Early nutrition is associated with marked long-term benefits for preterm infants.1 2 In view of the small gastric capacity and functional immaturity of the gastrointestinal tract, very low birth weight premature infants require total parenteral nutrition (TPN) to help achieve adequate nutritional intakes until full enteral feeds can be established.

Exposure of TPN to ambient light generates organic peroxides3 4 and hydrogen peroxide (H₂O₂)5 6 that represent an oxidative load7 which could be of significance in preterm infants who have immature antioxidant defences.8 Photo-sensitised riboflavin present in parenteral multivitamin preparations (MVP) catalyses electron transfer between electron donors such as vitamin C, amino acids or lipids6 and dissolved oxygen producing H₂O₂. Shielding TPN from light protects the solution from the generation of peroxides.9 10 The infusion of light-exposed (LE) MVP or H₂O₂ induces comparable oxidative responses in lungs of guinea pig pups.11 12 In the liver13 and plasma14 of these animals, the infusion of LE MVP is associated with increased triglyceride (TG) concentration suggesting that peroxides infused with TPN might interfere with lipid metabolism. Furthermore, oxidative stress impairs glucose uptake in muscle and fat15; therefore, we questioned whether the generation of oxidants in TPN could influence parameters frequently used to monitor the adequacy of the metabolic response.

The aforementioned studies and results from our trial to evaluate effects of photoprotection of TPN on clinical and biochemical endpoints16^–18 prompted us to test in a post hoc analysis whether photoprotection of TPN in preterm infants affects intermediary metabolism resulting in alterations in plasma glucose and TG concentrations.

METHODS

We conducted a prospective study in which preterm infants were allocated to LE and light-protected (LP) TPN to compare the effects of shielding TPN on clinical16 17 and on biochemical endpoints.18 Infants admitted to the NICU between 2001 and 2004 and requiring TPN were eligible for the study. Infants with multiple congenital anomalies, and those who were unstable (requiring vasopressors, inhaled nitric oxide or paralysis) or had sepsis were excluded. Allocation to TPN regimens was carried out in the pharmacy prior to commencement of the multivitamin/amino acid/dextrose parenteral nutrition (PN) solution using a computer-generated randomisation sequence. PN was initiated from birth with amino acid/dextrose solution providing 2.25 g/kg/d of amino acids. Following introduction of micronutrients, minerals, vitamins and lipids on day 2, TPN was increased daily to achieve full intakes of amino acids, glucose and fat. Lipid was initiated at 0.5 g/kg/d in infants with a birth weight <1000 g, and at 1 g/kg/d in infants >1000 g. PN was increased daily until achieving full TPN intake: 130 ml/kg/day of amino acid/dextrose solution + 1.5 ml/kg/day of multivitamins (MVP: Multi-12 Pediatric, Sandoz, Montréal, QC, Canada) and 20 ml/kg/day of 20% lipid solution (Intralipid 20%, Pharmacia Upjohn, LIEU) introduced into the venous line close to the site of infusion in the baby. Full TPN consisted of amino acids: 3.5–4 g/kg/d, glucose: 12–15 g/kg/d and fat: 3–4 g/kg/d. Light protection of TPN was started with the initiation of MVP and lipids in the regimen. Photoprotection was started from the moment of preparation in the pharmacy and continued throughout the delivery of the solution to the infant. Shielding from light was achieved using protective covering for the TPN bags and syringes and amber tubing (Codan Santa Ana, California, USA); therefore, in the LP group both the amino acid–dextrose solution as well as the lipid emulsion were shielded from light. This procedure decreases the amount of infused peroxides.10 As part of monitoring of TPN, TGs were routinely measured with each increase in lipid intake until reaching full TPN; blood glucose was measured daily when blood glucose was in normal range (>2.6 or <10) or with every change in glucose infusion when blood glucose was outside this range.

Enteral nutrition was initiated within 48 h of birth in stable preterm infants according to a protocol described previously.17 Infants who were not tolerating advancement of early enteral nutrition progressed to full TPN. Daily nutrient intake, parenteral and enteral, was monitored in LE and LP until full TPN was achieved, around days 7–9 after birth.

To test whether photoprotection of TPN in preterm infants affects plasma TGs, a post hoc analysis of TG concentrations was performed in a subgroup of preterm infants receiving LP or LE. In view of the influence of enteral nutrition on plasma TG, only infants on minimal intakes of enteral feeds (<5 ml/kg/d) were included in this analysis. Plasma accumulation of TG was obtained by calculating the slope of plasma TG concentrations (TG, mM) as a function of the amount of lipids received (g/kg/d) during the preceding 24 h. A similar post hoc analysis of blood glucose was performed.

Analytical procedures

TG concentrations were determined on 10 μl of plasma using a colorimetric commercial kit (VITROS Chemical Products TRIG kit, Ortho-Clinical Diagnostics Inc, Rochester, New York, USA). Blood glucose was obtained by point of care testing using the Sure StepFlex® (Life Scan, Canada Ltd, Burnaby, BC, Canada). In these subjects point of care testing correlated with laboratory glucose oxidase technique (y = 0.88x+0.1, r² = 0.95, n = 200).

Statistical analysis

Plasma TG, blood glucose and nutrient intakes (daily averages) were analysed by factorial ANOVA (days × light exposure). Student’s t test was used to compare slopes. Chi² was used to compare proportions of hypoglycaemia and hyperglycaemia determinations. Data are presented as mean ± SEM and the threshold of significance was set at p<0.05.

RESULTS

Sixty-two neonates allocated to LP and 66 randomised to LE were recruited to a study aimed at determining the effects of photoprotecting TPN on nutrient handing.16 17 Of that initial group, 32 LP subjects and 27 LE subjects received full TPN and were therefore included in the present secondary analysis. Mean gestational age (LE: 28±1 vs LP: 28±1 wk), birth weight (1.0±0.1 vs 1.0±0.1 kg), initial severity of illness index SNAP II score19 (LE: 24±4 vs LP: 18±2), did not differ between infants receiving full TPN. Figure 1 documents that there was no difference between LE and LP in the progression over time of the intravenous macronutrient intakes. Upon reaching full TPN mean (SD) intravenous intakes for LE vs LP on day 7 of life were as follows: glucose: 13.9 (0.5) vs 13.2 (0.4) g/kg/d; amino acids: 2.9 (0.1) vs 2.9 (0.1) g/kg/d; lipids: 2.5 (0.2) vs 2.7 (0.1) g/kg/d; MVP: 1.2 (0.1) vs 1.3 (0.1) ml/kg/d. Figure 2 shows that the volume of enteral feeds was <5 ml/kg/d and similar between both groups.

• Download figure
• Open in new tab
• Download powerpoint

Figure 1 Macronutrient intakes as a function of postnatal age. There was no difference in macronutrient intakes between LE and LP. Data expressed as mean ± SEM. LE, infants receiving light-exposed parenteral nutrition (open circle; sample size: 27–32). LP, infants receiving light-protected parenteral nutrition (dark circle; sample size: 20–27).

• Download figure
• Open in new tab
• Download powerpoint

Figure 2 Enteral feeding volumes as a function of postnatal age. Each circle represents a sample size of 14–20 infants. There was no difference between LE and LP. Data expressed as mean ± SEM. LE, infants receiving light-exposed parenteral nutrition (open circle). LP, infants receiving light-protected parenteral nutrition (dark circle).

Effect of light exposure on plasma triglyceride concentration

Plasma TG was compared for the first 9 days of life, while on full TPN. After that age the increasing volume of enteral feeds (>5 ml/kg/d) given every 1–2 h would interfere with TG concentrations. The linearity of the relationship between mean plasma TG and postnatal age (y = 0.11x + 0.47) was significant only in infants receiving LE (r² = 0.78, p<0.01) (fig 3).

• Download figure
• Open in new tab
• Download powerpoint

Figure 3 Plasma triglyceride concentrations as a function of postnatal age. The linear relation between TG and postnatal age was significant in the LE group (y = 0.11x + 0.47, r² = 0.78, p<0.01) but not in the LP group (y = 0.02x + 0.76, r² = 0.26). The factorial ANOVA demonstrated a significant (*p<0.05) interaction between LE and day of life. Plasma triglyceride concentrations were higher in LE group on days 8 (**: p<0.01) and 9 (*: p<0.05). Data expressed as mean ± SEM (sample size  =  8–16). LE, infants receiving light-exposed parenteral nutrition (open circle). LP, infants receiving light-protected parenteral nutrition (dark circle).

The influence of iv lipid intake on plasma TG was evaluated by calculating the individual slope of plasma TG (mM) on the amount of lipids received (g/kg) during the preceding 24 h. The mean (SEM) slope for each group was statistically different from zero (p<0.01) (LE  =  0.238 (0.062); LP = 0.095 (0.032)); the mean slope for the LE group was 2.5 times higher than for the LP group.

The significant (p<0.05) interaction between light exposure and postnatal age allowed us to compare postnatal days, individually. On days 8 and 9 of life, mean (SD) plasma TG was higher in the group receiving LE TPN (1.5 (0.3) vs 0.9 (0.1), p<0.01 and 1.4 (0.2) vs 0.9 (0.1), p<0.05, respectively).

Effect of light exposure on blood glucose concentration

Overall, mean (SEM) blood glucose measured over the first 9 days of life was significantly higher (p<0.001) in LE (6.6 (0.2) mM, n = 215) compared to LP (6.0 (0.1) mM, n = 240). Significant logarithmic relationships between mean daily blood glucose and postnatal age were found for both LE (y = 1.14Ln(x) + 4.75, r² = 0.62, p<0.05) and LP (y = 0.82Ln(x) + 4.86, r² = 0.64, p<0.05) (fig 4) documenting that blood glucose continued to rise with age in spite of the levelling off of glucose intake after day 5 of life (fig 1). There was no interaction between light exposure and days. The number of subjects presenting with hyperglycaemia (>10 mM) was not significantly different between LE (11/32) and LP (6/27). The number of data points in the hyperglycaemia range was significantly higher (p<0.05) in LE: 103/570 compared to LP: 45/512; however, the number of subjects requiring insulin because of hyperglycaemia was not significantly different (LE: n = 6/32 vs LP: n = 4/27). When subjects who required insulin were excluded from this analysis, in order to isolate the effect of photoprotection on blood glucose from any further outside influence, there was no statistically significant difference in mean (SEM) blood glucose between LE (6.1 (0.2) mM, n = 161) and LP (5.9 (0.1) mM, n = 212) over the first 9 days of life.

• Download figure
• Open in new tab
• Download powerpoint

Figure 4 Blood glucose concentrations as a function of postnatal age. The factorial ANOVA demonstrated that overall blood glucose concentrations were higher (p<0.05) in LE (y = 1.14 Ln(x) + 4.75, r² = 0.62, p<0.05) compared to LP (y = 0.82 Ln(x) + 4.86, r² = 0.64, p<0.05) and that there was no interaction with days of life. The correlations between average daily blood glucose and postnatal age were significant (p<0.05). No interaction was found between light exposure and days. Data expressed as mean ± SEM (sample size  =  9–15). LE, infants receiving light-exposed parenteral nutrition (open circle). LP, infants receiving light-protected parenteral nutrition (dark circle).

DISCUSSION

Failure to shield TPN from light contributed to high blood glucose and plasma TG compared to newborn infants receiving photoprotected TPN. The same phenomenon observed in animals receiving TPN,14 supports the hypothesis that light exposure of TPN has an effect on intermediary metabolism. In view of concerns of TPN-induced lipid dysregulation, plasma TG concentration is routinely monitored. An upper limit of 1.7 mM is considered safe in neonates.20 Once this threshold is reached, further increase in energy provided by the fat emulsion is curtailed until lipid clearance improves; therefore, shielding TPN from light provides a potential benefit for preterm infants. By avoiding hypertriglyceridaemia it will allow for increased intake of the lipid emulsion at a time when optimising the provision of energy is important to initiate and sustain growth.21

The two groups of infants (LE and LP) were similar in terms of gestational age, birth weight, and severity of illness, intravenous and enteral feeds; thus, the differences noted in the TG and blood glucose concentrations appear to be related to the degree of light exposure of TPN. Exposure of TPN to light generates oxidants in the form of peroxides and results in loss of antioxidant vitamins.4 Peroxide concentrations range from 255 to 400 μM in LE TPN solutions, while they range from 100 to 175 μM3 6 10 16 in photoprotected preparations. The differences in intermediary metabolism might be associated with differences in peroxides or to other light-induced byproducts generated in the TPN solution.22 23

The difference in blood glucose observed between LE and LP is statistically significant, but it is questionable whether this is of clinical relevance since there was no difference in the need for insulin or the number of subjects treated for hypoglycaemia; however, the higher blood glucose observed during LE fits with the observations that oxidative stress impairs glucose uptake in muscle and fat.15 Since this post hoc analysis involves a fairly small number of subjects it should be viewed as hypothesis generating. Although the actual mechanisms involved in producing these results are currently unclear, several speculations can be entertained. H₂O₂ inhibits insulin receptor binding and insulin receptor autophosphorylation24 proving that increased reactive oxygen species secretion into peripheral blood is involved in induction of insulin resistance25; therefore, LE TPN, which is associated with higher H₂O₂ content, might induce a state of insulin resistance as blood glucose continues to rise after day 5 of life although glucose intake levels off (fig 4).

The liver plays a central role in lipid homeostasis. It constitutes the major site of lipogenesis and very-low-density lipoprotein (VLDL) production involved in lipid transport.12 13 The light-induced hepatic triglyceride accumulation14 could result from enhanced lipogenesis and/or decreased mitochondrial beta oxidation of fatty acids26 27 and/or inhibition of triglyceride hydrolase and/or diminished VLDL secretion. The observed accumulation of plasma TG (fig 3) could be explained by a stimulation of lipogenesis, and/or a lower hepatic uptake. This is supported first by the fact that the difference in plasma TG was observed only after 1 week on TPN and second by the observation that the plasma TG/lipid intake ratio increased faster with LE. The absence of effect of light/peroxides on triglyceride hydrolase14 suggests that the initial step of transport of TG through the endoplasmic reticulum to the VLDL is not involved, which is clearly supported by elevated plasma TG levels. Plasma cholesterol would have informed on the impact of light on the clearance of lipoproteins in which case both TG and cholesterol would have been affected. The higher blood glucose associated with elevated TG is more characteristic of the metabolic syndrome, which is associated with insulin resistance. Conversely, with lipogenesis from glucose one would have expected to find a decrease in circulating glucose rather than the significantly higher blood glucose observed with LE.

There is a risk of selection bias when performing a non-pre-specified post hoc analysis on a subgroup. Therefore, a limitation of this report might stem from the fact that results are derived from a subgroup that represents close to half of the original trial group.16 In view of the confounding influence of enteral nutrition on plasma TG levels in patients who are fed every 2 h, we opted to test the effect of photoprotection on TG in the present subgroup of infants (fig 3) because they received full TPN with minimal enteral support (<5 ml/kg/d). It is reassuring however that we found for the original trial group (LE: n = 58; LP: n = 56) an effect of photoprotection on plasma TG that was in the same direction as in the subgroup on days 8 and 9 (data not shown). On the other hand, there was no difference in blood glucose between LE and LP in the original group.

Our study adds to the list of potential complications associated with failure to protect TPN from light exposure16 17 and adds credence to the positive impact that photoprotection of TPN may have on clinical endpoints, especially in this fragile population. Ethical limitations related to blood sampling in such a vulnerable population, favour investigation of the mechanisms underlying these findings in an animal model14; however, the aforementioned complications associated with failure of light protection of TPN and the impact of being able to increase energy intake early in life beg for further research in the form of a multicentre randomised trial to confirm the effects of photoprotection of TPN on clinical outcomes in preterm newborns.