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Clinical and Forensic Toxicology Roger L. Bertholf, Ph.D. Associate Professor of Pathology Chief of Clinical Chemistry & Toxicology Roger L. Bertholf,

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Presentation on theme: "Clinical and Forensic Toxicology Roger L. Bertholf, Ph.D. Associate Professor of Pathology Chief of Clinical Chemistry & Toxicology Roger L. Bertholf,"— Presentation transcript:

1 Clinical and Forensic Toxicology Roger L. Bertholf, Ph.D. Associate Professor of Pathology Chief of Clinical Chemistry & Toxicology Roger L. Bertholf, Ph.D. Associate Professor of Pathology Chief of Clinical Chemistry & Toxicology

2 Toxicology Disciplines Industrial Toxicology –Toxic exposures in the workplace –Product testing Molecular Toxicology Veterinary Toxicology Environmental Toxicology Clinical Toxicology Forensic Toxicology Industrial Toxicology –Toxic exposures in the workplace –Product testing Molecular Toxicology Veterinary Toxicology Environmental Toxicology Clinical Toxicology Forensic Toxicology

3 Clinical Toxicology The branch of toxicology that is concerned with human poisoning –Drug overdoses Pharmaceuticals Drugs of abuse –Toxic exposures Environmental Occupational Accidental The branch of toxicology that is concerned with human poisoning –Drug overdoses Pharmaceuticals Drugs of abuse –Toxic exposures Environmental Occupational Accidental

4 The Top Ten Poisoning... 1.Cleaning Products 2.Analgesics 3.Cosmetics 4.Plants 5.Cough/Cold Preparations 6.Hydrocarbons 7.Bites 8.Topicals 9.Foreign Bodies 10.Chemicals 1.Antidepressants 2.Analgesics 3.Sedative/Hypnotics 4.Street Drugs 5.Cardiovascular Drugs 6.Alcohols 7.Fumes 8.Chemicals 9.Asthma Medications 10.Cleaning Products Exposures Deaths

5 Pharmacokinetics Plasma drug concentration Time   t 1/2 Peak plasma concentration 

6 Pharmacokinetics Summary

7

8

9 The Autonomic Nervous System Sympathetic (Thoracocolumbar) Norepinephrine Parasympathetic (Craniosacral) Acetylcholine   22 11 CardiovascularSmooth muscle Insulin release Gluconeogenesis GI Vascular smooth muscle MuscarinicNicotinic

10 Cholinergic Poisoning Organophosphates, some mushrooms (Group III, clytocybe and inocybe species), betel nuts, pilocarpine, carbachol, acetylcholine Miosis, vasodilatation, bronchial secretions, bradycardia, increased bowel motility, urination, sweating Mnemonic: DUMB BELS (Diarrhea, Urination, Miosis, Bradycardia, Bronchorrhea, Emesis, Lacrimation, Salivation) Atropine + pralidoxamine is antidote Laboratory monitors AChE activity Organophosphates, some mushrooms (Group III, clytocybe and inocybe species), betel nuts, pilocarpine, carbachol, acetylcholine Miosis, vasodilatation, bronchial secretions, bradycardia, increased bowel motility, urination, sweating Mnemonic: DUMB BELS (Diarrhea, Urination, Miosis, Bradycardia, Bronchorrhea, Emesis, Lacrimation, Salivation) Atropine + pralidoxamine is antidote Laboratory monitors AChE activity

11 Anticholinergic Poisoning Belladonna, Jimsonweed, antihistamines, phenothiazines, certain mushrooms (Group V), scopolamine, tricylics, OTC sleeping pills Mnemonic: Red as a beet, dry as a bone, mad as a hatter, hot as a stone, bowel and bladder lose their tone, and the heart runs alone. Physostigmine is antidote Belladonna, Jimsonweed, antihistamines, phenothiazines, certain mushrooms (Group V), scopolamine, tricylics, OTC sleeping pills Mnemonic: Red as a beet, dry as a bone, mad as a hatter, hot as a stone, bowel and bladder lose their tone, and the heart runs alone. Physostigmine is antidote

12 Sympathetic Poisoning  : vasoconstriction, pupillary dilitation, coronary artery dilitation, decreased bowel motility, bladdar contraction  1 : tachycardia  2 : smooth muscle dilatation, insulin release, lipolysis, renin release, gluconeogenesis (miosis, vasodilatation, bronchodilatation, hyperglycemia, decreased bowel motility, bladder relaxation)  : vasoconstriction, pupillary dilitation, coronary artery dilitation, decreased bowel motility, bladdar contraction  1 : tachycardia  2 : smooth muscle dilatation, insulin release, lipolysis, renin release, gluconeogenesis (miosis, vasodilatation, bronchodilatation, hyperglycemia, decreased bowel motility, bladder relaxation)

13 Nicotinic Poisoning Insecticides, tobacco, black widow spider venom Tachycardia, hypertension, muscle fasciculations, weakness, paralysis d-turbocurarine is antidote Insecticides, tobacco, black widow spider venom Tachycardia, hypertension, muscle fasciculations, weakness, paralysis d-turbocurarine is antidote

14 Ethanol Most common (by far) toxic exposure Often associated with: –Trauma –Loss of consciousness –Other drug exposure Frequently involves medico-legal interventions Most common (by far) toxic exposure Often associated with: –Trauma –Loss of consciousness –Other drug exposure Frequently involves medico-legal interventions

15 Ethanol Pharmacodynamics Blood alcohol concentration (mg/dL, %) CNS impairment  Loss of inhibition Impaired motor coordination Slurred speech Staggering gait Loss of consciousness

16 Ethanol distribution Serum 95% H 2 O Cells 82% H 2 O EtOH Serum (or plasma) ethanol is 5 – 15% higher than whole blood ethanol

17 Enzymatic Ethanol Methods ADH is selective, but not specific for ethanol Other enzymes that involve NADH can potentially interfere ADH is selective, but not specific for ethanol Other enzymes that involve NADH can potentially interfere

18 Non-ethanol Alcohol Poisoning Alcohol toxicity is primarily related to metabolites –Ethanol  Acetaldehyde  Acetate –Isopropanol  Acetone –Methanol  Formaldehyde  Formic acid –Ethylene Glycol  Oxalate and Hippuric acid Non-ethanol alcohol exposures can be detected by an increase in the osmol gap Alcohol toxicity is primarily related to metabolites –Ethanol  Acetaldehyde  Acetate –Isopropanol  Acetone –Methanol  Formaldehyde  Formic acid –Ethylene Glycol  Oxalate and Hippuric acid Non-ethanol alcohol exposures can be detected by an increase in the osmol gap

19 The Osmol Gap Calculated Osmolality:

20 The Osmol Gap Colligative properties depend on the number of of dissolved particles –Boiling point –Freezing point Osmolality is usually determined by freezing point depression The difference between the calculated and measured osmolality is the Osmol Gap Colligative properties depend on the number of of dissolved particles –Boiling point –Freezing point Osmolality is usually determined by freezing point depression The difference between the calculated and measured osmolality is the Osmol Gap

21 The Osmol Gap Alcohol Toxic mg/dLMW Add to serum osmol (mosm/mg/dL) Ethanol Methanol Isopropanol Ethylene glycol

22 Osmol Gap Mnemonic Methanol Ethanol Diuretics (glycerol, mannitol, sorbitol) Isopropanol Ethylene glycol

23 Analgesic Poisoning Exposures Fatalities

24 Salicylate Poisoning Toxic symptoms develop at serum concentrations exceeding 250 mg/L Serum concentrations exceeding 1000 mg/L can be fatal Symptoms are tinnitus, hyperventilation, respiratory failure, convulsions, coma Lab results reveal mixed metabolic acidosis/respiratory alkalosis Acidification of urine enhances elimination Toxic symptoms develop at serum concentrations exceeding 250 mg/L Serum concentrations exceeding 1000 mg/L can be fatal Symptoms are tinnitus, hyperventilation, respiratory failure, convulsions, coma Lab results reveal mixed metabolic acidosis/respiratory alkalosis Acidification of urine enhances elimination

25 Acetaminophen Poisoning Toxic symptoms develop at serum concentrations exceeding 100 mg/L Serum concentrations exceeding mg/L result in severe liver damage Symptoms may not appear until hepatic failure is evident and irreversible Antidote is N-acetylcysteine Toxic symptoms develop at serum concentrations exceeding 100 mg/L Serum concentrations exceeding mg/L result in severe liver damage Symptoms may not appear until hepatic failure is evident and irreversible Antidote is N-acetylcysteine

26 Acetaminophen Metabolism

27 Carbon Monoxide Poisoning Can be deliberate or accidental –CO is odorless CO binds irreversibly to hemoglobin, displacing oxygen CO-Hb (carboxyhemoglobin) can be measured on a co-oximeter –Different max than O 2 -Hb (oxyhemoglobin) Hyperbaric oxygen may be indicated Can be deliberate or accidental –CO is odorless CO binds irreversibly to hemoglobin, displacing oxygen CO-Hb (carboxyhemoglobin) can be measured on a co-oximeter –Different max than O 2 -Hb (oxyhemoglobin) Hyperbaric oxygen may be indicated

28 Metal Poisonings Iron is most common (particularly in kids) –Ferritin –Deferoxamine is antidote Arsenic is most notorious –Acute vs. chronic –Inorganic vs. arsine gas –BAL (dimercaprol) is antidote Iron is most common (particularly in kids) –Ferritin –Deferoxamine is antidote Arsenic is most notorious –Acute vs. chronic –Inorganic vs. arsine gas –BAL (dimercaprol) is antidote

29 Metal Poisonings Lead –Most cases in children exposed to lead paint –Blood lead >10  g/L is considered risk –Monitor with  -aminolevulinic acid dehydratase –EDTA is antidote Mercury –Organic vs. inorganic –Neurotoxic, nephrotoxic, teratogenic Lead –Most cases in children exposed to lead paint –Blood lead >10  g/L is considered risk –Monitor with  -aminolevulinic acid dehydratase –EDTA is antidote Mercury –Organic vs. inorganic –Neurotoxic, nephrotoxic, teratogenic

30 Spot Tests for Metal Poisoning Reinsch Test: Copper wire turns... –Shiny silver: Mercury –Dull black: Arsenic –Shiny black: Bismuth –Dark purple sheen: Antimony Gutzeit test for arsenic –Acidification produces arsine gas, which discolors silver nitrate paper Iron reacts with potassium ferricyanide and ferrous sulfate to produce Prussian Blue Reinsch Test: Copper wire turns... –Shiny silver: Mercury –Dull black: Arsenic –Shiny black: Bismuth –Dark purple sheen: Antimony Gutzeit test for arsenic –Acidification produces arsine gas, which discolors silver nitrate paper Iron reacts with potassium ferricyanide and ferrous sulfate to produce Prussian Blue

31 Thin Layer Chromatography

32 TLC Stains Ninhydrin: 1 o or 2 o amines (sympathomimetics) Mercuric Sulfate: barbiturates, glutethimide, phenytoin (white ppt) Diphenylcarbazone: same as mercuric sulfate (blue or purple spot) Iodoplatinate: 3 o amines Dragendorf's reagent: methaqualone UV absorption at 254 nm: benzodiazepines, barbiturates, methaqualone Fluorescence at 366: Benzodiazepines, quinine, quinidine Ninhydrin: 1 o or 2 o amines (sympathomimetics) Mercuric Sulfate: barbiturates, glutethimide, phenytoin (white ppt) Diphenylcarbazone: same as mercuric sulfate (blue or purple spot) Iodoplatinate: 3 o amines Dragendorf's reagent: methaqualone UV absorption at 254 nm: benzodiazepines, barbiturates, methaqualone Fluorescence at 366: Benzodiazepines, quinine, quinidine

33 Medical vs. forensic drug testing Patient consent not required Identity of specimen is presumed Screening result is sufficient for medical decision Results are used for medical evaluation Patient consent not required Identity of specimen is presumed Screening result is sufficient for medical decision Results are used for medical evaluation Subject must consent to be tested Identity of specimen must be proved Only confirmed results can be considered positive Results are used for legal action

34 Illegal Drug Use in the U.S. (1998 Household Survey) 13.6 million Americans use illicit drugs –25 million in % of youths age use marijuana –14.2% in million Americans use cocaine –5.7 million in million Americans use illicit drugs –25 million in % of youths age use marijuana –14.2% in million Americans use cocaine –5.7 million in 1985

35 Types of drugs used

36 History of workplace drug testing 1960s – 1970s: The Department of Defense begins testing military personnel for illegal drug use. 1986: President Reagan establishes the “Federal Drug-Free Workplace”. 1988: Mandatory Guidelines for Federal Workplace Drug Testing Programs is published in the Federal Register. 1960s – 1970s: The Department of Defense begins testing military personnel for illegal drug use. 1986: President Reagan establishes the “Federal Drug-Free Workplace”. 1988: Mandatory Guidelines for Federal Workplace Drug Testing Programs is published in the Federal Register.

37 The “NIDA” program NIDA (now SAMHSA) requirements for drug testing were drafted by Research Triangle Institute The RTI established the National Laboratory Certification Program (NLCP) Drug testing for federal agencies (DOT, NRC, etc.) must be performed in a NLCP- certified laboratory NIDA (now SAMHSA) requirements for drug testing were drafted by Research Triangle Institute The RTI established the National Laboratory Certification Program (NLCP) Drug testing for federal agencies (DOT, NRC, etc.) must be performed in a NLCP- certified laboratory

38 Florida Drug-Free Workplace The Florida HRS (now AHCA) established a drug-free workplace program in 1990 Specifications for the State of Florida program are similar to federal requirements, but there are notable differences Employees of Florida Drug-Free Workplace- compliant businesses must be tested in AHCA-licensed laboratories The Florida HRS (now AHCA) established a drug-free workplace program in 1990 Specifications for the State of Florida program are similar to federal requirements, but there are notable differences Employees of Florida Drug-Free Workplace- compliant businesses must be tested in AHCA-licensed laboratories

39 Screening vs. Confirmation Low cost Fast Semi-quantitative High sensitivity Low specificity Low cost Fast Semi-quantitative High sensitivity Low specificity High cost Slow Quantitative High sensitivity High specificity

40 A confirmatory method should... Utilize the most accurate (specific) testing method available Have sensitivity equal to or better than the screening method Be economically feasible Be simple enough to standardize across many laboratories Produce results that are legally defensible Utilize the most accurate (specific) testing method available Have sensitivity equal to or better than the screening method Be economically feasible Be simple enough to standardize across many laboratories Produce results that are legally defensible

41 GC/Mass Spectrometry Injector GC Column Ionizer Mass Filter Detector Data System

42 Electron impact ionization e-e- e-e (+) From GC To MS Filament Collector Ion volume (or source) Focusing lens (-) Power supply

43 The “Right Hand Rule” Direction of current Direction of magnetic field

44 Magnetic sector mass spectrometer To detector + From ion source

45 Quadrupole mass spectrometer + From ion source To detector

46 Electron multiplier e-e e - + From mass filter Positive dynode Negative dynode Ammeter

47 Mass spectrum

48 Single ion monitoring (SIM) Time  m/z Full scan time = 1.0 sec (  sec/ion) 0.1s

49 Cocaine C 17 H 21 NO 4 MW=303.35

50 303 (M + ) [M-31] [M-121] + 82 (base peak) 121

51 Cocaine fragmentation (EI)

52 303 (M + ) [M-31] [M-121] + 82 (base peak) 121

53 Amphetamine/Methamphetamine

54 44 91

55 Amphetamine fragmentation

56 44 91

57 Methamphetamine fragmentation

58 58 91

59 TMS derivative of amphetamine

60 TMS-amphetamine fragmentation

61 Mass spectra of TMS-amphetamine

62 TMS-methamphetamine fragmentation

63 Mass spectra of TMS- methamphetamine

64 Methamphetamine metabolism

65 Cocaine HCl and free base forms

66 Cocaine metabolism

67 TMS derivative of benzoylecgonine

68 TMS-benzoylecgonine fragmentation

69 Mass spectra of TMS- benzoylecgonine

70 Opiates

71 Glucuronidation

72 Morphine hydrolysis

73 TMS derivative of codeine

74 Mass spectra of TMS-codeine

75 Heroin metabolism

76  9 -Tetrahydrocannabinol (THC)

77 THC-COOH detection THC-COOH glucuronide (  15%) THC-COOH TMS-THC-COOH Hydrolysis BSTFA


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