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Muscle high energy phosphates in chronic peripheral vascular disease
JOURNAL
OF SURGICAL
44, 277-283 (1988)
RESEARCH
Muscle High Energy Phosphates
in Chronic Peripheral Vascular Disease’
GEORGE J. TODD, M.D.,2 BARBARA VAN DE WIELE, M.D., J. ASKANAZI, M.D., KEIJI YOSHIKAWA, M.D., DAVID H. ELWYN, PH.D., JOHN M. KINNEY, M.D., AND KEITH REEMTSMA, M.D. Departments of Surgery and Anesthesiology, College of Physicians & Surgeons, Columbia University, New York, New York 10032 Submitted for publication February9, 1987 There is little information available concerning the alterations in skeletal muscle energy metabolism which occur in response to chronic arterial occlusive disease. In addition, the effect of arterial reconstruction on skeletal muscle energy metabolism in patients with peripheral vascular disease has not been defined. Needle biopsies were obtained from the quadriceps femoris muscle of 7 patients with aortoiliac diseaseand 15 patients with femoropopliteal diseaseand from the gastrocnemius muscle of 9 patients with femoropopliteal disease.Muscle samples were analyzed for ATP, ADP, AMP, phosphocreatine, creatine, and lactate. Eleven patients were rebiopsied after vascular reconstruction. Patients with rest pain had decreasedtotal adenine nucleotides, energy charge potential, and ATP/ADP ratios as compared to those of controls. ATP levels were significantly decreasedin muscle samples obtained distal to the arterial occlusion (i.e., quadriceps/aortoiliac, gastrocnemius/femoropopliteal) in patients with rest pain (compared with controls). ATP levels did not differ significantly from those of controls in muscle samples obtained from patients with claudication. However, energy charge potential was significantly decreasedin all patients with claudication regardlessof biopsy site and location of arterial occlusive disease. Normalization of muscle energy metabolism was not demonstrated following arterial reconstruction. We conclude that resting skeletal muscle energy metabolism is abnormal in patients with chronic arterial insufficiency and that progression of diseasetoward more severeischemia is associatedwith more marked derangement. Whether the possible beneficial effects of revascularization on muscle energy metabolism are masked by the concurrent effect of injury in the early postoperative period remains to be clarified. 0 1988 Academic Press, Inc.
the metabolic changeswhich occur in muscle tissue during chronic ischemia could assistin Maintenance of high energy phosphate the design of optimal, scientifically based stores in skeletal muscle depends on a baltreatment programs. ance between substrate and oxygen supplies This study examines resting levels of musand energy utilization. While it is known that cle high energy phosphates in patients with acute ischemia results in rapid depletion of arterial occlusive disease using Bergstrom’s high energy phosphate reserves in skeletal technique of percutaneous muscle biopsy muscle [I], little information is available [2]. Levels of high energy phosphates in the concerning the alterations in muscle energy affected extremity were determined before metabolism which occur in response to and after arterial reconstruction. In addition, chronic arterial occlusive disease. In addi- the relationship between high energy phostion, the effect of arterial reconstruction on phate levels and the clinical symptoms and changes in muscle energy metabolism in pa- distribution of arterial occlusive disease has tients with peripheral vascular disease has been examined. not been defined. A further understanding of INTRODUCTION
MATERIALS ’ Supported by U.S. Army Contract No. 49-193MD2552 and Public Health Service Grant No. 6Ml4546. 2 To whom reprint requests should be addressed at 161 Fort Washington Avenue, New York, NY 10032.
AND METHODS
Patients Thirty one patients with chronic arterial occlusive disease participated in the study.
277
0022-4804/88 $1.50 Copyright 0 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.
278
JOURNAL
OF SURGICAL
RESEARCH:
There were 15 male and 16 female patients with a mean age of 64.6 years (range 40-84 years). All patients underwent routine preoperative evaluation, including arteriography prior to vascular reconstruction. On the day of operation, after induction of anesthesia, percutaneous needle biopsies of the quadriceps femoris muscle of the affected extremity were performed in 22 patients. Needle biopsies of the gastrocnemius muscle of the affected extremity were performed in 9 patients. Thirteen patients underwent repeat biopsy of the quadriceps femoris muscle and one patient underwent repeat biopsy of the gastrocnemius muscle. The second biopsies were performed under local anesthesia (Xylocaine 1%). Repeat biopsies were performed between Postoperative Days 7 and 11, except for two patients who were rebiopsied 20 and 90 days after vascular reconstruction. Biopsies of the quadriceps femoris muscle were obtained from 15 patients with femoropopliteal disease and 7 patients with aortoiliac disease.Of the 15 patients with femoropopliteal disease,9 had rest pain or incipient gangrene and 6 had intermittent claudication. Of the 7 patients with aortoiliac disease, 5 had rest pain or incipient gangrene and 2 had intermittent claudication. Needle biopsies of the gastrocnemius were obtained only from patients with femoropopliteal disease.In this group, 4 had intermittent claudication and 5 had incipient gangrene or rest pain. Ten of thirty-one patients had diabetes mellitus. Controls Seven patients undergoing total hip replacement were chosen for the control group (four female, three male, mean age 59 years) [3]. These patients had previously been studied under similar conditions in our laboratory. They were similar in age and level of activity but demonstrated no clinical evidence of arterial occlusive disease. Preoperative evaluation was identical except that the patients with vascular disease underwent arteriographic evaluation. On the morning of operation, after induction of
VOL. 44, NO. 3, MARCH
1988
anesthesia, percutaneous muscle biopsies were obtained from the quadriceps femoris of the unaffected extremity. This study was approved by the Institutional Review Board of Columbia Presbyterian Medical Center. The nature and purpose of the study and possible risks involved were explained to all patients. Written informed consent was obtained. Muscle Biopsy and Analysis Muscle samples were obtained from the quadriceps femoris by the percutaneous needle biopsy technique described earlier [2]. In the operating room, following induction of anesthesia, a OS-cm incision was made in the skin of the lateral aspect of the thigh, 15-20 cm above the knee, and the needle was introduced into the muscle. Muscle samples of the gastrocnemius were obtained by introducing the needle into the gastrocnemius muscle through a surgical incision in the medial aspect of the leg. Repeat biopsies were performed under local anesthesia (Xylocaine 1%) with anesthetic infiltration restricted to the skin. Samples were frozen by plunging the needle into liquid freon maintained at its melting point. Samples were then transferred to liquid nitrogen and stored. Muscle samples were freeze-dried, extracted, and analyzed for ATP, ADP, AMP, phosphocreatine (PC), creatine (CR), and lactate by modification of the enzymatic methods of Bergemeyer [4]. Total adenine nucleotides (TAN), energy charge potential (ECP), and ATP/ADP ratio were calculated. RESULTS
Resting Levels of High Energy Phosphates in Chronic Ischemia High energy phosphate levels in muscle samples obtained from the quadriceps femoris and gastrocnemius at operation are presented in Table 1 according to distribution of the arterial occlusive disease and presenting symptoms. Significantly de-
1
82 + 7 25.7 f 16”
50 f 9 8.5 f 0.9’
66.6 + 1.2
4.1 f 0.2
25.0 f 1.9 0.949 f 0.003
CR
LAC
TAN EC 8.1 + 0.7 5.9 2 0.5d
Note. Values are expressed as Means + SE (mmole/kg DW). f, too low to measure. a P i 0.05, bP < 0.02, ‘P < 0.01, dP 4 0.001, patients vs controls.
ATP/ADP
9.9 zk 0.5
59 + 0.3
45 * 10
54 f 8
66.6 + 1.2
PC
21.2 t 1.8 0.924 + 0.004d
0.4 ? 0.29
+
0.13 + 0.02
0.13 f 0.05
0.15 f 0.23
AMP
23.6 + 2.3 0.935 f 0.005c
2.6 f 0.4
2.9 + 0.3
3.1 + O.ld
2.6 f 0.3
2.3 f 0.2
ADP
6.6 +- 0.5’
5.9 f l.lC
17.2 f 2.7” 0.892 + 0.4
8.1 + 0.2’
24 + 2.6 0.939 + 0.001”
8.6 f 2.7
5.7 -+ 1
21.9 + 1.1 0.934 + 0.004”
64+ 14 8.5 f 2
11’
27+
58 5 5’
11
62?
0.15 f 0.02
21.2 f 2.3 2.6 + 0.3
Claudication (n = 4)
16.5 f 2.4’ 2.5 k 0.5
Rest pain (n = 5)
6.7 + 0.07’
19.2 * 2.8 0.926 k 0.007’
49 f 9” 10.2 t 3.4
59 + 5
0.15 f 0.02
Femoropopliteal (n = 9)
Gastrocnemius
37 +- 12b
14.2 f 2.6c
18.9 zt 0.7
18.1 + 1.8
20.8 -t 2.2
Rest pain (n = 5)
22.5 f 1.9
Claudication (n = 2)
ATP
Clinical symptoms:
Rest pain (n = 9)
Aortoiliac (n = 7)
Claudication (n = 6)
Femoropopliteal (?I = 15)
Quadriceps femoris
Controls (n = 7)
Distribution of occlusive disease:
Biopsy Site:
HIGH ENERGY PHOSPHATE CONTENT IN SKELETAL MUSCLE BY BIOPSY SITE, CLINICAL SYMPTOMS, AND DISTRIBUTION OF ARTERIAL OCCLUSIVE DISEASE
TABLE
% u t; F iz
+
8
5
2
F 2 VI
8 %
E
2
280
JOURNAL OF SURGICAL RESEARCH: VOL. 44, NO. 3, MARCH 1988
TABLE 2 creased levels of ATP, as compared to those of controls, were seen in muscle samples obEFFECTOF REVASCULARIZATION(PREOP/POSTOP) tained from the gastrocnemius muscle of paGroup I Group II tients with femoropopliteal disease and rest (12= 5) (n = 6) pain and from the quadriceps femoris muscle of patients with aortoiliac diseaseand rest 23.8 f 1.1” 12.9 + 1.9 pain (P < 0.0 1). Significantly decreasedtotal ATP 14.5 * 2.5 16.2 + 1.7 adenine nucleotides, as compared to those of 3.35 * 0.20 2.37 + 0.26 controls, were seen in muscle samples ob- ADP ____ 2.60 + 0.30 2.43 f 0.36 tained from the quadriceps femoris of patients with aortoiliac diseaseand rest pain (P 0.395 F 0.29 0.082 +- 0.03 < 0.05). Energy charge potential was signifi- AMP 0.036 f 0.02 0.084 2 0.03 cantly decreased, as compared to that of 64 f 5 35k 11 controls, in all but one (aortoiliac/rest pain) PC 43 It 9 47 + 8 of the six groups of patients. The ATP to 67 f 4 27 f 11 ADP ratio was significantly decreased in all but one (femoropopliteal/claudication) of CR 50 * 9 3Ok 12 the six groups of patients. Both PC and CR 27.3 + 1.0” 15.7 + 1.8 were significantly decreased in muscle sam- TAN 18.8 4 2.0 17.2 + 2.9 ples obtained from the quadriceps muscle of 0.89 ~tr0.04 0.94 f 0.004b patients with aortoiliac disease. EC
Revascularization Eleven patients who underwent successful vascular reconstruction (i.e., patent bypass grafts) were rebiopsied on or between the seventh to the eleventh postoperative day. Second biopsies were obtained from the quadriceps femoris in 10 patients and from the gastrocnemius in 1 patient. Analysis of muscle samples obtained from these patients identified two separatepatient populations. Six patients demonstrated a relatively normal pattern of high energy phosphates in muscle samples obtained at operation. Five patients had markedly depressedATP levels. For the purpose of analysis and discussion we have labeled patients with low preoperative ATP levels (defined as an ATP value more than two standard deviations below the mean control level) as Group I and patients with a relatively normal preoperative pattern of high energy phosphates as Group II. Levels of high energy phosphates before and after revascularization are presented in Table 2. Prior to revascularization, ADP, TAN, PC, CR, and ATP/ADP were significantly lower and AMP was significantly
ATP ADP
0.93 + 0.004 5.7 f 0.1 __-
0.91 f 0.01 7.3 f 0.6
6.3 i 0.4
5.9 f 0.6
Note. Values are expressedas Means f SE (mmole/kg DW). 0 P < 0.05, preop vs postop. bP < 0.02, preop vs postop.
higher in Group I than in Group II. ATP levels in Group I patients rose from a mean of 12.9 to 16.2 M/kg DW. In Group II, ATP levels declined significantly (P < 0.02) from a mean of 23.8 w/kg DW to a mean of 14.5 mM/kg DW. TAN and ECP also decreased significantly following revascularization in Group II patients. In contrast, no significant change was demonstrated in values of TAN and ECP in Group I patients. Two patients with rest pain in whom femoropopliteal bypass was unsuccessful experienced exacerbation of symptoms of arterial insufficiency postoperatively. In one patient ATP and TAN levels were markedly depressedprior to vascular reconstruction. The second biopsy, obtained on Postoperative Day 8, following development of worsening
TODD ET AL.: MUSCLE PHOSPHATES IN VASCULAR DISEASE
281
rest pain and cutaneous gangrene of the foot, demonstrated a further decreasein ATP and TAN. The second patient had nearly normal ATP and TAN levels prior to operation and decreasedlevels of ATP and TAN in muscle samples obtained on Postoperative Day 20 following the development of gangrene of the foot. It should be noted that muscle samples were obtained from the quadriceps femoris in both of these patients. Finally, one patient with aortoiliac diseaseand rest pain prior to vascular reconstruction was biopsied 90 days following surgery. ATP and TAN levels, initially low, rose to near normal levels. These data are presented in Table 3.
unaffected unless shock is profound and prolonged [ 111.Production of local ischemia by total occlusion of lower extremity arterial flow by means of an upper thigh tourniquet depletes muscle high energy phosphates in humans [ 121and rabbits [ 131. There is little information available concerning alterations in resting skeletal muscle energy metabolism in patients with chronic ischemia. In the present study, we observed resting ATP levels that were not significantly different from those of controls in quadriceps muscle samples from patients with intermittent claudication. Energy charge potential, which reflects the relative concentrations of ATP, ADP, and AMP and is more DISCUSSION important to the cell than the size of the adeDepletion of high energy phosphate re- nylate pool [ 131,was significantly decreased servesin acute ischemia has been studied ex- as compared to that of controls in this group. tensively. Metabolically active organs such as However, in muscle samples obtained distal liver and kidney are rapidly depleted of to the arterial occlusion (i.e., gastrocnemius/ adenosine triphosphate during shock [ 5-91. femoropopliteal; quadriceps/aortoiliac), paSkeletal muscle high energy phosphates are tients with rest pain had significantly dedepleted in patients in cardiogenic shock [I] creased ATP, TAN, ECP, and ATP/ADP and in healthy humans performing heavy ratios as compared to those of controls. exercise [lo]. Animal experiments with a Phosphocreatine and creatine content exhibshock model have demonstrated that work- ited great variation in samples obtained prior ing skeletal muscle (diaphragm) is rapidly to vascular reconstruction; however, both depleted of high energy phosphates, whereas were significantly decreased in muscle from resting skeletal muscle (soleus) is basically patients with aortoiliac disease.
TABLE 3 HIGH ENERGY PHOSPHATE LEVELS IN Two PATIENTS WITH POSTOPERATIVE EXACERBATION OF SYMPTOMS OF ARTERIAL INSUFFICIENCY (1 AND 2) AND ONE PATIENT WITH POSTOPERATIVE RESOLUTION OF SYMPTOMS BIOPSIED 3 MONTHS AFTER VASCULAR RECONSTRUCTION (3)
Patient 1 (interval
8 days)
Patient 2 (interval 20 days) Patient 3 (interval 90 days)
ATP
ADP
AMP
TAN
ECP
14.1
1.3 2.0
0.356
15.8
0.94
0.07 I
14.1
0.92
2.2 1.4
0.124
22.0
0.95
0.107
13.1
0.94
12.1 19.7 11.6 9.3 18.9
Values are expressed as mmole/kg DW.
1.6 3.1
0.029
10.9
0.92
0.023
22.0
0.93
282
JOURNAL OF SURGICAL RESEARCH: VOL. 44, NO. 3, MARCH 1988
These results are similar to the findings of Pernow et al. [ 141 showing decreased ATP concentrations in quadriceps muscle obtained from patients with severeintermittent claudication although the decrease in the present study did not reveal statistical significance. Neither decreased ATP/ADP ratio and decreased ECP in muscle of patients with arterial insufficiency nor a correlation between ATP and TAN content and the severity of ischemia as evidenced by clinical symptoms has, to our knowledge, been previously demonstrated. Previous studies have shown increased in vitro succinic oxidase activity and increased in vitro incorporation of glucose into CO*, lactate, and glycogen in muscle samples obtained from quadriceps and gastrocnemius of patients with intermittent claudication [ 151, but not in patients with rest pain. It is interesting to note that these observations parallel the changes in high energy phosphate metabolism seen in this study. Significantly decreased levels of ATP without complete depletion of PC stores, as seenin the patients studied, are interesting in view of the widespread assumption, based on exercise studies of normal patients, that ATP levels in the resting state are maintained because of the ready availability of high energy bonds from creatine phosphate. The pattern in chronic ischemia, decreasedATP prior to PC depletion, parallels that observed in injury [ 161.The explanation for decreasedECP in quadriceps muscle of patients with femoropopliteal disease and decreased ECP, TAN, and ATP/ADP ratios in quadriceps muscle of patients with femoropopliteal disease and rest pain is somewhat difficult to discern. It may reflect the generalized nature of arteriosclerotic disease.It has been postulated that inactivity underlies reduced concentrations of PC and TAN in muscles of elderly subjects [ 171; however, it seems unlikely to contribute to these findings as ATP concentrations, ECP and, ATP/ADP ratios are normal in muscle from elderly subjects [ 171. In addition, control subjects in our study would be expected to have similarly
reduced activity levels secondary to hip disease. Neither consistent nor significant normalization of muscle high energy phosphates was demonstrated following arterial reconstruction. A significant depression of high energy phosphate levels was identified in Group II patients who had, prior to vascular reconstruction, relatively normal values. It should be noted that Group I patients were predominantly, though not exclusively, patients with aortoiliac disease and rest pain. Group II patients were predominantly patients with femoropopliteal diseaseand claudication in whom, following femoropopliteal bypass, the blood supply to the quadriceps femoris would be expected to be unchanged. Liaw et al. [ 161 have shown a progressive decrease, proportional to the severity of injury, of cellular levels of high energy phosphates in skeletal muscle of patients who have sustained major trauma. It seemslikely that the changes observed in Group II patients are related to injury and that possible beneficial effects of arterial reconstruction in Group I patients are masked by the concurrent effect of injury in the early postoperative period. In summary, peripheral arterial insufficiency is associated with abnormal skeletal muscle energy metabolism; progression of diseasetoward more severe ischemia is associated with more pronounced derangement of high energy phosphate metabolism. Whether the observed changes are due to inadequate or slow resynthesis of ATP due to oxygen or substrate deficit, functional disturbance of mitochondrial or cellular membranes, structural changes in muscle secondary to ischemia, or disturbance of the regulation of the purine nucleotide cycle remains to be elucidated. REFERENCES . Karlsson, J., Wiilerson, J. T., Leshin, S. J., Mullins, C. B., and Mitchell, J. H. Skeletal muscle metabolites in patients with cardiogenic shock or severe congestive heart failure. Stand. J. Clin. Lab. Invest. 35: 13, 1975.
TODD ET AL.: MUSCLE PHOSPHATES IN VASCULAR DISEASE 2. Bergstrom, J. Muscle electrolytes in man. &and. J. Clin. Lab. Invest. 14(Suppl. 68): 1, 1962. 3. Liaw, K. Y., Askanazi, J., Michelsen, C. B., Furst,
P. F., Elwyn, D. H., and Kinney, J. M. Effect of postoperative nutrition on muscle high energy phosphates. Ann. Surg. 195: 12, 1982. 4. Bergemeyer, H. U. (Ed.). Methods of Enzymatic Analysis. New York Academic Press, 1974. 5. Chaudry, I. H., Sayeed, M. M., and Baue, A. E. Alterations in high-energy phosphates in hemorrhagic shock as related to tissue and organ function. Surgery 79: 666, 1976. 6. Crowell, J. W., Jones, C. E., and Smith, E. E. Effect of allopurinol on hemorrhagic shock. Amer. J. Physiol. 216: 744, 1969. 7. LePage, G. A. Biological energy transformations during shock as shown by tissue analysis. Amer. J. Physiol. 146: 267, 1946. 8. Loiselle, J. M., and Denstedt, 0. F. Biochemical changesduring acute physiological failure in the rat.
II. The behavior of adenine and pyridine nucleotides of liver during shock. Canad. J. Biochem. 42: 21, 1964. 9. McShan, W. H., Potter, V. R., Goldman, A., Shipley, E. G., and Meyer, R. K. Biological energy transformations during shock as shown by blood chemistry. Amer. J. Physiol. 145: 93, 1945. 10. Karlsson, J. Lactate and phosphagen concentrations in working muscle of man. Acta Physiol. &and. (‘suppl.) 358: 1, 1971.
283
11. Chaudry, I. H., Sayeed, M. M., and Baue, A. E. Effect of hemorrhagic shock on tissue adenine nucleotides in conscious rats. Canad. J. Pharmacol. 52: 131,1974. 12. Harris, R. C., Hultman, E., Kauser, L., and Nordesjo, L. 0. The effect of circulatory occlusion on isometric exercise capacity and energy metabolism of the quadriceps muscle in man. Scund. J. Clin. Lab. Invest. 35: 87, 1975. 13. Imai, S., Riley, A. L., and Beme, R. M. Effect of
&hernia on adenine nucleotides in cardiac and skeletal muscle. Circ. Res. 15: 443, 1964. 14. Pemow, B., Saltin, J., Wahren, J., and Cronestrand, R. Muscle metabolism during exercise in patients with occlusive arterial disease:Effect of reconstructive surgery. Stand. J. C/in. Lab. Invest. 31 (Suppl. 128): 21, 1973. 15 Holm, J., Bjomtorp, P., and Schersten, T. Meta’ bolic activity in human skeletal muscle. Effect of peripheral arterial insufficiency. Eur. J. Clin. Invest. 2: 321, 1972.
16. Liaw, K. Y., Askanazi, J., Michelsen, C. B., Kantrowitz, L. R., Furst, P., and Kinney, J. M. Effect of injury and sepsis on high-energy phosphates in muscle and red cells. J. Trauma 20: 755, 1980. 17. Moller, P., Begstrom, J., Furt, P., and Hellstrom, K. Effect of aging on energy rich phosphagens in human skeletal muscles. Clin. Sci. 58: 553, 1980.
OF SURGICAL
44, 277-283 (1988)
RESEARCH
Muscle High Energy Phosphates
in Chronic Peripheral Vascular Disease’
GEORGE J. TODD, M.D.,2 BARBARA VAN DE WIELE, M.D., J. ASKANAZI, M.D., KEIJI YOSHIKAWA, M.D., DAVID H. ELWYN, PH.D., JOHN M. KINNEY, M.D., AND KEITH REEMTSMA, M.D. Departments of Surgery and Anesthesiology, College of Physicians & Surgeons, Columbia University, New York, New York 10032 Submitted for publication February9, 1987 There is little information available concerning the alterations in skeletal muscle energy metabolism which occur in response to chronic arterial occlusive disease. In addition, the effect of arterial reconstruction on skeletal muscle energy metabolism in patients with peripheral vascular disease has not been defined. Needle biopsies were obtained from the quadriceps femoris muscle of 7 patients with aortoiliac diseaseand 15 patients with femoropopliteal diseaseand from the gastrocnemius muscle of 9 patients with femoropopliteal disease.Muscle samples were analyzed for ATP, ADP, AMP, phosphocreatine, creatine, and lactate. Eleven patients were rebiopsied after vascular reconstruction. Patients with rest pain had decreasedtotal adenine nucleotides, energy charge potential, and ATP/ADP ratios as compared to those of controls. ATP levels were significantly decreasedin muscle samples obtained distal to the arterial occlusion (i.e., quadriceps/aortoiliac, gastrocnemius/femoropopliteal) in patients with rest pain (compared with controls). ATP levels did not differ significantly from those of controls in muscle samples obtained from patients with claudication. However, energy charge potential was significantly decreasedin all patients with claudication regardlessof biopsy site and location of arterial occlusive disease. Normalization of muscle energy metabolism was not demonstrated following arterial reconstruction. We conclude that resting skeletal muscle energy metabolism is abnormal in patients with chronic arterial insufficiency and that progression of diseasetoward more severeischemia is associatedwith more marked derangement. Whether the possible beneficial effects of revascularization on muscle energy metabolism are masked by the concurrent effect of injury in the early postoperative period remains to be clarified. 0 1988 Academic Press, Inc.
the metabolic changeswhich occur in muscle tissue during chronic ischemia could assistin Maintenance of high energy phosphate the design of optimal, scientifically based stores in skeletal muscle depends on a baltreatment programs. ance between substrate and oxygen supplies This study examines resting levels of musand energy utilization. While it is known that cle high energy phosphates in patients with acute ischemia results in rapid depletion of arterial occlusive disease using Bergstrom’s high energy phosphate reserves in skeletal technique of percutaneous muscle biopsy muscle [I], little information is available [2]. Levels of high energy phosphates in the concerning the alterations in muscle energy affected extremity were determined before metabolism which occur in response to and after arterial reconstruction. In addition, chronic arterial occlusive disease. In addi- the relationship between high energy phostion, the effect of arterial reconstruction on phate levels and the clinical symptoms and changes in muscle energy metabolism in pa- distribution of arterial occlusive disease has tients with peripheral vascular disease has been examined. not been defined. A further understanding of INTRODUCTION
MATERIALS ’ Supported by U.S. Army Contract No. 49-193MD2552 and Public Health Service Grant No. 6Ml4546. 2 To whom reprint requests should be addressed at 161 Fort Washington Avenue, New York, NY 10032.
AND METHODS
Patients Thirty one patients with chronic arterial occlusive disease participated in the study.
277
0022-4804/88 $1.50 Copyright 0 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.
278
JOURNAL
OF SURGICAL
RESEARCH:
There were 15 male and 16 female patients with a mean age of 64.6 years (range 40-84 years). All patients underwent routine preoperative evaluation, including arteriography prior to vascular reconstruction. On the day of operation, after induction of anesthesia, percutaneous needle biopsies of the quadriceps femoris muscle of the affected extremity were performed in 22 patients. Needle biopsies of the gastrocnemius muscle of the affected extremity were performed in 9 patients. Thirteen patients underwent repeat biopsy of the quadriceps femoris muscle and one patient underwent repeat biopsy of the gastrocnemius muscle. The second biopsies were performed under local anesthesia (Xylocaine 1%). Repeat biopsies were performed between Postoperative Days 7 and 11, except for two patients who were rebiopsied 20 and 90 days after vascular reconstruction. Biopsies of the quadriceps femoris muscle were obtained from 15 patients with femoropopliteal disease and 7 patients with aortoiliac disease.Of the 15 patients with femoropopliteal disease,9 had rest pain or incipient gangrene and 6 had intermittent claudication. Of the 7 patients with aortoiliac disease, 5 had rest pain or incipient gangrene and 2 had intermittent claudication. Needle biopsies of the gastrocnemius were obtained only from patients with femoropopliteal disease.In this group, 4 had intermittent claudication and 5 had incipient gangrene or rest pain. Ten of thirty-one patients had diabetes mellitus. Controls Seven patients undergoing total hip replacement were chosen for the control group (four female, three male, mean age 59 years) [3]. These patients had previously been studied under similar conditions in our laboratory. They were similar in age and level of activity but demonstrated no clinical evidence of arterial occlusive disease. Preoperative evaluation was identical except that the patients with vascular disease underwent arteriographic evaluation. On the morning of operation, after induction of
VOL. 44, NO. 3, MARCH
1988
anesthesia, percutaneous muscle biopsies were obtained from the quadriceps femoris of the unaffected extremity. This study was approved by the Institutional Review Board of Columbia Presbyterian Medical Center. The nature and purpose of the study and possible risks involved were explained to all patients. Written informed consent was obtained. Muscle Biopsy and Analysis Muscle samples were obtained from the quadriceps femoris by the percutaneous needle biopsy technique described earlier [2]. In the operating room, following induction of anesthesia, a OS-cm incision was made in the skin of the lateral aspect of the thigh, 15-20 cm above the knee, and the needle was introduced into the muscle. Muscle samples of the gastrocnemius were obtained by introducing the needle into the gastrocnemius muscle through a surgical incision in the medial aspect of the leg. Repeat biopsies were performed under local anesthesia (Xylocaine 1%) with anesthetic infiltration restricted to the skin. Samples were frozen by plunging the needle into liquid freon maintained at its melting point. Samples were then transferred to liquid nitrogen and stored. Muscle samples were freeze-dried, extracted, and analyzed for ATP, ADP, AMP, phosphocreatine (PC), creatine (CR), and lactate by modification of the enzymatic methods of Bergemeyer [4]. Total adenine nucleotides (TAN), energy charge potential (ECP), and ATP/ADP ratio were calculated. RESULTS
Resting Levels of High Energy Phosphates in Chronic Ischemia High energy phosphate levels in muscle samples obtained from the quadriceps femoris and gastrocnemius at operation are presented in Table 1 according to distribution of the arterial occlusive disease and presenting symptoms. Significantly de-
1
82 + 7 25.7 f 16”
50 f 9 8.5 f 0.9’
66.6 + 1.2
4.1 f 0.2
25.0 f 1.9 0.949 f 0.003
CR
LAC
TAN EC 8.1 + 0.7 5.9 2 0.5d
Note. Values are expressed as Means + SE (mmole/kg DW). f, too low to measure. a P i 0.05, bP < 0.02, ‘P < 0.01, dP 4 0.001, patients vs controls.
ATP/ADP
9.9 zk 0.5
59 + 0.3
45 * 10
54 f 8
66.6 + 1.2
PC
21.2 t 1.8 0.924 + 0.004d
0.4 ? 0.29
+
0.13 + 0.02
0.13 f 0.05
0.15 f 0.23
AMP
23.6 + 2.3 0.935 f 0.005c
2.6 f 0.4
2.9 + 0.3
3.1 + O.ld
2.6 f 0.3
2.3 f 0.2
ADP
6.6 +- 0.5’
5.9 f l.lC
17.2 f 2.7” 0.892 + 0.4
8.1 + 0.2’
24 + 2.6 0.939 + 0.001”
8.6 f 2.7
5.7 -+ 1
21.9 + 1.1 0.934 + 0.004”
64+ 14 8.5 f 2
11’
27+
58 5 5’
11
62?
0.15 f 0.02
21.2 f 2.3 2.6 + 0.3
Claudication (n = 4)
16.5 f 2.4’ 2.5 k 0.5
Rest pain (n = 5)
6.7 + 0.07’
19.2 * 2.8 0.926 k 0.007’
49 f 9” 10.2 t 3.4
59 + 5
0.15 f 0.02
Femoropopliteal (n = 9)
Gastrocnemius
37 +- 12b
14.2 f 2.6c
18.9 zt 0.7
18.1 + 1.8
20.8 -t 2.2
Rest pain (n = 5)
22.5 f 1.9
Claudication (n = 2)
ATP
Clinical symptoms:
Rest pain (n = 9)
Aortoiliac (n = 7)
Claudication (n = 6)
Femoropopliteal (?I = 15)
Quadriceps femoris
Controls (n = 7)
Distribution of occlusive disease:
Biopsy Site:
HIGH ENERGY PHOSPHATE CONTENT IN SKELETAL MUSCLE BY BIOPSY SITE, CLINICAL SYMPTOMS, AND DISTRIBUTION OF ARTERIAL OCCLUSIVE DISEASE
TABLE
% u t; F iz
+
8
5
2
F 2 VI
8 %
E
2
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JOURNAL OF SURGICAL RESEARCH: VOL. 44, NO. 3, MARCH 1988
TABLE 2 creased levels of ATP, as compared to those of controls, were seen in muscle samples obEFFECTOF REVASCULARIZATION(PREOP/POSTOP) tained from the gastrocnemius muscle of paGroup I Group II tients with femoropopliteal disease and rest (12= 5) (n = 6) pain and from the quadriceps femoris muscle of patients with aortoiliac diseaseand rest 23.8 f 1.1” 12.9 + 1.9 pain (P < 0.0 1). Significantly decreasedtotal ATP 14.5 * 2.5 16.2 + 1.7 adenine nucleotides, as compared to those of 3.35 * 0.20 2.37 + 0.26 controls, were seen in muscle samples ob- ADP ____ 2.60 + 0.30 2.43 f 0.36 tained from the quadriceps femoris of patients with aortoiliac diseaseand rest pain (P 0.395 F 0.29 0.082 +- 0.03 < 0.05). Energy charge potential was signifi- AMP 0.036 f 0.02 0.084 2 0.03 cantly decreased, as compared to that of 64 f 5 35k 11 controls, in all but one (aortoiliac/rest pain) PC 43 It 9 47 + 8 of the six groups of patients. The ATP to 67 f 4 27 f 11 ADP ratio was significantly decreased in all but one (femoropopliteal/claudication) of CR 50 * 9 3Ok 12 the six groups of patients. Both PC and CR 27.3 + 1.0” 15.7 + 1.8 were significantly decreased in muscle sam- TAN 18.8 4 2.0 17.2 + 2.9 ples obtained from the quadriceps muscle of 0.89 ~tr0.04 0.94 f 0.004b patients with aortoiliac disease. EC
Revascularization Eleven patients who underwent successful vascular reconstruction (i.e., patent bypass grafts) were rebiopsied on or between the seventh to the eleventh postoperative day. Second biopsies were obtained from the quadriceps femoris in 10 patients and from the gastrocnemius in 1 patient. Analysis of muscle samples obtained from these patients identified two separatepatient populations. Six patients demonstrated a relatively normal pattern of high energy phosphates in muscle samples obtained at operation. Five patients had markedly depressedATP levels. For the purpose of analysis and discussion we have labeled patients with low preoperative ATP levels (defined as an ATP value more than two standard deviations below the mean control level) as Group I and patients with a relatively normal preoperative pattern of high energy phosphates as Group II. Levels of high energy phosphates before and after revascularization are presented in Table 2. Prior to revascularization, ADP, TAN, PC, CR, and ATP/ADP were significantly lower and AMP was significantly
ATP ADP
0.93 + 0.004 5.7 f 0.1 __-
0.91 f 0.01 7.3 f 0.6
6.3 i 0.4
5.9 f 0.6
Note. Values are expressedas Means f SE (mmole/kg DW). 0 P < 0.05, preop vs postop. bP < 0.02, preop vs postop.
higher in Group I than in Group II. ATP levels in Group I patients rose from a mean of 12.9 to 16.2 M/kg DW. In Group II, ATP levels declined significantly (P < 0.02) from a mean of 23.8 w/kg DW to a mean of 14.5 mM/kg DW. TAN and ECP also decreased significantly following revascularization in Group II patients. In contrast, no significant change was demonstrated in values of TAN and ECP in Group I patients. Two patients with rest pain in whom femoropopliteal bypass was unsuccessful experienced exacerbation of symptoms of arterial insufficiency postoperatively. In one patient ATP and TAN levels were markedly depressedprior to vascular reconstruction. The second biopsy, obtained on Postoperative Day 8, following development of worsening
TODD ET AL.: MUSCLE PHOSPHATES IN VASCULAR DISEASE
281
rest pain and cutaneous gangrene of the foot, demonstrated a further decreasein ATP and TAN. The second patient had nearly normal ATP and TAN levels prior to operation and decreasedlevels of ATP and TAN in muscle samples obtained on Postoperative Day 20 following the development of gangrene of the foot. It should be noted that muscle samples were obtained from the quadriceps femoris in both of these patients. Finally, one patient with aortoiliac diseaseand rest pain prior to vascular reconstruction was biopsied 90 days following surgery. ATP and TAN levels, initially low, rose to near normal levels. These data are presented in Table 3.
unaffected unless shock is profound and prolonged [ 111.Production of local ischemia by total occlusion of lower extremity arterial flow by means of an upper thigh tourniquet depletes muscle high energy phosphates in humans [ 121and rabbits [ 131. There is little information available concerning alterations in resting skeletal muscle energy metabolism in patients with chronic ischemia. In the present study, we observed resting ATP levels that were not significantly different from those of controls in quadriceps muscle samples from patients with intermittent claudication. Energy charge potential, which reflects the relative concentrations of ATP, ADP, and AMP and is more DISCUSSION important to the cell than the size of the adeDepletion of high energy phosphate re- nylate pool [ 131,was significantly decreased servesin acute ischemia has been studied ex- as compared to that of controls in this group. tensively. Metabolically active organs such as However, in muscle samples obtained distal liver and kidney are rapidly depleted of to the arterial occlusion (i.e., gastrocnemius/ adenosine triphosphate during shock [ 5-91. femoropopliteal; quadriceps/aortoiliac), paSkeletal muscle high energy phosphates are tients with rest pain had significantly dedepleted in patients in cardiogenic shock [I] creased ATP, TAN, ECP, and ATP/ADP and in healthy humans performing heavy ratios as compared to those of controls. exercise [lo]. Animal experiments with a Phosphocreatine and creatine content exhibshock model have demonstrated that work- ited great variation in samples obtained prior ing skeletal muscle (diaphragm) is rapidly to vascular reconstruction; however, both depleted of high energy phosphates, whereas were significantly decreased in muscle from resting skeletal muscle (soleus) is basically patients with aortoiliac disease.
TABLE 3 HIGH ENERGY PHOSPHATE LEVELS IN Two PATIENTS WITH POSTOPERATIVE EXACERBATION OF SYMPTOMS OF ARTERIAL INSUFFICIENCY (1 AND 2) AND ONE PATIENT WITH POSTOPERATIVE RESOLUTION OF SYMPTOMS BIOPSIED 3 MONTHS AFTER VASCULAR RECONSTRUCTION (3)
Patient 1 (interval
8 days)
Patient 2 (interval 20 days) Patient 3 (interval 90 days)
ATP
ADP
AMP
TAN
ECP
14.1
1.3 2.0
0.356
15.8
0.94
0.07 I
14.1
0.92
2.2 1.4
0.124
22.0
0.95
0.107
13.1
0.94
12.1 19.7 11.6 9.3 18.9
Values are expressed as mmole/kg DW.
1.6 3.1
0.029
10.9
0.92
0.023
22.0
0.93
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These results are similar to the findings of Pernow et al. [ 141 showing decreased ATP concentrations in quadriceps muscle obtained from patients with severeintermittent claudication although the decrease in the present study did not reveal statistical significance. Neither decreased ATP/ADP ratio and decreased ECP in muscle of patients with arterial insufficiency nor a correlation between ATP and TAN content and the severity of ischemia as evidenced by clinical symptoms has, to our knowledge, been previously demonstrated. Previous studies have shown increased in vitro succinic oxidase activity and increased in vitro incorporation of glucose into CO*, lactate, and glycogen in muscle samples obtained from quadriceps and gastrocnemius of patients with intermittent claudication [ 151, but not in patients with rest pain. It is interesting to note that these observations parallel the changes in high energy phosphate metabolism seen in this study. Significantly decreased levels of ATP without complete depletion of PC stores, as seenin the patients studied, are interesting in view of the widespread assumption, based on exercise studies of normal patients, that ATP levels in the resting state are maintained because of the ready availability of high energy bonds from creatine phosphate. The pattern in chronic ischemia, decreasedATP prior to PC depletion, parallels that observed in injury [ 161.The explanation for decreasedECP in quadriceps muscle of patients with femoropopliteal disease and decreased ECP, TAN, and ATP/ADP ratios in quadriceps muscle of patients with femoropopliteal disease and rest pain is somewhat difficult to discern. It may reflect the generalized nature of arteriosclerotic disease.It has been postulated that inactivity underlies reduced concentrations of PC and TAN in muscles of elderly subjects [ 171; however, it seems unlikely to contribute to these findings as ATP concentrations, ECP and, ATP/ADP ratios are normal in muscle from elderly subjects [ 171. In addition, control subjects in our study would be expected to have similarly
reduced activity levels secondary to hip disease. Neither consistent nor significant normalization of muscle high energy phosphates was demonstrated following arterial reconstruction. A significant depression of high energy phosphate levels was identified in Group II patients who had, prior to vascular reconstruction, relatively normal values. It should be noted that Group I patients were predominantly, though not exclusively, patients with aortoiliac disease and rest pain. Group II patients were predominantly patients with femoropopliteal diseaseand claudication in whom, following femoropopliteal bypass, the blood supply to the quadriceps femoris would be expected to be unchanged. Liaw et al. [ 161 have shown a progressive decrease, proportional to the severity of injury, of cellular levels of high energy phosphates in skeletal muscle of patients who have sustained major trauma. It seemslikely that the changes observed in Group II patients are related to injury and that possible beneficial effects of arterial reconstruction in Group I patients are masked by the concurrent effect of injury in the early postoperative period. In summary, peripheral arterial insufficiency is associated with abnormal skeletal muscle energy metabolism; progression of diseasetoward more severe ischemia is associated with more pronounced derangement of high energy phosphate metabolism. Whether the observed changes are due to inadequate or slow resynthesis of ATP due to oxygen or substrate deficit, functional disturbance of mitochondrial or cellular membranes, structural changes in muscle secondary to ischemia, or disturbance of the regulation of the purine nucleotide cycle remains to be elucidated. REFERENCES . Karlsson, J., Wiilerson, J. T., Leshin, S. J., Mullins, C. B., and Mitchell, J. H. Skeletal muscle metabolites in patients with cardiogenic shock or severe congestive heart failure. Stand. J. Clin. Lab. Invest. 35: 13, 1975.
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