Chapter Fifteen: Evolution of MH Tests and Frustrations

Chapter 15 Sub-sections

The patterns of test development varied over the years until the principal focus zeroed in on skeletal muscle. MH tests in time established precision and accuracy. Kalow et al (1970) introduced what became the gold standard for testing of MH susceptibility, namely exposure of excised small skeletal muscle strips from the thigh to caffeine and halothane in a body temperature 37º C (98.6º F) tissue bath. They demonstrated that susceptible muscle responded with contractures (literally a muscle cramp). Ellis et al (1971) added halothane, without caffeine, as a test substance. Normal muscle doesn't develop a contracture until greater concentrations of drug are added to the muscle bath. In muscle susceptible to MH, contractures occur with much lower concentrations. This sensitivity to lower concentrations of test substance is the basis of contracture diagnosis, and is the clinical surrogate of MH.

For muscle biopsy testing, the patient needs anesthesia, either a general anesthetic that won't trigger MH, or a regional anesthetic such as a spinal, epidural, or specific nerve block in the leg. The muscle has to be carefully handled during excision so that it is viable in the laboratory, i.e., it responds with a twitch to electrical stimulation. It is dissected into thin strips and mounted in a tissue bath containing usual body saline solutions, attached to a strain gauge, to measure muscle contractile activity.

As time has passed, a variety of substances has been introduced to produce muscle contractures on biopsy specimens, thinking that the unique most accurate substance might be discovered and thus perfect the process of contracture testing. These include, in addition to the classic halothane and caffeine, ryanodine, 4-chloro-m-cresol, and potassium.

Various MH work demonstrated that porcine red blood cells showed changes that predicted MH susceptibility in pigs (Andresen, 1971; Hojny, 1973; Rasmusen et al, 1976). Further studies demonstrated muscle abnormalities in patients and families (Britt, Kalow et al, 1973: Isaacs et al, 1973; King et al, 1972; Kelstrup et al, 1974).

A more detailed early history of MH and its investigations can be accessed in other sources (Gronert, 1980; Gronert, Pessah, et al, 2005).

MH: Exaggeration of Normal Responses

Moulds (1975) presented still the best definition of an MH episode as an exaggeration of normal physiological responses rather than different or new responses, whether human or porcine.

The MH susceptible pig has a single point genetic mutation in all muscular breeds throughout the world (Fujii et al, 1991). This is an amazing finding: a single mutation explains their MH susceptibility. Since it is unlikely that this mutation arose de novo in swine of muscular build throughout the world, it is likely that potent breeding stock were shared via shipping sows or boars to various countries some 150 years ago. While humans share many mutations, the single point porcine mutation permits identification of muscular litters that lack the mutation. Testing of swine litters is valuable in controlling breeding, saving millions in pork losses at slaughter. Even normal swine become over-active with stress, and stressed MH susceptible swine can respond with an MH episode.

Abattoir (slaughterhouse) Triggering of MH and Major Pork Losses

Swine losses in North America during slaughter were as much as several hundred million dollars annually prior to identification of the MH mutation. At the abattoir, after stunning and exsanguination, the carcass was suspended from a hook, skinned, gutted, and cleaned while moved along an overhead track to the cooling room. Susceptible swine had a huge increase in metabolism related to the stresses of slaughter, and, in the 45 minutes or so needed to reach the cooler, the meat became hot, edematous, oozed water, and spoiled.

Testing litters for the single point mutation minimizes these losses by permitting breeding of muscular swine without the MH mutation. The laboratory director and the technical transfer person at the University of Toronto described application of the test program for screening of swine litters, using porcine muscle obtained from the University of Guelph. The test was patented, and assigned to the University of Toronto Innovations Foundation for licensing worldwide. Commercial testing began in a number of laboratories.

Summary of MH Testing

My reconsideration of MH testing, as a Monday morning quarterback, has the advantage of knowing that contracture responses are reliable and have genetic confirmation. The rigidity of an MH episode and the concomitant increase in serum creatine phosphokinase (CK) values suggested that the origin of MH was directly related to skeletal muscle (Britt et al, 1973). Family evaluations implied that plasma CK values might aid in detection of susceptible persons (King et al, 1972; Isaacs et al, 1973; Kelstrup et al, 1974), but values in non-stressed susceptible humans were inconsistent and inaccurate (Paasuke et al,1986). CK, myoglobin, and potassium are released from muscle during extreme stress. Large increases in CK are not in themselves harmful, and indicate that the external membrane of the muscle is more porous. Myoglobin and potassium, respectively, are toxic to kidney and heart function. Back to MH testing: Once skeletal muscle contracture responses gained value in a few laboratories, they were evaluated by others in succeeding years.

After 1980, there was solid evidence that contracture responses were a valid determination of susceptibility. But these findings originated in a few university laboratories, and each had slightly varying protocols for study of porcine and human susceptible and non-susceptible muscle (Gronert, 1979, 1980; Gronert, Thompson et al, 1980). It was mandatory that all centers use standardized protocols, so that threshold responses for development of muscle contracture could be compared everywhere. After several years of conferences, contracture protocols were standardized (Ellis, 1984; Larach, 1989) and the revised protocol was studied in a few individual centers to confirm accuracy (Melton et al, 1989). But all centers had to be involved in such confirmation, and, a few years later, multi-center studies across Europe and North America provided acceptable sensitivity and specificity (Ording et al, 1997; Allen et al, 1998).

Genetic analysis of patients with clinical problems confirms the validity of contracture studies (Tobin et al, 2001; Girard et al, 2001; Kraev et al, 2003; Loke et al, 2003). The research into contracture testing illustrates Sir Karl Popper's dictum that a theory can be refuted but not verified, i.e., you cannot prove that it is true (Pollock et al, 1985). With time and multiple unsuccessful attempts at refutation, the theory, in this case contracture testing, is considered established. Other MH tests failed scrutiny.

Mayo MH Research

We developed a series of comprehensive multi-organ studies to quantitate alterations in MH. The porcine model, with its purebred approach to inbreeding for muscularity, hybrid vigor, and sensitivity to stress, results in marked stress susceptibility. Anesthetics trigger MH with the potent volatile agents such as halothane, isoflurane, sevoflurane, desflurane, and/or the muscle relaxant succinylcholine. Non-anesthetic MH occurs in swine exposed to environmental stress, fighting, shipping, coitus, or slaughter. It also rarely occurs in humans.

The exaggerated porcine response to anesthesia was first reported in 1966 (Hall LW et al). Others followed, analyzing its patterns: Harrison et al, 1969; Berman et al, 1970; Nelson et al, 1973; Lister et al, 1974; Hall GM et al, 1975; Gronert, Milde et al, 1976; Gronert, Theye, 1976, 1976; Hall GM et al, 1976, 1976, 1976; van den Hende et al, 1976; Lucke et al, 1976; Wood et al, 1977; Gronert, Heffron et al, 1977; Gronert, Milde et al, 1977; Hall GM et al, 1977, 1977; Gronert, Theye et al, 1978; Hall GM et al, 1978; Gronert, Milde et al, 1980; Hall GM et al, 1980; Hall GM et al, 1982; Gronert, White, 1988. While swine and human MH episodes are similar in many ways, porcine data can only with caution be transferred to humans.

The porcine feature of MH was identified as the Porcine Stress Syndrome, well reviewed (Topel et al, 1968; Cassens et al (1975). For our studies, we needed purebred swine, such as Poland China, Pietrain, or Landrace.

Tricks in Obtaining MH Susceptible Swine

When we wished to begin porcine MH research, we needed a reliable source of MH susceptible swine, and this proved tricky. Our prior single MH pig from the burn study was perhaps a single exception among his litter, and we didn't even know where he'd come from. In our search for suitable pigs, we quickly discovered that pig farmers don't want visitors, particularly investigators. Swine are susceptible to infection and strangers can introduce problems. Even normal swine are excitable and panic when confronted. We began our search by visiting the University of Wisconsin in Madison in early 1973. Their muscle laboratory was generously funded by royalties from sales of warfarin (Coumadin®), now widely used to diminish blood clotting in prevention of strokes and heart attacks.

Some years prior, their scientists had noted that cows that ate spoiled hay developed a bleeding disorder, and that the responsible substance was warfarin, leading the way to applied human anticoagulant use. The university had a swine herd reportedly susceptible to MH. Pat and I drove there, towing our horse trailer for bringing swine home. I spoke on hyperkalemia to the anesthesia department and met the MH researcher Charles Williams. He and I had vigorous arguments concerning our contradictory opinions regarding MH at the welcoming anesthesia department dinner. Williams, a biochemist, concerned with intra-cellular reactions, and I, an applied physiologist --- that's what anesthesia is --- had differing perspectives.

We tested the Wisconsin pigs by restraining them and giving them halothane to breathe. None tested positive. We brought several back to Minnesota, but they were not appropriate for MH research. I contacted the swine caretaker at Madison and asked why they had not tested positive. He told me that they'd bred it out of their herd by noting which sow or boar produced susceptible litters, and controlled breeding now produced muscular pigs without the MH gene.

He advised us to visit a local swine breeder in Minnesota who prided himself on his highly muscular animals. He said to never admit that we're looking for animals with problems, as no breeder would admit to that, ever.

The Wisconsin swine breeder was correct. It was difficult to gain the confidence of purebred swine breeders. A local Poland China breeder agreed to permit testing if we paid twice the market price for any pigs that we either killed or decided to take for study. His pigs were appropriately muscular: Fig. 13: note the developed hams (and the 14 gauge ear veins). We did not specify why we were testing. The farmer was suspicious and didn't become friendly until we'd visited him on several occasions.

Figure 13
Fig. 13. Poland China swine. Photo by Esther Gallant, Ph. D., used with permission.

He soon realized that we wanted to study stress susceptibility in his pigs, as that was a recognized problem in muscular breeds. He frankly told us that it would be useless, since he'd never ever seen a sign of it in any of his pigs. For access, he had us wear clean jeans and wash our boots doubly in pans of cleaner at the barn door. We had to be quiet and move slowly and easily in getting near the pigs. Even normal swine are easily spooked.

Figure 14
Fig. 14. Note the stiff limbs, off the ground, in the air.

We brought to the barnyard a G tank of oxygen (a cylinder about five feet tall, weighing some 80 pounds), a halothane vaporizer, and a plastic mask formed from a Clorox bleach bottle. We had cut out the bottom of the bottle and lined it with soft rubber to fit around the pig's snout, and drilled the stopper for flowing oxygen-halothane into the bottle, with a reservoir bag between gas supply and mask. The first pig breathed the mixture while restrained, and within three minutes developed pronounced hind limb rigidity, indicative of MH susceptibility. Fig. 14.

We stopped the administration of anesthetic, and congratulated ourselves on our success. About 8-10 minutes later, during this self-congratulation, the farmer called our attention to the fact that the pig had rolled its eyes back up into its head, and said, “When they do this, they die.” (This from someone who had never seen a stress pig death)

Sure enough, the pig died within a minute or so. We checked a rectal temperature; it was 112º F (44.4º C). We had now identified two MH pigs for research and prematurely killed them both (remember, we had a pig in the hyperkalemic study who surprisingly developed MH, which we successfully treated; we then lost him due to high pressure blowout of his lungs). Fortunately, further barnyard testing was more successful. The barnyard death occurred in 1973, but, once we knew of dantrolene (Harrison, 1975), we brought both a homemade intravenous preparation of dantrolene and bicarbonate to the barnyard.

Development of Dantrolene

Dantrolene has an unusual history. Keith Ellis, Ph.D., then of Norwich Eaton Laboratories in upper New York state, helped evaluate it. It was originally designed to replace Furadantin as a new urinary antibiotic. In initial toxicity studies in rats --- toxicity studies require huge doses --- they observed that the rats were blitzed. They lay around the cage without moving, their muscles were relaxed, and yet they breathed just fine. Their respiration was unaffected, and yet all other muscles appeared very weak. What was going on? Might this be an unanticipated property that could be effective in muscle disorders?

These findings mandated studies of the site of action in muscle. Keith and colleagues determined that dantrolene altered excitation-contraction coupling within skeletal muscle, and that it didn't alter cardiac function (Ellis et al, 1974, 1976, 1976). Dantrolene is a miracle drug for an MH episode (Harrison, 1975; Gronert, Milde et al, 1976; Hall, 1980).

Until the FDA approved dantrolene for human use in 1979, we developed our own preparation for emergency use, as approved by the Mayo Human Subjects Committee. Since Mayo had about 70,000 surgeries annually, and the incidence of MH was about 1/50,000, we expected to see MH episodes, and wanted to be prepared. Sterile dantrolene crystals for use in an intravenous solution for MH therapy were prepared by Ed Mansfield of the Mayo Clinic Pharmacy (Gronert, Mansfield et al, 1978). This preparation proved useful for a boy with a rapidly progressing episode of MH (Gronert, 1983). We performed contracture studies in the boy's father, which were positive for MH, and later an MH mutation was detected in him and his two sons. This family and I have maintained contact since that episode.

Water solubility of the lipid soluble dantrolene is pH dependent, requiring an alkaline medium, and the oral capsule contains the usual excipients, e.g., starch, talc, magnesium stearate, and lactose. Mr. Mansfield dissolved the dantrolene at pH 10.3, and filtered the non-soluble excipients. He sterilized the resulting solution by passing it through a 0.45 µ millipore filter. He re-precipitated dantrolene by lowering the pH to 3.0. The resulting crystals were dried in a laminar flow hood and aseptically weighed into 500 mg lots. Dantrolene crystals are re-dissolved when needed by adding sodium hydroxide (for alkalinity, pH 9) and mannitol (to make the solution isotonic – otherwise it's hypotonic and may hemolyze red blood cells).

Each Mansfield-prepared 500 mg bottle cost us $10. We had originally requested dantrolene crystals from the pharmaceutical company for our research, but this was denied because we had no track record in dantrolene or MH research. Our own preparation eased the problems of our porcine MH research and our clinical care prior to NIH approval.

Research Approaches

We reviewed prior studies of MH in pigs (above) and began our own. Using pigs that tested either normal or susceptible, we first defined the response to halothane alone (Gronert, Theye, 1976) and then to succinylcholine alone (Gronert, Theye, 1976). The former approach showed that halothane gradually and progressively increased oxygen consumption and carbon dioxide production to a considerable degree, although not to as great a degree as is possible in extreme exercise. The latter approach – succinylcholine – showed that it affected the muscle immediately, with a sharp increase in oxygen consumption. This increase was brief and not great, as the action of succinylcholine is brief; circulation to muscle washes


Figure 15
Fig. 15. Whole body oxygen consumption during MH triggered by halothane and succinylcholine; top line: symptomatic treatment; middle line: no treatment; bottom line: symptomatic treatment plus dantrolene.

Figure 15
Fig. 16. Lactate values during MH; pigs not receiving dantrolene died.

Figure 15
Fig. 17. Hyperkalemia during MH triggered by halothane and succinylcholine. Untreated and symptomatically treated pigs died.

succinylcholine away from the muscle and it's hydrolyzed. However, a second dose of succinylcholine increases the likelihood of serious reaction from MH and death (Lucke et al, 1976).

The use of halothane and succinylcholine together acutely triggered MH. Fig. 15 - 17. (Figs. 1, 3, 4, in: Gronert GA, Milde JH, Theye RA: Dantrolene in porcine malignant hyperthermia. Anesthesiology 1976, June, number 1; 44:488-495, copyright owner Lippincott, Williams & Wilkins, www.LWW.com, used with permission.) We confirmed Harrison's 1975 article on the efficacy of dantrolene in treating porcine MH, with a broader study of various altered parameters and their return to normal (Gronert, Milde et al, 1976). Figs. 15-17 demonstrate this effectiveness in normalizing whole body oxygen consumption, lactate production, and plasma potassium values. These three figures illustrate the curative response to dantrolene in swine triggered into MH by halothane and succinylcholine. The three treatment groups are: Untreated, Treated - symptomatic treatment only, and Treated – symptomatic therapy plus dantrolene. Horizontal axis: time in mins; vertical axis: Fig 15: whole body oxygen consumption (dotO2); Fig 16: arterial lactate values; Fig 17: arterial K+ values. Symptomatic treatment included hyperventilation with oxygen, discontinuation of halothane, sodium bicarbonate to reverse metabolic acidosis, volume loading with cold fluids, and surface cooling. Pigs treated symptomatically or untreated died.

We delved into the mysteries of MH; various organ systems needed to be analyzed as to possible roles in MH. The sympathetic nervous system plays a significant part in an MH episode, with rapid heart rate and elevated blood levels of sympathetic amines; some investigators felt that it was a vital factor in starting an MH episode. The sympathetic system in a stress and/or emergency situation invokes the exaggerated fight, fright, or flight response that prepares you for battle, brings out fear, and/or has you ready to run. MH susceptible swine can be triggered into an episode, awake, while not anesthetized, during extreme excitement, and even normal swine are grossly excitable. We needed to know, however, whether the sympathetic response is essential for an MH episode to develop.