Radioimmunoassay for Insulin-Like Growth Factor II (IGF-II)

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1 Radioimmunoassay for Insul-Like Growth Factor II () KUMIKO ASAKAWA, NAOMI HIZUKA, KAZUE TAKANO, IzuMI FUKUDA, IzuMi SUKEGAWA, HIROSHI DEMURA AND KAZUO SHIZUME 1 Department of Internal Medice, Institute of Clical Endocrology Tokyo Women's Medical College Research Laboratory, The Foundation for Growth Science, Tokyo, 162, Japan1 Ab str act Insul-like growth factor II () levels human plasma were measured physiological pathological conditions by radioimmunoassay (RIA) biosynthetic. This RIA was specific for cross-reactivity IGF-I was 1%. The sensitivity was 15pg/tube 50% displacement at 50pg/tube. The tra- ter-assay coefficients of variation for were %, respectively. The plasma levels normal adults, hypopituitarism active acromegaly were }15.8, } } 24.3ng/ml, respectively. After human growth hormone (hgh) treatment hypopituitarism, slightly creased, but not significantly. After adenomectomy acromegaly, significantly decreased. These data dicate that concentrations plasma were partially GH dependent. This GH dependency was less than that of IGF-I. was low anorexia nervosa liver cirrhosis high renal failure. In two cases extrapancreatic tumorassociated hypoglycemia, plasma was creased to ng/ml, returned to normal after tumor resection. These data showed that was partly dependent on GH nutritional coditions that was the most likely cause of some cases of hypoglycemia extrapancreatic tumor. This specific sensitive RIA of would be useful evaluatg its physiological pathological role plasma tissue. Received February 6, 1990 Please address all correspondence to: Dr. Kumiko Asakawa, Department of Medice, Institute of Clical Endocrology, Tokyo Women's Medical College, 8-1, Kawada-cho, Shjuku-ku, Tokyo, 162, Japan Two ma forms of IGFs have been isolated from human plasma these two IGFs are structually similar (Rderknecht Humbel, 1976, 1978). Because of the presence of these two IGFs, efforts have been made to develop methods which specifically measure these peptides. Radioimmunoassay for is now available several laboratories (Moses et al., 1980; Zapf et al., 1981; Daughaday et al., 1981; Htz et al., 1982; Enberg et al., 1984). Recently was synthesized by re-

2 ASAKAWA et al. combant DNA technology. In this study we used biosynthetic for RIA of, measured plasma levels physiological pathological conditions man this sensitive specific RIA compared these results data from other laboratories. Materials Methods Pep tid es Biosynthetic human (lot No. T51- ZL7-92) porce sul were kdly provided by Eli Lilly Co. (Indianapolis, IN). Biosynthetic human IGF-I (lot No K) was donated by Fujisawa Pharmaceutical Co. (Osaka, Japan). Monoclonal antibody to rat was provided by Amano Pharmaceutical Co. (Nagoya, Japan) (Tanaka et al., 1985). Iodation of was iodated to a specific activity of MBq/ƒÊg by a modification of the chlorame T method (Zapf et al., 1981). The reaction products were separated on a 0.7 ~ 50cm Sephadex G-50 column. RIA For Radioimmunoassay was carried out at 4 Ž 0.03M phosphate buffer, ph 7.5, contag 0.25% bove serum album, 0.02% sodium azide, 0.02% protame sulphate, 0.01M EDTA. stard or unknown samples (50ƒÊl) were precubated anti- antibody (50ƒÊl) for 72h a total volume of 0.45 ml. At the end of precubation, 125I- (50ƒÊl: cpm) was added cubated for 16-18h. Separation of bound from unbound was performed by the double antibody method. Su bje cts Forty-eight healthy adult volunteers (24 males 24 females, yr) 36 normal children (25 males 11 females, 0-19 yr) were studied to determe the normal range of plasma. concentrations 28 active acromegaly (20 males 8 females), 24 hypopituitarism, 9 Turner's syndrome, 7 liver cirrhosis, 7 renal failure, 16 diabetes mellitus, 12 anorexia nervosa, 30 pregnant woman were measured. Another two tumor-associated hypoglycemia (Suzuki er al. 1989; Haruki et al. 1990) were studied to measure values plasma tumor extract. One patient had mesothelioma the abdomen the other patient had histiocytoma the postperitoneum. IRI concentrations were both low the former case the tumor contaed a large amount of mrna. The active acromegaly had creased plasma GH that was not suppressed below 5ng/ml by the oral admistration of glucose (75g), /or had paradoxical GH response to TRH /or GnRH. In the untreated hypopituitarism, the diagnosis of GH deficiency was established by the failure of peak plasma GH to rise above 5ng/ml after provocative stimuli such as sul-duced hypoglycemia. They have been treated the appropriate replacement therapy for other deficient pituitary hormones. The diagnosis for liver cirrhosis, renal failure diabetes mellitus was established by clical laboratory fdgs. EDTA-plasma was taken from each subject stored at-20 Ž until the assay. EDTAplasma was extracted by the acid-ethanol extraction method (Daughaday et al., 1980, 1987). Tumor tissue was extracted aceton-formic acid (Russel et al., 1989). Gel filtration of plasma at neutral ph One milliliter of EDTA plasma was chromatographed on a 1.5 ~90cm column of Sephacryl S M phosphate buffer contag 0.1M NaCl. One-milliliter aliquots were collected, lyophilized. They were extracted acid-ethanol assayed for IGF-I (Miyakawa et al. 1986). Re su Characteristics of RIA for Figure 1 shows the stard curve of the RIA for cross-reactivity of the antibody sul IGFs. The sensitivity of the RIA was 15pg/tube 50% displacement at 50pg/tube. No cross-reactivity of the antibody sul its

3 Vol.37, No.5 RIA FOR IGF- II RIA Fig. 1. Comparison of the dose response curves for human, human IGF- I, sul acidethanol plasma. extracted extracted plasma was observed. IGF-I crossreacted it at a potency 100 times less than. The tra- ter-assay coefficients of variation of the assay were 6.3% 9.3%. The dilution curve for the acid-ethanol extract of human plasma were parallel to that for the stard (Fig. 1). concentrations Extracted concentrations 48 normal adults ranged from to ng/ml a mean of } 15.8ng/ml (Mean }SEM) as shown Fig. 2. There was no difference between concentrations females those males (601.2 }22.7 vs }22.4ƒÊg/ml). In normal children, plasma was low fants but the concentrations 5-year-olds were almost same as those of normal adults (Fig. 3). In 20 hypopituitarism plasma ranged from to a mean of }24.3ng/ml, which was statistically lower than normal subjects. However, the concentrations Fig. 2. normal adults hypopituitarism or acromegaly.

4 610 ASAKAWA Fig. 3. children. lapped pituitary for had several years. before slightly difference II ranged mean from of these values subjects, acromegalic tumor 49.0ng/ml statistically to but they after did not a 2). The were normal Thirteen operations However, body weight anorexia In those from. IGF was treatment, different groups. underwent resection. plasma before overlapped. of significant (Fig. than ranges nervosa. plasma acromegalic greater (768 }62.2ng/ml). two women treatment no pregnant Table 1. before after treatment were those was }45.6ng/ml statistically for than acromegalic normal }67.6ng/ml. there con- treatment after treat- after } which before was treatment. treatment decrease 6 of 13 after liver diseases, renal failure, diabetes mellitus, anorexia nervosa or Turner's syndrome. The shaded area 'dicates the Six hgh higher between active received but 28 subjects. concentrations treatment, normal over- normal were dwarfs }50.8ng/ml The age hypopituitarism those centrations Fig. ment Endocrol. Japon. October 1990 et al. liver cirrhosis were }61.1ng/ml concentrations anorexia nervosa }40.9

5 Vol.37, No.5 RIA FOR ng/ml, respectively these values were lower than those of normal subjects (Fig. 4). Six anorexia nervosa received travenous hyperalimentation therapy (Table 1). In three (cases 1, 2, 3), whose body weight creased, IGF- II was high after treatment. However, two other (cases 4 5) whose body weight creased less than 2 kg one patient (case 6) who had a long history of emaciation, did not change. In renal failure plasma was } ng/ml, which was significantly higher than normal subjects. In pregnant women there was no difference first, second third trimesters (604.8 } 33.6, } } 31.9 ng/ml). IGF-I diabetes mellitus or Turner's syndrome did not differ from that of normal subjects (Fig. 4). The plasma IGF-I concentrations two extrapancreatic tumor associated hypoglycemia were measured. Before tumor resection, plasma was ng/ml Fig. 5. Changes plasma IGF-I tumor associated hypoglycemia after tumor resection. 150K 40K 150K 40K Fig. 6. Sephacryl S-200 gel filtration of plasma IGF-I at neutral ph.

6 ASAKAWA et al. Endocrol. Japon. October 1990 plasma IGF-I was 41.2 < 35ng/ml, respectively. After tumor resection, hypoglycemia disappeared. concentrations were normalized to ng/ml IGF-I was normalized to ng/ml (Fig. 5). values the tumor were ƒÊg/g tissue. Gel filtration pattern of normal plasma The Sephacryl S-200 gel filtration patterns of IGF-I were observed (Fig. 6). There were two peaks of both IGFs, approximately 150K 40K. The 40K peak of was higher than that of IGF-I. Discussion In the present study we reported IGF- II concentrations man physiological pathological conditions determed by a specific sensitive radioimmunoassay biosynthetic. This antibody to was specific for cross reactivity IGF-I was 1%. Zapf et al.(1981) Enberg et al.(1984) reported that their polyclonal antibody to human exhibited 10% cross-reactivity IGF-I their RIAs required correction for IGF-I content. Our RIA did not require it. Fifty percent displacement of 125I- our RIA was 50 pg/tube (100pg/ml) while it was 1-2ng/ml other RIAs. Therefore, we could detect even a very small amount of various samples this assay. active acromegaly was creased, then decreased after operation. hypopituitarism was low. When these were treated hgh, creased slightly, but not significantly. Our data suggest that might be partially GH dependent, but this GH dependency is less than that of IGF-I. It has been reported that no crease IGF- II was found acromegaly, whereas GH deficiency was accompanied by significantly lower concentrations (Zapf et al. 1981, Enberg et al., 1984). Htz et al.(1982) showed that GH deficient their creased acute chronic GH treatment. They also suggested that is partly GH dependent. However, our data differ from theirs. Zapf et al. have shown that the mean values acromegalic were the same as those normal subjects, but their data there were several a higher concentration of. Therefore, we suspect that some acromegaly might have higher than normal. The data for anorexia nervosa suggest that the concentration depends on nutritional conditions, but this dependency is also less than that of IGF-I. These results suggest that the concentration human plasma might be partially dependent on GH /or nutritional conditions, this dependency is less than that of IGF-I. The ma control for plasma needs to be clarified. Our data concerng liver diseases, renal failure, pregnant women are accordance those of other laboratories. In tumor associated hypoglycemia, plasma creased slightly IGF-I was low, the tumor was high. After tumor resection, IGF-I II were back to normal. We suspect that hypoglycemia is due to IGF- II that was produced by the tumor. There have been several reports of similar cases which the tumor secretes hypoglycemia occurs (Megyesi et al. 1974, Hyodo et al. 1977, Daughaday et al., 1981, 1988, Shapiro 1988). In these cases, the immunoreactive plasma concentration was normal or creased slightly the tumor had creased concentration of IGF- II mrna. Daughaday et al. (1980) reported that a tumor secreted high

7 Vol.37, No.5 RIA FOR molecular weight that a considerable loss of high molecular weight may have occurred durg the acidethanol extraction process, a loss of may have occurred storage. They also showed that these cases was higher radioreceptor assay than radioimmunoassay. Therefore the reason why the immunoreactive plasma concentration was not extremely high might be the loss of the molecular heterogeneity of. It is terestg to study the molecular weight, receptor reactivity bioactivity of our cases. There would be many tumor-associated hypoglycemia we could determe the cause some by means of the assay of IGFs. With this sensitive specific RIA of biosynthetic we were able to measure human plasma might measure small amounts of IGF- II tissue or cerebrospal fluid, which is usable evaluatg the role of. Acknowledgem ents We are greatly debted to Eli Lilly Co. (Indianapolis, IN) for supplyg biosynthetic sul, Fujisawa Pharmaceutical Co.(Osaka, Japan) for biosynthetic IGF-I, Amano Pharmaceutical Co.(Nagoya, Japan) for monoclonal antibody to. We thank Dr. M. Iwashita, Dr. Y. Suzuki Dr. K. Haruki for supplyg samples from pregnant extrapancreatic tumor hypoglycemia. This work was partly supported by Grants Aid for Scientific Research from the Mistry of Education, Science Culture, Japan (Nos ), Yoshioka Memorial Fund. References Enberg G. K. Hall (1984) Immunoreactive serum of healthy subjects growth hormone disturbances uraemia. Acta Endocrol. 107, Daughaday W. H., I. K. Mariz S. L. Blethen (1980) Inhibition of access of bound somatomed to membrane receptor immunobdg sites: a comparison of radioreceptor radioimmunoassay of somatomed native acid-ethanol-extracted serum. J. Cl. Endocrol. Metab. 51, Daughady W. H., B. Trivedi, M. Kapadia (1981) Measurement of sul-like growth factor II by a specific radioreceptor assay serum of normal dividuals, abnormal growth hormone secretion, tumor-associated hypoglycemia. J. Cl. Endocrol. Metab. 53, Daughaday W. H.(1987). Radioimmunoassay for sul-like growth factor II. Method Enzymol. 146, Daughaday W. H., M. A. Emanuele, M. H. Brooks, A. L. Barbato, M. Kapadia P. Rotwe (1988) Synthesis secretion of sul-like growth factor II by a leiomyosarcoma associated hypoglycemia. N. Engl. J. Med. 319, Haruki K., T. Wasada, Y. Hirata, N. Hizuka, M. Yamamoto, K. Ikejiri (1990) A case of producg histiocytoma associated hypoglycemia. The Proceedgs of Peptide Hormone Pancreas 10, press ( Japanese) Hyodo T., K. Megyesi, C. R. Kahn, J. P. McLean., H. G. Friesen (1977) Adrenocortical carcoma hypoglycemia: evidence for. production of nonsuppressible sul-like activity by the tumor. J. Cl. Endocrol. Metab. 44, Htz R. L. F. Liu (1982) A radioimmunoassay for sul-like growth factor II specific for the C-peptide region. J. Cl. Endocrol. Metab. 54, Megyesi K., C. R. Kahn, J. Roth, P. Gorden (1974) Hypoglycemia association extrapancreatic tumors: demonstration of elevated plasma NSILA-s by a new radioreceptor assay. J. Cl. Endocrol. Metab. 38, Miyakawa M., N. Hizuka, K. Takano, I. Tanaka, R. Horikawa, N. Honda K. Shizume (1986) Radioimmunoassay for sul-like growth factor I (IGF-I) usg biosynthetic

8 ASAKAWA et al. Endocrol. Japon. October 1990 IGF-I. Endocrol. Japon. 33, Moses A. C., P. Nissley, P. A. Short M. M. Rechler (1980) Immunological crossreactivity of multiplication-stimulatg activity polypeptides. Eur. J. Biochem. 103, Rderknecht E., R. E. Humbel (1976) Polypeptides nonsuppressible sul-like cell-growth promotg activities human serum: isolation, chemical characterization, some biological properties of forms I II. Proc. Natl. Acad. Sci. USA 73, Rderknecht E. R. E. Humbel (1978) Primary structure of human sul-like growth factor II. FEBS lett. 89, Russell W. L., M. C. Smith, R. R. Bowsher (1989) Development of a new extraction method RIA for the quantification of sul-like growth factor-2 (IGF-2) rat tissue. 71st American Endocre Society, Seattle, Abs No Shapiro T., K. Polonsky, M. Kew, A. Rubenste, G. Bell (1988) Tumor hypoglycemia is associated creased expression of the gene for sul-like growth factor II. Cl. Res. 36, 490A abstract. Suzuki Y., S. Sugaya, K. Matsuoka, S. Moraga (1989) A case of postperitoneal tumor associated hypoglycemia. Tounyoubyou 32:618 abstract No. 34. ( Japanese) Tanaka H., K. Nishikawa, Y. Yamada, T. Hayano, O. Asami, Y. Nakanishi K. Yamashita (1985) Proceedgs of the International symposium on Growth Differentiation of Cells Defed Environment. eds. H. Murakami, I. Yamane, D. W. Barnes, J. P. Mather, I. Hayashi G. H. Sato p 345, Sprger-Verlag, Berl. Zapf J., H. Waters, E. R. Froesch (1981) Radioimmunological determation of sullike growth factors I II normal subjects growth disorders extrapancreatic tumor hypoglycemia. J. Cl. Invest. 68,