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Progress in Growth Factor Research, Vol. 6. Nos. 2-4, pp. 457-463, 1995 Copyright © 1996. Published by Elsevier Science Ltd. All rights reserved

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CLINICAL INFORMATION ON SERUM IGFBP-3 LEVELS AND IGFBP-3 PROTEOLYTIC ACTIVITY

IN CHILDHOOD

Yukihiro Hasegawa,* T o m o n o b u Hasegawa,t Katsura Fujii,* Hideko Konii,* M a k o t o Anzo,* Taiji Aso,* Shinobu Kotoh,*

Yutaka Tsuchiya t

*Division of Endocrinology and Metabolism, Tokyo Metropolitan Kiyose Children's Hospital, 1-3-1 Umezono Kiyose, Tokyo 204, Japan

tDepartment of Pediatrics, Keio University School of Medicine, 35 Shinanomachi Shinjyuku, Tokyo 160, Japan

In this review paper, three pieces o f clinical information in childhood are presented: (1) IGFBP-3 may replace GH provocation tests in the diagnosis o f GH deficiency (GHD); (2) IGFBP-3 levels are regulated by IGF-I levels in a short period, and (3) ratio o f free IGF-I to total IGF-I is high in serum o f early infancy, similarly to serum o f pregnancy, only partially owing to the presence o f IGFBP-3 proteolytic activity. Each paper will be published soon.

Keyworfls: IGFBP-3 , IGFBP-3 proteolyt ic activity.

(1) IGFBP-3 MAY REPLACE GH PROVOCATION TESTS IN THE DIAGNOSIS OF GILD.

IGFBP-3 is well known to be GH-dependent. IGFBP-3 is thought to be one of the best screening parameters in the diagnosis of GHD in childhood.

The diagnosis of GHD has been performed by a combination of auxological criteria and laboratory data. Among laboratory data, results of GH provocation tests have been considered to be a gold standard. However, recent questions about the utility of GH provocation tests have been advocated (see review article of Ref. 4): (a) almost no normal values are present except for a report by Martin et al. [5], and usual criteria of GH peaks of 10 ng ml q are totally arbitrary; (b) the tests are not reproducible; (c) they do have false positives and false negatives; (d) various assays for GH are used in different hospitals, and (e) some children with normal GH reserve, based on results of GH provocation tests, have low IGF-I and low IGFBP-3, and grow well with a conventional GH treatment, similarly to patients with GHD.

Thus, because of the above limitations of GH provocation tests, simple compar- isons of IGFBP-3 measurements and results of GH provocation tests are not

457

458 Y. Hasegawa et al.

enough to establish the clinical utility of IGFBP-3 in the diagnosis of GHD. Here we established two groups of subjects, namely severe G H D patients and short chil- dren with probably normal GH secretion, to try to validate the usefulness of IGFBP-3 measurements in the diagnosis of GHD.

Subject (1)

From all the children who have visited our hospital with complaints of short stature for these three years (1992-1994; n = 156), we excluded the children with a stature higher than -2.5 SDS [6] and the children in whom only one G H provoca- tion test was done. Among the selected children, two groups were further set; severe G H D group (Group 1) and short children with normal G H secretion (Group 2). Group 1 (n = 27) was defined as either patients with abnormality in GH gene or Pit- 1 gene, or patients with abnormalities on magnetic resonance image such as invisi- ble stalk, hypoplastic anterior pituitary lobe, ectopic posterior pituitary lobe. Group 2 (n --- 28) was defined as short children who had at least one G H peak of more than 20 ng ml -~. Because a group with unequivocally normal G H secretion should have been selected, not the usual criteria of 10 ng ml -~ but the criteria of 20 ng ml -~ were used to define this group.

Methods (1)

The usual GH provocation tests were arginine and insulin tolerance tests. Among children who had at least one GH peak of more than 10 ng m1-1, the percentages where one GH peak of these two provocation tests was less than 10 ng ml -~ and the other GH peak was more than 10 ng ml -~ were 192/o and 25% for the arginine test and the insulin test respectively, [7]. IGFBP-3 was measured as previously described [8]. The 5th percentile for each age was used as the cutoff value of low IGFBP-3 for each age.

Results (1)

In Group 1, none had IGFBP-3 levels above the 5th percentile for each age. In Group 2, 27 out of 28 patients had IGFBP-3 levels more than the 5th percentile for each age. One subject in Group 2 had not only low IGFBP-3 (less than the 5th percentile) but also low total IGF-I levels (less than the 5th percentile).

Conclusion (1)

Taken together with the theoretical assumption that G H secretion is continuous from zero to normal, our results suggest that IGFBP-3 measurements are useful in the diagnosis of GHD. Furthermore, IGFBP-3 measurements may replace GH provocation tests in the diagnosis of GHD. In addition to the limitations of GH provocation tests, IGFBP-3 measurements have at least two advantages; one sampling can be reliable, and the measurements are functional assay for G H secre- tion, thus physiological.

It is not surprising that some functional tests are more useful than direct measurements of the hormone in question. Other examples are non-esterified fatty

Serum IGFBP-3 Levels and IGFBP-3 Proteolytic Activity 459

acid in the diagnosis of hyperinsulinism, blood sugar levels and glycosylated haemo- globin in the diagnosis of insulin-dependent diabetes, urine osmolatility in the diag- nosis of diabetes insipidus.

In practice, not only IGFBP-3 but also IGF-I should be used. Other possible factors in the diagnosis of GHD are free IGF-I and acid-labile subunit levels. The data of free IGF-I have been published [9, 10].

(2) IGFBP-3 LEVELS ARE REGULATED WITH IGF-I LEVELS IN A SHORT PERIOD.

If you measure IGFBP-3 and IGF-I in, for example, 300 children with various degrees of GH secretion, IGFBP-3 levels correlate well with IGF-I levels as was shown by many authors previously. However, over a long period (a range of months), IGFBP-3 levels were reported not to be upregulated by IGF-I adminis- tration subcutaneously in patients with GH receptor dysfunction [11, 12]. Here, regulation of IGFBP-3 levels by IGF-I (levels) in a short period, namely over a range of minutes, was studied.

Subject (2)

The first subjects were two short children who were evaluated for intradaily vari- ations of IGFBP-3 and IGF-I levels. The other subject was a patient with GH gene deletion who was given IGF-I subcutaneously. The levels of IGFBP-3 and IGF-I at 0, 3, 6, 12, and 24 h after the administration of IGF-I were studied. IGFBP-3 and IGF-I were measured as previously described [8].

Results (2)

In two children who were studied for intradaily changes of IGFBP-3 and IGF-I levels, both levels of IGF parameters correlated significantly with each other (r = 0.78 and 0.67 respectively, p < 0.01 for both). One of the significant correlations is shown in Fig. 1. As can be seen in this figure, concomitant decrease and increase in IGFBP-3 and IGF-I levels were observed consistent with the concept of the short- period regulation of IGFBP-3 levels by IGF-I levels.

To further validate the short-period regulation of IGFBP-3 levels by IGF-I (a range of minutes), time-course of IGFBP-3 and IGF-I levels was studied in a patient who was given different doses of IGF-I (60-150/.tg kg-~). Representative increase in IGFBP-3 levels after the administration of IGF-I is shown in Fig. 2, where concomitant increase in IGFBP-3 and IGF-I is observed. When several different doses of IGF-I were administered, the increase in IGFBP-3 levels was in a dose-dependent manner with the increase in IGF-I levels (data not shown).

Conclusion (2)

IGFBP-3 levels are regulated by IGF-I levels in a short period (a range of minutes), probably in order to keep free IGF-I as stable as possible. The origin of

460 Y. Hasegawa et al.

~ Total IGF-I 2 5 0 ~ ~ 114 IGFBP-3

~ 200

F I I I I I I I I 9.00 12.00 15.00 18.00 21.00 24.00 3.0t) 6.01)

2.5 ::1.

e~

2.0 --

FIGURE 1. Intradaily changes of I G F B P - 3 and IGF-I levels in a short child with normal G H secretion.

the increase in IGFBP-3 levels is unknown at present; one possible origin is cell- associated IGFBP-3 which is readily accessible to the circulation.

(3) THE RATIO OF FREE IGF-I TO TOTAL IGF-I LEVELS IS HIGH IN SERUM OF EARLY INFANCY, SIMILARLY TO SERUM OF

PREGNANCY, ONLY PARTIALLY OWING TO THE PRESENCE OF IGFBP-3 PROTEOLYTIC ACTIVITY.

IGFBP-3 proteolytic activity was originally found in serum of pregnant women [13-15]. Its existence is postulated to lead to the increase in plasma free IGF-I levels, which may help maternal tissues such as placenta and uterus to grow during pregnancy. Indeed, we have already reported that plasma free IGF-I levels are higher in pregnant women than in age-matched non-pregnant women [9, 16].

There are two periods during childhood when growth is more rapid than during other periods: one is puberty, and the other is early infancy. The rapid growth during puberty is at least partially related to increased secretion of GH and gonadal steroids. This time, we analysed free IGF-I levels, total IGF-I levels, and IGFBP-3 proteolytic activity in early infancy and puberty in order to clarify the role of IGF- I system in rapid growth in those periods.

Subject (3)

The subjects were 51 normal young infants (1-5 months), normal prepubertal children (n = 28; 4--7 years), patients with precocious puberty (n = 3), normal girls at early stages of puberty (n = 7), normal adults (n = 78; 22-40 years), and normal pregnant women (n = 69; 10--38 weeks, 22-35 years).

Methods (3)

Plasma free and total IGF-I were measured by IRMA according to a recent paper by our group [17]. Ratio of free IGF-I to total IGF-I (f/t; mean + standard

Serum IGFBP-3 Levels and IGFBP-3 Proteolytic Activity 461

800[ . ~ IGF-I (ng/ml) 6

600/ ~ 0 IGFBP-3 (rag/L) 5

I// i

0 10 20 30 Time (hr)

FIGURE 2. Increase of 1GFBP-3 and IGF-I levels after the administration of IGF-I for a patient with GH gene deletion.

deviation) was calculated in each group of the subjects. IGFBP-3 proteolytic activ- ity was analysed by the Western immunoblot technique [18].

Results (3)

Free IGF-I to total IGF-I ratios (fit) were 2.02 + 0.69% in normal young infants, 1.20 + 0.39% in normal prepubertal children, 0.92 +0.23% in normal adults, and 1.98 + 0.67% in normal pregnant women. The subjects of patients with precocious puberty and normal girls at early stages of puberty were analysed together as children with puberty, f/t in this children with puberty was 0.81 + 0.19%. There was a significant difference in f/t between normal young infants and either normal prepubertal children, children with puberty, or normal adults. There was a signifi- cant difference in f/t between normal pregnant women and either normal prepuber- tal children, children at puberty, or normal adults. There was not a significant difference in f/t between normal young infants and pregnant women.

Twelve out of 18 infants had mild IGFBP-3 proteolytic activity; the levels of IGFBP-3 proteolytic activity were one-third to a half of the activity in pregnant serum (30-50% of IGFBP-3 proteolytic activity in our methodology). None of 12 subjects from early infancy had quantitatively similar IGFBP-3 proteolytic activity to that observed in pregnant serum; IGFBP-3 proteolytic activity > 50% in our methodology [14]. None of normal children and adults examined (n = 12 in normal prepubertal children, n -- 5 in children at puberty, n = 15 in normal adults) had IGFBP-3 proteolytic activity; all had less than 10% of IGFBP-3 proteolytic activ- ity. All the pregnant women examined (n -- 22) had significant IGFBP-3 proteolytic activity (more than 50% in our assay).

F/t in early infancy did not correlate with IGFBP-3 proteolytic activity.

462 Y. Hasegawa et al.

Conclusion (3)

(1) F/t is high in early infancy, which may be related with rapid growth during that period.

(2) IGFBP-3 proteolytic activity contributes to the increase in f/t to some extent, however there should be other factors contributing to the increase in f/t during infancy; i) IGFBP-3 proteolytic activity in early infancy was not so high in early infancy as in pregnancy, with the similar degree of f/t in both early infancy and pregnancy, ii) f/t in early infancy did not correlate with IGFBP-3 proteolytic activity (in contrast, we have reported that f/t correlated significantly with IGFBP-3 proteolytic activity in the subjects with various degrees (0-100%) of IGFBP-3 proteolytic activity [19].

REFERENCES

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2. Hasegawa Y, Hasegawa T, Nishi Y, Tsuchiya. Regulation of IGFBP-3 levels by IGF-I in a short period of time. (Submitted).

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4. Rosenfeld RG, Albertsson-Wikland K, Cassorla F, Frasier SD, Hasegawa Y, Hintz RL, Lafranchi S, Lippe B, Loriaux L, Melmed S, Preece MA, Ranke MB, Reiter EO, Rogol AD, Underwood LE, Werther GA. Diagnostic controversy; the diagnosis of childhood growth hormone deficiency revis- ited. J Clin Endocrinol Metab. 1995; 80: 1532-1540.

5. Martin G, Domene HM, Barnes KM, Blackwell B J, Cassorla FG, Cutler Jr GB. The effects of estrogen priming and puberty on the growth hormone response to standardized treadmill exercise and arginine-insulin in normal girls and boys. J Clin Endocrinol Metab. 1994; 79: 537-541.

6. Suwa S, Tachibana K, Maesaka H, Tanaka T, Yokoya S. Longitudinal standards for height and height velocity for Japanese children from birth to maturity. Clin Pediatr Endocrinol. 1992; 1:5-1 I.

7. Hasegawa Y, Hasegawa T, Kotoh S, Tsuchiya Y. Ratio of false positive results of GH provocation tests (arginine and insulin) in control and normal short children. Clin Pediatr Endocrinol. 1993; 2 (suppl): 65-67.

8. Hasegawa Y, Hasegawa T, Aso T, Kotoh S, Nose O, Ohyama Y, et al. Clinical utility of insulin- like growth factor-binding protein-3 in the evaluation and treatment of short children with suspected growth hormone deficiency. Eur J Endocrinol. 1994; 131: 27-32.

9. Hasegawa Y, Takada M, Hasegawa T, Tsuchiya Y. Clinical utility of newly developed free insulin- like growth-factor-I measurements by IRMA. Clin Pediatr Endocrinol. (Abstract) 1994; 3 (suppl 5): 160-161.

10. Hasegawa Y, Hasegawa T, Takada M, Tsuchiya Y. Free IGF-I measurements in the diagnosis of GH deficiency. Eur J EndocrinoL 1995 (In press).

11. Rosenfeld RG, Rosenbloom AL, Guevara-Aguirre J. Growth hormone (GH) insensitivity due to primary GH receptor deficiency. Endocrinol Rev. 1994; 15 (3): 369-390.

12. Blum WL, Cotterill AM, Postel-Vinay MC, Ranke MB, Savage MO, Wilton P. Improvement of diagnostic criteria in growth hormone insensitivity syndrome: solutions and pitfalls. Acta Paediatr Suppl. 1994; 399: 117-124.

13. Fielder PJ, Thordarson G, Talamates F, Rosenfeld RG. Characterization of insulin-like growth factor binding proteins (IGFBPs) during gestation in mice: effects of hypophysectomy and an IGFBP-specific serum protease activity. Endocrinology. 1990; 127: 2270-2280.

14. Guidice LC, Farrell EM, Pham H, Lamson G, Rosenfeld RG. Insulin-like growth factor binding proteins in maternal serum throughout gestation and on the puerperium: effects of a pregnancy- associated serum protease activity. J Clin Endocrinol Metab. 1990; 71: 806-816.

15. Hosselopp P, Segovia B, Lassarre C, Roghani M, Bredon M, Binoux M. Evidence of enzymatic degradation of insulin-like growth factor-binding proteins in the 150K complex during pregnancy. J Clin Endocrinol Metab. 1990; 71: 797-805.

Serum IGFBP-3 Levels and IGFBP-3 Proteolytic Activity 463

16. Hasegawa T, Hasegawa Y, Takada M, Tsuchiya Y. The free form of insulin-like growth factor-I increases in circulation during normal human pregnancy. J Clin Endocrinol Metab. (In press).

17. Takada M, Nakanome H, Kohsida M, Hirose S, Hasegawa T, Hasegawa Y. Measurements of free IGF-I using immunoradiometric assay. J Immunoassay. 1994; 15 (3): 263-276.

18. Hasegawa Y, Hasegawa T, Koni N, Aso T, Tanaka N, Kotoh S, et al. Proteolytic activity of IGFBP-3 in various clinical conditions during childhood using Western immunoblot technique. Endocrine J. 1995; 42 (4): 569-576.

19. Hasegawa Y, Hasegawa T, Fujii K, Konii H, Takada Y. Regulation of ratio of plasma free to total insulin-like growth factor-I levels by insulin-like growth factor binding protein-3 proteolytic activ- ity. (Submitted).