Hypermethioninemia in Campania: Results from 10 years of newborn screening.

In the last years tandem mass spectrometry (MS/MS) has become a leading technology used for neonatal screening purposes. Newborn screening by MS/MS on dried blood spot samples (DBS) has one of its items in methionine levels: the knowledge of this parameter allows the identification of infant affected by homocystinuria (cystathionine ß-synthase, CBS, deficiency) but can also lead, as side effect, to identify cases of methionine adenosyltransferase (MAT) type I/III deficiency. We started an expanded newborn screening for inborn errors of metabolism in Campania region in 2007. Here we report our ten years experience on expanded newborn screening in identifying patients affected by hypermethioninemia. During this period we screened approximately 77,000 infants and identified two cases: one case of classical homocystinuria and one patient affected by defect of MAT I/III. In this paper we describe these patients and their biochemical follow-up and review the literature concerning worldwide newborn screening reports on incidence of CBS and MAT deficiency.

Targeted metabolomic profiling in rat tissues reveals sex differences.

Sex differences affect several diseases and are organ-and parameter-specific. In humans and animals, sex differences also influence the metabolism and homeostasis of amino acids and fatty acids, which are linked to the onset of diseases. Thus, the use of targeted metabolite profiles in tissues represents a powerful approach to examine the intermediary metabolism and evidence for any sex differences. To clarify the sex-specific activities of liver, heart and kidney tissues, we used targeted metabolomics, linear discriminant analysis (LDA), principal component analysis (PCA), cluster analysis and linear correlation models to evaluate sex and organ-specific differences in amino acids, free carnitine and acylcarnitine levels in male and female Sprague-Dawley rats. Several intra-sex differences affect tissues, indicating that metabolite profiles in rat hearts, livers and kidneys are organ-dependent. Amino acids and carnitine levels in rat hearts, livers and kidneys are affected by sex: male and female hearts show the greatest sexual dimorphism, both qualitatively and quantitatively. Finally, multivariate analysis confirmed the influence of sex on the metabolomics profiling. Our data demonstrate that the metabolomics approach together with a multivariate approach can capture the dynamics of physiological and pathological states, which are essential for explaining the basis of the sex differences observed in physiological and pathological conditions.

Targeted metabolomics in the expanded newborn screening for inborn errors of metabolism.

Inborn errors of metabolism are genetic disorders due to impaired activity of enzymes, transporters, or cofactors resulting in accumulation of abnormal metabolites proximal to the metabolic block, lack of essential products or accumulation of by-products. Many of these disorders have serious clinical consequences for affected neonates, and an early diagnosis allows presymptomatic treatment which can prevent severe permanent sequelae and in some cases death. Expanded newborn screening for these diseases is a promising field of targeted metabolomics. Here we report the application, between 2007 and 2014, of this approach to the identification of newborns in southern Italy at risk of developing a potentially fatal disease. The analysis of amino acids and acylcarnitines in dried blood spots by tandem mass spectrometry revealed 24 affected newborns among 45,466 infants evaluated between 48 and 72 hours of life (overall incidence: 1 : 1894). Diagnoses of newborns with elevated metabolites were confirmed by gas chromatography-mass spectrometry, biochemical studies, and genetic analysis. Five infants were diagnosed with medium-chain acyl CoA dehydrogenase deficiency, 1 with methylmalonic acidemia with homocystinuria type CblC, 2 with isolated methylmalonic acidemia, 1 with propionic acidemia, 1 with isovaleric academia, 1 with isobutyryl-CoA dehydrogenase deficiency, 1 with beta ketothiolase deficiency, 1 with short branched chain amino acid deficiency, 1 with 3-methlycrotonyl-CoA carboxylase deficiency, 1 with formimino-transferase cyclodeaminase deficiency, and 1 with cystathionine-beta-synthase deficiency. Seven cases of maternal vitamin B12 deficiency and 1 case of maternal carnitine uptake deficiency were detected. This study supports the widespread application of metabolomic-based newborn screening for these genetic diseases.

Serum metabolomic profiles suggest influence of sex and oral contraceptive use.

AIM: Considering that the effects of sex and oral contraceptives (OCs) on blood metabolites have been scarcely studied and the fact that protocol designs for clinical trials emphasise the use of contraception for women of childbearing potential, we examined if OCs and sex affect the serum levels of the physiologically relevant amino acids, carnitine and acylcarnitines, using metabolomics approaches. METHODS: Healthy adult men and women were enrolled. They were drug free with the exception of women taking cyclic format OCs (ethinylestradiol + different progestins). OCs-free women were analysed during the follicular phase, and amino acids, free carnitine and acylcarnitines were measured using HPLC or LC-MS/MS, respectively. RESULTS: Men had significantly higher leucine, isoleucine, methionine, asparagine, proline, valine, tyrosine, glutamine+glutamate, glutamate, histidine and citrulline than OCs-free women, while tryptophan was significantly lower in men. OCs use significantly decreased the levels of glycine, proline, histidine, lysine, hydroxyproline and ornithine and increased isoleucine levels when compared with non-user women. OCs use reduced, although not significantly, carnitine levels in women. Total esterified carnitines were higher in untreated women than in OCs users. Globally, the effect of OCs and sex was specific for the individual esterified carnitine. The observed metabolic changes were not attributable to renal or hepatic functions or to differences in body weight. CONCLUSIONS: The assessed parameters were specifically influenced by sex, highlighting the need to have reference values for women and men. The major novelty of this study is the demonstration that OCs specifically change the profiles of serum amino acids and carnitine, which suggests that OCs users and non-users should be represented in clinical trials.

Maternal vitamin B12 deficiency detected in expanded newborn screening.

OBJECTIVES: Besides the inherited form, vitamin B(12) deficiency may be due to diet restrictions or abnormal absorption. The spread of newborn screening programs worldwide has pointed out that non-inherited conditions are mainly secondary to a maternal deficiency. The aim of our work was to study seven cases of acquired vitamin B12 deficiency detected during our newborn screening project. Moreover, we aimed to evaluate vitamin B(12) and related biochemical parameters status on delivering female to verify the consequences on newborns of eventually altered parameters. DESIGN AND METHODS: 35,000 newborns were screened; those showing altered propionyl carnitine (C3) underwent second-tier test for methylmalonic acid (MMA) on dried blood spot (DBS). Subsequently, newborns positive to the presence of MMA on DBS and their respective mothers underwent further tests: serum vitamin B(12), holo-transcobalamin (Holo-TC), folate and homocysteine; newborns were also tested for urinary MMA content. A control study was conducted on 203 females that were tested for the same parameters when admitted to hospital for delivery. RESULTS: Approximately 10% of the examined newborns showed altered C3. Among these, seven cases of acquired vitamin B(12) deficiency were identified (70% of the MMA-positive cases). Moreover, our data show a high frequency of vitamin B(12) deficiency in delivering female (approximately 48% of examined pregnants). CONCLUSIONS: We suggest to monitor vitamin B(12) and Holo-TC until delivery and to reconsider the reference interval of vitamin B(12) for a better identification of cases at risk. Finally, newborns from mothers with low or borderline levels of vitamin B(12) should undergo second-tier test for MMA; in the presence of MMA they should be supplemented with vitamin B(12) to prevent adverse effects related to vitamin B(12) deficiency.

Optimization of an HPLC method for phenylalanine and tyrosine quantization in dried blood spot.

OBJECTIVES: Patients affected by Phenylketonuria (PKU) require lifelong management based on phenylalanine (Phe) and tyrosine (Tyr) restricted intake or tetrahydrobiopterin (BH4) administration. Frequent monitoring of blood concentration of both amino acids during treatment is the key point for clinicians to achieve the best long-term neuropsychological outcome. RESULTS: The present study develops and validates a rapid and simple method for Phe and Tyr quantization in dried blood spot (DBS) since this specimen has the advantage of being low invasive, easily withdrawn even at home and stable if mail-delivered. The validation studies showed the robustness of the method. CONCLUSIONS: Serum and DBS samples from PKU patients were analyzed and compared, finding a good correlation of Phe and Tyr concentrations between the two different matrixes.

Citrulline blood levels as indicators of residual intestinal absorption in patients with short bowel syndrome.

Plasma citrulline is known to be a marker of absorptive enterocyte mass in humans. We evaluated whether citrulline and other blood amino acids are indicators of residual small intestinal length and therefore potential predictors of dependence on parenteral nutrition in the long term. We studied 25 patients with short bowel syndrome (SBS) after at least 18 months since last digestive circuit modification; 24 of them were again evaluated 1 year later. Ten patients were weaned off parenteral nutrition and 15 were dependent on parenteral nutrition. Fifty-four healthy volunteers (28 women and 26 men) served as controls. Amino acid levels were determined on serum with high-performance liquid chromatography (HPLC) as well as on blood and serum with tandem mass spectrometry analysis. Five amino acids (citrulline, leucine, isoleucine, valine and tyrosine) were significantly lower in all SBS patients than in controls, whereas glutamine, measured only by HPLC, was significantly higher. Nevertheless, only serum citrulline measured with HPLC was significantly related to small bowel length. We conclude that HPLC remains the reference methodology to evaluate blood or serum amino acid levels in adult population with SBS.