Ongoing Anesthesiology Research Activities

This section is arranged as follows:

MH susceptibility is a ‘soft’ diagnosis, usually determined by family history of the disease, or by ‘suspicious’ metabolic responses to general anesthetics. Because MH-positive individuals do not always have episodes with each anesthesia, estimates of the frequency of MH in the general population are not thought to be very accurate. The current best estimates from large population studies have yielded values of 1 in 50,000 adults and 1 in 15,000 children. The difficulty with diagnosing MH has two consequences for military personnel; (1) it is possible to have the disease and not know it, creating obvious risks for military personnel deployed to the battlefield and (2) persons can be falsely labeled as MH susceptible, making them ineligible for deployment. The only way to definitively diagnose MH is to surgically remove a muscle segment from the thigh (Vasculus lateralis) and then to test its contracture sensitivity in vitro to halothane and/or caffeine (caffeine halothane contracture test, or CHCT). The CHCT criteria for positive MH diagnosis was standardized by the North American MH Registry (NAMHR) in 1987 and provides a diagnostic sensitivity of 97% and specificity of 78%. USUHS is one of only eight medical centers in the US to offer this diagnostic test, and is the only one affiliated with the military. Since 1987, a total 312 individuals have been evaluated at the USUHS MH Center. Of these, 226 have undergone diagnostic muscle biopsy and contracture testing, leading to a positive diagnosis in 111. The USUHS MH Center database includes clinical history, muscle histology/histochemistry and contracture test results on these patients.

• Developing a genetic screening program for MH
• Developing a minimally-invasive diagnostic test (e.g. blood test) for MH
• Carrying out basic research to determine the molecular causes of MH
• Providing medical students and graduate physicians the opportunity to do medical research on a clinically important disease.

 

 

 

Clinical service (Muldoon/Holman)          Back to Top

Since 1987, active duty military personnel and their dependents have been referred for evaluation and diagnostic biopsy to the anesthesiology laboratory at USUHS. A total of 312 individuals have been evaluated for the MH susceptibility at the USUHS MH Center. Clinical activities include clinical consultation, review of medical records, arrangement of muscle biopsy with surgeons, and supporting research protocols. Details of clinical activities are as follows:

1. The Clinical Coordinator of the Center, an experienced attending anesthesiologist, works closely with the Center Director (Chairperson, Anesthesiology) to evaluate all referrals, including civilians in the geographic area and military personnel and dependents worldwide.

2. After review of the medical records associated with the referral, a recommendation is made to the patient and/or parents with regard to biopsy and precautions to be taken.

3. If a biopsy is recommended and the patient consents, the appropriate surgeons are contacted, patient    referral is given, and scheduling arrangements are made. Most frequently, military patients have their biopsies performed at NNMC and civilians are referred to Children's National Medical Center.

4. The USUHS staff responsible for carrying out the CHCT and the genetic studies prepare for the arrival of biopsy specimens. Note: genetic screening protocols are optional for the patient undergoing the muscle biopsy and can be declined by the patient.

5. The Coordinator meets the patient and the Anesthesia/Surgery team preoperatively in the holding area, answers questions, and consents the patient/parents appropriately.

6. The Coordinator accompanies the operating team and patient to the operating room and assists with obtaining specimens for transport to USUHS.

7. After delivery of specimens to USUHS and completion of the CHCT, the Coordinator contacts the patient and gives a preliminary report if the initial testing.

8. After all studies have been completed, the Clinical Coordinator and Director go over the results and final recommendations are sent to the patient. Further patient counseling is arranged as necessary on a case by case basis.

9. Further studies may be requested to diagnose other family members. These are discussed with the patients and the family members and arrangements are made as appropriate.

Muscle contracture test                Back to Top

The criteria for diagnosing MH sensitivity using the caffeine halothane contracture test (CHCT) are strict. Isolated, perfused muscle fibers must show 0.2 g of tension for a 2-mM caffeine exposure or 0.7 g of tension following 3% halothane exposure. Both caffeine and halothane produce elevated intracellular calcium in the muscle fiber by interacting with the ryanodine receptor. Although the molecular mechanism of action for either agent is not yet known, each is assumed to stabilize the ryanodine receptor in an open conformation, allowing excess Ca^{+ +}efflux from the SR. The CHCT has a very high sensitivity (97%), meaning that few MH-susceptible persons are missed, but only a 78% specificity, meaning that there is 22% chance that a person will be labeled with a false positive result. Given the need to improve the CHCT, we are testing two other compounds known to bind to the ryanodine receptor, 4-chloro-m-cresol and ryanodine itself, to determine whether they would be useful adjunctive agents for the diagnosis of MH.

In order to be recognized as a diagnostic center to perform the 4-chloro-m-cresol and ryanodine tests, it is necessary to establish reference values for these tests with normal healthy control subjects. USUHS grant C08085-01 to LTC Joseph A. Bettencourt is currently supporting this research effort. Muscle specimens are obtained from normal volunteers undergoing elective lower extremity and abdominal surgery for contracture testing using 4-chloro-m-cresol and ryanodine. In order to verify that participating patients do not have MH or any other neuromuscular disease, values of creatine phosphokinase (CPK) are obtained at the time of enrollment.

Current Research Protocols                  Back to Top

Bettencourt, Joseph, LTC, Et al

Two new agents, 4-chloro-m-cresol (4-Cmc) and ryanodine have been used in Europe as supplemental tests in the diagnosis of MH. Both drugs activate the calcium release ryanodine (RYR1) receptor in skeletal muscle. These tests are believed to be useful additional tests to evaluate patients with suspected MH susceptibility, but there is insufficient data for these agents in a normal North American population. Under approved protocol with informed consent, 12 viable muscle strips were obtained from 6 patients between the ages of 42 and 57 years scheduled for elective surgery. Two patients were female and 4 were male. For 4-CmC, the skeletal muscle strips were exposed to increasing concentrations of the drug of 25 micromolar (uM), 50 uM, 75 uM, 100 uM, 125 uM, 150 uM, and 00 uM. Previous studies have consistently demonstrated that a concentration of 75 uM is the optimal threshold concentration for discriminating MHN from MHS muscle. This study demonstrated no increase in baseline contraction with any concentration of 4-CmC lower than 125 uM. Mean increases in baseline contraction tension with increasing concentrations were 125uM – 0.008g, 150 uM –0.125g and 200uM – 0/783g. For ryanodine, an increase in baseline contraction tension is produced in both MHS and MHN muscle, but the response in MHS muscle is quicker and more intense. Time to contraction is the discriminating variable with ryanodine test studies have identified a ryanodine concentration of 1 uM as providing optimum discrimination between MHN and MHS muscle. In this study, 10 viable muscle strips from 6 volunteers were evaluated. The 10 strips were observed for 30 minutes at a concentration of 1 uM with a mean increase in baseline contraction of 0.59g. The increases in tension for both 4-CmC and ryanodine do not meet the criteria for MHS by the European group. This data demonstrates that both 4-CmC and ryanodine may be excellent adjunctive agents in phenotyping patients as either MHS or MHN and establishes a normal baseline for these tests.

Conclusion:

1. Concentrations of 4CmC ranging from 25 – 125uM did not induce contractions in muscle strips from normal individuals.
2. Concentrations greater than 50uM, 4CmC induced contractions in strips from MHS individuals.
3. Ryanodine at concentrations of 0.5 and 1.0uM did not induce contractions in muscle specimens from control volunteers.
4. Concentrations of 1.0uM Ryanodine resulted in no overlap between control subjects and MHS muscle strips.
5. Results to date indicate that both drugs could be useful additional test agents.

MH susceptibility is associated with altered Ca²⁺ homeostasis in skeletal muscle, which in turn is associated with functional abnormalities of the ryanodine receptor type 1 (RYR1, Ca²⁺ release channel of skeletal muscle). RyRs belong to a class of Ca²⁺ -permeable ion channels that regulate release of Ca²⁺ from intracellular Ca²⁺ stores. Although it is known that mutations in RyRI are linked to the occurrence of MH, it is not yet known a) how anesthetics modify RyRI function (b) how mutations in RyRI affect calcium Ca²⁺ release from the sarcoplasmic reticulum (SR) or (c) how sustained elevations in intracellular Ca²⁺ [Ca²⁺]i lead to MH. RyRI is localized primarily to skeletal muscle, but is also present in cerebellar Purkinje cells. Recently Dr. Yoshi Sei found that RyRI is expressed in peripheral B-lymphocytes (Sei Y, et al., and J. Biol. Chem. 274. 5995-6002, 1999), which raises the possibility of using B cells to test for MH susceptibility. If RyRI function in human skeletal muscle and B cells are similar, then mutations in RyRI that lead to aberrant Ca²⁺ regulation in skeletal muscle will also lead to aberrant Ca regulation in B cells.

Although it is known that mutations in RyRI are linked to the occurrence of MH, it is not yet known (a) how anesthetics modify RyRI function (b) how mutations in RyRI affect Ca²⁺ release from the sarcoplasmic reticulum (SR) or (c) how sustained elevations in intracellular Ca²⁺ [Ca²⁺]i lead to MH. In skeletal muscle RyRIs are coupled to dihydropyridine (DHP) receptors to regulate release of Ca²⁺ from the SR. In non-muscle cells, RyRs regulate release of Ca²⁺ from microsomal stores, but the mechanism(s) of coupling between RyRs and surface receptors are less well understood. In order to better understand the molecular mechanism of RyRI action, and how mutations in RyRI produce functional changes that lead to MH susceptibility, we plan to (1) purify an endosomal fraction containing RyRI from B cells obtained from normal and MH-positive donors and reconstitute the receptor into planar lipid bilayers. (2) Characterize the ion channel properties of the reconstituted B cell RyRIs at the single channel level and compare them to muscle RyRIs (3) express RyRI receptors with known mutations in COS-1 cells, and measure Ca release through RyRIs and (4) immortalize B cells from MH patients to obtain sufficient numbers of cells to fully characterize the RyRI protein there. This program is supported by the Malignant Hyperthermia Association of the United States (MHAUS) and by USUHS grants 08091 and R8078 to Drs. McKinney and Sei, respectively.

This page was last updated on July 23, 2001

[ Home ] [ Malignant Hyperthermia ] [ National Capital Area Anesthesiology Residency Information ] [ Medical Student Rotation Information ] [ Ongoing Anesthesiology Research Activities ] [ Mission Statement ] [ Division of Critical Care Medicine ] [ Departmental and Affiliated Personnel ] 

  USUHS Home

USUHS Anesthesiology        Disclaimer

These web pages are maintained by:  Frank Olszewski.

E-mail suggestions about this website to:  folszewski@usuhs.mil