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A4236
October 14, 2014
1:00 PM - 3:00 PM
Room Hall B1-Area C
Responses to in Vitro Malignant Hyperthermia Bioassays Are Temperature Dependent
Andrew M. Parsons, M.D., Saiid Bina, Ph.D., Eugene R. Smith, B.S., John F. Capacchione, M.D., Sheila M. Muldoon, M.D.
Walter Reed National Military Medical Center, Bethesda, Maryland, United States
Introduction: Malignant hyperthermia (MH) is a pharmacogenetic disorder of skeletal muscle calcium regulation characterized by a hypermetabolic reaction with exposure to volatile anesthetics and/or succinylcholine. The diagnosis is based on an in vitro contracture bioassay performed on a skeletal muscle biopsy. In a previous in vitro study performed on MH susceptible (MHS) swine B lymphocytes, adenosine response to 4-chloro-m-cresol (4CmC) stimulation (a measure of ATP turnover) was significantly greater compared to B cells from normal swine (1), suggesting the use of B cells as a surrogate to muscle testing. Since muscle contracture and B cell stimulation cellular energetics both require temperature sensitive enzymatic reactions, we hypothesize that responses in these bioassays are temperature dependent.

Methods: Immortalized human B lymphocytes from 4 patients confirmed MHS by in vitro muscle contracture testing and RYR1 analysis were prepared as previously described (1) and treated with 4CmC (1mM) at 20, 30, 37 and 45°C to assay for adenosine content via high performance liquid chromatography. This assay was repeated multiple times for each patient. Skeletal muscle strips (n=4) from two MHS confirmed patients were placed in organ baths and baseline twitch was recorded at 37, 30 and 20°C. Halothane (3%)-induced contractures were measured at 37 and 20°C in two different sets of MHS confirmed muscle strips. Data were analyzed using one-way ANOVA, and presented as mean ± SEM. P<0.05 considered significant.

Results: Basal adenosine levels of B cells at 20 and 45°C were 0.760 ± 0.091 and 1.259 ± 0.119 µM (P=0.018, n=7), respectively. 4CmC (1mM) increased adenosine levels by 27 ± 5 and 78 ± 12% when B cells were incubated at 20 and 45°C, respectively (P=0.016, n=7). Skeletal muscle baseline twitch amplitude and halothane (3%)-induced contractures decreased significantly (> 90%) with decreasing temperature.

Conclusions: These preliminary data indicate that in vitro MH diagnostic bioassays are temperature dependent. Future temperature studies should compare non-MHS B cells and muscle responses to those that are MHS.

References:

1. Anesthesiology 2010; 113:917-24.

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