HYDROGEN PEROXIDE FORMATION BY ZINC OXIDE IN OINTMENTS

DISSERTATION

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philo sopty in the Graduate School of the Ohio State University

Hy

HECTOR ANTONIO LOZADA, B.3. in Phar., M.S. in Phar.

The Ohio State University

195S

Approved by

Adviser College of Pharmacy ii

TABLE OF CONTENTS

PAGE

ACKNOWLEDGEMENTS...... iv

PURPOSE OF THE RESEARCH...... v

INTRODUCTION...... 1

ZINC OXIDE...... 5

ZINC OXIDE AS A PHOTOSENSITIZER...... 10

ULTRAVIOLET L I G H T ...... IA

INFRARED ABSORPTION S P E C T R A ...... 16

SCME PROPERTIES OF THE OINTMENT B A S E S ...... 19

WHITE OIN T M E N T ...... 19

HYDROPHILIC OINTMENT...... 20

POLYETHYLENE GLYCOL OINTMENT ...... 21

HYDROUS WOOL F A T ...... 24

AQUAPHOR ...... 26

CHOLESTEROL...... 26

HYDROGEN PEROXIDE...... 28

APPARATUS AND MATERIALS...... 32

EXPERIMENTAL...... 37

PREPARATION OF S A M P L E S ...... 37

IRRADIATION OF S A M P L E S ...... 39

METHOD OF ASSAY...... 40

EXPERIMENTAL RE S U L T S ...... 43

DISCUSSION...... 66

CONCLUSIONS...... 84 iii

TABLE OF CONTENTS (CONT»D. )

PAGE

CONTEMPLATED FUTURE RESEARCH...... 86

BIBLIOGRAPHY...... 87

AUTOBIOGRAPHY...... 91 iv

ACKNOWLEDGEMENTS

My deepest and sincere appreciation to Dr. Earl P. Guth, Professor,

College of Pharmacy, for the guidance and assistance offered to me during the course of this research. His efforts have to a large extent contributed to the successful completion of this work.

I also want to make grateful acknowledgement to Dean Luis Torres

Diaz, University of Puerto Rico - College of Pharmacy, for his efforts in securing the financial assistance necessary for the continuation of my studies for the degree Doctor of Philosophy.

Sincere appreciation is extended to Dr. Loyd E. Iferris and

Dr. John W. Nelson, College of Pharmacy, for their invaluable assistance.

I also wish to acknowledge all the graduate students in the College of Pharmacy for their cooperation, with special gratitude to V<er

Morozowich.

Finally, all my love and devotion goes to ray dear wife, Lillian, for her moral support and understanding. v PURPOSE OF THE RESEARCH

Considerable work has been done in connection with the ability of photosensitized zinc oxide to produce hydrogen peroxide from water and oxygen. Blubaugh, Mathias, Minardi, Reese, and Young (6 , 4» 5» 3» 7)

contributed a good deal of knowledge in applying this phenomenon to pharmaceutical preparations containing zinc oxide or calamine. None of these investigators, however, worked with ointment bases containing

zinc oxide.

The main objectives of this investigation were -

1. To determine whether the official formula for Zinc Oxide

Ointment would produce hydrogen peroxide under the conditions described in this investigation.

2. To compare the amount of hydrogen peroxide produced by the official formula with the amount produced by using hydrous wool fat, aquaphor, hydrophilic ointment, and polyethylene glycol ointment as bases.

3. To determine the effect of temperature on hydrogen peroxide formation.

4. To determine any relation between hydrogen peroxide production and time of exposure to ultraviolet irradiation.

5. To report any changes in hydrogen peroxide production when using sodium formate as additive.

6. To report other information disclosed during the course of the investigation. HYDROGEN PEROXIDE FORMATION BY ZINC OXIDE IN OINTMENTS

INTRODUCTION

Ointments constitute one of the earliest types of medication employed by man. They were popular remedies in ancient Babylon and

Egypt. Fats of animals mixed with such things as resins, waxes, and powdered herbs were very commonly used dermatological preparations in ancient times. The Papyrus Ebers, which dates from about the sixteenth century before Christ, shortly prior to the time of Moses, contained a formula for a greaseless ointment which was made from Hartshorn beaten up with incense and flour and mixed with sweet ale. Ointments are defined by the Pharmacopeia of the United States (l) as ”semi-solid preparations usually containing medicinal substances and intended for external application to the body.”

Zinc oxide is one of the most popular and frequently used ingre­ dients in the treatment of dermatological conditions of varied nature.

It is official in the Pharmacopeia of the United States (l) and is also an ingredient of such official preparations as Calamine, Calamine Lotion,

Zinc Oxide Paste, Zinc Gelatin, Phenolated Calamine Lotion, Coal Tar

Ointment, and Zinc Oxide Ointment. The National Formulary (2) includes it in its formulas for Calamine Ointment, Zinc Oxide Paste with

Salicylic Acid, Compound Resorcinol Ointment, and Zinc Compounds and

Eugenol Cement. Among all these formulas, one of the most frequently used is that of Zinc Oxide Ointment. It has been used for many years as an astringent, protective and mildly antiseptic external preparation.

1 Physicians all over the country continue to prescribe this kind of preparation for treatment of skin conditions, although sometimes they make sane changes in the official base by substituting for it other fats, glycols, or emulsion type bases.

Reese, Mathias, Minardi, Blubaugh, and Young (3, 4* 5> 7) have studied the “Formation of Hydrogen Peroxide in Calamine Lotions," "The

Effect of Various Phenols on the Formation of hydrogen Peroxide by Zinc

Oxide," "The Effect of Time on the Hydrogen Peroxide Production by

Calamine Lotions," "A General Study of the Effects of lipids Upon the

Quantity of Peroxides Produced by Fhotosensitized Zinc Oxide," and "The

Effect of Selected Rienols on the Formation of Hydrogen Peroxide by

Calamine liniment N. F. IX," respectively. All these researchers worked with water suspensions of zinc oxide or with emulsions containing zinc oxide as a major ingredient. None of these studies were concerned with ointments containing zinc oxide. This study deals with this type of preparation.

Reese (3) found considerable variation in the production of hydrogen peroxide by different formulations of calamine lotion.

This amount of peroxide was related to the kind of ingredients present in the formula. Seme substances were found to increase the peroxide. Among these were polyethylene glycols, sorbitan esters, propylene glycol alginates and the two already known, glycerine and phenol. They all showed a pronounced effect on the production of hydrogen peroxide, with phenol being the most efficient of those studied. large amounts of ferric oxide were reported to be detrimental to the production of hydrogen peroxide unless there are ’’additives” present in the lotions. Fluorescent and incandes­ cent lamps were found to stimulate peroxide production. Sunlight proved to be an excellent promoter of peroxide formation even in cloudy weather.

The formation of hydrogen peroxide was not linear with respect to time, two hours yielding less than five times the amount formed in fifteen minutes. Reese also found that lotions which are bacteriostatic may be produced by adding 1 per cent of phenol and/or irradiation with ultra­ violet light. The phenol and the peroxide so formed appeared to have complemental synergism rather than additive action.

Mathias (4) reported significant difference in the amount of hydrogen peroxide produced by several commercial brands of calamine lotions upon irradiation with ultraviolet light. With one exception, he found no significant difference in the amount of peroxide produced by irradiation of a lotion prepared with native calamine and one made with prepared calamine. In his study of the effect of adding phenol, resorcinol, resorcinol monoacetate, and pyrogallol to the suspension, he concluded that the number of hydroxyl groups may have an effect upon the quantity of hydrogen peroxide produced by ultraviolet light. An increase in hydroxyl groups in the phenol brings an increase in ability to produce peroxide. Nevertheless, there appeared to be an optimum quantity of phenols beyond which there is no increase in hydrogen peroxide production. An increase of concentration beyond this limit showed a decrease in peroxide formation.

Minardi (5) studied the effect of time on the hydrogen peroxide production of calamine lotions. He concluded that after a period of fourteen weeks no significant increase is obtained upon irradiation with ultraviolet light. Another of his findings was the fact that sodium

carboxymethylcellulose and/or dioctyl sodium sulfosuccinate retarded the photochemical reaction by which hydrogen peroxide is produced from zinc

oxide and water.

Blubaugh (6 ), working with lipids, found that the addition of fixed oils greatly reduced the amount of peroxides produced by photo­

sensitized zinc oxide. Although he found a considerable variation in the reduction produced by different oils, no correlation could be made between this and the physical constants of the oils. Fixed oils also decreased the ability of sodium formate to act as an nadditiven in the reaction. Mineral oil did not markedly reduce the amount of peroxide produced by zinc oxide. Blubaugh observed a variation in peroxide production by using zinc oxide obtained from different sources, and he indicated that this might be connected with the method used in the manufacture of the zinc oxide. Squalene was shown to increase the peroxide production while addition of human blood plasma caused a reduction.

In his work with selected phenols, Young (7) concluded that an olive oil-calcium hydroxide solution emulsion has a depressing effect on the catalytic activity of zinc oxide in the peroxide production.

The amount of agitation was found to have a direct relationship on the amount of peroxide formed. The addition of various phenols to calamine liniment will alter its physical properties (i.e., viscosity) but increase the production of hydrogen peroxide. Contrary to the observations of Mathias (4) regarding the effect of the number of hydroxyl ions in the phenol upon the formation of hydrogen peroxide by calamine lotion, Young found that the number of hydroxyl ions does not appear to influence directly the amount of peroxide formed by calamine liniment. The age of the calamine liniment containing various phenols influences the peroxide forming ability of the liniments by either increasing or decreasing it. The amount of peroxide formed varies also with the concentration of the phenols in calamine liniment.

As pointed out before, none of these investigators worked with ointments, their main concern being with aqueous types of preparations and the emulsion types of preparations containing either zinc oxide or calamine. Also it was noticed that no information is given whatsoever about the possibility of the role of temperature on the peroxide forma­ tion by zinc oxide. It was thought that a study of these two new aspects of the problem was in order and the official formula (l) for

Zinc Oxide Ointment was used as the basis for this study.

ZINC OXIDE

Zinc Oxide is described in the Pharmacopeia of the United States,

XV Revision (l) as "very fine, odorless, amorphous, white or yellowish white powder, free from gritty particles. It gradually absorbs carbon dioxide from the air." When freshly ignited, it should contain not less than 99 per cent of zinc oxide. It is insoluble in water and alcohol but is soluble in dilute acids.

Feitknecht and Haberli (8 ) report that the solubility product of zinc cod.de varies with the method of preparation. They found that the

solubility product of a sample prepared by precipitation was 1.6 x 10“^ ;

zinc oxide prepared by drying zinc hydroxide at 100°C was 8.3 x 1 0 ~ ^ t

and zinc oxide prepared by decomposition of zinc carbonate at 1000°C was

■I r t 2.7$ x 10“ . This indicates that there are sane differences in the

zinc oxide due to the different methods of preparation.

Outcalt (9 ) reports that Le Clair and Sorel, in France, started

the commercial production of zinc oxide during the latter part of the

eighteenth century. Le Clair was a master painter and paint grinder

and is reported to have been the first to use zinc oxide in combination with basic carbonate of white lead in paint. The zinc oxide used by

Le Clair was made by burning slab zinc (spelter) with air, a process which is now known as the French process, the indirect process, or the two-step process for production of zinc oxide.

The manufacture of zinc oxide in the United States was started from an observation made by a workman. A white fume was given off from sane zinc ore which he U3ed to repair temporarily a break in a boiler flue. Weatherill and Jones in 1885 perfected a furnace to produce zinc

oxide by a direct or single step process, which has since been known as the American process (9)» Other furnaces have been developed and used in this connection since that time but seme of the original type

Weatherill furnaces are still operated in this country.

The French process involves burning of slab zinc metal (spelter) with air. In order to obtain a white pigment a very pure spelter must be used. If it contains lead it will burn to litharge giving a yellowish tint. To render the lead harmless, a method has been developed

in which the burning is done in air mixed with carbon dioxide, the lead

is changed to white lead and thus the yellow tinge is avoided. The zinc

is unaffected by the process (9)» The furnace used in this process is

essentially a retort which is externally heated, usually by producer gas,

but may also be heated by coal, oil or natural gas.. The zinc metal is

heated in a retort to the boiling point when the zinc vapors come in

contact with air and burn with a bright yellowish flame to produce zinc

oxide fumes. The zinc oxide fumes are then conducted through suitable

pipe lines to settling chambers.

Although the physical properties of the zinc oxide may be controlled within certain limits by the rate of oxidation, the particle shape of the

product is characteristically spherical. The Ralmerton process, which

employs an air blast at the point of oxidation (9 ), produces a smaller

particle size but the shape of the particle is still spherical.

The American process uses the principle of vaporization of zinc directly from zinc ore rather than from the metal. The various types of

American process furnaces have sufficiently different characteristics to warrant some brief description.

(1) The original or Eastern type of Weatherill furnace U3es anthracite coal to heat the layer of zinc ore mixed with anthracite coal.

This combined charge is allowed to burn until a temperature of about

1100°C is attained, at which temperature the carbon in the coal reduces the zinc from its ore, the rate being regulated by air blown through the

grates. The particles obtained are predominantly nodular. The physical properties of the zinc cocide are influenced by the temperature prevailing

at the oxidizing zone, by the length of the time the fume is maintained

at an elevated temperature, and by the amount of dilution of zinc oxide

fume in the gases of combustion. Because of this and the large volume

of gases of combustion that must be handled, the uniformity of the

properties is very poor and little control can be achieved.

(2) The Western Weatherill type of furnace uses bituminous coal.

The process yields large acicular particles. Uniformity of the finished

product is more difficult to be obtained than by using the previous

method.

(3) The mechanical furnace is an improvement upon the Eastern

type furnace. This process uses a moving grate fed with coal and zinc

ore. Good uniformity of the product is achieved.

(4) The Electro-thermic furnace differs greatly in smelting design from other American processes. The charge to the furnace is purified and sintered zinc ore and coke. The heat necessary for reduc­ tion is supplied by an electric current. Combustion gases are not present and a wide range of control over properties is achieved. It

combines in the zinc oxide the desirable properties of both the French and American processes. The zinc oxide thus obtained is of the high

chemical purity associated with the French process and the particle sizes and shapes characteristic of both processes (9 )»

(5) The Cornillat furnace is essentially of the French process type but uses semibituminous coal and internal heating. little control

over properties is achieved. (6 ) The Wet Process finds practically no commercial application at the present because of the high production cost. This process involves precipitation of the zinc from solutions in the form of zinc carbonate and subsequent calcination of the carbonate to obtain the oxide. Spherical particles are obtained and the particles are finer in size than those obtained by any other method (9).

(7) A by-product zinc oxide is produced on a small scale by

calcining zinc carbonate which is obtained as a by-product from industries where zinc dust is used as a reducing agent. The properties of this zinc oxide are of inferior grade.

Zinc oxide has been an official product in the Pharmacopeia of the

United States (10) since the first edition in 1820. The Pharmacopeia of the United States V, 1876 (11) and later the British Pharmacopeia of

I885 (12), recognized zinc oxide prepared from zinc carbonate by the Wet

Method. This process produces a zinc oxide the particles of which are

finer in size than those produced by the fume processes (i.e., Weatherill and Electro-thermal Processes) (9 ).

Zinc oxide has been widely used in medicine as well as in industry.

The medicinal properties of zinc oxide are primarily associated with dermatology because of its effectiveness in the treatment of many skin disorders. It is a component of dermatological formulas such as powders, pastes, ointments, and lotions. Among other conditions of the skin it

is included (13) in formulas for the treatment of seborrheic dermatitis, atopic dermatitis, eczematous dermatitis, lichen planus, and psoriasis.

It is also used in formulas (13) as a drying agent in conditions like 10 sudorrhea* lichen planus; as an antipruritic in many types of dermatitis and allergic urticaria. Although it has no well-defined pharmacologic action on the skin, its popularity may be attributed (14) to a combina­ tion of four qualities: it is protective, mildly astringent, possibly weakly antiseptic, and non-toxic. In dentistry, it has been used in treatment of Vincent’s angina (14) or trench mouth, and also in combina­ tion with zinc acetate, zinc stearate and rosin as a dental protective

(2 ), i.e., as a pulp capping or temporary filling.

Internally it has been used as an antispasmodic in chorea, epilepsy, and whooping cough (14). It has also been employed a3 a protective in diarrhea. Nevertheless, it is seldom used internally in modern medicine.

In industry zinc oxide has been employed in the production of paints and in the manufacture of synthetic rubber.

ZINC OXIDE AS A PHOTOSENSITIZER

The catalytic activity of zinc oxide in the photochemical formation of hydrogen peroxide was first reported by Baur and Neuweiler (15) in

1927. Ten years later Goodeve confirmed this action (16). All these investigations as well as that of Winter (17) have shown that hydrogen peroxide is formed at light activated zinc oxide surfaces in contact with oxygen water and miscellaneous compounds. Winter (17) stated that the zinc oxide prepared by the French process showed chalking in paint films, fluorescence with ultraviolet light, and photochemical activity leading to formation of hydrogen peroxide in aqueous solutions. Zinc 11 oxide prepared by the American process showed no chalking, a yellow fluorescence under ultraviolet light, and no photochemical activity.

The proposed over-all reaction (17, IS) for peroxide formation is as follows:

2H20 + 02 .* 2H202

This reaction involves a large increase in free energy. The light absorption region of zinc oxide limits the effective wave lengths for peroxide formation to those less than about 4000 A. Of the total energy of the sun which is incident at the earth*s surface about 4 per cent can be effective in promoting this reaction (19).

Chari and Qureshi (20) studied the reaction in some detail and found that sunlight, artificial ultraviolet, and visible light up to

4700 A. are effective in causing the reaction to proceed. The zinc oxides were prepared from zinc hydroxide precipitated from zinc sulfate and zinc nitrate, from the ignition of the carbonate, and from ignition of the nitrate. The oxide prepared from the carbonate was the most effective and the one prepared from ignition of the nitrate the least active. This finding is in accordance with previous reports of Smith and

Hawk (21) and Schleede, Ritcher, and Smith (22) in which they conclude that the oxide produced from zinc carbonate is more active than zinc oxide prepared by combustion of zinc or by thermal decomposition of the nitrate.

Huttig and Feher (23)» in 1937, from their study of zinc oxides prepared from a variety of starting materials, concluded that the catalytic activity of zinc oxide depends upon the nature of the material 12 from which it was obtained presumably because of the nature of the original crystals.

G a m (24)» in his dissertation in 1952» concluded that the causes of the differences in catalytic activity of zinc oxide appeared to be most likely because of impurities, lattice defects or metallic zinc. The principal cause is the different general methods of preparation producing differences in the surface characteristics of zinc oxide.

It has been shown that the presence of a small concentration of one of many water soluble, easily oxidized, organic compounds (often called

"stabilizers," "promoters" or "additives") such as sodium formate, potassium oxalate, phenol, etc., in the zinc oxide-water-oxygen mixture increases the rate of peroxide formation. Chari and Qureshi (20) tested nine compounds and phenol was the most efficient followed by glycerine and acetanilid.

In systems containing additive, zinc oxide is a photosensitizer for peroxide formation (16). This is evidenced by the facts that hydrogen peroxide is formed only in the region of light absorption of zinc oxide, i.e,, 2500 A., and that no apparent change is observed in zinc oxide since it can be re-used again without change in the efficiency of peroxide formation (19).

Oxygen is necessary in the system for hydrogen peroxide formation.

Although the rate of peroxide formation is greatly increased when the additives are present in the irradiated system, peroxide decomposition is also very rapid when the oxygen supply is low.

Yamahuzi, Nisioeda, and Imagawa (25), and Yamahuzi, Nisioeda and 13

Ifcrusi (26) studied the photochemical formation of hydrogen peroxide in the presence of biological sensitizers. The latter authors suggest that the formation proceeds by three reactions:

21^0 = H2O2 / 2H ( I) 2H / Os = H202 ( ID A / H20 / 02 = AO / H2O2 (III) where ’’A" in reaction (III) is the organic sensitizer. While the energy of light is sufficient to promote these reactions, the heat liberated in

(II) and (III) would accelerate reaction (I). These substances which are called ’‘stabilizers11 or ’’promoters” are actually reactants and undergo oxidation simultaneously with the hydrogen peroxide formation.

Experiments showed that oxalate and formate ions are oxidized to carbon­ ate; phenol is oxidized in part to catechol (19 )•

Example:

H20 / 02 / HC00- ^ H2 02 / HCO3 V*

ULTRAVIOLET LIGHT

In 1800, Sir William Herschel discovered that the solar spectrum extended out beyond the portion which was visible to the human eye. One year later, J. W. Ritter investigated the portion of the spectrum beyond the violet and showed that chemical action was caused by some kind of energy in this region, the region which is now called the ultraviolet.

Thomas Young, in 1804, showed that the invisible chemically active radiations beyond the violet end of the spectrum (subsequently called actinic rays) corresponded to some radiations of shorter wavelength than visible light (27). There is really no sharp dividing line between these forms of radiant energy. They are all manifestations of the same kind of electromagnetic radiation differing from each other only in the frequency.

From the viewpoint of biological and therapeutic effects these ultraviolet radiations have received much more attention than the visible and infrared energy. They produce fluorescence, photographic action and many known biological effects. Ultraviolet light is conveniently divided into three parts in reference to the visible spectrum (28):

1. Extreme ultraviolet region (wavelength shorter than 2000 A.) is the least penetrating of all radiant energy and is absorbed by most substances including air. It must be produced and studied in a vacuum by means of special photographic plates, gelatin emulsions being opaque to it.

2. Middle ultraviolet energy (between 2000 A. and 3000 A. ) is a very important spectral region from biological and therapeutic viewpoints. 15

Ordinary glass will not transmit it, but quartz is transparent through^ out this range. The solar spectrum barely extends into this region for its short-wave limit is near 2900 A. The solar energy between 29OO A. and 3100 A., however, is known to be very valuable because of its anti­ rachitic and germicidal action, in the production of vitamin D, in the production of erythema, and in the production of conjunctivitis in extreme cases. Ordinarily conjunctivitis is not produced by sunlight.

The maximum of germicidal action is in the neighborhood of 2600 A. but it can extend to 3100 A. The maximum for production of conjunctivitis seems to be near 2500 A. and for erythemal effect 3000 A. The produc­ tion of vitamin D by irradiation of ergosterol is apparently due to energy somewhere between 25OO to 3100 A.

3« Near ultraviolet energy (between 3000 and 3900 A.) produces fluorescence and photographic action. Ordinary glass is quite trans­ parent between 3900 and 3500 A., but this transparency decreases until most glasses of ordinary thickness are fairly opaque at 3100 A. Most substances that are transparent to visible radiant energy, including ordinary glasses, become quite opaque in this region. Quartz, seme special glasses, and water are special exceptions.

The source of ultraviolet light for this study was a DAZ0R

Floating Fixture Ultraviolet Lamp, Model N. U-58, equipped with a

General Electric UA~3» 360 Watt Quartz Photochemical "Uviarc” lamp.

The radiant energy output of this lamp is greatest at 2537 A., 3131 A., and 3564 A. (2 9 ). 16

INFRARED ABSORPTION SPECTRA

As pointed out previously, Sir William Herschel in 1800, discovered that there was an extension of the solar spectrum beyond the visible region. He found a rise in temperature as he passed from the violet to the red end of the spectrum by passing a thermometer through the various colors obtained by a spectrum that he fomed. Still more astounding was the fact that the thermometer showed a higher temperature in the dark space beyond the red than it did in any part of the visible spectrum (2 7 ).

This meant that there was some intense radiation beyond the visible region. Thi3 invisible region beyond the red end is now known as infrared radiation.

The degree of molecular motion possessed by a molecule is dependent upon the amount of energy that it possesses. If energy is given in the form of radiant energy, motion begins and it can take any one of or a combination of four types of motion depending upon the amount of energy supplied to it.

These four forms of motion are described (30) as follows, according to the energy requirements:

1. Translational - in which the molecule moves from one point in space to another. Radiant energy of a very low order (20 microns in wavelength) is sufficient to initiate this motion.

2. Rotational - in which the molecule rotates about seme central axis. Molecular motion of this type is initiated by radiation in the order of twenty microns.

3. Vibrational - in which atoms are displaced from their normal positions and oscillate back and forth or move sidewise with swinging motion within the molecule. The rotations involving small energies are superimposed on atomic displacements giving rise to absorption bands in the near infrared region of the spectrum which extends from 2 to 16 microns.

Electronic - in which an electron is displaced to a higher energy level within the molecule. Greater displacements and greater energies are involved in ultraviolet and visible absorption spectra.

Infrared absorption spectra are concerned then with molecules which are capable of rotation and vibration. However, there is an additional requirement. A compound to be active in the infrared region must be sufficiently asymmetrical to possess a dipole moment. Vibrations of two similar atoms against each other, as for example in nitrogen and oxygen molecules, will not result in a change in the electrical symmetry of the molecules and these molecules will not absorb in the infrared region (3 1 )»

The most significant portion of the infrared region of the spectrum for structure determination is the region from 2 to 8 microns, because it is in this region that individual bands are more or less characteristic of specific pairs or groups of atoms. Above 8 microns, the bands are due to vibrations and rotations in which all the atoms in the molecule take part.

The spectrographs of pure compounds are so highly specific that they have become accepted as the ,Tfingerprints of those compounds'* (31)* When a compound undergoes reaction the fingerprint changes to that characteris­ tic of a new compound but the groups that are present in the parent 18

compound plus those groups in the compound with which the parent compound

was reacted will quite often show their characteristic absorption bands.

As an example of the above, reference is made to Figures 4 and 5>

pages 75 and 76 respectively. In Figure 4* the infrared absorption

spectrograph of cholesterol shows peaks of absorption between

1375-1445 cm"-*-, 1345-1260 cm“^, and 1160-1055 cmT-*- due to the effect of

the presence of a hydrcayl group in position 3 of the cholesterol mole­

cule (32). However, in Figure 5 the infrared spectrograph of a mixture

of cholesterol and ,,axidized,, cholesterol is shown. In addition to those

peaks of absorption due to hydroxyl group we can notice a new peak of

absorption at 1700-1800 cm“^* due to partial oxidation to a ketone.

A further consideration in the use of infrared spectra for analyti­

cal work is the effect of the solvent used upon the spectrograph. The

ideal situation is that of a liquid compound in which the absorption

observed is due to molecules of the compound only. Since this is not usually the case, the bands due to the solvent used must be considered.

These factors then place the determination of the structure of a new

compound somewhat upon an empirical basis. There is some uncertainty present but when the information from the infrared spectrograph is com­ bined with other data valid conclusions as to the structure may be withdrawn.

In this investigation, the apparatus used was the Baird Associates Two-Beam Infrared Spectrophotometer, property of the Department of Chem­ istry, The Ohio State University. This is a self-recording instrument.

Sodium chloride crystals were used and the samples were prepared in a

Nujol mull. Nujol did not interfere in this particular case since interest was only to show oxidation of cholesterol, and the bands of

absorption of Nujol are fortunately distinguishable from those of choles­ terol or "oxidized” cholesterol.

SOME PROPERTIES OF THE OINTMENT BASES

White Ointment

The official formula for White Ointment, Pharmacopeia of the United

States XV (1), calls for 5 per cent of white wax in 95 per cent of white petrolatum. It is prepared by melting the wax in a suitable dish on a water bath, adding the white petrolatum until liquefied and letting it

congeal. It is an ointment base for several official ointments like ammoniated mercury ointment, sulfur ointment, and zinc oxide ointment

of the Fharmacopeia of the United States (1); boric acid ointment,

ethyl aminobenzoate ointment, mild mercurial ointment, phenol ointment, and yellow mercuric oxide ointment of the National Formulary (2).

Its characteristics are essentially those of white petrolatum, the white wax being added as a stiffening agent. Because of its good keep­ ing properties and physical nature, it has been largely employed for many years as a base for ointments which are white or light in color. The white petrolatum used in its preparation is a colloidal dispersion of aliphatic liquid hydrocarbons (C^q to C24 ) in solid hydrocarbons

(C25 to C^q) obtained from petroleum. This mixture is semisolid, and the disperser of the liquid phase in the solid phase is a non-crystalline, naturally occurring branched chain paraffinic type of hydrocarbon known as proto-substance. Without this, the oil will leak out or "sweat" (33)* 20

The white petrolatum differs from petrolatum only in color. Both contain a small amount of oC -tocopherol which is added as an antioxidant.

The white ointment and the white petrolatum have been used as mild dressings for blistered or excoriated surfaces. There has been sane objection to their use in this respect and as bases for ointments because they seem to retard healing and also because of their greasy nature.

Wool fat was formerly an ingredient of white ointment but it was emitted from the formula in the Pharmacopeia of the United States XIV because of the sensitization which it sometimes causes (14 )•

Hydrophilic Ointment

This is an official preparation in the Pharmacopeia of the United

States (1). It is a rather new type of ointment ba3e. Its formula contains white petrolatum, stearyl alcohol, propylene glycol, polyoxyl

40 stearate, methyl and propylparaben, and purified water. This oint­ ment is really an emulsion type of ointment base. The emulsion formed is of the oil in water type. It belongs to the general classification of hydrophilic or water washable ointment bases, most of which are 0/W type of emulsions. The advantages of this type of base are several: they are easily removed from the skin or clothing with water; they can be diluted with water and permit incorporation of aqueous ingredients; provide media to absorb serous discharges of wounds, and also allow the heat in inflamed areas to be more easily dissipated; they allow for a greater release of many medicaments incorporated in them, and they are more acceptable cosmetically. 21

hydrophilic ointment was first recognized in the Pharmacopeia of the

United States XIII, the then official formula differing from the present in containing glycerine in place of propylene glycol and 1 per cent sodium lauryl sulfate instead of polyoxyl 40 stearate (34)» In the Pharmacopeia of the United States XIV (35) the glycerine which caused the ointment to soften when it was triturated or used as a levigating agent (14 ) was replaced by the propylene glycol which corrected this defect. In the present formula (1 ), sodium lauryl sulfate has been replaced by the non­ ionic surfactant polyoxyl 40 stearate (3 6 ) to avoid skin irritation produced by the Pharmacopeia of the United States XIV formula because the sodium lauryl sulfate is anionic and is recognized as being a primary irritant. The p-hydroxy-benzoic acid esters are used as preservatives.

There is some evidence to indicate that the combination of both esters is more efficient in this respect than either one alone in equivalent concentrations. The stearyl alcohol stiffens the ointment and also acts as an adjuvant emulsifier. Propylene glycol serves as a humectant and also assists the parabens as preservatives (14 )•

Polyethylene Glycol Ointment

This ointment is official in the Pharmacopeia of the United States

XV (1) and consists of a mixture of 60 per cent of polyethylene glycol

400 and 40 per cent of polyethylene glycol 4000. The mixture is prepared by heating the polyethylene glycols 400 and 4000 to 65°C on a water bath and then stirring the mixture until it congeals. It is an ointment base of the water soluble type. It is the official base for the Benzoic and 22

Salicylic Acid Ointment of the Pharmacopeia of the United States XV (1) and for the Compound Undecylenic Acid Ointment of the National Formulary

X (2).

Polyethylene glycols are polymers represented by the general formula

HOCHg (CffjOCF^nCHjjOH. The ”nM ranges from 1 to a large number which produces solid, waxy materials having high molecular weights. The liquids polyethylene glycols cover the range of molecular weight from 150 to 700, while the solids vary from 1,000 to 10,000. The solid polyethylene glycols are sold under the trade name of Carbowaxes (Carbide & Carbon

Company). They are commonly prepared by either polymerization of ethylene oxide, using a trace of water, heat and pressure or by dehydra­ tion of ethylene glycol (3 3 )*

With the advent of the liquid and solid polyethylene glycols and the development of synthetic emulsifiers, the preparation of a wide variety of water miscible and washable ointment bases was made possible.

The idea of greasiness inseparably associated with ointments is not considered so today. Because these washable bases have seme advantages over oleaginous bases, there is an increasing tendency on the part of dermatologists to prescribe the water soluble bases as vehicles for cutaneous medicinal preparations. These series of polyethylene glycols possess certain physical properties combined with chemical inertness and heat stability (33) which favor their use as ingredients in washable ointment bases.

The official Polyethylene Glycol Ointment is a homogenous, white, semisolid base having the consistency of petrolatum. The Pharmacopeia 23 of the United States XV (1) indicates in its monograph that "if a firmer preparation is desired, not more than 400 On. of polyethylene glycol 400 may be replaced by an equal amount of polyethylene glycol 4000." It is completely soluble in water and does not stain clothing or bed linens.

However, because of its high solubility in water we cannot add more than

3 per cent of water to the ointment or otherwise the consistency of the base will be affected. This might be considered as a disadvantage in the use of thi3 base. Before incorporating zinc oxide, sulfur or other substance to the base, these should first be triturated with a small amount of glycerine, propylene glycol or polyethylene glycol 400.

The formula for this ointment base was first prepared by Zopf and his associates at the State University of Iowa, College of Pharmacy, in

1950 (37)• The ointment was made official in the Pharmacopeia of the

United States XIV (35).

A great deal of work has been done to determine any untoward effects of this type of ointment base, but the order of toxicity seems to be low and apparently no more capable of sensitizing the skin than many other materials commonly used in ointment bases (14). However, there have been reports of two cases of demonstrable sensitivity to polyethylene glycols in ointment bases (38 ). Also Sulzberger and Baer (39) expressed seme disappointment in polyethylene bases after longer usage, mentioning as disadvantage s,

(1 ) the possible irritation to diseased or dry skin,

(2 ) the possible allergic sensitization in a small percentage of cases and an increase of sensitizing potential of active ingredients, and 24

(3 ) less effectiveness in removing scales, particularly from the scalp, and reduced emollient and lubricating effect when compared with greasy bases.

Hydrous Wool Fat

This product is described in the Pharmacopeia of the United States

XV (1) as "wool fat containing not less than 25 per cent and not more than 30 per cent of water.” Wool fat is the purified, anhydrous, fat­ like substance obtained from the wool of sheep. Wool fat is also known as anhydrous lanolin, while the hydrous wool fat is also called lanolin.

Lanolin is included in the formulas of Compound Menthol Ointment and

Mild Mercurous Chloride Ointment in the National Formulary X (2).

The wool of sheep contains from 10 to 50 per cent of a grease which occurs as an external coating on the fiber. It is analogous in function and composition to the human sebum (14)« Lanolin contains the sterols cholesterol and oxycholesterol as well as triterpenes and aliphatic alcohols. About 7 per cent of the alcohols are found in the free state; the remainder occur as esters of the following fatty acids: carnaubic, cerotic, lanoceric, lanopalmitic, myristic, and palmitic acids. Some of them are found free (40). The content of free cholesterol has been reported as approximately 1 per cent, and the acetyl value is reported as 23*3 (4l)» The iodine value is between IS and 36 (1).

Wool fat, although often related to the fats, is more accurately classified chemically as a wax. True fats consist mainly of esters of glycerol and fatty acids, while lanolin is composed largely of esters of high molecular weight alcohols, as cholesterol and oxycholesterol. Common wool grease requires extensive purification to make it

suitable for medicinal use (40). This is accomplished with the aid of

solvents, bleaching agents and other suitable agents. After purification and addition of water, it appears as a brownish yellow, tenacious, unc­ tuous mass. lanolin is insoluble in water but soluble in chloroform and ether with separation of water.

Sulton and Gardiner reported that wool fat penetrated the human skin much less readily than did lard (14). However, Johnston and Lee (42), by using as a tracer radioactive sodium chloride dispersed as an aqueous solution in various ointment bases, found wool fat to produce better absorption through the skin than did a base of lard, petrolatum or of a washable type of ointment. The efficiency of wool fat, as that of any other base, in promoting release of the active medicament when applied locally, depends not only on the base itself but also on the nature of the medicament and the state of the skin to which the ointment is applied.

All these things have to be taken into consideration before stating any­ thing about the properties of a base in releasing a given drug.

Lanolin has been much used in the treatment of rough, dry skin, and similar conditions. While it is satisfactory for this purpose it has been largely superseded by newer formulations of the water in oil emulsion type in which the emulsifying agents present in wool fat are used as emulsifiers. It is largely used as a vehicle for ointments, especially when a liquid is to be incorporated. It is found in sane ointments of the National Formulary X (2), since it gives a distinctive quality to the ointment, increasing its absorption on topical application 26

and maintaining a uniform consistency for the ointment under most climat­

ic conditions.

Reports of sensitization to wool fat led to the deletion in the

Pharmacopeia of the United States XIV (35) of this substance from all

ointments, in which it was formerly used, on the recommendation of

dermatologists who had noticed this undesirable manifestation in seme of their patients using this animal wax. Sulzberger et al., (43) obtained results connecting the aliphatic alcohol fraction of wool fat with the development of sensitization. They also report that the hypersensitivity due to lanolin is of weak intensity when compared with other eczematous hypersensitivities.

Aquaphor

There is not too much to be said about aquaphor since it is a propietary ointment vehicle (Duke Laboratories) and its exact composition is not revealed. It is described in the Remington*s - Practice of

Pharmacy (40) as na hydrophilic ointment base containing high molecular hydroxyl animal fats." It is said to contain about 3 per cent of the free alcohols of wool fat including cholesterol and oxycholesterol.

Cholesterol

This is an official product in the Pharmacopeia of the United States.

It is also called cholesterin. Cholesterol occurs as white or faintly yellow, almost odorless, pearly leaflets or granules. It melts between

147°C and 150°C. Cholesterin is insoluble in water and sparingly soluble in alcohol; it is soluble in hot alcohol, acetone, ether, chloroform, petroleum ether, and in vegetable oils. It is stable under normal

storage conditions (3 3 )•

The term cholesterol literally means bile solid alcohol, since it was first isolated, in 1770» from human gallstones, of which it is generally the chief component. In 1815 it was shown to be unsaponifiable and was called cholesterin. later, the alcoholic nature of the compound was established, and since then it has been designated cholesterol.

It is a steroid alcohol which is widely distributed in the animal organism. In fact, it has been found in all animal tissues but does not occur in plant tissues. It is synthesized in the animal body. The amount of cholesterol in animal tissues varies widely but it is reported

(4 4 ) as existing in the plasma of humans in a concentration of 152/24 milligrams per one hundred milliliters. In the blood it occurs free and also in the form of esters. An excessive amount of this compound in the blood may result in a precipitation from its colloidal solution and subsequent deposition in the arterial wall. This phenomenon leads to the condition known as atherosclerosis.

The skin possesses the capacity to synthesize cholesterol at a rate comparable to that of the liver. Cholesterol is converted to 7**dehydro- cholesterol in the body and upon irradiation with ultraviolet light

(i.e., when skin is exposed to sun rays) it foras vitamin D3 . Choles­ terol is particularly abundant in brain and nerve tissue, adrenal glands, and egg yolk. Fish oils constitute one of the principal sources of cholesterol. Cue of the most important methods of commercial production 28 involves extracting the unsaponifiable matter in the spinal chord of cattle, with petroleum benzin.

In pharmacy, cholesterol has extensive use as an emulsifying agent and absorption base for the emulsification and incorporation of medicinal products in oils or fats.

The chemical formula for cholesterol is shown in Figure 3 page 74*

HYDROGEN PEROXIDE

Above its melting point (-1.7°C), 100 per cent hydrogen peroxide is a syrupy liquid which appears colorless when observed in thin layers and slightly blue in thick layers (45 )• The boiling point of absolute hydrogen peroxide is reported as 152°C (14)» It is a powerful oxidizing agent. The anhydrous hydrogen peroxide contains 47 per cent by weight of available oxygen. The pharmaceutical as well as the industrial applications of hydrogen peroxide solutions are related to its ability to yield oxygen. One of the chief advantages of its use in pharmacy and industry is the fact that it gives rise only to water and oxygen when it acts as an oxidizing agent. In commerce, hydrogen peroxide is available as an aqueous solution in concentrations varying from 3 per cent to 80 per cent by weight. The strength of hydrogen peroxide is frequently designated also by the volume of active oxygen that it yields under standard conditions. Every 3»3 volume units are equivalent to 1 per cent by weight (i.e., the official. (1) Hydrogen Peroxide Solution, which is 3 per cent by weight, will release approximately 10 times its volume of oxygen). 29

Hydrogen peroxide was first prepared by a French pharmacist, Thenard, in 1818. He obtained it by treating barium peroxide with hydrochloric acid. The greater portion of the hydrogen peroxide now available is prepared electrolytically (14, 40 ).

Pure concentrated (30 per cent or stronger) hydrogen peroxide solu­ tions are quite stable (40). The commercial products, however, rapidly deteriorate in the absence of a preservative or stabilizer. One of the most commonly employed stabilizers is acetanilid. Generally, a dosage of 1/5 grain of acetanilid per fluid ounce of the official solution is sufficient (45)• Chari and Qureshi (20) tried seme organic compounds for their ability to preserve hydrogen peroxide solutions and listed them in the order of decreasing stabilizing properties as follows: phenol, glycerol, acetanilid, ethyl alcohol, ethyl oxide, acetone, benzidine, salicylic acid, and oxalic acid.

Other factors that affect the stability of hydrogen peroxide solu­ tions are temperature, purity, acidity, and effect of ultraviolet light.

Stability decreases with an increase in temperature. Horkheimer (46) found that solutions of hydrogen peroxide retained their potency for six weeks at 16 to 18°C, but at 20 to 23°G they lose some strength over this same period. Kiss and Lederer (47) determined the rates of decomposition of hydrogen peroxide solution in the presence of various metallic ions and found that only Cu7^ and Fe7^ ions showed any considerable catalytic effect upon the decomposition. The presence of small quantities of mineral acids aids in the stabilization of hydrogen peroxide solutions, but with too much acid the stability is so great that it fails to liberate "nascent” oxygen and this impairs the use of the solution as an antisep­

tic. Alkalies, on the other hand, rapidly decompose hydrogen peroxide

solutions with the liberation of oxygen (40)« It is also well known that

ultraviolet rays of short wavelength (230-300 millimicrons) decompose

solutions of hydrogen peroxide at a measurable rate. Therefore, solu­ tions must be protected from exposure to direct sunlight.

Hydrogen peroxide is frequently used as a germicidal agent. Its ability to kill microorganisms depends upon the release of oxygen and will persist only as long as oxygen is being released. Although in relatively dilute solutions it will eventually destroy many of the pathogenic microorganisms, its action is extremely slow unless the solu­ tion is fairly concentrated. Heinemann (48) reached the conclusion from his experiments, that three teaspoonfuls of the official solution after six weeks of exposure will destroy 99 per cent of the bacteria present in a liter of drinking water. This is about 1:1000 solution of hydrogen peroxide. Kavanagh (49) studied several antibacterial substances against nine species of bacteria. The results he obtained are given below for hydrogen peroxide as compared with penicillin and streptomycin which were the most effective of the twenty two antibacterial substances that he used in his experiments. 31

TABLE I

Minimum Inhibitory Concentrations of Antibacterial Substances (in micrograms/ml)

Organism Dihydro Hydrogen Penicillin Strepto­ Strepto­ Peroxide mycin mycin G X

B. Mvcoides 0.25 31 30 30 0.01

B. Subtilis 0.5 k 0.03 0.06 0.25

B. Aureus 0.03 8 0.02 0.03 0.03..

E. Coli 0.25 10 14 lk . 0.25

K. Fheumoniae 0.13 3 110 2k0 0.13

P. Fischeri 200 .. 5 16 8 200

P. Aeurginosa A. 8 500 500 k

M. Phlei 0.25 31 U 29 0.25

M. Smegma 1 k . 45P A7P.... 1

It should be pointed out that from the data above, penicillin was more efficient than hydrogen peroxide against only three of the nine organisms studied.

Hydrogen peroxide has also been effective as a wound cleanser. Its value in this connection is probably due to removal of organic debris which forms a breeding place for microorganisms. 32

APPARATUS AND MATERIALS

A. APPARATUS

1» Pyrex Tubes - These were plain round bottom 40 ml. pyrex centrifuge tubes with a diameter of one inch. The lip of the original tube was heated on an oxygen-gas torch and fixed in such a way as to permit proper sealing of the tube with a rubber stopper. In other words the depression originally present in the tube was flattened in order to be able to stopper the tube properly.

2. Ultraviolet Source - DAZOR Floating Fixture Ultraviolet

Lamp, Model N. U-58. This lamp is equipped with a General Electric UA-3

360 Watt Quartz Photochemical "Uviarc" Lamp. The radiant energy output of the lamp is greatest at 2537 A., 3131 A. and 3564 A. A five minute period is necessary to reach maximum output. This time was allowed to each sample before starting counting the time. In case of restarting,

10 minutes were allowed.

3. Centrifuge - This is a Servall Superspeed Angle Centrifuge,

Type SS-1; 115 Volts AC; 50/60 cycles (Ivan Servall, Inc., New York).

The current to the centrifuge is controlled by a rheostat which was set at 45 and this measured approximately 78OO r.p.m. Samples were centri­ fuged for only five minutes.

4 . Sample Holder - This unit consists of three different parts as shown in Figure 1 page 34* • The base is made of stainless steel. The dimensions are 8” x 8" and 4 inches tall. Two wheels made also of stainless steel, having a diameter of 4 inches each, were welded to a central rod 12 inches long and 1/4 inch diameter. This central rod was 33

connected on its right end to a motor. The two wheels have four holes

each. These holes have a diameter of 9/8 inch, which is 1/8 inch larger

than the diameter of the tubes used with the samples. Four tubes can be

operated at a time by passing them, lying on the side, through the holes

in the wheels. The tubes were usually held in position with rubber

bands (see Figure 1, page 34)•

5. Welter Bath - This unit was prepared by me from a used can

of ether. The dimensions of the can are x 6^" and 4 inches tall.

With the can lying on one side, the top was removed and the can placed

in the space between the base of the sample holder and the spinning

wheels (see Figure 1, page 34).

On one side of the can, and very close to the top, a hole

was made and a rubber stopper was passed through it. The rubber stopper

has one hole and through this hole a glass tubing is passed. This,

connected by means of a rubber tubing to the tap, furnished the incoming water. On the opposite side of the can, and about one-half inch lower, another hole was made and a similar procedure was followed. This

carried the water out of the bath to the sink. A thermometer was placed

in the rubber tubing carrying the water to the bath. This was made possible through the use of a T glass tube. When temperatures other than room temperature were desired in the bath, the rubber tubing car­ rying the incoming water was connected to a Y glass tube and this in turn connected to the hot water and the cold water outlet. mixing the water appropriately and using the thermometer, a temperature can be

obtained which is constant in the range of / 1°C. The temperature of 34

Stainless Steel Base

Side view of the complete unit. The broken line shows the position of the water bath.

Detail of one Spinning Wheel

Figure 1. Sample Holder and Water Bath for Irradiation Experiments 35 the water inside the bath, was frequently rechecked. The depth of the

water in the bath was kept constant at about 1-3/4 inches.

6 . Motor For Spinning The Samples - This was a Cenco Stirrer

Variable Speed motor; 115 Volts, A.C.; 60 cycles; 'type NS1-12 (Central

Scientific Company, Chicago). The motor was always run at the lowest

speed (approximately 62 revolutions per minute). The motor was con­ nected to the central rod of the sample holder by means of a piece of

Tygon tubing.

7. Filtering Unit - This consists of a sintered glass filter of medium porosity mounted on a hard rubber base which fits into a

250 ml. filtration flask which in turn is connected to a suction pump.

8 . Infrared Spectrophotometer (see page 18).

B. MATERIALS

1. Zinc Cbd.de, Reagent Grade, B & A, General Chemical Division,

Allied Chemical & Dye Corporation, New York. This brand of zinc oxide was used almost exclusively since Rubin et al., (19) reported that in their work they found it to give the most consistent results and to be one of the most efficient.

2. Zinc Oxide, U. S. P. (Zoco Brand, Zinc Cbri.de Company of

Canada, limited, Montreal).

3. White Petrolatum - Distributed by the Qrr, Brown and Price

Company, Columbus, Ohio.

4. White Beeswax - U. S. P. (sunbleached) Laboratory Supply,

Ohio State University. 5. hydrophilic Ointment - U. S. P. - prepared with ingredients furnished by the Laboratory Supply, Ohio State University.

6 . Aquaphor - Laboratory Supply - Ohio State University.

7. Lanolin Hydrous - Qrr, Brown and Price Company, Columbus,

Ohio.

8 . Liquid Petrolatum - U. S. P. - Laboratory Supply, Ohio

State University.

9 . Polyethylene Glycol Ointment - U. S. P. - made from ingredients furnished by Laboratory Supply, Ohio State University.

10. Cholesterol - U. S. P. (Cholesterin - Merck and Company,

Inc., Rahway, New Jersey). EXPERIMENTAL

PREPARATION OF SAMPLES

The formula for Zinc Oxide Ointment, Pharmacopeia of the United

States XV (1), was selected as the basic formula for the study of the hydrogen peroxide production in ointments containing zinc oxide.

Zinc Oxide Ointment, U.S.P.

Zinc Oxide • . . 200 Gm. Liquid Petrolatum 150 On. White Ointment . < 650 Gto.

To make 1,000 On.

Freshly prepared ointments were used. The above formula was changed only in respect to the white ointment. Other bases were substi­ tuted for the white ointment base.

Four other bases were selected to carry out this study* These bases were selected on the basis of common usage, and also on properties, i.e., absorption base, emulsion base, water soluble base. The four bases thus selected were aquaphor, hydrophilic ointment, hydrous wool fat, and polyethylene glycol ointment.

The ointment in which aquaphor replaced white ointment was prepared by following the official (1) procedure: The zinc oxide was triturated in a clean mortar and levigated very carefully with the liquid petrolatum.

Then the aquaphor was added and the preparation thoroughly mixed. This general technique was followed for the preparation of other ointments except the one containing polyethylene glycol ointment as base.

For making the ointment in which hydrophilic ointment was used as 38 the base, the hydrophilic ointment was first prepared according to the specifications in the Pharmacopeia of the United States (1). All ingre­ dients used were of the highest quality available. The ointment containing zinc oxide was prepared following the general procedure described above.

In the case of polyethylene glycol, a somewhat different procedure was followed. In this case no liquid petrolatum was used as levigating agent since in practice a small amount of polyethylene glycol 400 can be used as levigating agent for the zinc oxide (14). The procedure followed for making the samples was as follows: A solution of polyethylene glycol in distilled water (or sodium formate solution) was made containing

1.6 On. of the polyethylene glycol ointment in each 25 ml. of solution.

Then to each 25 ml. of this solution were added exactly 0.4 Gm. of zinc oxide. This means that each sample containing polyethylene glycol oint­ ment as base contained 2 Gn. of ointment in 25 ml. of solution.

Controls were always run with the samples. The controls consisted of the same ingredients as the samples but emitting the zinc oxide. This permitted the determination of the effect of the base alone on the produc­ tion of hydrogen peroxide.

When sodium formate solution was used instead of distilled water, a

0.2 M. solution of the same was made and then 25 ml. of this solution were added to each sample and each control.

Before irradiation, samples were placed inside a pyrex tube (see description on page 32), In order to get a more or less uniform film of sample within the pyrex tube, sane of the ointment was placed inside the 39

tube and then carefully spread in a film by using a scoopula. The tube

was then cleaned from any ointment in the outside or near the opening,

and the pyrex tube with the ointment film inside was accurately weighed.

The weight of the sample was then obtained by difference. Twenty-five

ml. of either distilled water or sodium formate solution (0.2 M.) were

added to each tube. The tube was then stoppered with a rubber stopper.

The stopper was covered first with seme tin foil and finally with Saran

wrap in order to avoid any contact of the rubber with the contents of

the tube. Every tube was carefully tested for any leaks and finally a

band of adhesive tape (plastic) was put around the opening of the tube.

In the case of studies with polyethylene glycol ointment, the method described above was modified. Zinc oxide 0.4 On* was added to

each tube and then 25 ml. of the solution of polyethylene glycol in dis­

tilled water or sodium formate solution (see page 38)* The rest of the

procedure was the same.

IRRADIATION OF SAMPLES

The pyrex tubes containing the samples were placed in the sample

holder and secured by a rubber band. The water was then circulated

through the bath.

The ultraviolet lamp used for irradiation of the samples was placed

at a distance of 18" from the top of the sample holder. The sample holder

with the water bath inside was completely enclosed within aluminum sheet­

ing. The unit used was the same which was used and photographed by

Blubaugh (6 ). 40

All samples were irradiated for one hour except those used for the

study of the effect of time.

METHOD OF ASSAY

The so-called Kingzette or iodometric method developed by Kolthoff

(50) and later modified by Chari and Qureshi (20) was used for the

quantitative determination of hydrogen peroxide in the samples. Hydrogen

peroxide reacts with iodide in an acid medium to liberate iodine v/hich

can then be titrated with standard sodium thiosulfate solution using

starch T.S. (1) as the indicator.

H^Og / 21“ / 2l/ = I2 / 2H20

Ammonium molybdate catalyzes this reaction.

When the ointment base was of the greasy type (i.e., white ointment, aquaphor, and hydrous wool fat) and when the temperature of the water bath was below 45°C, the samples after irradiation were placed in water at / 60°C for 1-2 minutes or until the grease was melted. Then the tubes were shaken vigorously and cooled. This ensured complete extrac­ tion of the hydrogen peroxide from the samples. If the temperature was

45°C or over, this was unnecessary since the samples were melted and by

simply shaking the tube the same effect was obtained. All tubes were

cooled to roam temperature before the stopper was removed.

In the case of ointments containing hydrophilic ointment as base a modified procedure was followed due to the fact that an emulsion was produced within the tube. A solution containing 3 Gm. of ammonium

sulfate in each 10 ml. of solution was prepared. Ten ml. of this 41

solution were added to the emulsion and the samples were then centrifuged

for 5 minutes at 7800 r.p.m. This procedure broke the emulsion so that

it could be filtered. The fact of diluting the sample with 10 ml. of

this solution was taken into consideration in making the subsequent

calculations.

After these pre-treatments, the samples were filtered through clean

and dry sintered glass funnels (see page 35 )•

Ten ml. of the filtrate were then accurately measured with a pipet

and placed into 250 ml. iodine flasks provided with ground glass stoppers.

To this 10 ml. aliquot in the iodine flask, the following reagents were

added in the order indicated:

10 ml. of 2 M. sulfuric acid

10 ml. of 0.2 N. potassium iodide

5 ml. of 0.04 N. ammonium molybdate

The flasks were then stoppered, well shaken, and placed in the dark

for about 10 minutes. The iodine which was liberated from the solution was then titrated with 0.01 M. sodium thiosulfate, using a 10 ml. micro­

buret and freshly prepared starch solution as the indicator.

The procedure for the controls was exactly the same as that for the

samples containing zinc oxide.

The potassium iodide solution was prepared fresh each day. The

sodium thiosulfate solution was assayed before use.

The sintered glass filters were carefully cleaned after usage.

Petroleum ether or chloroform was passed through the filter to remove

greasy substances. Then the filter was washed with HC1 and finally with 42 distilled water. After the washing, the filters were dried at 100°C.

A colorimetric method using potassium permanganate and acid was also tried but was discontinued because most of the ointments gave inconsis­ tent results, and in some cases (i.e., aquaphor and polyethylene glycol ointments) the color developed after mixing the aliquot with the acid- permanganate solution was different frcm that of the permanganate solu­ tion. A brownish color was developed.

The titanium sulfate method (51) was used to determine qualitatively the presence of hydrogen peroxide in the samples. This method is reported to have a sensitivity of 1:1,800,000 (i.e., about 5 micrograms in 10 ml. of solution). EXPERIMENTAL RESULTS

PART I

Determination of Hydrogen Peroxide Production in Ointments Contain­ ing Zinc Cbri.de With Different Bases.

For this study, the general procedure for preparation, irradi­ ation, and assay of samples described previously was followed. Through­ out this part of the investigation, distilled water was used in the irradiation of the samples. In all cases, 25 ml. of distilled water were added just before irradiation. The samples were cooled with water at room temperature (i.e., 25°C /l°). All ointments used contained 20 per cent of zinc oxide, the only variable being the base. The irradiation time was one hour.

TABLE II

Zinc Oxide with White Ointment

Weight of Sample ml. of 0.01 N. Sodium in On. Thiosulfate

1.1593 0.08 1.2127 0.10 1.3940 0.08 1.2468 0.08 Average 1.2532 0.08

Controls

0.9616 0.08 1.0394 0.08 Average 1.0005 0.08

Micrograms of hydrogen peroxide produced 0 TABLE III

Zinc Cbd.de with hydrophilic Ointment

Weight of Sample in On. ml. of 0.01 N. Sodium Thiosulfate

2.2570 7.49 2.7415 10.57 2.3121 7.60 2.6507 7.9S Average 2.4903 8.41

Controls

3.2942 0 2.3130 0 Average 2.8036 0

Micrograms of hydrogen peroxide produced by 2 On. of ointment 1,107

TABLE IV

Zinc Cbd.de with hydrous Wool Fat

Weight of Sample in On. ml. of 0.01 N. Sodium Thiosulfate

1.5380 0.10 I.OI65 0.15 1.2053 0.15 I.7696 0.13 Average 1.3824 0.13

Controls

0.8222 0.20 1.2923 0.23 Average 1.0573 0.23

Micrograms of hydrogen peroxide produced by 2 Gm. of ointment -26 TABIE V

Zinc Oxide with Aquaphor

Weight of Sample in On. ml.. of 0.01 N. Sodium Thiosulfate

1.0140 0.18 1.5092 0.13 1.1104 0.13 0.9536 0.13 Average 1.1468 0.15

Controls

O.6856 0.30 1.1398 0.43 Average 0.9127 0.38

Micrograms of hydrogen peroxide produced by 2 ftn. of ointment -68

TABLE VI

Zinc Cbcide with Polyethylene Glycol Ointment

Samples (1.6 On. of Polyethylene ml. of 0.01 N. Sodium Glycol Ointment / 0.4 On. ZnO) Thiosulfate

1 6.48 2 6.58 3 6.30 4 6.50 Average 6.47

Controls

1 0.53 2 0.63 Average 0.58

Micrograms of hydrogen peroxide produced by 2 Gm. of ointment 1,003 46

TABLE VII

Hydrogen Peroxide Production of Zinc Oxide Ointments

lype of Base Micrograms of H2O2 Produced Used by 2 Gta. of Ointment

Aouaphor -68*

Hydrous Wool Fat -26*

White Ointment 0

Polyethylene Glycol Ointment 1.003

Hydrophilic Ointment 1.107

PART II

Relationship of Time of Irradiation to the Production of Hydrogen

Peroxide.

Two ointment formulas were used for this study namely Zinc

Oxide Ointment U. S. P. and Polyethylene Glycol Ointment U. S. P. with

20 per cent zinc oxide. Distilled water was used inside the tubes.

During the irradiation the samples were kept at 25°C by use of a water bath.

* Sterol molecule undergoes oxidation TABIE VIII

Zinc Oxide with White Ointment Irradiated 120 Minutes

Weight of Samples in On. ml. of 0.01 N. Sodium Thiosulfate

1.8546 0.08 1.7285 0.08 1.7005 0.08 1.5161 0.08 Average 1.7002 0.08

Controls

1.6534 0.08 1.5987 0.08 Average 1.6261 0.08

Micrograms of hydrogen peroxide produced 0

TABLE IX

Zinc Oxide with White Ointment Irradiated 240 Minutes

Weight of Samples in On. ml. of 0.01 N. Sodium Thios\ilfate

1.0220 0.23 0.9354 0.18 1.2489 0.18 1.4195 0.20 Average 1.1590 0.20

Controls

1.4620 0.18 1.5727 0.23 Average 1.5174 0.20

Micrograma of hydrogen peroxide produced 0 4$

TABLE X

Zinc Oxide with Polyethylene Glycol Ointment Irradiated 30 Minutes

Samples (1.6 On. of Polyethylene ml. of 0.01 N. Sodium Glycol Ointment / 0.4 Ga. ZnO) Thiosulfate

1 5.25 2 5.OS 3 5.65 4 5.50 Average 5-37

Controls

1 0.45 2 0.40 Average 0.43

Micrograms of hydrogen peroxide produced by 2 On. of ointment 841

For details of results obtained with Zinc Oxide with Poly­ ethylene Glycol Ointment irradiated for 60 minutes, see Table VI on page 45. TAB IE XI

Zinc Oxide with Polyethylene Glycol Ointment Irradiated 120 Minutes

Samples (1.6 On. of Polyethylene ml. of 0.01 N. Sodium Glycol Ointment / 0.4 On. ZnO) Thiosulfate

1 10.15 2 9-95 3 9.65 4 10.53 Average 10.07

Controls

1 0.85 2 O.78 Average 0.82

Micrograms of hydrogen peroxide produced by 2 Gn. of ointment 1,575

TABLE XII

Zinc Oxide with Polyethylene Glycol Ointment Irradiated 180 Minutes

Samples (1.6 On. of Polyethylene ml. of 0.01 N. Sodium Glycol Ointment / 0.4 On. ZnO) Thiosulfate

1 11.43 2 10.80 3 10.93 4 10.40 Average 10.89

Controls

1 1.18 2 1.10 Average 1.14 Micrograms of hydrogen peroxide produced by 2 Gn. of ointment 1,660 50

TABLE XIII

The Effect of Tine of Exposure to Ultraviolet Light on the Hydrogen Peroxide Production of Selected Zinc Oxide Ointments

Time of Exposure Amount of Hydrogen Base Used in Minutes Peroxide Produced in Micrograms

60 0

White Ointment 120 0

240 0

30 841

Polyethylene Glycol 60 1,003_ Ointment 120 1.575 .

240 1.660

PART III

The Effect of Temperature on the Production of Hydrogen Peroxide

For this study, four ointment bases were used. An ointment containing polyethylene glycol ointment as a base was studied through a range of six different temperatures. Ointments prepared with other bases were only studied under selected temperatures. The other bases used were acruaphor, white ointment, and hydrous wool fat. All temper­ atures indicated herewith are / 1°C. The irradiation time for all samples was one hour* Sodium formate solution was used as an "additive" in all samples since Blubaugh (6) had shown that sodium formate had the property of stabilizing hydrogen peroxide. 51

The samples were cooled by the use of ice water in the bath.

Ice cubes were placed in the bath and kept out of the path of the rotating sample holder by using a wire screen.

When temperatures other than room temperature were used, the samples were rotated in the heated water bath for 15 minutes before irradiation.

TABLE XIV

Zinc Oxide with White Ointment Irradiated at 25°C

Weight of Samplesi in On. ml. of 0.01 N. Sodium Thiosulfate

1.2556 0.08 1.0116 0.03 1.1925 0.08 0.6784 0.08 Average 1.0345 0.07

Controls

1.0387 0.08 0.8535 0.05 Average 0.9461 0.07

Micrograms of hydrogen peroxide produced by 2 Gm. of ointment 0 TAB IE XV

Zinc Oxide with White Ointment Irradiated at 45°C

Weight of Samples in Gn. ml. of 0.01 N. Sodium Thiosulfate

1.4550 0.40 1.1595 0.43 0.6551 O.65 1.0310 0.68 Average 1.0751 0.54

Controls

1.0184 0.15 0.9395 0.18 Average 0.9790 0.17

Micrograms of hydrogen peroxide produced by 2 Gn. of ointment 124

TABLE XVI Zinc Oxide with White Ointment Irradiated at 55°C

Weight of Samples in Gn. ml. of 0.01 N. Sodium Thiosulfate

0.9509 6.53 0.8816 6.55 1.0274 5.85 0.9500 6.45 Average 0.9525 6.35

Controls

1.0714 0.50 1.0216 0.60 Average I.O465 0.55

Micrograms of hydrogen peroxide produced by 2 Gn. of ointment 2,128 TABLE XVII Zinc Oxide with Aquaphor Irradiated at 55°C

Weight of Samples in Gm. ml. of 0.01 N. Sodium Thiosulfate

0.8267 0.45 0.7865 0.58 1.0080 O.63 1.0536 O.58 Average 0.9187 O.56

Controls

1.2854 0,58 1.2555 0.55 Average I.27O5 0.57

Micrograms of hydrogen peroxide produced by 2 On. of ointment 39

TABLE XVIII

Zinc Oxide with hydrous Wool Fat Irradiated at 55°C

Weight of Samples in Gn. ml. of 0.01 N. Sodium Thiosulfate

1.3345 1.45 1.4045 1.00 0.9025 0.90 0.9085 0.75 Average 1.1375 1.03

Controls

1.6690 1.48 0.8534 1.08 Average 1.2612 1.28 Micrograms of hydrogen peroxide produced by 2 Gn. of ointment 30 TABLE XIX

Zinc Oxide with Polyethylene Glycol Ointment Irradiated at 0°C

Samples (1.6 Gn. of Polyethylene ml. of 0.01 N. Sodium Glycol Ointment / 0.4 Gn. ZnO) Thiosulfate

1 11.30 2 9.85 3 10.42 4 10.45 Average 10.51

Controls

1 0.23 2 0.20 Average 0.22 Micrograms of hydrogen peroxide produced by 2 Gn. of ointment 1,752

TABLE XX

Zinc Oxide with Polyethylene Glycol Ointment Irradiated at 25°C

Samples (1.6 Gn. of Polyethylene ml. of 0.01 N. Sodium Glycol Ointment / 0.4 Gn. ZnO) Thiosulfate

1 12.40 2 11.83 3 12.90 4 12.38 Average 12.38

Controls

1 0.33 2 0.30 Average 0.32 Micrograms of hydrogen peroxide produced by 2 Gn. of ointment 2,054 TABLE XXI Zinc Oxide with Polyethylene Glycol Ointment Irradiated at 35°C

Samples (1.6 Gm. of Polyethylene ml. of 0.01 N. Sodium Glycol Ointment / 0.4 On. ZnO) Thiosulfate

1 15.15 2 15.00 3 14.03 4 14.68 Average 14.72 Controls

1 0.25 2 0.23 Average 0.24

Micrograms of hydrogen peroxide produced by 2 Gn. of ointment 2s466

TABLE XXII Zinc Oxide with Polyethylene Glycol Ointment Irradiated at 45°C

Samples (1.6 Gm. of Polyethylene ml. of 0.01 N. Sodium Glycol Ointment / 0.4 On* ZnO) Thiosulfate

1 15.65 2 15.S5 3 16.40 4 13.82 Average 15.43 Controls

1 0.33 2 0.28 Average 0.31 Micrograms of hydrogen peroxide produced by 2 On. of ointment 2,575 TABLE XXIII Zinc Oxide with Polyethylene Glycol Ointment Irradiated at 55°C

Samples (1.6 On. of Polyethylene ml. of 0.01 N. Sodium Glycol Ointment / 0.4 Gn. ZnO) Thiosulfate

1 14.27 2 14.50 3 14*43 4 13.23 Average 14.11 Controls o o o o 1 . . 2 Average O.25

Micrograms of hydrogen peroxide produced by 2 Gm. of ointment 2,360

TABLE XXIV Zinc Oxide with Polyethylene Glycol Ointment Irradiated at 67°C

Samples (1.6 Gn. of Polyethylene ml. of 0.01 N. Sodium Glycol Ointment / 0.4 Gn. ZnO) Thiosulfate

1 12.08 2 11.80 3 10.55 4 10.83 Average 11.32 Controls

1 0.25 2 0.28 Average 0.27 Micrograms of hydrogen peroxide produced by 2 Gn. of ointment 1,882 270C-

'O0) © %o

© TJ © 2300 1 fn M CD (Xi 3 o c ©bfl «no O U • %& . CM o & © 1900 00 o fn O

1500 25 35 45 55 67 Temperature in °C

Figure 2. Effect of Temperature on Production of hydrogen Peroxide by Zinc Oxide with Polyethylene Glycol Ointment 58 TABLE XXV

Effect of Temperature on the Production of hydrogen Peroxide Hy Various Ointments Containing ZnO

Amount of Hydrogen Base Used Temperature of the Peroxide Produced WAter Bath in °C in Micrograms

25 0

White Ointment ...... 4 5 .. 12U

55 2.128

0 _ 1,752

25 2.054

Polyethylene Glycol . 35 2.A66 Ointment 45 2,575

.... 5 5 ...... 2,360......

67 1,882. _

Aquaphor* 55 39 hydrous Wool Fat* 55 30

* Sines irradiation of ointments using these substances

as bases had shown negative peroxide values when irradiated

at room temperature, 55°C was chosen since at this tempera­

ture White Ointment and Polyethylene Glycol Ointment gave a

significant increase in peroxide. As will be shown, the

values recorded as peroxide do not truly represent the

amount of peroxide produced because it was determined that

the cholesterol molecule undergoes oxidation. 59

PART IV

Oxidation of Cholesterol

A solution containing approximately 4 per cent of hydrogen peroxide was prepared. Two grams of cholesterol were then added to 50 ml. of this solution in a 250 ml. Erlenmeyer flask. The flask was securely closed with a rubber stopper covered with tin foil and Saran wrap. The solution was then shaken in a mechanical shaker for one hour and stored inside the locker for one week. A blank containing no cholesterol was run at the same time. After one week, both the sample and the control solutions were filtered through ordinary filter paper and an aliquot of one ml. was taken for analysis. Two samples and two controls were assayed according to the official method in the Pharmacopeia of the United States (1) for

Rydrogen Peroxide Solution.

The results obtained are shown on Table XXVI page 60.

In another experiment, one gram of cholesterol was dissolved in

20 ml. of chloroform and this solution was mixed in a test tube with

10 ml. of a solution of 3 per cent hydrogen peroxide. This mixture was irradiated for one hour. After separation of the chloroform layer, the aqueous solution was analyzed and found to contain only 2.5 per cent of hydrogen peroxide. The control treated in the same way contained

2.68 per cent of hydrogen peroxide. 60

TABIE XXVI

Amount of hydrogen Peroxide Which Reacts With Cholesterol

Samples ml. of 0.1 N. KMnO^

1 20.61 2 20.83 Average 20.72

Controls

1 22.89 2 22.99 Average 22.94

Milligrams of hydrogen peroxide found in 50 ml. of control solution 1,951

Milligrams of hydrogen peroxide found in 50 ml. of solution treated with cholesterol 1,762

Milligrams of hydrogen peroxide which reacted with 2 Gm. of cholesterol 189

Subsequent experiments were undertaken to prove the presence of an oxidized form of cholesterol after treatment with hydrogen peroxide.

The procedure followed was to dissolve one gram of cholesterol in 20 ml. of chloroform, mix in a pyrex tube (see page 32) with 10 ml. of dis­ tilled water and 1 On. of zinc oxide, and irradiate the mixture at room temperature for one hour. The mixture was centrifuged, decanted, and the water layer wa3 separated from the chloroform layer using a separating funnel. The chloroform solution was filtered through a sintered glass funnel (see page 35)» The filtrate was evaporated on a steam bath. The residue was dried in a vacuum at roan temperature. 61

This dried residue was then subjected to infrared analysis using a Nujol mull and sodium chloride cells (see page 18). A control sample without zinc oxide was also processed. Infrared spectrograph of cholesterol irradiated in absence of zinc oxide is shown in Figure 4» page 75 while that of cholesterol irradiated in presence of zinc oxide is shown in

Figure 5, page 76.

The "oxidized” product gave a positive hydroxylamine hydrochloride test (32) whereas the control sample was negative. The melting point of the "oxidized” cholesterol was found to be 141-2°C while that of cholesterol was 148°C.

To further study the oxidation product of cholesterol the procedure for the isolation of the ketone was that using Girard reagent T (56 ).

The procedure consisted of dissolving 1-2 grams of the "oxidized" prod­ uct in 50 ml. of absolute alcohol to which 10 per cent of acetic acid had been added; then add 5-10 per cent of Girard reagent T. The mixture was refluxed for sixty minutes. After cooling the solution, distilled water containing sodium bicarbonate was added to precipitate the cholesterol and neutralize the acetic acid. The solution was then filtered by suction to remove the cholesterol. Concentrated sulfuric acid was added to obtain a concentration equivalent to 1 N ^SO^. The solution was then stoppered and allowed to precipitate. The mixture was stored in a refrigerator for 10 to 12 hours to permit complete precipitation. The precipitate was then filtered by suction and dried in a vacuum for three hours.

The infrared spectrograph of the ketone is shown in Figure 5 page 76. 62

This product was also tested with hydroxylamine hydrochloride (32) and

also with 2,4-dinitrophenylhydrazine (32) giving positive results. The dinitrophenylhydrazone obtained with the latter has a melting point

ranging from 146-l6l°C. Since the product had a wide melting range, the

2,4-dinitrophenylhydrazone obtained frcm the ketonic substance was

subjected to fractional crystallization. A few drops of water were ad­ ded to the test tube containing the hydrazone and the 2,4-dinitrophenyl­ hydrazine in order to obtain a heavier precipitate. The precipitate was extracted with ether and the ether removed. Alcohol was added to the residue and two fractions were obtained. A few drops of water were added to the alcohol-soluble fraction until a slight cloudiness was noticed. Then it was stored in the refrigerator overnight. The melting range of the dried precipitate obtained from this fraction was 172-182°C.

The precipitate consisted of reddish-brown crystals.

The alcohol insoluble fraction was dried in a vacuum and the melting point was found to be 194°C which is the melting point of 2,4-dinitro­ phenylhydrazine (57). This fraction was very soluble in ethyl acetate and aniline which are two good solvents for 2,4-dinitrophenyltydrazine

(57).

The ketone was partially soluble in alcohol and chloroform. The dinitrophenylhydrazone is soluble in alcohol and chloroform. Both products were insoluble in water. The ketone showed, however, a tendency to be hygroscopic. 63 PART V

Hydroxyl Value of Hydrous Wool Fat

In order to determine if any change in the hydroxyl value of

hydrous wool fat occurred after treatment with hydrogen peroxide solution,

the following procedure was used: An accurately weighed amount of hydrous

wool fat in a tared flask was dissolved in 50 ml. of chloroform. Fifty milliliters of approximately 2.68 per cent solution of hydrogen peroxide

were added and the mixture was stoppered and shaken for one hour. The

chloroform layer was separated from the aqueous layer and evaporated on

a steam bath. The sample was then acetylated by adding twice its weight

of acetic anhydride and refluxing for one hour. The acetylated product was then mixed with 500 ml. of hot water and boiled for 30 minutes, The wool fat was permitted to separate and the water was siphoned off the beaker. This washing operation was repeated three times. After the washing, the product was refluxed with 50 ml. of 0.5 N alcoholic potas­

sium hydroxide solution (1) for thirty minutes. The alcohol was

evaporated on a steam bath and the soap dissolved in 50 ml. of distilled water. Seventy-five milliliters of 0.5 N sulfuric acid were added to the flask and the mixture steam distilled until 600 ml. of distillate had been collected (52). A 100 ml. aliquot of the distillate was ti­ trated with 0.1 N potassium hydroxide using phenolphthalein as indicator.

Controls treated with 50 ml. of distilled water instead of hydrogen

peroxide solution, were run simultaneously with the samples. 64 TABLE XXVII

Hydroxyl Values of Hydrous Wool Fat and "Oxidized” Hydrous Wool Fat

Weight of Samples in On. ml. of 0.1 N KOH (average of two aliquot fractions)

7-915 18.90 7.068 14.88 Average Hydroxyl Value = 12.6

Controls

7.862 27.54 7.371 25.74 Average Hydroxyl Value = 19*4

PART VI

Pharmacology

Ointments containing 5 per cent and 13 per cent of "oxidized"

cholesterol in white petrolatum were prepared and tested on rabbits*

eyes which had been previously irritated with a 2 per cent solution of

dioctyl sodium sulfosuccinate (Aerosol O.T.). The rabbits were kept in the rabbit boxes for 15 minutes after the treatment. Then they were put

in their ordinary cages and left for ten to twelve hours (usually over­

night). After this time the irritated eye was treated with the ointment.

Ointments containing 5 per cent of cholesterol in white petrolatum, white

petrolatum alone, and a commercial 1 per cent hydrocortisone acetate

ointment (Cortef Acetate; Upjohn Company) were also tried. For each

trial two rabbits were used. Finally, an ointment containing 13 per cent of "oxidized” cholesterol in a base of polyethylene glycol ointment was tried.

All these ointments were prepared under laboratory conditions and were stored in ophthalmic, clean ointment tubes. Approximately 100 mgm.

of ointment was used in treating each eye.

The rabbits were inspected two, four, six, and twelve hours after the medication was applied.

None of the animals treated with ointments containing white petro­

latum alone, "oxidized" cholesterol in white petrolatum, cholesterol in white petrolatum or "oxidized" cholesterol in polyethylene glycol oint­ ment showed any improvement in the condition of the treated eyes twelve hours after medication was applied. The eyes which were used as control

likewise did not show improvement. The eyes of the rabbits treated with the commercial Cortef Acetate Ointment (Upjohn Company) did show a marked improvement. This observation was made six hours after medication was applied. 66

DISCUSSION

Rubin and co-workers (19), in 1953* reported that in a series of experiments they found that Zinc Oxide, Reagent Grade B & A (see page 35) was one of the most efficient and produced the most consistent results as photosensitizer for the production of hydrogen peroxide from water oxygen.

Although not reported in this dissertation, some preliminary exper­ iments were made with Zoco Brand Zinc Oxide (see page 35)• This is a brand of Zinc Oxide U. S. P. obtained by the French process. The values for the hydrogen peroxide produced by this zinc oxide were lower than those obtained by the B & A brand zinc oxide. These two factors influ­ enced the selection of the B & A Reagent Zinc Oxide for this work.

The iodcmetric method for the quantitative determination of hydrogen peroxide as modified by Chari and Qureshi (20) has been consistently used by other investigators (3-7; 1 9 ) and has been proved to be satis­ factory for the determination of small quantities of hydrogen peroxide.

The production of titratable iodine, however, may be influenced by the presence of other oxidizing agents in the sample. In order to overcome this difficulty, controls were run together with samples in all experi­ ments. The amount of hydrogen peroxide reported as produced by the sample is the difference between the amount produced by the sample and that produced by an amount of base equal to that in the sample. The latter information was obtained by the use of controls. 67

PART I

Petermination of Hydrogen Peroxide Production in Ointments Contain­ ing Zinc Oxide with Different Bases.

The hydrogen peroxide production of zinc oxide ointments con­ taining five different bases was studied under room temperature conditions. Since al1 the ointments contained the same amount of zinc oxide, differences in the amount of hydrogen peroxide produced are only attributable to the difference in the base used. Table VII, on page 46, shows a comparison of results obtained with ointments containing aquaphor, hydrous wool fat, white ointment, polyethylene glycol oint­ ment and hydrophilic ointment as bases.

Results indicate that hydrophilic ointment (emulsion type of base) produced the greatest amount of hydrogen peroxide (1,107 micro­ grams) followed very closely by the water soluble base, polyethylene glycol ointment (1,003 micrograms). White ointment, however, did not show production of any peroxide under these conditions.

The negative values recorded for peroxide in aquaphor and in hydrous wool fat ointments was totally unexpected since Blubaugh (6) had shown high peroxide values in liniments containing liquid petrolatum and zinc oxide. Since each of these substances contains cholesterol, the only possible explanation to the negative peroxide values was that either the peroxide was being rapidly decomposed or was being used in the oxidation of the cholesterol. Infrared studies, and chemical tests gave positive evidence that the cholesterol molecule was being oxidized and that a ketone group resulted. 68

The three greasy type ointment bases are shown to produce less measurable hydrogen peroxide than the water soluble base and the emulsion type of base.

Since the greasy bases did not melt during this experimentation, it was believed that this might be one of the reasons for the low yield in hydrogen peroxide production. The results of experiments run at higher temperatures will be discussed later.

None of the samples or controls of the three greasy bases gave a positive qualitative test (51) for hydrogen peroxide with titanium sulfate. This fact suggests the possibility that the reported small excess of hydrogen peroxide produced by the controls in the case of aquaphor and hydrous wool fat is probably due to the presence of oxi­ dizing substances, other than hydrogen peroxide, in the base.

On the other hand, samples containing hydrophilic ointment and polyethylene glycol ointment gave a positive qualitative test for hydrogen peroxide, while the controls gave a negative test.

PART II

Relationship of Time of Irradiation to the Production of Hydrogen

Peroxide.

Since no hydrogen peroxide was obtained from the official

Zinc Cbcide Ointment upon irradiation for one hour with ultraviolet light and at room temperature (see Table II, page 43)» it was considered that the time of exposure to ultraviolet light was insufficient for the production of hydrogen peroxide. Therefore, the time of exposure was 69 increased to two hours and to four hours. Irradiation time of poly­ ethylene glycol ointment containing zinc oxide was similarly increased.

A summary of the results of increased time of irradiation of both ointments is given in Table XIII, page 50.

The official Zinc Oxide Ointment (1) did not show any produc­ tion of hydrogen peroxide under the conditions of these experiments.

Upon examination of Table II, page 43, and Table VIII, page 47, it can be seen that even by increasing the time of irradiation from one to two hours no hydrogen peroxide was obtained. Table IX, page 47, shows that even after four hours of exposure there was no production of hydrogen peroxide. This was thought probably due to the fact that the layer of ointment surrounding the sample tube did not permit an effective pene­ tration of the ultraviolet radiation. Other experiments were then performed to determine if higher temperatures (i.e., melting the oint­ ment ) would effect the production of hydrogen peroxide. The results of elevated temperatures of irradiation are given in Tables XIV, XV, and

XVI, pages 51 and 52.

The slight increase in hydrogen peroxide shown in Table IX, page 47, as produced by both the samples and controls may be attributed to the fact that upon a longer period of irradiation some hydrogen peroxide is produced from decomposition of water.

Although the average weight of samples and controls in

Table VIII, page 47, is somewhat different no correction was made for the amount of base in the sample for two reasons: (1 ) the values of sodium thiosulfate used by samples and controls are quite similar and 70

(2 ) the qualitative test for hydrogen peroxide using titanium sulfate

(51) was negative in both the samples and the controls. This same

criterion was used in interpreting results in Table VIII, page 47*

The ointment prepared with polyethylene glycol ointment gave

more consistent results of hydrogen peroxide production. Table XIII,

page 50, shows that hydrogen peroxide is formed after 30 minutes of

irradiation. An increasing amount of hydrogen peroxide is produced as

the time of irradiation is increased from 30 minutes to 240 minutes.

However, no direct relation can be made between the increase in hydrogen

peroxide production and the time of irradiation. Sixty minutes of

exposure produce 1,003 micrograms of hydrogen peroxide or approximately

20 per cent more than 30 minutes of irradiation. After 120 (1,575 micrograms) and 240 (1,660 micrograms) minutes of exposure the amount

of hydrogen peroxide produced is approximately 50 per cent greater than

60 minutes. Table XIII, page 50, shows that there is an increase of

hydrogen peroxide as the time of irradiation is increased. Although

there is an increase in the total amount of hydrogen peroxide at the

end of 240 minutes as compared with that shown for 120 minutes, the

increase is less than 100 micrograms. This is not a great increase if

it is compared with the one produced between 60 and 120 minutes (i.e.,

572 micrograms). A probable reason for this is that the ultraviolet

light may also cause seme destruction of hydrogen peroxide as it is

produced.

All samples gave a positive qualitative titanium sulfate (51)

test for hydrogen peroxide while the controls gave negative tests. 71

PART III

The Effect of Temperature on the Production of Hydrogen Peroxide

Tills study was undertaken to determine if melting the greasy bases in the sample tube would show an increase in the production of hydrogen peroxide. Since high temperatures decompose hydrogen peroxide, a series of experiments was performed using 0.2 M sodium formate solu­ tion as a preservative. An ointment containing polyethylene glycol was also studied under six different temperatures.

Results obtained with all the bases used are summarized in

Table XXV, page 5&.

The ointment containing White Ointment did not show any production of hydrogen peroxide at room temperature (25°C) even in the presence of sodium formate solution.

At 45°C, two grams of ointment were calculated to produce

124 micrograras of hydrogen peroxide. The controls gave a negative qualitative test for hydrogen peroxide while the samples were positive.

It was noticed that at 45°C the ointment did not fuse and therefore the temperature was increased to 55°C.

At 55°C there was a great increase in the production of hydrogen peroxide. An increase of 10°C in temperature (from 45° to

55°C) produced an increase in hydrogen peroxide of 2,004 micrograms per 2 Gm. of ointment. This fact shows that the ointment must liquefy in order to obtain a significant amount of hydrogen peroxide production.

Qualitative tests for hydrogen peroxide were the same as those obtained at 45°G. 72

Since good results were obtained at a temperature of 55°C with white ointment as base, this same temperature was used for the study of ointments containing aquaphor and hydrous wool fat as bases. The results obtained with these two bases are given in Table XXV, page 58. These are

39 and 30 micrograms of hydrogen peroxide respectively for 2 On of oint­ ment. There is good reason to believe that the hydrogen peroxide pro­ duced in these two cases reacted with the cholesterol in the base and therefore, the values obtained are very low as compared with the ones obtained for white ointment base.

Results obtained with polyethylene glycol ointment as base are also shown in Table XXV, page 58. Here again there is an interesting situation. Polyethylene glycol ointment permits production of hydrogen peroxide in an appreciable amount (1,752 micrograms per 2 Gm. ointment) even at 0°C. The production of hydrogen peroxide increases with a rise in temperature up to a maximum of 2,575 micrograms per 2 Gn. of ointment obtained at 45°C. However, irradiation at temperatures above this

(i.e., 55° and 67°C) showed a gradual decrease in the detectable amount of hydrogen peroxide. A bar graph showing the effect of temperature on the polyethylene glycol ointments is shown in Figure 2, page 57*

The increase in hydrogen peroxide production going from 0°C through 45°C shows a gradual increase. Beginning with 55°C there is a gradual reduction in the amount of hydrogen peroxide produced. Never­ theless, the amount obtained is greater than at 0°C. It could be that at these high temperatures, greater amounts of hydrogen peroxide are produced but at the same time the decomposition seems to be proceeding also at a higher rate. 73

From the results obtained in this study, it seems that high temperatures of at least 55°C are necessary for greasy bases to melt and increase the production of hydrogen peroxide. Ointments made with aquaphor and with hydrous wool fat and irradiated at 55°C did not show a significant increase in peroxide production.

It should also be noticed that the presence of sodium formate in the sample enhances the preservation of hydrogen peroxide. This will be clearly seen by comparing the amount of hydrogen peroxide produced by

2 Qn. of ointment containing polyethylene glycol ointment and distilled water at room temperature (see Table VI, page 45) with that produced by the same amount of the same ointment under the same conditions but using sodium fomate solution instead of distilled water (see Table XX, page 54)• These amounts are 1,003 micrograms and 2,054 micrograms respectively.

PART IV

Oxidation of Cholesterol

Cholesterol is a natural product widely distributed in the animal kingdom. It is by far the oldest recognized and also the most important member of the sterol group. Cholesterol is present in most animal cells in varying concentrations. The high proportion of choles­ terol in the skin is of special interest because it presumably is a precursor of 7-dehydrocholesterol which is provitamin D^. It is believed that the activation of this precursor to vitamin D3 takes place at or near the surface of the skin when it is irradiated by s

CH( CH3 )- (CH2 ) y CH( CH^ )2

CH-

Cholesterol Cholestenone OH

CH( CH3 )- ( CHg )3-CH( CH3 )2

Activated Vitamin D3 with Open 7-dehydrocholesterol Ring and Oxidized (Vitamin D3) 0 OH Group

Figure 3» Chemical Formulas 75

WAVl NUM M tS IN C M ' WAV'! W U | | » i IN C M1 sooc 4000 jcoc jvoc zooo ivoo uoo noo i ?oo noo tooo *00 100 100

10

40

70

07 I 7 I 19 II 12 14 It 14 WAYt UNOTH

Cholesterol

Irradiated Cholesterol

Figure 4* Infrared Spectrographs 76

i t * 11 11 IS It WAV* tlNOTH

"Oxidized" Cholesterol

Ketonic Substance Obtained From "Oxidized" Cholesterol

Figure 5» Infrared Spectrographs 77

sunlight or other sources of ultraviolet light. Lanolin is an important

source of cholesterol.

From the formula for cholesterol in Figure 3, page 74, it will

be noted that the hydroxyl group is in position 3 of ring A.

Reference to Table XXVI, page 60, shows that cholesterol

reacts with hydrogen peroxide solution since there is a difference

between the amount of hydrogen peroxide retained by the samples.and that retained by the controls.

The infrared spectrograph (see Figure 5, page 76 ) of the product obtained by irradiating cholesterol, dissolved in chloroform, with zinc oxide and water for one hour at room temperature, shows that

seme oxidation of cholesterol occurs. The infrared spectrograph of cholesterol may be 3een in Figure 4, page 75 •

The characteristic bands of infrared absorption due to the presence of a secondary alcohol group in cholesterol may be noticed between 1,375-1,445 cnT1, 1,345-1,260 cm"1, and 1,160-1,055 cm"1 (32).

These peaks of absorption are clearly noticed in the spectrograph for cholesterol which is shown in Figure 4, page 75• It should be noticed also that the irradiation of cholesterol does not produce any change in these maxima of absorption since the spectrographs for cholesterol and irradiated cholesterol are practically the same. Therefore, it may be assumed that ultraviolet light alone does not cause any oxida­ tion of the sterol molecule.

On the other hand, when cholesterol is irradiated in the presence of zinc oxide and water, there is evidence of seme oxidation 78

occurring in the sterol molecule. Figure 5, on page 76, shows the

infrared spectrograph of the "oxidized" cholesterol. This spectrograph

shows a combination of absorption bands due to the non-oxidized and the

oxidized forns of cholesterol. The band of maximum absorption shown

between 1,700 cm“^ and 1,800 cm“^ is characteristic for ketones (32).

The small peak of absorption between 1,260 cm”^ and 1,300 cm”^ is also

characteristic of ketones. Ketosteroids are most effectively charac­

terized by the infrared absorption band between 1,650 cm~T and 1,800 cm“-*-

associated with the carbonyl stretching vibration (53)«

Upon oxidation, cholesterol gives various ketones, hydroxy

compounds, and acids, the products depending upon the oxidating agents

and conditions used (44). Wintersteiner and Bergstrom (54) isolated

7-ketocholesterol and 7-hydroxycholesterol from cholesterol which had

been aerated at 90°C. The chief reaction product is 7~ketocholesterol.

On mild oxidation with cupric oxide, cholesterol is converted

to the ketone, cholestenone, in which the double bond shifts from the

5:6 to the 4:5 position. Aluminum tertbutoxide and acetone, and chloro- magnesium ethoxide and acetone are also very satisfactory for the oxi­

dation to cholestenone (55). The chemical formula of cholestenone

appears in Figure 3, page 74.

From the results obtained in these series of experiments, it

can be concluded that cholesterol is oxidized to a certain extent under the conditions of this research. The infrared absorption spectrograph

of the "oxidized” cholesterol presents sufficient evidence to indicate that an oxidation has occurred. Further proof of this is the fact that 79 the “oxidized*’ cholesterol gives a positive hydroxylamine hydrochloride test (32)* This is a qualitative test for ketones ir which the carbonyl group reacts with hydroxy lamine hydrochloride liberating hydrochloric acid which changes the color of the solution. The oxime produced is not sufficiently basic to produce a hydrochloride.

1*20=0 / EjN-OH • HC1 — --- A R ^ N - O H / HC1 / H20

The lowering of the melting point from 148°G in cholesterol to 141-2°C in “oxidized1* cholesterol also suggests that a change in the molecule has taken place.

Isolation of the Ketone

The use of Girard reagent (56) for the isolation of ketoster- oids is well known,, This reagent is trimethylaminoacethydrazide hydro­ chloride and i3 used for the separation of ketones from non-ketonic material. It is prepared from ethyl chloroacetate, trimethylamine and hydrazine hydrate in ethanol. Ketones react with Girard reagent to produce compounds which are soluble in water and are known as hydrazones. 1 , EtOH / HAc _ 0=0 / t (CHo )oNCHoCONHNHo_/Cl- ■■ /_C=N-KHC0CH2-N(CH3 )o_/Cl P 1 2S04 / h2o »

This reagent is specific for ketones. The product obtained by treating ’’oxidized" cholesterol with Girard reagent T appears yellow at first but after drying in a vacuum it has a brown color and a resinous appearance.

The infrared spectrograph of the ketone is shown in Figure 5» page 76. Here again it is noticed that there is a band of maximum 80 absorption extending from 1,800 cm“^ to 1,550 cm”'*" which is character­ istic of ketones (32). However, although the spectrographs were repeated a few times using different samples, the resolution was never completely good. This fact indicates that it is not a single 'compound but a mix­ ture of different ketones. Absorption bands corresponding to hydroxyl groups (approximately at 1,365 cm“^ and 1,445 cm--*-) can also be seen in the spectrograph. This suggests the possibility of the existence of a compound having both functions, a keto group and an alcohol group.

The ketone gave a positive hydroxylamine hydrochloride test­ and also a 2,4-dinitrophenylhydrazone test (32). The latter also reacts with ketones to form hydrazones: t t C6 r-I3(N02 )2NHNH2 / P=0 ------>■ C6H3(N02 )2NHN=£ / h2 o

However, the range in the melting point of the 2,4-dinitro­ phenylhydrazone obtained from the ketone is so great that it definitely indicates that the compound is not pure.

Infrared analysis cannot be used to determine if the sterol molecule suffers any opening of the rings. Provitamin D^, 7-dehydro- cholesterol, upon irradiation with ultraviolet light produces vitamin D3 . This change from the provitamin to vitamin D3 includes also opening of ring B in 7-dehydrocholesterol producing a third double bond in vitamin D3 (see Figure 3, page 74). There is a possibility, therefore, that a phenomenon similar to this may occur with cholesterol producing a compound with a structure similar to the keto form of vitamin D3 as shown in Figure 3> page 74* 81

PART V

Hydroxyl Value of Hydrous Wool Fat

Wool fat is the fatty substance obtained from the wool of sheep. The content of free cholesterol in wool fat is reported as

1 per cent (41)» lanolin is analogous in composition to the human sebum (14)»

The acetyl value of a fat or fat like substance is defined as the number of milligrams of potassium hydroxide required for the neutralization of the acetic acid obtained on saponifying one gram of acetylated fat or wax.

The determination of the acetyl value is based on the prin­ ciple that hydroxyl groups in the fat will assimilate, on heating with acetic anhydride, acetyl groups according to the number of hydroxyl groups per molecule of compound. The chemical change that occurs consists in the replacing of the hydrogen at cm of the alcoholic hydrox­ yl group or groups by the radical of acetic acid. Therefore, the determination of the acetyl value indicates the amount of hydroxyl groups present in a sample.

The hydroxyl number of a wax is similar to the acetyl number, except that it corresponds to the acetyl value based on the original amount of wax taken instead of being based on the amount of the acety­ lated wax; consequently, the hydroxyl number is a trifle higher than the acetyl value.

The reported acetyl number of wool fat is 23*3 (41)» The values indicated in Table XXVII, page 64, demonstrate that there is a 82 reduction in the hydroxyl value of hydrous wool fat when it is treated with hydrogen peroxide under conditions of this research. A decrease in the hydroxyl number suggests oxidation of the free hydroxyl groups in hydrous wool fat.

The hydroxyl number of 19*4 Tor hydrous wool fat as reported in Table XXVII, page 64, is lower than the acetyl value of 23.3 reported in the literature (41)« This value reported here is based on the weight of hydrous wool fat and the 23.3 value is based on anhydrous wool fat.

The results obtained here substantiate the evidence that the cholesterol present in bases reacts with the hydrogen peroxide produced by photosensitized zinc oxide and water. This accounts for the low values for hydrogen peroxide found in ointments containing aquaphor and hydrous wool fat (see Table VII, page 46, and Table XXV, page 58).

PART VI

Pharmacology

There was no observed effect of the ointments containing cholesterol in the treatment of irritation in the eyes of rabbits.

Since the amount of the oxidized product produced in ointment bases by irradiation is estimated to be very low, an ointment containing a rather high percentage (13 per cent) of ’'oxidized” cholesterol and one con­ taining 5 per cent ’’oxidized” cholesterol were prepared. The irritated condition of the rabbit’s eyes did not show any improvement 12 hours after these ointments were applied. The same results were obtained with an ointment containing pure cholesterol and with white petrolatum alone. 83

White petrolatum is used extensively as a base for ophthalmic ointments, but because of its greasy nature, it was thought that proba­ bly this base would not promote the dispersion of the oxidized choles­ terol at an effective level. Therefore, polyethylene glycol ointment was used instead of white petrolatum in another series of experiments with ’’oxidized" cholesterol. However, not even this type of water soluble ointment base produced any improvement in the eyes of the rabbits 12 hours after the medication was applied.

In order to test this method for the treatment of irritations, a known anti-inflamatory commercial ointment (Cortef Acetate - 1 per cent, Upjohn Company) was tried on the rabbits. Six hours after medica­ tion with this commercial ointment, the irritation had subsided. Since no anti-inflamatory properties could be shown to be associated with the

"oxidized" cholesterol, it appears as if the principal change in the cholesterol molecule is the oxidation of the sterol group. Further testing must be done in order to determine if other changes in the cholesterol molecule occur as a result of oxidation and irradiation. 84

CONCLUSIONS

A considerate interpretation of the data obtained during the course of this investigation led to the following conclusions:

1. The official formula for Zinc Oxide Ointment does not produce hydrogen peroxide at room temperature upon irradiation with ultraviolet light.

2. A temperature of at least 55°C is necessary to produce an appreciable amount of hydrogen peroxide in the above ointment.

3. Sodium formate stabilizes the hydrogen peroxide that is formed.

4* Zinc oxide incorporated into hydrophilic ointment and poly­ ethylene glycol ointment produces a higher measurable amount of hydrogen peroxide than official Zinc Oxide Ointment.

5. There is an increase in the hydrogen peroxide production of zinc oxide with polyethylene glycol ointment when the temperature is increased from 0°C to 45°C.

6. The official Zinc Oxide Ointment does not produce any hydrogen peroxide even after four hours of irradiation with ultraviolet light at room temperature. However, an ointment containing polyethylene glycol ointment does show an increase in production of hydrogen peroxide with an increase in time of irradiation. The increase is more significant when going from 30 minutes to 120 minutes than when going from 120 minutes to 240 minutes.

7. Hydrogen peroxide is produced by irradiation of zinc oxide in aquaphor and in hydrous wool fat. This peroxide is utilized in the oxidation of the cholesterol present in the base3. The slight amount of measurable peroxide found when these products were irradiated at 55°C is probably excess peroxide.

8. The product obtained by oxidation of cholesterol with hydrogen peroxide seems to be a mixture of ketonic substances which were not identified. 86

CONTEMPLATED FUTURE RESEARCH

No attempt was made in this investigation to correlate the amount of zinc oxide present in the ointment with the hydrogen peroxide produced.

Although there is some indication that increased peroxide production can be obtained by increasing the quantity of zinc oxide in lotions (6) nothing definite has been done in this respect in relation to ointments.

If the production of hydrogen peroxide is related to the therapeutic efficiency of Zinc Cbd.de Ointment, then it may be possible to formulate a better zinc oxide ointment.

The most interesting finding of this investigation was the fact that cholesterol present in ointment bases reacts with the hydrogen peroxide. This suggests that since all vegetable and animal lipids contain sterols and since a variety of oils have been used in formula­ tions containing zinc oxide, studies be performed to determine the effect peroxide formation would have on the sterols contained in these lipids.

Since the pharmacological action of zinc oxide is not clear, it is believed that studies could be undertaken to determine if the sterols in the skin are affected in a similar manner as the cholesterol in the base is affected.

There are many propietary ointments in the market containing zinc oxide. A comprehensive study of these preparations, including hydrogen peroxide production, effect of other substances present, and probably bacteriostatic action, would be very informative. 87

BIBLIOGRAPHY-

1. The Pharmacopeia of the United States of America, Fifteenth Revision; Mack Publishing Co., Easton, Pa. (1955)*

2. The National Formulary, Tenth Edition; J. B. LLppincott Co., Philadelphia, Pa. (1955)-

3. Reese, D. R., ’’Formation of Mydrogen Peroxide in Calamine Lotions,” M. S. thesis, The Ohio State University (1953)*

4. Mathias, M. C., "The Effect of Various Phenols on the Formation of Hydrogen Peroxide by Zinc Cbcide,” M. S. thesis, The Ohio State University (1954).

5. Minardi, F. C., "The Effect of Time on the Hydrogen Peroxide Production by Calamine Lotions," M. S. thesis, The Ohio State University (1954)*

6. Blubaugh, F. C., "A General Study of the Effects of Lipids Upon the Quantity of Peroxides Produced by Photosensitized Zinc Oxide," Ph. D. dissertation, The Ohio State University (1955)*

7. Young, J. C., "The Effect of Selected Phenols on the Formation of Hydrogen Peroxide by Calamine Liniment N. F. IX," M. S. thesis, The Ohio State University (1955)*

8. Feitknecht, W., and Haberli, E., Helv. Chim. Acta 33:922 (1950).

9. Outcalt, H. E., Zinc Oxide Can. Chem. and Process Inds., 27:118 (1943)*

10. Pharmacopeia of the United States, First Edition, Wells and Lilly, Boston, Mass. (1820).

11. Ibid., Fifth Revision, J. B. Iippincott Co., Philadelphia, Pa. (1876).

12. British Pharmacopeia, Spottis Woode and Co., Gracechurch St., London (I885).

13. The Merck Manual, Ninth Edition, Merck & Co., Inc., New York (1956).

14. Osol, A., and Farrar, G. E., The Dispensatory of the United States of America, 25th Edition, J. B. Iippincott Co., Philadelphia, Pa. (1955). 88

15. Baur, E., and Neuweiler, C., Helv. Chim. Acta, 10:901 (1927).

16. Goodeve, C. F., Trans. Faraday Soc., 33:340 (1937).

17. Winter, G., Nature, 163:326 (1949).

18. Rollefson, G. K., and Burton, M., Photochemistry and the Mechanisms of Chemical Reactions, Prentice-Hall, Inc., New York (I946), p. 386.

19. Rubin, T. R., Calvert, J. G., Rankin, G. T., and Ma Nevin, W., J. Am. Chem. Soc., 75:2850 (1953).

20. Chari, C. N., and Qureshi, M., "Formation of Rydrogen Peroxide From Water. I. In Presence of Zinc Oxide," J. Indian Chem. Soc., 21:97, 297 (1944).

21. Smith, D. F.,.and Hawk, C. 0., "The Catalytic Decomposition of Methanol," J. Phys. Chem. 32:415 (1928).

22. Schleede, A., Ritcher, M., and Schmidt, W., Z. Anorg. allgem. Chem. 223:49 (1935).

23. Huttig, G., and Feher, I., Z. Anorg. allgem. Chem. 231:249 (1937).

24. Garn, P. D., "The Catalytic Activity of Zinc Oxide in the Fhotochemical Formation of Hydrogen Peroxide," dissertation, Ph. D., The Ohio State University (1952).

25. Yamahuzi, K., Nisioeda, N., and Imagawa, H., Biochem. Z., 301:404 (1939). 26. Yamahuzi, K., Nisioeda, N., and Ryusi, R., Biochem. Z., 303:260 (1939).

27. Koller, L. R., Ultraviolet Radiation, John Wiley and Sons, Inc., New York (1952).

28. Luckiesh, M., Applications of Germicidal, Erytheraal and Infrared Energy, D. Van Nostrand Co., Inc., New York (1946) p. 15*

29. Pamphlet, General Electric Engr. Division, Lamp Dept., Cleveland 12, Ohio.

30. Daniels, F., Outlines of Physical Chemistry, 8th Edition, John Wiley and Sons, Inc., New York (1948) p« 593-6.

31. Willard, H. H., Merritt, L. L., and Dean, J. A., Instrumental Methods of Analysis, Second Edition, D. Van Nostrand Co., Inc., New York (1951) PP* 51-53* 89

32. Shriner, R. L., Fuson, R. C., and Curtin, D. Y., The Systematic Identification of Organic Compounds, Fourth Edition, John Wiley and Sons, Inc., New York (I956).

33* Wilson, C. 0., and Gisvold, 0., Textbook of Organic Medicinal and Pharmaceutical Chemistry, Third Edition, J. B. Iippincott Co., Philadelphia (1956) p. 64.

34* The Pharmacopeia of the United States, Thirteenth Revision, Mac Publishing Co., Easton, Pa. (1947)*

35* Ibid., Fourteenth Revision, Mac Publishing Co., Easton, Pa. (1950).

36. Barr, M., Grim, W. M., and Tice, L. F., "An Improved Formula for Hydrophilic Ointment," J. A. Ph. A., Pract. ed. 15:758 (1954)*

37. Meyers, D. B., Nadkarni, M. V., and Zopf, L. C., "Formulation and Practical Applications of Carbowax Vehicles," J. A. Ph. A., Pract. ed. 11:32 (1950).

38. Strauss, M. J., Arch. Dermat. Syph., 61:420 (1950).

39. Sulzberger, M. B., and Baer, R. L., Yearbook of Dermatology and Syphilology 1953-54, p. 19*

40. Martin, E. W., and Cook, E. F., Remington*s Practice of Pharmacy, Eleventh Edition, The Mack Publishing Co., Easton, Pa. (1956).

41. Deuel, H. J., The lipids, Vol. I; Interscience Publishers, Inc., New York (1951) p. 368-370.

42. Johnston, G. W., and Lee, C. 0., "A Radioactive Method for Testing Absorption from Ointment Bases," J. A. Ph. A. 32:278 (1943).

43. Sulzberger, M. B., Warshaw, T., and Herrmann, F., J. Invest. Dermat. 20:33 (1953).

44. West, E. S., and Todd, W. R., Textbook of Biochemistry, The Macmillan Co., New York (1955).

45. Pamphlet, "Hydrogen Peroxide," (1951), Pennsylvania Salt Manuf. Co., 1000 Widener Bldg., Philadelphia 7, Pa.

46. Horkheimer, P., "Stability of Hydrogen Peroxide Solutions," Pharm. Ztg. 80:507 (1935). 90

47* Kiss. A., and Lsderer, R., "The Mechanism of the Catalytic Decomposition of Hydrogen Peroxide Solutions by Metal Ions," Rec. Trav. Chon. 46:453 (1927)*

48. Heinemann, P. G., J. A. M. A. 60:1603 (1913)*

49* Kavanagh, F., J. Bact. 54:761 (1947).

50. Kolthoff, I. M., "Iodcmetric Method for Hydrogen Peroxide," 2. Anal. Chem. 60:400 (1921).

51. Chemical, Clinico-Chemical Reactions, Tests, and Reagents, The Merck Index, Fifth Edition, Rahway, New Jersey.

52. Lewkowitsch, J., Chemical Technology and Analysis of Oils, Fats, and Waxes, Sixth Edition, Vol. 1, The Macmillan Co., Ltd., St. Martin’s Street, London (1921) p. 437*

53* Jones, R. N., Herling, F., and Katzenellenbogen, J. Am. Chon. Soc., 77:651-6 (1955).

54* Wintersteiner, 0., and Bergstrom, S., J. Biol. Chem. 137:785 (1941).

55. Newer Methods of Preparative Organic Chemistry, Interscience Publishers, Inc., New York (1948) p. 146-150.

56. Girard, A., and Sandulesco, G., Helv. Chim. Acta. 19:1095 (1936).

57. Handbook of Chemistry and Physics, 36th Edition, Chemical Rubber Publ. Co., Cleveland, Ohio (1954). 91

AUTOBIOGRAPHY

I, Hector Antonio Lozada, was born in Caguas, Puerto Rico, July 13,

1926. I received ray secondary school education in the public schools of Puerto Rico, and graduated from Caguas High School in May, 1945* My undergraduate training was obtained at the University of Puerto Rico,

College of Pharmacy, from which I received the degree Bachelor of

Science in Pharmacy in 1949* In that same year I entered the Philadel­ phia College of Pharmacy and Science, Philadelphia, Pennsylvania, and received the degree Master of Science in Pharmacy in 1950. I returned to Puerto Rico in 1950 and was appointed Instructor of Pharmacy at the

University of Puerto Rico, College of Pharmacy. In 1955, I signed a contract for a permanent position at the University of Puerto Rico and at the same time was promoted to Assistant Professor of Pharmacy. I was married in August, 1956. That same year I received a sabbatical leave from the University of Puerto Rico to come to The Ohio State

University and continue studies for the degree Doctor of Philosophy.

In 1957, I received a renewal of my scholarship from the University of

Puerto Rico until completion of the requirements for the degree Doctor of Philosophy at The Ohio State University.