Clinical Science and Molecular Medicine (1975) 48, 289-295.
Human platelet monoamine oxidase activity in iron-deficiency anaemia MOUSSA B. H. YOUDIM, H. F. WOODS, B. MITCHELL, D. G. GRAHAME-SMITH AND SHEILA CALLENDER (1) MRC Clinical Pharmacology Unit, and
(1)
Nuffield Department of Clinical Medicine, Radcliffe Infirmary, Oxford
(Received 15 August 1974)
Summary
amine oxidase activity in vitro when compared with normal animals (Symes,Sourkes, Youdim, Gregoriadis & Birnbaum, 1969). Recently these results have been confirmed by measuring total monoamine oxidase activity in vivo (Symes, Missala & Sourkes, 1971). These investigations also showed that restoring iron to the diet of iron-deficient rats restored monoamine oxidase activity to normal 1 week later. Because of the apparent importance of iron in determining monoamine oxidase activity we have studied platelet monoamine oxidase activity in patients with undoubted iron deficiency. Platelet monoamine oxidase activity has been determined in patients with psychiatric diseases with the implication that it may reflect brain monoamine oxidase activity (Murphy & Wyatt, 1972; Meltzer & Stahl, 1974) and thus it is important to determine factors such as iron status, which may influence monoamine oxidase activity in the platelet.
1. The activity of monoamine oxidase, when assayed with four substrates, was significantly lowered in platelets prepared from the blood of patients with iron-deficiencyanaemia. 2. Treatment with oral iron preparations restored platelet monoamine oxidase activity to normal in those patients whose serum iron concentrations also returned to normal. 3. Platelet monoamine oxidase activity remained low if treatment failed to restore serum iron concentration to within normal limits. Key words: monoamine oxidase, platelets, irondeficiency anaemia. Introduction The enzyme monoamine oxidase [monoamine-O, oxidoreductase (deaminating), EC 1.4.3.4] oxidatively deaminates noradrenaline, dopamine, 5hydroxytryptamine, phenylethylamine, tryptamine and certain primary and secondary amines occurring naturally in the central nervous system and other _tissues of mammals (Youdim, 1975). The purified enzyme contains sulphydryl groups; covalently bound flavin-adenine dinucleotide is a cofactor. Iron is present in the liver enzyme purified from rat and bovine tissues (Youdim, 1975; Youdim & Sourkes, 1966; Oreland, 1971). Previous studies have shown that rats made irondeficient have significantly lower liver tissue mono-
Patients and methods Patients
In the first part of the investigation platelet monoamine oxidase activity was determined in sixteen patients with undoubted iron-deficiency anaemia, who were selected by use of the following criteria: (a) haemoglobin concentration less than 10 g/dl; (b) mean corpuscular volume (MCV) less than 76 pm 3 ; (c) serum iron below 14'3 pmol/l for male patients and less than 10·7 pmol/l for female patients; (d) decreased saturation of iron-binding capacity. The control group of subjects consisted of
Correspondence: Dr M. B. H. Youdirn, MRC Clinical Pharmacology Unit, Radcliffe Infirmary, Oxford OX2 6HE. D
289
Moussa B. H. Youdim et af.
290
TABLE 1. Clinical details of the subjects studied The results are expressed as mean values±sEM. Normal (20) Female No. Mean age (years) Haemoglobin (g/dl) Mean corpuscular volume (pm 3 ) Serum iron (pmol/l) Total iron-binding capacity (pmol/l)
Platelet count (l03/mm 3)
out-patients and members of hospital staff. The clinical information concerning these two groups is shown in Table 1. In the second part of the investigation a further group of patients attending the anaemia clinic were examined to determine platelet monoamine oxidase activity in subjects with a wide range of serum iron concentrations. Methods Preparation of platelets. Blood (20 ml) was obtained by venepuncture and collected into 0·129 mol/l sodium citrate solution. Platelet-rich citrated plasma was obtained by centrifuging the citrated blood at 200 g for 10 min. This plasma was further centrifuged at 2000 g for 10 min; the platelet plug thus obtained was washed three times with 0·3 mol/l sucrose and then resuspended in 0·3 mcl/l sucrose by treatment for 20 s with low-energy sonic oscillation. All procedures were carried out at 4°C on blood samples obtained between 09.30 hours and 12.00 hours. Assay of monoamine oxidase activity. Monoamine oxidase activity in the platelet suspension was determined with [1-14C]dopamine,5-hydroxy-[l-14C]tryptamine, [1-14C]phenylethylarnine and [l_1 4C]tyramine
as substrates (Robinson, Lovenberg, Keiser & Sjoerdsma, 1968). This method measures the 14C_ labelled metabolites formed during oxidative deamination and activity isexpressed as d.p.m.jmg of platelet protein in 30 min incubation with the substrate. All substrates were used at a final concentration of 1 mmol/l. The protein contents of platelet suspensions
Male
17 3 53·7 53-3 13-9± 0'40 92'5± 1'80 22-6± 2'23 6H± 3'5 328 ±20'4
Iron-deficient (16) Female
Male
15 1 47·3 67·0 8'79± 0'24 64'5 ± 1'36 3-8 ± 0'32 87-9 ± 4'41 361 ±41'8
were determined by the method of Lowry, Rosebrough, Farr & Randall (1951) with human serum albumin as standard. The intra-assay variability is less than 5%. Blood counts, serum iron and total iron-binding capacity. Blood counts, serum iron and total ironbinding capacity were determined in a specimen of venous blood obtained at the same time as that used for the preparation of platelets. Blood counts were performed with a Coulter counter. Serum iron and total iron-binding capacity were measured by the method of Stookey (1970) and Ramsay (1957). Statistical methods. The significance of differences between mean values was assessed with Student's r-test,
Results Platelet monoamine oxidase activity in iron-deficiency anaemia
Monoamine oxidase activities in platelets obtained from patients with iron-deficiency anaemia and from normal subjects are shown in Table 2. It can be seen that there is a decrease of monoamine oxidase activity in platelets from iron-deficient patients with all four substrates tested. These changes are significant (P < 0'001) when tyramine, dopamine and 5hydroxytryptamine are the substrates and are of borderline significance (P < 0,05) when phenylethylamine is the substrate. The platelet monoamine oxidase activities for normal subjects listed in Table 2 are equivalent to 6'87, 7'52, 9·10 and 3·87 nmol of substrate deamin-
Monoamine oxidase activity in iron deficiency
ated per mg of protein in 30 min for tyramine, dopamine, 5-hydroxytryptamine and phenylethylamine respectively. These results are similar to those reported by other investigators (Murphy & Wyatt, 1972; Meltzer & Stahl, 1974; Collins & Sandler, 1971). Where possible, four substrates were studied because of the existence of multiple forms of the enzyme each having a different substrate specificity (Youdim, 1972; Youdim& Collins, 1975).
291
Effect of treatment with oral iron preparations
The effect of treatment with oral iron preparations on haematological response and platelet monoamine oxidase activity was studied in a group of seven irondeficient patients (five females and two males), who received ferrous sulphate (200 mg t.d.s.) for 38 days; post-treatment samples were obtained 4 days after cessation of therapy. Serum iron concentrations at
TABLE 2. Effect of iron deficiency on monoamine oxidase activity in human platelets towards various substrates Monoamine oxidase activities are expressed as d.p.m. of deaminated products formed per rng of platelet protein per 30 min and are mean values±sEM with the number of subjects in parentheses. Platelet monoamine oxidase activity
Tyramine
Dopamine
5-Hydroxytryptamine
Phenylethylamine
Normal subjects
2256±260 (20)
2508± 190 (19)
3033± 314 (20)
1289± 199 (20)
Iron-deficient patients
1386±206 (16)
1385± 162 (16)
1448±207 (16)
818± 180 (16)
Decrease (:Yo) p
39
45
52
37
Human platelet monoamine oxidase activity in iron-deficiency anaemia MOUSSA B. H. YOUDIM, H. F. WOODS, B. MITCHELL, D. G. GRAHAME-SMITH AND SHEILA CALLENDER (1) MRC Clinical Pharmacology Unit, and
(1)
Nuffield Department of Clinical Medicine, Radcliffe Infirmary, Oxford
(Received 15 August 1974)
Summary
amine oxidase activity in vitro when compared with normal animals (Symes,Sourkes, Youdim, Gregoriadis & Birnbaum, 1969). Recently these results have been confirmed by measuring total monoamine oxidase activity in vivo (Symes, Missala & Sourkes, 1971). These investigations also showed that restoring iron to the diet of iron-deficient rats restored monoamine oxidase activity to normal 1 week later. Because of the apparent importance of iron in determining monoamine oxidase activity we have studied platelet monoamine oxidase activity in patients with undoubted iron deficiency. Platelet monoamine oxidase activity has been determined in patients with psychiatric diseases with the implication that it may reflect brain monoamine oxidase activity (Murphy & Wyatt, 1972; Meltzer & Stahl, 1974) and thus it is important to determine factors such as iron status, which may influence monoamine oxidase activity in the platelet.
1. The activity of monoamine oxidase, when assayed with four substrates, was significantly lowered in platelets prepared from the blood of patients with iron-deficiencyanaemia. 2. Treatment with oral iron preparations restored platelet monoamine oxidase activity to normal in those patients whose serum iron concentrations also returned to normal. 3. Platelet monoamine oxidase activity remained low if treatment failed to restore serum iron concentration to within normal limits. Key words: monoamine oxidase, platelets, irondeficiency anaemia. Introduction The enzyme monoamine oxidase [monoamine-O, oxidoreductase (deaminating), EC 1.4.3.4] oxidatively deaminates noradrenaline, dopamine, 5hydroxytryptamine, phenylethylamine, tryptamine and certain primary and secondary amines occurring naturally in the central nervous system and other _tissues of mammals (Youdim, 1975). The purified enzyme contains sulphydryl groups; covalently bound flavin-adenine dinucleotide is a cofactor. Iron is present in the liver enzyme purified from rat and bovine tissues (Youdim, 1975; Youdim & Sourkes, 1966; Oreland, 1971). Previous studies have shown that rats made irondeficient have significantly lower liver tissue mono-
Patients and methods Patients
In the first part of the investigation platelet monoamine oxidase activity was determined in sixteen patients with undoubted iron-deficiency anaemia, who were selected by use of the following criteria: (a) haemoglobin concentration less than 10 g/dl; (b) mean corpuscular volume (MCV) less than 76 pm 3 ; (c) serum iron below 14'3 pmol/l for male patients and less than 10·7 pmol/l for female patients; (d) decreased saturation of iron-binding capacity. The control group of subjects consisted of
Correspondence: Dr M. B. H. Youdirn, MRC Clinical Pharmacology Unit, Radcliffe Infirmary, Oxford OX2 6HE. D
289
Moussa B. H. Youdim et af.
290
TABLE 1. Clinical details of the subjects studied The results are expressed as mean values±sEM. Normal (20) Female No. Mean age (years) Haemoglobin (g/dl) Mean corpuscular volume (pm 3 ) Serum iron (pmol/l) Total iron-binding capacity (pmol/l)
Platelet count (l03/mm 3)
out-patients and members of hospital staff. The clinical information concerning these two groups is shown in Table 1. In the second part of the investigation a further group of patients attending the anaemia clinic were examined to determine platelet monoamine oxidase activity in subjects with a wide range of serum iron concentrations. Methods Preparation of platelets. Blood (20 ml) was obtained by venepuncture and collected into 0·129 mol/l sodium citrate solution. Platelet-rich citrated plasma was obtained by centrifuging the citrated blood at 200 g for 10 min. This plasma was further centrifuged at 2000 g for 10 min; the platelet plug thus obtained was washed three times with 0·3 mol/l sucrose and then resuspended in 0·3 mcl/l sucrose by treatment for 20 s with low-energy sonic oscillation. All procedures were carried out at 4°C on blood samples obtained between 09.30 hours and 12.00 hours. Assay of monoamine oxidase activity. Monoamine oxidase activity in the platelet suspension was determined with [1-14C]dopamine,5-hydroxy-[l-14C]tryptamine, [1-14C]phenylethylarnine and [l_1 4C]tyramine
as substrates (Robinson, Lovenberg, Keiser & Sjoerdsma, 1968). This method measures the 14C_ labelled metabolites formed during oxidative deamination and activity isexpressed as d.p.m.jmg of platelet protein in 30 min incubation with the substrate. All substrates were used at a final concentration of 1 mmol/l. The protein contents of platelet suspensions
Male
17 3 53·7 53-3 13-9± 0'40 92'5± 1'80 22-6± 2'23 6H± 3'5 328 ±20'4
Iron-deficient (16) Female
Male
15 1 47·3 67·0 8'79± 0'24 64'5 ± 1'36 3-8 ± 0'32 87-9 ± 4'41 361 ±41'8
were determined by the method of Lowry, Rosebrough, Farr & Randall (1951) with human serum albumin as standard. The intra-assay variability is less than 5%. Blood counts, serum iron and total iron-binding capacity. Blood counts, serum iron and total ironbinding capacity were determined in a specimen of venous blood obtained at the same time as that used for the preparation of platelets. Blood counts were performed with a Coulter counter. Serum iron and total iron-binding capacity were measured by the method of Stookey (1970) and Ramsay (1957). Statistical methods. The significance of differences between mean values was assessed with Student's r-test,
Results Platelet monoamine oxidase activity in iron-deficiency anaemia
Monoamine oxidase activities in platelets obtained from patients with iron-deficiency anaemia and from normal subjects are shown in Table 2. It can be seen that there is a decrease of monoamine oxidase activity in platelets from iron-deficient patients with all four substrates tested. These changes are significant (P < 0'001) when tyramine, dopamine and 5hydroxytryptamine are the substrates and are of borderline significance (P < 0,05) when phenylethylamine is the substrate. The platelet monoamine oxidase activities for normal subjects listed in Table 2 are equivalent to 6'87, 7'52, 9·10 and 3·87 nmol of substrate deamin-
Monoamine oxidase activity in iron deficiency
ated per mg of protein in 30 min for tyramine, dopamine, 5-hydroxytryptamine and phenylethylamine respectively. These results are similar to those reported by other investigators (Murphy & Wyatt, 1972; Meltzer & Stahl, 1974; Collins & Sandler, 1971). Where possible, four substrates were studied because of the existence of multiple forms of the enzyme each having a different substrate specificity (Youdim, 1972; Youdim& Collins, 1975).
291
Effect of treatment with oral iron preparations
The effect of treatment with oral iron preparations on haematological response and platelet monoamine oxidase activity was studied in a group of seven irondeficient patients (five females and two males), who received ferrous sulphate (200 mg t.d.s.) for 38 days; post-treatment samples were obtained 4 days after cessation of therapy. Serum iron concentrations at
TABLE 2. Effect of iron deficiency on monoamine oxidase activity in human platelets towards various substrates Monoamine oxidase activities are expressed as d.p.m. of deaminated products formed per rng of platelet protein per 30 min and are mean values±sEM with the number of subjects in parentheses. Platelet monoamine oxidase activity
Tyramine
Dopamine
5-Hydroxytryptamine
Phenylethylamine
Normal subjects
2256±260 (20)
2508± 190 (19)
3033± 314 (20)
1289± 199 (20)
Iron-deficient patients
1386±206 (16)
1385± 162 (16)
1448±207 (16)
818± 180 (16)
Decrease (:Yo) p
39
45
52
37
