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Craft Technologies, Inc., Wilson, NC 27893
3To whom correspondence should be addressed. E-mail: ncraft{at}crafttechnologies.com
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMeasurement of VA indicators...A rapid VA field...REFERENCES |
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KEY WORDS: • vitamin A • assessment • dried blood spot • retinol binding protein • fluorometry • enzyme immunological assay • high performance liquid chromatography
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMeasurement of VA indicators...A rapid VA field...REFERENCES |
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Vitamin A (VA) is best known for its role in the vision cycle. Retinal
11-cis in the eye binds to the protein, opsin, to form rhodopsin
(Rando 1995
). After exposure to light, 11-cis retinal
isomerizes to trans-retinal, releasing opsin. The release of
opsin initiates an impulse along the optic nerve to send visual images
to the brain. When VA is lacking, night blindness results. Another
critical ocular function of VA is its necessity in cellular
differentiation. Severe, prolonged VAD results in keratinization of
many tissues, including the lens of the eye. This condition is known as
xerophthalmia, literally meaning dry eye, and can result in blindness.
VA in the blood is transported in association with retinol binding
protein (RBP) and transthyrethrin in approximately a 1:1:1
M ratio (Peterson 1971
). RBP in circulation
with VA is called holo-RBP and the portion without VA is called
apo-RBP. Apo-RBP is synthesized in the liver, and in individuals of
normal VA status, is not released in significant quantities unless VA
is available to form holo-RBP. In the blood of well-nourished
individuals, holo-RBP constitutes 
85% of total RBP. Although a
small amount of VA is present in lipoproteins as retinyl esters,
85–90% of the VA transported in the blood is bound to RBP
(Krasinski et al. 1989
).
Although there are noninvasive indicators of clinical VAD, such as
keratomalacia and Bitot’s spots, efforts are ongoing to identify
minimally invasive indicators of subclinical VAD. Table 1
lists the cutoff levels of common biological indicators of subclinical
VAD (World Health Organization 1994
). The most common
indicator of subclinical VAD is the measurement of serum retinol.
Because serum retinol is known to be depressed in response to infection
and inflammation, some researchers question the use of this indicator
alone (World Health Organization 1994
).
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).
How large is the problem that motivates the development of innovative
and minimally invasive techniques to measure VA status? It is estimated
that 2.8 million children age 4 y and under have clinical signs of
VAD (World Health Organization 1995
). This is just the
tip of the iceberg because 251 million children in > 60 countries
suffer from moderate to severe subclinical VAD. Subclinical VAD is
associated with increased risk of morbidity and mortality in children
and pregnant women (West et al. 1995
).
Thus, simple rapid inexpensive noninvasive methods of determining VA status would be beneficial to determine the location and the prevalence of VAD, in addition to monitoring the effects of programmatic interventions. One major improvement in methodology would be the use of a sample other than serum/plasma. The current collection process typically involves venipuncture. This process is poorly accepted by subjects who are having blood drawn, and there is risk of disease transmission to both the subject and the phlebotomist. Additionally, the blood must be centrifuged and the serum frozen until it is analyzed. Ideally, the test for VA status should require an easily obtainable sample that could be measured immediately, thereby eliminating the need for sample transportation, storage and laboratory analysis. We have taken two approaches to achieve this goal. First, we have developed tests to measure indicators of VA status in dried blood spots (DBS). This sample method is less invasive than venous blood collection, requires minimal preparation and may not require refrigerated storage. Second, we are developing a portable test that permits the determination of VA concentration from a drop of blood at the time of collection.
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Measurement of VA indicators in DBS |
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TOPABSTRACTINTRODUCTIONMeasurement of VA indicators...A rapid VA field...REFERENCES |
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DBS have been used for decades as a sample matrix to measure several
analytes in newborn infants (Garrick et al. 1973
,
Vladutiu et al. 1980
, Mizejewski et al. 1982
, McCabe et al. 1987
). Until recently, it
was not deemed possible to measure VA in DBS due to its instability.
However, this assumption was dismissed when Shi et al.
(1995
) demonstrated that holo-RBP could be measured
in DBS using capillary electrophoresis with laser-excited
fluorescence detection and that it correlated with serum retinol.
Recognizing that the VA was protected in the DBS while associated with
RBP meant that other, less sophisticated, techniques could also be used
if adequate sensitivity was present. Very recently, Craft et al.
(2000a
) reported the development of a high performance
liquid chromatography (HPLC) method to measure retinol in DBS.
Figure 1
illustrates the chromatographic separation of retinol extracted from
DBS. The correlation observed between retinol measured by HPLC in serum
and that in DBS of 17 healthy adult males is illustrated in
Figure 2
. Initially, 50-µL aliquots of blood were used to provide a known DBS
volume and a sample of adequate size to achieve the necessary
instrument sensitivity. Because this step would not be convenient
during field collection, later one-fourth-inch center punches from
spots of unknown volume were used, as is done with other DBS tests
(Orfanos et al. 1978
, Kirby et al. 1981
).
We determined that the measured concentration of retinol in a fixed
unknown volume of blood could be multiplied by a factor to convert it
to a value equivalent to serum retinol (Craft et al. 2000a
). To date, this factor has been determined by
measuring a subset of serum samples for which we have matching
DBS. The serum retinol concentrations are divided by the DBS retinol
concentrations to arrive at adjustment factors. The median of these
factors is then applied to all DBS samples from this population. This
factor adjusts for sample volume, extraction efficiency and storage
effects. The volume of the serum component in a typical one-fourth-inch
punch from a DBS has been determined to be 6.6 µL (O’Broin and Gunter 1999
). If extraction efficiency and storage effects
are established, this volume could be used in the calculation of DBS
retinol rather than a subset of matching serums.
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).
Figure 3
illustrates the correlation between venous serum retinol and capillary
DBS retinol. Although our intent was to work with subjects that were in
the low retinol range, the retinol levels of these subjects were all
adequate due to fortification of Guatemalan sugar with VA. Within
healthy normal volunteers the correlations
(r2) between plasma retinol and
DBS retinol ranged from 0.88 to 0.90.
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) with a sensitivity of 89% and a specificity of 98%. The second
subset was from 54 children in Nepal (Table 3
) with a sensitivity of 93% and specificity of 90%. A third subset of
samples was analyzed from mothers and children in Liberia (Table 4
) with a sensitivity of 73% and specificity of 100%. Projects
involving DBS retinol are currently underway in India, Laos, Mexico,
Nicaragua, Tanzania and Uganda.
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Another approach to measuring VA status in DBS has been to measure RBP
as a surrogate for VA. Although test kits have been available for many
years to measure RBP in serum and urine, a commercial test has not been
validated to test RBP in DBS. The Program for Appropriate Technologies
in Health has been developing an enzyme immunological assay (EIA) for
RBP in serum and DBS. EIA have been developed for scores of analytes to
which an antibody can be raised. In this situation, the antibody is to
RBP, which serves as a transporter of VA, rather than to the vitamin
itself. The tests are relatively simple, requiring only a small sample
due to the highly colored indicators that form when
antigen-antibody binding occurs. Although there is some question
regarding the stability of the retinol in DBS under various conditions,
proteins may remain immunologically active in DBS for long periods
(Mizejewski et al. 1982
). Although RBP might possibly
serve as a more robust marker of VA status in serum, our experience is
that it responds similarly to retinol during DBS storage (Erhart, J.,
Craft, N., personal communication).
The EIA kit under development includes three calibrants covering the
deficient to normal range for RBP. After elution of the DBS sample, the
assay involves incubation of the RBP with anti-RBP followed by a
single wash step. The incubation takes 30 min to perform.
Correlations (r2) between serum RBP and
retinol have been reported to range from 0.4 to 0.8 (Burri and Kutnink 1989
, Parviainen and Ylitalo 1983
).
However, reports are unavailable for correlations between DBS RBP and
serum retinol.
One major concern expressed regarding the use of DBS has been the potential variability among samples. Can samples be collected reproducibly and are serum volumes in a punch similar among subjects? Both the Program for Appropriate Technologies in Health and the Centers for Disease Control and Prevention have demonstrated that the adsorption process of the DBS collection cards adjusts for most of the difference between blood from anemic and normal subjects. Plasma was mixed with red blood cells to provide hematocrits ranging from 20 to 50%. The reconstituted blood was spotted on DBS cards and dried. A center punch was used to measure RBP by EIA. Although the hematocrits varied 2.5-fold, RBP measured in DBS prepared from these samples varied by < 5% (Tam, M., personal communication).
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A rapid VA field test |
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TOPABSTRACTINTRODUCTIONMeasurement of VA indicators...A rapid VA field...REFERENCES |
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The methodology is based on the enhanced fluorescence of VA when it is
bound to RBP. This is the basis of the Futterman fluorometric assay in
which serum is diluted with saline and the fluorescence of holo-RBP
is measured directly (Futterman et al. 1975
). The
weaknesses of the Futterman assay are: 1) it requires
processing of blood to obtain a serum sample, 2) hemolysis
and dietary components interfere with the fluorescence, and
3) it is performed using a laboratory fluorometer
(Marinovic et al. 1997
). Craft Technologies (Wilson, NC)
has modified a small commercial fluorometer (Hoefer Pharmacia Biotech,
San Francisco, CA) to provide the appropriate excitation and emission
wavelengths to measure holo-RBP. In addition, the fluorometer was
modified to operate from a direct current power supply (battery,
automobile cigarette lighter or alternating current transformer). These
modifications provided a portable fluorometer optimized for VA
fluorescence making the Futterman measurement possible in the field.
Figure 4
illustrates the linear range of fluorescence of a serum sample diluted
well into the deficient range for VA. To eliminate the other weaknesses
of this assay, antibodies to RBP have been coated on the interior of
high surface area capillary tubes. The goal is to collect whole blood
samples directly into the capillaries from a finger prick. Using these
capillaries will not only eliminate the need for centrifugation, but
also the antibodies on the interior surface of the capillaries will
remove the RBP from potential interfering substances, such as
hemoglobin and phytofluene. After incubation to allow the anti-RBP
to bind to RBP in the sample, the blood is flushed from the capillary
and the fluorescence of holo-RBP is measured directly.
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In summary, there is a dire need for innovative minimally invasive approaches to measure micronutrient deficiencies. We have approached VA assessment from two angles: 1) a readily obtainable less-invasive more easily transportable sample and 2) a portable field test to obtain data at the time of collection. Although neither of these provides the perfect noninvasive solution to testing for VAD, both correlate well with serum retinol concentrations.
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FOOTNOTES |
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2 Supported by the Micronutrient Initiative,
International Development Research Centre, Ontario, Canada; Task Force
Sight and Life, Basel, Switzerland; and the Office of Health and
Nutrition, U.S. Agency for International Development, Washington,
D.C.
4 Abbreviations used: VAD, vitamin A deficiency;
VA, vitamin A; RBP, retinol binding protein; DBS, dried blood spot;
HPLC, high performance liquid chromatography; EIA, enzyme immunological
assay.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMeasurement of VA indicators...A rapid VA field...REFERENCES |
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19. World Health Organization Global Prevalence of Vitamin A Deficiency, WHO/NUT/953 1995 World Health Organization Geneva, Switzerland
20. World Health Organization Indicators for Assessing Vitamin A Deficiency and Their Application in Monitoring and Evaluating Intervention Programmes, WHO/NUT/941 1994 World Health Organization Geneva, Switzerland
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