Quantitative measurement of porphobilinogen in urine by stable-isotope dilution liquid chromatography-tandem mass spectrometry.

BACKGROUND: Measurement of porphobilinogen (PBG) is useful in the diagnosis of the acute neurologic porphyrias. Currently used colorimetric assays lack analytical and clinical sensitivity and specificity. METHODS: We developed a liquid chromatography-electrospray tandem mass spectrometry (LC-MS/MS) method for the measurement of PBG in 1 mL of urine, using 5-(aminoethyl)-4-(carboxymethyl) 1H-2,4-[(13)C]pyrolle-3-propanoic acid ([2,4-(13)C]PBG; 2.75 microg) as internal standard. After solid-phase extraction, LC-MS/MS analysis was performed in the selected-reaction monitoring (SRM) mode. PBG and [2,4-(13)C]PBG were monitored through their own precursor and product ion settings (m/z 227 to 210 and m/z 229 to 212, respectively). The retention time of PBG and [2,4-(13)C]PBG was 1.0 min in a 2.3-min analysis. RESULTS: Daily calibrations (n = 6) between 0.1 and 2.0 mg/L were linear and reproducible. Inter- and intraassay CVs were 3.2-3.5% and 2.6-3.1%, respectively, at mean concentrations of 0.24, 1.18, and 2.15 mg/L. The regression equation for the comparison between an anion-exchange column method (y) and the LC-MS/MS method (x) was: y = 0.84x + 0.74 (S(y/x) = 5.8 mg/24 h; r = 0.85; n = 100). In 47 volunteers, PBG excretion was 0.02-0.42 mg/24 h, lower than reported reference intervals (up to 2.0 mg/24 h) based on colorimetric methods. In 85 samples with PBG < or =0.5 by LC-MS/MS, 8 (9.4%) had values > or =2.0 mg/24 h by the anion-exchange method (mean +/- SD, 4.3 +/- 1.8 mg/24 h). In 11 patients with confirmed diagnoses of acute porphyria and increased PBG by LC-MS/MS, 2 had values within the reported reference intervals by a quantitative anion-exchange method. CONCLUSIONS: The quantitative LC-MS/MS method for PBG measurement exhibits greater analytical specificity and improved clinical sensitivity and specificity than currently available methods.

Ascorbate levels in red blood cells and urine in patients with sickle cell anemia.

UNLABELLED: Ascorbic acid can be important in sickle cell anemia (SCA) because significant oxidative stress occurs in the disease. Ascorbate could contribute to reduction of the increased oxygen free radicals generated in sickle red blood cells (SRBC) and to the recycling of vitamin E in the cells, while renal loss could contribute to the low plasma levels. Evaluation of red blood cell (RBC) and urine ascorbate in SCA has not been reported. Results showed (1) ascorbate levels in SRBC were similar to those in normals; (2) urine ascorbate excretion was increased in 36% of patients; (3) plasma levels of ascorbate were decreased. CONCLUSIONS: (1) Ascorbate is present in SRBC, most likely due to ascorbate recycling, despite increased free-radical generation. (2) The increase in renal excretion may contribute to the low plasma levels of ascorbate. (3) The presence of ample ascorbate in SRBC and decreased plasma ascorbate suggests that ascorbate movement across the SRBC membrane may differ from normal RBC.