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  • Quantitative Phosphate Determinations

    Determination of Inorganic Phosphate
    in Biologic Samples

    A 1N perchloric acid Solutions/Reagents
    B 5mM sodium molybdate (Na2MoO4·2H2O, Mr 241.95)
    Important! Do not use ammonium molybdate!
    C isopropylacetate
    Standard 10mM KH2PO4 in 0.5Mperchloric acid
    Put 1.5 ml of Soln. B and 2.0 ml of Soln. C into phosphate-free test
    tubes. The sample,which should contain notmore than 100 nmoles
    phosphate, is mixed with an equal volume of Soln. A. Give 0.5 ml of
    this mixture to the above mixture of B and C. Shake the obtained
    mixture vigorously for 30 s and then spin in a centrifuge for a short
    period to separate the phases. To avoid the decomposition of labile
    organic phosphates, the extraction should be done at 0 ◦C or below.
    The molybdatophosphate complex remains in the organic
    phase, which is removed and read at 725 nm against a blank.
    Standards are made in the range of 5–100 nmoles phosphate
    per 0.5 ml.
    References
    Wahler BE,Wollenberger A (1958) Biochem Z 329:508
    Important! Use phosphate-free test tubes (cleaned with hot diluted
    HCl) or plastic disposables.
    This procedure is simpler andmore reliable than that by Fiske and
    Subbarow.
    Solutions/Reagents A 6N HCl
    B 2.5% ammonium molybdate (w/v) in ddH2O
    C 10% ascorbic acid (w/v) in ddH2O
    D 2% urea (w/v) in ddH2O
    E Reagent after ashing: Soln. B, C, and ddH2O are mixed in
    a ratio of 1:1:8
    E Reagent without previous ashing: Soln. A, B, C, and ddH2O
    are mixed in a ratio of 1:1:1:7
    E and E are stable only for 1 day.
    Standard 10mM KH2PO4
    conc. sulfuric acid
    conc. nitric acid
    Ashing
    Ashing must be done if phosphate is at least partially covalently
    bound as, for example, in nucleic acids, nucleotides, or phosphoproteins.
    Give 0.2 ml of conc. sulfuric acid to 1–2 ml of aqueous sample.
    Concentrate the liquid carefully in a hood at about 130 ◦C and then
    heat to 280 ◦C until white fog appears. After cooling, add one to
    two drops of conc. nitric acid and heat again until nitrous gases
    are visible. After cooling, add 2 ml of Soln. D, boil the solution for
    a short period, and fill up to 5.0 ml with ddH2O.
    Determination
    Mix 2.0 ml of the sample solution
    Determination
    Mix 2.0 ml of the sample solution after digestion or sample in
    Soln. A with Soln. E and E . Close the test tubes and incubate in
    the dark at 37 ◦C for 1.5–2 h. After that, read samples, blank, and
    standards at 750 nm.
    The standard curve is made in the range of 50–350 nmoles
    phosphate.
    100 nmoles phosphate = 9.497 μg PO4 = 3.097 μg P
    References
    Chen PS, Toribara TY (1956) Anal Chem 28:1756
    1.3.3 Phospholipid Determination
    Solutions/Reagents A chloroform/methanol 1:2 (v/v)
    chloroform
    Wet the lyophilized sample with 80 μl ddH2O. After that, add
    300 μl of Soln. A and homogenize the sample with a glass-Teflon
    homogenisator. After addition of 100 μl chloroform, centrifuge the
    mixture for phase separation. This extraction is repeated three to
    four times.
    Combine the organic phases and vaporize the organic solvents
    a nitrogen stream. The remaining solvent is removed in vacuum.
    Use the dry residue for digestion and phosphate determination
    described in Protocol 1.3.2.
    1 nmoles phosphate ≈ 85 ng phospholipid; 1 μg phosphate ≈ 8.3 μg phospholipid with an average Mr of 800.
    References
    Blight EG, DyerW(1959) Can J Biochem Physiol 37:911
    1.4 Monosaccharide Determination
    The quantitative determinations of the monosaccharides ribose
    and deoxyribose are given in Protocols 1.2.2 and 1.2.3, respectively.
    The following protocol is useful for all monosaccharides.
    80% phenol (w/v) in ddH2O (phenol must be colorless)
    conc. sulfuric acid
    Give 1.0 ml of the monosaccharide-containing solution (10–70 μg
    of saccharide) into a centrifuge tube and mix with 20 μl of Soln. A.
    Add 2.5 ml of conc. sulfuric acid onto the surface of the liquid
    (caution, corrosive!), then cool for 10 min and keep at 25–30 ◦C for
    10–20 min. After an additional 30 min at RT, read the absorption
    (hexoses) at 490 nm, pentoses at 480 nm.
    The standard curve is made fromthe appropriatemonosaccharide
    dissolved in water.
    Important! Because phenol is used, the waste has to be disposed
    according to the local regulations.
    Commercially available kits (e.g., Roche Glycan Quantitation Kit)
    are based on the oxidation of the carbohydrates with periodic acid
    and subsequent coupling of the formed aldehyde with a hydrazide
    (e.g., digoxigenin hydrazide). The formed conjugate is estimated Immunochemical
    immunochemically by ELISA.








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