http://groups.yahoo.com/group/aspartameNM/message/925

aspartame puts formaldehyde adducts into tissues, Part 1/2

full text Trocho & Alemany 6.26.98: Murray 12.22.2 rmforall



Trocho & Alameny (1998):

"These are indeed extremely high levels for adducts of formaldehyde, a

substance responsible of chronic deleterious effects (33), that has also

been considered carcinogenic (34,47). The repeated occurrence of claims

that aspartame produces headache and other neurological and

psychological secondary effects-- more often than not challenged by

careful analysis-- (5,9,10,15,48) may eventually find at least a partial

explanation in the permanence of the formaldehyde label, since

formaldehyde intoxication can induce similar effects (49).



The cumulative effects derived from the incorporation of label in the

chronic administration model suggests that regular intake of aspartame

may result in the progressive accumulation of formaldehyde adducts.



It may be further speculated that the formation of adducts can help to

explain the chronic effects aspartame consumption may induce on

sensitive tissues such as brain (6,9,19,50). In any case, the possible

negative effects that the accumulation of formaldehyde adducts can

induce is, obviously, long-term. The alteration of protein integrity

and function may needs some time to induce substantial effects.



The damage to nucleic acids, mainly to DNA, may eventually induce cell

death and/or mutations.



The results presented suggest that the conversion of aspartame methanol

into formaldehyde adducts in significant amounts in vivo should to be

taken into account because of the widespread utilization of this

sweetener. Further epidemiological and long-term studies are needed to

determine the extent of the hazard that aspartame consumption poses for

humans."



[ Notes by Rich Murray are in square brackets.



http://groups.yahoo.com/group/aspartameNM/message/910

formaldehyde & formic acid from methanol in aspartame:

Murray: 12.9.2 rmforall



It is certain that high levels of aspartame use, above 2 liters daily

for months and years, must lead to chronic formaldehyde-formic acid

toxicity, since 11% of aspartame (1,120 mg in 2L diet soda, 5.6 12-oz

cans) is 123 mg methanol (wood alcohol), immediately released into the

body after drinking (unlike the large levels of methanol locked up in

molecules inside many fruits), then quickly transformed into

formaldehyde, which in turn becomes formic acid, both of which in

time become carbon dioxide and water-- however, about 30% of the

methanol remains in the body as cumulative durable toxic metabolites of

formaldehyde and formic acid-- 37 mg daily, a gram every month.

If 10% of the methanol is retained as formaldehyde, that would give 12

mg daily formaldehyde accumulation, about 60 times more than the 0.2 mg

from 10% retention of the 2 mg EPA daily limit for formaldehyde in

drinking water.



Bear in mind that the EPA limit for formaldehyde in

drinking water is 1 ppm,

or 2 mg daily for a typical daily consumption of 2 L of water.



http://groups.yahoo.com/group/aspartameNM/message/835

RTM: ATSDR: EPA limit 1 ppm formaldehyde in drinking water July 1999

5.30.2 rmforall



This long-term low-level chronic toxic exposure leads to typical

patterns of increasingly severe complex symptoms, starting with

headache, fatigue, joint pain, irritability, memory loss, and leading to

vision and eye problems and even seizures. In many cases there is

addiction.  Probably there are immune system disorders, with a

hypersensitivity to these toxins and other chemicals.



Confirming evidence and a general theory are given by Pall (2002):

http://groups.yahoo.com/group/aspartameNM/message/909

testable theory of MCS type diseases, vicious cycle of nitric oxide &

peroxynitrite: MSG: formaldehyde-methanol-aspartame:

Martin L. Pall: Murray: 12.9.2 rmforall



Here is research in 1998 at a very low level of aspartame

ingestion, 10 mg/kg, for rats, which have a much greater tolerance for

aspartame than humans.  The same toxicity level for humans would be

about 1 mg/kg. Many headache studies in humans used doses of about 30

mg/kg daily.  A daily dose of 1120 mg aspartame, about 2 L diet soda,

used in many experimental tests on humans, is 19 mg/kg and supplies 123

mg methanol into the body, 2 mg/kg for a 60 kg body.  Many cases report

typical serious symptoms at this level.  This report shows that

aspartame causes binding of methanol's product, formaldehyde, a potent,

cumulative toxin, into tissues.



http://ww.presidiotex.com/barcelona/index.html  full text & graphs

Trocho C, Pardo R, Rafecas I, Virgili J, Remesar X,

Fernandez-Lopez JA, Alemany M  ["Trok-ho"]

Formaldehyde derived from dietary aspartame binds to tissue

components in vivo.  Life Sci 1998 Jun 26; 63(5): 337-49.

Sra. Carme Trocho, Sra. Rosario Pardo, Dra. Immaculada Rafecas,

Sr. Jordi Virgili, X. Remesar,  Dr. Jose Antonio Fernandez-Lopez,

Dr. Marià Alemany Fac. Biologia Tel.: (93)4021521, FAX: (93)4021559

Departament de Bioquimica i Biologia Molecular, Facultat de Biologia,

Universitat de Barcelona, Spain.  34-934021521  fax 34-934021559

Avinguda Diagonal, 645; 08028 Barcelona, Spain.

Maria Alemany, PhD (male)  alemany@...

http://www.bq.ub.es/grupno/grup-no.html



http://groups.yahoo.com/group/aspartameNM/message/864

Murray: Butchko, Tephly, McMartin: Alemany: aspartame formaldehyde

adducts in rats 9.8.2 rmforall

Prof. Alemany vigorously affirms the validity of the Trocho study

against criticism:

Butchko, HH et al [24 authors], Aspartame: review of safety.

Regul. Toxicol. Pharmacol. 2002 April 1; 35 (2 Pt 2): S1-93, review

available for $35, [an industry funded organ].  Butchko:

"When all the research on aspartame, including evaluations in both the

premarketing and postmarketing periods, is examined as a whole, it is

clear that aspartame is safe, and there are no unresolved questions

regarding its safety under conditions of intended use."

[They repeatedly pass on the ageless industry deceit that the methanol

in fruits and vegetables is as as biochemically available as that in

aspartame-- see the 1984 rebuttal by Monte.]



http://groups.yahoo.com/group/aspartameNM/message/911

RTP ties to industry critized by CSPI: Murray: 12.9.2 rmforall ]

************************************************************************



*****Summary:

Adult male rats were given an oral dose of 10 mg/kg aspartame C-14

labelled in the methanol carbon. At timed intervals of up to 6 hours,

the radioactivity in plasma and several organs was investigated.

Most of the radioactivity found (>98% in plasma, >75% in liver) was

bound to protein.



Label present in liver, plasma and kidney was in the range of 1-2% of

total radioactivity administered per g or mL, changing little with time.

Other organs (brown and white adipose tissues, muscle, brain, cornea and

retina) contained levels of label in the range of 1/12 to 1/10th of that

of liver.  In all, the rat retained, 6 hours after administration about

5% of the label, half of it in the liver.



The specific radioactivity of tissue protein, RNA and DNA was quite

uniform. The protein label was concentrated in amino acids, different

from methionine, and largely coincident with the result of protein

exposure to labelled formaldehyde. DNA radioactivity was essentially in

a single different adduct base, different from the normal bases present

in DNA. The nature of the tissue label accumulated was, thus, a direct

consequence of formaldehyde binding to tissue structures.



The administration of labelled aspartame to a group of cirrhotic rats

resulted in comparable label retention by tissue components, which

suggests that liver function (or its defect) has little effect on

formaldehyde formation from aspartame and binding to biological

components.



The chronic treatment of a series of rats with 200 mg/kg of non-labelled

aspartame during 10 days resulted in the accumulation of even more label

when given the radioactive bolus, suggesting that the amount of

formaldehyde adducts coming from aspartame in tissue proteins and

nucleic acids may be cumulative.



It is concluded that aspartame consumption may constitute a hazard

because of its contribution to the formation of formaldehyde adducts.



Key Words: aspartame, aspartame toxicity, formaldehyde, methanol*****



[  These results are consistent with four previous studies on aspartame,

methanol, formaldehyde, and formic acid in monkeys and primates.



McMartin (1979) admitted one datum that showed accumulation of

formaldehye in the midbrain from an acute toxicity dose of methanol, and

widespread accumulation of formic acid in five tissues.  He wrote:

"It is now generally accepted that the toxicity of methanol is due to

the formation of toxic metabolites (1,2), either formaldehyde or formic

acid."



Biochemical Pharmcacology 1979: 28; 645-649.

Lack of a role for formaldehyde in methanol poisoning in the monkey.

Kenneth E. McMartin, Gladys Martin-Amat, Patricia E. Noker

and Thomas R. Tephly

The Toxicology Center, Dept. of Pharmacology,

University of Iowa, Iowa City, Iowa 52242



*****Abstract [not given in PubMed]:

Methanol was administered [by nasogastric tube] either to untreated

cynomolgus monkeys [2-3.5 kg] or to a folate-deficient cynomolgus

monkey which exhibits exceptional sensitivity to the toxic effects of

methanol.

Marked formic acid accumulation in the blood and in body fluids and

tissues was observed.

No formaldehyde accumulation was observed in the blood and no

formaldehyde was detected in the urine, cerebrospinal fluid, vitreous

humor, liver, kidney, optic nerve, and brain in these monkeys at a time

when marked metabolic acidosis and other characteristics of methanol

poisoning were observed.

Following intravenous infusion into the monkey, formaldehyde was

rapidly eliminated from the blood with a half-life of about 1.5 min and

formic acid levels promptly increased in the blood.

Since formic acid accumulation accounted for the metabolic acidosis and

since ocular toxicity essentially identical to that produced in

methanol poisoning has been described after formate treatment, the

predominant role of formic acid as the major metabolic agent for

methanol toxicity is certified.

Also, results suggest that formaldehyde is not a major factor in the

toxic syndrome produced by methanol in the monkey.*****



So, this is an acute toxicity study, with little relevance for chronic

long-term, low-level exposure.

None of the five tissues showed any formaldehyde in this study,

except the midbrain, 0.14 mmol/kg wet weight tissue [units

converted from their 0.14 micromole/gm]-- just 1.5 times the detection

limit of .09 mmol/kg wet tissue weight (given on p. 648).

It is reasonable to surmise that more sensitive assays would have

found formaldehyde and formate bound to and reacted with a variety of

cellular substances in all tissues, as later found by Trocho (1998).



J. Nutrition 1973 Oct; 103(10): 1454-1459.

Metabolism of aspartame in monkeys.

Oppermann JA, Muldoon E, Ranney RE.

Dept. of Biochemistry, Searle Laboratories,

Division of G.D. Searle and Co. Box 5110, Chicago, IL 60680



They found that about 70% of the radioactive methanol in aspartame put

into the stomachs of 3 to 7 kg monkeys was eliminated within a day as

carbon dioxide in exhaled air and as water in the urine: page 1458

"That fraction not so excreted (about 30%) was converted to body

constituents through the one-carbon metabolic pool."  They did not

mention that this meant that about 30% of the methanol must transform

into formaldehyde and then into formic acid, much of which must remain

as toxic products in all parts of the body.  This study did not monitor

long-term use of aspartame.



The low oral dose of aspartame and for methanol was 0.068 mmol/kg,

about 1 part per million [ppm] of the acute toxicity level of 2,000

mg/kg, 67,000 mmol/kg, used by McMartin (1979).  Two L daily use of

diet soda provides 123 mg methanol, 2 mg/kg for a 60 kg person, a dose

of 67 mmole/kg, a thousand times more than the dose in this study.

By eight hours excretion of the dose in air and urine had leveled off

at 67.1 +-2.1% as CO2 in the exhaled air and 1.57+-0.32% in the urine,

so 68.7% was excreted, and 31.3% was retained. This data is the

average of 4 monkeys.



Xenobiotica 1982 Feb;12(2):119-24

Formaldehyde metabolism by the rat: a re-appraisal.

Mashford PM, Jones AR.

Dept. of Biochemistry, University of Sidney, Australia



Six rats were injected with a 4 mg/kg dose = 133 mmol/kg, and by 48

hours, 82% was in the exhaled air as CO2, and 7.5% in the urine = total

89.5% excreted, so 10.5% was retained in the body.



Biochem. Pharmacol. 13: 1137-1142 (1964).

The metabolic fate of formaldehyde-C14 intraperitoneally administered

to the rat.

W. Brock Neely.

Biochemical Research Labs, Dow Chemical Co., Midland, Michigan



In one rat, a 60.5 mg/kg dose = 2,000 mmol/kg was injected, and by 48

hours, 82.0% was in the exhaled air as CO2 and 13.9% was in the urine

= total 95.9% excreted, so 4% was retained in the body. ]

**********************************************************************



Aspartame is one of the most widely used artificial sweeteners. Its

peptide nature: aspartyl-phenylalanine methyl-ester facilitates its

intestinal hydrolysis and the absorption (1-3) of innocuous amino

acids together with small amounts of free methanol, far away from the

lower limits of toxicity for that compound (4). The use of large

amounts of aspartame in the diet, however, has been claimed to be the

cause of a number of ailments, like headaches (5) and other symptoms

(6-7), which are difficult to explain (8) from its known composition

and the easy blending of its building components in the overall host

metabolism. A number of studies have linked aspartame with neurologic

pathologies, but most of the results yielded negative or inconclusive

correlations (9-16). The acute toxicity of aspartame is believed to be

low (17), which has promoted a wide distribution of the product as a

potent hypocaloric and safe substitute of sugar (18-19).



Methanol is primarily oxidized in several tissues to formaldehyde and

formic acid (20-21), the latter being considered the main metabolite

responsible for the deleterious effects of acute methanol intoxication

in man (22), but also in experimental animals (23), in spite of the

marked resistance of the rat to formate (24-25).



The enzymes involved in methanol metabolism are alcohol dehydrogenase

(EC 1.1.1.1) and aldehyde dehydrogenase (EC 1.2.1.3), as well as the

microsomal oxidase pathway (26).



Acute methanol intoxication may produce blindness and hepatic loss of

function (27-28), since the retina, cornea and liver contain the

highest alcohol dehydrogenase activity (29-30). These tissues are, thus,

where one can expect, eventually, the largest accumulation of their

byproducts: formaldehyde and formate, in the event of intoxication.



It may be assumed that liver functional failure due to cirrhosis could

result in the loss of its role as barrier to intestinal methanol, and

thus, the effects of methanol intoxication on other tissues (i.e. the

retina) would be more marked. The cirrhotic rat may be, then, used as a

model of acute or chronic methanol toxicity.



Formaldehyde is a highly reactive small molecule which strongly binds to

proteins (31) and nucleic acids (32) forming adducts which are

difficult to eliminate through the normal metabolism pathways. As a

result, formaldehyde induces severe functional alterations (33),

including the development of cancer (34). The small amounts of

formaldehyde which can be potentially produced from dietary use of

aspartame have been often overlooked in its potential toxicity precisely

because of the limited amount eventually produced.



However, the administration of labelled aspartame to experimental

animals results in the incorporation of a significant proportion of the

label to proteins (35).  [Oppermann (1973)] The accumulation of label

has been postulated to be the consequence of label drift into amino

acids (essentially in the methionine methyl group) through the

one-carbon pool (35). This aspect has not been, however, proved nor

further investigated.



We have intended here to determine the extent of conversion of aspartame

methanol to formaldehyde and its eventual effect on the overall

physiologic function of the rat. In addition, we have probed whether the

aspartame methanol carbon presence in tissue components is due to the

eventual drift of label into methionine and nucleic acid components

through the one-carbon pool, or is the consequence of a direct reaction

with free formaldehyde forming stable adducts.



[ http://groups.yahoo.com/group/aspartameNM/message/870

Aspartame: Methanol and the Public Interest 1984:

Monte: Murray 9.23.2 rmforall



Rereading this prescient classic review from 1984, I find its findings

are supported in much recent research, so I am again  making the full

text widely available.

[I have put my comments or corrections in square brackets, and spaced

the text to ease the reader's task]



For instance, I had forgotten this, which answers the industry PR

"science" that fruits and vegetables

supply much more methanol than does aspartame:



"Fruit and vegetables contain pectin

with variable methyl ester content.

However, the human has no digestive enzymes for pectin (6, 25)

particularly the pectin esterase required

for its hydrolysis to methanol (26).



Fermentation in the gut may cause disappearance of pectin (6) but the

production of free methanol is not guaranteed by fermentation (3).  In

fact, bacteria in the colon probably reduce methanol directly to formic

acid or carbon dioxide (6)  (aspartame is completely absorbed before

reaching the colon). Heating of pectins has been shown to cause

virtually no demethoxylation; even temperatures of 120 deg C produced

only traces of methanol (3).  Methanol evolved during cooking of high

pectin foods (7) has been accounted for in the volatile fraction during

boiling and is quickly lost to the atmosphere (49).

Entrapment of these volatiles probably accounts for the elevation in

methanol levels of certain fruit and vegetable products

during canning (31, 33)."



Recent research [see links at end of post] supports his focus on the

methanol to formaldehyde toxic process:



"The United States Environmental Protection Agency in their Multimedia

Environmental Goals for Environmental Assessment recommends a minimum

acute toxicity concentration of methanol in drinking water at 3.9 parts

per million, with a recommended limit of consumption below 7.8 mg/day

(8). This report clearly indicates that methanol:



"is considered a cumulative poison

due to the low rate of excretion once it is absorbed.

In the body, methanol is oxidized to formaldehyde and

formic acid; both of these metabolites are toxic." (8)....



Recently the toxic role of formaldehyde (in methanol toxicity) has been

questioned (34).  No skeptic can overlook the fact that, metabolically,

formaldehyde must be formed as an intermediate to formic acid

production (54).



Formaldehyde has a high reactivity which may be why it

has not been found in humans or other primates during methanol

poisioning (59)....



If formaldehyde is produced from methanol and does have a reasonable

half life within certain cells in the poisoned organism the chronic

toxicological ramifications could be grave.



Formaldehyde is a known carcinogen (57) producing squamous-cell

carcinomas by inhalation exposure in experimental animals (22).  The

available epidemiological studies do not provide adequate data for

assessing the carcinogenicity of formaldehyde in man (22, 24, 57).



However, reaction of formaldehyde

with deoxyribonucleic acid (DNA) has resulted in irreversible

denaturation that could interfere with DNA replication and result in

mutation (37)...."



http://www.dorway.com/wmonte.txt

Dr. Woodrow C. Monte  Aspartame: methanol, and the public health.

Journal of Applied Nutrition 1984;  36 (1):  42-54.

(62 references)   Professsor of Food Science

Director of the Food Science and Nutrition Laboratory

Arizona State University,  Tempe, Arizona 85287

6411 South River Drive #61 Tempe, Arizona 85283-3337

602-965-6938    woody.monte@... [now retired in New Zealand]  ]



Materials and Methods:

Aspartame. Aspartame labelled (C14) in the

methanol carbon was custom-prepared by Amersham (Amersham, UK). The

product had a specific activity of 433 MBq/mmol, and a chromatographic

purity >98%. The standard dose given orally to the rats was 4.5 Mbq per

kg of rat weight, always supplementing unlabelled aspartame (Sigma, St.

Louis, MO USA) to give a specific activity of 55 Mbq/mmol.



Acute and chronic administration of aspartame to normal rats:

Sixteen-week-old healthy adult male Wistar rats, weighing initially

380-460 g, were used. The rats were housed in collective cages in a

controlled environment (21-22 deg. C; 70-75% relative humidity;

lights on from 08:00 to 20:00), and were fed a standard chow pellet

(B&K, Sant Vicent dels Horts, Spain) and tap water ad libitum.



Two groups of rats were selected. The first group NC (Normal-Chronic,

N=5) received a daily oral gavage of 0.68 mmol per kg of rat weight (200

mg per kg) of a water suspension (2.5 mL/kg) of non-radioactive

aspartame (Sigma). This treatment was continued for 10 days. On day 11,

the rats were administered an gavage of 4.5 Mbq per kg of rat weight

of labelled aspartame in 68 µmol of cold aspartame per kg, in the same

volume of the standard gavage. The second group NA (Normal-Acute, N=l2)

was given a single dose of 4.5 Mbq per kg of rat weight of labelled

aspartame in 68 µmol of cold aspartame per kg of rat weight. Prior to

the administration of the last (or only) dose, blood was extracted from

the tail vein and used for the measurement of biochemical parameters

using a Spotchem dry strip (panel 1 and 2) analysis system (Menarini,

Milano, Italy).



The rats chronically treated (NC group) were killed by decapitation 6

hours after the administration of the labelled aspartame gavage. The

rats in the NA group were killed by decapitation at 15 or 30 min and at

1, 2, 6 or 24 hours after the administration of the final labelled

aspartame load. All animals were dissected, and samples of blood plasma

(heparinized), liver, kidneys, brain, cornea, retina, hind leg striated

muscle, epididymal fat pads and interscapular brown adipose tissue were

cut, weighed (blotted when necessary), and frozen in liquid nitrogen.

The samples were preserved at -20 degrees C until processed.



Tissue samples were homogenized in water: methanol (4:1) in order to

limit the losses of free methanol, using an all-glass Tenbroek

homogenizer. Aliquots of the homogenates were immediately counted for

radioactivity using a water-miscible scintillation cocktail (Ecolite,

from ICN, Costa Mesa, CA USA. Plasma samples were counted directly

after mixing with the scintillation cocktail. In all cases, two

countings, 24-hours apart were performed. In all cases we obtained the

same countings; there were no samples showing a significant loss of

radioactivity (purportedly due to the eventual evaporation of methanol

to the head space of the vial). Thus, it was assumed that no significant

amounts of labelled methanol were present in the final homogenates.

Aliquots of the homogenates were precipitated with trifluoroacetic acid

to remove the protein from supernatants, and the two fractions were

then counted separately.



Acute and chronic administration of aspartame to liver-damaged rats:

Six week-old healthy adult male Wistar rats weighing initially

100--120 g were used. The rats were housed and fed under the same

conditions described above for the controls. The rats were made

cirrhotic by means of three i.p. injections per week of carbon

tetrachloride diluted 1:1 with corn oil (36). The rats received

0.4 mL injections during the first 2 weeks, then 0.6 mL until week 6

and finally 0.8 mL until week 10, when the period of treatment was

considered finished, when the rats weighed 340-380 g.



Two groups of liver-damaged rats were selected. The first group CC

(Cirrhotic-Chronic, N=5) received a daily oral gavage of non-radioactive

aspartame for 10 days, and on day 11 they received 4.5 Mbq/kg of

labelled aspartame as in the NC group. The second group CA

(Cirrhotic-Acute, N=11) was given a single dose of 4.5 Mbq/kg of

labelled aspartame in 68 µmol of cold aspartame per kg as in the NA

group. Tail vein blood was sampled from these animals, and its plasma

stored frozen; this was later used to measure biochemical parameters as

in group NA.



The CC chronically treated rats were killed by decapitation-- as in the

control series-- 6 hours after the administration of the labelled oral

bolus of aspartame, and those in the CA group were killed at 15 or 30

min and at 1, 2, 6 or 24 hours after receiving the labelled

aspartame load. Samples of blood plasma and tissues were weighed,

frozen and stored at -20 degrees C until processed. Some samples of

liver were preserved in 4% formaldehyde and later used for the

preparation of stained tissue sections in order to determine the

degree of hepatic alteration (37). Blood and tissue samples were

processed as described for normal rats.



Statistical comparison between means was determined with standard

two-way anova programs, as well as with the Student's t test.



Nucleic acids analysis:

Two additional adult rats were treated as in group NC, but they received

the gavage for only three days. The last gavage contained 37 Mbq of

radioactive aspartame. After killing, blood plasma and liver samples

were obtained and frozen. Liver tissue was used for the extraction and

purification of total RNA and DNA using the Tripure (Boehringer

Mannheim, Germany) isolation reagents system. These preparations yielded

pure fractions of DNA, RNA, and protein. Nucleic acids content was

determined by uv light absorption at 260/280 nm (38), and protein with

the Bradford method (39). The radioactivity of these fractions was

measured and used for the estimation of their specific radioactivity.

The pooled DNA samples of the two rats used were hydrolysed with 88%

formic acid at 170 deg. C in a sealed glass ampoule (40), and the

corresponding constituting bases separated through thin layer

chromatography on 0.1 mm thick cellulose plates (5716 Merck,

Darmstadt, Germany), run against standards of C-14 labelled adenosine,

guanine and thymine (all from ICN, Costa Mesa, CA USA) containing their

cold counterparts (from Sigma, St Louis, MO USA). The mobile phases used

were isopropanol: 25% ammonium hydroxide (4:1 by volume) and butanol:

acetic acid: water (4:1:1 by volume) (41). Spot radioactivity was

measured by exposure of the chromatograms with the Bio-Rad Molecular

Imaging Screen-BI (Bio-Rad, Hercules, CA USA) for several days. The

plates were later read with a Bio-Rad Molecular Imager System GS-525

two-dimensional array radioactivity counter; this instrument provided a

printed "photographic plate" of the bidimensional distribution of

radioactivity in the chromatogram. Labelled standards of DNA bases were

used to determine whether the hydrolysed sample presented any

radioactivity in their spots. Cytosine was not included as standard

since no carbon from 1C pool participates in its structure through the

whole process of pyrimidine synthesis.



The DNA digest from the liver of rats exposed to labelled aspartame was

also analysed through HPLC, using a Kontron (Milano, Italy) HPLC fitted

on line with a diode array detector 440 (Kontron) and an eluate

scintillation detector LB 507 A (Beckman, Fullerton CA USA). The

instrument was run with the Data System 450-MT2/DAD (Kontron) software.

We used a scx cationic interchange column (Kontron) (250x4 mm, 10 µm),

maintained at 25ºC, and total flow was 0.8 mL/min. An isocratic gradient

of 100% 10 mM ammonium phosphate buffer pH 5.56 was used. The

scintillation detector used a cocktail ultima-flo M (Packard, Meriden

IL USA) with a mixture ratio of 3:1. A series of standards of adenine,

thymine and guanine were run under the same conditions. In all cases

the radioactivity in the fractions was recorded.



Protein analysis:

The rats used for nucleic acid analysis provided

enough plasma samples for protein analysis; plasma proteins were

selected because they could not be contaminated with nucleic acids.

The plasma proteins (0.100 mL aliquots) were precipitated with 10%

trifluoroacetic acid. Aliquots of the precipitated proteins were then

hydrolyzed for 48 h at 110°C in 6N HCl in Teflon-sealed tubes with

occasional shaking (42). The digests were filtered to remove the black

Maillard adducts (which retained part of the radioactivity). The amino

acids in the digests were derivatized with dinitrofluorobenzene, and the

DNP-amino acids were separated by bidimensional thin layer

chromatography (43) on 0.15 mm thick silicagel plates (Polygram Sil

G/UV254, Mocherey-Nagel, Düren, Germany). The presence of label in amino

acid spots was measured as in the case of nucleic acids using the

Bio-Rad Molecular Imager. In separate runs, 14C-labelled methionine

(NEN, Boston, MA, USA) diluted with cold methionine (Sigma) was added to

rat plasma, digested, derivatized and processed as indicated above.

Thus, the DNP-methionine spot was identified; in any case, the position

of standard amino acids in the bidimensional chromatogram was known

(43). The derivatization method used prevented the contamination of the

plates by radioactive materials different from amino acids, since only

the DNP-derivatized compounds were recovered.



An aliquot of 0.2 mL of blood serum albumin (Sigma) dissolved in water

(100g/L) was incubated for 2 h at 37ºC with 0.02 mL of a labelled

substrate preparation, containing 1 nmol and 5 kBq of 14C-labelled:

a) aspartame, b) formaldehyde (Amersham), c) formic acid (Sigma), or d)

methanol (Amersham). The samples were then precipitated, washed with

10% trifluoroacetic acid and the precipitates counted for

radioactivity.



The protein exposed to formaldehyde retained a large proportion of the

initial radioactivity added. In the cases of aspartame, formic acid and

methanol, only background values were obtained in the washed protein

precipitates, showing that none of these procedures resulted in stable

label attachment to proteins. The samples of albumin exposed to

formaldehyde label were processed in parallel to the sample of plasma

(i.e. hydrolysis, derivatization, and thin layer chromatography).

[Continued in Part 2/2]

***********************************************************************