The role of the liver in the metabolism of vitamin D G. Ponchon, H. F. Deluca J Clin Invest. 1969;48(7):1273-1279. https://doi.org/10.1172/JCI106093. The metabolism of vitamin D 3 has been studied after intravenous injection of 10 IU of [1,2- 3 H]-vitamin D 3 to vitamin D- deficient rats. The disappearance of the radioactivity from the plasma follows a very peculiar pattern characterized by an early rapid disappearance followed by a rebound of radioactivity before assuming still a third rather slow disappearance rate. The “rebound” phenomenon is concomitant with a rapid release of the radioactivity from the liver and is accounted for by the appearance of 25-hydroxycholecalciferol and other metabolites in the blood. It is postulated that the liver is the major site of transformation of vitamin D 3 into 25-hydroxycholecalciferol. Research Article Find the latest version: https://jci.me/106093/pdf
Transcript
The role of the liver in the metabolism of vitamin D
The metabolism of vitamin D3 has been studied after intravenous injection of 10 IU of [1,2-3H]-vitamin D3 to vitamin D-deficient rats. The disappearance of the radioactivity from the plasma follows a very peculiar pattern characterized by anearly rapid disappearance followed by a rebound of radioactivity before assuming still a third rather slow disappearancerate. The “rebound” phenomenon is concomitant with a rapid release of the radioactivity from the liver and is accountedfor by the appearance of 25-hydroxycholecalciferol and other metabolites in the blood. It is postulated that the liver is themajor site of transformation of vitamin D3 into 25-hydroxycholecalciferol.
From the Department of Biochemistry, University of Wisconsin,Madison, Wisconsin 53706
A B S T R A C T The metabolism of vitamin D3 has beenstudied after intravenous injection of 10 IU of [1,2-3H]-vitamin Da to vitamin D-deficient rats. The disappear-ance of the radioactivity from the plasma follows a verypeculiar pattern characterized by an early rapid dis-appearance followed by a rebound of radioactivity beforeassuming still a third rather slow disappearance rate.The "rebound" phenomenon is concomitant with a rapidrelease of the radioactivity from the liver and is ac-counted for by the appearance of 25-hydroxycholecal-ciferol and other metabolites in the blood. It is postu-lated that the liver is the major site of transformationof vitamin D3 into 25-hydroxycholecalciferol.
INTRODUCTIONThere has been much recent investigation into earlybiochemical events after the administration of vitaminD to man and animals (1). In experiments in man, itwas noted that after the intravenous administration oflabeled vitamin Ds the plasma radioactivity falls rapidlyat first and then "rebounds" before assuming a regulardecay curve (2, 3). Previously this had not been notedin animals. Until now, the origin and significance ofthis "rebound" remained a matter of speculation. The"rebound" phenomenon has now been demonstrated inrats and is the result of radioactivity leaving predomi-nantly the liver to enter the plasma. This radioactivityis composed primarily of 25-hydroxycholecalciferol, therecently identified metabolite believed to be the meta-bolically active form of vitamin D3 (4, 5). The resultsfurther suggest that the liver is the main organ involvedin the conversion of vitamin D3 to 25-hydroxychole-calciferol.
The aim of the present study is to investigate the roleof the liver in the metabolism of vitamin D3 in the rat,
This work was published with the approval of the direc-tor of the Wisconsin Agricultural Experiment Station.
Received for publication 17 January 1969 and in revisedform 19 March 1969.
particularly in respect to the plasma "rebound" and theformation of 25-hydroxycholecalciferol.
METHODS
Tritiated vitamin D. [1,2-3H]-vitamin D3 with a specificactivity of 24,453 dpm/IU or 0.44 uc/,ug was prepared in thislaboratory as reported earlier (6). Radiochemical purity,achieved through column chromatography on silicic acid,was assessed by chromatographic methods and ultravioletspectrophotometry (6). The labeled vitamin Ds was dis-solved either in absolute ethanol or in plasma from vita-min-D deficient rats at a concentration of 200 IU/ml.
Animals. Holtzman, male weanling rats housed in indi-vidual hanging wire cages were fed for 2-3 months thevitamin D-free diet No. 11 (0.47% Ca, 0.30%o P) describedby Guroff, DeLuca, and Steenbock (7). At the time ofexperiments, the rats weighed an average of 230 g and werevitamin D deficient as revealed by a lack of growth andlow serum calcium values.
Experimental procedure. In the first series of experi-ments, the disappearance of the plasma radioactivity wasinvestigated after intravenous injection of labeled vitaminDs (series I). Doses of 10 IU [1,2-3H]-vitamin D3 in 0.05ml plasma were injected into the jugular vein of 13 ratsunder light ether anesthesia. The animals were placed inindividual restraining cages (8) during the periods of bloodsampling or in their original cages with food (diet No. 11)and water ad lib. 0.4 ml of heparinized blood samples fromthe tail vein was collected at specific time intervals afterdosage. Plasma was separated by centrifugation for radio-activity measurements. Hematocrits were performed onseveral occasions using heparinized microhematocrit tubes(Yankee, Clay-Adams, Inc., New York). The proportionof the injected labeled vitamin D3 present in the plasma ateach time period was obtained by multiplying the radio-activity of the 0.1 ml plasma sample by the estimated plasmavolume "P1 V." The total initial blood volume "initial BV"was assumed to represent 6% of the body weight, and theplasma "initial P1 V" as well as the red blood cell volumes"initial RBCV" were deduced on the basis of the hematocritvalue, usually 50 at the beginning of the experiments. Bloodand plasma volume estimates were corrected for the bloodlosses occurring by multiple blood samplings during theexperiments. The following calculations were used for thesecorrections: (a) Loss of red blood cells at the time of thefirst sampling = blood volume subtracted X Hct/100 = milli-liters of RBC lost. (b) This volume of lost RBCwas sub-
The Journal of Clinical Investigation Volume 48 1969 1273
tracted from the "initial RBCV" to give the "remainingRBCV." (c) At the beginning of the next blood sampling,knowing the new Hct value, the "remaining P1 V" was givenby "remaining RBCV" X (100- Hct)/Hct. The same calcu-lation was applied to each successive blood sample.
Bile duct cannulations were performed in two rats underether-pentobarbital (Nembutal, Abbott Laboratories, NorthChicago, Ill.) anesthesia. The bile was collected through apolyethylene catheter PE 10 (Intramedic, Clay-Adams, Inc.,New York). Blood samples were collected as usual from thetail vein.
In another group of rats (series II), 10 IU of [1,2-2H]-vitamin Ds in 0.05 ml 100%o ethanol was injected intra-venously via the jugular vein under light ether anesthesia.Animals were killed by exsanguination through cardiacpuncture under ether anesthesia at specific time intervalsafter dosage. One rat was sacrificed at each time. Theplasma was immediately separated by centrifugation from theheparinized blood. The livers and kidneys were removed,rinsed in 0.9%, aqueous NaCl, blotted on adsorbent paper,and weighed. The small intestines were dissected from themesentery and cut into segments which were rinsed in 0.9%aqueous NaCl after all intestinal contents were carefullyremoved. They were blotted on adsorbent paper and weighed.The femurs were dissected, weighed, and split longitudinallyto remove the bone marrow. Samples of striated musclessurrounding the femurs were freed of fat and ligaments,rinsed in 0.9% aqueous NaCl, blotted on adsorbent paper,and weighed. All plasma and tissue samples were kept frozenat -100C until use.
Total radioactivities in plasma and tissues of each ratwere determined in triplicate (femurs in duplicate). Totalplasma volume, skeleton, and striated muscle weights wereestimated to represent respectively 3, 15, and 30%o of bodyweight.
In the last series of experiments (series III) the distri-bution of radioactivity among vitamin D and its metabolitesin plasma and tissue was studied as a function of time afterinjection of vitamin D3-'H. Each of 30 rats was injectedintravenously with 10 IU of [1,2-3H]-vitamin Ds in 0.05 mlof plasma from vitamin D-deficient rats. Groups of four ratseach were bled and sacrificed as in the previous experimentsat various times after vitamin Ds-sH dosage (six rats werekilled at 4 hr). Plasma and livers were collected, and theirtotal radioactivities were measured in triplicate as in seriesI. In addition, pooled plasma and liver samples from eachgroup of rats were submitted to lipid extraction and chroma-tography to determine the relative and absolute amounts ofunchanged vitamin D3 and its metabolites.
Extraction and chromatography. Lipid extraction inchloroform-methanol was carried out according to the pro-cedure of Bligh and Dyer (9) slightly modified in thislaboratory (10). The radioactivities of the "chloroform-soluble," "aqueous-soluble," and "nonextractable" phaseswere measured in triplicate. The chloroform extracts weredried under N2 and redissolved in 5-10 ml of redistilledSkelly B (petroleum ether, bp 670C). They were chromato-graphed on silicic acid columns (reference 11, footnote 1)according to the following solvent system. (a) An expo-nential gradient 0-50%o diethyl ether in redistilled Skelly Bwas first obtained by superimposing a holding chamber con-taining 250 ml of 75%o diethyl ether in petroleum ether ona 250 ml Erlenmeyer mixing chamber containing 230 ml of100% Skelly B. (b) An exponential gradient from 50%o di-
1 Ponchon, G., and H. F. DeLuca. Data in preparation.
ethyl ether in Skelly B to 76% ether, 21%o Skelly B, and 3%methanol was next used by adding 200 ml of 5% methanol indiethyl ether to the empty holding chamber. (c) An expo-nential gradient from 76% ether, 21% Skelly B, and 3%methanol to 40% ether, 6% Skelly B, and 54% methanolwas obtained by adding 150 ml of 100% methanol to theempty holding chamber. The effluents were collected in 10-mlfractions directly in counting vials for radioactivity measure-ments.
Radioactivity measurements. 0.1 ml of each plasma sam-
ple was pipetted in triplicate into counting vials and driedunder an infrared lamp. To the vials were added 1 drop ofdistilled water and 0.5 ml of "NCS" solubilizer (Nuclear-Chicago Corporation, Des Plaines, Ill) (12). The stopperedvials were then incubated at 40'C for 12-24 hr to achievecomplete dissolution of the organic matter in the NCS.Toluene counting solution (100 mg of dimethyl 1,4-bis[2-(5-phenyloxazolyl)] benzene (POPOP) and 2.0 g of 2,5-di-phenyloxazole (PPO) per liter of toluene analytical reagentwas then added to each counting vial.
Total radioactivities in the various tissues were deter-mined in triplicate by the combustion technique of Kelly,Peets, Gordon, and Buyske (13) with a Thomas Ogg SafetyIgniter (Arthur H. Thomas Co., Philadelphia, Pa.), as de-scribed earlier (6). The resulting tritiated water was dis-solved and counted in combustion counting solution (6 gof PPO, 5 mg of dimethyl POPOP, 200 ml of absoluteethanol, and toluene to 1 liter).
Aliquots of the chloroform-soluble phases were monitoredfor radioactivity in toluene counting solution. Aliquots ofeach aqueous-soluble phase were dried in counting vials,redissolved in distilled water and NCS solubilizer, and mixedwith toluene counting solution. The nonextractable radio-activity was obtained by dissolving the residue from thelipid extractions in the NCS solubilizer and counting intoluene counting solution. The chromatographic fractionswere dried directly in the counting vials and counted intoluene counting solution. The "dose standards" were mea-
sured in triplicate in toluene counting solution after disso-lution in NCS solubilizer (D3-3H in 0.05 ml of plasma).
All radioactivity measurements were performed with a
Packard Tri-Carb 3003 liquid scintillation counter equippedwith an automatic external standardization system 3951 (14).Counting efficiencies were in the order of 30-35% with thetoluene counting solution and in the order of 15-25% withthe combustion counting solution.
RESULTS
Plasma radioactivity after i.v. injection of 10 IU of[1,2-3H]-vitamin D3 to vitamin D-deficient rats. (seriesI). Figs. 1 and 2 show the characteristic pattern ofplasma radioactivity after an intravenous dose of triti-ated vitamin D3. It may be described as three successivephases.
The initial phase does not last longer than 30 min andconsists of a very rapid disappearance of the plasmaradioactivity. 51.7 ±8.0% (reference 4, footnote 2) ofthe dose is present in the plasma compartment 5 minafter injection and only 34.9 ±4.1% (4) after 30 min.
The second phase occurs between 30 min and 8 hr
2 Mean + SEM (number of rats).
1274 G. Ponchon and H. F. DeLuca
i MEAN+SE
TIME AFTER INJECTION (DAYS)
FIGURE 1 Plasma radioactivity after i.v. injection of 10IU of [1,2-8H]-vitamin D3 to vitamin D-deficient rats(series I).
and is characterized by a rebound of the plasma radio-activity from a low level of 29.8 ±2.8% (2) at 90 minto a high of 38.4 ±0.6% (2) at 150 min. The shape ofthis peak is quite irregular. There are large individualvariations in its intensity and time of occurrence.
The third phase is represented by a very slow declineof the plasma radioactivity from 9.4 +0.5% (13) at 2days to 0.5 ±0.2% (2) after 3 wk.
The two rats with a bile duct cannulation showed alsoa rebound of plasma radioactivity between 30 min and 6hr after dose, although the radioactivity level waslower (Table I).
Plasma and tissue radioactivity after i.v. injection of10 IU of [1,2-8H]-vitamin D3 to vitamin D-deficientrats. (series II). The fraction of the radioactive dosepresent in plasma and tissues at various times afterinjection is shown in Fig. 3. The plasma and liverradioactivity curves exhibited a striking "mirror image."
56
48
40
} 32
L 24
16
8 i MEAN+SE
I I I I I120 1 3 6 8
TIME AFTER INJECTION (HOURS)
TABLE IPlasma Radioactivity after i.v. Injection of 10 I U of
[1, 2-3H]- Vitamin Da to Two Vitamin D-DeficientRats with Bile Duct Cannulation
Values as per cent of dose in body plasma compartment.
While the liver released most of its radioactive content(from 43.9% at 90 min down to 1.4% at 120 min)between 90 min and 150 min after an i.v. dose, theplasma radioactivity increased during the same period(from 12.1% at 100 min up to 24.6% at 180 min). Atthe same time the skeleton and muscles were losing someradioactivity, but in much smaller amounts.
From 150 min on, the liver and to a much smallerextent other tissues reaccumulated radioactivity. Simul-taneously, the plasma radioactivity fell to lower levels.
The lower levels of plasma radioactivity observed inthis series of experiments as compared to the valuesgiven above are undoubtedly due to the ethanol used asvehicle for the injection of the vitamin DS-8H instead ofplasma. Ethanol was used in this series of experimentsbefore its artifactual effect was established. The im-portance of the injection solvent in the study of themetabolism of vitamin D will be reported in anotherpublication (15). The main effect of ethanol is to accel-
100
FIGURE 2 Plasma radioactivity after i.v. injection of 10IU of [1,2-3H]-vitamin Ds to vitamin D-deficient rats(series I).
FIGURE 3 Plasma and tissue radioactivity after i.v. injectionof 10 IU of [1,2-8H]-vitamin Da to vitamin D-deficientrats (series II).
The Role of the Liver in the Metabolism of Vitamin D 1275
TABLE IIFraction of Dose in Plasma and Liver after i.v. Injection of 10 I U of [1, 2-'H]- Vitamin D3
in Plasma (Series III) to Vitamin D-Deficient Rats
Values expressed as per cent of dose.Mean ±SE (number of rats, if not specified = 4).Numbers in parentheses = number of rats.
erate the disappearance from the plasma of the vitaminDIS-H injected i.v. Hence it was decided to rely uponvitamin D-deficient plasma as vehicle for the injectionof the labeled vitamin D8 in the experimental series Iand III.
Distribution of vitamin D8 and its metabolites inplasma and liver after i.v. injection of 10 IU of [1,2-3H]-vitamin D8 to vitamin D-deficient rats. (seriesIII). The plasma radioactivity disappeared extremelyfast during the first period of 30 min, then leveled until6 hr before assuming a faster disappearance rate(Table II). The apparent absence of rebound is proba-bly due to individual variations in groups of rats killedat each time and to the smaller number of times periods.The liver reached its maximum uptake by 30 min andlost abruptly half of its radioactivity between 30 minand 2 hr. Afterwards, it reaccumulated some of the dosebefore the final decrease of radioactivity.
During lipid extraction of plasma and liver most ofthe radioactivity was soluble in the chloroform phase(Table III). Except at 15 min, around 99% of theplasma-3H was chloroform soluble. Liver extractsshowed an increased proportion of aqueous-soluble and
TABLE I IIPartitioning of Radioactivity During Lipid Extraction
Values are per cent of total sample radioactivity.
nonextractable tritium after 24 hr. The larger propor-tion of aqueous-soluble radioactivity seen in plasma andliver at 15 min might be due to some alteration of the[1,2-3H]-vitamin Ds at the time of injection. The chro-matographic patterns of unchanged vitamin D and itsmetabolites obtained in plasma and liver after 4 and 48hr are shown in Figs. 4 and 5. The major chromato-graphic fractions were expressed in per cent of chro-matographed radioactivity and per cent of dose inTables IV and V.
In plasma (Table IV), more than 90% of the radio-activity chromatographed corresponded to unchangedvitamin D (peak III) until 30 min after the i.v. dose.The 6-7% radioactivity eluted with a retention volumeclose to peak IV probably represented radiochemicalimpurities in the [1,22H]-vitamin D3 in plasma since itscontrol chromatography revealed 4.4% of the dose elutedin an identical position. In sharp contrast was the im-portant proportion of peak IV (25.6% of chromato-gram) present in the 2 hr plasma extract and risingfurther to reach 55.3% of chromatographed radio-activity after 48 hr. Expressed as per cent of dose themaximal concentration of peak IV in plasma wasreached by 24 hr after dose and decreased afterwards(9.9% of dose at 24 hr; 5.6% at 48 hr). Both at 24and 48 hr, peak IV was the major radioactive vitaminD metabolite in plasma. Peak V and more polar metabo-lites made their appearance later than peak IV andaccounted for a growing fraction of the radioactivityafter 24 hr. Peaks I and II never represented an impor-tant fraction of the radioactivity in plasma nor did theyshow any significant change with time. As a conse-quence of the appearance of peak IV, V, and more polarmetabolites, the proportion of unchanged vitamin D(peak III) decreased consistently from 2 hr on. Itsplasma concentration decreased so much that there wasless vitamin D than peak IV already at 24 hr.
In liver (Table V), the bulk of the radioactivity wasrepresented by unchanged vitamin D (peak III) until
1276 G. Ponchon and H. F. DeLuca
TABLE IVMajor Chromatographic Fractions in Plasma after i.v. Injection of 10 I U of [1, 2-3H]- Vitamin D,
in Plasma (Series III) into Vitamin D-Deflcient Rats
Chromatographicfractions 15 min 30 min 2 hr 4 hr 6 hr 24 hr 48 hr
IV 36.5 7.8 33.0 7.8 54.7 9.9 55.3 5.6V + more polar 6.1* 2.6* 6.9* 2.0* 25.6* 6.8*
fractions 3.3 0.7 3.0 0.7 15.8 2.8 28.6 2.9
Mean values from pooled samples of four rats.* Represents IV and V plus more polar fractions.
6 hr after dose. It was not until 24 hr that peaks IV,V, and more polar metabolites increased significantly.Maximum accumulation of peak IV was reached at 24hr (0.8% of dose), while peaks V and more polarmetabolites were still increasing after 48 hr. Note thatin contrast to the plasma there was practically as muchpeak V and more polar metabolites as peak IV both at24 and 48 hr. Peak I (vitamin D esters) showed atendency to accumulate slowly in the liver after 24 hr.Peak II exhibited no significant changes.
ml
30 MIN
I
02 1L0I
N6.9A
X1S7.4 X365
I It1 -=0.6 3.3t
Nj556348 HR T-U[
112&S
I 12.409 AJ
I A IC
FIGuRE 4 Silicic acid column chromatographic profiles oflipid extracts from plasma obtained after i.v. injection of10 IU of [1,2-3H]-vitamin Ds to vitamin D-deficient rats(series III).
DISCUSSIONThe disappearance of the radioactivity from the plasmaof rats given 0.25 jug of [1,2-8H]-vitamin Ds intra-venously showed a very characteristic pattern whichmay be described in three successive phases.
The first period of very fast disappearance of radio-activity occurred during the first 30 min after injection.It is probably the result of rapid dilution of the label inthe vascular and extravascular spaces combined with anuptake by various tissues such as liver, bones, muscles,kidneys, and small intestine (6, 16).
617
30 MIN
I0O4
N 717
4 HR
I IIA
N8.7 f-U
K2-3
N 5&3
48 HR
N Y-UffI x | | 13.71 J
6.7 72
I A B CIFIGURE 5 Silicic acid column chromatographic profiles oflipid extracts from liver obtained after i.v. injection of 10IU of [1,2-3H] -vitamin Da to vitamin D-deficient rats(series III).
The Role of the Liver in the Metabolism of Vitamin D 1277
TABLE VMajor Chromatographic Fractions in Liver after i.-v. Injection of 10 I U of [1, 2-3H]- Vitamin D3
in Plasma (Series III) into Vitamin D-Deflcient Rats
Chromatographicfractions 15 min 30 min 2 hr 4 hr 6 hr 24 hr 48 hr
V + more polar 5.5* 2.1*fractions 0 0 1.1 0.2 2.3 0.6 1.5 0.3 13.8 0.6 15.1 0.8
Mean values from pooled samples of four rats.* Represents IV and V plus more polar fractions.
The second phase exhibited a most intriguing re-bound in plasma radioactivity reaching a maximum be-tween 2 and 3 hr after dose. This secondary peak ofradioactivity varies with each animal in its timing andintensity. A similar phenomenon has been reported inman and remained so far unexplained (2, 3).
Since this rebound occurs in the two rats with bileduct cannulation, we may rule out the possibility of anintestinal reabsorption of radioactive bile products. Thesame was found by Avioli et al. in three patients withcomplete biliary fistula (2).
The third period corresponds to a very slow dis-appearance of plasma radioactivity after 2 days. It isevident that such a slow decrease must be considered inthe eventual estimation of the biological half-life ofvitamin D and its metabolites. A calculation based uponthe 1st 2 days after injection is likely to give muchshorter apparent half-lives than if computed over weeks.
The plasma rebound of radioactivity observed afterintravenous injection of labeled vitamin D3 is a promi-nent feature. It must be due to a shift of the radioactivematerial from the extravascular, probably intracellular,to the plasma compartment. This prompted us to look fora reciprocal change in the radioactivity of differenttissues.
A very definite loss of radioactivity from the liverwas found during the plasma rebound occurring between2 and 3 hr after dose. Although some radioactivity wasalso lost from the skeleton and muscle, it was clear thatthe liver was responsible for the major part of theplasma radioactivity rebound by releasing into theblood a large portion of the previously accumulateddose.
Individual rat metabolic variations probably accountfor some irregularity in the plasma and tissue radio-activity curves. The characteristic pattern was best ob-
served by repetitive blood sampling on a single animalrather than by data obtained from groups of rats killedat each time. Ethanol used as a vehicle for injection ofthe tritiated vitamin in one series of experiments (seriesII) is responsible for the lower plasma radioactivitieswhen compared to the data obtained after injection ofvitamin D--H in plasma vehicle (15).
On the basis of the very high and quick uptake ofvitamin D by the liver (6, 16-18) followed by a promptrelease of the accumulated dose, it is reasonable toassign an important role to the liver in the early meta-bolic events of the vitamin. One aspect might be theneed to clear the lipoprotein-bound vitamin from theplasma to favor a more specific protein binding (19).However, the vitamin binds readily to its plasma pro-tein carrier before the liver has released much radio-activity, and even when added to serum in vitro (19).Thus the obvious question of what is the nature of theradioactive substance released by the liver into theblood stream was raised. One important possibility isthat the released radioactivity represented vitamin Dmetabolites particularly 25-hydroxycholecalciferol (ref-erences 4 and 5, footnote 1) considered to be the activeform of vitamin D.
Chromatographic data show the appearance of largeamounts of 25-hydroxycholecalciferol (peak IV) inplasma at 2 hr after dose. That corresponds with themaximum rebound of plasma radioactivity. Since vita-min D3 (peak III) (6, 11) was sharply decreasing, andthe other chromatographic fractions were unchanged,the increase in plasma radioactivity must be due to theappearance of 25-hydroxycholecalciferol. It was duringthe same time that the liver was losing half of its 8Hradioactive content without significant change in itschromatographic profile.
It is interesting to note that the maximum accumula-
1278 G. Ponchon and H. F. DeLuca
tion of 25-hydroxycholecalciferol (peak IV) in plasmaoccurred by 24 hr, and that most of it was actuallyproduced during the first 4 hr. This is particularly rele-vant to the observation that the earliest physiologiceffects of vitamin D3 occur between 10 and 12 hr afteran intravenous dose (1) and supports the idea that25-hydroxycholecalciferol is the active principle ofvitamin D3.
More polar metabolites consisting mainly of peak Vappeared later in plasma and liver. These compoundshave not yet been characterized but have been shownbiologically inactive.'
The conclusion of these observations is that the liveris responsible for the very characteristic rebound ofplasma radioactivity occurring around 150 min afterintravenous injection of labeled vitamin Da to D-defi-cient rats. The radioactive dose accumulated in the liverduring the first 30 min is partly released into the bloodbetween 30 min and 4 hr. The rebound of radioactivityin the plasma is accounted for by the accumulation ofthe highly biologically active metabolite, 25-hydroxy-cholecalciferol. It is further suggested that the liver isthe major site of hydroxylation of vitamin Da (chole-calciferol) into 25-hydroxycholecalciferol. As soon as itis synthesized in the liver cells, 25-hydroxycholecal-ciferol is released into the plasma.
ACKNOWLEDGMENTSThis work was supported by grant No. AMO5800-07 fromthe U. S. Public Health Service.
REFERENCES1. DeLuca, H. F. 1967. Mechanism of action and metabolic
fate of vitamin D. In Vitamins and Hormones. R. S.Harris, I. G. Wool, and J. A. Loraine, editors. AcademicPress Inc., New York. 25: 315.
2. Avioli, L. V., S. W. Lee, J. E. McDonald, J. Lund, andH. F. DeLuca. 1967. Metabolism of vitamin D8-3H inhuman subjects: distribution in blood, bile, feces, andurine. J. Clin. Invest. 46: 983.
3. Avioli, L. V., F. Williams, J. Lund, and H. F. DeLuca.1967. Metabolism of vitamin Da-8H in vitamin D-resistantrickets and familial hypophosphatemia. J. Clin Invest.46: 1907.
4. Blunt, J. W., H. F. DeLuca, and H. K. Schnoes. 1968.25-hydroxycholecalciferol: a biologically active metabo-lite of cholecalciferol. Chem. Commun. 801.
5. Blunt, J. W., H. F. DeLuca, and H. K. Schnoes. 1968.25-hydroxycholecalciferol. A biologically active metabo-lite of vitamin Da. Biochemistry. 7: 3317.
6. Neville, P. F., and H. F. DeLuca. 1966. The synthesis of[1,2-VH] vitamin D3 and the tissue localization of 0.25 ,ug(10 IU) dose per rat. Biochemistry. 5: 2201.
7. Guroff, G., H. F. DeLuca, and H. Steenbock. 1963.Citrate and action of vitamin D on calcium and phos-phorus metabolism. Amer. J. Physiol. 204: 833.
8. Bollman, J. L. 1948. A cage which limits the activity ofrats. J. Lab. Clin. Med. 33: 1348.
9. Bligh, E. G., and W. J. Dyer. 1959. A rapid method oftotal lipid extraction and purification. Can. J. Biochem.Physiol. 37: 911.
10. Lund, J., and H. F. DeLuca. 1966. Biologically activemetabolite of vitamin Da from bone, liver, and bloodserum. J. Lipid Res. 7: 739.
11. Norman, A. W., J. Lund, and H. F. DeLuca. 1964.Biologically active forms of vitamin Ds in kidney andintestine. Arch. Biochem. Biophys. 108: 12.
12. Hansen, D. L., and E. T. Bush. 1967. Improved solubili-zation procedures for liquid scintillation counting ofbiological materials. Anal. Biochem. 18: 320.
13. Kelly, R. G., E. A. Peets, S. Gordon, and D. A. Buyske.1961. Determination of C' and H' in biological samplesby Shoniger combustion and liquid scintillation techniques.Anal. Biochem. 2: 267.
14. DeWachter, R., and W. Fiers. 1967. External standardi-zation in liquid scintillation counting of homogeneoussamples labeled with one, two, or three isotopes. Anal.Biochem. 18: 351.
15. Ponchon, G., and H. F. DeLuca. 1969. Ethanol-inducedartifacts in the metabolism of 'H-vitamin D3. Proc. Exp.Biol. Med. In press.
16. Norman, A. W., and H. F. DeLuca. 1963. The prepara-tion of H'-vitamins Da and D8 and their localization inthe rat. Biochemistry. 2: 1160.
17. Kodicek, E., E. M. Cruickshank, and D. R. Ashby. 1960.The metabolism of 'C-labelled vitamin D2 injectedintracardially into rats. Biochem. J. 76: 15P.
18. DeLuca, H. F., M. Weller, J. W. Blunt, and P. F.Neville. 1968. Synthesis, biological activity, and metabo-lism of 22,23-3H-vitamin D4. Arch. Biochem. Biophys.124: 122.
19. Rikkers, H., and H. F. DeLuca. 1967. An in vivo studyof the carrier proteins of 'H-vitamins Ds and D4 in ratserum. Amer. J. Physiol. 213: 380.
The Role of the Liver in the Metabolism of Vitamin D 1279
