THE EXCRETION OF l-MALIC ACID IN RELATION TO THE TRICARBOXYLIC ACID CYCLE IN THE KIDNEY* By P. VISHWAKARMAt AND W. D. LOTSPEICH (From the Department of Physiology, University of Cincinnati College of Medicine, Cincinnati, Ohio) (Submitted for publication May 27, 1958; accepted October 16, 1958) The tricarboxylic acid cycle, described by Krebs and Johnson (1), has become generally accepted as the final common pathway for the aerobic oxidation of carbohydrate, fat and protein in most tissues of a large number of animal species. For reference during the discussion that follows, this cycle is presented in its usual schematic form in Figure 1. PROTEIN CARBOHYDRATE FAT ACETYL-CoA + 2H OXALACETATEH20 MALATE CITRATE H20 f \ H20 FUMARATE Cis- ACONITATE SUCCINIC- 4l. H20 2H DEHYDROGENASE SUCCINATE ISOCITRATE H20O 2H L- KETOGLUTARATE OXALOSUCCINATE CO2 FIG. 1. DIAGRAMMATIC REPRESENTATION OF THE TRI- CARBOXYLIC ACID CYCLE This sequence of biological oxidations is pres- ent in mammalian kidney, and all of its inter- mediate organic acid substrates have been identi- fied at one time or another in urine of lower ani- mals and man (2). Under certain circumstances * This work was supported by grants (A-1381) from the National Institutes of Health, United States Public Health Service, and the American Heart Association and the Youngstown Area Heart Association. t Research Fellow in Physiology, on leave from State Medical Service of Bihar, India. the excretion of some of these acids shows re- markable variations. For instance, in metabolic alkalosis the rate of excretion of citrate and to a lesser extent of a-ketoglutarate is greatly elevated (3-6). On the other hand, in metabolic acidosis, or more precisely in conditions favoring intracel- lular acidosis such as potassium deficiency (4, 5) or the administration of Diamox@ (7), the rate of excretion of these same acids is reduced al- most to zero. The way in which fluctuating acid-base balance causes these changes in organic acid excretion is not certain. However, a large body of indirect evidence supports the concept that some of these acids, besides undergoing glomerular filtration and tubular reabsorption, are produced in the renal tubular cells and secreted into the urine. It is supposed that it is this tubular synthesis that is sensitive to pH changes within the cells and thus affects the changing rates of organic acid excre- tion. Such would appear to be the case for citrate and a-ketoglutarate although direct evi- dence for their tubular secretion is not available. In the case of malic acid, however, such evidence is available and constitutes the main subject of the present paper. In 1953 Craig, Miller, Owens and Woodward reported (8) that during the intravenous infusion of sodium salts of either a-ketoglutaric or suc- cinic acids in dogs there was both glomerular fil- tration and net tubular secretion of malic acid. Under these circumstances they found that the infusion of malonic acid, the succinoxidase in- hibitor, caused an abrupt change in the pattern of malic acid excretion to one of glomerular filtra- tion with net tubular reabsorption. It is the purpose of the present paper to report experiments on malic acid excretion in the dog that confirm and extend these findings of Craig and co-workers and include a description of the varying patterns of malic acid excretion during 414
Transcript
THE EXCRETIONOF l-MALIC ACID IN RELATION TO THETRICARBOXYLIC ACID CYCLE IN THE KIDNEY*
By P. VISHWAKARMAtAND W. D. LOTSPEICH(From the Department of Physiology, University of Cincinnati College of Medicine,
Cincinnati, Ohio)
(Submitted for publication May 27, 1958; accepted October 16, 1958)
The tricarboxylic acid cycle, described by Krebsand Johnson (1), has become generally acceptedas the final common pathway for the aerobicoxidation of carbohydrate, fat and protein in mosttissues of a large number of animal species. Forreference during the discussion that follows, thiscycle is presented in its usual schematic form inFigure 1.
PROTEIN CARBOHYDRATE FAT
ACETYL-CoA+
2H OXALACETATEH20
MALATE CITRATE
H20 f \ H20
FUMARATE Cis- ACONITATESUCCINIC- 4l. H20
2H DEHYDROGENASE
SUCCINATE ISOCITRATE
H20O 2H
L- KETOGLUTARATE OXALOSUCCINATE
CO2
FIG. 1. DIAGRAMMATICREPRESENTATIONOF THE TRI-CARBOXYLICACID CYCLE
This sequence of biological oxidations is pres-ent in mammalian kidney, and all of its inter-mediate organic acid substrates have been identi-fied at one time or another in urine of lower ani-mals and man (2). Under certain circumstances
* This work was supported by grants (A-1381) fromthe National Institutes of Health, United States PublicHealth Service, and the American Heart Association andthe Youngstown Area Heart Association.
t Research Fellow in Physiology, on leave from StateMedical Service of Bihar, India.
the excretion of some of these acids shows re-markable variations. For instance, in metabolicalkalosis the rate of excretion of citrate and to alesser extent of a-ketoglutarate is greatly elevated(3-6). On the other hand, in metabolic acidosis,or more precisely in conditions favoring intracel-lular acidosis such as potassium deficiency (4, 5)or the administration of Diamox@ (7), the rateof excretion of these same acids is reduced al-most to zero.
The way in which fluctuating acid-base balancecauses these changes in organic acid excretion isnot certain. However, a large body of indirectevidence supports the concept that some of theseacids, besides undergoing glomerular filtration andtubular reabsorption, are produced in the renaltubular cells and secreted into the urine. It issupposed that it is this tubular synthesis that issensitive to pH changes within the cells and thusaffects the changing rates of organic acid excre-tion. Such would appear to be the case forcitrate and a-ketoglutarate although direct evi-dence for their tubular secretion is not available.In the case of malic acid, however, such evidenceis available and constitutes the main subject of thepresent paper.
In 1953 Craig, Miller, Owens and Woodwardreported (8) that during the intravenous infusionof sodium salts of either a-ketoglutaric or suc-cinic acids in dogs there was both glomerular fil-tration and net tubular secretion of malic acid.Under these circumstances they found that theinfusion of malonic acid, the succinoxidase in-hibitor, caused an abrupt change in the pattern ofmalic acid excretion to one of glomerular filtra-tion with net tubular reabsorption.
It is the purpose of the present paper to reportexperiments on malic acid excretion in the dogthat confirm and extend these findings of Craigand co-workers and include a description of thevarying patterns of malic acid excretion during
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MALIC ACID EXCRETION IN THE KIDNEY
infusion of a number of the other cycle acids in-cluding fumarate and l-malate itself. These re-
sults will then be discussed in relation to the gen-
eral problem of organic acid excretion and thebiochemical function of the tricarboxylic acidcycle in the kidney.
METHODS
The experiments were performed on unanesthetized, hy-drated female mongrel dogs trained to lie on an animaltable while loosely restrained. Sodium salts of the acidsof the tricarboxylic acid cycle were administered by con-
stant intravenous infusion. Standard clearance tech-niques were used to quantify the rates of glomerular fil-tration and urinary excretion of the several acids studied.The creatinine clearance was used as a measure of glo-
merular filtration rate by methods described previously(9). Intravenous infusions were administered at a con-
stant rate of 3 ml. per minute. Malic acid was determinedessentially by the method of Hummel (10) which gave
excellent quantitative malate recoveries from both plasmaand urine, even in the presence of considerable amountsof each of the other cycle substrates. Succinate in tung-stic acid filtrates of plasma and in diluted urines was de-termined enzymatically after ether extraction accordingto the method described by Umbreit, Burris and Stauffer(11). Citrate was measured with slight modification ac-
cording to Natelson, Pincus and Lugovoy (12). Malatewas found to be completely filtrable from plasma over a
wide range of concentrations. The ultrafiltration studieswere carried out first with Visking cellophane bags inair. The results of these studies were compared andfound to agree with those from dialysis against phosphatebuffer as described by Taggart (13).
TABLE 1
Experiment illustrating pattern of 1-malic acid excretion and effect of malonate duringinfusion of sodium citrate in dog
Total Glomerular l-Malic acidconcurrent filtration
time rate Plasma Filtered Excreted Secreted Reabsorbed
The pattern of malate excretion during infusionof citrate, a-ketoglutarate, or succinate
Table I shows the details of an experiment il-lustrating the pattern of l-malate excretion duringthe infusion of sodium citrate. Because of itstoxicity when infused at high rates, citrate ad-ministration had to be limited. Nevertheless it isseen that its infusion produced a small but defi-nite elevation in plasma malate concentration tosome 3.0 to 3.4 pg. per ml.; endogenous malatelevels vary from 0.5 to 2 ,Ag. per ml. Further-more, in the first three periods malate excretionwas some 500 ,ug. per minute greater than itsfiltration, indicating that a rather large tubularsecretion of malate occurred during citrate infu-sion even in these relatively small amounts.
Effect of malonate. Since the secretion of mal-ate here must depend first on its synthesis fromcitrate via the tricarboxylic acid cycle steps shownin Figure 1, it would be expected that malonicacid, the succinoxidase inhibitor, would block thispart of the malate excretory process. Indeed,this is what happened. The high rate of tubularmalate secretion seen in the first three periods dis-appeared after malonate and there appeared in-stead a net tubular reabsorption of malate amount-ing to 128 to 105 ,ug. per minute (last threeperiods). Thus there appears to be, as Craig andassociates found, a bidirectional movement of mal-ate across the renal tubule in the dog; the re-
absorptive limb of the process becomes evidentwhen the net tubular secretion is blocked aftermalonate (8).
Experiments similar in design to that shown inTable I were performed with a-ketoglutarate andsuccinate. We wished to see whether the pat-tern of malate excretion seen with citrate was
also observed during 'infusion of these two sub-strates which would likewise have to utilize thesuccinoxidase system in order to give rise to mal-ate. Results of experiments of this type are shownwith a-ketoglutarate in Table II and succinate inTable III. It is apparent in both these experi-ments that the pattern of malate excretion dur-ing either a-ketoglutarate or succinate infusionis qualitatively similar to that seen when citratewas substrate. Plasma malate is elevated as, con-
sequently, is its rate of glomerular filtration.There is a large net tubular secretion which ineach case is changed to a net tubular reabsorptionafter the addition of malonate to the infusion.
With a-ketoglutarate and succinate there are,
however, striking quantitative differences in mal-ate excretion from that seen with citrate. Forinstance, with a-ketoglutarate and even more so
with succinate, plasma malate levels as well as
rates of tubular secretion and tubular reabsorption(after malonate) are considerably higher thanwith citrate. Whether the increasing size of thesefunctions with the three substrates bears any re-
lation to their progressive nearness to succinoxi-dase in the cycle is a possibility but not one sub-
TABLE III
Experiment illustrating pattern of 1-malic acid excretion and effect of malonate duringinfusion of sodium succinate in dog
Total Glomerular I-Malic acidconcurrent filtration
time rate Plasma Filtered Excreted Secreted Reabsorbed
min. ml./min. pg./ml. mg./min. mg./min. mg./min. mg./min.Prime-Succinic acid, 0.56 Gm./Kg. (neutralized with NaOH) + creatinine, 0.08 Gm./Kg.Infuse-Succinic acid, 5.1 mg./Kg./min. -(neutralized with NaOH) + creatinine, 0.7 mg./
ject to proof from these data. The relative small-ness of the malate parameters during citrate infu-sion is most likely related to the fact that itscardiac toxicity limits the amount that can begiven.
The experiments presented thus far are in com-plete agreement with the results of Craig and co-workers (8) and confirm their finding of a bi-directional malate movement in the tubule of thedog kidney, and extend that finding to includecitrate as well as a-ketoglutarate and succinate asmalate precursors in this phenomenon.
The pattern of malate excretion during infusionof fumarate or 1-malate
It next became of interest to study the natureof malate excretion during infusion of substrateslying beyond the malonate-sensitive succinoxi-dase system in the cycle. Experiments were per-formed with two of these: fumarate and l-malateitself.
In the first experiment (No. 20) shown inTable IV sodium fumarate was given. Thisproduced a marked elevation in plasma malate and,hence, in its glomerular filtration. In strikingcontrast to the earlier studies almost all the filteredmalate was reabsorbed; no net tubular secretionwas seen. In addition, the rate of tubular re-absorption of malate was much greater than that
observed during malonate infusion with eithercitrate, a-ketoglutarate, or succinate.
In the second experiment (No. 21) shown inTable IV sodium l-malate was infused in suf-ficient amounts to achieve plasma levels and fil-tered loads analogous to those achieved in thefumarate infusion studies. One can see that withmalate infusion, as with fumarate, there was like-wise glomerular filtration and net tubular reab-sorption of malate; no tubular secretion was seen.However, in striking contrast to the fumarate ex-periment, the reabsorptive rate of malate wasmuch lower, being only 4 to 6 mg. per minute,compared to 27 to 29 mg. per minute at analogousmalate loads during fumarate infusion.
Experiments like No. 21, Table II, were re-peated and rates of malate filtration, excretion andreabsorption during l-malate infusion were mea-sured over a wide range of plasma malate levels.The massed data of six such experiments arepresented in Figure 2. Although the curveswere drawn through the points by inspection, itis apparent that below filtered loads of 4 to 5mg. per minute reabsorption of filtered malate isessentially complete. However, as plasma levelrises a maximum reabsorptive rate (Tm malate)is reached and remains relatively constant over awide range of filtered malate loads. Thus in ex-hibiting a "Tm" (during l-malate infusion), the
L-MALIC ACID FILTERED Mg/MIN.FIG. 2. EXCRETION AND REABSORPTIONOF MALATE IN RELATION TO FILTERED MALATE LOAD
malate reabsorptive mechanism resembles those ofglucose, phosphate, sulfate and some amino acids.
Catalytic effect of fumarate on malate reabsorption
As was noted above in the experiments of TableIV, the rate of malate reabsorption during fuma-rate infusion is some five to six times higher thanit is with equivalent malate loads during sodiuml-malate infusion. In attempting to design fur-ther experiments to clarify the nature of this dif-ference. we were reminded of the catalytic "spark-ing" effect of fumarate in isolated tissues oxidiz-ing pyruvate (14, 15). We wondered whetherfumarate could exert a similar catalytic effect on
malate reabsorption by "sparking" the oxidativeenergy production in the renal tubular cells.
To test this hypothesis, experiments such as
that presented in Figure 3 were performed. So-
dium l-malate was infused at rates similar to thosein Experiment No. 21, Table IV. Analogousfiltered loads of malate were reached, and, as isseen in the first two bars in the bottom set, themalate reabsorptive rate was also similar at about4 mg. per minute. Now while maintaining thismalate infusion constant, sodium fumarate, in one-
sixtieth the molar amount of infused malate, was
added to the infusion and two more clearanceperiods were taken. With the fumarate additionthe rate of malate reabsorption doubled or tripledas can be seen in the second two bars of the lowerset. The massed data of two additional and simi-lar experiments are presented in Figure 4. Ineach of these, two control periods were followedby four periods with fumarate addition. Theblack dots show malate reabsorption during con-
trol periods; these group around the dashed linerepresenting Tm malate taken from Figure 2.
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418
MALIC ACID EXCRETION IN THE KIDNEY
zaa 12-
0:10-00
4-
bi
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4c
qc I c2
zi
202
w
(049'a'a
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2-J
MALATE MALATE+ FUMARATE 2-
MALATE MALATE+ FUMARATE
FIG. 3. AN EXPERIMENT ILLUSTRATING THE CATALYTICEFFECT OF FUMARATEON MALATEREABSORPTION
* MALATE0 MALATE+ FUMARATE
0
0
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0Tm MALIC
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12 16 20 24 28 32L-MALATE FILTERED (MGM/MIN)
FIG. 4. MASSEDDATA OF Two EXPERIMENTSSHOWINGEFFEcT OF FUMARATEONMALATEREABSORPTION
from the glomerular filtrate. These relations asvisualized at present are summarized schematicallyin Figure 5. The upper tubular cell represents themechanisms of malate reabsorption and the lowerone, those of malate synthesis and secretion. Plasmamalate level rises with infusion of citrate, a-keto-glutarate, succinate, fumarate, and, of course, l-mal-ate itself. The malate formed from these severalsubstrates in various tissues, or administered pre-formed, is freely filtrable and passes into the glo-
The open circles represent rates of malate re-absorption during fumarate addition. In all casesmalate reabsorptive rates were significantly higherduring periods when fumarate was infused alongwith the malate.
The word "catalytic" to describe this effect offumarate is used advisedly because these quan-tities of fumarate, of themselves, would neverlead to malate loads or reabsorptive rates such asthese. In addition, the effect is the more strik-ing because it occurs with an actual decline in thefiltered malate load (Figure 3), the result of ageneral reduction of plasma malate seen duringthe infusion of small doses of fumarate withl-malate.
DISCUSSION
The experiments presented here support thetheoretical conclusion that the tricarboxylic acidcycle is involved in the tubular synthesis and se-cretion of malic acid, as well as in its reabsorption
FIG. 5. SUMMARYOF THE PATHWAYSOF MALATE EX-CRETION IN THE KIDNEY
SUMMARYOF EXCRETORYPATHWAYSOF L-MALIC ACID IN THE KIDNEY
GlomerularFiltration
MALATE
FumorateIncreases
MMALATE(Preformed)
4 v-~~
4 ----___ ---_ _ _ _
Lumen Tubular Cell PeritubularSpace and Blood
Stream
419
4i2. VISHWAKARMAAND W. D. LOTSPEICH
merular filtrate where it undergoes tubular reab-sorption (upper cell). This mechanism exhibits aTmduring infusion of l-malate. It is unaffected bymalonate. Catalytic amounts of fumarate accele-rate the reabsorptive capacity of the tubules formalate some two- to threefold, and during the infu-sion of fumarate alone, malate reabsorption is fiveor six times as high as it is at equivalent loadsduring malate infusion. Substrates of the cyclesuch as citrate, a-ketoglutarate and succinate getinto the tubular cell (possibly from both sides asshown in the lower cell) and are synthesized thereinto malate by a malonate-sensitive mechanismpresumably involving the succinoxidase system.That citrate and a-ketoglutarate can gain entranceto the tubule cell from the lumen is known fromthe fact that tubular reabsorption of each acid hasbeen observed (16, 17). These observations onthe several mechanisms of malic acid excretion inthe kidney raise several important points aboutthe nature of the phenomena themselves and theirrelation to the larger problem of organic acid ex-cretion, its function in the overall economy of thebody, and the role of the tricarboxylic acid cyclein renal tubular function.
In measuring "reabsorption" or "secretion" bythe clearance technique, one simply measures netdifference between rate of glomerular filtration andurinary excretion of a substance. This measure-ment does not reveal multidirectional fluxes thatmay comprise the "net" movement, nor does it re-veal the fate of a substance reabsorbed or secretedby the tubules. Thus with a metabolite such asmalic acid the measurement of its "reabsorption"does not tell us whether it has been moved intactwithout utilization from glomerular filtrate toperitubular blood or whether it has been partly orcompletely "utilized" by the tubule cells. There-fore, the catalytic effect of fumarate on malate re-absorption could represent either a facilitation ofits actual transtubular movement, an acceleration ofits tubular "utilization," or both. To differentiatethese possibilities, studies will be required in whichone measures simultaneously excretion of malate byclearance techniques and its utilization by renalarteriovenous sampling. Although such studieshave not appeared for malate, Cohen (17) has re-ported them for other metabolites: lactate, aceto-acetate, pyruvate and a-ketoglutarate. These stud-ies have clearly shown that renal "utilization" and
"reabsorption" of certain metabolites occur simul-taneously and are independently affected by vari-ous experimental manipulations.
It seems, at first, strange that malate secretionis observed during infusion of citrate, a-ketoglu-tarate, or succinate but not during administrationof fumarate or malate. One must presume thatwith the former three substrates malate is syn-thesized in the tubule cells by reactions involvingsuccinoxidase and reaches sufficiently high con-centration there (above that in plasma and glo-merular filtrate) to pass along a concentrationgradient into luminal urine. The equilibriumconstants of both fumarase and malic dehydroge-nase strongly favor such accumulation of malatewithin the cell. The equilibrium constant of thefumarase step is 4.0 to 4.5 at pH 7.3 and 250 C.(18) in favor of malate, and the constant of thediphosphopyridine nucleotide (DPN) -linked malicdehydrogenase step at pH 7.2 and 220 C. is 2.33 x10-5 (19), again strongly in favor of malate.
When fumarate or malate is infused, however,plasma levels of malate are much higher. Thusperhaps under these circumstances concentrationdifferences between cell and lumen are not sogreat, tubular secretion does not occur, and thereabsorptive limb of the bidirectional malate sys-tem becomes predominant. Whether this explana-tion of the observed facts is correct must await fur-ther study, but at the moment it seems a plausibleformulation.
During infusion of citrate and a-ketoglutarate,glomerular filtration and net tubular reabsorptionof these acids have been observed (16, 17). Pre-liminary studies in our laboratory with succinatehave produced succinate to creatinine clearanceratios varying from 0.78 to 1.25, indicating thatthis acid is probably excreted predominantly byglomerular filtration when it is infused in thenormal dog.
In contrast, however, the pattern of excretion ofcitrate and a-ketoglutarate is different when otherintermediates of the tricarboxylic acid cycle areinfused or when acid-base balance is altered.And, as with malate, the evidence, although in-direct, supports the concept that they are syn-thesized from precursors in the tubule cells andsecreted into the urine. For instance, in 1937Orten and Smith (20) showed in dogs that theinfusion of succinate, malate, malonate, or maleate
42-0
MALIC ACID EXCRETION IN THE-KIDNEY
causes a remarkable increase in citrate excretion.A year later Krebs, Salvin and Johnson (21) re-
ported similar studies with the rabbit and rat andexplained Orten and Smith's results in terms of the"citric acid cycle" which by then had been clearlyformulated in Kreb's laboratory. Krebs and co-
workers (21) also explained Orten and Smith'smalonate effect on the basis of its succinoxidaseinhibition: citrate simply accumulated behind theblock in the cycle and appeared in large quantitiesin the urine. Furthermore, in the malonate-treatedanimal, succinate, the plasma level of which was
markedly increased, appeared in large quantitiesin the urine. In the case of citrate and a-keto-glutarate, however, their increased rate of ex-
cretion was not accompanied by any change intheir plasma levels. Thus the evidence indicatedthat tubular synthesis of citrate and a-ketoglu-tarate from the injected precursors must have oc-
curred, just as we have visualized it occurring dur-ing the secretion of malate in the experiments re-
ported in the present paper. Abundant confirma-tion of the observations of Orten and Smith (20)and Krebs, Salvin and Johnson (21) is presentedin the recent comprehensive paper by Nordmannand Nordmann (2).
All these studies make it appear likely that ci-trate, a-ketoglutarate, fumarate, malate and oxalo-acetate excretion during infusion of one of theseacids occurs variously by combined processes ofglomerular filtration and tubular secretion. Reab-sorption may also be occurring, as with malate, butthe net process appears to be tubular secretionunder these circumstances. Only in the case ofmalate, in the study of Craig and associates (8)and the present one, has such a mechanism beenshown experimentally. Similar studies with theseother members of the tricarboxylic acid cycle are
in progress.
It would appear that the organic aciduria ofmetabolic and respiratory alkalosis is also theresult of tubular synthesis and secretion of citrateand a-ketoglutarate. Milne, Scribner and Craw-ford (22) recently reported an observation stronglysupporting this concept. They found that the in-creased citrate excretion of rats given potassiumbicarbonate was associated with a considerable risein renal tissue citrate; in fact, they reported citratelevels comparable to those seen in kidney tissue af-ter fluoroacetate poisoning. One would visualize
the synthesis of citrate and a-ketoglutarate (andthus their urinary excretion) as being regulatedprimarily by intracellular pH. This in turn wouldbe a function of potassium concentration and as-sociated intracellular bicarbonate (23). Organicacid excretion would thus become a function of pHin the tubule cells rather than of urinary pH orgeneral changes in acid-base balance of the body.
In support of this idea is the fact that the"condensing enzyme" in tissues shows an equi-librium state much more in favor of citrate pro-duction as pH rises (19). Evans, MacIntyre,MacPherson and Milne (4) have shown that citrateand a-ketoglutarate excretion rises in potassium-depleted man given potassium chloride even in theface of a falling urinary pH. And Orten andSmith (20) as well as Cooke and co-workers (5)have stressed the independence of organic aciduriaand urinary pH. In like manner both the tubu-lar reabsorption and secretion of malate, in thepresent studies, appear to be independent of uri-nary pH and bicarbonate excretion. For example,in one experiment during the infusion of sodiuml-malate, urine pH gradually rose from 7.30 to 7.81and the rate of bicarbonate excretion, from 86 to182 uM per minute. But malate reabsorption re-mained relatively constant at 3.50 to 5.30 mg. perminute. Similar independence of malate secre-tion rate during succinate infusion has beenobserved.
The functions of organic acid excretion in rela-tion to overall body economy are not completelyclear. Cooke and associates (5) have suggestedthat organic acids may be secreted by the tubulesand function as part of a fixed anion (chloride)conserving mechanism in alkalosis analogous tothe fixed cation conservation mechanism that op-erates in acidosis. The decrease in chloride excre-tion that accompanied the increase in organic acidexcretion in Cooke's (5) studies is suggestive ofsuch a mechanism. However, as we have seen,the term "alkalosis" is probably too restrictive tocover the basic intracellular mechanism regulatingorganic acid excretion.
In addition to this function, Shorr and his as-sociates (24) emphasized that citrate excretionappears to play an important role in the solubiliza-tion of calcium in the urine and thus the preventionof renal calcinosis. Harrison and Harrison (7)in a different approach to the problem have ob-
421
P. VISHWAKARMAAND W. D. LOTSPEICH
served in rats that the administration of Diamox®causes a profound inhibition of citrate excretionwithout a concomitant reduction in urinary cal-cium. Citrate can chelate calcium and in its ab-sence calcium phosphate precipitates, with renalstones forming as a result. In line with the conceptof citrate synthesis presented above, the action ofDiamox® can be seen in terms of a reduction inintracellular citrate synthesis accompanying a re-duced potassium, bicarbonate and pH in the tu-bule cells.
Another function of organic acid excretionappears to be related to the hormonal regula-tion of the menstrual cycle. Shorr, Bernheimand Taussky (25) have observed that citrateexcretion is lowest during menstruation andrises in the immediate postmentrual period un-til, after a dip for a day or two in the mid-period, it rises to even higher rates that are sus-tained until the sharp decline just before the onsetof the next menstruation. Evidence supportingthe sex steroid control of these variations in ci-trate excretion derives from the fact that they canbe produced at will by estradiol injection inamenorrheic females. Testosterone given to hy-pogonadal males also affects citrate excretion butin the opposite direction. The functional basis forthese hormone-induced variations in citrate ex-cretion is not at all understood. They may bethe secondary result of a systemic change in acid-base balance or they may be the result of asteroid effect on tubular production of citrate.They occur without commensurate changes inplasma citrate levels.
The studies reported here have been mainlyconcerned with one of the substrates of the tri-carboxylic acid cycle. To what extent these find-ings with malic acid relate to the general prob-lem of organic acid excretion and its physiologicrole cannot be stated with certainty at present.However, several important facts have emerged.First, this study has clearly demonstrated that atleast one of the members of the tricarboxylic acidcycle can be secreted by the tubules in responseto infusion of certain other members of the samecycle. Secondly, the succinoxidase enzyme sys-tem is apparently an important component of thismechanism. Thirdly, the curious and interestingfumarate "catalysis" of malate reabsorption has
been described. This phenomenon suggests thatelements of the cycle are involved in both thetubular secretion and reabsorption of malate.
Thus the general concept presents itself that thetricarboxylic acid cycle, usually considered as anoxidative mechanism in energy metabolism, func-tions also in the kidney in the synthesis, reab-sorption and secretion of its own intermediates.This may be analogous to the way pyridoxalphosphate functions in amino acid transport (26)in a way quite separate from its role in inter-mediary metabolism of amino acids, or to the wayadenosine triphosphate functions in the red cellmembrane in phosphate transport (27) quiteseparately from its role in intracellular energymetabolism. Much additional work will be re-quired before this hypothesis can stand or fall onthe basis of solid observation.
SUMMARY
The mechanism of excretion of malic acid wasstudied in the dog during infusion of severaltricarboxylic acid cycle intermediates includingl-malic acid itself. With infusion of citrate, a-keto-glutarate and succinate there was glomerular fil-tration and net tubular secretion of malate. Witheach of these substrates following infusion of thesuccinoxidase inhibitor, malonic acid, the net tub-ular secretion of malate completely disappearedand glomerular filtration with net reabsorption ofmalate was seen instead. Thus it appears thatthe tubules are capable of synthesizing malatefrom precursors in the tricarboxylic acid cycle andsecreting it into the tubular urine. In contrast tothese findings, the infusion of fumarate or l-mal-ate leads only to malate filtration and reabsorption.Thus the tubules are capable of both secretion andreabsorption of malate under certain definite con-ditions. During l-malate infusion there was adefinite "Tm" for malate reabsorption that aver-aged 4 to 8 mg. per minute. During fumarateinfusion the capacity of the tubules to reabsorbmalate was greatly increased. This phenomenonwas shown to be the result of a "catalytic" effectof fumarate on malate reabsorption. The relationof these studies to the operation of the tricarboxylicacid cycle in the kidney and the whole problem oforganic acid excretion was discussed.
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MALIC ACID EXCRETION IN THE KIDNEY
ACKNOWLEDGMENTS
The authors wish to thank Mr. Seraphim Woronkowfor valuable technical assistance and Mr. John Pott-schmidt who helped on some of the work as a summer
research project while a medical student.
REFERENCES
1. Krebs, H. A., and Johnson, W. A. Role of citricacid in intermediate metabolism in animal tissues.Enzymologia 1937, 4, 148.
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