Scientists often turn metaphorical when groping for ways to describe their esoteric profession. Certainly that’s the tack Eric Johnson takes when trying to explain the work of his fellow biochemist and mentor Ed Schantz. Ed’s work is like fine wine making, says the 36-year-old Johnson. He can make this stuff in three weeks, but it’s taken him years of development to perfect his technique.
The stuff Schantz makes isn’t vino; it’s toxin. Specifically, it’s botulin, the toxin produced by the single-celled botulinum bacterium and the most poisonous substance in the world.
When ingested, botulin causes botulism, a form of food poisoning that can result in muscle paralysis and even death. But botulin has a good side also: in very small doses, it is an effective treatment for a whole class of human illnesses known as dystonias--uncontrollable muscle spasms that researchers suspect are caused by involuntary and excessive electrical impulses from the brain. These spasms cause eyelids to blink or clamp shut, voices to stop in throats, necks to twist into painful contortions. Many of these conditions, however, are today being controlled by injections of botulin. And every dose ever administered by a doctor was made by Schantz, an 84-year-old emeritus professor and biochemist at the Food Research Institute of the University of Wisconsin at Madison.
Because the various dystonias are relatively rare--Johnson cites one study that estimated the incidence at 391 per million population--the larger drug companies haven’t had much profit incentive to produce the toxin. Also, as Johnson notes, botulin is six million times more toxic than rattlesnake venom. A lot of drug companies don’t want to get near it for reasons of employee safety. Add to that the delicate craftsmanship and complexity required to produce the stuff and it becomes clear why Schantz’s ability to make a pure and effective toxin is so unusual, and why he is passing that craft on, in the tradition of a vintner, to Johnson.
It would be very difficult for anyone to learn the procedure from a book, Johnson says. Ed’s taught me how to do it; he’s the master. It really is like wine making. While all the steps are written down, there’s still a certain art to getting it right, a technique that Ed has refined over the years. Ed’s also a rather modest person, but he alone has supplied toxin to everyone around the world for their scientific work, and done so for little or no compensation. If you read the scientific literature from 1950 to 1980, they all say they obtained their toxins from Ed Schantz.
One reason botulin needs an artisan’s touch is that it is a very complex molecule. (Its molecular weight is roughly 80 times that of insulin, for example.) It consists of one large poisonous protein accompanied by a group of smaller stabilizing proteins that prevent the large one from changing chemically and losing its potency over time. The usefulness of the deadly poison to the bacterium itself has never been discovered, although some researchers have suggested that by killing an animal the bacterium might be trying to provide a nice home for its progeny, which can grow only in the absence of oxygen. The inside of a soup can also makes a nurturing environment, as long as it’s not subjected to too much heat. That explains why botulism was common back when people regularly canned their own food. Now that most people get their canned goods from a store, botulism has become extremely rare, as all commercial canners heat their products to the temperature necessary to kill the botulinum spores.
The toxin does its deadly work by attaching itself permanently to nerve endings. Once the botulin molecule is in place, neurotransmitters-- chemicals that transmit nerve signals--are blocked from reaching a muscle, and the muscle quickly becomes weakened or, if enough of the nerves are blocked, paralyzed. People who die of botulism generally do so because they suffocate after their chest muscles are paralyzed. Those who survive the disease often spend weeks on respirators and remain weakened for months, until new nerve endings can grow.
The process of producing the toxin in the laboratory has been considerably streamlined since the early days. When he began working on botulin, Schantz had a normal-size lab to spread his experiments out in. But now he manages to make all the toxin needed for research on a small bench in a combined lab-office not much bigger than a coat closet.
Schantz is a master of many branches of toxin farming and bacterial husbandry--he was the first to purify and describe the chemical structure of the red tide shellfish toxin--but botulin has always been his specialty. He began growing it almost 50 years ago, during World War II, while an Army officer stationed at Fort Detrick, Maryland, then the home of the U.S. biological-warfare program.
Army intelligence had indicated there was a danger of botulinum and other bacteria being used against our troops, Schantz says. I was assigned to work on whether a botulinum toxin could be used as a possible agent of war.
The answer to Schantz’s initial question was no. Botulin would not be an effective battlefield weapon because it doesn’t remain potent once it’s exposed to air, though Schantz points out it could still be useful for sabotage (in food that is eaten without heating, for example, or used as an aerosol in small areas). Johnson notes too that during the Gulf War the Army was gobbling up all the toxin it could. They’re very concerned about warfare that in some unknown way could employ botulinum, he says. They’re storing it and producing their own, hoping to develop an immunization and an antiserum for the troops.
After World War II, Schantz continued to work at Fort Detrick as a civilian. In 1972 he left government service and joined the Food Research Institute, which had various research relationships with the fort. By the time Schantz arrived at Wisconsin, botulin’s application to dystonias had already become a major part of his work.
This started around 1968, says Schantz. I was contacted by Alan Scott, a surgeon who’d been working with patients suffering from strabismus.
Strabismus, commonly known as cross-eye, is caused by an overactive eye muscle. Technically it is not a form of dystonia. But like the dystonias, nerves affected by strabismus carry too many signals to the muscles, causing them to contract. At the time Scott first contacted him, Schantz says, the usual treatment was surgical: cutting away a portion of the hyperactive muscle would weaken it, allowing the eye to line up correctly.
Scott asked if any of the toxins I worked with might inactivate the spastic or hyperactive muscles so the invasive surgery could be avoided.
Schantz suggested the botulinum toxin, which he had in a purified, crystalline form. His reasoning was that botulin could weaken the eye muscles in the same way it weakens the muscles of botulism sufferers. If a few of those nerves could be silenced, the muscles would relax. If the dose was carefully controlled and the injecting needle precisely targeted, the physician could exactly counterbalance the disease and restore normal control of the muscle to the patient.
Schantz and Scott began their collaboration by testing botulin on monkeys with cross-eye (which Scott induced with the same kind of surgery he used to correct strabismus in humans). The early results showed that the treatment might well work. Although the results would not be permanent--new nerve endings would eventually grow--Scott thought the treatment could be fine-tuned to provide significant relief. As Scott continued the experiments to establish the best dosages and the best places to target the toxin, Schantz worked on bringing his toxin-making procedures in line with Food and Drug Administration rules for substances given to humans.
Around 1978, says Schantz, the FDA gave Scott permission to go ahead with human volunteers, since it was turning out that the toxin was working well in monkeys. Scott was soon sending samples of the toxin to other physicians around the country, who used it on many more strabismus sufferers who volunteered for the experimental treatment. Scott also began trying botulin as a treatment for a type of dystonia called blepharospasm, which causes uncontrollable blinking.
For years blepharospasm--like other dystonias--was thought by most doctors to be psychosomatic in nature, and many sufferers endured a lot of misguided treatment and psychoanalysis. Ironically, says Schantz, after all these years of assuming the disease was psychosomatic, injecting a little bit of the toxin into the eyelids cleared it up. Doctors have since told me how delighted their patients were now that they had relief for something there had previously been no treatment for.
Scott and other physicians gradually increased the scope of their toxin attack. Chronic writer’s cramp and musician’s cramp, which is especially severe for violinists, are also dystonias that are now treated with botulin (although people with writer’s cramp are first urged to learn to write with their other hand). So is dysphonia, which affects the muscles of the pharynx and results in an extremely strained voice, as well as spasmodic torticollis, an especially painful and debilitating condition that causes the head to move about uncontrollably. Despite these successes, it wasn’t until December 1989 that the FDA finally declared that the use of botulin for treatment was no longer experimental but an established medical practice--and even then for only two of these diseases, blepharospasm and adult strabismus.
All this time, right up to the present, every dose of botulin was prepared from the 1979 harvest of a single crop of bacteria that the FDA had approved. It was pos-sible to get that kind of mile-age out of a single batch because the toxin is so powerful that an effective dose is usually about a millionth of a milligram. (The lethal dose for a human is about one ten-thousandth of a milligram.) Schantz can make as much as 60 milligrams from a three-gallon batch of the vigorous bacteria in a carboy, a type of glass jug that is used in biochemistry as well as home beer brewing.
Schantz and Johnson begin by placing some Clostridium botulinum cells into a blend of dextrose, digested milk protein, and brewer’s yeast extract. Since the bacteria aren’t competing with any other microorganisms, they can multiply quickly, turning the medium a cloudy brown in 18 to 24 hours.
The bacterium has an interesting natural life cycle, says Johnson. It makes a spore that’s distributed worldwide in water and soil. If you take soil samples around the world, you’ll find the spores exist naturally in ten to thirty percent of those samples. And when the spore gets into a food and conditions are right--these conditions include temperatures above 50 degrees, a certain acidity level, and an absence of oxygen--it’ll grow into a bacterium that’s rod-shaped. Then it has a choice; it either makes more spores, or, when nutrients become limited, it undergoes what we consider a mass lysis, or cell suicide. When we make the toxin, after the culture becomes cloudy to the point where you can’t see through it, it dissolves its own cell wall, for reasons we don’t understand. That’s when the toxin is released from the cell. Then the culture clears and you’ll see right through it.
In the lab it takes about three days to grow the culture of bacteria and produce the clear, botulin-containing liquid. But the next steps are rather complicated.
Once the toxin is dissolved in the fluid we have to isolate it, says Schantz. We do that by a process known as precipitation. Sulfuric acid is added to the solution; the higher acidity makes the toxin insoluble, and it settles out in a kind of muddy mixture.
The mud contains the botulin, along with other substances produced by the bacteria. While the mud would work fine as a poison, it wouldn’t be wise to inject such an impure substance into a dystonia patient. So the process of purification is repeated. The mud is redissolved in a salt solution and again precipitated with acid, then redissolved and precipitated yet again, this time using alcohol at a temperature of -5 degrees Celsius.
Each time they dissolve and precipitate, some of the unwanted bacterial products stay in the solution, and the precipitate gets closer to pure toxin. The final step is to redissolve the solution and add ammonium sulfate. This causes the botulin to crystallize into microscopic, glasslike needles that are composed of a very pure toxin.
Such a description doesn’t begin to cover the intricacies of the process, but neither would a more technical description. Like many other biological procedures, of which wine making is one, it is as much a matter of touch and lore as it is technical knowledge. The crystallization step is especially delicate, and it is where Schantz and Johnson’s artistry comes into play.
A lot of these things are judgment calls, says Schantz. You have to add just enough ammonium sulfate so that the toxin doesn’t precipitate out. Sometimes you’ll see just a little opalescence, then you have to refrigerate it for a few days and wait for the crystals to form. But you have to know when to add the ammonium sulfate and how much opalescence there should be. Otherwise you won’t get the pure crystals.
Schantz and Johnson don’t use modern techniques like columns or automated chemical reactors, which could conceivably make the process easier. In a column, chemicals are brought together and separated out by resins that bind to the molecules and then--theoretically--release them.
With a column you run the risk of getting traces of the resins in your toxin, Johnson says, and you also lose the stabilizing proteins that keep the toxin from losing its potency. The precipitation method avoids those problems. It’s a procedure virtually no one in the world uses anymore except us; it’s considered old-fashioned and not in vogue.
The British are using enzymes and columns to make a botulinum toxin product, but we don’t think the quality is as good; there have been some side effects. Of course, we’re being subjective too. A protein chemist might say modern techniques would work just as well; but all we know is that what we’ve made works the best so far.
The techniques Schantz and Johnson use are so common they could be improvised in someone’s kitchen. You could make this toxin yourself, says Johnson. But maintaining the high toxicity in the culture and the properties of the toxin as you purify it are what you have to have a lot of years to know how to do.
The batch they produced in 1979 was acquired in 1990 by Allergan Pharmaceuticals after Allergan purchased Oculinum, a small company that had been formed by Scott. Allergan just walked into it, notes Johnson, in the sense that they purchased a drug without having to invest the years of development and high research costs that accompany most drug research.
Schantz and Johnson won’t have to worry about making more toxin for medical use soon. The 1979 batch is expected to last about five more years. But they continually produce toxin for research. Neurologists and other researchers need it for developing treatments for diseases other than dystonias; one possible use may be in the treatment of muscle spasms associated with both cerebral palsy and multiple sclerosis. (One ongoing clinical trial with 12 children stricken with cerebral palsy has shown promise, Johnson notes.) And last May, Mitchell Brin, a neurologist at Columbia, reported some preliminary success in treating patients suffering from severe stuttering; he injected the toxin into their vocal cords. If his method proves effective in further tests, it could provide relief for roughly 2.5 million Americans.
So there will continue to be one or two small flasks containing the brown, murky botulinum culture on Schantz and Johnson’s workbench, or perhaps the clear solution of a later phase of toxin production. And the Food Research Institute will still have to ask certain prospective employees whether they’d be willing to develop an immunity to botulism as part of their job. It’s not a particularly complicated process--three doses of the botulinum toxin are injected into the new lab worker over a period of three months--though it does tend to put many people off.
But look at the bright side, the researchers say. You’d be able to eat from bulged-out cans.
