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Blood levels of cystine aminopeptidase (oxytocinase) in patients with toxic and simple goiter
CLINICA CHIMICA ACTA
BLOOD
LEVELS
PATIENTS
RUDOLF
OF CYSTINE
WITH
KLIMEK
AMINOPEPTIDASE
(OXYTOCINASE)
IN
TOXIC AND SIMPLE GOITER*
AND
Central Endocrinological (Received
491
EDMUND
MALOLEPSZY
Laboratory **, Medical
Academy,
Cracow
(Poland)
July 8, 1968)
SUMMARY
In 62 women with goiter the cystine aminopeptidase (oxytocinase) activity was assayed by chemical method, using L-cystine-di-I-naphthylamide as substrate. Levels of enzyme show statistically significant differences between women with simple, nontoxic (16 cases) and toxic goiter (46 cases). In the former these values are within limits observed in healthy persons, and in the latter they exceed the upper limit of normal by 70%. Assay of oxytocinase may prove useful in studies on the hormonal disorders connected with the hypothalamus.
Assay of levels of endogenous hormones produced in the hypothalamic nuclei has proved especially useful in the enzymatic diagnosis of hormonal disorders. In pregnant women, a rise in endogenous oxytocin and its derivatives has been found to. be associated with a rise in levels of the inactivating enzyme, oxytocinaselva. Production of this enzyme is induced also under the influence of exogenous oxytocin. Women requiring administration of this hormone during labour have significantly lower levels of oxytocinasemia than women who deliver without the aid of hormonal intervention (refs. 2, 3). In the recently described hypothalamic post-pregnancy syndrome4 the centers producing oxytocin, vasopressin and its derivatives are damaged. Retrospective and prospective studies have shown lower levels of oxytocinase in these women during pregnancy, compared with healthy women 5. Hence, assays of oxytocinase in pregnant women may be regarded as an indicator of endogenous synthesis of cyclopeptide hormones. This paper reports results of a study designed to show whether serum oxytocinase levels differ also in women with goiter but with or without clinical or laboratory symptoms of thyroid hyperfunction. Gilbert-Dreyfus et ~1.~showed that vasopressin infusions in humans can differentiate between primary andsecondary thyroid hypofunction. Conceivably, increased production of cyclopeptide hormones in the hypothalamus can lead to symptoms of thyroid hyperfunction accompanied by a rise in blood levels of oxytocinase, which should be absent if the hormone is not produced * Presented at the Meeting of Polish Endocrinological ** Head: Dot. Dr. med. Rudolf Klimek.
Society in Cracow, on 10th April, 1968. CL&. Chim. Acta,
22 (1968) 491-495
492
KLIMEK,
MAEOLEPSZY
in excess. Conversely, primary thyroid hyperfunction may stimulate the hypothalamus and induce oxytocinase production. On these assumptions, the following study was carried out consisting in determinations of oxytocinase levels in the blood of women with simple and hyperactive goiter. The finding of different activities in these clearly differentiated clinical conditions could throw light on feedback mechanisms between the hypothalamus and the thyroid gland, and might prove useful in the future in endocrinologic enzymatic diagnosis. METHODS AND MATERIAL
Cystine aminopeptidase (oxytocinase) activity was assayed by the chemical method described by Tuppy and Nesvadba, as modified by Klimek, using L-cystinedi-ig-naphthylamide as substrate. Total serum cholesterol was assayed by the method of Bloor, basal metabolism by the method of Krogh, and 1311uptake 24 h after oral administration of 40 ,uC of labeled iodine. The methods were described in previous communications1-5. Statistical analysis was made by means of Student’s t test. The study material consisted of 62 women, including 46 with hyperactive goiter and 16 with simple nontoxic goiter. All the women had normal ESR, urine analysis, blood morphology, chest roentgenograms, blood bilirubin levels and results of liver function tests, and aminotransferase and alkaline phosphatase levels. Only patients with goiter but without disorders of other endocrine glands were admitted to the study. No sedative drugs, hormonal preparations or thyreostatic drugs were given for three days preceding the examination. The mean age of the patients with nodular hyperactive goiter (group I) was 44.1 years (S.D. 13 years, range 17-69 years), and did not differ significantly from the mean age of the patients with simple nodular nontoxic goiter, which was 38 years (S.D. 12 years, range 17-73 years). RESULTS
Results of assays of total cholesterol, basal metabolism, TABLE
and maximum la11
I
RESULTS OF LABORATORY
ASSAYS
I
Group
toxic goiter No. of cases Total
mean
cholesterol
&
mg %
range
Basal metabolism % Maximum uptake %
S.D.
mean & S.D. range lslI f
Serum oxytocinase $mol/l/min Clin. Chim. Acta,
mean S.D. range
mean & S.D. range 22
(1968) 491-495
II simple goiter
Student’s t test
46
x6
62
105.05
157.45 33.8 100.0-223.0
p > 0.05
32.4 105.0-134.0
37,53 =7.9 +7-t72 67.77 8.48 56.0-88.0 0.66 0.17 0.4-1.3
7.0 7.7 --o-+18 35.67 9.90 18.0-52.0 0.42 0.12 o.z-o.6
t = 6.5 p < 0.005
t = 10.3 p < 0.005
t = 4.8 p < 0.005
CYSTINE AMINOPEPTIDASE
IN GOITER
493
uptake after 24 h, and oxytocinasemia levels are presented in Table I, showing arithmetic means, standard deviations, ranges and values by Student’s t test. As can be seen, apart from total cholesterol, the results of the assays differed significantly in the studied groups. Besides confirmation of known differences in basal metabolism and iodine uptake in these diseases, statistically significant different levels of oxytocinasemia were found. Figs. I, 2 and 3 illustrate in graphic form the results of the assays of basal metabolism, iodine uptake and oxytocinase, and also give the upper limits of normal values. In cases of simple goiter, all the results of iodine uptake and oxytocinase levels, and basal metabolism in 81.3 o/Oof cases, lie within normal limits. In toxic goiter, on the other hand, iodine uptake in all cases, basal metabolism in 88.1%, and oxytocinasemia in 70 o/o of cases are elevated. The coefficient of correlation between all the results of oxytocinase assays and
Fig.
I.
Basal metabolism.
%Of
cases
Fine line, simple goiter; heavy line, toxic goiter.
%Of
cases
‘“t
I
16%
I III
55 %
I
II
I
I
I
0.2
I
90 %
0.6
,umol/l/min
1.4
Fig. 2. Labelled iodine uptake in simple (fine line) and toxic (heavy line) goiter. Fig. 3. Oxytocinasemia
in simple (fine line) and toxic (heavy line) goiter. Clin.
Chim. Acta,
22
(1968)
491-495
494
KLIMEK,
MALOLEPSZY
basal metabolism was r = +0.22, and between oxytocinase and maximum iodine uptake Y = +0.25. The low values do not show significant correlation between the studied parameters, DISCUSSION
Apart from direct neurosurgical intervention, the functional state of the hypothalamus can be studied only indirectly by means of dynamic tests, e.g. the test with metopiron or nicotine, or by assay in the blood of enzymes connected with the physiologic or pathologic state of the hypothalamus, as already demonstrated for was called oxytocinase and Abderhalden enzymes 2~7~8.In previous studies, attention to the diagnostic evaluation of the state of the hypothalamus on the basis of oxytocinase assays in pregnant women. In addition, slight, but statistically significant elevation of blood oxytocinase levels, up to 1.3 ,umole/l/min, was found in patients with diseases of the heart, stomach and kidneys up1o.It is now known that cyclopeptide hormones produced in the hypothalamus play an essential role in these conditions and induce production of inactivating enzymes. In this study, statistically significant differences of oxytocinasemia were found between patients with nontoxic and toxic goiter. In the latter case, secondary or primary hypothalamic disorders are beyond doubt in the light of neuroendocrinologic data. The method of enzyme analysis employed in this study does not distinguish between primary and secondary disorders, but unequivocally reveals hyperfunction of centers in the hypothalamus producing cyclopeptide hormones in states of toxicosis. This leads to further studies under fixed clinical conditions with the purpose of determining which fraction of iso-oxytocinases is changed. As is known, under physiologic conditions in pregnant women levels of iso-oxytocinases vary in different stages of pregnancyll. CONCLUSIONS
I. Levels of cystine aminopeptidase show statistically significant differences between women with nontoxic and toxic goiter. In the former, the values of oxytocinase are contained within limits observed in healthy persons, and in the latter exceed the upper limit of normal by 70 %. 2. In both groups of patients statistically significant differences were found between basal metabolism and iodine uptake, in agreement with data in the literature, confirming the selection of the material. 3. Assay of oxytocinase may prove useful in studies on the pathogenesis of hormonal disorders connected with the hypothalamus, as well as in endocrinologic diagnosis. REFERENCES I R. KLIMEK, Gynaecologia,163 (1967) 48. 2 R. KLIMEK, Ginekologia Polska, 3g (1968) 3. 3 R. KLIMEK, in A. HORSKY AND 2. STENBERA (Eds.),Intra-Uterine cerpta Medica Found., Amsterdam, 1967. 4 R. KLIMEK, Rev. Franc. Endocrinol. Clin., 8 (1967) 43. 5 R. KLIMEK, ActualitLes Endocrinologiques, Paris, 8 (1968) 200.
CZin.Chim. Acta, 22 (1968) 491-495
Dangers to the Foetus, Ex-
CYSTINE
AMINOPEPTIDASE
IN GOITER
495
6 GILBERT-DREYFUS, I. C. SAVOIE, E. BERNARD-WEIL AND J. SEBAOUN, Presse Mid., 68 (1960) 1647. 7 J. GROCHOWSKI,R.KLIMEKAND H.D~oini,CIin. Chim. Acta, 14(1966) 405. 8 R. KLIMEK,M. PIETRZYCKAAND J. GROCHOWSKI, Clin.Chim. Acta, 7(1962) 398. g A. BIENIOWA AND R. KL~MEK, Neural.,Neurochir., Psychiatr., Polska, 4 (1963) 470. IO K. CICHECKA, R. KLIMEK AND W. KWIATKOWSKI, Przeglad Lekarski, 7 (1964) 347. II R. KLIMEK, CZin.Chim. Acta, 20 (1968) 233. Clin.Chim.
Acta, 22 (1968) 491-495
BLOOD
LEVELS
PATIENTS
RUDOLF
OF CYSTINE
WITH
KLIMEK
AMINOPEPTIDASE
(OXYTOCINASE)
IN
TOXIC AND SIMPLE GOITER*
AND
Central Endocrinological (Received
491
EDMUND
MALOLEPSZY
Laboratory **, Medical
Academy,
Cracow
(Poland)
July 8, 1968)
SUMMARY
In 62 women with goiter the cystine aminopeptidase (oxytocinase) activity was assayed by chemical method, using L-cystine-di-I-naphthylamide as substrate. Levels of enzyme show statistically significant differences between women with simple, nontoxic (16 cases) and toxic goiter (46 cases). In the former these values are within limits observed in healthy persons, and in the latter they exceed the upper limit of normal by 70%. Assay of oxytocinase may prove useful in studies on the hormonal disorders connected with the hypothalamus.
Assay of levels of endogenous hormones produced in the hypothalamic nuclei has proved especially useful in the enzymatic diagnosis of hormonal disorders. In pregnant women, a rise in endogenous oxytocin and its derivatives has been found to. be associated with a rise in levels of the inactivating enzyme, oxytocinaselva. Production of this enzyme is induced also under the influence of exogenous oxytocin. Women requiring administration of this hormone during labour have significantly lower levels of oxytocinasemia than women who deliver without the aid of hormonal intervention (refs. 2, 3). In the recently described hypothalamic post-pregnancy syndrome4 the centers producing oxytocin, vasopressin and its derivatives are damaged. Retrospective and prospective studies have shown lower levels of oxytocinase in these women during pregnancy, compared with healthy women 5. Hence, assays of oxytocinase in pregnant women may be regarded as an indicator of endogenous synthesis of cyclopeptide hormones. This paper reports results of a study designed to show whether serum oxytocinase levels differ also in women with goiter but with or without clinical or laboratory symptoms of thyroid hyperfunction. Gilbert-Dreyfus et ~1.~showed that vasopressin infusions in humans can differentiate between primary andsecondary thyroid hypofunction. Conceivably, increased production of cyclopeptide hormones in the hypothalamus can lead to symptoms of thyroid hyperfunction accompanied by a rise in blood levels of oxytocinase, which should be absent if the hormone is not produced * Presented at the Meeting of Polish Endocrinological ** Head: Dot. Dr. med. Rudolf Klimek.
Society in Cracow, on 10th April, 1968. CL&. Chim. Acta,
22 (1968) 491-495
492
KLIMEK,
MAEOLEPSZY
in excess. Conversely, primary thyroid hyperfunction may stimulate the hypothalamus and induce oxytocinase production. On these assumptions, the following study was carried out consisting in determinations of oxytocinase levels in the blood of women with simple and hyperactive goiter. The finding of different activities in these clearly differentiated clinical conditions could throw light on feedback mechanisms between the hypothalamus and the thyroid gland, and might prove useful in the future in endocrinologic enzymatic diagnosis. METHODS AND MATERIAL
Cystine aminopeptidase (oxytocinase) activity was assayed by the chemical method described by Tuppy and Nesvadba, as modified by Klimek, using L-cystinedi-ig-naphthylamide as substrate. Total serum cholesterol was assayed by the method of Bloor, basal metabolism by the method of Krogh, and 1311uptake 24 h after oral administration of 40 ,uC of labeled iodine. The methods were described in previous communications1-5. Statistical analysis was made by means of Student’s t test. The study material consisted of 62 women, including 46 with hyperactive goiter and 16 with simple nontoxic goiter. All the women had normal ESR, urine analysis, blood morphology, chest roentgenograms, blood bilirubin levels and results of liver function tests, and aminotransferase and alkaline phosphatase levels. Only patients with goiter but without disorders of other endocrine glands were admitted to the study. No sedative drugs, hormonal preparations or thyreostatic drugs were given for three days preceding the examination. The mean age of the patients with nodular hyperactive goiter (group I) was 44.1 years (S.D. 13 years, range 17-69 years), and did not differ significantly from the mean age of the patients with simple nodular nontoxic goiter, which was 38 years (S.D. 12 years, range 17-73 years). RESULTS
Results of assays of total cholesterol, basal metabolism, TABLE
and maximum la11
I
RESULTS OF LABORATORY
ASSAYS
I
Group
toxic goiter No. of cases Total
mean
cholesterol
&
mg %
range
Basal metabolism % Maximum uptake %
S.D.
mean & S.D. range lslI f
Serum oxytocinase $mol/l/min Clin. Chim. Acta,
mean S.D. range
mean & S.D. range 22
(1968) 491-495
II simple goiter
Student’s t test
46
x6
62
105.05
157.45 33.8 100.0-223.0
p > 0.05
32.4 105.0-134.0
37,53 =7.9 +7-t72 67.77 8.48 56.0-88.0 0.66 0.17 0.4-1.3
7.0 7.7 --o-+18 35.67 9.90 18.0-52.0 0.42 0.12 o.z-o.6
t = 6.5 p < 0.005
t = 10.3 p < 0.005
t = 4.8 p < 0.005
CYSTINE AMINOPEPTIDASE
IN GOITER
493
uptake after 24 h, and oxytocinasemia levels are presented in Table I, showing arithmetic means, standard deviations, ranges and values by Student’s t test. As can be seen, apart from total cholesterol, the results of the assays differed significantly in the studied groups. Besides confirmation of known differences in basal metabolism and iodine uptake in these diseases, statistically significant different levels of oxytocinasemia were found. Figs. I, 2 and 3 illustrate in graphic form the results of the assays of basal metabolism, iodine uptake and oxytocinase, and also give the upper limits of normal values. In cases of simple goiter, all the results of iodine uptake and oxytocinase levels, and basal metabolism in 81.3 o/Oof cases, lie within normal limits. In toxic goiter, on the other hand, iodine uptake in all cases, basal metabolism in 88.1%, and oxytocinasemia in 70 o/o of cases are elevated. The coefficient of correlation between all the results of oxytocinase assays and
Fig.
I.
Basal metabolism.
%Of
cases
Fine line, simple goiter; heavy line, toxic goiter.
%Of
cases
‘“t
I
16%
I III
55 %
I
II
I
I
I
0.2
I
90 %
0.6
,umol/l/min
1.4
Fig. 2. Labelled iodine uptake in simple (fine line) and toxic (heavy line) goiter. Fig. 3. Oxytocinasemia
in simple (fine line) and toxic (heavy line) goiter. Clin.
Chim. Acta,
22
(1968)
491-495
494
KLIMEK,
MALOLEPSZY
basal metabolism was r = +0.22, and between oxytocinase and maximum iodine uptake Y = +0.25. The low values do not show significant correlation between the studied parameters, DISCUSSION
Apart from direct neurosurgical intervention, the functional state of the hypothalamus can be studied only indirectly by means of dynamic tests, e.g. the test with metopiron or nicotine, or by assay in the blood of enzymes connected with the physiologic or pathologic state of the hypothalamus, as already demonstrated for was called oxytocinase and Abderhalden enzymes 2~7~8.In previous studies, attention to the diagnostic evaluation of the state of the hypothalamus on the basis of oxytocinase assays in pregnant women. In addition, slight, but statistically significant elevation of blood oxytocinase levels, up to 1.3 ,umole/l/min, was found in patients with diseases of the heart, stomach and kidneys up1o.It is now known that cyclopeptide hormones produced in the hypothalamus play an essential role in these conditions and induce production of inactivating enzymes. In this study, statistically significant differences of oxytocinasemia were found between patients with nontoxic and toxic goiter. In the latter case, secondary or primary hypothalamic disorders are beyond doubt in the light of neuroendocrinologic data. The method of enzyme analysis employed in this study does not distinguish between primary and secondary disorders, but unequivocally reveals hyperfunction of centers in the hypothalamus producing cyclopeptide hormones in states of toxicosis. This leads to further studies under fixed clinical conditions with the purpose of determining which fraction of iso-oxytocinases is changed. As is known, under physiologic conditions in pregnant women levels of iso-oxytocinases vary in different stages of pregnancyll. CONCLUSIONS
I. Levels of cystine aminopeptidase show statistically significant differences between women with nontoxic and toxic goiter. In the former, the values of oxytocinase are contained within limits observed in healthy persons, and in the latter exceed the upper limit of normal by 70 %. 2. In both groups of patients statistically significant differences were found between basal metabolism and iodine uptake, in agreement with data in the literature, confirming the selection of the material. 3. Assay of oxytocinase may prove useful in studies on the pathogenesis of hormonal disorders connected with the hypothalamus, as well as in endocrinologic diagnosis. REFERENCES I R. KLIMEK, Gynaecologia,163 (1967) 48. 2 R. KLIMEK, Ginekologia Polska, 3g (1968) 3. 3 R. KLIMEK, in A. HORSKY AND 2. STENBERA (Eds.),Intra-Uterine cerpta Medica Found., Amsterdam, 1967. 4 R. KLIMEK, Rev. Franc. Endocrinol. Clin., 8 (1967) 43. 5 R. KLIMEK, ActualitLes Endocrinologiques, Paris, 8 (1968) 200.
CZin.Chim. Acta, 22 (1968) 491-495
Dangers to the Foetus, Ex-
CYSTINE
AMINOPEPTIDASE
IN GOITER
495
6 GILBERT-DREYFUS, I. C. SAVOIE, E. BERNARD-WEIL AND J. SEBAOUN, Presse Mid., 68 (1960) 1647. 7 J. GROCHOWSKI,R.KLIMEKAND H.D~oini,CIin. Chim. Acta, 14(1966) 405. 8 R. KLIMEK,M. PIETRZYCKAAND J. GROCHOWSKI, Clin.Chim. Acta, 7(1962) 398. g A. BIENIOWA AND R. KL~MEK, Neural.,Neurochir., Psychiatr., Polska, 4 (1963) 470. IO K. CICHECKA, R. KLIMEK AND W. KWIATKOWSKI, Przeglad Lekarski, 7 (1964) 347. II R. KLIMEK, CZin.Chim. Acta, 20 (1968) 233. Clin.Chim.
Acta, 22 (1968) 491-495