Acylcarnitine Analysis Research Articles

Glutaric Aciduria Type II Presenting as Myopathy and Rhabdomyolysis in a Teenager

Late-onset glutaric aciduria type II has been described recently as a rare but treatable cause of proximal myopathy in teenagers and adults. It is an autosomal recessive disease affecting fatty acid, amino acid, and choline metabolism. This is usually a result of 2 defective flavoproteins: either electron transfer flavoprotein (ETF) or electron transfer flavoprotein-ubiquinone oxidoreductase (ETF:QO). We present a 14-year-old boy with a background of autistic spectrum disorder who presented with severe muscle weakness and significant rhabdomyolysis. Before the onset of muscle weakness, he was very active but was completely bedridden at presentation. Diagnosis was established quickly by urine organic acid and plasma acylcarnitine analysis. He has shown significant improvement after starting oral riboflavin supplementation and is now fully mobile. This case highlights that late-onset glutaric aciduria type II is an important differential diagnosis to consider in teenagers presenting with proximal myopathy and rhabdomyolysis and it may not be associated with hypoglycemia.

Present Status of Expanded Newborn Screening Project for Inborn Errors of Metabolism by Tandem Mass Spectrometry

In Japan, screening for six diseases including four inborn errors of metabolism has been performed since 1977 for all neonates to prevent severe mental handicaps or death. A rapid screening procedure for analysis of several amino acids and acylcarnitines in blood spots by tandem mass spectrometry was developed by Millington DS et al. in the early 1990s. Although it is called expanded (or extended) newborn screening, the procedure is insufficiently sensitive to or specific for several diseases. Screening for all diseases that can be screened using this procedure is suggested to be cost-ineffective. Many European countries target only two diseases: medium-chain acyl-CoA dehydrogenase deficiency and phenylketonuria; their prevalence in Caucasian populations is very high, but some countries target more than twenty diseases and others an intermediate number. A pilot study targeting 22 diseases suggests that the combined incidence is one per 9,000 (0.01%) in Japan. This primary screening requires secondary screening to confirm the disease using urine, and either organic acids with solvent extraction or metabolome without fractionation are analyzed by gas chromatography-mass spectrometry. There is no need for primary or secondary screening tests to be performed at the same laboratory because the skills required are quite different. Understanding of the methodological problems of tandem mass screening and amelioration of variation and false positivity rate of this screening method among laboratories are critical to the success of the screening system in Japan. GC/MS-based urine metabolomics is expected to become one of the primary screening methodologies for neonates/infants who are already ill.

Index of Suspicion

The reader is encouraged to write possible diagnoses for each case before turning to the discussion. We invite readers to contribute case presentations and discussions. Please inquire first by contacting Dr. Deepak Kamat at DKamat@med.wayne.edu. A 2-year-old boy with a history of eczema and food allergies to nuts and soy presents to the emergency department with a 10-day history of generalized edema. The swelling began in the periorbital area but soon progressed to involve his entire body. There have been no fevers or any recent illnesses other than 1 day of cough and a few watery stools. He has had no new exposures to foods or other substances. His family history is remarkable for multiple allergies and eczema. Physical examination reveals a temperature of 98.8°F (37.1oC), heart rate of 98 beats per minute, respiratory rate of 36 breaths per minute, blood pressure of 103/65 mm Hg, and an oxygen saturation of 97% on room air. His weight is 12.6 kg (34th percentile). He is noted to have a moderate amount of periorbital edema, decreased breath sounds at the lung bases bilaterally, a protuberant abdomen, and 2+ pitting edema on his lower extremities. The rest of his physical examination findings are within normal limits. Laboratory evaluation reveals a white blood cell count of 23,800/μL (23.8 × 109/L), with 37% neutrophils, 49% lymphocytes, 4% bands, 7% monocytes, and 3% eosinophils; hemoglobin level of 13.3 g/dL (133 g/L); and platelet count of 427 × 103/μL (427 × 109/L). Serum electrolyte, blood urea nitrogen, and creatinine levels are normal. Sodium level is 132 mEq/L (132 mmol/L). Serum albumin level is 1.7 g/dL (17 g/L) (reference range, 3.4-5.4 g/dL [34-54 g/L]). Total protein level is 3.8 g/dL (38 g/L). Aspartate aminotransferase, alanine aminotransferase, and coagulation profiles …

Three novel mutations in the carnitine–acylcarnitine translocase (CACT) gene in patients with CACT deficiency and in healthy individuals

Carnitine-acylcarnitine translocase (CACT) and carnitine palmitoyltransferase II (CPT2) are key enzymes for transporting long-chain fatty acids into mitochondria. Deficiencies of these enzymes, which are clinically characterized by life-threatening non-ketotic hypoglycemia and rhabdomyolysis, cannot be distinguished by acylcarnitine analysis performed using tandem mass spectrometry. We had previously reported the CPT2 genetic structure and its role in CPT2 deficiency. Here, we analyzed the CACT gene in 2 patients diagnosed clinically with CACT deficiency, 18 patients with non-traumatic rhabdomyolysis and 58 healthy individuals, all of whom were confirmed to have normal CPT2 genotypes. To facilitate CACT genotyping, we used heat-denaturing high-performance liquid chromatography (DHPLC), which helped identify five distinct patterns. The abnormal heteroduplex fragments were subjected to CACT-specific DNA sequencing. We found that one patient with CACT deficiency, Case 1, carried c.576G>A and c.199-10t>g mutations, whereas Case 2 was heterozygous for c.106-2a>t and c.576G>A. We also found that one patient with non-traumatic rhabdomyolysis and one healthy individual were heterozygous for c.804delG and the synonymous mutation c.516T>C, respectively. In summary, c.576G>A, c.106-2a>t and c.516T>C are novel CACT gene mutations. Among the five mutations identified, three were responsible for CACT deficiency. We have also demonstrated the successful screening of CACT mutations by DHPLC.

Quantification of plasma carnitine and acylcarnitines by high-performance liquid chromatography-tandem mass spectrometry using online solid-phase extraction

Carnitine is an amino acid derivative that plays a key role in energy metabolism. Endogenous carnitine is found in its free form or esterified with acyl groups of several chain lengths. Quantification of carnitine and acylcarnitines is of particular interest for screening for research and metabolic disorders. We developed a method with online solid-phase extraction coupled to high-performance liquid chromatography and tandem mass spectrometry to quantify carnitine and three acylcarnitines with different polarity (acetylcarnitine, octanoylcarnitine, and palmitoylcarnitine). Plasma samples were deproteinized with methanol, loaded on a cation exchange trapping column and separated on a reversed-phase C8 column using heptafluorobutyric acid as an ion-pairing reagent. Considering the endogenous nature of the analytes, we quantified with the standard addition method and with external deuterated standards. Solid-phase extraction and separation were achieved within 8 min. Recoveries of carnitine and acylcarnitines were between 98 and 105 %. Both quantification methods were equally accurate (all values within 84 to 116 % of target concentrations) and precise (day-to-day variation of less than 18 %) for all carnitine species and concentrations analyzed. The method was used successfully for determination of carnitine and acylcarnitines in different human samples. In conclusion, we present a method for simultaneous quantification of carnitine and acylcarnitines with a rapid sample work-up. This approach requires small sample volumes and a short analysis time, and it can be applied for the determination of other acylcarnitines than the acylcarnitines tested. The method is useful for applications in research and clinical routine.

Rapid quantitative analysis of carnitine and acylcarnitines by ultra-high performance–hydrophilic interaction liquid chromatography–tandem mass spectrometry

l-Carnitine and its acyl esters (acylcarnitines) play an important role in the metabolism of fatty acids. However, most of the present methods for the quantitative analysis of acylcarnitines have restrictions both in sample preparation and in chromatographic separation. Herein we present a validated method for determination of carnitine and eleven acylcarnitines in human serum and rat tissue biopsies by using ultra-high performance–hydrophilic interaction liquid chromatography–tandem mass spectrometry (UHP–HILIC–MS/MS). The procedure uses minimal sample preparation including only addition of organic solvent, labeled internal standard, incubation and centrifugation. The separation is performed without derivatization or addition of ion-pairing reagent within 7min on a hydrophilic interaction liquid chromatographic column with mass spectrometric detection. The method is linear in response over the concentration range from 20 to 600ng/ml for carnitine and acetylcarnitine and 5–200ng/ml for the other acylcarnitines, with correlation coefficients higher than 0.994. Recoveries were higher than 88% for most of the compounds. Limits of detection were 5ng/ml for carnitine and acetylcarnitine and approximately 0.5ng/ml for other acylcarnitines. The method was applied to the analysis of serum and tissue samples.

Expanded newborn screening and confirmatory follow-up testing for inborn errors of metabolism detected by tandem mass spectrometry

Newborn screening (NBS) of inborn errors of metabolism (IEM) is a coordinated comprehensive system consisting of education, screening, follow-up of abnormal test results, confirmatory testing, diagnosis, treatment, and evaluation of periodic outcome and efficiency. The ultimate goal of NBS and follow-up programs is to reduce morbidity and mortality from the disorders. Over the past decade, tandem mass spectrometry (MS/MS) has become a key technology in the field of NBS. It has replaced classic screening techniques of one-analysis, one-metabolite, one-disease with one analysis, many-metabolites, and many-diseases. The development of electrospray ionization (ESI), automation of sample handling and data manipulation have allowed the introduction of expanded NBS for the identification of numerous conditions on a single sample and new conditions to be added to the list of disorders being screened for using MS/MS. In the case of a screened positive result, a follow-up analytical test should be performed for confirmation of the primary result. The most common confirmatory follow-up tests are amino acids and acylcarnitine analysis in plasma and organic acid analysis in urine. NBS should be integrated with follow-up and clinical management. Recent improvements in therapy have caused some disorders to be considered as potential candidates for NBS. This review covers some of the basic theory of expanded MS/MS and follow-up confirmatory tests applied for NBS of IEM.

The Trans-Fatty Acid, Elaidic Acid, Inhibits Macrophage Fatty Acid Catabolism and Stimulates Expression of Inflammatory Mediators

Abstract 3277In recent decades, addition to the diet of synthetically hydrogenated vegetable oils has markedly increased human consumption of trans fatty acids. Epidemiological studies have linked this change in diet to current high rates of atherosclerotic cardiovascular disease. Despite recognition of this important connection, the basic mechanisms by which trans fatty acids contribute to the pathogenesis of atherosclerosis are still not well understood. In the present studies we examined the effects of trans fatty acids on macrophage functions and their possible role in the pathogenesis of atherosclerosis. Human macrophages, derived from peripheral blood mononuclear cells, were treated with the trans fat elaidic acid (C18:Δ9–10 trans), the corresponding cis fatty acid oleic acid (C18:Δ9–10 cis), or the saturated fatty acid stearic acid (C18:0). We examined changes in macrophage fat metabolism using GC/MS to measure cell fatty acid content and intermediates, and MS/MS to identify acylcarnitine derivatives, and assayed fatty acid oxidation using fatty acids radiolabeled at the [1–14C] position and the double bond at the [C9-C103H] position. After 44 hours treatment with 100 micromolar elaidic acid, macrophages showed an accumulation of multiple unsaturated fatty acid intermediates, both long-chain and short-chain, by GC/MS analysis, that were not observed in cultures containing either oleic or stearic acid. Using acylcarnitine analysis, we observed an increase in C12 and C18 intermediates in the macrophages exposed to trans fat (either as fatty acids or partially hydrogenated soy oil) compared to controls. These results suggest a block in acyl-CoA removal one group proximate to the trans bond. Beta-oxidation assays using carbon-1 radiolabeled oleic and elaidic acids revealed enhanced entry of the trans-fat into the catabolic cycle compared to the entry of the natural cis-fatty acid. Using carbon 9–10 radiolabeled oleic acid to study oleic acid catabolism, we discovered that in the presence of the trans fat, oxidation of the cis fat was diminished. Thus, in addition to the block in the catabolism of the trans fat itself, the degradation of the cis monounsaturated fatty acids are also impaired in the presence of the trans fat. We then examined the effects of inhibited fatty acid catabolism on macrophage function by examining changes in gene expression. Initial results from Affymetrix gene expression profiling, were confirmed using quantitative real time PCR. These studies revealed that exposure to trans fatty acid, compared to cis fatty acids, markedly upregulated macrophage expression of interleukin 1 beta, an inflammatory cytokine previously implicated in the pathogenesis of atherosclerosis. Also increased was expression of heparin-binding epidermal growth factor, previously implicated as a stimulus for vascular smooth muscle proliferation in atherosclerosis. The results overall suggest that the deleterious effects of trans fats may be linked to impaired macrophage fatty acid catabolism, contributing to lipid accumulation in the atheroma, and also to increased macrophage production of inflammatory mediators. Disclosures:No relevant conflicts of interest to declare.

Sudden unexpected infant death (SUDI) in a newborn due to medium chain acyl CoA dehydrogenase (MCAD) deficiency with an unusual severe genotype

Medium chain acyl CoA dehydrogenase deficiency (MCAD) is the most common inborn error of fatty acid oxidation. This condition may lead to cellular energy shortage and cause severe clinical events such as hypoketotic hypoglycemia, Reye syndrome and sudden death. MCAD deficiency usually presents around three to six months of life, following catabolic stress as intercurrent infections or prolonged fasting, whilst neonatal-onset of the disease is quite rare. We report the case of an apparently healthy newborn who suddenly died at the third day of life, in which the diagnosis of MCAD deficiency was possible through peri-mortem blood-spot acylcarnitine analysis that showed very high concentrations of octanoylcarnitine. Genetic analysis at the ACADM locus confirmed the biochemical findings by demonstrating the presence in homozygosity of the frame-shift c.244dup1 (p.Trp82LeufsX23) mutation, a severe genotype that may explain the unusual and very early fatal outcome in this newborn. This report confirms that inborn errors of fatty acid oxidation represent one of the genetic causes of sudden unexpected deaths in infancy (SUDI) and underlines the importance to include systematically specific metabolic screening in any neonatal unexpected death.

Extraction and analysis of carnitine and acylcarnitines by electrospray ionization tandem mass spectrometry directly from dried blood and plasma spots using a novel autosampler

Acylcarnitines are routinely analyzed by electrospray ionization tandem mass spectrometry (ESI-MS/MS) both in clinical diagnostic and public health newborn screening laboratories from plasma and dried whole blood spots (DBS) on filter paper. The use of DBS as a convenient method of collecting and storing samples for subsequent analysis of various biomolecules is increasing, thus prompting the development of new devices to recover and quantify such analytes in an automated manner. Acylcarnitines were extracted directly from DBS using a novel autosampler that sequentially loads DBS cards into a pneumatic clamp and then pumps a fixed volume of solvent containing appropriate internal standards through a section of the DBS card directly into a triple quadrupole mass spectrometer via ESI. Plasma was first spiked with internal standard then spotted onto filter paper for analysis. Acylcarnitines were analyzed in DBS, and both free and total carnitine were assayed in dried plasma spots (DPS). Results using the new autosampling technique were of equal quality to those obtained by punching a 3-mm diameter disk from a DBS or DPS, then extracting and analyzing the target analytes from conventional 96-well microtiter plates, with far reduced time per sample. Recovery for most analytes was >60% and reproducibility was generally within 20% (CV). The simplicity and robustness of the DBS autosampler make it an attractive alternative to conventional methods of analyzing DBS specimens, thus saving time and labor costs, especially in high-throughput applications. Although the method as described is for direct infusion analysis, the autosampler is easily coupled to column hardware for applications requiring liquid chromatography/mass spectrometry (LC/MS).

Acute severe encephalopathy related to human herpesvirus-6 infection in a patient with carnitine palmitoyltransferase 2 deficiency carrying thermolabile variants

We describe a male infant with carnitine palmitoyltransferase 2 (CPT2) deficiency who presented with acute encephalopathy related to human herpesvirus-6 (HHV-6) infection. He was hospitalized for pylexia and status epilepticus, diagnosed with acute encephalopathy, and treated with intensive supportive care including mechanical ventilation, support for hypothermia, and control of the intracranial pressure, that caused severe neurological sequelae. HHV-6 was detected in his cerebrospinal fluid, indicating HHV-6 related encephalopathy. In the acute phase, acylcarnitine analysis of blood suggested a defect of long chain fatty acid β-oxidation, and CPT2 deficiency was genetically confirmed. In addition, other gene alterations that have been previously reported as “thermolabile variants” were found. Some patients with the infantile form of CPT2 deficiency present with acute encephalopathy, but others do not develop encephalopathy. The correlation between phenotype and genotype has not been clarified. Our case may contribute to the elucidation of the genetic factor involved in acute encephalopathy in CPT2 deficiency.

Mouse Models for Methylmalonic Aciduria

Methylmalonic aciduria (MMA) is a disorder of organic acid metabolism resulting from a functional defect of methylmalonyl-CoA mutase (MCM). MMA is associated with significant morbidity and mortality, thus therapies are necessary to help improve quality of life and prevent renal and neurological complications. Transgenic mice carrying an intact human MCM locus have been produced. Four separate transgenic lines were established and characterised as carrying two, four, five or six copies of the transgene in a single integration site. Transgenic mice from the 2-copy line were crossed with heterozygous knockout MCM mice to generate mice hemizygous for the human transgene on a homozygous knockout background. Partial rescue of the uniform neonatal lethality seen in homozygous knockout mice was observed. These rescued mice were significantly smaller than control littermates (mice with mouse MCM gene). Biochemically, these partial rescue mice exhibited elevated methylmalonic acid levels in urine, plasma, kidney, liver and brain tissue. Acylcarnitine analysis of blood spots revealed elevated propionylcarnitine levels. Analysis of mRNA expression confirms the human transgene is expressed at higher levels than observed for the wild type, with highest expression in the kidney followed closely by brain and liver. Partial rescue mouse fibroblast cultures had only 20% of the wild type MCM enzyme activity. It is anticipated that this humanised partial rescue mouse model of MMA will enable evaluation of long-term pathophysiological effects of elevated methylmalonic acid levels and be a valuable model for the investigation of therapeutic strategies, such as cell transplantation.

Direct and quantitative analysis of underivatized acylcarnitines in serum and whole blood using paper spray mass spectrometry.

A simple protocol for rapid quantitation of acylcarnitines in serum and whole blood has been developed using paper spray mass spectrometry. Dried serum and whole blood containing a mixture of ten acylcarnitines at various concentrations were analyzed as spots from paper directly without any sample pretreatment, separation, or derivatization. The composition of the spray solvent was found to be a critical factor: for serum samples, spray solvent of methanol/water/formic acid (80:20:0.1) gave the best signal intensity while for blood samples which contain more matrix components, acetonitrile/water (90:10) was a much more suitable spray solvent. For the paper type and size used, 0.5 μL of sample provided an optimal signal for both serum and whole blood samples. For quantitative profiling, the limits of quantitation obtained from both serum and blood were much lower than the clinically validated cutoff values for diagnosis of fatty acid oxidation disorders in newborn screening. Linearity (R(2) > 0.95) and reproducibility (RSD ~10 %) were achieved in the concentration ranges from 100 nM to 5 μM for the C2 acylcarnitine, and for other acylcarnitines, these values were from 10 to 500 nM. Acylcarnitine profiles offer an effective demonstration of the fact that paper spray mass spectrometry is an appropriate, simple, rapid method with high sensitivity and high reproducibility applicable to newborn screening tests.

Metabolic autopsy with postmortem cultured fibroblasts in sudden unexpected death in infancy: Diagnosis of mitochondrial respiratory chain disorders

Mitochondrial respiratory chain disorders are the most common disorders among inherited metabolic disorders. However, there are few published reports regarding the relationship between mitochondrial respiratory chain disorders and sudden unexpected death in infancy. In the present study, we performed metabolic autopsy in 13 Japanese cases of sudden unexpected death in infancy. We performed fat staining of liver and postmortem acylcarnitine analysis. In addition, we analyzed mitochondrial respiratory chain enzyme activity in frozen organs as well as in postmortem cultured fibroblasts. In heart, 11 cases of complex I activity met the major criteria and one case of complex I activity met the minor criteria. In liver, three cases of complex I activity met the major criteria and four cases of complex I activity met the minor criteria. However, these specimens are susceptible to postmortem changes and, therefore, correct enzyme analysis is hard to be performed. In cultured fibroblasts, only one case of complex I activity met the major criteria and one case of complex I activity met the minor criteria. Cultured fibroblasts are not affected by postmortem changes and, therefore, reflect premortem information more accurately. These cases might not have been identified without postmortem cultured fibroblasts. In conclusion, we detected one probable case and one possible case of mitochondrial respiratory chain disorders among 13 Japanese cases of sudden unexpected death in infancy. Mitochondrial respiratory chain disorders are one of the important inherited metabolic disorders causing sudden unexpected death in infancy. We advocate metabolic autopsy with postmortem cultured fibroblasts in sudden unexpected death in infancy cases.

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.

Combined extraction of acyl carnitines and 26:0 lysophosphatidylcholine from dried blood spots: Prospective newborn screening for X-linked adrenoleukodystrophy

X-linked adrenoleukodystrophy (X-ALD) is a severe genetic disorder that affects the nervous system, and the adrenal cortex. Newborn screening for X-ALD has been proposed to allow improved diagnosis along with prospective monitoring and treatment for this severe disorder. Newborn dried whole blood spot (DBS) 26:0 lysophosphatidyl choline was validated as a diagnostic marker for X-ALD and other peroxisomal disorders of peroxisomal β-oxidation.In this study, we developed a new one step extraction procedure that simultaneously extracts acyl carnitines and the lysophosphatidyl cholines from DBS. Further analysis of these metabolites has been performed by two different high throughput LC–MS/MS methods. The 26:0 lysophosphatidyl choline levels in this study were consistent with previously published values and discriminate between healthy and abnormal profiles. There is a very minor modification to the original acyl carnitine extraction procedure and our data indicates that there is no significant effect on acyl carnitine levels in DBS.Our new method potentially can be complementary to the current newborn screening panel. It successfully combines the existing method for acyl carnitine analysis and 26:0 lysophosphatidyl choline that can be applied for prospective X-ALD newborn screening.

An improved LC‐MS/MS method for the detection of classic and low excretor glutaric acidemia type 1

Glutaric acidemia type I (GA1) is associated with elevated glutarylcarnitine (C5DC), typically measured as its butylester by acylcarnitine profile analysis using tandem mass spectrometry (MS/MS) and the precursor-product ion pair of m/z 388-85. This method neither distinguishes between C5DC and its isomer 3-hydroxydecanoylcarnitine (C10-OH) nor reliably detects the low-excretor variant of GA1, leading to both false-positive and false-negative results when testing for GA1. To overcome these limitations, we developed an LC-MS/MS method that discriminates C5DC from C10-OH by the use of precursor-product ion pairs specific for butylated C5DC (m/z 388-115) and underivatized C10-OH (m/z 332-85). The C5DC method was validated over the linearity range of 0.025-20 μM with a lower limit of quantification (LOQ) of 0.025 μM. Excellent precision and accuracy were also observed. We tested plasma samples from 10 patients with confirmed GA1 (including 3 with the low-excretor variant), 21 patients with mild elevations of C5DC or C10-OH by routine acylcarnitine analysis for which GA1 ultimately was excluded, and 29 normal controls. By using the m/z 388-115 ion pair, all cases of GA1, including the low-excretor variant, were reliably distinguished from normal controls. By using the m/z 388-85 pair, patients with ambiguous elevations of C5DC or C10-OH demonstrated clearly elevated levels of C10-OH (m/z 332-85) but normal C5DC (m/z 388-115), confirming that the apparent elevation of C5DC is due to interference by C10-OH. Our method results in excellent detection of GA1, including the low-excretor variant, and also provides a means to discriminate C5DC and C10-OH in follow-up testing and routine acylcarnitine studies.

A patient with mitochondrial trifunctional protein deficiency due to the mutations in the HADHB gene showed recurrent myalgia since early childhood and was diagnosed in adolescence

Mitochondrial trifunctional protein (MTP) is a multienzyme complex involved in the metabolism of long-chain hydroxyacyl-CoA, a product of the fatty acid β-oxidation cycle. MTP is an α4β4 hetero-octomer encoded by two different genes: HADHA (OMIM 600890) and HADHB (OMIM 143450). MTP deficiency induces three different types of presentation: (1) a lethal phenotype with neonatal onset (severe); (2) a hepatic phenotype with infant onset (intermediate); and (3) a neuromyopathic phenotype with late-adolescent onset (mild). While acylcarnitine analysis has revealed increased levels of long-chain hydroxyacylcarnitine in blood when an MTP deficiency exists, the neuromyopathic type is usually asymptomatic and does not always result in an abnormality in acylcarnitine analysis results. We report here the case of a 13-year-old girl with recurrences of intermittent myalgia since her early childhood, for whom the disorder had not been definitely diagnosed. Since she was referred to our hospital because of rhabdomyolysis, we have repeatedly performed blood acylcarnitine analysis and found slight increases in long-chain 3-OH-acylcarnitine levels, on the basis of which we made a chemical diagnosis of MTP deficiency. Immunoblot analysis of skin fibroblasts revealed loss of α- and β-subunits of MTP. In addition, analysis of the HADHB gene, which encodes long-chain 3-ketoacyl-CoA thiolase, one of the enzymes constituting MTP, identified compound heterozygous mutations of c.520C>T (p.R141C) and c.1331G>A (p.R411K).MTP deficiency is considered an extremely rare disorder, as only five cases (lethal phenotype, two patients; hepatic phenotype, two patients; and neuromyopathic phenotype, one patient) have thus far been reported in Japan. However, it is likely that the neuromyopathic phenotype of MTP deficiency has not yet been diagnosed among patients with recurrences of intermittent myalgia and rhabdomyolysis, as in our patient reported here.

Neonatal carnitine palmitoyltransferase II deficiency associated with Dandy-Walker syndrome and sudden death

Neonatal onset of carnitine palmitoyltransferase II (CPT II) deficiency is an autosomal recessive, often lethal disorder of the mitochondrial beta-oxidation of long-chain fatty acids. It is a rare multiorgan disease which includes hypoketotic hypoglycemia, severe hepatomuscular symptoms, cardiac abnormalities, seizures and lethargy, as well as dysmorphic features. Until now, only 22 affected families have been described in the literature.An increasing number of mutations are being identified in the CPT2 gene, with a distinct genotype–phenotype correlation in most cases. Herein we report a new case of neonatal CPT II deficiency associated with Dandy-Walker syndrome and sudden death at 13days of life. CPT II deficiency was suggested by acylcarnitine analysis of dried-blood on filter paper in the expanded newborn screening. Genetic analysis of the CPT2 gene identified the presence of a previously described mutation in homozygosity (c.534_558del25bpinsT).All lethal neonatal CPT II deficiency patients previously described presented severe symptoms during the first week of life, although this was not the case in our patient, who remained stable and without apparent vital risk during the first 11days of life.The introduction of tandem mass spectrometry to newborn screening has substantially improved our ability to detect metabolic diseases in the newborn period. This case illustrates the value of expanded newborn screening in a neonate with an unusual clinical presentation, combining hydrocephalus and sudden death, that might not commonly lead to the suspicion of an inborn error of metabolism.

Newborn Screening for Isovaleric Acidemia Using Tandem Mass Spectrometry: Data from 1.6 Million Newborns

Electrospray ionization-tandem mass spectrometry (ESI-MS/MS) has been used in the Bavarian newborn screening (NBS) program since 1999. The use of ESI-MS/MS has led to the inclusion of isovaleric acidemia (IVA) into NBS. We retrospectively evaluated data on more than 1.6 million newborns screened during 9.5 years. Acylcarnitines from whole blood spotted on filter paper were converted to their corresponding butyl esters, and the samples were analyzed by use of ESI-MS/MS with stable isotope labeled internal standards. A total of 24 individuals with IVA were detected by use of a multiparametric threshold criteria panel including isovalerylcarnitine (C5) and the ratios of C5 to octanoyl-, butyryl-, and propionylcarnitine. A cutoff set at the 99.99th percentile for isolated C5 or at the 99th percentile for C5 plus at least 2 ratios resulted in a positive predictive value for IVA screening of 7.0% and an overall recall rate of 0.024%. Adjusted reference ranges for age and birth weight were applied, and the incidence of IVA in the study population was calculated to be 1 in 67,000. Missed cases were not brought to our attention. IVA was also detectable in cord blood and early postnatal blood samples. IVA can be reliably detected in NBS through acylcarnitine analysis in dried blood spots by using multiparametric threshold criteria. Further improvement (positive predictive value 13.0%, recall rate 0.01%) can be achieved by using more stringent recall criteria. In view of the potentially life-threatening natural course of IVA in early life, presymptomatic diagnosis may thus prevent mortality and morbidity.