Outcomes of patients with combined methylmalonic acidemia and homocystinuria after treatment / 中华儿科杂志

<p><b>OBJECTIVE</b>Combined methylmalonic acidemia with homocystinuria is a common form of methylmalonic acidemia in China. Patients with this disease can progress to death without timely and effective treatment. This study aimed to analyze the treatment outcomes of patients with combined methylmalonic acidemia and homocystinuria.</p><p><b>METHOD</b>From September 2004 to April 2012, 58 patients with combined methylmalonic acidemia and homocystinuria (34 males and 24 females) were diagnosed and treated in our hospital. Fifty cases were from clinical patients including 42 early-onset cases and 8 late-onset cases. Their age when they were diagnosed ranged from 18 days to 30.8 years. The other 8 cases were from newborn screening. All the patients were treated with vitamin B12, betaine, folic acid, vitamin B6, and L-carnitine. The physical and neuropsychological development, general laboratory tests, the levels of amino acids, acylcarnitines, and homocysteine in blood, and organic acids in urine were followed up.</p><p><b>RESULT</b>The follow-up period ranged from 1 month to 7.1 years. Three cases died (all were early-onset cases). In the other patients after treatment, the symptoms such as recurrent vomiting, seizures, lethargy, and poor feeding disappeared, muscle strength and muscle tension were improved, and general biochemical abnormalities such as anemia and metabolic acidosis were corrected. Among the surviving 55 cases, 49 had neurological impairments such as developmental delay and mental retardation. The median levels of blood propionylcarnitine and its ratio with acetylcarnitine, serum homocysteine, and urine methylmalonic acid were significantly decreased (P < 0.01), from 7.73 µmol/L (ranged from 1.5 to 18.61 µmol/L), 0.74 (ranged from 0.29 to 2.06), 97.3 µmol/L (ranged from 25.1 to 250 µmol/L) and 168.55 (ranged from 3.66 to 1032.82) before treatment to 2.74 µmol/L (ranged from 0.47 to 12.09 µmol/L), 0.16 (ranged from 0.03 to 0.62), 43.8 µmol/L (ranged from 17 to 97.8 µmol/L) and 6.81 (ranged from 0 to 95.43) after treatment, respectively.</p><p><b>CONCLUSION</b>Patients with combined methylmalonic acidemia and homocystinuria respond to a combined treatment consisting of supplementation of hydroxycobalamin, betaine, folic acid, vitamin B6 and L-carnitine with clinical and biochemical improvement. But the long-term outcomes are unsatisfactory, with neurological sequelae in most patients.</p>

Application of succinylacetone levels measurement in the blood and urine in the diagnosis of tyrosinemia type 1 / 中华儿科杂志

<p><b>OBJECTIVE</b>To establish the diagnostic method of tyrosinemia type 1 and evaluate its value, the succinylacetone levels in the blood of suspected patients with tyrosinemia were tested by tandem mass spectrometry, and the succinylacetone in the urine was tested by gas chromatography-mass spectrometry.</p><p><b>METHOD</b>A total of 190 patients suspected of having tyrosinemia, were tested by tandem mass spectrometry for measurement of the level of succinylacetone in the blood, and detected by gas chromatography-mass spectrometry for measurement of the level of succinylacetone and organic acid in the urine. The method of measuring the level of succinylacetone in blood by tandem mass spectrometry as follows: After the diameter of 3 mm dry blood spots were punched into wells of 96-well plate, 100 µl 80% acetonitrile were added into each well, which contained hydrazine monohydrate and the internal standard of succinylacetone. The supernatant fluid were transferred to another 96-well plate and dried under heated nitrogen, after the plate was incubated for 30 min at 65°C. The residual hydrazine reagent was removed by addition of 100 µl methanol to each well and evaporated under heated nitrogen. The mobile phase (80% acetonitrile) was added to each well and 20 µl samples were tested by tandem mass spectrometry. The diagnostic terms were the clinical manifestation and the high level of succinylacetone in both blood and urine.</p><p><b>RESULT</b>Eleven patients were diagnosed as tyrosinemia type 1, with 9 males and 2 females. Their ages ranged from 2 months to 6 years. The succinylacetone levels in the blood of the patients were remarkably increased (7.26-31.09 µmol/L), with an average of (14.2 ± 7.8)µmol/L. Seven patients were tested for the level of succinylacetone in the urine by gas chromatography-mass spectrometry, and 4 were positive and 3 negative. Their tyrosine levels in the blood were 190-543 µmol/L(Normal: 20 - 100 µmol/L), with an average of (327.3 ± 125.8) µmol/L. All the patients presented the symptoms of hepatomegaly. Among them, 9 patients died and 2 patients were improved after treatment.</p><p><b>CONCLUSION</b>The higher levels of succinylacetone in the blood or urine is a remarkable evidence for the diagnosis of tyrosinemia type 1. Determination of succinylacetone in the dry blood spots using tandem mass spectrometry was a good method for diagnosis of tyrosinemia type 1. To test succinylacetone in urine by gas chromatography-mass spectrometry may yield a false-negative result for tyrosinemia type 1.</p>

Primary carnitine deficiency in 17 patients: diagnosis, treatment and follow up / 中华儿科杂志

<p><b>OBJECTIVE</b>Many children were found to have low free carnitine level in blood by tandem mass spectrometry technology. In some of the cases the problems occurred secondary to malnutrition, organic acidemia and other fatty acid oxidation metabolic diseases, and some of cases had primary carnitine deficiency (PCD). In the present article, we discuss the diagnosis of PCD and evaluate the efficacy of carnitine in the treatment of PCD.</p><p><b>METHOD</b>We measured the free carnitine (C0) and acylcarnitine levels in the blood of 270 000 neonates from newborns screening program and 12 000 children with suspected clinical inherited metabolic diseases by tandem mass spectrometry. The mutations of carnitine transporter protein were tested to the children with low C0 level and the diagnosis was made. The children with PCD were treated with 100 - 300 mg/kg of carnitine.</p><p><b>RESULT</b>Seventeen children were diagnosed with PCD, 6 from newborn screening program and 11 from clinical patients. Mutations were found in all of them. The average C0 level [(2.9 ± 2.0) µmol/L] in patients was lower than the reference value (10 µmol/L), along with decreased level of different acylcarnitines. The clinical manifestations were diverse. For the 6 patients from newborn screening, 4 were asymptomatic, 1 showed hypoglycaemia and 1 showed movement intolerance from 2 years of age. For the 11 clinical patients, 8 showed hepatomegaly, 7 showed myasthenia, 6 showed cardiomyopathy, 1 showed chronic abdominal pain, and 1 showed restlessness and learning difficulty. Among these patients, 14 cases were treated with carnitine. Their clinical symptoms disappeared 1 to 3 months later. The C0 level in the blood rose to normal, with the average from (4.0 ± 2.7) µmol/L to (20.6 ± 8.3) µmol/L (P < 0.01). However, the level was still lower than the average level of healthy children [(27.1 ± 4.5) µmol/L, P < 0.01].</p><p><b>CONCLUSION</b>Seventeen patients were diagnosed with PCD by the test levels of free carnitine and acylcarnitines in blood with tandem mass spectrometry, and gene mutation test. Large dose of carnitine had a good effect in treatment of the PCD patients.</p>

Rapamycin induces differentiation of glioma stem/progenitor cells by activating autophagy / 癌症

Glioma stem/progenitor cells (GSPCs) are considered to be responsible for the initiation, propagation, and recurrence of gliomas. The factors determining their differentiation remain poorly defined. Accumulating evidences indicate that alterations in autophagy may influence cell fate during mammalian development and differentiation. Here, we investigated the role of autophagy in GSPC differentiation. SU-2 cells were treated with rapamycin, 3-methyladenine (3-MA) plus rapamycin, E64d plus rapamycin, or untreated as control. SU-2 cell xenografts in nude mice were treated with rapamycin or 3-MA plus rapamycin, or untreated as control. Western blotting and immunocytochemistry showed up-regulation of microtubule-associated protein light chain-3 (LC3)-II in rapamycin-treated cells. The neurosphere formation rate and the number of cells in each neurosphere were significantly lower in the rapamycin treatment group than in other groups. Real-time PCR and immunocytochemistry showed down-regulation of stem/progenitor cell markers and up-regulation of differentiation markers in rapamycin-treated cells. Transmission electron microscopy revealed autophagy activation in rapamycin-treated tumor cells in mice. Immunohistochemistry revealed decreased Nestin-positive cells and increased GFAP-positive cells in rapamycin-treated tumor sections. These results indicate that rapamycin induces differentiation of GSPCs by activating autophagy.