Effects of la-Hydroxyvitamin D3, 1,25-Dihydroxyvitamin D3, 1,24,25-Trihydroxyvitamin D3, and 1,25,26-Trihydroxyvitamin D3 on Mineral Metabolism and 1,25-Dihydroxyvitamin D Concentrations in Dairy Cows 1

K. HOVE, 2 R. L. HORST, and E. T. L ITTLEDIKE National Animal Disease Center

Agricultural Research Service US Department of Agriculture

P.O. Box 70 Ames, IA 50010

ABSTRACT

Six mature, nonlactating, nonpregnant Jersey cows were treated with five 1-a-hydroxylated metabolites of vitamin D3 to compare relative potencies of the metabolites.

Blood plasma 1,25-dihydroxyvitamin D peaked at about 1000 pg/ml 12 h after intramuscular injection of 500 //g 1,25- dihydroxyvitamin D3 but returned to preinjection concentration within 3 to 4- days. Administration of 500 lag 1-a- hydroxyvitamin D3 intramuscularly led to a gradual increase in 1,25-dihydroxy- vitamin D concentration to 500 pg/ml 48 h after treatment. When 500 /ag of 1,25-dihydroxyvitamin D3 or la-hydrox- yvitamin D3 were given orally, plasma 1,25-dihydroxyvitamin D peaked at about 200 and 100 pg/ml, respectively. The decline from peak concentrations occurred more slowly after oral treatment than after intramuscular treatment. Plasma cal- cium was increased by 20 to 30% for a week or more, depending on 1,25- dihydroxyvitamin D concentrations in plasma. All treatments resulted in cal- ciuria.

Treatment with 1 or 3 mg doses of 1,24(R),25-trihydroxyvitamin D3 or both carbon-25 stereoisomers of 1,25,26-tri- hydroxyvitamin D3 induced smaller and less protracted changes in plasma minerals.

Received March 15, 1982. 1This investigation was supported in part by a

Public Health Service International Research Fellow- ship (No. 1 F 05 TW 02779--01).

2Agricultural University of Norway, Department of Animal Nutrition, P.O. Box 25, 1432 AS-NLH, Oslo, Norway.

Based on these criteria, the trihydrox- ylated vitamin D3 metabolites appeared only one-tenth as potent as 1,25-di- hydroxyvitamin D3 in the cow.

All la-hydroxylated metabolites of vitamin D3 tested affect mineral metab- olism in cows. Oral administration of vitamin D 3 metabolites may be a useful alternative to parenteral treatment.

INTRODUCTION

The rapidly expanding knowledge of metab- olism and actions of vitamin D has led to investigations on effectiveness of metabolites of vitamin D for prevention of bovine parturient paresis (milk fever). Although 25-hydrox- yvitamin D (25-OHD) acts more rapidly than vitamin D itself, hydroxylation of the i position to 1,25-dihydroxyvitamin D [1,25-(OH)2D] is required for the vitamin to exert its full effect (10, 15). Recent efforts to utilize vitamin D metabolites to control milk fever have con- concentrated on lu-hydroxyvitamin D3 (I~- OHD3) and, to some degree, 1,25-(OH)2D3 (6, 8, 11, 12, 20, 26, 31). Considerable information is available on the effects of 1o~-OHD3 and 1,25-(OH)2D3 on mineral metabolism in cattle and sheep (1, 2, 4, 5, 18, 27). Reports (8, 18) suggest that parenteral administration of 10t-OHD3 tO dairy cows prolongs increase in plasma 1,25-(OH)2D concentration more than administration of equivalent amounts of 1,25-(OH)2D3. We compare effects of la-OHD3 and 1,25-(OH)2D3 given orally (po) and parenterally (i.m.) on 1,25-(OH)2D3 con- centrations of blood plasma.

The 1,25-dihydroxyvitamin D can be me- tabolized in vitro to trihydroxylated compounds [ 1,24,25-(OH)3D3 and 1,25,26-(OH)3D3 ], which retain some of the biological activity of the parent 1,25-(OH)2D (3, 14, 19). Both

1983 J Dairy Sci 66:59--66 59

60 HOVE ET AL.

trihydroxylated metabolites recently have been isolated and identified from plasma of cows given large amounts of vitamin D3 (25). No information is available on actions of these trihydroxylated metabolites on mineral metab- olism in the cow. We compare effects of the l~-hydroxylated vitamin D3 compounds I~- OHD3, 1,25-(OH)2D3, 1,24,25-(OH)3D3, and 1,25,26-(OH)3D3 on plasma calcium (Ca), mag- nesium (Mg), hydroxyproline (OH-Pro), and phosphorus (Pi) concentrations and urinary calcium and magnesium excretion in non- lactating cows.

M A T E R I A L S A N D M E T H O D S

Six mature (> 5 yr of age), nonlactating, nonpregnant Jersey cows were kept in individual pens, and each was fed 5 kg alfalfa hay cubes and 2 kg of a beet pulp-based commercial dairy feed per day. The ration was divided into two equal parts and fed at 0800 and 1500 h. The daily intake of minerals (Ca, 66 g; Mg, 19 g; P, 22 g) was well above National Research Council (21) requirement. Treatments were given to individual cows in a random order. Four to five weeks elapsed between treatments with the various metabolites.

Treatments

Vitamin D metabolites were dissolved in 3 ml ethanol and injected i.m. The following treatments were given: 500 /lg la-OHD3 (4 cows); 500 pg 1,25-(OH)2D3 (4 cows); 1 mg 1,24(R),25-(OH)3D3 (4 cows); 1 mg 1,25(S),26- (OH)3D3 (5 cows); 3 mg 1,24(R),25-(OH)3D3 (2 cows); 3 mg 1,25(S),26-(OH)3D3 (2 cows); and 3 mg 1,25(R),26-(OH)3D3 (2 cows); Five hundred micrograms of either 1,25-(OH)2D3 (4 cows) or la-OHD3 (4 cows) were incorporated into fatty acid pellets (Rumen Kemi, Oslo, Norway) and mixed with the morning ration. The la-hydroxyvitamin D3 was a gift from Leo Pharmaceutical, Copenhagen, Denmark, and the other metabolites were gifts from Milan Uskokovia and John Partridge of Hoffmann-La Roche, Inc., Nutley, NJ.

Daily blood samples were taken by veni- puncture with heparinized syringes from the external jugular vein for 5 days before and 11 days after treatment. Additional blood samples were taken 6 and 12 h after treatment. Blood was centrifuged immediately and the plasma

1200 -

IO00-

== 800-

"T" 6O0-

~_ 200

0 . . . . . . . . .

-2 ]1

\

- - , . . . . ? I 2 3 4 5 6 7

0A¥S AFTER TREATI~ENT

Figure 1. Mean plasma concentrations of 1,25- (OH)~ D in 4 cows treated intramuscularly (e e) or orally (o o) with 500 ~g 1,25-(OH)aD 3 (mean -+ SE).

separated and frozen ( -15°C)unt i l assayed. Urine was collected continu ously with a catheter placed in the urinary bladder during 15 of the 23 treatment periods. Twenty-four-hour urine samples were collected, volumes were deter- mined, and a representative urine sample was taken and stored at -15°C. Urine collections began 1 day before treatment and were con- tinued 3 to 6 days after treatment. Creatinine clearance was used to estimate the glomemlar filtration rate, which varied between 40 and 60 liters/h.

Plasma and urine concentrations of Ca and Mg were determined by atomic absorption spectrophotometry, and standard colorimetric procedures (Technicon AutoAnalyzer) were used for inorganic phosphorus (Pi), creatinine, and OH-Pro (plasma only). Plasma concen- trations of 1,25-(OH)2D were determined as in (17). Concentrations of trihydroxylated me- tabolites in plasma will be reported elsewhere (16).

Urinary Ca was measured in forty-nine 24-h collections. Results were expressed as micro- moles of Ca excreted per liter of creatinine clearance. The transport constants for the tubular reabsorption of Ca were calculated according to Nordin (22) for the ultrafilterable fraction of plasma Ca 56%.

R ESU LTS

Treatments with 1 , 2 5 - ( O H ) 2 D ~ and 1 -~ -OHD 3

Mean plasma 1,25-(OH)2D concentrations of cows given 1,25-(OH)2D3 i.m. were 1050 pg/ml

Journal of Dairy Science Vol. 66, No. 1, 1983

ACTIVE VITAMIN D METABOLITES IN COWS 61

12 h after injection and returned to base within 4 to 7 days (Figure 1). In contrast, mean plasma 1,25-(OH)zD concentrations of cows given la-OHD3 i.m. increased to a plateau of 400 to 500 pg/ml 24 to 48 h after t reatment (Figure 2) and remained higher after day 2 than those of cows treated with 1,25-(OH)2D3 (Figures 1 and 2). Oral t reatment with either metaboli te induced much smaller increases in plasma 1,25-(OH)2D than the corresponding i.m. injections (Figures 1 and 2). Plasma con- centrations of 1,25-(OH)eD after oral dosing of 500 ~tg 1,25-(OH)2D3 were about twice those following I~-OHD3 treatment. After initial increases, the rate of decline in plasma 1,25- (OH)2D concentrations was slower after oral administration of 1,25-(OH)2D or I~-OHD3 than it was after i.m. administration (Figures 1 and 2).

Oral and i.m. t reatment with 1,25-(OH)2D3 led to comparable degrees of hypercalcemia throughout the sampling period. Mean plasma Ca increased .7 mmol/ l i ter (Figure 3) and remained 20 to 30% above pretreatment for about 5 days. Mean plasma Ca concentrations remained above pretreatment concentrations throughout the sampling period. Both treat- ments led to about a 20% reduction in plasma Mg concentrations (P<.01, t test). The reduction was of longer duration in animals receiving po treatment (Figure 3). Cows receiving the i.m. dose of 1,25-(OH)2D3 averaged plasma Pi increases of about 50% whereas plasma Pi increased only 25% after po treatment. Mean plasma Pi plateaued 3 days after t reatment and

130 T ~ n- .~ -,- o-o s00m ~,2~-(o%o~P~

,~ 120 . ~ ' ~ ~ .

90 , , , , , ~ , , , , ,

~" ,oo

~ 8(1 t

150 ,~

140

I10 ~ ~ ±

100

-I 0 I 2 3 4 5 6 7 8 9 I0 I1 DAYS AFTER TREATMENT

Figure 3. Relative changes in mean plasma con- centrations of calcium (Ca), magnesium (Mg), and inorganic phosphorus (Pi) in 4 cows after intramuscular (o o) (i.m.) and oral (o o) (po) treatment with 500 pg 1,25-(OH)2D s (mean -+ SE). One hundred percent is the mean concentration observed 4 to 5 days before treatment (Ca: 2.12 retool/liter; Mg: 0.84 mmol/liter; Pi: 1.79 mmol/liter). Shaded areas represent mean standard deviations (in percent) on observations taken from the same cows in the pre- injection period.

500

~4oo I

"-" 200 ~ " , . , i •

0 I I I I I I I I I I I I

-2 -I O I 2 3 4 5 6 7 6 9 I0 DAYS AFTER TREATMENT

Figure 2. Mean plasma concentrations of 1,25- (OH) 2 D in 4 cows treated intramuscularly (# e) or orally (o o) with 500 pg of I~-OHD 3 (mean -* SE).

tended to remain elevated throughout the sampling period (Figure 3).

Increases in mean plasma Ca were similar after t reatment with either la-OHD3 i.m. (Figure 4) or 1,25-(OH)zD3 i.m. or po (Figure 3). However;, mean plasma Ca concentrations increased only 10% following po I~-OHD3. Mean plasma Mg decreased significantly in cows given lcz-OHD3 either i.m. or po. By day 2, mean plasma Mg was 13% below preinjection (P<.02, t test) (Figure 4).

Mean plasma Pi concentrations peaked 5 days after i.m. la-OHD3 treatment. After oral la-OHD3 treatment, mean plasma Pi con- centrations were variable.

Journal of Dairy Science Vol. 66. No. 1. 1983

62 HOVEET AL.

Treatments With Trihydroxylated Metabolites

As in Figure 5A, 1-mg doses of 1,24(R),25- (OH)3D3 and 1,25(S),26-(OH)3D3 increased mean Ca concen t ra t ion 5 to 8% in plasma. By day 4, the Ca concen t ra t ion re turned to preinject ion. Mean plasma Mg decreased 10 to 13% for i to 5 days after t r ea tmen t (Figure 5A). Mean plasma Pi concent ra t ions increased 20% above p re t r ea tmen t for about 4 days (Figure 5A).

The 3-mg doses of 1 ,24(R) ,25-(OH)3D3, 1,25(S) ,26-(OH)3D~, and 1,25(R),26-(OH)3D3 caused larger increases in plasma Ca (Figure 5B)

o - 4 500~@ ~-(OH)D3iM 130

m HO _..1 Q .

", , - . - -

W

,.,.,. es

C ,

15o 2

140 -

130 -

I~0 - \

~o 1,~ ; ,oo-p 9oI'

!

0 I I I I I I I I I

2 3 4 5 6 7 8 9 I0 I I DAYS AFTER TREATMENT

Figure 4. Relative changes in mean plasma con- centrations of calcium (Ca), magnesium (Mg), and inorganic phosphorus (Pi) in 4 cows after intramuscular (e e) (i.m.) and oral (o o) (po) treatment with 500 vg 1-a-OHD~ (mean + SE). One hundred percent is the mean concentration observed 4 to 5 days before treatment (Ca: 2.29 mmol/liter; Mg: 0.82 mmol/liter; Pi: 1.65 mmol/liter). Shaded areas represent mean standard deviations (in percent) o n observations taken from the same cows in the preinjection period.

120 - A ~.. ,~ 1,2~{R)2~-{OHI3D ~

~ I 0 0 -

,,o]

sO I . . . . , , ,

~, ' ~ ~ 120 -

~ I00 -

9 0 -

l B w ~ 3 ~ I,z4(s)~q0a~3o s ~ .e l=s I ,~(R~q0xh03

x t"

?- . : f .."

i i i i i i i

DAYS AFTER TREATMENT

Figure 5. Relative changes in mean plasma con- centrations of calcium (Ca), magnesium (Mg), and inorganic phosphorus (Pi) after injections of I m g (A) or 3 mg (B) of different trihydroxylated metab- elites of vitamin D 3 . A: Mean + SE of 4 cows given 1,24(R),25-(OH)3D 3 (A A) and 5 cows given 1,25(S),26-(OH)3 D 3 (o o). B: Means of 2 cows per metabolite [1,24(R),25-(OH) 3D 3 (J A), 1,25(R),26-(OH)sD 3 (e e), 1,25(S),26-(OH)3D 3 ( c - - o ) ] . One hundred percent (hash-marked area) is the mean concentration observed 4 to 5 days before treatment (Ca: 2.23 mmol/liter; Mg: 0.86 mmol/liter; Pi: 1.58 mmol/liter). Shaded areas represent mean standard deviations (in percent) on observations taken from the same cows in the pre- injection period.

than did the 1-mg doses (Figure 5A). The dura t ion of mean plasma Ca response was about the same for bo th doses. Plasma Mg and Pi changes fol lowing the 3-mg doses were generally the same (Figure 5B) as those wi th the lower doses (Figure 5A) excep t for no increase in Pi concent ra t ions after 3 mg of 1,25(S),26-(OH) 3D3.

Plasma Concentrations of Hydroxyproline

Low concent ra t ions of OH-Pro which are typical for mature cows were seen in our animals (12 to 18 gruel / l i ter) . In some cows, small decreases (2 to 4 tamol/liter) were observed during the period o f hypercalcemia.

Journal of Dairy Science Vol. 66, No. 1, 1983

ACTIVE VITAMIN D METABOLITES IN COWS 63

Urinary Excretion of Calcium and Magnesium

The excretion of Ca was related to total plasma Ca in Figure 6. Treatments generally increased both the plasma Ca and urinary Ca excretion. The apparent t ransport maximum for Ca (TmCa) was 1.65 mmol/ l i ter of glome- rular filtrate (GF), and the K m for the transport (indicating the Ca concentrat ion in the ultra- filtrate required to half-saturate the transport mechanism) was 65 btmol/liter GF. Increasing plasma Ca from 2.20 to 2.65 mmol/ l i ter in- creased urinary Ca output 20-fold according to the calculated average calcium excretion line (Figure 6). The variation in urinary excretion on a given plasma Ca concentrat ion was con- siderable, however, and the distr ibution of the symbols in Figure 6 might indicate that the

45

40

55

50 ¸ L,I,-

. . . 25,

E

---~20. la,l

15-

l •o • o o

• _ ) C~)

" I I ' / "

I0 o o • • o

o

. ~ ~ o

2.0 2.2 2.4 2.6 2.8 3.0 Ca(mmol l l )

Figure 6. Relationship between mean plasma calcium concentration and urinary calcium excretion (Ca E) per liter of glomerular filtrate (GF) in 15 experiments with urinary collections. The symbols are observations from the pretreatment period, and from the 24-h periods following 1-~-OHD s -- e; 1,25-(OH)2D a -- o; 1,24(R),25-(OH)3D 3 -- A; and 1,25(S),26-(OH)sD s -- m. The solid line represents the calculated average calcium excretion (see Methods).

various metaboli tes differ in their effects on the relationship between Ca in plasma and urine.

Because changes in plasma Mg were too small to allow the same type of analysis as for Ca, we compared the renal handling of Mg in the pretreatment period with that in the actual 24-h period when plasma Mg was at a minimum. In both instances, the average urinary excretion of Mg was about 2.5 g/24 h (.09 + .05 mmol / liter GF) although plasma Mg was reduced by 12% on the average after injection of the vitamin D metaboli te. On the assumption that the ultrafil terable Mg fraction in plasma was a constant 65% (we assumed that 35% of plasma Mg was in bound form), we estimated that 15.8 + 1.9% (SE) of the filtered Mg load was excreted initially as compared to 17.9 + 2.9% in the 24-h period when plasma Mg was at a minimum. The two estimates were not different (P>.05, t test).

DISCUSSION

All of the l~-hydroxyla ted compounds tested have hypercalcemic, hyperphosphatemic, and hypomagnesemic activity after administra- tion to nonlactating, nonpregnant cows. Intra- muscular administration of 1~-OHD3 and 1,25-(OH)2D3 resulted in higher 1,25-(OH)zD concentrations in plasma than oral administra- tion. In agreement with (7, 18), i.m. ad ministration of I~-OHD3 prolonged increases in plasma 1,25-(OH)2D concentrations more than i.m. administration of 1,25-(OH)2D3. This is probably because I~-OHD3 must enter the 1,25-(OH)zD3 pool by 25-hydroxylat ion to become active. In our experiment, plasma 1,25-(OH)2D peaked at 450 pg/ml following administration of 500 /ag of 1-a-OHD3 i.m. Administrat ion of 400/2g by Carstairs et al. (8), however, yielded only peak plasma concen- tration of 140 pg/ml. The discrepancy between the two experiments is difficult to explain. Differences in puri ty of the administered I~-OHD3 or differences in carrier (ethanol vs. corn oil) may have been responsible. Also, animal differences could have been responsible as lactating Holstein cows were used in the study of Carstairs et al. (8). Regardless, oral ad- ministration of fat-protected I&-OHD3 or 1,25-(OH)2D3 resulted in about one-fifth the increase of plasma 1,25-(OH)eD3 compared with i.m. administration of these metabolites.

Journal of Dairy Science Vol. 66, No. 1, 1983

64 HOVE ET AL.

However, no information is available on in- testinal absorption of vitamin D metabolites in ruminants. Studies in the rat have shown that absorption of both 25-hydroxyvitamin D3 and the dihydroxylated compounds occur rapidly from the small intestine (22). Ruminal micro- organisms can degrade vitamin D (28, 29). Vitamin D metabolites were fed in a lipid vehicle designed to give incorporated substances protection against ruminal degradation. How- ever, intraruminal injections of ethanolic solutions of 500 /ag of 1-ot-OHD3 or 500 pg 1,25-(OH)2D3 to a fistulated cow, which should maximize degradation, gave plasma 1,25-(OH)2D3 cohcentrations comparable to those after oral dosing with fat-protected metabolites (data not shown). The comparable plasma 1,25-(OH)2D concentrations for metab- olites either fed in fat or given intraruminally in ethanol may indicate that incorporation of the metabolites in fat did not prevent significant microbial degradation.

Increased concentrations of Ca in plasma after treatment of cows with I~-OHD3 or 1,25-(OH)2D3 are documented (1, 2, 4, 5, 18, 27). Changes after injection of these two metabolites agree well with earlier findings. Almost identical Ca concentrations were observed after oral or i.m. doses of 1,25-(OH) 2D3, although oral treatment caused only one-fifth of the peak 1,25-(OH)2D concen- tration after i.m. treatment. This suggests that 1,25-(OH)2D3 given orally may have a direct effect on the gut to increase intestinal absorption of Ca. Thus, the oral route may have an ad- vantage over parenteral administration to prevent milk fever, because equivalent stimula- tion of gut absorption may be associated with lower plasma 1,25-(OH)2D3 concentration, and, thus, less feedback inhibition of renal l~-hydroxylase enzyme by 1,25-(OH)2D3 may be present. However, if equivalent hypercalcemic inhibition of renal la-hydroxylase is present, any advantage of the oral administration could be annulled. When plasma 1,25-(OH)2D was raised to only 100 pg/ml with oral I~-OHD3 treatment, however, increases in plasma Ca were much lower. A major effect of 1,25- (OH)~ D on Ca metabolism is mediated through activation of intestinal absorption. Although direct evidence is lacking from cows, results demonstrating enhanced Ca absorption after I~-OHD3 treatment of sheep (4) and 1,25-

(OH)2D3 treatment of goats (Hove, unpublished data) indicate that this is a common mechanism of action in ruminant species also. Calcium derived via bone resorption probably is only a minor contribution to the hypercalcemia because plasma hydroxyproline concentrations were reduced during the hyperealcemic period. This agrees with the results of Ca-kinetic data in sheep treated with I~-OHD3 (4). Some con- troversy still exists because increased urinary hydroxyproline excretion was reported by Capen et al. (5) in cows treated with 600 gg of 1,25-(OH)2 D3.

Both oral and parenteral administration of these vitamin D metabolites would require individualized handling of milk fever-prone cows in a herd. This would present a major practical problem under group management practices in many present-day herds. The oral route would require more of the metab- olites, because significant rumen degradation of these compounds is likely unless means are developed to protect these compounds from rumen contents. Oral administration of 1,25- (OH)2 D 3 may be advantageous if the compound has local effects on the gut. This may make it possible to stimulate intestinal absorption of Ca with doses of the compound in the physiologic range rather than the huge pharmacologic doses that currently are being given. If this is the case, then it would be possible to minimize the prolonged inhibition of renal l~-hydroxylase that follows the commonly used doses of most vitamin D compounds (unpublished).

The apparent transport maximum for Ca of 1.65 mmol/liter GF calculated from the urine observations is close to 1.88 mmol/liter GF calculated for man (23). The Km for reab- sorption, which indicates the Ca concentration in the ultrafiltrate required to half-saturate the transport mechanism, was much lower than the corresponding figure in man. This is consistent with the high rate of Ca reabsorption that characterizes the ruminant kidney (13).

When plasma Ca was elevated to about 2.7 mmol/liter, urinary Ca excretion increased about 20-fold, and urinary Ca excretion ranged from 1 to 2 g/day.

Our studies give no information on the rate of entry of Ca into the extracellular pool. In a study with sheep treated with I~-OHD3, Braithwaite (4) observed a fourfold increase in Ca absorption up to about 40% of dietary

Journal of Dairy Science Vol. 66, No. 1, 1983

ACTIVE VITAMIN D METABOLITES IN COWS 65

intake. Large amounts of Ca would have to be deposited in bodies of cows in our study - either in the bones or as soft tissue cal- cifications - if a comparable increase in Ca absorption did occur after la-OHD3 and 1,25-(OH)2 D3 treatment.

Both trihydroxylated compounds at the 1-mg dose resulted in smaller increments of plasma Ca that were of shorter duration than 1,25- (OH)2D3 or Icg-OHD3. This is the first reported evidence of in vivo biological activity of 1,25, 26-(OH)3D3. The naturally-occurring 25,26- (OH)2D3 is the 25 (S) stereoisomer (24), and it, thus, seems likely that the naturally-occurring 1,25,26-(OH)aDs is the 25(S) form. Because both the 25(R) and 25(S) stereoisomers of the molecule were equally potent in promoting hypercalcemia, our results indicate that the stereochemistry of the C25 atom in 1,25,26- (OH)aD3 is of minor importance for expressing the biological activity of 1,25,26-(OH)3D3. The trihydroxylated compounds appear only one- tenth as potent as 1,25-(OH)2D3 in causing hypercalcemia, as judged from the magnitude and duration of the response.

Doses of the trihydroxylated compounds were in pharmacologic quantities. We estimate the concentration of the trihydroxylated compounds is less than 10 pg/ml in normal cows. This is much less than 1,25-(OH)2Ds con- centrations and would be undetectable in our assays (unpublished).

All compounds tested caused some hypo- magnesemia. Similar changes in Mg concen- trations of plasma have been reported after treatment with la-OHD3 or 1,25-(OH)2Ds, but the cause of this hypomagnesemia is unclear (1, 6, 27).

Plasma Mg was at or above the renal threshold for Mg (.7 mmol/liter) (30) in our experiment, and changes in urinary Mg excretion were consequently rather insensitive to small varia- tions in plasma Mg. It, nevertheless, would be exppected that a 10 to 15% reduction in the filtered load of Mg would lead to a reduction in urinary Mg excretion. Our finding of a constant or even increased fractional excretion when plasma Mg was minimal could be explained by the concept of a common carrier mechanism for Ca and Mg in the kidneys (9) because the hypomagnesemia occurred simultaneously with raised Ca concentrations. More precise mea- surements of the urinary Mg excretion however,

should be undertaken during development of the hypomagnesemia to clarify this point.

Because 1,25-(OH)2D3 stimulates Pi ab- sorption in most species, it seems likely that the gradual increments in plasma Pi that occurred with increasing plasma 1,25-(OH)2D con- centrations were because of a dose-dependent stimulation of Pi absorption. Trihydroxylated metabolites of vitamin D all showed hyper- phosphatemic effects. The relatively large variations and persistent increases in plasma Pi after 10t-OHD3 or 1,25-(OH)2D3 treatment indicate the homeostatic control of Pi is less precise than the control of plasma Ca.

In conclusion, all the 1-hydroxylated com-

pounds tested induced hypercalcemia, hyper- phosphatemia, and hypomagnesemia in the cow. Maximal increments in plasma Ca appeared to occur when plasma 1,25-(OH)2D reached 250 pg/ml whereas higher 1,25-(OH)2D gave even larger increases in plasma Pi. Cows used in our study were not lactating or pregnant, and care, therefore, must be taken in applying our findings to pregnant or lactating cows, or to cows prone to milk fever where the drain of minerals is likely to influence results.

REFERENCES

1 Barlet, J. P. 1975. Influence du lc~-hydroxy chol~calcif~rol sur la calcemi6 et la phosphattmie des bovins. C. R. Acad. Sci. Paris 5. 281, Series D: 1497.

2 Bjorklund, N. E., G. Jonsson, and B. Pehrson. 1976. Effects and side effects of parenterally administered 1-ce-hydroxy cholecalciferol (1-c~- OH-D s) to cows. Page 1003 in Rep. and Abstr., 9th Int. Congr. Diseases in Cattle, Paris.

3 Boris, A., J. F. Hurley, and T. Trmal. 1980. In vivo mobilization of bone calcium in thyroparathy- roidectomized rats by l-a, 25-dihydroxychole- calciferol (calcitriol) and related compounds. J. Nutr. 110:2291.

4 Braithwaite, G. D. 1980. The effect of dose rate of 1-a-hydroxycholecalciferol on calcium and phos- phorous metabolism in sheep. Br. J. Nutr. 44:183.

5 Capen, C. C., G. F. Hoffsis, and A. W. Norman. 1977. Dose-response of 1,25-dihydroxycholecal- ciferol on serum calcium and phosphorous, urine electrolyte and hydroxyproline excretion in dairy cattle. Page 565 in Vitamin D biochemical, chemical and clinical aspects related to calcium metabolism. A. W. Norman, K. Schaefer, J. W. Coburn, H. F. DeLuca, D. Fraser, H. G. Grigolelt, and D. yon Herrath, ed. Walter deGruyter & Co., Berlin, New York.

6 Capen, C. C., G. F. Hoffsis, and A. W. Norman. 1979. 1,25-Dihydroxycholecalciferol and the pre-

Journal of Dairy Science Vol. 66, No. i, 1983

66 HOVE ET AL.

vention of parturient hypocalcemia and paresis ("milk fever") in pregnant dairy cows. In Vitamin D: basic research and its clinical applications. A. W. Norman, K. Schaefer, D. yon Herrath, H. G. Grigoleit, J. W. Coburn, H. F. DeLuca, E. B. Mawer, T. Suda, ed. Waiter deGruyter & Co., Berlin, New York.

7 Carstairs, J. A., N. A. Jorgensen, and H. F. DeLuca. 1980. Uptake and metabolism of 1,25-dihydrox- yvitamin D~ after intramuscular administration to the cow. J. Dairy Sci. 63 (Suppl. 1):87. (Abstr.)

8 Carstairs, J. A., M. K. Treutelaar, N. A. Jorgensen, and H. F. DeLuca. 1981. PotentiaI use of a com- bination of 1,25<tihydroxyvitamin D 3 [1,25- (OH)2D 3] and 1-c~-hydroxyvitamin D 3 [1-a- (OH)D 3] as a milk fever preventative. J. Dairy Sci. 64 (Suppl. 1):156. (Abstr.)

9 Chicco, C. F., C. B. Ammerman, R. M. Valesecchi, and N. A. Marcilese. 1978. Effect of dietary magnesium on calcium utilization by sheep. Acta. Cient. Venez. 29 (Suppl. 2):161. (Abstr.)

10 Fraser, D. R., and E. Kodicek. 1970. Unique biosynthesis by kidney of a biologically active vitamin D metabolite. Nature 228:764.

11 Gast, D. R., R. L. Horst, N. A. Jorgensen, and H. F. DeLuca. 1979. Potential use of 1,25-dihydrox- ycholecalciferol for prevention of parturient paresis. J. Dairy Sci. 62:1009.

12 Gast, D. R., J. P. Marquardt, N. A. Jorgensen, and H. F. DeLuca. 1977. Efficacy and safety of 1-c~- hydroxyvitamin D 3 for prevention of parturient paresis. J. Dairy Sci. 60:1910.

13 Hansard, S. L., C. L. Comar, and M. P. Plumlee. 1952. Absorption and tissue distribution of radio- calcium in cattle. J. Anim. Sci. 11:524.

14 Holick, M. F., A. Kleiner-Bossaller, H. K. Schnoes, P. M. Kasten, I. T. Boyle, and H. F. DeLuca. 1973. 1,24,25-Trihydroxyvitamin D~: A metabolite of vitamin D~ effective on intestine. J. Biol. Chem. 248:6691.

15 Holick, M. F., H. K. Schnoes, and H. F. DeLuca. 1971. Identification of 1,25-dihydroxycholecal- ciferol, a form of vitamin D 3 metabolically active in the intestine. Proc. Natl. Acad. Sci. 68:803.

16 Horst, R. L., K. Hove, E. T. Littledike, J. J. Part- ridge, T. A. Reinhardt, and M. R. Uskokovic. 1982. Plasma concentrations of 1,25-dihydroxyvitamin D, 1,24,25-trihydroxyvitamin D3, and 1,25,26- tr ihydroxyvitamin D 3 after parenteral administra- tion of these compounds to cows. J. Dairy Sci. (Submitted for publication).

17 Horst, R. L., E. T. Littledike, J. L. Riley, and J. L. Napoli. 1981. Quantitation of vitamin D and its metabolites and their plasma concentrations in five species of animals. Anal. Biochem. 116:189.

18 Hove, K., R. L. Horst, and E. T. Littledike. 1981. la(OH)D 3 and 1,25-(OH)2D 3 administration to cows: A comparison of perosal and intramuscular

treatment. J. Dairy Sci. 64 (Suppl. 1):156. (Abstr.) 19 Kleiner-Bossaler, A., and H. F. DeLuca. 1974.

Formation of 1,24,25-trihydroxyvitamin D 3 from 1,25-dihydroxyvitamin D 3. Biochim. Biophys. Acta 338:489.

20 McMurray, C. H., D. A. Rice, and P. S. McBride. 1980. Milk fever controls: Comparison of 1-alpha and vitamin D 3 in conjunction with induced parturition. Vet. Rec. 107:188. National Research Council. 1978. Nutrient re- quirements of dairy cattle. No. 3. Nutrient re- quirements of domestic animals. Natl. Acad. Sci., Washington, DC.

22 Nechana, H., D. Hoff, A. Harell, and S. Edelstein. 1977. The intestinal absorption of vitamin D and its metabolites. J. Mol. Med. 2:413.

23 Nordin, B.E.C. Page 290--294 in Calcium, phos- phate and magnesium metabolism. Churchill Livingstone, Edinburgh, London, New York.

24 Partridge, J. J., S.-J. Shiuey, N. K. Chadha, E. G. Baggiolini, J. F. Blount, and M. Uskokovic. 1981. Synthesis and structure proof of a vitamin D 3 metabolite, 25(S),26-dihydroxycholecalciferol. J. Am. Chem. Soc. 103:1253.

25 Reinhardt, T. A., J. L. Napoli, R. L. Horst, E. T. Littledike, and D. C. Beitz. 1981. Isolation and chemical characterization of 1,25,26-trihydrox- yvitamin D 3 and 1,24,25-trihydroxyvitamin D 3 from plasma of cows treated with large doses of vitamin D 3. J. Dairy Sci. 64 (Suppl. 1):157. (Abstr.)

26 Sachs, M., A. Bar, R. Cohen, Y. Mazur, E. Mayer, and S. Hurwitz. 1977. Use of la-hydroxycholecal- ciferol in the prevention of bovine parturient paresis. Am. J. Vet. Res. 38:2039.

27 Sansom, B. F., M. J. Vagg, and W. M. Allen. 1975. The effects of 1-hydroxycholecalciferol on the calcium and phosphorous metabolism of dairy heifers. Proc. Nutr. Soc. 35:55A. (Abstr.)

28 Sommerfeldt, J. L., R. L. Horst, E. T. Littledike, D. C. Beitz, and J. L. Napoli. 1981. Metabolism of orally administered [s i l l -v i tamin D 2 and [3H]- vitamin D 3 by dairy calves. J. Dairy Sci. 64 (Suppl. 1):157. (Abstr.)

29 Sommerfeldt, J. L., R. L. Horst, J. L. Napoli, D. C. Beitz, and E. T. Lirtledike. 1980. Evidence for in vitro production of vitamin D 2 and vitamin D 3 metabolites by rumen microbes. J. Dairy Sci. 63 (Suppl. 1):88. (Abstr.)

30 Storry, J. E., and J.A.F. Rook. 1963. Magnesium metabolism in the dairy cow. V. Experimental observations with a purified diet low in magnesium. J. Agric. Sci. 61:167.

31 Wittwer, F., and E.J.H. Ford. 1980. The effect of 1-~-hydroxycholecalciferol on the concentrations of calcium, magnesium and inorganic phosphate in the plasma of parturient cows. J. Dairy Res. 47:177.

21

Journal of Dairy Science Vol. 66, No. 1~ 1983