I H ‘\L I‘M} 11! | H H. ‘1 U ? f I ‘ 1 L | m H I \. H is I H u I i i II I‘ ‘ ‘I h V,-#._, — ‘ } w THE RELATIVE RATES OF REACTEONS ".N THE ULTRAVIOLET {RRADIATION OF ERCOSTEROL AT THE \‘fi’AV’ELENGTI—JS 254nm AND 365nm Thesis for the Degree of M. S. E‘ITCHIGAN STATE COLLEGE Mary Louise Dodge 1943 “This is to certify that the ; . ML momma“ .3 {ecu/hm ‘Jw Ute WM 32$“ 3 3x. LMMIL ‘ 2— S 4 WA presented by. has been accepted towards fulfilment of the requirements for MS; degree in We!) thug ‘ A/ M Major professor Date M 3" 43 TEE RELATIVE RATES OF REACTIONS IN THE ULTRA-FIGHT IRRADIATION 0F ERGOSTEROL AT THE WAVELENGTHS 254nm AND 365 an by my Louise Dodge A THESIS Submitted to the Graduate School of Michigan State College of Agriculture and Applied Science in partial fulfilment of the requirements for the degree or MASTER OF SCIENCE Department of Chemistry 1943 APPRECIATION The writer wishes to eXpreee her appreciation to Dr. D. T. Ewing for his helpful suggestions and counsel during the course of the studies undertaken. I. Introduction The ultraviolet irradiation of cholesterol and ergosterol has been studied very extensively with the primary purpose of finding the Optimum conditions for the production of Vitamin D. From numerous investigations it is generally concluded that the wavelengths which affect ergosterol in ultraviolet irradiation are those which are absorbed from 305 mu to 230 mu. Conflicting results have been reported as to the efficiency of the formation of Vitamin D by any of the wavelengths within the region of the absorption of ergo- sterol. A.study of the effectiveness of different wave- lengths as to their power to produce Vitamin D was made by Knudsen and Benford (l). The wavelengths studied and found effective were 2655, 2804, 2894, 2967, 3024, and 3128 A9. The most effective activation of ergosterol was obtained from light of the wavelength 2804 L°, which is the line that shows maximum absorption of ergosterol. Line 3128 A9 was least effective. 0n the other hand, Ken, Daniels, and Steenbock (2) found that the quantum efficiency was constant for_radiations at the 256, 265, 280, and 293 mu lines. This conclusion was based on the assumption that Vitamin D is the only product formed from ergosterol upon irradiation with monochromatic light. It is known, however, that the irradiation of ergosterol -1- produces several substances either in series and/or in parallel. Reerink.and van Wijk (3) found that irradiation with light of wavelength greater than 275 mu gives rise to a series of reaction products which deffers from that obtained by irradiation with light of wavelength 254mu. This was concluded from the fact that the changes in the absorption spectrum are different in the two cases. During the course of the irradiation of ergosterol several substances are formed before the Vitamin D is obtained and the latter is not stable to the activating energy but is transformed intocmher compounds (4). The following scheme, which is based primarily on the studies by Windaus and his co-workers, is given.for the reaction mechanism (5}. Ergosterol Lumisterol2 Pro-Tachysterolz Tachysterclz Vitamin D2 (Calciferol) Suprasterolz 1 Toxisterolz (Substance 248) Suprasterolz II. The photochemical process is irreversible; there is no equilibrium between the irradiation products. According to work done by Human and Steenbock (6), the upper limit of the ultraviolet zone capable of synthesizing Vitamin D lies between 303 and 515 mu. Ergosterol exhibits a little absorption power beyond this limit and Bills (7) states that there is reason to believe that still longer wavelengths play a part in the decom- position of ergosterol and/ or of its primary irradiation products. Studies by nmmerous other investigators indicate that the formation of products other than Vitamin D might be prevented by eliminating or filtering out the shorter wavelengths in the total Spectrum developed by the quartz' mercury vapor lamp. (8) From the above considerations, it would seem reason- able to conclude that irradiation of ergosterol at wave- lengths in the range 275 to 503 mu would be most effective in producing Vitamin D with the smallest amount of by- 2 products, that irradiation at shorter wavelengths (254 mu) would produce more of irradiation products other than Vitamin D and less of the Vitamin itself, and that irradiation of ergosterol at longer wavelengths ‘365 mu) might result in decomposition of the ergosterol but probably would not be at all effective in forming Vitamin D. With these considerations in mind, the present study of the irradiation of ergosterol was carried out at the wavelengths 254 mu and 565 mu. Ergosterol can be activated in the dry state, in vapor form, and in solutions. irradiation of ergosterol in the dry state is not very satisfactory because the Vitamin D is produced only on the surfaces of the crystals -3- and further irradiation destroys the Vitamin D formed before the middle of the crystal has been activated. Irradiation in the vapor phase has not been thoroughly investigated. The best method of irradiation is to expose solutions of ergosterol to the action of the ultra- violet light. The presence of dissolved oxygen in the solvent affects the absorption spectrum picture of actio vation to a considerable extent. ‘9) This is due, how- ever, to the altering of the by-products of the irradia- tion rather than to any affecting of the formation or destruction of Vitamin D. Bills, Honewell, and Cox th) took no special precautions to exclude traces of oxygen during irradiation in their study of the Specific solvent effect on activation. Activation curves of ergosterol irradiated in alcohol, cyclohexane, and other had the same general shape, but other permitted the greatest activation. The irradiation of ergosterol in this investigation was carried out in a peroxide-free other solution, but no attempts were made to carry out the procedure in an oxygen-free atmosphere. However, the solution being irradiated was in a cell completely filled and not in contact with the atmOSphere during the irradiation period. II. Experimental The apparatus used inthis study was a Bausch and Lamb ultraviolet sector photometer equipped with a Bausch and Lomb medium quartz optical system. A.Hilger hydrOgen discharge tube Operating at a potential of 5,000 volts was used as the source of the continuous ultraviolet spectrum. One-fourth centimeter quartz cells enclosed in metal cases held the solvent and the solution. The ergosterol solutions were irradiated by a Hanovia quartz mercury vapor lamp. Since no monochromator was available at the time this study was carried out, a system of filters had to be used to obtain the wavelengths desired for irradiation. Corning Filter No. 597, thickness 4.85 mm., was placed between the mercury vapor lamp and the ergosterol solution, so that irradiation could be carried out at wavelength 565 mu. This filter transmits, approximately, 0% of the mercury lines below 515 mu, 5% of the 315 mu line, 51% of the 554 mu.line, 80-85% of the 365-6 mu lines, 10-15% of the 405-8 mu lines, and 0% of the lines above 408 mu. A mixture of chlorine gas and bromine vapor contained in a five centimeter quartz cell enclosed in a metal case, filtered out all lines in the range 260-590 mu. The materials used were Eastman absolute alcohol and Eastman C. P. grade of ethyl ether for solvents; Eastman # 40, Anti-helation photographic plates; Eastman develOper p.11. The ergosterol used was sample # 95752 supplied by Parke Davis Company of Detroit, Michigan. Before each run of a series of irradiations, the ether was treated with ferrous sulfate, dried, and redis- tilled in order to insure‘fresh solutions of ergosterol in peroxide-file ether. All solutions used were of a concentration of 0.01024%. The concentration is expressed in terms of grams of solute per l00 cubic centimeters of solvent. The procedure may be outlined as follows: The solution of the ergosterol in absolute alcohol‘or in peroxide-free ether was made up to the desired concentra- tion and the one-fourth centimeter quartz cells were filled, one with solvent and the other with the ergosterol solution. The cell containing the solution was placed in contact with the filter which in turn was placed at the cpening to the quartz mercury vapor lamp. Irradiation was carried out for the desired length of time and then the cell containing the solution and the cell containing the solvent were used in the sector photometer in order to obtain the absorption Spectra of the irradiated solu- tion. Two plates yhowing the spectrum of the irradiated solution were made after each particular time of irradia- tion. One plate was made with the correct sector settings to give the general absorption Spectrum.of the solution (survey plate), and the second plate was made so as to show fine structure at the maximum points of absorption (structure plate). The plates were developed in D-ll developer for five minutes, fixed in hypo for fifteen minutes, and washed in running water thirty minutes before drying. Four series of irradiations of ergosterol were made. The first series was made on a solution of ergosterol in absolute alcohol. During irradiation no filter was used for this series; the entire epectrum of the mercury vapor lamp was utilized. Plates were made showing the absorp- tion spectrum of ergosterol in absolute alcohol irradiated for 0, 5, 10, 15, 20, 25, 55, and 60 minutes. A.second' series was made on a solution of ergosterol in peroxide- free other. No filter was used during the irradiation of this series. Plates were made showing the absorption spectrum of the ergosterol in peroxide-free ether irrad- iated for 0, 5, 10, 15, 20, 25, and 50 minutes. In the third series, a solution of ergosterol in peroxide-free ither was again.used. Corning Filter # 597 was used to give irradiation at wavelength 565 mu. Irradiations were carried out at intervals of 0, 50, 60, and 120 min- utes. The fourth series was made on a solution of ergos- terol in peroxide-free ether, using the chlorine- bromine filter for irradiation at wavelength 254 mu. The time -7- intervals for irradiation were 0, 5, 15, 50, 60, 90, 120, and 150 minutes.‘ 111. Results The next four pages show graphic figures of the results obtained. The graphs were drawn by plotting extinction values against wavelengths. The extinction values were calculated according to Beer's relationship: 1 108 10/1 Blggm ‘ cd Where 10 is equal to the incident light 6100 percent). 1 equals the percent of light transmitted. o equals the percent concentration of the solution. d equals the length of the cell in centimeters. Following is a detailed explanation of the figures shown. Figure 1. Series I. The ultraviolet irradiation of ergosterol in an absolute alcohol solution of concentration 0.01024%. No filter was used during the irradiation.of this series. Gr ph. Plate Number Time 2£_Irradiation A. 51 . ' 0 minutes 3 55 5 minutes 0 58 10 minutes D 40 15 minutes E 55 20 minutes I 45 25 minutes G 44 55 minutes 20-402-4xm - A ‘3 J’ a . I R '\ A AU!“ ' H ' \K“\ _x \q w 1* I \ e: 1' __ “pg“ L «as» n—W—fi . a... l I ‘____5' [E I T 1 f Y [c [-1 T T T I [TD 1 i f\\ _ / ._.' ”VERY '__i M- \ ~ i : ' e a: - nfiL—n—W—Wa—l “Wt-w :- an I 7; e T i i I I I i I Tr ¢ '\ ; f A g ' A/"l ? l in. / \ki ‘ ' T f _1 ' K\ j , \ s * / \ _ / _ j/ \i / \ WAVELENGTH F'GJ. Irradiation of Erqes‘terd in &\CO\|\ 30‘0“.“ WW“ onffihvfd Moreen; up" how. {I 20-402-4xm a] ’1 f/ " \k : // an J \ ,7 fir alas—W all I, \ / \ a. / \ / \ _. \ x” \ a. // ‘\ ... // \ Mill—$7.4 willingness ’ ‘\ ___. J K ; /// \ I- x/ \ .. // \ J/ \ £1.44th «We; / N / \ ”MW h WA v E L E N 61‘ H F'G. Z Irrahahou of En; ester“ \n ether Sowtun with uufflteved maroon; vapor \lnp 20-402-4xm WAVELENGTH F\6.3 Irradiation of [matted ‘m other behfieu a? umn‘flx‘th 55 5m» (.2 B K. i “x * U ' k '3‘- lb I“ (I p ‘ +~ ii ; is f“: -i __ . ' RM \ :r 9 will V \ 1 ,+—~ - “a- Li , - \l A u—-' —sl—~_f_ ”—14 as f. _ f—ss ‘WfiL—TLT—A. ,. rn zo-4nz-4x I! 4'. L 6 - I\\ R i \ /\ L j// \ - / \ \\ ~ \ WAVELENGTH \ in dhev ‘ ‘. f que‘teve “9’ ‘* 1"r'.‘?::*:..“.:....a... 15...). to o | Figure 11. Series II. The ultraviolet irradiation of ergosterol in a peroxide-free other solution of concentration 0.01024%. No filter was used during the irradiation of this series. Graph. Plate Number Time 2£_Irradiation A 48 0 minutes B 49 5 minutes 6 50 10 minutes D 51 15 minutes I 52 20 minut e s P 55 25 minutes G 54 50 minutes Figure 111. Series III. The ultraviolet irradiation of ergosterol in a peroxide-free other solution of concentration 0.01024%. Corning Filter #59? was used to give irradiatione at wavelength 565 mu. Graph. Plate Number_ Time 2; irradiation A. 59 0 minutes B 60 50 minutes 6 62 60 minutes D 65 120 minutes Figure IV. Series IV. The ultraviolet irradiation of ergosterol in a peroxide-free other solution of concentration 0.01024%. A chlorine-bromine filter was used to give irradiation at wavelength 254 mu. Graph Plate Number Time pf Irradiation a. 76 0 minutes B 78 5 minutes 6 80 15 minutes D 82 50 minutes E 85 60 minutes I 8? 90 minutes G 90 120 minutes H' 95 150 minutes IV. Discussion Attempts to characterize the individual products of irradiation and to determine the number of them by interpretation of absorption epeitra have been numerous but rather unsatisfactory. Confusing and conflicting results have been obtained. This can be readily under- stood, for with mixtures of substances having overlap- ping absorption bands, determinations of the position and height of the bands are unreliable. Methods of bioassay and chemical methods of isolation have been the principal means of clarifying results. it is now generally accepted that the absorption maxima for the products of irradiation of ergosterol are as follows; Ergosterol has absorption maxima at wavelengths 295.5, 282, 270, and 260 mu. Lumisterol has absorption maxima at wavelengths 265 and 280 mu. Tachysterol has a principal absorption band at 280 mu.and lesser bands at 268 and 294 mu. Calciferol shows strong ultraviolet absorption with a maximum at 265 mu. Toxisterol has a strong absorption maximum at 248 mu. Suprasterols l and -10- II show general absorption below 250 mu. Morton, Heilbron and Kamm (11) reported that the disappearance of ergosterol irradiated in dilute solutions was related directly to the time. According to Bills t12}, concentrated solutions do not show the linear relationship. He reasons that under no conditions is the time-disappearance curve actually a straight line, for the products of irradiation are numerous and some of them are sufficiently absorptive to act as filters against the remaining unchanged ergosterol. The results from the present study of irradiation at wavelength 254 mu would seem to support the latter view. There is strong evidence that lumisterol (Plate #78, absorption maxima at 270 and 280 mu) and tachy- sterol tPlate #80, absorption maxima at 280, 268, and 294 mu) are formed in a linear relationship to time, but further irradiation seems to give no direct sequence of formation of the other expected irradiation products. From Plate #90 it seems that calciferol and suprasterols are present, but that their absorption curves are mod- ified by the presence of lumisterol and] or tachysterol. ”These results indicate the correctness of our assumption that irradiation at the short wavelength 254 mu produces more of the irradiation products other than Vitamin D2 and less of the Vitamin D2 itself. Irradiation at 565 mu, however seems to follow a direct linear relationship to time. The decomposition -11- of ergosterol takes place at a much slower rate at this longer wavelength than at 254 mu or than it does when the entire mercury vapor spectrum is used for irradia- tion. As assumed at the beginning of this study, there is a decomposition of ergosterol when irradiation is carried out at 565 mu, and there is no evidence of the formation of any of the irradiation products of ergos- terol. The latter fact is in agreement with the Opinion that 515 mu.is the upper limit of the range of wavelengths which eill activate ergosterol to Vitamin D2. Irradiation of ergosterol in alcohol and in other solutions, using the entire mercury vapor spectrum for irradiation, gives evidence of the formation of the activation products of ergosterol, but the overlapping of absorption bands of the different products makes it difficult to ascertain just how rapidly and to what extent each of the products is formed. The fact that the absorption maxima are not clear cut would seem to indicate the presence of each of the individual conver- sion products in a mixture. Rider (15) thought such a mixture might be attributable to the effects of radia- tions of different wavelengths in the total Spectrum develOped by the quartz mercury vappr lamp. V. Summary 1. The activation products of ergosterol irradi- ated in the range of wavelengths 275-315 mu are given in a scheme to show the sequence of their formation. 2. The method is given for the irradiation of er- goeterol at the wavelengths 254 mu and 365 mu by the use of filters to produce the monochromatic light desired. 3. Irradiation of ergosterol at wavelength 254 mu produces more of the irradiation products other than Vitamin D2 and less of the Vitamin D2 itself. The dis- appearence of ergosterol is not a direct linear relation- ship to the time of irradiation, but is complicated by secondary reactions due to the presence of the numerous products of irradiation. 4. Irradiation of ergosterol at wavelength 565 mu brings about a general decomposition of ergosterol, but gives no evidence of the formation of any of the known products of activation. The disappearence of ergosterol is a direct linear relationship to the time of irradiation. 5. The general decomposition of ergosterol when irradiated at wavelength 365 mu is a much slower reaction than the formation of activation products from ergosterol irradiated at 254 mu. '1. Acknowledgement the writer is indebted to the Kappa Kappa Gamma Fraternity for a fellowship during the tenure of which the work here reported was carried out. -13- l. 2. 3. 4. 6. 7. 10. ll. 12. 13. Literature Cited Knudsen, A. and F. Benford, J. Biol Chem., 124, 28? t19581- Ken, S. K., F. Daniels and H. Steen book, J. Am. Chem. Soc., 29, 2575 (1928). Reerink, E. H. and A. van Wijk, Biochem. J., fig, 1294 (1929). Bills, C. E. and F. G. Brickwedde, Nature, 121, 452, (19281- Rosenberg, H. R., Chemistry and Physiology of the Vitamins, lntersciencei’ublishers, inc., New 101k, B0 YO. 1942. p. 272‘ Haman, R. W. and H. Steenbbck, 1nd. Eng. Chem., Anal. Ed., g, 291 {1936}. Bills, C. E., Physiol Rev., l2! 1 (1935 - Rider, T. H. et al., J. Am. Med.-Assoc., 106, 452 (19561o Rosenberg, H. 3., Chemistry and Physiology of the Vitamins, lnterscience Publishers, inc., New Xork, N. 1., 1942, p. 569a Bills, 0. E., E. M. Heneywell, and W. E. Cox, J. Biol. Chem., 22, 601 (1951}- Morton, R. A., I. M. Heilbron and E. D. Kamm, J. Chem. Soc., 2000-2005 (1927). 31118, C. E0, PhyBiOI. ROVO’ l2. 1 (1933)“ Rider, T. H. et al., J. Am. med. Assoc., 166, 452 (1956)- . 1— 7'7?— 5‘1 2 I" 2" 1 ’99, J“L ‘\ 4 ‘53 148294 .u'r’T. D645 Dodge cn>4~- {T541 . t . . I . ' u . . x , r‘mll} l'-|'l If»! 0 n D. | . CG Il-tl- I o I . ll-ull‘ I 0 e n. 1 on... - III a nu. .. a an. . .15 I. lulu-.. o s t In no .I.i)-..‘l. lute it u a ...n'.ll 5". it- I III I I I‘.‘l.c-‘I III "I--.- a.» l n v r. 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