Lymphocyte-based determination of susceptibility to malignant hyperthermia: a pilot study in swine.
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.


CONCLUSIONS
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.
Bina
Saiid
S
Department of Anesthesiology, Uniformed Services University of the Health Sciences, Bethesda, Maryland, USA. sbina@usuhs.mil
Capacchione
John
J
Muldoon
Sheila
S
Bayarsaikhan
Munkhuu
M
Bunger
Rolf
R
D000241:Adenosine / Q000378:metabolism; D058892:Adenosine Deaminase Inhibitors; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D004198:Disease Susceptibility; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007093:Imidazoles / Q000494:pharmacology; D007288:Inosine / Q000378:metabolism; D008214:Lymphocytes / Q000502:physiology*; D008274:Magnesium / Q000494:pharmacology; D008305:Malignant Hyperthermia / Q000175:diagnosis*; D009125:Muscle Relaxants, Central / Q000494:pharmacology; D010080:Oxazoles / Q000494:pharmacology; D010085:Oxidative Phosphorylation / Q000187:drug effects; D010865:Pilot Projects; D019837:Ryanodine Receptor Calcium Release Channel / Q000502:physiology; D012519:Sarcoplasmic Reticulum / Q000187:drug effects; D013552:Swine; D014475:Uncoupling Agents / Q000494:pharmacology
D058892:Adenosine Deaminase Inhibitors; D004791:Enzyme Inhibitors; D007093:Imidazoles; D009125:Muscle Relaxants, Central; D010080:Oxazoles; D019837:Ryanodine Receptor Calcium Release Channel; D014475:Uncoupling Agents; D007288:Inosine; C061387:azumolene; D000255:Adenosine Triphosphate; D008274:Magnesium; D000241:Adenosine; D002118:Calcium