Laboratory monitoring of patients with hereditary tyrosinemia type I.

BACKGROUND: The prognosis of patients with Hereditary Tyrosinemia Type 1 (HT-1) has greatly improved with early detection through newborn screening and the introduction of nitisinone (NTBC) therapy. A recent guideline calls for periodic monitoring of biochemical markers and NTBC levels to tailor treatment; however, this is currently only achieved through a combination of clinical laboratory tests. We developed a multiplexed assay measuring relevant amino acids, succinylacetone (SUAC), and NTBC in dried blood spots (DBS) to facilitate treatment monitoring. METHODS: Tyrosine, phenylalanine, methionine, NTBC and SUAC were eluted from DBS with methanol containing internal standards for each analyte and analyzed by liquid chromatography tandem mass spectrometry over 6.5 min in the multiple reaction monitoring positive mode. RESULTS: Pre-analytical and analytical factors were studied and demonstrated a reliable assay. Chromatography resolved an unknown substance that falsely elevates SUAC concentrations and was present in all samples. To establish control and disease ranges, the method was applied to DBS collected from controls (n = 284) and affected patients before (n = 2) and after initiation of treatment (n = 29). In the treated patients SUAC concentrations were within the normal range over a wide range of NTBC levels. CONCLUSIONS: This assay enables combined, accurate measurement of revelevant metabolites and NTBC in order to simplify treatment monitoring of patients with HT-1. In addition, the use of DBS allows for specimen collection at home to facilitate more standardization in relation to drug and dietary treatment.

The critical role of psychosine in screening, diagnosis, and monitoring of Krabbe disease.

PURPOSE: Newborn screening (NBS) for Krabbe disease (KD) is performed by measurement of galactocerebrosidase (GALC) activity as the primary test. This revealed that GALC activity has poor specificity for KD. Psychosine (PSY) was proposed as a disease marker useful to reduce the false positive rate for NBS and for disease monitoring. We report a highly sensitive PSY assay that allows identification of KD patients with minimal PSY elevations. METHODS: PSY was extracted from dried blood spots or erythrocytes with methanol containing d5-PSY as internal standard, and measured by liquid chromatography-tandem mass spectrometry. RESULTS: Analysis of PSY in samples from controls (N = 209), GALC pseudodeficiency carriers (N = 55), GALC pathogenic variant carriers (N = 27), patients with infantile KD (N = 26), and patients with late-onset KD (N = 11) allowed for the development of an effective laboratory screening and diagnostic algorithm. Additional longitudinal measurements were used to track therapeutic efficacy of hematopoietic stem cell transplantion (HSCT). CONCLUSION: This study supports PSY quantitation as a critical component of NBS for KD. It helps to differentiate infantile from later onset KD variants, as well as from GALC variant and pseudodeficiency carriers. Additionally, this study provides further data that PSY measurement can be useful to monitor KD progression before and after treatment.

Cyanide-Nitroprusside Colorimetric Assay: A Rapid Colorimetric Screen for Urinary Cystine.

BACKGROUND: Cystinuria is an autosomal recessive disorder resulting in poor proximal tubule reabsorption of cystine in the nephron, increasing the risk of cystine stone formation. A fast, inexpensive assay to screen for urinary cystine is needed because cystine stones are difficult to noninvasively differentiate from more common calcium-containing ones. Tandem mass spectrometry (MS/MS) is sensitive and specific but is labor-intensive and costly. Alternatively, a colorimetric assay is fast and cost-effective; however, creatinine interference is an issue. METHODS: A published cyanide-nitroprusside colorimetric assay was modified for a high-throughput microplate format. Creatinine interference was reduced using 0.1 mol/L PBS and a standard reaction time of 60 s and was further corrected using a formula derived from the slope of multiple creatinine standard curves. RESULTS: The limit of blank was determined to be 2.6 mg/L, the limit of detection 11.9 mg/L, and the limit of quantitation 15.3 mg/L. The analytic measurement range was established as 15.3-100 mg/L cystine. Intraassay and interassay CV was calculated to be 9.6% and 8.0%, respectively, for a high-level cystine concentration (83.6 mg/L). Low-level cystine (36.4 mg/L) intraassay and interassay CV was determined to be 18.1% and 17.6%, respectively. Passing-Bablok regression analysis of colorimetric vs LC-MS/MS results revealed a slope of 1.10 and y intercept of -7.14 mg/L, with an overall bias of 2% by Bland-Altman plot analysis. CONCLUSIONS: We analytically validated a rapid colorimetric assay suitable to quantify urinary cystine. The effect of thiol drugs on this assay remains to be determined.

Silent Tyrosinemia Type I Without Elevated Tyrosine or Succinylacetone Associated with Liver Cirrhosis and Hepatocellular Carcinoma.

Tyrosinemia type I (TYRSN1, TYR I) is caused by fumarylacetoacetate hydrolase (FAH) deficiency and affects approximately one in 100,000 individuals worldwide. Pathogenic variants in FAH cause TYRSN1, which induces cirrhosis and can progress to hepatocellular carcinoma (HCC). TYRSN1 is characterized by the production of a pathognomonic metabolite, succinylacetone (SUAC) and is included in the Recommended Uniform Screening Panel for newborns. Treatment intervention is effective if initiated within the first month of life. Here, we describe a family with three affected children who developed HCC secondary to idiopathic hepatosplenomegaly and cirrhosis during infancy. Whole exome sequencing revealed a novel homozygous missense variant in FAH (Chr15(GRCh38):g.80162305A>G; NM_000137.2:c.424A > G; NP_000128.1:p.R142G). This novel variant involves the catalytic pocket of the enzyme, but does not result in increased SUAC or tyrosine, making the diagnosis of TYRSN1 problematic. Testing this novel variant using a rapid, in vivo somatic mouse model showed that this variant could not rescue FAH deficiency. In this case of atypical TYRSN1, we show how reliance on SUAC as a primary diagnostic test can be misleading in some patients with this disease. Augmentation of current screening for TYRSN1 with targeted sequencing of FAH is warranted in cases suggestive of the disorder.

Anesthesia and organic aciduria: is the use of lactated Ringer's solution absolutely contraindicated?

BACKGROUND: Organic acidurias (OAs) are rare inborn errors of metabolism that can present with various neurologic manifestations, propensity for acute metabolic decompensation with anion-gap metabolic acidosis, developmental delay, poor feeding, and failure to thrive. OBJECTIVE: In this case series, we outline the anesthetic management and perioperative outcomes of OA patients. METHODS: We reviewed demographic characteristics, comorbidities, and perioperative course of patients with four different OAs who underwent anesthetic care at our institution between January 1, 2000, and December 31, 2013. RESULTS: Eleven patients with OA underwent 19 anesthetic procedures, of which 13 were <2 h in duration and seven were outpatient procedures. One patient with methylmalonic acidemia developed metabolic acidosis during a 10-h procedure with substantial blood loss but lacked evidence that this acidosis could be attributed to his underlying metabolic disease. The patients who received hydration with lactated Ringer's solution and/or nitrous oxide anesthetic had a perioperative course free of metabolic complication. Two patients died within 30 days of surgery from causes likely to be unrelated to anesthetic exposure. CONCLUSIONS: Our patients with various forms of metabolically compensated OAs tolerated anesthetics for surgical procedures without metabolic decompensation, even when lactated Ringer's solution was used for hydration. Measures to prevent protein catabolism and intraoperative events that may precipitate metabolic acidosis, in addition to close monitoring of acid-base status during more extensive procedures, must be part of perioperative treatment of these patients.

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.