Mark J. Magera

Combined newborn screening for succinylacetone, amino acids, and acylcarnitines in dried blood spots

BACKGROUND Tyrosinemia type I (TYR 1) is a disorder causing early death if left untreated. Newborn screening (NBS) for this condition is problematic because determination of the diagnostic marker, succinylacetone (SUAC), requires a separate first-tier or only partially effective second-tier analysis based on tyrosine concentration. To overcome these problems, we developed a new assay that simultaneously determines acylcarnitines (AC), amino acids (AA), and SUAC in dried blood spots (DBS) by flow injection tandem mass spectrometry (MS/MS). METHODS We extracted 3/16-inch DBS punches with 300 microL methanol containing AA and AC stable isotope-labeled internal standards. This extract was derivatized with butanol-HCl. In parallel, we extracted SUAC from the residual filter paper with 100 microL of a 15 mmol/L hydrazine solution containing the internal standard 13C5-SUAC. We combined the derivatized aliquots in acetonitrile for MS/MS analysis of AC and AA with additional SRM experiments for SUAC (m/z 155-137) and 13C5-SUAC (m/z 160-142). Analysis time was 1.2 min. RESULTS SUAC was increased in retrospectively analyzed NBS samples of 11 TYR 1 patients (length of storage, 52 months to 1 week; SUAC range, 13-81 micromol/L), with Tyr concentrations ranging from 65 to 293 micromol/L in the original NBS analysis. The mean concentration of SUAC in 13 521 control DBS was 1.25 micromol/L. CONCLUSION The inclusion of SUAC analysis into routine analysis of AC and AA allows for rapid and cost-effective screening for TYR 1 with no tangible risk of false-negative results.

Second-tier test for quantification of alloisoleucine and branched-chain amino acids in dried blood spots to improve newborn screening for maple syrup urine disease (MSUD).

BACKGROUND Newborn screening for maple syrup urine disease (MSUD) relies on finding increased concentrations of the branched-chain amino acids (BCAAs) leucine, isoleucine, and valine by tandem mass spectrometry (MS/MS). d-Alloisoleucine (allo-Ile) is the only pathognomonic marker of MSUD, but it cannot be identified by existing screening methods because it is not differentiated from isobaric amino acids. Furthermore, newborns receiving total parenteral nutrition often have increased concentrations of BCAAs. To improve the specificity of newborn screening for MSUD and to reduce the number of diet-related false-positive results, we developed a LC-MS/MS method for quantifying allo-Ile. METHODS Allo-Ile and other BCAAs were extracted from a 3/16-inch dried blood spot punch with methanol/H2O, dried under nitrogen, and reconstituted into mobile phase. Quantitative LC-MS/MS analysis of allo-Ile, its isomers, and isotopically labeled internal standards was achieved within 15 min. To determine a reference interval for BCAAs including allo-Ile, we analyzed 541 dried blood spots. We also measured allo-Ile in blinded samples from 16 MSUD patients and 21 controls and compared results to an HPLC method. RESULTS Intra- and interassay imprecision (mean CVs) for allo-Ile, leucine, isoleucine, and valine ranged from 1.8% to 7.4%, and recovery ranged from 91% to 129%. All 16 MSUD patients were correctly identified. CONCLUSIONS The LC-MS/MS method can reliably measure allo-Ile in dried blood spots for the diagnosis of MSUD. Applied to newborn screening as a second-tier test, it will reduce false-positive results, which produce family anxiety and increase follow-up costs. The assay also appears suitable for use in monitoring treatment of MSUD patients.

Determination of Total Homocysteine, Methylmalonic Acid, and 2-Methylcitric Acid in Dried Blood Spots by Tandem Mass Spectrometry

BACKGROUND Newborn screening (NBS) for inborn errors of propionate, methionine, and cobalamin metabolism relies on finding abnormal concentrations of methionine and propionylcarnitine. These analytes are not specific for these conditions and lead to frequent false-positive results. More specific markers are total homocysteine (tHCY), methylmalonic acid (MMA), and methylcitric acid (MCA), but these markers are not detected by current NBS methods. To improve this situation, we developed a method for the detection of tHCY, MMA, and MCA in dried blood spots (DBSs) by liquid chromatography-tandem mass spectrometry (LC-MS/MS). METHODS The analytes were extracted from a single 4.8-mm DBS punch with acetonitrile:water:formic acid (59:41:0.42) containing dithiothreitol and isotopically labeled standards (d(3)-MMA, d(3)-MCA, d(8)-homocystine). The extract was dried and treated with 3 N HCl in n-butanol to form butylesters. After evaporation of the butanol, the residue was reconstituted and centrifuged and the supernatant was subjected to LC-MS/MS analysis. Algorithms were developed to apply this method as an efficient and effective second-tier assay on samples with abnormal results by primary screening. RESULTS The 99th percentiles determined from the analysis of 200 control DBSs for MMA, MCA, and HCY were 1.5, 0.5, and 9.8 μmol/L, respectively. Since 2005, prospective application of this second-tier analysis to 2.3% of all NBS samples led to the identification of 13 affected infants. CONCLUSIONS Application of this assay reduced the false-positive rate and improved the positive predictive value of NBS for conditions associated with abnormal propionylcarnitine and methionine concentrations.

Streamlined determination of lysophosphatidylcholines in dried blood spots for newborn screening of X-linked adrenoleukodystrophy

BACKGROUND Pre-symptomatic hematopoietic stem cell transplantation is essential to achieve best possible outcomes for patients with the childhood cerebral form of X-linked adrenoleukodystrophy (X-ALD). We describe a high-throughput method for measurement of C20-C26 lysophosphatidylcholines (LPCs) and biochemical diagnosis of X-ALD using the same dried blood spots (DBS) routinely used for newborn screening. METHODS LPCs are extracted from 3-mm DBS punch with methanol containing an isotopically labeled LPC as internal standard. This extract is transferred to a 96-well plate, evaporated and then reconstituted in mobile phase for flow injection analysis tandem mass spectrometry (FIA-MS/MS) in selected reaction monitoring mode for measurement of four different LPCs (C20, C22, C24, C26) and the internal standard (d4-C26-LPC). Analysis time is 1.5min per sample. RESULTS The mean CVs from the intra- and inter-assay experiments for LPCs were 6.3-15.1% for C20-LPC, 4.4-18.6% for C22-LPC and 4.5-14.3% for C24-LPC. Limits of detection were determined for C20-LPC (LOD=0.03μg/mL), C22-LPC (0.03μg/mL), C24-LPC (0.03μg/mL) and C26-LPC (0.01μg/mL). Reference ranges were established from DBS of 130 newborns and 20 adults. Samples of patients with X-ALD (n=16), peroxisomal biogenesis disorders (n=8), and X-ALD carriers (n=12) were analyzed blindly and all were correctly identified. CONCLUSION Analysis of LPC species by FIA-MS/MS is a fast, simple and reliable method to screen for X-ALD and other peroxisomal disorders in DBS. To maximize specificity, abnormal results can be verified by a 2nd tier assay using LC-MS/MS.

Determination of homovanillic acid in urine by stable isotope dilution and electrospray tandem mass spectrometry.

We have developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the analysis of homovanillic acid (HVA), a biochemical marker for catecholamine and neurotransmitter metabolism. Urine specimens are spiked with 5 microg of a stable-isotope labeled internal standard, 13C(6)18O-HVA, and prepared by automated solid phase extraction. Residues were dissolved in acetonitrile: 0.05% aqueous acetic acid and analyzed by MS/MS in the selected reaction monitoring mode (HVA: m/z 181 to m/z 137; 13C(6)18O-HVA: m/z 189 to m/z 145) after separation using a Discovery RP Amide C16 column. Consecutive calibrations (n=7) between 0.52 and 16.7 mg/l exhibited consistent linearity and reproducibility. At a urine concentration of 0.51 mg/l, the signal-to-noise ratio for HVA was 21:1. Inter- and intra-assay CVs ranged from 0.3% to 11.4%, at mean concentrations ranging 1.8 to 22.7 mg/l. Recovery of HVA added to urine ranged between 94.7% and 105% (1.25 mg/l added), 92.0% and 102% (5.0 mg/l), and 96.0% and 104% (10 mg/l). LC-MS/MS is well suited to replace an HPLC method for routine HVA determination, by providing positive identification, faster turn around time, virtually no repeat analyses and a 44% reduction of personnel necessary to perform the testing.

Liquid Chromatography–Tandem Mass Spectrometry Method for the Determination of Vanillylmandelic Acid in Urine

The biochemical detection of catecholamine-secreting tumors relies on the determination of several metabolites, among them vanillylmandelic acid (VMA), a metabolite derived from epinephrine and norepinephrine via the intermediate 4-hydroxy-3-methoxyphenylglycol (1)(2). VMA may also be produced by the oxidative deamination of normetanephrine and metanephrine or by O-methylation of dihydroxymandelic acid, a minor metabolite of norepinephrine (2)(3). Our laboratory is actively involved in the application of stable-isotope-labeled internal standards and tandem mass spectrometry (MS/MS) to the determination of a wide variety of biochemical markers currently being tested with more conventional analytical platforms. This effort has substantially improved the efficiency and effectiveness of our services and reduced turnaround time in a high-volume testing environment. The following is a report of our development of a MS/MS method for the determination of VMA in urine. VMA was purchased from Sigma, and d,l-vanillylmandelic acid, ring-2H3 98% (d3-VMA; GC/MS for isotopic enrichment 99.7%) was purchased from Cambridge Isotope Laboratories. OasisTM HLB solid-phase extraction columns were obtained from Waters. All other chemicals and solvents were of the highest purity available from commercial sources and were used without further purification. Stock solutions of VMA and d3-VMA were prepared by dissolving 5 or 10 mg, respectively, in 10 mL of 0.1 mol/L HCl. A working solution of VMA (20 mg/L) was prepared by diluting stock solutions with 0.1 mol/L HCl. Calibrators were prepared in deionized water by the addition of working solution (20 mg/L) to VMA concentrations of 0 (blank), 1.04, 2.08, 4.17, 8.33, 12.50, 16.67, and 20.00 mg/L. Prepared calibrators were then treated as specimens …

Homogentisic acid interference in routine urine creatinine determination.

We report the artifactual elevation of homogentisic acid (HGA) in urine from alkaptonuric patients after replacing the creatinine method (Jaffe reaction) in our laboratory with an automated enzymatic method. Samples with elevated HGA by GC-MS had lower creatinine values as determined by the enzymatic method than by the Jaffe reaction. The low creatinine values were due to interference by HGA in the enzymatic method. The enzymatic method is unsuitable for creatinine determination in urine of patients with alkaptonuria.

Acute Increases in Methylated Arginines in Obstructive Sleep Apnea: Implications for Cardiovascular Risk

Background: Available data suggest that hemoglobin Ale (HbAlc) levels may be related to cardiovascular risk in the general non-diabetic population. We sought to test this hypothesis prospectively in a cohort of women without overt cardiovascular disease. Methods: We conducted a nested 1:2 cazcontrol study in the Women’s Health Study cohort. We Identified 464 cases of incident myocardial infarction, stroke, or coronary revascularization and 928 controls who remained free of cardiovascular events at the time of case diagnosis. The mean follow-up was 7 years. Results: 136 of the overall study population had a history of diabetes at enrollment or an overtly elevated baseline HbAlc (~-6.40%) and were excluded from the primary analyses. Among women without diabetes or an elevated baseline HbAlc, baseline mean levels of HbAlc were significantly higher among future cardiovascular cases than controls (5.47% * 0.27 vs 5.37% t 0.22; p<O.OOOl). The crude relative risks (RR) of incident cardiovascular events for increasing quartiles of HbAlc were 1, 0.98, 1.33, and 2.25 (95% Cl for highest vs lowest quartile 1.59-3.18). HbAlc levels were significantly correlated with several other traditional cardiovascular risk factors, including age, body mass index, systolic blood pressure, C-reactive protein, and total cholesterol: high density lipoprotein-cholesterol ratio. In fully adjusted models the predictive effect of HbAlc was attenuated and not significant (RR = 1 .OO for top vs bottom quartile, 95% Cl 0.65-l .54). In contrast. in the population including women with diabetes at enrollment, diabetes (RR 4.97, 95% Cl 2.81-8.77) remained a strong Independent determinant of cardiovascular risk I” fully adjusted analyses, while HbAl c levels did not (RR = 1 .ll for top vs bottom quartile, 95% Cl 0.73-1.71). Conclusion: HbAlc is associated with future cardiovascular risk among non-diabetic women, but this relationship is largely attributable to a strong correlation with other cardiovascular risk factors. In contrast, diabetes is a strong independent determinant of cardiovascular risk, even after adjustment for HbAl c levels.