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Plasma kineticsof vitamin A inhumans after a singleoral dose of [8,9,19-13C]retinylpalmitateD. v. Reinersdorff,lt*E.Bush,+and D. J. LiberatotDepartment of Vitamin and Nutrition Research,* F. Hoffmann-La Roche LTD, Basel, Switzerland, andDepartment of Drug Metabolism and Pharmacokinetics: Hoffmann-La Roche Inc., Nutley, NJ

Abstract The kinetics of vitamin A and its major metaboliteswere investigated in humans. Eleven healthy male subjectsingested 105 pmol (100,000 IU) f [8,9,19-13C]retinyl palmi-tate in an oily solution. Twenty-seven blood samples werecollected during the 1-week study. Plasma samples were ana-lyzed for retinyl esters and for [12C]- and [8,9,19-13C]retinol.Retinol isotopes were quantified using a newly developedGC-MS method. Total retinyl esters peaked at about 4.45pmol/L from 3.5 to 12 h after dosing. As a result of theperturbation of the tracee system, the plasma concentrationof [Wlretinol increased and then decreased as the concen-tration of [8,9,19-W]retinol ncreased, ndicating rapid distri-bution kinetics.A broad single peak (1.16f 0.32 pmol/L) wasobserved for [8,9,19-'3C]retinol at about 10 to 24 h postdose;this likely reflects hepatic secretion of [8,9,19-13C]retinol sso-ciated with retinol-binding protein. Then, declining levels ofthe tracer and increasing levels of the tracee were observed.At its peak, the ingested [8,9,19-13C]retinoleached about51%of the observed total plasma retinol concentration. Thispercentage dropped to 13.4% on day 7 indicating slow finalelimination from plasma. Our data support the concept thatthe liver follows the principle 'last in/first out' in maintainingvitamin A homeostasis.-v. Reinersdofi, D., E. Bush, and D.J. Liberato. Plasma kinetics of vitamin A in humans after asingle oral dose of [8,9,19-W]retinyl almitate.J. Lipid Res.1996.87: 1875-1885.Supplementary ke y words mass spectrometric method retinolretinyl esters stable isotopes vitaminA metabolism

Vitamin A is an essential nutrient for humans that isinvolved in important physiological functions includingvision, growth, reproduction, and the differentiationand maintenance of epithelial tissue (1 ,2). The absorp-tion, transport, storage, and metabolism of vitamin Aare highly regulated, involving a number of specificbinding proteins and enzymes (3).

In recent years, great progress has been made in thefield of vitamin A and retinoid research, especially re-garding receptor-mediated retinoid actions (4, 5) andretinoid-binding proteins (6, 7). In spite of this growinginterest in retinoids, many questions concerning plasmaretinol homeostasis (8) and whole body turnover of

vitamin A remain to be answered. Extensive tracerkinetic studies on whole-body vitamin A metabolism inthe rat have been conducted by Green an d colleagues(9-14). These studies quantified plasma retinol turnoverin the rat and demonstrated that there is multiple recy-cling of retinol in the body.To date, only a few retinol kinetic and metabolicstudies have been carried out in humans (15-18) andthere are limited data on plasma retinol turnover inhumans (8). Such studies require the use of a label inorder to distinguish the endogenous plasma concentra-tions from those resulting from the administered dose

In the present study, w e administered vitamin Alabeled with a stable isotope and measured the plasmakinetics of vitamin A and its major metabolites in hu-mans for 1week. Here we describe the contribution ofthe labeled retinol to the total RBP-retinol plasma poolafter a singleoral dose of 105pmol of [8,9,19-13C]retinylpalmitate, the study design, and a new method for theanalysis of stable isotope labeled versus unlabeledplasma retinol, and we discuss the results of the ob-served vitamin A plasma concentration versus time pro-files.

(19).

SUBJECTS AND EXPERIMENTAL DESIGNThe study was conducted at Clinical Research Associ-

ates, 50 Madison Ave., New York, NY, n compliancewith the principles of the 'Declaration of Helsinki' andafter approval of the study protocol by the InstitutionalEthics Review Committee. T he subjects were recruited

Abbreviations: C,,, maximal measured plasma concentration;HPLC, high performance liquid chromatography; IU, internationalunits; RBP, retinol-binding protein; tm,, time to reach maximalplasma concentration;UV ,ultraviolet spectroscopy.'To whom correspondence should be addressed.

Journalof Lipid Research Volume 37,1996 1875


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from an available populat ion of healthy male volunteersat the study location. All eleven healthy male subjects,24 to 40 years of age, give written informed consentbefore they were enrolled in the study.

All subjects body weights were within 15% of idealbody weight according to the Metropolitan Life Insur-ance Tables. Their health status was determined bymedical history, physical examination, and laboratorytests. All volunteers were non-smokers, had no historyof drug or alcohol abuse, were not using medication ofany kind, discontinued intake of any multivitamin prepa-rations 2 weeks prior to the study, and did not consumealcoholic beverages starting 3 days prior to blood collec-tion for lab tests and baseline sampling or during thestudy. The subjects consumed a vitamin A restricted dictthroughout the study according to a list which wasprovided to them. Therefore, their vitamin A intakeduring the remainder of the 1-week study was thoughtto be minor. In o rder to collect blood for determina tionof fasting plasma concentrations of vitamin A and itsmetabolites, subjects reported to the study site 24 h priorto ingestion of the vitamin A dose. They received alow-fat snack for supper and remained at the study siteovernight. After a 10-h overnight fast, a zero-h bloodsample was obtained. Then, a single oral dose of 105pmol (100,000 IU) of [8,9, 19-I3C]-labeled etinyl palmi-tate was administered with 7 parts (w/w) of coconut oilin a hard gelatin capsule. Simultaneously, each subjectconsumed 0.5 L of whole milk, which contained 20 g offat. No other food o r liquids were allowed until after t he4-h blood sample was taken. Low-fat meals were pro-vided after t he 4-h and 8-h blood samples. Blood sampleswere obtained at 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5,6, 7, 8, 9, 10, 11, 12, 18, 24, 48, 72, 96, 120 and 168 hafter [8,9,19-3C]retinyl palmitate administration. Thesubjects remained at t he study site until after collectionof the 24-h blood sample and returned after 8-11over-night fasts for collection of the final samples. At eachsampling time 15 mL of blood was collected intoheparinized Vacutainers by venipuncture of a forearmvein. Blood samples were immediately centrifuged for10 min at 2500 rpm and 10C.After the plasma had beentransferred into amber, siliconized scintillation vials,samples were immediately frozen at -70Cuntil analysis.All sample handling and analytical work was carried outunder yellow lights.

MATERIAL AND METHODSChemicals and solvents

Retinyl palmitate, all-trans retinol, oleoyl chloride,stearoyl chloride, and butylated hydroxytoluene (BHT)were purchased from Sigma Chemical Co., St. Louis,

MO, and nonanoyl chloride was from Aldrich ChemicalCompany, Milwaukee, WI. [8,9,19-1?C]-all-tram etinoland [2,3,6,7,10,1 -13C]-all-truns etinol were synthesixdby A. A . Liebmann and W. Burger, Department olIsotope Synthesis, Hoffmann-La Roche Inc., Nut ley , NJRetinyl oleate, retinyl stearate, and retinyl nonanoatewere synthesized in our laboratory using the procedureof Huang and Goodman (20). All solvents (HLPGgrade), Dulbeccos phosphate-buffered saline (PBS) 1x.sterile, without calcium an d magnesium were obtainedfrom Fisher Scientific Co., Springfield, NJ, and N,O-bis(trimethylsily1)-trifluoroacetamideBSTFA) was fromPierce, Rockford, 11,.Plasma analyses

Plasma samples were analyzed for retinyl esters,[W]-and [8,9,19-1:3C]retinol, I2C]- an d [8,9, 19-l~4C]-all-trun.s-retinoic acid, [I%]- an d [8,9, 19-1:3C]-13-cis-retinoiccid,[W]-and [S,9,19-~~C]-alI-truns-4-oxo-retinoiccid, arid[l2CC]- and [8,9,19-~3C]-13-czs4-oxo-retinoiccid. Thedata on vitamin A metabolites will be presented else-where.

For the reversed-phase HPLC analysis of individualplasma retinyl esters, plasma aliquots of 0.25 to 1.0 mLwere used. The retinyl ester calibration standards ( 1 to1000 ng/mL ) were prepared in a 5% dilution of a retinylester-poor plasma lot with PBS. Retinyl nonarioate wasadded as internal standard prior to extraction of sam-ples and standards with 1 mL ethanol containing 1 ppmBHT and 1 mL of n-hexane. The hexane was removedand the aqueous phase was reextracted with 2 niL ofn-hexane (21). The combined upper phases were driedunder reduced atmospheric pressure at 8 C and sam-ples were redissolved in 50 pL of 20% ethyl acetate inisopropanol containing 0.02% BHT. Ten-pL aliquotswere injected on to a Zorbax R,Cls (150by 46 mm, 5 pm)analytical column with C18 Brownlee guard cartridges(15 by 32 mm). The HPLC unit consisted of ;I WatersModel 590 pump with an automated gradient controller,a Hewlett-Packard Autosampler Series 1050, and an AB1783 programmable absorbance detector (Applied Bio-systems).

Retinyl nonanoate, oleate, palmitate, arid stearate(respective retention times of3.8 ,8.8,9.4, and 12.2 min)were separated using a modification of the solvent sys-tem described by Furr, Cooper, and Olson (22). Themobile phase of 20% methylene chloride in acetonitrilewas used a t flow gradient starting with a flow rate of 1.2niL/min for the first 6 min and then 2.0 mL/min forthe next 8 min. As the HPLC method was optimized forPast separation of the retinyl esters, the retinol peakelutes very close to the void volume. For siinultaneousquantification of retinol with this method, the solventconditions would need to be adjusted to ensure separa-

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tion from BHT. The W-signa l was monitored at 325 nmand automatically transferred via a Nelson Series 900Interface into a data base. Peak integration and quanti-fication was performed based on the peak height ratiosof each of the measured esters to the internal standardusing linear regression analysis. Each tray was internallyvalidated by analysis of plasma quality control samplescontaining known concentrations of retinyl esters. Thecorrelation coefficient for all of the regression lines wasyO.990. The interassay variation coefficient as deter-mined from spiked control plasma samples was 5.2%,and the limit of detection for the individual retinyl estersin plasma was 5 pmol.

A GC/MS method was developed for analysis ofplasma ['*C]- and [8,9,19-13C]-all-truns retinol.[2,3,6,7,10,1 -13C]-all-truns etinol (internal standard)was added to plasma aliquots (1 mL) prior to liquid-liq-uid extraction with 1 mL of methanol containing 1ppm BHT and 1 mL of n-hexane. The hexane wasremoved and the aqueous phase was reextracted with2 mL of n-hexane. After the hexane from the combinedupper layers had been brought to dryness under astream of nitrogen, the samples were reconstituted in50 pL of acetonitrile. The samples were vortexed, soni-cated for 10 min, and briefly centrifuged. The super-natant containing retinol was removed again into aclean tube and brought to dryness. Then, the solubili-zation in acetonitrile was repeated in another cleantube and the supernatant was removed and brought todryness. Using this procedure a substantial fraction ofthe extracted plasma lipids remained at the bottom ofeach tube and were eliminated due to their poor solu-bility in small volumes of acetonitrile. The sample pu-rification was necessary in order to avoid peak broad-

500000-

400000-9)0E: "-c4an

200000-

100000-112 163 184

ening on the GC column. The samples were then dis-solved in 50 pL BSTFA to derivatize retinol totrimethysilylretinol in an instant reaction. TMS-retinolproved to be a stable derivative that could be kept upto 4 weeks in the refrigerator. Retinol analysis wasperformed on a Hewlett-Packard mass spectrometerHP5989A MS-Engine interfaced to a HP5890 gas chro-matograph. Two-pL aliquots of the samples in BSTFAwere injected via cool on-column injection onto a 1-mpiece of 0.53 mm deactivated fused silica tubing (SGE,Austin Texas) connected to the stationary phase of15-m DB-1 capillary GC column with 0.25 mm ID and0.1 pm film thickness U&W Scientific, Folsom, CA).Helium was used as carrier ga s at a constant linearvelocity of 2 mL/min. The gradient program startedat an initial oven temperature of 35C with an initialinjector temperature of 38C. The oven and the injectortemperatures were then ballistically brought up to300C and held for 5 min. The interface temperaturewas kept constant at 275"C, the ion source at 250'C,and the quadrupole analyzer at 100C. Derivatized all-trans retinol eluted at 4.6 min from the GC columndirectly into the ion source. Ions were produced bynegative chemical ionization using methane/reagentgas at a gas pressure of about 1.5 Torr. Fragment ionscorresponding to anhydroretinol (16, 23, 24) (Fig.1)were measured in the selected ion monitoring modewith a dwell time of 20 msec/ion at the mass units ofm/z 268, 271 and 274 (Fig.2). Quantification of thesamples was based on the peak height ratios of [ *C]-and [8,9,19-13C]anhydroretino1o the internal standard[2,3,6,7,10,1 -13C]anhydroretinol. The reproducibilitywas found to be higher using the peak height ratiosrather than the ratios of the integrated peak areas.

, 373 43385 309/

I 268f r a g m e n t i o n :an h y d r o - r e n o l(M-H20)

m o l e c u l a r i o n :t r l m e t h y l s i l y l - r e t i n o l(M-1)

Fig. 1. Negative chemical ion mass spectrum of trimethylsilylretinol. The molecular ion (M-1) appears at m/ z357. The fragment ion, anhydroretinol, that is monitored for quantitative analysis appears at m/ z 268.

v . Reinendo$ Bush, and Liberato Plasma 1%-labeled vitamin A kinetics in humans 1877


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0

D3n4

0Cmaan4

0C05sn

400000-

300000-

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Io n 268.20 am unI\

Io n 271.20 amu4.56

Tlmo (mln)

Ion 274 .20 am u4.56

4.3 4.4 4.5 4.6 4.7 4.8 4.8T im e ( m i d

Fi g 2. GC-MS chromatogranis obtained from a plasma sample 24h after oral administration of [8,9,19-'X]retinyl palmitate. The massspectrometric detection was carried out in the single-ion monitoringmode at m/z 268, 271, and 274 for the unlabeled analyte, labeledanalyte, and internal standard, respectively.

In order to correct for natural abundance signaloverlaps, a multivariant regression analysis was per-formed using matrix notation (25). The peak heightratios and the corresponding concentrations of a seriesof standards containing varying amounts of both [ ' * C ] -and [8,9,19-'"C]retinol and a constant amount of[2,3,6,7,10,1 -'%]retinol (10 ng/mL) served as refer-ence for the unknowns. Because a deconvolution tech-nique was applied to separate the label from naturalabundance, the use of the more enriched [2,3,6,7,10,11-'%]-label would not have significantly lowered the sen-sitivity limits of the tracer. The calibration range for[8,9,19-13C]retinol anged from 1 to 625 ng/mL and for['*C]retinol from 250 to 850 ng/mL. Accuracy was

within 6%, and the interassay precision as determinedfrom spiked control plasma samples were 6.2%(unla-beled analyte) an d 3.6% (labeled analyte). Spiked con-trol plasma samples were also used in each tray forinternal validation of the assay. The purity of the stand-ards was confirmed on the same analytical system byindividual injection of the three derivatized retinol iso-mers and simultaneous monitoring of all relevantmasses. The contamination with the respective otheranalyte mass was found to be less than 0.25%.Statistics and kinetic calculations

The Student's t-test at the significance level of 5%wasused to test the difference between two groups of values.The data are presented as mean f SD. The plasmaconcentration versus time profiles are presented for onerepresentative subject, in order to demonstrate the char-acteristic plasma fea tures.

For labeled and unlabeled retinol, the data weretransformed as described by Cobelli et al. (26) in orderto correct for natural abundance associated with thetracer mass.

The data were analyzed by non compartmental meth-ods. Plasma peak concentrations (Cmax) nd times toreach maximal plasma concentrations ( mW) ere deter-mined directly from the data points. The areas underthe concentration versus time profiles from time to to t,,(AUCto-tn) were calculated using the linear trapezoidalrule. Half-times were estimated by linear regressionanalysis of the final observed slope of the concentrationversus time data (27).

RESULTSThe endogenous concentrations for total plasma ret-

inol (1.88 f 0.39 pmol/L) and for plasma retinyl esters(0.022f 0.013 pmol/L) were consistent with values forhealthy male adults reported in the literature (2 ,28-30).As the subjects were recruited from a healthy populationof volunteers and the results of the laboratory testsdemonstrated that they were healthy, it is reasonable toassume that their liver vitamin A levels were in thenormal sufficient range. The concentra tion versus timeprofiles for plasma retinyl esters and retinol are shownin Fig. 3 during the first 24 h after the ingestion of 105pmol of [8,9,19-'3C]retinyl palmitate and in Fig. 4 forthe entire study time. We assume that the retinyl esterpeak between 3 and 9 h corresponds to absorption ofthe vitamin A dose and clearance of the resultant ab-sorptive lipoproteins, whereas the profiles for retinolspecies reflect basically metabolized retinol carried byRBP.

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45004000 :3500 :3000 :

a 2500 1CYg 2000:8 15001000 ;500 :

Fig. 3. Plasma concentration versus time profiles of total retinyl esters, [8,9,19-1SC]retinol,I2C]retinol, nd total retinol (sum of [8,9,19-1sC]-and ['2C]retinol) during the first 24 h after oral administration of 105 p o l 8,9,19JSC]retinyl almitate in subject 5. Data from subject 5 werechosen to illustrate, because these data appeared to be representative for most of the subjects (Figs.3 to 5).

0'1?J 9,!'I

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* aI ---------, --q '%- - w--=&.. ',~2mL - - - - - * - - . . , , , A . . , - - - - - - - - - - ~.----.. --.- - - - - _e.--

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0-24 -12 0 12 24 36 48 60 72 84 96 108 120 132 144 156 16E

1time [h]- A - 12C]retinol -[8,~,19-13C]retlnol--[8,9,19-13C] +[12C] total retinol --otal retlnyl esters (UV-HPLC)

Fig. 4. Plasma concentration versus time profiles of total retinyl esters, [8,9,19-1sC]retinol. Wlretinol, and totalretinol (sum of [8,9,19-W]- and [l*C]retinol)during the 1-week study period after oral administration of 105 p o l[8,9,19-'SC]retinyl palmitate in subject 5.

v. Reinemdo@ Bush, and Liberato Plasma 13C-labeledvitaminA kinetics in humans 1879


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IARLE 1. Pattern of plasma retinyl esters (:,,,.,,uf total retinylesters (sum of individual retinyl esters).

Calculations based on concentrations normalized LO etinol

12:44567891011

74.6(i0.276.367.468.067.668.170.467.974.36 9 . 1

%18.725.619.028.026.82 5 327.324.926.619.13 1 :I

6. 75 .26.74.65.27.14.64.75.5t i t i5.0

Mean W .7 24.8 5.6SI) 3.9 4 .2 I .oC V . % 5.6 17.0 17.0~ ~ .

Retinyl estersWhen samples were analyzed for individual retinylesters, only three moieties showed significant absorp-

tion maxima at 325 nm: retinyl palmitate, rctinylstearate, and retinyl oleate. This was confirmed by run-ning an absorbance spectrum of a pooled plasma sampleat the concentration maxima of the retinyl esters (datanot shown). The distribution of total plasma retinylesters among these three fractions was remarkably simi-lar among subjects and averaged 69.7% retinyl palmi-

tate, 24.8% retinyl stearate, and 5.6% retinyl oleaic(Table 1) . The dynamic behavior of the three esters witssimilar as evident from the concentration versus tirnc.profiles (Fig. 5) . Thus, as expected, these data indicatt:that the individual ester species are metabolized i r iparallel. Therefore, we refer to the sum of these t1irc.cindividual esters as plasma retinyl esters.

The observed maximal plasma concentrations o f th t .retinyl esters ranged from 1.91 to 7.74 pmol/L (Table2 ) .The peak occurred 3.5-12 11 after vitamin A admini.stration. Thus, there was a substantial interinc1ividu;tlvariation in both the time and the magnitude of thc.plasma peak. Typically, there was a pronouncc d lag timrbefore the rapid increase in plasma concentration. A sh e plasma concentration approached the maximunt.the slope of the increase was reduced, creating almost Lplateau in four of the eleven subjects. I n t w o o f the.eleven subjects the maximum occurred as late as 12 I 1after dosing and these curves displayed multiple peak\before the main maximum. The semi-logarithmic plotof thc concentration versus time profiles of tlie plasmitretinyl esters revealed for all subjects at least two distinctphases in the decay within the first 24 h after dosing.Retinol

Plasma concentration versus time profiles fo r [S,S,l!L?C]retinol, [12C]retinol, and total retinol (sum of l i t -beled and unlabeled retinol) after administration o f thc:retinyl palmitate are shown in Figs. 3 and 4. Ilasnw

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00

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00 3 6 9 12 15 18 21 24 27 30 33 36 39 42 45 48

time [h]-total retinyl esters (UV-HPLC) - *- ret.palmitate ---ret.stearate --et.oleate

The plawia concentration veIsus time profiles o f indivitiu;il retiny1esters (retinyl palinitate, -oleate, md stearate) n suhjccr 5ig. 5.

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TABLE 2. Maximal plasma concentrations (C& and corresponding times (ha")of retinyl esters and retinol isotopes after oraladministration of 100,000 IU of [8,9,19-'SC]retinylpalmitateRetinyl Esters [8,9.19-"C]Retinol ['%]Retinol Total Retinol

Subject L a x t m a r Gnu tmax Cmax t m Cmax t m upmol /L h pm ol /L h P W L h p mo l/L h

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1.915.482.207.744.222.265.962.994.705.965.52

5.56.012.05.55.03.55.03.55.012.05.0

0.981.232.051.131.031.311.041.120.891.150.89

12.024.024.012.011.010.010.010.012.024.010.0

1.602.172.481.711.722.361 892.652.231.911.58

4.54.53.03.03.51.53.51.53.55.02.5

2.052.513.232.152.312.962.322.812.282.001.77

10.09.018.08.07.07.08.07.03.5

24.06.0Mean 4.45 6.2 1.16 14.5 2.03 3.3 2.40 9.8SD 1.90 3.0 0.32 6.2 0.37 1.2 0.44 5.9Median 4.70 5.0 1.12 12.0 1.91 3.5 2.31 8.0C.V.% 42.7 48.2 27.6 42.8 18.4 35.1 18.5 60.9

kinetics for the three moieties showed distinct charac-teris ics.

For labeled retinol, plasma concentrations increasedafter a lag phase of 1.5-5.5 h. This is similar to the delayobserved for the retinyl esters. Also, there was a positivecorrelation between the t m w for the retinyl esters versusthe t m a x for [8,9,19-l3C]retino1 r2=0.837). On average,[8,9,19-13C]retinol oncentrations peaked 14.5 h afterdosing, and 8.3f .0 h later than the peak of the retinylesters. The maximal plasma concentration of [8,9,19-13C]retinolwas 1.16f 0.32 pmol/L (Table 2). The slopeof the increase was much smoother than the retinylesters and the curves displayed a broad single peak inall subjects.

A perturbation in the plasma levels of endogenousunlabeled retinol was observed as early as 3.3 f 1.2 hafter dosing. This rise occurred before the increase inretinyl esters was observed. On average, these retinolpeak concentrations of 2.03 k 0.37 pmol/L were 0.15f0.06 pmol/L higher than their respective predose val-ues. After this maximum, declining concentrations of['*C]retinol were observed, as the concentrations of[8,9,19-'3C]retinol increased in plasma. While [8,9,19-'3C]retinol levels peaked, the ['2C]retinol values reacheda minimum. This was followed by an asymptotic increasetowards endogenous concentrations for ["%]retinol,while [8,9,19-13C]retinol evels declined.In all subjects, the total plasma retinol levels wereperturbed and elevated at 9.8 f 5.9 h and 0.52 zk 0.23pmol/L above baseline levels. As expected, the magni-tude of the perturbation was not correlated to the initialretinol concentration prior to dosing (r2= -0.31). Onaverage, the initial baseline concentration of totalplasma retinol was restored to 102f 16% at 168 h afterthe vitamin A dose.

Thus, overall, there was a consistent time course forappearance of the peak concentrations of retinyl estersand the retinol isotopes in plasma after retinyl palmitateadministration. First, unlabeled retinol concentrationspeaked, followed by retinyl esters, and total plasmaretinol. Finally, [8,9,19-13C]retinol reached maximalplasma concentration after a pronounced lag phase(Figs. 3 and 4).

At its peak (10-24 h after dosing), the ingested labeledretinol accounted for 51.4 f 6.6% of the observedplasma [12C]-+ [8,9,19-13C]retinol oncentration (Figs.3,4 and Table 3).By the end of the study period (168hafter dosing), the plasma concentrations of labeled ret-inol were still elevated considerably (0.27 t- 0.21pmol/L) and contributed on average 13.4% to theplasma retinol concentration. Based on our data, wepredict that the kinetic half-time (t1h) for elimination oflabeled retinol from plasma (estimated from the finalobserved slopes) is at least 5.0f 2.3 days.

In order to estimate the relative contribution of la-beled retinol to the retinol plasma pool, we calculatedthe area under the plasma versus time curves (AUC)between 0 and 168 h (Table 4) . During the observedtime period, an AUC of 86.2 f 38.5 bmol/L x h wasestimated for [8,9,19-13C]retinol and 242.6 f 38.7pmol/L x h for ['*C]retinol. Thus, the relative averagecontribution of [8,9,19-13C]retinol o the total retinolAUC0-168h was 25.8*7.6%. The AUCO-48h of the retiny1esters (22.0 f 11.2 pmol/L x h) was not correlated withthe AUCO-168 h of the labeled retinol (r2=0.13).

DISCUSSIONThe present study was performed to qualitatively and

quantitatively determine the kinetic behavior of plasma

v. Reinersdo$ Bush, and Liberato Plasma 1%-labeled vitamin Akinetics in humans 1881


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TABLE 3 . Percent distribution of labeled [8,9,19-1'iC]retinol nd unlabeled [12C]retinol etinol i n totalplasma-retinol [8,9,19-13C]-_ _ _[l?C]retinol t C,,,,, of [8,9,19-13C]retinol[8,9,19'"C]RctinoI C(1) ill c,,,,,,l' [H,S,lS-'"C]-+ ["C] as w of

....[ l'C]Kctinol l o t a l Relinol [R,O. IS-"(:]Rctinol

Subicc t c,,,. IH.9.19-13ClRetinol Retinol Total Rctinol1234567891011

0.981.232.051.131.031.311.041.120.891.150.89

Mean 1.16SD 0.32C.V.% 27.6

pmo l /L0.961.111.160.860.861.481.161.671.100.850.881.100.2724.4

vitamin A and its metabolites after an oral dose of 105pmol of vitamin A to healthy male subjects. The concen-tration versus time profiles of the retinyl esters and oflabeled, unlabeled, and total retinol showed very similarcharacteristics among the eleven subjects.Retinyl esters

The labeled and unlabeled isotopes of retinyl esterswere not distinguished in this study, because an appro-priate mass-spectrometric method was not available.However, it is reasonable to assume that the majority ofthe retinyl esters were newly absorbed labeled species.This assumption is based on current knowledge aboutvitamin A absorption (3, 6) . In humans of normal vita-min A status, the plasma concentrations of retinyl estersare below 0.1 pmol/L in the fasting state and onlyincrease transiently as a result of dietary vitamin Aabsorption ( 31, 32) . The large interindividual variationin the lag time between the ingestion of the vitamin Adose and the increase in the plasma retinyl ester concen-trations might be related to the dose formulation inwhich the vitamin A was administered (in coconut oil).Other possible explanations are influences on fat meta-bolism due to the low-fat supper and the 10-h fastingperiod prior to the vitamin A administration.

In all cases, retinyl ester levels in plasma peakedbefore labeled retinol. Also, a positive correlation wasfound between the tlnaxfor retinyl esters and the t,,,,, forlabeled retinol. This observation is consistent with whatis known about vitaminA metabolism and likely reflectshepatic secretion of [8,9,19-'"C]retinol associated withRBP (33) . The apparent biexponential decay of theplasma retinyl esters was similar to what Berr (31)foundin a study after intravenous infusion of retinyl palmitate-loaded autologus plasma. After separating the plasmainto different lipoprotein fractions, he found that dur-ing the initial rapid decay retinyl esters were associated

1.942.343.211.991.882.782.202.791 992.001.762.260.4720.6

%50.2852.6963.7756.6954.4847.0047.2940.2244.7957.5550.2451.366.6112.9

with chylomicron remnants and, thus, this process likelyreflected hepatic uptake. The second slower phase wasrelated to the clearance of retinyl esters associated withvery low density lipoproteins (VLDL),mostly of hepaticorigin.Retinol

The earliest increases in plasma retinol concentra-tions after an oral dose of 105 pmol vitamin A wereobserved in the unlabeled ['*C]retinol fraction. In mostsubjects, [2CIretinol concentrations increased even be-fore the retinyl ester and [8,9,19-%]retinol fractionsstarted to rise and reached a maximum before the otherfractions did.

As plasma levels are a result of the influx of mass intothe plasma compartment relative to the efflux of massfrom the plasma into tissue compartments, an increasein a compound's plasma concentration can only bemeasured if mass accumulates transiently in plasma (27) .This would be the case when the rate of influx intoplasma from intestinal absorption is high relative to therate of clearance from plasma.

During the initial absorption phase, the turnover ofthe plasma retinyl esters was probably already increased,due to starting influx of absorbed retinyl esters intoplasma on the one hand and efficient plasma clearanceon the other hand. This hypothesis is based on the rapidhepatic clearance rate of the retinyl esters contained inchylomicron remnants (half-time 19k 11 min; ( 31 ) ) .Asa result, no measurable increase in plasma retinyl esterswould be expected during the initial absorption phase.

In order to further interpret the observed kineticprofiles for plasma vitamin A we used some informationfrom a whole-body model for vitamin A dynamics in rats(9, 13,14).According to this model, two vitamin A poolscan be distinguished in the liver. The perisinusoidalstellate cells behave kinetically as a large slowly turning-

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TABLE 4. Area under the plasma concentration versus time curves (AUC(lo-I.) f retinyl esters and retinol isotopes. Percent distribution of[8,9,19-W]retinol-AUC n total plasma retinol-AUC

Subject

1234567891011MeanSDC.V.%

TotalRetinolAu c 1 n -1 6 ~l AUCK-IR ) AUc(o-168 ) AUC WIGR)

Retinyl Esters [8,9,14 15C] Retinol [12C]Retinol [8,9.19-%]- +[ W ] - [8,9.19-t5C]Retinol-AUCas 96ofTotal Retinol-AUC

9.4128.5516.9035.3918.3811.2321.4016.4117.1747.6119.8022.011.250.8

58.44116.55189.0788.0982.2072.3156.3764.2056.5588.0376.3586.238.544.7

Fmol/L x h214.41235.12273.44236.01220.57242.53253.68306.57298.89209.49177.95242.638.716.0

272.86351.67462.51324.10302.77314.83310.05370.77355.44297.52254.30328.856.417.1

%21.433.141.927.227.123.018.217.315.929.630.025.87.629.3

over pool and the hepatocytes as a small faster turning-over pool. Both pools contribute to the liver output ofRBP-bound retinol into plasma. The model predicts acontribution of dietary vitamin A to the liver output ofabout 9% in rats with marginally adequate vitamin Astatus. In the model, dietary vitamin A is taken up asretinyl esters into the hepatocyte pool and subsequentlysecreted as RBP-bound retinol into the plasma. If nodietary vitamin A is available, the liver output to plasmamust consequently be constituted from recirculatingRBP-retinol and , to a larger extent, from stellate cells.The turnover rates from these pools adjust dependingupon dietary vitamin A input in order to maintainplasma retinol homeostasis (9).

In our study, the uptake of retinyl esters by the livermight have resulted in an enhanced turnover rate ofretinol in the hepatocyte pool. This process could, inturn, have increased the secretion rate of RBP-retinolinto the plasma. These changes in the turnover rates arethought to be induced by a mass competition process inthe hepatocyte pool initiated by the incoming mass ofabsorbed labeled retinol. As the hepatocytes initiallycontained unlabeled retinol of endogenous origin, theinitial output into plasma was consequently constitutedfrom this material. This was reflected in plasma by theearly increase in the ['*C]retinol concentration prior tothe rise of the [8,9,19-'3C]retinol concentration. As theabsorption process continued, larger quantities of la-beled retinyl esters were taken up by the hepatocytesand the endogenous unlabeled vitamin A was more andmore diluted. Consequently, the relative contributionof labeled retinol to the liver RBP-retinol output into theplasma was transiently increased. This was reflected inthe plasma by increasing concentrations of the [8,9,19-13C]retinol,while ['*C]retinol concentrations declined(see Fig. 4). Our data support the hypothesis that the

liver follows the principle of 'last in/first out' in main-taining the plasma retinol homeostasis and keeping upthe vitamin A mass balance in the hepatocyte pool. Indoing so, the liver would first use the vitaminA providedby the recirculation and dietary sources to the hepato-cyte pool and recruit any additional amount from thestellate cell storage pool. O n the other hand, the fractionof dietary vitamin A not needed for maintaining homeo-stasis would be transferred to the stellate cells. As a ruleof thumb, this fraction is about 50% of the dose ab-sorbed in the normal physiological dosing range andnormal vitamin A liver status (34).

During the observed study period, 25.8 k 7.6% of theplasma RBP-retinol was contained as labeled retinolspecies and at its peak (10-24 h after dosing), theingested labeled retinol reached about 51% of the totalretinol plasma concentration (see Fig. 4 and Table 3).This is most likely due to the production of retinol fromretinyl esters in the liver and subsequent output of thisnewly produced RBP-bound retinol into the plasma (35).However, our initial results from compartmental mod-eling indicate that there was a small fraction of labeledretinol transferred to RBP from enterocytes or trans-ported in chylomicrons and then transferred to RBPthat contributed to the early rise of the [8,9,19-13C]reti-no1 concentration versus time profile. The majority ofthe labeled retinol mass in plasma was, according to ourmodel, derived from the normal pathway involving he-patic uptake of retinyl ester-containing chylomicronsand subsequent RBP-retinol release. This is consistentwith data reported by Goodman and colleagues (36)where they found a small fraction of unesterified retinolassociated with lymph after oral ingestion of labeledvitamin A.

After the administration of 105pmol of vitamin A, aplasma perturbation of total retinol was observed. By

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the end of the 1-week study period, these plasma levelsreturned back to baseline. This observation is consistentwith the concept of the homeostatic regulation ofplasma retinol when the vitamin A status is adequate.Also due to the homeostasis, the magnitudes of theperturbations were not correlated with the initial retinolconcentrations before dosing.

The final elimination of [8,9,19-13C]retinol fromplasma appeared to be very slow, as indicated by stillconsiderably elevated plasma levels 168h after the dose.Also, the curves did not display a monoexponentialterminal decay when plotted on a semi-logarithmic cale,suggesting that [8,9,19-'3C]retinol was not yet equili-brated among all tissues. This observation is consistentwith the concept of multiple recycling of each retinolmolecule among plasma, liver, and extrahepatic tissuesin the body as established by the work of Green et al.(9-14). Keep in mind that the subjects were on a lowvitaminA diet during the study to minimize interferenceof the equilibration process with additional unlabeledvitamin A. A retrospective compartmental analysis oftracer kinetic data obtained in humans by Goodman etal. (8) ndicate that the sampling time in future experi-ments should be at least 200 days. This would allowmixing of the tracer with the exchangeable tracee pools,so that the curves reach a terminal slope, permitting anaccurate calculation of the final elimination rate. Model-based compartmental analysis can accommodate stand-ardized dietary vitamin A intakes in such long-termstudies.

Overall, the absorbed vitamin A dose was traced intothe plasma pools of the retinyl esters and the RBP-reti-nol. The incorporation of the dietary labeled vitaminAinto the plasma RBP-retinol pool was characterized byfast distribution kinetics which were reflected in thetransient large increases in labeled retinol and in therapid declines of the unlabeled retinol plasma fraction.Whereas the rate of increase of labeled retinol probablyreflected the secretion rate of liver RBP-retinol, thedecay of the unlabeled retinol might give an estimate ofthe turnover rate of retinol in plasma. Currently, we areusing model-based compartmental analysis to quantifythese processes in more detai1.W

The authors thank Dr. A. A. Liebmann and his colleagues forsynthesizing all stable isotope labeled compounds used in thisstudy; Dr. K. Kohlhof (Clinical Research Associates, NewYork) for carrying out the clinical part of the study; Dr. C.Huselton (Hoffmann-La Roche, Nutley) for technical trainingat the mass-spectrometer; Drs. M. Green and J. B. Green(Pennsylvania State University) for stimulating discussions andeditorial help during the preparation of the manuscript.Manuscriptreceived 10February 1996 and in rmisedfonn 7June 1996.

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bstfa_retinol

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