Production of a Monoclonal Antibody for Sensitive Monitoring of Deoxycholic Acid Residues Anchored on Endogenous Proteins

Kobayashi, N.; Katayama, Hiroshi; Nagata, Masanori; Goto, Junichi

doi:10.2116/analsci.16.1133

Cited by 14 publications

(17 citation statements)

References 23 publications

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“…The R k values of peaks a, b, and c relative to that of the IS, 12-oxo-lithocholic acid, were identical to those of CA, DCA, and CDCA, respectively ( Table 1 ). Further confirmation of the identity of peak b was provided by ELISA, which demonstrated its high affinity to an anti-DCA monoclonal antibody (23). The amount of DCA in the sample, as measured by ELISA (0.06 nmol/g wet weight of brain) was comparable to the result obtained by LC/ESI-MS.…”

Section: Ca Dca and Cdca Are Present In The Rat Brainsupporting

confidence: 53%

“…To characterize DCA, the fraction having a chromatographic retention time corresponding to that of authentic DCA was collected and subjected to ELISA using a monoclonal antibody with high specificity to DCA and markedly low affinity to the other bile acids (23). AffiniPure rabbit anti-mouse IgG ϩ IgM antibody diluted 1:400 with 50 mM sodium phosphate buffer (pH 7.3) (buffer I) was distributed in each well of the EIA/RIA plates, which were left overnight at 4 Њ C. After washing three times with 50 mM sodium phosphate buffer (pH 7.3) containing 0.9% sodium chloride (buffer II…”

Section: Elisa For Dcamentioning

confidence: 99%

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

Mano

Goto

Uchida

et al. 2004

Journal of Lipid Research

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Using liquid chromatography/electrospray ionization mass spectrometry, we have found three unconjugated bile acids [cholic acid (CA), chenodeoxycholic acid (CDCA), and deoxycholic acid (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 in the brain. The most abundant of the three, it was present at a concentration of 1.6 nmol/g wet weight ( ‫ف‬ 15 mg of protein) of brain, corresponding to almost 30 times its serum concentration. CA and DCA were present at 1/30th the concentration of CDCA. Bile acids conjugated with amino acids, sulfuric acid, and glucuronic acid were not detected. These data clearly demonstrate that unconjugated CDCA and, to a lesser extent, CA and DCA, exists in the rat brain. Bile acids are synthesized in the liver from cholesterol by the action of hepatic enzymes and excreted into the small intestine via the bile duct. In the intestinal lumen, they assist lipolysis and the absorption of fats by forming mixed micelles and then return to the liver upon absorption in the ileum and proximal colon. Because of their efficient hepatic uptake, bile acids have low concentrations in the peripheral blood. Recent observations also indicate that the nuclear bile acid receptor, the farnesoid X receptor, regulates the bile acid pool by repressing the transcription of genes encoding hepatocyte transporters (1) as well as cholesterol 7 ␣ -hydroxylase (2, 3), which is the ratelimiting enzyme for bile acid biosynthesis.

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Section: Ca Dca and Cdca Are Present In The Rat Brainsupporting

confidence: 53%

Section: Elisa For Dcamentioning

confidence: 99%

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

Mano

Goto

Uchida

et al. 2004

Journal of Lipid Research

Self Cite

113

100

View full text Add to dashboard Cite

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“…Although the present antibody was raised against a ␤-alanine conjugate, (S)-ibuprofen bound to the ⑀-amino group of lysine on peptide fragments was extracted, because of its feeble antigenic recognition of the structure around the bridge portion (Table 1). Previous papers show an almost identical phenomenon (24,30). When bile acids are directly conjugated to BSA through its C-24 carboxy group, raised antibodies show affinity not only for the hapten but also for its glycine, taurine, and lysine conjugates.…”

Section: Figmentioning

confidence: 81%

The Enantioselective Immunoaffinity Extraction of an Optically Active Ibuprofen-Modified Peptide Fragment

Ikegawa

Isriyanthi

Nagata

et al. 2001

Analytical Biochemistry

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“…Affi-Gel-10 was purchased from Bio-Rad. Affinity gel immobilized antideoxycholate monoclonal antibody (16) was prepared according to a previously reported method (13). A ZipTip C 18 cartridge was purchased from Millipore (Milford, MA).…”

Section: Methodsmentioning

confidence: 99%

A Nonenzymatic Modification of the Amino-terminal Domain of Histone H3 by Bile Acid Acyl Adenylate

Mano

Kasuga

Kobayashi

et al. 2004

Journal of Biological Chemistry

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Although it has been proposed that the secondary bile acids, deoxycholic acid and lithocholic acid, increase the number of aberrant crypt foci in the colon and may act as colon tumor promoters, there is little evidence detailing their mechanism of action. Histones play an important role in controlling gene expression, and the posttranslational modification of histones plays a role in regulation of intracellular signal transduction. In particular, the amino-terminal tail domain of histone H3 is sensitive to several posttranslational modifications, and acetylation of this domain changes its electrostatic environment and results in the loss of native folding. Therefore, we studied the modification of ⑀-amino groups on human histone H3 by deoxycholyl adenylate, which is an active intermediate in deoxycholyl thioester biosynthesis. After incubation of recombinant human histone H3 with a smaller amount of acyl adenylate, followed by enzymatic digestion, the peptide fragment mixtures were analyzed by matrix-assisted laser desorption ionization mass spectrometry. These data showed the formation of only one adduct fragment, which corresponded to amino acids 3-8 with a deoxycholate adduct, suggesting that the ⑀-amino group of Lys 4 had the highest reactivity. This novel modification, formation of a bile acid adduct on the histone H3 amino-terminal tail domain through an active acyl adenylate, may relate to the carcinogenesis-promoting effects of secondary bile acids.

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Production of a Monoclonal Antibody for Sensitive Monitoring of Deoxycholic Acid Residues Anchored on Endogenous Proteins

Cited by 14 publications

References 23 publications

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

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

The Enantioselective Immunoaffinity Extraction of an Optically Active Ibuprofen-Modified Peptide Fragment

A Nonenzymatic Modification of the Amino-terminal Domain of Histone H3 by Bile Acid Acyl Adenylate

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