Oral Sucrose for Heel Lance Increases Triphosphate Use and Oxidative Stress in Preterm Neonates

Yayesh Asmerom, MS1, Laurel Slater, BSN, RNC1, Danilo S. Boskovic, PhD1, Khaled Bahjri, MD2, Megan S. Holden, BS1, Raylene Phillips, MD3, Douglas Deming, MD3, Stephen Ashwal, MD3, Elba Fayard, MD3, and Danilyn M. Angeles, PhD1

Objective To examine the effects of sucrose on pain and biochemical markers of (ATP) degradation and oxidative stress in preterm neonates experiencing a clinically required heel lance. Study design Preterm neonates that met study criteria (n = 131) were randomized into 3 groups: (1) control; (2) heel lance treated with placebo and non-nutritive sucking; and (3) heel lance treated with sucrose and non-nutritive sucking. Plasma markers of ATP degradation (, , and ) and oxidative stress (allantoin) were measured before and after the heel lance. Pain was measured with the Premature Infant Pain Profile. Data were analyzed by the use of repeated-measures ANOVA and Spearman rho. Results We found significant increases in plasma hypoxanthine and uric acid over time in neonates who received sucrose. We also found a significant negative correlation between pain scores and plasma allantoin concentration in a subgroup of neonates who received sucrose. Conclusion A single dose of oral sucrose, given before heel lance, significantly increased ATP use and oxidative stress in premature neonates. Because neonates are given multiple doses of sucrose per day, randomized trials are needed to examine the effects of repeated sucrose administration on ATP degradation, oxidative stress, and injury. (J Pediatr 2013;163:29-35).

remature neonates experience many painful procedures as part of their standard care in the neonatal intensive care Punit.1,2 To prevent or treat procedural pain, the use of oral sucrose is recommended by many national and international clinical guidelines on the basis of results from multiple randomized clinical trials in which investigators determined su- crose to be effective in reducing signs of pain.3 However, there are no studies to date in which investigators examine the effects of sucrose, a disaccharide of fructose and , on neonatal cellular adenosine triphosphate (ATP) , despite the well-documented relationship between fructose metabolism and reductions in ATP synthesis in adult animals,4 in children ages 11 months to 12 years,5 and in healthy adults.5,6 Inhibition of ATP synthesis, as a consequence of sucrose administration, may reduce a premature neonate’s already modest ATP stores.7 In addition, evidence shows that the administration of sucrose does not attenuate the tachycardia that often accompanies painful procedures.8 In neonatal pigs and newborn lambs, tachycardia significantly increased glucose oxidation and myocardial oxygen requirement,9 which paralleled significant reductions in /ATP and significant elevations in adenosine diphosphate (ADP) and inorganic .10 More importantly, sucrose may not be an effective analgesic as evidenced by its lack of impact on neonatal nociceptive circuits in the brain and spinal cord.11 Together, these considerations imply that oral sucrose administration may alter ATP metabolism and may have adverse cellular effects in neonates with limited stores. The aim of this study is to examine the effects of a single dose of oral sucrose on behavioral/physiological markers of pain and biochemical markers of ATP metabolism and oxidative stress in premature neonates experiencing a clinically required heel lance. The heel lance was chosen because it is a frequent painful procedure in the neonatal intensive care unit, as shown in 26 different clinical trials.12 Pain was quantified using the Premature Infant Pain Profile (PIPP),13 ATP metabolism was quan- tified by measuring plasma concentrations of (hypoxanthine, xanthine, and uric acid), which are well-documented markers of ATP use and breakdown, and oxidative stress, measured as plasma concentrations of allantoin, a well-accepted in vivo free radical marker.14 Methods

We conducted a prospective double-blind randomized controlled study at Loma Linda University Children’s Hospital neonatal intensive care unit. Study

From the Departments of 1Basic Sciences, 2Biostatistics, ADP Adenosine diphosphate and 3Pediatrics, Loma Linda University School of ATP Adenosine triphosphate Medicine, Loma Linda, CA GLUT Glucose transporter This study is funded by National Institutes of Health (RO1 NR011209). The authors declare no conflicts of interest. PIPP Premature Infant Pain Profile NNS Non-nutritive sucking 0022-3476/$ - see front matter. Copyright ª 2013 Mosby Inc. All rights reserved. http://dx.doi.org/10.1016/j.jpeds.2012.12.088

29 THE JOURNAL OF PEDIATRICS www.jpeds.com Vol. 163, No. 1 protocol and informed consent documents were approved by heel lance. Multiple studies showed that sucrose was most ef- the Loma Linda University Children’s Hospital Institutional fective when given approximately two minutes before heel Review Board. Subjects included in the study were premature lance.8,18-23 Neonates randomized to the placebo group re- infants #36.5 weeks’ gestation who: (1) weighed $800 g; (2) ceived an equal volume of sterile water to the anterior portion had a central catheter in place; and (3) required a heel lance. of the tongue along with a pacifier. The neonate’s face was Exclusion criteria included neonates: (1) with unstable oxy- videotaped by trained research staff to record facial action genation and hemodynamic status; (2) receiving opioids or at “0” minutes, during the heel lance and up to 30 seconds sedatives or any antiepileptic medications; (3) diagnosed post heel lance. with intraventricular hemorrhage $ grade 3; or (4) with fa- cial or multiple congenital anomalies that might alter the Pain Assessment pain response. The heel lance was performed for an accurate To assess pain, we used the PIPP, an instrument designed to measurement of glucose from neonates receiving assess acute pain in preterm neonates.13 This scoring system glucose-rich total parenteral nutrition through a central cath- includes seven items, each graded from 0 to 3. Two items eter. Parents of premature infants who met study criteria describe baseline characteristics of the neonate (gestational were approached for informed consent as soon after birth age and behavioral state), 2 items are derived from physio- as possible. With consent, subjects were randomized into 1 logic measurements (heart rate and oxygen saturation), and of 3 groups: (1) control; (2) placebo with non-nutritive 3 items describe facial actions (brow bulge, eye squeeze, and sucking (NNS, or pacifier); or (3) sucrose (Sweet-Ease; nasolabial furrow). Baseline pain was scored before the heel Children’s Medical Ventures, Phillips Healthcare, Andover, lance (0 minute) during a 30-second window. Procedural Massachusetts) with NNS (Figure 1; available at www. pain was scored from the time of heel lance to 30 seconds jpeds.com). Randomization was performed by a research after the lance. Facial actions were recorded with a digital pharmacist, who used a permuted block randomization camera with real-time counter that allowed for intensive table generated by the study statistician. slow motion stop frame, videocoding, and playback. Previ- The experimental procedure is described in Figure 2 ous work on validation of the PIPP score showed an ability (available at www.jpeds.com). Investigators collaborated to differentiate painful from non-painful or baseline with the clinical staff to obtain a sample of approximately events.13,24 0.8 mL of blood from a central catheter before (“0” minute) and 5 minutes after the heel lance to measure and Measurement of Purines allantoin levels. In control neonates who did not receive Purine metabolites were measured as previously published by a study drug or undergo a heel lance, similar samples were our laboratory.25 Specifically, plasma was removed, trans- collected at “0” and 5 minutes from baseline. The time ferred to separate Eppendorf tubes, and immediately centri- period of 5 minutes after heel lance for blood sample fuged in Eppendorf 5702R (Pittsburgh, Pennsylvania) collection was based on previous investigations, which centrifuge, for 30 minutes at 18 000 g. The supernatant was showed plasma levels of purines and organic transferred to Microcon centrifugal filter devices (Millipore hydroperoxides significantly increasing 5 minutes after Corp, Bedford, Massachusetts), 200 mL per device, and conditions such as incomplete ischemia.15 These data were spun for 90 minutes at 14 000 g, 4C. Filtrate was removed, validated by unpublished preliminary studies in our and 150 mL was transferred to an Eppendorf tube containing laboratory, where we found increases in plasma purines 1 10 7 mol of 2-aminopurine (internal standard). High- compared with baseline five minutes after heel lance, and performance liquid chromatography (Waters 996 PDA, 715 purine values that were less than baseline, 20-30 minutes Ultra Wisp Sample Processor; Millipore Corp) analysis was after heel lance. Blood samples were centrifuged within 5 done in the same day, or the tubes were frozen at 80C until minutes to separate the plasma which was then stored at analysis. Previous analysis via high-performance liquid chro- 80C. All samples were analyzed within 1 week of matography of plasma demonstrated that purines remained acquisition. stable with freezing. Three 45-mL injections were used for each sample onto Heel Lance Procedure and Administration a Supelcosil LC-18-S 15 cm 4.6 mm, 5-mm column of Study Drug (SGE, Austin, Texas), with the following isocratic conditions: The study drug was prepared immediately before the experi- 50 mM ammonium formate buffer, pH 5.5, flow rate 1.0 mL/ mental procedure by the research pharmacist and labeled as min. Hypoxanthine, xanthine, and uric acid were quantitated “study drug” to ensure blinding. The dose of sucrose was by obtaining peak areas at appropriate retention and wave- based on previously published studies in premature in- lengths.26 Once the peak area of 2-aminopurine at approxi- fants.12,16-18 Neonates randomized to the sucrose group re- mately 10.8 minutes and 305 nm was determined, the area ceived a single dose of 24% sucrose in the following ratios of hypoxanthine, xanthine, and uric acid to 2- volumes: 2 mL for neonates >2 kg, 1.5 mL for neonates 1.5- aminopurine were determined and converted to micromolar 2 kg, and 0.5 mL for neonates that were <1.5 kg. The study concentrations using standard curves. Samples were analyzed drug was administered slowly via syringe to the anterior in triplicates and values with a coefficient of variation of less tongue along with a pacifier (NNS) 2 minutes before the than 10% were included in the final analyses. The limits of

30 Asmerom et al July 2013 ORIGINAL ARTICLES detection for the purines are as follows: 1.58 mM hypoxan- Chicago, Illinois). Differences were considered significant thine, 1.32 mM xanthine, and 5.0 mM uric acid. at P < .05. Measurement of Allantoin. Allantoin was measured in Results plasma using an adaptation of the method developed by Gruber et al27 and Al-Khalaf and Reaveley.28 Plasma (50 m Of the 151 subjects who provided consent between the L) was transferred to an Eppendorf tube containing months of July 2009 and February 2012, 131 subjects were 10 m m 15 5 10 mol internal standard (50 L10 M[ N]-la- randomized into 1 of 3 groups: control (n = 42), heel lance beled allantoin). Spiked samples were simultaneously de- m and placebo (n = 45), or heel lance and 24% sucrose (n = 44; proteinized and extracted by the addition of 100 Lof Figure 1). All subjects randomized to the heel lance groups acetonitrile. These samples were then vortexed and centri- were given a pacifier (NNS) immediately before, during, fuged at 20 000 g, 4C for 5 minutes, and the supernatant m and after study drug administration. There were no was dried under N2. After drying, 50 L of MTBSTFA significant differences between the groups (Table I). (ie, N-methyl-N-tert-butyldimethylsilyltrifluoroacetamide) in pyridine (1:1 vol/vol) was added and the derivatization Effects of Oral Sucrose on Behavioral and reaction was facilitated by incubation at 50 C for 2 hours. Physiological Markers of Pain Analysis was performed on Agilent 6890N Network GC There were no significant differences in baseline pain score System connected to an Agilent 5973 Inert Mass Selective between the 3 groups (Table II). Sucrose significantly Detector (both Agilent Technologies, Inc, Santa Clara, attenuated the increase in pain score in response to heel California). Separation was performed using an Agilent lance, compared with placebo (Table II). The heart rate 122-5532G capillary column (25.7 m length, 0.25 mm inter- response to heel lance was greatest in the sucrose group nal diameter). Helium was used as the carrier gas at a flow (P < .001). Heart rate increased by 11% in the sucrose rate of 1.5 mL/min. Derivatized product (1 mL) was injected group, compared with 6% in the placebo group and 0.5% in split mode (split 20:1, split flow 29.4 mL/min, total flow in the control group. We observed no significant changes in 33.8 mL/min). The initial column temperature was set at mean oxygen saturation in response to heel lance in any of 100 C and held at that temperature for 2 minutes; it was in- the 3 groups. These data suggest that the lower pain scores creased to 180 C at a rate of 10 C/min. The column was in the sucrose group were attributable to significant held at this temperature for 4 minutes and then increased reductions in the behavioral components of the PIPP to 260 C at a rate of 20 C/min. This temperature was main- scoring tool and not from physiological markers of pain tained until the end of the run. Allantoin was quantified us- such as heart rate or oxygen saturation. ing selected ion monitoring mode with the 398.00 m/z ion being monitored for allantoin and the 400.00 m/z for DL- allantoin-5-13C;1-15N. The ion abundance ratios (398.00/ Effects of Oral Sucrose on Markers of ATP 400.00) were converted to micromolar concentrations by Metabolism (Purines) and Oxidative Stress use of a standard curve. (Allantoin) There were no significant differences in baseline purine and Statistical Analyses. A repeated-measures ANOVA with allantoin levels in any of the groups. However, although one between factor (type of intervention) and one within plasma purine and allantoin concentration decreased over factor (time) was used to compute the minimum sample time in subjects randomized to the control and placebo size needed for this study. The sample size was based on groups, we observed a significant increase over time in plasma the following assumptions: (1) the significance level was set hypoxanthine and uric acid in neonates who received sucrose to 0.05; and (2) required power was 80%. After adjusting before the heel lance (Figure 3, A and B). This effect persisted for a 10% dropout rate, we enrolled 42-45 participants per even when analysis was limited to subjects <33 weeks’ group for a total of 131 subjects. gestation at the time of sampling. Xanthine concentrations To analyze the data, assumptions of normality and equal remained stable over time in each of the 3 groups. Plasma variance were assessed. Demographic data for categorical allantoin concentration increased over time in those who variables were analyzed by use of the c2 test. Repeated- received sucrose; however, this effect was not statistically measures ANOVA for one between subject factor (group) significant (data not shown). and one within subject factor (time) were assessed to eval- uate the effect of the heel lance on plasma purines and Effects of Oral Sucrose on Plasma Allantoin allantoin concentrations over time. Interaction terms in in Neonates with a Minimal Pain Response to the general linear model were used for this purpose. The Heel Lance interaction terms assess the differences between the groups We found that 63% of neonates who received sucrose dem- over time. Correlations between purines, allantoin, and onstrated a minimal response to heel lance, defined as an in- biobehavioral markers (PIPP) were examined with the crease in PIPP score of <33%. When we examined the effect Spearman rho. All statistical analyses were performed using of sucrose in this subgroup, we found that plasma allantoin SPSS Statistics for Windows Version 20 (SPSS Inc, concentration increased significantly over time (Figure 3,

Oral Sucrose for Heel Lance Increases Adenosine Triphosphate Use and Oxidative Stress in Preterm Neonates 31 THE JOURNAL OF PEDIATRICS www.jpeds.com Vol. 163, No. 1

Table I. Subject demographics Control (n = 42) Heel lance, placebo-NNS (n = 45) Heel lance, Sweet-Ease/NNS (n = 44) F value P value EGA, weeks 30.5 2.6 30.3 3.2 30.1 3.1 0.218* .804 Birth weight, g 1456.4 502 1498.4 706 1374.1 552 0.499* .608 Apgar, 1 minute 5 35 36 2 0.961* .385 Apgar, 5 minute 7 27 27 2 1.008* .368 Sex, n (%) 1.175† .556 Male 25 (60%) 22 (49%) 22 (50%) Female 17 (40%) 23 (51%) 22 (50%) Race, n (%) 4.277† .831 White 15 (36%) 14 (31%) 19 (43%) Hispanic 15 (36%) 21 (47%) 15 (34%) African American 6 (14%) 7 (16%) 6 (14%) Asian 2 (4%) 2 (4%) 2 (4.5%) Other 4 (10%) 1 (2%) 2 (4.5%) Condition at time of sampling FiO2,% 0.25 0.07 0.26 0.09 0.23 0.03 1.783* .172 EGA, weeks 32.5 2.3 32.6 2.6 33.1 2.1 0.759* .470 SNAPPE-II 7.8 10.8 7.9 11.9 8.3 11.6 0.020* .980 † Mode of O2 delivery 6.861 .334 Spontaneous RA 19 19 24 Nasal cannula 6 11 11 NCPAP 6 6 1 NIPPV 11 0 8 Hemoglobin 13.1 2.4 12.7 2.3 12.5 2.2 0.846* .432 Hematocrit 38.7 6.5 37.6 6.4 36.9 5.7 0.844* .433

EGA, estimated gestational age; NCPAP, nasal continuous positive airway pressure; NIPPV, noninvasive intermittent positive pressure ventilation; RA, room air; SNAPPE-II, Score for Neonatal Acute PhysiologyPerinatal Extension-II. *Repeated-measure ANOVA. †c2 test.

C). When we examined the correlation between the percent use/depletion, and increased purine production is well change in PIPP pain score over time and the percent documented in adult animal and human literature change in allantoin concentration over time, we found (Figure 4).6,30,31 Sucrose is a disaccharide of glucose and a significant negative correlation (Spearman rho, 0.378, fructose. It is hydrolyzed by sucrase, an secreted by P = .014), suggesting that although sucrose significantly epithelial cells of the villi in the small intestine (Figure 4, decreased the pain scores, it also increased markers of A). Both glucose and fructose are rapidly absorbed from oxidative stress in this subgroup of premature neonates. the gastrointestinal tract through glucose transporter (GLUT) 5 (fructose) and sodium-glucose cotransporter/ Discussion GLUT2 (glucose) transporters in the apical membrane and transferred to the portal circulation via GLUT2 Although oral sucrose given before a single heel lance signif- transporters in the basolateral membrane of enterocytes icantly decreased behavioral markers of pain, consistent with (Figure 4, B). the findings of numerous clinical investigators,8,17,23,29 it also The expression of these GLUTs is up-regulated by exposure increased markers of ATP use, as evidenced by significant in- of intestinal lumen to fructose solutions32 or by previous expo- creases over time in plasma hypoxanthine and uric sure to corticosteroids.33 Once in circulation, glucose uptake is acid concentrations. The relationship between sucrose, ATP insulin-dependent while fructose uptake is independent of

Table II. Pain score, heart rate, and oxygen saturation Control (n = 42) Heel lance, placebo-NNS (n = 45) Heel lance, Sweet-Ease/NNS (n = 44) F value P value Pain score Mean (min-max) Baseline 3.9 (1-8) 3.8 (1-8) 3.8 (1-7) 0.233* .792 Procedural 5.9 (2-15) 6.3 (2-12) 4.6 (2-10) 6.216* .003† Heart rate Baseline 154.4 (12.8) 155.9 (14.4) 154.1 (13.3) 0.206* .814 Procedural 154.9 (13.9) 164.9 (14.6) 170.5 (14.7) 14.480* <.001z Oxygen saturation Baseline 96.5 (0.5) 96.2 (0.5) 96.2 (0.5) 0.123* .885 Procedural 96.2 (0.6) 95.8 (0.6) 96.4 (0.6) 0.235* .791

*Repeated-measures ANOVA. †Sweet-Ease group significantly lower than control or placebo groups. zControl group significantly lower than both heel lance groups.

32 Asmerom et al July 2013 ORIGINAL ARTICLES

(the enzyme that phosphorylates glucose). Moreover, activity is relatively unregulated, being limited only by fructose concentration.34 Fructose-1- phosphate is split by aldolase (aldolase B) into glyceraldehyde and dihydroxyacetone phosphate, a member of the sequence of intermediates. The third enzyme in the fructose pathway is , which catalyzes the of glyceraldehyde to glyceraldehyde-3- phosphate, another intermediate in the glycolytic pathway. These fructose-related biochemical reactions are signifi- cant because each phosphorylation step requires ATP.31 As ATP is consumed, it is degraded to ADP, leading to an in- crease in ADP concentration.4 Simultaneously, inorganic phosphate levels decrease because they are sequestered in fructose-1-phosphate or the mitochondria to generate ATP necessary to maintain fructose phosphorylation (Figure 4, D).4 As inorganic phosphate concentration decreases, oxidative phosphorylation is inhibited, reducing ATP synthesis and rapidly depleting ATP.4,6,30,31 ATP and ADP results in increased concentration of purines such as uric acid (Figure 4, E).5,6,35 These observations have been documented in adult animals as well as in children ages 11 months to 12 years5 and in healthy adults.5,6 We show similar effects in preterm neonates, in which a single dose of sucrose significantly increased plasma hypoxanthine and uric acid concentrations. We also found that neonates in the sucrose treatment group had the largest increase in heart rate compared with those in the control or placebo groups, providing additional evidence that sucrose does not attenuate the tachycardia that accompanies painful procedures, but may increase it. This in- crease in heart rate may be due to the stimulatory effect of su- crose on the sympathetic nervous system, as shown in rats36,37 and healthy young adults.38 Interestingly, in humans, the sympathetic response to sucrose ingestion was enhanced under conditions of acute moderate hypoxia.39 Together, these data suggest that the apparent analgesic effect of oral su- crose may come at a price, namely tachycardia, which in turn contributes to increased ATP use. An additional finding of this study is the effect of sucrose administration on plasma allantoin concentration in neo- nates with reduced pain responses (PIPP score increased by <33%). We found that in this subgroup, plasma allantoin concentration significantly increased over time, compared with neonates with a larger pain response (defined as having an increase in PIPP score of $34% compared with baseline; Figure 3, C). The demographics of this subgroup of Figure 3. A B, and Plasma hypoxanthine and uric acid con- neonates were not significantly different from those with centration increased over time in preterm neonates who re- a larger pain response. Although newborns with reduced ceived oral sucrose before a clinically required heel lance. C, In neonates with minimal pain response (<33% increase in pain responses had significantly lower baseline heart rates P PIPP with heel lance), plasma allantoin concentration in- (151 11.6 beats/min vs 159 14.4 beats/min, = .028), creased over time. they tended to have a greater percent change in heart rate with the heel lance procedure compared with those with larger pain responses (12 7% vs 9 7%, P = .290). These insulin.4 Inside the cell, fructose is rapidly phosphorylated into data suggest that changes in heart rate due to sucrose fructose-1-phosphate by the enzyme fructokinase (Figure 4, administration may be more predictive of oxidative stress C). The activity of fructokinase is 4-fold greater than than changes in facial or behavioral activity. Additional

Oral Sucrose for Heel Lance Increases Adenosine Triphosphate Use and Oxidative Stress in Preterm Neonates 33 THE JOURNAL OF PEDIATRICS www.jpeds.com Vol. 163, No. 1

Figure 4. Sucrose metabolism. A, Sucrose is hydrolyzed into fructose and glucose by the enzyme sucrase. B, Glucose and fructose are absorbed from the gastrointestinal tract, transferred to the portal circulation, and enter hepatocytes. C, Fructose is phosphorylated to form fructose-1-phosphate by the enzyme fructokinase. D, Fructose phosphorylation rapidly depletes the cell of ATP and inorganic phosphate. E, Decreases in ATP production, in addition to increased ATP utilization, results in increased hy- poxanthine, xanthine, and uric acid production. If this is combined with increased oxidative stress, uric acid can be oxidized to form allantoin. SGLT, sodium-glucose cotransporter; ISF, interstitial fluid; TCA, tricarboxylic acid cycle; IMP, monophosphate. studies are required to examine the relationship between increase in allantoin may not be always evident five minutes sucrose administration, pain reactivity, and oxidative stress. after the heel lance procedure. A limitation of this study is that although statistical power In this prospective, randomized, double-blind study, we of more than 80% was achieved for hypoxanthine and uric made the observation that a single dose of oral sucrose, given acid, it was not achieved for allantoin. A larger sample size before a heel lance, significantly increased markers of ATP may be required to adequately examine the effect of sucrose use and oxidative stress in premature neonates over time. treatment on allantoin. It is possible that a significant This finding suggests that the apparent analgesic effect of

34 Asmerom et al July 2013 ORIGINAL ARTICLES oral sucrose may come at a price, namely increased ATP use. 16. Ramenghi LA, Wood CM, Griffith GC, Levene MI. Reduction of pain re- Because neonates can be exposed to numerous painful proce- sponse in premature infants using intraoral sucrose. Arch Dis Child dures per day requiring multiple doses of sucrose, random- 1996;74:F126-8. 17. Acharya AB, Annamali S, Taub NA, Field D. Oral sucrose analgesia for ized trials should be performed to examine the effects of preterm infant venepuncture. Arch Dis Child Fetal Neonatal Ed 2004; repeated sucrose administration not only on markers of 89:F17-8. ATP breakdown and oxidative stress but also on cellular in- 18. McCullough S, Halton T, Mowbray D, Macfarlane PI. Lingual sucrose jury. If it is determined that the metabolic risks of using su- reduces the pain response to nasogastric tube insertion: a randomised crose in neonates is indeed greater than the known benefits of clinical trial. Arch Dis Child Fetal Neonatal Ed 2008;93:F100-3. 19. Stevens B, Ohlsson A. Sucrose for analgesia in newborn infants undergo- reducing behavioral indices of pain, additional studies need ing painful procedures. Cochrane Database Syst Rev 2000;CD001069. to be performed to identify alternative effective substances 20. Stevens B, Yamada J, Beyene J, Gibbins S, Petryshen P, Stinson J, et al. or methods to prevent or treat pain in neonates. n Consistent management of repeated procedural pain with sucrose in pre- term neonates: is it effective and safe for repeated use over time? Clin J We want to thank Desiree Wallace, PharmD and all the nurses and Pain 2005;21:543-8. physicians at Loma Linda University Children’s Hospital for their 21. Johnston CC, Stremler RL, Stevens BJ, Horton LJ. Effectiveness of oral support of this study. sucrose and simulated rocking on pain response in preterm neonates. Pain 1997;72:193-9. 22. Blass EM, Watt LB. Suckling- and sucrose-induced analgesia in human Submitted for publication Aug 22, 2012; last revision received Oct 30, 2012; newborns. Pain 1999;83:611-23. accepted Dec 27, 2012. 23. Gibbins S, Stevens B, Hodnett E, Pinelli J, Ohlsson A, Darlington G. Ef- Reprint requests: Danilyn M. Angeles, PhD, Division of Physiology, ficacy and safety of sucrose for procedural pain relief in preterm and Department of Basic Sciences, Loma Linda University School of Medicine, term neonates. Nursing Res 2002;51:375-82. Loma Linda, CA 92350. E-mail: [email protected] 24. Ballantyne M, Stevens B, McAllister M, Dionne K, Jack A. Validation of the premature infant pain profile in the clinical setting. Clin J Pain 1999; References 15:297-303. 25. Slater L, Asmerom Y, Boskovic DS, Bahjri K, Plank MS, Angeles KR, et al. 1. Johnston CC, Collinge JM, Henderson SJ, Anand KJ. A cross-sectional Procedural pain and oxidative stress in premature neonates. J Pain 2012; survey of pain and pharmacological analgesia in Canadian neonatal in- 13:590-7. tensive care units. Clin J Pain 1997;13:308-12. 26. Calderon TC, Wu W, Rawson RA, Sakala EP, Sowers LC, Boskovic DS, 2. Simons SH, van Dijk M, Anand KS, Roofthooft D, van Lingen RA, et al. Effect of mode of birth on purine and malondialdehyde in umbilical Tibboel D. Do we still hurt newborn babies? A prospective study of pro- arterial plasma in normal term newborns. J Perinatol 2008;28:475-81. cedural pain and analgesia in neonates. Arch Pediatri Adolesc Med 2003; 27. Gruber J, Tang SY, Jenner AM, Mudway I, Blomberg A, Behndig A, et al. 157:1058-64. Allantoin in human plasma, serum, and nasal-lining fluids as a bio- 3. Batton DG, Barrington KJ, Wallman C. Prevention and management of marker of oxidative stress: avoiding artifacts and establishing real pain in the neonate: an update. Pediatrics 2006;118:2231-41. in vivo concentrations. Antioxid Redox Signal 2009;11:1767-76. 4. Mayes PA. Intermediary metabolism of fructose. Am J Clin Nutr 1993; 28. Al-Khalaf DVPD, Reaveley JMCA. Assay of serum allantoin in humans by 58(5 Suppl):754S-65S. gas chromatography-mass spectrometry. Clin Chim Acta 2002;318:63-70. 5. Perheentupa J, Raivio K. Fructose-induced hyperuricaemia. Lancet 29. Johnston CC, Filion F, Snider L, Majnemer A, Limperopoulos C, 1967;2:528-31. Walker CD, et al. Routine sucrose analgesia during the first week of 6. Sahebjami H, Scalettar R. Effects of fructose infusion on lactate and uric life in neonates younger than 31 weeks’ postconceptional age. Pediatrics acid metabolism. Lancet 1971;1:366-9. 2002;110:523-8. 7. Gustafsson J. Neonatal energy substrate production. Indian J Med Res 30. Douard V, Ferraris RP. Regulation of the fructose transporter GLUT5 in 2009;130:618-23. health and disease. Am J Physiol Endocrinol Metab 2008;295:E227-37. 8. Harrison D, Johnston L, Loughnan P. Oral sucrose for procedural pain 31. Raivio KO, Kekomaki MP, Maenpaa PH. Depletion of liver nu- in sick hospitalized infants: a randomized-controlled trial. J Paediatr cleotides induced by D-fructose. Dose-dependence and specificity of the Child Health 2003;39:591-7. fructose effect. Biochem Pharmacol 1969;18:2615-24. 9. Ascuitto RJ, Joyce JJ, Ross-Ascuitto NT. Mechanical function and sub- 32. David ES, Cingari DS, Ferraris RP. Dietary induction of intestinal fruc- strate oxidation in the neonatal pig heart subjected to pacing-induced tose absorption in weaning rats. Pediatr Res 1995;37:777-82. tachycardia. Mol Genet Metab 1999;66:212-23. 33. Douard V, Cui XL, Soteropoulos P, Ferraris RP. Dexamethasone sensi- 10. Portman MA, Heineman FW, Balaban RS. Developmental changes in the tizes the neonatal intestine to fructose induction of intestinal fructose relation between phosphate metabolites and oxygen consumption in the transporter (Slc2A5) function. Endocrinology 2008;149:409-23. sheep heart in vivo. J Clin Invest 1989;83:456-64. 34. Heinz F, Lamprecht W, Kirsch J. of fructose metabolism in hu- 11. Slater R, Cornelissen L, Fabrizi L, Patten D, Yoxen J, Worley A, et al. Oral man liver. J Clin Invest 1968;47:1826-32. sucrose as an analgesic drug for procedural pain in newborn infants: 35. Narins RG, Weisberg JS, Myers AR. Effects of carbohydrates on uric acid a randomised controlled trial. Lancet 2010;376:1225-32. metabolism. Metabolism 1974;23:455-65. 12. Stevens B, Yamada J, Ohlsson A. Sucrose for analgesia in newborn in- 36. Walgren MC, Young JB, Kaufman LN, Landsberg L. The effects of var- fants undergoing painful procedures. Cochrane Database Syst Rev ious carbohydrates on sympathetic activity in heart and interscapular 2010;CD001069. brown of the rat. Metabolism 1987;36:585-94. 13. Stevens B, Johnston C, Petryshen P, Taddio A. Premature Infant Pain 37. Young JB, Weiss J, Boufath N. Effects of dietary monosaccharides on Profile: development and initial validation. Clin J Pain 1996;12:13-22. sympathetic nervous system activity in adipose tissues of male rats. Di- 14. Hicks M, Wong LS, Day RO. Identification of products from oxidation abetes 2004;53:1271-8. of uric acid induced by hydroxyl radicals. Free Radic Res Commun 1993; 38. Rousmans S, Robin O, Dittmar A, Vernet-Maury E. Autonomic nervous 18:337-51. system responses associated with primary tastes. Chem Senses 2000;25: 15. Lazzarino G, Vagnozzi R, Tavazzi B, Pastore FS, Dipierro D, Siragusa P, 709-18. et al. Mda, oxypurines, and relate to reperfusion in short- 39. Klemenc M, Maver J, Princi T, Flander P, Golja P. The effect of sucrose term incomplete cerebral-ischemia in the rat. Free Radic Biol Med ingestion on autonomic nervous system function in young subjects dur- 1992;13:489-98. ing acute moderate hypoxia. Eur J Appl Physiol 2008;104:803-12.

Oral Sucrose for Heel Lance Increases Adenosine Triphosphate Use and Oxidative Stress in Preterm Neonates 35 THE JOURNAL OF PEDIATRICS www.jpeds.com Vol. 163, No. 1

Figure 1. Enrollment flow chart. )Reasons why subjects were not sampled include: change in acuity or failure to meet study criteria after consent was obtained (n = 9); central line will not draw or were being infused preventing sampling (n = 5); and line was discontinued before sampling could be sched- uled (n = 6).

Figure 2. Study procedure.

35.e1 Asmerom et al