RESUMO
The construction of enzyme delivery systems, which can control enzymatic activity at a target site, is important for efficient enzyme-prodrug therapy/diagnosis. Herein we report a facile technique to construct a systemically applicable ß-galactosidase (ß-Gal)-loaded ternary complex comprising tannic acid (TA) and phenylboronic acid-conjugated polymers through sequential self-assembly in aqueous solution. At physiological conditions, the ternary complex exhibited a hydrodynamic diameter of â¼40 nm and protected the loaded ß-Gal from unfavorable degradation by proteinase. Upon cellular internalization, the ternary complex recovered ß-Gal activity by releasing the loaded ß-Gal. The intravenously injected ternary complex thereby delivered ß-Gal to the target tumor in a subcutaneous tumor model and exerted enhanced and selective enzymatic activity at the tumor site. Sequential self-assembly with TA and phenylboronic acid-conjugated polymers may offer a novel approach for enzyme-prodrug theragnosis.
Assuntos
Ácidos Borônicos/metabolismo , Nanopartículas/metabolismo , Neoplasias/metabolismo , Polímeros/metabolismo , Taninos/metabolismo , beta-Galactosidase/metabolismo , Animais , Ácidos Borônicos/química , Linhagem Celular Tumoral , Feminino , Hidrodinâmica , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Estrutura Molecular , Nanopartículas/química , Neoplasias/diagnóstico , Tamanho da Partícula , Polímeros/síntese química , Polímeros/química , Propriedades de Superfície , Taninos/química , beta-Galactosidase/administração & dosagem , beta-Galactosidase/sangueRESUMO
Objective: To investigate the potential role of ß-galactosidase in altering immunoglobulin G (IgG) galactosylation in serum of rheumatoid arthritis (RA).Methods: The expression level and activity of ß-galactosidase in serum and CD 19+ B cells were measured by enzyme-linked immune sorbent assay (ELISA). The effect of ß-galactosidase on the N-glycan changes in serum from mice intravenously treated with ß-galactosidase was observed by linear ion-trap quadrupole-electrospray ionization mass spectrometry (LTQ-ESI-MS). We established a collagen-induced arthritis (CIA) rat model to explore the biological function of ß-galactosidase in RA.Results: The expression level of ß-galactosidase in serum of 32 patients was elevated when compared with those of 30 healthy controls. The activity and expression level of ß-galactosidase in CD19+ B cells from RA patients was higher than those from healthy controls. The ratio of m/z 1142/937 was reduced in mice treated with ß-galactosidase when compared with normal mice. We found that ß-galactosidase was implicated in the development of inflammation by affecting body weight and elevating the expression level of interleukin-6, tumor necrosis factor-α, and rheumatoid factor in the serum.Conclusions: Our results suggested the high level of ß-galactosidase in B cells and serum of RA patients and revealed that altered ß-galactosidase may be implicated in the progression of inflammation.
Assuntos
Artrite Reumatoide/sangue , beta-Galactosidase/sangue , Animais , Artrite Experimental/metabolismo , Artrite Reumatoide/patologia , Feminino , Humanos , Imunoglobulina G/sangue , Interleucina-6/sangue , Masculino , Camundongos , Ratos , Fator Reumatoide/sangue , Fator de Necrose Tumoral alfa/sangueRESUMO
Acid sphingomyelinase (aSMase) is involved in the generation of metabolites that function as part of the sphingolipid signaling pathway. It catalyzes the breakdown of sphingomyelin into ceramide, a bioactive lipid that, among other roles, is involved in regulation of apoptosis. Dry drop blood test (DBS) and colorimetric 2-step enzymatic assay were used to assess the activity of human blood aSMase, beta-galactosidase, and beta-glucosidase, these enzymes are lysosomal hydrolases that catalyze the degradation of related sphingolipids, of sphingolipid signaling molecules. Blood was collected from a group of healthy volunteers and patients that were diagnosed with multiple myeloma (MM) in various stages of the disease. Additionally, activity of those enzymes in patients diagnosed with other hematological cancers was also assessed. We found that aSMase activity in the blood of patients with MM (at the time of diagnosis) was 305.43 pmol/spot*20 h, and this value was significantly lower (p < 0.030) compared to the healthy group 441.88 pmol/spot*20 h. Our collected data suggest a possible role of aSMase in pathogenesis of MM development.
Assuntos
Mieloma Múltiplo/sangue , Esfingolipídeos/sangue , Esfingomielina Fosfodiesterase/sangue , beta-Galactosidase/sangue , beta-Glucosidase/sangue , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Feminino , Humanos , Leucemia de Células Pilosas/sangue , Leucemia de Células Pilosas/diagnóstico , Leucemia de Células Pilosas/patologia , Leucemia Linfocítica Crônica de Células B/sangue , Leucemia Linfocítica Crônica de Células B/diagnóstico , Leucemia Linfocítica Crônica de Células B/patologia , Metabolismo dos Lipídeos , Linfoma de Zona Marginal Tipo Células B/sangue , Linfoma de Zona Marginal Tipo Células B/diagnóstico , Linfoma de Zona Marginal Tipo Células B/patologia , Masculino , Pessoa de Meia-Idade , Mieloma Múltiplo/diagnóstico , Mieloma Múltiplo/patologia , Estadiamento de Neoplasias , Leucemia-Linfoma Linfoblástico de Células Precursoras/sangue , Leucemia-Linfoma Linfoblástico de Células Precursoras/diagnóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Mielofibrose Primária/sangue , Mielofibrose Primária/diagnóstico , Mielofibrose Primária/patologiaRESUMO
Pregnancy is a stress factor culminating into mild endoplasmic reticulum (ER) stress, which is necessary for placental development. However, excessive or chronic ER stress in pre-eclamptic placentas leads to placental dysfunction. The precise mechanisms through which excessive ER stress impacts trophoblasts are not well understood. Here, we showed that ER stress reduces the number of lysosomes, resulting in inhibition of autophagic flux in trophoblast cells. ER stress also disrupted the translocation of lysosomes to the surface of trophoblast cells, and inhibited lysosomal exocytosis, whereby the secretion of lysosomal-associated membrane protein 1 (LAMP1) into culture media was significantly attenuated. In addition, we found that serum LAMP1 and beta-galactosidase levels were significantly decreased in pre-eclampsia patients compared to normal pregnant women, potentially indicating lysosomal dysfunction through ER stress in pre-eclamptic placentas. Thus, we demonstrated that excessive ER stress essentially disrupts homeostasis in trophoblasts in conjunction with autophagy inhibition by lysosomal impairment.
Assuntos
Autofagia , Estresse do Retículo Endoplasmático , Lisossomos/patologia , Pré-Eclâmpsia/patologia , Trofoblastos/patologia , Adulto , Biomarcadores/sangue , Biomarcadores/metabolismo , Linhagem Celular , Meios de Cultura/metabolismo , Exocitose , Feminino , Humanos , Proteínas de Membrana Lisossomal/sangue , Proteínas de Membrana Lisossomal/metabolismo , Lisossomos/metabolismo , Placentação , Pré-Eclâmpsia/sangue , Pré-Eclâmpsia/diagnóstico , Gravidez , Cultura Primária de Células , Trofoblastos/citologia , beta-Galactosidase/sangue , beta-Galactosidase/metabolismoRESUMO
INTRODUCTION: Recent studies have indicated that lysosomal dysfunction contributes to the development of idiopathic Parkinson's disease (PD). It is uncertain whether dysregulation of serum lysosomal acid hydrolase activity exists in sporadic PD patients compared with normal controls (NCs) and parkinsonian syndrome (PS) patients. METHODS: Sporadic PD patients without GBA1 mutations (nâ¯=â¯68) were matched with normal controls (nâ¯=â¯45), and parkinsonian syndrome patients (nâ¯=â¯32) in terms of family history, age, and sex. We measured the activities of lysosomal enzymes, α-galactosidase, ß-galactosidase, and ß-hexosaminidase and examined the possible correlations between lysosomal acid hydrolase activities with age in NCs, PD, and PS patients. RESULTS: ß-Galactosidase activity was significantly higher in the PD and PS than in the NC group (Pâ¯<â¯0.001). The ß-galactosidase to α-galactosidase and ß-hexosaminidase to ß-galactosidase activity ratios were more useful for distinguishing PD and PS patients from NCs (Pâ¯<â¯0.0001). Furthermore, α-galactosidase activity was significantly higher in PS patients than both PD and NC groups (pâ¯=â¯0.04). ß-Galactosidase and α-galactosidase activities exhibited a statistically significant negative correlation with age in NCs, and ß-hexosaminidase activity showed a positive correlation with age in PS. However, PD patients did not show any of these correlations. CONCLUSION: Our results suggest the presence of an unknown regulatory mechanism(s) of serum acid hydrolase activities with aging in the normal population and abnormalities in their regulation in PD and PS patients. However, the pattern of dysregulation in these two groups is different. Thus, serum lysosomal acid hydrolase activity can be used as a peripheral biomarker for PD.
Assuntos
Transtornos Parkinsonianos/sangue , alfa-Galactosidase/sangue , beta-Galactosidase/sangue , beta-N-Acetil-Hexosaminidases/sangue , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/sangue , Estudos de Coortes , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Doença de Parkinson/sangueRESUMO
PURPOSE: Adaptation of the colorimetric method for the determination of ß-d-galactosidase, ß-d-glucuronidase and α-l-fucosidase activities in serums from hemolyzed blood, the material currently being discarded. MATERIALS AND METHODS: The materials included serums from hemolyzed and non-hemolyzed blood, obtained from 26 healthy volunteers. The adaptation of the method involved precipitation of the proteins with trichloroacetic acid after incubating serums with substrates, but before determining the products of enzymatic reactions. RESULTS: In serums from hemolyzed and non-hemolyzed blood of the same persons, we found high correlations among the results obtained using hemolyzed blood (with adapted) and non-hemolyzed blood (with non-adapted) methods. CONCLUSION: We are able to determine the ß-d-galactosidase, ß-d-glucuronidase and α-l-fucosidase activities in serums from hemolyzed blood (with adapted) and non-hemolyzed blood (with non-adapted) methods, with the same accuracy and precision.
Assuntos
Glucuronidase/sangue , Hemólise , alfa-L-Fucosidase/sangue , beta-Galactosidase/sangue , Adulto , Feminino , Hemoglobinas/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Cinética , Masculino , Pessoa de Meia-Idade , Nitrofenóis/metabolismo , Adulto JovemRESUMO
The association of lysosomal dysfunction and neurodegeneration has been documented in several neurodegenerative diseases, including Alzheimer's Disease (AD). Herein, we investigate the association of lysosomal enzymes with AD at different stages of progression of the disease (mild and severe) or with mild cognitive impairment (MCI). We conducted a screening of two classes of lysosomal enzymes: glycohydrolases (ß-Hexosaminidase, ß-Galctosidase, ß-Galactosylcerebrosidase, ß-Glucuronidase) and proteases (Cathepsins S, D, B, L) in peripheral blood samples (blood plasma and PBMCs) from mild AD, severe AD, MCI and healthy control subjects. We confirmed the lysosomal dysfunction in severe AD patients and added new findings enhancing the association of abnormal levels of specific lysosomal enzymes with the mild AD or severe AD, and highlighting the difference of AD from MCI. Herein, we showed for the first time the specific alteration of ß-Galctosidase (Gal), ß-Galactosylcerebrosidase (GALC) in MCI patients. It is notable that in above peripheral biological samples the lysosomes are more sensitive to AD cellular metabolic alteration when compared to levels of Aß-peptide or Tau proteins, similar in both AD groups analyzed. Collectively, our findings support the role of lysosomal enzymes as potential peripheral molecules that vary with the progression of AD, and make them useful for monitoring regenerative medicine approaches for AD.
Assuntos
Doença de Alzheimer/sangue , Disfunção Cognitiva/sangue , Galactosilceramidase/sangue , beta-Galactosidase/sangue , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/enzimologia , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/sangue , Disfunção Cognitiva/enzimologia , Disfunção Cognitiva/patologia , Progressão da Doença , Feminino , Regulação da Expressão Gênica , Humanos , Lisossomos/enzimologia , Masculino , Medicina Regenerativa , Índice de Gravidade de Doença , Proteínas tau/sangueRESUMO
GM1 gangliosidosis (GM1) is an autosomal recessive lysosomal storage disease where GLB1 gene mutations result in a reduction or absence of lysosomal acid ß-galactosidase (ßgal) activity. ßgal deficiency leads to accumulation of GM1-ganglioside in the central nervous system (CNS). GM1 is characterized by progressive neurological decline resulting in generalized paralysis, extreme emaciation and death. In this study, we assessed the therapeutic efficacy of an adeno-associated virus (AAV) 9-mßgal vector infused systemically in adult GM1 mice (ßGal(-/-)) at 1 × 10(11) or 3 × 10(11) vector genomes (vg). Biochemical analysis of AAV9-treated GM1 mice showed high ßGal activity in liver and serum. Moderate ßGal levels throughout CNS resulted in a 36-76% reduction in GM1-ganglioside content in the brain and 75-86% in the spinal cord. Histological analyses of the CNS of animals treated with 3 × 10(11) vg dose revealed increased presence of ßgal and clearance of lysosomal storage throughout cortex, hippocampus, brainstem and spinal cord. Storage reduction in these regions was accompanied by a marked decrease in astrogliosis. AAV9 treatment resulted in improved performance in multiple tests of motor function and behavior. Also the majority of GM1 mice in the 3 × 10(11) vg cohort retained ambulation and rearing despite reaching the humane endpoint due to weight loss. Importantly, the median survival of AAV9 treatment groups (316-576 days) was significantly increased over controls (250-264 days). This study shows that moderate widespread expression of ßgal in the CNS of GM1 gangliosidosis mice is sufficient to achieve significant biochemical impact with phenotypic amelioration and extension in lifespan.
Assuntos
Sistema Nervoso Central/metabolismo , Gangliosidose GM1/genética , Terapia Genética , beta-Galactosidase/genética , Animais , Astrócitos/metabolismo , Astrócitos/patologia , Tronco Encefálico/metabolismo , Tronco Encefálico/patologia , Sistema Nervoso Central/patologia , Dependovirus/genética , Modelos Animais de Doenças , Gangliosídeos/metabolismo , Gangliosidose GM1/metabolismo , Gangliosidose GM1/terapia , Vetores Genéticos , Humanos , Camundongos , Medula Espinal/metabolismo , Medula Espinal/patologia , beta-Galactosidase/biossíntese , beta-Galactosidase/sangueRESUMO
We have reported a possible involvement of apurinic/apyrimidinic endonuclease 1 (APE1), one of the DNA repair pathways, in various nephropathy models and found that there is a close connection between APE1 and p53-dependent apoptosis. Therefore, we investigated the changes of APE in aging rat kidney since aging is the consequence of increased susceptibility to apoptosis and impaired repair. Characteristics of chronological aging were compared among 6-, 24- and 28-month-old male Sprague-Dawley rats. Serum blood urea nitrogen and creatinine were measured for renal function. Western blot assay was compared for p53, bax, cleaved caspase 3, rH2AX, and APE1. Immunohistochemical staining of 8-hydroxy-2'-deoxyguanosine (8-OHdG) and APE1 was performed. Cellular senescence was checked by beta-galactosidase staining. Compared with young rats, aged rats showed significant increase in creatinine level with cellular senescence in the proximal convoluted tubules confirmed by beta-galactosidase staining. All the checked variables were significantly increased with aging: 1) increased p53, bax, and caspase 3 may activate the apoptotic pathway, 2) increased rH2AX and 8-OHdG immunolocalization in the proximal convoluted tubules might mean augmented DNA damage, and 3) increased APE1 might be caused by the immunoreactivity in the distal convoluted tubules while decreased in the proximal convoluted tubules. These results suggested that APE1 might have little protective effects on p53-dependent apoptosis irrespective of DNA repair activities in aged renal proximal tubules. Therefore, researchers should use older animals than 24-month-old rats in future studies for investigating the relationship between the apoptosis and DNA repair in the aging kidneys.
Assuntos
DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Nefropatias/enzimologia , Rim/enzimologia , Fatores Etários , Animais , Apoptose/fisiologia , Nitrogênio da Ureia Sanguínea , Creatinina/sangue , Dano ao DNA , Reparo do DNA , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , Humanos , Rim/fisiologia , Nefropatias/sangue , Nefropatias/fisiopatologia , Masculino , Ratos , Ratos Sprague-Dawley , beta-Galactosidase/sangueRESUMO
A seven-month-old male child was brought in for an eye test for poor vision and nystagmus noticed from four months of age. The child had delayed milestones of development and multiple (six times) episodes of unexplained lower respiratory tract infection (from two months of age) treated by pediatricians at different centers without complete cure. Fundus examination showed bilateral cherry-red spots at the macula. There were diffusely distributed hyper-pigmented patches (Mongolian spots) on the back and extensor aspect of the extremities. The case was sent back to the pediatricians for a re-evaluation to rule out storage disorder. Lysosomal enzyme assay in the leucocytes showed a significantly reduced ß-galactosidase level (15.6 nmol/hr/mg protein in contrast to a normal range of 79.6 to 480.0). This confirmed the patient to be a case of lysosomal storage disease, the GM1 gangliosidosis (type I).
Assuntos
Gangliosidose GM1/diagnóstico , Macula Lutea/patologia , Pneumonia Aspirativa/diagnóstico , Consanguinidade , Gangliosidose GM1/enzimologia , Humanos , Lactente , Masculino , Recidiva , Transtornos da Visão/diagnóstico , beta-Galactosidase/sangueAssuntos
Hemangioma/patologia , Neoplasias Cutâneas/patologia , Adulto , Criança , Feminino , Hemangioma/enzimologia , Hemangioma/genética , Humanos , Neoplasias Cutâneas/enzimologia , Neoplasias Cutâneas/genética , alfa-Galactosidase/sangue , alfa-Galactosidase/genética , alfa-L-Fucosidase/sangue , beta-Galactosidase/sangueRESUMO
INTRODUCTION: Beta-galactosidase (GAL) is a lysosomal exoglycosidase involved in the catabolism of glycoconjugates through the sequential release of beta-linked terminal galactosyl residues. The stimulation of activity of exoglycosidases and other degradative enzymes has been noted in cancers as well as in alcohol and nicotine addiction separately. This is the first study to evaluate the activity of the serum senescence marker GAL in colon cancer patients with a history of alcohol and nicotine dependence, as a potential factor of worse cancer prognosis. MATERIAL AND METHODS: The material was serum of 18 colon cancer patients and 10 healthy volunteers. Ten colon cancer patients met alcohol and nicotine dependence criteria. The activity of beta-galactosidase (pkat/ml) was determined by the colorimetric method. Comparisons between groups were made using the Kruskal-Wallis analysis and differences evaluated using the Mann-Whitney U test. Spearman's rank correlation coefficient was used to measure the statistical dependence between two variables. RESULTS: The activity of serum GAL was significantly higher in colon cancer patients with a history of alcohol and nicotine dependence, in comparison to colon cancer patients without a history of drinking/smoking (p=0.015; 46% increase), and the controls (p=0.0002; 81% increase). The activity of serum GAL in colon cancer patients without a history of alcohol/nicotine dependence was higher than the activity in the controls (p = 0.043; 24% increase). DISCUSSION/CONCLUSION: Higher activity of beta-galactosidase may potentially reflect the accelerated growth of the cancer, invasion, metastases, and maturation, when alcohol and nicotine dependence coincide with colon cancer. For a better prognosis of colon cancer, alcohol and nicotine withdrawal seems to be required.
Assuntos
Alcoolismo/complicações , Alcoolismo/enzimologia , Biomarcadores Tumorais/sangue , Neoplasias do Colo/complicações , Neoplasias do Colo/enzimologia , Tabagismo/enzimologia , beta-Galactosidase/sangue , Idoso , Consumo de Bebidas Alcoólicas/sangue , Feminino , Voluntários Saudáveis , Humanos , Masculino , Pessoa de Meia-Idade , Invasividade Neoplásica/fisiopatologia , Prognóstico , Fumar/sangue , Tabagismo/complicaçõesRESUMO
This study investigates the miniaturization of the screening technique using dried blood spots on filter paper (DBS) to measure GBA and CT activities, and GBA and ß-galactosidase activities in leukocytes. 274 DBS from individuals with suspected GD were screened for 1.5 years. Of these, we confirmed the diagnosis in 13.5%. The miniaturization of the DBS and leukocyte techniques afforded to reduce costs and sample size appropriate for a reliable diagnosis.
Assuntos
Bioensaio , Doença de Gaucher/sangue , Doença de Gaucher/diagnóstico , Hexosaminidases/sangue , Leucócitos/metabolismo , Programas de Rastreamento , beta-Galactosidase/sangue , Coleta de Amostras Sanguíneas , Brasil , Estudos de Casos e Controles , HumanosRESUMO
Lyme disease (LD) is the most prevalent tick-borne disease in Europe. LD is caused by the spirochete Borrelia burgdorferi. LD is a chronic disease which can attack a number of organs: skin, heart, brain, joints. Chronic, low-grade inflammation involves general production of pro-inflammatory cytokines and inflammatory markers and is a typical feature of aging. So far, the best method of diagnosing LD is a time-consuming and expensive two-stage serological method. The aim of our study was to evaluate the activity of two lysosomal exoglycosidases: α-fucosidase (FUC) and ß-galactosidase (GAL) in the serum of patients with Lyme disease, as potential markers of LD. Due to the increasing number of patients with Lyme disease and a number of false results, new ways to diagnose this disease are still being sought. As elevated level of ß-galactosidase is a manifestation of residual lysosomal activity in senescent cells, the increase in its activity in serum during chronic Lyme disease might be a marker of a potentially accelerated senescence process. The study was performed on serum taken from cubital veins of 15 patients with Lyme disease and eight healthy subjects (control group). FUC and GAL activity was measured by the method of Chatterjee et al. as modified by Zwierz et al. In the serum of patients with Lyme disease, GAL activity significantly increased (p = 0.029), and the activity of FUC had a tendency to increase (p = 0.153), compared to the control group. A significant increase in GAL activity in the serum of patients with Lyme disease indicates an increased catabolism of glycoconjugates (glycoproteins, glycolipids, proteoglycans) and could be helpful in the diagnosis of Lyme disease, although this requires confirmation in a larger group of patients. As GAL is the most widely used assay for detection of senescent cells, an elevated level of ß-galactosidase might be a manifestation of accelerated senescence process in the course of Lyme disease.
Assuntos
Envelhecimento/sangue , Doença de Lyme/sangue , Doença de Lyme/enzimologia , alfa-L-Fucosidase/sangue , beta-Galactosidase/sangue , Adulto , Biomarcadores/sangue , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
OBJECTIVE: To explore the incidence of various types of mucopolysaccharidosis (MPS) and their clinical characteristics. METHODS: A total of 75 children highly suspected as having MPS underwent quantitative and electrophoretic analysis of urinary glycosaminoglycans (GAGs) and enzymatic analysis of seven types of MPS from January 2009 to December 2011. Fluorescence assay was used to measure the activities of α-L-iduronidase, iduronate-2-sulfatase, α-N-acetylglucosaminidase, galactosamine-6-sulfatase, ß-galactosidase, arylsulfatase B and ß-glucuronidase in the white blood cells. RESULTS: A total of 52 cases were confirmed with MPS based on clinical, radiological, and enzymatic examinations. The 52 cases, with a mean age of 4.0 ± 2.2 years, included 5 cases of MPS I (10%), 20 cases of MPS II (38%), 20 cases of MPS IVA (38%), 6 cases of MPS VI (12%) and 1 case of MPS VII (2%). No MPS IV B cases or MPS IIIB cases were found. Compared with healthy children of the same age, the GAG/Cr ratio was significantly elevated in 50 confirmed cases of MPS (two MPS IVA cases having no increased ratio). All children with increased urinary GAGs had a confirmed diagnosis of MPS. The age of onset was between 1 and 2 years after birth in most cases, and often complicated by hernia and valvular heart disease. Children with MPS I, MPS II, and MPS VI presented with ugly and unsmooth face, short stature, joint stiffness, and limitation of motion, while children with MPS IVA presented with short stature, skeletal dysplasia, and joint laxity. CONCLUSIONS: Type IVA and type II are the most common in MPS cases, followed by type VI and type I. MPS children are characterized by special appearances including ugly and unsmooth facial appearance, short stature and skeletal dysplasia. Quantitative analysis of urinary GAG, as a simple, rapid, and reliable method, is recommended for screening of MPS.
Assuntos
Mucopolissacaridoses/diagnóstico , Acetilglucosaminidase/sangue , Criança , Pré-Escolar , Creatinina/urina , Feminino , Glucuronidase/sangue , Glicosaminoglicanos/urina , Humanos , Iduronidase/sangue , Lactente , Imageamento por Ressonância Magnética , Masculino , Mucopolissacaridoses/enzimologia , Mucopolissacaridoses/patologia , beta-Galactosidase/sangueRESUMO
UNLABELLED: The enzymatic profile of lysosomal exoglycosidases in middle ear cholesteatoma has not been well known. The assessment of glycoconjugate catabolism may contribute to a better understanding of cholesteatoma pathogenesis. OBJECTIVE: The study aim was to evaluate catabolic processes of glycoproteins, glycolipids, and proteoglycans in cholesteatoma through outlining the concentration of N-acetyl-ß-hexosaminidase (HEX), ß-glucuronidase (GLUC), and ß-galactosidase (GAL) activity as well as in serum of cholesteatoma patients and healthy volunteers. STUDY DESIGN: Acquired cholesteatomas (n = 25) and normal retroauricular skin specimens (n = 25) were taken during surgery as well as serum from cholesteatoma patients and healthy volunteers. HEX, GAL, and GLUC activity was assessed on basis of p-nitrophenol release from derivatives of the substrate (HEX: N-acetylglucosamine i N-acetylgalactosamine, GAL from galactose, and GLUC from glucuronide). RESULTS: The mean concentration of activity of HEX 1142.39 pKat/ml, GAL 8.90 pKat/ml, and GLUC 14.06 pKat/ml was significantly higher compared with the concentration of enzyme activity in normal tissue: HEX 267.65 pKat/ml, GAL 3.44 pKat/ml, and GLUC 3.90 pKat/ml. In the serum of cholesteatoma patients, the mean concentration of enzyme activities were as follows: HEX 641.62 pKat/ml, GAL 4.55 pKat/ml, and GLUC 12.80 pKat/ml and were significantly higher compared with the concentration of HEX activity (215.75 pKat/ml), GAL (1.89 pKat/ml), and GLUC (5.51 pKat/ml) in the serum of the healthy control group. In cholesteatoma compared with the normal tissue, there is an increase of the glycoconjugate catabolism due to significantly higher concentration of HEX, GAL, and GLUC activity in cholesteatoma. Cholesteatoma causes systemic reaction due to the increase of HEX, GAL, and GLUC activity in patient serum.
Assuntos
Colesteatoma da Orelha Média/enzimologia , Glucuronidase/sangue , Lisossomos/enzimologia , beta-Galactosidase/sangue , beta-N-Acetil-Hexosaminidases/sangue , Adulto , Colesteatoma da Orelha Média/etiologia , Humanos , Pessoa de Meia-Idade , Pele/enzimologiaRESUMO
BACKGROUND: Dried blood spots (DBS) on filter paper is a valuable sampling technique in clinical chemistry, but the stability of enzymes used in the diagnosis of lysosomal storage diseases (LSDs) needs to be evaluated. METHODS: In a first experiment, blood from 20 subjects was collected using a syringe without additives and distributed into EDTA tubes, heparin tubes, and spotted on filter paper for the comparison of sampling effects. In a second experiment, blood from 30 healthy subjects was spotted on filter paper and analyzed for ß-galactosidase and total hexosaminidase activities after storage of the samples at different temperatures for up to 180 days. RESULTS: Initially, we observed that enzyme activities were the same, independent of the collection method. When DBS was stored at 37°C the activity of ß-galactosidase dropped to 85% of the initial value after 180 days (p<0.05). At all other temperatures (-20°C, 4°C and 25°C), the results were within the methodological error. Total hexosaminidase activity did not change significantly during the entire study period and at different storage temperatures. CONCLUSIONS: The two enzymes investigated in the present study may be stored for up to 17 days (ß-galactosidase) or 180 days (total hexosaminidase) until analysis without loss of activity.
Assuntos
Coleta de Amostras Sanguíneas/métodos , Temperatura , beta-Galactosidase/metabolismo , beta-N-Acetil-Hexosaminidases/metabolismo , Coleta de Amostras Sanguíneas/instrumentação , Humanos , Doenças por Armazenamento dos Lisossomos/sangue , Doenças por Armazenamento dos Lisossomos/enzimologia , Papel , Fatores de Tempo , beta-Galactosidase/sangue , beta-N-Acetil-Hexosaminidases/sangueRESUMO
Multiple epidemiological studies have shown that individuals affected by type-2 diabetes mellitus (T2DM) carry a 2-to-5-fold higher risk of developing Alzheimer's disease (AD) when compared to non-diabetic subjects. Thus, biochemical parameters that can be easily and routinely assessed for high-confidence evaluation of diabetic conditions leading to AD (AD-T2DM) are regarded as efficient tools aimed at early diagnosis and, in turn, timely AD treatment. In this regard, the activity of lysosomal glycohydrolases may of use, in light of the implication of these enzymes in early events that underlie AD pathology and an overt correlation, in diabetes, between altered metabolic homeostasis, abnormal glycohydrolase secretion in body fluids, and occurrence of diabetic complications. Based on marked up-regulation previously shown in a peripheral, cell-based model of AD, we selected ß-Galactosidase, ß-Hexosaminidase, and α-Mannosidase to discriminate T2DM from AD-T2DM subjects. A screen of 109, 114, and 116 patients with T2DM, AD and AD-T2DM, respectively, was performed by testing enzyme activities in both blood plasma and peripheral blood mononuclear cells. Compared to age-matched, healthy controls (n = 122), ß-Galactosidase and ß-Hexosaminidase activities markedly diverged across the three groups, whereas virtually unchanged values were observed for α-Mannosidase. In particular, plasma ß-Galactosidase and ß-Hexosaminidase levels were higher in patients with AD-T2DM compared to those with T2DM, suggesting different mechanisms leading to enzyme secretion. Statistical analyses based on ROC curves showed that both ß-Galactosidase and ß-Hexosaminidase activities, either intracellular or plasma-secreted, may be used to discriminate AD patients from controls and AD-T2DM from T2DM patients.