Determination of endogenous formaldehyde in plants (fruits) bound to L-arginine and its relation to the folate cycle, photosynthesis and apoptosis.

A very powerful nucleophilic reagent, hydralazine(1-hydrazino-phtalazine) proved to be suitable for determination of the endogenous formaldehyde level in biological samples. It was found that in different plants (vegetables, fruits, especially in red beet, cauliflower, kohlrabi, grapes) is a large amount of releasable endogenous formaldehyde (0.5-1.0 mM) bound to L-arginine mainly in the form of N(G)-trihydroxymethyl-L-arginine (TriHMA). N(G)-hydroxymethyl-L-arginines (HMA) were proved to transfer their hydroxymethyl groups to tetrahydrofolic acid producing N5,N10-methylene-tetrahydrofolate, the coenzyme of thymidylate synthase. HMA was found to inhibit the cell proliferation of HT-29 cell culture (human colon adenocarcinoma ATCC HT-B 38) causing apoptosis. Photosynthetic experiments produced confirmatory evidences that 14CH2O could be formed in photosynthesis already after 10 seconds of 14CO2 fixation in the seedlings of Zea mays L. (single cross) and the 14CH2O was immediately trapped by L-arginine mainly as TriHMA.

The hydrazine derivative aminoguanidine inhibits the reaction of tetrahydrofolic acid with hydroxymethylarginine biomolecule.

UNLABELLED: Aminoguanidine (AG), a hydrazine derivative is known to inhibit the formation of Advanced Glycosylation Endproducts (AGE) and AG has been proposed as an agent in prophylaxis of diabetic complications. However, treatment with hydrazine produced liver and lung tumors by formation of N7- and O6-methylguanine in the DNA of rodents. The hydrazine derivative, isonicotinic acid hydrazide induced pulmonary tumors in mice. N(G)-hydroxymethyl-arginine (HMA) was synthesized by our research group and it showed anticancer effect against experimental tumors. HMA was found earlier in human blood and urine, and recently in many plants (in fruits and vegetables). We could demonstrate a reaction (pH = 7.5, 37 degrees C, 1h) between HMA and tetrahydrofolate (THF) producing N5,N10-methylene-tetrahydrofolate (CH2-THF), the coenzyme of thymidylate synthase (TS). In model experiments AG proved to react with formaldehyde (HCHO) and to eliminate the C1-fragment of HMA, but not that of CH2-THF. In the presence of AG burst chemiluminescence and a higher speed of the formylation and methylation reactions were found in the AG, HCHO, hydrogen peroxide (H2O2) and L-lysine system than without AG. CONCLUSIONS: HMA as a biomolecule is one of the compounds which are responsible for the endogenous HCHO level. The biochemical function of HMA may be the direct supply of C1-fragment for the folate cycle. AG can disturb the above function of HMA. The reaction between AG and HCHO seems to be dangerous for biological systems because of the possible presence of L-lysine and H2O2. The burst chemiluminescence indicates excited molecules with extreme high energy producing uncontrolled formylation and methylation reactions. Considering the results of the experiments with AG its use as a medicament seems to be questionable.

Formaldehyde generators and capturers as influencing factors of mitotic and apoptotic processes.

There is a growing amount of evidence pointing to the fact that several endogenous and exogenous methylated compounds are potential formaldehyde generators in their biological reactions. N(G)-methylated lysines, N(G)-methylated as well as hydroxymethylated arginines, and 1'-methyl-ascorbigen have been examined in this respect. The apoptosis-inducing effect of formaldehyde molecules formed from methyl groups was earlier first published by our group. Dimedone, an artificial capturer molecule for formaldehyde, has been found to prevent the apoptosis-inducing effect of 1'-methyl-ascorbigen as well as N(G)-hydroxymethylated arginines. More recently resveratrol, present in grapes and wines, has been shown to have cardioprotective and cancer chemopreventive effect. Our group has been successful in demonstrating that this natural formaldehyde capturer molecule can also influence cell proliferation and apoptosis. The apoptosis-inducing or -preventing effect of formaldehyde generators and capturers seems to be dose-dependent and may be utilized in various disturbances of cell proliferation and active cell death.

Reduction of apoptosis of in vitro cultured lymphocytes of HIV-positive persons by N(G)-hydroxy-methylated-L-arginine and 1'-methyl-ascorbigen.

Some formaldehyde generating chemicals due to reduction of apoptosis in lymphocytes may slow down the progress of immune decline of HIV-infected individuals. N(G)-hydroxy-methylated-L-arginine (MAX) and 1'-methyl-ascorbigen (MeAsc) could enter this way the biochemical pathway of cells and affect the apoptotic process. Separated peripheral blood lymphocytes of five asymptomatic HIV-positive persons were cultured. Unstimulated, IL-2 stimulated and IL-2 stimulated plus 0.1, 1.0, 10.0 microg/ml MAX or MeAsc treated lymphocytes were investigated for apoptosis morphologically (HE) and by flow cytometrical DNA fragmentation method. IL-2 stimulation lowered the apoptotic rate in lymphocytes of HIV-positive persons related to unstimulated ones. MAX and MeAsc reduced the apoptotic activity of stimulated lymphocytes in the least or the middle doses while in the higher dose did not. MAX and MeAsc reduced the apoptotic activity of stimulated lymphocytes originated from HIV-positive patients in vitro. This compounds may have the same effect in vivo and may prolong the symptomless period of HIV-infected patients. The role of methylation and production of formaldehyde in this process is discussed.

Analogies and differences in the excited reactions of formaldehyde and D-glucose.

The investigations proved that D-glucose (as reducing sugar) can easily be activated in a ternary system (L-lysine: D-glucose: H2O2) similarly to formaldehyde at 20 degrees C, in pH = 7.4 forming chemiluminescence (CL) and singlet oxygen. The kinetic investigation showed that: CL lasted many hours (permanent emission) and had no bell-shaped curve differently from other aldehydes e.g. formaldehyde. The reason of the effect is that D-glucose exists mainly in ring form in water solution (Haworth ring form) and the open form (the aldehyde group) is slowly liberated during the excited reaction. These excited reactions may be important in human organism, because D-glucose and lysyl residues of proteins occur permanently in human body and endogenous formaldehyde and H2O2 may be liberated there, too.

Possibility of formation of excited formaldehyde and singlet oxygen in biotic and abiotic stress situations.

The results obtained prove that singlet oxygen and excited formaldehyde can be liberated from hydrogen peroxide and formaldehyde in free and bound L-lysine containing plant and other tissues, similarly to the model reactions. It can be concluded that these reactive molecules have extremely high reactivity and are possibly aggressive to cellular components like proteins, nucleic acids and to microbial systems within plant and other tissues. However, the manifestation of their activity depends on level of quenching systems in plant and other tissues. It seems that these special reactions and their products may be an important part of the resistance potential (e.g. natural disease resistance) in biological systems.

Studies on in vitro S-methylation of naturally occurring thiol compounds with N-methyl-N-nitrosourea and methyl methanesulfonate.

N-Methyl-N-nitrosourea (MNU) and methyl methanesulfonate (MMS) were found to rapidly methylate glutathione (GSH) in vitro yielding S-methyl glutathione, as verified and quantitated by high-performance liquid chromatography and thin-layer chromatography. Formation of S-methylcysteine in the acid-hydrolyzate of the methylated GSH further confirmed the formation of S-methyl glutathione. Other naturally occurring thiol compounds such as cysteine and homocysteine were also methylated by MNU. The observed pH dependency of GSH methylation by MNU suggests that the sulfide anion form of the thiol may represent the favored methyl acceptor. The high reactivity of GSH toward MNU and MMS may be of biological significance in that it could compete with macromolecular cellular components as a target for alkylation.

A hypothesis for the mechanism of tumor killing by NG-hydroxymethyl-L-arginines.

Arginine reacts with formaldehyde in a spontaneous equilibrium reaction yielding hydroxymethyl derivatives. They seem to have an direct and indirect inhibiting effect on cell proliferation.

The isolation of Nepsilon-formyl-L-lysine from the reaction between formaldehyde and L-lysine and its identification by OPLC and NMR spectroscopy.

From the reaction between l-lysine and formaldehyde, Nepsilon-formyl-L-lysine was isolated by means of ion-exchange column chromatography. The identification of Nepsilon-formyl-L-lysine was carried out by ion-exchange overpressured-layer chromatography (OPLC) and 1H NMR and 13C NMR spectroscopies. The m.p. and mixed m.p. values, the retention characteristics and the chemical shifts of the isolated product were identical with those of an authentic sample of Nepsilon-formyl-L-lysine.

Spontaneous N epsilon-methylation and N epsilon-formylation reactions between L-lysine and formaldehyde inhibited by L-ascorbic acid.

For the inhibition of spontaneous N epsilon-methylation and N epsilon-formylation reactions between L-lysine and formaldehyde, L-ascorbic acid proved to be most suitable. The inhibition was not complete unless the molar concentration of ascorbic acid exceeded that of formaldehyde. T.l.c., potentiometric titration, n.m.r. spectroscopy and radiometric analysis were applied in the study of the inhibition process. Formaldehyde was reduced by L-ascorbic acid to ethylene glycol.

Possible role of the control of arginase and methionine adenosyl transferase in the transport processes of endogenous formaldehyde and in hypermethylation.

The authors believe the normal formaldehyde equilibrium in the organism to be governed by a biological control mechanism, which is thought to be closely connected to L - arginine and - indirectly - to the arginase enzyme. Arginine reacts with formaldehyde in a spontaneous equilibrium reaction yielding methylol - derivatives thus mobilizing formaldehyde. The methylol - derivatives of L - arginine were found in serum and urine. The authors assume the methylol - derivatives of L - arginine to have an inhibiting effect on cell - proliferation.

Spontaneous N epsilon-methylation of L-lysine by formaldehyde.

It has been found that the conversion of L-lysine (1) into its corresponding N epsilon-methylated derivatives, mainly N epsilon-mono-methyl-L-lysine (5; MML), but also N epsilon, N epsilon-dimethyl-L-lysine (DML) and N epsilon, N epsilon, N epsilon-trimethyl-L-lisine (TML) takes place by treatment with formaldehyde in spontaneous reaction. The identification of N epsilon-methylated lysines was carried out by different chromatographic and spectroscopic methods. This spontaneous N epsilon-methylation of L-lysine by formaldehyde may also play an important role in living organisms.