Aminotransferase Isoenzymes Research Articles

Cardiac biomarkers alterations in rapid weight loss and high-intensity training in judo athletes: a crossover pilot study.

Studies evaluating alterations in cardiac biomarkers in rapid sport-associated weight loss (RWL) and high-intensity sport-specific training (HISST) are lacking. This pilot study aimed to examine the effects of RWL and HISST on heart rate, blood pressure, cardiac biomarkers, and left ventricular systolic function. Nine elite male judokas participated in the presented survey. The athletes underwent a baseline assessment and two testing protocols, the first phase with RWL where they had to lose 5% of their body weight simultaneously with HISST, and the second phase after 7 days, in which only HISST was performed. Participants underwent electrocardiogram, biomarker, and transthoracic echocardiogram evaluation after each phase. In the first phase (RWL and HISST) athletes, heart rate increased significantly, 58.11 (7.78) versus 79 (9.25), P=0.001; as well as cardiac biomarkers: lactate dehydrogenase isoenzyme 175.33 (31.22) vs. 238.56 (56), P=0.003; aspartate aminotransferase 16.56 (4.61) vs. 29 (9.96), P=0.027; creatine kinase isoenzyme-MB 13 (11.5;24) vs. 29.11 (10.05), P=0.004; and high sensitivity cardiac troponin 10 (0) vs. 14.49 (6.4), P=0.045. In the second phase, only HISST was associated with a significant increase in the alanine aminotransferase isoenzyme, 37.78 (11.22) vs. 26 (8.03), P=0.024, together with creatine kinase 472 (185;654) vs. 166.88 (56.57), P=0.01, compared to the initial measurement. RWL combined with HISST produced significant alterations in cardiac biomarkers without impairment of left ventricular systolic function.

Diagnostic significance of alanine aminotransferase isoenzymes in alcoholic and non-alcoholic fatty liver cancers.

Alanine aminotransferase (ALT) expression is highly elevated in the serum of patients with hepatocellular carcinoma. However, the role of ALT isoenzymes in the total ALT activity remains unclear. In this study, we systematically investigated the role of ALT isoenzymes in alcoholic and non-alcoholic fatty liver cancer. Materials and methods: The expression of ALT1 and ALT2 at the mRNA and protein levels in 25 paired primary liver cancer tissues was detected by reverse transcription quantitative PCR (RT-qPCR), Western blotting, and immunohistochemistry. Serum ALT activity was determined using an automated biochemical analyzer. The mRNA and protein expression levels of ALT1 and ALT2 were lower in the tissues of alcoholic and non-alcoholic fatty liver cancers than in the paracancerous tissues. Notably, ALT2 was highly expressed in non-alcoholic fatty liver cancer tissues compared with alcoholic fatty liver cancer tissues. Total serum ALT activity was mainly contributed by ALT1 in alcoholic fatty liver cancer, whereas ALT1 contributed only marginally more to the total ALT activity than ALT2 in non-alcoholic fatty liver cancer. ALT2/ALT1 ratio can well discriminate normal control group, alcoholic liver cancer and non-alcoholic liver cancer. ALT1 contributed more to the total ALT activity than ALT2 in both alcoholic and non-alcoholic fatty liver cancer. Serum ALT2 to ALT activity was higher in non-alcoholic fatty liver cancer than that in alcoholic fatty liver cancer. ALT2/ALT1 ratio has some diagnostic significance for alcoholic and non-alcoholic liver cancer.

Influence of Polyamines on Red Beet (Beta vulgaris L. ssp. vulgaris) Gynogenesis

The influence of polyamines (PAs), putrescine (Put) and spermidine (Spd) on the efficiency of gynogenesis in ovule cultures of red beet (syn. beetroot) (Beta vulgaris L. vulgaris) cultivar “Czerwona Kula” and breeding accessions no. 3/2010 and no. 7/2008 was investigated. The effect of Put on the process of plant regeneration from gynogenetic embryos was studied. The response to the applied PAs was strongly dependent on the genotype. In “Czerwona Kula”, an increase in the number of obtained embryos was achieved by using each of the two PAs in the B5 medium. The effect of Spd was stronger. Put added to the regeneration medium at the concentration of 0.5 mg L−1 increased the number of obtained plants. All shoots placed on the rooting medium supplemented with 160 mg L−1 Put formed roots. The distribution of ploidy and homozygosity of gynogenetic plants depended on the genotype. Of the tested genotypes, the highest number of haploid plants, 68%, was obtained in red beet “Czerwona Kula”. The highest percentage of homozygotes, 69% for the glucose phosphate isomerase (GPI, E.C.5.3.1.9) isoenzyme and 100% for the aspartate aminotransferase (AAT, E.C.2.6.1.1) isoenzyme, was obtained in the population of gynogenetic plants of cultivar “Czerwona Kula”.

Effects of lymphocyte, C-reactive protein and prealbumin levels on clinical typing and course of disease in children infected with novel coronavirus.

Objectives:To investigate the effects of lymphocyte (LY), C-reactive protein (CRP) and prealbumin (PA) levels on the clinical typing and course of disease in children infected with novel coronavirus (2019-nCoV) at the early stage.Methods:A total of 140 children with 2019-nCoV infection diagnosed in Shijiazhuang People’s Hospital and Hebei Provincial Chest Hospital from January 2021 to February 2021 were selected for this study. According to the clinical symptoms, laboratory results and imaging examination, the children were divided into asymptomatic infection group, mild infection group and common infection group. The levels of white blood cell (WBC), LY, CRP, PA, albumin (ALB), aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatine kinase (CK) and creatine kinase MB isoenzyme (CKMB) in the children were recorded on the 2nd d after the positive detection of 2019-nCoV nucleic acid.Results:There were 73(52.1%) children in the asymptomatic infection group, 35(25.0%) children in the mild infection group and 32(22.9%) children in the common infection group. LY level in the common infection group was lower than that in the asymptomatic infection group and the mild infection group (F= 3.152, both p< 0.05). CRP level in the common infection group was higher than that in the asymptomatic infection group and the mild infection group (F= 6.343, both p< 0.05). CRP level in the mild infection group was higher than that in the asymptomatic infection group (t= 2.052, p< 0.05). PA level in the common infection group and the mild infection group was lower compared with the asymptomatic infection group (F= 5.229, both p< 0.05). WBC, ALB, AST, ALT, CK and CKMB levels in the three groups showed no statistical significance (F= 1.803, F= 1.208, F= 2.391, F= 1.973, F= 0.401, F= 1.332, respectively, all p> 0.05). Correlation analysis demonstrated that LY and PA levels were negatively correlated with hospital stay (r= -0.265, r= -0.325, both p< 0.050), but CRP level was not correlated with hospital stay (r= -0.039, p> 0.05).Conclusion:CRP is correlated with the clinical typing of children with 2019-nCoV infection, while LY and PA levels may be closely correlated with the clinical typing and course of treatment of children with 2019-nCoV infection.

DenseNet-Based Classification of MRI Images for Detecting the Difference before and after Treating Liver Cancer

This study aimed to explore the evaluation of Adriamycin-loaded microspheres in the treatment of liver cancer under DenseNet-based magnetic resonance imaging (MRI) image classification algorithm. According to different treatment methods, the research objects were classified into a normal saline (saline) group, a doxorubicin raw material (DOX) group, and a chitosan cross-linked pectin-doxorubicin conjugate macromolecular (CS-PDC-M) group. DenseNet’s migration learning was employed to analyze the dynamic enhanced MRI characteristics and classify the MRI images. The CS-PDC-M-targeted nanotransfer system was examined with its apparent morphology, drug absorption, and cytotoxicity. Tumor volume was monitored using MRI, and alanine aminotransferase (ALT) and creatine kinase isoenzyme (CK-MB) values were detected. Results showed that the classification accuracy of liver cancer MRI image based on DenseNet model reached 80% at the arterial hepatobiliary stage. The DOX and CS-PDC-M group had obviously smaller tumor volume than that of the saline group P &lt; 0.05 with a statistical meaning. The mortality in the DOX group was 30%, while there was no death in the saline and CS-PDC-M groups. Compared with the saline and CS-PDC-M groups, ALT and CK-MB from the DOX group increased substantially P &lt; 0.05 . Therefore, DOX had an inhibitory effect on tumor but damaged the heart and liver. DOX was used to construct CS-PDC-M that could maintain the original treatment effect of DOX and inhibit its side effects on the body, so CS-PDC-M had a clinical application value. In conclusion, Adriamycin-loaded microspheres could not only maintain the original therapeutic effect of Adriamycin but also inhibit its toxic and side effects on the body. The DenseNet model was applied in the liver cancer MRI dynamic image classification algorithm, and the normalization algorithm could improve the accuracy of the liver cancer microvessel classification, thus promoting the diagnostic efficiency of liver cancer diagnosis, which had clinical application value.

Isolated cell-bound membrane vesicles (CBMVs) as a novel class of drug nanocarriers

BackgroundCell-bound membrane vesicles (CBMVs) are a type of membrane vesicles different from the well-known extracellular vesicles (EVs). In recent years, the applications of EVs as drug delivery systems have been studied widely. A question may arise whether isolated CBMVs also have the possibility of being recruited as a drug delivery system or nanocarrier?MethodsTo test the possibility, CBMVs were isolated/purified from the surfaces of cultured endothelial cells, loaded with a putative antitumor drug doxorubicin (Dox), and characterized. Subsequently, cellular experiments and animal experiments using mouse models were performed to determine the in vitro and in vivo antitumor effects of Dox-loaded CBMVs (Dox-CBMVs or Dox@CBMVs), respectively.ResultsBoth Dox-free and Dox-loaded CBMVs were globular-shaped and nanometer-sized with an average diameter of ~ 300–400 nm. Dox-CBMVs could be internalized by cells and could kill multiple types of cancer cells. The in vivo antitumor ability of Dox-CBMVs also was confirmed. Moreover, Quantifications of blood cells (white blood cells and platelets) and specific enzymes (aspartate aminotransferase and creatine kinase isoenzymes) showed that Dox-CBMVs had lower side effects compared with free Dox.ConclusionsThe data show that the CBMV-entrapped Doxorubicin has the antitumor efficacy with lower side effects. This study provides evidence supporting the possibility of isolated cell-bound membrane vesicles as a novel drug nanocarrier.

Effects of ulinastatin combined with furosemide on myocardial enzymes, renal function and adverse reactions in patients with acute renal failure after cardiopulmonary resuscitation

Objective To explore the effects of ulinastatin combined with intravenous pump injection of furosemide on myocardial enzymes, renal function and adverse reactions in patients with acute renal failure(ARF) after cardiopulmonary resuscitation(CPR). Methods From January 2016 to May 2018, 117 patients with ARF after successful CPR in the Second People's Hospital of Hefei were divided into observation group(n=59) and control group(n=58) using simple random method.The control group received routine treatment, while the observation group added ulinastatin combined with intravenous pump infusion of furosemide.Myocardial enzymology, renal function, metabolism, inflammatory index, adverse reaction and survival rate were compared. Results Three and 7 days after treatment, the hydroxybutyrate dehydrogenase(HDBH), isoenzymes of creatine kinase isoenzyme(CK-MB) and mitochondrial aspartate aminotransferase isoenzyme(m-AST) were decreased in the two groups, and compared with the control group, which of the observation group were lower [HDBH: (231.42±31.15)U/L vs.(268.59±34.87)U/L; F=12.01, P=0.00; CK-MB: (32.38±4.15)ng/mL vs.(37.57±3.96)ng/mL; F=15.12, P=0.00; m-AST: (25.18±4.24)U/L vs.(33.92±5.60)U/L; F=12.36, P=0.00]. After treatment, the blood urea nitrogen(BUN), 24h urine protein quantity and creatinine(Cr) in the two groups increased firstly and then decreased, and compared with the control group, those of the observation group were lower[BUN: (7.02±1.66)mmol/L vs.(8.47±1.38)mmol/L; F=11.24, P=0.00; Cr: (82.69±9.87)μmol/L vs.(90.18±10.37)μmol/L; F=10.39, P=0.00; 24h urine protein quantity: (15.43±2.17)mg vs.(18.62±3.14)mg; F=11.06, P=0.00]. Three and 7 days after treatment, the levels of blood lactic acid(Lac), hypersensitive C-reactive protein(hs-CRP) and tumor necrosis factor-α(TNF-α) were decreased in the two groups, and compared with the control group, those of the observation group were lower [Lac: (1.18±0.27)mmol/L vs.(2.17±0.34)mmol/L, F=16.29, P=0.00; hs-CRP: (4.89±0.81)mg/L vs.(6.17±1.10)mg/L, F=13.41, P=0.00; TNF-α: (72.18±7.62)ng/L vs.(83.16±7.79)ng/L, F=11.39, P=0.00]. The incidence rates of adverse reactions in the observation group and the control group were 5.09% and 1.72%, respectively, the difference was statistically significant(χ2=0.24, P=0.62). The survival rates in the observation group and the control group were 50 cases (84.75%) and 39 cases (67.24%) respectively, the difference was statistically significant(χ2=4.92, P=0.02). Conclusion Intravenous pump injection of furosemide combined with ulinastatin can protect the heart and kidney, and improve the survival rate of patients with ARF after CPR and it is worthy of popularizing. Key words: Cardiopulmonary resuscitation; Renal insufficiency; Blood urea nitrogen; Creatinine; Blood lactic acid; Ulinastatin; Furosemide

Clinical analysis of continuous blood purification in the treatment of severe sepsis in infants

Objective To investigate the clinical efficacy and safety of continuous blood purification (CBP) in the treatment of severe sepsis in infants. Methods A retrospective analysis of 40 infants with severe sepsis treated with CBP was performed at PICU of Hunan Children′s Hospital from January 2014 to July 2017, and 50 infants with severe sepsis who were not treated with CBP at the same period were enrolled as control group.The indicators included blood gas analysis, lactic acid (Lac), blood glucose, electrolytes, blood routine, C-reaction protein (CRP) and procalcitonin (PCT), alanine aminotransferase (ALT) and aspartate aminotransferase (AST), total bilirubin(TB), urea nitrogen (BUN), serum creatinine (Scr) and creatine kinase isoenzyme MB (CK-MB), pediatric critical illness score (PCIS). Results (1) After 3 days of treatment, the recovery of body temperature, heart rate, respiratory rate and blood pressure in CBP group were better than those in the control group, and the differences between two groups were statistically significant(P 0.05). (5) Two cases of thrombocytopenia, 2 cases of femoral vein thrombosis, 2 cases of hypovolemic shock, and 1 case of blood coagulation in filter happened in CBP gruop, all cases had no puncture site infection. Conclusion CBP can improve the vital signs, internal environment, inflammatory reaction and organ function of infants with severe sepsis, and the effect is better than that of traditional methods.The complications of CBP in infants with severe sepsis are relatively large, so we should strictly master the indications of CBP in the treatment of severe sepsis in children. Key words: Continuous blood purification; Severe sepsis; Infants; Clinical efficacy; Safety

Induced androgenesis as a biotechnology method for obtaining DH plants in Daucus carota L.

ABSTRACTThe influence of various factors on the efficiency of obtaining double haploid lines in anther cultures was examined in Daucus carota L. The impact of the genotype, the formation of donor plants and the growth conditions was investigated. The effects of various regeneration media were analysed. Acclimatisation of androgenic plants and their evaluation were also conducted. Androgenesis was induced on B5 medium containing 2,4-D and NAA at 0.1 g·L−1. Depending on the genotype, 1.2–305.3 embryos per 100 anthers were obtained. The highest number of embryos (5.5 per 100 anthers) was obtained from donor plants with the first-order shoots containing one umbel. Cultivation of donor plants under controlled conditions improved the number of embryos from 305 in the open field to 1764 in the growth chamber. Regeneration of plants from androgenetic embryos proceeded most effectively on B5 medium without hormones and amino acids. Regenerated plants adapted in over 60%. Ploidy analysis showed the presence of 92% of plants with a doubled chromosome set and 8% with a tetraploid number of chromosomes. Plants with a doubled chromosome set were 73% homozygous for the glucose phosphate isomerase isoenzyme and 100% homozygous for the aspartate aminotransferase isoenzyme, which confirms their gametic origin.

Analysis of different toxic impacts of Fipronil on growth, hemato-biochemistry, protoplasm and reproduction in adult cockerels

Fipronil (FIP), a broad-spectrum phenylpyrazole, is extensively used across the globe to control pests and as a veterinary drug. Incorrect use of FIP leads to contamination of soil and water ultimately causes toxic effects in non-target organisms. Therefore, the present experimental study was aimed to determine the deleterious effects of sub-lethal concentrations of FIP in adult cockerels. FIP mixed in corn oil was administered orally in different groups as follows: A = control, B = 1.5 mg/kg, C = 2.5 mg/kg, D = 3.5 mg/kg and E = 4.5 mg/kg body weight for 60 d. Signs of toxicity like anemic comb, tremors, gasping and watery droppings were evident in birds exposed to higher levels. The feed intake, body weight, absolute and the relative weight of different organs were significantly (p ≤ .05) decreased in birds exposed to higher concentrations. Histomorphometric analysis of small intestine in birds exposed to higher levels of FIP showed decreased villus height and crypt depth of duodenum, jejunum and ilium. Histologically, congestion in liver, pyknosis of hepatocytes and congestion in kidneys along with necrosis of tubular epithelial cells and increased urinary spaces were observed in birds. Microscopically, seminiferous tubules exhibited necrotic nuclei of the spermatids, arrest of process of spermatogenesis and admixture of necrotic cells in testes of exposed birds. Moreover, significantly (p ≤ .05) decreased hematological parameters such as erythrocytes counts, hemoglobin concentrations and hematocrit, while increased total leukocyte counts were recorded in birds. Furthermore, significantly (p ≤ .05) increased concentrations of serum urea, creatinine, alanine aminotransferase, aspartate aminotransferase and cardiac isoenzyme (CK-MB), while reduced serum testosterone levels were recorded in birds. Results revealed significantly (p ≤ .05) increased frequency of micronuclei in erythrocyte and bone marrow cells. The findings of our study demonstrated that FIP induces deleterious impacts on multiple tissues in birds even at low doses.

Development of monoclonal antibodies and immunochromatographic lateral flow device for rapid test of alanine aminotransferase isoenzyme 1

BackgroundAlanine aminotransferase (ALT) has been used as a sensitive marker for liver injury in people and in preclinical toxicity studies. But measurement of ALT isoenzymes, ALT1 and ALT2, was reported to be of more diagnostic value. The aim of this study is to develop an ideal pair of anti-ALT1 monoclonal antibodies (MAbs) of high specificity and affinity, and subsequently prepare a Immunochromatographic lateral flow device (LFD) for rapid test of ALT1 in human serums. MethodsThe complete coding sequence of ALT1 gene (1500 bp) was cloned from human hepatoma G2 cells (HepG2) and inserted into the expression vector pET-32a(+). ALT1 recombinant protein was routinely prepared by E. coli BL21 (DE3) expression and Ni2+ affinity purification. Balb/c mice were immunized with purified ALT1 and the splenocytes were fused with Sp2/0 myeloma cells. The positive clones, verified by indirect enzyme-linked immunosorbent assay (ELISA) using purified ALT1, were subcloned to single clones by limiting dilution process. A MAb pair was selected from the obtained MAbs according the sandwich ELISA pairing results and then used for lateral flow device (LFD) production. After evaluation of the sensitivity and specificity, the LFD strips were employed to test human serum samples with known ALT activity levels. ResultsALT1 recombinant protein was expectedly prepared by expression and purification. A total of 8 stable clones that produced antibodies specifically recognizing ALT1 protein were developed. After sandwich ELISA pairing, an ideal pair of anti-ALT1 MAbs, designated as BD7 and DG3, were selected and proved to be of high specificity, titer and affinity. Based on the MAb pair, LFD strips specifically for ALT1 rapid test were subsequently prepared. The detection threshold of the LFD strips was 12 U/L. No cross reaction was found. ConclusionsThe ALT1 LFD with high sensitivity and specificity was successfully developed. It is valuable for testing ALT1 protein in human sera and can be a beneficial complement for traditional ALT test.

Effects of leucine metabolism on T cell activation: a potential target for nutritional immunotherapy (IRC8P.439)

Abstract T cells are essential for cell-mediated defense against pathogens and cancer. Recent advances in understanding T cell regulation reveal that cell metabolism can greatly impact the final outcome of the immune response. A primary regulator of cell growth and proliferation, the mammalian target of rapamycin (mTOR), is critical for T cell activation and differentiation. Leucine is a nutritional activator of mTOR and its intracellular concentrations are regulated by the branched chain aminotransferase (BCAT) isoenzymes, cytosolic BCATc and mitochondrial BCATm. We showed recently that activated CD4+ T cells, lacking cytosolic leucine transamination (BCATc KO), had up-regulated mTOR signaling and glycolysis. To explore further the role of BCATc in T cell activation, we measured INFƴ production and proliferation in T cells from BCATc KO and wildtype mice. Loss of BCATc increased INFƴ by 2.8-fold in activated T cells but did not impact T cell proliferation. Knocking out the mitochondrial BCATm had a robust effect on T cell glycolytic rate (3.1-fold increase) upon activation but only mild effects on mTOR and INFƴ. The results suggest that loss of either enzyme is sufficient to increase T cell glycolysis and mTOR signaling but not T cell proliferation. Thus, BCATc and BCATm are critical players in T cell activation by providing negative feedback regulation of mTOR and glycolysis. Manipulating leucine metabolism may be one mechanism to modulate activation and differentiation of T cells.

Aspartate aminotransferase isoenzymes in cucumber roots as depending upon the mineral composition of the medium

Aspartate aminotransferase (GOT) isoenzymes separated by means of electrophoresis on cellulose acetate gel were examined. Five GOT isoenzymes were discovered in cucumber roots after several days of their growth. The activity of these isoenzymes was dependent upon the mineral composition of the medium. If the medium did not contain nitrogen, isoenzymes 1, 3, and 4 showed only slight activity. Activity of all of the isoenzymes in the roots of seedlings assimilating NH&lt;sub&gt;4&lt;/sub&gt;&lt;sup&gt;+&lt;/sup&gt; ions was higher than in the roots of seedlings assimilating N0&lt;sub&gt;3&lt;/sub&gt;&lt;sup&gt;-&lt;/sup&gt; ions. Higher activity was observed in all of the isoemzymes in the nitrate or ammonium medium lacking calcium, while only some showed higher activity at the lack of potassium.

Liver Aspartate Transaminase Isoenzymes as Biomarkers of Chronic Exposure to Chromium(VI)

Exposure to hexavalent chromium compounds is associated with the risk of lung cancer, dermatitis, gastrointestinal ulcers, and other tissue damages. The aim of this study was to compare liver isoenzyme and total serum activities of aspartate aminotransferase (AST) as cytotoxic biomarkers of acute and chronic cytotoxicity of Cr(VI). We investigated the extent of cell damage caused by chromium(VI) in acute (2.5 mg kg(-1)) daily doses administered over five days and chronic (0.25 mg kg(-1) and 0.5 mg kg(-1)) daily doses administered over 15 to 60 days by measuring total AST in serum and low molecular weight AST (LMW-AST) and high molecular weight AST (HMW-AST) activities in thirty liver fractions. We also evaluated the kinetic properties and electrophoretic mobility of the LMW- and HMW-AST isoenzymes in liver subcellular fractions. Liver LMW-AST and total serum AST activities significantly decreased after 15 days of exposure (P<0.05). With continued treatment, AST activity increased by 15.67% (P<0.05). Interestingly, changes in serum AST activity were similar to changes in the liver LMW-AST isoenzyme. Our results confirmed that total serum AST activity may serve as a reliable tissue biomarker for long-term exposures to Cr(VI), but they also suggest that the LMW-AST isoenzyme could be even more sensitive.

Differential redox potential between the human cytosolic and mitochondrial branched-chain aminotransferase

The human branched-chain aminotransferase (hBCAT) isoenzymes are CXXC motif redox sensitive homodimers central to glutamate metabolism in the central nervous system. These proteins respond differently to oxidation by H(2)O(2), NO, and S-glutathionylation, suggesting that the redox potential is distinct between isoenzymes. Using various reduced to oxidized glutathione ratios (GSH:GSSG) to alter the redox environment, we demonstrate that hBCATc (cytosolic) has an overall redox potential that is 30 mV lower than hBCATm (mitochondrial). Furthermore, the CXXC motif of hBCATc was estimated to be 80 mV lower, suggesting that hBCATm is more oxidizing in nature. Western blot analysis revealed close correlations between hBCAT S-glutathionylation and the redox status of the assay environment, offering the hBCAT isoenzymes as novel biomarkers for cytosolic and mitochondrial oxidative stress.

Relationship between Tei index and the myocardial enzymes in newborns after asphyxia LEI Xiao-yan,

Objective To study the relationship between Tei index and the myocardial enzymes in newborns with myocardial damage due to asphyxia. Methods Forty-four newborns with mild asphyxia,27 newborns with severe asphyxia and 20 healthy newborns hospitalized in our hospital from January 2005 to December 2008 were enrolled in this study.The peak E and peak A and E/A ratio at mitral valve,the ejection fraction,isovolumetric contraction time,isovolumetric relaxation time and Tei index were measured by conventional echocardiography,and aspartate aminotransferase,creatine kinase,lactate dehydrogenase,hydroxybutyrate dehydrogenase and creatine phosphate kinase isoenzyme,and cardiac troponin T were tested also.One-way ANOVA was used to analyze the difference between groups. Results Tei index,isovolumetric contraction time and isovolumetric relaxation time in the severe asphyxia newborns were 0.62±0.13,(47±7)ms and(52±8)ms,higher than those in the mild asphyxia newborns[0.51±0.14,(41±6)ms and(43±6)ms],those in the mild asphyxia newborns were higher than in the healthy newborns[0.39±0.12,(34±6)ms and (37±6)ms] (all P<0.01).Aspartate aminotransferase,lactate dehydrogenase,hydroxybutyrate dehydrogenase,creatine kinase and its isoenzymes and cardiac troponin T in the severe asphyxia newborns were higher than in the mild asphyxia newborns and healthy group(P<0.01).Tei index,isovolumetric contraction time and isovolumetric relaxation time were positively related to the myocardial enzymes.The positive relationships were more statistically significant in Tel index,creatine kinase,its isoenzymes and cardiac troponin T(r=0.762,0.821,0.778,P<0.01).Conclusions Combination of Tei index and myocardial enzymes can not only be applied to assess the cardiac function in newborns with myocardial damage due to asphyxia,but also to evaluate the degree of cardiac dysfunction accurately and dynamically. Key words: Asphyxia neonatorum; Stroke volume; Creatine kinase; Lactate dehydrogenases

Alanine aminotransferase isoenzymes: Molecular cloning and quantitative analysis of tissue expression in rats and serum elevation in liver toxicity #

The elevation of serum alanine aminotransferase (ALT) is regarded as an indicator of liver damage based on the presumption that ALT protein is specifically and abundantly expressed in the liver. However, ALT elevation is also observed in non-liver injury conditions (for example, muscle injury) and in apparently healthy people. Conversely, serum ALT activity is normal in many patients with confirmed liver diseases (for example, cirrhosis and hepatitis C infection). To improve the diagnostic value of the ALT assay and to understand the molecular basis for serum ALT changes in various pathophysiological conditions, we have cloned rat ALT isoenzyme ALT1 and ALT2 complementary DNAs (cDNAs), examined their tissue expressions at the messenger RNA and protein levels, and determined ALT1 and ALT 2 serum levels in response to liver damage in rodents. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis shows that ALT1 messenger RNA is widely distributed and mainly expressed in intestine, liver, fat tissues, colon, muscle, and heart, in the order of high to low expression level, whereas ALT2 gene expression is more restricted, mainly in liver, muscle, brain, and white adipose tissue. The tissue distribution pattern of ALT1 and ALT2 proteins largely agrees with their messenger RNA expression. Interestingly, hepatic ALT2 protein is approximately four times higher in male rats than in female rats. In addition, ALT isoenzymes distribute differentially at the subcellular level in that ALT1 is a cytoplasmic protein and ALT2 a mitochondrial protein, supporting bioinformatic prediction of mitochondrial localization of ALT2. Using animal models of hepatoxicity induced by carbon tetrachloride and acetaminophen, we found that both serum ALT1 and ALT2 protein levels were significantly elevated and correlated with ALT activity, providing, for the first time, the molecular basis for the elevated total serum ALT activity.

Expression, purification, and initial characterization of human alanine aminotransferase (ALT) isoenzyme 1 and 2 in High-five insect cells

Alanine aminotransferase (ALT) is a key enzyme for gluconeogenesis as well as a widely used serum marker for liver injury. We have identified two ALT isoenzymes, ALT1 and ALT2, which are encoded by separate genes. In this study, we described the expression, purification and initial characterization of human ALT1 and ALT2 proteins in High-five insect cells. Human ALT1 and ALT2 were expressed as His-tagged fusion proteins by recombinant baculovirus in insect cells and purified into homogeneity in one step by using immobilized Ni 2+-affinity chromatography. Tag-free ALT1 and ALT2 were obtained by cleavage of enterokinase digestion and used for initial characterization of the enzymes. The specific ALT activity of purified fusion or His-tag-removed ALT1 was about 15-fold higher than that of ALT2 and their enzymatic activities decreased quickly at 37 °C and −20 °C, but were well preserved at −80 °C. Nevertheless, the ALT1 and ALT2 activities remained stable in a buffer containing 25% glycerol. The pH profile was similar between hALT1 and hALT2 in that both enzymes remained fully active between pH 6.5 and 8.0. The purified ALT recombinant proteins can not only be used as a reference protein standard for the ALT assay in clinical chemistry, but also will be useful for understanding the biochemical and biological significance of the isoenzymes and for developing ALT isoform-specific assays for clinical or preclinical diagnostic use.

L-alanine:2-oxoglutarate aminotransferase isoenzymes from Arabidopsis thaliana leaves

The occurrence of four l-alanine:2-oxoglutarate aminotransferase (AOAT) isoenzymes (AOAT-like proteins): alanine aminotransferase 1 and 2 (AlaAT1 and AlaAT2, EC 2.6.1.2) and l-glutamate:glyoxylate aminotransferase 1 and 2 (GGAT1 and GGAT2, EC 2.6.1.4) was demonstrated in Arabidopsis thaliana leaves. These enzymes differed in their substrate specificity, susceptibility to pyridoxal phosphate inhibitors and behaviour during molecular sieving on Zorbax SE-250 column. A difference was observed in the electrostatic charge values at pH 9.1 between GGAT1 and GGAT2 as well as between AlaAT1 and AlaAT2, despite high levels of amino acid sequence identity (93 % and 85 %, respectively). The unprecedented evidence for the monomeric structure of both AlaAT1 and AlaAT2 is presented. The molecular mass of each enzyme estimated by molecular sieving on Sephadex G-150 and Zorbax SE-250 columns and SDS/PAGE was approximately 60 kDa. The kinetic parameters: Km (Ala)=1.53 mM, Km (2-oxoglutarate)=0.18 mM, kcat=124.6 s−1, kcat/Km=8.1 × 104 M−1·s−1 of AlaAT1 were comparable to those determined for other AlaATs isolated from different sources. The two studied GGATs also consisted of a single subunit with molecular mass of 47.3–70 kDa. The estimated Km values for l-glutamate (1.2 mM) and glyoxylate (0.42 mM) in the transamination catalyzed by putative GGAT1 contributed to indentification of the enzyme. Based on these results we concluded that each of four AOAT genes in Arabidopsis thaliana leaves expresses different AOAT isoenzyme, functioning in a native state as a monomer.

Hepatic Alanine-glyoxylate Aminotransferase Activity and Oxalate Metabolism in Vitamin B6 Deficient Rats

Hepatic Alanine-glyoxylate Aminotransferase Activity and Oxalate Metabolism in Vitamin B6 Deficient Rats