Lethal neonatal case and review of primary short-chain enoyl-CoA hydratase (SCEH) deficiency associated with secondary lymphocyte pyruvate dehydrogenase complex (PDC) deficiency.

Mutations in ECHS1 result in short-chain enoyl-CoA hydratase (SCEH) deficiency which mainly affects the catabolism of various amino acids, particularly valine. We describe a case compound heterozygous for ECHS1 mutations c.836T>C (novel) and c.8C>A identified by whole exome sequencing of proband and parents. SCEH deficiency was confirmed with very low SCEH activity in fibroblasts and nearly absent immunoreactivity of SCEH. The patient had a severe neonatal course with elevated blood and cerebrospinal fluid lactate and pyruvate concentrations, high plasma alanine and slightly low plasma cystine. 2-Methyl-2,3-dihydroxybutyric acid was markedly elevated as were metabolites of the three branched-chain α-ketoacids on urine organic acids analysis. These urine metabolites notably decreased when lactic acidosis decreased in blood. Lymphocyte pyruvate dehydrogenase complex (PDC) activity was deficient, but PDC and α-ketoglutarate dehydrogenase complex activities in cultured fibroblasts were normal. Oxidative phosphorylation analysis on intact digitonin-permeabilized fibroblasts was suggestive of slightly reduced PDC activity relative to control range in mitochondria. We reviewed 16 other cases with mutations in ECHS1 where PDC activity was also assayed in order to determine how common and generalized secondary PDC deficiency is associated with primary SCEH deficiency. For reasons that remain unexplained, we find that about half of cases with primary SCEH deficiency also exhibit secondary PDC deficiency. The patient died on day-of-life 39, prior to establishing his diagnosis, highlighting the importance of early and rapid neonatal diagnosis because of possible adverse effects of certain therapeutic interventions, such as administration of ketogenic diet, in this disorder. There is a need for better understanding of the pathogenic mechanisms and phenotypic variability in this relatively recently discovered disorder.

Triple therapy with pyridoxine, arginine supplementation and dietary lysine restriction in pyridoxine-dependent epilepsy: Neurodevelopmental outcome.

Pyridoxine-dependent epilepsy (PDE) is an epileptic encephalopathy characterized by response to pharmacologic doses of pyridoxine. PDE is caused by deficiency of α-aminoadipic semialdehyde dehydrogenase resulting in impaired lysine degradation and subsequent accumulation of α-aminoadipic semialdehyde. Despite adequate seizure control with pyridoxine monotherapy, 75% of individuals with PDE have significant developmental delay and intellectual disability. We describe a new combined therapeutic approach to reduce putative toxic metabolites from impaired lysine metabolism. This approach utilizes pyridoxine, a lysine-restricted diet to limit the substrate that leads to neurotoxic metabolite accumulation and L-arginine to compete for brain lysine influx and liver mitochondrial import. We report the developmental and biochemical outcome of six subjects who were treated with this triple therapy. Triple therapy reduced CSF, plasma, and urine biomarkers associated with neurotoxicity in PDE. The addition of arginine supplementation to children already treated with dietary lysine restriction and pyridoxine further reduced toxic metabolites, and in some subjects appeared to improve neurodevelopmental outcome. Dietary lysine restriction was associated with improved seizure control in one subject, and the addition of arginine supplementation increased the objective motor outcome scale in two twin siblings, illustrating the contribution of each component of this treatment combination. Optimal results were noted in the individual treated with triple therapy early in the course of the disease. Residual disease symptoms could be related to early injury suggested by initial MR imaging prior to initiation of treatment or from severe epilepsy prior to diagnosis. This observational study reports the use of triple therapy, which combines three effective components in this rare condition, and suggests that early diagnosis and treatment with this new triple therapy may ameliorate the cognitive impairment in PDE.

Tacrolimus and sirolimus in capillary dried blood spots allows for remote monitoring.

Therapeutic drug monitoring of tacrolimus and sirolimus plays a significant role in the clinical follow-up of transplant patients receiving IMS therapy. Success of transplant and favorable patient outcome relies on maintaining adequate therapeutic drug levels. The purpose of this research is to assess the clinical utility of remote collection of DBS for immunosuppressant monitoring and compare the IMS level in paired collections of venous whole blood and DBS. Sirolimus and tacrolimus levels were clinically correlated in capillary blood collected from a finger poke with venous whole blood from pediatric, post-transplant patients. The participants took the dried blood spot card home with them with a pre-addressed, postage-paid envelope and mailed it back to the laboratory. Overall, a small but statistically significant negative bias was observed (-0.6 ng/mL, p = 0.0011). A chart review was performed to assess whether clinical management would have changed, and none of the cases revealed a clinically significant change. Sirolimus in DBS also correlated with venous levels. Overall, a small but statistically negative bias was observed (-0.8 ng/mL, p = 0.029). In summary, analysis of IMS levels in DBS is possible, and the difference noted between capillary and venous blood is within the clinically acceptable limits.

Exposure to environmental tobacco smoke during pregnancy in rats yields less effect on indices of brain cell number and size than does postnatal exposure.

While there is evidence that human perinatal exposure to environmental tobacco smoke (ETS) can result in an increased risk of respiratory disorders and sudden infant death syndrome, evidence linking ETS exposure to neurodevelopmental handicaps is suggestive but less compelling. We previously noted that postnatal ETS exposure, rather than prenatal exposure, resulted in reduced concentration of hindbrain DNA and increased protein/DNA ratio when rat brain tissue was studied at 9 weeks postnatal age. We have now evaluated the effects of ETS exposure during pregnancy on brain development by assaying brain tissue at term. ETS exposure had no detectable effects on regional brain concentrations of DNA, protein and cholesterol or on protein/DNA and cholesterol/DNA ratios. While ETS exposure during pregnancy also had no detectable effects on the weights of the individual fetuses or on the weights of various organs, certain regions of the fetal skeleton demonstrated accelerated ossification. The findings of this study are contrasted to the developmental effects of both nicotine and ETS in Rhesus macaques. Additional studies designed specifically to assess the risk of prenatal ETS exposure on brain development in non-human primates and other precocial species are warranted.

Validation of a therapeutic range for nitisinone in patients treated for tyrosinemia type 1 based on reduction of succinylacetone excretion.

The drug nitisinone (NTBC; Orfadin, Vienna, Austria) has been used for the treatment of hereditary tyrosinemia type-1 since 1991. Nitisinone effectively blocks the metabolism of tyrosine to prevent the formation of the toxic compound succinylacetone (and precursor fumarylacetoacetate) in affected children. Monitoring of plasma drug levels and urine succinylacetone can be used to assess compliance and adequate dose of drug. We present retrospective data from patient monitoring for over 10 years that provide validation of a target therapeutic range for nitisinone of 40 to 60 µmol/L. The target nitisinone range is justified as valid based on reduction of succinylacetone excretion. There was no statistical significance in succinylacetone excretion in mmol/mol creatinine above a level of 40 µmol/L plasma NTBC (P > 0.05).

One-year pilot study on the effects of nitisinone on melanin in patients with OCA-1B.

BACKGROUND: Oculocutaneous albinism (OCA) results in reduced melanin synthesis, skin hypopigmentation, increased risk of UV-induced malignancy, and developmental eye abnormalities affecting vision. No treatments exist. We have shown that oral nitisinone increases ocular and fur pigmentation in a mouse model of one form of albinism, OCA-1B, due to hypomorphic mutations in the Tyrosinase gene. METHODS: In this open-label pilot study, 5 adult patients with OCA-1B established baseline measurements of iris, skin, and hair pigmentation and were treated over 12 months with 2 mg/d oral nitisinone. Changes in pigmentation and visual function were evaluated at 3-month intervals. RESULTS: The mean change in iris transillumination, a marker of melanin, from baseline was 1.0 ± 1.54 points, representing no change. The method of iris transillumination grading showed a high intergrader reliability (intraclass correlation coefficient ≥ 0.88 at each visit). The number of letters read (visual acuity) improved significantly at month 12 for both eyes (right eye, OD, mean 4.2 [95% CI, 0.3, 8.1], P = 0.04) and left eye (OS, 5 [1.0, 9.1], P = 0.003). Skin pigmentation on the inner bicep increased (M index increase = 1.72 [0.03, 3.41], P = 0.047). Finally, hair pigmentation increased by both reflectometry (M index [17.3 {4.4, 30.2}, P = 0.01]) and biochemically. CONCLUSION: Nitisinone did not result in an increase in iris melanin content but may increase hair and skin pigmentation in patients with OCA-1B. The iris transillumination grading scale used in this study proved robust, with potential for use in future clinical trials. CLINICALTRIALS: gov NCT01838655. FUNDING: Intramural program of the National Eye Institute.

Quantifying MMA by SLE LC-MS/MS: Unexpected challenges in assay development.

OBJECTIVES: Analysis of serum/plasma methylmalonic acid (MMA) is important for the diagnosis and management of methylmalonic acidemia in pediatric populations. This work focuses on developing and validating a liquid chromatography tandem mass spectrometry (LC-MS/MS) method to monitor methylmalonic acidemia using a simple method preparation. DESIGN AND METHODS: MMA and stable isotope labeled d3-MMA was extracted using supported liquid extraction (SLE). Assay imprecision, bias, linearity, recovery and carryover were determined. The relationship between MMA and propionyl acylcarnitine (C3-acylcarnitine) was also evaluated using historical paired results from 51 unique individuals. RESULTS: Baseline separation between MMA and succinic acid was completed in 7min. The assay was linear from 0.1 to 500µM. The intra-day and inter-day imprecision CV ranged from 4.1 to 13.2% (0.3 to 526µM) and 5.0 to 15.7% (0.3 to 233µM), respectively. Recovery ranged from 93 to 125%. The correlation with a national reference laboratory LC-MS/MS assay showed a Deming regression of 1.026 and intercept of -1.335. Carryover was determined to be <0.04%. Patient-specific correlation was observed between MMA and C3-acylcarnitine. CONCLUSION: This report describes the first LC-MS/MS method using SLE for MMA extraction. In addition, we illustrate the challenges encountered during this method development that should be assessed and resolved by any laboratory implementing a SLE LC-MS/MS assay designed to quantify analytes across several orders of magnitude.

Expression of human paraoxonase (PON1) during development.

BACKGROUND: Paraoxonase (PON1), a HDL-associated enzyme, protects against toxicity from specific organophosphorus compounds and oxidized lipids. Common polymorphisms in the PON1 gene have been identified and characterized in the coding region, 5' regulatory region and 3' UTR. The Q192R coding region polymorphism determines substrate-dependent differences in catalytic efficiency of hydrolysis. The -108CT polymorphism in the 5' regulatory region has a significant effect on PON1 expression, with the -108C allele expressing on average twice the level of plasma PON1 as the -108T allele. In addition to the effects of regulatory and coding region polymorphisms on PON1 levels and activity, plasma PON1 levels are also developmentally regulated. Since PON1 levels are important in determining resistance to specific organophosphorus compounds, the time course of appearance of PON1 in newborns is of great interest. RESULTS: We report here that PON1 levels plateau between 6 to 15 months of age, and that variability in the age at which PON1 levels plateau is quite variable among individuals. In mice and rats, plasma PON1 activity reaches a plateau at 3 weeks of age. In mice that lack endogenous PON1, human transgenes encoding either PON1(Q192) or PON1(R192) under the control of the human PON1 regulatory sequences exhibited a similar time course of expression as that seen in wild-type mice, indicating conservation of the developmental regulatory elements between mouse and human PON1.

Algorithm for locating analytes of interest based on mass spectral similarity in GC x GC-TOF-MS data: analysis of metabolites in human infant urine.

The developed algorithm reported herein, referred to as "DotMap," addresses the need to rapidly identify analyte peak locations in gas chromatography x gas chromatography-time of flight mass spectrometry (GC x GC-TOF-MS) data. The third-order structure of GC x GC-TOF-MS data is such that at each point in the GC x GC chromatogram, a complete mass spectrum is measured. DotMap utilizes this third-order structure to search for the location of a given spectrum of interest in a complete data set, or in a user selected portion of the complete data set. The algorithm returns a contour plot indicating the location of signal(s) with the most similar mass spectra to the analyte of interest. A spectrum from the region indicated is then subjected to an automated mass spectral search to give immediate feedback on the accuracy of the analysis. This algorithm was investigated with a trimethylsilyl (TMS) derivatized human infant urine sample that contained organic acid metabolites. One hundred percent of 12 selected TMS derivatized organic acid metabolites in human infant urine were located with the DotMap algorithm. A typical automated DotMap analysis takes 30 s on a 1.6 GHz PC with 1024 MB of RAM. Vanillic acid (TMS) was located by DotMap, but also exhibited overlap with other organic acids. The presence of vanillic acid (TMS) was confirmed by subjecting the appropriate GC x GC region to chemometric signal deconvolution by PARAFAC to yield pure component information suitable for subsequent quantification.

Improving the value of costly genetic reference laboratory testing with active utilization management.

CONTEXT: Tests that are performed outside of the ordering institution, send-out tests, represent an area of risk to patients because of complexity associated with sending tests out. Risks related to send-out tests include increased number of handoffs, ordering the wrong or unnecessary test, specimen delays, data entry errors, preventable delays in reporting and acknowledging results, and excess financial liability. Many of the most expensive and most misunderstood tests are send-out genetic tests. OBJECTIVE: To design and develop an active utilization management program to reduce the risk to patients and improve value of genetic send-out tests. DESIGN: Send-out test requests that met defined criteria were reviewed by a rotating team of doctoral-level consultants and a genetic counselor in a pediatric tertiary care center. RESULTS: Two hundred fifty-one cases were reviewed during an 8-month period. After review, nearly one-quarter of genetic test requests were modified in the downward direction, saving a total of 2% of the entire send-out bill and 19% of the test requests under management. Ultimately, these savings were passed on to patients. CONCLUSIONS: Implementing an active utilization strategy for expensive send-out tests can be achieved with minimal technical resources and results in improved value of testing to patients.

Analysis of vanillylmandelic acid and homovanillic acid by UPLC-MS/MS in serum for diagnostic testing for neuroblastoma.

BACKGROUND: Vanillylmandelic acid (VMA) and homovanillic acid (HVA) are typically measured in urine for the diagnosis and monitoring of neuroblastoma, a tumor in children <5 y. A protocol for evaluation of serum VMA and HVA has been utilized at our institution for approximately 25 y, originally validated using high performance liquid chromatography (HPLC) with an electrochemical detector. We recently validated a serum VMA/HVA method by ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). METHODS: After solvent extraction and clean up with Ultrafree centrifugal filters, samples were analyzed by UPLC-MS/MS in multiple reaction monitoring mode. RESULTS: The assay was linear between 2 and 1000 ng/ml for VMA and HVA. Within run and run to run CVs were <5% for VMA at all levels, <10% for HVA at high levels, and <20% at low levels. Correlation with the HPLC method was acceptable with a constant bias. The reference interval for VMA by UPLC-MS/MS was determined to be ≤20 ng/ml, and HVA≤30 ng/ml. Original patient data comparing urine to serum showed diagnostic agreement >80% for both VMA and HVA. CONCLUSION: Correlation of VMA and HVA was acceptable after adjustment of reference intervals. Collection of a single serum sample instead of 24-h urine collection saves time and improves accuracy of measurement due to difficulty of collecting a 24-h urine sample in infants and young children. UPLC-MS/MS also offers improved analyte specificity, improved signal to noise, and rapid analysis time.

Clinical validation and implementation of a multiplexed immunosuppressant assay in dried blood spots by LC-MS/MS.

BACKGROUND: Therapeutic drug monitoring of immunosuppressive drugs is important in transplant patients. We developed and validated liquid chromatography-mass spectrometry (LC-MS/MS) assay for simultaneous quantitation of tacrolimus (TaC), sirolimus (SrL), and cyclosporin A (CsA) in dried blood spots (DBSs) to offer patients home sample collection, avoiding travel for blood draws. METHODS: After extraction, samples were analyzed by LC-MS/MS in multiple reaction monitoring mode. RESULTS: The assay was linear between 1.2-40 ng/ml for TaC and SrL, and 30-1000 ng/ml for CsA. Inter- and intra-assay CVs were ≤14.8% for all 3 drugs. This method correlated well with the existing clinical whole blood assay, with coefficients of determination >0.95 for all 3 drugs. DBS quality control samples were stable for at least 30 days at -20, 4, and 25°C. Stability of patient DBS samples was at least 5 days at temperatures up to 60°C, except for SrL where degradation was observed at 60°C within 24 h. No effect of hematocrit level, blood spot volume or punch location was observed. CONCLUSION: Immunosuppressant levels measured in DBS correlate with whole blood LC-MS/MS assay and may contribute to successful outcome of organ transplant and patient satisfaction.

The use of acarbose inhibition in the measurement of acid alpha-glucosidase activity in blood lymphocytes for the diagnosis of Pompe disease.

PURPOSE: Acid alpha-glucosidase is present in various tissues, including blood cells. Historically, enzyme measurement in cultured fibroblasts, or muscle, has been the gold standard to confirm a diagnosis of Pompe disease, due to the possibility of alternate isoenzyme activity masking disease in white cell assays. Enzyme measurement in an isolated lymphocyte population with acarbose, an inhibitor of neutral alpha-glucosidase, has greatly improved the sensitivity and specificity of the test in blood cells allowing for more rapid laboratory testing for Pompe disease. METHODS: An assay for acid alpha-glucosidase was performed with and without inhibitor in lymphocytes from 14 patients with a clinical suspicion of infantile Pompe disease. Concurrent testing was performed in fibroblasts in an independent laboratory. RESULTS: Thirteen of 14 patients demonstrated a clear deficiency in lymphocytes with acarbose inhibition. One patient was indeterminate, although below normal activity, suggesting the need for confirmatory testing. Tissue enzyme activity in all was consistent with infantile Pompe disease, and corroborated enzyme activity seen in lymphocytes. There were no false positives for disease, making the positive predictive value of lymphocyte enzyme testing 100%. CONCLUSION: Enzyme assay using acarbose as an inhibitor, can be performed in isolated lymphocytes for rapid diagnosis of infantile Pompe disease.

Fisher ratio method applied to third-order separation data to identify significant chemical components of metabolite extracts.

This report is about applying a Fisher ratio method to entire four dimensional (4D) data sets from third-order instrumentation data. The Fisher ratio method uses a novel indexing scheme to discover the unknown chemical differences among known classes of complex samples. This is the first report of a Fisher ratio analysis procedure applied to entire 4D data sets of third-order separation data, which, in this case, is comprehensive two-dimensional gas chromatography coupled with time-of-flight mass spectrometry analyses of metabolite extracts using all of the collected mass channels. Current analysis methods for third-order separation data use only user-defined subsets of the 4D data set. First, in a validation study, the Fisher ratio method was demonstrated to objectively evaluate and determine the chemical differences between three controlled urine samples that differed by known spiked chemical components. It was determined that, out of more than 600 recognizable chemical components in a single sample, the six spiked components, along with only two other matrix components, differed most significantly in concentration among the control samples. In a second study, the Fisher ratio method was used in a novel application to discover the unknown chemical differences between urine metabolite samples from pregnant women and nonpregnant women. A brief list of the top 11 components that were most significantly different in concentration between the pregnant and nonpregnant samples was generated. Because the Fisher ratio calculation statistically differentiates regions of the chromatogram with large class-to-class variations from regions containing large within-class variations, the Fisher ratio method should generally be robust against biological diversity in a sample population. Indeed, application of principal component analysis in this second study failed due to biological diversity of the samples.