Clinical Biochemistry 43 (2010) 1411–1414

Contents lists available at ScienceDirect

Clinical Biochemistry

j ourna l homepage: www.e lsev ie r.com/ locate /c l inb iochem

Pediatric reference intervals for lymphocyte vitamin C (ascorbic acid)☆

Alex Levin a, Charmaine DeSouza b, Christian Zaarour c, Warren Walsh d, Man Khun Chan d,Zulfikarali Verjee d, Susan McIntyre e, Khosrow Adeli d,⁎a Pediatric Ophthalmology and Ocular Genetics, Wills Eye Institute, Philadelphia, USAb Child Health Evaluative Sciences, The Hospital for Sick Children, University of Toronto, Toronto, Canadac Department of Anesthesia, The Hospital for Sick Children, University of Toronto, Toronto, Canadad Department of Paediatric Laboratory Medicine, The Hospital for Sick Children, University of Toronto, Toronto, Canadae Congenital Heart Surgeons Society Data Center, The Hospital for Sick Children, University of Toronto, Toronto, Canada

☆ This study was funded in part by the Canadian HemResearch Program, Brandan's Eye Research Fund and thhave no real or perceived financial conflicts of intediscussed herein.⁎ Corresponding author. Department of Paediatric Lab

Atrium, The Hospital for Sick Children, 555 UniversityM5G 1X8. Fax: +1 416 813 6257.

E-mail address: khosrow.adeli@sickkids.ca (K. Adeli)

0009-9120/$ – see front matter © 2010 The Canadian Sdoi:10.1016/j.clinbiochem.2010.09.008

a b s t r a c t

a r t i c l e i n f o

Article history:

Received 14 May 2010Received in revised form 27 August 2010Accepted 11 September 2010Available online 19 September 2010

Keywords:Vitamin CLymphocytesAscorbic acidReference intervalsChildren

Objective: To establish pediatric reference intervals for lymphocyte vitamin C.Design and methods: This was a prospective study of 194 well children aged 0–7 years old of mixed

ethnicitywhohad blood drawn for the purpose of this study. Bloodwas collected during elective surgery undergeneral anesthesia and lymphocytes isolated and stored as frozen ascorbic acid lymphocyte lysates for laterHPLC analysis by previously described methodology. Reference intervals were established according to theClinical and Laboratory Standards Institute (CLSI) and the International Federation of Clinical Chemistry (IFCC)guidelines (C28-A3). Horn–Pesce robust method was used to estimate the 95% confidence interval and 95%reference interval.

Results: Reference intervals were independent of age or gender and shown to be 12.9–52.8 μg/108 cells(lymphocytes).

Conclusion: We have defined pediatric reference ranges for lymphocyte vitamin C in healthy, fasted

children at a relevant age group (0–7 years). The new reference interval can now be used to more reliablyexplore possible implications of variation of vitamin C levels on bleeding and other clinical signs.

© 2010 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Introduction

Ascorbic acid has an active role in many body metabolic processes.It is well established that, ascorbic acid acts in the manufacturing ofcollagen, strengthening of blood vessels, manufacturing of hemoglo-bin, secretion of adrenal hormones, protection against viral andbacterial infections, assimilation of iron, production of naturalantihistamine, production of interferon, neutralization of free radicals,re-cycling of vitamin E, andmanufacturing of lipoprotein lipase. It alsoacts as an “anti-cancer agent”. As a result of its important involvementin so many processes measurement of ascorbate would be a usefulclinical tool. Humans are vulnerable to vitamin C deficiency as we lackthe enzyme, L-gulono-gamma-lactone oxidase, needed to synthesizethis essential substance, making us totally dependent on dietary

ophilia Society, Care until Curee Foerderer Fund. The authorsrest relevant to the material

oratory Medicine, Room 3652,Avenue, Toronto, ON, Canada

.

ociety of Clinical Chemists. Publish

vitamin C. Although overt severe deficiency (scurvy) is rare, vitamin Cdeficiency may still occur in the Western world. A high prevalence ofsub-clinical scurvy has been suggested. Plasma vitamin Cwas found tobe depleted in 30%, and deficient (below 11 μmol/L) in 6% of cases ofpeople attending a Health Maintenance Organization (HMO) clinic inTempe, Arizona in 1998 [1].

Plasma vitamin C levels have been shown to be unreliable as theyvary with time of day, diet, gender and age. Plasma levels are alsoreduced by infection, stress, allergy, cancer, atherosclerosis, diabetes,smoking, intoxications, drug of abuse, and in women on birth controlpills. The accepted gold standard methodology is high performanceliquid chromatography (HPLC) although there remains a large inter-laboratory variability with an average coefficient of variance of 15%across multiple studies. Measurement error is also induced due to theinstability of vitamin C in serum. After only one day at roomtemperature, serum ascorbic acid measurements are significantlydiminished and are undetectable by the second day. Similarly, serumstored at 4 °C shows significant decreases in ascorbic acid levels after3–5 days. Lastly, serum and plasma measurements do not correlatevery well with tissue vitamin C levels. Generally, plasma levelsdiminish earlier than tissue levels in deficiency and replete sooner[2–4]. Lymphocyte vitamin C levels are more representative of tissue

ed by Elsevier Inc. All rights reserved.

Table 1Age-specific reference intervals (gender combined).

Analyte Age-specific reference intervals (gender combined)

0≤x≤1 1bx≤3 3bx≤5 5bx≤7

Vitamin C (μg/108) 13.1–70.0 8.4–41.4 10.9–47.2 8.7–56.6n 47 51 47 44Outliers 1 4 4 1

1412 A. Levin et al. / Clinical Biochemistry 43 (2010) 1411–1414

stores and are preferred, but not routinely available. We previouslyreported amethodology for HPLC analysis of lymphocyte vitamin C [5]and now report pediatric reference intervals.

Methods

This study was approved by the Research Ethics Board at TheHospital for Sick Children, Toronto. We initially targeted patients whowere being sent for routine blood work done by their attendingphysicians from medical and surgical clinics at The Hospital for SickChildren to have an additional simultaneous blood draw without aseparate venipuncture. Despite wide spread publicity of this researchstudy, theuseof in-clinic physicianandalliedhealth reminders, andpre-printed forms, recruitment proved to be largely unsuccessful. TheResearchEthics Board approvalwas subsequently amended to allow thecollection of 2 cm3 of blood on eligible systemically well childrenattending the hospital for elective surgery while they were undergeneral anesthesia. The anesthesiologist for that surgery explained thestudy to the parents, provided them with a consent package andobtained consent. These patients had been fasted for a length of timeprior to surgery depending on their age (clear liquids up to 2 h preanesthetic, breast milk up to 4 h, formula and milk up to 6 h preanesthetic, no solid food after midnight).

While their child was in the operating room, the substitutedecision maker completed a one page questionnaire requestinghistorical information of potential relevance (Appendix A) Hospitalcharts of enrolled patients, whomet the inclusion criteria (see below),were reviewed by our Research Assistant (CD) to ensure that thepatients met the inclusion criteria. Patients with values measuringbeyond the 95th or 5th percentiles were contacted by phone andasked additional question about their history to identify possiblefactors contributing to these results. These patients were also offeredthe opportunity to return as outpatients for a repeat blood draw.Results were otherwise not transmitted to the families.

Patients were enrolled without regard to race, gender or ethnicbackground. Children were recruited from May 1, 2004 to May 6,2008. Children were ineligible for the study if they had any of thefollowing conditions: gastro-intestinal disease, liver disease, knowndietary or nutritional abnormalities, active treatment for infection,taking medication known to affect vitamin C levels, cancer, upperrespiratory tract infection, fever, renal disease, taking supplementalvitamin C (other than daily multivitamin), known bleeding disorderor on anticoagulation therapy or scurvy. We also excluded patients forwhom the substitute decision maker did not understand English orrefused consent. Each patient was identified with a study numberindependent of their health record number but the numbers werelinked so that patients with extreme values could be contacted.

Reference intervals were established according to the Clinical andLaboratory Standards Institute (CLSI, formerly NCCLS) [6] and theInternational Federation of Clinical Chemistry (IFCC) [7] guidelines ondefining, establishing, and verifying reference (C28-A3). Results werereviewed to detect outliers using the Dixon's test prior to estimatingreference intervals. A one way ANOVA analysis was performed todetermine which age groups and gender groups could be combined.Since the number of sample was less than the recommended 120, theHorn–Pesce robust method was used to estimate the 95% CI(confidence interval) and 95% RI (reference interval) of distributionas upper and lower normal reference intervals.

Experimental protocols

Lymphocyte preparation

Bloodwas collected on ice in anappropriate 2.0 cm3purple topEDTAtube and transported to the Rapid Response Lab of the Department ofPaediatric LaboratoryMedicine (DPLM) at TheHospital for SickChildren

for lymphocyte lysates preparation, storage and analysis. Lymphocytelayer was extracted using cold Histopaque-1077 (Sigma-Aldrich, St.Louis, MO) and by centrifugation in a (Microfuge 18 Centrifuge(Beckman-Coulter, Fullerton, CA)) at 1500 rotations per minute (rpm)for 15 min. Cells were washed three times with cold phosphate buffersaline (PBS) solution (prepared in-house) and by centrifugation at2000 rpm for 5 min. Lymphocyte cells were resuspended in anappropriate volume of PBS with 10% metaphosphoric acid (Sigma-Aldrich, St. Louis,MO) to obtain a final volume of 1.0×106lymphocytes/mL. The suspension was sonicated on ice for 10 s using a Cell Sonicator(Ultrasonic Processor XL, Misonex Inc., Farmingdale, NY) The suspen-sion was centrifuged at 14,000 rpm for 10 min and the supernatantcontaining the ascorbic acid lymphocyte lysate sampleswas transferredimmediately to a cryo vial (Fisherbrand, Fisher Scientific, Pittsburgh, PA)and stored at −80 °C.

HPLC analysis

The HPLC method has previously been described [5]. One modifica-tion to the original procedurewas implemented. The lymphocyte lysatesamples were not spiked with the vitamin C standards. The frozensamples were thawed on ice and mixed by vortex. The thawed lysateswere then transferred to limited volume inserts, capped and placed onthe HPLC autosampler. 10.0 μL of each sample was injected onto thecolumn. Quantitationwas calculated directly froma six point calibrationcurve (0.25, 0.50, 1.00, 2.50, 5.00 and 10.00 μg/mL) by the HPLC Breezesoftware (Waters Corp., Milford,MA). Resultsweremultiplied by 100 toconvert the results from106 cells (final lysate concentration) to 108 cells(reporting concentration).

Results

We collected 196 samples of which 2 needed to be discarded. Fivepatients had results that were clearly outliers (3 with zero vitamin Cdetected and 2 with unusually high values). These values wereinconsistent with the well clinical status of these children and theresults were presumed spurious and not used in our analysis. Thechildren were not available for retesting except in one case whereretesting revealed values consistent with the reference rangesdetermined by this study. 186 (95%) samples were obtained in theoperating room and the remainder was drawn from the outpatientclinic (all outpatients with autism). The number of patients in eachage bracket is summarized in Table 1. The results of the parentquestionnaires are summarized in Tables 2 and 3.

The total number of data included in the reference intervalscalculation is 189. Results were reviewed to detect outliers usingDixon's test prior to estimating reference intervals, 10 outliers wereexcluded. Datawere partitioned into four age groups. A onewayANOVAanalysis was performed to determine which age and gender groupscould be combined. All partitioning decisions were also checked by theHarris–Boydmethodwhich is recommended for partitioning in the CLSI[6] C28-A3 guidelines. Table 1 shows the reference intervals of the fourage groups with genders combined. We found no age differences.Table 4 shows: a) the combined data for all age groups but with genderseparation and b) age and gender combined reference intervals. The

Table 2Subject ethnicity.

Ethnic group Number

Asian 18Black 13British 17Canadian 40European 21South Asian 16Italian 14Spanish 3Native Canadian 5Declined to state 47Total 194

Table 4Reference intervals by gender.

Vitamin C (μg/108) Gender separated Gender combined

F M

Reference interval 13.6–73.5 9.3–67.8 10.5–69.4n 72 117 189Outliers 2 1 3

1413A. Levin et al. / Clinical Biochemistry 43 (2010) 1411–1414

combined data with gender separation indicated no differencesbetween the sexes so the reference intervals for children aged 0 to7 years are age and gender combined.

The Horn–Pesce robust method [8] was used to estimate the 95%confidence interval (CI) and 95% reference interval (RI) of distributionas upper and lower normal reference intervals, according to the mostrecent draft version of the CLSI and IFCC C28-A3 guidelines. Thecombined reference interval (for both genders between 0 and 7 yearsof age) was determined to be 10.5–69.4 μg/108.

Discussion

We have established the first, pediatric reference intervals forlymphocyte ascorbic acid using HPLC reporting a range of 10.5–69.4 μg/108 for children aged 0–7 years regardless of gender. Usinglymphocytes to assess vitamin C status reduces variation due to dailycircadian rhythms and dietary patterns [2]. Defining pediatric referenceintervals for lymphocyte vitamin C will facilitate its routine use in thepediatric setting. We employed a HPLC methodology that hadpreviously been validated [5].

Although ethnic variation in ascorbic acid levels has previouslybeen suggested, our data set does not allow for further subgroupanalysis. The majority of our patients were Caucasian of Europeanancestry.We did not observe the gender differences reported in adults[2]. The overwhelming majority of our patients who providedinformation indicated that they were on no vitamin supplementation.We cannot rule out the possibility that those who were taking“mulitvitamins” may have had their ascorbic acid levels chronicallyaugmented by the supplementary intake. This should not impact onthe lower end of our established reference range. All but 8 of oursubjects were fasting. Although such dietary changes do not impact onlymphocyte ascorbic acid levels, the reference range we haveestablished should be considered a fasting level pending furtherinvestigation. If any error or bias is induced as a result of ourenrollment of preoperative patients, it would be towards lower levelswhich were not clinically significant as we are not aware that any of

Table 3Vitamin supplements.

Iron 3Multivitamina 51Vitamin D 7No supplement or declined to state 133Total 194

a Included Jamieson MultiVitamins (http://www.jamiesonlabs.com/en/products/supplements_list.aspx?GroupID=67), Infant TriVisol (http://www.drugstore.com/products/prod.asp?pid=17428&catid=52795&trx=GFI-0-RVP-52795&trxp1=52795&trxp2=17428&trxp3=1&trxp4=1&btrx=BUY-GFI-0-RVP-52795), and“Natural vitamin supplements” as well as nonspecified.

our patients had bleeding complications of their procedures. Thechildren we evaluated were well but some may have had recentintercurrent illness or other events such as vaccination which havebeen postulated as causes of temporary ascorbic acid serum levelreductions. These transient and temporary effects should not impacton lymphocyte levels but if so, this would exaggerate the lower limitof our reference values.With all factors considered, our sample of wellchildren should be representative of the “average” fasting childencountered in the course of pediatric practice.

The establishment of reference intervals allows for comparativemeasurement in relevant clinical circumstances. For example, it hasbeen theorized, based solely on serumvitamin C levels, that infantsmaydevelop transient vitamin C deficiency which would present withhemorrhagic features or even sudden death even in the absence of ameasurable coagulopathy using such routine tests as complete bloodcell counts, platelet levels, partial thromboplastoin time, and prothrom-bin time [9]. It has been hypothesized that such circumstances couldcreate clinical findings whichmight lead to the misdiagnosis of abusivehead injury including subdural and retinal hemorrhage [10,11]. This islargely based on rare observations of hemorrhaging in children withovert scurvy [12]. Likewise, it has been suggested that routine childhoodimmunization or illness can lead to elevated histamine levels andsecondary depression of ascorbic acid levels thus aggravating anypredisposition to ascorbic acid deficiency. These situations can depressbuffy coat ascorbic acid to a lesser degree [2]. Yet, there is little evidenceto support these claims [13], The use of serum levels may not haveclinical importance in terms of bleeding, especially since serum vitaminC is transferred to white blood cells and platelets in times of stress [14].Human retinal and infant vitreous levels of ascorbic acid are particularlyhigh [15,16]. Despite themismatch between the claims and the indirectevidence which refutes them, the establishment of pediatric referenceintervals for lymphocyte ascorbic acid now allows for such hypothesesto be more rigorously tested.

Appendix A. Questionnaire

Patient Name: ______________________________________________Patient Birthdate: ___________________________________________

1. Does your child take vitamins or iron? If so, please list

2. Does your child have any dietary restrictions? If so, please list

3. Is your child a vegetarian? If so, please describe what your childdoes not eat

4. Please add any special information about your child's diet whichyou think we should know:

(Optional) 5. Many lab tests have normal ranges which differ fromcountry to country and between ethnic groups. We would like toknow your child's ethnic background to help us learn if these kinds ofdifferences are important for vitamin C levels. If you agree, please list

1414 A. Levin et al. / Clinical Biochemistry 43 (2010) 1411–1414

your child's ethnic background (for example country from wheremost of family originates on each mother's side and father's side).

References

[1] Johnston C, Thompson M. Vitamin C status of an out-patient population. J Am CollNutr 1998;17:366–70.

[2] Wilson C. Clinical pharmacological aspects of ascorbic acid. Ann NY Acad Sci1975;25:354–76.

[3] Lykkesfeldt J. Determination of ascorbic acid and dehydroascorbic acid inbiological samples by high-performance liquid chromatography using subtractionmethods: reliable reduction with tris[2-carboxyethyl]phosphine hydrochloride.Anal Biochem 2000;282:89–93.

[4] Ng LL, Ngkeekwong FC, Quinn PA, Davies JE. Uptake mechanisms for ascorbate anddehydroascorbate in lymphoblasts from diabetic nephropathy and hypertensivepatients. Diabetologia 1998;41:435–42.

[5] Emadi-Konjin P, Verjee Z, Levin A, Adeli K. Measurement of intracellular vitamin Clevel in human lymphocytes by reverse phase High Performance LiquidChromatography (HPLC). Clin Biochem 2005;38:450–6.

[6] Clinical and Laboratory Standards Institute (CLSI). Defining, Establishing, andVerifyingReference Intervals in theClinical Laboratory; ApprovedGuideline— Third Edition CLSI

document C28-A3 (ISBN 1-56238-682-4). Clinical and Laboratory Standards Institute,940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898 USA, 2008.

[7] International Federation of Clinical Chemistry. Approved recommendation (1987) ontheory of reference values. Part 5. Statistical treatment of collected reference values.Determination of reference limits. J Clin Chem Clin Biochem 1987;25:645–56.

[8] Horn PS, Pesce AJ. Reference intervals. A user's guide. Washington, DC: AACCPress; 2005.

[9] Kalokerinos A. Every second child. Sydney: ThomasNelson (Australia) Limited; 1974.[10] Innis M. Vaccines, apparent life-threatening events, Barlow's disease, and

questions about “Shaken Baby Syndrome”. J Am Phys Surg 2006;11:17–9.[11] Scheibner V. Shaken baby syndrome diagnosis on shaky ground. J Australas Coll

Nutr Environ Med 2001;20:1–5.[12] Govan C,Walsh F. Symptomatology of subdural hematoma in infants and in adults.

Arch Ophthalmol 1947;37:701–15.[13] Fung E, Nelson E. Could vitamin C deficiency have a role in shaken baby

syndrome? Pediatr Int 2004;46:753–5.[14] Fain O.Musculoskeletal manifestations of scurvy. Joint Bone Spine 2005;72:124–8.[15] Hosoya K, Minamizono A, Katayama K, Terasaki T, Tomi M. Vitamin C transport in

oxidized form across the rat blood-retinal barrier. Investig Ophthalmol Vis Sci2004;45:1232–9.

[16] Sen A, Roy R, Mukherjee K. Ascorbic acid concentration in developing human fetalvitreous humor. Ind J Ophthalmol 1983;31:73–4.