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Increased acylphosphatase levels in erythrocytes from hyperthyroid patients
CIinica Chimica Actu, 183 (1989) 351-358
351
Elsevier
CCA 04518
Increased acylphosphatase levels in erythrocytes from hyperthyroid patients Paolo Nassi ‘, Gianfranco Liguri I, Chiara Nediani I, Niccol6 Taddei ‘, Patrizia Piccinni ‘, Donatella Degl’Innocenti I, Riccardo G. Gheri 2 and Giampietro Ramponi 1 Departimento di Scienze Biochimiche, University of Florence, Florence and ’ Servizio di Endocrinologia U.S.L. 1OD press0 Dipartimento di Fisiopatologia Clinica, University of Florence, Florence (Italy) (Received
10 July 1988; revision
Key words: Hyperthyroidism;
received 24 April 1989; accepted
Human
erythrocytes;
26 April 1989)
Acylphosphatase
level
Summary Acylphosphatase activity and content were measured in erythrocytes from hyperthyroid patients and healthy controls. In addition, the soluble enzymes glucose-6phosphate dehydrogenase, hexokinase, and the membrane bound (Naf + K+)ATPase and Ca2+-ATPase were assayed. Our results confirmed previous studies indicating a decrease of (Na+ + Kf)-ATPase and an increase of Ca2+-ATPase activity in hyperthyroid erythrocytes. While glucose-6-phosphate dehydrogenase was not significantly changed, hexokinase and acylphosphatase activities were signifiBoth activities and content of cantly higher in the hyperthyroid group. acylphosphatase returned to normal levels in erythrocytes from treated patients, when they were euthyroid. These findings suggest that an excess of thyroid hormones may stimulate acylphosphatase biosynthesis in erythroid cells and indicate a potential clinical usefulness of this enzyme in hyperthyroidism.
Introduction Acylphosphatase (EC 3.6.1.7) is a cytosolic animal tissues which acts on compounds with a natural acylphosphates hydrolyzed by the enzyme [l], carbamoylphosphate [2], succinoylphosphate
hydrolase widely distributed in carboxylphosphate bond. Among are 3-phosphoglyceroylphosphate [3], as well as the phosphorylated
Correspondence and requests for reprints to: Dr. P. Nassi, Universita di Firenze Viale Morgagni 50, 50134 Florence, Italy.
0009-8981/89/$03.50
0 1989 Elsevier Science Publishers
Dipartimento
B.V. (Biomedical
di Science
Division)
Biockimiche,
352
intermediate of sarcoplasmic reticulum Ca’ -ATPase [4]. We have reported the structural and fuctional properties of acylphosphatase that was purified to homogeneity from the skeletal muscle of various vertebrate species, including man [5], and from human erythrocytes [6]. Acylphosphatase from human skeletal muscle and that from human erythrocytes are isoenzymes, each consisting of the same number of aminoacids but differing significantly in primary structure: they exhibit similar substrate specificity and kinetic properties, except that the erythrocyte isoenzyme appears to have a higher catalytic power. As regards physiological function. since all the substrates of acylphosphatase are ‘high energy’ phosphorylated compounds, this enzyme seems to be mainly involved in energy metabolism and it is conceivable that changes in its levels may provide a biochemical basis for the regulation of energy expenditure. In this connection, it has been postulated that, by hydrolyzing 3-phosphoglyceroylphosphate, acylphosphatase may hasten glycolysis at the expense of ATP formation with an ‘uncoupling’ effect similar to that elicited experimentally by arsenate [7]. Furthermore. the hydrolysis of Cal+-ATPase phosphorylated intermediate suggests a possible involvement in the control of the efficiency of this active transport system. In agreement with the latter hypothesis we have observed, in muscle biopsies from patients with Duchenne muscular dystrophy, significant decreases for both Ca’ ‘-ATPase and acylphosphatase, as well as a high positive correlation between the levels of these enzymatic activities [S]. Human red blood cell (RBC) membrane Ca’+-ATPase is stimulated in vitro by physiological concentrations of thyroid hormone [9]. Clinical confirmation of this finding has been provided by studies demonstrating that membrane Ca*+-ATPase activity levels are increased and decreased. respectively, in RBCs from hyperthyroid and hypothyroid patients compared to those in RBCs from healthy subjects [lo]. These observations, together with the above mentioned properties of acylphosphatase led us to suppose that this enzyme might be an endogenous marker of thyroid action. The present study deals with acylphosphatase activity and content in RBCs of hyperthyroid patients and normal controls. The behaviour of acylphosphatase was compared to that of other soluble enzymes such as hexokinase and glucose-6phosphate dehydrogenase. Erythrocyte membrane Ca”-ATPase and (Nat + K ’ )ATPase activities were also determined. Experimental Materials Bovine serum albumin, enzymes (glucose-6-phosphate dehydrogenase, lactate dehydrogenase, pyruvate kinase), coenzymes and substrates (NADP, NADH, ATP, glucose, glucose-6-phosphate, phosphoenolpyruvate) were obtained from Boehringer, Mannheim, FRG. Ouabain was from E. Merck, Darmstadt, FRG. All other chemicals were the best commercially available. Benzoylphosphate was synthesized as described by Camici et al [ll]. Antibodies against human erythrocyte acylphosphatase were obtained and purified according to Berti et al. [12].
353
Patients The measurements were made in the RBCs from 14 untreated hyperthyroid patients (12 women and 2 men, median of ages 50 yr, range 44-64) and from 9 healthy individuals (7 women and 2 men, median of ages 44 yr, range 41-49). All individuals had fasted overnight before venepuncture. The diagnosis of hyperthyroidism was based on the patient’s history, clinical features and concentrations in plasma of thyroxine, tri-iodothyronine and TSH. RBC acylphosphatase levels were restudied in 8 hyperthyroid patients after 7 or more months of treatment with methimazole, when they were euthyroid based on clinical evaluation and the results of hormone tests. All the treated hyperthyroid patients had detectable TSH levels. Such patients were taking antithyroid medication at the time of retesting; methimazole, however, added in vitro at a concentration achieved clinically (10-4-10-5 mol/l), did not alter acylphosphatase activity of RBCs. Hormone assays Total thyroxine (T4) and tri-iodothyronine (Tj) were assayed by means of the automated procedure ARIA/HT (Becton Dickinson, Salt Lake City, UT, USA); free thyroxine (FT,) and tri-iodothyronine FT,) by kit methods (FT, kit, FT3 kit by Sclavo S.P.A., Cinisello Balsamo, Milan, Italy). TSH was determined by a sensitive @ immunoassay system (Allegro HS-TSH by Nichols Institute Diagnostic, San Juan Capistrano, CA, USA). Erythrocyte membrane preparation All the following operations were carried out at O-4” C. 10 ml of heparinized fresh blood were centrifuged at 2000 x g for 10 min. After plasma and buffy coat were removed, red cells were resuspended in 154 mmol/l NaCl and centrifuged twice at 2000 x g for 10 min. Lysis was achieved by adding 10 vol of 5 mmol/l Tris-HCl buffer, pH 7.4, containing 0.1 mmol/l EDTA. After magnetic stirring for 15 min the sample was centrifuged at 50000 X g for 30 min. The supernatant was used for soluble enzyme and hemoglobin measurements. The pellet was washed twice in 5 mmol/l Tris- HCl buffer, pH 7.4, containing 17 mmol/l NaCl, then twice in the same buffer without NaCl and finally centrifuged at 50000 X g for 15 min. Membranes so obtained were resuspended in 5 vol of 10 mmol/l Tris-HCl buffer, pH 7.4, and homogenized by a glass/glass pestle homogenizer. These suspensions were used for ATPase activity measurements. Soluble enzyme assays Acylphosphatase content in hemolysate was determined by a non-competitive enzyme-linked immunosorbent assay (ELISA) carried out using polyclonal antierythrocyte acylphosphatase antibodies [13]. Acylphosphatase activity was measured by a continuous optical method with benzoylphosphate as substrate, based on the difference in absorbance at 283 nm between benzoylphosphate and benzoate [14]. The unit of activity is defined as the amount of enzyme that liberates 1 pmol of
354
benzoate/min at pH 5.3. Enzymatic activities for hexokinase (EC‘ 2.7.1.1) and glucose-6-phosphate dehydrogenase (EC 1.1.1.49) were determined by continuous optical methods at 340 nm [15,16]. All the assays were performed at 25°C in a mode1 550s Perkin Elmer spectrophotometer, and activities were expressed as units (pm01 of substrate transformed/mm) per g hemoglobin (Hb). The ferricyanide-~ cyanide reagent as used to measure the hemoglobin concentration in hemolysates.
A TPase assays (Nat + K+)- and Ca’+-ATPase were determined using a coupled optical method at 340 nm and 37°C. The incubation mixture contained 25 mmol/l Tris-HC1 buffer, pH 7.4, 1 mmol/l ATP, 25 mmol/l KCl. 1 mmol/l MgCl,, 75 mmol/l NaCI, 0.5 mmol/l phosphoenolpyruvate, 0.2 mmol/l NADH, 2000 U/l of lactate dehydrogenase, 2000 U/l of pyruvate kinase and 200 ~1 of membrane suspension in a final volume of 2 ml. (Na+ + K’)-ATPase activity was calculated as the difference in ATP hydrolysis in the absence and in the presence of 1 mmol/l ouabain [17]. Ca”+-ATPase activity was calculated as the difference in hydrolysis of ATP with and without 0.15 mmol/l Ca’+ [IO]. ATPase activity was expressed as units (pmol of ATP hydrolyzed/min) per g of membrane protein. Protein concentration in the membrane suspension was determined with the biuret method of Beisenhertz et al. [18] using bovine serum albumin as the standard.
Statistical analysis The statistical significance of the data was assessed by Student’s t test. When the variance between two means were significantly different, an approximate t test was performed [19]. The correlation between two variables was assessed by the productmoment correlation coefficient r.
TABLE
I
Hormonal
assays of untreated.
Study group (reference
ranges)
Untreated (n =14)
hyperthyroid
Controls (n=9) Treated hyperthyroid (euthyroid) (n = 8) Results
are presented
treated
hyperthyroid
patients
and healthy
controls
T4 Ir&‘dl (5.5-12)
T,
FT, pg/ml (6.6-16)
FT,
TSH
w’dl (90-200)
pg/ml (2.X-5.6)
pU/ml (0.5-4.6)
19.2 f 4.8
305 +41
31.8 + 6.5
10.1 -_t1.9
i 0.04
7.6k1.3
138+1x
11.4i2.7
4.1 i0.7
2.7kO.8
9.7k1.6
151k15
12.15 2.5
4.4 i 0.8
2.2 kO.7
as mean f SD
355 10
l-
A
C
B o 6-
0.6
-
0.4
-
0 2-
-
1
-
1
2
1
2
Fig. 1. Activity (units per g hemoglobin) of acylphosphatase (A), hexokinase (B) and glucose-h-phosphate dehydrogenase (C) in erythrocytes of hyperthyroid patients (1) and normal controls (2). Results are presented as mean f SD. Differences were statistically significant for acylphosphatase (P < 0.001) and hexokinase (P < O.Ol), not significant for glucose-6-phosphate dehydrogenase.
Results Table I shows plasma hormone levels of hyperthyroid patients, healthy controls and of thyrotoxic patients after treatment, when they were euthyroid. Figure 1 presents the levels of soluble enzymes measured in RBCs of hyperthyroid patients and normal controls. Glucose-6-phosphate dehydrogenase, which we examined as a representative enzyme of hexose monophosphate shunt, was not significantly modified. Hexokinase, the rate limiting enzyme of erythrocyte glycoly-
A
4-
B
3-
2-
2
Fig. 2. Activity (units per g membrane protein) of (Na+ + K’ )-ATPase (A) and Ca”-ATPase (B) in erythrocytes of hyperthyroid patients (1) and healthy controls (2). Results are presented as mean+ SD. Differences were statistically significant (P -c0.001) for both activities.
356 TABLE
II
Acylphosphatahe normal controls
activity
and content
Study group
Untreated hyperthyroid (n =14) Controls (n=9) Treated hyperthyroid (euthyroid (n = 8) Results are presented as mean + SD. a P -c 0.001 vs. control levels. ’ P < 0.001 vs. untreated hyperthyroid
III erythrocytes
of untreated.
treated
hyperthyroid
Activity
Content
(U/g
(pu’g Hb)
Hh)
149.53 F 43.39 .’ 55.16+1x.41 61.7X-tl3.04
2.X3+0.12
patients
and
j’
2.09iO.17 h
2.14i0.15
h
levels.
sis [20] was significantly higher in the hyperthyroid group (mean + SD = 0.79 t_ 0.21 U/g Hb; P < 0.01) compared to healthy controls (0.54 k 0.16). Erythrocyte acylphosphatase activity in the hyperthyroid patients (149.53 + 43.39 U/g Hb) was about three times as high as in the controls (55.16 + 18.41); its increase therefore was much more pronounced than that of hexokinase, besides being statistically significant (P < 0.001). Figure 2 depicts (Nat -t K+)-ATPase and Ca’+- ATPase activities measured in erythrocyte membranes. (Na+ + Kf)-ATPase activity of hyperthyroid membranes was significantly lower (0.38 _t 0.11 U/g membrane protein; P < 0.001) than that of control membranes (0.68 & 0.06). Ca’+-ATPase activity, on the contrary. was significantly higher (2.49 + 0.48; P < 0.001) in hyperthyroid membranes compared to control values (1.41 + 0.16). Table II presents data about acylphosphatase activity and content observed in the erythrocytes of untreated, treated hyperthyroid patients and healthy controls. Both these parameters increased in hyperthyroid RBCs and normalized after treatment when the patients returned to the euthyroid state.
Discussion
Our findings about membrane bound enzymes are consistent with the results of other investigators and, for (Na+ + K+)-ATPase, they may be mainly ascribed to a decrease of the number of sodium pump sites [22]. The effect on Cal+-ATPase is debated; according to Dube et al. [lo], although thyroid hormone in vitro acts directly upon erythrocyte membrane, it is also possible that the action of endogenous iodothyronines on this enzyme is mediated by nuclear effects on erythroid precursors. As regards the soluble enzymes, the change of hexokinase activity agrees with previously reported increases of other glycolytic enzymes such as glyceraldehyde-3-
351
phosphate dehydrogenase and lactate dehydrogenase [21] and suggests a stimulation of this pathway aimed at satisfying increased energy needs of RBCs. At any rate, among the enzymes were examined, acylphosphatase showed the most dramatic change and the increase of its activity appears as one of the highest We verified if ever reported for erythrocyte enzymes in hyperthyroidism. acylphosphatase activity in the RBCs of hyperthyroid patients correlated with any of the biochemical indices of thyroid status, notably with plasma thyroid hormone levels. A significant correlation (r = 0.81) was found with FT, concentration. Correlation coefficients with other hormones were: r = 0.67 for T4, r = 0.74 for T3, r = 0.78 for FT,. As for the mechanisms responsible for these effects, we have previously observed that thyroxine inhibits acylphosphatase activity in vitro [5]. This fact, together with what is known about how thyroid hormones generally act, leads us to consider that a direct action on the enzyme is unlikely and to suggest that increased acylphosphatase levels are mainly due to an increased biosynthesis in the erythroid cells. The higher acylphosphatase content that we found in RBCs of hyperthyroid patients and the return to normal values for both activity and content of this enzyme in the treated euthyroid subjects may support this interpretation. The marked increase of erythrocyte acylphosphatase levels that we observed in the present study represent a novel finding in the biochemical abnormalities associated with hyperthyroidism. We think it is worthwhile, therefore, to probe more deeply into the changes of RBCs acylphosphatase in dysthyroid states and the mechanisms undergoing such effects. Our results suggest that further study into the potential clinical usefulness of this enzyme as a marker of the peripheral effects of thyroid hormones would be of value. Acknowledgements This study was supported by grants from the Consiglio and the Minister0 della Pubblica Istruzione (Fondi 60%).
Nazionale
delle Ricerche
References 1 Ramponi G, Treves C. Guerritore A. Hydrolitic activity of muscle acylphosphatase on 3-phosphoglyceroylphosphate. Experientia 1967;23:10-19. 2 Ramponi G, Melani F, Guerritore A. Azione dell’ acilfosfatasi sul carbamilfosfato. G Biochim 1961;10:189-196. 3 Berti A. Stefani M, Liguri G. Camici G, Manao G, Ramponi G. Acylphosphatase action on dicarboxylic acylphosphates. Ital. J B&hem 1977;26:377-378. 4 Stefani M, Liguri G, Berti A, Nassi P, Ramponi G. Hydrolysis by horse muscle acylphosphatase of (CaZC + Mg2+ )-ATPase phosphorylated intermediate. Arch Biochem Biophys 1981;208:37-41. 5 Nassi P, Liguri G, Landi N, et al. Acylphosphatase from human skeletal muscle: purification. some properties and levels in normal and myopatic muscles. Biochem Med 1985;34:166-175. 6 Liguri G, Camici G, Manao G, et al. A new acylphosphatase isoenzyme from human erythrocytes: purification characterization and primary structure. Biochemistry 1986;25:8089-8094. 7 Harary 1. The hydrolysis of I,3 diphosphoglyceric acid by acyl phosphatase. Biochim Biophys Acta 1957:434-436.
358 8 Landi N, Nassi P, Liguri G, Bobbi S. Sbrilli C, Marconi G. Sarcoplasmic reticulum Ca”-ATPase and acylphosphatase activities in muscle biopsies from patients with Duchenne muscular dystrophy. Clin Chim Acta 1986;158:245-251. 9 Davis FB, Davis PJ, Blas SD. Role of calmodulin in thyroid hormone stimulation in vitro of human erythrocyte Ca’+ -ATPase activity. J Clin Invest 1983;71:579-586. 10 Dube MP, Davis FB, Davis PJ, Schoenl M. Blas SD. Effects of hyperthyrotdism and hypothyroidism on human red blood cell Ca’+-ATPase activity. J Clin Endocrinol Metab 1986:62:253-257. 11 Camici G, Manao G, Cappugi G. Ramponi G. A new synthesis of benzoylphosphate. a substrate for acylphosphatase assay. Experientia 1976;22:705. 12 Berti A, Liguri G, Stefani M. Nassi P, Ramponi G. Purification of horse muscle acylphosphatase antibodies by affinity chromatography. Physiol Chem Phys 1982;14:307-311, 13 Liguri G, Nassi P, Degl’lnnocenti D, et al. Acylphosphatase levels of human erythrocytes durmg cell ageing. Mech Ageing Dev 1987;39:59-67. 14 Ramponi G, Treves C. Guerritore A. Continuous optical assay of acylphosphatase with benzoylphosphate as substrate. Experientia 1966:22:705. 15 Beutler E. Red cell metabolism. 3rd ed. Orlando: Grune & Stratton. Inc.. 1984;38-40. 16 Beutler E. Red cell metabolism, 3rd ed. Orlando: Grune & Stratton, Inc.. 1984;68-71. 17 Cole CH, Waddel RW. Alterations in intracellular sodium concentration and ouabaine-sensitive ATPase in erythrocytes from hyperthyroid patients. J Clin Endocrinol Metab 1976;42:1056-1063. 18 Beisenhertz G, Boltze HG, Bucher Th. et al. Diphosphofructose aldolase, Phosphoglyceraldehyde-dehydrogenase, Milchsaure-dehydrogenase und Pyruvat- kinase aus Kaninchen muskulatur in einem Arbeitsgang. Z Naturforsch 1953;8b:555-557. 19 Sokal RR, Rohlf J. Biometry. 2nd ed. New York: WH Freeman, 1981~41 l-412. 20 Magnani M. Stocchi V. Bossu M, Dacha M, Fornaini G. Decay pattern of rabbit erythrocyte hexokinase in cell ageing. Mech Ageing Dev 1979;11:209-217. 21 Swaminathan R, Segall NH. Chapman C. Morgan DB. Red blood cell composition in thyroid disease. Lancet 1976;ii:1382-1385. 22 Rubython EJ, Cumberbatch M. Morgan DB. Changes in the number and activity of sodium pumps in erythrocytes from patients with hyperthyroidism. Clin Sci 1983:64:44-447.
351
Elsevier
CCA 04518
Increased acylphosphatase levels in erythrocytes from hyperthyroid patients Paolo Nassi ‘, Gianfranco Liguri I, Chiara Nediani I, Niccol6 Taddei ‘, Patrizia Piccinni ‘, Donatella Degl’Innocenti I, Riccardo G. Gheri 2 and Giampietro Ramponi 1 Departimento di Scienze Biochimiche, University of Florence, Florence and ’ Servizio di Endocrinologia U.S.L. 1OD press0 Dipartimento di Fisiopatologia Clinica, University of Florence, Florence (Italy) (Received
10 July 1988; revision
Key words: Hyperthyroidism;
received 24 April 1989; accepted
Human
erythrocytes;
26 April 1989)
Acylphosphatase
level
Summary Acylphosphatase activity and content were measured in erythrocytes from hyperthyroid patients and healthy controls. In addition, the soluble enzymes glucose-6phosphate dehydrogenase, hexokinase, and the membrane bound (Naf + K+)ATPase and Ca2+-ATPase were assayed. Our results confirmed previous studies indicating a decrease of (Na+ + Kf)-ATPase and an increase of Ca2+-ATPase activity in hyperthyroid erythrocytes. While glucose-6-phosphate dehydrogenase was not significantly changed, hexokinase and acylphosphatase activities were signifiBoth activities and content of cantly higher in the hyperthyroid group. acylphosphatase returned to normal levels in erythrocytes from treated patients, when they were euthyroid. These findings suggest that an excess of thyroid hormones may stimulate acylphosphatase biosynthesis in erythroid cells and indicate a potential clinical usefulness of this enzyme in hyperthyroidism.
Introduction Acylphosphatase (EC 3.6.1.7) is a cytosolic animal tissues which acts on compounds with a natural acylphosphates hydrolyzed by the enzyme [l], carbamoylphosphate [2], succinoylphosphate
hydrolase widely distributed in carboxylphosphate bond. Among are 3-phosphoglyceroylphosphate [3], as well as the phosphorylated
Correspondence and requests for reprints to: Dr. P. Nassi, Universita di Firenze Viale Morgagni 50, 50134 Florence, Italy.
0009-8981/89/$03.50
0 1989 Elsevier Science Publishers
Dipartimento
B.V. (Biomedical
di Science
Division)
Biockimiche,
352
intermediate of sarcoplasmic reticulum Ca’ -ATPase [4]. We have reported the structural and fuctional properties of acylphosphatase that was purified to homogeneity from the skeletal muscle of various vertebrate species, including man [5], and from human erythrocytes [6]. Acylphosphatase from human skeletal muscle and that from human erythrocytes are isoenzymes, each consisting of the same number of aminoacids but differing significantly in primary structure: they exhibit similar substrate specificity and kinetic properties, except that the erythrocyte isoenzyme appears to have a higher catalytic power. As regards physiological function. since all the substrates of acylphosphatase are ‘high energy’ phosphorylated compounds, this enzyme seems to be mainly involved in energy metabolism and it is conceivable that changes in its levels may provide a biochemical basis for the regulation of energy expenditure. In this connection, it has been postulated that, by hydrolyzing 3-phosphoglyceroylphosphate, acylphosphatase may hasten glycolysis at the expense of ATP formation with an ‘uncoupling’ effect similar to that elicited experimentally by arsenate [7]. Furthermore. the hydrolysis of Cal+-ATPase phosphorylated intermediate suggests a possible involvement in the control of the efficiency of this active transport system. In agreement with the latter hypothesis we have observed, in muscle biopsies from patients with Duchenne muscular dystrophy, significant decreases for both Ca’ ‘-ATPase and acylphosphatase, as well as a high positive correlation between the levels of these enzymatic activities [S]. Human red blood cell (RBC) membrane Ca’+-ATPase is stimulated in vitro by physiological concentrations of thyroid hormone [9]. Clinical confirmation of this finding has been provided by studies demonstrating that membrane Ca*+-ATPase activity levels are increased and decreased. respectively, in RBCs from hyperthyroid and hypothyroid patients compared to those in RBCs from healthy subjects [lo]. These observations, together with the above mentioned properties of acylphosphatase led us to suppose that this enzyme might be an endogenous marker of thyroid action. The present study deals with acylphosphatase activity and content in RBCs of hyperthyroid patients and normal controls. The behaviour of acylphosphatase was compared to that of other soluble enzymes such as hexokinase and glucose-6phosphate dehydrogenase. Erythrocyte membrane Ca”-ATPase and (Nat + K ’ )ATPase activities were also determined. Experimental Materials Bovine serum albumin, enzymes (glucose-6-phosphate dehydrogenase, lactate dehydrogenase, pyruvate kinase), coenzymes and substrates (NADP, NADH, ATP, glucose, glucose-6-phosphate, phosphoenolpyruvate) were obtained from Boehringer, Mannheim, FRG. Ouabain was from E. Merck, Darmstadt, FRG. All other chemicals were the best commercially available. Benzoylphosphate was synthesized as described by Camici et al [ll]. Antibodies against human erythrocyte acylphosphatase were obtained and purified according to Berti et al. [12].
353
Patients The measurements were made in the RBCs from 14 untreated hyperthyroid patients (12 women and 2 men, median of ages 50 yr, range 44-64) and from 9 healthy individuals (7 women and 2 men, median of ages 44 yr, range 41-49). All individuals had fasted overnight before venepuncture. The diagnosis of hyperthyroidism was based on the patient’s history, clinical features and concentrations in plasma of thyroxine, tri-iodothyronine and TSH. RBC acylphosphatase levels were restudied in 8 hyperthyroid patients after 7 or more months of treatment with methimazole, when they were euthyroid based on clinical evaluation and the results of hormone tests. All the treated hyperthyroid patients had detectable TSH levels. Such patients were taking antithyroid medication at the time of retesting; methimazole, however, added in vitro at a concentration achieved clinically (10-4-10-5 mol/l), did not alter acylphosphatase activity of RBCs. Hormone assays Total thyroxine (T4) and tri-iodothyronine (Tj) were assayed by means of the automated procedure ARIA/HT (Becton Dickinson, Salt Lake City, UT, USA); free thyroxine (FT,) and tri-iodothyronine FT,) by kit methods (FT, kit, FT3 kit by Sclavo S.P.A., Cinisello Balsamo, Milan, Italy). TSH was determined by a sensitive @ immunoassay system (Allegro HS-TSH by Nichols Institute Diagnostic, San Juan Capistrano, CA, USA). Erythrocyte membrane preparation All the following operations were carried out at O-4” C. 10 ml of heparinized fresh blood were centrifuged at 2000 x g for 10 min. After plasma and buffy coat were removed, red cells were resuspended in 154 mmol/l NaCl and centrifuged twice at 2000 x g for 10 min. Lysis was achieved by adding 10 vol of 5 mmol/l Tris-HCl buffer, pH 7.4, containing 0.1 mmol/l EDTA. After magnetic stirring for 15 min the sample was centrifuged at 50000 X g for 30 min. The supernatant was used for soluble enzyme and hemoglobin measurements. The pellet was washed twice in 5 mmol/l Tris- HCl buffer, pH 7.4, containing 17 mmol/l NaCl, then twice in the same buffer without NaCl and finally centrifuged at 50000 X g for 15 min. Membranes so obtained were resuspended in 5 vol of 10 mmol/l Tris-HCl buffer, pH 7.4, and homogenized by a glass/glass pestle homogenizer. These suspensions were used for ATPase activity measurements. Soluble enzyme assays Acylphosphatase content in hemolysate was determined by a non-competitive enzyme-linked immunosorbent assay (ELISA) carried out using polyclonal antierythrocyte acylphosphatase antibodies [13]. Acylphosphatase activity was measured by a continuous optical method with benzoylphosphate as substrate, based on the difference in absorbance at 283 nm between benzoylphosphate and benzoate [14]. The unit of activity is defined as the amount of enzyme that liberates 1 pmol of
354
benzoate/min at pH 5.3. Enzymatic activities for hexokinase (EC‘ 2.7.1.1) and glucose-6-phosphate dehydrogenase (EC 1.1.1.49) were determined by continuous optical methods at 340 nm [15,16]. All the assays were performed at 25°C in a mode1 550s Perkin Elmer spectrophotometer, and activities were expressed as units (pm01 of substrate transformed/mm) per g hemoglobin (Hb). The ferricyanide-~ cyanide reagent as used to measure the hemoglobin concentration in hemolysates.
A TPase assays (Nat + K+)- and Ca’+-ATPase were determined using a coupled optical method at 340 nm and 37°C. The incubation mixture contained 25 mmol/l Tris-HC1 buffer, pH 7.4, 1 mmol/l ATP, 25 mmol/l KCl. 1 mmol/l MgCl,, 75 mmol/l NaCI, 0.5 mmol/l phosphoenolpyruvate, 0.2 mmol/l NADH, 2000 U/l of lactate dehydrogenase, 2000 U/l of pyruvate kinase and 200 ~1 of membrane suspension in a final volume of 2 ml. (Na+ + K’)-ATPase activity was calculated as the difference in ATP hydrolysis in the absence and in the presence of 1 mmol/l ouabain [17]. Ca”+-ATPase activity was calculated as the difference in hydrolysis of ATP with and without 0.15 mmol/l Ca’+ [IO]. ATPase activity was expressed as units (pmol of ATP hydrolyzed/min) per g of membrane protein. Protein concentration in the membrane suspension was determined with the biuret method of Beisenhertz et al. [18] using bovine serum albumin as the standard.
Statistical analysis The statistical significance of the data was assessed by Student’s t test. When the variance between two means were significantly different, an approximate t test was performed [19]. The correlation between two variables was assessed by the productmoment correlation coefficient r.
TABLE
I
Hormonal
assays of untreated.
Study group (reference
ranges)
Untreated (n =14)
hyperthyroid
Controls (n=9) Treated hyperthyroid (euthyroid) (n = 8) Results
are presented
treated
hyperthyroid
patients
and healthy
controls
T4 Ir&‘dl (5.5-12)
T,
FT, pg/ml (6.6-16)
FT,
TSH
w’dl (90-200)
pg/ml (2.X-5.6)
pU/ml (0.5-4.6)
19.2 f 4.8
305 +41
31.8 + 6.5
10.1 -_t1.9
i 0.04
7.6k1.3
138+1x
11.4i2.7
4.1 i0.7
2.7kO.8
9.7k1.6
151k15
12.15 2.5
4.4 i 0.8
2.2 kO.7
as mean f SD
355 10
l-
A
C
B o 6-
0.6
-
0.4
-
0 2-
-
1
-
1
2
1
2
Fig. 1. Activity (units per g hemoglobin) of acylphosphatase (A), hexokinase (B) and glucose-h-phosphate dehydrogenase (C) in erythrocytes of hyperthyroid patients (1) and normal controls (2). Results are presented as mean f SD. Differences were statistically significant for acylphosphatase (P < 0.001) and hexokinase (P < O.Ol), not significant for glucose-6-phosphate dehydrogenase.
Results Table I shows plasma hormone levels of hyperthyroid patients, healthy controls and of thyrotoxic patients after treatment, when they were euthyroid. Figure 1 presents the levels of soluble enzymes measured in RBCs of hyperthyroid patients and normal controls. Glucose-6-phosphate dehydrogenase, which we examined as a representative enzyme of hexose monophosphate shunt, was not significantly modified. Hexokinase, the rate limiting enzyme of erythrocyte glycoly-
A
4-
B
3-
2-
2
Fig. 2. Activity (units per g membrane protein) of (Na+ + K’ )-ATPase (A) and Ca”-ATPase (B) in erythrocytes of hyperthyroid patients (1) and healthy controls (2). Results are presented as mean+ SD. Differences were statistically significant (P -c0.001) for both activities.
356 TABLE
II
Acylphosphatahe normal controls
activity
and content
Study group
Untreated hyperthyroid (n =14) Controls (n=9) Treated hyperthyroid (euthyroid (n = 8) Results are presented as mean + SD. a P -c 0.001 vs. control levels. ’ P < 0.001 vs. untreated hyperthyroid
III erythrocytes
of untreated.
treated
hyperthyroid
Activity
Content
(U/g
(pu’g Hb)
Hh)
149.53 F 43.39 .’ 55.16+1x.41 61.7X-tl3.04
2.X3+0.12
patients
and
j’
2.09iO.17 h
2.14i0.15
h
levels.
sis [20] was significantly higher in the hyperthyroid group (mean + SD = 0.79 t_ 0.21 U/g Hb; P < 0.01) compared to healthy controls (0.54 k 0.16). Erythrocyte acylphosphatase activity in the hyperthyroid patients (149.53 + 43.39 U/g Hb) was about three times as high as in the controls (55.16 + 18.41); its increase therefore was much more pronounced than that of hexokinase, besides being statistically significant (P < 0.001). Figure 2 depicts (Nat -t K+)-ATPase and Ca’+- ATPase activities measured in erythrocyte membranes. (Na+ + Kf)-ATPase activity of hyperthyroid membranes was significantly lower (0.38 _t 0.11 U/g membrane protein; P < 0.001) than that of control membranes (0.68 & 0.06). Ca’+-ATPase activity, on the contrary. was significantly higher (2.49 + 0.48; P < 0.001) in hyperthyroid membranes compared to control values (1.41 + 0.16). Table II presents data about acylphosphatase activity and content observed in the erythrocytes of untreated, treated hyperthyroid patients and healthy controls. Both these parameters increased in hyperthyroid RBCs and normalized after treatment when the patients returned to the euthyroid state.
Discussion
Our findings about membrane bound enzymes are consistent with the results of other investigators and, for (Na+ + K+)-ATPase, they may be mainly ascribed to a decrease of the number of sodium pump sites [22]. The effect on Cal+-ATPase is debated; according to Dube et al. [lo], although thyroid hormone in vitro acts directly upon erythrocyte membrane, it is also possible that the action of endogenous iodothyronines on this enzyme is mediated by nuclear effects on erythroid precursors. As regards the soluble enzymes, the change of hexokinase activity agrees with previously reported increases of other glycolytic enzymes such as glyceraldehyde-3-
351
phosphate dehydrogenase and lactate dehydrogenase [21] and suggests a stimulation of this pathway aimed at satisfying increased energy needs of RBCs. At any rate, among the enzymes were examined, acylphosphatase showed the most dramatic change and the increase of its activity appears as one of the highest We verified if ever reported for erythrocyte enzymes in hyperthyroidism. acylphosphatase activity in the RBCs of hyperthyroid patients correlated with any of the biochemical indices of thyroid status, notably with plasma thyroid hormone levels. A significant correlation (r = 0.81) was found with FT, concentration. Correlation coefficients with other hormones were: r = 0.67 for T4, r = 0.74 for T3, r = 0.78 for FT,. As for the mechanisms responsible for these effects, we have previously observed that thyroxine inhibits acylphosphatase activity in vitro [5]. This fact, together with what is known about how thyroid hormones generally act, leads us to consider that a direct action on the enzyme is unlikely and to suggest that increased acylphosphatase levels are mainly due to an increased biosynthesis in the erythroid cells. The higher acylphosphatase content that we found in RBCs of hyperthyroid patients and the return to normal values for both activity and content of this enzyme in the treated euthyroid subjects may support this interpretation. The marked increase of erythrocyte acylphosphatase levels that we observed in the present study represent a novel finding in the biochemical abnormalities associated with hyperthyroidism. We think it is worthwhile, therefore, to probe more deeply into the changes of RBCs acylphosphatase in dysthyroid states and the mechanisms undergoing such effects. Our results suggest that further study into the potential clinical usefulness of this enzyme as a marker of the peripheral effects of thyroid hormones would be of value. Acknowledgements This study was supported by grants from the Consiglio and the Minister0 della Pubblica Istruzione (Fondi 60%).
Nazionale
delle Ricerche
References 1 Ramponi G, Treves C. Guerritore A. Hydrolitic activity of muscle acylphosphatase on 3-phosphoglyceroylphosphate. Experientia 1967;23:10-19. 2 Ramponi G, Melani F, Guerritore A. Azione dell’ acilfosfatasi sul carbamilfosfato. G Biochim 1961;10:189-196. 3 Berti A. Stefani M, Liguri G. Camici G, Manao G, Ramponi G. Acylphosphatase action on dicarboxylic acylphosphates. Ital. J B&hem 1977;26:377-378. 4 Stefani M, Liguri G, Berti A, Nassi P, Ramponi G. Hydrolysis by horse muscle acylphosphatase of (CaZC + Mg2+ )-ATPase phosphorylated intermediate. Arch Biochem Biophys 1981;208:37-41. 5 Nassi P, Liguri G, Landi N, et al. Acylphosphatase from human skeletal muscle: purification. some properties and levels in normal and myopatic muscles. Biochem Med 1985;34:166-175. 6 Liguri G, Camici G, Manao G, et al. A new acylphosphatase isoenzyme from human erythrocytes: purification characterization and primary structure. Biochemistry 1986;25:8089-8094. 7 Harary 1. The hydrolysis of I,3 diphosphoglyceric acid by acyl phosphatase. Biochim Biophys Acta 1957:434-436.
358 8 Landi N, Nassi P, Liguri G, Bobbi S. Sbrilli C, Marconi G. Sarcoplasmic reticulum Ca”-ATPase and acylphosphatase activities in muscle biopsies from patients with Duchenne muscular dystrophy. Clin Chim Acta 1986;158:245-251. 9 Davis FB, Davis PJ, Blas SD. Role of calmodulin in thyroid hormone stimulation in vitro of human erythrocyte Ca’+ -ATPase activity. J Clin Invest 1983;71:579-586. 10 Dube MP, Davis FB, Davis PJ, Schoenl M. Blas SD. Effects of hyperthyrotdism and hypothyroidism on human red blood cell Ca’+-ATPase activity. J Clin Endocrinol Metab 1986:62:253-257. 11 Camici G, Manao G, Cappugi G. Ramponi G. A new synthesis of benzoylphosphate. a substrate for acylphosphatase assay. Experientia 1976;22:705. 12 Berti A, Liguri G, Stefani M. Nassi P, Ramponi G. Purification of horse muscle acylphosphatase antibodies by affinity chromatography. Physiol Chem Phys 1982;14:307-311, 13 Liguri G, Nassi P, Degl’lnnocenti D, et al. Acylphosphatase levels of human erythrocytes durmg cell ageing. Mech Ageing Dev 1987;39:59-67. 14 Ramponi G, Treves C. Guerritore A. Continuous optical assay of acylphosphatase with benzoylphosphate as substrate. Experientia 1966:22:705. 15 Beutler E. Red cell metabolism. 3rd ed. Orlando: Grune & Stratton. Inc.. 1984;38-40. 16 Beutler E. Red cell metabolism, 3rd ed. Orlando: Grune & Stratton, Inc.. 1984;68-71. 17 Cole CH, Waddel RW. Alterations in intracellular sodium concentration and ouabaine-sensitive ATPase in erythrocytes from hyperthyroid patients. J Clin Endocrinol Metab 1976;42:1056-1063. 18 Beisenhertz G, Boltze HG, Bucher Th. et al. Diphosphofructose aldolase, Phosphoglyceraldehyde-dehydrogenase, Milchsaure-dehydrogenase und Pyruvat- kinase aus Kaninchen muskulatur in einem Arbeitsgang. Z Naturforsch 1953;8b:555-557. 19 Sokal RR, Rohlf J. Biometry. 2nd ed. New York: WH Freeman, 1981~41 l-412. 20 Magnani M. Stocchi V. Bossu M, Dacha M, Fornaini G. Decay pattern of rabbit erythrocyte hexokinase in cell ageing. Mech Ageing Dev 1979;11:209-217. 21 Swaminathan R, Segall NH. Chapman C. Morgan DB. Red blood cell composition in thyroid disease. Lancet 1976;ii:1382-1385. 22 Rubython EJ, Cumberbatch M. Morgan DB. Changes in the number and activity of sodium pumps in erythrocytes from patients with hyperthyroidism. Clin Sci 1983:64:44-447.