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Background: Isovaleric acidemia (IVA), a rare autosomal recessive disorder in leucine metabolism caused by defected IVD gene, is characterized by episodes of acute metabolic crisis and psychomotor development retardation. This study aimed to determine the clinical, biochemical, and mutation spectrum of patients with IVA from mainland China., Methods: Eight patients (three boys and five girls) from eight unrelated families were collected, IVD gene mutations and phenotypes were examined., Results: The patients were admitted because of vomiting, feeding difficulty, psychomotor retardation and "dirty sock" odor. Elevated blood isovaleryl (C5)-carnitine and urine isovalerylglycine were detected from all our patients. Fourteen mutations of the IVD gene were detected, eight of them are novel, c.145C>T (p.Q49Ter), c.359G>A (p.R120Q), c.424C>T (p.R142C), c.458T>C (p.L153P), c.466-1G>T, c.676_677insA (p.T226Nfs*13), c.1039G>A (p.A347T) and c.1076A>G (p.D359G). With this study, a total of 34 alleles were studied in the Chinese population. c.1208A>G (p.Y403C), the common mutation in Taiwan, accounts for 9/34 alleles (7 in previous reports and 2 in this study)., Conclusions: We described eight novel mutations detected from eight unrelated Chinese patients and provided evidence to support that the p.Y403C is the hotspot mutation in this population., (Copyright © 2019 Elsevier B.V. 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.

Background: Detection of amino acids (AA), acylcarnitines (AC), and guanidinoacetate (GAA) in dried blood spots by tandem mass spectrometry has made it possible to detect different inborn errors of metabolism in neonatal screening programs. Despite its proven sensitivity many issues related to sample preparation remain unsolved. Hematocrit has a profound effect on blood viscosity, and may thereby influence flux and diffusion properties of the blood. As newborn infants show a considerable interindividual variability of hematocrit levels, we investigated its effect on levels of AA and AC in dried blood spots., Methods: Blood samples with defined hematocrit levels (20%, 30%, 40%, 50%, 60%) were produced by diluting blood cells with plasma from a single donor. Forty dried blood spots were made for each hematocrit level and a central as well as a peripheral 3 mm disk was punched and analysed for AA, AC, and GAA, respectively., Results: Levels of most AA and GAA increased significantly with increasing hematocrit (p<0.001), while the effect of hematocrit on some AA was less pronounced. Total AC, free carnitine, some long, medium and short chain AC correlated positively with hematocrit levels (p<0.001). In samples with low hematocrit, levels of most AA and free carnitine were higher in the peripheral than in the central disk (p<0.0001)., Conclusions: Both hematocrit and position of the disk within the dried blood spot have a significant and sometimes additive effect on levels of AA, AC and GAA in dried blood spots. Theoretically, diagnoses may be missed depending on hematocrit and position of the disk.

Background: Guanidinoacetate (GAA) and creatine (Cr) are reliable biochemical markers of primary creatine disorders. The aim of this study was to develop and validate a method for the determination of GAA and Cr in dried blood spot through the use of stable isotope dilution and flow injection analysis (FIA)-ESI-MS/MS., Methods: Dried blood spots were extracted using methanol-water solution containing D3-Cr. After evaporation and formation of butyl esters, samples were analyzed using multiple reaction monitoring mode (m/z 174.2-->101.1 for GAA, 188.3-->90.1 for Cr and 191.3-->93.1 for D3-Cr)., Results: The analysis was very fast (1 min). The detection limits were 0.34 micromol/l of blood and 0.30 micromol/l of blood for Cr and GAA, respectively, and the response was linear over the range 0.25-12.5 micromol/l of blood for GAA and 3.57-624.7 micromol/l of blood for Cr. Recovery range was 93-101% for Cr and 94-105% for GAA and between-run CVs were 5.3% for GAA and 4.5% for Cr. Ion suppression effect was also studied. The method was applied to spots obtained from two patients affected by GAMT deficiency, four patients affected by AGAT deficiency (including a newborn) as well as 282 healthy subjects., Conclusions: The detection of GAA in dried blood spot by FIA-ESI-MS/MS is a highly reliable and high throughput method for the diagnosis of GAMT and AGAT deficiencies and a possible tool for newborn screening of both these tractable disorders.

Guanidinoacetate methyltransferase (GAMT) deficiency is a disorder of creatine metabolism characterized by low plasma creatine concentrations in combination with elevated guanidinoacetate (GAA) concentrations. Although rare, GAMT deficiency has been identified in children with seizures, extrapyramidal movements, developmental delay, myopathies and behavioral abnormalities. Treatment with creatine monohydrate has been proven to be effective. We describe an isotope dilution electrospray tandem mass spectrometry (ES-MS/MS) assay for the simultaneous determination of plasma GAA and creatine using multiple reaction monitoring (MRM), d(3)-creatine as the internal standard and derivatization of GAA and creatine as butyl-esters. We analysed plasma of 16 healthy adults and 20 healthy children as well as three affected children. Plasma GAA concentrations were 5.02+/-1.84 micromol/l (mean+/-S.D.) in adults, 3.91+/-0.76 micromol/l in children age 5-10 years and 11.57, 15.16, 14.36 micromol/l in children with GAMT deficiency. Plasma creatine concentrations were 34.7+/-15.25 micromol/l in adults, 58.96+/-22.30 micromol/l in children and 5.37, 8.15, 403.5 micromol/l in two untreated children and one treated child with GAMT deficiency, respectively. GAA can also be reliably measured from filter cards, which is sufficient to make the correct diagnosis while creatine is consistently falsely elevated probably secondary to liberation of red cell creatine. In nine healthy newborn infants, GAA concentrations from filter cards were 4.83+/-1.43 and 5.04+/-1.84 micromol/l in 16 healthy adults. We conclude that isotope dilution ES-MS/MS is ideal for rapid high-throughput diagnosis of GAMT deficiency both from plasma and filter paper cards. Using this technique neonatal screening is feasible for this treatable inborn error of creatine metabolism.

In normal human plasma the concentrations of the renal osmolyte, glycine betaine, are usually between 20 and 70 mumol/l, in adult males (median 44 mumol/l) higher than in females (34 mumol/l). Concentrations are lower in renal disease (median 28 mumol/l) and normal in diabetes. Urinary excretion of glycine betaine shows no sex difference and is frequently elevated both in renal disease and in diabetes (medians: normal, 6.2, renal 12.3 and diabetes, 39.7 mmol/mol creatinine). The elevation in diabetes does not strongly correlate with known renal disease, nor with either urinary microalbumin or plasma creatinine. There is no correlation with glycated haemoglobin. The positive correlation with the excretions of another renal osmolyte, sorbitol, was highly significant in diabetic subjects. In the diabetic group there was also a significant negative correlation between plasma glycine betaine and urine microalbumin.

A method for the measurement of plasma glycine by HPLC with electrochemical detection after derivatization with phenylisothiocyanate (PITC) is described. The absorption of glycine in eighteen women undergoing intrauterine glycine irrigation during transcervical resection of endometrium was assessed by measurement of plasma glycine before, immediately after and 24 h after surgery. The plasma glycine concentration was normal in all women before surgery (range 120-386 mumol/l) but had risen dramatically in some patients after surgery (range 180-24,800 mumol/l) before returning to normal levels over the following 24 h (range 173-553 mumol/l). No clinical consequences were observed despite the large increases in plasma glycine; mild hyponatraemia occurred in only one patient. These findings, albeit in a limited number of patients, support the hypothesis that the symptoms sometimes associated with the use of glycine buffers are most likely due to water overload with hyponatraemia rather than a toxic affect of glycine itself.

A new method of clinical analysis based on 1H spin echo NMR spectroscopy is presented. It is capable of providing information on six metabolites within viable erythrocytes, directly and without any preparative procedures prior to analysis except for cell separation and washing. Erythrocytes from patients with rheumatoid arthritis and Graves' disease are compared with cells obtained from healthy volunteers. The NMR detectable species in the cytosol of the cells are glutathione, ergothioneine, choline, creatine, glycine, lactate and to a lesser extent alanine and valine. Significant differences are observed between the ergothioneine pools in the rheumatoid group (P less than 0.01) compared to the control group. The glutathione: di-glutathione ratio can be assessed from the ratio, g2 to g4, taken from different signals in the glutathione molecule. The total concentration of glutathione present is easily assessed qualitatively but is more difficult to quantitate.

Serum concentrations of glycine and taurine conjugates of cholic, chenodeoxycholic, and deoxycholic acid were measured with a HPLC-enzymatic assay in 104 patients with hepatobiliary diseases. The capability of discriminating between four major diagnostic categories by the serum bile acid concentrations, alone and in conjunction with five commonly used biochemical liver tests, was evaluated by stepwise linear discriminant analysis. The serum bile acid pattern alone was inferior to the routine liver tests in separating the diagnostic groups. When the two sets of analytes were combined, the generalized group distance (Rao's V) was significantly increased, showing that the serum bile acid pattern contained discriminatory information in addition to the routine liver tests. The gain in correct reclassification of patients obtained by adding the serum bile acids, however, was only marginal.

The activity of the glycine cleavage system in liver of hyperglycinemia with organic acidemia was 1/6 to 1/20 tha of normal human livers. The reduced activity results from the reduction in concentration of the enzyme of the glycine cleavage system. All the protein components of the glycine cleavage system examined were reduced when the activity was determined seprately. H-protein was purified from patients' and control livers, and there was found no difference in their chromatographic properties.

A new technique has been developed for rapid and precise quantitation of glycine in micro samples of biological fluids. Glycine is measured as the n-butyl N-trifluoroacetyl (TAB) derivative by chemical ionization mass fragmentography, using both methane and isobutane as carrier/reagent gases, with the analogous derivative of 2,2-dideuterioglycine added as the internal standard. This technique is also readily applicable, with sensitivity to the femtomole level, to determinations of other amino acids, as, for example, in studies of congenital errors of metabolism.

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.

Recent experience with three children who had non-ketotic hyperglycinemia suggested that interpretation of blood glycine levels in children is complex. The elevations of blood glycine in these children were quite modest, and comparable to those of other children admitted to hospital with other diseases. Often we find elevations of blood glycine in children who have had some degree of starvation before blood was taken for amino acid analysis. In a "typical" patient, valine, leucine, isoleucine and occasionally threonine are depressed, while the glycine level is raised. As nutrition improves all of the amino acids return to normal. Our data suggest that blood glycine levels in children with an acute episode of a debilitating disease need to be interpreted with respect to the immediate state of nutrition of the children.