[Plasma metabonomics study of ischemic cerebral apoplexy rats treated with Tongsaimai pellets].

OBJECTIVE: To observe abnormal metabolic changes caused by ischemic cerebral apoplexy and the regulating action of Tongsaimai pellets on abnormal metabolism by analyzing the change of small molecules in plasma of ischemic cerebral apoplexy rat. To find the potential biomarkers, and to explore metabolic mechanisms of Tongsaimai pellets. METHOD: Rat models of middle cerebral artery occlusion was established with electric coagulation, and rats were divided into 4 groups, model group, sham-operation group, Tongsaimai pellets group and positive control group. Tongsaimai pellets and positive control group were orally administrated by 13.2 g x kg(-1) x d(-1) of crude drugs and 32 mg x kg(-1) x d(-1) of Nimodipine respectively, m odel and sham-operation group by equal volume of distilled water for a week. Plasma of model and sham-operation group were collected, and plasma of Tongsaimai pellets and positive control group were collected on the 1st, 3rd , 7th day after administration. Endogenous metabolites of four groups were determined with GC-MS. Partial least squares discriminant analysis (PLS-DA) was applied to analyze multivariate data and set up model, and T-test was used in significant statistical analysis. RESULT: Compared with sham-operation group rats, pyruvic acid, taurine and hydroxyproline obviously increased in model group rats, while lactic acid, glyceric acid, aminomalonic acid, fructose, tryptophan and leucine significantly decreased, so these metabolites were potential metabolic biomarkers. These endogenous metabolites except taurine got restoration in Tongsaimai group rats. CONCLUSION: Abnormal metabolite level in plasma can be certainly recovered by Tongsaimai pellets, and the treatment of Tongsaimai pellets can be connected with the regulation of related metabolic pathways.

Multiple sources of metabolic disturbance in ETHE1-related ethylmalonic encephalopathy.

Ethylmalonic encephalopathy (EE) is a rare metabolic disorder caused by dysfunction of ETHE1, a mitochondrial dioxygenase involved in hydrogen sulfide (H2S) detoxification. Patients present in infancy with psychomotor retardation, chronic diarrhea, orthostatic acrocyanosis and relapsing petechiae. High levels of lactic acid, ethymalonic acid (EMA) and methylsuccinic acid (MSA) are detected in body fluids. Several pathways may contribute to the pathophysiology, including isoleucine, methionine and fatty acid metabolism. We report on a 15-month-old male presenting with typical EE associated with a homozygous ETHE1 mutation. We investigated oral isoleucine (150 mg/kg), methionine (100 mg/kg), fatty acid loading tests and isoleucine-restricted diet (200 mg/day) for any effects on several metabolic parameters. Before loading tests or specific dietary interventions, EMA, C4-C5 acylcarnitines and most acylglycines were elevated, indicating functional deficiency of short chain acyl-CoA (SCAD) as well as all branched acyl-CoA dehydrogenases. Excretion of EMA and n-butyrylglycine increased following each of the loads, and isoleucine led to increased levels of derivative metabolites. An isoleucine-restricted diet for 8 days corrected some of the abnormalities but led to no obvious clinical improvement and only partial effects on EMA. A principal component analysis supports the inference that these dietary conditions have consistent effects on the global metabolic profile. Our results suggest that multiple pathways modulate EMA levels in EE. They might all interact with H2S toxicity. Prolonged dietary interventions involving the restriction for branched aminoacids, fatty acids and methionine could be discussed as auxiliary therapeutical strategies in EE.

[Simulation of the disease caused by Hantaan virus in white mice by a complex of virological, biochemical, and hematological parameters].

The essence of studies was that the disease is simulated in 12-day albino mice subcutaneously infected with Hantaan virus, strain 76-118 in a dose of 10 LD50. As an efficiency index, the study of drugs uses major (death protection coefficient, mean animal lifetime) and auxiliary (virological: pathogen accumulation in the brain tissues of deceased animals) parameters, biochemical (the levels of creatinine, urea, alanine aminotransferase, aspartate aminotransferase, malonic dialdehyde), hematological (count of leukocytes, leukogram) ones; as well as interferon status (the levels of circulatory interferon, leukocytic production of alpha- and gamma-interferons). The procedure for simulating the disease caused by Hantaan virus on an experimental animal is used to choose effective drugs against the pathogen of hemorrhagic nephrosonephritis.

[Changes of creatine phosphokinase and malondialdehyde in the serum and clinical use of large doses of vitamin C following open heart surgery].

The protective effect of a large dose Vit. C on ischemic myocardium was studied on 10 patients who underwent open heart operation and received 250 mg/kg Vit. C before the start of ECC (Group II). Another 10 similar patients receiving no Vit. C served as a control (Group I). Blood samples were taken from the brachial artery at different time intervals for determination of CPK-MB and MDA in both groups. Considerable increases of serum CPK-MB and MDA in both group I and II were noted. The differences of CPK-MB and MDA concentration between Group I and II were highly significant from 2 to 12 hours postoperatively. The effect of Vit. C in the reduction of the release of CPK-MB and MDA after open heart operation was attributed to its action as an oxygen free radical scavenger, thus decreasing the peroxidation of the lipids present in the cell membrane.

Sequential changes in plasma methylmalonic acid and vitamin B12 in sheep eating cobalt-deficient grass.

Methylmalonic acid (MMA) concentrations are elevated in plasma as a result of vitamin B12 deficiency. This study reports the sequential changes in plasma MMA in lambs maintained on a cobalt-deficient pasture compared with supplemented controls. The results indicate that MMA is elevated in the early stages of deficiency, preceding the onset of loss of production and clinical signs of disease. It remains elevated as long as the lambs are unsupplemented with cobalt (Co). The most striking clinical sign was a loss of body condition as opposed to weight. The defect in the methylmalonyl CoA mutase is obviously an early defect in cobalt deficiency.

Methylmalonic acid in the diagnosis of cobalt deficiency in barley-fed lambs.

Eight lambs were fed on a cobalt-deficient whole-barley diet supplemented with urea, vitamins and minerals. Four control lambs were fed on the same diet which had been further supplemented with Co. Plasma vitamin B12 levels in the Co-depleted group declined rapidly, falling below the normal range within 5 weeks. Differences between the live weights of the animals in the two groups approached statistical significance by week 14. However, methylmalonic acid (MMA) rose above normal levels in the Co-depleted group within 7 weeks. This suggested that an elevated plasma concentration of MMA is a comparatively early indicator of functional vitamin B12 deficiency. It is recommended that 10 mumol/l be the upper level of normality for plasma MMA concentration in barley-fed animals, in contrast with the level of 5 mumol/l for grass-fed animals. Changes in the plasma concentrations of MMA and ethylmalonic acid associated with feeding the barley-based diet per se did not significantly affect the validity of the gas-liquid chromatographic assay for MMA.

[Studies on the beneficial effect of levocarnitine chloride (LC-80) on organic acidemias, especially propionic acidemia and methylmalonic acidemia].

The beneficial effect of LC-80 in the therapy for organic acidemias, especially propionic acidemia and methylmalonic acidemia, was compared with those of its optical isomers, d-carnitine chloride (d-isomer) and dl-carnitine chloride (dl-isomer) in rat liver mitochondria. LC-80 at concentrations of 5 and 10 mM did not inhibit the mitochondrial function, while the d-isomer at a concentration of 5 mM significantly reduced the respiratory control ratio (RCR) of mitochondria. In addition, the dl-isomer at concentrations of 10 and 20 mM also significantly reduced RCR in a concentration-dependent manner. Thus, it seems likely that the d-isomer inhibits the mitochondrial function. On the other hand, the inhibition of mitochondrial function induced by a preincubation with propionate (4.76 mM) was significantly reversed by LC-80 (5 and 10 mM) in a concentration-dependent manner, while the d-isomer (5 mM) had no effect on the inhibitory effect of propionate. Moreover, although the dl-isomer (10 and 20 mM) significantly reversed the inhibitory effect of propionate as compared with the d-isomer, its effect was significantly weaker as compared with the effect of LC-80. The substrate specificity of rat liver mitochondrial carnitine acetyltransferase (CAT) was more potent with propionyl CoA than with acetyl CoA. Kinetic studies indicate that the d-isomer is a competitive inhibitor of CAT. These results suggest that LC-80 is useful in the clinical treatment of organic acidemias, whereas the d-isomer has a harmful effect in clinical application.

The influence of cod-liver oil diet on various lipid metabolism parameters, the thromboxane formation capacity, platelet function and the serum MDA level in patients suffering from myocardial infarction.

The influence of an eicosapentaenoic-acid-rich diet, containing 8.1 g cod-liver oil, given daily for two weeks on various parameters of lipid metabolism, the thromboxane formation capacity, platelet function and the serum MDA level was studied in 16 patients with myocardial infarction. The 18:2, 20:3 and 20:4 fatty acid portions of serum phospholipids were significantly decreased, whereas the 20:5 and 22:6 portions were significantly elevated. Serum total cholesterol, LDL cholesterol and the thromboxane formation capacity were significantly increased. Serum triglycerides, HDL cholesterol, the prostacyclin blocking activity of LDL, and several parameters of platelet function were unchanged, the platelet count was significantly depressed. As compared to those of healthy persons, the serum MDA levels in myocardial infarction patients were significantly increased both before and after cod-liver oil ingestion. A possible correlation between the elevated serum levels of MDA and total cholesterol, LDL cholesterol as well as thromboxane formation capacity and the cod-liver oil treatment in patients with myocardial infarction is discussed.

[Lipid peroxides and antioxidants in the blood of patients with arteriosclerotic circulatory disorders]. / Lipidperoxide und Antioxidantien im Blut von Patienten mit arteriosklerotischen Durchblutungsstörungen.

Malondialdehyde, vitamin E, glutathione peroxidase and selenium were analysed in the blood of 96 male patients with arteriosclerotic obstructive disease in the region of the leg. Of these 32 patients additionally had manifestations of a coronary or cerebral arteriosclerosis. In the parameters mentioned no significant differences were found between patients with general arteriosclerosis and isolated arteriosclerosis of the leg. Patients with arteriosclerosis and hyperlipoproteinaemia revealed higher values of malondialdehyde than patients without hyperlipoproteinaemia. No correlations were found between malondialdehyde and total cholesterol, triglycerides, vitamin E, glutathione peroxidase as well as selenium.

An evaluation of protein requirements in methylmalonic acidaemia.

A 3-month-old girl and a 13-month-old boy with vitamin B12-unresponsive methylmalonic acidaemia were studied to determine responses to varying levels of protein intake of growth, nitrogen balance and organic acid metabolism. A linear increase in the excretion of methylmalonic acid was observed in both patients above a critical level of protein intake. The inflection point was judged to reflect a ceiling above which amino acid intake exceeded requirements and catabolism was initiated. Below this point in each infant there was a plateau of minimal excretion of methylmalonic acid. Within this plateau level a reasonable rate of growth and metabolic stability were achieved at intakes between 0.70 and 0.75 and between 0.75 and 1.17 g protein kg-1, respectively, indicating that there is a range of protein tolerance and the importance of an individual approach to the provision of protein in patients with methylmalonic acidaemia. In the 3-month-old infant, nitrogen equilibrium was achieved at protein intakes above 0.6 g kg-1 and modest nitrogen retention was attained at a protein intake of 0.75 g kg-1, a level at which the excretion of methylmalonic acid was minimal and weight gain satisfactory. A protein intake of 1.25 g kg-1 was required to achieve a level of nitrogen retention often considered optimal for normal growth; however, this infant demonstrated an elevated excretion of methylmalonic acid and was close to clinical illness at this level of protein intake. The 13-month-old infant demonstrated a normal level of nitrogen retention, minimal excretion of methylmalonic acid, and a satisfactory rate of growth at protein intakes of 1.0-1.17 g kg-1. The values should prove useful guidelines for the management of infants requiring minimal intakes of protein. In studies carried out at 18-20 months of age, supplementation of the basic diet containing 0.75 g kg protein-1 with a mixture of amino acids not containing the precursors of methylmalonic acid was associated with increase of retention of nitrogen and increased concentrations of some essential amino acids in plasma, but effects on growth and the excretion of methylmalonic acid were not significant.

Cerebral CT findings in methylmalonic acid propionic acidemias.

The cerebral computed tomographic findings in two infants with methylmalonic acidemia and one infant and one adult with propionic acidemia are presented. Pertinent metabolic, clinical, and pathologic features of these genetic disorders of vitamin B12 (cobalamin) and biotin metabolism are reviewed briefly. Computed tomographic abnormalities consist of focal or diffuse deep cerebral hemisphere lucencies and diffuse loss of brain substance. These findings correlated well with the clinical and pathologic findings. The leukoencephalopathy in these uncommon autosomal recessive conditions appears to be due to ketoacidosis, which in some cases may be managed by vitamin supplementation.

The natural history of the inherited methylmalonic acidemias.

Six biochemical and genetic forms of methylmalonic acidemia have been defined previously: two (mut degrees and mut-) resulting from defects in the mutase apoenzyme, and four (cbl A, cbl B, cbl C, and cbl D) resulting from deficient adenosylcobalamin synthesis. We retrospectively surveyed the clinical presentation, response to cobalamin supplementation, and long-term outcome in the four most prevalent mutant classes by collecting detailed information on 45 patients (15 mut degrees, 5 mut-, 14 cbl A, and 11 cbl B). Most patients presented acutely with a common set of clinical and laboratory findings; however, there were significant differences between mutant classes: mut degrees patients presented earlier in infancy than did cbl A and cbl B patients; in response to cobalamin supplements, marked decreases in the concentration of methylmalonic acid in blood or urine were reported in most cbl A patients and in nearly half the cbl B patients, but not in mut degrees or mut- patients; and finally, most cbl A, cbl B, and mut- patients were still living, whereas most mut degrees patients died during the first few months of life. Our data indicate that genotypic classification of the methylmalonic acidemias has prognostic and therapeutic use as well as diagnostic value.

The recovery of platelet malondialdehyde production after a single intake of aspirin--the effect of dosage.

We studied the recovery pattern of platelet malondialdehyde (MDA) formation after a single intake of various doses of aspirin (ASA). In normal subjects, there were striking differences in the recovery pattern between large doses (1,000 or 500 mg) and small doses (100 or 50 mg) of oral ASA. The 2 day lag phase was present in the large dose group which showed the complete inhibition of the circulating platelets 2 hr after oral ASA but not in the small dose group which showed the incomplete inhibition. The difference of recovery time between these two groups was about 2 days. These data suggest that ASA's effect on megakaryocytes might affect the recovery pattern of MDA formation by the circulating platelets. In 2 patients with chronic idiopathic thrombocytopenic purpura, the 2 day lag phase was not observed in the recovery even after a large ASA dose.

Inhibition of bone marrow stem cell growth in vitro by methylmalonic acid: a mechanism for pancytopenia in a patient with methylmalonic acidemia.

A 7-week-old infant with methylmalonic acidemia had pancytopenia and hypoplastic bone marrow. The patient responded to large doses of vitamin B12 treatment, and within 3 wk, the blood counts and bone marrow cellularity returned to normal. To understand the mechanism of marrow depression in this infant, we examined the effect of the patient's plasma and methylmalonic acid itself on the in vitro growth of bone marrow-committed stem cells. The patient's plasma obtained before B12 treatment completely inhibited the marrow cell growth, whereas the posttreatment plasma showed no inhibition. Methylmalonic acid when added to the culture dishes in concentrations comparable to those reported in plasma of methylmalonic acidemia patients, inhibited growth of marrow stem cells in a concentration-dependent fashion. On the other hand, 16 to 18 hr incubation of cells in the same concentration of methylmalonic acid did not affect the recovery of viability of the cells. The observations suggest that methylmalonic acid is inhibitory to the proliferation of marrow stem cells. The mechanism of inhibition is yet to be elucidated.

Dietary therapy in two patients with vitamin B12-unresponsive methylmalonic acidemia.

The biochemical and therapeutic responses to dietary therapy were studied in a 25-month-old girl and a 1-month-old girl with methylmalonic acidemia (MMA-emia), which was unresponsive to vitamin B12. The minimum daily intake of protein which patients could tolerate and display a good development was between 1.0 and 1.2 g per kg body weight. Supplementation with amino acid mixture devoid of toxic amino acids was required to prevent protein malnutrition when daily protein intake was restricted to 0.6 g per kg body weight. Caloric intake should be sufficient, not only to promote growth but also to prevent a rise in MMA level, especially when a patient has ketoacidosis. It was found that MMA excretion per mg creatinine in random urine specimens correlated significantly with serum MMA and twenty four-hour output of MMA per kg body weight. Therefore measurement of MMA in a single urine specimen is useful for evaluating the in vivo accumulation of MMA.

Methylmalonic acidemia controlled with oral administration of vitamin B12.

A 3-month-old male infant had two episodes of fever, projectile vomiting, dehydration, generalized fine tremors and gross metabloic ketoacidosis. Methylmalonic acid was found in high concentration in both serum and urine, although the concentration of serum vitamin B12 was normal. A therapeutic trial of vitamin B12, administered parenterally, reduced greatly the methylmalonic aciduria. The patient has since been given vitamin B12 supplements continuously, initially 1 mg intramuscularly every other day, then 15 mg/d orally, and the protein in his diet was subsequently restricted. The most effected control of the methylmalonic aciduria was achieved with the combined regimen of oral vitamin therapy and dietary protein restriction. His physical and intellectual development have progressed normally and he has survived several acute respiratory tract infections without recurrence of metabolic acidosis.

Prenatal therapy of a patient with vitamin-B12-responsive methylmalonic acidemia.

Methylmalonic acidemia due to deficient synthesis of 5'-deoxyadenosylcobalamin was discovered in a mid-term fetus by culture of amniotic-fluid cells. Elevated concentrations of methylmalonic acid were also found in amniotic fluid and maternal urine. Treatment during the last nine weeks of gestation with large doses of vitamin B12 given to the mother reversed the increasing maternal excretion of methylmalonic acid, which was 23 mug per milligram of creatinine at 31 weeks' gestation. Just before delivery, the mother was excreting 5 mug, two to three times normal. At birth the methylmalonic acid content of the baby's urine (67 mug per milligram of creatinine) and serum (2.0 mug per milliliter) was only moderately elevated, and serum vitamin B12 concentration was very high. Acid levels rose in serum and urine in response to oral protein loading, but subsided after vitamin B12 administration. The infant is developing normally on a restricted protein diet alone at present. Prenatal therapy of methylmalonic acidemia is possible with large amount of vitamin B12 administered to the mother.

Studies of methylmalonyl coenzyme A carbonylmutase activity in methylmalonic acidemia. I. Correlation of clinical, hepatic, and fibroblast data.

Methylmalonyl-CoA carbonylmutase (mutase) activity was measured in fibroblast extracts from 15 patients with methylmalonic acidemia and in extracts of postmortem tissues from 6 of these children. Propionate oxidation and synthesis of 5-deoxyadenosylcobalamin (AdoCbl, the vitamin B12 coenzyme that is part of the mutase holoenzyme) were measured in intact fibroblasts. Mutase activity was low in the absence of added AdoCbl in fibroblast extracts from both control subjects and patients. When the assay included supplemental AdoCbl, mutase activity increased in the control subjects (to 24.0 pmol succinate/mg protein/min) and in extracts from eight of the patients (20.8 pmol/mg protein/min), but showed almost no change in extracts from the other seven patients (0.16 pmol/mg protein/min). We have defined the eight fibroblast lines that showed normal mutase activity in the presence of AdoCbl as "responsive lines" and the other seven lines as "nonresponsive." In the liver or kidney extracts of postmortem tissues, mutase activity responded to AdoCbl supplementation if fibroblast mutase activity from that patient had responded, and failed to respond if fibroblast activity failed to respond. Mean propionate oxidation in intact fibroblasts was much higher in control lines than in either responsive or nonresponsive lines (0.728 vs 0.097 vs 0.080 nmol CO2/10(6) cells/hr, respectively). AdoCbl synthesis was normal (0.27 pg AdoCbl/mg cells wet weight) in nonresponsive fibroblasts but was undetectable (less than 0.005 pg/mg cells) in the responsive lines. Thus, the deficiency of mutase activity in responsive fibroblast lines is due to the failure to synthesize significant amounts of AdoCbl, whereas the deficiency in nonresponsive lines is due to some other abnormality, presumably a defect in the mutase apoenzyme.