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aspartame puts formaldehyde adducts into tissues, full text Trocho & Alemany 6.26.98: Murray 12.22.2 Part 1/2 rmforall

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  • Rich Murray
    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
    Message 1 of 1 , Dec 22, 2002
      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]
      ***********************************************************************
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