Adenine nucleotide metabolism in human blood platelets: a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Clinical Biochemistry at Massey University

Abstract

At present, a skeletal muscle biopsy provides the most specific test for susceptibility to Malignant Hyperthermia (MH). This procedure is unsuitable for large scale screening of individuals and a simpler, less invasive test to distinguish MR-susceptible (MHS) people from those not possessing the genetic defect, is highly desirable.

Platelets contain a calcium-activated contractile system, a calcium-storing and releasing system, and an active ATP-generating system. It is thus logical to assume that the same processes occur in platelets as those in muscle during MR-induced accelerated metabolism. In this research, [8-14 c]adenine incorporation into blood platelet adenine nucleotides was investigated with a view to using differences between platelets from normal and MHS individuals as the basis for a clinical test.

It was assumed that under resting and/or halothane-stimulated conditions, nucleotide turnover in MHS platelets is significantly abnormal, and that the turnover abnormality is reflected in differences in adenine incorporation to platelet nucleotides via the salvage pathway.

MHS platelets took up less adenine and assimilated it into nucleotides at a slower rate than normal platelets. However, after two hours, 20% more labelled ATP was extracted from MHS platelets than normal, with a concomittant decrease in ADP levels. Halothane had little effect on normal platelets but caused a 10% decrease in incorporation into ATP in MHS platelets. AMP labelling was lower than normal in MHS platelets, indicating increased deamination of this nucleotide.

Specific radioactivities of nucleotides were not measured since [ 14 c]adenine distributes evenly among metabolic ATP, ADP, and AMP; therefore, the total radioactivities were used as a measure for the levels of adenine nucleotides within the metabolic pool.

From the limited number of individuals screened, results suggest that MHS platelets have a higher basal ATP turnover rate than normal. When challenged with halothane the adenylate energy charge decreased, causing an increased nucleotide turnover rate which in turn led to a decreased ATP level due to the increased deamination of AMP. The appearance of more hypoxanthine and inosine than normal in the extraplatelet medium is consistent with the above sequence of events.

The platelet-halothane bioassay displays a limited ability to distinguish between normal and MHS individuals and may have the potential to become a less invasive equivalent to the "ATP-depletion test" in muscle.