Human biomarkers of vitamin A status
This section is focused on biochemical biomarkers of vitamin A status. Information on dark adaptation testing and nightblindness assessment, which can be used to assess VAD in a population is available elsewhere (15, 16) (for more information, see the WHO VMNIS brief on Xerophthalmia and night blindness for the assessment of clinical vitamin A defciency in individuals and populations).
Also, ecological and demographic indicators can be useful for VAD in populations, such as socioeconomic status, food insecurity, infectious disease rates, infant mortality rate, access to safe drinking water, etc. can be useful for assessing VAD in populations (5, 17). Further, coverage rates of vitamin A intervention programmes such as food fortification and high-dose capsule distribution, can indicate uptake or success of interventions, and thereby the ongoing risk of deficiency. Recent reviews have summarised the various methods of human vitamin A status assessment (5, 14).
The WHO recommends using serum retinol concentration to assess vitamin A status of a population (5, 17).
Serum retinol concentration
Serum retinol has limitations as an indicator of VAD because it is homeostatically controlled, and decreases transiently during infection or inflammation. Nevertheless, the distribution of serum retinol concentrations within a population can be useful for assessing VAD (18).
It is important to measure biomarkers of inflammation (serum concentrations of C-reactive protein, α1-acid glycoprotein) to interpret serum retinol data. Regression analysis can be used to adjust serum retinol concentrations in populations with high rates of inflammation (19). Serum retinol cut-off values of <0.7 µmol/L and <0.35 µmol/L are used for moderate and severe deficiency, respectively, in children (20, 21). A serum retinol cutoff of <1.05 µmol/L is commonly used for moderate deficiency in adults (5). Serum retinol binding protein (RBP) concentration may be used as a proxy for serum retinol concentration.
The WHO recommends using at least two biological indicators of vitamin A status for assessing the public health significance or VAD, or one biological indicator in combination with at least four ecological and/or demographic population risk factors for VAD, such as infant mortality rate, under 5 mortality rate, immunisation coverage rates, dietary VA intake, etc. (17)
For more information on WHO recommendations, see Indicators for assessing vitamin A deficiency and their application in monitoring and evaluating intervention programmes.
Serum retinol can be measured by high-performance liquid chromatography (HPLC) (22), or by fluorometry using a portable device (e.g. the iCheck Fluoro device).
Serum retinol and carotenoids (and vitamin E) can be measured simultaneously by HPLC: see SOP from CDC.
Relative dose response tests
The modified relative dose response (MRDR) test is used to assess the adequacy of liver vitamin A stores (5). Briefly, a small oral dose of vitamin A2 (3,4 didehydroretinol) is administered to an individual and a blood sample is obtained 4-7 hours later for measurement of the serum concentrations of vitamin A2and vitamin A1(retinol) by HPLC (5, 23). The MRDR ratio is calculated as (plasma vitamin A2 concentration/vitamin A1 concentration). An MRDR ratio of ≥0.06 is used as the cutoff value to indicate inadequate liver vitamin A stores.
The relative dose response (RDR) test is similar, but two blood samples are required, and a test dose of vitamin A1(retinol) is used instead of vitamin A2. Briefly, after obtaining a fasting, baseline blood sample, a small oral dose of vitamin A1 is administered to an individual, and a second blood sample is obtained 5 hours later. The serum retinol concentration is measured by HPLC in both the baseline and 5-hour blood samples. The relative dose response is calculated as: [(plasma retinol concentration at 5 h – plasma retinol concentration at 0 h)/(plasma retinol concentration at 5 h)] x 100. An RDR of ≥20% is used as the cut-off value to indicate inadequate liver vitamin A stores (17).
Note that the MRDR and RDR tests do not provide quantitative estimates of liver vitamin A stores, and only provide information on adequacy/inadequacy of stores.
Retinol binding protein (RBP) (and biomarkers of inflammation)
Serum RBP concentration can be used as a proxy for serum retinol concentration for assessing VAD in a population (5). Serum retinol and serum RBP concentrations are well correlated generally. However, body mass index, kidney function, and perhaps other factors such as age and physiological status, may affect the relationship between serum retinol and serum RBP (5). It may be preferable to measure both serum retinol and RBP concentrations in a subset of a population to determine the relationship between serum retinol and RBP concentrations, and to establish population-specific cut-offs for RBP (24).
Serum RBP can be measured by a sandwich ELISA assay (25), or a multiplex immunoarray assay (26).
Cut-off values for RBP of <14.7 μg/mL (26)or <0.7 μmol/L (25)have been proposed for inadequate vitamin A status, although population specific cut-offs may be preferable, as mentioned above.
The sandwich ELISA and multiplex immunoarray assays that are used for measurement of RBP can be used to measure multiple serum proteins simultaneously, including biomarkers of inflammation (C-reactive protein, α1-acid glycoprotein) (see links above).
Retinol isotope dilution technique
The retinol isotope dilution (RID) technique is the only assessment method that provides a quantitative estimate of total body stores of vitamin A (14).
The RID technique is based on the principle of isotope dilution. Briefly, stable isotope-labelled vitamin A (13C-retinyl acetate or 2H-retinyl acetate) is administered orally, and total body stores of vitamin A are estimated based on the plasma ratio of isotopically-labelled retinol to non-labelled retinol, which is measured at a single time point or multiple time points depending the method used, and an appropriate prediction equation (14, 27). The equations for estimating total body stores of vitamin A are evolving, and simplified equations have been developed recently that are based on compartmental modelling of plasma retinol kinetic data (28, 29). Recent reviews provide detailed information on the equations that have been used to estimate total body vitamin A stores using the RID method (14, 30). Total body stores of vitamin A can be converted to liver vitamin A concentrations using assumptions regarding liver weight as a percentage of body weight, and the percentage of total body vitamin A that is stored in the liver (5). Because the RID method provides a quantitative estimate of liver vitamin A concentration, it could potentially be used to assess vitamin A status across the continuum of deficient to excessive liver vitamin A stores. However, firm cut-off values have not yet been established for deficient or excessive vitamin A status based on estimated liver vitamin A concentrations. However, proposed cut-off values are available elsewhere (5, 27). The current capabilities and limitations of the RID technique are discussed in a recent review (14). Additional details on the application of compartmental modelling of plasma retinol kinetic data to develop equations for estimating total body vitamin A stores can be found in recent publications (28, 29, 31).
Mass spectrometry (LC/MS/MS (32), GC/isotope ratio/MS (33)) is used to measure stable isotope labelled vitamin A in plasma or serum. The mass spectrometry methods require specialised lab equipment and appropriately-trained personnel. Consultation with experts is recommended to discuss issues related to dosing, sensitivity of the analytical method, and timing and number of blood samples to be collected.
Breast milk retinol
Breast milk retinol concentration provides an assessment of both maternal and infant vitamin A status. Breast milk retinol concentration reflects maternal vitamin A intake and status, and provides information on the risk of inadequate vitamin A intake in predominantly or exclusively breastfed infants (34). Vitamin A is present in the fat portion of the milk, and milk fat varies within a feeding episode (lowest in foremilk, highest in hind milk) and throughout the day, therefore milk samples should be collected and processed carefully (34).
Two breast milk collection protocols are commonly used to assess the vitamin A concentration (34). Full milk samples are collected at a standardized time of day (usually mid-morning) by using a manual or electric breast pump to express all of the milk from a breast that has not been used to feed the child for at least one hour. Casual milk samples are collected 30 seconds after the child initiates breastfeeding by removing the child from the breast and hand-expressing a small amount of milk (5-10 mL) into a container (35). Milk fat can separate easily from the aqueous portion; therefore, milk samples must be well-mixed prior to aliquotting for measurement of the vitamin A concentration. Full milk samples are mixed thoroughly and a small sample (~3-5 mL) is aliquotted for analysis of the vitamin A content. The remaining milk is returned to the mother to feed to her baby with a spoon. Full milk samples are representative of the milk an infant consumes during a feeding episode. Similarly, casual milk samples are mixed thoroughly before being aliquotted for measurement of the vitamin A concentration (34).
Breast milk vitamin A concentration can be measured by HPLC (36, 37, 38), or by fluorometry using a portable device (39). The vitamin A content of full milk samples is expressed as μmol/L and/or as nmol/g milk fat. The vitamin A content of casual milk samples is expressed as nmol/g milk fat because retinol is found in milk fat, and the fat content of casual samples can be highly variable.
Milk fat can be measured by using the crematocrit method (40). A crematocrit centrifuge is commercially available (EKF Crematrocrit PlusTM). Cutoff values for low breast milk vitamin A concentrations are <1.05 μmol/L (full milk samples) or <28 nmol/g milk fat (full or casual milk samples).
Serum retinol on dried blood spots
Dried blood spots have been used to measure retinol concentration, although because results are inconsistent possibly due to poor extraction and/or instability of retinol, DBS are not commonly used. However, recent data suggest that retinol (41) and RBP (42) can be measured reliably from DBS in well-nourished populations. More data are needed to determine whether the DBS methods are reliable in populations with low serum retinol and RBP concentrations. DBS methods are advantageous in field studies because the samples do not require centrifugation or refrigeration, however, more information is needed on stability of DBS in various environmental conditions. Though DBS specimens offer many advantages, samples must be carefully prepared and stored. See the OpeN-Global page on Common Methods for advice and further information on DBS samples, the BOND review on vitamin A for vitamin A-specific information, and page 4 for quality control issues.
Serum retinyl esters
The fasting serum retinyl ester concentration can be used to assess excessive vitamin A status (5). Serum retinyl esters increase transiently after consumption of vitamin A, but are low in the fasted state. In contrast, in patients with hypervitaminosis A, fasting retinyl esters are elevated (43). It is essential to obtain fasting blood when using serum retinyl ester concentration as an indicator of excessive vitamin A status. The serum retinyl ester concentration can be measured by HPLC (44). A firm cut-off value for serum retinyl esters has not yet been established. However, a cut-off value of >10% of total serum vitamin A as retinyl esters has been proposed (5).
Note that it can be challenging to obtain fasting blood samples from young children in community settings.