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Background: Several acylcarnitines used as primary markers on dried blood filter papers (DBS) for newborn screening lack specificity and contribute to a higher false positive rate. The analysis of urine acylglycines is useful in the diagnosis of inborn errors of metabolism (IEM) including medium chain acyl-CoA dehydrogenase deficiency (MCADD), isovaleric acidemia, and beta-ketothiolase deficiency (BKTD). Currently, no method for analyzing acylglycines from DBS has been published., Methods: Acylglycines were extracted from two 3.2 mm DBS punches and butylated using Butanol-HCl. Ultra Performance Liquid Chromatography (UPLC-MS/MS) with run time of 10 min permits resolution and quantitation of 15 acylglycines; including several isobaric. Method development was completed. Reference intervals (n = 573) were established for four birth weight groups. Furthermore, samples from patients with a confirmed IEM (n = 11), and false positive screens (n = 78) were analyzed to validate the interpretation obtained from the newly established reference intervals., Results: Calibration curves were linear from 0.005 to 25.0 μM. Ion suppression was evaluated as minimal (2 to 10%). Samples from known patients were used to validate the reference intervals. For C5OH-related disorders, tiglylglycine (TG), TG/acetylglycine (AG) ratio, 3methylcrotonylglycine (3MCG) and 3MCG/AG ratio increased specificity. Propionylglycine (PG) and PG/acetylglycine ratio were two discriminatory markers in the investigation of C3-related disorders. Hexanoylglycine (HG), octanoylglycine (OG), suberylglycine (SG), and the ratios HG/AG, OC/AG and SG/AG were excellent markers of MCADD deficiency., Conclusion: This method shows potential application as a second tier screen in order to reduce the false positive rate for a number of IEM targeted by newborn screening., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

A 4-year-old female with history of developmental regression and autistic features was diagnosed with guanidinoacetate methyltransferase deficiency at age 21 months. Upon treatment, she showed improvements in her developmental milestones, sensorial-neural hearing loss and brain atrophy on cranial-MRI. The creatine/choline ratio increased 82% in basal ganglia and 88% in white matter on cranial MR-spectroscopy. The CSF guanidinoacetate decreased 80% after six months of ornithine and creatine supplementation and an additional 8% after 18 months of additional arginine restricted diet. We report the most favorable clinical and biochemical outcome on treatment in our patient., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Background: Recent neonatal screening for isovaleric acidemia by tandem mass spectrometry based on dried blood-spot levels of C5-acylcarnitines, including isovalerylcarnitine and its isomer, pivaloylcarnitine, which is derived from pivalate-generating antibiotics, has caused many false-positive results. We have developed a method to overcome this interference., Methods: The amounts of isovalerylglycine were determined by a stable-isotope dilution electrospray tandem mass spectrometric analysis, using multiple-reaction monitoring with product ions of m/z 132, which were generated predominantly from quasi-molecular ions of isovalerylglycine butylester but apparently not from those of pivaloylglycine butylester., Results: Isovalerylglycine concentrations in dried blood spots of control newborns were 0.17+/-0.03 nmol/ml, and those of patients with isovaleric acidemia ranged from 1.3 to 80.0 nmol/ml. Those of the newborns treated with antibiotics, which caused high C5-acylcarnitine levels (1.9+/-1.7 nmol/ml) in dried blood spots, were 0.22+/-0.05 nmol/ml., Conclusions: Our data showed that the present method is useful in eliminating the false-positive results due to antibiotics use in newborn screening for isovaleric acidemia.

Primary disorders of creatine metabolism have been only recently described. We report new molecular and biochemical findings obtained from a child affected by guanidinoacetate methyltransferase deficiency. This patient presented with neurological regression, epilepsy, and a movement disorder during the first year of life. HPLC analysis showed high concentrations of guanidinoacetic acid in urine, plasma, and CSF. Molecular analyses of cDNA and genomic DNA revealed two novel mutations, a G insertion following nucleotide 491 of the cDNA (c.491insG) in exon 5 and a transversion at nt -3 in intron 5 (IVS5-3C>G). The c.491insG mutation causes a frameshift and a premature stop codon at the end of the exon. The IVS5-3C>G mutation prevents the splicing of the last exon of the gene precluding the complete maturation of the transcript and, most likely, causes rapid degradation of the mRNA., (Copyright 2000 Academic Press.)

Two infants with the neonatal type of nonketotic hyperglycinemia that had manifested as early neonatal consciousness disturbance are presented. Transient hyperammonemia had been detected in both initially. High levels of glycine in plasma and cerebrospinal fluid disturb the nervous system, causing variable manifestations of this disease. Both cases were complicated by intracranial hemorrhage, which has never before been reported. After treatment with sodium benzoate and dextromethorphan, some neurologic improvement was observed, although the glycine levels did not lower. Recent clinical trials are reviewed, and because of the unfavorable outcomes, the special need for prenatal diagnosis is highlighted.

Acyl glycines are normally minor metabolites of fatty acids; however, the excretion of certain acyl glycines is increased in several inborn errors of metabolism. Therefore measurement of the metabolites in body fluids can be used to diagnose these metabolic disorders. The chemical synthesis of a range of acyl glycines is described, together with that of their 13C2-isotopomers for use as internal standards. An analytical method for the measurement of hexanoyl, octanoyl, 3-phenylpropionyl and suberyl glycines in urine, employing gas chromatography-mass spectrometry with negative-ion chemical ionisation was adapted to measure a larger range of acyl glycines in a single blood spot on a standard Guthrie card. Diagnoses of a case of medium-chain acyl-CoA dehydrogenase deficiency and a case of isovaleric acidaemia were confirmed using a single blood spot from each patient.

Regarding a case of beta-ketothiolase deficiency revealed by ketoacidosis with hyperglycinemia, the authors show the way to diagnose and to treat this disease. Ketoacidosis without hyperglycemia or lactacidemia suggested this diagnosis. Gas chromatography-mass-spectrography revealed unusual urinary excretion of metabolic products of isoleucine. The enzymological study of fibroblasts confirmed the diagnosis. The treatment of acute episodes consisted of acidosis control and exclusive glucides intake before diagnosis. Afterwards, a controlled proditic diet and L-carnitine must be given and fasting must be avoided.

A four-month-old boy with beta-ketothiolase deficiency is described in this report. Presenting symptoms and signs were vomiting, irritability and acidotic respiration. Laboratory investigations revealed hyperglycinemia, metabolic acidosis and ketosis. Subsequent urinary GC-MS analysis of the patient's urine sample showed the typical pattern of beta-ketothiolase deficiency. Our experience with this case indicates that accurate diagnosis and early treatment of inborn errors might be lifesaving.

Two cases of propionicacidemia are reported. The first patient was a child aged 16 months with psychomotor retardation, severe disturbances of wakefulness, and vomiting; the second case concerned a new-born baby with metabolic acidosis and neurological disorders. In both cases the diagnosis of propionicacidemia was made after the discovery of ketonuria, and raised blood ammonia and glucose levels. Appropriate dietary measures led to great improvement in the first case, and nearly normal psychomotor development in the second case at 7 months of age. Problems related to biotin sensitivity, leucine intolerance, and raised blood ammonia levels are discussed.

In patients with non-ketotic hyperglycinaemia and two patients with urea cycle disorders treated with varying doses of sodium benzoate there was linear correlation between intake of benzoate and excretion of hippurate. Patients with non-ketotic hyperglycinaemia excreted significantly more benzoate in the form of hippurate than patients with urea cycle disorders (74 +/- 7.0 vs 41 +/- 3.6%). The plasma concentration of glycine decreased following benzoate treatment only in the patients with non-ketotic hyperglycinaemia. The observed difference between the two groups in the excretion of hippurate seems to support the concept that glycine availability may be limiting in benzoate therapy for some patients.

The molecular nature of the glycine cleavage system was investigated in 16 patients with nonketotic hyperglycinemia (NKH). The overall activity of the glycine cleavage system was found to be decreased in all of the liver and brain tissue studied. It was undetectable or extremely low in the neonatal type of NKH, whereas there was some residual activity in the infantile type of NKH. Thus the clinical phenotypes do seem to relate to the degree of the defect in the glycine cleavage system. In the neonatal type, a specific defect in P protein was found in 9 cases and a specific defect in T protein in 2 cases. In the infantile type, a partial defect in T protein was found in 2 cases. Differential diagnosis between NKH and ketotic hyperglycinemia is described. A feasibility of prenatal diagnosis of NKH by chorionic villus biopsy is provided.

Two mentally retarded adults with nonketotic hyperglycinemia had biochemical findings similar to those of the infantile form of the disease. Our patients differ from other adult patients and may represent the survival to adulthood of individuals with a mild form of infantile nonketotic hyperglycinemia.

Measurement of methylmalonyl-CoA mutase and propionyl-CoA carboxylase activities in lysates from fibroblasts derived from control, nonketotic hyperglycinemia, propionic acidemia, and both vitamin B12-responsive and -nonresponsive variants of methylmalonic acidemia showed only one abnormality: a 59% decrease in carboxylase activity in the nonketotic hyperglycinemic lysates (P less than 0.01). When fibroblasts from all cell types were grown on valine-supplemented (24 mM) media, mutase activity was generally inhibited. As for carboxylase activity, control lines were inhibited 35% as compared to controls without valine and propionic acidemia activity was undetectable. On the other hand, carboxylase activity in both methylmalonic acidemia variants was increased 40% and nonketotic hyperglycinemia carboxylase activity was increased 80% (P less than 0.01) when grown on valine-supplemented media. Isoleucine could not substitute for valine in producing increased carboxylase activity in these mutants. Glycine cleavage activity in fresh rat liver homogenates (11.1 micronmol/gm protein/90 min) did not vary significantly when 24 mM valine was added to the reaction (9.9 micronmol/mg protein/90 min). Therefore, the hyperglycinemia observed in both ketotic and nonketotic forms is probably not caused by a direct effect of valine on the glycine cleavage reaction. These data suggest that the presence of increased amounts of propionic acid in serum or urine does not necessarily rule out the possibility of nonketotic hyperglycinemia due to the decreased activity of the carboxylase enzyme.

The biochemical characteristics of a patient with most of the clinical symptoms of nonketotic-hyperglycinemia have been studied. Despite the extremely low plasma/cerebrospinal fluid glycine ratio of the patient, typical of the nonketotic syndrome, at autopsy we found no enhancement of glycine levels in brain and glycine cleavage enzyme activities in the brain and liver that were similar to those of a control newborn. In order to ascertain the basic defect responsible for glycine accumulation in the cerebrospinal fluid and for the neurological damage, we examined the possible existence of altered glycine transport systems in nervous cells. Our findings indicate that (1) low and high affinity, Na+-dependent transport systems for glycine can be detected in both rat and control human postmortem tissue and (2) the low affinity glycine transport system is absent in the brain of the patient, whereas an alteration of transport systems occurs in the patient's spinal cord, probably involving the high-affinity component. These alterations could account for the clinical features of the patient. Since the feasibility of determining glycine transport parameters in postmortem tissue has been established, we think it would be of interest to investigate such systems in cases where the etiology of hyperglycinemia is not clear.

The tendency towards metabolic acidosis developing during simple infections lead to the detection of hyperglycinemia which was shown to be caused by the rare inborn error of metabolism, which was shown to be a methylmalonic acidemia, in identical twins. Under a protein restricted diet and vitamin-B12-injections once a week, all clinical symptoms disappeared so that vitamin-B12-dependency became evident. Under this therapeutic regimen methylmalonic aciduria was well under control.

Repeated biochemical studies of the erythrocytes of a patient with pyroglutamic acidemia have shown a varying biochemical disorder in these cells. Whereas earlier studies demonstrated absence of glutathione and massive amino acid loading in erythrocytes, these cells later contained small but readily measurable amounts of glutathione and had a relatively normal amino acid content. The marked increase in amino acids after acid hydrolysis of erythrocytes had also disappeared. Transport studies showed a significant increase in the active transport of glycine, while the transport of other amino acids was comparable to that of normal cells. We are unable to detect any activity of gamma-glutamyl transpeptidase in human erythrocytes. Our observations suggest a role for glutathione in the transport of amino acids by erythrocytes, but at present no definite conclusion can be drawn with regard to the participation of the gamma-glutamyl cycle in this process. The biochemical variability in our patient's erythrocytes is unexplained and should be searched for in other patients with pyroglutamic acidemia.

We diagnosed a 22-year-old man with psychomotor retardation, rare seizures, hyperglycinemia, and hyperglycinuria as an atypical variant of nonketotic hyperglycinemia (NKH). Despite this clinical phenotype and a CSF/plasma ratio confirming a mild variant, measurement of hepatic glycine cleavage activity and the P-protein component indicated the more severe neonatal variant.

An oral loading test with L-valine (100 mg/kg body weight) in 3 patients with non-ketotic hyperglycinemia was accompanied by drowsiness and hyperreflexia of the patients. Metabolic studies revealed a slightly lower rate of disappearance of valine from blood in 2 of the patients. In a third patient, the curve was not different from controls. Gas chromatographic analysis for short-chain fatty acids in serum carried out during the valine loading test did not show increased concentrations. Urine collected during the valine loading test did not show excretion of N-acylglycine derivatives. As neither of the branched chain amino acid transaminases proved to be inhibited by glycine, inhibition by glycine of the uptake of valine by the tissues became likely. This uptake has been measured in rat liver slices. Glycine was found to be a competitive inhibitor of valine uptake, with a Ki of 4.9 mM. It is concluded that the tendency to a decreased rate of valine disappearance from blood in non-ketotic hyperglycinemia could be due to an inhibition of valine uptake by the high plasma glycine concentration. The relationship of the inhibited valine uptake in liver slices of rats with the clinical symptoms in non-ketotic hyperglycinemia patients after a valine load remains to be established.

The molecular nature of the glycine cleavage system was investigated in eight patients with typical (neonatal) and two patients with atypical (late onset) nonketotic hyperglycinemia (NKH). The overall activity of the glycine cleavage system was found to be decreased in all of the liver and brain tissue studied, but it was undetectable or extremely low in typical NKH, whereas there was some residual activity in atypical NKH. Six patients with typical NKH had a specific defect in the P protein, and one a defect in the T protein; the activity of the T protein was defective in one patient with atypical NKH.