Expresión disminuida de caspasa 3 asociada al polimorfismo del gen del antígeno-4 asociado a linfocito T-citotóxico (CTLA4) en pacientes chilenos con diabetes tipo 1 / Decreased caspase 3 expression and cytotoxic T lymphocyte antigen-4 polymorphism in Chilean patients with type 1 diabetes

Background: Several polymorphisms of the CTLA4 gene have been associated with autoimmune diseases. The activation of induced cell death is the major event and caspase 3 represents the main protein for the apoptotic machinery, especially in lymphocytes. Aim: To correlate CTLA4 polymorphisms with caspase 3 expression in peripheral blood mononuclear cells (PBMC) simulating in vitro the glucose effect. Material and Methods: CTLA4 polymorphisms were determined by restriction fragment length polymorphisms (RFLPs). PBMC from 21 patients with type 1 diabetes aged 8.5 ± 4.3 years and 21 healthy subjects aged 18.3 ± 1.8 years, were stimulated under normal (5 mM) and toxic (14 mM) glucose conditions to assess its effect on the expression and activity of caspase 3. Relative abundance of caspase 3 mRNA was measured by semi quantitative RT-PCR and its activity, by a colorimetric assay. Results: When stimulated with 14mM glucose, PBMC of G allele carriers with type 1 diabetes had significantly lower relative mRNA abundance of caspase 3 (median value = 0.12, range 0.01-0.70 AU) compared with non-carriers (median value = 0.81, range 0.06-1.09 AU). When the incubation was carried out with the lower glucose concentration, a similar profile of caspase 3 activity was observed in diabetic patients carrying G allele (median value = 0.57, range 0.13-1.20 AU) as compared with non-carriers (median value = 0.89, range 0.14-5.50 AU). No significant changes after stimulating with glucose, were observed in PBMCs of the control group. Conclusions: PBMC of recently diagnosed patients with T1D, carrying the G allele in + 49A/G polymorphisms of CTLA4, have a decreased expression and activity of caspase 3.

Butyrylcholinesterase and diabetes mellitus in the CHE2 C5- and CHE2 C5+ phenotypes / Butirilcolinesterase e diabetes melito nos fenótipos CHE2 C5- e CHE2 C5+

OBJECTIVE:To investigate the relationship between butyrylcholinesterase (BChE) activities (total and band specific) and diabetes mellitus. SUBJECTS AND METHODS: BChE activities (BChEA, AC 4/5, AC OF and RC5) were analyzed in 101 type 1 (DM1) and in 145 type 2 (DM2) diabetic patients, in relation to phenotype, weight and incidence of metabolic syndrome (MS) in these patients. The C4/5 and C5 complex were separated from other molecular forms (C OF) using an acid agar gel. RESULTS: The BChE activity (BChEA) and the absolute activities of C4/5 (AC4/5) and C OF (AC OF) showed a high positive correlation coefficient to weight in the CHE2 C5- group, while the relative activity of C5 complex (RC5) showed a negative correlation to weight. CONCLUSIONS: The present study suggests that the positive correlation of the BChE activities to diabetes mellitus and to insulin resistance may depend on the CHE2 locus variability. High values of BChE activities were associated with insulin resistance only in CHE2 C5- diabetic patients, while in CHE2 C5+ diabetic patients, the presence of C5 complex, especially in a relatively high proportion, leads to less fat storage and better protection against metabolic syndrome.

OBJETIVO: Investigar a associação entre as atividades (total e banda específica) da butirilcolinesterase (BChE) e diabetes melito. SUJEITOS E MÉTODOS: As atividades da BChE (BChEA, AC4/5, AC OF e RC5) foram analisadas em 101 pacientes diabéticos do tipo 1 (DM1) e 145 do tipo 2 (DM2) em relação aos fenótipos, ao peso e à incidência da síndrome metabólica. Os complexos C4/5 e C5 foram separados das outras formas moleculares (C OF), usando gel de ágar ácido. RESULTADOS: A atividade da BChE (BChEA) e as atividades absolutas de C4/5 (AC4/5) e de C OF (AC OF) mostraram altos coeficientes de correlações positivos com peso no grupo de CHE2 C5-, enquanto a atividade relativa do complexo C5 (RC5) mostrou correlação negativa com o peso. CONCLUSÕES: O presente estudo sugere que as correlações positivas das atividades da BChE com diabetes melito e com a resistência à insulina podem depender da variabilidade do loco CHE2. Altos valores nas atividades da BChE estão associados com a resistência à insulina somente nos pacientes diabéticos CHE2 C5-, enquanto nos pacientes diabéticos CHE2 C5+ a presença do complexo C5, especialmente em alta proporção relativa, leva a um menor estoque de gordura e à maior proteção contra a síndrome metabólica.

Inhibitory effect of gundelia extract on urinary alpha-amylase activity of type-I diabetes mellitus

alpha-amylase is an enzyme that degrades starch into maltose and glucose by hydrolyzing alpha-1,4-glucan bonds. It is known that the enzyme is found to be elevated in patients suffering from diabetes mellitus. A potent extracts of locally distributed wild plant, the Gundelia was found to inhibit elevated activity of alpha-amylase with dose responses. Samples of 50 diabetes mellitus type-I has been investigated for urinary alpha- amylase activity. An inhibitory dose of Gundelia extract were used for the inhibition of the elevated enzyme activity. The data obtained were compared with that of healthy controlled samples [30].The adopted protocol was a colorimetric determination[Anonymous,1980]. alpha-amylase activity was found to be elevated[281.70 +/- 10.03 IU/24hr] in patient's serum compared with that of controlled sample [43.38 +/- 3.33 IU/24hr]. The activity of the enzyme was found to be inhibited [160.11 +/- 250.4 IU/hr] in patients serum using dose response of 15mg/ml of Gundelia extract. The study were comprehensive to determined the physical parameters of the enzyme activity and a values of Vmax and Km were obtained. It was known that Gundelia is used in prevention and treatment of liver diseases. The plant has a role in the body as an antioxidant factor. It has a hypolipemic effect, therefore, a use of such plant extract could have a hypoglycemic activities on patients with DM-I

Prevalência do anti-TPO e anti-21-hidroxilase no paciente diabético tipo 1 / Prevalence of anti-thyroid peroxidase and anti-adrenal 21-hidroxylase in type 1 diabetes patients

Ainda não está definida a estratégia ideal para rastrear a doença de Addison em pacientes com diabetes melito tipo 1 (DMT1). Objetivo: O objetivo deste estudo foi determinar a prevalência do anticorpo anti-21-hidroxilase (AC anti-21OH) em pacientes DMT1 de etnia diversificada e investigar sua associação à disfunção adrenal e autoimunidade tireoidiana. Métodos: Quarenta indivíduos foram avaliados, submetidos à entrevista e à dosagem de AC antitireoperoxidase (anti-TPO), anti-21OH, TSH, T4 livre e cortisol. AC anti-21OH foi encontrado em 7,5% (n = 3)dos casos, sem disfunção adrenal associada. Resultados: Positividade para anti-21OH não ocorreu exclusivamente em pacientes com anti-TPO (+). Este foi detectado em 25% dos casos e associado a níveis de TSH mais elevados (p = 0,034) e à idade mais avançada (p = 0,009). Conclusões: Embora nossa frequência de anti-TPO (+) seja similar à da literatura, a presença de anti-21OH (+) foi superior. Entretanto, esses AC não foram associados à disfunção hormonal, o que parece não justificar o rastreamento universal da doença de Addison.

There is still no consensus about the best strategy to screen Addison’s disease (AD) in type 1 diabetes mellitus (T1DM) patients. Objective: The aim of this study was to determine the frequency of anti-21-hydroxilase (anti-21OH) in a multiethnic T1DM population and investigate if its presence is associated with any adrenal dysfunction or thyroid autoimmunity. Methods: Forty individuals underwent an interview and blood was drawn for anti- thyroperoxidase (anti-TPO), anti-21OH, TSH, free T4 and cortisol measurement. Results: Anti-21OH was found in 7.5% (n = 3), none with adrenal dysfunction. This antibody was not exclusively seen in patients with anti-TPO (+). Anti-TPO was positive in 25% and associated with higher TSH levels (p = 0.034) and older age (p = 0.009). Conclusions:Although the frequency of anti-TPO in this sample was similar to previous studies, a higher prevalence of anti-21-OH was found. However, no coexisting adrenal dysfunction was detected, which does not support universal screening for AD in this group.

Atividade da enzima acetil-hidrolase do fator ativador de plaquetas (PAF-AH) em pacientes com diabete melito tipo 1 / Platelet-activating factor acetylhydrolase (PAF-AH) activity in patients with type 1 diabetes mellitus

OBJETIVO: Avaliar a atividade da acetil-hidrolase do fator ativador de plaquetas (PAF-AH) e sua relação com variáveis clinicodemográficas, com o controle metabólico, os níveis de apolipoproteínas A e B e a suscetibilidade da lipoproteína de baixa densidade (LDL) à oxidação in vitro em pacientes com DM tipo 1 (DM 1). MÉTODOS: Foram avaliados 42 pacientes com DM1 (27 mulheres) e 48 não-diabéticos (16 mulheres), pareados por sexo, idade e índice de massa corporal (IMC). Os exames realizados foram: glicemia de jejum (GJ) e pós-prandial (GPP), lipidograma, ácido úrico (AU), hemoglobina glicosilada (HbA1c) e coeficiente de oxidação da lipoproteína de baixa densidade (LDL) por espectrofotometria. A análise da atividade da PAF-AH foi realizada por espectrofotometria (Cayman Chemical). RESULTADOS: A análise da atividade da PAF-AH mostrou haver maior atividade enzimática nos pacientes com DM 1 do que nos não-diabéticos (0,0150 ± 0,0051 versus 0,0116 ± 0,0041; p < 0,001). Nos pacientes com DM 1, encontramos correlação direta entre a atividade da PAF-AH e a idade e a LDL, e inversa entre a PAF-AH e a HbA1c e a lipoproteína de alta densidade (HDL). CONCLUSÃO: A PAF-AH, na amostra estudada, apresentou maior atividade nos pacientes com DM 1, fator que pode estar relacionado ao maior risco de desenvolver doenças cardiovasculares apresentados por portadores dessa doença. Ainda são necessários novos estudos para avaliar a real participação dessa enzima no risco de desenvolvimento das doenças ateroscleróticas nos pacientes com DM 1.

OBJECTIVE: To evaluate platelet-activating factor acetylhydrolase (PAF-AH) activity and its relationship with clinical and demographic variables, metabolic control, apolipoprotein A and B levels and the susceptibility of low-density lipoprotein (LDL) to in vitro oxidation in patients with type 1 diabetes mellitus (DM 1). METHODS: Forty two patients with DM 1 (27 females) and 48 control subjects (16 females) matched for gender, age and body mass index (BMI) were evaluated. The following tests were performed: fast plasma glucose (FG) and postprandial plasma glucose (PPG), lipid profile, uric acid (UA), glycosylated hemoglobin (HbA1c), and low-density lipoprotein (LDL) oxidation rate using colorimetric assay. The PAF-AH activity was analyzed using colorimetric assay (Cayman Chemical). RESULTS: The analysis of PAF-AH activity showed a higher enzyme activity in patients with DM 1 than in control subjects (0.0150 ± 0.0051 vs. 0.0116 ± 0.0041; p < 0.001). In patients with DM 1, a direct correlation between PAF-AH activity and age and LDL, and an inverse correlation between PAF-AH and HbA1c and high-density lipoprotein (HDL) were found. CONCLUSION: In the sample studied, PAF-AH showed a higher activity in patients with DM 1, a factor that may be related to the higher risk of developing cardiovascular diseases observed in these patients. Further studies are necessary to evaluate the real participation of this enzyme in the risk of development of atherosclerotic diseases in patients with DM 1.

Función pancreática exocrina en diabetes mellitus: Determinación de elastasa fecal / Pancreatic exocrine function in diabetes mellitus: Determination of fecal elastase

Background: One of the complications of diabetes mellitus is the development of pancreatic exocrine insufficiency. Aim: To study pancreatic exocrine function in diabetics patients. Material and methods: Seventy two diabetic patients were included in the protocol, but two were withdrawn because an abdominal CAT scan showed a chronic calcified pancreatitis, previously undiagnosed. Fecal elastase was measured by ELISA and the presence of fat in feces was assessed using the steatocrit. Results: Mean age was 60±12 years and 67 (96%) patients had a type 2 diabetes. Fecal elastase was normal (elastase >200 µg/g) in 47 (67%) patients, mildly decreased (100-200 µg/g) in 10 (14%) and severely decreased in 13 (19%). There was a significant association between elastase levels and time of evolution of diabetes (p=0.049) and between lower elastase levels and the presence of a positive steatocrit (p=0.042). No significant association was found between elastase levels and other chronic complications of diabetes such as retinopathy, nephropathy, neuropathy, microangiopathy or with insulin requirement. Conclusions: One third of this group of diabetic patients had decreased levels of fecal elastase, that was associated with the time of evolution of diabetes. Patients with lower levels of elastase have significantly more steatorrhea. Among diabetics it is possible to find a group of patients with non diagnosed chronic pancreatitis.

Urinary N-acetyl-beta-D-glucosaminidase activity in type I diabetes mellitus.

We measured urinary albumin excretion rate (AER) and N-acetyl-beta-D-glucosaminidase (NAG) activity in relation to disease duration, acetylated hemoglobin (HbA1c), hypertension and puberty in 44 children and adolescents with type 1 diabetes mellitus. AER and Urinary NAG activity were significantly higher in the patients compared to controls (AER 19.4 +/-; 35.8 vs 4.7 +/- 4.4, NAG activity 5.6 +/- 0.6U vs. 1.6 +/- 0.2U). Microalbuminuria was present in seven patients (15.9%), all of whom were pubertal. There was no correlation between AER and urinary NAG activity. There was a significant direct correlation between AER and disease duration (P <0.05), HbA1c (P < 0.05), diastolic blood pressure (P <0.05) and puberty (P <0.05). None of the microalbuminuria related variables was significantly correlated with urinary NAG activity. Puberty was an independent factor for elevated urinary NAG activity. This study shows that urinary NAG is elevated in children and adolescents with type 1 diabetes mellitus, but is not associated with AER related factors except for puberty. Urinary NAG activity does not appear to be a useful marker for early detection of diabetic nephropathy in children and adolescents with type 1 diabetes mellitus.

Lipoproteína de baja densidad rica en triglicéridos y actividad de lipasa hepática en pacientes diabéticos insulino-dependientes / Low density lipoprotein rich in triglycerides and hepatic lipose activity in insulin-dependent diabetic patients

Genetic hepatic lipase (HL) deficiency is associated with low density lipoprotein (LDL) rich in triglycerides (TG), whose affinity for B:E receptors is decreased. In rats, experimental hypoinsulinemia produces HL deficiency. However, the relation between human insulin-dependent Diabetes Mellitus (IDDM), HL activity and the characteristics of LDL have not been studied. The objective of our study is to evaluate the relation between HL activity and the chemical composition of LDL in treated IDDM patients. Subjects were 15 IDDM patients and 15 controls (C), matched for sex and body mass index (BMI). The IDDM patients were classified by the WHO criteria, were free of nephropathy and hypothyroidism, and received no medication except insulin. Controls were clinically healthy and normolipidemic with no family history of diabetes. The IDDM group was divided into two subgroups: subgroup IDDM-A (n = 9) with HL values > 4.3 and IDDM-B (n = 6) with HL < than 4.2 mu-moles glycerol/ml h. The HL in IDDM was lower than in C (p < 0.001). Table 1 shows clinical data. Blood samples were drawn after 12 h fasting. Percentage of HbAlc and plasma concentrations of glucose, total cholesterol, LDL-cholesterol, HDL-cholesterol and TG were assayed. LDL was separated by sequential ultracentrifugation at densities of 1.019-1.063 g/ml and its chemical composition was analyzed. The most relevant results were: plasma TG concentration was higher in IDDM than in C (p < 0.05) (Table 2), although average values DMID not exceed the reference values of 200 mg/dl. The TG-LDL were higher in IDDM than in C: 24.8 +/- 2.7 vs 17.5 +/- 1.1 mg/dl plasma, media +/- SE, (p < 0.02). This difference reflected the values of IDDM-B, whose plasma concentrations of TG-LDL were higher than in C: 32.3 +/- 3.6 vs 17.5 +/- 1.1 mg/dl (p < 0.001), and also higher than in IDDMA (p < 0.02). (Table 3). The chemical composition of LDL in IDDM-B contained a higher percentage of TG than C: 8.5 +/- 0.7 vs 6.8 +/- 0.3 percent (p < 0.05), a lower percentage of cholesterol than IDDM-A: 39.0 +/- 1.7 vs 45.2 +/- 2.2 percent (p < 0.05) and also a larger percentage of proteins than IDDM-A: 28.9 +/- 1.9 vs 20.8 +/- 1.0 percent (p < 0.01). The correlations between TG/cholesterol and HL activity in IDDM were r = -0.53 (p < 0.05) and in IDDM-B, r = -0.81 (p = 0.05). The noteworthy result of this study is the modification of the LDL particle in IDDM, rich in TG in patients with low HL activity. Anomalies in the chemical composition of LDL like those described decrease the uptake of this particle by its physiological B:E receptors. It has recently been demonstrated that LDL is an indissoluble association of lipids and apoproteins, and that both act simultaneously to hold the apoB in a spatial position that expresses normal epitopes. It has been described that particles of LDL rich in TG and poor in cholesterol, shows low affinity for LDL receptors in human fibroblasts. Also in IDDM the interaction of LDL rich in TG with B:E receptors is decreased. This might be one more mechanism contributing to the accelerated atherosclerosis of these patients. Our results suggest that there may be a threshold of HL activity for the complete hydrolysis of the TG of LDL, for the normalization of the TG/cholesterol relation and for the conformation of typical LDL particles.