Nutrition: obtaining reliable biomarker data to gstudy the health status of populations

Christine M. Pfeiffer, Ph.D.Chief, Nutritional Biomarkers Branch, DLS

AAAS 2011 Annual MeetingScience Without Borders Session

February 18, 2011

National Center for Environmental HealthDivision of Laboratory Sciences

Outline

• Introduction to CDC’s Nutritional Biomarkers Branch

• Introduction to an important U.S. health survey, NHANES

• Vitamin D as an example:

– Evolution of measurement techniques used for NHANES

– Consequences of method changes on long-term monitoring of l ti t dpopulation trends

– Programs & partnerships across different sectors to improve measurements, make them comparable across the globe, and create a lasting impact on public health

2

CDC’s Nutritional Biomarkers Branch

WATER-SOLUBLE VITAMINS & METABOLITES

FAT-SOLUBLE VITAMINS & NUTRIENTSMETABOLITES

• Folate• Vitamin B6• Vitamin B12

Vit i C

NUTRIENTS• Vitamin A• Vitamin E• Carotenoids

• Vitamin C• Homocysteine• Methylmalonic acid• Iron-status indicators

• Vitamin D• Polyunsaturated fatty acids

DIETARY BIOACTIVE COMPOUNDS• Phytoestrogens• Caffeine and metabolites• Polyphenols

GOAL: Improve the laboratory diagnosis and detection of nutrition-related diseases

yp

3

U.S. NHANESCross-sectional nationally representative survey

• Conducted by the National Center for Health Statistics, CDC

• Designed to collect information about the health and diet of people in the United St tStates

• Approximately 5,000 persons examined per yearU i i th t it bi h• Unique in that it combines a home interview with health tests that are done in a Mobile Examination Center

4

U.S. NHANESSampling Design

Stage 1Counties

Stage 2Segments

Counties

Stage 3Stage 3Households

Stage 4SPsSPs

5

National Report on Biochemical Indicators of Diet and Nutrition in the U. S. Population

1999 20021999-2002

6

Reference Interval Tables by Age and Sex

www.cdc.gov/nutritionreport7

Vitamin D Examplep

• Why is it important to measure biological levels of vitamin D?y p g

• What measurement techniques are suitable for NHANES?

• What happens if measurement techniques have to be changed• What happens if measurement techniques have to be changed mid-stream?

• How do we make measurements comparable across the globe?• How do we make measurements comparable across the globe?

• What do we need to ensure high quality measurements?

8

Vitamin D: Nutrient of the Day

9

What is Vitamin D?

• Fat-soluble vitamin that helps the body absorb calcium

• Primarily needed for bone growth and bone remodeling

• Other roles in human heathOther roles in human heath

– Modulates neuromuscular and immune function

R d i fl ti– Reduces inflammation

10

Main Sources of Vitamin D

Sunlight: UVB exposureg pVitamin D3 – cholecalciferol

FoodVitamin D3 – cholecalciferol, animal sources

S l t

Vitamin D2 – ergocalciferol, plants (mushrooms)

SupplementsVitamin D2 and vitamin D3

CDC. MMWR Recomm Rep 1992 Sep 11;41(RR-14):1-7

11

Vitamin D Metabolism

• Vitamin D is rapidly taken up and transported to the liver

• Vitamin D2 and vitamin D3 are metabolized

o First, in the liver to the circulating form of 25-hydroxyvitamin D or 25(OH)D

o Then in the kidney to the active form of 1,25-dihydroxyvitamin D

CDC. MMWR Recomm Rep 1992 Sep 11;41(RR-14):1-7

12

Vitamin D and Health

• In children, there is a high risk of rickets with low levels of 25(OH)D

• In adults, there is a risk of osteomalacia with inadequate levels of 25(OH)D

• Low levels of 25(OH)D have been associated with increased risk for numerous other health outcomes

• These associations are primarily based on ecologic/observational studies

13

Why is it Important to Measure Biological Levels of Vitamin D?

Because the level of serum 25(OH)D is the result of the cumulative exposure to various sources of vitamin D, it is considered to be the best biomarker to assess vitamin statusconsidered to be the best biomarker to assess vitamin status.

14

What Measurement Techniques are Suitable for NHANES?

Hi h t ibl• Highest possible accuracy

• Best possible precision

Good sensitivity because only small sample volumes are available• Good sensitivity because only small sample volumes are available

• High enough throughput to handle minimum 5,000 samples per year

• Stable over time to avoid method fluctuations• Stable over time to avoid method fluctuations

• Has to be affordable and sustainable over multiple years

anes

15

History of Measurement of 25(OH) D in NHANES

• Radioimmunoassay (RIA) developed in the mid-1980s – this was “state-of-the-art” at the time

o NHANES III (1988–1994): Original DiaSorin RIA

o NHANES 2000–2006: Reformulated DiaSorin RIA

• Liquid chromatography coupled to tandem mass spectrometry• Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) – this is “state-of-the-art” now

o NHANES 2007 and forward

anes

16

Undesirable Features of the Classical Immunoassay Methodology

Suboptimal performance

characteristicsDefinition Impact on population

monitoring

SpecificityCompounds other than

25(OH)D may alter results

Difficult to obtain accurate testing results

PrecisionGreater variation in

laboratory measurements

Difficult to identify small changes in the population over

time

Robustness Fluctuations in assay performance over time

Difficult to interpret changes in the population over time

17

Advantages of the New Analytical MethodologyIsotope Dilution LC-MS/MS

Separation of 25(OH)D3 from epimer of 25(OH)D3

C:\Xcalibur\...\data090720a18 7/20/2009 4:50:35 PM SRM 972 L4

RT: 0.00 - 17.50 SM: 5G

1200000

11.27

10.42

NL: 1.32E6TIC F: + p sid=-7.00 SRM ms2 401.30@-11.00 [ 382 80-383 80]

Mass transitionm/z: 401/383

(NIST SRM 972 Level 4)

200000

400000

600000

800000

1000000

Inte

nsity

0.11 2.360.504 803 26 3 76 16 13

382.80-383.80] MS data090720a18

25(OH)D3 epi-25(OH)D3

0 2 4 6 8 10 12 14 16Time (min)

0

200000 4.803.26 3.76 16.136.08 7.29 7.72 9.728.22 12.65 13.15 15.14

RT: 0.25 - 17.50 SM: 5G

1200002.37 16.19

2.190 52

NL: 1.86E5TIC F: + p sid=-7.00 SRM ms2Mass transition

40000

60000

80000

100000

Inte

nsity

0.52 3.773.11

1.04 4.166.11

4.815.81

11.597.35 12.62 13.5610 79 13 85

SRM ms2 413.30@-11.00 [ 394.80-395.80] MS data090720a18

25(OH)D2(m/z): 413/395

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17Time (min)

0

20000

10.79 13.857.746.36 9.889.5114.56

18

Advantages of the New Analytical MethodologyIsotope Dilution LC-MS/MS

Specificity Less possibility for compounds other than 25(OH)D to alter results

Precision <8% day-to-day variation

RobustnessIn-house calibration with 25(OH)D2 and 25(OH)D3, and calibration verification with NIST reference material

19

How to Make Past and Current Data Comparable?

In 2009, the NIH Office of Dietary Supplements and the CDC NCHS sponsored a roundtable on vitamin D issues in NHANES

Future methodology should be LC-MS/MS A subset of the samples analyzed with the DiaSorin RIA will be re-

analyzed by LC MS/MS to bridge the past and the futureanalyzed by LC-MS/MS to bridge the past and the future

Yetley EA et al. J. Nutr. Nov 2010

20

Bridge to the Past will be Imperfect due to the Suboptimal Specificity of the Immunoassay Data

80%Difference Plot

40%

60%

80%

LC-M

S/M

S

0%

20%

RIA

-LC

-MS/

MS)

/

Identity

Bias (-9.7%)

-40%

-20%

(Dia

Sorin

95% CI

95% Limits of agreement(-44.0% to 24.5%)

95% CI

-80%

-60%

0 50 100 150 200 250 300LC MS/MS (nmol/L)LC-MS/MS (nmol/L)

21

Comparison of Data across Several Labs or Countries is Very Resource Intensive

Canada Survey

Countries is Very Resource-Intensive

y

Current Situation:

• Exchange samples

UK Survey

US Survey

for each method comparison separately

• Adjust survey data for• Adjust survey data for differences in calibration

Germany Survey

Ireland S

• Compare adjusted survey data

SurveySurvey

22

Comparison of Data across Several Labs or Countries is Easy after Standardization

Canada Survey

Countries is Easy after Standardization

Survey Future situation:

• One-time exchange of samples to achieve

UK Survey

US Survey

Reference

samples to achieve common calibration

• No further comparison Reference por adjustment of survey data needed

GermanySurvey

IrelandSurveySurvey

23

Standardization of Measurements – Vitamin D Example

“Pure Compound” Reference Material (NIST SRM 2972)

Secondary Reference Material(NIST SRM 972)NIST

Mass Spectrometry-Based Reference Method

Calibrate Calibrationverification

NIST, CDC

Panel of Sera with Assigned Values

Assign Value

NIH, CDC, NIST Gent

CDC Vitamin D Standardization Coordinating Center

“Routine” Assays

Calibrate

NIST, Gent Coordinating Center

NIH, CDC, NIST, Gent, PH labs, C i l kit

Patient Samples

Assign Value

Commercial kit producers

Users Patient SamplesUsers

24

Maintaining Quality Measurements

UK DEQASClinical & research labsClinical & research labs,

commercial kit producers

CDC LC-MS/MS Candidate

Reference Method

NIST VitDQAPClinical & research labs, commercial kit

CAP Bone & Growth Survey

Clinical labs, commercialReference MethodproducersClinical labs, commercial

kit producers

NIH-CDC Vit D Standardization Program

Selected PH labs

25

What do we Need to Ensure Continued High Quality Measurements?

• Use method anchored to accuracy-based traceability chain

• Establish and follow detailed SOPs

• Train staff well in all procedures

• Include internal quality control samples in every run

• Participate in external quality assurance programs

• Use reference materials on a regular basis

26

Ulti t l f i d tUltimate goals of improved measurements:

• Better diagnosis and treatment:

• Better assessment of the vitamin D status of the individual and populationsand populations

• More reliable prevalence estimates of inadequate levels

• Help reduce or prevent negative health outcomes:

• Better estimation of optimal levels of 25(OH)D for various p ( )health outcomes

27

Acknowledgments

• CDC/NCEH:

o Rosemary Schleicher and her vitamin D laboratory team

o Hubert Vesper - CDC Vitamin D Standardization Coordinating CenterCenter

• CDC/NCHS: Anne Looker, David Lacher, Cliff Johnson

• NIH/ODS: Beth Yetley, Chris Sempos, Paul Coates

• NIST: Karen Phinney Steven Wise

28

• NIST: Karen Phinney, Steven Wise