Quantitation of plasma and urine 3-hydroxyglutaric acid, after separation from 2-hydroxyglutaric acid and other compounds of similar ion transition, by liquid chromatography-tandem mass spectrometry for the confirmation of glutaric aciduria type 1.

BACKGROUND: Glutaric aciduria type 1, a deficiency of glutaryl-CoA dehydrogenase, causes an accumulation of neurotoxic metabolites glutaric acid and 3-hydroxyglutaric acid (3-HGA). Testing of these analytes is routinely done by GC-MS but seldom account for interference from isomers or compounds with similar ion transitions. We developed a liquid chromatography tandem mass spectrometry method that accurately measures 3-HGA in urine and plasma specimens, while utilizing similar reagents and instrumentation used for the routine performance of amino acid and acylcarnitine analysis in determining the diagnosis of several metabolic disorders. METHOD: Plasma and urine samples were added aliquots of the deuterated 3-HGA internal standard and acetonitrile. The protein-free supernatant was brought to dryness, and the residue derivatized using 3 M HCL in 1-butanol with heating. The dried derivative was then reconstituted in 50% methanol-water solution and aliquot transferred to an HPLC vial for analysis by LC-MS/MS. Separation was performed using a C8 HPLC column under flow gradient conditions of 0.2% formic acid in water and methanol, respectively. Ionization was by ESI and detection of selected precursor-product ion transitions by multiple reaction monitoring (MRM) in positive mode. RESULTS: The butyl-ester derivative of 3-HGA eluted at 7.82 min while 2-hydroxyglutaric acid (2-HGA) eluted at 8.21 min. This was equivalent to a separation factor of 1.05 and a resolution of 1.03, respectively. The 3-HGA calibration curve was linear over the range 6.20-319 ng mL-1 (r2 = 0.9996), and the reportable range determined by the linearity was found to be 1.54-384 ng mL-1. The calculated limits of detection and quantitation were 0.348 and 1.56 ng mL-1, respectively. Intra- and Inter-assay %CVs for quality control plasma and urine samples ranged from 2 to 18%, with recoveries of 66-115%. The method correlated to the gold standard GC-MS method for both serum (r2 ≥ 0.996) and urine analysis (r2 ≥ 0.949). The concentration of 3-HGA in normal, non-GA1 individuals was ≤25.2 ng mL-1 (in plasma) and ≤ 35.0 µmol mmol-1 of creatinine (in urine). CONCLUSIONS: This LC-MS/MS method accurately quantified plasma and urine 3-HGA concentration after successful resolution from 2-HGA and other compounds with similar ion transitions. This method is suitable for confirmatory testing of 3-HGA, as a follow-up to an abnormal newborn screen test result, with concern for GA type 1.

Urinary isoflavone and lignan phytoestrogen levels and risk of uterine fibroid in Jamaican women.

BACKGROUND: Isoflavones and lignans are phytoestrogens, and therefore, are able to bind to and activate estrogen receptors. The resultant estrogenic or antiestrogenic effect is dependent on the concentration of these phytoestrogens relative to endogenous estrogens and the site of their action, among others. Thus, isoflavones and lignans act as selective estrogen receptor modulators; having a beneficial effect in some tissues while simultaneously causing deleterious changes in others. OBJECTIVE: This case-control study investigates the relationship between urinary concentrations of genistein, daidzein, equol, and enterolactone, and the presence of uterine leiomyomas (fibroids) in Jamaican women. DESIGN: Phytoestrogen concentration in spot urine samples from 157 uterine fibroid cases and 171 fibroid-free controls diagnosed by ultrasonography, were assessed by Time-resolved Fluoroimmnoassay. Statistical evaluations were performed using SPSS 12.0. RESULTS: The median concentration of urinary enterolactone was significantly different between uterine fibroid cases and controls (p=0.029). However, this was not observed to affect risk of uterine fibroid, as trends across quartiles of urine enterolactone did not differ significantly between cases and controls. Median urinary genistein (p=0.510), daidzein (p=0.838), equol (p=0.621), total isoflavones (0.510) and total phytoestrogens (p=0.084) were similar for both groups. Binary logistic regression analysis of quartiles of urine genistein, daidzein, equol, enterolactone, total isoflavones, and total phytoestrogens showed no association with uterine fibroid. CONCLUSIONS: Uterine fibroid cases had a higher median urine concentration of enterolactone compared with controls. However, this was not observed to affect ones risk of fibroid. Neither was urine genistein, daidzein, equol total isoflavones, and total phytoestrogens observed to be associated with risk of uterine fibroid.

Comparison of cystatin C and Beta-2-microglobulin kinetics in children on maintenance hemodialysis.

Middle-molecules (MM) are not monitored in children on hemodialysis (HD), but are accumulated and increase the risk of cardiovascular disease and mortality. Molecular properties of Cystatin C (CyC), 13 kDa, potentially make it a preferred MM marker over Beta-2-Microglobulin (B2M), 12 kDa. We compared CyC and B2M kinetics to investigate if CyC can be used as preferred MM marker. CyC (mg/L) and B2M (µg/mL) were measured in 21 low-flux HD sessions in seven children. Blood samples were taken at HD start (pre), 1 and 2 hours into HD and at end of HD (post) for all sessions and 60 minutes after the first HD (Eq). PreCyC (9.85 ± 2.15) did not differ (P > 0.05) from postCyC (10.04 ± 2.83). PostB2M (38.87 ± 7.12) was higher (P < 0.05) than preHD B2M (33.27 ± 7.41). There was no change in CyC at 1 and 2 hours into HD, while B2M progressively increased. CyC or B2M changes did not significantly correlate with spKt/V (2.09 ± 0.86), ultrafiltration (4.61 ± 1.98%) or HD duration (218 ± 20 minutes). EqCyC was not different from postCyC (11.07 ± 3.14 vs. 10.71 ± 2.85, P > 0.05), while EqB2M was lower than postB2M (36.48 ± 7.68 vs. 41.09 ± 8.99, P < 0.05). MMs as represented by B2M and CyC are elevated in children on standard HD. Intensified HD modalities would be needed for their removal. B2M is affected by the dialytic process with a rise during HD independent of ultrafiltration and decrease 1 hour after, while CyC remains unchanged. We suggest that CyC be used as preferred marker of MM removal and as a marker of adequacy of intensified HD regimens.

Cystatin C in children on chronic hemodialysis.

BACKGROUND: Cystatin C (CyC) concentration has been suggested as a marker of middle-molecule accumulation, hemodialysis (HD) adequacy and for estimating residual renal function (RRF), but it has not been studied in pediatric HD. High CyC is associated with increased cardiovascular disease (CVD). We investigated CyC kinetics and the effect of RRF on CyC in a pediatric HD population. METHODS: A total of 21 HD sessions and 20 interdialytic periods were analyzed in seven patients, age 5-19 years, of whom four were anuric (A) and three were non-anuric (NA). CyC was measured before (preHD) and after (postHD) three standard HD sessions in 1 week and prior to the first session of the following week. RESULTS: We found no difference (p=0.67) in CyC concentration between preHD CyC (9.85 ± 2.15 mg/l; A vs. NA, p=0.37) and postHD CyC (10.04 ± 2.83 mg/l; A vs NA, p=0.28). The weekly average preHD CyC median concentration was 10.14 mg/l (A vs. NA, p=0.87) and correlated with age (r=0.808, p=0.03) and height measurement (r=0.799, p=0.03), but not with RRF, single-pool Kt/V, ultrafiltration, HD duration or blood liters processed. CONCLUSIONS: Cystatin C is very elevated in children on HD. It does not rise between HD sessions, is not removed by standard HD and remains at steady state; therefore, elimination is extrarenal. Low RRF does not affect CyC elimination. CyC increases with age and height. If a high CyC concentration can be proven to have a causative role in the development of CVD, routine intensified HD regimens in children may be indicated for its removal.

Urinary phytoestrogens and risk of prostate cancer in Jamaican men.

We evaluated the relationship of spot urinary concentrations of phytoestrogens with total prostate cancer and tumor grade in a hospital-based case-control study in Jamaica. Urine samples were analyzed for genistein, daidzein, equol (isoflavones), and enterolactone (lignan) among newly diagnosed cases (n = 175) and controls (n = 194). Urinary concentrations of enterolactone (lignan) were higher among cases. There were no significant differences in median concentrations of isoflavone excretion. Compared with non-producers of equol (reference tertile), men who produced equol were at decreased risk of total prostate cancer (tertile 2: OR, 0.42; CI, 0.23-0.75) (tertile 3: OR, 0.48; CI, 0.26-0.87) (p (trend), 0.020) and high-grade disease (tertile 2: OR, 0.31; CI, 0.15-0.61) (tertile 3: OR, 0.29; CI, 0.13-0.60) (p (trend), 0.001). Higher concentrations of enterolactone were positively related to total prostate cancer (OR, 1.85; CI, 1.01-3.44; p (trend), 0.027) as well as high-grade disease (OR, 2.46; CI, 1.11-5.46; p (trend), 0.023). There were no associations between urinary excretion of genistein and daidzein with risk of prostate cancer. Producers of equol (isoflavone) may be at reduced risk of total- and high-grade prostate cancer whereas enterolactone may increase the likelihood of disease.