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.

Adherence to clinic recommendations among patients with phenylketonuria in the United States.

OBJECTIVE: Assess current management practices of phenylketonuria (PKU) clinics across the United States (US) based on the key treatment metrics of blood phenylalanine (Phe) concentrations and blood Phe testing frequency, as well as patient adherence to their clinic's management practice recommendations. METHODS: An online survey was conducted with medical professionals from PKU clinics across the US from July to September 2015. Forty-four clinics participated in the survey and account for approximately half of PKU patients currently followed in clinics in the US (Berry et al., 2013). RESULTS: The majority of PKU clinics recommended target blood Phe concentrations to be between 120 and 360µM for all patients; the upper threshold was relaxed by some clinics for adult patients (from 360 to 600µM) and tightened for patients who are pregnant/planning to become pregnant (to 240µM). Patient adherence to these recommendations (percentage of patients with blood Phe below the upper recommended threshold) was age-dependent, decreasing from 88% in the 0-4years age group to 33% in adults 30+ years. Patient adherence to recommendations for blood testing frequency followed a similar trend. Higher staffing intensity (specialists per 100 PKU patients) was associated with better patient adherence to clinics' blood Phe concentrations recommendations. CONCLUSION: Clinic recommendations of target blood Phe concentrations in the US are now stricter compared to prior years, and largely reflect recent guidelines by the American College of Medical Genetics and Genomics (Vockley et al., 2014). Adherence to recommended Phe concentrations remains suboptimal, especially in older patients. However, despite remaining above the guidelines, actual blood Phe concentrations in adolescents and adults are lower than those reported in the past (Walter et al., 2002; Freehauf et al., 2013). Continued education and support for PKU patients by healthcare professionals, including adequate clinic staffing, are needed to improve adherence. Future research is needed to understand how to improve adherence to reduce the number of patients lost to follow-up, as the findings of this and similar surveys do not address how to keep patients in clinic.

Minimal ureagenesis is necessary for survival in the murine model of hyperargininemia treated by AAV-based gene therapy.

Hyperammonemia is less severe in arginase 1 deficiency compared with other urea cycle defects. Affected patients manifest hyperargininemia and infrequent episodes of hyperammonemia. Patients typically suffer from neurological impairment with cortical and pyramidal tract deterioration, spasticity, loss of ambulation, seizures and intellectual disability; death is less common than with other urea cycle disorders. In a mouse model of arginase I deficiency, the onset of symptoms begins with weight loss and gait instability, which progresses toward development of tail tremor with seizure-like activity; death typically occurs at about 2 weeks of life. Adeno-associated viral vector gene replacement strategies result in long-term survival of mice with this disorder. With neonatal administration of vector, the viral copy number in the liver greatly declines with hepatocyte proliferation in the first 5 weeks of life. Although the animals do survive, it is not known from a functional standpoint how well the urea cycle is functioning in the adult animals that receive adeno-associated virus. In these studies, we administered [1-13C] acetate to both littermate controls and adeno-associated virus-treated arginase 1 knockout animals and examined flux through the urea cycle. Circulating ammonia levels were mildly elevated in treated animals. Arginine and glutamine also had perturbations. Assessment 30 min after acetate administration demonstrated that ureagenesis was present in the treated knockout liver at levels as low at 3.3% of control animals. These studies demonstrate that only minimal levels of hepatic arginase activity are necessary for survival and ureagenesis in arginase-deficient mice and that this level of activity results in control of circulating ammonia. These results may have implications for potential therapy in humans with arginase deficiency.

AAV-based gene therapy prevents neuropathology and results in normal cognitive development in the hyperargininemic mouse.

Complete arginase I deficiency is the least severe urea cycle disorder, characterized by hyperargininemia and infrequent episodes of hyperammonemia. Patients suffer from neurological impairment with cortical and pyramidal tract deterioration, spasticity, loss of ambulation and seizures, and is associated with intellectual disability. In mice, onset is heralded by weight loss beginning around day 15; gait instability follows progressing to inability to stand and development of tail tremor with seizure-like activity and death. Here we report that hyperargininemic mice treated neonatally with an adeno-associated virus (AAV)-expressing arginase and followed long-term lack any presentation consistent with brain dysfunction. Behavioral and histopathological evaluation demonstrated that treated mice are indistinguishable from littermates, and that putative compounds associated with neurotoxicity are diminished. In addition, treatment results in near complete resolution of metabolic abnormalities early in life; however, there is the development of some derangement later with decline in transgene expression. Ammonium challenging revealed that treated mice are affected by exogenous loading much greater than littermates. These results demonstrate that AAV-based therapy for hyperargininemia is effective and prevents development of neurological abnormalities and cognitive dysfunction in a mouse model of hyperargininemia; however, nitrogen challenging reveals that these mice remain impaired in the handling of waste nitrogen.

Urinary phenylacetylglutamine as dosing biomarker for patients with urea cycle disorders.

UNLABELLED: We have analyzed pharmacokinetic data for glycerol phenylbutyrate (also GT4P or HPN-100) and sodium phenylbutyrate with respect to possible dosing biomarkers in patients with urea cycle disorders (UCD). STUDY DESIGN: These analyses are based on over 3000 urine and plasma data points from 54 adult and 11 pediatric UCD patients (ages 6-17) who participated in three clinical studies comparing ammonia control and pharmacokinetics during steady state treatment with glycerol phenylbutyrate or sodium phenylbutyrate. All patients received phenylbutyric acid equivalent doses of glycerol phenylbutyrate or sodium phenylbutyrate in a cross over fashion and underwent 24-hour blood samples and urine sampling for phenylbutyric acid, phenylacetic acid and phenylacetylglutamine. RESULTS: Patients received phenylbutyric acid equivalent doses of glycerol phenylbutyrate ranging from 1.5 to 31.8 g/day and of sodium phenylbutyrate ranging from 1.3 to 31.7 g/day. Plasma metabolite levels varied widely, with average fluctuation indices ranging from 1979% to 5690% for phenylbutyric acid, 843% to 3931% for phenylacetic acid, and 881% to 1434% for phenylacetylglutamine. Mean percent recovery of phenylbutyric acid as urinary phenylacetylglutamine was 66.4 and 69.0 for pediatric patients and 68.7 and 71.4 for adult patients on glycerol phenylbutyrate and sodium phenylbutyrate, respectively. The correlation with dose was strongest for urinary phenylacetylglutamine excretion, either as morning spot urine (r = 0.730, p < 0.001) or as total 24-hour excretion (r = 0.791 p<0.001), followed by plasma phenylacetylglutamine AUC(24-hour), plasma phenylacetic acid AUC(24-hour) and phenylbutyric acid AUC(24-hour). Plasma phenylacetic acid levels in adult and pediatric patients did not show a consistent relationship with either urinary phenylacetylglutamine or ammonia control. CONCLUSION: The findings are collectively consistent with substantial yet variable pre-systemic (1st pass) conversion of phenylbutyric acid to phenylacetic acid and/or phenylacetylglutamine. The variability of blood metabolite levels during the day, their weaker correlation with dose, the need for multiple blood samples to capture trough and peak, and the inconsistency between phenylacetic acid and urinary phenylacetylglutamine as a marker of waste nitrogen scavenging limit the utility of plasma levels for therapeutic monitoring. By contrast, 24-hour urinary phenylacetylglutamine and morning spot urine phenylacetylglutamine correlate strongly with dose and appear to be clinically useful non-invasive biomarkers for compliance and therapeutic monitoring.

The response of patients with phenylketonuria and elevated serum phenylalanine to treatment with oral sapropterin dihydrochloride (6R-tetrahydrobiopterin): a phase II, multicentre, open-label, screening study.

This study aimed to evaluate the response to and safety of an 8-day course of sapropterin dihydrochloride (6R-tetrahydrobiopterin or 6R-BH4) 10 mg/kg per day in patients with phenylketonuria (PKU), who have elevated blood phenylalanine (Phe) levels, and to identify a suitable cohort of patients who would respond to sapropterin dihydrochloride treatment with a reduction in blood Phe level. Eligible patients were aged > or = 8 years, had blood Phe levels > or = 450 micromol/L and were not adhering to a Phe-restricted diet. Suitable patients were identified by a > or = 30% reduction in blood Phe level from baseline to day 8 following sapropterin dihydrochloride treatment. The proportion of patients who met these criteria was calculated for the overall population and by baseline Phe level (< 600, 600 to < 900, 900 to < 1200 and > or = 1200 micromol/L). In total, 485/490 patients completed the study and 20% (96/485) were identified as patients who would respond to sapropterin dihydrochloride. A reduction in Phe level was observed in all subgroups, although response was greater in patients with lower baseline Phe levels. Wide variability in response was seen across all baseline Phe subgroups. The majority of adverse events were mild and all resolved without complications. Sapropterin dihydrochloride was well tolerated and reduced blood Phe levels across all PKU phenotypes tested. Variability in reduction of Phe indicates that the response to sapropterin dihydrochloride cannot be predicted by baseline Phe level.

Arginase induction by sodium phenylbutyrate in mouse tissues and human cell lines.

Hyperargininemia is a urea cycle disorder caused by mutations in the gene for arginase I (AI) resulting in elevated blood arginine and ammonia levels. Sodium phenylacetate and a precursor, sodium phenylbutyrate (NaPB) have been used to lower ammonia, conjugating glutamine to produce phenylacetylglutamine which is excreted in urine. The elevated arginine levels induce the second arginase (AII) in patient kidney and kidney tissue culture. It has been shown that NaPB increases expression of some target genes and we tested its effect on arginase induction. Eight 9-week old male mice fed on chow containing 7.5 g NaPB/kg rodent chow and drank water with 10 g NaPB/L, and four control mice had a normal diet. After one week all mice were sacrificed. The arginase specific activities for control and NaPB mice, respectively, were 38.2 and 59.4 U/mg in liver, 0.33 and 0.42 U/mg in kidney, and 0.29 and 1.19 U/mg in brain. Immunoprecipitation of arginase in each tissue with AI and AII antibodies showed the activity induced by NaPB is mostly AI. AII may also be induced in kidney. AI accounts for the fourfold increased activity in brain. In some cell lines, NaPB increased arginase activity up to fivefold depending on dose (1-5 mM) and exposure time (2-5 days); control and NaPB activities, respectively, are: erythroleukemia, HEL, 0.06 and 0.31 U/mg, and K562, 0.46 and 1.74 U/mg; embryonic kidney, HEK293, 1.98 and 3.58 U/mg; breast adenocarcinoma, MDA-MB-468, 1.11 and 4.06 U/mg; and prostate adenocarcinoma, PC-3, 0.55 and 3.20 U/mg. In MDA-MB-468 and HEK most, but not all, of the induced activity is AI. These studies suggest that NaPB may induce AI when used to treat urea cycle disorders. It is relatively less useful in AI deficiency, although it could have some effect in those patients with missense mutations.

Prenatal diagnosis for arginase deficiency: a case study.

Arginase deficiency is a rare, autosomal recessive, disorder of the urea cycle characterized by mild hyperammonaemia, hyperargininaemia, dibasic aminoaciduria and orotic aciduria, associated with progressive spastic tetraplegia, seizures, psychomotor retardation, and growth failure. We report a family who presented with their daughter at 4 years 11 months of age with an acute encephalopathy. Initial laboratory results revealed hyperammonaemia (160 micromol/L; normal 0-34), hyperargininaemia (512 micromol/L; normal 23-86) and orotic aciduria. A diagnosis of arginase deficiency was confirmed by enzyme assay, and treatment with a modified protein-restricted diet along with sodium benzoate therapy was initiated. Over time, intellectual development has been normal, but the child developed spasticity in her lower extremities. Subsequently, the mother presented at 6 weeks of pregnancy seeking prenatal diagnosis. Prenatal testing for arginase deficiency has only been reported in one other case. Arginase is not expressed in cultured amniotic fluid cells or chorionic villus samples. Testing for arginase activity assay in red blood cells, isolated by cordocentesis, was performed and predicted an unaffected fetus. The result was confirmed by postnatal enzyme analysis of red cells from the newborn. On the basis of our experience, prenatal diagnosis of arginase deficiency by cord red blood cell arginase activity assay appears possible.

Expression of arginase isozymes in mouse brain.

The two forms of arginase (AI and AII) in man, identical in enzymatic function, are encoded in separate genes and are expressed differentially in various tissues. AI is expressed predominantly in the liver cytosol and is thought to function primarily to detoxify ammonia as part of the urea cycle. AII, in contrast, is predominantly mitochondrial, is more widely expressed, and is thought to function primarily to produce ornithine. Ornithine is a precursor in the synthesis of proline, glutamate, and polyamines. This study was undertaken to explore the cellular and regional distribution of AI and AII expression in brain using in situ hybridization and immunohistochemistry. AI and AII were detected only in neurons and not in glial cells. AI presented stronger expression than AII, but AII was generally coexpressed with AI in most cells studied. Expression was particularly high in the cerebral cortex, cerebellum, pons, medulla, and spinal cord neurons. Glutamic acid decarboxylase 65 and glutamic acid decarboxylase 67, postulated to be related to the risk of glutamate excitotoxic and/or gamma-aminobutyric acid inhibitoxic injury, were similarly ubiquitous in their expression and generally paralleled arginase expression patterns, especially in cerebral cortex, hippocampus, cerebellum, pons, medulla, and spinal cord. This study showed that AI is expressed in the mouse brain, and more strongly than AII, and sheds light on the anatomic basis for the arginine-->ornithine-->glutamate-->GABA pathway.

Laboratory evaluation of urea cycle disorders.

The urea cycle disorders (UCDs) represent a group of inherited metabolic diseases with hyperammonemia as the primary laboratory abnormality. Affected individuals may become comatose or die if not treated rapidly. Diagnosis of a UCD requires a high index of suspicion and judicious use of the laboratory. It is important to rule out other conditions causing hyperammonemia that may require different treatment. The astute clinician may suspect a specific UCD in the appropriate clinical setting, but only laboratory results can confirm a specific diagnosis. The importance of the laboratory in helping the clinician to differentiate among various causes of hyperammonemia, in confirming a specific UCD, in carrier testing, and in prenatal diagnostic testing is highlighted in this review.

Psychosocial issues and coping strategies in families affected by urea cycle disorders.

A survey was sent to the American members of the National Urea Cycle Disorders Foundation to ascertain the types and extent of stress imposed on families who have a child with a urea cycle defect. Forty percent of the surveys were returned. The greatest sources of stress were financial, fear of death, and the restrictions imposed by the diet. Other than removal of the economic stress and uncertainty, the results did not suggest that any specific support systems required augmentation. Instructions to mitigate frustrations occurring in emergency situations would, however, be a great help to families.

Arginase activity in human breast cancer cell lines: N(omega)-hydroxy-L-arginine selectively inhibits cell proliferation and induces apoptosis in MDA-MB-468 cells.

L-Arginine is the common substrate for two enzymes, arginase and nitric oxide synthase (NOS). Arginase converts L-arginine to L-ornithine, which is the precursor of polyamines, which are essential components of cell proliferation. NOS converts L-arginine to produce NO, which inhibits proliferation of many cell lines. Various human breast cancer cell lines were initially screened for the presence of arginase and NOS. Two cell lines, BT-474 and MDA-MB-468, were found to have relatively high arginase activity and very low NOS activity. Another cell line, ZR-75-30, had the highest NOS activity and comparatively low arginase activity. The basal proliferation rates of MDA-MB-468 and BT-474 were found to be higher than the ZR-75-30 cell line. N-Hydroxy-L-arginine (NOHA), a stable intermediate product formed during conversion of L-arginine to NO, inhibited proliferation of the high arginase-expressing MDA-MB-468 cells and induced apoptosis after 48 h. NOHA arrested these cells in the S phase, increased the expression of p21, and reduced spermine content. These effects of NOHA were not observed in the ZR-75-30 cell line, which expresses high NOS and relatively low arginase. The effects of NOHA were antagonized in the presence of L-ornithine (500 microM), which suggests that in MDA-MB-468 cell line, the arginase pathway is very important for cell proliferation. Inhibition of the arginase pathway led to depletion of intracellular spermine and apoptosis as observed by terminal deoxynucleotidyl transferase (Tdt)-mediated nick end labeling assay and induction of caspase 3. In contrast, the ZR-75-30 cell line maintained its viability and its L-ornithine and spermine levels in the presence of NOHA. We conclude that NOHA has antiproliferative and apoptotic actions on arginase-expressing human breast cancer cells that are independent of NO.

Induction of arginase II in human Caco-2 tumor cells by cyclic AMP.

The objective of this study was to elucidate the mechanism by which cyclic AMP increases arginase activity in cultured human Caco-2 tumor cells. Caco-2 cells were incubated for 24 h in the presence of 8-bromo cyclic AMP or forskolin, and the cells were harvested, lysed, and assayed for total arginase activity. Both test agents increased arginase activity by twofold, and this was attributed to the induction of the arginase II isoform. Both arginase II mRNA and protein showed increased expression in response to 8-bromo cyclic AMP and forskolin, and these effects were inhibited by H-89 (protein kinase A inhibitor), enhanced by okadaic acid (phosphatase inhibitor), and enhanced by 1-methyl-3-isobutylxanthine (cyclic nucleotide phosphodiesterase inhibitor). Cyclic GMP did not appear to be involved in arginase II induction. These observations indicate that cyclic AMP stimulates arginase II gene expression by mechanisms involving activation of protein kinase A and consequent activation of appropriate transcription factors.

Molecular diagnosis and inborn errors of metabolism: a practitioner's view.

Recombinant DNA technology has altered completely the face of genetics and its clinical practice. cDNAs for enzymes involved in inborn errors were among the first cloned, but the impact of this advance on our subspecialty has been more limited. This discussion addresses the uses and "abuses" of recombinant DNA technology in the field, and areas of greater or lesser usefulness in current practice, and the future, are discussed.

Molecular basis of hyperargininemia: structure-function consequences of mutations in human liver arginase.

Hyperargininemia is a rare autosomal recessive disorder that results from a deficiency of hepatic type I arginase. At the genetic level, this deficiency in arginase activity is a consequence of random point mutations throughout the gene that lead to premature termination of the protein or to substitution mutations. Given the high degree of sequence homology between human liver and rat liver enzymes, we have mapped both patient and nonpatient mutations of the human enzyme onto the structure of the rat liver enzyme to rationalize the molecular basis for the low activities of these mutant arginases. Mutations identified in hyperargininemia patients affect the structure and function of the enzyme by compromising active-site residues, packing interactions in the protein scaffolding, and/or quaternary structure by destabilizing the assembly of the arginase trimer.

The human arginases and arginase deficiency.

Arginase is the final enzyme in the urea cycle. Its deficiency is the least frequently described disorder of this cycle. It results primarily in elevated blood arginine, and less frequently in either persistent or acute elevations in blood ammonia. This appears to be due to a second arginase locus, expressed primarily in the kidney, which can be recruited to compensate, in part, for the deficiency of liver arginase. The liver arginase gene structure permitted study of the molecular pathology of patients with the disorder and the results of these studies and the inferences about the protein structure are presented. The conserved regions among all arginases allowed the cloning of AII, the second arginase isoform. It has been localized to the mitochondrion and is thought to be involved in ornithine biosynthesis. It shares the major conserved protein sequences, and structural features of liver arginase gene are also conserved. When AI and AII from various species are compared, it appears that the two diverged some time prior to the evolution of amphibians. The evidence for the role of AII in nitric oxide and polyamine metabolism is presented and this appears consonant with the data on the tissue distribution.