Identification of cholesteryl nitrate as a product of the reaction between NO2 and cholesterol monomolecular films

Chem. Phys. Lipids 6 (1971) 225-234 © North-Holland Publishing Company

IDENTIFICATION AS A PRODUCT

OF CHOLESTERYL

OF THE REACTION

CHOLESTEROL

NITRATE

B E T W E E N NO2 A N D

MONOMOLECULAR

FILMS

AIDA M. KAMEL, NORMAN D. WEINER and ALVIN FELME1STER College of Pharmacy, Rutgers University, Newark, New Jersey 07104, and College of Pharmaceutical Sciences, Columbia University, New York, New York 10023

The effects of exposure of monomolecular films of cholesterol, dihydrocholesterol, 5-cholesten-3-one, 7-hydroxycholesterol, and 7-ketocholesterol to air and NO2 were studied. Whereas exposure to air resulted in expansion of the films in all cases, exposure to NOz resulted in condensation of all of the films with the exception of 5-cholesten-3-one. The expansion effects were determined to be the result of the formation of various autoxidation products of cholesterol. The condensation effects were caused by the formation and subsequent desorption of cholesteryl nitrate. Identification of the products was based on surface pressure-surface area and thin layer chromatographic evidence.

Introduction In a series o f papers on the effects o f air p o l l u t a n t s on m o n o m o l e c u l a r films o f various m e m b r a n e c o m p o n e n t s , we r e p o r t e d that nitrogen dioxide interacted strongly with films o f cholesterol. 1) This interaction a p p e a r s to be greater than those o f NO2 with various p h o s p h o l i p i d s 2) or certain lipidp r o t e i n c o m p l e x e s ) ) F u r t h e r m o r e , this interaction is c o m p l i c a t e d by the fact that m o n o l a y e r s o f cholesterol a p p e a r to be quickly autoxidized, even u n d e r a m b i e n t conditions. 4) W h e n cholesterol m o n o m o l e c u l a r films are exposed to air for periods o f time ranging from 45 minutes to several hours an expansion effect is observed, i.e., each molecule occupies a larger area at all surface pressures, z) It was p o s t u l a t e d that this expansion effect was the result o f a u t o x i d a t i o n o f the cholesterol. This p o s t u l a t i o n was s u p p o r t e d by two observations. Firstly, o x i d a t i o n p r o d u c t s were identified by thin hayer c h r o m a t o g r a p h i c analysis o f films exposed to air for 35 to 90 minutes. 4) Secondly, when each o f these o x i d a t i o n products was spread as a m o n o m o l e c u l a r film, the resultant surface pressure-surface area ( n - A ) curves were always m o r e e x p a n d e d t h a n that o f freshly spread cholesterol. 5) On the other hand, when cholesterol m o n o m o l e c u l a r films were exposed to a t m o s p h e r e s c o n t a i n i n g nitrogen d i o x i d e for similar periods o f time, a 225

226

A I D A M. K A M E L , N O R M A N D . W E I N E R A N D A L V I N F E L M E I S T E R

condensation effect was observed, i.e., each molecule appears to occupy a smaller area at all surface pressures. This condensation effect was attributed to an NOz-cholesterol interaction and the subsequent desorption of the products formed, i) The extent of desorption was a function of a number of variables such as time of exposure, concentration of N O 2 and the surface pressure of the film prior to exposure. A limiting effect corresponding to a 60-75% loss of cholesterol was observed after exposure of the films to 95 ppm of N O 2 for 90 minutes and to 175 ppm of NO2 for 60 minutes, respectively. Bergstrom et al. 6) previously reported a limiting effect during the autoxidation of aqueous dispersions of cholesterol. They attributed this effect, which corresponded to a loss of 70% cholesterol, to an inhibitory action exerted by the oxidation products. Although the reaction mechanism for the autoxidation of cholesterol is not fully understood, various reaction pathways have been proposed.7). It is well accepted, however, that the mechanism is complex and many reaction products result. In contrast, no studies have been reported on the reaction mechanism of N O 2 with cholesterol, although it has been suggested s) that the reaction products formed on exposure of cholesterol, in aqueous dispersion, to NO2 containing atmospheres, may be, in part, the result of the interaction of NO2 with various air oxidation products of cholesterol. It is clear that the products formed on autoxidation of cholesterol can not produce the condensation effects observed on exposure of cholesterol films to N O 2. It was the purpose of this study to identify the product(s) of the NOz-cholesterol interaction which would account for these observed condensation effects. Furthermore, in order to gain insight into the mechanisms of the reactions which occur when monomolecular films of cholesterol are exposed to NO2 containing atmospheres, investigations were conducted on the effects of air and NO2 on films of cholesterol and several of its oxidation products. Postulations that have been made as a result of interpretation of g - A data have been confirmed by the use of TLC. Materials and methods

The cholesterol, dihydrocholesterol, 7-ketocholesterol, and 5-cholesten-3one were purchased from Mann Research Labs. New York, N.Y. The 7-hydroxycholesterol (a mixture of the ~ and fl isomers) was obtained by hydrolyzing a mixture of the benzoate esters (K & K Laboratories, Plainview, New York) in alcoholic KOH solution at 50 ° for five hours. The cholesteryl nitrate was purchased from Eastman Organic Chemicals, Rochester, New York. Spectroscopic grade hexane was used to prepare

IDENTIFICATION OF CHOLESTERYL NITRATE

227

solutions of the steroids. All other chemicals were of reagent grade. Previously deionized water was distilled from an all glass still prior to use. Silica gel, precoated aluminum sheets (20 x 20 cm, 250/~), Brinkman Instruments, Inc., Westbury, New York, were used for all chromatographic separations. A mixture of 0.5% nitrogen dioxide (99.5% pure) and 99.997% prepurified grade nitrogen, Matheson Company, East Rutherford, N.J., was used as a source of N O 2. The gas mixture was allowed to pass through a flow meter at a rate of 100 ml/min and directed into a short length of perforated Teflon tubing which was affixed to the underside of a Lucite trough cover as described previously. 2) This served to maintain the desired gaseous atmosphere over the film. The film balance used to study the n - A characteristics of the film has been described previously. 2) Surface pressures were measured by the Wilhelmy plate method. Solutions of cholesterol, dihydrocholesterol, 7-ketocholesterol, 7-hydroxycholesterol, and 5-cholesten-3-one in hexane were spread on a 0.065 M phosphate buffer at pH 7.0. In one set of experiments compression was initiated immediately (i.e., about 3 rain) or after a 60 minute exposure to the atmosphere. In another set of experiments, the NO2 gas mixture was allowed to flow over the film for 60 minutes. At the end of this time period, the flow of gas was discontinued and compression of the film was initiated. In all cases surface pressure readings were obtained at various films areas. In the mixed film studies, hexane solutions of cholesterol and cholesteryl nitrate at mole fractions of 0, 0.25, 0.50, 0.75, and 1.0 were spread onto the subphase. In all cases, the same total number of molecules were spread (6.3 × 1016 molecules), and the calculated initial surface area per molecule after spreading was the same as that for the pure steroid films (50 Aa/mole cule). Compression of the mixed films was initiated immediately (3 minutes) after spreading as well as after one hour exposure to NO2. T L C Studies Films of cholesterol were removed after exposure to either air or NO2 for 60 minutes. Removal of the films was accomplished by applying suction through a glass tube drawn to a fine tip and collecting the lipid material along with a small volume of the subphase in a 50 ml side arm flask. In order to obtain a large enough sample, four successive cholesterol films were collected and pooled. The lipids were then extracted into chloroform. After drying over anhydrous sodium sulfate, the chloroform solution was evaporated to a small volume ( ~ 100 id) and this total volume was spotted on to a chromatographic sheet. The chromatographs were developed 2-dimensionally in an Eastman Chromagram sandwich-type unit using first a solvent of acetone: chloroform

228

AIDA M. KAMEL, N O R M A N D. W E I N E R A N D A L V I N FELMEISTER

40

50

0

w 20

Z >a

IO

0

20 40 60 AREA/MOLECULE, A=

80

Fig. 1. Surface pressure-surface area (n-A) curves of monomolecular films of cholesterol prior to exposure (©), after 60 minutes exposure to air (rn), and after 60 minutes exposure to NO2 (A).

(10:90, v/v) followed by a solvent o f cyclohexane: c h l o r o f o r m (70: 30, v/v). The spots were visualized by spraying with a s a t u r a t e d solution o f a n t i m o n y trichloride in c h l o r o f o r m followed by heating in an oven at 105 ° for 5 minutes. Identification o f c o m p o u n d s are based on Ry values a n d colors relative to the s t a n d a r d s tested. 8) Results and discussion

Fig. 1-5 show the ~ - A curves o f m o n o m o l e c u l a r films o f cholesterol, dihydrocholesterol, 5-cholesten-3-one, 7-hydroxycholesterol, and 7-ketocholesterol, following exposure to air and to NO2. In all cases, e x p o s u r e to air resulted in m o r e e x p a n d e d 7r-A curves, due to the air o x i d a t i o n o f the film c o m p o n e n t s ) ) W i t h the exception o f 5-cholesten-3-one, the ~ - A curves

229

I D E N T I F I C A T I O N OF C H O L E S T E R Y L N I T R A T E

40.

50"

(,3 W 20

z>-

-

(

v

20

40 60 A R E A / M O L E C U L E , A2

80

Fig. 2. Surface pressure-surface area (~r-A) curves of monomolecular films of dihydrocholesterol prior to exposure (©), after 60 minutes exposure to air (D), and after 60 minutes exposure to NO2 (zx). o f all the films exposed to NO2 exhibited a condensation effect, due to the desorption of reaction products from the film surface. 1) The 7r-A curve of the 5-cholesten-3-one, after exposure to nitrogen dioxide showed an expansion equal to that observed on exposure to air (fig. 3), indicating that only air oxidation o f this c o m p o u n d occurs under both conditions. A comparison o f figs. 1 & 2 shows that both cholesterol and dihydrocholesterol exhibit about the same degree o f condensation on exposure to N O > It appears then, that the interaction o f NOz with both o f these sterols leads to products with the same surface properties. Furthermore, it is evident that the site of attack by the nitrogen dioxide must be the 3-hydroxy g r o u p and not the 5,6 double bond, since only the former site is c o m m o n to both o f these compounds. The fact that no condensation was observed

230

AIDA M. KAMEL, NORMAN D.WEINER AND ALVIN FELME[STER

30

o

20 Z )0

10

0

20

40 60 AREA/MOLECULE, A2

Fig. 3. Surfacepressure-surface area (n-A) curves of monomolecularfilms of 5-cholesten3-one prior to exposure (O), after 60 minutes exposure to air (D), and after 60 minutes exposure to NOz (gx). when 5-cholesten-3-one was exposed t o N O 2 adds further support to the postulation that the 3-hydroxy group is required for the observed condensation effect. As is shown by the ~-A curves of 7-hydroxychotesterol and 7-ketocholesterol (fig. 4, 5), two common air oxidation products of cholesterol, the addition of either a hydroxy or a keto group at position 7 does not influence the condensation effect produced by the interaction of cholesterol with N O 2. The data thus clearly demonstrate that the N O 2 interaction, which leads to desorption, involves a chemical attack at the 3-hydroxy position. In order for the reaction between the sterol and N O 2 tO lead to desorption, the product or products must be insoluble in the aqueous subphase, and have essentially no capacity to interact with the subphase and thereby form a stable monomolecular film. It should be pointed out that desorption via an increase in polarity and subsequent dissolution in the aqueous subphase

231

I D E N T I F I C A T I O N OF C H O L E S T E R Y L N I T R A T E

40

30

~D

zc Z >0

I0

o

20

40 60 AREA/MOLECULE, A z

s'o

Fig. 4. Surface pressure-surface area (~z-A) curves of m o n o m o l e c u l a r films of 7-hydroxycholesterol prior to exposure (©), after 60 minutes exposure to air ([3), a n d after 60 m i n u t e s exposure to NO,, (A).

would seem quite unlikely since even the most polar of the autoxidation products of cholesterol, cholestan-3fl, 5~, 6/%triol, forms a stable, slightly expanded, monomolecular film and exhibits no tendency to desorb from the surface. 5) Esterification of cholesterol resulting in the formation ofcholesteryl nitrate is consistent with this postulation as well as the experimental observations. Nitrate esterification of the hydroxy group would be expected to markedly reduce its subphase interaction and consequently the spreading tendency of cholesterol. If cholesteryl nitrate was, in fact, the species formed upon attack of cholesterol by N O 2 , o n e would expect that this compound would not form a stable film. Furthermore, it would be expected that cholesteryl nitrate

232

AIDA M. KAMEL, NORMAN D. WEINER AND ALVIN FELMEISTER

40

30

L) ~" 2 0 o) wJ z >..

I0

0

I

20

40 60 A R E A / M O L E C U L E , Az

80

Fig. 5. Surface pressure-surface area (n-A) curves of monomolecular films of 7-ketocholesterol prior to exposure (©), after 60 minutes exposure to air ([]), and after 60 minutes exposure to NOz (A). would not form stable mixed films with cholesterol. The anticipated surface proPerties o f cholesteryl nitrate were confirmed e x p e r i m e n t a l l y ; when a hexane solution o f this c o m p o u n d was placed on the a q u e o u s subphase, no surface pressure could be detected upon compression, even at very small a r e a s / m o l e c u l e (15 A2/molecule). W h e n mixtures o f various m o l e fractions o f cholesterol a n d cholesteryl nitrate were spread on the subphase, the resultant n - A curves were representative o f the cholesterol fraction only. Thus, it a p p e a r s that even in the presence o f cholesterol all o f the nitrate ester was c o m p l e t e l y d e s o r b e d from the surface at all pressures tested. Identical effects were observed when mixed films o f cholesteryl nitrate a n d lecithins were spread. C o n f i r m a t i o n o f the f o r m a t i o n o f cholesteryl nitrate u p o n exposure of

IDENTIFICAT[ON OF CHOLESTERYL NITRATE

233

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+L

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Figs. 6A and 6B. Two-dimensional chromatograms of cholesterol films exposed to air and NO2, respectively. Key: cholestan-3p, 5a, 6/? trio! (1); 7-hydroxycholesterols (2); 7-ketocholesterol (3); cholesterol (4); unidentified (5); 4, 6-cholestadien-3-one (6); 6-cholesten-3, 5-diol (7); unidentified (8); and cholesteryl nitrate (9). cholesterol films to N O 2 w a s obtained by the use o f TLC. Figs. 6A and 6B show the products observed on two-dimensional thin layer chromatographic analysis o f films exposed to air and to NOz, respectively. Under both conditions significant air oxidation occured. However, the cholesterol film exposed to N O 2 shows the formation o f significant amounts o f cholesteryl nitrate.

234

AIDAM.KAMEL,NORMAND.WEINERANDALVINFELMEISTER

In view of the confirming evidence, it can be concluded that cholesteryl nitrate is one of the products formed on exposure of m o n o m o l e c u l a r film of cholesterol to NO 2. Based on previous studies, autoxidation of the cholesterol fraction of cell membranes, in vivo, would be expected to have little or no effect on membrane permeability, s) On the other hand, the in vivo interaction of N O 2 with cholesterol resulting in the formation of cholesteryl nitrate would be expected to greatly affect the permeability of the m e m b r a n e , since the ester desorbs, even in the presence of cholesterol and phospholipids. It is conceivable then, that this interaction may account, in part, for effects such as p u l m o n a r y edema, observed when animals are exposed to high concentrations of NO2 e.g.9).

Acknowledgement This study is supported by G r a n t N u m b e r AP788 National Air Pollution Control A d m i n i s t r a t i o n , C o n s u m e r Protection and E n v i r o n m e n t a l Health Service, Public Health Service.

References 1) 2) 3) 4) 5) 6) 7)

A. M. Kamel, A. Felmeister, and N. D. Weiner, J. Pharm. 52 (1970) 2807 A. Felmeister, M. Amanat, and N. D. Weiner, Environ. Sci. Technol. 2 (1968) 40 A. Felmeister, M. Amanat, and N. D. Weiner, Arch. Biochem. Biophys. 126 (1968) 962 A. M. Kamel, N. D. Weiner, and A. Felmeister, J. Colloid Interf. Sci. 35 (1971) 163 A. M. Kamel, A. Felmeister, and N. D. Weiner, J. Lipid Res. 12 (1971) 155 S. Bergstrom and O. J. Wintersteiner, J. Biol. Chem. 141 (1941) 597 S. Bergstrom and B. Samuelsson, Autoxidation and Antioxidants, Volume 1, ed. W. O. Lundberg, lnterscience Publishers, New York (1961) pp. 240-241 8) A. M. Kamel, N. D. Weiner, and A. Felmeister, submitted to Lipids 9) B. L. Steadman, R. A. Jones, D. E. Rector, and J. Siegel, Toxicol. Appl. Pharmacol. 9 (1966) 160