THE RENALEXCRETIONOF INOSITOL IN NORMALANDDIABETIC HUMANBEINGS1

BY WILLIAM H. DAUGHADAYAND J. LARNER2WITH THE TECHNICAL ASSISTANCE OFELLABETH HOUGHTON

(From the Departments of Medicine and Biological Chemistry, Washington University Schoolof Medicine, and Barnes and Homer Phillips Hospitals, St. Louis, Mo.)

(Submitted for publication July 2, 1953; accepted October 28, 1953)

The presence of increased amounts of inositol inthe urine of patients with diabetes mellitus hasbeen known for almost a century (1-3). The in-osituria in diabetes mellitus has generally been at-tributed to polyuria because the excretion of inosi-tol has also been reported to be high in patientswith diabetes insipidus. Some experimental sup-port for this hypothesis has been provided byNeedham (4), who observed a moderate increasein the excretion of inositol by rats made polyuricby feeding salt while on a low inositol diet.

Understanding of the metabolism of inositol hasbeen delayed in part by the lack of specific andsensitive analytical methods. A new analyticalprocedure was made possible by the observationof Eastcott (5) that certain strains of yeast re-quire inositol for optimal growth. The resultantmicrobiological assay has permitted the estimationof microgram quantities of inositol with satisfac-tory accuracy. In this repoft the inosituria of dia-betes is confirmed using the yeast microbiologicalassay, and observations in man relating this abnor-mality to glucosuria rather than polyuria arepresented.

METHODS

Inositol in urineInositol was measured by the method of Atkin, Wil-

liams, Schultz, and Frey (6). Unknown solutions weretested in duplicate at three dosage levels. The turbidityproduced by the growth of Saccharomyces carlsbergensisin the tubes containing the unknown solution was com-pared to that developed in tubes containing known amountsof inositol and incubated simultaneously. Duplicate tubescontaining seven different concentrations of inositol wereprepared for each assay. Treatment with Deeminite 8was introduced for the studies presented in Tables III and

1 This work was supported by a grant-in-aid from theNutrition Research Foundation.

2 Fellow of the Life Insurance Medical Research Foun-dation: Present address, Division of Bio-Chemistry, Uni-versity of Illinois, Urbana.

3 Obtained from A. S. Aloe and used without purifica-tion.

IV to remove pigments, inhibitory substances 4 and virtu-ally all salts. The procedure used for free inositol was asfollows: A dilution of urine was made so that the inositolcontent was between 2 and 8 micrograms per milliliter.To 50 ml. of such diluted urine was added 7 Gm. of Deemi-nite. After shaking intermittently for 30 minutes theslurry was filtered and the filtrate was used directly forassay.

Glucose added to normal urine was without significanteffect on inositol measurements (Table I). Representa-tive recoveries from a normal urine of relatively high anda urine of low inositol content are given in Table II. Thevery low concentration of inositol in certain urines pre-sented special problems of assay. Maximum values wereoccasionally noted in the tubes containing the least amountof test solution suggesting the presence of inhibitors.Purification of the inositol in such a urine has been car-ried out to determine whether inhibitors present in theurine had masked large amounts of inositol. Saturatedlead acetate was added to diluted urine at a pH of about6. The resultant precipitate was discarded. The pH ofthe supernatant solution was then adjusted to about 10with concentrated ammonium hydroxide. After standingover night in an ice box the precipitate was collected andwashed with absolute alcohol. Inositol was eluted fromthe precipitate with water acidified to pH 3 with sulfuricacid. The resulting eluate was desalted with Deeminiteand assayed for inosit-ol. No evidence of unrecognizedinositol was obtained and the purified solutions did notshow inhibitory effects in the assay. Ninety-two per centof added inositol was recovered from urine by this pro-cedure (Table II).

The inositol of human urine exists almost exclusivelyin the free form as determined by assay before and afterprolonged hydrolysis according to the method of Woolley(8). Loss of some inositol occurs under such drasticconditions and only from 50 to 93 per cent of added inositolwas recovered. For these reasons most samples were as-sayed without hydrolysis.

Inositol in plasmaTotal plasma inositol was measured by refluxing 4 ml.

of plasma with an equal volume of concentrated hydro-4 Choline has been shown to inhibit the microbiologic

assay for inositol (7, 8). Because choline is present inonly trace amounts in urine and Deeminite removes 99per cent of choline added to distilled water, choline is notconsidered a significant inhibitor in these assays.

326

THE RENAL EXCRETION OF INOSITOL

TABLE I

Effect'of urine glucose on inositol assay *

Urinesample Glucose added Inositol found

ml. mg. per cent pg./sample mg./day

5 0 0 190 1035 50 1 198 1085 150 3 179 935 300 6 169 91

* The indicated amounts of glucose were added to 5 ml.of urine A. After diluting to 50 ml. with water the samplewas desalted with Deeminite and analyzed for inositol.

chloric acid for 12 to 14 hours on a sand bath. The digestwas evaporated to dryness at 1000 C. under reduced pres-sure with a stream of air. The residue was taken up in20 ml. of water and was decolorized with Deeminite.Using the above method 75 per cent of inositol added toplasma was recovered.

Free inositol was determined in plasma by dialyzing4 ml. of plasma in a cellophane bag against 25 ml. of dis-tilled water at 80 overnight. Aliquots of the dialysatewere analyzed for inositol. Calculations were made on theassumption that the concentration after dialysis was equalinside and outside the bag. Eighty per cent of the inositoladded to plasma could be recovered by this method.

Clearance studiesThe renal clearance of endogenous creatinine was used

as an index of glomerular filtration. Creatinine wasmeasured by the method of Folin and Wu (9). Glomeru-lar filtration determined in this manner was adequate forthe purposes of this study. The inositol clearance was cal-culated from the free (dialyzable) plasma inositol and thefree urine inositol values. Fifteen-minute urine collectionperiods were used and a sample of heparinized blood wasobtained five minutes after the start of each collection pe-

riod. One minute before the end of each period, 20 ml.of distilled water and 20 ml. of air were injected throughthe catheter. The contents of the bladder were expressedby suprapubic pressure.

General technics

The subjects for these studies were healthy medicalstudents and patients on the wards of the Barnes Hos-pital and the Homer G. Phillips Hospital. Normal sub-jects were allowed to eat unrestricted diets and the dia-betic patients ate standard diabetic diets. Strict dietarycontrol of inositol intake was not attempted because in-formation concerning the inositol content and availabilityof inositol in common foods is not at hand. A goodmixed diet has been estimated to contain about 1 Gm. ofinositol per day (10). Analysis of diabetic diets in thislaboratory gave values between 300 and 900 mg. per day.

Toluene was used as a preservative for urine duringcollection. Aliquots of urine were refrigerated until as-

say. Urine sugar was measured by the method of Somogyi(11).

RESULTS

Inositol excretion in normal subjects

The daily inositol excretion in the urine by 11non-diabetic subjects was measured. The resultsof 21 analyses gave a mean excretion of 37 mg. per

day with a range of 8 to 144 mg. per day (Figure1). Because of the frequent assertion that urineinositol is related to urine volume, the excretion ofinositol has been plotted against the urine volumein Figure 2. The effect of oliguria produced bywater restriction and polyuria by excessive hydra-tion has been studied in two normal subjects. A

TABLE 11

Recovery of inositol added to urine

InositolAliquot added Preparation Inositol Found

Urine ml. pg. of sample p&g. aliquot mg./day

A 2 0 Standard 72 982 100 Standard 158

86% Recovery

B 5 0 Standard 23(?) 13(?)5 100 Standard 138

115% Recovery

10 0 Pb Precipitation* <5t10 200 Pb Precipitation* 189

92%Recovery

50 0 Pb Precipitation* 80 3.9

* Purified by basic lead precipitation method described under Methods.t Inositol content too low for accurate assay.

'327

328 WILLIAM H. DAUGHADAYAND J. LARNER

TABLE III

The plasma level and urinary excretion of inositol after the oral ingestion of 3 grams of inositol-normal and diabetic subjects

Plasma inositol, mg./100 ml. Urine inositol, mg./day Urine glucose, Gm./day

0* 2 hrs.* 4hrs.* 24 hrs.* Day I Day 2t Day 3 Day 1 Day 2t Day 3

Normalsubjects

1 0.8 - 0.9 0.9 37 50 35 - -2 0.8 1.9 1.0 69 181 81 - - -3 1.2 1.6 0.8 103 127 964 1.1 - 1.7 0.9 101 146 86 - -5 1.2 2.5 1.6

Mean 1.02 1.72 1.02 78 101 75

Diabeticsubjects

1 0.3 1.3 2.2 1.1 305 660t 471- >41 63 1312 0.8 1.4 1.9 1.2 304 563 82 43 403 0.7 3.8 3.3 0.9 698 1299 533 135 147 102

Mean 0.60 2.16 2.47 1.07 436 841 502 86 84 91

* Hours after the ingestion of 3 Gm. of inositol.t Inositol administered on Day 2.t The urine collected during the first four hours after inositol administration was lost.

TABLE IV

The renal clearance of inositol

Normal Diabetic Normal and glucosePatient .......... LK tM ES SW MJ BB LRAge.......... 20 25 35 45 48 18 25Surface area, M' .......... 1.74 1.40 1.69 1.82 1.48 1.52

Control period I-15 minutesInositol, plasma, mg. % 0.62 0.52 0.56 0.58 0.85 0.31 0.48Inositol, urine, mg. 0.09 0.12 17.2 10.2 0.21 0.09 0.10Glucose, blood, mg. % - 517 389 75 79 95Glucose, urine, Gm. 9.5 2.6 .005 .003 .010Clearance-Creatinine, ml./min. 96 78 165* 113 92 57 93Clearance-Inositol, ml./min. 0.9 1.5 205* 117 1.7 1.9 1.4

Control period II-15 minutesInositol, plasma, mg. % 0.95 0.32 0.52Inositol, urine, mg. 0.32 0.15 0.10Clearance-Creatinine, ml./min. 88 79 103Clearance-Inositol, ml./min. 2.3 3.3 1.3

Loading period I-15 minutes Inositol loading Glucose loadingInositol, plasma, mg. % 56 75 57 58 1.2 0.44 0.53Inositol, urine, mg. 764 697 1452 1215 9.6 5.3 5.5Glucose, blood, mg. % 497 375 393 568 782Glucose, urine, Gm. - 5.8 2.1 3.6 2.8 6.8Clearance-Creatinine, ml./min. 76 65 71 105 85 72 93Clearance-Inositol, ml./min. 91 62 170 139 53 80 69

Loading period II15 minutes Inositol lcading Glucose loadingInositol, plasma, mg. % 51 70 53 48 1.4 0.54 0.66Inositol, urine, mg. 852 621 884 716 9.0 4.2 9.6Glucose, blood, mg. % - - 506 355 334 550 588Glucose, urine, Gm. - 4.1 1.6 2.5 2.5 6.2Clearance-Creatinine, ml./min. 135 59 60 97 78 74 104Clearance Inositol, ml./min. 112 60 111 100 43 52 97

* The high clearances in this period can be attributed to faulty urine collection. The ratio of inositol to creatinineclearance should be unaffected.

THE RENAL EXCRETION OF INOSITOL

INOSITOL EXCRETION BY NON-DIABETICS Inositol excretion in diabetes

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FIG. 1. THE URINARY EXCRETION OF INOSITOL BY 11NON-DIABETIC SUBJECTSON RESTRICTED DIETS

three-fold increase in urine volume produced onlyan insignificant increase in urine inositol. Muchmore inositol was found in the urine of diabeticsubjects with glycosuria in excess of 75 Gm. a daythan in the urine of non-diabetic subjects at com-parable urine volumes (Figure 2).

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Repeated observations have been made of thedaily inositol excretion of seven hospitalized pa-tients with uncontrolled diabetes mellitus. From280 to 851 mg. per day of inositol were excretedduring-severe glycosuria. One representative sub-ject (Figure 3) was given decreasing doses ofinsulin until the urinary excretion of glucose in-creased to more than 100 Gm. per day. The inosi-tol in the urine was measured both with and with-out hydrolysis and the results agreed within theaccuracy of the method. During the period of un-controlled diabetes, large amounts of inositol werepresent in the urine. Upon reinstitution of insulintreatment, the excretion of inositol fell to the nor-mal range.

A second representative case, (Figure 4) ex-hibited severe glycosuria during a period of insulinresistance. Before insulin therapy became effec-tive between 479 and 851 mg. of inositol were ex-creted daily. With improved diabetic control, theinositol excretion fell. The urine volume in bothof these cases did not exceed 3.8 liters despite gly-cosuria in excess of 100 Gm. per day.

Inositol load experimentsThe effect of the oral ingestion of inositol on

plasma concentration and urinary excretion hasbeen studied. After a control day, a single dose of 3

+

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INOSITOL

+

MG/DAY

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0 1 2 3 4 5URINE VOLUME LITERS PER DAY

FIG. 2. THE DAILY ExCRETION OF INOSITOL BY NON-DIABETIC ( 0 ) AND DIABETIC (+) SUBJECTS PLOTTEDAGAINST URINE VOLUME

The broken lines connect values from the same indi-vidual during water restriction and over-hydration.

DAYS 1 2 3 4 5 6 7 8 9 10

INSULIN. UNITS Lo0 o |o 120150440 404401401461URINE, LITERS 1. 2.3 3.0 2.5*1.8 1 2. 5

FIG. 3. THE DAILY EXCRETION OF FREE AND HYDRO-LYZABLE INOSITOL BY A REPRESENTATIVE PATIENT WITHUNCONTROLLEDDIABETES

The excretion of glucose is shown in the shaded columns.

4t

329

* *

WILLIAM H. DAUGHADAYAND J. LARNER

900-

800-

700-

600-

500-INOSITOL

400-MG/ DAY

300-

200

100-

DAYS 1 2 3 4 5 6 7 8 9 10 l1 12 13 14 15 16 17I NSULI N. UNI TS 12 50 10510 0 95 95I 115i 125 201210 120 130 3 1URI NE, L I T ERS I .9 2.3 2.91 2.8 3.8 2.7 2.3 2.3 2.0 I 2.7 2.02. J8

FIG. 4. THE DAILY ExcRETIoN OF INOSITOL BY A SECONDREPRESENTATIVEPATIENTWITH UNCONTROLLEDDIABETES

The excretion of glucose is shown in the shaded columns.

Gm. of inositol dissolved in 200 ml. of tap water wasgiven by mouth on the morning of the second day.This dose of inositol was estimated to be from threeto five times the normal dietary intake. Breakfastwas omitted, but, thereafter, the subjects were al-lowed to eat their regular diet. The inositol excre-tion was measured on the control day and on thetwo days after inositol ingestion. The inositol ex-cretion by normal subjects, Table III, increased 23mg. per day following inositol ingestion. The in-crease in inositol excretion by the diabetic patientsaveraged 405 mg. per day. The plasma levels ofinositol of diabetic subjects before inositol load-ing were within the same range as those of the non-diabetic. The plasma inositol rose in both normaland diabetic subjects.

Inositol has been given intravenously to threecontrol and five diabetic patients. Glycosuria wassevere in all but one diabetic patient who only hadmild glycosuria. A solution of 0.2 per cent inosi-tol in 0.9 per cent sodium chloride was adminis-tered in a dose of 20 mg. per Kg. over a period offour hours.

The plasma inositol levels following intravenousadministration of inositol were essentially similarwhen measured either by the hydrolysis or dialysistechniques. Only the results obtained by the latter

method have been used in plotting Figure 5. Theplasma inositol level rose 200 to 400 per cent inboth diabetic and control subjects during the infu-sion and promptly fell after the end of the infusion.

The diabetic patients excreted much more inosi-tol than the control subjects on the preinfusion day.The inositol increment on the day of inositol infu-sion amounted to 36 per cent of the administereddose in the diabetic and only 13 per cent in thenormal subjects. On the day after inositol infu-sion, the excretion in the urine had returned nearlyto the control level in both groups.

The renal clearance of inositol

The clearance of endogenous creatinine andinositol have been measured in two diabetic andtwo non-diabetic patients. Endogenous inositolclearance was very low in the two normal sub-jects and high in the two diabetic subjects, approxi-mately equivalent to creatinine clearance (TableIV). The inositol clearance at high plasma levelsof inositol has been measured. Seventy millilitersof 10 per cent inositol was given rapidly by vein.The loading dose was followed by the injection of asustaining solution containing one per cent inositolin isotonic saline given at a rate of 4 ml. per minute.After a five-minute equilibration period inositol

330

0---I

THE RENAL EXCRETION OF INOSITOL

THE INTRAVENOUS ADMINISTRATIONOF INOSITOL

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I I2 4 6 24 HOURS

INOSITOL, 20 tAGI JER KG- Y-

4-

/ A_

7It

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\IN %%.4

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CONTROL INFUSION POST-ICONTROL I NFUSION POST- INF.

nAYS

FIG. 5. CHANGESIN PLASMA DIALYZADLE INOSITOLAND URINE INOSITOL ExcRETIoN BEFORE AND AFTER THE

INTRAVENOUSADMINISTRATION OF 20 MG. PER KG. BODYWEIGHTOF INOSITOL FOR FOURHOURS

Values obtained from diabetic patients are indicated by(+), those for normal patients by (0).

and creatinine clearances were determined. Withplasma inositol levels between 51 and 75 mg. per

100 ml. the clearance of inositol rose to levels com-

parable to creatinine clearance in both normal anddiabetic subject.

The effect of glucose loading on inositol excre-

tion has been studied in three non-diabetic sub-

jects. One half-hour prior to the clearance meas-

urements the patients received 3 Gm. of inositol bymouth. The inositol clearance of these subjectswas low during the control period (Table IV).Glucose loading was performed by the rapid intra-venous administration of 50 per cent glucose in a

dose of 2 ml. per Kg. of body weight for M. J. and2% ml. per Kg. of body weight for the other twosubjects. When the priming dose of glucose hadbeen given, a sustaining solution of 20 per centglucose was started at a rate of 4 ml. per min. andwas continued for two 15-minute clearance periods.A sustained hyperglycemia and glycosuria wereachieved in these patients. A 30-fold increase ininositol clearance occurred during glucose infusion.

DISCUSSION

The present observations confirm earlier reportsof an increased urinary excretion of inositol inhuman diabetes mellitus. Since urine volume cor-related poorly with inositol excretion in non-dia-betic subjects, other causes of the inosituria havebeen sought.

These studies indicate that an alteration of renalfunction is responsible for the inosituria of dia-betes. Filtered inositol is almost completely reab-sorbed at physiologic plasma levels in normal sub-jects. With plasma levels of inositol between 51and 75 mg. per 100 ml., the ability of the tubule toreabsorb inositol is exceeded, and inositol clearanceapproaches the rate of filtration. A striking differ-ence exists in patients with diabetes mellitus.Large amounts of inositol are excreted even atnormal levels of plasma inositol. The renal clear-ance of inositol in non-diabetic subjects rose greatlywhen a massive load of glucose was presented tothe renal tubules. It would seem clear that glucoseincreases the excretion of inositol probably byinhibiting the reabsorption of inositol by the hu-man renal tubule.

A comparable situation exists in the reabsorptionof xylose by the renal tubule. Glucose and phlori-zin have been found to block the reabsorption ofxylose suggesting that glucose and xylose mayshare the same transport mechanism ( 12). Phlori-zin also inhibits the reabsorption of inositol by therenal tubules in rats (13). At low plasma levelsthe reabsorption of inositol by the renal tubules ismuch more complete than the reabsorption ofxylose. As the plasma level is raised the renalclearance of both substances approaches the rateof glomerular filtration. Further study is neces-sary to establish the relation between inositol andglucose transport mechanisms.

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331

A.

WILLIAM H. DAUGHADAYAND J. LARNER

SUMMARY

1. The urine of 11 non-diabetic subjects con-tained an average of 37 mg. of inositol per day(range 8 to 144 mg.). Urine volume correlatedpoorly with inositol excretion.

2. Seven uncontrolled diabetic patients had aurinary excretion of from 280 to 851 mg. per dayof inositol. The inosituria of diabetes disappearedafter the control of glycosuria.

3. A small rise in the plasma level of inositoloccurred following the ingestion of 3 Gm. of inosi-tol in both diabetic and non-diabetic subjects.The average increase in urinary excretion on theday of inositol ingestion was 23 mg. in the controlgroup and 405 mg. in the diabetic group.

4. Diabetic subjects excreted three times moreinositol in their urine than did non-diabetic sub-jects following the slow intravenous administrationof 20 mg. per Kg. of inositol but the levels ofplasma dialyzable inositol were the same in thetwo groups.

S. The renal clearance of inositol in normal sub-jects at physiologic plasma levels of inositol waslow. A 3000 per cent increase in inositol clear-ance occurred following glucose loading at normalplasma inositol levels. At high plasma inositollevels clearance rose to the level of endogenouscreatinine clearance. The renal clearance of inosi-tol in diabetic subjects was high even at normalplasma inositol levels.

6. It is concluded that a renal tubular mecha-nism for the reabsorption of inositol exists andthat tubular transport of inositol is inhibited byhigh glucose loads. The inosituria of diabetes mel-litus can be attributed to an increased inositolclearance produced by glycosuria and not bypolyuria.

ACKNOWLEDGMENTS

The authors wish to acknowledge the assistance ofMrs. M. Heady, laboratory technician, and Miss S. Wood,research nurse, in the performance of this study.

The pyrogen-free inositol for parenteral use was gen-erously provided by Dr. H. J.. Byrne of the CommercialSolvents Corporation, Terre Haute, Indiana.

REFERENCES

1. Vohl, H., Uber des Auftreten des Inosits in HambeiNieren-krankheiten und die Verwandlung des Dia-betes mellitus in Diabetes inositus. Arch. Physiol.Heilh., 1858, n.f. 2, 17, 410.

2. Neukomm, J., Ueber das vorkommen von Leucir,Tyrosin und anderer Umsatzstaffe im MenschlichenK6rper bei Krankheiten. Zurich, Otell, Fiussli u.Comp., 1859, p. 48 (Dissertation) (quoted by Need-ham, J.).

3. Gallois, F.-N., De Pinosurie, Paris, J.-B. Bailliire,1864, p. 61 (quoted by Needham, J.).

4. Needham, J., Studies on inositol. II. The synthesis ofinositol in the animal body. Biochem. J., 1924, 18,891.

5. Eastcott, E. V., Wildiers' bios, the isolation and identi-fication of "Bios I." J. Phys. Chem., 1928, 32, 1094.

6. Gy6rgy, P., ed., Vitamin Methods, Volume I, NewYork, Academic Press, 1950.

7. Taylor, W. E., and McKibbin, J. M., The determina-tion of lipide inositol in animal tissues. J. Biol.Chem., 1953, 201, 609.

8. Woolley, D. W., The determination of inositol. Biol.Symposia, 1947, 12, 279.

9. Folin, O., and Wu, H., A system of blood analysis.J. Biol. Chem., 1919, 38, 81.

10. Williams, R. J., The approximate vitamin require-ments of human beings. J. A. M. A., 1942, 119, 1.

11. Somogyi, M., A rapid method for the estimation ofurine sugar. J. Lab. & Clin. Med., 1941, 26, 1220.

12. Smith, H. W., The Kidney: Structure and Functionin Health and Disease, New York, Oxford Uni-versity Press, 1951.

13. Daughaday, W. H., and Larner, J., Unpublishedexperiments.

332