14HIGHLY SENSITIVE METABOLIC FLUX ANALYSIS USING C MICROTRACERS

Morrison MC, Stevens L, Kleemann R,Verheij ER, Vaes W.

COMBINED WITH ACCELERATOR MASS SPECTROMETRY

Department of Metabolic Health Research, the Netherlands
Organisation for Applied Scientific Research (TNO), Leiden,
the Netherlands.

A PROOF OF CONCEPT STUDY ON DE NOVO LIPOGENESIS

Assessment of DNL using a 14C-acetate microtracer

300120✱180Time after dosing (min)302010200100240120120180180240240120120120180180180240240240800

8006006004004002002000C label in perfusatelipid fration (Bq/mL perfusate)

1450

50000 3060 240
90 120 240
30 60
90 120Timeafter(min)dosing (min)Time after
dosing

C18:0

800
600
400
200030 60 90 120 240

Time after dosing (min)D20040402020000030
600 240
903012060240
90 120 240
30 60 90 120

60

60

0

Cholesterol
505080

80

EE

150
100
50

Timeafter(min)dosing(min)Timeafter
dosing (min)Time after
dosing

40
30
20
10

Cholesterol AUC(Bq/mL perfusate)

C16:0100

100

C18:0

Cholesterol AUC(Bq/mL perfusate)

1000

C

Myristic acid AUC(Bq/mL perfusate)

C16:00004030201000 3060 240
90 120 24030 6090 120

Timeafter(min)dosing (min)Time after
dosing

ELC/MS analysis followed by AMS enrichment analysis showed 14C enrichment
in C16:0, C18:0 and C18:1 fatty acids, again confirming DNL. Furthermore, 14C
from acetate was also observed in the cholesterol fraction, showing that thismethodology can also be applied for analysis of new cholesterol synthesis.
Cholesterol AUC(Bq/mL perfusate)

Stearin AUC(Bq/mL perfusate)

50806040204030201000De novo lipogenesis activity was assessed in Ldlr-/-.Leiden
MASLD mice1 fed a standard chow or a MASLD-inducing highfat diet (HFD) with/without the DNL inhibitor firsocostat (ACCi),
as well as in an ex vivo porcine liver perfusion model2.

2402401000Stearin AUC(Bq/mL perfusate)t=60 minutes

C18:1

in plasma and liver reflect DNL activity

18018010000 3060 240
90 120 240
30 6090 120

DD

Stearin AUC(Bq/mL perfusate)

DNL inhibitor firsocostat (inhibits ACC),
strongly reduces 14C incorporation inplasma lipid fraction as expected.

BPalmatin AUC(Bq/mL perfusate)

firsocostat

FFDvehicle

002. Stevens et al., Evaluation of Normothermic Machine Perfusion of Porcine Livers as a Novel Preclinical Model to Predict Biliary
Clearance and Transporter-Mediated Drug-Drug Interactions Using Statins. Drug Metab Dispos. 2021.

120120Similar to observations in the Ldlr-/-.Leiden MASH mouse model, 14C fromacetate was also incorporated into the plasma lipid fraction in the ex vivo liver
perfusion model. In addition, enrichment of 14C was also observed in the bile
collected from the ex vivo liver.
0Methods

1. Gart et al., Translational characterization of the temporal dynamics of metabolic dysfunctions in liver, adipose tissue and thegut during diet-induced NASH development in Ldlr-/-.Leiden mice. Heliyon. 2023.

60 60

10150
Time
after
dosing(min)
150
Time
after
dosing(min)

100

Incorporation of 14C from 14C-acetate into total lipid extracts(from plasma and liver samples), bile, fatty acids (plasma andliver), and cholesterol was assessed by LC/MS and AMS.

200200Time
after(min)dosing (min)Time after
dosingincreased in Ldlr-/-.Leiden mice with
MASH (FFD) relative to chow.

14C fatty acids

C bile (Bq)
C label in perfusate
lipid fration (Bq/mL perfusate)

14606060

CC25 20 00 01000

1000

Time
after
dosing
(min)
Time
after
dosing
(min)
Time
after
dosing(min)

Timeafter
afterdosing
dosing(min)
(min)
Time

14

1460
600 000 00

B

B

Myristic acid AUC(Bq/mL perfusate)

40600000

25 25

Myristic acid AUC(Bq/mL perfusate)

014C incorporation in lipid fraction isProvide proof of concept for aapproach toassess activity of the de novo lipogenesis (DNL) pathway. DNL is
an important metabolic pathway that is deregulated in various
disease states, including metabolic dysfunction-associatedsteatotic liver disease (MASLD).

255

50 4

Palmatin AUC(Bq/mL perfusate)✱C Perfusate
in perfusate
labelC(Bq/mL14 perfusate)bile (Bq)perfusate)(Bq/mL
lipid fration C

Ldlr-/-.Leiden
MASH model575 6410 50

1050

C100 8615 751575

14100chow

14C-microtracer

1414C Perfusate

1520100CB
125 10820100

C bile (Bq)
(Bq/mL perfusate)

20
14C from acetate is incorporated into lipid fraction inend of dosing

12525102512525plasma (and liver) of Ldlr-/-.Leiden MASH miceAC B

end of dosing

Palmatin AUC(Bq/mL perfusate)

end of dosing

0AimA
B

A

mBq per ml plasma(lipid extract)

An alternative isotope is 14C, which has an extremely low naturalabundance (1 in 1012). In combination with analysis by extremely
sensitive accelerator mass spectrometry (AMS), this enables thedetection of very small amounts of 14C-labeled product/biomarker
at very low (microtrace) amounts of labelled substrateadministration.

DNL is a pathway in which non-lipid
precursors (primarily carbohydrates) areconverted into lipids. The process begins
with acetyl-Coa and can be assessed using
a 14C-acetate tracer.

C Perfusate(Bq/mL perfusate)

An isotope that is frequently used for such analyses is 13C, which
is analyzed by isotope ratio mass spectrometry. The high naturalabundance of 13C (~1%) requires the use of large amounts oflabelled substrates, especially in clinical studies.

Metabolic flux analysis in ex vivo liver for DNL, bile andcholesterol synthesis using 14C microtracers.

14

Metabolic flux measurements play an important role in advancing
our understanding of (patho)physiology, disease mechanisms
and the development of new therapeutics. By incorporating
stable or radioactive isotopes into specific molecules (tracers),
the distribution and fate of the isotope can be followed – thereby
providing insight into the movement and metabolictransformation of biomolecules.

mBq per ml plasma(lipid extract)

Introduction

30 60 90 120 240

Time after dosing (min)030 60 90 120 240

Time after dosing (min)4. Conclusion
Fatty acid profiling analysis (LC/MS) combined with AMS enrichment analysis
showed that the 14C signal from acetate is predominantly found in palmitate(C16:0, the primary end product of DNL) and in lesser amounts also in fatty
acids that result from further processing of palmitate (C18:0 and C18:1) thus
confirming that the observed incorporation of 14C from acetate into the lipid
fraction of plasma and liver is indeed a reflection of DNL.

This highly sensitive 14C microtracer approach combined with AMS analysis canbe used for assessment of metabolic fluxes in various experimental models at
much lower amounts of tracer used than in conventional tracer methodologies.
This allows study of metabolic pathways without disrupting the pathway ofinterest by adding large quantities of precursor. This opens up opportunities tostudy the activity of (metabolic) pathways in which only very small amounts ofproduct are produced.

martine.morrison@tno.nl

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