Identity of calcitonin extracted from normal human thyroid glands with synthetic human calcitonin-(1–32)

Biochimica et Biophysica Acta, 707 (1982) 59-65

59

Elsevier Biomedical Press

BBA31308

IDENTITY OF CALCITONIN EXTRACTED FROM NORMAL HUMAN THYROID GLANDS WITH SYNTHETIC HUMAN CALCITONIN-(I-32) PAUL H. TOBLER a, ALBERT JOHL b.t, WALTER BORN a, RENE MAIER b and JAN A1 FISCHER a..

Research Laboratory for Calcium Metabolism, Departments of Orthopaedic Surgery and Medicine, 8008 Zurich and b Ciba-Geigy Ltd., Pharmaceuticals Division, 4002 Basle (Switzerland) (Received March 19th, 1982)

Key words: Calcitonin; HPLC; Gel filtration," (Human thyroid)

Calcitonin in human thyroid glands obtained at autopsy from normal subjects was extracted with 2 M acetic acid. The extracts were additionally purified by adsorption to Sep-Pak C ts cartridges, and calcitonin was identified after gel filtration analysis, reverse-phase high-pedormance liquid chromatography (HPLC), thin-layer chromatography and isoelectric focusing. The purification steps were monitored by radioimmunoassay, and partially purified calcitonin was used for biological and physicochemical comparison with synthetic human calcitonin-(l-32) and its MetS-sulfoxide form. On gel filtration analysis a predominant peak coeluted with the synthetic hormone, and on HPLC two discrete peaks with the retention times of monomeric and dimeric human calcitonin were found. Thin-layer chromatography allowed the detection of two peaks with the Rf of human calcitonin-(l-32) and of its sulfoxide, respectively. The pl (7.9) of the predominant peaks of synthetic calcitonin were identical. Our findings provide strong evidence that the predominant forms of human caleitonin extracted from normal thyroid glands correspond to synthetic calcitonin-(l-32) and to dimeric calcitonin.

Introduction Human calcitonin is a 32-amino-acid peptide hormone which was isolated from tumour tissue of patients with medullary carcinoma of the thyroid [1,2]. Its amino acid sequence was elucidated [3] and the structure confirmed by total synthesis [4]. Because the normal human thyroid contains rather small amounts of it, the hormone was originally isolated from medullary carcinoma of the thyroid, and its structure in normal thyroids has not been identified. Nevertheless, bioactive [5,6] and immunoreactive calcitonin has been detected in normal human thyroids [7-11], but the identity of * To whom correspondence should be addressed at: Klinik Balgrist, Forchstrasse 340, CH-8008 Zurich, Switzerland. t Deceased. 0167-4838/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press

calcitonin from normal thyroid tissue and synthetic human calcitonin-(1-32) has not been established. Medullary carcinomas contain 100-10000 times more calcitonin than normal thyroid glands. Human calcitonin-(1-32)was therefore synthesized according to the structure of a calcitonin derived ~ o m carcinoma tissue. Since this synthetic hormone is widely used in patients with disorders of skeletal metabolism [12], it was felt that it would be interesting to provide evidence that this form corresponds to that originating from normal human thyroids. In view of the very low amounts of calcitonin present in normal thyroids isolation to homogeneity and structural analysis are impracticable. With the recent advent of sophisticated peptide separation methodology, using high-performance liquid chromatography (HPLC), we have been able to

60 provide evidence that calcitonin extracted from the human hypothalamus and the pituitary gland corresponds to monomeric human calcitonin-(1-32) and its sulfoxide [13]. It seemed therefore pertinent to use HPLC and additional methods of peptide separation, such as gel filtration, thin-layer chromatography and isoelectric focusing, and the hypocalcaemic rat bioassay to provide evidence that calcitonin extracted from normal human thyroids corresponds to the synthetic hormone used therapeutically in man. Materials and Methods

Peptides. Synthetic calcitonin-(1-32) (monomeric form), extracted purified dimeric human calcitonin from a medullary carcinoma of the thyroid, and synthetic human parathyroid hormone-(1-34) have been donated by W. Rittel, Ciba-Geigy AG, Basle, Switzerland, [3H]calcitonin-(1-32) by R. Wade, Ciba-Geigy Ltd., Horsham, U.K. [14], and extracted bovine parathyroid hormone-(1-84) by the Medical Research Council, U.K. Bovine serum albumin was purchased from Sigma Company, St. Louis, MO, U.S.A. and human serum albumin from Behring Werke, Hoechst Pharma AG, Zurich. Preparation of thyroids and extraction. Thyroids (20-30 g) obtained at autopsy were dissected not later than 12 h postmortem, and were, after determination of wet weight, frozen at -80°C. Patients with malignant tumours and chronic renal insufficiency were excluded from the study. The frozen thyroids were minced and placed in 10 vol. 2 M acetic acid, and transferred to a boiling water bath for 5 min. Subsequently the tissue was homogenized for 5 rain using an Ultra Turrax (18K pestle) homogenizer (IKA-Werk, Staufen, G.R.F.). The homogenate was frozen in liquid nitrogen, thawed, and centrifuged at 48000 X g at 4°C for 30 min. Aliquots of 20 ml of the clear supernatants were passed 20 times through Sep-Pak C~8 catridges (Waters Assoc., Milford, MA, U.S.A.), activated with 10 ml methanol, and equilibrated with 20 ml 0.1% trifluoroacetic acid in water (v/v). The cartridges were washed with 40 ml 0.1% trifluoroacetic acid and the calcitonin eluted in 15 ml 80% methanol in water containing 0.1% trifluoroacetic acid. The methanol was evaporated and the sam-

ples lyophilized after the addition of 10 ml 0.1 M acetic acid. Lyophilized extracts were dissolved in 1 ml 0.1 M acetic acid for further analysis. Gel filtration. For gel filtration of thyroid extracts columns of Bio-Gel P-150 (100-200 mesh, Bio-Rad Laboratories, Richmond, CA, U.S.A.), 1.6 x 100 cm, have been used by ascending flow (flow rate 3.8-5.2 ml/h) at 4°C in 0.2M ammonium acetate, pH 4.7, containing 0.5 g bovine serum albumin/l, and fractions were collected in 1.9- to 2.6-ml volumes. The void volume (V0) was estimated by the elution position of the largest mol. wt. proteins determined spectrophotometrically at 280 nm, and the salt volume (V~) with Nal31I. The elution position of the calcitonin component, designated K d, is defined as elution volume of the immunoreactive substance or labelled marker minus Vo/(V~ minus V0). Tracer amounts of radioiodinated human serum albumin, bovine parathyroid hormone-(1-84), human parathyroid hormone-(1-34), human calcitonin-(1-32) and Na131I were added to each extract as calibrating substances. Radioactivity was measured in an automatic gamma-well spectrometer (model MR 252, Kontron AG, Zurich, Switzerland). Recovery of immunoreactive calcitonin ranged from 60 to 80%. High-performance liquid chromatography. The high-performance liquid chromatography (HPLC) system consisted of a programmer (model 420, Altex, Berkeley, CA, U.S.A.) and two pumps (model 110 A, Altex). Samples were injected via a septumless valve (model 7125, Rheodyne, Berkeley, CA, U.S.A.) fitted with a 0.5 ml injector loop. Acetonitrile (HPLC-grade S) and heptafluorobutyric acid (sequencer grade) were obtained from Rathburn Chemicals (Walkerburn, U.K.). Doubleglass-distilled water from the deionized laboratory water supply was pumped through a 0.22/~m filter (Millipore, Bedford, MA, U.S.A.) and subsequently through a Radial-PAK C~8 cartridge (Waters Assoc., Milford, MA, U.S.A.) to remove traces of organic impurities. Reverse-phase HPLC was performed on a Nucleosil Ci8 (10 /~m, 250 X 4.6 mm, Macherey-Nagel GmbH, Diiren, G.R.F.) column by gradient elution according to a slightly modified method of Bennett et al. [15]. In short, solvent A consisted of 0.1 M heptafluorobutyric acid in water and solvent B of 20% solvent A in acetonitrile. The column was equilibrated at room

61 temperature with 25% solvent B (20% acetonitrile). Extracts containing 150-500 ng immunoreactive calcitonin and tracer amounts of [ 3H]calcitonin-(132) and its sulfoxide (30000-60000 dpm) not interfering in the radioimmunological determinations were injected in a volume of 0.5 ml. After a loading phase of 3 min at initial conditions to concentrate the peptides on the column head [16], calcitonin was eluted with a linear gradient from 25 to 73% solvent B (20-58.4% acetonitrile) over 90 rain at a flow rate of 1.5 ml/min. 1.5-ml fractions were collected in siliconized tubes. 0.3-ml aliquots were analysed for 3H-radioactivity by liquid scintillation spectroscopy (model MR 300, Kontron AG, Zurich, Switzerland) in Rotiszint 22 (Carl Roth KG, Karlsruhe, G.R.F.). and the remaining fractions analysed radioimmunologically and in the hypocalcaemic rat bioassay. The recovery of immunoreactive human calcitonin (with S.E.M.) on HPLC was 77.4±5.6% (range 68.095.0%). Analytical isoelectricfocusing. Analytical isoelectric focusing was performed on polyacrylamide gel plates (LKB, Stockholm, Sweden), pH range 3.59.5 with an LKB Multiphor unit. During the experiments the power was set to 25 W. The voltage increased from approx. 250 to 1350 V while the current at the same time (1.5 h) dropped from about 50 to 20 mA. The actual pH values were measured with a surface pH electrode (type LOT 403-30-M8, Ingold, Zurich, Switzerland) immediately after termination while the plate still rested on the cooling surface of the Multiphor unit. The gels were subsequently frozen and the lanes sliced into 2.5-mm pieces, and calcitonin was eluted in 0.5 ml radioimmunoassay buffer. Thin-layer chromatography. Thin-layer chromatography was performed on silica gel 60 F254plates (20 × 20 × 0.025 cm, Merck, Darmstadt, G.R.F.) with 50 to 100 ng immunoreactive calcitonin and developed in ethylacetate/water/ acetic acid (48 : 27 : 24, v/v). Human calcitonin-(132) and its sulfoxide were used as calibrating substances. Subsequently gels were scratched from the plate in 2-mm strips, and calcitonin extracted with methanol/water/trifluoroacetic acid (80: 19: 1, v/v), the solvent evaporated, and reconstituted in radioimmunoassay buffer. Radioimmunoassay. Immunoreactive hormone

was determined in a previously described homologous human calcitonin-(1-32) assay [13,17]. The antibodies (goat 6A obtained on day 143) used are predominantly directed to determinants located in the COOH-terminal parts of the molecule. Bioassay. The hypocalcaemic rat bioassay was performed according to the method of Kumar et al. [18] in female rats. Results

On gel filtration analysis of thyroid extracts a broad peak with a K d of 0.73 coeluting with synthetic human calcitonin-(1-32) ( K d 0.74) was predominantly recognized (Fig. 1). Moreover, a shoulder was visible eluting in the position of dimeric calcitonin ( K d 0.62). 131I-labelled human calcitonin-(1-32) consistently eluted 0.1 K d units after the peak of immunoreactive synthetic hormone. On H P L C two peaks of immunoreactive calcitonin were regularly seen (Fig. 2). The earlier

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Fig. I. Elution profile o f a Sep-Pak C]s extract of i m m u n o r e a c tive h u m a n calcitonin of a n o r m a l h u m a n thyroid a f t e r chrom a t o g r a p h y o n Bio-Gel P-150. R a d i o i o d i n a t e d h u m a n s e r u m a l b u m i n ([]25I]HSA), b o v i n e p a r a t h y r o i d h o r m o n e - ( l - 8 4 )

([|31I]bPTH-(1-84)), human parathyroid hormone-(1-34) ([125I]hPTH-(I-34)),and [t31I]hCT-(l-32)and Nal3]I were added as calibrating substances to an extract containing 40 ng immunoreactive calcitonin. The elution positions of dimeric human calcitonin (hCT-D) and of human calcitonin-(1-32)are also indicated. 1.6× 100 cm column, 0.2 M ammonium acetate, pH 4.7, and bovine serum albumin (0.5 mg/ml) as eluent, reversed flow (flow rate 4.2 ml/h, 2.1-ml fractions).

62

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Fig. 3. Hypocalcaemic activity measured 50 rain after intravenous injection of a calcitonin extract of a normal human thyroid additionally purified on reverse-phase HPLC (for details see Fig. 2). • corresponds to immunoreactive human calcitonin-(1-32), and • to dimeric human calcitonin (each point represents the mean of three rats±S.E.M.) and • the dose-response line of human calcitonin-(l-32) (each point represents the mean of 10 rats--+S.E.M.).

2O

70

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TIME,min Fig. 2. Reverse-phase HPLC profile of a Sep-Pak CIScalcitonin extract of a normal human thyroid. The column (Nucleosil 10 C is) was eluted with a linear gradient of acetonitrile containing heptafluorobutyric acid as hydrophobic anion-pairing reagent (. . . . ) (for details see Materials and Methods). Effluent fractions were analysed for immunoreactive calcitonin ( L • ) and for 3H-radioactivity (© and arrows). The arrows indicate elution positions of human [3H]calcitonin-(1-32) sulfoxide ([3H]hCT-(l-32)sox, retention time 39-40 rain) and human [3H]calcitonin-(i-32) (retention time 48-50 min). A, Extract of normal thyroid; B, human calcitonin-(l-32) ( • ) and dimeric human calcitonin (•); C, human [3H]calcitonin-(l-32) sulfoxide and [3H]calcitonin-(l-32).

eluting peak had the retention time of the reduced, biologically active form of [3H]calcitonin-(1-32), which coelutes with synthetic calcitonin-(1-32). Interestingiy, extracted calcitonin was almost fully recovered in the non-oxidized form. The second peak eluted in the region of dimeric calcitonin; this peak and the dimeric form were quantitatively converted into m0nomeric human calcitonin-(1-32) and its sulfoxide by incubation in 1 M ammonia for 1 h at 45°C (not shown) [1,3]. In five individual

extractions the overall recovery of [3H]calcitonin(1-32) initially added to the frozen thyroids amounted to 78.6---5.7% (mean---S.E.M.) with a range of 61.9% to 94.8%. The mean calcitonin content of human thyroids amounted to 140.0 ± 36.7 n g / g wet weight (range 62.5-250 n g / g wet weight). The hypocalcaemic activity of the two peaks coeluting with the monomer and the dimer, respectively, was comparable to synthetic human calcitonin-(1-32) (Fig. 3). The MetS-sulfoxide form of human calcitonin-(1-32) was biologically inactive in amounts as high as 100 ng. On thin-layer chromatography (Fig. 4) the predominant peak of immunoreactive calcitonin had an R e of 0.554, which corresponds to human calcitonin-(1-32) (Rf 0.551). A smaller peak with an R f of 0.503 was visible in the region of synthetic calcitonin-(1-32) sulfoxide (R r 0.513). Two minor peaks (Rf 0.529 and 0.682) have not been identified. On isoelectric focusing (Fig. 5) the predominant peak of immunoreactive hormone had a p l of 7.9 (range: 7.8-8.2 p I units), which was the same for

63

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tO. Fig. 4. Tl'fin-layer chromatography of an extract of a normal human thyroid on a precoated silica gel 60 F254 plate (20 × 20 X 0.025 cm). Solvent system: ethylacetate/water/acetic acid, 48 : 27 : 24 (v/v). Solvent front 15.5 cm from origin. Hatched areas: R f of human calcitonin-(1-32) sulfoxide (hCT-(I-32)~ x) and human calcitonin-(1-32) determined on the same plate. Immunoreactive calcitonin was measured in extracts .of 2-ram slices.

-5

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L~" I both dimeric and monomeric forms and for human calcitonin-(1-32) sulfoxide. The peaks with pI 6.8 in extracts of normal thyroids and with p l 7.2 in dimeric human calcitonin have not been further analysed. Discussion

Human calcitonin-(1-32)has been isolated from C-cell tumours [1,2]. Moreover, bioactive and immunoreactive calcitonin has been detected in normal thyroid tissue in very much lower amounts than in C-cell tumours [5-8,21-24,26]. Clark et al. [8] for the first time demonstrated that immunodilution curves of a tissue extract of normal thyroid tissue and of synthetic human calcitonin-(1-32) were superimposable, suggesting immunological identity; similar results have subsequently been obtained with fragments and analogues of human calcitonin, and with calcitonin from different

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Fig. 5. Isoelectric focusing of a Sep-Pak Cls calcitonin extract of a normal human thyroid. Anaytical isoelectric focusing was performed on polyaerylamide gel plates. The pH range was 3.5-9.5. After the run the gels were frozen and the lanes sliced into 2.5-mm pieces, and calcitonin was eluted in radioimmunoassay buffer. • represents immunoreactive calcitonin, and O the pH. A, Extract of normal thyroid; B, human calcitonin-(1-32); C, dimeric human calcitonin.

species with different structures and physicochemical properties [17,19,20]. Calcitonin in plasma and in tissue extracts is predominantly measured radioimmunologically. Antibodies to human calcitonin have usually been generated to extractive hormone of medullary

64 carcinoma of the thyroid or to synthetic human hormone [8-13,17], since calcitonin from normal thyroid tissue was not available in sufficient quantity. The possibility can therefore be considered that homologous radioimmunoassays using synthetic human calcitonin-(1-32) based on the structure of calcitonin derived from C-cell tumours do not necessarily recognize calcitonin in normal subjects, if it is not structurally identical to the tumour cell hormone. Moreover, the synthetic compound is widely used in the treatment of metabolic bone disease [12]. In view of the very low amounts of calcitonin present in normal human thyroid glands its isolation and structural analysis is hardly feasible. On gel filtration analysis a predominant component of calcitonin extracted from normal human thyroids has been demonstrated to co-elute with synthetic human calcitonin-(1-32) (Fig. 1) [21-24]. Moreover, a minor Scalcitonin form coeluting with dimeric hormone has also been recognized. Salmon calcitonin-(1-32) differing in 16 amino acids from human calcitonin-(1-32) and the human hormone exhibit the same elution volume on gel filtration analysis [25]. On isoelectric focusing the predominant components in normal thyroids have a pI of 7.9, which is the same for both monomeric and dimeric human calcitonin (Fig. 5) [26]. These techniques are only marginally useful to address the question of identity of synthetic hormone and extractive calcitonin from normal human thyroids. However, with the reverse-phase HPLC and the thin-layer chromatography systems used in the present study human calcitonin-(1-32) can be separated from a biologically inactive derivative with a single modification of an oxidized methionine residue. The predominant immunoreactive calcitonin components extracted from normal human thyroids show retention behaviour on HPLC and R f on thin-layer chromatography identical to synthetic human calcitonin-(1-32) and its sulfoxide. As expected, the former form was shown to be biologically active and the latter biologically inert. In earlier extractions with cysteine-urea-HCl according to the method of Rasmussen et al. [27] our yield of immunoreactive calcitonin was 3-6times lower than with the method described in the present paper, and 30-50% of calcitonin was in the sulfoxide form. With the presently used rapid

and mild extraction method oxidized biologically inactive calcitonin accounted for less than 10% of the extracted hormone. It seems therefore that the sulfoxide form is artefactually formed during the extractions. It remains to be demonstrated as to whether formation of the sulfoxide form in peripheral organs represents a degradative pathway of human calcitonin-(1-32) in vivo. In view of the higher biological potency of the human Val 8calcitonin-(1-32) analogue with a valine in place of the methionine as compared to the normal hormone, the conversion of human calcitonin-(132) into its biologically inactive sulfoxide form might be physiologically important [28]. On HPLC, excellent resolution of human calcitonin analogues differing in only one or two amino acids from human calcitonin-(1-32) has been achieved (not shown). As a special precaution we have added [3H]calcitonin-(1-32) and its sulfoxide as calibrating substances to the frozen thyroid glands in amounts not interfering in the radioimmunological measurements. The retention behaviour of the tritiated forms was identical to that of the non-labelled hormones. The elution volume of the radioiodinated hormone, on the other hand, was markedly different from non-labelled compound on both HPLC and gel filtration analysis. A minor component was detected on HPLC with the retention time of dimeric human calcitonin. Following incubation in 1 M ammonia [1,3] the peak coeluting with the dimer was quantitatively converted into monomer and its sulfoxide indicating that we have detected dimeric human calcitonin besides the monomeric human calcitonin-(1-32). In no instance was the monomeric [3H]calcitonin-(1-32)converted into dimer during the extractions, suggesting that dimeric calcitonin is indeed present in C-cells and is not artefactually formed during the isolation procedure [1,2]. In conclusion, the predominant calcitonin forms extracted from normal human thyroid glands correspond to monomeric and dimeric human calcitonin originally isolated from C-cell tumours [1,2]. Based on their indistinguishable behaviour on HPLC and thin-layer chromatography, human calcitonins of normal human thyroids and of medullary carcinoma of the thyroid are identical.

65

Acknowledgements W e are i n d e b t e d to P r o f e s s o r Chr. H e d i n g e r ( D e p a r t m e n t of P a t h o l o g y , U n i v e r s i t y of Z u r i c h ) a n d to P r o f e s s o r Ph. H e i t z ( D e p a r t m e n t o f Pathology, U n i v e r s i t y of Basle) for the h u m a n t h y r o i d g l a n d s . T h i s w o r k was s u p p o r t e d b y Swiss N a t i o n a l Science F o u n d a t i o n g r a n t s 3.941-0.78 a n d 3.813-0.81. T h i s research was c a r r i e d o u t in p a r t i a l f u l f i l m e n t of the r e q u i r e m e n t s for Dr. sc. nat., Federal Institute of Technology, Zurich.

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