Iron Metabolism Ciba Foundatlon Copyright 0 1977 Ciba Foundation
Monoamine metabolism and platelet function in iron-deficiency anaemia WOODS, H. F., YOUDIM, M. B. H.,*t BOULLIN, D.* and CALLENDER, S.3 Department of Pharmacology and Therapeutics, Academic Division of Medicine, University of Shefield, * MRC Clinical Pharmacology Unit and University Department of Clinical Pharmacology, and 3 Nufield Department of Medicine, RadcliffeInfirmary, Oxford
Abstract The evidence that iron plays a role in the metabolism and the function of monoamine neurotransmitters in iron-deficient rats led us to investigate platelet monoamine metabolism and platelet function in iron-deficient patients. In patients with iron-deficiency anaemia, platelet monoamine oxidase (MAO) activity was significantly lowered when dopamine and 5-hydroxytryptamine (5-HT) were substrates. Treatment with oral iron(I1) (ferrous) sulphate restored platelet M A 0 activity to normal when the serum iron concentration returned to normal. Examination of the physicochemical properties of the platelet enzyme showed that iron deficiency causes an increased sensitivity of M A 0 to heat inactivation and inhibition by irreversible M A 0 inhibitor drugs. However, the K, of M A 0 for monoamine substrates was unchanged while V,,, was lowered. These results suggest that a smaller amount of active M A 0 protein is present and binding studies with the 1%-labelled M A 0 inhibitor, L-deprenil, confirmed this. Iron may be necessary for the synthesis of M A 0 apoenzyme or as a cofactor for an enzyme which attaches the flavin adenine dinucleotide covalently to the apoenzyme or may itself function as a cofactor. Platelets from patients with iron-deficiency anaemia showed abnormal aggregation responses to dopamine and 5-HT. This abnormality, which was corrected after treatment of the anaemia, appears to be related to a change in the platelet itself since the aggregation response of platelets from iron-deficient subjects suspended in normal plasma was also abnormal. The physiological and clinical implications of these findings are discussed.
The control of tissue monoamine concentrations i n mammals depends, in part, on the activity of the enzyme monoamine oxidase (monoamine-02 oxidoreductase [deaminating], EC 1.4.3.4; MAO). This enzyme oxidatively deaminates dopamine, 5-hydroxytryptamine, noradrenaline, phenylethylamine, tryptamine and other primary and secondary amines which may occur in mammalian f Present address: Department of Pharmacology, School of Medicine, Israel Institute of
Technology, Haifa
227
228
H . F. WOODS
et al.
tissues. The enzyme and its cofactor requirements have been extensively investigated (see Youdim 1975) and these studies reveal that the purified enzyme contains thiol groups and covalently bound flavin adenine dinucleotide (FAD) which is a cofactor for thereaction(1). In 1966,Youdim & Sourkes showed that RCOOH RCHz-NHz
+
FAD 02-4
t
RCHO
+
NH3
+
HzOz
iron was present in the purified rat liver enzyme and this was later confirmed for pig liver by Oreland (1971). The activity of the rat liver enzyme measured in vitro was lowered when the animal was made iron deficient (Symes et al. 1969) and measurements of monoamine oxidase activity in vivo have confirmed this finding (Symes et al. 1971). Treatment of the animals with iron restored the tissue monoamine oxidase activity to normal. If these findings apply to man, they might be expected to have widespread implications for monoamine homeostasis. Platelet monoamine oxidase activity has been used as a peripheral ‘marker’in patients with schizophrenia or bipolar depression on the basis that it might be an indicator of brain M A 0 activity. The presence of iron deficiency might modify these interpretations. These considerations led us to examine, first, the platelet M A 0 activity in patients with iron-deficiency anaemia and, secondly, platelet aggregation responses as a measure of platelet function in platelet-rich plasma obtained from patients with iron-deficiency anaemia. We shall discuss these results with regard to their clinical implications in iron deficiency. PATIENTS AND METHODS
Patients
We studied patients of both sexes who were diagnosed as having an irondeficiency anaemia using the diagnostic criteria : (1) haemoglobin concentration less than 10 g/dl; (2) mean corpuscular volume less than 76 pm3; (3) serum iron concentration below 14.3 pmol/l for male patients and below 10.7 pmol/l for female patients. In these patients there was a decreased saturation of ironbinding capacity. The control groups consisted of out-patients with no haematological abnormalities and members of the hospital staff (Table 1). As part of the investigation reported here, we investigated a further series of patients
MONOAMINE METABOLISM AND PLATELETS IN IRON-DEFICIENCY ANAEMIA
229
TABLE 1 Clinical details of the subjects studied. The results are expressed as mean values iS.E.M. Paranieter
Number Mean age (yr) Haenioglobin (g/dl) Mean corpuscular volume (pm3) Serum iron concentration (pniol/l) Total iron-binding capacity (crmol/l) Platelet count (103/mm3)
Norriiul (20) Iron-deficient (16) _ _ ~ Female Male Female Mule 17 3 53.3 53.7 13.9 f 0.40 92.5 i 1.80 22.6 f 2.23 67.1 3.5
328 -C 20.4
1 15 67.0 47.3 8.79 =k 0.24 64.5 3= 1.36 3.8 f 0.32 87.9 & 4.41 361 =k 41.8
attending the anaemia clinic to examine platelet monoamine oxidase activity in subjects having a range of serum iron concentrations. Methods Preparation of platelets. Platelets were harvested from 20 ml of venous blood collected without stasis into 129 mM-sodium citrate solution. A sonicated suspension of platelets was then prepared by the method described by Youdim et al. (1975). In all cases the samples were obtained between 0930 and 1200 h on the day of investigation. Determination of platelet monoamine oxidase activity. We determined the M A 0 activity of the platelet suspension using the radioactive assay of Robinson et al. (1968) with [1-Wldopamine, 5-hydro~y[l-~~C]tryptamine, [1-14C]tyramine and phenyl[ l-14C]ethylamine as substrates. In addition, the photofluorimetric method of Kraml(l965) was used in some cases. The substrate concentrations were 1 mmol/l with the exception of the kynuramine which was 0.1 mmol/l. Usually four substrates were studied because of the existence of multiple forms of M A 0 each having a different substrate specificity (Youdim 1972). Determination of enzyme properties. When the Km,heat inactivation and pH optimum had been determined and the inhibitor and binding studies with [Wldeprenil completed (see Youdim et al. 1976), platelet suspensions from a second group of iron-deficient patients and a second control group of subjects were pooled. We chose the groups of subjects to resemble as closely as possible those studied during the determination of the effect of iron deficiency on platelet M A 0 activity.
230
H. F. WOODS
et al.
Platelet aggregation studies. We examined platelet aggregation in platelet-rich citrated plasma prepared from venous blood collected without stasis into 129m~-sodiumcitrate solution. The blood was collected from a group of irondeficient patients and normal control subjects selected according to the criteria listed above. The methods used to determine the aggregation responses to adenosine diphosphate (ADP) and 5-hydroxytryptamine (5-HT) were those described by Boullin et al. (1975a, b). When we investigated the site of the platelet abnormality, we separated platelets and resuspended them using the method described by Boullin et al. (1975~). Haematological data. We used a specimen of venous blood collected at the same time as that from which the platelets were prepared to measure the serum iron and total iron binding capacity. Blood counts were done with a Coulter counter. RESULTS
Platelet M A 0 activity in iron-deficiency anaemia The platelet M A 0 activity in iron-deficient and control subjects is shown in Fig. 1 ; there was a lowering of activity in platelets obtained from iron-deficient subjects towards the four substrates. The changes were highly significant when tyramine, dopamine and 5-hydroxytryptaminewere the substrates but of borderline significance for phenylethylamine. In a larger series of patients drawn from the clinic, we examined the relationship between the serum iron concentration and platelet M A 0 activity towards the four substrates shown in Fig. 1. These results showed that when the subjects were grouped on the basis of normal or low serum iron concentration there was a significant difference in M A 0 activity between the two groups for all substrates tested. When the subjects were grouped on the basis of normal or low haemoglobin concentration, however, there was no significant difference between the groups for the substrates with the exception of 5-hydroxytryptamine for which the difference was of borderline significance (Youdim et al. 1975). Sourkes & Missala (1976), using our data, confirmed this finding by calculating the partial correlation coefficients among the three variables : platelet M A 0 activity, serum iron concentration and haemoglobin concentration. This has led us to calculate the linear correlation coefficients between platelet M A 0 activity and serum iron concentration and those between platelet M A 0 activity and haemoglobin concentration for the four substrates tested. An example of these correlations is given in Fig. 2 for 5-hydroxytryptamine and in Table 2 which lists the correlation coefficients for all four substrates. These results show that for 5-hydroxytryptamine and phenylethylamine there is
23 1
MONOAMINE METABOLISM AND PLATELETS IN IRON-DEFICIENCY ANAEMIA oop.
5H1
Phen.
c .-
E
ac
W c
2 0
r ._
-" m
T :loo0 3 0
p
Monoamine metabolism and platelet function in iron-deficiency anaemia WOODS, H. F., YOUDIM, M. B. H.,*t BOULLIN, D.* and CALLENDER, S.3 Department of Pharmacology and Therapeutics, Academic Division of Medicine, University of Shefield, * MRC Clinical Pharmacology Unit and University Department of Clinical Pharmacology, and 3 Nufield Department of Medicine, RadcliffeInfirmary, Oxford
Abstract The evidence that iron plays a role in the metabolism and the function of monoamine neurotransmitters in iron-deficient rats led us to investigate platelet monoamine metabolism and platelet function in iron-deficient patients. In patients with iron-deficiency anaemia, platelet monoamine oxidase (MAO) activity was significantly lowered when dopamine and 5-hydroxytryptamine (5-HT) were substrates. Treatment with oral iron(I1) (ferrous) sulphate restored platelet M A 0 activity to normal when the serum iron concentration returned to normal. Examination of the physicochemical properties of the platelet enzyme showed that iron deficiency causes an increased sensitivity of M A 0 to heat inactivation and inhibition by irreversible M A 0 inhibitor drugs. However, the K, of M A 0 for monoamine substrates was unchanged while V,,, was lowered. These results suggest that a smaller amount of active M A 0 protein is present and binding studies with the 1%-labelled M A 0 inhibitor, L-deprenil, confirmed this. Iron may be necessary for the synthesis of M A 0 apoenzyme or as a cofactor for an enzyme which attaches the flavin adenine dinucleotide covalently to the apoenzyme or may itself function as a cofactor. Platelets from patients with iron-deficiency anaemia showed abnormal aggregation responses to dopamine and 5-HT. This abnormality, which was corrected after treatment of the anaemia, appears to be related to a change in the platelet itself since the aggregation response of platelets from iron-deficient subjects suspended in normal plasma was also abnormal. The physiological and clinical implications of these findings are discussed.
The control of tissue monoamine concentrations i n mammals depends, in part, on the activity of the enzyme monoamine oxidase (monoamine-02 oxidoreductase [deaminating], EC 1.4.3.4; MAO). This enzyme oxidatively deaminates dopamine, 5-hydroxytryptamine, noradrenaline, phenylethylamine, tryptamine and other primary and secondary amines which may occur in mammalian f Present address: Department of Pharmacology, School of Medicine, Israel Institute of
Technology, Haifa
227
228
H . F. WOODS
et al.
tissues. The enzyme and its cofactor requirements have been extensively investigated (see Youdim 1975) and these studies reveal that the purified enzyme contains thiol groups and covalently bound flavin adenine dinucleotide (FAD) which is a cofactor for thereaction(1). In 1966,Youdim & Sourkes showed that RCOOH RCHz-NHz
+
FAD 02-4
t
RCHO
+
NH3
+
HzOz
iron was present in the purified rat liver enzyme and this was later confirmed for pig liver by Oreland (1971). The activity of the rat liver enzyme measured in vitro was lowered when the animal was made iron deficient (Symes et al. 1969) and measurements of monoamine oxidase activity in vivo have confirmed this finding (Symes et al. 1971). Treatment of the animals with iron restored the tissue monoamine oxidase activity to normal. If these findings apply to man, they might be expected to have widespread implications for monoamine homeostasis. Platelet monoamine oxidase activity has been used as a peripheral ‘marker’in patients with schizophrenia or bipolar depression on the basis that it might be an indicator of brain M A 0 activity. The presence of iron deficiency might modify these interpretations. These considerations led us to examine, first, the platelet M A 0 activity in patients with iron-deficiency anaemia and, secondly, platelet aggregation responses as a measure of platelet function in platelet-rich plasma obtained from patients with iron-deficiency anaemia. We shall discuss these results with regard to their clinical implications in iron deficiency. PATIENTS AND METHODS
Patients
We studied patients of both sexes who were diagnosed as having an irondeficiency anaemia using the diagnostic criteria : (1) haemoglobin concentration less than 10 g/dl; (2) mean corpuscular volume less than 76 pm3; (3) serum iron concentration below 14.3 pmol/l for male patients and below 10.7 pmol/l for female patients. In these patients there was a decreased saturation of ironbinding capacity. The control groups consisted of out-patients with no haematological abnormalities and members of the hospital staff (Table 1). As part of the investigation reported here, we investigated a further series of patients
MONOAMINE METABOLISM AND PLATELETS IN IRON-DEFICIENCY ANAEMIA
229
TABLE 1 Clinical details of the subjects studied. The results are expressed as mean values iS.E.M. Paranieter
Number Mean age (yr) Haenioglobin (g/dl) Mean corpuscular volume (pm3) Serum iron concentration (pniol/l) Total iron-binding capacity (crmol/l) Platelet count (103/mm3)
Norriiul (20) Iron-deficient (16) _ _ ~ Female Male Female Mule 17 3 53.3 53.7 13.9 f 0.40 92.5 i 1.80 22.6 f 2.23 67.1 3.5
328 -C 20.4
1 15 67.0 47.3 8.79 =k 0.24 64.5 3= 1.36 3.8 f 0.32 87.9 & 4.41 361 =k 41.8
attending the anaemia clinic to examine platelet monoamine oxidase activity in subjects having a range of serum iron concentrations. Methods Preparation of platelets. Platelets were harvested from 20 ml of venous blood collected without stasis into 129 mM-sodium citrate solution. A sonicated suspension of platelets was then prepared by the method described by Youdim et al. (1975). In all cases the samples were obtained between 0930 and 1200 h on the day of investigation. Determination of platelet monoamine oxidase activity. We determined the M A 0 activity of the platelet suspension using the radioactive assay of Robinson et al. (1968) with [1-Wldopamine, 5-hydro~y[l-~~C]tryptamine, [1-14C]tyramine and phenyl[ l-14C]ethylamine as substrates. In addition, the photofluorimetric method of Kraml(l965) was used in some cases. The substrate concentrations were 1 mmol/l with the exception of the kynuramine which was 0.1 mmol/l. Usually four substrates were studied because of the existence of multiple forms of M A 0 each having a different substrate specificity (Youdim 1972). Determination of enzyme properties. When the Km,heat inactivation and pH optimum had been determined and the inhibitor and binding studies with [Wldeprenil completed (see Youdim et al. 1976), platelet suspensions from a second group of iron-deficient patients and a second control group of subjects were pooled. We chose the groups of subjects to resemble as closely as possible those studied during the determination of the effect of iron deficiency on platelet M A 0 activity.
230
H. F. WOODS
et al.
Platelet aggregation studies. We examined platelet aggregation in platelet-rich citrated plasma prepared from venous blood collected without stasis into 129m~-sodiumcitrate solution. The blood was collected from a group of irondeficient patients and normal control subjects selected according to the criteria listed above. The methods used to determine the aggregation responses to adenosine diphosphate (ADP) and 5-hydroxytryptamine (5-HT) were those described by Boullin et al. (1975a, b). When we investigated the site of the platelet abnormality, we separated platelets and resuspended them using the method described by Boullin et al. (1975~). Haematological data. We used a specimen of venous blood collected at the same time as that from which the platelets were prepared to measure the serum iron and total iron binding capacity. Blood counts were done with a Coulter counter. RESULTS
Platelet M A 0 activity in iron-deficiency anaemia The platelet M A 0 activity in iron-deficient and control subjects is shown in Fig. 1 ; there was a lowering of activity in platelets obtained from iron-deficient subjects towards the four substrates. The changes were highly significant when tyramine, dopamine and 5-hydroxytryptaminewere the substrates but of borderline significance for phenylethylamine. In a larger series of patients drawn from the clinic, we examined the relationship between the serum iron concentration and platelet M A 0 activity towards the four substrates shown in Fig. 1. These results showed that when the subjects were grouped on the basis of normal or low serum iron concentration there was a significant difference in M A 0 activity between the two groups for all substrates tested. When the subjects were grouped on the basis of normal or low haemoglobin concentration, however, there was no significant difference between the groups for the substrates with the exception of 5-hydroxytryptamine for which the difference was of borderline significance (Youdim et al. 1975). Sourkes & Missala (1976), using our data, confirmed this finding by calculating the partial correlation coefficients among the three variables : platelet M A 0 activity, serum iron concentration and haemoglobin concentration. This has led us to calculate the linear correlation coefficients between platelet M A 0 activity and serum iron concentration and those between platelet M A 0 activity and haemoglobin concentration for the four substrates tested. An example of these correlations is given in Fig. 2 for 5-hydroxytryptamine and in Table 2 which lists the correlation coefficients for all four substrates. These results show that for 5-hydroxytryptamine and phenylethylamine there is
23 1
MONOAMINE METABOLISM AND PLATELETS IN IRON-DEFICIENCY ANAEMIA oop.
5H1
Phen.
c .-
E
ac
W c
2 0
r ._
-" m
T :loo0 3 0
p
