De-imFAR phase II project: a study protocol for a cluster randomised implementation trial to evaluate the effectiveness of de-implementation strategies to reduce low-value statin prescribing in the primary prevention of cardiovascular disease.

INTRODUCTION: This study aims to reduce potentially inappropriate prescribing (PIP) of statins and foster healthy lifestyle promotion in cardiovascular disease (CVD) primary prevention in low-risk patients. To this end, we will compare the effectiveness and feasibility of several de-implementation strategies developed following the structured design process of the Behaviour Change Wheel targeting key determinants of the clinical decision-making process in CVD prevention. METHODS AND ANALYSIS: A cluster randomised implementation trial, with an additional control group, will be launched, involving family physicians (FPs) from 13 Integrated Healthcare Organisations (IHOs) of Osakidetza-Basque Health Service with non-zero incidence rates of PIP of statins in 2021. All FPs will be exposed to a non-reflective decision assistance strategy based on reminders and decision support tools. Additionally, FPs from two of the IHOs will be randomly assigned to one of two increasingly intensive de-implementation strategies: adding a decision information strategy based on knowledge dissemination and a reflective decision structure strategy through audit/feedback. The target population comprises women aged 45-74 years and men aged 40-74 years with moderately elevated cholesterol levels but no diagnosed CVD and low cardiovascular risk (REGICOR<7.5%), who attend at least one appointment with any of the participating FPs (May 2022-May 2023), and will be followed until May 2024. We use the Reach, Effectiveness, Adoption, Implementation and Maintenance (RE-AIM) framework to evaluate outcomes. The main outcome will be the change in the incidence rate of PIP of statins and healthy lifestyle counselling in the study population 12 and 24 months after FPs' exposure to the strategies. Moreover, FPs' perception of their feasibility and acceptability, and patient experience regarding the quality of care received will be evaluated. ETHICS AND DISSEMINATION: The study was approved by the Basque Country Clinical Research Ethics Committee and was registered in ClinicalTrials.gov (NCT04022850). Results will be disseminated in scientific peer-reviewed journals. TRIAL REGISTRATION NUMBER: NCT04022850.

Validity and reliability of the transculturally adapted Spanish version of the Implementation Leadership Scale (ILS).

BACKGROUND: There is a need for pragmatic and reliable measures of sound factors that affect evidence-based practice (EBP) adoption and implementation in different languages and cultural environments. The Implementation Leadership Scale (ILS) is a brief and efficient measurement tool of strategic leadership for EBP implementation. The objective of this study was to assess the psychometric properties of the Spanish version of the ILS. METHODS: The process of translation of the original ILS into Spanish consisted of forward translation, panel meeting, and back-translation. Scale face and content validity compared to that of the original version were assessed and ensured before agreement on the final version. Psychometric properties were examined in 144 healthcare professionals (family physicians, pediatricians, practice and pediatric nurses) involved in implementation or improvement research projects. ILS factor structure was tested by confirmatory factor analysis (CFA). Reliability was assessed by internal consistency analysis. The Pearson correlation between the ILS and the Organizational Support dimension of the Organizational Readiness for Knowledge Translation (OR4KT) questionnaire in the subsample of pediatricians and pediatric nurses (n = 52) was estimated for convergent validity analysis. RESULTS: The CFA results indicated that the original four theorized first-order factors with a second-order Implementation Leadership factor fit the data well (χ2 = 107.70; df = 45; p < 0.001). All standardized first- and second-order factor loadings were statistically significant. Fit indexes showed acceptable figures (GFI = 0.90; CFI = 0.97; RMSEA = 0.10; SRMR = 0.053). Cronbach's alpha coefficient for the four dimensions of ILS ranged from 0.90 to 0.97, while the reliability estimated for the total scale was 0.95. Results of convergent validity revealed high correlation (r = 0.56) between the ILS and the OR4KT's Organizational Support dimension. CONCLUSION: The CFA results demonstrated that the tested first- and second-order factor structure of the 12-item Spanish version of the ILS is consistent with the factor structure of the original tool. The availability of the ILS will allow Spanish-speaking researchers to assess and advance understanding of the implementation leadership construct as a predictor of organizational implementation context.

Applying the behavior change wheel to design de-implementation strategies to reduce low-value statin prescription in primary prevention of cardiovascular disease in primary care.

Introduction: A substantial proportion of individuals with low cardiovascular risk receive inappropriate statin prescription for primary prevention of cardiovascular disease (CVD) instead of the evidence-based recommendations to promote healthy lifestyle behaviors. This study reports on the structured process performed to design targeted de-implementation strategies to reduce inappropriate prescription of statins and to increase healthy lifestyle promotion in low cardiovascular risk patients in Primary Care (PC). Methods: A formative study was conducted based on the Theoretical Domains Framework and the Behavior Change Wheel (BCW). It comprised semi-structured interviews with PC professionals to define the problem in behavioral terms; focus groups with Family Physicians and patients to identify the determinants (barriers and facilitators) of inappropriate statin prescription and of healthy lifestyle promotion practice; mapping of behavioral change interventions operationalized as de-implementation strategies for addressing identified determinants; and consensus techniques for prioritization of strategies based on perceived effectiveness, feasibility and acceptability. Results: Identified key determinants of statin prescription and healthy lifestyle promotion were: the lack of time and clinical inertia, external resources, patients' preferences and characteristics, limitation of available clinical tools and guidelines, social pressures, fears about negative consequences of not treating, and lack of skills and training of professionals. Fourteen potential de-implementation strategies were mapped to the identified determinants and the following were prioritized: 1) non-reflective decision assistance strategies based on reminders and decision support tools for helping clinical decision-making; 2) decision information strategies based on the principles of knowledge dissemination (e.g., corporative diffusion of evidence-based Clinical Practice Guidelines and Pathways for CVD primary prevention); 3) reflective decision-making restructuring strategies (i.e., audit and feedback provided along with intention formation interventions). Conclusions: This study supports the usefulness of the BCW to guide the design and development of de-implementation strategies targeting the determinants of clinicians' decision-making processes to favor the abandonment of low-value practices and the uptake of those recommended for CVD primary prevention in low-risk patients. Further research to evaluate the feasibility and effectiveness of selected strategies is warranted. Clinical trial registration: Sanchez A. De-implementation of Low-value Pharmacological Prescriptions (De-imFAR). ClinicalTrials.gov, Identifier: NCT04022850. Registered July 17, 2019. In: ClinicalTrials.gov. Bethesda (MD): U.S. National Library of Medicine (NLM). Available from: https://www.clinicaltrials.gov/ct2/show/NCT04022850.

Profile of sodium phenylbutyrate granules for the treatment of urea-cycle disorders: patient perspectives.

Urea-cycle disorders are a group of rare hereditary metabolic diseases characterized by deficiencies of one of the enzymes and transporters involved in the urea cycle, which is necessary for the removal of nitrogen produced from protein breakdown. These hereditary metabolic diseases are characterized by hyperammonemia and life-threatening hyperammonemic crises. Pharmacological treatment of urea-cycle disorders involves alternative nitrogen-scavenging pathways. Sodium benzoate combines with glycine and phenylacetate/phenylbutyrate with glutamine, forming, respectively, hippuric acid and phenylacetylglutamine, which are eliminated in the urine. Among the ammonia-scavenging drugs, sodium phenylbutyrate is a well-known long-term treatment of urea-cycle disorders. It has been used since 1987 as an investigational new drug, and was approved for marketing in the US in 1996 and the EU in 1999. However, sodium phenylbutyrate has an aversive odor and taste, which may compromise patients' compliance, and many patients have reported difficulty in taking this drug. Sodium phenylbutyrate granules are a new tasteless and odor-free formulation of sodium phenylbutyrate, which is indicated in the treatment of urea-cycle disorders. This recently developed taste-masked formulation of sodium phenylbutyrate granules was designed to overcome the considerable issues that taste has on adherence to therapy. Several studies have reported the clinical experience of patients with urea-cycle disorders treated with this new tasteless formulation of sodium phenylbutyrate. Analysis of the data indicated that this taste-masked formulation of sodium phenylbutyrate granules improved quality of life for urea-cycle disorder patients. Furthermore, a postmarketing report on the use of the product has confirmed the previous observations of improved compliance, efficacy, and safety with this taste-masked formulation of sodium phenylbutyrate.

Influence of phenylketonuria's diet on dimethylated arginines and methylation cycle.

Phenylketonuria's (PKU) treatment based on low natural protein diet may affect homocysteine (Hcys) metabolic pathway. Hcys alteration may be related to the methylation of arginine to asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA), which both modify nitric oxide production. The aim of this work is to evaluate the status of Hcys formation methylation cycle and ADMA and SDMA levels in patients with PKU in order to establish a potential relationship.Forty-two early diagnosed PKU patients under dietary treatment and good adherence to their diets were enrolled in this cross-sectional study. Their nutritional and biochemical profile, as well as Hcys synthesis status, ADMA and SDMA levels were analyzed and compared with a control group of 40 healthy volunteers. ADMA and SDMA were determined by high-performance liquid chromatography system coupled to triple quadrupole mass spectrometer.In this study, 23 classic PKU, 16 moderate PKU, and 3 mild HPA were enrolled. The median age was 10 years old. Median ADMA, SDMA, and Hcys concentration levels (5.1 µM [2.3-25.7], 0.35 µM [0.18-0.57], 0.43 µM [0.26-0.61], respectively) were lower in patients with PKU (P < .001 for ADMA and SDMA) whereas vitamin B12 and folate levels (616 pg/mL [218-1943] and 21 ng/mL [5-51], respectively) were higher comparing with controls. Statistically significant correlations were found between ADMA, and Phe (r = -0.504, P = .001) and Hcys (r = -0.458, P = .037) levels. Several nutrition biomarkers, such as prealbumin, 25-hydroxy vitamin D, selenium, and zinc, were below the normal range.Our study suggests that patients with PKU suffer from poor methylation capacity. Restriction of natural proteins in addition to high intake of vitamin B12 and folic acid supplementation in the dietary products, produce an impairment of methylation cycle that leads to low Hcys and ADMA levels. As a result, methylated compounds compete for methyl groups, and there is an impairment of methylation cycle due to low Hcys levels, which is related to the lack of protein quality, despite of elevated concentrations of cofactors.

Lipid profile status and other related factors in patients with Hyperphenylalaninaemia.

BACKGROUND: The mainstay of treating patients with phenylketonuria (PKU) is based on a Phe-restricted diet, restrictive in natural protein combined with Phe-free L-amino acid supplements and low protein foods. This PKU diet seems to reduce atherogenesis and confer protection against cardiovascular diseases but the results from the few published studies have been inconclusive. The aim of our study was to evaluate the relationship between the lipid profile and several treatment-related risk factors in patients with hyperphenylalaninaemia (HPA) in order to optimize their monitoring. METHODS: We conducted a cross-sectional multicentre study. A total of 141 patients with HPA were classified according to age, phenotype, type of treatment and dietary adherence. Annual median blood phenylalanine (Phe) levels, Phe tolerance, anthropometric measurements, blood pressure (BP) and biochemical parameters [(triglycerides, total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low density lipoprotein-cholesterol (LDL-C), apolipoprotein A (ApoA), apolipoprotein B (ApoB), vitamin B12, total homocysteine (tHcy), Methionine (Met), high sensitivity C-Reactive Protein (hsCRP)] were collected for each patient. RESULTS: Plasma TC levels were lower in patients with PKU than in the mild-HPA group (150 ± 31 vs. 164 ± 22 mg/dL), and there was a weak inverse correlation between plasma TC and Phe levels. HDL-C, LDL-C, ApoA and ApoB levels were lower in the PKU group than in mild-HPA. Patients with PKU had higher systolic BP than the mild-HPA group and there was found a quadratic correlation between median Phe levels and systolic BP (p = 6.42e(-5)) and a linear correlation between median Phe levels and diastolic BP (p = 5.65e(-4)). In overweight or obese PKU patients (24.11 %), biochemical parameters such as TC, triglycerides, LDL-C, tHcy, hsCRP and BP were higher. By contrast, HDL-C was lower in these patients. CONCLUSION: Our data show a direct correlation between lipid profile parameters and good adherence to the diet in PKU patients. However, lipid profile in overweight or obese patients displayed an atherogenic profile, in addition to higher hsCRP concentrations and BP. Our study contributes to a better understanding of the relationship between phenotype and treatment in patients with HPA, which could be useful in improving follow-up strategies and clinical outcome. TRIAL REGISTRATION: Research Ethics Committee of Santiago-Lugo 2015/393. Registered 22 September 2015, retrospectively registered.

Potential renoprotective effects of piceatannol in ameliorating the early-stage nephropathy associated with obesity in obese Zucker rats

Obesity-associated nephropathy is considered to be a leading cause of end-stage renal disease. Resveratrol supplementation represents a promising therapy to attenuate kidney injury, but the poor solubility and limited bioavailability of this polyphenol limits its use in dietary intervention. Piceatannol, a resveratrol analogue, has been suggested as a better option. In this study, we aimed to provide evidence of a preventive action of piceatannol in very early stages of obesity-associated nephropathy. Thirty obese Zucker rats were divided into three experimental groups: one control and two groups orally treated for 6 weeks with 15 and 45 mg piceatannol/kg body weight/day. Enzyme-linked immunosorbent assays (ELISA) were used to determine renal and urinary kidney injury molecule-1 (Kim-1), renal fibrosis markers (transforming growth factor β1 and fibronectin) and renal sirtuin-1 protein. Oxidative stress was assessed in the kidney by measuring lipid peroxidation and nitrosative stress (thiobarbituric acid reactive substrates and 3-nitrotyrosine levels, respectively) together with the activity of the antioxidant enzyme superoxide dismutase. Renal fatty acids profile analysis was performed by thin-layer and gas chromatography. Piceatannol-treated rats displayed lower levels of urinary and renal Kim-1. Renal fibrosis biomarkers and lipid peroxidation exhibited a tendency to decrease in the piceatannol-treated groups. Piceatannol treatment did not modify superoxide dismutase activity or sirtuin-1 protein levels, while it seemed to increase the levels of polyunsaturated and omega-6 polyunsaturated fatty acids in the kidneys. Our findings suggest a mild renoprotective effect of piceatannol in obese Zucker rats and the need of intervention at early stages of renal damage (AU)

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Molecular epidemiology, genotype-phenotype correlation and BH4 responsiveness in Spanish patients with phenylketonuria.

Phenylketonuria (PKU), the most common inborn error of amino acid metabolism, is caused by mutations in the phenylalanine-4-hydroxylase (PAH) gene. This study aimed to assess the genotype-phenotype correlation in the PKU Spanish population and the usefulness in establishing genotype-based predictions of BH4 responsiveness in our population. It involved the molecular characterization of 411 Spanish PKU patients: mild hyperphenylalaninemia non-treated (mild HPA-NT) (34%), mild HPA (8.8%), mild-moderate (20.7%) and classic (36.5%) PKU. BH4 responsiveness was evaluated using a 6R-BH4 loading test. We assessed genotype-phenotype associations and genotype-BH4 responsiveness in our population according to literature and classification of the mutations. The mutational spectrum analysis showed 116 distinct mutations, most missense (70.7%) and located in the catalytic domain (62.9%). The most prevalent mutations were c.1066-11G>A (9.7%), p.Val388Met (6.6%) and p.Arg261Gln (6.3%). Three novel mutations (c.61-13del9, p.Ile283Val and p.Gly148Val) were reported. Although good genotype-phenotype correlation was observed, there was no exact correlation for some genotypes. Among the patients monitored for the 6R-BH4 loading test: 102 were responders (87, carried either one or two BH4-responsive alleles) and 194 non-responders (50, had two non-responsive mutations). More discrepancies were observed in non-responders. Our data reveal a great genetic heterogeneity in our population. Genotype is quite a good predictor of phenotype and BH4 responsiveness, which is relevant for patient management, treatment and follow-up.

Potential renoprotective effects of piceatannol in ameliorating the early-stage nephropathy associated with obesity in obese Zucker rats.

Obesity-associated nephropathy is considered to be a leading cause of end-stage renal disease. Resveratrol supplementation represents a promising therapy to attenuate kidney injury, but the poor solubility and limited bioavailability of this polyphenol limits its use in dietary intervention. Piceatannol, a resveratrol analogue, has been suggested as a better option. In this study, we aimed to provide evidence of a preventive action of piceatannol in very early stages of obesity-associated nephropathy. Thirty obese Zucker rats were divided into three experimental groups: one control and two groups orally treated for 6 weeks with 15 and 45 mg piceatannol/kg body weight/day. Enzyme-linked immunosorbent assays (ELISA) were used to determine renal and urinary kidney injury molecule-1 (Kim-1), renal fibrosis markers (transforming growth factor ß1 and fibronectin) and renal sirtuin-1 protein. Oxidative stress was assessed in the kidney by measuring lipid peroxidation and nitrosative stress (thiobarbituric acid reactive substrates and 3-nitrotyrosine levels, respectively) together with the activity of the antioxidant enzyme superoxide dismutase. Renal fatty acids profile analysis was performed by thin-layer and gas chromatography. Piceatannol-treated rats displayed lower levels of urinary and renal Kim-1. Renal fibrosis biomarkers and lipid peroxidation exhibited a tendency to decrease in the piceatannol-treated groups. Piceatannol treatment did not modify superoxide dismutase activity or sirtuin-1 protein levels, while it seemed to increase the levels of polyunsaturated and omega-6 polyunsaturated fatty acids in the kidneys. Our findings suggest a mild renoprotective effect of piceatannol in obese Zucker rats and the need of intervention at early stages of renal damage.

Dimethylarginines as biomarkers for the kidney transplant management in methylmalonic aciduria.

Methylmalonic aciduria (MMA) is an inborn error of metabolism associated with many complications despite treatment. Chronic renal failure is the most common problem, and patients may eventually require kidney transplant. Therefore, it is worth investigating whether living donor kidney transplant offers a better option than deceased kidney donors; and the value of novel vascular risk biomarkers in the assessment of transplanted MMA patients. We report a case of a 26-year-old man with MMA, who progressed to end-stage renal disease and received kidney transplant from a heterozygous next-of-kin living donor at 20 years of age. Although post-transplant urinary levels of methylmalonic acid decreased, this reduction was lower than previously reported for deceased donors. No episodes of metabolic decompensation were observed after transplantation. During his clinical progress, vascular complications appeared; and finally, pancreatitis was the cause of death. After kidney transplant, we evaluated novel vascular risk factors, such as asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA), which were used as early biomarkers of progression and metabolic management for this transplanted patient. This case report illustrates the disadvantage of transplantation with an allograft from a heterozygous living donor, and the utility of vascular risk biomarkers in renal transplant assessment.

Sanfilippo syndrome: Overall review.

Mucopolysaccharidosis type III (MPS III, Sanfilippo syndrome) is a lysosomal storage disorder, caused by a deficiency in one of the four enzymes involved in the catabolism of glycosaminoglycan heparan sulfate. It is characterized by progressive cognitive decline and severe hyperactivity, with relatively mild somatic features. This review focuses on clinical features, diagnosis, treatment, and follow-up of MPS III, and provides information about supplementary tests and differential diagnosis. Given that few reviews of MPS III have been published, several studies were compiled to establish diagnostic recommendations. Quantitative urinary glycosaminoglycan analysis is strongly recommended, and measurement of disaccharides, heparin cofactor II-thrombin complex and gangliosides is also used. Enzyme activity of the different enzymes in blood serum, leukocytes or fibroblasts, and mutational analysis for SGSH, NAGLU, HGSNAT or GNS genes are required to confirm diagnosis and differentiate four subtypes of MPS III. Although there is no global consensus for treatment, enzyme replacement therapy and gene therapy can provide appropriate results. In this regard, recent publications on treatment and follow-up are discussed.

6R-tetrahydrobiopterin treated PKU patients below 4 years of age: Physical outcomes, nutrition and genotype.

BACKGROUND AND AIMS: Phenylalanine-restricted diets have proven effective in treating phenylketonuria. However, such diets have occasionally been reported to hinder normal development. Our study aimed to assess whether treating 0-4-year-old phenylketonuric patients with 6R-tetrahydrobiopterin might prevent growth retardation later in life. METHODS: We conducted a longitudinal retrospective study which examined anthropometric characteristics of phenylketonuric patients on 6R-tetrahydrobiopterin therapy (22 subjects), and compared them with a group of phenylketonuric patients on protein-restricted diets (44 subjects). Nutritional issues were also considered. We further explored possible relationships between mutations in the PAH gene, BH4 responsiveness and growth outcome. RESULTS: No significant growth improvements were observed in either the group on 6R-tetrahydrobiopterin treatment (height Z-score: initial= -0.57 ± 1.54; final=-0.52 ± 1.29; BMI Z-score: initial=0.17 ± 1.05; final=0.18 ± 1.00) or the diet-only group (height Z-score: initial=-0.92 ± 0.96; final= -0.78 ± 1.08; BMI Z-score: initial=0.17 ± 0.97; final=-0.07 ± 1.03) over the 1-year observation period. Furthermore, we found no significant differences (p>0.05) between the two groups at any of the time points considered (0, 6 and 12 months). Patients on 6R-tetrahydrobiopterin increased their phenylalanine intake (from 49.1 [25.6-60.3] to 56.5 [39.8-68.3] mgkg(-1)day(-1)) and natural protein intake (from 1.0 [0.8-1.7] to 1.5 [1.0-1.8] g kg(-1)day(-1)), and some patients managed to adopt normal diets. Higher phenylalanine and natural protein intakes were positively correlated with better physical outcomes in the diet-only group (p<0.05). No correlation was found between patient genotype and physical outcomes, results being similar regardless of the nutritional approach used. We did not detect any side effects due to 6R-tetrahydrobiopterin administration. CONCLUSIONS: Our study indicates that treating 0-4-year-old phenylketonuric patients with 6R-tetrahydrobiopterin is safe. However, poor developmental outcomes were observed, despite increasing the intake of natural proteins. Genotype could be a valid predictor of tetrahydrobiopterin-responsiveness, since patients who carried the same genotype responded similarly to the 6R-tetrahydrobiopterin loading test. On the other hand, harbouring 6R-tetrahydrobiopterin responsive genotypes did not predispose patients to better physical outcomes.

Quantification of arginine and its methylated derivatives in healthy children by liquid chromatography-tandem mass spectrometry.

Asymmetric dimethylarginine (ADMA) is a competitive inhibitor of nitric oxide synthase, which is responsible for most of the vascular nitric oxide (NO) produced. NO is an important physiological mediator of vascular tone and structure in normally functioning endothelial cells. We report the optimization of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous determination of arginine (Arg) and its derivatives in biological samples. Chromatographic separation and mass detection were performed by reverse phase chromatography coupled with tandem mass spectrometry. For sample preparation, plasma proteins were removed by centrifugal filters. Positive electrospray ionization was performed and analytes were detected by multiple reaction monitoring. Inter- and intra-day repeatability, accuracy, recovery, and limits of detection and quantification were evaluated to validate the method. Plasma and urine levels were measured in healthy children to establish control values: 52.2-124.7 µM for Arg, 0.06-0.16 µM for MMA, 0.42-1.10 µM for ADMA and 0.41-0.96 µM for SDMA in plasma. Quantification of Arg and its methylated derivatives by LC-MS/MS can be carried out without the need of organic solvents for sample preparation, and be used as a valuable tool in research on endothelial dysfunction.

A new case of maternal phenylketonuria treated with sapropterin dihydrochloride (6R-BH4).

PURPOSE: A woman with phenylketonuria (PKU) was diagnosed through neonatal screening, her PAH mutation was p.V388M/p.I65T, for which she received treatment with phenylalanine restriction, and was administered oral sapropterin dihydrochloride (6R-BH(4)) from the age of thirty. The purpose of this article is to describe the treatment with BH4 during her pregnancy and to evaluate a plan for its use. METHODS: The patient had an unplanned pregnancy at 34 years of age, for which she received a phenylalanine-free supplement enriched with essential fatty acids, vitamins and trace elements. RESULTS: The dose of 6R-BH(4) was reduced from 500 mg/day to 100 mg/day until its suspension in the 28th week of gestation, and was well tolerated. Blood phenylalanine control was easily accomplished during this pregnancy, and no nutritional deficiency was seen. CONCLUSION: The pregnancy had a normal outcome, and so we consider that adaptation of the dose of 6R-BH(4) to the prenatal periods aided a greater efficiency and a lower risk in the treatment of maternal PKU.

Tetrahydrobiopterin therapy vs phenylalanine-restricted diet: impact on growth in PKU.

BACKGROUND: Treatment of phenylketonuria based upon strict vegetarian diets, with very low phenylalanine intake and supplemented by phenylalanine-free formula, has proven to be effective in preventing the development of long-term neurological sequelae due to phenylalanine accumulation. On the other hand, such diets have occasionally been reported to hinder normal development, some individuals presenting with growth retardation. Tetrahydrobiopterin therapy has opened up new treatment options for a significant proportion of phenylketonuric patients, enabling them to eat normal diets and be freed from the need to take synthetic supplements. However, little is known about how this therapy affects their physical development. METHODS: We conducted a retrospective longitudinal study examining anthropometric characteristics (height, weight, body mass index and growth speed Z-scores) in a cohort of phenylketonuric patients on tetrahydrobiopterin therapy (38 subjects) comparing their characteristics with those of a group of phenylketonuric patients on phenylalanine-restricted diets (76 subjects). Nutritional issues were also considered, to further explore the possibility of higher natural protein intake being associated with better physical development. Data were collected every six months over two different periods of time (two or five years). RESULTS: No improvement was observed in the aforementioned anthropometric variables in the cohort on tetrahydrobiopterin therapy, from prior to starting treatment to when they had been taking the drug for two or five years. Rather, in almost all cases there was a fall in the mean Z-score for the variables during these periods, although the changes were not significant in any case. Further, we found no statistically differences between the two groups at any considered time point. Growth impairment was also noted in the phenylketonuric patients on low-phenylalanine diets. Individuals on tetrahydrobiopterin therapy increased their natural protein intake and, in some instances, this treatment enabled individuals to eat normal diets, with protein intake meeting RDAs. No association was found, however, between higher protein intake and growth. CONCLUSION: Our study identified growth impairment in patients with phenylketonuria on tetrahydrobiopterin, despite higher intakes of natural proteins. In fact, individuals undergoing long-term tetrahydrobiopterin treatment seemed to achieve similar developmental outcomes to those attained by individuals on more restricted diets.

An orchestrated program regulating secretory pathway genes and cargos by the transmembrane transcription factor CREB-H.

CREB3 proteins comprise a set of ER-localized bZip transcription factors defined by the presence of a transmembrane domain. They are regulated by inter-compartmental transport, Golgi cleavage and nuclear transport where they promote appropriate transcriptional responses. Although CREB3 proteins play key roles in differentiation, inflammation and metabolism, a general framework relating their defining features to these diverse activities is lacking. We identify unique features of CREB3 organization including the ATB domain, which we show it is essential for transcriptional activity. This domain is absent in all other human bZip factors, but conserved in Drosophila CREBA, which controls secretory pathway genes (SPGs). Furthermore, each of the five human CREB3 factors was capable of activating SPGs in Drosophila, dependent upon the ATB domain. Expression of the CREB3 protein, CREB-H, in 293 cells, upregulated genes involved in secretory capacity, extracellular matrix formation and lipid metabolism and increased secretion of specific cargos. In liver cells, which normally express CREB-H, the active form specifically induced secretion of apolipoproteins, including ApoA-IV, ApoAI, consistent with data implicating CREB-H in metabolic homeostasis. Based on these data and other recent studies, we propose a general role for the CREB3 family in regulating secretory capacity, with particular relevance to specialized cargos.

Different mechanisms of recognition and ER retention by transmembrane transcription factors CREB-H and ATF6.

CREB-H and activating transcription factor 6 (ATF6) are transmembrane transcription factors that, in response to endoplasmic reticulum (ER) stress, traffic to the Golgi where they are cleaved by specific proteases, producing the N-terminal domains that effect appropriate transcriptional responses. We show that unlike in ATF6 whose lumenal tail binds BiP and contains determinants for stress sensing and Golgi transport, in CREB-H the lumenal tail is not involved in ER retention, not required for Golgi transport and does not bind BiP. The main determinant for CREB-H ER retention resides in a membrane-proximal cytoplasmic determinant that is conserved in related members of the CREB-H family, but lacking in ATF6. We refine requirements within the ER-retention motif (ERM) and show that ERM-ve variants exhibited constitutive Golgi localization and constitutive cleavage by the Golgi protease, S1P. The ERM also conferred ER retention on a heterologous protein. Furthermore, deletion of the lumenal tail of CREB-H had no effect on ER retention of parental CREB-H or Golgi localization of ERM-ve variants. Importantly, when the lumenal tail of ATF6 was transferred into an ERM-ve variant, the chimera was now retained in the ER. Together, these data demonstrate novel and qualitatively distinct mechanisms of trafficking and stress signalling in CREB-H compared to ATF6.

Purification and properties of NrtC and NrtD, the ATP-binding subunits of the ABC nitrate/nitrite transporter of Phormidium laminosum.

A genomic region from the thermophilic, filamentous, nondiazotrophic cyanobacterium Phormidium laminosum including nrtC and nrtD was cloned and sequenced. These genes encode NrtC and NrtD, the ATP-binding subunits of the ABC bispecific transporter of nitrate/nitrite NRT. We report a different nrtC sequence from the one previously reported (Merchán et al., Plant Mol. Biol. 28:759-766, 1995) and we identified the presence of nrtD gene downstream nrtC in the nirA operon. Each gene was expressed in E. coli cells as a hexahistidine-tagged fusion protein. The recombinant proteins (His(6)NrtC and His(6)NrtD) were purified, and their ability to catalyze the hydrolysis of ATP and other nucleosides triphosphate was characterized. Both subunits showed its maximum ATPase activity at 45-50 degrees C and pH 8.0, and similar K(m) (0.49 and 0.43 mM) and V(max) (0.085 and 0.114 U mg(-1) protein, respectively) values were calculated. The native NrtC subunit purified from nitrogen-starved cells of P. laminosum also hydrolyzed ATP in vitro in the absence of other components of NRT. These findings indicated that NrtC and NrtD are responsible for ATP-hydrolysis to energize the active transporter NRT. The effect of some activators (Mg(2+)) and inhibitors (ADP) on the ATPase activity of the subunits was assessed as well as the effect of some potential regulatory metabolites on His(6)NrtC. The existence in vitro of homodimers of either NrtC or NrtD but not heterodimers of both subunits was confirmed by matrix assisted laser desorption ionization-time of flight mass spectrometry and/or electrophoresis in non-denaturing conditions. Finally, the existence in vivo of NrtC-NrtD heterodimers is discussed.

The nitrate/nitrite ABC transporter of Phormidium laminosum: phosphorylation state of NrtA is not involved in its substrate binding activity.

Most cyanobacteria take up nitrate or nitrite through a multisubunit ABC transporter (ATP-binding cassette) located in the cytoplasmic membrane. Nitrate and nitrite transport activity is instantaneously blocked by the presence of ammonium in the medium. Previous biochemical studies reported the existence of phosphorylation/dephosphorylation events of the nitrate transporter (NRT) related to the presence of ammonium-sensitive kinase/phosphatase activities in plasma membranes of the cyanobacterium Synechococcus elongatus PCC 6301. In this work, we have analyzed the biochemical properties of the periplasmic nitrate/nitrite-binding subunit (NrtA) of NRT from the thermophilic nondiazotrophic cyanobacterium Phormidium laminosum. Our results show that cyanobacterial NrtA is phosphorylated in vivo. However, substrate binding activity in vitro is not affected by the phosphorylation state of the protein, ruling out the possibility that phosphorylation/dephosphorylation of NrtA is involved in the regulation of the nitrate/nitrite uptake by NRT transporter. Moreover, NrtA is present as multiple isoforms showing the same molecular mass but different isoelectric points ranging from pI 5 to 6. Mass spectrometric characterization of NrtA isoforms shows that the protein is phosphorylated at residue Tyr203, and contains several methionine sulphoxide residues which account for the observed isoforms. Both phosphorylated and non-phosphorylated forms of NrtA are active in vitro, showing comparable binding affinity for nitrate and nitrite. Both substrates behave as pure competitive inhibitors with a binding stoichiometry of one molecule of anion per NrtA monomer.