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Immunoheterogeneity of serum calcitonin in heroin addicts
Drug and Alcohol Dependence, 22 (19881165Elsevier
Scientific
Publishers
Ireland
167
165
Ltd.
Immunoheterogeneity
of serum calcitonin
W. Spagnolli, G. DeVenuto, M. Mattarei,
in heroin addicts
A. DalPiaz, 0. Merza and R. Miori
Divisione di Medicina 1 and aLaboratorio di Biochimica ed Ematologiu, Istituti Ospedalieri (Received
July llth,
Trento (Italy1
19881
Serum calcitonin (CT) concentrations were determined in 96 heroin addicts. CT levels were assayed by radioimmunological method employing two antisera, one vs. the 17 - 32 fraction (IMN) of the human CT, the other vs. the ll- 32 fraction (B71. In heroin addicts the CT mean values were significantly higher (P < 0.0011 than in normal subjects (141 f 16.0 and 292 2 21.7 pgl ml with IMN-antiserum and with B7-antiserum, respectively, in heroin addicts; 64 f 7.8 and 189 f 21.7 pglml in controls). Serum CT levels assayed with B7-antiserum were notably higher (P < 0.001) both in controls and in heroin addicts, Our results draw attention to the heterogeneity of high ICT values found in heroin addicts. Key words: heroin addicts; calcitonin;
immunoreactive
calcitonin-like
material
Introduction
Patients and Methods
Evidence has been accumulated suggesting a possible role of calcitonin (CT) in the neuroendocrine regulation [l - 31. A close relationship between endogenous opioids and CT has already been reported. Indeed, experimental and clinical studies demonstrated a central morphine-like analgesic effect of CT which is not antagonized by naloxone [4,5]. It has been found that CT, administered in pharmacological doses, significantly increases the plasma concentration of /I-endorphin [6]. Furthermore, recent data show that chronic consumption of opiates increase CT serum levels of heroin addicts [7,8]. It is difficult to evaluate the latter observation, owing to problems in measuring this hormone in the blood. The radioimmunoassay is not specific enough for the monomeric form of CT, a 32 aminoacid peptide, because of circulating immunoreactive CT-like materials (ICT) [9,10]. Employing two antisera directed towards different fractions of the sequence of human CT, we measured serum CT in a group of heroin addicts.
Subjects were 96 heroin addicts (69 men), aged 17- 35 years, who had been abusers for more than 2 years. Thirty-two of them declared a daily i.v. intake of street heroin < 500 mg; the others referred larger doses. Urine samples collected at the moment of withdrawal of blood gave positive results for free morphine in all patients (Taxi Lab., Marion Laboratories, U.S.A.). Serum transaminases and/or alkaline phosphatase were slightly elevated in 53 subjects none of them showed liver or kidney failure. We also examined 33 normal subjects matched for age and sex, without history of opioid or psychoactive drug intake. Serum ICT was assayed by the radioimmunological method (RIA) employing two antisera, one vs. the 17-32 fraction of the sequence of human CT (Immunonuclear-Nuclear Corporation, Stillwater U.S.A., IMN), the other vs. the 11-32 fraction (‘home made’ reagent, B71 [ll]. In the normal subjects our ICT values for the latter anti-serum agree with those referred by
0376-8716/88/$03.50 0 1988 Elsevier Printed and Published in Ireland
Scientific
Publishers
Ireland Ltd.
B7-antiserum
1
m
Cont’ols (? 331
P I
ESU
0 001
7
I
k?rcn 3OaCtS
r 25)
ICT serum values in heroin addicts and in normal Fig. 1. controls, assayed with two different antisera.
Thashjian [12] and by Heynen and Franchimott [13]. Routine tests were performed employing common automatized methods. Significance of differences was calculated using the Student’s t-test for paired and unpaired data. Results Serum ICT concentrations in heroin addicts were significantly higher than in controls (P < 0.001) both with IMN and B7 antisera. Serum ICT mean concentrations (& S.E.) in heroin addicts were 141 f 16.0 pglml and 292 f 21.7 pglml for antierum IMN and antiserum B7, respectively. Corresponding values in controls were 64 f 7.8 pglml and 189 f 13.6 pglml, respectively (Fig. 11. Serum ICT levels assayed with B7 antiserum were notably higher P < 0.0011 both in normal subjects and in heroin addicts. We did not find any significant difference of the serum levels of calcium and phosphate between the two groups examined.
nificantly higher than in normals with antisera utilized. These data were obtained from a large group of subjects and confirm those reported by others [7,8]. We also found that the ICT concentrations assayed with B7 antiserum were significantly higher, both in heroin addicts and in controls, than ICT values obtained with IMN antiserum. the differences among investigators in the serum calcitonin values are well known. The reasons for this are probably related to the heterogeneity of circulating calcitonin and the differences in the relative binding affinity of antisera in RIA [9,101. Our results suggest that the antisera we used probably detected not only the biologically active monomeric calcitonin, but also forms of different molecular weight such as precursors, subunits, metabolites and carrier proteins. It is, therefore, not to be excluded that in serum of heroin addicts there are biologically inactive forms of ICT, although Tagliaro et al., in a small group of their subjects found an increase of the monomeric form of calcitonin employing pre-RIA purifying techniques [14]. The high ICT concentration found in heroin addicts is not easy to explain. Opiates themselves might be involved in the increase of ICT [14]. This explanation might be corroborated by the observation that serum ICT decreased after heroin withdrawal [8]. In conclusion, our results obtained with two different antisera highlight the heterogeneity of the high ICT serum levels found in heroin addicts. References 1 2
3 4
Discussion Serum ICT levels in heroin addicts were sig-
5
R. Isaac et al., J. Endocrinol. Metab., 50 (198011011. R. Gaggi et al., Mechanisms underlying anorexia induced by calcitonin peptides, in: A. Pecile (Ed.). Calcitonin 84, Excerpta Medica, Milan, 1984, p. 261. L.J. Deftos et al., J. Clin. Endocrinol. Metab.. 47 (1978) 457. C.E. Fiore et al., Alcune attivita extrasceletriche della calcitonina: effetto antalgico e neuromodulatore, in: C. Gennari and G. Segre (Eds.1, The effects of calcitonin in man, Masson, Milano, 1983, p. 291. P.C. Braga et al., Life Sci., 22 (19781971.
167 6
C. Gennari, Calcitonin and bone metastases of cancer, in: A. Pecile (Ed.), Calcitonin 80, Excerpta Medica, Amsterdam, 1981, p. 277. 7 F. Tagliaro et al., J. Endocrinol. Invest., 7 119841331. 8 W. Spagnolli et al.,Drug Alcohol Depend., 20 (19871143. 9 L.J. Deftos et al., J. Clin. Endocrinol. Metab., 40 119751 409. 10 D. Ziliotto et al., Calcitonin serum levels in normal and
11 12 13 14
pathological conditions, in: A. Pecile (Ed.). Calcitonin 84, Exeerpta Medica, Milan, 1984, p. 89. F. Tagliaro et al., G. It. Chim. Clin., 7121(1982)147. A.H. Tashjian et al., N. Engl. J. Med., 283 (1970)890. G. Heynen and P. Franehimont, Eur. J. Clin. Invest., 4 (19741213. F. Tagliaro et al., Drug Alcohol Depend., 16 (1985)181.
Scientific
Publishers
Ireland
167
165
Ltd.
Immunoheterogeneity
of serum calcitonin
W. Spagnolli, G. DeVenuto, M. Mattarei,
in heroin addicts
A. DalPiaz, 0. Merza and R. Miori
Divisione di Medicina 1 and aLaboratorio di Biochimica ed Ematologiu, Istituti Ospedalieri (Received
July llth,
Trento (Italy1
19881
Serum calcitonin (CT) concentrations were determined in 96 heroin addicts. CT levels were assayed by radioimmunological method employing two antisera, one vs. the 17 - 32 fraction (IMN) of the human CT, the other vs. the ll- 32 fraction (B71. In heroin addicts the CT mean values were significantly higher (P < 0.0011 than in normal subjects (141 f 16.0 and 292 2 21.7 pgl ml with IMN-antiserum and with B7-antiserum, respectively, in heroin addicts; 64 f 7.8 and 189 f 21.7 pglml in controls). Serum CT levels assayed with B7-antiserum were notably higher (P < 0.001) both in controls and in heroin addicts, Our results draw attention to the heterogeneity of high ICT values found in heroin addicts. Key words: heroin addicts; calcitonin;
immunoreactive
calcitonin-like
material
Introduction
Patients and Methods
Evidence has been accumulated suggesting a possible role of calcitonin (CT) in the neuroendocrine regulation [l - 31. A close relationship between endogenous opioids and CT has already been reported. Indeed, experimental and clinical studies demonstrated a central morphine-like analgesic effect of CT which is not antagonized by naloxone [4,5]. It has been found that CT, administered in pharmacological doses, significantly increases the plasma concentration of /I-endorphin [6]. Furthermore, recent data show that chronic consumption of opiates increase CT serum levels of heroin addicts [7,8]. It is difficult to evaluate the latter observation, owing to problems in measuring this hormone in the blood. The radioimmunoassay is not specific enough for the monomeric form of CT, a 32 aminoacid peptide, because of circulating immunoreactive CT-like materials (ICT) [9,10]. Employing two antisera directed towards different fractions of the sequence of human CT, we measured serum CT in a group of heroin addicts.
Subjects were 96 heroin addicts (69 men), aged 17- 35 years, who had been abusers for more than 2 years. Thirty-two of them declared a daily i.v. intake of street heroin < 500 mg; the others referred larger doses. Urine samples collected at the moment of withdrawal of blood gave positive results for free morphine in all patients (Taxi Lab., Marion Laboratories, U.S.A.). Serum transaminases and/or alkaline phosphatase were slightly elevated in 53 subjects none of them showed liver or kidney failure. We also examined 33 normal subjects matched for age and sex, without history of opioid or psychoactive drug intake. Serum ICT was assayed by the radioimmunological method (RIA) employing two antisera, one vs. the 17-32 fraction of the sequence of human CT (Immunonuclear-Nuclear Corporation, Stillwater U.S.A., IMN), the other vs. the 11-32 fraction (‘home made’ reagent, B71 [ll]. In the normal subjects our ICT values for the latter anti-serum agree with those referred by
0376-8716/88/$03.50 0 1988 Elsevier Printed and Published in Ireland
Scientific
Publishers
Ireland Ltd.
B7-antiserum
1
m
Cont’ols (? 331
P I
ESU
0 001
7
I
k?rcn 3OaCtS
r 25)
ICT serum values in heroin addicts and in normal Fig. 1. controls, assayed with two different antisera.
Thashjian [12] and by Heynen and Franchimott [13]. Routine tests were performed employing common automatized methods. Significance of differences was calculated using the Student’s t-test for paired and unpaired data. Results Serum ICT concentrations in heroin addicts were significantly higher than in controls (P < 0.001) both with IMN and B7 antisera. Serum ICT mean concentrations (& S.E.) in heroin addicts were 141 f 16.0 pglml and 292 f 21.7 pglml for antierum IMN and antiserum B7, respectively. Corresponding values in controls were 64 f 7.8 pglml and 189 f 13.6 pglml, respectively (Fig. 11. Serum ICT levels assayed with B7 antiserum were notably higher P < 0.0011 both in normal subjects and in heroin addicts. We did not find any significant difference of the serum levels of calcium and phosphate between the two groups examined.
nificantly higher than in normals with antisera utilized. These data were obtained from a large group of subjects and confirm those reported by others [7,8]. We also found that the ICT concentrations assayed with B7 antiserum were significantly higher, both in heroin addicts and in controls, than ICT values obtained with IMN antiserum. the differences among investigators in the serum calcitonin values are well known. The reasons for this are probably related to the heterogeneity of circulating calcitonin and the differences in the relative binding affinity of antisera in RIA [9,101. Our results suggest that the antisera we used probably detected not only the biologically active monomeric calcitonin, but also forms of different molecular weight such as precursors, subunits, metabolites and carrier proteins. It is, therefore, not to be excluded that in serum of heroin addicts there are biologically inactive forms of ICT, although Tagliaro et al., in a small group of their subjects found an increase of the monomeric form of calcitonin employing pre-RIA purifying techniques [14]. The high ICT concentration found in heroin addicts is not easy to explain. Opiates themselves might be involved in the increase of ICT [14]. This explanation might be corroborated by the observation that serum ICT decreased after heroin withdrawal [8]. In conclusion, our results obtained with two different antisera highlight the heterogeneity of the high ICT serum levels found in heroin addicts. References 1 2
3 4
Discussion Serum ICT levels in heroin addicts were sig-
5
R. Isaac et al., J. Endocrinol. Metab., 50 (198011011. R. Gaggi et al., Mechanisms underlying anorexia induced by calcitonin peptides, in: A. Pecile (Ed.). Calcitonin 84, Excerpta Medica, Milan, 1984, p. 261. L.J. Deftos et al., J. Clin. Endocrinol. Metab.. 47 (1978) 457. C.E. Fiore et al., Alcune attivita extrasceletriche della calcitonina: effetto antalgico e neuromodulatore, in: C. Gennari and G. Segre (Eds.1, The effects of calcitonin in man, Masson, Milano, 1983, p. 291. P.C. Braga et al., Life Sci., 22 (19781971.
167 6
C. Gennari, Calcitonin and bone metastases of cancer, in: A. Pecile (Ed.), Calcitonin 80, Excerpta Medica, Amsterdam, 1981, p. 277. 7 F. Tagliaro et al., J. Endocrinol. Invest., 7 119841331. 8 W. Spagnolli et al.,Drug Alcohol Depend., 20 (19871143. 9 L.J. Deftos et al., J. Clin. Endocrinol. Metab., 40 119751 409. 10 D. Ziliotto et al., Calcitonin serum levels in normal and
11 12 13 14
pathological conditions, in: A. Pecile (Ed.). Calcitonin 84, Exeerpta Medica, Milan, 1984, p. 89. F. Tagliaro et al., G. It. Chim. Clin., 7121(1982)147. A.H. Tashjian et al., N. Engl. J. Med., 283 (1970)890. G. Heynen and P. Franehimont, Eur. J. Clin. Invest., 4 (19741213. F. Tagliaro et al., Drug Alcohol Depend., 16 (1985)181.