Myocardial Catecholamines in Hypertrophic and Dilated (Congestive) Cardiomyopathy: A Biopsy Study

Transcription

1 834 lacc Vol. 2. No.5 November Catecholamines in Hypertrophic and Dilated (Congestive) Cardiomyopathy: A Biopsy Stdy CHUICHI KAWAI, MD, FACC, YOSHIKI YUI, MD, TSUNEO HOSHINO, MD, SHIGETAKE SASAYAMA, MD, FACC, AKIRA MATSUMORI, MD Kyoto. Japan A high performance liqid chromatographic method was sed to determine myocardial norepinephrine and epinephrine concentrations in 66 biopsyspecimens obtained from the right or left ventricle dring rotine diagnostic cardiac catheteriation of 45 patients with dilated (congestive) or hypertrophic cardiomyopathy, or with heart disease other than cardiomyopathy, sch as acte perimyocarditis, postmyocarditis and constrictive pericarditis. The validity of catecholamine determination in a 2 to 6 mg biopsy specimen to assess overall ventriclar myocardial catecholamines was demonstrated. Norepinephrine concentrations in the myocardim were inversely correlated with the grade of hypertrophy in patients with congestive cardiomyopathy or heart disease other than cardiomyopathy, bt not in patients with hypertrophic cardiomyopathy. The fact that the myo- cardial norepinephrine concentration was always lower in the left than in the right ventricle of the same patient may be explained by the simple diltion of sympathetic nerve endings in the left ventricle. There were some cases of hypertrophic cardiomyopathy in which the concentration of myocardial norepinephrine was exceptionally high, althogh its mean vale was not significantly higher than that in patients with other types of heart disease who served as a control grop withot cardiomyopathy. Some patients with dilated cardiomyopathy had lower levels of myocardial norepinephrine than wold be expected for the degree of interstitial fibrosis and the severity of heart failre. The mean plasma norepinephrineand epinephrine levels were significantly elevated in patients with dilated cardiomyopathy. The term" noradrenosis" was proposed by Pearse (1) in 1964 to describe a significant increase in the level of florescent noradrenaline in myocardial specimens removed at operation from the otflow tract of the left ventricle in patients with hypertrophic obstrctive cardiomyopathy. However, the hypothesis of an increased noradrenaline content and agmented sympathetic nerve spply to the interventriclar septal myocardim in cases of hypertrophic obstrctive cardiomyopathy has not been sbstantiated. In 1971, Van Noorden et a1. (2) failed to confirm the presence of noradrenaline by microspectroflorimetric methods in samples of myocardim obtained from the area of greatest obstrction dring open heart srgery in cases of hypertrophic obstrc- From the Third Division, Department of Internal Medicine, Faclty of Medicine, Kyoto University, Kyoto, Japan. This stdy was spported in part by Research Grants for Cardiomyopathy and for Cardiovasclar Diseases (56-c) from the Mirstry of Health and Welfare, and Scientific Research Grants 35738(1978), 44444, (1979), 58766(198), 4445(1981) from the Ministry of Edcation, Science and Cltre, Tokyo, Japan. Manscript received Febrary I, 1983; revised manscript received Jne 6, 1983, accepted Jne 8, Address for reprints: Chichi Kawai, MD, Third Division, Department of Internal Medicine, Kyoto University Hospital, 54 Kawaracho Shogoin, Sakyok, Kyoto 66, Japan by the Amencan College of Cardiology tive cardiomyopathy. However, the myocardial noradrenaline content of patients with congestive cardiomyopathy has never been reported, becase these patients rarely ndergo cardiac srgery. This report describes the myocardial catecholamines in biopsy specimens removed from the right and left ventricles dring diagnostic cardiac catheteriation in an attempt to elcidate the role of myocardial catecholamines and sympathetic nervos activity in the pathogenesis of cardiomyopathy. Methods Patients. Biopsy specimens were obtained dring rotine diagnostic cardiac catheteriation after premedication with 5 mg of diaepam (Cercine). Specimens were taken from the right ventriclar side of the septm or left ventriclar free wall, or both, by the Konno-Sakakibara (3) or Kawai (4) biopsy catheter in 34 patients with cardiomyopathy (1 congestive [dilated] and 24 hypertrophic) and in 11 patients with other types of heart disease (2 with acte perimyocarditis, 6 with postmyocarditis, 1 with constrictive pericarditis, 1 with coronary arteriovenos fistla and 1 with /83/$3.

2 lacc Vol. 2, No 5 November 1983:834-4 KAWAI ET AL 835 Reliability, reprodcibility andsensitivity of themethod for determining myocardial catecholamine concentration. There was a linear relation between florescence intensity (peak height) and catecholamine concentration from oto 1, pg, the highest concentration tested. Within-rn and day to day coefficients of variation were 1. and 1,6% for norepinephrine and 1. and 1.8% for epinephrine, respectively (n = 1), A significant correlation (p <.1) was observed between measrements (n = 1) in the two assays for both norepinephrine (correlation coefficient [r] =.98) and epinephrine (r =.94). Analytical recovery of norepinphrine and epinephrine on the basis of the recovery rate from samples fortified with 1 pg of each standard catecholamine was abot 1%. With this chromatographic system, the lowest detection limit for each catecholamine in the myocardim is abot 1 pg. Figre 1 (right panel) shows a representative catecholamine chromatogram of a myocardial biopsy specimen from the left ventricle of a patient with hypertrophic cardiomyopathy. The left panel demonstrates the standard sample containing 2 pg of norepinephrine and epinephrine, Conalcoholism), The se of any catecholamine or vasodilating agent was forbidden for either therapetic or diagnostic prposes for a least 24 hors before the procedre, The diagnosis of congestive (dilated) and hypertrophic cardiomyopathy was made according to the definition and classification proposed by the World Health Organiation/ International Society and Federation of Cardiology task force (5). The last grop of patients served as a control grop withot cardiomyopathy. All the patients gave written consent before the stdy. Catecholamine determination in myocardial biopsy specimens. Biopsy specimens, weighing approximately 2 to 6 mg, were divided into two parts: one for histologic stdy and the other for catecholamine determination by high performance liqid chromatography which has been developed recently in or instittion (6-9), The other section of the biopsy specimen was qickly weighed and froen in liqid nitrogen. The specimen was homogenied in 1 JLI of freshly prepared cold.4 N perchloric acid/mg wet weight of the tisse containing 5 mm redced gltathione in a microhomogenier sbmerged in an ice bath and then centrifged in a refrigerated centrifge at 4 C for 2 mintes at 3, g. An aliqot (1 to 1 JLI) of the clear spernatant was directly injected into the doble colmn, which was composed of an pper anion exchange colmn of CDR 2 (qarternary ammonim anion exchanger with average particle sie 9 JLm) (Mitsbishi Chemical Co.) and a lower cation exchange colmn of Zipax SCX (D Pont Instrments Co.). The mobile phase was.7 N, sodim phosphate (NaH 2P4 ) with a flow rate of.8 mllmin. The separated catecholamines then entered a continos flow system described previosly (6), and the reagents for the trihydroxyindole method were added seqentially at a constant flow rate with the se of a proportioning pmp. Becase perchloric acid cased no adverse effect on the detector system (trihydroxyindole reaction), tisse extracts were sed withot netraliation. Finally, the florescent prodcts were applied to a highly sensitive spectroflorophotometer. The plasma catecholamine concentrations were determined by the high peformance liqid chromatographic method described in a previos report (7), The blood samples for the determination of catecholamines were drawn throgh an indwelling catheter in a peripheral vein after the patient had been resting for at least 3 mintes on a day close to the cardiac catheteriation stdy. Histologic stdy. For histologic stdies, the biopsy specimen was fixed in 1% formalin, dehydrated with graded ethanol and embedded in paraffin. The paraffin block was ct in 4 JL sections by a microtome, and the sections were stained with hematoxylin-eosin, elastic Van Gieson, periodic acid-schiff or Masson trichrome, The shortest diameters of cardiocytes were measred only in ncleated transverse sections, More than 3 myocardial cells in each specimen were measred by an oclar mi- crometer disc with a linear scale, and the average cell diameter of each specimen was calclated, Fibrosis was graded as follows: Grade signified no apparent fibrosis with the exception of small islets of fibros tisse arond the capillaries, Focal and minimal fibrosis was grade I and moderate to severe fibrosis of nearly 3 to 5% of the area of the entire specimen was classified as grade II. All the histologic stdies were performed by observers who were ignorant of the sorce of the specimen. Statistical analysis. Paired or npaired t tests and analyses of variance followed by mltiple comparison tests were sed in the statistical analysis, Vales are expressed as the mean ± standard deviation. Statistical significance is defined as a probability (p) vale of less than,5, Figre 1. A representative catecholamme chromatogram of biop Sled myocardim obtained from a patient with hypertrophic cardiomyopathy (HeM) (right) and that of a standard sample containing a known amont of catecholarnines (left), Epi = epinephrine; NE = norepinephrine, ----F = t -- l =--,=- -- -=-- - -=-- -=- Epi I--- -O f- f- -c-::-=- c-::- f- 21?Q_,_ Reslts I---f- - r-- - f-- " f::=:- f- - -I--- -I ====

3 836 KAWAI ET AL. JACC Vol. 2. No 5 November 1983:834-4 centrations of norepinephrine and epinephrine in this specimen are 92 and 34 pg/mg wet weight, respectively. These vales are lower for norepinephrine and higher for epinephrine than is sal in hypertrophic cardiomyopathy. Catecholamine concentrations in myocardial biopsy specimens (Tables I to 4). Myocard ial and plasma catecholamine concentrations, diameters ofmyocytes and grade of fibrosis in each sbgrop of patients are shown in Tables I to 4. The mean norepinephrine concentration in the left ventriclar myocardim was significantly lower (p <.5) in congestive than in hypertrophic cardiomyopathy. There was no significant difference in the mean norepinephrine concentration in either the right or left ventriclar myocardim between the patients with hypertrophi c cardiomyopathy and those with other heart disease. The mean myocardial epinephrine concentration showed no significant difference among the three sbgrops. Biopsy specimens were removed fr om both the right and left ventricle in 14 patients. Norepinephrine concentration was always significantly higher in the right than in the left ventricle (p <.1). The mean diameter of the cardiocytes was 17 ± 3 JLm in the right ventricle and 22 ± 3 JLm in the left. This differen ce was also statistically significant (p <.1). Plasma catecholamine concentrations in patients of each sbgrop (Tables I to 4). The mean plasma norepinephrine and epinephrine concentrations in patients with heart disease other than cardiomyopathy (noncardiomyopathy grop) were 177 ± 47 and 16 ± 3 pg/ml, respectively, which are within the normal range by or method. In patients with congestive cardiomyopathy, the concentrations were 42 ± 151 and 78 ± 58 pg/rnl, respectively, which were significantly higher than those in patients with hypertrophi c cardiomyopathy (27 ± 58 and 22 ± 14 pg/ml, respectively) (p <.1 for norepinephirine and <.5 for epinephrine). The mean plasma norepinephrine and epinephrine levels were also significantly higher in patients with congestive cardiomyopath y than in patients with other heart disease (p <.1 for norepinephrine and <.5 for epinephrine). Relation between myocardial norepinephrine concentration and diameter of myocardial cells (Fig. 2 to 4). The myocardial norepinephrine concentration showed a close inverse correlation (I' = -.86, P <.5; regression eqation: Y == 4,223-21X) with the diameter of the myocardial cells obtained from the patients with heart disease other than cardiomyopathy (Fig. 2). The inverse relation was also statistically significant, bt less so in patients with congestive cardiomyopathy (I' = -.55, P <.5; regression eqation : Y = 1,416-4X) (Fig. 3). There was no significant inverse correlation in patients with hypertrophic card iomyopathy (Fig. 4). There were two patients (Cases 12 and 13) with hypertrophic cardiomyopathy with exceptionally high levels of myocardial norepinephrine, regardless of the mean fiber width of the myocardial cells. The mean diameters of cardiocytes in the right ventricle were significantly larger in the patients with congestive and hypertrophic cardiomyop athy than in those with other heart disease (p <.5) (Table 4). Relation between myocardial norepinephrine concentration and grade of interstitial fibrosis (Fig. 5). Generally, as the grade of interstitial fibrosis increases, myocardial norepinephrine concentration decreases. There were six specimens, two from the left ventricle and for from the right ventricle in five patients with congestive cardiomyopathy, in which low concentrations of myocardial nor- Table 1. Reslts in 1 Patients With Congestive Cardiomyopathy Catecholamines Plasma (pglmg wet weight) Diameter of Myocytes Grade of Catecholammes NYHA Fnctional Class NE Epi (rn) Fibrosis (pg/rnl) Age (yr) On EF Case &Sex RV LV RV LV RV LV RV LV NE Epi Admission At Biopsy (%) I 44M 365 III 26 II IV II 27 2a 36M II III II 4 2b M II II F II II II 5 58M II III II 42 6a 37M I II I I 23 6b M 1, I IV II 8 47M I II II II M IO II III III M 1, I II II 37 EF = ejection fraction; Epi = epmephrinc; F = female; I = slight fibrosis; II = moderate to severe fibrosis; LV = left ventricle; M = male; NE = norepinephrine; NYHA = New York Heart ASSOCiation; RV = right ventricle; - = not measred; = no fibrosis.

4 lacc Vol 2, No 5 November KAWAI ET AL 837 Table 2. Reslts in 24 Patients With Hypertrophic Cardiomyopathy Catecholarrnes (pg/mg wet weight) Diameter of Plasma Myocytes Grade of Catecholamines NE Epl (rn) Fibrosis (pg/ml) Age (yr) Case &Sex RV LV RV LV RV LV RV LV NE Epi la 55M 1, I Ib 1,2 2 2 I 2 52M 1,95 1, M I M 2, M 1, I M 1, I 7 37M I 8 52M 1,516 1, I a 37M 1,238 1, b 1, I 1 44M I II M II 12 57M 1,76 (4,81) 25 (2) 14 (18) II M (7,93) 5 1 (15) 18 II I 14 5M I M I M II 17 47M I a 55M 1, II b 19 58M 1, II M II M II F 1, M 1,2 1, II I M 1, II II Figres in parentheses indicate vales not inclded for statistical analysis. Abbreviations as in Table 1. epinephrine were present despite minimal interstitial fibrosis. The mean vale of norepinephrine concentration in these specimens was 657 ± 25 pg/mg wet weight, definitely lower than that in the specimens from the patients with hypertrophic cardiomyopathy and other heart disease with minimal interstitial fibrosis. The mean vale in the latter grop was 1,959 ± 142 pg/mg wet weight. The average diameter of the myocardial cells was 19 pm in the former and 14 pm in the latter grop. Discssion Methodologic problems. The recent introdction of highly sensitive, specific techniqes for measring catecholamines has allowed s to determine the concentration of myocardial norepinephrine and epinephrine in a small piece of biopsy specimen obtained from the right or left ventricle dring rotine diagnostic cardiac catheteriation. Whether catecholamine levels in a small piece of biopsy specimen represent those of the entire ventricle is a sbstantial problem in biopsy stdies. Norepinephrine concentrations in the specimens removed from different sites of the right or left ventricle in the same patient showed little variation (coefficient of variation 7.6%, n = 6, Tables I to 3). However, some heterogeneities of myocardial innervation, particlarly in hearts that are heavily fibrosed, may case an inevitable risk that the biopsy specimen does not represent myocardial catecholamine concentrations throghot the ventricle. Moreover, there may be a sampling error in that the histologic featres of one sample and the catecholamines measred in a separate sample in the same patient might not match. In previos stdies (1, 11), very low concentrations of norepinephrine were demonstrated in atrial tisses and ventriclar mscles removed dring operation on patients with heart failre. The norepinephrine concentration was also markedly depressed in papillary mscles removed from the left ventricle of patients ndergoing mitral valve replacement who had been in severe left ventriclar failre (12). The role of catecholamines appears to be important in determining the pathogenesis of cardiomyopathy. Determination of the norepinephrine concentration in the myo-

5 838 KAWAI ET AL l ACC Vol. 2. No 5 November Tab le 3. Reslts in II Patie nts With Other Heart Diseases Catecholamines Diameter of (pg/rng wet weight) Myocytes NE Epi ( rn) Grad e of Fibrosis Plasma Catecholamines (pg/rnl) Age (yr) Case &Sex RV LV RV LV RV LV RV LV NE Epl Acte Perimyocard is IBM 1, M 1, Postrnyocarditis 3 34F II M II M a 45M II b II 7 25M I 8 57F 1, Constricti ve Pericarditis 9 53M 2,69 32 II Coronary Arteriovenos FIstla 1 44F 1, Alcoholism I I 42M 1, Abbrevtations as in Tab le I. cardim removed at operation, however. may be meaningless becase of the inflence of anesthesia and other nonphysiologic proced res. Biopsy specimens obtained dring rotine diagnostic cardiac catetheriation certainly provide informa tion nder far more physiologic circmstances. Morever, myocardial catecholarnines can be investig ated only in biopsy specimens in patients with congestive cardiomyopathy, in whom cardiac srgery is rarely indicated. nor epinephrin e concentration in congestive cardiomyopathy. The mean myocardial norepinephrine concen tration in mg wet weight of tisse in the left ventriclar myocardim was lower in patients with conges- Table 4. Norepinephrine Concentrations, Diameters of Cardiocytes and Plasma Catecholamines (mean ± sta ndard deviation) Norepinephrine Card iocyte Plasma (pg/rng Diameter Catecholamines wet weight) ( rn) (pg/ml) RV LV RV LV NE Epi CCM 739 ± == 311] 18 ±4 J 24 ± 3 2 ±151]j 78±58 (n = 11) (n = 6) * (n = 1) t (n = 1) * HCM 195 ± :!: ± 3} * 22 ± 3 27 :!: 58 t 22 ± 14 ' ( n = 21) (n = 12) (n = 19) (n = 19) Other 1182 ± ± :!: ± 3 (n = 12) (n = 2) (n = 9) (n = 9) Total n = 44 n = 2 *p<.5. t p<o.ooi. CCM = congestive cardiomyopathy; Epi epmephnne; HCM = hypertrophic cardiomyopathy; NE = norepinephrine. other = other diseases; other abbreviations as in Table 1

6 JACC Vol. 2, No 5 November KAWAI ET AL. 839 '" ;; 2, '" E "' "' 1, "' o -c Ci '" (n=14,r=- 86, P<O 5) Y= X "... '" 1 2 MEAN FIBER WIDTH OF MUSCULATURE (M) Figre 2. Relation between myocardial norepinephrine concentration andmean fiber width ofcardiocytes in II patients (14 biopsy specimens) with heart diseases other than cardiomyopathy (noncardiomyopathy grop). LV = left ventriclar and RV = right ventriclar biopsy. tive than in those with hypertrophic cardiomyopathy. However, the present stdy failed to show larger stores of myocardial norepinephrine in patients with hypertrophic cardiomyopathy than in those with noncardiomyopathic heart disease. Decreased myocardial levels ofnorepinephrine stores in congestive cardiomyopathy may well be the reslt of heart failre. This is spported by the fact that the mean plasma norepinephrine and epinephrine levels were significantly higher in patients with congestive cardiomyopathy than in those with hypertrophic cardiomyopathy or other heart disease. It is conceivable, therefore, that patients with congestive cardiomyopathy were at least in latent heart failre, althogh there was no obvios evidence of heart failre at the time of the stdy. Mechanism of myocardial norepinephrine depletion in heart failre. The mechanism of cardiac norepinephrine depletion in heart failre is still controversial. In dogs with Figre 3. Relation between myocardial norepinephrine concentration andmean fiber width ofcardiocytes in 1patients (17 biopsy specimens) with congestive cardiomyopathy (CCM). Abbreviations as in Figre 2. s: ;; ); 2 '"E "' ii' "' 1, "' : o Z '" V=1416-4X (n=17. r=-55 p<oos) 1 2 MEAN FIBER WIDTH OF MUSCULATURE (J.lM) 3 '" ;; 2, '" E rn.e "' 1, :: o "' '"s '" o e (n=35.ns).8 I.. o r!, 1 2 MEAN FIBER WIDTH OF MUSCULATURE( M ) Figre 4. Relation between myocardial norepinephrine concentration andmean fiber width ofcardiocytes in24patients (35 biopsy specimens) with hypertrophic cardiomyopathy (H[O)CM). right ventriclar failre, the redction of cardiac norepinephrine concentration was shown not to be the reslt of a simple diltion of sympathetic nerve endings in a hypertrophied mscle mass, becase the total ventriclar content of norepinephrine was lower in both the hypertrophied right and nonhypertrophied left ventricle (13). Marked redction in the activity of tyrosine hydroxylase was considered to be responsible for the cardiac norepinephrine depletion throgh decreased biosynthesis (14). On the other hand, in cardiomyopathic Syrian hamsters the rate constant for cardiac norepinephrine trnover was mch higher than in the control grop. Ths, in the late stage of hamster cardiomyopathy, Figre 5. Relation between myocardial norepinephrine concentration andthegrade of interstitial fibrosis in45 patients (66 biopsy specimens). Abbreviations as before. '" ;; 8.T 5.t 3, '" E ;;, 2, ;;; "' :: r 1, '"s '" f.. I 481 GRADE OF FI BROSIS H(O)CM-RV H(O)CM-LV. : I :- Fj gim:.c A '8. oe. A.c [] CO

7 84 KAWAI ET AL lacc Vol 2, No 5 November the increase in cardiac sympathetic tone is responsible for the decrease in cardiac norepinephrine (15). Or reslts may also indicate that the myocardial norepinephrine level in no way provides s with a biochemical index of myocardial sympathetic activity. In the hamster with cardiomyopathy, moreover, there is an increase rather than a decrease of tyrosine hydroxlase activity dring the development of heart failre (16). Althogh there is clear evidence of depletion in cardiac norepinephrine in heart failre, the specific mechanism responsible for it has not been clarified. Role of myocardial hypertrophy and interstitial fibrosis. The present stdy indicates that the diameter of the myocardial cells (grade of myocardial hypertrophy) and the grade of interstitial fibrosis shold always be taken into accont in evalating myocardial norepinephrine concentrations, becase the redction of norepinephrine concentration in the myocardim may be the reslt of a simple diltion of sympathetic nerve endings in a hypertrophied mscle mass (17) or an increase in interstitial fibrosis, or both. The lower vale of the myocardial norepinephrine concentration in the left than in the right ventricle may be explained by the simple diltion of sympathetic nerve endings in the left ventriclar myocardial cells, which have larger diameters than those in the right. It is worthwhile to note that the simple diltion theory holds tre in patients with heart disease other than cardiomyopathy, whose myocardim showed fairly normal histologic featres. In patients with hypertrophic cardiomyopathy, there was no significant inverse correlation between the diameter of the myocardial cells and myocardial norepinephrine concentrations. This is partly becase there were some patients wich exceptionally high concentrations of myocardial norepinephrine, regardless of the cell diameter and the grade of interstitial fibrosis. In some patients with congestive cardiomyopathy, however, the level of myocardial norepinephrine was lower than wold be expected for the degree of hypertrophy, interstitial fibrosis or severity of heart failre. Implications. These findings sggest that abnormal myocardial norepinephrine (a depletion in congestive cardiomyopathy and an increase in some cases of hypertrophic cardiomyopathy) may be related to the development of cardiomyopathy. The reglation of beta-adrenergic receptor density in the myocardim and, therefore, sensitivity to catecholamine shold also be clarified for a better nderstanding of its role in abnormal myocardial catecholamine activity in cardiomyopathy. The reslts of two recent important stdies (18,19) on receptor density in failing hearts are conflicting. The obvios differences between these two stdies appear to be the dration and case of heart failre and the species examined. Frther stdies in this field are essential to elcidate more precisely the role of myocardial catecholamine in the pathogenesis of cardiomyopathy. We thank Alice S. Cary, MD for pertinent advice on the manscript. References I. Pearse AGE. The histochemistry and electron microscopy of obstrctive cardiomyopathy. In: Wolstenhome GEW, O'Connor M, eds. Cardiomyopathies. London: J&A Chrchill, 1964: Van Noorden S, Olsen EGJ, Pearse AGE. Hypertrophic obstrctive cardiomyopathy, a histological, histochemical, and ltrastrctral stdy of biopsy material. Cardiovasc Res 1971;5: Konno S, Sakakibara S. Endo-myocardial biopsy. Chest 1963;44: Kawai C, Kitara Y. New endomyocardial biopsy catheter for the left ventricle. Am J Cardiol 1977;4: Report of the WHO/ISFC task force on the definition and classification of cardiomyopathies. Br Heart J 198;44: Yi Y, Kimra M, Itokawa Y, Kawai C Ultramicro method for the determination of ptcograrn amonts of norepinephnne and epinephnne by high-performance liqid chromatography. J Chromatogr 1979;177: Yi Y, Fjita T, Yamamoto T, Itokawa Y, Kawai C. liqid-chromatographic determination of norepinephrine and epinephrine in hman plasm Clin Chern 198;26: Yi Y, Itokawa Y, Kawai C. A rapid and highly sensitive method for determination of picogram levels of norepmephnne and epinephrine in tisses by high-performance liqid chromatography. Anal Biochem 198,18: YUIY, Kawai C. Comparison of the sensitivity of varios post-colmn methods for catecholarnme analysis by high-performance liqid chromatography J Chromatogr 1981;26: DeQattro V, Nagats T, Mende A, Verska J. Deterrnmants of cardiac noradrenalme depletion in hman congestive failre. Cardiovasc Res 1973;7: II. Chidsey CA, Branwald E, Morrow AG. Catecholamine excretion and cardiac stores of norepinephnne in congestive heart failre. Am J Med 1965;39: Chidsey CA, Sonnenblick EH, Morrow AG, Branwald E Norepinephnne stores and contractile force of papillary mscle from the failing hman heart. Circlation 1966;33: Chidsey CA, Kaiser GA, Sonnenblick EH, Spann JF Jr, Branwald E. Cardiac norepinephrine stores in expenmental heart failre in the dog. J Clm Invest 1964;43: Pool PE, Covell JW, Levitt M, Gibb J, Branwald E. Redction of cardiac tyrosme hydroxylase activity in experimental congestive heart failre: its role in depletion of cardiac norepinephnne stores. Ctrc Res 1967;2: Sole MJ, Lo C, Laird, Sonnenblick EH, Wrtman RJ. Norepinephrine trnover in the heart and spleen of the cardiomyopathic Syrian hamster. Circ Res 1975; Sole MJ, Kamble AB, Hssain MN. A possible change in the ratelimiting step for cardiac norepinephrine synthesis m the cardiornyopathic Syrian hamster. Circ Res 1977; Borchard F. The Adrenergic Nerves of the Normal and the Hypertrophied Heart: Biochemical, Histochemical, Electron-Microscopic and Morphometric Stdies. Stttgart:George Thieme Pblishers, 1978: Karliner JS, Barnes P, Brown M, Dollery D. Chronic heart failre in the ginea pig increases cardiac a- and {3-adrenoceptors. Er J Pharmacol 198;67: Bristow MR, Ginsbrg R, Minobe W, et al. Decreased catechol amme sensitivity and {3-adrenergic-receptor density in failing hman hearts. N Engl J Med 1982;37:25-11.