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Nutritional Anemia, Calcium Phosphorus and Nitrogen Balance and Bone Composition of Rats Fed Raw versus Pasteurized Milk*
N U T R I T I O N A L A N E M I A , CALCIU1V[ P H O S P H O R U S A N D N I T R O G E N B A L A N C E A N D B O N E C O M P O S I T I O N OF R A T S FED RAW VERSUS PASTEURIZED MILK ~ 1t. A. L A S B Y AND L. S. PALtrIER
Division of Agricultural Biochemistry, University of Minnesota, St. Paul. PART I. NUTRITIONAL ANEMIA
Two groups of workers ]lave concerned themselves with the relative values of sole diets of raw or pasteurized milk in producing nutritional anemia in the rat. Scott and E r r (1) r e p o r t results showing t h a t a special milk, when fed raw, allowed a better gain in weight and greater increase in red cell count and hemoglobin than when the same milk was fed a f t e r holding pasteurization. Some commercially pasteurized milk gave inferior results to those obtained with the special pasteurized milk. I t is not stated whether the rats were fed individually or in groups and what their food consumptions were. Neither are a n y copper and iron analyses of the milk reported. Krauss, Erb, and W a s h b u r n (2) report results c o n t r a r y to those of Scott and E r f . They fed milk f r o m the same bulk to two groups of rats, one group receiving raw milk and the other the same milk a f t e r pasteurization. The animals in both groups developed nutritional anemia as characterized by a marked decrease in hemoglobin and erythrocyte count and the decreases were at the same rate in both groups. No data are given for the food intake or growth. EXPERIMENTAL
Methods. The procedure of E l v e h j e m and K e m m e r e r (3) was followed in rendering the rats anemic. The young were weaned at three weeks, and then placed on the e x p e r i m e n t a l diet of milk alone for an eight weeks period. The rats were housed in individual galvanized wire cages with ½. inch mesh screen bottoms. The two series of experiments conducted were similar except in the milk fed. They were carried out b y the paired feeding method using males only. Both members of any one p a i r were litter mates and as similar as possible as to weight at the beginning of the experimental period. Those on the sole milk diet received no water. The control group consisted of litter mates fed on the stock diet and w a t e r ad lib. The rats in both series were weighed weekly and food consumption recReceived for publication November 6, 1934. The data presented are t a k e n in large p a r t from the thesis of H. A. Lasby presented to the Graduate School of the University of Minnesota in partial fulfillment of the requirements for the degree of Doctor of. Philosophy. All pertinent individual data are available for reference in the Division of Agricultural Bioche~rei~try. P a p e r l~o. 1320 Journal Series, Minnesota Agricultural Experiment Station. 181
182
H. A. LASBY AND L. S. PALMER
ords wer.e kept. Hemoglobin determinations were made at the time of weaning and weeldy thereafter. The method used was t h a t of Newcomer (4). Blood was obtained by cutting with a razor the tip of the tail, previously dipped in hot w a t e r to promote the flow of blood. Calcium chloride was first used to stop the flow of blood but later collodion was substituted, as it proved to be more satisfaotory. Series I. Seven p a i r s and three controls were used. The milk fed was obtained fresh daily, except Sunday, f r o m the University of Minnesota A g r i c u l t u r a l E x p e r i m e n t Station D a i r y herd. The experiment was conducted during May, J u n e and J u l y when the cows were on pasture. No special containers were used for ,the collection of the milk. I t was b r o u g h t up f r o m the barns in a tin pail f r o m which it was t r a n s f e r r e d to glass containers for storage and pasteurization. A portion of each d a y ' s milk was fed raw to one group ahd a portion was pasteurized in glass and fed as such to the other. F o r pasteurization, t h e milk was placed in stoppered 1 l. E r l e n m e y e r flasks, which were then immersed to the neck in a w a t e r bath. A t e m p e r a t u r e of 620-63 ° C. was maintained in the flask for 30 minutes. Subsequently the flasks and milk were cooled as r a p i d l y as possible in running cold water and stored, as was the raw milk, in a cold room at 40 ° C. No a t t e m p t was made to standardize the fat content of the milk fed. Feedings were made morning and afternoon. Definite quantities of milk were measured by pipette into glazed porcelain ramekins placed on the Screen bottom of the cage. A n y milk unconsumed at the next feeding was measured. Series I I . Eleven pairs and three controls were started. The milk used was commercial 1 raw and pasteurized milk obtained during F e b r u a r y and March, thus r e p r e s e n t i n g late winter feeding. Both were obtained f r o m the same bulk. I t contained 3.5 p e r cent b u t t e r fat. The pasteurization had been carried out in Y u n d t - G r i d l e y stainless steel tanks b y the holding process at 143.5 ° F. (62 ° C.) for 30 minutes. Q u a r t aliquots were taken each d a y and delivered for consumption the following day, except S a t u r d a y , when two quarts of F r i d a y ' s raw and pasteurized milk were delivered for S a t u r d a y ' s and S u n d a y ' s use. Chemical analyses. Analyses for iron and copper were made on the raw a n d pasteurized milk. I n series I r a n d o m samples were taken every week and analyzed. I n Series I I , 100 cc. aliquots were taken daily and the analyses made on weekly composites. The method used for iron was t h a t of E l v e h j e m and H a r t (5), K e n n e d y (6), and E l v e h j e m (7). I n Series I the copper was determined by the method of E l v e h j e m and Lindow (8) This milk was furnished through the courtesy of the Franklin Cooperative Creamery Association of Minneapolis. We acknowledge our indebtedness to them for it, and especially to Ass 't. Supt. Waiter Ahlstrom, for insuring the taking of samples and their
delivery.
183
NUTRITIONAL ANEMIA
as modified by Gebhardt and Sommers (9) and in Series I I by the method of Cherbuliez and Ansbacher (10) and Ansbacher, Remington and Culp (11). The latter was believed to be more accurate a n d certainly less tedious. Results. Series I. The results obtained are presented graphically in figure 1. The mean hemoglobin value for the raw milk group decreased
!,,o
"
Growff,
a~
'~F
Her, o?/o~,,
Food Con~p~,~n
Mean I
Control Mean i
I
I
5 wh. periocl~
FIG. 1. Hemoglobin content of, blood, body weight and milk consumption of individual rats fed raw and laboratory pasteurized summer milk from Station Dairy herd on pasture, together with mean values and the means of the blood hemoglobin and body weight of controls fed stock diet. The continuous lines represent raw milk feeding and the dotted lines pasteurized milk. ~he death of an animal before the end of the eight weeks period is starred. from 7.80 to 2.31 gms. per 100 cc. while that of the pasteurized group decreased from 8.08 to 1.88 in eight weeks. The mean weight of the rats fed raw milk increased from 33.14 to 83.5 gms. in contrast to an increase from 33.71 to 78.5 gins. for those fed pasteurized milk. The data were ti'eated statistically to test for the significance of the differences between the means of hemoglobin and the weight values obtained from the t w o groups of rats. Using Fischer's application of ' S t u d e n t s " distribution of t and the 5 per cent level of significance the data in table 1 show that the differences for each successive weekly interval were not significant except for the fifth week hemoglobin values. This c a n n o t be considered important inasmuch
184
H . A . LASBY AND L. S. :PALMER TABLE 1
Statistical significance of weekly differenves in mean hemoglobin content of blood and body weight of rats fed raw and pasteurized milk in paired feeding tests CALCULATIONS
FROM HEMOGLOBIN
CALCULATIONS
FROM BODY WEIGI-IT
Week
t
P
t
.P
0 1 2 3 4 5 6 7 8
1.9277 1.4279 1.0463 0.8717 0.3036 3.4662 0.5587 1.3859 1.9765
0.0780 .1786 .3194 .4020 .7704 .0062 .5914 .1086 .0966
0.8738 0.8356 0.3999 1.5515 0.5104 0.4004 0.1887 ~.0751 1.7363
0.4018 .4228 .6962 .1582 .6212 .6976 .8542 .0914 .1320
............ ............ ............ ............ ............ ............ ............ ............ ............
as no s t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e o c c u r r e d i n e i t h e r t h e p r e c e d i n g or s u c c e e d i n g week. T h e c o p p e r a n d i r o n a n a l y s e s of t h e r a w a n d p a s t e u r i z e d m i l k gave r e s u l t s i n d i c a t i n g t h a t t h e s a m e q u a n t i t y of m e t a l s wa~ p r e s e n t in each. S i x i r o n a n a l y s e s on r a w m i l k m a d e d u r i n g t h e e i g h t weeks p e r i o d v a r i e d f r o m 1.2.3 to 1.48 mgms. p e r l i t e r w i t h a m e a n of 1.35 a n d on t h e same m i l k a f t e r p a s t e u r i z a t i o n f r o m 1.20 to 1.48 mgms. p e r l i t e r w i t h a m e a n of 1.35. S i x c o p p e r a n a l y s e s m a d e on t h e same m i l k v a r i e d f r o m 0.11 to 0.17 mgms. p e r l i t e r w i t h a m e a n of 0.14 while those on t h e p a s t e u r i z e d m i l k v a r i e d f r o m 0.12 to 0.16 mgms. p e r l i t e r w i t h a m e a n of 0.14 mgm. Series I I . S i n c e the c o n d i t i o n s u s e d in l a b o r a t o r y p a s t e u r i z a t i o n , a r e n o t those u n d e r w h i c h m a r k e t m i l k is t r e a t e d , a series was r u n in o r d e r to d e t e r m i n e w h e t h e r c o m m e r c i a l p a s t e u r i z a t i o n h a d a n y effect on the s e v e r i t y of the a n e m i a p r o d u c e d b y f e e d i n g it as the sole d i e t to rats. T h e r e s u l t s o b t a i n e d a r e p l o t t e d in F i g u r e 2. P a i r n u m b e r e i g h t was o m i t t e d f r o m a l l c a l c u l a t i o n s b e c a u s e t h a t m e m b e r of t h e p a i r b e i n g f e d p a s t e u r i z e d m41k e s c a p e d f r o m t h e cage one n i g h t a n d t h e r e was a subseq u e n t rise in h e m o g l o b i n . I t m a y be seen t h a t t h e r e was a m u c h w i d e r s p r e a d b e t w e e n t h e hemog l o b i n a n d g r o w t h c u r v e s of t h e two r a t s of a p a i r t h a n in those of S e r i e s I. I n all cases t h a t m e m b e r of a p a i r r e c e i v i n g p a s t e u r i z e d m i l k g r e w b e t t e r a n d h a d m o r e h e m o g l o b i n . I f t h e r a t on r a w m i l k h a d a h i g h e r h e m o g l o b i n o r w e i g h e d m o r e at t h e b e g i n n i n g of t h e e x p e r i m e n t , t h e r e was a subseq u e n t l y p o o r e r g r o w t h or a g r e a t e r d e c r e a s e in h e m o g l o b i n s t h a n on past u r i z e d milk, r e s u l t i n g in l o w e r v a l u e s at t h e e n d of the e x p e r i m e n t . I n all eases t h e m i l k c o n s u m p t i o n w a s c o n t r o l l e d b y t h a t m e m b e r of t h e p a i r r e c e i v i n g r a w milk. T a b i e 2 shows the s t a t i s t i c a l s i g n i f i c a n c e of t h e w e e k l y d i f f e r e n c e s of the m e a n values. I t is seen t h a t t h e differences i n t h e m e a n h e m o g l o b i n v a l u e s
185
NUTRITIONAL ANEMIA
Growth 7
'I"
/
Hemoqlob,n
IMean
I
~eon
iC~h'o/ ,)" w k pemods
:FIO. 2. H e m o g l o b i n c o n t e n t of blood~ b o d y w e i g h t a n d milk c o n s u m p t i o n of indiv i d u a l r a t s f e d commercially r a w a n d p a s t e u r i z e d milk f r o m w i n t e r f e e d i n g , t o g e t h e r w i t h m e a n v a l u e s a n d t h e m e a n s of blood h e m o g l o b i n a n d b o d y w e i g h t of controls f e d stock diet. T h e c o n t i n u o u s lines r e p r e s e n t r a w milk f e e d i n g , a n d t h e d o t t e d lines p a s t e u r ized milk. T h e d e a t h of a n a n i m a l b e f o r e t h e end of t h e e i g h t weeks p e r i o d is starred.
were statistically significant at the end of the second week and t h e r e a f t e r to the end of the experiment. The fact t h a t the differences of the fi~st two weeks were highly insignificant does not mitigate against the significance found subsequently. A lapse of time before the a p p e a r a n c e of the difference would be expected since the difference in the concentrations of copper and iron as shown by chemical analyses were small. W h e n the mean weekly TABLE 2 Statistical significance of weekly differences in mean hemoglobin content of blood and body weight of rats fed commercial raw and pasteurized rail# in paired feeding tests CALCULATIONSFROM HEMOGLOBIN
CALCULATIONSFROM BODYWEIGHT
Wee#
t
P
t
t)
0 1 2 3 4
0.2822 0.7104 2.0342 2.4231 2.8503 6.3123 3.1061 4.7452
0.7828 .4866 .0576 .0~84 .0130 .0000 .0112 .0090
0.3419 0.9687 0.6187 2.0437 1.0779 1.2947 0.5347 3.7983
0.8134 .3456 .5434 .0572 .2966 .2264 .6080 .0194
5 6
7
186
]=I. A. L A S B ¥ A N D L. S. P A L M E R
weights of the groups are considered, it m a y be seen t h a t those fed pasteurized milk had consistently g r e a t e r mean weights t h a n those fed the raw milk. However, Table 2 shows t h a t the difference was significant only in the t h i r d and seventh weeks. The significance in the third week was border line and since t h a t in the seventh was based on only four individuals it is doubtful t h a t it should be considered as t r u l y significant. The greater differences observed in this series are no doubt due to the iron and copper intakes of the rats fed the pasteurized milk. However, it seems that although this g r e a t e r intake was sufficient to produce significant differences in the hemoglobin values, slightly more would be necessary to produce equally significant differences in growth. Chemical analyses of the commercial pasteurized milk showed that it differed in copper and iron content f r o m the r a w milk. E i g h t analyses f o r iron made during the eight weeks period showed for r a w milk a range f r o m 0.85 to 1.49 mgms. per liter with a mean of 1.13 while for pasteurized milk the range was f r o m 1.06 to 1.64 mgms. per liter with a mean of 1.34. W h e n the same milk was analyzed for copper, the raw milk showed a range f r o m 0.15 to 0.24 mgms. per liter with a mean of 0.19 and the pasteurized milk ranged f r o m a content of 0.23 to 0.32 mgms. per liter with a mean of 0.27. I t seems evident t h a t the milk picked up copper and iron in the milk p l a n t a~though the stainless steel pasteurizing tanks could not have been responsible. I t is this additional quantity of the metals in the pasteurized milk which is reflected in the higher hemoglobin and greater growth of the rats fed the commercially pasteurized milk. As the experimental period progressed all rats in both series developed an appearance characteristic of nutritional anemia. I n case of piebald rats the black f u r became g r a y except in small area on the head. A f t e r the rats had been on the milk diet for some time it was noted t h a t the incisor teeth were growing out without the characteristic brown pigment on the outer surfaces. I n some the d a r k coloration was still visible on the distal ends of the teeth. This lack of tooth p i g m e n t seems to be a characteristic result of milk diets. Gross autopsies p e r f o r m e d revealed that all organs and muscle tissue were bloodless in appearance. The caecum was almost inv a r i a b l y enlarged and often the heart. I n Series I no difference could be noted in the a p p e a r a n c e and behavior of those fed raw milk and those fed pasteurized milk. I n Series I I , however, there was a marked difference in the dispositions of the two groups. Those fed pasteurized milk were more active and eager for food, while the others tended to be inactive and huddled in the corners of the cages. I n all cases the animals fed raw milk controlled the food consumption of the pair. The m o r t a l i t y in this series was much g r e a t e r t h a n in Series I. Of the eleven pairs started, only one survived the eight weeks experimental period. I n the ten p a i r s in which early death occurred, nine of the ten rats d y i n g were fed r a w milk. W h y the mortality
NUTRITIONAL ANEMIA
187
in this series was higher t h a n in Series I is uncertain. However, it m a y have been due both to a smaller f a t content if the commercial milk which was standardized to 3.5 p e r c e n t b u t t e r f a t content, resulting in a lower caloric intake t h a n in Series I, and to the fact t h a t this milk which was secured for s t u d y in the latter p a r t of the winter had a lower v i t a m i n content, p a r t i c u l a r l y v i t a m i n A, as the result of the t y p e of winter feeding practiced on the f a r m s where the milk was produced. PART II. CALCIUM~ PHOSPI:IORUS~ AND NITROGEN METABOLISM
T h a t the heat t r e a t m e n t of milk alters certain of the milk salts is now definitely established. W h e t h e r this change alters their nutritive value is still unanswered. Considerable e x p e r i m e n t a l n u t r i t i o n a l work has been done on the comparison of the retention of the calcium, phosphorus, and nitrogen of raw and pasteurized milk. Most of the results are inconclusive and few can pass uncriticized. F o r an accurate comparison the r a w and pasteurized milk fed m u s t come f r o m the same bulk and a sufficient n u m b e r of animals m u s t be used to insure the required degree of accuracy in the subsequent evaluation of the results. The effect of other articles of diet on the retention of these minerals is not known. There also a p p e a r s to be species differences so t h a t results obtained f r o m one species are not necessarily applicable to anather. Daniels, Stuessy, and F r a n c i s (12) c o m p a r e d the growth of r a t s fed milk which had undergone various heat treatments. Ellis and Mitchell (13) carried out carefully controlled calcium balance studies with rats fed raw or pasteurized milk plus a bas~l ration containing a small a m o u n t of calcium. They f o u n d t h a t the animals fed raw milk gained more in weight and showed a higher calcium content of the carcase t h a n those fed pasteurized milk. I n both cases the results were statistically significant. However, when the average calcium retention was as high as 98 per cent for rats on raw milk and 92 per cent for rats on pasteurized milk the calcium intake was p r o b a b l y too low to be of any practical importance. Krauss, Erb, and W a s h b u r n (2) measured the ability of rats to use calcium and phosphorus f r o m raw pasteurized milk b y analyzing the f e m u r s of rats a f t e r a period of feeding on the milk as a sole diet. No differences in the percentage of ash, calcium and phosphorus were found. EXPERIMENTAL
Methods. Male rats 10 weeks of age, a n d weighing f r o m 225 to 292 gms. were selected f r o m an inbred strain selected f o r high efficiency of food utilization during growth. A t this age the rats selected were still storing calcium, their normal growth curve was still a s t r a i g h t line, and their efficiency was known. The paired feeding method was used. Male litter mates were paired as n e a r l y as possible for weight. The s t u d y included
188
H . A. L A S B Y A N D L. S. P A L M E R
six pairs f r o m two litters. One member of each pair was fed raw milk and the other pasteurized milk f r o m the same bulk. The milk fed was f r o m the University of Minnesota A g r i c u l t u r a l E x p e r i m e n t Station herd. The exp e r i m e n t was conducted in late September and early October. The method of pasteurizing was the same as t h a t used in Series I of the anemia studies. Seven d a y collection periods were used. The r a t was always placed on the p a r t i c u l a r diet to be used three d a y s before collections were begun. Three successive collection periods were used for each pair. The order of feeding for one m e m b e r of a p a i r was raw, pasteurized, and raw, and for the other pasteurized, raw, and pasteurized. This alternation was used in order to minimize the possible influence of the order of feeding the two milks. The milk was fed twice a day, morning and late afternoon. To the morning feeding there was added 1 cc. of a slightly acidulated solution of ferric chloride and copper sulfate made up so t h a t 1 cc. contained 0.4 mgms. of Fe and 0.32 mgms. of Cu in order to p r e v e n t nutritional anemia. The milk thus supplemented was completely, consumed by both members of a pair before more was fed. The milk was measured out b y a pipette and any unconsumed a f t e r 24 hours was measured. The cages used were especially designed for metabolism studies. The r a t s were weighed weekly and food consumption records kept. The urine and feces were collected a f t e r each period and analyzed for calcium, phosphorus, and nitrogen. Similar analyses were made on aliqnots of weekly composite samples of the r a w and pasteurized milk fed. The milk was preserved with formaldehyde until the analyses were made. F i f t e e n pairs of balance determinations were made using six pairs of rats. B y subtracting the total amount of a mineral excreted in the urine and feces f r o m the intake as supplied b y the milk, the balance was obtained. F o r the sake of comparison, the balance values were then calculated on the basis of the percentage of the intake retained. I n order to compare the percentages of the intake of the respective minerals which were retained on the diets of raw and pasteurized milk, the mean values were calculated. Chemical analyses. The calcium and phosphorus determinations were made on the samples b y the method of Morris, Nelson, and P a l m e r (14). The nitrogen was determined by semi-micro K j e l d a h l method of Cavett (15). Results. The results obtained are presented in Table 3. The significance of the difference was then determined as in P a r t I. The mean percentages of intake retained together with the corresponding probabilities are given in Table 4. There was no significant difference between the retentions on the diets of raw and pasteurized milk. The slight differences which were found favored the pasteurized milk.
TABLE 3 Calcium, phosphorus and n i t r o g e n balances PAIR NO.
RAT NO.
1.
1728 1730 1729
2.
1731 3.
1733 1735
4.
1766 1767 't
5.
1765 1764"
6.
1767' 1769
])IET
1. 1. 1. 2. 1. 2. 1. 2. 3. 1. 2. 3. 1. 2. 3. 1. 2. 3. 1. 2. 3. 1. 2. 3. 1. 2. 3. 1. 2. 3.
raw past. raw past. past. raw raw past. raw past. raw past. raw past. raw past. raw past. raw past. raw past. raw past. raw past. raw past. raw past.
Ca
Ca INTAKE
TOTAL Ca EXCRETION
Ca BALANCE
mgm. 539.8 531.7 383.2 631.8 371.0 625.6 532.7 637.9 609.7 537.7 631.6 620.1 625.0 604.5 603.8 576.3 624.4 622.1 580.8 612.3 639.8 561.6 628.4 643.1 498.0 483.4 538.5 382.8 480.2 546.6
mgm. 344.4 394.4 262.8 478.5 239.2 478.7 300.1 378.8 450.1 297.1 354.0 385.0 393.8 487.9 490.4 267.6 436.5 453.1 333.4 422.2 479.4 318.9 483.0 466.4 335.2 346.1 491.2 267.9 352.2 409.4
mgm. 195.4 137.3 120.4 153.3 131.8 146.9 232.6 259.1 159.6 140.6 2.67.6 235.1 231.2 116.6 113.4 308.7 187.9 169.0 247.4 190.1 160.4 242.7 145.4 176.7 162.8 137.3 147.3 114.9 128.0 137.2
INTAKE RETAINED
per cent 63.8 74.2 68.6 75.7 64.5 76.5 56.4 59.4 73.8 73.9 57.6 62,.1 60.3 80.7 81.2 46.4 69.9 72.7 57.4 69.0 74.9 56.8 76.9 72.5 67.4 71.6 72.7 70.0 73.3 74.9
~ INTAKE
m gm . 435.7 423.4 302.9 477.9 291.3 486.2 429.9 486.2 465.9 428.1 490.9 458.5 413.9 464.7 474.6 298.7 445.6 457.0 384.6 470.7 502.9 388.6 448.4 472.4 329.8 371.6 423.3 2,64.9 342.7 401.5
$OTAL P
p
P
EXCRETION
BALANCE
INTAKE RETAINED
N INTAKE
N EXCRETION
N BALANCE
INTAKE RETAINED
mgm. 184.8 155.8 90.8 190.7 98..7 205.9 156.7 261.0 195.5 170.4 256.5 213.9 174.1 188.6 209.9 157.2 192.7 202.5 159.9 202.9 2~16.8 197.4 162.4 174.2 118..3 110.2 182.7 50.2 117.1 149.2
pe r cent 57.6 63.2 70.1 60.1 66.1 57.7 63.6 45.9 58.1 60.2 47.8 53.4 57.9 59.4 55.8 60.6 56.8 55.7 58.4 56.9 56.9 75.0 81.3 83.0 73.0 69.8 75.5 89.8 58.8 80.7
mg~. 2633.8 2553.6 1995.5 3068.0 1807.5 2984.8 2598.8 3097.5 2930.1 2582.1 3013.5 2985.3 2391.4
mgm. 2141.8 1814.6 1531.9 2470.2 1519.1 2268.7 2756.1 1960.9 2255.0 1833.2 2358.8 2414.3 1852.1
mgm. 492.0 737.2 463.6 597.8 288.4 716.1 842.7 1136.6 675.1 748.9 654.7 571.0 539.3
per cent 81.3 71.1 78.1 80.5 84.1 76.0 67.6 ~63.3 77.0 71.0 78.3 80.9 77.5
2376.0
1725.0
651.0
72.6
2~22.2 2,593.5 2756.7 2231.3 2650.5 2787.6 1905.5 2047.5 2320.2 1578.5 2025.4 2369.4
1775.9 2235.1 2304.9 1673.7 2150.9 2313.3 2391.0 1429.8 1751.1 1417.8 1190,9 1912.9
446.3 358.4 451.8 557.6 499.6 474.3 514.5 617.7 569.1 160.7 834.5 256.5
79.9 86.2 83.6 75.0 81.3 83.0 73.0 69.8 75.5 89.8 58.8 80.7
m gm . 250.9 267.6 212.1 " 287.2 192.6 280.3 273.2 221.5 270.4 257.7 234.4 244.6 239.8 276.1 264.7 241.5 252.9 254.5 224.7 267.8 286.1 191.2 286.0 298.2 211.5 261.4 240.6 214.7 2°~5.6 252.3
TOTAL
N
OO
190
H . A. L A S B Y A N D L. S. P A L M E R BONE ANALYSES
Methods. F o r the purpose of determining whether the calcium and phosphorus of r a w and pasteurized milk were equally available for bone formation, analyses were made on bones of the rats used in Series IT of the a n e m i a studies. T h e femurs and tibias were removed immediately after death, stripped of adhering flesh, and stored u n t i l the analyses were made. TABLE 4
Mean percentage retention of calcium, phosphorus and nitrogen from raw and pasteurized witlc and 1~ value of differences R E T E N T I O N ON RAW MILK
R E T E N T I O N ON PASTEURIZED MILK
per cent
per cent
68.9
68.3
0.6009
..................................
58.5
60.5
0.1120
.......................................
76.0
77.5
0.1616
ELEMENT
Calcium
..........................................
Phosphorus l~itrogen
P
A t t h a t time ,the f e m u r and tibia of one leg f r o m each animal were selected, crushed in filter paper, and e x t r a c t e d with alcohol for 48 hours and with ether for 24 hours. The percentage of ash in the bones was determined and the ash analyzed for calcium and phosphorus according to the method of Morris, Nelson, and P a l m e r (14). Weekly aliquots of the milk were also analyzed for calcium and phosphorus by the same method. The mean calcium values of the weekly aliquots of r a w and pasteurized milk were 11.96 mgm. and 12.01 mgm. per liter respectively. The corresponding phosphorus values were 8.92 mgm. and 8.96 mgm. per litter of raw and pasteurized milk. Results. Results of the bone analyses of the eleven pairs of rats are p r e s e n t e d in Table 5. These data were treated statistically by the method previously referred to. A probability of 0.0'54 was obtained t h a t the percentage of ash in the bones of rats fed r a w milk was significantly different f r o m t h a t of the rats fed pasteurized milk. This is a border line value. The percentage of ash was then correlated with the age of the rat, since the age might influence the percentage of ash and the rats used were of different ages depending on the time during the e x p e r i m e n t a l period at which death occurred. A correlation coefficient equal to + 0.7638 was obtained but this was not significant for the n u m b e r of samples correlated." Since the f e m u r and tibia of the other hind leg of the rats were also available, the length of these w a s obtained by means of calipers. The means of these data also are shown in T a b l e 5. The total length of the two bones was then correlated with the percentage of ash. This was done because the rats fed pasteurized milk grew better but their bones showed a lower ash content t h a n did the bones
NUTRITIONAL TABLE
191
ANEMIA 5
Calcium and phosphorus content and length dry-extravted f e m u r plus tibia o f rats dying f r o m anemia after consuming commercial raw and pasteurized milk BONE COMPOSITION
TOTAL LENGTH FEMUR PLUS TIBIA
~ILK
Total Ash
l~aw
.................................
Pasteurized
............
Ca
per cent
per cent
per vent
58.93
22.13
9.38
5.326
56.99
21.67
9.16
5.389
mm.
of the rats fed r a w milk. Hence, if a longer bone showed a lower ash conL tent, it should contain more organic and less mineral matter. The correlation coefficient between the length of ehe leg bones and the ash content in the raw g r o u p was + 0.6238 and in the pasteurized group + 0.2493, neither of which are significant. I t seems, therefore" t h a t the difference in the mean percentage of ash in the bones of the two groups is p r o b a b l y not significant. SUMMARY AND CONCLUSIONS E x p e r i m e n t s designed to compare the severity of nutritional anemia developed in comparative groups of rats when one group was fed r a w milk and the other the same milk a f t e r pasteurization have been carried out. The resul.ts of the experiment show: 1. W h e n the pasteurization of milk is carried out in glass in the laboratory, the severity of the anemia and the growth of the rats are not significantly different on diets of raw or pasteurized milk. 2. The iron and copper eontenes of milk are not affected b y pasteurization in glass in the laboratory. 3. W h e n the pasteurization of milk is carried out commercially, the anemia developed m a y be less severe t h a n on r a w milk and the g r o w t h better. 4. The iron a n d copper contents of milk commercially pasteurized m a y be g r e a t e r t h a n .that of the milk before the heat' t r e a t m e n t due to contact with the metallic equipment in use in commercial milk plants. E x p e r i m e n t s designed to compare the calcium, phosphorus, and nitrogen balance metabolism of paired rats, one of which received a sole diet of r a w milk and the other a sole diet of the same milk a f t e r holding pasteurization in glass in the laboratory, have been carried out. The results of these experiments lead to the following conclusions: 1. The calcium retentions are the same on the two diets. 2. The phosphorus retentions are slightly but not significantly g r e a t e r on pasteurized milk. 3. The nitrogen retentions are slightly b u t not significantly g r e a t e r on pasteurized milk.
192
H. A. LASBY AND L. S. PALMER
A n a l y s e s of the p e r c e n t a g e of ash, c a l c i u m a n d p h o s p h o r u s have been m a d e on d r y - e x t r a c t e d f e m u r - t i b i a bones of r a t s fed a sole diet of r a w or c o m m e r c i a l l y p a s t e u r i z e d milk. T h e f o l l o w i n g r e s u l t s were f o u n d : 1. T h e bones of the r a t s fed r a w m i l k h a d a h i g h e r p e r c e n t a g e of ash t h a n those of the rats fed p a s t e u r i z e d milk. T h e difference was p r o b a b l y n o t significant. 2. The m e a n c a l c i u m a n d p h o s p h o r u s c o n t e n t s of the bones of the rats fed r a w m i l k were s l i g h t l y b u t n o t s i g n i f i c a n t l y h i g h e r t h a n those of the rats fed p a s t e u r i z e d milk. The ash, calcium, a n d p h o s p h o r u s differences while n o t s i g n i f i c a n t m a y have b e e n due, i n p a r t , to t h e b e t t e r g r o w t h of the rats fed the p a s t e u r i z e d milk. 3. The m e a n c a l c i u m a n d p h o s p h o r u s c o n t e n t s of the m i l k before a n d a f t e r p a s t e u r i z a t i o n were the same. I~EFEEENCES (I) SCOTT, E., AND E~, 210 (1931).
L.A.
Ohio tests prove natural milk is best.
Jersey Bull. 50:
(2) KRAUSS,W. E., EI~B, J. 1=[., AND WASHBURN,R.G. The effect of pasteurization on some of the nutritive properties of milk. Ohio Agr. Exp. Sta. Bull. 518 (1933). (3) ELVEHJEM, C. A., AND KRMMERER, A.R. An improved technique for the production of nutritional anemia. J. Biol. Chem. 93:189-195 (1931). (4) N~WCOMEa,H.S. Absorption spectra of acid hematin, oxyhemoglobin, and carbon monoxide hemoglobin. A new hemoglobinometer. J. Biol. Chem. 37:465-496 (1919). (5) ELVEHJ~M, C. A., AND H ~ , E.B. Quantitative methods for the determination of iron in biological materials. J. Biol. Chem. 67:43-51 (1926). (6) KENNEDY,1~. P. The quantitative determination of iron in tissues. J. Biol. Chem. 74:385-391 (1927). (7) ELVEHJEI~,C.A. A note on the determination of iron in milk and other biological materials. J. Biol. Chem. 86:463-467 (1930). (8) ELVEHJZ~, C. A., AND LI~T,OW, C.W. The determination of copper in biological materials. J. Biol. Chem. 81:435-443 (1929). (9) GEDHARDT~H. T., ANDSOMMERS,I~. H. Determination of copper in dairy products. J. Ind. Eng. Chem. Anal. Ed. 3:34-26 (1931). (10) CH]~vm~z, E., AND ANSP,ACH~, S. Sur le dosage du cuivre dans des matieres organiques. I4elv. Claim. Acta 13:187-194 (1930). (11) ANSBACHE~, S., I~.~MINGTON, l~. E., AND CULP, F. B. Copper determination in organic matter. J. Ind. Eng. Chem. Anal. Ed. 3:314-317 (1931). (12) DANIELS,A. L., STUESSY, S., AND FRANCIS, E. The nutritive value of boiled milk. Am. J. Dis. Children 11:45-54 (191{}). (13) ELLIS, M., AN]) MITOHELL, tI. H. Effect of pasteurization of milk on the utilization of its calcium for growth in the rat. Am. J. Physiol. 1 0 4 : 1 - 9 (1933). (14) l~oP~IS, H. P., l ~ s o ~ , J. W., AND PALI~E~, L.S. Quantitative determination of calcium, magnesium, and phosphorus in feedstuffs and cattle excreta. J. Ind. Eng. Chem. Anal. Ed. 3:164-167 (1931). (15) CAVETT,J . W . An improved micro Kjeld~&~method. J. Lab. Clin. Med. 17:79-82
(1931).
Division of Agricultural Biochemistry, University of Minnesota, St. Paul. PART I. NUTRITIONAL ANEMIA
Two groups of workers ]lave concerned themselves with the relative values of sole diets of raw or pasteurized milk in producing nutritional anemia in the rat. Scott and E r r (1) r e p o r t results showing t h a t a special milk, when fed raw, allowed a better gain in weight and greater increase in red cell count and hemoglobin than when the same milk was fed a f t e r holding pasteurization. Some commercially pasteurized milk gave inferior results to those obtained with the special pasteurized milk. I t is not stated whether the rats were fed individually or in groups and what their food consumptions were. Neither are a n y copper and iron analyses of the milk reported. Krauss, Erb, and W a s h b u r n (2) report results c o n t r a r y to those of Scott and E r f . They fed milk f r o m the same bulk to two groups of rats, one group receiving raw milk and the other the same milk a f t e r pasteurization. The animals in both groups developed nutritional anemia as characterized by a marked decrease in hemoglobin and erythrocyte count and the decreases were at the same rate in both groups. No data are given for the food intake or growth. EXPERIMENTAL
Methods. The procedure of E l v e h j e m and K e m m e r e r (3) was followed in rendering the rats anemic. The young were weaned at three weeks, and then placed on the e x p e r i m e n t a l diet of milk alone for an eight weeks period. The rats were housed in individual galvanized wire cages with ½. inch mesh screen bottoms. The two series of experiments conducted were similar except in the milk fed. They were carried out b y the paired feeding method using males only. Both members of any one p a i r were litter mates and as similar as possible as to weight at the beginning of the experimental period. Those on the sole milk diet received no water. The control group consisted of litter mates fed on the stock diet and w a t e r ad lib. The rats in both series were weighed weekly and food consumption recReceived for publication November 6, 1934. The data presented are t a k e n in large p a r t from the thesis of H. A. Lasby presented to the Graduate School of the University of Minnesota in partial fulfillment of the requirements for the degree of Doctor of. Philosophy. All pertinent individual data are available for reference in the Division of Agricultural Bioche~rei~try. P a p e r l~o. 1320 Journal Series, Minnesota Agricultural Experiment Station. 181
182
H. A. LASBY AND L. S. PALMER
ords wer.e kept. Hemoglobin determinations were made at the time of weaning and weeldy thereafter. The method used was t h a t of Newcomer (4). Blood was obtained by cutting with a razor the tip of the tail, previously dipped in hot w a t e r to promote the flow of blood. Calcium chloride was first used to stop the flow of blood but later collodion was substituted, as it proved to be more satisfaotory. Series I. Seven p a i r s and three controls were used. The milk fed was obtained fresh daily, except Sunday, f r o m the University of Minnesota A g r i c u l t u r a l E x p e r i m e n t Station D a i r y herd. The experiment was conducted during May, J u n e and J u l y when the cows were on pasture. No special containers were used for ,the collection of the milk. I t was b r o u g h t up f r o m the barns in a tin pail f r o m which it was t r a n s f e r r e d to glass containers for storage and pasteurization. A portion of each d a y ' s milk was fed raw to one group ahd a portion was pasteurized in glass and fed as such to the other. F o r pasteurization, t h e milk was placed in stoppered 1 l. E r l e n m e y e r flasks, which were then immersed to the neck in a w a t e r bath. A t e m p e r a t u r e of 620-63 ° C. was maintained in the flask for 30 minutes. Subsequently the flasks and milk were cooled as r a p i d l y as possible in running cold water and stored, as was the raw milk, in a cold room at 40 ° C. No a t t e m p t was made to standardize the fat content of the milk fed. Feedings were made morning and afternoon. Definite quantities of milk were measured by pipette into glazed porcelain ramekins placed on the Screen bottom of the cage. A n y milk unconsumed at the next feeding was measured. Series I I . Eleven pairs and three controls were started. The milk used was commercial 1 raw and pasteurized milk obtained during F e b r u a r y and March, thus r e p r e s e n t i n g late winter feeding. Both were obtained f r o m the same bulk. I t contained 3.5 p e r cent b u t t e r fat. The pasteurization had been carried out in Y u n d t - G r i d l e y stainless steel tanks b y the holding process at 143.5 ° F. (62 ° C.) for 30 minutes. Q u a r t aliquots were taken each d a y and delivered for consumption the following day, except S a t u r d a y , when two quarts of F r i d a y ' s raw and pasteurized milk were delivered for S a t u r d a y ' s and S u n d a y ' s use. Chemical analyses. Analyses for iron and copper were made on the raw a n d pasteurized milk. I n series I r a n d o m samples were taken every week and analyzed. I n Series I I , 100 cc. aliquots were taken daily and the analyses made on weekly composites. The method used for iron was t h a t of E l v e h j e m and H a r t (5), K e n n e d y (6), and E l v e h j e m (7). I n Series I the copper was determined by the method of E l v e h j e m and Lindow (8) This milk was furnished through the courtesy of the Franklin Cooperative Creamery Association of Minneapolis. We acknowledge our indebtedness to them for it, and especially to Ass 't. Supt. Waiter Ahlstrom, for insuring the taking of samples and their
delivery.
183
NUTRITIONAL ANEMIA
as modified by Gebhardt and Sommers (9) and in Series I I by the method of Cherbuliez and Ansbacher (10) and Ansbacher, Remington and Culp (11). The latter was believed to be more accurate a n d certainly less tedious. Results. Series I. The results obtained are presented graphically in figure 1. The mean hemoglobin value for the raw milk group decreased
!,,o
"
Growff,
a~
'~F
Her, o?/o~,,
Food Con~p~,~n
Mean I
Control Mean i
I
I
5 wh. periocl~
FIG. 1. Hemoglobin content of, blood, body weight and milk consumption of individual rats fed raw and laboratory pasteurized summer milk from Station Dairy herd on pasture, together with mean values and the means of the blood hemoglobin and body weight of controls fed stock diet. The continuous lines represent raw milk feeding and the dotted lines pasteurized milk. ~he death of an animal before the end of the eight weeks period is starred. from 7.80 to 2.31 gms. per 100 cc. while that of the pasteurized group decreased from 8.08 to 1.88 in eight weeks. The mean weight of the rats fed raw milk increased from 33.14 to 83.5 gms. in contrast to an increase from 33.71 to 78.5 gins. for those fed pasteurized milk. The data were ti'eated statistically to test for the significance of the differences between the means of hemoglobin and the weight values obtained from the t w o groups of rats. Using Fischer's application of ' S t u d e n t s " distribution of t and the 5 per cent level of significance the data in table 1 show that the differences for each successive weekly interval were not significant except for the fifth week hemoglobin values. This c a n n o t be considered important inasmuch
184
H . A . LASBY AND L. S. :PALMER TABLE 1
Statistical significance of weekly differenves in mean hemoglobin content of blood and body weight of rats fed raw and pasteurized milk in paired feeding tests CALCULATIONS
FROM HEMOGLOBIN
CALCULATIONS
FROM BODY WEIGI-IT
Week
t
P
t
.P
0 1 2 3 4 5 6 7 8
1.9277 1.4279 1.0463 0.8717 0.3036 3.4662 0.5587 1.3859 1.9765
0.0780 .1786 .3194 .4020 .7704 .0062 .5914 .1086 .0966
0.8738 0.8356 0.3999 1.5515 0.5104 0.4004 0.1887 ~.0751 1.7363
0.4018 .4228 .6962 .1582 .6212 .6976 .8542 .0914 .1320
............ ............ ............ ............ ............ ............ ............ ............ ............
as no s t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e o c c u r r e d i n e i t h e r t h e p r e c e d i n g or s u c c e e d i n g week. T h e c o p p e r a n d i r o n a n a l y s e s of t h e r a w a n d p a s t e u r i z e d m i l k gave r e s u l t s i n d i c a t i n g t h a t t h e s a m e q u a n t i t y of m e t a l s wa~ p r e s e n t in each. S i x i r o n a n a l y s e s on r a w m i l k m a d e d u r i n g t h e e i g h t weeks p e r i o d v a r i e d f r o m 1.2.3 to 1.48 mgms. p e r l i t e r w i t h a m e a n of 1.35 a n d on t h e same m i l k a f t e r p a s t e u r i z a t i o n f r o m 1.20 to 1.48 mgms. p e r l i t e r w i t h a m e a n of 1.35. S i x c o p p e r a n a l y s e s m a d e on t h e same m i l k v a r i e d f r o m 0.11 to 0.17 mgms. p e r l i t e r w i t h a m e a n of 0.14 while those on t h e p a s t e u r i z e d m i l k v a r i e d f r o m 0.12 to 0.16 mgms. p e r l i t e r w i t h a m e a n of 0.14 mgm. Series I I . S i n c e the c o n d i t i o n s u s e d in l a b o r a t o r y p a s t e u r i z a t i o n , a r e n o t those u n d e r w h i c h m a r k e t m i l k is t r e a t e d , a series was r u n in o r d e r to d e t e r m i n e w h e t h e r c o m m e r c i a l p a s t e u r i z a t i o n h a d a n y effect on the s e v e r i t y of the a n e m i a p r o d u c e d b y f e e d i n g it as the sole d i e t to rats. T h e r e s u l t s o b t a i n e d a r e p l o t t e d in F i g u r e 2. P a i r n u m b e r e i g h t was o m i t t e d f r o m a l l c a l c u l a t i o n s b e c a u s e t h a t m e m b e r of t h e p a i r b e i n g f e d p a s t e u r i z e d m41k e s c a p e d f r o m t h e cage one n i g h t a n d t h e r e was a subseq u e n t rise in h e m o g l o b i n . I t m a y be seen t h a t t h e r e was a m u c h w i d e r s p r e a d b e t w e e n t h e hemog l o b i n a n d g r o w t h c u r v e s of t h e two r a t s of a p a i r t h a n in those of S e r i e s I. I n all cases t h a t m e m b e r of a p a i r r e c e i v i n g p a s t e u r i z e d m i l k g r e w b e t t e r a n d h a d m o r e h e m o g l o b i n . I f t h e r a t on r a w m i l k h a d a h i g h e r h e m o g l o b i n o r w e i g h e d m o r e at t h e b e g i n n i n g of t h e e x p e r i m e n t , t h e r e was a subseq u e n t l y p o o r e r g r o w t h or a g r e a t e r d e c r e a s e in h e m o g l o b i n s t h a n on past u r i z e d milk, r e s u l t i n g in l o w e r v a l u e s at t h e e n d of the e x p e r i m e n t . I n all eases t h e m i l k c o n s u m p t i o n w a s c o n t r o l l e d b y t h a t m e m b e r of t h e p a i r r e c e i v i n g r a w milk. T a b i e 2 shows the s t a t i s t i c a l s i g n i f i c a n c e of t h e w e e k l y d i f f e r e n c e s of the m e a n values. I t is seen t h a t t h e differences i n t h e m e a n h e m o g l o b i n v a l u e s
185
NUTRITIONAL ANEMIA
Growth 7
'I"
/
Hemoqlob,n
IMean
I
~eon
iC~h'o/ ,)" w k pemods
:FIO. 2. H e m o g l o b i n c o n t e n t of blood~ b o d y w e i g h t a n d milk c o n s u m p t i o n of indiv i d u a l r a t s f e d commercially r a w a n d p a s t e u r i z e d milk f r o m w i n t e r f e e d i n g , t o g e t h e r w i t h m e a n v a l u e s a n d t h e m e a n s of blood h e m o g l o b i n a n d b o d y w e i g h t of controls f e d stock diet. T h e c o n t i n u o u s lines r e p r e s e n t r a w milk f e e d i n g , a n d t h e d o t t e d lines p a s t e u r ized milk. T h e d e a t h of a n a n i m a l b e f o r e t h e end of t h e e i g h t weeks p e r i o d is starred.
were statistically significant at the end of the second week and t h e r e a f t e r to the end of the experiment. The fact t h a t the differences of the fi~st two weeks were highly insignificant does not mitigate against the significance found subsequently. A lapse of time before the a p p e a r a n c e of the difference would be expected since the difference in the concentrations of copper and iron as shown by chemical analyses were small. W h e n the mean weekly TABLE 2 Statistical significance of weekly differences in mean hemoglobin content of blood and body weight of rats fed commercial raw and pasteurized rail# in paired feeding tests CALCULATIONSFROM HEMOGLOBIN
CALCULATIONSFROM BODYWEIGHT
Wee#
t
P
t
t)
0 1 2 3 4
0.2822 0.7104 2.0342 2.4231 2.8503 6.3123 3.1061 4.7452
0.7828 .4866 .0576 .0~84 .0130 .0000 .0112 .0090
0.3419 0.9687 0.6187 2.0437 1.0779 1.2947 0.5347 3.7983
0.8134 .3456 .5434 .0572 .2966 .2264 .6080 .0194
5 6
7
186
]=I. A. L A S B ¥ A N D L. S. P A L M E R
weights of the groups are considered, it m a y be seen t h a t those fed pasteurized milk had consistently g r e a t e r mean weights t h a n those fed the raw milk. However, Table 2 shows t h a t the difference was significant only in the t h i r d and seventh weeks. The significance in the third week was border line and since t h a t in the seventh was based on only four individuals it is doubtful t h a t it should be considered as t r u l y significant. The greater differences observed in this series are no doubt due to the iron and copper intakes of the rats fed the pasteurized milk. However, it seems that although this g r e a t e r intake was sufficient to produce significant differences in the hemoglobin values, slightly more would be necessary to produce equally significant differences in growth. Chemical analyses of the commercial pasteurized milk showed that it differed in copper and iron content f r o m the r a w milk. E i g h t analyses f o r iron made during the eight weeks period showed for r a w milk a range f r o m 0.85 to 1.49 mgms. per liter with a mean of 1.13 while for pasteurized milk the range was f r o m 1.06 to 1.64 mgms. per liter with a mean of 1.34. W h e n the same milk was analyzed for copper, the raw milk showed a range f r o m 0.15 to 0.24 mgms. per liter with a mean of 0.19 and the pasteurized milk ranged f r o m a content of 0.23 to 0.32 mgms. per liter with a mean of 0.27. I t seems evident t h a t the milk picked up copper and iron in the milk p l a n t a~though the stainless steel pasteurizing tanks could not have been responsible. I t is this additional quantity of the metals in the pasteurized milk which is reflected in the higher hemoglobin and greater growth of the rats fed the commercially pasteurized milk. As the experimental period progressed all rats in both series developed an appearance characteristic of nutritional anemia. I n case of piebald rats the black f u r became g r a y except in small area on the head. A f t e r the rats had been on the milk diet for some time it was noted t h a t the incisor teeth were growing out without the characteristic brown pigment on the outer surfaces. I n some the d a r k coloration was still visible on the distal ends of the teeth. This lack of tooth p i g m e n t seems to be a characteristic result of milk diets. Gross autopsies p e r f o r m e d revealed that all organs and muscle tissue were bloodless in appearance. The caecum was almost inv a r i a b l y enlarged and often the heart. I n Series I no difference could be noted in the a p p e a r a n c e and behavior of those fed raw milk and those fed pasteurized milk. I n Series I I , however, there was a marked difference in the dispositions of the two groups. Those fed pasteurized milk were more active and eager for food, while the others tended to be inactive and huddled in the corners of the cages. I n all cases the animals fed raw milk controlled the food consumption of the pair. The m o r t a l i t y in this series was much g r e a t e r t h a n in Series I. Of the eleven pairs started, only one survived the eight weeks experimental period. I n the ten p a i r s in which early death occurred, nine of the ten rats d y i n g were fed r a w milk. W h y the mortality
NUTRITIONAL ANEMIA
187
in this series was higher t h a n in Series I is uncertain. However, it m a y have been due both to a smaller f a t content if the commercial milk which was standardized to 3.5 p e r c e n t b u t t e r f a t content, resulting in a lower caloric intake t h a n in Series I, and to the fact t h a t this milk which was secured for s t u d y in the latter p a r t of the winter had a lower v i t a m i n content, p a r t i c u l a r l y v i t a m i n A, as the result of the t y p e of winter feeding practiced on the f a r m s where the milk was produced. PART II. CALCIUM~ PHOSPI:IORUS~ AND NITROGEN METABOLISM
T h a t the heat t r e a t m e n t of milk alters certain of the milk salts is now definitely established. W h e t h e r this change alters their nutritive value is still unanswered. Considerable e x p e r i m e n t a l n u t r i t i o n a l work has been done on the comparison of the retention of the calcium, phosphorus, and nitrogen of raw and pasteurized milk. Most of the results are inconclusive and few can pass uncriticized. F o r an accurate comparison the r a w and pasteurized milk fed m u s t come f r o m the same bulk and a sufficient n u m b e r of animals m u s t be used to insure the required degree of accuracy in the subsequent evaluation of the results. The effect of other articles of diet on the retention of these minerals is not known. There also a p p e a r s to be species differences so t h a t results obtained f r o m one species are not necessarily applicable to anather. Daniels, Stuessy, and F r a n c i s (12) c o m p a r e d the growth of r a t s fed milk which had undergone various heat treatments. Ellis and Mitchell (13) carried out carefully controlled calcium balance studies with rats fed raw or pasteurized milk plus a bas~l ration containing a small a m o u n t of calcium. They f o u n d t h a t the animals fed raw milk gained more in weight and showed a higher calcium content of the carcase t h a n those fed pasteurized milk. I n both cases the results were statistically significant. However, when the average calcium retention was as high as 98 per cent for rats on raw milk and 92 per cent for rats on pasteurized milk the calcium intake was p r o b a b l y too low to be of any practical importance. Krauss, Erb, and W a s h b u r n (2) measured the ability of rats to use calcium and phosphorus f r o m raw pasteurized milk b y analyzing the f e m u r s of rats a f t e r a period of feeding on the milk as a sole diet. No differences in the percentage of ash, calcium and phosphorus were found. EXPERIMENTAL
Methods. Male rats 10 weeks of age, a n d weighing f r o m 225 to 292 gms. were selected f r o m an inbred strain selected f o r high efficiency of food utilization during growth. A t this age the rats selected were still storing calcium, their normal growth curve was still a s t r a i g h t line, and their efficiency was known. The paired feeding method was used. Male litter mates were paired as n e a r l y as possible for weight. The s t u d y included
188
H . A. L A S B Y A N D L. S. P A L M E R
six pairs f r o m two litters. One member of each pair was fed raw milk and the other pasteurized milk f r o m the same bulk. The milk fed was f r o m the University of Minnesota A g r i c u l t u r a l E x p e r i m e n t Station herd. The exp e r i m e n t was conducted in late September and early October. The method of pasteurizing was the same as t h a t used in Series I of the anemia studies. Seven d a y collection periods were used. The r a t was always placed on the p a r t i c u l a r diet to be used three d a y s before collections were begun. Three successive collection periods were used for each pair. The order of feeding for one m e m b e r of a p a i r was raw, pasteurized, and raw, and for the other pasteurized, raw, and pasteurized. This alternation was used in order to minimize the possible influence of the order of feeding the two milks. The milk was fed twice a day, morning and late afternoon. To the morning feeding there was added 1 cc. of a slightly acidulated solution of ferric chloride and copper sulfate made up so t h a t 1 cc. contained 0.4 mgms. of Fe and 0.32 mgms. of Cu in order to p r e v e n t nutritional anemia. The milk thus supplemented was completely, consumed by both members of a pair before more was fed. The milk was measured out b y a pipette and any unconsumed a f t e r 24 hours was measured. The cages used were especially designed for metabolism studies. The r a t s were weighed weekly and food consumption records kept. The urine and feces were collected a f t e r each period and analyzed for calcium, phosphorus, and nitrogen. Similar analyses were made on aliqnots of weekly composite samples of the r a w and pasteurized milk fed. The milk was preserved with formaldehyde until the analyses were made. F i f t e e n pairs of balance determinations were made using six pairs of rats. B y subtracting the total amount of a mineral excreted in the urine and feces f r o m the intake as supplied b y the milk, the balance was obtained. F o r the sake of comparison, the balance values were then calculated on the basis of the percentage of the intake retained. I n order to compare the percentages of the intake of the respective minerals which were retained on the diets of raw and pasteurized milk, the mean values were calculated. Chemical analyses. The calcium and phosphorus determinations were made on the samples b y the method of Morris, Nelson, and P a l m e r (14). The nitrogen was determined by semi-micro K j e l d a h l method of Cavett (15). Results. The results obtained are presented in Table 3. The significance of the difference was then determined as in P a r t I. The mean percentages of intake retained together with the corresponding probabilities are given in Table 4. There was no significant difference between the retentions on the diets of raw and pasteurized milk. The slight differences which were found favored the pasteurized milk.
TABLE 3 Calcium, phosphorus and n i t r o g e n balances PAIR NO.
RAT NO.
1.
1728 1730 1729
2.
1731 3.
1733 1735
4.
1766 1767 't
5.
1765 1764"
6.
1767' 1769
])IET
1. 1. 1. 2. 1. 2. 1. 2. 3. 1. 2. 3. 1. 2. 3. 1. 2. 3. 1. 2. 3. 1. 2. 3. 1. 2. 3. 1. 2. 3.
raw past. raw past. past. raw raw past. raw past. raw past. raw past. raw past. raw past. raw past. raw past. raw past. raw past. raw past. raw past.
Ca
Ca INTAKE
TOTAL Ca EXCRETION
Ca BALANCE
mgm. 539.8 531.7 383.2 631.8 371.0 625.6 532.7 637.9 609.7 537.7 631.6 620.1 625.0 604.5 603.8 576.3 624.4 622.1 580.8 612.3 639.8 561.6 628.4 643.1 498.0 483.4 538.5 382.8 480.2 546.6
mgm. 344.4 394.4 262.8 478.5 239.2 478.7 300.1 378.8 450.1 297.1 354.0 385.0 393.8 487.9 490.4 267.6 436.5 453.1 333.4 422.2 479.4 318.9 483.0 466.4 335.2 346.1 491.2 267.9 352.2 409.4
mgm. 195.4 137.3 120.4 153.3 131.8 146.9 232.6 259.1 159.6 140.6 2.67.6 235.1 231.2 116.6 113.4 308.7 187.9 169.0 247.4 190.1 160.4 242.7 145.4 176.7 162.8 137.3 147.3 114.9 128.0 137.2
INTAKE RETAINED
per cent 63.8 74.2 68.6 75.7 64.5 76.5 56.4 59.4 73.8 73.9 57.6 62,.1 60.3 80.7 81.2 46.4 69.9 72.7 57.4 69.0 74.9 56.8 76.9 72.5 67.4 71.6 72.7 70.0 73.3 74.9
~ INTAKE
m gm . 435.7 423.4 302.9 477.9 291.3 486.2 429.9 486.2 465.9 428.1 490.9 458.5 413.9 464.7 474.6 298.7 445.6 457.0 384.6 470.7 502.9 388.6 448.4 472.4 329.8 371.6 423.3 2,64.9 342.7 401.5
$OTAL P
p
P
EXCRETION
BALANCE
INTAKE RETAINED
N INTAKE
N EXCRETION
N BALANCE
INTAKE RETAINED
mgm. 184.8 155.8 90.8 190.7 98..7 205.9 156.7 261.0 195.5 170.4 256.5 213.9 174.1 188.6 209.9 157.2 192.7 202.5 159.9 202.9 2~16.8 197.4 162.4 174.2 118..3 110.2 182.7 50.2 117.1 149.2
pe r cent 57.6 63.2 70.1 60.1 66.1 57.7 63.6 45.9 58.1 60.2 47.8 53.4 57.9 59.4 55.8 60.6 56.8 55.7 58.4 56.9 56.9 75.0 81.3 83.0 73.0 69.8 75.5 89.8 58.8 80.7
mg~. 2633.8 2553.6 1995.5 3068.0 1807.5 2984.8 2598.8 3097.5 2930.1 2582.1 3013.5 2985.3 2391.4
mgm. 2141.8 1814.6 1531.9 2470.2 1519.1 2268.7 2756.1 1960.9 2255.0 1833.2 2358.8 2414.3 1852.1
mgm. 492.0 737.2 463.6 597.8 288.4 716.1 842.7 1136.6 675.1 748.9 654.7 571.0 539.3
per cent 81.3 71.1 78.1 80.5 84.1 76.0 67.6 ~63.3 77.0 71.0 78.3 80.9 77.5
2376.0
1725.0
651.0
72.6
2~22.2 2,593.5 2756.7 2231.3 2650.5 2787.6 1905.5 2047.5 2320.2 1578.5 2025.4 2369.4
1775.9 2235.1 2304.9 1673.7 2150.9 2313.3 2391.0 1429.8 1751.1 1417.8 1190,9 1912.9
446.3 358.4 451.8 557.6 499.6 474.3 514.5 617.7 569.1 160.7 834.5 256.5
79.9 86.2 83.6 75.0 81.3 83.0 73.0 69.8 75.5 89.8 58.8 80.7
m gm . 250.9 267.6 212.1 " 287.2 192.6 280.3 273.2 221.5 270.4 257.7 234.4 244.6 239.8 276.1 264.7 241.5 252.9 254.5 224.7 267.8 286.1 191.2 286.0 298.2 211.5 261.4 240.6 214.7 2°~5.6 252.3
TOTAL
N
OO
190
H . A. L A S B Y A N D L. S. P A L M E R BONE ANALYSES
Methods. F o r the purpose of determining whether the calcium and phosphorus of r a w and pasteurized milk were equally available for bone formation, analyses were made on bones of the rats used in Series IT of the a n e m i a studies. T h e femurs and tibias were removed immediately after death, stripped of adhering flesh, and stored u n t i l the analyses were made. TABLE 4
Mean percentage retention of calcium, phosphorus and nitrogen from raw and pasteurized witlc and 1~ value of differences R E T E N T I O N ON RAW MILK
R E T E N T I O N ON PASTEURIZED MILK
per cent
per cent
68.9
68.3
0.6009
..................................
58.5
60.5
0.1120
.......................................
76.0
77.5
0.1616
ELEMENT
Calcium
..........................................
Phosphorus l~itrogen
P
A t t h a t time ,the f e m u r and tibia of one leg f r o m each animal were selected, crushed in filter paper, and e x t r a c t e d with alcohol for 48 hours and with ether for 24 hours. The percentage of ash in the bones was determined and the ash analyzed for calcium and phosphorus according to the method of Morris, Nelson, and P a l m e r (14). Weekly aliquots of the milk were also analyzed for calcium and phosphorus by the same method. The mean calcium values of the weekly aliquots of r a w and pasteurized milk were 11.96 mgm. and 12.01 mgm. per liter respectively. The corresponding phosphorus values were 8.92 mgm. and 8.96 mgm. per litter of raw and pasteurized milk. Results. Results of the bone analyses of the eleven pairs of rats are p r e s e n t e d in Table 5. These data were treated statistically by the method previously referred to. A probability of 0.0'54 was obtained t h a t the percentage of ash in the bones of rats fed r a w milk was significantly different f r o m t h a t of the rats fed pasteurized milk. This is a border line value. The percentage of ash was then correlated with the age of the rat, since the age might influence the percentage of ash and the rats used were of different ages depending on the time during the e x p e r i m e n t a l period at which death occurred. A correlation coefficient equal to + 0.7638 was obtained but this was not significant for the n u m b e r of samples correlated." Since the f e m u r and tibia of the other hind leg of the rats were also available, the length of these w a s obtained by means of calipers. The means of these data also are shown in T a b l e 5. The total length of the two bones was then correlated with the percentage of ash. This was done because the rats fed pasteurized milk grew better but their bones showed a lower ash content t h a n did the bones
NUTRITIONAL TABLE
191
ANEMIA 5
Calcium and phosphorus content and length dry-extravted f e m u r plus tibia o f rats dying f r o m anemia after consuming commercial raw and pasteurized milk BONE COMPOSITION
TOTAL LENGTH FEMUR PLUS TIBIA
~ILK
Total Ash
l~aw
.................................
Pasteurized
............
Ca
per cent
per cent
per vent
58.93
22.13
9.38
5.326
56.99
21.67
9.16
5.389
mm.
of the rats fed r a w milk. Hence, if a longer bone showed a lower ash conL tent, it should contain more organic and less mineral matter. The correlation coefficient between the length of ehe leg bones and the ash content in the raw g r o u p was + 0.6238 and in the pasteurized group + 0.2493, neither of which are significant. I t seems, therefore" t h a t the difference in the mean percentage of ash in the bones of the two groups is p r o b a b l y not significant. SUMMARY AND CONCLUSIONS E x p e r i m e n t s designed to compare the severity of nutritional anemia developed in comparative groups of rats when one group was fed r a w milk and the other the same milk a f t e r pasteurization have been carried out. The resul.ts of the experiment show: 1. W h e n the pasteurization of milk is carried out in glass in the laboratory, the severity of the anemia and the growth of the rats are not significantly different on diets of raw or pasteurized milk. 2. The iron and copper eontenes of milk are not affected b y pasteurization in glass in the laboratory. 3. W h e n the pasteurization of milk is carried out commercially, the anemia developed m a y be less severe t h a n on r a w milk and the g r o w t h better. 4. The iron a n d copper contents of milk commercially pasteurized m a y be g r e a t e r t h a n .that of the milk before the heat' t r e a t m e n t due to contact with the metallic equipment in use in commercial milk plants. E x p e r i m e n t s designed to compare the calcium, phosphorus, and nitrogen balance metabolism of paired rats, one of which received a sole diet of r a w milk and the other a sole diet of the same milk a f t e r holding pasteurization in glass in the laboratory, have been carried out. The results of these experiments lead to the following conclusions: 1. The calcium retentions are the same on the two diets. 2. The phosphorus retentions are slightly but not significantly g r e a t e r on pasteurized milk. 3. The nitrogen retentions are slightly b u t not significantly g r e a t e r on pasteurized milk.
192
H. A. LASBY AND L. S. PALMER
A n a l y s e s of the p e r c e n t a g e of ash, c a l c i u m a n d p h o s p h o r u s have been m a d e on d r y - e x t r a c t e d f e m u r - t i b i a bones of r a t s fed a sole diet of r a w or c o m m e r c i a l l y p a s t e u r i z e d milk. T h e f o l l o w i n g r e s u l t s were f o u n d : 1. T h e bones of the r a t s fed r a w m i l k h a d a h i g h e r p e r c e n t a g e of ash t h a n those of the rats fed p a s t e u r i z e d milk. T h e difference was p r o b a b l y n o t significant. 2. The m e a n c a l c i u m a n d p h o s p h o r u s c o n t e n t s of the bones of the rats fed r a w m i l k were s l i g h t l y b u t n o t s i g n i f i c a n t l y h i g h e r t h a n those of the rats fed p a s t e u r i z e d milk. The ash, calcium, a n d p h o s p h o r u s differences while n o t s i g n i f i c a n t m a y have b e e n due, i n p a r t , to t h e b e t t e r g r o w t h of the rats fed the p a s t e u r i z e d milk. 3. The m e a n c a l c i u m a n d p h o s p h o r u s c o n t e n t s of the m i l k before a n d a f t e r p a s t e u r i z a t i o n were the same. I~EFEEENCES (I) SCOTT, E., AND E~, 210 (1931).
L.A.
Ohio tests prove natural milk is best.
Jersey Bull. 50:
(2) KRAUSS,W. E., EI~B, J. 1=[., AND WASHBURN,R.G. The effect of pasteurization on some of the nutritive properties of milk. Ohio Agr. Exp. Sta. Bull. 518 (1933). (3) ELVEHJEM, C. A., AND KRMMERER, A.R. An improved technique for the production of nutritional anemia. J. Biol. Chem. 93:189-195 (1931). (4) N~WCOMEa,H.S. Absorption spectra of acid hematin, oxyhemoglobin, and carbon monoxide hemoglobin. A new hemoglobinometer. J. Biol. Chem. 37:465-496 (1919). (5) ELVEHJ~M, C. A., AND H ~ , E.B. Quantitative methods for the determination of iron in biological materials. J. Biol. Chem. 67:43-51 (1926). (6) KENNEDY,1~. P. The quantitative determination of iron in tissues. J. Biol. Chem. 74:385-391 (1927). (7) ELVEHJEI~,C.A. A note on the determination of iron in milk and other biological materials. J. Biol. Chem. 86:463-467 (1930). (8) ELVEHJZ~, C. A., AND LI~T,OW, C.W. The determination of copper in biological materials. J. Biol. Chem. 81:435-443 (1929). (9) GEDHARDT~H. T., ANDSOMMERS,I~. H. Determination of copper in dairy products. J. Ind. Eng. Chem. Anal. Ed. 3:34-26 (1931). (10) CH]~vm~z, E., AND ANSP,ACH~, S. Sur le dosage du cuivre dans des matieres organiques. I4elv. Claim. Acta 13:187-194 (1930). (11) ANSBACHE~, S., I~.~MINGTON, l~. E., AND CULP, F. B. Copper determination in organic matter. J. Ind. Eng. Chem. Anal. Ed. 3:314-317 (1931). (12) DANIELS,A. L., STUESSY, S., AND FRANCIS, E. The nutritive value of boiled milk. Am. J. Dis. Children 11:45-54 (191{}). (13) ELLIS, M., AN]) MITOHELL, tI. H. Effect of pasteurization of milk on the utilization of its calcium for growth in the rat. Am. J. Physiol. 1 0 4 : 1 - 9 (1933). (14) l~oP~IS, H. P., l ~ s o ~ , J. W., AND PALI~E~, L.S. Quantitative determination of calcium, magnesium, and phosphorus in feedstuffs and cattle excreta. J. Ind. Eng. Chem. Anal. Ed. 3:164-167 (1931). (15) CAVETT,J . W . An improved micro Kjeld~&~method. J. Lab. Clin. Med. 17:79-82
(1931).