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Muscle ischaemia in peripheral vascular disease studied by 31P-magnetic resonance spectroscopy
Eur J VascSurg4, 637-642 (1990)
Muscle Ischaemia in Peripheral Vascular Disease Studied by al P-Magnetic Resonance Spectroscopy Linda J. Hands, Mohammed H. Sharif, Geoffrey S. Payne, Peter J. Morris and George K. Radda Magnetic Resonance Facility and Nuffield Department of Surgery, University of Oxford, John Radcliffe Hospital, Oxford, U.K. We have used 31phosphorus magnetic resonance spectroscopy (31P-MRS) to study foot muscle metabolism in patients with peripheral vascular disease. Sixteen patients with calf claudication, 32 patients with rest pain and 13 control subjects had spectra collectedfrom the foot muscle, Extensor digitorum brevis, ankle pressures measured and, in most cases, transcutaneous 02 and C02 recordings made over the foot. The intracellular pH and the ratio of inorganic phosphate to phosphocreatine (Pi/PCr) obtained from the MR spectra were signifi'cantly higher (p < 0.005 and p < 0.02, respectively) in the muscle of patients with rest pain and were particularly high in those with gangrene or ulceration. Ankle pressures and transcutaneous 02 and C02 measurements failed to distinguish those patients with advanced peripheral ischaemia. These results suggest that MRS measurements of metabolic changes in foot muscle are usefid in the detection and quantitation of significant distal ischaemia. Key Words: Magnetic resonance spectroscopy; Vascular disease; Foot muscle.
Introduction The patient with vascular disease presents with pain, ulceration or gangrene, all of which arise in distal ischaemic tissues. Yet most investigations are directed towards the pathological anatomy and haemodynamics of the more proximal major blood vessels, factors obviously important in assessment of possible surgical reconstruction. The state of the distal tissue is important for the patient in terms of symptoms, healing after amputation and the likelihood of salvage if blood flow is restored. Moreover, monitoring of changes in these tissues gives an objective measurement of the response to treatment. Tissue viability depends on maintenance of cell function which, in turn, relies on adequate energy production within the cell. 31p-magnetic resonance spectroscopy provides a means of studying cellular energy metabolism non-invasively, particularly in muscle, and has already been used to investigate the metabolic changes in calf muscle associated with claudication. 1-3 It provides a measure of the cellular Please address all correspondence to L. J. Hands, John Radcliffe Hospital, Oxford, OX3 9DU, U.K. 0950-821X/90/060637+06 $03.00/0© 1990Grune&StrattonLtd.
energy status from the relative concentrations of the phosphorus containing compounds important in energy metabolism and also allows intracellular pH to be measured. In this study we have extended the use of 31p_ MRS to the foot muscle, Extensor digitorum brevis, to assess metabolic change in the periphery associated with ischaemia ranging from claudication to rest pain.
Subjects Control subjects
Thirteen subjects, aged 41-79 years (median 71 years), including six females, were chosen on the basis of a similar mobility to the patients with severe peripheral vascular disease. All were recovering from surgery and had been bed or chair bound for 3 days-2 weeks (median 5 days). Two were diabetic, two were current. smokers and three were ex-smokers. Ten were taking simple analgesics such as coproxamol and five were on anti-hypertensive medication ([3-blockers, two; nifedipine, one; diuretics, three). None had any symptoms
638
L.J. Hands et al.
of peripheral vascular disease and all had normal leg pulses. Significant peripheral vascular disease was further excluded by measuring the ankle Doppler pressures after a 1 min exercise test 4 and these showed no fall from resting values which were above brachial pressure.
partial pressures were recorded after at least 15 min recording. 02 measurements were made in three control subjects, ten patients with claudication and 19 with rest pain. CO2 measurements were performed in three control subjects, eight patients with claudication and 13 with rest pain. CO 2
Patients
Collection of MRS data
Forty-eight patients with angiographic evidence of peripheral vascular disease affecting aorto-iliac, superficial femoral and distal arteries were studied. They were divided into two groups on the basis of their symptoms: 1. Thirty-two patients had unequivocal ischaemic rest pain in the foot. This diagnosis was based on a history of foot pain at rest at night, relieved by making the leg dependent, and signs of ischaemia in the foot (mottled skin, poor capillary return, gangrene or ulceration). Thirteen patients had ischaemic ulceration or gangrene, six were diabetic, six were current smokers and 17 were ex-smokers. All were taking simple analgesics, 12 were on anti-hypertensive medication ([3blockers, one; nifedipine, four; diuretic, nine) and three were taking perhexiline. 2. Sixteen patients had calf claudication but no foot symptoms. One of these was diabetic, four were current smokers and nine were ex-smokers. Two were taking simple analgesics and seven were on antihypertensive medication (nifedipine, two; diuretics, Local ethical committee approval for the study was obtained and all patients gave informed consent.
Subjects lay supine with the lower leg resting on a contoured foam block and the foot unsupported. A 23 m m diameter surface coil, mounted on a 1 mm thick perspex plate, was taped directly to the skin overlying Extensor digitorum brevis (EDB). The foot was earthed with a strip of aluminium foil around the ankle to reduce interference from airborne noise. The coil was tuned to the phosphorus frequency (32.7 MHz) with secondary tuning to the proton frequency (80.8 MHz) achieved by the transmission line method of Gordon and Timms. 5 A signal was collected from EDB with the foot at the centre of a 60 cm free bore 1.9 T superconducting magnet interfaced to a Bruker Biospec NMR spectrometer. The magnet was shimmed using the proton signal, usually to a linewidth of between 20 and 25 Hz. The phosphorus signal was collected using a radiofrequency pulse width of 20 ~s (at 20 W) with a repetition rate of 2 s. This combination yielded the maximum signal from phosphocreatine per unit time. In most cases 256 scans were sufficient to produce reasonable signal to noise in the spectrum although occasionally 512 scans were necessary. Total patient examination time was about 30 min.
Methods
Analysis of MRS data6
Ankle pressures
Figure 1 shows spectra obtained from both a normal and an ischaemic foot. Relative concentrations of phosphocreatine (PCr), inorganic phosphate (Pi), phosphomonoesters (PME), phosphodiesters (PDE) and ATP were obtained by triangulation of the appropriate peak ([3-ATP peak for ATP) and the results expressed as ratios. Correction for the saturation effects was made by comparing spectra obtained at a 2 s repetition rate with a fully relaxed spectra (using a pulse interval of 20 s) in seven subjects. This gave correction factors of 1.68_+ 0.28 for PCr/ATP and 1.57 + 0.41 for Pi/ATP. Correction factors for PME/ ATP and PDE/ATP were assumed to be the same as for Pi/ATP.
five).
All control subjects and patients had resting ankle Doppler pressures measured on the side to be studied.
Transcutaneous 02 and C02 measurements These were performed using a TCM3 monitor (Radiometer, Copenhagen). The patient lay supine for 20 min, the combined 02 and CO2 electrode was then attached to the skin overlying Extensor digitorum brevis and the plateau values of transcutaneous 02 and Eur J VascSurgVol4, December1990
31p-MRS in Peripheral Vascular Disease
(a)
4
(b)
2
5
I I0
I 5
| 0
6
I -5 -
7
I -I0
1 --15
I -20
p.p.m.
Fig. 1. (a) 31p-MRSspectrum from normal Extensor digitorum brevis; (b) 31P-MRSspectrum from Extensordigitorum brevis in patient with rest pain showing increase in inorganic phosphate concentration and reduction in phosphocreatine concentration. 1, phosphomonoesters; 2, inorganic phosphate; 3, phosphodiesters; 4, phosphocreatine; 5, y ATP;6, c~ATP; 7, 1~ATP. The intracellular pH was calculated from the chemical shift (o-) of the inorganic phosphate (Pi) relative to that of the PCr peak using the following formula: 6 pH = 6.75 + log(or - 3.27)/(5.69 - or)
639
higher Pi/PCr ratios than either the control group (p < 0.02) or the claudication group (p < 0.002) (Fig. 2a) but there was no significant difference between the latter two groups. The five highest values were obtained from patients with ulceration or gangrene, although in five other patients with similar changes from w h o m the ratio could be obtained it fell within the normal range (control m e a n + 2 S.D.). The median pH increased with the level of ischaemia (controls 7.11, claudication group 7.17 and rest pain group 7.19). The rest pain group had a significantly higher pH than the control group (p < 0.005) although there was considerable overlap between the two groups. However only one patient with ulceration or gangrene had a pH within the normal control range (mean + 2 S.D.) although two with these changes had a p H significantly lower t h a n that of control subjects (Fig. 2b). As might be expected from this, Pi/PCr tended to rise with increasing pH. However, w h e n Pi/PCr reached its highest levels, pH appeared to fall, at least in two of the patients (Fig. 3). There was a tendency for both PME/ATP a n d PDE/ATP to increase with increasing ischaemia but this did not reach a significant difference between the groups, although there was a significant positive correlation between both ratios a n d the pH (PME/ATP p < 0.0001, r -- 0.53; PDE/ATP p < 0.005, r = 0.40). Age, smoking history, medication, mobility and distribution of disease appeared to have no significant influence on the MRS results. There were too few diabetic patients in each group to determine w h a t effect this had on the metabolic changes. There was a significant difference between the ankle pressures of the rest pain group and both the control subjects (p<0.0001) a n d the claudication group (p<0.005). However, the ankle pressure did not provide a means of distinguishing those patients with ulceration and gangrene (Fig. 2c). There was a significant negative correlation b e t w e e n pH and ankle pressure over all the groups (p = 0.005, r = -0.43). Transcutaneous 02 a n d CO2 measurements did not differ significantly b e t w e e n the groups.
Statistical analysis The M a n n - W h i t n e y U test was used to determine significant differences between the groups.
Results
The group of patients with rest pain had significantly
Discussion
The changes in foot muscle metabolism detected by MRS occurred mainly with advanced ischaemia. The Pi/PCr ratio, in particular, increased only in those patients with rest pain. Phosphocreatine is the high energy store of muscle, whereas inorganic phosphate accumulates from ATP breakdown. The failure of the Eur J Vasc Surg Vol 4, December1990
640
L.J. Hands et aL
2.0
A - pat ents with u l c e r a t i o n / g a n g r e n e
1.5
~3 O. h--
1,0
z~
A
A
0.5
°i°
A A
I Controls
0,0
A
I
Patients w i t h cloudicotion
Patients with rest pain
(o) 7,5 • - patients with ulceration/gangrene 7.4
7,3 -17.2 A A 7.1 A
A
A
A
7.0
6,9 Controls
Patients with claudicafion
Patients with rest pain
(b) 200 • -patients
with ulceration/gangrene
150 -rE E
A A
.~ I 0 0
A
A
A
A
A
A A
A
A A
A
• •
o
'~
50
Controls
Patients with claudication
A A~A A
A A
Patients with rest pain
(c)
Fig. 2. (a) Inorganic p h o s p h a t e / p h o s p h o c r e a t i n e (Pi/PCr) ratios in each g r o u p ; (b) p H v a l u e s in each group; (c) ankle p r e s s u r e s in each group. Eur J Vasc Surg Vol 4, December 1990
31 P - M R S in Peripheral V a s c u l a r D i s e a s e
cell to maintain more of the phosphate in the high energy form of phosphocreatine reflects an impaired energy status, which presumably only develops with more advanced ischaemia either because there is a large reserve in energy production or because the cell is able to compensate for its reduced oxygen/substrate supply until extreme levels of ischaemia occur. However the pH changed at a much earlier stage. It tended to be higher in the patients with claudication than in the control subjects. It was significantly raised in the patients with rest pain and showed a significant negative correlation with ankle pressure in all the patients. There was also a significant positive correlation over all the patients between pH and the PME/ ATP and PDE/ATP ratios, probably related to increasing ischaemia and not necessarily interdependent. The PME peak includes signal from sugar phosphate intermediates of glycolysis which have been shown to accumulate in ischaemic muscle. 7 Phosphodiester levels probably relate to phospholipid breakdown and may represent damage caused by ischaemia. 8 Extracellular fluid becomes acidic in severely ischaemic limbs 9 and it is therefore surprising that the intracellular pH measured by MRS moves in the opposite direction. There are at least two possible explanations for this apparent paradox. Acute ischaemia is k n o w n to cause a fall in membrane potential. 10If the same occurs in chronic ischaemia then the intracellular pH, normally held lower than that of extracellular fluid, will tend to drift up towards pH 7.4. An alternative explanation is that intracellular buffering capacity increases with the fall in temperature associated with ischaemia. 11 Measurement of the degree of ischaemia once rest
7.5
641
pain has developed is difficult. The only objective evidence we had for increasing ischaemia was the presence of ulceration or gangrene. Such changes may be precipitated in a vulnerable extremity by increased external pressure and are hardly an absolute measure of ischaemia. This may explain why all the ulcerated/ gangrenous feet did not show extreme metabolic changes. Nonetheless, the Pi/PCr ratios and the intracellular pH do provide a means of distinguishing particularly severe ischaemia within the rest pain group, which the ankle pressures and transcutaneous oxygen/carbon dioxide measurements failed to do. For example, a Pi/PCr ratio greater than 0.75 was associated with ulceration and/or gangrene; presumably in feet where the ratio approaches this value such changes are imminent. At the other end of the rest pain spectrum, there were only five patients in the rest pain group who had unreconstructable disease but did not require an amputation because of the severity of their disease; four had an EDB pH and Pi/PCr within the normal range (mean + 2 S.D.), the fifth had an elevated pH and was managed by lumbar sympathectomy. Ankle pressure measurements do not provide the same discrimination because of the wide range of foot ischaemia associated with a pressure at or near zero. The current methods in widespread clinical use for assessing distal ischaemia, namely ankle pressure measurements and transcutaneous 02 measurements, are both, to some extent, removed from changes at a cellular level. It is these metabolic alterations which dictate whether tissue will survive and it seems that 31p-MRS parameters approach measurements of these changes more closely and so have
• -potients with ulcerotion/gangrene
7.4
7.3 JX -r Q.
7.2 ~X ,x
7.1
/~aa ~
a
7.0
6.9
= 0.0
I 0.5
i
1 1,0
i
I 1.5
2.0
Pi/PCr Fig. 3. Relationship between Pi/PCr and pH in Extensor digitorum brevis in controls and patients with peripheral vascular disease.
Eur J Vasc Surg Vol 4, December 1990
642
L.J. Hands et aL
potentially more importance for accurate assessment of cellular viability.
Acknowledgements The British Heart Foundation, The Medical Research Council and the Department of Health and Social Security provided financial support for this work.
References 1 KELLERU,OBERHANSLIR,HuBERP, etaI. Phosphocreatinecontent and intracellular pH of calf muscle measured by phosphorus NMR spectroscopy in occlusive arterial disease of the legs. Eur J Clin hwest 1985; 15: 382-388. 2 ZATINA MA, BERKOWITZHD, GROSS GM, MARIS JM, CHANCE B. 31p-nuclear magnetic resonance spectroscopy: non-invasive biochemical analysis of the ischemic extremity. J Vasc Surg 1986; 3: 411420. 3 HANDS LJ, BOREPJ, GALLOWAYG, MORRISPJ, RADDAGK. Muscle
Eur J Vasc Surg Vol 4, December 1990
metabolism in patients with peripheral vascular disease investigated by 31p-NMR spectroscopy. Clin Sci 1986; 71: 283-290. 4 LAING SP, GREENHALGH RM. Standard exercise test to assess peripheral arterial disease. BMJ 1980; 280: 13-16. 5 GORDONRE, TIMMS WE. An improved tune and match circuit for Boshimming in intact biological samples. J Magn Res 1982; 46: 322 324. 6 TAYLORDJ, BORE PJ, STYLESP, GADIAN DG, RADDA GK. Bioenergetics of intact h u m a n muscle. A 31p nuclear magnetic resonance study. Mo! Biol Med 1983; 1; 77-94. 7 THRELFALLCJ, STONERHB. The effects of hindlimb ischaemia on the products and intermediates of glycolysis in rat tissues. Biochemical J 1961; 79: 553-562. 8 RADDAGK, RAJAGOPALANB, TAYLORDJ. Biochemistry in vivo: an appraisal of clinical magnetic resonance spectroscopy. Magn Reson Quarterly 1989; 5: 122-151. 90'DONNELL TF, CLOWESGHA, BROWSENL, RYANNT. A metabolic approach to the evaluation of peripheral vascular disease. Surg Gynecol Obstet 1977; 144: 51-57. 10 JENNISCHEE, ENGERE, MEDEGARDA,APPELGRENL, HALJAMAEH. Correlation between tissue pH, cellular transmembrane potentials and cell energy metabolism during shock and ischaemia. Circulatory Shock 1978; 5: 251-260. 11 WHrTE FN. A comparative physiological approach to hypothermia. J Thorac Cardiovasc Surg 1981; 82: 821-831.
Accepted I August 1990
Muscle Ischaemia in Peripheral Vascular Disease Studied by al P-Magnetic Resonance Spectroscopy Linda J. Hands, Mohammed H. Sharif, Geoffrey S. Payne, Peter J. Morris and George K. Radda Magnetic Resonance Facility and Nuffield Department of Surgery, University of Oxford, John Radcliffe Hospital, Oxford, U.K. We have used 31phosphorus magnetic resonance spectroscopy (31P-MRS) to study foot muscle metabolism in patients with peripheral vascular disease. Sixteen patients with calf claudication, 32 patients with rest pain and 13 control subjects had spectra collectedfrom the foot muscle, Extensor digitorum brevis, ankle pressures measured and, in most cases, transcutaneous 02 and C02 recordings made over the foot. The intracellular pH and the ratio of inorganic phosphate to phosphocreatine (Pi/PCr) obtained from the MR spectra were signifi'cantly higher (p < 0.005 and p < 0.02, respectively) in the muscle of patients with rest pain and were particularly high in those with gangrene or ulceration. Ankle pressures and transcutaneous 02 and C02 measurements failed to distinguish those patients with advanced peripheral ischaemia. These results suggest that MRS measurements of metabolic changes in foot muscle are usefid in the detection and quantitation of significant distal ischaemia. Key Words: Magnetic resonance spectroscopy; Vascular disease; Foot muscle.
Introduction The patient with vascular disease presents with pain, ulceration or gangrene, all of which arise in distal ischaemic tissues. Yet most investigations are directed towards the pathological anatomy and haemodynamics of the more proximal major blood vessels, factors obviously important in assessment of possible surgical reconstruction. The state of the distal tissue is important for the patient in terms of symptoms, healing after amputation and the likelihood of salvage if blood flow is restored. Moreover, monitoring of changes in these tissues gives an objective measurement of the response to treatment. Tissue viability depends on maintenance of cell function which, in turn, relies on adequate energy production within the cell. 31p-magnetic resonance spectroscopy provides a means of studying cellular energy metabolism non-invasively, particularly in muscle, and has already been used to investigate the metabolic changes in calf muscle associated with claudication. 1-3 It provides a measure of the cellular Please address all correspondence to L. J. Hands, John Radcliffe Hospital, Oxford, OX3 9DU, U.K. 0950-821X/90/060637+06 $03.00/0© 1990Grune&StrattonLtd.
energy status from the relative concentrations of the phosphorus containing compounds important in energy metabolism and also allows intracellular pH to be measured. In this study we have extended the use of 31p_ MRS to the foot muscle, Extensor digitorum brevis, to assess metabolic change in the periphery associated with ischaemia ranging from claudication to rest pain.
Subjects Control subjects
Thirteen subjects, aged 41-79 years (median 71 years), including six females, were chosen on the basis of a similar mobility to the patients with severe peripheral vascular disease. All were recovering from surgery and had been bed or chair bound for 3 days-2 weeks (median 5 days). Two were diabetic, two were current. smokers and three were ex-smokers. Ten were taking simple analgesics such as coproxamol and five were on anti-hypertensive medication ([3-blockers, two; nifedipine, one; diuretics, three). None had any symptoms
638
L.J. Hands et al.
of peripheral vascular disease and all had normal leg pulses. Significant peripheral vascular disease was further excluded by measuring the ankle Doppler pressures after a 1 min exercise test 4 and these showed no fall from resting values which were above brachial pressure.
partial pressures were recorded after at least 15 min recording. 02 measurements were made in three control subjects, ten patients with claudication and 19 with rest pain. CO2 measurements were performed in three control subjects, eight patients with claudication and 13 with rest pain. CO 2
Patients
Collection of MRS data
Forty-eight patients with angiographic evidence of peripheral vascular disease affecting aorto-iliac, superficial femoral and distal arteries were studied. They were divided into two groups on the basis of their symptoms: 1. Thirty-two patients had unequivocal ischaemic rest pain in the foot. This diagnosis was based on a history of foot pain at rest at night, relieved by making the leg dependent, and signs of ischaemia in the foot (mottled skin, poor capillary return, gangrene or ulceration). Thirteen patients had ischaemic ulceration or gangrene, six were diabetic, six were current smokers and 17 were ex-smokers. All were taking simple analgesics, 12 were on anti-hypertensive medication ([3blockers, one; nifedipine, four; diuretic, nine) and three were taking perhexiline. 2. Sixteen patients had calf claudication but no foot symptoms. One of these was diabetic, four were current smokers and nine were ex-smokers. Two were taking simple analgesics and seven were on antihypertensive medication (nifedipine, two; diuretics, Local ethical committee approval for the study was obtained and all patients gave informed consent.
Subjects lay supine with the lower leg resting on a contoured foam block and the foot unsupported. A 23 m m diameter surface coil, mounted on a 1 mm thick perspex plate, was taped directly to the skin overlying Extensor digitorum brevis (EDB). The foot was earthed with a strip of aluminium foil around the ankle to reduce interference from airborne noise. The coil was tuned to the phosphorus frequency (32.7 MHz) with secondary tuning to the proton frequency (80.8 MHz) achieved by the transmission line method of Gordon and Timms. 5 A signal was collected from EDB with the foot at the centre of a 60 cm free bore 1.9 T superconducting magnet interfaced to a Bruker Biospec NMR spectrometer. The magnet was shimmed using the proton signal, usually to a linewidth of between 20 and 25 Hz. The phosphorus signal was collected using a radiofrequency pulse width of 20 ~s (at 20 W) with a repetition rate of 2 s. This combination yielded the maximum signal from phosphocreatine per unit time. In most cases 256 scans were sufficient to produce reasonable signal to noise in the spectrum although occasionally 512 scans were necessary. Total patient examination time was about 30 min.
Methods
Analysis of MRS data6
Ankle pressures
Figure 1 shows spectra obtained from both a normal and an ischaemic foot. Relative concentrations of phosphocreatine (PCr), inorganic phosphate (Pi), phosphomonoesters (PME), phosphodiesters (PDE) and ATP were obtained by triangulation of the appropriate peak ([3-ATP peak for ATP) and the results expressed as ratios. Correction for the saturation effects was made by comparing spectra obtained at a 2 s repetition rate with a fully relaxed spectra (using a pulse interval of 20 s) in seven subjects. This gave correction factors of 1.68_+ 0.28 for PCr/ATP and 1.57 + 0.41 for Pi/ATP. Correction factors for PME/ ATP and PDE/ATP were assumed to be the same as for Pi/ATP.
five).
All control subjects and patients had resting ankle Doppler pressures measured on the side to be studied.
Transcutaneous 02 and C02 measurements These were performed using a TCM3 monitor (Radiometer, Copenhagen). The patient lay supine for 20 min, the combined 02 and CO2 electrode was then attached to the skin overlying Extensor digitorum brevis and the plateau values of transcutaneous 02 and Eur J VascSurgVol4, December1990
31p-MRS in Peripheral Vascular Disease
(a)
4
(b)
2
5
I I0
I 5
| 0
6
I -5 -
7
I -I0
1 --15
I -20
p.p.m.
Fig. 1. (a) 31p-MRSspectrum from normal Extensor digitorum brevis; (b) 31P-MRSspectrum from Extensordigitorum brevis in patient with rest pain showing increase in inorganic phosphate concentration and reduction in phosphocreatine concentration. 1, phosphomonoesters; 2, inorganic phosphate; 3, phosphodiesters; 4, phosphocreatine; 5, y ATP;6, c~ATP; 7, 1~ATP. The intracellular pH was calculated from the chemical shift (o-) of the inorganic phosphate (Pi) relative to that of the PCr peak using the following formula: 6 pH = 6.75 + log(or - 3.27)/(5.69 - or)
639
higher Pi/PCr ratios than either the control group (p < 0.02) or the claudication group (p < 0.002) (Fig. 2a) but there was no significant difference between the latter two groups. The five highest values were obtained from patients with ulceration or gangrene, although in five other patients with similar changes from w h o m the ratio could be obtained it fell within the normal range (control m e a n + 2 S.D.). The median pH increased with the level of ischaemia (controls 7.11, claudication group 7.17 and rest pain group 7.19). The rest pain group had a significantly higher pH than the control group (p < 0.005) although there was considerable overlap between the two groups. However only one patient with ulceration or gangrene had a pH within the normal control range (mean + 2 S.D.) although two with these changes had a p H significantly lower t h a n that of control subjects (Fig. 2b). As might be expected from this, Pi/PCr tended to rise with increasing pH. However, w h e n Pi/PCr reached its highest levels, pH appeared to fall, at least in two of the patients (Fig. 3). There was a tendency for both PME/ATP a n d PDE/ATP to increase with increasing ischaemia but this did not reach a significant difference between the groups, although there was a significant positive correlation between both ratios a n d the pH (PME/ATP p < 0.0001, r -- 0.53; PDE/ATP p < 0.005, r = 0.40). Age, smoking history, medication, mobility and distribution of disease appeared to have no significant influence on the MRS results. There were too few diabetic patients in each group to determine w h a t effect this had on the metabolic changes. There was a significant difference between the ankle pressures of the rest pain group and both the control subjects (p<0.0001) a n d the claudication group (p<0.005). However, the ankle pressure did not provide a means of distinguishing those patients with ulceration and gangrene (Fig. 2c). There was a significant negative correlation b e t w e e n pH and ankle pressure over all the groups (p = 0.005, r = -0.43). Transcutaneous 02 a n d CO2 measurements did not differ significantly b e t w e e n the groups.
Statistical analysis The M a n n - W h i t n e y U test was used to determine significant differences between the groups.
Results
The group of patients with rest pain had significantly
Discussion
The changes in foot muscle metabolism detected by MRS occurred mainly with advanced ischaemia. The Pi/PCr ratio, in particular, increased only in those patients with rest pain. Phosphocreatine is the high energy store of muscle, whereas inorganic phosphate accumulates from ATP breakdown. The failure of the Eur J Vasc Surg Vol 4, December1990
640
L.J. Hands et aL
2.0
A - pat ents with u l c e r a t i o n / g a n g r e n e
1.5
~3 O. h--
1,0
z~
A
A
0.5
°i°
A A
I Controls
0,0
A
I
Patients w i t h cloudicotion
Patients with rest pain
(o) 7,5 • - patients with ulceration/gangrene 7.4
7,3 -17.2 A A 7.1 A
A
A
A
7.0
6,9 Controls
Patients with claudicafion
Patients with rest pain
(b) 200 • -patients
with ulceration/gangrene
150 -rE E
A A
.~ I 0 0
A
A
A
A
A
A A
A
A A
A
• •
o
'~
50
Controls
Patients with claudication
A A~A A
A A
Patients with rest pain
(c)
Fig. 2. (a) Inorganic p h o s p h a t e / p h o s p h o c r e a t i n e (Pi/PCr) ratios in each g r o u p ; (b) p H v a l u e s in each group; (c) ankle p r e s s u r e s in each group. Eur J Vasc Surg Vol 4, December 1990
31 P - M R S in Peripheral V a s c u l a r D i s e a s e
cell to maintain more of the phosphate in the high energy form of phosphocreatine reflects an impaired energy status, which presumably only develops with more advanced ischaemia either because there is a large reserve in energy production or because the cell is able to compensate for its reduced oxygen/substrate supply until extreme levels of ischaemia occur. However the pH changed at a much earlier stage. It tended to be higher in the patients with claudication than in the control subjects. It was significantly raised in the patients with rest pain and showed a significant negative correlation with ankle pressure in all the patients. There was also a significant positive correlation over all the patients between pH and the PME/ ATP and PDE/ATP ratios, probably related to increasing ischaemia and not necessarily interdependent. The PME peak includes signal from sugar phosphate intermediates of glycolysis which have been shown to accumulate in ischaemic muscle. 7 Phosphodiester levels probably relate to phospholipid breakdown and may represent damage caused by ischaemia. 8 Extracellular fluid becomes acidic in severely ischaemic limbs 9 and it is therefore surprising that the intracellular pH measured by MRS moves in the opposite direction. There are at least two possible explanations for this apparent paradox. Acute ischaemia is k n o w n to cause a fall in membrane potential. 10If the same occurs in chronic ischaemia then the intracellular pH, normally held lower than that of extracellular fluid, will tend to drift up towards pH 7.4. An alternative explanation is that intracellular buffering capacity increases with the fall in temperature associated with ischaemia. 11 Measurement of the degree of ischaemia once rest
7.5
641
pain has developed is difficult. The only objective evidence we had for increasing ischaemia was the presence of ulceration or gangrene. Such changes may be precipitated in a vulnerable extremity by increased external pressure and are hardly an absolute measure of ischaemia. This may explain why all the ulcerated/ gangrenous feet did not show extreme metabolic changes. Nonetheless, the Pi/PCr ratios and the intracellular pH do provide a means of distinguishing particularly severe ischaemia within the rest pain group, which the ankle pressures and transcutaneous oxygen/carbon dioxide measurements failed to do. For example, a Pi/PCr ratio greater than 0.75 was associated with ulceration and/or gangrene; presumably in feet where the ratio approaches this value such changes are imminent. At the other end of the rest pain spectrum, there were only five patients in the rest pain group who had unreconstructable disease but did not require an amputation because of the severity of their disease; four had an EDB pH and Pi/PCr within the normal range (mean + 2 S.D.), the fifth had an elevated pH and was managed by lumbar sympathectomy. Ankle pressure measurements do not provide the same discrimination because of the wide range of foot ischaemia associated with a pressure at or near zero. The current methods in widespread clinical use for assessing distal ischaemia, namely ankle pressure measurements and transcutaneous 02 measurements, are both, to some extent, removed from changes at a cellular level. It is these metabolic alterations which dictate whether tissue will survive and it seems that 31p-MRS parameters approach measurements of these changes more closely and so have
• -potients with ulcerotion/gangrene
7.4
7.3 JX -r Q.
7.2 ~X ,x
7.1
/~aa ~
a
7.0
6.9
= 0.0
I 0.5
i
1 1,0
i
I 1.5
2.0
Pi/PCr Fig. 3. Relationship between Pi/PCr and pH in Extensor digitorum brevis in controls and patients with peripheral vascular disease.
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potentially more importance for accurate assessment of cellular viability.
Acknowledgements The British Heart Foundation, The Medical Research Council and the Department of Health and Social Security provided financial support for this work.
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Accepted I August 1990