A prospective, methodology study to identify potential biomarkers of myelin dynamics in CSF using metabolic labeling with stable isotopes in healthy volunteers

Multiple sclerosis (MS) is the most important inflammatory disease of the
central nervous system in the Western world.
This inflammatory disease is characterized by demyelination that results from
breakdown of myelin with a subsequent relative failure of the
spontaneous remyelination process. Current treatment of multiple sclerosis is
aimed at inhibition of the inflammatory component causing the demyelination.
Enhancement of remyelination may improve recovery after an exacerbation.
The myelin sheath that surrounds most axons in the brain is a spiral structure
that consists of extensions of the plasma membrane of oligodendrocytes. In the
adult brain, the components of myelin have different turnover rates. Several
dynamic changes due to the degradation and synthesis of the lipid and protein
components of myelin occur during demyelination and remyelination, respectively.
The ability to monitor the processes of demyelination and remyelination will be
necessary to be able to quantify the response therapeutic interventions
targeting remyelination, but may also be valuable to track disease activity in
demyelinating disorders.
This study aims to develop a metabolic labeling methodoly using
non-radioactive, stable isotope labeling combined with mass spectrometric
analyses to measure the turnover kinetics of the myelin specific lipids,
galactosylceramides (GalC), and sulfatides in human CSF. For this purpose,
healthy human subjects are administered with D2O (deuterated water, 2H2O or
*heavy water*), wherein the stable isotope deuterium (2H) is incorporated into
biomolecules including lipids that were newly synthesized during the duration
of label exposure. After degradation, these molecules will be measured in CSF
that will be obtained through lumbar punctures.

Primary Objective
• To demonstrate feasibility of measuring deuterium incorporation into
galactosylceramides within the quantitation limits of the assay, in human CSF
after chronic dosing of D2O.
• To develop a mathematical model to allow estimation of the kinetics of myelin
turnover based on myelin breakdown products in CSF after chronic labeling with
deuterium.

Secondary Objectives
• To determine the fractional synthesis rate of sulfatide (fSulft).
• To determine the D2O enrichment in body water using urine.
• To measure total concentration of galactosylceramide and sulfatide in the CSF.
• To determine the intra-individual variability of galactosylceramide and
sulfatide in CSF of healthy subjects.
• Evaluate the safety and tolerability of drinking D2O for a longer period of
time.

Exploratory Objectives
• Evaluate dynamic proteomics of other proteins and lipids in the CSF and
plasma samples.
• Determine galactosylceramides in urine.

Study in 6 healthy volunteers to investigate if D2O administration results in
deuterium labeling of myelin. Quantification of absorption will be determined
in CSF, plasma and urine.
• 6 subjects that meet the recruitment criteria will be instructed to consume
two doses of 60 mL 70% D2O every day for ten weeks. Urine samples will be
collected once a week to measure the percentage D2O in body water.
• the subjects will receive 4 lumbar punctures (LP) for CSF and concurrent
venous punctures for plasma collection. These are scheduled 35, 70, 94 and 167
days after start of the study.

Possible burdens:
- Bruising at the place of venapunction
- The possible side-effects from the lumbar puncture (for example headache, low
bloodpressure, nausea and dizziness).
- Findings during tests (i.e. positive test result for hepatitis B, hepatitis C
or HIV).
- Drinking heavy water can cause dizziness. However, because the amounts are
small, we don't expect this side-effect (based on literature).

The healthy volunteers will not benefit from this study. The methodology can be
used in future studies to prove the effectiveness of pharmaceutical compunds
improving remyelinization, benefiting MS patients.