Reaffirmation That Metabolism of Polyamines by Bovine Plasma Amine Oxidase Occurs Strictly at the Primary Amino Termini*

Younghee Lee and Lawrence M. SayreDagger

From the Department of Chemistry, Case Western Reserve University, Cleveland, Ohio 44106

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

Oxidation of the biologically important polyamines spermine and spermidine by plasma amine oxidase (PAO) was specified many years ago to occur at the terminal primary rather than internal secondary amine positions. However, the finding of sequential enzymatic conversion of spermine to spermidine and then to putrescine (1,4-butanediamine) is superficially suggestive of metabolism at the secondary amine positions, and a recent publication (Houen, G., Bock, K., and Jensen, A. L. (1994) Acta Chem. Scand. 48, 52-60) claimed that the original interpretation of preferential "terminal" deamination does not stand up to scrutiny with modern methods of analysis. We herein demonstrate that the findings cited in support of secondary amine deamination can arise artifactually from spontaneous elimination/addition reactions following initial metabolism at the terminal positions of 3-(aminopropyl)amines. We further find no evidence for the ability of PAO to metabolize the secondary amine position in homospermidine, which is devoid of such complicating side reactions. Our results support the original claimed specificity of PAO for the primary amino termini of polyamines, all of which are consistent with the general finding that the quinone-dependent copper amine oxidases specifically metabolize primary amines.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

In the early days of clarification of the substrate specificity of the soluble amine oxidase present in mammalian plasma (PAO),1 the Tabor group (1) demonstrated that the naturally occurring polyamines spermine and spermidine are metabolized at the primary amino termini, in contrast to several bacterial amine oxidases that metabolize polyamines at the secondary nitrogen atoms (2, 3). The soluble plasma enzyme (EC 1.4.3.6), now known as one class of a family of copper-containing amine enzymes, is thereby distinguished from the flavin-dependent amine oxidases present in mammalian mitochondria, which although primarily involved in metabolism of primary amines, can also dehydrogenate certain secondary and tertiary amines (4). With the recognition that the copper-containing amine oxidases metabolize amines through a transamination process mediated by an active site 2,4,5-trihydroxyphenylalanine quinone cofactor derived post-translationally from a conserved tyrosine (5), the restriction to primary amine substrates might be viewed as a simple consequence of the need to form a neutral Schiff base with the quinone cofactor in the first step of the enzymatic mechanism. However, secondary amines could theoretically also fulfill a transamination mechanism via initial formation of an iminium quinone-derived intermediate. In fact, there have been isolated claims of the ability of certain secondary amines to be processed by the copper-containing plasma amine oxidases (6, 7). Nonetheless, in these cases (N-methyl-beta -aminopropionitrile and beta ,beta '-iminodipropionitrile), we suspected that the commercial materials contained traces of the corresponding primary amine beta -aminopropionitrile, a known substrate/inactivator of PAO (8) and inactivator of lysyl oxidase (9), and we showed that successive purification cycles of the secondary amines eliminated the production of H2O2 during their incubation with PAO.2

It was thus very surprising to us to see the provocative claim by Houen et al. (10) that the metabolism of polyamines (spermine and spermidine) by plasma amine oxidases occurs principally at the secondary rather than primary amine centers. These workers provided data on the sequential conversion of spermine to spermidine and then to putrescine, which they claimed resulted from regiospecific oxidation at the two internal secondary amine positions of spermine, releasing 1 and then 2 eq of 3-aminopropanal (Scheme 1). Upon attempted isolation, the latter is unstable with respect to elimination to acrolein and ammonia and could only be identified qualitatively by ion-exchange chromatography (before and after borohydride reduction) and by 1H NMR spectroscopy.


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Scheme 1.  

A review of the literature published since the initial studies by the Tabor group reveals that the formation of spermidine and then putrescine had been consistently observed (11-13), although Bachrach and co-workers (14) interpreted this to be a consequence not of secondary amine oxidation but of spontaneous elimination of acrolein by the labile aminopropionaldehyde products of terminal deamination (Scheme 2).


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Scheme 2.  

One piece of evidence obtained by the Tabor group in support of metabolism of polyamines at the terminal rather than internal amino groups was their quantitation of the released ammonia (1). In retrospect, this alone would be insufficient distinguishing evidence since the same amount of ammonia would be generated according to the Scheme 1 proposal of Houen et al. (10). However, the Tabor group also endeavored to identify the nature of the released aldehyde product(s) and finding these to be thermally labile, ultimately achieved a consistent result by using short treatments with relatively high amounts of enzyme, in which case the metastable dialdehyde product of double terminal amine oxidation could be trapped by a prompt NaBH4 post-treatment (Scheme 3) (1). A good yield of the stable N,N'-bis(3-hydroxypropyl)-1,4-butanediamine, identified by comparison to an authentic standard, required a short incubation time (15 min) with a relatively high concentration of enzyme prior to NaBH4 quenching. In retrospect, these results are understandable in terms of the spontaneous acrolein elimination competing kinetically with enzymatic deamination (Scheme 2) when lower enzyme concentrations are used.


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Scheme 3.  

In their work, Houen et al. (10) also considered the terminal deamination pathway claimed by Tabor et al. (1), but ruled it out on the basis of their failure to detect the requisite N,N'-bis(3-hydroxypropyl)-1,4-butanediamine and their identification of 3-aminopropanal instead. Moreover, in their hands, the spermine right-arrow spermidine right-arrow putrescine conversion pattern was unaffected by a NaBH4 post-treatment. Houen et al. (10) stated that the early conclusion of terminal deamination relied on what would be considered today as inadequate methods of product characterization, whereas they now had access to more modern methods (NMR, HPLC). However, it seems clear to us that despite citing the seminal Tabor group paper (1), Houen et al. (10) did not recognize how rapidly the borohydride quench had to be carried out to avert elimination of acrolein by the initial aminoaldehyde products. In fact, they seemed unaware that their own experimental conditions (low enzyme concentration and longer reaction times) had been described by Tabor et al. (1) to be inadequate to achieve deamination at the second amino terminus during the time that the aminoaldehyde generated at the first amino terminus still survived. Thus, Houen et al. (10) had no chance to observe the borohydride-trapped product of double terminal deamination.

Independent of the above controversy, additional evidence for PAO metabolism of secondary amines presented by Houen et al. (10) was their observed time-dependent production of benzaldehyde from N-ethylbenzylamine, using the bovine plasma enzyme (BPAO). However, their graph showed that there was no oxidation at all for the first 6 h. The low level of benzaldehyde formed at later times could thus reflect autoxidation of this benzylic secondary amine (15), possibly aided by copper released upon deterioration of the enzyme after long term incubation.

A key complication surrounding the mechanistic controversy is that if oxidation by PAO occurs exclusively at the primary amino termini as originally claimed (Scheme 2), then it is not immediately obvious why Houen et al. (10) were able to trap 3-aminopropanal, predicted according to their secondary amine oxidation route (Scheme 1). Although Houen et al. (10) thereby concluded that spermidine was undergoing internal deamination, we suspected that 3-aminopropanal might be arising instead from combination of the ammonia and acrolein formed in the terminal deamination pathway (Scheme 2).

In the present investigation, we first confirm the formation of 3-aminopropanal from the reaction of ammonia and acrolein employing concentrations that would be present according to the conditions used by Houen et al. (10) for oxidation of spermidine. Second, we used homospermidine, also a naturally occurring polyamine (16), as an alternative tool to study the site of metabolism by BPAO, since extension of one methylene group would abrogate the acrolein elimination side reaction that results in the above-described mechanistic conundrum. Our results reaffirm the initial conclusions of the Tabors and Bachrach that PAO metabolism of polyamines occurs strictly at the primary amino termini.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

General Methods-- NMR spectra were obtained on a Varian Gemini 300 instrument (1H NMR at 300 MHz; 13C NMR at 75 MHz), with chemical shifts being referenced to the solvent peak. High resolution mass spectra (HRMS, electron impact) were obtained at 20-40 eV on a Kratos MS-25A instrument. All solvents, reagents, and organic fine chemicals were the most pure available from commercial sources. Acrolein and 1-pyrrolidinebutyronitrile were freshly distilled before use. Doubly distilled water was used for all experiments. BPAO (100 units/g of protein) and catalase were purchased from Sigma). All evaporations were conducted at reduced pressure using a rotary evaporator. Deuterated buffers were prepared by first pH-adjusting aqueous phosphate solution, evaporating, and redissolving the salts in D2O.

1H NMR Monitoring of the Reaction of Ammonia with Acrolein-- An NMR experiment was carried out by mixing acrolein (0.28 µl, 2 mM) and NH4OH (2.6 µl of 30% aqueous solution, 20 mM) in 2 ml of 100 mM phosphate buffer (D2O, pH 7.2). The 1H NMR spectrum was recorded periodically over a 3-day period. Signals corresponding to 3-aminopropanal hydrate at delta  3.10 and 5.21 (first apparent after 3 h) grew as the signals corresponding to starting acrolein decreased. After 24 h, little change in the spectrum occurred, except for the new appearance of an unknown singlet at delta  3.68. When the [NH4+] was reduced to become stoichiometric with acrolein (2 mM), as would be predicted by Scheme 2, unknown 1H NMR signals at delta  3.69 and 5.16 were observed. In their experiments, Houen et al. (10) started with 0.2 mM spermidine (in 100 mM pH 7.2 phosphate buffer), which would ultimately give 0.2 mM acrolein. Our experiments used a 10-fold higher concentration to permit 1H NMR monitoring. The [NH4+] present in the Houen et al. (10) experiment would also be only 0.2 mM if the only source of ammonia were its generation stoichiometric with acrolein, in which case our model 1H NMR experiment failed to detect aminopropanal. However, Houen et al. (10) used partially purified BPAO, which we suspect contained some (NH4)2SO4. Indeed, in the experiment where Houen et al. (10) detected aminopropanal by ion-exchange chromatography (Fig. 5 of Ref. 10), the NH3 peak present at time = 0 was nearly as large as it was at time = 20 h. Although we can thus only guess at the [NH4+] in their experiment, we presume that it was in excess of [acrolein], as in our experiment.

Acetyl Derivatization of the Reaction of Ammonia with Acrolein-- A mixture of acrolein (140 µl, 2 mM) and NH4OH (1.3 ml of 30% aqueous solution, 20 mM) in 1 liter of phosphate buffer (100 mM, pH 7.2) was stirred at room temperature for 3.5 h. Then NaBH4 (3.7 g, 100 mmol) was added with stirring, and after 20 min, acetic anhydride (20 ml, 200 mmol) was added dropwise while maintaining the reaction mixture at pH 10.5 by addition of 1 M NaOH solution. After stirring an additional 30 min, the reaction mixture was concentrated until the first signs of a precipitate appeared. The aqueous layer was extracted with ethyl acetate (100 ml × 3). The combined organic extract was dried (Na2SO4) and concentrated, and the resulting residue was placed under high vacuum. After most of the CH3CONH2 by-product was sublimed under vacuum, the remaining residue was dissolved in CDCl3 for 1H NMR analysis. The chemical shifts of the major (60%) compound present (delta  1.67 (p, J = 5.7 Hz, 2H), 1.99 (s, 3H), 3.38 (q, J = 5.6 Hz, 2H), 3.63 (t, J = 5.8 Hz, 2H)) match the 1H NMR spectrum of 3-(N-acetyl)aminopropanol previously reported (17). The minor (30%) compound present, displaying 1H NMR signals at delta  1.82 (p, 2H), 1.98 (s, 6H), 3.27 (q, 2H), and 4.11 (t, 2H), is assigned as the N,O-diacetyl derivative of 3-aminopropanol. The remaining 1H NMR peaks (corresponding to 10% of the total integral) were not identified.

Reaction of BPAO with Homospermidine-- To a solution of homospermidine·3HCl (25 mg, 93 mmol), prepared as described (18), in 25 ml of phosphate buffer (100 mM, pH 7.2), were added 250 µl of catalase (4000 unit/ml in 100 mM phosphate buffer, pH 7.2) and 500 µl of BPAO (dialyzed for 3 h against phosphate buffer to remove (NH4)2SO4), and the mixture was incubated for 2 h at room temperature. The incubation solution was placed in an ice bath, and 3 ml of freshly prepared NaBH4 (200 µM in 1 M aqueous NaOH) was added. After stirring the mixture for 20 min at room temperature, acetic anhydride (1 ml) was added slowly while maintaining the solution at pH 10.5 by dropwise addition of 0.5 M NaOH. Upon completion of the addition, the pH was raised to 12.5 for 20 min and then lowered to 10.5 by dropwise addition of dilute HCl. The solution was evaporated under reduced pressure until most of the water was removed. The remaining residue was extracted with EtOAc (50 ml × 2), and the organic layer was dried (Na2SO4) and concentrated to afford a viscous oil. 1H NMR analysis showed a clean mixture of triacetylated homospermidine 5 and 1-(4-acetamidobutyl)pyrrolidine (6) in a 5:1 ratio (NMR signals of the independently synthesized samples are given below). A duplicate reaction incubated for 20 h before NaBH4 reduction and treated as described above afforded the same mixture of 5 and 6 in a 1.3:1 ratio.

N,N-Bis(4-acetamidobutyl)acetamide (5)-- A mixture of homospermidine·3HCl (54 mg, 0.2 mmol), Ac2O (190 µl, 2 mmol), and triethylamine (279 µl, 2 mmol) in CH2Cl2 (10 ml) was stirred overnight at room temperature. The volatile material was removed under reduced pressure, and the residue was treated with concentrated Na2CO3 (1 ml). Extraction with EtOAc (20 ml × 2) and concentration of the organic layer gave a crude mixture that was purified by flash column chromatography using EtOAc:MeOH (1:1) as eluant to afford the triacetyl derivative 5 of homospermidine: 1H NMR (CDCl3) delta  1.24-1.50 (8H), 1.80 (s, 6H), 1.90 (s, 3H), 3.00-3.17 (8H), 7.24 (bt, NH, 2H); 13C NMR (CDCl3) delta  21.5, 23.0(2C), 24.9, 26.0, 26.4, 26.8, 38.6, 38.8, 45.0, 48.3, 170.3, 170.7 (2C); HRMS calculated for C14H27N3O3 m/z 285.2054, found 285.2048. The asymmetry seen in the NMR spectrum most likely reflects intramolecular hydrogen bonding between the carbonyl of the tertiary amide group and one of the two terminal secondary amide groups.

1-(4-Acetamidobutyl)pyrrolidine (6)-- A solution of AlCl3 (5 g, 38 mmol) in anhydrous ether (100 ml) was added to LiAlH4 (1.45 g, 38 mmol) in anhydrous ether (100 ml). The mixture was stirred under argon for 10 min followed by addition of 1-pyrrolidinebutyronitrile (4.6 g, 33 mmol) in anhydrous ether (50 ml). After heating at reflux for 3 h, the mixture was cooled to O °C and quenched with aqueous 30% KOH (50 ml). Ether (200 ml) was added, and the reaction mixture was heated at reflux for an additional 30 min. The organic layer was decanted, dried (Na2SO4), and concentrated to give a crude oil that was distilled under reduced pressure to yield 3.7 g (79%) of 1-(4-aminobutyl)pyrrolidine: 1H NMR (CDCl3) delta  1.45 (m, 4H), 1.72 (m, 4H), 1.88 (bs, NH2, 2H), 2.39 (t, J = 6.71 Hz, 2H), 2.44 (m, 4H), 2.65 (t, J = 6.71 Hz, 2H); 13C NMR (CDCl3) delta  23.4(2C), 26.4, 31.9, 42.1, 54.2(2C), 56.4; HRMS calculated for C8H18N2 m/z 142.1471, found 142.1469. A mixture of the latter amine (142 mg, 1 mmol), Ac2O (0.94 ml, 10 mmol), and triethylamine (1.39 ml, 10 mmol) in CH2Cl2 (30 ml) was stirred overnight at room temperature. The volatile material was removed under reduced pressure, and the residue was treated with concentrated Na2CO3 (1 ml). Extraction of the aqueous solution with EtOAc (30 ml × 2) and concentration of the organic layer gave pure 6 (152 mg, 83%): 1H NMR (CDCl3) delta  1.59 (4H), 1.81 (m, 4H), 2.50 (t, J = 6.90 Hz, 2H), 2.56 (m, 4H), 3.23 (dt, J = 6.90 and 5.67 Hz, 2H), 6.83 (bs, NH, 1H); 13C NMR (CDCl3) delta  23.3, 23.4(2C), 26.4, 27.7, 39.4, 54.0(2C), 55.9, 170.0; HRMS calculated for C10H20N2O m/z 184.1577, found 184.1577.

    RESULTS
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Results
Discussion
References

Direct 1H NMR Observation and Trapping of the Product of Reaction of NH3 with Acrolein-- Houen et al. (10) followed the oxidation reaction of spermidine by BPAO in phosphate buffer (D2O, pH 7.2) using 1H NMR monitoring at various time points (up to 200 h) and observed the production of putrescine and 3-aminopropanal, with concomitant decrease of spermidine. Because 3-aminopropanal undergoes deuterium-proton exchange at C-2 under the reaction condition owing to enolization (Equation 1 


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Equation 1.
), only two signals were observed, a singlet at delta  3.10 assigned to the C-3 proton and a singlet at delta  5.24 assigned to the C-1 position existing as the carbonyl hydrate (Equation 1).

We incubated acrolein (2 mM) in 100 mM phosphate buffer (D2O, pH 7.2) in the presence of 20 mM ammonium (from NH4OH), which we considered (see "Experimental Procedures") to simulate the initial product condition of the enzymatic conversion of spermidine (10), and the 1H NMR spectrum was recorded over a 3-day period. Two singlets at delta  3.10 and 5.21 grew as the signals corresponding to starting acrolein decreased. Although the chemical shifts of these two signals are consistent with those of 3-aminopropanal as assigned by Houen et al. (10) we felt the need to confirm the structure by isolation of the known 3-acetamidopropanol (17) when the reaction mixture was treated with NaBH4 followed by acetic anhydride trapping, as shown in Equation 2 


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Equation 2.
.

Reaction of Homospermidine with Bovine Plasma Amine Oxidase-- As an alternative approach to resolving the question of primary versus secondary amine metabolism of polyamines by BPAO, we investigated homospermidine as a substrate to avert the complication of acrolein elimination following deamination. Being a symmetrical polyamine, oxidation of homospermidine at the primary amino group would give 4-(4-aminobutylamino)butanal (1) (and eventually 4,4'-iminodibutanal (2)), whereas oxidation at the secondary amino group would instead give 4-aminobutanal (3) and putrescine (4) (Scheme 4). This study would unfortunately not be free of all complications, since aminobutyraldehydes are known to cyclize (19, 20). For this reason, and to avoid any complicating intermolecular amine-carbonyl side reactions, we planned to conduct our product analysis following a sequential NaBH4/Ac2O quench prior to workup.


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Scheme 4.   q (quench) = NaBH4; Ac2O, pH 10.5; pH 12.5 then pH 10.5. 

Homospermidine was incubated with BPAO and catalase in 0.1 M pH 7.2 phosphate buffer at room temperature. After 2 h, the mixture was quenched with NaBH4 at 0 °C. Excess acetic anhydride was added to the mixture while maintaining the pH at 10.5; then the pH was raised to 12.5 for 20 min, in order to hydrolyze any existing O-acetyl groups, and then lowered back to 10.5. Following workup, 1H NMR analysis of the crude product revealed a clean mixture of triacetylated homospermidine 5 and 1-(4-acetamidobutyl)pyrrolidine (6) in a ratio of 5 to 1 (see Scheme 4). A duplicate reaction incubated for 20 h before NaBH4 reduction and treated as described above afforded the same mixture of 5 and 6 in a ratio of 1.3 to 1, consistent with more extensive enzymatic conversion of starting homospermidine at the later time point.

Isolation of 6 provides direct evidence for primary rather than secondary amine metabolism by BPAO according to Scheme 4. Compound 6 arises because the aldehyde 1 formed from deamination of the terminal amino group apparently exists totally as 1-(3-aminobutyl)pyrrolinium prior to the NaBH4/Ac2O quench; the diacetylated derivative of N-(3-hydroxybutyl)-1,4-butanediamine otherwise expected by direct quenching of aldehyde 1 was not observed. This finding is precedented by the identification of 1-(3-aminopropyl)-2-pyrroline in the oxidation products of spermidine by plant diamine oxidase (21). The other possible product expected from metabolism of the terminal amino group, 1-(4-hydroxybutyl)pyrrolidine (7) was not seen at all in the reaction mixture. This indicates that there is still substantial unreacted substrate, so that the enzyme has not yet had a chance to carry out the second stage of oxidation. If the enzyme processed homospermidine at the secondary amino group, compounds 8 and/or 9 would have been isolated under the reaction condition and workup treatment. The complete absence of these materials indicates that metabolism at the secondary amino group did not occur. The structures of 5 and 6 were confirmed by independent synthesis of authentic samples (see "Experimental Procedures").

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

We have demonstrated that 3-aminopropanal, observed as key evidence of oxidation of polyamines (spermine and spermidine) at the secondary amino group by BPAO, could also be generated under the conditions of oxidation at the primary amino group. This is because primary amine deamination of N-(aminopropyl) groups is followed by nonenzymatic elimination of acrolein, which can combine with the NH3 in solution. Our study on the metabolism of homospermidine, which is devoid of such complicating side reactions, revealed that BPAO processed this compound exclusively at the primary amino terminus. Therefore, the claim by Houen et al. (10) that the metabolism of polyamines by PAO occurs principally at the secondary amine centers appears to be an artifact. It should be pointed out that if PAO really were a secondary amine metabolizer, it would be necessary to explain the regiochemical course in the case of unsymmetrical substrates; viz. in the case of spermine and spermidine, it would be unclear why the reaction gave 3-aminopropanal each time rather than 1,3-propanediamine and the corresponding 4-aminobutanal. Both regiochemistries have been observed for spermidine using one or another secondary amine metabolizer (21). In the case of PAO, our results instead are in line with the earlier interpretation by the Tabor group (1) that metabolism of polyamines occurs at terminal amino groups.

In addition, we have been unable to substantiate any other claims (6, 7) that PAO can metabolize secondary amines. Since model studies confirm that secondary amines can undergo transaminative processing via a quinone iminium intermediate (22), the restriction of PAO to oxidation of primary amines must reflect a steric exclusion issue rather than an intrinsic chemical requirement. The steric explanation is not surprising in that, even for primary amines, PAO exhibits a strict preference for metabolism of unbranched primary amines to aldehydes---metabolism of branched primary amines to ketones has not been reported (23). Based on this knowledge, we were surprised to find that the secondary amine 3-pyrroline and its 3-aryl derivatives induce mechanism-based inactivation of BPAO (22). Although we could not detect any productive turnover in these cases, the observed inactivation indicates that BPAO can at least initially process these sterically "tied back" secondary amines. As structural information on the copper amine oxidases continues to be revealed (24), substrate structure requirements will eventually be understood in terms of the shapes and dimensions of the active site cavities.

    FOOTNOTES

* This work was supported by Grant GM 48812 from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed. Tel.: 216-368-3704; Fax: 216-368-3006; E-mail: lms3{at}po.cwru.edu.

1 The abbreviations used are: PAO, plasma amine oxidase; BPAO, bovine plasma amine oxidase; HRMS, high resolution mass spectrometry.

2 F. Wang and L. M. Sayre, unpublished studies.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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