Quantification of acylglycines in human urine by HPLC electrospray ionization-tandem mass spectrometry and the establishment of pediatric reference interval in local Chinese.

Urinary organic acids, plasma amino acids and acylcarnitine profile analyses are the main tools used to diagnose inborn errors of metabolisms (IEMs). However, without metabolic decompensation, these parameters are often not helpful. On the other hand, in cases of IEM, acylglycines are consistently raised even when patients appear to be in remission. This study aims to set-up a simple liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) method for the determination of urine acylglycines, complementary to organic acid and acylcarnitine profiles, for the diagnosis of IEM. In addition, local reference intervals for various acylglycines are established by using this method. Acylglycines were isolated by solid-phase extraction, derivatized with n-butanol, separated by HPLC, and detected by ESI-MS/MS. Acylglycines were quantified with deuterated internal standards. Mean recoveries of acylglycines ranged from 90.2 to 109.3%. Within- and between-run imprecisions for all acylglycines have CVs less than 10%. Linear regression coefficients were greater than 0.99. Reference intervals were established according to CLSI guidelines by analyzing 204 samples from apparently healthy individuals less than 18 years of age. The distributions of AG in the "normal" urine were skewed towards the right. After log transformation, all the results were normally distributed. Partitioning into age group reference intervals was not indicated, according to the Harris and Boyd approach. In this context, a single reference interval for each acylglycine could be used. This method of urine acylglycines analysis is a powerful diagnostic tool, complementary to urine organic acids and plasma acylcarnitine profiling, for detecting certain inborn errors of metabolism.

Urine organic acid analysis for inherited metabolic disease using gas chromatography-mass spectrometry.

Urine organic acid analysis is an essential component of the workup of the patient suspected to have an inborn error of metabolism (IEM). Urine contains several hundred different organic acids, which arise from a multitude of different sources including both normal and abnormal metabolism. They may also arise from drugs and drug metabolism or from xenobiotics and dietary supplements. In addition to the diagnosis of inborn errors of metabolism, the identification of organic acids in a urine sample has a wide range of potential applications, including toxicology and poisonings. The method described below extracts the acidic fraction from urine samples, derivatizes the extracted compounds, and identifies intermediate metabolites by GC-MS. The method utilizes electron impact ionization gas chromatography-mass spectrometry (GC-MS) with total ion collection.

Methylmalonic and propionic aciduria.

Methylmalonic and propionic aciduria (PA) are the most frequent forms of branched-chain organic acidurias. These autosomal recessive disorders result from deficient activity of methylmalonyl-CoA mutase and propionyl-CoA carboxylase, respectively. Clinically, acute or chronic neurologic signs are caused by the accumulation of toxic compounds proximal to the metabolic block. Phenotype varies from severe neonatal-onset forms with high mortality and poor outcome to milder forms with a later onset. In both cases the clinical course is dominated by the risk of relapses of life-threatening episodes of metabolic decompensation and of severe organ failure. Despite improvement of treatment, the overall outcome remains disappointing with no major differences between the two diseases. The diagnosis is based on the presence of characteristic compounds in body fluids as detected by organic acid analysis in urine and acylcarnitine profile in blood. Therapy is based on low-protein high-energy diet, carnitine supplementation, and metronidazole. Some patients with methylmalonic aciduria (MMA) respond to pharmacological doses of vitamin B12. Given the poor long-term prognosis, liver transplantation has been recently attempted as an alternative therapy to conventional medical treatment to cure the underlying metabolic defect. Nevertheless, the overall experience to date does not clearly demonstrate its effectiveness in preventing further deterioration or improving survival and quality of life. The recent implementation of neonatal screening by electrospray tandem mass spectrometry has decreased early mortality and improved the short-term outcome, without changing the detection rate of both diseases in the screening population compared to clinically detected cases. However, the limited number of patients and the short duration of their follow-up do not yet permit drawing final conclusions on its effect on the long-term outcome of methylmalonic and propionic acidemia.

Effect of supplementation with L-carnitine at a small dose on acylcarnitine profiles in serum and urine and the renal handling of acylcarnitines in a patient with multiple acyl-coenzyme A dehydrogenation defect.

We studied the effects of L-carnitine supplementation at a small dose on the profiles of acylcarnitines in serum and urine, as well as the renal handling of acylcarnitines, in a patient with multiple acyl-coenzyme A dehydrogenation defect. After supplementation with L-carnitine at a dose of 20 mg/kg/day, the concentration of each acylcarnitine measured both in the serum and in the urine had increased significantly, with the exception of that of an acylcarnitine with a carbon chain length (C) of 8 (C8 acylcarnitine). The magnitude of increase in the concentrations of the acylcarnitines in the serum was not associated with chain length, whereas in the urine, the magnitude tended to be greater in proportion to the shortness of the chain length. The fractional excretions of C2-C5 acylcarnitines exceeded 100%, indicating that they were produced in, or transported across, renal tubular epithelial cells and secreted into the urine. These results indicate that supplementation with a relatively small amount of L-carnitine can enhance the renal excretion of accumulated short-chain-length acylcarnitines through tubular excretion, in addition to basic glomerular filtration.

[Late-onset riboflavin-responsive multiple acyl-CoA dehydrogenase deficiency (glutaric aciduria type II)].

OBJECTIVE: Glutaric aciduria type II, or multiple acyl-CoA dehydrogenase deficiency is an autosomal recessively inherited defect of mitochondrial energy metabolism. The authors report two cases of late-onset glutaric aciduria type II, and evaluate the procedures for the diagnosis and treatment of this rare disease. METHODS: The clinical and biochemical characteristics of 2 patients with late-onset glutaric aciduria type II were documented. Case 1 presented with lipid storage myopathy for 3 years. Case 2 presented with intermittent episodes of non-ketotic hypoglycemia and muscle weakness for 9 years. The diagnosis of the 2 cases was confirmed with gas chromatography/mass spectrometry analysis of urine samples. Riboflavin supplementation and a low-fat, low-protein, high-carbohydrate diet were initiated as soon as the diagnosis was made. RESULTS: Organic acid analysis on both untreated cases revealed massive glutaric acid with elevated concentrations of isovalerylglycine, isobutyrylglycine, ethylmalonic acid, adipic acid, suberic acid and other dicarboxylic acids. The clinical manifestations were improved remarkably after the administration of riboflavin and diet control. Consistent improvements of sera enzymes and urine organic acids were observed during the course of treatment. CONCLUSION: Patients with unexplained myopathy, metabolic acidosis or hypoglycemia should be carefully screened for inherited metabolic disorders. Riboflavin in conjunction with appropriate diet control is an effective therapeutic regime for patients with late-onset glutaric aciduria type II.

Practical management of combined methylmalonicaciduria and homocystinuria.

Combined methylmalonicaciduria and homocystinuria is a disorder of intracellular cobalamin metabolism that remains a challenge to the physician unfamiliar with the diagnosis. We have followed six patients with combined methylmalonicaciduria and homocystinuria (four males, two females, age 4.2-24 years) for a median of 6.5 years (range 4-9 years). Age at diagnosis was between 18 days and 14 months in early-onset cases (n = 4) and 15 and 19 years in late-onset cases. Predominant clinical features include microcephaly, hydrocephalus, seizures, and white-matter changes on magnetic resonance imaging in early-onset cases. The white-matter changes may be secondary to impaired methylation owing to a lack of readily available methyl groups. Spastic quadriparesis and diplegia are long-term sequelae in late-onset cases. Management consists of hydroxycobalamin intramuscular injections, oral folate, betaine, and carnitine supplementation. Dietary protein restriction may be necessary when metabolic control remains difficult. The implementation of an emergency regimen should alleviate episodes of metabolic decompensation and reduce the rate of hospital admissions.

Evaluation of urinary acylglycines by electrospray tandem mass spectrometry in mitochondrial energy metabolism defects and organic acidurias.

We analyzed the urinary acylglycine excretion in 26 patients with mitochondrial energy metabolism disorders and in 55 patients with organic acidurias by electrospray tandem mass spectrometry (ESI-MS/MS), monitoring precursor ions of m/z 90. Urinary concentrations of the different acylglycines were quantified using deuterated internal standards. Normal values for the most important acylglycines were established. In MCAD and MAD (neonatal form) deficiencies, typical excretion patterns of urinary acylglycines were found in all the samples. In isovaleric aciduria, propionic aciduria, and 3-methylcrotonylglycinuria typical glycine conjugates were always found. Methylmalonic aciduria (mutase deficiency), multiple carboxylase deficiency, and 3-hydroxy-3-methylglutaric aciduria revealed pathological acylglycine profiles, even if not specific for the disease. In all these diseases acylglycine excretion seems to be less influenced by the clinical status than organic acid excretion. This method is a useful diagnostic tool for these metabolic disorders, complementary to organic acids and acylcarnitine profiles.

Acylcarnitine removal in a patient with acyl-CoA beta-oxidation deficiency disorder: effect of L-carnitine therapy and starvation.

Carnitine levels and acylcarnitine profiles in a patient with mild multiple acyl-CoA dehydrogenase deficient beta-oxidation were compared with control results. Whereas blood and urine total carnitine levels were moderately decreased, blood esterified carnitine levels in the patient were about 2-fold higher than in controls. Urinary acylcarnitine profiles presented with a larger variety of carnitine esters than in controls and included propionylcarnitine, butyrylcarnitine, 2-methylbutyrylcarnitine, hexanoylcarnitine and octanolycarnitine. Total carnitine levels in body fluids were similarly affected by chronic oral L-carnitine administration in patient and controls. By contrast, esterified carnitine level increase was 2-fold more important in controls than in patient. Whereas no qualitative changes in urinary acylcarnitine profiles were induced by L-carnitine therapy in controls, several alterations of these profiles were observed in the patient. The effect of starvation on metabolites was also studied, especially beta-oxidation rates assessed by free fatty acids to 3-hydroxybutyric acid ratios in blood from the patient in the untreated and L-carnitine treated states. In the L-carnitine-supplemented patient, the effect of starvation on the time course of carnitine levels and acylcarnitine profiles could also be documented. The ability of chronic oral L-carnitine administration to remove relatively less important amounts of acylcarnitines in the patient than in controls is further discussed, as well as qualitative alterations of acylcarnitine profiles induced by this therapy in the pathological condition.

High performance liquid chromatography (HPLC) method for confirming thin layer chromatography (TLC) findings in inborn errors of metabolism children in Malaysia.

High performance liquid chromatography (HPLC) with phenylisothiocyanate (PITC) is recently used for confirming the diagnosis of inborn errors of metabolism (IEM) especially amino acid disorders in Malaysian children. The method of HPLC used is a precolumn derivatization of amino acids with phenylisothiocyanate and is separated by reversed phase chromatography using 3.9 x 300 mm free amino acid columns and is detected by a UV/Vis detector. The samples are obtained from cases suspected of inborn errors of metabolism, especially of amino acid disorders, which are detected clinically by pediatricians. Initially, samples from patients suspected of inborn errors of metabolism, either urine or serum, are run on one-dimensional thin layer chromatography and supplementary chemical tests to detect the abnormal bands and associated abnormalities respectively. Positive samples are further run on HPLC to determine the specific amino acids abnormality. An examples of a case of maple syrup urine disease is discussed, based on the thin layer chromatography findings and HPLC findings.

Ethylmalonic aciduria: an organic acidemia with CNS involvement and vasculopathy.

Five infants from 3 families, one Egyptian, two Yemeni, are described with a progressive encephalopathy, four of whom have been studied in detail. All patients showed vascular lesions of the skin, characterized by waxing and waning petechiae and ecchymoses. Acrocyanosis was present in three patients. All patients showed retinal lesions characterized by tortuous veins. Protracted diarrhea was not a consistent finding, although they had metabolic crisis in association with diarrhea. They did not show failure to thrive. The neurologic symptoms were indicative of a progressive pyramidal tract disease. Three patients died following sudden emergence of severe basal ganglia, putaminal and head of caudate lesions. In one patient the CT changes in brain were suggestive of infarction. The patients who died manifested pulmonary congestion, or wet lung, and respiratory difficulties during the terminal stage of the disease. In all patients before and during the terminal event, mild-to-moderate hematuria, and in two RBC in CSF, was observed. In one patient there was mild hemoperitoneum at the terminal event. The urine organic acids indicated increased excretion of ethylmalonic, methylsuccinic, glutaric, and adipic acids. The patients invariably showed lactic acidosis, but no ketosis, during and in between the acidotic attacks of the disease. The acylcarnitine profile in blood of two patients showed a pronounced increase in C4 and C5 carnitine esters. In three patients, biopsies from petechiae indicated absence of an immune event, showing only fresh hemorrhage. An immunologic study in one patient was normal for the suppressor:cytotoxic lymphocyte ratio and concentration of interleukin-2 receptor during and in between hemorrhagic attacks. The cytochrome c oxidase activity in fibroblasts was normal. The rate of oxidation of glucose, leucine, isoleucine, valine, propionate and butyrate by fibroblasts was normal. The disease is not responsive to treatment with riboflavin, ascorbic acid, vitamin E, glycine, or carnitine. One patient remained stable on prolonged large doses of methylprednisolone. The biochemical defect leading to ethylmalonic aciduria in this disease remains unknown.

Ethylmalonic/adipic aciduria: effects of oral medium-chain triglycerides, carnitine, and glycine on urinary excretion of organic acids, acylcarnitines, and acylglycines.

A 9-y-old girl with ethylmalonic/adipic aciduria was hospitalized to determine the possible therapeutic efficacy of oral carnitine and glycine supplementation. To provoke a mild metabolic stress, her diet was supplemented with 440 mg/kg/d of medium-chain triglycerides. She was treated successively with carnitine (100 mg/kg/d) for 5 d, neither carnitine nor glycine for 2 d, and then glycine (250 mg/kg/d) for 6 d. Consecutive 12-h urine collections were obtained throughout the entire period. The urinary excretion of eight organic acids, four acylglycines, and four acylcarnitines, which accumulate as a result of a metabolic block of five mitochondrial acyl-CoA dehydrogenases, were quantitatively determined by capillary gas chromatography, stable isotope dilution gas chromatography/mass spectrometry, and radioisotopic exchange HPLC, respectively. The excretion of each group of metabolites was calculated as the mean percentage of total output (mumol/24 h) during the four phases of the protocol (organic acids/acylglycines/acylcarnitines = 100.0%): 1) regular diet (3 d); 88.1/10.8/1.1; 2) medium-chain triglyceride supplementation (4); 82.5/15.6/1.9; 3) medium-chain triglycerides plus carnitine (5); 79.2/8.2/12.6; and 4) medium-chain triglycerides plus glycine (6); 81.0/18.7/0.3. Comparison between total and individual excretion of acylglycines and acylcarnitines indicates that oral glycine supplementation enhanced the conjugation and excretion of fatty acyl-CoA intermediates as efficiently as carnitine. We propose that oral glycine supplementation should be considered in the treatment of other inborn errors of metabolism associated with abnormal urinary excretion of acylglycines.

Phenylacetylglutamine may replace urea as a vehicle for waste nitrogen excretion.

Phenylacetylglutamine (PAG), the amino acid acetylation product of phenylacetate (or phenylbutyrate after beta-oxidation) was evaluated as a waste nitrogen product in patients with inborn errors of urea synthesis. A boy with carbamyl phosphate synthetase deficiency receiving a low nitrogen intake excreted 80-90% of administered phenylacetate or phenylbutyrate as PAG. The amount of PAG nitrogen excreted varied from 38-44% of his dietary nitrogen, similar to the relationship between urea nitrogen and dietary nitrogen found in normal subjects receiving low dietary nitrogen. With few exceptions, neither phenylacetate nor phenylbutyrate accumulated in plasma. Treatment with relatively high dose phenylacetate or phenylbutyrate (0.5-0.6 g/kg/d) resulted in normal daytime levels of glutamine. These data suggest that PAG may replace urea as a waste nitrogen product when phenylbutyrate is administered at a dose that yields PAG nitrogen excretion equal to 40-44% of a low nitrogen intake.

[Hypocarnitinemia in patients affected by a primary defect of ammonia metabolism treated with sodium benzoate]. / Hypocarnitinémie chez les patients atteints d'un défaut primaire du métabolisme de l'ammoniaque traités avec le benzoate de sodium.

We have studied the plasma and urinary levels of free and esterified carnitine in 18 patients affected by a primary defect of ammonia metabolism, which had been managed with or without a therapy of sodium benzoate. None of these patients presented with any acute neurologic or digestive symptoms during the study. Our group of non-treated patients showed an increase in the levels of plasma esterified carnitine and an elevation of urinary concentration of free carnitine, while the levels of urinary esterified carnitine clearly approached the superior limits of normal values. The group treated with sodium benzoate showed a more profoundly disturbed plasma and urinary carnitine profile: a significantly lower plasma and urinary free carnitine, accompanied by a clearly increased esterified/free carnitine ratio. We did not find any evidence of a relationship between the plasma levels of free or esterified carnitine and the protein intake or the plasma ammonia concentration. We are proposing a hypothesis to explain the hypocarnitinemia seen in our patients being treated with benzoate, along with other modifications observed in the carnitine profile. We believe that a supplement of carnitine could be beneficial in the management of some of these patients.

[Rationale for the use of sodium benzoate in clinical hepatology]. / Benzoato de sodio. Fundamentos para su uso clínico en hepatología.

Sodium benzoate is widely used in the Alimentary Industry at low doses for its antimicrobial action. It has also been used as a liver function test. The principle is to evaluate the liver capacity for conjugation of glycine to benzoic acid and to form hippuric acid which is excreted in the urine. In hyperammonemic syndromes, secondary to enzymatic deficiency of the urea cicle, sodium benzoate has the property to act as an alternative way of nitrogen excretion to urinary hippurate instead of urea. Recently, it has been proposed as a therapeutic alternative in cirrhotic patients with portal systemic encephalopathy. Historical, biochemical and clinical data which constitute the principles to validate its clinical application in Hepatology are reviewed in this manuscript.

Magnetic resonance spectroscopy in the recognition of metabolic disease.

Magnetic resonance (MR) is rapidly entering many fields of clinical medicine following a long history as a powerful tool in physics and chemistry. The non-invasive and non-destructive property of this technique has enabled the chemical shift in higher magnetic fields to be exploited to identify and quantitate metabolites in both in vitro and in vivo analysis. High resolution proton spectroscopy of body fluids has been shown to be complementary with established analytical techniques, while the development of whole body large bore magnets is enabling both the study of structure and metabolism in humans in vivo. Phosphorus MR spectroscopy has provided a method of monitoring ATP production and utilisation in situ in both perfused preparation and intact tissue. In human muscle it has been possible to test established theories of tissue energy metabolism. It provides a unique method with which to evaluate the state of tissue oxidative metabolism. The opportunities afforded by other nuclei are being studied, but the low sensitivity of the MR technique forces limitations. Recent technical advances in tissue localization have as yet only been applied in a limited way. The use of MR in metabolic disease will be considered with specific reference to disorders of skeletal muscle metabolism.

Riboflavin-responsive ethylmalonic-adipic aciduria.

A patient presenting with a condition resembling Reye's syndrome was found to have a urinary organic acid excretion pattern similar to those previously described in a single patient with ethylmalonic-adipic aciduria. The present patient responded clinically to riboflavin supplementation and his fibroblasts, when cultured in riboflavin-depleted medium, showed an abnormal reduction in the rate of butyrate oxidation.

Propionylcarnitine excretion in propionic and methylmalonic acidurias: a cause of carnitine deficiency.

Two patients with propionic acidemia (PA) and two patients with methylmalonic aciduria (MMA) had low plasma free carnitine and increased short-chain acylcarnitines. Urinary excretion of free carnitine was decreased, while the excretion of short-chain acylcarnitines, mostly propionylcarnitine , was increased. Carnitine supplementation markedly increased the short-chain acylcarnitine fractions of both plasma and urine. Total carnitine content was decreased in skeletal muscle biopsies obtained from two of the patients. It is suggested that in these organic acidurias mitochondrial propionylcarnitine , formed from free carnitine and excess propionylCoA exchanges with free cytosolic carnitine: propionylcarnitine is then lost in the urine, causing secondary carnitine deficiency in the tissues.