Journal of Neurochemistry Raven Press, Ltd., New York 0 1991 International Society for Neurochemistry

Transfemn and Iron Uptake by the Brain: Effects of Altered Iron Status Eve M. Taylor, Andrew Crowe, and Evan €3. Morgan Department of Physiology, The University of Western Australia, Nedlands, Western Australia, Australia

Abstract: Transfemn (Tf) and iron uptake by the brain were measured in rats using 59Fe-1251-Tf and '311-albumin(to correct for the plasma content of 59Feand "'I-Tf in the organs). The rats were aged from 15 to 63 days and were fed (a) a low-iron diet (iron-deficient) or, as control, the same diet supplemented with iron, or (b) a chow diet with added carbony1 iron (iron overload), the chow diet alone acting as its control. Iron deficiency was associated with a significant decrease and iron overload with a significant increase in brain nonheme iron concentration relative to the controls. In each dietary treatment group, the uptake of Tf and iron by the brain decreased as the rats aged from 15 to 63 days. Both Tf and iron uptake were significantlygreater in the irondeficient rats than in their controls and lower in the iron-loaded rats than in the corresponding controls. Overall, iron deficiency produced about a doubling and iron overload a halving of the uptake values compared with the controls. In contrast to that in the brain, iron uptake by the femurs did not decrease

with age and there was relatively little difference between the different dietary groups. "'I-Tf uptake by the brains of the iron-deficient rats increased very rapidly after injection of the labelled proteins, within 15 min reaching a plateau level which was maintained for at least 6 h. The uptake of "Fe, however, increased rapidly for 1 h and then more slowly, and in terms of percentage of injected dose reached much higher values than did "'I-Tf uptake. It is concluded that, after the age of 15 days in the rat, there is a decline in the rate of uptake of iron by the brain, probably attributable to a decrease in the number of Tf receptors on brain capillary endothelial cells, and that the expression of these receptors is highly responsive to the iron status of the animal. Key Words: Iron uptake-Transfemn receptors-Iron deficiency-Iron overload-Blood-brain barrier. Taylor E. M. et al. Transfemn and iron uptake by the brain: Effects of altered iron status. J. Neurochem. 57, 1584- 1592 ( 1 99 I).

Because of its involvement in heme and nonheme iron-containing enzymes, iron is required by all cells of the body, including those of the brain. Therefore, the question of how the iron is transported across the blood-brain barrier from the plasma where it is bound to its transport protein, transferrin (Tf), is one of considerable interest and importance. Some light has been shed on this problem by the recent observations that Tf receptors are present on brain capillary endothelial cells (Jefferies et al., 1984; Risau et al., 1986; Pardridge et al., 1987) and by the demonstration of Tf endocytosis by these cells in vitro (Pardridge et al., 1987) and in vivo (Fishman et al., 1987). These observations have led to the hypothesis that the brain acquires its iron from the blood plasma by transcytosis of the Tf-Fe complex across the brain capillary endothelial cells (Pardridge, 1986, 1988; Pardridge et al., 1987; Fishman et al., 1987).

During an earlier investigation of iron transport into the brain of the rat, we observed that the maximal rate of transfer occurred in animals aged 15 days (Taylor and Morgan, 1990). At this age, rats have a low level of storage iron (Leslie and Kaldor, 197 1; Linder et al., 1972; Siimes et al., 1980) and exhibit a "physiological anemia" which is partially responsive to iron therapy (Siimes et al., 1980). This raises the question whether iron transport into the brain is affected by storage iron levels and, if so, the mechanism involved. The aim of the present study was to investigate these questions using rats of different ages with normal iron stores, iron deficiency, or iron overload.

Received March 25, 1991; accepted April 7, 1991. Address correspondenceand reprint requests to Dr. E. H. Morgan at Department of Physiology, The University of Western Australia, Nedlands, Western Australia 6009, Australia.

Abbreviations used: C, chow-fed C + Fe, chow-fed with added carbonyl iron; I.D., iron-deficient; 1.D. + Fe, iron-deficient supplemented with iron; PIT, plasma iron turnover; PVE, plasma volume equivalent; Tf, transfemn.

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MATERIALS AND METHODS Materials 59Fe (FeC13, 10-13 mCi/mg), 12'1 (NaI, 4-12 mCi/mg), and I3'I (NaI, 4-12 mCi/mg) were purchased from Amersham

IRON STATUS AND BRAIN IRON UPTAKE International (Amersham, U.K.). Rat Tf and rat albumin were isolated from plasma and labelled with 59Feplus 1251 and I3'I, respectively, as in earlier work (Gardiner and Morgan, 1974; Trinder et al., 1988). Carbonyl iron was obtained from Sigma Chemical (St. Louis, MO, U.S.A.).

Animals The experiments were performed with rats of a Wistar strain. Iron deficiency was produced by feeding distilled water and a low-iron diet modified from that of Diplock et al. (1967) by the omission of iron and the substitution of American Institute of Nutrition vitamin mixture 76 for the vitamin mixture originally described. The iron content of this diet was 5-10 rng/kg. Controls for the iron-deficient rats received tap water and the same diet to which was added ferrous ammonium sulfate (1.3 gfkg). Iron excess was achieved by feeding tap water and 1.5% (wtfwt) carbonyl iron mixed with a standard rat chow diet which contained 170 mg of Fefkg (Milne Feeds Ltd., Perth, Western Australia). The controls for these animals received the same diet without added iron. All diets were commenced in pregnancy (day 12-15 after observing spermatozoa in the vagina) and were continued during suckling and after weaning. The diets and the corresponding rats will be referred to as I.D. (iron-deficient), I.D. Fe (iron-deficient supplemented with iron), C (chow-fed), and C Fe (chow-fed with added carbonyl iron).

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Experimental procedure Rats aged 15, 2 1, 28, 42, and 63 days were studied. They were injected via a lateral tail vein with a mixture of difemc 59Fe-'251-Tfand 13'I-albumindissolved in 0.15 MNaCl. The volume of injected solution varied from 50 to 100 pl and the amounts of Tf and albumin from 100 to 400 pg and 1001,000 pg, respectively, depending on the age of the rats. A sample of blood (-50 pl) was collected into a heparinized microhematocrit tube by incision of the ventral tail vein 3 min after the injection. After centrifugation, the hematocrit was recorded and an aliquot of the plasma counted for radioactivity in order to allow calculation of the plasma volume. At varying times afier the injection, the rats were anesthetized with intraperitoneal sodium pentobarbitone. The abdomen and chest were opened and a sample of blood collected from the right ventricle with a heparinized syringe and a 25gauge needle. The right ventricle was then incised and the animal perfused through the left ventricle with heparinized 0.15 M NaCl at room temperature (-20°C) using a 20-ml syringe and 23-gauge needle. The volume perfused vaned from 20 to 40 ml, depending on the size of the rats. The brain, liver, and femurs were removed. Radioactivity was counted in the two femurs, in cells from a sample of blood after washing three times with ice-cold 0.15 M NaCl and from samples of plasma, and in brain after homogenization with 0.15 M NaC1. The plasma and brain samples were precipitated with 10%trichloroacetic acid and centrifuged, and radioactivity was counted separately in the precipitates and supernatants in order to determine protein-bound radioactivity. The specific uptake of Tf in the brain was calculated as previously described (Taylor and Morgan, 1990), using the tissue and plasma protein-bound '3'1-albumin values to correct for the content of plasma. The results are expressed as the percentage of injected dose of '251-Tfand as plasma volume equivalent (PVE) to the amount of specifically bound Tf. The brain uptake of 59Fewas corrected for that due to Tf (by use of the protein-bound 1251 values) and 59Fe-labelled

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blood cells (by use of the hematocrit and the I3'I-albumin and washed cell 59Fe radioactivity values). Because of the efficiency of the perfusion procedure, the corrections for labelled red cells were small, amounting to less than 5% of the total 59Feuptake in all cases. Similar calculations were performed for the femurs, except that with the femurs total 13'1 activity, not protein-bound activity, was used. The final 59Fe uptake values were expressed as the percentage of the injected dose of radioactivity. They represent the amount of 59Fereleased from Tf and taken up by the brain in excess of that present on Tf in the brain. The validity of using '311-albuminas a means of estimating brain plasma volumes was assessed by determining the apparent tissue plasma volumes at varying times from 0.25 to 2 h after injection and by comparing the values obtained with 1251-polyvinylpyrrolidone (average MW, 40,000) with those of 13'I-albumin. No difference in the tissue plasma values obtained with the two markers was observed, and no change in the 1311-albuminvalues occurred during the time period studied (Taylor and Morgan, 1990). These studies confirm the absence of significant specific uptake of albumin by brain capillary endothelium (Pardridge et al., 1985) and the slow rate of transfer of albumin across the blood-brain barrier (Olsson et al., 1968), and indicate the validity of its use in the types of experiments described in this article.

Analytical methods The plasma iron concentration was determined by the method advocated by the International Committee for Standardisation in Haematology (1978), and tissue nonheme iron as described by Kaldor (1954). Plasma Tf was assayed by radial immunodiffusion using a monospecific antibody produced in rabbits. The specificity of the antibody was demonstrated by Ouchterlony double immunodiffusion against rat serum and purified rat Tf. Plasma iron turnover (PIT) was determined by measuring 59Fein plasma collected from the ventral tail vein at approximately 5, 15, 30, and 60 min after injection of the labelled Tf and was calculated as described by Bothwell et al. (1979). Radioactivity was counted in a three-channel y-scintillation counter (LKB-Wallace 1282 Compu-gamma). Statistical evaluation of the results was performed by analysis of variance.

RESULTS The iron status of the rats in the four dietary treatment groups was assessed by measurement of hematocrit, plasma iron and Tf concentrations, and liver nonheme iron concentrations. The I.D. diet produced manifest iron deficiency by the time suckling rats were 15 days of age, as indicated by the low hematocrit, plasma iron, and liver nonheme iron values (Fig. 1). This persisted throughout the period studied in the present investigation, i.e., up to 63 days of age. The carbonyl iron-supplemented diet (C f Fe) also produced evidence of iron loading by 15 days of age, with elevated plasma iron and nonheme iron values (Fig. 1). The plasma Tf concentrations changed relatively little with age. The mean f SEM values were as follows: I.D., 8.46 mg/ml; I.D. Fe, 5.18 mg/ml; C, 5.15 mg/ ml; and C Fe, 4.83 mg/ml.

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The results for Tf-free tissue uptake of 59Feby the brain 2 h after the injections (Fig. 3) showed the same trend with respect to age in all four groups of animals, falling progressively from the highest levels in the youngest animals studied (15 days old) to the oldest (63 days old). The values were much higher in the I.D. rats than in their controls, whereas those of the C Fe animals were lower than in the corresponding controls. The changes with respect to time and the differences between the treatment groups were all highly significant ( p < 0.001). The specific uptake of Tf by the brain (Fig. 4) showed a very similar pattern to that of "Fe uptake, decreasing with age and much higher in the I.D. and lower in the C Fe rats than in the relevant controls. Again, these changes and differences were highly significant (p < 0.001). The results in Fig. 4 are expressed in terms of PVE to the amount of specifically bound Tf. This mode of expression is preferred over that of percentage of injected dose of '"I-Tf, because the latter is influ-

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30 40 AGE (Days)

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'1 B

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0 10

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300:

-M

* I.D.+Fe

250

-

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-0-r

8 200

P

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150-

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100-

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I.D.

200-

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0

A * I. D. + Fe

' 50:

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o! 10 10

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FIG. 1. Hematocrit (A), plasma iron concentration (B), and liver nonheme iron concentration(C) in rats fed I.D., I.D. Fe, C , and C Fe diets. The diets were fed from the times that the dams were pregnant, through the suckling period, and then until the ages indicated in the figure (see text for details). The liver results are expressed as rg/g wet weight. Each value is the mean t SE for four to 12 rats. The liver nonheme iron results for the C Fe rats are presented at one-tenth the real results (e.g., the mean value at 63 days is 3,300 pg/g).

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Both iron deficiency and iron loading caused reductions in the rate of growth of the rats compared with the relevant controls, but in the case of the I.D. rats, this was statistically significant only at 63 days ( p < 0.05; Fig. 2A). After the age of 15 days, these changes were accompanied by significant (p < 0.01), but relatively much smaller, decreases in the growth of the brain (Fig. 2B). Iron deficiency also caused significant (p < 0.01) reductions in brain nonheme iron levels (Fig. 2C). With the iron-loaded rats, the brain nonheme iron content at the different ages did not differ significantly from that of the controls (p < 0.05) although, as a consequence of the smaller brain size, the nonheme iron concentrations were slightly, but significantly (p < 0.05),higher (Fig. 2C). J. Neurochem., Val. 5 7, No. 5, I991

I

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0.75 d 0.50 0.25

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FIG. 2. Body weight (A), brain weight (B), and brain nonherne iron concentration(C) in I.D., I.D. Fe, C , and C Fe rats of different ages. The brain nonherne iron concentration is expressed as r g / g wet weight. Each value is the mean k SE for four to 12 rats.

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IRON STATUS AND BRAIN IRON UPTAKE

*

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C+Fe

g 4 b 3

+ 6

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1587

etary groups at any age. These values when expressed as PVEs were 2.7 pg/brain for all treatments at age 15 days and vaned only from 1.7 to 3.1 pg/brain at all of the other ages. Iron uptake by the femurs was measured for comparison with brain uptake, because the mechanism of uptake of Tf-Fe by the bone marrow cells is established as being due to receptor-mediated endocytosis (Morgan, 1981), as postulated for the brain (Fishman et al., 1987; Pardridge et al., 1987). However, the patterns of uptake of 59Fewere different from those obtained with the brain. Iron uptake in terms of percent dose of injected 59Feshowed no significant change with age in the I.D., I.D. Fe, and C Fe rats, but increased significantly in the C rats ( p < 0.05; Fig. 5). Overall, there was no significant difference between the I.D. and its control animals ( p > 0.05), whereas the values for the C Fe rats were lower than for their controls ( p < 0.001). PIT was estimated in groups of four to six rats at each age. The patterns of disappearance of 59Fefrom the plasma are illustrated for the 15-day-oldrats in Fig. 6. Patterns similar to these were observed in the older age groups. With the I.D. Fe, C , and C Fe rats, the 59Fedisappeared from the plasma in a single exponential manner for at least 60 min and extrapolated back to zero time at close to 100%of the initial value calculated from the injected dose and the plasma volume determined with 1311-albumin. However, with the I.D. rats, an exponential type of disappearance curve was not established until 10-1 5 min after the injection and, when extrapolated to zero time, these curves intersected the ordinate at 55-75% of the calculated initial value. Hence, in these rats there was a rapid loss of 2545% of the injected s9Fe within the first 10-15 min, followed by a slower exponential decrease. The slopes

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A G E (Days)

FIG. 3. Uptake of Tf-free 59Feby the brain 2 h after the intravenous injection of 5gFe-1Z51-Tf to I.D., I.D. Fe, C, and C Fe rats of different ages. The 59Fevalues were calculated by correcting the total brain "Fe values for the 59Feattributable to its content of '"I-Tf (see text for details). Each value is the mean ? SE for four to 12 rats.

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enced by the increase in plasma volume which occurs as the rat grows from 15 to 63 days of age. This causes the specific activity of plasma Tf and the uptake values to fall in proportion to the plasma volume increase. The PVE values, by contrast, are not influenced by the differencesin plasma volume between the different age or treatment groups. There were no significant differences in the mean amounts of l3'I-a1bumin in the brains of the four di1

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FIG. 4. Specific Tf uptake by the brain of I.D., I.D. Fe, C.and C Fe rats of different ages 2 h after the intravenousinjection of 59Fe-1251-Tf. The specific Tf uptake values were calculated by correcting the total brain lZ51-Tffor the 1251-Tfattributable to plasma as determined with 1311-albumin.The results are expressed as the PVE to the amount of lZ5lin the brain after the correction for plasma 1251-Tf.Each value is the mean -t SE for four to 12 rats.

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FIG. 5. Tf-free 59Feuptake as percentage of injecteddose of 59Fe by the two femurs of ID.,I.D. Fe, C , and C Fe rats of different ages 2 h after the intravenous injection of 59Fe-'251-Tf. Each value is the mean k SE of four to 12 rats.

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1

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uptake observed in the I.D. and C rats of these age groups. Brain and femur uptake measurements were made at different times from 0.25 to 6 h after injection of the 59Fe-'251-Tf.As shown in Fig. 8, iron uptake by the brain as percent dose increased rapidly during the first hour after injection and then more slowly until 6 h. As was found before, iron uptake was greater in 15day-old than 2 1-day-old rats and at each age was greater in the I.D. than in the C rats. By contrast, Tf uptake occurred very rapidly to reach a level by 15 min after injection which was maintained until 6 h. In terms of percentage of injected dose of lZ5I-Tf,the brain uptake values were only about 20% as high as the 6-h 59Fe uptake results for each age and treatment group. From these results, it is clear that at the 2-h time point chosen for the studies described above, the Tf uptake values had reached a plateau or steady-state level and the uptake of 59Fehad achieved nearly maximal values. Tf and iron uptake by the femurs was also measured in the time-course investigation. The general pattern of changes was similar to that described for the brain. The Tf PVE values reached a high level within 0.25 h of injection, and plateau levels were then maintained for the 6-h duration of the measurements. In both treatment groups, 59Feuptake rose rapidly at first and then more slowly to achieve maximal values by 2 h after injection (results not shown).

I.D+W I. D.

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FIG. 6. Disappearance of 59Fefrom the plasma of a 15-day-old

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I.D., 1.D. Fe. C. and C Fe rat following the intravenousinjection of 59Fe-'251-Tf. The percentageof injected dose of 59Fein the plasma was calculated from the plasma 59Fe,the injected dose of 59Fe, and the plasma volume as determined with '3'l-alb~min.

of the exponential phases of each of these curves were used to calculate the fractional PIT in terms of plasma pools per day. These results and the values for iron uptake by the brain calculated from them are summarized in Table 1. The relationships between iron uptake and specific Tf uptake by the brain were assessed by correlating the iron and Tf uptake in absolute units. For this purpose, iron uptake for each animal was calculated as the product of its brain 59Feuptake value as percent dose and the mean absolute PIT for the age group corresponding to the animal. Tf uptake was calculated from the PVE value and the plasma Tf concentration of each animal. For each of the I.D., C, and C Fe groups of animals, highly significant correlations (p < 0.00 I), but with different slopes, were obtained (Fig. 7). The results for the I.D. Fe rats did not differ significantly from those of the C rats, but, for clarity, they have been omitted from Fig. 7. The 15- and 2 I -day-old rats were chosen for more detailed study because of the high levels of Tf and iron

DISCUSSION

The results of this investigation demonstrate that the developmentally related change in Tf and iron uptake by the brain of the rat, decreasing from high levels at 15 days of age to much lower levels by 63 days (Taylor and Morgan, 1990), is affected by the iron status of the animals. Both the developmental changes and the effects of iron deficiency appear to be specific for the brain, because they were not seen with the femurs which are representative of the major iron-utilizing organ, the bone marrow. The specific uptake of Tf by the brain is probably a function of the number of Tf receptors on brain capillary endothelial cells. Such receptors have been dem-

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TABLE 1. PIT and uptake of T f - f e iron by the brain (Uptake) in I.D. + Fe, I.D.. C, and C Fe rats between 15 and 63 days of age

+

I.D.

+ Fe

I.D.

C + Fe

C

Age (days)

PIT

Uptake

PIT

Uptake

PIT

Uptake

PIT

Uptake

15 21 28 42 63

29.5 & 2.8 25.0 k 2.0 20.2 k 1.0 45.7 f 16.8 20.6 k 0.8

0.22 0.04 0.03 0.06 0.01

42.9 t 2.7 55.0 k 4.3 61.3 t 3.3 56.3 f 5.0 57.2 t 6.8

0.68 0.47 0.17 0.08 0.04

27.3 f 1.9 34.7 f 2.5 28.2 f 2.6 29.9 1.5 26.4 t 1.5

0.29 0.08 0.04 0.03 0.01

26.1 -+ 1.4 25.1 f 0.9 23.3 k 1.8 23.8 f 1.5 17.1 t 1.2

0.12 0.05 0.03 0.01 0.00

+

The results are expressed in terms of plasma pools of iron per day. The PIT values are the means k SE for five to 16 animals. The brain uptake values were calculated for each group of animals as the product of the mean values for PIT and the Tf-free 59Feuptake by the brain 2 h after the injection, expressed as a fraction of the 59Feinjected dose.

J. Neurochem.. Vol. 57, No 5 , 1991

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ferric and diferric Tf. In the control and iron-deficient, but not the iron-loaded animals, there was a more rapid tissue uptake of iron injected in the diferric than in the monoferric form. Hence, in the present work, the relative distribution of 59Febetween brain and femurs is probably representative of the natural situation, and the PIT determinations are likely to be correct for the iron-loaded rats and overestimated in the controls. However, based on the results of Huebers et al. ( 1981, 1985), this overestimation should be relatively small, less than 20% of the correct value. In the case of the iron-deficient rats, the slower exponential disappearance of 59Fefrom the plasma between 15 and 60 min after injection was used to calculate PIT. The clearance of 59Fefrom plasma collected from iron-deficient rats during this period after injection of difemc 59Fe-Tfinto iron-deficient rats has been shown to be similar to that of 59Feon monoferric Tf, because of conversion of the injected diferric Tf into the monofemc form in vivo (Huebers et al., 1981). Hence, our estimates of PIT are probably representative of the true PIT values. Thus, the calculations of iron uptake in absolute units (Table 1, Fig. 7) based on the PIT values and the uptake of 59Feby the brain 2 h after the injections when the brain uptake had reached nearly maximal values should be close to the correct values in all four groups of rats. Although the I2%Tf was injected in the diferric form, the calculated specific uptake of Tf 2 h after the injections probably represents the uptake of native Tf in each animal. By this time after injection, most of the 59Fehas been cleared from the plasma and replaced by nonradioactive iron from tissue sources, converting the '251-Tfinto the same type of mixture of diferric Tf, monoferric Tf, and apotransferrin as is naturally pres-

p& A

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TRANSFERRIN UPTAKE o@

FIG. 7. Relationship between Tf-free iron uptake and specific Tf uptake by the brains of I.D., C, and C Fe rats. The results are for individual animals of different ages from 15 to 63 days. The iron uptake for each animal was calculated as the product of its Tf-free brain 59Feuptake as percent dose and the mean PIT for the age group corresponding to the animal. Tf uptake was calculated from the PVE value and plasma Tf concentrations of each animal. The equations for the regression lines are as follows: I.D., y = 0.0828 0 . 0 0 5 9 0 ~(r = 0.494); C,y = 0.109 0 . 0 1 3 7 ~(r = 0.715); and C Fe, y = 0.0312 0 . 0 2 7 6 ~(r = 0.781).

+

+

+

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+

onstrated previously at these sites in adult rats (Jefferies et al., 1984). The present results suggest that their numbers are much greater in younger rats than in adults, that at each age the numbers are influenced by the iron status of the animal, and that they function to mediate iron transfer from the blood plasma to the brain. The results of the time-course studies showed that 12'I-Tf uptake by the brain reached a plateau level soon after injection of the labelled protein and that this level was maintained past 2 h, the time chosen for the other experiments. The Tf was injected in the diferric form and, therefore, was not in the same state as the circulating Tf, especially in the iron-deficient rats. Diferric Tf has a higher affinity for its receptor than monofemc Tf, whereas apotransfemn has the least affinity (Kornfeld, 1969; Huebers et al., 1985).However, only &ferric and monoferric Tf can donate iron to cells and, because of their higher affinity for the receptors than that of apotransferrin and their high concentrations in plasma relative to the K, of the Tf receptor, they are probably the only forms of the protein taken up by receptormediated processes. In the case of the iron-loaded rats, iron-containing plasma Tf would be mainly in the diferric form; with the control rats it would be a mixture of diferric and monoferric Tf, and with the iron-deficient rats mainly monofenic. Huebers et al. (198 1) have investigated the metabolism of iron injected as difemc or monofemc Tf into control, iron-loaded, and irondeficient rats. They found that the distribution of the iron between the body tissues was similar for mono-

21 DAY

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* *

C:Fe 1.5

I.D. :Fe

--t- C:Tf

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FIG. 8. Specific 1251-Tfand Tf-free 59Fe uptake by the brains of

15- and 21-day-old C and I.D. rats at different times after the intravenous injection of 5gFe-1251-Tf. Each value is the mean SE for five 15-day-old rats and three 21day-old rats.

*

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ent in the circulation. This, plus the rapid attainment of a steady state between plasma and tissue Tf, justifies the calculation of amount of Tf uptake by the brain as the product of the PVE value and plasma concentration. The results so obtained would be a function of the number of Tf receptors on brain capillary endothelial cells and may even be a direct estimate of the number of receptor binding sites if all of the Tf taken up by the capillary wall is recirculated receptor-bound back to the plasma and none is passed on to the brain interstitium. The failure of brain uptake of '251-Tfto increase during the 6-h period of the time-course experiments indicates that little or no Tf is transferred in this manner or that, if it is, the protein is very rapidly catabolized within the brain. Whether or not this is true, it may still be concluded that the greater uptake of Tf by the brain in iron-deficient rats and lower uptake in the iron-loaded ones, relative to the controls, is evidence that Tf receptor function in brain capillaries (probably due to changes in receptor number) is influenced by iron supply. Moreover, this effect is expressed much more strongly in the brain than in femoral bone marrow. Regulation of the expression of Tf receptors by iron supply has been demonstrated with several types of cells in culture by incubating them in the presence of iron chelators in order to reduce iron supply or in media containing additional amounts of iron (Ward et al., 1982, 1984; Louache et al., 1984; Pelosi et al., 1986). The present investigation provides evidence that Tf receptor expression in brain endothelial cells is regulated in a similar manner. This may be due to an effect of intracellular iron on the stability of Tf receptor mRNA (Owen and Kuhn, 1987). The question arises as to whether the age-related changes in Tf uptake are also a consequence of this type of regulatory mechanism. Maximal Tf uptake by the brain occurred in the control rats when iron stores, as indicated by liver nonheme iron and plasma iron levels, were lowest, and Tf uptake decreased as the iron status improved. However, storage and plasma iron levels remained low in the iron-deficient rats as they grew older, yet Tf uptake by the brain decreased. Hence, it is possible that another factor, in addition to the availability of iron to endothelial cells, plays a role in the regulation of receptor numbers. This may be an effect of the rate of cell proliferation in the brain or of the endothelial cells themselves, because such cell proliferation is occurring rapidly in the first 2-3 weeks of life in the rat (Altmann, 1969; Robertson et al., 1985). The rate of proliferation of many types of cells in culture has been shown to influence Tf receptor expression (Lamck and Cresswell, 1979;Trowbridge and Omary, 1981; Sutherland et al., 1981) although the mechanism by which this is effected is uncertain. The changes in Tf uptake by the brain which accompanied alterations in age or iron status of the animals were paralleled by similar changes in iron uptake (Figs. 3 and 4). This suggests that iron uptake was deJ. Neurochem., Val. 57. No. 5. 1991

pendent on the number of Tf receptors present on the brain capillary endothelial cells. This dependence is emphasized by the significant relationships observed between Tf uptake and brain iron uptake calculated from the PIT and 59Feuptake data (Fig. 7). That the slopes of the regression lines relating Tf and iron uptake varied between treatment groups is likely to be an expression of the effect of the degree of saturation of plasma Tf with iron on iron uptake by the tissues of the rat (Huebers et al., 1981). Ben-Shachar et al. (1988) have shown previously that iron deficiency causes selective changes in the permeability of the blood-brain barrier for insulin, glucose, and certain amino acids. In particular, insulin transfer was increased, which raises the possibility that iron deficiency could induce a nonspecific increase in the permeability of the blood-brain bamer to proteins. If so, this could be the cause of the increase in transport of Tf-Fe observed in the present work. However, this is unlikely, because the brain 59Feuptake values presented here have been corrected for the amount of I2%Tf present in the brain. Moreover, iron deficiency did not lead to any increase in the amount of '251-albumin present in the brain, and the rate of transcapillary exchange of albumin is very similar to that of Tf (Morgan, 1969). Hence, a more likely explanation for the effects of iron deficiency on iron transport into the brain is the one presented above, i.e., an increase in Tf receptor numbers and rate of endocytosis of Tf-Fe by brain capillary endothelium. The Tf receptors on brain capillary endothelial cells undoubtedly mediate iron transport into the brain by receptor-mediated endocytosis of Tf. This type of process has been demonstrated in brain endothelial cells (Fishman et al., 1987; Pardridge et al., 1987), as well as in many other types of cells (Morgan, 1981). However, the question remains as to whether the intact TfFe complex is transported into the brain or whether the iron alone crosses the blood-brain bamer with recycling of the Tf back to the plasma. The present experiments provide suggestive, although not definitive, evidence on this question. Thus, in the 15-day-old rats, an amount of iron approximately equivalent to 30% and 70% of the plasma pool was transmitted to the brain per day in chow-fed and iron-deficient rats, respectively. If this had been accompanied by an equivalent loss of Tf to the brain, it would have caused a large loss of Tf from the plasma, much greater than the normal turnover of plasma Tf which is only about 0.25 plasma pools per day in adult rats (Morgan, 1966), and is approximately the same in normal 15-day-old rats (Taylor and Morgan, 1990). Hence, it is unlikely that transcytosis of the Tf-Fe complex into the brain occurs. More probably the Tf recycles from endothelial cells back to the plasma after releasing its iron within the cells, whereas the iron is transported into the brain. The observation that dietary iron deficiency from an early age leads to decreased brain storage iron levels confirms earlier studies (Dallman et al., 1975; Dallman

IRON STATUS AND BRAIN IRON UPTAKE

and Spirito, 1977; Youdim et al., 1980, 1989; BenShachar et al., 1986, 1988) and emphasizes the susceptibility of the developing brain to the effects of iron deficiency. This may lead to decreased growth of the brain as observed in this work and to biochemical and functional abnormalities (Pollitt and Leibel, 1982; Pollitt et al., 1986; Yehuda and Youdim, 1988; Youdim et al., 1989). Iron overload, on the other hand, did not produce statistically significant changes in brain nonheme iron content and only small increases in nonheme iron concentration. The investigation was limited to rats up to the age of 63 days. Whether iron supplementation for longer periods of time would have led to increased brain iron content is yet to be determined. However, from the present work it is clear that the brain is protected to a large degree from iron overload, probably because of the presence of the relatively impermeable blood-brain bamer and the limited maximal capacity of the process of receptor-mediated endocytosis of Tf by the brain capillaries. Similar conclusions can be drawn from the work of Dallman et al. (1975) and Ben-Shachar et al. (1986) who showed that brain iron concentrations increase relatively slowly and rise only to the normal level during rehabilitation of iron-deficient rats with iron. The fact that brain iron levels can be restored is probably a consequence of the increased capacity for iron transport by the blood-brain bamer which is induced by iron deficiency. The brain contrasts markedly with the liver, where rehabilitation leads to rapid recovery of iron levels and iron supplementation causes large increases in storage iron levels. The protection of the brain from iron overload may be important in preventing the potentially toxic effects of excess iron from affecting brain function. Acknowledgment: This work was supported by grants from the National Health and Medical Research Council of Australia and the L. S. Prior bequest t o the Faculty of Medicine, University of Western Australia.

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