METHOD AND APPARATUS FOR OBSERVING RATES OF REACTION OF OXYGEN IN LIVING TISSUES

• US 3,830,222 A
• Filed: 07/07/1972
• Issued: 08/20/1974
• Est. Priority Date: 07/07/1972
• Status: Expired due to Term

• Current Assignee: Britton Chance
• Original Assignee: Britton Chance
• Inventors: Chance, Britton
• Primary Examiner(s): Howell, Kyle L.
• Application Number:
  US05/269,580
• Time in Patent Office: 774 Days
• Field of Search: 128/2A,2R,2.1R,2.1Z 424/9 356/39
• US Class Current: 600/334
• CPC Class Codes: G01N 21/631 using photolysis and investigating photolysed fragments

Abstract

A system for providing flash photolysis activation of COinhibited cytochrome oxidase in living tissue in the presence of oxygen. In a typical procedure employing cardiac tissue this initiates oxidation of reduced pyridine nucleotide (PN) and flavoprotein (Fp), with a high rate of response. The fractional extent of the photolysis response of PN and Fp indicates the fraction of the total mitochondrial population containing cytochrome a3CO to which oxygen has diffused at the time of the photolysis flash, thereby providing an indication of the effectiveness of oxygen diffusion in the tissue without destruction of the tissue. A double flash is used to evaluate the extent of photolysis, one flash occurring a few seconds after perfusing the tissue with oxygen, followed by another flash a few seconds later. The readout is obtained on a storage oscilloscope, using a double beam, time-shared photometer assembly with a compensating photomultiplier. The flash lamps are triggered by a pulse from the compensating photomultiplier, with a delay to fire the flash lamps at an appropriate phase angle.

Claims

2. The apparatus of claim 1, and wherein the photometer device comprises a fluorometer including means to excite the tissue sample with radiant energy and means to measure the fluorescence of the tissue caused by said last-named radiant energy.

3. The apparatus of claim 1, and wherein the photometer device comprises a time-sharing system delivering time-spaced excitation beams of different wavelength to the sample, generating corresponding time-spaced optical responses of the tissue sample, whereby responses to photolysis of a plurality of components in the tissue sample may be sequentially measured.

4. The apparatus of claim 1, and wherein the photometer device comprises a time-sharing cyclic fluorometer including means to excite the tissue sample with time-spaced excitation beams of different wavelength and means to measure corresponding time-spaced fluorescence responses of the tissue sample, whereby the responses of a plurality of components in the tissue sample may be simultaneously measured.

5. The apparatus of claim 1, and means to excite the tissue sample with a second pulse of photolyzing radiant energy subsequent to the first pulse for evaluating the extent of photolysis.

6. The apparatus of claim 1, wherein the anoxygenating material includes carbon monoxide.

7. The apparatus of claim 1, and wherein the photometer device comprises a time-sharing system delivering time-spaced excitation beams to the sample, generating corresponding time-spaced optical responses of the tissue sample, said means to excite the sample with the pulse of photolyzing radiant energy comprising a flash lamp means adapted to be located adjacent the sample, and circuit means to momentarily energize the flash lamp means in the interval between a pair of said time-spaced excitation beams.

8. The apparatus of claim 7, and wherein the photometer device includes means to generate a noise-compensating electrical pulse coincidentally with each excitation beam, said circuit means including means to energize said flash lamp means after a predetermined time interval following a noise-compensating electrical pulse.

9. The apparatus of claim 29, and a second photolysis flash lamp means adapted to be located adjacent the sample, and wherein said circuit means includes means to momentarily energize the second flash lamp means after a second predetermined time interval following the momentary energization of the first-named flash lamp means, for evaluating the extent of photolysis produced by the first-named flash lamp means.

10. A method of determining the effectiveness of oxygen diffusion in intact animal tissue comprising the steps of perfusing a sample of the intact tissue with an anoxygenating material which inhibits oxidation of the tissue sample, then perfusing the sample with oxygen to initiate oxygenation thereof, continuously measuring the progress of oxidation with time, and then rapidly terminating the inhibiting effect sHortly after oxygenation has commenced by illuminating the sample with a photolyzing flash of radiant energy of sufficient intensity to destory the inhibiting effect to thereby allow the sample to rapidly react with the oxygen, whereby to obtain a readout in accordance with the extent to which the oxygen has diffused in the tissue sample at the time of the photolyzing flash.

11. The method of claim 10, and wherein the oxidation-inhibiting material is carbon monoxide.

12. The method of claim 10, and illuminating the sample with a second photolyzing radiant energy flash shortly after the first flash, for evaluating the extent of photolysis.

13. The method of claim 12, and wherein the first photolyzing flash is applied approximately 1.5 seconds after the start of the perfusion of the sample with oxygen and the second photolyzing flash is applied between 1 and 2 seconds after the first flash.

14. The method of claim 10, and wherein the photolyzing flash is applied approximately 1.5 seconds after the start of the perfusion of the sample with oxygen.