An unexpected role for leucyl aminopeptidase in UV tolerance revealed by a genome-wide fitness assessment in a model cyanobacterium.

UV radiation (UVR) has significant physiological effects on organisms living at or near the Earth's surface, yet the full suite of genes required for fitness of a photosynthetic organism in a UVR-rich environment remains unknown. This study reports a genome-wide fitness assessment of the genes that affect UVR tolerance under environmentally relevant UVR dosages in the model cyanobacterium Synechococcus elongatus PCC 7942. Our results highlight the importance of specific genes that encode proteins involved in DNA repair, glutathione synthesis, and the assembly and maintenance of photosystem II, as well as genes that encode hypothetical proteins and others without an obvious connection to canonical methods of UVR tolerance. Disruption of a gene that encodes a leucyl aminopeptidase (LAP) conferred the greatest UVR-specific decrease in fitness. Enzymatic assays demonstrated a strong pH-dependent affinity of the LAP for the dipeptide cysteinyl-glycine, suggesting an involvement in glutathione catabolism as a function of night-time cytosolic pH level. A low differential expression of the LAP gene under acute UVR exposure suggests that its relative importance would be overlooked in transcript-dependent screens. Subsequent experiments revealed a similar UVR-sensitivity phenotype in LAP knockouts of other organisms, indicating conservation of the functional role of LAPs in UVR tolerance.

Marine Invertebrates: A Promissory Still Unexplored Source of Inhibitors of Biomedically Relevant Metallo Aminopeptidases Belonging to the M1 and M17 Families.

Proteolytic enzymes, also known as peptidases, are critical in all living organisms. Peptidases control the cleavage, activation, turnover, and synthesis of proteins and regulate many biochemical and physiological processes. They are also involved in several pathophysiological processes. Among peptidases, aminopeptidases catalyze the cleavage of the N-terminal amino acids of proteins or peptide substrates. They are distributed in many phyla and play critical roles in physiology and pathophysiology. Many of them are metallopeptidases belonging to the M1 and M17 families, among others. Some, such as M1 aminopeptidases N and A, thyrotropin-releasing hormone-degrading ectoenzyme, and M17 leucyl aminopeptidase, are targets for the development of therapeutic agents for human diseases, including cancer, hypertension, central nervous system disorders, inflammation, immune system disorders, skin pathologies, and infectious diseases, such as malaria. The relevance of aminopeptidases has driven the search and identification of potent and selective inhibitors as major tools to control proteolysis with an impact in biochemistry, biotechnology, and biomedicine. The present contribution focuses on marine invertebrate biodiversity as an important and promising source of inhibitors of metalloaminopeptidases from M1 and M17 families, with foreseen biomedical applications in human diseases. The results reviewed in the present contribution support and encourage further studies with inhibitors isolated from marine invertebrates in different biomedical models associated with the activity of these families of exopeptidases.

Expression, Characterisation, Homology Modelling and Molecular Docking of a Novel M17 Family Leucyl-Aminopeptidase from Bacillus cereus CZ.

Leucyl-aminopeptidase (LAP), an important metallopeptidase, hydrolyses amino acid residues from the N-terminus of polypeptides and proteins, acting preferentially on the peptide bond formed by N-terminus leucine. A new leucyl-aminopeptidase was found in Bacillus cereus CZ. Its gene (bclap) contained a 1485 bp ORF encoding 494 amino acids with a molecular weight of 54 kDa. The bcLAP protein was successfully expressed in E. coli BL21(DE3). Optimal activity is obtained at pH 9.0 and 58 °C. The bcLAP displays a moderate thermostability and an alkaline pH adaptation range. Enzymatic activity is dramatically enhanced by Ni2+. EDTA significantly inhibits the enzymatic activity, and bestatin and SDS also show strong inhibition. The three-dimensional model of bcLAP monomer and homohexamer is simulated byPHYRE2 server and SWISS-MODEL server. The docking of bestatin, Leu-Trp, Asp-Trp and Ala-Ala-Gly to bcLAP is performed using AutoDock4.2.5, respectively. Molecular docking results show that the residues Lys260, Asp265, Lys272, Asp283, Asp342, Glu344, Arg346, Gly372 and His437 are involved in the hydrogen bonding with the ligands and zinc ions. There may be two nucleophilic catalytic mechanisms in bcLAP, one involving His 437 or Arg346 and the other involving His437 and Arg346. The bcLAP can hydrolyse the peptide bonds in Leu-Trp, Asp-Trp and Ala-Ala-Gly.

Characterization and heterologous expression of a novel Co2+-dependent leucyl aminopeptidase Amp0279 originating from Lysinibacillus sphaericus.

This study aims to explore the potential aminopeptidases of Lysinibacillus sphaericus based on the unique metabolic characteristics of this species which cannot metabolize carbohydrates and may have a strong ability to metabolize amino acids. Fifteen peptidase-encoding genes predicted in L. sphaericus C3-41 have been heterologously expressed in Escherichia coli BL21, and of these genes, only Amp0279 shows a high ability to hydrolyze L-leucine-4-nitroanilide (Leu-pNA). Phylogenetic analysis, 3D-structure modeling, and enzyme assays indicated that Amp0279 should be a novel Co2+-dependent aminopeptidase belonging to the M29 family. The optimal conditions of Amp0279 were determined to be 50 °C and pH 8.0 with the addition of 100 µM Co2+, and under this condition, the specific activity of Amp0279 matched that of Flavourzyme® (3.54 × 104 vs. 3.37 × 104 U/mg for the protein ingredient of Flavourzyme®). Amp0279 is mainly expressed in the middle sporulation phase in wild-type L. sphaericus or in Bacillus subtilis under the control of the sporulation-dependent strong promoter pcry8E, which is carried by the recombinant vector pHT315-8E21b. Furthermore, the secretory expression systems based on B. subtilis and Corynebacterium glutamicum were used to enhance the soluble expression of Amp0279. Obvious expression and enzymatic activity were detected from the crude supernatant media of both host bacteria without further concentration and purification. Moreover, expression can occur in the vegetative phase in B. subtilis under the control of the Pgrac promoter. KEY POINTS: • A novel Co2+-dependent leucyl aminopeptidase Amp0279 originating from L. sphaericus was characterized. • The activity of Amp0279 as a leucyl aminopeptidase matches that of Flavourzyme® under optimal conditions. • B. subtilis- and C. glutamicum-based expression systems are built to promote secretory (soluble) Amp0279 expression.

Purification and characterization of a Cys-Gly hydrolase from the gastropod mollusk, Patella caerulea.

A magnesium-dependent cysteinyl-glycine hydrolyzing enzyme from the gastropod mollusk Patella caerulea was purified to electrophoretic homogeneity through a simple and rapid purification protocol. The molecular masses of the native protein and the subunit suggest that the enzyme has a homohexameric structure. Structural data in combination with kinetic parameters determined with Cys-Gly and compared with Leu-Gly as a substrate, indicate that the purified enzyme is a member of the peptidase family M17. The finding that an enzyme of the peptidase family M17 is responsible also in mollusks for the breakdown of Cys-Gly confirms the important role of this peptidase family in the glutathione metabolism.

Network of ion-pairs is responsible for the large differences in the thermal stability of two structurally similar aminopeptidases.

Aminopeptidases are an important class of enzymes for protein metabolism. Leucyl aminopeptidase (PepL) preferably removes leucine from the N-terminus of small peptides. PepL of Lacticaseibacillus casei was observed to be thermally unstable, while a structurally similar aminopeptidases T (AmpT) of Thermus thermophilus is highly stable. To understand the molecular interaction responsible for large difference in their stability, molecular dynamics simulations were carried out to study thermal stability of PepL and AmpT in 300 K to 450 K temperature range over 100 ns. PepL sampled a larger conformational space with a rugged free-energy landscape, while AmpT navigated a smoother energy landscape to reach global minimum. The RMSD, RMSF, radius of gyration and principal component analysis suggested large movements in PepL than in AmpT with increase in temperature. Analysis of residue-interaction network revealed AmpT possessing a greater number of low, medium and high energy contacts in comparison to PepL. AmpT showed a higher abundance of ion-pair clusters and ionic residues per cluster compared to PepL, moreover retained a greater number of high energy contacts at elevated temperatures. These findings showed that the inherently lower stability of PepL originates from a comparatively smaller number of contacts and can be pivotal in engineering PepL for higher stability.

Identification and Validation of Compounds Targeting Leishmania major Leucyl-Aminopeptidase M17.

Leishmaniasis is a neglected tropical disease; there is currently no vaccine and treatment is reliant upon a handful of drugs suffering from multiple issues including toxicity and resistance. There is a critical need for development of new fit-for-purpose therapeutics, with reduced toxicity and targeting new mechanisms to overcome resistance. One enzyme meriting investigation as a potential drug target in Leishmania is M17 leucyl-aminopeptidase (LAP). Here, we aimed to chemically validate LAP as a drug target in L. major through identification of potent and selective inhibitors. Using RapidFire mass spectrometry, the compounds DDD00057570 and DDD00097924 were identified as selective inhibitors of recombinant Leishmania major LAP activity. Both compounds inhibited in vitro growth of L. major and L. donovani intracellular amastigotes, and overexpression of LmLAP in L. major led to reduced susceptibility to DDD00057570 and DDD00097924, suggesting that these compounds specifically target LmLAP. Thermal proteome profiling revealed that these inhibitors thermally stabilized two M17 LAPs, indicating that these compounds selectively bind to enzymes of this class. Additionally, the selectivity of the inhibitors to act on LmLAP and not against the human ortholog was demonstrated, despite the high sequence similarities LAPs of this family share. Collectively, these data confirm LmLAP as a promising therapeutic target for Leishmania spp. that can be selectively inhibited by drug-like small molecules.

Safety evaluation of the food enzyme leucyl aminopeptidase from the genetically modified Aspergillus oryzae strain NZYM-BU.

The food enzyme leucyl aminopeptidase (EC 3.4.11.1) is produced with the genetically modified Aspergillus oryzae strain NZYM-BU by Novozymes A/S. The genetic modifications do not give rise to safety concerns. The food enzyme is free from viable cells of the production organism and its DNA. It is intended to be used in five food manufacturing processes. Dietary exposure to the food enzyme TOS was estimated to be up to 1.508 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by means of a repeated dose 90-day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 4,928 mg TOS/kg bw per day, the highest dose tested, which, when compared with the estimated dietary exposure, resulted in a margin of exposure of at least 3,268. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and no match was found. The Panel considered that the risk of allergic reactions by dietary exposure cannot be excluded, but the likelihood is low. Based on the data provided, the Panel concluded that the food enzyme does not give rise to safety concerns under the intended conditions of use.

Safety evaluation of the food enzyme leucyl aminopeptidase from the non-genetically modified Aspergillus sp. strain AE-MB.

The food enzyme leucyl aminopeptidase (EC 3.4.11.1) is produced with the non-genetically modified Aspergillus sp. strain AE-MB by Amano Enzyme Inc. The food enzyme is considered free from viable cells of the production organism. It is intended to be used in five food manufacturing processes: processing of dairy products for the production of (1) flavouring preparations; processing of plant- and fungal-derived products for the production of (2) protein hydrolysates; processing of meat and fish products for the production of (3) protein hydrolysates, (4) modified meat and fish products and processing of (5) yeast and yeast products. Dietary exposure to the food enzyme-total organic solids (TOS) was estimated to be up to 2.273 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by means of a repeated dose 90-day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 183 mg TOS/kg bw per day. The calculated margin of exposure for each age group was 135 (infants), 81 (toddlers), 83 (children), 109 (adolescents), 160 (adults) and 184 (the elderly). A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and no matches were found. The Panel considered that the risk of allergic reactions by dietary exposure cannot be excluded, but the likelihood is low. The safety of the food enzyme could not be established given the derived margins of exposure. Therefore, the Panel concluded that this food enzyme could not be considered safe under the intended conditions of use.

Cloning, purification and preliminary crystallographic analysis of the Helicobacter pylori leucyl aminopeptidase-bestatin complex.

Helicobacter pylori is an important human pathogenic bacterium associated with numerous severe gastroduodenal diseases, including ulcers and gastric cancer. Cytosolic leucyl aminopeptidase (LAP) is an important housekeeping protein that is involved in peptide and protein turnover, catabolism of proteins and modulation of gene expression. LAP is upregulated in metronidazole-resistant H. pylori, which suggests that, in addition to having an important housekeeping role, LAP contributes to the mechanism of drug resistance. Crystals of H. pylori LAP have been grown by the hanging-drop vapour-diffusion method using polyethylene glycol as a precipitating agent. The crystals belonged to the primitive triclinic space group P1, with unit-cell parameters a = 97.5, b = 100.2, c = 100.4 Å, α = 75.4, ß = 60.9, γ = 81.8°. An X-ray diffraction data set was collected to 2.8 Šresolution from a single crystal. Molecular-replacement results using these data indicate that H. pylori LAP is a hexamer with 32 symmetry.

Designing dual inhibitors against potential drug targets of Plasmodium falciparum -M17 Leucyl Aminopeptidase and Plasmepsins.

Malaria is one of the major diseases of concern worldwide, especially in the African regions. According to a recent WHO report, 95% of deaths that occur due to malaria are in the African regions. Resistance to present antimalarial drugs is increasing rapidly and becoming a problem of concern. M17 Leucyl Aminopeptidase (PfM17LAP) and vacuolar Plasmepsins (PfPM) are two important enzymes involved in the haemoglobin degradation pathway of Plasmodium falciparum. PfM17LAP regulates the release of amino acids and PfPM mediates the conversion of haemoglobin proteins to oligopeptides. These enzymes thus play an essential role in the survival of malaria parasites inside the human body. In the present study, we used in-silico molecular docking, simulation and Molecular Mechanics Poisson-Boltzmann Surface Area (MMPBSA) studies to find potential dual inhibitors of PfPM and PfM17LAP using the ChEMBL antimalarial library. Absorption, distribution, metabolism, excretion and toxicity (ADMET) profiling of the top ten ranked molecules was done using the BIOVIA Discovery Studio. The present investigation revealed that the compound CHEMBL426945 is stable in the binding site of both PfPM and PfM17LAP. In this study, we have reported novel dual-inhibitors that may act better than the present antimalarial drugs.Communicated by Ramaswamy H. Sarma.

Safety evaluation of the food enzyme leucyl aminopeptidase from non-genetically modified Aspergillus oryzae strain NZYM-EX.

The food enzyme leucyl aminopeptidase (EC 3.4.11.1) is produced with the non-genetically modified microorganism Aspergillus oryzae strain NZYM-EX by Novozymes A/S. The food enzyme is free from viable cells of the production organism. It is intended to be used in eight food manufacturing processes: processing of dairy products for the production of (1) flavouring preparations, (2) modified milk proteins; processing of plant- and fungal-derived products for the production of (3) protein hydrolysates, (4) soy sauce; processing of meat and fish products for the production of (5) protein hydrolysates; processing of cereals and other grains for the production of (6) baked products, (7) brewed products; (8) processing of yeast and yeast products. Dietary exposure to the food enzyme total organic solids (TOS) was estimated to be up to 0.577 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by means of a repeated dose 90-day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 440 mg TOS/kg bw per day, the highest dose tested, which, when compared with the estimated dietary exposure, resulted in a margin of exposure of at least 763. A search for similarity of the amino acid sequence of the food enzyme to known allergens was made and no match was found. The Panel considered that the risk of allergic reactions by dietary exposure cannot be excluded, but the likelihood is low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

Urinary leucine aminopeptidase 3 in population environmentally exposed to airborne arsenic.

INTRODUCTION: Environmental arsenic contamination is a major toxicological problem worldwide due to its carcinogenic and nephrotoxic potential. AIM: The purpose of this observational study was to determine the suspected association between urinary arsenic (uAs) and urinary leucine (or leucyl) aminopeptidase 3 (uLAP3) to evaluate uLAP3 as a candidate biomarker of exposure to airborne arsenic. MATERIALS AND METHODS: A total of 918 adults occupationally and/or environmentally exposed to airborne arsenic were enrolled in the study. Baseline information (age; sex; history of smoking; alcohol, fish and seafood consumption) was gathered. Total uAs concentrations [µg/L] of 918 subjects, as well as the sum of arsenic species (ΣiAs) in 259 subjects, were obtained. Urinary LAP3 was measured by an immune-enzymatic assay using an ELISA kit. Urinary creatinine concentration was assessed with the IB/lAB/1289 research protocol (version II, 2015-09-17). The values of uAs and uLAP3 were recalculated per unit of creatinine. The association between uAs and uLAP3 was assessed using a logistic regression model adjusted for confounders. RESULTS: The study identified a positive correlation between the logarithm of uAs and the logarithm of uLAP3 in the study population (r = 0.1737, p < 0.0000) and between urinary creatinine and uLAP3 concentration not adjusted for creatinine level (r = 0.1871, p < 0.001). In the logistic regression model, there was also an association between increased (≥15 µg/L) uAs and decreased (below the 25th quartile) uLAP3 [OR uLAP3 = 1.22 (95% CI 1.03 to 1.44, p < 0.02)]. CONCLUSIONS: These data suggest that urinary LAP3 may be a potential biomarker of arsenic exposure, which warrants further study.

KBE009: A Bestatin-Like Inhibitor of the Trypanosoma cruzi Acidic M17 Aminopeptidase with In Vitro Anti-Trypanosomal Activity.

Chagas disease, caused by the kinetoplastid parasite Trypanosoma cruzi, is a human tropical illness mainly present in Latin America. The therapies available against this disease are far from ideal. Proteases from pathogenic protozoan have been considered as good drug target candidates. T. cruzi acidic M17 leucyl-aminopeptidase (TcLAP) mediates the major parasite's leucyl-aminopeptidase activity and is expressed in all parasite stages. Here, we report the inhibition of TcLAP (IC50 = 66.0 ± 13.5 µM) by the bestatin-like peptidomimetic KBE009. This molecule also inhibited the proliferation of T. cruzi epimastigotes in vitro (EC50 = 28.1 ± 1.9 µM) and showed selectivity for the parasite over human dermal fibroblasts (selectivity index: 4.9). Further insight into the specific effect of KBE009 on T. cruzi was provided by docking simulation using the crystal structure of TcLAP and a modeled human orthologous, hLAP3. The TcLAP-KBE009 complex is more stable than its hLAP3 counterpart. KBE009 adopted a better geometrical shape to fit into the active site of TcLAP than that of hLAP3. The drug-likeness and lead-likeness in silico parameters of KBE009 are satisfactory. Altogether, our results provide an initial insight into KBE009 as a promising starting point compound for the rational design of drugs through further optimization.

PepA binds to and negatively regulates esrB to control virulence in the fish pathogen Edwardsiella piscicida.

As an important marine fish pathogen, Edwardsiella piscicida infects a broad range of fish species and causes substantial economic losses. The EsrA-EsrB two-component system is essential for the expression of type III and type VI secretion systems (T3/T6SSs), the key virulence determinants in the bacterium. In this study, a pull-down assay with the esrB promoter as bait was performed to identify the upstream regulators of esrB. As a result, PepA, a leucyl aminopeptidase, was identified as a repressor of EsrB and T3/T6SS expression. PepA bound to the esrB promoter region and negatively regulated the production of T3/T6SS proteins in early stages. Moreover, PepA was found to affect the in vivo colonization of E. piscicida in turbot livers through the regulation of EsrB expression. Collectively, our results enhance the understanding of the virulence regulatory network and in vivo colonization mechanism of E. piscicida. One sentence summary: PepA regulates EsrB expression in Edwardsiella piscicida.

Development of a High-Throughput Screening Assay to Identify Inhibitors of the Major M17-Leucyl Aminopeptidase from Trypanosoma cruzi Using RapidFire Mass Spectrometry.

Leucyl aminopeptidases (LAPs) are involved in multiple cellular functions, which, in the case of infectious diseases, includes participation in the pathogen-host cell interface and pathogenesis. Thus, LAPs are considered good candidate drug targets, and the major M17-LAP from Trypanosoma cruzi (LAPTc) in particular is a promising target for Chagas disease. To exploit LAPTc as a potential target, it is essential to develop potent and selective inhibitors. To achieve this, we report a high-throughput screening method for LAPTc. Two methods were developed and optimized: a Leu-7-amido-4-methylcoumarin-based fluorogenic assay and a RapidFire mass spectrometry (RapidFire MS)-based assay using the LSTVIVR peptide as substrate. Compared with a fluorescence assay, the major advantages of the RapidFire MS assay are a greater signal-to-noise ratio as well as decreased consumption of enzyme. RapidFire MS was validated with the broad-spectrum LAP inhibitors bestatin (IC50 = 0.35 µM) and arphamenine A (IC50 = 15.75 µM). We suggest that RapidFire MS is highly suitable for screening for specific LAPTc inhibitors.

M17 aminopeptidases diversify function by moderating their macromolecular assemblies and active site environment.

The family of M17 aminopeptidases (alias 'leucine aminopeptidases', M17-LAPs) utilize a highly conserved hexameric structure and a binuclear metal center to selectively remove N-terminal amino acids from short peptides. However, M17-LAPs are responsible for a wide variety of functions that are seemingly unrelated to proteolysis. Herein, we aimed to investigate the myriad of functions attributed to M17. Further, we attempted to differentiate between the different molecular mechanisms that allow the conserved hexameric structure of an M17-LAP to mediate such diverse functions. We have provided an overview of research that identifies precise physiological roles of M17-LAPs, and the distinct mechanisms by which the enzymes moderate those roles. The review shows that the conserved hexameric structure of the M17-LAPs has an extraordinary capability to moderate different molecular mechanisms. We have broadly categorized these mechanisms as 'aminopeptidase-based', which include the characteristic proteolysis reactions, and 'association-driven', which involves moderation of the molecule's macromolecular assembly and higher order complexation events. The different molecular mechanisms are capable of eliciting very different cellular outcomes, and must be regarded as distinct when the physiological roles of this large and important family are considered.

Facile electrochemical detection of botulinum neurotoxin type E using a two-step proteolytic cleavage.

Facile electrochemical methods for measuring protease concentration or protease activity are essential for point-of-care testing of toxic proteases. However, electrochemical detection of proteases, such as botulinum neurotoxin type E (BoNT/E), that cleave a peptide bond between two specific amino acid residues is challenging. This study reports a facile and sensitive electrochemical method for BoNT/E detection. The method is based on a two-step proteolytic cleavage using a target BoNT/E light chain (BoNT/E-LC) and an externally supplemented exopeptidase, L-leucine-aminopeptidase (LAP). BoNT/E-LC cleaves a peptide bond between arginine and isoleucine in IDTQNRQIDRI-4-amino-1-naphthol (oligopeptide-AN) to generate isoleucine-AN. Subsequently, LAP cleaves a bond between isoleucine and AN to liberate a free electroactive AN species. The liberated AN participates in electrochemical-chemical-chemical (ECC) redox cycling involving Ru(NH3)6(3+), AN, and a reducing agent, which allows a high signal amplification. Electrochemical detection is carried out without surface modification of indium-tin oxide electrodes. We show that dithiothreitol is beneficial for enhancing the enzymatic activity of BoNT/E-LC and also for achieving a fast ECC redox cycling. An incubation temperature of 37°C and the use of phosphate buffered saline (PBS) buffer resulted in optimal signal-to-background ratios for efficient BoNT/E detection. BoNT/E-LC could be detected at concentrations of approximately 2.0 pg/mL, 0.2, and 3 ng/mL after 4h, 2h, and 15 min incubation in PBS buffer, respectively, and approximately 0.3 ng/mL after 2-h incubation in bottled water. The method developed could be applied in fast, sensitive, and selective detection of any protease that cleaves a peptide bond between two specific amino acid residues.

Application of cholyglycine in common hepatic diseases / 中国医师杂志

Objective To observe the changes of glycocholic acid (CG) and evaluate the diagnostic value of CG combined with total bile acid (TBA) and leucine aminopeptidase (LAP) in various liver diseases.Methods From October 2016 to March 2017,210 serum samples of healthy people,asymptomatic hepatitis B virus (HBV) infected,hepatitis,biliary obstruction,hepatocirrhosis and primary hepatocellular carcinoma patients were collected.CG and LAP were detected by corresponding kits,and liver function,coagulation function and other indicators of patients were collected and analyzed statistically.Results The serum level of CG were elevated in the 4 liver disease groups and differed statistically from the normal group or the asymptomatic HBV infected group.CG level was positively correlated with LAP (r =0.380,P ＜ 0.01).In liver function indexes,CG was correlated with total bilirubin (TB),direct bilirubin (DB),TBA and alkaline phosphatase (AKP).At the same time,CG was correlated with fibrinogen(Fib),thrombin time(TT).LAP and TBA were introduced into regression equation Y =-0.835 + 0.157X1 +0.312X2 (X1:LAP,X2:TBA,R2 =0.685) as final variables in multivariate linear regression to analyse the influencing factors of CG.Receiver operator characteristic (ROC) curve analysis showed that CG had the strongest ability to diagnose liver diseases in combination with LAP.Conclusions The change of CG level is of great significance in all kinds of liver diseases.The combination of LAP has the strongest ability to diagnose liver diseases.

Application of leucine aminopeptidase in biliary obstructive diseases / 中国医师杂志

Objective To investigate the role of leucine aminopeptidase in biliary obstructive dis-ease, and to evaluate the value of leucine aminopeptidase and its combination with alkaline phosphatase (AKP), glutamine transferase and 5′-nucleotidase (5′-NT) in diagnosis and treatment of bile duct obstruc-tion. Methods A total of 181 cases were collected, who were diagnosed as healthy, asymptomatic HBV carriers and patients with hepatitis, biliary obstruction, liver cirrhosis and primary liver cancer samples at Xiangya Hospital of Central South University from October 2016 to March 2017. The leucine aminopeptidase (LAP), AKP, gamma glutamyl transpeptidase (GGT) and 5′-NT were detected with the corresponding kits, and analyzed with different statistical methods. Results The highest level of LAP was in patients with biliary obstruction, compared to other groups, and the differences were statistically significant (P<0. 05). In biliary obstruction group, LAP and AKP, GGT and 5′-NT were correlated ( r=0. 690, P<0. 01; r=0. 864, P<0. 01;r=0. 735, P<0. 01). Receiver operating characteristic (ROC), curve area (AUC) of LAP, 5′-NT, GGT and AKP in the diagnosis of biliary obstruction were 0. 945 and 0. 898, 0. 942 and 0. 916;the AUC of LAP combined with 5′-NT, GGT and AKP was 0. 966;and the AUC of LAP combined with 5′-NT was 0. 968. Conclusions LAP can be used as a preliminary index of differential diagnosis of biliary obstructive disease, and its diagnostic value could be improved when combined with 5-NT.