tures as those for plasma (see the 500MHz Ή NMR spectrum for packed red blood cells in Figure 11). The large water resonance is superimposed on a broad envelope of resonances, in this case from the protons of hemoglobin and the red cell membrane. The tech niques discussed above can be used to selectively eliminate the interfering resonances and obtain high-resolution Ή NMR spectra for the small intracel lular compounds (6, 9). To illustrate, a 500-MHz spin-echo X H NMR spectrum of intact red blood cells is shown in Figure 12. This spec trum was measured with a spin-spin relaxation period of 0.134 s. The hemo globin and membrane resonances are eliminated, and the water resonance is significantly reduced in intensity. By
increasing the length of the spin-spin relaxation period, the water resonance can be further reduced in intensity (6, 28). Most of the resonances in the spinecho spectrum have been assigned to small intracellular compounds; the as signments are given in Figure 4. The spin-echo method has been used ex tensively in Ή NMR studies of chemi cal and metabolic processes in intact red blood cells. Figure 13 shows a 500-MHz CPMG X H NMR spectrum of intact red blood cells. This spectrum, which is for a dif ferent sample of red blood cells, was measured with a longer spin-spin re laxation period (0.27 s) to completely eliminate the water resonance. Com pared with the spin-echo spectrum in Figure 12, the resonances in this spec-
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Figure 11. The 500-MHz 1H NMR spectrum of human red blood cells. The spectrum was measured by the single-pulse method using a flip angle of ~ 5 ° . Sample preparation involved separating the red blood cells from plasma and white cells by centrifugation.
Figure 12. A portion of the 500-MHz spin-echo 1H NMR spectrum of intact red blood cells. The spectrum was measured with pulse sequence C in Figure 3, using r = 0.067 s; 128 transients were co-added. The red blood cells used in the measurement of this spectrum were washed once with an equal volume of isotonic saline solution after separation from the plasma. To obtain good resolution, the blood was bubbled with oxygen to ensure that the hemoglobin was in the diamagnetic oxygenated form.
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ANALYTICAL CHEMISTRY, VOL. 60, NO. 24, DECEMBER 15, 1988 · 1387 A
