Presence of protein-bound unconjugated bile acids in the cytoplasmic fraction of rat brain

• DOI: 10.1194/jlr.m300369-jlr200
• Publication Date: 2004-02-10T00:33:06Z
• Authors: Nariyasu Mano; Takaaki Goto; Masashi Uchida; Koji Nishimura; Masayuki Ando; Norihiro Kobayashi; Junichi Goto
• Keywords: Deoxycholic acid; Chenodeoxycholic acid

Using liquid chromatography/electrospray ionization mass spectrometry, we have found three unconjugated bile acids [cholic acid (CA), chenodeoxycholic (CDCA), and deoxycholic (DCA)] in the rat brain cytoplasmic fraction. CDCA was detected only upon extraction with high concentrations of guanidine, indicating that it is bound noncovalently to protein brain. The most abundant three, present at a concentration 1.6 nmol/g wet weight (∼15 mg protein) brain, corresponding almost 30 times its serum concentration. CA DCA were 1/30th CDCA. Bile conjugated amino acids, sulfuric acid, glucuronic not detected.These data clearly demonstrate and, lesser extent, DCA, exists detected. These are synthesized liver from cholesterol by action hepatic enzymes excreted into small intestine via duct. In intestinal lumen, they assist lipolysis absorption fats forming mixed micelles then return ileum proximal colon. Because their efficient uptake, low peripheral blood. 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Nishio Nakai Nomura Unno Naitoh Matsuno Yawo novel gene family liver-specific transporter LST-1.J. 274: 17159-17163Abstract (495) To expand these previous findings, study examines existence using HPLC combined electrospray spectrometry (LC/ESI-MS) immunological method monoclonal antibody. Unconjugated purchased Nacalai Tesque, Inc. (Kyoto, Japan), except ursodeoxycholic generously donated Mitsubishi Pharma Co. (Tokyo, Japan). labeled stable isotopes 12-oxo-lithocholic used internal standards (ISs) acid-conjugated our laboratory previously methods (20Goto Miura Inada Nambara Nagakura Studies steroids: CCXXXVIII. Determination gas chromatography-mass negative ion chemical detection.J. 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Analysis LC/MS JMS-LCmate (JEOL, Tokyo, Japan) double-focusing magnetic spectrometer equipped ESI probe under detection mode. resolution set 750 3000, voltages electrospray, orifice, lens −2.5 kV, −15 V, −80 respectively. temperatures orifice desolvating plate 150 250°C, Liquid chromatographic separation Inertsil ODS-2 μm, 2.1 mm inner diameter × mm; GL Science, flow rate 220 μl/min. High-resolution selected monitoring (SIM) polyethylene glycol calibration standard added postcolumn additive Samples analyzed low-resolution (m/z 391) high-resolution 391.2848) SIM 20 mM ammonium acetate (pH 7.0 adjusted ammonia-acetonitrile) (2:1, v/v), mobile phase. Peak area ratios calculated Areasample/AreaIS each condition. compare relative retention factors (Rks) target compounds those standards, peaks chromatogram authentic collected IS, Rks IS (tR − t0/tRIS t0) determined different conditions [20 4.0, 5.0, 7.0)-acetonitrile 53:47, 11:9, 2:1 (v/v), respectively]. Male female Wistar rats (200–250 g) fed commercial pellet diet ad libitum used. Venous after decapitation, whole brains (about 1.9 g weight) saline homogenized 7 vol ice-cold 100 Tris-HCl buffer 7.4) containing 0.25 M sucrose. homogenate centrifuged 9,000 min 105,000 60 min, final supernatant fluid stored below 4°C. procedures carried out 0–4°C. An aliquot (200 μl, dropwise 500 μl 1.65 8.6) 0.03 EDTA 7.3 guanidine hydrochloride gently min. After addition 3,7-[18O-2H]2CDCA mixture 5 ml 1,800 10 evaporated vacuo, dissolved 300 0.1 solution, subjected analysis ions deprotonated molecules 375, 391, 407 mono-, di-, tri-hydroxylated respectively) glycine-conjugated 432, 448, 464 taurine-conjugated 482, 498, 514 ions. limit pg (∼12 fmol), signal-to-noise ratio amount. (100 μl) diluted 0.5 potassium phosphate 7.0) IS. passed solid-phase precoated BSA, water, eluted water-ethanol (1:9, v/v). eluate redissolved 200 analysis. recovery investigated 18O-labeled CA, CDCA, spiked samples avoid endogenous acids. evaluate intra-day variation, prepared six fraction (0.3, 3.0, ng) same day them procedure described above. inter-day variation evaluated over 6 days. characterize time ELISA specificity markedly affinity (23Kobayashi Katayama Nagata Goto Production sensitive residues anchored proteins.Anal. 2000; 16: 1133-1138Crossref (13) 1:400 50 sodium 7.3) (buffer I) distributed plates, left overnight washing 0.9% chloride II), wells blocked skim milk II 37°C h. 0.05% (v/v) Tween III), anti-DCA 1:5,000 0.1% gelatin IV) added. incubation room temperature h, solutions aspirated off III. (4 either fractions above added, incubated 3 washing, measured colorimetrically substrate 0.04% o-phenylenediamine dihydrochloride 0.018% H2O2. enzymatic reaction terminated adding H2SO4. absorbance 492 nm MPR A4I microplate reader (Tosoh, commonly unfold proteins (24Tanford C. Protein denaturation.Adv. 1968; 23: 121-282Crossref (2441) LC/ESI-MS This procedure, large quantity gave rise remarkable results (Fig. 1A)in major peak (peak c) observed, along minor (peaks b) when used, coated (25Yoshida Morita Masujima Imai direct plasma onto reverse phase column determination drugs.Chem. 1982; 30: 2287-2290Crossref (63) alone (26Mitamura K. Yatera Shimada Quantitative 3-sulfate ionization-mass spectrometry.Anal. 15: 951-955Crossref (32) chromatograms resulting extracts displayed b, respectively 1B, C). None procedures, however, yielded any amino, sulfuric, conjugates. Under various conditions, spectrum isotopic distribution c identical 2). Furthermore, exact (C24H39O4) c, MS, matches characteristic behavior attributable number position hydroxyl group nucleus, useful identification trace biological fluids (27Nambara High-performance chromatography.in: Setchell K.D.R. 4. York1988: 43-64Crossref Thus, a, phases pH values. Rk values (Table 1). Further confirmation identity b provided ELISA, amount sample, (0.06 brain) comparable result LC/ESI-MS. identify respectively.TABLE 1Relative fractionRelative Retention Factor ValuesBile Acid PeakpH 4.0aTwenty millimolar 4.0)/acetonitrile (53:47).pH 5.0bTwenty 5.0)/acetonitrile (11:9).pH 7.0cTwenty 7.0)/acetonitrile (2:1).Peak a0.370.330.57CA0.370.330.57Peak b1.681.691.88DCA1.681.701.89Peak c1.471.541.68CDCA1.471.541.68CA, acid; acid.a Twenty (53:47).b (11:9).c (2:1). Open table new tab acid. exist cerebral flow, simple diffusion responsible Therefore, compared here levels. As shown Table 2, fraction, significantly lower No fluid. 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