Proc. Natl. Acad. Sci. USAVol. 87, pp. 583-587, January 1990Biophysics

3H nuclear magnetic resonance study of anaerobic glycolysis inpacked erythrocytesRICHARD D. NEWMARK*t, SUN UNt, PHILIP G. WILLIAMS4§, PAUL J. CARSONf, HIROMI MORIMOTOt§,AND MELVIN P. KLEIN:*Research Medicine and Radiation Biophysics Division, tChemical Biodynamics Division, and tNational Tritium Labeling Facility, Lawrence BerkeleyLaboratory, Berkeley, CA 94720

Communicated by Melvin Calvin, October 16, 1989 (received for review May 3, 1989)

ABSTRACT The utility and power of 3H NMR spectros-copy as a technique for monitoring biological systems in vivo isillustrated with glucose metabolism in erythrocytes. Use ofC-1-tritiated glucose allowed us to monitor the disappearanceof the a and P tritons, with the production of lactate and'H3HO (HTO), as well as some intermediates. Spin latticerelaxation times (T,) were measured to avoid T, distortion ofthe spectral intensities. Detection of the formation of 1 mMtritiated water in the presence of 110 M H20 protons anddeuterons allows the eventual fate of the label in the pentoseshunt to be observed in vivo.

3HNMR Spectroscopy. In these and earlier studies using raterythrocytes (1-3) we have found 3H NMR spectroscopy tobe a powerful technique for the study of metabolic pathwaysin vivo. The results that will be presented show that 3H hasmany advantages as an isotopic label and that NMR spec-troscopy is able to follow the progress of this label throughmetabolic intermediates.Most isotopic studies of complex metabolic pathways rely

on radioisotope counting methods that trade specificity forsensitivity-i.e., they measure total isotope concentration atsome stage of an experiment. 3H is increasingly popular as aradiolabel due to its high specific activity (4)-i.e., 461 timesthat of 14C. This means tritiated substrates may be detectedat much lower chemical concentrations than 14C-labeledradiochemicals, and the half-life of tritium is sufficiently longso as not to interfere with the study at hand.The nuclei most widely studied by NMR spectroscopy are

'H, 13C, and 31p, with 19F, 15N, and a number of quadrupolarnuclei such as 2H, 23Na, and 39K utilized to a lesser extent (5).In comparison with radioisotope methods, the NMR exper-iment maintains a high level of molecular specificity, but thisstrength is mitigated by the relative lack of sensitivity of thetechnique. Very few nuclei are suitable as isotopic tracers inbiological NMR spectroscopy: 13C and 2H are the mostcommonly used.

Tritium is both an NMR and a radioactive tracer. As anNMR nucleus 3H has all of the advantages of 1H: spin 1/2, highgyromagnetic ratio (y), and similar chemical shifts and cou-plings (4, 6). The absence of naturally occurring 3H (<10-16%of H isotopes) avoids confusion with regard to possiblesources of the NMR signal, and with an appropriate choice ofthe labeling site the 3H atom is not subject to easy exchange.The analysis of 3H spectra is very similar to standard 1HNMR spectroscopy, and it is simplified both by the largebody of 'H chemical shift information that is available in theliterature and by the intrinsic simplicity of the spectra that areobtained.The low sensitivity of NMR spectroscopy requires that

large quantities of 3H are necessary for NMR experiments

compared to biochemical tracer studies using radioisotopecounting methods. Typically, 4 MBq (37 MBq = 1 mCi) perresonance line is the minimum required for reasonable de-tection at 7 tesla (T). For a typical metabolic study using a1.5-ml sample in a 10-mm tube, the total radioactivity in thesample can amount to several GBq (100 mCi). However,tritium is an 18 ke V 3- emitter, and this soft radiation iseasily contained. The care and handling of tritium for NMRis well documented, and established procedures are routinelyused by several groups (7, 8). For the experiments discussedin this paper the only special precaution used was the additionof a commercially available Teflon insert for the standardglass sample tube to protect against spillage should the glasstube break. A slight disadvantage of this safety precautionwas a degradation of the minimum line width that wasobtainable, from 0.2 to 1-2 Hz.Although 3H has been utilized extensively as a radioiso-

topic tracer, it has received relatively little use as an NMRlabel in biological systems, presumably due to its radioac-tivity. However, there are a number of instances wheretritiated products have been isolated from a biological reac-tion and analyzed by 3H NMR spectroscopy (9-16). Exam-ples of this type of study include the stereochemistry ofproton elimination in the biosynthesis of tritiated cy-cloartenol (11) and the behavior of enzyme-substrate com-plexes involving the tritiated enzyme porphobilinogen deam-inase (15). We now describe 3H NMR experiments on a livingsystem.

Glycolysis. Glycolysis has served as the prototype formulti-enzyme metabolic pathways for several decades. Al-though these pathways are discussed as ifeach step were wellunderstood, many of the individual steps remain controver-sial (17). The regulation of an individual metabolic step canhave a profound influence on the difference between healthyand diseased states, yet nearly all of the basic and contem-porary work on metabolic pathways is based on in vitro orindirect studies.Mammalian erythrocytes provide an ideal system in which

to study the fate of a nuclear spin label in metabolic pathways,and glycolysis has been well characterized in vitro. Some 90%of glucose is utilized in the Embden-Meyerhoff pathway,while the remaining 10% of the glucose flux is thought to bethrough the pentose phosphate shunt (Fig. 1). NMR spectros-copy has been used to study in vivo glycolysis in severaldifferent systems (17-21), including erythrocytes. Isolatedenzyme and in vivo studies have relied on the use of 13C labelsand naturally abundant protons (22) as probes of the metabolicpathway. None of these studies have been able to monitordirectly the fate of a label associated with the pentose shunt.

Abbreviations: HTO, 'H3HO; 2,3-DPG, 2,3-diphosphoglycerate;DSS, 2,2-dimethyl-2-silapentane-5-sulfonate; FID, free inductiondecay.tTo whom reprint requests should be sent at: Mail Stop 55-121,Lawrence Berkeley Laboratory, 1 Cyclotron Road, Berkeley, CA94720.

583

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Proc. Natl. Acad. Sci. USA 87 (1990)

CH20H

0 OHOH

HO T

OH5-D-Glucose

CH20H

0 T/OHOH

OHa-D-Glucose

CH20PO32-

0 T

HO-O

OHa-D-Glucose 6-phosphate

CH20PO32- CH20PO32-

CHTOPO32- 0 CHTOPO32- 0 CHTOH

C= .o HO

I ~~~~~~~HCH2oH t H O

hydroxyacetone HO HOPhosphate

CHTOI

CHOH1 I

/0 \0 HGlycerald3-Phosp]

HMP (Pentose) ShuntNADPT (GST )

NADP GSSG( H20 )

HO

CH20PO32 HTO

0

HOOH

6-Phosphoglucono-8-lactone

a-D-Fructose 1,6-diphosphate a-D-Fructose 6-phosphate

P32- CHTOPO32-CHTOPO32- CHTOPO32-

CHOHI CHOPO32 CHOH

4p \ 2- CO2- CO2-ehyde 1,3-Diphosphoglycerate 2,3-Diphosphoglycerate 3-Phosphoglyceratehate

CH2T

.P U=oI

2-

Pyruvate

CHT

op32-CO2.3o2-

Phosphoenolpyruvate

H20 CHTOHW!S CHOPO32

I2 -2-Phosphoglycerate

FIG. 1. Metabolic pathways associated with the Embden-Meyerhoff pathway for glycolysis and the initial stages of the hexosemonophosphate (pentose phosphate) shunt. The relevant structures have been schematically represented such that the expected fate of the 3H(T) label may be followed. GS, glutathione.

Deuterium has been used as a nuclear spin label to study thefate of protons in these pathways (29), but the studies werecomplicated by the presence of naturally occurring deuterium.The low y of deuterium means that the NMR detectionsensitivity is reduced by a factor of 7 relative to tritium. Thesensitivity and resolution of the observed species are furtherreduced by the large linewidths that result from the efficientrelaxation induced by the quadrupolar nucleus, although theshorter spin lattice relaxation times (T, values) that are typicalof deuterium nuclei help to recover some of the sensitivity perunit time.

EXPERIMENTALLabeling of Glucose. An approximately 21.6-mg (0.12

mmol) sample of glucose was dissolved in sodium phosphatebuffer, pH 7.0, and allowed to react with carrier-free tritiumgas over a 5% Pd/BaSO4 catalyst for 2 hr at ambienttemperature (23). After reaction the excess tritium gas wasremoved by evacuation, the sample was flushed with nitro-gen, and the product was washed with methanol, thenlyophilized twice from water to remove any residual labiletritium. After lyophilization the glucose was dissolved in thedeuterated pH 8 phosphate buffer that was used to wash theerythrocytes and then analyzed by HPLC on a Waters CHOcolumn, with both radioactivity and refractive index detec-tion. From the specific activity of the product (150-300GBq/mmol-i.e., 4-8 Ci/mmol) we deduce that replacementoccurs to approximately 14-28%, with significant variabilitybetween preparations. The proton-decoupled 3H NMR spec-trum of the labeled glucose contains two lines, from the a andanomers at 5.12 and 4.54 ppm [relative to 2,2-dimethyl-

2-silapentane-5-sulfonate (DSS)], respectively, and total im-purities amount to less than 2%.

Cell Preparation. The erythrocytes were obtained fromcanine whole blood, 5-10 ml, which was collected in a syringewith 500 units (0.5 ml) of heparin to retard coagulation. Aftertransfer to a plastic centrifuge tube, the blood was spun for10 min at 1500 X g to determine the hematocrit (typically 49%

1%), and the plasma, leukocytes, and buffy coat were thenremoved by pipette. The packed erythrocytes were washedwith a similar volume ofa pH 8 isotonic phosphate buffer (116mM NaCI/47.4 mM Na2HPO4/2.7 mM NaH2PO4), repacked,and washed again. The cells were washed twice more with asimilar pH 8 phosphate buffer made up in 2H20, to act as alock solvent. The resulting cells, with hematocrit greater than90%, were kept refrigerated until use, though never morethan 1 week.Packed erythrocytes, 1.5 ml in the deuterated phosphate

buffer, were added to 100 1,l of 0.3 M C-1-tritiated glucose,yielding a final glucose concentration of 18.75 mM. Sampleswere prepared in stoppered Teflon tubes (Wilmad, no. 6010)and inverted several times to thoroughly mix the solutions.The Teflon tubes were placed inside standard 10-mm glassNMR tubes for NMR spectroscopic study.NMR Spectroscopy. NMR spectroscopy was carried out on

an IBM Instruments AF-300 spectrometer (3H at 320 MHz,1H at 300 MHz), using a multinuclear, 3H{1H}, 10-mm probe.

A high-quality 3H band-stop, 1H band-pass filter [Cir-Q-Tel(Kensington, MD), FBT/20-300/3-6/50-3A/3A] was placedin the proton decoupling line of the instrument and the 3Hsignal was passed through a 3H band-pass, 1H band-stopfilter. Referencing of chemical shifts was achieved by gen-eration of a ghost 3H DSS signal from internal DSS in the 1HNMR spectrum (6).

Dil

CH2TICHOHICO2Lactate

584 Biophysics: Newmark et al.

Proc. Natl. Acad. Sci. USA 87 (1990) 585

The 3H coil had a 14-Asec 90° pulse width. All freeinduction decays (FIDs) were acquired with a 14-Asec pulse,a 1.37-sec acquisition time (8192 points), and an additional18.63-sec recycle delay time. Proton broad-band decouplingwas achieved with a WALTZ-16 (30) composite pulse se-quence (0.46W rms), which was on for the entire experiment.The probe was thermally regulated at 297 K. Since thechemical shift of water is extremely temperature sensitive(0.03 ppm/K) and there was very little variation in theposition of the HTO peak, we estimate the temperaturestability of the sample to be better than ±0.5 K. The 3H FIDcould be clearly observed after one pulse. Each spectrumrepresents 90 FIDs accumulated over 30 min and transformedwith 3 Hz of Lorentzian broadening; 130 spectra wereobtained over 65 hr. The unbroadened linewidths at half-height were approximately 6 Hz. These conditions werechosen to obtain fully relaxed spectra, without T1 influenceon peak intensities. The sample was spun at 10 Hz, and lessthan 5% additional cell packing could be observed when thesample was removed from the magnet after 65 hr. The Z andZ2 gradients were autoshimmed continuously during theentire experiment.

RESULTSAs can be seen in the stacked plot in Fig. 2, all of the tritiumsignal is initially in the a and f8 anomers of the C-1-labeledglucose. The 40:60 intensity ratio between the a- and (3-glucose peaks, at 5.12 and 4.54 ppm, respectively, is in goodagreement with previously reported values (24). The firstspectral acquisition was initiated 10 min after the addition ofthe glucose to the packed erythrocytes, and within 2 hrmetabolic intermediates and lactate began to appear.The resonance at 4.77 ppm is due to the formation of

tritiated water (HTO). At physiological temperatures (310 K)the HTO and (B-glucose resonances have degenerate chemi-

cal shifts at 4.54 ppm. Since we were interested in each ofthese peaks in isolation we chose to conduct the experimentsat 297 K, where they are resolved. At 313 K the glucose iscompletely consumed within about 10 hr, whereas at 297 Kthe consumption of glucose takes approximately 48 hr. Therelatively high pH of the buffer (pH = 8) was chosen topartially compensate for the decrease in the reaction rates atthe lower temperature.The peak that appears at 1.24 ppm is due to the formation

of lactate, where the tritium appears at the C-3 position, onthe methyl group. The only intermediate that is observed witha substantial concentration is 2,3-diphosphoglycerate (2,3-DPG), at 3.98 ppm, where the tritium atom is on C-3. This isin agreement with Minakami and Yoshikawa (25), who haveshown the effect of high pH on the production of 2,3-DPG.The identity of the 3.98-ppm resonance was confirmed byexamination of the proton spectra of 2,3-DPG in the deuter-ated buffered phosphate solution. An expanded view of thelast one-dimensional spectrum that was obtained, shown inFig. 3, reveals that at least two other intermediates appear inlow but dectectable concentrations, at 2.27 and 3.52 ppm.Although the identities of these resonances have not beenconfirmed, it is very likely that they are associated withpyruvate (protons at 1.58, 1.70, and 2.42 ppm relative to DSS)and 1,3-dihydroxyacetone phosphate (protons at 3.56, 4.40,and 4.78 ppm), respectively, as both of these intermediatesare known to pool in erythrocytes (26).The T1 values of the major tritium resonances were mea-

sured at 297 K by using an inversion recovery pulse se-quence. The measurements were made midway through a48-hr experiment, after half of the glucose had been con-sumed. The glucose T1 values were measured again in thedeuterated phosphate buffer without the packed erythro-cytes. The delay times of the inversion-recovery sequencewere distributed randomly to avoid artifacts due to theconsumption of the glucose. A nonlinear least-squares fitting

A

0(.1) >,(1 4.0 .2. 0 1.()C(hemical Shift. ppm

FIG. 2. Stacked plot of the one-dimensional 3H NMR spectra, which were acquired every 30 min (every other spectrum is shown). The thicklines in the stacked plot represent increments of 10 hr. All FIDs were acquired fully relaxed at 297 K, using a 900 pulse, a 20-sec recycle time,and 8192 points over 10 ppm. Each spectrum represents 90 FIDs transformed with 3 Hz of Lorentzian broadening; 130 spectra were obtainedover 65 hr. 2,3DPG, 2,3-diphosphoglycerate.

Biophysics: Newmark et al.

Proc. Natl. Acad. Sci. USA 87 (1990)

.

4.0 3.0 2.0Chemical shift, ppm

1.0

FIG. 3. Expanded view of the final one-dimensional spectrumthat appears in Fig. 2. The expanded view shows the appearance ofthe intermediates 1,3-dihydroxyacetone phosphate and pyruvate, aswell as traces of other unidentified intermediates that appear atconcentrations less than 100 ,uM.

routine was used to fit a single exponential to the data fromthese measurements to extract the various T1 values, and theresult ofthese calculations along with the relative uncertaintyin each measurement, can be found in Table 1. All of the T1data were fit very well by single exponentials.There is almost a factor of8 difference in T1 values between

the a-glucose in the phosphate buffer and the intracellular2,3-DPG. This range of T1 values indicates that care must beexercised with regard to collecting quantitative data in thissystem. The 20-sec recycle delay that was used to obtain thespectra in Fig. 2 was chosen to be 5 times the longest T1, sothat the spectra were fully relaxed. Since the nuclear Over-hauser enhancements, arising from proton decoupling, areknown to be similar to within 2% for all of the significantchemical species (27), the integrated intensities of the peakscan be used as quantitative estimates of the relevant con-centrations.

Table 1. T1 values for the primary tritiated constituents of themetabolic pathway at 297 K

T1, sec

Chemical PhosphateResonance shift, ppm Erythrocytes buffera-Glucose 5.12 1.91 ± 0.01 3.80 ± 0.01/3-Glucose 4.54 1.29 ± 0.01 2.13 ± 0.01HTO 4.77 3.02 ± 0.022,3-DPG 3.98 0.51 ± 0.06Lactate 1.24 2.50 ± 0.02The uncertainties represent one standard deviation computed from

a nonlinear least-squares fit of the data to a single exponential.The integrated intensities of each of the major resonances

have been plotted as a function of time in Fig. 4. Some of theintegrals (e.g., the HTO and }-glucose) are not as accurate asmight be obtained from line-fitting techniques, due to over-lap. As can be seen in Fig. 2, the a-glucose, 2,3-DPG, andlactate resonances are all well isolated, and the integratedintensities of these lines provide a reasonable estimate of thequantity of each of these species.The initial ratio of the intensities of the a- and B-glucose

resonances is 40:60, reflecting anomeric equilibration. Inagreement with Oxley et al. (21), this ratio does not varysignificantly during the time course of the experiment (seeInset in Fig. 4), indicating that the rate of anomeric conver-sion is faster than the rate of glucose consumption.

DISCUSSIONA primary motivation of this study was to demonstrate theease and utility of 3H NMR spectroscopy as a technique tostudy metabolic pathways in vivo with isotopic labels. Theresults that have been presented in Figs. 2 and 4 show thathigh-quality kinetic data are readily obtained. Note that thereplacement of a specific 1H with 3H allows the fate of thatproton to be traced with the sensitivity of proton NMR, butwithout the background of numerous other signals. It shouldalso be stressed that the tritium atom need not be bound toa specific isotope for detection, as required in 13C-1Hinverse experiments, where the magnetization associatedwith the 13C nucleus is detected at the 1H frequency. Animportant consequence of this last point is that millimolarHTO can be detected in the presence of 110 M protons (in

1 ra//-Glucose ratio e - eTotal 5 major lines

o.s

\. ,8-Glucose ° 10

* T. , . ~ ,- i. iv0 10 20 30 40 50 60

2.ime.

4,

030 40 Lactate.,hr

HTO

2,3-DPG0 10 20 30 40 50 60

Time, hr

FIG. 4. Integrated intensities of the major resonances that appear in the stacked plot shown in Fig. 2. (Left) Integrated intensities of the totalspectrum, the two glucose peaks, and the lactate peak. (Right) Sum of the integrated intensities of the five major peaks (a- and P-glucose, HTO,2,3-DPG, and lactate), the HTO, and the 2,3-DPG. The integral of the total spectral intensity in Left demonstrates conservation of spin to within10%, while the integral of the sum of the major resonances drops by approximately 20%, indicating the amount of signal spread throughout anumber of trace intermediates. (Inset) Plot of the ratio of the a- and /-glucose anomers as a function of time. The invariance of this ratio indicatesthat the rate of anomeric conversion is fast compared with the rate of a-glucose consumption.

100

80 F60 -

._ct

._

_440

20

80

60

40

20

70

586 Biophysics: Newmark et al.

r--.,W I. .%Pwb.I' -%%"16i-' .

..... ...-0%0-%,

Total 0-10 ppm

^

Proc. Natl. Acad. Sci. USA 87 (1990) 587

H20), a feature that has been utilized in this and ongoingstudies to gain insight into the activity of the pentose shuntin vivo.

On the basis of 14C radiotracer experiments, it is thoughtthat between 2% and 10% of the glucose flux goes through thepentose shunt. As shown in Fig. 1, the pentose shunt pro-vides one unequivocal path for a tritium label starting on

glucose to be incorporated into water. Consequently, theintensity of the HTO resonance must reflect the activity ofthe pentose shunt. After the glucose has been completelyconsumed, approximately 15% of the final tritium signalexists as water; the rest being lactate (40%), residual 2,3-DPG(11%), and a few other minor intermediates. HTO may beproduced from other pathways during anaerobic glycolysis,and further work is necessary to determine the preciseamount of HTO that is produced from the pentose shunt.The introduced tritium may be explicitly accounted for at

every stage of the experiment, with 80% present as HTO,2,3-DPG, or lactate after consumption of all the glucose. Theother 20% of the tritium is spread through a number of otherintermediates that are either barely observable (e.g., 1,3-dihydroxyacetone phosphate and pyruvate) or not at allobservable. There has been some question as to the NMRvisibility of different metabolites such as lactate (29). Asshown in Fig. 4 Left, conservation of spin is maintainedthroughout the experiment (within 10%), which would sug-

gest that the metabolites which are observed in these exper-

iments are completely NMR visible.We should also note that the results obtained from use of

rat (1, 2) and dog erythrocytes gave similar rates of glycol-ysis, and the major intermediates were the same for exper-

iments conducted under similar conditions.In other similar experiments using short recycle delays (i.e,

3 sec, on the order of the glucose T1 in the buffer) the glucoseresonance seemed to increase steadily for several hours priorto the expected decrease associated with glycolysis. Whenthe same experiments were repeated under fully relaxedconditions the increase in the integrated intensity was notobserved. We believe that the increase in signal intensityunder these partially saturated conditions is due to thetransport of glucose across the cell membrane, where theglucose is subject to faster T1 relaxation. Hence, the shorterT1 causes a more intense signal to be observed as the fractionof glucose within the cell increases.The results that have been presented are meant to be

illustrative rather than exhaustive. A number of aspects ofglucose metabolism in erythrocytes can be addressed withthis technique: quantitation of metabolic fluxes (e.g., pentose

shunt activity) or the activity and influence of blocking agents

and other additives. These processes may be best monitoredor analyzed by starting with other labeled glucose molecules,which are relatively easily prepared (28). In addition, a

number of further applications of in vivo 3H NMR spectros-

copy may be envisaged, since the technique is obviously not

limited to the study of glycolysis in erythrocytes.We have demonstrated that the metabolism of the C-1

proton of glucose is readily traced by 3H labeling and NMRspectroscopy. We have employed glucose at 2-3 times phys-iological concentrations for these studies, but with only 15%of the C-1 protons replaced by tritium (i.e. about 3 mMtritiated glucose). Use of a more highly tritiated substrate(e.g., one tritium in each molecule) would allow observationof metabolites below the 1 mM level with similar ease,

especially if the studies were conducted at higher field.

We thank Dr. K. M. Brennan, Dr. S. Ebbe, and Mr. C. Stalnakerfor assistance in obtaining the whole blood and Mr. N. Warren for

participating in some of the early experiments. We are particularly

grateful for the plotting software used to generate the stacked plots,which was provided by Mr. Brian L. Knittel. R.D.N. was supportedby National Institutes of Health Grant HL07367. The NationalTritium Labeling Facility is supported by the Biotechnology Re-sources Program, Division of Research Resources, National Insti-tutes of Health, under Grant P41 RR01237. All work was conductedat the Lawrence Berkeley Laboratory (Department of Energy Con-tract DE-AC03-76F00098 to the University of California).

1. Un, S. (1987) Dissertation (Univ. of California, Berkeley).2. Carson, P. J. (1986) Dissertation (Univ. of California, Berke-

ley).3. Williams, P. G., Morimoto, H., Gehring, K. B., Nikaido, H.,

Carson, P. J., Un, S., Klein, M. & Wemmer, D. E. (1989) inSynthesis and Applications of Isotopically Labeled Com-pounds: Proceedings of the Third International Symposium,eds. Baillie, T. A. & Jones, J. R. (Elsevier, Amsterdam), pp.487-492.

4. Evans, E. A., Warrell, D. C., Elvidge, J. A. & Jones, J. R.(1985) Handbook of Tritium NMR Spectroscopy and Applica-tions (Wiley, Chichester).

5. Gupta, R. K. (1987) NMR Spectroscopy of Cells and Orga-nisms (CRC, New York), Vols. 1 and 2.

6. Bloxsidge, J. P., Elvidge, J. A., Jones, J. R., Mane, R. B. &Saljoughian, M. (1979) Org. Magn. Reson. 12, 574-578.

7. Bloxsidge, J. P. & Elvidge, J. A. (1983) Prog. Nucl. Magn.Reson. Spectrosc. 16, 99-114.

8. Williams, P. G. (1988) Fusion Technol. 14, 840-844.9. Al-Rawi, J. M., Elvidge, J. A., Thomas, R. & Wright, B. J.

(1974) J. Chem. Soc. Chem. Commun., 1031-1032.10. Elvidge, J. A., Jaiswal, D. K., Jones, J. R. & Thomas, R.

(1977) J. Chem. Soc. Perkin Trans. 1, 1080-1083.11. Altman, L. J., Han, C. Y., Bertolino, A., Handy, G., Laun-

gani, D., Muller, W., Schwartz, S., Shanker, D., de Wolf,W. H. & Yang, F. (1978) J. Am. Chem. Soc. 100, 3235-3257.

12. Libor, S., Bloxsidge, J. P., Elvidge, J. A., Jones, J. R.,Woods, L. F. J. & Wiseman, A. (1980) Biochem. Soc. Trans.8, 98-99.

13. Aberhart, D. J. & Tann, C.-H. (1980) J. Am. Chem. Soc. 102,6377-6380.

14. Abraham, E., Pui, C. P., White, R. L., Crout, D., Lutstorf, M.,Phillip, P. J. & Derome, A. E. (1983) J. Chem. Soc. Chem.Commun., 723-724.

15. Evans, J. N. S., Burton, G., Fagerness, P. E., MacKenzie,N. E. & Scott, A. 1. (1986) Biochemistry 25, 905-912.

16. Frenzel, T., Beale, J. M., Kobayashi, M., Zenk, M. H. &Floss, H. G. (1988) J. Am. Chem. Soc. 110, 7878-7880.

17. Thorburn, D. R. & Kuchel, P. W. (1985) Eur. J. Biochem. 150,371-386.

18. Ugurbil, K., Brown, T. R., den Hollander, J., Glynn, J. A. &Shulman, R. G. (1978) Proc. Natl. Acad. Sci. USA 75, 3742-3746.

19. Shulman, R. G., Brown, T. R., Ugurbil, K., Ogawa, S., Cohen,S. & den Hollander, J. (1979) Science 205, 160-166.

20. den Hollander, J., Brown, T. R., Ugurbil, K. & Shulman, R. G.(1979) Proc. Natl. Acad. Sci. USA 76, 6096-6100.

21. Oxley, S. T., Porteous, R., Brindle, K. M., Boyd, J. & Camp-bell, 1. D. (1984) Biochim. Biophys. Acta 805, 19-24.

22. Brindle, K. M., Brown, T. R., Campbell, I. D., Grathwohl, C.& Kuchel, P. W. (1979) Biochem. J. 180, 37-44.

23. Evans, E. A., Sheppard, H. C., Turner, J. C. & Warrell, D. C.(1974) J. Labelled Compd. 10, 569-587.

24. Angyal, S. J. (1984) Adv. Carbohydr. Chem. Biochem. 42,15-68.

25. Minakami, S. & Yoshikawa, H. (1966) J. Biochem. 59, 145-150.26. Lehninger, A. L. (1977) Biochemistry (Worth, New York).27. Bloxsidge, J. P., Evans, E. A., Jones, J. R. & Mane, R. B.

(1977) J. Chem. Res. Synop., 258-259.28. Elvidge, J. A., Jones, J. R., Mane, R. B., Chambers, V. M.,

Evans, E. A. & Warrell, D. C. (1978) J. Labelled Compd.Radiopharm. 15, 141-151.

29. Hotchkiss, R. S., Song, S.-K., Ling, C. S., Ackerman, J. H. &Karl, I. E. (1990) Am. J. Physiol., in press.

30. Shaka, A. J. & Keeler, J. (1987) Prog. NucI. Magn. Reson.Spectrosc. 19, 47-129.

Biophysics: Newmark et al.