Effective Use of Analytical Laboratories for the Diagnosis of Toxicologie Problems in Small Animal Practice

Toxicology of Selected Pesticides, Drugs, and Chemicals

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Effective Use of Analytical Laboratories for the Diagnosis of Toxicologic Problems in Small Animal Practice

Robert H. Poppenga, DVM, PhD,* and W. Emmett Braselton, ]r, PhDt

As is evident from the preceding article, a toxicologic diagnosis is based upon (1) a knowledge of the circumstances surrounding a particular case, (2) a knowledge of the clinical symptomatology, (3) receipt and evaluation of proper specimens by a qualified laboratory, and (4) judicious interpretation of the laboratory results, based upon all the factors involved in the case. For a large percentage of cases submitted to a diagnostic laboratory, the referring veterinarian has primary responsibility for the first two components, the referring veterinarian and the laboratory diagnostician share the responsibility for the third, and the latter individual is ultimately responsible for the fourth. The failure to have all the necessary ingredients can result in a wrong or missed diagnosis. As with veterinary medicine as a whole, diagnostic laboratories are becoming highly sophisticated. Many veterinary toxicology laboratories now have the capability to detect a large number of suspected toxicants in feed, tissue, and environmental samples at extremely low concentrations. The ability to detect toxicants at such low levels has often outpaced the ability of the diagnostician to interpret the analytical findings. The objective of this article is to provide the practicing veterinarian with guidelines for using veterinary analytical laboratories to maximize the probability of correct toxicologic diagnoses. From the Department of Pharmacology and Toxicology and The Animal Health Diagnostic Laboratory, Michigan State University College of Veterinary Medicine, East Lansing, Michigan *-\.ssistant Professor -=-Professor \'eterinary Clinics of North America: Small Animal Practice-Vol. 20, No. 2, March 1990

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CASE HISTORY A proper history is an essential first ingredient for a successful toxicologic diagnosis. A good clinical history may provide specific clues or may only point the veterinarian and the diagnostician in a general direction. An in-depth discussion of the components of a good history can be found in the article on field investigations of small animal toxicoses. It is important that the veterinarian not be misled by the perceptions of the owner. In many cases, owners are convinced that their animal has been "poisoned." This statement can lead many veterinarians to consider only a toxicologic etiology in lieu of other infectious or noninfectious causes of illness. Alternatively, the veterinarian should be careful in suggesting to a client that his or her animal appears to have been poisoned, when inadequate evidence exists to support such a conclusion. Clinical Signs and Postmortem Examination Clinical signs may lead the veterinarian to suspect a particular toxicant. For example, the cholinesterase-inhibiting organophosphorus (OP) and carbamate insecticides often cause characteristic clinical signs which can be remembered by the mnemonic DUMBELS for diarrhea, urination, miosis, bronchospasm, emesis, lacrimation, and salivation, respectively. 5 Alternatively, clinical signs may be rather non-specific and therefore of less help. Clinical pathologic results are important to include in the submitted history. As an example, the presence of metabolic acidosis and high anion and osmolal gaps suggest ethylene glycol or methanol toxicoses and may influence the choice of analytical tests. 2 • 10 In many toxicoses, the only clinical sign is death. A thorough postmortem examination is essential in such circumstances. This may help eliminate nontoxicologic etiologies or perhaps narrow the list of possible toxicants. It should be kept in mind that many toxic agents may cause nonspecific lesions or no lesions at all. Often, when a postmortem examination is done in the clinic, tissue samples are collected for either histologic or toxicologic examination but not both. Two sets of tissue samples from animals with suspected toxicoses should be routinely saved. One set should be preserved in 10 per cent buffered formalin for histologic evaluation and another set frozen for possible toxicologic analysis. A common and often unforgiving mistake is failure to submit brain, spinal cord, or peripheral nervous tissue when signs referable to the central or peripheral nervous system are present. A prudent and cost-effective procedure in unexplained deaths is to submit a full set of tissues for histologic examination following gross examination and to keep a second set frozen for later toxicologic analysis pending the histologic findings. It is always easier to dispose of unneeded frozen tissues than to collect tissues from an animal that has already been buried or otherwise discarded. SAMPLE COLLECTION AND SUBMISSION The previous article by Galey and Hall discusses a general sample collection protocol for investigation of possible toxicoses when live and/or

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dead animals are involved. Additional comments on selected samples can be found in Table l. Noteworthy are the references to urine and crop contents, which are samples that are often overlooked but may sometimes be the most important in obtaining a diagnosis.

LABORATORY ANALYSIS It is advisable to be familiar with the testing procedures of the diagnostic laboratory that you use. Although most laboratories offer a wide range of analytical tests, no one laboratory can run all possible analyses. If a particular test is not offered by an individual laboratory, it may be poSsible to forward the sample to a referral laboratory. Laboratory personnel are often knowledgeable about which laboratories have the capability and expertise to run an unusual test. The frequency of particular toxicoses varies for different regions of the country and thus the need for investing the resources and manpower to develop specific testing capabilities also varies. It is not cost-effective for diagnostic laboratories to develop analytical procedures for which they receive few requests. Since many analytical procedures can detect the presence of certain compounds over a wide range of tissue concentrations, toxicology laboratories are not necessarily restricted to detection of high, potentially toxic concentrations of these agents. For example, some toxicology laboratories may offer therapeutic drug monitoring services or determinations of tissue mineral concentrations in order to rule out deficiency diseases. It is always wise to consult with the laboratory you use regarding the available range of analytical services. The following discussion describes some of the analytical methods available, specific testing procedures most often requested, and some of the most frequent mistakes made in submitting samples for specific tests. It is important to note that for certain toxicants it is not necessary to quantify tissue concentrations; the presence of detectable toxicant in tissues along with compatible clinical signs is sufficient to yield a diagnosis. Alternatively, for agents that are ubiquitous in the environment, quantification of tissue concentrations may be critical for proper differentiation of a toxicosis from a background exposure.

Metal Analyses Currently, the most widely used method for determination of major, minor, and trace elements in biological samples is atomic absorption spectroscopy (AAS). The method is based on the principle that individual elements in the atomic ground state absorb light of a unique and characteristic wavelength to go to an excited state. Quantification is accomplished by measurement of the amount of energy absorbed in the process. Individual lamps are used to generate the specific wavelengths unique to each element, so that a single element is measured during each determination. Cntil recently, the usual procedure for atomization involved aspiration of the sample, in solution, into an air-acetylene flame. This procedure is not adequate for determination of some trace and ultratrace elements, and

Table I. Comments on Selected Specimens for Submission to Veterinary Toxicology Laboratories

1:-Q

~

0'> SPECIMEN*

SAMPLE AMOUNT AND HANDLING INSTRUCTIONS

SPECIAL CONSIDERATIONS

Urine

50 ml Store in nonbreakable, screw-top container Store and submit frozen

Underutilized sample The pharmacokinetics of a particular compound need to be considered Possible to identify compounds in urine that cannot be found in other tissue samples (i.e,, serum or liver) Probably best utilized to indicate exposure; more difficult to interpret quantitative results Required quantity may be difficult to obtain from some small animals

Kidney

50-100 g Store and submit frozen

Best specimen to submit in certain heavy metal toxicoses (lead, arsenic, inorganic mercury) Tissue of choice for calcium determinations in suspected ethylene glycol toxicoses Can also be used for organic screening tests

Hair

l-10 g Plastic Zip-Joe bags

May be used for determining previous exposure to heavy metals Exposure history over time may be determined (most recent exposure reflected by concentrations of metal in proximal hair shafts and earlier exposures reflected by concentrations of metals in hair collected more distally) Not used for evaluating acute metal toxicoses Removing external contamination is a problem With the exception of a few heavy metals such as lead, interpretation of findings is difficult May be used to confirm topical exposure to pesticides

Vomitus, stomach and GI contents

50-100 g Store and submit frozen

May .find highest concentrations of rapidly acting toxicant in these specimens Owner should be instructed to save vomitus if animal vomited at home If gastric lavage undertaken in hospital, save initial lavage aspirate

Crop contents

All available Store and submit frozen

Often overlooked specimen for establishing a toxicologic diagnosis in avian species May not be enough sample present to perform meaningful tests Can combine with other upper GI tract contents to obtain needed amount

Bedding

Submit a minimum 100 g representative sample

May be important sample for rodent or exotic animal species Submit sample from cage and from original container if possible

Brain

Make a mid-sagittal cut for dog or catsubmit half of brain For small rodent or bird, submit entire brain Store and submit frozen

Cholinesterase values may vary among different brain regions; most laboratories use entire half in order to assure consistency of results Often used to assess significance of exposure to organochlorine pesticides, particularly in wildlife species Limited tissue available from small species such as rodents and birds; therefore, must balance amount available with analyses requested (histologic versus toxicologic)

Liver biopsy (ante mortem)

'12-1 g minimum Submit frozen and formalin-fixed samples

Used to assess presence of copper-storage disease in certain breeds of dogs Results reported on a dry weight basis

Liver (postmortem)

50-100 g Store and submit frozen

Perhaps best specimen for general mineral or organic screening tests

Samples of medications or commercial products suspected of being ingested

No firm rule for amount to submit Generally 2-3 tablets or capsules of a medication 50-100 g of a solid material 20-100 ml of a liquid preparation (mix before sampling)

May be useful to lab for confirming identity or assessing suitability of a test procedure for a specific compound Include a copy of label ingredients

Water

1 liter minimum

Often require large volume, owing to small amounts of potential toxicants present May be extremely important sample for aquarium fish problems

Tissue surrounding suspected injection site

10 g Store and submit frozen

May provide a specimen containing a concentrated deposit of drug or chemical

*Do not submit specimens in containers that have previously been used to store other materials (particularly old medication containers). If multiple specimens are submitted, package and label each specimen separately.

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newer methods of atomization, utilizing flameless devices (e. g., graphite furnace) have become available. Flameless AAS, with attendant background correction techniques, extends the detection limits for specific elements such as lead, down to or below part per billion (ppb) concentrations. Flameless AAS has the added advantage that determinations can be made on much smaller sample aliquots, thus permitting analyses where sample size is severely limited, such as in certain species of birds or small exotic animals. Diagnosis of animal disease problems associated with either toxic concentrations of heavy metals, or deficiencies of trace minerals is often predicated on analytical determinat,ion of a number of major, minor, or trace elements. Thus, using single-element techniques such as AAS, which often require completely separate sample preparation procedures for each element, can result in a time-consuming and expensive progression of tests. Several techniques are now available for simultaneous multielemental quantitative analysis. One of these, inductively coupled argon plasma atomic emission spectroscopy (ICP-AES), is particularly applicable to veterinary toxicology. 9 Elements are atomized and elevated to excited states in a hightemperature (8,000 to l0,000°K) argon plasma and emit characteristic wavelengths oflight as they decay back to lower energy states. Wavelengths are dispersed by a diffraction grating and simultaneously measured or rapidly viewed in sequence. Instruments commercially available at present make it feasible to simultaneously screen for up to 60 elements in a single sample, although in practice 20 to 30 elements are all that are usually required. Because of the nature of the plasma, chemical interferences are almost nonexistent, greatly simplifying the sample preparation procedure. Self-absorption at high analytic concentrations, which limits the linearity of AAS techniques, is greatly reduced in the argon plasma, permitting analyses to be carried out over a much broader concentration range without additional dilution steps. A disadvantage of ICP-AES is that it does not have the sensitivity inherent in flameless AAS, so that some ultratrace determinations are not possible. Sample size can also be a limiting factor. Specific metals of veterinary toxicologic significance are listed in Table 2, along with relevant comments concerning the appropriate samples for submission and other considerations for the individual metals. Organic Analyses Capabilities for rapid identification of toxic organic chemicals in a variety of biological matrices are of increasing importance in toxicologic diagnosis. Since clinical signs for different toxicoses often overlap or are not observed, a presumptive diagnosis may sometimes be an inadequate guide in the selection of confirmatory tests; therefore, a battery of tests may be required, which can result in great expenditure of time and money. Also, when classical methods are directed toward identification of individual compounds or chemical classes, the available samples may be consumed before arriving at a diagnosis. The technique of gas chromatography-mass spectrometry (GC-MS), combined with computerized searching of mass spectral library databases, provides the opportunity to screen for a variety of toxicants in a single test,

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Table 2. Comments and Specimen Submission Guidelines for Selected Metals APPROPRIATE

SPECIMEN(S) AND HANDLING

TEST

Arsenic

INSTRUCTIONS

Urine, stomach contents, fresh liver and kidney Submit frozen

SPECIAL CONSIDERATIONS

Arsenic is eliminated rapidly from the body; therefore, if several days have elapsed between exposure and collection of specimens, detectable tissue

SUBMISSION MISTAKES

Submission of serum or

whole blood samples

concentrations may not be found

Calcium Fresh kidney Submit frozen

Useful adjunctive test to confirm ethylene glycol toxicoses

Copper

See Table 1

Liver: Biopsy in 10% buffered formalin and fresh frozen

Serum copper concentrations do

not accurately reflect liver copper

specimen

concentrations in

suspected cases of copper storage disease in dogs Lead

1-2 ml whole blood (heparin preferred anticoagulant if ASV*) Bone: Submit 4-5 g Suspected lead source (paint chips, improperly glazed pottery used as animal food or water bowl, used motor oil from vehicles using leaded gasoline, etc.) Fresh liver and kidney Submit tissues frozen, with the exception of whole blood Formalin-fixed liver has been reported to be an acceptable

Depending on particular analytical procedure used, 100 fJ,l of whole blood may be enough-consult laboratory Lead associates with red blood cell; therefore, whole blood is needed Other tests such as free erythrocyte protoporphyrin in blood or delta-amino-

Submission of plasma or serum samples

levulinic acid determination in urine are used as sensitive indicators of lead exposure in humans but have not been as

widely used in veterinary medicineconsult laboratory for availability and ability to interpret results Bone concentrations may be important in situations in which whole animal carcasses

are being fed to exotic animal species

specimen ~lercury

1-2 ml whole blood, Toxicoses not common in companion animals fresh liver, kidney, May be important consideration in wild animals where diet consists of a large brain Submit tissue frozen, proportion of fish with the exception of Specific analytical procedure used may be important since relatively low blood whole blood concentrations are associated with

toxicoses (ICP-AES may not be sensitive enough) Tissue distribution is dependent on whether inorganic or organic form of mercury has

been ingested; therefore, good to submit multiple tissues Thallium Urine, fresh liver, and kidney Submit frozen

Toxicosis still occasionally seen

Zinc

Rubber blood collection tube stoppers may leach zinc, thus artificially elevating serum

1-2 ml serum Fresh liver and kidney Submit frozen

Detection of any amount in tissue is

regarded as significant

zinc values

Probably will not interfere with ante mortem diagnosis of zinc toxicosis, owing to degree of zinc elevations associated with toxicosis

If primary concern is a zinc deficiency, do not use inappropriate

storage tubes for serum; use plastic-

capped or royal blue top vacutainer tubes

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and has gained widespread acceptance in human medicine, especially for rapid identification of drugs in overdose victims. Major drawbacks in veterinary toxicology have been (1) high initial cost of GC-MS instrumentation; (2) a lack of simplified procedures for extraction and purification of a wide variety of potential toxicants, including drugs, environmental contaminants, and pesticides of numerous chemical classes prior to their introduction into the GC-MS instrument; and (3) difficulty in chromatographing the diverse groups of compounds on a single GC column. Recent developments in mass spectrometry and personal computer-based data systems have, however, drastically reduced the cost of instruments, such that commercial table-top GC-MS systems are now available to many veterinary toxicology laboratories. Current mass spectral database libraries contain spectra of over 40,000 compounds. Also, the advent of chemically inert capillary columns allows high resolution of a wide variety of chemicals in a single run. Newly developed extraction techniques and purification procedures provide the necessary approaches to rapid extraction and cleanup of many of the complex biological samples encountered in veterinary toxicology. A final advantage of the GC-MS technique is that the combination of gas chromatography retention time and electron-impact mass spectrum, when comparable to the known reference compound, provides unequivocal identification of that substance, and is rarely challenged in a court of law. Consequently, veterinary toxicology laboratories are beginning to utilize GC-MS as a screening procedure rather than strictly as a means of verification. 6• 7 • 8 To illustrate the broad capability of the GC-MS screening procedure, Table 3 lists the variety of drugs, pesticides, environmental contaminants and other chemicals that have been identified in animal tissues, body fluids, and food by the GC-MS screen at Michigan State University's Animal Health Diagnostic Laboratory. The GC-MS technique is limited, however, to compounds that can be volatilized and chromatographed in the gas phase. Large molecular weight and highly polar compounds are largely precluded from this type of analysis; however, some compounds not normally amenable to GC-MS screening methods may be chemically "derivatized" and determined by GC-MS if they are suspected from the clinical history. Included are steroids, phenoxyacetic acid herbicides, and certain mycotoxins. Although not often available in veterinary toxicologic laboratories, methods apart from GC, such as high performance liquid chromatography (HPLC) are beginning to be used to introduce nonvolatile compounds into a mass spectrometer. Compounds and classes of compounds not usually amenable to GCMS screens include metals, salts, many antibiotics and anticoagulants, mycotoxins, bacterial toxins, venoms, and mushroom toxins. Individual testing methods, dependent on the particular compound (or class) of interest, are utilized for these chemicals. For chromatography of relatively polar compounds in aqueous solvents, reversed phase HPLC is a highly useful complement to GC and has become the method of choice for determination of many of the chemicals that could not be determined, or only determined with difficulty, by GC. Thin-layer chromatographic (TLC)

Tuhlc :J. C:ompowul.\' Organochlorines Aldrin Chlordane DDT, DDE, DDD Dieldrin Endosulfan Endrin Heptachlor Heptachlor epoxide Hexachlorophene Lindane Nonachlor Octachlor Polychlorinated biphenyls Polychlorinated terphenyls Tetrachloroethane Toxaphene Trichlorobenzene

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ill Anima/Tissues and Stomach Contents by a GC-MS Screen at Michigan State University, November, 1984 to April, 1989

Organophosphorus Insecticides Chlorpyrifos Coumaphos Diazinon Disulfoton Ethion F ensulfothion Fonofos Malathion Phorate Phorate sulfoxide Phosphamidon Ronnel Stirofos Terbufos

Carbamate Insecticides

Drugs

Aldicarb Carbaryl Carbofuran Methomyl Propoxur

Acepromazine Aminopromazine Atropine Caffeine Camphor Chloral hydrate Chlorpheniramine Cotinine Diazepam Diethylcarbamazine Diphenhydramine Diphenylhydantoin Dipyrone Embutramide (T-61) Guaifenesin Ibuprofen Ketamine Lidocaine Methampyrone Metrazole Nicotine Nordazepam Phenmetrazine Phenothiazine Phenylbutazone Phthalyl sulfacetamide Procaine Prochlorperazine Tetrahydrocannabinol Theobromine Theophylline Xylazine

Fungicides and Herbicides Atrazine Captan Carboxin Chlorothalonil MCPA Metolachlor Trifluralin Phenols Chloroxylenol Clorophene 2,5-Dichlorophenol Pentachlorophenol o- Phenylphenol

Drugs (Barbiturates) Amytal Mephobarbital Pentobarbital Phenobarbital Primidone Secobarbital Thiamylal Thiopental

Miscellaneous Compounds Anthracene Benzo(a)pyrene Bergapten BHA Chrysene Coumarin Dichloroaniline Ethoxyquin Fuel oil Metaldehyde MGK 264 (insecticide synergist) Mineral oil Molecular sulfur Nicotinic acid Nitrapyrin N,N-diethyl-m-toluamide (DEET) Phenanthrene Piperonyl butoxide Psoralen Pulegone (Pennyroyal oil) Pyrene Strychnine Vitamin A Warfarin

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techniques, because of their low cost and adaptability to both polar and nonpolar compounds, are also still widely used in veterinary toxicology. An additional advantage of TLC, at times, is that compounds not easily seen by the detection systems available for HPLC, can be visualized by unique color reagents, or by charring procedures that show the presence of any organic compound. GC, particularly with selective detection systems such as electron-capture (ECD), and nitrogen/phosphorus detectors (NPD), is still utilized extensively in determination of specific chemical classes. Examples include the use of GC-ECD for determination of organochlorine pesticides and pollutants, and NPD for determination of OP pesticides and nitrogen-containing drugs. It is important to keep in mind, however, that positive determinations by a single chromatographic technique should be verified by a second, independent procedure. This is especially desirable if the case might be involved in litigation.

Insecticides

Cholinesterase-Inhibiting Insecticides. This group includes the OP and carbamate insecticides. Stomach or crop contents and (less often) liver and skin or hair are the samples most likely to yield positive results when submitted in an attempt to determine the presence of a specific compound. Some OPs and most carbamates are rapidly metabolized and eliminated from tissues, and it is not unusual to fail to detect them except in cases of acute death following exposure. Cholinesterase determinations on whole-blood or brain samples are often valuable adjunctive tests in cases of suspected OP toxicosis, owing to the relatively tight and persistent binding of the enzyme and resultant inhibition of its activity by these compounds. Carbamates only transiently bind cholinesterase and therefore depression of enzyme activity is not as consistent as in the case of OP toxicoses. Following exposure to carbamates, it is possible to have initially inhibited enzyme activity that returns to normal during shipment of a sample to the laboratory. 11 It is important to recognize that different methodologies for measuring cholinesterase activity generally make interlaboratory comparisons of cholinesterase values inappropriate. 1· 3 In addition, there are marked species differences with regard to normal cholinesterase activities in a given tissue, and the sensitivity among species of the cholinesterase enzymes in red blood cells and plasma to inhibition by OPs or carbamates varies. Acetylcholinesterase activity also varies in different regions of the brain. Generally, one half of the brain, divided mid-sagittally, is submitted to the laboratory for analysis in order to minimize sample variation. Some laboratories require the caudate nucleus and base their interpretations on only this portion of the brain. Because of the above considerations, it is important for the laboratory performing the analyses to have a database of normal versus depressed levels for a range of species, and, generally, these must be determined in each laboratory, using the method they routinely employ. Cholinesterase activity should not be the only criterion used for evaluating a case of suspected OP or carbamate toxicosis. It is best to check with your laboratory when submitting any specimen for the first time. Pyrethrin and Pyrethroid Insecticides. This group of insecticides is

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being increasingly used for a variety of purposes. Many of these insecticides can be detected in tissues such as the brain and liver. Unfortunately, interpretation of detected tissue concentrations is still very difficult in assessing suspected toxicoses. Until more information is available relating tissue concentrations to adverse effects, analytical tests will be valuable only for confirming exposures and for serving as a developing database of values assoCiated with clinical signs or death. On occasion, synergists such as piperonyl butoxide or MGK 264 are detected in tissue samples. Since these compounds are frequently a component of insecticidal formulations containing pyrethrins or pyrethroids and less frequently a component of rotenone and carbamate formulations, their presence may be a clue to previous exposure to these categories of insecticides. Organochlorine lnsectides. Although not as commonly encountered today as several years ago, occasional exposures of small companion animals to organochlorine (OC; chlorinated hydrocarbon) insecticides still occur. These compounds are extremely lipid-soluble and tend to concentrate in highest amounts in adipose tissue. Other organs such as the brain and liver can accumulate OCs, but concentrations tend to be much lower than in adipose tissue. The OC insecticides are still commonly detected at toxic concentrations in certain wild animals, particularly birds. From a live animal, the specimens of choice for OC analysis are body fat and serum. Brain, liver, and body fat can be submitted from dead animals. Because of the possibility of redistribution to fat that may occur to a limited extent in acute exposures or to a very great degree in chronic exposures, brain concentrations of the OC compounds, which correlate with the occurrence of neurologic signs, are of greatest importance in diagnosis. Body fat and liver concentrations are often much more difficult to interpret. It is important to note whether results are reported on a tissue wet weight basis or tissue fat basis, since the numerical values from the same tissue are considerably different. Metaldehyde. Metaldehyde is used as a molluscicide. Since metaldehyde use varies in different geographic regions, the incidence of toxicosis also varies, being more common in coastal and low-lying areas. Metaldehyde is broken down in the stomach to various aldehydes, which may give a formaldehyde odor to the contents. Stomach contents should be submitted in suspected poisonings. Some laboratories now have the capability of detecting metaldehyde in liver and blood as well. When the parent metaldehyde is not detected, the presence of other aldehydes in the stomach contents may be diagnostically significant. Rodenticides

Anticoagulants. Anticoagulant rodenticides remain a common toxicologic problem in pet animals. When toxicosis is suspected in a live animal, whole blood or plasma can be submitted for analysis. Serum samples are not recommended, owing to the possible partial adsorption of the anticoagulant onto the clot. 4 If the animals dies, analysis of liver tissue or unclotted blood is generally recommended. Stomach contents are often submitted in lieu of other tissues, but they are often not the best choice.

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It is important to keep in mind that clinical signs may not become apparent for 2 to 4 days after exposure and that originally ingested material is unlikely to remain in the stomach. Strychnine. Strychnine can be detected in vomitus, gastric lavage fluid, or urine from a live animal. Stomach contents, liver, and urine can be submitted from a dead animal. Since many instances of strychnine poisoning in pet animals are malicious in origin, it is important to submit suspect bait, if available, for analysis. Cholecalciferol. Cholecalciferol is becoming a more widely used rodenticide, and the incidence of serious poisoning has rapidly grown. Although an elevated serum calcium level is a useful diagnostic clue, it may be difficult to rule out other causes of hypercalcemia. More work needs to be done to develop a reliable ante mortem diagnostic procedure to differentiate these conditions. The presence of marked elevations of 250H-D3 in serum may be of value, although methods for its detection need to be refined prior to widespread use by veterinary diagnostic laboratories. Ethylene Glycol Ethylene glycol (EG) is rapidly metabolized over a period of several hours to a day following exposure. Although many laboratories have the capability for detecting the parent compound, it has often been eliminated from the body by the time the problem is recognized or death has occurred. However, in our experience, with a sufficiently sensitive method, it is often possible to detect EG even though the animal is in acute renal failure. Detection of EG metabolites is also possible, but it is wise to check with the laboratory first. In-house detection kits for EG are available and should aid in recognition of recent exposure (EGT Test Kit, PRN Pharmacal, Inc, Pensacola, FL 32504). In our experience, the EGT test is capable of detecting a concentration in blood of 500 ppm (50 mg/dl) or greater. Depending on the length of time between exposure of the animal and collection of serum, and given the relatively high detection limit of the test, a negative finding may not rule out previous exposure to EG. Also, to the authors' knowledge, the ability of other substances to interfere with the test procedure has not been independently determined. A valuable aid to the diagnosis of EG poisoning is the measurement of kidney calcium levels. Renal calcium concentrations commonly surpass 4,000 ppm following EG toxicoses. By using a multielement screening procedure to detect elevated kidney calcium concentrations, we have diagnosed EG toxicosis in instances in which it was not originally suspected. Veterinary and Human Medications The wide variety of compounds in this category makes generalization most difficult. If a particular drug compound is of interest, it is always a good idea to check with the diagnostic laboratory first. Frequently, toxicology laboratories receive requests for tissue analysis to determine the presence of tranquilizers, barbiturates, or euthanasia agents. Whereas many can be detected in tissues, interpretation of the results is often impossible due to lack of information relating tissue concentrations with particular adverse sequelae. Interpretation, therefore,

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is often restricted to a confirmation of exposure. Illicit drug screening is often an overlooked area in veterinary diagnosis. Serum and especially urine are most often the specimens of choice for ante mortem evaluation. Stomach contents, liver, kidney, and urine should be submitted from a dead animal. In addition, if subcutaneous or muscle tissues are noted to have lesions suggestive of an injection site, the affected tissues should also be submitted for analysis. The most common mistake with regard to submission of samples for drug analysis is the failure to submit a variety of samples, including serum, urine, and tissues. Depending on the pharmacokinetics of a given compound and the time frame of the case, it may be possible to detect the parent drug or its metabolites in only one or a few of the submitted samples. INTERPRETATION OF RESULTS

As indicated above, all the information regarding the case may be important for proper interpretation of the analytical results. UnfOrtunately, detected amounts of a toxicant in submitted samples may not provide a definitive answer. Other information, including the time frame and magnitude of exposure, clinical signs, clinical pathologic measurements, gross and histologic examination, and adjunctive testing such as cholinesterase determinations may all be critical to a diagnosis. Often it is impossible to interpret a given tissue concentration because data are not available correlating these with specific adverse effects. In such situations, the diagnostician can only use such phrases as "compatible with toxicosis" or "laboratory results indicate exposure to." SUMMARY

The increasing sophistication of toxicologic analyses offered by veterinary diagnostic laboratories provides the practitioner with a valuable resource for the diagnosis of companion and exotic animal toxicoses. The availability of such testing is a valuable service that can be offered to veterinary clientele. Appropriate and timely toxicologic testing may permit more successful treatment of affected patients and protect animals and humans from hazardous exposure that might occur if a responsible toxicant goes unrecognized. Perhaps the most critical point to keep in mind, however, is that no matter how sophisticated the toxicologic laboratory is, a correct diagnosis is dependent upon the submission of appropriate biologic and environmental samples. REFERENCES L Elleman GL, Courtney KD, Featherstone RM: A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem Pharmacal 7:88, 1961

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2. Grauer GF, Thrall MA, Henre BA, eta!: Early clinicopathologic findings in dogs ingesting ethylene glycol. Am J Vet Res 45:2300, 1984 3. Michel HO: An electrometric method for determination of red blood cell and plasma cholinesterase activity. J Lab Clin Med 34:1564, 1949 4. Mount ME, Woody BJ, Murphy MJ: The anticoagulant rodenticides. In: Kirk RW (ed): Current Veterinary Therapy IX: Small Animal Practice. Philadelphia, WB Saunders, 1986, pp 15~165 5. Ellenhorn MJ, Barceloux DC: Medical Toxicology: Diagnosis and Treatment of Human Poisoning. New York, Elsevier, 1988, pp 1067-1103 6. Ray AC, Post LO, Hewlett TP, et a!: A survey of compounds identified in a veterinary toxicology laboratory using GC/MS. Vet Hum Toxicol 23:418, 1981 7. Schock RJ, Braselton WE: Investigation of the utility of bonded phase packed columns for the identification of organic toxicants by gas chromatography-mass spectrometry (GC-MS). American Association of Veterinary Laboratory Diagnosticians, 25th Annual Proceedings, p 453, 1982 8. Smith M, Lewis D: A potpourri of pesticide poisonings in Alberta in 1987. Vet Hum Toxicol, 30:118, 1988 9. Stowe HD, Braselton WE, Slanker, M, et a!: Multielemental assays of canine serum, liver, and kidney by inductively coupled argon plasma emission spectroscopy. Am J Vet Res 47:822, 1986 10. Thrall MA, Grauer GF, Mero KN: Clinicopathologic findings in dogs and cats with ethylene glycol toxicoses. JAm Vet Med Assoc 1:37, 1984 11. Wilhem K, Reiner E: Effect of sample storage on human blood cholinesterase activity after inhibition by carbamates. Bull Wild Hlth Org 48:235, 1973 12. Zook BC, Kopito, MS, Carpenter JL: Lead poisoning in dogs: Analysis of blood, urine, hair, and liver for lead. Am J Vet Res 33:903, 1972

Address requests for reprints to: Robert H. Poppenga, DVM, PhD Department of Pharmacology and Toxicology Life Sciences Building Michigan State University East Lansing, MI 48824-1317