Some Biochemical Constituents in Urine of Vitamin A-Deficient Holstein Calves1

SOME I~IOCHEMICAL CONSTITUENTS IN URINE OF VITAMIN A-DEFICIENT HOLSTEIN CALVES 1 C. G. WOELFEL, M. C. CALHOUN, J. E. I~OUSSEAU, JR., A~D H. D. EATON Animal Industries Department AND

S. W. NIELSEN Animal Diseases Department Storrs (Conn.) Agricultural Experiment Station, Storrs SUJ~)iARY

To study possible effects of hypovitaminosis-A on urine composition, 30 male Holstein calves, average age 53 days and live weight 158 lb, and partially depleted of their vitamin A reserves, were fed a vitamin A depletion ration supplemented with artificially dehydrated alfalfa leaf meal to provide a deficient or adequate carotene intake, 15 or 150 ~g per pound of live weight daily. Cerebrospinal fluid pressm'es were higher in the deficient group accompanied by lower vitamin A concentrations in the plasma and liver. Specific gravity, osmolality, and total solids of urine were significantly lower in the calves receiving a 15/~g intake. Concentrations and total daily outputs of urea nitrogen and creatinine were slightly less in the urine of the deficient calves. To further study urine composition, repeat collections were made with five pairs of calves. I n the deficient calves, average osmolality was again less, as well as concentrations and total daily outputs of sodium and chloride. Potassium was unaffected, but concentrations and total daily outputs of phosphorus were greater. The latter element was the only criterion which was significantly different. Kidney calcium concentrations were slightly higher. Histopathological change in the kidney of vitamin A-deficient animals of many species has been observed (27). Similar findings have been noted in the bovine, with the occurrence being more frequent with prolonged deficiency (17, 28). Urinary calculi have been noted in various chronic vitamin A-deficient animals (27). I n the bovine, the evidence (10) does not support a direct effect of vitamin A deficiency on the occurrence of urinary calculi. However, urate deposition in the kidneys and ureters was observed in hypovitaminotic-A chicks (11). Of possible added interest was the recent observation (23), in goats, fed diets low in vitamin A activity, of increased calcium deposition in t h e kidney without evidence of caleuii formation. Evidence for the effects of vitamin A deficiency on renal function or urinary constitu:Received for publication April 25, 1963. 1Scientific Contribution No. 32, Agricultural Experiment Station, University of Connecticut, Storrs. This study was supported in part by grantin-aid funds provided by a PHS research grant, B2108(C3) from the National Institute of Neurological Diseases and Blindness, Public Health Service. These data are part of a thesis to be presented to the Graduate School of the University of Connecticut by the senior author in partial fulfillment of the requirements for the degree of Doctor of Philosophy.

ents, or both, is controversial. As early as 1932, E]vehjem and Neu (11) reported marked increases in blood uric acid of vitamin A-deficient chicks, thus indicating impaired renal clearance of this urinary constituent. Later,. Herrin and Nicholes (18) reported decreased inulin and urea clearances in young vitamin A-deficient dogs which returned to normal levels upon administration of either vitamin A or carotene. I n opposition to the above reports, Stoewsand and Scott (32) could demonstrate no consistent effect of vitamin A deficiency on blood uric acid levels in chicks exhibiting signs of ataxia typical of hypovitaminosis-A, and Moore and Sykes (26) could find no indication of urinary chemical changes in vitamin A-deficient calves exhibiting marked elevations in eerebrospinal fluid pressure. Because of the reported structural and, possibly, functional changes of the kidney associated with hypovitaminosis-A, the present study was undertaken to provide preliminary inforn~ation on some biochemical constituents in urine of vitamin A-deficient calves. Also, the calcium content of the kidney in this deficiency was explored. Some of these statistics have been presented in an abstract (34). EXPERI:I~ElqTAL PROCF~DURE

Animals and feeding. Thirty male Holstein calves one day of age were transported during the period from November, 1960--January, 947

948

c.G.

W O E L F E L E T AL

1961, from various state institution herds to the Animal Nutrition Research barn. Upon arrival, each calf was placed in an individual sawdust-bedded tie stall in a portion of the barn where neither light nor temperature was controlled, and given one 500 mg oblet of chlortetracyeline (American Cyanamid's Aureomycin) for three consecutive days as a prophylaxis for scours. One gelatin capsule containing 100,000 U S P units of vitamin A (Nopcay 250, Type M, Lot V7115, guarantee 250,000 U S P units of vitamin A per gram) was administered on the first day only. All calves were raised to approximately the 35th day (standard deviation of six) of age on limited whole milk, limited starter, and ad lib. chopped alfalfa hay and water, as in previous studies (16). On the 35th day of age, each calf received a vitamin A depletion ration such that the anticipated gain in live weight was 10 lb per seven-day period (16). One week after the blood plasma vitamin A concentration had decreased to, or reached less than, 12 ~g per 100 ml, each calf's depletion ration was supplemented with either a 15-~g or 150-~g carotene intake per pound live weight per day from artificially dehydrated alfalfa leaf meal for 17 wk. Based on previous data (16), the lower carotene intake was considered deficient and the higher intake adequate with regard to vitamin A status. The calves, numbered consecutively upon arrival at the barn, were assigned to deficient or adequate carotene intakes at random in replicates of two (based on arrival time). The average age at the start of carotene supplementation was 53 days, with a standard deviation of six. Either one or three days following the 17 wk of carotene supplementation, during which the same feeding regime was continued, each calf was slaughtered.

The calculation of the amount of alfalfa leaf meal to be fed to each calf and its incorporation into the depletion ration, and treatment of scours, were similar to that cited previously (16). Observations and analyses. All feeds fed were weighed to the nearest 0.1 lb. Samples for proximate analyses and carotene content (3) were taken every 4 wk throughout the experiment and results are reported in Table 1. Live weight for each calf was obtained at successive seven-day periods and linear growth measurements, height, heart girth, and girth of paunch, were taken to the nearest ~ - i n . at the start and termination of carotene supplementation. The minimum and maximum daily barn temperatures during carotene supplementation period were 13 and 19 C, respectively, with standard deviations of four and five, and light intensities for each calf, both artificial and natural, measured at 4-wk intervals at midday, averaged 4.7 foot-candles with a standard deviation of 2.3. Blood samples were taken by puncture of the jugular vein, eitrated and analyzed for vitamin A and carotenoids (21) weekly during partial vitamin A depletion and on the seventh day of the 4th, 8th, 12th, and 16th wk of carotene supplementation. During the 14th wk similar blood samples were drawn and analyzed for whole blood urea and ammonia by the aeration method of Van S]yke and Cullen and plasnm creatine and creatinine by the Jaffe reaction (15). Twenty-four-hour urine collections were made essentially as described by Horn et al. (19) during the 14th wk of carotene supplementation. Once-per-day feeding, that is, feeding initially the alfalfa mixed in a small quantity of

TABLE 1 Chemical composition of feeds Per cent dry matter

Per cent dry matter Crude protein

:Ether extract

Crude fiber

NFE

Ash

61.3 ± 1.0 45.7 -- 1.1 62.4 +--- 0.9 38.7 ± 0.8

6.3 ± 0.2 7.3 ± 0.4 6.6 +--- 0.2 11.9 ± 0.3

Carotene

(mg/lb) Calf starter ~

90.0 23.2 2.9 6.3 _ 1.5 +_ 1.4 ± 0.2 ± 0.3 Chopped alfalfa 91.8 16.2 1.5 29.3 hay ~ ± 1.2 ± 0.6 ± 0.3 ± 1.4 Vitamin A deple89.4 16.2 2.4 12.4 tion ration b ± 0.5 ± 0.3 ± 0.3 +-- 0.8 Alfalfa leaf 91.2 23.5 4.2 21.7 meal e ___ 0.7 -+- 0.4~ ± 0.2 +-- 0.8 a Average values for three samples with its standard error. b Average values for seven samples with its standard error. Average values for six samples with its standard error.

2,.72 +

0.II 6.41

± 0.26 0.12 +--- 0.01

70.62 ± 3".15

CONSTITUENTS IN URINE

vitamin A depletion ration to insure complete consumption of the alfalfa and followed by feeding the remainder of the daily vitamin A depletion ration allowance, was continued when calves were in the metabolism stalls. W a te r was also allowed ad lib. from a water bowl mounted in the stall. The 24-hr urine sample, collected in a 2.5o'al polyethylene carboy containing 10 ml of toluene, was mixed thoroughly and its specific gravity and volume determined. Immediately, a representative sample was obtained for the determination of p i t , total solids by difference after vacuum desiccation of the frozen sample for 32 hr, and total nitrogen by the I(jeldahl method (15). Centrifuged samples were used for osmolality (2) employing a Fiske osmometer (Model G), which measures freezing point depressions but is calibrated to read osmotic pressure units, total protein by the gravimetrie method of Folin and Denis (29), urea and ammonia nitrogen by the aeration method of Van Slyke and Cullen, and creatine and ereatinine by the Jaffe reaction (15).

949

Cerebrospinal fluid pressures were measured manometrically during the 15th wk of carotene supplementation (33). Because of the lowered specific gravity, osmolality, and total solids, without appreciable change in nitrogenous constituents of the urine of the deficient calves of the first few replicates, repeat collections were made on five deficient and five adequate calves during the 17th wk of carotene supplementation. In addition to measuring specific gravity and osmolality, chloride was determined, employing an automatic chloride titrator (Amineo-Cotlove No. 4-4420) (8), and sodium and potassium by flame photometry (1) on centrifuged urine samples. After wet ashing (13), total phosphorus content was measured by the eolorimetric procedure of Fiske and Subbarrow (12) and calcium by flame pbotomet~T (6). At slaughter, a gross examination of all organs was performed and tissue samples placed in 10% neutral buffered formalin for histopathological examination, these results to be presented separately (28). The liver was ground

TABLE 2 Effect of deficient, 15. ~g, and adequate, 150 ~g, carotene intake upon feed consmnption, scours, and growth of Holstein calves ~ Carotene intake b 15 ~

150~g

Standard deviation per calf

Age (days) Initial c 53 54 6 Days free of feed refusals % 98.3 99.8 ...... Arcsin ~/% 85 89 4 Ration consumed % 99.7 100.0 ...... Arcsin V % 87.8 89.6 1.9 Days free of scours % 96.6 99.6 ...... Aresin ~/% 83 88 6 Growth Live weight Initial (lb) 161 156 19 Gain (lb/day) 1.74 1.82 0.12 Adj. gain (lb/day) a 1.73 1.83 0.11 Height at withers Initial (in.) 33.2 32.4 1.5 Gain (in./day) 0.063 0.0.65 0.009 Adj. gain (in./day) a 0.065 0.064 0.007 Heart girth Initial (in.) 35.8 35.1 1.5 Gain (in./day) 0.102 0.111 0.0'08 Girth of paunch Initial (in.) 41.7 41.0 2.2 Gain (in./day) 0.135 0.138 0.018 Adj. gain (in./day) d 0.136 0.137 0.017 a Average values for 15 calves per carotene intake grouping. All statistics refer to the carotene supplementation period or to data collected the day prior to this period, initial values. b ttg/lb live wt/day. The day prior to carotene supplementation. d Adjusted by covariance for initial measurement.

950

C.G.

W O E L F E L ET AL

amount due to spillage of water f r o m the water bowl on the day of collection. No weighbacks occurred the week p r i o r to collection or the week after. A slightly higher incidence of scours occurred in the deficient calves (P < 0.05, when the per cent days free of scours f o r each calf was transformed to the arcsin ~ / % ) . Also, two calves in the deficient g r o u p exhibited rectal temperatures equal to or greater than 39.5 C, which required t r e a t m e n t with sulfamethazine, as compared to none among the adequate calves. Slightly smaller daily gains in live weight (P < 0.01) were observed in the deficient calves.

in a H o b a r t food chopper and the kidney homogenized in a Servall omnimixer, and samples f r o m both tissues held at --1S C f o r subsequent analyses. Carotenoids and vitamin A were determined in the liver by a modification of the G a l l u p - H o e f e r method (5), and kidney dry nmtter and ash according to A.O.A.C. procedures (3). A n additional sample was ashed (24) f o r the measurement of calcium by flame photometry (9). The analyses of variance and covariance (7, 31) consisted of variation due to replicates (pairs) of calves, to carotene intakes (adequate versus deficient), and to the interaction of these two sources (error). RESULTS

Feed consumption, hearth and growth (Table

Ccrebrospinal fluid pressure a~d carotenoid and vitamirb A concentrations (Table 3). Cere-

2). Ration consumption in those calves fed the deficient carotene intake was slightly less, as was also the number of days free of feed refusals (P < 0.05 when the percentage values of both criteria f o r each calf were transformed to the arcsin x / % ) . These feed refusals did not occur at the time when calves were in the metabolism stalls, with the exception of two calves of the deficient group, one weighing back 0.S lb and the other an undetermined

brospinal fluid pressure obtained the 15th wk of carotene supplementation was considerably greater (P < 0.001 when each calf's pressure was transformed to its common logarithm) in the deficient calves than in the adequate group, thus corroborating the existence of hypovitaminosis A in the fornler group of calves (26). As anticipated, plasma and liver concentrations of carotenoids and vitamin A (P < 0.001 f o r the f o u r criteria) were considerably greater

TABLE 3 Effect of deficient, 15 ~g, and adequate, 150 t~g, carotene intake upon some physiological measurements and vitamin A status of Holstein calves ~ Carotene intake b 15 ~g

150 ~g

Standard deviation per calf

Oerebrospinal fluid pressure c

(ram of saline) Actual 241 79 Common logarithm 2.37 1.88 Plasma carotenoids (t~g/lO0 ml) Initial a 6 6 Average " 12 63 Terminal ~ 14 76 Plasma vitamin A (/~g/lO0 ml) Initial a 6.8 5.6 Average ~ 5.8 26.3 Terminal ~ 6.2 28.3 Liver, terminal g Weight (kg) 3.16 3.15 Carotenoids (~9/100 g) Actual 22 86 Common logarithm 1.32 1.93 Vitamin A (t~g/lO0 g) Actual 7.8 682.0 Common logarithm 0.84~ 2.80 a Average values for 15 calves per carotene intake grouping. b ~g/lb live wt/day. c The 15th wk of carotene supplementation. d The day prior to carotene supplementation. e Average for carotene supplementation. The 16th wk of carotene supplementation. At slaughter upon completion of carotene supplementation.

0.13 3 8 13 3.7 2.1 3.4 0.25 0.11 0.21

CONSTITUENTS

IN

in the calves f e d the 150 t~g carotene i n t a k e t h a n in those f e d the 15 t~g intake. B l o o d and urine constituents and k i d n e y clearance ( T a b l e 4). N i t r o g e n o u s blood cons t i t u e n t s indicative of k i d n e y f u n c t i o n , whole blood urea, a n d a m m o n i a as well as p l a s m a creatinine, exhibited little difference between t r e a t m e n t groups. L o w e r osmolality ( P < 0.01), specific g r a v i t y ( P < 0.01), a n d p e r cent t o t a l solids ( P < 0.01) in u r i n e occurred in the deficient calves. These differences were still evident a f t e r a d j u s t m e n t b y covariance f o r u r i n e volume ( P < 0.05, P < 0.01, P <

951

URINE

0.01, r e s p e c t i v e l y ) . U r i n e p r o t e i n concentration was low a n d n o t significantly r e l a t e d to carotene intake, a v e r a g i n g 2.5 m g p e r 100 ml in the deficient calves a n d 4.1 m g i n the adeq u a t e calves, w i t h s t a n d a r d d e v i a t i o n p e r calf of 4.3 rag. W h i l e no significant differences occ u r r e d in the r e m a i n d e r of the u r i n e constituents, u r e a n i t r o g e n a n d c r e a t i n i n e t e n d e d to be lower in c o n c e n t r a t i o n a n d daily o u t p u t in the deficient calves. K i d n e y clearance of u r e a a n d creatinine, based on the 2 4 - h r u r i n e collection a n d blood or p l a s m a c o n c e n t r a t i o n , did not exh i b i t statistically significant differences between

TABLE 4 Effect of deficient, 15 ~g, and adequate, 150 tLg, carotene intake upon some blood constituents indicative of kidney function, some physical characteristics and nitrogenous constituents in urine and kidney clearances of Holstein calves a Carotene intake b 15/~g

150/tg

Standard deviation per calf

Blood constituents (,mg/lO0 ml) Whole blood urea nitrogen 5.85 5.79 0.13 Whole blood amonia nitrogen 1.44 1.62 1.4 Plasma creatinine 0.56 0.55 0.14 Urine constituents Total volume (1) 3.9 3.7 0.6 Specific gravity 1.0294(1.0297) 1.0331 (1.0329) 0.0031 (0.0028) Osmolality (milliosmols/t~gH~O) 797 (804) 895 (888) 89 (83') Total solids (g/iOOg) 5.5 (5.5) 6.2 (6.2) 0.6 (0.5) pit 7.91 8.09 0.39 Concentration of nitrogenous constituents (mg/lO0 ml) -Total nitrogen 723 (731) 761 (753) 103 (96) Urea nitrogen 304 327 80 Ammonia nitrogen 80 80 38 Creatinine 121 (123) 131 (]29) 20 (18) Creatine 38 38 24 Distribution of nitrogenous constituents (%) Urea nitrogen 42 42 7 Ammonia nitrogen 11 10 5 Creatinine 16.8 17.5 2.8 Creatine 5.2 4.9 3.1 Daily output (g/24 hr) Total solids 221 238 29 Total nitrogen 28.3 28.2 4.2 Urea nitrogen 11.9 12.2 3.2 Ammonia nitrogen 3.2 3.0 1.5 Creatinizm 4.7 4.8 0.7 Creatine 1.5 1.4 0.9 Kidney clearances Urea ~ 44 46 15 Creatinine ¢ 182 192 42 Urea/creatinine ratio d 0.25 0.24 0.08 Average values for 15 calves per carotene intake grouping observed the 14th wk of carotene supplementation. Values in psrentheses are adjusted by covariance for urine volume. b t~g/lb live wt/day.

r ( s ~) (s,)7F(100)7 | L-(L-W~.J' where

¢ Clearance = L-

U~ equals urine concentration of x, Ut ----urine

flow in milliliter per minnte, P~--= the whole blood (urea) or plasma (creatlnine) concentrations, and L W = live weight. d Ratio = (urea c l e a r a n e e ) / ( e r e a t i n i n e clearance).

952

c.G.

W O E L ~ E L ET AL

carotene i n t a k e g r o u p s , b u t were slightly lower in deficient calves, due to t h e i r lower concent r a t i o n s in the urine. The r a t i o of u r e a clearance to the c r e a t i n i n e clearance showed no difference.

Repeat collection of urine for mineral a~alysis (Table 5). Because of t h e lowered specific g r a v i t y , osmolality, a n d t o t a l solids, w i t h o u t a p p r e c i a b l e c h a n g e i n t h e n i t r o g e n o u s cons t i t u e n t s of the u r i n e of the calves receiving the deficient carotene intake, r e p e a t u r i n e collections were carried out on five calves p e r caro-

tene g r o u p i n g to d e t e r m i n e possible differences i n some of t h e m i n e r a l constituents. A s in the first series of collections, t h e u r i n e specific g r a v i t y a n d osmolality were lower in the deficient calves. C o n c e n t r a t i o n a n d total daily o u t p u t of sodium a n d chloride were also less i n the deficient g r o u p . P h o s p h o r u s was h i g h e r b o t h in c o n c e n t r a t i o n ( P < 0.05) a n d daily o u t p u t ( P < 0.10), while p o t a s s i u m exhibited i n a p p r e c i a b l e difference. The r a t i o of sodium to p o t a s s i u m was lower i n t h e deficient g r o u p .

Kidney dry matter; ash and calcium content

TABLE 5 Effect of deficient, 15 ~g, and adequate, 150 ~g, carotene intake upon some physical characteristics and mineral constituents in urine, from repeat collections of Holstein calves ~ Carotene intake b 15 ~g

150 ~g

Standard deviation per calf

Total volume (1) 4.2 4.0 0.7 Specific gravity 1.0309 ( 1.0313 ) 1.0327 (1.03.24.) 0.0035 (0.0025) Osmolality (milliosmols/kgH~O) 885 (895) 948 (937) 104 (81) pH 8.54 8.52 9.25 Concentrations of mineral constituents (mg/lO0 ~d) Calcium 0.9 0.9 0.4 Phosphorus 81 49 21 Sodium 320 393 85 Potassium 814 (827) 841 (828) 124 (90) Chloride 454 538 87 Daily output (g/24 I~r) Calcium 0.04 0.03 0.0.2 Phosphorus 3.4 2.0 1.0 Sodium 13.5 15.7 4.4 Potassium 33.9 33.0 4.8 Chloride 19.3 21.0 4.3 Na/K Ratio 0.40 0.48 0.13 Average values for five calves per carotene intake grouping observed the 17th wk of carotene supp]ementatlon. Values in parentheses are adjusted by covariance for urine volume. b ~ g / l b live wt/day. TABLE 6 Effect of deficient, 15 tLg, and adequate, 150 ~g, carotene intake upon dry matter, ash, and calcium contents of kidneys of Holstein calves a

15 ~g

150 ~g

Standard deviation per calf

291 19.8

289 19.6

38 0.6

Carotene intake b Weight per kidney (g) Dry matter (g/iO0 g) Ash Fresh basis (g/lO0 g) Dry matter basis

(g/100 g dry matter)

Calcium Fresh basis (mg/lO0 g) Dry matter basis

1.22

1.23

0.03

6.17

6.26

0.17

9.0

8.3

1.9

(mg/lO0 g dry matter) 45 42 10 Ash basis (mg/lO0 g ash) 731 675 144 a Average values for right and left kidneys for each of 15 calves per carotene intake grouping upon slaughter at the completion of carotene supplementation. b ~g/lb live wt/day.

953

CONSTITUENTS IN URINE

(Table 6). While there was higher average calcium content in the kidneys of the deficient calves, the difference was not statistically significant.

Gupta. Other ration constituents, possibly tocopherol, could be involved in the greater calcium content of the kidneys observed in the hypovitaminotic-A animals.

DISCUSSION

The significantly lower specific gravity, osmolality, and total solids of the urine of the calves fed the deficient carotene intake are tentatively interpreted as indicative of a decreased renal concentrating capacity (30). I t is recognized that had these measurements been made on urine collected after a period of fasting, the differences may well have been greater (20). Results of this study would tend to confirm the observation by the two groups of Wisconsin workers (11, 18) that kidney function is affected in vitamin A deficiency. However, the results are not directly comparable, especially with respect to vitamin A status of the animals. Although, with exception of phosphorus, none of the differences observed in the mineral content of urine between deficient and adequate calves were of sufficient magalitude to be statistically significant, the apparent lower sodium concentration and daily output in the urine of the vitamin A deficient calves were of particular interest. This is because of the importance of sodiunl in electrolyte balance, and the presence of edenm in bovine vitamin A deficiency as evidenced by anasarca (22), edema of the optic disc (25) and, possibly, elevated cerebrospinal fluid pressure (26). Additional studies of mineral metabolism in vitamin A deficiency are needed to more fully understand the importance of the above findings with respect to the altered urinary mineral concentration and daily output. I t is recognized that these apparent effects on mineral concentration and daily output may not be directly due to vitamin A deficiency but may be a result of the increased cerebrospinal fluid pressure affecting the central nervous system. This appears to be especially so in the case of sodium (4, 14). While the presence of higher concentrations of calcium in the kidneys of the deficient calves would appear to agree with the recent report of Majumdar and Gupta (23), more definitive experiments are needed before the involvement of vitamin A deficiency in renal calcinosis can be accepted. This is because in neither expel~iment were all ration variables, except carotene intake, held constant, alfalfa used as a source of carotene in the present experiment, and slaughter house specimens obtained from goats previously fed undefined rations used as controls in the experiment of Majmndar and

ACKNOWLEDG~ENTS

The authors express their appreciation to B. A. Donohue and T. Watts for their care and feeding of the animals; to A. Hoza and Mrs. Mae Miller for technical assistance; and to Dr. H. J. Fisher, Connecticut Agricultural Experiment Station, New Haven, for proximate analyses of feeds fed. The Nopco Chemical Co., Harrison, New Jersey, supplied the vitamin A supplement administered to the calves at one day of age and Dr. R. E. Messersmith, Agricultural Experiment Station, American Cyanamid Co., Princeton, New Jersey, the chlortetracycline (Aureomycin) oblets and sulfamethazine (Sulmet) used in the prevention and treatment of scours. We also appreciate the constructive criticism and suggestions made by various staff members at this Station during the preparation of the manuscript. We welcome this opportunity to acknowledge our indebtedness to Dr. L. A. Moore for suggesting this study. REFERENCES

(1) A~ONY~Ous. Baird Associates Flame Photometer Manual. Baird Associates, Inc. Cambridge, Mass. (2) ANONYI~IOUS. Instruction Manual Fiske Osmometer, Model D. Fiske Associates, Inc. Hathorne, Mass. (3) ASS(~CrAvION O~ OF~rCL~n AGP~CU~TVr~. CHEmiSTS. Official Methods of Analysis. 8th ed. Washington, D. C. 1955. (4) BI~IIAgAYER,H. The Adrenocortical Response to Increased Intracranial Pressure in Man. Mem. Soc. Endocrinol. No. 9. Progr. in Endocrinology. I. p. 60. The Cambridge University Press. 1960. (5) B V N ~ , R. H., ROUSSE~V, J. E., jm, EATOI~, I-I. D., AI'~D BEALI~ G.

(6) (7) (8)

(9) (10)

Estimation

of Vitamin A and Carotenoids in Calf Liver. J. Dairy Sei., 37: 1473. 1954. BUlCaII~L-MAICTI,1~., AND RAMLaEZ-MUNO'Z,J. Flame Photometry. Elsevier Publishing Co., New York. 1957. Cocmr~N, W. G., A~]) Cox, G. M. Experimental Designs. 2nd ed. John Wiley and Sons, Inc., New York. 1957. CO~0VE, E., TaA~TnO~, H. V., AND BOW~AN, t¢. L. An Instrument and Method for Automatic, Rapid, Accurate and Sensitive Titration of Chloride in Biological Samples. J. Lab. Clin. Med., 51: 461. 1958. D ~ o r ~ Y , B. A. Determination of Bovine Serum Calcium with a Simple Flame Photometer. J. Dairy Sci., 42: 872. 1959. EnA~, C. J., HA~, W. E., ANn DY~, I. A. An Attempt to Induce Urinary Calculi Formation in Steers with the Effects on Certain Blood and Urine Constituents. J. Animal Sci., 18: 383. 1959.

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C. G. %VOELFEL ET AL

(11) ELVEHJE~, C. A., AND NEU, V. F. Studies in Vitamin A Avitaminosis in Chicks. J. Biol. Chem., 97: 71. 1932. (12) FISK~, C. H., A~D SuB~r~ow, Y. The Colorimetrie Determination of Phosphorus. J. Biol. Chem., 66: 3q5. ]92'5. (13) GEVa~ITZ, tI. W. Digeesting ]3iological Material for Calcium and Phosphorus Analysis. Ind. Eng. Chem., Anal. Ed., 7:167. 1935. (14) GILBEI~T, G. J., AND GLASE~, G. H. On the Nervous System Integration of Water and Salt Metabolism. Arch. Neurol., 5: 179. 1961. (15) HAWK, P. ]3., OSER, B. n., AND SUMI~F,Iq,SON, W. H. Practical Physiological Chemistry. 13th ed. The ]3lakiston Co., Inc., New York. 1954. (16) HAZZ~..aO,D. G., G~Iro, A. P., Jm, RO~rSSEAU, J. E., Jm, WOFLm~L, C. G., EATON, H. D., NmLSm¢, S. W., AND GOSShE~, D. G. Effect of Level of Ration Intake and Duration of Vitamin A Deficiency upon Some Biochemical Constituents in Serum, Cerebrospinal Fluid, and Aqueous Humor of Holstein Calves Fed Fixed Carotene Intakes. J. Dairy Sci., 45: 91. 1962. (17) H~I,~BOLDT, C. F., JU~GRFAm, E. L., EATON, H. D., _ ~ M o o ~ , L. A. The Pathology of Experimental Hypovitaminosis A in Young Dairy Animals. Am. J. Vet. Research, 14: 343. 1953. (18) HE~RIN, ~. C., AND NICHOLES, H. J. The Influence of Vitamin A upon Urea and Inulin Clearance in the Dog. Am. J. Physiol., 125: 786. 1939. (19) Itom'¢, L. H., JR., RAY, M. L., Am) N~u)~A~¢~¢, A. L. Digestion and Nutrient-]3alance Stalls for Steers. J. Animal Sci., 13: 20. 1954. (20) JACOBSON, M. H., LEVY, S. E., KAU~.~N, R. M., GALLIN~K, W. E., AND DONNELLY~ 0. W. Urine Osmolality. Arch. Int. Med., 110: 83. 1962. (21) K~BL~, M. S. The Photocolorimetric Determination of Vitamin A and Carotene in Human Plasma. J. Lab. Clin. Med., 24: 1055. 1939. (22) MM)S~N, L. L., ANI) E ~ I ~ , I. P. Some Observations on Beef Cattle Affected with

(23)

(24)

(25)

(26)

(27) (28) (29)

(30) (31)

(3'2)

(33)

(34)

Generalized Edema or Anasarca Due to Vitamin A Deficiency. J. Nutrition, 34: 6.03. 1947. MAJU~I)AR, ]3. N., AN]) GUP~A, ]3. N. Vitamin A Deficiency and Urinary Calculi in Goats. Annals Biochem. Exptl. Med., 19: 249. 1969. MrCHA~S, G. D., AlvI)SRSO~, C. T., MAre G•br, S., ARrl) ]~II~Sl~D, L. W. A Method for the Colorimetric Determination of Calcium and Magnesium in Small Amounts of Urine, Stool, and Food. J. Biol. Chem., 180: 175. 1949. Moorm, L. A. Some Ocular Changes and Deficiency Manifestations in Mature Cows Fed a Ration Deficient in Vitamin A. J. Dairy Sci., 24: 893. 1941. Moom~, L. A., AND SYKES, J-. F. Terminal Cerebrospina] Fluid Pressure Values in Vitamin A Deficiency. Am. J. Physiol., 134: 436. 1941. MOOR~, T. Vitamin A. Elsevier Publishing Company, New York. 1957. Nngt,s~-, S. W. Unpublished data. Storrs (Conn.) Agr. Expt. Sta. 1961-63. P ~ R s , J. P., AND VA~ SLYK~ D. D. Quantitative Clinical Chemistry, Vol. II. Methods. The Williams and Wilkins Company, Baltimore, Md. 1956. S~ITtt, H. W. Principles of Renal Physiology. Oxford University Press, N. Y. 1956. SNE~mCOR, G. W. Statistical Methods. 5th ed. The Iowa State College Press, Ames. 1966. S'2OE~VSAN~),G. S., ~ I ) SCOTT, M. L. Effect of Protein on Utilization of Vitamin A in the Chick. Proc. Soc. Exptl. Biol. Med., 106: 635. 1961. SYKES, J. F., AND M o o ~ , L. A. The Normal Cerebrospinal Fluid Pressure and a Method for Its Determination in Cattle. Am. J. Vet. Research, 3: 364. 194'2. WOEILFEI*,C. G., CALHOUN, M. C., I:~OUSSE~U, J. E., JR., EAT0~, H. D., NmSSF~¢, S. W., AiqD KERSTING, E. J. Some Biochemical Constituents in the Urine of Vitamin A Deficient Holstein Calves. J. Animal Sci., 20: 957. (Abstract.) 1961.