renal excretion of hexitols (sorbitol, man- nitol, and ... · dog and man* ry willie w. smith,...

RENAL EXCRETION OF HEXITOLS (SORBITOL, MAN- NITOL, AND DULCITOL) AND THEIR DERIVATIVES (SORBITAN, ISOMANNIDE, AND SORBIDE) AND OF ENDOGENOUS CREATININE-LIKE CHROMOGEN IN DOG AND MAN*

RY WILLIE W. SMITH, NORMA FINKELSTEIN, AND HOMER W. SMITH

(Prom the Department of Physiology, New York iiniversity College oJ

Medicine, New York)

(Received for publication, May 18, 1940)

Inulin is the only substance hitherto reported which fulfils the physiological specifications for the measurement of the rate of glomerular filtration in man (34). The only other substance advo- cated for this purpose is creatinine (28). In the dog, the exogenous creatinine clearance is identical with the inulin clearance, and both clearances are independent of plasma concentration, which facts are good evidence that, in this animal, both clearances are at the level of filtration. But in man, the exogenous creatinine clearance exceeds the simultaneous inulin clearance by 40 per cent OI more (19, 24, 30, 33), and convincing evidence has been advanced by Shannon that this is attributable to the tubular excretion of creatinine. The endogenous creatinine clearance measured by the enzymatic method of J!Iiller and Dubos is identical wit,11 the inulin clearance in some instances, but in ot,hers it substantially exceeds the inulin clearance, a cir’cumstancc consonant with the behavior of exogenous creatinine (21). The apparent clearance of endogenous chromogenic substance, either with or without correction, has been recommended as a measure of glomerular filtration, but, such endogenous clearances cannot in our opinion be accepted for this purpose (vide injra).

* This investigation has been supported in part by a grant from the Commonwealth Fund.

We are indebted to the Atlas Powder Company for supplying the hexit,ol derivatives used in this work.

231

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232 Renal Excretion of Hexitols

In view of the theoretical and practical importance of this problem, we have continued our efforts to obtain supplementary evidence on the mechanism of excretion of inulin in the human kidney by searching for additional substances which fulfil the specifications for glomerular excretion without tubular participation. In this search we have been guided generally by the facts that all electrolytes, the excretion of which has been examined, undergo either tubular excretion (phenol red, hippuran, diodrast, iopax, neoiopax) or t,ubular reabsorption (sodium, potassium, chloride, nitrate, thiocyanatc, sulfate, uric acid, etc.). Though t,his circumstance does not negate the possibility that an electro- lyte of suitable properties might escape both tubular excretion and reabsorption, any search in this direction must, at the present time, go forward empirically. We have therefore confined ow investigation for the most part to compounds related t,o the carbo- hydrat,cs, since no one of the simple carbohydrates, at, least, is known to bc excreted by the tubules in any species (37), while the process of tubular reabsorption in this group is apparently related to spatial configuration. The present report deals chiefly with the excretion of certain of the hexitols and their derivatives, with supplementary data on the excretion of cndogenous creatininc- like chromogens.

Results

Table I summarizes our observations on the simult,aneous clearances of the hexitols and their anhydrides in the dog. The agrce- ment of the simultaneous clearances of sorbitol, mannitol, dulcitol, and sorbitan with the creatinine or inulin clearance is such as to leave no doubt that the three hexitols and the first anhydride, sorbitan, on the one hand, and creatinine and inulin, on the other, arc excreted by identical mechanisms.

The marked discrepancy between the crcatinine clearance and the simultaneous clearances of isomannide and sorbide equally leaves no doubt, since these substances are completely filtrable from the plasma, that about half of the filt.ered material is rrab- sorbed from the glomerular filtrate by the tubules. That this reabsorption does not involve the tubular mechanism for the reabsorption of glucose is indicated by the fact that elevation of the plasma glucose to levels al; which this mechanism is saturated (32)


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Smith, Hnkelst,ein, and Smith

-. . ..~

Subject

TABLE I Renal Clearances of Hexitols in Dogs -__-__

Sorbitol

0.9LO.99 0.96 0.96-1.03 0.99 0.93-0.99 0.97 0.93-0.97 0.95

Average 0.97 0.97 -__

Mannitol .-___ -

Vicky 0.96-1.00 0.98 0.94--1.00 0.98 Red 0.914.94 0.92 0.91-0.96 0.94

0.91--0.92 0.92 0.96-0.98 0.97

Average . . . . .._................,,.._,,,,,.,,,_..., 0.94 0.96

Dulcitol

Average . . . . . . . . . . . . .._.__._....._._....._.._._... --__

Sorbitan -

Clemi Vicky

Average . . . . ..__....__.........._.......,.,,,,,_,_ ~-~__ -__

Isomannide

Vicky 7- 6-39 6 126-175 31 0.43-0.48 0.45 ll--2O-39 5 142-200 31 0.45-0.49 0.47

Red 7-11-39 4 107-147 28 0.39-0.46 0.41 3 408-448 32

/ 3 1555568* 43 0.44-0.54 0.48 0.54-0.58 0.56

Average 0.47 -.-__--.~

Sorbide

Red / 4- 8-401 5 I 90-110 / 63 1 0.52.-0.571 0.55 1 I -

* Plasma glucose, 428 to 458 mg. per cent.


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does not alter the isomannide-creatinine clearance ratio (see Table I). The fact that raising the plasma level of isomannide itself does not greatly raise this clearance ratio argues against, though it does not definitely disprove, the possibility that the reabsorption of isomannide is an “active” process, in the sense in which the reabsorption of glucose is an active process.

No suggestion can be offered as to why isomannide and sorbide should be reabsorbed by the tubules when sorbitol, mannitol, dulcitol, and sorbitan are not, other than to point out that the first two compounds are second anhydrides, whereas sorbitan is a first anhydride, and the other four compounds are hexahydric alcohols.

Table II summarizes our data on man. As in the dog, the sorbitol, mannitol, and sorbitan clearances are clearly identical with the inulin clearance. The dulcitol clearance in man is consistently slightly below the inulin clearance; whether this is attributable to some systematic analytical error or to a species difference we have been unable to determine, but in view of the identity of these clearances in the dog (Table I), we discount the latter interpretation.

The excretion of isomannide and sorbide in man was not examined, since suitably pure material was not available.

DISCUSSION

The problem of measuring accurately the rate of glomerular filtration in man has assumed increased importance as the possi- bilities of evaluating tubular activity have evolved (14, 27, 29, 31, 32, 37, 38). The only convincing method of demonstrating the processes of tubular excretion or reabsorption is by the comparison of simultaneous urine-plasma ratios in individual nephrons or, in the case of the mammalian kidney, by comparison of the over-all clearances. Evidence obtained by examining the effects of chang- ing the plasma concentration of a single substance on the absolute rate of its excretion, though capable of revealing tubular excretion or reabsorption when such evidence is positive, cannot when it is negative add more than inferential support to the evidence obtained by the comparative method.

The earlier comparative data on renal clearances in man has been reviewed elsewhere (37). Since that review, only one new line of evidence of this nature has become available: Shannon and


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Smith, Finkelstein, and Smith 237

Fisher (32) have shown that during hyperglycemia in the dog, when the glucose reabsorptive mechanism of the tubules is saturated, the clearance of xylose (which is normally reabsorbed to a slight extent) is identical with the creatinine clearance. In unpub- lished studies, Shannon and Ranges have now shown this to be equally true in man. In the matter of absolute rates of excretion, Miller, Alving, and Rubin (20), using the micromethod (I), have shown that the inulin clearance in any one subject is independent of the plasma concentration of inulin at levels ranging from 5 to 85 mg. per cent, supplementing the demonstration by Shannon and Smith (34) of constant clearance between 50 and 400 mg. per cent.

The present demonstration that the clearances of sorbitol, mannitol, dulcitol, and sorbitan arc identical with the creatinine (and therefore inulin) clearance in the normal dog supplements the already abundant evidence that the creatinine and inulin clearances are at the level of glomerular filtration in this animal. Because of analytical difficulties, the clearances of several substances cannot be determined simultaneously, but since we may reasonably equate the clearances of substances which have been separately compared with either the creatinine or inulin clearance we may say that the inulin, creatinine, sorbitol, mannitol, dulcitol, sorbitan, and xylose clearances (the last named during hyperglycemia only) are identical in this animal.1

The conclusion that the inulin clearance is at the level of glomerular filtration in man has hitherto rested upon (a) absence of evidence indicating tubular excretion, i.e. absence of any change in t’he inulin clearance at widely varying plasma levels (5 to 400 mg. per cent) ; (b) positive evidence of tubular excretion of creatinine in man, consisting of the depression of the creatinine

1 The ferrocyanide clearance should be included in this list, though the significance of this clearance, first studied by Van Slyke, Hiller, and Miller (45), remains subject to some doubt, since Miller and Winkler (23) have found that ferrocyanide is reabsorbed in man. On this point Miller and Winkler remark, “We are inclined to interpret our results as representing a species difference between dog and man. It should, however, be pointed out that the levels of serum ferrocyanide in our human experiments are distinctly lower than those in the dog experiments of Van Slyke, Killer, and Miller, and that in this respect the clearances obtained in their experiments were obtained under conditions differing from ours.”


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clcarancc and of the creatinine-inulin clearance ratio on elevation of the plasma creatinine concentration; and (c) the identity of the inulin and creatinine clearances in phlorhizinized man (20, 30, 33, 34). To this evidence is now added the demonstration that the clearances of inulin, sorbitol, mannitol, and sorbit.an are identical, while that of dulcitol is so slightly below the inulin clearance that the discrepancy may be due to systematic error. That this identity is a consequence of the identical reabsorption of all these substances is highly improbable.2

It is of interest to note that the three hexitols, though excreted alike by the kidney, are metabolized to markedly different degrees. Todd, Myers, and West (44) conclude that sorbitol increases the blood glucose in the dog, and Carr and Forman (2) find that it increases the liver glycogen in the rat. According to Todd et al. (44) less than 50 per cent of this hexitol is excreted in the urine in 24 hours after intravenous injection. Considerable metabolism is also indicated in Waters’ report (46). We find that after the intravenous administration of 9 gm. of sorbitol and 8 gm. of inulin in normal man, only 32 per cent of the sorbitol appeared in the urine, whereas 98 per cent of the inulin was excreted.

Dulcitol, on the other hand, does not elevate blood glucose 01

the R.Q. (3) and it is reported by Ishihara, Kimura, Miyaja, Shen- taku, and Sugiyama (15) not to increase liver glycogen in the rat, while Carr and Krantz (3) state that it reduces tissue glycogen and only slightly elevates liver glycogen. In a single observation on a normal man receiving 4 gm. of dulcitol intravenously, WC found 87 per cent to be excreted in the urine in 10 hours. Apparently the metabolism of this substance is slight.

Also slight, according to t,hc available cvidencc, is the mct)abo- lism of mannitol. Carr, Musser, Schmidt, and Krantz (5) report that mannitol does not increase the R.Q. of fasting rats, although it increases the blood sugar in rabbits; according to Todd, Myers, and West (44) it does not increase the blood sugar in dogs, and these authors find, in accord with Silberman and Lewis (35), that when it is given per OS there is but slight conversion to liver glycogen, although such conversion is evident on long feeding, according to Carr and Krantz (4). In two observations in which

2 Since this paper was completed, Steinitz (41) has reported the identity of the sucrose and inulin clearances in man.


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10 gm. of mannitol and 5 gm. of inulin were given intravenously to normal men, we were able to recover from urine collected during 10.5 hours following the injection 81 per cent of the administered mannitol and 95 per cent of the inulin in one case, and in the other, 89 per cent of the mannitol and 97 per cent of the inulin.

Isomannide is apparently not metabolized to any great extent (17, 16).

It may be noted that no toxic symptoms were apparent in these patients after they had been given as much as 80 gm. of sorbitol or mannitol, 50 mg. of dulcitol, or 70 gm. of sorbitan by intravenous administration over a period of 2 hours.

Clearances of Endogenous Substances Giving the Ja$e Reaction

The apparent creatinine clearance based upon the rndogenous sllbstancc or substances which yield color with alkaline picrate has been recommended as a measure of glomerular filtration by Popper, Mandel, and Mayer (26), Popper and Mandel (25), Findley (IO), and Steinitz and Tiirkand (43). We have consequently included here observations on the apparent clearance of these endogenous chromogens in man, as determined simultaneously with the inulin clearance.

Popper, Mandel, and Mayer (26) recommend precipitating plasma proteins by picric acid, since this reduces the chromogen content of the filtrate, and we have compared their method with the BaC03-Fe2(S04)3 method of Steiner, Urban, and West (40). The first method, as applied by us, consists of the drop by drop addition of 4 cc. of plasma to 12 cc. of saturated picric acid; the mixture is heated in a boiling water bath for 12 to 15 seconds and filtered through Schleicher and Schiill Ko. 597 filter paper. 10 cc. of cooled filtrate are transferred to an Evelyn colorimcter tube and alkalinized with 0.5 cc. of 10 per cent NaOH. The light absorption is determined after 20 minutes with a No. 520 filter, and the chromogen content read from a standard curve prepared as in the analysis of urine. Urine is diluted to approximately a urine-plasma ratio of 1.0 and 3 cc. of the diluted solution are added to 9 cc. of saturated picric acid solution in the calorimeter tube; 0.6 cc. of 10 per cent KaOH is added and light absorption is read after 10 to 20 minutes.

In t,he Steiner, Urban, and West method, 4 cc. of 17 per cent


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Renal Excretion of Hexitols

Fe2(S04)3 are added to a mixture of 4 cc. of plasma and 32 cc. of water contained in a 125 cc. Erlenmeyer flask. 6 gm. of precipitated BaC03 are added3 and the flask is stoppercd and shaken until all evolution of CO2 has stopped. The mixture is centrifuged and filtered through washed cotton or filter paper, and 4 drops of saturated Na2S04 are added. After the mixture has st,ood for 15 minutes, the BaSO4 is centrifuged out and t,he fluid is filtered again. 10 cc. of this filtrat,e are transferred to an Evelyn calorimeter tube and 5 cc. of alkaline picratc (5 volumes of saturated picric acid plus 1 voluma of 10 per cent NaOH) are added. Light absorption is read with a No. 520 filter exactly 10 minutes later. Urines are diluted to approximately the same concentration as the plasma filtrates and analyzed as above without) prc- cipitation. Standard curves arc prepared wit)h IO cc. of aqueous creatinine solution handled in t,he same manner as the urines.

We find that, both of the above methods give quantitative r(l- covery of creatinine when this is added to plasma in concent,ra- tions of 0.25 mg. per cent or more, if the concentration of the cndogenous chromogenic substance as determined by the respcc- tive methods is deducted from the total chromogenic substance. A detailed report of these recoveries appears to be superfluous.

The two methods of precipitation, however, give markedly different recoveries of endogenous chromogen from both plasma and urine. In a series of fourteen samples of human plasma, the chromogen present in the picric acid filtrates ranged from 50 to 79.2 (average 66.6) per cent of that present in the iron filtrate. Popper, Mandel, and Mayer (26) report their picric acid filtrates contain about 50 per cent as much chromogen as the Folin picric acid (12), and Ferro-Luzzi and coworkers (8, 9) report that> Somogyi’s (39) Zn precipitate method yields substantially lower chromogen values than does the Folin and Wu tungstate filtrate (13). Chesley and Chesley (7), using the tungstate filt,rate, find that the endogenous chromogen clearance has about the same magnitude as the urea clearance, which fact, ma.y be attributed to the high plasma chromogen values given by this filtrate. In urine, although the difference between methods of precipitation is less marked, there is still not ident’it)y of behavior; we find in

3 The BaC03 should be tested to be certain that, with the quantity of Fe,(SOa)a used, the filtrate will not. turn red litmus paper blue.


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twenty-one samples that the picric acid filtrate yielded from 85 to 102 (average 92) per cent of the chromogen given by the iron filtrate.

It is clear that these various methods of protein precipitation, at least two of which we have found give 100 per cent recovery of added creatinine, do not yield the same chromogen content in either plasma or urine, and therefore the chromogen in plasma and urine cannot all be creatinine.

Apparent clearances” of chromogen in man, as determined by both methods, are given in Table III. These observations have been made during the measurement of the renal plasma flow by the diodrast clearance, when diodrast was present in the plasma to the extent of 0.9 to 1.8 mg. per cent. Diodrast gives no color in the Jaffe reaction, and in our opinion does not influence the apparent clearance of chromogen. The observations have been extended to include high plasma levels of diodrast (20 to 40 mg. per cent), utilized for the measurement of the maximal rate of tubular excretion of diodrast, with a view to determining whether or not “saturation” of the tubules with diodrast affects the apparent chromogen clearance. Inasmuch as the chromogen-inulin clearance ratio invariably falls during the determination of the maximal rate of tubular excretion of diodrast, we infer that part of the urine chromogen is excreted by the tubules.

The data of Table III show that the apparent chromogen clearance as calculated from the picrate filtrate is higher, whether compared with the inulin clearance or with the apparent chromogen clearance as calculated from the iron filtrate. This is to be ex- pected, since the picrate filtrate gives a lower chromogen yield from the plasma than does the iron filtrate.

Popper and Mandel(25) have argued, on the basis of comparison with the xylose clearance, that the apparent chromogen clearance, as determined by them, is at the level of glomerular filtration. Apart from the now demonstrated active tubular reabsorption of xylose (34, 37), the data advanced to support this conclusion arc unconvincing, since the chromogen-xylose clearance ratios pre-

4 We say apparent clearances, since physiologically the term clearance should be applied only to discrete chemical entities and not to the excretion of unidentified substances.


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Smith, Finkelstcin, and Smith 243

sented by Popper and Mandel vary from 0.77 to 4.5, and average 1.35.

The chromogen-inulin clearance ratios reported by Steinitz and Tiirkand (43) (using the picrate filtrate) for normal subjects average 1.03, but they range from 0.73 to 1.17; in subjects with glomerulonephritis, the range is from 1.04 to 1.73, with an average of 1.37. We have not examined this ratio in glomerulonephritis, but in our normal subjects this ratio is variable and generally exceeds 1.0.

It is clear from the data of Table III and the other investiga- tions cited that the apparent clearance of endogenous chromogenic substance differs markedly when different methods of precipitation of plasma protein are used, and that with no method so far reported is the endogenous chromogen clearance consistently identical with the inulin clearance. The nature of the substances in plasma which participate as chromogens in the Jaffc reaction is still a matter of debate; although creatinine is present among these chromogens (21), the participation of one or more other substances is no doubt the source of variability in the clearance in normal subjects and the even greater variability in subjects with renal disease. Whatever the empirical usefulness of this test, so long as the nature of the substance or substances involved is unknown, its use in the quantitative exploration of renal func- tion, and especially in the evaluation of tubular activity (42), is likely to be a source of considerable error.

SUMMARY

1. Methods for the quantitative determination in the blood and urine of the hexitols, sorbitol, mannitol, and dulcitol; of the monoanhydride, sorbitan; and of the dianhydrides, isomannide and sorbide, are described.

2. The clearances of sorbitol, mannitol, dulcitol, and sorbitan are identical with the simultaneous creatinine or inulin clearances in the dog.

3. The clearances or sorbitol, mannitol, and sorbitan are identical with the simultaneous inulin clearance in man. The dulcitol- inulin clearance ratio averages 0.94; it is believed that the discrepancy between these two clearances in man is possibly attributable to systematic analytical error.


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4. Sorbitol and mannitol are cleared from the plasma at the same rate as inulin both when the glomerulus is normal and when it is filtering protein.

5. Isomannide and sorbide undergo tubular reabsorption in the dog; the excret’ion of these substances in man was not examined.

6. Data are given on the metabolism of the hexitols and their derivatives in man.

7. The data presented here are strong supportive evidence that t’he inulin clearance is at the level of glomerular filtration in both dog and man.

8. The excretion of endogenous chromogenic substance (Jaffc reaction), as determined in picric acid and iron filtrates of plasma, has been compared with the excretion of inulin in man. The inadequacy of the so called “endogenous creatinine” clearance as a measure of glomerular filtration is discussed.

Methods

The methods of administration of the various substances examined here, and of clearance determination, have been similar to those previously described from this laboratory (6, 14, 38). For administration to dogs the hexitols were used without repurifi- cation, but for human administration they were filtered through a Seitz E. K. No. 3 asbestos filter and administered in 7.5 to 10 per cent solution. Heparin is used as an anticoagulant.

Fermentation and Precipitation of Plasma and Urine (Zinc Filtrate)

When the plasma inulin concentration ranges from 10 to 25 mg. per cent, removal of inulin is unnecessary; the hexitol equivalent of inulin is 1.0 and appropriate correction can be made in both plasma and urine. Fermentable sugar must, however, be removed. For this purpose, bakers’ yeast is washed six to eight times until the supernatant fluid is clear, and suspended in approximately 20 per cent solution. This solution will keep in the ice box for a week, but must invariably be centrifuged and resus- pended on the day of use, and the extracellular water content determined with a hematocrit tube. 2 cc. of plasma or diluted urine are treated with 6 cc. of yeast suspension for 15 minutes at room temperature and then centrifuged. From 2 to 5 cc. of the


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supernatant fluid are transferred to a 50 cc. Erlenmeyer flask and, if further dilution is desired, 5 cc. or more of water are added. Precipitation is effected by the addition of 6 cc. of Zn solution and, after thorough mixing, 2 cc. of 0.75 N NaOH. The Zn solution consists of 66.7 gm. of ZnS04.7HzO and 167 cc. of 1.0 N

H&O4 made up to 2000 cc., and so adjusted with HzS04 or NaOH that 20 cc. require between 7.14 and 7.25 cc. of 0.75 N NaOH to neutralize to phenolphthalein. (If a 1:lO dilution is wanted routinely, the requisite quantity of water may be incorporated in the Zn solution.) The mixture is well shaken and after 10 minutes transferred to a centrifuge tube, centrifuged, and then filtered through washed cotton.

With high plasma concentrations of inulin, this substance must be converted to fructose and absorbed by yeast before hexitol determination. 10 cc. of l.he filt)rat,e are hydrolyzed in a cali- brated tube by the addition of 1 cc. of 1.0 N H&X>4 and heating in a boiling water bath for 15 minutes. After cooling, 1 drop of 0.02 per cent phenol red solution is added and the sample is neu- tralized with 1 cc. of 1.0 N KOH, the end-point being carefully adjusted by the drop by drop addition of 1 per cent NazC03. The mixture is then made up to 15 cc. and 9 cc. of the resulting solution are mixed with 1 cc. of packed yeast and the mixture agitated frequently for 40 minutes. Fructose is removed by yeast more slowly than is glucose, but if the concentration of inulin in the sample does not exceed 15 mg. per cent, and if plasma and urine samples are adjusted to contain about equal concentrat.ions, no appreciable error results from failure of the yeast to absorb all but slight traces of the fructose or other carbohydrates derived from the hydrolysis of inulin.

Analysis of Sorbitol, Mannitol, and Dulcitol (Periodate Method)

This method is similar to that described by Silberstein, Rappa- port, and Reifer (36), and involves the oxidation of glucose-free filtrate with a known excess quantity of KIO4, and determination of the excess KIOd by liberation of 12 and ISazSz03 titration. We have found it advantageous to substitute the Somogyi (39) ZnSO,H&04 filtrate, as described above, for the Na8iOj of Silberstein et al. and to carry out the liberation and titration of 1~ in an acid rather than neutral solution.


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A HI04 solution is prepared by adding 2 parts of 5 per cent HzS04 to 3 parts of 0.1 per cent KIOh. Duplicate samples consisting of 2 cc. of filtrate are pipetted into Pyrex ignition tubes (24 X 200 mm.) and exactly 5 cc. of the acid Ii104 solution are added to each tube. The tubes are closed with glass tears to effect condensation and are placed in boiling water for exactly 20 minutes. During this time the hexitol, if its concentrat,ion does not exceed 20 mg. per cent, will be completely oxidized to formic acid and formaldehyde (11, 18). After cooling, about 0.5 gm. of KI crystals is added to the samples one at a time and the liberated iodine titrated with 0.005 N EazSx03, 1 drop of a 1 per cent soluble starch solution being added near the end-point The Na$z03 is standardized against 0.001 N KIOs.

Two dummy samples of the I~104 solution, with 2 cc. of water snbst,ituted for the filtrate, are boiled simultaneously with each group of unknowns and titrated to obtain the standard KIO, titer. Since the KIOI-KI mixture is titrated in acid solution, all the iodine liberated by the unreduced KIO4 is titrated with Na&&03, and consequently in the analysis of the unknowns the Naz&03 equivalent of the dummy KIOI is diminished by slightly less than one-quarter (for example from 19.5 to 15.7 cc.). In view of this fact, and to afford maximal accuracy in titration, we have utilized a special burette of the Rang type, the upper portion consist)ing of a bulb graduated to deliver 15 cc. between two marks, the lower portion consisting of a 5 cc. burette 15 inches in length and graduated in 0.02 cc. Duplicate samples should check wit>hin 0.06 cc.”

The difference between the st’andard KIOJ titer and the titer of the unknown, multiplied by 9.2/2, and by the appropriate dilu- t,ion factor, gives the mg. per cent of hcxitol in the original sample. (The figure 9.2 is a conversion factor which takes into account the incomplete oxidation of t,hc hexitol and is applicable to sorbitol, mannitol, and dulcitol.)

5 In approximat,ely neutral solution, KIOd liberates only 1.0 equivalent of iodine, and Silbcrstein et al. (36) recommend that the titration of the KIOa + KI mixture be carried out, in neutral solution. This has the advantage of eliminating the residual titration figure of 15 cc., as described above, but WC find the adjustment of the mixture to neutralit,y difficult and uncertain, and prefer to titrate the total KIOd iodine in acid solution. The use of a special burette, as described above, circumvents the single disadvantage of this procedure


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The plasma “hexitol” blank (about 10 mg. per cent) must bc determined by analysis of a control sample of plasma as described above. When the urine is treated with yeast before precipitation, the blank is negligible. A small quantity of hexitol is lost on the yeast, but in so far as this loss is uniform in blood and urine samples when these are handled alike, it introduces no error in the calculations of the clearances. Creatinine does not con- tribute to the blank.

In a series of recoveries of sorbitol at 50 to 200 mg. per cent from sixteen samples of dog and human plasma the average deviation in recovery was 1.6 mg. per cent, with a maximal deviation of 4.6 mg. per cent. Four of these samples contained 30 to 60 mg. per cent creatinine, which did not increase the error of recovery.

Sorbitol and mannitol do not penetrate the red blood cells of human blood in vitro, as shown by the fact that 95 to 96 per cent of added hexitol is recoverable from plasma 2, 15, and 33 minutes after the addition of known quantities to whole blood.‘j A small apparent loss by this method is explicable from failure to obtain a plasma-free hematocrit. The in vitro penetration of the other compounds was not examined.

Neither sorbitol, mannitol, isomannidc, nor sorbide is bound by human plasma proteins, as shown by ultrafiltration and dialysis as described by Shannon (29). Allowing for a protein concentration of 6 gm. per 100 cc., and deducting the blank as determined in a control ultrafiltrate from the same plasma, we recovered 98 to 1.00 per cent of the hcxitols from the ultrafiltrate at a concentration of 125 to 150 mg. per cent. The other compounds wcrc not examined.

Analysis of So&lan, Tsomannide, and Solhide (C&c Szdjdc Method) 7

l’lasma and urine arc precipitated and trcat,cd with yc>ast as in the periodate method described above. The oxidizing agent is

6 This is also true of diodrast (38), but White and his coworkers (47, 48) have shown that to some extent this substance penetrates the red blood cells of both dog and man in vivo. A plausible explanation of this fact is that during the circulation of the blood through the capillary bed the permeability of the red cells is increased by distortion.

f A similar method in which ceric sulfate is utilized for the dctermina- tion of isomannide has been used by Krantz and Carr (16), the details of


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prepared according to the description of Miller and Van Slyke (22). 10 cc. of 0.1377 N ceric sulfate are diluted to 100 cc. with 5 per cent HzS04. 2 cc. of filtrate are pipetted into tubes, 5 cc. of the ceric sulfate solution are added, and the tubes are closed with tears and placed in a boiling water bath for 30 minutes. Two dummy samples are boiled with each group of unknowns. After cooling, 0.5 gm. of KI is added to each tube and the liberated 1~ is titrated with 0.005 N Na&03, with an ordinary burette dclivcring 15 cc. Standard curves, prepared by the analysis of aqueous solutions of sorbitan, etc., in the above manner, are used for the conversion of cc. of ceric sulfate to concentration of the hexitol derivative. Such curves are nearly straight lines and can bc extrapolated to zero ordinates. Plasma and urine blanks, which arc considerably higher than in t,he KIOd method, are determined on control samples, the latter being calculated as mg. per minute and deducted from all experimental periods. The sorbitan equivalent of inulin is 1.0.

Inulin determinations were made on the above Zn filtrates cithcr by the method of Smith et al. (38), or by the micromethod of Rlving, Rubin, and Miller ((1) and personal communication). The determination of creatinine is described in the body of the paper. Diodrast iodine was determined by the method described by Smith et al. (38).

We wish to thank Dr. Irwin Wellen, Dr. Catharine A. Welsh, and Miss Anna Rosenthal of the Department of Obstetrics and Gynecology, and Dr. William Goldring, Dr. Herbert Chasis, and Dr. Rlbcrt Erdmann, Jr., of the Department of Medicine for c~linic~al assistance in making these observations.

BIBLIOGRAPHY

I. hiving, A. S., Itubin, J., and Miller, B. I’., .J. Niol. Che~a., 127, 60!) (1939).

2. Carr, C. J., and Forman, S. E., J. Biol. Chem., 128, 425 (1939). 3. Carr, C. J., and Krante, J. C., dr., J. Biol. Chem., 107, 371 (1934). 4. Carr, C. J., and Krantz, J. C., Jr., J. Biol. Chem., 124, 221 (1938).

which were kindly communicated to us by Professor Krantz. The modifi- ca.tions which WC have made have been dcsigncd to dccreasc the blank and increase the uniformity of oxidation.


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Smith, Finkelstein, and Smith

5. Carr, C. J., Musser, R., Schmidt, J. E., and Krantz, J. C., Jr., J. Biol. Chem., 102, 721 (1933).

6. Chasis, I-I., Ranges, H. A., Goldring, W., and Smith, H. W., J. Clin. Inv., 17, 683 (1938).

7. Chesley, L. C., and Chesley, E. R., J. Clin. Inv., 19, 475 (1940). 8. Ferro-Luzzi, G., Z. ges. ezp. Med., 94, 708 (1934). 9. Ferro-Luzzi, G., Saladino, A., and Santamaura, S., 2. ges. exp. Med.,

96, 250 (1935). 10. Findley, T., Jr., Am. J. Physiol., 123, 260 (1938). 11. Fleury, P., and Lange, J., Compt. rend. Acad., 196, 1395 (1932). 12. Folin, O., J. Biol. Chem., 17, 475 (1914). 13. Folin, O., and Wu, H., J. Biol. Chem., 38, 81 (1919). 14. Goldring, W., Chasis, H., Ranges, H. A., and Smith, H. W., J. Clin.

Inv., in press (1940). 15. Ishihara, T., Ximura, T., Miyaja, S., Shentaku, T., and Sugiyama, G.,

Arb. med. F&.&t Okayama, 6, 545 (1938). 16. Krantz, J. C., Jr., and Carr, C. J., Proc. Sot. Exp. Biol. and Med., 39,

577 (1938). 17. Krantz, J. C., Jr., Evans, W. E., Jr., and Carr, C. J., Quart. J. Phurm.

and Pharmacol., 8,213 (1935). 18. Malaprade, L., Bull. Sot. chim., 43,683 (1928); Compt. rend. Acad., 186,

382 (1928). 19. McCance, R. A., and Widdowson, E. M., J. Physiol., 91, 222 (1937);

Luncet, 233, 247 (1937); J. Physiol., 96, 36 (1939). 20. Miller, B. F., Alving, A. S., and Rubin, J., J. C&n. Inv., 19, 89 (1940). 21. Miller, B. F., and Dubos, R., J. Biol. Chem., 121, 447, 457 (1937). 22. Miller, B. F., and Van Slyke, D. D., Proc. Am. Sot. Biol. Chem., J.

Biol. Chem., 1.14, p. lxxi (1936); J. Biol. Chem., 114, 583 (1936). 23. Miller, B. F., and Winkler, A. W., J. Clin. Inv., 16, 489 (1936). 24. Miller, B. F., and Winkler, A. W., J. Clin. Inv., 17, 31 (1938). 25. Popper, H., and Mandel, E., Ergebn. inn. Med. u. Kinderh., 63, 686

(1937). 26. Popper, II., Mandel, E., and Mayer, H., Biochem. Z., 291, 354 (1937). 27. Ranges, H. A., Chasis, H., Goldring, W., and Smith, H. W., Proc. Am.

Physiol. Sot., Am. J. Physiol., 126, P 603 (1939). 28. Rehberg, P. B., Biochem. J., 20, 447 (1926). 2!). Shannon, J. A., Am. J. Physiol., 113, 602 (1935). 30. Shannon, J. A., J. CZin. Inv., 14, 403 (1935). 31. Shannon, J. A., Physiol. Rev., 19, 63 (1939). 32. Shannon, J. A., and Fisher, S., Am. J. Physiol., 122, 765 (1938). 33. Shannon, J. A., and Ranges, H. A., Proc. Am. Physiol. Sot., Am. J.

Physiol., 126, P 626 (1939). 34. Shannon, J. A., and Smith, II. W., J. Clin. Inv., 14, 393 (1935). 35. Silberman, A. K., and Lewis, H. B., Proc. Sot. Exp. Biol. and Med.,

31, 253 (1933-44). 36. Silberstein, F., Rappaport, F., and Reifer, I., Klin. Woch., 16, 1506

(1937).


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Renal Excretion of Hexitols

37. Smith, H. W., The physiology of the kidney, New York (1937). 38. Smith, H. W., Goldring, W., and Chasis, H., J. Clin. Inv., 17,263 (1938). 39. Somogyi, M., J. Biol. Chem., 86, 655 (1930). 40. Steiner, A., Urban, F., and West, E. S., J. Biol. Chem., 98, 289 (1932). 41. Steinita, K., Am. J. Phy.sioZ., 129, 252 (1940). 42. Steinitz, K., J. CZin. Inv., 19, 299 (1940). 43. Steinitz, K., and Tiirkand, H., II. Medizinischen Universitiitsklinik,

Istanbul, 1005 (1939); J. CZin. Inv., 19, 285 (1940). 44. Todd, W. R., Myers, J., and West, E. S., J. BioZ. Chem., 127, 275 (1939). 45. Van Slyke, D. D., Hiller, A., and Miller, B. F., Am. J. Physiol., 113,

629 (1935). 46. Waters, E. T., Proc. XVI Internat. Physiol. Cong., Kongressbericht II,

Zurich, 122 (1938). 47. White, H. I,., Findley, T., Jr., and Edwards, J. C., Proc. Sot. Exp.

BioZ. and Med., 43, 11 (1940). 48. White, H. I,., and Heinbecker, P., Proc. Sot. Exp. Bid. and Med., 43,

8 (1940).


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Homer W. SmithWillie W. Smith, Norma Finkelstein and

DOG AND MANCREATININE-LIKE CHROMOGEN IN

SORBIDE) AND OF ENDOGENOUS(SORBITAN, ISOMANNIDE, AND

DULCITOL) AND THEIR DERIVATIVES(SORBITOL, MANNITOL, AND

RENAL EXCRETION OF HEXITOLS

1940, 135:231-250.J. Biol. Chem.

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