Oral Sucrose for Heel Lance Increases Adenosine TriphosphateUse 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 adenosine triphosphate (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-nutritivesucking. Plasmamarkers of ATP degradation (hypoxanthine, xanthine, and uric acid) and oxidative stress (allantoin)were measured before and after the heel lance. Pain was measured with the Premature Infant Pain Profile. Datawere analyzed by the use of repeated-measures ANOVA and Spearman rho.ResultsWe found significant increases in plasma hypoxanthine and uric acid over time in neonates who receivedsucrose.We also found a significant negative correlation between pain scores and plasma allantoin concentration ina subgroup of neonates who received sucrose.Conclusion A single dose of oral sucrose, given before heel lance, significantly increased ATP use and oxidativestress in premature neonates. Because neonates are givenmultiple doses of sucrose per day, randomized trials areneeded to examine the effects of repeated sucrose administration on ATP degradation, oxidative stress, and cellinjury. (J Pediatr 2013;163:29-35).

Premature neonates experience many painful procedures as part of their standard care in the neonatal intensive careunit.1,2 To prevent or treat procedural pain, the use of oral sucrose is recommended by many national and internationalclinical 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 effectsof sucrose, a disaccharide of fructose and glucose, on neonatal cellular adenosine triphosphate (ATP) metabolism, despite thewell-documented relationship between fructose metabolism and reductions in ATP synthesis in adult animals,4 in children ages11 months to 12 years,5 and in healthy adults.5,6

InhibitionofATPsynthesis, as a consequenceof sucrose administration,may reduce a premature neonate’s alreadymodestATPstores.7 In addition, evidence shows that the administration of sucrose does not attenuate the tachycardia that often accompaniespainful procedures.8 In neonatal pigs and newborn lambs, tachycardia significantly increased glucose oxidation and myocardialoxygen requirement,9 which paralleled significant reductions in phosphocreatine/ATP and significant elevations in adenosinediphosphate (ADP) and inorganic phosphate.10 More importantly, sucrose may not be an effective analgesic as evidenced byits lack of impact on neonatal nociceptive circuits in the brain and spinal cord.11 Together, these considerations imply thatoral sucrose administrationmay alterATPmetabolismandmayhave adverse cellular effects inneonateswith limited energy stores.

The aim of this study is to examine the effects of a single dose of oral sucrose on behavioral/physiological markers of pain andbiochemical markers of ATP metabolism and oxidative stress in premature neonates experiencing a clinically required heellance. The heel lance was chosen because it is a frequent painful procedure in the neonatal intensive care unit, as shown in26 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 purines (hypoxanthine, xanthine, and uric acid), which are well-documentedmarkers of ATP use and breakdown, and oxidative stress, measured as plasma concentrations of allantoin, a well-acceptedin vivo free radical marker.14

ADP

ATP

GLUT

PIPP

NNS

Methods

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

From the Departments of 1Basic Sciences, 2Biostatistics,and 3Pediatrics, Loma Linda University School ofMedicine, Loma Linda, CA

This study is funded by National Institutes of Health (RO1NR011209). The authors declare no conflicts of interest.

0022-3476/$ - see front matter. Copyright ª 2013 Mosby Inc.

All rights reserved. http://dx.doi.org/10.1016/j.jpeds.2012.12.088

Adenosine diphosphate

Adenosine triphosphate

Glucose transporter

Premature Infant Pain Profile

Non-nutritive sucking

29

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protocol and informed consent documents were approved bythe Loma Linda University Children’s Hospital InstitutionalReview Board. Subjects included in the study were prematureinfants#36.5 weeks’ gestation who: (1) weighed$800 g; (2)had a central catheter in place; and (3) required a heel lance.Exclusion criteria included neonates: (1) with unstable oxy-genation and hemodynamic status; (2) receiving opioids orsedatives or any antiepileptic medications; (3) diagnosedwith intraventricular hemorrhage $ grade 3; or (4) with fa-cial or multiple congenital anomalies that might alter thepain response. The heel lance was performed for an accuratemeasurement of blood glucose from neonates receivingglucose-rich total parenteral nutrition through a central cath-eter. Parents of premature infants who met study criteriawere approached for informed consent as soon after birthas possible. With consent, subjects were randomized into 1of 3 groups: (1) control; (2) placebo with non-nutritivesucking (NNS, or pacifier); or (3) sucrose (Sweet-Ease;Children’s Medical Ventures, Phillips Healthcare, Andover,Massachusetts) with NNS (Figure 1; available at www.jpeds.com). Randomization was performed by a researchpharmacist, who used a permuted block randomizationtable generated by the study statistician.

The experimental procedure is described in Figure 2(available at www.jpeds.com). Investigators collaboratedwith the clinical staff to obtain a sample of approximately0.8 mL of blood from a central catheter before (“0” minute)and 5 minutes after the heel lance to measure purine andallantoin levels. In control neonates who did not receivea study drug or undergo a heel lance, similar samples werecollected at “0” and 5 minutes from baseline. The timeperiod of 5 minutes after heel lance for blood samplecollection was based on previous investigations, whichshowed plasma levels of purines and organichydroperoxides significantly increasing 5 minutes afterconditions such as incomplete ischemia.15 These data werevalidated by unpublished preliminary studies in ourlaboratory, where we found increases in plasma purinescompared with baseline five minutes after heel lance, andpurine values that were less than baseline, 20-30 minutesafter heel lance. Blood samples were centrifuged within 5minutes to separate the plasma which was then stored at�80�C. All samples were analyzed within 1 week ofacquisition.

Heel Lance Procedure and Administrationof Study DrugThe study drug was prepared immediately before the experi-mental procedure by the research pharmacist and labeled as“study drug” to ensure blinding. The dose of sucrose wasbased on previously published studies in premature in-fants.12,16-18 Neonates randomized to the sucrose group re-ceived a single dose of 24% sucrose in the followingvolumes: 2 mL for neonates >2 kg, 1.5 mL for neonates 1.5-2 kg, and 0.5 mL for neonates that were <1.5 kg. The studydrug was administered slowly via syringe to the anteriortongue along with a pacifier (NNS) 2 minutes before the

30

heel lance. Multiple studies showed that sucrose was most ef-fective when given approximately two minutes before heellance.8,18-23 Neonates randomized to the placebo group re-ceived an equal volume of sterile water to the anterior portionof the tongue along with a pacifier. The neonate’s face wasvideotaped by trained research staff to record facial actionat “0” minutes, during the heel lance and up to 30 secondspost heel lance.

Pain AssessmentTo assess pain, we used the PIPP, an instrument designed toassess acute pain in preterm neonates.13 This scoring systemincludes seven items, each graded from 0 to 3. Two itemsdescribe baseline characteristics of the neonate (gestationalage and behavioral state), 2 items are derived from physio-logic measurements (heart rate and oxygen saturation), and3 items describe facial actions (brow bulge, eye squeeze, andnasolabial furrow). Baseline pain was scored before the heellance (0 minute) during a 30-second window. Proceduralpain was scored from the time of heel lance to 30 secondsafter the lance. Facial actions were recorded with a digitalcamera with real-time counter that allowed for intensiveslow motion stop frame, videocoding, and playback. Previ-ous work on validation of the PIPP score showed an abilityto differentiate painful from non-painful or baselineevents.13,24

Measurement of PurinesPurine metabolites were measured as previously published byour laboratory.25 Specifically, plasma was removed, trans-ferred to separate Eppendorf tubes, and immediately centri-fuged in Eppendorf 5702R (Pittsburgh, Pennsylvania)centrifuge, for 30 minutes at 18 000 g. The supernatant wastransferred to Microcon centrifugal filter devices (MilliporeCorp, Bedford, Massachusetts), 200 mL per device, andspun for 90 minutes at 14 000 g, 4�C. Filtrate was removed,and 150 mL was transferred to an Eppendorf tube containing1 � 10�7 mol of 2-aminopurine (internal standard). High-performance liquid chromatography (Waters 996 PDA, 715Ultra Wisp Sample Processor; Millipore Corp) analysis wasdone in the same day, or the tubes were frozen at�80�C untilanalysis. Previous analysis via high-performance liquid chro-matography of plasma demonstrated that purines remainedstable with freezing.Three 45-mL injections were used for each sample onto

a Supelcosil LC-18-S 15 cm � 4.6 mm, 5-mm column(SGE, Austin, Texas), with the following isocratic conditions:50 mM ammonium formate buffer, pH 5.5, flow rate 1.0 mL/min. Hypoxanthine, xanthine, and uric acid were quantitatedby obtaining peak areas at appropriate retention and wave-lengths.26 Once the peak area of 2-aminopurine at approxi-mately 10.8 minutes and 305 nm was determined, the arearatios of hypoxanthine, xanthine, and uric acid to 2-aminopurine were determined and converted to micromolarconcentrations using standard curves. Samples were analyzedin triplicates and values with a coefficient of variation of lessthan 10% were included in the final analyses. The limits of

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detection for the purines are as follows: 1.58 mM hypoxan-thine, 1.32 mM xanthine, and 5.0 mM uric acid.

Measurement of Allantoin. Allantoin was measured inplasma using an adaptation of the method developed byGruber et al27 and Al-Khalaf and Reaveley.28 Plasma (50mL) was transferred to an Eppendorf tube containing5 � 10�10 mol internal standard (50 mL 10 mM [15N]-la-beled allantoin). Spiked samples were simultaneously de-proteinized and extracted by the addition of 100 mL ofacetonitrile. These samples were then vortexed and centri-fuged at 20 000 g, 4�C for 5 minutes, and the supernatantwas dried under N2. After drying, 50 mL of MTBSTFA(ie, N-methyl-N-tert-butyldimethylsilyltrifluoroacetamide)in pyridine (1:1 vol/vol) was added and the derivatizationreaction was facilitated by incubation at 50�C for 2 hours.Analysis was performed on Agilent 6890N Network GCSystem connected to an Agilent 5973 Inert Mass SelectiveDetector (both Agilent Technologies, Inc, Santa Clara,California). Separation was performed using an Agilent122-5532G capillary column (25.7 m length, 0.25 mm inter-nal diameter). Helium was used as the carrier gas at a flowrate of 1.5 mL/min. Derivatized product (1 mL) was injectedin split mode (split 20:1, split flow 29.4 mL/min, total flow33.8 mL/min). The initial column temperature was set at100�C and held at that temperature for 2 minutes; it was in-creased to 180�C at a rate of 10�C/min. The column washeld at this temperature for 4 minutes and then increasedto 260�C at a rate of 20�C/min. This temperature was main-tained until the end of the run. Allantoin was quantified us-ing selected ion monitoring mode with the 398.00 m/z ionbeing monitored for allantoin and the 400.00 m/z for DL-allantoin-5-13C;1-15N. The ion abundance ratios (398.00/400.00) were converted to micromolar concentrations byuse of a standard curve.

Statistical Analyses. A repeated-measures ANOVA withone between factor (type of intervention) and one withinfactor (time) was used to compute the minimum samplesize needed for this study. The sample size was based onthe following assumptions: (1) the significance level was setto 0.05; and (2) required power was 80%. After adjustingfor a 10% dropout rate, we enrolled 42-45 participants pergroup for a total of 131 subjects.

To analyze the data, assumptions of normality and equalvariance were assessed. Demographic data for categoricalvariables were analyzed by use of the c2 test. Repeated-measures ANOVA for one between subject factor (group)and one within subject factor (time) were assessed to eval-uate the effect of the heel lance on plasma purines andallantoin concentrations over time. Interaction terms inthe general linear model were used for this purpose. Theinteraction terms assess the differences between the groupsover time. Correlations between purines, allantoin, andbiobehavioral markers (PIPP) were examined with theSpearman rho. All statistical analyses were performed usingSPSS Statistics for Windows Version 20 (SPSS Inc,

Oral Sucrose for Heel Lance Increases Adenosine Triphosphate U

Chicago, Illinois). Differences were considered significantat P < .05.

Results

Of the 151 subjects who provided consent between themonths of July 2009 and February 2012, 131 subjects wererandomized into 1 of 3 groups: control (n = 42), heel lanceand placebo (n = 45), or heel lance and 24% sucrose (n = 44;Figure 1). All subjects randomized to the heel lance groupswere given a pacifier (NNS) immediately before, during,and after study drug administration. There were nosignificant differences between the groups (Table I).

Effects of Oral Sucrose on Behavioral andPhysiological Markers of PainThere were no significant differences in baseline pain scorebetween the 3 groups (Table II). Sucrose significantlyattenuated the increase in pain score in response to heellance, compared with placebo (Table II). The heart rateresponse to heel lance was greatest in the sucrose group(P < .001). Heart rate increased by 11% in the sucrosegroup, compared with 6% in the placebo group and 0.5%in the control group. We observed no significant changes inmean oxygen saturation in response to heel lance in any ofthe 3 groups. These data suggest that the lower pain scoresin the sucrose group were attributable to significantreductions in the behavioral components of the PIPPscoring tool and not from physiological markers of painsuch as heart rate or oxygen saturation.

Effects of Oral Sucrose on Markers of ATPMetabolism (Purines) and Oxidative Stress(Allantoin)There were no significant differences in baseline purine andallantoin levels in any of the groups. However, althoughplasma purine and allantoin concentration decreased overtime in subjects randomized to the control and placebogroups, we observed a significant increase over time in plasmahypoxanthine and uric acid in neonates who received sucrosebefore the heel lance (Figure 3, A and B). This effect persistedeven when analysis was limited to subjects <33 weeks’gestation at the time of sampling. Xanthine concentrationsremained stable over time in each of the 3 groups. Plasmaallantoin concentration increased over time in those whoreceived sucrose; however, this effect was not statisticallysignificant (data not shown).

Effects of Oral Sucrose on Plasma Allantoinin Neonates with a Minimal Pain Response toHeel LanceWe found that 63% of neonates who received sucrose dem-onstrated a minimal response to heel lance, defined as an in-crease in PIPP score of <33%. When we examined the effectof sucrose in this subgroup, we found that plasma allantoinconcentration increased significantly over time (Figure 3,

se and Oxidative Stress in Preterm Neonates 31

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* .804Birth weight, g 1456.4 � 502 1498.4 � 706 1374.1 � 552 0.499* .608Apgar, 1 minute 5 � 3 5 � 3 6 � 2 0.961* .385Apgar, 5 minute 7 � 2 7 � 2 7 � 2 1.008* .368Sex, n (%) 1.175† .556Male 25 (60%) 22 (49%) 22 (50%)Female 17 (40%) 23 (51%) 22 (50%)

Race, n (%) 4.277† .831White 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 samplingFiO2, % 0.25 � 0.07 0.26 � 0.09 0.23 � 0.03 1.783* .172EGA, weeks 32.5 � 2.3 32.6 � 2.6 33.1 � 2.1 0.759* .470SNAPPE-II 7.8 � 10.8 7.9 � 11.9 8.3 � 11.6 0.020* .980Mode of O2 delivery 6.861† .334

Spontaneous RA 19 19 24Nasal cannula 6 11 11NCPAP 6 6 1NIPPV 11 0 8

Hemoglobin 13.1 � 2.4 12.7 � 2.3 12.5 � 2.2 0.846* .432Hematocrit 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 AcutePhysiology�Perinatal Extension-II.*Repeated-measure ANOVA.†c2 test.

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C). When we examined the correlation between the percentchange in PIPP pain score over time and the percentchange in allantoin concentration over time, we founda significant negative correlation (Spearman rho, �0.378,P = .014), suggesting that although sucrose significantlydecreased the pain scores, it also increased markers ofoxidative stress in this subgroup of premature neonates.

Discussion

Although oral sucrose given before a single heel lance signif-icantly decreased behavioral markers of pain, consistent withthe findings of numerous clinical investigators,8,17,23,29 it alsoincreased markers of ATP use, as evidenced by significant in-creases over time in plasma hypoxanthine and uricacid concentrations. The relationship between sucrose, ATP

Table II. Pain score, heart rate, and oxygen saturation

Control (n = 42) Heel lance, placebo-NNS (n = 45

Pain scoreMean (min-max)Baseline 3.9 (1-8) 3.8 (1-8)Procedural 5.9 (2-15) 6.3 (2-12)

Heart rateBaseline 154.4 (12.8) 155.9 (14.4)Procedural 154.9 (13.9) 164.9 (14.6)

Oxygen saturationBaseline 96.5 (0.5) 96.2 (0.5)Procedural 96.2 (0.6) 95.8 (0.6)

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

32

use/depletion, and increased purine production is welldocumented in adult animal and human literature(Figure 4).6,30,31 Sucrose is a disaccharide of glucose andfructose. It is hydrolyzed by sucrase, an enzyme secreted byepithelial cells of the villi in the small intestine (Figure 4,A). Both glucose and fructose are rapidly absorbed fromthe gastrointestinal tract through glucose transporter(GLUT) 5 (fructose) and sodium-glucose cotransporter/GLUT2 (glucose) transporters in the apical membraneand transferred to the portal circulation via GLUT2transporters in the basolateral membrane of enterocytes(Figure 4, B).The expression of these GLUTs is up-regulated by exposure

of intestinal lumen to fructose solutions32 or byprevious expo-sure to corticosteroids.33Once in circulation, glucose uptake isinsulin-dependent while fructose uptake is independent of

) Heel lance, Sweet-Ease/NNS (n = 44) F value P value

3.8 (1-7) 0.233* .7924.6 (2-10) 6.216* .003†

154.1 (13.3) 0.206* .814170.5 (14.7) 14.480* <.001z

96.2 (0.5) 0.123* .88596.4 (0.6) 0.235* .791

Asmerom et al

Figure 3. A and B, Plasma hypoxanthine and uric acid con-centration increased over time in preterm neonates who re-ceived oral sucrose before a clinically required heel lance. C,In neonates with minimal pain response (<33% increase inPIPP with heel lance), plasma allantoin concentration in-creased over time.

July 2013 ORIGINAL ARTICLES

insulin.4 Inside the cell, fructose is rapidly phosphorylated intofructose-1-phosphate by the enzyme fructokinase (Figure 4,C). The activity of fructokinase is 4-fold greater than

Oral Sucrose for Heel Lance Increases Adenosine Triphosphate U

glucokinase (the enzyme that phosphorylates glucose).Moreover, fructokinase activity is relatively unregulated,being limited only by fructose concentration.34 Fructose-1-phosphate is split by aldolase (aldolase B) intoglyceraldehyde and dihydroxyacetone phosphate, a memberof the glycolysis sequence of intermediates. The thirdenzyme in the fructose pathway is triokinase, which catalyzesthe phosphorylation 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 AsATP is consumed, it is degraded to ADP, leading to an in-crease in ADP concentration.4 Simultaneously, inorganicphosphate levels decrease because they are sequestered infructose-1-phosphate or the mitochondria to generate ATPnecessary to maintain fructose phosphorylation (Figure 4,D).4 As inorganic phosphate concentration decreases,oxidative phosphorylation is inhibited, reducing ATPsynthesis and rapidly depleting ATP.4,6,30,31 ATP and ADPcatabolism results in increased concentration of purinessuch as uric acid (Figure 4, E).5,6,35 These observations havebeen documented in adult animals as well as in childrenages 11 months to 12 years5 and in healthy adults.5,6 Weshow similar effects in preterm neonates, in which a singledose of sucrose significantly increased plasma hypoxanthineand uric acid concentrations.We also found that neonates in the sucrose treatment

group had the largest increase in heart rate compared withthose in the control or placebo groups, providing additionalevidence that sucrose does not attenuate the tachycardia thataccompanies 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 inrats36,37 and healthy young adults.38 Interestingly, in humans,the sympathetic response to sucrose ingestion was enhancedunder 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 turncontributes 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 allantoinconcentration significantly increased over time, comparedwith neonates with a larger pain response (defined as havingan increase in PIPP score of $34% compared with baseline;Figure 3, C). The demographics of this subgroup ofneonates were not significantly different from those witha larger pain response. Although newborns with reducedpain responses had significantly lower baseline heart rates(151 � 11.6 beats/min vs 159 � 14.4 beats/min, P = .028),they tended to have a greater percent change in heart ratewith the heel lance procedure compared with those withlarger pain responses (12 � 7% vs 9 � 7%, P = .290). Thesedata suggest that changes in heart rate due to sucroseadministration may be more predictive of oxidative stressthan changes in facial or behavioral activity. Additional

se and Oxidative Stress in Preterm Neonates 33

Figure 4. Sucrose metabolism. A, Sucrose is hydrolyzed into fructose and glucose by the enzyme sucrase. B, Glucose andfructose are absorbed from the gastrointestinal tract, transferred to the portal circulation, and enter hepatocytes. C, Fructose isphosphorylated to form fructose-1-phosphateby theenzyme fructokinase.D, Fructosephosphorylation rapidlydepletes thecell ofATP and inorganic phosphate. E, Decreases in ATP production, in addition to increased ATP utilization, results in increased hy-poxanthine, xanthine, anduric acidproduction. If this is combinedwith increasedoxidative stress, uric acid canbeoxidized to formallantoin. SGLT, sodium-glucose cotransporter; ISF, interstitial fluid; TCA, tricarboxylic acid cycle; IMP, inosine monophosphate.

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studies are required to examine the relationship betweensucrose administration, pain reactivity, and oxidative stress.

A limitation of this study is that although statistical powerof more than 80% was achieved for hypoxanthine and uricacid, it was not achieved for allantoin. A larger sample sizemay be required to adequately examine the effect of sucrosetreatment on allantoin. It is possible that a significant

34

increase in allantoin may not be always evident five minutesafter the heel lance procedure.In this prospective, randomized, double-blind study, we

made the observation that a single dose of oral sucrose, givenbefore a heel lance, significantly increased markers of ATPuse and oxidative stress in premature neonates over time.This finding suggests that the apparent analgesic effect of

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July 2013 ORIGINAL ARTICLES

oral sucrose may come at a price, namely increased ATP use.Because neonates can be exposed to numerous painful proce-dures per day requiring multiple doses of sucrose, random-ized trials should be performed to examine the effects ofrepeated sucrose administration not only on markers ofATP breakdown and oxidative stress but also on cellular in-jury. If it is determined that the metabolic risks of using su-crose in neonates is indeed greater than the known benefits ofreducing behavioral indices of pain, additional studies needto be performed to identify alternative effective substancesor methods to prevent or treat pain in neonates. n

We want to thank Desiree Wallace, PharmD and all the nurses andphysicians at Loma Linda University Children’s Hospital for theirsupport of this study.

Submitted for publication Aug 22, 2012; last revision received Oct 30, 2012;

accepted Dec 27, 2012.

Reprint requests: Danilyn M. Angeles, PhD, Division of Physiology,

Department of Basic Sciences, Loma Linda University School of Medicine,

Loma Linda, CA 92350. E-mail: dangeles@llu.edu

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se and Oxidative Stress in Preterm Neonates 35

Figure 1. Enrollment flow chart. )Reasonswhy subjects werenot sampled include: change in acuity or failure to meet studycriteria after consent was obtained (n = 9); central line will notdraw or lipids were being infused preventing sampling (n = 5);and line was discontinued before sampling could be sched-uled (n = 6).

Figure 2. Study procedure.

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35.e1 Asmerom et al