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Xenon is a perspective gas for creation of oxygen free environment for different applications of biomaterials. To use xenon in suspensions and products properly it is necessary to know the molecular mechanisms of its interactions with water and cells. This work reports the study of bacterial suspensions of Escherichia coli in the presence of xenon using nuclear magnetic resonance (NMR). The work studied how the spin-lattice relaxation times of water protons in suspension change under xenon conditions. Xenon is able to form clathrate hydrates with water molecules at a temperature above the melting point of ice. The work studied NMR relaxation times which reflect the rotation freedom of water molecules in suspension. Lower relaxation times indicate reduced rotational freedom of water. Single exponential behavior of spin-lattice relaxation of protons in the suspensions of microorganisms has been registered. A recovery of longitudinal magnetization in cell suspensions with xenon clathrates has been characterized by two peaks in T1-distribution. Fast relaxing T1-component was related to the intracellular water and depended on the amount of xenon clathrates. The obtained results elucidate how the NMR method can monitor the process of clathrate formation and how the xenon atoms and hydrates interact with cells.
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International Journal of Biochemistry and Biophysics 5(1): 26-36, 2017
DOI: 10.13189/ijbb.2017.050104
Xenon-Water Interaction in Bacterial
Suspensions as Studied by NMR
Victor Rodin1,*, Alexander Ponomarev2, Maxim Gerasimov3, Leonid Gurevich4
1Institute of Organic Chemistry, Johannes Kepler University Linz, Altenbergerstraße 69, 4040 Linz, Austria
2Department of Medical Cell Technologies, Ural State Medical University, Ekaterinburg, Russian Federation
3Department of Clinical and Experimental Medicine, Linköping University, Linköping, Sweden
4Department of Physics and Nanotechnology, Aalborg University, Aalborg, Denmark
*Corresponding Author:
Copyright© 2017 by authors, all rights reserved. Authors agree that this article remains permanently open access under the
terms of the Creative Commons Attribution License 4.0 International License
Abstract Xenon is a perspective gas for creation of
oxygen free environment for different applications of
biomaterials. To use xenon in suspensions and products
properly it is necessary to know the molecular mechanisms
of its interactions with water and cells. This work reports
the study of bacterial suspensions of Escherichia coli in the
presence of xenon using nuclear magnetic resonance
(NMR). The work studied how the spin-lattice relaxation
times of water protons in suspension change under xenon
conditions. Xenon is able to form clathrate hydrates with
water molecules at a temperature above the melting point of
ice. The work studied NMR relaxation times which reflect
the rotation freedom of water molecules in suspension.
Lower relaxation times indicate reduced rotational freedom
of water. Single exponential behavior of spin-lattice
relaxation of protons in the suspensions of microorganisms
has been registered. A recovery of longitudinal
magnetization in cell suspensions with xenon clathrates has
been characterized by two peaks in T1-distribution. Fast
relaxing T1-component was related to the intracellular water
and depended on the amount of xenon clathrates. The
obtained results elucidate how the NMR method can
monitor the process of clathrate formation and how the
xenon atoms and hydrates interact with cells.
Keywords Bacterial Suspensions, Water, Xenon,
Clathrates, Nuclear Magnetic Resonance
1. Introduction
Despite their inertness, noble gases possess a number of
interesting properties, including their anaesthetic effect
[14]. They are very soluble in lipids and may bind to both
membrane lipids and proteins [35]. Because of the high
correlation between lipid solubility and anaesthetic potency
(known as the Meyer-Overton correlation) it was suggested
that the molecular mechanisms of general anaesthetics are
associated with the interactions between anaesthetic agents
and a hydrophobic interior of the lipid bilayers [2, 4, 5].
Although different theories have been discussed in the
literature for long time, the exact mechanism of anaesthetic
action of noble gases is still a mystery [5, 6]. It is not clear
which of the different mechanisms suggested is most likely
to be the main mechanism of action of anaesthetic agents.
Xenon is the most potent anaesthetic of all the noble gases.
The high polarizability of xenon compared to the other noble
gases increases its affinity to hydrophobic environments.
Thus xenon can be substantially partitioned into the
hydrophobic core of the membrane bilayer. The solubility of
xenon in human serum albumin solutions, hemoglobin
solutions, human blood, and other biological solutions and
homogenates was studied in the Ref. [7]. For example, at 298
K the amount of xenon dissolved in 1 g human albumin at
0.1013 MPa of total pressure was found to be 0.2382 ml.
According to the data [3] a blood-gas partition coefficient for
xenon is 0.115 whereas the solubility in water measured
identically was 0.096.
The anaesthetic gas xenon is attractive for many
applications in biotechnology and medicine [810]. Xenon is
also the perspective gas for preservation of biological
materials including different enzymes and cells [1116].
This noble gas is able to replace lighter gases of air, e.g.
oxygen and nitrogen [17, 18]. This inert gas was already
applied for many biological and medical systems in creation
of oxygen free environment, in particular, for bacterial cell
suspensions and erythrocytes of blood [9, 10, 1924]. The
authors of Refs. [19, 20] developed the method of platelet
storage in the presence of xenon. In order to use the xenon in
the storage of biological suspensions and biotechnological
products properly it is necessary to know the molecular
mechanisms of its interactions with cell membrane and cell
components [5, 11, 13, 21]. Nowadays many questions are
still opened.
International Journal of Biochemistry and Biophysics 5(1): 26-36, 2017 27
One important feature of xenon is its ability to create
clathrate hydrates from water molecules at relatively “soft”
conditions when the pressure is still about 0.40.7 MPa and
temperature circa 276278 K [1214, 21]. When forming
hydrates, lower hydrate formation pressures are desirable in
practice [21, 25, 26]. It is known that crystalline hydrates are
formed by many simple gases that do not interact strongly
with water, and mostly the gas molecules or atoms occupy
“cages” formed by the network of water molecules. The
majority of these gas hydrates adopt one of two possible
cubic cage structures and therefore are called clathrate
hydrates [21, 25, 26]. Although high pressure is often
needed to hold gas atoms inside the water “cages”, xenon
gas is an exception from the rule. Xenon hydrates are the
clathrates of low pressure. They can be stabilized at about
276280 K [10, 12, 25].
Clathrate hypothesis of anaesthesia of L.Pauling [27, 28]
suggested the existence of clathrate microcrystals formed by
anaesthetic gas and water molecules even at physiological
conditions (at least momentarily): normal atmospheric
pressure and temperature of cells. Some suggestions of
clathrate theory of L.Pauling have been checked later in Refs.
[2733]. First studies on formation of xenon clathrates in
cellular suspensions of microorganisms have been initiated
in Refs. [9, 13, 14, 34, 35]. Those publications showed how
the methods of magnetic resonance can produce valuable
information about the changes in bacterial medium under gas
pressure. Later publications [21, 3640] reported more NMR
results on xenon clathrates. The use of paramagnetic
additives expanded the possibilities of NMR relaxation in
studying cell suspensions with gas hydrates [21, 36]. NMR
was also applied for studying clathrate formation in two-
component and complex systems [9, 3740]. Nevertheless
many questions about the role of xenon and its clathrates in
cell suspensions still remain open.
The most intensively investigated prokaryotic model
organism in biophysics is the Escherichia coli (E.coli), K-12
strain [41, 42]. It is considered as very important
representative of cellular suspensions in biotechnology.
E.coli is a common gram-negative rod-shaped bacterium,
found in normal human (and other animals) bacterial flora. It
is about 2.0 µm in length and its diameter is 0.251.0 µm
[41]. These microorganisms are facultative anaerobic
bacteria which can be easily grown in a laboratory.
The current work reports NMR-study of bacterial
suspensions of E.coli under xenon pressure addressing how
the xenon and its hydrates participate in the interactions with
cells and affect the state of intra- and intercellular water. The
biomaterials with crystals of xenon clathrates are
“transparent” for radio waves. This allows using the methods
of radio spectroscopy (i.e., NMR) to analyze the structural
and phase state and molecular-dynamic properties of water
in the cell suspensions in the presence of a clathrate phase.
The paper presents how NMR water proton spin-lattice
relaxation times in E.coli suspensions vary under xenon
2. Experimental Materials and Methods
2.1. Materials
The cells of E.coli have been grown in flasks with volume
of 0.75 liter using beef-extract broth or mineral liquid
medium with glucose. A sterilized phosphate buffered saline
(PBS) was added. The cells at the stationary phase of growth
were used (age of 1416 hrs). The samples with cells were
centrifuged at 3000 rpm for 15 min. The precipitate was
washed twice in distilled water, PBS and was used in
subsequent experiments as in Refs. [9, 21, 36]. A
concentration of the biomaterial (cellular suspensions) under
study was expressed in g of dry mass per g H2O and by a
number of cells per 1 ml [21].
2.2. Custom-made Thermostat Chamber with Xenon Kit
We designed and built thermostat chamber with
transparent front door from polymethyl methacrylate. Inside
the chamber a system to transfer the gas to tubes with
samples was built. The gas cylinder (with medical grade
xenon) was connected to the special kit with tubes filled by
suspensions / biomaterials [21, 27, 36]. The tubes with
samples were under gas pressure as described in earlier
publications [21, 37]. Mostly the pressure circa 0.81.0 MPa
or less was enough to produce stable clathrates at 278280 K.
In some experiments “dry” ice (carbon dioxide) was used to
touch the bottom of the tubes for the initiation of clathrate
formation according to described procedure [21, 36, 43].
After saturation of the samples with gas and xenon clathrate
formation the tubes could be detached from the kit in the
thermostat chamber and be used in NMR measurements at
above melting point of common ice but not exceeded 286 K.
2.3. NMR Methods in Studying Water Clathrates of
The NMR studies of cell suspensions and suspensions
with clathrate crystals were performed at a proton resonance
frequency of 90 MHz. Some experiments have been carried
out also at 20 MHz. In the single-pulse 1H NMR experiments,
the free induction decay (FID) signals were measured. The
FID consists of a 9 pulse which rotates the magnetization
onto the transverse plane where it decays with characteristic
time. The initial part of the FID is hidden in the dead-time
zone of the spectrometer. The length of the 90° pulse was 8
µs. The FIDs on the samples with clathrates have been
measured according to the procedure of earlier publications
[27, 37, 43]. The signal of total time-domain is the sum of
the signals of the water protons and the protons of clathrate
crystals: S = Swat + Sclath. In the study of water dispersions of
xenon clathrates [21,37] it was shown that spin-spin
relaxation time T2 of the fast relaxing component (protons of
clathrates) did not exceed 25 µs. The structure of clathrate
ice in studied solutions and suspensions at above melting
point of common hexagonal ice was relatively “softer” than
that in common ice with T2 ~ 10 µs. For ice protons the most
28 Xenon-Water Interaction in Bacterial Suspensions as Studied by NMR
part of FID was hidden in the dead-time zone. Only the tail
of the transverse magnetization decay might be observable in
the experiment. Although the part of FID for the clathrates is
a bit longer than the FID for common ice, the situation with
FID in clathrates is comparable with that in ice. To monitor
the water signal only, the contribution of the protons of
clathrate ice should be subtracted. In such a case, normally,
the measurements were carried out at experimental
conditions when transverse magnetization of clathrate
protons decayed very fast to zero (whereas the transverse
magnetization of liquid water has not decayed) and only
protons of water could be measured in the study [37,43]. On
the base of this approach a fraction of water built in clathrate
structures was determined using the difference of the proton
signals of liquid water in suspensions before and after
clathrate formation [21].
The spin-lattice relaxation time T1 was measured by the
inversion recovery (IR) method using the pulse sequence
(180°t90°) at the pulse sequence repetition time ≥ 5T1 [45,
46]. The period of 5T1 was waited between the consecutive
scans of pulse sequence in order to ensure that the fully
recovered signal was acquired. When t is varied then
experimental dependence of magnetization Mt = f (t) can be
obtained and fitted by the law Mt = M0i·[1exp(t/T1i)],
where i is the number of relaxation component. The analysis
of relaxation data was conducted after transferring the raw
data from spectrometer computer to desk computer using
in-house MatLab® codes. In these NMR studies there was an
aim to model experimental data as a sum of several
relaxation components or distribution of NMR-relaxation
times. To fit the data inverse Laplace transform (ILT) in one
direction was commonly applied [4750]. The kernel
function in ILT was associated with Mt data obtained from
IR NMR experiments.
3. Results and Discussion
3.1. Spin-lattice Relaxation of Water Protons in
Suspensions of E.coli before and after Xenon
Clathrate Formation
The T1experiments discovered that the recovery of
longitudinal magnetization of measurable protons in all
studied samples of cell suspensions (concentration range of
0.0010.24 g dry mass /g H2O) can be fitted by single
exponential curve and characterized by a single value of the
spin-lattice relaxation time T1. However a recovery of
longitudinal magnetization of protons in bacterial
suspensions with clathrates of xenon has bi-exponential
behavior. T1 values of the two exponential components differ
by a factor of 6 to 10. Alternatively, the treatment of the
recovery of longitudinal magnetization using inverse
Laplace transform resulted in a distribution of spin-lattice
relaxation times T1 in the kind of 2 peaks. Fig.1 shows
T1-distributions obtained by one- dimensional ILT applied to
the data sets for original bacterial suspension and E.coli with
xenon clathrates.
The two peaks registered for the suspension of E.coli with
xenon clathrates (Fig.1) had maximums at 0.25 and 1.2 s.
The integral intensities of these peaks corresponded to 66%
and 34 % of measurable protons. The single peak observed in
T1-distribution in suspension of E.coli without clathrates was
at T1 ≈ 0.70 s (Fig.1). This was in line with previously
published NMR relaxation data on cellular suspensions [21,
40] which showed fast exchange between the protons of
intra- and intercellular water, i.e., single exponential T1
-relaxation [5153]. The permeability of the cell membrane
to water is quite high (for instance, in red blood cells
exchange time of the order of 10 ms [53]). Water proton
relaxation times in E.coli bacteria showed one exponential
behavior, i.e., two sites (two compartments: intracellular and
extracellular spaces) are in a fast exchange limit. Thus a
researcher needs to reduce one of the relaxation times to less
than exchange time in order to remove this condition and to
generate two exponential behavior [51, 52]. In the presence
of paramagnetic ions of manganese in the extracellular water
shortening the relaxation times outside the cells it was
possible to record two separate NMR signals in the
population of cells with non-damaged cellular membranes
[36, 51, 52].
The relaxation rates of the water molecules in cell
suspensions are governed by several important factors, in
particular, the strength of local magnetic interactions
between water nuclei, the molecular motion and proton
exchange rates. Nuclear magnetic inter- and intramolecular
dipole-dipole coupling describes the interactions between
water nuclei [5153]. The magnetic interactions are partially
averaged within the hydration layer by the processes
depending on the interactions of water with
macromolecules/cell surface/clathrates. These are proton
transfer, dynamic orientation and diffusion of water
molecules through regions of different orientations [26, 40,
44, 53]. The NMR relaxation times are sensitive to molecular
motions in the range of 10-8 to 10-12 s [45, 46, 53]. In liquid
solutions water molecules tumble at a rate of about 10-12 s
[45]. This motion is considerably slowed down when water
molecules interact with biological macromolecules/cell
surface. For example, the correlation time is of the order of
10-12 s for the water molecules non-involved in the
association through hydrogen bonds with surface of cells.
The rotational motion of water molecules associated via
hydrogen bonds with polar groups of the macromolecules is
reduced so that their correlation time is even sometimes of
the order of 10-6 s [53]. Under the conditions of rapid
exchange between the hydration and bulk water in
suspensions and macromolecular solutions, single relaxation
rate (1/T1) is observed.
Single exponential relaxation of protons in suspensions of
E.coli confirmed fast exchange between intracellular and
intercellular water on the NMR time scale. Formation of
xenon clathrates affected the exchange rate and resulted in
the measurement of two separate compartments. The
population of fast relaxing T1-component was associated
with the fraction of intracellular water. It depended on the
amount of xenon clathrates.
Fig.2 shows how the amount of clathrate ice, i.e. the
amount of water molecules bounded within the structure of
International Journal of Biochemistry and Biophysics 5(1): 26-36, 2017 29
xenon clathrates, depends on the concentration of cells in
suspension. The fraction of clathrates has been calculated
according to the procedure [3740] which compares the
measurements of the FID in original bacterial suspension
and suspension with xenon clathrates. The transition of
liquid water into solid phase of clathrates resulted in a
change of spin-spin relaxation times T2 of protons. T2 ~ 12 s
for free mobile water whereas T2 ~ 10 µs for protons of solid
phase. Such a difference in T2-values gives an opportunity to
separate proton signals of liquid and solid phases much
better. The results of Fig.2 are in line with the previously
published data on the xenon clathrates formation in solutions
of biomacromolecules [12, 27, 31, 32, 34]. It was shown [31,
32, 34] that a water fraction bound in clathrate structures was
due to abundance of hydrophobic groups on the surface of
proteins. Probably some groups on the cellular surfaces also
participate in the formation of xenon hydrates stabilizing
clathrate lattice from water molecules [21,28,31]. As a result
the amount of stable clathrate structures was increasing with
cell concentration in studied range. One observed that the
amplitude of fast relaxing T1component (relatively to total
water content in cell suspension) increased nonlinearly with
an increase in the initial cell concentration. Apparently this is
due to the advent of solid phase of water (clathrates) and
subsequent displacement of the microorganisms in the free
volume of the suspension.
Fig.3 shows calculated fraction of intracellular water
(open circles) and the dependence of the population of the
fast relaxing T1-component on cell concentration
recalculated to the signal of liquid water. This recalculation
shows the true concentration of the cells remaining in the
liquid phase of the suspension after the formation of
clathrates. The dependence of the population of this
T1-component looks almost linear, and this assumes that the
fast component of T1 is due to the protons of intracellular
water. The slow T1-component can be attributed then to the
extracellular water protons. This assignment is further
supported by a decrease of the population of the slow
component with increasing concentration of cells. A fraction
of the extracellular water protons which are not in clathrate
structures decreased. According to this analysis, the cell
concentration increased relatively to volume of liquid phase
after clathrate formation. A fraction of intracellular water
(Fig. 3, open circles) was calculated on the base of summary
volume of cells and had a linear dependence on
concentration of cells.
0.1 1 10
T1 (s)
Intensity (a.u.)
Figure 1. T1-distribution of 1H obtained by one-dimensional ILT on the recovery of longitudinal magnetization data set measured in the suspension of
E.coli with concentration of 0.07 g dry mass/g H2O (open circles) and in the same sample after xenon clathrate formation at 1.2 MPa (solid line). T=280 K.
Frequency is 90 MHz.
The integral intensities of 2 peaks in the suspension of E.coli with xenon clathrates (from left to right) are 66% and 34 % of measurable protons. The signals
in each T1-experiment have been normalized per maximal signal registered after total recovery of longitudinal magnetization.
30 Xenon-Water Interaction in Bacterial Suspensions as Studied by NMR
0 2 4 6 8
C (x1010 cell/ml)
water in clathrates (%)
Figure 2. The dependence of the amount of H2O molecules bounded within the clathrate structures (i.e., protons of solid phase with T2 ~ 10 µ s) on the
concentration of cells in the suspensions of E.coli with xenon clathrates. The results were obtained from NMR (FID) measurements taking into account the
procedure of Refs. [37, 38]. T = 280 K. Pressure is 1.2 MPa. Frequency is 20 MHz.
0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35
C (g dry mass / g H2O)
Intensity (%)
Figure 3. The relative amplitude of fast relaxing T1-component (vertical line segments) and calculated fraction of intracellular water (open circles) in the
liquid phase of E.coli suspensions with xenon clathrates as a function of cell concentration (C). Solid line is the linear fit to the data (open circles) calculated
on the base of summary volume of cells.
T = 280 K. Pressure is 1.2 MPa. Frequency is 90 MHz.
International Journal of Biochemistry and Biophysics 5(1): 26-36, 2017 31
It is worth to emphasize that the dependence of
intracellular water amount on the concentration of cells in
suspension (Fig.3, open circles) ideally should be a straight
line coming through the origin of the coordinates and the
meaning which can be obtained at tightly packed cells, i.e.,
when extracellular water would be completely removed. The
experimental curve for the population of the fast relaxing
T1-component is positioned lower than that based on the total
volume of the cells (Fig.3). This could be explained by the
osmotic compression of the cell due to an increase in the
concentration of solutes in extracellular space upon xenon
clathrate formation and a decrease in the amount of
remaining liquid water. This is in line with the published data
on paramagnetic doping (when the Mn2+ ions are introduced
into the extracellular water to separate the proton signals of
intracellular and extracellular compartments) in studying the
xenon clathrate formation in the extracellular compartments
of bacterial suspensions [21, 36].
The behavior of T1-components in E.coli suspensions with
xenon hydrates depended on the amount of clathrate
microcrystals. This suggests that the proton exchange
between the extracellular water and the protons of solid
clathrate lattice could also affect T1-component of the
extracellular water. This is in line with some published
NMR-data [37, 39, 43, 44, 51]. Ref. [44] showed that the T1
of protons of ice was increasing with decreasing the
temperature from 273 K to 213 K. For example, at T=273 K
the T1 of protons of ice was 0.5 s increasing to 3 s at 250 K. In
contrast to ice the T1 of protons of liquid water decreased
with decreasing the temperature of water [43, 44, 51].
According to the published data on dispersions of xenon
clathrates in pure water [37, 39], the temperature dependence
of T1 for water protons showed a minimum. A proton
exchange between liquid water and solid clathrate phase
resulted in relaxation behavior of the protons of water
molecules displaced among clathrate microcrystals in
dispersion. Upon this influence dependence of T1 on
temperature for the protons of water remaining in liquid state
became similar to that for the protons of ice. Thus the
protons of liquid water near the crystals of clathrate ice could
experience spin-lattice relaxation with increased values of T1
exceeded typical values for liquid water. However an
interpretation of longitudinal relaxation in E.coli suspension
with xenon hydrates could be much harder than it was for
clathrate dispersions of xenon in pure water because of
additional relaxation sinks and hydration of cell surfaces. For
phase of clathrate crystals at the temperatures above 273 K
T1 is probably of the order of few seconds (the estimate is
based on the mobility of water molecules in ice at 213273 K
and in clathrate crystals at 275280 K [25, 26, 30, 38, 44,
54]). Then for the protons of liquid water remaining in
extracellular compartment and dispersed between hydrate
crystals and bacterial cells we can expect T1 to be of the order
of 1 s. The amount of water molecules participating in the
exchange with clathrate phase was increasing with growth of
the amount of hydrate crystals in suspensions as surface of
boundaries of water-clathrate ice was growing up. This
resulted in changing spin-lattice relaxation time T1 for slow
component of longitudinal magnetization recovery. In one
particular example for E.coli suspension with xenon
hydrates, T1 doubled compared to the initial spin-lattice
relaxation constant of T1=0.54 s at a cell concentration of
0.23 g dry mass / g H2O.
According to the data of Ref. [44], the proton spin-lattice
relaxation times in carefully purified ice have been measured
for the temperature range of 213273 K. The authors of this
work considered a relationship of T1 with rotational jump
time τj as follows: T1=2.14×105×τj. The dependence of T1
against inverse temperature has been characterized by single
apparent activation energy of Ea ≈59 kJ/mol [44] (or Ea ≈56.3
kJ/mol according to the data published in Ref. [40]) and T1 in
ice was frequently dependent as 02 [44]. So the
measurements of T1 at different resonance frequencies could
also clarify some details in frequently connecting motion of
protons in suspensions with xenon hydrates. From the data of
Refs. [37, 43] the temperature dependence of T1 (an
Arrhenius plot [21,40] in the range of 278286 K) for the
protons of water molecules displaced among clathrate
microcrystals in dispersion gave the apparent activation
energy of Ea =26.4±3.7 kJ/mol. NMR data of Ref. [55]
presented apparent activation energy for xenon hydrates as
Ea =24.7±3.3 kJ/mol.
In order to estimate correlation times τc (as characteristics
of molecular motions based on NMR relaxation time
measurements) in E.coli suspensions with xenon clathrates
we applied the approach of earlier works [45, 46, 51, 53, 56]
assuming the motions involved in intramolecular relaxation
and using the expressions reported for 1/T1 in terms of the
correlation times:
21 00
1cc JJC
( , ) 1c
 
where ω0 is the Larmor angular frequency in a constant
magnetic field B0. It is related to the resonance frequency ν0
by the relation ω0=2πν0. C = 2.51010 s2 is the constant of
dipole binding of water molecules.
The use of Eq.(1) can give first estimation of the
relaxation behavior of the protons of remaining water
molecules displaced among clathrate microcrystals in
bacterial suspensions assuming some values of correlation
times. According to published data [5658] T1 values for the
unfrozen water in some biomaterials (e.g., solutions of
agarose or lysozyme) indicate that mobility of water
remaining in liquid state can be described by a distribution of
correlation times with single peak centered at τc ~ 10-9 s. For
the conditions (temperature, pressure) when xenon still did
not form clathrates in suspension each water molecule
spends part of its time as isotropic water with single
correlation time. The correlation between motion of protons
from the water molecules and from macromolecules in
principle gives rise to the process of cross-relaxation or
spin-diffusion. The observed relaxation rates of water
protons can be influenced by cross-relaxation rates.
Spin-diffusion occurs by way of mutual exchange of
32 Xenon-Water Interaction in Bacterial Suspensions as Studied by NMR
spin-magnetization between water protons of the hydration
water and protons on macromolecules / cell surface. The
contribution of cross-relaxation can become very important
under the conditions of slow molecular diffusion.
The mobility of water remaining in suspensions after
xenon hydrate formation might be comparable with that for
unfrozen water in the solutions of biomolecules. This allows
a calculation of T1 at two experimental resonance
frequencies (90 and 20 MHz) at the value of τc, for instance,
of 2.010-9 s. The results show reasonable difference (34
times) in ratio of T1-values for 90 and 20 MHz although the
value T1≈28 ms (at 90 MHz) looks very small in comparison
with experimental T1-values for extracellular water (0.51.2
s) in bacterial suspension with xenon clathrates. For changed
value of correlation time (one order) to τc=2.010-10 s the
calculation of T1 according to Eq.(1) would result in
T1=6063 ms (these simulated values are still small in
comparison with T1 from experiments) showing very small
difference between simulated T1 values for 90 and 20 MHz.
The estimation for next change to τc=2.010-11 s was also
unsuccessful to match the experimental meanings of T1 for
two resonance frequencies by the calculated values. This
suggests that the motions with correlation times τc ~10-9 s (or
less for one-two orders) are not likely to be main input in
bacterial suspensions with xenon clathrates. So the approach
with one correlation time based on literature data should be
definitely changed to distribution of τc to fit the experiments
in E.coli suspension with clathrates of xenon.
3.2. Factors Defining Mobility of Water among Xenon
Clathrates in Bacterial Suspension
The approach of Ref. [56] which studied bound water in
frozen erythrocytes by NMR could be probed on bacterial
cells adopting the usual formulations of the relationship
between relaxation times and correlation times and assuming
as well a log-normal distribution of correlation times with
distribution function:
dP ])(exp[
)( 2
Here Z=ln(τ/τ*) and τ* is the median of the correlation
time distribution and β is the distribution width. The authors
of Ref. [56] assumed that β is independent of temperature
and τ* is dependent of temperature following to Arrhenius
expression with activation energy for motion corresponding
to the correlation times. The spin-lattice relaxation rate 1/T1
is then [56]:
)2(1 )(2
)(1 )(
For the evaluation of the relaxation rates it is necessary to
estimate the integrals in Eq.(3). In Ref. [56] this was done
numerically using the relation [57]:
dP 12
)(1 )(
for a log-normal distribution where X =ln(ω0·τ*).
The approach to fit relaxation data in bacterial
suspensions with xenon clathrates is based on certain
assumptions (water molecules are undergoing to
translational and rotational motions governed by a
distribution of correlation times) which allow several
adjustable parameters for each correlation time: dipole
constant C, β, τ* and activation energy stemming from
temperature dependence of τ*. In principle these parameters
could be determined by a computer fit of the data using an
iterative method. In reality, the dipole constant of water, in
other words, the rigid-lattice second moment is in the range
from 1.51010 s2 for isolated molecule of water to 2.61010
s2 for crystals of ice [56]. There is indefinite choice for the
constant of dipole binding as the relaxation data on T1 were
obtained for the remaining water in E.coli suspensions after
clathrate formation experiencing an exchange with solid
phase of clathrate ice.
Fitting the data to the model with one log-normal
distribution of correlation times gives the values for the most
of the parameters as widely varied. The authors of Ref. [56]
for the remaining liquid water in frozen red blood cell (RBC)
suspension proposed a model which describes NMR
relaxation data by a distribution with two peaks. Additional
parameters for the fraction of water molecules under first
peak and for the fraction of water molecules under second
peak were introduced in order to fit the distribution of τc.
Analysing the published model for RBC suspensions we
suppose that for clathrates of xenon in cell suspensions more
consistent model could be developed further. Some
additional experiments should involve relaxation of xenon
atoms and introduction of parameters responsible for the
presence of xenon in suspensions. An idea to explain a cell
metabolism during/after xenon clathrate formation could be
developed to add the details to total pattern of cell changes.
An increase of salt concentration in extracellular water at
xenon clathrate formation causes shrinking the cells by loss
of free water. When the cells compressed the anion and
cation flux across the membrane can be controlled and
coupled to the cell metabolism involving a regulation of
water structure. The remaining intracellular water can be
more highly structured resulting in decreased relaxation
times (i.e., increased rates R1=1/T1) with cell concentration
(Fig.4, Fig.5).
For the future modelling the suspensions with xenon it is
necessary also to take into account the changes produced by
xenon dissolved in cell suspension. It is possible to consider
extracellular water as saline with a concentration 0.9% by
weight. According to the data [59] the solubility of xenon in
saline water is low, in particular, Ostwald coefficient is equal
to 0.0926 (at standard temperature and 0.101 MPa of gas
pressure). However the T1 value of xenon is quite long (66 s,
when measured at magnetic field B0 =9.4 T) due to dipolar
couplings with proton spins [59].
International Journal of Biochemistry and Biophysics 5(1): 26-36, 2017 33
0 1 2 3 4 5 6 7 8
C (x1010 cell/ml)
R1, slow (s-1)
Figure 4. Spin-lattice relaxation rate R1=1/T1 of slow relaxing component of longitudinal magnetization of water protons in liquid phase of E.coli
suspensions with xenon clathrates as a function of cell concentration. T = 280 K. Pressure is 1.2 MPa. Frequency is 20 MHz
0 1 2 3 4 5 6 7 8
C (x1010 cell/ml)
R1, fast (s-1)
Figure 5. Spin-lattice relaxation rate R1=1/T1 of fast relaxing component of longitudinal magnetization of water protons in liquid phase of E.coli suspensions
with xenon clathrates as a function of cell concentration. T = 280 K. Pressure is 1.2 MPa. Frequency is 20 MHz
34 Xenon-Water Interaction in Bacterial Suspensions as Studied by NMR
An increase of the cell concentration in E.coli suspensions
with xenon clathrates resulted in decrease of slow (and fast)
components of longitudinal relaxation time T1 (Figs.4, 5).
This could be associated with increasing viscosity because of
the concentration of solutes (inside and outside cells)
increased. This is also could be considered as a result of
increasing exchange between the protons of liquid water and
the protons of macromolecular surface structures of cell in
the case of spin-diffusion. Last suggestion is rather unlikely
as published data indicated that spin-diffusion affects the
relaxation of protons at high concentration of proteins and
macromolecules [45, 6062]. A temperature dependence of
slow T1 component in the range of 277289 K (Arrhenius
plot) resulted in apparent activation energy Ea=27.2±2.0
kJ/mol which exceeds Ea-value for pure water (~21 kJ/mol
[40]). Probably, a mobility of water molecules in liquid
phase of cell suspensions at xenon clathrate formation
decreased not only due to increasing cell concentration and
compress of cytoplasm but significantly due to dissolution of
xenon gas in cells in the amount enough to create separate
clathrate cages but not enough to build up a space network of
completed clathrate structures. According to the NMR data
on 129Xe dissolved in physiological saline at 0.1 MPa and
injected into RBC suspension gas-exchange processes
between extra- and intracellular compartments occur on the
time scale of a few tens milliseconds [59,63]. The mixing of
xenon between physiological saline and RBCs resulted in
accumulation of 129Xe NMR signals inside cells with
intensity comparable to that in extracellular compartment for
just the period of ~1 s [59]. However an affinity of xenon to
build up the clathrate structures in extra- and intracellular
spaces at the temperatures above melting point of ice can be
strongly depending on increased gas pressure and other
conditions in cells and outside cells including changed
metabolism (flux of water and ions via membrane),
availability of free liquid water and accumulation of xenon in
hydrophobic part of membrane.
4. Conclusions
Structural-dynamic changes in bacterial cell properties
induced by xenon have been studied by proton spin-lattice
relaxation measurements. The inert gas xenon is able to form
clathrate hydrates from water molecules at pressure circa
0.40.7 MPa and temperature about 276278 K. An amount
of water bound in solid phase of xenon clathrates can be
estimated from NMR data. The results clarified how NMR
method can monitor a process of clathrate formation and
how the xenon atoms and its hydrates interact with cells. The
formation of xenon hydrates affected exchange dynamics of
water between two compartments and resulted in apparent
two T1-components.The results of T1-measurements showed
that the mobility of intra- and intercellular water can be
changed at above melting point of common ice with the aid
of xenon hydrates. The ability of xenon hydrates to lower
water proton relaxation time inside the cells may reflect their
propensity to structure and influence on exchange between
extracellular and intracellular water.
Detailed interpretation of correlation times as the
characteristics of molecular motions in compartments of
bacterial suspension in the presence of clathrate ice is not
easy because of the lack of suitable mathematical models.
However, the results showed that the surface of cells
participates in clathrate formation. It affects the dynamics of
the water molecules and determines their correlation times.
The changes in mobility of extracellular water at xenon
hydrate formation result in changes in cell properties.
Molecular mechanisms of these changes (affecting
membrane permeability and activity of proteins and lipids)
must be further explored involving different NMR facilities.
There are many NMR methods for probing the
compartments of heterogeneous, in particular, cellular
systems. NMR has a useful role in characterizing the
molecular properties of separate spaces of cellular
suspensions giving a possibility to probe water, solutes and
surfaces non-invasively. This allows keeping the
biomaterials under xenon conditions further without
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Different NMR methodologies have been considered in studying water as a part of the structure of heterogeneous biosystems. The current work mostly describes NMR techniques to investigate slow translational dynamics of molecules affecting anisotropic properties of polymers and biomaterials. With these approaches, information about organized structures and their stability could be obtained in conditions when external factors affect biomolecules. Such changes might include rearrangement of macromolecular conformations at fabrication of nano-scaffolds for tissue engineering applications. The changes in water–fiber interactions could be mirrored by the magnetic resonance methods in various relaxations, double-quantum filtered (DQF), 1D and 2D translational diffusion experiments. These findings effectively demonstrate the current state of NMR studies in applying these experiments to the various systems with the anisotropic properties. For fibrous materials, it is shown how NMR correlation experiments with two gradients (orthogonal or collinear) encode diffusion coefficients in anisotropic materials and how to estimate the permeability of cell walls. It is considered how the DQF NMR technique discovers anisotropic water in natural polymers with various cross-links. The findings clarify hydration sites, dynamic properties, and binding of macromolecules discovering the role of specific states in improving scaffold characteristics in tissue engineering processes. Showing the results in developing these NMR tools, this review focuses on the ways of extracting information about biophysical properties of biomaterials from the NMR data obtained.
The chapter considers the achievements of NMR for biomedical engineering and estimates development of these methods to unveil the details of processes associated with recovering cells and tissues. NMR methods give the information about translational dynamics of molecules and macromolecules in complex systems including natural porous biosystems in different physicochemical environment. NMR relaxation, pulse field gradient (PFG) NMR in one- and two-dimensional realisations, and double-quantum-filtered (DQF) NMR techniques are analysed with brief theory and the examples in studying normal and disease state of cells and tissues, regulation of cell processes and monitoring changes in the cell/tissue microstructure. NMR & MRI methods are the leading non-invasive characterization tools, which can provide different MR parameters in studying the properties of tissue-engineered constructions. DQF NMR technique collects the information about local and macroscopic order in heterogeneous systems and it is effectively applied in the tissues with anisotropic motion of molecules. It is considered how DQF NMR has been applied to study collagen tissues with different quantity of covalent intermolecular cross-links. The apparent diffusion coefficient (Dapp) could be applied in studying biomedical engineering applications. The change in Dapp and NMR relaxation parameters (T1, T2) correlated well with the growth of engineered tissues. A restricted distance and permeability coefficient could be monitored as important MR parameters of fibers and tissue characteristics. Various classes of nanoparticles applied for drug delivery and other destinations in biotechnology have been studied effectively by NMR techniques on different nuclei. Hopefully the material of the chapter can help to establish a bridge between researchers specialised in particular MR techniques and cell and tissue engineers. Biological, Physical and Technical Basics of Cell Engineering pp 339-363 | Cite as Magnetic Resonance in Studying Cells, Biotechnology Dispersions, Fibers and Collagen Based Tissues for Biomedical Engineering Introduction Molecular, cellular and tissue engineering establishes the ways to improve the health of many people by restoring and maintaining cell and tissue functions [7, 13, 15, 23] as well as generating alternative tissues for reconstructive surgery. It is important to understand a mechanism of cellular interactions, a role of surface contacts and predict how cell growth, and how cell/tissue differentiation is affected by the environment [32, 34]. Cellular surroundings, both chemical and physical, can have strong effects on behavior, growth, differentiation and storage of cells [7, 32, 34, 37, 39, 54, 69]. The interdisciplinary approach of cell and tissue engineering provides a real basis for the investigation and fabrication of new biomedical devices with a broader perspective on quantifying efficacy and establishing clinical applications [7, 13, 23, 34, 37, 69]. Studying physical factors or creating special environmental conditions for the cells and tissues became very important. Many physical methods have been applied and developed to restore, maintain, and to supply the cell and tissue functions. Nuclear magnetic resonance (NMR) methods are also applied in this area [12, 15, 17, 20, 21, 26, 36, 38, 39, 42, 53, 54]. It is important to understand the NMR achievements, apply those for biomedical engineering and estimate development further to unveil the details of recovering cell and tissue functions [21, 38, 39, 42, 54, 69]. Nowadays the NMR methods study the processes inside living cells [12, 17, 26]. The techniques clarify the intracellular protein-protein interactions responsible for most biological functions [7, 26, 39, 54]. NMR now can analyze living prokaryotic cells in details [12, 17, 26, 54]. This chapter discusses mostly those NMR methods, which give the information about translational dynamics of molecules and macromolecules in complex systems including natural porous biomaterials. NMR relaxation, pulse field gradient (PFG) NMR, NMR imaging and multiple-quantum-filtered (MQF) NMR techniques are studying normal and disease cells and tissues, regulation of cell behaviour and fermentation processes resulting in special cell/tissue properties based on dynamics of molecules and diffusion characteristics [10, 16, 31, 38, 39, 41, 42, 51, 57]. Giving non-invasive characterization of engineered connective tissues, these tools consider a possibility to tabulate differences in the MR properties of tissue-engineered constructions [16, 21, 38, 57]. PFG NMR, NMR relaxation and MQF NMR methods are potential techniques in a study of molecular dynamics and diffusion properties in the anisotropic systems [31, 42]. An anisotropic molecular tumbling does not average all dipolar interactions to zero. The residual proton-proton dipolar interactions (RDIs) between macromolecular protons and water protons produce second-rank tensor that is responsible for the observed double-quantum-filtered (DQF) spectrum [16, 42, 51, 57]. There is no DQF signal in isotropic systems. The DQF spectra of connective tissues may represent a sum of spectra arising from a number of different non-interacting sites. If to consider local residual interaction at each site and orientation of local symmetry axis relative to the external field, this gives effective characterization of the total water motion in each site. DQF technique collects the information about local and macroscopic order in the systems with effective anisotropic motion of molecules [10, 31]. Proton and sodium MQF spectroscopy and proton spin-lattice (T1) and spin-spin (T2) relaxation times were used to study the chondrogenesis and osteogenesis of collagen contained tissues [21]. The apparent diffusion coefficient (Dapp) was also applied in studying tissue engineering materials [20, 21, 38]. PFG experiment can be carried out at different diffusion times or applying the gradients in two orthogonal directions to natural-oriented tissues. This can clarify anisotropy and restriction diffusion and estimate the restricted distance and permeability coefficient [42, 49]. Dapp values and NMR relaxation parameters (T1, T2) correlate well with the growth of engineered tissues and help to characterize the scaffolds [21]. MQF spectroscopy shows that the tissue-engineered cartilage has no order or preference in collagen orientation [21]. The chapter considers also two-dimensional correlation NMR spectroscopy as the distributions of diffusion coefficients in two orthogonal directions in the anisotropic systems. These 2D NMR methods can reveal the correlation of the diffusion motion of molecules along either collinear or orthogonal directions of applied pulse gradients of magnetic field. The approach is promising in studying tissues with anisotropic structure revealing microscopic local anisotropy in the presence of global isotropy. 2 Theory and Methods. Main NMR Techniques and Experiments In order to apply MR parameters for biomedical engineering properly, more MRS/MRI experiments with different combination of scaffolds and cells and understanding the details of measurements are needed [20, 21, 38]. The efforts to develop NMR tools to the tissue characterization are depending on how cell and tissue engineers are involved in MR experiments, as well as how deep is their understanding the basics of MR methods. As discussed in [21] and functional MRI/MRS for regenerative medicine meetings, the collaboration between MR community and regenerative medicine community is important for successful transfer of biomedical engineering achievements to practice. Therefore additional explanation of MR parameters and factors affected those should be presented in more details. This section introduces NMR methods and explains basic experimental approaches. Some models and experimental techniques are described to help in the realisation and interpretation of NMR relaxation and NMR diffusion experiments in one- and two-dimensions. A technique of double-quantum-filtered (DQF) NMR is presented for the study of molecular anisotropic motion. The magnetic properties of atomic nuclei, basic principles of NMR phenomenon and NMR applications in various fields are described in publications [1, 4, 5, 6, 11, 19, 40, 42]. Electrons, protons and neutrons in atoms can be imagined as spinning on their axes. In some atoms for example, 12C these spins are paired against each other and the nucleus of the atom has no overall spin. However, in other atoms, such as 1H, 13C, and 31P, the nucleus does possess an overall spin. The nuclei with spin experience NMR phenomenon, which occurs when the nuclei with nonzero spin are placed in a static magnetic field and a second oscillating magnetic field is orthogonally applied [1, 4, 11, 19]. According to quantum mechanics, this nuclear spin is characterised by a nuclear spin quantum number, I [1, 5, 6, 40]. A nucleus of spin I will have 2I + 1 possible orientations in magnetic field B0. For a spin-half nucleus (I = 1/2) an interaction with a magnetic field results in two energy levels [1, 5, 19, 40]. When external magnetic field B0 = 0 these orientations are of equal energy. In the case of nonzero B0, the energy levels split. Each level is given by a magnetic quantum number, m, which is restricted to the values from −I to I in integer steps. Thus, for a spin-half nucleus, there are only two values of magnetic quantum number m, +1/2 and −1/2 [11, 19, 40]. The energy state with m = +1/2 is denoted as α and is characterized with the lowest energy. This state is often described as ‘spin up’ notation. The state with m = −1/2 is denoted β, and it is described as ‘spin down’ notation. The m-values (+1/2, −1/2) express the parallel and antiparallel orientations of the nuclear spin with respect to the static magnetic field. The details of energy levels for two or more spins in molecule can be found elsewhere [4, 11, 19]. The initial populations of the energy levels of the nucleus in a magnetic field are determined by thermodynamics according to the Boltzmann distribution. The lower energy level will contain slightly more nuclei than the higher level when nuclei are in the state of equilibrium. With action of an electromagnetic radiation, it is possible to excite the nuclei from the low level into the higher level. The difference in energy between the energy levels ΔE = Eβ − Eα determines the frequency of radiation, which is needed for this excitation. When positive charged nucleus is spinning, this generates a small magnetic field. The nucleus therefore possesses a magnetic moment µ. This magnetic moment is proportional to its spin I, Planck’s constant h, and the constant γ, which is called the gyromagnetic ratio [1, 19]. γ for nucleus is a ratio of magnetic dipole moment to its angular momentum. γ is a fundamental nuclear constant which has a different value for every nucleus [4, 40]. The energy E m of each level m is proportional to the strength of the magnetic field at the nucleus B0, magnetic quantum number m and γh. The transition energy ΔE will be also proportional to B0. When the static magnetic field B0 is increased, the transition energy increases too. It is possible to imagine a nucleus (I = 1/2) in a magnetic field. This nucleus is in a base state. It is at the lower energy level and its magnetic moment does not oppose the applied field. The nucleus is spinning on its axis. In the presence of a magnetic field, this axis of rotation will precess around the magnetic field. The frequency of precession is called the Larmor frequency. The Larmor frequency is identical to the transition frequency. The angle of precession, φ, (the angle between the direction of the applied field and the axis of nuclear rotation) will change when energy is absorbed by the nucleus. The absorption of energy leads to a higher energy state. Due to the difference in population of levels in the state of equilibrium, there is a macroscopic magnetization M. It is oriented along the direction of the static magnetic field B0 (Z-axis). NMR-studies can be done in time-domain (for example, studying translational dynamics) and in frequency-domain using one-dimensional and two-dimensional NMR techniques (for example, studying structure of proteins) [5, 19, 40]. Fourier transformation is used to do transfer between time-domain and frequency-domain. The Fourier transform (FT) is a mathematical technique for converting data of time-domain to data in frequency-domain (giving a spectrum), and an inverse Fourier transform (IFT) converts data from the frequency-domain to the time-domain. The alternative magnetic field B1(t) = B1m.cos(ωt) is applied perpendicularly to constant magnetic field B0. Behaviour of the spin system in crossing magnetic fields is considered on the base of movement of magnetization vector M. Basic equations for the vector of the bulk magnetization M in outer magnetic field are the differential Bloch equations with the spin-lattice and spin-spin relaxation terms [5, 11, 19, 40]. These equations are much easier in a rotating coordinate system X′Y′Z′, which rotates with frequency ω around B0 (Z, Z′-axes) in the direction of nuclear precession [11, 40]. In rotation frame, vector M rotates around magnetic field B1 (X′-axis) with angle frequency ω = γB1. In the absence of magnetic field B1, magnetization M is along the direction of outer magnetic field B0. The angle θ for rotation of magnetization M during precession time tp is presented as θ = γB1tp [11]. During time tp, if vector M turns by θ = 90°, then this rotation is called as a 90°- or π/2-pulse (duration time tp is called the pulse length). When θ = 180° then this rotation is called as a π-pulse. An application of π/2-pulse to the magnetization M (using magnetic field B1) results in vector M being along the Y′-axis and the intensity of measurable signal (along Y′-axis) has maximal value. During the time (because of relaxation processes) a projection of magnetization vector M to the Y′-axis will decrease. This detected signal is called as a Free induction decay (FID). The signal intensity depends on the population difference between the two energy levels considered. The system is irradiated with a frequency, whose energy is equal to the difference in energy between these two energy levels. Upward transitions absorb energy and downward transitions release energy. The probability for transitions in either direction is the same. The number of transitions in either direction is determined by multiplying the initial level population by a probability. The nuclei, which are in a lower energy state can absorb radiation. At absorption of radiation, a nucleus jumps to the higher energy state. When the energy level populations are the same, the number of transitions in either direction will also be the same. When there is no further absorption of radiation, the spin system will be saturated. However, a possibility of saturation means that the relaxation processes would occur to return nuclei to the lower energy state. The sample in which the nuclei are held is called the lattice. All nuclei in the sample that are not observable are considered as lattice. Nuclei in the lattice are in vibrational and rotational motion, which creates a complex magnetic field. The magnetic field caused by motion of nuclei within the lattice is called the lattice field. The components of the lattice field, which are equal in frequency and phase to the Larmor frequency of the considered nuclei can interact with nuclei in the higher energy state, and cause them to lose energy and return back to the lower (equilibrium) state. The equilibrium is considered as a state of polarization with magnetization M0 directed along the longitudinal magnetic field B0. The restoration of the equilibrium is named longitudinal (spin-lattice) relaxation. The relaxation time constant, T1 describes the average lifetime of nuclei in the higher energy state. T1 is typically in the range of 0.1–20 s for protons in non-viscous liquids and other dielectric materials at room temperatures. A larger T1 indicates a slower or more inefficient spin relaxation [1, 4, 11]. The efficiency of spin-lattice relaxation depends on factors that influence molecular movement in the lattice, such as viscosity and temperature. The longitudinal relaxation times are often measured using the inversion recovery (IR) pulse sequence (180° − t − 90°) at repetition time ≥5T1 [40, 42, 47, 48, 52]. The period of 5T1 is waited in order to ensure that the fully recovered signal was acquired. When t is varied then experimental dependence of magnetization Mt = f (t) can be obtained and fitted by the law Mt = ∑M0i·[1–2·exp(−t/T1i)], where i is the number of relaxation component. An aim is to model experimental data as a sum of several relaxation components or distribution of NMR-relaxation times. To fit the data inverse Laplace transform (ILT) in one direction is applied [42, 53]. The kernel function in ILT is associated with Mt data from IR NMR experiments. Figure 1 shows T1 distributions obtained by one-dimensional ILT on the data sets of IR NMR experiments for bacterial suspension of E. coli and E. coli under xenon conditions. In original suspension, an exchange between intra- and extracellular water is too fast on NMR scale and gives one T1 component. The formation of xenon hydrates affected exchange dynamics of water between intra- and extracellular water compartments resulted in apparent two T1 components/peaks. Open image in new windowFig. 1 Fig. 1 T1 distribution of 1H in the suspension of E. coli with concentration of 0.07 g dry mass/g H2O (open circles) and in the same sample with xenon at 1.2 MPa (solid line) [53]. T = 280 K, ν = 90 MHz. All intensities have been normalized per maximal signal An interaction between neighbouring nuclei with identical frequencies but differing magnetic quantum states is described by spin-spin (transverse) relaxation. These nuclei can exchange quantum states. A nucleus in the lower energy level will be excited, while the excited nucleus relaxes to the lower energy state. The populations of the energy states do not change, but the average lifetime of a nucleus in the excited state will decrease. This can result in broadening resonance lines. More details are presented in publications [1, 4, 11, 19, 40]. Transverse relaxation is characterized by time constant T2. Spin-spin relaxation is a process whereby nuclear spins come to thermal equilibrium between themselves. At room temperatures, the T2 values for different materials are usually in the range from 10 µs to 10 s. It is always true for T2 ≤ T1. Transverse relaxation is unlike the longitudinal relaxation. It is sensitive to the interaction, which results in dephasing nuclear spins [39, 42]. For measurement of T2, it is possible to use Han pulse sequence (90° − τ − 180°). This sequence creates a signal of spin echo at t = 2τ [11, 19, 40]. When interval 2τ is varied, an experimental dependence M2τ = f(2τ) can be obtained and modelled to calculate the T2 [42]. The modification of Han method is Carr-Purcell (CP) pulse sequence (90° − τcp − 180° − 2τcp − 180° − 2τcp − 180° − …) which contains π-pulses train [11]. The Carr-Purcell-Meiboom-Gill (CPMG) sequence is the modification of CP-method: uses phase shift of π-pulses (by 90°) with respect to initial 90° pulse [40, 52]. CPMG decays are modelled by a sum of relaxation components [39, 43]. Alternatively, a distribution of T2 components, which could reconstruct spin-echo decay from CPMG experiment, should be found. To extract the function f(T2), the inverse Laplace transform is used. The probability density f(T2) is calculated from the Eq. (1) for the spin-echo signal M t presented according to Refs. [2, 6]:
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An improved method for storing platelets and compositions that contain stored platelets for use in transfusions. The method entails obtaining a platelet concentrate from blood obtained from an individual and holding the platelet concentrate at refrigerated temperatures under an atmosphere having a pressure of from 3.5 to 5 bars comprising more than 65% xenon and for at least one week. Also provided is a refrigerated composition that contains a platelet concentrate, wherein the platelet concentrate contains xenon, and wherein the platelet concentrate has been isolated from an individual for at least seven days.
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Purpose: To evaluate the dependency of the (129) Xe-red blood cell (RBC) chemical shift on blood oxygenation, and to use this relation for noninvasive measurement of pulmonary blood oxygenation in vivo with hyperpolarized (129) Xe NMR. Methods: Hyperpolarized (129) Xe was equilibrated with blood samples of varying oxygenation in vitro, and NMR was performed at 1.5 T and 3 T. Dynamic in vivo NMR during breath hold apnea was performed at 3T on two healthy volunteers following inhalation of hyperpolarized (129) Xe. Results: The (129) Xe chemical shift in RBCs was found to increase nonlinearly with blood oxygenation at 1.5 T and 3 T. During breath hold apnea, the (129) Xe chemical shift in RBCs exhibited a periodic time modulation and showed a net decrease in chemical shift of ∼1 ppm over a 35 s breath hold, corresponding to a decrease of 7-10 % in RBC oxygenation. The (129) Xe-RBC signal amplitude showed a modulation with the same frequency as the (129) Xe-RBC chemical shift. Conclusion: The feasibility of using the (129) Xe-RBC chemical shift to measure pulmonary blood oxygenation in vivo has been demonstrated. Correlation between (129) Xe-RBC signal and (129) Xe-RBC chemical shift modulations in the lung warrants further investigation, with the aim to better quantify temporal blood oxygenation changes in the cardiopulmonary vascular circuit. Magn Reson Med, 2016. © 2016 The Authors Magnetic Resonance in Medicine published by Wiley Periodicals, Inc. on behalf of International Society for Magnetic Resonance in Medicine. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
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A new method of preservation of nucleated cells in an electric refrigerator with the use of xenon is proposed. After slow freezing of an object and cryoanabiosis at −80°C for 1 day, cell membranes of more than 60% leukocytes were resistant to a vital dye. In 85% of granulocytes, the baseline level of lysosomal cationic proteins was retained, the lipid peroxidation intensity decreased, and the antioxidant activity of enzyme systems regulating the content of peroxides was also reduced. Cryopreservation of biological objects in inert gases is a promising direction in clinical practice and can become an alternative to the conventional method using liquid nitrogen.
to arrive at some temporary consensus model or models; and to present reliable physical data pertaining to water under a range of conditions, i.e., "Dorsey revisited," albeit on a less ambitious scale. I should like to acknowledge a debt of gratitude to several of my col­ leagues, to Prof. D. J. G. Ives and Prof. Robert L. Kay for valuable guidance and active encouragement, to the contributors to this volume for their willing cooperation, and to my wife and daughters for the understanding shown to a husband and father who hid in his study for many an evening. My very special thanks go to Mrs. Joyce Johnson, who did all the cor­ respondence and much of the arduous editorial work with her usual cheerful efficiency. F. FRANKS Biophysics Division Unilever Research Laboratory ColworthjWelwyn Colworth House, Sharnbrook, Bedford March 1972 Contents Chapter 1 Introduction-Water, the Unique Chemical F. Franks I. lntroduction ........................................ . 2. The Occurrence and Distribution of Water on the Earth 2 3. Water and Life ...................................... 4 4. The Scientific Study of Water-A Short History ........ 8 5. The Place of Water among Liquids . . . . . . . . . . . . . . . 13 . . . . . Chapter 2 The Water Moleeule C. W. Kern and M. Karplus 1. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 . . . . . . . . . . 2. Principles of Structure and Spectra: The Born-Oppenheimer Separation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 . . . . . . . . . . . . 3. The Electronic Motion ............................... 26 3.1. The Ground Electronic State of Water ............ 31 3.2. The Excited Electronic States of Water ........... 50 4. The Nuclear Motion ................................. 52 5. External-Field Effects ................................. 70 5.1. Perturbed Hartree-Fock Method . . . . . . . . . . . . . . . 74 . . .
To medically use and store proteins like enzymes, long-term maintenance of their activity must be considered. We examined the effectiveness of several methods for preserving the activity of three model-protein solutions. Solutions of catalase, L-lactate dehydrogenase, and carbonic anhydrase were used to form gas hydrates with xenon and natural gas. These enzyme aqueous solutions showed inhibitory effects on hydrate formation, and exhibited significant differences in induction time as well. The hydrates formed of enzyme solutions with xenon or natural gas are expected to have a better preservation effect than storage at room temperature and in liquid nitrogen. Changes in the activity of enzymes stored under different conditions were measured in relation to storage time. Storage in hydrate was good for maintaining the activity of catalase and L-lactate dehydrogenase. For carbonic anhydrase, the activity at room temperature was generally similar to that after storage in gas hydrate, but storing it in liquid nitrogen produced better results. For certain enzymes, storage in gas hydrates is expected to be a more effective method of maintaining activity than protein storage methods like freeze-drying, which causes mechanical damage to the protein.
solvent molecules;ray diffraction techniques;water molecule;hydrocarbons;platonic solids
Due to solute impurities and freezing-point depression in polycrystalline ice, a complicated and dynamic network of liquid water forms within the solid ice matrix at the boundaries between ice crystal grains. Impurity concentrations, temperature and pressure influence this network structure and impact physical, transport and rheological properties of ice. However, the nature of this internal network structure is not fully understood. Here we utilize nuclear magnetic resonance (NMR) measurements of diffusion and magnetic relaxation to study the geometry and interconnectivity of the liquid-filled network in laboratory ice, formed from a 7 g L–1 NaCl solution, and its evolution due to recrystallization processes. Additionally, we apply these NMR measurements to observe the impact on ice microstructure of an ice-binding protein (IBP) excreted by the V3519-10 organism (Flavobacteriaceae family) isolated from the Vostok ice core in Antarctica. Recrystallization inhibition was observed as a function of IBP concentration. This work demonstrates the utility of advanced NMR techniques for applications to ice microstructure and has broader implications for understanding geophysical properties of cryospheric systems.