, together with skin and subcutaneous fat, both gastrocnemius and soleus muscles in an approximately estimated ratio of 2 to 1. The use of an inductively coupled coil (Fig. 1) minimized di-electric losses of the signal due to tissue conductivity. The 1H spectra for the assessment of water and fat were obtained from four free induction decays (FID) at a frequency of 100 MHz. Excitation time was 200 ixs, relaxation delay 1 s. Such a short relaxation time allowed detection of water and fat peaks only.

The 31p spectra were obtained on 40 FID. The shimming was performed on the proton signal. A pseudo 90 ° pulse angle, de- fined as the angle providing maximal signal intensity for 31p, was

chosen. Radio-frequency pulses lasted 40 ~s whereas the relaxation delay was 15 s giving a total acquisition time for each spectrum of 10 min.

A reference phosphorus signal (a known amount of 200 mmol' 1-1 methylene diphosphonate water solution in a glass ca- pillary) was inserted at the centre of the coil. Such external reference was used to evaluate the homogeneity of the magnetic field and thus to correct the signals arising from the muscle for changes in the geometry of the system. This allowed calibration of the 31p spectra, the peak areas of which were normalised to that of the reference tracing.

Since the investigated hemispherical tissue volume is constant, the muscle mass detected, and therefore the size of the 31p_

NMR signal will decrease with increasing thickness of the subcutaneous fat layer, i.e. the distance between the coil and the muscle mass. Therefore a separate set of experiments was carried out on a muscle phantom (2 kg of pork meat) whereby the relation- ship was established between the phosphorus signal, a function of the detected muscle volume, and the distance between the coil and the muscle mass. This distance was changed by inserting wooden plates of increasing thickness between the coil and the phantom. The acquisition of the spectra was performed as for the experiments on humans.

The assumption was made that free phosphorus metabolites in subcutaneous fat and in the skin are negligible and therefore do not contribute to the size of the signal.

Statistical analysis. Analyses of variance (ANOVA) were used for groups A, B, C to determine the effect of factor "age" on the parameters measured. For significant F-coefficients from ANOVA, posthoc analyses were carried out using the Fisher's protected least significant difference test to establish the differ- ences between the means. Groups D and E were treated sepa- rately, each group being compared with group A using Student's t-test for unpaired observations. The accepted level of signifi- cance was set at 5%.

Results A n e x a m p l e o f a 3 1 p - N M R s p e c t r u m w i t h t h e e x t e r n a l r e f e r e n c e p e a k is s h o w n in Fig. 2. T h e r e l a t i o n s h i p b e - t w e e n i n t e n s i t y o f t h e p h o s p h o r u s signals a n d t h e dis- t a n c e b e t w e e n t h e coil a n d t h e m u s c l e p h a n t o m is s h o w n in Fig. 3. T h e c o i l - t o - m u s c l e d i s t a n c e f o r hu- m a n s ( h a l f o f t h e s k i n f o l d t h i c k n e s s ) w a s also f o u n d t o c o r r e l a t e s a t i s f a c t o r i l y w i t h h o m o l o g o u s 1 H - N M R s p e c t r o s c o p y e s t i m a t e s o f f a t ( r = 0 . 6 9 , P < 0 . 0 1 ,

Fig. 4).

T h e r e s u l t s as g i v e n in Fig. 3 a l l o w e d t h e n o r m a l i z a - t i o n o f [ A T P ] a n d [PC] t o a g i v e n m u s c l e v o l u m e . T o a c h i e v e this, t h e o b s e r v e d [ A T P ] a n d [PC] v a l u e s f o r a n y m e a s u r e d s k i n f o l d t h i c k n e s s w e r e c o r r e c t e d to t h e p e a k a r e a t h a t w o u l d h a v e b e e n o b s e r v e d if s k i n f o l d t h i c k n e s s h a d b e e n 0 m m . T h e m e a s u r e d [ A T P ] a n d [PC] v a l u e s n o r m a l i s e d f o r m u s c l e v o l u m e a r e s u m m a r i s e d in T a b l e 2. N o sig- n i f i c a n t d i f f e r e n c e c o u l d b e f o u n d a m o n g g r o u p s . D i s c u s s i o n W e a r e u n a w a r e o f p r e v i o u s h u m a n 3 1 p - N M R S s t u d i e s in w h i c h a d i r e c t c o m p a r i s o n o f [ A T P ] a n d [PC] o b - t a i n e d f r o m d i f f e r e n t s u b j e c t s h a s b e e n p e r f o r m e d . I n fact, t h o s e s t u d i e s in w h i c h t h e a u t h o r s h a v e n o t

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109 PC e x f e r n a l r e f e r e n c e I [ I I [ I 25 20 15 I0 5 ATP c~ i i i i i i i 0 -5 - I 0 -15 -20 -25 PPM

Fig. 2. Example 3aphosphorus-nuclear magnetic resonance (31p_ N M R ) spectrum. PC, Phosphocreatine; ATP, adenosine 5'-triphosphate; ppm, parts per million

1.0 • ~ 0.8 • 0 . 6 • 62 • 0 4

".

%.

0.0 • ' 110 ' , , , 0 20 30 40 50

Distance from coil axis (mm)

Fig. 3. Surface of inorganic phosphate (P~) peak area as a function of the distance from the coil axis. The P~ values have been normalised with respect to the condition of no interface between coil and muscle (distance of coil axis of 0 mm) The symbols rep- resent experimental values obtained on pig muscle after interpos- ing wooden plates of increasing thickness between the coil and the muscle surface

looked at absolute high energy phosphates values (e.g. Meyer 1988, 1989; Meyer et al. 1985; Miller et al. 1988; Mole et al. 1985) were mainly concerned with the changes in [PC] within the same subject as a function of some independent variables, such as the imposed

: D ,< g) t,, m "g I1) ,,i,- (/3 1 0 0 - 8 0 - 6 0 - 4 0 - 2 0 - 0 y = 2 1 . 6 4 + 3 2 . 5 8 x r = 0 . 6 9 n = 4 3 & & A . A " A A & . . - " ZXz~ /x 0 0.3 0.6 0.9 1.2 1.5 S k i n f o l d f h i c k n e s s (cm)

Fig. 4. Surface area of lipids on the ~H-nuclear magnetic resonance spectrum as a function of skinfold thickness at the site of the measurements

mechanical load, or changes of the muscle metabolic status following physical or chemical changes. For such studies, a knowledge of muscle high energy phosphate concentration, even in arbitrary units, is not a prere- quisite.

However, there are conditions, such as for example, the measurement of the kinetics of PC hydrolysis at the onset of constant load exercise in humans (see Binzoni et al. 1992) in which the knowledge of [ATP] and [PC], even in arbitrary units, is essential for the comparison of the rate of PC hydrolysis among different subjects. In such cases the important result is not the relative change of a given phosphorus metabolite as a function of time but only its absolute concentration difference. For the latter purpose a knowledge of the change, even in arbitrary (but identical) units, is required.

The [PC] and [ATP] values reported in this study are given in arbitrary units, and not in millimoles per litre, although an external phosphate reference was inserted at the centre of the coil. Despite the amount of phosphate inside the reference being known, a direct proportionality between the reference phosphate content and total muscle phosphates cannot be established, because the phosphorus nuclei in the former are in a physico-chemical configuration different from that in the latter. Indeed, the method would allow the assessment of the absolute concentrations of phosphates (in millimoles per litre) only if the external reference were uniformely distributed inside the muscle mass. It goes without saying that this condition cannot be ful- filled.

Despite its limitations, the present method provides for the first time a tool for estimating with sufficient precision the actual muscle volume detected by the coil (Fig. 3). This allows the standardization of the [ATP] and [PC] values obtained to a specific volume of muscle (approximately 70 ml in this study), from which a direct comparison among subjects can be made.

The present method was indeed based on an empir- ical calibration. The proposed method has been proved to work satisfactorily, as the internal variability of the

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f o u r m e a s u r e m e n t s o b t a i n e d in e a c h of 47 subjects did n o t e x c e e d 8 % . This m a y still a p p e a r to be r a t h e r a r o u g h estimate, b u t is b e t t e r b y far t h a n t h a t o b t a i n e d using b i o c h e m i c a l m e t h o d s b a s e d o n c h e m i c a l assays of e x t r e m e l y small n e e d l e b i o p s y samples, especially if o n e c o n s i d e r s t h a t b y t h e latter t e c h n i q u e n o t i m e d m e a s u r e m e n t s can be p e r f o r m e d .

T h e results o b t a i n e d b y the p r e s e n t m e t h o d s h o w e d t h a t n e i t h e r age n o r training affected the c o n c e n t r a t i o n o f high e n e r g y p h o s p h a t e s in h u m a n muscle. T h e find- ing o f an e q u a l l y high e n e r g y p h o s p h a t e c o n c e n t r a t i o n in s e d e n t a r y individuals a n d in athletic subjects con- firms p r e v i o u s results f r o m the literature t h a t h a v e b e e n b a s e d o n c h e m i c a l assays ( E r i k s s o n 1980; Saltin a n d G o l l n i c k 1983; T h o r s t e n s s o n et al. 1975). H a v i n g e x p a n d e d the m e a s u r e m e n t o f [ A T P ] a n d [PC] o v e r a wide age r a n g e s h o u l d p r o v e e x t r e m e l y useful f o r fu- t u r e studies o f p e a k m u s c l e p o w e r o f b o t h n o r m a l individuals (see t h e a c c o m p a n y i n g p a p e r , F e r r e t t i et al. (1994)) a n d f o r f u t u r e studies o n patients with p a t h o - logical m u s c l e conditions.

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