Matrix-Assisted Ionization on a Portable Mass Spectrometer: Analysis Directly from Biological and Synthetic Materials.

Matrix-assisted ionization (MAI)-mass spectrometry (MS) eliminates the need for high voltage, a heat source, lasers, and compressed gases in the ionization process and uses minimal solvents in sample preparation, thus making MAI ideal for field-portable mass spectrometers. The broad applicability of MAI is demonstrated by simple, rapid, and robust positive and negative detection mode analyses of low and high mass compounds including some pesticides, dyes, drugs, lipids, and proteins (186 Da to 8.5 kDa) from various materials including urine, biological tissue sections, paper, and plant material on a low pumping capacity, single-quadrupole mass spectrometer. Different sample introduction methods are applicable, including the use of a pipet tip or glass melting point tube, allowing integration of sample preparation with sample introduction for increased analytical utility and ease of operation, even when sampling directly from surfaces.

Reproducibility and quantification of illicit drugs using matrix-assisted ionization (MAI) mass spectrometry.

Matrix-assisted ionization (MAI) mass spectrometry (MS) is a simple and sensitive method for analysis of low- and high-mass compounds, requiring only that the analyte in a suitable matrix be exposed to the inlet aperture of an atmospheric pressure ionization mass spectrometer. Here, we evaluate the reproducibility of MAI and its potential for quantification using six drug standards. Factors influencing reproducibility include the matrix compound used, temperature, and the method of sample introduction. The relative standard deviation (RSD) using MAI for a mixture of morphine, codeine, oxymorphone, oxycodone, clozapine, and buspirone and their deuterated internal standards using the matrix 3-nitrobenzonitrile is less than 10% with either a Waters SYNAPT G2 or a Thermo LTQ Velos mass spectrometer. The RSD values obtained using MAI are comparable to those using ESI or MALDI on these instruments. The day-to-day reproducibility of MAI determined for five consecutive days with internal standards was better than 20% using manual sample introduction. The reproducibility improved to better than 5% using a mechanically assisted sample introduction method. Hydrocodone, present in a sample of undiluted infant urine, was quantified with MAI using the standard addition method.

High-throughput characterization of small and large molecules using only a matrix and the vacuum of a mass spectrometer.

Matrix assisted ionization vacuum (MAIV) rapidly generates gas-phase analyte ions from subliming solid-phase matrix:analyte crystals for analysis by mass spectrometry (MS). Ionization from the solid-phase allows the use of a variety of surfaces for introducing matrix:analyte samples to the vacuum of a mass spectrometer, including common laboratory materials, such as disposable pipet tips, filter paper, tooth picks, and nylon mesh. MAIV is shown here to be capable of analyses as fast as 3 s per sample with achievable sensitivities in the low femtomole range. MAIV-MS coupled with ion mobility spectrometry (IMS)-MS and tandem mass spectrometry (MS/MS) is shown to be especially powerful for analysis and characterization of a wide range of molecules ranging from small molecules such as drugs and metabolites (∼300 Da) to intact proteins (25.6 kDa). Automated sample introduction is demonstrated on two different commercial mass spectrometers using a programmable XYZ stage. A MAIV high-throughput nontargeted MS(E) approach is also demonstrated utilizing IMS for rapid characterization of small molecules and peptides from standard solutions, as well as drug spiked human urine.

Amlodipine prevents apoptotic cell death by correction of elevated intracellular calcium in a primary neuronal model of Batten disease (CLN3 disease).

CLN3 disease (Spielmeyer-Vogt-Sjogren-Batten disease) is a severe pediatric neurodegenerative disorder for which there is currently no effective treatment. The disease is characterized by progressive neuronal death, which may be triggered by abnormal intracellular calcium levels leading to neuronal apoptosis. Previously, we demonstrated reversal of the calcium effect in a neuroblastoma cell line using amlodipine and other calcium channel antagonists. In the present studies, we developed a CLN3 siRNA-inhibited primary rat neuron model to further study etoposide-induced calcium changes and apoptosis in CLN3 disease followed by recovery experiments with amlodipine. Our results show that intracellular calcium is significantly elevated in siRNA-inhibited cortical neurons after potassium chloride-induced depolarization. We were also able to show that amlodipine, a predominantly L-type dihydropyrimidine calcium channel antagonist can reverse the aberrant calcium elevations in this model of the disease. We performed an in situ TUNEL assay following etoposide-exposure to siRNA inhibited primary neurons, and apoptotic nuclei were detected providing additional evidence that increased neuronal apoptosis is associated with increased calcium levels. Amlodipine also reduced the absolute number of apoptotic cells in this experimental model.

Diagnosis of a patient with a kinetic variant of medium and short-chain 3-hydroxyacyl-CoA dehydrogenase deficiency by newborn screening.

Medium and short-chain 3-hydroxyacyl-CoA dehydrogenase deficiency is a rare cause of impaired mitochondrial fatty acid oxidation. We present a case report of a patient with hyperinsulinism and homozygosity for a novel mutation causing a kinetic variant of the enzyme. The diagnosis was initially inferred by abnormal newborn screening acylcarnitine analysis with elevated C4-hydroxyacylcarnitine.

Short-chain 3-hydroxyacyl-coenzyme A dehydrogenase associates with a protein super-complex integrating multiple metabolic pathways.

Proteins involved in mitochondrial metabolic pathways engage in functionally relevant multi-enzyme complexes. We previously described an interaction between short-chain 3-hydroxyacyl-coenzyme A dehydrogenase (SCHAD) and glutamate dehydrogenase (GDH) explaining the clinical phenotype of hyperinsulinism in SCHAD-deficient patients and adding SCHAD to the list of mitochondrial proteins capable of forming functional, multi-pathway complexes. In this work, we provide evidence of SCHAD's involvement in additional interactions forming tissue-specific metabolic super complexes involving both membrane-associated and matrix-dwelling enzymes and spanning multiple metabolic pathways. As an example, in murine liver, we find SCHAD interaction with aspartate transaminase (AST) and GDH from amino acid metabolic pathways, carbamoyl phosphate synthase I (CPS-1) from ureagenesis, other fatty acid oxidation and ketogenesis enzymes and fructose-bisphosphate aldolase, an extra-mitochondrial enzyme of the glycolytic pathway. Most of the interactions appear to be independent of SCHAD's role in the penultimate step of fatty acid oxidation suggesting an organizational, structural or non-enzymatic role for the SCHAD protein.

Flow injection tandem mass spectrometric measurement of ceramides of multiple chain lengths in biological samples.

A method is presented for the measurement of ceramide species in biological fluids using flow injection tandem mass spectrometry. Ceramides are important signaling compounds in a number of cell:cell interactions including apoptosis and neurodegeneration. Because of the large number of potential fatty acid constituent moieties on ceramide molecules, a method which accurately distinguishes different chain-length species was required. The present method does not require HPLC separation and is designed to be applicable to high throughput analysis required for clinical studies. We provide a reference range for all measurable ceramide species in normal human plasma and an example of the utility of the assay in providing biomarkers in an in vitro apoptotic cell death study using murine hematopoietic cells treated with daunorubicin.

Measurement of plasma amino acids by Ultraperformance® Liquid Chromatography.

BACKGROUND: Since the early 1960s, quantitative amino acid analysis (AAA) has traditionally been performed using ion-exchange chromatography with post-column ninhydrin derivatization. This established technology has many advantages, including relatively low cost of operation and ease of use. However, analysis times of 120+ min and high maintenance requirements make this technology unsuitable for the modern clinical laboratories with a requirement for rapid turnaround times. The work described here is a summary of our experiences with a new approach to full profile analysis of physiological amino acids. METHODS: Amino acids were derivatized in batches with a proprietary reagent, AccQTag®, which reacts with primary and secondary amines. The derivatized amino acids were separated using Ultraperformance® Liquid Chromatography (UPLC). In a prospective method comparison, quantitative plasma amino acid data obtained from approximately 170 patient samples using both the UPLC method and a traditional ion-exchange chromatography amino acid analyzer were evaluated. RESULTS: The data obtained from the two methods were found to agree well. Correlation coefficients for the most important amino acids seen in inborn errors of metabolism, such as phenylalanine, tyrosine and branched chain amino acids varied from 0.8658 to 0.9932 with minor slope biases. This approach also reduced the run time from 120 to 45 min per sample using a sample volume of 0.1 mL, compared to the 0.5 mL volume required for ion-exchange chromatography. CONCLUSIONS: This new approach for the full profile analysis of physiological amino acids has been shown to be a viable substitute for current ion-exchange methodologies. It provides substantial benefits including significant reductions in runtime and necessary sample volume for the investigation and monitoring of patients with metabolic disorders and for nutritional management of a variety of patients.

PTC124 improves readthrough and increases enzymatic activity of the CPT1A R160X nonsense mutation.

Deficiency of carnitine palmitoyltransferase 1A (CPT1A) results in impaired hepatic long-chain fatty acid oxidation and ketogenesis. We have previously described a patient with a severe CPT1A phenotype who is homozygous for the nonsense mutation 478 C > T (R160X). It has been known for some time that gentamicin can promote readthrough of nonsense codons. Recently, a new compound (PTC124) with less clinical toxicity than gentamicin has been indicated as a therapy for patients with nonsense mutations for multiple genetic diseases. The study is designed to investigate whether PTC124 can promote readthrough of the R160X CPT1A mutation and increase normal sized CPT1 protein expression and activity in the patient's skin fibroblasts. Our study demonstrated that after both PTC 124 and gentamicin treatment, there was an increase in CPT1 activity in patient fibroblasts to levels that are similar to that of the mild Inuit P479L variant. Our results provide additional evidence for proof of principle that PTC124 is a potential therapeutic agent for treating patients with any genetic condition that results from a nonsense mutation.

Screening for calcium channel modulators in CLN3 siRNA knock down SH-SY5Y neuroblastoma cells reveals a significant decrease of intracellular calcium levels by selected L-type calcium channel blockers.

BACKGROUND: Defects of the CLN3 gene on chromosome 16p12.1 lead to the juvenile form of neuronal ceroid-lipofuscinosis (JNCL, Batten Disease), the most common recessive inherited neurodegenerative disorder in children. Dysregulation of intracellular calcium homeostasis in the absence of a functional CLN3 protein (CLN3P, Battenin) has been linked to synaptic dysfunction and accelerated apoptosis in vulnerable neuronal cells. Prolonged increase of intracellular calcium concentration is considered to be a significant trigger for neuronal apoptosis and cellular loss in JNCL. METHODS: We examined the potential effect of 41 different calcium channel modulators on intracellular calcium concentration in CLN3 siRNA knock down SH-SY5Y neuroblastoma cells. RESULTS: Six drugs belonging to the group of voltage dependent L-type channel blockers show significant lowering of the increased intracellular calcium levels in CLN3 siRNA knock down cells. CONCLUSIONS: Our studies provide important new data suggesting possible beneficial effects of the tested drugs on calcium flux regulated pathways in neuronal cell death. Therapeutic intervention in this untreatable disease will likely require drugs that cross the blood-brain barrier as did all of the positively screened drugs in this study. GENERAL SIGNIFICANCE: Better comprehension of the mechanism of neurodegeneration in rare recessive disorders, such as neuronal ceroid-lipofuscinoses, is likely to help to better understand mechanisms involved in more complex genetic neurodegenerative conditions, such as those associated with aging.

Intermediate levels of neuronal palmitoyl-protein Delta-9 desaturase in heterozygotes for murine Batten disease.

We recently demonstrated reduced activity of a novel palmitoyl-protein Delta-9 desaturase in neuronal tissues from mice with the cln3 Juvenile Neuronal Ceroid-Lipofuscinosis (Batten disease) gene ablated. In this follow-up study we have been able to obtain tissues from heterozygous cln3 mice and report that the enzyme activity in brain and pancreas from the heterozygotes is intermediate at 40% of the wild-type activity and consistent with recessive inheritance. Neuronal tissues from the CLN1 knock-out mouse demonstrated normal enzymatic activity pointing to the specificity of the desaturase function to CLN3. Non-neuronal tissues did not have measurable desaturase activity in wild-type or knock-out mice using this assay system. This may be due to lack of sensitivity of our assay system in these tissues or failure to activate the enzyme in these tissues. This is the first report of a heterozygous abnormality in Batten disease and provides important confirmation that this is the function of the CLN3 protein in neuronal tissues.

A rapid ultra performance liquid chromatography tandem mass spectrometric method for measuring amino acids associated with maple syrup urine disease, tyrosinaemia and phenylketonuria.

BACKGROUND: Patients with inherited disorders of amino acid metabolism including maple syrup urine disease, tyrosinaemia and phenylketonuria on dietary management require frequent monitoring of disease-relevant plasma amino acids in order to optimize therapeutic benefit. Poorly controlled maple syrup urine disease in particular may result in catastrophic metabolic decompensation. Most methods for monitoring amino acid concentrations are time-consuming and have clinically impractical turnaround times, particularly when the required time to run standards and control samples is taken into account. METHODS: We have analysed plasma amino acids using standard ion-exchange chromatography with ninhydrin detection in an amino acid analyser and compared the data with that obtained for the same samples using ultra-performance liquid chromatography (UPLC) separation with detection by tandem mass spectrometry. RESULTS: The two methodologies compared very well for the measurement of six important amino acids with correlation coefficients greater than 0.96 for all. The time for sample preparation was longer for the UPLC methodology as batched derivatization and evaporation is required but UPLC-tandem mass spectrometry generated sample results every 8 min while conventional ion-exchange chromatography took almost 1 h per sample. CONCLUSION: UPLC-tandem mass spectrometry generates data that compares well with existing 'gold standard' methodologies but significantly reduces sample turnaround time. Decreasing the turnaround time for amino acid analyses is very likely to improve clinical care for patients with amino acid disorders as dietary adjustments can be made sooner.

Juvenile neuronal ceroid-lipofuscinosis (Batten disease): a brief review and update.

Juvenile neuronal ceroid-lipofuscinosis (JNCL, Batten disease, Spielmeyer-Vogt-Sjogren disease, CLN3) is the most common inherited, autosomal recessive, neurodegenerative disorder in man. Like the other neuronal ceroid-lipofuscinoses, it is characterized by progressive loss of vision, seizures, and loss of cognitive and motor functions, leading to premature demise. JNCL is caused by mutations of CLN3, a gene that encodes a hydrophobic transmembrane protein, which localizes to membrane lipid rafts in lysosomes, endosomes, synaptosomes, and cell membrane. While the primary function of the CLN3 protein (CLN3P) may be debated, its absence affects numerous cellular functions including pH regulation, arginine transport, membrane trafficking, and apoptosis. We have recently suggested that the unifying primary function of CLN3P may be in a novel palmitoyl-protein Delta-9 desaturase (PPD) activity that in our opinion could explain all of the various functional abnormalities seen in the JNCL cells. Another group of researchers has recently shown a correlation between the CLN3P expression and the synthesis of bis(monoacylglycerol)phosphate (BMP) and suggested that CLN3P may play a role in the biosynthesis of BMP. In this review, following an introduction to the neuronal ceroid-lipofuscinoses, we provide a brief overview and an update of the most recent research in JNCL, specifically that related to the function of CLN3P.

CLN3P, the Batten's disease protein, is a novel palmitoyl-protein Delta-9 desaturase.

OBJECTIVE: Batten's disease, one of the most common recessively inherited, untreatable, neurodegenerative diseases of humans, is characterized by progressive neuronal loss and intraneuronal proteolipid storage. Although the gene for the disorder was cloned more than a decade ago, the function of the encoded protein, CLN3P, has not been defined thus far. METHODS: Sequence analysis using the Pfam server identified a low stringency match to a fatty acid desaturase domain in the N-terminal sequence of CLN3P. We developed a fatty acid desaturase assay based on measurement of desaturase products by gas chromatography/mass spectrometry. RESULTS: We show that CLN3P is a novel palmitoyl-protein Delta-9 desaturase, which converts membrane-associated palmitoylated proteins to their respective palmitoleated derivatives. We have further demonstrated that this palmitoyl-protein Delta-9 desaturase activity is deficient in cln3(-/-) mouse pancreas and is completely ablated in neuroblastoma cells by RNA inhibition. INTERPRETATION: We propose that palmitoyl-protein desaturation defines a new mechanism of proteolipid modification, and that deficiency of this process leads to the signs and symptoms of Batten's disease.

Molecular assay for detection of the common carnitine palmitoyltransferase 1A 1436(C>T) mutation.

BACKGROUND: Carnitine palmitoyltransferase 1A (CPT1A) deficiency is a metabolic disorder that occurs at a key checkpoint of fatty acid metabolism. A new form of CPT1A deficiency caused by a mutation at nucleotide 1436 (C>T), resulting in an amino acid substitution of leucine for proline at position 479 (P479L), has been isolated in Canadian First Nations and Inuit populations. The present study offers a molecular method for assessing CPT1A 1436 (C>T) mutation status. METHODS: CPT1A-deficient fibroblasts from four patient fibroblast cell lines and ten patient peripheral blood spots were all analyzed by polymerase chain reaction (PCR) coupled to restriction endonuclease (RE) treatment. Genomic DNA was PCR-amplified and treated with an RE specific for normal DNA. CPT1A 1436 (C>T) mutations were identified by resistance to RE treatment. RESULTS: The RE-PCR assay identified homozygosity for the 1436 (C>T) mutation in four fibroblast cell lines and nine blood spots with CPT1A enzyme deficiency. In addition, the assay identified one blood spot that corresponded to the heterozygous genotype. CONCLUSIONS: RE-PCR assay for the 1436 (C>T) mutation provides a rapid assay for the diagnosis of CPT1A deficiency resulting from this mutation. The assay will have utility in screening populations with a high prevalence of this genotype.

Reye-like syndrome resulting from novel missense mutations in mitochondrial medium- and short-chain l-3-hydroxy-acyl-CoA dehydrogenase.

Medium- and short-chain l-3-hydroxy-acyl-CoA dehydrogenase (M/SCHAD) deficiency is a recessively inherited disorder of fatty acid oxidation. Currently, only four patients from three families have been reported in the literature. All these patients presented with hypoglycemia associated with hyperinsulinism (HI). This association suggests that there is a role for M/SCHAD in regulating the pancreatic secretion of insulin. We present a fifth patient whose presentation was similar to Reye syndrome, a feature in common with most of the previously recognized disorders of fatty acid oxidation but with no clinical evidence of HI. Sequencing of the HAD1 gene on chromosome 4 revealed compound heterozygosity for two novel missense mutations, 170A>G, resulting in D45G, and 676T>C, resulting in Y214H. The mutant enzymes were expressed and subjected to kinetic analysis. Y214H has no detectable activity, whilst D45G, which resides in the cofactor-binding pocket, has an altered K(m) for NADH (96 microM versus 24 microM for the wild-type). This represents the first kinetic M/SCHAD mutant, which explains the high residual activity in skin fibroblasts. The lack of obvious HI in this patient may be related to the high residual activity and indicates that HI associated with M/SCHAD deficiency may only be present with complete deficiency. The spectrum of M/SCHAD phenotype should be broadened to include acute liver disease.

Over-expression of CLN3P, the Batten disease protein, inhibits PANDER-induced apoptosis in neuroblastoma cells: further evidence that CLN3P has anti-apoptotic properties.

Juvenile neuronal ceroid-lipofuscinosis (JNCL) or Batten/Spielmeyer-Vogt-Sjogren disease (OMIM #204200) is one of a group of nine clinically related inherited neurodegenerative disorders (CLN1-9). JNCL results from mutations in CLN3 on chromosome 16p12.1. The neuronal loss in Batten disease has been shown to be due to a combination of apoptosis and autophagy suggesting that CLN3P, the defective protein, may have an anti-neuronal death function. PANDER (PANcreatic-DERived factor) is a novel cytokine that was recently cloned from pancreatic islet cells. PANDER is specifically expressed in the pancreatic islets, small intestine, testis, prostate, and neurons of the central nervous system, and has been demonstrated to induce apoptosis. In this study, we over-expressed CLN3P in SH-SY5Y neuroblastoma cells and monitored the effects on PANDER-induced apoptosis. CLN3P significantly increased the survival rate of the SH-SY5Y cells in this system. This study provides additional evidence that the function of CLN3P is related to preventing neuronal apoptosis.

Bile acylcarnitine profiles in pediatric liver disease do not interfere with the diagnosis of long-chain fatty acid oxidation defects.

BACKGROUND: Plasma acylcarnitine measurement is an important diagnostic tool for inherited disorders of fatty acid and organic acid metabolism. Biliary excretion has been shown to be the primary route of excretion for acylcarnitines and analysis of bile acylcarnitine profiles may provide greater sensitivity for detecting metabolic disorders. Disorders of fatty acid oxidation frequently present with deranged liver function and the effect of hepatic disease on biliary acylcarnitine excretion are unknown. METHODS: We measured biliary acylcarnitine levels in pediatric patients aged 6 months to 1 year undergoing open liver biopsy with prospectively determined non-metabolic liver disease in order to determine the effect of the liver disease on acylcarnitine excretion. Bile was collected in syringes and was transported immediately and stored at -70 degrees C until the time of testing. The disease patient population consisted of 2 patients with known defects in long- and short-chain fatty acid oxidation (long-chain L-3-hydroxy acyl-CoA dehydrogenase: LCHAD and short-chain L-3-hydroxy acyl-CoA dehydrogenase: SCHAD). The sample from the LCHAD patient was collected at autopsy and the patient with SCHAD deficiency was subsequently diagnosed as part of the prospective study and removed from the unknown etiology group. Acylcarnitine profiles were obtained for each specimen as butylated derivatives using tandem mass spectrometry. RESULTS: The non-metabolic liver disease had no effect on the diagnostic value of bile acylcarnitine levels for detecting LCHAD deficiency. The concentrations of bile long-chain acylcarnitine species analyzed from patients with non-metabolic liver disease were far lower than the levels seen in LCHAD deficiency which also demonstrated a characteristic pattern of 3-hydroxyacylcarnitine excretion. In SCHAD deficiency, for which pathognomonic markers have not yet been established, bile analysis did not improve the diagnostic ability. CONCLUSION: The analysis of bile acylcarnitines for the diagnosis of long-chain fatty acid oxidation defects will provide unbiased information even in the presence of severe non-metabolic liver disease.