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European Journal of Heart Failure 2002 4(5):587-591; doi:10.1016/S1388-9842(02)00088-0
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© 2002 European Society of Cardiology

Myocardial lactate dehydrogenase patterns in volume or pressure overloaded left ventricles

Cornelia Pipera,*, Dieter Horstkottea, Anne-Katrin Bockb, Edmund Wudelc, Heinz-Peter Schultheißb and Andrea Dörnerb

a Department of Cardiology, Heart Center North Rhine-Westphalia, Ruhr University of Bochum Georgstrasse 11, 32545 Bad Oeynhausen, Germany
b Department of Cardiology, Benjamin Franklin Hospital, Free University of Berlin Berlin, Germany
c Department of Diagnostic Radiology and Nuclear Medicine, Benjamin Franklin Hospital, Free University of Berlin Berlin, Germany

* Corresponding author. Tel.: +49-5731-971258; fax: +49-5731-972194. E-mail address: cpiper{at}hdz-nrw.de

Received August 2, 2001; Revised October 25, 2001; Accepted January 17, 2002


   1. Background
Top
 1. Background
2. Aims
 3. Methods and results
 4. Results
 5. Conclusion
 References
 
In patients with left ventricular hypertrophy [1–4] and congestive heart failure [5–9] myocardial energy metabolism is adapted to the chronic burden. The pyridine nucleotide redox system (NAD/NADH system) is a sensitive indicator for the functional status of cellular oxidation [2,10]. As the NAD/NADH system is closely linked with the lactate dehydrogenase system the latter can be used to evaluate the NAD/NADH system and thus the myocardial energy status [11]. Myocardial lactate dehydrogenase activity and isoenzyme patterns can be considered a sensitive method to analyze changes of the enzyme status in patients with various heart diseases [7,12,13].


   2. Aims
Top
 1. Background
 2. Aims
3. Methods and results
 4. Results
 5. Conclusion
 References
 
We assessed myocardial LDH activity and LDH isoenzyme pattern of patients with aortic stenosis, aortic or mitral regurgitation, analyzed whether severity of left ventricular volume or pressure overload correlates with severity of myocardial dysfunction, and whether the LDH system can be used as an early biochemical indicator for myocardial maladaptation.


   3. Methods and results
Top
 1. Background
 2. Aims
 3. Methods and results
4. Results
 5. Conclusion
 References
 
3.1. Patients
Twenty-four patients, 13 men and 11 women with a mean age of 64±12 (34–86) years with chronic pressure (aortic stenosis: AS, n=12) or volume overload (aortic regurgitation: AR, n=6; mitral regurgitation: MR, n=6) but without terminal heart failure were examined. All patients underwent cardiac catheterization. No patient revealed signs of significant (>50%) coronary artery disease. After informed consent, we obtained endomyocardial biopsies from the interventricular septum during right heart catheterization. Myocarditis was excluded according to the Dallas classification [14].

The clinical and hemodynamic characteristics of the patients enrolled are listed in Table 1. We calculated aortic valve obstruction from the transvalvar pressure loss (PLaorta), and volume overload from the transvalvar regurgitant fraction (RF) [15]. Analysis of echocardiography was used to calculate the maximal systolic circumferential wall stress (WS; g/cm2) [15].


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  		Table 1 Clinical, invasive and non-invasive hemodynamic characteristics of patients with aortic stenosis (AS), aortic or mitral regurgitation (AR/MR), of patients undergoing heart transplantation because of end-stage heart failure caused by ischemic or dilated cardiomyopathy (EXPL) and of patients with suspected but excluded heart disease (controls)

 
In addition, endomyocardium was taken from the right ventricular septum of 10 hearts explanted from patients with end-stage heart failure due to dilated (DCM; n=4) or ischemic cardiomyopathy (ICM; n=6) (Table 1). Endomyocardial samples from six individuals without left ventricular dysfunction, who underwent cardiac catheterization for suspected myocarditis and in whom coronary artery and inflammatory heart disease had been excluded served as controls.

3.2. Gated equilibrium blood pool imaging
To evaluate the myocardial adaptation to the chronic volume or pressure overload, changes of the left ventricular ejection fraction (EF) at rest and under dynamic exercise were measured using gated equilibrium blood pool imaging. The planar triggered studies of the left ventricle were performed with a gamma camera (Sopha DSX) and analyzed with a fully automated method for EF determination [16]. The red blood cells were in vivo labeled after administration of 1mg SN(II)Cl with 10 MBq Tc-99m per kg. With the patient lying supine, the head of the gamma camera was positioned in a modified left anterior oblique view which was parallel to the interventricular septum. An increase in EF≤5% in AS or AR and ≤10% in MR or a decrease in EF under exercise conditions (25 W below the individual symptom-limited maximal exercise) was considered exhausted myocardial adaptation, an increase in EF >5% in AS or AR and >10% in MR, as maintained adaptation [14,17–19]. To further evaluate the left ventricular diastolic function, peak left ventricular filling rates at rest (PFR), were measured and expressed as end diastolic volume/second (EDV/s) (see Table 1) [20].

3.3. Cardiopulmonary exercise
To objectify the cardiac condition we also measured the maximal oxygen uptake (VO2 max) during a stepwise increased symptom-limited bicycle exercise test (Table 1) [21].

3.4. Assessment of myocardial lactate dehydrogenase activity
Biopsies were homogenized in 300 µl ice-cold 0.01 M phosphate buffer (pH 7.5), supplemented with 1 mM phenylmethanesulfonyl fluoride using an Ultra-Turrax T25 (Janke and Kunkel, Staufen, Germany). Homogenization was performed for 15 s at 13 500 rev./min. The homogenate was subsequently centrifuged for 30 min at 15 000 rev./min (Eppendorf centrifuge 5403) at 4 °C. Protein content was determined using the BCA protein assay (Fa. Pierce, Bonn, Germany).

For the LDH activity determination 500 ng protein was mixed with 500 µl reaction buffer (0.6 mM sodium pyruvate, 0.18 mM NADH; and 50 mM phosphate buffer, pH 7.5) and was incubated for 1 min at room temperature. The decrease in the extension rate was measured at 340 nm for 3 min in a Beckman spectrophotometer DU® (Krefeld, Germany). The LDH activity was calculated according the formula {Delta}E/minxF (units/l) (F=8095) and was standardized to the protein concentration. LDH activity was finally expressed as units/mg soluble protein.

3.5. Gel electrophoresis
The five LDH isoenzymes were separated according to their electrical charge on a 1% agarose gel, 1.4% AMPD bicine barbitate aspartate buffer, 0.1% sodium azide. Soluble protein (400 ng) was applied onto the gel and the electrophoresis was performed in a Paragon electrophoresis chamber (Beckman, Krefeld, Germany) for 35 min at 100 V. The gel was incubated in LD substrate solution (208 mM lithium L-lactate; 5.6 mM NAD; 2.4 mM p-nitroblue tetrazolium chloride and 0.33 mM N-methylphenazonium methosulfate) for 30 min at 45 °C. The gel was washed in 5% acetic acid for 1 min and was subsequently pressed between filter papers for 3 min. After an additional incubation of 5 min in 5% acetic acid the gel was dried. The relative percentages of each isoenzyme specific band was calculated using a densitometer (Fig. 1).


Figure 1
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  		Fig. 1 Gel-electrophoretic separation of LDH-1, LDH-2, LDH-3, LDH-4, LDH-5 isoenzymes as duplicates in endomyocardial biopsies from a control patient (C), three patients with VHD and an explanted terminal failing heart (EXPL).

 
3.6. Statistical analysis
All data are given as mean±S.D. For statistical analysis non-parametric tests were used. For comparison of more than two groups, the Dunn's test was utilized to assess two by two differences. Dunn's test is based on the pairwise comparison of mean ranks. All analyses were performed using StatView 4.0, except for the Dunn's test (SAS 8.1). A P-value of <0.05 was considered significant.


   4. Results
Top
 1. Background
 2. Aims
 3. Methods and results
 4. Results
5. Conclusion
 References
 
This study was designed to analyze whether changes in the cardiac function of patients with valvar heart disease (VHD) are associated with an altered activity and/or isoenzyme shift of the myocardial lactate dehydrogenase (LDH). Compared with the control group (LDH activity: 7.4±1.7 units/mg) the mean LDH activity was distinctly higher in patients with left ventricular pressure overload (AS, 11.7±5.5 units/mg) and volume overload (AR and MR, 9.8±3.8 units/mg). The highest average LDH activity (16.6±7.8 units/mg) however, was found in hearts explanted from patients with end-stage heart failure (P<0.05) (Fig. 2). In comparison with the control group the average amount of LDH-1 isoenzyme was significantly decreased (P<0.05), while the average amount of LDH-5 isoenzyme was distinctly and of LDH-M subunits significantly (P<0.05) increased in myocardium from explanted hearts. An LDH isoenzyme shift towards LDH-5, respectively, towards LDH-M as found in end-stage heart failure was not seen in patients with VHD. In patients with AS, AR, or MR neither the mean amount of LDH-1 isoenzyme was found to be decreased nor that of LDH-5 isoenzymes or of LDH-M subunits were found to be increased (Fig. 3).


Figure 2
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  		Fig. 2 Box plots showing a significant elevation of myocardial LDH activities in explanted hearts (EXPL) from patients with end-stage heart failure and a distinct but not significant one in patients with aortic stenosis (AS), aortic or mitral regurgitation (AR/MR) compared with those found in controls (C).

 


Figure 3
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  		Fig. 3 In patients with valvar heart disease compared to controls (C) no significant changes were seen in the percentage of LDH-1, LDH-5 or LDH-M isoenzymes between patients with AS or AR/MR and C. However, in EXPL the average level of LDH-1 decreased while that of LDH-5 and LDH-M increased, resulting in a LDH-isoenzyme shift towards LDH-5/LDH-M compared with controls.

 
According to the increased LDH activity in end-stage heart failure we observed in patients with VHD a negative correlation between LDH activity and cardiac index (r=–0.45) as well as between LDH activity and maximal oxygen uptake (r=–0.35) (Fig. 4). LDH activity was not only elevated in impaired systolic function but also parallel to increasing left ventricular peak filling rates (r=0.55), which are indicative for impaired left ventricular relaxation (Fig. 5).


Figure 4
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  		Fig. 4 A distinct increase in myocardial LDH activity was found in patients with VHD parallel to decreasing maximal oxygen consumption (VO2 max).

 


Figure 5
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  		Fig. 5 Increased myocardial LDH activity was correlated with impaired left ventricular diastolic function measured as increased peak filling rates in patients with AS, AR and MR.

 
In patients with VHD, increasing LDH activities were found parallel to the increase in left ventricular workload, with a good correlation to increased pressure (r=0.66), and a poor one to increased volume overload (r=0.14). Systolic wall stress and changes of left ventricular EF during dynamic exercise, parameters accepted as indicators for myocardial adaptation/maladaptation to increased workload, were not correlated with increasing LDH activity or LDH isoenzyme shift.


   5. Conclusion
Top
 1. Background
 2. Aims
 3. Methods and results
 4. Results
 5. Conclusion
References
 
Our data indicate that the level of LDH activity is increased not only in human hearts explanted for end-stage heart failure, but also in patients with valvar heart disease parallel to decreasing systolic and diastolic ventricular function. A significant shift of the LDH isoenzymes was found in end-stage heart failure but not in patients with VHD of different hemodynamic severity. This might indicate that with progression of left ventricular myocardial dysfunction, LDH activity increases before a shift of the LDH isoenzymes occurs. Such a shift may not be seen, until the myocardium is severely or terminally damaged. However, it was found to be reversible if heart failure was medically recompensated [7].

LDH activity but not LDH isoenzyme shift may be used to detect early energetic imbalance and may be an early marker for an inadequate myocardial adaptation to chronic overload [15,22,23]. According to our results LDH activities ≥12 units/mg possibly indicate the beginning of an exhausted myocardial adaptation, as mean peak filling rates at rest [2.8±0.6 (EDV/s)] were distinctly higher in valvar heart patients with LDH activities ≥12 units/mg than in those with lower LDH activities [2.1±0.9 (EDV/s)]. If these findings are validated in prospective studies [24,25] increased myocardial LDH activity (possibly ≥12 units/mg) can be considered as an early indicator for an energetic exhaustion of myocardial adaptation to chronic left ventricular overload and may eventually be used to improve the timing of valve surgery in order to gain full reversibility of myocardial function early after valve surgery.


   Acknowledgements
 
This study was supported by a grant from the Franz-Loogen-Foundation. Furthermore, we would like to thank Mr Rito Bergemann from the Institute of Medical Outcome Research (IMOR) for the statistical analyses.


   References
Top
 1. Background
 2. Aims
 3. Methods and results
 4. Results
 5. Conclusion
 References
 

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