FORENSIC MEDICINE COURSE

FORENSIC TOXICOLOGY

&

FORENSIC ALCOHOLOGY

KRZYSZTOF S. BOROWIAK

HISTORICAL ROOTS OF TOXICOLOGY

FORENSIC TOXICOLOGYAs medicine science was develope in the end of

XVIII C. (London, Vien, Berlin). The first Forensic Department in Poland was founded in

Krakow in the 1805

CLINICAL TOXICOLOGY

First Poisining Centre in

Washington D.C.

(1957)

OCCUPATIONAL AND

INDUSTRIAL

TOXICOLOGY

60. of XX C.

EXPERIMENTAL

TOXICOLOGY

- Geno

- Immuno

- new products

FORENSIC CLASSIFICATION OF TOXINS

POISONS

VOLAITABLE WITH WATER STEAM

Detectioned with:

GC, GC-MS or Spectrometry

ORGANIC NOT VOLAITABLE SUBSTANCES ISOLATED WITH SOLID 0PHASE OR LIQUID EXTRACTION, detectioned with GC, HPLC, Immuno-methods, spectrometry

NON-ORGANIC SUBSTANCES (METALS) WHEN MINERALISATION IS NECCESSARY, DETECTION WITH AAS

FORENSIC CLASSIFICATION OF TOXINS (2)

POISONS

EFFECTS CAN BE OBSERVED IN AUTHOPSY

ANATHOMOPATOLOGICAL CHANGES ARE NOT OBSERVED

Blood toxins:

- blocking oxygen transport or metabolism

Heavy metals

With characteristic odourCorrosive

and irritants

•Drug of abuse

• chemicals

•Drugs and pharmaceutics

• others

MOST FREQUENT POISONS DETECTED IN THE POST MORTEM SAMPLES

DRUGS

ALCOHOLS

DRUG ABUSE

SOLVENTS

Pesticides

ABBREVATION – IN POLISH FORENSIC STATISTIC ON THE 3RD PLACE THE CARBON MONOXIDE IS LOCATED

45%

55%

15%

5%

THE COLLECTION ROUTES AND TYPE OF POST MORTEM SAMPLES

• Blood ( 2 x 10 mL) – alcohols & drug abuse, drugs & other organics, • Urine (25-50 mL) – alcohols, drug metabolites, drug of abuse • Brain tissue (50g) – psychotropic substances• Kidney (1) – nophrotoxicity estimation and elimination • Liver (50-100 g) – hepatotoxicity and high conc. of metabolites• Intenstine with contents (50g) • Stomach with separated colected contents• Lungs (50 g) – excelent matherial for solvents and gasses int.

When the results of authopsy are unknown and we suspect neccessary of multi-direct toxicological examination

THE COLLECTION ROUTES AND TYPE OF POST MORTEM SAMPLES

• BLOOD COLLECTED FOR ETHANOL AND OTHER ALCOHOLS DETERMINATION SHOULD BE TAKEN FROM SURFACE VESSELS (VEINS)

• BLOOD COLLECTED FOR CARBOXYHEMOGLOBIN SHOULD BE TAKEN FROM DEEP LOCATED

VESSELS• VIALS WITH SAMPLES COLLECTED FOR

VOLAITABLE SUBSTANCES DETERMINATION SHOULD BE

PERFECT CLOSED

ALL BIOLOGICAL SAMPLES SHOULD BE PRESERVED IN TO ABSOLUTLY CHEMICALY CLEAN VIALS,

BOTLES AND JARS

IMPORTANT TO REMEMBER

DIFFERENTIATE OF DEATH CASE

Observation during authopsy: pink-redish colour of body surface, internal organ surface and blood

That can be resulted from:

• death in the course of carbon monooxide fatal intoxication (COHb > 40% detected in the blood)

• death in the course of cianides intoxication

(bitter almond smell)

• death in the course of low temperatures action (freezing)

Differentiate course of death

Observation durring authopsy: gray-blue colour of corps and organs surfaces, chocolade colour of blood

Result from:

• Death in course of methemoglobinemic substances intoxication (nitrates, nitrites and anilin e.g. – MtHb > 30%)

• deep anoxemia, metabolic acidosis

• putrification of body

OTHER CHARACTERISTIC CHANGES ILLUSTRATED EFFECTS OF POISON

INFLUENCE

TYPE OF INJURY EXAMPLES

• CAUSTIC POISONS (CORROSIVE – ACUTE LYE BURNS, CORROSION OF GASTRIC MUCOSA, HYPEREMIA, HAEMORAGIAE, NECROSIS) - (STRONG ACIDS, ALKALIAS, HOME SANITARY-CLEANING AGENTS)

• SKIN BLISTERS (BARBITURATES) • RENAL DAMAGES & NECROSIS – METALS & METALOIDS TETRACYCLINES, BABRBITURATES, METHANOL,

GLYCOLS, • PULMONARY & GI CHANGES (OEDEMA,HYPEREMIA,

HEMORRAGIAE, IRRITANTS,) – GASES, ARSEN OXIDE, INSECTICIDES, PETROLEUM DISTILATE PRODUCTS (GASOLINE, KEROSENE....)

• HYPEREMIA – PINK-RED BLOOD AND ORGAN SURFACES – C=O

• SKIN AND BLOOD CYANOSIS – METHEMOGLOBINEMIC AGENTS

MECHANISMS OF CELL DEATH FOLLOWING DRUG INDUCED LIVER INJURY (By J.G.O’Grady)

DRUG

COVALENT BINDING TO CELLULAR STRUCTURES

Covalently modified P-450

Covalently modified proteins

Haptenic epitopes

Auto-antigen epitopes

Reactive metabolites Covalently modified

DNA

Mutations,

cancerogenesis

DEATH OF CELLNeoantigens Immune system

MORPHOLOGIC CLASSIFICATION OF DRUG-RELATED CHRONIC LIVER INJURY

TYPE OF INJURY EXAMPLES • CHRONIC HEPATITIS HALOTANE, ISONIAZIDE,

DANTROLENE, SULPHONAMIDES,• CHRONIC CHOLESTASIS TCA, FENOTHIAZINES,

BARBITURATES, PHENYTOIN, TOLBUTAMIDE,

• CHRONIC STEATOSIS ETHANOL, METHOTREXATE, ANTINEOPLASTIC AGENTS,

• PHOSPHOLIPIDOSIS AMIODARONE, THIORIDAZINE,• VASCULAR (occlusion, ESTROGENS, ANABOLICS, hypertension, peliosis)• NEOPLASM ESTROGENS, ANABOLICS, VINYL

MORPHOLOGIC CLASSIFICATION OF DRUG-RELATED ACUTE LIVER INJURY

(HEPATITIS)

TYPE OF INJURY EXAMPLES___________________________________________

• HEPATOCELLULAR (cytolytic) • zonal necrosis - CCL-4, halothane, Acetaminophen

• steatosis (makro and mikrovesicular) – ethanol,

methotrexate, tetracyclines, valproic acid, • CHOLESTATIC • hepatocanalicular -Amoxiciline, chlorpromazine,• Canalicular – estrogens, steroids, anabolics

• VASCULAR (vein occlusion) estrogens, anabolics.

MEDICO-LEGAL ASPECTS OF ETHANOL

TYPE OF CRIMES AND ACCIDENTS STRONGLY ASSOCIATED WITH USE OF ETHANOL :

• MAJOR TRAUMA• MOTOR VEHICLE COLLISIONS• FIRES, BURNS AND FALLS• DOMESTIC VIOLENCE, CHILD ABUSE• SUICIDES ATTEMPT• TRAUMA AND INJURES OUTCOM COMPLICATIONS

• Ethyl alcohol is most important constituent of different „alcoholic” beverages (Long-drinks), and basic solvent used

in several medicinal preparations.

• Ethanol is most frequent abused intoxicating substance companioned of human from several hundred years.• It is most commonly encountered toxic substance in the forensic toxicology also.

CLINICAL AND MEDICO-LEGAL CLASSIFICATIONOF ETHANOL

ACTION

> 4,0 - 4,5 ‰ PARALYTIC PHASE (V)3,0 - 4,0 ‰ NARCOTIC PHASE (IV)

2,0-3,0 ‰ EXCITATION PHASE (III)

1,0-2,0 ‰ EUPHORIC PHASE (II)

0,2-1,0 ‰ DYSPHORIC ACTION (I)

< 0,2 - 0,5 > ‰ – STATUS AFTER USAGE OF ALCOHOL - OFFENSE § 87 KW

> 0,5 ‰ DRUNKNESS STATUS - CRIME ACCORDING TO § 178 CRIME CODE

2000-2004 YEARS

• 240 from 760 clinical examination (alcohols)

• 975 drivers examined

• 343 under alcohol influence

• near 110 under drug abuse (THC, AMPH.)

• 84 - both substances W 2005 - 2007

• Ca. 2800 drivers and victims of accidents were examined (alcohol + drug abuse): 800 -1200 per year

• 2007 - under influence of drug abuse 221 examined (from 595)

• THC, AMPH, MDMA, Cocaine

STATISTIC DATA OF PAM FOR. DEPARTM.

AMOUNT OF ETHANOL IN THE DIFFRERENT ALCOHOLIC BEVERAGES

• GLASS OF VODKA 100g = 32g PURE SPIRIT

• 1-2 GLAS OF 12-14% VINE 500 mL = 32g PURE SPIRIT

• 4 GLAS OF PORTER BEER (ALE, GUINESS – 8-9%) = 32g PURE SPIRIT

INTAKE ANY OF ABOVE LONG DRINKS CAUSED OVERCROSS THE LEGAL LIMIT OF ETHANOL IN

BLOOD

OVER 0.5 PROMILLE (0.5 mg/mL)

ETHANOL CONCENTRATION IN SOME COMMERCIAL PRODUCTS

PRODUCT ETHANOL

C [%]

„PROOF” AMOUNT

in [g]BEER 2-6 4-12 1,6-4,8

WINES 10-20 20-40 8-16

DESTILATED SPIRIT (vodka,

brandy, cognac)40-60 80-120 32-48

COUGH MEDICINES

3-25 6-50 2,4-20

MOUTHWASH 16-27 32-54 12,8-21,6

COLOGNES 40-60 80-120 32-48

Drinking and driving-the limits in Europe

BULGARIA, HUNGARY, CZECHY, SLOVAKIA, FSRR

PORTUGAL, YUGOSLAVIA, NEDERLAND, BELGIUM, FINLAND, GERMANY, UK

ENGLAND,SPAIN, FRANCE, SWITZERLAND, AUSTRIA

IRELAND

POLAND *, SWEDEN, NORWAY

1,0

0,8

0,5

0,2

0,0

‰ (PROMILLE 1/1000) = MG/ML

‰ x 10 = MG/L

INFLUENCE OF DIFFERENT FACTORS ON THE BODY-KINETIC OF ETHANOL

EXTERNAL AGENTS

• TIME-DEPEND AND FREQUENTNES OF DRINKING

• KIND OF BEVERAGES

• DIET

• ENVIROMENTAL TEMPERATURE

• BIOMETOROLOGY STATUS

CONSTITUAL AGENTS

• RASE

• SEX

• AGE

• HEALTH STATUS

• GENETIC PREDISPOSITION

THEREFORE EFFECTS OF ETHANOL INFLUENCE ARE PERSONAL, INDIVIDUAL AND CHANGEBLE IN TIME

HEPATIC CELLULAR OF ETHANOL BIOTRANSFORMATION

ETHANOL

ALCOHOLDEHYDROGENASE

ACETYTALDEHYDE

ACETALDEHYDE

DEHYDROGENASE

ACETATE

CYTOCHROMEP450 2E1

MEOS

ENDOPLASMIC RETICUKUM

CYTOPLASME OF

HEPATOCYTE

MITOCHONDRIUM

XANTHINE OXIDASEACETALDEHYDE OXYIDASE

OXIDANT STRESS

INACTIVE METABOLITES

CURVES ILLUSTRATED DIFFERENT BODY-KINETIC OF ETHANOL

30 60 90 120 (t) min.

0,0 5 %

0,1 %

0,15 % ETHANOL DEFFICIT

AVERAGE EMPTY FULL CUMULATION

β

β

β

/ R / - DISTRIBUTION COEFFICIENT

ELIMINATION COEFFICIENT

ABSORPTION

EFFECTS OF THE EXCESSIVE ALCOHOLS INTAKE ON THE LIVER TISSUE

ETHANOL INTAKE,MORE THAN 40 UNITS A WEEK

METABOLISM ABNORMALITIES

TRIGLICERIDES, SUGARS, GLIKANES

FATTY LIVER PROCESS

ALCOHOLIC HEPATITIS

ALCOHOLIC CIRRORIS

FIBROTIC INJURYFATTY LIVER

70%

30%

1 UNIT = 10 g PURE (spirit) ALCOHOL (100%)

100 ml of vodka (40%) included 32 g of pure alcohol

THE RETROSPECTIVE AND PROSPECTIVE ALCOHOLOMETRIC CALCULATION

EXPERT’S OPPINION FOR COURTS, PROSECUTORS AND INSURANCE

COMPANIES

WHEN SHOULD BE PERFORMED ?

• WHEN DRIVER-PERPETRATOR OF ROAD FATAL ACCIDENT WAS ESCAPED

• WHEN ETHANOL ESTIMATION WAS PERFORMED SOME TIME AFTER ACCIDENT

• WHEN WHITNESES HAVE DIFFERENT OPPINIONS ON THE SOBRIETY OF DRIVER

• WHEN THERE IS POSSIBILITY OF OVERDRINKING OF ETHANOL POST FACTUM

• WHEN POSSIBLE ADVERSE OR PATHOLOGIC AFTER ALCOHOL REACTIONS MUST BE RESPECT

RETROSPECTIVE ALCOHOLOMETRIC CALCULATION

THEORETIC, CALCULATED CONCENTRATION OF BLOOD ETHANOL, BASED ON THE RESULTS OF ALCOHOLOMETRIC EXAMINATION

C(t) = C(a) + B (60) x T

When: C(t) – ethanol concentration in blood in the road accident moment (time)

C(a) – ethanol concentration estimated in blood in the alcoholometric examination

B (60)- elimination valuue factor (0,07-0,28 mg/h) for expert apply limited (0,1-0,2 mg/h)

T - time difference from accident to examination (in hours) for example 3 hours

RETROSPECTIVE ALCOHOLOMETRIC CALCULATION continued

THE OBLIGATORY CONDITIONS LEAD TO ATTEMPT OF RETROSPECTIVE CALCULATION

• THE ETHANOL ELIMINATION PHASE SHOULD BE NOTED (DECREASING RESULT IN THE FOLLOWING EXAMINATIONS e.g. 1.0 ; 0.8 ; 0.7 mg/mL)

• TIME PERIOD FROM ACCIDENT TO EXAMINATION NOT OVER 5-7 HOURS

• THE ESTIMATED IN BLOOD CONCENTRATION - NOT LESS THAN 0.5 mg/mL

PROSPECTIVE ALCOHOLOMETRIC CALCULATION

APPLIED FOR VERYFICATION OF PENETRATOR WITNESS OR OTHER PARTICIPANTS OF ROAD

ACCIDENT BASED ON THE DECLARED AMOUNT OF INTAKE ETHANOL

C(o) = A x P x R• C(o) – [mg/ml] theoretical, calculated concentration of

blood ethanol based on the known amount and kind of drinken ethanol (declared by driver or whitnes)

• A - amount of intake ethanol expressed in the grams of pure (100%) ethanol (e.g. 100 ml 40% vodka = 32 g pure ethanol)

• P - body weight (kg) standard range 65-85 kg, at present more frequently BMC (body mas index) is used

• R - body distribution coefficient (male - 0.7 ; female – 0.6)

ANALYTICAL METHODS OF ETHANOL DETERMINATION

THE PHYSICO-CHEMICAL METHODS DETERMINATION OF ETHANOL IN THE BREATH AIR (used mainly by Police,

industry security...)

Oxygen Ethanol, carbon dioxide

Vessel-pulmonary membrane

HENRY LAW (1803) - gases diffusion balance; CUSHING R. (1910) non-active diffussion

INFRARED SPECYFIC DETECTORS FOR ETHANOL

ANALYTICAL METHODS OF ETHANOL DETERMINATION cont.

Oxygen Ethanol, carbon dioxide

Vessel-pulmonary membrane

•PERSONAL (ENSURE) – No specyfic, only for drivers autocontrol, may be used also as screening test

•PROFFESSIONAL (Police) – different type of detectors

ALCOMAT (Drager), ALCOTEST 7110, ALCOMETR A.2.0

(estimation of infra-red abssorbance)

ALCOTEST 7310,7410,ALERT, AVAT IV (electrochemical)

ANALYTICAL METHODS OF ETHANOL DETERMINATION cont.

Aldehyde and acid acetic, carbon dioxide

•GAS CHROMATOGRAPHY (GC) the best procedure: specyfic, fast, multi-detectable, sensitive

•GC-MS, GC headspace – mostly use at present

•SPECTROMETRIC/ENZYMATIC ( applied ADH for estimation of ethanol) measurement with UV/VIS spectrometers

•IMMUNOENZYMATIC (FPIA, EMIT) ABBOT, VIVA

Laboratory tube with blood, urine, stomach contents...

ETHANOL +Oxidation agentor enzymes