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SMCR: A new approach to recoveringtemporal metabolic signal modulation inNMR spectroscopic datasets
Selena Richards
Application to a life-long caloricrestriction study in dogs
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Caloric Restriction• Natural intervention
• Increase longevity• Reduce onset and prevalence of late
life diseases• McCay et al. 1930’s
• Yeast, worms, fruit flies, rodents anddogs
• Nestle Research Centre• CR in Labrador Retrievers
• 1.8 years longer median lifespan• Osteoarthritis and neoplastic diseases
• Biochemical and physiologicalprocess unknown• Retardation of cellular, DNA and
macromolecular damage
Caloric RestrictedCaloric RestrictedControl Fed
Aged 6 years Aged 6 years
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Dynamic biofluid
• Inter-subject variability is large• Severely complicates retrieval of statistically valid
biomarkers• Variation in recency of dietary intervention and
differences between individuals metabolic phenotype• Identify an individuals healthy state
• Advantages• Readily available & relatively non-invasively• Rich source of nutrients transit from one organ to
another• Reflect current state of health and disease
• Nutritional metabonomics (1H NMR blood serum)• Distinctive biomarkers associated with dietary intervention
• Dynamic concentration changes in lipids, lipoproteins and ketone bodies assystem maintains homeostasis
• Mask subtle systematic changes associated with diet
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Objectives
YoungYoung
OldOld
Aging
11H NMR metabolic profileH NMR metabolic profile
Expression Expression of diabetesof diabetes
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Objectives
YoungYoung
OldOld
Aging
11H NMR metabolic profileH NMR metabolic profile
Expression Expression of diabetesof diabetes
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ObjectivesM
etab
olic
con
stitu
ents
δ ppm
1
2
3
Lipidic
SugarProtein
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sugarsLife-long trajectoryLife-long trajectory
Objectives
Old
Time (years)0 13
lipidic
proteins
Young Middle aged
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Self Modeling Curve Resolution
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• Underlying Principle of SMCR• Bilinear model
• Initial Estimates• Quantitative Iterative Target Transformation Factor Analysis
(QITTFA)• Starting estimates approximate the final solution• Advantages
• Refinement of Initial estimates prior to ALS• Absence of unstructured variance
• Five Steps of the QITTFA routine
SMCR Theory
sT1 sT2= +c1 c2
E = ||D - C ST||
D + E
Spectrotype 1 Spectrotype 2
Richards, S. E.; Walmsley, A. D. Journal of Chemometrics 2007, 22, 63-80.
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Initial Estimates (QITTFA)1. Needle Spectrum
Uni
t int
ensi
ty
5.0 ppm
δ ppm
in1 = (1, 0 0,….0)
2. Singular Value Decomposition
vT1= t1D vT2
t2
4. Needle Output Spectra Constrained
in’1 = out’1
inte
nsity
out1 = in1 V* V*T
3. Needle Output Spectra
inte
nsity
NegativityNegativity
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Initial Estimates (QITTFA)
δ ppmout No.
5. Selection of Output Spectra(Initial Estimates)
Needle Output Matrix
out1 = in1 V* V*T
out2 = in2 V* V*T
outm = inm V* V*T
Which is the purest Which is the purest outout (initial estimate)? (initial estimate)?
out450
δ ppm
I
SIMPLISMA
Initial estimate spectrum No. 1Initial estimate spectrum No. 1
Purity spectrumPurity spectrum
p
out450
Out no.
!+=
j
j
jx
sp
α
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Validation of SMCR
Rotational AmbiguityRotational AmbiguityD D == C C SSTTDD = ( = (CCTT) () (TT-1-1SSTT))
Intensity AmbiguityIntensity Ambiguity nnDD= = ΣΣ (1/ (1/kkiiccii) () (kkiissiiTT)) i=1 i=1
E = ||D - C ST||
ST = C+ D
C = D (ST)+
Constrain? Constrain?
Alternating Least Squares (ALS)Alternating Least Squares (ALS)
Constrained ( Constrained ( --- ) ) Unconstrained (Unconstrained (- -))
δ ppm
I
δ ppm
I
Poor matchPoor matchActive constraints
Lack of fit (LOF)Lack of fit (LOF)
!
LOF(%) =100
dij" ˆ d
ij( )2
ij
#
dij2
ij
#
Good matchGood matchFew active constraints
!
r2(%) =100"
ˆ d ij
2
ij
#
dij
2
ij
#
% Variance explained% Variance explained
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Experimental
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Experimental Design & Analysis
• Animal Handling• 48 dogs
• paired gender and weaning weight• Randomly assigned (CF/CR)• Initiated 8 weeks• CR (75% of CF)
• NMR spectroscopy• 400 µl of saline (10% D2O)in 200 µl blood plasma• Bruker DRX 600 NMRspectrometer
• 600.13 MHz for 1H• NOESY
• Binned Data (0.005 ppm)
Caloric RestrictedCaloric RestrictedControl Fed
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Experimental Design & Analysis
• Multivariate Metabolic Trajectory• PCA Trajectory plots• Reveal patterns and trends in the data• Scaled to UV
• SMCR Analysis• QITTFA Initial Estimates
• Non-negativity constraints, maximum iteration 500• ALS
• Non-negativity constraints in C and S and normalization of S
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Results and Discussion
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Multivariate Metabolic Trajectory (PCA)
Metabolites associated with the first two PC indicators of diet NOT aging
Control fed (CF)
Caloric Restricted (CR)
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Initial Estimates
Lipoprotein fatty acyl(CH2)n
CH=CH
C=CH-CH2-CH=C
-CH2-CO
-CH2-C=
Lipoprotein fatty acyl(CH3)
Cholesterol in HDL
Phosphatidylcholine-N(CH3)3
Phosphatidylcholine-OCH2
Phosphatidylcholine-NCH2
Dominantly Lipidic
GlucoseGlucose
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Initial Estimates
Sugar-protein
Lactate
Lactate Citrate
Glucose
Alanine
N-Acetyl glycoprotein
Albumin Lysylgroups
Mainly albuminCH3
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Final SMCR SolutionDominantly Lipidic
Sugar-protein
Lipoprotein fatty acyl(CH2)n
CH=CH
C=CH-CH2-CH=C
-CH2-CO
-CH2-C=
Lipoprotein fatty acyl(CH3)
Cholesterol in HDL
Phosphatidylcholine-N(CH3)3
Phosphatidylcholine-OCH2
Phosphatidylcholine-NCH2
Glucose Glucose
Lactate
LactateCitrate
Glucose
Alanine
N-Acetyl glycoprotein
Albumin Lysyl groups Mainly albumin CH3
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Final SMCR ResultsContour plot of the R2 Correlation Coefficients
Cholesterol in HDL
Lipoproteins
Phosphatidylcholine
Glucose
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Semi-quantitative trajectory
Period 1 (puppies) Period 2 (middle aged) Period 3 (elderly)
Dominantly lipidic (CF)
Dominantly lipidic (CR)
Sugar-protein (CF)
Sugar-protein (CR)
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Conclusion
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Conclusion
• Identified predominant sources of variation• No a priori information
• Pinpointed age groups where aging and dietbecame significant• Age groups which were phenotypically different
• Addition of new chemometric tool• Metabonomics toolbox
• Diverse application with other biomedical problems• Subtle time dependent changes
Richards, S. E.; Wang, Y.; Lawler, D.; Kochhar, S.; Holmes, E.; Lindon, J. C.; Nicholson, J. K. Analytical Chemistry 2008, 80, 4876-4885
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Acknowledgments
• Imperial College• Yulan Wang• Elaine Holmes• John Lindon• Jeremy Nicholson• Anthony Maher• Olaf Beckonert
• Nestle Research Centre• Dennis Lawler• Sunil Kochhar• Ziad Ramadan
• Funding• Nestle Research Centre