Current NMR Strategies for Biomarker Discovery - Proteomic and Metabolomic Approaches to Biomarker Discovery - page 90

quench metabolic activity by more ef cient

protein denaturation but can cause degradation
of acid-labile metabolites such as fructose-2-6-
bisphosphate, AcCoA, NADPH, and NADH. 47
Martineau and coworkers compared

to ensure that a representative region is selected.
This approach will also allow the NMR results to
be directly compared to histopathology.

ve extrac-
tion methods for breast cancer cells: 4:1 ACN/
H 2 O, MeOH, 4:1 MeOH/H 2 O, 2:2:1.8 MeOH/
CHCl 3 /H 2 O, and PCA. They concluded that
the MeOH/CHCl 3 /H 2 O method was the most
reproducible and extracted a higher range of
metabolites. 45
Perchloric acid extraction is still preferred by
a number of investigators where metabolism
must be quickly quenched, that is, brain tissues
and cells. For liver tissue extraction, the 2:2:1.8
MeOH/H 2 O/CHCl 3 extraction method was
shown to be superior in reproducibility to other
extractions methods such as 1:1 and 2:1 MeOH/
H 2 O, 1:1 and 4:1 ACN/H 2 O, and PCA. 48

Internal and External Chemical Shift
Standards

Internal Standards
Internal or external standards are used for
spectral referencing and quantitation. The two
most widely used internal proton chemical shift
reference standard for urine samples are
3-trimethylsilyl-propionate sodium salt (TSP)
and 3-trimethylsilyl-1-propane sulfonic acid
sodium salt (DSS). The deuterated versions,
DSS-d 6 and TSP-d 4 , are preferred because only
one sharp singlet is observed from the three
methyl groups. The chemical shift of TSP is
known to be pH, concentration, and temperature
dependent, which reemphasizes the need for
careful pH control. 53 e 55 Both TSP and DSS (to
a lesser extent) are known to interact with macro-
molecules and are thus not suitable for serumand
plasma samples where protein content is
high. 55,56 Some researchers have used NMR
peaks from endogenous metabolites (such as
formate, lactate, and glucose) already present in
the sample. Another reference standard d 4,
4-dimethyl-4-silapentane-1-ammonium tri

Intact Tissue for HR-MAS
In vivo NMR spectroscopy of intact biological
tissues is studied by high-resolution magic angle
spinning (HR-MAS). An introduction to
HR-MAS and detailed discussions on sample
collection, storage, and preparation can be found
in a review by Sitter et al. 49 Beckonert et al. have
also provided a sample protocol for intact tissue
preparation for HR-MAS. 50 The sample prepara-
tion stage involves minimal

uor-
oacetate (DSA), whose structure is similar to
DSS but has minimal interactions with peptides
and proteins d is not very hydroscopic and has
minimal pH and concentration dependence. 57,58
With these attributes, DSA can be used as
a universal internal standard for all biological

tissue handling.

Tissue samples are usually first rinsed with

D 2 O saline to remove blood and to improve
water suppression, then placed in a Zirconia
rotor. The D 2 O serves as the NMR lock solvent.
Rinsing the tissue has, however, been shown to
lead to metabolite leakages and could result in
up to 50% loss of water soluble metabolites. 51
Different tissues have different biochemical
stability issues. 52 For tissues with high metabolic
rates, such as brain tissue, the biggest challenge
is to minimize enzyme dependent post-mortem
metabolic changes. For cancer research, tumor
heterogeneity and necrosis core must be consid-
ered when excising a tissue section for analysis

fluids. The partially deuterated form DSA-d 6
((CH 3 ) 3 SiCD 2 CD 2 CD 2 NH 3 þ CF 3 COO e ) appears
as a singlet at d H 0.016 measured relative to TSP
( d H 0.000) at pH 7.4. 58 Tetramethylsilane (TMS)
is the preferred standard for organic solvents.
For HR-MAS studies, an internal metabolite
such as the anomeric proton of a -glucose at
d 5.23, b -glucose at d 4.65, acetate at d 1.93, or
alanine at d 1.48 is used for referencing instead