Newborn screening for homocystinurias: Recent recommendations versus current practice.

PURPOSE: To assess how the current practice of newborn screening (NBS) for homocystinurias compares with published recommendations. METHODS: Twenty-two of 32 NBS programmes from 18 countries screened for at least one form of homocystinuria. Centres provided pseudonymised NBS data from patients with cystathionine beta-synthase deficiency (CBSD, n = 19), methionine adenosyltransferase I/III deficiency (MATI/IIID, n = 28), combined remethylation disorder (cRMD, n = 56) and isolated remethylation disorder (iRMD), including methylenetetrahydrofolate reductase deficiency (MTHFRD) (n = 8). Markers and decision limits were converted to multiples of the median (MoM) to allow comparison between centres. RESULTS: NBS programmes, algorithms and decision limits varied considerably. Only nine centres used the recommended second-tier marker total homocysteine (tHcy). The median decision limits of all centres were ≥ 2.35 for high and ≤ 0.44 MoM for low methionine, ≥ 1.95 for high and ≤ 0.47 MoM for low methionine/phenylalanine, ≥ 2.54 for high propionylcarnitine and ≥ 2.78 MoM for propionylcarnitine/acetylcarnitine. These decision limits alone had a 100%, 100%, 86% and 84% sensitivity for the detection of CBSD, MATI/IIID, iRMD and cRMD, respectively, but failed to detect six individuals with cRMD. To enhance sensitivity and decrease second-tier testing costs, we further adapted these decision limits using the data of 15 000 healthy newborns. CONCLUSIONS: Due to the favorable outcome of early treated patients, NBS for homocystinurias is recommended. To improve NBS, decision limits should be revised considering the population median. Relevant markers should be combined; use of the postanalytical tools offered by the CLIR project (Collaborative Laboratory Integrated Reports, which considers, for example, birth weight and gestational age) is recommended. tHcy and methylmalonic acid should be implemented as second-tier markers.

Phototherapy-induced blistering reaction and eruptive melanocytic nevi in a child with transient neonatal porphyrinemia.

Neonatal blue-light phototherapy induced a blistering reaction followed by eruption of melanocytic nevi on the exposed skin surface of a child with transient neonatal porphyrinemia. New nevi are still developing 4 years after the triggering event. The role of phototoxicity-induced epidermal injury, that of porphyrins and the influence of neonatal blue-light therapy, in this unique phenomenon are discussed.

[Maternal and neonatal vitamin B12 deficiency detected by expanded newborn screening]. / Anyai és újszülöttkori B12-vitamin-hiány felismerése kiterjesztett újszülöttkori szuréssel.

INTRODUCTION: Infant vitamin B12 deficiency can manifest as a severe neurodegenerative disorder and is usually caused by maternal deficiency due to vegetarian diet or pernicious anaemia. Its early recognition and treatment can prevent potentially serious and irreversible neurologic damage. Biochemically, vitamin B12 deficiency leads to an accumulation of methylmalonic acid, homocysteine, and propionylcarnitine. Expanded newborn screening using tandem mass spectrometry may identify neonatal and maternal vitamin B12 deficiency by measurement of propionylcarnitine and other metabolites in the dried blood spot sample of newborns. AIM: To summarize our experiences gained by screening for vitamin B12 deficiency. METHOD: Clinical and laboratory data of vitamin B12-deficient infants diagnosed in Szeged Screening Centre were retrospectively analysed. RESULTS: In Hungary, expanded newborn screening was introduced in 2007. Since then approximately 395 000 newborns were screened in our centre and among them, we identified four newborns with vitamin B12 deficiency based on their screening results. In three cases an elevated propionylcarnitine level and in the fourth one a low methionine level were indicative of vitamin B12 deficiency. We also detected an additional vitamin B12-deficient infant with neurological symptoms at 4 months of age, after a normal newborn screening, because of elevated urinary methylmalonic acid concentration. Vitamin B12 deficiency was secondary to maternal autoimmune pernicious anaemia in all the five infants. As a result of the recognized cases the incidence of infant vitamin B12 deficiency in the East-Hungarian region was 1.26/100 000 births, but the real frequency may be higher. Conslusions: Optimizing the cut off values of current screening parameters and measuring of methylmalonic acid and/or homocysteine in the dried blood spot, as a second tier test, can improve recognition rate of vitamin B12 deficiency. Orv Hetil. 2017; 158(48): 1909-1918.

The fate of tyrosinaemic Hungarian patients before the NTBC aera.

Before the introduction of the NTBC treatment (Orfadine) from two tyrosinemic Hungarian families 1-3 tyrosinemic homozygous male patients died of hepatocellular carcinoma and one patient of hepatocellular carcinoma combined with clear cell renal adenocarcinoma. From the third tyrosinemic family one homozygous girl patient has been treated with NTBC (Orfadine), IMTV-AM, she is symptom-free. Her molecular genetic mutations analysis in the FAH gene detected a common intronel mutation, affecting splicing and of predicted severe effect, IVS6-1 g > t/IVS6-1 g > t with systemic name c.456-1 g > t/c.456-1 g > t (Prof. Magdalena Ugarte).

A new severe acute necrotizing pancreatitis model induced by L-ornithine in rats.

OBJECTIVE: Intraperitoneal administration of large doses of L-arginine is known to induce severe acute pancreatitis in rats. We therefore set out to determine whether metabolites of L-arginine (L-ornithine, L-citrulline, and nitric oxide) cause pancreatitis. DESIGN: The authors conducted an in vivo animal study. SETTING: This study was conducted at a university research laboratory. SUBJECTS: Study subjects were male Wistar rats. INTERVENTIONS: Dose-response and time course changes of laboratory and histologic parameters of pancreatitis were determined after L-arginine, L-ornithine, L-citrulline, or sodium nitroprusside (nitric oxide donor) injection. MEASUREMENTS AND MAIN RESULTS: Intraperitoneal injection of 3 g/kg L-ornithine but not L-citrulline or nitroprusside caused severe acute pancreatitis; 4 to 6 g/kg L-ornithine killed the animals within hours. Serum and ascitic amylase activities were significantly increased, whereas pancreatic amylase activity was decreased after intraperitoneal injection of 3 g/kg L-ornithine. The increase in pancreatic trypsin activity (9-48 hrs) correlated with the degradation of IkappaB proteins and elevated interleukin-1beta levels. Oxidative stress in the pancreas was evident from 6 hrs; HSP72 synthesis was increased from 4 hrs after L-ornithine administration. Morphologic examination of the pancreas showed massive interstitial edema, apoptosis, and necrosis of acinar cells and infiltration of neutrophil granulocytes and monocytes 18 to 36 hrs after 3 g/kg L-ornithine injection. One month after L-ornithine injection, the pancreas appeared almost normal; the destructed parenchyma was partly replaced by fat. Equimolar administration of L-arginine resulted in lower pancreatic weight/body weight ratio, pancreatic myeloperoxidase activity, and histologic damage compared with the L-ornithine-treated group. L-ornithine levels in the blood were increased 54-fold after intraperitoneal administration of L-arginine. CONCLUSIONS: We have developed a simple, noninvasive model of acute necrotizing pancreatitis in rats by intraperitoneal injection of 3 g/kg L-ornithine. Interestingly, we found that, compared with L-arginine, L-ornithine was even more effective at inducing pancreatitis. Large doses of L-arginine produce a toxic effect on the pancreas, at least in part, through L-ornithine.

Congenital syringocystadenoma papilliferum.

We present a boy with a congenital, ulcerated nodule on the scalp. At birth, the lesion was considered to be the result of a traumatic injury, but a biopsy at the age of 6 months pointed to a diagnosis of syringocystadenoma papilliferum. We draw attention to the difficulty of identifying head lesions in young children from clinical signs alone.

Pre-treatment and post-treatment with alpha-tocopherol attenuates hippocampal neuronal damage in experimental cerebral hypoperfusion.

Alpha-tocopherol, a potent antioxidant, has been widely investigated as a dietary supplement with which to reduce the risk of atherosclerosis, and has recently been considered as a potential supplement to moderate oxidative neuronal damage in Alzheimer's disease patients. Since alpha-tocopherol appears beneficial in vascular and neurodegenerative disorders, we set out to identify its neuroprotective action in a rat model of chronic cerebral hypoperfusion-induced brain injury. The bilateral common carotid arteries of male Wistar rats were permanently occluded (2VO). Sham-operated animals served as controls. Half of the animals were pre- or post-treated repeatedly with alpha-tocopherol (5x100 mg/kg daily, i.p.), the other half receiving only soybean oil, the alpha-tocopherol vehicle. One week after the onset of 2VO, the spatial learning capacity of the animals was assessed in the Morris water maze. After testing, hippocampal slices were stained with cresyl violet in order to examine the pyramidal cell layer integrity. The density of microtubule-associated protein-2 (MAP-2)-positive dendrites and the OX-42-labeled microglial activation level were determined immunocytochemically. Finally, alpha-tocopherol was determined in the peripheral tissues, blood and brain. Alpha-tocopherol moderated the 2VO-induced learning impairment. The various forms of alpha-tocopherol treatment, and particularly the post-treatment, prevented the 2VO-induced pyramidal cell death and the activation of microglia in the hippocampus CA1 region, and the degeneration of MAP-2-positive dendrites in the CA3 region. The alpha-tocopherol concentration was elevated in the peripheral tissues and the blood, but not in the brain. The data indicate that alpha-tocopherol, particularly when administered as post-treatment, is neuroprotective in chronic cerebral hypoperfusion.

Concurrent Confirmation and Differential Diagnosis of Congenital Adrenal Hyperplasia from Dried Blood Spots: Application of a Second-Tier LC-MS/MS Assay in a Cross-Border Cooperation for Newborn Screening.

BACKGROUND/AIMS: Newborn screening for congenital adrenal hyperplasia (CAH) is generally performed using 17- hydroxyprogesterone dissociation-enhanced, lanthanide fluorescence immunoassay (DELFIA®). The primary screening results must be confirmed due to high false-positive rates; however, the need to obtain a separate specimen can hamper early recognition, differential diagnosis and treatment. We aimed to develop a single liquid chromatography-tandem mass spectrometry (LC-MS/MS) method that allows both the confirmation and differential diagnosis of CAH using the same dried blood spot (DBS) as in primary screening. METHODS: An LC-MS/MS assay for cortisol, 21-deoxycortisol, 11-deoxycortisol, 4-androstenedione and 17-hydroxyprogesterone was developed, validated and applied to a total of 163 DBS samples tested positive in primary newborn screening in a cross-border cooperation. RESULTS: Excellent baseline resolution and reliable determination of all analytes were achieved in DBS samples following simple sample preparation without derivatization. Of a total of 163 DBS samples tested positive in primary screening, the 21-hydroxylase-deficient form of CAH was confirmed in 1 sample. CONCLUSIONS: The present LC-MS/MS assay was successfully applied as a second-tier test in a cross-border cooperation for newborn screening. The assay allows concurrent confirmation and differential diagnosis of CAH and can be performed on the same DBS samples as in primary screening, enabling early diagnosis and treatment.

Methotrexate inhibits the glyoxalase system in vivo in children with acute lymphoid leukaemia.

The inhibition of glyoxalase I leads to antitumour activity through the accumulation of methylglyoxal. Our earlier observations suggested that methotrexate (MTX) may affect the glyoxalase system. This prompted a serial study of the drug on this metabolic pathway. Ten children with acute lymphoid leukaemia (ALL), admitted to our department between January 2002 and July 2003, were enrolled. Plasma D-lactate was assayed before, 24 and 72 h after the start of four consecutive MTX infusions (5 g/m(2)/24 h) in each patient. Inhibition of glyoxalase I was tested in vitro, using human erythrocyte lysates and yeast enzyme. The elevated initial plasma D-lactate levels (P<0.02) fell significantly (P<0.001) in response to 24 h MTX infusions. In vitro, MTX, folic and folinic acids inhibited the activity of glyoxalase I. Thus, MTX seems to affect the alpha-oxoaldehyde metabolism in vivo, as a likely consequence of glyoxalase I inhibition. This action probably contributes to the anticancer activity and toxicity of the drug.

Oxidative stress in juvenile essential hypertension.

OBJECTIVE: Oxidative stress, an antioxidant/pro-oxidant imbalance, in patients with juvenile essential hypertension was measured via several biochemical parameters. As the blood pressure is associated with the body mass index (BMI), results were compared with those on BMI-matched controls. DESIGN AND SETTING: A prospective observational study at a university teaching hospital. PATIENTS: Children and adolescents with essential hypertension (mean standard deviation: age 14.4 +/- 3.1 years, BMI 25.0 +/- 6.9 kg/m(2), n = 52) before any treatment, and controls with a similar BMI distribution (age 14.3 +/- 4.3 years, BMI 24.4 +/- 6.6 kg/m(2), n = 48). METHODS: Measurements were made of the plasma levels of (1) nitrites + nitrates, an indirect measure of available nitric oxide; (2) lipid peroxidation end-products, as malondialdehydes and free thiols; and (3) the redox status of the red blood cell glutathione, as a new oxidative stress parameter. RESULTS: There were decreased plasma levels of nitrates and increased levels of lipid peroxidation end-products in the hypertensive patients, resulting in a consistent increase in the plasma lipid peroxidation/nitric oxide ratio as compared with the controls with the same BMI (P <0.01). This ratio additionally correlated directly with both the systolic and diastolic blood pressures for the overall patient population (P <0.001). A significant glutathione depletion in the red blood cells resulted in an elevated ratio of oxidized/reduced forms with a reduced antioxidant protective capacity in the hypertensive patients versus the BMI-matched controls (P <0.001). CONCLUSIONS: The presence of systemic oxidative stress was proven in hypertensive children and adolescents, irrespective of their BMI.

Newborn screening for lysosomal storage disorders in hungary.

Even though lysosomal storage disorders (LSDs) are considered to be orphan diseases, they pose a highly relevant cause for morbidity and mortality as their cumulative prevalence is estimated to be 1:4,000. This is especially important as treatment in form of enzyme replacement therapy, substrate reduction therapy or stem cell transplantation is amenable for some LSDs. It is plausible that an early start of treatment might improve the overall prognosis and, even more important, prevent irreversible damage of key organs. To get a more precise insight into the real frequency of some LSDs in the general population, we screened 40,024 samples from the Hungarian newborn screening (NBS) program in Szeged for Fabry disease (FD), Gaucher disease (GD), Pompe disease (PD), and Niemann-Pick A/B (NPB) disease using tandem mass spectrometry. Altogether, 663 samples (1.66%) were submitted for retesting. Genetic confirmation was carried out for 120 samples with abnormal screening results after retesting, which identified three cases of GD, three cases of FD, nine cases of PD, and two cases with NPB. In some cases, we detected up to now unknown mutations - one in NPB and seven in PD - which raise questions about the clinical consequences of a NBS in the sense of late-onset manifestations. Overall, we conclude that screening for LSDs by tandem MS/MS followed by a genetic workup in identified patients is a robust, easy, valid, and feasible technology in newborn screening programs. Furthermore, early diagnosis of LSDs gives a chance to early treatment, but needs more clinical long-term data especially regarding the consequence of private mutations.

Determination of glutathione and glutathione disulfide in biological samples: an in-depth review.

Glutathione (GSH) is a thiol-containing tripeptide, which plays central roles in the defence against oxidative damage and in signaling pathways. Upon oxidation, GSH is transformed to glutathione disulfide (GSSG). The concentrations of GSH and GSSG and their molar ratio are indicators of cell functionality and oxidative stress. Assessment of redox homeostasis in various clinical states and medical applications for restoration of the glutathione status are of growing importance. This review is intended to provide a state-of-the-art overview of issues relating to sample pretreatment and choices for the separation and detection of GSH and GSSG. High-performance liquid chromatography, capillary electrophoresis and gas chromatography (as techniques with a separation step) with photometric, fluorimetric, electrochemical and mass spectrometric detection are discussed, stress being laid on novel approaches.

Enhanced methylglyoxal formation in the erythrocytes of hemodialyzed patients.

Methylglyoxal (MG) contributes significantly to the carbonyl stress in uremia; however, the reason for its increased concentration is not clear. Thus, the present study was aimed to investigate the formation and degradation of MG in the erythrocytes of hemodialyzed (HD) patients with end-stage renal disease. In 22 nondiabetic patients on long-term HD, erythrocyte MG and d-lactate levels, glyoxalase activities, and whole blood reduced glutathione content were determined. The data were compared with those from 22 healthy controls. Erythrocyte MG and d-lactate production were also investigated in vitro under normoglycemic (5 mmol/L) and hyperglycemic (50 mmol/L) conditions. The erythrocyte MG levels were elevated (P < .001) in the HD patients. The blood reduced glutathione content and glyoxalase I activity were similar to the control levels, but the glyoxalase II activity was significantly (P < .005) increased. In the normoglycemic in vitro model, production of both MG (P < .001) and d-lactate (P < .002) was significantly enhanced in the HD erythrocytes relative to the controls. During hyperglycemia, the MG formation and degradation rates were further increased (P < .001). The present study demonstrated an increased formation of MG in the erythrocytes of HD patients. This seemed to be related to a glucose metabolism disturbance of the cells. The degradation system of MG was also activated; still, it was not able to counteract the high rate of MG formation. The alterations and imbalance of these metabolic processes may contribute to the carbonyl overload and stress in the HD patients.

Roles of paraoxonase and oxidative stress in adolescents with uraemic, essential or obesity-induced hypertension.

BACKGROUND/AIMS: Paraoxonase 1 (PON1) is associated with high-density lipoproteins in the plasma, and is capable of hydrolysing oxidized lipids and preventing the oxidation of low-density lipoproteins. Oxidative stress and the PON1 (activity and Q192R polymorphism) were analysed in adolescent patients with essential (n = 49) or obesity-induced hypertension (n = 79), uraemic patients (n = 20), and also in obese normotensive patients (n = 60) and age-matched controls (n = 57). METHODS: The PON1 activity was measured via paraoxon hydrolysis. The PON1 genotype was determined by real-time PCR. The levels of oxidized and reduced glutathione, the end-products of nitric oxide, cysteine, homocysteine and lipid peroxidation in the plasma were measured and related to the PON1 status. RESULTS: There were no significant differences between the patient groups and the control group in the genotype distributions and the allele frequencies of the Q192R polymorphism. The PON activity was significantly lower (p < 0.001) in the uraemic hypertensive group than in the controls. The MDA concentration was significantly higher in the uraemic hypertensive (p < 0.001) and obese hypertensive (p < 0.05) patients. The plasma NOx concentrations were significantly lower (p < 0.001) and the ratio MDA/NOx were significantly higher in all four patient groups. The GSH levels were significantly lower in the patients with hypertension (p < 0.001) and obesity-induced hypertension (p < 0.05) than in the controls, while the GSSG level (p < 0.01) and the ratio GSSG/GSH (p < 0.05) was significantly higher in the uraemic hypertensive group. The plasma homocysteine level was significantly higher (p < 0.001) in the uraemic hypertensive patients as compared with the controls. CONCLUSIONS: We found no significant correlation between the biochemical parameters and neither genotypes nor enzyme activities. The PON1 status and the levels of certain biochemical parameters are independently associated with the hypertension in hypertensive and obese hypertensive patients, and the elevated levels of lipid peroxides and plasma homocysteine may contribute to the increased risk of cardiovascular complications in patients on haemodialysis.

Alpha-tocopherol and NADPH in the erythrocytes and plasma of multiple sclerosis patients. Effect of interferon-beta-1b treatment.

OBJECTIVES: To investigate the influence of interferon-beta-1b (INF-beta-1b) therapy on blood antioxidants (alpha-tocopherol and NADPH) in multiple sclerosis (MS). PATIENTS AND METHODS: Patients with relapsing-remitting MS (n = 14) have been studied during 6 months of INF-beta-1b therapy. alpha-Tocopherol was determined by HPLC and UV or electrochemical detection; NADPH was quantified spectrophotometrically. RESULTS: The erythrocyte alpha-tocopherol level was reduced (p < 0.001) before treatment, but had regained the control level by 6 months of therapy. The plasma alpha-tocopherol/lipid ratios were constant during therapy. Plasma triglyceride levels were transiently increased (p = 0.0270) after 1 month of treatment. INF-beta-1b had also induced a transient decrease in NADPH after 1 month, but thereafter the level returned to approximately the initial value (p = 0.0248). CONCLUSION: INF-beta-1b seems to exert a sparing effect toward the erythrocyte alpha-tocopherol content. The fall in NADPH in parallel to the rise in plasma triglycerides suggests stimulation of fatty acid synthesis by INF-beta-1b.

Aplasia cutis congenita after methimazole exposure in utero.

We describe a patient who was exposed to the antithyroid drug methimazole during the first 6 weeks of gestation and was born prematurely with scalp and skull defects associated with facial asymmetry. A review of the literature seems to support the hypothesis that methimazole is a potential teratogen. Although the risk of birth defects is low with clinically applied doses of the drug, it cannot be regarded as safe and should therefore be avoided in the treatment of pregnant women.

Heme oxygenase 1 expression in young uremic patients on hemodialysis.

Oxidative stress plays an important role in the cardiovascular complications in end-stage renal disease (ESRD) patients on long-term hemodialysis (HD). Heme oxygenase-1 (HO-1) inhibits inflammatory events and protects against oxidative stress and endothelial injury. Therefore, we followed the effects of single HD sessions on HO-1 expression. A competitive reverse transcriptase PCR method was used to estimate HO-1 induction before and immediately after HD and 48 h later in 17 young uremic patients. We also measured the concentrations of plasma hemoglobin and bilirubin as indicators of hemolysis, the ferroxidase activity, and the erythrocyte-derived reduced and oxidized glutathione levels as oxidative stress markers, and the homocysteine levels as an independent risk factor. We found significant differences in HO-1 expression patterns in the patients, depending on the duration of HD treatment. Short-term HD [ n=7, median 19 months (9, 29 quartiles)] resulted in an elevated HO-1 expression, which was not further upregulated during HD. Long-term HD [ n=10, median 97 months (53, 150 quartiles)] led to downregulation of baseline HO-1 expression in ESRD patients. In these patients, a single HD session results in erythrocyte injury and a transient one- to five-fold elevation of HO-1 expression. The chronic downregulation of the baseline expression of HO-1 in long-term HD patients resulted in recurring oxidative stress during each HD session, which may contribute to accelerate the progression of atherosclerosis.

Erythema nodosum in association with celiac disease.

We present a 16-year-old girl with a 4-year history of chronic persistent erythema nodosum. Recurrently low serum iron values suggested the possibility of a malabsorption syndrome. The presence of antitransglutaminase and antiendomysium antibodies and the jejunal biopsy specimen findings showed an underlying celiac disease. On a strict gluten-free diet, the skin lesions resolved and the girl has since remained symptom free for 9 months. Thus celiac disease can be a triggering factor for erythema nodosum. In the chronic form of the skin lesions, serologic testing for this specific enteropathy may be justified.