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Jonathan Popovitch's Log Jonathan Popovitch Assignment




Jonathan Popovitch Chemistry Information Retrieval December 4, 2009 

Absinthe has been viewed as a toxic beverage that induces hallucinations, and in some cases cause people to cut their own ears off. With a very popular artist, Vincent van Gogh, this was the case. Absinthe is liquor that is distilled, and then infused with various herbs and spices to give its traditional taste and green color. Absinthe was originally begun as a wine being infused with wormwood, which is what gives the beverage the possible hallucinogenic properties; this began in as early as 500 BCE [13]. By the early 1700’s the absinthe trend caught on in Europe. Manufacturers began bottling their product and distributing it throughout France, Spain, Germany, and other countries. By the early 1900’s the American legislature noticed the effects of absinthe and labeled it as an illegal drug/beverage. This was all due to the speculation that thujone acted much like marijuana, only being ingested instead of being inhaled. The law was passed in 1912, to make it officially banned in the United States [13].

The way that customers indulged in this beverage was by taking 1 portion of the green liquor, and then a slotted spoon was placed over top of the beverage with a sugar cube placed in the center of the spoon. Water was then dripped on top of the sugar cube in order to dissolve it, the amount of water was roughly 5 portions of water to 1 portion of absinthe. This then turned the green beverage into a while milky solution, which is known as the ouzo effect [20]. Adding the sugar water to the beverage was to sweeten up the taste of the very harsh high alcohol taste of the drink, the water also caused some of the alcohol soluble ingredients to precipitate out; for example anise, star anise, and fennel [19]. Some people believed that copper and antimony were added in order to enhance the color. There are different styles of absinthe that an individual could choose from for example;  The controversial ingredient in the beverage is the wormwood. The Artemisia species all have very sharp, and thin leaves, the plant its self looks like a shrub or weed; they also belong to the same family as the flower dasies. The plant can grow as tall as five feet, and grow in both the northern and southern hemispheres [16]. Although there are many different species of wormwood (Artemisia absinthum, Artemisia, pontica, Artemisia alba, Artemisia californica, ect.) the main species used is the Artemisia absinthium (Grand wormwood) due to its potent bittersweet aroma, as well as its thujone content [23]. Some species of wormwood have little to no thujone content, and are sometimes used to create a less powerful tasting/effective product. Historically absinthium has been used as a pain reliever for gastric pain, assist with fever reduction, used as an antiseptic, and has been made into teas to help pregnant women with their discomfort [21]. Recently the extract from the plant has been used to make a nonalcoholic beverage with very low thujone levels, which was removed by a chemical process. In the past few years absinthe has began to gain popularity once again, which is proven through the increasing sales through the internet. Historically absinthe drinkers were very artistic, whether it was an artist, poets, writers, or musicians. Many younger people seek that “green fairy” hallucination; the mystery behind the beverage is also enticing. Since the beverage contains a slight stimulant there is a derivation between the sensations of being intoxicated from the alcohol, and enlightened from the chemical reaction. Since the chemical reaction causes the sensation of excitement, and the alcohol is a depressant and sedative, it is a comparable bodily reaction to alcohol and caffeine.[4] Nowadays the trend is catching back on with younger adults. Surprisingly enough the individuals experimenting with the beverage are among the same groups that drank absinthe close to 200 years ago [19].
 * Blanche- which was a clear liquor and did not have that signature green color
 *  Verte -this was the traditional “green” drink everyone knows, it had an additional step after being distilled, herbs were steeped in to give the color and flavor
 * Absenta- the Spanish version of absinthe, it had a sweeter taste to it than the Verte
 * Hausgemacht absinthe- German home-made, lacked the quality of the professionally made beverages
 * Bohemian absinth- this was absinthe minus the anise, fennel or other herbs
 * Red absinthe- this is normal Verte absinthe, but instead of being steeped with the normal mixture of herbs to give the green color, these herbs turn it red [20] [19] [16]

Thujone is a ketone in which can form two different stereoisometric structures; the α-thujone and the β-thujone. In the course of chemical testing on GABA receptors, it was determined that α-thujone is 2.3 times more active than β-thujone. The oil is known to the world as the psychedelic “drug” that is associated with absinthe [22]. It was speculated in the past that thujone had a pharmacological effect on the central nervous system by the thujone activating the CB1 canabinoid receptor [18] [14], but that is not a fact. Cannabinoids, like tetrahydracannabinol (THC) found in marijuana, effect the central nervous system and depressants, which was mistaken as having the same reaction as absinthe. The ketone is a GABA inhibitor, which means that it slows or stops activity to the neurotransmitter gamma-aminobutyric acid [2] receptor chloride channel blocker. This behavior follows the pattern of picrotoxin, which is also isolated from plants, these acts as a convulsant. Thujone is considered to be convulsant, due to extremely high doses causing seizures. The most active areas in the human body that metabolize the chemical are in the brain as well as the liver. When individuals used to indulge in the alcohol they reported in having an up-beat sensation, this is due to the thujone being an analepic stimulant (mood elevator and antidepressant) much like picrotoxin. [15]

In high doses in laboratory animals (guinea pigs) convulsions began at concentrations far beyond and physical reach. In the past there have been concentrations studies to determine the “convulsion dose” of thujone, in a rat the CD was 100mg/kg by body weight [16]. For a man who weighed 155 pounds that would mean that he would have to ingest about 7.03 grams of thujone. Considering that the average beverage is 21mg/L, that individual would have to consume about 335 Liters of a high percentage alcohol. That man would die much sooner from alcohol poisoning than have a convulsion from the thujone content in absinthe. This data of the convulsion dose is very outdated. Recently (2003) there was a toxicology study to determine the LD50. The study in mice showed that with 30mg/kg there were no deaths that occurred, at 60mg/kg every mouse subject died, so the LD50 was the midpoint ironically at 45mg/kg [18]. Before the mice died, they would convulse within 2 minutes quickly leading to death. The group conducting the research noticed that there was an increase in the LD50 when the mouse was given ethanol prior to the thujone treating; the lethal dose then was back up to 100mg/kg.

Thujone has been proven to have convulsant properties, and many insecticides show the same properties. Theoretically since thujone has many of the similar properties it can be considered an insecticide. With any insecticide it may have harmful properties to many different body parts in different species. In humans like previously stated thujone is metabolized in the liver, more specifically the microsome NADPH systems, as well as in the in the brain. There is also an interaction between the thujone and the cytochrome P450, which causes oxidation. “Three different P450 inhibitors block the metabolism of α- and β-thujones” [6]. The P450 system is able to metabolise the α-thujone into 7-hydroxy-α-thujone, along with the stereo isomer 4-hydroxy-α-thujone and 4-hydroxy-β-thujone. While testing rats, it was determined that a major metabolite was 4-hydroxy-α-thujone and 4-hydroxy-β-thujone in the microsome-NADPH system, where as for the P450 system only the 4-hydroxy-α-thujone was present[6].

In 2007 a Louisianan scientist by the name of Ted Breaux, argued that the side effects of absinthe could be related to the concentration of thujone. Ted also assisted in determining the content of thujone in absinthe[22]. The procedure that is now commonly used is to put the absinthe through a Gas Chromatograph-Mass spectrometer in order to detect both steroisomers of thujone, this method is that the Alcohol and Tobacco Tax and Trade Bureau use[1]. In order to prepare a sample standard, pure mixture of α-thujone, methanol, and cyclododecanone are used all in known concentrations [10].

In order to determine what the concentrations of thujone was the past absinthe, many historical samples were obtained from numerous distilleries around Europe. For the bottles to be able to be tested they must first pass a series of 9 qualification tests:  Each sample was subjected to the same preparation in order to be given consistent results. The thujone was extracted by a liquid-liquid extraction using 1,1,2-trichloro-1,2,2-trifluoroethane, then a known concentration of the cyclodecanone is added and shaken for 1 min. This mixture is then centrifuged for 5 mins at 3000 rotations per min, finally the lower organic layer is drawn off and ready for the GC-MS. The GC-MS used a helium gas carrier at a flow rate of 1.0ml/min. When the mass spectrum was taken it was in the style of EI (electron impact). The retention time for the α-thujone was 17.2mins, where as the β-thujone came out at 17.7mins, the cyclodecanone emerged at 25.1 mins. [10] . This method is also used to determine other materials that could be present in the absinthe; such as fenchone, pinocamphone, methanol, copper, and antimony [11]. Fenchone is sometimes found in absinthe due to impurities in the wormwood being used, and is supposed to be regulated at less than 5mg/L, which is strange due to the LD­­50 in mice being 6160mg/kg. Pinocamphone, has an isomer of isopinocamphone which is a powerful convulsant and a lethal dose of 0.05ml/kg, so it is understandable that the French government wants to keep the pinocamphone concentrations under 20mg/L. Methanol can contaminate the absinthe during which ever process the manufacturer uses to produce their alcohol, but during distillation since it has a lower boiling point than ethanol, it would boil off first. Copper and antimony have been suspected of being added to the beverage to give the tint of color that people are looking for, and inorganic salts can be harmful in large amounts [11].
 * Full sized bottles of 1 liter or full half-sized bottle 0.5 liter
 * Sealed from the original manufacturer
 * Cork was tested for consistent with age of bottle
 * No signs of tampering, and exposed to low light or heat
 * <span class="wiki_link"> Origins of each bottle is known (wine cellar or storehouse)
 * <span class="wiki_link">Original labels still on bottle
 * <span class="wiki_link">The cork did not disintegrate and still had air tight seal
 * <span class="wiki_link">Content was consistent with vintage absinthe
 * <span class="wiki_link">All brands tested were the most popular brand, with the highest quality [11].

After the ban was lifted in Europe in 1988, one thing came into question, does the new absinthe still have the same concentration of the “hallucinogen”? According to historical documents the concentration of vintage absinthes was 260mg/L of thujone [5]. The problem with this reported value is that it is theoretically calculated with no data to back it up, also at the time of this calculation there was no data to disprove it. When the Gas Chromatography-Mass Spectroscopy method above was implemented the true concentrations of thujone in absinthe was revealed. Many samples of vintage absinthes were taken from all over Europe that followed the nine restrictions above, as well as many of the modern day absinthes so they could be compared. A total of 35 samples were taken, twelve of which have been made in the past ten years. The average thujone content in the vintage drinks was 25.4 mg/L, and out of all the samples only six of the pre-banned bottles and one modern bottle had a higher content than the restriction of 35mg/L [11]. The thujone concentration varied greatly though out the distillation process, the drink may have started with an initial concentration of around 260mg/L, however after numerous distillations the resulting concentrations was more realistically around 21mg/L[8]. The extremely high value from history was not done through testing or sampling, it was originally done by mathematicians, after testing only a very select few of the vintage absinthes had a higher thujone content between 60-101 mg/L, which is much below the imaginary number of the past[9]. Some factors that may have affected the concentration of thujone in the absinthe could have been; <span class="wiki_link"> Some skeptics may say, “What about Ultra Violet contamination? It spoils beer and other alcohols why wouldn’t it degrade the absinthe over time?” there has been a study to answer that exact question. There were two separate methods that the group looked at it; Ultra Violet irradiation of the alcohol in both clear and green bottles, and test absinthes made from 2001-2005 and see if their content is still consistent with the day they were bottled. When testing by the UV method, a clear bottle, and a vintage green bottle, was filled with the same amount of absinthe. Each was subjected to the same amount of UV light for 10, 20, 40, 80, and 200 hours being re-tested through the GC-MS at each time interval. The results from that test showed that the α-thujone was barely affected where as the β-thujone was degraded by close to 18% in the clear bottle after 200 hours. After about 20 hours into the test the group noticed that the absinthe was losing its green tint, and by 60 hours the solution was clear and colorless, they hypothesized that this was due to the antioxidants present in the liquor, along with the chlorophyll that was more than likely responsible for its green color. The absinthe was also subjected to pH testing to determine when the thujone degrades or converts to the other steroisometric structure. At the pH’s of 2.5 and 6.5 no reaction occurred, but at the pH of 11.5 the α-thujone epimerized into the β-thujone, resulting in close to twice as much β-thujone as original. Temperature was also tested, and no change was noticed between 0­oC and 100oC. The samples collected and stores from 2001-2005, showed no change whatsoever. [12]
 * <span class="wiki_link">Variation in the concentration of oil in the wormwood
 * <span class="wiki_link">Drying time
 * <span class="wiki_link">Distillation equipment
 * <span class="wiki_link">Standardizing something that cannot be tasted [7]

There are many myths surrounding absinthe, most of which have already been disproven. Absinthism is an infamous “medical” syndrome used to describe a person who drinks an enormous amount of absinthe. Some of the symptoms are sleeplessness, termers, convulsions, nightmares, and the most famous hallucinations [19]. It was never known whether it was the absinthe that causes theses symptoms or the alcohol that is in the beverage. Since absinthe is a high alcohol content drink (can be 75% or higher which means 150 proof alcohol) people hear there is a possible toxin/hallucinogen, they look right to the thujone for the symptoms [13]. In the 19th century some tests were implemented to see the acute and chronic effects of too much absinthe. Some acute effects included: vertigo, seizures, nervous debility, and hallucinatory delirium. The chronic use resulted in the following: mania, softening of the brain, general paralysis, and psychosis. Most of the symptoms are the same as alcoholism, so the tests were inconclusive. A French scientist named Magnan began animal testing with pure wormwood extract and pure alcohol. His tests consisted of injecting the oil directly into the stomachs of animals to see the effects, as well as trapping animals in a chamber with vaporized extract, the outcome was that the wormwood will in fact cause you to have seizures and die. Even in the 19th century these tests were considered cruel and un-realistic.[17] In modern times there are more sophisticated methods of determining a chemicals toxicity. The bitter taste of the wormwood extract activated the taste receptor, and it is sensitive enough to assist in protection against taking in a toxic amount [17], this is only if you are a sober person who can realize what it is you taste. In the 1800’s there was a case of a man who drank the wormwood extract in its pure form, and became deathly ill, with the symptoms of foaming at the mouth and convulsions. A similar case occurred when a man drank aroma therapeutic oils in attempt to hallucinate. In each drink of absinthe there is a negligible amount of thujone to even show signs of acute pharmacological symptoms [17].

A behavioral study was done with 25 people ranging in age from 19 to 58 years old. Each subject would have three absinthe drinks to choose from; no α-thujone, 10mg/L, and 100mg/L. The individuals would then consume three of their drinks (all drinks had the same alcohol content), after they finished their drinks they began to get tested on their attentiveness. Their attention was tested by a series of vision tests, which doubled as a reaction time test, followed by a questionnaire that would determine the persons mood change. The results show that in the vision test the contestants that had the higher thujone content beverages had more false reactions than the lower content and no thujone participants. However the results show that the reaction time of the people who had the high thujone content to be the highest, but the lower content group actually decreased their reaction time to lower than the no thujone group. [3]

In conclusion, absinthe is a misunderstood alcoholic beverage. Absinthe is made from Artemisia absinthium (wormwood) and does contain α and β thujone, which in high doses by its self can cause seizures and possibly death. The drink is a very high alcohol proof spirit, and has all of the side effects that a normal alcohol has with it, that means dizziness, vomiting, seizures, hallucinations, and a bad headache in the morning.

References [1]Alcohol and Tobacco Tax and Trade Bureau. (2007). Screening of distilled spirits for thujone by gas chromatography-mass spectrometry.http://www.wormwoodsociety.org/index.php?option=com_content&task=view&id=258&Itemid=215 [2]Bielenberg, J. (2007). Thujone. Medizinische Monatsschrift Fuer Pharmazeuten, 30(9), 322. ISSN/ISBN: 0342-9601 http://www.ncbi.nlm.nih.gov/pubmed/17912872 [3]Dettling, A., Skopp, G., Schuff, A., & Grass, H. (2004). Absinthe: Attention performance and mood under the influence of thujone. Journals of Studies of Alcohol,, 573. http://www.thujone.info/Performance-Mood.pdf [4]Gambelunghe, C., & Melai, P. (2002). Absinthe: Enjoying a new popolarity among young people? Forensic Science International, 130(2-3), 183-186. doi:DOI: 10.1016/S0379-0738(02)00374-2 [5]Gimpel, M., Hoenersch, Y., Altmann, H., Wittowski, R., & Fauhl-Hassek, C. (2006). Absinthe: Thujone content of absinthe spirits using historical recipes. Deutsche Lebensmittel-Rundshau, 102(10), 457. http://www.cababstractsplus.org/abstracts/Abstract.aspx?AcNo=20063216487 [6]Höld, K., Sirisoma, N., & Casida, J. (2001). Detoxification of α- and β-thujones (the active ingredients of absinthe): Site specificity and species differences in cytochrome P450 oxidation in vitro and in vivo. Journal of Agricultural and Food Chemistry, (14), 589. doi:10.1021/tx000242c [7]Lachenmeier, D. (2007). Absinthe - history of dependence to thujone or to alcohol?(75), 306. doi:10.1055/s-2007-959210 [8]Lachenmeier, D., & Kuballa, T. (2007). Behaviour of thujone during distillation and possible concentration ranges in pre-ban absinthe. Journal of the Science of Food and Agriculture, 87(11), 2147. doi:10.1002/jsfa.2978 [9]Lachenmeier, D., & Nathan-Maister, D. (2007). Systematic misinformation about thujone in pre-ban absinthe.6(103), 255. http://www.erowid.org/references/refs_view.php?A=ShowDocPartFrame&ID=7110&DocPartID=6451 [10]Lachenmeier, D. W., Emmert, J., Kuballa, T., & Sartor, G. (2006). Thujone—Cause of absinthism? Forensic Science International, 158(1), 1-8. doi:DOI: 10.1016/j.forsciint.2005.04.010 [11]Lachenmeier, D. W., Nathan-Maister, D., Breaux, T., Kuballa, T., Sohinus, E., & Schoeberl, K. (2008). Chemical composition of vintage preban absinthe with special reference to thujone, fenchone, pinocamphone, methanol, copper, and antimony concentrations. Journal of Agricultural and Food Chemistry, 56, 3073. doi:10.1021/jf703568f [12]Lachenmeier, D. W., Nathan-Maister, D., Kuballa, T., & Breaux, T. (2009). Long-term stability of thujone, fenchone, and pinocamphone in vintage preban absinthe. Journal of Agricultural and Food Chemistry, 57, 2782. doi:10.1021/jf803975m [13]Lux, E. F. (2007). The absinthe enigma; resurgence of a legendary spirit. Erowid Extracts,, 12. http://www.erowid.org/chemicals/absinthe/absinthe_writings4.shtml [14]Meschler, J. P., & Howlett, A. C. (1999). Thujone exhibits low affinity for cannabinoid receptors but fails to evoke cannabimimetic responses. Pharmacology Biochemistry and Behavior, 62(3), 473-480. doi:DOI: 10.1016/S0091-3057(98)00195-6 [15]Olsen, R. (2000). Absinthe and gamma-aminobutyric acid receptors. Proceedings of the National Academy of Sciences of the United States of America, 97(9), 4417. http://www.pnas.org/content/97/9/4417.full [16]Oxygenee, L.Accurate, authoritative information about THUJONE, the controversial active ingredient in ABSINTHE. Retrieved November 12, 2009http://thujone.info/ [17]Padosch, S., Lachenmeier, D., & Kroner, L. (2006). Absinthism: A fictitious 19th century syndrome with present impact. Substance Abuse Treatment, Prevention, and Policy, 1(1), 14. doi:DOI:10.1186/1747-597X-1-14 [18]Patocka, J., & Plucar, B. (2003). Pharmacology and toxicology of absinthe. Journal of Applied Biomedicine,, 199. http://journals.indexcopernicus.com/abstracted.php?icid=496438 [19]Robinson, B., & Stone, G.Absinthe education. Retrieved November 12, 2009http://www.wormwoodsociety.org/ [20]Wikipedia.Absinthe. Retrieved October 15, 2009http://en.wikipedia.org/wiki/Absinthe [21]Wikipedia.Artemisia absinthium. Retrieved October 15, 2009http://en.wikipedia.org/wiki/Absinth_Wormwood [22]Wikipedia.Thujone. Retrieved October 15, 2009http://en.wikipedia.org/wiki/Thujone [23]Wormwood. (2009). Retrieved December/1, 2009, from http://www.drugs.com/npp/wormwood.html