Nuclear Magnetic Resonance Spectroscopy: Theory and Applications
505
susceptible to fracture [58]. Similar results were observed in human patients bone
samples suffering from osteoporosis [59]. When a lot of studies started coming on
bone samples it became essential to optimized the optimum storage and transport
conditions for cortical bone samples so that we do not get variations in ssNMR
parameters such as
31 P longitudinal relaxation (T 1 ),
13 C ssNMR spectra and
1 H
NMR to measure water content [52, 54, 60, 61]. Surprisingly storing in PBS which
is normal for micro-CT related preservation of bones changes ssNMR parameters,
specially
31 P longitudinal relaxation time (T 1 ) [54] a comparative study showing
correlation of micro-CT parameters and ssNMR parameters showed that most of the
parameters correlate specially total water, phosphorus relaxation and inter-atomic
organic to inorganic Interface [53].
Most of the research in history and even now on collagen protein has been carried
out overexpressed and purified collagen protein in absence of the mineral part. Now
we know that mineralized form of type 1 collagen from mineralized cortical bones
are significantly different from the collagen type 1 which is extracted from Kangaroo
tail. This difference was observed by comparing Hydrated native collagen in bone
matrix has entirely different
1 H–
1 H dipolar coupling network [62]. Collagen stability
is widely accepted being attributed to Hyp mediated hydrogen bonding network with
water molecules [63]. Recently there were reports rejecting the old hypothesis and
considering stereoelectronic effects as prime contributor for the stability of collagen
protein [64]. Singh et al., measured the backbone dynamics of collagen protein as a
function of dehydration by recording
15 N spectra which gives resonances from Gly
and Pro/Hyp in natural abundance (0.37%) by paramagnetic doping to reduce the
T 1 of
1 H nuclei hence the recycle delay [65]. This study showed that the backbone
dynamics of Pro/Hyp got more affected after removal of water as compared to Gly
[65]. Furthermore, it was shown that cortical bones have water compartments and
other than playing role in stability of collagen protein they also play crucial role in
load bearing capability of bones. Exchange of water takes place between different
water compartments.
One of the main challenge in the study of bones by NMR spectroscopy is low
natural abundance of most commonly used nuclei such as
13 C (1.1%), and
15 N
(0.37%), hence efforts have been done to increase the sensitivity of the NMR signal by
following different approaches [65, 66]. As we know that the experiment time in NMR
depends upon T 1 and it is advised that relaxation delay (d1, which is used before each
scan to allow proper relaxation of the spins from x–y plane to z-axis) should be 5 times
T 1 . By reducing T 1 we can reduce total experimental time. Paramagnetic doping using
Cu-EDTA and Gadolinium-DTPA helped to reduce the total experimental time [65,
67, 68]. Dynamic nuclear polarization (DNP) based sensitivity enhancement helped
to detect CH-p interaction in native mineralized collagen protein inside bones for the
first time [69]. DNP has also been applied in cartilage samples to detect structural
changes in collagen protein upon pigmentation in Alkaptonuria [70]. Similar level
of sensitivity enhancement has been achieved using the latest BioSolids CryoProbe
technology for ssNMR [71]. Another approach involved the development of heavy
mouse which had bones enriched in
13 C and
15 N nuclei up to 20% by growing
them on
13 C and
15 N labeled diet [72]. This resulted in discovery of poly (adenosine
505
susceptible to fracture [58]. Similar results were observed in human patients bone
samples suffering from osteoporosis [59]. When a lot of studies started coming on
bone samples it became essential to optimized the optimum storage and transport
conditions for cortical bone samples so that we do not get variations in ssNMR
parameters such as
31 P longitudinal relaxation (T 1 ),
13 C ssNMR spectra and
1 H
NMR to measure water content [52, 54, 60, 61]. Surprisingly storing in PBS which
is normal for micro-CT related preservation of bones changes ssNMR parameters,
specially
31 P longitudinal relaxation time (T 1 ) [54] a comparative study showing
correlation of micro-CT parameters and ssNMR parameters showed that most of the
parameters correlate specially total water, phosphorus relaxation and inter-atomic
organic to inorganic Interface [53].
Most of the research in history and even now on collagen protein has been carried
out overexpressed and purified collagen protein in absence of the mineral part. Now
we know that mineralized form of type 1 collagen from mineralized cortical bones
are significantly different from the collagen type 1 which is extracted from Kangaroo
tail. This difference was observed by comparing Hydrated native collagen in bone
matrix has entirely different
1 H–
1 H dipolar coupling network [62]. Collagen stability
is widely accepted being attributed to Hyp mediated hydrogen bonding network with
water molecules [63]. Recently there were reports rejecting the old hypothesis and
considering stereoelectronic effects as prime contributor for the stability of collagen
protein [64]. Singh et al., measured the backbone dynamics of collagen protein as a
function of dehydration by recording
15 N spectra which gives resonances from Gly
and Pro/Hyp in natural abundance (0.37%) by paramagnetic doping to reduce the
T 1 of
1 H nuclei hence the recycle delay [65]. This study showed that the backbone
dynamics of Pro/Hyp got more affected after removal of water as compared to Gly
[65]. Furthermore, it was shown that cortical bones have water compartments and
other than playing role in stability of collagen protein they also play crucial role in
load bearing capability of bones. Exchange of water takes place between different
water compartments.
One of the main challenge in the study of bones by NMR spectroscopy is low
natural abundance of most commonly used nuclei such as
13 C (1.1%), and
15 N
(0.37%), hence efforts have been done to increase the sensitivity of the NMR signal by
following different approaches [65, 66]. As we know that the experiment time in NMR
depends upon T 1 and it is advised that relaxation delay (d1, which is used before each
scan to allow proper relaxation of the spins from x–y plane to z-axis) should be 5 times
T 1 . By reducing T 1 we can reduce total experimental time. Paramagnetic doping using
Cu-EDTA and Gadolinium-DTPA helped to reduce the total experimental time [65,
67, 68]. Dynamic nuclear polarization (DNP) based sensitivity enhancement helped
to detect CH-p interaction in native mineralized collagen protein inside bones for the
first time [69]. DNP has also been applied in cartilage samples to detect structural
changes in collagen protein upon pigmentation in Alkaptonuria [70]. Similar level
of sensitivity enhancement has been achieved using the latest BioSolids CryoProbe
technology for ssNMR [71]. Another approach involved the development of heavy
mouse which had bones enriched in
13 C and
15 N nuclei up to 20% by growing
them on
13 C and
15 N labeled diet [72]. This resulted in discovery of poly (adenosine