Free d -serine concentration in normal and Alzheimer human brain

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Free D-Serine Concentration in Normal and Alzheimer Human Brain YOKO NAGATA,.1 MAURO BORGHI,t GEORGE H. FISHER:I: AND ANTIMO D'ANIELLO§

*Department of Life Science, Himeji Institute of Technology, Shosha, Himeji 671-22, Japan tDepartment of Medical Jurisprudence, Policlinico Le Scotte, University of Siena, Italy :l:Department of Chemistry, BatTy University, Miami Shores, FL 33161 §Department of Biochemistry, Stazione Zoologica "',4. Dohm" Villa Communale, Naples, Italy [Received 28 January 1995; Revised 26 April 1995; Accepted 1 May 1995] ABSTRACT: We have analyzed both free L- and o-serine in frontal cortex of normal and Alzheimer human brain by high-performance liquid chromatography (HPLC). There was no significant difference between the two brains. In normal brain, L- and oserine concentrations were 666 ± 222 and 66 _+41 nmol/g of wet tissue, respectively, and the ratio of D-isomer to L-isomer (D/L) was 0.099 _+ 0.031. In Alzheimer brain, the concentrations were 750 ± 150 and 66 +_ 40 nmoVg, respectively, and the D/L ratio was 0.086 ± 0.040. Thus, it was shown that the free o-serine concentr~tion in the Alzheimer brain was comparable to that in the normal brain.

Alzheimer Brain Bank, St. Paul, Minnesota; from the State of Florida Brain Bank at the University of Miami School of Medicine, Department of Neurology, Miami, Florida; and from the Dade County, Florida, Medical Examiner. All brains were of male subjects of the ages 50 to 82. The brains were frozen immediately after autopsy and kept frozen until dissected. Postmortem times ranged from 3 to 24 h, with an average of 12 to 16h. Free amino acids were isolated from superior frontal cortex as follows: 1 g of dissected brain tissue was homogenized with 20 ml of 5% trichloroacetic acid and centrifuged at 60,000 g in a table centrifuge for 30 rain at 4°C. The supernatant was applied to a cation exchange column (Dowex 50W-x8, 200-400 mesh, H÷). After washing the column with 15-20 ml of 0.01 M HC1, the amino acids were eluted with 15 ml of 4 M NFLOH. The eluate was collected in a petri dish from which the ammonia was evaporated on a hot plate at 40-50°C in a hood, or the eluate was dried by using a rotary evaporator. The residue was analyzed for D- and L-serine by the procedure described below. The analysis of D- and L-serine was carded out taking care to eliminate any external contamination. Details of the analytical method that gives a 20-pmol detecion limit are described elsewhere [13]. Briefly, a sample dissolved in a small amount of distilled water was derivatized with 1-fluoro-2,4-dinitrophenyl5-L-alanine amide (FDAA) to determine D- and L-serine. FDAA is a reagent that produces diastereomers of amino acids [10]. The FDAA-amino acids were separated on a silica gel-plate by twodimensional thin-layer chromatography. FDAA-serine recovered from the plate was analyzed by HPLC with a reversed-phase column, Nova-Pak C18 (150 × 3.9 mm 1D, Waters, Milford, MA, USA). The FDAA-amino acid was eluted with a linear gradient of acetonitrile in 50 mM triethylamine-phosphate buffer (pH 3.5) from 10% to 25% over 20 rain at a flow rate of 1.0 ml/ min at 27°(2. The eluate was monitored at 340 nm with a D-2500 Chromato-Integrator (Hitachi, Tokyo, Japan), and peak areas of FDAA-D- and -L-serine were obtained automatically. Amounts of D- and L-serine were calculated based on the peak areas and standard curves [ 13]. FDAA was obtained from Pierce (Rockford, IL, USA), and solvents of HPLC-grade were from Nacalai Tesque (Kyoto, Ja-

KEY WORDS: D-Serine, Alzheimer disease, Human brain, Frontal cortex.

INTRODUCTION Neutral D-amino acids are present in microorganisms, some insects, marine invertebrates, and higher plants [18]. We have found substantial amounts of free D-serine, D-alanine, and D-proline in human plasma from patients with renal diseases [12] and mouse tissues, such as serum, liver, and kidney of a mutant mouse lacking D-amino acid oxidase [14]. With regard to brain, free D-aspartate and b-alanine were found in normal and Alzheimer human brain [6]. We have detected free D-serine at a high concentration (approximately DIE = 0.4) in the cerebra, but not in the cerebella of mouse, rat, and bull, and showed a reverse relationship between the presence of D-serine and D-amino acid oxidase (EC 1.4.3.3) activity in the tissues [11,15]. D-Serine is a potent activator of N-methyl-D-aspartate (NMDA) receptor complex by binding to the strychinine-insensitive,glycine modulatory site [2,4,5,17]. However, nothing is known about the physiological role of endogenous brain D-serine. We investigated the concentration of free D-serine in the Alzheimer cerebral cortex whether the distribution of D-serine was related to the dysfunction of Alzheimer brain, under the purpose of obtaining a clue for the physiological function of D-serine in normal brain.

MATERIALS AND METHODS Neuropathologically confirmed Alzheimer brains and normal brains were obtained from the St. Paul-Ramsey Medical Center To whom requests for reprints should be addressed. 181

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TABLE 1 CONCENTRATIONS OF FREE I- AND J)-SERINE, AND THE o/~ RATIOS IN FRONTAL CORTICES OF NORMAL AND ALZHEIMER BRAINS Normal (nmol/g wt)

Alzheimer (nmol/g wt)

Sample"

Age

I.

I)

Idl

Sample~'

Age

I

J~

tdt

I 2 3 4 5 6 7 8 mean ± SD

50 50 61 63 63 67 71 71 62 ± 8

480 722 1170 480 540 624 635 678 666 ± 222

58 53 163 36 69 59 31 60 66 ± 41

(I. 121 0.073 0.139 0.075 0.128 0.094 0.049 0.089 0.099 ÷ 0.031

I 2 3 4 5 ~ 7

71 71 73 73 78 82 82

969 858 851 703 523 659 690

150 55 76 40 67 36 39

0.155 0.064 0.089 0.057 0.127 (I.055 0.056

mean ± SD

76 ± 5

750 ± 150

66 ± 40

0.086 ± 0.040

~'All samples were of male subjects.

pan). Thin-layer chromatography plates, 20 × 20 cm Kieselgel 60, were obtained from Merck (Darmstadt, Germany). Other chemicals used in this study were of analytical grade.

RESULTS AND D I S C U S S I O N The results on the frontal cortices are shown in Table 1. The postmortem time of the brain sample may not have had a significant effect on the data because the samples may have included almost no D-amino acid oxidase as descibed below. The oxidase is the only mammalian enzyme known to catabolize D-serine. The average DL ratios of free serine were 0.086 _+ 0.040 and 0.099 _ 0.031, respectively, in Alzheimer and normal brains. There was no statistically significant difference as judged by t-test between the two values. It is surprising, however, that both normal and Alzheimer brains contain such a high level of o-serine (66 nmol/g). In spinal cord and medulla oblongata of an Alzheimer subject, the level of free D-serine was much less: 8.6 and 28 nmol/ g in the spinal cord and medulla oblongata, respectively, and the D/L ratios were, respectively, 0.015 and 0.013. The low o-serine concentration in these brain areas coincides with our observation on normal mouse (unpublished data). This study demonstrated the presence of free o-serine in human cerebrum. This result is consistent with our previous finding [15[ that free o-serine was present in mammalian (mouse, rat, and bull) forebrain at a high concentration. The presence of free D-serine at a similar concentration to the result of this study was also communicated recently, on normal h u m a n frontal cortex [8] and normal and Alzheimer human temporal cortices [ l ]. The function of free o-serine observed in this study is unknown. In vitro experiments showed that D-serine was a potent agonist of glutamate binding to N M D A receptor [4,5,9,19]. Recently, a view that the endogenous free o-serine in the brain was a possible excitatory transmitter, an intrinsic ligand for the glycine site of the N M D A receptor was proposed based on colocalization (concentration) of the receptor and D-serine in rat brain [7]. It was shown in this experiment that the free D-serine concentration was not altered in Alzheimer frontal cortex. Also any change was not observed in the D-serine level with senescence in normal h u m a n frontal cortex [8]. These results indicate that the free D-serine concentration remains relatively constant, in spite of decrease of cerebral function in the Alzheimer and senescent h u m a n brains. The distribution of free o-serine in the brain may be accounted for D-amino acid oxidase activity [3,11,15] rather than the distribution of N M D A receptor: D-Ser-

ine concentration was remarkably higher in the cerebrum than cerebellum, and the enzyme activity was considerably lower in the cerebrum than cerebellum in mice [11]: o-Serine concentration was increased to almost the cerebrum-level in the cerebellum of a mutant mouse lacking the oxidase activity [11], A study on the distribution of o-serine binding activity is a matter for further investigation. The free o-serine detected in this experiment may not be of bacterial origin in analogy with mice; the brain o-serine level of germ-free mice did not differ from that of specific pathogen-free mice [15]. o-Alanine ingested with drinking water was detected in the mouse cerebrum at concentrations comparable to those observed in the serum [16]. Because D-serine is a small amino acid like alanine, it could pass through b l o o d - b r a i n barrier, and oral uptake seems to be a possible route. ACKNOWLEDGEMENTS GHF thanks the NIH-MBRS Grant #45455, the Research Corporation Cottrell College Science Grant Program, and the Barry University Alzheimer's Research Fund for financial support. This project was also partially funded under an agreement with the Aging and Adult Services Program Office, Department of Health and Rehabilitative Services, State of Florida. Neurological Brain Bank.

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D-SERINE IN N O R M A L A N D A L Z H E I M E R B R A I N

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