Lactic Acidosis as a Result of Iron Deficiency

CLEMENT A. FINCH, PHILIP D. GOLLNICK, MICHAEL P. HLASTALA, LOUISE R. MILLER,ERICK DILLMANN, and BRUCE MACKLER, Departmenit of Aledicitne, Divisiotnsof Hemlatology anid Resljiratory Diseases, Departmetnts of Pediatrics antdthe Centerfor Child Development atd Menttal Retardationi, Departmentof Physiology anrd Biophysics, Uniiversity of WVa.shtinlgtonI, Seattle,Witashinigtoni 98195; anid Departtmienit of Phylsical Educationi, Washinigtoni StateUniversity, Pullmnta, WVashington 99164

A B S T R A C T Iroin-deficien1t rats halve an1 im11pairedwork performancice, even when their aniemiiia is correcte(dby exchanige transfusioin. Muscle activity is associate(lwith a higher blood lactate concenltrationi thani isobserved in iron-replete aniimals. The accumulationof lactate is a result of excessive productioni aslactate clearancie fromn the 1)loo0( was shown to beunaffecte(l. By acljusting the work loa(l to a lowerlevel, it was possible to (livide iron-deficient animilalsinto two groups, oine capal)le of continued treadlmiillrunninig and aniother in which aniimals stoppe(d before20( min. In the former, blood lactate conicenitrationireache(d a plateau at moderate levels, whereas itcoontiniued to increase in the latter until the anIimialstopped runninig. Levels ofa-glycerophosphate oxidasein skeletal mulscle mitochondria were found( to be mulchlower in the secon(d group (P < 0.001). Lactateinfusioin into normiial animals was showin to interferewith work performiiance, anid muainteinance of a normiialpH in iron-deficieint anid iron-replete animiials didinotprevent the impairment in work associated with highbloo(1 lactate concentrations. Additional evidenlce Wcasobtained that energy substrate (blood glucose anidfree fatty acids, muscle glycogen) was adequate in iron-deficient animiials. Oxygein tension in their venacaval blood was higher than in controls. Fuirthermore,the in situ behavior of electrically stimulatedgastrocnemius and soleus muscles appeared similar tothat of control animals. Because the stimulation of thesingle muscle in the iron-deficient animal did notresult in appreciable elevation of blood lactate andldid not show impaired contractility further supportedthe hypothesis that the elevation ofblood lactate causedthe decreased work performance. It is concluded thatiron deficiency by a depletion in the iron-containingmitochondrial enzyme, a-glycerophosphate oxidase,

Received for publication 10 July 1978 acnd in revised form12 February 1979.

imppairs glycolvsis, resultinig in excess lactatte formation,which at high levels leads to cessattion of physicalactivity.

INTRODUCTION

In a previous publication we have described animpaire(d runninig performiiance by iron-deficienit rats(1). The effect of anemia was eliminiate(d by a(djustinlgthe hemoglobin of iron-deficient and control anim-alsto a uniformii coneenitrationi of hemoglobin whichperimlitte(l Imlaxiimiumii work. Given parenteral iron,dleficienit animilcals regainede a normiial runniinlg abilitywvithin 3-4 d. The cause of the runnling dlisal)ility wasbelieved to be a decrease in the mitochonidrial enzynmesystem, a-glycerophosphate oxidase. Evidleniee for thisassociatioin was circumstantial, based on the similartime relation ship between the depletioni ofthis enzymeandl its repletion with iron therapy and the impairmiientan(l recovery of work capacity. In this study furtherevidence is presentedl supporting the causal role of thea-glycerophosphate oxidase systemii, andl a metabolicexplanation for the imppaired work performance isprovided.

NIETHOD S

Male Sprague-Dawley rats were obtained at 4 wk of age,1 wk after weaniing. Rats to be made iron deficient weregiven a low-iron dliet prepared in our laboratory,' whichcontained 5-10 mg iron/kg. By the end of the first monith onthis diet the hemoglobin concentration of those animalsreceiving it had fallen to =6 g/dl. The plasma iron and totaliron b)inding capacity averaged 45*4.82 and 850±15.5 ,g/dlplasima, respectively. Two types of control animnals were

MIade according to ICN Nutritional Biochemicals, Cleve-land, Ohio.

2 Throughout this article variations are expressed asstandard error.

J. Clin. Invest. (© The American Society for Clinical Investigation, Inc. * 0021-9738/79/07/0129/09 $1.00 129Volumne 64 July 1979 129-137

employed. The first (type 1 control [C1])3 was given PurinaLaboratory Chow (Ralston Purina Co., St. Louis, Mo.) whichcontained 382 mg iron/kg. The mean plasma iron (non-fasting) in this group of animals was 195+8 ,ug/dl, and theiron binding capacity averaged 463+13.2 ug/dl. Their meanhemoglobin before exchange transfusion was 14.2+0.14 g/dl.The second type of control animals (type 2 control [C2]) wasgiven the iron-deficient diet described above but receivedweekly intraperitoneal injections of 5 mg of iron dextran(Pharmacia Fine Chemicals, Div. of Pharmacia Inc.,Piscataway, N. J.). The mean plasma iron and total ironbinding capacity of these animals 1 wk after the last ironinjection was 142.8+12.3 and 534.6+20.9 Ag/dl plasma. Theirmean hemoglobin concentration was 12.6+3 g/dl blood. Allanimals were allowed to eat and drink ad libitum. Only thosewhose weights were between 200 and 250 g were used.The protocol for the work performance studies included

practice on the treadmill 7, 6, and 5 d before the actual study,insertion of a vena caval catheter 4 d before, and an exchangetransfusion carried out 1-4 h before the actual "run." Workperformance was evaluated on a small animal treadmill asdescribed elsewhere (1) (model 42-15, Quinton Instruments,Seattle, Wash.). Two different levels of work were employed.For the "fast run," the slope was initially set at 12.50, and thebelt was run at a speed of 18.76 m/min. As the animal ran,the slope but not the rate was increased (2). In the sameanimals, earlier training runs were carried out at rates of13.4, 16.16, and 18.76 m/min with the slope set at 12.5° onsuccessive days. For the "slow run" the slope was 00, and aspeed of 10.72 m/min was used. Neither rate nor slope waschanged at the time of the study. However, the training ondays 7, 6, and 5 before the slow run study was carried outat a rate of 8.04, 10.72, and 10.72 m/min, respectively. Theend point in the determination of running time in allexperiments was that point at which the animal spent over50% of the time on the electrically charged plate at thebottom of the running belt.On the day of the study the hemoglobin of all animals was

adjusted by exchange through the vena caval catheter ofblood components freshly drawn from normal animals (anemicanimals being exchanged with erythrocytes and normnal animalswith plasma) so as to achieve a common hemoglobin levelof _10 g/dl blood. Details of the catheter placement andexchange transfusion have been previously described (1).To determine the effect of lactic acidosis, a 0.8-M solution

of L-lactic acid was infused intravenously in normal animalsat a rate of 0.13 ml/min; in other animals a 1.2-M solutionof sodium bicarbonate was infused intravenously at the samerate, either with or without simultaneous lactate infusion. Theinfusions were carried out through an inlying caval catheterwhile animals were exercising on the treadmill. L-['4C]Lactatesolution in tracer amounts was also injected into animals atrest to evaluate turnover. In all these studies blood sampleswere drawn from a second caval catheter distal to that of thecatheter used for injection.The use of inlying catheters permitted the removal of

venous and arterial blood at a precise time, either in theresting animal or at the termination of exercise. Certainresting or exercising animals were killed by intravenousphenobarbital. As soon as they fell over, a leg was quicklyskinned and the muscle frozen with tongs precooled inliquid nitrogen. The muscle was cut out and dropped intoliquid nitrogen. It was then pulverized with a mortar andpestle precooled with liqiuid nitrogen and stored, frozen at-60°C. The freezing of the muscle was accomplished within

3Abbreviations used in this paper: Cl, type 1 control(s);C2, type 2 control(s).

0.6+0.04 min of the time the animal was anesthetized. Inthese studies muscle glycogen was determined by themethods of Passonneau and Lauderdale (3) and Lo et al. (4)whereas L-lactate was determined by the method ofHohorst (5).a-Glycerophosphate oxidase activity of the whole muscle

was determined as follows: a 0.5-g aliquot from the thighmuscles was placed in 3.5 ml of a solution of 0. 15 M sucroseand 0.025 M Tris, pH 7.5, at 3-5°C. The muscle washomogenized for 30 s with a Tekmar homogenizer (TekmarCo., Cincinnati, Ohio). This was followed by homogenizationover 3-5 min with a glass Teflon (E. I. Du Pont de Nemours& Co., Inc., Wilmington, Del.) motor-driven homogenizer atfull speed. A final homogenization was carried out by handwith a ground glass homogenizer to break up connectivetissue clumps. The assays for enzymatic activity were carriedout with a chamber volume of 1.75 ml in an oxygen polaro-graph at 25°C. The chamber contents included 1.12 mlof a solution of 0.15 M sucrose and 0.025 M Tris, pH 7.5,0.1 ml of 0.2 M potassium phosphate buffer, pH 7.5, and0.5 ml of muscle homogenate. After a constant blank rate ofoxygen utilization was established, assays were begunby the addition of 0.03 ml of 0.5 M a-glycerophosphate.Observations were made on an in situ preparation of the

gastrocnemius and soleus muscles ofnormal and iron-deficientanimals with equalized hemoglobin concentrations. Theanimals were prepared under anesthesia provided byintravenous injections of sodium pentobarbital. The muscleswere isolated without disturbing the circulation. The leg wasstabilized by drilling a hole through the femur and securingthe drill bit in a steel holder on the board upoIn which theanimals were mounted in a supine position. The ankle wasfixed with the knee joint at a 900 angle. The distal tendonof the muscle was looped through a brass ring, secured witha silk ligature, and connected directly to a Statham force trans-ducer (Statham Instruments, Inc., Oxnard, Calif.). The nerveto the muscle was cut, and electrodes were fixed to theproximal and distal ends of the muscle. Muscle length was ad-justed until a single stimulus evoked a maximal contraction.Stimuli of 14 V with a 0.76 ms duration were administeredat 5 Hz. Tension was monitored with a storage oscilloscopeand recorded with a Beckman R611 polygraph (BeckmanInstuments, Inc., Fullerton, Calif.). The body temperature ofthe animals was maintained at 370C with a water-circulatedheating pad. Mineral oil at 370C was continuously dripped onthe muscle. Blood samples were obtained from the indwellingcatheter before and after stimulation. Samples of the restedand stimulated muscles were taken with the freeze clampmethod (6). These were stored in dry ice until analyzed.Blood and muscle lactate, glucose, and glycerol weredetermined with fluorometric methods (7, 8).Blood analyses included hemoglobin concentration by the

cyanmethemoglobin technique and plasmiia iron and ironbinding capacity as reported by The International Committeefor Standardization in Hematology (9) and by Cook (10);glucose was determined by the oxidase method (TechniconManual, Technicon Corp., Tarrytown, N. Y.); free fatty acidsas described by Dole (11); and pyruvate and L-lactate by themethods of Hohorst (5) and Biucher et al. (12). The P504 wasdetermined by the mixing techni(lue (13); and P02, Pco2, andpH were measured with a microblood gas analyzer (Radiom-eter model BMS3, Radiometer Co., Copenhagen, Denmark).Base excess was determined from the Siggaard-AindersonNomogram. Oxygen content was determined with a LexO2Conoxygen analyzer.

Statistical methods emiiployedl includled the Studlen-t's

4 P50 iS corrected to pH = 7.4, Pco2 = 40 torr, temperature= 37°C.

130 Finch, Gollnick, Hlastala, Miller, Dillmann, and Mackler

unpaired t test and covariance analysis as described bySnedecor and Cochran (14).

RESULTS

Oxygen1 supply an(l gas exchatnge in the exercisinJgiron-deficient animnal. Studies were carried out onvenla caval blood to (letermiinie the adequacy of oxygensupply. The P50 of eight iron-deficienit animilcals whosehemoglobins had been adljustedl 1-4 h before to9.93±0.14 g/dl was 39.4±1.1 torr, whereas thecorrespondinig values for eight (C1) conitrols withhemoglobins aIt 10.3±)0.14 g/dl was 39.2±-0.6 torr. Meainarterial P02 before acnd immlilediately after a 2-mimfast runi in six ironi-deficienit but nionianiemic'i.animilalsdid not show any signiificanit dlifferenice fromi controlanimncals (Table I). However, the greater drop in pHobserved in iron-deficient anliimals would result in agreater in vivo availability of oxygen during exercise.The meian arterial Po2 of resting animiicals, i.e., 69 aindl72 torr, although they appear low, are conisistenit withprevious reports in the literature (15, 16).Measurements of vena caval 1)lood0 gases in animilcals

at rest and during exercise are also summllllcarized inTable I. In the exercising iron-deficienit group higherin vitro Po2 andl lower Pco2 vatlues as comiipared with

Po0 (torr)

FIGURE 1 02 content (C) of vena caval blood in resting andexercising animals. The Po2 values are corrected for in vivoconditions and are presumed to reflect average tissue oxygentension.

control animilals were demiionistrated, as well as a higherconcenitrationi of blood lactate and a lower pH. Thebase excess after exercise was lower in the iron-deficient group because ofthe larger content of lactate inthe blood. This resulted in a imuch higher in vivo venousPo2 because of the decrease in oxygen affinity (Fig. 1).

TABLE IBlood Gases in Iron-Deficient anid Control Animilals before aind after a 2-mnit Fast Run

Restinig After 2-min runiniing

Conitrol Iron-deficient Control Iron-deficient

Arterial blood*Hemoglobin before exchange

transftusion, gldl 13.44+0.32 5.52±0.19Hemoglobin after exchatnge

transfusion, gldl 10.13±0.07 9.79±0.07Po2, torr 72.2±2.6 68.9±1.1 85.7±3.2 85.6±1.8Pco2, torr 36.6±2.1 31.3± 1.3 26.4± 1.6 21.6±1.4§pH 7.477±0.011 7.443±0.020 7.346±0.13 7.138+0.032"Lactate, ml\l 1.07±0.05 1.04±0.13 7.82±0.50 15.3±1.8§Base excess, me(/lliter +3.4±0.6 -2.1 ± 1.0§ -10.1±1.0 -20.9±1.0"02 content, mlIldl 12.0±0.4 11.8±0.18 11.5±0.5 11.3±0.3

Vena caval bloodtHemoglobin before exchange

transfusion, gldl 14.80± 1.10 5.30±0.30Hemoglobin after exchange

transftusion, gldll 9.90±0.10 9.80±0.10Po2, torr 28.70±1.20 28.40±1.50 17.10±2.50 35.40±3.00'PCm2, torr 39.40±0.08 37.10±1.30 47.30±1.80 42.90±+1.30pH 7.46±0.01 7.44±0.01 7.18±0.03 7.00±0.02"Lactate, mnM,\ 0.94±0.05 2.24±0.49§ 10.90±1.30 15.68±1.93Base excess, ineqlliter +4.00±1.10 + 1.41±0.64 - 10.20±1.19 -20.40±1.16"

* Six animals studied in each group.Eight animals studied in each group.

§ Difference between ironi-deficient and control animals significant to P < 0.05.H Difference between iron-deficient and control animals significant to P < 0.001.

Lactic Acidosis as a Result of Irotn Deficiencij 131

A final study was carried out in which 12 exchainge-transfused iron-deficient animals who were able tocontiniue over 15 min of the slow run showed, at15 min, a venous Po2 of 30.1+2.5 torr and a meainvenous Pco2 of 25.7±1.3 torr. On the other hand, eightexchanige-transfused iron-deficient animu-als who ranless than 15 mni, at the time they stopped running,showed a miieani Po2 of 42.5±3.9 torr and a meani Pco2of 32.0+2.1 torr. The differences between the twogroups were significanit (P < 0.05) for the Pco2 only.

Lactic acidosis. Additionial studies of lactate andpyruvate concenltratioIns after 2 mmi of the fast run areshown in Table II. Pvruvate levels were compparablein the two groups and( did nlot change with exercise.On the other hand, blood lactate at rest was higherin the iron-deficient animlals, anld the difference wasexaggerated with exercise, the lactate levels of iron-deficient anlimals being abtout four timnes those ofcontrols.To examiine the cause of the increasedl lactate conl-

centratioIn, L-[14C]lactate was inljected in restinganimiials, and its disappearanice curve is shown inFig. 2A. Disappearanice rates appear generally similardespite the larger 1)1ooc( lactate pool in the iron-deficient atnimals. A comparisoni of lactate clearancewas also mncade in animiials (luring exercise. Here it wasnecessary to infuse lactate into norm-tial animilalsimme(liately before runninig to achieve a lactateincrease comiiparable to that seeni in iron-deficientanimiials. Blood lactate conceintration moniitored duringtwo successive 10-mmiin perio(ds after the ainimalsstoppe(l running is shown in Fig. 2B. Elevated bloodlactate concentrations fell at least as rapidly andperhaps more so in iroin-deficienlt as in control aniiaials.The excess accumnulationi of blood lactate could be

shown more clearly when the work load was re(lucedto a poiInt where som11e iron-deficienit animilals couldrun for prolonged periods. UInder the condition of theslow runi, normiial ainimals ran easilx for several hourswith little increase in 1)lood0 lactate concenitration. At

100 a

` 70

4) 60-

040

0 10 20 30 40 50 60TIME AFTER INJECTION

(min)

C-K1

K4Ks

0Z0-.

TIME AFTER END of RUN (min)

FIGURE 2 (A) The turnover of L-['4C]lactate in resting rats. Eachpoint represents an average of values in five animals. Allpoints are expressed as a percentage of the activity incirculation at 5 min. The initial disappearance rate in iron-deficient animlals (dashed linie) is about one-halfthat ofcontrolanimals (solid line). (B) The rate of decrease in plasmalactate beginning immediately after 2 min of the fast run. Theniormal animals (solid line) were infused with lactic acidbefore and during the run to achieve a similar blood lactateelevation. Vertical bars in both figures represent +1 SE.

20 min their average lactate had increased from0.77±0.05 to 0.93+(0.07 mM, and their initial pH of7.46±0.01 was unchanged. Under the same conditions,12 of 27 iron-deficient animals with adljusted hemo-globins were able to run for only 6-12 min, 7 animals

TABLE IIBlood Characteristics before and after a 2-min Fast Run*

Resting Exercised (2-min fast run)

Iron-deficient Iron-deficientControls (Cl) diet Controls (Cl) diet

Hemoglobin before transfusion,gidl twhole blood 13.90+0.20 6.00±0.70 14.50±0.50 4.70±0.20

Hemoglobin concentration after exchangetransfu-sion, gidl wchole blood 9.70+0.10 10.10+0.10 9.80±0.10 9.80±0.10

Lactate, mM 0.78±0.07 1.76±0.164 4.57±0.50 18.35+1.384Pyruvate, mM 0.11±0.01 0. 19±0.02§ 0.30±0.03 0.22±0.02

* Different groups of animals resting and exercised (6 animals).4 Difference between iron-deficient and control animals significant to P < 0.001.§ Difference between iron-deficient and control animals significant to P < 0.05.

132 Finch, Gollnick, Hlastala, Miller, Dillmann, and Mackler

for 15 min, and 8 ran for prolonged periods, well inexcess of 20 min (Table III). Lactate levels wereinversely proportional to running time. Most con-spicuous was the progressive increase in lactate anddepression in pH observed in those iron-deficientanimals who were unable to run beyond 15 min (Fig. 3)as comiipared with either controls or iron-deficientanimals who ran over 30 mnii (P < 0.001).The relationship of work performance to a-glycero-

phosphate activity. The separationi of exchanige-transfused iron-deficient animiials into two groups onthe basis of their running ability permitted a furtherexaminatioin of the relationship between a-glycero-phosphate oxidase activity of muscle mitochondria andwork capacity. Two groups of ainimals were identified.Group 1 consisted of aniimals who had been oIn ainiron-deficient diet with a mean hemoglobiin of 5.6+0.3g/dl before exchange transfusion and who ran 15 min orless on the slow run. Group 2 consisted of iron-deficieintanimals with a meian hemoglobin of 5.5+0.4 g/dl l)eforeexchange who rain more thain 70 min oIn the slow run.Mean concentrations of the enzyme for the twogroups are showin in Table IV. A highly siginificanitdifference in the a-glycerophosphate oxidase activitybetween the two groups wvas found despite theiridentical deficit in circulating hemoglobin beforethe study.Lactate vs. acidosis as a cause of wvork imilpairmtietit.

Data thus far suggested that tissue changes in irondeficienicy, perhaps related to a decrease in a-glycero-phosphate oxidase activity resulted in a lactic acidosisand impairmnent of runniing. To determine whetherlactic acid would produce this saimie reductioin inrunning time in normncal animiicals, L-lactate wvasinfused for a period of 15 min at rest, aind the infusionwas continued durinig a fast run on the treadmiiill. Areduction in runniing time resulted at a mieani lactateconcentration of 18.4 mM, simiiilar to the level reachedin iron-deficient animilals (Table V).

12 -''ss -73

1123761

4 5s 70

9 1f

RUNNING TIME ( min

FIGURE 3 Chacnges in blood lactate and pH concentrationin iron-deficient animals unable to run 20 min (slow run) aredisplayed. Each (lot and vertical line represent the meantSEof values obtained in six animiials. Normal animals run at thesamiie rate had a mleani plasma lactate of0.8+0.2 mMI at 15 min.

The iuierease in lactate was associated with aproportionatl decrease in pH, so that it was not clearwhich was initerferinig with work performanciee. Ac-cordingly, bicarbonate and(l lactate were simiiultanieouslyinfused inito niormllal rats during a fast runi until theystopped runninig (Tatble V). Despite the miiainitenianiceof a normal venous bloo10 pH, a high bloodl lactate wasassociatedl with a malcrke(d reductionl in runninig timiie.The higher lactatte coneenitrationi reachedl by animaicllsof group 2, inifuisedl with lactatte at the samcle rate asanimlals of group 1, was presumiied to b)e the effect ofan arteriatl blood alkalosis. Ironi-deficienit aniimalslls whoshowe(d at miarked inierease in lactiate (lutinig runniinlgbut in whomii the decrease in pH was prevenited 1y a

bicarbonate inifusioni also showed nio imiiprovemiient inwork performanltlce (Table VI).Energy substrate. Although the iniereatse in lactatte

rather thanl the acid pH seeme(l responsible for cessationi

TABLE IIIVetna Caval Blood Values Obtaineed at the Enid of a Slotw Runl Grouped According

to thze Wl'ork Performatnce of the Animals*

Hemoglobin HemoglobinNunuber of l)efore exchanige after exchanige

Animiials animiials transitisioll tranisf'usioll pH Lactate

gldl m Al

Controls (C2) 10 12.0±0.3 10.1±0.1 7.46±0.012 0.93±0.07Iron-deficient (ran <15 min) 12 5.0±0.2 9.9±0.1 6.96±0.0304 16.20±0.834Iron-deficient (ran 15 min) 7 5.0±0.3 9.8±0.1 6.93±0.0504 17.40±1.004Iron-deficient (ran >15 min) 8 5.4±0.2 9.9±0.1 7.36±0.030 3.70±0.80

* Animals which stopped running before 15 min were sampled through the inlyinlg catheter at thetime they stopped; animals running longer were sampled at 15 min while they wvere running.t Difference between iron-deficient and control animals significant to P < 0.001.

Lactic Acidosis as a Result of Ironi Deficietncil 133

TABLE IVa-Glycerophosphate Oxidase Activity in Control and Iron-Deficient Rats

Segregated According to Runnitn£g Ability

Number of Running Specific TotalAnimals animals time activity* activityt

minS

Control (C1) 5 >30 0.0032+0.0003 0.60±0.01Iron-deficient (good runners) 5 >30 0.0025±0.0001§ 0.45+0.03Iron-deficient (poor runners) 5 9-19 0.0011±0.0000' 0.20±0.00'

* Specific activity = ,umol a-glycerophosphate oxidized/min per mg protein.Total activity = ,umol a-glycerophosphate oxidized/min per g tissue.

§ Difference between iron-deficient and control animals significant to P < 0.05."Difference between iron-deficient and control animals significant to P < 0.001.

of activity, it was also desirable to establish that energysubstrate was adequate. To examine this, blood fromnanimals running 2 min under conditions of the fast runwas examined. The mean plasmiia glucose of six iron-deficient animals whose original hemoglobin of 5.6 ghad been increased to 10.0 g/dl bloodl was 8.7±0.6 mMas compared with 10.4±0.5 in six Cl animiials whohad also been exchange transfusedl. Although thedifference was significant (P < 0.05), the glucoseconcentration of iron-deficient animilals appearedadequate. Corresponding mean values for free fattyacids of 12 iron-deficient animals was 335±29 ,ueq/literand for 12 control animals was 376±46 gseq/liter(P > 0.05). The glycogen content of thigh muscles insix iron-deficient exchange-transfused animals exercisedfor 2 min by the fast run was 29.8±4.1 as comnparedwith Cl values of 32.1±2.1 g/100 mnM of glucose U/kgwet muscle tissue (P > 0.05).

In situ studies of muscle futnction. Electricalstimulation of the in situ isolated muscles did notreveal any major defect in the contractile or fatigueproperties (Table VII). These studies examined both

the highly aerobic soleus mu;sele andl the mixed-fibered gastrocnemius muscle to insure that an effectspecific to one fiber type was not occurring. Calculationsof mean tension-time units (the average tensionimultiplied by the time of coontraction) in the normiialand iron-deficient animals were virtually ideentical withvalues of 98.8 and 98.2 for the gastrocnemi-ius anid301 and 299 for the soleus muscle. The lack of anlydifference in tension-time units between the stimulatedmuscles rules out such a specificity. Chemiiical studiescarried out on the soleus muscle showed no significantdifference between muscle ATP in iron-deficient ascompared with Cl animals at rest (6.53±0.39 vs.5.13±0.75 mM/kg, P > 0.05) anid after stimulation(3.55±0.35 vs. 3.25±0.55 mM/kg, P > 0.05). Likewise,muscle creatine phosphate after stimulation was similarin iron-deficient and control animuals (3.29+0.90 vs.3.04±1.23 mM/kg), but a difference appeared to existbetween resting values of 6.15±0.18 in iron-deficientvs. 10.75± 1.46 mM/kg in C2, P <0.01. Blood lactatelevels changed <0.5 mM in all animals stimulated,presumably because of the small mass of muscle

TABLE VEffect of Lactic Acid Infusion in Normal Rats on Running Time (Fast Run)

Blood values at end of run

Animals Running time Lactate pH Po2 Pco2

min mM torr torr

Group 1*Base-line run C1 18.00+1.4 11.55+0.79 7.204±0.028 24.9+2.24 37.5+1.64Lactate run 7.00+ 1.4§ 18.37± 1.56"1 7.000+0.054"1 23.6+2.01 39.5+2.80

Group 2tBase-line run C1 19.90± 1.4 10.58+0.81 7.263+0.027 26.3+3.20 36.9+0.32Lactate + bicarbonate run 7.95± 1.5§ 27.40± 1.44§ 7.435+0.027"1 26.3+1.60 34.0+4.80

* These six animals had a mean hemoglobin before exchange transfusion of 11.8+0.5 g/dl and were exchanged to 10.0+0.1 g/dl.These six animals had a mean hemoglobin concentration of 13.5+0.3 g/dl before and 10.0+0.1 g/dl after exchange transfusion.

§ Difference between iron-deficient and control animals significant to P < 0.001."Difference between iron-deficient and control animals significant to P < 0.05.

134 Finch, Gollnick, Hlastala, Miller, Dillmann, and Mackler

TABLE VIEffect ofBicarbonate Infusion on Running Times of Six Iron-Deficient Rats (Slow Run)

Blood values at end of runRunningtime Lactate pH Po, Pco,

mtn mM torr torr

Base-line 12.3+1.5 15.52+±0.78 7.04+0.04 46.4+±4.5 34.1+2.6Bicarbonate 12.6±0.6 18.05±1.95 7.47±0.04* 33.5±4.1 35.2+2.2

* Significance between bicarbonate and base-line runs of P < 0.001.

contracting. Muscle lactate of control animals wasincreased from 1.83+0.43 resting to 11.67+4.31 mMwith stimulated, whereas muscle lactate ofiron-deficientanimals increased from 2.79+0.6 to 5.7+ 1.11 mM. Thegreater lactate concentration of the soleus muscle inexercising controls as compared with that of iron-deficient animals (P > 0.05) was unexplained, butneither reached the high levels seen in the runninganimal.

DISCUSSION

Iron deficiency causes depletion of iron-containingcellular compounds (17-20). The degree of thisdepletion is a function of a number of factors including

TABLE VIISummary of Contractile Data for the Stimulated Muscle

of Normal and Iron-Deficient Rats*

Control (Cl) Iron-deficient

Group 1Gastrocnemius

stimulationPeak tension, g 262.50+22.40 212.50+11.604Peak tension, g/g 176.90±+13.90 197.40±6.82Time to 50% of peak

tension, min 0.75±0.05 0.93+0.07tTime to 25% peak

tension, min 3.86±1.78 2.80±0.78Group 2

Soleus stimulationPeak tension, g 13.00+0.84 10.30±0.634Tensions after 30

min, g 6.90+0.04 9.80±0.211

* The six animals in group 1 had a mean hemoglobin of13.8±0.6 g/dl before and 10.1±0.1 after exchange transfusion.The six iron-deficient animals in group 1 had a meanhemoglobin of 5.2+0.2 before and 9.9+0.1 after exchange.Group 2 normal animals had a mean hemoglobin of 13.5+0.3before and 10.1±0.1 after exchange. Group 2 iron-deficientanimals had a mean hemoglobin of 5.6±0.2 before and9.9±0.1 after exchange.4 Difference between iron-deficient and control animalssignificant to P < 0.05.

age of the animal and severity and duration of thedeficiency (21). In a previous study (1) the feedingof an iron-deficient diet to 4-wk-old rats for 1 moreduced blood hemoglobin concentration and musclemyoglobin to between 40 and 50% of basal levels,whereas cytochrome a, b, and c and a-glycerophosphateoxidase activities were reduced to 50 and 60%. In thisstudy a similar degree of depletion was produced. Itwas felt advisable to have two types of control animals:Cl animals were on a normal diet containing abundantiron, whereas C2 animals were on the identical dietused for iron-deficient animals but were givenparenteral iron weekly. Although some minor differenceswere observed between these two controls, probablyrelated to the lesser amount of available iron in theC2 animal, these were insignificant when comparedwith the differences between controls and iron-deficientanimals. An additional feature of the experimentalmodel was the use of exchange transfusion to eliminatethe effects of anemia. It was found that tissue irondysfunction persisted for at least 1 wk after thehemoglobin of iron-deficient animals had been raisedby exchange transfusion (1).Two intensities of physical activity were employed

to examine work performance. The fast run consistedof an incremental work load designed to test maximalwork capacity. Even control animals were unable tocontinue much beyond 20 min, and the iron-deficientnonanemic animal usually began to rest intermittentlyby the end of the 2nd min. The slow run was anattempt to determine that level of activity at whichcontrol animals and some iron-deficient animals mightattain an equilibrium state, in which energy productioncould be sustained for long periods at a level adequatefor the work output required. This permitted a separationof iron-deficient animals into those with severe vs. mildimpairment in work capacity.A marked curtailment in work performance by

iron-deficient animals had been previously demon-strated, and it was proposed that the critical featurewas a decrease in the mitochondrial enzyme system,a-glycerophosphate oxidase (1). Evidence was circum-stantial in that the concentration of the enzyme in themuscle paralleled changes in work performance. Other

Lactic Acidosis as a Result of Iron Deficiency 135

iron compounds in muscle such as myoglobin andcytochromes were excluded as a cause of muscledysfunction because their concentrations, althoughdecreased, did not improve with iron therapy duringthe time required to normalize running. The relation-ship between the a-glycerophosphate oxidase systemand work performance was documented in a somewhatdifferent way in this study. Iron-deficient animals withthe same degree ofanemia before exchange transfusion,after their hemoglobin was adjusted to 10 g/dl, weresegregated on the basis of their running time. A muchlower a-glycerophosphate oxidase activity was foundin those animals with poor running times as comparedwith iron-deficient animals who could continue theslow run beyond 20 min (P < 0.001). Thus, a-glycero-phosphate oxidase deficiency is either the cause of thefunctional abnormality or is closely coupled with it. Onthe other hand, the dissociation between the pre-exchange hemoglobin concentration and work per-formance indicates that hemoglobin concentration perse is an unreliable indicator of the severity of tissueiron deficiency.The metabolic effects of iron deficiency within the

skeletal muscle cell are undoubtedly complex. Oxidativephosphorylation is decreased along the pyruvate,malate, succinate, and a-glycerophosphate oxidasepathways (1). a-glycerophosphate oxidase itself hastwo effects, one involving oxidative phosphorylationwithin the mitochondria and the other a couplingwith cytoplasmic NADH to regenerate cytoplasmicNAD (22). Excess lactate could then result fromconversion of pyruvate to lactate as a means ofregenerating NAD and could also come from impairedmitochondrial phosphorylation with accumulation ofpyruvate. Because the metabolic reserve of the cellis not known, one can only speculate that the recoveryof a-glycerophosphate activity makes it possible forthe muscle to function at maximiium work capacitydespite other residual mitochondria deficiencies.The relationship between lactic acidosis and the

cessation of running was impressive. By employingthe slow run it was possible to select some iron-deficient animals who could run over extended periodsof time and who did not develop high blood lactateconcentrations, whereas other animals had a progres-sive elevation and discontinued work at a lactate levelof about 18 mmol, a level similar to that found iniron-deficient animals in the fast run and to normalanimals infused with lactate at a point when theystopped running. Although this strongly suggested thatlactic acidosis forced a discontinuation of activity,other possibilities needed to be considered. A decreasein oxygen supply as a result of anemia (23) or ofdecreased cardiac output (24) can produce lacticacidosis, but the former was excluded by exchangetransfusion and the latter was excluded by blood gas

studies. In fact, there was a considerable increase inthe in vivo oxygen tension of arterial and venousblood, largely attributable to the lower pH (Bohreffect) in the iron-deficient animal. Other nutrientsrequired for normal muscle metabolism, includingblood glucose and free fatty acids, as well as muscleglycogen, appeared adequate at the time that activityceased, and at any rate a deficiency would beexpected to reduce blood lactate (25).One way of insuring adequate substrate and at the

same time limiting the accumulation of lactic acid wasby the use of an in situ muscle preparation. Hereblood flow to the working muscle could be maximallyincreased, while the production of lactate within thesingle muscle would be small compared with thedisposal capacity of the body. In this setting bothsoleus and gastrocnemius muscles of the iron-deficient animals performed as well as those ofcontrols.Such results would seem to exclude structuralabnormalities of the muscle as a cause of impairedperformance and would still be consistent with ahypothesis that lactic acidosis was required to impairmuscle work. Lactate concentrations in these musclesafter stimulation were less than those observed withtreadmill exercise, perhaps because of a lower workrequirement but also because of the greater gradientbetween muscle and blood in the in situ musclepreparation.One of the questions raised by these studies is the

way in which lactic acidosis impairs work performance.Muscles are known to function well at an intracellularpH calculated to be as low as 6.4 (26). Because themaintenance of a normal blood pH did not improverunning time either in the iron-deficient or normalanimals infused with lactate, some effect of lactateitself rather than acidosis was likely responsible.Admittedly, blood rather than muscle pH was monitored,but the similar impairment of work performance withinfusion of lactate into normal animals along with therapid escape of hydrogen ions from muscles (27)directs suspicion to the lactate concentration.This is not to say that severe acidosis created by

lactate excess is without effect. Certainly there isample evidence of its ability to impair cardiac function(24), to impair vasomotor regulation (28), and to impairthe catabolism of lactate itself -in the liver (29). Ourobservation, however, differs from the usual descriptionsof lactic acidosis in that it occurs in the face ofincreased physical activity, and focuses rather onstriate muscle performance.At the present time the clinical implications of these

observations are conjectural. It is not known whetherhumans with chronic iron deficiency have limitationsin work performance based on a similar tissueabnormality. Although it may be difficult in man toseparate the known decrease in work performance

136 Finch, Gollnick, Hlastala, Miller, Dillmann, and Mackler

associated with anemila (30) from la similar tissuelesion, divergences mnay occur between the effect ofiron deficiency on1 hemoglobin concentration ascompared with its effect on tissue enzymes. It is alsopossible that iron deficiency may contribute to othertypes of lactic acidosis described in main (24).

ACKNOWLEDGMENT

This vork wcas supported in pait by National Institutes ofHealth gracnts HL-06242, HL-18527, HL-12174, RCDAHL-00182, GM-23006, HD-05961, and HD-02274.

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Lactic Acidosis as a Result of Iron Deficientcy 137