metabolism, blood levels and rate of excretion of - diabetes

Metabolism, Blood Levels and Rate of Excretionof Acetohexamide in Human Subjects

/. A. Galloway, M.D., R. E. McMahon, Ph.D., H W. Culp, M.S., F. / . Marshall, Ph.D.,and E. C. Young, M.D., Indianapolis

SUMMARY

The metabolism of acetohexamide was studied in normaland diabetic subjects, utilizing acetohexamide-C-14 and hy-droxyhexamide-C-14 in two types of experiments. In one,blood levels were determined by an isotopic dilution analysis(IDA). In the other, following the oral and intravenousadministration of acetohexamide-C-14, radiocarbon levels inthe blood and urine and the nature of the urinary metabo-lites were elucidated. IDA showed an average half-life foracetohexamide of 1.6 hrs. (range 0.8 to 2.4) and an averagehalf-time for its principal metabolic product, hydroxyhexa-mide, of 5.3 hrs. (range 3.7 to 6.4). Half-time for total radio-carbon was 5.3 hrs. (range 3.5 to 11). Urinary recovery ofradioactivity after oral doses averaged 71.6 per cent intwenty-four hours.

Of the urinary metabolites of acetohexamide (AH) re-covered, about 65 per cent was L-hydroxyhexamide (HH)and the remainder consisted chiefly of 4-trans-hydroxy-acetohexamide (HAH), 4-trans-hydroxyhydroxyhexamide(HHH), and unchanged acetohexamide with small quanti-ties of other hydroxylated isomers. Fecal excretion wasmeasured in one patient following the oral administrationand found to be 15 per cent. The observation that, evenfollowing intravenous injection, urinary recovery was only85 per cent suggested that biliary excretion represents thesecondary route of elimination of acetohexamide and/orits metabolites. The radioactivity which was not recoveredin the urine at the end of 48 hrs., e.g., on the average25 per cent, was probably excreted in the stools, resultingfrom the failure of a small quantity of the tablet to beabsorbed and from biliary excretion. The blood and urinedata agree with those reported by Sheldon, Anderson andStoner and confirm the report by Smith, Vecchio andForist that the half-life of acetohexamide (AH) plus hy-droxyhexamide (HH) is comparable to that of tolbutamide.In spite of this relatively short half-life, limited bloodsugar data indicate definite hypoglycemic effects up to24 hrs. after a single 1.0-gm. dose. These findings indicatethat the duration of action of AH and its metabolites arenot necessarily related to drug levels. DIABETES 16:118-27,February, 1967.

From The Lilly Laboratories for Clinical Research, MarionCounty General Hospital, and The Lilly Research Laboratories,Indianapolis, Indiana.

Acetohexamide, a sulfonylurea synthesized by the LillyResearch Laboratories, has proved to be an effective hypo-glycemic agent.1 Chemically it is N-(p-acetylphenyl-sulfonyl)-N'-cyclohexylurea (figure i ) .

The earliest report on the metabolism of acetohexa-mide was that of Welles, Root, and Anderson,2 whofound that the principal metabolite was hydroxyhexa-mide which had hypoglycemic activity. It appearedlikely that this metabolite contributed significant-ly tothe over-all hypoglycemic response induced by the ad-ministration of acetohexamide. More recently, McMahon,Marshall, and Culp3 have conducted a more completestudy of the fate of acetohexamide in the rat and inhumans. In tracer studies, they found that the metaboli-cally formed hydroxyhexamide had the L(-) config-uration and that it was effective in lowering bloodsugar in rats. Culp and McMahon4 have described asoluble enzyme from rabbit kidney which is capableof reducing acetohexamide to L (-)-hydroxyhexamidewhen TPNH is added as cofactor. The significance ofthis enzyme in the intact animal has not yet beenassessed.

McMahon et al.3 found that in addition to hydroxy-hexamide a number of minor metabolites were alsoformed. These were compounds in which the cyclo-hexane ring had been hydroxylated. The two most im-portant of these minor metabolites in the human were4'-trans-hydroxyacetohexamide and 4'-trans-hydroxyhy-droxyhexamide. Figure i shows the structure of aceto-hexamide and its principal human metabolites.

By the use of a modified Toolan-Wagner procedure,Smith, Vecchio, and Forist5 measured the half-lives ofboth acetohexamide and metabolically-formed hydroxy-hexamide. They found that following absorption fromthe gastrointestinal tract, acetohexamide was convertedto hydroxyhexamide at an average half-life of 1.3 hrs.Hydroxyhexamide, in turn, disappeared from the bloodwith a half-life of 4.6 hrs.

118 DIABETES, VOL. 16, NO. 2

J . A. GALLOWAY, M.D., R. B. MC MAHON, PH.D., H. W. CULP, M.S., F. J . MARSHALL, PH.D., AND E. C. YOUNG, M.D.

MATERIALS AND METHODS

H 3 C

S O ? N H C O N H -

ACETOHEXAMIDE

AH

HH

H3C

H3C^C

HO'

L(-)-HYDROXYHEXAMIOE

V SO,NHCONH-

s-HYDROXYACETOHEXAMIDE

-SO,NHCONH -

HYDROXYHYDROXYHEXAMIDE

HAH

H H H

Note: Present in small quantities are the 4'-cis, 3'-cis, and 3'-transisomers of the minor metabolites.

FIG. I. Metabolic products of acetohexamide found in urineof humans.

The purpose of the present investigation was tomake a thorough study of the dynamics of the absorp-tion, metabolism, and excretion of acetohexamide andits metabolites. Radiocarbon labeling was used to fa-cilitate the work.

Seven volunteers, two men and five women, wereincluded in the study (table i ) . Although H.W. andhis daughter, N.H., have strong family histories, andeach has had a positive cortisone glucose tolerancetest, for the purpose of this study they are consideredas normals. The remainder of the patients were maturity-onset diabetics who, with the exception of B.A., hadbeen treated with acetohexamide for periods of onemonth to four years prior to the initiation of thestudy. Prior to, and during the course of the investiga-tion, B.A., was treated with n o U. of Lente Insulindaily. The patients had normal renal function exceptfor L.S. and F.L. who, during the eighteen months theexperiments were being performed, demonstrated occa-sional mild elevations of blood urea nitrogen (20 to22 mg. per 100 ml.) but without tubular abnormali-ties as indicated by the phenolsulfonphthalein test.However, there were no blood urea nitrogen abnor-malities on the days of the experiments. Furthermore,the blood levels and excretion patterns of these twopatients were nearly identical to all but one (B.A.)of the remaining subjects. For these reasons, the occa-sional elevations in the blood urea nitrogen levels werenot regarded as being significant.

The following types of protocols were utilized (table1):

I. Acute (Patients received single one-gram doses of

TABLE 1

Data and types of experiments carried out for each patient in acetohexamide study

PatientB.A.R.B.N.H.F.L.S.R.L.S.H.W.

Age

57502474356742

Sex

FFFMFFM

Lengthof timediabetesmellitusknown

11 yrs.3 mos.06 yrs.1 mo.4 yrs.0

Height(inches)

60626562616471

Weight(pounds)

163167140136212163202

Idealbody

weight

130135132133131142168

Approx.per cent Types of experiments

over I. Acute* II. ChronicfIBW A B A B

252462

621520

x x

X X

*I. Acute (Patients received single one-gram doses of acetohexamide with no treatment for at least three days before orafter the test.)A. Acetohexamide, two 500-mg. tablets (commercial material), by mouth followed by serial determination of

blood levels of acetohexamide and hydroxyhexamide using the isotopic dilution analysis (IDA).B. Labelled acetohexamide, two 500-mg. tablets (prepared to simulate commercial material), each containing 25 nc.

acetohexamide-C-14, by mouth, followed by serial determinations of total radiocarbon in blood and stools, andof quantities of metabolites of acetohexamide in urine.

f l l . Chronic (Patients treated several days before and after the day of the experiment with single one-gram doses of aceto-hexamide in the mornings.)A. Same as I. (B).B. One gram of acetohexamide-C-14 containing 25 /tc. of acetohexamide-C-14, injected intravenously.

FEBRUARY, 1967 119

METABOLISM, BLOOD LEVELS AND RATE OF EXCRETION OF ACETOHEXAMIDE IN HUMAN SUBJECTS

acetohexamide with no treatment for at least three daysbefore or during the test.)

A. Acetohexamide, two 500-mg. tablets (commercialmaterial), by mouth followed by serial determinationof blood levels of acetohexamide and hydroxyhexamideusing the isotopic dilution analysis (IDA). Bloodsamples were taken at o, 0.5, 1, 1.5, 2, 3, 4, 6, 8, and10 hrs.

B. Labeled acetohexamide, two 500-mg. tablets (pre-pared to simulate commercial material), each contain-ing 25 JJUC. acetohexamide-C-14, by mouth followed bycollection of blood samples at o, 0.5, 1, 1.5, 2, 3, 4,6, 8, 9, 10, 11, 13, 15, 17, 20, 24, 26, and 28 hrs.Pooled urine specimens were collected every 3 hrs. forthe first 12 hrs., then every 12 hrs. for the next fourand one half days. An attempt was made to collectand count stools for 72 hrs. after administration ofthe labeled drug, but in only one instance was thiseffort completely satisfactory.II. Chronic (Patients treated several days before andafter the day of administration of labeled acetohexa-mide, given in single one-gram doses in the mornings.)

A. Same as I. (B) above.B. The intravenous administration of one gram of

acetohexamide-C-14 containing 25 /*c. of acetohexamide-C-14, followed by collection of blood samples (1 ml.with a 26-gauge needle) at o, 2.5, 5, 10, 20, 30 mins.and 1, 1.5 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 16, 18,20, 22, 24, 26, 28, 32, 36, 40, 44, 48, 54, 60, 66, 72,78, 84, 90, 96, 108, 120, 132, and 144 hrs. An in-dwelling catheter was placed in the bladder for the first28 hrs. of the experiment. Urine samples were takenhourly for the first 12 hrs., every 2 hrs. for the next 16hrs., then every 4 hrs. for 20 hrs., every 6 hrs. for 48hrs., and every 12 hrs. for the final two days of a six-day collection period.

The preparation of acetohexamide-C-14, labeled inthe urea carbonyl carbon, has been described by Mc-Mahon et al.3

The tablets containing acetohexamide-C-14 were pre-pared in the following manner: 6.4 gm. of unlabeledacetohexamide was dissolved in ethanol with approxi-mately 100 mg. of acetohexamide-C-14 with an initialspecific activity of 3.3 ju,c./mg. After evaporation ofthe solvent the resulting labeled material was com-bined with normal tableting excipients and processedaccording to standard manufacturing procedures; thefinal step was compression into tablets of 500 mg., eachcontaining 25 /AC. of radioactivity.

Isotopic dilution analysis {IDA) for acetohexamideand hydroxyhexamide

Twenty micrograms each of acetohexamide-C-14 andhydroxyhexamide-C-14 (prepared by the reduction ofacetohexamide-C-14) were added to 2 ml. of plasmaand incubated overnight at 37°. The plasma was thenextracted under the usual Spingler conditions.6 Extract-ed acetohexamide and hydroxyhexamide were chromato-graphed on thin layer plates (Silica gel GF-254, E.Merck) using a chloroform acetic acid (9:1) system.The spots represented by acetohexamide and hydroxy-hexamide were eluted with butanone-2. The solventwas removed and the residue taken up in 5 ml. ofnormal amyl acetate. A 1 per cent aliquot was removedand counted by a liquid scintillation method to deter-mine radiocarbon recovery. Acetohexamide and hydroxy-hexamide present were determined by the Spinglermethod.6

The amounts of acetohexamide and hydroxyhexa-mide were estimated by the following formula:

vg. of AH or HHobserved by Spin- Xgler method

total dpm added 2o ug.total dpmrecovered

= fig./2 ml. plasmafig./2 ml. X 0.05 = mg. per cent

Method for radiocarbon countingThe radiocarbon content of plasma samples was de-

termined by a modified Schoniger combustion proce-dure.7 Urine radiocarbon content was determined byliquid scintillation counting. The amounts of variousmetabolites present in each urine sample were estimatedby thin layer chromatography of the raw urine usingthe procedures described above in the isotopic dilutionmethod and elsewhere by McMahon et al.3 By use ofmodel substances it was possible to identify and quanti-tate the various metabolites of acetohexamide in humanurine.

RESULTS

Table 2 shows the values obtained for the bloodlevels of acetohexamide and hydroxyhexamide for peri-ods up to 10 hrs. in acute experiments in which theIDA was used for determination of the drug levels.The means of these values are plotted against time infigure 2. In this, the mean half-life for acetohexamideis 1.6 hrs. with a range of 0.8 to 2.4 hrs. The meanhalf-life for hydroxyhexamide is 5.3 hrs. with a rangeof 3.7 to 6.4 hrs. Peak levels for the acetohexamide

1 2 0 DIABETES, VOL. 16, NO. 2

J . A. GALLOWAY, M.D., R. E. MCMAHON, PH.D., H. W. CULP, M.S., F. J . MARSHALL, PH.D., AND E. C. YOUNG, M.D.

TABLE 2

Isotopic dilution analysis results: blood levels of acetohexamide and hydroxyhexamide following the oral administration of 1gm. of acetohexamide (reported in fig./ml.)

Patient/time

B.A.

R.B.

N.H.

F.L.

S.R.

L.S.

MEAN

AcetoHydroxyTotalRatio







0

000

A:H

000

A:H

000

A:H

000

A:H

000

A:H

000

A:H

000

A.H

0.5hr.

159

241.7-1

261238

2.2:1

171431

1.2:1

—

—

000

—

426

2:1

12.47.419.81.7:1

1hr.

101929

1:1.9

312253

1.4:1

312455

1.3:1

—

372461

1.5:1

2111321.9:1

2620461.3:1

1.5hrs.

243458

1:1.4

292756

1.1:1

5252

1041:1

—

—

6638

1041.7:1

583290

1.8:1

45.836.682.41.3:1

2hrs.

296392

1:2.2

344478

1:1.3

4164

1051:1.6

4341841:1

313667

1:1.2

5365

1181:1.2

38.552.290.71:1.4

3hrs.

136073

1:4.6

174158

1:2.4

256085

1:2.4

_

—

3624601.5:1

2872

1001:2.6

23.851.475.21:2.2

4hrs.

25355

1:26.5

73037

1:4.3

236487

1:2.8

174966

1:2.9

293665

1:1.2

66268

1:10.3

1449631:3.5

6hrs.

03636

0-36

424281:6

1050601:5

(7hrs.)

42933

1:7.3

2050701:2.5

84654

1:5.8

8.441.249.61:4.9

8hrs.

03737

0:37

01616

0:16

44246

1:10.5

(9hrs.)

12627

1:26

522271:4.4

63339

1:5.5

3.030.033.01:10

10hrs.

02222

0:22

01010

0:10

918271:2

(11hrs.)

722291:3.1

914231:1.6

62632

1:4.3

4.818.022.81:3.8

Peaklevel

tig./ml.

2963

3444

5264

4349

6650

5872

4760.3

Peaktime(hrs.)

22

22

1.52

24

1.56

1.53

1.83.2

Half-life

(hrs.)

0.85.5

1.23.7

1.86.4

1.75.4

1.86.2

2.44.8

1.65.3

occur 1.5 to 2 hrs. and for the hydroxyhexamide 2 to6 hrs. after the administration of acetohexamide.

The data for the total radiocarbon levels, followingthe acute administration of 1 gm. of acetohexamide-C-14,are listed in table 3, and the mean levels are comparedwith the IDA results in figure 2. It will be noted thatthe mean half-life of acetohexamide plus all of itsmetabolic products is 5.3 hrs. with a range of 3.5 to

11 hrs. The semilog plot of the mean radiocarbon levelsin the blood in figure 3 shows the decay of blood levelsto be a first-order reaction with no tailing off to sug-gest protein binding or other delay in excretion, as hasbeen reported with chlorpropamide.8-9 In figure 4, theblood radiocarbon levels in two patients following acuteand chronic oral administration of acetohexamide arecompared. In patient F.L., the rate of decay of blood

TABLE 3

Blood radiocarbon levels for protocol I. (B)

Patient/time

B.A.R.B.F.L.S.R.L.S.H.W.Mean

FEBRUARY,

0

1.1

00

00.3

1967

0.5hr.4410.90.30

31.73119.7

1hr.

56.846.61553.258.87050.1

1.5hrs.73

10043.979.674.581.575.4

2hrs.66.1775197.391.179.277

4hrs.61.541.534.970.453.34851.6

6hrs.5626.325.650.937.53538.6

7hrs.55.621.621.635.931.530.232.7

9hrs.46.713.217.324.531.521.925.9

11hrs.41.17.7

14.215.729.113.920.3

13hrs.37.44.7

11.811.723

9.916.4

15hrs.30.2

2.69.97.6

22.66.2

13.2

17hrs.26.0

3.1—6.2

17.75.6

11.7

20hrs.22.0

0.86.15.1

136.38.9

24hrs.20.40.6

2.98.46.17.7

26hrs.18.40.33.72.5

35.6

28hrs.14.50.13.03.251.84.6

36hrs._

4.0

—4.0

1 2 1

100i


MEANS» - - « Total Sulfonylurea IDA

•( — — Total Sulfonylurea (i.e., total radio-/ ' . carbon) after administration of ace-

» • tohexamide—C"- Acetohexamide IDA

Hydroxyhexamide IDA

9 11 13

H O U R S

FIG. 2. A comparison of mean blood sulfonylurea levels determined by isotopic dilution analysis, and total radiocarbon after the oraladministration of acetohexamide, I.O gm.

radioactivity was slightly greater following the acuteadministration of the labeled dose than when the la-beled compound was interposed in the chronic treat-ment schedule. In patient L.S. the reverse occurred.Thus, these data suggest that blood levels in patientson the same dosage schedule may vary from day to dayat given times and are not appreciably affected bywhether or not treatment has been in progress beforeand after the trace dose is given.Urine data

The urine radiocarbon recovery data are recorded intable 4 and are summarized in figure 5. It is of interestthat the mean total urinary radiocarbon recovery forsix patients at the end of 24 hrs. was 71.6 per cent and

rose only to 77.2 per cent at the end of 48 hrs. Of theurinary metabolites of acetohexamide recovered, about65 per cent was L-hydroxyhexamide (HH) and the re-mainder consisted chiefly of 4-trans-hydroxyacetohexa-mide (HAH), 4-trans-hydroxyhydroxyhexamide (HHH),and unchanged acetohexamide with small quantities ofother hydroxylated isomers.

Following intravenous administration of acetohexa-mide-C-14, the radiocarbon recovery in patient F.L.was 80.4 per cent at 24 hrs., 84.3 per cent at 48 hrs.,and rose to only 85 per cent at the end of six days.In patient L.S., 82.4 per cent of the dose was recoveredat 24 hrs., 85.6 per cent at 48 hrs., and only 85.9 percent at the end of six days.

TABLE 4

Protocol I. (B)

Radiocarbon recoveries in urine of patients receiving acetohexamide-C-14 (one gram orally on an acute basis)

Patient/timeB.A.R.B.F.L.S.R.L.S.H.W.Mean

0-3hrs.5.5

15.112.618.017.012.713.5

Per cent0-6hrs.18.050.429.038.345.540.036.9

of dose0-8hrs.22.361.645.965.457.557.051.6

recovered in urine0-12hrs.30.171.549.676.062.767.659.6

0-24hrs.47.278.364.485.276.678.071.6

0-48hrs.54.579.969.590.486.382.377.2

Per centdose

asHH0-24hrs.14.159.345.157.048.457.746.9

Per centof urine

metabolitesasHH

24hrs.29.975.770.0

• 66.963.274.063.3

122 DIABETES, VOL. 16, NO. 2

10O

J. A. GALLOWAY, M.D., R. E. MC MAHON, PH.D., H. W. CULP, M.S., F. J. MARSHALL, PH.D., AND E. C. YOUNG, M.D.

RADIOCARBON PLASMA LEVELS

PATIENT: F.I.

FIG. 3. A semilog plot of the mean total radiocarbon plasmalevels of six patients following oral administration ofacetohexamide C-14, 1.0 gm.

DISCUSSION

The half-life and actual drug levels which resultedfrom this study are in substantial agreement with thoseof earlier workers.5-10 Peak concentrations of AH werefound in the blood at an average time of 1.8 hrs. andthen decayed with a mean half-time of 1.6 hrs. AH dis-appearance resulted mainly from its conversion to HHalthough some AH was eliminated through the kidneys.HH, in turn, reached a peak concentration at an aver-age time of 3.2 hrs. This compound disappeared fromthe blood, mainly by urinary excretion, with an averagehalf-life of 5.3 hrs. The over-all sulfonylurea half-lifethen is very similar to that reported for tolbutamide.2

The mean half-time obtained in the present studies by

1 GM. ACETOHEXAMIDE-C" (CHRONIC) 10/8/64

I GM. ACETOHEXAMIDE-C14 (ACUTE) «/4/64

1 GM. ACETOHEXAMIDE-C'* (CHRONIC) 10/7/64

1 GM. ACETOHEXAMIDE-C14 (ACUTE) 4/18/64

12HOURS

FIGURE 4b

the total radiocarbon method (5.3 hrs.) is also con-sistent with this conclusion.

The actual blood levels for the one-gram doses used

FEBRUARY, 1967 1 2 3

tx.±1

OoUJ

a.oCQa.oOaa.h-

zUJoccUJQ.

90-i

JO-

70-

60-

50

40

30

20

10

MEAN• -H .W.x - R . B .O - L . S .• - B . A.* - F . L.• - S . R.

X

o /

7/

//I

A./ *O/ •

1

A

X

//

•

•

X

O y^

*

•

X

0_ ^ — •

*

•

•

o

X

*

•


of AH are compared. In all of these experiments thepatients were given their usual diabetic diets dividedequally in three meals offered at approximately 0.5, 5,and 10 hrs. after the drug was given. It will be notedthat in eleven experiments with diabetics 24 hrs. afterthe administration of AH, when total drug levels wereless than 10 mcg./ml. (1 mg. per cent), the bloodglucose was reduced in seven instances (experiments 1,2» 3> 6, 7, 8, and 10), was the same in two (experi-ments 9 and 11), and was increased in two (experiments12 and 13). Of interest, however, is the fact that inthese latter two instances the blood sugar at 9 hrs. afteradministration of AH was more than 10 per cent belowthat of the fasting level. Patients N.H. (experiment 5)and H.W. (experiment 14) indicate the relative re-sistance of normal subjects to the hypoglycemic activityof AH.

In one experiment (No. 6) , when the one-gram dosewas repeated, there was further improvement of thesubsequent fasting blood sugar. "We believe that betterblood glucose control could have been achieved hadthe doses of AH been repeated daily for several days.

Sheldon et al. have reported the range of therapeuti-cally effective serum concentrations of AH (and metabo-lites) to be lower than those for tolbutamide andchlorpropamide. Our data and those of Sheldon et al.indicate that the duration of action of AH and itsmetabolites is not necessarily related to drug levels assuggested by Smith, Vecchio and Forist.5

In radioactive studies reported in this paper, recoveryof radioactive metabolites in the urine averaged 65 percent in 24 hrs. This checks reasonably well with the re-sults reported by Sheldon, Anderson, and Stoner10 whofound an average of 60 per cent (±19.8) in ninety-eightpatients. The modified Spingler procedure which wasused probably would not have measured some of themore polar metabolites (i.e., HAH and HHH). In thepresent study HH was found to be the predominanturinary metabolite. In the earlier work some AH wasalso found to be present while later samples contained,in addition, a mixture of the various hydroxylatedmetabolites (figure 1).

The finding of several metabolites indicates thatacetohexamide is not unique among the sulfonylureas.Although Fajans11 reported that with sulfur-35 thecarboxylic acid metabolite of tolbutamide was the onlyone found in the urine of six patients. Thomas andIkeda,12 using tritiated tolbutamide, found that twothirds of the material in the urine was carboxy metabo-lite and one third was the hydroxymethyl metabolite. John-

3 6 8 12 24 48

TIME IN HOURS

FIG. 5. Rates of radiocarbon recovery from the urine in sixpatients following the oral administration of aceto-hexamide C-14, 1.0 gm.

in this study are similar to those reported by Sheldon,Anderson and Stoner.10 In figure 2 and tables 2 and 3,the drug levels 3 to 5 hrs. after a dose were in therange of 65 to 50 mcg./ml. The mean drug levels re-ported by Sheldon et al.10 (Sheldon, figure 2, page364), using the method of Spingler,6 were in therange of 120 and 70 mcg./ml. with a mean of 95, 3to 5 hrs. after a one-gram dose. The differences be-tween the Spingler and radiocarbon data could well bedue to differences in methodology as well as to thetime of ingestion of food in relation to the taking ofthe acetohexamide.

Because the purpose of these studies was solely toelucidate the metabolism of AH, and a fixed dose of1.0 gm. of this agent was being used, there was noprospective attempt to correlate blood glucose withdrug levels. Consequently, patients were selected onthe basis of their willingness to participate in thestudies and not for their responsiveness to AH. More-over, blood sugars were obtained irregularly in mostof the experiments, and not at all in some. Therefore,the blood glucose data on hand were collected andanalyzed retrospectively. This is presented in table 5,in which the available blood sugars the day before, theday of, and the day after the administration of 1.0 gm.

1 2 4 DIABETES, VOL. 16, NO. 2

J . A. GALLOWAY, M.D., R. E. MC MAHON, PH.D., H. W. CULP, M.S., F. J. MARSHALL, PH.D., AND E. C YOUNG, M.D.

TABLE 5

Blood sugar levels taken from patients at intervals stated following the oral administration of 1.0 gm. of acetohexa-mide or acetohexamide-C-14 by mouth. Blood glucose was determined by the modified Somogyi-Nelson method. Patientsreceived .drug at 0 hrs. and were fed at 0.5, at 5 and at 10 hrs., e.g., drug was given at 7:00 a.m. and patients were fed at

about 7:30 a.m., noon, and 5:00 p.m.

Experimentnumber

1

2

3

4

5

6

7

8

9

10

11

12

13

14

B.A. 3/31/644/1/644/2/64

5/10/645/11/645/12/64

R.B. 3/15/643/16/643/17/64

6/14/646/15/646/16/64

N.H. 3/31/644/1/644/2/64

F.L. 2/2/642/3/642/4/642/5/64

8/3/648/4/648/5/64

10/7/6410/8/6410/9/64

S.R. 3/24/643/25/643/26/64

5/4/645/5/645/6/64

L.S. 3/31/644/1/644/2/64

4/28/644/29/644/30/64

10/6/6410/7/6410/8/64

H.W. 6/14/646/15/646/16/64

0

8168

196166

12312177

63

565874

971047681

116123101

8265

180180

137125

116116

106128146

96123

66

Hours after administration of drug2 4 9 10 15 16 Other

163

220

66

31

179168

146

195

141

199215

183

160

208143

40

133

1972751666481

49

7938

110180108

192138174

123148

125127

138

92

123

189123168

202

5360

17876

124

76

11091

118

123

18094

10485

123

127108126

88

6060

100

6644

100

71

305

174180177

118121104108

143110

116

115

116

149136160

71

7069

162

12:30 a.m.64

Drug leveldata in:

Table2

Table3

Table2

Table3

Table2

Table2

Table3

Table2

Table3

Table2

Table3

Table3

Comments

Insulin-dependent diabetic. On100 U. Lente Insulin daily.

Maturity-onset

Nondiabetic at

Maturity-onset

Maturity-onset

Maturity-onset

Nondiabetic at

diabetic.

time of study.

diabetic.

diabetic.

diabetic.

time of study.

son et al.8 reported that chlorpropamide is essentiallyunchanged. Three metabolites have been reported fortolazamide: 4-carboxy tolazamide, 4-hydroxymethyl to-lazamide, and hydroxylated tolazamide.13

The excretion and recovery rates were in close agree-ment for all patients except B.A., without whose

values the mean recovery rates would have risen to76.5 per cent and 81.8 per cent at the end of 24 and 48hrs., respectively. The most likely explanation for theanomalous data from B.A. is that urine collectionswere faulty; however, blood levels during the same ex-periment were consistent with the delayed urinary ex-

FEBRUARY, 1967 125


cretion rate. Fifteen per cent of the radioactivity re-covered from this patient was found in the stools. Itis noteworthy that, in the same patient, the half-timeof AH and HH as determined by the isotopic dilutionanalysis (table 2) was well within the range of thatin the other patients. These findings amplify the pointelucidated by the comparison rates of excretion of F.L.and L.S. (see above)—namely, that day-to-day varia-tion of blood levels of sulfonylureas occurs in the samepatient.

An attempt was made to obtain stools for 72 hrs.after the administration of labeled drug but, becauseof problems of collection and of inadvertent discardingof specimens before counts could be made, a satisfac-tory determination of fecal radioactivity was made inonly one patient, B.A., who, as mentioned above, ex-creted 15 per cent of the total dose in the stools.

The possibility that biliary excretion represents a sec-ondary route of elimination of acetohexamide and/orits metabolites is suggested by (1) our observationthat, six days after intravenous injection in two patients,urinary recovery was only 85 per cent and (2) theresults of animal studies which showed that no C-14-O2was present in the expired air. (Stools were not col-lected in these two experiments.) Reports with labeledtolbutamide,11-12 chlorpropamide,8 and tolazamide13 sug-gest that biliary excretion is not unique for acetohexa-mide. For example, although Fajans11 reports that infour of six patients nearly 100 per cent of orally ad-ministered tolbutamide-S-35 was recovered in the urine,in the remaining two patients the recovery was 90.6and 80 per cent. In these two subjects, failure to find100 per cent of the administered radioactive drug wasattributed to loss of urine samples. Thomas and Ikeda,12

using tritiated tolbutamide, reported only 85 per centrecovery of the orally administered labeled drug inthe urine in 48 hrs.

Johnson et al.8 reported that fecal excretion afterthree days was less than 3.0 per cent in a single pa-tient who received chlorpropamide-S-35 intravenouslywhile on chronic treatment orally. The long half-lifeof chlorpropamide suggests that, if stool analysis isused to assess the role of the biliary tract in excretion,collections would have to be made for a number ofdays after the drug's intravenous administration. Inone patient who received 400 rag. of tritiated tolaza-mide orally, 8.9 per cent of the radioactive drug wasfound in the feces by the end of 72 hrs.13 In our ex-periments and in those cited above, the administeredradioactive material not recovered in the urine was

probably excreted in the stools as a result of biliary-excretion and of the failure of a small quantity of thetablet to be absorbed. This latter factor was probablyenhanced by the fact that the labeled drug we usedwas pressed into a tablet resembling the commercialmaterial. To the best of our knowledge, in the studieswith tolbutamide and chlorpropamide, solutions or cap-sules were used.

Thus, the data presented here on acetohexamide, to-gether with the information available on tolbutamide,chlorpropamide, and tolazamide, suggest but do notdefinitely establish that, for all four of the sulfonylureasmentioned, the biliary tract is a minor pathway forexcretion. Further experiments are needed with labeledsulfonylureas administered orally and intravenously toanimals and/or human subjects with cannulated com-mon bile ducts to assess the importance of biliary ex-cretion and possible recirculation to the liver afterbiliary excretion.

ACKNOWLEDGMENT

The authors gratefully acknowledge the cooperationand assistance of Leo Oliner, M.D., Chief, RadioisotopeService, Veterans Administration Hospital, Indianapo-lis, Indiana; W. R. Kirtley, M.D., Director, MedicalResearch Division, Eli Lilly and Company, Indianapolis;Allan M. Peabody, M.D., Mr. R. H. Carmichael, andthe nursing staff of The Lilly Laboratory for ClinicalResearch, Marion County General Hospital, Indian-apolis; and Mr. W. M. Miller, Biochemistry-Microbi-ology Research Division, Eli Lilly and Company, In-dianapolis.

REFERENCES1 Marshall, F. J., Sigal, M. V., Jr., Sullivan, H. R., Cesnick,

G, and Root, M. A.: Further studies on N-arylsulfonyl-N'-alkylureas. J. Med. Chem. 6:60, 1963.

2 Welles, J. S., Root, M. A., and Anderson, R. C : Metabolicreduction of i-(p-acetylbenzenesulfonyl)-3-cyclohexylurea (ace-tohexamide) in different species. Proc. Soc. Exper. Biol. Med.107:583, 1961.

3McMahon, R. E., Marshall, F. J., and Culp, H. W.: Thenature of the metabolites of acetohexamide in the rat and inthe human. J. Pharmacol. Exp. Ther. 149:272, 1965.

4 Culp, H. W., and McMahon, R. E.: The NADPH2 de-pendent reduction of acetohexamide and related carbonyl com-pounds by a rabbit kidney enzyme. Fed. Proc. 24(2) \426y

1965.5 Smith, D. L., Vecchio, T. J., and Forist, A. A.: Metabo-

lism of antibiadetic sulfonylureas in man. I. Biological half-lives of the p-acetylbenzenesulfonylureas, V-18536 and aceto-hexamide and their metabolites. Metabolism 14:229, 1965.

6 Spingler, H.: Uber eine Moglichkeit zur colorimetrischeaBestimmung von N-(4-Methyl-Benzolsulfonyl)-N'-Butyl-Harn-

126 DIABETES, VOL. 16, NO. 2:

J . A. GALLOWAY, M.D., R. E. MC MAHON, PH.D., H. W. CULP, M.S., F. J . MARSHALL, PH.D., AND E. C. YOUNG, M.D.

stoff in Serum. Klin. Wchnschr. 35:533, 1957.7Kelly, R. G., Peets, E. A., Gordon, S., and Buyske, D. A.:

Determination of C-14 and H-3 in biological samples bySchoniger Combustion and liquid scintillation technics. Anal.Biochem. 2:267, 1961.

8 Johnson, P. C, Hennes, A. R., Driscoll, T., and West,K. M.: Metabolic fate of chlorpropamide in man. Ann. N.Y.Acad. Sci. 74:459, 1959.

9 Johnson, P. C, West, K. M., and Masters, F.: Albuminbinding of chlorpropamide. Metabolism 9:1111, i960.

10 Sheldon, J., Anderson, J., and Stoner, L.: Serum concen-tration and urinary excretion of oral sulfonylurea compounds.Relation to diabetic control. Diabetes 14:362, 1965.

11 Fajans, S. S.: Discussion of the paper: Metabolic fate ofchlorpropamide in man (Johnson, P. C, et al.). Ann. N.Y.Acad. Sci. 74:471, Mar. 30, 1959.

12Thomas, R. C, and Ikeda, G. J.: The metabolic fate oftolbutamide in man and in the rat. J. Med. Chem. 9:507, 1966.

13 Clinical Research Files, Upjohn Company, Kalamazoo,Michigan.

Maternal Carbohydrate Disorder andCongenital Malformations

Victoriano N. Navarrete, M.D., Isabel H. Torres, M.D., Irene R. Rivera, M.D.,Velvl P. Shor, M.D., and Pedro M. Grdcia, M.D., Mexico City

SUMMARYIn 152 consecutively observed mothers who had delivered

malformed infants in their last pregnancy, 10 per centhad abnormal standard glucose tolerance tests and 45 percent abnormal triamcinolone glucose tolerance tests. Thesefindings are in contrast to a 3.3 per cent prevalence ofabnormal triamcinolone glucose tolerance tests in a con-trolled group selected for absence of findings suggestingdiabetes. The observation suggests that a relation existsbetween carbohydrate disturbance and the delivery of mal-formed infants. DIABETES 16:127-30, February, 1967.

Among 137,000 babies born in Mexico City at theHospital for Gynecology and Obstetrics No. 1* duringthe period 1958 to 1963 there were 1,787 malformedinfants, an incidence of 1.3 per cent.1 Similar fre-quencies have been reported in New York,2 England,3

and Japan.4 These incidences are not wholly compara-ble, however, for they were gathered from birth cer-tificates and not obtained directly from the hospitalrecord information. Furthermore, there were undoubt-edly differences in interpretation of the term "malfor-mation." Presumably the figures derived from hospitaldata are more accurate than those obtained from birthcertificates.5"9

Many different causes have been proposed for con-genital malformations and have included genetics,10"17

environmental influence,18'19 viral disease,20'24 radia-tion,25"27 toxoplasmosis,28'29 drugs,30'32 and certain meta-

From the Internal Medicine Department, Hospital de Gineco-Obstetricia No. 1, "I.M.S.S.," Mexico City, D.F., Mexico.

•Instituto Mexicano del Seguro Social.

bolic disorders, particularly diabetes mellitus. In re-gards to the last, Downing and Goldberg observed afamily history positive for diabetes in 32 per cent ofchildren operated upon for cardiac septal defects,33 andWarren and LeCompte in postmortem examinationsfound congenital malformations in 18 per cent of in-fants of diabetic mothers.34 In a previous communica-tion we reported that congenital malformations werepresent in 11.7 per cent of the offspring of a group ofwomen with an abnormal triamcinolone glucose toler-ance test.35 Both Hoet36 and Torres37 have claimedthat the prevalence of congenital malformations in theinfants of diabetic mothers is decreased when gooddiabetic control is carried out throughout the preg-nancy. In spite of these findings and claims, Rubinand Murphy38 and Warkany and Kalter9 state that arelation of diabetes mellitus to the development of con-genital malformation remains to be determined.

It is the purpose of the present paper to describea high prevalence of abnormal carbohydrate tolerance inmothers giving birth to infants with congenital mal-formation.

MATERIALS AND METHODSObservations were made on 212 ambulatory and in-

fection-free women divided into two groups:Group I. All women (152) without known diabetes

who, from 1964 to the present time, delivered in thishospital a malformed infant in the previous pregnancy.

Group II. Sixty women, similar in parity and age,without history suggesting prediabetes or early diabetes.

Fifty-six per cent of the women with malformedinfants were from fifteen to thirty years of age and 44

FEBRUARY, 1967 127

metabolism, blood levels and rate of excretion of - diabetes

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