PART I. NEUROCI-IEMISTRY

Ascorbic Acid Distribution Patterns in Human Brain

A Comparison with Nonhuman Mammalian Species"

ARVIN F. O K E , ~ LESLIE MAY,' AND RALPH N. ADAMS~

Department of Chemistry University of Kansas

Lawrence, Kansas 66044

Department of Chemistry Indiana University

Bloomington, Indiana 47901

b

INTRODUCTION

Neural tissue has been recognized to contain ascorbic acid ( AA) concentrations that rank among the highest of mammalian biological tissue. Autoradiographic studies of labeled AA' and endogenous assays of regional specific area^'^ suggest that varying neuroanatomical locations concentrate AA in differing degrees. Certain brain locations, including the hippocampus and hypothalamus, consistently show high AA values in both man and animals when compared with other structures within the central nervous system (CNS).

have traced the entrance of AA from blood to brain. Ascorbate molecules leave the blood and enter ventricular fluids through the choroid plexus. These molecules presumably then diffuse passively across ventricular walls into neighboring CNS tissue. Beyond this, it is not known if brain tissue in close proximity to ventricular walls concentrates high AA levels possibly at the expense of those structures more distant to it. Is it only incidental that these structures lay juxtaposed to the ventricular system containing cerebral spinal fluid? Does the brain in fact resemble a diffusing gradient away from ventricular walls? To address these questions a topographical mapping was undertaken of human brain and small animal brains.

In a series of experiments, Spector and

'The support of this work by the NIH through grant no. NS08740 is gratefully acknowledged. Human tissue for this research was in part obtained from the National Neurological Research Bank, VA Wadsworth Medical Center, Los Angeles, Calif., which is sponsored by the NINCDS/ NIMH, the National Multiple Sclerosis Society, the Hereditary Disease Foundation, and the Veterans Administration.

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TISSUE PREPARATION AND ASCORBIC ACID ANALYSIS

Small animal brains were quickly removed and stored at -70°C until just prior to tissue slicing, which followed procedures described earlier.'' Coronal slices ca. 1 mm in thickness were gridded into a matrix ca. 1.5 X 1.5 mm. Grid lines were recorded on reproduced graphics of the slice.

For human brain analysis, a coronal slice containing the hippocampus was selected. This frozen slice was photographed before and after a 4 X 4 mm to 5 X 5 mm grid matrix was etched onto the surface. The latter size matrix replaced the former when it became apparent the greater detail provided little added pattern information. Matrix units within the area of the hippocampus were further subdivided.

Analysis of tissue AA utilized electrochemical detection before and after the addition of ascorbic acid oxidase (AAO). Two techniques, both incorporating flow injection analysis, have been used. An initial method switched a sample between two sepharose beds, one of which had AAO bound to the sepharose moiety." A later method bypassed the sepharose and utilized an automatic sampling unit that permitted sampled before and after the addition of solution AAO. In both cases, the difference between peak heights was the AA determination.

Tissue sonicated in pH 5.5 acetate buffer was followed by 5-min centrifugation at 10,OOO rpm. Samples were kept on ice and quickly analyzed, usually within 20 min.

TOPOGRAPHIC DISTRIBUTION OF ASCORBIC ACID IN SMALL ANIMAL BRAINS

The pattern of distribution of AA throughout the full rostra1 to caudal extent of rat brain is represented in FIGURE 1. A consistent pattern was observed. Telencephalic structures that make up the cortical mantle (ie.. cerebral cortex, hippocampus, and amygdala) contained the highest concentrations: levels up to 600 pg/g of tissue wet weight. Just underneath the cortical mantle (and/or the boundary imposed by the corpus callosum) AA concentrations generally shifted suddenly to levels reduced by approximately 50%. Neural tissue making up the core portion of the forebrain appears therefore to be circumscribed by a wall of dense AA. Within these core diencephalic structures, only the hypothalamus and especially the anterior medial regions of the hypothalamus contain values approximating those seen in the cortical mantle.

As we have reported in an earlier study,' CNS tissues with rostro-caudal extensions show decreasing AA concentrations toward the posterior limits. This is clearly seen when the percentage of the AA values in the most anterior position of a region is plotted against varying distances posterior to it (FIG. 2). Anterior-posterior (AP) decrements of ca. 30% are maintained within such structures as the caudate-putamen, thalamus, and the hippocampus. The hypothalamus has a more drastic decreasing gradient of ca. 60%. Neocortical values indicate a progressive 20% AP decrement. The cortex of the cerebellum, although anatomically quite separate and distinct from the cerebral neocortex, has an ascorbate value which suggests it is an extension of the decreasing values evident in the neocortex.

The portrait of ascorbate distribution in rat brain thus consists of diencephalic and mesencephalic core tissue with low concentration surrounded by a high telen- cephalic border. Nestled within this pattern are dense pockets whose boundaries closely

FIGURE 1. (A-F) Topographic density pattern of ascorbic acid content in rat brain throughout the rostral-caudal extent. Shaded areas on right half of slice represent cerebral ventricles. A, amygdala, Aq. aqueduct of Sylvius; Cb, cerebellum; CC, corpus callosum; CR caudate-putamen; CX, n m r t e x ; H, hippocampus; Hy, hypothalamus; RE reticular formation; T, thalamus.

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delineate the topography of an underlying neuroanatomical structure, as is the case in the amygdala, hippocampus, and hypothalamus. Most regions have an AP de- creasing gradient.

Preliminary data on guinea pig AA distribution provides a pattern closely ap- proximating that of the rat. Although a full three-dimensional topographical mapping has not been done, selected sections concur with the results from rat. FIGURE 3 is a chimeric composite with one hemisphere representing rat and the other representing guinea pig. The guinea pig hemisphere has been sufficiently reduced so as to appear

100

90

80 I- t W 0 70 a

0 60

50

W

40

0 1 2 3 4 5 6 7 8

m m F R O M ANTERIOR L I M I T S

FIGURE 2. Ascorbic acid content of varying distances within the neocortex (CX), hippocampus ( H ) , caudate-putamen (CP), hypothalamus (Hy) , and thalamus (7') of rat brain along the rostral- caudal extension expressed as a percentage of the value found in the most anterior limits.

falsely equivalent in size with the rat. The ascorbate distribution on one side is almost a mirror image of the other, suggesting equivalent pattern layouts.

Similarly, the bovine brain (FIG. 4) demonstrates 'concentric zones of differing ascorbate concentrations. An enriched cortical mantle is separated from core dien- cephalic structures by a depleted zone (i.e., internal capsule) of fibers projecting to and from the cortical gray matter.

Ascorbic acid distribution within these vertebrate species suggests a high degree of internal organization. Gradients are maintained. Neuroanatomical regions are dis- tinguished by AA concentration boundaries. Furthermore, the three species sampled share a uniform pattern.

OKE el 01.: ASCORBIC ACID IN HUMAN BRAIN

FIGURE 3. Chimeric representation of topographic density pattern of ascorbic acid content in closely matched sections of rat and guinea pig brains. For shading see FIGURE 1.

BOVINE B R A I N

FIGURE 4. Topographic density pattern of ascorbic acid for bovine brain. For shading see FIGURE 1.

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TOPOGRAPHIC DISTRIBUTION OF ASCORBIC ACID IN HUMAN BRAIN

Distribution patterns of AA in 10 human brains fell within one of three patterns. The most prevalent one seen (7 of 10 cases) was a dorsal-to-ventral (DV) increasing gradient (FIG. 5A). Concentration shifts for the most part disregard neuroanatomical landmarks. Cortical gray matter, consisting of cell bodies and projection terminals to them, do not necessarily contain any more AA than the white myelinated fibers of passage to them. With the exception of the hippocampus, no subcortical neuroana- tomical grouping showed delineation due to ascorbate levels, a condition much unlike that seen with classical neurotransmitters. The ascorbate tissue value for any neuronal region, outside of the hippocampus, was dependent solely on the DV axis and not on neuroanatomical substrate.

FIGURE 6 represents the quantitative profile for seven brains in this group. A mean (+SEM) value is given for each row of the grid matrix. Any unit within the grid which contains hippocampal tissue is excluded from calculations. Typically, dorsal row AA content for the DV gradient group was less than 100 pg/g. Comparatively,

FIGURE 5. Topographic density of ascorbic acid for three distribution patterns in human brain. (A) Dorso-ventral increasing gradient pattern. (B) Uniform low density pattern. (C) Uniform high density pattern. A, amygdala; Cd, caudate nucleus; GP, globus pallidus; R putamen; Pul, pulvinar, T. thalamus.

OKE et al.: ASCORBIC ACID IN HUMAN BRAIN 7

ventral rows show mean values between 150-200 pg/g. Any single brain within this group will quite commonly contain a two- to threefold DV increase.

A second pattern noted in 2 of 10 cases was (hippocampus exempted) a uniformly low content of AA. FIGURE 5B represents one such case. Row means, ranging from 82 pg/g to 122 pg/g are within 25% of the total slice mean of 94 pg/g. The other instance showed a slice mean of 106 pg/g, with row means ranging from 87 pg/g to 120 pg/g (see FIG. 6) .

The third pattern is seen in FIGURE 5C. High row means extend almost the full DV axis. Row means noted in FIGURE 6 ranging from 102 to 164 pg/g are within 25% of the slice mean (140 pg/g).

As previously stated, the one neuroanatomical locus maintaining consistent AA values from brain to brain is the hippocampus, specifically the area within it called the dentate gyrus. FIGURE 7 shows three reconstructed drawings from the hippocam- pus, the area represented inside the box within the insert. FIGURES 7A and 7B are hippocampi from brains with DV gradients. FIGURE 7C comes from a brain uniformly low in AA. The high values in the dentate gyrus fall between 275-325 pg/g in all brains. Concentrations there did not in any way indicate the pattern distribution of the slice. The dentate gyrus, therefore, appears to maintain a high level of AA irrespective of surrounding tissue concentration.

Pattern variability is seemingly not correlated with known variables. TABLE 1 lists age, sex, and time from death to autopsy for each case. Age or sex do not appear to be related to any particular grouping. Death-to-autopsy does suggest a distinct group difference. Although it represents only one case, the unusually short 3-h death-to- autopsy interval is substantially lower than those seen in either of the other groups. It is conceivable that pattern variations result simply from postmortem oxidative decay inequalities, ie., are due to regionally different rates of AA decay. Vulnerability to oxidation would be greatest in the dorsal areas, followed by ventral regions, then the hippocampus. Accordingly, the group showing the greatest decay would logically also have the longest death-to-autopsy intervals. This is clearly not the case.

Assuming AA decay rates are equivalent in both human and small animal brains, a rat brain still within the skull was allowed to remain at room temperature for 18 h before removal. This condition is presumably more severe than an equivalent death- to-autopsy period since in the latter case the environment is not room temperature but 4°C. FIGURE 8 is a comparison of AA pattern distribution of a selected section in a rat brain immediately removed and frozen with the 18-h delayed brain. No indication of AA decay is seen as a consequence of the time delay. Nor is there a noticeable loss in pattern distinctiveness. We conclude that pattern variability is not a function of regional decay rates.

The full nature of these varying patterns of AA distribution within human brain requires a more extensive sampling than is available at the present time. Further, it is not known whether any particular pattern is maintained throughout the full ros- trocaudal extent of the brain, although additional slices for a limited number of brains suggest the pattern seen in the midbrain region is also observed in frontal and caudal slices. Within any particular brain, these patterns may remain static or may be reflective of a point in time of continuous changing flux due to metabolic demands and dietary

The extensive grid mapping carried out in this study is, we believe, of considerable significance in quantitative chemical neuroanatomy studies. It provides information that is not obtained by sampling methods that utilize whole anatomical nuclei or pooled homogenates of brain regions. Within subnuclei, or throughout the brain extent, it provides gradients and patterns that may be of considerable significance. Secondly, as an a priori sampling mode, it is unbiased and can be used to establish if chemical

supply.

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+ + +

0 0 0

1....1....1 DV

* +

+ + + + + + + -0- + +

DV

+ - + +

-b- --c + --c + + *

C....I....I u u DV DV DV

+ * + + + + + + + +

-0- +

C....L....r 100 200

Ascorbic Acid

C rrgm/gm 1

u 111..111..1 LOW LOW HIGH

Row Means [+SEMI FIGURE 6. Row means ( * SEM) of ascorbic acid from human coronal slice. Group identi- fication is: DK dormventral increasing gradient; LOW, unifrom low density; HIGH, uniform high density.

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A

C

FIGURE 7. Topographic density pattern of ascorbic acid in human hippocampus (boxed area) for (A,B) dorso-ventral gradient group and (C) uniform low group.

FIGURE 8. Topographic density pattern of ascorbic acid in a coronal section of rat brain. Left side contains values from a brain frozen immediately following removal. Right side represents values following 18 h at room temperature. For shading see FIGURE 1.

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TABLE 1. Group, Age, Sex and Death-to-Autopsy Time for Human Brain Ascorbic Acid Analysis

Group Age Sex Death-to-Autopsy (h)

Dorsal-ventral 22 F gradient 34 F

37 M 45 M 46 M 50 F 74 M

29 18 29 22 24 63 76

Uniform High density 56 M 23

74 M 37 Low density 58 M 3

and classical neuroanatomy are similar or disparate. A somewhat surprising example of the latter is seen in the present work in which the AA distribution in rat, guinea pig, and bovine brains does conform to classical neuroanatomical delineations, but human brain AA patterns appear markedly independent of brain structure with the exception of the hippocampus. The latter in humans has an AA distribution anatom- ically specific and its invariant high level may be of functional significance.

ACKNOWLEDGMENTS

M. Leeson and J. Comack carried out parts of the ascorbate assays included in this study.

REFERENCES

1. HORNIG, D. 1975. Distribution of ascorbic bid, metabolites and analogues in man and animals. Ann. N. Y. A d . Sci. 258 103-115.

2. UALAKSHMI, R., J. MALATHY & C. V. RAMAKRISHNAN. 1967. Effect of dietary protein content on regional distribution of ascorbic acid in the rat brain. J. Neurochem. 1 4

3. SCHAUS, R. 1957. The ascorbic acid content of human pituitary, cerebral cortex, heart,

4. KRATZING, C. C., J. D. KELLEY & B. A. OELRICHS. 1982. Ascorbic acid in neural tissues.

5. MILBY, K., A. OKE & R. N. ADAMS. 1982. Detailed mapping of ascorbate distribution in

6. MEFPORD, I. N., A. OKE & R. N. ADAMS. 1981. Regional distribution of ascorbate in

7. SPECTOR, R. & A. V. LORENZO. 1973. Ascorbic acid homeostasis in the central nervous

161- 167.

and skeletal muscle and its relation to age. Am. J. Clin. Nutr. 5 39-41.

J. Neurochem. 39 625-627.

rat brain. Neurosci. Lett. 28: 15-20.

human brain. Brain Res. 212: 223-226.

system. Am. J. Physiol. 225 757-763.

OKE er al.: ASCORBIC ACID IN HUMAN BRAIN 11

8. SPECTOR, R. & A. V. LORENZO. 1974. Specificity of ascorbic acid transport system of the central nervous system. Am. J. Physiol. 226 1468-1473.

9. SPECTOR, R. 1981. Penetration of ascorbic acid from cerebrospinal fluid into brain. Exp. Neurol. 7 2 645-653.

10. OKE, A., R. KELLER & R. N. ADAMS. 1978. Dopamine and norepinephrine enhancement in discrete rat brain regions following neonatal 6-hydroxydopamine treatment. Brain Res. 148: 245-250.

BRADBERRY, C. W., R. T. BORCHARDT & C. J. DECEDUE. 1982. Immobilization of ascorbic acid oxidase. FEBS Lett. 146: 348-352.

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DISCUSSION OF THE PAPER

M. LEVINE (National Institutes of Health, Bethesda, M d . ) : Do you have any

A. F. OKE ( University of Kansas, Lawrence, Kans. ): No, I don’t. E. J. DILIBERTO ( Wellcome Reseorch Laboratories, Research Triangle Park, N.C. ):

Has anybody looked at different brain areas to see if that pattern is really reflecting a difference in the mechanism of uptake into the different neurons?

additional data on the distribution of ascorbate within the nervous tissue itself?

A. F. OKE: No, we haven’t. S. D. VARMA ( University of Maryland at Baltimore, Baltimore, Md. ): Do you

think that the high concentration of ascorbate in the brain is due to an active transport across the blood-brain barrier?

A. F. OKE: Spector’s work indicates that ascorbic acid enters the brain not through the blood-brain barrier but through the choroid plexus of the ventricles and then passively diffuses. In fact that was one of the initial directions we had in looking at this, we wanted to find out if there is a concentration gradient away from the ventricular fluids of the brain. We are looking at a homeostatic mechanism here rather than an instantaneous input of the species.

S. D. VARMA: Well if the same kind of transport is not involved and ascorbic acid is really permeable, then why should you have a high concentration in one compart- ment, much higher than the blood or the cerebral spinal fluid?

A. F. OKE: That’s an intriguing question: Why does the brain concentrate in some areas? Why does the hippocampus tend to find its allotment and maintain its allotment, while the rest of the tissue may suffer, or the rest of the tissues is trying to gain its allotment? We can’t answer those questions well.

E. J. DILIBERTO: Did you say that you felt that the ascorbic acid was freely permeable?

S. D. VARMA: There are many reports that dehydroascorbic acid is actively trans- ported and then is reduced by glutathione to ascorbic acid.

E. J. DILIBERTO: But I’m not sure if that’s really been shown for the brain. In fact the reduction system that I think is most important there is the semidehydroas- corbic reductase. It’s not glutathione dependent.

M. CHEN ( University of Washington, Seattle, Wash. ): I think your finding is very interesting. I just wonder about those patterns in the ten individuals. Do you have any clinical data about how they died and what the status of their death was? How much stress had they been under, and were you able to separate them into different groups? Do you know the cause of death?

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A. E. OKE: Yes, we do have the cause of death and although that was not listed, the majority of them were from pulmonary distress and heart attack. Certainly before any of these questions can be answered a lot more work will have to be done.

B. LADU ( University of Michigan Medical School, Ann Arbor, Mich. ): I wonder about the dietary ascorbic acid intake on your subjects, and certainly that could be tested with the guinea pigs on different levels of ascorbic acid in order to see how that affects the uniformity or nonuniformity of the distribution.

A. F. OKE: Yes, that's something that we certainly plan on doing. A. B. KITABCHI ( University of Tennessee, Memphis, Tenn. ): It has always been

stated that 'of the three organs, adrenal, ovaries and pituitary, adrenal and pituitary concentrate ascorbic acid to a larger degree. Did you evaluate posterior versus anterior pituitary in these subjects?

A. F. OKE: No, I did not. M. CHEN: Did you have any diabetics in the ten subjects? A. F. OKE: No.