Comparison of non-collagen protein and total creatine as reference for determination of energy stores in endomyocardial biopsies

Objective: The aim was to establish a reliable reference system for biochemical measurements in endomyocardial biopsies. Methods: Myocardial tissue samples were obtained from pigs before and after cardioplegic arrest and reperfusion. Non-collagen protein content was evaluated as a non-specific reference system and compared with total creatine content representing a specific myocardial reference system. The influence of base strength, extraction temperature, and extraction time on protein yields was determined in tissue precipitates redissolved in NaOH. Interference from protein of collagenous origin was excluded by hydroxyproline determinations. Variability of myocardial ATP content in relation to non-collagen protein and total creatine was compared in endomyocardial biopsies taken before and after cardioplegic arrest and reperfusion. Results: The two methods showed comparable analytical precision. Apart from an interference in 1.0 mol·litre−1 NaOH for extended extraction periods at high temperatures, myocardial protein yields increased with increasing base strength, extraction temperature, and extraction time. During cardioplegic arrest and reperfusion heart weight increased due to oedema. Simultaneously, myocardial non-collagen protein content decreased. No change in total creatine was found during cardioplegic arrest but there was a significant loss of creatine after reperfusion. Comparison of variability in myocardial ATP content with non-collagen protein or total creatine as reference systems revealed no difference. Conclusions: Determination of non-collagen protein can be optimised with standardised conditions for protein extraction in tissue precipitates. Employment of total creatine as a reference system does not reduce variability of myocardial metabolite determinations in endomyocardial biopsies compared with non-collagen protein. Loss of myocardial creatine may in itself provide additional information about myocardial injury but this makes it unsuitable as a reference system for measuring metabolic changes during reperfusion. Multiple biopsies seem necessary for estimation of myocardial energy stores. Cardiovascular Research 1993; 27: 2113-2117