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Department of Nutritional Sciences, University of Wisconsin-Madison, Madison, WI 53706-1571
3To whom correspondence should be addressed.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • vitamin A • retinol • stable isotopes • rats • isotope ratio mass spectrometry
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, Tanumihardjo et al. 1995
and 1996
) and the United States
(Duitsman et al. 1995
, Spannaus-Martin et al. 1997
) among those from a lower socioeconomic class are
compromised. For women, the reasons for this compromised status include
low dietary intakes and increased demands during pregnancy and
lactation. For children, inadequate dietary intake and multiple
childhood infections can deplete vitamin A liver reserves, increasing
the risk of morbidity (Sommer et al. 1984
). During times
of acute infection, retinol is excreted in large amounts in the urine
(Alvarez et al. 1995
, Mitra et al. 1998
,
Stephensen et al. 1994
).
Vitamin A surveys are important in determining which population groups
are at risk of deficiency, but they cannot determine vitamin A status.
Although clinical signs of deficiency have been used in the past in
large national surveys, i.e., Bitot’s spots and xerophthalmia,
marginal vitamin A status is much harder to diagnose. Dietary surveys
alone are not usually sufficient to determine the vitamin A status of
groups because of multiple factors that affect the absorption and
utilization of the pro–vitamin A carotenoids and preformed vitamin A
(DePee and West 1996
).
Unfortunately, vitamin A status assessment is not as simple as
determining plasma or serum concentrations of retinol. Serum
concentrations of retinol are homeostatically controlled and do not
begin to decline until liver reserves are dangerously low
(Underwood 1984
). In addition, in times of infection,
serum concentrations are transiently reduced due to the acute phase
response (Willumsen et al. 1997
). In a healthy
individual, the liver contains 
80–90% of the total body reserves
(TBR)4
of vitamin A, mostly in the form of retinyl esters. A reserve of
0.070 µmol of retinol/g of liver has been defined as an adequate
amount for humans. The reported range of liver reserves in
well-nourished healthy American adults is 0.44–0.74 µmol/g
(Underwood 1984
). However, liver biopsies of humans are
justifiable only under certain instances, and therefore indirect
methods to determine liver reserves are needed.
Vitamin A assessment methods that will accurately determine the liver
reserves of a population with the smallest number of samples and are
minimally invasive are still in development. Biochemical assessment
techniques that have been used include the relative dose-response
(RDR) and modified relative dose-response (MRDR) tests
(Tanumihardjo et al. 1994
) and the deuterated retinol
dilution (DRD) assay (Furr et al. 1989
, Haskell et al. 1997
and 1998
). The RDR and MRDR tests operate on the
same principle. As liver reserves become depleted,
apo-retinol-binding protein accumulates in the liver. A
challenge dose of either retinyl ester in the RDR or dehydroretinyl
acetate in the MRDR is administered, and the response of the retinol or
dehydroretinol–holo-retinol-binding protein complex is
measured in the serum 5 h after dosing. The major difference in
practice is that two blood samples are needed for the RDR (before and
after dosing) and only one is needed for the MRDR (after dosing).
However, the dose-response tests lack usefulness in defining the
actual liver reserve of vitamin A and will only distinguish between a
moderately inadequate and an adequate vitamin A status. They also are
not able to determine whether an individual is in a subtoxic or toxic
vitamin A state.
The DRD assay has been successfully applied in several population
groups, but large doses of vitamin A (70–140 µmol in adults) must be
used because gas chromatography/mass spectrometry (GC-MS) analysis is
not very sensitive (Furr et al. 1989
, Haskell et al. 1998
). A recent improvement in the sensitivity of MS was
made by using electron capture negative chemical ionization for
detection. However, the retinol still must be derivatized, and a
relatively large dose, 35 µmol, of retinyl acetate is administered to
adults (Ribaya-Mercado et al. 1999
).
We developed an isotope dilution method in the rat model that does not
have the same pitfalls as the DRD method. The improved methodology was
first suggested by Parker et al. (1997)
for use in
ß-carotene studies. Our application involves the use of stable
13C-labeled retinol and the very sensitive
GC/combustion/isotope ratio MS (GC-C-IRMS) method (Goodman and Brenna 1995
). In this regard, physiological doses of retinol
and routine venipuncture techniques can be used. The method holds
considerable promise for use in determining TBR of vitamin A in human
population groups and in sensitivity to changes in liver concentration
of vitamin A.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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10,11,14,15-13C4-Retinyl acetate was
synthesized according to a modification of the method of Bergen et al. (1988)
. Briefly, starting with ß-ionone (Aldrich
Chemical, Milwaukee, WI), a commercially available precursor, the
backbone of the retinol moiety was synthesized through reaction with
the carbanion of 13C2-labeled
triethylphosphonoacetate (Aldrich Chemical). Next, acetone was added
under basic conditions. A second reaction was performed with the
carbanion of 13C2-labeled
triethylphosphonoacetate. The resulting ethyl ester was reduced to the
alcohol and esterified to 13C4-retinyl acetate
with acetic anhydride. The synthetic vitamin was purified carefully on
8% water–deactivated alumina with relatively innocuous, highly
volatile organic solvents, such as hexanes and diethyl ether. The
resulting preparation was characterized with the use of UV-VIS
spectroscopy, TLC, HPLC and GC-MS.
A small amount of the 10,11,14,15-13C4-retinyl acetate was saponified with ethanolic potassium hydroxide and mixed with HPLC-purified unlabeled retinol at 1, 2 and 3% of the total. The mixtures were analyzed with GC-C-IRMS to standardize the technique.
Animals and diet.
Weanling female Sprague-Dawley rats (n = 47)
were obtained from Harlan Sprague-Dawley through Laboratory Animal
Resources at Iowa State University, Ames, IA. Rats were housed
individually in plastic cages in an environmentally controlled room
with a 12-h light cycle. Rats were fed tap water and food ad libitum.
The diet was purchased from ICN (Aurora, OH) and was the pelleted
variety of the modified vitamin A–deficient diet (Table 1
). Animal procedures were reviewed and approved by the Iowa State
University Institutional Animal Care and Use Committee.
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The rats were assigned to one of three dietary groups. After 4 d of acclimation to the vitamin A–free diet and the supplementation procedure, the rats were orally supplemented daily with retinyl acetate dissolved in 100 µL of corn oil: group L (n = 15) received 35 nmol/d, group M (n = 16) received 70 nmol/d and group H (n = 16) received 350 nmol/d. After supplementation for 17 d, to obtain baseline measurements, three rats from each group were killed by exsanguination while under ether anesthesia. The remaining rats each received via displacement pipette a single oral dose of 52 nmol of 13C4-retinyl acetate dissolved in corn oil. No additional supplemental vitamin A was fed to the rats after the 13C4-retinyl acetate was administered. Three rats from each group were killed on d 1, 2 and 4. Finally, on d 10, the remaining rats from group L (n = 3), group M (n = 4) and group H (n = 4) were killed. After they were killed, the serum was prepared and stored along with the whole liver at -70°C until analysis with HPLC and GC-C-IRMS as described later.
Preparation of serum samples for GC-C-IRMS.
Serum samples (0.75–1.5 mL) were treated with an equal volume of
ethanol to denature proteins. Retinyl acetate was used as an internal
standard to determine extraction efficiencies. Three hexane extractions
were performed and pooled, evaporated completely dry and redissolved in
100 µL of methanol. The sample was then cooled at -70°C for 10
min to precipitate unwanted lipids. After brief centrifugation, the
supernatant was drawn into a syringe and injected into the first HPLC
system. The system consisted of a 15-cm, 5-µm C-18 HPLC column with
90:10 methanol/water pumped at 1 mL/min by a Waters 510 HPLC pump
(Milford, MA). After passage through an ISCO V-4 detector (Lincoln, NE)
set at 325 nm, the retinol peak was collected and dried under argon.
The residue was redissolved in 100 µL of methanol and reinjected into
a second HPLC system. The second purification was performed on a 30-cm,
5-µm, C-18 HPLC column with 98:2 methanol/water flowing at 0.8 mL/min
using a second Waters 510 HPLC pump and ISCO V-4 detector. Both HPLC
systems were connected to a dual-channel Shimadzu integrator
(Kyoto, Japan). The first HPLC system was washed after every second
injection with 50:50 methanol/methylene dichloride. The doubly purified
retinol was collected in a glass conical screw-top tube and stored
overnight at -70°C in 100 µL of methanol to which 0.1% butylated
hydroxytoluene had been added. The next morning, the retinol solution
was vacuum dried and redissolved in 5 µL of hexanes for injection
onto the GC-C-IRMS system. Dependent on the amount of retinol present,
1.5–3 µL was directly injected onto the GC column of a 5890A
Hewlett-Packard GC device (Wilmington, DE) fitted with a Fisons VG
Isotech Isochrom GC-C interface to the Fisons/VG Isotech Optima
IRMS device (Manchester, U.K.). The isotope ratio of each sample was
determined by comparison with a working standard of CO2
gas.
Preparation of liver samples.
The liver analysis was performed on two separate occasions. The liver contained little contamination from blood because the blood was collected from the pumping heart with a Vacutainer, which is able to drain the liver. First, the samples were extracted without internal standard to avoid 12C interference in the GC-C-IRMS analysis of the retinol after saponification. Liver (1 g) was blotted and extracted with 50 mL of methylene dichloride after being ground with anhydrous sodium sulfate. An aliquot (2 mL) was dried and redissolved in 200 µL, of which 40 µL was injected onto a 30-cm, C-18 Waters Resolve column for retinyl ester determination. A separate aliquot was dried and saponified with alcoholic potassium hydroxide at 45°C. The retinol was then extracted and purified in the same manner as the serum for analysis with GC-C-IRMS. A separate aliquot of liver (0.3–0.5 g) was analyzed at a later date using retinyl acetate as an internal standard. Although hydrolysis of the retinyl esters had occurred during long-term storage of the liver samples, the total retinol content was fairly constant. The results reported include the retinol and all identifiable retinyl ester peaks for measured values.
Determination of measured and calculated values of TBR of vitamin A.
The measured TBR were determined by multiplying the retinol equivalents
of the liver (nmol/g) by the weight of the whole liver and then
dividing by either 50% (for group L) or 80% (for groups M and H),
depending on the assigned dietary group and how much vitamin A is
anticipated to be stored in the liver. The following mass balance
equation (eq. 1
; Goodman and Brenna 1992
) was used to
calculate TBR from the enrichment of the retinol in the serum with
13C:
 | (1) |
where a is the amount of dose absorbed and stored
(determined experimentally to be 0.5 of that administered,
Bausch and Rietz 1977
, Hughes et al. 1976
), and b represents the baseline reserves of
vitamin A.
 |
 |
and
 |
The Fa for the dose is 0.200 because 4 of the 20 C atoms are labeled with 13C. Fb is determined before dosing using the appropriate dietary group at time 0, and Fc is determined after dosing with 13C4-retinyl acetate.
Statistical analysis.
Data are presented as means ± SD. Linear regression was used to compare measured and calculated values both within the groups and between the groups. The effect of the dietary group was examined by using one-way ANOVA. The interaction of dietary group and day was determined by two-way ANOVA tables using SAS Version 8 (SAS Institute, Cary, NC).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). As expected, the group with the highest liver reserve (group H) had a
much higher serum-to-liver ratio at the outset of the experiment
compared with groups L and M (P < 0.01). This
approached 1.0 as vitamin A was withdrawn from the diet and serum
enrichment equilibrated with liver vitamin A stores in all groups
studied. The difference between the groups lasted until d 4, when the
differences were no longer apparent (P = 0.26).
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illustrates the change in atom percent excess
[(Fpostdose -
Fb) x 100] with time in the three
groups. At all times studied, there was a distinct, significant
difference among the dietary groups (P < 0.0001). The
physiological dose reached equilibrium between 4 and 10 d.
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shows the relationship between the calculated and measured TBR of
vitamin A of the rats at equilibrium (n = 11). The
relationship across the dietary groups was not different from unity,
with a slope of 1.0 and a correlation coefficient of 0.98 (P
< 0.0001). However, the relationship within each dietary group
was not significant (P = 0.67). The calculated mean TBR
were 0.49 ± 0.03, 0.82 ± 0.007 and 3.72 ± 0.40 µmol
for groups L, M and H, respectively. In comparison, the measured mean
TBR values were 0.50 ± 0.045, 0.69 ± 0.10 and 3.6 ± 0.29 µmol, respectively. Overall, the calculated and measured
estimations were within 7% of each other, but this differed by dietary
group. That is, the difference between measured and calculated TBR was
1.7 ± 2.8% for group L, where the assumption was made that 50%
of TBR were stored in the liver. The differences between the measured
and calculated values were -17 ± 17% and -3.2 ± 12% for
groups M and H, respectively, where the assumption was made that 80%
of the vitamin A was stored in the liver in the measured values.
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. At the onset of the experiment, rats in dietary group L had the
highest serum retinol even though they had the lowest TBR of retinol,
but this changed at 4 d after supplementation ceased. Groups M and
H exhibited similar changes in serum retinol concentration even though
there was a fivefold difference in their TBR of vitamin A. The
interaction between group and day tended to be significant
(P = 0.074).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In both the calculated and measured TBR of vitamin A, there are
necessary or practical assumptions. The major assumption in the
calculated value is the percentage of the dose that is absorbed and
stored. The measured and estimated values that have been reported in
the literature are 50–75% (Bausch and Reitz 1977
,
Kelley and Green 1998
). In these calculations, 50% was
used based on the following argument. Because we know the dietary
intake of the rats during the experiment, we can estimate the
absorption coefficient in these animals to test our assumption of 50%.
Although we did not measure the TBR of the rats at the beginning of the
experiment before supplementation began, Gardner and Ross (1993) measured the liver reserves of rats during a normal
growth cycle. By applying their data to the present experiment, we can
estimate the TBR of the rats at the onset of the experiment. The mean
weight was 57.5 ± 4.0 g in this experiment, and therefore
the estimated liver weight is 3.2 g (extrapolated from
Gardner and Ross 1993). By using the liver retinol value
that they obtained at age 18 d (60 nmol/g) and assuming that it
is 80% of TBR, we estimated the initial TBR of our rats to be 0.24
µmol. The amount of vitamin A that the rats consumed was 0.59, 1.2
and 5.9 µmol during the supplementation period. If we take the final
mean calculated values (i.e., 0.49 ± 0.03, 0.82 ± 0.007 and
3.72 ± 0.40 µmol), subtract the extrapolated initial TBR of
0.24 µmol and divide by the total amount of vitamin A that was
administered during the experiment, the values are 42, 48 and 59% for
groups L, M and H, respectively. Moreover, by assuming that 50% of the
administered amount of vitamin A during the experiment was absorbed and
stored daily (i.e., 0.30, 0.60 and 3.0 µmol) and by adding the 0.24
µmol that was present at enrollment, we estimated the TBR of the rats
to be 0.54, 0.84 and 3.24 µmol, respectively. Thus, the calculated
values agree very well with the estimated TBR of the rats on the basis
of 50% daily absorption and storage of the administered doses and
initial TBR at enrollment. That is, the difference between the
estimated and calculated values is 9.5, 2.4 and 13.8% (using the mean
of the estimated and calculated for comparison). Had we estimated that
75% of the isotopic dose was absorbed and stored, the calculated
values would have been much larger than the measured values and
unrealistic compared with the total amount of retinol that was
administered during the experiment. That is, the mean calculated values
for the groups would have been 0.74, 1.23 and 5.6 µmol, which is 125,
103 and 95% of the total vitamin A administered during the experiment.
This would mean that all of the retinol administered during the
experiment was retained, and this certainly is not the case.
The measured vitamin A TBR (0.50 ± 0.045, 0.69 ± 0.10 and 3.6 ± 0.29 µmol) are 85, 58 and 60% of the total vitamin A administered. The measured values differ from the estimated TBR by 7.3, 19 and 11% for groups L, M and H, respectively. The measured values are reasonable considering that an estimation was made that 50, 80 and 80% of the total vitamin A was in the liver in groups L, M and H, respectively, for the measurement of TBR. The amount of vitamin A stored in the livers of the rats in this experiment most likely ranges between 50 and 90%. Although 50% is reasonable for group L, it is not a reasonable assumption for groups M and H, where greater storage of the daily doses likely occurred. We chose 80% for groups M and H, which is probably intermediate between the actual values. For group M, the calculated values from the 13C-retinol isotope dilution test probably better reflect the actual TBR than the measured TBR. In practice, the values calculated from the 13C/12C at equilibrium of the dose may better reflect TBR than the measured because of the assumptions necessary in the measured liver values themselves.
The third assumption necessary in isotope dilution testing concerns how
the isotope ratio of vitamin A in the serum relates to the isotope
ratio in the liver. This value has been shown to be 0.65–0.80 in
previous studies in both rats (Hicks et al. 1984) and
humans (Haskell et al. 1997
). After allowing 10 d
for equilibration in this study, the serum-to-liver ratio was 1.01
± 0.04. This value shows that the isotopic dose was equally
distributed between the liver and serum pool. Thus, we did not need to
correct the calculated TBR for any discrepancy in this dilution. This
value was 1.0 for two reasons: 1) the rats consumed a
vitamin A–free diet for the duration of the study after dosing with
13C4-retinol, and therefore
dietary retinol did not continue to dilute the plasma pool, and
2) the dose of
13C4-retinol was
physiological, and therefore a large amount of the label was not stored
in the liver at the time of dosing. Even with the assumptions that
needed to be made in the calculated and measured values, the
relationship was indeed linear (Fig. 2)
. Using linear regression, the
slope of the line was 1.01 and the correlation coefficient was 0.98
(P < 0.0001). This included rats (n = 11) from the three dietary groups after the isotopic dose had reached
equilibrium (d 10) with the vitamin A stored in the liver. The
appropriate human studies will have to be performed to determine how
these values may differ in individuals, i.e., the serum-to-liver
isotope ratio and calculated-to-measured values.
The dietary strategy used in the present experiment was to maintain
serum retinol concentrations but to limit the amount of retinol stored
in the liver in at least one dietary group. In that regard, there was
no difference overall in the serum retinol concentrations of the groups
even though there was a 2- to 10-fold difference in dietary retinol.
The 13C4-retinyl acetate
isotope dilution assay was very good in determining the TBR of vitamin
A of the rats over a very broad range in this controlled experiment.
For comparison, the MRDR test most likely would not have been able to
distinguish a difference in the vitamin A status of these three groups
of rats even though there was a broad difference in their TBR of
retinol. The "breakpoint" for the MRDR test to react positively in
rats is 25 nmol/g of liver (Tanumihardjo et al. 1988
and 1990
), and all of the rats in this study had a
greater value.
A disadvantage of this 13C method is that it
requires extensive sample preparation. The retinol must be carefully
purified before injection into the GC-C-IRMS to provide consistent
results. Extreme care was taken in the sample preparation before
introduction into the gas chromatograph, which included a lipid
precipitation and two HPLC purifications. Although we did not
investigate the use of solid phase extraction techniques, others have
had good results with conventional GC-MS analysis of retinol
(Dueker et al. 1993
). This may be one way to decrease
the time needed for purification or to allow the use in laboratories
that do not have access to two HPLC systems.
The 13C-retinol isotope dilution assay holds considerable promise in estimating the TBR of human population groups. We are currently developing this method for use in humans to accurately assess vitamin A status.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Supported by U.S. Department of Agriculture/NRICGP Grant 97-35200-4290. The author conducted the experimental work while working as a scientist in the laboratory of James A. Olson at Iowa State University.
4 Abbreviations used: DRD, deuterated retinol dilution; H, high vitamin A; L, low vitamin A; M, moderate viatmin A;
MRDR, modified relative dose-response; RDR, relative dose response; TBR, total body reserves.
Manuscript received March 29, 2000. Initial review completed May 11, 2000. Revision accepted August 3, 2000.
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 S. A. Tanumihardjo Assessing Vitamin A Status: Past, Present and Future J. Nutr., January 1, 2004; 134(1): 290S - 293. [Abstract] [Full Text] [PDF]
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M. van Lieshout, C. E West, and R. B van Breemen Isotopic tracer techniques for studying the bioavailability and bioefficacy of dietary carotenoids, particularly {beta}-carotene, in humans: a review Am. J. Clinical Nutrition, January 1, 2003; 77(1): 12 - 28. [Abstract] [Full Text] [PDF]
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S. A. Tanumihardjo Vitamin A and Iron Status Are Improved by Vitamin A and Iron Supplementation in Pregnant Indonesian Women J. Nutr., July 1, 2002; 132(7): 1909 - 1912. [Abstract] [Full Text] [PDF]
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