The blood is a suspension of cells, mainly red cells or erythrocytes, dispersed in a ... Erythrocyte suspensions were prepared from whole blood : a test tube of.
Photoacoustic Spectroscopy of Human Blood: Oxidation and Sedimentation Studies P. Poulet, M. Ouzafe, and J. Chambron Institut de Physique Biologique, Faculte de Medecine, 4, rue Kirschleger, F-67085 Strasbourg Cedex, France
1 INTRODUCTION The blood is a suspension of cells, mainly red cells or erythrocytes, dispersed in a fluid, the plasma. Erythrocytes contain the hemoglobin, the protein which assures oxygen transport from the lungs to the tissues and the elimination of carbon dioxide. We demonstrate that the oxygen-hemoglobin interaction can be studied on a red cell suspension using photoacoustic detection. In addition it is possible to measure the sedimentation of red cells in the plasma in a very short time by recording the time-evolution of the photoacoustic signal produced by a whole blood sample. 2 PHOTOACOUSTIC SPECTROSCOPY OF HEMOGLOBIN Erythrocyte suspensions were prepared from whole blood : a test tube of heparinized blood was separated into two parts, one being reduced with sodium dithionite in slight excess. The two samples were then centrifuged and red cell suspensions were pumped in a syringe through the plasma. The photoacoustic cell was purged with nitrogen prior to the study of deoxygenated hemoglobin (Hb) and the sample was introduced anaerobically through a needle across the gas valve of the cell. The study of oxidized hemoglobin (Hb0 2) was realized with the cell filled with air. Photoacoustic spectra of Hb and HbO were analyzed by the RG theory applied to thermally thick samples /1/. 2The absorption spectra calculated from photoacoustic experiments are presented in Fig. 1. They exhibit the Soret band in the near ultraviolet and the Q bands in the visible range. A more precise verification of the validity of the theoretical model used, which neglects scattering effects, has been performed. The photoacoustically measured optical absorption spectra, -~sing 2 ~ 1 thermal diffusivity of the red cell suspension of about 1.37 10 em s , agree well with the absorption spectra calculated from the estimated concentration of hemoglobin in red cells and the molar extinction coefficients of Hb and Hb0 2. A more complete discussion of the above agreement and the use of the RG theory was reported in /2/. It justifies the use of theoretical models neglecting scattering effects for the study of hemoglobin oximetry and blood sedimentation. 3 HEMOGLOBIN OXIMETRY The hemoglobin affinity for oxygen is described by the saturation curve of hemoglobin which shows the ratio Hb0 2/(Hb + Hb0 2) as a function of the partial oxygen pressure P0 2. 538 P. Hess et al. (eds.), Photoacoustic and Photothermal Phenomena © Springer-Verlag Berlin Heidelberg 1988
50,---------------,
400
500
Fig. 1 : Photoacoustic absorption spectra of hemoglobin (---) and oxyhemoglobin ( ).p: optical absorption coefficient, a : thermal diffusivity
600
WAVELENGTH (nm)
A modified photoacoustic cell containing a polarographic electrode enables the simultaneous measurement of PO and of the photoacoustic signal at 470 nm (where the difference between th~ absorption of hemoglobin and oxyhemoglobin is maximum) during a slow increase of the PO from zero (cell purged with 95 % N2, 5 % co,) up to 400 mmHg, by oxygen ~iffusion through a Millipore filter placed between the photoacoustic cell and a second gas volume filled with oxygen and water. This arrangement represents a Helmholtz resonator with a resonance frequency of 150 Hz and a gain of about 3. The recorded photoacoustic signals were then linearized (versus optical absorption coefficient) by using the RG theory applied to a thermally thick sample and the saturation curve was calculated (Fig. 2).
z
100
t-
80
0
60
~
40
~
20
t-
l)
>-
X 0
20
40
60
80
100
Fig. 2 : Saturation curve of adult human hemoglobin (5% CO , 37•c. Signal amplitude as meas~red ( ... ) and 1i neari zed (-).
PARTIAL OXYGEN PRESSURE (mmHg)
The sigmoid shape of the saturation curve is due to the cooperative effects between the four globin chains of the hemoglobin molecule /3/. The effect of saturation on the PA signal shifts the saturation curve to the left. For linearized values, P50, the pressure at which 50% of hemoglobin is oxidized, is about 26 mmHg (standard value 26.6) at 37•c. The Hill number /2,3/, expressing the cooperative effects is about 2.5 (standard value 2.8). 4 BLOOD SEDIMENTATION Photoacoustic experiments suspensions. Measurements cells in plasma /4,5/. The thickness f and diameter 0
reported above were performed on red cell on whole blood exhibit the sedimentation of red sedimentation rate of disk-shaped erythrocytes of is given by /2/ 539
v
e0
=
(p- p 0 )
I 7.65
~
;
(1)
is the plasma density, p the red-cell density. ~ the viscosity. The p tRickness of the plasma layer after a sedimentation time t is equal to vt. The sample can thus be considered as a two-layer stratified sample with an upper non-absorbing layer of plasma and a deeper, absorbing, thermally thick layer containing red cells. The evolution of the photoacoustic signal is given by /2, 6/ - amplitude P(t) = P(o) 0(t) = 0(o)
- phase
(2)
exp(-vt/~s),
(3)
vt/~s
is the thermal diffusion length in plasma.
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The time evolution of the PA amplitude and phase are displayed in Fig. 3 and Fig. 4. Signals were recorded at various frequencies on samples from the same female blood kept on citric acid. 200
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