Microdetermination methods for substances of low and high molecular weights: Enzymatic cycling and...
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$20 MICRODETERMINATION METHODS FOR SUBSTANCES OF LOW AND HIGH MOLECULAR WEIGHTS: ENZYMATIC CYCLING AND IMMUNOENZYMATIC CYCLING ASSAY. TAKAHIKO KATQ± De~artmgnt Rf Biochemistry± Institute of Brain Research~ Universitx of !R~ZR Facultx Rf [Enzymatic Cycling] 10 -*3 moles of galactose, for instance, is converted by galactose dehydrogenase into an equal amount of NADH in the presence of excess NAD ÷ • After destroying NAD* , the NADH is added to the reaction mixture containing alcohol and malate dehydrogenases (ADH and MDH) with excess concentrations of ethanol and oxalaeetate. The NADH is oxidized by MDH and successively reduced by ADH back to NADH, producing equal amounts of malate and acetaldehyde (NAD cycling). After this cyclic reaction is repeated I0,000 times, malate accumulates to an amount 10,000-fold the original galactose, is converted to NADH by MDH reaction (indicator reaction) and fluorometrically determined. There are three kinds of enzymatic cycling reactions, i.e. NAD, NADP and CoA cyclings, which are routinely used to determine small molecuies (sugars, amino acids, nucleotides, etc.) and reaction products of enzymes contained in individual neurons isolated from brains. [Immunoenzymatie Cycling Assay] A polyclonal antibody (Ab) is raised against a high-molecular-weight substance as antigen (Ag; eg: horse ferritin) and the IgG is physically adsorbed to the surface of glass beads I00 ~ m in diameter. Ferritin (10 -21 moles, i.e. 630 moleeules) is bound to a single glass bead via Ag-Ab reaction in 2.6 nl of reaction mixture. In succession, the conjugate of Fab' of the IgG and ~ -D- galactocerebrosidase from E. Coli is similarly bound to the glass bead in 54 nl of the solution. After washing, the glass bead is added to 15 nl of 2-nitro- phenyl-~ -D-galactoside solution and 10 -*4 moles of galaetose, which are in proportional amount to the original ferritin, are formed by the galactosidase. The galactose is converted to NADH and amplified lO0,000-fold by NAD cycling for assay as described above. As examples of the application of these methods, choline acetyltransferase activity was determined using CoA cycling in indivi- dual motor neurons from animal spinal cords, and the distribution of ornithine aminotransferase molecules was demonstrated in the layers of mouse retinas. GC/MS STUDY ON METABOLISM OF TETRAHYDROISOQUINOLINE, AN ENDOGENOUS MPTP-LIKE SUBSTANCE, IN BRAIN. TOSHIMITSU NIWA .I , MITSUO YOSHIDA 2 , TOSHIHARU NAGATSU 3 , IDepartment of Medicine, Naqoya University Branch Hospital, Naqoya 461, Janan, ZDepartment of Neurology, Jichi Medical School, Tochiqi-ken 329-04, Japan, 3Department of Biochemistry, Naqoya University School of Medicine, Naqoya 466, Japan 1,2,3,4-Tetrahydroisoquinoline (TIQ) has been recently discovered in rat brain [I], and in parkinsonian and normal human brains [2]. Repeated administration of TIQ to marmosets produced parkinsonian symptoms with reduction in tyrosine hydroxylase, dopamine and total biopterin concentrations in the substantia nigra [3]. To determine if subcutaneously administered TIQ can pass through the blood- brain barrier and if TIQ can be metabolized in the brain and liver, we analyzed the brains and livers of TIQ-administered marmosets by gas chromatography mass spectrometry (GC/MS). TIQ was quantified by GC/MS in brains and livers of marmosets which showed parkinsonism after daily subcutaneous injection of TIQ at a dose of 50 mg/kg. A mixture of d3-TIQ/d~-TIQ was used as an internal standard. TIQ was highly increased in the brains and livers of the TIQ-treated marmosets, without detectable metabolite of TIQ. TIQ was present as an endogenous amine at very low concentrations in the brains and livers of saline-treated marmosets. The concentration of TIQ in the brains of TIQ-injected marmosets was highly increased to the same degree as in their livers. This means that TIQ can pass easily through the blood-brain barrier. No metabolite of TIQ, such as 1,2-dihydroisoquinoline, could be detected in the brains and livers of TIQ-treated marmosets. This suggests that TIQ is not a substrata of monoamine oxidase. In conclusion, TIQ can pass easily through the blood-brain barrier, cannot be metabolized in the brain and liver, and the accumulated TIQ in the brain may produce parkinsonism. References I M. Kohno, S. Ohta and M. Hirobe, Biochem. Biophys. Res. Commun., 140 (1986) 448. 2 T. Niwa, N. Takeda, N. Kaneda, Y. Hashizume and T. Nagatsu, Biochem. Biophys. Res. Comuun., 144 (1987) 1084. 3 T. Nagatsu and M. Yoshida, Neurosci. Lett., 87 (1988) 178.
Transcript of Microdetermination methods for substances of low and high molecular weights: Enzymatic cycling and...
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MICRODETERMINATION METHODS FOR SUBSTANCES OF LOW AND HIGH MOLECULAR WEIGHTS: ENZYMATIC CYCLING AND IMMUNOENZYMATIC CYCLING ASSAY. TAKAHIKO KATQ± De~artmgnt Rf Biochemistry± Institute of Brain Research~ Universitx of !R~ZR Facultx Rf
[Enzymatic Cycling] 10 -*3 moles of galactose, for instance, is converted by galactose dehydrogenase into an equal amount of NADH in the presence of excess NAD ÷ • After destroying NAD* , the NADH is added to the reaction mixture containing alcohol and malate dehydrogenases (ADH and MDH) with excess concentrations of ethanol and oxalaeetate. The NADH is oxidized by MDH and successively reduced by ADH back to NADH, producing equal amounts of malate and acetaldehyde (NAD cycling). After this cyclic reaction is repeated I0,000 times, malate accumulates to an amount 10,000-fold the original galactose, is converted to NADH by MDH reaction (indicator reaction) and fluorometrically determined. There are three kinds of enzymatic cycling reactions, i.e. NAD, NADP and CoA cyclings, which are routinely used to determine small molecuies (sugars, amino acids, nucleotides, etc.) and reaction products of enzymes contained in individual neurons isolated from brains. [Immunoenzymatie Cycling Assay] A polyclonal antibody (Ab) is raised against a high-molecular-weight substance as antigen (Ag; eg: horse ferritin) and the IgG is physically adsorbed to the surface of glass beads I00 ~ m in diameter. Ferritin (10 -21 moles, i.e. 630 moleeules) is bound to a single glass bead via Ag-Ab reaction in 2.6 nl of reaction mixture. In succession, the conjugate of Fab' of the IgG and ~ -D- galactocerebrosidase from E. Coli is similarly bound to the glass bead in 54 nl of the solution. After washing, the glass bead is added to 15 nl of 2-nitro- phenyl-~ -D-galactoside solution and 10 -*4 moles of galaetose, which are in proportional amount to the original ferritin, are formed by the galactosidase. The galactose is converted to NADH and amplified lO0,000-fold by NAD cycling for assay as described above. As examples of the application of these methods, choline acetyltransferase activity was determined using CoA cycling in indivi- dual motor neurons from animal spinal cords, and the distribution of ornithine aminotransferase molecules was demonstrated in the layers of mouse retinas.
GC/MS STUDY ON METABOLISM OF TETRAHYDROISOQUINOLINE, AN ENDOGENOUS MPTP-LIKE SUBSTANCE, IN BRAIN. TOSHIMITSU NIWA .I , MITSUO YOSHIDA 2 , TOSHIHARU NAGATSU 3 ,
IDepartment of Medicine, Naqoya University Branch Hospital, Naqoya 461, Janan, ZDepartment of Neurology, Jichi Medical School, Tochiqi-ken 329-04, Japan, 3Department of Biochemistry, Naqoya University School of Medicine, Naqoya 466, Japan
1,2,3,4-Tetrahydroisoquinoline (TIQ) has been recently discovered in rat brain [I], and in parkinsonian and normal human brains [2]. Repeated administration of TIQ to marmosets produced parkinsonian symptoms with reduction in tyrosine hydroxylase, dopamine and total biopterin concentrations in the substantia nigra [3]. To determine if subcutaneously administered TIQ can pass through the blood- brain barrier and if TIQ can be metabolized in the brain and liver, we analyzed the brains and livers of TIQ-administered marmosets by gas chromatography mass spectrometry (GC/MS).
TIQ was quantified by GC/MS in brains and livers of marmosets which showed parkinsonism after daily subcutaneous injection of TIQ at a dose of 50 mg/kg. A mixture of d3-TIQ/d~-TIQ was used as an internal standard. TIQ was highly increased in the brains and livers of the TIQ-treated marmosets, without detectable metabolite of TIQ. TIQ was present as an endogenous amine at very low concentrations in the brains and livers of saline-treated marmosets. The concentration of TIQ in the brains of TIQ-injected marmosets was highly increased to the same degree as in their livers. This means that TIQ can pass easily through the blood-brain barrier. No metabolite of TIQ, such as 1,2-dihydroisoquinoline, could be detected in the brains and livers of TIQ-treated marmosets. This suggests that TIQ is not a substrata of monoamine oxidase. In conclusion, TIQ can pass easily through the blood-brain barrier, cannot be metabolized in the brain and liver, and the accumulated TIQ in the brain may produce parkinsonism. References I M. Kohno, S. Ohta and M. Hirobe, Biochem. Biophys. Res. Commun., 140 (1986) 448. 2 T. Niwa, N. Takeda, N. Kaneda, Y. Hashizume and T. Nagatsu, Biochem. Biophys. Res. Comuun., 144 (1987) 1084. 3 T. Nagatsu and M. Yoshida, Neurosci. Lett., 87 (1988) 178.
