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Urinary insulin II. Excretion in children and adults
Urinary Insulin II. Excretion in Children and Adults By SAMIR
S.
NAJJAR
The overnight urinary excretion of immunoreactive insulin (IRI) was determined in healthy children and adults, and in offsprings of diabetic parents. There was no correlation between the weight and the urinary IRI excretion except in children weighing 40 kg and less. Adults excreted more IRI during the day than during the night. There was a marked day to day variation in the urinary IRf excretion in normal adults which
AND LEILA STEPHAN
limits the interpretation and comparative use of this determination. Elevated levels of urinary insulin however may still be useful in detecting individuals with elevated plasma IRI. Four such instances were found among 78 offsprings of a diabetic parent. The plasma IRI response to a glucose load in two of these individuals was similar to that found in prediabetics. (Metabolism 19: No. 4, April, 301-308, 1970)
HE EXCRETION OF IMMUNOREACTIVE INSULIN (IRI) in the urine of adults,lm3children4 and newborns” has been recently demonstrated. The purpose of this communication is to report on certain aspects of urinary excretion of IRI that have not been previously described, to further extend the observations on urinary excretion of IRI in children, and to present some data that are not in agreement with what has been published on the subject.
T
MATERIALS AND METHODS The IRI in the urine was assayed by the double antibody radioimmunoassay of Morgan and Lazarow as modified by Soeldner and Stone.6 The method has been described in detail elsewhere.7 Each sample was assayed in duplicate and the values of urinary IRI in this study were the average of at least two different determinations. Aliquots of a pool of postprandial human serums were frozen and were used as controls with each urinary assay. The concentration of TRI in the pooled serum varied from 37.5 to 44 @/ml., attesting for the reproducibility of the assay. Urine from normal, healthy children and adults was collected overnight. The urine collection was started after emptying the bladder at least two hours after the last evening meal; no food was allowed until the collection was completed the next morning. These subjects were referred to as “normal controls” and the urinary IRI excretion as “basal.” Several adults voided another urine specimen the same morning while still fasting, this is referred to as the “second voided” specimen. Twenty-four-hour urine collections were performed on six adults. The specimen was divided into two: a day specimen collected from 8 a.m. to 10 p.m. and a night specimen collected from 10 p.m. to 8 a.m. There was no attempt to limit food intake during or before the night period. Four healthy adults collected daily overnight specimens for one week. No particular
___ From the Department of Pediatrics, School of Medicine, American University of Beirut, Beirut, Lebanon. Received for publication December 19, 1969. SAMIR S. NAJJAR, M.D.: Associate Professor, Deparfment of Pediatrics, American Vniversity of Beirut, Beirut, Lebanon. LEILA STEPHAN, B.S.: Research Assistant, Department of Pediatrics, American University of Beirut, Beirut, Lebanon, METABOLISM, VOL. 19, No. 4 (APRIL), 1970
301
302
NAJJARANDSTEPHAN
Adults Age in years Fig, l.-Overnight “basal” urinary expressed in cU per hour.
IRI excretion
in normal
children
and adults
20f 16..c z 2 12” 3’ I
8-
n
4
_
Adults Age in years Fig. 2.-Overnight “basal” urinary IRI excretion expressed in terms of urinary creatinine.
dietary or exercise regimen was imposed
on them. fashion as the normal controls. Ramdom morning specimens were collected on was diabetic. Those who showed elevated levels of lection of urine under the sxme conditions as those
in normal
They collected
children
and adults
the urine in the same
78 children who had one parent who urinary IRI excretion had a repeat colused for the normal controls.
RESULTS
Overnight or ‘Basal” IRi Excretion The overnight urinary excretion of IRI in normal children and adults is shown in Fig. 1 expressed in ;.:U per hour and in Fig. 2 expressed in f*U per milligram of urine creatinine. There was a tendency for th: hourly excretion of insulin to increase with age, however, the scatter was wide. The mean amount excreted by seven children 1 to 5 years old was 149.3 /&J/hour (SD _t 62.45), by 20
URINARY
- _____
INSULIN
Table
EXCRETION
“Basal” and “Second
l.-
Subject
fiU IRI/hr.
1 2
215 30
Voided”
635
Urinary JRI Excretion
_ ___.
“Second Voided” pU IRI/mg. Creatinine
WUIRIlhr.
300
3.8 3.8 2.7 4.3 5.4
-
0.25 2.5 5.4
100
303
AND ADULTS
Overnight pU lRI/mg. Creatinine ~___ 4.0 4.0
-
3 4 5
IN CHILDREN
100 330 470
~.
children 6 to 15 years old 172 $/hour (SD t 57.62) and by 17 adults 225 &J/hour (SD i 200). The difference between the three groups was not statistically significant. When expressed in terms of urine creatinine the amount of IRI excreted decreased with age for a mean of 12.99 ,JJ/mg. (SD i: 5.39) in 12 children 1 to 5 years of age to 7.80 ,&/mg. (SD * 3.3 1) in 3 1 children 6 to 15 years of age and 3.61 $J/mg. (SD * 4.47) in 21 adults. The difference among the children was not significant but the difference between children and adults was significant (p < 0.02 for the l- to 5-year-olds and p < 0.05 for the 6- to 15-year-olds). Prolonging the overnight fast by few hours did not appreciably change the “basal” IRI excretion. The concentration of IRI in “basal” and “second voided” specimens was essentially the same (Table 1) . Urinary Excretion of IRI in Relation to Body Size (Fig. 3)
There was no correlation between the “basal” IRI excretion and the weight of the subjects of the group as a whole (R = 0.240). Such a correlation, however, was found in children weighing 40 kgm and less (R = 0.478, p = 0.01). Day-to-day Variation in Urinary IRI Excretion
The results of the day-to-day variation of urinary IRI excretion are shown in Tables 2 and 3. There were wide variations in the overnight excretion of the 500
400
F
l
R =.0.240
0
0 m 0
10
’
0 ’
18
’
0
l
’
26
’
’
34
’
’
42
’
WEIGHT Fig. 3.-Correlation
’
50
’
’
56
’
’
66
’
IN KG.
of urine IRI and body weight.
’
74
0 ’
’
62
304
NAJJAR
Table 2 .-Day-to-day Subject
Day
1 2 3 4
1
4*, ~.~3~ 30 625 -
-Subject
Day
1
STEPHAN
Variation in 24-hour Urinary IRI Excretion Expressed in aU per Hour ____ ~~ ~ __ 2
~.
31 617 298
Table S.-Day-to-day
AND
3 915_
__
4
495 60 526 130
160 350 98
5
6
7
1240 225 660 195
346 480 175
620 140 275 -
~-
Variation in 24-hour Urinary IRI Excretion Expressed in aIJ per mg. of Creatlnine __~____ 2
3
4
5
6
I
1 2 3
6.8 1.0 10.4
12.0 0.25 9.4
13.7 2.7 5.7
7.5 0.5 8.2
16.4 3.9 8.6
5.8 7.3
6.2 3.3 4.0
4
-
3.8 _.___
-
1.8
2.7
2.2
-
same subject whether expressed in $-J/hour or in ,uU mg. of urine creatinine. Although “basal” excretion of IRI seems to vary over a wide range, there was a tendency for “high excretors” (e.g., subject 1) and “low excretors” (e.g., subject 2) to have rather consistent excretion rates. Diurnal Variation in Urinary IRI Excretion
More IRI was excreted per hour and per milligram of creatinine during the day than during the night. The results obtained in six subjects who collected urine on two or three consecutive days are shown in Table 4. These results also illustrate the day-to-day variation in insulin excretion. This variation is found both in the day and in the night specimens. Table Subject Day
1
2
1 2 3 1 n
L
3
4
5
6
1 2 3 1 2 3 1 2 3 1 2 3
pU/hr.
920 1100 750 730 1400 700 1450 840 1000 1800 750 1100 1500 470 550 1180 1000
4.-Diurnal Day
Excretion
of Urinary
IRI Night
pU/mg.
Creatinine
11.5 13.6 9.3 6.60 15.30 9.4 15.0 8.2 11.8 21.6 5.2 12.2 20.0 4.8 6.9 14.2 10.6
pU/hr.
pU/mg.
70
Creatinine
0.86 1.25 1.75 1.9 2.0 6.3 2.5 4.0 3.38 1.92
100 130 120 116 490 75 330 400 125 Negligible 340 100 540 390 285 200
4.4 1.6 5.0 6.0 4.0 3.0
URINARY
INSULIN
20 -
EXCRETION
IN CHILDREN
305
AND ADULTS
.
z 165 .c ;i 2 12-
.
”
.
? 3 t
8-
a -
4-
O1”“““‘“““l” 2 4
i
to
8 . . . 6 Age
8 in
10
12
14
16
years
.. .
. Adults
Age unknown
Fig. 4.-Random morning or “basal” urinary IRI excretion in offsprings of one diabetic parent expressed in terms of urinary creatinine. Shaded area represents normal values, Mean * SD, Fig. 2.
Urinary IRI Excretion
in Children of a Diabetic Parent
Seventy-four of 78 children of a diabetic parent had urinary IRI levels on a randomly voided morning specimen or on a repeat overnight specimen collected in a similar fashion to the normal controls that were within the normal range for age (Fig. 4). Four had levels consistently elevated. The average values found in these four children were 70.0 &J/mg. creatinine in a 16-year-old girl, 20.9 yU/mg. creatinine in a 19-year-old girl, and 58.8 and 42.0 ,J_J/mg. in 9- and 12-year-old brothers, respectively. None had proteinuria. A glucose tolerance test using 100 Gm. of glucose orally was performed on the two girls. Their blood sugar levels following the glucose load were within normal, however, their plasma IRI levels were greatly elevated (Fig. 5). The girls were not obese. Their fathers had maturity onset type diabetes mellitus and were insulin dependent. The urinary IRI excretion was not determined during the glucose tolerance test and a glucose-cortisone tolerance test was not performed. DISCUSSION
Overnight collection of urine started two hours after the last evening meal probably represents the nearest to basal insulin excretion since it is least affected by food intake, the most potent physiologic stimulus for insulin secretion. The two-hour interval was chosen because we’ and others” have shown that most of the insulin secreted in response to a glucose load is excreted in this time interval. Further support to the adequacy of this interval is provided by the absence of a significant difference between the “basal” and the “second-voided” urinary lR1 excretion rates. This finding also lends more support to the basal characteristic of the overnight excretion rate. Rubenstein et al.” reported on the overnight urinary IRI excretion in healthy adults. The urine was collected in the same fashion as in our normal controls. They obtained values very similar to ours. The mean hourly excretion was 215 PU and the mean excretion expressed per milligram of urine creatinine was 3.6. The values we obtained for the 24-hour urinary IRI excretion (calculated by add-
306
NAJJAR
AND STEPHAN
Fig. 5.-Plasma IRI and plasma glucose levels during glucose tolerance test in two subjects with elevated urinary IRI excretion.
TIME
IN
MINUTES
ing the amounts excreted in the day and the night specimens) were also similar to those previously reported by others.1,4 No values for overnight urinary IRI excretion in children is available in the literature. McArthur and Stimmler4 reported on the 24-hour urinary excretion in 16 children, the youngest of whom was eight years old. Our study comprises a larger group of children and includes younger ages. Overnight excretion is probably a better index for comparison since it is nearest to basal conditions and is not affected by food intake which can be quite variable in quantity, quality, and frequency during a 24-hour period. Our results showed a tendency to an increase in the hourly excretion of IRI with age. However, the scatter was wide and there was no statistical significance to the difference between the various age groups. The urinary IRI excretion expressed in terms of urinary creatinine decreased with age and the difference between children and adults was statistically significant. This probabIy represents the increased excretion of urinary creatinine with ages rather than an actual decrease in urinary IRI excretion. Rubenstein et al.? and Stimmler and McArthurq found a correlation between body weight and urinary IRI excretion, we could establish such a correlation
URINARY INSULIN EXCRETION IN CHILDREN AND ADULTS
307
only in children weighing 40 Kg. and less. No explanation can be offered for this discrepancy. Lambert and Hoetg claimed that serum insulin levels in healthy women are markedly elevated at midnight. Gagliardino lo found that rats excreted more IRI in the urine from 5 p.m. to 8 a.m. than from 8 a.m. to 5 p.m. irrespective of dietary manipulations. This pattern of diurnal variation was not found in our subjects who, on the contrary, excreted more IRI during the day than during the night. This is consistent with the data of Lestradet’l who found that the plasma levels of IRI in man followed closely the glycemic curve reaching its maximum between 12 and 14 hours and its minimum at 5 hours. The day to day variation in the overnight or “basal” urinary IRI excretion in the same individual was great. The same is true for the excretion rates during the day and the night. The individual values, however, remained within the normal range for adults except for case 1 in Tables 2 and 3. There is no apparent explanation for this variation in insulin excretion which has not been previously documented. It probably does not represent a technical artifact because the assays were repeated on several occasions with little or no change in the results. It could possibly result from prior priming of the beta cells of the pancreas by unequal physiologic stimuli, or it could be the result of inconstant nocturnal stimuli or suppressors of insulin secretion such as the nocturnal bouts of growth hormone release described by Hunter and Riga1.l” Recently Takahashi et al.‘” failed to demonstrate insulin peaks during sleep similar to those of growth hormone. It may reflect the claim of Chamberlain and Stimmler3 that a very small and variable proportion of the filtered IRI appears in the urine. This, however, is at variance with the finding of Rubenstein et all& of a constant renal clearance of insulin irrespective of its blood level. The daily variation in the urinary IRI excretion, in addition to the small renal clearance of IRI,‘i,14 puts great limitations on the value of individual determinations of urinary IRI, particularly when used for comparative purposes. When such values are definitely well beyond the range of normal for age, and in the absence of renal disease, they may be helpful in detecting individuals with elevated plasma IRI (Fig. 5). Of the 78 offsprings of one diabetic parent, four had very high values of urinary IRI. The two girls that were examined had a normal glucose tolerance test. The levels of their plasma IRI in response to an oral glucose load however were elevated and were similar to those found in prediabetics, mainly elevated values at one and two hours when compared to normal controls.‘” It is difficult to speculate at this time on the significance of this finding. If these two girls should develop diabetes mellitus in the future, overnight urinary IRI excretion may then become a useful screening test for the early detection of prediabetic individuals. This necessarily awaits further study and documentation. REFERENCES 1. Jorgensen, K.R.: Immunoassay of insulin in human urine. Acta Endocr. (Kobenhavn) 51:400, 1966. 2. Rubenstein, A.H.. Lowy, C., Welborn, T.A., and Russell Frazer, T.: Urine insulin
in normal subjects. Metabolism 16:234, 1967. 3. Chamberlain, M.H., and Stimmler, L.: The renal handling of insulin. J. Clin. Invest. 46:911, 1967.
308 4. McArthur, R.G., and Stimmler, L.: Urinary insulin excretion in healthy children and siblings of childhood-onset diabetics. Lancet 2: 1236, 1966. 5. Lowy, C., and Schiff, D.: Urinary excretion of insulin in the newborn. Lancet 1:225, 1968. 6. Soeldner, J.S., and Slone, D.: Critical variables in the radioimmunoassay of serum insulin using the double antibody technic. Diabetes 14:771, 1965. 7. Najjar, S., and Stephan, L.: Urinary insulin. I. Methodology. Lebanese Medical Journal 22:95, 1969. 8. Graystone, J.E.: Creatinine excretion during growth. In Cheek, D.B. (Ed.): Human Growth. Philadelphia, Lea and Febiger, 1968, p. 182. 9. Lambert, A.E., and Hoet, J.J.: Diurnal pattern of plasma insulin concentration in the human. Diabetologia 2:69, 1966.
NAJJAR
AND STEPHAN
10. Gagliardino, J. J. : Physiologic variations of insulin output in urine. Metabolism 17: 139, 1968. 11. Lestradet, H., and Tichet, J.: Variation circadienne de l’insulinemie. Proceedings of the European Society for Paediatric Encodrinology, Vienna, 1968 (Abstract 57). 12. Hunter, W.M., and Rigal, W.M.: The diurnal pattern of plasma growth hormone concentration in children and adolescents. J. Endocr. 34:147, 1966. 13. Takahashi, Y., Kipnis, D.M., and Daughaday, W.H.: Growth hormone secretion during sleep. J. Clin. Invest. 47:2079, 1968. 14. Rubenstein, A.H., Lowy, C., and Russell Frazer, T.: Radioimmunoassay of insulin in urine. Diabetologia 3:453, 1967. 15. Ricketts, H.T., Cherry, R.A., and Kusteins, L.: Biochemical studies of “prediabetics.” Diabetes 15:880, 1966.
S.
NAJJAR
The overnight urinary excretion of immunoreactive insulin (IRI) was determined in healthy children and adults, and in offsprings of diabetic parents. There was no correlation between the weight and the urinary IRI excretion except in children weighing 40 kg and less. Adults excreted more IRI during the day than during the night. There was a marked day to day variation in the urinary IRf excretion in normal adults which
AND LEILA STEPHAN
limits the interpretation and comparative use of this determination. Elevated levels of urinary insulin however may still be useful in detecting individuals with elevated plasma IRI. Four such instances were found among 78 offsprings of a diabetic parent. The plasma IRI response to a glucose load in two of these individuals was similar to that found in prediabetics. (Metabolism 19: No. 4, April, 301-308, 1970)
HE EXCRETION OF IMMUNOREACTIVE INSULIN (IRI) in the urine of adults,lm3children4 and newborns” has been recently demonstrated. The purpose of this communication is to report on certain aspects of urinary excretion of IRI that have not been previously described, to further extend the observations on urinary excretion of IRI in children, and to present some data that are not in agreement with what has been published on the subject.
T
MATERIALS AND METHODS The IRI in the urine was assayed by the double antibody radioimmunoassay of Morgan and Lazarow as modified by Soeldner and Stone.6 The method has been described in detail elsewhere.7 Each sample was assayed in duplicate and the values of urinary IRI in this study were the average of at least two different determinations. Aliquots of a pool of postprandial human serums were frozen and were used as controls with each urinary assay. The concentration of TRI in the pooled serum varied from 37.5 to 44 @/ml., attesting for the reproducibility of the assay. Urine from normal, healthy children and adults was collected overnight. The urine collection was started after emptying the bladder at least two hours after the last evening meal; no food was allowed until the collection was completed the next morning. These subjects were referred to as “normal controls” and the urinary IRI excretion as “basal.” Several adults voided another urine specimen the same morning while still fasting, this is referred to as the “second voided” specimen. Twenty-four-hour urine collections were performed on six adults. The specimen was divided into two: a day specimen collected from 8 a.m. to 10 p.m. and a night specimen collected from 10 p.m. to 8 a.m. There was no attempt to limit food intake during or before the night period. Four healthy adults collected daily overnight specimens for one week. No particular
___ From the Department of Pediatrics, School of Medicine, American University of Beirut, Beirut, Lebanon. Received for publication December 19, 1969. SAMIR S. NAJJAR, M.D.: Associate Professor, Deparfment of Pediatrics, American Vniversity of Beirut, Beirut, Lebanon. LEILA STEPHAN, B.S.: Research Assistant, Department of Pediatrics, American University of Beirut, Beirut, Lebanon, METABOLISM, VOL. 19, No. 4 (APRIL), 1970
301
302
NAJJARANDSTEPHAN
Adults Age in years Fig, l.-Overnight “basal” urinary expressed in cU per hour.
IRI excretion
in normal
children
and adults
20f 16..c z 2 12” 3’ I
8-
n
4
_
Adults Age in years Fig. 2.-Overnight “basal” urinary IRI excretion expressed in terms of urinary creatinine.
dietary or exercise regimen was imposed
on them. fashion as the normal controls. Ramdom morning specimens were collected on was diabetic. Those who showed elevated levels of lection of urine under the sxme conditions as those
in normal
They collected
children
and adults
the urine in the same
78 children who had one parent who urinary IRI excretion had a repeat colused for the normal controls.
RESULTS
Overnight or ‘Basal” IRi Excretion The overnight urinary excretion of IRI in normal children and adults is shown in Fig. 1 expressed in ;.:U per hour and in Fig. 2 expressed in f*U per milligram of urine creatinine. There was a tendency for th: hourly excretion of insulin to increase with age, however, the scatter was wide. The mean amount excreted by seven children 1 to 5 years old was 149.3 /&J/hour (SD _t 62.45), by 20
URINARY
- _____
INSULIN
Table
EXCRETION
“Basal” and “Second
l.-
Subject
fiU IRI/hr.
1 2
215 30
Voided”
635
Urinary JRI Excretion
_ ___.
“Second Voided” pU IRI/mg. Creatinine
WUIRIlhr.
300
3.8 3.8 2.7 4.3 5.4
-
0.25 2.5 5.4
100
303
AND ADULTS
Overnight pU lRI/mg. Creatinine ~___ 4.0 4.0
-
3 4 5
IN CHILDREN
100 330 470
~.
children 6 to 15 years old 172 $/hour (SD t 57.62) and by 17 adults 225 &J/hour (SD i 200). The difference between the three groups was not statistically significant. When expressed in terms of urine creatinine the amount of IRI excreted decreased with age for a mean of 12.99 ,JJ/mg. (SD i: 5.39) in 12 children 1 to 5 years of age to 7.80 ,&/mg. (SD * 3.3 1) in 3 1 children 6 to 15 years of age and 3.61 $J/mg. (SD * 4.47) in 21 adults. The difference among the children was not significant but the difference between children and adults was significant (p < 0.02 for the l- to 5-year-olds and p < 0.05 for the 6- to 15-year-olds). Prolonging the overnight fast by few hours did not appreciably change the “basal” IRI excretion. The concentration of IRI in “basal” and “second voided” specimens was essentially the same (Table 1) . Urinary Excretion of IRI in Relation to Body Size (Fig. 3)
There was no correlation between the “basal” IRI excretion and the weight of the subjects of the group as a whole (R = 0.240). Such a correlation, however, was found in children weighing 40 kgm and less (R = 0.478, p = 0.01). Day-to-day Variation in Urinary IRI Excretion
The results of the day-to-day variation of urinary IRI excretion are shown in Tables 2 and 3. There were wide variations in the overnight excretion of the 500
400
F
l
R =.0.240
0
0 m 0
10
’
0 ’
18
’
0
l
’
26
’
’
34
’
’
42
’
WEIGHT Fig. 3.-Correlation
’
50
’
’
56
’
’
66
’
IN KG.
of urine IRI and body weight.
’
74
0 ’
’
62
304
NAJJAR
Table 2 .-Day-to-day Subject
Day
1 2 3 4
1
4*, ~.~3~ 30 625 -
-Subject
Day
1
STEPHAN
Variation in 24-hour Urinary IRI Excretion Expressed in aU per Hour ____ ~~ ~ __ 2
~.
31 617 298
Table S.-Day-to-day
AND
3 915_
__
4
495 60 526 130
160 350 98
5
6
7
1240 225 660 195
346 480 175
620 140 275 -
~-
Variation in 24-hour Urinary IRI Excretion Expressed in aIJ per mg. of Creatlnine __~____ 2
3
4
5
6
I
1 2 3
6.8 1.0 10.4
12.0 0.25 9.4
13.7 2.7 5.7
7.5 0.5 8.2
16.4 3.9 8.6
5.8 7.3
6.2 3.3 4.0
4
-
3.8 _.___
-
1.8
2.7
2.2
-
same subject whether expressed in $-J/hour or in ,uU mg. of urine creatinine. Although “basal” excretion of IRI seems to vary over a wide range, there was a tendency for “high excretors” (e.g., subject 1) and “low excretors” (e.g., subject 2) to have rather consistent excretion rates. Diurnal Variation in Urinary IRI Excretion
More IRI was excreted per hour and per milligram of creatinine during the day than during the night. The results obtained in six subjects who collected urine on two or three consecutive days are shown in Table 4. These results also illustrate the day-to-day variation in insulin excretion. This variation is found both in the day and in the night specimens. Table Subject Day
1
2
1 2 3 1 n
L
3
4
5
6
1 2 3 1 2 3 1 2 3 1 2 3
pU/hr.
920 1100 750 730 1400 700 1450 840 1000 1800 750 1100 1500 470 550 1180 1000
4.-Diurnal Day
Excretion
of Urinary
IRI Night
pU/mg.
Creatinine
11.5 13.6 9.3 6.60 15.30 9.4 15.0 8.2 11.8 21.6 5.2 12.2 20.0 4.8 6.9 14.2 10.6
pU/hr.
pU/mg.
70
Creatinine
0.86 1.25 1.75 1.9 2.0 6.3 2.5 4.0 3.38 1.92
100 130 120 116 490 75 330 400 125 Negligible 340 100 540 390 285 200
4.4 1.6 5.0 6.0 4.0 3.0
URINARY
INSULIN
20 -
EXCRETION
IN CHILDREN
305
AND ADULTS
.
z 165 .c ;i 2 12-
.
”
.
? 3 t
8-
a -
4-
O1”“““‘“““l” 2 4
i
to
8 . . . 6 Age
8 in
10
12
14
16
years
.. .
. Adults
Age unknown
Fig. 4.-Random morning or “basal” urinary IRI excretion in offsprings of one diabetic parent expressed in terms of urinary creatinine. Shaded area represents normal values, Mean * SD, Fig. 2.
Urinary IRI Excretion
in Children of a Diabetic Parent
Seventy-four of 78 children of a diabetic parent had urinary IRI levels on a randomly voided morning specimen or on a repeat overnight specimen collected in a similar fashion to the normal controls that were within the normal range for age (Fig. 4). Four had levels consistently elevated. The average values found in these four children were 70.0 &J/mg. creatinine in a 16-year-old girl, 20.9 yU/mg. creatinine in a 19-year-old girl, and 58.8 and 42.0 ,J_J/mg. in 9- and 12-year-old brothers, respectively. None had proteinuria. A glucose tolerance test using 100 Gm. of glucose orally was performed on the two girls. Their blood sugar levels following the glucose load were within normal, however, their plasma IRI levels were greatly elevated (Fig. 5). The girls were not obese. Their fathers had maturity onset type diabetes mellitus and were insulin dependent. The urinary IRI excretion was not determined during the glucose tolerance test and a glucose-cortisone tolerance test was not performed. DISCUSSION
Overnight collection of urine started two hours after the last evening meal probably represents the nearest to basal insulin excretion since it is least affected by food intake, the most potent physiologic stimulus for insulin secretion. The two-hour interval was chosen because we’ and others” have shown that most of the insulin secreted in response to a glucose load is excreted in this time interval. Further support to the adequacy of this interval is provided by the absence of a significant difference between the “basal” and the “second-voided” urinary lR1 excretion rates. This finding also lends more support to the basal characteristic of the overnight excretion rate. Rubenstein et al.” reported on the overnight urinary IRI excretion in healthy adults. The urine was collected in the same fashion as in our normal controls. They obtained values very similar to ours. The mean hourly excretion was 215 PU and the mean excretion expressed per milligram of urine creatinine was 3.6. The values we obtained for the 24-hour urinary IRI excretion (calculated by add-
306
NAJJAR
AND STEPHAN
Fig. 5.-Plasma IRI and plasma glucose levels during glucose tolerance test in two subjects with elevated urinary IRI excretion.
TIME
IN
MINUTES
ing the amounts excreted in the day and the night specimens) were also similar to those previously reported by others.1,4 No values for overnight urinary IRI excretion in children is available in the literature. McArthur and Stimmler4 reported on the 24-hour urinary excretion in 16 children, the youngest of whom was eight years old. Our study comprises a larger group of children and includes younger ages. Overnight excretion is probably a better index for comparison since it is nearest to basal conditions and is not affected by food intake which can be quite variable in quantity, quality, and frequency during a 24-hour period. Our results showed a tendency to an increase in the hourly excretion of IRI with age. However, the scatter was wide and there was no statistical significance to the difference between the various age groups. The urinary IRI excretion expressed in terms of urinary creatinine decreased with age and the difference between children and adults was statistically significant. This probabIy represents the increased excretion of urinary creatinine with ages rather than an actual decrease in urinary IRI excretion. Rubenstein et al.? and Stimmler and McArthurq found a correlation between body weight and urinary IRI excretion, we could establish such a correlation
URINARY INSULIN EXCRETION IN CHILDREN AND ADULTS
307
only in children weighing 40 Kg. and less. No explanation can be offered for this discrepancy. Lambert and Hoetg claimed that serum insulin levels in healthy women are markedly elevated at midnight. Gagliardino lo found that rats excreted more IRI in the urine from 5 p.m. to 8 a.m. than from 8 a.m. to 5 p.m. irrespective of dietary manipulations. This pattern of diurnal variation was not found in our subjects who, on the contrary, excreted more IRI during the day than during the night. This is consistent with the data of Lestradet’l who found that the plasma levels of IRI in man followed closely the glycemic curve reaching its maximum between 12 and 14 hours and its minimum at 5 hours. The day to day variation in the overnight or “basal” urinary IRI excretion in the same individual was great. The same is true for the excretion rates during the day and the night. The individual values, however, remained within the normal range for adults except for case 1 in Tables 2 and 3. There is no apparent explanation for this variation in insulin excretion which has not been previously documented. It probably does not represent a technical artifact because the assays were repeated on several occasions with little or no change in the results. It could possibly result from prior priming of the beta cells of the pancreas by unequal physiologic stimuli, or it could be the result of inconstant nocturnal stimuli or suppressors of insulin secretion such as the nocturnal bouts of growth hormone release described by Hunter and Riga1.l” Recently Takahashi et al.‘” failed to demonstrate insulin peaks during sleep similar to those of growth hormone. It may reflect the claim of Chamberlain and Stimmler3 that a very small and variable proportion of the filtered IRI appears in the urine. This, however, is at variance with the finding of Rubenstein et all& of a constant renal clearance of insulin irrespective of its blood level. The daily variation in the urinary IRI excretion, in addition to the small renal clearance of IRI,‘i,14 puts great limitations on the value of individual determinations of urinary IRI, particularly when used for comparative purposes. When such values are definitely well beyond the range of normal for age, and in the absence of renal disease, they may be helpful in detecting individuals with elevated plasma IRI (Fig. 5). Of the 78 offsprings of one diabetic parent, four had very high values of urinary IRI. The two girls that were examined had a normal glucose tolerance test. The levels of their plasma IRI in response to an oral glucose load however were elevated and were similar to those found in prediabetics, mainly elevated values at one and two hours when compared to normal controls.‘” It is difficult to speculate at this time on the significance of this finding. If these two girls should develop diabetes mellitus in the future, overnight urinary IRI excretion may then become a useful screening test for the early detection of prediabetic individuals. This necessarily awaits further study and documentation. REFERENCES 1. Jorgensen, K.R.: Immunoassay of insulin in human urine. Acta Endocr. (Kobenhavn) 51:400, 1966. 2. Rubenstein, A.H.. Lowy, C., Welborn, T.A., and Russell Frazer, T.: Urine insulin
in normal subjects. Metabolism 16:234, 1967. 3. Chamberlain, M.H., and Stimmler, L.: The renal handling of insulin. J. Clin. Invest. 46:911, 1967.
308 4. McArthur, R.G., and Stimmler, L.: Urinary insulin excretion in healthy children and siblings of childhood-onset diabetics. Lancet 2: 1236, 1966. 5. Lowy, C., and Schiff, D.: Urinary excretion of insulin in the newborn. Lancet 1:225, 1968. 6. Soeldner, J.S., and Slone, D.: Critical variables in the radioimmunoassay of serum insulin using the double antibody technic. Diabetes 14:771, 1965. 7. Najjar, S., and Stephan, L.: Urinary insulin. I. Methodology. Lebanese Medical Journal 22:95, 1969. 8. Graystone, J.E.: Creatinine excretion during growth. In Cheek, D.B. (Ed.): Human Growth. Philadelphia, Lea and Febiger, 1968, p. 182. 9. Lambert, A.E., and Hoet, J.J.: Diurnal pattern of plasma insulin concentration in the human. Diabetologia 2:69, 1966.
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10. Gagliardino, J. J. : Physiologic variations of insulin output in urine. Metabolism 17: 139, 1968. 11. Lestradet, H., and Tichet, J.: Variation circadienne de l’insulinemie. Proceedings of the European Society for Paediatric Encodrinology, Vienna, 1968 (Abstract 57). 12. Hunter, W.M., and Rigal, W.M.: The diurnal pattern of plasma growth hormone concentration in children and adolescents. J. Endocr. 34:147, 1966. 13. Takahashi, Y., Kipnis, D.M., and Daughaday, W.H.: Growth hormone secretion during sleep. J. Clin. Invest. 47:2079, 1968. 14. Rubenstein, A.H., Lowy, C., and Russell Frazer, T.: Radioimmunoassay of insulin in urine. Diabetologia 3:453, 1967. 15. Ricketts, H.T., Cherry, R.A., and Kusteins, L.: Biochemical studies of “prediabetics.” Diabetes 15:880, 1966.