Low Carnitine Levels Research Articles

Skeletal Muscle Atrophy in Chronic Kidney Disease (CKD)

Background: 
 Skeletal muscle atrophy and dysfunction occur with chronic kidney disease (CKD) progression leading to morbidity, mortality, and falls. Skeletal muscle dysfunction may be due to impaired fatty acid (FA) oxidation and enhanced pyruvate oxidation as demonstrated in preliminary metabolomic data. We performed multiple techniques to assess the extent of muscle dysfunction and associated pathways, including: systematic review and meta-analysis, assays of disease progression and FA metabolism, expression of markers associated with skeletal muscle FA metabolism and pyruvate oxidation. 
 
 Methods: 
 Meta-analysis: Multiple databases were used to identify relevant studies of muscle atrophy in preclinical and clinical models. 
 Experimental Study: 1)CKD rats and 2)Normal littermates (N=12/gr) at 35 weeks. Extensor digitorum longus (EDL) and soleus were harvested at sacrifice. Serum Biochemistry: Plasma BUN, calcium and phosphorus were analyzed using colorimetric assays. Carnitine Assay: Plasma carnitine levels was measured using ELISA kit. Protein Expression: Western blots with the lysate of EDL and soleus to determine the activity levels of PDH and PDK4. 
 
 Results: 
 A total of 4685 studies were screened in the meta-analysis, of which 646 were relevant. Subsequent steps are to perform full text review and data extraction. Animal studies: BUN and phosphorus were significantly increased in CKD compared to normal. Carnitine levels were significantly decreased in CKD rats compared to normal. PDH was not significantly different in the EDL or soleus. PDK4 is yet to be performed. 
 
 Conclusions: 
 The extent to which muscle atrophy occurs will be identified in the meta-analysis. Elevated BUN confirmed disease and carnitine assay confirmed low carnitine levels in CKD. Identifying low carnitine has led to an interventional study of carnitine supplementation to determine if there was improved FA oxidation. Testing of PDK4 is needed to determine significance of pyruvate regulation. Reviewing the literature and understanding the mechanism of skeletal muscle atrophy in CKD will allow future targeted therapeutics.

Primary carnitine deficiency in two sisters with intractable epilepsy and reversible metabolic cardiomyopathy: Two case reports.

Primary carnitine deficiency (PCD) is a disorder of the carnitine cycle that results in defective fatty acid oxidation. When carnitine cannot be transported into the cells, fatty acid oxidation is impaired, resulting a variety of symptoms, such as chronic muscle weakness, cardiomyopathy, hypoglycemia and liver dysfunction. The clinical manifestations and outcomes of different cases with PCD vary among patients. The present case report focused on two sisters with PCD. The younger sister presented with intractable epilepsy, and the older sister presented with reversible metabolic cardiomyopathy. Potential mutations in the SLC22A5 gene were investigated within the family, and a nonsense mutation [c.760C>T (p.R254X)] was identified in four family members. The two sisters harbored homozygous mutations, whereas their parents presented heterozygous mutations. Metabolic disease screening revealed low plasma free carnitine levels (<5 µmol/l) in the two sisters. The plasma free carnitine levels of their parents were normal, and they were asymptomatic. PCD in the two patients was managed using oral levocarnitine. The metabolic cardiomyopathy of the older sister improved following 3 months of treatment. However, the epilepsy of the younger sister was recurrent with oral antiepileptic therapy lasting one year and eight months, and epilepsy was finally controlled following right cerebral resection. The present case report demonstrated that the clinical manifestations presented by patients with PCD within the same family were different. The results indicated that treatment with levocarnitine supplementation should be initiated as soon as possible before irreversible organ damage occurs. In addition, metabolic decompensation and cardiac muscle functions were improved following carnitine supplementation. The resection of the severely diseased unilateral brain combined with carnitine supplementation and antiepileptic therapy may be an effective treatment for PCD with intractable epilepsy complications.

Association Between the Serum Carnitine Level and Ammonia and Valproic Acid Levels in Patients with Bipolar Disorder.

Valproic acid (VPA) is not only an antiepileptic drug but also a mood stabilizer for patients with bipolar disorder. Long-term VPA therapy can cause carnitine deficiency, which may result in an increase in the blood ammonia level, in patients with epilepsy. However, information about this effect in patients with bipolar disorder is limited. The aim of this study was to investigate the associations between the serum VPA level and the carnitine and ammonia levels in psychiatric adult patients with epilepsy. The subjects were 182 consecutive Japanese adult patients (mean age 54.3 ± 19.5 years) diagnosed with bipolar disorder and treated with VPA. The serum VPA level, carnitine fraction, and plasma ammonia level were measured. Furthermore, the free carnitine and acylcarnitine fractions were measured using an enzyme cycling method. Sixty-nine patients (38%) had a low free carnitine level. There were significant differences in sex, height, VPA dose, serum VPA level, total carnitine level, acylcarnitine level, and acylcarnitine/free carnitine ratio between patients with a low free carnitine level and those with a normal range of free carnitine. The simple and multiple regression analyses revealed that the VPA dose and the serum VPA level were inversely and significantly correlated with the free carnitine level. The plasma ammonia level was correlated with the VPA dose, serum VPA level, and acylcarnitine level but not with the free carnitine level. These findings suggest that carnitine deficiency is associated with the VPA dose and the serum VPA level in patients with bipolar disorder. However, it is unlikely that carnitine deficiency is associated with hyperammonemia in patients with bipolar disorder.

Screening 3.4 million newborns for primary carnitine deficiency in Zhejiang Province, China

Testing for primary carnitine deficiency (PCD) has been implemented in many newborn screening (NBS) programs, but few large-scale studies on NBS for PCD have been reported in China. This study aimed to assess the incidence and biochemical, clinical, and genetic characteristics of PCD discovered by NBS. Dried blood spots from newborns were analyzed by tandem mass spectrometry (MS/MS) and suspected positive patients were further tested using molecular genetic analysis. Infants who carried two variants in SLC22A5 or those with extremely low free carnitine levels during recall were referred for follow-up and treatment. Over 3.4 million newborns were screened and 113 newborns were diagnosed with PCD, yielding a positive predictive value of 1.93%. In addition, 63 mothers with PCD were identified. The incidence of PCD in newborns and mothers in Zhejiang was 1:30,182 and 1:54,137, respectively. Thirty-seven distinct variants were identified in SLC22A5 of which 10 were novel. c.1400C > G (p.S467C) was the most prevalent variant in both newborns and mothers with PCD, while c.760C > T (p.R254*), which is reportedly common in other Chinese regions, was rarely detected in maternal PCD patients. This study reports the largest series of patients with PCD detected by NBS and identifies 10 novel variants, expanding the variant spectrum of SLC22A5.

Novel Mutations in OCTN2 Identified in Newborn Screening Exhibit Reduced Function and Lead to Carnitine Transporter Deficiency

Newborn screening programs have revolutionized the diagnosis and treatment of genetic disorders. A compelling example is found in SLC22A5 encoding OCTN2, a bi‐directional plasma membrane transporter, which acts in the influx carnitine and efflux of drugs. Newborns with low plasma carnitine levels and biallelic SLC22A5 variants are diagnosed with Carnitine Transporter Deficiency (CTD), a potentially fatal disorder that can be treated with L‐carnitine supplementation. Here, we sought to validate the role of novel OCTN2 variants in CTD via functional characterization of mutant transporters in vitro.In a recent study (PMID 31364285), three novel OCTN2 missense variants were identified in newborn screening. Following site‐directed mutagenesis and transient transfection in HEK293T cells, we performed isotopic uptake assays using 14C‐carnitine. Sodium phenylbutyrate, a chemical chaperone previously shown to increase function of other OCTN2 variants, was used to treat transfected cells. Welch’s two‐sample t‐test was used to determine significance.Two novel missense variants, p.Asp165Glu and p.Gly473Val, exhibited substantial reduction of carnitine transport, retaining less than 5% of wildtype transporter function (p = 9.63*10−6 and 1.02*10−5, respectively). In contrast, only moderate reduction was observed for p.Met433Thr, which retained 61% wildtype function (p = 0.021). Sodium phenylbutyrate showed trends (which were not significant) toward increasing the function of all three variants, suggesting that intracellular errors of protein folding, processing, or trafficking may contribute to their reduced function.Our studies validate the role of three novel variants in CTD, none of which have been associated previously with the disorder. The studies are consistent with the idea that even partial loss‐of‐function alleles may be disease‐causing for CTD. Finally, we suggest that sodium phenylbutyrate represents a potential modulator of OCTN2 function, with further studies warranted to confirm effects on function and membrane localization of OCTN2 variants.Support or Funding InformationThis work is supported by the Chan Zuckerberg Biohub.

Multi-omics Integration Analysis Robustly Predicts High-Grade Patient Survival and Identifies CPT1B Effect on Fatty Acid Metabolism in Bladder Cancer.

The perturbation of metabolic pathways in high-grade bladder cancer has not been investigated. We aimed to identify a metabolic signature in high-grade bladder cancer by integrating unbiased metabolomics, lipidomics, and transcriptomics to predict patient survival and to discover novel therapeutic targets. We performed high-resolution liquid chromatography mass spectrometry (LC-MS) and bioinformatic analysis to determine the global metabolome and lipidome in high-grade bladder cancer. We further investigated the effects of impaired metabolic pathways using in vitro and in vivo models. We identified 519 differential metabolites and 19 lipids that were differentially expressed between low-grade and high-grade bladder cancer using the NIST MS metabolomics compendium and lipidblast MS/MS libraries, respectively. Pathway analysis revealed a unique set of biochemical pathways that are highly deregulated in high-grade bladder cancer. Integromics analysis identified a molecular gene signature associated with poor patient survival in bladder cancer. Low expression of CPT1B in high-grade tumors was associated with low FAO and low acyl carnitine levels in high-grade bladder cancer, which were confirmed using tissue microarrays. Ectopic expression of the CPT1B in high-grade bladder cancer cells led to reduced EMT in in vitro, and reduced cell proliferation, EMT, and metastasis in vivo. Our study demonstrates a novel approach for the integration of metabolomics, lipidomics, and transcriptomics data, and identifies a common gene signature associated with poor survival in patients with bladder cancer. Our data also suggest that impairment of FAO due to downregulation of CPT1B plays an important role in the progression toward high-grade bladder cancer and provide potential targets for therapeutic intervention.

First Case Report of Primary Carnitine Deficiency Manifested as Intellectual Disability and Autism Spectrum Disorder

Systemic primary carnitine deficiency (PCD) is a genetic disorder caused by decreased or absent organic cation transporter type 2 (OCTN2) carnitine transporter activity, resulting in low serum carnitine levels and decreased carnitine accumulation inside cells. In early life, PCD is usually diagnosed as a metabolic decompensation, presenting as hypoketotic hypoglycemia, Reye syndrome, or sudden infant death; in childhood, PCD presents with skeletal or cardiac myopathy. However, the clinical presentation of PCD characterized by autism spectrum disorder (ASD) with intellectual disability (ID) has seldom been reported in the literature. In this report, we describe the clinical features of a seven-year-old girl diagnosed with PCD who presented atypical features of the disease, including a developmental delay involving language skills, concentration, and attention span, as well as autistic features and brain alterations apparent in magnetic resonance imaging. We aim to highlight the difficulties related to the diagnostic and therapeutic approaches used to diagnose such patients. The case reported here presented typical signs of PCD, including frequent episodes of hypoglycemia, generalized muscle weakness, decreased muscle mass, and physical growth deficits. A molecular genetic study confirmed the definitive diagnosis of the disease (c.1345T>G (p.Y449D)) in gene SLC22A5, located in exon 8. PCD can be accompanied by less common clinical signs, which may delay its diagnosis because the resulting global clinical picture can closely resemble other metabolic disorders. In this case, the patient was prescribed a carnitine-enriched diet, as well as oral carnitine at a dose of 100 mg/kg/day. PCD has a better prognosis if it is diagnosed and treated early; however, a high level of clinical suspicion is required for its timely and accurate diagnosis.

Serum carnitine levels of children with epilepsy: Related factors including valproate

ObjectiveThis study measured the serum carnitine levels in patients with epilepsy and determined the factors contributing to low carnitine levels. MethodsWe measured the serum carnitine levels in 94 consecutive patients with epilepsy, including the free carnitine (FC) and acylcarnitine fractions, using an enzyme cycling method. We defined a low FC as a serum FC level < 36 μmol/L. Age, body mass index (BMI), standard deviation score of BMI (BMI-SDS), use of valproate, cognitive disorder, and feeding problems differed between patients with low and normal FC. In patients taking valproate, the associations of the serum FC level with the platelet count and serum ammonia and amylase levels were analyzed. ResultsUnivariate analysis showed that a low BMI and BMI-SDS, the use of valproate, and cognitive disorder were more frequent in patients with a low FC. Logistic regression analysis revealed that a low BMI-SDS and cognitive disorders were independently associated with a low FC. Among the patients taking valproate, a low BMI-SDS and age were associated with a low FC. The serum FC and ammonia levels were inversely correlated, whereas no correlation was observed between the serum FC level and platelet count or serum amylase level. ConclusionA low BMI and cognitive disorders were related to a low FC in patients with epilepsy and the serum carnitine levels should be monitored in these patients.

Biochemical, Molecular, and Clinical Characterization of Patients With Primary Carnitine Deficiency via Large-Scale Newborn Screening in Xuzhou Area.

Background: Primary carnitine deficiency (PCD) is attributed to a variation in the SLC22A5 (OCTN2) gene which encodes the key protein of the carnitine cycle, the OCTN2 carnitine transporter. PCD is typically identified in childhood by either hypoketotic hypoglycemia, or skeletal and cardiac myopathy. The aim of this study was to the clinical, biochemical, and molecular characteristics of PCD patients via newborn screening with tandem mass spectrometry (MS/MS).Methods: MS/MS was performed to screen newborns for inherited metabolic diseases. SLC22A5 gene mutations were detected in the individual and/or their family member by DNA mass array and next-generation sequencing (NGS).Results: Among the 236,368 newborns tested, ten exhibited PCD, and six others were diagnosed with low carnitine levels caused by their mothers, who had asymptomatic PCD. The incidence of PCD in the Xuzhou area is ~1:23,637. The mean initial free carnitine (C0) concentration of patients was 6.41 ± 2.01 μmol/L, and the follow-up screening concentration was 5.80 ± 1.29 μmol/L. After treatment, the concentration increased to 22.8 ± 4.13 μmol/L.Conclusion: This study demonstrates the important clinical value of combining MS/MS and NGS for the diagnosis of PCD and provides new insight into the diagnosis of PCD and maternal patients with PCD using C0 concentration and SLC22A5 mutations.

Renal dysfunction is rare in Fukuyama congenital muscular dystrophy

BackgroundThe leading cause of death in patients with Fukuyama congenital muscular dystrophy (FCMD) is congestive heart failure or respiratory dysfunction, which is same as that in Duchenne muscular dystrophy (DMD). Recent studies reported that renal dysfunction is a common complication and an increasing cause of death in advanced DMD. It can be attributable to circulatory instability or inappropriate use of drugs for treating cardiac dysfunction. MethodsWe retrospectively evaluated renal function in 38 genetically diagnosed patients with FCMD (range, 1.3–32.9 years; mean age, 13.7 ± 6.9 years) using cystatin C. We examined possible relationships of cystatin C with blood natriuretic peptide and creatinine levels along with cardiac echocardiography findings. ResultsTwenty-five patients were treated for cardiac dysfunction. Elevated cystatin C level was detected only in two, who also showed proteinuria, glycosuria, hematuria, and extremely high β2-microglobulin levels on urine tests, and were thus diagnosed with renal tubular cell damage. Because both patients were treated for intractable epilepsy with various antiepileptic drugs, including valproic acid (VPA), and had low serum carnitine levels, renal tubular cell damage was considered as an adverse effect of VPA. Unlike patients with DMD, no patient with FCMD had renal dysfunction. Such a rare occurrence of renal dysfunction can be attributable to mild cardiac dysfunction, short disease duration, and careful and early fluid management. ConclusionRenal dysfunction is rare in patients with FCMD; however, renal tubular cell damage should be ascertained, particularly in those undergoing VPA treatment for epilepsy.

Evaluation ofserum free carnitine/acylcarnitine levels and left ventricular systolic functions in children with idiopathic epilepsy receiving valproic acid

ObjectivesIn the study, the effect of valproic acid on serum free/acylcarnitine levels and left ventricular systolic function in pediatric patients with idiopathic epilepsy receiving valproic acid was investigated. Patients and MethodsPatients receiving valproic acid treatment for six months between January 2012 and December 2012 were evaluated. Blood samples were obtained from the participants twice (pretreatment and the sixth month of treatment) and serum-free and acylcarnitine levels (from C2 to C18:1-OH) were measured using tandem mass spectrometry. Cardiac functions (ejection fraction, shortening fraction, cardiac output, left ventricular systolic and diastolic diameters, left atrial diameter, aortic diameter, cardiac output, and myocardial performance index) were evaluated by echocardiography simultaneously. ResultsA total of fourty patients, 23 female (57.5%) and 17 male (42.5%), with the diagnosis of idiopathic epilepsy and receiving valproic acid monotherapy were studied. Comparison of serum-free and acylcarnitine levels measured pretreatment and sixth month of treatment revealed a decrease in average C0 and C5:1 (respectively p < 0.001, p = 0.013) and an increase in C2, C3, C5-OH, C8:1 and C4-DC levels (respectively p < 0.001, p < 0.001, p = 0.019, p = 0.013, p < 0.001). Other serum acylcarnitine levels did not change significantly (p > 0.05). No difference was observed in concurrent echocardiographic measurements of left ventricular systolic function (p > 0.05). ConclusionThe study demonstrated that valproic acid treatment results in low levels of free carnitine and changes in some acylcarnitine subgroups but has no influence on left ventricular systolic function.

Carnitine levels and mutations in the SLC22A5 gene in Faroes patients with Parkinson’s disease

IntroductionMitochondrial dysfunction, oxidative stress and energy production have been implicated in the etiology of Parkinson’s disease (PD). Several agents are under investigation for potential neuroprotective effects including acetyl-l-carnitine (ALC). ObjectiveTo investigate whether low carnitine levels and mutations in the SLC22A5 gene encoding the carnitine transporter are associates with PD risk in the Faroe Islands where the prevalence of both PD and carnitine transporter deficiency (CTD) is high. MethodsWe conducted a case-control study with 121 cases and 235 randomly selected controls, matched by gender and age. Blood spots were analyzed for free and total carnitine levels by QUATTRO LC triple quadrupole mass spectrometry (MS/MS) and sequencing performed for five genetic mutations in the SLC22A5 gene with ABI PRISM 3130. ResultsPD cases had significantly lower levels of free and total carnitine levels compared with controls (P < .001). However, stratifying according to mutation status, the lower levels of carnitine was only evident among the non-mutation carriers. Specifically, no difference was found in c.95A > G mutation frequency in the SLC22A5 gene among cases and controls (p = .70). ConclusionLow carnitine levels seem to be associated with PD, but only in individuals without the c.95A > G mutation rendering the carnitine transporter less efficient. Thus, the difference in carnitine levels is not caused by a higher frequency of c.95A > G mutation carriers in cases. The cause may be dietary or due to different gut microbiota among cases.

A fatty acid analogue targeting mitochondria exerts a plasma triacylglycerol lowering effect in rats with impaired carnitine biosynthesis.

L-carnitine is important for the catabolism of long-chain fatty acids in the mitochondria. We investigated how the triacylglycerol (TAG)-lowering drug 2-(tridec-12-yn-1-ylthio)acetic acid (1-triple TTA) influenced lipid metabolism in carnitine-depleted, 3-(2,2,2-trimethylhydrazinium)propionate dehydrate (Mildronate; meldonium)-treated male Wistar rats. As indicated, carnitine biosynthesis was impaired by Mildronate. However, TAG levels of both plasma and liver were decreased by 1-triple TTA in Mildronate-treated animals. This was accompanied by increased gene expression of proteins involved in mitochondrial activity and proliferation and reduced mRNA levels of Dgat2, ApoB and ApoCIII in liver. The hepatic energy state was reduced in the group of Mildronate and 1-triple TTA as reflected by increased AMP/ATP ratio, reduced energy charge and induced gene expression of uncoupling proteins 2 and 3. The increase in mitochondrial fatty acid oxidation was observed despite low plasma carnitine levels, and was linked to strongly induced gene expression of carnitine acetyltransferase, translocase and carnitine transporter, suggesting an efficient carnitine turnover. The present data suggest that the plasma TAG-lowering effect of 1-triple TTA in Mildronate-treated rats is not only due to increased mitochondrial fatty acid oxidation reflected by increased mitochondrial biogenesis, but also to changes in plasma clearance and reduced TAG biosynthesis.

Impact of L-Carnitine on Bisphenol A-Induced Kidney Damage in Rats

The present study aimed to explore the protective effects of L-carnitine on the oxidative stress induced by Bisphenol A (BPA) in rat kidneys. Forty male adult rats were allocated into four groups (10 rats each); placebo, BPA (50 mg/kg bwt per os), L-carnitine (500 mg/kg bwt i.p), and the BPA/L-carnitine-treated group. After 70 days, blood and kidneys were collected for measurement of renal function, oxidative/antioxidative status, histopathological analyses, and gene expression of nrf2. BPA significantly increased serum urea and carnitine levels suggesting renal damage evidenced by hydropic degenerative changes and hydronephrosis. The level of malondialdehyde (MDA) in kidney was elevated along with decline in renal total antioxidant capacity (TAC) and reduced glutathione (GSH) concentrations following challenged with BPA. Notably, the expression of nrf2 mRNA transcript in kidney tissues was down-regulated after BPA-induced toxicity. L-carnitine treatment ameliorates the damaged effects of BPA on kidneys as indicated by low levels of serum urea and carnitine, and renal MDA. Further, L-carnitine enhances the antioxidants (TAC, and GSH), with up-regulation of nrf2 expression in kidney tissues. Our results suggest the renoprotective activity of L-carnitine in BPA-induced kidney injuries through induction of antioxidant system and modulation of oxidative stress

Biochemical phenotyping unravels novel metabolic abnormalities and potential biomarkers associated with treatment of GLUT1 deficiency with ketogenic diet.

Global metabolomic profiling offers novel opportunities for the discovery of biomarkers and for the elucidation of pathogenic mechanisms that might lead to the development of novel therapies. GLUT1 deficiency syndrome (GLUT1-DS) is an inborn error of metabolism due to reduced function of glucose transporter type 1. Clinical presentation of GLUT1-DS is heterogeneous and the disorder mirrors patients with epilepsy, movement disorders, or any paroxysmal events or unexplained neurological manifestation triggered by exercise or fasting. The diagnostic biochemical hallmark of the disease is a reduced cerebrospinal fluid (CSF)/blood glucose ratio and the only available treatment is ketogenic diet. This study aimed at advancing our understanding of the biochemical perturbations in GLUT1-DS pathogenesis through biochemical phenotyping and the treatment of GLUT1-DS with a ketogenic diet. Metabolomic analysis of three CSF samples from GLUT1-DS patients not on ketogenic diet was feasible inasmuch as CSF sampling was used for diagnosis before to start with ketogenic diet. The analysis of plasma and urine samples obtained from GLUT1-DS patients treated with a ketogenic diet showed alterations in lipid and amino acid profiles. While subtle, these were consistent findings across the patients with GLUT1-DS on ketogenic diet, suggesting impacts on mitochondrial physiology. Moreover, low levels of free carnitine were present suggesting its consumption in GLUT1-DS on ketogenic diet. 3-hydroxybutyrate, 3-hydroxybutyrylcarnitine, 3-methyladipate, and N-acetylglycine were identified as potential biomarkers of GLUT1-DS on ketogenic diet. This is the first study to identify CSF, plasma, and urine metabolites associated with GLUT1-DS, as well as biochemical changes impacted by a ketogenic diet. Potential biomarkers and metabolic insights deserve further investigation.

PAX7 mutation in a syndrome of failure to thrive, hypotonia, and global neurodevelopmental delay.

PAX7 encodes a transcription factor essential in neural crest formation, myogenesis, and pituitary lineage specification. Pax7 null mice fail to thrive and exhibit muscle weakness, dying within 3weeks. We describe a human autosomal-recessive syndrome, with failure to thrive, severe global developmental delay, microcephaly, axial hypotonia, pyramidal signs, dystonic postures, seizures, irritability, and self-mutilation. Aside from low blood carnitine levels, biochemical and metabolic screen was normal, with growth hormone deficiency in one patient. Electromyography was normal, with no specific findings in brain MRI/MRS yet nondemonstrable neuropituitary, a finding of unclear significance. Muscle biopsy showed unaffected overall organization of muscle fibers, yet positive fetal alpha myosin staining, suggesting regeneration. Homozygosity mapping with whole-exome sequencing identified a single disease-associated mutation in PAX7, segregating as expected in the kindred with no homozygosity in 200 ethnically matched controls. Transfection experiments showed that the PAX7 splice-site mutation putatively causes nonsense-mediated mRNA decay affecting onlyPAX7 isoform 3. This isoform, expressed specifically in brain, skeletal muscle and testes, is the sole Pax7 variant normally found in mice. The human muscle phenotype is in line with that in conditional Pax7 null mutant mice, where initial aberrant histological findings resolve postnatally through muscle regeneration.

Biochemical characteristics of newborns with carnitine transporter defect identified by newborn screening in California.

Carnitine transporter defect (CTD; also known as systemic primary carnitine deficiency; MIM 212140) is due to mutations in the SLC22A5 gene and leads to extremely low carnitine levels in blood and tissues. Affected individuals may develop early onset cardiomyopathy, weakness, or encephalopathy, which may be serious or even fatal. The disorder can be suggested by newborn screening. However, markedly low newborn carnitine levels can also be caused by conditions unrelated to CTD, such as the low carnitine levels often associated with normal pregnancies and some metabolic disorders occurring in the mother. In order to clarify the biochemical characteristics most useful for identification of CTD in newborns, we examined California Department of Public Health newborn screening data for CTD from 2005 to 12 and performed detailed chart reviews at six metabolic centers in California. The reviews covered 14 cases of newborn CTD, 14 cases of maternal disorders (CTD, 6 cases; glutaric aciduria, type 1, 5; medium-chain acyl CoA dehydrogenase deficiency, 2; and cobalamin C deficiency, 1), and 154 false-positive cases identified by newborn screening. Our results show that newborns with CTD identified by NBS exhibit different biochemical characteristics, compared to individuals ascertained clinically. Newborns with CTD may have NBS dried blood spot free carnitine near the lower cutoff and confirmatory plasma total and free carnitine levels near the normal lower limit, particularly if obtained within two weeks after birth. These findings raise the concern that true cases of CTD may exist that could have been missed by newborn screening. CTD should be considered as a possible diagnosis in cases with suggestive clinical features, even if CTD was thought to be excluded in the newborn period. Maternal plasma total carnitine and newborn urine total carnitine values are the most important predictors of true CTD in newborns. However, biochemical testing alone does not yield a discriminant rule to distinguish true CTD from low carnitine in newborns due to other causes. Because of this biochemical variability and overlap, molecular genetic testing is imperative to confirm CTD in newborns. Additionally, functional testing of fibroblast carnitine uptake remains necessary for cases in which other confirmatory testing is inconclusive. Even with utilization of all available diagnostic testing methods, confirmation of CTD ascertained by NBS remains lengthy and challenging. Incorporation of molecular analysis as a second tier step in NBS for CTD may be beneficial and should be investigated.

Microbiota-dependent metabolite and cardiovascular disease marker trimethylamine-N-oxide (TMAO) is associated with monocyte activation but not platelet function in untreated HIV infection

BackgroundHIV infection is associated with increased risk of cardiovascular disease beyond that explained by traditional risk factors. Altered gut microbiota, microbial translocation, and immune activation have been proposed as potential triggers. The microbiota-dependent metabolite trimethylamine-N-oxide (TMAO) predicts myocardial infarction (MI) in the general population and has recently been shown to induce platelet hyperreactivity. In the present study, we investigated if TMAO was associated with platelet function, microbial translocation, and immune activation in both untreated and combination anti-retroviral therapy (cART) HIV infection.MethodsTMAO and the pre-cursors betaine, choline, and carnitine were quantified by mass-spectrometry in plasma samples from a previously established cross-sectional cohort of 50 untreated and 50 cART treated HIV-infected individuals. Whole-blood impedance aggregometry, C-reactive protein, sCD14, and lipopolysaccharide were assessed as measures of platelet function, inflammation, monocyte activation, and microbial translocation, respectively.ResultsTMAO was not associated with platelet aggregation response after stimulation with four different agonists, or with overall hypo- or hyperreactivity in untreated or treated HIV-infected individuals. In contrast, sCD14 a marker of both monocyte activation and microbial translocation was independently associated with TMAO in untreated HIV-infection (R = 0.381, P = 0.008). Lower levels of carnitine [32.2 (28.4–36.8) vs. 38.2 (33.6–42.0), P = 0.001] and betaine [33.1 (27.3–43.4) vs.37.4 (31.5–48.7, P = 0.02], but similar TMAO levels [3.8 (2.3–6.1), vs. 2.9 μM (1.9–4.8) P = 0.15] were found in cART treated compared to untreated HIV-infected individuals, resulting in higher ratios of TMAO/carnitine [0.12 (0.07–0.20) vs. 0.08 (0.05–0.11), P = 0.02] and TMAO/betaine [0.11 (0.07–0.17) vs. 0.08 (0.05–0.13), P 0.02].ConclusionsIn contrast to recent studies in HIV-uninfected populations, the present study found no evidence of TMAO-induced platelet hyperreactivity in HIV infected individuals. Microbial translocation and monocyte activation may affect TMAO levels in untreated individuals. Furthermore, the elevated ratios of TMAO/betaine and TMAO/carnitine in cART-treated individuals could possibly suggest a role of cART in TMAO metabolism.

Neonatal Screening for Primary Carnitine Deficiency: Lessons Learned from the Faroe Islands

Primary carnitine deficiency is caused by the defective OCTN2 carnitine transporter encoded by the SLC22A5 gene. A lack of carnitine impairs fatty acid oxidation resulting in hypoketotic hypoglycemia, hepatic encephalopathy, skeletal and cardiac myopathy, and arrhythmia. This condition can be detected by finding low levels of free carnitine (C0) in neonatal screening. Mothers with primary carnitine deficiency can also be identified by low carnitine levels in their infant by newborn screening. Primary carnitine deficiency is rare (1:40,000–1:140,000 newborns) except in the Faroe Islands (1:300) due to a founder effect. A specific mutation (c.95A&gt;G, p.N32S) is prevalent, but not unique, with three additional mutations (c.131C&gt;T/p.A44V, a splice mutation c.825-52G&gt;A, and a risk-haplotype) recently identified in the Faroese population. In the Faroe Islands, several adult patients suffered sudden death from primary carnitine deficiency leading to the implementation of a nationwide population screening (performed after 2 months of age) in addition to universal neonatal screening. While most affected infants can be identified at birth, some patients with primary carnitine deficiency might be missed by the current neonatal screening and could be better identified with a repeated test performed after 2 months of age.

Impact of l-carnitine supplementation on metabolic profiles in premature infants.

To describe the influence that of l-carnitine supplementation on acylcarnitine (AC) profiles and hospital outcomes in premature infants. This study is a secondary analysis of previously reported work. Metabolic profiles were obtained using standard newborn techniques on infants born between 23 and 31 completed weeks of gestation. The profiles were drawn within the first 24 h after birth and on approximately days 7, 28 and 42 of life, or at the time of discharge. A single, central, contract laboratory analyzed and managed the samples. We studied 995 patients; none was subsequently diagnosed with an inborn error of metabolism. l-Carnitine was added to parenteral nutrition in 390 (39%) study subjects; 592 (60%) did not receive supplementation. Non-supplemented infants were more likely to develop low levels of free carnitine (FC; <7 μm) on day 28; (41% vs 5%, P<0.01); and FC values were lower on day 7. Despite higher levels of FC and fewer patients with significant carnitine deficiencies, we found no evidence that l-carnitine supplementation was associated with improved short-term hospital outcomes. l-Carnitine supplementation is common in prematurely born neonates and is associated with higher carnitine levels, but is not associated with improved short-term hospital outcomes.