Two Fatalities Involving Two Fatalities Involving Substituted Phenols:Substituted Phenols:

Dinitrophenol and DisophenolDinitrophenol and Disophenol

Ray Gary Ray Gary San Diego County Medical ExaminerSan Diego County Medical Examiner

2,42,4--DinitrophenolDinitrophenol(DNP)(DNP)

What is DNP?What is DNP?Yellow crystalline solid; molecular weight = 184.11 Yellow crystalline solid; molecular weight = 184.11

Used commercially in the production of dyes, wood Used commercially in the production of dyes, wood preservatives, photographic developers, explosives and preservatives, photographic developers, explosives and pesticides.pesticides.

In the 1930’s DNP was used extensively as a diet pill In the 1930’s DNP was used extensively as a diet pill due to its ability to increase basal metabolic rate.due to its ability to increase basal metabolic rate.

Noted side effects include: increased sweating, Noted side effects include: increased sweating, increased heart rate, weight loss, and a marked increase increased heart rate, weight loss, and a marked increase in body temperature.in body temperature.

How does DNP work?How does DNP work?

UncouplerUncoupler of oxidative of oxidative phosphorylationphosphorylation

Inhibits the synthesis of ATP in mitochondriaInhibits the synthesis of ATP in mitochondria

ATP is burned at a rapid rateATP is burned at a rapid rate

Body temperature increases dramaticallyBody temperature increases dramatically

Fat is burned as energyFat is burned as energy

2,42,4--DNP effects on humansDNP effects on humans

Reproductive damageReproductive damageSkin / eye irritationSkin / eye irritationNose / throat irritationNose / throat irritationLiver and Kidney damageLiver and Kidney damageCardiac disturbancesCardiac disturbancesDigestive and respiratory tract irritationDigestive and respiratory tract irritationCauses cataractsCauses cataractsDeathDeath

DNP throughout historyDNP throughout historyEarly 1900’s, European and American scientists found Early 1900’s, European and American scientists found that DNP caused a rise in body temp in lab animalsthat DNP caused a rise in body temp in lab animalsWWI, French munitions workers experienced weight loss WWI, French munitions workers experienced weight loss from absorption through skinfrom absorption through skinWWII, Hitler used DNP to keep prisoners warm during WWII, Hitler used DNP to keep prisoners warm during winter without the need for heating the buildingswinter without the need for heating the buildings1933, Stanford University study declared that 2,41933, Stanford University study declared that 2,4--DNP DNP could cause amazing weight losscould cause amazing weight loss1938, DNP use for the treatment of obesity was banned 1938, DNP use for the treatment of obesity was banned by the FDAby the FDA

DNP popularity in the 1930’sDNP popularity in the 1930’s

Stanford University releases weight loss findings in Stanford University releases weight loss findings in 19331933

~100,000 people took~100,000 people took DNP during the next 15 monthsDNP during the next 15 monthsMost people lost an average of 1Most people lost an average of 1--2.5 lbs/week with no 2.5 lbs/week with no change in diet or exercisechange in diet or exerciseMany reports of adverse reactions, including cataracts Many reports of adverse reactions, including cataracts and deathand death1935 epidemic of cataracts began to occur 1935 epidemic of cataracts began to occur –– only in only in womenwomen

DNP and metabolic rateDNP and metabolic rateDoseDose Avg. increaseAvg. increase

(mg/kg/day)(mg/kg/day) Basal Metabolic Rate Basal Metabolic Rate 1.21.2 13 %13 %2.3 2.3 20 %20 %3.53.5 31 %31 %4.74.7 50 %50 %5.95.9 56 %56 %

*One individual had 82 % increase in BMR after taking *One individual had 82 % increase in BMR after taking 3.3 mg/kg/day for 182 days.3.3 mg/kg/day for 182 days.

DNP and metabolic rate cont.DNP and metabolic rate cont.

Another study found that 3Another study found that 3--5 mg/kg/day 5 mg/kg/day resulted in raising metabolism 25resulted in raising metabolism 25--30 %30 %

Could lose 20 lbs of fat in 6 weeks Could lose 20 lbs of fat in 6 weeks ––with NO with NO change in diet or exercisechange in diet or exercise

Animal Studies on DNPAnimal Studies on DNP

SpeciesSpecies 100% survival dose100% survival dose 100% lethal dose100% lethal doseratsrats 20 mg/kg20 mg/kg 60 mg/kg60 mg/kgdogsdogs 20 mg/kg20 mg/kg 30 mg/kg30 mg/kg

2,42,4--DNP deathsDNP deaths

1933 San Francisco newspaper reported the 1933 San Francisco newspaper reported the death of a Vietnamese Dr. who overdosed on death of a Vietnamese Dr. who overdosed on DNPDNP

Body temperature was 110 ºFBody temperature was 110 ºF

2,42,4--DNP deaths cont.DNP deaths cont.

80 kg male took 46 mg/kg followed by second 80 kg male took 46 mg/kg followed by second dose one week laterdose one week later

Temp of 105.7 Temp of 105.7 ººFFPulse of 146 Pulse of 146 bpmbpmDeath occurred 11 hours after ingestionDeath occurred 11 hours after ingestionAutopsy showed signs similar to heatstrokeAutopsy showed signs similar to heatstrokeRigor mortis set in 10 min. after deathRigor mortis set in 10 min. after deathBody temp rose to 115 Body temp rose to 115 ººF after deathF after death

2,42,4--DNP deaths cont.DNP deaths cont.

Woman took 7 mg/kg/day for 5 daysWoman took 7 mg/kg/day for 5 daysAdmitted to hospital in comatose conditionAdmitted to hospital in comatose conditionTemp. of 101.8 Temp. of 101.8 ººFFPulse of 140 Pulse of 140 bpmbpmAutopsy revealed:Autopsy revealed:

Hemorrhagic lungs, spleen and spinal cordHemorrhagic lungs, spleen and spinal cordDegeneration of renal tubules and liver cellsDegeneration of renal tubules and liver cellsSegmentation and fragmentation of cardiac Segmentation and fragmentation of cardiac musclesmuscles

2,42,4--DNP deaths cont.DNP deaths cont.

Woman took 2.66 mg/kg/day for 14 daysWoman took 2.66 mg/kg/day for 14 daysTemp. of 102 Temp. of 102 ººFFPulse of 148 Pulse of 148 bpmbpmBecame comatose and diedBecame comatose and died

Teenage girl took 1.0 mg/kg/day for 46 daysTeenage girl took 1.0 mg/kg/day for 46 daysTemp. of 105.6 Temp. of 105.6 ººFFHospitalization and then deathHospitalization and then death

2,42,4--DNP deaths cont.DNP deaths cont.

17 yr. old female (75 kg)17 yr. old female (75 kg)Ingested 12Ingested 12--15 “diet pills” in suicide attempt15 “diet pills” in suicide attemptTemp. at admission was 98.8 ºFTemp. at admission was 98.8 ºFPulse of 150 Pulse of 150 bpmbpmTemp. rose to 104 ºFTemp. rose to 104 ºFNumerous attempts to lower body tempNumerous attempts to lower body temp

Cold compresses, ↑ AC, acetaminophen, Cold compresses, ↑ AC, acetaminophen, benzosbenzos

Died ~6 hours after admissionDied ~6 hours after admissionPostmortem serum DNP = 315 mg/LPostmortem serum DNP = 315 mg/L

Review of DNP deathsReview of DNP deaths

Deaths occurred in people who ingested 3Deaths occurred in people who ingested 3--46 46 mg/kg/day for short periodsmg/kg/day for short periods

Deaths occurred in people who ingested 1Deaths occurred in people who ingested 1--4 4 mg/kg/day for longer periodsmg/kg/day for longer periods

No deaths reported in studies where subjects No deaths reported in studies where subjects were given oral doses were given oral doses << 1.3 mg/kg/day for 1.3 mg/kg/day for <14 days<14 days

Bodybuilders and 2,4Bodybuilders and 2,4--DNPDNP

DNP regarded as “Mother of all fat burners”DNP regarded as “Mother of all fat burners”“Most effective tool available today for the loss of “Most effective tool available today for the loss of body fat”body fat”Can be purchased online ($7/200 mg or $10/300 mg)Can be purchased online ($7/200 mg or $10/300 mg)Recommendations when taking DNP include:Recommendations when taking DNP include:

Sleep with fan pointed towards head at night Sleep with fan pointed towards head at night Wear light colored / loose fitting clothingWear light colored / loose fitting clothingWash bedding daily because of profuse sweatingWash bedding daily because of profuse sweatingConsume lots of water (up to 8 liters per day)Consume lots of water (up to 8 liters per day)Supplements are essential to counter negative Supplements are essential to counter negative effects of DNPeffects of DNP

How does DNP stack up?How does DNP stack up?

Weight loss agentWeight loss agent boost in metabolismboost in metabolism

ECA Stack (ephedrine,ECA Stack (ephedrine, 3 %3 %caffeine, aspirin)caffeine, aspirin)

ClenbuterolClenbuterol / / CytomelCytomel StackStack 55--10 %10 %

2,42,4--DinitrophenolDinitrophenol 50 %50 %

CircumstancesCircumstances

28 year old male college student (6 ft, 212 lbs)28 year old male college student (6 ft, 212 lbs)Found with fever, laying in bathtub full of iceFound with fever, laying in bathtub full of iceTaken to fire dept. then transported to hospitalTaken to fire dept. then transported to hospitalAdmitted to hospital suffering from confusion, Admitted to hospital suffering from confusion, hyperthermia and muscle rigidityhyperthermia and muscle rigiditySubject was conscious upon admission, but died Subject was conscious upon admission, but died soon aftersoon after

CircumstancesCircumstances cont.cont.

Deceased had eaten clam chowder despite Deceased had eaten clam chowder despite shellfish allergiesshellfish allergies

Roommate stated deceased was using Roommate stated deceased was using bodybuilding supplementsbodybuilding supplements

Medication bottles, syringes and yellow powder Medication bottles, syringes and yellow powder turned over to Medical Examiner’s Officeturned over to Medical Examiner’s Office

Supplements and steroids were from MexicoSupplements and steroids were from Mexico

Hospital AdmissionHospital AdmissionBlood sugar Blood sugar 123123PulsePulse 140140Blood pressure Blood pressure 180/20180/20WBCsWBCs 15,80015,800Platelets Platelets 5454PotassiumPotassium 11.211.2SGOTSGOT 624,624, SGPTSGPT 713713Hospital Tox screenHospital Tox screen NegativeNegativeTemperatureTemperature 106ºF106ºF

Autopsy findingsAutopsy findings

JaundicedJaundicedLeft ventricular hypertrophy of 1.6 cmLeft ventricular hypertrophy of 1.6 cmMulti organ congestion Multi organ congestion Severe pulmonary edemaSevere pulmonary edemaSevere cerebral edemaSevere cerebral edemaHepatic necrosisHepatic necrosis150 150 mlsmls blood in stomach, hemorrhagic gastritisblood in stomach, hemorrhagic gastritisEvidence of CPR (fractured ribs and sternum)Evidence of CPR (fractured ribs and sternum)

ToxicologyToxicology

Routine Tox screening included:Routine Tox screening included:AlcoholsAlcohols GCGC--FIDFIDDrugs of AbuseDrugs of Abuse ELISAELISABasic drug screenBasic drug screen GC/MSGC/MSAcid/Neutral screenAcid/Neutral screen HPLCHPLC--DADDADUrine drug screenUrine drug screen ToxiToxi--LabLab

**antemortemantemortem blood samples used where possibleblood samples used where possible

ToxicologyToxicology resultsresultsAlcoholsAlcohols NegativeNegativeDrugs of AbuseDrugs of Abuse NegativeNegative

Basic drug screenBasic drug screen DiphenhydramineDiphenhydramine 0.13 mg/L0.13 mg/L

LidocaineLidocaine <0.05 mg/L<0.05 mg/LAcid/Neutral screenAcid/Neutral screen Unknown peakUnknown peakUrine drug screenUrine drug screen Unknown spot in acid extractUnknown spot in acid extract

DiphenhydramineDiphenhydramineYellow powder identified by GC/MS as:Yellow powder identified by GC/MS as:2,42,4--Dinitrophenol (DNP)Dinitrophenol (DNP)

2,42,4--Dinitrophenol ExtractionDinitrophenol Extraction

500 500 µµl specimen (calibrator, control, or sample)l specimen (calibrator, control, or sample)100 100 µµl internal standard solution (l internal standard solution (phenobarbphenobarb))500 500 µµl of 1 N HCl and vortexl of 1 N HCl and vortex8 8 mlsmls 11--chlorobutane and mix for 30 min.chlorobutane and mix for 30 min.Centrifuge and decant into fresh tubesCentrifuge and decant into fresh tubesEvaporate at 37ºCEvaporate at 37ºCReconstitute with 200 Reconstitute with 200 µµl mobile phase (0.031 M l mobile phase (0.031 M Pot. Phosphate buffer / Pot. Phosphate buffer / acetonitrileacetonitrile; 85:15); 85:15)Inject onto HPLC columnInject onto HPLC column

HPLC instrument parametersHPLC instrument parameters

2,42,4--Dinitrophenol by HPLCDinitrophenol by HPLC

2,42,4--DNP by HPLCDNP by HPLC--DADDAD

2,42,4--DNP concentrationsDNP concentrations

AM bloodAM blood 28 mg/L28 mg/LPeripheral bloodPeripheral blood 31 mg/L31 mg/LPeripheral serumPeripheral serum 29 mg/L29 mg/LGastric contentsGastric contents 850 mg850 mgUrineUrine 53 mg/L53 mg/LVitreousVitreous 3.4 mg/L3.4 mg/LBileBile presentpresentLiverLiver presentpresent

ConclusionsConclusionsDNPDNP popularity is growingpopularity is growing

Attractive to individuals who wish to drop weight Attractive to individuals who wish to drop weight with NO change in diet or exercise with NO change in diet or exercise –– “magic pill”“magic pill”

Very dangerousVery dangerousNo antidoteNo antidoteNarrow therapeutic windowNarrow therapeutic windowCan be bought online without prescription despite Can be bought online without prescription despite

FDA regulationsFDA regulations

DisophenolDisophenol(2,6(2,6--diiododiiodo--44--nitrophenol)nitrophenol)

What is Disophenol?What is Disophenol?

2,62,6--diiododiiodo--44--nitrophenol; MW = 391nitrophenol; MW = 391AnthelminticAnthelmintic used to treat parasitic infestations used to treat parasitic infestations in dogs and livestock in dogs and livestock Similar in structure and action to 2,4Similar in structure and action to 2,4--DNPDNPUncouplerUncoupler of oxidative of oxidative phosphorylationphosphorylationNot approved by FDA for human consumptionNot approved by FDA for human consumptionNo known cases of human fatality involving No known cases of human fatality involving Disophenol until nowDisophenol until now

2,4-Dinitrophenol

DNP

2,6-Diiodo-4-Nitrophenol

Disophenol

CircumstancesCircumstances

61 year old Caucasian female61 year old Caucasian femaleAdmitted to hospital with complaints of Admitted to hospital with complaints of vomiting, diarrhea, dizziness and joint painvomiting, diarrhea, dizziness and joint painDecedent left ER but was convinced to return Decedent left ER but was convinced to return by hospital staffby hospital staffNurses noted rapid rise in body temperatureNurses noted rapid rise in body temperatureCooling measures were unsuccessfulCooling measures were unsuccessfulCardiac arrest and death ~9 hours after Cardiac arrest and death ~9 hours after admissionadmissionDecedent exhibited rigor mortis type stiffness Decedent exhibited rigor mortis type stiffness within minutes of cardiac arrestwithin minutes of cardiac arrest

Circumstances cont.Circumstances cont.

Self diagnosed Self diagnosed GongylonemaGongylonema infestationinfestationParasitic infestation of oral mucosaParasitic infestation of oral mucosaVery uncommon in humansVery uncommon in humansDecedent may have been delusionalDecedent may have been delusionalClaims to have identified 3 mating pairs of wormsClaims to have identified 3 mating pairs of wormsNo evidence of infestationNo evidence of infestationBiopsy and postmortem examination both negativeBiopsy and postmortem examination both negative

Ingested Disophenol (2,6Ingested Disophenol (2,6--diiododiiodo--44--nitrophenol)nitrophenol)AnthelminticAnthelmintic no longer available in U.S.no longer available in U.S.Used to treat hookworms in caninesUsed to treat hookworms in canines

Disophenol “Prescription”Disophenol “Prescription”

After 2 years and multiple After 2 years and multiple M.D.sM.D.s, decedent , decedent found someone to prescribe Disophenol even found someone to prescribe Disophenol even though it’s not available in the U.S.though it’s not available in the U.S.“R“Rxx”: take five 100 mg capsules, repeat dose ”: take five 100 mg capsules, repeat dose one week laterone week laterCapsules from Mexico: actually contained 500 Capsules from Mexico: actually contained 500 mg eachmg eachDecedent ingested five 500 mg capsules (5 X Decedent ingested five 500 mg capsules (5 X recommended dose)recommended dose)

Autopsy resultsAutopsy results

Pulmonary congestion and edemaPulmonary congestion and edemaHepatic necrosisHepatic necrosisNo gross evidence of parasitic infectionNo gross evidence of parasitic infectionNo microscopic evidence of parasitic infectionNo microscopic evidence of parasitic infectionMild upper gastrointestinal hemorrhageMild upper gastrointestinal hemorrhage

ToxicologyToxicology

Routine Tox screening included:Routine Tox screening included:AlcoholsAlcohols GCGC--FIDFIDDrugs of AbuseDrugs of Abuse ELISAELISABasic drug screenBasic drug screen GC/MSGC/MSAcid/Neutral screenAcid/Neutral screen HPLCHPLC--DADDAD

**antemortemantemortem blood samples used for all screensblood samples used for all screens

ToxicologyToxicology resultsresults

AlcoholsAlcohols NegativeNegativeDrugs of AbuseDrugs of Abuse NegativeNegativeBasic drug screenBasic drug screen NegativeNegativeAcid/Neutral screenAcid/Neutral screen AcetaminophenAcetaminophen 7.7 mg/L7.7 mg/L

Unknown peak*Unknown peak*

Blue/white capsule identified by GC/MS as:Blue/white capsule identified by GC/MS as:Disophenol (2,6Disophenol (2,6--diiododiiodo--44--nitrophenol)nitrophenol)

*Unknown peak in Acid/Neutral screen later identified as*Unknown peak in Acid/Neutral screen later identified asDisophenolDisophenol

Disophenol extractionDisophenol extraction

0.25 0.25 mlsmls sample, calibrator or control into sample, calibrator or control into microtubesmicrotubes0.50 0.50 mlsmls extraction solvent (extraction solvent (acetonitrileacetonitrile + + MPPH internal standard)MPPH internal standard)Let stand 10 minutesLet stand 10 minutesCentrifuge at high speedCentrifuge at high speedTransfer supernatant to Transfer supernatant to autosamplerautosampler vialsvialsInject on HPLCInject on HPLC

HPLC Instrument Parameters

HPLC Calibration Curve

Disophenol by HPLC-DAD

Disophenol quantitationDisophenol quantitation

AM blood (~8 hrs before death)AM blood (~8 hrs before death) 564 mg/L564 mg/LAM blood (~1.5 hrs before death)AM blood (~1.5 hrs before death) 296 mg/L296 mg/LPeripheral bloodPeripheral blood 138 mg/L138 mg/LCentral bloodCentral blood 126 mg/L126 mg/LBileBile 36 mg/L36 mg/LLiverLiver 97 mg/L97 mg/LVitreousVitreous 5.3 mg/L5.3 mg/L

ConclusionsConclusions

Cause of death:Cause of death: acute acute disophenoldisophenolintoxicationintoxication

Manner of death:Manner of death: accidentaccident

Significance:Significance: Only known case of Only known case of disophenoldisophenol fatality in humanfatality in human