Perinatal Reduced Blood Concentrations of Free Carnitine and Acylcarnitines in Infants with Cystic Fibrosis.

OBJECTIVE: Cystic fibrosis (CF) is a multisystemic inherited disease. The aim of this study was to determine free carnitine (FC) and acylcarnitine concentrations in CF newborns with various mutations of the CFTR gene perinatally. STUDY DESIGN: FC/acylcarnitines were determined in dried blood spots via liquid chromatography-tandem mass spectrometry (LC-MS/MS) on the third day of life of full-term normal (n = 50) and CF (n = 28) newborns. For infants with elevated immunoreactive trypsinogen values, FC/acylcarnitines were quantified again 48 hours later, followed by mutational analysis of CFTR gene via Sanger sequencing. RESULTS: Initial FC and sums of acylcarnitine concentrations were statistically significantly lower in CF patients than in controls and even lower 48 hours later. The mutations F508del and 621 + 1G > T were predominantly identified among CF patients. CONCLUSION: Low FC and acylcarnitine concentrations were measured perinatally in CF patients, for all CFTR mutations detected. Carnitine supplementation of breastfeeding mothers could be beneficial.

The phenylketonuria patient: A recent dietetic therapeutic approach.

Phenylalanine hydroxylase (PAH) deficiency, commonly named phenylketonuria (PKU) is a disorder of phenylalanine (Phe) metabolism inherited with an autosomal recessive trait. It is characterized by high blood and cerebral Phe levels, resulting in intellectual disabilities, seizures, etc. Early diagnosis and treatment of the patients prevent major neuro-cognitive deficits. Treatment consists of a lifelong restriction of Phe intake, combined with the supplementation of special medical foods, such as Amino Acid medical food (AA-mf), enriched in tyrosine (Tyr) and other amino acids and nutrients to avoid nutritional deficits. Developmental and neurocognitive outcomes for patients, however, remain suboptimal, especially when adherence to the demanding diet is poor. Additions to treatment include new, more palatable foods, based on Glycomacropeptide that contains limited amounts of Phe, the administration of large neutral amino acids to prevent phenylalanine entry into the brain and tetrahydrobiopterin cofactor capable of increasing residual PAH activity. Moreover, further efforts are underway to develop an oral therapy containing phenylalanine ammonia-lyase. Nutritional support of PKU future mothers (maternal PKU) is also discussed. This review aims to summarize the current literature on new PKU treatment strategies.

Birth weight related essential, non-essential and conditionally essential amino acid blood concentrations in 12,000 breastfed full-term infants perinatally.

Essential, non-essential and conditionally essential amino acid blood concentrations play a critical role in newborns. We aimed to quantitate most of these amino acids in the blood of full-term breastfed infants, perinatally and correlate the obtained values with their birth weight. Breastfed full-term infants (n = 12,000; 6000 males, 6000 females) with birth weight 2000-4000 g were divided into 4 equal groups: Group A, 2000-2500 g; B, 2500-3000 g; C, 3000-3500 g and D, 3500-4000 g. Blood samples as Dried Blood Spots (DBS) were collected on the 3rd day of life and analyzed via liquid chromatography-tandem mass spectrometry (LC-MS/MS) protocol. Blood concentrations of the amino acids, Phenylalanine, Leucine, Glutamine, Ornithine, Alanine, Tyrosine and Glycine in full-term breastfed newborns, were found to be related to their birth weight, perinatally. On the contrary, no relationship between birth weight and blood concentrations of the amino acids Valine, Methionine, Citrulline and Arginine was found. Due to the number of the samples, data from this study could be applied as neonatal screening reference values for full-term breastfed newborns in relation to their birth weight.

Mutational analysis of GALT gene in Greek patients with galactosaemia: identification of two novel mutations and clinical evaluation.

Classical galactosaemia is an inborn error of metabolism due to the deficiency of the enzyme galactose-1-phosphate uridylyltransferase (GALT). The aim of the study was to identify the underlying mutations in Greek patients with GALT deficiency and evaluate their psychomotor and speech development. Patients with GALT deficiency (n = 17) were picked up through neonatal screening. Mutational analysis was conducted via Sanger sequencing, while in silico analysis was used in the cases of novel missense mutations. Psychomotor speech development tests were utilized for the clinical evaluation of the patients. Eleven different mutations in the GALT gene were detected in the patient cohort, including two novel ones. The most frequent mutation was p.Q188R (c.563 A > G). As for the novel mutations, p.M298I (c.894 G > A) was identified in four out of 32 independent alleles, while p.P115S (c.343 C > T) was identified once. Psychomotor evaluation revealed that most of the patients were found in the borderline area (Peabody test), while only two had speech delay problems. The WISK test revealed three patients at borderline limits and two were at lower than normal limits. The mutational spectrum of the GALT gene in Greek patients is presented for the first time. The mutation p.Q188R is the most frequent among Greek patients. Two novel mutations were identified and their potential pathogenicity was estimated. Regarding the phenotypic characteristics, psychomotor disturbances and speech delay were mainly observed among GALT-deficient patients.

Clinical diagnostic Next-Generation sequencing: the case of CFTR carrier screening.

A 23-mutation panel for CFTR carrier screening is recommended to women of reproductive age by the American College of Obstetricians and Gynecologists. In the present study the optimized efficiency regarding the carrier rate of Next-Generation sequencing (NGS) technology is compared to the one of limited mutation detection panels. A total of 824 consequent cases were subjected to the commercial Cystic Fibrosis Genotyping Assay. Some 188 negative samples randomly selected from the initial group of probands were further subjected to an extended mutation panel characterized by 92% detection rate, as well as to massive parallel sequencing. Twenty-two probands subjected to the commercial assay proved to carry one mutation included in the ACOG panel (carrier rate 0.0267). The latter panels revealed the presence of mutations not included in the ACOG panel in four probands, resulting to an increase of carrier rate of 0.0106 in the case of in-house panel and an increase of rate of 0.0213 if NGS was used. The above data seem to support the implementation of NGS in the routine CFTR carrier screening.

Glucose-6-Phosphate Dehydrogenase (G6PD) deficiency in Greek newborns: the Mediterranean C563T mutation screening.

Glucose-6-Phosphate Dehydrogenase (G6PD) gene is located at the X-chromosome at Xq28 and the disease is recessively inherited predominantly in males. More than 400 variants have been proposed based on clinical and enzymatic studies. The aim of the current study was to identify C563T mutation in G6PD-deficient newborns and to correlate the enzyme residual activity with the presence of the mutation. Some 1189 full-term neonates aged 3-5 days old were tested for G6PD activity in dried blood spots from Guthrie cards using a commercial kit. DNA extraction from Guthrie cards and mutation identification among the deficient samples were performed with current techniques. A total of 92 (7.7%) newborns were G6PD-deficient. In 46 (50%), the mutation C563T was identified. The residual activity in C563T hemizygote males (n = 28) was statistically significantly lower (1.23 ± 0.93 U/g Hb) than that in non-C563T G6PD-deficient males (n = 25) (4.01 ± 1.20 U/g Hb, p < 0.0001) and in controls (13.6 ± 2.9 U/g Hb, p < 0.0001). In C563T heterozygote females, the estimated enzyme activity was lower than that determined in non-C563T females. Male C563T hemizygotes suffer from G6PD deficiency and severe neonatal jaundice. G6PD activity showed statistically significant correlation with total bilirubin blood levels.

Dried Blood Spots in Neonatal Studies: A Computational Analysis for the Role of the Hematocrit Effect.

Dried blood spot (DBS) microsampling is extensively employed in newborn screening (NBS) and neonatal studies. However, the impact of variable neonatal hematocrit (Ht) values on the results can be a source of analytical error, and the use of fixed Ht for calibration (Htcal) is not representative of all neonatal subpopulations. A computational approach based on neonatal demographics was developed and implemented in R® language to propose a strategy using correction factors to address the Ht effect in neonatal DBS partial-spot assays. A rational "tolerance level" was proposed for the Ht effect contribution to the total analytical error and a safe Ht range for neonatal samples, where the correction of concentrations can be omitted. Furthermore, an "alert zone" for a false positive or negative result in NBS was proposed, where the Ht effect has to be considered. Results point toward the use of Htcal values closely representative of populations under analysis and an acceptable level of percentage relative error can be attributed to the Ht effect, diminishing the probability of correction. Overall, the impact of the Ht effect on neonatal studies is important and future work may further investigate this parameter, correlated to other clinical variables potentially affecting results.

Novel H2S-Releasing Bifunctional Antihistamine Molecules with Improved Antipruritic and Reduced Sedative Actions.

Hydrogen sulfide (H2S) is an endogenous gasotransmitter with anti-inflammatory actions that also reduces itching. To test whether a combination of an antihistamine with a H2S donor has improved antipruritic efficacy, bifunctional molecules with antihistamine and H2S-releasing pharmacophores were synthesized and tested in vitro and in vivo. H2S release from the hybrid molecules was evaluated with the methylene blue and lead acetate methods, and H1-blocking activity was assessed by determining tissue factor expression inhibition. All new compounds released H2S in a dose-dependent manner and retained histamine blocking activity. Two compounds with the highest potency were evaluated in vivo for their antipruritic as well as sedative action; they proved to possess higher efficacy in inhibiting histamine-induced pruritus and decreased sedative effects compared to the parent compounds (hydroxyzine and cetirizine), suggesting that they exhibit superior antipruritic action and limited side effects that likely arise from the H2S-releasing moiety.

Homologous Recombination Deficiency Score Determined by Genomic Instability in a Romanian Cohort.

The Homologous Recombination Deficiency (HRD) Score, determined by evaluating genomic instability through the assessment of loss of heterozygosity (LOH), telomeric allelic imbalance (TAI), and large-scale state transitions (LST), serves as a crucial biomarker for identifying patients who might benefit from targeted therapies, such as PARP inhibitors (PARPi). This study aimed to investigate the efficacy of HRD testing in high-grade serous ovarian carcinoma, tubal, and peritoneal cancer patients who are negative for somatic BRCA1 and BRCA2 mutations and to evaluate the impact of HRD status on Bevacizumab and PARPi therapy response. A cohort of 100 Romanian female patients, aged 42-77, was initially selected. Among them, 30 patients had unsuitable samples for HRD testing due to insufficient tumor content or DNA integrity. Using the OncoScan C.N.V. platform, HRD testing was successfully performed on the remaining 70 patients, with 20 testing negative and 50 testing positive for HRD. Among the HRD-positive patients, 35 were eligible for and benefited from PARPi maintenance therapy, resulting in a median progression-free survival (PFS) increase from 4 months to 8.2 months. Our findings support the importance of HRD testing in ovarian cancer patients, demonstrating the potential therapeutic advantage of PARPi therapy in HRD-positive patients without somatic BRCA1/2 mutations.

Enhanced interpretation of newborn screening results without analyte cutoff values.

PURPOSE: To improve quality of newborn screening by tandem mass spectrometry with a novel approach made possible by the collaboration of 154 laboratories in 49 countries. METHODS: A database of 767,464 results from 12,721 cases affected with 60 conditions was used to build multivariate pattern recognition software that generates tools integrating multiple clinically significant results into a single score. This score is determined by the overlap between normal and disease ranges, penetration within the disease range, differences between conditions, and weighted correction factors. RESULTS: Ninety tools target either a single condition or the differential diagnosis between multiple conditions. Scores are expressed as the percentile rank among all cases with the same condition and are compared to interpretation guidelines. Retrospective evaluation of past cases suggests that these tools could have avoided at least half of 279 false-positive outcomes caused by carrier status for fatty-acid oxidation disorders and could have prevented 88% of known false-negative events. CONCLUSION: Application of this computational approach to raw data is independent from single analyte cutoff values. In Minnesota, the tools have been a major contributing factor to the sustained achievement of a false-positive rate below 0.1% and a positive predictive value above 60%.

The relationship between neurotransmission-related amino acid blood concentrations and neuropsychological performance following acute exercise.

Amino acid neurotransmitters, including glutamate, phenylalanine, tyrosine, alanine, and glycine, underlie the majority of the excitatory and inhibitory neurotransmission in the nervous system, and acute exercise has been shown to modulate their concentrations. We aimed to determine whether any correlation exists between the above-mentioned amino acid blood concentrations and the neuropsychological performance after an acute exercise intervention. Sixty basketball players were randomly assigned to one of two experimental conditions: exercise or inactive resting. All participants underwent a comprehensive neuropsychological assessment and blood samples were taken on a Guthrie card before and after the end of the experimental conditions. Amino acid blood concentrations were significantly elevated and cognitive performance significantly improved post-exercise on specific neuropsychological assessments. Significant intervention × group interaction effects were apparent for Trail Making Test part-B [F(1,58) = 20.46, p < .0001, η2 = .26] and Digit Span Backwards [F(1,58) = 15.47, p < .0001, η2 = .21] neuropsychological assessments. Additionally, regression analysis indicated that tyrosine accounted for 38.0% of the variance in the Trail Making Test part-A test. These results suggest that elevated blood concentrations of neurotransmission-related amino acids are associated with improved neuropsychological performance after a single bout of high-intensity exercise.

Mutational analysis for biotinidase deficiency of a Greek patients' cohort ascertained through expanded newborn screening.

Late-onset multiple carboxylase deficiency, also known as biotinidase (BTD) deficiency, is an autosomal recessively inherited disorder of biotin metabolism. Its early diagnosis and treatment seems that it can even fully prevent its various clinical manifestations. Mutations in the BTD gene scattered throughout its coding region have been detected in patients ascertained either through newborn screening or clinically. From March 2010 up to June 2011, 18 954 Greek neonates were subjected to biochemical determination of BTD activity through a semiquantitative fluoroimmunoassay. Subsequently, the first cohort of our 'suspected' samples was further tested for the presence of aberrations associated either with partial or profound BTD deficiency through sequencing of the coding region of the BTD gene, including splice-site junctions. On the basis of the molecular data derived from the study of our first cohort of 'suspected' samples, a panel of four mutations, most frequently encountered in the Greek population, was created, and a rapid, reliable and cost-effective real-time-based genotyping assay for the detection of these mutations was developed. This is the first report about the BTD mutational spectrum in Greece, and it could be a beneficial utility in the differential clinical diagnosis of BTD deficiency.

Validation of a novel LC-MS/MS method for the quantitation of colistin A and B in human plasma.

A liquid chromatography/tandem mass spectrometry (LC-MS/MS) method, characterized by complete automation and high-throughput, was developed for the determination of colistin A and B in human plasma. All sample preparation procedures were performed by using 2.2 mL 96-deep-well plates, whereas robotic liquid-handling workstations were utilized for all liquid transfer steps, including solid-phase extraction (SPE). The whole preparation procedure was very rapid, whereas the method had a very short chromatographic run time of just 2 min. Sample analysis was performed by reversed phase LC-MS/MS, with positive electrospray ionization, using multiple reaction monitoring. The absence of available purified colistin A and B standards led to the development of a novel LC method with evaporative light-scattering detector for the determination of their stoichiometries in the standard mixture, along with its purity. The proposed bioanalytical method was fully validated and it was proven to be selective, accurate, precise, reproducible and suitable for the determination of colistin A and B in human plasma. It was applied successfully to a pharmacokinetic study for the determination of both analytes in samples of patients.

Advantages of automation in plasma sample preparation prior to HPLC/MS/MS quantification: application to the determination of cilazapril and cilazaprilat in a bioequivalence study.

An HPLC/MS/MS method characterized by complete automation and high throughput was developed for the determination of cilazapril and its active metabolite cilazaprilat in human plasma. All sample preparation and analysis steps were performed by using 2.2 mL 96 deep-well plates, while robotic liquid handling workstations were utilized for all liquid transfer steps, including liquid-liquid extraction. The whole procedure was very fast compared to a manual procedure with vials and no automation. The method also had a very short chromatographic run time of 1.5 min. Sample analysis was performed by RP-HPLC/MS/MS with positive electrospray ionization using multiple reaction monitoring. The calibration curve was linear in the range of 0.500-300 and 0.250-150 ng/mL for cilazapril and cilazaprilat, respectively. The proposed method was fully validated and proved to be selective, accurate, precise, reproducible, and suitable for the determination of cilazapril and cilazaprilat in human plasma. Therefore, it was applied to a bioequivalence study after per os administration of 2.5 mg tablet formulations of cilazapril.

Quantitation and evaluation of perinatal medium-chain and long-chain acylcarnitine blood concentrations in 12,000 full-term breastfed newborns.

OBJECTIVES: Medium-chain (MCA) and long-chain acylcarnitine (LCA) blood concentrations play a significant role in the fatty acid (FA) oxidation process, especially during the first days of life. Identification of their abnormal concentrations, via expanded newborn screening, can lead to the diagnosis of FA oxidation disorders. This study aimed to demonstrate MCA and LCA concentrations in Dried Blood Spots (DBS) of full-term breastfed infants, in relation to their birth weight (BW) perinatally. METHODS: Breastfed full-term infants (n = 12,000, 6,000 males, 6,000 females) with BW 2,000-3,999 g were divided into four equal groups: Group A, 2,000-2,499 g, B 2,500-2,999 g, C 3,000-3,499 g, and D 3,500-3,999 g. Samples were collected as DBS and acylcarnitines were determined via a liquid chromatography tandem mass spectrometry method. RESULTS: MCA and LCA blood concentrations were determined significantly lower in group A (low birth weight infants) in both sexes. Infants with BW > 3,500 g (group D), were characterized by lower levels of C10, C10:1, C14, C14:1 acylcarnitines and higher levels of C16 and C18:1 acylcarnitines, as compared to the other groups of this study. CONCLUSIONS: Concentration patterns in full-term breastfed newborns in relation to sex and mainly BW found in this study could be very helpful for neonatologists, especially for newborns of group A.

Expanded newborn screening in Greece: 30 months of experience.

In Greece, the National Newborn Screening Program was initiated in 1974 and is performed by the Institute of Child Health (ICH). However, there is a complete absence of conditions that have high rates of mortality and a relatively high prevalence listed in the Catalogue of Disorders screened by the ICH. Our laboratory has expanded the existing NBS program to include newborn screening for inborn errors of metabolism, screening for cystic fibrosis (the most common congenital disorder in the Greek population), congenital adrenal hyperplasia, and for biotinidase deficiency. From July 2007 to December 2009, 45,000 dried blood spots (DBS) were collected from infants born in Athens, Greece, and were analyzed. We present a report of our 30-month experience in the newborn screening area. The samples were tested for amino acidopathies, fatty acid oxidation disorders (FAOD), and organic acid metabolic disorders by applying flow injection analysis-electrospray ionization-tandem mass spectrometry (FIA-ESI-MS/MS); for cystic fibrosis by immunoreactive trypsinogen (IRT) measurement (time-resolved fluoroimmunoassay); for congenital adrenal hyperplasia by fluoroimmunoassay to measure the 17 hydroxy-progesterone level; and for biotinidase deficiency using a colorimetric method and a semiquantitative fluoroimmunoassay to determine biotinidase activity. Sample analysis resulted in establishing cutoff values for the respective disease markers for the first time in the Greek population. Four infants were identified with cystic fibrosis, two with congenital adrenal hyperplasia, two with phenylketonuria (PKU), one with medium-chain acyl CoA dehydrogenase deficiency (MCADD), and one with biotinidase deficiency. To the best of our knowledge, this is the first article reporting the status of expanded newborn screening in Greece.

Dried plasma spots as an alternative sample collection technique for the quantitative LC-MS/MS determination of gabapentin.

Dried plasma spots were employed as an alternative sample collection technique for the quantitative determination of gabapentin in human plasma, using an automated liquid chromatography tandem mass spectrometry method. After the methanolic extraction of single plasma, 1/8-in. disks which contained only 1.98 microL plasma volume, were placed in 96-well format plates, then gabapentin and its internal standard, 4-aminocyclohexanecarboxylic acid, were derivatized with n-butanol/HCl (3 M) and detected as butyl esters. The assay exhibited excellent linearity over the concentration range of 40.0-10.0 x 10(3) ng/mL, which is suitable for the determination of gabapentin after per os administration of a single tablet. Variations in intra- and inter-assay accuracy and precision were within internationally accepted criteria. Homogeneity of plasma spots was proven, whilst butyl ester stability for both analytes was estimated and found very satisfactory. The quantitative analysis of Gabapentin with dried plasma spot specimens seems to be a prominent and advantageous technique, especially when applied to pharmacokinetic studies, where plasma sampling procedure becomes rapid and required plasma volumes negligible.

Validation of a novel, fully automated high throughput high-performance liquid chromatographic/tandem mass Spectrometric method for quantification of pantoprazole in human plasma.

An automated high-throughput HPLC/MS/MS method was developed for the quantitative determination of pantoprazole in human plasma. Only 100 microL plasma was placed in 2.2 mL 96 deep-well plates, and both pantoprazole and omeprazole (IS) were extracted from human plasma by liquid-liquid extraction, using diethyl ether-dichloromethane (70:30, v/v) as the organic solvent. Robotic liquid-handling workstations were used for all liquid transfer and solution preparation steps and resulted in a short sample preparation time. After vortexing, centrifugation, and freezing, the supernatant organic solvent was evaporated and reconstituted in a small volume of reconstitution solution. Sample analysis was performed by utilizing the combination of RP-HPLC/MS/MS, with positive-ion electrospray ionization and multiple reaction monitoring detection. The chromatographic run time was set at 1.8 min with a flow rate of 0.6 mL/min on a Nucleosil octylsilyl (C8) analytical column. The method was proven to be sensitive, specific, accurate, and precise for the determination of pantoprazole in human plasma. The method was applied to a bioequivalence study after per os administration of a 40 mg pantoprazole gastric retentive tablet.

Perinatal free carnitine and short chain acylcarnitine blood concentrations in 12,000 full-term breastfed newborns in relation to their birth weight.

BACKGROUND: Free carnitine (C0) and short chain acylcarnitine (SCA) blood concentrations play a significant role in fatty acid oxidation process during the first days of life. The aim of this study was to demonstrate C0 and SCA concentrations in Dried Blood Spots (DBS) of full term breastfed infants in relation to their birth weight (BW) perinatally. METHODS: Breastfed full term infants (n = 12,000, 6000 males, 6000 females) with BW 2000-4000 g were divided into 4 equal groups: Group A, 2000-2500 g, B 2500-3000 g, C 3000-3500 g and D 3500-4000 g. Blood samples in the form of DBS were collected on the 3rd day of life and analyzed via a liquid chromatography tandem mass spectrometry (LC-MS/MS) protocol. RESULTS: BW-related C0 and SCAs were found as follows: C0 was determined to be statistically significantly higher in group A (BW 2000-2500 g) in both males and females. Lower acetylcarnitine (C2) and hydroxybutyrylcarnitine (C4OH) blood concentrations were detected in group A of both sexes, whereas butyrylcarnitine (C4) concentrations were found to be lower in the same group of males only. Furthermore, high concentrations of C2 and C4OH were shown in group D (BW 3500-4000 g) in both sexes. SCA sum of means ± SD values in males and females of group A were statistically significantly lower as compared to other study groups. CONCLUSION: Due to the number of the samples, data from this study could be applied as neonatal screening reference values for full term breastfed newborns in relation to their birth weight.

Birth weight related blood concentrations of the neurotransmission amino acids glutamine plus glutamate, phenylalanine and tyrosine in full-term breastfed infants perinatally.

Background The amino acids glutamine plus glutamate, phenylalanine and tyrosine are implicated in neurotransmission. We aimed to evaluate these amino acid blood concentrations in full-term breastfed infants with different birth weight (BW) perinatally. Methods Breastfed full-term infants (n = 6000, males 3000, females 3000) BW 2000-4000 g were divided into four equal groups. Both males and females Groups A, 2000-2500 g, B 2500-3000 g, C 3000-3500 g, D 3500-4000 g. Blood samples on Guthrie cards, were taken on the 3rd day of life and quantified via a liquid chromatography tandem mass spectrometry (LC-MS/MS) method. Results Glutamine plus glutamate mean values were found to be statistically significantly different between males vs. females in all the studied groups. The highest values were determined in both males and females in group D. Statistically significantly higher values of phenylalanine appeared in group D vs. other groups. Tyrosine mean values were calculated to be statistically significantly different in both sexes in group A compared to other groups. Conclusions Differences of glutamine plus glutamate, phenylalanine and tyrosine levels among full-term newborns with different BW are presented for the first time in the literature. Newborns with BW 3000-4000 g are benefited by having higher concentrations of the mentioned neurotransmission related amino acids. Neonatal screening reference values for these amino acids in relation to BW could be established, not only for preterm and low BW infants but also for full-term newborns with BW >3000 g.