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rmforall
12-21-2005, 11:48 PM
http://groups.yahoo.com/group/aspartameNM/message/1263
many studies on endothelial injury (diabetic neuropathy) by adducts of
formaldehyde derived from methylamine from many of the same sources
as also supply methanol (formaldehyde), including aspartame:
PH Yu et al: DJ Conklin et al: Murray 2005.12.04

Gubisne-Haberle D, Yu PH, et al, September, 2004 full text:

"Methylamine,
a major metabolite of creatine (Mitchell and Zhang, 2001), can also be
derived from adrenaline,
lecithin,
dietary sources,
and cigarette smoke (Yu, 1998).

SSAO converts methylamine into formaldehyde,
hydrogen peroxide,
and ammonium,
all of which are cytotoxic.

Aminoacetone is derived from threonine and glycine, and its deamination
leads to the production of cytotoxic methylglyoxal.

Formaldehyde and methylglyoxal are extremely reactive chemicals,
which can interact with free amino or amide groups
to form Schiff's base,
subsequently methylene bridges, and produce irreversible covalently
cross-linked complexes between proteins, as well as between proteins
and single-stranded DNA (Bolt, 1987).

Increased protein aggregation and deposition is associated
with the aging process and with numerous pathological conditions,
such as the formation of plaques in the pancreas, brain, kidney,
and other organs."


Conklin DJ, et al, February 2004:

"Methylamine is both an exogenous
(present in cigarette smoke and wine and foods)
and an endogenous amine, and it is a metabolic end product
of diverse compounds, including epinephrine, carbamate insecticides,
creatine, nicotine, and sarcosine (35, 38, 48)."

"For example, MA excretion is elevated in rats after the administration
of SSAO inhibitors (34) and in humans after the consumption of
creatinine, certain fish and seafood,
and some fruits and vegetables (38)."

Gronvall JL, et al, August, 2000:

"Under the influence of semicarbazide-sensitive amine oxidase (SSAO),
methylamine is deaminated to formaldehyde, which can react
with various macromolecules and form irreversible adducts.
We hereby present an autoradiographic method of visualising SSAO
activity by measuring the in vivo formation of such adducts
from 14C-methylamine. [ in mice ]

Our results revealed high concentrations of radioactive deposits in the
intestinal wall, brown adipose tissue, spleen and bone marrow."


Relevant aspartame (methanol, formaldehyde, formic acid) studies are
listed at the end of this post.

Rich Murray, MA Room For All rmforall@comcast.net
505-501-2298 1943 Otowi Road Santa Fe, New Mexico 87505

http://groups.yahoo.com/group/aspartameNM/messages
group with 148 members, 1,263 posts in a public, searchable archive
http://RoomForAll.blogspot.com http://AspartameNM.blogspot.com

Dark wines and liquors, as well as aspartame, provide
similar levels of methanol, above 100 mg daily, for
long-term heavy users, 2 L daily, about 6 cans.

Methanol is inevitably largely turned into formaldehyde,
and thence largely into formic acid.
It is the major cause of the dreaded symptoms of "next
morning" hangover.

http://groups.yahoo.com/group/aspartameNM/message/1237
ubiquitous potent uncontrolled co-factors in nutrition research are
formaldehyde from wood and tobacco smoke and many sources,
including from methanol in dark wines and liquors, in pectins
in fruits and vegetables, and in aspartame: Murray 2005.12.04

http://groups.yahoo.com/group/aspartameNM/message/1250
aspartame causes cancer in rats at levels approved for humans,
Morando Soffritti et al, Ramazzini Foundation, Italy &
National Toxicology Program
of National Institute of Environmental Health Sciences
2005.11.17 Env. Health Pers. 35 pages: Murray

http://groups.yahoo.com/group/aspartameNM/message/925
aspartame puts formaldehyde adducts into tissues, Part 1/2
full text Trocho & Alemany 1998.06.26: Murray 2002.12.22

"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."

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. Mari├* 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@porthos.bio.ub.es
http://www.bq.ub.es/grupno/grup-no.html
************************************************** ****

Atherosclerosis. 1996 Feb; 120(1-2): 189-97.
Formaldehyde produced endogenously via deamination of methylamine.
A potential risk factor for initiation of endothelial injury.
Yu PH, Zuo DM. yup@sask.usask.ca
Department of Psychiatry, University of Saskatchewan,
Saskatoon, Canada.

Methylamine can be converted by semicarbazide-sensitive amine
oxidase (SSAO) to formaldehyde and hydrogen peroxide,
which have been proven to be toxic towards cultured endothelial cells.

We investigated whether or not these deaminated products
from methylamine can exert potentially hazardous toxic effects in vivo.

Long lasting residual radioactivity in different tissues was detected
following administration of [14C]-methylamine in the mouse.

Approximately 10% of the total administered radioactivity could
even be detected 5 days after injection of [14C]-methylamine.

Eighty percent of the formation of irreversible adducts can be
blocked by a highly selective SSAO inhibitor,
(E)-2-(4-fluorophenethyl)-3-fluoroallylamine hydrochloride
(MDL-72974A).

The residual radioactivity was primarily associated with
the insoluble tissue components and the soluble macromolecules.

Radioactively labelled macromolecules were fragmented following
enzymatic proteolysis.

Results suggest that the formaldehyde derived
from methylamine interacts with proteins in vivo.

In the streptozotocin-induced diabetic mice,
both SSAO activity and the formation of residual radioactivity
were found to be significantly increased in the kidney.

Chronic administration of methylamine enhances blood prorenin level,
which strongly suggests that uncontrolled deamination of methylamine
may be a risk factor for initiation of endothelial injury,
and subsequent genesis of atherosclerosis. PMID: 8645360
************************************************** *****

Diabetologia. 1997 Nov; 40(11): 1243-50.
Aminoguanidine inhibits semicarbazide-sensitive amine oxidase activity:
implications for advanced glycation and diabetic complications.
Yu PH, Zuo DM.
Department of Psychiatry,
University of Saskatchewan, Saskatoon, Canada.

Aminoguanidine, a nucleophilic hydrazine,
has been shown to be capable of blocking the formation
of advanced glycation end products.
It reduces the development of atherosclerotic plaques
and prevents experimental diabetic nephropathy.
We have found that aminoguanidine
is also quite potent at inhibiting semicarbazide-sensitive amine oxidase
(SSAO) both in vitro and in vivo.
The inhibition is irreversible.
This enzyme catalyses the deamination
of methylamine and aminoacetone,
which leads to the production of cytotoxic formaldehyde
and methylglyoxal, respectively.
Serum SSAO activity was reported to be increased
in diabetic patients and positively correlated
with the amount of plasma glycated haemoglobin.
Increased SSAO has also been demonstrated
in diabetic animal models.
Urinary excretion of methylamine is substantially increased
in the rats following acute or chronic treatment with aminoguanidine.
Urinary methylamine levels were substantially increased
in streptozotocin (STZ)-induced diabetic rats
following administration of aminoguanidine.
The non-hydrazine SSAO inhibitor
(E)-2-(4-fluorophenethyl)-3-fluoroallylamine hydrochloride
(MDL-72974A) has been shown to reduce urinary excretion
of lactate dehydrogenase (an indicator of nephropathy)
in STZ-induced diabetic rats.

Formaldehyde not only induces protein crosslinking,
but also enhances the advanced glycation of proteins in vitro.

The results support the hypothesis that increased
SSAO-mediated deamination may be involved
in structural modification of proteins
and contribute to advanced glycation in diabetes.

The clinical implications for the use of aminoguanidine
to prevent glycoxidation have been discussed. PMID: 9389414
************************************************** *****

J Neural Transm Suppl. 1998; 52: 201-16.
Deamination of methylamine and angiopathy; toxicity of formaldehyde,
oxidative stress and relevance to protein glycoxidation in diabetes.
Yu PH.
Neuropsychiatry Research Unit, College of Medicine,
University of Saskatchewan, Saskatoon, Canada.

Semicarbazide-sensitive amine oxidase (SSAO) is located
in the vascular smooth muscles, retina, kidney and the cartilage tissues,
and it circulates in the blood.

The enzyme activity has been found to be significantly increased
in blood and tissues in diabetic patients and animals.

Methylamine and aminoacetone are endogenous substrates for SSAO.

The deaminated products are formaldehyde
and methylglyoxal respectively, as well as H2O2 and ammonia,
which are all potentially cytotoxic.

Formaldehyde and methylglyoxal are cytotoxic towards endothelial cells.

Excessive SSAO-mediated deamination may
directly initiate endothelial injury and plaque formation,
increase oxidative stress,
which can potentiate oxidative glycation, and/or LDL oxidation
and damage vascular systems.

Formaldehyde is also capable of exacerbating advanced glycation,
and thus increase the complexity of protein cross-linking.

Uncontrolled SSAO-mediated deamination may be
involved in the acceleration of the clinical complications in diabetes.
Publication Types: Review PMID: 9564620
************************************************** *****

Clin Nephrol. 1998 May; 49(5): 299-302.
Impairment of methylamine clearance in uremic patients
and its nephropathological implications.
Yu PH, Dyck RF.
Department of Psychiatry, Royal University Hospital of Saskatchewan,
University of Saskatchewan, Saskatoon, Canada.

The urinary levels of methylamine were analyzed
by an HPLC/fluorometric method following derivatization of the amine
with O-phthaldialdehyde (OPA).
The excretion of methylamine in the uremic patients
was found to be dramatically reduced.
The impairment of clearance of methylamine explains why this amine
was substantially increased in the serum of uremic patients.
Increased deamination of methylamine would enhance
formaldehyde and oxidative stresses, i.e. in the blood vessels,
and cause vascular damage.
This may be related to the increased risk of angiopathy associated
with renal failure, and accelerate the progression of renal failure.
PMID: 9617493
************************************************** *****

Life Sci. 1998; 63(23): 2049-58.
Deamination of methylamine and aminoacetone increases aldehydes
and oxidative stress in rats.
Deng Y, Boomsma F, Yu PH.
Neuropsychiatry Research Unit,
University of Saskatchewan, Saskatoon, Canada.

Semicarbazide-sensitive amine oxidase (SSAO)-mediated
deamination of methylamine and aminoacetone in vitro
produces carbonyl compounds, such as formaldehyde and
methylglyoxal, which have been proposed to be cytotoxic
and may be responsible for some pathological conditions.
An HPLC procedure was developed to assess different aldehydes,
which were derivatized with 2,4-dinitrophenylhydrazine (DNPH).
We have demonstrated in vivo deamination of methylamine and
aminoacetone by examining the excretion of formaldehyde and
methylglyoxal, respectively, in rats.
Following chronic administration of methylamine,
the urinary level of malondialdehyde (MDA),
an end product of lipid peroxidation,
was also found to be substantially increased.
A selective SSAO inhibitor blocked the increase of MDA.
The results support the idea that increased SSAO-mediated deamination
of methylamine and aminoacetone can be a potential cytotoxic risk factor.
PMID: 9839528
************************************************** *****

Neurochem Res. 1998 Sep; 23(9): 1205-10.
Increase of formation of methylamine and formaldehyde in vivo after
administration of nicotine and the potential cytotoxicity.
Yu PH.
Department of Psychiatry, University of Saskatchewan, Saskatoon,
Canada. yup@sask.usask.ca

Methylamine is a constituent of cigarette smoke and the major end
product of nicotine metabolism.
Smoking or nicotine can induce the release of adrenaline,
which is in turn deaminated by monoamine oxidase,
also producing methylamine.

We found that the urinary level of methylamine was significantly elevated
following administration of nicotine (25 mg/Kg, i.p.).

Semicarbazide-sensitive amine oxidase (SSAO) inhibitors
further increased the excretion of methylamine induced by nicotine.

Following administration of L-(-)-[N-methyl-3H]nicotine
long-lasting irreversible radioactive adducts were detected in
different mouse tissues and
such adduct formation could be blocked by selective SSAO inhibitors.

These adducts are probably cross-linked oligoprotein complexes
cross-linked by formaldehyde.

The findings support the idea that nicotine can enhance
SSAO/methylamine-mediated increase of formaldehyde and
oxidative stress and this could in part contribute the adverse effect
of health associated with smoking. PMID: 9712192
************************************************** *****

Atherosclerosis. 1998 Oct; 140(2): 357-63.
Endogenous formaldehyde as a potential factor of vulnerability of
atherosclerosis: involvement of semicarbazide-sensitive
amine oxidase-mediated methylamine turnover.
Yu PH, Deng YL. yup@sask.usask.ca
Department of Psychiatry,
University of Saskatchewan, Saskatoon, Canada.

The mouse is known to be highly resistant to atherosclerosis.
However, some inbred mouse strains are vulnerable to atherosclerosis
when they are fed a high-cholesterol, high-fat diet.
Increased deamination of methylamine (MA) and
the subsequent production of formaldehyde
has been recently shown to be a potential risk factor of atherosclerosis.
In the present study semicarbazide-sensitive amine oxidase
(SSAO)-mediated MA turnover in C57BL/6 mouse,
a strain very susceptible to atherosclerosis,
has been assessed in comparison to a moderate, i.e. BALB/c,
and resistant, i.e. CD1, mouse strains.
Kidney and aorta SSAO activities were found
to be significantly increased in C57BL/6
in comparison to BALB/c and CD1 mice.
A significant increase of urinary MA and formaldehyde
were detected in C57BL/6. [14C]MA
following intravenous injection would be quickly metabolized by SSAO.
The labeled formaldehyde product would cross link with proteins.
C57BL/6 exhibits significantly higher labeled protein adducts
than BALB/c and CD1 in response to [14C]MA.
The results indicated that mice vulnerable to atherosclerosis
possess an increased SSAO-mediated MA turnover.
The increase of production of formaldehyde, possibly other aldehydes,
may induce endothelial injury or be chronically involved
in protein cross-linking and subsequent angiopathy.
PMID: 9862279
************************************************** *****

Diabetologia. 2002 Sep; 45(9): 1255-62. Epub 2002 Aug 8.
Involvement of semicarbazide-sensitive amine oxidase-mediated
deamination in atherogenesis in KKAy diabetic mice
fed with high cholesterol diet.
Yu PH, Wang M, Deng YL, Fan H, Shira-Bock L. yup@usask.ca
Neuropsychiatry Research Unit,
University of Saskatchewan, Saskatoon, Saskatchewan, Canada.
Fan, H. huf289@duke.usask.ca
Room A120 Medical Research Building
(306) 966-2552 (306) 966-8830

AIMS/HYPOTHESIS:
Semicarbazide-sensitive amine oxidase has been recognised
to be a potential risk factor in vascular disorders associated
with diabetic complications and to be related to mortality in patients
suffering from heart disease.
This enzyme, associated with the vascular system,
catalyses the deamination of methylamine and aminoacetone,
and also acts as an adhesion molecule related to leucocyte trafficking
and inflammation.
The deaminated products include the toxic aldehydes, formaldehyde and
methylglyoxal, respectively, hydrogen peroxide and ammonia.
MATERIALS AND METHODS:
In this study, the KKAy mouse, a strain possessing features
closely resembling those of Type II (non-insulin-dependent)
diabetes mellitus has been used to substantiate the hypothesis.
Vascular lesions were induced via chronic feeding
of a high cholesterol diet.
RESULTS:
Both MDL-72974A, a selective mechanism-based
semicarbazide-sensitive amine oxidase inhibitor and
aminoguanidine effectively inhibited aorta semicarbazide-sensitive amine
oxidase activity,
and caused a substantial increase in urinary methylamine,
and a decrease in formaldehyde and methylgloxal levels.
Inhibition of semicarbazide-sensitive amine oxidase
also reduced oxidative stress, as shown by a reduction of
malondialdehyde excretion.
Both MDL-72974A and aminoguanidine reduced albuminuria,
proteinuria and the number of atherosclerotic lesions in animals fed
with a cholesterol diet over a period of treatment for 16 weeks.
CONCLUSION/INTERPRETATION:
Increased semicarbazide-sensitive amine oxidase-mediated
deamination could be involved in the cascade of atherogenesis
related to diabetic complications. PMID: 12242458
************************************************** *****

Anal Biochem. 2003 Jul 15; 318(2): 285-90.
A novel sensitive high-performance liquid
chromatography/electrochemical procedure for measuring formaldehyde
produced from oxidative deamination of methylamine
and in biological samples.
Yu PH, Cauglin C, Wempe KL, Gubisne-Haberle D.
Neuropsychiatry Research Unit, Department of Psychiatry,
University of Saskatchewan, Saskatoon, Saskatchewan,
Canada S7N 5E4. yup@usask.ca

Formaldehyde is a well-known environmental toxic hazard.
It is also a product of oxidative deamination of methylamine
catalyzed by semicarbazide-sensitive amine oxidase (SSAO).
Increased SSAO-mediated deamination has been implicated
in some pathophysiological conditions, such as diabetic complications.
The measurement of formaldehyde in the enzymatic reactions and
in vivo production using conventional methods was not
straightforward due to limitations of selectivity and sensitivity.
A novel high-performance liquid chromatography
(HPLC)/electrochemical procedure for the measurement of
formaldehyde has been developed.
The measurement is based on the formation of adducts between
formaldehyde and dopamine.
These adducts can be selectively purified and concentrated
using a batch method of alumina absorption,
separated by HPLC, and electrochemically quantified.
The method is highly selective and substantially more sensitive,
i.e., detection of picomole levels of formaldehyde,
than the conventional methods.
The procedure not only facilitates the assessment of SSAO
activity in vitro but also is useful for assessing formaldehyde
in tissues and biological fluids. PMID: 12814633
************************************************** *****

http://ajpheart.physiology.org/cgi/content/full/286/2/H667 free full text

Am J Physiol Heart Circ Physiol. 2004 Feb; 286(2): H667-76.
Vasoactive effects of methylamine in isolated human blood vessels:
role of semicarbazide-sensitive amine oxidase, formaldehyde,
and hydrogen peroxide.
Conklin DJ, Cowley HR, Wiechmann RJ,
Johnson GH, Trent MB, Boor PJ.
Department of Medicine, University of Louisville, Louisville, KY 40202,
USA. dj.conklin@louisville.edu

It is hypothesized that methylamine (MA) and semicarbazide-sensitive
amine oxidase (SSAO) activity are involved in the cardiovascular
complications in human diabetics.

To test this, we

1) determined the acute vasoactive effects of
MA (1-1,000 micromol/l) in uncontracted and
norepinephrine (NE 1 micromol/l)-precontracted human blood vessels
used for coronary artery bypass grafts [left internal mammary artery
(LIMA), radial artery (RA), and right saphenous vein (RSV)];

2) tested whether MA effects in LIMA and RSV were dependent on
SSAO activity
using the SSAO inhibitor semicarbazide (1 mmol/l, 15 min);

3) determined the effects of MA metabolites formaldehyde and hydrogen
peroxide in LIMA and RSV;

4) tested whether the MA response was nitric oxide, prostaglandin,
or hyperpolarization dependent;

5) measured the LIMA and RSV cGMP levels after MA exposure;

and 6) quantified SSAO activity in LIMA, RA, and RSV.

In NE-precontracted vessels,
MA stimulated a biphasic response in RA and RSV (rapid contraction
followed by prolonged relaxation) and dominant relaxation in LIMA
(mean +/- SE, % relaxation 55.4 +/- 3.9, n == 30).

The MA-induced relaxation in LIMA was repeatable, nontoxic,
and age independent.

Semicarbazide significantly blocked MA-induced relaxation
(% inhibition 82.5 +/- 4.8, n == 7) and
SSAO activity (% inhibition 98.1 +/- 1.3, n == 26) in LIMA.

Formaldehyde (% relaxation 37.3 +/- 18.6, n == 3) and
H(2)O(2) (% relaxation 55.6 +/- 9.0, n == 9) at 1 mmol/l
relaxed NE-precontracted LIMA comparable with MA.

MA-induced relaxation in LIMA was nitric oxide, prostaglandin,
and possibly cGMP independent and blocked by hyperpolarization.

We conclude that vascular SSAO activity may convert
endogenous amines, like MA, to vasoactive metabolites.
PMID: 14715500

"A CURRENT HYPOTHESIS STATES that chronic methylamine
(MA) exposure induces vascular injury and promotes vascular disease,
including atherosclerosis, in humans via semicarbazide-sensitive amine
oxidase-mediated (SSAO) metabolism of MA to injurious metabolites:
formaldehyde, H2O2, and ammonia (NH3) (19, 50-52).

Recent clinical and experimental studies support such a relationship.
Altered plasma MA levels, MA excretion, and elevated plasma SSAO
activity are present in human diseases associated with chronic vascular
pathology [e.g., diabetes mellitus and uremia
(Refs. 3, 5, 6, 27, and 49, for reviews, see Refs. 19 and 51)].

In the case of Type I diabetes, SSAO plasma levels increase
at the onset of disease (6) and plasma SSAO activity
positively correlates with the amount of glycosylated hemoglobin,
an indicator of the severity of complications in human diabetics (5, 43).

Similarly, plasma SSAO activity is elevated within 2 wk after
streptozotocin-induced diabetes in rats (22).

Thus much circumstantial evidence links MA levels and SSAO
activity to the development of vascular pathology in diabetic humans,
and recently the suggestion has been made that therapeutic inhibition
of SSAO could slow the progression of vascular disease
(17, 19, 51, 53).

MA is a ubiquitous primary amine derived from a multitude of sources
and is preferentially metabolized by SSAO compared with other amine
oxidases (16, 33, 47).

Methylamine is both an exogenous
(present in cigarette smoke and wine and foods)
and an endogenous amine, and it is a metabolic end product
of diverse compounds, including epinephrine, carbamate insecticides,
creatine, nicotine, and sarcosine (35, 38, 48).

MA metabolism in rats and humans appears to be due largely to
SSAO activity (16, 34).

For example, MA excretion is elevated in rats after the administration
of SSAO inhibitors (34) and in humans after the consumption of
creatinine, certain fish and seafood, and some fruits and vegetables (38).

Moreover, MA is metabolized by vascular homogenates,
including the rat aorta and human umbilical artery,
to formaldehyde (9, 39).

Finally, SSAO inhibitors prevent MA toxicity
in cultured endothelial cells (47).

Thus present evidence supports the concept that MA,
exogenous and endogenous, is converted to metabolites,
formaldehyde and H2O2, by endogenous SSAO activity.

The plasma and tissue forms of the SSAO (EC 1.4.3.6 [EC] )
enzymes are distinct from the monoamine oxidases (MAO),
diamine oxidases, and polyamine oxidases (33).

The copper-containing SSAOs share common features including
insensitivity to MAO inhibitors (e.g., clorgyline, deprenyl),
preference for aliphatic amines and the aromatic benzylamine,
and inhibition by carbonyl-containing compounds,
such as semicarbazide, for which the most current name is derived
(33, 47).

SSAO activity is present in all mammalian cardiovascular tissues tested,
including plasma/serum, the aorta, and the heart with the most
concentrated SSAO activity present in the mammalian aorta,
including those of the human and rat (11, 13, 14, 32, 33, 37, 39, 44).

This high level of SSAO activity in the cardiovascular tissues implies
functionality, although a specific function for the SSAO enzyme
has yet to be established (7, 33, 51).

To better understand the contribution of MA and vascular SSAO
activity to human vascular physiology and pathophysiology,
we studied the acute vascular effects of MA in isolated human blood
vessels of patients undergoing coronary artery bypass surgery.

We tested whether MA-induced vascular effects were dependent
on blood vessel SSAO activity by using the SSAO inhibitor
semicarbazide.

Additionally, we assessed the role of age, endothelial function,
and contribution of nitric oxide and prostaglandins in the MA effects
in the left internal mammary artery (LIMA).

Finally, we measured the SSAO activity present
in the three human blood vessel types examined,
the LIMA, radial artery (RA), and right saphenous vein (RSV)."

"DISCUSSION

This is the first report of the acute vasoactive effects of MA in
isolated human blood vessels.

Several of our findings clearly appear at odds with the proposed role
of MA as a potent vascular toxicant in humans.

We find the most prominent effect of MA in isolated human
blood vessels is a generally robust yet benign relaxation.

This relaxation, highly expressed in LIMA, is dependent on vascular
SSAO activity and, more importantly, is reversible and repeatable.

It is quite distinct from the SSAO-dependent, yet quite injurious,
actions of allylamine, a wellknown cardiovascular toxicant,
or from effects of the ,-unsaturated aldehyde acrolein
in isolated rat TA, rat coronary arteries, and human blood vessels,
where both agents produce vasospasm
in rat coronary arteries (12, 13).

Our present findings in isolated human blood vessels,
while somewhat surprising, suggest that vascular SSAO activity
(and possibly MA) may be a source
of vasoactive signaling molecules that generally relax blood vessels.

MA Responses in Human CABG Vessels
Are Dependent on Vascular SSAO Activity

MA effects in isolated human LIMA are clearly dependent
on vascular SSAO activity.

Data from several experiments support this conclusion.

The pretreatment of isolated LIMA with semicarbazide
(1 mmol/l, over 15 min before the addition of MA)
significantly inhibits MA responses.

The inhibition was specific for SSAO activity,
because semicarbazide pretreatment had no direct or indirect
effects on vascular function.

Moreover, semicarbazide pretreatment and posttreatment inhibition
of MA relaxation in LIMA precludes
a nonspecific mechanism of action of MA
[e.g., MA does not directly block adrenergic receptors (31)].

We show here, for the first time, that three human blood vessels
used as CABG vessels possess similar levels of relatively abundant,
age-independent (age range == 45-81 yr) amine oxidase activity
that is inhibited by semicarbazide in a concentration-dependent
manner (Refs. 13 and 37,
although our reported inhibition in human saphenous vein
appears more sensitive,
IC50 == 500 vs. 14.7 ┬Ámol/l, Ref. 37 vs. present study, respectively).

Recently, we found similarly abundant SSAO activity
in homogenized human coronary arteries of accident victims
aged 7-71 yr (13).

Human aortic SSAO activity was 10 times more concentrated
than in human coronary arteries, LIMA, RA, RSV, and other
human blood vessels but was also age independent (23, 32).

Similarly the age of rats (12-14 vs. 52 wk) had little effect
on TA responses to MA exposure in our present study.

So whereas age and perhaps atherosclerosis (23) appear
to have little effect on SSAO expression in the blood vessel wall,
the factors important in the regulation of overall SSAO expression
(e.g., genetic, diabetes, environmental, etc.)
remain to be identified (5, 6, 19, 51).

Dependence on SSAO activity is a hallmark
of allylamine cardiovascular toxicity in vivo, in isolated blood vessels,
and in cultured cardiovascular cells (2, 8, 52).

The cellular toxicity of MA, in contrast, has a more complicated history
because MA toxicity is orders of magnitude less toxic than allylamine
in cultured cardiovascular cells (14, 30, 52).

Whereas allylamine and MA are relatively similar substrates
for homogenized rat aortic SSAO activity
[Kms == 145.2 and 246.7 ┬Ámol/l, respectively (47)],
human blood vessels have strikingly different Kms for MA
[e.g., cerebral microvessels, 22 ┬ÁM (Ref. 11),
umbilical artery, 832 ┬ÁM (see Ref. 33 for a review)].

While the disparity in toxicity between these two amines
is likely due to the metabolism of allylamine to acrolein and
MA to the less toxic formaldehyde,
it is likely that affinity and catalytic rates and, thus,
specificity of various SSAOs for different amines vary
dramatically from site to site (11, 33, 51).

However, biological activity is not predictable solely based
on substrate availability and Km.

In our present study, for example, rat aortic responses were
minimal in the presence of 1 mmol/l MA
despite the fact that the rat aorta possessed high SSAO activity
and an adequate affinity (9, 32).

Nonetheless, substrate specificity and distribution of SSAOs
are likely important to the potential contribution of MA
to differential cardiovascular pathology in humans
[e.g., atherosclerosis, retinopathy, nephropathy,
and coronary artery disease (51)].

Mechanism of Action of MA Responses in Human LIMA

Because effects of MA in LIMA and RSV appear dependent
on SSAO activity, it follows that the effects of MA
are likely due to one or more of MA's metabolites:
formaldehyde, H2O2, and NH3.

The MA responses in LIMA and RSV were similar,
qualitatively and quantitatively,
to both formaldehyde- and H2O2-induced responses.

For example, H2O2 (1 mmol/l) pretreatment inhibits
subsequent NE contractions in rabbit aorta,
whereas in precontracted vessels H2O2 induces
contractions, relaxations, or biphasic responses
dependent on the blood vessel type (25, 28, 40).

More specifically, H2O2 induced biphasic responses
in rat TA and human RA and RSV
are similar in appearance to our MA responses
in isolated RA and RSV (Ref. 28 and present study).

In the arteries, however, weak contractions
are similar for MA and formaldehyde exposures,
whereas H2O2 is much more efficacious.

Notably, vasospasm occurs more often in human RA and RSV
compared with LIMA, and our general reactivity findings
are consistent with these data (10, 24).

Additionally, MA, formaldehyde, and H2O2 effects generally
are reversible at 1 mmol/l in the rabbit aorta, rat TA,
and human blood vessels (Refs. 25, 28, and 40 and present study).

Finally, the apparent EC50s for MA, formaldehyde, and H2O2
in all three human vessels are very similar
(approximate range == 200-300 ┬Ámol/l),
although formaldehyde responses are only observed at 1 mmol/l.

Thus we conclude that both formaldehyde and H2O2
produced by SSAO metabolism of MA contribute to the
MA-induced vascular responses in human LIMA.

MA-, formaldehyde-, and H2O2-induced relaxation in LIMA
may be dependent on vascular smooth muscle cell membrane
hyperpolarization via activation of K+ channels
because HI K+ precontraction decreases their ability
to stimulate relaxation.

Hyperpolarization is used in H2O2-induced relaxation
of the rat aorta and porcine coronary artery (4, 28),
and K+ channels may act as redox-sensitive targets for H2O2
as proposed in the mechanism
of hypoxic pulmonary vasoconstriction and
hypoxia-induced vasodilation (see Ref. 1 for a review).

Moreover, H2O2 is considered an EDHF
in human mesenteric arteries
but not in the carbachol-induced relaxation in human RA (21, 36).

In addition, the relaxation (and generally low toxicity)
observed with 1 mmol/l formaldehyde in human blood vessels,
while surprising, is not without precedent
because formaldehyde (660 ┬Ámol/l) relaxes NE-precontracted
but not 25 mmol/l KCl-depolarized rabbit aortas
by inhibition of Ca2+ influx and NE inactivation in vitro (42).

However, we cannot rule out that the
strong depolarizing stimulus used in the present study
(i.e., 100 mmol/l KCl) may have inhibited relaxation pathways
stimulated during NE-precontraction
by MA, formaldehyde, and H2O2.

Regardless of which specific metabolite is involved,
MA-induced relaxation clearly appears independent of
endothelial NO or prostanoid release in human LIMA.

This conclusion is supported by the observation
that inhibition of endothelial nitric oxide synthase activity
with L-NAME significantly reduced the ACh relaxation
but had no effect on the MA relaxation.

Yet, if the endothelium is involved,
it perhaps is releasing an EDHF in response to MA metabolites.

Hamilton et al. (21) propose that blood vessels with reduced
EDRF capacity compensate with enhanced EDHF production.

Because no inverse relationship was observed
between ACh and MA relaxations in LIMA from patients
with significantly reduced EDRF,
we favor the more likely role of formaldehyde or H2O2
generated at the vascular smooth muscle cell plasma membrane
[ i.e., the location of SSAO (44) ]
acting as autocrine and/or paracrine factor(s).

It is unclear whether MA-induced relaxations are dependent
on increased cGMP.

While the MA-induced relaxation in RSV appears
cGMP independent,
there is a weak positive association between cGMP levels
and the % relaxation to MA in LIMA.

However, even though many relaxations are mediated by cGMP,
it is possible that the formation of formaldehyde and/or H2O2
at the vascular smooth muscle cell plasma membrane directly
relax(es) vessels by activation of K+ channels, thiol oxidation,
inhibition of Ca2+ influx, adrenergic inactivation,
or some combination of these mechanisms.

Implications for Human Health:
Pharmacological and Toxicological Considerations

In the present study, MA at 1-1,000 ┬Ámol/l had
no easily observable adverse effects in isolated human blood vessels.

However, it is clear that massive MA exposure can be lethal in humans.
As a result of an accidental spill of purified liquid MA,
35 Chinese persons of 7-71 yr of age
and a nearly equal male-to-female distribution were hospitalized
7 to 8 h postexposure with 6 resulting fatalities (46).

Overt toxicity in these patients included significant
cardiovascular symptoms of tachycardia and
low or "unmeasurable" blood pressure and pulse.
These symptoms are consistent with the scenario
of severe systemic hypotension, accompanied by reflex tachycardia,
declining cerebral perfusion, and ultimate coma
(10 of 35 people suffered light to deep coma).

On the basis of the MA-induced prolonged and robust relaxation
in LIMA, RA, and RSV blood vessels in our study,
one might predict severe hypotension would follow
a systemic, high-level MA dose
[ assuming the MA relaxation occurs in the resistance vessels --
a testable hypothesis that is supported by the ubiquitous presence
of SSAO activity in human conductance and resistance blood vessels
(11, 13, 23, 32, 39) ].

With the exception of an acute toxic exposure,
reaching a toxic level dosage of MA in humans is unlikely.

Although MA is present in a variety of common exogenous sources,
including wine, cigarette smoke, a variety of foods,
and as a metabolite of nicotine,
it is unlikely that anyone could reach acute toxic doses by these paths
(35, 38, 48), because the estimated normal human plasma
[MA] == under 1-5 ┬Ámol/l and the uremic human plasma
[MA] == 10-20 ┬Ámol/l (3, 45).

Even though [MA] in plasma is elevated in diabetic humans,
especially those with chronic renal dysfunction,
it is unlikely to exceed 50 ┬Ámol/l (27, 49).

Some red wines possess up to 5 mg/l MA (35),
which could elevate plasma MA levels by 30 ┬Ámol/l
in a 70-kg male assuming the consumption of 1 liter of wine
and 100% absorption.

Our data suggest that exposure of isolated human blood vessels
to 80 ┬Ámol/l MA would produce a very modest vascular relaxation.

It has been suggested that treatment of human diabetics
with a SSAO inhibitor, such as aminoguanidine,
may provide vascular protection by inhibiting SSAO activity,
diminishing amine metabolism,
subsequent aldehyde and adduct formation,
and reducing advanced glycation end products (AGEs)
(17, 20, 37, 48).

However, it is unclear how a SSAO inhibitor would affect the
variety of cardiovascular and noncardiovascular pools
of SSAO activity,
including adipose and gastrointestinal tract smooth muscle,
where the function of SSAO also remains undetermined (18).

For example, the SSAO protein homolog vascular adhesion
protein-1 (VAP-1) expressed in endothelial and vascular
smooth muscle cells mediates lymphocyte binding (26, 41).

Whereas previous studies detect little to no SSAO activity
in endothelial cell cultures (50),
purified human and bovine brain microvessels possess
concentrated SSAO activity with relatively high affinity for MA (11).

Thus endothelial cell VAP-1 may contribute
to overall vascular SSAO enzymatic activity
and also to nonenzymatic functions associated with amine binding,
a pathway we cannot rule out in the present study.

Moreover, there are no specific inhibitors for each tissue-specific
pool of SSAO activity
(Refs. 17 and 37 and L. Sayre, unpublished observations),
and thus we should tread cautiously because the effects
of SSAO inhibition or overexpression of SSAO
in developing rats are detrimental to vascular tissue (19, 29)
and remain unknown in the adult,
although treatment of Parkinson's patients
with carbidopa or hydralazine may indicate limited systemic toxicity
(for reviews, see Refs. 19 and 51)."
************************************************** *****

J Pharmacol Exp Ther. 2004 Sep; 310(3): 1125-32. Epub 2004 May 5.
Protein cross-linkage induced by formaldehyde
derived from semicarbazide-sensitive amine oxidase-mediated
deamination of methylamine.
Gubisne-Haberle D, Hill W, Kazachkov M, Richardson JS, Yu PH.
Diana Gubisne-Haberle, Wayne Hill, Mychaylo Kazachkov,
J. Steven Richardson, and Peter H. Yu
Neuropsychiatry Research Unit, University of Saskatchewan,
Saskatoon, SK S7N 5E4, Canada.
Kazachkov, M. myk809@duke.usask.ca Room A136
Medical Research Building (306) 966-8823 (306) 966-8830
Richardson, JS js.r@usask.ca Room A127
Health Sciences Building (306) 966-6301 (306) 966-6220

Semicarbazide-sensitive amine oxidase (SSAO)
catalyzes the conversion of methylamine to formaldehyde.

This enzyme is located on the surface of the cytoplasmic membrane
and in the cytosol of vascular endothelial cells,
smooth muscle cells, and adipocytes.

Increased SSAO activity has been found in patients
with diabetes mellitus, chronic heart failure,
and multiple types of cerebral infarcts
and is associated with obesity.

Increased SSAO-mediated deamination
may contribute to protein deposition, the formation of plaques,
and inflammation, and thus may be involved in
the pathophysiology of chronic vascular and neurological disorders,
such as diabetic complications, atherosclerosis, and Alzheimer's disease.

In the present study, we demonstrate the induction of cross-linkage
of formaldehyde with the lysine moiety of peptides and proteins.

Formaldehyde-protein adducts were reduced
with sodium cyanoborohydride, hydrolyzed in hydrochloric acid,
and the amino acids in the hydrolysates were derivatized
with fluorenylmethyl chloroformate and
then identified with high-performance liquid chromatography.

We further demonstrate that incubation of methylamine
in the presence of SSAO-rich tissues, e.g., human brain meninges,
results in formaldehyde-protein cross-linkage
of particulate bound proteins as well as of soluble proteins.

This cross-linkage can be completely blocked
by a selective inhibitor of SSAO.

Our data support the hypothesis that the SSAO-induced
production of formaldehyde may be involved in the alteration
of protein structure, which may subsequently cause protein
deposition associated with chronic pathological disorders.
PMID: 15128865
************************************************** *****

Toxicology. 2005 Jun 1; 210(2-3): 235-45.
Cytotoxic effect of formaldehyde with free radicals via increment
of cellular reactive oxygen species.
Saito Y, Nishio K, Yoshida Y, Niki E.
Human Stress Signal Research Center (HSSRC),
National Institute of Advanced Industrial Science and Technology
(AIST), 1-8-31 Midorigaoka, Ikeda, Osaka 563-8577, Japan.
yoshiro-saito@aist.go.jp ; kazukun@jg7.so-net.ne.jp ;
etsuo-niki@aist.go.jp ;

It is well known that formaldehyde (HCHO) and reactive oxygen
species (ROS), such as free radicals,
are cytotoxic as well as potentially carcinogenic.

Although the individual effects of these reactants on cells
have been investigated,
the cytotoxicity exerted by the coexistence of HCHO and
reactive radicals is poorly understood.

The present study using Jurkat cells demonstrated that the coexistence
of HCHO with water-soluble radical initiator,
2,2'-azobis-[2-(2-imidazolin-2-yl)propane] dihydrochloride (AIPH)
dramatically decreased cell viability, and that under such conditions
scant cell death was observable induced by either of the reactants alone.

Based on the results of phosphatidylserine exposure and caspase
activation, this observed cell death, in fact, was apparently necrotic
rather than apoptotic.

To understand the mechanisms of the cell toxicity of HCHO and AIPH,
we assessed two kinds of oxidative stress markers
such as cellular glutathione (GSH) content and cellular ROS,
and the DNA-protein cross-links,
which formed as the result of HCHO treatment.

A marked decrease in total cellular GSH was observed
not only in the case of the coexistence conditions
but also with AIPH alone.

Dichlorodihydrofluorescein (DCF) assay revealed
that cellular ROS were synergistically increased before cell death.

The formation of DNA-protein cross-links was observed
in the presence of HCHO and AIPH,
and the extent was similar to HCHO alone.

Co-incubation with semicarbazide, which inactivates HCHO,
prevented this cell death induced by a combination of HCHO
and AIPH.

Semicarbazide also exhibited an inhibitory effect
on the synergistic increment of cellular ROS and
the formation of DNA-protein cross-links.

These results suggest that the free radicals from AIPH
induced GSH reduction,
while HCHO resulted in the formation of DNA-protein cross-links,
eventuating in a synergistic, incremental increase of cellular
ROS and cell death brought about by this combination.
PMID: 15840437
************************************************** ******

http://ajpendo.physiology.org/cgi/content/full/286/4/E634 free full text
Am J Physiol Endocrinol Metab. 2004 Apr;286(4):E634-41.
Epub 2003 Dec 2.

Involvement of SSAO-mediated deamination in adipose glucose
transport and weight gain in obese diabetic KKAy mice.
Yu PH, Wang M, Fan H, Deng Y, Gubisne-Haberle D.
Peter H. Yu, Michael Wang, Hui Fan, Yulin L. Deng,
and Diana Gubisne-Haberle.
Neuropsychiatry Research Unit, University of Saskatchewan,
Saskatoon, Saskatchewan, Canada S7N 5E4.

Semicarbazide-sensitive amine oxidase (SSAO) is located on
outer surfaces of adipocytes and endothelial and
vascular smooth muscle cells.
This enzyme catalyzes deamination of methylamine and aminoacetone,
leading to production of toxic formaldehyde and methylglyoxal,
respectively, as well as hydrogen peroxide and ammonium.
Several lines of evidence suggest that increased SSAO activity
is related to chronic inflammation and vascular disorders related
to diabetic complications.
We found that a highly potent and selective SSAO inhibitor,
(E)-2-(4-fluorophenethyl)-3-fluoroallylamine (FPFA),
was capable of reducing numbers of atherosclerotic lesions
as well as weight gain in obese KKAy mice fed an atherogenic diet.
SSAO inhibitors cause a moderate and long-lasting hyperglycemia.
Such an increase in serum glucose is a result of reduction of glucose
uptake by adipocytes.
SSAO-mediated deamination of endogenous methylamine
substrates induces adipocyte glucose uptake and lipogenesis.
Highly selective SSAO inhibitors can effectively
block induced glucose uptake.
The results suggest that increased SSAO-mediated deamination
may be concomitantly related to obesity and vascular disorders
associated with type 2 diabetes. PMID: 14656718
************************************************** *****

http://jpet.aspetjournals.org/cgi/content/full/310/3/1125 free full text

Abstract:

J Pharmacol Exp Ther. 2004 Sep; 310(3): 1125-32.
Epub 2004 May 5.
Protein cross-linkage induced by formaldehyde derived from
semicarbazide-sensitive amine oxidase-mediated deamination
of methylamine.
Gubisne-Haberle D, Hill W, Kazachkov M, Richardson JS, Yu PH.
Diana Gubisne-Haberle, Wayne Hill, Mychaylo Kazachkov,
J. Steven Richardson, and Peter H. Yu yup@usask.ca
Neuropsychiatry Research Unit,
University of Saskatchewan, Saskatoon, SK S7N 5E4, Canada.


Semicarbazide-sensitive amine oxidase (SSAO) catalyzes
the conversion of methylamine to formaldehyde.

This enzyme is located on the surface of the cytoplasmic membrane
and in the cytosol of vascular endothelial cells,
smooth muscle cells, and adipocytes.

Increased SSAO activity has been found in patients
with diabetes mellitus, chronic heart failure, and multiple types
of cerebral infarcts and is associated with obesity.

Increased SSAO-mediated deamination may contribute to protein
deposition, the formation of plaques, and inflammation,
and thus may be involved in the pathophysiology of chronic
vascular and neurological disorders, such as diabetic complications,
atherosclerosis, and Alzheimer's disease.

In the present study, we demonstrate the induction of cross-linkage
of formaldehyde with the lysine moiety of peptides and proteins.

Formaldehyde-protein adducts were reduced with sodium
cyanoborohydride, hydrolyzed in hydrochloric acid,
and the amino acids in the hydrolysates were derivatized
with fluorenylmethyl chloroformate
and then identified with high-performance liquid chromatography.

We further demonstrate that incubation of methylamine
in the presence of SSAO-rich tissues, e.g., human brain meninges,
results in formaldehyde-protein cross-linkage of particulate bound
proteins as well as of soluble proteins.

This cross-linkage can be completely blocked by a selective
inhibitor of SSAO.

Our data support the hypothesis that the SSAO-induced
production of formaldehyde may be involved in the alteration
of protein structure,
which may subsequently cause protein deposition associated
with chronic pathological disorders. PMID: 15128865

Journal of Pharmacology And Experimental Therapeutics
First published on May 5, 2004; DOI: 10.1124/jpet.104.068601
0022-3565/04/3103-1125-1132$20.00
JPET 310:1125-1132, 2004

Full Text (PDF)
All Versions of this Article: jpet.104.068601v1
310/3/1125 most recent

CELLULAR AND MOLECULAR

Protein Cross-Linkage Induced by Formaldehyde
Derived from Semicarbazide-Sensitive Amine Oxidase-Mediated
Deamination of Methylamine.
Diana Gubisne-Haberle,
Wayne Hill,
Mychaylo Kazachkov,
J. Steven Richardson, 306 966-6301
and Peter H. Yu. yup@usask.ca 306 966-8816
Neuropsychiatry Research Unit, Departments of Psychiatry
(D.G.-H., W.H., M.K., J.S.R., P.H.Y.) and Pharmacology (J.S.R.),
University of Saskatchewan, Saskatoon, Saskatchewan, Canada
Received March 18, 2004; accepted May 5, 2004.
http://www.usask.ca/
http://www.usask.ca/psychiatry/66%20faculty%20pages/facultyYu.html
Professor, Neuropsychiatry Research Unit
Telephone: (306) 966-8816
Facsimile: (306) 966-8830
E-mail: yup@sask.usask.ca
Office: Room 128 Medical Research Building
http://www.usask.ca/psychiatry/facultytable2.html

Materials and Methods
Results
Discussion
References

Materials and Methods:

Semicarbazide-sensitive amine oxidase (SSAO) is located both on the
outer membrane surface and in the cytoplasm of endothelial and
vascular smooth muscle cells (Lyles, 1966; Blicharsky and Lyles, 1990)
and of adipocytes (Conforti et al., 1995).

Recently, this enzyme has drawn considerable interest regarding its
potential physiological and pathological roles
(Salmi and Jalkanen, 2001; Yu et al., 2003).

SSAO also circulates in blood
(van Dijk and Boomsma, 1998; Yu et al., 1994).

Serum SSAO activity is increased in patients with a variety of vascular
disorders, such as diabetes (Nilsson et al., 1968; Yuen et al., 1987;
Boomsma et al., 1995; Garpenstrand et al., 1999),
multiple types of cerebral infarction (Ishizaki, 1990), and
congestive heart failure (Boomsma et al., 1997).

The enzyme activity is also positively correlated with body mass index
(Meszaros et al., 1999; Weiss et al., 2003).

High blood SSAO has been identified to be a potential prognostic risk
factor of mortality for the heart patient (Boomsma et al., 2000).

SSAO, a copper-containing enzyme with topaquinone as the cofactor,
is distinctly different from mitochondrial monoamine oxidase (MAO)
(Holt et al., 1998).

SSAO catalyzes the deamination of aliphatic amines,
including methylamine and aminoacetone, whereas MAO deaminates
catecholamines, serotonin, and long-chain aliphatic amines.

Methylamine,
a major metabolite of creatine (Mitchell and Zhang, 2001), can also be
derived from adrenaline,
lecithin,
dietary sources,
and cigarette smoke (Yu, 1998).

SSAO converts methylamine into formaldehyde,
hydrogen peroxide,
and ammonium,
all of which are cytotoxic.

Aminoacetone is derived from threonine and glycine, and its
deamination leads to the production of cytotoxic methylglyoxal.

Formaldehyde and methylglyoxal are extremely reactive chemicals,
which can interact with free amino or amide groups
to form Schiff's base,
subsequently methylene bridges, and produce irreversible covalently
cross-linked complexes between proteins,
as well as between proteins and single-stranded DNA (Bolt, 1987).

Increased protein aggregation and deposition is associated with the
aging process and with numerous pathological conditions, such as the
formation of plaques in the pancreas, brain, kidney, and other organs.

It would therefore be interesting to know whether formaldehyde and
methylglyoxal produced from SSAO-catalyzed deamination of
methylamine and aminoacetone, respectively,
are involved in the process of protein cross-linkage.

In animal studies, chronic methylamine treatment causes an increase in
oxidative stress and vascular damage (Deng et al., 1998),
while inhibiting SSAO activity reduces atherogenesis (Yu et al., 2002).

SSAO-mediated deamination has been shown to be toxic
toward cultured endothelial cells,
and selective SSAO inhibitors can prevent such toxicity
(Yu and Zuo, 1993).

Massive exposure to methylamine after an industrial accident
was lethal to humans (Yang et al., 1995).

It has been hypothesized that the continuous presence of an elevated
level of SSAO-mediated aldehydes may be responsible for the chronic
inflammation and plaque formation associated with the cerebral
vasculature, where SSAO is located (Yu, 2001).

The present study demonstrates that formaldehyde
derived from the action of SSAO on methylamine
cross-links proteins.

Materials.

Benzylamine, methylamine, lysine, N-methyl-lysine,
N,N-dimethyl-lysine, N,N,N-trimethyl-lysine,
fluorenylmethyl chloroformate (FMOC-Cl),
bovine serum albumin (BSA), clorgyline, and (-)-deprenyl
were purchased from Sigma-Aldrich (St. Louis, MO);
[7-14C]benzylamine and [14C]methylamine were obtained
from American Radiolabeled Chemicals (St. Louis, MO) and
PerkinElmer Life and Analytical Sciences (Boston, MA), respectively.
H-Lys-Leu-OH.HBr was from Bachem California (Torrance, CA).
MDL-72974A
[(E)-2-4-fluorophenethyl-3-flouroallylamine HCl]
was previously provided by Marion-Merrel-Dow Inc. (Cincinnati, OH).
The other chemicals were of analytical grade.

Preparation of SSAO-Rich Tissues.

Samples of aorta from Wistar rats (200 g),
small intestine from male CD 1 Swiss mice (28-30 g),
and human brain meninges were used.
All procedures involving animals were approved
by the Ethics Committee of the University of Saskatchewan.
After dissection, the tissues were rinsed thoroughly with saline (0.9%
NaCl), sliced into small pieces,
and homogenized with a Polytron homogenizer
(PT-10-35) for 1 min on ice in chilled
0.02 M phosphate buffer (pH 7.4) [1:10% (w/v)].
The crude homogenates were centrifuged at 900g for 10 min,
and the low-speed supernatants
were further centrifuged at 100,000g for 30 min.
The resulting supernatants and membrane fractions
were stored at -70┬░C
and used as the enzyme samples in the study.
To obtain a higher concentration of SSAO, we concentrated (50x)
the high-speed supernatant with the Minicon B15 sample concentrator
(Millipore, Mississauga, ON, Canada).
Tissue slices (250 x 250 ┬Ám) were prepared from saline-washed
meninges, aorta, or small intestine with a McIwain tissue chopper
(Michle Laboratory Engineer, Surry, UK).

Determination of SSAO Activity.

SSAO activity was assessed by a radioenzymatic procedure
using 14C-labeled benzylamine as the substrate (Yu and Zuo, 1993).
The SSAO enzyme preparations were incubated with clorgyline
(10-6 M) and (-)-deprenyl (10-6 M) at 37┬░C for 20 min
to ensure that any MAO activity was completely blocked.
Aliquots of the enzyme preparation were then incubated with 50 ┬Ál of
[14C]benzylamine (4 x 10-4 M, 1 ┬ÁCi/ml) in a final volume of 200 ┬Ál in
phosphate buffer (0.1 M, pH 7.4) at 37┬░C for 30 min.
The enzyme reaction was terminated by adding 250 ┬Ál of 2 M citric acid.
The oxidized products were extracted into 1 ml of toluene/ethyl acetate
[1:1 (v/v)], of which 600 ┬Ál was transferred to a counting vial
containing 10 ml of ACS scintillation cocktail
(American Radiolabeled Chemicals).
Radioactivity was assessed in an LS-7500 liquid scintillation counter
(Beckman Coulter, Inc., Fullerton, CA).

Cross-Linkage of Formaldehyde with Proteins.

To test formaldehyde-induced protein cross-linkage,
we used a bipeptide, Lys-Leu-OH, and BSA.
The peptide or BSA (2 mg/ml) was incubated in the presence of 10 mM
formaldehyde in 0.02 M phosphate buffer (pH 7.4) at 37┬░C for 4 h,
followed by further incubation with 10 mM NaCNBH3 for 24 h.
The reaction mixture regarding BSA-formaldehyde adducts was then
dialyzed extensively
(Spectr/Por cellulose membrane,
molecular weight cuttoff 3.5 kDa; Spectrum Laboratories,
Rancho Dominguez, CA)
with three changes of 0.2 M phosphate buffer (pH 8.0).
The formaldehyde-peptide or formaldehyde-protein adducts
were hydrolyzed in 6 N HCl at 110┬░C for 24 h.
The amino acids and altered amino acids were derivatized with
FMOC-Cl and assessed by an HPLC-UV procedure (Bank et al., 1996).

Precolumn Derivatization of Amino Acids with FMOC-Cl.

The hydrolyzed samples were neutralized with 10 M NaOH.
To each 100-┬Ál sample,
50 ┬Ál of potassium borate buffer (0.8 M, pH 10)
was added, and the solution was vigorously vortexed for 1 min.
One hundred microliters of FMOC-Cl solution (10 mM in dehydrated
acetonitrile) was added,
and the solution was immediately vortexed for 1 min.
One milliliter of hexane was then added, and the reaction mixture was
vigorously shaken for 45 s, centrifuged, and the hexane phase
containing excess reagent was discarded.
The extraction was repeated twice.
A 10-┬Ál aliquot of acetic acid [20% (v/v)] was added,
the tube was mixed, and the sample was applied to HPLC analysis.

Chromatography.

The HPLC system consisted of a Shimadzu LC-10AD VP
delivery system, a DGU-14A degasser, a SIL-10AD VP
auto-injector (Man-Tech, Guelph, ON, Canada),
and an integrator (Spectra Physics, San Jose, CA).
The separation was performed using a reverse-phase
Ultrasphere LP analytical column
(4.6 x 250 mm, 5 ┬Ám; Beckman Coulter, Toronto, ON, Canada).
Elution was either isocratic with 0.05 M sodium-acetate buffer
[pH 5.0; 43% (v/v)] in acetonitrile (flow rate 1.4 ml/min),
or a ternary gradient system,
where solvent A was 20 mM citric acid containing
5 mM tetramethylammonium chloride
adjusted to pH 2.85 with 20 mM sodiumacetate,
solvent B was composed of 80% (v/v) 20 mM sodium-acetate
solution containing 5 mM tetramethylammonium
(adjusted to pH 4.5 with concentrated phosphoric acid)
and 20% (v/v) methanol,
and solvent C was acetonitrile.
The gradient program is according to Bank et al. (1996).
The separation was performed at room temperature.
Spectrophotometric detection was conducted
using a -Max model 481 LC spectrophotometer (Millipore)
at a wavelength of 265 nm.
Data represent the average of at least three analyses.

Formaldehyde-Protein Adducts
Derived from SSAO-Mediated Reactions.

For the detection of cross-linkage of proteins by formaldehyde
derived from the deamination of methylamine,
SSAO-rich tissue homogenates
[200 ┬Ál; 1:10 (w/v) in 0.02 M phosphate buffer, pH 7.4]
were incubated with methylamine (10-3 M) at 37┬░C for 4 h.
Then, 1 ml of 10 mM NaCNBH3 was added, and the samples
were mixed and further incubated at 37┬░C for 24 h.
For controls, tissue homogenates were preincubated
(37┬░C for 30 min) with the specific SSAO inhibitor
MDL-72974A (1 x 10-6 M).

In other experiments, [14C]methylamine was used to trace the
deamination and the subsequent cross-linkage of formaldehyde
with proteins in vitro and in vivo.

The tissue homogenates were ultracentrifuged
(100,000g for 30 min).
Because SSAO is present in both the soluble
and the particulate fractions,
both fractions were used in cross-linkage experiments.
The soluble fractions (high-speed supernatant) were further purified
by gel filtration via a Sephadex G-25 PD-10 column
(Amersham Biosciences AB, Uppsala, Sweden).

For the in vivo studies, a 100-┬Ál injection of [14C]methylamine
(2 ┬ÁCi) was administered via the tail vein,
and tissues were collected after different time periods.

HPLC-Mass Spectrometry.

N-methylated lysine-FMOC derivatives were analyzed
by reversed phase HPLC
(Alliance model 2695; Waters, Milford, MA)
coupled to electrospray mass spectrometry
(Quattro UltimaTM; Micromass, Manchester, UK).
The HPLC fractions (1.4 ml), which were photometrically detected,
were collected.
Amino acid derivatives in these fractions were then extracted
with 1:1 ethyl acetate/toluene,
and the organic fraction was transferred to a new vial and dried under
nitrogen.
These extracted fractions were then reconstituted with 1 ml of 1:1
acetonitrile/water.
Ten-microliter aliquots of these extracted fractions were injected onto
an analytical column (100 x 2.1 mm, 4-┬Ám Genesis C18;
model FK10960EJ; Jones Chromatography, Hengoed, UK)
by an integrated HPLC pump and auto-sampler
model 2695; Waters)
at a flow rate of 0.20 ml/min with an isocratic mobile phase of 75:25
acetonitrile/water.
Mass spectrometric analysis was conducted in both
positive and negative ion MS1-mode (m/z 50-850).
Source temperature was 120┬░C, and capillary voltage
was 2.53 kV with a cone voltage of 45 V.
Spectra were matched to those of standards where possible.

Results:

Methylamine Induced Production of Formaldehyde-Protein Adducts.

As can be seen in Fig. 1, the administration of [14C]methylamine
to mice causes a long-lasting radioactive deposition in different tissues
that is substantially reduced by the highly selective
SSAO inhibitor MDL-72974A.

This is consistent with the notion that formaldehyde derived from the
deamination of methylamine induces protein cross-linkages.

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Fig. 1. Residual radioactivity detected in different mouse tissues after
administration of [14C]methylamine.

The animals were pretreated with saline or SSAO inhibitor
MDL-72974A (5 mg/kg i.p.) 2 h before administration of
[14C]methylamine (5 ┬ÁCi, 64 pmol/mouse, via tail vein injection).

The residual radioactivity in different tissues was assessed 48 h after
administration of the 14C-labeled methylamine.

The radioactivity (dpm per gram of fresh tissue) was measured
in a Beckman Coulter scintillation counter.
Values are means ┬▒ standard error of the mean of five animals.
Statistical comparison in different groups of experiments was performed
using a one-way analysis of variance,
followed by Newman-Keuls multiple comparisons; *, p 0.01.

Lysine as a Target for Cross-Linkage of Formaldehyde and Proteins.

The interaction of formaldehyde with proteins is rather complicated.
To demonstrate that formaldehyde, derived from the deamination of
methylamine, cross-links with proteins, a simplified model is required.
In the present study, lysine was selected as the reaction target,
although formaldehyde reacts with several other amino acid residues in
proteins as well.
The interaction of formaldehyde with a bipeptide, H-Lys-Leu-OH, was
initially used.

Figure 2 shows chromatograms demonstrating that formaldehyde induces
H-Lys-Leu-OH cross-linkage (Fig. 2C).

This cross-linkage is unstable upon acid hydrolysis (i.e., in 6 N HCl at
110┬░C for 24 h),
because only lysine and leucine residues are detected in the hydrolysates
either pretreated or untreated with formaldehyde (Fig. 2, D and E).
This is expected, because formaldehyde first forms relatively unstable
Schiff's bases between two free amino groups of lysine.
However, when the Schiff's bases
are reduced by sodium cyanoborohydride,
N-methyl-lysine and formyl-lysine peaks are detected (Fig. 2E).
N-Methyl-lysine was therefore used as a marker for
formaldehyde-induced protein cross-linkage
in the subsequent experiments.

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Fig. 2. Cross-linkage of formaldehyde with bipeptide H-Lys-Leu-OH.

Amino acids derived from Lys-Leu-OH with or without cross-linkage
with formaldehyde and/acid hydrolysis were derivatized
with FMOC-Cl and separated by HPLC
and assessed by UV detection ( == 265 nm).
A, amino acid standards.
B, H-Lys-Leu-OH alone.
C, H-Lys-Leu-OH (6 mM) + formaldehyde (10 mM).
D, HCl hydrolysates of H-Lys-Leu-OH,
E, HCl hydrolysates of H-Lys-Leu-OH-formaldehyde adduct.
F, HCl hydrolysates of Lys-Leu-OH-formaldehyde adduct
pretreated NaCNBH3 (10 mM).


We also demonstrated the cross-linkage of formaldehyde
with BSA, a protein containing 14% lysine residues.
Based on the detection of N-methyl-lysine in the acid hydrolysates of
formaldehyde-BSA adducts lysine was found to be a major target for
formaldehyde (Fig. 3).
However, under the present chromatography condition,
dimethyl-lysine and trimethyllysine peaks were not detected
due to interference of amino acids in the BSA hydrolysate.
The extent of cross-linkage is dose-dependent, namely,
cross-linkage can be detected at low micromolar formaldehyde
and becomes saturated around 5 mM formaldehyde.
The sensitivity of the spectrometric FMOC method regarding
N-methyl-lysine is around 5 ng/injection.
To achieve higher sensitivity, we traced the reactions with 14C-labeled
formaldehyde and used mass spectrometry.
This substantially enhanced the sensitivity of detection and therefore has
been used in subsequent experiments.

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Fig. 3. Cross-linkage of formaldehyde with BSA.

A, BSA (2 mg/ml) alone.
B, BSA in the presence of formaldehyde (1 mM).
The reaction mixtures were incubated at pH 7.4 for 4 h.
Adducts were pretreated NaCNBH3 (10 mM) before HCl hydrolysis.
Amino acids derived from the formaldehyde-BSA adducts
in the hydrolysates were derivatized with FMOC-Cl,
separated by HPLC, and assessed by UV detection ( == 265 nm).
Under these chromatographic conditions, the FMOC derivatives of
N-methy-lysine, lysine, and tyrosine
are separated from other amino acids.

Identification of Methylated Lysine
in the Formaldehyde-Protein Adducts.

The methylated lysine residues collected from the HPLC fractions were
identified by HPLCMS.
As can be seen in Fig. 4, the mass spectra of the FMOC derivatives of
lysine, N-methyl-lysine, N,N-dimethyl-lysine,
and N,N,N-trimethyl-lysine are characterized by intense ion peaks
at 591, 605, 619, and 411, respectively.
The masses are consistent with the attachment of two FMOC moieties
to each residue except for trimethylamine, which has only one FMOC.
Other major constituents shown in the mass scans consist of fragments
that have lost one or both FMOC moieties.
Further analyses of the mass spectra of the molecular ions
reveal fragments corresponding to lysine, methyl-lysines
as well as lysines with one FMOC remaining.
The results unambiguously identify methylated lysine as a result of
formaldehyde-protein interaction.

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Fig. 4. FMOC derivatization and identification of lysine
and N-methylated lysines by mass spectrometry.

Spectra indicate that the molecular ions of the FMOC
derivatives of lysine,
methyl-lysines linked with two moieties of FMOC
are 590, 604, and 618, respectively.
Only one FMOC moiety was attached to trimethyl-lysine.

Cross-Linkage of Formaldehyde,
Derived from SSAO-Catalyzed Deamination of Methylamine, with BSA.

In this preliminary experiment, mouse intestine, which possess very high
SSAO activity, was used.
The tissues were homogenized and extracts were centrifuged
and concentrated using a protein concentrator
(Amicon Corp., Danvers, MA).
Then, the enzyme extracts were incubated
in the presence of methylamine (1 mM)
and BSA (2 mg/ml) at 37┬░C for 4 h,
and N-methyl-lysine in the hydrolyzed proteins was detected.

Approximately, 2% of the total lysine residues were methylated.
In the controls, i.e., tissue extracts + BSA in the absence of methylamine,
no methyl-lysine was detected.
When the tissue extracts were pretreated with the highly selective SSAO
inhibitor MDL-72974A,
formaldehyde-induced cross-linkage was not detected.
The spectrophotometric HPLC/FMOC method does not allow
the assessment of N,N-dimethyl-lysine and N,N,N-trimethyl-lysine,
because the corresponding peaks
cochromatograph with other amino acids.
However, radioactive N-methyl-lysine, N,N-dimethyl-lysine, and
N,N,N-trimethyl-lysine can be traced
if 14C-labeled methylamine was used as substrate.
As shown in Fig. 5, the radioactivity in HPLC fraction 18 is due to
N,N-dimethyllysine and N,N,N-trimethyl-lysine,
whereas fraction 28 corresponds to N-methyl-lysine.
The amount of methyl-lysine produced is approximately 3-fold that of
N,N-dimethyl-lysine and N,N,N-trimethyl-lysine combined.

The SSAO inhibitor MDL-72974A again effectively blocks
the generation of methylated lysine.

Radioactivity was also detected in fraction 3.
Although MDL-72974A also inhibits the formation of these radioactive
products, these compounds have not been identified.

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Fig. 5. SSAO-mediated formation of formaldehyde-protein adducts
of the mouse intestine.

Concentrated extracts of mouse small intestine tissue
were incubated with [14C]methylamine (1 x 10-3 M; 1 ┬ÁCi)
in the presence or absence of MDL-72974A (1 x 10-6 M)
at 37┬░C for 4 h,
and then with 10 mM NaCNBH3 at 37┬░C for 24 h.
After dialysis (0.2M Na2HPO4 for 24 h) and hydrolysis
(6 N HCl, at 110┬░C for 24 h),
the samples were separated by HPLC
(as described under Materials and Methods),
fractions of 1.4 ml/min were collected,
and the radioactivity was determined.
A, chromatographic separation patterns of lysine FMOC-derivatives
(100 ng/50-┬Ál injection volume).
B, radioactivity detected in the HPLC fractions.
Fraction 18 contains N,N-dimethyl-lysine and
N,N,N-trimethyl-lysine, and fraction 28 represents the N-methyl-lysine.

Evidence of SSAO-Mediated Formaldehyde-Protein Cross-Linkage in
Human Tissue.

Tissue slices (250-┬Ám2 pieces) of human brain meninges,
which are also rich in SSAO,
were incubated with 14C-labeled methylamine (1 mM)
in the presence or absence of the SSAO inhibitor MDL-72974A.
BSA was not included in this experiment.
After incubation for 60 min at 37┬░C, the tissue preparations were
homogenized and then separated into soluble and particulate fractions
by centrifugation at 100,000g for 30 min.
The soluble fractions were subjected to gel filtration
via a PD-10 Sephadex G-25 M column.
Column fractions 7 to 12 contain the major radioactivity
of small molecules, which are excess methylamine
and unbound formaldehyde (Fig. 6).
Labeled macromolecules were detected in the void volume.

When SSAO activity was blocked with MDL-72974A,
no labeled macromolecules were found and the radioactivity
in small molecule fractions increased.

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Fig. 6. SSAO-mediated formation of formaldehyde-protein
adducts in the soluble fraction from human meninges.

Human brain meninges tissue slices were
incubated with [14C]methylamine (10-3 M; 1 ┬ÁCi)
in the presence or absence of a selective SSAO inhibitor
MDL-72974A (1 x 10-6 M) at 37┬░C for 4 h.
The tissue slices were then homogenized using a Polytron
and centrifuged at 100,000g for 30 min.
A liquot of the supernatant was applied to a small
Sephadex G-25 column (PD-10), and 1-ml fractions were collected.
Radioactivity in different fractions was assessed.

A substantial amount of radioactivity was found to deposit in the
particulate fractions.
MDL-72974A reduces the deposit of radioactivity in these fractions.
The radioactively labeled protein adducts in the particulate preparations
were treated with sodium cyanoborohydride,
followed by hydrolysis in HCl and derivatization with FMOC-Cl.
The HPLC profile is shown in Fig. 7.
Radioactively labeled methylated lysines (i.e., in fractions 18 and 28)
and methylarginine (fraction 16) were detected.
Unknown radioactively labeled compounds in fractions 3, 12, and 20
were also detected.

The formation of these radioactively labeled products was reduced by
MDL-72974A.

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Fig. 7. SSAO-mediated formation of formaldehyde-protein adducts
in the particulate bound fraction and identification of the formaldehyde
cross-linkage sites.

Human meninges tissue slices were incubated
with [14C]methylamine (1 x 10-3 M; 1 ┬ÁCi)
in the presence (closed circle) or absence (open square)
of a selective SSAO inhibitor MDL-72974A (1 x 10-6 M)
at 37┬░C for 4 h.
The reaction mixtures were subsequently incubated
in the presence of 10 mM NaCNBH3 at 37┬░C for 24 h.
After dialysis against 0.2 M phosphate buffer (pH 7.2) for 24 h,
the samples were hydrolyzed in 6 N HCl at 110┬░C for 24 h.
The samples were then derivatized with FMOC-Cl
and separated by HPLC as described in text.
Fractions of 1.4 ml/min were collected,
and the radioactivity was determined.
N-methylated lysines and arginine are indicated in the figure.

Discussion:

Formaldehyde is produced via SSAO-catalyzed oxidative
deamination of methylamine (Yu, 1990).

Methylamine is a readily available endogenous substrate
derived from e.g., creatine and adrenaline
(Yu et al., 1997; Yu and Deng, 2000).

When radioactively labeled methylamine was administered to rodents,
irreversibly bound methylamine metabolites were detected by an
autoradiographic examination (Gronvall et al., 1998).

The formation of such protein adducts was blocked by selective SSAO
inhibition.

To test the involvement of formaldehyde derived from methylamine
in protein cross-linkage,
it is necessary to assess formaldehyde-protein cross-linkage sites.

Although it is known that formaldehyde forms methylene bridges
between protein residues
and produces intramolecular and intermolecular cross-linkage
and possibly advanced protein aggregation
(Francel-Conrat and Olcott, 1948),
the identification of such an interaction is not well established,
especially under the condition of low formaldehyde concentrations.

We have therefore developed a simple method for identification of
formaldehyde-protein cross-linkage.

Under physiological conditions (i.e., 37┬░C, pH 7.4),
formaldehyde readily interacts
with the bipeptide H-Lys-Leu-OH and with BSA.

Although cross-linkage of formaldehyde with H-Lys-Leu-OH occurs,
after acid hydrolysis only lysine and leucine residues are produced.

N-Methyl-lysine was detected
only when the molecule was reduced by NaCNBH3.

The -amino amino group of lysine reacts with formaldehyde to form
hydroxymethylamine,
which is unstable to either extensive dialysis or acidic hydrolysis.

Hydroxymethylamine combines slowly with free amino, amid, guanidyl,
phenolic, or imidazole groups of amino acids in the same
or another protein molecule to form Schiff's base,
which would not be detected after hydrolysis of the proteins unless the
Schiff's base is reduced to stable intra- or intermolecular methylene
cross-links (Francel-Conrat and Olcott, 1948).

N-Methyl-lysine is a very useful marker to substantiate
SSAO-mediated formaldehyde-protein cross-linkage.

Inhibition of SSAO activity also causes
increase in labeled hydrophilic compounds,
which are radioactively labeled
and eluted in fractions of the solvent front.

They are probably free formaldehyde,
or chemically altered formaldehyde,
released from sites loosely bound to proteins.

Blood vessels and small intestine tissues possess
very high SSAO activity.

They were used for SSAO-induced formaldehyde-protein
cross-linkage studies.

Methylamine,
after incubation with SSAO-containing tissue extracts or slices,
is converted into formaldehyde
and thus reacts with lysine residues and induces protein cross-linkage.

SSAO blocks such reactions.

After incubation of 14C-labeled methylamine with tissue slices of
SSAO-rich human brain meninges,
the majority of the radioactivity was associated
with the particulate bound proteins.

The lysine residue seems to be the primary target for formaldehyde
interaction.

It is interesting to note that the generation of formaldehyde-protein
adducts in the meninges tissue slices occurs in the absence of BSA.

This suggests that endogenous proteins in or on meninges are readily
available for interaction with formaldehyde
derived from SSAO-mediated reactions.

It is unclear whether it is the membrane-bound,
or the cytoplasmic SSAO that is primarily responsible
for the production of the formaldehyde involved
in the production of formaldehyde-protein adducts.

It seems to be reasonable to suggest that formaldehyde
generated in the immediate compartment adjacent
to the outer membrane SSAO sites would favor
the interaction of formaldehyde with membrane proteins.

If deamination of methylamine takes place within the cells,
formaldehyde would be quickly metabolized by cytosolic aldehyde
dehydrogenase.

Furthermore this newly generated formaldehyde
be quickly diffused and diluted by the cytoplasm.

We have shown that formaldehyde
at concentrations lower than 10 ┬ÁM become more difficult
to cross-link with proteins.

Both of these factors would reduce
the interaction of formaldehyde with
proteins and this suggests that the cytoplasmic SSAO
may play a minor role in the production
of formaldehyde induced protein deposition.

The membrane-bound SSAO was independently identified
as an endothelial surface vascular adhesion protein-1
involved in lymphocyte trafficking (Salmi et al., 2002).

Formaldehyde generated extracellularly
via SSAO-catalyzed deamination of methylamine
would lead to the formylation and methylation of adjacent
membrane-bound proteins and
could even play a role in lymphocyte adhesion
(Salmi and Jalkanen, 2002).

Although this formaldehyde-induced alteration of proteins may not be
sufficient to cause acute toxicity,
the effect of chronic elevated levels of formaldehyde-protein
cross-linkage may cause an accumulation of protein deposits,
the formation of extracellular plaques,
and result in the slow, silent damage to vascular tissues associated with
various pathological conditions,
such as diabetic complications and Alzheimer's disease
(Yu et al., 2003).

It is known that some proteins undergo post-translational
methylated by protein methyltransferase
using S-adenosylmethionine as the methyl group donor
(Paik and Kim, 1980).

The modification of proteins by methyl groups
can alter protein function due to changes in charges,
steric relations,
or hydrophobicity at the site of the attached methyl group
(Alleta et al., 1998).

The present study demonstrates an additional mechanism,
namely, formylation and subsequent methylation, of proteins,
which may result in altered function of proteins.

Recently, it has been demonstrated that formaldehyde derived from
SSAO-mediated deamination of methylamine
dilates human blood vessels (Conklin et al., 2004)
and may play a role in the regulation of blood pressure.

In conclusion, the present study demonstrates
that formaldehyde produced by SSAO-catalyzed
deamination of methylamine,
forms adducts with cytoplasmic as well as membrane-bound proteins.

Such cross-linkage may affect vascular function
or cause pathological consequences.

Future investigations are required in to understand the physiological
and pathological implications of protein alterations
induced by the endogenous production of formaldehyde.

Footnotes

This work was supported by grants from the
Canadian Institute of Health Research,
Saskatchewan Health, and Saskatchewan Alzheimer's Society.
doi:10.1124/jpet.104.068601.

ABBREVIATIONS:

SSAO, semicarbazide-sensitive amine oxidase;
MAO, monoamine oxidase;
FMOC, fluorenylmethyl chloroformate;
BSA, bovine serum albumin;
MDL-72974A, (E)-2-4-fluorophenethyl-3-fluoroallylamine HCl;
HPLC, high-performance liquid chromatography.

Address correspondence to: Dr. Peter H. Yu,
Neuropsychiatry Research Unit,
University of Saskatchewan, Saskatoon, SK S7N 5E4, Canada.
E-mail: yup@usask.ca

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

Life Sci. 1998; 63(9): 759-68.
Autoradiographic imaging of formaldehyde adducts in mice: possible
relevance for vascular damage in diabetes.
Gronvall JL, Garpenstrand H, Oreland L, Ekblom J.
Department of Neuroscience (Pharmacology),
Uppsala University, Biomedical Center, Sweden.

The activity of semicarbazide-sensitive amine oxidase (SSAO) has been
reported to be elevated in blood from diabetic patients.
It has been suggested that the enzyme is involved in the development of
complications such as retinopathies, nephropathies and neuropathies,
which are associated with advanced diabetes, possibly by the formation
of toxic metabolites.
Under the influence of SSAO,
methylamine is deaminated to formaldehyde
which is known to react with various macromolecules.
It has therefore been proposed that specific inhibition of SSAO
could be of therapeutic value for treatment of diabetic patients.
The present results provide evidence that treatment
with an SSAO inhibitor potently reduces the levels of irreversible adducts.
In this study, 14C-methylamine was given intraperitoneally
to NMRI mice, and the tissue distribution of irreversibly bound
methylamine metabolites was estimated by an autoradiographic method.

Such radioactive residues occurred in high concentrations in the intestinal
wall, brown adipose tissue, spleen and bone marrow.

By inhibiting SSAO irreversibly with hydralazine before giving
14C-methylamine to the mice,
it was possible to determine the resynthesis rate of SSAO in different
tissues.
A complete recovery of SSAO activity
was seen in the intestinal wall after 6 days,
whereas only about 60% was recovered in adipose tissue after 14 days.
This suggests that factors controlling the synthesis of SSAO
differ in these tissues,
or that these tissues express different forms of enzymes.
PMID: 9740313
************************************************** *****

Neurobiology (Bp). 2000; 8(2): 167-77.
An autoradiographic method of visualising semicarbazide-sensitive amine
oxidase activity in mouse tissue sections.
Gronvall JL, Garpenstrand H, Oreland L, Ekblom J.
Department of Neuroscience (Pharmacology), Biomedical Centre,
Uppsala University, Sweden. jenny.gronvall@medfarm.uu.se

Under the influence of semicarbazide-sensitive amine oxidase (SSAO),
methylamine is deaminated to formaldehyde, which can react
with various macromolecules and form irreversible adducts.
We hereby present an autoradiographic method of visualising SSAO
activity by measuring the in vivo formation of such adducts
from 14C-methylamine.

Our results revealed high concentrations of radioactive deposits in the
intestinal wall, brown adipose tissue, spleen and bone marrow.

Hydralazine is a potent SSAO inhibitor and pretreatment with this
irreversible inactivator resulted in a nearly complete loss of radioactive
deposits in the tissues.

By giving 14C-methylamine at different time-points after irreversible
inhibition of SSAO,
it was also possible to determine the resynthesis rate of SSAO.
Interestingly, the recovery rate of SSAO after such inactivation
was tissue-specific.
The possible therapeutic value of a specific SSAO inhibitory drug
has been discussed. PMID: 11061213
************************************************** *****

"Since the creatine turnover rate in a healthy adult is about 2 g a day
and one gets about 1 g of creatine daily from diet taking a low dose of
creatine such as 1 gram a day might result in only a very low increase
in formaldehyde levels since endogenous production of creatine would
probably decrease somewhat in response to the creatine."

Med Sci Sports Exerc. 2005 Oct; 37(10): 1717-20.
Effect of oral creatine supplementation on urinary methylamine,
formaldehyde, and formate.
Poortmans JR, Kumps A, Duez P, Fofonka A,
Carpentier A, Francaux M.
Higher Institute of Physical Education and Physical Therapy,
Free University of Brussels, Brussels, Belgium.
jrpoortm@ulb.ac.be; pduez@ulb.ac.be;

PURPOSE: It has been claimed that oral creatine supplementation
might have potential cytotoxic effects on healthy consumers
by increasing the
production of methylamine and formaldehyde.

Despite this allegation, there has been no scientific evidence
obtained in humans to sustain or disprove
such a detrimental effect of this widely used ergogenic substance.

METHODS: Twenty young healthy men ingested 21 g
of creatine monohydrate daily for 14 consecutive days.

Venous blood samples and 24-h urine were collected
before and after the 14th day of supplementation.

Creatine and creatinine were analyzed in plasma and urine,
and methylamine, formaldehyde, and formate
were determined in 24-h urine samples.

RESULTS: Oral creatine supplementation increased
plasma creatine content 7.2-fold (P 0.001)
and urine output 141-fold (P 0.001)
with no effect on creatinine levels.

Twenty-four-hour urine excretion of methylamine and formaldehyde
increased, respectively,
9.2-fold (P == 0.001) and 4.5-fold (P == 0.002)
after creatine feeding,
with no increase in urinary albumin output
(9.78 +/- 1.93 mg x 24 h(-1) before,
6.97 +/- 1.15 mg x 24 h(-1) creatine feeding).

CONCLUSION: This investigation shows that short-term, high-dose
oral creatine supplementation enhances the excretion
of potential cytotoxic compounds,
but does not have any detrimental effects on kidney permeability.
This provides indirect evidence of the absence of microangiopathy in
renal glomeruli. PMID: 16260971
************************************************** *****

This study suggests care in interpreting the statistical measures of
genotoxicity from formaldehyde and other toxins, using the popular, fast,
inexpensive, automated Comet assay.

Mutagenesis. 2003 Mar; 18(2): 159-66.
Statistics of the Comet assay: a key to discriminate between genotoxic
effects.
Duez P, Dehon G, Kumps A, Dubois J. pduez@ulb.ac.be
Institut de Pharmacie, Laboratoire de Chimie Bioanalytique,
de Toxicologie et de Chimie Physique Appliquee, CP 205/1, Belgium.

The alkaline Comet assay is a widely used single cell gel electrophoresis
technique for the quantification of DNA strand breaks, crosslinks and
alkali-labile sites induced by a series of physical and chemical agents.

DNA migration in an electric field, supposed proportional to strand
breakage, is a proposed estimation of genotoxicity.

Breaks are quantified from geometric and fluorescence measurements
by image analysis of comet-shaped DNA,
often reported parameters being tail DNA and tail moment.

Although a variety of statistical approaches have been used in the
literature, most of these do not take into account
the distribution patterns of comet data.

In order to investigate a methodology for statistically demonstrating
a comet effect, two different experiments,
a reproducibility study and a trend analysis,
were undertaken on a murine lymphoma cell line (P388D1)
photodynamically stressed after induction of porphyrins
with delta-aminolaevulinic acid.

This treatment results in significant heterogeneity of DNA damage,
producing values ranging from 0 to 100% tail DNA in the same sample.

The comparison of distribution curves for stressed and non-stressed
samples shows that none of the application conditions are verified,
either for parametric tests (which require normal distributions),
or non-parametric tests (which assume essentially similar distributions).

Meaningful statistics (median and 75th percentile) were consequently
extracted from repeated experiments and found suitable
for comparing stress conditions in an ANOVA and in a trend analysis;
the 75th percentile is theoretically more sensitive but tends to more
rapidly saturate at extensive stress levels.

We conclude that a trend analysis of median comet metrics
from repeated experiments at different stress levels
certainly an efficient way to statistically demonstrate a genotoxic effect.

Whether the considered comet parameter is tail DNA or tail moment
had no influence on the conclusions of our experiments,
which were carried up to stress levels
leading to a median 70% tail DNA. PMID: 12621072
************************************************** *****

Sunday, December 4, 2005

Any unsuspected source of methanol, which the body always quickly
and largely turns into formaldehyde and then formic acid, must be
monitored, especially for high responsibility occupations, often with
night shifts, such as pilots and nuclear reactor operators.


http://groups.yahoo.com/group/aspartameNM/message/1237
ubiquitous potent uncontrolled co-factors in nutrition research are
formaldehyde from wood and tobacco smoke and many sources,
including from methanol in dark wines and liquors, in pectins
in fruits and vegetables, and in aspartame: Murray 2005.12.04

http://groups.yahoo.com/group/aspartameNM/message/1250
aspartame causes cancer in rats at levels approved for humans,
Morando Soffritti et al, Ramazzini Foundation, Italy &
National Toxicology Program
of National Institute of Environmental Health Sciences
2005.11.17 Env. Health Pers. 35 pages: Murray


As a medical layman, I suggest that evidence mandates immediate
exploration of the role of these ubiquitious, potent formaldehyde
sources as co-factors in epidemiology, research, diagnosis,
and treatment in a wide variety of disorders.

Folic acid, from fruits and vegetables, plays a role by powerfully
protecting against methanol (formaldehyde) toxicity.

Many common drugs, such as aspirin, interfere with folic acid,
as do some mutations in relevant enzymes.

The majority of aspartame reactors are female.

In mutual service, Rich Murray
************************************************** *****

Rich Murray, MA Room For All rmforall@comcast.net
505-501-2298 1943 Otowi Road Santa Fe, New Mexico 87505

http://groups.yahoo.com/group/aspartameNM/messages
group with 148 members, 1,262 posts in a public, searchable archive
http://RoomForAll.blogspot.com http://AspartameNM.blogspot.com

Dark wines and liquors, as well as aspartame, provide
similar levels of methanol, above 100 mg daily, for
long-term heavy users, 2 L daily, about 6 cans.

Methanol is inevitably largely turned into formaldehyde,
and thence largely into formic acid.
It is the major cause of the dreaded symptoms of "next
morning" hangover.

Fully 11% of aspartame is methanol -- 1,120 mg aspartame
in 2 L diet soda, almost six 12-oz cans, gives 123 mg
methanol (wood alcohol). If 30% of the methanol is turned
into formaldehyde, the amount of formaldehyde, 37 mg,
is 18.5 times the USA EPA limit for daily formaldehyde in
drinking water, 2.0 mg in 2 L average daily drinking water,

185 times the New Jersey limit,
615 times the California and Maine limits,
1850 times the Maryland limit.

The 1999 July EPA 468-page formaldehyde profile admits that
four states substantially exceed the federal EPA limit:

Environmental Protection Agency 2.00 mg in 2 L daily
drinking water

California and Maine------------ 0.06 mg
Maryland---------------------- 0.02 mg
New Jersey-------------------- 0.20 mg

http://groups.yahoo.com/group/aspartameNM/message/1108
faults in 1999 July EPA 468-page formaldehyde profile:
Elzbieta Skrzydlewska PhD, Assc. Prof., Medical U. of
Bialystok, Poland, abstracts -- ethanol, methanol,
formaldehyde, formic acid, acetaldehyde, lipid peroxidation,
green tea, aging: Murray 2004.08.08 2005.07.11

http://groups.yahoo.com/group/aspartameNM/message/835
ATSDR: EPA limit 1 ppm formaldehyde in drinking water July
1999: Murray 2002.05.30 rmforall

Aspartame is made of phenylalanine (50% by weight) and
aspartic acid (39%), both ordinary amino acids, bound
loosely together by methanol (wood alcohol, 11%).
The readily released methanol from aspartame is within hours
turned by the liver into formaldehyde and then formic acid,
both potent, cumulative toxins.

http://groups.yahoo.com/group/aspartameNM/message/1106
hangover research relevant to toxicity of 11% methanol in
aspartame (formaldehyde, formic acid): Calder I (full text):
Jones AW: Murray 2004.08.05 2005.09.28

Since no adaquate data has ever been published on the exact
disposition of toxic metabolites in specific tissues in
humans of the 11% methanol component of aspartame, the many
studies on morning-after hangover from the methanol impurity
in alcohol drinks are the main available resource to date.

Jones AW (1987) found next-morning hangover from red wine
with 100 to 150 mg methanol (9.5% w/v ethanol, 100 mg/L
methanol, 0.01%, one part in ten thousand).

http://groups.yahoo.com/group/aspartameNM/message/1182
Joining together: short review: research on aspartame
methanol, formaldehyde, formic acid) toxicity: Murray
2005.07.08 rmforall

http://groups.yahoo.com/group/aspartameNM/message/1071
research on aspartame (methanol, formaldehyde, formic acid)
toxicity: Murray2004.04.29 rmforall

http://groups.yahoo.com/group/aspartameNM/message/1250
aspartame causes cancer in rats at levels approved for humans,
Morando Soffritti et al, Ramazzini Foundation, Italy &
National Toxicology Program
of National Institute of Environmental Health Sciences
2005.11.17 Env. Health Pers. 35 pages: Murray

http://groups.yahoo.com/group/aspartameNM/message/1226
USA National Institutes of Health National Toxicology
Program aids eminent Ramazzini Foundation, Bologna, Italy,
in more results on cancers in rats from lifetime low levels
of aspartame (methanol, formaldehyde), Felicity Lawrence,
www.guardian.co.uk: (http://www.guardian.co.uk:) Murray 2005.09.30

http://groups.yahoo.com/group/aspartameNM/message/1186
aspartame induces lymphomas and leukaemias in rats, full plain text,
M Soffritti, F Belpoggi, DD Esposti, L Lambertini: Ramazzini
Foundation study 2005.07.14: main results agree with their previous
methanol and formaldehyde studies: Murray 2005.09.03

http://groups.yahoo.com/group/aspartameNM/message/1189
Michael F Jacobson of CSPI now and in 1985 re aspartame
toxicity, letter to FDA Commissioner Lester Crawford;
California OEHHA aspartame critique 2004.03.12; Center for
Consumer Freedom denounces CSPI: Murray 2005.07.27

http://groups.yahoo.com/group/aspartameNM/message/1143
methanol (formaldehyde, formic acid) disposition: Bouchard M
et al, full plain text, 2001: substantial sources are
degradation of fruit pectins, liquors, aspartame, smoke:
Murray 2005.04.02 rmforall

http://groups.yahoo.com/group/aspartameNM/message/1141
Nurses Health Study can quickly reveal the extent of aspartame
(methanol, formaldehyde, formic acid) toxicity: Murray 2004.11.21
[ Any scientist can get access to this data for free by submitting a proper
research proposal.
No one has admitted mining the extensive data on diet soda use
and many symptoms for decades for about 100,000 nurses. ]

http://groups.yahoo.com/group/aspartameNM/message/1213
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: Murray 2005.09.09

http://groups.yahoo.com/group/aspartameNM/message/939
aspartame (aspartic acid, phenylalanine) binding to DNA:
Karikas July 1998: Murray 2003.01.05 rmforall
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
http://www.chem.uoa.gr gkokotos@atlas.uoa.gr
K.H. Schulpis inchildh@otenet.gr G.J. Reclos reklos@otenet.gr

http://groups.yahoo.com/group/aspartameNM/message/1088
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

http://groups.yahoo.com/group/aspartameNM/message/1067
eyelid contact dermatitis by formaldehyde from aspartame,
AM Hill & DV Belsito, Nov 2003: Murray 2004.03.30

Thrasher (2001): "The major difference is that the Japanese
demonstrated the incorporation of FA and its metabolites into the
placenta and fetus.
The quantity of radioactivity remaining in maternal and fetal tissues
at 48 hours was 26.9% of the administered dose." [ Ref. 14-16 ]

Arch Environ Health 2001 Jul-Aug; 56(4): 300-11.
Embryo toxicity and teratogenicity of formaldehyde. [100 references]
Thrasher JD, Kilburn KH. toxicology@drthrasher.org
Sam-1 Trust, Alto, New Mexico, USA.
http://www.drthrasher.org/formaldehyde_embryo_toxicity.html full text

http://groups.yahoo.com/group/aspartameNM/message/1052
DMDC: Dimethyl dicarbonate 200mg/L in drinks adds
methanol 98 mg/L [ becomes formaldehyde in body ]: EU Scientific
Committee on Foods 2001.07.12: Murray 2004.01.22

http://groups.yahoo.com/group/aspartameNM/message/925
aspartame puts formaldehyde adducts into tissues, Part 1/2
full text Trocho & Alemany 1998.06.26: Murray 2002.12.22

http://groups.yahoo.com/group/aspartameNM/message/1224
Aspartame disease: an FDA-approved epidemic, H. J. Roberts,
MD 2004: Murray 2005.09.30

http://groups.yahoo.com/group/aspartameNM/message/1233
Aspartame -- the shocking story, The Ecologist, 2005 Sept.,
p. 35-51, full text: Murray 2005.09.30: the correct author,
Pat Thomas, What Doctors Don't Tell You www.wddty.co.uk (http://www.wddty.co.uk) :
2005.10.11

http://groups.yahoo.com/group/aspartameNM/message/1131
genotoxicity of aspartame in human lymphocytes 2004.07.29
full plain text, Rencuzogullari E et al, Cukurova University,
Adana, Turkey 2004 Aug: Murray 2004.11.06

http://groups.yahoo.com/group/aspartameNM/message/1228
NM EIB votes 4-2 for 5-day aspartame toxicity hearing July,
2006, requesting a Hearing Officer and a medical expert from
Environmental Dept. and legal advice from NM Attorney
General: Murray 2005.10.04

http://groups.yahoo.com/group/aspartameNM/message/1237
ubiquitous potent uncontrolled co-factors in nutrition research are
formaldehyde from wood and tobacco smoke and many sources,
including from methanol in dark wines and liquors, in pectins
in fruits and vegetables, and in aspartame: Murray 2005.12.04
************************************************** *****