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Ertapenem (Invanz) is a newly developed carbapenem β-lactam antimicrobial agent. The drug usage in pediatric age needs an accurate drug monitoring for effective patient management. The aim of this study was to evaluate the use of dried blood spot (DBS) specimens to measure ertapenem concentration during treatment. The analysis was performed by UPLC-MS/MS operating in multiple reaction monitoring (MRM) mode. The calibration curve in matrix was linear in the concentration range of 0.5-100 mg/L with correlation coefficient value higher than 0.997. Performance parameters of this method like lower limit of detection (LLOD, 0.2 mg/L), lower limit of quantification (LLOQ, 0.5 mg/L), matrix effect (20%), intra- and inter-day imprecision (CV within than 15%) and accuracy (between 94 and 155%) of drug concentrations have been evaluated. The drug stability at different temperatures was tested for one month, to evaluate the risks of sample delivery at different climatic conditions. The reported method allows now ertapenem analysis and offers many advantages for patients including the possibility of collecting samples at home. This new assay is both precise and accurate and is especially suitable for therapeutic drug monitoring and pharmacokinetic studies in neonates in whom obtaining larger blood samples is not convenient or possible., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Fabry disease: polymorphic haplotypes and a novel missense mutation in the GLA gene. Fabry disease (FD) is an X-linked lysosomal storage disorder with a heterogeneous spectrum of clinical manifestations that are caused by the deficiency of α-galactosidase A (α-Gal-A) activity. Although useful for diagnosis in males, enzyme activity is not a reliable biochemical marker in heterozygous females due to random X-chromosome inactivation, thus rendering DNA sequencing of the α-Gal-A gene, alpha-galactosidase gene (GLA), the most reliable test for the confirmation of diagnosis in females. The spectrum of GLA mutations is highly heterogeneous. Many polymorphic GLA variants have been described, but it is unclear if haplotypes formed by combinations of such variants correlate with FD, thus complicating molecular diagnosis in females with normal α-Gal-A activity. We tested 67 female probands with clinical manifestations that may be associated with FD and 110 control males with normal α-Gal-A activity. Five different combinations of GLA polymorphic variants were identified in 14 of the 67 females, whereas clearcut pathogenetic alterations, p.Met51Ile and p.Met290Leu, were identified in two cases. The latter has not been reported so far, and both mutant forms were found to be responsive to the pharmacological chaperone deoxygalactonojirimycin (DGJ; migalastat hydrochloride). Analysis of the male control population, as well as male relatives of a suspected FD female proband, permitted the identification of seven different GLA gene haplotypes in strong linkage disequilibrium. The identification of haplotypes in control males provides evidence against their involvement in the development of FD phenotypic manifestations., (© 2011 John Wiley & Sons A/S.)

The California bay laurel or Umbellularia californica (Hook. & Arn.) Nutt., is known as the 'headache tree' because the inhalation of its vapours can cause severe headache crises. However, the underlying mechanism of the headache precipitating properties of Umbellularia californica is unknown. The monoterpene ketone umbellulone, the major volatile constituent of the leaves of Umbellularia californica, has irritating properties, and is a reactive molecule that rapidly binds thiols. Thus, we hypothesized that umbellulone stimulates the transient receptor potential ankyrin 1 channel in a subset of peptidergic, nocioceptive neurons, activating the trigeminovascular system via this mechanism. Umbellulone, from µM to sub-mM concentrations, selectively stimulated transient receptor potential ankyrin 1-expressing HEK293 cells and rat trigeminal ganglion neurons, but not untransfected cells or neurons in the presence of the selective transient receptor potential ankyrin 1 antagonist, HC-030031. Umbellulone evoked a calcium-dependent release of calcitonin gene-related peptide from rodent trigeminal nerve terminals in the dura mater. In wild-type mice, umbellulone elicited excitation of trigeminal neurons and released calcitonin gene-related peptide from sensory nerve terminals. These two responses were absent in transient receptor potential ankyrin 1 deficient mice. Umbellulone caused nocioceptive behaviour after stimulation of trigeminal nerve terminals in wild-type, but not transient receptor potential ankyrin 1 deficient mice. Intranasal application or intravenous injection of umbellulone increased rat meningeal blood flow in a dose-dependent manner; a response selectively inhibited by systemic administration of transient receptor potential ankyrin 1 or calcitonin gene-related peptide receptor antagonists. These data indicate that umbellulone activates, through a transient receptor potential ankyrin 1-dependent mechanism, the trigeminovascular system, thereby causing nocioceptive responses and calcitonin gene-related peptide release. Pharmacokinetics of umbellulone, given by either intravenous or intranasal administration, suggest that transient receptor potential ankyrin 1 stimulation, which eventually results in meningeal vasodilatation, may be produced via two different pathways, depending on the dose. Transient receptor potential ankyrin 1 activation may either be caused directly by umbellulone, which diffuses from the nasal mucosa to perivascular nerve terminals in meningeal vessels, or by stimulation of trigeminal endings within the nasal mucosa and activation of reflex pathways. Transient receptor potential ankyrin 1 activation represents a plausible mechanism for Umbellularia californica-induced headache. Present data also strengthen the hypothesis that a series of agents, including chlorine, cigarette smoke, formaldehyde and others that are known to be headache triggers and recently identified as transient receptor potential ankyrin 1 agonists, utilize the activation of this channel on trigeminal nerves to produce head pain.

Orotic acid (OA), a marker of hereditary orotic aciduria, is usually used for the differential diagnosis of some hyperammonemic inherited defects of urea cycle and of basic amino acid transporters. This study was aimed to establish age related reference intervals of OA in urine, and for the first time in plasma, and dried blood spot (DBS) from 229 apparently healthy subjects aged from three days to 40 years. The quantification of OA was performed by a previously implemented method, using a stable isotope dilution with 1,3-[(15)N(2)]-orotic acid and hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS). The method has proved to be sensitive and accurate for a quantitative analysis of OA also in DBS and plasma. According to previous studies, urinary OA levels (mmol/mol of creatinine) decrease significantly with age. The upper limits (as 99th %ile) were of 3.44 and 1.30 in groups aged from three days to 1 year (group 1) and from 1 year to 12 years (group 2), respectively; in teenagers (from 13 to 19 years; group 3) and adults (from 20 to 40 years; group 4) urinary levels became more stable and the upper limits were of 0.64 and 1.21, respectively. Furthermore, OA levels in DBS (μM) also resulted significantly higher in subjects of group 1 (upper limit of 0.89) than in subjects of groups 2, 3 and 4 (upper limits of 0.24, 0.21, and 0.29, respectively). OA levels in plasma (μM) were significantly lower in subjects of group 3 (upper limit of 0.30) than in subjects of groups 1, 2, and 4 (upper limits of 0.59, 0.48, and 0.77, respectively). This method was also employed for OA quantification in plasma and DBS of 17 newborns affected by urea cycle defects, resulting sensitive and specific enough to screen these disorders., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Tyrosinemia type 1 is caused by deficiency of fumarylacetoacetate hydrolase. The enzymatic defect impairs the conversion of fumarylacetoacetate to fumarate, causing accumulation of succinylacetone which induces severe liver and kidney dysfunction along with mutagenic changes and hepatocellular carcinoma. Treatment is based on nitisinone (NTBC), an enzymatic inhibitor which suppresses succinylacetone production. NTBC, which has dramatically changed the disease course improving liver and kidney functions and reducing risk of liver cancer, causes a side effect of the increase of tyrosine levels. Treatment is therefore based on the combination of NTBC with a protein-restricted diet to prevent the potential toxicity of excessive tyrosine accumulation. Long-term therapy requires a careful monitoring in blood of NTBC levels along with other disease biomarkers, which include succinylacetone, and a selected panel of circulating aminoacids. We have developed a straightforward and fast MS/MS method for the simultaneous determination of NTBC, succinylacetone, tyrosine, phenylalanine, and methionine on a dried blood spot requiring a 2 min run. A single assay suitable for quantitative evaluation of all biochemical markers is of great advance over conventional methods, especially in pediatric patients, since it reduces laboratory costs and blood sampling, is less invasive and particularly suitable for pediatric patients, and allows easier storage and shipping.

The human HADH gene encodes the short-chain-L-3-hydroxyacyl-CoA dehydrogenase, the enzyme which catalyzes the third step of the β-oxidation of the fatty acids in the mitochondrial matrix. Loss-of-function mutations in the HADH gene lead to short-chain-L-3-hydroxyacyl-CoA dehydrogenase deficiency, an autosomal recessive genetic defect of unknown prevalence with a wide spectrum of phenotypic variability. As in other metabolic diseases, the diagnostic relevance of the biochemical evaluations, plasma acylcarnitines, and urinary organic acids, are crucially dependent on the clinical conditions of the patient during specimen collection.This paper describes the eighth patient carrying a HADH gene mutation, a new homozygous deletion c.565delG leading to an early stop codon (p.V116Wfs124X), in an infant with hyperinsulininemic hypoglycemia, displaying abnormal patterns of plasma acylcarnitines and urinary organic acids. We conclude that, when the residual catalytic activity of the mutated enzyme is seriously reduced, the biochemical hallmarks of the disease, namely plasma 3-hydroxybutyrylcarnitine and urinary 3-hydroxyglutaric acid, are invariably present.

Background: Guanidinoacetate (GAA) and creatine are reliable biochemical markers for primary and secondary creatine defects. We describe a method by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for simultaneous determination of plasma GAA and creatine. We analyzed 283 healthy subjects from 0 to 63 years old to obtain age-related control values., Methods: Plasma samples were extracted with acetonitrile containing 13C2-GAA and d3-creatine. Samples were analyzed by LC-MS/MS in positive ionisation mode, after derivatization to butyl-esters. Optimal chromatographic separation was achieved using a column Supelcosil™ LC-4.6mm with isocratic elution in 5min., Results: Run time was 5min. Standard curves were linear from 0.05 to 200μmol/L for creatine and from 0.02 to 40μmol/L for GAA. Limit of detection (LOD) and limit of quantitation (LOQ) were respectively 0.005 and 0.05μmol/L for creatine; LOD and LOQ were 0.002 and 0.02μmol/L respectively for GAA. Intra and inter-assay CVs for creatine and GAA were <8%. Recovery experiments adding 50 and 100μmol/L creatine and 10 and 20μmol/L GAA were 102.1% and 101.2%, for creatine; 102.95% and 96.45% for GAA. The method was applied to 283 plasma controls from healthy subjects to obtain control values in three specific age ranges: 0-12, 13-20, >20 years old., Conclusion: A rapid and high sensitive LC-MS/MS method was developed and validated for determination of creatine and GAA in plasma and it could also be applied to other biological materials, such as CFS and urines. This method is useful for diagnoses of primary and also for secondary creatine defects that may occur in inherited metabolic diseases in which precursors of creatine biosynthesis are involved., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Artemisinin (qinghaosu), a unique endoperoxide sesquiterpene lactone isolated from Artemisia annua L., is a very active antimalarial drug, including severe and cerebral malaria. However, its therapeutical efficacy is limited due to its scarce bioavailability. In this article, artemisinin-loaded conventional and polyethylene glycol (PEGylated) liposomes were proposed as carriers to increase biopharmaceutical properties of the drug. Encapsulation efficacy was determined by high-performance liquid chromatography/diode array detection/electrospray ionization-mass spectrometry, dimensional analysis was performed by dynamic light scattering, and morphology was performed by trasmission electron microscopy. After dialysis, both liposomal formulations showed an encapsulation efficacy of more than 70%; mean diameter of all the artemisinin-loaded vesicles was approximately 130-140 nm. The polydispersity index of the formulations ranged from 0.2 to 0.3 and resulted as suitable for intraperitoneal (i.p.) administration. Pharmacokinetic profile and the main pharmacokinetic parameters of the carriers were evaluated in healthy mice i.p. Free artemisinin was rapidly cleared from plasma and hardly detected 1 hour after administration. Conversely, both liposomal formulations showed much longer blood-circulation time than free artemisinin; artemisinin was still detectable after 3 and 24 hours of administration, respectively, for conventional and PEGylated liposomes. AUC(0-24 h) values were increased by approximately 6 times in both of the liposomal formulations, in comparison with free artemisinin. A strong effect of formulation on the half-life of artemisinin was enhanced by more than 5-fold by the incorporation of PEG into liposomes. Liposomes loaded with artemisinin, especially the long-circulating vesicles, could really represent a new strategy for developing smart, well-tolerated, and efficacious therapeutic nanocarriers to treat tumors, but could also be very useful to treat parasitic disease.

Background: Adenosine deaminase (ADA)-severe combined immunodeficiency (SCID) is an SCID caused by a defect in the enzyme adenosine deaminase. It is usually fatal in infancy because of severe recurrent infections. When diagnosis is made, permanent damage caused by infections or by metabolites is often present. Gene therapy, bone marrow transplantation, or enzyme therapy might be effective if performed early. ADA-SCID complies with all the criteria for inclusion in a newborn screening program. However, screening methods are still expensive or provide a non-negligible number of indeterminate results., Objective: The aim of the present study was to develop a simple, reliable, and inexpensive method for diagnosis of ADA-SCID by using dried blood spot (DBS) samples taken at birth. Cost per test was calculated, including the cost for reagents, equipment, and operators., Methods: DBS samples from 4 patients with genetically confirmed ADA-SCID and 12,020 DBS samples from healthy newborns were examined. Adenosine and 2'-deoxyadenosine were tested by using tandem mass spectrometry (PCT EP2010/070517)., Results: The mean levels of adenosine and 2'-deoxyadenosine were 7.8 ± 3.1 and 8.5 ± 6.0 μmol/L, respectively, in affected children; adenosine was found at 0.23 ± 0.09 μmol/L, whereas 2'-deoxyadenosine was never detected in healthy control subjects (adenosine: P < 10(-6) [95% confidence limit, 7.59-7.78] and 2'-deoxyadenosine: P < 10(-6) [95% confidence limit, 8.65-8.82] for control subjects vs patients with ADA-SCID). No indeterminate or false-positive results were found. Cost per test was €0.01 ($0.013). A pilot population-based newborn screening for ADA-SCID has started in Tuscany, Italy., Conclusion: Tandem mass spectrometry can be used for diagnosis of one of the most frequent form of SCID at a negligible cost., (Copyright © 2011 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.)

Purpose: The blood-brain barrier discriminates the access of several molecules to the brain. This hampers the use of some drugs, as doxorubicin, potentially active for treatment of brain tumors. We explored the feasibility of active modification of the blood-brain barrier protection, by using morphine pretreatment, to allow doxorubicin accumulation in the brain in an animal model., Methods: Rats were pretreated with different doses of intraperitoneal morphine before injection of doxorubicin (12 mg/kg). Quantitative analysis of doxorubicin was performed by mass spectrometry. Acute heart and kidney damage was analyzed by measuring doxorubicin accumulation, LDH activity and malondialdehyde plasma levels., Results: The concentration of doxorubicin was significantly higher in all brain areas of rats pretreated with morphine than in control tissues (P < 0.001). This was evident only at therapeutic morphine dose (10 mg/kg, three times over 24 h), while lower doses (2.5 and 5 mg/kg) were not associated with doxorubicin accumulation. Pretreatment with morphine did not induce an elevation of LDH activity or of lipid peroxidation compared to controls., Conclusion: Our data suggest that morphine pretreatment is able to allow doxorubicin penetration inside the brain, by modulating the blood-brain barrier. This is not associated with acute cardiac or renal toxicity. These preliminary results will enable us to generate novel therapeutic approaches to refractory or recurrent brain tumors, and might be useful in other human diseases of the central nervous system in which molecules usually stopped by the blood-brain barrier may have a therapeutic impact.

The genome of the silkmoth Bombyx mori contains 44 genes encoding odorant-binding proteins (OBPs) and 20 encoding chemosensory proteins (CSPs). In this work, we used a proteomic approach to investigate the expression of proteins of both classes in the antennae of adults and in the female pheromone glands. The most abundant proteins found in the antennae were the 4 OBPs (PBP, GOBP1, GOBP2, and ABP) and the 2 CSPs (CSP1 and CSP2) previously identified and characterized. In addition, we could detect only 3 additional OBPs and 2 CSPs, with clearly different patterns of expression between the sexes. Particularly interesting, on the other hand, is the relatively large number of binding proteins (1 OBP and 7 CSPs) expressed in the female pheromone glands, some of them not present in the antennae. In the glands, these proteins could be likely involved in the solubilization of pheromonal components and their delivery in the environment.

The biological role of GPI anchors is of paramount importance; however, we are still far from fully understanding the structure-function relationship of these molecules. One major limiting factor has been the tiny quantities available from natural sources; obtaining homogeneous and well-defined GPI structures by synthesis, is both a challenge and an attractive goal. We report here the convergent synthesis of the essential core of the human GPI anchor 1, exploiting a common precursor to obtain the trisaccharidic donor 2 and a novel protecting groups sequence. The final product, prepared for the first time, is biologically active., (Copyright © 2011. Published by Elsevier Inc.)

Purpose: Therapeutic hypothermia has recently been introduced to treat term newborns with hypoxic-ischemic encephalopathy, of whom more than half have seizures. Phenobarbital is widely used to treat neonatal seizures, but it is unknown whether its pharmacokinetics is affected by hypothermia. We evaluated the influence of hypothermia on phenobarbital pharmacokinetics in asphyxiated newborns., Methods: Nineteen term asphyxiated newborns treated with mild whole body hypothermia, started within 6 h after birth and protracted for 72 h, received phenobarbital for clinical seizures. Treatment schedule consisted of a loading dose of 20 mg/kg, titrated to response, up to a maximum dose of 40 mg/kg, followed by a maintenance dose of 2.5 or 1.5 mg/kg every 12 h. Phenobarbital concentrations were measured on 28 dried blood spots in each newborn., Key Findings: Eighteen newborns showed plasma concentrations within the reference range after receiving a loading dose of 20 mg/kg. In the remaining newborn, who had received a loading dose of 35 mg/kg, phenobarbital concentrations exceeded the upper reference limit. Phenobarbital concentrations reached a virtual steady state in all newborns. Pharmacokinetic parameters were then calculated. Minimum and maximum concentration (24.7 ± 8.8 and 30.63 ± 10.3 mg/L), average plasma concentration (27.37 ± 9.4 mg/L), and half-life (173.9 ± 62.5 h) were considerably higher than reported in literature for normothermic newborns. Pharmacokinetic parameters did not differ significantly between infants receiving different maintenance doses., Significance: Phenobarbital administered to newborns under whole body hypothermia results in higher plasma concentrations and longer half-lives than expected in normothermic newborns., (Wiley Periodicals, Inc. © 2011 International League Against Epilepsy.)

Purpose: To achieve clinical validation of cutoff values for newborn screening by tandem mass spectrometry through a worldwide collaborative effort., Methods: Cumulative percentiles of amino acids and acylcarnitines in dried blood spots of approximately 25–30 million normal newborns and 10,742 deidentified true positive cases are compared to assign clinical significance, which is achieved when the median of a disorder range is, and usually markedly outside, either the 99th or the 1st percentile of the normal population. The cutoff target ranges of analytes and ratios are then defined as the interval between selected percentiles of the two populations. When overlaps occur, adjustments are made to maximize sensitivity and specificity taking all available factors into consideration., Results: As of December 1, 2010, 130 sites in 45 countries have uploaded a total of 25,114 percentile data points, 565,232 analyte results of true positive cases with 64 conditions, and 5,341 cutoff values. The average rate of submission of true positive cases between December 1, 2008, and December 1, 2010, was 5.1 cases/day. This cumulative evidence generated 91 high and 23 low cutoff target ranges. The overall proportion of cutoff values within the respective target range was 42% (2,269/5,341)., Conclusion: An unprecedented level of cooperation and collaboration has allowed the objective definition of cutoff target ranges for 114 markers to be applied to newborn screening of rare metabolic disorders.

Celiac disease (CD) is an autoimmune disorder caused by a permanent sensitivity to gluten in genetically susceptible individuals. Accurate diagnosis of CD at an early stage and its treatment with a gluten-free diet (GFD) are important for optimum treatment and prognosis. Recently, by employing a noninvasive metabonomic approach, we have shown that CD has a well-defined metabonomic signature. Here we address potential CD patients, defined as subjects who do not have, and have never had, a jejunal biopsy consistent with clear CD, and yet have immunological abnormalities similar to those found in celiac patients. Sixty-one overt CD patients at diagnosis, 29 patients with potential CD, and 51 control subjects were examined by (1)H NMR of their serum and urine: out of 29 potential CD patients, 24 were classified as CD and 5 as control subjects. Potential CD largely shares the metabonomic signature of overt CD. Most metabolites found to be significantly different between control and CD subjects were also altered in potential CD. Our results demonstrate that metabolic alterations may precede the development of small intestinal villous atrophy and provide a further rationale for early institution of GFD in patients with potential CD, as recently suggested by prospective clinical studies.

Hyperammonemia is a true neonatal emergency with high toxicity for the central nervous system and developmental delay. The causes of neonatal hyperammonemia are genetic defects of urea cycle enzymes, organic acidemias, lysinuric protein intolerance, hyperammonemia-hyperornithinemia- homocitrullinemia syndrome, transient hyperammonemia of the newborn, and congenital hyperinsulinism with hyperammonemia. In some of these conditions the high blood ammonia levels are due to the reduction of N-acetylglutamate, an essential cofactor necessary for the function of the urea cycle, or to the reduction of carbamoyl-phosphate synthase-I activity. In these cases, N-carbamylglutamate (carglumic acid) can be administered together with the conventional therapy. Carglumic acid is an analog of N-acetylglutamate that has a direct action on carbamoyl-phosphate synthase-I. Its effects are reactivation of the urea cycle and reduction of plasma ammonia levels. As a consequence it improves the traditional treatment, avoiding the need of hemodialysis and peritoneal dialysis. In this review we evaluate the possible field of application of carglumic acid and its effectiveness and safety.

Rufinamide (RUF) is a new antiepileptic drug with efficacy in several types of seizures. The aim of this study was to evaluate the use of dried blood spot (DBS) specimens to determinate RUF levels during treatment. Therapeutic drug monitoring of RUF could be useful in routine clinical practice. Advantages of DBS include short collection time, low invasiveness, ease and low cost of sample collection, transport and storage. The analysis was performed in selected reaction monitoring (SRM) mode. The calibration curve in matrix was linear in the concentration range of 0.008-0.8 mg/L (0.48-47.60 mg/L in DBS) of rufinamide with correlation coefficient value of 0.996. In the concentration range of 0.48-47.6 mg/L, the coefficients of variation in DBS were in the range 1.58-4.67% and the accuracy ranged from 89.73% to 107.32%. The sensitivity and specificity of tandem mass spectrometry allow now high throughput rufinamide analysis. This new assay has favourable characteristics being highly precise and accurate. The published HPLC-UV methods also proved to be precise and accurate, but required not less than 0.2-0.5 mL of plasma and are therefore unsuitable for sample collection in neonates in whom obtaining larger blood samples is not convenient or possible., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Purpose: Oxygen-induced retinopathy (OIR) is a model for human retinopathy of prematurity (ROP). In OIR mice, this study determined whether blockade of β-adrenergic receptors (β-ARs) with propranolol influences retinal levels of proangiogenic factors, retinal vascularization, and blood-retinal barrier (BRB) breakdown., Methods: Propranolol was administered subcutaneously and picropodophyllin (PPP) intraperitoneally. Intravitreal injections of vascular endothelial growth factor (VEGF) were performed. Messengers of β-ARs, VEGF, its receptors, IGF-1 and IGF-1R were measured with quantitative RT-PCR. VEGF content was determined with ELISA. β-ARs, hypoxia-inducible factor (HIF)-1α, occludin, and albumin were measured with Western blot. Retinal localization of β3-ARs was determined by immunohistochemistry. Retinopathy was assessed by scoring fluorescein-perfused retinas, and plasma extravasation was visualized by Evans blue dye., Results: Hypoxia did not influence β-AR expression, except that it increased β3-AR protein with dense β3-AR immunoreactivity localized to engorged retinal tufts. Hypoxia upregulated VEGF, IGF-1, their receptors, and HIF-1α. Propranolol dose-dependently reduced upregulated VEGF and decreased hypoxic levels of IGF-1 mRNA and HIF-1α. Blockade of IGF-1R activity with PPP did not influence propranolol's effects on VEGF. Retinal VEGF in normoxic mice or VEGF in brain, lungs, and heart of the OIR mice were unaffected by propranolol. Propranolol ameliorated the retinopathy score, restored occludin and albumin, and reduced hypoxia-induced plasma extravasation without influencing the vascular permeability induced by intravitreal VEGF., Conclusions: This is the first demonstration that β-AR blockade is protective against retinal angiogenesis and ameliorates BRB dysfunction in OIR. Although the relevance of these results to infant ROP is uncertain, the findings may help to establish potential pharmacologic targets based on β3-AR pharmacology.

Tyrosinemia type I is a genetic disorder characterized by accumulation in the blood and urine of the toxic metabolite succinylacetone (SUAC), not detectable in healthy samples. In many countries, newborns are screened for tyrosinemia type I using tyrosine as a primary marker. Unfortunately, tyrosine accumulation may take longer to occur and it may be not obvious when specimens are collected, in the first few days of life, as for newborn screening. In 2008, we reported changes to simultaneously measure acylcarnitines, amino acids, and SUAC during expanded newborn screening. We established the usefulness of this method after identifying a first asymptomatic newborn affected by tyrosinemia type I. Now we report a second infant with positive SUAC screening result (14.1 μmol/L, n.v. < 2) and normal tyrosine concentration (74 μmol/L; n.v. < 250). We also performed molecular analysis of FAH gene in both patients after diagnosis at newborn screening. They had consanguineous parents and were both homozygous for two known disease-causing mutations of the FAH gene. The outcome of patients detected in the MS/MS screening is significantly favorable. We also report our results of newborn screening for tyrosinemia type I before and after inclusion of SUAC as a primary marker for this disease.

The analysis of organic acids in urine is commonly included in routine procedures for detecting many inborn errors of metabolism. Many analytical methods allow for both qualitative and quantitative determination of organic acids, mainly in urine but also in plasma, serum, whole blood, amniotic fluid, and cerebrospinal fluid. Liquid-liquid extraction and solid-phase extraction using anion exchange or silica columns are commonly employed approaches for sample treatment. Before analysis can be carried out using gas chromatography-mass spectrometry, organic acids must be converted into more thermally stable, volatile, and chemically inert forms, mainly trimethylsilyl ethers, esters, or methyl esters.

Background: Despite new therapeutic approaches have improved the prognosis of newborns with retinopathy of prematurity (ROP), an unfavourable structural and functional outcome still remains high. There is high pressure to develop new drugs to prevent and treat ROP. There is increasing enthusiasm for anti-VEGF drugs, but angiogenic inhibitors selective for abnormal blood vessels would be considered as an optimal treatment.In an animal experimental model of proliferative retinopathy, we have recently demonstrated that the pharmacological blockade of beta-adrenoreceptors improves retinal neovascularization and blood retinal barrier breakdown consequent to hypoxia. The purpose of this study is to evaluate the propranolol administration in preterm newborns suffering from a precocious phase of ROP in terms of safety and efficacy in counteracting the progression of retinopathy., Methods/design: Preterm newborns (gestational age at birth lower than 32 weeks) with stage 2 ROP (zone II-III without plus) will be randomized, according to their gestational age, to receive propranolol added to standard treatment (treatment adopted by the ETROP Cooperative Group) or standard treatment alone. Propranolol will be administered until retinal vascularization will be completely developed, but not more than 90 days. Forty-four participants will be recruited into the study. To evaluate the safety of propranolol administration, cardiac and respiratory parameters will be continuously monitored. Blood samplings will be performed to check renal, liver and metabolic balance. To evaluate the efficacy of propranolol, the progression of the disease, the number of laser treatments or vitrectomies, the incidence of retinal detachment or blindness, will be evaluated by serial ophthalmologic examinations. Visual function will be evaluated by means of behavioural standardized tests., Discussion: This pilot study is the first research that explores the possible therapeutic role of beta blockers in ROP. The objective of this research is highly ambitious: to find a treatment simple, inexpensive, well tolerated and with few adverse effects, able to counteract one of the major complications of the prematurity. Any favourable results of this research could open new perspectives and original scenarios about the treatment or the prevention of this and other proliferative retinopathies., Trial Registration: Current Controlled Trials ISRCTN18523491; ClinicalTrials.gov Identifier NCT01079715; EudraCT Number 2010-018737-21.

Objective: To investigate whether topiramate associated with mild or deep hypothermia in asphyxiated term infants is safe in relation to the short-term outcome., Study Design: We report on 27 consecutive asphyxiated newborns who were treated with whole body hypothermia and 27 additional consecutive newborns with hypothermia who were co-treated with oral topiramate, once a day for 3 consecutive days, at 2 different doses., Results: Newborns were divided in 6 groups according to the depth of hypothermia and the association with higher or lower topiramate dosage. A statistical comparison of the groups identified some differences in biochemical and hemodynamic variables, but no adverse effects attributable to topiramate were detected. There were no statistically significant differences in the groups in short-term outcomes, survival rate at discharge, or incidence of pathologic brain magnetic resonance imaging., Conclusion: Although the number of newborns in this study was limited, the short-term outcome and the safety data appear to support the evaluation of topiramate in clinical trials to explore its possible additive neuroprotective action., (Copyright (c) 2010 Mosby, Inc. All rights reserved.)

Cell suspension cultures of Vitis vinifera L., particularly after elicitation, have shown the ability to biosynthesize several stilbenoids. In this work the application of specific tandem mass spectrometry (MS/MS) experiments and the integration with data from UV-vis spectra, available only for the main compounds of the extract, allowed the detection of over 20 different stilbenoids. Up to eight monoglicosides, both monomers and dimers, a high number of dimer forms (up to 17), belonging to the group of viniferins and to their oxidized derivatives, were identified in the extract of Malvasia cell cultures. Moreover, the selectivity and sensitivity of the method allowed detection also of a few cis derivatives (up to 3) present in very low concentrations and presumably produced by the light exposure during treatment of the sample. To the best of the authors' knowledge, there are no literature data on MS/MS experiments targeted to simultaneously study monomeric and dimeric stilbenoids, both as free and glycosilated forms, in a complex mixture from V. vinifera cell suspension cultures., (Copyright 2010 John Wiley & Sons, Ltd.)

In propionic aciduria and methylmalonic aciduria, hyperammonemia as a symptom of metabolic decompensation is one of the major clinical problems. Hyperammonemia is a true neonatal emergency with high mortality and neurological complications in most survivors. It requires a rapid and vigorous treatment in order to normalize the ammonia concentration as fast as possible. We report on two full-term neonates, one with propionic aciduria and the other with methylmalonic aciduria, whose plasma ammonia concentrations responded dramatically to oral N-carbamylglutamate. N-carbamylglutamate, added to the classic treatment, quickly normalized plasma ammonia levels in both patients and avoided the need of hemodialysis or peritoneal dialysis. A particularly sudden fall of ammonia was obtained in one patient through beginning N-carbamylglutamate treatment precociously., (Copyright 2009 S. Karger AG, Basel.)

Advantages of dried blood spot include low invasiveness, ease and low cost of sample collection, transport, and storage. We used tandem mass spectrometry (LC-MS/MS) to determine phenobarbital levels on dried blood spot specimens and compared this methodology to commercially available particle enhanced turbidimetric inhibition immunoassay (PETINIA) in plasma/serum samples. The calibration curve in matrix using D(5)-phenobarbital as internal standard was linear in the phenobarbital concentration range of 1-100 mg/L (correlation coefficient 0.9996). The coefficients of variation in blood spots ranged 2.29-6.71% and the accuracy ranged 96.54-103.87%. There were no significant differences between the concentrations measured using PETINA and LC-MS/MS (both had similar precision and accuracy) however, LC-MS/MS allows at least 1.5 times higher throughput of phenobarbital analysis and additionally offers ease of sample collection which is particularly important for newborns or small infants.

Background: Insulin-resistant hyperglycaemia may occasionally complicate the clinical course of organic acidaemias., Study Design: Clinical observation., Results: Two term infants, one suffering from acute early-onset methylmalonic acidaemia, the other suffering from acute early-onset propionic acidaemia, presented acutely with dehydration, ketoacidosis, and hyperammonaemia. Urinary organic acid, plasma amino acids, and blood and plasma acylcarnitine analysis allowed the diagnosis of methylmalonic and propionic acidaemias. The detection of the novel c.481G>A (p.Gly161Arg) and the known c.655A>T (p.Asn219Tyr) MUT gene mutations identified the first patient as affected by methylmalonic acidaemia mut type. The high increase of propionylcarnitine after carnitine administration in both patients suggested a greatly elevated metabolic intoxication. Both newborns showed insulin-resistant hyperglycaemia. Patient 1 died, but patient 2, after a strong reduction of glucose administration, survived. To our knowledge, this is the only patient with this complication who survived., Conclusion: Insulin-resistant hyperglycaemia complicating neonatal onset of methylmalonic and propionic acidaemias is probably a marker of a serious disease. One patient with this complication survived after a strong reduction of glucose administration. Even if this is probably only a partial intervention, we hypothesize that in this situation a reduction of glucose administration can reduce almost the risk of persistent hyperglycaemia. Further studies are required to confirm our hypothesis.

Purpose: Therapeutic hypothermia reduces mortality and neurologic impairment in neonates with hypoxic-ischemic encephalopathy. Topiramate exerts a neuroprotective effect in asphyxiated neonatal animal models. However, no studies have investigated the association of hypothermia and topiramate, because topiramate pharmacokinetics during hypothermia and the optimal administration schedule are unknown. The influence of hypothermia on topiramate pharmacokinetics was evaluated in asphyxiated neonates treated with prolonged whole-body hypothermia and topiramate., Methods: Thirteen term newborns were treated with mild or deep whole body hypothermia for 72 h; all received oral topiramate, 5 mg/kg once a day for the first 3 days of life, and seven had concomitant phenobarbital treatment. Topiramate concentrations were measured on serial dried blood spots., Results: Topiramate concentrations were within the reference range in 11 of 13 newborns, whereas concentrations exceeded the upper limit in 2 of 13, both newborns on deep hypothermia. Topiramate concentrations reached a virtual steady state in nine newborns, for whom pharmacokinetic parameters were calculated. Values of topiramate maximal and minimal concentration, half-life, average concentration, and area under the time-concentration curve resulted in considerably higher values than those reported in normothermic infants. With respect to normothermic infants, time of maximal concentration was mildly delayed and apparent total body clearance was lower, suggesting slower absorption and elimination. Pharmacokinetic parameters did not differ significantly between infants on deep versus mild hypothermia and in those on topiramate monotherapy versus add-on phenobarbital., Conclusion: Most neonates on prolonged hypothermia treated with topiramate 5 mg/kg once a day exhibited drug concentrations within the reference range for the entire treatment duration.

A CE/UV method was developed to separate by a micellar system the four DNA bases and other five purinic-pyrimidinic compounds (5-methyl-cytosine, uracil, xanthyne, hypoxanthyne and 5-bromo-uracil). Selectivity, precision, accuracy and sensitivity were assessed and proved to be suitable for the analysis of the primary structure of DNA. This method was adopted to study 16 aged samples including two Egyptian mummies, formaldehyde-fixed paraffin-embedded tissues and other forensic specimens. Lower relative values of the four canonical unmodified DNA bases (uDNAb) and more complex pherograms were found in the aged samples when compared with the modern controls. The results of the CE analysis, together with those obtained by classical molecular methods (agarose gel electrophoresis, DNase I and RNase A assays, and UV spectrophotometry), were finally evaluated for assessing the reliability of STR typing. Since samples with low uDNAb showed no amplification or unreliable STR profiles, the uDNAb value is discussed as a further quality criterion in the evaluation of the genetic data obtained from aged samples.

The aim of this study was to set up a robust method suitable for large-scale studies (screening) with a minimized preparation process and with reduced running costs, for measuring five enzyme activities on dried blood spots by a new and simplified tandem mass spectrometry-based method. After incubation, all 5 reaction mixtures, carried out separately, were stopped, combined together, and centrifuged. The cleaning-up of the injected mixture was performed through a fast online trapping step preceding a liquid chromatography/tandem mass-spectrometry measurement. This method takes only 4 min as analysis run time and without any purification following the enzymatic reaction. We assessed the effectiveness of this approach in assaying the enzymatic activities on dried blood spots from 10 patients affected by "Pompe", 6 by "Gaucher", 12 by "Fabry", 3 by "Niemann-Pick" A/B, and 2 by "Krabbe" diseases. Reference values were established on 5000 healthy newborns and 300 healthy adults. All affected patients showed enzymatic activities below the normal range. In heterozygous carriers (18 for Fabry, 10 for Pompe, and 4 for Gaucher disease) the activities were slightly lower than in control subjects. The results show that the method set out in its simplicity, low costs, and low processes preparations can be fully applicable to a mass screening.

In an expanded newborn screening program for inborn errors of metabolism by LC-MS/MS in Tuscany, six newborns out of 169,000 showed decreased blood citrulline levels. In one of them, molecular analysis of the OTC gene identified the known p.Trp265Leu mutation, which is correlated with late-onset ornithine transcarbamylase deficiency (OTCD). Hypocitrullinemia is not a reliable marker for OTCD newborn screening, especially for late-onset forms that may exhibit normal citrulline levels. However, when hypocitrullinemia is detected in a newborn in whom intestinal dysfunction and prematurity have been excluded, OTCD should be investigated first because of the OTCD incidence (1:14,000) and the small size of the OTC gene coding sequence.

We performed a study on 25 common carotid arteries (CCA) affected by atherosclerotic plaques, comparing them to 25 normal CCA. We researched the presence and localisation of atrial natriuretic factor (ANF) and oxytocin (OX) by immunohistochemistry. We found ANF abundantly expressed in plaques, mainly localising in intima and subintima layers; by contrast normal vessels were scarcely positive or negative; finally, OX resulted negative or weakly positive in both atherosclerotic and normal vessels. Our results show for the first time ANF presence in atherosclerotic plaques of CCA and are in agreement with other studies, mainly performed in vitro, that hypothesise the involvement of this molecule in the modulation of atherosclerotic inflammatory lesions and its use as anti-inflammatory agent.

A new analytical approach using matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) for the study of honeybee venom is shown. In vitro and in vivo models simulating the bee sting have been developed using live honeybees and, as the envenomation sites, pig ears and rat legs; MALDI MSI has been used to map, over time, the diffusion and distribution of three venom allergens (Api m 1, Api m 4, and Api m 6) and two venom toxins (apamine and mast cell degranulating peptide). In conjunction with other classical biochemical techniques and high resolution mass spectrometry (HRMS), structural data have been obtained that contribute to current understanding of honeybee venom composition. Initial data have also been obtained demonstrating the feasibility of mapping the organism's response to the sting. The opportunity to monitor venom diffusion and the organism's response at the same time might open new pathways for in vivo preclinical studies in designing and testing new venom immunotherapy (VIT).

Topiramate (TPM) is a new antiepiletic drug with efficacy in several types of seizures. Therapeutic drug monitoring of TPM is essential for effective patient management. The aim of this study was to evaluate the use of dried blood spot (DBS) specimens to determinate the TPM levels during the treatment. Advantages of DBS include short collection time, low invasiveness, ease and low cost of sample collection, transport and storage. Performance comparison between this method and the commercially available fluorescence-polarization immunoassay (FPIA) was made. The analysis was performed in selected reaction monitoring (SRM) mode. The calibration curve in matrix using D(12)-topiramate was linear in the concentration range of 0.0166-1.66mg/L (0.5-50mg/L in DBS) of topiramate with correlation coefficient value of 0.9985. In the concentration range of 0.5-50mg/L, the coefficients of variation in DBS were in the range 2.13-11.85% and the accuracy ranged from 93.93% to 110.67%. There was no significant differences between the concentrations (ranging 0.5-50mg/L) measured both FPIA on venous samples and LC-MS/MS assay on simultaneous DBS samples. The sensitivity and specificity of tandem mass spectrometry allow now high throughput topiramate analysis (the improvement was three times in comparison with FPIA). This new assay has favourable characteristics being highly precise and accurate. FPIA also proved to be precise and accurate, but is not always suitable for the sample collection in neonates in whom obtaining larger blood samples is not convenient or possible.

We report on our 6-year experience of expanded newborn screening by tandem mass spectrometry in Tuscany (Italy), the first Italian Region to screen all newborns for more than 40 inborn errors of metabolism: organization, diseases observed and updates on methods to reduce false-positive and false-negative tests are described. Blood collection is recommended between 48 and 72 h of life. Blood spots are sent daily by courier to laboratory. When a positive result occurs, two subsequent procedures are followed: for disorders with possible acute metabolic decompensation, the baby is immediately recalled and clinical examinations and confirmatory tests are performed; for the other disorders, the nursery provides for a second blood spot. If the test is positive, clinical examinations and confirmatory tests are performed. In both cases, if confirmatory tests are positive, a treatment and a follow-up programme are started. Up to now, spots from 160 000 infants have been analysed and 80 affected patients have been identified (disorders of amino acids, organic acids and fatty acids metabolism). We describe adjustments to cut-off values, the introduction of a second-tier test for propionic acidaemia and for methylmalonic aciduria, the inclusion of succinylacetone in the panel of metabolites, and protocols for premature infants and for newborns on parenteral nutrition or transfused. These changes resulted in a reduction in recalls from 1.37% to 0.32% and consequently of working time and parental stress. Avoiding false-negatives by using more specific markers and minimizing the false-positive rate with second-tier testing is important for a successful newborn screening programme.

In expanded newborn screening programs by liquid chromatography/tandem mass spectrometry false negatives for tyrosinemia type I are a significant problem. We describe a method for inclusion of succinylacetone in order to avoid false negatives. We studied spots from 13,000 neonates born in Tuscany (January-May 2007) and ten spots from six patients with tyrosinemia type I. The traditional screening method was modified by adding dioxooctanoid acid (or 13C2-succinylacetone) as an internal standard to the methanolic solution of deuterated acylcarnitines and amino acids. A hydrazine solution was added to the mixture. The times of extraction, butylation and drying were only slightly prolonged. Specific multiple reaction monitoring for derivatized and labelled succinylacetone and dioxooctanoic acid was carried out. The assays were linear up to 100 micromol/L for succinylacetone. Intra- and inter-day imprecision data were in the range of 1.34% to 7.09% and 3.50% to 4.49%. Limits of detection and of quantification were 0.2 micromol/L and 0.4 micromol/L, respectively. Recovery ranged from 97.02% to 100.29%. Succinylacetone levels in samples from unaffected neonates were very close to the detection limit. Of the 46 recalls, eight (17.4%) were for abnormal tyrosine levels and all these cases had succinylacetone levels within the normal range (<2.4 micromol/L). In ten spots from six affected patients succinylacetone values ranged from 3.3 to 35.0 micromol/L. Including succinylacetone in newborn screening programs for amino acids and acylcarnitines avoids false-negative results for tyrosinemia type I. Newborn screening laboratories should consider implementing these modifications.

Malonic aciduria is a rare autosomal recessive disorder caused by deficiency of malonyl-CoA decarboxylase, encoded by the MLYCD gene. We report on a patient with clinical presentation in the neonatal period. Metabolic investigations led to a diagnosis of malonyl-CoA decarboxylase deficiency, confirmed by decreased activity in cultured fibroblasts. High doses of carnitine and a diet low in lipids led to a reduction in malonic acid excretion, and to an improvement in his clinical conditions, but at the age of 4 months he died suddenly and unexpectedly. No autopsy was performed. Molecular analysis of the MLYCD gene performed on the proband's RNA and genomic DNA identified a previously undescribed mutation (c.772-775delACTG) which was homozygous. This mutation was present in his mother but not in his father; paternity was confirmed by microsatellite analysis. A hypothesis of maternal uniparental disomy (UPD) was investigated using fourteen microsatellite markers on chromosome 16, and the results confirmed maternal UPD. Maternal isodisomy of the 16q24 region led to homozygosity for the MLYCD mutant allele, causing the patient's disease. These findings are relevant for genetic counselling of couples with a previously affected child, since the recurrence risk in future pregnancies is dramatically reduced by the finding of UPD. In addition, since the patient had none of the clinical manifestations previously associated with maternal UPD 16, this case provides no support for the existence of maternally imprinted genes on chromosome 16 with a major effect on phenotype.

Background: The expansion of newborn screening programs has increased the number of newborns diagnosed with inborn errors of metabolism in the presymptomatic phase, but it has also increased the number of costly, stress-producing false-positive results. Because propionylcarnitine (C3) is one of the analytes most frequently responsible for false-positive results, we aimed to develop a rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to identify free methylmalonic (MMA) and 3-OH propionic (3OH-PA) acids in blood spots., Methods: We studied newborn screening spots from 250 healthy controls; 124 from infants with abnormal C3, of whom only 5 (4%) were truly affected; 124 from infants with altered isolated methylmalonylcarnitine; and 4 from clinically diagnosed patients. Whole blood was eluted from a 3.2-mm dried blood spot by a CH(3)CN/H(2)O 7:3 and 5 mL/L formic. This extract was injected into a LC-MS/MS equipped with pneumatically assisted electrospray without derivatization. Total analysis time was 5 min per sample., Results: The assays were linear up to 3300 nmol/L for both metabolites. Intra- and interassay imprecision data were 3.6%-8% and 3.1%-6%, respectively, for MMA and 5.2%-20% and 3.6%-17% for 3OH-PA. Limit of detection and limit of quantitation were 1.95 and 4.2 micromol/L, respectively, for MMA and 8 and 10 micromol/L for 3OH-PA. The recoveries were 92.9%-106.1%. No deterioration was noted on the columns after 500 chromatographic runs. If the new method had been used as a 2nd-tier test for the 124 samples, only the 5 true positives would have been recalled for additional samples, and the positive predictive value would have been 100%., Conclusions: This method has the potential to markedly reduce false-positive results and the associated costs and anxiety. It may also be suitable for diagnosing and routinely monitoring blood spots for methylmalonic aciduria and propionic acidemia.

Introduction: Morphological anomalies of the extracranial internal carotid artery (ICA) cause symptomatic cerebrovascular insufficiency in 4-16% of the cases. The aim of the present study is to evaluate macroscopic and microscopic features of a group of extracranial ICA anomalies, specifically kinking, coiling, and tortuosity, eventually affecting the surgical approach., Materials and Methods: From January 2003 to December 2005, 10 out of 169 (6%) revascularized patients (pts) were operated upon because of an ICA anomaly. They were all but two symptomatics. Seven pts were treated by ICA transection and end-to-side reimplantation of the ICA at the level of the carotid bulb; three pts were treated by ICA resection and end-to-end anastomosis. In all the cases a segment of ICA was resected; in three cases one more segment was also obtained from a common carotid artery (CCA) and these specimens were histologically examined. Patients were followed-up through a 3-year period., Results: No pts died and none suffered of neurologic events. Duplex scan and arteriographic postoperative control showed the correct surgical reconstruction. Matching preoperative clinical findings with presence or absence of significant atherosclerotic stenotic lesion, we found out a positive cerebral CT in one pt (20%) in both groups; fluent neurological deficit was preeminent in pts with pure ICA anomalies (40% vs. 0%) (P = 0.2); pts with pure ICA anomalies were significantly younger than 65 years old (80% vs. 0%) (P = 0.03) and males were more involved by pure ICA anomalies (60% vs. 40%) (P = 0.1). The histological examination of ICA specimens showed a reduction of elastic fibers and muscular cells with a compensative increase of connective fibers., Conclusions: At our knowledge this is the first study focused on ICA anomalies like kinking, coiling, and tortuosity, comparing histologic features of CCA and ICA specimens coming from the same affected carotid axis. Our results, although preliminary, show elastic and muscular tissue substituted by loose connective tissue, configuring a metaplasia of tunica media limited to the ICA. Our hypothesis is that extracranial ICA, being a segment of transition between an elastic vessel (CCA) and a muscular vessel (intracranial ICA), is particularly subject to metaplastic transformation, analogously to other transition zones in human body. Our purpose is now to confirm by ultrastructural and molecular biology techniques, in a wider series, the presence of this metaplasia, since this could condition also the revascularization techniques.

Purines and pyrimidines are the basic constituents of DNA and RNA and constitute the basis of at least 50 other important compounds that serve equally vital but separate roles as integral components of intracellular mononucleotide pools. They maintain the supply of these basic components to the different nucleotide pools through an extremely efficient mechanism involving the degradation and recycling of the daily waste products of normal cell turnover. We have developed an LC-MS/MS diagnostic and routine monitoring method for known defects due to both purine and pyrimidine metabolism in a single analysis. Precision tests were made by spiking several urine samples with different creatinine concentrations. For nonspiked low-creatinine urine, intraday precision was in the range of 0.1-9.8% and interday precision was between 1.6 and 14.1%. For nonspiked high-creatinine urine, intraday precision was in the range 0.5-17.2% and interday precision was between 1.5 and 29%. Limit-of-detection (LOD) was in the range 0.1-10 micromol/l and limit-of-quantification (LOQ) in the range of 0.2-15 micromol/l. The current 'dilute and shoot' approach monitors many metabolites, and utilizes a reverse phase chromatographic analysis with a detection requiring 17 min of analysis time. Tandem mass spectrometry and isotope dilution technique enable the accurate quantitation of more than 30 metabolites in one analysis., (Copyright 2006 John Wiley & Sons, Ltd.)

We describe two patients affected by Barth syndrome. Their symptoms became manifest on respectively the third and first day of their lives. Clinical presentation included poor sucking, lethargy, hypotonia, hypothermia and cardiomyopathy. Laboratory findings such as hypoglycaemia, metabolic acidosis, elevated transaminases, hyperlactacidaemia and mild hyperammonaemia pointed to an inborn error of energy metabolism with possible mitochondrial involvement. Molecular analysis of the TAZ (G4.5) gene showed the c.877G > A mutation leading to the G197R amino acid substitution in patient 1, and the new splice donor c.829 + 1G > A genetic lesion in patient 2.

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disorder characterized by severe gastrointestinal dysmotility, cachexia, ptosis, ophthalmoparesis, peripheral neuropathy and leukoencephalopathy. The disease is due to a thymidine phosphorylase defect. This enzyme catalyses the phosphorolysis of thymidine to thymine and deoxyribose 1-phosphate. For this reason, increased levels of thymidine in plasma and urine are found in MNGIE patients. Haemodialysis can reduce circulating plasma thymidine levels and can be beneficial in some MNGIE patients. We developed a fast analytical method based on HPLC-ESI-MS/MS capable of identifying pyrimidine nucleotides (thymine, cytosine, uracil) and nucleosides (thymidine, citidine, uridine) in plasma and urine after direct dilution of the samples without pre-treatment. In the patient studied, we observed a significant reduction of plasmatic and urinary thymidine levels during and after dialysis. However, we noted a progressive reduction of the initial thymidine level after some dialytic trials. This method will be useful not only for thymidine level follow-up during dialysis in MNGIE patients but also for the improvement of the diagnosis or diagnostic suspect in other pyrimidine defects such as dihydropyrimidine dehydrogenase deficiency, dihydropyrimidinase deficiency and ureidopropionase deficiency.

Two new antibacterial peptides, denominated as Dominulin A and B, have been found on the cuticle and in the venom of females of the social paper wasp Polistes dominulus. The amino acidic sequence of the two peptides, determined by mass spectrometry, is INWKKIAE VGGKIL SSL for Dominulin A (MW = 1854 Da) and INWKKIAEIGKQVL SAL (MW = 1909 Da) for Dominulin B. Their presence on the cuticle was confirmed using MALDI-TOF by means of micro-extractions and direct analyses on body parts. The presence in the venom and the primary structure of the dominulins suggest their classification in the mastoparans, a class of peptides found in the venom of other Aculeate hymenoptera. Their antimicrobial action against Gram+ and Gram- bacteria fits in the range of the best natural antimicrobial peptides. Dominulins can represent an important defense of the colony of Polistes dominulus against microbial pathogens.

This work is focused on the recovery and structural characterisation of bioactive compounds from SOR (solid olive residue), a low cost and widely available by-product in the Mediterranean basin. In the light of the interesting biological activities attributed both to secoiridoid and phenylpropanoids, the aim of this work was to carry out a systematic tandem mass spectrometric study for the identification and characterisation of these two class of compounds extracted from SOR derived from Coratina, a cultivar widely diffused in the south of Italy. Five phenylpropanoids, in addition to verbascoside, and two new secoiridoids were identified., (Copyright (c) 2006 John Wiley & Sons, Ltd.)

Genetic and biochemical prenatal diagnosis was performed at 11 weeks of gestation in a family with a proband affected by mut methylmalonic aciduria (MMA) and homozygotes for the MUT gene c.643G>A (p.Gly215Ser) mutation. Both chorionic villus and amniotic fluid samples were used. The presence of high levels of methylmalonic acid and propionylcarnitine determined by gas chromatography/mass spectrometry and LC/MS/MS analysis, respectively, and the identification of the p.Gly215Ser at a homozygous level in foetal DNA allowed a certain, rapid and early diagnosis. To our knowledge, this is the first mut MMA prenatal diagnosis carried out by genetic and biochemical approach.

Objectives: We report on the first prenatal molecular diagnosis of holocarboxylase synthetase (HLCS) deficiency in the fourth pregnancy of an at-risk family. This disorder is a rare autosomal recessive inborn error of metabolism, leading to a multiple carboxylase defect (MCD). HLCSD diagnosis was performed postmortem in the proband on DNA from autoptic biological material. Molecular analysis of the proband's entire HLCS gene by direct sequencing identified the R508W amino acid change, at the homozygous status., Methods: Fetal DNA was isolated from chorionic villus sampling at 11 weeks of gestation. Direct sequencing of exon 6 of the fetal HLCS gene was performed., Results: The R508W mutation was identified in the fetal DNA at the homozygous level. The genetic lesion was confirmed on abortive tissue., Conclusion: Molecular diagnosis has several advantages over enzymatic activity assay of carboxylases in chorionic villi or amniocytes. It can be performed earlier, is faster, and the response time is shorter., (Copyright 2005 John Wiley & Sons, Ltd)

Solid olive residues (SOR) are byproducts of the olive-milling process, but they have an increasing importance in the pharmaceutical industry due to their rich content of biophenols. Such compounds are studied widely for their antioxidant and antimicrobial activities, but there is a lack of information about their quantitative recovery. This research highlighted the key role played both by the selection of the cultivar and by the degree of olive fruit ripening on the phenolic content on the SOR. The extraction methods were selected to reach the best quantitative results mainly using a safe food solvent. In light of the results the Soxhlet extraction with ethanol could be proposed as preferential for a higher recovery of verbascoside and its analogues.