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    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.    

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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:     •

N-­‐acetylcysteine o

Supports and  restores  normal  glutathione  levels  

o

Supports liver  detoxification  

Glycine o

Milk Thistle  Extract   o

Supports liver  detoxification  

o

Contains natural  antioxidants  

Vitamin C   o

Regenerates the  natural  antioxidant,  glutathione  

o

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.      

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Introduction

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.  

(ii)

Insight into  the  key  pathways  in  the  human  body  involved  in  the  processing  and  elimination   (metabolism)  of  alcohol  

(iii)

Presentation of  the  scientific  evidence  supporting  the  involvement  of  and  benefits  derived   from  the  ingredients  in  the  Tibi  Tonic  recipe.  

(iv)

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).      

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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).  

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Glutathione (GSH)  

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).      

13  


4

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)

Phase II

Oxygenated metabolites

O2

P450

conjugation

Water soluble metabolites (mercapturates glucuronates)

hepatotoxic Cysteine + glutamic acid + glycine

Glycine + benzoic acid Glucose

UDP-Glucose

less or not hepatotoxic

glutathione excretion

hippuric acid UDP-Glucaric acid

UDP-Glucuronic 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.  

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5

N-­‐acetylcysteine (NAC)  

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).  

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6

Milk Thistle  

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.  

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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)    

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Safety data  

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).    

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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).      

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Conclusions

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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Tibi Tonic science dossier  

Scientific review by Dr. David C. Clark of vitamin supplement Tibi Tonic

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