Determination of serum uric acid using high-performance liquid chromatography (HPLC)/isotope dilution mass spectrometry (ID-MS) as a candidate reference method.

The singlet oxygen generation of a photosensitizer compound (PS) is an important property for photodynamic treatments. The PS quantum yield of 1 O 2 ( Φ Δ ) is usually obtained by the time-resolved measurement of 1 O 2 phosphorescence emission. However, the equipment employed is quite expensive. In the present study, the methodology previously proposed by us for Φ Δ dosimetry using uric acid (UA) as a chemical-probe was systematically evaluated. The PS photo-excitation was achieved with a polychromatic light source (LED) that generates 1 O 2 which attacks the UA. The level of UA oxidation is proportional to a parameter called chemical photodynamic efficiency, which is correlated to Φ Δ by comparing it to a standard compound. The photobleaching reaction of the PS is considered in the calculation. Several parameters that influence this measurement have been investigated with different PS (phenothiazines, xanthenes, chlorins, and benzoporphyrin dyes) in water, ethanol, and aqueous P-123 polymeric surfactant. Excellent results were obtained when standard compounds belonging to the same class of the photosensitizer and the same solvent were used. The chemical method of 1 O 2 evaluation with UA is reliable, exact, precise, easy to perform and low cost, although limited by the standard compound employed as Φ Δ Std .

Singlet oxygen dosimetry using uric acid as a chemical probe: Systematic evaluation

Urate is the terminal product of purine metabolism in primates, including humans. Urate is also an efficient scavenger of oxidizing species and is thought to be an important antioxidant in human body fluids. Allantoin, the major oxidation product of urate, has been suggested as a candidate biomarker of oxidative stress because it is not produced metabolically. Although urate is converted to allantoin under strongly alkaline pH, such conditions have been used in the past to facilitate extraction of allantoin. We evolved a method for the determination of allantoin concentrations in human plasma and serum by gas chromatography-mass spectrometry without such artifact. With this method, we show that alkaline conditions do indeed cause breakdown of urate, leading to significant overestimation of allantoin concentration in human samples. By using our alternative method, serum samples from 98 volunteers were analyzed, and allantoin levels were found to be significantly lower than was previously reported. The in vivo utility and sensitivity of our method was further evaluated in human nasal-lining fluids. We were able to demonstrate an ozone-induced increase in allantoin, in the absence of increases in either ascorbate or glutathione oxidation products.

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Allantoin in human plasma, serum, and nasal-lining fluids as a biomarker of oxidative stress: avoiding artifacts and establishing real in vivo concentrations.

A new isotope dilution mass spectrometric method for uric acid is described. A known weight of [1,3-15N2]uric acid is added to a known weight of serum, and the mixture is allowed to equillibrate. The serum is put through an anion-exchange resin, and the isolated uric acid is converted to the tetrakis-(tert-butyldlmethylsilyl) derivative of uric acid. For measurement, the derivative is injected into a gas chromatograph interfaced with a low-resolution, magnetic sector mass spectrometer. Isotope ratio measurements are made from the abundances of the [M - tert-butyl]+ ions at m/z 567 and 569. Bias is investigated by measuring the uric acid level in the same samples under different chromatographic conditions and with different ionization techniques. If these confirmatory measurements agree with the principal measurements, we have strong evidence for the absence of measurement bias. Uric acid was determined in three lyophilized human serum pools by this method. For Standard Reference Material (SRM) 909, four sets of six samples each were prepared. For Candidate SRM 909a, which consisted of two pools, each with a different level of uric acid, six sets of two samples of each level were prepared. The coefficient of variation for a single measurement ranged from 0.34% to 0.42%, while the relative standard error of the mean ranged from 0.08% to 0.14%. The results from the confirmatory measurements demonstrated that there was no significant bias in the measurements. The combination of high precision and absence of significant bias in the results qualifies this method as a candidate definitive method as defined by the National Committee for Clinical Laboratory Standards.

Determination of serum uric acid by isotope dilution mass spectrometry as a new candidate definitive method

Determination of uric acid in human urine by ion chromatography with conductivity detector

We examined the stability of uric acid in dilute aqueous ammonium hydroxide solution by mass spectrometry. Uric acid decomposes in ammonium hydroxide even as dilute as 15 mmol/L when the mole ratio of ammonium hydroxide to uric acid is 50:1. There are at least four products of the decomposition, two of which have been identified as allantoin and urea. The slope of the decomposition curve indicates that uric acid is destroyed at an initial rate of 2-3% per hour. In ammonium hydroxide at a concentration of 1 mmol/L and a mole ratio of ammonium hydroxide to uric acid of less than or equal to 3.4, uric acid is not detectably decomposed. Evidently, any method for determination of uric acid that involves treating the analyte with ammonium hydroxide before analysis may destroy it. Therefore, a published method described as being "definitive" for uric acid (J Clin Chem Clin Biochem 1985; 23:129-35) could produce incorrect results because it involves storing the uric acid in 15 mmol/L ammonium hydroxide at a mole ratio of ammonium hydroxide to uric acid of greater than 120:1.

M J Welch

Papers

The stability of uric acid in ammonium hydroxide.