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Nutrition Department, The Pennsylvania State University, University Park, PA;
*
The Program in International Nutrition and Department of Nutrition, University of California at Davis, Davis, CA; and
Johns Hopkins University School of Hygiene and Public Health, Departments of International Health and Molecular Microbiology and Immunology, Baltimore, MD, and The Papua New Guinea Institute of Medical Research, Maprik, Papua New Guinea
2To whom correspondence should be addressed. E-mail: fxr5{at}psu.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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0.36 selectively detected five of seven patients (71% sensitivity) with hepatic VA stores 69.9 nmol/g of tissue (i.e., 20 µg/g), indicative of marginal VA deficiency. Using this cut-off value, 28% (n = 245) of children from Papua New Guinea had marginal VA deficiency before supplementation. After 7 mo, a low ratio persisted in 29% (n = 92) of placebo-treated children but in only 11% (n = 83) of those receiving VA supplements (
2, P < 0.01). At the end of the study, 13 mo after initiation or 4 mo after the last dose of VA, the percentage of children with a low ratio was still lower (2, P < 0.02) in the VA group, 42.5% (n = 113) than in the placebo group, 58.6% (n = 118). These results demonstrate that a cut-off value 0.36 is indicative of marginal VA deficiency and can be used as an indirect method of VA assessment.
KEY WORDS: • humans • liver • malaria • vitamin A status
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). In addition, the survival of children and pregnant women improves with VA supplements (2
,3
). These findings underscore the importance of accurately establishing the VA status of those at risk and in need of VA supplements. There are several methodologies available to assess VA status of populations; however, their interpretation during infection/inflammation is not clear, and some are complex and expensive.
Previously, it was demonstrated in rats that the molar ratio of plasma retinol-binding protein to transthyretin (RBP:TTR) selectively diagnosed VA deficiency during inflammation (4
). Several biologic properties of the RBP:TTR ratio support its use as an indicator of VA status. The formation and secretion of holo-RBP into plasma requires adequate hepatic VA stores; otherwise, RBP is poorly secreted and accumulates in the liver (5
). Transthyretin is important in maintaining circulatory levels of holo-RBP. It forms a large transport complex, thus reducing the glomerular filtration of the smaller-sized holo-RBP (5
). Studies of the binding and dissociation capacities of these proteins showed that retinol’s selectivity for RBP might originate from its interaction with TTR, which enhances retinol’s uptake by peripheral tissues (6
,7
). Burri et al. (8
), using reverse-phase HPLC and molecular exclusion techniques, demonstrated that the retinol-RBP-TTR concentration was a better linear predictor of hepatic VA stores than unbound retinol, or retinol bound only to RBP.
Moreover, the structural and functional interactions between these proteins persist even during infection or malnutrition. Assessment of their mutual dependency, i.e., correlation coefficients (r) and determination coefficients (r2), showed that TTR explains 70–80% of the variance of RBP and vice versa in patients with hepatic disease, surgery or trauma, in children with malaria or measles infection or in malnourished children (9
–12
). Vahlquist et al. (13
) demonstrated that a constant ratio between these proteins is maintained by the formation of the complex, which reduces the metabolic rate of RBP and maintains the pool distribution of these proteins fairly equally.
Although the stoichiometry of these proteins is such that 1 mol of RBP binds 1 mol of TTR, the total molar concentration of plasma TTR exceeds that of RBP by 2.2–2.5 times (14
). In calculating the RBP:TTR ratio, the numerator represents the concentration of circulating RBP, 
85–90% of which is bound to the retinol-RBP-TTR complex (8
), and the denominator represents the concentration of circulating TTR, of which 45% is bound to the retinol-RBP-TTR complex (14
). This makes the ratio highly responsive to a reduction in circulating RBP secondary to lowering of hepatic VA stores. For all these reasons, we evaluated the sensitivity and specificity characteristics of the RBP:TTR ratio to determine a cut-off level indicative of marginal VA deficiency.
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SUBJECTS AND METHODS |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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These were 15 Bangladeshi patients who participated in a study of the assessment of VA status using hepatic VA stores (15
). The patients were 21–65 y old and were scheduled for elective abdominal surgery. They were recruited from two surgical wards at the Dhaka Medical College Hospital during a 10-mo period. Patients were eligible if they were free from infectious diseases, afebrile, and had no recent history of malabsorption, liver or kidney disease (15
). Written informed consent was obtained from subjects, and the Human Subjects Review Committee of the University of California-Davis and the Ethical Committee of the Bangladesh Medical Research Council approved all study procedures (15
). At surgery, a wedge biopsy of liver tissue (200 mg) was obtained from the inferior central portion of the right lobe by the attending surgeon. For determination of hepatic VA concentrations (i.e., including retinol and retinyl esters), a 25-mg liver sample was dehydrated with anhydrous sulfate, and total VA was determined by HPLC after saponification with KOH solution and extraction with hexanes (15
). Blood samples were collected between December, 1992 and September, 1993. Plasma samples were stored at -20°C and had been thawed and frozen 4–5 times before determinations of serum RBP and TTR in 1999. Plasma samples, 50–100 µL, were stored at -70°C upon their arrival at the Pennsylvania State University.
Reference values from healthy Canadian infants, children and adolescents.
Serum RBP and TTR concentrations were determined using a Behring LN Nephelometer (Behring Diagnostics, Montreal, Canada) in samples collected from clinically healthy individuals, and reference intervals were derived by nonparametric methods after determining that analyte concentrations differed significantly among age groups (16
).
Children from a malaria-endemic area.
This population consisted of a subset of children 6 mo to 6 y of age, who participated in a randomized, double-blind, placebo-controlled trial to assess the effect of VA supplementation on malaria-related morbidity (17
). The study was conducted between July, 1995, and August, 1996 in the North Wosera District, East Sepik Province, Papua New Guinea. After enrollment and randomization to VA or placebo treatment, children received their respective treatments (i.e., 210 µmol of VA as retinyl palmitate, or peanut oil as a placebo) every 3 mo for 9 mo. All procedures were approved by the Institutional Review Board of the Johns Hopkins School of Medicine and the Papua New Guinea Medical Research Advisory Committee. At baseline, 40 and 34% of the children were positive for Plasmodium falciparum and/or P. vivax, respectively, and by the end of the study, the infection rate had increased to 55 and 33% (17
). Moreover, 67% of children had an enlarged spleen, indicating high levels of chronic malaria morbidity in the population. Concomitantly, the plasma retinol concentration of these children was 0.64 ± 0.23 µmol/L (mean ± SD) in the placebo group and 0.69 ± 0.24 µmol/L in the VA group at baseline (P > 0.05) (17
). By the end of the study, the mean plasma retinol concentration was 0.67 ± 0.27 µmol/L in the placebo group, and 0.76 ± 0.25 µmol/L in the VA group (P = 0.003) (17
). Previously, the inflammatory status of this population had been determined in a subsample of children with low-to-normal retinol concentrations (11
). Children with plasma retinol <0.70 µmol/L had higher levels of acute phase proteins including C-reactive protein and 
-1 acid glycoprotein (AGP) than those with normal retinol concentrations and children with enlarged spleens had lower levels of plasma retinol and higher levels of AGP (11
). Thus, 67% of these children had chronic inflammation by the end of the study (11
,16
). Because of the high rates of malarial infection and the associated low concentrations of plasma retinol, this population offered a unique opportunity to assess the response of the RBP:TTR ratio to VA supplementation.
Blood samples were collected before treatment allocation, at 7 mo (i.e., 1 mo after the second dose) and at thirteen mo, i.e., 3–4 mo after the last dose of VA (17
). Venous blood was kept in a dark box at ambient temperature for no >6 h before centrifugation (1500 x g for 5 min) and storage of plasma at -70°C (18
). Available samples were placed in liquid nitrogen and transported to Baltimore, MD, and retinol concentrations were measured by HPLC (17
). Aliquots of 50–100 µL were received on dry ice and stored at -70°C at the Pennsylvania State University facilities. These plasma samples had been thawed and frozen 2 or 3 times before the determination of plasma concentrations of RBP and TTR in 1999.
Determination of plasma RBP and TTR by single radial immunodiffusion.
Plasma RBP and TTR concentrations were measured by radial immunodiffusion. Gel-plates were prepared in our laboratory as previously described (11
). For TTR, a calibrator from DAKO (Carpinteria, CA) was titrated and a reference serum sample from the College of American Pathologists (Northfield, IL) was used as an external standard. For RBP, calibrators and reference standards were from the Binding Site (San Diego, CA). The mean ± SEM within-run precision (CV) of the RBP measurements in plasma, based on the reproducibility of the RBP concentration of the reference, was 8.6 ± 1.5%. The within-run precision of the TTR measurements in plasma, based on the reproducibility of the TTR concentration of the reference, was 5.24 ± 1.1%. The within-run accuracy of RBP based on the external reference value was 6.0 ± 4.0%, and for TTR based on its respective external standard, 8.6 ± 4.1%.
Statistical analysis.
Exploratory and confirmatory analyses were conducted to determine the distribution of variables and their associations. The RBP:TTR ratio was calculated for individual samples. Sensitivity and specificity characteristics of the RBP:TTR ratio were examined on the basis of a 2 x 2 decision matrix. The rows indicated selected cut-off values of the RBP:TTR ratio (e.g., screening marginal VA deficiency), and the columns represented the hepatic VA concentration indicative of marginal VA deficiency and normal VA stores. On the basis of this matrix, sensitivity (%) was defined as the quotient of true positive over all those with marginal hepatic VA stores and specificity (%) as the quotient of true negatives over all those with normal hepatic VA stores. The effect of VA supplementation on the RBP:TTR ratio was assessed between VA and placebo-treated children at each of the sampling times (i.e., baseline, 7 and 13 mo). These comparisons included the RBP:TTR ratio as a continuous or as a categorical variable (i.e., the percentage of children classified as marginally VA deficient or normal on the basis of a selected cut-off value). Nonparametric tests (Wald-Wolfowitz test, Mann-Whitney U tests, 2) were used to assess the differences between groups with significance accepted at a P-value 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Patients with marginal hepatic VA stores had significantly lower concentrations of plasma RBP than those with sufficient VA stores; however, plasma concentrations of TTR did not differ between the groups. The association between plasma concentrations of RBP and the RBP:TTR ratio was examined to assess a possible cut-off value (Fig. 1
). There was a positive direct linear association between plasma RBP concentrations and the RBP:TTR ratio (r = 0.69, P < 0.001). Plasma RBP concentrations associated with marginal VA deficiency were directly proportional to a low RBP:TTR ratio (panel A), as indicated by a line crossing a RBP:TTR ratio of 0.36. In contrast, there was no association between concentrations of plasma TTR and the ratio (panel B, r = 0.03, P > 0.43). Clearly, these results confirmed previous observations (4
) that a low RBP:TTR ratio reflects the disproportional reduction of RBP to TTR. The association between low plasma RBP concentration and a low ratio was examined further in relation to marginal hepatic VA stores (panel C). Five of eight patients (62%) with low plasma RBP (1.70 µmol/L) had ratios 0.36 and marginal hepatic VA stores.
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0.36 for the RBP:TTR molar ratio is presented in Table 2
. The sensitivity of this cut-off value was 71.4% and its specificity was 50%, with a positive predictive value of 78%, whereas a cut-off value 0.30 had a very low sensitivity (14%). The probability of selecting five of seven patients with low hepatic VA stores by chance alone was 0.08, based on a binomial distribution and an estimated 0.40 rate of VA deficiency in this population (15
).
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. Of the original subsample of 245 children whose plasma was evaluated at baseline, sufficient plasma was available for determination of RBP:TTR ratios at 7 mo post-treatment in 71% of these children, and 94% could be examined at 13 mo. At baseline, before children received placebo or VA supplements, the distribution of the RBP:TTR ratio did not differ between the groups, and 30% of children had RBP:TTR ratios 0.36. Seven months after receiving placebo or VA, the RBP:TTR was significantly greater in the VA group than in the placebo group (Table 3
). More importantly, the percentage of children with a RBP:TTR ratio 0.36 was significantly lower than in the placebo-treated group. At 13 mo, VA-treated children still had significantly higher RBP:TTR ratios than placebo-treated children; consequently, the proportion of children with low ratios was still significantly lower in the VA group compared with the placebo group.
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. The 2.5th and 97.5th percentiles of the distribution of RBP increased from infants to adolescents, as did TTR. However, the distribution of the ratio remained constant across these age groups as indicated by values at the 2.5th and 97.5th percentiles. It is noteworthy that the RBP:TTR ratio associated with the 2.5th percentile in these age groups ranged between 0.35 and 0.38, which includes 0.36. This suggests that 2.5% of infants, children or adolescents would have marginal VA deficiency, as would have been expected in a normal population.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). These findings suggested the possibility of using the RBP:TTR ratio to assess VA status in individuals with inflammation (4
). In the present investigation, we evaluated the sensitivity and specificity characteristics of the RBP:TTR ratio to determine its validity as an indirect method of VA status.
The sensitivity of the RBP:TTR ratio in detecting VA deficiency was considered using a paradigm proposed by the National Academy of Science and the NRC for the evaluation of biomarkers (19
). A biomarker’s sensitivity is given by its intrinsic relation to a specific function. For the RBP:TTR ratio, this function is a reduced secretion of RBP due to low hepatic VA stores (5
). In humans, a hepatic VA concentration of 69 nmol/g of tissue (< 20 µg/g) is indicative of marginal VA deficiency (18
,20
). In the present evaluation, we found that patients with marginal VA deficiency had significantly lower concentrations of plasma RBP (median, 1.7 µmol/L) than patients with normal hepatic VA stores (median, 2.2 µmol/L). Thus, a cut-off value of 0.36 was chosen using exploratory data analysis to indicate that it was associated with low plasma concentrations of RBP (Fig. 1)
. Using this cut-off value, five of seven adult patients with liver stores 69 nmol/g were selectively identified. On the other hand, a cut-off value of 0.30 had very low sensitivity and specificity in agreement with findings reported by Filteau et al. (21
). Recently, Donnen et al. (22
) observed that the RBP:TTR ratio could not be used to determine the difference in VA status of children with protein energy malnutrition between those receiving VA supplements and placebo during nutritional rehabilitation (23
). However, the VA status of these children did not differ as indicated by similar increases in their serum retinol concentrations 7 d after receiving placebo or VA (22
). It is probable that their retinol increased secondary to VA in the diet (e.g., palm oil, fish) consumed during the 7 d of rehabilitation, before the time when the RBP:TTR was evaluated (22
,23
).
Although the validity study suggested a close association between a cut-off value of 0.36 and having marginal VA deficiency, there are some caveats in the interpretation of these results. These include the effect of long-term storage on determinations of RBP and TTR concentrations, a limited sample size of 15 patients and the reliability of needle biopsies in measuring VA stores. Although it is recommended that blood constituents be analyzed soon after sample collection, several studies have shown that serum or heparinized plasma concentrations of retinol and its transport proteins are extremely stable to storage at -20°C from 1 mo up to 8 y (24
–27
). Moreover, the RBP:TTR ratio offers an advantage under conditions such as long-term storage or during repeated freezing and thawing of samples even after 5 freeze/thaw cycles (28
) because the ratio is calculated from determinations of RBP and TTR concentrations in the same sample; thus, weighing RBP by TTR helps to control for dehydration or hemodilution.
Validity studies usually have a trade-off between accuracy and precision. The first human study evaluating the relative-dose response (RDR) test for VA status was conducted in 8 adult patients with cirrhosis of the liver (29
). This group was used because the accuracy of night blindness as an indicator of VA deficiency in cirrhotic patients was unequivocal (29
,30
). The RDR helped diagnose VA deficiency in 5 of 8 cirrhotic patients, a sensitivity of 62.5%, and it has since been in use as an indicator of VA status (31
). To further validate the RDR, Amedee-Manesme et al. (32
) conducted a study in 12 surgical adult patients with known hepatic VA stores, and Furr et al. (33
) confirmed that the VA concentrations determined from liver biopsies correlated with total VA stores. Indeed, there is a close correlation (r = 0.96) between hepatic VA determinations obtained with a needle biopsy and those obtained with gross liver samples (34
). Although the present study is limited in precision because of the small sample size, the use of hepatic concentrations to validate the RBP:TTR ratio underscores the importance of its findings.
Moreover, the evaluation of the RBP:TTR was further tested among children with high rates of malaria infection, suffering from spleen enlargement and inflammation (11
,17
). The proportion of children with a cut-off value 0.36 was significantly lower in children receiving VA supplements compared with children receiving placebo. This difference remained significant even 4 mo after receiving the last dose of VA. Nonetheless, the proportion of children with marginal VA deficiency increased in the VA group at the 13-mo compared with the 7-mo evaluation (Table 3
). This might reflect the small residual effect of VA supplements in changing VA status at 4 mo postdosing. Tanumihardjo (35
) calculated that a single oral dose of 210 µmol of VA can maintain liver VA stores of children for at least 1 mo postdosing, whereas by 4 mo, VA stores would have decreased.
The changes in the ratio at the13-mo evaluation (Table 3
) also suggested a worsening in the VA status of these children. Possible factors contributing to poor VA status in children from the Wosera district may include a generally poor diet, as reflected in a 25–29% prevalence of stunting (36
,37
), high rates of asexual P. falciparum and long duration of parasitemia (38
). Additionally, there is a seasonal effect on micronutrient intake, whereby iron intake in July is lower than in April (39
). Indeed, Shankar et al. (17
) found that hemoglobin concentrations decreased by 10 g/L in children receiving VA or placebo by the end of the study. Thus, it is possible that seasonal variations in VA intake as observed for iron might enhance the detrimental effect of malaria-related morbidity on the VA status of this population.
Studies on the prevalence of VA deficiency in this region have shown mixed results. Using clinical signs of VA deficiency such as Bitot’s spots, VA deficiency has been estimated at a low rate of 0.19% (1
). On the other hand, low retinol concentrations are prevalent in this population. Shankar et al. (17
) found a 7.0% rate of VA deficiency (retinol < 0.35 µmol/L), and others have reported a 91% rate of subclinical VA deficiency (retinol < 0.70 µmol/L) (1
). Thus, a rate of 30% marginal VA deficiency as determined at baseline using the RBP:TTR ratio is likely to represent the VA status of this population because this rate is compatible with a low prevalence rate of xerophthalmia.
Although these results demonstrated the internal validity of a cut-off value 0.36 in children, it is important to note that the sensitivity of this cut-off value was evaluated in a population of adult patients. Children, however, acquire adult hepatic VA stores between 1 and 4 y of age (20
). Moreover, the RBP:TTR ratio is not affected by age differences, as shown in Table 4
. In summary, these analyses show that a cut-off value of 0.36 for the RBP:TTR ratio can be used to assess the efficacy of VA supplements and selectively detect subjects with marginal VA deficiency in targeted populations.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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3 AGP, -1 acid glycoprotein; BMI, body mass index; RBP:TTR, molar ratio of retinol-binding protein to transthyretin; RDR, relative-dose response; VA, vitamin A.
Manuscript received 24 May 2002. Initial review completed 22 June 2002. Revision accepted 9 September 2002.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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35. Tanumihardjo, S. A. (2001) Can lack of improvement in vitamin A status indicators be explained by little or no overall change in vitamin A status of humans?. J. Nutr. 131:3316-3318.
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