Lactose and Galactose Content in Spanish Cheeses: Usefulness in the Dietary Treatment of Patients with Galactosaemia.

In galactosaemia, a strict galactose-free diet is necessary to prevent or resolve acute symptoms in infants. However, because the body produces up to 10 times more galactose than is found in a galactose-restricted diet, excessively restrictive diets should be avoided in children and adults to prevent nutritional deficiencies. Since cheese is a nutritional source of the calcium necessary for bone health, the latest international guidelines on the management of classical galactosaemia (2017) allow the consumption of cured cheeses with less than 25 mg of galactose/100 g and recommend that each country verifies the adequacy of the cheeses, since most mature cheeses do not always have a lower galactose content. In total, 32 cheese samples were purchased (19 Spanish and 13 international cheeses), and their lactose and galactose contents were analysed using ion chromatography with pulsed amperometric detection (IC-PAD). Five Spanish cheeses contained less than 25 mg of galactose/100 g: García Baquero semi-cured cheese; Hacendado, Gran Reserva and Mahón cured cheeses; and García Baquero Reserva 12-month cured cheese. In addition, eight international cheeses were confirmed as suitable: Comté, Gouda, Gruyere, Maasdam, Parmigiano, Edam, Emmental, and some samples of Cheddar. In addition to the well-known low-galactose Swiss and Dutch cheeses, according to the current results, five Spanish cheeses can be safely consumed. The greater availability of types of cheese favours better bone health in patients with galactosaemia.

Identification of Clinical Variants beyond the Exome in Inborn Errors of Metabolism.

Inborn errors of metabolism (IEM) constitute a huge group of rare diseases affecting 1 in every 1000 newborns. Next-generation sequencing has transformed the diagnosis of IEM, leading to its proposed use as a second-tier technology for confirming cases detected by clinical/biochemical studies or newborn screening. The diagnosis rate is, however, still not 100%. This paper reports the use of a personalized multi-omics (metabolomic, genomic and transcriptomic) pipeline plus functional genomics to aid in the genetic diagnosis of six unsolved cases, with a clinical and/or biochemical diagnosis of galactosemia, mucopolysaccharidosis type I (MPS I), maple syrup urine disease (MSUD), hyperphenylalaninemia (HPA), citrullinemia, or urea cycle deficiency. Eight novel variants in six genes were identified: six (four of them deep intronic) located in GALE, IDUA, PTS, ASS1 and OTC, all affecting the splicing process, and two located in the promoters of IDUA and PTS, thus affecting these genes' expression. All the new variants were subjected to functional analysis to verify their pathogenic effects. This work underscores how the combination of different omics technologies and functional analysis can solve elusive cases in clinical practice.

Switching to Glycerol Phenylbutyrate in 48 Patients with Urea Cycle Disorders: Clinical Experience in Spain.

Background and objectives: Glycerol phenylbutyrate (GPB) has demonstrated safety and efficacy in patients with urea cycle disorders (UCDs) by means of its clinical trial program, but there are limited data in clinical practice. In order to analyze the efficacy and safety of GPB in clinical practice, here we present a national Spanish experience after direct switching from another nitrogen scavenger to GPB. Methods: This observational, retrospective, multicenter study was performed in 48 UCD patients (age 11.7 ± 8.2 years) switching to GPB in 13 centers from nine Spanish regions. Clinical, biochemical, and nutritional data were collected at three different times: prior to GPB introduction, at first follow-up assessment, and after one year of GPB treatment. Number of related adverse effects and hyperammonemic crisis 12 months before and after GPB introduction were recorded. Results: GPB was administered at a 247.8 ± 102.1 mg/kg/day dose, compared to 262.6 ± 126.1 mg/kg/day of previous scavenger (46/48 Na-phenylbutyrate). At first follow-up (79 ± 59 days), a statistically significant reduction in ammonia (from 40.2 ± 17.3 to 32.6 ± 13.9 µmol/L, p < 0.001) and glutamine levels (from 791.4 ± 289.8 to 648.6 ± 247.41 µmol/L, p < 0.001) was observed. After one year of GPB treatment (411 ± 92 days), we observed an improved metabolic control (maintenance of ammonia and glutamine reduction, with improved branched chain amino acids profile), and a reduction in hyperammonemic crisis rate (from 0.3 ± 0.7 to less than 0.1 ± 0.3 crisis/patients/year, p = 0.02) and related adverse effects (RAE, from 0.5 to less than 0.1 RAEs/patients/year p < 0.001). Conclusions: This study demonstrates the safety of direct switching from other nitrogen scavengers to GPB in clinical practice, which improves efficacy, metabolic control, and RAE compared to previous treatments.

Vitamin C and folate status in hereditary fructose intolerance.

BACKGROUND: Hereditary fructose intolerance (HFI) is a rare inborn error of fructose metabolism caused by the deficiency of aldolase B. Since treatment consists of a fructose-, sucrose- and sorbitol-restrictive diet for life, patients are at risk of presenting vitamin deficiencies. Although there is no published data on the status of these vitamins in HFI patients, supplementation with vitamin C and folic acid is common. Therefore, the aim of this study was to assess vitamin C and folate status and supplementation practices in a nationwide cohort of HFI patients. METHODS: Vitamin C and folic acid dietary intake, supplementation and circulating levels were assessed in 32 HFI patients and 32 age- and sex-matched healthy controls. RESULTS: Most of the HFI participants presented vitamin C (96.7%) and folate (90%) dietary intake below the recommended population reference intake. Up to 69% received vitamin C and 50% folic acid supplementation. Among HFI patients, 15.6% presented vitamin C and 3.1% folate deficiency. The amount of vitamin C supplementation and plasma levels correlated positively (R = 0.443; p = 0.011). Interestingly, a higher percentage of non-supplemented HFI patients were vitamin C deficient when compared to supplemented HFI patients (30% vs. 9.1%; p = 0.01) and to healthy controls (30% vs. 3.1%; p < 0.001). CONCLUSIONS: Our results provide evidence for the first time supporting vitamin C supplementation in HFI. There is great heterogeneity in vitamin supplementation practices and, despite follow-up at specialised centres, vitamin C deficiency is common. Further research is warranted to establish optimal doses of vitamin C and the need for folic acid supplementation in HFI.

Lymphocyte Medium-Chain Acyl-CoA Dehydrogenase Activity and Its Potential as a Diagnostic Confirmation Tool in Newborn Screening Cases.

The determination of acylcarnitines (AC) in dried blood spots (DBS) by tandem mass spectrometry in newborn screening (NBS) programs has enabled medium-chain acyl-coA dehydrogenase deficiency (MCADD) to be identified in presymptomatic newborns. Nevertheless, different confirmatory tests must be performed to confirm the diagnosis. In this work, we have collected and analyzed the NBS results and confirmatory test results (plasma AC, molecular findings, and lymphocyte MCAD activity) of forty individuals, correlating them with clinical outcomes and treatment, with the aim of obtaining useful diagnostic information that could be applied in the follow-up of the patients. Our results led us to classify patients into two groups. The first group (14 cases) had high increased octanoylcarnitine (C8) levels, biallelic pathogenic variants, and severe impaired enzyme activity (<10% of the intra-assay control (IAC)); all of these cases received nutritional therapy and required carnitine supplementation during follow-up, representing the most severe form of the disease. The second group (16 patients) was a heterogeneous group presenting moderate increases in C8, biallelic likely pathogenic/pathogenic variants, and intermediate activity (<41% IAC). All of them are currently asymptomatic and could be considered as having a milder form of the disease. Finally, eight cases presented a normal−mild increase in plasma C8, with only one pathogenic variant detected, and high−intermediate residual activity (15−100%). Based on our results, we confirm that combined evaluation of acylcarnitine profiles, genetic findings, and residual enzyme activities proves useful in predicting the risk of future metabolic decompensation, in making decisions regarding future treatment or follow-up, and also in confirming the clinical effects of unknown clinical variants.

Transferrin Isoforms, Old but New Biomarkers in Hereditary Fructose Intolerance.

Hereditary Fructose Intolerance (HFI) is an autosomal recessive inborn error of metabolism characterised by the deficiency of the hepatic enzyme aldolase B. Its treatment consists in adopting a fructose-, sucrose-, and sorbitol (FSS)-restrictive diet for life. Untreated HFI patients present an abnormal transferrin (Tf) glycosylation pattern due to the inhibition of mannose-6-phosphate isomerase by fructose-1-phosphate. Hence, elevated serum carbohydrate-deficient Tf (CDT) may allow the prompt detection of HFI. The CDT values improve when an FSS-restrictive diet is followed; however, previous data on CDT and fructose intake correlation are inconsistent. Therefore, we examined the complete serum sialoTf profile and correlated it with FSS dietary intake and with hepatic parameters in a cohort of paediatric and adult fructosemic patients. To do so, the profiles of serum sialoTf from genetically diagnosed HFI patients on an FSS-restricted diet (n = 37) and their age-, sex- and body mass index-paired controls (n = 32) were analysed by capillary zone electrophoresis. We found that in HFI patients, asialoTf correlated with dietary intake of sucrose (R = 0.575, p < 0.001) and FSS (R = 0.475, p = 0.008), and that pentasialoTf+hexasialoTf negatively correlated with dietary intake of fructose (R = -0.386, p = 0.024) and FSS (R = -0.400, p = 0.019). In addition, the tetrasialoTf/disialoTf ratio truthfully differentiated treated HFI patients from healthy controls, with an area under the ROC curve (AUROC) of 0.97, 92% sensitivity, 94% specificity and 93% accuracy.

Quantification of urinary derivatives of Phenylbutyric and Benzoic acids by LC-MS/MS as treatment compliance biomarkers in Urea Cycle disorders.

PURPOSE: Salts of phenylacetic acid (PAA) and phenylbutyric acid (PBA) have been used for nitrogen elimination as a treatment for hyperammonaemia caused by urea cycle disorders (UCD). A new analytical method for PBA measurement in urine which helps to evaluate the drug adherence has been implemented. METHODS: Urine specimens from UCD patients receiving PBA were analysed by tandem mass spectrometry to measure urine phenylacetylglutamine (PAGln). Some clinical and biochemical data for each patient were collected. RESULTS: Our study included 87 samples from 40 UCD patients. The PAGln levels did not correlate with height, weight or age. However, the PAGln values showed correlation with PBA dose (r = 0.383, P = 0.015). Plasma glutamine and ammonia levels presented a positive correlation (r = 0.537, P < 0.001). The stability for PAGln in urine was determined at different storage temperatures. CONCLUSIONS: We have developed a simple method for the determination of PAGln in urine, which acts as useful biomarker of effective drug delivery. PAGln in urine is stable at room temperature at least for 15 days, and for several months when frozen at -20 °C. This procedure is useful for the optimization and monitorization of the drug dose allowing the use of spot urine samples.

The role of attentional biases to appetitive stimuli in childhood overweight.

Overweight during childhood constitutes a high-risk factor for adult obesity. An abnormal attention to food stimuli (i.e., a bias) has been suggested as an underlying mechanism to the onset and/or maintenance of obesity. Previous literature supports the existence of a biased attention toward food stimuli in adults with obesity. However, it is unknown whether this attentional bias occurs in high-risk children for adult obesity. We aimed to examine attentional biases to food at different stages of attention processing in overweight children. A dot-probe task was applied to 25 children with overweight and 25 healthy-weight children (8-12 years old). Attentional preference to or avoidance of pleasant food stimuli, which were displayed simultaneously with pleasant non-food stimuli (matched in valence and arousal), was examined at 100-ms (initial visual orienting), 500-ms (attention engagement), and 1500-ms (maintained attention) presentation rates. Both children with overweight and healthy-weight children showed an attentional bias toward food images at a 100-ms presentation rate. However, unlike healthy-weight children, those with overweight showed an attentional preference toward food images at 500- and 1500-ms presentation rates. A biased initial orienting to food cues can be found regardless of weight. However, a biased attention engagement and a biased maintained attention toward food cues are characteristics of children with overweight. Therefore, as in adults, children at risk of adult obesity have an abnormal attentional processing of food stimuli.

Newly validated biomarkers of brain damage may shed light into the role of oxidative stress in the pathophysiology of neurocognitive impairment in dietary restricted phenylketonuria patients.

Despite a strict dietary control, patient with hyperphenylalaninemia or phenylketonuria may show cognitive and/or behavioral disorders. These comorbid deficits are of great concern to patients, families, and health organizations. However, biomarkers capable of detecting initial stages of neurological damage are not commonly employed. The pathogenesis of phenylketonuria is complex in nature. Increasingly, the role of oxidative stress has gained acceptance and biomarkers reflecting oxidative damage to the brain and easily accessible in peripheral biofluids have been validated using mass spectrometry techniques. In the present review, the role of oxidative stress in the pathogenesis of phenylketonuria and hyperphenylalaninemia has been updated. Moreover, we report on newly validated brain-specific lipid peroxidation biomarkers and inform on their relevance in the detection and monitoring of neurological damage in phenylketonuric patients. In preliminary studies, a correlation between lipid peroxidation biomarkers and neurological dysfunction in patients with PKU was reported. However, there is a need of adequately powered trials to confirm the validity of these biomarkers for early detection of brain damage, initiation of treatment, and reliably monitor evolving disease both in phenylketonuria and hyperphenylalaninemia.

Carbohydrate status in patients with phenylketonuria.

BACKGROUND: In patients with phenylketonuria (PKU), a low-phenylalanine (Phe) diet supplemented with low-protein foods and a Phe-free amino acid mixture favors a dietary intake rich in carbohydrates, but little is known about how these molecules are metabolized in this setting. The objective of the present study was to analyze carbohydrate metabolism in patients with hyperphenylalaninemia. METHODS: We conducted a multicenter cross-sectional study to investigate biochemical markers of basal and postprandial carbohydrate metabolism in PKU patients according to age, Phe tolerance, waist circumference and body mass index (BMI), diet, tetrahydrobiopterin (BH4) supplementation, and adherence to treatment. Basal biomarkers and anthropometric parameters were also evaluated in patients with mild hyperphenylalaninemia (MHPA) and in healthy controls. RESULTS: A total of 83 patients aged 4-52 years were studied; 68.7% had PKU and 31.3% had MHPA. 68 healthy controls of similar sex and age were also evaluated Metabolic control was adequate in 71.9% of PKU patients. Fasting glucose levels (mean 80.77 ± 8.06 mg/dL) were high in just one patient, but fasting insulin levels, with a mean of 12.74 ± 8.4 mIU/L, were altered in 15 PKU patients (26.3%) and markedly higher than in patients with MPHA (p = 0.035). Fasting insulin levels and Homeostasis Model Assessment Insulin Resistance (HOMA-IR) were significantly higher than in healthy controls and correlated with body mass index, waist circumference, age, and also showed statistically significant differences according to diagnosis and Phe tolerance (p < 0.05). Patients under BH4 therapy had lower insulin levels and HOMA-IR. A higher mean carbohydrate intake from AA mixtures was observed in classic PKU patients. The caloric intake in the form of carbohydrates was also higher in PKU than MHPA patients (p = 0.038) and it was correlated with basal insulin (rho = 0.468, p = 0.006), HOMA-IR (rho = 0.423, p = 0.02), BMI (rho 0.533, p = 0.002), and waist circumference (rho 0.584, p = 0.0007). CONCLUSIONS: This study shows that PKU patients are at risk of carbohydrate intolerance and insulin resistance, more evident in adults and overweight patients, probably related to their higher caloric intake in form carbohydrate content. A higher dependency of AA mixtures was demonstrated in PKU patients.