Journal of Clinical InvestigationVol. 42, No. 2, 1963

CHLORPROPAMIDE-INDUCEDLEUCINE HYPOGLYCEMIA*

By C. LUCASt AND G. REAVENt(From the Department of Medicine, Stanford University School of Medicine, Palo Alto, Calif.)

(Submitted for publication August 20, 1962; accepted October 24, 1962)

Previous reports from this laboratory havedemonstrated that L-leucine-induced hypoglycemiacan be experimentally produced in normal dogs dur-ing periods of either exogenous (1) or endoge-nous (2) hyperinsulinism. On the basis of theseexperiments, it was suggested that L-leucine actsto inhibit hepatic glucose output and that "leucinehypoglycemia" occurs in situations where increasedhepatic glucose output is necessary for maintenanceof euglycemia. To extend these observations ofexperimental leucine hypoglycemia, it seemed de-sirable to investigate the effect of L-leucine inother situations characterized by hypoglycemia,hyperinsulinism, or both. Consequently, we havemeasured the blood glucose response to L-leucineat varying intervals after both the acute andchronic administration of chlorpropamide.1 Inaddition, we have attempted to ascertain in con-trolled experiments whether or not L-leucine byitself has any effect on blood glucose concentra-tion. Finally, we have attempted to demonstratethat L-leucine inhibits hepatic glucose output bycomparing hepatic-vein-femoral-vein (HV-FV)blood glucose concentration differences in dogs.with and without pretreatment with chlorpropa-mide.

METHODS

A. Effect of L-leucine on peripheral blood glucose con-centration with and without chlorpropantide pre-treatment

Nine mongrel dogs were used in these experiments,and the hypoglycemic response to L-leucine was testedin each dog under all five of the following experimentalconditions: 1) 2 hours, 2) 12 hours, and 3) 24 hours af-

*Work supported in part by grant A-4293 from theNational Institute of Arthritis and Metabolic Diseases,U. S. Public Health Service, Bethesda, Md.

t Recipient of training grant 2A-5021-(C5), U. S.Public Health Service.

i: Address requests for reprints to: Dr. G. Reaven,Department of Medicine, Stanford University, Palo Alto,Calif.

1 I-propyl-3- (p-chloro-benzenesulfonyl) urea.

ter a single oral dose of chlorpropamide, 4) 24 hours af-ter 3 consecutive days -of the same oral dose of chlor-propamide, given once daily, and 5) without chlorpropa-mide pretreatment.

Chlorpropamide was given in a dose of 250 or 375 mg.All dogs weighing less than 10 kg were arbitrarily given250 mg, and 375 mg was used in dogs weighing over10 kg. Each experiment was performed as follows. Adog was anesthetized with pentobarbital, and two bloodsamples were drawn 15 minutes apart for glucose deter-mination, with the average of these two values used asa baseline. Then 30 mmoles of L-leucine in 200 ml ofdistilled water or an equal volume of 0.45 per cent NaClwas given by an intravenous infusion lasting approxi-mately 15 minutes. Blood was then drawn for measure-ment of glucose concentration 30 and 60 minutes afterL-leucine or saline had been administered. The resultshave been expressed as percentage of fall in glucoseconcentration (100 X maximal fall observed in blood glu-cose concentration divided by the baseline value). Thepercentage of fall in blood glucose due to L-leucine inany given dog under each experimental condition is thenexpressed as follows: maximal fall in blood glucose afterleucine X 100/average baseline minus maximal fall inblood glucose after saline X 100/average baseline. Bloodglucose was measured by the Somogyi method as modi-fied by Nelson (3).

Serum chlorpropamide levels were measured in eightindividual experiments by the method of Toolan andWagner (4).

B. Effect of L-leucine on hepatic metabolism

1. Effect of L-leucine on hepatic-vein and fentoral-veinblood glucose concentration in dogs without prior pre-treatment. Five experiments were performed with thefollowing protocol. After the induction of anesthesiawith pentobarbital, a radio-opaque catheter was passedpercutaneously into the vena cava from the femoral veinand inserted in a hepatic vein under fluoroscopic con-trol. A second catheter was placed in the other femoralvein for collection of peripheral venous blood. Afterthree blood samples were drawn from each catheterover a 15-minute period, each dog received a rapid in-travenous infusion of 30 mMof L-leucine in 200 ml ofdistilled water. Paired blood samples were then drawnfrom each catheter every 10 minutes for the next 60 min-utes. The location of the catheter in the hepatic veinwas established before each sample was taken. Bloodglucose was measured by the iodometric technique ofSomogyi (5, 6).

230

CHLORPROPAMIDE-INDUCEDLEUCINE HYPOGLYCEMIA

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FIG. 1. MEANHYPOGLYCEMICRESPONSETO L-LEUCINE OF

ALL DOGSTESTED (9). I-bars indicate SE of 2.

2. Effect of L-leucine or saline on hepatic-vein andfemoral-vein blood glucose concentration in dogs pre-treated with a single oral dose of chlorpropamide. Four-teen experiments were performed, seven with L-leucine andseven with saline. In this series of experiments, eachdog was pretreated with a single oral dose of chlorpropa-mide 4 hours before the beginning of the experiment. Inevery other respect, the experiments were like those de-scribed in section B1.

3. Effect of L-leucine on hepatic-vein, fentoral-vein, andfemoral-artery blood glucose concentration in dogs withportacaval shunt. Three experiments were performedon dogs in which end-to-side portacaval shunts had beenperformed several weeks previously. In each of theseexperiments the dogs were studied 4 hours after a singleoral dose of chlorpropamide. In addition to the previ-ously described catheters in the hepatic and femoral veins,a catheter was also passed percutaneously into the fe-moral artery, and blood was sampled from all three ofthese catheters for determination of blood glucose con-

centration. In this series of experiments, hepatic bloodflow was also estimated by the bromsulphalein (BSP)extraction method of Bradley, Ingelfinger, Bradley, andCurry (7). Bromsulphalein was measured by a modi-fication (8) of the method of Rosenthal and White (9).In other respects, the protocols for these experimentswere as described previously.

RESULTS

A. Effect of L-leucine on peripheral blood glucoseconcentration with and without chlorpropamidepretreatment

The blood glucose response of each dog toL-leucine under the five experimental conditionsis listed in Table I, and the mean hypoglycemic

response of all dogs is illustrated in Figure 1.The data were pooled and a single standard errorof 4.10 was calculated for each mean. Three con-clusions may be drawn from these experiments.First, L-leucine by itself produces a modest butstatistically significant fall in blood glucose con-centration (p < 0.03). Second, the mean hypo-glycemic response to L-leucine 2 and 12 hoursafter a single oral dose of chlorpropamide is sig-nificantly greater than that due to L-leucine alone.Finally, the mean hypoglycemic response to L-leu-cine in the same dogs receiving chlorpropamide ona chronic basis is significantly less than that 2 to 12hours after one acute dose, and no greater thanthat produced by L-leucine alone. The compari-son between means was made by the method of the5 per cent least significant difference (10), inwhich means that differed by more than 11.7 weresignificant at the 0.05 level.

It appears unlikely that the diminished responseto L-leucine 24 hours after 3 consecutive days ofchlorpropamide pretreatment results from a de-crease in the degree of hypoglycemia produced bychlorpropamide. First, baseline blood glucoseconcentration was as low after the chronic ad-ministration of chlorpropamide as it was after oneacute dose. Second, the serum levels of chlor-propamide 24 hours after 3 days of chlorpropamideadministration were as high as those measured 2hours after chlorpropamide (Table II). Althoughthere was no significant difference between themeasured chlorpropamide levels, the leucine effectwas more impressive in the 2-hour group.

TABLE II

Correlation between per cent fall in blood glucose concentra-tion after L-leucine administration and serum level of

chlorpropamide

Time L-leucine given

24 Hours after 32 Hours after dayswof chlor-

chlorpropamide propamide

Serum SerumFall in level Fall in levelblood chlor- blood chlor-

Dog no. glucose propamide glucose propamide

% AgIml % oglml911 43 107 27 174

21* 31 123 15 91574* 23 106 -20t 106

42* 30 75 7 120

* Experiments performed at different times from those in Table I.t Negative number signifies rise.

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232

I _-

CHLORPROPAMIDE-INDUCEDLEUCINE HYPOGLYCEMIA

TABLE III

Effect of L-leucine alone on hepatic-vein (HV)-femoral-vein (FV)blood glucose concentration differences

Baseline(time in minutes) Minutes after L-leucine administration

Dog -15 -7 0 10 20 30 40 50 60

mg/100 ml mg/100 ml

404 HV' 85 88 91 86 83 78 80 79 90FV 83 84 85 85 81 77 72 74 85HV-FV +2 +4 +6 +1 +2 +1 +8 +5 +5

901 HV 86 88 84 84 78 78 73 73 70FV 72 75 81 84 78 70 72 66 63HV-FV +14 +13 +3 0 0 +8 +1 +7 +7

657 HV 82 92 90 77 76 77 78 95 92FV 78 80 80 72 72 69 71 71 73HV-FX +4 +12 +10 +5 +4 +8 +7 +24 +19

450 HV 90 76 92 82 81 82 81 82 87FV 74 81 81 79 78 77 74 74 80HV-FVT +16 -5 +11 +3 +3 +5 +7 +8 +7

609 HV 89 86 92 85 83 83 82 80 81FV 82 88 87 83 81 78 79 74 72HV-FV +7 -2 +5 +2 +2 +5 +3 +6 +9

B. The effect of L-leucine on hepatic mnetabolismn1. L-Leucine alone. L-Leucine produced a small

but consistent decrease in blood glucose concen-tration of every dog, studied (Table III). Al-though there was considerable variation from timeto time and from dog to dog, it appeared as ifHV-FV glucose concentration differences tendedto narrow during the first 20 minutes after ad-ministration of L-leucine. This observation issomewhat more apparent when the mean responseof all dogs is examined (Figure 2). Mean HV-FV glucose concentration differences 10 and 20

100

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MORAL-VEIN GLUCOSECONCENTRATIONDIFFERENCES. Av-erage response of 5 dogs not pretreated with chlorpro-pamide is shown.

minutes after L-leucine were compared with meanbaseline values and found to be significantly de-creased (t test, p < 0.01). Although the meanHV-FV glucose concentration difference was de-creased at these times, it remained significantlypositive.

2. L-Leiucine given 4 hours af ter chlorpropamnide.The effect of L-leucine or saline on blood glu-cose concentration of each dog studied is seen inTable IV. L-Leucine consistently lowered bloodglucose concentration of both hepatic and femoralveins, whereas saline had no significant effect.

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FIG. 3. EFFECT OF L-LEUCINE ON HEPATIC-VEIN-FE-MORAL-VEIN GLUCOSECONCENTRATIONDIFFERENCES. Av-erage response of 7 dogs pretreated with chlorpropamideis shown.

233

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C. LUCAS AND G. REAVEN

TABLE IV

Effect of L-leucine on the hepatic-vein (HV)-femoral vein (FV) blood glucose concentration difference (HV-FV) in dogspretreated with chlorpropamide*

BaselineBlood (time in minutes) Minutes after L-leucine administration

glucoseDog concentration -15 -7 0 +10 +20 +30 +40 +50 +60

mg/100 ml63 73 6058 59 59

+5 +14 +1

53 56 4946 47 50

+7 +9 -1

74 75 7467 65 66

+7 +10 +8

609 HV 72 73 71FV 72 64 64HV-FV 0 +9 +7

657 HV 71 71 69FV 70 69 66HV-FV +1 +2 +3

190 HV 88 83 96FV 81 83 81HV-FV +7 0 +15

450 HV 82 81 81F-V 52 62 72HV-FV +30 +19 +9

574 HV 77 79 82FV 70 71 73HV-FV +7 +8 +9

901 HV 74 74 71FV 69 75 72HV-FV +5 -1 -1

760 HV 78 77 75FV 75 73 72HV-FV +3 +4 +3

609 HVFVHV-FV

657 HVFVHV-FV

764 HVFVHV-FV

450 HVFVHV-FV

63 70 6559 59 60

+4 +11 +5

57 55 5451 53 55

+6 +2 - 1

83 86 8878 77 78

+5 +9 +10

82 84 8676 77 79

+6 +7 +7-

mg/100 ml59 34 37 41 34 3644 52 45 29 25 29

+15 -18 -8 +12 +9 +7

46 43 41 35 52 5850 37 33 32 36 40

-4 +6 +8 +3 +16 +18

63 58 42 54 58 6756 54 52 49 52 60

+7 +4 -10 +5 +6 +7

63 60 54 60 61 6056 54 52 54 50 56

+7 +6 + 2 +6 +11 +4

67 61 46 43 54 4762 51 56 59 56 56

+5 +10 -10 -16 -2 -9

79 72 54 57 73 9178 69 62 70 60 84

+1 +3 -8 -13 +13 +7

53 49 47 41 67 5054 50 43 37 50 60

-1 -1 +4 +4 +17 -10

Minutes after saline administration

78 84 85 87 86 8275 77 80 81 81 77

+3 +7 +5 +6 +5 +5

78 81 80 81 82 7574 -74 73 68 68 64

+4 +7 +7 +13 +14 +11

73 72 65 67 66 6666 68 64. 63 63 63

+7 +4 +1 +4 +3 +3

69 66 69 68 69 6761 62 60 63 61 60

+8 +4 +9 +5 +8 +7

55 51 55 57 52 5551 48 48 48 47 43

+4 +3 +7 +9 +5 +12

92 89 87 88 87 8779 80 79 83 79 80

+13 +9 +8 +5 +8 +17

84 87 92 93 93 9679 78 82 86 83 81

+5 +9 +10 +7 +10 +15

The mean response of all dogs receiving L-leu- First, the hypoglycemic response to L-leucine 4cine after chlorpropamide pretreatment is illus- hours after chlorpropamide is much greater thantrated in Figure 3. Although there was again that resulting from L-leucine alone. Second, al-considerable variation;from time to time and from though the effect of L-leucine on HVand FV glu-dog to dog, certain conclusions seem warranted. cose concentration appears similar during the

402 HVFVHV-FV

901 HVFVHV-FV

383 HVFVHV-FV

234

CHLORPROPAMIDE-INDUCEDLEUCINE HYPOGLYCEMIA

first 20 minutes, regardless of pretreatment, cer-

taim differences becomne apparent 30 and 40 min-utes after the administration of L-leucine. In ani-mals receiving L-leucine alone, the HV blood glu-cose concentration tended to increase from then011.

In contrast is the continued, accelerated fall inHV glucose concentration during the augmentedhypoglycemic response after pretreatment withchlorpropamide. Under these conditions, themean HV-FV glucose concentration differencecontinued to decrease until, 30 and 40 minutes af-ter the administration of L-leucine, the normal posi-tixe hepatic balance was abolished, and then themean HV-FV glucose concentration differencewxAas not statistically significantly different fromzero (t test). The mean response to saline 4hours after chlorpropamide is illustrated in Fig-ure 4, which. shows that HV-FV blood glucoseconcentration differences did not vary appreciably,remained positive throughout, and were statisti-cally different from zero (p <0.001).

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FIG. 4. EFFECT OF SALINE ON HEPATIC-VEIN-FEMORAL-VEIN GLUCOSECONCENTRATIONDIFFERENCES. Average re-

sponse of 7 dogs pretreated with chlorpropamide isshown.

3. L-Leucblie 4 hours after chlorpropainide indogs with portacaval shunt. These experimentswere performed in an effort to distinguish the ef-fect of L-leucine on hepatic metabolism from any

possible effect it might have on adipose tissue

TABLE V

Effect of L-leucine on hepatic-vein (HV), hepatic-artery (HA), and femoral-vein (FV) blood glucose concentration in dogswith end-to-side portacaval shunts and pretreated with chlorpropamide

Baseline(time in minutes) Minutes after L-leucine administration

Dog no. -20 -10 0 10 20 40 60

379 Concentration, mg/100 ml

HV 72 75 70 48 44 28 18HA 60 62 58 47 39 24 14FV 55 57 54 42 38 22 12HV-HA +12 +13 +12 + 1 +5 +4 +4HV-FV +17 +18 +16 +6 +6 +6 +6

Estimated hepatic blood flow, ml/min 169 162 147 160 155 147 157Estimated net hepatic glucose output, mg/min* 20.28 21.06 17.64 1.60 7.75 5.88 6.28

378 Concentration, mg/100 ml

HV 80 78 75 60 54 45HA 72 71 70 59 56 46FV 63 60 62 58 55 46HV-HA +8 +7 +5 +1 -2 -1HV-FV +17 +18 +13 +2 -1 -1

Estimated hepatic blood flow, ml/min 182 170 196 200 186 160Estimated net hepatic glucose output, mg/min* 14.56 11.90 9.80 2.0 -3.72 -1.60

377 Concentration, mg/100 ml

HV 69 68 67 57 36 36HA 63 62 62 60 38 40FV 57 57 56 51 34 35HV-HA +6 +6 +5 -3 -2 -4HV-FV +12 +11 +11 +6 +2 +1

Estimated hepatic blood flow, ml/min 237 328 359 371 342 335Estimated net hepatic glucose output, mg/min* 14.22 19.68 17.95 -11.13 -6.84 -13.40

* Negative value indicates absence of net hepatic glucose output.

235

C. LUCAS AND G. REAVEN

drained by the splanchnic bed. In dogs with por-

tacaval shunt, the blood glucose concentration dif-ference between the hepatic vein and the periph-eral arteries and veins is primarily a function ofhepatic metabolism alone. Under these conditions,it is assumed that blood glucose concentration inthe femoral artery is identical with that in thehepatic artery. The results of these experimentsare seen in Table V and are similar to those ob-served under the same conditions in intact dogs,i.e., hypoglycemia produced by L-leucine was ac-

companied by a marked fall in HV-FV and HV-HA (hepatic-artery) blood glucose differences.In addition, estimated hepatic blood flow was shownnot to change appreciably after L-leucine adminis-tration, and consequently net hepatic glucose out-put was markedly reduced during hypoglycemia.The observation that L-leucine does not affecthepatic blood flow was also confirmed in 3 otherdogs by use of a noncannulating, square-wave,

electromagnetic flowmeter ( 11 ) .2 Consequentlyit appears unlikely that the changes in HV-FVblood glucose concentrations in intact dogs couldhave resulted from increased splanchnic glucoseutilization, and they indicate further that the ef-fect of L-leucine is to inhibit mechanisms for themaintenance of hepatic glucose output.

DISCUSSION

Any theory attempting to explain the mechanismof leucine hypoglycemia must take into accounttwo fundamental characteristics of this phenome-non. First, although the results we have de-scribed demonstrate that L-leucine can lowerslightly the blood glucose concentration of nor-

mal subjects, significant hypoglycemia has onlybeen produced in certain patients with idiopathicinfantile hypoglycemia (12), or insulin-secretingtumors of the pancreas (13), or in normal men

(14) and dogs pretreated with insulin (1) or

chlorpropamide. Second, the response to L-leu-cine is not uniform in these situations and cannotbe demonstrated in all patients with these clinicalsyndromes (15-19), nor, as we have shown, inall dogs pretreated with chlorpropamide. Sinceall the situations in which L-leucine had produceda significant hypoglycemic response seemed to

2 These measurements were obtained with the aid ofDr. S. Kountz, Department of Surgery, Stanford Uni-versity School of Medicine, Palo Alto, Calif.

share in common hyperinsulinism, or hypogly-cemia, or both, we had suggested earlier that L-leu-cine "decreases the hyperglycemic response tohypoglycemia, specifically, hepatic glucose output'(1). The present demonstration that net hepaticglucose output is markedly reduced during hypo-glycemia produced by L-leucine supports this the-sis. On the other hand, even if it is assumed thatL-leucine produces hypoglycemia by inhibitinghepatic glucose output during hypoglycemic stress,it is difficult to explain the variable response toL-leucine in what appear to be identical situations.

An attempt to solve this dilemma has beenmade by differentiating between patients who are"leucine-sensitive" and those who are "leucine-insensitive" (20). Implicit in this distinction isthe assumption that these two categories are re-flections of different disease processes, and thatpatients can be separated on the basis of theirresponse to L-leucine. Since the etiology or etiolo-gies of idiopathic infantile hypoglycemia are ob-scure, the merit of a distinction based on the pa-tient's response to L-leucine is difficult to evalu-ate. On the other hand, the distinction between"leucine-sensitive" and "leucine-insensitive" seemsto have little meaning when applied to patientswith insulin-secreting tumors of the pancreas, andavoids the question of why L-leucine produceshypoglycemia only in some patients with organichyperinsulinism (15-17). This problem is notconfined to clinical situations, and it is as difficultto account for the variable hypoglycemic responseto L-leucine that has been observed in experi-mental leucine hypoglycemia. The administrationof L-leucine to normal dogs, as to normal men,does not elicit significant hypoglycemia. In con-trast, if L-leucine is administered within 2 to 12hours after a single oral dose of chlorpropamide,normal dogs, like "leucine-sensitive" patients,demonstrate a significant hypoglycemic response.Furthermore, when L-leucine was given to normaldogs who had received chlorpropamide chroni-cally, there was only a modest fall in blood glucoseconcentration, similar to that described in "leu-cine-insensitive" patients. Although the analo-gies are obvious, it is clear that they are onlyanalogies. On the other hand, the conditions un-der which experimental leucine hypoglycemia hasbeen studied were varied purposefully, and it ispossible that a more detailed analysis of these ex-

236

CHLORPROPAMIDE-INDUCEDLEUCINE HYPOGLYCEMIA

periments may prove helpful in explaining theconsiderable variation in the hypoglycemic effectof L-leucine.

In the experiments described, two basic pat-terns of response to L-leucine were demonstrated.A normal dog receiving L-leucine exhibited a smallfall in blood glucose concentration. The same dog,given the same amount of L-leucine 2 hours aftera single oral dose of chlorpropamide, demonstrateda significantly greater hypoglycemic response. Inthe first instance, normal dogs are not reacting toany hypoglycemic stimulus, and it might be pre-dicted that the ability of L-leucine to inhibit hepaticglucose output would result in only a minor fallin blood glucose concentration. The degree, how-ever, of hypoglycemic stress in a normal dog 2hours after 250 to 375 mg of chlorpropamide iscertainly greater, and in this case, inhibition ofhepatic glucose output could possibly result in amuch greater fall in blood glucose concentration.On the other hand, it is clear that the degree ofhypoglycemia produced by L-leucine is not directlyrelated to the severity of the hypoglycemic stimu-lus alone. For example, dogs receiving chlor-propamide chronically were, if anything, reactingto a more profound hypoglycemic stimulus thanwere dogs 2 hours after a single oral dose ofchlorpropamide. Yet the administration of L-leu-cine to dogs after chronic chlorpropamide pre-treatment elicited a fall in blood glucose con-centration no greater than that observed afterL-leucine alone. Perhaps the explanation for thediminished effect of L-leucine in normal dogs afterchronic pretreatment with chlorpropamide is thatthe counter-regulatory mechanisms for the main-tenance of euglycemia had been so well stimulatedthat it was more difficult to produce hypoglycemiain this case. Obviously there are insufficient datato explain precisely why L-leucine acts differentlyunder certain conditions. On the other hand, itis clear that different hypoglycemic responses toL-leucine can occur in the same subject underdifferent conditions, and it is difficult in the lightof these observations to view "leucine sensitivity"as an "inborn error of metabolism." It seemsmore reasonable to assume that the specific meta-bolic action of L-leucine is relatively constant un-der all conditions and that only the resultant fallin blood glucose concentration varies from situ-ation to situation. Finally, it would appear that

a more thorough understanding of the variousmechanisms by which the liver maintains eugly-cemia is essential for ultimate understanding ofthe phenomenon of leucine hypoglycemia.

SUMMARY

The administration of L-leucine alone to nor-mal dogs slightly lowered blood glucose concen-tration, which was greatly increased after suitablepretreatment with chlorpropamide. Experimentsperformed on both intact dogs and dogs withportacaval shunt have indicated that this aug-mented hypoglycemic response to L-leucine afterchlorpropamide is accompanied by a marked de-crease in positive hepatic glucose balance. It issuggested that L-leucine acts similarly in all situa-tions to inhibit hepatic glucose output and that theresultant fall in blood glucose concentration willvary considerably, depending on the status of otherhomeostatic mechanisms for maintenance of eugl--cemia at the time L-leucine is administered.

ACKNOWLEDGMENT

The authors wish to express their appreciation to Mr.James Black of the Charles Pfizer Company for hisgenerous supply of chlorpropamide, to Miss NancyChristiansen for her technical assistance, and to Dr.Rupert Miller for his helpful statistical suggestions.

REFERENCES

1. Reaven, G., and Lucas, C. The use of insulin in theproduction of L-leucine-induced hypoglycemia innormal dogs. J. clin. Invest. 1962, 41, 654.

2. Reaven, G., Lucas, C., and Winkelman, C. Hypo-glycemic effect of L-leucine during periods of en-dogenous hyperinsulinism. Diabetes. In press.

3. Nelson, N. A photometric adaptation of the So-mogyi method for the determination of glucose.J. biol. Chem. 1944, 153, 375.

4. Toolan, T. J., and Wagner, R. L., Jr. The physicalproperties of chlorpropamide and its determinationin human serum. Ann. N. Y. Acad. Sci. 1959, 74,449.

5. Somogyi, M. Determination of blood sugar. J.biol. Chem. 1954. 69, 160.

6. Somogyi, M. Notes on sugar determination. J.biol. Chem. 1952, 19, 195.

7. Bradley, S. E., Ingelfinger, F. J., Bradley, G. P.,and Curry, J. J. The estimation of hepatic bloodflow in man. J. clin. Invest. 1945, 24, 890.

8. Henry, R. J., Chiamori, N., and Ware, A. G. De-termination of bromsulfonphthalein in serum bymeans of acetone precipitation of proteins. Amer.J. clin. Path. 1959, 32, 201.

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C. LUCAS AND G. REAVEN

9. Rosenthal, S. M., and White, E. C. Clinical ap-plication of the bromsulphalein test for hepaticfunction. J. Amer. med. Ass. 1925, 84, 1112.

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