The influence of renal disease on the insulin I131 disappearance curve in man

The Influence of Renal Disease on the Insulin 1131 Disappearance Curve in Man By JOHN

P. O’BRIEN AND ALTON R. SHARPE, JR.

Insulin 1’“’ disappearance curves were done in 3 normal patients donating a kidney to an uremic reIative. There was no significant difference in the slopes of the curves done before and 2 weeks after nephrectomy. Clearance studies done before and 2 weeks after nephrectomy showed a significant reduction in inulin, PAH and creatinine clearance. Insulin 1131 disappearance curves were also done in 4 uremic patients receiving a renal homotransplant. They were done while the patient was uremic

and again 2 weeks after renal transplantation when renal function was normal. A significant delay in the disappearance of insulin 11”’ was noted in the uremic state. After transplantation, the slope of the insulin I’“1 disappearance curve approximated that seen in the normal patients (donors). It is suggested that with the onset of renal disease, insulin-degradation is delayed so that it circulates undegraded for a prolonged period of time. (Metabolism 16: No. 1, January, 76-83, 1967)

A MELIORATION of diabetes has been noted with the onset of the fi nephrotic syndrome.‘-3 Some investigators” have attributed this phenomenon to anorexiv and diminished food intake accomqanying the later stages of this syndrome, but it seems doubtful that this could account for the impressive reduction in insulin dosage that has been re.ported in some cases.’ Indeed, a fully satisfactory explanation has not been forthcoming. In an experimental approach to the problem, previous investigators4s5 have demonstrated a somewhat analogous sequence of events in the alloxan diabetic rat, in which, despite controlled food intake, glycosuria and hyperglycemia are significantly reduced following the induction of nephrosis with nephrotoxic serum. Ricketts and co-workers” have noted a significant difference in the half in diabetic nephrotic and nondiabetic neyhrectolife of plasma insulin P mized rats. Normally, insulin can be graded in all tissues but the liver and the kidney are the most active.’ The degree of localization of radioinsulin in the normal kidney8 suggests that renal damage may result in less inactivation of radioinsulin, thus leaving more circulating, active hormone available. Renal and hepatic function in animals has been shown to have an important influence on From the Departments of Medicine, Radiology and Surgery, Richmond, Virginia. Supported by P.H.S. Grant HE 08203 (Clinical Transplant 65-04 (Clinical Research Center). Received for publication Aug. 25, 1966. JOHN P. O’BRIEN, M.D.: Instructor in Clinical Research Associate in Transpkntation, Department of Richmond, Virginia. Currently on Military Leave with Associate Professor of RadioZogy (Nuclear Medicine) Medical College of Virginia, Richmond, Va. 76

Medical Center)

College und NIH

of Virginia, MO 1 FR

Medicine, Department of Medicine; Surgery, Medical College of Virginia, USAF. ALTON R. SHARPE, JR., M.D.: and Assistant Professor of Medicine,

RENALDISEASE

Table l.-Laboratory

Data from Uremic Subjects Before

K.G.

Chronic

R.P.

Cortical

Necrosis

E.R.

Chronic

Giomerulonephritis

M.C.

Chronic

Glomerulonephritis

In

thought icantlv.‘” determine

Crsotinine

I BUN

CCR

Transdantotion Creatinine Cl%VXlC~

Creatinine

Pyelonephritis

the disappearance esis.

BUN

Diagnosis

Patient

After

Transplantation

curve of free insulin half time”,“-”

patients,

however,

to he the

The following whether

to that observed

the

only factor

influence

which

of

prolongs

study was undertaken

a delay in insulin

in patients

receiving

and supports

previous insulin

previous

insulin

is

signif-

with renal disease

occurs

therapv

therapy

disappearance

in patients

disappearance

this hypoth-

to

that is comparable

with insulin.

METHOD

Ilenlthq

Patients and Uremic Patients

WC studied 7 patients, details of whom are given in Table 1. None had a family history of diabetes or an abnormal ghrcose tolerance. Three patients were healthy people with normal renal function who were donating a kidney to a uremic relative. They were studied before and 2 weeks after nephrectomy. Clearance studies (C,,,C,,,,,,C,,) were also done in these kidnev donors before and 2 weeks after nephrectomv. Four patients were uremics who were admitted for renal transplantation. These patients were studied while uremic and again 2 weeks after transplantation.

Procedure The patients fasted overnight and were then studied while lying warm and comfortable in a hospital bed. An 18 gauge Cournard needle was inserted into an antecubital vein in one arm and blood samples obtained at 10. 15, 20. 30, 60, 120, 180 and 240 minutes after injection of 25 IL<‘,(less than 0.5 units) of insldin It”r which was injected intravenonslv into the other arm. Blood samples removed.

were

Radioactivity

allowed

to clot

at room

temperature,

in 1 ml. of sermlr was measured

centrifuged

and

in a well type scintillation

the

sera

detector.

Protein was then precipitated bv the addition of 10 ml. of 10 per cent cold trichloroacetic acid and centrifuged. After 2 further washes with 3 per cent cold trichloroacetic acid, the radioactivitv of the precipitated fraction was measured. With this method, the degradation products are removed in the supernate while the radioactivity precipitate represents the undegraded insulin. free and bound.r:<

of the trichloroacetic

acid

RESULTS

The plasma followed

disappearance

by a slower

second

dog.14 This disappearance example

is shown

for insulin

and was similar

curve was similar

in Figure

phase of disappearance

curve phase

1. Analysis

follows

I’“’

had an initial to that

in both groups

of the variance

a first order relationship

rapid

described studied.

phase for the

A typical

shows that the second such that, Log y = (Y

+ ,6x where y is the serum concentration of the radioinsulin expressed in pU/ml. and x is the time after injection in minutes. The constant cx is the logarithm of

78

O’BRIEN

I.0

L 0

I

I

50

100 TIME

Fig. l.-Normal

insulin disappearance

I

150 IN MINUTES

I

200

AND SHARPE

I

250

curve showing rapid and slow phase.

the insulin concentration at zero time, while /3 is a rate constant that is characteristic of the disappearance rate during the slow second phase.15 The slope P is determined in each case by the logarithmic plot of the data by the method of least squares and values for this constant are shown in Table 2. When the slopes for the initial phase are compared with those for the second phase, initial phase slopes are significantly faster than are the second phase slopes. The initial phase is difficult to study in patients because of the necessity of obtaining multiple blood samples in rapid order. However, the rate of disappearance in the rapid phase largely determines the labelled insulin level for the early phases of the second phase of the curve. The point of intersection of the 2 parts of the curve would logically be the point of most significance in assessing the rapidity of the first phase, but since this point is not readily determinable only because the second phase slope is so much slower than is the first phase.16 The insulin P1 disappearance curves typical of the types obtained in the normal patients undergoing nephrectomy are illustrated in Figure 2. There were no significant differences in the curves done before and 2 weeks after nephrectomy. The results of the clearance studies done in these patients are shown in Table 3. Figure 3 shows the insulin I la1 disappearance curves typical of the types obtained in the uremic patients undergoing renal homotransplantation. There was a significant difference between the slopes of the curves in the uremic

RENAL

DISEASE

Table 2.-Logarithm of the Plasma Insulin ZJJ1Conczntration in U/ml. at Time Zndicated DONOR5 Ccl%

-

1.

30 Min. -

H.T.

-

60 Min.

BEFORE NEPHRECTOMY

120 Min. -

-

180 Min.

240 Min. -

p

-

a

I .2a41

1.0792

.7782

.7702

.bo21

- .002738

I .2334

2. A.W.

.5Q31

.845l

.b@21

.4771

.3010

-.002913

~9928

3. H.D.

I .0792

.9542

.?702

.602l

.&I21

-.m375

1.1024

Mwn

-.0x2675

1.1095

S. E.

+.cm584

DONORS

GXClS

-

1.

-

30

Min.

-

f

.ob955

TWO WEEKS AFTER NEPHRECTOMY

60 Min.

120 Min.

180 Min. --

240 Min.

p

a

.7782

.bm1

- .002679

1.2433

-

H.T.

1.2041

1.0414

2. A.W.

I .2304

I.1462

.845l

.6990

.bO21

-.003132

1.2992

3. H.D.

I .4150

I .2041

I .a(14

.9031

.9031

-.ccQ3b6

1.3914

M-I-l

-.@I2726

1.3113

5. E.

t. 0002224

2 .04318

.9031

UREMIC PATIENTS C0SOl

~ 30 Min.

- 60 Min.

1. E.R.

I ~3979

1.3222

2. M.C.

1.3424

I .2304

-

BEFORE TRANSPLANTATION

- 120 Min.

- 180 Mtn.

- 240 Min.

p

I .2553

I .2041

1.1761

-.001013

1 D3988

I .2041

.0792

-.a31525

1.3634

.O

-

a

3.

K.G.

I .4314

I .3424

I .3010

.2553

.2041

-.ooo9743

I .4296

4.

R.P.

I .2380

1.1818

I.0899

.Ol70

.9823

-.&II236

1.2576

-.001302

1~3527

‘I

+.0001512

f

P

a

Meon S. E.

UREMIC PATIENTS

COSCIS

-

-

30 Min.

-

60 Min.

.03057

TWO WEEKS AFTER TRANSPLANTATION 120 Min.

180 Min.

240 Min.

I .0792

.9u31

.6021

.4771

.4771

-.W2936

I .0776

2. M.C.

1.1461

.9542

.8451

.7781

~4771

-.ca2766

1.1887

3.

K.G.

1.2041

I.1461

.7781

.602l

.bo21

-.CY33210

I a2710

4.

R.P.

I .0792

I .0414

-8451

.7782

.69w

-.@I11887

I.1263

I.

E.R.

Meall

-.cO2700

1.1659

S. E. ____~~__

2.0002859

+ .04176

80

O’BRIEN AND SHARPE

30

-

m

AFTER

#EPHRECTOMY r1.99 BEFORE NEPHRECTOM r=.96 CONFUENCE

I

I

30

60

I .o

I

I

TIME

L MIT5

I

240

160

120

Y

IN MINUTES

Fig. 2.-Typical insulin disappearance curve obtained in healthy donors before and after nephrectomy. 30 -

BEFORE 1RANSPiANTATlON

20 s c El0

.;a

30

CONF/OENCE

60

L/M/T.5

1

1

120

180

240

TIME IN MINUTES

Fig. 3.-Typical insulin disappearance curve obtained in the same uremic patient before and after transplantation. state and those 2 weeks after transplantation, indicating a delay in insulin I’“’ disappearance in the uremic state. The curves obtained 2 weeks after transplantation when renal function was normal were similar to those obtained in the normal donors. DISCUSSION

As has been pointed out earlier, insulin degradation may take place in essentially all tissues, the kidney being the most active.i Support for this is in the observation that a great deal of injected radioinsulin is localized in the normal kidney.8 It has shown that insulin breakdown is greatly impaired by nephrectomyG and that in laboratory animals, the status of renal function is an important factor in influencing the characteristics of the insulin 11”’ disappear-

RENAL

81

DISEASE

Table 3.-Clearance ___ -._

Cases

Studies of Donors are Shown which were Done

before and 2 Weeks after Nephrectomy

lnulln Clmarance beform ofter naphrectomy nophroctomy

PAH Clearance after before nophrectomy nephrectomy

Creatinine before nophractomy

ClearonCb after nephrectomy

l..H.T.

130

75

556

302

146

92

2.

A.W.

110

67

574

389

161

W

3.

H.D.

102

73

520

335

130

89

ante curve.s-11 The results of our studies agree with the animal work and demonstrate the same influence in man. Since the kidney plays such an important role in the degradation of insulin, it is suggested that renal disease affects this degradation process adversely with the result that insulin remains in the blood stream undegraded for a prolonged period of time. It has been stated that this occurs either by decreasing the effective tissue where degradation takes place or uremia, per se, may have some adverse effect on the enzyme systems degrading insulin.” However, our uremic patients were prepared for transplantation with multiple hemodialyses and each patient was tested at various levels of nitrogen retention (Table 1). We found no correlation between the severity of the uremic state and the prolongation of the disappearance curve. It appears that once the renal mass is decreased to the point where uremia occurs, insulin degradation is impaired and correcting the uremia with dialysis would not change the delay in insulin disappearance, whereas correcting the deficit in effective renal mass by transplantation returned the insulin disappearance curve to normal. In our healthy patients, renal mass was decreased by 50 per cent with significant reduction in clearance (Table 3) and no change in the insulin 1131 disappearance curve. This would seem to indicate that renal mass must be decreased to the point of renal failure before insulin degradation is affected adversely. The insulin disappearance curve could be prolonged by the presence of insulin antibodies.ls Bolinger feels that this is dependent on previous insulin medication.‘s None of our patients had received exogenous insulin prior to this study. The insulin binding 20.21 that has been described in diabetic patients could prolong the insulin disappearance curve. None of our patients were diabetic nor was there any family history of diabetes. Whether insulin binding is significantly present is under current investigation. The validity of the present study can be questioned on the basis that insulin I’“l does not behave in the body in the same manner as endogenous insulin. Previous investigators 16~22have shown a loss of hypoglycemic activity with the iodination of insulin. However, this was not supported in the work of Ferrebee et a1.23 who demonstrated a comparable hypoglycemic effect of insulin after iodination. The different results obtained by the various groups can be explained by differences in the percentage of insulin tyrosine molecules iodinated. In our studies, the insulin 1131 was prepared by mild iodination so that

8.2

O’BRIEN AND

SHARPE

there was not more than one atom of iodine per good molecular weight of insulin. The insulin 1131 so prepared behaved like unlabeled pure insulin in dialysis, chromatography, electrophoresis, precipitation with trichloroacetic acid and in biological assays of insulin activity.24 An important factor in determining the characteristics of the disappearance curve of labeled insulin from the plasma is the size of the endogenous or unlabeled insulin pool. l6 It is necessary to determine the size of this pool so that the ratio of labeled to unlabeled insulin can be calculated. Several methods of determining blood insulin have been described26s26 but their inherent error makes them useless in determining the size of the endogenous insulin pool. Unless the ratio of labeled to unlabeled insulin is known, the disappearance curve of labeled insulin cannot be considered the actual disappearance of endogenous insulin. I6 However, from the selection of patients in this study, one would not expect a great variance in the endogenous insulin pool. REFERENCES 1. Zubrod. C. G., Eversole, S. L., and Dana, G. W.: Amelioration of diabetes and rarity of acidosis in patients with Kimmelstiel-Wilson Lesions. New Eng. J. Med. 245:518, 1951. 2. Runyan, J. W., Jr., Hurwitz, D., and Robbins, S. L.: Effect of KimmelstielWilson syndrome on insulin requirements in diabetes. New Eng. J. Med. 252:388, 1955. 3. Epstein, F. H., and Zupa, V. J.: Clinical correlates of the Kimmelstiel-Wilson lesion. New Eng. J. Med. 254:896, 1956 4. Kalant, N., Clamen. N., and Hoffman. M. M.: Effect of experimental nephrosis on alloxan diabetes in rats. Diabetes 7: 140. 1958. 5. Creutzfelat, W., Fredricks. H., Malsch. D.. and Moench, A.: Experimentelle Untersuchungen zur Auswirkung einer Wierenschadigung auf den Diabetes Mellitus. Arch. Exp. Path. u Pharmak. 236:392, 1959. 6. Ricketts, H. T., Wildberger. H. L.. and Regut, L.: Role of the kidney in disposal of insulin in rats. Diabetes 12: 155. 1963. 7. Williams, R. H.. Hay, J. S.. and Tjaden, M. D.: Degradation of insulin I131 and glucagon I-131 and factors influencing it. Ann. N. Y. Acad. Sci. 74:513, 1959. 8. Elgee, N. J., Williams, R. H., and Lee,

N. D.: Distribution studies with insulin vest. 33:1252, 1954.

and degradation I-131. J. Clin. In-

9. Lee, N. D., and Williams, R. H.: Intracellular localization of labelled thyroxine and labelled insulin in mammalian liver. Endocrinology 54:5, 1954. 10. Bolinger, R. E., and Slinker, B. J.: Effect of carbon tetrachloride on distribution and disappearance of labelled insulin on rabbit serum. Proc. Sot. Exp. Biol. Med. 88:574, 1955. 11. Palmer, D. L.. and Bolinger, R. E.: Effect on nephrectomy and splanchnicectomy on plasma disappearance OF labelled insulin in rabbit. Proc. Sot. Exp. Biol. Med. 107:809, 1961. 12. Goodman. L. S.. and Gilman, A.: Pharmacological Basis of Therapeutics: A Text Book of Pharmacology, Toxicology and Therapeutics for Physicians and Medical Students. 2nd Ed. New York, Macmillian. 1955. 1931. p. 1629. 13. Mahon, W. A., Steinke, J., McKann, G. M., and Mitchell, M. L.: Measurement of I-131 insulin like activity in cerebrospinal fluid of man. Metabolism 11:416. 1962. 14. Haugaard, N., Vaughan, M.. Haugaard. E. S., and Stadie, W. C.: Studies of radioiodine labelled insulin. J. Biol. Chem. 208:549. 1954.

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DISEASE

15. Steel, R. G. D., and Torrie, J. H.: Principles and Procedures of Statistics. New York, McGraw-Hill Book Co., Inc., 1960, p. 161. 16. Bolinger, R. E., and Grady, H. J.: Plasma insulin I-131 in diabetic patients. Ann. Int. Med. 48:753. 1958. 17. O’Brien. J. P.. and Sharpe, A. R., Jr.: Abnormal carbohydrate metabolism in renal failure. Metabolism 14:1294, 1965. 18. Berson, S. A., Yallow, R. S., Bauman. A., Rothchild, M. A., and Newerly. K.: Insulin I-131 metabolism in human subjects: demonstration of insulin binding globulin in the circulation of insulin treated subjects. J. Clin. Invest. 3.5:170. 1956. 19. Bolinger, R. E., Morris. J. H., McKnight, F. G., and Diederich. D. A.: Disappearance of I-131 labelled insulin from plasma as a guide to management of diabetes. New Eng. J. hied. 270:767, 1964.

20. Antoniades,

21.

22. 23.

24.

H.

N..

Beigelman,

P.

M..

Pennell, R. B.. Thorn, G. W., and Oncley, J. L.: Insulin-like activity of human plasma constituents. III. Eluinsulin-like activity tion of from cationic exchange resins. Metabolism 7:266, 1958. Ibid. Separation of human plasma protein concentrate with insulin activity. Science 127:593. 1958. Jensen, H. F.: Insulin. London, Oxford University Press, 1938. Ferrebee, J. W.. Johnson, B. B., Mithoefer. J, C., and Gardella, J. W.: Insulin and adrenocorticotropin labelled with radioiodine. Endocrinology 48:277. 1951. Medical Division of Abbott Laboratories-personal Communication.

25. Bornstein, J.: The insulin content of the blood plasma. Diabetes 2:23, 1953. 26. Valiance-Owen, J.. Hnrlock, B., and Please, N. W.: Plasma insulin activity in diabetes. Brit. Med. J. 2:583, 1955.