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(Received for publication, September 20, 1994; and in revised form, March 16, 1995) From the
All-trans-retinoic acid (RA) activates ligand-dependent
transcription factors that regulate retinoid-responsive gene
expression. It is assumed that all-trans-RA is formed within
cells through in situ oxidation of retinol derived from the
circulation. However, the circulation contains low levels of
all-trans-RA (approximately 0.2-0.7% of that of plasma
retinol). Our studies investigated the extent to which plasma
all-trans-RA contributes to tissue pools of this retinoid and
explored factors responsible for regulating its uptake by tissues and
cells. Rats were continuously infused, to steady state, with
all-trans-[ Retinoids (vitamin A) are essential for vision, reproduction,
growth and differentiation, and maintenance of the general health of
the organism(1, 2, 3) . Retinoic acid and
possibly some of its metabolites are the active retinoid forms
responsible for mediating most of the nonvisual functions of this class
of compounds. Both 9-cis- and all-trans-retinoic acid
interact in the nucleus with ligand-dependent transcription factors to
regulate expression of retinoid-responsive genes(4) . Two
distinct classes of nuclear receptors for retinoic acid have been
described; these include the retinoic acid receptors (RAR Ultimately, all retinoids in the body are
derived from the diet. The bulk of dietary retinoid, consumed as either
preformed vitamin A or provitamin A (carotenoids), is taken up in the
intestine and packaged as retinyl ester, along with other dietary
lipids, in chylomicrons (6, 7, 8) . The
majority of this retinyl ester is delivered to the liver (6, 7, 8) . A small portion of the dietary
retinyl ester in chylomicrons is taken up by some extrahepatic tissues,
particularly bone marrow, kidney, and adipose tissue, where the retinyl
ester may serve as a source of retinoid for meeting tissue
needs(7) . Within the liver, the newly arrived dietary retinoid
is either stored as retinyl ester in hepatic stellate cells or secreted
into the circulation as retinol bound to its specific plasma transport
protein, retinol-binding protein (RBP)(7, 9) . It is
generally thought that tissue retinoid needs are satisfied primarily
through the delivery of retinol to cells via RBP. Plasma retinol is
internalized by cells from RBP through a process involving the action
of CRBP(9, 10) . Within the cell, the retinol can be
oxidized to retinoic acid through enzymatic processes (7) which
have also been proposed to involve CRBP(11, 12) . In addition to the transport process described above, other pathways
for the delivery of retinoid to tissues may operate within the body. A
small fraction of dietary retinoid is converted to retinoic acid in the
intestine (or may arrive as such in the diet) and is absorbed via the
portal system as retinoic acid bound to
albumin(7, 8, 12) . In plasma, retinoic acid
circulates bound to albumin(12) . The fasting plasma level of
all-trans-retinoic acid is very low, being in the range of
4-14 nmol/liter in humans (about 0.2-0.7% of plasma retinol
levels) (13, 14) and 7.3-9 nmol/liter in
rats(15, 16) . It is presently not clear to what
extent plasma retinoic acid contributes to tissue pools of this
retinoid. Our studies reported below address this question. In
addition, they explore whether retinoic acid accumulation by tissues
from plasma is specific and regulated in response to physiologic state
and whether the lipid content of a cell influences either the rate of
uptake or half-life of retinoic acid within a cell.
For some studies, rats were given
a bolus injection of a dose consisting of 5 µCi of
all-trans-[ In preliminary experiments developing and characterizing the
methodologies used for our studies, it quickly became obvious that
plasma retinoic acid was being taken up by most tissues and was very
rapidly being metabolized. We observed that after the stoppage of the
circulation of a perfused rat (i.e. upon sacrifice) the
metabolism of retinoic acid by tissues continued. Thus, a critical
point in the design of these experiments was to allow very little time
to elapse between the stoppage of circulation and the dissection of
tissues. This ensured that our measures provided accurate estimations
of the contribution which plasma retinoic acid makes to tissue pools.
With this in mind and as part of our experimental design, we limited
the maximum period of time between stoppage of the circulation and
flash freezing of the final dissected tissue to 5 min. Only
approximately 10 tissues could be dissected and processed (weighed and
frozen) in 5 min. Because of this experimental limitation, we chose to
study only the 10 tissues listed above.
For standards,
authentic all-trans- and 9-cis-retinoic acid were
obtained as a kind gift of Dr. Art Levin (Hoffmann-La Roche, Inc.,
Nutley, NJ) and authentic 13-cis-retinoic acid was obtained
from Sigma. Low limits of detections for all-trans-,
13-cis-, and 9-cis-retinoic acid in our HPLC assay
were estimated to be respectively, 0.7, 1.0, and 0.7 ng/g tissue.
The fractional catabolic rate (FCR) in plasma pools per hour was
determined from the following equation:
where W is the weight of the rat in grams and rat
plasma volume is assigned to be 0.04 ml/g total body
weight(27) . The absolute catabolic rate (ACR) in ng/h was
determined from the following equation:
Since we were concerned that residual plasma contamination of
our perfused tissues might influence our calculations, we determined
the amount of plasma contamination in the perfused tissues of four
representative rats (which were different from those employed for
retinoic acid continuous infusion). For this purpose, a bolus dose of For the bolus studies, only three to five samples
could be obtained from each animal. Therefore, the data from all
animals on the same diet had to be pooled. With this in mind, different
sampling times were used for different animals so that, taken together,
data were available at seven to eight distinct time points for each
diet. The data were expressed as percent of injected dose and were
fitted by a two-pool model to estimate plasma FCR(30) . The
tissue data were expressed as percent of injected dose per g of tissue
at 10 min after injection. Retinoic acid and oleic acid levels were
compared by paired t tests.
Figure 1:
Representative normal phase HPLC
profiles for retinoic acid from tissue extracts prepared from the same
rat for liver (panel A), brain (panel B), adipose
tissue (epididymal fat) (panel C), and testis (panel
D). The preparation of the extracts and the HPLC procedures were
carried out as described under ``Experimental Procedures.''
The upper portion of each panel gives the profile detected by
measurement of UV absorbance at 350 nm and the lower portion the profile of
The tissue levels of
all-trans-retinoic acid are given in Table 1. The tissue
with the highest mean level of all-trans-retinoic acid was the
pancreas, and the lowest mean level was observed in the brain.
All-trans-retinoic acid was present in all tissues examined
for each of the eight rats; however, as seen in Fig. 1, only
trace amounts of 13-cis- or 9-cis-retinoic acid were
detectable in any tissue extract. Although we did not specifically plan
to determine the levels of these cis isomers in tissues, we
can see no reason why our extraction and HPLC procedures would
selectively allow determination of all-trans-retinoic acid and
not of the two cis isomers. This suggests to us that
13-cis- and 9-cis-retinoic acid were present in the
tissues examined at very low levels relative to those of
all-trans-retinoic acid and that there was little or no
isomerization of the infused
all-trans-[
From
serial plasma samples and measurement of plasma
all-trans-[
From our data obtained with
continuously infused animals, it was also possible to calculate the FCR
and ACR for all-trans-retinoic acid in our experimental
animals. These are provided in Table 3. These values are
consistent with the conclusion that all-trans-retinoic acid is
rapidly turning over in the rat. The FCR indicates that the plasma pool
of all-trans-retinoic acid is turned over approximately every
2 min.
Figure 2:
Plasma clearance curves for bolus doses of
all-trans-[
The animals receiving the bolus
doses were sacrificed after 10 min, and levels of
Figure 3:
Uptake and metabolism of
all-trans-[
It has long been known that, under normal physiologic and
dietary conditions, all-trans-retinoic acid is present in the
circulation, albeit at very low levels (at 0.2-0.7% of those of
plasma
retinol)(7, 13, 14, 15, 16) .
However, no information is presently available regarding the
physiologic role of plasma retinoic acid. The studies reported here
were designed to investigate the possible role of circulating
all-trans-retinoic acid in providing tissues with this
retinoid. Our data indicate that, for the brain and liver, greater than
three-fourths of the all-trans-retinoic acid present in these
tissues is derived from the circulation. In marked contrast, less than
1% of the all-trans-retinoic acid present in the testis is
derived from the circulation. As seen in Table 2, most of the 10
tissues we examined took up significant amounts of
all-trans-retinoic acid from the circulation. Thus,
circulating all-trans-retinoic acid plays an important role
for making this retinoid available to tissues. The values obtained
for the fractional catabolic and absolute catabolic rates for
all-trans-retinoic acid indicate that this retinoid is rapidly
turning over in the whole animal. The data obtained from the continuous
infusion studies indicate that plasma pools of
all-trans-retinoic acid are being turned over every 2 min. For
these studies, it is possible that some of the infused
all-trans-[ Because our studies indicate that most tissues are able to
take up all-trans-retinoic acid from the circulation, we
wanted to gain insight into the physiologic processes and mechanisms
responsible for this uptake. In particular, we wanted to understand
whether retinoic acid accumulation by tissues is specific and
regulatable in response to physiologic state or whether it is
nonspecific and not regulated. Since both retinoic acid and fatty acids
are transported in the circulation bound to albumin, we first asked
whether retinoic acid and oleic acid are cleared from the circulation
via common processes. As seen in Fig. 2and Table 4,
plasma oleic acid is cleared more rapidly than plasma retinoic acid. In
addition, retinoid nutritional status was found to influence the rate
of retinoic acid but not the rate of oleic acid clearance from the
plasma. Overall, these data suggest that retinoic acid accumulation by
cells and tissues occurs through processes which are tissue and cell
type specific and which are responsive to physiologic (e.g. nutritional) state. It seemed possible that uptake of retinoic
acid by cells might depend simply on the general ability of the cell to
take up and store lipid. Adipose tissue plays an important role in
whole body retinyl ester storage(23) , expresses nuclear
receptors for retinoic acid(23, 36) , and influences
the tissue distribution of retinoic acid and its derivatives, through
sequestration of the retinoids, when these compounds are given in large
pharmacologic doses(37) . Together these observations might
suggest that the lipid-rich adipose tissue can play an important role
in retinoic acid accumulation and storage. To address this possibility,
we asked whether the lipid content of a cell influences either the rate
of uptake of all-trans-retinoic acid by, or half-life within,
a cell. Our studies with BFC-1 A striking finding of our studies was
that the testis, which possesses all-trans-retinoic acid at a
level similar to the other tissues (see Table 1), derives almost
none of this retinoid from the circulation. Since 1925, it has been
known that a rat maintained in the total absence of dietary retinol,
but supplemented with all-trans-retinoic acid in the diet,
will be generally healthy, but blind and
sterile(1, 38) . Because retinaldehyde is the active
retinoid in the visual cycle and since all-trans-retinoic acid
cannot be reduced to retinaldehyde, the inability of dietary
supplementation with all-trans-retinoic acid to restore vision
is understood (39) . It is known that the lesion in
reproduction arises from a failure of spermatogenesis, which shows an
obligatory requirement for retinol in the diet. However, the
biochemical basis for the inability of all-trans-retinoic acid
to substitute for the obligatory retinol needed during spermatogenesis
has not been understood. A recent study indicated that when a large
dose of all-trans-retinoic acid (5 mg) was injected
intratesticularly into a retinoid-deficient rat in which
spermatogenesis was blocked, spermatogenesis rapidly
resumed(40) . This suggests that retinoic acid can promote
spermatogenesis. Our data indicate that a barrier exists in the testis
for the uptake of all-trans-retinoic acid from the
circulation. This would explain why dietary all-trans-retinoic
acid cannot substitute for retinol to maintain spermatogenesis. The
biochemical nature of the testis barrier to all-trans-retinoic
acid is not clear. It is known that retinoic acid, at physiologic pH,
readily traverses membranes and it is generally thought that retinoic
acid enters cells by passive diffusion (41, 42) .
Thus, it would seem unlikely that a physical barrier in the testis,
such as the lack of a cell surface receptor for retinoic acid, could
account for the inability of the plasma to contribute to testis
all-trans-retinoic acid pools. Alternatively, it is possible
that a metabolic barrier, consisting of enzymes that rapidly metabolize
all-trans-retinoic acid, functions to prevent entry of
all-trans-retinoic acid into certain testicular cells from
plasma. To investigate this possibility, we isolated three cell
fractions from testes of rats continuously infused to steady state with
all-trans-[ In summary, our studies show that plasma all-trans-retinoic
acid plays an important role, under normal physiologic conditions, in
the delivery of retinoic acid to tissues. They provide an explanation
for an observation that is over 65 years old, regarding why dietary
all-trans-retinoic acid (i.e. delivered by the
circulation) will not substitute for dietary retinol and maintain
spermatogenesis in rats. Studies with preadipocytes and adipocytes
indicate that the cellular processes responsible for accumulating
all-trans-retinoic acid are cell type-specific, and studies of
the uptake by tissues of bolus doses of all-trans-retinoic
acid indicate that these processes are also tissue-specific. Moreover,
the uptake of all-trans-retinoic acid is responsive to
physiologic (e.g. nutritional) state. Together, these data
indicate that the delivery of all-trans-retinoic acid by
plasma to tissues is a dynamic and important process for maintaining
tissue retinoic acid pools.
Volume 270,
Number 30,
Issue of July 28, pp. 17850-17857, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
H]RA. From measures of
specific activities of all-trans-[H]RA
at steady state, we determined that the preponderance of
all-trans-RA in brain and liver was derived from the
circulation. For six other tissues, approximately 10-30% of the
retinoid was derived from the circulation, but pancreas and testis
derived very little from the circulating pool. In other studies, we
showed that retinoid nutritional status influences clearance of a bolus
dose of all-trans-RA and that neither the rate of cellular
all-trans-RA uptake nor its intracellular half-life is
influenced by cellular lipid levels. Taken together, our data indicate
that plasma all-trans-RA contributes to tissue pools of this
retinoid and that specific and physiologically responsive cellular
processes mediate its uptake.
, -
,
-)
()and the retinoid X receptors
(RXR, -, -)(4) . Response elements for these
transcription factors have been characterized for a diverse group of
genes including those for laminin B1, RAR
, cellular
retinol-binding protein, type I (CRBP), apolipoprotein A-I, oxytocin,
and phosphoenolpyruvate carboxykinase(3, 4) . Review
of the literature indicates that retinoids mediate expression of over
150 genes(5) .
Animals and Surgical Procedures
Male
Sprague-Dawley rats (Charles River Laboratories, Wilmington, MA)
ranging in weight from 400 to 500 g and maintained on a commercial chow
diet (Ralston Purina Inc., St. Louis, MO), without any source of
carotenoid, were employed for all of our studies. Rats were
anesthetized with a mixture of ketamine and xylazine prior to jugular
vein cannulation. Polyethylene catheters were inserted into both the
right and left jugular veins and externalized by routing through
subcutaneous dorsal tunnels(17) . The catheters were fixed with
dental cement to the cranial area, and the lines were filled with
either 200 units of sodium heparin in saline or a mixture of 50 units
of sodium heparin and 40% polyvinylpyrrolidone in saline to ensure that
they remained patent. The catheterized rats awoke within 1-3 h
after surgery and were allowed to recover for at least 1 day prior to
the start of the experiments. Animals were allowed free access to water
and chow throughout the studies.Continuous and Bolus Infusion Studies
To study the
contribution of plasma retinoic acid to tissue retinoic acid pools,
physiologic doses of
all-trans-[H]retinoic acid (50.6
Ci/mmol, DuPont NEN) were infused continuously into control animals.
The retinoic acid dose consisted of 1-1.5 µCi of
all-trans-[H]retinoic acid dispersed in
1.0 ml of 1% fatty acid-free rat albumin (the physiologic carrier of
retinoic acid in the circulation) (12) in phosphate-buffered
saline (PBS; 10 mM sodium phosphate, 150 mM sodium
chloride, pH 7.2). The infusate was pumped through one jugular cannula
at a flow rate of 0.78 ml/h. The infusion rate was chosen, based on
preliminary studies, to provide measurable radioactivity in tissues
without significantly perturbing the plasma retinoic acid pool. Three
to five serial blood samples (each of approximately 0.5 ml) were taken
from the second jugular cannula at 1-h intervals to establish that the
plasma all-trans-[H]retinoic acid levels
had reached a steady state. Immediately following the final sampling
and usually 5 h after the start of infusion, the animals were
sacrificed with a mixture of ketamine and xylazine, and tissues (brain,
liver, kidney, epididymal fat, perinephric fat, seminal vesicle,
epididymis, pancreas, and testis) were quickly perfused with ice-cold
PBS and excised for HPLC analysis of retinoic acid levels. Urine was
collected throughout the infusion period so that the hydration status
of the animal could be monitored.H]retinoic acid (50.6
Ci/mmol) and 0.5 µCi of [
C]oleic acid (55.5
mCi/mmol) in 1.0 ml of 1% fatty acid-free rat albumin in PBS through
one jugular cannula. Between 1.5 and 10 min after injection, three to
five serial blood samples (each of approximately 0.5 ml) were taken
from the second jugular cannula for analysis of
[H]retinoic acid and
[C]oleic acid remaining in the circulation. At
10 min, the injected animals were anesthetized with ketamine and
xylazine; the whole body was perfused with ice-cold PBS; and brain,
liver, kidney, epididymal fat, perinephric fat, seminal vesicles,
epididymis, pancreas, and testis were taken for extraction and analysis
of tissue levels of [H]retinoic acid and
[C]oleic acid as described in the section below. Extraction of Retinoic Acid from Plasma and
Tissues
All extraction and analytical procedures were carried
out in a darkened room using brown glass tubes to protect the retinoids
from exposure to light. Plasma samples were diluted with equal volumes
of PBS prior to extraction. Tissues were homogenized in PBS (2 ml of
PBS/g of tissue) using three 15-s pulses of a Brinkmann Polytron PT 300
homogenizer (Brinkmann Instruments), at setting 5 on the homogenizer.
An internal standard consisting of a known amount of
all-trans-7-(1,1,3,3,-tetramethyl-5-indanyl)-3-methyl-octa-2,4,6-trienoic
acid (kindly provided by Dr. A. Levin, Hoffmann-LaRoche, Inc., Nutley
NJ) was added in 0.1 ml of ethanol to each plasma or tissue sample in
order to monitor the recovery of retinoic acid during the extraction
and HPLC procedures(18) . Retinoic acid was extracted from the
tissue homogenates and plasma using a modification of the procedure
described by Tang and Russell(19, 20) . This
extraction procedure is quite gentle and does not cause
retinoyl--glucuronide hydrolysis. Briefly, plasma and tissue
homogenates (3 ml) were extracted twice with chloroform/methanol (2:1),
and the chloroform extracts were combined and concentrated, under a
gentle stream of N
, to a final volume of less than 1 ml.
This retinoid-containing chloroform extract was then applied to 100 or
500 mg aminopropyl solid phase extraction columns (Baxter Labs. Inc.,
Chicago) that had previously been equilibrated with hexane. Under these
chromatographic conditions, most lipids are retained by the column. The
neutral lipids were eluted first from the column with 5 ml of
chloroform/isopropanol (2:1). After the neutral lipids were eluted, the
retinoic acid was eluted from the aminopropyl column with 5 ml of 2%
acetic acid in diethyl ether containing 0.01% butylated hydroxytoulene
as an antioxidant. The acetic acid/diethyl ether eluates were
collected, evaporated to dryness under a gentle stream of
N, and redissolved in HPLC mobile phase
(hexane/acetonitrile/acetic acid, 99.5:0.4:0.1) for injection onto the
HPLC.HPLC Analysis
All-trans-retinoic acid
levels were determined by normal phase HPLC employing two silica
columns linked in tandem. The silica columns consisted of a 3.9
150-mm Waters 5 µ Resolve (Waters Associates, Milford, MA) followed
by a 4.6
150-mm 3 µ Supelcosil LC-Si (Supelco Inc.,
Bellefonte, PA). The first column was preceded by a Waters silica
Guard-PAK guard column. For chromatography, we employed an isocratic
system where the mobile phase consisted of hexane/acetonitrile/acetic
acid (99.5:0.4:0.1) flowing at 1.8 ml/min. The mobile phase was made
fresh daily and filtered and degassed immediately prior to use. The
solvent was delivered by a Varian Star 9010 pump (Varian Instruments,
Sugarland, TX). We routinely injected 90 µl of sample onto the
columns using a Varian Star 9095 Autosampler. Retinoic acid mass was
detected at 350 nm using a Spectra-Physics Spectra 100 variable
wavelength detector (Spectra Physics Inc., Piscataway, NJ).
All-trans-retinoic acid levels were quantitated from the
integrated area under its peak using a standard curve, constructed with
authentic standards of all-trans-retinoic acid of known mass.
All-trans-[
H]retinoic acid was detected
and quantitated with an in-line Berthold LB506C-1 radioactivity monitor
(EG& Berthold, Nashua, NH). This normal phase system was selected
because it allowed for maximal detection of H counts/min
compared to other normal phase and reverse phase HPLC systems, where
more quenching of radioactivity was observed.Nutritional Studies
For some studies, rats were
maintained for 10 weeks on one of two different nutritionally complete
purified diets which supplied different levels of retinoid. The
purified diets assessed were a nutritionally complete control diet
which contained 2.4 µg of retinol/g of diet and a totally
retinoid-deficient but otherwise complete diet. These diets and their
use have been described in detail in the
literature(21, 22) . After 10 weeks on the diets,
bolus infusion studies were carried out on these animals as described
above.Adipocyte Cell Cultures
To determine whether lipid
content of cells and tissues influences retinoic acid uptake and
retention we investigated the ability of cultured preadipocytes and
adipocytes to take up all-trans-retinoic acid from the culture
medium. BFC-1 preadipocytes and adipocytes were cultured and
maintained according to procedures we have previously
described(23, 24, 25) . Preadipocytes were
cultured in 75-cm plastic tissue culture flasks. For our
studies of retinoic acid uptake and metabolism, the preadipocytes were
used on the day they reached confluence. Similarly, BFC-1
adipocytes were cultured in 75-cm flasks and were used when
greater than 75% of the cells present in the flask had undergone
adipose conversion as evidenced by the presence of characteristic lipid
droplets within the cells. Five µCi of
all-trans-[H]retinoic acid in 5 µl
of ethanol were dispersed in 1.0 ml of fatty acid-free rat albumin in
PBS, and 0.1 ml of the dispersion was added directly to 3 ml of the
appropriate culture medium(23, 24, 25) . The
final concentration of the all-trans-retinoic acid in the
culture medium was 3 10
M. After
the labeled medium was provided to the cells, they were incubated at 37
°C in a humidified atmosphere containing 5% CO
for
periods of up to 6 h. After incubation, the medium was removed for
analysis of retinoic acid. The cells were washed three times with
ice-cold PBS and scraped from the plate into a small volume of PBS. The
medium and cells were immediately frozen in liquid N and
stored at -70 °C for up to 1 week prior to analysis for
retinoic acid levels. Retinoic acid was extracted from the cells and
medium and analyzed as described in the above sections. The half-life
of all-trans-retinoic acid in preadipocytes and adipocytes was
calculated from the slope obtained by regression analysis of a linear
plot of total system (cells + medium) all-trans-retinoic
acid levels and time (for times ranging from 1 through 6 h after
addition of all-trans-retinoic acid to the medium). For these
plots, at each time of analysis, cellular and medium
all-trans-retinoic acid levels were determined in triplicate,
and the average value was used for half-life calculation.Mathematical Modeling and Statistical Methods
The
specific activity of [H]retinoic acid present at
steady state in each tissue (and plasma) was determined from individual
measurements of radiolabeled retinoic acid (dpm/g of tissue) and
retinoic acid mass (pmol or ng/g of tissue) carried out for each
tissue. The purpose of the continuous infusion of
all-trans-[H]retinoic acid was to
establish a steady state among the infused retinoic acid, plasma
retinoic acid, and tissue retinoic acid pools. Since the pool of
retinoic acid present within each tissue must be composed of the
retinoic acid synthesized endogenously by the tissue from retinol
oxidation (and possibly from retinoyl--glucuronide hydrolysis (26) but not carotenoid cleavage, because the rats employed for
our studies received no carotenoid in the diet) and the retinoic acid
taken up from the circulation, the contribution that plasma retinoic
acid makes to a tissue pool is simply the ratio of the specific
activity of all-trans-[H]retinoic acid
present in the tissue to plasma
all-trans-[H]retinoic acid specific
activity:
I-RBP, prepared as described previously(28) , was
intravenously injected in PBS into the rats through a jugular vein
catheter and allowed to circulate for 3-5 min. This time period
is sufficiently short so that very little
I-RBP would be
expected to be taken up by cells within the rat tissues(29) .
The animals were then sacrificed, and tissues were perfused and excised
as described above for the experimental animals. Levels of
I in the tissues and plasma were measured directly using
an LKB RIAGAMMA (LKB-Pharmacia Instruments, Piscataway, NJ). The
maximum residual plasma contamination of each tissue was calculated
assuming that the level of
I measured in each tissue
arose solely from the residual plasma present in the tissue. From these
measurements, we determined that the residual plasma contamination for
each tissue was both very small (<1% of the total
I
counts/min present in the circulation) and very reproducible for the
four rats. Our calculations of the percent contribution which plasma
retinoic acid makes to tissue pools were made both uncorrected and
corrected for possible residual plasma content of the tissues. The two
sets of calculated values were found to be very similar, and for no
tissue were differences of greater than 5% observed. The data in this
report are given without correcting for average residual plasma present
in each tissue.
Steady State
In preliminary experiments with
approximately 20 rats, we demonstrated that, for our experimental
conditions, a steady state in plasma retinoic acid and tissue retinoic
acid pools was established within 2.5 h after the start of continuous
retinoic acid infusion. Repeated plasma samples (usually three or four)
were taken over 6 h (for some animals this period ranged up to 24 h)
after the start of infusion and plasma
all-trans-[H]retinoic acid specific
activity was measured. When computer-determined slopes of plots of
plasma retinoic acid specific activities for different animals were
statistically analyzed, they were not found to be significantly
different from zero, the slope of a horizontal line. Thus, when plasma
all-trans-[H]retinoic acid specific
activity was plotted as a function of time, for times greater than 2.5
h, there was no consistent trend with time. We concluded from this
analysis that a steady state was reached within 2.5 h after the start
of infusion. This conclusion was also supported by measures of H counts/min levels in urine collected over defined time
intervals which began 2.5 h after infusion started. Although none of
the H counts/min present in the urine were present as
retinoic acid, the total H counts/min determined in the
timed urine samples were reproducibly found to be equal to the total
all-trans-[H]retinoic acid infused into
the animal over the time interval of urine collection. The observation
that the radioactivity infused into an experimental animal equals that
being excreted by the animal would indicate that the whole body pool of
retinoic acid is at isotopic steady state. These two observations, when
taken together, fully support the conclusion that plasma and tissue
retinoic acid pools are in a steady state condition within 2.5 h after
the start of all-trans-[H]retinoic acid
infusion. This conclusion is further supported by our data which
indicate that the fractional catabolic rate of plasma
all-trans-retinoic acid is very large (see below).Tissue Distribution
We employed sensitive
analytical procedures which enabled us to measure simultaneously
all-trans-retinoic acid mass and radioactivity in all tissues
examined. To obtain the simultaneous profiles, a UV absorbance detector
was linked in series with an in-line radiation monitor. The extraction
procedure did not hydrolyze retinoyl--glucuronides to retinoic
acid(19, 20) . Levels were measured in the brain,
liver, kidney, epididymal and perinephric fat, seminal vesicle, spleen,
epididymis, pancreas, testis, and plasma for eight rats which were
continuously infused for 5-6 h with
all-trans-[H]retinoic acid.
Representative HPLC profiles for the simultaneous determination of
retinoic acid mass (UV absorbance) and radioactivity for extracts
prepared from liver, brain, epididymal fat, and testis of one
experimental animal are shown in Fig. 1. The upper portion of
each profile gives the UV absorbance and the lower portion H counts/min. As can be seen from the markers in Fig. 1, the normal phase HPLC procedure we employed was able to
resolve 13-cis-, 9-cis-, and
all-trans-retinoic acid. In our system, pure authentic
all-trans-didehydroretinoic acid eluted approximately 2 min
after all-trans-retinoic acid.
H
counts/min.
H]retinoic acid.
H]retinoic acid specific
activity, we established that a steady state was reached in each of the
eight experimental animals. At steady state, the extent to which plasma
contributes to the retinoic acid pool within a tissue is simply the
ratio of tissue retinoic acid specific activity to plasma specific
activity (). Table 2gives the plasma retinoic acid
contribution to retinoic acid pools in the 10 tissues investigated. The
values presented in Table 2indicate that retinoic acid pools in
two tissues (brain and liver) are derived almost entirely from the
plasma; two other tissues, pancreas and testis, derive almost no
retinoic acid from the circulation. Other tissues (kidney, epididymal
fat, perinephric fat, seminal vesicle, spleen, and epididymis) derive a
minority of their retinoic acid pools from the circulation. It should
be noted that the mathematical model used for calculating these percent
contribution values assumes that tissue all-trans-retinoic
acid pools are kinetically homogeneous. We are not aware of any data
which contradict the validity of this assumption, nor do any of our
data suggest that this is not the case.
Bolus Studies for Different Diets
Since the FCR
indicates that the turnover of plasma retinoic acid is very rapid, we
also carried out studies to assess the rate of plasma clearance of
all-trans-[H]retinoic acid given as a
bolus dose. As part of these studies, we asked both whether plasma free
fatty acids (which like retinoic acid circulate bound to albumin) are
cleared from the plasma at the same rate as retinoic acid and whether
retinoid nutritional status influences plasma clearance of
all-trans-retinoic acid and/or free fatty acids. For this
purpose, a bolus dose consisting of tracer amounts of
all-trans-[H]retinoic acid and
[C]oleic acid bound to rat albumin was injected
intravenously into rats maintained on one of two purified diets which
differed only in retinoid content. The diets consisted of a
nutritionally complete control diet providing 2.4 µg of retinol/g
of diet and a totally retinoid-deficient diet but otherwise
nutritionally complete diet. The animals receiving the totally
retinoid-deficient diet, at the time of their use for these studies,
all had plasma retinol levels ranging between 1.8 and 4.2 µg/dl
compared to a range of 21.4-26.3 µg/dl for the control fed
animals. The results of these bolus studies are presented in Fig. 2along with fitted curves. The calculated FCRs are given in Table 4. As can be seen from Fig. 2and Table 4,
plasma retinoic acid is cleared rapidly from the circulation but at a
slower rate than oleic acid. Also, in total retinoid deficiency, the
rate of clearance of plasma all-trans-retinoic acid is
increased significantly but the rate of oleic acid clearance remains
unchanged. The FCR estimated from the bolus data for the control-fed
group is approximately 41% of that estimated from the continuous
infusion studies of chow-fed rats. Although the two FCR values for
all-trans-retinoic acid estimated using these two independent
approaches are not in very close agreement, they do both indicate that
all-trans-retinoic acid is being rapidly turned over in the
circulation. Similar rates for plasma clearance of
all-trans-retinoic acid have also been observed when large
pharmacologic doses were injected intravenously into
rats(31, 32) . The rate of clearance of plasma oleic
acid that we observed in our studies is very similar to the rate
reported for the physiologic clearance of fatty acids from the
circulation of rats(33) .
H]retinoic acid (upper
panels) and [C]oleic acid (lower
panels) given simultaneously to rats maintained on a control (left panels) or a totally retinoid-deficient (right
panels) diet. For this figure, curves were fitted starting at 100%
at zero time. Individual measures of
all-trans-[H]retinoic acid or
[C]oleic acid from which the curves were fitted
are indicated () in each panel. All procedures were carried out
as described under ``Experimental
Procedures.''
H and C counts/min in total lipid extracts prepared from tissues
were assessed. The distribution of H and C
counts/min in tissues is given in Table 5. These tissues
differentially took up retinoic acid and oleic acid. As would be
expected, the brain took up very little oleic acid from the
circulation; but as can be seen in Table 5, retinoic acid uptake
was substantial in the brain. On the contrary, the liver took up
relatively high levels of both retinoic acid and oleic acid. Brain,
seminal vesicle, epididymis, and testis, regardless of dietary group,
took up significantly more (p < 0.05) of the dose of
retinoic acid than of the oleic acid dose. Liver, kidney, epididymis,
and pancreas from retinoid-deficient animals were found to take up
significantly more (p < 0.05) retinoic acid than
corresponding tissues from animals fed the control diet. The
observation that tissues differentially take up
all-trans-retinoic acid given in a bolus trace dose validates
our finding in Table 2that plasma all-trans-retinoic
acid contributes to tissue pools in a tissue dependent manner.
Cell Studies
To gain additional insight into the
cellular processes and mechanisms responsible for retinoic acid uptake
and clearance, we carried out studies which asked both if cells of
common origin take up and metabolize retinoic acid at similar rates and
if cellular lipid content influences the uptake and accumulation of the
fat soluble retinoic acid. Since the BFC-1 preadipocytes can be
induced to differentiate into lipid-laden adipocytes upon addition of
insulin and thyroid hormone to the culture
medium(24, 25) , we investigated
all-trans-[H]retinoic acid uptake and
its rate of metabolism in both BFC-1 preadipocytes and adipocytes.
As seen in Fig. 3,
all-trans-[H]retinoic acid is rapidly
taken up from the culture medium by both BFC-1 preadipocytes and
adipocytes. Levels of
all-trans-[H]retinoic acid within the
preadipocytes and adipocytes reach a maximum within 30 min after
addition of all-trans-[H]retinoic acid
to the medium. Thus, it would appear that neither the amount nor the
rate of uptake of retinoic acid by cells is markedly influenced by the
lipid content of the cells. Interestingly though, the half-life of the
retinoic acid within the cells is markedly different. From Fig. 3A, we estimate the half-life of retinoic acid in
the BFC-1 adipocytes to be approximately 1.25 h, whereas the
half-life in BFC-1 preadipocytes is approximately 5 h. Because
neither BFC-1 preadipocytes nor BFC-1 adipocytes express
cellular retinoic acid-binding protein (CRABP)(24) , an
intracellular retinoid-binding protein which is thought to play a role
in the oxidative metabolism of retinoic acid(7) , it would
appear that metabolism of retinoic acid in these cells in not dependent
on CRABP presence and that these two cell types have markedly different
capacities for retention and metabolism of retinoic acid.
H]retinoic acid by BFC-1
preadipocytes (open circles) and adipocytes (closed
circles). Confluent cultures of BFC-1 preadipocytes and
adipocytes were provided with 0.5 µCi of
all-trans-[H]retinoic acid (at a final
concentration of 3 10
M) in the
appropriate culture medium at 0 h. At the indicated times, cells (Panel A) and medium (Panel B) were taken for HPLC
analysis of all-trans-[
H]retinoic acid
as described under ``Experimental
Procedures.''
H]retinoic acid did not enter
the circulation or was metabolized rapidly in a nonspecific fashion.
(Any loss of infusate has no effect on plasma contribution
calculations.) In fact, however,
all-trans-[H]retinoic acid accounted for
most of the plasma radioactivity at early time points after the start
of infusion. Also, even if 75% of the infusion is lost, the FCR becomes
7.5 instead of 30 pools/h, still indicating very rapid turnover. In the
bolus studies, 20-40% of the injected
all-trans-[H]retinoic acid was in plasma
at 1 min, indicating substantial integrity of the dose. The literature
indicates that plasma and tissue retinol turns over in the rat at a
much slower rate than retinoic acid(34, 35) . If the
average tissue concentration of all-trans-retinoic acid is
taken as 4 ng/g (see Table 1), this would mean that 2000 ng of
all-trans-retinoic acid is present in the entire body of a
500-g rat. Since the ACR for all-trans-retinoic acid is 192
ng/h, this indicates that, for control rats, the whole body pool of
all-trans-retinoic acid is replaced approximately once every
10 h. preadipocytes and adipocytes (Fig. 3) indicate that cellular lipid content does not influence
the rate of uptake of all-trans-retinoic acid by cells.
Considering their high lipid content, BFC-1 adipocytes might have
been expected to retain or sequester retinoic acid more readily than
cells containing less lipid, but we found that the adipocytes
catabolize all-trans-retinoic acid far more rapidly than the
relatively lipid poor BFC-1 preadipocytes. It would appear for
these two cell types that the half-life of retinoic acid within the
cell is controlled by mechanisms unrelated to cellular lipid content.
In addition, we have shown that all-trans-retinoic acid is
present both in epididymal and perinephric adipose tissue in
concentrations similar to those in most other tissues. Hence, although
adipose tissue may take up and harbor large quantities of retinol,
retinyl ester and retinoic acid given in very large pharmacological
doses to rodents(37) , our data indicate that, although adipose
tissue represents a large tissue pool for all-trans-retinoic
acid, it does not seem to be a major storage site of
all-trans-retinoic acid under normal physiologic circumstances
and that the lipid content of a cell does not influence retinoic acid
uptake from the circulation.H]retinoic acid. These cell
fractions consisted of a Sertoli + germ cell fraction, an
interstitial (Leydig) cell fraction, and a peritubular cell
fraction(43) . In three independent experiments, 72.5, 59.7,
and 39.5% of the total H counts/min present in the testes
were found in the peritubular cell fraction (data not shown). The
remainder of the H counts/min were found to be distributed
nearly equally between the Sertoli + germ cell fraction, the
interstitial cell fraction, and extracellularly in the isolation
medium. Based on these preliminary studies, it would seem that the
peritubular cells may serve as the cellular site for a metabolic
barrier that prevents circulating retinoic acid from entering the
interior of the seminiferous tubules where spermatogenesis occurs.
We thank Letitia Wong Yen Kong for her advice and
assistance with the surgical procedures.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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