14C-Pathway probing as dynamic biomarker read-out:

De Novo Lipogenesis using

14C-labeled acetate and Accelerator Mass Spectrometry

Elwin R. Verheij, Lianne Stevens, Martine C. Morrison, Robert Kleemann, and Wouter H.J Vaes
TNO, Leiden, The Netherlands

INTRODUCTION

The application of isotopes is a proven concept in biomarker research to measure the dynamics ofendogenous processes. Good examples, amongst others, are the administration of D2O to determine
collagen production (analysis of OD-proline in collagen), 13C or 2H labelled amino acid to measureproduction of specific proteins, 41Ca to monitor changes in bone mineral, etc. to assess the efficacy ofinterventions. Here we present the use of 14C acetate to investigate de novo lipogenesis (DNL) andcholesterol biosynthesis. The advantages of 14C as opposed to 13C are 1) the natural background of
14C is extremely low, and 2) it enables us to harness the extreme sensitivity of Accelerator Mass
Spectrometry (AMS).

EX VIVO LIVER PERFUSION MODEL

EXPERIMENTAL

A microtracer equivalent of 14C-acetate was administered to mice (30 Bq) and blood was collected
after 5h. Bligh and Dyer extraction was used to measure the total amount of 14C in the plasma lipid
fraction. Fatty acids were obtained by saponification of the lipid fraction using 4M KOH followed byextraction with n-hexane. The fatty acids were separated and fractionated by HPLC to obtain a 14Cfatty acid profile by AMS analysis.
In an ex-vivo liver (pig) experiment 14C-acetate was administered by portal infusion. Plasma samples
were collected up to 4 h and analysed in similar fashion as the mouse plasma samples.

14C detected in bile
125

4100

321060120180240

Time after dosing (min)150

AUC: 13,9 kBq
10075

DPM

14C bile (Bq)50

Presence of 14C inplasma lipid fraction

corrected for bile production

end of dosing

14C Plasma (Bq)

DE NOVO LIPOGENESIS

14C detected in Plasma

5050

2500601201800240

Time after dosing (min)060120180240

Time after dosing (min)LC-MS-AMS CONFIRMED INCORPORATION OF 14C IN
FATTY ACIDS
PLASMA (MOUSE)
C16:0

C18:1

PROOF OF CONCEPT EXPERIMENTPLASMA (LIVERPERFUSION MODEL)t = 0 min
C18:0
C16:0
C16:0

LIVER (MOUSE)
C16:0

C18:1

Mice were challenged with different dietary conditions:• LFHC:
low-fat, high carb (starch)
n=5• HFS:
high-fat, moderate carb (sucrose) n=5• HFF:
high-fat, moderate carb (fructose)
n=514CFROM ACETATE IS INCORPORATED INTO LIPID
FRACTION IN PLASMA AND LIVER
CORRELATION105

C incorporation into lipids (AU)in plasma

15

✱

25
20
15
10
51414

✱✱

C incorporation into lipids(fold-change over background)

20

PLASMA - LIVER

PLASMA✱

14

C incorporation into lipids(fold-change over background)

LIVER✱0LFHC

HFS

HFF0

LFHC

HFSHFF25LFHC
HFS20HFF1510R2=0.87
p = <0.001

50014102030

C incorporation into lipids (AU)in liver

DNL activity is quantifiable in liver tissue and in plasma• Strong correlation between plasma and liver → plasma can be used as a proxy for liver
DNL
Method is sensitive enough to detect dietary effects on DNL activity• As expected, DNL activity is highest in the low-fat, high-carb diet group and DNL issuppressed by diets high in fat with fructose or sucrose

INHIBITION OF 14C INCORPORATION BY DNL INHIBITOR
PLASMA
✱✱

4-58%

0firsocostat

2vehicle

14C/12C ratio (x 1E+11)

6Treatment with the DNL inhibitor
firsocostat (ACCi) resulted in a14Cstrong
reductionofincorporation into total plasma
lipids as expected.

t = 60 min

C18:0

C16:0
C18:1 C18:0

C18:1

C18:0

Subsequent fatty acid profiling analysis (quantitation by AMS, identification by LC/MS) andenrichment analysis of the different fractions collected 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 (elongation and/or desaturation: C18:0and C18:1) thus confirming that the observed incorporation of 14C from acetate into the lipid fractionof plasma and liver is indeed a reflection of DNL.

CONCLUSIONS
The administration of 14C-acetate, in vivo and ex vivo models, followed by AMS analysis results in theincorporation of the 14C label in the primary fatty acid products of DNL (including elongation anddesaturation). The results of the experiments with different diets and the administration of a DNL
inhibitor agree very well with the expected outcome, thus proving the method allows quantitative
measurement of DNL activity. Latest experiments also showed the incorporation of the 14C label incholesterol. The 14C-label in the liver perfusion model was excreted in bile. Identification of themetabolites containing the label is in progress.
The next step is to apply this approach in a study with human volunteers. Extrapolation of the amountof 14C administered in the mouse experiments (30 Bq) suggests that a dose of about a few µCi
should be sufficient to measure DNL activity in human volunteer studies. This amount of radioactivity
is the same order of magnitude as for drug microtracer studies, and thus would not require any
dosimetry study to be applied in a clinical setting.
The administration of 14C-labeled substrate followed by isolation of the specific 14C-end-product of ametabolic pathway, either by extraction of UPLC isolation, can be applied generically to quantify thedynamics of enzymatic processes. The principle can be used in many other applications, not only forconversion of small molecules but also the de novo synthesis rate of specific proteins byadministering a 14C labelled amino acid. Isolation of the target protein can be achieved with, forexample, immunoaffinity capture technologies.
Advantages of this approach are:1) It is a generic biomarker technology to measure the dynamics of metabolic pathways (fluxes),protein production, or other processes with very high specificity.
2) Accelerator mass spectrometry is among the most sensitive analysis techniques enabling themeasurement of biomarkers at extremely low concentrations.
3) Due to the extremely low and de facto constant background of 14C the measured end-productconcentration is not superimposed on inter and/or intra subject background variation.
Theoretically, this should increase the statistical power of study outcomes.