European Journal of Clinical Nutrition (2014) 68, 1300–1304 © 2014 Macmillan Publishers Limited All rights reserved 0954-3007/14 www.nature.com/ejcn
ORIGINAL ARTICLE
Characterization of the vitamin A transport in preterm infants after repeated high-dose vitamin A injections AC Longardt1, B Schmiedchen2, J Raila2, FJ Schweigert2, M Obladen1, C Bührer1 and A Loui1 BACKGROUND/OBJECTIVES: Preterm infants have low vitamin A stores at birth, and parenteral administration of high-dose vitamin A reduces pulmonary morbidity. The aim was to characterize vitamin A transport and status. SUBJECTS/METHODS: Prospective study of 69 preterm infants (median birth weight 995 g, gestational age 28 weeks), in which 51 received 5000 IU vitamin A three times per week intramuscular (i.m.) for 4 weeks and 18 infants without i.m. vitamin A served as controls. Serum retinol, retinyl palmitate, total retinol-binding protein 4 (RBP4), retinol-unbound RBP4 (apo-RBP4) and transthyretin concentrations were determined at days 3 (D3) and 28 (D28) of life. RESULTS: D3 retinol concentrations were low for the entire group (382 (285/531) nmol/l; median/interquartile range) and unrelated to gestational age. D28 retinol was unchanged in controls (382 (280/471) nmol/l), but increased in the vitamin A group (596 (480/825) nmol/l; P o0.001). A similar pattern was observed for RBP4. The calculated retinol-to-RBP4 ratio rose in vitamin A infants (D3: 0.81 (0.57/0.94), D28: 0.98 (0.77/1.26); P o 0.01) but not in controls. In the vitamin A group, the retinol-to-RBP4 ratio was 41 in 15% of all infants on D3 and in 45% of infants on D28, but was ⩽ 1 in all, but one, controls on D28. CONCLUSIONS: In preterm infants receiving a 4-week course of high-dose i.m. vitamin A, serum retinol concentrations increased by 55%, with molar concentrations of retinol exceeding those of RBP4 in 45% of the infants suggesting transport mechanisms other than RBP4. European Journal of Clinical Nutrition (2014) 68, 1300–1304; doi:10.1038/ejcn.2014.202; published online 15 October 2014
INTRODUCTION An adequate vitamin A supply is essential for normal fetal and postnatal development. During pregnancy, retinol reaches the fetus via the placenta, whereas after birth it is ingested as retinyl ester, transported to the liver and stored in stellate cells of the liver. The liver secretes retinol into plasma bound by retinol-binding protein (RBP4) at a 1:1 molar ratio. RBP4 is synthesized mainly in the liver. Holo-RBP4, consisting of apo-RBP4 and retinol, binds to transthyretin (TTR), which prevents losses through glomerular filtration. Fetal serum concentrations of the holo-RBP4-TTR complexes are kept constant during gestation up to 36 weeks. In term infants, retinol concentrations in cord blood were found to be 38% higher than in preterm infants.1 Lung immaturity accounts for much of the mortality and morbidity in very-low-birth-weight (VLBW) infants (birth weight o1500 g). The final step of pulmonary differentiation is facilitated by vitamin A. VLBW infants have less bronchopulmonary dysplasia (BPD) when given high-dose vitamin A intramuscular (i.m.) for 4 weeks.2 Because vitamin A is administered intramuscularly, willingness to apply this regimen is rather poor, as reported by Mactier.3 However, enterally administered vitamin A had no effect in these infants.4,5 The mechanisms behind this observation are not completely understood, but might be due to alterations of vitamin A transport in the immature gastrointestinal tract. In this study, we aimed to characterize vitamin A transport and status in VBLW infants who had or had not received vitamin A i.m., by measuring serum concentrations of retinol, retinyl palmitate, RBP4, TTR, the ratio of retinol-to-RBP4 and the percentage of apo-RBP4.
SUBJECTS AND METHODS Study design During the study period (from June 2007 to June 2008), 150 VLBW infants were admitted to the neonatal intensive care unit of the Charité University Hospital, Berlin. Fifteen infants died within 3 days. One hundred and one infants with absence of major renal or gastrointestinal malformations or refractory shock were eligible to participate. Infants considered to be at risk for BPD at day 3 of life (D3), defined as need for positive pressure support (artificial ventilation or continuous positive airway pressure) and/or supplemental oxygen, received vitamin A i.m. (water-miscible retinyl palmitate (15 mg retinol/ ml); AQUASOL A Parenteral, AstraZeneca LP, Westborough, MA, USA) at a dose of 5000 IU three times a week for 4 consecutive weeks according to Tyson et al. (vitamin A group, n = 63).2 Infants not considered at risk for BPD, did not receive vitamin A i.m. and served as control group (n = 38). Anyhow both groups received some vitamin A with fat-soluble vitamins (Vitalipid Infant, Baxter, Unterschleißheim, Germany) with the parenteral nutrition. This study was originally designed to test the hypothesis that low retinol serum concentrations can be diagnozed using urine measurements. To reach a confidence interval of ± 0.1 and an estimated area under the curve of 0.8, a sample size of 91 patients was needed. Serum retinol, retinyl palmitate, total RBP4, retinol-unbound RBP4 (apo-RBP4) and TTR were scheduled to be measured on day 3 of life (D3, baseline, before commencement of any i.m. vitamin A administration) and on day 28 (D28, after near completion of the 4-week course of i.m. vitamin A). Here we report for the first time on 69 out of 101 included infants (51 infants in the vitamin A group and 18 infants in the control group) with complete data sets. In the remaining infants, sample volumes on D3, D28 or both had been too low to measure all parameters. This study was approved by the Ethics Institutional Review Board of the Charité (No. EA2/053/07), written informed consent was obtained from the parents.
1 Department of Neonatology, Charité University Medical Center, Berlin, Germany and 2Department of Physiology and Pathophysiology, Institute of Nutritional Science, University of Potsdam, Potsdam, Germany. Correspondence: Dr AC Longardt, Klinik für Neonatologie, Charité Universitätsmedizin Berlin, Augustenburger Platz 1, Berlin D-13344, Germany. E-mail:
[email protected] Received 19 December 2013; revised 21 May 2014; accepted 17 July 2014; published online 15 October 2014
Vitamin A transport in preterm infants AC Longardt et al
1301 Nutrition The infants were fed according to current nutritional recommendations.6 Intravenous administration of fat-soluble vitamins (Vitalipid Infant, Baxter) including vitamin A (0.13 mmol retinyl palmitate/ml) was started on day 5 of life and discontinued upon adequate advancement of oral feeding. Enteral nutrition consisted either of mother’s own milk supplemented with a human milk fortifier (Aptamil FMS, Milupa, Friedrichsdorf, Germany) or a preterm formula (Beba preterm formula, Nestlé, Frankfurt/Main, Germany). The children received different amounts of milk and fortifier corresponding to their individual nutritional situation.
Sample and data collection Serum was obtained from blood samples (0.3 ml), taken before vitamin A injection, by centrifugation (3000 g; 10 min) on D3 and D28. Samples were frozen at − 80 °C until analyzed within 3 months. Clinical data were recorded from charts. The observational period was 35 days of life. Nutritional data were recorded for the full length of hospital stay in order to get valid data for the use of human milk and fortifier.
Measurements Retinol and retinyl palmitate concentrations were determined chromatographically (HPLC, Waters GmbH, Eschborn, Germany) as described.7 RBP4 and TTR concentrations were measured using ELISA.7 For RBP4 and TTR, the intra-assay coefficient of variation and inter-assay variation is o10%. For retinol and retinyl palmitate, both variations are o4%. The detection limits of the assays are 2.0 ng/ml for retinol, 2.4 ng/ml for retinyl palmitate, 612 pg/ml for TTR and 4011 pg/ml for RBP4. The relative percentage of holo-RBP4 and apo-RBP4 was assessed using nondenaturating polyacrylamide gel electrophoresis with subsequent immunoblot analysis.8
Statistical analyses Statistical analysis was performed using SPSS software 19.0 (IBM/SPSS Inc., Chicago, IL, USA). Continuous variables were described as median and interquartile range. Groups were compared using the Mann–-Whitney U-test or the χ2 test with Yates correction. Time points were compared by the Wilcoxon test. Correlation analyses were performed using Spearman rank correlation. Significance level was 0.05.
RESULTS Infants’ clinical data are shown in Tables 1 and 2. The vitamin A group consisted of a higher number of small for gestational age infants, had a lower median gestational age, needed more oxygen supplementation and mechanical ventilation, developed BPD and retinopathy of prematurity more frequently, and received parenteral nutrition for a longer period compared with the control group (Table 2). The patients’ selection because of low sample volumes had no considerably influence on the clinical characteristics and results reported here (data not shown).
Table 1.
Baseline (D3) serum concentrations of retinol, retinyl palmitate, RBP4, TTR and apo-RBP4 are given in Table 3. None of these parameters were related to gestational age (data not shown). In 40 of 69 infants, retinyl palmitate was below the detection level explaining the median of 0 in Table 3. The serum concentrations on day 28 (D28) are shown in Table 4. In the vitamin A group, median serum concentrations of retinol rose by 270 nmol/l, whereas RBP4 concentrations rose by 207 nmol/l. Median concentrations of retinyl palmitate rose by 222 nmol/l in the vitamin A group, and by 54 nmol/l in the control group (Table 4). The correlations between the retinol-to-RBP4 ratio and retinol on D3 in the entire group and on D28 separated for both groups are shown in Figure 1. There was a significant correlation between the retinol-to-RBP4 ratio and retinol on D3 (Rs = 0.410, P o 0.001) but not on D28 in either the vitamin A or the control group. Moreover, at D3, there was no correlation between the retinol-toRBP4 ratio and the percentage of apo-RBP4. At D28, all but one of the infants of the control group had a retinol-to-RBP4 ratio o 1 despite increasing apo-RBP4 concentrations, whereas in the vitamin A group, 45% of the infants had a retinol-to-RBP4 ratio 41. DISCUSSION The study results demonstrate that serum retinol concentrations in VLBW infants are consistently low, compared with term newborns, across the range of 24–32 weeks of gestation.1 After serial high-dose injections of retinyl palmitate, circulating retinyl esters increased markedly. Moreover, the molar retinol-to-RBP4 ratios exceeded 1.0 in a considerable proportion of infants, suggesting that retinol transport in serum is also achieved by transport proteins other than RBP4. The data are in line with the notion that blood concentrations of retinol and RBP4 are kept rather constant throughout fetal life, rising only at the end of pregnancy.1 Lung immaturity in VLBW infants requires exogenous surfactant, mechanical ventilation and oxygen supplementation. Vitamin A injections have been shown to reduce the duration of oxygen dependence. This strategy, however, is not commonly practised as i.m. injections are considered too painful to be justified by the relatively modest improvement in respiratory outcome.3 In addition, the parenteral administration of vitamin A to VLBW infants at dosages that exceed recommended daily allowances has been a matter of concern. Retinol is bound to RBP4 and transported from the liver to peripheral tissues as holo-RBP4. In adults, up to 20% of RBP4 is unbound to retinol (apo-RBP4). Values of the apo-RBP4-to-holo-RBP4
Infant clinical characteristics
Clinical characteristics Gestational age (weeks+days) Birth weight (g) Head circumference (cm) Length (cm) Male gender (n; %) Antenatal steroids (n; %) Small for gestational age (n; %) Mechanical ventilation (days) Supplemental oxygen (days) Bronchopulmonary dysplasia (n; %) Intraventricular hemorrhage 4II° (n; %) Retinopathy of prematurity ⩾ II° (n; %)
Vitamin A group (n = 51) 27+2 950 28 40 29 48 5 3 16 13 6 12
(25+2/28+4) (740/1080) (26/30) (37/42) (57) (94) (10) (0/22) (3/41) (25) (12) (24)
Control group (n = 18) 29+1 1248 30 43 6 16 6 0 0 0 1 0
(28+4/30+0) (1015/1370) (29/31) (42/45) (33) (89) (33) (0/0) (0/1) (0) (6) (0)
P valuea o 0.001 o0.001 0.001 o0.001 0.086 0.462 0.019 o0.001 o0.001 0.017 0.453 0.020
All values are shown as n (%) or median (interquartile range). aMann–Whitney U-test, χ2 test.
© 2014 Macmillan Publishers Limited
European Journal of Clinical Nutrition (2014) 1300 – 1304
Vitamin A transport in preterm infants AC Longardt et al
1302 Table 2.
Infant nutrition and growth
Nutrition
Vitamin A group (n = 51)
Parenteral nutrition Amino acids (days) Lipids (days) Trace elements (days) Vitamins (days)
15 13 10 9
Enteral nutrition Mother’s own milk 450% (days) Full enteral nutrition 4130 ml/kg/d (day of life) Fortifier use (n; %)
56 (34/77) 17 (14/21) 51 (100)
Vitamin A administration Cumulative vitamin A i.v. (IU) Cumulative vitamin A i.m. (IU) Cumulative parenteral vitamin A (IU)
(12/17) (10/16) (8/13) (7/13)
11 10 8 7
1510 40 28 13.4 1.0 0.6 32+0
(9/13) (8/12) (6/11) (5/9)
0.002 0.002 0.008 0.008
29 (18/40) 16 (13/18) 18 (100)
1898 (1449/2530) 60 000 (55 000/60 000) 61 840 (57 300/62 944)
Growth Body weight at day of life 35 (g) Body length at day of life 35 (cm) Head circumference at day of life 35 (cm) Weight gain at day of life 1–35 (g/d) Length gain at day of life 1–35 (cm/week) Head growth at day of life 1–35 (cm/week) Corrected gestational age at day of life 35 (weeks+days)
P valuea
Control group (n = 18)
(1147/1695) (37/42) (26/30) (10.4/ 18.4) (0.8/1.2) (0.5//0.7) (30+0/33+0)
0.002 0.127 0.005
1530 (1150/2990) 0 (0/0) 1530 (1150/2990) 1813 43 30 19.0 1.0 0.5 34+0
0.389 o 0.001 o 0.001 o0.001 0.001 0.001 0.004 0.486 0.858 o0.001
(1625/2060) (42/45) (29/31) (15.9/ 20.4) (0.8/1.4) (0.4/0.8) (33+0/34+0)
Abbreviations: i.m., intramuscular; i.v., intravenous. All values are n (%) or median (interquartile range), nutritional data are given for the full length of hospital stay, growth data are given on day of life 35 and growth velocities from day of life 1–35. aMann–Whitney U-test, χ2 test.
Table 3.
Centiles of retinol, RBP4, TTR, retinyl palmitate, apoRBP4 and retinol/RBP4 ratio in serum on day 3 of life
Centiles Serum values Retinol (nmol/l) RBP4 (nmol/l) TTR (nmol/l) Retinyl palmitate (nmol/l) apoRBP4 (%) Retinol/RBP4 ratio
Valid n
3
5
10
25
Median
75
90
95
97
69 69 69 69 67 69
101 255 146 0 0 0.2
140 275 167 0 1.9 0.2
220 323 225 0 7.7 0.4
285 363 596 0 24.5 0.6
382 506 938 0 37.9 0.7
531 713 1434 37 58.5 0.9
611 930 1913 85 73.9 1.3
796 1745 2346 412 78.2 1.6
1173 2865 2545 919 82.9 1.7
Abbreviations: apo-RBP4, retinol-unbound RBP4; RBP4, retinol-binding protein 4; TTR, transthyretin.
Table 4.
Serum concentrations of retinol, RBP4, TTR, retinyl palmitate, apoRBP4 and retinol/RBP4 ratio on day 28 of life Vitamin A group (n = 51) Retinol (nmol/l) RBP4 (nmol/l) TTR (nmol/l) Retinyl palmitate (nmol/l) apoRBP4 (%) Retinol/RBP4 ratio
596 641 1280 222 18.6 0.98
(480/825) (442/904) (895/1958) (131/373) (13.3/27.9) (0.77/1.26)
Control group (n = 18) 382 417 1488 80 23.0 0.80
(280/471) (384/698) (1073/2254) (55/107) (18.8/32.0) (0.67/0.93)
P valuea o 0.001 0.045 0.254 o 0.001 0.062 0.020
Abbreviations: apo-RBP4, retinol-unbound RBP4; RBP4, retinol-binding protein 4; TTR, transthyretin. All values are shown as median (interquartile range). aMann–Whitney U test, χ2 test.
ratio 41 indicate that there is more retinol-unbound than retinol-bound RBP4. On D3, we compared the retinol-to-RBP4 ratio to apo-RBP4 and to retinol. In all, 15% of the VLBW infants showed a retinol-to-RBP4 ratio 41, suggesting a substantial amount of RBP4-unbound retinol. On D28 in the vitamin A group, European Journal of Clinical Nutrition (2014) 1300 – 1304
but not in the controls, almost half of the infants had a retinol-toRBP4 ratio 41, suggesting that most of the retinol is not bound to RBP4. A possible explanation could be that the i.m. dose of retinyl palmitate is too high or too often given to be handled in this very immature organism. Whether the excess retinol is transported via nonspecific mechanisms or bound to unidentified specific proteins is a matter of speculation. We speculate that retinol and retinyl ester may be nonspecific, transported also by lipoproteins, as this has been shown in adults after they got a meal.9 Because the VLBW infants got fortified human milk every 2 or 3 h and therefore had no fasting periods, such transport way is conceivable. Only one study described the kinetics of i.m. administered retinyl palmitate in adults with severe malaria,10 but no information concerning the metabolism of retinyl palmitate were given. Moreover, impaired kidney function does affect serum RBP4 concentrations. Increased concentrations of apo-RBP4 indicate decreased glomerular filtration and hence decreased renal uptake and catabolism in the proximal tubule.11 Hence, high concentrations of apo-RBP4, as we observed on D3 but not on D28, are in line with the notion in the literature that in VLBW infants glomerular filtration is low during the first 3–4 days of life and © 2014 Macmillan Publishers Limited
Vitamin A transport in preterm infants AC Longardt et al
1303 3.0
Day 3
Day 28
3.0
Retinol / RBP4
2.5
All infants (n=69)
n.s.
1.5 1.0 0.5
2.0
Rs= 0.410, p< 0.001
1.5
0 3.0
1.0
2.5
0.5 0 0
500
1000
1500
Retinol (nmol/ l)
Retinol / RBP4
Retinol / RBP4
2.5
2.0
Vitamin A group (n=51)
Control group (n=18)
2.0
n.s. 1.5 1.0 0.5 0 0
500 1000 Retinol (nmol/ l)
1500
Figure 1. Serum retinol-to-RBP4 ratio in correlation to serum retinol on day 3 and day 28 of life; on day 28, separated for the vitamin A group (n = 51) and the control group (n = 18). On D3, a positive correlation between serum retinol-to-RBP4 ratio and retinol serum concentration was noted (Rs = 0.410, Po 0.001). NS, not significant.
then gradually increases.12,13 In rats suffering from acute renal insufficiency, serum retinol concentrations were found to decrease even after a high dose of retinyl palmitate. Administration of apo-RBP4 induced an increase of retinol. Hence, an upregulating effect and a positive feedback signal of apo-RBP4 have been suspected.14 Data on vitamin A in VLBW infants are rather sparse in the literature. Galinier et al.1 reported data from cord blood and found 55% higher retinol concentrations in VLBW infants than we found on day 3 of life. Vitamin A blood concentrations after birth were published in nine studies. Weinman et al. investigated retinol concentrations on D7 and D28 in 124 infants with a gestational age of 24–36 weeks.15 On D7, VLBW infants had retinol concentrations twice as high as those measured by us on D3. It is questionable that this could be due to the fact that Weinman’s infants received vitamin A enterally via milk during the first 7 days. Weinman’s infants got much lower values of vitamin A enterally during the first week of life (987 ± 344 IU/kg/d)15 than the infants of Wardle’s study (5000 IU/d)5. In both studies, no effect of the supplementation on the serum retinol concentrations had been shown. Another explanation could be the higher gestational age of their cohort, as the fetus starts to produce RBP4 at around 34 gestational weeks with a sharp increase in retinol concentrations thereafter.16 Even on D28, retinol concentrations in our cohort were half as high as those measured by Weinman et al.15 There are eight further studies,2,5,17–22 which analyzed vitamin A status in the first day of life. Six studies2,17,19,21,23,24 analyzed the effect of i.m. vitamin A supplementation. A comparison of these studies is limited, because of the marked differences in study design. Little is known about fetal vitamin A metabolism.25 It is known that intrauterine vitamin A is transferred via the placenta mainly during the third trimester. Thus, infants born before that time have insufficient vitamin A storage. Fetal RBP4, important for the fetal vitamin A transport,26 is produced in the fetal liver in the last quarter of gestation.27 Galinier described that concentrations for retinol, RBP4 and TTR in cord blood were significantly lower in infants with a mean gestational age of 32 weeks compared with those of 39 weeks.1 In our cohort, no correlation between gestational age and retinol or RBP4 concentrations was found, but all infants were born before 33+1 weeks gestation, that is, before the major retinol increase and hepatic RBP4 synthesis takes place. © 2014 Macmillan Publishers Limited
Several authors defined vitamin A deficiency as moderate at retinol concentrations o 700 nmol/l and severe at concentrations o350 nmol/l.2,15,17,18,28 These cutoff levels were defined for term newborn infants or children.29 A definition of vitamin A deficiency in VLBW infants does not exist. As the transplacental vitamin A transfer mostly occurs late in pregnancy,15,27 preterm infants born before 28 weeks are expected to have reduced retinol concentrations and to need higher vitamin A supplementation.1 Data concerning the resorption of i.m. administered vitamin A do not exist. High serum retinyl palmitate concentrations are considered to be a possible sign of vitamin A intoxication. Reports on vitamin A toxicity, however, in infants are rare. Acute vitamin A toxicity has been reported in adults with a single-dose exceeding 660 000 IU, or 10 000 IU/kg.30 Toxic effects in infants under 6 months receiving 20 000 IU/week for 4 weeks have been described,31 but clinical symptoms are not given and the treatment dose was higher than ours. A 3-month-old infant who received 62 000 IU/day enteral vitamin A for 80 days showed symptoms such as severe anemia and thrombocytopenia. A retinyl palmitate-to-retinol ratio of 53% (normal value given by Perotta 0.1–4.7%) and of RBP4 of 24 mg/ml (normal value given by Perotta 20–30 mg/ml)32 were measured. After 4-week vitamin A supplementation (2143 IU/day i.m.), we measured a median (interquartile range) retinyl palmitate-toretinol ratio of 37% (20%/55%). In our control group, however, the D28 retinyl palmitate-to-retinol ratio of 20% (17%/ 26%) still exceeded the ‘normal range’ according to Perotta et al.32 This indicates that the ‘ranges’ given by Perrotta et al. are not comparable to those of our cohort. Although we measured high retinyl ester concentrations, no clinical signs of intoxication, such as bulging fontanels, lethargy or liver damage were noted. Such symptoms, however, are unspecific and difficult to assess in preterm infants.33,34 These findings are comparable to those reported by others.2,17,21,22 Our infants showed no signs of renal failure but low glomerular filtration related to their degree of prematurity were reported in the literature,12,13 which may partly explain the high retinyl palmitate concentrations. CONCLUSIONS VLBW infants display low serum concentrations of retinol and high concentrations of retinol-unbound RBP4 on D3. After 4 weeks of European Journal of Clinical Nutrition (2014) 1300 – 1304
Vitamin A transport in preterm infants AC Longardt et al
1304 high-dose i.m. vitamin A administration, there is only a modest increase of circulating retinol, which exceeds RBP4-mediated binding capacity in a considerable fraction of infants, suggesting other transport proteins besides RBP4. The dose of retinyl palmitate may be too high or too often given. These data call for the establishment of vitamin A reference values and for a definition of vitamin A deficiency, which is specific to VLBW infants. Current supplementation regimes for preventing BPD in these infants by high-dose parenteral vitamin A administration exceeding the recommended daily allowances should proceed with caution and should be revised. CONFLICT OF INTEREST The authors declare no conflict of interest.
ACKNOWLEDGEMENTS We thank all parents of the preterm infants who enabled this study, B Metze for data processing and A Carney for editorial assistance. This work was supported by a grant from Else Kröner-Fresenius-Stiftung, Subsidy Contract Number 2010 A151.
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