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.