Oxidative Destruction of Vitamin D J. C. FRITZ, J. L. HALPIN, J. H. HOOPER, AND E. H. KRAMKE
Evidence is presented indicating that vitamin D is not stable when added to various dry carriers, as it must of necessity be added to mixed feed. Oxidation is shown to be the cause of this destruction, and any condition which promotes oxidation, such as increased surface area, accelerates the destruction. Temperature coefficients and other data show that the destruction is a chemical reaction. The various forms of vitamin D, including crystalline activated ergosterol and crystalline activated 7-dehydrocholesterol, as well as natural sources, are all susceptible to destruction. Various methods for stabilization of vitamin D have been tested. Effective protection is obtained by a protective coating which prevents air contact. Inert gas packing and the use of antioxidants retard the rate of destruction.
The Borden Company, Elgin, Ill.
vv
HEX animals spend most of their lives indoors, it is essential that vitamin D be included in the food. The importance of vitamin D has been emphasized in connection with the present war conditions. Conservation of food supplies is our first nutritional problem (7). The supply of many sources of vitamin D has been seriously curtailed. Other species of fish can, however, be used to replace the vitamin D formerly secured from the cod. When the problem is ope of supplying vitamin D to poultry which cannot use calciferol efficiently for bone formation, the cost is out of proportion to the percentage of the diet actually made up by vitamin D carriers. Since most poultry feeds would contain 5 pounds or more per ton of a product containing 400 A. 0. A. C. chick units of vitamin D per gram, the cost of this addition would be not less than two dollars per ton of feed. It is obvious that any condition or set of conditions which requires the use of a margin above the birds’ actual requirements constitutes an economic waste. One of these wasteful factors is loss of potency during storage. Vitamin D was formerly considered to be stable under ordinary conditions. Recent observations, however, have indicated that a substantial loss of potency may occur both before and after the addition of vitamin D carriers to feeds. Some preliminary observations on the stability of vitamin D were summarized in a previous report from this laboratory
.
(5). Ewing (4) discussed the stability of vitamin D and concluded that loss of potency may be very rapid under some conditions.
TABLE I. EFFECTOF VARIOUSDRYCARRIERSON VITAMIND STABILITY 7 -
Carrier Kaolin Fuller’s earth Calcium carbonate Calcium carbonate Mineral mixture Mineral mixture Mineral mixture Sodium chloride Manganese sulfate Sand Sucrose Lactose Dried Dried Dried Dried Dried Dried Dried
whey whey whey whey whey skim milk skim milk
Soybean meal So1vent Solvent Expeller Expeller Expeller Ground r a w soybeans Ground yellow corn Wheat middlings Corn distillers’ solubles Molasses distillers’ solubles Dried stick Liver meal
1 wk.
.. .0. .. ..
1 ma.
4 ma.
6 ma.
0 0 R7 50
....
0 0
.. ..
84
.. .. 12 0 .. 6 . . . . . . . . . . . . . . . I
.
100 100 100
100 92
100
. . 100 .. 100 . . . . .
.. . . . .95. .. 90
. . . . . .
0 0 0 0
0
..
..
2 ma. 0
31
91
.. .. .. .. ..
yo of Original Potency Found after Storage
...... . . . . . . . . . . . . 30 . . . . . . . . . 0
. .. .
. .. .
20
0
0 0
...... ......
... . .. .
. .. .
. d .
. . . . . .. .. .. .. .. . . . . .
. . . . . . . 0. . . ... .. 0
...
......
0 12 100
...... 92 68 ... 58
84 95 90
... ... ... ... ... ... .94. . . ... ..
60
100
... 99
...
80
87
100
100
91 .... .. ...
7s 67 100 73
. . . . . . . . .
100 98
100 79
8 ma.
..
10 ma. 12 mo. 15 ma.
.. .. .. .. ..
.. ..
.. ..
.... ..
..
.. ..
..
..
..
.. ..
..
.. i .
.. .
I
..
55
....
.. .. .. .. ..
....
.. .. .. ..
78
..
..
94
95
.. .. .. ..
..
.. ..
.. .. ..
.. .. .. .. ..
979
.. .. ..
....
.. .. .. ..
.. .. .... ..
.. .... .. .. .... .. .. .. .. .. .. .. .. .. .. ..
74
.. .. .. ..
..
.. ..
.. ..
80
90
.. .. ..
.. .. ..
..
Evidence of Destruction I n poultry feeding practice it is dustomary to add the concentrated source of vitamin D to a dry carrier. Since it is rather difficult to disperse uniformly a few tenths per cent of. a high-potency oil in a heterogeneous mixture, it is customary to make a premix of the oil on some suitable ingredient. This may be done either in the feed plant or by manufacturers who supply vitamin D impregnated ingredients to the feed mixer. The selection of the carrier to which the vitamin D concentrate is to be added has a marked influence upon the subsequent stability of the vitamin. Table I shows the results of a series of tests in which a highpotency fish liver concentrate was added to various dry carriers. The assays were by the A. 0. A. C. prophylactig technique ( I ) . Most of the values reported represent the average of two or more individual assays using twenty-two chicks per
980
INDUSTRIAL A N D E N G I N E E R I N G C H E M I S T R Y
Vol. 34, No. 8
is a typical curve with potency plotted against time. Under a given set of conditions the destruction of vitamin D is a function of time. Materials which tend t o give the vitamin D increased surface a t which oxidation may occur cause a rapid loss of potency. Protection results from elimination of air contact. Further evidence that the destruction is due to oxidation is afforded by observations on the effect of gas packing. We have already noted that the vitamin D was quickly lost when oils carrying this factor were added to washed sand. It is highly improbable that silicon dioxide would have any harmful effect, Microscopic studies show that the oil carrier is distributed in a thin film over the surface of the sand particles, and the oil is quickly dried into a tough film. Figure 2 gives the results of gas packing a portion of a sample of this type.
TABLE 11. EFFECT OF STORAGE ON VITAMIN D ADDEDAS FISH OIL TO POULTRY FEEDS
pen, and in general the results are considered reproducible within *10 per cent. It will be noted that the cereal carriers protect the vitamin D. Milk products show some destructive action. The vitamin D activity is quickly lost when the oil is added t o minerals or crystalline sugars. The most rapid destruction was found to occur when the oil was added to adsorbent materials. The practical significance of these observations is summarized in Table 11,which shows the effect of storage on vitamin D in mixed feeds. Most of these feeds are commercial products and as such are referred to only by laboratory number. The results are variable. Some feeds show marked loss of vitamin D during three-month storage a t room temperature; others show no loss during six-month storage. These data agree with the observations of Bird (2)who also found marked loss of vitamin D from mixed feeds during storage a t room temperature. Chemical @nalysesdid not offer any explanation for the variable stability of the feeds studied. Feeds with charcoal or high miheral content were among the most destructive. However, there were some exceptions to this general observation. The question might logically be raised as to whether there was any difference in the stability of vitamin D from different sources. Table I11 shows the results of adding vitamin D from various sources to materials found to have a destructive action. The evidence shows that all sources of vitamin D are susceptible to destruction. Whether any apparent differences in the rates are significant must await further work. I n this study some of the tests were made by the A. 0. A. C. chick method and others by the U. S. P. rat curative technique (9). The latter method was used for all work with activated ergosterol.
Method of Destruction While no one theory can explain all of the observations, the evidence indicates that oxidation is a primary cause of destruction. The rate of destruction is quite rapid in the case of vitamin D on certain carriers. The data presented here do not show much evidence of a lag or induction period in this destruction. Figure 1
A. 0. A. C . Units Vitamin D/100 G. Feed Fresh 3 mo. 6 mo. 12 mo. 25 25 24 78 72 21 26 60 53 .. 60 32 29 .. 90 86 42 57 24 .. 82 88 80 44 49 .. .. 83 60 .. .. 99 98 .. 80 50 40 .. 41 52 80 135 127 133 133 ..
Feed No. A. 0. A. C. t)asal B-79 13-80 B-94 B-107 B-128 B-130 B-131 B-132 B-133 B-143 B-194 B-195 B-199 B-212 B-213 B-214 B-237 B-238 B-240 B-241 B-393 B-394
....
..
.. ..
..
..
When the vitamin D oil is added to sand, the activity is quickly lost from the unprotected portion. I n another portion of the same sample the air was largely replaced by nitrogen, and this procedure afforded considerable protection. The protection was incomplete. Gas analysis showed that 3 per cent of oxygen remained in the gas-packed containers. It may, therefore, be assumed that destruction in the protected portion was due to oxygen not completely removed. From the foregoing it would be assumed that temperature would play a part in the destruction of vitamin D under the conditions described. Samples stored a t 0" C. showed stability, while comparable samples stored a t room temperature showed marked loss of vitamin D activity. This marked increase in the rate of destruction from zero t o room temperature indicates a chemical rather than a physical reaction.
OF STABILITY OF VITAMIN D FROM DIFFERENTSOURCES TABLE 111. COMPARISON
Source of Vitamin D
Units Vitamin D per Gram--Original 1 m o . 2 mo. 3 mo. 4 mo. 5 mo. 6 mo. 10 ... 0 .. . .. 51 .. . 33 ... 18 440 430 , 141 33 0 .. 100 0 .. . . .. 100 0 .. . ..
Reference cod liver oil Tuna liver oil Nonsaponifiedfraotion,finhoil Activated ergosterol
Dry Carrier Dried whey Dried whey Dried whry Sucrose Oyster shell
D-activated animal sterol8
Dried whey Mineral mixt.
533 100
.. . ., .
Activated 7-dehydrocholesterol
Kaolin Oyster shell Sand Dried whey
100 100 100 133
0 92 87 104
I
..
.. . ._. 400 ... 20 .. . .. . ..0. ... .... . . 110 63
..
..
... .. .
.... ..
.. ..
293
..
..
70
... ...
.. .... ..
0 20 72
44 100
..
..
August, 1942
INDUSTRIAL AND ENGINEERING CHEMISTRY
981
oils and the biological activity of these samples showed a definite relationship.
Stabilization Studies A number of procedures for stabilization of vitamin D were investigated. Two of these have already been discussed a t some length-storage under inert gas and storage a t 1ow t e m p e r a t u r e. While these methods are practical in the 'case of human foods, they are not practical when applied to animal and poultry feeds. Under these conditions the cost would be prohibitive although such methods are deserving of FIGURE 2 (Left).EFFECTOF GAS PACKIXG ON F I G U R E 3 (Right). STABILITY OFVITAMIND IN WHEY STABILITY OF VITAMIN D OIL ADDEDTO SAND OIL HOMOQENIZED WITH CONDENSED consideration where highSOLUBLES OR ADDEDTO DRYWHEYSOLUBLES potency concentrates are stored in a manufacturing dant. Since the evidence indicates that-much, if not all, of the deNo effect of diffused daylight could be observed. Samples struction can be attributed to oxidation, it is logical that prostored in opaque containers showed the same rate of vitamin D tection from air woqld afford protection of the vitamin D. Inloss as did samples stored in clear glass bottles. vestigation shows that this is a true assumption. Tests were A number of premixes made by the addition of fish liver oil made along the lines suggested by the use of calcium stearate to a cereal carrier were litter combined with mineral mixtures, coating to protect potassium iodide from oxidation (6). Caland the subsequent stability was noted. Typical observacium stearate was found similarly to protect vitamin D cartions are summarized in Table IV. The protection afforded riers when they were subsequently mixed with materials was variable. In the case of vitamin D premixed with ground otherwise destructive of the vitamin. Other coating agents yellow corn, rapid loss of potency resulted when this premix were also found to be effective. The principal requirement was combined with mineral salts. No adequate explanation is was that the impregnated particles should be completely surapparent. rounded by a relatively impervious 'coating. Experience to date indicates that the nature of the insulation has little effect on the protection afforded. Typical examples of such TABLEIV. EFFECTOF ADDINGDESTRUCTIVE MATERIALSTO protection are noted in Table VI. CEREALCARRIERSPREVIOUSLY IMPREGNATED WITH FISHLIVER OILS Another application of the same principle is noted when an Material Added, -Units Vitamin D/Gramoil-in-water emulsion is formed, using materials high in minerCereal Carrier % Original 1 mo. 2 mo. 3 mo. 4 mo. als known to have a destructive action when used as dry carGround corn CaCOa, 10 507 0 0 .. ... riers for vitamin D. Figure 3 shows the relative stability of Mineral mixt 25 516 0 0 .. ... Dried whey, SO 580 0 0 .. .. . the vitamin D when a fish oil is emulsified with a high mineral Soybean meal CaCOs 25 375 375 370 .. 375 solution as contrasted with the addition to these minerals in Mined1 mixt.. 25 375 225 70 dry form. Dried whey, 50 250 222 175 '20 If the destruction of vitamin D is due to oxidation, the use Distillers' sol. Dried whey, 75 100 85 28 7 ... of antioxidants should afford protection. A test was set up in which a high-potency fish liver oil was added to dried whey, with and without the addition of antioxidants. Olcott (8) A number of observations indicate that the presence of fat found hydroquinone to be among the more effective antioxiwhich becomes rancid during the storage period accelerates destruction of vitamin D. Crystalline vitamin Ds proved to be somewhat more stable when dissolved in diethyl ether and TABLEv. STABILITY OF ACTIVATED 7-DEHYDROCHOLESTEROL added to minerals than the same material dissolved in corn DISSOLVED IN ETHER OR DISSOLVED IN CORNOIL BEFORE ADDIoil and added to the minerals (Table V). It seems apparent TION TO DESTRUCTIVE MATERIALS that the development of rancidity in the oil present has a detUnits Vitamin D/GramSolvent Carrier Original 1 mo. 2 m o . 4 mo. 6 mo. rimental effect upon vitamin D. That the mere presence of fat in itself is not harmful is shown by tests of vitamin D oils Ethyl ether CaCOa 133 92 17 0 .. Dried whey 133 104 .. 100 '72 added to ground raw soybeans. This carrier contained 17 per Sand 100 87 .. 44 20 Kaolin 100 0 .. ... .. cent of fat, and the added vitamin D was very stable. Soybean oil is generally recognized as containing natural antioxidants (5). Crude soybean oil shows marked resistance to oxidation. A comparison between the Kreis test applied to 15 0 .. diethyl ether extracts of samples containing added vitamin 7 -
INDUSTRIAL AND ENGINEERING CHEMISTRY
982
TABLE VI.
EFFECT OF
COATING SOYBEAN MEALIMPREGNATED WITH VIT.4MIN IMMEDIATELY BEFORE ADDINGDESTRUCTIVE MATERI.4LS Destructive Material Added Later Dried whey Dried whey
Coating Agent None cla stearate
-
1 mo.
...0
96 100
73 100
0 100
... 100
...
8 100
0 100
...
100
None Hydrogenated fat
Dried whey Dried whey
... ...
None Hydrogenated fat
Mineral mixt. Mineral mixt.
45 100
None Hydrogenated f a t Hydrogenated f a t Hydrogenated fat
Mineral Mineral Mineral Mineral
None iMolasses
Mineral mixt. Mineral mixt.
mixt. mixt. mixt. mixt.
Per Cent of Original Potency 2 mo. 4 ma. 6 mo. 8 mo. 10 ma. 12 mo.
30 100
70 100
...
...
D OIL
...
...
94
...
15 86
... ...
...
..
...
.. .. .. .. .. .. .. .. .. ..
... ...
75
50
.,.
...
... 81 ...
100
... ...
.. ..
.. ..
18 86
..
., .. ~. .. .. .. .. ..
.. .. ,.
..
72
80
.. ..
..
Vol. 34, No. 8
The addition of hydroquinone to the fish oil prior to addition of the oil to minerals did not protect the vitamin D from subsequent destruction. A limited number of observations showed the same rate of vitamin D loss, regardless of whether the oil did or did not contain hydroquinone. The limited quantity of hydroquinone which could be used in this manner may have made this use less effective than the addition of dry hydroquinone to the carrier before addition of the oil.
Acknowledgment dants in his studies on the autoxidation of lard. Hydroquinone was used in finely powdered form a t a 2 per cent level. Since powdered sodium thiosulfate has been found to protect potassium iodide in feeds, this compound was similarly tried a t a 2 per cent level. The work of Dahle and Nelson (3) and others indicated that oat flour contains antioxidants. Twentyfive per cent of oat flour was tested. The results are summarized in Table VII. Partial protection was afforded by the oat flour and by the hydroquinone but not by the thiosulfate under the conditions of this test.
T.4BLE
VII.
EFFECT OF ANTIOXIDANTS ON VITAMIND . Antioxidant,
None Hydroquinone, 2 Sodium thiosulfate, 2 Oat flour, 25
STABILITY O F
yo of Original Potency after 3 Ma. 0 39 0
64
The authors express their appreciation to Xutrition Research Laboratories for the gift of the activated ergosterol used in this study, and to Winthrop Chemical Company, for the gift of the crystalline vitamin D,.
Literature Cited (1) ilssoc. of Official Agr. Chem., Official and Tentative Methods of Analysis, 5th ed., 1940. (2) Bird, H . R., personal communication, 1941. (3) Dahle, C. D . , and Nelson, D. H., J. Dairy Sci., 24, 29-39 (1941). (4) Ewing, W. R., Handbook of Poultry Nutrition, pp. 454-8 (1941). (5) Fritz, J. C., Archer, W. F., and Barker, D. K., Ann. Meeting Poultry Sci. Assoc., Stillwater, Okla., 1941. (6) Johnson, F. F., and Frederick, E. R., Science, 92, 315-16 (1940). (7) -Murlin, J. R., Federation of Am. SOC.for Expt>l.Biol., Boston, 1942. (8) Olcott, H. S., J.Am. Chem. SOC.,56,2492 (1934). (9) U. S. Pharmacopoeia XI, 2nd Supplement, 1939. PRESENTED in a program on “Vitamins” before a joint session of the Divisions of Biological Chemistry and of Agricultural and Food Chemistry a t the 103rd Meeting of the A J I E R I C ACHEJIICAL N SOCIETY, Memphis, Tenn.
Phosphine and Sludge Digestion W I L L E M R U D O L F S AND GLENN W. S T A H L New Jersey Agricultural Experiment Station, Rutgers University, Yew Brunswick, N. J.
ETTLED sewage solids may contain from 0.75 to 3.0 per cent phosphorus as P206, whereas in digested sludge the content ma.y vary from 1.0 to 4.0 per cent on a dry solids basis. Sludge obtained from chemically treated sewage may contain considerably more. The apparent increase in phosphorus in the sludge after digestion is un‘doubtedly due to the destruction of organic matter by biological activities. Since living things require phosphorus and the organic matter undergoes profound changes, it is probable that during these changes the organic phosphorus compounds pass through several stages before the phosphorus is “mineralized” arid left in the residue. It is conceivable that in the course of these changes a portion of the phosphorus is produced in gaseous form and evolves from the sludge mixed with the other gases formed. Two hydrides of phosphorus, pK3 and (pHz),, which eshibit properties of interest in connection with hazards, are known. The first, phosphine, is an extremely poisonous gas, the formation of which has been reported during the bacterial reduction of organic and inorganic phosphorus compounds.
S
Rawn, Banta, and Pomeroy ( I O ) reported it as a constituent of digestion tank gases. The second, hydrogen hemiphosphide, is a spontaneously flammable substance which usually occurs as an impurity in phosphine, especially when the latter is prepared under alkaline conditions. If phosphine is produced with regularity from phosphorus compounds during sludge digestion, it is possible that apparently spontaneous digestion tank explosions might be due to the presence of such small traces of hydrogen hemiphosphide as commonly occur along with phosphine. For explosion it is necessary that oxygen be present. Oxygen for explosive combination with the gases may be present when digested sludge is drawn and air allowed to enter a tank or when digester gases leak into confined spaces where air is present. Kumerous investigators have been interested in the production of phosphine from both organic and inorganic materials, but many contradictions in the results obtained are found in the literature. One of the earliest reports on the production of phosphine is that of Selmi (II), who found that during the anaerobic decomposition of egg yolk, albumin.
