IRRADIATED MILK Vitamin
D Potency as a Function of Energy Input
BRIAN O’BRIEN, H. DOUGLAS McEWEN, AND KENNETH MORGAREIDGE The Institute of Optics and the Department of Biochemistry of The University of Rochester, Rochester, N. Y.
The antirachitic activation of whole milk by direct action of ultraviolet energy from carbon arcs is studied. Assuming monomolecular reactions, a theory is derived which provides a means of predicting the amount of energy input to the arc necessary to produce a given vitamin D potency in t h e treated milk. For average milk samples the theory predicts that, if all the provitamin could be quantitatively converted to active vitamin D without subsequent destruction, a potency of the order of 2.0 I. U. per gram would be possible. In actual practice a potency of 1.65 I. U. can be achieved in the bubble-film type of irradiator under the operating conditions here studied. With increasing energy inputs, anti-
rachitic potency increases to a maximum value at about 300 joules per gram and then falls off with irradiation doses in excess of this value. Samples receiving more than 300 joules lose potency on storage about in proportion to the excess radiant energy ; those receiving less than 300 joules are stable with respect t o vitamin content. This effect is in line with the theory. In the light of the theory, the experimental data show t h a t , if any intermediate compound is formed in the conversion of provitamin t o vitamin, the second half of the process (the conversion of the intermediate to vitamin) requires only relatively small amounts of radiant energy.
T
HE practical value of endowing dairy products with substantial antirachitic potency has resulted in a considerable amount of work from several laboratories during the past few years. Three methods of achieving potency in ordinary whole milk have been employed: feeding a source of vitamin D (such as irradiated yeast) to lactating cows, adding a vitamin-containing supplement directly to the milk, and irradiating an otherwise untreated product direct with ultraviolet energy. Little is known at present concerning the form of provitamin occurring naturally in milk. Since studies on the relative rat and chick potencies of irradiated milk tend to place it in the same class with cod liver oil (i. e., the active vitamin is derived from a sterol of animal rather than plant origin), some workers prefer to regard the milk vitamin as the activated form of 7-dehydrocholesterol. Actually there is little experimental proof as yet. As a preliminary study in this direction as well as for its practical value, it was considered desirable to explore the energetics of the activation of whole milk under accurately controlled irradiation conditions. As a first objective it seemed important to answer three questions: What is the maximum potency obtainable in whole milk by direct irradiation under specified conditions? What are the laws governing the conversion of provitamin to active vitamin? What are the effects of relatively heavy irradiation on the stability or actual destruction of the antirachitic activity?
Experimental Technic The milks employed in this study were drawn from various parts of the western and central New York milksheds throughout a period of more than a year. Whole fresh samples were irradiated by the Little Falls, N. Y., plant of the Cherry-Burre11 Corporation, using the bubble-film type of irradiator. Additional samples, irradiated in the same kind of machine but in other localities, were used to supplement the data on the lower energy inputs. Milk flow and electrical quantities were measured accurately. The present report is limited to samples irradiated with “U” carbons of the National Carbon Company (22-mm. upper and 13-mm. lower electrodes). Results which have been obtained with other types and makes indicate that the “U” carbon operates at a fair average efficiency. Moreover it is widely used in milk irradiation so that the data presented here may be considered representative. Sixty-cycle alternating current a t 60,90,and 120 amperes was used a t 50 volts across the arc. The ballast was reactive and approximately equal to the arc impedance, so that with the carbons used the harmonics were of sufficiently low amplitude to permit computation of the power input as nearly equal to the arc volt-amperes. The energy input to the arc was calculated in joules per gram of milk irradiated, and the resulting antirachitic potencies are expressed in terms of International 839
840
INDUSTRIAL AND ENGINEERING CHEMISTRY
units of vitamin D per gram. The ultraviolet output of such an arc a t constant voltage is not quite a linear function of the current. This introduces a small systematic shift at energy values below 10 joules per gram where arc currents of 90 or 60 amperes were used. At 15 joules per gram and higher, the arc current was maintained a t 120 amperes throughout the experiments. Energy inputs up to 2100 joules per gram were employed without producing any gross physical changes in the milk. The characteristic flavor change produced by irradiation was very marked a t the 2100-joule level. These high energy values reqresent about four hundred times the power input usually employed in commercial irradiation. Under proper irradiation conditions, unchanged flavor was noted in milks which received as much as 75 joules per gram. Antirachitic potencies were determined by means of the standard curative assay technic in albino rats, combined with the radiographic method of estimating healing. In every assay, international standard solution of irradiated ergosterol was fed for direct comparison (8). Ordinarily ninety-six animals were used for feeding four samples and the international standard solution. The observations given here represent about two thousand assay animals. Refinements in the radiographic technic (to be reported elsewhere) yield a precision such that the maximum error in the determination of the potency of a single sample is not greater than *15 per cent. The experimental data are best presented with reference to the energy necessary to produce maximum potency. It was found that, for average milk samples irradiated under the conditions outlined above, arc inputs in the neighborhood of 300 joules per gram resulted in a maximum antirachitic activity of the order of 1.6 International units per gram. The average fat content of the milks was 3.5 per cent. . Occasionally samples with no higher fat content exhibited a potency at moderate energy inputs which was 50 per cent or more above the average for that energy. These few samples came from a single district, and it appears probable that their provitamin content was likewise above average. The manner in which potency increases up to the maximum as the e n e r g y i n p u t is increased is shown i n Figure 1. Each circle represents one assay of a sample which had received the corresponding amount of irradiation. The solid curve was calculated from the theory as wiIl be shown JOULES PER GRAM INPUT TO ARC in the succeeding section. As the energy inFIGURE1. INCREASE O F POTENCY WITH ENERGY INPUT put is increased above Circles represent the actual potenciea of that necessary to inmilk Remules which had been irradiduce maximum antiated, oorr'esponding to the arc energy inputs shown. The solid ourve is not rachitic potency (about an empirical "best fit" but 1s calculated from theory. 300 joules per gram), the actual activity decreases progressively. This effect is shown in Figure 2. The immediate destruction of antirachitic potency is not the only effect of overirradiation. I n both Figures 1 and 2 the open circles represent potencies determined by feeding the samples from 2 to 10 days following irradiation. In Figure 2, the crosses show the potency determinations on the same samples after storage a t 2" C. for 20 additional days. The solid and dotted curves of Figure 2, like the solid line of Figure 1, are not simply an empirical "best fit" of the experimental points but are
VOL. 30, NO. 7
calculated from the theory as outlined below, the only assignable constants being the reaction rates. It is evident that, for energy inputs in excess of 300 joules per gram, there has been an acceleration of the spontaneous decomposition of the active principle. For example, a t the highest energy input level (2100 joules per gram) the potency immediately after irradiation was about 1.O International .
la,
a
502 -
'
-
.
'
1
~
I
j
I
I
J
l
I
l
I
I
l
I
unit (I. U.) per gram for an average sample. Following the 20-day storage period, the sample assayed only 0.4 I. U. per gram. However, samples receiving less than 300 joules per gram showed substantially the same potency both before and after storage. It is obvious that this finding is of much practical significance in the production of high-potency milks.
Theoretical Discussion It is possible to postulate, on purely theoretical grounds, certain fundamental relations which should hold for the activation of the natural provitamin D of milk. I n the absence of any definite evidence to the contrary, it may be assumed for this purpose that the chemistry of the transformation from provitamin to vitamin in milk consists of a series of reactions analogous to those which have been shown to occur on the irradiation of ergosterol with the resultant production of calciferol (vitamin Dz). In this discussion, then, we will assume that the series of reactions which take place in milk activation involve three distinct processes. First, the naturally present provitamin is photochemically transformed into an inactive intermediate material (corresponding to tachysterol or lumisterol in the ergosterol transformation). The second reaction then takes place, converting the inactive intermediate into the active vitamin. On continued irradiation, the third reaction takes place through which the active vitamin is degraded to inactive product or products. If we represent these substances, from original provitamin to inactive end product, as A , B, C, and D, respectively, we may then state the series of reaction as: A+B+C+D
It appears most probable that the reactions A to B and B to C, where C is the active vitamin, are monomolecular. There is less evidence on the nature of the reaction C to D ,but this also will be treated as monomolecular. As will be seen, quantitative agreement with the present experimental results is quite good. Now, let z = concentration of A y = concentration of B z = concentration of C w = concentration of D I = reaction rate A to B per unit concentration of A
INDUSTRIAL AND ENGINEERIKG CHEMISTRY
JULY, 1938
m = reaction rate B to C per unit concentration of B n = reaction rate C to D per unit concentration of C
We may then define the initial conditions as x
=
xo,and y
= z = t~ =
0
The formation of B from A is given by
which integrates to x
=
xo ePzt
Next, C is formed from B: dY = Ex dt
- my
Substituting from Equation 2 ,
4~ dt
lxo e-lt - my
(4)
which integrates to
The formation of
stance A (the provitamin) to substance B , the remainder of the irradiation process being required to convert the intermediate, B , to the active component, C. This seems improbable, particularly in view of what is known concerning t h e ergosterol t r ansf ormation. When rn is very much greater than 1, we need recognize only two reactions, both photochemical processes, and may write an expression of the theory which agrees very well with the experimental data. Let r be the rate constant for the over-dl converFIGURE3. GENERAL FORMS sion of the provitamin to OF CURVES FOR x, y, z, w the active vitamin, and let s be the rate constant for the conversion of the active material into inactive endproducts. Equation 5 then becomes
D from C is given by
:;
-=
p
where P E P,,,.
my - nz
Substituting from Equation 5 ,
-
dz __ - mlxo dt m - 1
(e--lt
-
e-mt)
-
nz
which integrates to
=
(e
dw= dt
nz
Substitut,ing from Equation 8 and integrating, =
nmlzc
[IZ(m1--l ) ( ne-lt
-
1)
1m(m - Z)(n - m ) 1 - e-"' n(n - l ) ( n - m)
Atipresent no direct experimental means are available for determining the concentration of either B or D. However, examination of the expressions for 5,y, and x (Equations 2, 5, and 8) shows that they give rise to curves of the general forms shown in Figure 3. Furthermore, their relation is such that if the value for 1 is either much greater or much less than m, the form of the expression for z converges to that for y, and the expression for y converges to that for 2. Figures 1 and 2 show that the experimental data fall along a curve similar to that given by the expression derived for y (Equation 5 ) . Since we know that energy is required to initiate this series of reactions, it must be assumed that m is very much greater than 1. This is equivalent to saying that substantially all of the ultraviolet energy required for the initial process is used up in the production of B from A , whereas C may be formed from B spontaneously or, a t least, on the absorption of very small amounts of radiant energy. The alternate assumption, that m is very much less than 1, is numerically satisfactory but leads to the conclusion that the first small quantity of radiant energy converts substantially all of sub-
-
e--sE)
= potency, I. U. per gram = energy (radiation intensity X =
time)
potency which would be obtained if t h e conversion of provitamin to vitamin were 100 per cent and s were equal to 0.
The solid curves in Figures 1 and 2 provide a fair approximation to the experimental points. This was calculated by assigning the following values to the constants :
s
The concentration of D is given by
--rE
s-r
P,,,. r
w
841
. "
= 1.95 I. U. = 0.0111 = 0.00037
per gram
Obviously the theory can be made to explain the loss of activity on storage only if P,,,. and r remain constant while s increases in value. That it does this is shown by the dotted curve in Figure 2 where s has been given the value 0.00081. Differentiating Equation 11, we have the rate of change of potency as
For small energy inputs this converges to
For the above values of P,,,. =
and r, 0.0216 I. U. per joule
Haman and Steenbock ( 1 ) found that the ultraviolet energy necessary to produce vitamin Dz (calciferol) from pure ergosterol is 900 ergs per I. U. for energies up to 104 ergs per mg., or potencies up to about 11 I. U. per mg. of ergosterol for this case is 40,000 I. E.per mg. irradiated, Since P,,, (by definition), it is evident that their experiments were carried out a t very small energy inputs compared to that necessary to produce maximum potency, or again, the case covered by Equation 13. A comparison with the present
VOL. 30, NO. 7
INDUSTRIAL AND ENGINEERING CHEMISTRY
a42
results is of interest, even thodgh the provitamin of milk is not ergosterol. The great difference (a factor of about 5 x 106) between the results of Haman and Steenbock and those of the present writers is not surprising when we remember that only a small part of the electrical input to a carbon arc gppears as ultraviolet radiation of wave length less than 3100 A., and, further, that a large part of the absorption of such wave lengths by milk is due to substances (chiefly protein) other than the provitamin.
Acknowledgment The experimental portion of this work was supported by the Cherry-Burrell Corporation. The writers are indebted
to Loomis Burrell and to C. B. Dalzell of that organization for their generous cooperation. They are also indebted to the Wisconsin Alumni Research Foundation with whose support the compilation of results was made possible.
Literature Cited
,
(1) Haman, R. W., and Steenbock, Harry, IND.ENG.CHEM.,Anal. Ed., 8, 291 (1936). (2) O’Brien, Brian, and Morgareidge, Kenneth, Proc. SOC.Ezptl. Bzol. Med., 32, 113 (1934). REC~IVE March D 24, 1938. This paper is an extension of material reported under the same title before the 31st Annual Meeting of the American 6ooiety of Biological Chemists, Memphis, Tenn., April 21 to 24, 1837.
CORRESPONDENCE Thermodynamics in Hydrocarbon Research SIR:In a recent paper Thomas, Egloff, and Morrell ( 4 ) referred to some of my earlier work (1,d ) in which thermodynamic data are applied to the problems of cracking and hydrogenation. Explaining in detail the limitations and deficiencies inherent in any thermodynamic treatment of such a problem which is truly kinetic in nature, I stated ( 2 )that not thermodynamic considerations but the principles of reaction kinetics are of prime importance for the actual procedure of these reactions; the latter are in no way connected with the energy content as calculated from thermodynamic data. Moreover, in another paper (3) I explained: “It should be clearly understood that thermodynamic caIculations may be applied only with care to problems of this type, since reaction rates primarily depend upon the energy of activation which is not related to the thermodynamic probability of some equilibrium between the initial and the final state of the system. Thermodynamics, therefore, affords a decision only about the possibility but not about the actual occurrence or the course of some definite chemical reaction.” It is my desire to stress the viewpoint taken by Egloff and his associates, particularly since their quotation of my results suggests that there must have been some possibility of misinterpretation, although I hoped that my opinion was made perfectly clear in my earlier publications. GEORQR. SCHULTZE PHYSICAL-CEEMICAL INSTITUTE OF BERLIN, GERMANY UNIVIURSITY December, 1937
..... SIR: From Schultze’s publications it is apparent that he fully realizes the limitations of the thermodynamic method. According to our interpretation, Schultze does not always stay within these limitations. For example, he discusses (1) the general cracking equation:
+
C m + nHz(m+ .)+z = C ~ H Z ~ +Cn’L Z
+
His conclusions are quoted: “Of all the ( m n - 2) different decomposition possibilities that comply with the equation, that reaction is most probable for which the resulting olefin is as large
+
as possible (i. e., n = m n - 1). This explains the formation of methane as the predominant constituent of the gas from
cracking.” We do not believe that such a conclusion can be reached and justified on the basis of thermodynamics alone. CHARLES L. THOMAS GUETAV ECLOFF J. C. MORRELL UNIVERSAL OIL PRODUCTS COMPANY CHICAQO ILL. Deoembir 23, 1937
Literature Cited (1) Schultze, Angew.*Chcm., 49,268, 284 (1936). (2) Schultze, Oel,Kohle. Erdoel, Teer, 12,267 (1936). (3) SchultEe, 2. Elektrochem., 42,674 (1936). (4) Thomas, Egloff, and Morrell, IND.ENQ.CHW 29, 1260 (1837).
Correction Our attention has been called to several errors in our recent paper on “Thermodynamics in Hydrocarbon Research” [IND. ENG.CHEM.,29,1260 (1937)l: METHANE(page 1261). AHoZo8should read -18,070 instead of +18,070. ETHANE(page 1261). A H O mshould read -20,600 rather than
-2060.
WPENTANE (page 1262). AHoag8for the liquid should read -42,230 instead of -2570. ISOPENTANE (page 1262). The first sentence should reed: ANozss = -38,080 for gaseous and S O 1 9 8 = 69.5 for liquid ieropentane, instead of A H ” Z S= ~ -38,080 and S0m = 59.5 for liquid
isopentane. ~-HEPTENE (page 1263). AF0zsa ehould read 20,720 instead of 8920. IBOBUTENE (page 1265). A F ’ m should read 14,240 instead of 14,290. In all these cases the figures are correct in the original Table 11, so that the values given there are unchanged. L. THOMAS CHARLES GUSTAV EGLOFF J. C. MORRELL
