NMR Metabolomics:

Present and Future for Clinical Diagnosis of Cardiovascular Diseases

Núria Amigó Grau, PhD
November 2022, Riyadh
ORIGIN

366 M
Diabetic
patients
(2030)

573 M
Obese
adults
(2030)

65% p e o p l e
with By 2030, almost 23.6
million people will
metabolic disorders die from CVDs*
dyes from CVD

*from World Health

Organization
 ORIGIN

LDL concentration in 136.905 hospitalizations with CVD

More than the

50% of the patients
with a CV accident
present healthy levels
of LDL cholesterol.

Sachdeva. et al., American Heart Journal (2009)

 ORIGIN

Fewer LDL Particles More LDL Particles

Lower Risk Higher Risk

LDL-C 115 mg/dL OK LDL-C 115 mg/dL OK

LDL-P 800 nmol/L OK LDL-P 1200 nmol/L ↑

The lipid targets recommended by the American Association of

Clinical Endocrinologists for patients with T2DM

Mora S. et al., Circulation (2009)

Otvos JD et al. Journal of Clinical Lipidology (2011).
Otvos JD et al. Journal of Clinical Lipidology (2011).
Garber A.J. et al., Endocrine Practice 19 (2013)
Nordestgaard, B. G. & Nielsen, L. B. Current Opinion in Lipidology. 1994
Langlois MR, et al. Clin Chem. 2018
 v

Langlois MR, et al. Clin Chem. 2018

 v

Langlois MR, et al. Clin Chem. 2018

Langlois MR, et al. Clin Chem. 2018
 2100 nmol/l partículas
v
v
v

VLDL-P LDL-P VLDL-P LDL-P

200 nmol/l 1900 nmol/l 50 nmol/l 2050 nmol/l
LIPIDS, LIPOPROTEINS, AND METABOLITES AND RISK OF MYOCARDIAL INFARCTION AND
STROKE

Holmes MV et al. J Am Coll Cardiol. 2018

Lusis AJ, Pajukanta P. Nat Genet. 2008
ORIGIN
LIPOPROTEIN ANALYSIS BY NMR

• NMR spectroscopy: allows the structural analysis of a simple

compound or a mixture.

Lipoprotein
Envelopes

Free Induction Decay (FID)

LIPOPROTEIN ANALYSIS BY NMR

Lipoprotein resonating groups and their NMR signals:

LIPOPROTEIN ANALYSIS BY NMR
 LIPOPROTEIN ANALYSIS BY NMR (1D
APPROACH)

Otvos J.D. et al., Clinical Laboratory 48 (2002) 171-180

LIPOPROTEIN ANALYSIS BY NMR (2D
APPROACH)
DOSY-NMR Signal attenuation of the VLDL and HDL fractions.
LIPOPROTEIN ANALYSIS BY NMR (2D
APPROACH)

TEM images of VLDL particles A) 50k B) 40k C)

DOSY peak map showing the diffusion coefficients. 200k and D) 120k magnifications.
 TECHNOLOGY

The Liposcale test measures the size and the

lipidic content of main lipoprotein types
(VLDL, LDL and HDL) and the particle
concentration of nine subtypes (large, medium
and small of each main type).

FAST
(5 min)

Automated Reproducible
IVD-CE
ISO 13.485

High-
Intact sample
throughput
Mallol, R. et al., Journal of Lipid Research (2015)
NON-NMR LIPOPROTEIN PROFILING

Ultracentrifugation Density separation + measurement of LDL ApoB by radioimmunoassay

VAP + light scattering Deconvolution into subfractions of the direct cholesterol quantitation of lipoproteins
separated by flotation rate (a function of size and hydrated density). Plasma
lipoproteins were separated by single vertical spin density-gradient
ultracentrifugation of diluted plasma samples.

IM + light scattering electrospray procedure to obtain direct lipoprotein particle counts as a function of
particle size.

Electrophoresis (GGE, agarose gel), LDL-size polyacrylamide gradient gel electrophoresis

 HORTEGA STUDY 1.287 PATIENTS >12 YEARS

Liposcale (n=1,287) Standard Bioquemistry (n=1,287)

Weighted Median Weighted Median
(p25, p75) (p25, p75)
Cholesterol (C), mg/dL
Total cholesterol-c 199 (173.8, 228.2) Total Cholesterol, mg/dL 200 (173, 223)
VLDL-c 13.3 (6.1, 23.3)
IDL-c 7.7 (4.5, 11.5)
LDL-c 113.5 (94.5, 137.1) LDL, mg/dL 111.2 (89.8, 135.5)
HDL-c 59.5 (48.1, 71.9) HDL, mg/dL 51.2 (42, 61.2)
Triglycerides (TG),
mg/dL
Total triglycerides-tg 110 (74.5, 164.6) Triglycerides, mg/dL 141 (106, 203)
Pichler, G., Amigo, N., Tellez-Plaza, M., Pardo-Cea, M. A., Dominguez-Lucas, A., Marrachelli, V. G., ... & Redon, J.
(2018). LDL particle size and composition and incident cardiovascular disease in a South-European population: The
Hortega-Liposcale Follow-up Study. International Journal of Cardiology, 264, 172-178.

Spearman’s correlation coefficients for NMR (Tot-c Lip, VLDL-c Lip…) and standard lipoproteins
(Tot-c std, LDL-c Std…) determination.
 HORTEGA STUDY 1.287 PATIENTS >12 YEARS

Spearman’s correlation coefficients for NMR

(Tot-c Lip, VLDL-c Lip…) and standard
lipoproteins (Tot-c std, LDL-c Std…)
determination.

Pichler, G., Amigo, N., Tellez-Plaza, M., Pardo-Cea, M. A.,

Dominguez-Lucas, A., Marrachelli, V. G., ... & Redon, J.
(2018). LDL particle size and composition and incident
cardiovascular disease in a South-European population:
The Hortega-Liposcale Follow-up Study. International
Journal of Cardiology, 264, 172-178.
NON-NMR LIPOPROTEIN PROFILING

Ion mobility
NON-NMR LIPOPROTEIN PROFILING

VAP + light Plasma lipoproteins were separated by single

scattering: vertical spin density-gradient
(Atherotech) ultracentrifugation of diluted plasma
samples.

3000

2500
f(x) = 1.16833863230028 x − 291.933673352805
R² = 0.95010111419857
LDL-P (nmol/L) VAP Diagnostics

2000

1500
Series1
Linear (Series1)
1000

500

0
500.00 1000.00 1500.00 2000.00 2500.00 3000.00

LDL-P (nmol/L) Liposcale NMR method

 BEYOND LIPOSCALE
COMPANY EVOLUTIONTEST

Liposcale-Clinical market
Liposcale ® Industrial International
Constitution R&D lines development-Research
Development expansion
market
2012 2014 2016 2020 2022

Glycoprotein Metabolomics profiling Liposcale® Test

Liposcale® Test profiling -Urine, cells, tissues,
faeces.
HOSPITALS

CLINICAL LABORATORIES (Liposcale test sales)

• High throughput screening technology
• Competitive
• Quantitative
• Robust & scalable
• Automated
• Fast
• Discovery / targeted approach simultaneously
 Threonine
HDL-C Cholesterol Tyrosine
IDL-C Histidine
LDL-C VLDL-C Glutamate
LIPOPROTEINS
Aminoacids Leucine Alanine

Triglycerides Isoleucine Valine

IDL-TG
VLDL-TG
LDL-TG Glycine Glutamine
HDL-TG
METABOLISM
VLDL-P
Particle number KETONE BODIES
L-VLDL-P Acetate Creatinine
LDL-P
M-VLDL-P Acetone
Blood plasma 3-Hydroxybutyrate
L-LDL-P
S-VLDL-P HDL-P
M-LDL-P INFLAMMATION Lactate Creatine
L-HDL-P Size
S-LDL-P GlycF Glycerol
VLDL-Z Glucose
M-HDL-P
GlycB
LDL-Z
S-HDL-P
GlycA
HDL-Z
 HDL-C Cholesterol
INFLAMMATION
IDL-C
LDL-C VLDL-C
LIPOPROTEINS

Triglycerides
IDL-TG
VLDL-TG
LDL-TG
HDL-TG

VLDL-P
Particle number
L-VLDL-P
LDL-P
M-VLDL-P
L-LDL-P Blood plasma
S-VLDL-P HDL-P
M-LDL-P INFLAMMATION
L-HDL-P Size
S-LDL-P GlycF
VLDL-Z
M-HDL-P
GlycB
LDL-Z
S-HDL-P
GlycA
HDL-Z
 Human Serum/Plasma Glycoprotein Analysis by 1H-NMR
An Emerging Method of Inflammatory Assessment

Fuertes-Martín R et al. JCM 2020. 9: 354

 Higher levels of Glyc A and B associated to Subclinical
atherosclerosis in FH

IMT Atherosclerotic plaque

PWV

Malo AI et al. Atherosclerosis 330 (2021) 1–7

Fuertes-Martín, et al. Journal of Clinical Medicine 9.2 (2020).C
 Threonine
HDL-C Cholesterol Tyrosine
IDL-C Histidine
LDL-C VLDL-C Glutamate
LIPOPROTEINS
Aminoacids Leucine Alanine

Triglycerides Isoleucine Valine

IDL-TG
VLDL-TG
LDL-TG Glycine Glutamine
HDL-TG
METABOLISM
VLDL-P
Particle number KETONE BODIES
L-VLDL-P Acetate Creatinine
LDL-P
M-VLDL-P Acetone
Blood plasma 3-Hydroxybutyrate
L-LDL-P
S-VLDL-P HDL-P
M-LDL-P INFLAMMATION Lactate Creatine
L-HDL-P Size
S-LDL-P GlycF Glycerol
VLDL-Z Glucose
M-HDL-P
GlycB
LDL-Z
S-HDL-P
GlycA
HDL-Z
Data analysis

Functional
interpretation
 Genomics Transcriptomics Proteomics Metabolomics

- Phenotype + Phenotype

4: AGCT 4: AGCU 20: Aminoacids >1000 compounds

"Genomics and proteomics tell you what might happen, but metabolomics tells you what actually did happen."
Bill Lasley, University of California (UC), Davis.
 Regressions Clustering

The
Risk stratification metabolome Pattern recognition

Classification models Predictive models

Diagnostic test Response & prognostic test

Precision medicine
 BEYOND LIPOSCALE TEST
TEAM

NURIA DANIEL R. ROMEU ENRIQUE SARA SAMINO NEUS MARTINEZ.LYDIA CABAU. CARLA MERINO
AMIGÓ CTO OZCARIZ Quality manager CSO Project Manager Lab Manager
CEO, Co- Computer engineer, COO Biochemist, PhD. Bioinformatics & Chemist, PhD. Biochemist, PhD.
Founder security & software Biochemist Regulatory biostatistics, PhD.
Biophysicist, developer. Lab manager. officer.
PhD
PARTNERS & INSTITUTIONS
XAVIER CORREIG Co-Founder & Technology
Advisor
Director of the Metabolomics Platform
Rovira I Virgili University (Tarragona, Spain)

ROGER MALLOL
Co-Founder
LLUÍS MASANA Co-Founder & Clinical Advisor
Computer engineer, Director of the Research Unit in Lipids and Atherosclerosis
PhD. Sant Joan University Hospital (Reus, Spain)
 Summary

1. Beyond LDL-C levels, cardiovascular associated lipid alterations are present in a

high percentage of apparently healthy individuals.
2. NMR spectroscopy can simultaneously characterize lipid and inflammatory residual
risk:
• Advanced lipoprotein profiling by NMR spectroscopy can be used to better understand
lipoprotein metabolism and to identify pro-atherosclerotic patterns.
• Advanced glycoprotein profiling by NMR spectroscopy can be used to identify patients with low
grade inflammation and increased risk for CVD.
3. NMR based on metabolomics is a high throughput methodology, scalable,
quantitative, robust and compatible with clinical requirements.
namigo@biosferteslab.com
LIPOPROTEIN ANALYSIS BY NMR (2D APPROACH)

Mallol R. et al., Journal of Lipid Research. 2015

 LIPOPROTEIN ANALYSIS BY NMR (2D
APPROACH)

 K ·Dcoefficient ·G 2
I ( g )  I 0e

K BT
rmolecule 
6  Dcoefficient
Mallol R. et al., Journal of Lipid Research (2015)
 LIPOPROTEIN ANALYSIS BY NMR (2D
APPROACH)

VITAGE (n=177)

25/33
Mallol R. et al., Journal of Lipid Research (2015)
 LIPOPROTEIN ANALYSIS BY NMR (2D
APPROACH)

Mallol R. et al., Journal of Lipid Research (2015)

LIPOPROTEIN ANALYSIS BY NMR (2D
APPROACH)
1st Tertile LDL-TG/LDL-C ratio
3000 2500
r=0.86 r=0.84
2500
2000

LDL-P (nmol/L)
2000

LDL-P (nmol/L)
1500
1500
1000
1000

500 500

0 0
20 40 60 80 100 20 40 60 80 100
LDL-ApoB (mg/dL) LDL-ApoB (mg/dL)
2nd Tertile LDL-TG/LDL-C ratio 3rd Tertile LDL-TG/LDL-C ratio
2500 2500
r=0.86 Liposcale Test (r=0.88)

2000 2000
LDL-P (nmol/L)

LDL-P (nmol/L)
1500 1500

1000 1000

500 500

0 0
20 40 60 80 100 20 40 60 80 100
LDL-ApoB (mg/dL) LDL-ApoB (mg/dL)
 LIPOPROTEIN ANALYSIS BY NMR (2D
APPROACH)

Vitage set (n=177): Briefly, the lipoproteins fractions were separated by sequential preparative ultracentrifugation, using a Kontron 45.6 fixed-angle
rotor in a Centrikon 75 (Kontron Instruments, Italy). The lipoprotein fractions isolated were VLDL (d < 1.006 g/ml), IDL (d = 1.006– 1.019 g/ml),
LDL (d = 1.019–1.063 g/ml), and HDL (d = 1.063–1.21 g/ml). Their cholesterol and triglyceride content were quantified using standard enzymatic
assays adapted to the Cobas-Mira-Plus autoanalyzer (SPINREACT S.A.U., Spain).
Size ranges obtained from 1D and 2D NMR approaches

Ranges MEAN Ranges (min, max)

LipoScience (Ø, nm) Liposcale (Ø, nm) (Ø, nm)

VLDL-Large >60 VLDL-Large 82 68,5 95,5

VLDL-Medium 35-60 VLDL-Medium 55 47 68,5

VLDL-Small 27-35 VLDL-Small 39 32,5 47

IDL 23-27 LDL-Large 26 24 32,5

LDL Large 21.2 -23 LDL-Medium 22 20,5 24

LDL Small 18-21.2 LDL-Small 19 17,5 20,5

HDL-Large 8.8-13 HDL-Large 12 10,5 13,5

HDL-Medium 8.2-8.8 HDL-Medium 9 8,5 10,5

HDL-Small 7.3-8.2 HDL-Small 8 7,5 8,5

 LIPOPROTEIN ANALYSIS BY NMR (2D
APPROACH)

Vitage set (n=177): Briefly, the lipoproteins fractions were separated by sequential preparative ultracentrifugation, using a Kontron 45.6 fixed-angle
rotor in a Centrikon 75 (Kontron Instruments, Italy). The lipoprotein fractions isolated were VLDL (d < 1.006 g/ml), IDL (d = 1.006– 1.019 g/ml),
LDL (d = 1.019–1.063 g/ml), and HDL (d = 1.063–1.21 g/ml). Their cholesterol and triglyceride content were quantified using standard enzymatic
assays adapted to the Cobas-Mira-Plus autoanalyzer (SPINREACT S.A.U., Spain).