igh-Voltage Cathode Rays
Effect of
on Ascorbic Aci IN VITRO AND IN SITU EXPERIMENTS BERNARD E. PROCTOR
AND
JOHN P. O'RIE.4R.4
Massachusetts Znstitute of Technology, Cambridge, .Muss.
Because ascorbic acid (titamin C) is an unstable \itamin, the change in ascorbic acid content of a food is used to indicate the extent to which essential food nutrients are destroyed by processing. As part of a research program to determine whether ionizing radiations have potential use in sterilizing foods, the effects of highvoltage cathode rays on ascorbic acid in orange juice (in situ experiments) and on pure ascorbic acid in 0.25yo oxalic acid (in vitro experiments) were studied. In all cases, loss of ascorbic acid o n irradiation was less in situ than in vitro. Loss of total ascorbic acid was much less than that of I-ascorbic acid. Loss of the latter was negligible when the medium was irradiated in the €rozen state. Dilute solutions of ascorbic acid w-ere more sensitive to irradiation than more concentrated solutions. Chemical and spectrophotonietric data are presented to show that dilretogulonie acid was probably not formed on irradiation.
in this study were calculated CI on1 a consideration of the depths, cross-sectional areas, and densities of the samples The d o ~ a y e s reported represent the aveiage doses absorbed In all experiments, the rate of radiation dosage was the samethat is, a current of 5 pa. a t 3 m . e . v . ,or approximately 1.3 X 106 rep per minute. The dosage. are expiessed a8 roeritgen-equivalents-physical (rep), as described by Evans (9). A Beckman quartz spectrophotometer, Model DU, was used to determine the ultraviolet absorption spectra of ascorbic acid. 3IETHQDS O F Ah-ALYSIS
Reduced and total ascorbic acid v a s determiued by the Hochberg, Melnick, and Oser ( 1 7 )photocolorinictric modification of the Bessey method ($), except that 0.5% oxalic acid was substituted for the metaphosphoric acid (15). Total ascorbic acid !vas also determined by the 2.4-diiiitrophenylhydrazine method of Roe and Oestei,lirig (B), except that oxalic acid was again substituted for the metaphosphoric acid. An Evelyn photoelectric colorimetcr was used for the assays.
KURIBER of reports (12, 13,19,20)have appeared recently on the effects of ionizing radiations on several vitaniins known to be essential for humans. The research covered by these reports was carried out as part of a program to determine if ionizing radiations have potential use as sterilizing agents for foodstuffs, for i t has been shown that x-rays can destroy microorganisms in foods ( 7 , 2 1 ) . One of the most unstable of the vitaniins is ascorbic acid (vitamin C). For this reason many investigators, in studying the effect of processing on foods, have used the change in ascorbic acid content of a food as an index of thc extent t o which the essential nutrients in the food have been destroyed by the processing method. ?\lost of the voluminous data relating to the effect of conventional processing procedures on ascorbic acid is based on determinations of the reduced form of the vitamin, Zascorbic acid, because it is the most sensitive to oxidation and because the method for its assay is simple and rapid. It is now well known that dehydroascorbic acid, the first oxidation product of vitamin C, is also biologically active (Id). Therefor?, a true picture of the effect of processing on ascorbic acid can be obtained only by determining the effect on "total" vitamin C (1ascorbic acid and dehydroascorbic acid) as determined bj- the Hochberg, Melnick, and Oser method ( I ? ' ) This paper presents data obtained on the effects of high voltage cathode rays on both total ascorbic acid and the reduced form when irradiated in situ and in vitro under a variety of conditions.
E x P m m m s r r . - I ~PROCEDURE
For the in situ experiments, the juice of Florida oranges purchased 011 the open markft was expressed with a glass juicer and strained through coarse cheesecloth. -1 su6cient volume of juice was prepared so that all samples in a Eries came from the same batch of juice. For the in vitro experiments, U.8.P. Reference Standard ascorbic acid was prepared in 0.257, oxalic mid for each series O f irradiations. This procedure was adopted t'o minimize the aut,oxidation of the ascorbic acid in the intervals bctween preparation and sampling. Irradiation was carried out on 35-ml. samples dispensed in glass crystallizing dishes of S-em. internal diarncter, which were covered with a single layer of aluminum foil 0.00065-inch in thickness. The dishes n-ere placed directly below and at a distance of 45 cm. from the exit window of the cathode ray tube. All samples were irradiated a t room temperature ( 2 5 " * 2 " C.) unless noted otherwise. The samples were slurried with four parts of 0.5% oxalic acid immediately after irradiation. The slurries were held in a refrigerator prior to analysis, which was done within a few hours. RESULTS A N D DISCUSSION
In Situ Irradiation. The effect of irradiation with dosagcs of 100,000 to 500,000 rep on the I-ascorbic acid content of orange juice is shown in Table I. d s destruct,iori of &ascorbic acid ia not synonymous with loss of total biological act'ivity of the vitamin (I+$),the irradiation n-as repeated on a different batch of juice, and analyses were made for both total ascorbic acid and the reduced form. The results are presented in the upper portion of Table 11. All three methods of analysis were carried out on
EQUIPMENT
An electrostatic generator of the Van de Graaff type ( 2 4 ) vias used as a source of cathode rays. This generalor is capable of producing a continuous supply of monoenergetic electrons in a magnetically focused beam, the cross-sectional density of which is accurately known. As the distribution of ionization energy has been well established (24, 26), the cathode ray dosages used 718
RAYSON ASCORBIC TABLE I. EFFECTOF 3-M.E.V. CATHODE ACIDIN FRESH ORANGE JUICE Sample
No. Confrol 1 2 3 4 5
a
Total Dose, Repa 100;000 200,000 300,000 400,000 500,000
I-Ascorbic Acid Amount Retentix mg./100 A. % 31.0 100 28.9 93.2 25.1 81.0 23.5 75.8 22.0 71.0 21.0 67.7
Roentgen-equivalents-physical.
OF 3-M.E.V. CATHODE RAYSON ASCORBIC TABLE11. EFFECT ACIDAS MEASUREDBY INDOPHENOL AND DINITROPHENYG HYDR-4ZINE
Sample
Total Dosage, Repa
METHODS
l-A~soorbio Total Ascorbic Acid , Acid Dinitro(IndoIndophenylphenol) phenol hydrazine
m1-.
2
200;ooo 500,000
------Mg./100 51.8 44.5 38.4
Control 1 2
200,000 500,000
-Retention, %100 100 85.8 95.5 74 2 89.2
Orange juice Control 1
54.5 52.1 48.6
54 4 50.8 47.5 100 93.5 87.3
U.S.P.ascorbic
acid in 0.2501, oxalic acid Control 11 12 13
119
INDUSTRIAL AND ENGINEERING CHEMISTRY
March ,1951
200.0Qo 500,000 1,000,000
Control 11 200,000 12 500,000 13 1,000,000 Roentgen-equivalents-physical.
--Mg./100 57.2 43.2 28.4 18.4
57.8 50.8 40.0 24.9
d-
--Retention, %100 100 75.5 88.0 49.7 69.2 32.2 43.0
60.5 52.0 36.5 22.0 100 85.9 60.3 36.4
aliquots from the same irradiated sample, a t each dosage level. The destruction of total ascorbic acid was less a t these dosages in comparison with the destruction of the reduced form only. The values obtained by the dinitrophenylhydraaine method are slightly less than those obtained by reduction with hydrogen sulfide and determination of the vitamin in the reduced form. As 2,3-diketogulonic acid, the product resulting from the opening of the lactone ring of dehydroascorbic acid, cannot easily be reduced to I-ascorbic acid, the indophenol method measures only the reduced form and the dehydroascorbic acid present before reduction. Hence it appears unlikely that diketogulonic acid is one of the end products found in the in situ irradiation of ascorbic acid. In Vitro Irradiation. To compare the effects of cathode rays on ascorbic acid in situ and in vitro, U.S.P. ascorbic acid in a stabilizing solution of 0.25y0 oxalic acid was irradiated in the same manner as the orange juice. The initial concentration employed was approximately 55 mg. %, measured as Z-ascorbic acid, in order that the concentration would be the same in each case. The ascorbic acid content was measured by the three methods previously described. The results are shown in the lower portion of Table 11. The destruction of pure ascorbic acid as measured by all three methods was in each instance markedly greater than the destruction of ascorbic acid when orange juice was irradiated. These results are in agreement with results: obtained by other investigators (1,6, IO,86), who have noted that pure compounds are more radiosensitive when natural protective substances have been removed. Also, the values obtained for total ascorbic acid by the dye method are again higher than those obtained by coupling the oxidized forms with 2,4-dinitrophenylhydraaine. This difference is more marked in the in 'vitro case. The irradiation of pure ascorbic acid was repeated several times, and the results were the
same-that is, in the controls the total ascorbic acid content as determined by the coupling reaction was slightly higher than the total dye value, but on irradiation it dropped below that of the dye value. One possible explanation of this result is that the oxalic acid may have been affected by irradiation and its product reacted with the dye to give an erroneously high dye value. To ascertain if this had occurred, the 0.25% oxalic acid solution was irradiated alone with dosages as high as 1,000,000 rep, but the irradiated solution had no measurable effect on the dye value. It appears that whatever effect radiation has on oxalic acid, it does not interfere with the ascorbic acid assay. The cause of the discrepancy between the two methods was not investigated further, but subsequent procedures were conducted to determine if diketogulonic acid appeared in the irradiated solution. According to Morton (18),Z-ascorbic acid has a typical ultraviolet absorption spectrum with a maximum a t 240 to 2 4 2 ~ a t a p H of 3.3 or lower. Herbert et al. (16)reported that the first oxidation product of ascorbic acid (dehydroascorbic acid) shows no trace of selective absorption. However, they found that on mutarotation of dehydroascorbic acid, a moderately intense absorption band appeared a t 295p. The compound absorbing in this region was thought t o be diketogulonie acid. As the analytical results cited in Table I1 appear t o indicate that no diketogulonic acid is found on irradiation of I-ascorbic acid, it was decided to check the accuracy of these results by determining the effect of irradiation on the ultraviolet absorption spectrum of ascorbic acid. A peak a t 295p, coincident with the disappearance of the 242p band, would indicate the production of diketogulonic acid, whereas the lack of a 295p band would tend t o confirm the analytical data. The results (Figure 1) indicate no detectable peak a t 295p for diketogulonic acid. As a further check, the ultraviolet absorption spectra of solutions of pure ascorbic acid a t various stages of oxidation were determined, to ascertain if a band a t 295p was produced. A solution of U.S.P. ascorbic acid (55 mg. %)was made up in 0.25y0
I. CONTROL
2bO
2kO
Z b
$80
h0
WAVELENGTH (mu) Figure 1. Effect of 3-M.E.V. Cathode Rays on Ultraviolet Absorption Spectra of Solutions of Ascorbic Acid (50 Mg. %) i n 0.25% Oxalic Acid
INDUSTRIAL AND ENGINEERING CHEMISTRY
720
oxalic acid and was refluxed at 100" C. for a total of 76.5 hours. Washed air was bubbled through the solution continuously by means of a long, thin glass tube, which ran inside the condenser into the solution. Samples were removed periodically and the absorption spectra determined. The results are shown in Figure 2. A slight peak a t 285 to 295, appeared coincidently with the disappearance of the 245, maximum. This result indicates that under prolonged oxidation of the ascorbic acid a t 100" C., a substance appeared having a maximum absorption a t 2 9 5 ~ . This substance either was not formed a t all during electron irradiation of the ascorbic acid or, if it was formed, was immediately destroyed so as not to be detectable chemically or spectrophotometrically. Effect of Concentration. A-umerous investigators have shown that the percentage destruction of a chemical compound by ionizing radiations is not the same for concentrated and dilute solutions. Instead, the relative effect of irradiation a t a given level on a concentrated solution is much less than on a dilute solution. This is in agreement with the "activated solvent" theory ( d T ) , according to 4 hich the absolute amount of a compound destroyed by a given dosage of irradiation should be independent of concentration. This will hold true only for simple one-stage destruction. In the more complex case, in which the reaction products are also radiosensitive, the linear relationship vr-ill not hold, as Dale has pointed out (6). T o determine whether this effect of concentration exists in ascorbic acid, solutions of various concentrations of the pure vitamin in 0.25% oxalic acid were irradiated u-ith equal dosages of cathode rays. The results, shown in the upper portion of Table 111, are in accordance with other reported work on various vitamins, enzymes, and inorganic chemicals (5, 6, 11, 12, 19, 20, 26).
I n further investigations of the effect of concentrationnamely, that the more dilute the solution the greater is the percentage destruction of a radiosensitive solute-several cans of commercially frozen orange juice concentrate were purchased and the contents thawed and mixed. One portion of the concen-
230
240
250
260
270
280
290 300 310
WAVELENGTH (mu)
Figure 2. Ultraviolet Absorption Spectra of Solutions of Ascorbic Acid (50 Mg. yo in 0.25qo Oxalic Acid) at Various Stages of Oxidation
Vol. 43, No. 3
TABLE 111. EFFECT O F 3-M.E.V. CATHODE RAYSON &ASCORBIC A C I D A T VARIOUS C O N C E N T R A T I O N S
Sample U.S.P. ascorbic acid in 0.25% oxalic acid A (control) A- 1
Total Dosage, Repa
I-ilscorbic Acid Retention, Mg./100 nil. 76
245 245 23 1
100 100 94.5
8-2
~00,000 000,000
1 (control) 2 3
200,000 500,000
53.0 38.3 24.8
100 77.2 46.8
200,000 500,000
25.9 13.4 5.8
100 51.8 22.4
200,000 500,000
4.8 0.64 Trace
100 13.3
196 187 177 152
100
4 (control) 5 6
C (control)
c-1 C-2
Orange juice (concentrated) 3C (control) 4C 5C 6C Orange juice (diluted 1:4) 1D (control)
600,000 1,000,000 2,000,000
600,000 Roentgen-equivalents-physical. 2D
49.0 31.4
...
95.5
90.3 77.5 100 64.0
trate was irradiated directly. Another portion was diluted with 3 parts of water and then irradiated. The results are shown in the lower part of Table 111. Comparison of the data in Tables I, 11, and 111 indicates that a t a given dosage of irradiation the percentage destruction of I-ascorbic acid in the diluted juice was approximately the same as that in the samples of fresh orange juice, but the percentage destruction in the concentrated juice was much less, Effect of Temperature. Svedberg and Brohult ( 2 3 ) found that haemocyanin and serum albumin were made inhomogeneous when irradiated by ultraviolet light and alpha particles at room temperature but that the same compounds were protected when irradiated at the temperature of liquid air. Brasch and Huber ( 3 , 4)have reported that color and flavor changes do not occur when foods are irradiated in the frozen state. Both orange juice and solutions of pure ascorbic acid were irradiated in the frozen state to determine the degree of protection afforded by this technique. The dishes containing the samples were placed on a level layer of pulverized dry ice and left there until the contents were thoroughly frozen, usually for 5 minutes. Irradiation caused no visible thawing of the samples. Duplicate samples in the liquid state were irradiated at room temperature. The results of irradiation of orange juice in the liquid and the frozen state are shown in Table IV. The I-ascorbic acid content of the orange juice x a s completely protected against destruction by irradiation when the juice was in the frozen state. Still more striking is the fact that pure ascorbic acid, even at the low concentration of 8 mg. %, was almost completely protected when irradiated in the frozen state (Table V). Obviously there are several possible explanations for the protective effect of subfreezing temperatures, such as concentration of the solute, crystallization of the solute, prevention of the formation of free radicals, or restriction of the mobility of free radicals if they are formed. According to TTeiss ( 2 7 ) and others, the primary action of irradiation is to produce highly reactive, free radicals in the solvent, and these free radicals in turn react with the solute. From this theory, it would appear that the mobility of these free radicals would be a limiting factor in a radiation-induced reaction, and any physical change in the medium that tends to limit that mobility would also tend to minimize the over-all effect of the irradiation.
INDUSTRIAL AND ENGINEERING CHEMISTRY
March 1951
72 1
5. Loss of ascorbic acid was negligible when the medium was in the frozen state. OF 3-M.E.V. CATHODE RAYSON 1-ASCORBIC irradiated TABLE IV. EFFECT 6 . Chemical and spectrophotometric, data are presented t o ACID IN UNFROZEN AND FROXEN ORAXGEJUICE show that 2,3-diketogulonic acid was not present in the irI-Ascorbic Acid radiated solution within a few hours after irradiation. Total Dose, Rep"
Mg./900 ml. Unfrozen Frozen 29.5 ... 100,000 28.3 29.? l + l 200,000 ... 28.0 2 2' 300,000 23.0 28.8 3' 400,000 21.8 29.6 4' 4 500,000 19.3 30.8 6 6' 28.8 5' 750,000 14.9 7 7' 1,000,000 12.2 29.6 a Roentgen-equivalents-physical.
Sample No. Contrql
+ + + +
+
Retention, % Unfrozen Frozen 100 96.0 i6i:3 96.5 is:0 97.7 73.8 100.2 65.4 104.3 50.4 97.7 41.3 100.2
TABLE V. EFFECT OF 3-M.E.V. CATHODE RAYSON L-ASCORBIC ACID IN UNFROZEN A N D FROZEN SOLUTIONS OF 0.25% OXALIC ACID NO.
Contrql 1 + 1 2 2' 3 3' 4' 4 5 5'
Total Dose, Rep a
I-Ascorbic Acid Retention, % Mg./100 ml. Unfrozen Frozen Unfrozen Frozen
8.0 100,000 3.63 200,000 2.31 300,000 1.32 400,000 0.95 500,000 0.85 760,000 0.61 1,000,000 0.59 Roentgen-equivalents-physical.
++ + +
;'+ ;:
8.73 8.62 8.41 8.46 8.33 8.16 8.12 7.93
100 45.4
28.9 16.5 11.8 10.6 7.6 7.4
100 98.8 96.3 97.0 95.4 93.5 93.0 90.8
Experiments of a preliminary nature conducted by the authors indicate that at low temperatures the destruction of ascorbic acid by cathode rays is not lessened in 65% until the solvent becomes 'decidedly viscous. Irradiation of ascorbic acid in 0.25% oxalic acid a t several temperatures between -20" and +25" C. showed a gradual decrease in destruction of the vitamin as the freezing point was approached, and then a rapid decrease in destructive effect between +5" and -5" C., followed b y a leveling off of the effect below - 5 " C. These results seem to indicate that a t least one important factorin the protective effectof low temperatures is the restriction of the mobility of free radicals caused by an increase in viscosity. Svedberg and Brohult ( 2 3 ) and Errera ( 8 ) have used the effect of low temperature to distinguish between direct and indirect action of radiations. If the'effect is the same a t subfreezing temperatures as a t room temperature, the action is probably direct. If there is a decreased effect at the lower temperature, this would indicate an indirect action of some sort. SUMMARY AND CONCLUSIONS
1. ~ ~ ~ ~by dhighi voltage ~ t i ~ ~ ra caused a considerable loss in the reduced form of vitamin l-ascorbic acid, both in orange juice and in solutions of the pure vitamin. 2' In both cases loss much less in terms Of vitamin C than in terms of the reduced form only. 3. Ascorbic acid in solutions of equivalent concentration was much more resistant t o cathode ray irradiat,ion in situ than in vitro. 4. Dilute solutions Of ascorbic acid were relatively more sensitive to irradiation than were concentrated solutions,
6,
ACKNOWLEDGMENT
The authors wish to thank John G. Trump, Kenneth A4. Wright, and the late Arthur M. Clarke of the department of electrical engineering, Massachusetts Institute of Technology, for their cooperation in making the electrostatic generator ' available for this study. The junior author wishes to express his appreciation to Standard Brands, Inc., for the graduate fellowship in food technology under which his work in this research has been conducted. LITERATURE CITED
(1) Anderson, R.
S.,and Harrison, B., J . Gen. Physiol., 27, 69
(1943). (2) Bessey, 0. A., J . Biol. Chem., 126,771 (1938). (3) Brasch, A., and Huber, W., Science, 105,112 (1947). (4) Ibid., 108,536 (1948). (5) Dale, W.M., Biochem. J . , 34, 1367 (1940). (6) Ibid., 36,80 (1942). (7) Dunn, C. G., Campbell, W. L., Fram, H., and Hutchins, A., J . Applied P h y s . , 19, 605 (1948). (8) Errera, M., Cold Spring Harbor S y m p o s i a on Quantitative Biology, 12, 60 (1947). (9) Evans, R. D., Nu.cleonics, 1, No. 2, 32 (1947). (10) Forssberg, A.,Nature, 159,308 (1947). (11) Fricke, H., Hart, E. J., and Smith, H. P., J. C h e m . P h y s . , 6 , 229 (1938). (12) Goldblith, s. A., and Proctor, B. E., Nucleonics, 5, 2, 50 (1949). (13) Goldblith, 8. A., Proctor, B. E., Hogness, J. R., and Langham, W. H., J. Biol. Chem., 179, 1163 (1949). (14) Gould, B. S.,and Schwachman, H., Ibid., 151,439 (1943). (15) Guild, L. P., Lockhart, E. E., and Harris, R. S., Science, 107, 226 (1948). (16) Herbert, R. W., Hirst, E. L., Percival, E. G. V., Reynolds, R. J. W., and Smith, F., J . Chem. SOC.,1933,1270. (17) Hochberg, M., Melnick, D., and Oser, B. L., IND.ENG.CHEM,, ANAL.ED., 15,182 (1943). (18) Morton, R. A., "Application of Absorption Spectra to Study of Vitamins, Hormones, and Coenzymes," 2nd ed., London, Adam Hilger, Ltd., 1942. (19) Proctor, B. E., and Goldblith, S.A., Nucleonics, 3, NO. 2, 32 (1948). (20) Ibid., 5 , No. 3, 56 (1949). (21) Proctor, B. E., Van de Graaff, R. J., and Fram, H., Research Rept., U. S. Army Quartermaster Contract Projects, p. 217 (July 1942-June 1943), Dept. Food Tech., Mass. Inst. Technology, Cambridge, Mass. (22) Roe, J. H., and Oesterling, M. J., J . Biol. Chem., 152, 511 (1944).. (23) Svedberg, T., and Brohult, S.,N a t u r e , 143,938 (1939). (24) Trump, J. G.9 and Van de Graaff, R. J., J. AWL. PhW.3 19, 599 (1948). (25) Trump, J. G,, Wright, K. *., and Clarke, A. M., Ibid., 21,345 (1950). (26) Tytell, A. A., and Kersten, H., Proc. SOC.E z p t l . Biol. M e d . , 48, 521 (1941). (27) Weiss*
J'3
Nature* 1532748
RECEIVEDJune 9, 1950. Presented before the Division of Agricultural and Food Chemistry, 117th Meeting of the AMERICAN CHEMICAL SOCIETY, Philadelphia, Pa.
