24
OLSON MEMORIAL LECTURE – CARIG WORKSHOP 2011
James A Olson Memorial Lecture –
CARIG Workshop at Experimental Biology 2011:
Isotope Dilution Assessment
of Vitamin A Status
Harold C Furr
University of Wisconsin, Department of Nutritional
Sciences, Madison, Wisconsin 53706 USA
Methods for estimation of vitamin A status
Vitamin A deficiency is still a major public health problem in
many parts of the world. The clinical, social, and economic consequences of vitamin A deficiency are well known, and will not
be discussed here. But this problem creates a need for appropriate methods for estimating human vitamin A status. Hence
a number of methods exist for assessing vitamin A status, each
of which is useful in only certain ranges of vitamin A status.
(Figure 1) However, it is worth keeping in mind that, whenever
there are so many different methods for doing something, not
one of those methods is fully satisfactory – each of these methods has disadvantages.
It is generally accepted (and numerous studies have agreed)
that most of the body’s vitamin A is in the liver, especially in
the well-nourished subject (animal or human).1 Therefore determining vitamin A concentration in the liver is unequivocally the
best method of assessing vitamin A status. From considerations
of the estimated length of time for protection from vitamin A
deficiency, liver concentration at which plasma RBP is saturated
with retinol and catabolism of vitamin A, Olson2 defined vitamin A status in terms of liver vitamin A concentration, and suggested that liver vitamin A >0.07 μmol/g (20 μg/g) is adequate,
0.035 to 0.07 μmol/g (10 to 20 μg/g) is considered marginal,
and <0.035 μmol/g (10 μg/g) is considered deficient. But, in
practice, obtaining liver samples from human subjects is difficult
and can be readily achieved only by biopsy of subjects undergoing abdominal surgery or by autopsy after death.
Isotope dilution should provide a quantitative measure of
vitamin A status with minimal invasiveness. This article sum-
marizes some of the principles and applications of isotope dilution. The principle of isotope dilution is quite simple: addition
of a known quantity of tracer (which has some distinguishable
marker) to the unknown pool, followed by removal of a sample
and measurement of the concentration of tracer; simple calculation should provide the mass of the initial pool:
Concentration₁ x Volume₁ = Concentration₂ x Volume₂
It is assumed that plasma retinol mixes thoroughly with other
body pools of vitamin A (or at least with liver vitamin A pools),
so that measuring the ratio of tracer to tracee in plasma allows
estimation of liver vitamin A pool size. (Figure 2)
Terminology and techniques in isotope dilution
Tracer: Molecules of the substance of interest (vitamin A), which
incorporate a distinguishable characteristic but which should
be (if possible) biochemically indistinguishable from the unlabeled mass. Ideally, the mass of tracer should be very low, so
that it does not perturb vitamin A metabolism. Isotopic labels
are most used, but Burri and Jacobs3 used chlorinated analogs of
vitamin A. The modified relative dose response (MRDR) can be
thought of as an isotope-dilution technique using the vitamin A
analog 3,4-didehydroretinol, and the MRDR response shows the
hyperbolic relationship characteristic of dilution.4
⇢
SIGHT AND LIFE | VOL. 25 (2) | 2011
“ Determining vitamin A concentr ati on in the liver is unequivocally
the best method of assessing
vitamin A st atu s ”
Dr Harold C Furr
25
26
OLSON MEMORIAL LECTURE – CARIG WORKSHOP 2011
figure 1: The relationship of vitamin A status indicators to liver reserves of vitamin A. (CIC, conjunctival impression cytology; RAG,
retinoyl β-glucuronide; RBP:TTR, retinol binding protein to transthyretin molar ratio; RDR, relative dose response; MRDR, modified
relative dose response.) From Tanumihardjo.47
indicator
deficient
sub-clinical
adequate
sub-toxic
toxic
xerophthalmia
night blindness
dark adaptometry
cic
serum retinol
rag-hydrolysis
rbp:ttr
breast milk retinol
rdr
mrdr
isotope dilution
liver sample
Radioactive tracers use ³H or ¹⁴C incorporated into the vitamin A
molecule. Each has been used in animal models and in early human studies and usually employs liquid scintillation counting for
quantitation. Accelerator mass spectrometry allows the use of
minute amounts of ¹⁴C which pose no discernable risk to human
subjects,5 but is expensive to implement. Non-radioactive tracers use ²H or ¹³C labels and mass spectrometry to determine the
ratio of labeled to non-labeled vitamin A.6-8 Quadrupole mass
spectrometers have been used to measure ²H retinol; they are
relatively inexpensive and rugged instruments, but do not have
as much sensitivity as other techniques. Isotope-ratio combustion mass spectrometers provide high sensitivity for measuring
¹³C/¹²C ratios,6 thus allowing lower doses of tracer; but sample
preparation is more demanding.
Tracee: The molecule of interest (vitamin A) which is to be quantitated. Concentrations of vitamin A in plasma and liver can be
measured by conventional techniques (HPLC or fluorescence).
Mixing (“equilibration”): In early writings on isotope dilution
as a means of quantitating vitamin A pools, it was assumed that
“In the well-nourished individual,
some 80 to 90% of total body
vitamin A is in the liver”
the tracer would completely equilibrate with the tracee, and that
a certain period of time would be necessary for equilibration.
However, kinetic considerations show that true equilibration of
tracer with tracee can occur only if there is no dietary input of
vitamin A after the administration of the tracer, and this is usually not realistic in practice. Dietary input of vitamin A continuously dilutes the tracer, as both tracer and tracee are catabolized
and lost.
Pools (compartments): Vitamin A in the body (animal or human)
is present in a number of discrete compartments, distinguishable by tissue and even by cell type. It is difficult to distinguish
all the compartments by kinetic models, however. In the wellnourished individual, some 80 to 90% of total body vitamin A is
in the liver, so the usual validation of isotope dilution estimates
is by comparison with the vitamin A content of the liver.
Introduction of isotope dilution for
estimating vitamin A status
The first publication on the use of isotope dilution to assess vitamin A status was by Rietz et al9 at Hoffmann-La Roche, describing the use of radioactive vitamin A to estimate vitamin A stores
in rats. Subsequent publications10,11 expanded on the initial
experiments, and a paper by Bausch and Rietz12 summarized
a number of experiments, using both radioactive and stable
isotope-labeled tracers, in both humans and animals. Good correlation was obtained with direct analysis of vitamin A in liver in
the animal studies; in the human studies, a single subject, there
OLSON MEMORIAL LECTURE – CARIG WORKSHOP 2011
SIGHT AND LIFE | VOL. 25 (2) | 2011
figure 2: Simple dilution calculation, for one unit of tracer
added to a tracee pool of varying size. Note that the tracer/
tracee ratio gives highest precision at low tracee pool
size (most important for detecting deficient and marginal
vitamin A status).
0.012
0.01
Tracer/tracee ratio
0.008
0.006
0.004
0.002
The first study in human subjects to directly compare measured
liver vitamin A content with the predictions of isotope dilution
was performed in the laboratory of Prof James Olson at Iowa
State University.16,17 Robert Bergen synthesized the labeled vitamin A; Andrew Clifford and Daniel Jones performed the mass
spectrometric analyses of D/H ratio at the University of California; Olivier Amedee-Manesme, a visiting pediatrician from
France, was the liaison with the local hospital where the study
was performed; and Dale Anderson was a surgeon in the local
hospital in Ames, Iowa, who obtained the liver biopsies. We
used a rather substantial oral dose of ²H-vitamin A, 45 mg for
each subject, to ensure that there would be sufficient tracer label
to measure with confidence. When we had the resulting data in
hand, I spent weeks trying to make sense of them; they did not
fit the Bausch and Rietz equation well. One Friday afternoon, Dr
Olson and I discussed the problem; he offered to take the data
home over the weekend. On Monday morning, he came in with
the “Olson equation”, which provided a good fit for almost all the
data: (Figure 3)
0
0
2000
4000
6000
8000
10.000
Tracee
was good agreement with total vitamin A as estimated by the
use of both radioactive and stable isotope labels.
Their equation contained an “experimental factor” (“0.5" in
the equations) for loss of tracer, attributed to inefficiency of absorption or catabolic loss; this experimental factor was not examined for dependence on time nor on vitamin A status, but was
taken to be a constant for either radioactive or stable isotope
tracer studies:12
For radioactive tracer:
0.5 * test dose (dpm)
Vitamin A liver stores =
Specific activity of vitamin A
in plasma
For stable isotope tracer, the vitamin A liver stores were
calculated by use of the ratio of tracer-to-tracee in plasma
retinol (as determined by mass spectrometry); their
calculation included a correction (“+ 1”) for the mass of
tracer dose administered.
For stable isotope tracer:
Vitamin A liver stores =
0.5* test dose* [(H-retinol/D-retinol) + 1]
⇢ A similar relationship was observed in rat studies by
Huque13,14 and by Cullum et al.15
Total liver reserves = F * dose * [S * a * [(H:D) - 1]]
where
F is a factor to express efficiency of storage (taken to be 0.5
from the work of Rietz)
H:D is the isotope ratio of non-deuterated (tracee) to
deuterated (tracer) retinol in plasma
S is the ratio of specific activity of retinol in plasma to
that in liver (taken as 0.65 from rat studies)
a = e-kt is the fraction of absorbed tracer dose remaining
in liver at time t after dosing, using the estimated rate of
catabolism of vitamin A obtained by Sauberlich et al18
(k = (ln 2) / 140 days = 0.00495 per d); this was assumed
to be constant, independent of the size of vitamin A stores.
⇢ This relationship was subsequently corroborated by
Haskell et al19 in Bangladeshi subjects; a linear relationship
between measured liver vitamin A content and total body
vitamin A stores as calculated by the Olson equation was
found. Haskell et al found the ratio of plasma to liver
specific activities to be 0.80, and they estimated mean
recovery of tracer in liver to be 0.378; multiplied together
in the Olson equation, the product of these factors is 0.30,
which was statistically not different from the product of
F x S (0.5 x 0.65 = 0.325) used originally by Olson.
27
27
28
OLSON MEMORIAL LECTURE – CARIG WORKSHOP 2011
figure 3: Relationship between calculated and measured
amounts of vitamin A (μmol /g wet weight) in the livers of 11
generally healthy human adults. The solid line is the line of
identity (1:1 correspondence). From Furr et al.16
1.2
Cord plasma retinol [μmol/l]
1.0
0.8
0.6
0.4
0.2
0
0
0.1
0.2
0.3
0.4
0.5
0.6
Measured liver vitamin A [μmol/g]
“The first study in human subjects
to directly compare measured
liver vitamin A content with the
predictions of isotope dilution was
performed in the laboratory of
Prof James Olson”
Applications of isotope dilution assessment
Ribaya-Mercado et al20 showed that the isotope dilution technique could indeed detect changes in total body stores of vitamin A in elderly subjects in Guatemala. Haskell et al21 found
that the isotope dilution technique could detect changes in total vitamin A body stores in male adult subjects in Bangladesh,
subsequent to long-term consumption of different amounts of
vitamin A. These two studies provided early corroboration of the
technique. The improvement in vitamin A status in Nicaraguan
schoolchildren after a sugar fortification program was evaluated
by Ribaya-Mercado et al.22
The kinetics of an oral dose of tracer vitamin A was examined
in preschool children in Peru.23 There was also an apparent correlation between serum tracer/tracee ratio at three days after
dosing with the total body stores, as estimated from the ratio at
longer times of mixing.
Conversion of carotenoids to vitamin A
and estimates of bioequivalency
Tang et al24,25 were the first to use isotope dilution to demonstrate that carotenoids from green and yellow vegetables can
maintain liver vitamin A stores, from a study in Chinese children.
Lin et al26 used double-label techniques (tracer retinol labeled
with ²H₆, and ²H₆-β-carotene which yielded ²H₃-retinol on cleavage) to verify that the extent of conversion of β-carotene to vitamin A is quite variable among human subjects, a finding corroborated by Wang et al.27 Ribaya-Mercado et al28 subsequently
found that the extent of conversion of provitamin A-carotenoids
to vitamin A varies inversely with vitamin A stores as determined by isotope-dilution, but is independent of serum retinol
concentration. Tang et al29 used the double-label approach to
show that β-carotene can be cleaved to vitamin A in tissues after
absorption. And Ribaya-Mercado et al30 showed an increase in
vitamin A pools in Filipino children after dietary consumption of
carotene-rich foods.
Haskell et al31 determined the bioequivalence of β-carotene
from sweet potato (13:1) and Indian spinach (10:1), compared
with that from isolated β-carotene in capsules (6:1). You et al32
did not estimate vitamin A status, but did use labeled retinol
to estimate the intestinal absorption and conversion of an oral
dose of carotenoids from red palm oil. Wang et al33 measured
a conversion factor of 4.5 for spirulina β-carotene in Chinese
adults. The vitamin A equivalency of labeled β-carotene in capsules was determined to be approximately 3.36, whether administered in an oil matrix or a vegetable diet, but the absorption
efficiency of β-carotene was much greater from the oil than from
the vegetables.34
Estimation of human dietary requirements of vitamin A
The dietary intake of vitamin A (preformed plus provitamin Acarotenoids) was correlated with vitamin A stores by RibayaMercado et al35 in an elderly Filipino population. They calculated that a daily intake of 400 μg RAE for women, or 500 μg
RAE for men, is sufficient to maintain adequate vitamin A stores.
(This may be compared with estimates of 567 μg RAE for women,
707 μg RAE for men, calculated by Olson from other considerations).36 Recently, Haskell et al37 used isotope-dilution estimates of vitamin A pool size to calculate an estimated average
requirement of approximately 350 μg RAE per day to maintain a
liver pool of 0.047 μmol/g for Bangladeshi men of small stature;
maintaining a higher vitamin A pool would, of course, require
a greater intake. Clearly, this approach is useful for estimating
Recommended Dietary Intakes for vitamin A.
SIGHT AND LIFE | VOL. 25 (2) | 2011
Alternative isotope dilution calculations
A practical problem with the Olson equation is the implied
need for a lengthy mixing period between administration of
tracer and collection of a blood sample for determination of
isotope ratio. From the human studies by Bausch and Rietz12
and Sauberlich et al,18 it was concluded that 21 days were necessary for complete mixing. However, tracer:tracee ratios at
three and six days after dosing were shown by Ribaya-Mercado
et al to correlate well to those at 21 days.20 Three-day predictive equations were determined by Tang et al,38 Haskell et al23
and Ribaya-Mercado et al.39 Although these equations differ in
form, happily they give similar results.
Duncan et al40 developed empirical two-component exponential regression equations to correlate rat liver vitamin A
stores with plasma isotope dilution data for 4, 4.4, 5 and 5.4
days for rats given tracer intravenously. Similarly, Adams and
Green41 developed empirical biexponential regression equations to correlate rat liver vitamin A stores with plasma isotope
dilution data at 3 days, 4, 4.4, 5, 5.4, and 6 days after oral administration of tracer dose. An extension of this approach for
application to humans has been developed (Duca and Green,
personal communication).
OLSON MEMORIAL LECTURE – CARIG WORKSHOP 2011
Reviews
Among the generally available reviews of isotope dilution for assessment of vitamin A status are those of Wasantwisut,42 Tanumihardjo et al,43 Furr et al,44 Haskell et al45 and Furr et al.46
Acknowledgments
The author happily acknowledges the support of Prof James
Olson in helping understand and implement the application of
isotope dilution for assessment of human vitamin A status, and
is also grateful for collaborations with many colleagues.
Correspondence: Harold C Furr, 115 West Rainbow Drive,
Bridgewater, VA 22812-1735 USA E-mail: [email protected]
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The kinetics of vitamin A metabolism in children has been examined in only one study, among 12–24 month children.23 So
far, the Olson equation as determined for adults has been used
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are essential for developing suitable prediction equations to allow the extension of this technique to children.
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