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1.
Braz. j. biol ; 84: e251970, 2024. tab, graf
Artigo em Inglês | LILACS, VETINDEX | ID: biblio-1345559

RESUMO

Abstract In order to better understand the ossification processes in anurans our study was carried out on tadpoles and adults of Lithobates catesbeianus. In this sense, we characterized the kinetic properties of alkaline phosphatase with p-nitrophenylphosphatase (pNPP) and pyrophosphate (PPi) and evaluated the activities of tartrate-resistant acid phosphatase and acid phosphatase. The enzyme extracts were obtained from tadpoles and adult femurs, which were divided into epiphysis and diaphysis. After homogenization, the samples were submitted to differential centrifugation to obtain cell membranes and, further, to phospholipase C (PIPLC) treatment, to remove membrane-bound proteins anchored by phosphatidylinositol. The average of specific activity for pNPP hydrolysis (at pH 10.5) by alkaline phosphatase released by phosphatidylinositol-specific phospholipase C (PIPLC) from Bacillus cereus among different bone regions at different animal ages was 1,142.57 U.mg-1, while for PPi hydrolysis (at pH 8.0), it was 1,433.82 U.mg-1. Among the compounds tested for enzymatic activity, the one that influenced the most was EDTA, with approximately 67% of inhibition for pNPPase activity and 77% for PPase activity. In the case of kinetic parameters, the enzyme showed a "Michaelian" behavior for pNPP and PPi hydrolysis. The Km value was around 0.6mM for pNPPase activity and ranged from 0.01 to 0.11mM for PPase activity, indicating that the enzyme has a higher affinity for this substrate. The study of pNPP and PPi hydrolysis by the enzyme revealed that the optimum pH of actuation for pNPP was 10.5, while for PPi, which is considered the true substrate of alkaline phosphatase, was 8.0, close to the physiological value. The results show that regardless of the ossification type that occurs, the same enzyme or isoenzymes act on the different bone regions and different life stages of anurans. The similarity of the results of studies with other vertebrates shows that anurans can be considered excellent animal models for the study of biological calcification.


Resumo Para melhor compreender o processo de ossificação em anuros, nosso estudo foi conduzido em girinos e adultos de Lithobates catesbeianus. Nesse sentido, as propriedades cinéticas da fosfatase alcalina com p-nitrofenilfosfato (pNPP) e pirofosfato (PPi) foram caracterizadas, e as atividades enzimáticas das fosfatases ácida e ácida tartarato resistente foram avaliadas. Os extratos enzimáticos foram obtidos de fêmur de girinos e adultos, divididos em epífise e diáfise. Após a homogeneização as amostras foram submetidas à centrifugação diferencial para obter membrana celular e, em seguida, ao tratamento com fosfolipase C (PIPLC), para remover as proteínas de membrana ancoradas por fosfatidilinositol. A média da atividade específica da fosfatase alcalina, liberada pela PIPLC de Bacillus cereus, para a hidrólise de pNPP (pH 10,5) nas diferentes regiões do fêmur e idades dos animais foi de 1.142,57 U.mg-1, enquanto para a hidrólise do PPi (pH 8,0) foi de 1.433,82 U.mg-1. Entre os compostos testados para a atividade enzimática, o de maior influência foi o EDTA, inibindo aproximadamente 67% e 77% das atividades de pNPPase e PPase, respectivamente. Quanto aos parâmetros cinéticos, a enzima apresentou comportamento Michaeliano para a hidrólise dos dois substratos. O valor de Km foi de 0,6 mM para a atividade de pNPPase e variou de 0,01 a 0,11 para a atividade de PPase, indicando uma maior afinidade por esse substrato. O estudo da hidrólise de pNPP e PPi revelou que o pH ótimo aparente de atuação foi de 10,5 para o pNPP e 8,0 para o PPi, próximo ao fisiológico, sendo que esse é considerado o substrato natural da fosfatase alcalina. Os resultados demonstram que, apesar do tipo de ossificação que ocorre, a mesma enzima ou isoenzimas, atuam nos diferentes locais do osso e estágios de vida dos anuros. A similaridade dos estudos com os realizados com outros vertebrados apontam que os anuros podem ser considerados excelentes modelos animais para o estudo da calcificação biológica.


Assuntos
Animais , Osteogênese , Fosfatase Alcalina/metabolismo , Rana catesbeiana , Osso e Ossos/metabolismo , Cinética
2.
Biol Pharm Bull ; 46(1): 95-101, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36596529

RESUMO

To prevent denosumab-induced hypocalcemia in patients with renal dysfunction, combination therapy with 1α,25-dihydroxy-vitamin D3 (active vitamin D) is recommended. We previously developed a risk prediction model for hypocalcemia in patients with cholecalciferol/calcium (natural vitamin D). However, the prescription status and the risk factors of patients with active vitamin D have not been identified, so we designed this retrospective observational study using a large practice database covering June 2013 to May 2020 to analyze prescription status and risk factors. Patients were classified according to vitamin D type. After that, factors associated with development of hypocalcemia in patients with active vitamin D were explored. Univariate analysis was conducted to compare patient backgrounds between the hypocalcemia and non-hypocalcemia groups. Receiver operating characteristic analysis was conducted to evaluate the predictive potential of the extracted factors. Of the 33442 patients who received denosumab, 22347 and 3560 patients were co-administered natural and active vitamin D, respectively. Patients with active vitamin D had significantly lower renal function (estimated glomerular filtration rate (eGFR) median: 74.0 vs. 69.7 mL/min/1.73 m2), but some patients (23.6%) with sufficient renal function (eGFR ≥90) were also receiving active vitamin D. Of the 3560 patients with active vitamin D, non-hypocalcemia (n = 166) and hypocalcemia (n = 17) groups who met the study criteria were analyzed. Renal function was lower in the hypocalcemia group, and alkaline phosphatase gave the best discrimination. High aspartate aminotransferase (AST), renal dysfunction, high alkaline phosphatase (ALP), and low hemoglobin may be significant factors in risk prediction for hypocalcemia in patients with active vitamin D.


Assuntos
Conservadores da Densidade Óssea , Hipocalcemia , Nefropatias , Humanos , Denosumab/efeitos adversos , Conservadores da Densidade Óssea/efeitos adversos , Fosfatase Alcalina , Hipocalcemia/induzido quimicamente , Vitamina D , Cálcio , Vitaminas , Fatores de Risco , Nefropatias/induzido quimicamente , Prescrições
3.
Biochem Med (Zagreb) ; 33(1): 010705, 2023 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-36627975

RESUMO

Introduction: The presence of macroenzymes in blood can cause diagnostic confusion. Therefore, confirming the presence of macroenzymes is important to reduce unnecessary (non-)invasive investigations. Polyethylene glycol (PEG) precipitation is a simple and fast first-line method for the detection of macroenzymes. However, there is no consensus on the upper reference limit for the PEG-precipitable activity (%PPA) of monomeric enzymes. The aim of this study was to verify a PEG precipitation protocol for the detection of macroenzymes in our laboratory by establishing upper reference limits (URLs) and determining imprecision for eight enzymes after PEG precipitation. In addition, we aimed to clinically verify the URLs using samples containing macroenzymes as identified by electrophoresis. Materials and methods: Per enzyme, at least 40 leftover blood samples from adult patients with either normal or increased enzyme activities were diluted 1:1 with 25% PEG 6000 and 1:1 with 0.9% NaCl. Mixtures were incubated for 10 min at 37°C and centrifuged. Supernatant enzyme activity was measured on Cobas c702 and the %PPA was calculated. Results: The following URLs were obtained: 26% PPA for amylase, 29% PPA for alkaline phosphatase (ALP), 61% PPA for alanine aminotransferase, 48% PPA for aspartate aminotransferase, 24% PPA for creatine kinase (CK), 55% PPA for gamma-glutamyltransferase, 65% PPA for lactate dehydrogenase, and 56% PPA for lipase. The within-lab imprecision was < 15%. Regarding the clinical verification, the two historical samples with proven macroCK showed a %PPA of 69% and 43%, respectively, and a sample with proven macroALP had a %PPA of 52%. Conclusion: In this study, URLs for monomeric enzyme activities after PEG precipitation for eight different enzymes were established. The URLs are suitable for clinical use, but are only partially in line with other studies. Therefore, our data highlight the importance of establishing laboratory-specific upper reference limits for %PPA to allow a correct interpretation.


Assuntos
Fosfatase Alcalina , Creatina Quinase , Adulto , Humanos , Aspartato Aminotransferases , L-Lactato Desidrogenase , Polietilenoglicóis
4.
Biochem Med (Zagreb) ; 33(1): 010703, 2023 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-36627977

RESUMO

Introduction: In order to deliver high quality results, detection and elimination of possible analytical interferences, such as lipaemia, is crucial. The aim of this study is to evaluate the efficacy of high-speed centrifugation in eliminating lipaemic interference and to define own lipaemic index (LI) for the studied biochemical analytes. Materials and methods: Evaluated analytes were: albumin, alkaline phosphatase, alanine-aminotransferase (ALT), aspartate-aminotransferase (AST), calcium, creatinine, gamma-glutamyltransferase (GGT), glucose, phosphates, total proteins, urea and total bilirubin. Those analytes and LIs have been analysed in duplicate in the Roche Diagnostics-c8000 analyser in samples centrifuged at 3000 rpm/10 minutes in the SL16 (Thermo Scientific, Waltham, USA) centrifuge and according to an own high-speed centrifugation protocol (12,900 rpm/15 minutes) in the MicroCL17R (Thermo Scientific, Waltham, USA) centrifuge. Lipaemia has been measured in each sample. The efficiency of high-speed centrifugation is verified by the Wilcoxon test (P < 0.05). In cases where significant differences are observed, our own LI is calculated. For ALT and AST, it is verified by McNemar test (P < 0.05). For creatinine, both Wilcoxon and McNemar test were applied. Results: There were statistically significant differences in analyte concentration before and after high-speed centrifugation for: albumin, creatinine, GGT, glucose, phosphates, urea and total bilirrubin. Own LI is calculated. McNemar test shows statistically significant diferences in the proportion of delivered results before and after high-speed centrifugation in ALT, AST and creatinine. Conclusions: This study confirms the efficacy of high-speed centrifugation protocol for all the considered analytes, excepting calcium, alkaline phosphatase and total proteins.


Assuntos
Cálcio , Hiperlipidemias , Humanos , Creatinina , Fosfatase Alcalina , Centrifugação , Glucose , Alanina Transaminase , Albuminas , Fosfatos
5.
Z Gastroenterol ; 61(1): 50-59, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36623543

RESUMO

BACKGROUND: Reference intervals for basic liver laboratory diagnostic rely on manufacturers' information, remaining unchanged for more than 20 years. This ignores known age and sex dependencies. METHODS: We performed a retrospective cross-sectional study to compare the age-dependent distribution of flagged and non-flagged laboratory findings between reference limits from 3 distinct sources: manufacturer, published reference study, and the truncated maximum likelihood method applied on a cohort of inpatients aged 18-100 years. Discordance rates adjusted for the permissible analytical uncertainty are reported for serum levels of albumin (n= 150,550), alkaline phosphatase (n= 433,721), gamma-GT (n=580,012), AST (n= 510,620), and ALT (n= 704,546). RESULTS: The number of flagged findings differed notably between reference intervals compared, except for alkaline phosphatase. AST and alkaline phosphatase increased with age in women. Overall discordance for AP, AST, and ALT remained below 10%, respectively, in both sexes. Albumin decreased with age which led to discordant flags in up to 22% in patients ≥70 years. GGT and ALT peaked in 50-59-year-old men with up to 23.5% and 22.8% discordant flags, respectively. CONCLUSION: We assessed the impact of different reference limits on liver related laboratory results and found up to 25 % discordant flags. We suggest to further analyse the diagnostic and economic effects of reference limits adapted to the population of interest even for well-established basic liver diagnostics.


Assuntos
Fosfatase Alcalina , Fígado , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Albuminas , Estudos Transversais , Valores de Referência , Estudos Retrospectivos , Idoso , Adolescente , Adulto Jovem , Adulto , Idoso de 80 Anos ou mais
6.
Nutrients ; 15(2)2023 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-36678218

RESUMO

Background: Milk proteins (MPs) and their derivative whey proteins (WPs) are important components of human diet that might prevent bone loss. We aimed to investigate the effects of MP on the bones of postmenopausal women, along with the effects of WP on osteoblast cells. Methods: We conducted a feasibility controlled clinical study with 62 postmenopausal women who were asked to consume an MP-enriched ice cream. We also investigated the effect of WP on the ERK1/2 and AKT pathways, RUNX2, alkaline phosphatase, RANKL/OPG ratio, and COL1A of Saos-2. Results: After 12 weeks, we found a greater bone mineral density and bone alkaline phosphatase reduction in women who consumed the MP-enriched ice cream compared to the control group (p = 0.03 and p = 0.02, respectively). In Saos-2 cells, WP upregulated ERK1/2 and AKT pathways (p = 0.002 and p = 0.016), cell proliferation (p = 0.03), and osteoblast differentiation markers, along with downregulating RANKL/OPG (p < 0.001). Moreover, the inhibition of ERK1/2 by PD184253 reverted the effects on both the RUNX2 and ALP mRNA expression and cells proliferation (p = 0.028, p = 0.004, and p = 0.003, respectively) when treated with WP. Conclusions: WP upregulates cell proliferation, RUNX2, and alkaline phosphatase through the activation of the ERK1/2 pathways on Saos-2. These mechanisms probably contribute to preventing bone loss in postmenopausal women.


Assuntos
Doenças Ósseas Metabólicas , Sorvetes , Humanos , Feminino , Proteínas do Leite/farmacologia , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Fosfatase Alcalina , Estudos de Viabilidade , Proteínas Proto-Oncogênicas c-akt , Osteoprotegerina , Ligante RANK
7.
Anal Chem ; 95(4): 2356-2365, 2023 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-36645297

RESUMO

Alkaline phosphatase (ALP) and interleukin-1beta (IL-1ß) are crucial salivary biomarkers for the diagnosis of periodontal disease that harms the periodontal tissue along with tooth loss. However, there has been no way of sensitive and portable detection of both biomarkers in saliva with multivariate signal readout. In this work, we design the multicolorimetric ALP and IL-1ß sensing platform based on geometrical transformation of silver nanoplate transducer. By utilizing enzymatic activity of ALP that dephosphorylates p-aminophenol phosphate (p-APP) to p-aminophenol (p-AP), localized surface plasmon resonance properties of silver nanoplate vary with ALP and show a distinct color change from blue to yellow based on a controlled seed transformation from triangular to hexagonal, rounded pentagonal, and spherical shape. The multicolor sensor shows an ALP detection range of 0-25 U/L with a limit of detection (LOD) of 0.0011 U/L, which is the lowest range of LOD demonstrated to date for state-of-the-art ALP sensor. Furthermore, we integrate the sensor with the conventional ELISA to detect IL-1ß for multicolor signaling and it exhibits a linear detection range of 0-250 pg/mL and an LOD of 0.066 pg/mL, which is 2 orders of magnitude lower than the monochromic conventional ELISA (LOD of 3.8 pg/mL). The ALP multicolor sensor shows high selectivity with a recovery of 100.9% in real human saliva proving its reliability and suitability for the readily accessible periodontal diagnosis with multivariate signal readout.


Assuntos
Doenças Periodontais , Prata , Humanos , Reprodutibilidade dos Testes , Fosfatase Alcalina/análise , Doenças Periodontais/diagnóstico , Corantes , Biomarcadores , Limite de Detecção
8.
J Enzyme Inhib Med Chem ; 38(1): 2163394, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36629454

RESUMO

Deposition of hydroxyapatite (HA) or alkaline phosphate crystals on soft tissues causes the pathological calcification diseases comprising of end-stage osteoarthritis (OA), ankylosing spondylitis (AS), medial artery calcification and tumour calcification. The pathological calcification is symbolised by increased concentration of tissue non-specific alkaline phosphatase (TNAP). An efficient therapeutic strategy to eradicate these diseases is required, and for this the alkaline phosphatase inhibitors can play a potential role. In this context a series of novel quinolinyl iminothiazolines was synthesised and evaluated for alkaline phosphatase inhibition potential. All the compounds were subjected to DFT studies where N-benzamide quinolinyl iminothiazoline (6g), N-dichlorobenzamide quinolinyl iminothiazoline (6i) and N-nitrobenzamide quinolinyl iminothiazoline (6j) were found as the most reactive compounds. Then during the in-vitro testing, the compound N-benzamide quinolinyl iminothiazoline (6g) exhibited the maximum alkaline phosphatase inhibitory effect (IC50 = 0.337 ± 0.015 µM) as compared to other analogues and standard KH2PO4 (IC50 = 5.245 ± 0.477 µM). The results were supported by the molecular docking studies, molecular dynamics simulations and kinetic analysis which also revealed the inhibitory potential of compound N-benzamide quinolinyl iminothiazoline (6g) against alkaline phosphatase. This compound can be act as lead molecule for the synthesis of more effective inhibitors and can be suggested to test at the molecular level.


Assuntos
Fosfatase Alcalina , Inibidores Enzimáticos , Simulação de Acoplamento Molecular , Cinética , Fosfatase Alcalina/metabolismo , Inibidores Enzimáticos/química , Benzamidas/farmacologia
9.
Biosens Bioelectron ; 222: 114984, 2023 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-36493720

RESUMO

Herein, we develop a CRISPR/Cas12a-based magnetic relaxation switching (C-MRS) biosensor for ultrasensitive and nucleic acid amplification-free detection of methicillin-resistant Staphylococcus aureus (MRSA) in food. In this biosensor, mecA gene in MRSA was recognized by CRISPR-RNA, which will activate the trans-cleavage activity of Cas12a and release the fastened alkaline phosphatase (ALP) on the particle. The freed ALP can then use to hydrolyze substrate to produce ascorbic acid that trigger the click reaction between magnetic probe. The transverse relaxation time of the unbound magnetic probe can be measured for signal readout. By incorporating collateral activity of CRISPR/Cas12a, on-particle rolling circle amplification, and ALP-triggered click chemistry into background-free MRS, as low as 16 CFU/mL MRSA can be detected without any nucleic acid pre-amplification, which avoids carryover contamination, but without compromising sensitivity. Moreover, this C-MRS biosensor could distinguish 0.01% target DNA from the single-base mutant. Recovery in eggs, milk and pork ranged from 75% to 112%, 82%-104%, and 81%-91%, respectively, revealing its satisfactory accuracy and applicability in the complex food matrix. The developed C-MRS biosensor fleshes out the CRISPR toolbox for food safety and provides a new approach for the sensitive and accurate detection of foodborne drug-resistant bacteria.


Assuntos
Técnicas Biossensoriais , Staphylococcus aureus Resistente à Meticilina , Ácidos Nucleicos , Staphylococcus aureus Resistente à Meticilina/genética , Sistemas CRISPR-Cas/genética , Fosfatase Alcalina , Corantes , Ovos , Fenômenos Magnéticos , Técnicas de Amplificação de Ácido Nucleico
10.
Anal Chem ; 95(2): 1454-1460, 2023 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-36538530

RESUMO

Organic photoelectrochemical transistor (OPECT) bioanalytics has recently appeared as a promising route for biological measurements, which has major implications in both next-generation photoelectrochemical (PEC) bioanalysis and futuristic biorelated implementations. Via biological dissociation of materials, bioetching is a useful technique for bio-manufacturing and bioanalysis. The intersection of these two domains is expected to be a possible way to achieve innovative OPECT bioanalytics. Herein, we validate such a possibility, which is exemplified by alkaline phosphatase (ALP)-mediated bioetching of a CoOOH/BiVO4 gate for a signal-on OPECT immunoassay of human immunoglobulin G (HIgG) as the model target. Specifically, target-dependent bioetching of the upper CoOOH layer could result into an enhanced electrolyte contact and light accessibility to BiVO4, leading to the modulated response of the polymeric poly(3,4-ethylenedioxythiophene):poly(styrene sulfonate) (PEDOT:PSS) channel that could be monitored by the channel current. The introduced biosensor achieves sensitive detection of HIgG with high selectivity and sensitivity. This work features bioetching-enabled high-efficacy OPECT bioanalysis and is anticipated to serve as a generic protocol, considering the diverse bioetching routes.


Assuntos
Técnicas Biossensoriais , Pontos Quânticos , Humanos , Fosfatase Alcalina/química , Pontos Quânticos/química , Óxidos , Imunoensaio/métodos , Técnicas Biossensoriais/métodos , Técnicas Eletroquímicas/métodos
11.
Calcif Tissue Int ; 112(2): 233-242, 2023 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-36571614

RESUMO

Alkaline phosphatases (ALPs) are a group of isoenzymes, situated on the external layer of the cell membrane; they catalyze the hydrolysis of organic phosphate esters present in the extracellular space. Zinc and magnesium are significant co-factors for the biological activity of these enzymes. Although ALPs are available in various body tissues and have distinct physiochemical properties, they are true isoenzymes since they catalyze a similar reaction. In the liver, ALP is cytosolic and present in the canalicular membrane of the hepatocytes. ALPs are available in placenta, ileal mucosa, kidney, bone, and liver. However, most of the ALPs in serum (over 80%) are delivered from liver and bone and in more modest quantities from the intestines. Despite the fact that alkaline phosphatases are found in numerous tissues all through the body, their exact physiological function remains largely unknown.


Assuntos
Fosfatase Alcalina , Isoenzimas , Gravidez , Feminino , Humanos , Isoenzimas/metabolismo , Placenta/metabolismo , Osso e Ossos/metabolismo , Intestinos , Fígado/metabolismo
12.
FASEB J ; 37(1): e22701, 2023 01.
Artigo em Inglês | MEDLINE | ID: mdl-36520031

RESUMO

Calcification of the medial layer, inducing arterial stiffness, contributes significantly to cardiovascular mortality in patients with chronic kidney disease (CKD). Extracellular nucleotides block the mineralization of arteries by binding to purinergic receptors including the P2Y2 receptor. This study investigates whether deletion of the P2Y2 receptor influences the development of arterial media calcification in CKD mice. Animals were divided into: (i) wild type mice with normal renal function (control diet) (n = 8), (ii) P2Y2 R-/- mice with normal renal function (n = 8), (iii) wild type mice with CKD (n = 27), and (iv) P2Y2 R-/- mice with CKD (n = 22). To induce CKD, animals received an alternating (0.2-0.3%) adenine diet for 7 weeks. All CKD groups developed a similar degree of chronic renal failure as reflected by high serum creatinine and phosphorus levels. Also, the presence of CKD induced calcification in the heart and medial layer of the aortic wall. However, deletion of the P2Y2 receptor makes CKD mice more susceptible to the development of calcification in the heart and aorta (aortic calcium scores (median ± IQR), CKD-wild type: 0.34 ± 4.3 mg calcium/g wet tissue and CKD-P2Y2 R-/- : 4.0 ± 13.2 mg calcium/g wet tissue). As indicated by serum and aortic mRNA markers, this P2Y2 R-/- mediated increase in CKD-related arterial media calcification was associated with an elevation of calcification stimulators, including alkaline phosphatase and inflammatory molecules interleukin-6 and lipocalin 2. The P2Y2 receptor should be considered as an interesting therapeutic target for tackling CKD-related arterial media calcification.


Assuntos
Fosfatase Alcalina , Lipocalina-2 , Insuficiência Renal Crônica , Túnica Íntima , Calcificação Vascular , Animais , Camundongos , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Cálcio/metabolismo , Lipocalina-2/genética , Lipocalina-2/metabolismo , Insuficiência Renal Crônica/complicações , Insuficiência Renal Crônica/genética , Insuficiência Renal Crônica/metabolismo , Túnica Íntima/metabolismo , Túnica Íntima/patologia , Regulação para Cima , Calcificação Vascular/etiologia , Calcificação Vascular/genética , Calcificação Vascular/metabolismo
13.
Eur J Pharm Sci ; 181: 106366, 2023 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-36565891

RESUMO

Fosamprenavir is a phosphate ester prodrug that, upon dissolution, is cleaved to the poorly soluble yet readily absorbable parent drug amprenavir. In this study, a novel cell-free in vitro setup with quasi-continuous monitoring of the dynamic dissolution/bio-conversion/permeation of fosamprenavir was designed and tested. It consists of side-by-side diffusion cells, where the donor and acceptor compartments are separated by the biomimetic barrier PermeaPad®, and sampling from the donor compartment is accomplished via a microdialysis probe. Externally added bovine alkaline phosphatase induced bioconversion in the donor compartment. Microdialysis sampling allowed to follow the enzymatic conversion of fosamprenavir to amprenavir by the bovine alkaline phosphatase in an (almost) real-time manner eliminating the need to remove or inactivate the enzyme. Biomimetic conversion rates in the setup were established by adding appropriate amounts of the alkaline phosphatase. A substantial (6.5-fold) and persistent supersaturation of amprenavir was observed due to bioconversion at lower (500 µM) concentrations, resulting in a substantially increased flux across the biomimetic barrier, nicely reflecting the situation in vivo. At conditions with an almost 10-fold higher dose than the usual human dose, some replicates showed premature precipitation and collapse of supersaturation, while others did not. In conclusion, the proposed novel tool appears very promising in gaining an in-depth mechanistic understanding of the bioconversion/permeation interplay, including transient supersaturation of phosphate-ester prodrugs like fosamprenavir.


Assuntos
Pró-Fármacos , Animais , Bovinos , Humanos , Fosfatase Alcalina , Biomimética , Ésteres , Microdiálise , Organofosfatos , Fosfatos , Pró-Fármacos/metabolismo , Solubilidade
14.
ACS Appl Bio Mater ; 6(1): 164-170, 2023 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-36525564

RESUMO

Drug-resistant breast cancers such as Triple negative breast cancer (TNBC) do not respond successfully to chemotherapy treatments because they lack the expression of receptor targets. Drug-resistant anti-cancer treatments require innovative approaches to target these cells without relying on the receptors. Intracellular self-assembly of small molecules induced by enzymes is a nanotechnology approach for inhibiting cancer cell growth. In this approach, enzymes will induce the self-assembly of small molecules to nanofibers, which leads to cell death. Here, we investigate the self-assembly of a modified small peptide induced by two different phosphatases: alkaline phosphatase (ALP) and eye absent tyrosine phosphatase (EYA). ALPs are expressed in many adult human tissues and are critical for many cellular functions. EYAs are embryonic enzymes that are over-expressed in drug-resistant breast cancers. We synthesized a small diphenylalanine-based peptide with a tyrosine phosphate end group as the substrate of phosphatase enzymes. Peptides were synthesized with solid phase techniques and were characterized by HPLC and MALDI-TOF. To characterize the self-assembly of peptides exposed to enzymes, different techniques were used such as scattering light intensity, microscopes, and phosphate detection kit. We then determined the toxicity effect of the peptide against normal breast cancer cells, MCF-7, and drug-resistant breast cancer cells, MDA-MB-231. The results showed that the EYA enzyme is able to initiate self-assembly at lower peptide concentration with higher self-assembling intensity compared to ALP. A significant decrease in the TNBC cell number was observed even with a low peptide concentration of 60 µM. These results collectively support the exploration of enzyme self-assembly to treat TNBC.


Assuntos
Nanofibras , Neoplasias de Mama Triplo Negativas , Humanos , Fosfatase Alcalina , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/metabolismo , Linhagem Celular Tumoral , Nanofibras/química , Peptídeos/farmacologia , Monoéster Fosfórico Hidrolases/farmacologia , Monoéster Fosfórico Hidrolases/uso terapêutico , Proliferação de Células
15.
J Pediatr Endocrinol Metab ; 35(2): 223-229, 2022 Feb 23.
Artigo em Inglês | MEDLINE | ID: mdl-34610231

RESUMO

OBJECTIVES: Nutritional rickets (NR) is still an important problem and one which increasing influxes of immigrants are further exacerbating. This study evaluated cases of mostly immigrant children followed up with diagnoses of NR in our pediatric endocrinology clinic. METHODS: Details of 20 cases diagnosed with NR between 2017 and 2020 were retrieved from file records. RESULTS: Twenty (11 male) cases were included in the study. Three (15%) were Turkish nationals and the others (85%) were immigrants. Hypocalcemia and hypophosphatemia were detected in 17 and 13, respectively. Alkaline phosphatase (ALP) values were normal in two cases, while ALP and parathyroid hormone (PTH) values were elevated in all other cases, and PTH levels were very high (473.64 ± 197.05 pg/mL). 25-hydroxyvitamin D levels were below 20 ng/mL in all cases. Patients with NR received high-dose long-term vitamin D or stoss therapy. Six patients failed to attend long-term follow-up, while PTH and ALP levels and clinical findings improved at long-term follow-up in the other 14 cases. CONCLUSIONS: The elevated PTH levels suggest only the most severe cases of NR presented to our clinic. Clinically evident NR is therefore only the tip of the iceberg, and the true burden of subclinical rickets and osteomalacia remains unidentified. Public health policies should therefore focus on universal vitamin D supplementation and adequate dietary calcium provision, their integration into child surveillance programs, adequate advice and support to ensure normal nutrition, exposure to sunlight, and informing families of the increased risk not only for resident populations but also for refugee and immigrant children.


Assuntos
Emigrantes e Imigrantes , Raquitismo/prevenção & controle , Adolescente , Fosfatase Alcalina/metabolismo , Cálcio na Dieta/administração & dosagem , Criança , Pré-Escolar , Suplementos Nutricionais , Feminino , Humanos , Lactente , Masculino , Hormônio Paratireóideo/sangue , Raquitismo/sangue , Raquitismo/epidemiologia , Vitamina D/administração & dosagem
16.
Bone ; 154: 116204, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34547524

RESUMO

Hypophosphatasia (HPP) is the heritable dento-osseous disease caused by loss-of-function mutation(s) of the gene ALPL that encodes the tissue-nonspecific isoenzyme of alkaline phosphatase (TNSALP). TNSALP is a cell-surface homodimeric phosphomonoester phosphohydrolase expressed in healthy people especially in the skeleton, liver, kidneys, and developing teeth. In HPP, diminished TNSALP activity leads to extracellular accumulation of its natural substrates including inorganic pyrophosphate (PPi), an inhibitor of mineralization, and pyridoxal 5'-phosphate (PLP), the principal circulating form of vitamin B6 (B6). Autosomal dominant and autosomal recessive inheritance involving >450 usually missense defects scattered throughout ALPL largely explains the remarkably broad-ranging severity of this inborn-error-of-metabolism. In 1985 when we identified elevated plasma PLP as a biochemical hallmark of HPP, all 14 investigated affected children and adults had markedly increased PLP levels. However, pyridoxal (PL), the dephosphorylated form of PLP that enters cells to cofactor many enzymatic reactions, was not low but often inexplicably elevated. Levels of pyridoxic acid (PA), the B6 degradation product quantified to assess B6 sufficiency, were unremarkable. Canonical signs or symptoms of B6 deficiency or toxicity were absent. B6-dependent seizures in infants with life-threatening HPP were later explained by their profound deficiency of TNSALP activity blocking PLP dephosphorylation to PL and diminishing gamma-aminobutyric acid synthesis in the brain. Now, there is speculation that altered B6 metabolism causes further clinical complications in HPP. Herein, we assessed the plasma PL and PA levels accompanying previously reported elevated plasma PLP concentrations in 150 children and adolescents with HPP. Their mean (SD) plasma PL level was nearly double the mean for our healthy pediatric controls: 66.7 (59.0) nM versus 37.1 (22.2) nM (P < 0.0001), respectively. Their PA levels were broader than our pediatric control range, but their mean value was normal; 40.2 (25.1) nM versus 39.3 (9.9) nM (P = 0.7793), respectively. In contrast, adults with HPP often had plasma PL and PA levels suggestive of dietary B6 insufficiency. We discuss why the B6 levels of our pediatric patients with HPP would not cause B6 toxicity or deficiency, whereas in affected adults dietary B6 insufficiency can develop.


Assuntos
Hipofosfatasia , Adolescente , Adulto , Fosfatase Alcalina/metabolismo , Osso e Ossos/metabolismo , Criança , Humanos , Hipofosfatasia/diagnóstico , Mutação/genética , Vitamina B 6 , Vitaminas
17.
BMC Neurol ; 22(1): 450, 2022 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-36463106

RESUMO

BACKGROUND: Stroke is one of the leading causes of disability worldwide. Recently, stroke prognosis estimation has received much attention. This study investigates the prognostic role of aspartate transaminase/alanine transaminase (De Ritis, AAR), alkaline phosphatase/alanine transaminase (ALP/ALT), and aspartate transaminase/alkaline phosphatase (AST/ALP) ratios in acute ischemic stroke (AIS). METHODS: This retrospective cohort study involved patients who experienced their first-ever AIS between September 2019 and June 2021. Clinical and laboratory data were collected within the first 24 hours after admission. Functional and mortality outcomes were evaluated 90 days after hospital discharge in clinical follow-up. Functional outcome was assessed by a modified Rankin Scale (mRS). The correlation between the laboratory data and study outcomes was evaluated using univariate analysis. In addition, regression models were developed to evaluate the predictive role of AST/ALP, ALP/ALT, and AAR ratios on the study outcomes. RESULTS: Two hundred seventy-seven patients (mean age 69.10 ± 13.55, 53.1% female) were included. According to univariate analysis, there was a weak association between 3-months mRS, and both AST/ALT (r = 0.222, P < 0.001), and AST/ALP (r = 0.164, P = 0.008). Subsequently, higher levels of these ratios and absolute values of AST, ALT, and ALP were reported in deceased patients. Based on regression models adjusted with co-variable (age, gender, underlying disease, and history of smoking) AST/ALT and AST/ALP ratios had a significant independent association with 3-month mRS (CI:1.37-4.52, p = 0.003, and CI: 4.45-11,547.32, p = 0.007, respectively) and mortality (CI: 0.17-1.06, adjusted R2 = 0.21, p = 0.007, and CI: 0.10-2.91, p = 0.035, adjusted R2 = 0.20, respectively). CONCLUSIONS: Elevated AST/ALP and AAR ratios at admission were correlated with poorer outcomes at 3 months in patients with first-ever AIS. Prospective studies in larger cohorts are required to confirm our findings and to evaluate further whether the AST/ALP and De Ritis ratios may represent a useful tool for determining the prognosis of AIS patients.


Assuntos
AVC Isquêmico , Acidente Vascular Cerebral , Humanos , Feminino , Masculino , AVC Isquêmico/diagnóstico , Fosfatase Alcalina , Alanina Transaminase , Estudos Prospectivos , Prognóstico , Estudos Retrospectivos , Aspartato Aminotransferases , Acidente Vascular Cerebral/diagnóstico
18.
Pestic Biochem Physiol ; 188: 105220, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36464385

RESUMO

Extensive usage of synthetic pesticides has proved to be destructive to all living being and the resurgence of pest resistance. Compounds derived from certain plants are usually safer compared to chemical control of pest. The present study thus intended to use Thymus vulgaris essential oil (EO) and two of its derivatives including thymol and carvacrol in order to see their deleterious effects on Glyphodes pyloalis (Walker). We also studied the oil components. This pest has recently become a serious concern for the silk industry. Our results showed that the thyme EO contain several components including thymol (26.9%), ρ-Cymene (14.54%), linalool (13.39%) and carvacrol (5.7%). Our toxicity tests revealed an estimated LD50 values for thyme EO, thymol and carvacrol 2.82, 32.18 and 56.54 µg/larva, respectively. However, the thyme EO was more toxic than its two tested compounds. The activity of certain detoxifying enzymes such as α- and ß-esterase, glutathione S-transferase and cytochrome P450 were significantly inhibited by thymol-treated larvae compared to the control group. Similarly, the activity of alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase and alkaline phosphatases enzymes in thymol-treated larvae decreased while the activity of acid phosphatases increased. Our results suggest that thyme EO and its components have potential for the control of G. pyloalis larvae in mulberry orchards, where no synthetic chemicals are allowed.


Assuntos
Morus , Óleos Voláteis , Thymus (Planta) , Animais , Óleos Voláteis/toxicidade , Timol/toxicidade , Larva , Fosfatase Alcalina
19.
BMC Complement Med Ther ; 22(1): 321, 2022 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-36464690

RESUMO

BACKGROUND: Diazinon (DZN), a widely used chemical herbicide for controlling agricultural pests, is an important organophosphorus pesticide and an environmental pollutant which induces toxic effects on living organisms during long-term exposure. Thymoquinone (TQ) is a phytochemical bioactive compound with antioxidant and anti-inflammatory properties. We aimed to evaluate the protective effects of TQ against DZN-induced hepatotoxicity through alleviating oxidative stress and enhancing cholinesterase (ChE) enzyme activity. METHODS: Rats were randomly divided into six groups (n = 8); a negative control group receiving corn oil; a group only receiving DZN (20 mg/kg/day); a group treated with TQ (10 mg/kg/day), and three treatment groups as TQ + DZN, receiving different doses of TQ (2.5, 5, and 10 mg/kg/day). All experimental animals were orally treated for 28 consecutive days. The levels of superoxide dismutase (SOD), glutathione (GSH), malondialdehyde (MDA), alanine transaminase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and lactic acid dehydrogenase (LDH) were determined. In addition, ChE activity and histopathological changes were evaluated. RESULTS: The results showed that DZN decreased GSH level (p < 0.01) and SOD activity (p < 0.01) in parallel to an increase in MDA level (p < 0.01) and increased the activity of AST, ALT, ALP, and LDH (p < 0.01) in comparison to the negative control group. Our findings demonstrated that TQ administration could diminish hepatotoxicity and reduce oxidative damage in DZN-treated rats, which could be linked to its antioxidant and free radical scavenging properties. It was also observed that TQ 10 mg/kg remarkably increased the activity of acetylcholinesterase, butyrylcholinesterase, and SOD enzymes, elevated GSH, decreased MDA, and reduced pathological alternations of the liver induced by DZN. CONCLUSION: Thymoquinone 10 mg/kg increased the activity of plasma and blood cholinesterases and reduced DZN-induced alternations of the liver. Improvement of butyryl- and acetylcholinesterase activity suggests that maybe TQ supplement could be beneficial as pre-exposure prophylaxis among farm workers spraying pesticides.


Assuntos
Doença Hepática Induzida por Substâncias e Drogas , Hepatite , Praguicidas , Animais , Ratos , Diazinon/toxicidade , Acetilcolinesterase , Antioxidantes/farmacologia , Butirilcolinesterase , Compostos Organofosforados , Praguicidas/toxicidade , Glutationa , Superóxido Dismutase , Fosfatase Alcalina , Doença Hepática Induzida por Substâncias e Drogas/tratamento farmacológico , Doença Hepática Induzida por Substâncias e Drogas/prevenção & controle
20.
Front Endocrinol (Lausanne) ; 13: 914872, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36465647

RESUMO

Purpose: Exercise therapy and key regulators of bone quality exert anti-hyperglycemic effects on type 2 diabetes mellitus (T2DM) mice. A number of programs have been reported to have an effect on bone disease in T2DM. Major unanswered questions concern the potential correlation of exercise with the improvement of bone quality in T2DM mice and how the nonlinear optical properties of bone are correlated with changes to its crystal structure. Methods: Subjects were randomly divided into six groups: 1) control (C) group, which was fed a normal diet (n = 8); 2) T2DM quiet group, which was given a high-fat diet and quiet (n = 8); 3) T2DM plus swimming (T2DM+S) group, which received T2DM and swim training (n = 8); 4) T2DM plus resistance exercise (T2DM+RE) group, which was given T2DM and resistance exercise (n = 8); 5) T2DM plus aerobic exercise (T2DM+AE) group, with T2DM and medium-intensity treadmill exercise (n = 8); and 6) T2DM plus high-intensity interval training (T2DM+HIIT), with T2DM and high-intensity variable-speed intervention (n = 8). The levels of runt-related transcription factor 2 (RUNX2), osterix (OSX), and alkaline phosphatase (ALP), as well as the bone microstructure and morphometry, were measured at the end of the 8-week exercise intervention. Results: Compared with the C group, the bone microstructure indexes [bone mineral density (BMD), bone volume/tissue volume (BV/TV), cortical thickness (Ct.Th), and connectivity density (Conn.D)], the bone biomechanical properties (maximum load, fracture load, yield stress, and elastic modulus), and the osteogenic differentiation factors (RUNX2, OSX, and BMP2) of the T2DM group were significantly decreased (all p < 0.05). Compared with the T2DM group, there were obvious improvements in the osteogenic differentiation factor (OSX) and Th.N, while the separation of trabecular bone (Tb.Sp) decreased in the T2DM+AE and T2DM+HIIT groups (all p < 0.05). In addition, the bone microstructure indicators BV/TV, tissue mineral density (TMD), Conn.D, and degree of anisotropy (DA) also increased in the T2DM+HIIT group, but the yield stress and Ct.Th deteriorated compared with the T2DM group (all p < 0.05). Compared with the T2DM+S and T2DM+RE groups, the BV/TV, trabecular number (Tb.N), Tb.Sp, and Conn.D in the T2DM+AE and T2DM+HIIT groups were significantly improved, but no significant changes in the above indicators were found between the T2DM+S and T2DM+RE groups (all p < 0.05). In addition, the BMD and the expression of ALP in the T2DM+AE group were significantly higher than those in the T2DM+HIIT group (all p < 0.05). Conclusion: There was a significant deterioration in femur bone mass, trabecular bone microarchitecture, cortical bone geometry, and bone mechanical strength in diabetic mice. However, such deterioration was obviously attenuated in diabetic mice given aerobic and high-intensity interval training, which would be induced mainly by suppressing the development of T2DM. Regular physical exercise may be an effective strategy for the prevention of not only the development of diabetes but also the deterioration of bone properties in patients with chronic T2DM.


Assuntos
Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 2 , Osteoporose , Camundongos , Animais , Humanos , Subunidade alfa 1 de Fator de Ligação ao Core , Diabetes Mellitus Tipo 2/terapia , Osteogênese , Fêmur , Osteoporose/etiologia , Osteoporose/terapia , Terapia por Exercício , Fosfatase Alcalina
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