OBJECTIVETo determine the levels of blood spot carnitine and acylcarnitine in children aged 0-15 years by tandem mass spectrometry, offer basic data for evaluating carnitine nutritional status and diagnosing metabolic diseases of organic acid and fatty acid.

METHODSThe concentration of carnitine and acylcarnitines were measured in blood spot by tandem mass spectrometry using underivatized samples. The samples included those from 1376 perinatal neonates, 49 neonates above 1 week of life, 64 children aged up to 1 year and 401 children aged 1 year to 15 years. A few premature infants and low birth weight infants were involved in perinatal neonates without selection. Other samples were taken from mainly outdoor patients for little surgical preoperative examination. Patients suffering from fever, diarrhea, liver disease, severe fat-metabolic diseases were excluded from this study.

RESULTSThe concentrations of carnitine (C(0)); short-chain acylcarnitines (SC-AC), including acetyl (C(2)), propionyl (C(3)), malonyl (C(3)DC), butyryl (C(4)), methylmalonyl (C(4)DC), isovaleryl (C(5)), glutaryl (C(5)DC); middle-chain acylcarnitines (MC-AC), including hexanoyl (C(6)), hexanediol (C(6)DC), octylenoyl (C(8:1)), octanoyl (C(8)), decadienoyl (C(10:2)), decanoyl (C(10:1)), decanoyl (C(10)); total carnitine and acylcarnitines (TCAC)were lower in neonate, highest in 1-3 months of age, higher in 6-12 months of age, and kept at the same level between 2 and 15 years of age. The concentrations of total long-chain acylcarnitines (LC-AC), including lauren (C(12:1)), lauroyl (C(12)), tetradecanoyl (C(14:1)), tetradecanoyl (C(14)), 3-hydroxy-tetradecanoyl (C(14)OH), hexadecenoyl (C(16:1)), hexadecanoyl (C(16)), 3-hydroxy-hexadecanoyl (C(16)OH), 3-hydroxy-hexadecanoyl (C(16:1)OH), octadecadienoyl (C(18:2)), octadecenoyl (C(18:1)), octadecanoyl (C(18)), 3-hydroxy-octadecenoyl (C(18:1)OH), and 3-hydroxy-octadecanoyl (C(18)OH) were the highest in neonate, decreased gradually, and kept the same level between 2 and 15 years of age. The concentrations of C(0) (23.387 ± 7.702) µmol/L, (30.064 ± 8.252) µmol/L, (25.021 ± 6.630) µmol/L, of LC-AC (4.998 ± 1.557) µmol/L, (2.854 ± 0.821) µmol/L, (2.459 ± 0.553) µmol/L, of TCAC (43.497 ± 12.632) µmol/L, (49.013 ± 12.497) µmol/L, (39.656 ± 9.257) µmol/L were significantly different among the groups of neonate, up to 1 year and above 1 year (P < 0.01). The concentrations of C(0) (24.115 ± 7.715) µmol/L and TCAC (43.65 ± 5.252) µmol/L in perinatal male neonates were higher than that (22.696 ± 7.246) µmol/L, TCAC (41.90 ± 5.038) µmol/L in female neonates. The C(0)/TCAC ratio of neonatal group (54.0% ± 7.1%) was significantly lower than that in the children group (62.1% ± 6.1%, P < 0.05), LC-AC/TCAC (33.5% ± 6.0%), MC-AC/TCAC (1.3% ± 0.3%), SC-AC/TCAC (11.6% ± 2.5%)ratios of neonatal group were higher than that of children group respectively (30.1% ± 4.9%; 0.9% ± 0.6%; 6.5% ± 2.3%, P < 0.05).

CONCLUSIONSConcentrations and profiles of carnitine and acylcarnitines change significantly during the first year of life, the age should be considered as a factor when evaluating carnitine nutritional status and diagnosing metabolic diseases of organic acid and fatty acid. Concentrations of carnitine and acylcarnitines were a little higher in male neonates than in female.

Adolescent ; Carnitine ; analogs & derivatives ; blood ; Child ; Child, Preschool ; Female ; Humans ; Infant ; Infant, Newborn ; Male ; Tandem Mass Spectrometry ; methods

OBJECTIVETo investigate the fat emulsion tolerance in preterm infants of different gestational ages in the early stage after birth.

METHODSA total of 98 preterm infants were enrolled and divided into extremely preterm infant group (n=17), early preterm infant group (n=48), and moderate-to-late preterm infant group (n=33). According to the dose of fat emulsion, they were further divided into low- and high-dose subgroups. The umbilical cord blood and dried blood filter papers within 3 days after birth were collected. Tandem mass spectrometry was used to measure the content of short-, medium-, and long-chain acylcarnitines.

RESULTSThe extremely preterm infant and early preterm infant groups had a significantly lower content of long-chain acylcarnitines in the umbilical cord blood and dried blood filter papers within 3 days after birth than the moderate-to-late preterm infant group (P<0.05), and the content was positively correlated with gestational age (P<0.01). On the second day after birth, the low-dose fat emulsion subgroup had a significantly higher content of short-, medium-, and long-chain acylcarnitines than the high-dose fat emulsion subgroup among the extremely preterm infants (P<0.05). In the early preterm infant and moderate-to-late preterm infant groups, there were no significant differences in the content of short-, medium-, and long-chain acylcarnitines between the low- and high-dose fat emulsion subgroups within 3 days after birth.

CONCLUSIONSCompared with moderate-to-late preterm infants, extremely preterm infants and early preterm infants have a lower capacity to metabolize long-chain fatty acids within 3 days after birth. Early preterm infants and moderate-to-late preterm infants may tolerate high-dose fat emulsion in the early stage after birth, but extremely preterm infants may have an insufficient capacity to metabolize high-dose fat emulsion.

Carnitine ; analogs & derivatives ; blood ; Fat Emulsions, Intravenous ; analysis ; metabolism ; Gestational Age ; Humans ; Infant, Newborn ; Infant, Premature ; metabolism

Medium- and short-chain acyl-CoA dehydrogenase deficiency is a disorder of fatty acid β-oxidation. Gene mutation prevents medium- and short-chain fatty acids from entry into mitochondria for oxidation, which leads to multiple organ dysfunction. In this study, serum acylcarnitines and the organic acid profile in urea were analyzed in two children whose clinical symptoms were hypoglycemia and metabolic acidosis. Moreover, gene mutations in the two children and their parents were evaluated. One of the patients was a 3-day-old male who was admitted to the hospital due to neonatal asphyxia, sucking weakness, and sleepiness. The serum acylcarnitine profile showed increases in medium-chain acylcarnitines (C6-C10), particularly in C8, which showed a concentration of 3.52 μmol/L (reference value: 0.02-0.2 μmol/L). The analysis of organic acids in urea gave a normal result. Sanger sequencing revealed a reported c.580A>G (p.Asn194Asp) homozygous mutation at exon 7 of the ACADM gene. The other patient was a 3-month-old female who was admitted to the hospital due to cough and recurrent fever for around 10 days. The serum acylcarnitine profile showed an increase in serum C4 level, which was 1.66 μmol/L (reference value: 0.06-0.6 μmol/L). The analysis of organic acids in urea showed an increase in the level of ethyl malonic acid, which was 55.9 (reference value: 0-6.2). Sanger sequencing revealed a reported c.625G>A (p.Gly209Ser) homozygous mutation in the ACADS gene. This study indicates that screening tests for genetic metabolic diseases are recommended for children who have unexplained metabolic acidosis and hypoglycemia. Genetic analyses of the ACADM and ACADS genes are helpful for the diagnosis of medium- and short-chain acyl-CoA dehydrogenase deficiency.
Acyl-CoA Dehydrogenase ; deficiency ; genetics ; Carnitine ; analogs & derivatives ; blood ; Female ; Humans ; Infant ; Infant, Newborn ; Lipid Metabolism, Inborn Errors ; genetics ; Male ; Mutation ; Urea ; analysis

BACKGROUNDData of classical inborn errors of metabolism (IEM) of amino acids, organic acids and fatty acid oxidation are largely lacking in Hong Kong, where mass spectrometry-based expanded newborn screening for IEM has not been initiated. The current study aimed to evaluate the approximate incidence, spectrum and other characteristics of classical IEM in Hong Kong, which would be important in developing an expanded newborn screening program for the local area.

METHODSThe laboratory records of plasma amino acids, plasma acylcarnitines and urine organic acids analyses from year 2005 to 2009 inclusive in three regional chemical pathology laboratories providing biochemical and genetic diagnostic services for IEM were retrospectively reviewed.

RESULTSAmong the cohort, 43 patients were diagnosed of IEM, including 30 cases (69%) of amino acidemias (predominantly citrin deficiency, hyperphenylalaninemia due to 6-pyruvoyl-tetrahydropterin synthase deficiency and tyrosinemia type I), 5 cases (12%) of organic acidemias (predominantly holocarboxylase synthetase deficiency) and 8 cases (19%) of fatty acid oxidation defects (predominantly carnitine-acylcarnitine translocase deficiency). The incidence of classical IEM in Hong Kong was roughly estimated to be at least 1 case per 4122 lives births, or 0.243 cases per 1000 live births. This incidence is similar to those reported worldwide, including the mainland of China. The estimated incidence of hyperphenylalaninemia was 1 in 29 542 live births.

CONCLUSIONSOur data indicate that it is indisputable for the introduction of expanded newborn screening program in Hong Kong. Since Hong Kong is a metropolitan city, a comprehensive expanded newborn screening program and referral system should be available to serve the neonates born in the area.

Acids ; urine ; Amino Acids ; blood ; Carnitine ; analogs & derivatives ; blood ; Hong Kong ; epidemiology ; Humans ; Infant, Newborn ; Metabolism, Inborn Errors ; blood ; diagnosis ; epidemiology ; urine ; Neonatal Screening ; methods ; Tandem Mass Spectrometry

OBJECTIVE: To investigate the genetic characterization of 3-hydroxyisovalerylcarnitine (C5-OH) metabolic abnormality in neonates. METHODS: Fifty two newborns with increased C5-OH, C5-OH/C3 and C5-OH/C8 detected by tandem mass spectrometry during neonatal screening were enrolled in the study. Genomic DNA was extracted from the whole blood samples of 52 cases and their parents. Seventy-nine genes associated with genetic and metabolic diseases including , were targeted by liquid capture technique. Variation information of these genes was examined by high-throughput sequencing and bioinformatic analysis, and then was classified based on the American College of Medical Genetics and Genomics (ACMG) standards and guidelines. The genetic types were classified as wild-type, -maternal-mutation, -paternal-mutation and -mutation. Wilcoxon rank-sum test was performed for the increased multiples of C5-OH calculated in neonatal screening. RESULTS: Twenty one variants (14 novel) were identified in 37 cases, 6 variants (5 novel) in 4 cases. The increased multiple of C5-OH calculated in -maternal-mutation and -mutation groups were significantly higher than that in wild-type group (all <0.05), while there was no significant difference between MCCC1-paternal-mutation group and wild-type group (>0.05). CONCLUSIONS Mutations on and genes are the major genetic causes for the increased C5-OH in neonates, and maternal single heterozygous mutation can contribute to the moderately to severely increased C5-OH.
Carbon-Carbon Ligases ; genetics ; Carnitine ; analogs & derivatives ; metabolism ; Female ; Genetic Testing ; Genetic Variation ; Humans ; Infant, Newborn ; Male ; Mutation ; Neonatal Screening ; Urea Cycle Disorders, Inborn ; genetics

BACKGROUNDMethylmalonic aciduria (MMA) is the most frequent disease of organic aciduria in China. Various biochemical strategies are followed for the prenatal diagnosis of MMA. However, since fetuses affected by MMA have decreased excretion of methylmalonic acid, the difficulties of prenatal biochemical diagnosis are obvious. Gas chromatography mass spectrometry (GC/MS) and tandem mass spectrometry (ESI/MS/MS) have allowed us to identify the disease in affected fetuses. The aim of this study was to determine the value of analysis of organic acids and total homocysteine in amniotic fluid in prenatal diagnosis of MMA.

METHODSThe clinical diagnoses and outcomes of nine probands with MMA and the prenatal diagnoses based on biochemical analysis of nine fetuses at risk for MMA were investigated. Amniotic fluid samples from pregnancies at risk for MMA and metabolically normal pregnancies were obtained at 16 - 24 weeks of gestation. Methylmalonic acid and methylcitric acid were measured by GC/MS, propionylcarnitine was analyzed by ESI/MS/MS, and total homocysteine was determined by fluorescence polarization immunoassay.

RESULTSIn two pregnancies, high levels of methylmalonic acid, methylcitric acid, propionylcarnitine, and total homocysteine indicated combined MMA and homocysteinemia in the fetuses. One of the mothers continued pregnancy and received cobalamin supplement as prenatal treatment, and the other terminated her pregnancy. In one pregnancy, significantly elevated levels of methylmalonic acid, methylcitric acid, and propionylcarnitine, and normal level of total homocysteine was found indicating isolated MMA in the fetus; abortion was performed on this case. In the other six pregnancies, all the levels of the above mentioned metabolites were normal suggesting that the fetuses were not affected by MMA. The diagnoses were confirmed after delivery by testing urinary organic acids and plasma total homocysteine.

CONCLUSIONSThe metabolic abnormalities of MMA occur early in gestation. The level of total homocysteine in amniotic fluid may be an additional indicator of fetal combined MMA and homocysteinemia. Determination of total homocysteine level in amniotic fluid may become a convenient and reliable method for prenatal diagnosis of the disease.

Amniotic Fluid ; chemistry ; Carnitine ; analogs & derivatives ; analysis ; Citrates ; analysis ; Female ; Gas Chromatography-Mass Spectrometry ; Homocysteine ; analysis ; blood ; Humans ; Male ; Methylmalonic Acid ; urine ; Pregnancy ; Prenatal Diagnosis ; methods ; Spectrometry, Mass, Electrospray Ionization

OBJECTIVETo evaluate the effects of valsartan and carnitine on cardiomyocyte Calpain-1 and Bcl-xl expressions of dogs with chronic alcohol intake-induced cardiomyopathy.

METHODSDogs were randomly assigned into 4 groups (n = 7 each): (1) alcohol fed (free access to 5%, 1(st) week; 10% 2(nd) week; 500 ml 25% bolus plus free access to 5% from 3 to 24 weeks, A); (2) alcohol + valsartan (5 mg×kg(-1)×d(-1), B); (3) alcohol + carnitine (300 mg×kg(-1)×d(-1), C); (4) Control (D). After six months, all animals were assessed for left ventricular (LV) function by echocardiography. The Bad and Bcl-xl protein expressions were evaluated by immunohistochemistry. The expression of Calpain-1 protein was determined with Western blot. Myocardial morphology was quantified on HE stained slices and under electron microscopy. The terminal deoxynucleotidyl transferase-mediated nick end-labeling (TUNEL) was performed for apoptosis analysis.

RESULTSCompared with group D, LVEDD and LVESD were significantly increased while EF and FS significantly decreased in group A. In alcohol fed group, expressions of Bad and Calpain-1 protein were significantly increased while Bcl-xl protein expression was downregulated, all changes could be significantly attenuated by intervention with valsartan and carnitine (all P < 0.05).

CONCLUSIONThese data suggest that alcohol could promote cardiac myocyte apoptosis, reduce cardiac function and aggravate myocardial remodeling which valsartan and carnitine could reduce alcoholic cardiomyopathy by downregulating Calpain-1 and Bad protein expression and upregulating expression of Bcl-xl protein.

Animals ; Apoptosis ; drug effects ; Calpain ; metabolism ; Cardiomyopathy, Alcoholic ; metabolism ; pathology ; Carnitine ; pharmacology ; Disease Models, Animal ; Dogs ; Myocytes, Cardiac ; drug effects ; metabolism ; Tetrazoles ; pharmacology ; Valine ; analogs & derivatives ; pharmacology ; Valsartan ; bcl-Associated Death Protein ; metabolism ; bcl-X Protein ; metabolism

OBJECTIVEThe new technology of tandem mass spectrometry is exerting a significant impact on the diagnostics of inborn metabolic errors, and allows to detect a number of these disorders in a single step. The aim of the present study was to establish a dry blood filter paper method for amino acid and acylcarnitine profiles test using tandem mass spectrometry and to apply the method for selective screening in high risk children with inborn error of metabolism.

METHODThe study group consisted of 104 high risk cases of inborn error of metabolism from 5 pediatric hospitals in Shanghai and Beijing were tested from November 2002 to June 2003; 77 were males and 27 females, the means age was 4.8 +/- 4.2 years. These patients had mental retardation, slow development, psychological abnormalities, muscle hypotonia, jaundice, hepatosplenomegaly, recurrent vomiting, and convulsion. Laboratory examinations suggested metabolic acidosis, hypoglycemia, hyperammonemia and hyperlactacidemia. Phenylketonuria was excluded in this study by routine phenylalanine screening. The control group consisted of 308 children, 170 males and 138 females. The blood was collected on filter paper, punched and extracted into methanol solution with stable isotope labeled internal standards, then derivatized with butanolic-HCl. After preparation, the samples were analyzed by tandem mass spectrometry (API 2000).

RESULTSTen of 104 patients (9.6%) were positive in our selective screening program, including one with tyrosinemia, one with homocystinuria, one with hyperornithinemia, two with methylmalonic acidemia, one with propionic acidemia, one with 3-hydroxy-3-methylglutaryl-CoA lyase deficiency, two with medium chain acyl-CoA dehydrogenase deficiency, and one with carnitine palmitoyl transferase type II deficiency.

CONCLUSIONThe authors established a fast, accurate and sensitive tandem mass spectrometry method for amino acid and acylcarnitine profiles analysis, nearly 30 metabolic diseases including amino acid disorders, organic acid disorders and fatty acid oxidation disorders could be detected, most of the diseases that cause death and disability represent preventable entities by early diagnosis and treatment. The results indicated the importance of selective screening for high risk patients with inborn error of metabolism.

Adolescent ; Amino Acids ; blood ; Carnitine ; analogs & derivatives ; blood ; Child ; Child, Preschool ; China ; Female ; Humans ; Infant ; Male ; Mass Screening ; Mass Spectrometry ; methods ; Metabolism, Inborn Errors ; blood ; diagnosis ; Pilot Projects ; Risk Factors

OBJECTIVEWe report the first case of acute encephalopathy induced by vaccination in an infant with methylmalonic aciduria cblA in China.

METHODThe clinical presentation, blood acylcarnitines analysis, urine organic acids analysis and gene studies of the patient were summarized.

RESULTThe proband, a boy, was admitted at the age of 15 months because of recurrent vomiting, acidosis and development delay for 8 months. The previously healthy boy presented vomiting and coma just one hour after hepatitis B vaccination at the age of seven months. Moderate dehydration, electrolyte disturbance and metabolic acidosis had been found. Although his acute metabolic crisis had been corrected soon after intravenous transfusion, psychomotor retardation and recurrent vomiting had been observed. When he was 15 months old, vomiting and lethargy occurred again 3 hours after DTaP vaccination. He was weakened as the illness became worse and got coma with dyspnea 7 days later. He was hospitalized with the suspected diagnosis of viral encephalitis. Blood acylcarnitines analysis, urine organic acids analysis and gene study had been performed for the etiologic investigation.His blood propionylcarnitine (16.3 µmol/L vs. normal range 1.0-5.0 µmol/L) and propionylcarnitine/free carnitine ratio (0.27 vs. normal range 0.03 to 0.25) increased. Markedly elevated urinary methylmalonic acid (388.21 mmol/mol creatinine vs. normal range 0.2 to 3.6 mmol/mol creatinine) and normal plasma total homocysteine supported the diagnosis of isolated methylmalonic aciduria. Two mutations, c.650 T>A (p.L217X) and c.742 C>T (p.Q248X), were identified in his MMAA gene, confirmed the diagnosis of cblA. Each parent carried one of the two mutations. Progressive clinical and biochemical improvement has been observed after hydroxylcobalamin injection, protein-restricted diet with the supplements of special formula and L-carnitine. He is currently 2 years and 7 months old with normal development and general condition.

CONCLUSIONA boy with cblA was firstly detected after the acute encephalopathy induced by vaccination in China. It is important to pay more attention to the patients with metabolic crisis or organ damage after vaccination. Metabolic studies are keys to the diagnosis of potential diseases and improve the outcome.

Amino Acid Metabolism, Inborn Errors ; complications ; Brain Diseases ; chemically induced ; Carnitine ; analogs & derivatives ; Diet, Protein-Restricted ; Hepatitis B Vaccines ; adverse effects ; Humans ; Infant ; Male ; Methylmalonic Acid ; urine ; Mutation ; Vaccination ; adverse effects ; Vitamin B Complex ; Vomiting

PURPOSE: Our previous high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry study identified bladder cancer (BCA)-specific urine metabolites, including carnitine, acylcarnitines, and melatonin. The objective of the current study was to determine which metabolic pathways are perturbed in BCA, based on our previously identified urinary metabolome. MATERIALS AND METHODS: A total of 135 primary BCA samples and 26 control tissue samples from healthy volunteers were analyzed. The association between specific urinary metabolites and their related encoding genes was analyzed. RESULTS: Significant alterations in the carnitine-acylcarnitine and tryptophan metabolic pathways were detected in urine specimens from BCA patients compared to those of healthy controls. The expression of eight genes involved in the carnitine-acylcarnitine metabolic pathway (CPT1A, CPT1B, CPT1C, CPT2, SLC25A20, and CRAT) or tryptophan metabolism (TPH1 and IDO1) was assessed by RT-PCR in our BCA cohort (n=135). CPT1B, CPT1C, SLC25A20, CRAT, TPH1, and IOD1 were significantly downregulated in tumor tissues compared to normal bladder tissues (p<0.05 all) of patients with non-muscle invasive BCA, whereas CPT1B, CPT1C, CRAT, and TPH1 were downregulated in those with muscle invasive BCA (p<0.05), with no changes in IDO1 expression. CONCLUSION: Alterations in the expression of genes associated with the carnitine-acylcarnitine and tryptophan metabolic pathways, which were the most perturbed pathways in BCA, were determined.
Aged ; Biomarkers/metabolism ; Carcinoma, Transitional Cell/genetics/*metabolism/pathology ; Carnitine/*analogs & derivatives/genetics/metabolism ; Case-Control Studies ; Female ; Humans ; Male ; Metabolic Networks and Pathways/*physiology ; Middle Aged ; RNA, Messenger/metabolism ; Real-Time Polymerase Chain Reaction ; Urinary Bladder Neoplasms/genetics/*metabolism/pathology