Reaction of ozone with nicotinamide and its derivatives

ARCHIVES

OF

BIOCHEMISTRY

Reaction

AND

of Ozone

with

J. B. MUDD, Department

of Biochemistry

161,

BIOPHYSICS

408-419

Nicotinamide

F. LEH,

AND

and Statewide Air California, Riverside, Received

(1974)

September

and

Its Derivatives

T. T. McMANUS Pollution California

Research 9,%07

Center,

University

of

20, 1973

Reaction of ozone with NADH eliminated the 340 nm absorbance. The 260 nm absorbance increased initially and then slowly declined. Equimolar amounts of NADH and ozone reacted, Products were separated by ion exchange chromatography and were shown to contain only traces of NAD. The products were not active in the alcohol dehydrogenase assay and did not form cyanide complexes. There was no reaction of NAD with ozone. NADPH and NADP showed the same reactivities as NADH and NAD. The reactions were not influenced bypH in the range 4.6-9.0. When NADH and tryptophan, or NADH and methionine, or NADH and glutathione (at low concentration) were competitors for available ozone, NADH was preferentially oxidized. Treatment of aqueous solutions of 1,4-dihydro-l-methyl nicotinamide with ozone caused changes in the absorbance spectrum consistent with ozonolysis of the 5,6 double bond. The product has been isolated after ozonolysis in methylene chloride

and characterized consistent

with

by uv, ir, mass spectroscopy, the

and elemental

analysis. Results were

reaction: H

H

Further ozonolysis cleaved the 2,3 double bond. Reaction of NADH with ozone resulted ih analogous reactions. The adenine moiety was resistant to ozonolysis, but this reaction was pH dependent, being greater at neutral and alkaline pH. BThe primary pollutants in automobile exhausts include unburned hydrocarbons and oxides of nitrogen (1.) In the presence of oxygen a series of photochemical reactions of the primary pollutants produces oxidant secondary pollutants (2). The major oxidant pollutant is ozone, and next in importance is peroxyacetyl nitrate. Together, these compounds may rise to concentrations as high as 0.8 ppm in the Los Angeles basin. Ambient concentrations of ozone cause the formation of lesions on many types of vegetation (3). Exposure of experimental animals to ozone concentrations of 0.5 ppm results in drastic lowering of enzymes of the pulmonary alveolar macrophage, such as lysozyme, acid phosphatase, and p-glucuro-

nidase (4). While these experiments show ambient levels of ozone to be toxic, they do not show what is the initial biochemical event. Enzymes may be inactivated directly by reaction with susceptible amino acid residues (5). On the other hand, a primary point of attack may be ozonization of unsaturated fatty acids and initiation of lipid peroxidation (6-8). Many other biochemical compounds may be expected to react with ozone. Among reduced nicotinamide derivatives these, appear to be particularly susceptible. We have previously reported the oxidation of reduced nicotinamide derivatives by ozone, emphasizing that the oxidation product had no biological activity (9), in contrast to the 408

Copyright All rights

Q 1974 by Academic Press, of reproduction in any form

Inc. reserved.

OZONIZATION

products of the oxidation of NADH and NADPH by peroxyacyl nitrates, which appear to be NAD and NADP (10). A recent paper has also reported that the oxidation of NADH by ozone produces inactive derivat’ives (ll), but another report suggests that the oxidation product from NADPH is NADP (12). A study of the reaction of ozone with the acid addition product of NADH was made by Cornforth et al. (13) for the purpose of clarifying the absolute stereochemistry at position 4. The sequence of reactions was described :

Ozonolysis of (I) gave two major products (II) and (III).

,A N/CHo

N’

4

F;

(IL)

(Iu)

CHO

Further oxidation of these intermediates produced succinic acid. In this paper we describe some characteristics of the reaction of ozone with NADH and the characterization of reaction products. MATERIALS

AND

METHODS

Ozone was generated in an oxygen stream flowing at 20 ml/min either by a silent electric discharge or by a mercury lamp. Ozone concentration in the gas stream was varied by the voltage applied and was monitored by bubbling the gas stream through a neutral solution of potassium iodide. The liberated iodine was assayed spectrophotometrically at 350 nm. The silent electric discharge apparatus was used for dosages in the range 0.1-2.0 rmoles/min, and the mercury lamp for dosages in the range 0.1-100 nmoles/min. Carbonyl-labeled [%]NAD was obtained from Amersham/Searle Corporation. [14C]NADH was prepared from this compound in a reaction mixture containing 2.22 X 106 dpm [l(C]NAD, 5.5 mg NAD (Sigma Chemical Company), 0.3 ml ethanol, 300 rmoles Tris-WC1 (pH 9.2)) yeast alcohol dehy-

OF NADH

409

drogenase (Boehringer Mannheim Corporation, New York), and water in a final reaction volume of 3.0 ml. Aliquots of 0.1 ml were used to follow the progress of the reaction by measuring absorbance at 340 nm with a Cary 15 spectrophotometer. At the completion of the reaction, 7.0 ml water was added, and the mixture was separated by ion exchange chromatography on DEAE-cellulose (14). The effluent was monitored at 260 nm and 340 nm, and the [14C]NADH Eractions were pooled and lyophilized. The residue was dissolved in 10.5 ml water, and ten l.O-ml aliquots were separately lyophilized until used. NADH was obtained from Sigma Chemical! Company, St. Louis, MO; l-methyl nicotinamide: was obtained from Calbiochem, La Jolla, CA.. Nieotinamide-adenine dinucleotidase was purchased from Sigma Chemical Company. All soIvents were distilled and dried over molecular sieves. Analysis of amino acids (tryptophan, methionine, methionine sulfoxide) was by ion exchange chromatography using a Beckman 120B amino acid analyzer. Thiol groups were measured by the method of Ellman (15). Assay of NAD and NADH oxidation products with alcohol dehydrogenase was as described above. Assay of NADP and NADPH oxidation products used glucose-6-phosphate dehydrogenase partially purified from spinach leaves. Assay of NAD and NADP as cyanide complexes followed the method of Ciotti and Kaplan (16). Visible and ultraviolet absorbance spectra were recorded with a Cary 15 spectrophotometer. Infrared spectra were obtained either in chloroform solution or in the solid state as KBr pellets using a Perkin-Elmer 621 spectrophotometerMass spectra were taken on a Finnigan 1015 instrument under the following conditions: ionizing voltage, 60 V; ionizing current, 70 mA; t,emperature range, 50-120°C. The method of Stock et al. (17) was used to prepare 1,4-dihydro-l-methyl nicotinamide, except that the compound was extracted from the aqueous reaction mixture with methylene chloride rather than ether. The product was generally an oily solid but could be crystallized from ethyl acetate, mp 85.5-86°C (18). An Aerograph Hi-Fi 600D chromatograph was used in the identification of degradation products. For volatile acids such as formic acid and acetic acid, a method derived from Jackson (19) was adopted using a column of 10 ft with internal diameter of 2 mm and packed with a mixture of polyethylene glycol (1 part), suberic acid (1 part), and acid washed Chromosorb W 6&80 mesh (10 parts). Dibasic acids such as malonic and succinic acids were determined as their esters using a col-

410

MUDD,

LEH, AND McMANUS

umn packed with diethylene glycol succinate (20%) on Chromosorb W (60-80 mesh) (Dummel and Kun (20)). The following values of retention {relative to solvent) were obtained: (1) methanol., 1.0; formic acid, 7.0; acetic acid, 13.3, (2) methylene chloride, 1.0; methyl malonate, 3.2; methyl succinate, 5.7. Nitrogen flow, 50 ml/mini oven temperature, 130°C; injector temperature, 160°C.

I

I

I

T

I 0,

I

Al

nmoles/min

RESULTS

Concentration efects on the reaction of ozone with NADH. Figures la and b show the oxidation of NADH by ozone, determined by the decrease in absorbance at 340 nm. The initial concentration of NADH was varied from 10 PM (run No. 9) to 0.9 mM (run No. 1). At the higher initial concentrations of NADH, 1 mole of NADH was oxidized per mole of ozone. At the lower concentrations of NADH (runs 8 and 9) the data show more than 1 mole of NADH oxidized per mole of ozone, but the control of ozone concentration in these runs was more difficult, and the actual stoichiometry is in doubt. The notable point is that even at low concentrations of NADH and low concentrations of ozone, the oxidation of NADH is a significant reaction. Stagesin the oxidation of NADH by ozone. The oxidation of NADH to NAD is accompanied by both a decrease in absorbance att 340 nm and an increase in absorbance at 260 nm. Figure 2 shows the results of an experiment where measurements were made at both of these wavelengths and in addition the amount of ozone passing through the NAD solution was monitored. The 340 nm absorbance changes were recalculated as micromoles oxidized, and the initial stages of oxidation give an equivalence with the amount of ozone introduced. While NADH is still present in the ozonized solution, very little ozone passes through into the trap. The amount of ozone passing into the trap

increases after the 340 nm absorbance has been eliminated. Monitoring at 260 nm gives predictable results up to the point that the 340 nm absorbance has been eliminated, but after that point the 260 nm absorbance declines slowly and may be taken as indicative of oxidation of the adenine moiety. InJluence of pH on the reaction of ozone

6 p moles

0 8 -’

/

0,

I

I 03,

B0.e

nmoles/min 6 7

IO 2

:

a:2

-07

_

\.

,umoles

o6

$

05

“? z

1”’

03

FIGS. la and b. Oxidation of NADH with different concentrations of ozone. Reaction mixtures consisted of 5.0 ml 0.1 M phosphate buffer (pH 7.2) containing varying amounts of NADH. Samples were bubbled with varying concentrations of ozone in oxygen at a flow rate of 20 ml/ min. Aliquots were taken at various times to assay progress of the reaction. The duration of gas flow varied between 11 min (Expt 1) to 110 min (Expt 9). Amounts of ozone administered were calculated from the reaction time and the ozone concentration. Data points show the amount of NADH remaining in the solution and the amount of ozone administered.

with NADH. Figure 3 shows that changes in pH from 4.6 to 9.1 have very little influence on the amount of NADH reacted per mole of ozone, and furthermore there was apparently no buffer effect. Resolution of the ozonolysis products. NAD and NADH can be resolved on a column of DEAE-cellulose.

This

method

was also used

in the separation of NADH ozonolysis products. The results of a chromatographic separation are shown in Fig. 4. 14C-Labeled

OZONIZATION

411

OF NADH

NADH was used in an attempt to obtain preliminary characterization of the products. The results of such an experiment are shown in Figs. 5a and b. Figure 5a shows the 260 nm absorbance of the various fractions, and it is clear that the major product by this criterion is compound C. Figure 5b shows the distribution of radioactivity of the same samples. Again, compound C is the major product, but the contributions of compound A and B are apparently greater in terms of t

I 0.2

I 0

04

0.6 3.8 p moles 03

I I.0

I 1.2

I 1.4

FIQ. 2. Stages of NADH oxidation. The apparatus consisted of two tubes connected in series. The reaction mixture in the first tube consisted of 350 nmoles NADH in 5.0 ml 0.1 M phosphate buffer (pH 7.2). The second tube contained a mixture of 2.0 ml 0.1 M phosphate buffer (pH 7.2) and 3.0 ml 0.1 M KI. The ozone containing oxygen stream was passed through the vessels at a flow rate of 20 ml/min. The ozone flow was 31.2 nmoles/min at the beginning and 30.0 nmoles/min at the end of the experiment. At timed intervals the solution in the first vessel was scanned from 220 to 400 nm, and the solution in the second vessel was measured at 359 nm to determine the amount of ozone not dissolved or reacted in the first vessel.

2d 5

0 A . A

0.5

z 2

I

ACETATE PHOSPHATE TRIS BORATE

I

I

I

I

I

I

4.0

5.0

6.0

7.0

8.0

9.0

I

PH

3. Effect of pH on the oxidation of NADH by ozone. Reaction mixtures consisted of 3.0 ml 0.067 M buffer containing 320 nmoles of NADH. The reaction mixtures were bubbled with an ozone containing oxygen stream at 20 ml/min. The ozone dosage was 9.5 nmoles/min at the beginning of the experiment and 10.7 nmoles/min at the end. Readings at 340 nm were made at 0,2, 4, 6, and 8 min in order to calculate the rate of oxidation. FIG.

40

32 24 FRACTION

16 NUMBER

6

0

FIG. 4. Elution diagram for NADH and oxidation products. Samples of NADH before (a) and after (b) treatment with ozone were separated by chromatography on DEAE-cellulose as described in the legend to Fig. 5.

412

MUDD,

0

0.2

0.4 ,umoles

0.6

LEH, AND McMANUS

0

0.8

O3

02

L. 04 ,omoles

06

OS

o3

Fra. 5. Reaction of [car60nyZJ4C]NADH with ozone. Reaction mixtures consisted of 6.0 ml 10 mM Tris-HCl (pH 7.2) containing 630 nmoles [W]NADH (142,000 dpm). Samples were exposed to ozone flowing at 10 nmoles/min in an oxygen stream. Samples were exposed for specific times and analyzed for NADH oxidation spectrophotometrically before being applied to a DEAE-cellulose column (0.9 X 17 cm). The mixing chamber contained 550 ml 10 mM Tris-KC1 (pH 7.2) and the reservoir 500 ml 0.35 M (NH4)HCOa in 10 mM Tris-HCl (pH 7.2). Flow rate was 0.75 ml/min. Fractions of 3 ml were collected after the effluent had been continuously monitored at 260 nm. Peak areas were pooled and analyzed for radioactivity by liquid scintillation counting and for absorbance (arbitrary units) at 260 nm. (A) Ab sorbance at 260 nm (B) radioactivity.

radioactivity than they were in terms of 260 nm absorbance. The data for the experiment concerning recoveries of radioactivity and 260 nm absorbance are given in Table I. Recoveries of radioactivity are relatively good in the early stages of oxidation. The total absorbance at 260 nm increased throughout the experiment, in agreement with the data presented in l’ig. 2. Chemical and biochemical properties of the ozonization products. In further efforts to characterize the oxidation products, the following analyses were made. A sample of NADH (approx 5 mg) was dissolved in 5.0 ml 10 mu Tris-Cl (pH 7.2), and a O.l-ml aliquot taken for spectral analysis. The ratio of 260 nm to 340 nm absorbance was 2.38, and the absorbance at 340 nm indicated the presence of 6.54 pmoles NADH in the original 5.0 ml. The NADH solution was exposed to ozone bubbling through solution at 20 ml/min for 2 hr and at a rate of 54 nmoles Os/min (total 03, 6.5 pmoles). An aliquot of 0.1 ml was again diluted to 3.0 ml for spectral analysis: 340 nm absorbance was zero, and 260 nm absorbance had increased 1.5-fold compared to the control. The remaining 4.8 ml were applied to a column of DEAE-cellulose (0.9 X 17 cm) with 550 ml 10 mM Tris-Cl

TABLE

I

RESOLUTION OF [l%]NADH OXIDATION PRODIJCTS~

03 (nmoles) 0 100 260 460 770

NADH

oxidized (nmoles) 0 108 254 422 630

AND

Total dpm recovered

Total 260 nm absorbance

141,500 141,100 141,600 118,300 124,600

3.83 5.40 5.29 6.57 6.87

0 Reaction conditions were as described for Fig. 5. Data in this Table were derived from the dat,a in Figs. 5a and b. Absorbance is in arbitrary units.

(pH 7.2) in the mixing chamber and 550 ml 0.35 M NHaHCOs in 10 mM Tris-Cl (pH 7.2) in the reservoir. The eluate was monitored continuously at 260 nm, and 4.3-ml fractions were collected. Compound A was in fractions 4 and 5, compound B in fractions 8-10, and compound C in fractions 20-24. Fractions 4 and 5 (Compound A) were lyophilized, and the residue was redissolved in 1.0 ml water. An aliquot of 0.9 ml was added to a 3.0-ml reaction mixture for assay with alcohol dehydrogenase. Before the addition of enzyme the solution had absorb-

OZONIZATION

ante at 260 nm of 0.453 and 340 nm of 0.117. The compound was inactive in the alcohol dehydrogenase assay. Authentic NAD was added, and the predicted rate of reduction was observed. Therefore, compound A is not KAD, but it is not an inhibitor of alcohol dehydrogenase. Fractions S-10 (Compound B) were lyophilized, and the residue was dissolved in 1.0 ml water. An aliquot of 0.3 ml was taken for the alcohol dehydrogenase assay. Before the addition of enzyme the solution had absorbance at 260 nm of 1.69 and at 340 nm of 0.036. Addition of enzyme initiated KADH formation at a rate expected if the absorbance at 260 nm were entirely due to NAD. A second aliquot was taken with ASG0 corresponding to 280 nmoles of I\‘AD. Cyanide was added to t’his aliquot, and the absorbance was measured at 327 nm. The increase in absorbance of 0.550 corresponds to 2X0 nmoles of KAD. Therefore, Compound B is pure NAD. 1;ractions 20-24 were pooled from the center of the Compound C peak. An aliquot of 0.9 ml was tested directly in the alcohol dehydrogenase assay. Before addition of the enzyme the solution had absorbance at 260 nm of 1.415 and at 340 nm of 0.007. Addition of alcoho1 dehydrogenase caused no change in the absorbance at 340 nm. Addition of authentic NAD gave the predicted rate of absorbance change, and this showed that compound C was not an inhibitor of the alcohol dehydrogenase. The cyanide assay confirmed that compound C is not NAD. NADPH is also oxidized by ozone, but the products have not been assayed. Oxonization of NAD and NADP. Treatment of NAD solutions with ozone caused no change in the ability to act as coenzyme in the alcohol dehydrogenase reaction, nor in the formation of cyanide complexes. Treatment of KADP solutions with ozone caused no change in the ability to act as coenzyme in the assay with glucose-6-phosphate dehydrogenase, nor in the formation of cyanide complexes. Reaction of ozone with tryptophan. When ozone comes into contact with living tissue, there are a number of compounds that can be oxidized. These include the unsaturated

OF NADH

04

413

r -

FIG. 6. Reaction of ozone as a function of concentration. Reaction mixtures tryptophan contained varying initial concentrations of tryptophan in 5.0 ml 0.1 M phosphate buffer (pH 7.2). Ozone in oxygen was bubbled through the solutions at 20 ml/min. Ozone dosage was 18 nmoles/ min. At time intervals the samples were removed and scanned spectrophotometrically from 220 to 4Ml nm. The decrease in absorbance at 279 nm is plotted: similar results are obtained if the increase in absorbance at 300 nm is plotted.

fatty acids of lipid components, and amino acids, either in peptide linkage or as free amino acids. It was of some interest to compare the reactivity of ozone with NADH on the one hand and protein components on the other. The reactivity of tryptophan with ozone is shown in Fig. 6. It is clear that in the 0.1 rniM range the amount of tryptophan reacting depends on the amount of ozone introduced to the system, but at lower ooncentrations of tryptophan, the initial stages of the reaction depend on the tryptophan concentration to a much greater extent than was the case for NADH. We would predict from this result that in mixtures of NADH and tryptophan that NADH would be preferentially oxidized. This was verified by the experiment depicted in Fig. 7. When tryptophan was present with NADH, the oxidation of NADH was unaffected. Oxidation of tryptophan did not take place until the NADH was almost depleted. Reaction of ozone with NADH plus methion&e. Previous studies had shown that methionine was particularly susceptible to oxidation by ozone and that the quantitative product was methionine sulfoxide. (The oxidation products from tryptophan

414

MUDD, r

I

I

I

I

I

LEH, AND McMANUS I

I

/” ; 2 0 H

0.3-

0’ ./

02

frp (-NADH)-

/

% E q

0.1

J:pl”~*-~p

-

0.0 -

/A I 0.0

/

0’ o~-o-o--o-o I 0 I

I 0.2 p moles

I 0.3 O3

I 0.4

trp

(t NADH) I 0.5

FIG. 7. Simultaneous exposure of NADH and tryptophan to ozone. Reaction mixtures contained 200 nmoles tryptophan and 502 nmoles NADH either separately or together in 5.0 ml 0.1 M phosphate buffer @H 7.2). The solutions were exposed to a stream of ozone in oxygen at 20 ml/ min. Ozone dosage was 30 nmoles/min. Samples were analyzed for NADH by measuring changes in absorbance at 340 nm. Subsequently 2.0 ml 0.2 M citrate buffer (pH 2.2) was added to the sample, and a 3.5-ml aliquot of the mixture was used for tryptophan analysis by ion exchange chromatography using the Beckman 120B amino acid analyzer.

are multitudinous and have not been characterized.) Figure 8 shows the results of an experiment in which NADH and methionine were simultaneously exposed to ozone. As in the case of tryptophan, NADH is equally well oxidized in the presence or absence of methionine. NADH prevents the oxidation of methionine to a large extent, but whether NADH is present or not, the methionine oxidation product is methionine sulfoxide. Reaction of ozone with NADH plus glutathione. Thiol groups are particularly susceptible to oxidation by ozone. Competition between thiol groups and NADH for ozone was also especially interesting because of the requirement for thiol groups in many enzymically catalyzed reactions. Experiments illustrated in Fig. 9 show that the competition between NADH and GSH is dependent on the concentrations of the reactants. In Fig. 9a the initial concentrations of NADH and GSH were 65 PM and 108 PM, respectively, and in this case oxidation of NADH was preferred over GSH (until NADH was

depleted). In Fig. 9b the initial concentrations of NADH and GSH were both 1.45 mM, and in this case GSH was readily oxidized while oxidation of NADH was somewhat inhibited. Oxidation of GSH was approximately 1.5 moles/mole of ozone; whereas, in the absenceof NADH the oxidation is closer to 2 moles/mole ozone. Reaction of ozone with I ,4-dihydro-lmethyl nicotinamide. At pH 7.2 in 0.1 M Tris-Cl buffer the treatment of 1,4-dihydrol-methyl nicotinamide with ozone caused characteristic changes in the uv spectrum (Fig. 10). These changes included initially a decrease in absorbance at 365 nm and an increase in the initially significant absorbance at 291 nm. On further ozonization the 291 nm absorbance decreasedand new peaks of absorbance appeared at 271 and 261 nm. On prolonged ozonization all the bands at 271, 291, and 261 disappeared. The absorbance at 291 nm may be attributed to an addition compound and may account for as much as 7 % of the nicotinamide. Separation of products on a column of DEAE-cellulose permitted the isolation of a compound with maximum absorbance at 261 m-n, and this was attributed to N-methyl nicotinamide

0.4 -

2 0 03; Lz . L

02-

2 3

Ol-

0.0 I

, 0.0

I 0.1

I I 02 0.3 ,umoles 03

I 04

1 05

I

FIG. 8. Simultaneous exposure of NADH and methionine to ozone. Reaction mixtures contained 200 nmoles methionine and 480 nmoles NADH either separately or together in 5.0 ml 0.1 M phosphate buffer (pH 7.2). Ozone was introduced at the rate of 42 nmoles/min in an oxygen stream flowing at 20 ml/min. Samples were analyzed as described in the legend to Fig. 7.

OZONIZATION

“‘l-,

a

415

OF NADH

I ’ ’ ’ ’ ’ ’ b’ ‘*

t

\

GSH \

0.0 L-L-U

l---O

0.2

0.0

1. I~I~I~OI~

0.4

0

p moles

2

4

JO

6

8

0,

FIG. 9. Simultaneous exposure of NADH and GSH to ozone. Reactions contained the indicated amounts of NADH and GSH in 5.0 ml 0.1 M phosphate buffer (pH 7.2). In (a) the ozone dosage was 33 nmoles/min. At the end of the exposure period a 1.0 ml aliquot was taken for analysis of thiol by the method of Ellman (15). The remainder of the solution was assayed for NADH by absorbance at 340 nm. Since the solutions were exposed for 15 min to a gas flow of 20 ml/min, a control using oxygen without ozone was used. Data for that control are indicated by (-.-. ). The expected oxidation of NADH in the absence of GSH is indicated by (- - -). In (b) the ozone dosage was 1.62 pmoleslmin. Aliquots of 0.1 ml were taken for thiol and NADH assay. The expected oxidation of NADH in the absence of GSH is indicated by (- - -).

240

260

280

300

320

WAVELENGTH,

340

360

380

FIG. 10. Spectrophotometric measurement of the reaction methylnicotinamide. Solutions contained initially 0.127 mM amide in 0.1 M Tris HCl (pH 7.2), volume 3.0 ml. The solutions at a flow rate of 20 ml/min. Ozone delivery was 4.8 bmoles/min. posed to the ozone for various times. (A) no ozone, (2) 10 set, (10) 4 min, (12) 12 min. Spectra were recorded in a Cary 15

corresponding to 2% of the nicotinamide in the reaction mixture. In order to determine the structure of the compound with maximum absorbance at’ 271 nm, the ozonolysis reaction was done in a solution of methylene chloride. Under

400

420

nm

of ozone with 1,4-dihydro-l1,4-dihydro-l-methylnicotinwere exposedto ozone/oxygen The spectra shown were ex(4) 20 set, (6) 30 set, (8) 50 set, spectrophotometer.

these conditions formation of the acid addition compound should be minimal. The 1,4dihydro-l-methyl nicotinamide (8.2 mg, 52.2 pmoles) was exposed to an equimolar amount of ozone. During the addition of ozone, pale yellow crystals were formed in

416

MUDD,

LEH,

the reaction mixture, precluding the measurement of reaction by following absorbance changes. This product was isolated and recrystallized from methanol-ether, giving colorless crystals (5.3 mg, 60%). The crystals were washed with ether (3 X 3 ml) and dried. Elemental analysis for carbon and hydrogen was compared with results calculated on the expectation that the 5,6 double bond had been broken with the production of 2 aldehyde groups. Calcd for C,Hlo03N2: Found :

AND

TABLE REACTIONS

1,4Dihydrol-methylnicotinamide

WITH

OZONE

la

Expt

OS/ DMN

CornpoundIV reacted (mole)

2b On/Cornpound IV

!22 (mole) 0.120 0.200 0.240 0.400 0.560 0.800 0.950 1.200 1.600 2.000 2.400 4.799

CONH,

was supported by its infrared spectrum which showed C==O stretching absorbance at 1694 and 1625 cm-’ corresponding to the carbonyl bond in aldehyde and amide. Other characteristic aldehyde vibrations appeared at 2700, 2830, 1460, and 907 cm-’ which can be attributed, to the C-H stretching, C-C stretching and hydrogen deformation of the CHO group (21). The amount of ozone administered and the amounts of reaction are shown in Table II. The results show that only a fraction of the administered ozone reacts with the 1,4dihydro-l-methyl nicotinamide. In contrast, compound IV, with an absorbance maximum at 271 nm, reacts more readily than the original 1,4-dihydro-l-methyl nicotinamide. It is, therefore, clear that the compound with the 271 nm absorbance maximum cannot accumulate when 1,4-dihydro-l-methyl nicotinamide is treated with ozone. The absorbance spectra shown in Fig. 10 show that the compounds having absorbance maxima at 291 and 271 decrease on prolonged ozonization. The spectroscopic changes are consistent with ozonolysis at the 2,3 double bond of the acid addition compound (I) and the primary ozonolysis

II

I,‘&DIHYDRO-l-METHYLNICOTIh’-

Expt

03

hole)

A sample of the product was introduced to the mass spectrometer and its molecular weight was indicated by a parent ion mass of 170. Identification of this compound as I.4 u

(IV1

OF

AMIDE:

C, 49.4; H, 5.88 C, 50.3; H, 5.95

CH3

McMANUS

0.046

4.34

0.092 0.128 0.180 0.206 0.255 0.296 0.327

4.35 4.39 4.45 4.51 4.70 5.41 6.12

0.060

2.02

0.117 0.181 0.248 0.293

2.06 2.22 2.42 2.73

0.346 0.385

3.47 4.16

0.405 0.427

5.93 11.24

0 Expt 1. Reaction mixtures contained 1,4dihydro-1-methylnicotinamide in 0.1 M Tris-HCI buffer (pH 7.2) in a final volume of 3.0 ml. Samples were exposed to a stream of ozone/oxygen at a flow rate of 20 ml/min. Ozone delivery was 2.4 pmoles/min. Reaction was followed spectrophotometrically. * Expt 2. Compound IV was prepared by ozonization of 1,4-dihydro-1-methylnicotinamide in methylene chloride as described in the text. This compound was used in a reaction mixture as described for Expt 1.

product (IV). Likely products from the prolonged ozonolysis would be succinic and malonic acids. To confirm these expectations, an ozonolysis mixture was treated with hydrogen peroxide in order to obtain carboxylic acids, and the mixture was methylated and analyzed by gas liquid chromatography. Methyl malonate and methyl succinate accounted for 60 % of the ozonized N-methylnicotinamide. In this experiment 1,4-dihydro-l-methyl nicotinamide (27.9 mg, 202 moles) was dissolved in methanol (3 ml) and reacted with ozone until the absorption at 271 nm was eliminated. A mixture of 30% hydrogen peroxide (2 ml) and sulfuric acid (0.2 ml) was added immediately, and 2 hr later the mixture was

OZONIZATION

heated under reflux 1 hr and left overnight. The next day 5 ml water was added, and excess peroxide was dissipated by adding sodium hydrogen sulfite. The mixture was adjusted to pH 2 with hydrochloric acid and then extracted three times with 3-ml portions of ether. The evaporated extract (19.6 mg) was assayed after formation of the methyl esters by gas liquid chromatography. The mixture was found to contain malonic acid (16.7 mg) and a trace of succinic acid. The amount of succinic acid produced in bhis series of reactions can bc varied by changing the reaction conditions. When the ozonolysis reaction was done in aqueous buffered solution (0.01 M Tris-HCl, pH 7.2) malonic and succinic acids were obtained in the ratio of 3: 1, whereas the reaction at pH 4.6 (0.1 M acetate buffer) yielded succinic acid only. Thus, the products reflect the amount of acid addition product formed. In a separate experiment the ozonolysis mixture was extracted with ether directly. This ether extract was examined by gas liquid chromatography: there was no cvidence for volatile acids. ReactioTLof oxonewith NADH. The changes in the absorbance spectrum of NADH when treated with ozone are simple compared with the reaction of 1,4-dihydro-l-methylnicotinamide. In the initial stages there is a decrease in absorbance at 340 nm and an increase at 260 nm. There was no evidence for the formation of an acid addition compound under the conditions used in our experiments. In one experiment 3 mg NADH was dissolved in 0.5 ml 0.01 M Tris-HCI buffer (pH 7.2) and treated with an cquimolar amount of ozone. The product was t’reated with KADase, and the products of this reaction separated on a DEAE-cellulose column. The compound corresponding to the modified nicotinamide was collected and purified by extraction with methanol. (It is noteworthy that NADH is not susceptible to hydrolysis by NADase.) In the mass spectrometer a parrnt ion of mass = X56 was observed. It is, therefore, suggestedthat ozonolysis of NADH is similar to t,hc oxidation of the 1,4-dihydro-l-methylnicotinamide to the extent that the 5,6 double

417

OF NADH

bond is broken with t’he production of dialdehyde. Reaction of ozone with NADH shows 1: 1 stoichiometry even at low concentrations of NADH and ozone (11). This contrasts with the react’ion of ozone with 1,4-dihydro-lmethylnicotinamide when an excessof ozone was needed to oxidize the nicotinamide. Reaction of ozonewith adenine. Reaction of ozone with XADH showed initially a decrease in absorbance at 340 nm and an increase at 260 nm. However, prolonged exposure to ozone caused the 260 nm absorbance to decrease again. Results of an experiment in which adenine was exposed to ozone are shown in Fig. 11. The adenine ring was least reactive at pH 4.6 but even at pH 7.2 and 8.7 the reactivity was 103-lo4 lessthan 1,4-dihydro-I-methylnicotinamide. DISCUSSION

Although all of the experiments reported in t’his paper were conducted in vitro, the results may be of greatest interest when considered in light of the toxic effects of ozone on biological systems. It is quite clear that ozone can react with a number of biologically important molecules, and the question is which of these is most susceptible, and which chemical reaction initiates the toxic response. The results in this paper show that NADH and KADPH are readily oxidized by ozone, and the products do not have a biological activity. On the other hand XAD and XADP are relatively resistant. Other things being equal, physiological conditions which cause the preponderance of the reduced forms may be predicted to be more susceptible to ozone. Results with NADH show that the oxidation by ozone holds to a constant time times concentration relationship. The stoichiometry is the samedown to concentrations of ozone found in the Los Angeles atmosphere, and NADH concentrations found in sometissues (22). The spectrophotomctric changes on treatment of NADH with ozone indicate that the adenine moiety is relatively resistant, and this is verified by experiment (Fig. 11). Among the amino acids, the most susceptible arc cysteinc, t’ryptophan, and methionine

418

MUDD,

LEH, AND McMANUS

of the dehydrogenases tested, other toxic reactions cannot be excluded. Nasr et al. (11) have reported that the NADPH/ NADP ratio in epithelial cells of trachea of rats exposed to ozone was not changed, but the absolute concentrations were not given. They concluded that other compounds were oxidized in preference to NADPH. In the reactions of ozone with 1,4-dihydro-1-methylnicotinamide, the absorbantes at 291, 261, and 271 nm are to be accounted for. The absorbance at 291 nm is the normal acid addition product studied by Stock et al. (17) and Cornforth et al. (13). OZONE, /moles The absorbance at 261 nm is attributed to FIG. 11. Reaction of adenine hemisulfate with the nicotinamide moiety, but this accounts ozone. Reaction vessels contained 69.9 PM adenine for only a small amount of the products. sulfate in buffered aqueous solution of final volume Bechara and Cilento (24) have studied the 3.9 ml. Solutions were exposed to ozone/oxygen at a flow rate of 20 ml/min. Ozone delivery was oxidation of nicotinamide derivatives in the N ,N ,N’ ,N’-tetramethyl-l3.5 pmoles/min. Spectra were recorded with a presence of phenylenediamine and oxygen. In the case Cary 15 spectrophotometer and the change in of the N-benzyl model the products showed absorbance was used to calculate reaction of the adenine. Symbols: (1) pH 4.6 acetate buffer, (2) absorbance at 290 nm characteristic of the pH 7.2 Tris buffer, (3) pH 8.7 Tris buffer. 5,6 addition compound and at 265 nm characteristic of the pyridinium compound. (5). A comparison has been made of the Since the compound absorbing at 261 nm oxidation of these amino acids with oxida- in our system made only a small contribution of NADH. In all cases NADH was tion to the products, the free radical mechpreferentially oxidized. Under physiological anism operative in the system of Bechara and Cilento (24) is not likely to be functionconditions in animals it seemsobvious that the first point of attack will be in the lung ing in the ozonolysis reaction. Treatment of tissue. The data of Coffin and coworkers the 1,4-dihydro-1-methylnicotinamide with show that pulmonary alveolar macrophage oxygen in the absence of a free radical initicells are broken open with the release of ator gave no change in absorbance at 261 enzymes normally confined to the lysosomes nm. The absorbance at 271 nm reported in (4). At the present time it is not possible to this paper has been attributed to the dialdegive NADH a role in this process. The ob- hyde resulting from the ozonolysis of the 5,6 servation is more likely to lead one to a double bond of the nicotinamide ring. Furconsideration of the effects of ozone on the ther ozonolysis breaks the double bond at integrity of membrane structure. Reaction 2,3 in a reaction analogous to that described with amino acid residues of membrane by Cornforth et al. (13). The studies reported in this paper show protein or unsaturated fatty acid residues of membrane lipid have been considered by that the reaction of the adenine moiety of various authors (5-S). However, there is no NADH is relatively unimportant. The reconclusive evidence that either of these action that doestake place is pH dependent, reactions is responsible for the toxic reac- with the greatest resistance of adenine being tions. Indeed human erythrocytes are quite at acid pH. The pH effect on the reaction may be interpreted as resulting from the resistant to ozone while being easily lysed of the Nl of the adenine ring. by ozonized phosphatidyl choline, a finding protonation which indicates the importance of molecules The pK corresponding to the protonation not actually in the membrane (23). Although of the Nl position was reputed to be approximately 4 (25). The protonated adenine the products of ozone oxidation of NADH probably is a tautomeric structure in which and NADPH do not seem to be inhibitors

OZONIZATION

the positive change is distributed throughout the adenine ring. Further evidence for such a structure is provided by nuclear magnetic resonance studies of purine ribosides (26) and x-ray crystallographic data for adenine hydrochloride (27, 28), according to which the proton is located on Nl rather than NlO of the amino group. Therefore, the observed change in reaction with ozone may arise from the electrostatic repulsion of the ozone due to the protonation of the adenine ring. Menzel has reported that treatment of NADH with ozone produces NAD (12), a result in conflict with ours. In his experiments, the product was reducible by sodium dithionite and was reactive in enzymic analysis with glyceraldehyde-3-phosphate dehydrogenase. We have attempted to reduce the ozonolysis product obtained from 1,4-dihydro-1-methylnicotinamide with a three-fold excess of dithionite, and care was taken to isolate the product from dithionite (which absorbs light strongly in the 280360 nm range) by extraction with methylene chloride. Spectral analysis of the product showed that approximately 2% of the ozonolysis product was the l-methylnicotinamide. Nasr et al. (11) obtained results consistent with ours; they concluded that oxidation of NADPH by ozone was more than a simple oxidation to NADP. REFERENCES

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