Reference Materials for the Nutrition Community

The Future of Reference Materials - Science and Innovation

23 November 2010

Joseph M. Betz1, Mary Frances Picciano1, Karen Phinney2

1Office of Dietary Supplements, National Institutes of Health 2National Institute for Standards and Technology

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NIH is the Nation’s Medical Research Agency

www.nih.gov

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Reference Materials and ODS

ODS Analytical Methods and Reference Materials Program- multi-year project with NIST

•  “Suites” of Standard Reference Materials –  Supplement raw materials & finished products –  Certified and Reference values for marker compounds,

nutrients, contaminants

•  Calibration Standards www.nist.gov

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Outcomes

Available•  Botanicaloils(tocopherols)•  Fishoil(FAs,tocopherols)•  Ginkgo•  Bi:erorange•  Sawpalme:o•  Mul?vitamin/mineraltablets•  Botanical&fishoils(Ω3’s,6’s)•  Greentea•  Berries•  Vit.Dinhumanserum

In Process •  St.John’swort•  Soy•  Blackcohosh•  Kudzu•  Redclover•  Turmeric•  Pomegranate•  Ginsengs(Asian,Siberian)

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Foodswithvaluesassignedfornutrients(proximates,vitamins,carotenoids,elements,fa:yacids,aminoacidsasappropriate)

• SRM1849Infant/AdultNutri?onalFormula• SRM1946LakeSuperiorFishTissue• SRM2384BakingChocolate• SRM2385SlurriedSpinach• SRM2387PeanutBu:er• SRM3287Blueberry(Fruit)plus5moreinprogress

NIST SRM for Nutrition (available prior to ODS program)

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• 909c‐Glucose,urea,uricacid,cholesterol,crea?nine,totalprotein,electrolytes

• 968e‐Fat‐SolubleVitamins,Carotenoids,Cholesterol,tocopherols,re?nol,beta‐carotene

• 2670‐ToxicElementsinUrine:14elements• 3950‐VitaminB6inHumanSerum

• 3951‐VitaminB12inHumanSerum

NIST SRM for Nutrition

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CaseStudy:

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Vitamin D Status of the US Population?

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Lookeretal.AmJClinNutr2008;88:1519‐27.

•  1988-94 vs. 2000 - 2004: − Reformulation (introduction of antibody to

improve binding) of RIA assay kit shifts in assay results between these two time periods

− Adjusted for assay drifts to compare time trends in 25(OH)D and identify contributing factors

25(OH)D Assay Concerns (Looker et al., 2008)

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•  Adjustments for assay differences: − Looker et al. (2008):

•  Overall mean 25(OH)D lower in 2000-2004 than in 1988-94 •  Assay changes accounted for much of the difference

•  No adjustment for assay differences: − Ginde et al. (2009):

•  Marked decrease in 25(OH)D in 2000-2004 from 1988-1994 •  Growing epidemic of vitamin D insufficiency

− Saintonge (2009): •  Vitamin D deficiency is increasing from 1988-1994 to 2006 •  National fortification and public health strategy

Adjustment Affects Policy Conclusions

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• SRM972‐VitaminDmetabolitesinHumanSerum− Suiteoffour1.0mlvialswithcer?fiedandreference

valuesfor25(OH)D2,25(OH)D3,and3‐epi‐25(OH)D3

• SRM2972‐25(OH)D2&25(OH)D3Calibra?onSolu?ons− SRM2972isasetofethanoliccalibra?onsolu?onsand

hascer?fiedvaluesfor25(OH)D2and25(OH)D3

• Valueassignmentbyisotope‐dilu?onLC‐MSandLC‐MS/MS

• RMdrivesscience‐previouslythought3‐epionlyoccurredininfants

NIST SRM- July 2009

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•  Ques%onsaboutequivalenceofLC/MSassayresponseto25(OH)D2and25(OH)D3

−  Immunoassaysmeasure“total”25(OH)D

− OlderLC/MSmethodsdidnotresolve3‐epimer

•  3‐epimersof25(OH)DmaybiasMS‐basedmethods

25(OH)D3 3‐epi‐25(OH)D3

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NIST LC-MS Methodology for 25(OH)D

25(OH)D2‐[2H3]25(OH)D3‐[2H6]

25(OH)D3

3‐epi‐25(OH)D3

25(OH)D2

Taietal.(2010)AnalChem82:1942‐1948

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Level165±15nmol/L25‐hydroxyvitaminD3(“normal”)

Level2Blendof“normal”serumandhorseserumtoobtainapproximatelyhalfthelevelof25‐hydroxyvitaminD3inthe“normal”pool(35±5nmol/L)

Level3“Normal”serumspikedwithequivalentamountof25‐hydroxyvitaminD2

Level4“Normal”serumspikedwith3‐epi‐25‐hydroxyvitaminD3

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CommutabilityisdefinedastheequivalenceofthemathemaZcalrelaZonshipsbetweentheresultsofdifferentmeasurementproceduresforareferencematerialandforrepresentaZvesamplesfromhealthyanddiseasedindividuals.

Vesperetal.,Clin.Biochem.Rev.,28(2007)139

Reasonsareferencematerialmaynotbecommutable:

•  Materialhandling,processing(lyophiliza?on,filtering,etc.)

•  Supplementa?onwithna?veornon‐na?veanalytes

•  Differencesbetweensamplesfromhealthyanddiseasedindividuals

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OtherRMneeds

Marketedproducts‐matrices•  Delayedreleasedosageforms‐ionexchangeresins,etc.

•  Oil‐filledgelcaps‐vegetarianandanimalgela?n

•  Microencapsulatedmaterials‐Carrageenan,gela?n,etc.

•  “Nano”ingredientsinfinishedproducts•  “Unusual”matrixsourcefornutrientsandnutrientelements‐e.g.iodineinkelpvsKI

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OtherRMneeds

Clinicalmaterialsandcalibra?onstandards•  Folate,othercomplexvitamersinserumforLC/MS

•  Biomarkersofnutrientexposurevs.markersofNutrientstatus‐e.g.Omega‐3fa:yacidsinserumvs.redbloodcellmembrane

•  Isotopelabelednutrientsandmetabolitesascalibra?onstandardsforMSdetec?on

•  Serumbasedcalibra?onsolu?ons

Yetleyetal.(2010)JNutr140:2030S‐2045S

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Non-RM Activities

NIST Vitamin D metabolite Laboratory Quality Assurance Program & Dietary Supplement Laboratory Quality Assurance Program

•  NIST sends blinded material, practice sample –  Participants perform analyses and return data to NIST –  NIST provides feedback on blinded results, including

suggestions for method optimization

•  To participate (no cost), send e-mail to vitdqap@nist.govorDSQAP@nist.gov

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“Reference Materials are the most efficient means to transfer accuracy to a field” -John Eckfeldt, MD, Ph.D. Professor of Laboratory Medicine, University

of Minnesota Medical Center

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www.ods.od.nih.gov E-mail: ods@nih.gov

E-mail: betzj@mail.nih.gov

Joseph M. Betz

http://dietary-supplements.info.nih.gov/FactSheets/AMRMProgramWebsite.asp