本文へ移動
Amino Acid Balance in Human Myocardium
 
FFECTS OF EXERCISE AND PACING LOADS ON MYOCARDIAL AMINO ACID BALANCE IN PATIENTS WITH NORMAL AND STENOTIC CORONARY ARTERIES WITH SPECIAL REFERENCE TO BRANCHED CHAIN AMINO ACIDS 
Yoshiharu Yamada, MD, Tadashi Ishihara, MD, Masataka Fujiwara, MD Shigemi Tamoto, MD, Ichiro Seki, MD and Nakaaki Ohsawa, MD
 The First Department of Internal Medicine, Osaka Medical College, Osaka,Japan.
 Mailing address:Yoshiharu Yamada,MD, First Department of Internal Medicine, Osaka Medical College, Daigakucho 2-7,Takatsuki city ,Osaka, Japan
 Telephone number 0726(83)1221
Japanese Circulation Journal Vol.57 No.4 April1993 pages272-282
Arterial and coronary sinus differences(A-S) of alanine, glutamate, isoleucine, leucine, valine and phenylalanine were measured in 7 control subjects and 12 patients with coronary artery disease(CAD) at rest and during exercise, and in 8 controls and 21 CAD patients at restand during pacing. Lactate, great cardiac vein flow and oxygen weresimultaneously measured; however, none of these parameters distinguished CAD from controls. Alterations of alanine and glutamate duringeach load were, for the most part, consistent with previous studies, i.e. greater release of alanine and uptake of glutamate in the ischemicgroup. A-S of isoleucine, leucine and valine showed significantpositive correlation to that of alanine(r=0.59,r=0.89,r=0.77respectively during exercise and r=0.57,r=0.58,r=0.64 , respectively during pacing). A-S of isoleucine, leucine and valine showedsignificant positive correlation to each arterial concentration only during exercise(r=0.54,r=0.62,r=0.63 respectively), not during pacing. Although uptake of each branched chain amino acid(BCAA) wasnot significant, the mean A-S of each BCAA was positive at rest in bothcontrols and CAD and declined during each load. A-S of leucine was significantly smaller in CAD than controls during exercise(0.7±7.0 vs6.8±4.1μmol/l, P<0.05) and those of leucine and valine were significantly smaller in CAD patients with ischemic electrocardiographicchange than in those without electrocardiographic change during pacing(0.1±5.9 vs 6.1±5.5, P<0.05; -3.1±10.1 vs 9.9±6.8μmol/l,P<0.01, respectively). These results suggest that BCAA, especially leucine and valine, tend to be taken up by human myocardium physiologically and show characteristic alterations under myocardial stress together with alanine and glutamate.
Glucose, fatty acid and lactate are the major oxidative substrates in the myocardial tissues. Although the essential role of amino acids isincorporation in heart protein, amino acid metabolism is closely linked to pyruvate or intermediates of the Krebs cycle by a transamination reaction in skeletal or cardiac muscle, so that amino acidmetabolism is considered to alter aaccording to the changes of carbohydrate or lipid utilization. Many in vivo studies have demonstrated alanine accumulation1-5 and glutamate depletion6 in myocardialtissue under the condition of oxygen deprivation, and these findings are attributed to myocardial adaptation to anaerobic metabolism. Myocardial amino acid metabolism in humans was first studied byCarlsten7 et al, who reported that there was no net myocardial uptakeor release other than the release of alanine. Mudge et al8 reported that glutamate uptake at rest and alanine release during pacing in eight patients who had normal coronary anatomy were significant, and that glutamate uptake and alanine release both at rest and duringpacing were significant in eleven patients with coronary artery disease. They further found that glutamate uptake and alanine release were greater in patients with coronary artery disease than in patients with normal coronary anatomy both at rest and during pacing. More recently, Thomassen et al9-12 examined the relation between alanine, glutamate and carbohydrate metabolism and suggested that glutamate plays an important role in maintaining a glycolytic flux in humans. On the other hand, in vitro studies demonstrated that branched chain amino acids(BCAA), which are mainly metabolized extrahepatically, are taken up by the myocardial tissues as oxidative substrates under conditions of limited glucose utilization13,14. Furthermore, an in vivo study showed that there was significant myocardial uptake of isoleucine and leucine in the basal state and that the plasma concentration of BCAA determined the rate of myocardial BCAA uptake15. On the other hand, in a descript
Methods
Patient
Fourty-eight consecutive patients who underwent diagnostic coronary angiography were the subjects of this study; they consisted of 15 control subjects with chest pain syndrome who had angiographically normal coronary arteries, and 33 patients with coronary artery disease demonstrating more than 75% luminal stenosis of the left anterior descending artery (CAD). Of these 48 patients, exercise load was undertaken for 7 controls and 12 CAD patients and pacing load for the remaining 8 controls and 21 CAD patients. The 7 control subjects in the exercise group consisted of 3 males and 4 females, mean age 59±9(48-69), and the 8 control subjects in the pacing group consisted of 1 male and 7 females, mean age 64±5(55-71). CAD patients of each group were further subdivided during each load into two groups, those without ischemic electrocardiographic change[ECG(-)] and those with electrocardiographic change[ECG(+)]. Features of the CAD patients are shown in Table I. All patients were without evidence of valvular disease or diabetes, and antianginal agents including β-blocker were withdrawn 7 days before the study. All patients gave written informed consent for participation in this study.
Exercise protocol
After overnight fasting, a 7F Webster catheter was introduced into the pulmonary artery via an antecubital vein in the supine position and the cardiac output was obtained by the Fick direct method; then the catheter was repositioned in the mid portion of the coronary sinus under fluoroscopic guidance and the position was checked by injection of a small amount of contrast medium. For arterial blood samples and pressure recordings, a short polyethylene catheter was inserted into the brachial artery by the Seldinger technique. After a 20-minute resting period, great cardiac vein flow(GCVF) was obtained by the N2O desaturation method17 with simultaneous sampling of arterial and coronary sinus blood, and electrocardiographic recordings were obtained for resting values. Then exercise load was performed in a supine bicycle ergometer(Godalta) at 50 Watt・50 r.p.m. for 15 minutes, and exercise values of all variables were obtained in the same fashion after 10 minutes of exercise.
 Pacing protocol
 A 7F Webster catheter and arterial catheter were introduced by the same method as in the exercise protocol, and a 7F thermodilution catheter was added into the pulmonary artery for cardiac output. GCVF was obtained by the thermodilution technique and other variables were obtained for resting values. Then atrial pacing was carried out at 130 p.p.m. by the distal two electrodes of the Webster catheter and the pacing values of variables were obtained at 3 minutes of pacing. No patients received atropine-sulphate at pacing. In both exercise and pacing groups, ST-T segment of lead V4,V5 was measured at 0.04 msec after j-point, and development of ST depression more than -1.0mm from the basal state or pseudonormalization of T wave during load were considered to be ischemic.
Chemical and hemodynamic measurement
Arterial and coronary sinus blood samples were collected into heparinized ice-cooled glass tubes and lactate was measured by the hydroxydiphenyl method. For amino acid determination, heparinized samples were immediately placed into ice and centrifuged at 3000 r.p.m. for 15 minutes within 30 minutes of collection. The plasma was deproteinized by addition of sulfosalicylic acid and centrifuged at 3000 r.p.m. for 15 minutes. The protein-free plasma extract was quickly frozen, stored at -20゜C and analyzed within 7 days. Individual amino acid determinations were made by an automatic amino acid analyzer (HITACHI L-8500) with lithium citrate buffer system. Myocardial substrate uptake was calculated by arterial concentration(A) minus coronary sinus concentration(S), and myocardial extraction ratio by A-S/A(%). Cardiac index(CI) was measured by the Fick direct method in the exercise group and by the thermodilution method in the pacing group. Oxygen content of blood samples(vol%) was measured by the Van-Slyke Neill method18 in the exercise group and by calculation according to the formula Hb×1.34× O2 saturation in the pacing group. Myocardial oxygen consumption was calculated by O2(A-S)×GCVF(ml/100g/min in the exercise group, ml/min in the pacing group)
Data analysis
All variables were expressed as mean±SD. Relations between myocardial uptake(A-S) of each individual amino acid were determined by linear regression analysis in all subjects of each group, and comparisons between controls and CAD or [ECG(-)] and [ECG(+)] in each group were assessed with Student's t test. A p value of less than 0.05 wasconsidered to be significant.
RESULTS
 Electrocardiographic change In the exercise group, 8 of 12 CAD patients demonstrated ischemic electrocardiographic change during exercise. 6 of these 8 showed ST depression, while the remaining 2 showed seudonormalization of T wave. In the pacing group, 13 of 21 CAD patients demonstrated ischemic electrocardiographic change during pacing. 11 of these 13 showed ST depression, while 2 showed pseudonormalization of T wave. Although the significance of pseudonormalization of T wave as myocardial ischemia is controversial, the total number of such patients is small and there are reports that T wave reversion seen in cases of previous myocardial infarction may reflect myocardial ischemia19, so we classified such findings as ischemic in the present study.
Hemodynamics, oxygen and lactate metabolism
Cardiac index(CI), great cardiac vein flow(GCVF), coronary sinus oxygen content(Cs02), myocardial oxygen consumption(MVO2), lactate uptake(lac(A-S)) and lactate extraction ratio(LER) are shown in Table II. None of these parameters distinguished CAD from controls or ECG(+) from ECG(-). In the exercise group, the mean A-S of lactate increased, accompanied by a raised arterial concentration in both controls and CAD, while LER decreased in CAD and ECG(+) during exercise. In the pacing group, both mean A-S of lactate and LER decreased in CAD and ECG(+) during pacing, and 7 of 21 CAD patients demonstrated lactate production during pacing.
Relation between arterial concentration and myocardial extraction
In the resting values from all 48 subjects, including controls and CAD, myocardial uptake of lactate was correlated positively with arterial concentration(r=0.41,p<0.01) and that of glutamate and leucine was also correlated(r=0.71 p<0.01,r=0.29 p<0.05 respectively), while other amino acids showed no significant correlation. During each load, myocardial uptake of lactate showed significant positive correlation to arterial concentration both during exercise(r=0.50 p<0.05) and pacing(r=0.42 p<0.05). Among the amino acids, (A-S) of glutamate showed significant positive correlation with arterial concentration during exercise and pacing. With regard to BCAA, A-S of total BCAA and each BCAA showed significant positive correlation with arterial concentration only during exercise(total BCAA:r=0.65 p<0.01, isoleucine:r=0.54 p<0.05, leucine:r=0.62 p<0.01, valine: r=0.63 p<0.01) as represented in Figure 1.
Transmyocardial amino acid balance
Correlation coefficient between myocardial uptake of each BCAA, alanine and phenylalanine during exercise and pacing is shown in Table III. A-S of isoleucine, leucine and valine showed siginificant positive correlation with that of alanine, while there were no significant correlation with that of phenylalanine during each load. There was significant positive correlation between the uptake of each BCAAduring both loads. Individual myocardial amino acid balance before and during exercise and pacing is summarized in Table IV and BCAA balance during each loadis shown in Figure 2. In the exercise group, mean A-S of alanine was positive in controls(13.8±28.7μmol/l) and negative in CAD(-15.9±34.2μmol/l) at rest. During exercise, (A-S) of alanine decreased in both controls and CAD, and it was significantly smaller in CAD compared to controls(-22.9±33.5 vs 3.0±18.6μmol/l). In the pacing group, mean (A-S) of alanine was negative in both controls(-1.6±13.8μmol/l) and CAD(-6.4±15.6μmol/l) at rest, and became more negative in CAD patients during pacing and it was significantly smaller in CAD than controls(-12.7±15.1 vs 1.2±8.8μmol/l). Among the CAD patients, A-S of alanine was significantly smaller in ECG(+) than ECG(-) during pacing(-17.6±16.4 vs -3.9±8.7μmol/l.) A-S of glutamate was significantly different from zero before and during both exercise and pacing except in some cases, so glutamate showed significant myocardial uptake. In the pacing group, glutamate uptake decreased during pacing in both controls and CAD, and it was significantly greater in CAD than controls both before and during pacing(31.9±15.0 vs 21.5±9.6, 20.5±8.4 vs 12.4±8.7μmol/l, respectively). None of the BCAA showed significant uptake or release in controls or CAD before or during each load; however, the mean A-S value at rest of each BCAA was positive in both controls and CAD, indicating that they tended to be taken up by the heart at rest. In the exercise group, the mean A-S of these amino acids in controls was greater than in CA
DISCUSSION
 Previous study of myocardial amino acid metabolism in humans has been investigated mainly in chronic ischemic heart disease7-12,16 , in which certain characteristic alterations were observed, such as a significant uptake of glutamate and release of alanine in myocardial ischemia. Glutamate uptake in the myocardial tissue is postulated to be a contribution to anaerobic energy production16,20-26 or to the malate-aspartate shuttle for maintenance of glycolytic flux27,28. Alanine is produced from glycolytic pyruvate by a transamination reaction1,2,29, which is postulated to be a nitrogen transfer from myocardial tissue. In the present study, only glutamate uptake was significant and no other amino acid examined was taken up or released in significant amounts. Although these insignificant results except for glutamate uptake were due to wide variation of A-S values in each individual subject, observations of greater uptake of glutamate and greater release of alanine in CAD patients than in controls were for the most part consistent with the findings of previous reports. On the other hand, BCAA, being different from other amino acids, are mainly metabolized extrahepatically and are taken up by skeletal or cardiac muscle as an oxidative substrate in vitro. Despite the well-recognized role of BCAA in animals, description of BCAA balance in human myocardium has been given only by Brodan et al16, who found that there was uptake of isoleucine and leucine, although not in significant amounts, during pacing in patients with coronary artery disease. Mudge et al8 reported that the balance of each BCAA was not significant and they did not make further reference to BCAA. Against such a background, the present study was focused on BCAA, to examine transmyocardial balance and the significance of these amino acids in the human myocardium. In our results, although the uptake of each BCAA was not statistically significant, the mean A-S of each BCAA at rest was positive in both controls and CAD patients. A-S values tended to decline dur
REFERENCES
 1.Taegtmeyer H,Ferguson AG,Lesch M:Protein degradation and amino acid metabolism in autolyzing rabbit myocardium. Exp Mol Pathol 1977;26:52-62
 2.Taegtmeyer H,Peterson NB,Ragavan VV,Ferguson AG,Lesch M:De novo alanine synthesis in isolated oxygen-deprived rabbit myocardium. J Biol Chem 1977;252:5010-5018
 3.Freminet A,Leclerc L,Poyart C,Huel C,Gentil M:Alanine and succinate accumulation in the perfused rat heart during hypoxia. J Physiol 1980;76:113-117
 4.Freminet A:Carbohydrate and amino acid metabolism during acute hypoxia in rats: blood and heart metabolites. Comp Biochem Physiol 1981;70B:427-433
 5.Tischler ME,Goldberg AL:Production of alanine and glutamine by atrial muscle from fed and fasted rats. Am J Physiol 1980;238:E487-E493 6.Wiesner RJ,Deussen A,Borst M,Schrader J,Grieshaber MK:Glutamate degradation in the ischemic dog heart:contribution to anaerobic energy production. J Moll Cell Cardiol 1989;21:49-59
 7.Carlsten A,Hallgren B,Jagenburg R,Werko L:Myocardial metabolism of glucose,lactic acid,amino acids and fatty acids in healthy human individuals at rest and at different work loads. Scandinav.J.Clin.£Lab.Investigation 1961;13:418-428
 8.Mudge GH,Mills RM,Taegtmeyer H,Gorlin R,Lesch M:Alterations of myocardial amino acid metabolism in chronic ischemic heart disease.J Clin invest 1976;58:1185-1192
 9.Thomassen AR,Nielsen TT,Bagger JP,Henningsen P:Myocardial exchanges of glutamate, alanine and citrate in controls and patients with coronary artery disease.Clin Sci 1983;64:33-40
 10.Thomassen AR,Nielsen TT,Bagger JP,Thuesen L:Myocardial glutamate and alanine exchanges related to carbohydrate metabolism in patients with normal and stenotic coronary arteries.Clin Physiol 1984;4:425-434 11.Thomassen AR,Mortensen PT,Nielsen TT,Falstie-Jensen N:Altered plasma concentrations of glutamate,alanine and citrate in the early phase of acute myocardial infarction in man.Eur Heart J 1986;7:773-778 12.Thomassen AR,Bagger JP,Nielsen TT,Henningsen P:Altered global myocardial substrate preference ar rest and during pacing in coronary artery disease with stable angina pectoris.Am J Cardiol 1988;62:686-693 13.Buse MG,Biggers JF,Drier C,Buse JF:The effect of epinephrine, glucagon, and the nutritional state on the oxidation of branched chain amino acids and pyruvate by isolated hearts and diaphragms of the rat. J Biol Chem 1973; 248: 697-706
 14. Buse MG,Biggers JF,Friderici KH,Buse JF:Oxidation of branched chain amino acids by isolated hearts and diaphragms of the rat.J Biol Chem 1972;247:8085-8096
 15.Schwartz RG,Barrett EJ,Francis CK,Jacob R,Zalet BL:Regulation of myocardial amino acid balance in the conscious dog.J Clin.Invest. 1985;75:1204-1211
 16.Brodan V,Fabian J,Andel M,Pechar J:Myocardial amino acid metabolism in patients with chronic ischemic heart disease. Basic Res Cardiol 1978;73:160-170
 17.Kety SS,Schmidt CF:Nitrous oxide method for quantitative determination of cerebral blood flow in man:Theory,procedure and normal values.J Clin Invest 1948;27:476-483
 18.Van Slyke DD,Neill JM:Determination of gases in blood and other solutions by vacuum extraction and manometric measurement. J Biol Chem 1924;61:523-573
 19.Noble RJ,Rothbaum DA,Knoebel SB,McHenry PL,Anderson GJ:Normalization of abnormal T waves in ischemia.Arch Intern Med 1976;136:391-395
 20.Pesarenko OI,Solomatina ES,Ivanov VE,Studneva IM,Kapelko VI,Smirnov VN:On the mechanism of enhanced ATP formation in hypoxic myocardium caused by glutamic acid. Basic Res Cardiol 1985;80:126-134
 21.Pisarenko OI,Lepilin MG,Ivanov VE:Cardiac metabolism and performance during L-glutamic acid infusion in postoperative cardiac failure. Clinical science 1986; 70: 7-12
 22.Pisarenko OI,Portnoy VF,Studneva IM,Arapov AD,Korostylev AN:Glutamate-blood cardioplegia improves ATP preservation in human myocardium. Biomed Biochim Acta 1987;6:499-504
 23.Knapp WH,Helus F,Ostertag H,Tillmanns H,Kubler W:Uptake and turnover of L-(13N)- glutamate in the normal human heart and in patients with coronary artery disease. Eur J Mucl Med 1982;7:211-215
 24.Bittl JA,Shine KI:Protection of ischemic rabbit myocardium by glutamic acid. Am J Physiol 1983;245:H406-H412
 25.Owen TG,Hochachka PW:Purification and properties of dolphin muscle aspartate and alanine transaminases and their possible roles in the energy metabolism of diving mammals. Biochem J 1974;143:541-553 26.Hochachka PW,Owen TG,Allen JF,Whittow GC:Multiple end products of anaerobiosis in diving vertebrates. Comp Biochem Physiol 1975;50B:17-22 27.Digerness SB,Reddy WJ:The malate-aspartate shuttle in heart mitochondria. J Moll Cell Cardiol 1976;8:779-785
 28.Safer B:The metabolic significance of the malate-aspartate cycle in heart. Circulation Res 1975;37:527-533
 29.Chochinov RH,Perlman K,Moorhouse JA:Circulating alanine production and disposal in healthy subjects. Diabetes 1978;27:287-295
 30.Barrio JR,Baumgartner FJ,Henze E,Stauber MS,Egbert JE,MacDonald NS,Schelbert HR,Phelps ME,Liu FT:Synthesis and myocardial kinetics of N-13 and C-11 labeled branched chain L-amino acids. J.Nucl.Med. 1983;24:937-944
 31.Morgan HE,Earl DCN,Broadus A,Wolpert EB,Giger KE,Jefferson LS:Regulation of protein synthesis in heart muscle. J Biol Chem 1970;246:2152-2162
 32.Abumrad NN,Rabin D,Wise KL,Lacy WW:The disposal of an intravenously administered amino acid load across the human forearm.Metab.Clin.Exp.1982;31:463-470
 33.Tischler ME,Goldberg AL:Amino acid degradation and effect of leucine on pyruvate oxidation in rat atrial muscle. Am J Physiol 1980;238:E480-E486
 
やまだクリニック
内科・循環器内科
〒563-0047
大阪府大阪府池田市室町7-12
TEL.072-752-1001
■内科疾患一般の診療を行います。必要に応じて専門医療機関をご紹介いたします。
■循環器疾患(心臓疾患)の診療 診断と治療には特に力を入れています。
また最近多い糖尿病の治療にもある程度踏み込んで診療を行います。
■介護保険事業ともかかわり、主治医の意見書作成や各介護事業者の方との連携をはかります
■訪問介護事業者の方との連携をはかり、安心して自宅で療養出来る一助を担います
■予防接種事業 池田市より委託を受けた定期予防接種の一部を行います
TOPへ戻る