SLC25A51 is a mammalian mitochondrial NAD+ transporter.

Mitochondria require nicotinamide adenine dinucleotide (NAD+) to carry out the fundamental processes that fuel respiration and mediate cellular energy transduction. Mitochondrial NAD+ transporters have been identified in yeast and plants1,2, but their existence in mammals remains controversial3-5. Here we demonstrate that mammalian mitochondria can take up intact NAD+, and identify SLC25A51 (also known as MCART1)-an essential6,7 mitochondrial protein of previously unknown function-as a mammalian mitochondrial NAD+ transporter. Loss of SLC25A51 decreases mitochondrial-but not whole-cell-NAD+ content, impairs mitochondrial respiration, and blocks the uptake of NAD+ into isolated mitochondria. Conversely, overexpression of SLC25A51 or SLC25A52 (a nearly identical paralogue of SLC25A51) increases mitochondrial NAD+ levels and restores NAD+ uptake into yeast mitochondria lacking endogenous NAD+ transporters. Together, these findings identify SLC25A51 as a mammalian transporter capable of importing NAD+ into mitochondria.

RNA editing of SLC22A3 drives early tumor invasion and metastasis in familial esophageal cancer.

Like many complex human diseases, esophageal squamous cell carcinoma (ESCC) is known to cluster in families. Familial ESCC cases often show early onset and worse prognosis than the sporadic cases. However, the molecular genetic basis underlying the development of familial ESCC is mostly unknown. We reported that SLC22A3 is significantly down-regulated in nontumor esophageal tissues from patients with familial ESCC compared with tissues from patients with sporadic ESCCs. A-to-I RNA editing of the SLC22A3 gene results in its reduced expression in the nontumor esophageal tissues of familial ESCCs and is significantly correlated with lymph node metastasis. The RNA-editing enzyme ADAR2, a familial ESCC susceptibility gene identified by our post hoc genome-wide association study, is positively correlated with the editing level of SLC22A3 Moreover, functional studies showed that SLC22A3 is a metastasis suppressor in ESCC, and deregulation of SLC22A3 facilitates cell invasion and filopodia formation by reducing its direct association with α-actinin-4 (ACTN4), leading to the increased actin-binding activity of ACTN4 in normal esophageal cells. Collectively, we now show that A-to-I RNA editing of SLC22A3 contributes to the early development and progression of familial esophageal cancer in high-risk individuals.

Mitigation of acute kidney injury by cell-cycle inhibitors that suppress both CDK4/6 and OCT2 functions.

Acute kidney injury (AKI) is a potentially fatal syndrome characterized by a rapid decline in kidney function caused by ischemic or toxic injury to renal tubular cells. The widely used chemotherapy drug cisplatin accumulates preferentially in the renal tubular cells and is a frequent cause of drug-induced AKI. During the development of AKI the quiescent tubular cells reenter the cell cycle. Strategies that block cell-cycle progression ameliorate kidney injury, possibly by averting cell division in the presence of extensive DNA damage. However, the early signaling events that lead to cell-cycle activation during AKI are not known. In the current study, using mouse models of cisplatin nephrotoxicity, we show that the G1/S-regulating cyclin-dependent kinase 4/6 (CDK4/6) pathway is activated in parallel with renal cell-cycle entry but before the development of AKI. Targeted inhibition of CDK4/6 pathway by small-molecule inhibitors palbociclib (PD-0332991) and ribociclib (LEE011) resulted in inhibition of cell-cycle progression, amelioration of kidney injury, and improved overall survival. Of additional significance, these compounds were found to be potent inhibitors of organic cation transporter 2 (OCT2), which contributes to the cellular accumulation of cisplatin and subsequent kidney injury. The unique cell-cycle and OCT2-targeting activities of palbociclib and LEE011, combined with their potential for clinical translation, support their further exploration as therapeutic candidates for prevention of AKI.

Carnitine transport and fatty acid oxidation.

Carnitine is essential for the transfer of long-chain fatty acids across the inner mitochondrial membrane for subsequent ß-oxidation. It can be synthesized by the body or assumed with the diet from meat and dairy products. Defects in carnitine biosynthesis do not routinely result in low plasma carnitine levels. Carnitine is accumulated by the cells and retained by kidneys using OCTN2, a high affinity organic cation transporter specific for carnitine. Defects in the OCTN2 carnitine transporter results in autosomal recessive primary carnitine deficiency characterized by decreased intracellular carnitine accumulation, increased losses of carnitine in the urine, and low serum carnitine levels. Patients can present early in life with hypoketotic hypoglycemia and hepatic encephalopathy, or later in life with skeletal and cardiac myopathy or sudden death from cardiac arrhythmia, usually triggered by fasting or catabolic state. This disease responds to oral carnitine that, in pharmacological doses, enters cells using the amino acid transporter B(0,+). Primary carnitine deficiency can be suspected from the clinical presentation or identified by low levels of free carnitine (C0) in the newborn screening. Some adult patients have been diagnosed following the birth of an unaffected child with very low carnitine levels in the newborn screening. The diagnosis is confirmed by measuring low carnitine uptake in the patients' fibroblasts or by DNA sequencing of the SLC22A5 gene encoding the OCTN2 carnitine transporter. Some mutations are specific for certain ethnic backgrounds, but the majority are private and identified only in individual families. Although the genotype usually does not correlate with metabolic or cardiac involvement in primary carnitine deficiency, patients presenting as adults tend to have at least one missense mutation retaining residual activity. This article is part of a Special Issue entitled: Mitochondrial Channels edited by Pierre Sonveaux, Pierre Maechler and Jean-Claude Martinou.

The impact of Organic Anion-Transporting Polypeptides (OATPs) on disposition and toxicity of antitumor drugs: Insights from knockout and humanized mice.

It is now widely accepted that organic anion-transporting polypeptides (OATPs), especially members of the OATP1A/1B family, can have a major impact on the disposition and elimination of a variety of endogenous molecules and drugs. Owing to their prominent expression in the sinusoidal plasma membrane of hepatocytes, OATP1B1 and OATP1B3 play key roles in the hepatic uptake and plasma clearance of a multitude of structurally diverse anti-cancer and other drugs. Here, we present a thorough assessment of the currently available OATP1A and OATP1B knockout and transgenic mouse models as key tools to study OATP functions in vivo. We discuss recent studies using these models demonstrating the importance of OATPs, primarily in the plasma and hepatic clearance of anticancer drugs such as taxanes, irinotecan/SN-38, methotrexate, doxorubicin, and platinum compounds. We further discuss recent work on OATP-mediated drug-drug interactions in these mouse models, as well as on the role of OATP1A/1B proteins in the phenomenon of hepatocyte hopping, an efficient and flexible way of liver detoxification for both endogenous and exogenous substrates. Interestingly, glucuronide conjugates of both the heme breakdown product bilirubin and the protein tyrosine kinase-targeted anticancer drug sorafenib are strongly affected by this process. The clinical relevance of variation in OATP1A/1B activity in patients has been previously revealed by the effects of polymorphic variants and drug-drug interactions on drug toxicity. The development of in vivo tools to study OATP1A/1B functions has greatly advanced our mechanistic understanding of their functional role in drug pharmacokinetics, and their implications for therapeutic efficacy and toxic side effects of anticancer and other drug treatments.

Oxaliplatin-induced neurotoxicity is dependent on the organic cation transporter OCT2.

Oxaliplatin is an integral component of colorectal cancer therapy, but its clinical use is associated with a dose-limiting peripheral neurotoxicity. We found that the organic cation transporter 2 (OCT2) is expressed on dorsal root ganglia cells within the nervous system where oxaliplatin is known to accumulate. Cellular uptake of oxaliplatin was increased by 16- to 35-fold in cells overexpressing mouse Oct2 or human OCT2, and this process was associated with increased DNA platination and oxaliplatin-induced cytotoxicity. Furthermore, genetic or pharmacologic knockout of Oct2 protected mice from hypersensitivity to cold or mechanical-induced allodynia, which are established tests to assess acute oxaliplatin-induced neurotoxicity. These findings provide a rationale for the development of targeted approaches to mitigate this debilitating toxicity.

Exacerbation of mood symptoms associated to primary and secondary carnitine deficiency: a case report.

Rarely screened in psychiatric patients, primary and/or secondary Carnitine deficiency could be influencing and/or mimicking the mood symptoms of our patient population. The brain and specifically neurons are highly vulnerable to impairments in oxidative metabolism, which can lead to neuronal cell death and disorders of neurotransmitters causing changes in cognition and behavior. For this reason, identification of this disorder is important since its treatment could result in symptom improvement and better quality of life of our patients. We present a case where exacerbation of mood symptoms was associated to primary and secondary Carnitine deficiency.

Ablation of both organic cation transporter (OCT)1 and OCT2 alters metformin pharmacokinetics but has no effect on tissue drug exposure and pharmacodynamics.

Organic cation transporter (OCT)1 and OCT2 mediate hepatic uptake and secretory renal clearance of metformin, respectively. Pharmacokinetic/pharmacodynamic (PK/PD) implications of simultaneous impairment of both transporters, such as by systemic pan-OCT inhibition, have not been studied directly. At present metformin PK/PD, distribution, and excretion were studied in Oct1/Oct2-knockout mice. Metformin clearance was reduced 4.5-fold from renal blood flow to unbound glomerular filtration rate, and volume of distribution was reduced 3.5-fold in Oct1/Oct2-knockout mice. Oral bioavailability was not affected (F = 64 ± 4 versus 59 ± 11; knockout versus wild type). Liver- and kidney-to-plasma concentration ratios were decreased in Oct1/Oct2-knockout mice 4.2- and 2.5-fold, respectively. The 2.9-fold increase in oral metformin exposure and reduced tissue partitioning yielded little to no net change in tissue drug concentrations. Absolute kidney exposure was unchanged (knockout/wild type = 1.1 ± 0.2), and liver exposure was only modestly decreased (knockout/wild type = 0.6 ± 0.1). Oral glucose area under the curve (AUC) lowering by metformin was not impaired in Oct1/Oct2-knockout mice at the five dose levels tested (ED50 = 151 versus 110 mg/kg; glucose lowering at highest dose = 42 ± 1 versus 39 ± 4%; knockout versus wild type); however, higher systemic metformin exposures were necessary in knockout mice to elicit the same effect (half-maximal efficacious AUC = 70 versus 26 µg x h/ml). Despite major changes in metformin clearance and volume of distribution in Oct1/Oct2-knockout mice, tissue drug exposure and PD were not affected. These findings challenge the presumption that systemic OCT inhibition will affect metformin pharmacology.

New mechanistic explanation for the localization of ulcers in the rat duodenum: role of iron and selective uptake of cysteamine.

Cysteamine, a coenzyme A metabolite, induces duodenal ulcers in rodents. Our recent studies showed that ulcer formation was aggravated by iron overload and diminished in iron deficiency. We hypothesized that cysteamine is selectively taken up in the duodenal mucosa, where iron absorption primarily occurs, and is transported by a carrier-mediated process. Here we report that cysteamine administration in rats leads to cysteamine accumulation in the proximal duodenum, where the highest concentration of iron in the gastrointestinal tract is found. In vitro, iron loading of intestinal epithelial cells (IEC-6) accelerated reactive oxygen species (ROS) production and increased [(14)C]cysteamine uptake. [(14)C]Cysteamine uptake by isolated gastrointestinal mucosal cells and by IEC-6 was pH-dependent and inhibited by unlabeled cysteamine. The uptake of [(14)C]cysteamine by IEC-6 was Na(+)-independent, saturable, inhibited by structural analogs, H(2)-histamine receptor antagonists, and organic cation transporter (OCT) inhibitors. OCT1 mRNA was markedly expressed in the rat duodenum and in IEC-6, and transfection of IEC-6 with OCT1 siRNA decreased OCT1 mRNA expression and inhibited [(14)C]cysteamine uptake. Cysteamine-induced duodenal ulcers were decreased in OCT1/2 knockout mice. These studies provide new insights into the mechanism of cysteamine absorption and demonstrate that intracellular iron plays a critical role in cysteamine uptake and in experimental duodenal ulcerogenesis.

Deficiency of multidrug and toxin extrusion 1 enhances renal accumulation of paraquat and deteriorates kidney injury in mice.

Multidrug and toxin extrusion 1 (MATE1/solute carrier 47A1) mediates cellular transport of a variety of structurally diverse compounds. Paraquat (PQ), which has been characterized in vitro as a MATE1 substrate, is a widely used herbicide and can cause severe toxicity to humans after exposure. However, the contribution of MATE1 to PQ disposition in vivo has not been determined. In the present study, we generated Mate1-deficient (Mate1-/-) mice and performed toxicokinetic analyses of PQ in Mate1-/- and wild-type (Mate1+/+) mice. After a single intravenous administration of PQ (50 mg/kg), Mate1-/- mice exhibited significantly higher plasma PQ concentrations than Mate1+/+ mice. The renal PQ concentration was markedly increased in Mate1-/- mice compared with Mate1+/+ mice. The subsequent nephrotoxicity of PQ were examined in these mice. Three days after intraperitoneal administration of PQ (20 mg/kg), the transcript levels of N-acetyl-ß-D-glucosaminidase (Lcn2) and kidney injury molecule-1 (Kim-1) in the kidney were remarkably enhanced in the Mate1-/- mice. This was accompanied by apparent difference in renal histology between Mate1-/- and Mate1+/+ mice. In conclusion, we demonstrated that Mate1 is responsible for renal elimination of PQ in vivo and the deficiency of Mate1 function confers deteriorated kidney injury caused by PQ in mice.

Diagnoses of newborns and mothers with carnitine uptake defects through newborn screening.

Carnitine uptake defect (CUD) is an autosomal recessive fatty acid oxidation defect caused by a deficiency of the high-affinity carnitine transporter OCTN2. CUD patients may present with hypoketotic hypoglycemia, hepatic encephalopathy or dilated cardiomyopathy. Tandem mass spectrometry screening of newborns can detect CUD, although transplacental transport of free carnitine from the mother may cause a higher free carnitine level and cause false negatives during newborn screening. From Jan 2001 to July 2009, newborns were screened for low free carnitine levels at the National Taiwan University Hospital screening center. Confirmation tests included dried blood spot free acylcarnitine levels and mutation analyses for both babies and their mothers. Sixteen newborns had confirmation tests for persistent low free carnitine levels; four had CUD, six had mothers with CUD, and six cases were false positives. All babies born to mothers with CUD had transient carnitine deficiency. The six mothers with CUD were put on carnitine supplementation (50-100mg/kg/day). One mother had dilated cardiomyopathy at diagnosis and her cardiac function improved after treatment. Analysis of the SLC22A5 gene revealed that p.S467C was the most common mutation in mothers with CUD, while p.R254X was the most common mutation in newborns and children with CUD. Newborn screening allows for the detection of CUD both in newborns and mothers, with an incidence in newborns of one in 67,000 (95% CI: one in 31,600-512,000) and a prevalence in mothers of one in 33,000 (95% CI: one in 18,700-169,000). Detection of CUD in mothers may prevent them from developing dilated cardiomyopathy.

Stability of acylcarnitines and free carnitine in dried blood samples: implications for retrospective diagnosis of inborn errors of metabolism and neonatal screening for carnitine transporter deficiency.

OBJECTIVE: Electrospray ionization-tandem mass spectrometry (ESI-MS/MS) is increasingly used in newborn screening programs. Acylcarnitine profiles from dried blood spots (DBS) are used to detect fatty acid oxidation disorders, carnitine cycle disorders, and organic acidurias. Stored dried blood is also a valuable source for postmortem investigations to unravel the cause of unexplained death in early childhood. However, diagnostic uncertainties arising from the unknown stability of acylcarnitines and free carnitine during prolonged storage have not yet been studied in a systematic manner. METHODS: Whole blood spiked with acylcarnitines was stored either at -18 degrees C or at room temperature up to 1000 days. At regular time intervals 3.2 mm spots of these samples were extracted with 150 microL of methanol. Free carnitine and acylcarnitines were converted to their corresponding butyl esters and analyzed by ESI-MS/MS. RESULTS: At -18 degrees C acylcarnitines are stable for at least 330 days. If stored for prolonged periods at room temperature (>14 days), acylcarnitines are hydrolyzed to free carnitine and the corresponding fatty acids. The velocity of decay is logarithmic and depends on the chain length of the acylcarnitines. Short-chain acylcarnitines hydrolyze quicker than long-chain acylcarnitines. CONCLUSION: The data indicate that stored filter cards should only be used for retrospective quantitation of acylcarnitines if appropriate correction for sample decay during storage is applied. Free carnitine increases upon storage but can reliably be quantitated under standardized derivatization conditions. Furthermore, carnitine transporter (OCTN2) deficiency can reliably be diagnosed by examining acylcarnitine profiles, which can supplement free carnitine levels as a discriminatory marker.

Decreased anxiety in mice lacking the organic cation transporter 3.

The organic cation transporter 3 (OCT3; synonymous: extraneuronal monoamine transporter, EMT, Slc22a3) encodes an isoform of the organic cation transporters and is expressed widely across the whole brain. OCTs are a family of high-capacity, bidirectional, multispecific transporters of organic cations. These also include serotonin, dopamine and norepinephrine making OCTs attractive candidates for a variety of neuropsychiatric disorders including anxiety disorders. OCT3 has been implicated in termination of monoaminergic signalling in the central nervous system. Interestingly, OCT3 mRNA is however also significantly up-regulated in the hippocampus of serotonin transporter knockout mice where it might serve as an alternative reuptake mechanism for serotonin. The examination of the behavioural phenotype of OCT3 knockout mice thus is paramount to assess the role of OCT3. We have therefore subjected mice lacking the OCT3 gene to a comprehensive behavioural test battery. While cognitive functioning in the Morris water maze test and aggression levels measured with the resident-intruder paradigm were in the same range as the respective control animals, OCT3 knockout animals showed a tendency of increased activity and were significantly less anxious in the elevated plus-maze test and the open field test as compared to their respective wild-type controls arguing for a role of OCT3 in the regulation of fear and anxiety, probably by modulating the serotonergic tone in limbic circuitries.

Plasma carnitine ester profile in homozygous and heterozygous OCTN2 deficiency.

The carnitine ester spectrum was studied using ESI tandem mass spectrometry in a 2.5-year-old male Roma child with homozygous deletion of 844C of the SLC22A5 gene, presenting with hepatopathy and cardiomyopathy. Besides the dramatic decrease of plasma free carnitine (1.38 vs 32.7 mumol/L in controls) all plasma carnitine esters were severely decreased in the proband: the total esters were 31.4% of the controls. In three heterozygous siblings the free carnitine level was 62.3% of the normal controls, while the levels of the individual carnitine esters ranged between 15.5% and 163% (average 70.9%). The heterozygous parents exhibited the same pattern. The proband was supplemented with 50 mg/kg per day of L-carnitine oral solution. After 2 months of treatment, his hepatomegaly, elevated transaminases and the pathological cardiac ultrasound parameters normalized. The plasma free carnitine rose to 12.8 mumol/L (39% of the controls). All of the carnitine esters also increased; however, the individual esters were still 8.5-169.7% of the controls (average 55.5%). After 13 months of treatment there was a further increase in free carnitine (15.9 mumol/L) as well as in the level of the individual esters, ranging between 16.1% and 140.3% of the controls (average 66.9%). The data presented here show that, besides the dramatic decrease of free carnitine, the carnitine ester metabolism is also affected in OCTN2 deficiency; the replenishment of the pools under treatment is slow. Despite an impressive clinical improvement, the carnitine metabolism can be still seriously affected.

Altered aminergic neurotransmission in the brain of organic cation transporter 3-deficient mice.

Organic cation transporters (OCTs) are carrier-type polyspecific permeases known to participate in low-affinity extraneuronal catecholamine uptake in peripheral tissues. OCT3 is the OCT subtype most represented in the brain, yet its implication in central aminergic neurotransmission in vivo had not been directly demonstrated. In a detailed immunohistochemistry study, we show that OCT3 is expressed in aminergic pathways in the mouse brain, particularly in dopaminergic neurons of the substantia nigra compacta, non-aminergic neurons of the ventral tegmental area, substantia nigra reticulata (SNr), locus coeruleus, hippocampus and cortex. Although OCT3 was found mainly in neurons, it was also occasionally detected in astrocytes in the SNr, hippocampus and several hypothalamic nuclei. In agreement with this distribution, OCT3/Slc22a3-deficient mice show evidence of altered monoamine neurotransmission in the brain, with decreased intracellular content and increased turnover of aminergic transmitters. The behavioral characterization of these mutants reveal subtle behavioral alterations such as increased sensitivity to psychostimulants and increased levels of anxiety and stress. Altogether our data support a role of OCT3 in the homeostatic regulation of aminergic neurotransmission in the brain.

Expression patterns of the organic cation/carnitine transporter family in adult murine brain.

UNLABELLED: Organic cation/carnitine transporters transport carnitine, drugs, and xenobiotics (e.g. choline, acetylcarnitine, betaine, valproic acid), and are expressed in muscle, heart, blood vessels, kidney, gut, etc. OBJECTIVE: To characterize expression patterns of mOctn1, -2 and -3 in murine brain. METHODS: We applied our transporter-specific antibodies to mOctn1, -2 and -3, followed by 2 0 antibody and DAB peroxidase detection to serial adult murine brain sections counterstained with hematoxylin. RESULTS: All three transporters showed strong expression in the external plexiform layer of the olfactory bulb and in olfactory nerve, the molecular layer and neuronal processes of input fibres extending vertically in motor cortex, in the dendritic arborization of the cornu ammonis and dendate gyrus (hippocampus), neuronal processes in the arcuate nucleus (hypothalamus), choroid plexus cells, and neuronal cell bodies and dendrites of cranial nerve nuclei V and VII. In the cerebellum, all three transporters were strongly expressed in dendritic processes of Purkinje cells, but Octn1 and -2 were expressed more strongly than Octn3 in Purkinje cell bodies. In spinal cord, Octn1, -2 and -3 were prominent in axons and dendritic end-arborizations of spinal cord neurons in both ascending and descending white matter tracts, whereas Octn3 was also strongly expressed in grey matter, specifically in anterior horn cell bodies. Octn3 was weakly expressed in glomerular layer neuronal cell bodies of olfactory bulb. CONCLUSIONS: hOCTN2 deficiency presents with carnitine-responsive cardiomyopathy, myopathy and hypoglycemic, hypoketotic coma with strokes, seizures and delays. In mouse, Octn1, -2 and -3 are expressed in many regions throughout the central nervous system with a pattern suggestive of roles in modulating cerebral bioenergetics and in acetylcholine generation for neurotransmission in olfactory, satiety, limbic, memory, motor and sensory functions. This distribution may play a role in the pattern of neurological injury that occurs in hOCTN2 deficiency during catabolic episodes of hypoglycemic, hypoketotic encephalopathy and which may manifest with cognitive impairment, hypotonia and seizures.

Effect of carnitine deprivation on carnitine homeostasis and energy metabolism in mice with systemic carnitine deficiency.

BACKGROUND/AIMS: Juvenile visceral steatosis (jvs-/-) mice lack the activity of the carnitine transporter OCTN2 and are dependent on carnitine substitution. The effects of carnitine deprivation on carnitine homeostasis and energy metabolism are not known in jvs-/- mice. METHODS: jvs-/- mice were studied 3, 6 and 10 days after carnitine deprivation, and compared to jvs-/- mice substituted with carnitine, wild-type (jvs+/+) and jvs+/- mice. Carnitine concentrations were assessed radioenzymatically. RESULTS: Compared to wild-type mice, carnitine-treated jvs-/- mice had decreased plasma beta-hydroxybutyrate levels and showed hepatic fat accumulation. The carnitine levels in plasma, liver and skeletal muscle were decreased by 58, 16 and 17%, respectively. After ten days of carnitine deprivation, the plasma carnitine concentration had fallen by 87% (to 2.3 mumol/l) and the tissue carnitine levels by approximately 50% compared to carnitine-treated jvs-/- mice. Carnitine deprivation was associated with a further drop in plasma beta-hydroxybutyrate and increased hepatic fat. Skeletal muscle glycogen stores decreased and lactate levels increased with carnitine deprivation, whereas tissue ATP levels were maintained. CONCLUSIONS: In jvs-/- mice, tissue carnitine stores are more resistant than carnitine plasma concentrations to carnitine deprivation. Metabolic changes (liver steatosis and loss of muscle glycogen stores) appear also early after carnitine deprivation.

Histopathologic abnormalities of the lymphoreticular tissues in organic cation transporter 2 deficiency: evidence for impaired B cell maturation.

Immunohistology of lymphoreticular tissues of a fatal case of organic cation transporter 2 deficiency revealed inhibited proliferation with increased apoptosis in the germinal centers, resulting in "burned out" follicles. This is indicative of impaired antigen driven B cell affinity maturation. Defective humoral immune response might explain the recurrent infections in untreated organic cation transporter 2 deficiency.

Deficiency of the carnitine transporter (OCTN2) with partial N-acetylglutamate synthase (NAGS) deficiency.

A patient with recurrent episodes of hyperammonaemia (highest ammonia level recorded 229 micromol/L, normal 9-33) leading to altered levels of consciousness was diagnosed with partial N-acetylglutamate synthase (NAGS) deficiency (9% residual activity) at age 5 years and was treated with ammonia-conjugating agents (Ucephan 250 mg/kg per day and later sodium phenylbutyrate 200-250 mg/kg per day) for 15 years. A chronically low serum carnitine level (pretreatment plasma free carnitine 4 nmol/L, normal 37 +/- 8 nmol/L; total carnitine 8 nmol/L, normal 46 +/- 10) was assumed to be secondary and was treated with supplemental carnitine (30-50 mg/kg per day). Hypoglycaemia (blood sugar 35 mg/dl, normal 70-100), cardiomegaly, and fatty liver were also noted at diagnosis. The patient died unexpectedly at age 20 years. In retrospect, it was learned that the patient had stopped his carnitine without medical consultation several weeks prior to his death. Additional molecular investigations identified two mutations (R254X and IVS3 + 1G > A) in the patient's OCTN2 (SLC22A5) gene, consistent with a diagnosis of primary carnitine deficiency due to carnitine transporter defect. R245X is a founder mutation in Southern Chinese populations. It is unknown whether the original NAGS deficiency was primary or secondary, but molecular analysis of the NAGS gene failed to identify mutations. Urea cycle enzyme expression may be affected by fatty acid suppression of an AP-1 binding site in the promoter enhancer region of the urea cycle gene. Regardless, it is clear that the NAGS abnormality has led to delay of recognition of the OCTN2 defect, and modified the clinical course in this patient.

Carnitine transporter and holocarboxylase synthetase deficiencies in The Faroe Islands.

Carnitine transporter deficiency (CTD) and holocarboxylase synthetase deficiency (HLCSD) are frequent in The Faroe Islands compared to other areas, and treatment is available for both disorders. In order to evaluate the feasibility of neonatal screening in The Faroe Islands we studied detection in the neonatal period by tandem mass spectrometry, carrier frequencies, clinical manifestations, and effect of treatment of CTD and HLCSD. We found 11 patients with CTD from five families and 8 patients with HLCSD from five families. The natural history of both disorders varied extensively among patients, ranging from patients who presumably had died from their disease to asymptomatic individuals. All symptomatic patients responded favourably to supplementation with L: -carnitine (in case of CTD) or biotin (in case of HLCSD), but only if treated early. Estimates of carrier frequency of about 1:20 for both disorders indicate that some enzyme-deficient individuals remain undiagnosed. Prospective and retrospective tandem mass spectrometry (MS/MS) analyses of carnitines from neonatally obtained filter-paper dried blood-spot samples (DBSS) uncovered 8 of 10 individuals with CTD when using both C(0) and C(2) as markers (current algorithm) and 10 of 10 when using only C(0) as marker. MS/MS analysis uncovered 5 of 6 patient with HLCSD. This is the first study to report successful neonatal MS/MS analysis for the diagnosis of HLCSD. We conclude that CTD and HLCSD are relatively frequent in The Faroe Islands and are associated with variable clinical manifestations, and that diagnosis by neonatal screening followed by early therapy will secure a good outcome.