Recent reports on the negative effect of transfusions on the long-term outcome of prematurity related to bronchopulmonary dysplasia (1,2), changes in brain structure (3), or neurocognitive development (4) support the need for strategies to cut down on the number of transfusions received early in life. In preterm infants, the number of red blood cell (RBC) transfusions is highest in the first 2 weeks of life (5,6). In this population, the prescription of early blood transfusions is influenced by initial haematocrit, birth weight, blood letting, postnatal age, and illness severity (7). Illness severity affects the need for blood transfusions through increased blood test monitoring and possibly the enhanced risk of haemolysis resulting from infusions of therapeutic solutions (8). Furthermore, oxidants may cause cell damage (9) as well as haemolysis (10) with ensuing potential need for transfusions. Because of an imbalance between immature antioxidant defences and elevated oxidant loads associated with lifesaving therapies, preterm infants may indeed be subjected to a significant oxidant stress (11).
In premature infants who cannot be fed via the intestinal tract until the gut matures, total parenteral nutrition (TPN) is used to provide essential nutrients for growth, including antioxidant vitamins. Exposure of TPN to ambient light generates oxidation products, such as peroxides originating from the lipid emulsion (12), from vitamins (13), and/or from interactions between nutrients and ambient light (14). RBCs from healthy neonates incubated in vitro with a lipid emulsion (0.44% w/v), a component of TPN, caused a depletion of reduced glutathione, enhanced generation of products of lipid peroxidation, and led to haemolysis (10). In view of the generation of both lipid peroxides and hydrogen peroxide in TPN solutions (15), this in vitro study raises questions about the potential effect of a complete TPN solution on haemolysis measured in vivo. Indeed, hydrogen peroxide derived from multivitamins contributes 80% of total peroxides generated in TPN (16). Hydrogen peroxide is a known contributor to haemoglobin degradation in RBC (17), and oxidative stress is the primary cause of RBC aging leading to their elimination (18,19).
Shielding TPN from light reduces the load of total peroxides (20) as well as lipid peroxides (12), affecting beneficially a variety of clinical outcomes such as plasma triglyceride levels (21), blood pressure (22), and bronchopulmonary dysplasia (23). We hypothesised that using this noninvasive approach to reduce the generation of peroxides in TPN may protect against haemolysis. The aim of the present study was to evaluate whether shielding TPN from light decreases haemolysis sufficiently to observe a functional benefit on the risk of early transfusions in extremely-low-birth-weight premature infants.
To evaluate in vivo the effect of a complete TPN admixture on haemolysis, an animal model (24) was used to ensure a comparison with an enteral source of nutrition and timely access to blood samples. A retrospective analysis of data collected in premature infants (11,25) was performed to evaluate the effect of photoprotection of TPN on transfusions.
Animal Experiments on Haemolysis
Three groups of 3-day-old guinea pigs (Charles River, St Constant, Canada) were compared:
1. Control animals (n = 4) were fed a regular chow diet from day 3 of life (2041-Teklad Global High Fibre Guinea Pig Diet, Harlan, Canada).
2. TPN(+)light (n = 4): animals received exclusively TPN (dextrose, amino acids, multivitamins, lipids) between days 3 and 7 of life via a catheter fixed in the external jugular vein. Complete intravenous nutrient admixture was provided as previously reported (24,26). TPN solution infused at a constant rate of 220 mL · kg−1 · day−1 was changed daily and provided without photoprotection.
3. TPN(−)light (n = 4): same as for TPN(+)light, except that the solution was photoprotected with opaque shielding of bag and extension set from time of admixture to delivery.
Animals were housed in a controlled environment for temperature and 12-hour light/dark cycle. At 1 week of age, after 4 days on the tested diets, animals were sacrificed under anaesthesia followed by cardiac puncture to obtain blood for determination of haemolysis. The study was approved by the institutional review board of the Ste-Justine Research Centre for the care and handling of animals, in accordance with the guidelines of the Canadian Council of Animal Care. Total plasma haemoglobin concentration, an index of haemolysis, was measured according to the method of Shim and Jue (27). Plasma was obtained after centrifugation (4 minutes at 12,000g) of fresh blood. One hundred microlitres of plasma was mixed to 900 μL of buffer (2.7 mmol/L KCl, 1.5 mmol/L KH2PO4, 8 mmol/L Na2HPO4, 136.9 mmol/L NaCl; pH 7.4). Sodium hydrosulfite (B.D.H. Inc, Toronto, Canada) was added to each sample (0.5 mg/sample), gently mixed, and incubated for 1 minute. During that period, free haemoglobin and the haemoglobin bound to haptoglobin is converted to cyanomethaemoglobin. The change in the Soret peak of absorption of oxyhaemoglobin (418 nm) and deoxyhaemoglobin (432 nm), before and after the addition of the reducing agent (sodium hydrosulfite), was used to determine the amount of haemoglobin in plasma after subtracting the blank values obtained with 1000 μL of buffer without plasma. Total peroxide concentration was measured in TPN solutions using the FOX assay (28) based on a H2O2 standard curve.
Retrospective Analysis of Infant Transfusion Data
To evaluate the effect of light protection of TPN on the number of transfusions received by premature infants, an exploratory case control, retrospective analysis was performed on data collected from 2 previous studies on the biochemical effects of shielding TPN from light (11,25). In both trials, preterm infants were randomly allocated to receive, from birth, TPN solutions, either light-exposed (LE) or light-protected (LP). During both trials (11,25) the progression to full TPN was achieved following the same standardised written protocol. Nutrient intakes were similar in each group within 5% of each other during the period infants were receiving TPN. During both trials (11,25) the same RBC transfusion criteria (29) were applied in accordance with a written unit policy that was not modified during this time. Compliance with RBC transfusion guidelines was not tested.
Trials differed by the modality of multivitamin administration in the following ways:
1. In the first crossover study, the multivitamin preparation (MVP) was mixed with the amino acid-dextrose solution, and lipids were infused separately (AA+MVP) (25). This preparation generates a mixture of lipid and hydrogen peroxides (15). Of a total of 77 infants, 39 started with LE and 38 started with LP. A crossover occurred in a specific number of infants after 7 days receiving full TPN. In the LE group, 27 received only the LE regimen, whereas 12 received LE followed by 3 days of LP. In the LP group, 12 received only the LP regimen, whereas 26 received LP followed by 3 days of the LE regimen.
2. In the second noncrossover study, multivitamins were mixed with the lipid solution, and the amino acid-dextrose solution provided separately (LIP+MVP) (11). This preparation decreases the generation of lipid peroxides (15). Of a total of 56 infants, 37 received the LE regimen and 19 received the LP regimen.
The primary endpoint of the present retrospective analysis was the number of RBC transfusions received before discharge or transfer. The risk of transfusion was established as the cumulative number of postnatal days divided by the cumulative number of transfusions received during the hospital stay. The potential confounding factors were length of exposure to peroxides through TPN, severity of illness reflected in Score for Neonatal Acute Physiology (SNAP) (30), haematocrit at birth, birth weight, gestational age, and modality of TPN. This retrospective analysis was approved by the clinical research ethics board of the University of British Columbia.
Results were expressed as mean ± standard error of the mean. The level of significance was set at P < 0.05. In the preclinical study, differences in total plasma haemoglobin concentrations between diets were tested using an analysis of variance with the linear mixed model procedure (Proc Mixed, SAS, version 9.1) and diet groups were compared using contrast statements (control vs TPN[−]light vs TPN[+]light). In the clinical study, a longitudinal binomial regression model was used to adjust for potential covariates in the comparison of transfusion counts between LE and LP. The model was analysed with and without those infants participating in a crossover between LE and LP or LP and LE regimens.
In the preclinical animal study, peroxide concentrations in TPN solutions were higher (P < 0.05) in the LE regimen (TPN[+]light = 365 ± 15 μmol/L) compared with the LP regimen (TPN[−]light = 209 ± 9 μmol/L). These concentrations are similar to those reported in TPN regimens received by premature infants in the clinical setting (20). Total plasma haemoglobin concentration was higher (P < 0.05) in animals receiving TPN compared with enterally fed controls (Fig. 1). There was no statistical difference in haemolysis associated with exposure to light between TPN[+]light and TPN[−]light.
The retrospective analysis showed that populations of infants receiving LE (n = 76) or LP (n = 57) regimens had similar clinical characteristics: birth weight (824 ± 219 vs 896 ± 217 g), gestational age (26 ± 2 vs 26 ± 2 weeks), male sex (52% vs 60%), birth haematocrit (0.44 ± 0.07 vs 0.46 ± 0.07), days of TPN (13 ± 1 vs 13 ± 1 day; range 6–21 days), and SNAP (21 ± 17 vs 20 ± 11). The osmolarity of TPN solutions reported by pharmacy ranged from 875 (peripheral line) to 1200 mOsm/L (central line). The risk of transfusion showed a significant interaction (P < 0.05) between LE, LP, and postnatal age (Fig. 2), suggesting that babies who received the LP TPN regimen may have a lower risk of requiring transfusions during TPN infusion. While receiving TPN, 47 of 76 of infants were transfused (71 transfusions) in the LE group, and 42 of 57 infants were transfused (42 transfusions) in the LP group; however, during the entire hospitalisation, the mean number of transfusions in LP (4.58 ± 0.63) was 7% higher than in LE (4.29 ± 0.51). The absence of a significant difference may be due to the low statistical power (60%). The following covariables had a significant effect on the number of transfusions: birth haematocrit (P < 0.001), birth weight (P < 0.001), gestational age (P < 0.001), and SNAP (P < 0.001). When adjusting for these covariables, the cumulative effect showed that the variable photoprotection of TPN was not significant. Running this model without the 39 infants participating in a short crossover between LP and LE regimens did not modify this conclusion. Furthermore, there was no dose-response relation with days of TPN.
Results from the preclinical component of the present study suggest that infusion of neonatal TPN solutions may be detrimental for erythrocyte survival. The higher concentration of plasma haemoglobin measured in animals receiving TPN (Fig. 1) suggests a greater degree of haemolysis. In view of previous in vitro observations reporting that lipid peroxides as well as H2O2 induce erythrocyte fragility, one is tempted to associate the elevated haemolysis observed in TPN with the generation of peroxides in these solutions. It is possible we did not find differences in haemolysis with light shielding in the preclinical study as animals were housed in a 12-hour light/dark cycle, particularly if there is an association with the length of exposure as with the effect of peroxides on endothelial cell viability (31).
In premature infants, the risk of transfusions is highest in the first weeks of life, a time when TPN is administered (Fig. 2). This figure suggests that during the time these babies receive TPN, there may be a lower risk of requiring transfusions in the LP group as shown statistically by a significant interaction between postnatal age and LE/LP; however, when adjusting for the multiple covariables influencing the risk of transfusion, the variable photoprotection was no longer significant. Sick premature infants receive infusions of other preparations such as antibiotics, diuretics, volume expanders, prostaglandin inhibitors, and vasoactive drugs that could affect RBC fragility because of supraphysiological osmolalities. TPN represents by far the largest volume and the one solution presenting the intravascular milieu with the longest exposure to potential toxic effects.
The absence of a demonstrable protective effect against the risk of transfusion by the LP solution, which generates a 50% lower concentration of peroxides, appears to speak against our hypothesis; however, these results could be interpreted as evidence that even the lower concentration of peroxides (200 μmol/L) may be sufficient to induce a maximum haemolytic response in this population that has low antioxidant defences, especially in blood (32,33). Nagababu et al (17) showed a heme degradation of RBC by 100 μmol/L H2O2 when the activities of glutathione peroxidase and catalase were inhibited. From the preclinical study in newborn guinea pigs, it appears that the effect of exposure to TPN on haemolysis occurs after 3 to 4 days. If the duration of exposure to reactive oxygen species did shorten RBC survival and influence the risk of transfusions, one would expect to find a dose-response relation with the duration of exposure to TPN and the peroxide load as shown with cell viability (31). It would be of interest to further investigate whether earlier oxidant injury during TPN infusion that shortened RBC survival and accelerated RBC removal/renewal would continue for many days or weeks following the interruption of TPN or the oxidant stimulus.
Limitations of the present study include potential confounding factors such as RBC transfusion criteria, osmolarity of infused solutions (34), and selection bias. The same written RBC transfusion policy was applied during each trial included in this case-control retrospective analysis (11,25). If a drift in compliance with written RBC transfusions criteria occurred over time, the same modifications should have affected LE as well as LP groups in each trial. Because we are comparing TPN regimens randomly assigned between LE and LP, modifications in compliance over time would unlikely introduce a bias. Furthermore, it would be highly unlikely that large differences in osmolarity between LE and LP regimens would occur and influence haemolysis or need for transfusions. In vitro studies show no effect of osmolarity on haemolysis within the range of osmolarities (500–1200 mOsm/L) of neonatal TPN solutions prepared for peripheral and central lines (16). We did, however, find increased haemolysis when osmolarities reached values in excess of 2300 mOsm/L, which are not consistent with safe clinical practice. The presence of a subset of patients undergoing a crossover did not contribute to change the conclusion of the statistical analysis. This may be explained by the fact that the crossover was limited to 3 days of the 13-day average duration on TPN for the overall population. Finally, it is also worth considering whether the absence of a significant difference in the number of transfusions may be due to the low statistical power (60%); however, because we found results (LP > LE) with a trend opposite from the initial hypothesis, it is unlikely that there is an influence of light exposure on the number of transfusions outside the duration of TPN or even during the period of light protection during the first 2 weeks of life (Fig. 2).
In summary, peroxides are generated in TPN solutions at concentrations above the range reported to cause haemolysis in vitro. Because TPN is associated with haemolysis in animal studies, the pathogenic role of infused peroxides is suspected. If this phenomenon is of clinical significance, the ensuing lower haemoglobin level would necessitate RBC transfusions; however, decreasing the peroxide load by half by shielding TPN from light did not enable us to decrease haemolysis in animal studies or measurably reduce the need for early transfusions in extremely-low-birth-weight premature infants. Nevertheless, based on the results of our preclinical studies, strategies to decrease the concentration or duration of exposure to peroxides may prove useful. Novel approaches to stimulate antioxidant defences include providing, enterally or parenterally, molecules such as hexapeptides found in human milk to prevent the induction of oxidative stress (35). By doing so, we hope to decrease the overall risks to premature infants of RBC transfusions per se (36) because increased redox potential is associated with increased need for transfusion (11).
The authors gratefully acknowledge the professional advice provided by Rollin Brant (Child and Family Research Institute, University of British Columbia, Vancouver, Canada) for the statistical analysis of clinical data.
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