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Miscellaneous Observations on the A.O.A.C. Vitamin D Assay
Miscellaneous Observations on the A.O.A.C. Vitamin D Assay JAMES C. FRITZ AND HOBART R. HALLORAN
Borden's Nutritional Research Laboratory, Elgin, Illinois (Received for publication February 20, 1943)
M
UCH has been written and more has been said regarding the tentative A.O.A.C. chick assay method for vitamin D (1940). Some of the suggestions for improving the accuracy were studied by the Animal Vitamin Research Council (Baird and Barthen, 1941). Most investigators feel that there is definite need for improvement, but many believe that the greatest variation in response is within the chicks. The suggestion that two or more groups of chicks be used to test each sample should increase the accuracy of the result. There is, of course, a limit to the number of chicks which can be reasonably used. Since any attempt to breed or select strains for more uniform vitamin D requirements will be a long-time experiment, we cannot afford to overlook other parts of the method which might be modified to give better accuracy or to decrease the labor and time required to conduct these assays. During the course of assay work involving the use of about 70,000 chicks, a number of observations were made on the various steps in the method. While these ideas are not all original with the authors, we believe a resume will be of interest to others who are using this method. . EFFECT OF DIET ON CALCIFICATION
Many laboratories have adapted modified diets for use in routine assays. A new basal diet used in the A.V.R.C collaborative work produced results which were judged to be slightly superior to those obtained with the regular basal diet (Baird and Barthen, 1941). Loy, DeWitt, and
Knudsen (1941) found advantages in the use of a new diet. The work reported on the assay procedure by Johnson (1942) was based on a modified diet. While these diets differed among themselves, all were attempts to secure more nearly normal growth of chicks. An ideal basal diet would be one which was adequate in all respects except vitamin D, and which would permit optimum growth and development when supplemented with vitamin D. There is definite evidence that the degree of calcification is governed by the levels and ratio of calcium and phosphorus as well as by the level of vitamin D. The work of Nowotarski and Bird (1943) has stressed the influences of dietary calcium and phosphorus levels on the vitamin D requirement. Good calcification with very little vitamin D is undesirable in the assay diet. To guard against this possibility most test diets carry relatively low levels of calcium and a calcium:phosphorus ratio near 1:1. However, test rations with normal levels of calcium and phosphorus. have been successfully used for assay purposes and these usually permit more satisfactory growth of chicks. Table 1 shows observations made with seven vitamin D assay diets used by the authors. These show clearly that under some conditions good results can be obtained with diets containing fairly normal levels of calcium and phosphorus. Apparently some of the ingredients available contain less than the usual calcium content. In the A.O.A.C. basal diet as prepared by this laboratory, the calcium:phosphorus ratio is
1314]
315
MISCELLANEOUS OBSERVATIONS ON THE A.O.A.C. VITAMIN D ASSAY TABLE 1.—Effect
of assay diet on growth and bone ash A.O.A.C. Diet A D i e t B D i e t C D i e t D Diet E* D i e t F
Ingredient
percent 58.0 25.0
Ground yellow corn Wheat middlings Wheat bran Dehydrated alfalfa meal Casein Yeast Gelatin 50 percent meat scraps Calcium carbonate Tricalcium phosphate Potassium phosphate Iodized salt MnS0 4 -4H 2 0
— 12.0 2.0
— —
2.0
—
1.0 0.2 gm/kg.
Riboflavin
Percent calcium Percent phosphorus Percent bone ash (day-old chicks) 3wks. 0.0 A.O.A.C. units/100 3 wks. 5.0 A.O.A.C. units/100 3 wks. 10.0 A.O.A.C. units/100 3 wks. 15.0 A.O.A.C. units/100 3 wks. 20.0 A.O.A.C. units/100 3 wks. 25.0 A.O.A.C. units/100 3 wks. 30.0 A.O.A.C. units/100 Av. gms. gain/3 weeks 0.0 A.O.A.C. units/100 5.0 A.O.A.C. units/100 10.0 A.O.A.C. units/100 15.0 A.O.A.C. units/100 20.0 A.O.A.C. units/100 25.0 A.O.A.C. units/100 3O.O A.O.A.C. units/100
gms. gms. gms. gms. gms. gms. gms.
grams grams ' grams grams grams grams grams
percent percent percent percent percent 83.0 77.0 75.0 75.0 48.0 — — — — 25.0 .5.0 5.0 5.0 — — 2.0 12.0 12.0 12.0 12.0 15.0 2.0 2.0 2.0 2.0 4.0 1.0 1.0 1.0 1.0 — — 2.0 2.0 — — — 2.0 2.0 2.0 2.0 2.0 2.0 — — — — 1.0 1.0 1.0 1.0 1.0 0.2 0.2 0.2 0.2 0.2 gm/kg. gm/kg. gm/kg. gm/kg. gm/kg. 0.05 gm/100 lbs.
0.83 0.86
0.82 0.79
34.71 31.36
34.75
—
33.06 37.31 38.26 39.18 40.91 84
—
89 125 116 123 127
—. — —
40.41 41.75
— — — —
94 106
—
0.93 0.87
1.75 0.90
32.91 34.18 38.69 43.34 45.32 45.78
37.06
83 90 107 114 120 128
—
— —
34.61 38.37 43.34 45.32 47.90 47.90
30.39
38.55
— —• —
—
1.0 0.2 gm/kg. 0.05 gm/100 lbs. 1.37 1.04
—. — —
131
—
1.0 2.0
1.84 0.98
35.91
—.
—
2.0 15.0 4.0 1.0
0.89 1.36
43.37
121
percent 49.0 25.0
—
— — —
66
—
100
—
118 162 181 197 209 214
—
35.37 37.64 43.20
— 94
—
124 135 135
—
* This diet was not fed simultaneously with the others.
less than 1:1. Replacement of the middlings by additional ground yellow corn (diet A) gave higher bone ash with a uniform level of vitamin D. The work of Jones (1939) indicates that there is no specific anticalcifying factor in grains. Emslie and Migicovsky (1941) have found evidence that a calcium:phosphorus ratio of less than 1:1 tends to depress bone ash regardless of the level of vitamin D supplied. On the other hand a ratio of 2:1 tends to give high levels of calcification regardless of the level of vitamin D supplied. These workers also believe that with the
same ratio you can get different results by altering the calcium and phosphorus content of the ration. These results on the calcium:phosphorus ratio are clearly illustrated by the observations made with diets C and D. The addition of calcium increased rate of growth and the level of bone ash at any given level of vitamin D. Conversely, the addition of phosphorus depressed both growth rate and bone ash at any given level of vitamin D. Diet E was not fed simultaneously with the other diets shown in Table 1. Therefore, these results cannot be directly compared,
316
JAMES C. FRITZ AND HOBART R. HALLORAN TABLE 2.—Effect
Pen
1186 1187 1204 1205 1206 1207 1208 1209 1210 1211
of B-G complex source on bone ash
Basal Diet
Modified A.O.A.C.—2 percent Modified A.O.A.C.—2 percent A.O.A.C.—2 percent yeast A.O.A.C.—-2 percent yeast Modified A.O.A.C.—4 percent Modified A.O.A.C.—4 percent Modified A.O.A.C.—4 percent Modified A.O.A.C.—4 percent Modified A.O.A.C.—4 percent Modified A.O.A.C.—4 percent
yeast yeast yeast yeast condensed whey solubles condensed whey solubles Butyl alcohol residue Butyl alcohol residue
but they are included as an example of a diet with a normal calcium:phosphorus ratio which did give a wide range in bone ash due to varying levels of vitamin D. Following long experience with several of these diets, the best and most consistent results have been obtained with diet B. This diet is a relatively simple mixture which presents no difficulties in compounding and contains no ingredients likely to carry vitamin D. Both growth and bone ash are stimulated by the addition of vitamin D. The addition of vitamin A does not seem to influence the rate of growth of the chicks. Factors other than vitamin D, calcium, and phosphorus may influence calcification. Hammond, Miller, and McClure (1942) observed that heat treatment of a diet interfered with the ability to produce normal bone ash in chicks even when supplemented with 40 A.O.A.C. units of vitamin D per 100 grams of diet. Higher levels
Grams gain in three weeks
Percent bone ash Pen av.
82 74 76 67 88 81 82 91 98 101
33.06 33.66 31.87 33.39 32.57 34.32 33.72 35.12 35.77 35.79
Diet av. 33.36 32.63 33.44 34.42 35.78
of vitamin D or a combination of paraaminobenzoic acid, choline, and pantothenic acid improved the calcification. Table 2 shows the effect upon the level of bone ash when the source of the vitamin B-G complex in the basal ration is varied. Each of the five diets was supplemented with 10 A.O.A.C. units of vitamin D per 100 grams. The source of the vitamin D was reference cod liver oil. EFFECT OF VITAMIN D UPON GROWTH
The effect of vitamin D upon growth of chicks was suggested as a criterion for use, along with bone ash measurements, by Carver, Heiman, and Cook (1939). The conditions specified included a preliminary depletion period and rigorous culling of both heavy and light chicks when assembling the assay groups. Table 3 shows the average results on a large number of chicks used in reference groups on routine assays in this laboratory. While there is a definite effect
TABLE 3.—Average weights of White Leghorn chicks at three weeks on the A.O.A.C. test diet plus varying levels of vitamin D A.O.A.C. units vitamin D per 100 grams Average weight of chicks (grams) Number of pens averaged above Range of pen average weights
0
5
10
15
20
25
30
115
123
137
148
148
162
175
28 92-137
11 105-135
23 113-169
31 116-193
38 109-188
12 123-197
5 143-202
MISCELLANEOUS
OBSERVATIONS ON THE A.O.A.C. VITAMIN D ASSAY
317
TABLE 4.—Ash content of tibiae at four weeks Percent bone ash Pen No.
Right tibiae
Supplement & Est. Units/100 grams Left tibiae
412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437
None Ref. C.L.O. to supply 10 units Ref. C.L.O. to supply 15 units Ref. C.L.O. to supply 20 units Ref. C.L.O. to supply 25 units Ref. C.L.O. to supply 30 units No. 1067, est. to supply 15 units No. 1067, est. to supply 20 units No. 1067, est. to supply 25 units No. 1067, est. to supply 30 units No. 1176, est. to supply 15 units No. 1176, est. to supply 15 units No. 1176, est. to supply 20 units No. 1176, est. to supply 20 units No. 1195, est. to supply 15 units No. 1195, est. to supply 15 units No. 1195, est. to supply 20 units No. 1195, est. to supply 20 units No. 1196, est. to supply 15 units No. 1196, est. to supply 15 units No. 1196, est. to supply 20 units No. 1196, est. to supply 20 units No. 1197, est. to supply 15 units No. 1197, est. to supply 15 units No. 1197, est. to supply 20 units No. 1197, est. to supply 20 units
upon growth, this effect cannot be used for assay purposes without careful selection of the chicks. Observations on weight, however, serve as a valuable check upon possible errors in the ash determination. For example, if a group of chicks shows comparatively good growth but the bone ash results are low, a repetition of that assay is certainly indicated. Table 3 shows the average weights of White Leghorn chicks at three weeks on the A.O.A.C. basal diet with varying levels of vitamin D. It will be noted that there is a general increase in weight with increasing levels of vitamin D, but that there is considerable overlapping of the weight range for the different levels of vitamin D. PREPARATION OF BONES
Various workers have recommended the removal of cartilage caps in the preparation of leg bone samples for ash determination
27.96 27.99 29.00 32.08 33.54 36.20 29.71 33.02 34.19 40.04 30.34 30.13 33.51 31.55 31.84 30.89 33.16 32.77 31.62 30.84 33.43 33.15 31.93 30.85 33.07 33.68
Djaphyses
Epiphyses
40.02 40.58 41.67 46.00 46.31 48.12 41.73 44.92 46.87 49.89 43.59 43.41 45.25 44.43 44.61 43.68 44.14 45.28 43.16 42.22 45.68 44.94 44.26 42.84 45.91 44.88
7.82 7.74 9.58 10.27 10.28 11.46 8.96 11.20 11.59 13.13 10.02 10.01 10.60 9.93 9.01 9.49 10.74 11.66 10.38 9.51 10.92 10.57 10.87 8.95 10.36 11.80
(St. John, Kempf, and Bond, 1933; Harshaw, Fritz, and Titus, 1934; Johnson, 1942). The principal object sought was easier and more uniform removal of flesh from the bones. Table 4 shows the ash content of tibiae from a number of lots of chicks. These birds received the A.O.A.C. basal diet and as will be noted, the ash content was rather low and high levels of vitamin D were required to produce calcification approaching normal levels. The ash content is shown for the left tibiae with cartilage attached, for the shaft only of the right tibiae, and for the cartilage caps from the right tibiae. Table 5 shows the effect of the cartilage caps on the percentage of ash. The difference between the ash content of the left tibiae with cartilages and of the right tibiae without cartilages was greater than that observed by Johnson (1942). It should, however, be noted that the birds
318
JAMES C. FRITZ AND HOBART R. HAIXORAN TABLE 5.—Effect of ash level on difference with and without cartilage caps
Approx. units vitamin D per 100 grams 0 10 15 20 25 30
Av. Average percent ash wt. chicks at Right four Left tibiae weeks tibiae shaft
Difference
grams 131 141 155 164 166 189
12.06 12.59 12.41 12.20 12.72 10.88
27.96 27.99 30.71 32.94 33.87 38.12
40.02 40.58 43.12 45.14 46.59 49.00
studied by Johnson were on a modified ration and made greater gains in weight. There is some indication from the data in Table 5 to indicate that the heavier birds may show less difference due to cartilages. It is not clear whether such a difference, if it exists, is due to the weight of the chicks or to the degree of calcification. Table 6 shows the effect of these cartilage caps on the assay results. It will be seen TABLE 6.—Effect of Cartilage Caps on Assay
Sample
1067 1176 1195 1196 1197
Av. difference in percent bone ash due to cartilage 11.61 12.79 12.26 11.74 12.09
A.O.A.C. units vit. D found/gram Left tibiae 47,600.0 460.0 498.0 501.0 507.0
Right tibiae shaft only 44,900.0 458.0 470.0 447.0 468.0
that the differences in interpretation with or without the cartilage caps are within usual limits of biological variation. By usual cleaning methods, the bone samples can be prepared much more rapidly if the cartilage caps are removed. Johnson (1942) estimates that about one minute per bone is required when the cartilage caps are removed while about two minutes per
bone are required by the accepted method. It is, however, obvious that any operation requiring this amount of time is impractical when large numbers of assays by this method are required. One means to speed up the cleaning of the bones is to thoroughly cook the flesh without preliminary dissection. In this laboratory, the legs are removed from the chicks by cutting through the femur with scissors and then cutting through the joint between the tibia and metatarsus. No cleaning whatever is done at this stage. The tibia-metatarsus joint can be conveniently located. There is, however, chance of cutting the end of the tibiae if the operator attempts to cut between the proximal end of the tibia and the distal end of the femur without preliminary dissection. Group composite samples of the leg sections containing the tibiae are placed in boiling water for four minutes. While this may seem a long period of time, it has been clearly demonstrated that immersion in boiling water for periods up to six minutes did not influence the level of ash in chick bones. It must also be recalled that a part of the four minutes elapses before the bone itself reaches the temperature of the boiling water. At the end of four minutes, the bones are removed and cooled by immersion in cold water. After this treatment, the flesh is readily removed without the use of scissors or other cutting instruments. With a little experience, the operator can remove the flesh and clean the bone thoroughly in approximately 10 seconds. With four technicians, pens of 20 to 25 chicks can be weighed, killed, legs removed, and bones cleaned in an average time of approximately seven minutes per group. This procedure results in bones cleaned as carefully as is obtained by the slower and more laborious methods usually employed. The cartilage caps are retained so that the ash content can be compared readily with the usual figures in the literature.
MISCELLANEOUS OBSERVATIONS ON THE A.O.A.C. VITAMIN D ASSAY EXTRACTION
When bones are extracted by the usual methods it is apparently necessary to crush the samples in order to permit complete extraction of fat. Table 7 shows the results obtained following extraction for 20 hours TABLE 7.—Ask content of crushed and uncrushed bones
319
solvent placed in the extractor. In order to eliminate fire hazard, several laboratories are known to have used carbon tetrachloride as the fat solvent. This was the extraction method used in connection with the A.V.R.C. collaborative work (Baird and Barthen, 1941). Table 8 shows a comparison of bone ash TABLE 8.—Comparison of fat solvents
Ash content of tibiae Pen No. 1S2S 1526 1527 1528 1529 1530 1531 1532 1533 1534 1535 1536 1537 1538 1539 1540 1541 1542 1543 1544
Leftcrushed 31.47 35.65 34.04 35.24 38.46 38.91 40.55 41.10 40.50 42.38 42.51 41.53 42.89 43.36 42.14 44.14 42.84 42.32 36.04 39.47 Average Differeiice
Right— uncrushed 31.36 35.20 33.06 33.84 36.74 37.45 38.58 39.82 37.98 39.19 40.38 38.33 41.05 40.57 40.22 40.86 40.71 40.82 34.61 37.88 -1.85
with ethyl alcohol followed by 20 hours with diethyl ether. It will be noted that in all pens the uncrushed bones were found to have a lower percentage of ash. The only logical explanation is that a quantity of fat remained in the uncrushed samples. The A.O.A.C. method does not specifically describe the method to be used for fat extraction. Some laboratories adhere to the 20 hours with alcohol followed by 20 hours with ether as suggested in the A.O.A.C. book of methods. Other laboratories use ethyl alcohol or isopropyl alcohol only for periods varying from 40 to 72 hours. At least one laboratory uses two portions of ethyl alcohol. The first is discarded after eight hours' extraction and a fresh lot of
Extracting procedure Pen No.
20 hrs. alcohol + 2 0 hrs. ether
72 hrs.
287 288 289 290 291 292 293 294 295 296 297 298 309 310 311 312 313 314 315 316 337 338 339 340 341 342 343 344 345 346
26.50 31.43 36.95 39.36 35.97 32.57 39.43 35.70 35.86 38.25 41.57 38.41 38.20 41.80 43.37 45.57 44.20 45.46 45.28 47.53 30.92 32.45 33.14 36.94 32.26 33.54 32.18 32.34 39.45 35.62
27.29 28.77 35.27 39.02 34.53 31.26 38.79 33.91 34.31 36.90 41.72 38.45 35.65 39.37 40.81 41.51 41.73 43.43 45.22 46.45 25.05 26.82 28.19 32.37 28.45 28.73 29.30 27.71 34.76 32.65
Average
37.41
34.95
ecu
following extraction using 20 hours with ethyl alcohol followed by 20 hours with ethyl ether as contrasted to 72 hours' continuous extraction with carbon tetrachloride. In this test, the left tibiae were placed in alcohol and the extraction started immediately after dissection. The right tibiae were air dried to reduce moisture content before extraction with a commercial grade
320
JAMES C. FRITZ AND HOBART R. HALLORAN 15
44
"5
13 »
12' -»
D r y . F a t - F r e e Bones
-o
Green Bones 11
10
32
o
a
10
15
20
AOAC u n i t s V i t . V p e r 100 grams
9
FIG. 1. Vitamin D response curves comparing ash content of dry, fat-free bones and of green bones.
of carbon tetrachloride. Ninety percent of the paired lots showed higher bone ash following extraction with alcohol and ether. The average difference was nearly 2.5 percent bone ash. Examination of the carbon tetrachloride extract of the bones showed a high acid content and the presence of substantial quantities of calcium. It seems clear that under the conditions here used, the carbon tetrachloride broke down into free hydrochloric acid. This, in turn, extracted calcium and other minerals. Carbon tetrachloride can be used successfully for bone extraction according to Hubbell (1940). She, however, found it necessary to use several precautions. Only c.p. carbon tetrachloride should be used. The bones should be carefully dried in an oven to remove all moisture before they are
placed in the solvent. Calcium oxide is placed in the boiling flask to neutralize any acid which might be formed and to serve as a dehydrating agent. ASH DETERMINATION
Another precaution which must be observed is that one hour at 8S0°C. may not be adequate to completely ash large bones. In a few instances where larger chicks were examined for bone ash content, a further loss of weight was found when the bones were re-heated after one hour at 850°C. In all cases, complete ashing together with less chance for loss due to violent combustion followed placing the bones into the muffle furnace at about S00°C, raising the temperature rapidly to 850°C, and holding at this point for one hour.
321
MISCELLANEOUS OBSERVATIONS ON THE A.O.A.C. VITAMIN D ASSAY TABLE 9.—Comparison of ash in green bones and in dry, fat-free bones
Percent of ash Pen No. 759 760 761 762 764 769 770 773 774 775 776 778 780 . 777 779 781 782 783 784 785 786 787 788 789 790 791 792 793 794 795 796 797 798 799 800 801 802 803 804 805
Sample/100 grams
Negative Control Ref. CLO, 43.5 mgtas. Ref. CLO, 87.0 mgms. Ref. CLO, 130.5 mgms. Ref. CLO, 174.0 mgms. No. B-107, 25 grams No. B-108, 25 grams No. 1425, 37.5 mgms. No. 1425, 33.3 mgms. No. B-61, 100 mgms. No. 186, 0.8571 mgms. No. 186, 0.7500 mgms. No. 186, 0.6667 mgms. No. B-63, 30.0 mgms. No. B-64, 30.0 mgms. No. B-112, 0.075 mgms. No. 115, 37.5 mgms. No. B-94, 25 grams No. B-94, 23 grams No. B-85, 200 mgms. No. B-112, 0.075 mgms. Negative control Ref. CLO, 87.0 mgms. Ref. CLO, 130.5 mgms. Ref. CLO, 174.0 mgms. No. 226, 0.75 mgms. No. 226, 0.6818 mgms. No. B-137, 0.07317 mgms. No. B-137, 0.07317 mgms. No. B-136, 37.5 mgms. No. 216, 37.5 mgms. No. B-129, 25 grams No. B-130, 25 grams No. B-131, 25 grams No. B-132, 25 grams No. B-133, 25 grams No. B-134, 25 grams No. B-126, 25 grams No. B-127, 25 grams : No. B-128, 25 grams
Green bones
Dry" fat-free bones
9.09 10.39 11.79 13.62 14.59 11.39 10.68 14.47 13.20 10.93 14.88 14.00 12.69 12.50 13.20 14.43 14.48 13.59 11.81 11.08 15.19 9.89 11.58 13.59 15.67 14.23 14.07 14.11 13.98 11.59 12.28 9.62 13.78 15.78 13.48 15.13 14.66 9.02 12.28 15.40
32.98 36.14 38.79 42.00 43.94 37.65 35.41 45.171 41.62/ 37.29 44.621 43.31 41.41 39.95 42.22 44.61 44.72 42.92\ 38.55/ 38.35 46.65 32.92 38.62 42.36 44.88 43.92\ 42.96/ 42.69\ 43.71/ 36.73 40.36 32.62 41.82 45.00 40.50 45.11 43.57 30.98 39.22 45.76
Average difference in interpretation of samples
Direct ashing of the bone samples can be used successfully either to conserve solvents or to save time. The procedure used at times in this laboratory is essentially as follows: The freshly dissected group composite samples which we shall term "green bones" are weighed, placed in a tared ash crucible, and placed in a cold muffle furnace. The temperature is raised rapidly to 850°C. and held at this point for one hour. Figure 1 shows a compari-
A.O.A.C. units per gram Percent difference
Green bones
Dry fat-free bones
0 115 115 115 115 0.34 0.24 463
0 115 115 115 115 0.32 0.18 480
70
75
-
7
22,000
22,600
-
3
400 467 252,000 507 0.50
400 500 267,000 533 0.53
-
0 7 6 5 6
37.5 >267,O0O 0 115 115 115 22,460
46 >267,000 0 115 115 115 23,000
215,900
226,000
267 320 0 0.61 0.82 0.58 0.73 0.68 0 0.48 0.77
190 320 0 0.57 0.82 0.49 0.83 0.69 0 0.42 0.90
— — — — — +6
+33 - 4
-18
-
— — — — —
-
2 5
+41 0 0
+ 70 +18 -12 - 2 0 +14 -14
+1.1%
son of the response curves secured from the green right tibiae, and from the dry, fatfree left tibiae. While the percentage of ash was, of course, much lower, the response curves were similar. The percentage of ash in the green bones and the range due to varying intake of vitamin D is essentially the same as that found by Baird and MacMillan (1942) when they studied the use of toes rather than tibiae in the vitamin D determination. It should be noted, however,
322
JAMES C. FRITZ AND HOBART R. HALLORAN
that these workers ashed toe samples which were previously extracted with fat solvents. Table 9 shows that the difference in interpretation of the assays is, in most cases, as close as would be expected from the use of duplicate pens of chicks using the same ashing procedure. It will also be noted that the two cases showing differences which may be considered outside the limits of biological variation represented single pen determinations. This technic provides assay data on the samples within three hours after the chicks are killed. SUMMARY
1. The composition of the basal diet has a marked influence upon calcification which results from the addition of a specific level of vitamin D. The mineral content and possibly the source of the B-G complex influence the degree of calcification. 2. On an otherwise normal diet, the level of vitamin D regulates the growth of chicks receiving the diet. 3. In the preparation of bone samples, saving of time can result either from removal of the cartilage caps or by cooking the leg sections containing the bone for four minutes before removing the flesh. 4. Bones should be crushed before extraction of the fat. A variety of solvents are currently used. If carbon tetrachloride is used, precautions must be taken to prevent this solvent from breaking down into free acid. 5. The ashing of green bones permits interpretation of assays as accurately as does the ashing of dry, fat-free bones. This technic results in considerable saving of time and fat solvents.
REFERENCES
Association of Official Agricultural Chemists, 1940. Official and tentative methods of analysis. 5th ed., Washington, D.C. Baird, F. D., and C. L. Barthen, 1941. A collaborative study of the A.O.A.C. chick method of assay for vitamin D. Jour. A.O.A.C 24:961973. Baird, F. D., and M. J. MacMillan, 1942. Use of toes rather than tibiae in the A.O.A.C. chick method of vitamin D determination. Jour. A.O.A.C. 25:518-524. Carver, J. S., V. Heiman, and J. W. Cook, 1939. A curative method for the assay of vitamin D with chicks. Poultry Sci. 18:288-294. Emslie, A. R. G., 1941 and 1942. Personal communications. Some of these data were given by B. B. Migicovsky at the 1941 meeting of the Canadian Physiological Society. Hammond, J. C , D. Miller, and H. E. McClure, 1942. Interference of heated diets with calcification in chickens. Poultry Sci. 21:185-188. Harshaw, H. M., J. C. Fritz, and H. W. Titus, 1934. The normal development of the leg bones of chickens with respect to their ash content. Jour. Agr. Res. 48:997-1008. Hubbell, R. B. 1940. Personal communication. Johnson, S. R., 1942. Modification in the chick vitamin D assay procedure. Poultry Sci. 2 1 : 329-332. Jones, J. H., 1939. A comparison of cereal and non-cereal diets in the production of rickets. Jour. Nutr. 18:507-516. Loy, H. W., Jr., J. B. DeWitt, and L. F. Knudsen, 1941. Observations on the chick method for the assay of vitamin D. II. A modified basal ration. Jour. A.O.A.C* 24:432-440. Nowotarski, J. S., and H. R. Bird, 1943. Dietary calcium and phosphorus levels and the vitamin D requirement of the chick. Poultry Sci. 22:7278. St. John, J. L., C. Kempf, and L. Bond, 1933. Observations on the bone ash method of determining . effectiveness of vitamin D supplements. Poultry Sci. 12:34-36.
Borden's Nutritional Research Laboratory, Elgin, Illinois (Received for publication February 20, 1943)
M
UCH has been written and more has been said regarding the tentative A.O.A.C. chick assay method for vitamin D (1940). Some of the suggestions for improving the accuracy were studied by the Animal Vitamin Research Council (Baird and Barthen, 1941). Most investigators feel that there is definite need for improvement, but many believe that the greatest variation in response is within the chicks. The suggestion that two or more groups of chicks be used to test each sample should increase the accuracy of the result. There is, of course, a limit to the number of chicks which can be reasonably used. Since any attempt to breed or select strains for more uniform vitamin D requirements will be a long-time experiment, we cannot afford to overlook other parts of the method which might be modified to give better accuracy or to decrease the labor and time required to conduct these assays. During the course of assay work involving the use of about 70,000 chicks, a number of observations were made on the various steps in the method. While these ideas are not all original with the authors, we believe a resume will be of interest to others who are using this method. . EFFECT OF DIET ON CALCIFICATION
Many laboratories have adapted modified diets for use in routine assays. A new basal diet used in the A.V.R.C collaborative work produced results which were judged to be slightly superior to those obtained with the regular basal diet (Baird and Barthen, 1941). Loy, DeWitt, and
Knudsen (1941) found advantages in the use of a new diet. The work reported on the assay procedure by Johnson (1942) was based on a modified diet. While these diets differed among themselves, all were attempts to secure more nearly normal growth of chicks. An ideal basal diet would be one which was adequate in all respects except vitamin D, and which would permit optimum growth and development when supplemented with vitamin D. There is definite evidence that the degree of calcification is governed by the levels and ratio of calcium and phosphorus as well as by the level of vitamin D. The work of Nowotarski and Bird (1943) has stressed the influences of dietary calcium and phosphorus levels on the vitamin D requirement. Good calcification with very little vitamin D is undesirable in the assay diet. To guard against this possibility most test diets carry relatively low levels of calcium and a calcium:phosphorus ratio near 1:1. However, test rations with normal levels of calcium and phosphorus. have been successfully used for assay purposes and these usually permit more satisfactory growth of chicks. Table 1 shows observations made with seven vitamin D assay diets used by the authors. These show clearly that under some conditions good results can be obtained with diets containing fairly normal levels of calcium and phosphorus. Apparently some of the ingredients available contain less than the usual calcium content. In the A.O.A.C. basal diet as prepared by this laboratory, the calcium:phosphorus ratio is
1314]
315
MISCELLANEOUS OBSERVATIONS ON THE A.O.A.C. VITAMIN D ASSAY TABLE 1.—Effect
of assay diet on growth and bone ash A.O.A.C. Diet A D i e t B D i e t C D i e t D Diet E* D i e t F
Ingredient
percent 58.0 25.0
Ground yellow corn Wheat middlings Wheat bran Dehydrated alfalfa meal Casein Yeast Gelatin 50 percent meat scraps Calcium carbonate Tricalcium phosphate Potassium phosphate Iodized salt MnS0 4 -4H 2 0
— 12.0 2.0
— —
2.0
—
1.0 0.2 gm/kg.
Riboflavin
Percent calcium Percent phosphorus Percent bone ash (day-old chicks) 3wks. 0.0 A.O.A.C. units/100 3 wks. 5.0 A.O.A.C. units/100 3 wks. 10.0 A.O.A.C. units/100 3 wks. 15.0 A.O.A.C. units/100 3 wks. 20.0 A.O.A.C. units/100 3 wks. 25.0 A.O.A.C. units/100 3 wks. 30.0 A.O.A.C. units/100 Av. gms. gain/3 weeks 0.0 A.O.A.C. units/100 5.0 A.O.A.C. units/100 10.0 A.O.A.C. units/100 15.0 A.O.A.C. units/100 20.0 A.O.A.C. units/100 25.0 A.O.A.C. units/100 3O.O A.O.A.C. units/100
gms. gms. gms. gms. gms. gms. gms.
grams grams ' grams grams grams grams grams
percent percent percent percent percent 83.0 77.0 75.0 75.0 48.0 — — — — 25.0 .5.0 5.0 5.0 — — 2.0 12.0 12.0 12.0 12.0 15.0 2.0 2.0 2.0 2.0 4.0 1.0 1.0 1.0 1.0 — — 2.0 2.0 — — — 2.0 2.0 2.0 2.0 2.0 2.0 — — — — 1.0 1.0 1.0 1.0 1.0 0.2 0.2 0.2 0.2 0.2 gm/kg. gm/kg. gm/kg. gm/kg. gm/kg. 0.05 gm/100 lbs.
0.83 0.86
0.82 0.79
34.71 31.36
34.75
—
33.06 37.31 38.26 39.18 40.91 84
—
89 125 116 123 127
—. — —
40.41 41.75
— — — —
94 106
—
0.93 0.87
1.75 0.90
32.91 34.18 38.69 43.34 45.32 45.78
37.06
83 90 107 114 120 128
—
— —
34.61 38.37 43.34 45.32 47.90 47.90
30.39
38.55
— —• —
—
1.0 0.2 gm/kg. 0.05 gm/100 lbs. 1.37 1.04
—. — —
131
—
1.0 2.0
1.84 0.98
35.91
—.
—
2.0 15.0 4.0 1.0
0.89 1.36
43.37
121
percent 49.0 25.0
—
— — —
66
—
100
—
118 162 181 197 209 214
—
35.37 37.64 43.20
— 94
—
124 135 135
—
* This diet was not fed simultaneously with the others.
less than 1:1. Replacement of the middlings by additional ground yellow corn (diet A) gave higher bone ash with a uniform level of vitamin D. The work of Jones (1939) indicates that there is no specific anticalcifying factor in grains. Emslie and Migicovsky (1941) have found evidence that a calcium:phosphorus ratio of less than 1:1 tends to depress bone ash regardless of the level of vitamin D supplied. On the other hand a ratio of 2:1 tends to give high levels of calcification regardless of the level of vitamin D supplied. These workers also believe that with the
same ratio you can get different results by altering the calcium and phosphorus content of the ration. These results on the calcium:phosphorus ratio are clearly illustrated by the observations made with diets C and D. The addition of calcium increased rate of growth and the level of bone ash at any given level of vitamin D. Conversely, the addition of phosphorus depressed both growth rate and bone ash at any given level of vitamin D. Diet E was not fed simultaneously with the other diets shown in Table 1. Therefore, these results cannot be directly compared,
316
JAMES C. FRITZ AND HOBART R. HALLORAN TABLE 2.—Effect
Pen
1186 1187 1204 1205 1206 1207 1208 1209 1210 1211
of B-G complex source on bone ash
Basal Diet
Modified A.O.A.C.—2 percent Modified A.O.A.C.—2 percent A.O.A.C.—2 percent yeast A.O.A.C.—-2 percent yeast Modified A.O.A.C.—4 percent Modified A.O.A.C.—4 percent Modified A.O.A.C.—4 percent Modified A.O.A.C.—4 percent Modified A.O.A.C.—4 percent Modified A.O.A.C.—4 percent
yeast yeast yeast yeast condensed whey solubles condensed whey solubles Butyl alcohol residue Butyl alcohol residue
but they are included as an example of a diet with a normal calcium:phosphorus ratio which did give a wide range in bone ash due to varying levels of vitamin D. Following long experience with several of these diets, the best and most consistent results have been obtained with diet B. This diet is a relatively simple mixture which presents no difficulties in compounding and contains no ingredients likely to carry vitamin D. Both growth and bone ash are stimulated by the addition of vitamin D. The addition of vitamin A does not seem to influence the rate of growth of the chicks. Factors other than vitamin D, calcium, and phosphorus may influence calcification. Hammond, Miller, and McClure (1942) observed that heat treatment of a diet interfered with the ability to produce normal bone ash in chicks even when supplemented with 40 A.O.A.C. units of vitamin D per 100 grams of diet. Higher levels
Grams gain in three weeks
Percent bone ash Pen av.
82 74 76 67 88 81 82 91 98 101
33.06 33.66 31.87 33.39 32.57 34.32 33.72 35.12 35.77 35.79
Diet av. 33.36 32.63 33.44 34.42 35.78
of vitamin D or a combination of paraaminobenzoic acid, choline, and pantothenic acid improved the calcification. Table 2 shows the effect upon the level of bone ash when the source of the vitamin B-G complex in the basal ration is varied. Each of the five diets was supplemented with 10 A.O.A.C. units of vitamin D per 100 grams. The source of the vitamin D was reference cod liver oil. EFFECT OF VITAMIN D UPON GROWTH
The effect of vitamin D upon growth of chicks was suggested as a criterion for use, along with bone ash measurements, by Carver, Heiman, and Cook (1939). The conditions specified included a preliminary depletion period and rigorous culling of both heavy and light chicks when assembling the assay groups. Table 3 shows the average results on a large number of chicks used in reference groups on routine assays in this laboratory. While there is a definite effect
TABLE 3.—Average weights of White Leghorn chicks at three weeks on the A.O.A.C. test diet plus varying levels of vitamin D A.O.A.C. units vitamin D per 100 grams Average weight of chicks (grams) Number of pens averaged above Range of pen average weights
0
5
10
15
20
25
30
115
123
137
148
148
162
175
28 92-137
11 105-135
23 113-169
31 116-193
38 109-188
12 123-197
5 143-202
MISCELLANEOUS
OBSERVATIONS ON THE A.O.A.C. VITAMIN D ASSAY
317
TABLE 4.—Ash content of tibiae at four weeks Percent bone ash Pen No.
Right tibiae
Supplement & Est. Units/100 grams Left tibiae
412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437
None Ref. C.L.O. to supply 10 units Ref. C.L.O. to supply 15 units Ref. C.L.O. to supply 20 units Ref. C.L.O. to supply 25 units Ref. C.L.O. to supply 30 units No. 1067, est. to supply 15 units No. 1067, est. to supply 20 units No. 1067, est. to supply 25 units No. 1067, est. to supply 30 units No. 1176, est. to supply 15 units No. 1176, est. to supply 15 units No. 1176, est. to supply 20 units No. 1176, est. to supply 20 units No. 1195, est. to supply 15 units No. 1195, est. to supply 15 units No. 1195, est. to supply 20 units No. 1195, est. to supply 20 units No. 1196, est. to supply 15 units No. 1196, est. to supply 15 units No. 1196, est. to supply 20 units No. 1196, est. to supply 20 units No. 1197, est. to supply 15 units No. 1197, est. to supply 15 units No. 1197, est. to supply 20 units No. 1197, est. to supply 20 units
upon growth, this effect cannot be used for assay purposes without careful selection of the chicks. Observations on weight, however, serve as a valuable check upon possible errors in the ash determination. For example, if a group of chicks shows comparatively good growth but the bone ash results are low, a repetition of that assay is certainly indicated. Table 3 shows the average weights of White Leghorn chicks at three weeks on the A.O.A.C. basal diet with varying levels of vitamin D. It will be noted that there is a general increase in weight with increasing levels of vitamin D, but that there is considerable overlapping of the weight range for the different levels of vitamin D. PREPARATION OF BONES
Various workers have recommended the removal of cartilage caps in the preparation of leg bone samples for ash determination
27.96 27.99 29.00 32.08 33.54 36.20 29.71 33.02 34.19 40.04 30.34 30.13 33.51 31.55 31.84 30.89 33.16 32.77 31.62 30.84 33.43 33.15 31.93 30.85 33.07 33.68
Djaphyses
Epiphyses
40.02 40.58 41.67 46.00 46.31 48.12 41.73 44.92 46.87 49.89 43.59 43.41 45.25 44.43 44.61 43.68 44.14 45.28 43.16 42.22 45.68 44.94 44.26 42.84 45.91 44.88
7.82 7.74 9.58 10.27 10.28 11.46 8.96 11.20 11.59 13.13 10.02 10.01 10.60 9.93 9.01 9.49 10.74 11.66 10.38 9.51 10.92 10.57 10.87 8.95 10.36 11.80
(St. John, Kempf, and Bond, 1933; Harshaw, Fritz, and Titus, 1934; Johnson, 1942). The principal object sought was easier and more uniform removal of flesh from the bones. Table 4 shows the ash content of tibiae from a number of lots of chicks. These birds received the A.O.A.C. basal diet and as will be noted, the ash content was rather low and high levels of vitamin D were required to produce calcification approaching normal levels. The ash content is shown for the left tibiae with cartilage attached, for the shaft only of the right tibiae, and for the cartilage caps from the right tibiae. Table 5 shows the effect of the cartilage caps on the percentage of ash. The difference between the ash content of the left tibiae with cartilages and of the right tibiae without cartilages was greater than that observed by Johnson (1942). It should, however, be noted that the birds
318
JAMES C. FRITZ AND HOBART R. HAIXORAN TABLE 5.—Effect of ash level on difference with and without cartilage caps
Approx. units vitamin D per 100 grams 0 10 15 20 25 30
Av. Average percent ash wt. chicks at Right four Left tibiae weeks tibiae shaft
Difference
grams 131 141 155 164 166 189
12.06 12.59 12.41 12.20 12.72 10.88
27.96 27.99 30.71 32.94 33.87 38.12
40.02 40.58 43.12 45.14 46.59 49.00
studied by Johnson were on a modified ration and made greater gains in weight. There is some indication from the data in Table 5 to indicate that the heavier birds may show less difference due to cartilages. It is not clear whether such a difference, if it exists, is due to the weight of the chicks or to the degree of calcification. Table 6 shows the effect of these cartilage caps on the assay results. It will be seen TABLE 6.—Effect of Cartilage Caps on Assay
Sample
1067 1176 1195 1196 1197
Av. difference in percent bone ash due to cartilage 11.61 12.79 12.26 11.74 12.09
A.O.A.C. units vit. D found/gram Left tibiae 47,600.0 460.0 498.0 501.0 507.0
Right tibiae shaft only 44,900.0 458.0 470.0 447.0 468.0
that the differences in interpretation with or without the cartilage caps are within usual limits of biological variation. By usual cleaning methods, the bone samples can be prepared much more rapidly if the cartilage caps are removed. Johnson (1942) estimates that about one minute per bone is required when the cartilage caps are removed while about two minutes per
bone are required by the accepted method. It is, however, obvious that any operation requiring this amount of time is impractical when large numbers of assays by this method are required. One means to speed up the cleaning of the bones is to thoroughly cook the flesh without preliminary dissection. In this laboratory, the legs are removed from the chicks by cutting through the femur with scissors and then cutting through the joint between the tibia and metatarsus. No cleaning whatever is done at this stage. The tibia-metatarsus joint can be conveniently located. There is, however, chance of cutting the end of the tibiae if the operator attempts to cut between the proximal end of the tibia and the distal end of the femur without preliminary dissection. Group composite samples of the leg sections containing the tibiae are placed in boiling water for four minutes. While this may seem a long period of time, it has been clearly demonstrated that immersion in boiling water for periods up to six minutes did not influence the level of ash in chick bones. It must also be recalled that a part of the four minutes elapses before the bone itself reaches the temperature of the boiling water. At the end of four minutes, the bones are removed and cooled by immersion in cold water. After this treatment, the flesh is readily removed without the use of scissors or other cutting instruments. With a little experience, the operator can remove the flesh and clean the bone thoroughly in approximately 10 seconds. With four technicians, pens of 20 to 25 chicks can be weighed, killed, legs removed, and bones cleaned in an average time of approximately seven minutes per group. This procedure results in bones cleaned as carefully as is obtained by the slower and more laborious methods usually employed. The cartilage caps are retained so that the ash content can be compared readily with the usual figures in the literature.
MISCELLANEOUS OBSERVATIONS ON THE A.O.A.C. VITAMIN D ASSAY EXTRACTION
When bones are extracted by the usual methods it is apparently necessary to crush the samples in order to permit complete extraction of fat. Table 7 shows the results obtained following extraction for 20 hours TABLE 7.—Ask content of crushed and uncrushed bones
319
solvent placed in the extractor. In order to eliminate fire hazard, several laboratories are known to have used carbon tetrachloride as the fat solvent. This was the extraction method used in connection with the A.V.R.C. collaborative work (Baird and Barthen, 1941). Table 8 shows a comparison of bone ash TABLE 8.—Comparison of fat solvents
Ash content of tibiae Pen No. 1S2S 1526 1527 1528 1529 1530 1531 1532 1533 1534 1535 1536 1537 1538 1539 1540 1541 1542 1543 1544
Leftcrushed 31.47 35.65 34.04 35.24 38.46 38.91 40.55 41.10 40.50 42.38 42.51 41.53 42.89 43.36 42.14 44.14 42.84 42.32 36.04 39.47 Average Differeiice
Right— uncrushed 31.36 35.20 33.06 33.84 36.74 37.45 38.58 39.82 37.98 39.19 40.38 38.33 41.05 40.57 40.22 40.86 40.71 40.82 34.61 37.88 -1.85
with ethyl alcohol followed by 20 hours with diethyl ether. It will be noted that in all pens the uncrushed bones were found to have a lower percentage of ash. The only logical explanation is that a quantity of fat remained in the uncrushed samples. The A.O.A.C. method does not specifically describe the method to be used for fat extraction. Some laboratories adhere to the 20 hours with alcohol followed by 20 hours with ether as suggested in the A.O.A.C. book of methods. Other laboratories use ethyl alcohol or isopropyl alcohol only for periods varying from 40 to 72 hours. At least one laboratory uses two portions of ethyl alcohol. The first is discarded after eight hours' extraction and a fresh lot of
Extracting procedure Pen No.
20 hrs. alcohol + 2 0 hrs. ether
72 hrs.
287 288 289 290 291 292 293 294 295 296 297 298 309 310 311 312 313 314 315 316 337 338 339 340 341 342 343 344 345 346
26.50 31.43 36.95 39.36 35.97 32.57 39.43 35.70 35.86 38.25 41.57 38.41 38.20 41.80 43.37 45.57 44.20 45.46 45.28 47.53 30.92 32.45 33.14 36.94 32.26 33.54 32.18 32.34 39.45 35.62
27.29 28.77 35.27 39.02 34.53 31.26 38.79 33.91 34.31 36.90 41.72 38.45 35.65 39.37 40.81 41.51 41.73 43.43 45.22 46.45 25.05 26.82 28.19 32.37 28.45 28.73 29.30 27.71 34.76 32.65
Average
37.41
34.95
ecu
following extraction using 20 hours with ethyl alcohol followed by 20 hours with ethyl ether as contrasted to 72 hours' continuous extraction with carbon tetrachloride. In this test, the left tibiae were placed in alcohol and the extraction started immediately after dissection. The right tibiae were air dried to reduce moisture content before extraction with a commercial grade
320
JAMES C. FRITZ AND HOBART R. HALLORAN 15
44
"5
13 »
12' -»
D r y . F a t - F r e e Bones
-o
Green Bones 11
10
32
o
a
10
15
20
AOAC u n i t s V i t . V p e r 100 grams
9
FIG. 1. Vitamin D response curves comparing ash content of dry, fat-free bones and of green bones.
of carbon tetrachloride. Ninety percent of the paired lots showed higher bone ash following extraction with alcohol and ether. The average difference was nearly 2.5 percent bone ash. Examination of the carbon tetrachloride extract of the bones showed a high acid content and the presence of substantial quantities of calcium. It seems clear that under the conditions here used, the carbon tetrachloride broke down into free hydrochloric acid. This, in turn, extracted calcium and other minerals. Carbon tetrachloride can be used successfully for bone extraction according to Hubbell (1940). She, however, found it necessary to use several precautions. Only c.p. carbon tetrachloride should be used. The bones should be carefully dried in an oven to remove all moisture before they are
placed in the solvent. Calcium oxide is placed in the boiling flask to neutralize any acid which might be formed and to serve as a dehydrating agent. ASH DETERMINATION
Another precaution which must be observed is that one hour at 8S0°C. may not be adequate to completely ash large bones. In a few instances where larger chicks were examined for bone ash content, a further loss of weight was found when the bones were re-heated after one hour at 850°C. In all cases, complete ashing together with less chance for loss due to violent combustion followed placing the bones into the muffle furnace at about S00°C, raising the temperature rapidly to 850°C, and holding at this point for one hour.
321
MISCELLANEOUS OBSERVATIONS ON THE A.O.A.C. VITAMIN D ASSAY TABLE 9.—Comparison of ash in green bones and in dry, fat-free bones
Percent of ash Pen No. 759 760 761 762 764 769 770 773 774 775 776 778 780 . 777 779 781 782 783 784 785 786 787 788 789 790 791 792 793 794 795 796 797 798 799 800 801 802 803 804 805
Sample/100 grams
Negative Control Ref. CLO, 43.5 mgtas. Ref. CLO, 87.0 mgms. Ref. CLO, 130.5 mgms. Ref. CLO, 174.0 mgms. No. B-107, 25 grams No. B-108, 25 grams No. 1425, 37.5 mgms. No. 1425, 33.3 mgms. No. B-61, 100 mgms. No. 186, 0.8571 mgms. No. 186, 0.7500 mgms. No. 186, 0.6667 mgms. No. B-63, 30.0 mgms. No. B-64, 30.0 mgms. No. B-112, 0.075 mgms. No. 115, 37.5 mgms. No. B-94, 25 grams No. B-94, 23 grams No. B-85, 200 mgms. No. B-112, 0.075 mgms. Negative control Ref. CLO, 87.0 mgms. Ref. CLO, 130.5 mgms. Ref. CLO, 174.0 mgms. No. 226, 0.75 mgms. No. 226, 0.6818 mgms. No. B-137, 0.07317 mgms. No. B-137, 0.07317 mgms. No. B-136, 37.5 mgms. No. 216, 37.5 mgms. No. B-129, 25 grams No. B-130, 25 grams No. B-131, 25 grams No. B-132, 25 grams No. B-133, 25 grams No. B-134, 25 grams No. B-126, 25 grams No. B-127, 25 grams : No. B-128, 25 grams
Green bones
Dry" fat-free bones
9.09 10.39 11.79 13.62 14.59 11.39 10.68 14.47 13.20 10.93 14.88 14.00 12.69 12.50 13.20 14.43 14.48 13.59 11.81 11.08 15.19 9.89 11.58 13.59 15.67 14.23 14.07 14.11 13.98 11.59 12.28 9.62 13.78 15.78 13.48 15.13 14.66 9.02 12.28 15.40
32.98 36.14 38.79 42.00 43.94 37.65 35.41 45.171 41.62/ 37.29 44.621 43.31 41.41 39.95 42.22 44.61 44.72 42.92\ 38.55/ 38.35 46.65 32.92 38.62 42.36 44.88 43.92\ 42.96/ 42.69\ 43.71/ 36.73 40.36 32.62 41.82 45.00 40.50 45.11 43.57 30.98 39.22 45.76
Average difference in interpretation of samples
Direct ashing of the bone samples can be used successfully either to conserve solvents or to save time. The procedure used at times in this laboratory is essentially as follows: The freshly dissected group composite samples which we shall term "green bones" are weighed, placed in a tared ash crucible, and placed in a cold muffle furnace. The temperature is raised rapidly to 850°C. and held at this point for one hour. Figure 1 shows a compari-
A.O.A.C. units per gram Percent difference
Green bones
Dry fat-free bones
0 115 115 115 115 0.34 0.24 463
0 115 115 115 115 0.32 0.18 480
70
75
-
7
22,000
22,600
-
3
400 467 252,000 507 0.50
400 500 267,000 533 0.53
-
0 7 6 5 6
37.5 >267,O0O 0 115 115 115 22,460
46 >267,000 0 115 115 115 23,000
215,900
226,000
267 320 0 0.61 0.82 0.58 0.73 0.68 0 0.48 0.77
190 320 0 0.57 0.82 0.49 0.83 0.69 0 0.42 0.90
— — — — — +6
+33 - 4
-18
-
— — — — —
-
2 5
+41 0 0
+ 70 +18 -12 - 2 0 +14 -14
+1.1%
son of the response curves secured from the green right tibiae, and from the dry, fatfree left tibiae. While the percentage of ash was, of course, much lower, the response curves were similar. The percentage of ash in the green bones and the range due to varying intake of vitamin D is essentially the same as that found by Baird and MacMillan (1942) when they studied the use of toes rather than tibiae in the vitamin D determination. It should be noted, however,
322
JAMES C. FRITZ AND HOBART R. HALLORAN
that these workers ashed toe samples which were previously extracted with fat solvents. Table 9 shows that the difference in interpretation of the assays is, in most cases, as close as would be expected from the use of duplicate pens of chicks using the same ashing procedure. It will also be noted that the two cases showing differences which may be considered outside the limits of biological variation represented single pen determinations. This technic provides assay data on the samples within three hours after the chicks are killed. SUMMARY
1. The composition of the basal diet has a marked influence upon calcification which results from the addition of a specific level of vitamin D. The mineral content and possibly the source of the B-G complex influence the degree of calcification. 2. On an otherwise normal diet, the level of vitamin D regulates the growth of chicks receiving the diet. 3. In the preparation of bone samples, saving of time can result either from removal of the cartilage caps or by cooking the leg sections containing the bone for four minutes before removing the flesh. 4. Bones should be crushed before extraction of the fat. A variety of solvents are currently used. If carbon tetrachloride is used, precautions must be taken to prevent this solvent from breaking down into free acid. 5. The ashing of green bones permits interpretation of assays as accurately as does the ashing of dry, fat-free bones. This technic results in considerable saving of time and fat solvents.
REFERENCES
Association of Official Agricultural Chemists, 1940. Official and tentative methods of analysis. 5th ed., Washington, D.C. Baird, F. D., and C. L. Barthen, 1941. A collaborative study of the A.O.A.C. chick method of assay for vitamin D. Jour. A.O.A.C 24:961973. Baird, F. D., and M. J. MacMillan, 1942. Use of toes rather than tibiae in the A.O.A.C. chick method of vitamin D determination. Jour. A.O.A.C. 25:518-524. Carver, J. S., V. Heiman, and J. W. Cook, 1939. A curative method for the assay of vitamin D with chicks. Poultry Sci. 18:288-294. Emslie, A. R. G., 1941 and 1942. Personal communications. Some of these data were given by B. B. Migicovsky at the 1941 meeting of the Canadian Physiological Society. Hammond, J. C , D. Miller, and H. E. McClure, 1942. Interference of heated diets with calcification in chickens. Poultry Sci. 21:185-188. Harshaw, H. M., J. C. Fritz, and H. W. Titus, 1934. The normal development of the leg bones of chickens with respect to their ash content. Jour. Agr. Res. 48:997-1008. Hubbell, R. B. 1940. Personal communication. Johnson, S. R., 1942. Modification in the chick vitamin D assay procedure. Poultry Sci. 2 1 : 329-332. Jones, J. H., 1939. A comparison of cereal and non-cereal diets in the production of rickets. Jour. Nutr. 18:507-516. Loy, H. W., Jr., J. B. DeWitt, and L. F. Knudsen, 1941. Observations on the chick method for the assay of vitamin D. II. A modified basal ration. Jour. A.O.A.C* 24:432-440. Nowotarski, J. S., and H. R. Bird, 1943. Dietary calcium and phosphorus levels and the vitamin D requirement of the chick. Poultry Sci. 22:7278. St. John, J. L., C. Kempf, and L. Bond, 1933. Observations on the bone ash method of determining . effectiveness of vitamin D supplements. Poultry Sci. 12:34-36.