Technical Brochure -‐ Tibi Tonic Prepared for BROOKLAWN ENTERPRISES LLC By David C. Clark Ph.D. June 2011 DISCLAIMER: This report (Report) has been produced independently on the request of Brooklawn Enterprises LLC . The views expressed in this Report are not necessarily the views of Brooklawn Enterprises LLC. The information, statements, statistics and commentary (together the ‘Information’) contained in this Report have been prepared by Brooklawn Enterprises LLC from publicly available material and from copyright material for which licenses for internal business use by Brooklawn Enterprises LLC have been purchased. Copies of copyright material linked to the Report cannot be distributed. Brooklawn Enterprises LLC does not express an opinion as to the accuracy or completeness of the information provided, the assumptions made by the parties that provided the information or any conclusions reached by those parties. Brooklawn Enterprises LLC has based this Report on information received or obtained, on the basis that such information is accurate. Brooklawn Enterprises LLC does not accept any responsibility and cannot be held liable for any person’s use of or reliance on the information and opinions contained herein. 1
Biography of the author: David C. Clark Ph.D. David Clark has a B.Sc. in Biochemistry and received his Ph.D. from studies on microtubule proteins at the National Institute for Medical Research in London in 1981. After several postdoctoral research positions in this field, an important target for anti-‐cancer drugs, he moved in 1984 to academic food research specializing in protein-‐stabilized foams and emulsions in a tenured research position at the independent, government-‐supported, Institute of Food Research (IFR) in Norwich, UK. He spent 11 years at IFR in a variety of roles culminating in his appointment as Head of Food Biophysics Department comprising 100+ staff and research students. During the course of his academic career, Dr Clark published more than 100 peer reviewed scientific articles. He held a variety of senior R&D/QA positions in a major food multinational, where he guided the development of several new bioactive ingredients including C12 Peption®, with blood pressure lowering activity and Cysteine Peption®, a protein hydrolysate, enriched in the amino acid cysteine, that exhibited detoxification properties by elevating glutathione levels in the liver. Dr. Clark has presented his work on cysteine at several international conferences and his team won several prestigious awards. A novel cysteine-‐based ingredient developed by his group was a finalist in the Health Ingredients Europe Most Innovative Ingredient competition in 2004. At the end of 2006, Dr. Clark became President and CEO of DMV International Nutritionals in New York, USA, where his responsibilities included development, production and commercialization of nutritional ingredients including protein hydrolysates and bioactives. He has recently founded a consultancy company, Bovina Mountain Consulting LLC based and is engaged in a number of projects providing regulatory, R&D and marketing support to an assortment of clients in Europe and the US.
Executive Summary Although the vast majority of alcohol is consumed in moderation, many individuals who drink in this responsible manner experience a range of post consumption symptoms that are individually or collectively referred to as a hangover. In this technical paper, the science behind the hangover has been reviewed. This subject has attracted many popular articles, although many do not reference the existing peer reviewed, scientific literature on this subject. This oversight has been addressed in this paper. In addition, the scientific basis of a new beverage, Tibi Tonic, is described. Tibi Tonic is formulated not to reduce the level or initial effect of the alcohol consumed but rather to support the body’s natural pathways for processing alcohol and to accelerate the elimination of the toxins that are responsible for hangover symptoms. The process from consumption of alcohol-‐containing beverages to the processing of alcohol by the body and the consequences of moderate to high consumption are reviewed. Perhaps surprisingly, the biochemical basis and cause of hangover symptoms is still not fully understood but leading theories are presented. Some of the medical literature is useful in providing insights into the natural mechanism of alcohol detoxification through execution of well-‐designed, placebo-‐controlled studies. These articles provide some background to the mechanism of action of a number of ingredients that have been carefully selected to create Tibi Tonic, a formulation for the avoidance of hangover symptoms. Tibi Tonic does not affect or reduce the initial pleasurable effect of consumption of alcohol as it does not in any way impede the uptake of alcohol from the gastrointestinal tract into the bloodstream, nor does it prevent the effect of alcohol on the nervous or motor systems. Rather, it assists the body and speeds up the clearing out of the hangover-‐inducing by-‐products of the body’s natural processing of alcohol. Tibi Tonic allows the pleasurable effects of moderate and responsible alcohol consumption to be enjoyed to the full extent, while the consumer can be confident that they will enjoy a restful sleep and wake up refreshed without a hangover. The critical roles of glutathione in alcohol detoxification and other cellular processes are outlined and a section is devoted to explaining the synthesis of glutathione and particularly, the limiting effect of availability of cysteine. The primary mechanism behind the processing of alcohol and toxins produced during its metabolism is explained. In addition, other competing pathways for alcohol metabolism are described. 3
The contributions made by the natural, active ingredients in the Tibi Tonic formulation are explained in depth and their specific beneficial effects may be summarized as follows: •
Supports and restores normal glutathione levels
Supports liver detoxification
Milk Thistle Extract o
Supports liver detoxification
Contains natural antioxidants
Vitamin C o
Regenerates the natural antioxidant, glutathione
Mitigates against tissue damage associated with low levels of glutathione
Vitamin B6 o
Improves sleep quality without affecting daytime sleepiness
Protects against symptoms associated with alcohol-‐induced hangover
Thiamin (Vitamin B1) o
Supports Increased alcohol dehydrogenase activity
There is a wealth of strong, scientific data supporting the effectiveness of N-‐acetylcysteine, including its bioavailability, and conversion into the crucial natural antioxidant, glutathione. The mechanism of action of N-‐acetylcysteine in detoxification is thoroughly understood due to its clinical use in treating overdoses of acetaminophen (the active ingredient in Tylenol®). There have been a number of studies into the effectiveness of Milk Thistle extract in cases of mild ethanol consumption, although the strongest evidence of its efficacy comes from clinical studies relating to chronic liver disease resulting from excessive alcohol consumption or hepatitis B and/or C. The antioxidant properties of milk thistle extract supplement those of glutathione. During the processing of alcohol by the body, highly reactive and damaging oxidizing intermediates are formed. The antioxidant properties of milk thistle extract supports the neutralization of these toxic components. 4
Interrupted or poor quality sleep is often experienced by individuals, who have consumed only limited quantities of alcohol. Evidence supporting the role of glycine, another Tibi Tonic ingredient, in the improvement of subjective sleep quality is presented. Glycine, an amino acid, is a neurotransmitter and suppresses involuntary muscle movement and twitches, which often disturb rapid eye movement (REM) sleep. The benefits of intake of additional vitamins specifically, Vitamin C, Thiamin (Vitamin B1) and Vitamin B6 in the alleviation of hangover symptoms has long been know. The Tibi Tonic formulation includes a proprietary mix of these vitamins. During alcohol processing in the body, glutathione is consumed or is converted to the inactive oxidized form, glutathione disulphide. Vitamin C is a very important factor in the regeneration of active glutathione from its inactive, oxidized form. Thiamin (Vitamin B1) has been implicated in the metabolic processing of acetaldehyde, a toxic by-‐product of the body’s natural processing of alcohol. In addition, the activity of one of the critical enzymes that the body uses to process alcohol, alcohol dehydrogenase is directly linked to Thiamin levels in the body. The mechanism of action of Vitamin B6 in reducing hangover symptoms is not fully understood. However, significant benefits were reported as the outcome of a placebo-‐controlled study. Safety data derived from clinical trials and other sources have been assessed and have not revealed any safety concerns or significant adverse effects, relating to the ingredients in the Tibi Tonic formulation if consumed in excess of the recommended 2 servings. For example, studies have shown that N-‐ acetylcysteine will only be converted into glutathione when the body has a shortage of the latter antioxidant. When the intake of N-‐acetylcysteine exceeds demand, the body will use the excess N-‐ acetylcysteine as an energy source.
This technical brochure provides a review of the science behind the symptoms of hangover that occurs following moderate and responsible consumption of alcohol. The role of ingredients in Tibi Tonic, in supporting and accelerating the body’s natural mechanisms for processing and elimination of alcohol in the hours after consumption and thereby avoiding the symptoms associated with a hangover is presented in the following order: (i)
A review of the science behind a hangover.
Insight into the key pathways in the human body involved in the processing and elimination (metabolism) of alcohol
Presentation of the scientific evidence supporting the involvement of and benefits derived from the ingredients in the Tibi Tonic recipe.
A review of Tibi Tonic ingredient safety
The active ingredients that were the focus of study in this Technical Brochure were N-‐acetylcysteine, Milk Thistle extract, Glycine, Thiamin (Vitamin B1), Vitamin B6, and Ascorbic acid (Vitamin C).
The science behind a hangover
The hangover, that dreaded phenomenon that manifests itself in symptoms such as headache, thirst, tiredness, lack of concentration, difficulty sleeping, diarrhea, sensitivity to light and sound, nausea, increased heart rate and blood pressure has been with us ever since humans started to drink alcohol containing beverages. Alcohol in this context refers to ethyl alcohol or ethanol. The best form of hangover cure is prevention through abstention – in general the promise of what is to come the morning after does encourage avoidance or at least moderation of intake. However, even moderate intake can result in hangover. In the US, absenteeism and poor job performance costs $148 billion annually (the equivalent of $2000 annually per working adult). Most of this cost (87%) is incurred by light to moderate drinkers, who consume up to 3 standard drinks per day in the case of men and 1 drink in the case of women (Wiese et al, 2000). Hangovers are most common (70%) in this segment of consumers. Perhaps surprisingly, there is no evidence suggesting that alleviation of hangover symptoms leads to further alcohol consumption (Earlywine, 1993), whereas the discomfort caused by such symptoms may cause sufferers to resort to another alcoholic drink – the so-‐called ‘hair of the dog that bit me’. Scientific understanding of what comprises a hangover is still a matter of some discussion but a good starting point is to review how the body processes alcohol once it is consumed and absorbed. It is reported that the body can process about 7 g of alcohol per hour. Given that a 12oz beer contains about 14 g of alcohol, it takes about 2 hours to clear that amount of ethanol from the body. The body starts to process the alcohol as soon as it is absorbed and continues to do so after consumption stops, processing alcohol and eliminating the waste products that are generated from that process. Alcohol is a foreign, toxic substance (referred to technically as a xenobiotic) and the body will process and break it down (i.e. metabolize) to ensure its removal from the blood stream. This processing takes place mainly in the liver. One of the intermediates of alcohol metabolism is acetaldehyde, a powerful muscle poison, which is roughly 30 times more toxic than alcohol. It induces rapid heartbeat, sweating, and nausea. Acetaldehyde is produced from alcohol by the action of the enzyme, alcohol dehydrogenase (ADH). A second enzyme, aldehyde dehydrogenase (ALDH), in combination with glutathione (GSH), catalyzes the conversion of the acetaldehyde to acetate, which is non-‐toxic. Glutathione is the most widely present antioxidant in the body. Its functionality relates to the presence of a sulfhydryl group. The liver is very effective at processing aldehyde for as long as there are glutathione reserves but these seemingly become depleted rather rapidly during alcohol metabolism. The rate of alcohol processing can vary between individuals for a variety of reasons ranging from whether food has been consumed before or with the alcohol to various genetic factors (Reed 1978). As with lactose-‐intolerance, peoples of certain ethnic backgrounds express critical enzymes used in metabolizing alcohol at reduced levels. For example, Asians have been shown to have deficiency in one of the aldehyde dehydrogenase isoenzymes (ALDH2), which is critical in the metabolism of alcohol. This genetic factor means that aldehyde, including acetaldehyde accumulates to higher levels in their blood stream, resulting in higher 7
susceptibility to alcohol intake with a number of unpleasant side effects, including facial flushing, tachycardia (increased heart rate), hypotension and vomiting (Wall et al., 1997). The process of alcohol metabolism consumes significant energy. The first energy source tapped by the body is blood glucose, which as it becomes depleted is replaced by fresh glucose mobilized from glycogen stores in the liver. These in turn become depleted quite quickly, placing further demands on the liver to restore them. Basically, the liver cannot meet the peak demand and the body becomes hypoglycemic. The brain is fueled primarily by blood glucose and hypoglycemia certainly contributes to the classic hangover symptoms of decreased attention, lack of concentration, weakness and fatigue during the morning after. Meanwhile, the liver is working very hard and it is for this reason that dosing oneself with acetaminophen (commonly known as Tylenol®) to alleviate the symptoms of a hangover is not advised. This over the counter (OTC) drug is toxic to the liver and generates further strain on the system. This is because the mechanism used by the body to eliminate acetaminophen uses the same pathways and material that the body needs to deal with the alcohol. Indeed, it is widespread medical practice for the treatment of acetaminophen overdose by administration of N-‐acetyl-‐cysteine. This compound is a precursor of glutathione, which is critical in the detoxification of acetaminophen, just as it is with alcohol. After going to bed, the consumer can enter a deep and apparently restful sleep but with many subjects the sleep is of low quality and becomes interrupted (Roehrs and Roth, 2001). Research shows that the poor, interrupted sleep is not linked with any particular type of alcoholic beverage but rather the alcohol itself interferes with the glutamine synthesis pathway. A consequence is the tell tale symptoms of increased heart rate, erratic pulse and anxiety resulting in disturbed sleep (Hilpurn, 2010). Consumption of alcohol to higher than legally defined levels impairs subjectively and objectively measured sleep in young adults (Rohsenow, (2010). He reported that after alcohol consumption, sleep was disrupted, characterized by lower sleep efficiency (more time awake during the night) and less REM sleep. When subjects did sleep, their sleep was deeper – comprising more slow wave sleep. Sleep disrupting effects did not account for impaired cognitive performance the next day. Impaired sleep did correlate with hangover symptoms. Those who spent less time sleeping (sleep efficiency) felt worse hangover symptoms the following morning. So poor sleep results in the fatigue the following day, which is often associated with a hangover. Alcohol is not the only xenobiotic or foreign substance/toxin consumed with alcoholic beverages. Other components, referred to collectively as congeners are also consumed with the alcoholic beverage. Levels of congeners are reported to be highest in dark liquors such as bourbon and whisky and lower in vodka and white rum (Rohsenow et al., 2010). Compounds that fall into the congener class include polyphenols, tannins, methanol, histamine, furfurals and others. Research (Rohsenow et al., 2010) has shown that on average the darker liquors produce the most severe hangover symptoms but results vary from individual to individual. Congener content was not correlated with alteration in level of intoxication as assessed by cognitive ability but did relate to how people felt the next day (extent of hangover symptoms). Hangover symptoms (headache, fatigue, nausea and thirst) peak when blood alcohol (Rohsenow et al., 2007) and acetaldehyde (Ylikahri et al., 1974) reaches 0 g%, hence the interest in 8
investigation of congeners. Methanol and its metabolism to formaldehyde and formic acid has been implicated as one of the most potent congeners (Calder, 1997). (Authors note: Be aware that the relative methanol contents of whiskey and vodka stated in this article were subsequently corrected to 26 mg/l and 3.9 mg/l respectively). Beer does not contain congeners, however, its carbonation increases the rate of alcohol transfer to the blood. Red wines are rich in tannins. Numerous anti-‐hangover interventions have been proposed over the years with mixed success. Wiese et al (2000) reviewed a number of interventions that provide at least partial relief from some hangover symptoms. The absence of a real metric for hangover measurement may in part account for the paucity of good clinical studies and the comparison of the effectiveness of treatments challenging. Most investigators resort to some form of subjective scoring system coupled with some form of psychomotor test of reaction time, coordination and attention span. Trials with simple carbohydrates (glucose or fructose solution (0.5-‐1.0 g/kg)) delivered a 50% reduction in mistakes in a choice recognition test but hangover severity was not reduced. Tolfenamic acid, a prostaglandin inhibitor (i.e. a non-‐steroid anti-‐inflammatory drug) was associated with a small improvement in hangover symptoms when administered prophylactically. Levels of prostaglandin E2 and thromboxane B2 were lowered suggesting implication of these cytokines with hangover severity. Prophylactic Vitamin B6 (pyritinol) reduced hangover symptoms by approximately 50% when dosed at a total level of 1200mg. Alcoholics, even before showing signs of liver disease have been shown to be deficient in many B vitamins including Vitamin B6, pyritinol (Lieber, 2004).
The identification of the key role played by glutathione (GSH) in the metabolism of alcohol merits further attention to this critically important antioxidant. Glutathione is present in all animal cells (Sen, 1997) often at up to millimolar (mM) concentrations. The primary roles of GSH are as detoxicant, antioxidant and cysteine reservoir. Disease states lower GSH concentration and restoration of normal levels has been demonstrated to be beneficial. Availability of cysteine is critical for synthesis of GSH to proceed. N-‐acetyl-‐cysteine and alpha-‐lipoic acid are pro glutathione agents, in the sense that they promote GSH synthesis when the body needs it. Glutathione, discovered by Kendall in 1929, is composed of the tripeptide, glu-‐cys-‐gly and has since been demonstrated to be involved in a wide range of processes including: Ø Detoxification of electrophilic xenobiotics or toxins Ø Regulation of the immune response Ø Is required for cell proliferation Ø Is required for leukotriene and prostaglandin synthesis Ø Is central to the thiol-‐disulphide exchange equilibria and as such manages the body’s redox state Ø GSH peroxide dependent metabolism of hydrogen peroxide Ø Direct scavenging of reactive oxygen species (ROS) Ø Regeneration of Vitamin C and Vitamin E Ø Modulates cellular processes including DNA synthesis The side chain thiol group is central to most of the physiological properties of glutathione. Glutathione or L-‐γ-‐glutamyl-‐L-‐cysteinylglycine, as it is chemically named, is synthesized intracellularly in a tightly regulated, two step process. Both steps consume energy, requiring the presence of adenosine triphosphate (ATP) and magnesium (Mg2+)is also required (Meister 1974). The first step involves the enzyme γ-‐glutamylcysteine synthetase (GCS), sometimes referred to as glutamate-‐cysteine ligase, which catalyses the formation of the dipeptide Glu-‐Cys (Deneke and Fanburg, 1989). It is notable that it is the
γ-‐carboxylic acid group of glutamic acid that is conjugated with the amino of cysteine to form an unusual peptide or amide bond. This confers considerable stability to the γ-‐glu-‐cys dipeptide, making it resistant to digestion by normal intracellular peptidases (DeLeve and Kaplowitz, 1991). Indeed, γ-‐glutamyl transpeptidase is the only enzyme that has been identified with the capability to hydrolyze this γ-‐ 10
glutamyl bond. GCS is regulated by feedback inhibition by GSH itself. So as GSH levels rise, the activity of the enzyme is reduced. Inhibition by GSH is competitive with respect to glutamate (Richman and Meister, 1975). The second step involves the addition of glycine to the dipeptide, which is catalyzed by glutathione synthetase. The C-‐terminal glycine protects against cleavage by γ-‐glutamyl cyclotransferase. This means that GSH is only digested extracellularly. GSH is also generated intracellularly from the oxidized form of glutathione, which comprises two GSH molecules linked by a disulfide bond – and is referred to as glutathione disulfide (GSSG). The reduction of this dimer to regenerate two GSH molecules is catalyzed by glutathione disulfide reductase in the presence of NADPH. Nimni et al. (2007) evaluated dietary intakes of a random sample of the US population with specific attention to sulfur balance. They concluded that a significant proportion of the population received insufficient sulfur in the diet. Methionine and cysteine are required for protein synthesis and this need can be met under most circumstances by methionine alone due to the body’s ability to convert methionine into cysteine. In turn, dietary intake of cysteine reduces the dietary need for methionine. The recommended daily allowance (RDA) for methionine (combined with cysteine) is 14 mg/kg body weight/day (US Food and Nutrition Board, National Research Council, 1989) and has not been revised since 1989. So for a person of 70kg body weight, the recommended daily allowance is 1.1g of methionine/cysteine per day, although performers of this study suggest that it would be safer to take approximately 2 g/day. The original data used in the calculation of the RDA was determined by Rose and Wixom (1955). More recently, Tuttle et al (1965), using individuals recruited from the VA Hospital, identified a minimal need of 2.1 g/day, with some subjects requiring more than 3 g per day. In the study of Nimni et al. (2007), it was shown that those individuals who could be classified as ‘ health conscious’ in terms of the diet composition (i.e. low red meat consumers) had the lowest sulfur amino acid content in their diet. The only group that had a lower intake was senior citizens. The dietary protein requirement to achieve nitrogen-‐equilibrium in the elderly is greater than 0.8g/kg body weight/day. Values around 1 g/kg body weight/day (BW/d) have been suggested (Kurpad and Vaz, 2000). It is recommended that 15% of energy requirement should come from protein in the diet. To meet this in elderly subjects with an average body weight of 70kg requires an intake of 75-‐85g protein per day. This would deliver on average 3.5-‐4g of sulfur amino acid per day. Arresting GSH synthesis by administration of buthionine sulfoximine results in very low GSH in cells of the rat and guinea pig, causing multi organ damage and death. Lung, liver and kidney showed damage within days of arresting synthesis (Meister 1988). Administration of ascorbate (vitamin C) reversed these effects. Decreased tissue GSH is caused by: Ø Limited GSH synthesis Ø Enhanced GSH utilization Ø Limited intracellular reduction of GSSG 11
The main effector of GSH synthesis is the availability of the rate limiting substrate, cysteine (Stipanuk et al., 2004; Droge et al., 1992). A diurnal variation of levels of cellular GSH correlates with feeding times in rodents (Edwards & Westerfield , 1952; Beck et al., 1958; Tateishi et al., 1974; Tateishi et al., 1977) suggesting increased intake of cysteine or precursors, for example N-‐acetyl-‐cysteine, promotes higher GSH levels. The effectiveness of administration of cysteine itself is limited, as it exhibits a relatively high level of toxicity as demonstrated in mice (Birnbaum et al 1957), which show weight loss and ultimate death, when high levels of cysteine is added to a basal amino acid diet. These effects are likely due to the reactivity of the free sulfhydryl group of cysteine, which readily reacts with aldehydes, such as pyridoxal and can also chelate divalent cations. In N-‐acetyl-‐cysteine, the reactivity of the free sulfhydryl group is reduced due to the reduction in deprotonation of that group compared to cysteine due to the presence of the N-‐acetyl group. In the case of the other two amino acids that comprise glutathione (GSH), there is no evidence suggesting that glycine levels in any way limit in vivo synthesis of glutathione. However, under certain conditions, glutamic acid may affect the rate of synthesis (Martensson and Meister, 1989; Huang et al., 1993). The liver has the highest organ content of GSH. Cysteine levels in the liver, the primary site of GSH synthesis are determined by dietary intake of cysteine, cystine (Cys2) and methionine, the essential sulfur-‐containing dietary amino acid (Tateishi et al., 1977). In addition, membrane transport of cysteine and the rate of methionine to cysteine conversion also modulate cysteine levels (Lu, 1999). The body does have a tendency to preserve cysteine reserves for acute phase protein synthesis. GSH reserves can be depleted due to its utilization resulting from exposure to reactive oxygen species (ROS) during oxidative stress induced by aerobic exercise (Sen CK and Hanninan O., 1994; Sen CK 1995; Sen et al., 1992). Decreased levels of GSH in red blood cells are observed in protein energy malnutrition cases as well as in cases of AIDS, cancer and alcoholism. Cysteine and methionine are not stored in the human body. Excessive intake is oxidized, producing sulfate, which is excreted in the urine. Alternatively, sulfur is stored in GSH. It has been shown by isotopic studies that 7 molecules of sulfur are incorporated in GSH for every 10 sulfur molecules that are consumed in protein synthesis. When sulfur amino acid intake is deficient, GSH synthesis is sacrificed, to allow continuation of protein synthesis (Grimble and Grimble, 1998). Direct administration of GSH orally is not effective in raising GSH levels of the body, with the exception of levels in intestinal cells, due to the ineffective intercellular transportation of GSH (Meister, 1991). Intra-‐peritoneal administration of GSH in rats results in rapid appearance of GSH in the plasma but it is then quickly cleared, showing feedback control (Bauman et al., 1988). Cysteine in its reduced (-‐SH) form is unstable and has been shown to be toxic to cultured cells and newborn rats and mice. The toxicity is linked to the pro-‐oxidant potential of the free amino acid form. Therefore there is a need for safe cysteine sources. Cysteine-‐enriched whey fractions (Immunocal®) and whey protein hydrolysates (Cysteine PeptionTM) have been developed and it has been shown that the 12
cysteine delivered in this form does boost levels of glutathione on the liver (Dudek and Sprong, 2005). In a consumer study, consumption of cysteine enriched hydrolysates were reported to have beneficial effects with respect to energy levels and sleep, indications which may be linked to the role played by GSH in detoxification (Dudek and Clark, 2005).
GSH and xenobiotic processing
Traditionally, metabolic pathways for xenobiotics or toxin clearance by the body are divided into Phase I and Phase II reactions (Klotz and Ammon, 1998) (Fig.4.1). The ‘functionalizing’ Phase I reactions are accomplished by the cytochrome P-‐450 superfamily. This process competes with the alcohol dehydrogenase and catalase reactions to produce oxygenated alcohol metabolites, which are damaging to the liver. These oxygenated metabolites are cleared by one of the three pathways that comprise the Phase II processes: (i) enzymatic catalysis resulting in conjugation with glucuronic acid; (ii) conjugation with hippuric acid formed by the reaction between glycine and benzoic acid and (iii) conjugation with glutathione produced as described above from cysteine, glutamic acid and glycine. In the latter case, glutathione sulfur transferases catalyze reaction between the sulfhydryl (-‐SH) group of GSH and potential alkylating agents – acetaldehyde in the case of alcohol processing, neutralizing their electrophilic sites, increasing their water solubility and making them more suitable for excretion. This metabolic process which occurs e.g. during elimination of overload of the drug acetaminophen, causes a reduction in the systemic level of GSH. Phase I
Xenobiotic (organic chemical, fat soluble)
Water soluble metabolites (mercapturates glucuronates)
hepatotoxic Cysteine + glutamic acid + glycine
Glycine + benzoic acid Glucose
less or not hepatotoxic
hippuric acid UDP-Glucaric acid
Figure 4.1: Schematic showing Phase I and Phase II pathways The Phase 1 process or alternatively the action of the enzyme, alcohol dehydrogenase results in production of ‘reactive oxygen species’ (ROS). These components create oxidative stress in the system, which is undesirable, hence, the need for rapid removal of ROS by the Phase II pathways, including the pathway involving GSH (Das and Vasudevan, 2007). A useful review of ROS, the effect on the liver and maintenance of the redox status can be found in Novo and Parola (2008). 14
Stamatoyannopoulos et al. (1975) implicated an atypical alcohol dehydrogenase (ADH) occurring in about 85% of Japanese as accounting for their marked and immediate sensitivity to alcohol. This genetic variant of ADH has several times the activity of the form of ADH common amongst Caucasians. It was hypothesized that the high activity form resulted in a rapid build up of highly toxic acetaldehyde in Japanese and other Mongoloids. It has been shown that these findings amongst other races including Oriental and Ojibwa Indians correlates with a higher incidence of alcoholism (Reed et al., 1976). There is contradictory information about the relative presence of different variants of ALDH, the enzyme that metabolizes aldehyde, in Japanese liver. Stamatoyannopoulos et al (1975) detected what they determined to be ‘normal’ pattern of slow and fast migrating forms of ALDH in electrophoretic studies of Japanese livers from autopsy. However, Goedde et al., (1979) reported that whilst they observed similar incidence of ADH anomalies in 85% of Japanese livers examined at autopsy they also found that ALDH varied, with 52% of subjects showing only a single ALDH form, corresponding to the slow migrating form. This ALDH variability was equally prevalent in subjects exhibiting the different ADH forms. In contrast, sixty-‐eight postmortem German subjects showed only the normal pattern of fast and slow ALDH forms. The slow migrating form of ALDH has a lower aldehyde-‐processing activity, suggesting higher concentrations of aldehyde would build in those Japanese subjects with the only the slow migrating form. This may account for the lower threshold for intoxication amongst Asians but remains to be proven. It has been shown independently that females possess less ADH than men and this accounts for their higher susceptibility to intoxication (Schenker, 1997) The effects of ethanol consumption manifest themselves in a number of changes in the body, many of which have been associated with oxidative stress. One of the earliest effects of ethanol consumption is a change in the structure of mitochondria, which appear distorted and enlarged. These changes are associated with the generation of fatty liver in the rat in cases of chronic ethanol exposure, suggesting that hepatic energy metabolism is compromised. Other forms of histological damage and cell necrosis have been reported (Bailey and Cunningham, 1999) consistent with that seen resulting from challenges from drugs such as acetaminophen i.e. Tylenol® (Dudek and Sprong, 2005). Production of cytokines, such as tumor necrosis factor-‐alpha (TNF-‐α) is one of the earliest events in many types of liver injury. This triggers the production of other cytokines that recruit inflammatory cells and kill hepatocytes. Dudek and Clark (2005) reported alcohol-‐induced (40g alcohol in the form of red wine) increases in F2-‐ isoprostanes (F2IP), a product of lipid peroxidation, were lowered in cysteine-‐enriched hydrolysate-‐ treated subjects compared to placebo. Higher levels of markers of lipid peroxidation and inflammation, indicated by C-‐reactive protein were found in subjects receiving the placebo. Levels of free fatty acids and fatty acid ethyl esters in organs including liver, kidney and brain and triglycerides and HDL in plasma have been associated with increased ethanol consumption. Though the mechanism is complex it is thought to involve the elevated presence of ROS (Das and Vasudevan, 2007). Thiol containing proteins are also susceptible to reaction with ROS, which can compromise their functionality. GSH can react with electrophiles spontaneously or enzymatically. Processing of the conjugate begins with cleavage of γ-‐glutamyl moiety by γ-‐glutamyl transferase (GGT) leaving the cysteinyl-‐glycine conjugate. The Cys-‐Gly linkage is subsequently cleaved by a dipeptidase leaving the cysteinyl conjugate. 15
This is followed by N-‐acetylation of the cysteinyl group producing mercapturic acid. This latter step usually occurs in the kidney. This metabolic process results in the destruction of a single GSH molecule for every ethanol molecule processed by this pathway. Of greater consequence is the excretion of the cysteine-‐derived sulfur atom, which can only be replaced by appropriate dietary or supplemental intake. So, the detoxification of every alcohol molecule by this pathway causes the irreversible consumption of one glutathione molecule. This lost glutathione must be replaced. Aging is associated with a decline in GSH levels and an impairment of GSH biosynthesis in many tissues (Vogt and Richie, 2007) and has been studied in mouse models. Liver GSH levels reduced by more than 50% within 6 hours of administration of a dose of ethanol of 2-‐5g/kg. Levels remained low after 24hr in both young and old mice but the old mice showed the lowest levels of GSH. If GSH levels were depleted before ethanol administration, a higher level of ethanol toxicity was observed (Strubelt et al., 1987). On the other hand, when GSH levels are enhanced by addition of its precursors, depletion of GSH by ethanol is prevented and toxicity is diminished (Sprince et al., 1974). Waly et al. (2011) also reported that ethanol treatment reduced GSH levels in rat liver and brain. Oxidative stress can also be induced by a range of extreme conditions including (i) inadequate intake of foodstuffs containing antioxidants, (ii) excessive intake of pro-‐oxidants (e.g. ethanol), (iii) exposure to noxious chemicals or UV light, (iv) injury and wounds and/or (v) intense exercise. Under such conditions, the body’s endogenous antioxidant systems become overwhelmed (Kerksick and Willoughby, 2005). It has been proposed that increased intake of antioxidants or precursors of antioxidants (e.g. glutathione, N-‐acetyl-‐cysteine, α-‐lipoic acid, Vitamin A, E and C) can reduce oxidative stress (Sen and Packer, 2000). Burgunder et al. (1989) studied the effect of orally administered N-‐acetylcysteine (30mg/kg on plasma sulfhydryl levels. Plasma levels reached a peak concentration within 45-‐60 minutes and disappeared with a half life of 1.3 hours. Free cysteine increased but total cysteine and free and total GSH were unchanged in the plasma. Plasma cysteine and GSH decreased when 2g of acetaminophen (Tylenol®) was administered. In contrast, when 2 g N-‐acetylcysteine was administered with the acetaminophen, plasma cysteine and GSH were raised. The data showed that NAC increases circulating cysteine by reacting with cystine to form mixed disulfides. NAC had no effect on plasma glutathione levels in the absence of stress on glutathione reserves. However, NAC supported glutathione synthesis when the demand for GSH was increased, as during the metabolism of acetaminophen. This finding elegantly demonstrates that the body will only utilize N-‐acetylcysteine when there is demand e.g. caused by GSH consumption. This is a positive finding with respect to the safety of consumption of N-‐acetylcysteine.
Summary of benefits: •
Supports and restores normal glutathione levels
Supports liver detoxification
The pharmacokinetics and bioavailability of reduced and oxidized NAC have been investigated (Olssen et al, 1988). These authors found that the bioavailability of 400mg of orally administered reduced N-‐ acetylcysteine was found to be 4.0%. The terminal half-‐life for total N-‐acetylcysteine was 6.25 hours after oral administration with a bioavailability of 9.1%. This treatment of the data in terms of total N-‐ acetylcysteine (reduced and oxidized forms) is probably the most correct manner. Nevertheless, the oral bioavailability is rather low even for total N-‐acetylcysteine. The earlier study of Borgstrom et al. (1986) reported even lower levels but differences in methodology make the 2 studies difficult to compare. N-‐acetylcysteine in plasma can be present in its reduced form as well as various oxidized forms. It can be oxidized to a disulfide, N, N’-‐diacetylcystine and it can form mixed disulfides by reacting with other low molecular weight thiols such as cysteine and GSH. N-‐acetylcysteine can be oxidized by reaction with thiol groups of plasma proteins (Olssen et al., 1988). Earlier studies using radiolabelled N-‐acetylcysteine in animals and man showed that cysteine and cystine were the major metabolites of NAC. Sulfate was the major urinary product along with taurine and unchanged N-‐acetylcysteine. N-‐acetylcysteine and alpha-‐lipoic acid are proven pro-‐GSH agents (Sen et al., 1997; Packer et al., 1995; Borgstrom et al., 1986). Indeed, lipoate is a more effective pro-‐glutathione agent than N-‐acetylcysteine. N-‐acetylcysteine enters cells and is hydrolyzed to release cysteine. N-‐acetylcysteine is widely used as a mucolytic agent (breaks down mucus) to treat chronic bronchitis, in cancer therapies, as an adjunct treatment for schizophrenia (Berk et al., 2008), as an antidote to acetaminophen (Tylenol®) over dosage (Burgunder et al., 1989) and other liver toxins such as amatoxin from poisoning mushrooms. The mucolytic properties are linked to the ability of its free sulfhydryl exchanging with and breaking the inter and intra molecular disulfide bridges that stabilize the mucus proteins and confer the elastic properties to this material. Much attention has been given to N-‐acetylcysteine due to its use in treating acetaminophen (Tylenol®) toxicity (Smilkstein MJ et al., 1988). It is well known that N-‐acetyl-‐p-‐benzoquinoneimine, a toxic metabolite of acetaminophen is detoxified by hepatic GSH. An overdose of acetaminophen rapidly results in the generation of N-‐acetyl-‐p-‐benzoquinoneimine (NAPQI) via the Phase I cytochrome P450 activation process. This material, rapidly overwhelms glutathione-‐S-‐transferase and eventually exhausts the reactants needed in the Phase II reactions namely, GSH, UDP-‐glucuronic acid and inorganic sulfate. NAPQI is the component that causes hepatocyte damage, liver necrosis, conjugates with critical liver 17
proteins and enzymes and can lead ultimately death. Prompt administration of NAC in cases of acetaminophen overdose can prevent liver damage (Acharya and Lau-‐Cam, 2010). N-‐acetylcysteine treatment of acetaminophen overdose only works when administered promptly (Yang et al., 2009). It has been shown that prolonged or late treatment with high doses of NAC following acetaminophen overdose can be detrimental and cause interference with liver regeneration. Hepatic concentrations of active sulfate, in the form of PAPS (adenosine-‐3'-‐phosphate 5'-‐phosphosulfate) were also decreased and could be restored to normal by supplementation with methionine (Glazenburg et al., 1983). The effectiveness of NAC, taurine and hypotaurine in promotion of antioxidant activity has been compared in rats (Acharya and Lau-‐Cam, 2009). Acetaminophen administered alone caused a decrease in GSH, GSSG and the activity of a number of liver function markers. Pre-‐administration of NAC, Taurine or hypotaurine before delivery of acetaminophen provided a measurable protective effect, with hypotaurine being the most effective, followed by NAC. Taurine offered the least additional protection. Acetaminophen is used by approximately 50 million adults in the US per week. Reports of hepatic failure and death following intended therapeutic use of acetaminophen by patients who consume alcohol has been published. Some practitioners recommend that the maximum dose of 4g per day be lowered or that acetaminophen use be avoided completely in alcoholic patients. The US FDA requires that labels of non prescriptive analgesic drugs containing acetaminophen carry warnings for patients who consume more than 3 alcoholic drinks daily to discuss acetaminophen use with their physician (Kuffner et al., 2007). The concern arises from the fact that both alcohol and acetaminophen share the same detoxification pathways and that they can deplete critical reactants needed for these pathways in a cumulative manner. However, this subject is controversial and some authors claim more evidence is still required before a full understanding is obtained (Prescott, 2000). The label recommendation for use of acetaminophen is limited to a maximum intake of 4 g per day but often higher levels are taken. Studies have shown that 35% of the daily 4 g dose is excreted as a conjugate with sulfur (mercapturic acid conjugate i.e. the toxin-‐GSH conjugate), 3% is in the larger form conjugated with cysteine and the rest is excreted with glucuronic acid (Lin and Levy, 1983). Addition of 0.5% methionine/cysteine to the daily diet can overcome the methionine deficiency, induced in acetaminophen (1%) treated rats. This acetaminophen dosage is equivalent to the 4g/day maximum recommended dose of acetaminophen for humans. The protective effect contributed by other cysteine derivatives including s-‐allyl cysteine (SAC), s-‐ethyl cysteine (SEC), s-‐methyl-‐cysteine (SMC) and s-‐propyl cysteine (SPC) have been studied in mice (Yan and Yin, 2007). These compounds are naturally found in Allium plants, such as garlic and onion. Pre-‐intake of these agents significantly attenuated alcohol-‐induced lipid oxidation, GSH depletion along with reduction in the presence of C-‐reactive protein, a marker for inflammation (Dudek and Clark, 2005).
Summary of benefits: •
Supports liver detoxification
Contains natural antioxidants
Milk thistle is a thistle of the genus Silybum Adans., a flowering plant of the daisy family, native to the Mediterranean region. The name milk thistle is derived from the milky sap that exudes from cut stems and the mottled white patches on its leaves. The extract of the seeds of milk thistle have been used by herbalists for thousands of years to treat liver conditions. Indeed, extracts of milk thistle appear to have been as an alternative medical remedy by the Ancient Greeks. In recent years, milk thistle derivatives have been reviewed and studied scientifically and in clinical studies to seek confirmation and an understanding of these medicinal properties (Gazak et al, 2007). The extract from the seeds contains approximately 65-‐80% silymarin, a flavolignan (flavonolignans) and 20-‐35% fatty acids, including linoleic acid (Greenlee et al., 2007). Silymarin is a complex mixture of polyphenolic compounds. This variability in the composition of the extract and inconsistent quality of design of research studies have doubtless played a role in the variable conclusions about its medical efficacy. In addition, whilst there are many references in the popular press about the putative effectiveness of milk thistle in preventing or alleviating hangover symptoms, the effort to establish clinical proof of efficacy has focused on patients suffering from chronic diseases implicating the liver. Flavonolignans have been shown to protect animals against various heptotoxic drugs including acetaminophen (Muriel et al., 1992), cisplatin, vincristine and cyclosporine, as well as radiation, iron overload, phaloidin, carbon tetrachloride and thioacetamide. As with NAC, milk thistle has been reported as being effective against poisonous mushrooms including the death cap variety, Amanita phalloides.The hepatoprotective action of milk thistle may include inhibition of lipid peroxide formation, scavenging of free-‐radicals (ROS), and changing cell membrane properties. Lipid peroxidation frequently accompanies liver damage from alcoholic and non-‐alcoholic causes. Placebo-‐controlled clinical studies have shown the efficacy of milk thistle extract in reducing aminotransferases in alcoholic liver disease and conclusions from a systematic review indicate usefulness of silymarin for liver cirrhosis (Saller et al., 2008). It has been shown to reduce liver toxicity associated with chemotherapy in children with acute lymphoblastic leukemia (Ladas et al., (2010). Other reported benefits include improvement of glycemic index in type 2 diabetics and utility in patients co-‐ infected with HIV and Hepatitis C. Rambaldi et al. (2005) conducted a review of randomized clinical trials conducted with Milk Thistle extract to assess its effectiveness in patients with alcoholic liver disease and/or hepatitis B and/or C liver 19
diseases. In total 13 studies were assessed. The quality of the trials was mixed with only 46% considered double-‐blind. Milk thistle intervention versus placebo had no significant effect in all-‐cause mortality. Liver-‐related mortality was significantly reduced by milk thistle in all trials except for the highest quality ones. They did observe a potential benefit of milk thistle on mortality from alcoholic liver disease but this was not confirmed in two other studies. Milk thistle was found to significantly improve levels of γ-‐ glutamyl transferase, a critical enzyme in the production of GSH. Milk thistle was not associated with a significant increase in risk of adverse effects. Tamayo and Diamond (2009) conducted a similar review. They commented that whilst Rambaldi et al. were not able to establish evidence to support a positive effect of milk thistle extract, their observation that milk thistle extract reduced all cause mortality in patients with alcoholic liver disease, without Hepatitis C was striking. The clinical studies they reviewed spanned the time period 2001-‐2007 and milk thistle extract was used at doses in the range of 120-‐1368 mg/day. Adverse effects were only reported in one study which attributed headaches (n=3) to milk thistle. A strict dose dependency curve has not been reported, nor have short term, high intake safety trials been conducted yet in a healthy population.
Other components and interactions
Summary of benefits: •
Glycine: Improves sleep quality without increasing daytime sleepiness
Vitamin C: Regenerates the natural antioxidant, glutathione
Vitamin C: Mitigates against tissue damage associated with low levels of glutathione
Thiamin (Vitamin B1): Supports Increased alcohol dehydrogenase activity
Vitamin B6: Protects against symptoms associated with alcohol-‐induced hangover
Yamadera et al., (2007) investigated the effects of the non-‐essential amino acid glycine on subjective sleep quality and as assessed by polysomnography in a placebo-‐controlled single-‐blind crossover trail. The subjects investigated had a history of continuous experience of unsatisfactory sleep. A dose of glycine (3g) was consumed one hour before going to bed. Glycine improved subjective sleep quality and efficacy. In addition, glycine lessened daytime sleepiness and improved performance in memory recognition tests. In contrast to hypnotic drugs, such as benzodiazepines used in the treatment of sleep disorders, glycine did not alter sleep architecture resulting in, for example increased rapid eye movement (REM) phases. Higher doses of glycine, up to 9g produced no serious adverse effects and did not induce acute daytime sleepiness. In preparation for the study, the subjects were instructed to avoid additional ingestion of alcohol beyond their normal intakes. Vitamin C supplementation (500 mg/day for 2 weeks), was shown to increase GSH levels in red blood cells by 50% in non-‐smoking subjects (Johnson et al., 1993). In addition, Vitamin C is effective in regeneration of GSH from its oxidized form, glutathione disulfide. Takabe et al., (1983) have studied the role of Thiamin in the catabolism of alcohol and acetaldehyde in rabbits. They found that when Thiamin is administered (0.5mg/kg body weight) before alcohol, it showed an increase in blood concentration over the first 3 hours, which then dropped such that it was lower than pre-‐administration levels after 12 hours and was subsequently restored to normal levels after 72 hours. This was interpreted as evidence for the involvement of Thiamin in alcohol metabolism. However, the ethanol depletion curve from the blood of rabbits fed ethanol and Thiamin were similar to those fed ethanol alone. This suggests that Thiamin is not directly involved in the metabolism of ethanol itself but it is postulated that it may be involved in processing of its metabolites. Intravenous administration of acetaldehyde induced a similar trend in Thiamin levels. Thus the data would be consistent with a role for Thiamin in acetaldehyde processing. The authors concluded that the reduction in Thiamin concentration with time was not due to alcohol impeding Thiamin uptake. Abe et al., (1979) showed that alcohol dehydrogenase activity in the liver decreased in Thiamin-‐deficient and ethanol administered rats. Studies have shown that N-‐acetylcysteine taken with Thiamin has a protective effect against aldehyde toxicity in rats (Sprince et al., 1974). 21
Wiese et al. (2000) quote that rehydration, prostaglandin inhibitors (drugs that inhibit inflammatory responses in the body) and Vitamin B6 may be effective interventions. Alcoholics even without liver disease tend to have clinical and/or laboratory signs of deficiencies in certain vitamins, particularly Thiamin (Vitamin B1), B2 (riboflavin), B6 (pyridoxine), and C (ascorbic acid), as well as folic acid. The severity of these deficiencies correlates with the amount of alcohol consumed and with the corresponding decrease in vitamin intake (Lieber, 2004)
N-‐acetylcysteine The impact of 30 day oral dosing with NAC at a level of 600 or 1200 mg/kg/day against negative controls on rats has been studied by Arfsten et al. (2004). The authors measured the effect of this sustained dose on organ histopathology, tissue GSH and total glutathione transferase activity levels. There was no difference in body weights between treated and control groups. There were no lesions discovered following histopathological investigation of the lungs, stomach, small intestine, liver, kidneys, spleen and thymus. Serum alanine aminotransferase activities were slightly elevated in the treated cohort. N-‐ acetylcysteine-‐ treated animals had increased GSH levels in the range 24-‐131% in skin, kidney and other tissues. The highest dose in this study would be equivalent to a dose of 84g/day in a human with an average body weight of 70 kg. N-‐acetylcysteine is used as a mucolytic agent and typical doses used in this human drug application is 600-‐1500 mg/day (AMR 2003) but is has been administered at doses as high as 2-‐4 g/day (AMR 2002). N-‐acetylcysteine has very low acute toxicity in humans (Gosselin et al, 2004) and is generally well tolerated up to doses of 1-‐2g/day. Acute side effects reported in association with N-‐acetylcysteine administration range from gastrointestinal upset, vomiting, fatigue to anaphylactic allergic reactions (Pendyala et al, 2001, Pendyala & Craven 1995; Tenenbein, 1984; Ziment, 1988). One attractive aspect of N-‐acetylcysteine is that the body will only use it to produce glutathione when there is a demand for glutathione and when levels are depleted. This provides additional safety. If the N-‐ acetylcysteine is not needed it will be catabolized for energy and the sulfate will be eliminated from the body. Subjects suffering from Diabetes mellitus should first consult a physician before using N-‐acetylcysteine. Similarly persons who suffer from kidney or bladder stones, kidney disease, particularly those on dialysis and liver disease should seek medical advice before using N-‐acetylcysteine. Also consumers who have allergies to eggs, milk or wheat should only take N-‐acetylcysteine under a doctor’s supervision (Anon., 2011). Contraindications reported for N-‐acetylcysteine include nausea, vomiting, and diarrhea or constipation. Rarely, it can cause rashes, fever, headache, drowsiness, low blood pressure, and liver problems. N-‐ acetylcysteine is possibly safe when taken by mouth for pregnant or breast-‐feeding women. N-‐ acetylcysteine crosses the placenta, but there is no evidence so far linking it with harm to the unborn child or mother. However, N-‐acetyl cysteine should only be used in pregnant women when clearly needed, such as in cases of acetaminophen toxicity (WebMD, 2011).
Milk Thistle extract In most clinical trials silymarin has been administered at doses of 400-‐500mg per day but higher doses have been tested. The most widely reported adverse effect is gastrointestinal distress (Venkataramanan et al., 2000). Laxative effects have been reported infrequently (Adverse drug reactions advisory committee, 1999). Sweating, nausea, vomiting and weakness have also occasionally been reported. In the review of clinical studies conducted by Tamayo and Diamond (2007) milk thistle extract was used at doses in the range of 120-‐1368 mg/day. Adverse effects were only reported in one study which attributed headaches (n=3) to milk thistle. A strict dose dependency curve has not been reported, nor have short term, high intake safety trials been conducted yet in a healthy population. Asymptomatic liver toxicity has been observed in a recent cancer patient trial but only with very high doses of silybin-‐ phytosome between 10-‐20g/day. At high doses (>1.5g/day) a laxative effect is possible caused by increased bile secretion. Mild allergic reactions were observed but were not serious (Monograph, 1999). Milk thistle was not associated with a significant increased risk of adverse effects in the study of clinical trials conducted by Rambaldi et al. (2005). Other components Glycine is considered a non hazardous substance according to European legislation recorded in Directive 67/548/EC. The oral LD50 for glycine is 7930 mg/kg in rat (MSDS for glycine, 2011). High doses of glycine, up to 9g, produced no serious adverse effects and did not induce acute daytime sleepiness (Yamadera et al., 2007).
The Tibi Tonic recipe comprises ingredients that have a role in supporting liver detoxification and restful sleep. The formulation once optimized for taste, resulted in an appealing drink shot. In this dossier, the science behind the ingredients selected and their active dosage has been reviewed in detail. There is compelling evidence of a benefit derived from inclusion of all the active ingredients in the recipe, however the evidence varies in strength. There is strong evidence supporting the effectiveness of glycine in improving sleep quality without effecting daytime sleepiness. Sleep disturbance is a major contributing factor to hangover symptoms, and affects many who indulge in moderate alcohol consumption. Glycine is also implicated in an alternative Phase II pathway for elimination of ROS species. The evidence supporting N-‐acetylcysteine efficacy is by far the strongest with data on bioavailability, conversion into glutathione and evidence showing that glutathione is consumed during alcohol metabolism. There have been several clinical studies on the effect of milk thistle extract, but these mainly address chronic conditions. There is certainly belief that milk thistle does reduce hangover symptoms via antioxidant-‐based detoxification mechanisms but the search for high quality clinical data to support anecdotal observation and case studies has proven elusive. The inclusion of Vitamin C in the Tibi Tonic formulation ensures regeneration of the critical natural antioxidant, glutathione from its inactive oxidized form. In addition, Vitamin C will also support maintenance of the activity of antioxidants supplied via Milk Thistle extract. The sulfur containing vitamin, Thiamin (Vitamin B1) has been implicated in alcohol metabolism, through enzyme activation or possibly in the elimination of alcohol metabolites. In addition, there is significant evidence of deficiency in this and other B vitamins in alcoholics, suggesting consumption of these vitamins does occur during alcohol processing by the body. -‐OoO-‐
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