Evidence favoring the sarcomatous origin of an insulin-like substance in a case of fibrosarcoma with hypoglycemia

Evidence Favoring the Sarcomatous Origin of an Insulin-Like Substance in a Case of Fibrosarcoma with Hypoglycemia* ROBERT VOLPH, M .D ., JOHN EVANS, M .D ., D .PHTL ., DONALD W . CLARKE, PH .D ., NICHOLAS FORBATH, M .D . and ROBERT EHRLICH, M .D . Toronto, Ontario, Canada ATIENTS manifesting "Whipple's triad" of

fibrosarcoma in which homogenates of the tumor possessed large amounts of insulin-like activity as determined by its ability to stimulate the conversion of labeled glucose into carbon dioxide and glycogen ; the conversion to glycogen is considered to be specific for insulin . These investigators also found that the insulin-like activity of the tumor was easily suppressed by aa sulfhydryl inhibitor and by specific antibeef-insulin guinea pig antibody. They thus infer that the tumor is indeed producing insulin . Nevertheless the results of their immunoassays for plasma insulin did not show any elevation, not even the usual rise following an injection of glucose . Because of the conflict in these and other data in many of the published cases, the mechanism(s) of the hypoglycemia remain obscure . Theories advanced to explain this manifestation include (1) hepatic insufficiency [34] ; (2) decreased hepatic inactivation of insulin [20] ; (3) reflex hyperinsulinism due to torsion of the pancreatico-duodenal vein by the weight of the tumor, with pancreatic venous stasis [35] ; (4) blocking of sympathetic impulses to the liver by pressure on the splanchnic nerves and celiac plexus by the tumor [33,36,37] ; (5) high carbohydrate consumption by the tumor [15,20,38, 391 ; (6) secretion by the tumor of insulin or insulin-like substances [13,15,16,18,22,28-32] ; (7) elaboration of an amino acid or similar substance causing hypoglycemia by an action unlike that of insulin [11] ; and (8) elaboration by the tumor of a substance which stimulates pancreatic beta cell activity [40] or which makes the tissues more responsive to pancreatic insulin .

P attacks occurring in the fasting state associated with blood sugar levels below 50 mg, per

100 ml ., and relieved by the administration of glucose, were previously considered to have pancreatic insulinomas . The possibility that nonpancreatic tumors might produce hypoglycemia was first proposed by Doege [1] and Anderson [2] . In 1961 Lowbeer [3] summarized forty-nine cases of this type and since that time forty-one further examples of the syndrome have been added [4-26,76,77] . Of the ninety cases now recorded, large tumors of mesodermal origin (0 .7 to 9 kg .) were found in forty-three ; they were located variously in the retroperitoneal space, in the mediastinum, in the pelvis, attached to the diaphragm, or within the liver . Other tumors reported to produce hypoglycemia include carcinomas of the adrenal cortex (thirteen cases) [76], pseudomyxoma peritonei (two cases), primary hepatic carcinoma (twentynine cases) [77], and gastrointestinal carcinoma (three cases) [16,27] . Bioassays of insulin-like activity in the tumors and plasma have yielded conflicting results ; values have been increased in some reports 113,15,16,18,22,28-32,781 and negative in others [11,14,17,21,33] . Oleesky et al . [18] reported that radioimmunoassay for insulin in the serum in one such case gave a very high result ; however, Schonfield [15] and Tranquada [16] et al ., while finding definite insulin-like activity in their cases, noted that immunoassay technics gave negative results for insulin . Boshell et al . [22] recently reported a case of a retroperitoneal

• From the Departments of Medicine, Physiology, and Paediatrics, University of Toronto, and the Medical Service, St . Joseph's Hospital, Toronto, Ontario, Canada . This study was presented in part at the annual meeting of the Royal College of Physicians and Surgeons of Canada, Edmonton, Alberta, January 17-19, 1963 . Manuscript received April 8, 1964.

540

AMERICAN JOURNAL OF MEDICINE

Sarcomatous Origin of an Insulin-Like Substance-Volpe el al . Recent reports tend to favor the view that most of the tumors are elaborating insulin or insulin-like substances, although it is evident that any one mechanism will not necessarily be operative in all cases . Observations in the case of fibrosarcoma with hypoglycemia to be described appear to support the view that this tumor is elaborating a substance which acts like insulin ; it may also be related immunologically, although is evidently not identical with insulin . CASE REPORT

Mrs . S . B ., born in 1908, was first admitted to St . Joseph's Hospital, Toronto, because of constipation and an abdominal mass in 1954 . A large tumor was removed front the transverse mesocolon, and a transverse colectomy was performed . The pathologic specimen measured 15 by 12 by 8 cm . and, on histologic examination, proved to be a spindle cell fibrosarcoma . The patient recovered without complication and continued well until 1959 when she was readmitted because of abdominal pain and recurrence of the large abdominal mass . At laparotomy a large tumor was found in the region of the right ovary, sigmoid colon and peritoneum . Again histologic examination showed a spindle cell fibrosarcoma . Cobalt radiation was given following operation . Except for mild, recurrent pain in the right upper quadrant she remained well until August 1961 when she noted moderate ankle edema . In October 1961 she experienced severe pain in the right upper quadrant, which radiated into the right flank, and was partially relieved when she sat up and bent forward . She also complained of right shoulder tip pain, anorexia, nausea, vomiting and constipation . After these symptoms had been present for three days the patient became confused and stuporous and was finally admitted to the hospital in a comatose state . In the emergency department she was given 5 per cent glucose intravenously and rapidly regained full consciousness . During the first few days in the hospital the patient had repeated bouts of confusion, slurred speech and weakness associated with sweating, tremor and tachycardia ; at times these culminated with the patient lapsing into a stupor . These episodes all occurred during fasting, were associated with subnormal blood sugar values, and were quickly relieved by the oral or intravenous administration of glucose . Within a few days of admission the abdominal and shoulder tip pain decreased, the nausea and vomiting stopped and the patient's appetite returned . Physical examination in October 1961 revealed a well developed, well nourished depressed middle-aged woman in considerable discomfort, The cardiac apex rate was regular at 88 per minute with a blood pressure of 130/80 mm . Hg . The thyroid gland was enlarged, firm, smooth, mobile, and the right lobe was larger than the left . The abdomen was slightly dis-

VOL . 38, APRIL 1965

X41

tended and the old surgical scars were noted .'l enderness was noted in the right upper quadrant and right flank but there were no signs of peritoneal irritation . The right lobe of the liver was firm, tender and enlarged 6 cm . below the right costal margin . Minimal pitting edema was noted at the right ankle . Neurologic examination was within normal limits between hypoglycemic attacks . The remainder of the physical examination was noncontributory Laboratory investigations indicated hypoglycemia . when the patient was in the fasting state and during episodes of confusion, abnormal bromsulfalein retention, and minor alterations in the serum protein fractions . Urinalyses were normal . 'File hemoglobin was 11 .0 gm . per cent, with a mildly hypoehromic, microcytic smear . The erythrocyte count was 3,7011,000 per cu . mm . ; the leukocyte count 7 .500 per cu . nun . . with a normal differential . Serum sodium was 140 mEq . per L . ; potassium, 3 .6 mEq . per L . ; chloride, 109 mEq . per L- ; serum phosphorus (fasting), 3 .0 mg . per 100 ml . ; carbon dioxide combining power, 26 .5 mEq . per L . ; and blood urea nitrogen, 6 mg . per 100 nil . The 24 hour urinary 17-ketosteroid level was 6 .1 mg . per day, the 17-hydroxycorticoid level 3 .9 mg . per day (both normal) . Bromsulfalcin retention was 14 per cent in 45 minutes ; serum bilirubin, 1 minute 0 .07 mg, per cent, total 0 .08 tug, per cent ; serum glutamic oxaloacetic transaminase, 21 units ; cephalin-cholesterol flocculation, negative ; alkaline phosphatase, 16 .8 King-Armstrong units ; albumin : globulin ratio, 3 .7 :3 .2 (electrophoresis showed a high gamma globulin of 18 per cent) . The urine was negative for bile and urobilinogen . The protein-bound iodine was 5 .4 µ g . per cent [411 (normal in our laboratory is 3 .5 to 6 .5 µg . per cent) ; 24 hour thyroidal list uptake, 39 per cent (normal 10 to 40 per cent) ; erythrocyte uptake of 1 131-labeled triiodothyronine, 16 .1 per cent (normal 12 .5 to 18 .5 per cent 1421) ; thyroid antibody (tanned red cell hemagluttination, complement fixation test), negative . Roentgenologic examination of the upper gastrointestinal tract demonstrated displacement of the stomach by the enlarged liver . A photoscan with 1"'labeled rose bengal showed the presence of large "cold areas" within the liver, compatible with large tumor deposits . (Fig . 1 .) Special studies were carried out in an effort to elucidate the nature of the hypoglycemia (see section "Special Investigations") . The episodes of hypoglycemia were satisfactorily controlled by the oral administration of glucose although it was necessary to awaken the patient at 3 A .M . to administer sweetened orange juice . At laparotomy on November 16, 1961, the right lobe of the liver was greatly enlarged and found to enclose a huge tumor approximately 12 cm . in diameter . The left lobe of the liver appeared normal . The pancreas was within normal limits and no other



542

Sarcomatous Origin of an Insulin-Like Substance-Volpe et al .

FIG . 2 . High power photomicrograph of the 1961 biopsy specimen of the tumor, showing a spindle-cell fibrosarcoma identical to the tumors removed in 1954 and 1959. The cells are large and vesic fated, there is considerable collagen formation, and the tumor is very vascular. No structures comparable to acini can be seen, and mitotic figures are rare .

Scan of the liver (November 1961) using 1'nlabeled rose bengal . The large indented "cold" areas represent the tumor invasion within the liver . FIG . 1 .

BLOOD

250

100

PLASMA

200

80

INSULIN

150

60

S .B . 4179-54

IlWf1NOASSAY uu/mt . 40 50

20

0

1

2

3

4

5

HOURS

Oral glucose tolerance test . The fasting blood glucose level is low, but the 1, 2 and 3 hour levels are above normal, indicating a diabetic-type curve . By 5 hours, however, the blood glucose level is down to 52 mg . per 100 ml . Insulin immunoassay levels do not rise significantly following the glucose load. FIG. 3 .

tumor deposits were recognized within the abdomen . A biopsy specimen of the hepatic tumor showed spindle cell fibrosarcoma, apparently identical with the tumors removed in 1954 and 1959 . (Fig. 2 .) No evidence of acinar formation was noted and mitoses were infrequent . Insufficient tissue was removed for insulin assay . The patient recovered well from the laparotomy but right upper quadrant pain persisted and the liver continued to enlarge gradually . A course of 3,500 r of cobalt radiation directed to the hepatic tumor did not diminish the tendency towards hypoglycemia,

reduce the size of the liver or relieve the abdominal pain . During the final eighteen months of the patient's life, the liver gradually enlarged and the abdominal pain became more constant and severe . Dexamethazone, 1 .5 mg ., was given at bed-time to prevent nocturnal hypoglycemia, in addition to sweetened orange juice at 3 A.M. The patient gained 10 pounds and moderate dependent edema and ascites developed . Numerous small papillomatous skin lesions appeared which, on histologic examination, proved to be fibromas . The patient's general health deteriorated AMERICAN JOURNAL OP MEDICINE



Sarcomatous Origin of an Insulin-Like Substance-Volpe c al .

543

8r0LC gm, IoIOUlumicr I

very slowly and on October 22, 1963, she died . Autopsy was not performed . BLOOD GLUCOSE

SPECIAL INVESTIGATIONS

Blood Sugar (True Glucose-Autoanalyzer Modification of Hoffman Technic [43]) . The blood sugar levels were determined at various times . During attacks, they ranged from 25 to 38 mg . per 100 ml . ; in the morning, while the patient was in the fasting state (after 5 to 7 hours of fasting), they ranged from 36 to 68 mg . per 100 ml . ; after a 22 hour fast, the blood sugar level was 36 mg . per 100 ml . and after a 9 hour fast, 40 mg . per 100 ml . A standard oral glucose tolerance test (Fig . 3) was also performed . The patient was given a 300 gm . carbohydrate diet for ten days before the test . The fasting value was low but the diabetictype curve suggested deficient insulin secretion in response to the glucose load . Two further oral glucose tolerance tests were performed at intervals ; the resultant curves were very similar . Intravenous Tolbutarnide Test (Fig . 4) . The blood sugar was measured at intervals following intravenous injections of 1 gm . of tolbutamide . Since tolbutamide increases the release of insulin from beta cells of the islets of Langerhans [4446], blood glucose levels fall to an average of 50 per cent of the value before injection within 30 minutes in normal subjects . In patients with insulinomas the response is greater, whereas in diabetic subjects blood sugar decreases much less [47-49] . In our patient the fall in the blood glucose level was slight, resembling the pattern seen in diabetic subjects . (Fig . 4 .) Such lack of response has been described previously in patients with nonpanereatic tumors associated with hypoglycemia [22,32] . This test, therefore, may also be interpreted to indicate decreased responsiveness of the pancreatic beta cells . Insulin Tolerance. Test (Fig . 5) . A fasting insulin tolerance test was performed using regular insulin 0 .1 units per kg . body weight intravenously . As shown in Figure 5, the patient was found to be normally responsive to insulin . Intramuscular Glucagon* Test (Fig . 6) . In order to determine the availability of hepatic glycogen, 3 .0 mg . glucagon hydrochloride was administered intramuscularly when the patient was in the fasting state . There was a striking hyperglycemic response, with the blood glucose level rising to 266 mg . per 100 ml . in 2 hours . Un* Glucagon was obtained from the anapolis, Indiana . VOL .

38,

APRIL

1965

Eli Lilly

Go ., Indi-

V.

~~ 40 -

mg.

5 . 9- c, r9 . ,4

per

come .

20-

C`-I J I . C 1 5 30 45 60

i . . 90 MINUTES

.I 120

180

Intravenous tolbutamide test . There is an insignificant fall in blood glucose levels following the intravenous injection of tolhutamide . This subnormal result is similar to that seen in diabetic subjects, and suggests beta cell hyporesponsiveness to tolbutamide . FIG . 4.

F

1

7.0 u . req . insulin 1,V,

60{ BLOOD GWGOSE

mg . per 100 .1 .

40\

5 . 8, 4179-54

20

0

30

60

90 MINUTES

120

150

180

FIG . 5 . Insulin tolerance test . The curve shows a normal response to exogenous insulin .

OL0

30

60

90 120 MINUTES

150

180

FIG . 6 . Intramuscular glucagon test . Following the injection of 3 .0 mg . glucagon intramuscularly (fasting) there was a very pronounced hyperglycemic effect, demonstrating both adequate hepatic glycogen storage and prompt glycogenolysis under this stimulus .



5 44

Sarcomatous Origin of an Insulin-Like Substance- Volpe et al . 808 .Ogm .leucinep .o .

BLOOD

7,5

60r'

BLOOD 5 .0 .4179-54

GLUCOSE

GLUCOSE

50 mg. per 100 .1 .

40

L

Mg . per 100 m1.

epinephrine 1 :1000 25

S .B .4179-54

0 .5 cc subculoneous

00 2

3

HOURS

30

L 60

, 90

i 120

150

J 180

MINUTES

Subcutaneous epinephrine test . Epinephrine administration resulted in a prompt rise in blood glucose levels, again demonstrating adequate glycogen storage and release . FIG . 7 .

ASSAYS

Time

Blood Glucose (mg . per 100 ml .)

Fasting

40

hour postprandial . . . .

202

Fasting

51

1

OF

Leucine tolerance test . L-leucine, 8 .0 gm ., was given orally in the fasting state . There was no significant fall in the blood glucose levels following the ingestion of leucine. FIG . 8 .

TABLE I INSULIN AND INSULIN-LIKE ACTIVITY Results (per ml .)

Type of Assay

Rat diaphragm glucose uptake [50,511 Rat diaphragm glucose uptake [50,511 Radioimmunoassay

Normal Range (per ml .)

125

milliunits

40-80

207

milliunits

130-800

8

microunits

<25

microunits microunits

microunits

[52,531 30

min . postprandial . . .

158

Radioimmunoassay .

12

microunits

~

100-300

microunits

[52,531

NOTE : The radioimmunoassay values obtained for fasting and postprandial insulin in this patient are not significantly different from each other by this method .

fortunately, the dosage and route of administration do not allow direct comparison with the

there was no significant fall in blood glucose levels consequent to leucine ingestion .

findings of others . This response, however, is indicative of adequate hepatic glycogen storage, and is consistent with the observations in similar tests in other cases of fibrosarcoma with hypoglycemia [11,18,29] . Furthermore the increase

Plasma Insulin Assays . Plasma was bioassayed for insulin-like activity by a modification of the rat diaphragm glucose uptake method of Vallance-Owen et al . 150,511 . The values obtained are shown in Table I . It may be noted

in blood glucose was greater than obtained by us in one patient with insulinoma, and in two normal persons tested in an identical manner . Subcutaneous Epinephrine Test (Fig . 7) . Five tenths of a cubic centimeter of epinephrine 1 :1000 solution was administered subcutaneously ; as shown in Figure 7 the injection of the epinephrine resulted in a definite and prolonged increase in blood sugar . L-Leucine Tolerance Test (Fig. 8) . Eight grams of L-leucine was given orally while the patient was in the fasting state . As depicted in Figure 8,

that the results are greatly elevated, both in the fasting and the postprandial state . Thus, despite the great difficulties encountered in this type of bioassay, and the wide range of normal results reported [511, it can be safely stated that the values were elevated in this case . After an initial attempt to neutralize this insulin-like activity by anti-human-insulin (provided by Dr. P . J . Moloney), the insulin-like activity remained equally high . However, when this was repeated on two further serum samples from this patient, these same observations held true AMERICAN JOURNAL

OF

MEDICINE



Sarcomatous Origin of an Insulin-Like Substance- -- Volpe' et al . in one sample whereas in the other, partial (but not total) neutralization was observed . Radioimmunoassays of plasma insulin were performed by the Morgan and Lazarow modification [5 2 ] of the method of Yalow and Berson [53] . Some of the results are noted in Table t, whereas others are shown in Figures 3, 9 and 10 . There was considerable variation in these values during the period of study . During a glucose tolerance test (January 31, 1963), the fasting plasma insulin was 8 yp per ml ., a value considered within normal (normal fasting 0 to 25 µµ per ml .) . However, there was no significant rise following the ingestion of glucose (normal 30 minute postcibal insulin level 100 to 300 gµ per ml .) . (Fig . 3 .) The fasting plasma insulin during the 1963 studies was often undetectable (e .g ., during March 1963), within normal limits in April, and elevated above normal during the periods when she was receiving human growth hormone and ovine prolactin . However, a final control fasting assay carried out six clays after the end of the prolactin period (and after every other evidence of prolactin activity had disappeared) was 88 µp per ml . This value is above normal, but constitutes the only clear-cut fasting elevation of the insulin immunoassay in a control period . Dr . John C . Floyd, University of Michigan, also carried out insulin immunoassays of plasma samples (August 1963), reporting less than 10 qi per ml . during fasting and after the ingestion of food . However, the frozen samples were warm when they reached Ann Arbor . Dr . John Davidson, Department of Physiology, University of Toronto, also performed an insulin assay on this patient's fasting serum (June 1963), using the method of Davidson and Haist [54] . In this method the serum is extracted with acid alcohol and dialyzed . The assay system used employed an analysis of the regression of the glycogen content of mouse hemidiaphragm against the log-dose of insulin in a validated four-point parallel slope arrangement [55] . The total serum insulin was found to be 2 .98 milliunits per ml . (95 per cent confidence limits 1 .96 to 4 .50 milliunits per ml .) This value is well within the normal range of 0 .8 to 7 milliunits per ml . [54] . Pool Size and the Rates of Production and Utilization of Glucose . The method of successive measured injections of tracer [.56] was used . The experimental procedure consisted of three injections of 25 µc . of uniformly labeled C14-glucose . Each injection was followed by a 60 to 90 minute VOL . 38, APRIL 1965

.543

sampling period . Samples were taken at 10 to 20 minute intervals and plasma glucose concentration and specific activity of plasma glucose were determined in each sample . Glucose from plasma was isolated by paper chromatography according to Depocas [57] . The slopes of the specific activity versus time curve and plasma glucose concentration versus time curve were calculated with the aid of the IBM 7090 computer . The glucose pool (N) (= amount of glucose in the body with which the injected tracer rapidly intermixes), the apparent volume of distribution of glucose or glucose space = (N/plasma glucose concentration), the rate of appearance of glucose in the central compartment, i .e ., endogenous glucose production (Ra), and the glucose utilization of the whole body (Rut) were calculated according to the usual technics [56] . With the patient in the fasting state, plasma sugar was 17 .5 mg . per cent and the glucose pool was correspondingly small, 4 .94 gin . (average of normal control subjects 14 .1 t 1 .31 gm .) [58] . The glucose space was 28 .3 L . or 40 .9 per cent of the body weight, considerably higher than the 26 .4 t 1 .1 per cent of body weight in our normal control subjects [58] . A likely cause for the high glucose space was the huge tumor . The rate of endogenous glucose production was low, 1 .53 tng . per kg . per minute (average of normal control subjects 1 .91 t 0 .053 rng . per kg . per minute) [3] . The rate of glucose utilization was 1 .50 mg . per kg . per minute (average of normal control subjects 1 .95 t 0 .03 mg . per kg . per minute) [58] . Both the rates of endogenous glucose production and glucose utilization were rather remarkable . A much higher rate of glucose production and a much lower rate of glucose utilization would be expected at such an extremely low plasma sugar level . The patient showed no signs of hypoglycemia until the end of the first experimental period when she began to twitch and sweat profusely, although she remained conscious . The plasma sugar was 15 .1 mg . per cent at that time . These signs were quickly relieved by the intravenous injection of 300 mg . per kg . glucose . A second tracer injection was given 20 minutes later . The glucose pool was found to he 19 .7 gm . and the glucose space was 20 .4 L . or 29 .6 per cent of the body weight at that time . The latter was smaller than in the fasting state . This indicates poor intermixing, likely due to the huge tumor mass .



546

Sarcomatous Origin of an Insulin-Like Substance Volpe' et al . CLUGAGON

FASTING 200 BLOOD 150 GLUCOSE 100 Mg . %

50 0

PLASMA INSULIN pp/ml . BODY WEIGHT Kg . PLASMA FREE FATTY ACID microeq ./1 . BLOOD LACTATE

20 0 72 70 68 1000 500 0 20 1 .00 .

4 6 8 10 12 14 16 18 20 22 24 26 28 2 4 6 8 10 12 FEBRUARY 1963 MARCH FRO. 9 . The metabolic effects of glocagon . Glucagon hydrochloride, 3 mg ., given intramuscularly twice daily caused hyperglycemia, but rebound hypoglycemic reactions are not shown on the chart . Insulin remained undetectable by immunoassay . There was a decrease in fasting plasma FFA, and a slight, but insignificant increase in blood lactate . Calcium and nitrogen balances are not shown on the graph, but were negative during the period of glucagon administration . Endogenous glucose production stopped completely after the intravenous glucose load . We observed this phenomenon in some normal subjects and in some patients with mild diabetes [58] . The rate of glucose utilization at the time of the second tracer injection was 5 .22 mg . per kg . per minute . This was a very high value considering that the plasma sugar was only 96 .7 mg. per cent, approximately 100 per cent higher than in normal subjects at a similar plasma sugar level . The constant K (i .e ., the slope of the log plasma sugar concentration versus time curve) after the intravenous glucose load was 0 .01875, within the normal range . Ninety minutes after the second tracer injection a third dose of tracer was given . The patient soon began to show symptoms and signs of severe hypoglycemia . (Plasma sugar concentration was 16 .1 mg . per cent .) Glucose had to be given intravenously which made evaluation of the third period impossible . Glucagon Study (See Fig. 9) . Glucagon hydro-

chloride (Lilly) was administered intramuscularly in doses of 3 .0 I .U . twice daily (7 A .M . and 7 P .M .) between February 12 and 23, 1963 . Parameters studied included fasting blood glucose (8 A .M .), fasting plasma insulin immunoassay, fasting plasma free fatty acid [59], fasting blood lactate [60,67] and urinary nitrogen when the patient was given a diet containing 10 gm . of nitrogen . Clinically, the patient tolerated the drug poorly, experiencing marked nausea, with occasional vomiting ; she had difficulty in consuming her entire diet . Furthermore, she manifested rebound hypoglycemia frequently toward the latter part of the period during which she was receiving glucagon, necessitating glucose injections and rendering the maintenance of balance conditions even more difficult . The patient tended to lose some weight during the glucagon period, in conjunction with a negative nitrogen balance . The fasting blood sugar levels were indeed elevated following the injections of glucagon, but such values as depicted AMERICAN JOURNAL OF MEDICINE



547

Sarcornatous Origin of an Insulin-Like Substance-Vo/pe el al . o . P, 1 mg/da ;1

H .G .H .

5mg ./day

- 0.P.

FAST INC BLOOD G .C.COSE mg .

FASTING PLASMA

100 -

50 _

INSULIN fV /ml . BODY

74

WEIGHT Kg .

FASTING

70

800

PLASMA F .F .A .

microeq ./t BLOOD

700

\ v.

600 1

` -

8

LACTATE 4 mg .

2

6

10

14

18

22

26

30

MAY 1963

'he administration of human growth hormone led to an increase in fasting blood glucose, insulin and FFA . The negative calcium and positive nitrogen balances are not depicted . Ovine prolactin induced similar, but less marked changes (see text) . IlGll = human growth hormone . O.P . = ovine prolactin . FIG . 10 . '1

are not representative of the dynamic shifts of blood glucose during the day ; the rebound hypoglycemic values are not shown . The plasma free fatty acid values declined significantly during the glucagon period, whereas there was an apparent slight but not significant increase in blood lactate . Plasma insulin was not detectable by immunoassay during this period . Following the cessation of glucagon administration, these changes were reversed . Human Growth Hormone and Ovine Prolactin Studies (Fig . 70) . The patient was given a balanced diet containing 11 .0 gm . of nitrogen, and 440 mg . calcium . After suitable control periods, human growth hormone was administered (5 mg . intramuscularly daily) for six days . After a second control period, ovine prolactin (5 mg . intramuscularly daily) was given for four days . It was then discontinued for two days, and restarted for an additional three vot .

38, APRIL 1965

days at a dosage level of 15 mg . daily . Blood was taken with the patient in the fasting state for determinations of glucose, insulin immunoassay ; plasma free fatty acid and blood lactate levels . Urine and stool specimens were collected in order to determine calcium and nitrogen balance . During the periods on human growth hormone and ovine prolactin, the patient had a somewhat enhanced sense of well-being . Following the administration of human growth hormone there was a weight gain associated with a positive nitrogen balance . There was a marked rise in the fasting blood glucose level (and this remained elevated when blood was taken later during the day prior to meals) . There also was a significant increase in plasma free fatty acid concomitant with the administration of growth hormone, and the plasma insulin (by immunoassay) increased significantly . These changes



548

Sarcomatous Origin of an Insulin-Like Substance.

were reversed a few days after cessation of human growth hormone therapy . Ovine prolactin was initially given intramuscularly in daily doses of 5 mg . This dose is considered to be less potent than an equal amount of human growth hormone . However, it did induce a positive nitrogen balance, and a slight gain in weight . Plasma insulin levels remained above control values . While fasting blood glucose levels remained low during the first three days on this dosage schedule, they increased in the succeeding three days . IJnfortunately, the plasma free fatty acid level was determined only twice during this period, and was low . This was at a time when the fasting blood glucose level was low, and both may represent "rebounds" following the earlier growth hormone period . The intervening control period may not have been long enough . The daily dose of prolactin was increased to 15 mg . for three days . The fasting blood glucose values remained above control values, as did the plasma insulin . Plasma free fatty acid increased significantly . During the entire study the blood lactate determinations showed no changes of significance . COMMENTS

The findings in this case suggest that the fibrosarcoma was producing and secreting a substance which acted in a manner similar to insulin to produce a hypoglycemic effect . It is of interest that this patient's primary tumor (1953) and its first recurrence did not result in hypoglycemia, although the tumors were large . This may be explained by the fact that these tumors were within the portal circulation, and that there perhaps may be some hepatic inactivation of the hypoglycemic factor . The tumors reported to produce hypoglycemia have had access to the systemic circulation . Beta Cell Hyporesponsiveness . There is no evidence that the tumor was elaborating a substance which stimulated the pancreatic beta cells ; actually, the finding of the diabetic-type of glucose tolerance curve, with no increase in the insulin levels after the administration of glucose, and the flat intravenous tolbutamide test both suggest beta cell hyporesponsiveness . In the light of these two test results, it might be suggested that the tumor was constantly elaborating an insulin-like substance, which thus induced a resting state of the pancreatic beta cells . (The administration of exogenous insulin

Volp e et al .

is known to suppress beta cell function [62] .) Comparable glucose tolerance and intravenous tolbutamide curves were recorded by Hayes et al . [11], although they did not interpret them in quite the same light . Similar flat intravenous tolbutamide test results have also been reported by Boshell et al . [22] and Floyd et al . [32] . Lack of Hypersensitivity to Insulin . The normal, but not excessive, decrease in blood sugar following the intravenous administration of regular insulin rules out the possibility that the tumor was elaborating a substance which enhances the action of insulin . Response to Leucine . One of the suggestions raised by Hayes et al . [11] was that the tumor might be elaborating an amino acid which induces hypoglycemia, perhaps in a manner similar to leucine-induced hypoglycemia . However, L-leucine administration did not cause hypoglycemia in our patient or in other similar patients studied [7,21,63] . One possible exception is a leucine tolerance test performed by Oleesky et al . [18] in which there appeared to be some fall in blood sugar levels following the administration of leucine . However, no control test with saline solution was performed, and the blood sugar levels may have dropped to a similar degree over a similar period of time . Of course, the absence of a response to test meals of L-leucine does not rule out some other amino acid abnormality as the basis for the hypoglycemia in these patients . The studies of Silverstein et al . [64] of the hypoglycemic factor in experimentally-induced leukemia in mice are of considerable interest in this regard ; these investigators found certain fractions of the neoplasma extracts had potent hypoglycemic activity . They found desoxyribonucleic acid and desoxycytidine to be the most potent hypoglycemic fractions, but the significance of these observations is not yet evident . More recently these investigators have suggested that metabolites of tryptophan might play a role in the pathogenesis of the hypoglycemia in patients with fibrosarcoma [79] . Lack of Depletion of Glycogen Stores . The hyperglycemic effect of epinephrine and intramuscularly administered glucagon demonstrates at least adequate hepatic glycogen storage . The possibility that either inadequate hepatic glycogen stores or insufficient glycogenolysis is responsible for the hypoglycemia is ruled out by these responses . It may be noted that the results of liver function tests were only slightly deranged, AMERICAN JOURNAL OF MEDICINE



Sarcomatous Origin of an Insulin-Like Substance-Volpe et al . and quite adequate normal liver parenchyma was visualized at laparotomy . None of the cases reported to date have been accepted as being due to inadequate available glycogen [3,21] . The fasting serum lactate level was normal . Most of the lactic acid present is derived from glycolysis, and is removed from the blood by the liver . Thus an increase in circulating lactate would have indicated a . high rate of glycolysis, an inefficient liver, or both . Uptake of Glucose, by Tumor . The suggestion that hypoglycemia is a result of a rapid uptake of glucose by the tumor itself has not been confirmed by pertinent studies [28,36] . Furthermore, this possibility is belied by the slow rate of growth of this and other reported similar tumors [3] ; the lack of mitotic figures in the histologic picture is further evidence of the slow growth rate . It is true that the studies of glucose metabolism employing C 1 "-labeled glucose showed a high rate of glucose utilization relative to the low blood sugar level, but this could be ascribed to an insulin-like effect on this and other tissues rather than to increased glucose uptake by the turner alone . (It should however be noted that Landau et al_ 120] recently made a strong case for this mechanism from a study of a hepatoma with hypoglycemia .) Depletion of hepatic glycogen stores might be expected in such a situation, whereas the evidence is to the contrary in our patientC14 -Glucose Studies . The C'"-glucose studies herein recorded indicate that the hypoglycemia in this patient was due to both a markedly diminished rate of glucose production and a relatively high rate of glucose utilization by the tissues . Similar results have also been obtained in two patients with insulinoma (unpublished data) . Butterfield et al . [7] have similarly reported inhibition of glucose production in a patient with fibrosarcoma and hypoglycemia . A Immoral insulin-like factor released by the tumor and acting on the liver and peripheral tissues might he responsible for both of the effects observed . A Immoral inhibition of hepatic glucose release during hypoglycemia is thus more likely than hepatic insufficiency, particularly since there is ample evidence of normal hepatic glycogen . Evidence for Increased Tumor Production of an Insulin-Like Substance. It thus seems attractive to accept the theory that the tumor is elaborating a substance which acts in a manner similar to insulin . The labeled glucose, studies, the evidence VOL .

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of pancreatic beta cell hyporesponsiveness, the low normal fasting serum phosphorus level and the possibly excessive hepatic glycogen storage are all consistent with this hypothesis . The finding of markedly increased plasma insulinlike activity (by the rat diaphragm method) both in the fasting state and postprandially further support this view . Since the insulin immunoassay results arc generally low, it would seem evident that the hypoglycemic tumor product is not true insulin . However the partial neutralization by antihuman insulin (on one occasion only) and a slight elevation of the fasting plasma insulin immunoassay on another single occasion suggests that the substance might have an immunologic determinant in common with insulin, and thus may have some chemical similarities . These views, of course, are speculative, and the observations may he artefactual . At any rate, the results are consistent with the thesis that the turner was constantly liberating an insulin-like substance, distinct from true insulin, throughout the day, without respect to physiologic demands, and at a fairly constant rate . The constant flow of this substance apparently suppressed pancreatic beta cell activity so that there was no increase in plasma insulin after glucose ; this would then explain the glucose tolerance and tolbutamide curves already noted . The patient's fasting hypoglycemia likewise resulted from a constant flow of the abnormal "hormone" which had an excessive insulin-like activity for fasting requirements, thus inhibiting hepatic glycogenolysis . Further evidence is obviously still necessary to determine the chemical structure of the hypoglycemic tumor product, and thus its relationship to insulin . Unfortunately, no studies could be made on the tumor itself, because no autopsy was performed . Effects of Glucagon, Human Growth Hormone and Ovine Prolactin . The administration of glucagon induced rapid albeit transient hyperglycemia, a result of rapid hepatic glycogenolysis [65] . As already noted, this indicates normal hepatic glycogen reserve in this patient . Despite this rise in blood glucose levels, plasma insulin remained nondetectable during the period of glucagon administration, As has been previously reported [66], administration of glucagon resulted in a slight negative nitrogen balance, but no ketonuria was demonstrated . There was no significant increase in blood lactate . Previous studies have shown no increase in blood lactate, indicating



550

Sarcomatous Origin of an Insulin-Like Substance -Volpe el al .

that glucagon does not affect muscle phosphorylase (67] . In addition there was a decrease in fasting plasma free fatty acid, a known glucagon effect [68] . This is considered to be due to decreased rate of free fatty acid release, a recognized effect of glucagon . This may be a secondary effect of the glucagon-induced hyperglycemia, since the free fatty acid level is very much dependent on the rate of glucose utilization, Human growth hormone induced the well known effects of positive nitrogen balance and negative calcium balance [69] . In addition, there was a marked rise in fasting blood glucose levels, as previously described in patients with hypopituitarism [70], and hypoglycemia [77] . There was a concomitant rise in fasting plasma free fatty acid, a very sensitive effect of human growth hormone . This is a manifestation of the lipolytic effect of growth hormone [72] . The increased availability of free fatty acid could spare glucose by acting as a substrate for some metabolic processes [73] . The apparent rise in the fasting insulin immunoassays was of considerable interest . Randle and Young [74] were able to show an increase in insulin-like activity in the plasma of normal cats after growth hormone administration, but Mahon et al . [71] were not able to show any effect of human growth hormone on insulin-like activity in their hypoglycemic patients . Insulin levels by immunoassay have not been reported after growth hormone in similar patients . A rise in the insulin level might be responsible for the transient fall in blood glucose levels observed immediately after the injection of growth hormone by Beck et al . [69] . It thus may be that pancreatic beta cells are stimulated by growth hormone, whether directly or indirectly ; this effect may alternatively be due to a decrease in insulin degradation, or a combination of both . The effects of ovine prolactin were similar to those of human growth hormone [75] with respect to nitrogen and calcium balance and insulin values, but less marked . The fasting blood glucose level tended to be elevated with this treatment, although this effect was not pronounced during the second (15 mg . daily) prolactin period . Fasting plasma free fatty acid was actually low during the first period (5 mg . per day) but this likely was a rebound effect tram the previous growth hormone period . It later rose during the second prolactin period . These studies again demonstrate the similarities

in the response of human subjects to human growth hormone and ovine prolactin [75] . Although human growth hormone or prolactin might have been considered as long-term therapy for hypoglycemia, such treatment was thought unnecessary on clinical grounds ; the patient's hypoglycemia could too readily be controlled by simpler means, i .e ., sugar ingestion or injections at appropriate times . SUMMARY AND

CONCLUSIONS

A fifty-three year old woman suffering from a large intrahepatic spindle cell fibrosarcoma is described, in whom hypoglycemia was a complicating feature . Blood glucose values during attacks varied between 25 and 38 mg . per 100 ml . ; the attacks occurred in the fasting state, and were quickly terminated by glucose administration . An intravenous insulin tolerance test showed a normal sensitivity to insulin . L-leucine was given orally, but failed to induce hypoglycemia . Both epinephrine and glucagon administration caused prompt increases in blood glucose, indicating adequate glycogen storage, and a normal response to these glycogenolytic stimuli . A glucose tolerance test showed a diabetictype curve, although by 5 hours the blood glucose level as 52 mg . per 100 ml . This curve suggested a hypoactive response of the beta cells of the islets of Langerhans to a glucose load . An intravenous tolbutamide test did not produce a significant decrease in blood glucose, again indicating a hypoactive beta cell response, and incidentally serving to rule out insulinoma . The patient's plasma showed a high insulinlike activity by the rat diaphragm glucose uptake method, both in the fasting and postprandial states . By contrast, radioimmunoassays for plasma insulin yielded similar low values in both the fasting and postprandial periods . Neutralization by anti-human-insulin of the plasma insulinlike activity (using rat diaphragm glucose uptake technic) was unsuccessful in two of the patient's plasma samples ; there was partial neutralization in a third . The data suggest that the tumor elaborated an insulin-like substance constantly, without regard 'to physiological requirements . This material may be related immunologically, but is certainly not identical with insulin . It would appear that the constant elaboration of this substance resulted in pancreatic beta cell suppression, as suggested by the failure of the serum AMERICAN JOURNAL OF MEDICINE



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of an Insulin-Like Substance-Volpe et al .

insulin to increase postprandially, and the inadequate response to glucose and tolbutamide administration .

Acknowledgmenl: Grateful acknowledgment is made to Dr. Charles Knowlton, who referred this patient, and who carried out the surgical procedures mentioned ; to Dr . Laurence Mautner and Dr . James Henning for their review of the pathologic material ; to Dr . Michael Pflun, Dr . Bernard Ferreira and Dr . Margaret Clowslcy for assistance in the investigation of this patient ; to Dr. W . John Simpson who performed the liver scan ; to Dr . Beale and Dr. J . William Meakin of the Princess Margaret Hospital, Toronto, who helped in the management of this patient ; to the Department of Art as applied to Medicine, University of Toronto, who provided most of the charts ; to Miss Marlene Bliss and Miss Ann Pitton, Department of Medical Photography, St . Joseph's Hospital, who prepared the photographs ; to the nurses and dietitians attached to the Metabolic Unit, St . Joseph's Hospital, and to Prof . K . .1 . R . Wightman and Dr . Stefan Fajans for reviewing the typescript . We are also indebted to Dr . Maurice Rabcn, who supplied the human growth hormone ; to the Endocrinology Study Section, U .S. National Institutes of Health and to Dr . A . Wilhelmi, Emory University, Atlanta, Georgia, for the ovine prolactin ; and to Hoechst Pharmaceuticals, Montreal, P .Q . for the intravenous tolbutamide . The other drugs utilized in the study were obtained commercially . REFERENCES

K . W . Fihrosarcoma of the mediastinurn . Ann . Sung ., 92 : 955, 1930 . ANDERSON, H . B . Tumor of adrenal gland with fatal hypoglycemia . Am . f. 3T . Sc ., 180 : 71, 1930 . LowBEER, L . Hypoglycemia producing cxtrapancreatic neoplasms : a review . Am . J. Clin . Path ., 35 : 233, 1961 . ANDREw . D ., GORANOW, 1 . and KRASTINOw, G . Hypoglycemia in intrathoracic fibroma . Endokvinolagie, 38 : 167, 1959 . Boss, J . If . Spontane Hypoglykamie als Folgc cities nichtinsulin-produzierenden Tumors der Nebennicrenrcgion . Schweiz . 7,tschr. f. dig . Path ., 22 : 232, 1959 . ROSSaAN . E . M . Mediastinal neurofibrosarcoma causing hypoglycemia . Arch . Int, Med ., 104 : 640, 1959 . BUETERFIE.LD . W . J. H ., KINDER, C . H . and MAHLER, R . F . Hypoglycemia associated with sarcoma . (Letter to editor .) Lancet. 1 : 703, 1960 .

1 . DoEGF,

2. 3. 4. 5.

6.

VOL .

38,

APRIL

1965

551

S . l3oesv ROS, G. A . Hypoglycemia in nictastatic fibrosarcoma of liver . Brit . Al . J., 1 : 836, 1960 . 9 . GOLD, G. L. and SmsNIDER, B . 1 . Sonic unusual syndromes associated with neoplastic disease . Ann, Int . Med ., 51 : 890, 1959 . 10 . PEDERSF.N, L, LUND, F . and RINGSTED, J . Hypoglycemia in the presence of massive librosarcoma (mesenchymoma) . Acta endocrinol ., 34 : 148, 1960 . 11 . HAVES, D . M ., SPURR, C . L ., FELTS, J . H. and MILLER, E . C . . .JR . Vo n Recklinghausen's disease with massive infra-abdominal tumor and spontaneous hypoglycemia . Metabolic studies before and after perfusion of the abdominal cavity with nitrogen mustard . Metabolism, 10 : 183, 1961 . 12 . DUNCAN, G . G . and SCHLESS. G . L . Massive retroperitoncal fibrosarcoma, spontaneous hypoglycemia, and generalized splanchnomegaly . Report of case . Metabolism, 10 : 200, 1961 . 13 . WHITNEY . J . E . and HELLER, B . 1 . Increased insulinlike activity of serum in patient with spontaneous hypoglycemia associated with retroperito heal fibrosarcoma Am . J. Med., 30 : 633 . 1961 . 14 . SFAl1FFER, J . M ., GRANVILLE . G . E . and LAW, S . W . Recurrent hypoglycemia and retroperitoneal librosa'coma . Not England J . M,d, 263 : 979, 1961 . 15 . SGRONFELD, A ., BABOTT, D . and GUNDERSEN, K . Hypoglycemia and polycythemia associated with primary hepatoma . d'eno EnYiand J. ., Med 263 : 231, 1961 . 16 . TRANQUADA, R . E ., BLNDER, A . B . and BFIGRLMAN, P . M . Hypoglvremia associated with carcinoma of the eecum and syndrome of testicular ferninization . New England .1. Med., 266 : 1302 . 1962 . 17 . GARrmLn, C . R ., BELCHER, 11 . V. and SHUTTLEWORTH, .I . S . Fibrous mcsothclioma with hypoglycemic psychosis and coma . J .tM .A ., 181 : 380, 1962 . 18 . OLEESKv, S ., BAILEY, I ., SAHOLS, L . and Bn .RUS, D. A librosarcoma with hypoglycemia and a high scrum-insulin level . Lancet, 2 : 378, 1962, 19 . ROGERS, J . C . T . and HOGSRWORTH, J, H . Large fibrogenic tumors and hypoglycemia . J.A .M .A., 178 : 1132, 1961 . 20 . LANDAU, B . R ., WR.Ls, N ., CRAIG, J . %V„ LEONARDS, J . R . and MORIWAKI, T . The mechanism of }lepatoma-induced hypoglycemia . Cancer, 15 : 1188, 1962. 21, FAST, B . B. Hypoglycemia associated with massive intraabduminal mesenchymal tumor. Winnipeg C'lin . Quart ., 15 : 104, 1962 . 22 . BosriELL, B . R ., KIRSCHENFELD, J . .1 . and SOTERES, F . S . Extrapancrcatic insnln-secreting tumor . New England J. Med., 270 : 338, 1964 . 23 . VAN DOLDER, G . Spontane hypoglycemic hij een patient met een grout longgeswel . Nederl . tijdschr . geneesk., 103 : 2030, 1961 . 24 . BUG.ARO, L . and M.ALIPIERO, S . On a case of spontaneous hypoglycemia associated with primary hepatic carcinoma . Eriuli Med ., 17 : 479, 1962 . 25 . FIRENSCHFR, 7 . F and BI .oM, P . S . Hypoglycemic en librosarcoorn . Nederf . Sijdschr. geneerk ., 107 : 1076, 1963. 26 . HRISKAI ..A, H . and GVLLING, M. Sarkoomaan liittyva hypoglykcmia . Duododm, 79 : 543, 1963 .



552

Sarcomatous Origin of an Insulin-Like Substance-Volpe

27. GONZALEZ, F . M ., GOLD, G . L . and SIINIDER, B . I . Gastrointestinal carcinoma and concomitant hypoglycemia . Ann . Int. Med., 58 : 149, 1963 . 28 . AUGUST, J . T. and HIATT, H . H . Severe hypoglycemia secondary to a nonpancreatic fibrosarcoma with insulin activity. New England J. Med ., 258 : 17, 1958 . 29 . VOLK, B. W ., GOLDNER, M . G. and WAINFIELD, B . Spontaneous hypoglycemia with abdominal spindle cell sarcomata. Geriatrics, 15 : 479, 1960 . 30. FIELD, J. B ., KEEN, H ., JOIINSON, P. and HERRING, B. Insulin-like activity of non-pancreatic tumors associated with fibrosarcoma . J. Clin . Endocrinol ., 23 : 1229, 1963 . 31 . PERKOFF, G. T . and SIMONS, E . L . Hypoglycemia in a patient with a fibrous tumor. Arch . Int . Med., 112 : 589, 1963 . 32 . FLOYD, J. C ., PowER, L., RULE, J ., FAJANS, S . S . and CONN, J. W. Insulin and ILA in tissue extracts and plasma of patients with nonpancreatic tumors associated with hypoglycemia . (Abstract .) Clin . Rer ., 11 : 297, 1963 . 33 . SECKEL, H . P . D . Postmortem glycogenolysis in hyperinsulinism and glycogen disease . J. Clin . Invest., 18 : 723, 1939 . 34. NADLER, W. H. and WOLFER, J . A. Hepatogenic hypoglycemia associated with primary liver carcinoma . Arch. Inn . Med., 44 : 700, 1929 . 35. LAYZER, H . On the relation of functional hyperinsulinism to visceroptosis ; report of twenty-one consecutive cases . Am . J, Digest. Dis., 18 : 300, 1951 . 36. SELLMAN, J. C ., PERROFF, G . T ., NULL, F. C, KIMMEL, J . R . and TAYLOR, F . H. I-Iypoglycemia associated with massive intraabdominal mesothelialcell sarcoma . New England J. Med., 260 : 847, 1959 . 37 . EVANS, C . L ., TSAI, C . and YOUNG, F . A . Behaviour of liver glycogen in experimental animals . J. Physiol., 73 : 67, 1931 . 38 . McFADYEAN, A . J. S . and Youxo, T. T . Hypoglycemia in primary carcinoma of liver . Arch . fat. Med., 98 : 720, 1956 . 39 . KLEIN, H . and KLEIN, S . P . Spontaneous hypoglycemia associated with massive hepatoma ; review of current concepts and report of case . Arch. Int. Med ., 103 : 273, 1959 . 40 . BERSON, S . A . and YALow, R . S . Plasma insulin in health and disease . Am- .1. Med., 31 : 874, 1961 . 41 . BARKER, S . B ., HUMPHREY, M. J . and SOLEY, M . A . The clinical determination of the serum proteinbound iodine . J. Clin . Invest., 30 : 55, 1951 . 42 . WALFISII, P . G., BRITTON, A ., VOLPIL, R . and EZRIN, C, Experience with an invitro test of thyroid function-the red blood cell uptake of I-triiodothyroninelabeled with radioactive iodine . Canal. M . A . J., 84 : 637, 1961 . 43 . HOFFMAN, W. S. A rapid photo-electric method for the determination of glucose in blood and urine . J. Biol. Chem ., 120 : 51, 1937 . 44 . PFEIFFER, E. F,, PFEIFFER, M ., DITSCHUNErr, H . and AHN, C . S . Clinical and experimental studies of insulin secretion following tolbutamide and Metahexamide administration . Ann . New York Acad. Sc., 82 : 479, 1959 . 45 . VALLANCR-OWFN, .I ., .TOPv .IN, G. F . and FRAZER, R .

cell

el al .

Tolbutamide control of diabetes mellitus . Lancet, 2 :584, 1959 . 46 . YALow, R . S., BLACK, H ., VILLAZON, M. and BERsoN, S. A . Comparison of plasma insulin levels following administration of tolbutamide and glucose. Diabetes, 9 : 356, 1960. 47 . FAJANS, S . S ., SCIINEIDER, J . M ., SCHTEINGART, D . E . and CONN, J. W. Diagnostic value of sodium tolbutamide in hypoglycemic states . J. Clin . Endocrinal., 21 : 371, 1961 . 48 . UNGER, R. H . and MADISON, L . L . Comparisons of response to intravenously administered sodium tolbutamide in mild diabetic and non-diabetic subjects . J. Clin . Invest., 37 : 627, 1958 . 49 . KAPLAN, N. Tolbutamide tolerance tests in carbohydrate metabolism evaluation, Arch . Inn. Med., 107 : 212, 1961 . 50 . VALLANCE-OWEN, J. and HURLOCK, B . Estimation of plasma insulin by rat diaphragm method . Lancet, 1 : 68, 1954. 51 . VALLANOE-OWEN, J., HURLOcK, B . and PLEASE, N . W . Estimation of plasma insulin. Lancet, 1 : 983, 1954. 52 . MORGAN, C . R . and LAZARow, A . Immunoassay of insulin : two antibody system . Diabetes, 12 : 115, 1963 . 53. YALow, R . S . and BERSON, S . A. Immunoassay of endogenous plasma insulin in man . J. Clin . Invest., 39 : 1157, 1960 . 54. DAVIDSON, J . K . and HAIST, R . E . A new method for recover of insulin activity from blood serum . Lancet, 2 : 656, 1963 . 55 . WAROLAW, A . C . and MOLONEY, P. J . The assay of insulin with anti-insulin and mouse diaphragm . Canal. J. Biochem . & Physiol ., 39 : 695, 1961 . 56. WRENSHALL, G. A . and HETENYI, G., JR. Successive measured injections of tracer as a method for determining characteristics of accumulation and turnover in higher animals with access limited to blood . Tests in hydrodynamic systems and initial observations on insulin action in dogs. Metabolism, 8 : 531, 1959 . 57 . DEPOCns, F . Turnover of plasma glucose in anaesthetized warm- and cold-acclimated rats exposed to cold. Canad. J. Biochem . & Physiol ., 37 : 175, 1959 . 58 . FORBATH, N . and HETENYI, G ., JR . Endogenous glucose production and utilization before and after an intravenous glucose load in normal and diabetic subjects. Abstracts of the Meeting of the Canadian Society for Clinical Investigation, January 15, 1964. Canal. M. A . J., 90 : 472, 1964. 59 . DOLE, V. P . and MEINERTZ, Ii . Microdetermination of long chain fatty acids in plasma and tissues. J. Biol . Chem ., 235 : 2595, 1960 . 60 . HORECKER, B . L . and KORNEERC, A . The extinction coefficient of the reduced band of pyridine nucleotides. J. Biol . Chem ., 175 : 385, 1948 . 61 . HORN, If . D . and BRUNS, F . H . Quantitative Bestimmung von L(+)-Milchsaure mit Milchsauredehydrogenase. Biochem . et Biophys . Acta, 21 : 378, 1956. 62 . LOOOTHETOPOULAS, J., KRAICER, J . and BEST, C . H . Granulation and reactive zinc in the cells of the islets of Langerhans, Effect of prolonged insulin therapy . Diabetes, 10 : 367, 1961 . AMERICAN JOURNAL OF MEDICINE



Sarcornatous Origin of an Insulin-Like Substance-Volpe 63 . MC.NAUGIITON_ M . C . and PRIEST, E . A. F . Hypoglycemia associated with Ieiomyosarcoma . Lancet,

1 : 204 . 1960 . 64. SILVERSTEIN, M . N ., WAKIM, K . G., BARN, R . C . and BA, RD, E . D . Additional preliminary observations on the hypoglycemic factor . Pror . Staff Meet . Mayo Clin . . 37 : 95, 1962 . 65 . BERGEN, S . S . and VAN ITALEIE, T . B. Clncagon:n, interim report . Metobolism, 9 : 132, 1960 . 66 . SALTER, J . M ., EZRIN, C ., LAIDLAW, J. C . and GORNALL, A . G . Metabolic effects of glucagon in human subjects . Metabolism, 9 : 753, 1960 . 67 . SUTHERLAND, E . W ., WosILArT, W . D . and RAIL, T . W . '1 be action of glucagon on liver phosphory last, In : Ciba Colloquia on Endocrinology, vol . 9, p . 179 . Boston, 1956 . Little, Brown and Co. 68 . BIERMAN, E . L., DOLE, V. P. and ROBERTS, T . N . An abnormality of nonesterihcd fatty acid metabolism in diabetes mellitus . Diabetes, 6 : 475, 1957. 69 . BECK, .1 . C ., MCGARRV, E . E ., DYRENEURTH, I ., MORGEN . R . 0 ., BIRD, E . D. and VENNING, E . H, Primate growth hormone studies in man . M'toboli en, 9 : 699 . 1960 . 70 . IKKOS, I). . LUFT, R . and CEMZRLL, C . A. Th effect of human growth hormone in man . Lancet, 1 :

720.1958 71 . MAHON . W . A., MITCHELL, M . L., STEINKE, J. and RABEN, M . S . Effect of human growth hormone on hypoglycemic states_ New England J. Med., 267 : 1179, 1962 .

VOL .

38, APRIL

1965

el al .

553

72 . RAPEN, M . S . Human growth hormone . Recent Progr . Hormone Res ., 15 : 71, 1959 . 73 . RANDIF, P. .i ., HALES, C, M ., GARLAND, P . B. and NP.wSHO1 .ME, E. A. The glucose-fatty acid cycle : its role in insulin sensitivity and the metabolic disturbauccs of diabetes mellitus Lancet, 1 : 785,

1963 . 74 . RANDLE, P. J . and YOUNG, P . G. 'the influence of pituitary growth hormone on plasma insulin activity. J. Endocrinvl., 13 : 335, 1956 . 75 . MCGARRV, E . R . and BECK, J. C . Some metabolic effects of ovine prolactin in man . Lancet . 2 : 915,

1962 . 76 . EYMONTT, M . J ., GWINUP, G ., KRUGER, F . A ., MAYNARD . D . E . and HAMWI, G . J . CushinK s syndrome with hypoglycemia caused by adrenocortical carcinoma . J. Clin . Endocrimol ., 25 : 46, 1965 . 77 . GINSBERG, D. M . Hypoglycemia associated with extrapanereatic neoplasms . In : Advances in Internal Medicine, vol . 12, pp . 33- 62 . Edited by Dock, W . and Snapper, I . New York. 1964. Year Book Medical Publishers. 78 . WHITNEY, J . E . and MASSEY, G . G . Apparent insulin activity in a fibrosarcoma associated with spontaneous hypoglycemia . .7. Clin . Endocrinol ., 21 :

541 . 1961 . 79 . SILVERSTEIN, M . N., WAKIM, K . G . and BARN, R . C . The role of tryptophan and its metabolites in hypoglycemia associated with neoplasm . C1in . Res.,

12 : 357, 1964 .