Biology Reference
In-Depth Information
nine months and reported that there were no
signi
a number of factors including the sample
spH,
ionic strength, solute and multivalent cation
concentration, and temperature. NMR peaks
belonging to the same metabolite can behave
differently due to ionizable groups, chemical
exchange, and interactions with other compo-
nents in the mixture. The chemical shifts of
metabolites when bound to metal ions are
usually shifted and broadened. Ethylenedia-
minetetraacetic acid (EDTA) is a strong metal
chelating agent that can be used to chelate
Ca
2
þ
and Mg
2
þ
ions.
30
e
32
Urine has a wide pH range and ionic
strength, which frequently lead to chemical shift
deviations that result in spectral misalignments,
making it dif
'
cant differences between the spectra of
both samples.
25
Saude et al. monitored changes
in raw urine samples from a single male and
female volunteers prepared with and without
sodium azide and stored at 22
C, 4
C, and
e
80
C for a period of four weeks.
26
Metabolic
changes attributed to bacterial activities were
observed in citrate, hippurate, glycine, benzoate,
and others, especially for raw urine samples
stored at room temperature.
27,28
Bacterial
contamination, especially for rodent studies, is
often unknown until NMR data collection, by
which time it is too late for remediation proce-
dures; therefore urine samples should be stored
at
e
80
C with 0.02% w/v sodium azide.
9
Sample preparation is equally important and
researchers continue to optimize conditions for
speci
cult to directly compare a series
of spectra. A typical
1
H NMR spectrum of
human urine is shown in
Figure 1
.
33
The pH
range of urine is 4.5
e
8, depending on diet,
health of the individual, bacterial contamina-
tion, and other factors.
34
Phosphate buffer,
which has a pKa close to physiological pH
and contributes no proton and carbon NMR
signals, is added to reduce urinary pH varia-
tions. The simplest and fastest method is to
dilute the urine samples in a predetermined
urine-to-buffer ratio. However, there is no
consensus on the exact ratio. For human urine,
a urine-to-buffer ratio of 2:1 with a buffer stock
of 0.2 M and 0.9 M has been recommended.
24,25
Taking both pH and ionic strength as well as
dilution effect into account, Xiao et al. deter-
mined that a urine-to-buffer ratio of 10:1 with
the buffer stock at 1.5 M was adequate to keep
the pH within the range of 7.1 to 7.7 for normal
human urine.
35
With the exception of citrate
and histidine, the chemical shifts of most metab-
olites with ionizable groups were controlled
within 0.002 ppm. When preparing buffer solu-
tion over 0.5 M, K
2
HPO
4
is chosen for its better
solubility over Na
2
HPO
4
. Some researchers
prefer
c applications. Each sample preparation
technique has its own advantages and draw-
backs and may not be universally applicable to
all studies. Investigators must decide what
compromises need to be made based on their
individual experimental designs and informa-
tion sought. For instance, lyophilization fol-
lowed by reconstitution in D
2
O results in the
loss of labile hydrogen atoms such as the methy-
lene group of creatinine.
29
Hopefully, in the near
future, optimized protocols will emerge for the
various types of samples studied by NMR spec-
troscopy and be universally adopted. Until then,
current techniques are discussed in detail so that
we can progress toward this goal.
Biological Fluids without
Macromolecules
Sample preparation for NMR analysis is
known for its simplicity. The two requirements
are a deuterated solvent
field-frequency
lock and a chemical shift reference. NMR
frequencies are reported on a chemical shift scale
that is independent of magnetic
for
finer pH adjustment by manual adjust-
ment with NaOH and HCl.
14,36
Although we
generally think of phosphate as a buffer, it is
a salt. It is well known that salt concentration
field strength.
The chemical shifts of metabolites and other
small molecules in solution are in
uenced by
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