The effect of serum on the hypodynamic frog heart

Hutton, Therese (1989) The effect of serum on the hypodynamic frog heart. Doctoral thesis, Lancashire Polytechnic.

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An investigation was made of the effects of serum and serum fractions on the isolated spontaneously beating frog heart
Initial experiments were performed to develop a sensitive bioassay preparation to test for inotropic agents in serum. The isolated spontaneously beating frog heart was found to be sensitive, reliable and responded consistently to serum and its components.
During the development of the bioassay it was found that the heart became weak after prolonged perfusion. An investigation was then made to determine the mechanism underlying the development of the hypodynamic (weak) condition of the isolated heart.
It was found that a heart which was immersed in static oxygenated Ringer's solution did not become hypodynamic. Continuous perfusion (washing) of the external surface of the heart led to partial development of the hypodynamic state. However when external perfusion was combined with internal perfusion, the onset of the hypodynamic state was faster reaching a steady-state of around 50% of the original tension after 150 minutes. During this steady state the preparation was used to investigate the inotropic effects of serum, serum fractions and several metabolic substrates including pyruvate, lactate and creatine on the heart.
A comparative study was made in order to determine the oxygen consumption and the levels of endogenous lactate, pyruvate and creatine phosphate in the fresh and hypodynamic hearts. The levels of lactate, pyruvate and creatine phosphate were significantly decreased in the hypodynamic hearts comparea with normal preparations. Oxygen consumption was unchanged and contractile force decreased by 47% in the hypodynamic hearts. These initial results show that the development of the weak condition of the heart may be associated with a metabolic deficiency of the
Perfusion of the isolated spontaneously beating hypodynamic frog heart with dialysed serum taken from several animal species evoked both positive inotropic and chronotropic responses in the presence of cy•adrenergic (phentolamine),/3-adrenergic (propranolol) and cholinergic antagonists (atropine). These effects were dose-dependent.
The cardioactive component(s) of serum were separated using several fractionation techniques. These included gel filtration on C50 and C200 Sephadex, filtration on Amicon microconcentrators and ultrafiltration on PM30 and XM100A membranes. During the separation procedures the fractions were tested for inotropic activity compared with dialysed serum. A fraction corresponding to serum albumin in molecular weight (60000-63000) evoked a positive inotropic effect on the heart. Commercial serum albumin evoked no net effect on the heart. Serum and its fractions were subjected to physical and chemical treatments including boiling, pH changes, chymotrypsin and lipase treatment and lipid extraction in an attempt to determine the nature of the cardioactive component(s).
During the various stages of treatment the fractions were tested on the heart for bioactivity. Boiling, pH changes and chymotrypsin treatment failed to denature the cardioactive component of serum corresponding to serum albumin
in molecular weight. After these treatments a positive inotropic response was still retained. Similar responses were obtained with a lipid extract of serum. However, the activity of these fractions disappeared after treatment with the enzyme lipase suggesting that that the cardioactive component has lipid like properties. No attempt was made to identify and characterise this unknown lipid due to lack of time.
In conclusion, the present study demonstrates that serum contains a lipid—like cardioactive fraction which may be associated with serum albumin. It evokes positive inotropic and chronotropic effects on the isolated hypodynamic frog heart.

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