- Email: [email protected]
The effects of iron deficiency on brain biogenic monoamine biochemistry and function in rats
002X-3908/80/0301-0259802.00/0
THE EFFECTS OF IRON DEFICIENCY ON BRAIN BIOGENIC MONOAMINE BIOCHEMISTRY AND FUNCTION IN RATS M. B. H. YOUDIM*.A. R. GREEN,M. R. BLOOMFIELD. D. G. GRAHAME-SMITH
B. D. MITCHELL, D. J. HEAL and
MRC Unit and University Department of Clinical Pharmacology, Radcliffe Infirmary. Oxford OX2 6HE. England
(.&YqJrrd
15 Octohrr
1979)
Summary-Rats were made iron-deficient by feeding them on a semi-synthetic diet containing milk powder. low in iron. Control rats were fed the same diet with an iron-supplement. When the rats were iron deficient (haemoglobin 6.9 + 0.6g/dl) a study was made of the iron distribution in the brain. Distribution was similar in control and iron-deficient rats with the highest concentration in the caudate nucleus, hypothalamus and cerebellum, Sub-cellular distribution studies showed the highest concentration to be in the crude mitochondrial fraction. The iron-deficient group had an approx. 60% decrease in brain iron stores. Measurement was also made of the monoamine metabolising enzymes (MAO) in iron-deficient rats. Despite large decreases in peripheral MAO activity no decrease in central MAO activity was observed in these rats. Nor was there any decrease in brain tryptophan hydroxylase or aldehyde dehydrogenase activity. In very severe iron-deficient (haemoglobin 4.5 + 0.6g/dl) rats, brain MAO activity still decreased only by 15.70”,, while heart MAO activity decreased by over 60?:,, while there was still no change in tryptophan hydroxylase activity. Iron deficient rats showed decreased behavioural responses following increased brain 5-hydroxytryptamine (5-HT) synthesis or after administration of the 5-HT agonist 5-methoxy N,N-dimethyltryptamine. They also showed decreased behavioural responses following administration of tranylcypromine plus L-DOPA. methamphetamine or apomorphine. No change in brain tryptophan or 5-HT synthesis was observed although endogenous S-HT content was decreased. No changes were seen in brain dopamine or noradrenaline content, nor in the activity of dopamine-sensitive adenylate cyclase. Brain protein synthesis was also unaltered. Eight days after restoration of the iron plus diet the behavioural responses were normalised. but in,jection of ferrous gluconate (acutely) failed to restore the behaviour. Reasons for the altered post-synaptic monoamine behavioural responses being unlikely to be the result of anaemia are discussed and the relevance of decreased monoamine function in iron-deficiency is also discussed, particularly with regard to the high incidence of this nutritional disorder.
Despite
the fact that
iron
deficiency
is the most
com-
in the world (Kessner and Kalk, 1973; Garby, 1973) there has been little work on the effects of iron deficiency on brain metabolism and function. It has been known for some time that iron is present in relatively large amounts in the brain. particularly in the basal ganglia (Cumings. 1948; 1968; Hallgren -and Sourander. 1958; Harrison. Netsky and Brown, 1968) and its concentration there appears to be independent of the stores in the liver (Hallgren and Sourander, 1958; Dallman. 1974). The importance of iron in protein synthesis, as a co-factor for haem and non-haem iron containing enzymes and its structural role in many membranes and on membrane bound proteins is well documented (Jacobs and Worwood, 1974). Nutritional iron appears to be important for monoamine oxidase activity and the activity of this enzyme mon
nutritional
disorder
* Present address: Israel Institute of Technology--Technion. School of Medicine. Department of Pharmacology. I2 Haaliya Street, Bat-Galim. POB 9649, Haifa. Israel.
has been shown to be lowered in the tissues of humans and rats with iron deficiency anaemia (Symes. Sourkes, Youdim, Birnbaum and Gregoriadis 1969; Youdim, Woods, Mitchell, Grahame-Smith and Callender, 1975a; Youdim, Grahame-Smith and Woods, 1976; Voorhess, Stuart, Stockman and Oski, 1975; Symes, Missala and Sourkes, 1971) returning to normal only when the serum iron concentration returns to normal (Youdim et al., 1975a: Voorhess et al., 1975; Symes rt al., 1971). It has also been suggested that iron is a cofactor for tyrosine hydroxylase (Sourkes, 1972) and tryptophan hydroxylase (Lovenberg, Jequier and Sjoerdsma, 1968; Ichiyama. Nakamura, Nishizuka and Hayaishi. 1970; Youdim. Hamon and Bourgoin, 1975b). enzymes involved in the formation of catecholamines and %hydroxytryptamine respectively. The effects of iron deficiency in rats on the activity of various monoamine metabolising enzymes in the brain and the behavioural responses of the animals to increased brain 5hydroxytryptamine (5-HT) and dopamine (DA) receptor stimulation has been investi259
M. B. H. YOU~IMet al.
260
gated. The results suggest that while there is little alteration of either enzyme activity or rates of amine synthesis in iron deficient animals there is nevertheless a marked reduction of monoamine functional activity as measured by drugs that act pre- and postsynaptically. METHODS
Preparation
of iron dejcient
rats
Male Sprague-Dawley derived rats (initial weight 80 + 20 g, mean + SEM) were made iron-deficient by feeding a semi-synthetic diet of distilled water and milk powder low in iron (McCall, Newman, O’Brien, Valberg and Witts. 1962). Control rats were given tap water and the same milk powder with ammonium ferrous sulphate (1.3 mg/g diet) added. The iron deficient group were given an ad lihitum diet and the iron-plus group had their food intake restricted to that of the ironaeficient group. Investigations were performed on animals that had been on the diet for 5 weeks at which time they were iron-deficient as judged by their blood haemoglobin concentration [control rats: 14.1 x 0.44g/dl (II = 16); iron deficient rats: 5.55 k 0.42 g/d1 (n = 22)]. Measurement
of enzyme activities
Aldehyde dehydrogenase activity was measured in brain, liver and heart homogenates by the method of Dietrich (1966) using 3-indoleacetaldehyde as substitute. Tryptophan hydroxylase activity was measured in the brain stem supernatant preparation using tetrahydrobiopterin as a co-factor by the method of Tong and Kaufman (1975). The activity of monoamine oxidase in tissue homogenates was measured using as a substrate either kynuramine (Youdim. 1975) or [‘%I]-tyramine (Tipton and Youdim, 1976). Succinate dehydrogenase activity was determined by the method of Pennington (1961). Determination
qf’brain
monoamines
5-Hydroxytryptamine was assayed fluorometrically by the method of Curzon and Green (1970) and dopamine and noradrenaline by the method of Chang (1964). The rate of 5-hydroxytryptamine synthesis was examined by measuring the rate of 5-HT accumulation following monoamine oxidase inhibition as described by NelT and Tozer (1968). Determination
of’ bruin amino acids
Tryptophan was measured by the method Denckla and Dewey (1967) and tyrosine by method of Waalkes and Udenfriend (1957). Estimation
Greengard (1972) and cyclic AMP measured by the method of Brown. Albano. Ekins. Sgherzi and Tampion (1971). Protein protein
determination
qf cyclic .4 MP and aden~~lnte cyclase
Brain homogenates were prepared from normal and iron deficient rats as described by Green, Heal, Grahame-Smith and Kelly (1976). The homogenates were incubated with increasing concentrations of dopamine as described by Kebabian, Petzold and
of the role of‘
Bovine serum albumin was used as standard for determination of protein by the procedure of Lowry. Rosebrough, Farr and Randall (1951). Protein synthesis was examined by determining the rate of incorporation of [‘4C]-tyrosine into brain protein as described by Grahame-Smith (1972). Iron determination
Non-haem iron was measured in tissue homogenates by a slightly modified procedure of Ramsay (1957) using o-phenanthroline to chelate the iron. The preparation was heated for 6Omin at 100°C in the presence of 10% (w/v) trichloroacetic acid before the iron determination. Measurement
of monoamine
function
and behauioural
hyperactivity
During the last few years various animal models have been developed to assess monoamine functional activity, namely the response to transmitter which is qeleased, stimulates the receptor and produces a behavioural response. Much of this work has already been reviewed (Green and Grahame-Smith, 1976). The function of 5-HT was examined by observing the behavioural changes which occur following the intraperitoneal injection of tranylcypromine (20 mg/kg) followed 30min later by L-tryptophan (100 mg/kg). This procedure increases the rate of 5-HT synthesis and results in the appearance of various behavioural changes, one feature of which is hyperactivity, which is measured on LKB Animex activity meters. Previous studies have suggested that the degree of hyperactivity is proportional to the rate of increase in 5-HT synthesis and “spill-over” into functional activity in the brain (Grahame-Smith, 1971a; Green and Grahame-Smith. 1976). The functional activity of brain dopamine was examined by the administration of tranylcypromine (20 mg/kg) and L-DOPA a procedure which increases brain (50 mg/kg).
Table
1. Distribution
Brain areas
of the
and measurement
synthesis
Whole brain Cerebellum Brain stem Caudate nucleus Amygdalla Cerebral cortex Hypothalamus
of non-haem rat brain
iron in various areas of
11g Fe,img protein 0.074 0.075 0.052 0.092 0.058 0.050 0.076
* + + + f * +
The results shown are the means + SEM mals. Whole brain non-haem concentration cient animals was 0.034 + 0.006 (6).
0.008 0.008 0.008 0.036 0.005 0.003 0.030 from
6 ani-
in iron defi-
Effects of iron deficiency in rat brain
261
dopamine concentration and results in the rats disyielding nuclear fraction (PI), crude mitochondrial playing hyperactivity which can also be measured on fraction (P2) and a microsomal-supernatant fraction (PI + S) demonstrated the presence of non-haem iron activity meters (Everett, Weigland and Rinaldi, 1963; in all fractions P,:8%; P,:55%; P3 + S:37%. The Green and Kelly, 1976). crude mitochondrial fraction contained the highest Postsynaptic 5-HT-mediated responses were concentration of non-haem iron/mg protein. measured by examining the degree of hyperactivity Iron-deficient rats had a total brain non-haem iron which follows administration of the putative agonist 5-methoxy N,N-dimethyltryptamine (Grahame-Smith. ’ concentration of 0.030 + 0.006 pg/mg protein, showing a 60% fall in non-haem iron. However the per1971b) while postsynaptic dopamine function was centage distribution between the fractions was the measured by examining the locomotor activity which same as in the control rats showing that iron defifollows administration of either the dopamine releasciency has no specific effects on the subcellular distriing drug methamphetamine or the agonist apomorbution of iron. phine. The behavioural changes following administration Monoamine metabolising enzymes during iron defof the various treatments outlined above were moniciency tored in groups of 3 animals in cages placed on LKB In rats with haemoglobin values of 6.9 f 0.6g/dl, Animex activity meters (sensitivity and tuning 30 PA). no decrease in brain tryptophan hydroxylase was Results show the data obtained from at least 3 separfound (Fig. 1). Nor was brain MAO activity lowered, ate experiments and are compared using the Student’s f-test. even though a large decrease in the activity of this enzyme was observed peripherally in the liver, heart RESULTS and adrenal gland (Fig. 1). The MAO activity was not Distribution of non-haemiron in the brain decreased in the spleen. Aldehyde dehydrogenase acIt was found that normal rat brain contained a tivity was unaltered in brain, liver and heart (Fig. 1). Therefore, severe iron deficiency was induced in mean concentration of 0.074 + 0.008 pg non-haem another group of rats by keeping them on the ironiron/mg protein and the regional distribution is deficient diet for 9 weeks. This resulted in haemoshown in Table 1. Higher values were seen in the globin concentrations of 4.5 + 0.6 g/dl. There was still caudate nucleus, hypothalamus, and cerebellum. no decrease in tryptophan hydroxylase activity in the Fractionation of control rat brains in 0.32 M sucrose,
Monoamine
oxidase
Aldehyde pehydroge~;
Tryptophan Hydraxyla:
Succinic Dehydragenase
Haemaglobin
Fig. I. The effect of iron deficiency on rat tissue monoamine metabolising enzymes. Results show the % activity of the enzymes compared to control animals. Stippled columns show enzyme activities in animals made iron deficient by placing them on iron deficient diet for 5 weeks (haemoglobin value in stippled column on right). Cross-hatched columns show enzyme activities in rats kept on the iron deficient diet for 9 weeks (haemoglobin value in lined column on right). Control haemoglobin value is also shown. Control monoamine oxidase activity values were as follows (all values expressed in nmol product formed/mg protein/min at 37°C). Brain 1.06 k 0.11; Liver 2.74 f 0.14; Heart 0.78 k 0.05; Adrenal gland 0.49 & 0.03; Spleen 0.13 + 0.04 (n < IO). Aldehyde dehydrogenase activity control values were as follows (expressed in same. units as MAO activity, see above): Brain 0.71 + 0.04; Liver 1.62 + 0.25; Heart 0.49 &-0.03 (n = 4). Tryptophan hydroxylase activity (using tetrahydrobiopterin (BH,) as cofactor) in the brain stem of control animals (expressed in nmol product formed/mg protein/ min at 37°C) was 0.16 f 0.02 (n = 6). Succinic dehydrogenase activity control values (expressed in nmol product formed per mg protein per min at 37°C) were Brain 13.40 + 1.81; Liver 54.50 f 3.10; Heart 20.1 I f 2.10 (n = 6). *P < 0.05; l*P < 0.02; tP < 0.01; $P < 0.001.
262
M. B. H.
8 x
YOUDIM
et u/
60-
-
Fig. 2. The total movements of control (stippled columns) and iron deficient (cross-hatched columns) rats during the 90 min after t_-tryptophan (100mg/kg) or methamphetamine (2 mg/kg) 60 min after L-DOPA) (50 mg/kg) and 40 min after 5-methoxy NJ-dimethyltryptamine (2 mg/kg) or apomorphine (2 mg,kg). The t_-tryptophan. L-DOPA and 5-MeODMT injected rats were pretreated 30 min before with tranylcypromine (20mg/kg). Also shown is the total activity of control and iron deficient rats during the overnight period (I 8 hr 00 min- 10 hr 00 min) the animals being on a 12 hr light 12 hr dark period (18 hr 00 min-06 hr 00 min). Results show mean + SEM of 3 separate observations. Different from control rats *P < 0.01; **p < 0.025; tP < 0.05.
brain. nificant
There
were.
decreases
however. of brain
small (155ZO”,,) but sigMAO
and
succinate
de-
hydrogenase activities (Fig. I). In other tissues MAO activity showed an even greater decrease than seen previously. A similar reduction in the activity of the iron-containing flavoprotein succinate dehydrogenase in the periphery was also seen (Fig. I). Thus. brain MAO and succinate dehydrogenase activities were little altered in animals in which the non-
haem
iron
even
though
the
of the brain activities
pheral
tissues
were
I$kfs
of’ irorl d
were
depleted
by 60”/,,
of these
enzymes
in peri-
markedly
hrcGt 5-H T ant! llopurnirtr
diminished.
011 the Jitrrctionul urtd mortoc~mine
of rat brain
5-hydroxytryp-
Fe deficient
Control 3.33 f 0.26 (4)
3.69 + 0.13(5)
0.3X & 0.02 (6)
0.31 + 0.02 (9)*
0.51 * 0.02 (4)
0.42 * 0.01 (5)*
0.13
0.1 I
m gl Brain 5-HT (/cg 5-HT,g) Brain 5-HT 60 min after tranylcypromine l/(g 5-HT’g) Rate of 5-HT svnthesis (kcp 5-HT gei hr-I) 5-HT accumulation after tranylcypromine/L-Tryp (1.195-HT/g)
acti&!:
0.95 + 0.07
(I I )
0.91 + 0.06 (7)
Tryptophan and S-HT results expressed as /rg compound/g brain (wet wt). Results expressed as mean f SEM with number of observations in brackets. The rate of 5-HT synthesis determined by the a,ccumulation of 5-HT over control during 60 min following tranylcypromine (Neff and Tozer, 1968). Haemoglobin values in these animals given in Table 3. 5-HT accumulation measured 60 min after L-tryptophan (100mg/kg). tranylcypromine (20 mg/kg) being given 30 min before tryptophan. * Different from control P < 0.01.
oj
synthesis
To assess changes in monoamine function behavioural models were used (see Methods).
Table 2. The effect of iron deficiency on the synthesis tamine (5-HT)
Brain tryptophan
stores
various
Etfects of iron deficiency
263
in rat brain
Table 3. Effect of iron deficiency on brain catecholamine concentrations Control Brain dopamine (pg/g) Brain noradrenaline (pg/g) Brain dopamine after tranylcypromine/DOPA Brain noradrenaline after tranylcypromine/DOPA Haemoglobin (g/dl)
Fe deficient
2.03 + 0.24(6) 0.19 + 0.01 (4) 5.72 & 0.32 (5)
1.74 k 0.13 (8) 0.20 + 0.01 (5) 5.75 & 0.57 (6)
0.54 k 0.07 (6)
0.55 k 0.04 (6)
14.1 *0.44(16)
5.55 + 0.42 (22)*
All results expressed as pg catecholamine/g brain (wet wt). Results expressed as mean + SEM with number of observations in brackets. Accumulation of dopamine and noradrenaline measured 60 min after L-DOPA (60 mg/kg), tranylcypromine (20 mg/kg) having been given 30 min before the L-DOPA. * Different from control P < 0.001.
Following tranylcypromine/L-tryptophan administration the iron-deficient rats showed markedly less hyperactivity than the control animals (Fig. 2). However the accumulation of S-HT in the brain following tranylcypromine/L-tryptophan was the same in both groups as was the rate of synthesis (Table 2), supporting the finding that iron deficiency does not affect tryptophan hydroxylase activity. While the concentration of tryptophan in the brain was not changed by iron deficiency the endogenous concentration of brain 5-HT was decreased (Table 2). As the synthesis of 5-HT was not inhibited it seemed probable that the decreased activity was due to a decreased post-synaptic response to 5-HT. This view was strengthened by the observation that the
behavioural response to the suggested 5-HT agonist 5-methoxy, N,N-dimethyltryptamine, was diminished significantly in the iron-deficient animals (Fig. 2). Since a group of dopaminergic neurones lies between the 5-HT neurones initiating the hyperactivity response and those mechanisms responsible for its behavioural expression (Green and GrahameSmith, 1974) it was possible that the altered 5-HT response was due to alterations in dopamine function. Alterations in dopamine function are indeed present in iron-deficient animals since their response to tranylcypromine/L-DOPA administration was markedly diminished (Fig. 2). Again this was not due to altered transmitter synthesis, because dopamine and noradrenaline concentrations and accumulation after tranylcypromine and L-DOPA administration were similar in both groups (Table 3). It was also possible to demonstrate by other approaches that iron-deficiency inhibited dopaminemediated behavioural responses. It was found that the locomotor responses of these animals to either the dopamine-releasing drug methamphetamine (2 mg/kg) or the putative dopamine agonist apomorphine (2 mg/kg) was inhibited (Fig. 2). Eflecr of iron deficiency on the brain adenylate cyclase system
,cL Dopomine
J
10-d
(M )
Fig. 3. The response of dopamine sensitive adenylate cyclase to dopamine in homogenates of normal (0) and irondeficient (m) rats. Homogenates prepared from normal and iron deficient rats were incubated with increasing concentrations of dopamine as described in Methods. Basal activity of two groups, control: 68.0 f 1.4 pmol/2 mg tissue (wet wt) per 2.5min; iron deficient 62.6 rt 2.4pmol/2mg tissue (wet wt) per 2.5 min (6 animals in each group). N.P.19/3+
The response with apomorphine suggests that irondeficiency causes an inhibition of the behavioural response to increased functional activity of DA by an action at or beyond the post-synaptic site. This does not appear to be due to an alteration in the sensitivity of the dopamine-sensitive adenylate cyclase system, since the response of caudate nucleus dopamine sensitive adenylate cyclase in vitro to increasing concentrations of dopamine was the same in both the control and iron-deficient rats (Fig. 3). Effect of an iron rich diet to iron deficient rats on the monoamine mediated behavioural responses
When the iron-deficient rats were given the ironrich (control) diet for 8 days the hyperactivity responses to tranylcypromine/L-tryptophan and tranylcypromine/L-DOPA returned to normal (Fig. 4) as
M. B. H. YOUVIMrr al.
264 (A)
(8)
t. -Trp
L -DCf’A Time
( min I
Fig. 4. The behavioural effect of injecting L-tryptophan or L-DOPA after a dose of tranylcypromine hyperactivity in iron-deficient rats fed iron plus diet. Iron deficient rats were placed on iron plus diet 8 days (0) and then injected with tranylcypromine (20mg/kg) followed 30min later with either L-tryptophan (100 mg/kg) or (b) L-DOPA (50 mg/kg). Control rats (iron plus diet only) also shown Activity was measured as movements/min.
did the responses to amphetamine and apomorphine. At this time the haemoglobin value had also increased (12.2 &- 0.64 g/dl). In contrast, an intraperitoneal injection of a large dose of iron (ferrous gluconate 20 mg/kg; “Jectofer”) did not restore the hyperactivity response to tranylcypromine and L-tryptophan 48 hr later. Overnight
locomotor
activity
Glover and Jacobs (1974) reported that iron-deficiency in rats resulted in an altered pattern of spontaneous activity. The animals were less active during the dark period and more active during the light period than control rats. These findings were confirmed with respect to dark period activity (Fig. 2). Protein
synthesis
Previously, Grahame-Smith (1972) found that inhibiting protein synthesis in rat by injection of cycloheximide resulted in attenuated hyperactivity responses to tranylcypromine/L-tryptophan and to S-MeODMT. The possibility was therefore examined that the changed behavioural responses of the irondeficient rats was due to a change in the rate of brain protein synthesis. No statistically significant differences between the rate of incorporation of the radioactivity into protein were observed between the control and iron-deficient groups. DISCUSSION
The brain non-haem good agreement with Manies (1975) and the ties between rat and trations found in
iron concentration data was in that of Dalhnan, Simes and distribution indicated similarihuman since in man concenbasal ganglia are high
on for (a) (0).
(0.15-0.48 mg/g brain, fresh wet wt-see Hallgren and Sourander, 1958; Cumings, 1948, 1968; Harrison et al., 1968) and are of the same order as the concentration found in the liver (Hallgren and Sourander. 1958; Dallman, 1974). The percentage distribution following fractionation of rat brain was also similar to that seen in human brain (Hallgren and Sourander, 1958) with the mitochondrial fraction containing the highest, concentration (Colburn and Mass. 1965: Rajan, Colburn and Davis, 1976). Iron has been implicated as a cofactor for tryptophan hydroxylase (Lovenberg er al., 1968; Ichiyama et al., 1968; Youdim et al., 1975b; Usdin, Wiener and Youdim. 1978) and tyrosine hydroxylase (Sourkes, 1972; Usdin et al., 1978; Musacchio, 1975) and has been suggested to be necessary for the synthesis of the active form of MAO (Youdim et al., 1976; Youdim, 1975). A deficiency of iron might therefore be expected to result in a lowered activity of these enzymes which would in turn alter monoamine synthesis and perhaps function. However. even in animals with a 60% depletion of non-haem iron both brain succinate dehydrogenase and MAO activities were little altered, even when the activities of these enzymes in peripheral stores were markedly lowered. These results are reminiscent of those from riboflavin deficiency studies. Although it is known that MAO (Youdim, 1976) and succinate dehydrogenase (Singer, 1976) contain FAD as co-factor, riboflavin deficiency in rats does not alter the activities of these enzymes in the brain (Youdim, 1976) although a significant decrease in enzyme activity is seen in the liver. The present results suggest that either iron is still present in sufficient quantities to act as a co-factor, or that the enzymes do not require iron for full activity.
265
Effects of iron deficiency in rat brain In the case of MAO and succinate dehydrogenase the latter interpretation seems unlikely in view of the changes in enzymic activities seen in peripheral tissues and the reported presence of iron in purified enzyme preparations (Youdim, 1976; Salach, 1979). The activities of brain enzymes which may be iron dependent appear, therefore, to be very resistant to the effect of iron-deficiency. Although tyrosine hydroxylase activity was not measured, Quick and Sourkes (1977) have shown that the activity of this enzyme in adrenal gland does not decrease in iron deficiency and our data on catecholamine synthesis in iron-deficient rats support this view. Finally, if iron is involved in tyrosine and tryptophan hydroxylases, it may be bound in some covalent form that simple nutritional deficiency will not displace. An increase in brain 5-HT content in iron-deficient animals has recently been reported by Mackler, Parson, Miller, Inamdar and Finch (1978). In contrast a significant decrease in the endogenous brain 5-HT content was found in the current study and may well be due to decreased 5-HT binding and storage at the nerve endings since ferrous iron has been shown to be involved selectively in this process (Tamir, Klein and Rapport, 1976). The behavioural studies demonstrated that irondeficiency markedly inhibits both 5-HT and dopaminemediated behaviours. The data on amine synthesis suggests that the alteration in behaviours is due to a postsynaptically mediated change and this was confirmed by the use of appropriate agonists. It is probable that this change is not occurring at the receptor site itself since adenylate cyclase activity did not change. It is more likely that the effect of iron deficiency involves changes “beyond” the receptor. The mechanism may therefore involve alterations in the function of other modulatory transmitter systems involved in the behaviours resulting from increased monoamine function. Alterations in monoaminemediated behaviour by altering the concentration or function of other neurotransmitters have been widely reported (e.g. Green, Tordoff and Bloomfield, 1976; Cott and Engel, 1977). One major problem of interpretation of the behavioural data is that the behaviour examined involves locomotor activity and iron-deficiency produces anaemia. However, it is unlikely that the changes observed are due to oxygen deficit. If this were so then the activity would not merely be less but would decrease further as the experiment progressed. However it was observed that the lowered hyperactivity response not only continues for over 2 hr after injection of the tranylcypromine/L-tryptophan but indeed increases with time. Also administration of a further injection of 5-MeODMT (3 mg/kg) 15 min after the first dose does not increase the locomotor response further but does result in the rats remaining active for a considerable period of time (over 90 min). Secondly Dallman (1974) has reported that isolated
muscle from iron-deficient rats is able to contract normally and Edgerton, Bryant and Gillespie (1972) were unable to demonstrate a relationship between. physical performance and muscle content of myoglobin and cytochromes. Furthermore, the behavioural response returned to normal after 8 days whereas Dallman and Schwartz (1965) have shown that the reversal of skeletal muscle deficiency of myoglobin and cytochrome may require a period six times longer than that required to correct the haemoglobin concentration (which was still not quite restored in the present animals after 8 days on the iron-rich diet). It is therefore likely that the lowered activity response is neither due to anaemia nor altered muscle function. The data of Dallman et al. (1975) suggests that nonhaem iron stores in the brain are only slowly restored in iron-deficient rats given an iron-plus diet. The present data on the effect of the iron-plus diet supports the conclusion that restoration of normal behavioural responses involves changes other than merely restoring whole body iron, and to data with ferrous gluconate suggests that the iron has to be incorporated in some way before there is restoration of monoaminemediated responses. The relatively few studies on the effects of iron deficiency on behaviour in animals and man have mainly been concerned with psychological test performances and the methodology has been questioned in several instances (see review by Pollitt and Leibel, 1976). However it is interesting that some of the changes observed did not appear to be due to the anaemia per se, but rather other metabolic correlates of iron depletion (Pollitt and Leibel, 1976). The neurotransmitters 5_hydroxytryptamine, dopamine and noradrenaline are involved in various neurological and psychiatric disorders such as depression, schizophrenia and Parkinson’s Disease and presumably therefore are intimately concerned in the regulation of mood and neuronal activity. The present results therefore indicate that the behavioural changes seen in people with iron-deficiency may not be a matter simply of the anaemia but also the result of neurotransmitter changes in the brain. Such changes merit further investigation in view of the prevalence of iron-deficiency in the world, particularly in young children (Pollitt and Leibel, 1976) and its possible long term effects on brain neurotransmitter systems.
REFERENCES
Brown, B. L., Albano, J. D. M., Ekins, R. P., Sgherzi, A. M. and Tampion, W. (1971) A simple and sensitive satura. tion assay method for the method of measurement of adenosine 3’5’ cyclic monophosphate. Biochem. J. 121:
561-562. Chang, C. C. (1964). A sensitive method for spectrophotofluorometric assay of catecholamines. Int. J. Neuropharmat. 3: 643649. Colburn, R. W. and Mass, J. W. (1965). Adenosine triphosphate-metanorepinephrine ternary complexes and catechol&ine binding. Nature, Lond. 208: 3742.
M. B. H. YOUIXMet al.
266
Cott, J. and Engel, J. (1977). Suppression by GABAergic drugs of the locomotor stimulation induced by morphine, amphetamine and apomorphine: evidence for both pre- and post-synaptic inhibition of catecholamine systems. J. Neural Transm. 40: 253-268. Cumings, J. N. (1948). Copper and iron content of brain and liver in normal and hepato-lenticular degeneration. Brain 71: 41@415 (1948). Cumings, J. N. (1968). Trace metals in the brain and Wilson’s Disease. J. C/in. Path. 21; l-7. Curzon, G. and Green, A. R. (1970). Rapid method for the determination of 5-hydroxytryptamine and 5-hydroxyindoleacetic acid in small regions of rat brain. Br. J. Pharmat. 39: 653-655.
Dallman. P. R. (1974). Tissue effects of iron deficiency. In: Iron in Biochemistry and Medicine (Jacobs, A. and Worwood, M., Eds), pp. 437-475. Academic Press, London. Dallman. P. R. and Schwartz. H. C. (1965). Distribution of cytochrome C and myoglobin in rats with dietary irondeficiency. Pediatrics 35: 677-685. Dallman, P. R.. Simes, M. A. and Manies, E. C. (1975). Brain iron: persistent deficiency following short term iron deprivation in the young rat. Br. J. Haemat. 31: 209-2
15.
Dietrich. R. A. (1966). Tissue and subcellular distribution of mammalian aldehyde-oxidising capacity. Biochem. Pharmac.
15: 191 I-1922.
Denckla, W. D. and Dewey. H. K. (1967). The determination of tryptophan in plasma, liver and urine. J. Lab. C/in. Med. 69: 16&169. Edgerton, V. R., Bryant, S. L. and Gillespie, C. A. (1972). Iron deficiency anaemia and physical performance and activity of rats. J. Nutr. 102: 381-395. Everett, G. M.. Weigland, R. G. and Rinaldi. R. U. (1963). Pharmacologic studies of some non-hydrazine MAO inhibitors. Ann. N.Y. Acad. Sci. 107: 1068&1077. Garby, L. (1973). Iron deficiency: definition and prevalence. In: Clinics in Haemarolo~p, (Callender, S. T.: Ed.), Vol. 2. VD. 245-257. W. B. Saunders. London. Glover. J.-gnd Jacobs. A. (1974). Actiiity pattern of irondeficient rats. Br. Med. .I. 2: 627-628. Grahame-Smith, D. G. (1971a). Studies in Go on the relationship between brain tryptophan. brain S-HT synthesis and hyperactivity in rats treated with a monoamine oxidase inhibitor and L-tryptophan. J. Neurochem. 18: 1053-1066.
Grahame-Smith, D. G. (197lb). Inhibitory effect of chlorpromzine on the syndrome of hyperactivity produced by L-tryptophan or 5-methoxy-N-dimethyltryptamine in rats treated with a monoamine oxidaie inhibitor. Br. J. Pharmac.
43: 856864.
Grahame-Smith, D. G. (1972). The prevention by protein synthesis inhibitors of brain protein synthesis of the hyperactivity produced in rats by monoamine oxidase inhibition and the administration of L-tryptophan or S-methoxy,N.N-dimethyltryptamine. J. Neurochem. 19: 2409-2422.
Green, A. R. and Grahame-Smith, D. G. (1974). The role of brain dopamine in the hyperactivity syndrome produced by increased 5-hydroxytryptamine synthesis in rats. Neuropharmacology
13: 949-959.
drome produced by increased S-hydroxytryptamine synthesis in rats. J. Neural Transm. 39: 103-l 12. Green, A. R., Heal, D. J., Grahame-Smith, D. G. and Kelly, P. H. (1976). The contrasting actions of TRH and cycloheximide in altering the effects of centrally acting drugs: evidence for the non-involvement of dopamine sensitive adenylate cyclase. Neuropharmacology 15: 591-599. Hallgren, B. and Sourander, P. (1958). The effect of age on the nonhaem iron in the human brain. J. Neurochem. 3: 41-51.
Harrison, W. W., Netsky, M. G. and Brown, M. D. (1968). Trace elements in human brain, copper. zinc. iron and magnesium. Clin. Chim. Acta 21: 55-60. Ichiyama, A.. Nakamura, S., Nishizuka, Y. and Hayaishi. 0. (I 970). Tryptophan-5-hydroxylase in mammalian brain. Adv. Pharmac. 6A: 5-17. Jacobs, A. and Worwood. M. (Eds) (1974). Iron in Biochemistry and Medicine. Academic Press. London. Kebabian, J. W., Petzold, G. L. and Greengard, P. (1972). Dopamine-sensitive adenyl cyclase in the caudate nucleus of the rat brain and its similarity to the “dopamine receptor”. Proc. natn. Acad. Sci., U.S.A. 69: 2143-2149. Kessner. J. and Kalk, L. (1973). Strategy for Evaluating Health SeruiceS. Institute of Medicine, National Academy of Sciences, Washington DC. Lovenberg, W., Jequier, E. and Sjoerdsma, A. (1968) Tryptophan hydroxylation in mammalian systems. Adtr. Pharmat. 6A: 21-36. Lowry. 0. H., Rosebrough, N. H., Farr, A. and Randall. R. L. (1951) Protein measurement with the Folin phenol reagent. .I. Biol. Chem. 193: 265-276. Mackler. B.. Parson. R.. Miller. L. R.. Inamdar. A. R. and Finch, C. A. (1978). Iron deficiency in the rat. Biochemical studies of brain metabolism. Pediat. Rex 12: 2 17-220.
McCall, M. G., Newman, G. E.. O’Brien, J. R. P., Valberg. L. S. and Witts, L. J. (1962). Studies in iron metabolism. I. The experimental production of iron deficiency in the growing rat. Br. J. Nurr. 16: 297-304. Musacchio, J. M. (1975). Enzymes involved in the biosynthesis and degradation of catecholamines. In: Handbook of Psychopharmacology, Amines (Iversen, L. L..
Vol 3: Biochemistry
oj Biogenic
Iversen, S. D. and Snyder, S. H.. Eds), pp. l-26. Plenum Press, New York. Neff, N. H. and Tozer. T. N. (1968). 1n viro measurement of brain serotonin turnover. Adc. Pharmac. 6A: 97-109. Pennington, R. J. (1961) Biochemistry of dystrophic muscle mitochondrial succinate-tetrazolium reductase and adenosine triphosphate. Biochem. J. 80: 649454. Pollitt, E. and Liebel, R. L. (1976). Iron deficiency and behavior J. Pediar. 88: 372-381. Quik. A. and Sourkes, T. L. (1977). The effect of chronic iron deficiency on adrenal tyrosine hydroxylase activity. Can. J. Biochem.
55: 60-65.
Rajan, K. S., Colburn, R. W. and Davis, J. M. (1976). Distribution of metal ions in the subcellular fractions of several rat brain areas. Life Sci. 18: 423431. Ramsay, W. N. M. (1957). The determination of the total iron binding capacity of serum. C/in. Chim. Acfa. 2: 221-226.
Green, A. R. and Grahame-Smith, D. G. (1976). Effects of Salach, J. (1979). Monoamine oxidase from beef liver mitodrugs on the processes regulating the functional activity chondria: simplified isolation procedure properties and of brain 5-hydroxytryptamine. Nature, Land. 260: determination of its cysternyl Ravin content. Archs Bio487-49 1. them. Biophys. 192: 128-137. Green, A. R. and Kelly, P. H. (1976). Evidence conSinger, T. P. (1976). Flauins and Flaooproteins. Elsevier. cerning the involvement of 5-hydroxytryptamine in Amsterdam. the locomotor activity produced by amphetamine or Sourkes, T. L. (1972) Psychopharmacology. In: Basic tranylcypromine plus L-DOPA. Br. J. Pharmac. 57: 1 Neurochemistry (Albers, R. W., Siegel, G. J., Katzman, R. 141-147. and Agranoff, B. W.. Eds), pp. 581-606. Little Brown. Green, A. R., Tordoff, A. F. C. and Bloornfteld, M. R. Boston. (1976) Elevation of brain GABA concentrations with Symes, A. L., Sourkes. T. L., Youdim, M. B. H., Birnbaum. amino-oxyacetic acid; effect on the hyperactivity synH. and Gregoriades, G. (1969). Decreased monoamine
Effects of iron deficiency in rat brain oxidase activity in liver of iron-deficient
Waalkes, T. P. and Udenfriend, S. (1957). A flurometric method for the estimation of tyrosine in plasma and Svmes. A. L.. Missala. K. and Sourkes, T. L. (1971). Irontissues. J. Lab. Clin. Med. 50: 733-736. and riboflavin-dependent metabolism of a monoamine in Youdim, M. B. H. (1975). Assay and purification of brain the rat in oi~o. Science, Wash. 174: 153-155. monoamine oxidase. In: Research Methods in NeuroTamir. H., Klein. A. and Rapport, M. B. (1976). Serotonin chemistry, (Marks, M. and Rodnight, R., Eds), Vol. 3, pp. binding protein: enhancement of binding by Fe*+ and 167-208. Plenum Press, New York. inhibition of binding by drugs. J. Neurochem. 26: Youdim. M. B. H. (1976). Rat liver mitochondrial mono871-878. amine oxidase; an iron requiring flavoprotein. In: F/aTipton, K. F. and Youdim, M. B. H. (1976). Assay of oins and Flavoproteins (Singer, T. P.. Ed.), pp. 651-665. monoamine oxidase. In: Monoamine Oxidase and its Elsevier. Amsterdam. Inhibition. Ciba Foundation Series (Wolstenholme, Youdim, M. B. H.. Woods, H. F., Mitchell, B., GrahameG. E. W. and Knight, J., Eds), Vol. 32, pp. 393-402. Smith. D. G. and Callender. S. T. (1975a). Human Elsevier. Amsterdam. platelet monoamine oxidase activity in ironldeficiency Tong, J. H. and Kaufman, S. (1975). Tryptophan hydroxy- I anaemia. Chin. Sci. Mol. Med. 48: 283-295. lase: purification and some properties of the enzyme Youdim. M. B. H.. Hamon. M. and Bouraoin. S. (1975b). from rabbit hindbrain. J. biol. Chem. 250: 41524158. Properties of partially purified pig brain stem ‘tryptoUsdin. E.. Wiener. N. and Youdim. M. B. H. (1978). Strucphan hydroxylase. J. Neurochem. 25: 407-414. ture and function of Monoamine Enzymes. Dekker. New Youdim, M. B. H., Grahame-Smith, D. G. and Woods, H. F. York. (1976). Some properties of human platelet monoamine Voorhess. M. C., Stuart, M. H.. Stockman, J. A. and Oski, oxidase in iron-deficiency anaemia. C/in. Sci. Mol. Med. F. A. (1975). Iron-deficiency anaemia and increased uri50: 479-485. nary norepinephrine excretion. J. Pediat. 86: 542-547. Biochem. 47: 999-1003.
rats. Can. J.
267
THE EFFECTS OF IRON DEFICIENCY ON BRAIN BIOGENIC MONOAMINE BIOCHEMISTRY AND FUNCTION IN RATS M. B. H. YOUDIM*.A. R. GREEN,M. R. BLOOMFIELD. D. G. GRAHAME-SMITH
B. D. MITCHELL, D. J. HEAL and
MRC Unit and University Department of Clinical Pharmacology, Radcliffe Infirmary. Oxford OX2 6HE. England
(.&YqJrrd
15 Octohrr
1979)
Summary-Rats were made iron-deficient by feeding them on a semi-synthetic diet containing milk powder. low in iron. Control rats were fed the same diet with an iron-supplement. When the rats were iron deficient (haemoglobin 6.9 + 0.6g/dl) a study was made of the iron distribution in the brain. Distribution was similar in control and iron-deficient rats with the highest concentration in the caudate nucleus, hypothalamus and cerebellum, Sub-cellular distribution studies showed the highest concentration to be in the crude mitochondrial fraction. The iron-deficient group had an approx. 60% decrease in brain iron stores. Measurement was also made of the monoamine metabolising enzymes (MAO) in iron-deficient rats. Despite large decreases in peripheral MAO activity no decrease in central MAO activity was observed in these rats. Nor was there any decrease in brain tryptophan hydroxylase or aldehyde dehydrogenase activity. In very severe iron-deficient (haemoglobin 4.5 + 0.6g/dl) rats, brain MAO activity still decreased only by 15.70”,, while heart MAO activity decreased by over 60?:,, while there was still no change in tryptophan hydroxylase activity. Iron deficient rats showed decreased behavioural responses following increased brain 5-hydroxytryptamine (5-HT) synthesis or after administration of the 5-HT agonist 5-methoxy N,N-dimethyltryptamine. They also showed decreased behavioural responses following administration of tranylcypromine plus L-DOPA. methamphetamine or apomorphine. No change in brain tryptophan or 5-HT synthesis was observed although endogenous S-HT content was decreased. No changes were seen in brain dopamine or noradrenaline content, nor in the activity of dopamine-sensitive adenylate cyclase. Brain protein synthesis was also unaltered. Eight days after restoration of the iron plus diet the behavioural responses were normalised. but in,jection of ferrous gluconate (acutely) failed to restore the behaviour. Reasons for the altered post-synaptic monoamine behavioural responses being unlikely to be the result of anaemia are discussed and the relevance of decreased monoamine function in iron-deficiency is also discussed, particularly with regard to the high incidence of this nutritional disorder.
Despite
the fact that
iron
deficiency
is the most
com-
in the world (Kessner and Kalk, 1973; Garby, 1973) there has been little work on the effects of iron deficiency on brain metabolism and function. It has been known for some time that iron is present in relatively large amounts in the brain. particularly in the basal ganglia (Cumings. 1948; 1968; Hallgren -and Sourander. 1958; Harrison. Netsky and Brown, 1968) and its concentration there appears to be independent of the stores in the liver (Hallgren and Sourander, 1958; Dallman. 1974). The importance of iron in protein synthesis, as a co-factor for haem and non-haem iron containing enzymes and its structural role in many membranes and on membrane bound proteins is well documented (Jacobs and Worwood, 1974). Nutritional iron appears to be important for monoamine oxidase activity and the activity of this enzyme mon
nutritional
disorder
* Present address: Israel Institute of Technology--Technion. School of Medicine. Department of Pharmacology. I2 Haaliya Street, Bat-Galim. POB 9649, Haifa. Israel.
has been shown to be lowered in the tissues of humans and rats with iron deficiency anaemia (Symes. Sourkes, Youdim, Birnbaum and Gregoriadis 1969; Youdim, Woods, Mitchell, Grahame-Smith and Callender, 1975a; Youdim, Grahame-Smith and Woods, 1976; Voorhess, Stuart, Stockman and Oski, 1975; Symes, Missala and Sourkes, 1971) returning to normal only when the serum iron concentration returns to normal (Youdim et al., 1975a: Voorhess et al., 1975; Symes rt al., 1971). It has also been suggested that iron is a cofactor for tyrosine hydroxylase (Sourkes, 1972) and tryptophan hydroxylase (Lovenberg, Jequier and Sjoerdsma, 1968; Ichiyama. Nakamura, Nishizuka and Hayaishi. 1970; Youdim. Hamon and Bourgoin, 1975b). enzymes involved in the formation of catecholamines and %hydroxytryptamine respectively. The effects of iron deficiency in rats on the activity of various monoamine metabolising enzymes in the brain and the behavioural responses of the animals to increased brain 5hydroxytryptamine (5-HT) and dopamine (DA) receptor stimulation has been investi259
M. B. H. YOU~IMet al.
260
gated. The results suggest that while there is little alteration of either enzyme activity or rates of amine synthesis in iron deficient animals there is nevertheless a marked reduction of monoamine functional activity as measured by drugs that act pre- and postsynaptically. METHODS
Preparation
of iron dejcient
rats
Male Sprague-Dawley derived rats (initial weight 80 + 20 g, mean + SEM) were made iron-deficient by feeding a semi-synthetic diet of distilled water and milk powder low in iron (McCall, Newman, O’Brien, Valberg and Witts. 1962). Control rats were given tap water and the same milk powder with ammonium ferrous sulphate (1.3 mg/g diet) added. The iron deficient group were given an ad lihitum diet and the iron-plus group had their food intake restricted to that of the ironaeficient group. Investigations were performed on animals that had been on the diet for 5 weeks at which time they were iron-deficient as judged by their blood haemoglobin concentration [control rats: 14.1 x 0.44g/dl (II = 16); iron deficient rats: 5.55 k 0.42 g/d1 (n = 22)]. Measurement
of enzyme activities
Aldehyde dehydrogenase activity was measured in brain, liver and heart homogenates by the method of Dietrich (1966) using 3-indoleacetaldehyde as substitute. Tryptophan hydroxylase activity was measured in the brain stem supernatant preparation using tetrahydrobiopterin as a co-factor by the method of Tong and Kaufman (1975). The activity of monoamine oxidase in tissue homogenates was measured using as a substrate either kynuramine (Youdim. 1975) or [‘%I]-tyramine (Tipton and Youdim, 1976). Succinate dehydrogenase activity was determined by the method of Pennington (1961). Determination
qf’brain
monoamines
5-Hydroxytryptamine was assayed fluorometrically by the method of Curzon and Green (1970) and dopamine and noradrenaline by the method of Chang (1964). The rate of 5-hydroxytryptamine synthesis was examined by measuring the rate of 5-HT accumulation following monoamine oxidase inhibition as described by NelT and Tozer (1968). Determination
of’ bruin amino acids
Tryptophan was measured by the method Denckla and Dewey (1967) and tyrosine by method of Waalkes and Udenfriend (1957). Estimation
Greengard (1972) and cyclic AMP measured by the method of Brown. Albano. Ekins. Sgherzi and Tampion (1971). Protein protein
determination
qf cyclic .4 MP and aden~~lnte cyclase
Brain homogenates were prepared from normal and iron deficient rats as described by Green, Heal, Grahame-Smith and Kelly (1976). The homogenates were incubated with increasing concentrations of dopamine as described by Kebabian, Petzold and
of the role of‘
Bovine serum albumin was used as standard for determination of protein by the procedure of Lowry. Rosebrough, Farr and Randall (1951). Protein synthesis was examined by determining the rate of incorporation of [‘4C]-tyrosine into brain protein as described by Grahame-Smith (1972). Iron determination
Non-haem iron was measured in tissue homogenates by a slightly modified procedure of Ramsay (1957) using o-phenanthroline to chelate the iron. The preparation was heated for 6Omin at 100°C in the presence of 10% (w/v) trichloroacetic acid before the iron determination. Measurement
of monoamine
function
and behauioural
hyperactivity
During the last few years various animal models have been developed to assess monoamine functional activity, namely the response to transmitter which is qeleased, stimulates the receptor and produces a behavioural response. Much of this work has already been reviewed (Green and Grahame-Smith, 1976). The function of 5-HT was examined by observing the behavioural changes which occur following the intraperitoneal injection of tranylcypromine (20 mg/kg) followed 30min later by L-tryptophan (100 mg/kg). This procedure increases the rate of 5-HT synthesis and results in the appearance of various behavioural changes, one feature of which is hyperactivity, which is measured on LKB Animex activity meters. Previous studies have suggested that the degree of hyperactivity is proportional to the rate of increase in 5-HT synthesis and “spill-over” into functional activity in the brain (Grahame-Smith, 1971a; Green and Grahame-Smith. 1976). The functional activity of brain dopamine was examined by the administration of tranylcypromine (20 mg/kg) and L-DOPA a procedure which increases brain (50 mg/kg).
Table
1. Distribution
Brain areas
of the
and measurement
synthesis
Whole brain Cerebellum Brain stem Caudate nucleus Amygdalla Cerebral cortex Hypothalamus
of non-haem rat brain
iron in various areas of
11g Fe,img protein 0.074 0.075 0.052 0.092 0.058 0.050 0.076
* + + + f * +
The results shown are the means + SEM mals. Whole brain non-haem concentration cient animals was 0.034 + 0.006 (6).
0.008 0.008 0.008 0.036 0.005 0.003 0.030 from
6 ani-
in iron defi-
Effects of iron deficiency in rat brain
261
dopamine concentration and results in the rats disyielding nuclear fraction (PI), crude mitochondrial playing hyperactivity which can also be measured on fraction (P2) and a microsomal-supernatant fraction (PI + S) demonstrated the presence of non-haem iron activity meters (Everett, Weigland and Rinaldi, 1963; in all fractions P,:8%; P,:55%; P3 + S:37%. The Green and Kelly, 1976). crude mitochondrial fraction contained the highest Postsynaptic 5-HT-mediated responses were concentration of non-haem iron/mg protein. measured by examining the degree of hyperactivity Iron-deficient rats had a total brain non-haem iron which follows administration of the putative agonist 5-methoxy N,N-dimethyltryptamine (Grahame-Smith. ’ concentration of 0.030 + 0.006 pg/mg protein, showing a 60% fall in non-haem iron. However the per1971b) while postsynaptic dopamine function was centage distribution between the fractions was the measured by examining the locomotor activity which same as in the control rats showing that iron defifollows administration of either the dopamine releasciency has no specific effects on the subcellular distriing drug methamphetamine or the agonist apomorbution of iron. phine. The behavioural changes following administration Monoamine metabolising enzymes during iron defof the various treatments outlined above were moniciency tored in groups of 3 animals in cages placed on LKB In rats with haemoglobin values of 6.9 f 0.6g/dl, Animex activity meters (sensitivity and tuning 30 PA). no decrease in brain tryptophan hydroxylase was Results show the data obtained from at least 3 separfound (Fig. 1). Nor was brain MAO activity lowered, ate experiments and are compared using the Student’s f-test. even though a large decrease in the activity of this enzyme was observed peripherally in the liver, heart RESULTS and adrenal gland (Fig. 1). The MAO activity was not Distribution of non-haemiron in the brain decreased in the spleen. Aldehyde dehydrogenase acIt was found that normal rat brain contained a tivity was unaltered in brain, liver and heart (Fig. 1). Therefore, severe iron deficiency was induced in mean concentration of 0.074 + 0.008 pg non-haem another group of rats by keeping them on the ironiron/mg protein and the regional distribution is deficient diet for 9 weeks. This resulted in haemoshown in Table 1. Higher values were seen in the globin concentrations of 4.5 + 0.6 g/dl. There was still caudate nucleus, hypothalamus, and cerebellum. no decrease in tryptophan hydroxylase activity in the Fractionation of control rat brains in 0.32 M sucrose,
Monoamine
oxidase
Aldehyde pehydroge~;
Tryptophan Hydraxyla:
Succinic Dehydragenase
Haemaglobin
Fig. I. The effect of iron deficiency on rat tissue monoamine metabolising enzymes. Results show the % activity of the enzymes compared to control animals. Stippled columns show enzyme activities in animals made iron deficient by placing them on iron deficient diet for 5 weeks (haemoglobin value in stippled column on right). Cross-hatched columns show enzyme activities in rats kept on the iron deficient diet for 9 weeks (haemoglobin value in lined column on right). Control haemoglobin value is also shown. Control monoamine oxidase activity values were as follows (all values expressed in nmol product formed/mg protein/min at 37°C). Brain 1.06 k 0.11; Liver 2.74 f 0.14; Heart 0.78 k 0.05; Adrenal gland 0.49 & 0.03; Spleen 0.13 + 0.04 (n < IO). Aldehyde dehydrogenase activity control values were as follows (expressed in same. units as MAO activity, see above): Brain 0.71 + 0.04; Liver 1.62 + 0.25; Heart 0.49 &-0.03 (n = 4). Tryptophan hydroxylase activity (using tetrahydrobiopterin (BH,) as cofactor) in the brain stem of control animals (expressed in nmol product formed/mg protein/ min at 37°C) was 0.16 f 0.02 (n = 6). Succinic dehydrogenase activity control values (expressed in nmol product formed per mg protein per min at 37°C) were Brain 13.40 + 1.81; Liver 54.50 f 3.10; Heart 20.1 I f 2.10 (n = 6). *P < 0.05; l*P < 0.02; tP < 0.01; $P < 0.001.
262
M. B. H.
8 x
YOUDIM
et u/
60-
-
Fig. 2. The total movements of control (stippled columns) and iron deficient (cross-hatched columns) rats during the 90 min after t_-tryptophan (100mg/kg) or methamphetamine (2 mg/kg) 60 min after L-DOPA) (50 mg/kg) and 40 min after 5-methoxy NJ-dimethyltryptamine (2 mg/kg) or apomorphine (2 mg,kg). The t_-tryptophan. L-DOPA and 5-MeODMT injected rats were pretreated 30 min before with tranylcypromine (20mg/kg). Also shown is the total activity of control and iron deficient rats during the overnight period (I 8 hr 00 min- 10 hr 00 min) the animals being on a 12 hr light 12 hr dark period (18 hr 00 min-06 hr 00 min). Results show mean + SEM of 3 separate observations. Different from control rats *P < 0.01; **p < 0.025; tP < 0.05.
brain. nificant
There
were.
decreases
however. of brain
small (155ZO”,,) but sigMAO
and
succinate
de-
hydrogenase activities (Fig. I). In other tissues MAO activity showed an even greater decrease than seen previously. A similar reduction in the activity of the iron-containing flavoprotein succinate dehydrogenase in the periphery was also seen (Fig. I). Thus. brain MAO and succinate dehydrogenase activities were little altered in animals in which the non-
haem
iron
even
though
the
of the brain activities
pheral
tissues
were
I$kfs
of’ irorl d
were
depleted
by 60”/,,
of these
enzymes
in peri-
markedly
hrcGt 5-H T ant! llopurnirtr
diminished.
011 the Jitrrctionul urtd mortoc~mine
of rat brain
5-hydroxytryp-
Fe deficient
Control 3.33 f 0.26 (4)
3.69 + 0.13(5)
0.3X & 0.02 (6)
0.31 + 0.02 (9)*
0.51 * 0.02 (4)
0.42 * 0.01 (5)*
0.13
0.1 I
m gl Brain 5-HT (/cg 5-HT,g) Brain 5-HT 60 min after tranylcypromine l/(g 5-HT’g) Rate of 5-HT svnthesis (kcp 5-HT gei hr-I) 5-HT accumulation after tranylcypromine/L-Tryp (1.195-HT/g)
acti&!:
0.95 + 0.07
(I I )
0.91 + 0.06 (7)
Tryptophan and S-HT results expressed as /rg compound/g brain (wet wt). Results expressed as mean f SEM with number of observations in brackets. The rate of 5-HT synthesis determined by the a,ccumulation of 5-HT over control during 60 min following tranylcypromine (Neff and Tozer, 1968). Haemoglobin values in these animals given in Table 3. 5-HT accumulation measured 60 min after L-tryptophan (100mg/kg). tranylcypromine (20 mg/kg) being given 30 min before tryptophan. * Different from control P < 0.01.
oj
synthesis
To assess changes in monoamine function behavioural models were used (see Methods).
Table 2. The effect of iron deficiency on the synthesis tamine (5-HT)
Brain tryptophan
stores
various
Etfects of iron deficiency
263
in rat brain
Table 3. Effect of iron deficiency on brain catecholamine concentrations Control Brain dopamine (pg/g) Brain noradrenaline (pg/g) Brain dopamine after tranylcypromine/DOPA Brain noradrenaline after tranylcypromine/DOPA Haemoglobin (g/dl)
Fe deficient
2.03 + 0.24(6) 0.19 + 0.01 (4) 5.72 & 0.32 (5)
1.74 k 0.13 (8) 0.20 + 0.01 (5) 5.75 & 0.57 (6)
0.54 k 0.07 (6)
0.55 k 0.04 (6)
14.1 *0.44(16)
5.55 + 0.42 (22)*
All results expressed as pg catecholamine/g brain (wet wt). Results expressed as mean + SEM with number of observations in brackets. Accumulation of dopamine and noradrenaline measured 60 min after L-DOPA (60 mg/kg), tranylcypromine (20 mg/kg) having been given 30 min before the L-DOPA. * Different from control P < 0.001.
Following tranylcypromine/L-tryptophan administration the iron-deficient rats showed markedly less hyperactivity than the control animals (Fig. 2). However the accumulation of S-HT in the brain following tranylcypromine/L-tryptophan was the same in both groups as was the rate of synthesis (Table 2), supporting the finding that iron deficiency does not affect tryptophan hydroxylase activity. While the concentration of tryptophan in the brain was not changed by iron deficiency the endogenous concentration of brain 5-HT was decreased (Table 2). As the synthesis of 5-HT was not inhibited it seemed probable that the decreased activity was due to a decreased post-synaptic response to 5-HT. This view was strengthened by the observation that the
behavioural response to the suggested 5-HT agonist 5-methoxy, N,N-dimethyltryptamine, was diminished significantly in the iron-deficient animals (Fig. 2). Since a group of dopaminergic neurones lies between the 5-HT neurones initiating the hyperactivity response and those mechanisms responsible for its behavioural expression (Green and GrahameSmith, 1974) it was possible that the altered 5-HT response was due to alterations in dopamine function. Alterations in dopamine function are indeed present in iron-deficient animals since their response to tranylcypromine/L-DOPA administration was markedly diminished (Fig. 2). Again this was not due to altered transmitter synthesis, because dopamine and noradrenaline concentrations and accumulation after tranylcypromine and L-DOPA administration were similar in both groups (Table 3). It was also possible to demonstrate by other approaches that iron-deficiency inhibited dopaminemediated behavioural responses. It was found that the locomotor responses of these animals to either the dopamine-releasing drug methamphetamine (2 mg/kg) or the putative dopamine agonist apomorphine (2 mg/kg) was inhibited (Fig. 2). Eflecr of iron deficiency on the brain adenylate cyclase system
,cL Dopomine
J
10-d
(M )
Fig. 3. The response of dopamine sensitive adenylate cyclase to dopamine in homogenates of normal (0) and irondeficient (m) rats. Homogenates prepared from normal and iron deficient rats were incubated with increasing concentrations of dopamine as described in Methods. Basal activity of two groups, control: 68.0 f 1.4 pmol/2 mg tissue (wet wt) per 2.5min; iron deficient 62.6 rt 2.4pmol/2mg tissue (wet wt) per 2.5 min (6 animals in each group). N.P.19/3+
The response with apomorphine suggests that irondeficiency causes an inhibition of the behavioural response to increased functional activity of DA by an action at or beyond the post-synaptic site. This does not appear to be due to an alteration in the sensitivity of the dopamine-sensitive adenylate cyclase system, since the response of caudate nucleus dopamine sensitive adenylate cyclase in vitro to increasing concentrations of dopamine was the same in both the control and iron-deficient rats (Fig. 3). Effect of an iron rich diet to iron deficient rats on the monoamine mediated behavioural responses
When the iron-deficient rats were given the ironrich (control) diet for 8 days the hyperactivity responses to tranylcypromine/L-tryptophan and tranylcypromine/L-DOPA returned to normal (Fig. 4) as
M. B. H. YOUVIMrr al.
264 (A)
(8)
t. -Trp
L -DCf’A Time
( min I
Fig. 4. The behavioural effect of injecting L-tryptophan or L-DOPA after a dose of tranylcypromine hyperactivity in iron-deficient rats fed iron plus diet. Iron deficient rats were placed on iron plus diet 8 days (0) and then injected with tranylcypromine (20mg/kg) followed 30min later with either L-tryptophan (100 mg/kg) or (b) L-DOPA (50 mg/kg). Control rats (iron plus diet only) also shown Activity was measured as movements/min.
did the responses to amphetamine and apomorphine. At this time the haemoglobin value had also increased (12.2 &- 0.64 g/dl). In contrast, an intraperitoneal injection of a large dose of iron (ferrous gluconate 20 mg/kg; “Jectofer”) did not restore the hyperactivity response to tranylcypromine and L-tryptophan 48 hr later. Overnight
locomotor
activity
Glover and Jacobs (1974) reported that iron-deficiency in rats resulted in an altered pattern of spontaneous activity. The animals were less active during the dark period and more active during the light period than control rats. These findings were confirmed with respect to dark period activity (Fig. 2). Protein
synthesis
Previously, Grahame-Smith (1972) found that inhibiting protein synthesis in rat by injection of cycloheximide resulted in attenuated hyperactivity responses to tranylcypromine/L-tryptophan and to S-MeODMT. The possibility was therefore examined that the changed behavioural responses of the irondeficient rats was due to a change in the rate of brain protein synthesis. No statistically significant differences between the rate of incorporation of the radioactivity into protein were observed between the control and iron-deficient groups. DISCUSSION
The brain non-haem good agreement with Manies (1975) and the ties between rat and trations found in
iron concentration data was in that of Dalhnan, Simes and distribution indicated similarihuman since in man concenbasal ganglia are high
on for (a) (0).
(0.15-0.48 mg/g brain, fresh wet wt-see Hallgren and Sourander, 1958; Cumings, 1948, 1968; Harrison et al., 1968) and are of the same order as the concentration found in the liver (Hallgren and Sourander. 1958; Dallman, 1974). The percentage distribution following fractionation of rat brain was also similar to that seen in human brain (Hallgren and Sourander, 1958) with the mitochondrial fraction containing the highest, concentration (Colburn and Mass. 1965: Rajan, Colburn and Davis, 1976). Iron has been implicated as a cofactor for tryptophan hydroxylase (Lovenberg er al., 1968; Ichiyama et al., 1968; Youdim et al., 1975b; Usdin, Wiener and Youdim. 1978) and tyrosine hydroxylase (Sourkes, 1972; Usdin et al., 1978; Musacchio, 1975) and has been suggested to be necessary for the synthesis of the active form of MAO (Youdim et al., 1976; Youdim, 1975). A deficiency of iron might therefore be expected to result in a lowered activity of these enzymes which would in turn alter monoamine synthesis and perhaps function. However. even in animals with a 60% depletion of non-haem iron both brain succinate dehydrogenase and MAO activities were little altered, even when the activities of these enzymes in peripheral stores were markedly lowered. These results are reminiscent of those from riboflavin deficiency studies. Although it is known that MAO (Youdim, 1976) and succinate dehydrogenase (Singer, 1976) contain FAD as co-factor, riboflavin deficiency in rats does not alter the activities of these enzymes in the brain (Youdim, 1976) although a significant decrease in enzyme activity is seen in the liver. The present results suggest that either iron is still present in sufficient quantities to act as a co-factor, or that the enzymes do not require iron for full activity.
265
Effects of iron deficiency in rat brain In the case of MAO and succinate dehydrogenase the latter interpretation seems unlikely in view of the changes in enzymic activities seen in peripheral tissues and the reported presence of iron in purified enzyme preparations (Youdim, 1976; Salach, 1979). The activities of brain enzymes which may be iron dependent appear, therefore, to be very resistant to the effect of iron-deficiency. Although tyrosine hydroxylase activity was not measured, Quick and Sourkes (1977) have shown that the activity of this enzyme in adrenal gland does not decrease in iron deficiency and our data on catecholamine synthesis in iron-deficient rats support this view. Finally, if iron is involved in tyrosine and tryptophan hydroxylases, it may be bound in some covalent form that simple nutritional deficiency will not displace. An increase in brain 5-HT content in iron-deficient animals has recently been reported by Mackler, Parson, Miller, Inamdar and Finch (1978). In contrast a significant decrease in the endogenous brain 5-HT content was found in the current study and may well be due to decreased 5-HT binding and storage at the nerve endings since ferrous iron has been shown to be involved selectively in this process (Tamir, Klein and Rapport, 1976). The behavioural studies demonstrated that irondeficiency markedly inhibits both 5-HT and dopaminemediated behaviours. The data on amine synthesis suggests that the alteration in behaviours is due to a postsynaptically mediated change and this was confirmed by the use of appropriate agonists. It is probable that this change is not occurring at the receptor site itself since adenylate cyclase activity did not change. It is more likely that the effect of iron deficiency involves changes “beyond” the receptor. The mechanism may therefore involve alterations in the function of other modulatory transmitter systems involved in the behaviours resulting from increased monoamine function. Alterations in monoaminemediated behaviour by altering the concentration or function of other neurotransmitters have been widely reported (e.g. Green, Tordoff and Bloomfield, 1976; Cott and Engel, 1977). One major problem of interpretation of the behavioural data is that the behaviour examined involves locomotor activity and iron-deficiency produces anaemia. However, it is unlikely that the changes observed are due to oxygen deficit. If this were so then the activity would not merely be less but would decrease further as the experiment progressed. However it was observed that the lowered hyperactivity response not only continues for over 2 hr after injection of the tranylcypromine/L-tryptophan but indeed increases with time. Also administration of a further injection of 5-MeODMT (3 mg/kg) 15 min after the first dose does not increase the locomotor response further but does result in the rats remaining active for a considerable period of time (over 90 min). Secondly Dallman (1974) has reported that isolated
muscle from iron-deficient rats is able to contract normally and Edgerton, Bryant and Gillespie (1972) were unable to demonstrate a relationship between. physical performance and muscle content of myoglobin and cytochromes. Furthermore, the behavioural response returned to normal after 8 days whereas Dallman and Schwartz (1965) have shown that the reversal of skeletal muscle deficiency of myoglobin and cytochrome may require a period six times longer than that required to correct the haemoglobin concentration (which was still not quite restored in the present animals after 8 days on the iron-rich diet). It is therefore likely that the lowered activity response is neither due to anaemia nor altered muscle function. The data of Dallman et al. (1975) suggests that nonhaem iron stores in the brain are only slowly restored in iron-deficient rats given an iron-plus diet. The present data on the effect of the iron-plus diet supports the conclusion that restoration of normal behavioural responses involves changes other than merely restoring whole body iron, and to data with ferrous gluconate suggests that the iron has to be incorporated in some way before there is restoration of monoaminemediated responses. The relatively few studies on the effects of iron deficiency on behaviour in animals and man have mainly been concerned with psychological test performances and the methodology has been questioned in several instances (see review by Pollitt and Leibel, 1976). However it is interesting that some of the changes observed did not appear to be due to the anaemia per se, but rather other metabolic correlates of iron depletion (Pollitt and Leibel, 1976). The neurotransmitters 5_hydroxytryptamine, dopamine and noradrenaline are involved in various neurological and psychiatric disorders such as depression, schizophrenia and Parkinson’s Disease and presumably therefore are intimately concerned in the regulation of mood and neuronal activity. The present results therefore indicate that the behavioural changes seen in people with iron-deficiency may not be a matter simply of the anaemia but also the result of neurotransmitter changes in the brain. Such changes merit further investigation in view of the prevalence of iron-deficiency in the world, particularly in young children (Pollitt and Leibel, 1976) and its possible long term effects on brain neurotransmitter systems.
REFERENCES
Brown, B. L., Albano, J. D. M., Ekins, R. P., Sgherzi, A. M. and Tampion, W. (1971) A simple and sensitive satura. tion assay method for the method of measurement of adenosine 3’5’ cyclic monophosphate. Biochem. J. 121:
561-562. Chang, C. C. (1964). A sensitive method for spectrophotofluorometric assay of catecholamines. Int. J. Neuropharmat. 3: 643649. Colburn, R. W. and Mass, J. W. (1965). Adenosine triphosphate-metanorepinephrine ternary complexes and catechol&ine binding. Nature, Lond. 208: 3742.
M. B. H. YOUIXMet al.
266
Cott, J. and Engel, J. (1977). Suppression by GABAergic drugs of the locomotor stimulation induced by morphine, amphetamine and apomorphine: evidence for both pre- and post-synaptic inhibition of catecholamine systems. J. Neural Transm. 40: 253-268. Cumings, J. N. (1948). Copper and iron content of brain and liver in normal and hepato-lenticular degeneration. Brain 71: 41@415 (1948). Cumings, J. N. (1968). Trace metals in the brain and Wilson’s Disease. J. C/in. Path. 21; l-7. Curzon, G. and Green, A. R. (1970). Rapid method for the determination of 5-hydroxytryptamine and 5-hydroxyindoleacetic acid in small regions of rat brain. Br. J. Pharmat. 39: 653-655.
Dallman. P. R. (1974). Tissue effects of iron deficiency. In: Iron in Biochemistry and Medicine (Jacobs, A. and Worwood, M., Eds), pp. 437-475. Academic Press, London. Dallman. P. R. and Schwartz. H. C. (1965). Distribution of cytochrome C and myoglobin in rats with dietary irondeficiency. Pediatrics 35: 677-685. Dallman, P. R.. Simes, M. A. and Manies, E. C. (1975). Brain iron: persistent deficiency following short term iron deprivation in the young rat. Br. J. Haemat. 31: 209-2
15.
Dietrich. R. A. (1966). Tissue and subcellular distribution of mammalian aldehyde-oxidising capacity. Biochem. Pharmac.
15: 191 I-1922.
Denckla, W. D. and Dewey. H. K. (1967). The determination of tryptophan in plasma, liver and urine. J. Lab. C/in. Med. 69: 16&169. Edgerton, V. R., Bryant, S. L. and Gillespie, C. A. (1972). Iron deficiency anaemia and physical performance and activity of rats. J. Nutr. 102: 381-395. Everett, G. M.. Weigland, R. G. and Rinaldi. R. U. (1963). Pharmacologic studies of some non-hydrazine MAO inhibitors. Ann. N.Y. Acad. Sci. 107: 1068&1077. Garby, L. (1973). Iron deficiency: definition and prevalence. In: Clinics in Haemarolo~p, (Callender, S. T.: Ed.), Vol. 2. VD. 245-257. W. B. Saunders. London. Glover. J.-gnd Jacobs. A. (1974). Actiiity pattern of irondeficient rats. Br. Med. .I. 2: 627-628. Grahame-Smith, D. G. (1971a). Studies in Go on the relationship between brain tryptophan. brain S-HT synthesis and hyperactivity in rats treated with a monoamine oxidase inhibitor and L-tryptophan. J. Neurochem. 18: 1053-1066.
Grahame-Smith, D. G. (197lb). Inhibitory effect of chlorpromzine on the syndrome of hyperactivity produced by L-tryptophan or 5-methoxy-N-dimethyltryptamine in rats treated with a monoamine oxidaie inhibitor. Br. J. Pharmac.
43: 856864.
Grahame-Smith, D. G. (1972). The prevention by protein synthesis inhibitors of brain protein synthesis of the hyperactivity produced in rats by monoamine oxidase inhibition and the administration of L-tryptophan or S-methoxy,N.N-dimethyltryptamine. J. Neurochem. 19: 2409-2422.
Green, A. R. and Grahame-Smith, D. G. (1974). The role of brain dopamine in the hyperactivity syndrome produced by increased 5-hydroxytryptamine synthesis in rats. Neuropharmacology
13: 949-959.
drome produced by increased S-hydroxytryptamine synthesis in rats. J. Neural Transm. 39: 103-l 12. Green, A. R., Heal, D. J., Grahame-Smith, D. G. and Kelly, P. H. (1976). The contrasting actions of TRH and cycloheximide in altering the effects of centrally acting drugs: evidence for the non-involvement of dopamine sensitive adenylate cyclase. Neuropharmacology 15: 591-599. Hallgren, B. and Sourander, P. (1958). The effect of age on the nonhaem iron in the human brain. J. Neurochem. 3: 41-51.
Harrison, W. W., Netsky, M. G. and Brown, M. D. (1968). Trace elements in human brain, copper. zinc. iron and magnesium. Clin. Chim. Acta 21: 55-60. Ichiyama, A.. Nakamura, S., Nishizuka, Y. and Hayaishi. 0. (I 970). Tryptophan-5-hydroxylase in mammalian brain. Adv. Pharmac. 6A: 5-17. Jacobs, A. and Worwood. M. (Eds) (1974). Iron in Biochemistry and Medicine. Academic Press. London. Kebabian, J. W., Petzold, G. L. and Greengard, P. (1972). Dopamine-sensitive adenyl cyclase in the caudate nucleus of the rat brain and its similarity to the “dopamine receptor”. Proc. natn. Acad. Sci., U.S.A. 69: 2143-2149. Kessner. J. and Kalk, L. (1973). Strategy for Evaluating Health SeruiceS. Institute of Medicine, National Academy of Sciences, Washington DC. Lovenberg, W., Jequier, E. and Sjoerdsma, A. (1968) Tryptophan hydroxylation in mammalian systems. Adtr. Pharmat. 6A: 21-36. Lowry. 0. H., Rosebrough, N. H., Farr, A. and Randall. R. L. (1951) Protein measurement with the Folin phenol reagent. .I. Biol. Chem. 193: 265-276. Mackler. B.. Parson. R.. Miller. L. R.. Inamdar. A. R. and Finch, C. A. (1978). Iron deficiency in the rat. Biochemical studies of brain metabolism. Pediat. Rex 12: 2 17-220.
McCall, M. G., Newman, G. E.. O’Brien, J. R. P., Valberg. L. S. and Witts, L. J. (1962). Studies in iron metabolism. I. The experimental production of iron deficiency in the growing rat. Br. J. Nurr. 16: 297-304. Musacchio, J. M. (1975). Enzymes involved in the biosynthesis and degradation of catecholamines. In: Handbook of Psychopharmacology, Amines (Iversen, L. L..
Vol 3: Biochemistry
oj Biogenic
Iversen, S. D. and Snyder, S. H.. Eds), pp. l-26. Plenum Press, New York. Neff, N. H. and Tozer. T. N. (1968). 1n viro measurement of brain serotonin turnover. Adc. Pharmac. 6A: 97-109. Pennington, R. J. (1961) Biochemistry of dystrophic muscle mitochondrial succinate-tetrazolium reductase and adenosine triphosphate. Biochem. J. 80: 649454. Pollitt, E. and Liebel, R. L. (1976). Iron deficiency and behavior J. Pediar. 88: 372-381. Quik. A. and Sourkes, T. L. (1977). The effect of chronic iron deficiency on adrenal tyrosine hydroxylase activity. Can. J. Biochem.
55: 60-65.
Rajan, K. S., Colburn, R. W. and Davis, J. M. (1976). Distribution of metal ions in the subcellular fractions of several rat brain areas. Life Sci. 18: 423431. Ramsay, W. N. M. (1957). The determination of the total iron binding capacity of serum. C/in. Chim. Acfa. 2: 221-226.
Green, A. R. and Grahame-Smith, D. G. (1976). Effects of Salach, J. (1979). Monoamine oxidase from beef liver mitodrugs on the processes regulating the functional activity chondria: simplified isolation procedure properties and of brain 5-hydroxytryptamine. Nature, Land. 260: determination of its cysternyl Ravin content. Archs Bio487-49 1. them. Biophys. 192: 128-137. Green, A. R. and Kelly, P. H. (1976). Evidence conSinger, T. P. (1976). Flauins and Flaooproteins. Elsevier. cerning the involvement of 5-hydroxytryptamine in Amsterdam. the locomotor activity produced by amphetamine or Sourkes, T. L. (1972) Psychopharmacology. In: Basic tranylcypromine plus L-DOPA. Br. J. Pharmac. 57: 1 Neurochemistry (Albers, R. W., Siegel, G. J., Katzman, R. 141-147. and Agranoff, B. W.. Eds), pp. 581-606. Little Brown. Green, A. R., Tordoff, A. F. C. and Bloornfteld, M. R. Boston. (1976) Elevation of brain GABA concentrations with Symes, A. L., Sourkes. T. L., Youdim, M. B. H., Birnbaum. amino-oxyacetic acid; effect on the hyperactivity synH. and Gregoriades, G. (1969). Decreased monoamine
Effects of iron deficiency in rat brain oxidase activity in liver of iron-deficient
Waalkes, T. P. and Udenfriend, S. (1957). A flurometric method for the estimation of tyrosine in plasma and Svmes. A. L.. Missala. K. and Sourkes, T. L. (1971). Irontissues. J. Lab. Clin. Med. 50: 733-736. and riboflavin-dependent metabolism of a monoamine in Youdim, M. B. H. (1975). Assay and purification of brain the rat in oi~o. Science, Wash. 174: 153-155. monoamine oxidase. In: Research Methods in NeuroTamir. H., Klein. A. and Rapport, M. B. (1976). Serotonin chemistry, (Marks, M. and Rodnight, R., Eds), Vol. 3, pp. binding protein: enhancement of binding by Fe*+ and 167-208. Plenum Press, New York. inhibition of binding by drugs. J. Neurochem. 26: Youdim. M. B. H. (1976). Rat liver mitochondrial mono871-878. amine oxidase; an iron requiring flavoprotein. In: F/aTipton, K. F. and Youdim, M. B. H. (1976). Assay of oins and Flavoproteins (Singer, T. P.. Ed.), pp. 651-665. monoamine oxidase. In: Monoamine Oxidase and its Elsevier. Amsterdam. Inhibition. Ciba Foundation Series (Wolstenholme, Youdim, M. B. H.. Woods, H. F., Mitchell, B., GrahameG. E. W. and Knight, J., Eds), Vol. 32, pp. 393-402. Smith. D. G. and Callender. S. T. (1975a). Human Elsevier. Amsterdam. platelet monoamine oxidase activity in ironldeficiency Tong, J. H. and Kaufman, S. (1975). Tryptophan hydroxy- I anaemia. Chin. Sci. Mol. Med. 48: 283-295. lase: purification and some properties of the enzyme Youdim. M. B. H.. Hamon. M. and Bouraoin. S. (1975b). from rabbit hindbrain. J. biol. Chem. 250: 41524158. Properties of partially purified pig brain stem ‘tryptoUsdin. E.. Wiener. N. and Youdim. M. B. H. (1978). Strucphan hydroxylase. J. Neurochem. 25: 407-414. ture and function of Monoamine Enzymes. Dekker. New Youdim, M. B. H., Grahame-Smith, D. G. and Woods, H. F. York. (1976). Some properties of human platelet monoamine Voorhess. M. C., Stuart, M. H.. Stockman, J. A. and Oski, oxidase in iron-deficiency anaemia. C/in. Sci. Mol. Med. F. A. (1975). Iron-deficiency anaemia and increased uri50: 479-485. nary norepinephrine excretion. J. Pediat. 86: 542-547. Biochem. 47: 999-1003.
rats. Can. J.
267