A is for Arsenic (17 page)

Before 1921, there were two schools of thought about how signals were transmitted between nerves, and between nerves and muscles. At these connections there is a gap called the synapse. It was known that electrical signals travelled along nerves, but could electrical impulses also be responsible for signals crossing the synapse? If it was not an electrical impulse, could it be chemical signals traversing the gap? The experiment that would decide which was correct came to scientist Otto Loewi (1873–1961) in a dream. He hastily jotted down the idea in the middle of the night and went back to sleep, but the next morning he found he could not read his writing, and couldn't remember his dream. The next day was the longest of his life as he tried to recall his moment of inspiration. Fortunately the following night
he had the same dream
. This time he got up and went straight to the lab.

Loewi dissected out two frogs' hearts and placed them, still beating, in Ringer's solution (see page
here
) in separate dishes. To the first heart he applied an electrical current that slowed the heart. He then took the fluid surrounding the first heart, and transferred it to the dish containing the second. The second heart slowed too. The electric stimulation of nerves in the first heart had triggered the release of chemicals that could affect the action of nerves in another heart. Transmission of signals across the synapse therefore had to be as a result of chemicals. Now that the general method of getting signals across synapses was understood, the details would be much easier to work out.

The most pressing issue was to establish which chemical was being released by the nerves. Loewi and his team knew two things: that whatever it was, it disappeared very quickly; and
that its effects were blocked by atropine. After testing various substances known to act on the nerves, including muscarine, pilocarpine, choline and acetylcholine, they found that acetylcholine matched all the criteria – swift disappearance from the body, and blockable by atropine. Further experiments, published in 1926, revealed that the reason acetylcholine seemed to disappear was that it was being broken down by an enzyme, cholinesterase. Loewi and his collaborator E. Navratil found that eserine inhibited this enzyme, stopping the breakdown of acetylcholine and allowing it to continue to interact with receptors. The use of eserine allowed scientists to study the effects of acetylcholine more closely, before it was broken down and disappeared.

The use of eserine had provided valuable information about the mechanism of nerve-signal transmission. This work was recognised by the Nobel Committee in 1936 when it awarded Loewi the Nobel Prize for Medicine and Physiology. As Loewi pointed out in his Nobel Prize lecture, the operational mechanism of an alkaloid had been determined for the first time. The initial work on eserine led to our understanding of how many other compounds, such as the organophosphates used as nerve gases and insecticides, inhibit cholinesterase in humans and insects (see page
here
).

Acetylcholine – one of a number of chemicals known as neurotransmitters – is released predominantly by nerves in the parasympathetic nervous system or PN, the ‘rest and digest' system that regulates fluids such as tears and saliva (though it is also found in both the sympathetic and central nervous systems). Eserine therefore mainly affects the PN. Another of the PN's roles is stimulating contractions of smooth muscle – in the gastrointestinal tract (allowing food to be squeezed through the gut), in the urinary tract and in the eye, as well as decreasing heart rate and relaxing the smooth muscles of blood vessels.

The breakdown of acetylcholine after it has performed its task is vital. To stop receptors from being constantly activated, and to allow repeat signals to be sent to the same site, the
acetylcholine must be removed from the binding site so the receptor is ready to receive more signals. The body uses enzymes, cholinesterases, to break up the acetylcholine, using water to cleave chemical bonds and leave two compounds, acetate and choline, neither of which will interact with the receptor site; in other words, going back to our ‘lock and key' analogy (see page
here
), the key is removed from the lock, leaving it ready for the next one to enter.

The human body has two types of cholinesterase enzymes: acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). AChE acts almost exclusively on acetylcholine, and is predominantly found in the muscles and brain. The body contains more of the BChE enzyme, though, which is distributed throughout the body. The BChE lock can be opened by several different molecular keys, whereas the AChE lock is specific to the acetylcholine one. BChE acts on a range of compounds, including aspirin, cocaine and heroin, breaking them down and limiting the amount of cholinergic
⁵⁰
toxins that can reach the brain.

Eserine binds to AChE as if it were acetylcholine, but the structural differences between the two compounds means a different chemical reaction then takes place. The eserine is broken up by the enzyme but only slowly, and in the process a fragment of the eserine structure known as a carbamate unit is transferred to the active site of the enzyme. With the carbamate present, the enzyme cannot carry out its normal function and is effectively inactive. It is as though a key has broken in the lock and a fragment of it has got stuck, so that no other keys will fit in the lock until the fragment is removed. The enzyme can be regenerated by having the carbamate removed by another enzyme, but this process is also slow. While the AChE is out of action, acetylcholine will continue to interact with the nerve receptors, and will stimulate them.

Eserine is classified as a ‘reversible cholinesterase inhibitor' because the enzyme can regain its function. There is an 81 per cent
recovery of AChE after two hours and 100 per cent within 24 hours. By contrast, other AChE-inhibiting compounds, such as the organophosphate nerve gas sarin, bind permanently; eserine can therefore prevent sarin poisoning, if it is administered at the appropriate time, by temporarily blocking AChE until the body has had a chance to eliminate most of the sarin. Eserine is also more soluble in fats than many other AChE inhibitors, so is able to cross the blood-brain barrier to prevent damage to the brain in the event of poisoning by sarin or similar compounds.

As well as its use in cases of atropine and sarin poisoning, eserine was once suggested as a treatment for tetanus,
⁵¹
and as an antidote for strychnine and curare poisoning. The most successful of these treatments was with curare and the drugs derived from it. Curare is the plant-derived arrow poison Agatha Christie's pharmacist Mr P. carried around in his pocket (see page
here
), and along with its related compounds, it has found wide uses in medicine. These compounds cause the relaxation of muscles by blocking acetylcholine receptors, which is often very useful during surgical procedures.

The similarity between curare poisoning and an inherited condition, myasthenia gravis, led the physician Mary Broadfoot Walker (1888–1974) to test eserine on one of her patients. Myasthenia gravis produces fluctuating muscle weakness, with muscles becoming weaker with increased activity but improving after periods of rest. Symptoms can develop suddenly, and are intermittent. The muscles that control eye movements, facial expressions, chewing and swallowing are usually the worst affected, but movement of the limbs and the muscles that control breathing can also undergo periods of weakness. When Walker conducted her experiments in 1934, the cause of myasthenia was not known, but one theory held
that patients were not producing enough acetylcholine to act on the receptors in the muscles. Injections of eserine produced a dramatic, though temporary, improvement in two patients, indicating that although the individuals were producing acetylcholine, it was failing to act on the muscles. Further studies have shown that myasthenia gravis patients produce antibodies circulating in the body that block acetylcholine receptors. Today the condition is treated by a combination of immunosuppressant drugs and anticholinesterases (such as neostigmine) that prevent the breakdown of acetylcholine and allow it to act on the receptors for a longer period of time.

Some real-life cases

Since the missionaries put a stop to ordeal trials in West Africa, deliberate eserine poisonings have been rare outside of Agatha Christie's novels, which makes you wonder where she found her inspiration. The poison itself is difficult to obtain, and even if someone had a prescription for it, consuming a whole bottle would be unlikely to be fatal, though it might make them very ill. In 1968 a biochemistry student attempted suicide by swallowing 1g of eserine salicylate, which he had stolen from a laboratory. He developed severe abdominal pains after ten minutes, followed by terrifying hallucinations that induced him to seek help. Although atropine was administered it made his condition worse, as he did not display the characteristic slow heartbeat, and atropine only increased his heart rate. Atropine is not a
true
antidote for eserine poisoning because the compounds act on different sites in the body. He was subsequently given aldoximes, which reactivated the AChE enzymes that were inhibited by the eserine in his body. He went on to make a full recovery.

I have only managed to find one other case of deliberate eserine poisoning, but it is not clear who did the actual deed. The case was reported in Austria, long after the publication of
Crooked House
and
Curtain
. A man of around 50 years of age was transferred to hospital suffering from diarrhoea and vomiting. He was released after a week of successful treatment.
A month later he was readmitted with the same symptoms. Toxicological analysis of the stomach content detected eserine, and calculations based on 450ml of stomach contents suggested that he had ingested approximately 100mg.

After two months in hospital the patient's condition changed dramatically for the worse, and medical staff were unable to save him. The cause of death was given as cardiogenic shock, but the deterioration in his body brought about by the eserine was thought to be the underlying reason for his death. An inquiry subsequently looked into the origin of the eserine the patient had ingested. Re-analysis of his stomach contents revealed eserine to be the only alkaloid present in the body in detectable quantities. Had the patient been poisoned with Calabar beans, other alkaloids would be expected to have been present.
⁵²

Pharmaceutical preparations of eserine, sold under the name ‘anticholium', also contain very small but detectable quantities of geneserine (the second most common alkaloid in Calabar beans). Anticholium is used to treat atropine poisoning, and is available in 5ml ampoules. The patient would have had to drink 50 ampoules of anticholium to reach the levels of eserine found in his stomach. This seems unlikely; together with the fact that no other compounds (such as geneserine) were found in the stomach, it was concluded that he had been poisoned with the pure chemical.

Agatha and eserine

The victim in the 1949 novel
Crooked House
, Aristide Leonides, is 85. One day, after his usual injection of insulin, he has a sudden seizure. The family can do nothing for him and call a doctor, but by the time he arrives Aristide is dead. He was not a well man, suffering from diabetes, a weak heart and glaucoma, but his death is very sudden and quite unexpected. The
symptoms Agatha Christie mentions in the novel are ‘difficulty in breathing' and ‘a sudden seizure', but in reality there were likely to have been others. Agatha was far too discreet to detail any involuntary urination or defecation, but other symptoms such as a slow pulse and convulsions might have been mentioned without embarrassment to the reader. Anyway, the symptoms are enough to make the doctor suspicious and demand a post-mortem.

The police investigating Aristide's death admitted that little was known about the post-mortem appearances of eserine. There may be congestion in the brain, lungs and gastrointestinal tract, although these signs might be found in cases of poisoning by a range of substances. When combined with the symptoms displayed by Aristide just before death the finger of suspicion would point towards one of the cholinesterase inhibitors, but there are many of those besides eserine. However, the fact that Aristide had a cholinesterase inhibitor in the form of eserine in his medicine cabinet – and an empty bottle of eserine eyedrops was found in the rubbish bins of Aristide's crooked house – gave the pathologist a good indication of which poison to look for at the post-mortem.

Plant alkaloids such as eserine can be extracted from tissue using the method first developed by Stas in 1850 (see page
here
). The isolated compound could then be identified by chromatography; in 1949, when the novel was written, chromatographic techniques were beginning to be used more widely. This would enable a toxicologist to see whether a victim had been poisoned by eserine from a medical prescription or by ingesting the bean, as the bean would contain other alkaloids that would also appear in the chromatography. This technique can also determine the amount of a particular compound in a sample. In
Curtain
, analysis of the alkaloids extracted from Mrs Franklin's body showed that several alkaloids of the Calabar bean were present, proving that she had been poisoned with one of the extracts used in her husband's research rather than by prescription medication. In
Crooked House
the post-mortem results confirmed that Aristide had died of eserine poisoning
from his medication, as there was an absence of other Calabar bean alkaloids.