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Sunday, August 29, 2010

Aspartame Studies in Food Chem Toxicol. 2007 Jun 16


Food Chem Toxicol. 2007 Jun 16;  
The effect of aspartame metabolites on the suckling rat
frontal cortex acetylcholinesterase. An in vitro study.

Simintzi I,

Schulpis KH,

Angelogianni P,

Liapi C,

Tsakiris S.

Department of Experimental Physiology, Medical School,

University of Athens,

P.O. Box 65257, GR 15401 Athens, Greece.


Aspartame (ASP) consumption is suggested to be implicated with

muscarinic dysfunction.


The aim of this work was to evaluate the effect of ASP

and its metabolites

on acetylcholinesterase (AChE) activity

in rat frontal cortex and pure enzyme.


Rat frontal cortex homogenate or pure enzyme AChE

(eel E. Electricus) were incubated with ASP and each of ASP components,

phenylalanine (Phe), aspartic acid (asp), and methanol (MeOH)

for 1h at 37 degrees C.


AChE was measured spectrophotometrically.


The results showed that incubation of rat tissue or pure enzyme

with the sum of ASP metabolites,

as reported to be found in the CSF

after 150 or 200mg/kg ASP consumption,

inhibited frontal cortex and pure AChE about -11% to -29% (p<0.001).


Asp, Phe or MeOH concentrations

related to their CSF levels after ingestion of abuse or toxic ASP doses,

when separately incubated with frontal cortex or pure AChE,

resulted in a significant decrease of the enzyme activities.


In conclusion:

ASP compounds may directly and/or indirectly act

on the frontal cortex AChE.


High or toxic doses of the sweetener

remarkably decreased the enzyme activity.


If this in vitro finding comes into human reality,

it may be suggested that cholinergic symptoms

are related to the consumption of the above ASP doses.

PMID: 17673349

second study by expert Greek team of neurotoxicity in infant rats

by aspartame (or its parts, methanol, phenylalanine, aspartic acid),

KH Schulpis et al, Toxicology 2007.05.18


Toxicology. 2007 May 18; [Epub ahead of print]

l-Cysteine and glutathione restore the reduction of rat hippocampal Na(+),

K(+)-ATPase activity induced by aspartame metabolites.

Simintzi I,

Schulpis KH,

Angelogianni P,

Liapi C,

Tsakiris S.

Department of Experimental Physiology,

Medical School, Athens University,

P.O. Box 65257, GR-15401 Athens, Greece.


Studies have implicated aspartame (ASP) ingestion in neurological problems.


The aim of this study was to evaluate hippocampal Na(+),K(+)-ATPase

and Mg(2+)-ATPase activities

after incubation with ASP or each of ASP metabolites,

phenylalanine (Phe), methanol (MeOH) and aspartic acid (asp) separately.


Suckling rat hippocampal homogenates or pure Na(+),K(+)-ATPase

were incubated with ASP metabolites.


Na(+),K(+)-ATPase and Mg(2+)-ATPase activities

were measured spectrophotometrically.


Incubation of hippocampal or pure Na(+),K(+)-ATPase with ASP concentrations

(expected in the cerebrospinal fluid (CSF))

after ASP consumption of 34, 150 or 200 mg/kg

resulted in hippocampal enzyme activity reduction of 26%, 50% or 59%,

respectively, whereas pure enzyme was remarkably stimulated.


Moreover, incubation with hippocampal homogenate

of each one of the corresponding in the CSF ASP metabolites

related to the intake of common, high/abuse doses of the sweetener,

inhibited Na(+),K(+)-ATPase, while pure enzyme was activated.


Hippocampal Mg(2+)-ATPase remained unaltered.


Addition of l-cysteine (cys) or reduced glutathione (GSH)

in ASP mixtures, related with high/toxic doses of the sweetener,

completely or partially restored the inactivated membrane

Na(+),K(+)-ATPase,

whereas the activated pure enzyme activity returned to normal.


CSF concentrations of ASP metabolites

related to common, abuse/toxic doses of the additive

significantly reduced rat hippocampal Na(+),K(+)-ATPase activity,

whereas pure enzyme was activated.


Cys or GSH completely or partially restored both enzyme activities.

PMID: 17602817


Kleopatra H. Schulpis, MD, PhD. Institute of Child Health,

Aghia Sophia Children's Hospital, GR-11527 Athens (Greece)

expert Greek group finds aspartame (or its parts, methanol,

phenylalanine, aspartic acid) harm infant rat brain enzyme activity,

KH Schulpis et al, Pharmacol. Res. 2007.05.13


Pharmacol Res. 2007 May 13; [Epub ahead of print]

The effect of aspartame on acetylcholinesterase activity in

hippocampal homogenates of suckling rats.

Simintzi I,

Schulpis KH,

Angelogianni P,

Liapi C,

Tsakiris S.

Department of Experimental Physiology,

Medical School, University of Athens,

P.O. Box 65257, GR-15401 Athens, Greece.


BACKGROUND:

Neurological disturbances have been implicated

with aspartame (ASP)consumption,

and the cholinergic system

with acetylcholinesterase (AChE) seems actively involved.


AIM:

To evaluate the effect of ASP and its metabolites

on rat hippocampal AChE activity.


METHODS:

Hippocampal homogenate or pure enzyme AChE (eel E. electricus) was

incubated with the sum or each of ASP components, phenylalanine (Phe),

aspartic acid (asp) and methanol (MeOH) for 1 h at 37 degrees C.


AChE activity was measured spectrophotometrically.


RESULTS:

Incubation of rat tissue or pure enzyme with the sum of ASP

metabolites in concentrations in CSF

(the concentrations were calculated according to the CSF/plasma

concentration ratios)

following 150 or 200 mgkg(-1) of ASP consumption,

resulted in significant enzyme activity reductions of 25 and 31%

for hippocampal AChE and 11% (p<0.01) and 19% for pure enzyme,

respectively.


Aspartic acid concentrations of 0.42 or 0.56 mM

significantly reduced the enzyme activities by 13 and 20%

for hippocampal AChE and 15 and 18% for pure enzyme, respectively.


Phe concentrations of 0.042 or 0.083mM decreased the enzyme activity

by 12% (p<0.01) and 20% (p<0.001) for hippocampal AChE

and 15 and 18% (p<0.001) for pure enzyme, respectively.


Methanol concentrations of 0.60 or 0.80 mM remarkably inhibited

hippocampal AChE by about 18 and 22%

and pure enzyme by about 14 and 20%, respectively.


CONCLUSIONS:

Low concentrations of ASP components

had no effect on hippocampal and pure AChE activity,

whereas high or toxic concentrations

remarkably decreased both enzyme activities.


Muscarinic symptoms may be related

to the latter concentrations of ASP metabolites.

PMID: 17580119


Kleopatra H. Schulpis, MD, PhD. Institute of Child Health,

Aghia Sophia Children's Hospital, GR-11527 Athens (Greece)


Metab Brain Dis. 2006 Mar; 21(1): 21-8. Epub 2006 May 6.

The effect of in vitro homocystinuria on the suckling rat hippocampal

acetylcholinesterase.

Schulpis KH,

Kalimeris K,

Bakogiannis C,

Tsakiris T,

Tsakiris S.

Inborn Errors of Metabolism Department,

Institute of Child Health Research Center,

Aghia Sophia Children's Hospital, Athens, Greece.


Homocystinuria is due to enzymatic deficiencies

resulting in elevated blood levels of homocysteine (Hcy),

homocystine (Hci),

and/or methionine (Met)

and the clinical presentation of mental retardation, seizures, and

cardiovascular disease.


Since these symptoms may be closely implicated with

acetylcholinesterase (AChE) activity,

we aimed to investigate whether this metabolic disorder affects the

hippocampal AChE activity

in 21 days suckling Wistar rat hippocampus.


Various concentrations of Hcy, Hci (0.05-0.5 mM), or Met (0.05-2 mM)


as well as Mixture A (Mix A) (0.3 mM (Hcy)+0.2 mM (Hci)+1.0 mM (Met) =

in vitro cystathionine beta-synthase deficiency homocystinuria),


Mix B1 (Hcy 0.3 mM + Hci 0.2 mM = in vitro severe

methylenetetrahydrofolate reductase deficiency homocystinuria)


or Mix B2 (Hcy 0.1 mM+Hci 0.05 mM = in vitro mild

methylenetetrahydrofolate reductase deficiency homocystinuria)


were preincubated with homogenized hippocampii or with eel

Electrophorus electricus pure AChE.


AChE was evaluated spectrophotometrically.


Hcy or Met stimulated hippocampal AChE by 50% (p < 0.001)

at low concentrations of the amino acids (up to 0.3-0.5 mM),

whereas Hci inhibited the enzyme by 40% (p < 0.001).


Mix A, Mix B1, or Mix B2 activated hippocampal AChE

by 40, 30, (p < 0.001), and 12% (p < 0.01), respectively.


In contrast, the S-containing amino acids, Mix A, Mix B1, Mix B2

failed to affect the pure AChE activity.


CONCLUSIONS:

a) The presence of -SH group in Hcy and Met

may result in hippocampal AChE stimulation

and the redox isomer Hci in the inhibition of the enzyme,

probably by producing free radicals,


and b) The SH-amino acids seem to affect

the hippocampal enzyme indirectly,

possibly by lipid(s)-protein modifications(s)

and Hci by inducing oxidative stress,

since no effect was observed on pure AChE activity.

PMID: 16773467

all three aspartame metabolites harm human erythrocyte

[red blood cell] membrane enzyme activity,

KH Schulpis et al, two studies in 2005,

Athens, Greece, 2005.12.14:

2004 research review, RL Blaylock


"High or abuse concentrations of ASP hydrolysis products significantly

decreased the membrane enzyme activity,

which was completely or partially prevented by L-cysteine

or reduced GSH."


[ Definition of Erythrocyte]

Erythrocyte:

A cell that contains hemoglobin and can carry oxygen to the body.

Also called a red blood cell (RBC).

The reddish color is due to the hemoglobin.

Erythrocytes are biconcave in shape,

which increases the cell's surface area

and facilitates the diffusion of oxygen and carbon dioxide.

This shape is maintained by a cytoskeleton

composed of several proteins.

Erythrocytes are very flexible

and change shape when flowing through capillaries.

Immature erythrocytes, called reticulocytes,

normally account for 1-2 percent of red cells in the blood. ]


Eur J Clin Nutr. 2005 Dec 14; [Epub ahead of print]

The effect of L-cysteine and glutathione on inhibition of

Na(+), K(+)-ATPase activity by aspartame metabolites

in human erythrocyte [red blood cell] membrane.

Schulpis KH, Kleopatra H. Schulpis, MD, PhD.

Institute of Child Health, Aghia Sophia Children's Hospital,

GR-11527 Athens (Greece) +30 1 7708291, Fax +30 1 7700111

Papassotiriou I, 

Tsakiris T,

Tsakiris S. Stylianos Tsakiris.

1 Institute of Child Health, Research Center,

'Aghia Sophia' Children's Hospital, Athens, Greece.


Background:

Reports have implicated Aspartame

(N-L-a-aspartyl-L-phenylalanine methyl ester, ASP)

in neurological problems.


Aim:

To evaluate Na(+), K(+)-ATPase activities in human erythrocyte

[red blood cell] membranes

after incubation with the ASP metabolites,

phenylalanine (Phe),

methanol (MeOH) and

aspartic acid (Asp).


Methods:

Erythrocyte [red blood cell] membranes

were obtained from 12 healthy individuals and

were incubated at 37 degrees C for 1 h

with the sum or each of the ASP metabolites separately,

which are commonly measured in blood after ASP ingestion.


Na(+), K(+)-ATPase and Mg(2+)-ATPase activities were measured

spectrophotometrically.


Results:

Membrane Mg(2+)-ATPase activity was not altered.


The sum of ASP metabolite concentrations corresponding

to 34, 150 or 200 mg/kg of the sweetener ingestion

resulted in an inhibition of the membrane

Na(+), K(+)-ATPase by -30, -40, -48%, respectively.


MeOH concentrations of 0.14, 0.60 or 0.80 mM

decreased the enzyme activity by -25, -38, -43%, respectively.


Asp concentrations of 2.80, 7.60 or 10.0 mM

inhibited membrane Na(+), K(+)-ATPase by -26, -40, -46%,

respectively.


Phe concentrations of 0.14, 0.35 or 0.50 mM

reduced the enzyme activity by -24, -44, -48%, respectively.


Preincubation with L-cysteine or reduced glutathione (GSH)

completely or partially restored

the inhibited membrane Na(+), K(+)-ATPase activity

by high or toxic ASP metabolite concentrations.


Conclusions:

Low concentrations of ASP metabolites had no effect

on Na(+), K(+)-ATPase activity.


High or abuse concentrations of ASP hydrolysis products significantly

decreased the membrane enzyme activity,

which was completely or partially prevented by L-cysteine

or reduced GSH. [reduced glutathione]


European Journal of Clinical Nutrition advance online publication,

14 December 2005; doi:10.1038/sj.ejcn.1602355. PMID: 16391576

aspartame (methanol, phenylalanine, aspartic acid) effects, detailed

expert studies in 2005 Aug and 1998 July, Tsakiris S, Schulpis KH,

Karikas GA, Kokotos G, Reclos RJ, et al, Aghia Sophia Children's

Hospital, Athens, Greece

[ The lowest dose level tested, 34 mg aspartame per kg body weight,

well below the FDA daily human limit of 50 mg/kg, 16 12-oz cans,

caused enzyme activity reduction by -33% in human red blood cell

membranes. ]


However, a missed opportunity in both studies is that the inevitable,

extremely and cumulatively toxic products of methanol in the human

body, formaldehyde and formic acid, which are responsible for the

toxicity of methanol, were not independently tested.


" It is concluded that low concentrations of ASP metabolites had no

effect on the [human red blood cell] membrane enzyme activity,

whereas high or toxic concentrations partially or remarkably decreased

the [human red blood cell] membrane AChE activity, respectively.

Additionally, neurological symptoms, including learning and memory

processes, may be related to the high or toxic concentrations of the

sweetener metabolites. " ]


Pharmacol Res. 2005 Aug 26; [Epub ahead of print]

The effect of aspartame metabolites on human [red blood cell]

erythrocyte membrane acetylcholinesterase activity.

Tsakiris S,

Giannoulia-Karantana A,

Simintzi I,

Schulpis KH.

Department of Experimental Physiology, Medical School,

University of Athens, P.O. Box 65257, GR-154 01 Athens, Greece.


Stylianos Tsakiris. 


Giannoulia-Karantana A. First Department of Pediatrics,

Aghia Sophia Children's Hospital, University of Athens, Greece.


Kleopatra H. Schulpis, MD, PhD. Institute of Child Health,

Aghia Sophia Children's Hospital, GR-11527 Athens (Greece)

Tel. +30 1 7708291, Fax +30 1 7700111 

[ Papoutsakis T. ]


Papadopoulos G. Department of Biochemistry and Biotechnology,

University of Thessaly, Ploutonos 26, 41221 Larisa, Greece

Abstract:


Studies have implicated aspartame (ASP) with neurological problems.

The aim of this study was to evaluate acetylcholinesterase (AChE)

activity in human erythrocyte [red blood cell] membranes

after incubation with the sum of ASP metabolites,

phenylalanine (Phe),

methanol (met) and

aspartic acid (aspt),

or with each one separately.


Erythrocyte [human red blood cell] membranes were obtained from

12 healthy individuals and were incubated with ASP hydrolysis

products for 1h at 37 degrees C.

AChE was measured spectrophotometrically.


Incubation of membranes with ASP metabolites corresponding

with 34 mg/kg, 150 mg/kg or 200 mg/kg of ASP consumption

resulted in an enzyme activity reduction by -33%, -41%, and -57%,

respectively.


Met concentrations 0.14 mM, 0.60 mM, and 0.80 mM decreased

the enzyme activity by -20%, -32% or -40%, respectively.


Aspt concentrations 2.80 mM, 7.60 mM or 10.0 mM inhibited

membrane AChE acitivity by -20%, -35%, and -47%, respectively.


Phe concentrations 0.14 mM, 0.35 mM or 0.50 mM reduced the

enzyme activity by -11%, -33%, and -35%, respectively.


Aspt or Phe concentrations 0.82 mM or 0.07 mM, respectively,

did not alter the membrane AChE activity.


It is concluded that low concentrations of ASP metabolites had

no effect on the membrane enzyme activity,

whereas high or toxic concentrations partially or remarkably

decreased the membrane AChE activity, respectively.

Additionally, neurological symptoms, including learning and memory

processes, may be related to the high or toxic concentrations of the

sweetener metabolites. PMID: 16129618


http://groups.yahoo.com/group/aspartameNM/message/939

aspartame (aspartic acid, phenylalanine) binding to DNA:

Karikas July 1998

Karikas GA, Schulpis KH, Reclos GJ, Kokotos G

Measurement of molecular interaction of aspartame and

its metabolites with DNA. Clin Biochem 1998 Jul; 31(5): 405-7.

Dept. of Chemistry, University of Athens, Greece

Murray, full plain text & critique:

chronic aspartame in rats affects memory, brain cholinergic

receptors, and brain chemistry, Christian B, McConnaughey M

et al, 2004 May: 2004.06.05

Pharmacol Biochem Behav. 2004 May; 78(1): 121-7.

Chronic aspartame affects T-maze performance, brain cholinergic

receptors and Na(+),K(+)-ATPase in rats.

Christian B, McConnaughey K, Bethea E, Brantley S,

Coffey A, Hammond L, Harrell S, Metcalf K, Muehlenbein D,

Spruill W, Brinson L, McConnaughey M.

Department of Pharmacology, Brody School of Medicine,

East Carolina University, Greenville, NC 27858, USA;

North Carolina School of Science and Mathematics,

Durham, NC 27811.

Mona M. McConnaughey, Ph.D. Research Assistant Professor

Department: PHARMACOLOGY & TOXICOLOGY

Office: Brody Medical Science 6E-120A 252-744-2756

This study demonstrated that chronic aspartame consumption in rats

can lead to altered T-maze performance and increased muscarinic

cholinergic receptor densities in certain brain regions.

Control and treated rats were trained in a T-maze to a particular side

and then periodically tested to see how well

they retained the learned response.

Rats that had received aspartame (250 mg/kg/day)

in the drinking water for 3 or 4 months showed a significant increase

in time to reach the reward in the T-maze,

suggesting a possible effect on memory due to the artificial

sweetener.

Using [(3)H]quinuclidinyl benzilate (QNB) (1 nM) to label muscarinic

cholinergic receptors and atropine (10(-6) M) to determine nonspecific

binding in whole-brain preparations,

aspartame-treated rats showed a 31 % increase in receptor numbers

when compared to controls.

In aspartame-treated rats, there was a significant increase in

muscarinic

receptor densities in the

frontal cortex, midcortex, posterior cortex, hippocampus, hypothalamus

and cerebellum of

80 %, 60 %, 61 %, 65 %, 66 % and 60 %, respectively.

The midbrain was the only area where preparations from

aspartame-treated rats showed a significant increase

in Na(+),K(+)-ATPase activity.

It can be concluded from these data that long-term consumption

of aspartame can affect T-maze performance in rats and alter

receptor densities or enzymes in brain. PMID: 15159141

May 31: Coca-Cola and the much larger Cargill Inc.,

after years of secret development, with 24 patents,

will soon sell rebiana (stevia) in drinks and food

in the many nations where it is approved as a sweetener --

for decades a major sweetener in Japan, China, Korea, Taiwan,

Thailand, Malasia, Saint Kitts, Nevis,

Brazil, Peru, Paraguay, Uruguay, and Israel,

and an approved supplement in USA, Australia, and Canada,

according to Wikipedia.