mode of action of pharmacological doses of cholecalciferol during

Mode of Action of Pharmacological Doses ofCholecalciferol during Parturient Hypocalcemiain Dairy Cows1

TIMOTHY A. REINHARDT2 AND HARRY R. CONRAD3

Ohio Agricultural Research and Development Center,Wooster, OH 44691

ABSTRACT A high-calcium diet (130 g calcium, 35 g phosphorus/day) and ahigh-phosphorus diet (45 g calcium, 70 g phosphorus/day) with and without aninjection of 10 x IO6lu of cholecalciferol were studied in pregnant cows. Cholecalciferol injections 7 days prepartum resulted in a 150% rise in circulating 1,25-dihydroxycholecalciferol 24 hours post-injection. By day 1 prepartum the 1,25-dihydroxycholecalciferol levels returned to pre-injection levels. The levels of1,25-dihydroxycholecalciferol in the plasma of control cows did not change untilparturition at which time the hormone increased from 100-200 pg/ml to 600 pg/ml.This was followed by a decrease to 200 pg/ml by day 2 postpartum. The injectionof cholecalciferol in cows eating high-phosphorus diets inhibited the rise ofplasma 1,25-dihydroxycholecalciferol seen in noninjected cows. Cholecalciferolinjections reduced the mean concentration of 1,25-dihydroxycholecalciferolduring the 7-day sampling period. Changes in plasma calcium, phosphorus,magnesium and hydroxyproline levels were within the range reported by otherinvestigators. The initial rise in plasma 1,25-dihydroxycholecalciferol followingcholecalciferol injections and the general reduction in circulating levels of thishormone during the prepartal and parturition periods suggest that these changesare primary factors in the preventive effects of cholecalciferol injections onparturient hypocalcemia. J. Nutr. 110: 1589-1596, 1980.INDEXING KEY WORDS calcium â€¢phosphorus â€¢cholecalciferol â€¢1,25-dihydroxycholecalciferol â€¢parturient paresis

Parturient hypocalcemia is a metabolic (4). However, recent studies have dem-disease associated with parturition and onstrated that the kidney is responsive tothe initiation of lactation. It is character- this hypocalcemia (5, 6). It has thereforeized by hypocalcemia, hypophosphatemia been proposed that parturient hypocal-and hypermagnesemia. This failure of the cemia results from an unexplained targetcalcium homeostatic mechanism has been organ resistance to one of the calciumassociated with many factors (1, 2). mobilizing hormones (5, 7, 11).

Recent theories as to the cause of par- There have been many different at-turient paresis are related to the metabolism tempts to prevent parturient hypocalcemiaof vitamin D. In normal metabolism vita- with widely differing results (1). Dietarymin D is converted to 25-hydroxychole- modification of calcium and/or phos-calciferol (25-OHD) and then to 1,25- phorus feeding or injecting pharmaco-(OH)2D in the kidney prior to exerting logical doses of vitamin D have been theits potent calcium mobilizing action on - -bone and intestine (3). A failure of Received for publication 14 January 1980.

i I . i i . Â« . ff^ â€¢ ' Approved for publication as Journal Article No. 1-80. Ohio Agri-thÃ¨ Kidneys tO blOSyntneSIZe SUlhCient cultural Research and Development Center, Wooster, OH 44691.1,25(OH)2D was suggested as a possible P^ÔÂ^Â scino A"imalDi!e"e Cen'er'u-s-aA"explanation for parturient hypocalcemia STOwhomreprintrequestsshouldbÂ«sent.

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1590 T. A. REINHARDT AND H. R. CONRAD

TABLE 1

Composition of grain mixtures

High-calcium grain concentrate High-phosphorus grain concentrate

CorngroundSoybeanmealLimestone2Dynamate3UreaSalt4Selenium

supplement533.424.434.42.71.91.51.7Corn

cobs, ground, No.4CorngroundSoybeanmealMonosodiumphosphate*Monoammoniumphosphate7DynamateSaltSelenium

supplement31.319.724.69.19.42.71,51.7

1Percentage on as fed basis. 2Analysis: CaCO3 95% min., Ã‡a38% min. 3Analysis: S 22% min.,K 18% min., Mg 11% min. Dynamate, International Minerals and Chemical Corp., Liberty, IL. *Analysis:NaCl 95% min., Cu 0.35% min., I 0.007% min., Co 0.007% min. The Morton Salt Co., Rittman, OH.52.55 g Na2SeO3 mixed in 17 Ib. H2O. â€¢Analysis: P 25% min., Na 19% min., Monsanto, St. Louis, MO.7Analysis: P 24% min., N 11% min. Monofos, International Minerals and Chemical Corp. Liberty, IL.

most successful (1,2,8). However, dietarymodification is not a practical alternativein some feeding regimes. Under theseconditions, where the disease is a problem, the cholecalciferol treatment appearsto be a useful and economical solutionfor prevention of parturient paresis. Because dietary calcium and phosphorusand vitamin D treatment had been shownto play such an important role in the etiology and incidence of parturient hypocal-cemia (1, 2, 8), we conducted an experiment designed to determine if a differenceexists between high dietary calcium orhigh dietary phosphorus-induced parturient hypocalcemia. The second objectivewas to elicit the role of pharmacologicaldoses of cholecalciferol in preventingparturient hypocalcemia in these twodietary regimes.

MATERIALS AND METHODS

The experimental design is a three-factor randomized block design, consistingof diet, vitamin D treatment and time ofsampling. Holstein-Friesian and Jerseycows (pregnant-nonlactating) were evenlydistributed among the treatments on thebasis of their previous history of parturient hypocalcemia, age and breed.

Two types of diets, high-calcium andhigh-phosphorus, were fed with and without an injection of 10 x IO6IU of cholecalciferol (CC, donated by DiamondShamrock, Cleveland, OH) in an ethanol

carrier, for a combination of four treatments (five cows per treatment). The dietsconsisted of 29% chopped orchard grasshay, 65% corn silage and 6.0% grain mixcontaining either a high-calcium or high-phosphorus level (table 1). These dietswere fed as complete mixed rations. Thecorn silage and grain were mixed togetherand then the chopped orchard grass wasadded and mixing was continued to complete the ration. The diets were fed adlibitum starting at least 20 days prepartum.Analysis of the diets showed that cowson the high-calcium diet consumed in thedaily ration 130 g calcium and 35 g phosphorus and that cows on the high-phosphorus diet consumed 45 g calcium and70 g phosphorus daily. Cows designatedfor CC injections received them 5-7 daysbefore the predicted calving date. Bloodsamples were taken just before the CCinjection and 24 hours later. Samples werealso obtained from all cows at 2 and 1days prepartum, the day of parturitionand day 1 and 2 postpartum. All bloodsamples, except those at parturition, wereobtained in the morning 4 hours post-feeding. Blood samples (80 ml) were collected in ice-cold heparinized tubes andcentrifuged. The plasma was stored at-20Â°until analyzed.

Plasma calcium and magnesium weredetermined using atomic absorption spec-troscopy (Model 82-270, Jarrel Ash Co.,Waltham, MA). Plasma inorganic phos-

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CHOLECALCIFEROL AND PARTURIENT PARESIS 1591

TABLE 2

Plasma calcium levels from cows fed high-calcium and high-phosphorus diets withand without injections of cholecalciferol

Plasma calcium(mg/dl)Days

beforeTreatmentHigh

calciumHigh calcium + CCHigh phosphorusHigh phosphorus + CC-(5-7)9.2'8.1'-(4-6)10.0a

10.0a7.8"

9.6a-210.6

9.27.99.7-18.1

9.98.38.6Parturition08.4a

7.5a6.7"8.5aDays16.2

7.57.27.6after28.3

8.07.77.97-day

means28.8

Â± .03C8.8 Â±0.3C7.5 Â± .03d8.6 Â±0.3C

1Cows were injected between the 5th and 7th day before parturition. 24-hour post-injection sampleswere taken between days 4 and 6. 2Least square means and SEMfor the 7-day sampling period. a'b Meansin columns with different superscripts differ (P < 0.10). c'd Means in columns with different superscriptsdiffer (P < 0.01).

phorus was determined as describedpreviously (9). Plasma hydroxyprolinewas determined by the method of Dabevand Struck (10).

Plasma l,25dihydroxycholecalciferol(donated by Hoffmann-LaRoche, Nutley,NJ) was determined by the method ofHorst et al. (6) with one modification. Thechick intestinal mucosa cytosol and dex-tran-coated charcoal were prepared usinga buffer containing 50 mMpotassium phosphate, 300 mM KC1, 2 mM dithiothreitoland 10% glycerol (v/v), pH 7.5. Cytosolprepared in this buffer and stored undernitrogen at â€”76Â°was stable for at least6 months.

Data was analyzed by analysis ofvariance.

RESULTS

Paresis did not occur in any of the cowsused in this study; therefore none of thecows were treated for parturient hypo-calcemia during the experimental period.However, normal hypocalcemia occurredin all cows within 24 hours after parturition. No differences were observed between plasma calcium levels of cows onthe high-calcium diet with and withoutan injection of CC (table 2). On high-phosphorus diets the injection of cholecalciferol resulted in higher plasma calciumlevels on days-(4-6) and day 0(P < 0.10)when compared to noninjected cows onthe high-phosphorus diet. However, theseelevated plasma calcium levels were not

TABLE3Plasma phosphorus levels from cows fed high-calcium and high-phosphorus diets with

and without injections of cholecalciferol

Plasma phosphorus (mg/dl)

Days before Parturition Days after

Treatment -(5-7) -(4-6) -2 -1 0 1 7-day means2

HighcalciumHighcalcium +CCHigh

phosphorusHighphosphorus + CC5.6'â€”6.6'4.85.36.76.74.45.85.66.24.25.16.06.53.33.84.85.73.13.94.55.43.34.95.05.94.0

Â±0.2a4.9Â±0.2b5.5Â±0.2C6.0Â±0.2"

1Cows were injected between the 5th and 7th day before parturition. 24-hour post-injection sampleswere taken between days 4 and 6. 2Least square means and SEMfor the 7-day sampling period.a~dMeans in columns with different superscripts differ (P < 0.01).

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TABLE 4

Plasma magnesium levels from cows fed high-calcium and high-phosphorus diets withand without injections of cholecalciferol

Plasma magnesium(mg/dl)Days

beforeHigh

HighHighHighTreatmentcalcium

calcium + CCphosphorusphosphorus + CC-(5-7)1.7'1.5'-(4-6)1.8

1.71.71.4-22.0

1.51.51.5-11.5

1.61.61.5Parturition01.8

1.81.82.0Days12.4

2.01.61.8after22

111.1.7.6.97-day2.0

1.71.61.6means*Â±

0.1"Â±0.1"Â±O.lbÂ±0.1"

1Cows were injected between the 5th and 7th day before parturition. 24-hour post-injection sampleswere taken between days 4 and 6. 2Least square means and SEMfor the 7-day sampling period.â€¢â€¢"Means with different superscripts differ (P < 0.10).

different from the plasma calcium valuesfound in high-calcium treatments whenthe same days were compared. Whencomparing the 7-day mean plasma calcium levels, the high-phosphorus diet (inthe absence of CC injection) induced agreater overall degree of hypocalcemia(P < 0.01) than seen in the other threetreatments (table 2).

Plasma phosphorus was generally elevated in cows receiving the high-phosphorus diets when comparing 7-daymeans (P < 0.01) [table 3]. Plasma phosphorus was also higher in cows receivingCC than those that did not when comparing 7-day means (P < 0.01).

Plasma magnesium levels were not different between treatment groups at anysingle time (table 4). However, the mean

plasma magnesium levels for the 7-daysampling period were elevated on thehigh-calcium treatment (P < 0.10) compared to magnesium levels on the otherthree treatments (table 4).

Plasma hydroxyproline levels were unaffected by diet and/or cholecalciferolat all times except day 2 postpartum. By2 days postpartum, plasma hydroxyprolinelevels were higher in cows receiving thehigh-calcium diet versus those that received this diet plus cholecalciferol (P< 0.05). The reverse was true on the high-phosphorus diet. On day 2 postpartumcows that were injected with cholecalciferol has plasma hydroxyproline levelshigher than those that were not injected(P < 0.05) [table 5].

The injection of 10 x 10Â«IU of CC re-

TABLE5Plasma hydroxyproline levels from cows fed high-calcium and high-phosphorus with

and without injections of cholecalciferol

Plasma hydroxyproline (fig/ml)

Days before Parturition Days after

Treatment -(5-7) -(4-6) -2 -1 0 1 7-day means2

HighcalciumHighcalcium +CCHigh

phosphorusHighphosphorus + CCâ€”1.461â€”1.48'1.261.511.571.541.291.401.521.801.251.611.631.431.291.451.411.751.561.491.701.892.31Â»1.70Â»1.60"2.16a1.44

Â±0.091.47Â±0.071.49Â±0.091.67Â±0.09

1Cows were injected between the 5th and 7th day before parturition. 24-hour post-injection sampleswere taken between days 4 and 6. 2 Least square means and SEM for the 7-day sampling period."â€¢bMeans with different superscripts differ (P < 0.05).

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High Co Diet

DAYS

Fig. 1 High Ca diet. The concentration of 1,25-dihydroxycholecalciferol in the plasma of dairy cowsfed a high-calcium diet with (â€¢)and without (â€¢)an injection of 10 x IO6units of cholesterol. Day 0represents parturition, while negative numbersrepresent days prior to and positive numbers represent days after parturition. The arrow indicates dayof cholecalciferol injection. Means are given Â±SEM.

suited in an increase of approximately150% in circulating l,25dihydroxychole-calciferol levels 24 hours post-injectionin both the high-calcium + CC treatment(P < 0.05) and the high-phosphorus + CCtreatment (P < 0.05) [figs. 1, 2]. By dayâ€”1. l,25dihydroxycholecalciferol levelsin injected cows returned from day -(4-6)elevated levels to pre-injection levels(P < 0.05). The levels of l,25dihydroxy-cholecalciferol in the plasma of cows notinjected with CC did not change untilparturition at which time the plasma levelsof l,25dihydroxycholecalciferol increasedapproximately 300% to 600 pg/ml (P< 0.01). This was followed by a markeddecrease (P < 0.01) by day 2 postpartumto 200 pg/ml of 1,25-dihydroxycholecalcif-erol (figs. 1, 2).

Cows on the high-phosphorus + CCtreatment also had significantly elevatedlevels of 1,25-dihydroxycholecalciferol atparturition when compared to day -1 (P< 0.05) but this plasma level of 1,25-

dihydroxycholecalciferol was significantlylower than the parturition levels of 1,25-hydroxycholecalciferol found in cows notinjected with CC (P < 0.01). On the high-calcium + CC treatment the trend wasthe same but nonsignificant.

The mean plasma levels of 1,25-dihydroxycholecalciferol for the entire timeperiod were compared between animalsreceiving CC injection (180 Â±19 pg/ml)and those that were not injected (237Â±22 pg/ml). The injection of CC wasassociated with a marked reduction in themean amount of 1,25-dihydroxycholecalciferol circulating during the entire period (P < 0.07).

DISCUSSION

The results demonstrate that injectionsof pharmacological doses of cholecalciferol(CC) are able to override the tightly controlled kidney 25-hydroxycholecalciferol1-hydroxylase enzyme. The rate of 1,25-

800 â€¢

TOO -

500-

300-

Om

200-

wo-

High PO4 DieÂ»

A + chohcokibrol

â€¢-chobcokibrol

-(5-7) "-(Â«-*" -2 I Â» 1 Ã•

t DAYS

Fig. 2 High PO4diet. The concentration of 1,25-dihydroxycholecalciferol in the plasma of dairy cowsfed a high-phosphorus diet with (A) and without (â€¢)an injection of 10 x IO6units of cholecalciferol. Day0 represents parturition, while negative numbersrepresent days prior to and positive numbers represent days after parturition. The arrow indicates dayof cholecalciferol injection. Means are given Â±SEM.

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dihydroxycholecalciferol synthesis is narrowly controlled apparently by the interaction of parathyroid hormone, calcium, phosphorus and 1,25-dihydroxy-cholecalciferol itself (11, 12). However,this 1,25-dihydroxycholecalciferol bio-synthetic control mechanism appears tohave been temporarily overwhelmed bypharmacological doses of CC. By 24 hourspost-injection of 10 x IO6 IU of CC, thecirculating levels of 1,25-dihydroxycholecalciferol increased 150-200% (figs. 1, 2);this increased biosynthesis of 1,25-dihydroxycholecalciferol in response to pharmacological CC injection does not appear to be affected by diet. Thus theresults are amenable with the hypothesisthat pharmacological injections of CCmay prevent parturient hypocalcemia bystimulating increased biosynthesis of1,25-dihydroxycholecalciferol. Conrad etal. (13) showed that pharmacologicaldoses of CC result in increased intestinal absorption and soft tissue deposition of calcium in CC treated cattile.Also Rowland et al. (14) demonstratedthat pharmacological does of CC increased osteoclastic bone rÃ©sorption indairy cows. The data presented here support and extend these earlier observationsby presenting evidence that CC injectionsincrease circulating 1,25-dihydroxycholecalciferol in the prepartum period. Thus,the increased intestinal absorption, bonerÃ©sorption and soft tissue deposition ofcalcium seen in CC treated dairy cattleprobably results from increased levels of1,25-dihydroxycholecalciferol.

The increased circulating levels of 1,25-dihydroxycholecalciferol (250 pg/ml) associated with CC injection return to pre-injection levels (80 pg/ml) by day 1 prepartum (figs. 1,2). These data suggest thatthe loss of control over 1,25-dihydroxycholecalciferol biosynthesis is temporary.Presumably the increased 1,25-dihydroxycholecalciferol levels are able to feed backon the parathyroid gland and kidney toreduce biosynthesis as previously shown(12). Consistent with previous studies (5,6) we found that parturition and initiationof lactation resulted in significant increases in circulating 1,25-dihydroxycholecalciferol (figs. 1 and 2). The injec

tion of CC suppressed the magnitude of1,25-dihydroxycholecalciferol increasewhen cows are fed a high-phosphorus diet(P < .01). It is reasonable to think thatrenal 1-hydroxylase enzyme may remainsuppressed through parturition as a resultof the increased 1,25-dihydroxycholecalciferol prepartum. Also the administrationof pharmacological levels of CC maydiminish the amount of parathyroid hormone (PTH) secreted by animals fed high-phosphorus diets (15) thus removing oneof the stimuli for the biosynthesis of 1,25-dihydroxycholecalciferol. In addition, theraised plasma phosphorus levels associated with this treatment may also contribute to this inhibition (3). On high-calcium+ CC treatment, a suppressed increase incirculating 1,25-dihydroxycholecalciferollevels at parturition was also observed.However, the 1,25-dihydroxycholecalciferol levels on this treatment were notsignificantly lower than those observedfor the high-calcium diet suggesting a dietby CC interaction due to lower plasmaphosphorus levels.

The injection of CC promotes a rise in1,25-dihydroxycholecalciferol levels prepartum (P < 0.05); however when theplasma 1,25-dihydroxycholecalciferol levels for the entire time period are averaged,the injection of CC is associated with amarked reduction in the amount of 1,25-dihydroxycholecalciferol the cows are exposed to during this critical prepartumperiod. Noninjected cows have a meanplasma 1,25-dihydroxycholecalciferol levelof 237 Â±22 pg/ml for the 7-day time period versus 180 Â±19 pg/ml for animalsreceiving the CC injection (P < 0.07).Horst et al. (5, 6) have shown that cowsdevelop parturient hypocalcemia havesignificant higher plasma 1,25-dihydroxycholecalciferol levels at parturition thancows which do not contract the disease.They concluded that the biosynthesis of1,25-dihydroxycholecalciferol, in responseto parturient hypocalcemia, is normal inaffected cows; therefore, they suggestedthat a target organ insensitivity to 1,25-dihydroxycholecalciferol may be responsible for parturient hypocalcemia in affected animals.

In formulating an expanded hypothesis

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of etiology we considered the fact that parturient hypocalcemia can be effectivelyprevented with pharmacological doses ofCC (2,8). The data presented here demonstrate that a specific effect of CC injectionis a reduction in the circulating 1,25-dihy-droxycholecalciferol levels at parturition.Unaffected animals have lower 1,25-dihy-droxycholecalciferol levels at parturitionand the prevention of parturient hypocalcemia by CC injection also results inlower 1,25-dihydroxycholecalciferol levels at parturition. This suggested thatoverproduction of 1,25-dihydroxycholecalciferol levels at parturition may in someway promote the development of parturient hypocalcemia. Although 1,25-dihydroxycholecalciferol is known to promotea net influx of calcium into the body, itsoverproduction in the parturient dairycow may promote a net efflux of calciumfrom the body large enough to induce asevere hypocalcemia. This hypothesis issupported by recent work in which wedemonstrated that the bovine mammarygland contains a specific high affinitybinding protein "receptor" for 1,25-dihy

droxycholecalciferol (16).This observationsuggests that 1,25-dihydroxycholecalciferol may function in the mammary glandin a similar manner to its function in theintestine (12), and thus it may promotethe normal transport of calcium into thisgland in support of lactation. Thereforeoverproduction of 1,25-dihydroxycholecalciferol may promote transport of calcium to this gland in excess ofthat requiredfor lactation.

Bone rÃ©sorption,using plasma hydroxy-proline as an indicator, tended to be depressed during the prepartal period on thehigh-calcium diet (table 5). However,plasma hydroxyproline levels were significantly higher at 2 days postpartum on thehigh-calcium diet when compared to thehigh-calcium + CC treatment. These results suggest that the demand for and response of bone calcium rÃ©sorptionin cowson high-calcium diets is important inmaintaining calcium homeostasis aroundparturition (7, 13). The prepartum effectsof CC appear to decrease these demandsfor bone calcium on high-calcium diets.

Cows on high-phosphorus diets did not

show a significant increase in bone rÃ©sorption (table 5) by 2 days prepartum. In fact,it was the high-phosphorus + CC dietwhich showed a significant increase inplasma hydroxyproline. Thus it is thoughtthat on high-phosphorus diets, the modeof action of CC injections may be differentthan it is on high-calcium diets. The promotion of bone rÃ©sorptionmight be partof this difference.

The mean plasma values for calcium,phosphorus and magnesium for all timeswere within the ranges reported by others(6, 7, 17). The injection of pharmacological levels of CC did not promote a rise inplasma calcium in cows receiving a high-calcium diet. This is consistent with previous observations (9).The reason that CCinjections did promote a rise in plasmacalcium levels in cows receiving the high-phosphorus diet is unknown. It is noteworthy that these animals were hypocalcÃ©miebefore the injection and theplasma calcium only returned to normallevels post-injection.

The important point to be made withrespect to pharmacological doses of CCin the prevention of parturient hypocalcemia, is that CC injections result ina dramatic rise in 1,25-dihydroxycholecalciferol levels during the prepartum period. It is suggested that the increasedplasma 1,25-dihydroxycholecalciferol incows receiving CC results in a large influxof readily available calcium to supportlactation demands, at a time when thetarget tissues of 1,25-dihydroxycholecalciferol are highly responsive. Followingthis spike in 1,25-dihydroxycholecalciferol levels, CC injections are associatedwith a depressed increase in 1,25-dihydroxycholecalciferol levels at parturition.Thus CC injections may prevent overproduction of 1,25-dihydroxycholecalciferolat parturition and a hypothetical increasedinflux of calcium to the mammary glandgreater than needed for lactation.

These observations do not rule out thehypothesis that parturient hypocalcemiaresults from target organ insensitivity tocalcium mobilizing hormones at parturition (5-7), but support the contention thatparturient hypocalcemia in dairy cattle isa complicated multietiological disease.

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LITERATURE CITED

1. Jorgensen, N. A. (1974) Combating milkfever. J. Dairy Sci. 57, 933-944.

2. Hibbs, J. W. (1950) Milk fever (parturientparesis) in dairy cowsâ€”a review. J. Dairy Sci.33, 758.

3. DeLuca, H. F. (1974) Vitamin D: the vitaminand the hormone. Fed. Proc. 33, 2211.

4. FrÃ¤ser, D. R. & Kodicek, E. (1970) Uniquebiosynthesis by kidney of a biologically activevitamin D metabolite. Nature 228, 764-766.

5. Horst, R. L., Eisman, J. A., Jorgensen, N. A. &DeLuca, H. F. (1977) Adequate response ofplasma 1,25 dihydroxy-vitamin D to parturitionin paretic (milk fever) dairy cows. Science 196,196-197.

6. Horst, R. L., Shepard, R. M., Jorgensen, N. A. &DeLuca, H. F. (1979) The determination ofthe vitamin D metabolites on a single plasmasample: changes during parturition in dairycows. Arch. Biochem. Biophys. 192, 512-523.

7. Yarrington, J. T., Capen, C. C., Black, H. E. &Re, R. (1977) Effects of a low calciumprepartal diet on calcium homeostatic mechanisms in the cow: morphologic and biochemicalstudies. J. Nutr. 107, 2244-2256.

8. Julien, W. E., Conrad, H. R., Hibbs, J. W. &Christ, W. L. (1977) Milk fever in dairy cows.VIII. Effect of injected vitamin D3 and calciumand phosphorus intake on incidence. J. DairySci. 60, 431-436.

9. Hollis, B. W., Conrad, H. R. & Hibbs,J. W. (1977) Changes in plasma 25-hydroxy-cholecalciferol and selected blood parametersafter injection of massive doses of cholecalciferol

or 25-hydroxycholecalciferol in non-lactatingdairy cows. J. Nutr. 107, 606-612.

10. Dabev, D. & Struck, H. (1971) Microliterdetermination of free hydroxyproline in bloodserum. Buy4" m. Med. 5, 17-21.

11. DeLuca, H. F. (1977) Vitamin D metabolism.Clin. Endocrino!. 7, 1S-17S.

12. Norman, A. W. & Henry, H. L. (1979) VitaminD to 1,25-dihydroxycholecalciferol: evolution ofa steroid hormone. Trends Biochem. Res. 4,14-18.

13. Conrad, H.R.& Hansard, S.L. (1957) Effectsof massive doses of vitamin D on the physiological behavior of calcium in cattle. J. Appi.Physiol. 10, 98-102.

14. Rowland, G. N., Capen, C. C., Young, D. M. &Black, H. E. (1972) Microradiographicevaluation of bone from cows with experimentalhypervitaminosis D, diet-induced hypocalce-mia, and naturally occurring parturient paresis.Cale. Tiss. Res. 9, 179-193.

15. Capen, C. C., Cole, C. R., Hibbs, J. W. & Wag-ner, A. R. (1966) Bioassay and quantitativemorphologic analysis of the parathyroid glands,and serum and urine changes of cows fed highlevels of vitamin D. Am. J. Vet. Res. 27, 1177-1186.

16. Reinhardt, T. A. & Conrad, H. R. (1980) Specific binding protein for 1,25-dihydroxy vitaminD-3 in bovine mammary gland. Arch. Biophys.Biochem. (In press).

17. Beitz, D. C., Burkhart, D. J. & Jacobson, N. L.(1974) Effects of calcium to phosphorus ratioin the diet of dairy cows on the incidence ofparturient paresis. J. Dairy Sci. 57, 49-55.

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mode of action of pharmacological doses of cholecalciferol during

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