Lymphocyte-based determination of susceptibility to malignant hyperthermia: a pilot study in swine
Saiid Bina1, John Capacchione, Sheila Muldoon
1Department of Anesthesiology, Uniformed Services University of the Health Sciences, Bethesda, Maryland, USA. sbina@usuhs.mil
Background:
Malignant hyperthermia susceptibility (MHS) is diagnosed by an invasive in vitro caffeine-halothane contracture test (CHCT) carried out on biopsied skeletal muscle tissue. We are presenting a novel blood test approach for malignant hyperthermia testing in a swine model. Our main aim was to determine whether adenosine production from lymphocytes after 4-chloro-m-cresol (4CmC) stimulation distinguishes homozygous swine carrying the Arg615Cys mutation in the ryanodine receptor type 1 (RyR1) gene (MHS swine) from normal swine.
Methods:
Lymphocytes were isolated from arterial blood (40 ml) obtained from MHS (n = 7) and normal (n = 7) swine. Cells were suspended in Hank's balanced salt solution and treated with 4CmC (0-10 mm) at 37°C in the presence of adenosine deaminase inhibitor. After termination and purification of samples, aliquots (50 μl) were assayed for adenosine content using high performance liquid chromatography.
Results:
Baseline adenosine levels before stimulating lymphocytes with 4CmC were 0.025 ± 0.004 and 0.041 ± 0.006 μm (mean ± SEM) in lymphocytes from normal and MHS swine, respectively (P = 0.125). Maximum responses were achieved at 1 mm 4CmC for both cell-line groups. Adenosine levels after stimulation with 4CmC (1 mm) were 0.185 ± 0.009 and 0.397 ± 0.038 μm in lymphocytes from normal and MHS swine, respectively (P = 0.0035). There was no overlap between adenosine levels in stimulated lymphocytes from MHS and normal swine.
Conclusion:
4CmC stimulation of porcine lymphocytes induces increased adenosine formation in MHS cells relative to those from normal swine; evaluation of adenosine formation in response to RyR1 agonists in human lymphocytes is needed.
