Coronary sinus lactate measurements in assessment of myocardial ischemia

Coronary Sinus Lactate Measurements in Assessment of Myocardial Ischemia Comparison with Changes in Lactate/Pyruvate and Beta-Hydroxybutyrate/ Acetoacetate Ratios and with Release of Hydrogen, Phosphate and Potassium Ions from the Heart LIONEL H. OPIE, MD, MRCP, FACC PATRICIA OWEN, BSc MICHAEL THOMAS, MD, MRCP ROLAND SAMSON, MB, ChB, FCP (SA)

London, England Cape Town, South Africa

From the Medical Research Council Cardiovascular Research Unit, Department of Medicine, Royal Postgraduate Medical School, Hammersmith Hospital, London W12 OHS, England; and Ischaemic Heart Disease Laboratory, Department of Medicine, Groote Schuur Hospital and University of Cape Town, South Africa. The Ischaemic Heart Disease Laboratory is supported by the Medical Research Council of South Africa, the Chris Barnard Fund, the University of Cape Town and the International Sugar Research Foundation. Address for reprints: Lionel H. Opie, MD, Department of Medicine, University of Cape Town, South Africa.

The value of measurements of lactate in coronary venous blood as a sign of myocardial anaerobiosis is reassessed. Lactate was measured after coronary arterial Iigation in dogs in (1) local venous blood draining from ischemic tissue, (2) coronary sinus blood, and (3) myocardial tissue. There was an estimated lactate concentration gradient of 2- to 4-fold from ischemic tissue (epicardial biopsy specimens) to local venous blood, whereas the gradient from epicardial tissue to coronary sinus blood was 8- to 16-fold. Differences in pyruvate concentrations between ischemic tissue and local venous or coronary sinus blood were not marked. Increases in tissue lactate concentration and in lactate/pyruvate values in anaerobic tissue occurred simultaneously. Whether such tissue changes are reflected in coronary venous blood depends on the degree to which the heart cell membrane impairs egress of lactate or pyruvate, and on the venous sampling site. With coronary sinus sampling, the production of small ischemic lesions (less than 10 percent of the volume of the whole heart) caused a readily detected decrease of lactate extraction by the heart, whereas the changes in the lactate/pyruvate ratio across the heart were less marked. When lesions were larger or when highly selective coronary venous sampling techniques were used, lactate/pyruvate changes were readily detectable; changes in the ratio beta-hydroxybutyrate/acetoacetate were not very helpful in detecting ischemia. Although lactate changes in coronary venous blood are a very sensitive index of regional myocardial ischemia after coronary arterial ligation, a knowledge of changes in lactate/pyruvate ratios, potassium ion, inorganic phosphate and hydrogen ion allows a more complete description of intracellular events. These additional measurements should also help to exclude unusual circumstances in which lactate discharge from the heart occurs in the presence of apparently normal oxygenation.

Since the introduction of coronary sinus catheterization in man by Bing et al. z in 1947, the change from uptake to output of lactate by the human heart has been used by many clinical workers as an index of impaired myocardial tissue oxygenation or inadequate coronary blood flow, or both. A further refinement of this approach has b e e n to look for decreased lactate extraction. 2 Huckabee s and others 4,5 found that the lactate/pyruvate ratio in coronary venous blood increased during myocardial hypoxia. On the assumption that lactate and pyruvate freely penetrated cell membranes, alterations in the ratio between reduced and oxidized forms of free nicotinamide adenine dinucleotide (NAD+/NADH) in the heart cell were thought to be reflected in altered coronary venous lactate/pyruvate ratios. Actual quantitation of the degree of myocardial hypoxia was attempted by calculations of (1) the difference in redox state across the heart (AEh)4; or (2) "excess lactate," that is,

September 7, 1973 The American Journalot CARDIOLOGY Volume32

295

CORONARY SINUS LACTATE AND MYOCARDIAL iSCHEMIA--OPIE El" AL.

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FIGURE 1. Effect of coronary arterial occlusion on blood lactate concentrations (mean values 4- standard error Of the mean in 8 to 44 experiments) in arterial, local venous and coronary sinus blood and on epicardial tissue lactate content (see Table I). Note (1) the relation between decreased lactate extraction, detected in coronary sinus blood, and lactate formation in local venous blood and (2) the relation between tissue lactate content and blood lactate concentration.

Forty-five greyhounds and 24 mongrel dogs were anesthetized by administration of barbiturates. The greyhounds were prepared for study of arteriovenous differences across ischemic and nonischemic myocardium as previously described12; mongrel dogs were subjected to the same procedures with the exception of local venous cannulation, which usually could not be performed. Blood from the ischemic zone was sampled by a small polyethylene catheter inserted directly into the local vein. Blood from the nonischemic zone was drained by a coronary

sinus catheter; the tip of the coronary sinus catheter was usually situated at the junction of the great coronary vein and the coronary sinus. An anterolateral (diagonal) or apical branch of the left anterior descending coronary artery was ligated since such ligations result in relatively stable preparations 13 in which cardiac function is probably well maintained. 14 Blood samples from the femoral artery, coronary sinus and local vein were taken at 5 to 10 minute intervals before and up to 120 minutes after arterial ligation. Production of myocardial ischemia was confirmed by the development of a circumscribed cyanosed area in the territory of the ligated artery and by epicardial S - T segment mapping. 15 The size of the ischemic tissue was estimated by the extent of visible cyanosis, the degree of epicardial S - T segment elevation and the volume of the heart served by the ligated artery, x6 Epicardial tissue biopsies were performed with a bone rongeur chilled in liqUid nitrogen. About 500 mg of tissue was obtained from a shallow crater on the epicardium approximately 3 m m deep. After an epicardial biopsy specimen was taken from the center of the ischemic area and a control specimen was taken from the free wall of the left ventricle far away from the lesion, the experiment was terminated. In control hearts without coronary arterial ligation both biopsy specimens were taken from tissue determined to be nonischemic because of the absence of S - T segment elevation. Transmural biopsy specimens were obtained by a dental drill technique, 17 modified to give a core of tissue 3 mm in diameter and weighing about 180 mg, or by rapid aortic transection of the heart arrested in 0.25 mM of icecold sucrose after aortic transection. 16 Biopsy specimens were plunged into liquid nitrogen or freon, pulverized, deproteinized in perchloric acid-acetone-ethylenediaminetetraacetic acid (EDTA), 16 and analyzed for pyruvate (see later) and lactate (after being kept in the frozen state). Blood or medium pyruvate, acetoacetate, lactate, beta hydroxybutyrate and plasma free fatty acids, potassium ion, inorganic phosphate and glucose were assayed as previously describedJ 2 Blood pyruvate was assayed enzymatically immediately after neutralization of the deproteinized sample. Tissue pyruvate was also assayed enzymatically within 30 minutes of deproteinization of the frozen tissue extract; 85 to 100 percent of added standard was recovered. Apparent tissue lactate,

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f o r m a t i o n of l a c t a t e at a r a t e greater t h a n t h a t accounted for b y p y r u v a t e f o r m a t i o n . 6 B o t h concepts rapidly gained ground until m a t h e m a t i c a l a n d theoretical a r g u m e n t s discredited t h e theory of "excess l a c t a t e . " 7,s T h u s , Gorlin 2 d r o p p e d p y r u v a t e m e a s u r e m e n t s in the a s s e s s m e n t of m y o c a r d i a l hypoxia. A E h still continues to be used, and the c o n c e p t of arteriovenous difference in l a c t a t e / p y r u v a t e ratio has als0 b e e n introduced. 9 Recent studies in the perfused r a t h e a r t h a v e shown e x p e r i m e n t a l c i r c u m s t a n c e s in which l a c t a t e a n d p y r u v a t e do not equilibrate freely l°,11 across the h e a r t cell m e m b r a n e . It is therefore i m p o r t a n t to c o m p a r e blood a n d tissue l a c t a t e a n d p y r u v a t e values in ischemic a n d n o n i s c h e m i c m y o c a r d i u m a n d to reassess t h e value of coronary venous change in l a c t a t e or l a c t a t e a n d p y r u v a t e c o n c e n t r a t i o n s in relation to tissue changes. We s t u d i e d an open chest dog h e a r t p r e p a r a t i o n 12 in which values d e t e r m i n e d in blood draining from the ischemic a n d nonischemic tissue could be comp a r e d with tissue values o b t a i n e d b y biopsy. C o m parisons were m a d e b e t w e e n l a c t a t e release a n d other possible indexes of i s c h e m i a such as changes in the l a c t a t e / p y r u v a t e or ketone body ratio a n d release of hydrogen, p h o s p h a t e or p o t a s s i u m ions. P a r ticular a t t e n t i o n was p a i d to t h e effect of i s c h e m i a on l a c t a t e a n d p y r u v a t e values in cardiac tissue a n d coronary venous blood. Methods

September 7, 1973

The American Journal of CARDIOLOGY

CORONARY SINUS LACTATE AND MYOCARDIAL ISCHEMIA--OPIE ET AL.

TABLE I Lactate and Pyruvate Values in Heart Tissue Before and After Coronary Arterial Ligation A. Measured Values NonischemicEpicardialTissue

Ischemic EpicardialTissue

Time After Ligation (min)

Lactate (#mole/g)

Pyruvate (#mole/g)

L/P Content Ratio

Lactate (#mole/g)

Pyrovate (#mole/g)

--10 (preligation) 10

1.17 ±0.20 (10) 1.32 ±0.14 (8) 1.19 ~0.07 (6) 1.65 ±0.20 (4) 1.72 ±0.28 (8)

0.054 ±0.004 (10) 0.052 ±0.013 (5) 0.065 ±0.011 (7) 0.067 ±0.025 (4) 0.092 ±0.018 (15)

22 ±3 (10) 27 ±4 (5) 21 ±4 (6) 33 ±8 (4) 26 ±5 (8)

1.17

0.054

20

60

120

3.63t ±0.55 (8) 2.26* ±0.29 (6) 2.99t ±0.30 (4) 2,90" ±0.63 (8)

0.054 ±0.010 (8) 0.044 ±0.007 (7) 0.069 ±0.014 (4) 0.066 ±0,010 (14)

IschemicTransmural Tissue Lactate (#mole/g) 1.87 ~0.28 (6) 10.51t ±1.40 (8) 14.42t ~:3.52 (7) 13.86t ±3.59 (8) 13.71" ±4.96 (3)

B. Calculated Values Measuredvs CalculatedL/P Ratios in EpicardialIschemicTissue

CalculatedIntracellularValuesin EpicardialIschemicTissue Time After Ligation LactateConcentration (min) (raM)

LactateGradient

PyruvateConcentration (raM) PyruvateGradient

Measured Ratio

Calculated Ratio

1.94

5.0

0.079

1.5

22

25

5.80

3.4

0.074

1.2

76t ±12

78

20

3.40

1.9

0.055

0.8

60

4.75

3.1

0.097

1.3

--10 (preligation) 10

(8) 57t ±9 (6) 47*

62

49

~8

(4) 120

4.60

3.5

0.089

1.0

56* ±13

52

(8) * P <0.05; t P <0.005 for comparison with nonischemic control. Nonischemic control for tissue biopsy specimens ---- simultaneously sampled nonischemic tissue; nonischemic control for local venous blood = initial venous values. Measured tissue values = combined content of extracellular and intracellular values (#mole/g) in biopsy sample. Calculated tissue values (in mM) are derived from the means of the measured values by allowing for extracellular lactate and pyruvate concentrations and for tissue water content, n Lactate or pyruvate gradient = difference between calculated lactate or pyruvate concentration in epicardial tissue and in local venous blood. For possible sources of error, see Results section. Ischemic transmural tissue was obtained from a different series ofdogs (see text).

pyruvate and l a c t a t e / p y r u v a t e ratios were obtained by relating the overall tissue value to the frozen weight of the tissue (that is, tissue content). Calculated tissue concentrations of lactate and pyruvate were derived as for the intracellular phosphate concentrations11,16: Intracellular lactate concentration = (lactate space extracellular space) (perfusate lactate concentration)/ total water - extracellular space

where lactate space = (apparent heart lactate content Lumole/g frozen weight]) x 1000/perfusate lactate (#mole /liter). The assumptions underlying this calculation are considered elsewhereJ 1 The extracellular space was taken as 175 # l i t e r / g wet weight from the d a t a of Danforth et al. is In agreement with the findings of Griggs et al., 19 there was no detectable formation of e d e m a in the ischemic tissue.

September 7, 1973

The American Journal of CARDIOLOGY

Volume 32

297

CORONARY SINUS LACTATE AND MYOCARDIAL I S C H E M I A - - O P I E ET AL.

Tissue activity of beta hydroxybutyrate dehydrogenase was determined in two greyhound dogs by the method of Lehninger et al. 2° Blood pH, PC02 and PO2 values were measured with the Instrumentation Laboratories electrode. The gas measurements were made at 37 C. Calibration was checked before and after each measurement.

known. Furthermore, transmural biopsy specimens from the center of the infarct (highest S-T segment elevation) showed lactate values 1.5 to 5 times higher than in the epicardial biopsy specimens, in keeping with the heterogeneity of ischemic tissue as shown by measurements of lactate across the heart wall. 19 The transmural biopsy specimens of ischemic tissue were studied in a series of dogs different from the series that underwent epicardial biopsy, but the preparations were identical except that local venous samples were not undertaken. In 23 biopsy specimens obtained by the drill technique in our dogs, epicardial lactate values were directly compared with endocardial values in ischemic tissue; endocardial values were 2 to 3.5 times higher. However, in 21 biopsy specimens obtained by the drill technique from nonischemic tissue in 11 other dogs, epicardial and endocardial lactate values were the same. Thus, two factors produced the higher values for transmural biopsy specimens of ischemic tissue. First, these specimens were taken from the center of the infarction whereas the epicardial biopsy produced a large fiat area of tissue extending beyond the center. Second, at one localized point in the ischemic tissue there were substantial transmural gradients as shown by the specimens obtained by drill technique. After 10 minutes of ischemia the apparent lactate concentration difference (not allowing for extracellular lactate in the biopsy specimens) between the central transmural tissue and local venous blood was about 6 and between this tissue and coronary sinus blood about 19. True concentration gradients could not be calculated. P y r u v a t e values (Fig. 2}: After coronary arterial ligation, local venous pyruvate values increased and pyruvate extraction levels progressively decreased (Table II). Pyruvate values in epicardial tissue were steady throughout and could not be differentiated from coronary sinus values. There was no detectable gradient from the calculated intracellular pyruvate concentration in epicardial tissue to that in local venous blood (Table I).

Results A total of 69 experiments involving ligation of a coronary artery were performed, with 45 cannulations of the local vein and 25 of the coronary sinus. The estimated volume of the heart served by the arterial ligation was 7.3 ± 0.5 percent. Results in greyhound and mongrel dogs did not differ and are grouped together. Blood lactate (Fig. 1}: Features of note are (1) basal lactate extraction; (2) substantial lactate discharge into local venous blood after arterial ligation, with the concentration exceeding the control venous value by 300 to 400 percent; and (3) diminished lactate extraction but no discharge in coronary sinus blood (Tables I and II). Tissue lactate (Table I): In the period before ligation, the tissue lactate content exceeded the arterial lactate concentration by about 1.6 times and the coronary venous lactate concentration by about 3 times. After ligation, the ischemic epicardial tissue lactate level was 2.1 (10 minutes after ligation), 1.2 (20 minutes), 2 (60 minutes) and 2.2 times (120 minutes) greater than the local venous lactate level. Thus, the apparent gradient from tissue to venous blood decreased during ischemia. The true concentration gradients from tissue to venous blood in both control and ischemic states were higher because of the contribution of extracellular lactate to the apparent values for tissue lactate. Thus, the gradient between lactate and local venous blood could be approximately 5 in nonischemic tissue and 2 to 3.5 in ischemic tissue. However, since even local venous blood may represent a mixture of blood draining from tissue with various degrees of ischemia as well as from normal tissue, the true value for extracellular lactate concentration in ischemic tissue is not . . . . ~ Femoral artery o Localvein Coronary sinus = = Epicardiit tissue o

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298

September 7, 1973

The American Journal of CARDIOLOGY

Volume 32

FIGURE 2. Effect of coronary arterial occlusion on blood pyruvate concentrations in arterial, local venous and coronary sinus blood, and on epicardial tissue pyruvate content.

CORONARY SINUS LACTATE AND MYOCARDIAL ISCHEMIA--OPIE E T A L .

L a c t a t e / p y r u v a t e ratios (Fig. 3): After coronary arterial ligation local venous lactate/pyruvate ratio and apparent lactate/pyruvate values in ischemic tissue both increased to about 3 times the preligation level. Local venous lactate/pyruvate exceeded the arterial ratio throughout the period after ligation;

100 to 120 minutes after ligation, apparent ischemic tissue lactate/pyruvate ratios were still clearly increased, but local venous lactate/pyruvate ratio was only slightly increased (1.4 times the arterial value). Coronary sinus lactate/pyruvate ratios tended to increase (Table II) but did not significantly exceed ar-

TABLE II Comparison of Changes in Lactate Extraction with Other Indexes of Ischemia in Local Venous and Coronary Sinus Blood After Coronary Arterial Ligation in the Dog

Time After Ligation (min)

Extraction Extraction Lactate Pyruvate

Venous aL/P% of L/P Art.

aL/P

aEh

apH

apCO~

Pi

K+

Gluc0se

FFA

Venous BHOB B/A

0 ±2

0 ±1

8 ±2

44 ±4

19 ±4

1.0 ±0.07

(18) 7

(9) 1.0

±2 (17) 201. ±2 (16) 231" ±3 (15) 241. ±2 (14) 20 ±5 (7)

±4 ±5 (23) (9) 46 10 ±2 ±5 (22) (8) 42 12 ±4 ±6 (20) (8) 42 10 ±3 ±6 (14) (8) 42 10 ± 2 ±10 (6) (4)

±0.10 (9) 1.1 ±0.13 (8) 1.3 ±0.16 (8) --1.4" ±0.16 (8) --1.6" ±0.19 (13)

49 ±5

1.1 ±0.08

A. Local Vein (45 experiments) --10

10

20

40

60

90-120

40 ±3

35 ±6

9.6 ±1.0

(46) --1951-

(24) 16

(24) 32.71.

±26 (44) --194i±28 (38) --1941. ±35 (33) --1301. ±26 (29) --721" ±19 (17)

±8 (25) 9" ±8 (24) 15 ±8 (19) 6* ±12 (14) --6* ±14 (8)

±2.9 (25) 33.01±3.2 (24) 27.51" ±3.3 (1.9) 23.31" ±4.2 (14) 15.1" ±1.7 (8)

--7 ±6

--0.7 ±0.7

0.0 ±4.1

0.047 ±0.007

(24) (24) (24) --2671- --22.51- --15.71±41 (25) --269? ±40 (24) --2191" ±39 (19) --1511" ±32 (14) --631" ±14 (8)

±2.8 (25) --22.71 ±2.9 (24) --18.11. ±3.0 (19) --13.91" ±3.8 (14) --5.81" ±1.4 (8)

±1.4 (25) --16.3t ±2.3 (24) --15.31. ±2.5 (19) --12.7" ±2.3 (14) --6.2 ±1.2 (8)

--13 ±1

(14) (12) (28) (19) 0.1301- --241. --411- --151±0.017 (14) 0.1461" ±0.018 (14) 0.1141" ±0.015 (13) 0.0841" ±0.010 (12) 0.071 ±0.012 (8)

±2 ±3 ±2 (13) (28) (19) --231" --311" --101" ±2 ±4 ±1 (12) (21) (17) --18" --141. --4 ±2 ±2 ±2 (11) (18) (14) --18" --10" --4 ±2 ±3 ±2 (10) (15) (11) --15 --6* --8 ±4 ±2 ±4 (6) (6) (4)

(17) (24) 181- 43

B. Coronary Sinus (25 experiments) --10

41 ±5

35 ±7

10.2 +1.5

--9 ±11

--1.1 ±1.3

0.6 -+-1.3

0.040 ±0.010

--12 ±2

5:~ ±1

4:~ ±1

9 ±2

10

(23) 20* ±6

(17) 28 ±10

(18) 12.8 ±1.8

(17) --24 ±11

(17) --3.5 ±1.9

(17) --1.3 ±1.7

(13) 0.057 ±0.008

(11) --10 ±2

(13) 01 ±1

(12) 1 ±1

(10) (10) 10 54 ±2 ±4

(10) (7) 23 1.2 ±6 ±0.21

20

(24) 21t ±4

(19) 26 ±9

(19) 13.0¶ ±1.7

(19) --21 ±10

(19) --2.0 ±1.3

(19) --1.3 ±1.4

(13) 0.063¶ ±0.011

(12) --12 ±2

(13) 1 ±2

(13) 2 ±1

(12) 9 ±2

(13) 52 ±3

(7) (7) 16 1.3 ±4 ±0.13

(25) 20t

(19) 26

(19) 11.2

(19) --14

(19) --0.6

(19) --0.7

(13) (11) 0.065¶ --10

(12) 4

(11) 3

(13) 10

(12) 56

±5

±7

±11

±1.1

±1.4

±2

±1

±1

±2

±4

(23)

(17)

(10)

(13)

(12)

(12)

(12)

191. ±5

30 ±9

12.3 ±1.7

--25 ±9

--2.5 ±1.1

--1.5 ±1.2

0.063¶ ±0.011

--12 ±2

3 ±1

3 ±1

12 ±3

(20) 27* ±4

(12) 24 ±5

(12) 10.4 ±1.2

(12) 2 ±10

(12) --0.8 ±1.4

(12) 1.0 ±4.0

(11) 0.035 ±0.010

(9) --12 ±2

(7) --1 ±4

(8) 3 ±3

(13)

(7)

(7)

(7)

(7)

(7)

(7)

(5)

(2)

(3)

40 60

90-120

±1.2

(17)

(17)

(17)

±0.012

(17)

(12)

19 ±6

(7) 7 ±7

(8) 1.2 ±0.08

(6)

(6)

50 ±4

11 ±7

1.3 ±0.15

(10) 11 ±5

(8) 41 ±2

(6) (6) 14 1.91±8 ±0.21

(6)

(4)

(4)

(8)

Figures in parentheses indicate number of observations. * P <0.05 for grouped comparison with control values (--10 minutes); t P <0.005 for grouped comparison with control values (--10 minutes); ::1:P <0.05 for grouped comparison between control values in coronary sinus and local vein; ¶ P <0.05 for paired comparison with control values in coronary sinus. Art. = arterial; B/A = beta hydroxybutyrate/acetoacetate; BHOB = beta hydroxybutyrate; a = arteriovenous difference; aEh = difference in the redox state of the blood lactate dehydrogenase system across the heart; extraction = arteriovenous difference as percent of arterial value; FFA = free fatty acids; L/P = lactate/pyruvate ratio; Pi = inorganic phosphate. Extraction of FFA was decreased in local venous blood compared with coronary sinus blood after ligation (P <0.05); 10 minutes after ligation the local venous concentration of BHOB was greater than coronary sinus value (P<0.05).

September 7, 1973

The American Journal o! CARDIOLOGY

Volume 32

299

CORONARY'SINUS LACTATE AND MYOCARDIAL ISCHEMIA--OPIE ET AL.

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terial values. Calculated and measured epicardial tissue lactate/pyruvate ratios were similar. The difference in the redox state of the blood lactate dehydrogenase system across the heart (AEh) and "excess lactate" were calculated by the formulas of Gudbjarnason et al. 4 and Huckabee, 6 respectively. These indexes changed in direction similar to that of the lactate/pyruvate ratios. (Data for "excess lactate" are not given because of objections to the mathematical validity of the calculation. 7) Comparison of changes in various indexes of ischemia in local venous and coronary sinus blood (Table II): In local venous blood all the indexes measured changed significantly after the onset of ischemia except that extraction of free fatty acids was decreased only in comparison with coronary sinus values and not with preligation values and the ketone body ratio. The change from lactate extraction in the control period to lactate discharge after !igation was a very definite phenomenon and was accompanied by major changes in the lactate/pyruvate ratio and values calculated therefrom. The local venous pH level decreased after ligation and ApH (difference between arterial and local venous pH) increased about 3 times above the value before ligation. APC02 increased to nearly double the preligation value within 10 minutes. Inorganic phosphate and potassium loss were readily detectable 10 minutes after ligation. 16 Glucose arteriovenous differences doubled. Beta-hydroxybutyrate extraction values decreased by about half (see also footnote to Table II). The ratio beta-hydroxybutyrate/acetoacetate rate rose compared with preligation values but not when compared with simultaneous arterial or coronary sinus values. In coronary sinus blood, lactate extraction decreased significantly, from about 40 percent to about 20 percent after onset of ischemia. Changes in the

300

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7, 1973

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Journal of CARDIOLOGY

FIGURE 3. Effect of coronary arterial occlusion on blood lactate/pyruvate ratios in arterial, local venous and coronary sinus blood and in epioardial tissue. Changes in coronary sinus lactate/pyruvate ratios are less easy to detect than reductions in lactate extraction (compare with Fig. 1 ).

lactate/pyruvate ratio and AEh across the heart were obscured by a large variation between animals and by reduced pyruvate extraction (Table I). Inorganic phosphate extraction was decreased significantly 10 and 20 minutes after ligation, thus supporting previous evidence that changes in inorganic phosphate are a more sensitive index of myocardial ischemia than potassium changes in this system. 16 Nevertheless, there was only a 5 percent change in inorganic phosphate extraction when these small ischemic lesions were monitored by coronary sinus changes. Beta-hydroxybutyrate extraction was unchanged. Beta-hydroxybutyrate dehydrogenase activity: This value was 0.32 in the left ventricle and 0.26 in the right ventricle (umole substrate converted/min per g fresh weight at 25 C; three biopsies in two normal greyhound hearts in situ). Sonicated and frozenthawed values were similar, and were about 20 percent of the values found in rat heart. 2o R a t e of development of venous metabolic changes (Fig'. 4): The highest rate of change in local venous blood after the production of ischemia was the increase in inorganic phosphate (0.65 mN/2 min); the next highest rates of change occurred in lactate (0.53 m M / 2 min) and potassium (0.43 m N / 2 rain). S - T segment elevation developed about as rapidly as phosphate loss. Glucose arteriovenous difference (not shown in Fig. 4) only increased substantially in the 2 to 5 minute period after ligation. Changes in blood pH, PCO2 and PO2: Before ligation, the mean arterial pH was 7.39 -~ 0.01 (26 dogs), the mean local venous pH was 7.36 ± 0.01 (25 dogs), and coronary sinus pH was 7.35 -~ 0.01 (25 dogs). After ligation, the arterial pH remained constant. The changes in venous pH (like those of lactate, potassium and phosphate le) were dependent on the size of the lesion. In 18 small lesions (estimated

Volume

32

C O R O N A R Y S I N U S LACTATE A N D M Y O C A R D I A L I S C H E M I A - - O P I E

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ligation was 64 4- 4 mm Hg, and the coronary sinus PCO2 increased to 56 4- 2 mm Hg. The differences in pH, PCO2 and PO2 across the ischemic myocardium in 14 experiments (average size 10 percent) are shown in Figure 5 and compared with lactate arteriovenous differences in the same experiments. Patterns of release of lactate and hydrogen ion were similar but of shorter duration than the increase in PCO2. Possible sources of error: Our results are open to criticism on several counts. First, it is virtually impossible to compare blood and tissue samples simultaneously. However, venous sampling from two sites by two workers was rapidly followed by biopsies that were completed in less than I minute. The overall delays involved were not too significant compared with the rate of lactate changes in blood (Fig. 1).

size 6 percent 4- 0.5 percent), the local venous pH 20 minutes after ligation was 7.28 4- 0.02; coronary sinus pH did not change. In eight large lesions (estimated size 19 percent 4- 2 percent) the local venous pH measured 10 minutes after arterial ligation decreased to 7.21 4- 0.02; coronary sinus pH values only decreased to 7.30 4- 0.01. Before arterial occlusion, the arterial PCO2 level was 37 4- 1 mm Hg (25 dogs), the local venous PCO2 was 50 4- 1 mm Hg (25 dogs), and the coronary sinus PCO2 was 49 4- 1 mm Hg (24 dogs). After ligation, there was no change in the arterial PCO2. but in 18 small lesions the PCO2 in local venous blood increased to 59 4- 3 mm Hg within 10 minutes of occlusion and then gradually decreased to normal 80 minutes after occlusion; coronary sinus PCO2 values did not change. In seven large lesions, the local venous PCO2 10 minutes after

pH units

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Volume

32

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1/` F I G U R E 5. Effect of c o r o n a r y arterial ligation pH, PCO2 and PO2 across the i s c h e m i c myocardium (arterial-local venous differences) in 14 dogs with a m e a n e s t i m a t e d size of lesion of 10 percent. Note the close c o r r e s p o n d e n c e between changes in lactate a n d pH.

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CORONARY SINUS LACTATE AND MYOCARDIAL ISCHEMIA--OPIE ET A L

Second, an open chest preparation with a certain amount of manipulation of the heart was required. Third, it was impossible to obtain sequential tissue biopsy samples in the same dog although sequential blood samples were easily obtained. Thus, the number of blood samples exceeded the number of tissue samples and strictly paired comparisons were not made. Fourth, the transmural drill biopsy technique can damage endocardial tissue and increase endocardial lactate values. However, in control nonischemic tissue endocardial and epicardial lactate values were similar, and acceptable values for phosphocreatine, adenosine triphosphate and inorganic phosphate were found in both epicardial and endocardial tissue (unpublished data). Fifth, pyruvate may be difficult to measure despite rapid enzymatic assay immediately after deproteinization and neutralization of tissue or blood. Poor assay of blood pyruvate should lead to a falsely high arterial lactate/pyruvate ratio, but our arterial ratios were lower than those of Griggs et al. 19 If our values for blood pyruvate are acceptable, then our tissue values should also be acceptable because our finding of no pyruvate gradient from tissue to blood is in good agreement with data on pyruvate kinetics studied in the perfused rat heart (at physiologic circulating pyruvate concentrations) .lo ,11 Finally, in attempting to calculate the true intracellular lactate concentration, one must allow for the extracellular space and extracellular lactate concentration; these variables cannot be measured in poorly perfused tissue. The actual lactate concentrations in fluid bathing severely ischemic cells may be much greater than in local venous blood and there may be little or no true lactate gradient in ischemia. Anoxia decreases the tissue extracellular lactate gradient in the perfused rat heart. 1~ However, there is a definite gradient for lactate between coronary sinus venous blood and ischemic tissue in the dog.

the significance of lactate release into coronary venous blood as a sign of myocardial ischemia in comparison with other metabolic changes in coronary venous blood. When small segments of the heart are made ischemic, the metabolic change most readily detected in coronary sinus blood is decreased lactate extraction, which corresponds to lactate discharge into local venous blood (Fig. 1, Table II). Thus, no increase in sensitivity could be gained from the additional measurement of pyruvate and consideration of lactate/pyruvate ratios or values such as the difference in the redox state of the lactate dehydrogenase system in arterial and coronary venous blood (AEh) or the difference in lactate/pyruvate ratio across the heart. 4,9 This conclusion supports the recent practice of Gorlin 2 and Krasnow et al., 5 of omitting pyru-

vate measurements in the metabolic assessment of myocardial ischemia in man. Our data do n o t indicate t h a t every instance of decreased lactate extraction or lactate discharge by the heart is evidence of ischemia or hypoxia of the myocardium. Lactate discharge can occur from the apparently well oxygenated heart in (1) the neonatal heart in situ2Z; (2) states in which the extracellular fluid contains little or no lactate as in the isolated heart perfused with glucose as the only substrate11; (3) the transplanted heart22; (4) intermittently in some apparently normal awake dogs with chronically implanted coronary sinus catheters9; and (5) occasionally in apparently normal patients. 23 To determine whether lactate discharge from the heart indicates tissue hypoxia, the clinical circumstances should be taken into account. For example, the occurrence of lactate changes in coronary sinus blood during angina pectoris induced by atrial pacing or exercise would be abnormal and would indicate myocardial tissue hypoxia provided that changes in free fatty acid extraction are excluded. The simultaneous occurrence of hemodynamic abnormalities or of S-T segment changes would also indicate that lactate discharge was abnormal. Additional biochemical measurements may also be helpful (see later). Lactate discharge or decreased lactate uptake is the result of increased glycolysis and a relative decrease in the rate of pyruvate entry into the Krebs cycle. The source of the increased glycolysis in ischemia is probably both increased glucose uptake and glycogen breakdown. An increased cytoplasmic lactate/pyruvate ratio reflects an increased cytoplasmic ratio between reduced and oxidized forms of free nicotine adenine dinucleotide (NADH/NAD+), which is frequently but not always the result of anaerobiosis. 9 Thus, theoretically, neither lactate nor lactate/pyruvate changes by themselves are diagnostic of ischemia. The pyruvate concentrations in coronary venous blood and tissue during ischemia are similar; it follows that changes in the lactate/pyruvate ratio in tissue or blood will largely reflect changes in lactate concentration. Although calculations of AEh of the lactate/pyruvate system across the heart during ischemia suggest that accurate quantitation of myocardial hypoxia is possible by blood measurements, extrapolation of lactate values or of lactate/pyruvate ratios from coronary venous blood to ischemic tissue is misleading. This is because the substantial concentration gradients for lactate developing from ischemic tissue to local venous blood and from local venous blood to coronary sinus blood show that lactate/pyruvate ratios measured in venous blood cannot accurately reflect lactate/pyruvate ratios in ischemic tissue. Considerations of the lactate/pyruvate

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Discussion

Comparison between lactate and lactate/pyruvate changes: Our studies were designed to evaluate

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ratio can help to distinguish physiologic from pathologic lactate discharge 9,21 but do not provide a more sensitive index of ischemia than lactate changes, and also do not allow calculation of cytoplasmic ratios of free NADH/NAD+ in ischemic heart tissue. Lactate gradients: The existence during ischemia of a lactate gradient from endocardial to epicardial tissue, and from tissue to coronary venous blood is in agreement with the findings of Griggs et al. 19 In nonischemic tissue, there is no transmural lactate gradient (our results and those of Griggs et al.Z9), but we give evidence for a tissue-coronary venous blood gradient. Griggs et al. 19 failed to find such a gradient in one study in which the arterial lactate concentration and lactate/pyruvate ratio were over double the values obtained in an earlier study by the same group. 24 In the earlier study 24 arterial lactate and lactate/pyruvate values corresponded to ours, and there must have been a tissue-coronary venous lactate gradient in normal myocardium in contrast to the absence of such a lactate gradient in the later study. 19 The existence of tissue-venous lactate gradients is well documented in the isolated rat heart 1°,11 in which the intracellular lactate concentration exceeds the arterial value until the latter exceeds 4 mM. 1° Glaviano2~ also found a lactate gradient between blood and tissue in the dog heart but reversed the direction of the gradient by an infusion of lactate. Thus, the higher arterial lactate value in Griggs' later study 19 might have altered tissue-blood lactate interrelations. All published data 1°,11,19,24 and our results suggest that pyruvate values in heart tissue and coronary venous fluid are similar (provided the extracellular concentration is not unphysiologically high11). Use of beta hydroxybutyrate/acetoacetate ratio: Because the ketone body redox couple (beta hydroxybutyrate/acetoacetate) is thought to be in equilibrium with the mitochondrial NADH/NAD+ pool, 26 there should be an increased ratio of beta hydroxybutyrate/acetoacetate in coronary venous blood 2 and a decreased extraction of beta hydroxybutyrate by the heart during hypoxia or ischemia. We found only small changes in this ratio (Table II), which is not surprising because of the low activity of beta hydroxybutyrate dehydrogenase in the dog heart. The apparent ketone body concentrations in heart tissue are extremely low and the ketone bodies are mainly extracellular. 27 Ketone body ratios are not a sensitive index of ischemia in the dog although decreased ketone body extraction may be better (see Addendum).36 Decreased pH and increased PCO2: The development of an extracellular acidosis could indicate increased formation of C02 or hydrogen ion within the heart cell. C02 retention is unlikely because of the rapid rate of diffusion. The rate of oxidative res-

piration is decreased in ischemia, hence increased C02 formation is probably a consequence of increased hydrogen ion formation. Thus, the extracellular acidosis we found probably reflects increased intracellular hydrogen ion formation associated with anaerobic glycolysis. This conclusion is supported by the close association of lactate and pH changes in coronary venous blood 2s (Fig. 5). Rapidity and significance of coronary venous changes in phosphate and potassium: Activation of the rate-limiting enzyme of glycolysis, phosphofructokinase, occurs in anaerobic glycolysis. Such activation is a complex phenomenon, resulting from decreased inhibition by adenosine triphosphate as well as activation by inorga.nic phosphate, adenosine diphosphate and adenosine monophosphate. 29 Therefore, there is unlikely to be a constant molar relation between formation of inorganic phosphate and lactate in the tissue; the possibility of unequal rates of diffusion from ischemic tissue would make a molar relation in venous blood even less likely. The varying relation between loss of potassium ion and phosphate in our experiments 12,1e makes it unlikely that the fixed molar relation observed by Case 3° between potassium ion, inorganic phosphate and lactate loss would hold for all varieties of ischemia. A.brupt cessation of flow in one coronary artery in our experiments differs from the gradual decrease of flow to the whole left ventricle in Case's experiments. Since we found substantial potassium ion loss and S-T segment elevation rather than depression, it appears unlikely that there is a direct relation between S-T segment depression and potassium ion loss, as previously suggested by Case et al. 31 Nevertheless, potassium ion loss occurs in a close temporal relation to electrocardiographic changes (whether depression or elevation of the S-T segment) and at the same time that anaerobic glycolysis starts and high energy phosphate compounds are broken down. Major depletion of tissue levels of potassium ion does not occur for some hours after coronary arterial ligation, and develops progressively over days. In contrast, major depletion of high energy phosphate compounds occurs within 5 to 60 minutes of coronary arterial ligation. Hence, it may be expected that release of potassium ion would be a better late sign of prolonged ischemia than release of phosphate. During early regional ischemia, however, release of inorganic phosphate is a more sensitive indicator than release of potassium. 16 Glucose changes: An increased arteriovenous difference for glucose indicates an increased glucose uptake relative to that of free fatty acid, and a shift from lipid to carbohydrate metabolism by the ischemic tissue: 12,82 As in patients with angina pectoris induced by pacing, 2,3s an increased glucose arterio-

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CORONARY SINUS LACTATE AND MYOCARDIAL ISCHEMI/~BOPIE ET AL.

venous difference is associated with lactate discharge (local venous changes, Table II) rather than decreased 'lactate extraction (coronary sinus changes, Table II). Glucose changes are, therefore, less sensitive than lactate changes in the detection of myocardial ischemia. The large variation in control arteriovenous differences for glucose across the human heart also argue against the use of glucose changes in the assessment of myocardial ischemia in man. a3 Application to h u m a n disease: Our conclusions are based on experiments with coronary arterial ligation which may differ from myocardial ischemia in man. Similar large gradients for lactate but not pyruvate have been found in other ~experimental circumstances such as the anoxic 11 or the ischemic rat heart (unpublished data). The development of gradients of lactate between tissue and blood during attacks of angina pectoris would explain the localized lactate release shown by Gorlin 2 with the use of selective coronary sinus catheterization in man. Furthermore, a close correspondence between loss of lactate, hydrogen ion, potassium ion and phosphate has been found by Case and co-workers 3°,31 in a v a r i e t y of conditions, including patients with pacing-induced angina. Although the patterns of change in coronary venous metabolites in pacing-induced angina differ from those we found after abrupt coronary arterial occlusion, it appears probable that our conclusions could be applied to the evaluation of coronary artery. disease .in man. 2,s3 Thus, decreased lactate extraction detected in coronary sinus blood is likely to be a more sensitive index of myocardial ischemia in man

than changes in lactate/pyruvate ratios, as also suggested by the data obtained by Neill a5 during pacing of patients with coronary artery disease. Coronary venous lactate changes need not always be indicative of myocardial tissue anaerolJiosis, and the additional measurements of pyruvate may provide greater certainty (but not sensitivity) of diagnosis provided no quantitation of the degree of ischemia is attempted. Similarly, it is to be expected that the additional measurement of other venous metabolic changes (hydrogen ion, inorganic phosphate and potassium) would also give greater accuracy in the biochemical diagnosis of myocardial ischemia, besides allowing a fuller understanding of intracellular events.

Acknowledgment We thank Professor J. P. Shillingford and the Medical Research Councils of Great Britain and South Africa for encouragement and support and Dr. D. H. Williamson, Metabolic Research Laboratory, Radcliffe Infirmary, Oxford, for enzyme assays.

Addendum After this paper was submitted for publication, Whereat and Chan s6 published data showing decreased extraction of beta-hydroxybutyrate and acetoacetate after the onset of acute myocardial infarction in the dog, but those changes were much less effective in separating ischemic from control animals than were changes in lactate extraction. The ratio beta-hydroxybutyrate/acetoacetate in coronary sinus blood did not differ from that in arterial blood in control dogs or those with infarction. As in our study, blood values for ketone bodies were very low.

References 1. Bing RJ, Vandam LD, Gregoire F, et ah Catheterization of coronary sinus and middle cardiac vein in man. Proc Soc Exp Biol Med 66:239-240, 1947 2. Gorlin R: Assessment of hypoxia in the human heart. Cardiology 57:24-34, 1972 3. Huckabee WE: Relationship of pyruvate and lactate during anaerobic metabolism. V. Coronary adequacy. Amer J Physiol 200:1169-1176, 1961 4. Gudbjarnason S, Hayden Re, Wendt VE, et al: Oxidation reduction in heart muscle. Theoretical and clinical considerations. Circulation 26:937-945, 1962 5. Krasnow N, Neill WA, Messer JV, et ah Myocardial lactate and pyruvate metabolism. J Clin Invest 41:2075-2085, 1962 6. Huckabee WE: Relationships of pyruvate and lactate during anaerobic metabolism. I. Effects of infusion of pyruvate or glucose and of hyperventilation. J Clin Invest 37:244-254, 1958 7. Harris P, Bateman M, Gloster J: Relations between the cardiorespiratory effects of exercise and the arterial concentration of lactate and pyruvate in patients with rheumatic heart disease. Clin Sci 23:531-543, 1962 8. Olson RE: "Excess lactate" and anaerobiosis. Ann Intern Med 59:960-963~ 1963 9. Opie LH, Marchetti G, Merle L: Myocardial metabolism of lactate and pyruvate in the awake dog. In, Coronary Blood Flow in Man. Methods and Significance in Myocardial Dis-

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10. 11.

12.

13. 14.

15. 16.

ease (Maseri A, ed). Torino, Minerva Medica, 1972, p 411-424 Henderson AH, Craig RJ, Gorlin R, et ah Lactate and pyruvate kinetics in isolated perfused rat hearts. Amer J Physiol 217:1752-1756, 1969 Opie LH, Mansford KRL: The value of lactate and pyruvate measurements in the assessment of the redox state of free nicotinamide-adenine dinucleotide in the cytoplasm of perfused rat heart. Europ J Clin Invest 1:295-306, 1971 Owen P, Thomas M, Young V, et al: Comparison between metabolic changes in local venous and coronary sinus blood after acute experimental coronary arterial occlusion. Amer J Cardiol 25:562-570, 1970 Thomas M. Shulman G, Opie LH: Arteriovenous potassium changes and ventricular arrhythmias following coronary artery occlusion. Cardiovasc Res 4:327-333, 1970 Hood WB Jr., McCarthy B, Lown B: Myocardial infarction following coronary ligation in dogs. Hemodynamic effects of isoproterenol and acetyl strophanthidin. Circ Res 21:191199, 1967 Maroko PR, Kjekshus JK, Sobel BE, et al: Factors influencing infarct size following experimental coronary artery occlusions. Circulation 43:67-82, 1971 Opie LH, Thomas T, Owen P, et al: Increased coronary venous inorganic phosphate concentrations during experimental myocardial ischemia. Amer J Cardiol 30:503-513, 1972

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17. Pool PE, Norris GF, Lewis RM, et al: A biopsy drill permitting rapid freezing. J Appl Physiol 24:832-833, 1968 18. Danforth WH, McKinsey JJ, Stewart JT: Transport and phosphorylation of glucose by the dog heart. J Physiol 162:367-384, 1962 19. Griggs DM Jr., Tchokoev VV, Chen Ch!n Chi: Transmural differences in ventricular tissue substrate levels due to coronary restriction. Amer J Physiol 222:705-709, 1972 20. Lehninger AL, Sudduth HC, Wise JB: D-~'-hydroxybutyric dehydroxygenase of mitochondria. J Biol Chem 235:24502455, 1960 21. Zlatos L, Barta E: Myocardial oxygen supply in puppies in the earlY postnatal period. J Molec Cell Cardiol 4:329-336, 1972 22. Gudbjarnason S: The use of glycolytic metabolism in the assessment of hypoxia in human hearts. Cardiology 57:3546, 1972 23. Lassers BW, Kaijser L, Carlson LA: Myocardial lipid and carbohydrate metabolism in healthy, fasting men at rest: studies during continuous infusion of 3H-palmitate. Europ J Clin Invest 2:348-358, 1972 24. Griggs DM Jr., Tchokoev VV, De Clue JW: Effect of betaadrenergic receptor st'imulation on regional myocardial metabolism: importance of coronary vessel patency. Amer Heart J 82:492-502, 1971 25. Glaviano VV: Distribution and gradient of lactate between blood and heart muscle. Proc Soc Exp Biol Med 118:11551158, 1965 26. Williamson DH, Lund P, Krebs HA: The redox state of free

27. 28. 29. 30. 31. 32. 33. 34. 35. 36.

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nicotinamide-adenine dinucleotide in the cytoplasm and mitochondria of rat liver. Biochem J 103: 514-527, 1967 Adler-Kastner L, Keller B, Kraupp O, et al: The myocardial oxaloacetate level in normal and chronic alloxan-diabetic rats in vivo. J Molec Cell Cardio/4:391-400, 1972 Obeid A, Smulyan H, Gilbert R, et al: Regional metabolic changes in the myocardium following coronary artery ligation in dogs. Amer Heart J 83:189-196, 1972 Mansour TE: Studies on heart phosphofructokinase: purification, inhibition and activation. J Biol Chem 238:22852292, 1963 Case RB" Ion alterations during myocardial ischemia. Cardiology 56:245-262, 1971//2 Case R B, Roselle HA, Crampton RS: Relation of ST-depression to metabolic and hemodynamic events. Cardiologia (Basel) 48:2-41, 1966 Owen P, Thomas M, Opie L" Relative changes in free fatty acid and glucose utilisation by ischaemic myocardium after coronary arterial occlusion. Lancet 1:1187-1190, 1969 Most AS, Gorlin R, Soeldner JS: Glucose extraction by the human myocardium during pacing stress. Circulation 45:92-96, 1972 Parker SO, Chiang MA, West RO et al: The effect of ischemia and alteratiohs of heart rate on myocardial potass!um balance in man. Circulation 42:205-217, 1970 Neill WA: M);ocardial hypoxia and anaerobic metabolism in coronary heart disease. Amer J Cardiol 22:507-515, 1968 Whereat AF, Chan A: Effects of hypoxemia and of acute coronary occlusion on myocardial metabolism in dogs. Amer J Physiol 223:1398-1406, 1972

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