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The half-life of endogenous serum immunoreactive insulin in man
The Half-life
of Endogenous
Serum Immunoreactive
Insulin in Man By R. F. WILLLAMS, R. E. GLEASON ANDJ. S. SOELDNER Estimates of the half-life of endogenous serum insulin were made following the end of prolonged glucose perfusions in normal subjects who achieved peak levels of glucose that ranged between 131 and 221 mg. per cent. The estimates were 16 minutes if pancreatic insulin
production is assumed to cease following the end of the glucose infusion, or nine minutes if basal insulin secretion is assumed to continue following the end of the glucose infusion. (Metabolism 17: No. 11, November, 1025-1029,1968)
IFFERENCES
in the estimated half-life (T%) of endogenous as well as exogenous serum immunoreactive insulin (IRI) have been described’-5 One preliminary report has suggested that after intravenous glucagon-glucose stimulation resulting in severe hyperglycemia, the T% of endogenous IRI approximates 11 minutes. 4 It has also been suggested that exogenous lslI labeled human insulin, when injected into man has a significantly shorter disappearance rate than had been previously demonstrated using 1311 labeled insulin derived from other species. Therefore, it appeared important to evaluate serum IRI disappearance following stimulation by glucose alone at peak levels of blood glucose that were only mildly hyperglycemic.
D
MATERIALS ANDMETHODS Seven normal healthy males, all weighing less than 110 per cent of ideal body weight and ranging in age from 21 to 35 years were studied. None had a history of diabetes and all had normal intravenous, oral and cortisone-primed oral glucose tolerance tests. All had responded in normal fashion to intravenous tolbutamide. Each subject ingested a 300 Gm. carbohydrate diet for three days prior to each test. After an overnight fast, a 15 per cent solution of glucose in water was infused into an antecubital vein via an 18-gauge, l-inch needle using a Harvard Multi-Speed Transmission Pump at rates calibrated to deliver 30, l-70, 250, 500, and 1000 mg. of glucose per minute during five conFrom
the Elliott P. Jo&n
Research
Laboratory
in the Department
of Medicine,
Harvard
Medical School, and the Peter Bent Brigham Hospital, and the Diabetes Foundation, Inc.. Boston, Mass. Supported by USPHS Grants #AM-09748-03 and AM-05077-12, and by the John A. Hartford Foundation, New York, N. Y. Received for publication April 4, 1968. ROBERT F. WILLIAMS, M.D.: Research Fellow, Elliott P. loslin Research Laboratory; Research Fellow, Department of Medicine, Harvard Medical School and The Peter Bent Brigham Hospital, Boston, Mass. RAY E. GLEASON, PH.D.: Research Associate in Mathematical Biology, Elliott P. Ioslin Research Laboratory; Department of Medicine, Harvard Medical School and The Peter Bent Brigham Hospital, Boston, Mass. J. STUART SOELDNER, h4.D.: Research Associate, Elliott P. Joslin Research Laboratory; Associate in Medicine, Department of Medicine, Harvard Medical School and The Peter Bent Brigham Hospital, Boston, Mass. 1025
1026
WILLIAMS, GLEASON AND SOELDNER
l
Blood Glucose
0 IRI Total A I RI Increment over Fosting Level
& =meontS.E.M.
!4 =16?4.5Min(S.D.)
190
20
200
220 230 MINUTES
240
250
Fig. I.-Mean blood glucose and mean serum immunoreactive prolonged glucose infusion in seven normal males tested twice.
270
250
insulin following
secutive 3%minute periods. Each perfusion was preceded and followed by a one-hour control period using normal saline as the perfusate. Venous blood samples were- withdrawn from the opposite arm using an indwelling 1%guage, l-inch needle, the patency of which was maintained by a slow saline infusion. Samples were obtained at S- or lominute intervals after allowing a reflux of 2 or 3 drops of blood from the needle hub to Specimens obtained for blood glucose obviate dilutional errors by the saline solution. were collected in tubes containing a powdered potassium oxalate and sodium fluoride mixture. Blood glucose determinations were performed the morning of the test using a Technicon AutoAnalyzer employing the methods of Hoffman.6 Blood samples for IRI were allowed to remain at 4°C until the following day when they were centrifuged and the serum decanted and frozen (-20°C) until analyzed by a modification of the double antibody technique.7 Each subject was tested twice. The second test was preceded by a rapid intravenous glucose tolerance test (0.5 Cm./Kg. body weight) performed at the 30-minute point of the initial one-hour control period. The individual disappearance rates of serum IRI were estimated during the period following the end of the glucose perfusion when levels of both insulin and glucose declined rapidly. These disappearance rates were calculated using both the total level of serum IRI and the increment above the fasting level. Decline of serum IRI appeared to fit an exponential decay curve better when increments above the fakng level were used. Tj/,values were calculated by simple regression analysis (method of least squares) of In IRI (y) upon time (x) for each individual test as well as for the grouped data. The calculations included only those values occurring at consecutive time intervals where the decline foilowing the cessation of glucose perfusion best fit a single exponential.
RESULTS Glucose this level
Ievels reached for the
last
their peak
ten minutes
(170 of the
2
29 mg. glucose
per cent)
and sustained
perfusion
(Fig.
1).
The
HALF-LIFE
greatest
OF
ENDOGENOIJS
decline
of glucose.
in serum
There
of fasting
blood
cose or serum preceded
IRI
There
or serum
achieved
comparing
or the mean
glucose
following
levels
perfusion
cessation
the mean
levels
of peak blood
alone
to the
glu-
perfusion
load. half-lives
of glucose
of serum
difference
alone
IRI
between
or when
derived
by regression
the half-lives
the perfusion
calculated
was preceded
by
approximated
16
load.
The estimated minutes
mean
T’& using
absolute
from both tests in each subject.
IRI values
were utilized,
mean
values
011
to those based
was observed
line
absolute
rates
above
based
fasting
upon
IRI values
time
above fasting
did
not
Using
on absolute and
differ
R.B.),
serum
Student’s serum
IRI, a significant
In two of 14 tests (EM.
of IRI
serum
IRI values
increments
was nine minutes.
disappearance
increments
(P < 0.001).
the regression
serum
When
the T1/2 estimate
“t” test in comparing
when
with
30 minutes
differences
IRI,
was no significant
the perfusion
a glucose
within
no significant
1 lists the individual
analysis. after
were
1027
IRI
IRI occurred
glucose
by a glucose
Table
SEHUM
IRI
difference the slope of
significantly
from
zero
were used. This was due to the variability
in
the serum IRI levels seen in these two subjects. DISCUSSION
These data suggest that after prolonged stimulation by glucose alone, the mean TX of endogenous serum immunoreactive insulin ranges between nine and 16 minutes. These results parallel those of Samols who used glucose-glucagon infusions,4 and P)rskov and Christensen who used human insulin labeled with 13113 and Martin et al. who used porcine insulin.” It has been recently demonstiated cant
increases
minutes The seven
that, after rapid in the IRI
and reached
half-life
pearance
which
rates
study,
( Fig.
The regression exponential
insulin
ever, using constant
lymph
ranged
of serum
to achieve
from
insulin,
in three
signifito seven
after the injection. was
estimates
approximately of the
disap-
15 to 40 mirmtes.l,R
IRI were within
the physiologic
and the IRI levels were maintained equilibration
with the extravascular
upon the total levels of serum
insulin and
a continued
of serum
determination basal
with
some previous which
This results
the increment
of hyperglycemia
occurred
15 to 20 minutes
produced
and suggests
perfusion.
more accurate
lymph
of porcine
1).
line based
decline
of the glucose
duct
with
the elevations
of time adequate
compartment
injection
equilibration
the IRI was endogenously
for a period
tain
after contrasts
of Ia11 labeled
In the present range,
of thoracic
peak levels between
calculated
minutes,”
intravenous
insulin
secretion
in an overestimation IRI above
fasting
of the Tl/z and furthermore output
is present
hyperinsulinemia.
IRI departs after
from an the end
of the T1/2. How-
appears
to allow
suggests
that
in the face of moderate
for a a cer-
degrees
29.3
1.8 4.6
Mean
SEM SD
2.4 6.5
100.7
98 107 93 106 97 95 109
2.7 7.1
76.7
82 80 87 70 64 76 78
1.6 4.2
17.1
17 13 17 15 27 16 15
10 26
176
205 194 149 194 131 197 162
BG mg. %
1.4 3.6
14.9
11.7* 17.6 10.3 21.5 15.6* 15.4 11.9
IRI
of TX
1.7 4.4
77.4
82 79 83 75 69 76 78
1.9 5.1
16.1
22 11 17 23 9 19 12
Fftstulg IRI BG rU/ml. mg. %
impossible.
1.1 2.8
9.3
7.4 11.1 5.4 13.6 -_t 10.6 7.5
InIX. IRI
11 28
165
221 157 134 176 158 180 133
BG mg. %
IVGTT
259
80
123 79 101 89 39 65 70
+ Perfusion k ‘a IRI JlU/ml.
1.8 1.7
17.1
15.4 14.3 10.2 19.1 23.1 23.8 14.1
IRI
T $min.
(ZRZ) at Various Zntervals and Estimates of Insulin Half-life
T ?/i-mm.
Insulin
making calculation
I8 47
109
221 82 109 89 77 107 82
IRI ~U/ml.
Perfusion Peak
and Serum lmmunoreactive
Fsstmg BG IRI &AJ/ml. mg. %
of Blood Glucose (BG)
*Slope of “b” not significant from 0. tIRI values fell below fasting levels within ten minutes,
35 33 31 30 26 29 21
EM MG ER FB RB GW CJ
Pt.
Age Yrs.
Table I.-Levels
1.1 2.7
8.7
5.8 9.2 4.0 10.0 10.6 12.4 8.8
IWX. IRI
(‘W)
!z
P
”$
HALF-LIFE
OF
ENDOGENOUS
SERUM
1029
IRI
REFERENCES 1. Eerson, S. A., Yalow, R. S., Bauman, N., Rothschild, M. S., and Newerly, K.: InsulinIl:
J. S., and Cahill, Jr., G. F.: Diffusion of glucose, insulin, inulin, and evans blue protein into thoracic duct lymph of man. J. Clin. Invest. 46:903, 1967. 6. Hoffman, W. S.: A rapid photoelectric method for determination of glucose in hlood and urine. J. Biol. Chem. 120:51, 1937. 7. Soeldner, J. S., and Slone, D.: Critical variables in the radio-immunoassay of serum insulin using the double antibody technic. Diabetes 14:771, 1965. 8. Bolinger, R. E., and Stephens, R. R.: Comparison of disappearance from plasma of insulin Irsr and of insulin-like activity. Prac. Sot. Exp. Biol. Med. 116:812, 1964.
of Endogenous
Serum Immunoreactive
Insulin in Man By R. F. WILLLAMS, R. E. GLEASON ANDJ. S. SOELDNER Estimates of the half-life of endogenous serum insulin were made following the end of prolonged glucose perfusions in normal subjects who achieved peak levels of glucose that ranged between 131 and 221 mg. per cent. The estimates were 16 minutes if pancreatic insulin
production is assumed to cease following the end of the glucose infusion, or nine minutes if basal insulin secretion is assumed to continue following the end of the glucose infusion. (Metabolism 17: No. 11, November, 1025-1029,1968)
IFFERENCES
in the estimated half-life (T%) of endogenous as well as exogenous serum immunoreactive insulin (IRI) have been described’-5 One preliminary report has suggested that after intravenous glucagon-glucose stimulation resulting in severe hyperglycemia, the T% of endogenous IRI approximates 11 minutes. 4 It has also been suggested that exogenous lslI labeled human insulin, when injected into man has a significantly shorter disappearance rate than had been previously demonstrated using 1311 labeled insulin derived from other species. Therefore, it appeared important to evaluate serum IRI disappearance following stimulation by glucose alone at peak levels of blood glucose that were only mildly hyperglycemic.
D
MATERIALS ANDMETHODS Seven normal healthy males, all weighing less than 110 per cent of ideal body weight and ranging in age from 21 to 35 years were studied. None had a history of diabetes and all had normal intravenous, oral and cortisone-primed oral glucose tolerance tests. All had responded in normal fashion to intravenous tolbutamide. Each subject ingested a 300 Gm. carbohydrate diet for three days prior to each test. After an overnight fast, a 15 per cent solution of glucose in water was infused into an antecubital vein via an 18-gauge, l-inch needle using a Harvard Multi-Speed Transmission Pump at rates calibrated to deliver 30, l-70, 250, 500, and 1000 mg. of glucose per minute during five conFrom
the Elliott P. Jo&n
Research
Laboratory
in the Department
of Medicine,
Harvard
Medical School, and the Peter Bent Brigham Hospital, and the Diabetes Foundation, Inc.. Boston, Mass. Supported by USPHS Grants #AM-09748-03 and AM-05077-12, and by the John A. Hartford Foundation, New York, N. Y. Received for publication April 4, 1968. ROBERT F. WILLIAMS, M.D.: Research Fellow, Elliott P. loslin Research Laboratory; Research Fellow, Department of Medicine, Harvard Medical School and The Peter Bent Brigham Hospital, Boston, Mass. RAY E. GLEASON, PH.D.: Research Associate in Mathematical Biology, Elliott P. Ioslin Research Laboratory; Department of Medicine, Harvard Medical School and The Peter Bent Brigham Hospital, Boston, Mass. J. STUART SOELDNER, h4.D.: Research Associate, Elliott P. Joslin Research Laboratory; Associate in Medicine, Department of Medicine, Harvard Medical School and The Peter Bent Brigham Hospital, Boston, Mass. 1025
1026
WILLIAMS, GLEASON AND SOELDNER
l
Blood Glucose
0 IRI Total A I RI Increment over Fosting Level
& =meontS.E.M.
!4 =16?4.5Min(S.D.)
190
20
200
220 230 MINUTES
240
250
Fig. I.-Mean blood glucose and mean serum immunoreactive prolonged glucose infusion in seven normal males tested twice.
270
250
insulin following
secutive 3%minute periods. Each perfusion was preceded and followed by a one-hour control period using normal saline as the perfusate. Venous blood samples were- withdrawn from the opposite arm using an indwelling 1%guage, l-inch needle, the patency of which was maintained by a slow saline infusion. Samples were obtained at S- or lominute intervals after allowing a reflux of 2 or 3 drops of blood from the needle hub to Specimens obtained for blood glucose obviate dilutional errors by the saline solution. were collected in tubes containing a powdered potassium oxalate and sodium fluoride mixture. Blood glucose determinations were performed the morning of the test using a Technicon AutoAnalyzer employing the methods of Hoffman.6 Blood samples for IRI were allowed to remain at 4°C until the following day when they were centrifuged and the serum decanted and frozen (-20°C) until analyzed by a modification of the double antibody technique.7 Each subject was tested twice. The second test was preceded by a rapid intravenous glucose tolerance test (0.5 Cm./Kg. body weight) performed at the 30-minute point of the initial one-hour control period. The individual disappearance rates of serum IRI were estimated during the period following the end of the glucose perfusion when levels of both insulin and glucose declined rapidly. These disappearance rates were calculated using both the total level of serum IRI and the increment above the fasting level. Decline of serum IRI appeared to fit an exponential decay curve better when increments above the fakng level were used. Tj/,values were calculated by simple regression analysis (method of least squares) of In IRI (y) upon time (x) for each individual test as well as for the grouped data. The calculations included only those values occurring at consecutive time intervals where the decline foilowing the cessation of glucose perfusion best fit a single exponential.
RESULTS Glucose this level
Ievels reached for the
last
their peak
ten minutes
(170 of the
2
29 mg. glucose
per cent)
and sustained
perfusion
(Fig.
1).
The
HALF-LIFE
greatest
OF
ENDOGENOIJS
decline
of glucose.
in serum
There
of fasting
blood
cose or serum preceded
IRI
There
or serum
achieved
comparing
or the mean
glucose
following
levels
perfusion
cessation
the mean
levels
of peak blood
alone
to the
glu-
perfusion
load. half-lives
of glucose
of serum
difference
alone
IRI
between
or when
derived
by regression
the half-lives
the perfusion
calculated
was preceded
by
approximated
16
load.
The estimated minutes
mean
T’& using
absolute
from both tests in each subject.
IRI values
were utilized,
mean
values
011
to those based
was observed
line
absolute
rates
above
based
fasting
upon
IRI values
time
above fasting
did
not
Using
on absolute and
differ
R.B.),
serum
Student’s serum
IRI, a significant
In two of 14 tests (EM.
of IRI
serum
IRI values
increments
was nine minutes.
disappearance
increments
(P < 0.001).
the regression
serum
When
the T1/2 estimate
“t” test in comparing
when
with
30 minutes
differences
IRI,
was no significant
the perfusion
a glucose
within
no significant
1 lists the individual
analysis. after
were
1027
IRI
IRI occurred
glucose
by a glucose
Table
SEHUM
IRI
difference the slope of
significantly
from
zero
were used. This was due to the variability
in
the serum IRI levels seen in these two subjects. DISCUSSION
These data suggest that after prolonged stimulation by glucose alone, the mean TX of endogenous serum immunoreactive insulin ranges between nine and 16 minutes. These results parallel those of Samols who used glucose-glucagon infusions,4 and P)rskov and Christensen who used human insulin labeled with 13113 and Martin et al. who used porcine insulin.” It has been recently demonstiated cant
increases
minutes The seven
that, after rapid in the IRI
and reached
half-life
pearance
which
rates
study,
( Fig.
The regression exponential
insulin
ever, using constant
lymph
ranged
of serum
to achieve
from
insulin,
in three
signifito seven
after the injection. was
estimates
approximately of the
disap-
15 to 40 mirmtes.l,R
IRI were within
the physiologic
and the IRI levels were maintained equilibration
with the extravascular
upon the total levels of serum
insulin and
a continued
of serum
determination basal
with
some previous which
This results
the increment
of hyperglycemia
occurred
15 to 20 minutes
produced
and suggests
perfusion.
more accurate
lymph
of porcine
1).
line based
decline
of the glucose
duct
with
the elevations
of time adequate
compartment
injection
equilibration
the IRI was endogenously
for a period
tain
after contrasts
of Ia11 labeled
In the present range,
of thoracic
peak levels between
calculated
minutes,”
intravenous
insulin
secretion
in an overestimation IRI above
fasting
of the Tl/z and furthermore output
is present
hyperinsulinemia.
IRI departs after
from an the end
of the T1/2. How-
appears
to allow
suggests
that
in the face of moderate
for a a cer-
degrees
29.3
1.8 4.6
Mean
SEM SD
2.4 6.5
100.7
98 107 93 106 97 95 109
2.7 7.1
76.7
82 80 87 70 64 76 78
1.6 4.2
17.1
17 13 17 15 27 16 15
10 26
176
205 194 149 194 131 197 162
BG mg. %
1.4 3.6
14.9
11.7* 17.6 10.3 21.5 15.6* 15.4 11.9
IRI
of TX
1.7 4.4
77.4
82 79 83 75 69 76 78
1.9 5.1
16.1
22 11 17 23 9 19 12
Fftstulg IRI BG rU/ml. mg. %
impossible.
1.1 2.8
9.3
7.4 11.1 5.4 13.6 -_t 10.6 7.5
InIX. IRI
11 28
165
221 157 134 176 158 180 133
BG mg. %
IVGTT
259
80
123 79 101 89 39 65 70
+ Perfusion k ‘a IRI JlU/ml.
1.8 1.7
17.1
15.4 14.3 10.2 19.1 23.1 23.8 14.1
IRI
T $min.
(ZRZ) at Various Zntervals and Estimates of Insulin Half-life
T ?/i-mm.
Insulin
making calculation
I8 47
109
221 82 109 89 77 107 82
IRI ~U/ml.
Perfusion Peak
and Serum lmmunoreactive
Fsstmg BG IRI &AJ/ml. mg. %
of Blood Glucose (BG)
*Slope of “b” not significant from 0. tIRI values fell below fasting levels within ten minutes,
35 33 31 30 26 29 21
EM MG ER FB RB GW CJ
Pt.
Age Yrs.
Table I.-Levels
1.1 2.7
8.7
5.8 9.2 4.0 10.0 10.6 12.4 8.8
IWX. IRI
(‘W)
!z
P
”$
HALF-LIFE
OF
ENDOGENOUS
SERUM
1029
IRI
REFERENCES 1. Eerson, S. A., Yalow, R. S., Bauman, N., Rothschild, M. S., and Newerly, K.: InsulinIl:
J. S., and Cahill, Jr., G. F.: Diffusion of glucose, insulin, inulin, and evans blue protein into thoracic duct lymph of man. J. Clin. Invest. 46:903, 1967. 6. Hoffman, W. S.: A rapid photoelectric method for determination of glucose in hlood and urine. J. Biol. Chem. 120:51, 1937. 7. Soeldner, J. S., and Slone, D.: Critical variables in the radio-immunoassay of serum insulin using the double antibody technic. Diabetes 14:771, 1965. 8. Bolinger, R. E., and Stephens, R. R.: Comparison of disappearance from plasma of insulin Irsr and of insulin-like activity. Prac. Sot. Exp. Biol. Med. 116:812, 1964.