FULL PAPER Magnetic Resonance in Medicine 00:00–00 (2014)

Relaxation and Exchange Dynamics of Hyperpolarized 129 Xe in Human Blood Graham Norquay,1 General Leung,1 Neil J. Stewart,1 Gillian M. Tozer,2 Jan Wolber,1,3 and Jim M. Wild1* ppm), make hyperpolarized (HP) 129Xe an attractive probe for in vivo MR studies of gas exchange in the lungs (2–4), as well as the perfusion of distal tissues and tumors (5,6). Integral to the design and feasibility of in vivo dissolved HP 129Xe MR experiments is an accurate knowledge of the spin-lattice relaxation rate of 129Xe in blood, as it is necessary for accurate modeling of the 129 Xe signal evolution while xenon is carried in the blood to the target tissues and organs of interest. The 129Xe relaxation rate in blood has been studied in previous NMR experiments performed by several groups. In a study conducted at a field strength of 4.7 T with hyperpolarized 129Xe, Albert et al. (7) found that the 129 Xe spin-lattice relaxation time, T1, in red blood cells (RBCs) increased with blood oxygenation, measuring T1 values of 4 s and 13 s in deoxygenated and oxygenated blood, respectively. The same group also performed measurements with thermally polarized 129Xe samples and found the 129Xe-RBC T1 in deoxygenated and oxygenated blood samples to be lower, with values of 2.7 6 0.22 s and 7.88 6 0.16 (8). Wolber et al. (9), using a field strength of 1.5 T, also reported an increase in 129Xe T1 with blood oxygenation (2.88 6 0.27 s deoxygenated and 5.71 6 0.35 s oxygenated blood) and found the T1 (1/T1) to increase (decrease) nonlinearly with blood oxygenation (10). In addition, both groups found the 129Xe T1 to be highest in blood that had been equilibrated with carbon monoxide; Albert et al. (8) reported a value of 11 6 2 and Wolber et al. (9) reported a value of 7.84 6 0.47 s. In a study conducted by Tseng et al. (11) with blood-foam at a field strength of 4.7 T, the opposite dependence of T1 on blood oxygenation was observed when compared with Albert et al. (7) and Wolber et al. (9). The T1 was reported to decrease from 40 s in deoxygenated blood to 20 s in oxygenated blood, and it was deduced that interactions between xenon and paramagnetic bubbles of oxygen gas in the blood was the principal cause of spin-lattice relaxation. The interior of the bubbles, within the blood-foam, provides a residency space for gaseous xenon and oxygen, and the bubble wall provides a surface compartment in which the oxygenexposed xenon can dissolve. Xenon gas and paramagnetic oxygen gas in the bubbles (undergoing nuclear-electron dipole-dipole T1 relaxation with a dependence inversely proportional to pO2) can readily exchange with the dissolved xenon in this regime and the effect of oxygen on the 129Xe T1 may have been overestimated as such. In the present study, 129Xe-blood relaxation was examined over the widest range of blood oxygenations to date (sO2 values of 0.06 – 1.00). The experimental design used in this study eliminated gas bubble formation while also removing the need to add xenon-saturated saline into the

Purpose: 129Xe-blood NMR was performed over the full blood oxygenation range to evaluate 129Xe relaxation and exchange dynamics in human blood. Methods: Hyperpolarized 129Xe was equilibrated with blood and isolated plasma, and NMR was performed at 1.5 T. Results: The 129Xe relaxation rate was found to increase nonlinearly with decreasing blood oxygenation. Three constants were extrapolated: rsO2 ¼ 11.1, a “relaxivity index” characterizing the rate of change of 129Xe relaxation as a function of blood oxygenation, and 1=T1oHb ¼ 0.13 s1 and 1=T1dHb ¼ 0.42 s1, the 129Xe relaxation rates in oxygenated blood and deoxygenated blood, respectively. In addition, rate constants, ka ¼ 0.022 ms1 and kb ¼ 0.062 ms1, were determined for xenon diffusing between red blood cells (RBCs) and plasma (hematocrit ¼ 48%). The 129Xe-O2 relaxivity in plasma, rO2 ¼ 0.075 s1 mM1, and the 129Xe relaxation rate in isolated 0 plasma (without dissolved O2), 1=T1;b ¼ 0.046 s1, were also calculated. Finally, intrinsic 129Xe-RBC relaxation rates, oHb dHb ¼ 0.19 s1 and 1=T1;a ¼ 0.84 s1, in oxygenated blood 1=T1;a and deoxygenated blood, respectively, were calculated. Conclusion: The relaxation and exchange analysis performed in this study should provide a sound experimental basis upon which to design future MR experiments for dissolved xenon transport from the lungs to distal tissues. Magn Reson Med C 2014 Wiley Periodicals, Inc. 000:000–000, 2014. V Key words: hyperpolarized gases; relaxation; blood; chemical exchange

129

Xe spectroscopy; T1

INTRODUCTION The nuclear polarization of 129Xe can be increased by four to five orders of magnitude by using the technique of spin-exchange optical pumping (1). This hyperpolarization enables the detection of 129Xe with NMR even at very small 129Xe concentrations. The solubility of xenon in various biological tissues and fluids, coupled with its large associated range of chemical shifts (hundreds of 1 Unit of Academic Radiology, Department of Cardiovascular Science, University of Sheffield, Sheffield, South Yorkshire, UK. 2 Department of Oncology, University of Sheffield, Sheffield, South Yorkshire, UK. 3 GE Healthcare, Amersham, Buckinghamshire, UK. Grant sponsors: Cancer Research UK and EPSRC, with additional funding from MRC and Department of Health (England); Grant numbers: C1276/ A1034 and EP/D070252/1. *Correspondence to: Jim M. Wild, Ph.D., Unit Academic of Radiology, University of Sheffield, Floor C, Royal Hallamshire Hospital, Glossop Road Sheffield S10 2JF, United Kingdom. E-mail: [email protected] Received 6 December 2013; revised 25 July 2014; accepted 29 July 2014 DOI 10.1002/mrm.25417 Published online 00 Month 2014 in Wiley Online Library (wileyonlinelibrary. com). C 2014 Wiley Periodicals, Inc. V

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blood samples (hence eliminating associated dilution effects) (7,9). Herein, we discuss the physical mechanisms governing the variation in 129Xe relaxation rate with blood oxygenation and present a quantitative analysis to determine parameters that underpin 129Xe relaxation and exchange mechanisms in whole blood samples.

It is assumed that the 129Xe resonance frequency at each site, xa and xb , is distinct, with a frequency difference given by Dx 5 jxa 2 xb j; the magnetization dynamics of 129Xe nuclei undergoing exchange between plasma and RBCs may then be described by coupled rate equations by extending previous NMR analysis of water protons diffusing between plasma and RBCs (15):

THEORY

  dMa ðtÞ 5 2 R1;a 1 ka Ma ðtÞ 1 kb Mb ðtÞ dt   dMb ðtÞ 5 2 R1;b 1 kb Mb ðtÞ 1 ka Ma ðtÞ: dt

For 129Xe nuclei freely diffusing between RBCs and plasma within whole blood samples, a two-site exchange process is considered: ka

RBC PLASMA kb

[1]

where ka and kb denote the exchange rates of 129Xe diffusing from RBCs to plasma and vice versa. For dynamic equilibrium, the fractional populations of 129Xe in RBCs and plasma, pa and pb , are related to the residency times sa ð ka2 1 Þ and sb ð kb2 1 Þ by pa 5

sa sb and pb 5 ; sa 1 sb sa 1 sb

[2]

where pa 1pb 5 1, and the time taken, sex , for the system to establish equilibrium is given by (12,13) 1 1 1 5 1 5 kex 5 ka 1 kb ; sex sa sb

[3]

where kex is defined as the exchange rate constant. A combination of Equations 2 and 3 yields sex 5 pa sb 5 pb sa :

[4]

The ratio of residency times of 129Xe in the plasma and RBCs can be estimated by recasting Equation 4 with fractional magnetizations in place of fractional populations: Ma sb 5 Mb sa )

Mb sb 5 ; Ma sa

[5]

where Ma and Mb are the fractional 129Xe magnetizations in RBCs and plasma. With knowledge of the residency time ratio and the equilibrium time constant, sex , which was calculated by Bifone et al. (14) to be 12 ms, Equation 3 can be rearranged so that the individual 129Xe residency times in RBCs and plasma are    sa sb 1 1 sex and sb 5 1 1 sex : sa 5 sb sa

Here, R1,a ( 1/T1,a) and R1,b ( 1/T1,b) are the intrinsic NMR relaxation rates of dissolved 129Xe nuclei in RBCs and plasma. Such systems have been considered in detail by Woessner (16), and the general solutions to Equation 8 are Ma ðtÞ 5 A1 exp ð2/1 tÞ 1 A2 exp ð2/2 tÞ Mb ðtÞ 5 B1 exp ð2/1 tÞ 1 B2 exp ð2/2 tÞ;

The magnetization ratio, Mb =Ma , will depend on the hematocrit (HCT) of the blood samples and the relative xenon solubilities in plasma and RBCs, so that the xenon residency times vary with HCT according to     Mb db 1 2 HCT sb db sb HCT 5 ) ; [7] 5 5 HCT Ma da sa da sa 1 2 HCT where db =da is the plasma-RBC partition coefficient for xenon.

[9]

where 2/6 5 ðR1;a 1 R1;b 1 ka 1 kb Þ 6 ½ðR1;a 2 R1;b 1 ka 2 kb Þ2 1 4ka kb 1=2

[10]

and the coefficients A and B depend on the fractional volume of the RBCs and plasma (and the solubility of xenon in each compartment), as well as on /6 . The 129Xe exchange rates, ka and kb , were measured by Bifone et al. (14) in (slightly diluted) whole blood to be of the order of tens of Hz, whereas the 129Xe relaxation rates, R1,a and R1,b, are in the sub-Hz range, based on measurements by Wolber et al. (9) and Albert et al. (7). In the limit of ka ; kb >> R1;a ; R1;b , Equation 10 can be approximated as 2/6 5 ðR1;a 1 R1;b 1 ka 1 kb Þ 6 ka 1 kb ;

[11]

so that the fast and slow decay rate constants, /1 and /2 , are /1 5

R1;a 1 R1;b 1 ðka 1 kb Þ 2

[12]

R1;a 1 R1;b : 2

[13]

and /2 5



[6]

[8]

Considering the interpulse delay time range used in this study (0.15 – 0.5 s), inserting the fast decay rate constant, /1 , into Equation 9 (due to the large value of ka 1 kb ) causes A1 exp ð2/1 tÞ and B1 exp ð2/1 tÞ ! 0, so that the solutions to Equation 8 become Ma ðtÞ 5 A2 exp ð2/2 tÞ Mb ðtÞ 5 B2 exp ð2/2 tÞ;

[14]

suggesting that in the case of ka ; kb >> R1;a ; R1;b , the Xe magnetizations in the plasma and RBC

129

Relaxation and Exchange Dynamics of Hyperpolarized

129

Xe

3

the design of Chann et al. (18). The laser emission was centered on a wavelength of 794.77 nm (Rb D1 resonance) and the full-width-half-maximum was measured to be 0.09 6 0.01 nm. For the generation of all HP 129Xe samples, a gas mixture of 3% isotopically enriched xenon (86% 129Xe), 10% N2, and 87% He (Spectra Gases, UK) was flowed through the cell using a mass flow controller (Aalborg, Cache, Denmark), and collected cryogenically using a freeze-out process (17). A gas flow rate through the cell of 300 sccm was maintained during the freeze-out, resulting in the production of  200 mL of gaseous xenon (129Xe polarization of 10% – 15%) over a period of 20 min. The cell temperature and pressure was maintained at 373 6 1 K and 2 bars, respectively, throughout the spin-exchange optical pumping process.

FIG. 1. Photograph of the xenon-blood exchange apparatus (left) and cross-section of the exchange module (right). Xenon (c) is pushed through the hollow-fiber tubes of the exchange module (b1 and b2) unidirectionally at a rate of  1 mL/s, while the blood (d) is passed into and out of the membrane and the sample volume (3-mL syringe enclosed within a custom-built solenoid RF coil [a]). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

compartments decay with a common rate constant, /2 , which we refer to as the observable 129Xe relaxation rate, R1 ( 1/T1), in whole blood samples. In this study, the 129Xe relaxation rate, 1/T1,b, in the plasma pool was assumed, via dipole-dipole interactions with dissolved paramagnetic molecular oxygen, to change linearly with oxygen concentration, [O2], according to 1 1 5 rO2 ½O2  1 0 ; T1;b T1;b

[15]

0 where rO2 is the 129Xe-O2 relaxivity in plasma and 1/T1;b 129 is the Xe relaxation rate in the absence of dissolved oxygen. The intrinsic 129Xe relaxation rate for xenon dissolved in RBCs, 1/T1,a, can be obtained by combining Equation 13 (where /2 5 1=T1 , R1,a 5 1/T1,a, and R1,b 5 1/T1,b) and Equation 15, yielding

 0 0 2T1;b 2 T1 rO2 ½O2 T1;b 11 1 2 1 5 2 5 : 0 T1 T1;b T1;a T1 T1;b

[16]

METHODS Spin-Exchange Optical Pumping Spin-exchange optical pumping was performed using a custom-built, 129Xe spin-exchange optical pumping polarizer (17). This polarizer consisted of a Helmholtz coil (diameter, 80 cm; B0,  3 mT); a cylindrical Pyrex optical cell (length, 25 cm; diameter, 5 cm) containing < 1 g of rubidium, located at the isocenter of the B0 field, and housed within a nonmagnetic, ceramic hot-air oven with optical glass windows; and an external cavity diode laser, which was constructed in-house following

Blood Sample Preparation and Analysis To prepare all blood samples, whole blood was withdrawn by a clinician from three self-consenting volunteers by venipuncture and transferred into lithium heparin vacuum containers approximately 2 to 3 hours prior to the start of the NMR experiments. All blood samples were allowed to equilibrate to a temperature of 20 6 2  C (the temperature at which the scanner room is maintained). Prior to conducting the NMR experiments, the xenon was first dissolved into the blood. To ensure effective mixing, the xenon and blood were passed through an exchange module (19,20) (Superphobic MicroModule 0.5 3 1 G680 Contactor; Membrana, North Carolina, USA), which provided an exchange surface area of 100 cm2. To perform the mixing, 10 mL of xenon gas was passed unidirectionally through the inside of thin-walled hollow-fiber tubes within the exchange module (Fig. 1) at a rate of  1 mL/s, while the blood was passed back and forth over the outside of the tubes for 10 s to ensure a sufficient concentration of xenon was dissolved into the blood. Using this mixing technique enables microscopic mixing, while also eliminating the formation of gas bubbles in the blood. Immediately after the mixing process, 2 mL of the xenon-blood mixture was drawn up into a 3-mL syringe contained within a custom-built solenoid coil, and the sample was then ready for NMR signal acquisition. A clinical blood gas analyzer (ABL80; Radiometer, Crawley, United Kingdom) was used to analyze the blood sample and determine the necessary physiological parameters such as sO2, pO2, pCO2, HCT, and pH. Immediately after acquiring NMR spectra (see the NMR Spectroscopy section), approximately 0.1 mL of blood was withdrawn directly from the NMR sample syringe and taken to the blood gas analyzer for analysis. Whereas NMR measurements were performed on blood samples at 20  C, the blood gas analyzer derives blood sO2 from the measured pO2, pH, and pCO2 assuming a blood sample temperature of 37  C. Therefore, the sO2 values of the samples measured at 20  C were corrected using the numerical technique of Kelman (21) that accounts for shifts in the standard oxygen dissociation curve in blood due to changes in temperature, pH, pO2, and pCO2 (Fig. 2). In order to increase the blood sO2 from its venous value (0.60 – 0.80), upon finishing each NMR

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5-mL lithium heparin vacuum containers for  5 hours to ensure the plasma had separated sufficiently, allowing 6 mL of plasma to be extracted from the four tubes using a 10-mL syringe. The concentration of molecular O2 in plasma ½O2  was calculated as ½O2  5 dpl pO2 ;

[17]

where dpl 5 1.63 3 1023 mm Hg21 mM is the solubility coefficient of O2 in isolated plasma at 20  C (23) and pO2 is the O2 partial pressure in the plasma, measured with the blood gas analyzer. To lower the plasma pO2 value, a stock solution of sodium dithionite (same concentration to that used in the whole blood samples) was added to the plasma; to increase the plasma pO2 value, room air was added into the exchange module and mixed with plasma, as described above. FIG. 2. Derived blood oxygenation, sO2, versus measured blood oxygen partial pressure, pO2. The blood oxygenation was derived numerically (see the Methods section) from blood pO2, pH, and pCO2 values measured using a blood gas analyzer. The blue and red circles correspond to oxygenation values derived using numerical methods based on Kelman (21) assuming blood sample temperatures of 20  C (the sample temperature at which the NMR was performed) and 37  C, respectively, and the black triangles correspond to oxygenation values derived by the blood gas analyzer (which assumes a sample temperature of 37  C). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

acquisition, 1 – 3 mL of O2 was passed through the exchange module at a rate of  0.2 mL/s and exchanged with the blood in the same way as it was for xenon. After mixing the blood and O2, the exchange module was flushed with  20 mL of N2 gas (without passing blood through the system) to remove the O2 from the xenon gas spaces. To decrease the blood oxygen saturation to values lower than 0.60, a saline suspension of sodium dithionite (Na2O4S2) was mixed with blood external to the exchange module. Stock solutions were prepared at concentrations of 100 mg of Na2O4S2 per 1 mL of saline (0.9% w/v sodium chloride), and the suspension was added to the blood at a concentration of 0.05 mL saline per 1 mL blood using a 1-mL syringe for mixing. The sodium dithionite reduced the partial pressure of extracellular molecular oxygen within the blood sample, thereby causing depletion of oxygen in the RBCs (22); with this technique, it was possible to reduce the blood sO2 to a value of 0.06. It is worth noting that as a result of the slight dilution, the sodium dithionite samples had lower levels of HCT, with the lowest recorded value being 38%. This HCT is still within the range of HCT values observed in the different volunteers’ blood samples (37% – 57%); therefore, we assume the small dilution to be negligible with respect to changes in 129Xe relaxation. Finally, a single blood sample was equilibrated with carbon monoxide using the same mixing procedure as described above for oxygen. Plasma Sample Preparation To obtain isolated plasma, 20 mL of whole blood, withdrawn as described above, was stored vertically in four

NMR Spectroscopy For all 129Xe-blood NMR spectroscopy experiments, a 1.5 T MR scanner (Signa HDx; GE Healthcare, Milwaukee, Wisconsin, USA) was used in conjunction with a custom-built, six-turn solenoid RF coil (length, 4 cm; diameter, 2 cm) tuned to resonate at 17.66 MHz. Prior to performing T1 measurements, excitation flip angles were determined for blood samples by applying a series of rectangular hard pulses (pulse width, 500 ls) centered on the 129Xe-RBC resonance frequency ( 220 ppm away from the 129Xe gas resonance) with interpulse delay times (TR) of 40 ms. For all T1 acquisitions, the receiver bandwidth was set to 2.5 kHz and the number of sample points was set to 512 for 500 ms TR values and to 256 for 150 ms TR values; for flip angle calibrations, in order to achieve very short TR, the bandwidth and sample points were set to 10 kHz and 64, respectively. A zerothorder phase correction was performed to enable signal analysis of the absorption spectra (Fig. 3). The measured 129Xe signal decrease was fitted to (cos a)n21, where a is the flip angle to be calibrated and n is the RF pulse number. Separate flip angle calculations were performed for each experiment and all values fell within the range 13.5 to 15 . After calibrating the flip angle, the blood samples were remixed with HP 129Xe and a T1 measurement sequence was applied consisting of 15 – 20 pulses with TR values of 150 ms and 500 ms for blood sO2 ranges of 0.06 – 0.80 and 0.80 – 1.00, respectively. 129Xe T1 calculations were made by fitting the decay in the 129Xe-RBC and 129Xe-plasma NMR signals to the relationship Sn 5 S0 sinaðcos aÞn 2 1 expð2ðn 2 1ÞTR=T1 Þ;

[18]

where S0 is the signal intensity at time t 5 0. The signals that were fitted to Equation 18 were determined by integrating over the 129Xe-RBC and 129Xe-plasma resonance peaks in the phased absorption spectrum. Figure 4 shows the typical 129Xe-blood decay spectra, with an inset showing fits to the 129Xe-RBC and 129Xe-plasma signal decays. A nonlinear least-squares fitting routine using the Levenberg-Marquardt method was implemented in

Relaxation and Exchange Dynamics of Hyperpolarized

129

Xe

FIG. 3. 129Xe spectrum in blood. The peaks at 196 and 220 ppm correspond to 129Xe dissolved within blood plasma and RBCs, respectively. All data analyses were performed using the absorption part of the 129Xe-blood signal, obtained by performing zerothorder phase corrections on the real and imaginary parts of the Fourier transformed spectra. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

MATLAB (Mathworks, Natick, Massachusetts, USA) and was used for all T1 calculations. The same NMR parameters above were used for T1 acquisitions on the isolated plasma samples, apart from the TR, which was set to 4 s. The ratio of 129Xe-plasma and 129Xe-RBC magnetizations, Mb /Ma , was estimated by integrating the area under the RBC and plasma resonances and averaging over 20 pulses under dynamic equilibrium where TR >> sex (Fig. 5). RESULTS Measurements of the 129Xe relaxation times and rates in human whole blood samples were made over a blood oxygenation range of sO2 5 0.06 – 1.00. Blood gas analysis parameters are given in Table 1 alongside 129Xe T1 values corresponding to venous blood (sO2 range 0.60 – 0.80), arterial blood (sO2 range 0.95 – 1.00), and blood outside the physiological range (sO2 < 0.60). (Note that all blood samples were obtained through venipuncture [see the Methods section], and we hereafter refer to blood as venous or arterial for sO2 ranges of 0.60 – 0.80 and 0.95 – 1.00, respectively.) In addition, a T1 relaxation value for blood equilibrated with CO has been included. 129 Xe-blood NMR spectra obtained from all samples exhibited two distinct resonance peaks from xenon dissolved in blood plasma and RBCs. While the 129Xe peak in plasma, located 196 ppm downfield from the 129Xe gas reference at 0 ppm, was found to be fixed in center frequency over the full range of blood oxygenation values, the 129Xe-RBC chemical shift increased from 220 ppm in approximately fully deoxygenated blood (sO2 5 0.06) to 224 ppm in completely oxygenated blood (sO2 5 1.00). This shift has been observed previously and can be explained by a decrease in 129Xe chemical shift shielding constant with increasing blood oxygenation (9,24).

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FIG. 4. Decaying spectra from 129Xe dissolved in blood acquired with 20 hard pulses of 500 ls width (and interpulse delay of 0.5 s). The inset shows a fit performed on the decreasing 129Xe NMR signal (integrals of 129Xe-RBC and 129Xe-plasma absorption peaks) in order to establish 129Xe-RBC (red triangles) and 129Xe-plasma T1 values (blue squares). The decaying spectra represent a blood sample with sO2 5 0.98. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

A typical data set showing the decay in signal from Xe-blood spectra over a number of NMR acquisitions is shown in Figure 4. The T1 values calculated from the 129 Xe signal decay in RBCs were found to be the same, within experimental error, as the T1 values calculated from the 129Xe signal decay in plasma, thus confirming the prediction of Equation 14 (for ka ; kb >> R1;a ; R1;b ) that the 129Xe magnetizations in plasma and RBCs in whole blood samples decay with a common observable T1. The overall 129Xe-blood relaxation rate, 1/T1, was found to be nonlinearly dependent on blood oxygenation, over an sO2 range of 0.06 – 1.00, as shown in Fig. 6c and d. The nonlinear relationship between 1/T1 and sO2 can be fitted to the empirical equation 129

FIG. 5. Ratio of 129Xe-plasma magnetization to 129Xe-RBC magnetization over a 20-pulse acquisition. The dashed blue line is the mean value magnetization ratio over all pulse acquisitions for a blood sample with a hematocrit of 0.48 at 20  C. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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Table 1 129 Xe Relaxation Times, T1, and Rates, 1/T1, Calculated from n Samples for a Variety of Blood Oxygenation, sO2, Values Samplesa A1 A2 A3 V2 D2 CO P

T1 (s)

1=T1 (s21)

n

sO2b

pO2 (mm Hg)

pCO2 (mm Hg)

pH

ctHbc (mM)

3.4–7.8 3.7–7.2 3.6–6.1 2.8 2.2, 2.3 7.9 11.4–20.4

0.29–0.13 0.27–0.14 0.28–0.16 0.36 0.45, 0.43 0.13 0.09–0.05

11 11 4 1 2 2 7

0.96–1.00 0.95–1.00 0.97–0.99 0.80 0.06, 0.47 — —

33–133 36–80 47–84 31 6, 18 — 2–303

27–74 10–50 23–48 65 89, 77 — 7–96

7.13–7.5 6.96–7.6 7.14–7.4 6.93 6.94, 6.92 — 6.8–7.5

9–12 7.6–10.6 8.1– 8.7 7.8 7.4, 7.8 — —

a

A1–A3: arterial (defined here to be the range sO2 ¼ 0.95  1.00) blood from three volunteers. V2: a single venous blood sample (defined for sO2 ¼ 0.60  0.80). D2: blood samples outside the physiological blood oxygenation (sO2 < 0.60). CO: a blood sample equilibrated with carbon monoxide. P: samples of isolated plasma solution. b Values were derived numerically from pO2, pCO2, and pH values that were measured on a blood gas analyzer (see Methods section and Fig. 2). c The concentration of hemoglobin in the blood.

1 1 5 j½1 2 expðrsO2 sO2 Þ 1 dHb ; T1 T1

[19]

where j is a scaling constant, rsO2 we define here to be a relaxivity index characterizing the rate of change of 129Xe relaxation as a function of blood oxygenation and 1=T1dHb is the relaxation rate for fully deoxygenated blood. The values of these constants, obtained by fitting Equation 19 to the 129Xe relaxation rate values measured in plasma and

RBCs (Fig. 6c, d) were derived to be j 5 4.6 3 1026 s21, rsO2 5 11.1 and 1=T1dHb 5 0.42 s21 (T1dHb 5 2.4 s). In addition, by setting sO2 equal to 1.00 in Equation 19, one can obtain the relaxation rate of 129Xe in fully oxygenated blood, 1=T1oHb 5 0.13 s21 (T1oHb 5 7.7 s). The measured values of 129Xe T1 range from 2.2 s in approximately fully deoxygenated blood (sO2 5 0.06) to 7.8 s in fully oxygenated blood (sO2 5 1.00). The rate of change of 129Xe relaxation with respect to oxygenation is

FIG. 6. 129Xe relaxation rates from NMR experiments performed on six different blood samples taken from three separate volunteers covering the full blood oxygenation range. a, b: Predicted intrinsic 129Xe relaxation rates in RBCs, 1/T1,a, and plasma, 1/T1,b, respectively; 1/T1,a was calculated using Equation 20, and 1/T1,b was calculated using Equation 15. c: Measured 129Xe relaxation rates in RBCs (red triangles) and plasma (black triangles). An empirical function (Eq. 19) was fitted to the data, where the following constants were determined: j 5 4.6 3 1026 s21, rsO2 5 11.1, and 1=T1dHb 5 0.42 s21 (T1dHb 5 2.4 s). The solid gray triangle represents the 129Xe relaxation rate for a blood sample that was equilibrated with carbon monoxide. d: All data plotted together.

Relaxation and Exchange Dynamics of Hyperpolarized

129

Xe

7

whole blood to be negligible as for the highest oxygen concentration of 0.22 mM, the relaxation contribution from oxygen, rO2 ½O2  5 0.017 s21, is significantly smaller than the lowest experimentally measured 129Xe relaxation rate of 1/T1 5 0.13 s21 in whole blood equilibrated with CO. Using the result that the observed 129Xe relaxation rate, 1/T1, in whole blood samples is the same in RBCs and plasma over the full blood oxygenation range, it is possible to express the intrinsic 129Xe relaxation rate for xenon dissolved in RBCs, 1/T1,a, as a function of sO2, in terms of the earlier derived relaxation constants, by combining Equations 16, 17, and 19, yielding h i  0 1 0 2T1;b jð1 2 exp ðrsO2 sO2 ÞÞ 1 T dHb 11 2 rO2 dwb pO2 T1;b 1 1 5 : 0 T1;b T1;a

[20] FIG. 7. 129Xe relaxation rates from NMR experiments performed on isolated plasma samples over a range of oxygen concentrations, [O2]. A linear fit (with a coefficient of determination R2 5 0.95) was performed on the data using the boxed equation, allowing determination of the 129Xe-O2 relaxivity in plasma, rO2 5 0.075 s21 mM21 and the 129Xe relaxation rate in the absence of 0 dissolved molecular oxygen, 1=T1;b 5 0.046 s21.

relatively slow in the sO2 range 0.06 – 0.90, with the T1 increasing from a value of 2.2 s to only 2.8 s; above sO2 5 0.90, the rate of change of relaxation with blood oxygenation is much more rapid. The average range of 129 Xe T1 in arterial blood samples from three separate volunteers (A1 – A3 in Table 1) was calculated to be 3.6 6 0.2 s (sO2 5 0.95) to 7.0 6 0.9 s (sO2 5 1.00). Blood equilibrated with CO was found to have a 129Xe T1 of 7.9 6 0.1 s, in good agreement with the highest T1 value of 7.8 s measured in fully oxygenated blood. The 129Xe relaxation rate in isolated plasma, 1/T1,b, was found to increase linearly with increasing dissolved O2 concentration, as predicted by Equation 15, which was fitted to the data to obtain the 129Xe-O2 relaxivity in plasma, rO2 5 0.075 s21 mM21, and the 129Xe spin-lattice relaxation rate in isolated plasma in the absence of molecular oxy0 0 gen, 1/T1;b 5 0.046 s21 (T1;b 5 21.7 s) (Fig. 7). The concentration of O2 in plasma within whole blood can be calculated by using Equation 17 with a previously measured O2-plasma solubility coefficient (for whole blood), dwb 5 1.69 3 1023 mm Hg21 mM, assuming an Hb concentration value of 9.3 mM (15 g/dL) and sample temperature of 20  C (25). The highest pO2 value in whole blood samples used throughout this study was measured to be 133 mm Hg, which corresponds to an O2 concentration of 0.22 mM. One can ascertain, therefore, the contribution of dissolved unbound molecular O2 to 129Xe relaxation in

This relationship is shown graphically in Figure 6a. Intrinsic relaxation rates for 129Xe in RBCs in fully oxyoHb genated blood, 1=T1;a 5 0.19 s21, and fully deoxygendHb ated blood, 1=T1;a 5 0.84 s21, were calculated by setting sO2 to 1.00 and 0.00, respectively, in Equation 20. The measured ratios of 129Xe-plasma to 129Xe-RBC magnetizations were used to calculate xenon-RBC and xenon-plasma of residency times/exchange rates (Eqs. 5 and 6) and xenon plasma-RBC partition coefficients (Eq. 7) for a range of HCT values. The mean xenon plasma-RBC partition coefficient calculated for blood at 20  C over an HCT range of 0.39 to 0.54 was calculated to be 0.36 6 0.04, which is in reasonable agreement with the xenon plasma-RBC partition coefficient of  0.3 calculated previously from red cell suspensions at 20  C by Chen et al. (26). See Table 2 for calculated values of residency times/exchange rates and xenon plasma-RBC partition coefficients.

DISCUSSION As first demonstrated by Pauling and Coryell (27), the fully deoxygenated form of hemoglobin—deoxyhemoglobin, Hb4 (electron spin S 5 2)—is paramagnetic, whereas the fully oxygenated form—oxyhemoglobin, Hb4O8 (S 5 0)—and carboxyhemoglobin (Hb with carbon monoxide bound to it) are both diamagnetic. In addition, it was shown by Coryell et al. (28) that blood sO2 measurement is essentially an ensemble average measure of the superposition of the two extreme hemoglobin states, Hb4 and Hb4O8, with only a very small contribution from the three intermediate hemoglobin states (Hb4O2, Hb4O4, and Hb4O6). Furthermore, Coryell et al. determined that the relative fractions of hemoglobin in the fully

Table 2 Ratio of 129Xe-Plasma Magnetization to 129Xe-RBC Magnetization, Mb =Ma ; Residency Times, sb and sa , and Exchange Rate Constants, kb and ka , for Xenon in Plasma and RBCs; and Calculated Xenon Plasma-RBC Partition Coefficients, db =da , for Three HCT Values HCT

Mb =Ma

sb (ms)

kb (ms21)

sa (ms)

ka (ms21)

db=da

0.39 0.48 0.54

0.56 0.36 0.34

18.7 16.3 16.1

0.053 0.061 0.062

33.5 45.3 47.3

0.03 0.022 0.021

0.36 0.33 0.4



All values presented in the table correspond to whole blood samples at a temperature of 20 C.

8

deoxygenated and fully oxygenated state vary approximately linearly with blood oxygenation. The observed nonlinear decrease in the 129Xe relaxation rate with increasing blood oxygenation (decreased net paramagnetism of the hemoglobin molecules) agrees with the findings of Wolber et al. (10), in which a nonlinear relationship between the 129Xe relaxation rate and blood oxygenation was also observed with the same direction of trend albeit over a smaller oxygenation range. Evaluation of the relaxation mechanisms contributing to the change in 129Xe relaxation with RBC oxygenation is substantially more complicated than evaluation of the change in 129Xe relaxation due to varying O2 concentration in plasma, hence a rigorous, fully quantitative treatment was beyond the scope of this study. However, we expand on the possible relaxation mechanisms herein. Two relaxation mechanisms are considered: 1) dipoledipole spin-lattice relaxation from 129Xe-Hb4 (in the case of deoxyhemoglobin) and 129Xe-1H interactions and 2) cross-relaxation of 129Xe to protons at the xenon-Hb binding sites via the spin-induced nuclear Overhauser effect (SPINOE) (29). Both of these mechanisms depend very strongly on the proximity of 129Xe to the neighboring proton spins and paramagnetic Hb4 (30), thus accurate knowledge of the xenon-Hb binding sites is needed to evaluate the relative dipole-dipole and crossrelaxation contributions to the 129Xe relaxation. A number of studies evaluating xenon binding in hemoglobin have been conducted to date. X-ray crystallography experiments reporting xenon binding in blood performed by Schoenborn et al. identified only one xenon binding site in both sperm whale myoglobin (31) (proximal to the iron heme) and horse hemoglobin (32) (distal to the iron heme). Xenon has most recently been used to examine CO and O2 within human hemoglobins (33,34), and Savino et al. (35), who closely examined hydrophobic cavity patterns in deoxygenated human hemoglobin, identified a total of 12 xenon binding sites per Hb4 tetramer: four located in the a1 chain, three in the a2 chain, two in the b1 chain, and three in the b2 chain. (The xenon binding sites in oxygenated hemoglobin were not considered in this study.) The Hb molecule undergoes a significant structural change as it makes the transition from the deoxyhemoglobin conformation to the oxyhemoglobin conformation (36); it is thus possible—and indeed likely—that the number and locations of xenon binding sites are different in deoxyhemoglobin when compared with oxyhemoglobin. Without knowledge of the locations of xenon atoms in oxyhemoglobin, it is not possible to accurately estimate the relative changes in dipole-dipole and cross-relaxation interaction strengths. Despite this, it is possible to make some assumptions in order to gain insight into the mechanisms driving the nonlinear change in the 129Xe relaxation rate with blood oxygenation. Let us assume the following: 1) dipoledipole 129Xe-Hb4 interaction is the dominant relaxation process (over 129Xe-1H dipole-dipole and 129Xe-1H SPINOE interactions); 2) the xenon-Hb binding site locations do not change with blood oxygenation (i.e., the 129Xeheme distances do not change); and 3) based on the find-

Norquay et al.

ings of Coryell et al. (28), the concentration of paramagnetic Hb4 changes approximately linearly with blood oxygenation. If these assumptions were valid, one would expect to observe a linear change in the 129Xe relaxation rate with blood oxygenation. However, the observation of a nonlinear change in 129Xe relaxation rate with blood oxygenation suggests that if dipole-dipole 129Xe interactions with paramagnetic Hb4 were the dominant relaxation process, the number/location of xenon-Hb binding sites must change with blood oxygenation. Considering 129 Xe cross-relaxation to protons, polarization transfer in SPINOE is most rapid when xenon is temporarily bound, thus if the xenon-Hb binding sites do indeed change with blood oxygenation, one would expect a concomitant change in the 129Xe-1H cross-relaxation rate. As highlighted above, knowledge of the xenon-Hb binding sites in oxyhemoglobin is crucially required before attempting a quantitative description of the complicated underlying relaxation mechanisms responsible for the change in 129 Xe relaxation rate with blood oxygenation.

CONCLUSIONS A nonlinear dependence of the 129Xe longitudinal relaxation rate on blood oxygenation has been observed over the widest range of blood oxygenation values to date. The most rapid rate of change in 129Xe blood relaxation rate is seen in the blood oxygenation range sO2 0.90 to 1.00. One would therefore expect 129Xe relaxation to be highly sensitive to changes in arterial blood oxygenation and less sensitive to changes in venous blood oxygenation. The quantitative relaxation and exchange analysis performed in the present study has implications for future studies of xenon transport from the lungs to distal tissues, organs, and tumors and should provide a sound experimental basis upon which to design novel MR experiments for these purposes. Given the knowledge of average blood oxygenation in the circulation and the transit time to the target tissue of interest, the signal evolution of 129Xe can be modeled based on these findings. REFERENCES 1. Happer W, Miron E, Schaefer S, Schreiber D, Vanwijngaarden WA, Zeng X. Polarization of the Nuclear Spins of Noble-Gas Atoms by Spin Exchange with Optically Pumped Alkali-Metal Atoms. Phys Rev A 1984;29:3092–3110. 2. Patz S, Muradyan I, Hrovat MI, Dabaghyan M, Washko GR, Hatabu H, Butler JP. Diffusion of hyperpolarized (129)Xe in the lung: a simplified model of (129)Xe septal uptake and experimental results. New J Phys 2011;13:015009. 3. Chang YV. MOXE: a model of gas exchange for hyperpolarized 129Xe magnetic resonance of the lung. Magn Reson Med 2013;69:884–890. 4. Stewart NJ, Leung G, Norquay G, et al. Experimental validation of the hyperpolarized 129Xe chemical shift saturation recovery technique in healthy volunteers and subjects with interstitial lung disease. Magn Reson Med 2014. doi: 10.1002/mrm.25400. 5. Peled S, Jolesz FA, Tseng CH, Nascimben L, Albert MS, Walsworth RL. Determinants of tissue delivery for 129Xe magnetic resonance in humans. Magn Reson Med 1996;36:340–344. 6. Kilian W, Seifert F, Rinneberg H. Dynamic NMR spectroscopy of hyperpolarized (129)Xe in human brain analyzed by an uptake model. Magn Reson Med 2004;51:843–847. 7. Albert MS, Balamore D, Kacher DF, Venkatesh AK, Jolesz FA. Hyperpolarized 129Xe T1 in oxygenated and deoxygenated blood. NMR Biomed 2000;13:407–414.

Relaxation and Exchange Dynamics of Hyperpolarized

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Relaxation and exchange dynamics of hyperpolarized 129Xe in human blood.

(129) Xe-blood NMR was performed over the full blood oxygenation range to evaluate (129) Xe relaxation and exchange dynamics in human blood...
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