Proceedings of the National Academy of SciencesVol. 67, No. 2, pp. 1071-1078, October 1970

Mechanism of Thyroid Calorigenesis:Role of Active Sodium Transport*

Faramarz Ismail-Beigit and Isidore S. EdelmanCARDIOVASCULAR RESEARCH INSTITUTE AND DEPARTMENTS OF MEDICINE AND BIOCHEMISTRY-

BIOPHYSICS, UNIVERSITY OF CALIFORNIA, SAN FRANCISCO, CALIF. 94122

Communicated by Julius H. Comroe, Jr., July 17, 1970

Abstract. The hypothesis that thyroid calorigenesis is mediated by stimula-tion of active Na+ transport was tested by measuring the Qo, of liver slices andskeletal muscle (diaphragm) from thyroxine- and triiodothyronine-injectedthyroidectomized and normal rats in media fortified with ouabain (10-3 M) and/or free of Na+ or K+. In both tissues, more than 90% of the increase in QO,produced by injections of thyroid hormone in euthyroid rats was derived fromincreased energy utilization by the Na+ pump. In triiodothyronine-treatedthyroidectomized rats, activation of Na+ transport accounted for 90% or more ofthe increment in Qo, in liver and 40% or more of the increment in diaphragm.Intracellular Na+, K+, and Cl- concentrations were measured in euthyroid andhyperthyroid liver and diaphragm. The transmembrane Na+ and K+ concen-tration differences were significantly increased in both tissues by the administra-tion of triiodothyronine. These results indicate that thyroid hormone activatesNa+ extrusion and K+ accumulation either by increasing the local concentrationof ATP or by direct stimulation of the Na+ pump.

In homoiothermic vertebrates, thyroid hormones [thyroxine (T4) and triiodo-thyronine (T3) ] regulate oxygen consumption (Qoj), which is the primary deter-minant of heat production. This effect in adult animals is exerted on virtuallyall tissues, with the exception of brain, spleen, smooth muscle, and gonads, and ispreserved in vitro.1-5 That hormonal augmentation of respiration under physio-logical conditions results from uncoupling of mitochondrial oxidative phos-phorylation has been contradicted by the available evidence.4-7 Short of severethyrotoxicosis, mitochondria from hyperthyroid homoiotherms have higherrespiratory and phosphorylative activity than those from euthyroid controls, butthe same levels of respiratory control.

Tata8 has summarized the evidence that induction of RNA and protein synthe-sis mediates the action of thyroid hormones during the anabolic phase of theresponse. Associated with the induction of protein synthesis is an increase in thenumber and size of mitochondria in skeletal muscle. However, the calorigenicaction of thyroid hormone cannot be explained by an increase in the number andrespiratory capacity of the mitochondria of target tissues, since in the coupledstate the local concentration of phosphate acceptor (ADP) will determine theQo2, when substrate and 02 are not rate-limiting. Regardless of the mechanism

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of the increase in Qo2, an increase in mitochondrial production of ATP cannot besustained without a corresponding increase in ATP utilization. The inferencethat release of ADP mediates the effect on metabolism implies hormonal activa-tion of one or more of the energy-using processes (for example, contraction,transport), thereby maintaining a higher rate of cycling of ADP -- ATP -- ADP.

Induction of an activator or component of one of the ATP-utilizing processespresumably results in a sustained increase in Qo,. The requirements for thehypothetical endergonic process as the site of the calorigenic effect are twofold:(1) the process should be common to all of the target cells, and (2) the processmust have the capacity to use ATP at a rate that is sufficient to account for theeffect of the hormone on respiration. One process that meets these requirementsis transmembrane active Na+ transport. We therefore tested the hypothesisthat activation of Na+ transport mediates the calorigenic action of thyroidhormone. The dependence of the thyroid-induced increment in Qo, on activeNa+ transport and the accompanying changes in intracellular ion compositionwere measured.Methods and Results. Effect of ouabain on Qo, of liver slices: Male

Sprague-Dawley rats (initial body weights = 150-200 g) were maintained onPurina chow ad libitum and were injected intraperitoneally with 150 jig of Na-L-thyroxine per 100 g body weight daily for 10-14 days. Untreated rats paired forweight served as euthyroid controls. The rats were killed by decapitation andthe livers were transferred to iced, oxygenated, modified Na+-Ringer's solution[Na+ = 135, K+ = 5, Mg2+ = 0.5, Ca2+ = 1.0, Cl- = 139,H2PO4- = 5.0, Trisbase = 5.0, glucose = 10 (in mM); pH = 7.40 and osmolality = 290 mOsm/liter]. Liver slices, 290 um thick, were prepared immediately with a tissuechopper and transferred to a Warburg respirometer (Aminco, Silver Springs,Md.) and Qo, was measured in 30-min intervals as described previously.9 Oua-bain, a specific inhibitor of (Na+ + K+)-ATPase, was used to inhibit the Na+pump.10 Ouabain has no direct effect on respiration of isolated mitochondria."'In a preliminary dose-response study, Na+-dependent respiration was almostmaximally depressed at 10-3 M ouabain, as reported previously.'2 Simultaneousmeasurements of Qo2 were made [with and without ouabain (10-3 M) ] in liverslices from euthyroid and hyperthyroid (T4-injected) rats. Protein content ofeach flask was determined by the method of Lowry et al. 13

Table 1 shows that injection of T4 for 10-14 days raised the Qo, from an aver-age of 9.4 to 19.0, l oxygen per mg protein per hr. Energy expenditure for Na+transport [(Qo2)tl rose from 1.9 to 9.2 (same units). Moreover, inhibition of

TABLE 1. Effect of ouabain on Qo, of liver slices (I T4)*.Ouabain Euthyroid (n = 8) Euthyroid + T4 (n = 10)

(Ml oxygen per mg protein per hr)None 9.4 ± 0.7 19.0 ± 1.410-8M 7.5±0o.7 9.8±1.4t(Qos)t = 1.9 4 0.2 9.2 ± 0.8

* Mean ± SE, n =-number of rats. Each flask was read at 30-min intervals for 1-2 hr.t (Qo2)t denotes ouabain-sensitive Qo3, assumed to represent the respiratory energy expended in

transmembrane active Na + transport.

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Na+ transport virtually eliminated the thyroxine-induced increase in respiration,as the differences in Qo, were small and not statistically significant in the presenceof ouabain (7.5 ± 0.7-vs. 9.8 ± 1.4).

Effect of ouabain on the response to triiodothyronine: To minimize thecontribution of long-term, morphogenetic effects, we studied the dependence onNa+ transport of the response to the other principal thyroid hormone, Ts, over a7-day period. Two sets of experiments were completed: (1) hypothyroid rats(body weight 230 g), 1 month after thyroidectomy, were either injectedintraperitoneally with 50 1Ag of T3 per 100 g body weight every other day for threedoses (i.e., total dose = 150 ug/100 g), or were untreated for the same period.(2) Paired normal rats (body weights - 215 g) were injected with T3 according tothe same schedule or untreated for the same period. Liver slices were preparedand Qo, was measured as described above. The dry weight of tissue in each flaskwas determined by-removing the slices, blotting briefly with filter paper and heat-ing at 910C for 24 hr in tared aluminum cups.

Table 2 shows that T3 produced a 100% increase in Qo, of liver slices from

TABLE 2. Effect of ouabain on Qo, of liver slices (i T,).*Ouabain Hypothyroid Hypothyroid + Ta Euthyroid Euthyroid + Ts

(n = 20) (n = 20) (n = 10) (n = 10)None 5.6± 0.2 11.4± 0.6 7.6 0.2 12.4 ±0.410-3 M 3.9 i 0.2 4.1± 0.3 5.0 ± 0.3 5.4 ± 0.4(Qo2)t = 1.7 ±t 0.2 7.3 ± 0.6 2.6 ±-0.1 7.0 ± 0.3

* Units are ul oxygen per mg dry weight per hr; means ± SE.

hypothyroid rats and a 63% increase in slices from euthyroid rats, and that bothtreated groups reached equivalent hyperthyroid levels. Concomitantly, (Qo)tincreased by 330% in slices from the hypothyroid animals and by 170% in slicesfrom euthyroid animals. As seen with T4, the T3-dependent increase in Qo. wasabolished by ouabain both in hypothyroid and in euthyroid rats. Under theseconditions, therefore, most of the T3-induced increase in Qo, is derived from anincrease in the energy expended in active Na+ transport, provided that ouabaininhibited the Na+ pump selectively.

Effect of ouabain on Qo, in Na+-free media (liver slices): To assess theselectivity of the effect of ouabain on Qo,, we prepared liver slices from thyroid-ectomized and euthyroid rats either untreated or injected with T3, as describedabove. Rates of respiration were determined in Na+-free modified Ringer'ssolution by substituting isomolar concentrations of sucrose for the NaCl in thestandard solution described above.

Addition of ouabain (10-i M) had no appreciable effect on Qo, in this system(Table 3). The action of ouabain on Qo, in standard media is therefore attribut-

TABLE 3. Effect of ouabain on Qo, of liver slices in sucrose Ringer's (i To).*Ouabain Hypothyroid Hypothyroid + Ts Euthyroid Euthyroid + T.

(n = 10) (n = 10) (n = 10) (n = 10)None 2.2 ± 0.1 3.0 ±t 0.2 3.6 ± 0.2 4.0 ± 0.410-3M 2.0 ± 0.1 2.4 ± 0.2 3.4 ± 0.2 3.6 + 0.3AQo2 0.2 ±t 0.1 0.6 ± 0.3 0.2 ± 0.3 0.4 ± 0.5

* Units as in Table 2; means 4+ SE.

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able to selective inhibition of Na+ transport. It is also noteworthy that the ratesof respiration of tissues from untreated and T3-treated rats were about equal.The absolute Qo2 values in the sucrose-Ringer's were significantly lower than thecorresponding values in a Na+-Ringer's with ouabain (compare Table 2), pre-sumably a secondary effect of either depletion of intracellular Na+ or intracellularpenetration of sucrose.

Quantitative relationship between Qo, and (Qo,)t (liver): Quantitativeestimates of the activity of Na+ transport as a metabolic pacemaker werecalculated from the data in Table 2. In hypothyroid rats, TL increased the frac-tion of total Qo, devoted to Na+ transport from 0.29 to 0.66 and in euthyroid ratsfrom 0.34 to 0.57. Furthermore, the metabolic demands of the Na+ pump ac-counted for 100% of the increase in Qo, in the transition from the hypothyroidto the hyperthyroid state and 92% of the increase in the transition from theeuthyroid to the hyperthyroid state. These estimates are minimal values as evenat 10-s M, ouabain does not inhibit all Na+ transport activity. 12

Effect of ouabain on Qo, of diaphragm: The role of activation of Naa trans-port as a mediator of thyroid calorigenesis was also tested in skeletal muscle,a principal target tissue. Thyroidectomized (body weights - 125 g) and eu-thyroid rats (body weights a 100 g) were either untreated or injected with T3 asdescribed above. The diaphragms were dissected from the rib attachments andfreed of the central connective tissue. Rates of respiration of half segments ofeach hemidiaphragm were measured simultaneously as described above. Toobtain maximum ouabain inhibition of Na+ transport, the solutions weremodified by substituting NaH2PO4 for all of the KH2PO4, thereby removing all ofthe K+. Dry weight of each hemidiaphragm was determined as described above.As shown in Table 4, 'T3 increased the Qo, of hypothyroid diaphragm by 69% and

TABLE 4. Effect of ouabain on Qo. of diaphragm (= T,).*Hypothyroid Euthyroid

Hypothyroid + Ts Euthyroid + TsMedium Ouabain (n = 12) (n = 11) (n = 13) (n = 12)

(A) Na+ Ringer's None 4.5 -- 0.2 7.6 -- 0.4 7.7 ±4 0.2 10.4 =- 0.3(B) K+-free Ringer's None 4.2 4± 0.2 6.5 4± 0.3 7.4 4- 0.2 9.4 4± 0.3(C) K+-freeRinger's 10-'M 3.6 -- 0.2 5.4 -± 0.2 6.5 ±- 0.3 6.8 -- 0.2(A-C) (Qo2)t = 0.9 ±t 0.2 2.2 4± 0.5 1.2 ±L 0.3 3.6 4± 0.3

* Units as in Table 2; means 4 SE.

of euthyroid diaphragm by 35%. Maximal inhibition of Na+ transport (ouabain+ K+-free media) substantially reduced the T3-dependent increase in Qo2 inhypothyroid muscle. In the inhibited state the T3-dependent increment in Qo,was 1.8 (i.e., 5.4 - 3.6) whereas in the control tissues this increment was 3.1(i.e., 7.6 - 4.5). In euthyroid muscle, inhibition of Na+ transport almostabolished the T3-induced increment in respiration. T3 raised (Qo,)t by 144%in hypothyroid and by 200% in euthyroid muscle.

Effect of ouabain on Qo. in Na+-free media (diaphragm): The specificity ofouabain as an inhibitor of Na+ transport was evaluated by measuring rates of res-piration in Na+-free media prepared by substituting choline chloride for the NaClin the standard solutions. The results are given in Table 5 and show that ouabain

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TABLE 5. Effect of ouabain on Qo2 of diaphragm in choline-Ringer's (±iT3).*Hypothyroid Hypothyroid + T, Euthyroid Euthyroid + T,

Ouabain (n = 9) (n = 9) (n = 15) (n = 15)None 3.6 -- 0.3 6.3 == 0.2 5.6 == 0.2 5.7 ±t 0.310-3M 3.5 -- 0.3 6.2 ±t 0.2 5.5 4t 0.3 6.2 it 0.3AQo2 0.1 +t 0.4 0.1 At 0.3 0.1 4t 0.4 -0.5 4t 0.4

* Units as in Table 2; means i SE.

had no detectable effect on Qo, of hypothyroid or euthyroid diaphragm whetheruntreated or after treatment with T3. As an additional control the thyroid-ectomized untreated rats were injected with diluent on the same schedule as theT3-treated animals. As with the liver slices, therefore, the inhibitory effect ofouabain on diaphragm is attributable to selective inhibition of active Na+ trans-port. Moreover, the rates of respiration of diaphragm from untreated and T3-treated euthyroid rats were the same (i.e., 5.6 h 0.2 vs. 5.7 4 0.3) in Na+-freemedia. T3-treated diaphragm from hypothyroid rats, however, maintainedsignificantly higher rates of respiration than the untreated tissue.

Quantitative relationship between Qo, and (Qo2)t (diaphragm): The quan-titative estimates of the contribution of Na+ transport to thyroid calorigenesis inskeletal muscle were calculated from the data in Table 4. In hypothyroid rats,T3 increased the fraction of total Qo, devoted to Na+ transport from 0.19 to 0.29and in euthyroid rats from 0.16 to 0.35. As a minimum, the metabolic demandsof the Na+ pump accounted for 43% of the increment in Qo, in the transition fromthe hypothyroid to the euthyroid state and 91% of the increment in Qo, in thetransition from the euthyroid to the hyperthyroid state. These results, there-fore, confirm the mediating role of transmembrane Na+ transport in thyroidcalorigenesis.

Effect of T3 on intracellular composition of liver: To elucidate the nature ofthyroid stimulation of Na+ transport, we measured intracellular ion concentra-tions. Slices prepared from untreated and T3-treated euthyroid rats were incu-bated in standard media containing ["C ]inulin for 40 min at 370C. The sliceswere dried at 91'C for 24 hr, weighed, and extracted with 0.1 N HNO3 for 48 hrat 250C. The extracts were analyzed for 14C in a Packard liquid scintillationspectrometer,'4 for Na+ and K+ by atomic absorption spectrometry,'5 and forCl- by potentiometric titration. 16

Table 6 shows that intracellular Na+ and Cl- concentrations were lower and

TABLE 6. Effect of T, on intracellular composition of liver slices.ICF*

mg H20/mg [Na+] [K+] [Cl-dry wt. .- meq/liter cell water - [Na+]/ [K+]

Euthyroid(n = 15) 1.86 4±0.057 95.2 ±2.8 114 ±t3.6 75.1 ±2.5 0.86 ±t0.05tEuthyroid + Ts 1.77 ± 0.032 71.4 ±t 4.0 147 ± 3.6 56.2 ± 3.1 0.49 ± 0.03(n= 15)P >0. 10 <0.005 <0.005 <0.005 <0.005

Liver slices were incubated at 37 IC for 40 min prior to analysis. Values are means + SE.* ICF denotes relative amount of intracellular fluid, which was computed as the difference between

total water content and the [14C]inulin space and is expressed in the units: mg water per mg dryweight of tissue.

t Injected with 50 fg Ts per 100 g rat three times on alternate days.

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K + concentration higher in slices from T3-treated animals than in the euthyroidcontrols. As a result the Na+/K+ ratio decreased to 57% of the euthyroidvalue. Similar analyses were made on rat diaphragm after constant intravenousinfusion of [4C ]sucrose as the extracellular marker. The results in Table 7

TABLE 7. Intracellular composition of diaphragm.*ICFt [Na+] [K+]

mg H20/mg dry wt. meq/liter cell water-- [Na+]/[K+]Euthyroid (n = 8) 3.023 + 0.086 23.9 + 1.4 150.1 + 3.2 0.160 i 0.010tEuthyroid + T3 (n = 7) 2.594 + 0.048 20.9 +4 1.3 161.7 + 2.6 0.129 + 0.008P <0.005 >0. 10 <0.025 <0. 025* Analyzed without prior incubation in vitro. Values are means i SE.t ICF was computed from the total water content less the [14C ]sucrose space. In unanesthetized

rats, [14C]sucrose was infused intravenously into the tail vein at a constant rate for 60 min prior toremoval of the diaphragm.

$ Injected with 50 ug Ts per 100 g rat three times on alternate days.

establish that T3 significantly reduced the intracellular content of water, in-creased intracellular K+ concentration, and lowered the Na+/K+ ratio. Theeffects noted in both liver and skeletal muscle are indicative of a sustained in-crease in Na+ transport activity and a consequent increase in the steady-statedifferences in transmembrane concentrations of Na+ and K+.

Discussion. The coupling between energy metabolism and active Na+ trans-port is a consequence of the energy dependence of the transport process. Asemphasized by Whittam, 17 the magnitude of the energy demands of the Na+ pumpdetermines its importance as a metabolic pacemaker. In normal mammaliantissues, 20-45% of the resting Qo, is expended in transmembrane Na+ transport.17In homoiothermic regulation of body temperature, therefore, the Na+ pumpconstitutes one of the primary heat sources. The significance of the Na+ pumpas a heat source was recognized by Nissan et al.,18 who provided evidence that thehypermetabolism of dehydration was a direct consequence of stimulation of theNa+ pump because of the rise in extracellular Na+ concentration. Our resultsindicate that activation of the Na+ pump accounts for virtually all of the increasein Qo, in the transitions hypothyroid -- euthyroid --. hyperthyroid states in liverand in the transition from the euthyroid to the hyperthyroid state in skeletalmuscle. In addition, activation of the Na+ pump accounts for more than 40% ofthe increase in respiration in the transition from the hypothyroid to the euthyroidstate in skeletal muscle.Whittam and Blond'9 concluded that the Na+ pump regulates mitochondrial

Qo1 in proportion to the rate of generation of ADP. The increase in the oubain-inhibitable fraction of Qo, implies coupled oxidative phosphorylation, as well asan increase in ADP release by the Na+ pump. Thus, at the dosages used in thisstudy, our results indicate that thyroid hormones do not uncouple mitochondrialoxidative phosphorylation. In the uncoupled state, the fraction of the Qo,devoted to ATP-dependent cellular work should fall. If (Qo,)t is a constantindex of ATP-dependent Na+ transport work, then uncoupling of oxidativephosphorylation should decrease the ratio (Qo2)t: Qo2. The results summarizedabove show that administration of T3 increased the fraction of (Qo2)t: Qo, by 150-

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200% in both liver and skeletal muscle. In addition, the results summarized inTables 6 and 7 indicate that thyroid hormone decreased the intracellular Na+/K+ratio, which is contrary to the expected effects of uncoupling at the mitochondriallevel.

Thyroid hormone-dependent activation of the Na+ pump could be a result of(1) a rise in intracellular Na+ concentration, perhaps as a result of increasedmembrane permeability to Na+ and consequent stimulation of the Na+ pump;(2) a change in the coupling ratio between the chemical reaction (i.e., ATP split)and transport work, such that the rate of ATP hydrolysis is increased for a givenrate of Na+ transport. As a result there would be a fall in the transmembraneNa+ and K+ gradients; (3) activation of mitochondrial ATP synthesis, resultingin stimulation of the Na+ pump by a local increase in ATP concentration; (4)direct activation of the Na+ pump (e.g., synthesis of more transport units with arise in Vm.x or a reduction in Km for ATP or intracellular Na+). The first twopossibilities, as either the sole or the predominant mechanism of the mediatingrole of Na+ transport in thyroid calorigenesis, are excluded by the results inTables 6 and 7. These data are consistent with either mechanism 3 or 4, whichshould produce a lowered intracellular Na+/K+ ratio. Moreover, increasedmembrane potentials, and Na+ and K+ gradients, have been noted in mammaliantissues during growth and development (which requires normal thyroid func-tion).20The key to the mechanism of thryoid activation of Na+ transport and its role in

calorigenesis may be contained in the findings of Valcana and Timiras,2' whostudied the effects of hypothyroidism on the electrolyte content and (Na+ + K+)-activated ATPase activity in the brain of the neonatal rat. In contrast to themature animal, the developing brain responds to thyroid hormone calorigenicallyas well as morphogenetically. Valcana and Timiras2' found that thyroid depri-vation resulted in an increase in the Na+ and Cl- and a decrease in the K+ andMg2+ content of the cerebral cortex and cerebellum. Moreover, the specificactivity of the transport enzyme declined in proportion to the decline in theelectrolyte gradients. Thus, thyroid activation of the transport enzyme may bedirectly involved in the metabolic response.Our results also raise the possibility that regulation of the Na+ pump may

contribute directly to changes in rates of metabolism in a variety of physiologicalstates (e.g., adaptation to cold, fever, catecholamine release, etc.).

We are indebted to Dr. Luke L. H. Chu who was instrumental in initiating this project.The authors are also grateful to Florette Yen and Allison Meader for their able technicalassistance and to Dr. Peter Lipton for many helpful discussions.

Abbreviations: T3, triiodothyronine; T4, thyroxine.* Financial support was provided by USPHS grant no. HE-06285. A preliminary report

of part of this work was presented at the 54th Annual Meeting of the Federation of AmericanSocieties for Experimental Biology: Ismail-Beigi, F., and I. S. Edelman, Fed. Proc., 29, 582Abs (1970).

t During the tenure of a Bay Area Heart Association Research Fellowship. This study is apart of the work to be submitted in partial fulfillment of the requirements for the Ph.D. degreein Biophysics at the University of California, San Francisco, Calif.

1 Barker, S. B., and H. M. Klitgaard, Amer. J. Physiol., 170, 81 (1952).

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2 Nielson, R. R., R. F. Loizzi, and H. M. Klitgaard, Amer. J. Physiol., 200, 55 (1961).8 Barker, S. B., in The Thyroid Gland, ed. R. Pitt-Rivers and W. R. Trotter (London:

Butterworths, 1964), vol. 1, p. 199.4Fletcher, K., N. B. Myant, and D. D. Tyler, J. Physiol., 162, 345 (1962).5 Tata, J. R., L. Ernster, 0. Lindberg, E. Arrhenius, S. Pedersen, and R. Hedman, Biochem.

J., 86, 408 (1963).6 Schafer, G., and L. Nagel, Biochim. Biophys. Acta, 162, 617 (1968).7 Stocker, W. W., F. J. Samaha, and L. J. De Groot, Amer. J. Med., 44, 900 (1968).8 Tata, J. R., in Regulatory Functions of Biological Membranes, BBA Library Serise, ed. J.

Jarnefelt (Amsterdam: Elsevier Pub. Co., 1968), vol. 11, p. 222.9 Umbreit, W. W., R. H. Burris, and J. F. Stauffer, Manometric Techniques (Minneapolis,

Minn.: Burgess Publishing Co., 1957).10 Skou, J. C., Physiol. Rev., 45, 596 (1965).11 Blond, D. M., and R. Whittam, Biochemistry, 92, 158 (1964).12 Whittam, R., and J. S. Willis, J. Physiol., 168, 158 (1963).13 Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem., 193, 265

(1951).14 Fanestil, D. D., and I. S. Edelman, Proc. Nat. Acad. Sci. USA, 56, 872 (1966).15 David, D. J., Analyst, 85, 779 (1960).16 Cotlove, E., Methods Biochem. Anal., 12, 277 (1964).17 Whittam, R., in The Cellular Functions of Membrane Transport, ed. J. F. Hoffman (New

Jersey: Prentice-Hall, 1964), p. 139.18 Nissan, S., A. Aviram, J. W. Czaczkes, L. Ullman, and T. D. Ullmann, Amer. J. Physiol.,

210, 1222 (1966).19 Whittam, R., and D. M. Blond, Biochem. J., 92, 147 (1964).2o Hazelwood, C. F., and B. L. Nichols, Johns Hopkins Med. J., 125, 119 (1969).21 Valcana, T., and P. S. Timiras, J. Neurochem., 16,935 (1969).

Correction. In the article "The Anomalous Deuterium Isotope Effect on theChemical Shift of the Bridge Hydrogen in the Enol Tautomer of 2,4-Pentane-dione," by Sunney I. Chan, L. Lin, Dale Clutter, and Phoebe Dea, which ap-peared in the April 1970 issue of Proc. Nat. Acad. Sci. USA, 65, 816-822, thefollowing correction should be made: line 25 (p. 819), the figure 2000 shouldread 5000.

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