Determination of the intracellular pH of intact erythrocytes by 1H NMR spectroscopy

ANALYTICAL

121,423-432

BIOCHEMISTRY

Determination

DALLAS Department

(1982)

of the Intracellular pH of Intact Erythrocytes by ‘H NMR Spectroscopy L. RABENSTEIN

of Chemistry,

University Received

A.

AND ANVARHUSEIN of Alberta,

Edmonton,

December

Alberta

ISAB

T6G 2G2,

Canada

18, 198 1

A method is described for determining the intracellular pH of intact erythrocytes by ‘H NMR. The determination is based on the pH dependence of the chemical shifts of resonances for carbon-bonded protons of an indicator molecule (imidazole) in intact cells. The imidazole is introduced into the erythrocytes by incubation in an isotonic saline solution of the indicator. The pH dependence of the chemical shifts of the imidazole resonances is calibrated from ‘H NMR spectra of the imidazole-containing red cell lysates whose pH is varied by the addition of acid or base and measured directly with a pH electrode. To reduce in intensity or eliminate the much more intense envelope of resonances from the hemoglobin, the ‘H NMR measurements are made by either the spin-echo Fourier transform technique or by the transfer-ofsaturation by cross-relaxation method.

High-resolution nuclear magnetic resonance (NMR) is a nondestructive technique with which many delicately balanced cellular processes and parameters can be observed directly (1). For example, intracellular pH (pH’) can be determined from the chemical shifts of NMR-active nuclei in molecules with partially ionized acidic groups (2-10). Measurement of pHi by NMR was first reported by Moon and Richards, who used the pH dependence of the chemical shifts of the 3’P resonances of intracellular inorganic phosphate and 2,3-diphosphoglycerate to determine pH’ of erythrocytes (2). The pH dependence of the chemical shift of the resonance for inorganic phosphate has since been used to measure pHi in. a variety of 3’P NMR studies of intact cellular systems (2-5,7-10). Although 3’P NMR has been used in most NMR studies of intact cells, ‘H NMR has also been used (6,11-14) and can be expected to be used with increasing frequency since almost all intracellular compounds contain hydrogen. In such studies, it is often of interest to monitor simultaneously pHi 423

and the cellular process, e.g., the rate of glucose metabolism. In ‘H NMR studies of human erythrocytes, pH’ can be estimated from the chemical shifts of the hemoglobin histidine resonances (6). However, this measurement is not straightforward because of the additional dependence of the histidine chemical shifts on the oxy-deoxy status of the hemoglobin (15). In this paper, we describe an alternative method for the determination of pH’ of intact erythrocytes by ‘H NMR. The method is based on the pH dependence of the chemical shifts of resonances from an indicator molecule (imidazole) which is introduced into the erythrocytes by incubation. MATERIALS

AND METHODS

Cells. The erythrocytes were obtained from venous blood which was collected in vacutainers (Becton, Dickinson, and Co.) containing EDTA solution. The whole blood was centrifuged at 5000 rpm at 4°C for 15 min, and then in most experiments the packed cells were washed three times by sus0003-2697/82/060423-10$02.00/O Copyright 0 1982 by Academic Press, Inc. All rights of reproduction in any form reserved.

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RABENSTEIN

pension in two times their volume of either a D20 solution or an HZ0 solution containing 0.154 M NaCl and 0.005 M glucose. This procedure with the DzO wash is sufficient to replace most of the intracellular Hz0 with DzO. To introduce the imidazole into the cells, the packed cells were then resuspended in their own volume of either an H20 or D20 solution containing 0.154 M NaCl, 0.005 M glucose and 0.005 M imidazole, the pH of which had been adjusted to -7.4. After incubation for 1 h, intracellular imidazole was readily observable by ‘H NMR. Although the intracellular imidazole concentration was not determined it is estimated to be less than 0.003 M from the intensity of the resonances for the imidazole remaining in the incubation medium. In some experiments, imidazole was added to the whole blood, and after incubation for 1 h the blood was centrifuged and the packed cells examined by ‘H NMR. In some experiments, NMR measurements were made on packed cells as a function of the external pH of the suspension from which they were isolated. The procedure used was to divide the suspension into two parts; to one part HCl was added (DC1 if the cells had been washed and suspended in DzO solution), the pH of the external solution was measured, and portions of the suspension were withdrawn at appropriate pH values. The suspension was centrifuged, and the packed cells transferred to the NMR tube. To the other part, NaOH (or NaOD) solution was added and NMR samples withdrawn as the pH was increased. To convert the hemoglobin of the oxy form, either a suspension of the cells or the hemolysate was bubbled with 5% CO*-95% OZ. Imidazole indicator calibration experiments. To calibrate the pH dependence of

the chemical shifts of the carbon-bonded protons of intracellular imidazole, ‘H NMR measurements were made on hemolysates containing imidazole as a function of pH. The hemolysates were prepared by taking packed cells containing imidazole, prepared

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as described above, through three freezethaw cycles. The hemolysate was then divided into two parts; to one part HCl (or DCl) was added, the pH of the hemolysate was measured, and NMR samples withdrawn. To the other part, NaOH (or NaOD) solution was added and NMR samples were withdrawn as the pH was increased. There was some precipitation as the pH was increased or decreased. Calibration measurements were made at 25 and at 37’C. pH measurements made with the glass electrode on hemolyzed erythrocytes which had been washed with saline-D,0 solution are reported as the meter reading and have not been corrected for deuterium isotope effects. Such readings will be indicated by pH*. Phosphate calibration experiments. To calibrate the pH dependence of the chemical shift of the 3’P resonance of intracellular inorganic phosphate, 3’P NMR measurements were made on hemolysates as a function of pH. The hemolysates were prepared by taking washed, packed cells through three freeze-thaw cycles. The hemolysate was divided into two portions and the pH adjusted as described above. Two calibration curves were prepared, one for cells washed with Hz0 solution and one for cells washed with D20 solution. NMR measurements. ‘H NMR spectra were measured at 400 MHz on a Bruker WH-400/DS spectrometer. Spectra were measured in the pulsed Fourier transform mode using several pulse sequences, as described below. Quandrature detection was used with spectral widths of 5000 Hz. Eight K of data points were generally used for acquisition of the free induction decay, with an acquisition time of 0.8 19 s and a 2-s delay between completion of acquisition and the next pulse. Although 300 transients were generally collected for each spectrum, good spectra could be obtained from 10 transients, reducing the instrument time from 14 min to less than 30 s. In measurements made on cells which had been washed with D20, the

‘H NMR

SPECTROSCOPIC

DETERMINATION

spectrometer was locked to the D,O resonance; in measurements made on cells which had been washed with H20, the spectrometer was operated in the unlocked mode. Chemical shifts are reported relative to the methyl resonance of sodium 2,2-dimethyl-2silapentane-5-sulfonic acid, based on the resonance for the a-CH2 protons of the Gly residue of GSH having a chemical shift of 3.76 ppm. 3’P NMR spectra were measured at 16 1.9 MHz on the Bruker WH-400/DS spectrometer. Chemical shifts were measured relative to HjP04 contained in a coaxial capillary. To extract the ‘H resonances of imidazole from the more intense envelope for the carbon-bonded protons of hemoglobin, two pulse techniques with which the hemoglobin envelope is either reduced in intensity or completely eliminated were used. Most of the measurements were made with the spin-echo Fourier transform technique (90’%- 180”72 acquisition, where 72 represents a delay interval). With this technique, considerable spectrum simplification results due to the rapid spin-spin relaxation rates of the hemoglobin carbon-bonded protons relative to those of the small molecules (6, 16-18). In some measurements, the hemoglobin envelope was suppressed by applying a presaturation pulse at 0.0 or at 8.0 ppm prior to the observation pulse (19). The entire hemoglobin envelope is reduced in intensity because the saturation is selectively transferred by cross-relaxation effects to protons linked by dipole-dipole coupling to those giving the resonances at the frequency of the saturation pulse (20). Glucose metabolism experiments. Erythrocytes were prepared from freshly drawn venous blood by washing once with KrebsRinger (without glucose) in Hz0 (2 1). Then half the cells were incubated in glucose-free Krebs-Ringer containing 0.005 M imidazole (pH -7.4) while the other half of the cells were incubated in glucose-free Krebs-Ringer. After incubation for 1 h, each portion of cells washed with and then resuspended in glu-

OF ERYTHROCYTE

pH

425

case-free Krebs-Ringer at a 4: 1, cells:KrebsRinger ratio. A 0.5-ml aliquot of the suspension was transferred to an NMR tube, 8 ~1 of 0.51 M glucose added, and the time course for lactate production at 37°C measured by monitoring the intensity of the lactate resonance relative to the intensity of the ergothioneine methyl resonance ( 19) as a function of time. Spin-echo spectra were measured using a 72 = 0.060 s; under these conditions the lactate methyl doublet is of a negative sense while the ergothioneine methyl singlet is positive (6). The chemical shifts of the imidazole resonances were also followed for the imidazole-containing erythrocytes. Each spectrum measured in the time course study required 82 s of signal averaging; the times used in plotting the time course data are the midpoints of the acquisition time intervals. RESULTS

The measurement of pHi by NMR is based on the pH dependence of the chemical shifts of resonances from nuclei which are located near partially ionized acidic groups. To be suitable as an ‘H NMR indicator of pHj, the molecule must have the following features: (a) a pK, which is near physiological pH, (b) an ‘H resonance which is resolvable and is due to protons located sufficiently close to the acidic group for good chemical shift sensitivity to pH, (c) the ‘H resonance should be a singlet, or a first-order multiplet if it is spin-coupled to other protons, so as to be easily observed by the spinecho technique (17) (d) the ‘H resonance should have a long spin-spin relaxation time relative to that of Hz0 so that it can be observed at long values of 72 in the spin-echo technique ( 18), and (e) proton exchange involving its acidic groups must be rapid on the NMR time scale so that an exchangeaveraged resonance is observed, the chemical shift of which will be determined by the relative concentrations of the acid and base forms and their respective chemical shifts.

426

RABENSTEIN

To determine if there are ‘H resonances from any compounds naturally present in erythrocytes, other than those from the imidazole groups of the hemoglobin histidine residues, which would be suitable for use as pHi indicators, 400 MHz ‘H NMR spectra were measured for packed erythrocytes which had been washed with isotonic DzO-saline solution and then resuspended in D,O-saline solution whose pH* was varied from 6.4 to 9.2. At pH intervals of approximately 0.5 pH unit, a sample of the suspension was removed, centrifuged, and the packed cells were transferred-to an NMR tube. ‘H NMR spectra were measured by the spin-echo method (17) using r2 values of 0.015 and 0.060 s, and by the transfer-of-saturation technique with the saturation pulse at 8.0 ppm (18). No resonances other than those of the imidazole protons of the hemoglobin histidine residues were found to have the necessary pH dependence. For this reason, we have chosen to use an indicator molecule which is introduced into the erythrocytes by incubation. In addition to the general requirements listed above, for an added molecule to be suitable as a pHi indicator, it must also cross the erythrocyte membrane. Imidazole as a PHi indicator. Figure 1 shows 400 MHz ‘H NMR spectra for packed erythrocytes which had been washed with a D20 solution of isotonic saline, suspended in a D,O solution of isotonic saline containing imidazole at pH* -7.4, and then separated by centrifugation after 1 h of incubation. Spectrum A is that obtained by the single-pulse technique, spectrum B is that obtained by the selective-transfer-ofsaturation method with the saturation pulse set at 0.0 ppm (18), and spectrum C is the spin-echo spectrum obtained with r2 = 0.060 s ( 17). The resonance for the proton on C2 of imidazole (C2-H) is at 8.139 ppm, and that for the protons on C4,5 (C4,5-H) is at 7.245 ppm. It is apparent from spectrum A that it is difficult to observe the imidazole signals with the single-pulse technique, as expected due to the large number of hemoglobin imidazole resonances which are

AND

ISAB

A

...s-?

L-i

>--”

4

B

~

L.J

-A

C

I, 100

I

( SO

I

,--I 6.0

4.0 PPm

/ 2.0

t

/ 0.0

I

FIG. 1. 400 MHz ‘H NMR spectra of packed erythrocytes which had been washed three times in a D1O solution of isotonic saline, suspended in a D20 solution of isotonic saline containing imidazole at pH+ 7.4 for 1 h, and then repacked. Spectrum A was measured by the single-pulse sequence; spectrum B by the selectivetransfer-of-saturation method with the saturation pulse at 0.0 ppm; and spectrum C by the spin-echo method with rz equal to 0.060 s.

also observed in the single-pulse spectrum. However, the imidazole signals are easily observed in spectra B and C.

‘H NMR

SPECTROSCOPIC

DETERMINATION

PH’

8.91

790

580

10

8

6

4

2

0

wm FIG. 2. 400 MHz ‘H NMR spectra of packed erythrocytes containing imidazole as a function of pH* at 25°C. The spectra were measured by the spin-echo method, with TV equal to 0.060 s.

The resonances for C2-H and C4,5-H of imidazole are pH dependent, as shown by the spectra in Fig. 2. These are spin-echo spectra for packed erythrocytes which had been washed with a DzO solution containing imidazole. After incubation for 1 h, the pH* was adjusted and portions of the suspension were removed, centrifuged, and the packed

OF

ERYTHROCYTE

pH

421

cells transferred to the NMR tube. The resonances for C2-H and C4,5-H are in the regions 7.7-8.7 and 7.1-7.5 ppm, respectively, depending on pH*. To confirm that the imidazole resonances in Figs. 1 and 2 are due to intracellular imidazole, the experiments shown in Fig. 3 were performed. Spectra A-D in Fig. 3 are for imidazole in D,O saline solution to which was added the Cu*+ complex of cyclohexanediaminetetraacetic acid (CyDTA).’ The Cu(CyDTA)*- complex is paramagnetic and, as spectra A-D demonstrate, it causes the imidazole resonances to broaden and disappear, presumably through ion pair interaction with the positively charged imidazole. Spectra E-G were obtained from an experiment in which erythrocytes were incubated in a saline-D,0 solution containing imidazole, washed and resuspended in salineD20 solution, and then Cu(CyDTA)‘complex was added to the suspension. Since the Cu (CyDTA)*- complex does not cross the erythrocyte membrane (22), the lack of any broadening of the imidazole resonances indicates the imidazole is in the intracellular region. Spectra H-K were obtained from a similar experiment except that the erythrocytes were suspended in a phosphate-containing solution to make the extracellular pH different from pHi. Under these conditions, the resonances from intracellular and extracellular imidazole have different chemical shifts (spectrum H). As CU(C~DTA)~is added, only one set of resonances, those due to extracellular imidazole, broaden and disappear. Calibration of the pH dependence of the chemical shifts of imidazole. The pH de-

pendence of the chemical shifts of the intracellular imidazole was calibrated by measuring the chemical shifts of imidazole in hemolysates whose pH had been measured directly with a glass electrode. Calibration data for imidazole in hemolysates which had been washed with D20 solution are shown ’ Abbreviation tetraacetic acid.

used:

CyDTA,

cyclohexanediamine-

428

RABENSTEIN

AND ISAB

I

8.0

7.0

8.5

7.5

8.5

L

I

,

,

,

,

,

,

,

7.5

PPm

FIG. 3. The imidazole region of the 400 MHz ‘H NMR spectra of: (A-D) 0.005 M imidazole in salineglucose solution containing (A) 0.0, (B) 6.0 X IO-‘, (C) 2 X 10m4,and (D) 3.9 X lo-“ M Cu (CyDTA)*-; (E-G) erythrocytes which had been incubated in 0.005 M imidazole-containing isotonic saline-glucose solution and then resuspended in isotonic saline-glucose solution (4 parts cells: I part medium) containing (E) 0.0, (F) I.4 X 10W4,and (G) 2.8 X 10m4M Cu(CyDTA)‘-; and (H-K) erythrocytes which had been incubated in 0.005 M imidazole-containing isotonic saline-glucose solution and then resuspended in isotonic saline-glucose-Na2HPOd solution containing (H) 0.0, (I) 4.2 X IO-‘, (J) 8.3 X 10m5,and (K) 1.6 X 1O-4 M Cu(CyDTA)‘-.

in Fig. 4. Data from two separate experiments are shown. Similar calibration data were obtained for imidazole in hemolysates of erythrocytes in which Hz0 was the solvent. pHi can be determined from the observed chemical shift by using calibration curves of the type shown in Fig. 4. An alternative procedure, which is more precise, is based on the relationship between the observed chemical shift and the pH,

,

t11

where pK, is for imidazole in the particular medium, a0 is the measured chemical shift of either the C2-H or C4,5-H of imidazole in the test sample, and 8nr and 6, are the chemical shifts of C2-H or C4,5-N of the protonated and deprotonated forms of imidazole. The constants in Eq. [ 1] (pK,, dHl, and 6r) were obtained from the calibration

‘H NMR SPECTROSCOPIC I

DETERMINATION

I

6.0

t-

70 PH.

80

90

FIG. 4. Chemical shift vs pH calibration curves for the C2-H and C4,5-H ‘H resonances of imidazole in hemolysates. The results from two separate experiments are shown. In the two experiments, the imidazole was introduced into the erythrocytes by suspension in isotonic solutions containing 0.005 M imidazole (solid circles) and 0.010 M imidazole (solid triangles). The curves through the points are theoretical curves calculated with the parameters listed in Table 1, 25°C.

data by nonlinear least-squares curve fitting, and are listed in Table 1. Uncertainties in the form of linear estimates of the standard TABLE CALIBRATIONPARAMETERSFORTHE~H

Medium

OF ERYTHROCYTE

pH

429

deviation were also calculated for each parameter by the nonlinear least-squares curvefitting program (23). The averages of the standard deviations for the pK, values and the limiting chemical shifts were 0.02 and 0.008 ppm, respectively. Accuracy of the method. The accuracy of pHi values obtained by this method depends on several factors, including the accuracy of the calibration data and the accuracy of the chemical shift measurements. Results obtained in this work indicate that, using the instrument parameters and calibration data described above, the pHi of erythrocytes after they have been incubated with imidazole can be measured to an accuracy of kO.02 pH unit. This estimate of the accuracy level was arrived at in two ways: (i) by comparison of the pHi obtained from ‘HNMR measurements on packed erythrocytes and from glass electrode measurements on the same samples after being hemolyzed and (ii) by comparison of the pHT values obtained for the same sample of imidazole-containing erythrocytes by ‘H NMR and the well-established 3’P NMR method (2,9,10). The procedure used for the measurement of pHT by both ‘H and 3’P NMR involved first washing the cells with a saline-glucose solution in D,O and then oxygenating the cells. The 3’P spectrum of a portion of the 1

DEPENDENCEOFIMIDAZOLECHEMICALSHIFTS'

Temperature (“C)

Indicator proton(s)

6

4

PK.

(P?%

(mm)

C2-H C4,5-H

7.2@

8.681 7.474

1.766

7.17b

Lysate of erythrocytes washed with saline-D20

25

Lysate of erythrocytes washed with saline-H20

25

C2-H C4,5-H

7.08 7.00

8.636 7.457

7.761 7.106

Lysate of erythrocytes washed with saline-D20

31

C2-H C4,5-H

7.10 7.06

8.682 1.477

7.157 7.107

Lysate of erythrocytes washed with saline-H20

37

C2-H C4,5-H

7.01

8.638 7.451

1.760

6.99

7.113

7.104

a Obtained from nonlinear least-squares curve fitting of pH vs chemical shift data to Eq. [ 11. b See text for a discussion of the different pK, values obtained from the different indicator proton resonances.

430

RABENSTEIN

packed cells was then measured. A second portion was incubated in isotonic saline-glucase-D,O solution containing 3 rnM imidazole for 1 h, after which the suspension was centrifuged and both 3’P and ‘H NMR spectra measured on the packed cells. The same pH7 (7.43 t- 0.02) was obtained by 3’P NMR for packed erythrocytes with and without imidazole, and the same pH: (7.40 + 0.02) was obtained by 3’P NMR and ‘H NMR for the imidazole-containing erythrocytes. Although the purpose of the present study is to develop an ‘H NMR technique with which pHi can be measured while simultaneously monitoring cellular processes, and not the development of a technique for the routine measurement of pHi, measurements have been made to establish the value of pHi (HZ0 medium) as determined by this method. Erythrocytes were washed with an HZ0 solution of isotonic saline-glucose, and then oxygenated while suspended for from 30 to 60 min in a pH 7.4 H,O solution of isotonic saline-glucose-O.005 M imidazole. An average pHi value of 7.29 +- 0.07 was found from the intracellular imidazole chemical shifts. This compares favorably with values determined by other methods (9,24,25). pHi was also measured by this method as a function of the extracellular pH, over the pH, range of 7.0 to 7.7. The measured pHi values agree within kO.05 pH unit with those predicted from the pH, values using the relation given by Bromberg et al. (26). DISCUSSION

A variety of methods have been described for the measurement of intracellular pH, including microelectrodes inserted directly into cells, the distribution of weak acids, bases, or visible dyes across the cell membrane, measurements on homogenates, NMR chemical shift measurements, and the rates of pH-dependent intracellular reactions (24,25). The methods most often used

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to measure pHi of erythrocytes are the distribution of weak acids or bases and measurements on hemolysates with glass electrodes. The NMR method provides a direct measurement of the intracellular pH; the method is based on chemical shifts of resonances from molecules that are observed directly in the intracellular region of intact cells. The chemical shift measurement can be made with high precision, and by choosing an indicator molecule having the proper pK, and NMR characteristics, the chemical shift will provide a sensitive measure of pHi. The method also should be highly accurate, provided the pH dependence of the indicator chemical shifts is carefully calibrated using a solution whose composition is identical to that within the cells. The results indicate an accuracy of at least +0.02 pH unit with imidazole as the indicator. In principle, a calibration curve could be prepared knowing just the p& of the indicator molecule in the intracellular medium and the chemical shifts of its protonated and deprotonated forms. This method has the disadvantage, however, that errors will result if there are any interactions between the indicator molecules and the contents of the cells which might affect the chemical shift. In this work, this source of error has been avoided by preparing a calibration curve directly from experimental data by measuring the chemical shifts of the indicator molecules in hemolysates as a function of pH. The calibration data also provide information about interactions involving the indicator. For example, we find that the chemical shift-pH titration curves for imidazole both in hemolysates and in isotonic saline solutions can be fit by a single acid-base equilibrium. However, different pK, values are obtained from the two different resonances for imidazole in hemolyzed erythrocytes at 25°C (Table l), suggesting that there is some additional interaction which involves imidazole and other cellular constituents, and that the interaction affects the

‘H NMR

SPECTROSCOPIC

0 --

40

DETERMINATION

60

Time (min)

FIG. 5. Time courses for the production of lactate after the addition of glucose to Krebs-Ringer suspensions of erythrocytes which had been incubated in glucose-free Krebs-Ringer (0) and glucose-free KrebsRinger containing 5 mM imidazole (pH 7.4) (0).

two resonances differently. Because of this, the pK,, values in Table 1 must be regarded as calibration constants and not acid-dissociation constants. This effect is much less at 37°C (Table 1). The curves through the points in Fig. 4 are theoretical curves calculated assuming a single equilibrium at 25°C with the parameters in Table 1. There are no literature values available for comparison with the calibration constants in Table 1, however, pK, values measured by NMR for imidazole in isotonic-H,0 solution can be compared to literature values measured under similar conditions. The NMR method yields pK, values of 7.13 (C2-H) and 7.12 (C4,5-H) at 25°C and 6.91 (C2-H and C4,5-H) at 37°C. For comparison, Tanford and Wagner report values of 7.12 (25°C) and 6.93 (37°C) for an ionic strength of 0.15 M (27). The ‘H NMR method for measuring erythrocyte pHi was developed SO that pHi and cellular processes can be monitored simultaneously. The ‘H NMR spectrum is measured by the pulsed Fourier transform

OF ERYTHROCYTE

pH

431

technique, which gives simultaneously the entire spectrum. Thus, the indicator resonances are obtained at the same time as resonances from other small molecules in the cell, making it possible to continuously measure pHi while simultaneously monitoring other resonances. For example, the rate of lactate production can be followed by measuring the intensity of the methyl resonance at 1.28 ppm for lactic acid as a function of time (6). By adding imidazole to the cells, pHi can also be measured simultaneously. We find that the added imidazole has little affect on the rate of lactate production, as illustrated by the results in Fig. 5. In Fig. 5, the ratio of the height of the lactate resonance at 1.28 ppm (11) to the height of the ergothioneine methyl resonance at 3.25 ppm (el) ( 18), is plotted as a function of time. It is of interest to note that, in the experiments shown in Fig. 5, pHi was found to decrease by -0.06 pH unit. ACKNOWLEDGMENTS We express our appreciation to the Hematology Division of the Department of Laboratory Medicine, University of Alberta Hospital, for providing the blood samples. This research was supported by an operating grant to D.L.R. from the Natural Sciences and Engineering Research Council and a Postdoctoral Fellowship to A.A.I. from the Alberta Heritage Foundation for Medical Research.

REFERENCES 1. Radda, G. K., and Seeley, P. J. (1979) Annu. Rev. Physiol. 41, 749-769. 2. Moon, R. B., and Richards, J. H. (1973) J. Biol. Chem. 248, 7276-7278. 3. Hoult, D. I., Busby, S. J. W., Gadian, D. G., Radda, G. K., Richards, R. E., and Seeley, P. J. (1974) Nature (London) 252, 285-287. 4. Salhany, J. M., Yamane, T., Shulman, R. G., and Ogawa, S. (I 975) Proc. Nat. Acad. Sci. USA 72, 49664970. 5. Burt, C. T., Glonek, T., and Barany, M. (1976) J. Biol. Chem. 251, 2584-2591. 6. Brown, F. F., Campbell, I. D., Kuchel, P. W., and Rabenstein, D. L. ( 1977) FEBS Left. 82, 12-16. 7. Ogawa, S., Shulman, R. G., Glynn, P., Yamane, T., and Navon, G. (1978) Biochem. Biophys. Acta 502, 45-50.

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8. Pollard, H. B., Shindo, H., Cruetz, C. E., Pazoles, C. J., and Cohen, J. S. (1979) J. Viol. C/rem. 254, 1170-l 177. 9. Lam, Y.-F., Lin, A., and Ho, C. (1979) Blood 54, 196-209. 10. Hollis, D. P. (1980) in Biological Magnetic Resonance (Berliner, L. J., and Reuben, J., eds.), Vol. 2, pp. l-44, Plenum, New York. Il. Brindle, K. M., Brown, F. F., Campbell, I. D., Grathwohl, C., and Kuchel, P. (1979) Biochem. J. 180, 37-44. 12. Isab, A. A., and Rabenstein, D. L. ( 1979) FEBS Lett. 106, 325-329. 13. Rabenstein, D. L., and Isab, A. A. (1980) FEBS Lett. 121, 61-64. 14. Rabenstein, D. L., Backs, S. J., and Isab, A. A. (198 1) J. Amer. Chem. Sot. 103, 2836-2841. IS. Brown, F. F., and Campbell, 1. D. ( 1976) FEBS Lett. 65, 322-326. 16. Rabenstein, D. L. (1978) Anal. Chem. 50, 1265A1276A. 17. Rabenstein, D. L., and Nakashima, T. T. (1979) Anal. Chem. 51, 1465A-1474A.

AND ISAB 18. Rabenstein. D. L., and lsab, A. A. (1979) J. Mug. Rex 36, 28 l-286. 19. Rabenstein, D. L., Isab, A. A., and Brown, D. W. (1980) J. Mug. Rex 41, 361-365. 20. Akasaka, K., Konrad, M., and Goody, R. S. (1978) FEBS Lett. 96, 287-290. 21. Krebs, H. A., and Henseleit, K. (1932) 2. Physiol. Chem. 210, 33-37. 22. Rabenstein, D. L., and Isab, A. A., unpublished results. 23. Dye, J. L., and Nicely, V. A. (197 I ) J. Chem. Ed. 48,433-448. 24. Waddell, W. J., and Bates, R. G. (1969) Physiol. Rev. 49, 285-329. 25. Cohen, R. D., and Iles, R. A. (1975) Crit. Rev. Clin. Lab. Sci. 6, IOl- 143. 26. Bromberg, P. A., Theodore, J., Robin, E. D., and Jensen, W. N. (1965) J. Lab. Clin. Med. 66, 464-475. 27. Tanford, C., and Wagner, M. L. (1953) J. Amer. Chem. Sot. 75, 434-435.