A STUDY, BY SIMULTANEOUSCLEARANCETECHNIQUES, OFSALICYLATE EXCRETIONIN MAN. EFFECT OF ALKA-

LINIZATION OF THE URINE BY BICARBONATEADMINISTRATION; EFFECT OF

PROBENECID1

By ALEXANDERB. GUTMAN,T. F. YO, AND JONAS H. SIROTA 2

(From the Department of Medicine, The Mount Sinai Hospital, and the Department of Medi-cine, Columbia University College of Physicians and Surgeons, New York, N. Y.)

(Submitted for publication December 6, 1954; accepted January 26, 1955)

In the course of a study of the uricosuric effectof salicylate in gouty subjects, and its potentiationby bicarbonate administration (1), it became evi-dent that interpretation of the findings would re-quire further investigation into the processes regu-lating salicylate excretion in man. These are ex-ceedingly complex, as indicated in recent reviewsof the subject by Gross and Greenberg (2) andP. K. Smith (3).

According to Kapp and Coburn (4), about 80per cent of administered salicylate is recoverableas the salicyl radicle in the urine of normal man.The remainder is converted in part to gentisicacid and a gentisic-salicyluric acid compound.Some 20 per cent of the salicyl radicle recoveredin the urine in man appears as free salicylate, atleast when salicylate is given orally; the remainderis excreted in conjugated form, combined eitherwith glycine to form salicyluric acid (55 to 60 percent) or with glucuronic acid (20 to 25 per cent).Salicyl conjugates are not detectable in the blood(5).

Upon administration of sodium bicarbonate, therate of urinary excretion of salicylate is greatlyenhanced (6-8). With increasing alkalinity ofthe urine, the proportion of free salicylate risesfrom approximately 20 per cent (after oral ad-ministration) to 60 to 70 per cent or more of thetotal urinary salicylate (6-8).

The prevailing view (9) is that, in man, freesalicylate is filtered at the glomerulus and in partreabsorbed in the tubules. A variable proportionof the free salicylate, under ordinary circumstances

1 This work was supported by grants-in-aid from theNational Institute of Arthritis and Metabolic Diseases,National Institutes of Health; The Arthritis and Rheu-matism Foundation, New York Chapter; and the Ameri-can Heart Association.

2 Present address: San Jose, Calif.

the major proportion, is conjugated in the tubulesand is then presumably excreted, as salicyl con-jugate, by the tubules. Alkalinization of the urineby concomitant administration of sodium bicarbo-nate is believed to enhance salicylate clearance bydiminishing reabsorption of salicylate by thetubules.

There appears to be no record in the literatureof simultaneous renal clearance studies of salicyl-ate, inulin, and p-aminohippurate in man. Thepresent report summarizes results obtained bythese techniques, including observations on theeffect of alkalinization of the urine by concomitantintravenous infusion of sodium bicarbonate. Theeffect of probenecid (Benemid@) on salicylateclearance was also studied.

METHODS

The experiments were conducted in gouty subjects sinceour initial interest was in concomitant changes in urateclearance (1). The 14 patients (ages 28 to 54) selectedfor study were in the non-tophaceous stage of the dis-order and gave no clinical or laboratory indication ofovert cardiovascular or renal disease. All had been main-tained on a constant diet low in purine and restricted inprotein (60 to 75 gnL per day) preceding study and werein the morning post-absorptive state at the time of theexperiment. Urine flow not less than 3 ml. per min.(usually 6 to 10 ml. per min.) was maintained throughoutthe period of study by liberal ingestion of water.

Glomerular filtration rate and renal plasma flow wereestimated as previously described (10), using standardmethods (11). Priming and sustaining infusions of inu-lin and p-aminohippurate in isotonic saline solution wereadministered intravenously by means of a Bowman infu-sion pump at rates calculated to maintain plasma con-centrations of 30 mg. per cent and 2 mg. per cent, re-spectively. Thirty minutes were allowed for equilibra-tion. Three control inulin clearance periods, each of 15to 20 minutes' duration, were first obtained in each sub-ject. After the third control clearance period, sodiumsalicylate was administered intravenously to 10 subjects.

711

ALEXANDERB. GUTMAN, T. F. YU, AND JONAS H. SIROTA

Patients A. S., J. H., D. R, and A. M. received 4.5 gm.in 500 ml. saline solution at a rate of 4 ml. per min. un-til the end of the experiment. As this slow rate of infu-sion did not always yield satisfactorily sustained plasmasalicylate levels, the remaining subjects first received apriming dose of 3.0 gm. sodium salicylate in 100 ml. salinesolution at a rate of 8 to 10 ml. per min., then a sustain-ing infusion of 2.0 gm. in 500 ml. saline solution at arate of 4 ml. per mir. After allowing 20 to 30 minutesfor equilibration, three 20-minute clearance periods, wereobtained. Then intravenous administration of 7.5 percent sodium bicarbonate was begun and, after allowing20 to 30 minutes for equilibration, urine and blood sam-ples were collected for three or more additional 20-minute periods. Patients A. S., J. H., D. R., and A. M.first received 3.75 gm. sodium bicarbonate within 3 to 5minutes, followed by infusion of 11.25 gmn. more slowlyover a span of approximately one hour, in combina-tion with the sustaining solution of sodium salicylate.Patients H. G., S. L., L. C., J. S., B. R., and H. W.were first given 11.25 gm. sodium bicarbonate by rapidinjection (12 to 15 minutes), then 3.75 gm. (in CaseH. W., 7.5 gm.) sodium bicarbonate more slowly over aperiod of one hour, in each instance together with thesustaining solution of sodium salicylate.

Urine samples were secured by means of an in-dwellingcatheter and at the end of each clearance period thebladder was emptied completely after washing with 20ml. saline solution. Blood samples were drawn at themid-points of the first and third collection periods duringthe control, salicylate, and salicylate and bicarbonate in-fusions. Representative plasma concentrations of inulin,p-aminohippurate and total salicylate for each clear-ance period were obtained by plotting the concentrationsin the samples semi-logarithmically against time and ex-trapolating back 2.5 minutes (approximate urine delaytime) from the mid-time of each urine collection period.

In four additional subjects the clearance studies wereperformed in the same way except that, instead of bi-carbonate, probenecid (20 mg. of the sodium salt per kg.body weight) was infused at the end of the third salicyl-ate clearance period.

Inulin in plasma and urine was determined by themethod of Schreiner (12), p-aminohippurate by themethod of Smith, Finkelstein, Aliminosa, Crawford, andGraber (13). The pH of the urine was measured at thebedside, immediately after delivery, by a Cambridge glasselectrode pH meter. Salicylate was determined by themethod of Brodie, Udenfriend, and Coburn (5) inplasma, plasma ultrafiltrate, urine before hydrolysis("free" urinary salicylate) and after acid hydrolysis("total" urinary salicylate). Hydrolysis was performedas described by Williams and Leonards (7), autoclaving1 to 2 ml. diluted urine with 0.5 ml. 6N HCI for threehours at a pressure of 20 lbs. per sq. inch. In the caseof "free" salicylate, the values are too high because of thepresence and chromogenicity of salicyluric acid, whichgives 35 per cent as much color with the ferric nitratereagent as salicylic acid; the glucuronide is not chromo-genic (5). No correction (6, 8) was attempted, how-

ever, because under the conditions of the acute experi-ments most of the salicyl radicle (except in Case A. M.)was excreted in the form of unconjugated salicylate evenbefore sodium bicarbonate was administered.

Ultrafiltration of the plasma salicylate was carried outby means of a Simms-Sanders "surge" apparatus (14).The concentration of ultrafiltrable salicylate in plasmawas expressed in terms of plasma water by use of theformula of McLean and Hastings (15), a serum proteinconcentration of 6 gm. per cent being assumed.

In calculating "free" salicylate clearances, urinary ex-cretion of "free" salicylate (UV) was represented bymg. "free" salicylate excreted per minute, as determinedin unhydrolyzed urine; plasma concentration (P) wasexpressed as mg. salicylate per ml. plasma ultrafiltrate.For "total"salicylate "clearances," UV values were rep-resented by mg. "total" salicylate excreted per minute, asdetermined in hydrolyzed urine. P values were againrepresented by mg. salicylate per ml. plasma ultrafiltratesince the plasma concentrations of salicyl conjugates aretoo low for precise measurement by available techniques.Excretion of gentisic acid and related degradation prod-ucts was not considered in calculating salicylate clearancebecause there is no evidence that these substances areformed in the kidney.

RESULTS

Total and ultrafiltrable plasma salicylate levels(Table I)

Sodium salicylate was infused at a rate suffi-cient to maintain plasma salicylate levels above15 mg. per cent in most experiments. The markedfall in plasma salicylate concentration which ordi-narily occurs when the salicylate clearance is in-creased by administration of an alkalinizing agent(16) is masked under the conditions of these ex-periments by the continued infusion of sodiumsalicylate. The changes in plasma total salicylate(Ps) recorded in Table I therefore simply reflectthe rate of salicylate injection.

Most of the salicylate present in plasma is knownto be bound to plasma proteins (6, 7, 17-20),hence is not filtered at the glomerulus. The re-sults of 50 plasma ultrafiltrations carried out inthe course of this study are summarized in Figure1. The linear distribution of points indicates thatthe ratio of ultrafiltrable plasma salicylate to totalplasma salicylate is relatively constant, 30 - 10 percent (mean 34 per cent) in terms of plasma water,over the range 2.0 to 35 mg. per cent total plasmasalicylate. At levels above this the curve swingssharply upward, as described by previous investi-gators (6, 7, 19), and as suggested by the one

712

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FIG. 1. RELATION OF PLASMA ULTRAFILTRATE SALICYL-ATE TO PLASMATomAL SALICYLATE

All concentrations are expressed in terms of plasmawater. Solid dots represent values obtained when so-

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point in this range in Figure 1. At such highplasma salicylate levels the capacity of the plasmaproteins to bind salicylate apparently is exceededand the excess circulates as free salicylate.

Administration of sodium bicarbonate does notaffect the ultrafiltrability of the plasma salicylate(7), as shown in Figure 1.

Effect of salicylate on glomerular filtration rateand renal plasma flow (Table I)

C1,, in the control period was within the normallimits of variation except for somewhat loweredvalues in Cases H. W., A. S., H. G., and J. S.(Table I). The mean Cl. of the control periodswas 109 ml. per min. Infusion of sodium salicyl-ate alone or combined with sodium bicarbonatedid not cause any consistent change, the mean Cl.of these periods being 112 and 116 ml. per min.,respectively. CpAH also showed no consistentchange in the salicylate and salicylate + NaHCO.

periods, the means being 490 ml. per min. in thecontrol period and 493 and 535 ml. per min., re-spectively, in the two experimental periods.

Salicylate clearance; effect of sodium bicarbonateinfusion (Table I)

The "free" salicylate clearance ranged from 8.3to 62.7 ml. per min. (mean 29.7 ml. per min.), the"total" salicylate "clearance" from 18 to 79 ml.per min. (mean 44.2 ml. per min.). Low salicyl-ate clearance values occurred in association withthe more acid urinary pH levels.

Upon infusion of sodium bicarbonate, the "free"and "total" salicylate clearance invariably rose.In those clearance periods in which the urine pHexceeded 7.0, the range in "free" salicylate clear-ance was 49.8 to 195 ml. per min. (mean 100 ml.per min.), that of "total" salicylate "clearance"

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RATIOS TO URINE PH BEFOREANDAFTER ALKALINIZATIONOF THE URINE BY SODIUM BIcARBONATE INFUSION

Solid dots represent "free" salicylate, hollow dots"total" salicylate.

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ALEXANDERB. GUTMAN, T. F. YU, AND JONAS H. SIROTA

was 49 to 191 ml. per min. (mean 113 ml.per min.). Alkalinization of the urine by infu-sion of sodium bicarbonate effected an approxi-mately three-fold mean rise in salicylate clearance.

The mean ratio of "free" to "total" salicylateexcreted in the urine rose from 0.71 to 0.91 whenthe urine was alkalinized by infusion of sodiumbicarbonate. The initial ratio, 0.71, was consider-ably higher than the ratio of approximately 0.25observed after oral administration of salicylate.This high initial ratio is ascribed to the sustainedintravenous infusion of sodium salicylate at arate rapid enough to maintain high plasma salicyl-ate levels and UV values, and presumably in ex-cess of the rate and capacity of tubular conjuga-tion; and the fact that the initial urine pH ex-ceeded 6.0 in most instances (Table I).

The clearance ratios, C"ire" s/Cin and C"tot,,a,, s/C1., show the same general trends as the salicylateclearance, since Cin did not change consistently.Figure 2 brings out more clearly the effect of al-kalinization of the urine upon "free" and "total"salicylate clearance in relation to the inulin clear-ance. The "free" salicylate/inulin clearance ratioexceeded 1.0 in 11 of 15 observations at urinarypH levels above 7.5; in 3 of 12 observations aturinary pH levels of 7.0 to 7.5; and only once in

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34 observations at urinary pH levels below 7.0.A clearance ratio equal to or greater than 1.0 wasobtained in at least one salicylate + bicarbonatecollection period in six of eight patients whoseurinary pH was brought to levels exceeding 7.3.

Effect of sodium bicarbonate infusion on "net"tubular reabsorption of "free" salicylate

Table I includes a recalculation of the clearancedata to indicate the "net" tubular reabsorption of"free" salicylate (filtered load minus UV) at dif-ferent levels of plasma salicylate, and the effect ofalkalinization of the urine upon this quantity.These relationships are brought out more clearlyin Figure 3 in which the data in individual casesare plotted as lines connecting points representing"net" tubular reabsorption of "free" salicylate be-fore and after infusion of sodium bicarbonate. Inthe few cases with initial urine pH below 6.0,more than 90 per cent of the filtered "free" salicyl-ate is reabsorbed. As the urine pH rises the "net"tubular reabsorption declines, very sharply as therange of alkalinity is approached and reached.At urine pH levels in excess of 7.5, negative valuesare obtained, reflecting accumulation utilizing theenergy of a pH gradient and/or tubular excre-tion of "free" salicylate.

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FIG. 3. RELATION OF "Nrr" REBSORTONOF "FE" SALICYLATE AT VAY-ING SALICYLATE LoADS To UiiNE PH

Each line connects points relating to a single patient, identified by a num-ber corresponding to the case number in Table I. Hollow dots representvalues obtained when sodium salicylate alone was infused, solid dots whensodium salicylate and sodium bicarbonate were infused concurrently.

716

SALICYLATE EXCRETION IN MAN

Effect of probenecid on salicylate clearance

Infusion of probenecid depressed urinary excre-

tion of "total" and "free" salicylate in each of thefour patients studied (Table II). The mean fallin salicylate/inulin clearance ratios was 33 per

cent for "total" salicylate and 40 per cent in the

case of "free" salicylate. These declines occurreddespite a slight increase in pH of the urine. Inaccord with previous findings (10), CpAH was de-pressed, from 498 to 380 ml. per min. and from495 to 355 ml. per min. in the two cases studied(Table II). Ci. is not significantly affected byprobenecid (10).

DISCUSSION

In the present study, standard simultaneousclearance techniques for the measurement of dis-crete renal functions were applied to the renal ex-

cretion of salicylate in an attempt to assess therelative importance of each phase of a complexfour-phase system involving filtration at the glo-merulus, tubular reabsorption, tubular conjuga-tion, and tubular excretion. There are additionalexcretory processes implicating extra-renal oxi-dation of salicylate to gentisic acid and relatedcompounds, and renal excretion of these metabolicproducts, but they will not be considered in thisdiscussion.

Before proceeding to an analysis of each phaseof the renal excretion of salicylate and salicylconjugates, certain limitations in procedure andinterpretation should be pointed out. In the case

of "total" salicylate, currently available analyticalmethods do not permit precise measurement of in-

dividual salicyl conjugates in the urine or theirdetection in plasma. The values cited for UV

and P consequently do not fulfill the requirementsfor calculation of valid clearance data and will notbe so considered. In the case of "free" salicylate,the values for P probably include undetectablysmall quantities of salicyl conjugates and the UVvalues do not take into account the (weak) chro-mogenicity of salicyluric acid. The error in-volved is small, however, in view of the high ra-

tios of "free" to "total" salicylate obtained, par-

ticularly upon alkalinization of the urine. Thedata for filtration and excretion of "free" salicyl-ate would therefore appear to be generally applica-

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ALEXANDERB. GUTMAN, T. F. YU, AND JONAS H. SIROTA

ble and from these data it may be possible to drawinferences as to the processes of tubular partici-pation not amenable to direct measurement.

Tubular reabsorption of free salicylate

It is apparent from the data in Table I that inman, under the ordinary circumstances of excre-

tion of acid urine, the filtered load of "free" salicyl-ate greatly exceeds the quantity of "free" (and"total") salicylate simultaneously appearing inthe urine. In these circumstances most of the"free" salicylate presumably is reabsorbed in thetubules.

As the urine pH rises after administration ofsodium bicarbonate (Figure 2), the salicylateclearance increases (6-8, 19-21). This is not as-

sociated with an increase in the ultrafiltrablefraction of the plasma salicylate (Figure 1), or

with enhancement of the glomerular filtration rate(Table I), hence cannot be ascribed to augmenta-tion of the quantity of salicylate filtered at the glo-merulus. It has been inferred (3, 8, 21) thattubular reabsorption of salicylate decreases as theurinary pH rises.

Our data are entirely consistent with this viewsince the "net" tubular reabsorption of "free"salicylate was found to decrease sharply as theurine pH rose with administration of bicarbonate(Figure 3). Nevertheless, it should be pointedout that the quantity of salicylate reabsorbed inthe tubules cannot be determined precisely by thetechniques employed because of limitations inmeasurements and the possibility of concomitanttubular excretion.

At present, one can only speculate as to whethertubular reabsorption of salicylate involves "active"as well as "passive" transport, and whether theinhibiting effect of bicarbonate administration can

be explained solely on the basis of changes inH+ gradient and related factors in the tubularurine. Penetration of salicyl into cells being a

function of pK, the renal tubular epithelium pre-

sumably is less permeable to dissociated than tonon-dissociated salicylate. Smith (3) has sug-

gested that the decreased salicylate reabsorptionat increasing urine pH can be accounted for bythe increased ratio of ionized to non-ionized sali-cylate in the tubular urine over the pH range inquestion, 5.0 to 8.0. As Dalgaard-Mikkelsen (20)

points out, however, the pK of salicylic acid isabout 3 and there is consequently little change indissociation over this pH range, as is clearlybrought out in titration curves recorded by Kappand Coburn (4). Berliner (22) suggests that ac-tive transport of salicylate nevertheless need notbe postulated: "If relatively complete imperme-ability to the ionized form exists and if equilibriumbetween tubule lumen and peritubule fluid occurs,the concentration of un-ionized salicylate mustapproach equality within and without the tubule.Under such conditions the observed changes in ex-cretion (including excretion, in alkaline urine, ofamounts in excess of those filtered) would bepredicted." There are no data available, however,to indicate whether such a mechanism adequatelyaccounts for the rate and magnitude of tubularreabsorption of salicylate without further postula-tion of specific enzyme transport systems. Itshould be pointed out that tubular enzyme systemsmay be presumed to play an at least indirect rolein tubular reabsorption of salicylate; for example,in the regulation of H+ exchange and through theacceleration of tubular transfer by the concentra-tion gradient produced by conjugation of free sali-cylate within the tubule cell.

Tubular excretion of free salicylate and salicylconjugates

When tubular reabsorption of free salicylate issufficiently suppressed by administration of so-dium bicarbonate, salicylate/inulin clearance ra-tios in excess of 1.0 are obtained (Figure 2).In one of our patients (H. W., who received thelargest injection of bicarbonate) the "free" salicyl-ate/inulin clearance ratio consistently reached 1.8or more when the urinary pH was greater than7.5; in five other cases the ratio rose to 1.0 to 1.2.Similar findings are reported by Bj0rneboe, Dal-gaard-Mikkelsen, and Raaschou (19) in man andby Davis and Smith (21) in the dog, both groupsusing thiosulfate clearance as a measure of glo-merular filtration rate; however, the latter investi-gators did not consider the results to be of specialsignificance. In rabbits, Dalgaard-Mikkelsen (20)obtained salicylate clearances five times greaterthan the creatinine clearance when the urine was

sufficiently alkalinized.Tubular excretion of free salicylate may thus

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SALICYLATE EXCRETION IN MAN

become manifest when the urine is alkaline. "Sub-stantial" tubular excretion of salicyluric acid (o-hy-droxyhippuric acid) has been shown by Smithand his co-workers (13) to occur in man withoutalkalinization of the urine. It would therefore ap-pear that tubular excretion of salicylate, at least inconjugated form, regularly occurs at all levels ofurinary pH but ordinarily is masked by pre-ponderant tubular reabsorption of free salicylate(19, 20).

Conjugation of salicylate

Under the ordinary circumstances of oral ad-ministration of salicylate and excretion of acidurine, approximately 80 per cent of the salicyleliminated is in conjugated form-some 55 to 60per cent combined with glycine and 20 to 25 percent as glucuronide (4). The proportion ofsalicyl conjugate falls sharply as the urine pHrises when bicarbonate is ingested together withsalicylate. The same general behavior is notedwhen salicylate is given intravenously and bicarbo-nate is then also injected (Table I), but the initialratio "free" salicylate/"total" salicylate was higherin most of our cases, for reasons already suggested.

Information as to the precise sites and mecha-nisms of salicylate conjugation in man is wanting.It has been inferred that the low plasma concen-tration of salicyl conjugates, too low to be meas-ured by currently available methods, implies thatconjugation occurs almost exclusively in the kid-neys. This assumption is not warranted, how-ever, since the available data are consistent withthe possibility that both salicyluric acid and salicylglucuronide may be derived, in part, from the liveror other extra-renal sources.

That the kidney is nevertheless an importantsite at least of salicyluric acid formation may beinferred from what is known about biosynthesisof an analog, hippuric acid. The classic studiesof Bunge and Schmiedeberg (23) demonstratedthat in the dog conjugation of benzoic acid withglycine takes place in the kidney. Perfusion ex-periments with human kidneys showed that therenal tissue of man also is capable of this transfor-mation (24). Recently, the renal and hepaticenzyme systems responsible for hippurate syn-thesis have been studied extensively and it hasbeen shown that the benzoyl thioester of coenzyme

A is the energy-rich intermediate (25). GlycineN-acylase has been shown to transfer the benzoylgroup from acyl coenzyme A to glycine (26). Therole of glycine N-acylase in respect to salicyluricacid formation has not been elucidated, however,because the activator systems in the species stud-ied thus far are incapable of forming the o-hy-droxybenzoyl thioester of coenzyme A. No dataare available as to the scope of activity of equiva-lent enzyme systems in human kidney.

Effect of probenecid

Beyer and his associates (27) have shown thatprobenecid inhibits the conjugation of p-amino-benzoic acid (PAB) and of p-aminosalicylic(PAS) with glycine; in the case of PAB, di-rectly or indirectly at the level of acetate activation(28, 29). Conjugation of PAB with glucuronicacid apparently is not interfered with by probene-cid (30). PABgiven concurrently with salicylatedecreases urinary excretion of salicylate, appar-ently by competitively inhibiting formation ofsalicyluric acid (31).

It might be expected by analogy with such ex-periments that probenecid would interfere withconjugation of salicylate with glycine in the renaltubules, thus causing a reduction in urinary excre-tion of salicyl compounds. In the four experi-ments cited in Table II of this study, probenecid,infused at a rate sufficient to effect a distinct fallin CpAH, caused a significant decline in salicylate/inulin clearance ratio in each instance. The effectwas somewhat more marked in respect to "free"salicylate, however, and indeed the ratio of "free"to "total" salicylate excreted in the urine invariablyfell. The significance of this result is not clear; tu-bular secretion of "free" salicylate may be inhibitedby probenecid.

SUMMARY

1. Simultaneous renal clearance studies of sali-cylate, inulin, and p-aminohippurate were carriedout in 10 human subjects, and the effect of alka-linization of the urine by concomitant injection ofbicarbonate was determined.

2. Ci. and CPAHwere not consistently affectedby infusion of sodium salicylate alone or in com-bination with sodium bicarbonate. Administra-

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ALEXANDERB. GUTMAN, T. F. YU, AND JONAS H. SIROTA

tion of sodium bicarbonate had no influence on theproportion of plasma salicylate which is ultra-filtrable.

3. The "free" salicylate/inulin clearance ratiosranged from 5 to 60 per cent with a mean of 28.3per cent. As the urinary pH rose with infusion ofsodium bicarbonate, the salicylate clearance in-creased to a mean approximately three-fold andthe "net" tubular reabsorption of "free" salicylatedecreased correspondingly. When the urine pHexceeded 7.5 the salicylate excreted usually ex-ceeded that filtered, implying tubular excretion of"free" salicylate and salicyl conjugates under thesecircumstances.

4. The effect of probenecid on salicylate clear-ance was studied in four cases. Probenecid causeda mean decline of 33 per cent and 40 per cent in"total" and "free" salicylate/inulin clearance ra-tios, respectively.

5. The data indicate that renal elimination ofadministered salicylate is a complex four-phaseprocess involving filtration at the glomerulus, tu-bular reabsorption, tubular conjugation and tu-bular excretion. Extra-renal conjugation and oxi-dation also occur.

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