P R O D U C T I O N R E S E A R C H PAPERS

Sequential Sampling and Analysis of Renal Hydroxylase Activities of Cattle Given l~ -Hydroxyv i tamin D3

E. T. LITTLEDIKE, 1 G. W. ENGSTROM? and MAX SACHS 3 National Animal Disease Center

Agricultural Research Service US Department of Agriculture

Ames, IA 50010

ABSTRACT

A new method was developed for sequential sampling of bovine renal cortex. This method results in minimum hemorrhage and adhesions and provides sufficient renal cortex tissue for assay of 25-hydroxyvitamin D lc~-, 24-, and 23- hydroxylase activities. Application of this procedure in calves and pregnant cows treated with la -hydroxyvi tamin D3 is described. The success of these experi- ments suggests these techniques could be used to follow enzyme activities that con- trol crucial aspects of vitamin D metabo- lism in normal peripartum cows and cows with milk fever or other diseases of mineral metabolism.

INTRODUCTION

1,25-Dihydroxyvitamin D [1,25-(OH)2D] is considered to be the physiologically active form of the prohormones 25-hydroxyvitamin D3 (25-OHD3) and vitamin D3. Control of 1,25- dihydroxyvitamin D3 [1,25-(OH)2D3] produc- tion is tightly regulated by renal l~-hydroxy- lase (2, 5, 7, 9, 10). Other renal hydroxylases (23- and 24-hydroxylases) regulate production of the other major vitamin D metabolites. Characterization of the major factors that con- trol 1~- or 23- and 24-hydroxylase activity is essential to understand vitamin D metabolism and diseases of mineral metabolism. There are

Received June 21, 1985. 1 US Department of Agriculture, National Program

Staff, Building 005, Room 206, Beltsville Agricultural Research Center-West, Beltsville, MD 20705.

2 To whom correspondence should be addressed. 323 Mose Shapiro St., Netanya 42240, Israel.

no reports of techniques to sample and mea- sure lc~-, 24-, and 23-hydroxylase activities in bovine kidney. Sequential sampling of the kidney from cattle is necessary to characterize renal hydroxylase activity during development of diseases of calcium or magnesium metabo- lism, i.e., rickets, parturient paresis, and grass tetany. Also, determining the effects of treat- ment of cattle with vitamin D or its metabolites on renal hydroxylase activity is important, because these vitamin D compounds are being investigated to prevent parturient paresis (1, 6, 15, 19).

There are a variety of biopsy needles com- mercially available for needle biopsy specimens from kidneys and other organs (3). Sumner- Smith et al. (20) surgically obtained large sam- ples of the bovine kidney tissue by using a partial nephrectomy technique. However, we know of no procedure or instrument available for taking serial 100 to 200 mg samples from the bovine kidney without the undesirable effects of major surgery and general anesthesia.

A technique was needed to take serial samples of bovine kidney of sufficient size to measure the activities of the renal hydroxylase enzymes involved in vitamin D metabolism. The technique must result in minimum damage (hemorrhage) and minimum postsampling adhe- sions so that four to five sequential samples can be taken from the same kidney. The bovine kidney has tough, fibrous interlobular divisions and a strong fat ty peritoneal covering, which tends to occlude biopsy instruments. Accord- ingly, a biopsy tool was needed that combined both cutting and aspiration capabilities.

This report describes 1) a biopsy tool that is satisfactory for sequentially obtaining 100 to 200-mg samples of bovine kidney, and 2) the renal 25-OHD l&-, 24-, and 23-hydroxylase activities of cattle before and after t reatment with lc~-OHD3.

1986 J Dairy Sci 69:990-997 990

ENZYME ACTIVITY IN BOVINE RENAL BIOPSIES 991

MATERIALS AND METHODS

Biopsy Instrument

The biopsy toot (Figure 1A)was made of stainless steel tubing and consisted of four separate pieces. The guide tube (Figure 1C) was .9 cm o.d., .8 cm i.d., and 30 cm long with a knurled knob 2.5 cm long and 1.9 cm in diame- ter on the proximal end. Through this was passed a cutting-suction tube (Figure 1B) that was .7 cm o.d., .5 cm i.d., and 39 cm in length. The proximal end of the cutting-suction tube had a knurled knob 1.2 cm long and 1.9 cm in diameter fitted so that 1.8 cm of the proximal end of the cutting tube extended past the knob. A solid side arm extended about 3 cm from one side of the knob and a suction side port (.8 cm o.d. and .6 cm i.d.) extended 3.3 cm on the opposite side of the knob. The cutting-suction tube had two opposing flattened surfaces on its distal end. The blade attachment area was removed from two Bard Parker (BD) ~7 a sur- gical handles and soldered to either side of the flattened surfaces of the cutting-suction tube. Two BD #11 stainless steel surgical blades were ground to a width of .5 cm and fitted over the two-blade attachment areas of the cutting suc- t ion tube. A nipple-type rubber bulb (2 cc) was fitted over the proximal end of the cutting- suction tube to seal it and act as a vacuum indicator.

A rubber "0" ring (1 cm o.d., .6 cm i.d.) was slipped over the cutting tube and placed an appropriate distance from the proximal end of the tube so that 1.3 cm of the surgical blades protruded from the guide tube when the unit was assembled for use.

A solid .5 cm x 40 cm extrusion rod (Figure 1A) with a 1.8 x 2.5-cm knurled knob was used to push surgical type gel foam through the cut- ting-suction tube. A trocar .8 cm x 35 cm with a knurled knob (1.8 x 2.5 cm) at its proximal end and with the distal end sharpened to a four- sided beveled point was put through the guide tube to introduce the guide tube when a blind

4Mention of a trade name, proprietary product, or vendor does not constitute a guarantee or warranty by the US Department of Agriculture and does not imply its approval to the exclusion of other products or ven- dors that may be suitable.

percutaneous approach was used in young cattle.

Preparation of Cows and Calves for Surgery

Cows were sedated with 25 mg of xylazine i.m. They were also given a Cakala paraverte- bral block of the 13th thoracic and first and second lumbar nerves on the right side using injections of 2% lidocaine solution. Calves were given 10 mg xylazine i.m. and 2% lidocaine solution as a local anesthetic at the site of entry.

Surgical Procedure

A 12-cm skin incision was made in the right paralumbar fossa beginning 10 cm below the transverse process of the 1st lumbar vertebrae and proceeding diagonally in a caudoventral direction. The external abdominal oblique mus- cle was bisected and the internal abdominal oblique and transverse abdominus muscles were incised along their axes. The peritoneum was cut, the right kidney palpated, and a site on the external surface free of fat was selected for sampling. The sampling site was selected slightly toward the periphery of the kidney to avoid the central renal pelvis and larger blood vessels and ureter. The guide tube was placed in position and the cutting-suction tube was in- serted such that the knives penetrated the cortex of the kidney. The cutting tube then was pressed firmly against the "0" ring to form a seal and vacuum (20 mm Hg) applied by a suc- tion device (Model 128, C. M. Sorensen Co., Inc., Long Island City, NY). The clear polyvinyl suction tube between the suction pump jar and the cutting tube had previously been partially restricted with a clamp. The clamp had been closed just enough (approx. 75%) to prevent suction of the kidney sample into the jar. When the rubber pipette device collapsed, indicating a vacuum was present, the cutting tube was rotated once (360 °) in a clockwise direction and then once in a counterclockwise direction. Just enough force was applied to the cutting tube to maintain the vacuum. Usually the sam- ple was removed after the initial two rotations of the cutting tube. If not, one additional rota- tion in either direction was made. Once the sample was visible at the partially clamped area of the tube, the suction was stopped, the cutting tube was withdrawn, and the sample

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992 LITTLEDIKE ET AL.

removed from the polyvinyl tube. The biopsy hole was plugged by inserting gel foam (Gel- foam sterile sponge or resorbable gelatin sponge, Upjohn Co., Kalamazoo, MI) down the guide tube using the extruder rod. The guide tube was removed and digital pressure over the site of biopsy was maintained for several minutes to facilitate clotting and prevent bleed- ing. The peritoneum, muscles, and skin were closed in layers.

Little difficulty was experienced in going through the same incision two or three times at 2-d intervals. A second incision caudal and more vertical was made to avoid previous ad- hesions if more than two or three sequential biopsy specimens were needed. If, on visual in- spection, the right kidney appeared abnormal or all the safe, easily accessible areas had been sampled, the left kidney was sampled. The left

I I I

kidney was located by palpation just medial to the right kidney. Its location was variable. It was located either slightly caudal, alongside, or slightly in front of the right kidney. The left kidney could be biopsied on its medial surface near either pole using the same procedures pre- viously described.

During the sampling procedures, if too much force was applied to the cutting and guide tube, the cortical area of the kidney was penetrated and the medulla and some of the bigger renal vessels were encountered. In this case, a foam plug (Gelfoam sterile sponge or absorbable gelatin sponge, Upjohn Co., Kalamazoo, MI) was installed and digital pressure applied. The bleeding could be s topped at the surface of the kidney, but bleeding would occasionally con- t inue into the renal pelvis, and bloody urine was evident for several hours. Packed cell

t ~ rr T I ~ '~ ~

B

I C

i • i ( iii ¸ r l • i f i

Figure 1. Biopsy instrument. A) Three pieces of apparatus; solid extrusion rod (top), cutting suction tube (middle), guide tube (bottom). B) Close-up of distal ends of cutting suction tube. C, D) Close-up of distal end of assembled cutting and guide tube.

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ENZYME ACTIVITY IN BOVINE RENAL BIOPSIES 993

volume decreases were evident if the bleeding continued for several hours. However, all cows recovered quickly even after prolonged bleed- ing. Combiotic (30 ml/d) was given for 5 d if excessive bleeding or microbial contamination were thought to have occurred.

In young cattle (under 200 kg), a single incision (5 cm) was made about 5 cm vertical from the first lumbar vertebrae and about 8 cm behind the last rib. The trocar was used to introduce the guide tube into the peritoneal cavity. The trocar was removed and the kidney located by advancement of the guide tube. If necessary, a finger could be inserted through the incision and the area on the kidney for biopsy located. Usually, one or more flattened areas near either pole of the kidney could be located by feel with the guide tube or finger. The cutting-suction tube was advanced through the guide tube, the sample removed, and the hole sealed using the usual procedures.

Sterols

Crystalline vitamin D3 was purchased from Sigma Chemical Company, St. Louis, MO. Crystalline 25-OHD 3 was obtained from the Upjohn Company, Kalamazoo, MI. The 1,25- (OH)~[26,27-3H]D3 and 24,25-(OH)2126,27- 3H] D3 were prepared in our laboratory by the method of Horst et al. (12). Concentrations of standard vitamin D metabolites were deter- mined in ethanol (Ema x -+ 18,200) at 265 nM with a Beckman DB scanning spectrophotome- ter (Beckman Instruments, Palo Alto, CA). l~-Hydroxyvi tamin D3 was obtained from M. R. Uskokovic (Hoffmann-La Roche, Inc., Nutley, N J).

Preparation of Kidney Homogenate for Enzyme Assay

Biopsy samples of renal cortex (90 to 250 rag) were placed in ice-cold .25 M sucrose, damp dried, weighed, and used to prepare a 20% (wt/vol) homogenate in ice-cold buffered sucrose (.05 M Hepes buffer, pH 7.4 at 37°C in .25 M sucrose). An experiment was done to evaluate which homogenizer was most effective in breaking cell walls. A small biopsy tool was used to take multiple samples (90 to 150 mg) from a calf's kidney over a 5-rain period. The samples were pooled into four groups. Homoge- nization of one of the four groups of samples

was done with each of the following: a Potter- Elvehjem teflon glass homogenizer (size A or B), a Brinkmann Polytron Model PT 10/35 homogenizer, or a glass-glass hand-operated Tenbroeck tissue grinder (Table 1). In subse- quent experiments, homogenization was done only with the Potter-Elvehjem or Brinkmann Polytron homogenizer. Two to four samples from each of the four homogenates were tested in the 25-OHD-hydroxylase assay system (4). Crude homogenate was transferred to 1.5-ml polypropylene microcentrifuge tubes and cen- trifuged in a Beckman Microfuge 12 at top speed (12,000 x g) for 10 min in a cold room (4°C). The supernatant was decanted and the pellet resuspended with a thin glass rod and a Potter-Elvehjem homogenizer in a volume of fresh ice-cold, buffered sucrose equal to that of the decanted supernatant.

Assay of Renal Cortical 1~-, 24-, and 23-Hydroxylases

A modification of (4) was used with the small biopsy size tissue samples. Ten-milliliter (1.3 × 10 cm) glass tubes were used as incuba- tion flasks. Each tube contained 2 mM MgC12, 30 mM Hepes buffer (pH 7.4 at 37°C), 30 mM L-malate, 150 mM sucrose, and kidney cortex homogenate equivalent to 20 mg tissue. Tubes containing .5-ml volume were placed in a test tube rack, oxygenated 30 s, placed in a 37°C water bath shaker (80 cycles/min) for a 5-min temperature equilibration, and the reaction started by addit ion of 10/ag of 25-OHD3 sub- strate in 1 /al ethanol. Incubation time was 15 rain and the reaction was s topped by addit ion of 1.5 ml of a 2:1 mixture of methanol:chloro- form (vol/vol). Measured amounts (about 1000 cpm in 50 /al ethanol) of radioisotopically- labeled 1,25-(OH)2126,27-3H]D3 and 24,25- (OH)2-[26,27-3H]D3 were added to each incu- bation tube and to two liquid scintillation vials to determine recovery efficiency of the vitamin D metabolites from extraction and chromatog- raphy procedures used in the assay. Recoveries for 1,25-(OH)2D3, 24,25-(OH)zD3, and 23,25- (OH)2D3 ranged from 50 to 75%. The (OH2)D metaboli te fraction from Sephadex LH-20 column was separated by high-performance liquid chromatography. 1,25-Dihydroxyvitamin D 3 was resolved with two different chromato- graphic systems: a straight phase Zorbax Sil

Journal of Dairy Science Vol. 69, No. 4, 1986

994 LITTLEDIKE ET AL.

co lumn (.45 x 25 cm, Dupoint ) with a 90:10 hexane : i sopropanol (vol/vol) solvent system, and a straight-phase Zorbax Sil NH2 co lumn (.45 x 25 cm, Dupont ) with an 86:14 hexane: isopropanol (vol /vol) solvent system. 24,25- Dihydroxyv i t amin D3 (8, 11) and 23,25- (OH)2D3 (13, 16, 21) fract ions were resolved with two dif ferent solvent systems on straight- phase Zorbax Sil columns (.45 x 25 cm, Dupont) . The first solvent system was 90 :10 hexane : i sopropanol (vol/vol) and the second 86 :10 :4 (hexane :me thy lene chlor ide: isopro- panol (vol/vol/vol) . Quant i ta t ion of vi tamin D metabol i tes was done by the radioligand pro- tein binding assay (14) except that the binding protein used for 1,25-(OH)2D3 binding was f rom bovine thymus (18), whereas that for 23,25-(OH)2D3 and 24,25-(OH)2D3 was vita- min D-replete, rat plasma-binding protein (12). Sensit ivity of the analytical me thod for deter- minat ion of 1,25-(OH)2D3, 24,25-(OH)2D3, and 23,25-(OH)2D3 in cow-calf k idney homoge- nate incubat ion mixtures was the same as that for 1,25-(OH)2D 3 (7 pg/ml) and 24,25-(OH)2- D 3 and 23,25-(OH)2D3 (.2 ng/mi) in plasma.

Animals and Diets

Two pregnant (first tr imester) , lactating (under 5 kg/d) 4-yr-old Jersey cows housed at

the National Animal Disease Center were given 4.5 kg of a comple te feed mix, 4.5 kg of cracked corn, and brome grass hay (free choice) each day. This provided the cows with an excess (20 to 30%) of energy and protein and a 1.5- to 2-fold excess of calcium and phosphorus relative to National Research Council require- ments (17).

Three calves were injected i.m. with 100 /.tg of l a -OHD3 in 1 mI o f e thanol and renal biopsies done at 1 or 2, 4, and 8 d after the injections.

Cows were injected i.m. with 350 /ag of 10~- OHD3 in 1 ml o f e thanol immedia te ly after the initial renal biopsy.

RESULTS A N D DISCUSSION

The ins t rument shown in Figure I was suc- cessfully used to sample kidneys of calves and cows sequential ly. Samples be tween 100 and 200 mg were easily obtained, which was ade- quate for renal hydroxylase act ivi ty determina- tion. The sampling technique, if proper ly done, resulted in minimal hemorrhage and adhesions. Thus, several samples could be obta ined at one t ime or the sampling could be spread over several days or weeks. The cut t ing aspiration characteristics o f the ins t rument were con-

TABLE 1. Comparison of homogenization methods 1 on renal 25-hydroxyvitamin D 3 (OHD 3) lc~-hydroxylase activity. 2

Homogenizers l~-Hydroxylase

- - ( p m o l / m i n / g ) - - (n) SE

Potter-Elvehjem, 3 size A 52.8 3.4 34 Potter-Elvehjem, 3 size B 48.2 6.6 2 Tenbroeck 3 .5 .5 4 Polytron s 50.0 5.5 2

1 Multiple biopsy samples (90 to 150 mg each) were taken from the kidneys of a calf and the cortisol tissue was removed, cut into small pieces, and separated into four groups for homogenization.

~Assay tubes contained 400 ~tl of incubation medium, 10 /ag 25-OHD 3 in 1 ~tl ethanol and 100 ~ll of a 20% homogenate equivalent to 20 mg renal tissue. Radioligand protein binding assay used for quantification of enzyme product.

3 Tissue grinders (homogenizers) from Thomas Scientific, Philadelphia, PA. 4Two tO four aliquots of each of the four homogenates were assayed and the mean -+ SE calculated. s Homogenizer from Brinkman Instruments.

Journal of Dairy Science Vol. 69, No. 4, 1986

T A B L E 2. Rena l lc~-, 23-, and 2 4 - h y d r o x y t a s e ac t iv i t i es o f t w o p r e g n a n t ( f i rs t t r imes t e r ) Je r sey cows before and af ter t r e a t m e n t w i t h l c~ -hydroxyv i t amin D3.1 Z

Time N af ter t t y d r o x y l a s e ac t iv i ty 2,3

in jec t ion lc~- 23- 24- > ('3

(d) - - ( p m o l / m i n / g ) - - (n) - - ( p m o l / r n i n / g ) - - (n) - - ( p m o l / m i n / g ) - - (n)

,< 19.7 .2 84 5.1 1.1 6 52 1 g

3.1 1.1 8 22 .9 5.6 7 1371 54 7 3.9 .5 6 7 5 . 6 29.9 5 1390 638 6 O < 4.2 .1 8 121.9 3.0 6 796 435 6

e~

o

W

< o ox xo

Z o

x~

1 In j ec t ed i.m. wi th 350 ~ig l c~ -hydroxyv i t amin Da i m l n e d i a t e l y a f te r in i t ia l renal b iopsy , m Z

Tissue samples o b t a i n e d by b iopsy p r o c e d u r e ranged f rom 90 to 2 5 0 mg. H o m o g e n a t e w&s p repa red wi th a Po t t e r -E lveh j em t issue g r inde r (size A). I n c u b a t i o n > t i m e was 15 min at 37°C.

3 Assay tubes c o n t a i n e d 4 0 0 ~1 of i n c u b a t i o n m e d i u m , 10 ~g 2 5 - h y d r o x y v i t a m i n D 3 in 1 ~1 e thano l , and h o m o g e n a t e equ iva l en t to 25 mg renal t issue. Radio- l igand p ro t e in b ind ing assay used for q u a n t i t a t i o n .

V~ 4 Sum of n u m b e r o f rep l ica tes d o n e on b iopsy sample f rom each cow. Means ± SE were of two values. The values were the means of the rep l ica tes as a s ingle

value for each cow at each t ime.

xo xo tal

996 LITTLEDIKE ET AL.

TABLE 3. Application of biopsy procedure to calf renal 25-hydroxyvitamin D 3 (25-OHD s) lc~-hydroxylase. 1

Calf l~-Hydr°xylase2 no. 2d 4 d 8d

.X SE (n) X SE (n) X SE (n) 1 1.87 .03 63 3.01 .12 6 22.43 3.5 6 2 1.89 .10 6 3.06 .25 6 8.89 1.5 6

1 Two calves (136 kg Holstein steers) given one i.m. injection of l~-hydroxyvitamin D 3 (100 gg) 2 d before first biopsy sample time (A).

2 Assay tubes contain 400 #1 of incubation medium, 10 /ag 25-OHD 3 in 1 /al ethanol, and homogenate equi- valent to 20 mg renal tissue. Radioligand protein-binding assay used for quantitation of 1,25-(OH)2D 3.

3 Number of aliquots of homogenate assayed from each renal biopsy.

sidered essential for successful sampling o f renal tissue.

A compar ison of results obtained with dif- ferent homogeniza t ion techniques is presented in Table 1. Both of the motor-dr iven Potter- Elvehjem teflon-glass homogenizers and the Poly t ron homogenizer gave comparable values. In contrast , the Tenbroeck hand-operated glass- glass homogenizer gave much lower values, presumably because it was no t as effect ive in breaking the cell walls of bovine and renal co r t ex tissue.

l a - H y d r o x y v i t a m i n D3 (350 Og) was in- jected into each o f two pregnant cows short ly after the first biopsy sample was taken. Table 2 shows the changes in la- , 23-, and 24-hydroxy- lase activities over a period of 8 d post inject ion. T rea tmen t with l a -OHD3 caused a rapid reduc- t ion in l a -hydroxy lase activity. There was a s imultaneous rapid and large increase in 24- hydroxylase activity, which was sustained for 4 d and was decreasing at 8 d. The act ivi ty of 23-hydroxylase also increased after t rea tment , but the amoun t o f increase was less than the 24-hydroxylase activity increase and was still increasing at 8 d. Similar results were obtained f rom two calves, each given one inject ion of 100 /~g of I~-OHD3. Renal biopsy samples (100 to 200 mg) were obtained f rom each calf 2 at 4 and 8 d after t rea tment . The results (Table 3) show that inject ion of I~-OHD3 caused a marked reduc t ion in l~-hydroxylase act ivi ty wi thin 2 d, which persisted for at least 4 d.

The combina t ion of the sampling device wi th a microadapta t ion of the renal hydroxy- lase activity assay allowed demons t ra t ion of

severe acute and prolonged inhibi t ion of the most impor tan t renal enzymat ic regulat ion step in vi tamin D metabol i sm fol lowing l a -OHD3 administrat ion. This finding has impor tan t ramificat ions since l a -OHD3 is a promising c o m p o u n d for the prevent ion of par tur ient paresis in dairy cows (1, 6, 15, 19, 22). The prolonged inhibi t ion o f renal l a -hydroxy lase would make the cows ex t remely susceptible to hypoca lcemia once the effects of the l a -OHD3 were removed. The longitudinal changes of two o ther impor tan t renal enzymes fol lowing l a - OHD3 were also character ized using these tech- niques. This t echn ique promises to be valuable to characterize the regulat ion of vi tamin D metabol i sm under a variety of physiologic and disease states.

A C K N O W L E D G M E N T S

The authors grateful ly acknowledge the expert technical assistance of Beverly Mullen, Terrie Wierenga, Derrel Hoy, and Jesse Goff.

REFERENCES

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3 Dougherty, R. W. 1981. Experimental surgery in farm animals. Iowa State Univ. Press, Ames.

4 Engstrom, G. W., R. L. Horst, T. A. Reinhardt, and E. T. Littledike. 1984. 25-Hydroxyvitamin D lc~- and 24-hydroxylase activities in pig kidney

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ENZYME ACTIVITY IN BOVINE RENAL BIOPSIES 997

homogenates : effect of vitamin D deficiency. J. Nutr . 114:119.

5 Fraser, D. R., and E. Kodicek. 1970. Unique bio- synthesis by kidney of a biologically active vitamin D metaboli te. Nature (London) 228:764.

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8 Haddad, J. G., Jr., C. Min, M. Mendelsohn, E. Slatopolsky, and T. J. Hahn. 1977. Competi t ive protein-binding radioassay of 24,25-dihydroxy- vitamin D in sera f rom normal and anephric sub- jects. Arch. Biochem. Biophys. 182:390.

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10 Henry, H. L., R. J. Midgett, and A. W. Norman. 1974. Regulat ion o f 25-hydroxyvi tamin D3-1- hydroxylase in vivo. J. Biol. Chem. 249:7584.

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12 Horst, R. L., E. T. Littledike, J. L. Riley, and J. L. Napoli. 1981. Quant i ta t ion of vitamin D and its metaboli tes and their plasma concentrat ions in five species o f animals. Anal. Biochem. 116:189.

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14 Horst, R. L., R. M. Shepard, N. A. Jorgensen, and H. F. DeLuca. 1979. The determinat ion of the

vitamin D metaboli tes on a single plasma sample: changes during parturi t ion in dairy cows. Arch. Biochem. Biophys. 192:512.

15 McMurray, C. H., D. A. Rice, and P. S. McBride. 1980. Milk fever controls: comparison of 1~ and vitamin D 3 in conjunct ion with induced parturi- tion. Vet. Rec. 107:188.

16 Napoli, J. L., B. C. Pramanik, J. J. Partridge, M. R. Uskokovic, and R. L. Horst. 1982. 23S,25- Dihydroxyvi tamin D 3 as a circulating metabol i te of vitamin D 3. J. Biol. Chem. 257:9634,

17 National Research Council. 1978. Nutr ient require- ments o f dairy cattle. 5th rev. ed. Natl. Acad. Sci., Washington, DC.

18 Reinhardt , T. A., R. L. Horst, J. W. Orf, and B. W. Hollis. 1984. A microassay for 1,25-dihydroxy- vitamin D no t requiring high performance liquid chromatography: application to clinical studies. J. Clin. Endocrinol. Metab. 58:91.

19 Sachs, M., A. Bar, R. Cohen, Y. Mazur, E. Mayer, and S. Hurwitz. 1977. Use of l~-hydroxychole- calciferol in the prevention of bovine partur ient paresis. Am. J. Vet. Res. 38:2039.

20 Sumner-Smith , G., J. Archibald, J. DeBoer, J. S. Dingwall, and W. U. McDonnell. 1971. Compara- tive experimental surgery: the development of techniques for the partial removal o f parenchyma- tous organs, Dep. Clin. Stud., Ont. Vet. Coll, Univ. Guelph, Ont., Canada. Pages 1 0 3 0 - 1 0 3 1 in 19th Congr. Mundial de Medicina, Veterinaria y Zoo- technica. Vol. 3.

21 Tanaka, Y., J. K. Wichmann, H. K. Schnoes, and H. F. DeLuca. 1981. Isolation and identification of 23,25-dihydroxyvi tamin D3, an in vivo metaboli te o f vitamin D 3. Biochemistry 20:3875.

22 Wittwer, F., and E.J.H. Ford. 1980, The effect of lc~-hydroxycholecalciferol on the concentrat ions of calcium, magnes ium and inorganic phosphate in the plasma of parturient cows. J. Dairy Sci. 47: 177.

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