Anal. Chem. 1999, 71, 3887-3893
In Situ Fiber-Optic Oxygen Consumption Measurements from a Working Mouse Heart Yadong Zhao,† Angela Richman,‡ Charles Storey,‡ Nina B. Radford,‡ and Paul Pantano†,*
Department of Chemistry, The University of Texas at Dallas, Richardson, Texas 75083, and Department of Internal Medicine and Radiology, The Mary Nell and Ralph B. Rogers Magnetic Resonance Center, The University of Texas Southwestern Medical Center, Dallas, Texas 75235
Luminescence-based imaging-fiber oxygen sensors (IFOSs) were utilized for the in situ measurement of oxygen consumption from intact perfused mouse hearts. IFOSs were fabricated using a technically expedient, photoinitiated polymerization reaction whereby an oxygensensitive polymer matrix was immobilized in a precise location on an imaging fiber’s distal face. The oxygensensing layer used in this work comprised a transition metal complex, Ru(Ph2phen)32+, entrapped in a gaspermeablephotopolymerizablesiloxanemembrane(PS802). The transduction mechanism was based upon the oxygen collisional quenching of the ruthenium complex luminescence; detection was performed utilizing an epi-fluorescence microscope/charge coupled device imaging system. IFOS measurements from working mouse hearts were validated through concurrent, blind, ex situ blood gas analyzer (BGA) measurements. The BGA and IFOS methodologies were utilized successfully to measure oxygen concentrations in aortic and pulmonary artery perfusates from the working mouse heart before and after isoproterenol administration. Coupled with coronary-flow measurements, these data were used to calculate myocardial oxygen consumption. Regression analysis of measurements of myocardial oxygen consumption showed that there was a strong correlation between the values generated by the BGA sampling and those obtained via in situ IFOS methods. To our knowledge, this research represents the first report of in situ fiber-optic sensor monitoring of oxygen content from the intact, beating mouse heart. In clinical and biological settings, arterial blood oxygen content analysis is the only reliable means to confirm that a proper oxygen supply is being delivered to tissue.1 For example, cardiac tissue relies almost exclusively on the oxidation of substrates for energy generation. Therefore, the determination of myocardial oxygen consumption provides a measure of the heart’s total metabolism. Myocardial oxygen consumption measurements can also be used as a physiological tool to monitor the hemodynamic status of the heart in that it is influenced by a number of parameters including wall stress, contractility, and heart rate.2 * To whom correspondence should be addressed; (email)
[email protected]. † The University of Texas at Dallas. ‡ The University of Texas Southwestern Medical Center. (1) Collison, M. E.; Meyerhoff, M. E. Anal. Chem. 1990, 62, 425A-37A. 10.1021/ac9903003 CCC: $18.00 Published on Web 08/03/1999
© 1999 American Chemical Society
Myocardial oxygen consumption calculations require the measurement of three critical variables: arterial O2 content, venous O2 content, and coronary flow rate.3 Traditionally, the first two variables are measured in vitro or in vivo in the intact, beating heart by withdrawing one sample of blood or perfusate from the pulmonary artery and another sample from arterial circulation. Oxygen partial pressure in these samples is determined using an off-line blood gas analyzer (BGA). Most laboratory BGAs employ an amperometric Clark type electrode to determine oxygen partial pressure.1 Although Clark type electrodes are moderately rapid in response, simple to use, and relatively inexpensive, they have some limitations. Clark type electrodes consume oxygen, require a steady-state oxygen supply, and are easily poisoned by H2S, proteins, and various organic compounds.4 For biological in vivo or in vitro applications, particularly in rodent hearts, serial determinations of myocardial oxygen consumption are problematic because a significant amount of perfusing medium is required cumulatively for Clark type electrode sampling. In addition, obtaining samples without room-air contamination from the very small (20 ft) imaging fiber length employed. The long fiber length was required to accommodate future remote measurements from perfused rodent hearts located inside the bore of a NMR spectrometer. Figure 2-top shows an IFOS luminescence image immediately after the IFOS was fabricated. The luminescence intensity was very heterogeneous, that is, the central portion of the sensing area showed relatively weak luminescence intensities relative to the edges. This is a very interesting phenomenon since the polymer matrix had a hemispheric shape (Figure 1) such that the radiation path length transversing the sensing layer was greatest in the central portion of the imaging fiber. In addition, the excitation radiation profile of the epi-fluorescence microscope imaging system employing E-Plan microscope objectives is most intense in the central area. Assuming dye was loaded uniformly in the polymer matrix, the IFOS luminescence image should have displayed higher intensities in the central region since there is more polymer in the center of the imaging fiber. This is the exact opposite of what was observed with a newly fabricated IFOS (Figure 2, top). Fortunately, it was discovered that storing the IFOS in water for >1 week afforded a more uniform IFOS luminescence profile (Figure 2, bottom), and an ∼3-fold improvement in the I0/I ratio. Nonlinear Stern-Volmer responses are normal for most luminescence-based fiber-optic oxygen sensors. In this work, all IFOS calibration curves (Figure 4) were fitted using the following nonlinear Stern-Volmer equation:
I0/I ) 1/{f01/(1 + Ksv1PO2) + f02/(Ksv2PO2)} Here, I0 is the IFOS’s luminescence intensity in the absence of oxygen, I is the IFOS’s luminescence intensity at a given dissolved oxygen concentration, PO2 is the oxygen partial pressure, the f0i’s are the fraction of the total emission from each component under unquenched conditions, and the Ksvi’s are the associated SternVolmer quenching constants for each component. In this twosite model, it is assumed that the sensing dye molecules can exist in two distinctly different sites (e.g., internal- or peripheralimmobilized polymer regions), with each site having its own characteristic Stern-Volmer quenching constant.34,36,41-44 Analytical Chemistry, Vol. 71, No. 17, September 1, 1999
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Figure 5. Representative IFOS time response. An IFOS (∼175 µm sensing layer thickness) was exposed to room-temperature oxygenpurged, nitrogen-purged, and air-saturated KH buffers. For a given IFOS, the same image ROI was utilized for all measurements, and background-subtracted luminescence images were acquired at 20-s intervals.
IFOS stability was examined by testing IFOS shelf lifetime, dye leaching, and photobleaching. The IFOS shelf life was examined by recalibrating IFOSs over a 32-day period (Figure 4). It can be seen that the response was very stable over this time period (supported by n > 10 IFOSs). While the shelf life experiments indicated that dye leaching was not significant, separate leaching experiments were performed to support these results (data not shown). In a typical IFOS photobleaching experiment, the IFOS was immersed in KH buffer, the excitation radiation was directed continuously through the IFOS for 20 min, and background-subtracted IFOS luminescence intensities were measured at 1-min intervals. The total luminescence intensity drop due to photobleaching was less than 2% over a 5-min interval (supported by n > 5 IFOSs, data not shown). This level of photobleaching was acceptable since >80 images could be acquired during this excitation-exposure period, and this number of images was much greater than the minimum number of measurements required for one set of mouse heart oxygen concentration measurements (including pre- and post-calibrations). IFOS Temporal Response. A typical IFOS time response curve is presented in Figure 5. The IFOS was exposed sequentially to air-, nitrogen-, and oxygen-saturated KH buffers (supported by n ) 4 IFOSs). When the gas phase was changed from pure nitrogen to pure oxygen, the 90% response time (t90), defined as the time for the luminescence intensity to reach 90% of the steadystate response, was e20 s (note that 20 s was the time interval between 3-s-long CCD acquisitions). However, the IFOS response time going from pure oxygen to pure nitrogen was quite slow (t90 > 2 min). In other words, the IFOS recovery from high oxygen concentrations was slow, and the IFOS oxygen quenching response was fast. This may be caused by different diffusion rates and different solubilities of oxygen and nitrogen in the KH buffer and in the polymer matrix; in general, the permeability coefficient for oxygen is 2-5 times greater than that for nitrogen in dimethylsiloxane-type polymers.45 Slow temporal responses can (42) Turro, N. J. Modern Molecular Photochemistry; Benjamin Cummings: Menlo Park, 1978. (43) Hartmann, P.; Leiner, M. J. P.; Lippitsh, M. E. Sens. Actuators, B 1995, 29, 251-57. (44) Sacksteder, L.; Demas, J. N.; DeGraff, B. A. Anal. Chem. 1993, 65, 348083.
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Figure 6. Oxygen consumption data acquired from the BGA and IFOS techniques for seven mouse hearts before and after isoproterenol (iso) administration. Table 1. Hemodynamic Variables (Mean ( SD)a in the Working Mouse Heart before and after Isoproterenol Administration parameter
baseline
after isoproterenol
heart rate (bpm) coronary flow (mL/min) rate pressure product (mmHg‚bpm) myocardial O2 consumption (BGA) (µmol/min) myocardial O2 consumption (IFOS) (µmol/min)
362 ( 56 3.1 ( 0.1 21 146 ( 3878
498 ( 42b 3.4 ( 0.4c 29 652 ( 3372b
1.45 ( 0.3
2.06 ( 0.6c
1.45 ( 0.3
2.07 ( 0.6c
a Standard deviations were calculated from n ) 7 hearts; two BGA and two IFOS measurements were acquired for each baseline and isoproterenol sample. b p < 0.0001. c p < 0.001.
also be attributed to the thick IFOS polymer matrix (to 175 µm thick). While thin (
