Absorbed Poisons

It is possible to absorb a fatal dose of a poison, even through protective gloves. Toxicologists played an important role in the investigation of the illness and death of Karen Wetterhahan, a professor in the Dartmouth College Department of Chemistry, in February 1997. Wetterhahan, 48, was using dimethyl mercury (CH3-Hg-CH3) in her research. As described in a report issued by the U.S. Occupational Health and Safety Administration (OSHA), the incident occurred while she was working in a fume hood and wearing latex gloves.

During the transfer of liquid from one container to another, she apparently spilled a small amount (a few drops at most) on the back of one of the gloves. Wetterhahan reported that she took off the gloves and gave the incident no further thought. Later studies proved that the organic liquid quickly and easily penetrated the latex examination gloves, delivering the lethal dose via skin absorption. The fume hood likely protected her from exposure from inhalation, which could have occurred given that dimethyl mercury evaporates relatively easily.

The first neurological symptoms appeared months later, when Wetterhahan reported numbness, tingling, and difficulty speaking. Given the symptoms and her area of research, her medical teams suspected mercury poisoning. The suspicion was confirmed by further testing, including hair analysis. Mercury levels were recorded at 234 ppb (parts per billion, or ug/L) in her urine and at 4,000 ppb in her blood months after the exposure. Despite therapy she died nearly 10 months after the incident. As a result of this accident chemists and researchers around the world were warned to use protective gloves other than latex when handling dimethyl mercury. Undoubtedly lives were saved. Had it not been for advances in forensic chemistry and toxicology, the cause of her death might never have been revealed.

Absorption

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Absorption

Subs intro

Distribution —Elimination

Distribution —Elimination

Storage

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Transformations in the body. Once ingested, the drug or poison is processed by the body through metabolism.

the rate of elimination, a large dose may not produce toxic effects. Again, consider the aspirin example. If the adult woman took an aspirin tablet (325 mg) every day for a year to help prevent heart attacks, this would correspond to a dose of 137 grams, more than 10 times the LD50 calculated above. Aspirin is eliminated quickly, however, and it is taken in small doses over time. Thus, taking 137 grams in this way is not dangerous.

Although simplified, this example illustrates the difference between chronic exposure (longer times, lower amounts) and acute exposure (higher doses, shorter times) to toxins. Forensic toxicologists more often deal with acute poisoning, in which large amounts of the poison are ingested at one sitting or over a brief period. However, chronic poisonings are not uncommon. Arsenic, the historical favorite, was usually administered in small doses over time, as it accumulates gradually. In this way the symptoms appear incrementally and mimic those of common diseases. Similarly, many environmental toxins are pollutants found in small amounts in soil, air, and water. Arsenic is a trace pollutant found in drinking water in the western United States as well as in areas of Pakistan and Bangladesh. People who drink the water will accumulate arsenic in their bodies and eventually exhibit the same symptoms as those poisoned intentionally with arsenic powder. Other examples of environmental toxins are DDT, dioxin, and metals such as lead and mercury. These substances can occur as trace environmental contaminants that build up in a person's system as a result of low-level, chronic exposure.

Other factors that define toxicity depend on the person who has ingested the material. Age can be critical, with children and the elderly generally being more susceptible to toxic effects than other age groups. General health is important, as are genetic effects. Some people are inherently sensitive to or tolerant of substances based on their genes. Thus, toxicity of a substance is the cumulative effect of a variety of factors.

PHARMACOKINETICS AND TOXICOKINETICS

Pharmacology is the study of how drugs (pharmaceuticals) are transformed in the body. Pharmacology can be roughly divided into two aspects: pharmacodynamics and pharmacokinetics. The term dynamics refers to movement or motion, while kinetics refers to speed or rate of movement. A simple distinction between the two terms is that pharma-codynamics focuses on the way a drug affects a person over time, while pharmacokinetics traces the route and rate of transformations of a drug once it is ingested.

Toxicologists are interested in pharmacokinetics because it provides a means of studying the concentrations of drugs and metabolites that may be found in blood and tissues as well as how a drug is metabolized and excreted over time. The term toxicokinetics is also sometimes used to describe the same phenomena when the substance ingested is toxic; it is considered a subdiscipline of pharmacokinetics. Elements of toxi-cokinetics include

• modes of ingestion

• rates of absorption and distribution

• biotransformations and their rates

• relative concentrations of drugs and metabolites in different tissues

• rates of elimination

All of these processes are important in determining what a toxicolo-gist looks for in submitted samples. Additionally, understanding of the

Simplified view of toxicokinetics. Once a drug or poison gets into the body, there are three stages that it passes through. First is absorption, such as in the stomach or through the lungs if the substance is a gas. Next, the bloodstream distributes the material. Finally, it is eliminated. The metabolism and elimination steps may take hours, days, or even longer.

toxicokinetics of a substance can help re-create events or answer such questions as how much of a drug or poison was taken and when.

When a toxic substance or drug is introduced into the body, three general processes occur. For any detectable concentration of the substance to be found in blood, it must first be absorbed into the bloodstream. How and where this occurs depends on the nature of the substance and on the mode of ingestion. As an example, consider the act of drinking a glass of wine, which typically would contain around 10 percent ethanol (ethyl alcohol). Ingestion occurs by swallowing, and absorption into the bloodstream occurs primarily in the small intestine. As absorption proceeds, blood concentration of ethanol increases, and ethanol is delivered to all tissues of the body, including the brain. This phase is referred to as distribution. Any toxic effects (or therapeutic effects in the case of a drug) will depend on a sufficient concentration of the substance reaching the target organ, site, or structure. In the case of ethanol, a water-soluble compound, it can be distributed throughout the body, favoring tissues that have the highest water content. Toxic effects (also called intoxication) can occur in any organ, and in the case of alcohol, these will be seen in the brain and liver. If the concentration

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64 drugs, poisons, and chemistry becomes high enough in the brain, observable toxic effects will be seen, as alcohol is a central nervous system depressant. If concentrations continue to rise, drunkenness is the observable result. In the case of ethanol it takes about an hour for the maximum concentration to be reached in the blood.

Once a substance is distributed, bodily processes start to act on it and may result in the chemical conversion of some or all of the original substance into other compounds (metabolites). Collectively these transformations are called biotransformations, since they occur within a biological system. Operating concurrently with metabolism is the process of elimination, which removes a substance and its metabolites from the body. For the ethanol in the glass of wine, approximately 10 percent of it will pass unchanged into the urine, sweat, or breath. The remaining 90 percent is metabolized in the liver, where toxic effects

half-life

Time since ingestion

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Time since ingestion

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Half-life. If a person ingested 100 mg of a drug with a half-life of one hour, at the end of that hour, 50 mg of the unchanged drug would remain in his or her system. After two hours, 25 mg would remain, and so on. Half-life measures the persistence of a substance in the body.

Multiple transformations. As the original drug breaks down according to its half-life, the concentration of the resulting metabolite increases. It, too, has a half-life and will degrade, forming another breakdown product, and so on.

can occur if a person has chronic exposure to alcohol. Eventually the alcohol is converted to carbon dioxide and water, but this process is relatively slow and can take hours, depending on how much alcohol was consumed. It also occurs in steps. For alcohol the first step is the conversion of ethanol (CH3-CH2OH) to acetaldehyde (CH3-CHO). In the metabolic process some steps are fast, and some are relatively slow. The slowest step in any transformation is referred to as the rate-limiting step.

The rate of elimination or conversion of a substance can be expressed as its half-life (ty). The half-life of a toxin is the time required to remove or transform one-half of the total available. In general it takes about 10 half-lives for the concentration of a substance to fall to the point that it is essentially gone. A metabolite of cocaine, for example, that has a half-life of a day in urine will become undetectable after about 10 days. The longer the half-life of a substance, the longer it will remain in a tissue or fluid. If the method of removal of a substance is conversion

66 drugs, poisons, and chemistry into another, such as the conversion of alcohol into acetaldehyde, then the cycle starts again, with the new substance undergoing absorption, distribution, and elimination, with the rate of elimination expressed by another half-life. When any substance is ingested, it initiates a complex series of related transformations and eliminations that can result in the final excretion of a substance that is entirely different from the original. With a glass of wine, most of the alcohol, which is water-soluble liquid, is eliminated after several steps, leading to exhalation of carbon dioxide, an insoluble gas.

TYPES OF SAMPLES AND ANALYSIS

The types of samples that a forensic toxicologist may be asked to analyze are principally blood, urine, and tissue samples. There are, however, many other samples that occasionally must be tested. As discussed in preceding sections, drugs and poisons are distributed to the body by the bloodstream, and where those substances or their metabolites concentrate depends on the drug, how it breaks down, half-lives, and other factors. Also important is the time elapsed since ingestion, given that materials will partition into different fluids and tissues over time. Toxi-cologists are also interested in the relative concentrations of materials in different samples. After a person drinks a glass of wine, for example, the concentration of alcohol will increase in the blood and then decrease as it is metabolized in the liver or excreted unchanged. The difference between the concentration of the alcohol in the blood and the urine can be used to backtrack to an estimated time of ingestion.

Other drugs, like cocaine, and poisons, such as arsenic, can leave traces elsewhere, as in the saliva and even in the hair. Hair can be particularly useful, providing a permanent record of ingestion for some drugs and toxins. Hair grows from the root, and once the hair has grown slightly away from the root, it is isolated from blood flow and further metabolic changes. While in contact with the blood at the root, substances can be trapped in the hair and then frozen in place there. As hair grows, that section moves away from the root at a steady rate. By cutting a length of hair and analyzing it in sections it is sometimes possible to find drugs, poisons, and their metabolites and to estimate when the dose was taken.

Relative concentrations. After a substance is ingested and distributed, its concentration in the blood will start to fall as concentrations in other body "compartments" increase.

AREAS OF FORENSIC TOXICOLOGY

Forensic toxicologists are trained not only in sample analysis but also in the complex processes of ingestion and its modes: biotransformation and metabolism, absorption, and elimination. This knowledge allows toxicologists to re-create the original ingestion in much the same way the analysis of physical evidence allows forensic scientists to re-create the events at a crime scene. But before there can be re-creation, there must be analysis.

For most forensic toxicology analyses the first test performed is an immunoassay screening or combination of screenings. Immunoassay results will narrow down what may be present, but alone the results are inconclusive and will require more work. TLC may also be used. The next step is usually an instrumental method, such as GC-MS, to confirm the identity of compounds, find others not tested for in the immuno-assay, and determine the concentrations of drugs and metabolites of interest that are found. With this information the toxicologist can recreate what the person ingested and roughly when they ingested it. Very often there will be a mix of substances found, so the task of re-creating the ingestion event can be complex. Forensic toxicology can be roughly divided into these three areas: human performance toxicology, postmortem toxicology, and workplace toxicology.

Human performance toxicology refers primarily to alcohol intoxication. There are many kinds of alcohols, such as isopropanol (isopro-pyl alcohol, or rubbing alcohol) and methanol (wood alcohol). In the forensic lab the target alcohol is ethanol (ethyl alcohol). Ethanol is the type of alcohol found in beer, wine, liquors, and in some medications. In liquor, such as gin and vodka, the amount of ethanol is reported as its "proof." The proof of a sample is twice the percentage of ethanol it contains. Thus, an 86-proof whiskey would be 43 percent ethanol by volume. Wine is about 3-5 percent ethanol, but since wine is not classified as a hard liquor, the term proof does not apply.

The intoxication that results from ethanol depends on how much a person ingests over a given period of time, his or her size, the person's stomach contents, and other factors. The degree of intoxication is reported in terms of the blood alcohol concentration (BAC). The unit of BAC used in a legal setting is usually percentage by volume. In most states the legal limit is 0.08 percent, meaning that anyone whose blood has a concentration of alcohol that is 0.08 percent (0.08 g of ethanol per 100 ml of blood) or greater is considered to be legally intoxicated. There are noticeable effects at lower concentrations, however. At levels of approximately 0.03 percent, the person may feel euphoric and have impaired judgment. These effects increase with increasing intoxication. Once BAC levels approach 0.4 percent, coma can result, and levels of around 0.45 percent can result in death. According to the National Highway Transportation Safety Administration, intoxicated drivers kill about 18,000 people per year, roughly one death every 30 minutes. This does not include accidents involving other vehicles such as off-road vehicles, snowmobiles, trains, etc. Data from a 2001 survey (the latest available) reports that nearly a quarter of the drivers responding had driven within two hours of drinking alcohol. Approximately 1,400 college students die each year as a result of ethanol consumption. For a substance most people would consider nontoxic, alcohol kills an alarming number of people.

Headspace Toxicology

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Laboratory analysis for alcohol in blood. Ethanol in the blood is driven into the headspace, which is sampled and tested.

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Laboratory analysis for alcohol in blood. Ethanol in the blood is driven into the headspace, which is sampled and tested.

The job of the toxicologist in these cases is to analyze the BAC. When the operator of a vehicle is suspected of being drunk, trained personnel draw a blood sample as soon as possible. Time is important because, as explained in the preceding sections, the concentration of the alcohol in the body is constantly changing. If too much time elapses between the incident and the taking of the blood sample, the BAC results will be less representative of intoxication at the time of the incident. A forensic toxi-cologist analyzes the blood sample, and the concentration of alcohol is reported as grams per deciliter or as a percentage of total blood volume, as described in the preceding section.

The analysis of ethanol is performed using a headspace sampling technique. The blood sample is placed in a small vial such that there is an empty portion (headspace) remaining. The vial is sealed and heated gently, which forces ethanol out of the blood and into the headspace. A small portion of the headspace is sampled and introduced into a gas chromatograph, which detects the ethanol. The data is used to calculate the BAC.

Alveolar sac in lung

Alveolar sac in lung

Breath alcohol. The concentration of ethanol in exhaled breath can be related to the concentration of ethanol in the blood through Henry's law.

Before a blood sample is taken, a police officer must first have reason to believe that a person is intoxicated. For example, the officer might observe someone driving erratically and stop them. Once the driver has been pulled over, the officer would perform tests that may include a breath alcohol test. This test does not provide the exact blood alcohol level, but it can provide enough information to determine if further action, such as drawing blood, is necessary.

Breath alcohol measurements rely on a relationship called Henry's law, which states that the concentration of a substance above a liquid is proportional to the concentration of that substance dissolved in the liquid. In this case the liquid is the blood flowing through the lungs in the areas where oxygen is taken in and carbon dioxide is expelled. Since ethanol evaporates easily, it will also move into the air at this interface. According to Henry's law, the concentration of ethanol in blood is about 2,100 times as high as the concentration in the air above it. Thus, measurement of the ethanol in someone's exhaled breath can be related to the concentration of ethanol in his or her blood. Small portable devices are used to measure breath alcohol in the field. Breath alcohol measurement is not as reliable as analyzing a blood sample, but the results are adequate to determine if there is probable cause to assume that someone is intoxicated.

Another area of human performance toxicology is in sports. This is not forensic toxicology in the traditional sense, since many of the drugs that scientists test in these cases are not illegal. In Olympic competition the World Anti-Doping Agency and the United States Anti-Doping Agency maintain lists of the banned substances, as do most professional sports organizations. The lists include illegal drugs, steroids, and substances that enhance the transportation of oxygen, some OTC drugs such as ephedrine (based on the amount found), and other substances. Finally, some toxicologists work with animals such as racehorses, looking for performance-enhancing substances.

Although testing for alcohol and performance-enhancing substances is becoming more familiar, the public most often associates forensic toxicology with its other major division, postmortem toxicology. Samples for this type of work are obtained during autopsy and consist of bodily fluids, organs, and bones. The table indicates typical

Urine is used frequently in drug testing for sports and in the workplace. This image shows urine samples for doping analysis at a doping institute in Kreischa, Germany, October 30, 2003. (Ralf Hirschberger/DPA/Landov)

materials, those that are routinely collected. Other samples may be taken depending on what type of drugs or poisons might be present. In cases where a body is not found until long after death there may be no blood or flesh left. In these situations more durable materials such as bone may be all that is available. If a victim is trapped in a fire and dies, similar problems will arise due to the burn damage inflicted on the body.

A relatively new branch of postmortem toxicology is entomotoxi-cology, referring to entomology, the study of insects. Suppose a person who has taken a large dose of a drug such as cocaine wanders into the woods and dies in a place where the body is not found for weeks. Decomposition will mean that there is no blood or tissue that could be

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