A liquid crystals-based sensing platform for detection of α-amylase coupled with destruction of host-guest interaction.

A liquid crystals (LCs)-based sensing platform was constructed for simple, convenient, inexpensive and label-free detection of α-amylase, associated with disruption of host-guest interaction between sodium dodecyl sulphate (SDS) and ß-cyclodextrin (ß-CD). In the presence of SDS/ß-CD solution, a bright optical image was observed corresponding to the tilted anchoring of LCs at the aqueous/LCs interface. While a black optical appearance was captured when the pre-incubated mixture containing SDS/ß-CD complex and ≥0.0001 mg/mL α-amylase was transferred onto the fluid interface. The reason for this difference is that α-amylase could hydrolyze ß-CD and subsequently destroy the host-guest interaction between SDS and ß-CD. SDS molecules escaping from the cavity of ß-CDs were adsorbed at the aqueous/LCs interface, which evoked the homeotropic state of LCs. In the case of α-amylase below 0.0001 mg/mL, a bright optical image was observed. Based on these, detection of α-amylase could be achieved and its detection limit was ∼0.0001 mg/mL (15 U/L). Moreover, this sensing platform was successfully utilized to monitor α-amylase in the body fluids, such as urine and saliva, indicating its potentiality in the relevant clinical diagnosis.

Progress of pancreatitis disease biomarker alpha amylase enzyme by new nano optical sensor.

A new nano optical sensor binuclear Pd-(2-aminothiazole) (urea), Pd(atz,ur) complex was prepared and characterized for the assessment of the activity of alpha amylase enzyme in urine and serum samples for early diagnosis of Pancreatitis disease. The assessment of alpha amylase activity is carried out by the quenching of the luminescence intensity of the nano optical sensor binuclear Pd(atz,ur) complex at 457nm by the 2-chloro-4-nitrophenol (2-CNP) which produced from the reaction of the enzyme with 2-chloro-4-nitrophenyl-α-d-maltotrioside (CNPG3) substrate. The remarkable quenching of the luminescence intensity at 457nm of nano Pd(atz,ur) doped in sol-gel matrix by various concentrations of the 2-CNP was successfully used as an optical sensor for the assessment of α-amylase activity. The calibration plot was achieved over the concentration range 8.5×10(-6) to 1.9×10(-9)molL(-1) 2-CNP with a correlation coefficient of (0.999) and a detection limit of (7.4×10(-10)molL(-1)). The method was used satisfactorily for the assessment of the α-amylase activity over activity range (3-321U/L) in different urine and serum samples of pancreatitis patients. The assessment of the alpha amylase biomarker by the proposed method increases its sensitivity (96.88%) and specificity (94.41%) for early diagnosis of pancreatitis diseases.

Role of Electromagnetic Field Exposure in Childhood Acute Lymphoblastic Leukemia and No Impact of Urinary Alpha- Amylase--a Case Control Study in Tehran, Iran.

Childhood acute lymphoblastic leukemia (ALL) is one of the most common hematologic malignancies which accounts for one fourth of all childhood cancer cases. Exposure to environmental factors around the time of conception or pregnancy can increase the risk of ALL in the offspring. This study aimed to evaluate the influence of prenatal and postnatal exposure to high voltage power lines on the incidence of childhood ALL. It also examines the role of various factors such as environmental factors and alpha-amylase as a marker in the development of leukemia. This cross-sectional case control study was carried out on 22 cases and 100 controls who born and lived in low socioeconomic families in Tehran and were hospitalized for therapeutic purposes in different hospitals of rom 2013-2014. With regard to the underlying risk factors; familial history and parental factors were detected as risk factors of ALL but in this age, socioeconomic and zonal matched case control study, prenatal and childhood exposure to high voltage power lines was considered as the most important environmental risk factor (p=0.006, OR=3.651, CI 95% 1.692-7.878). As the population study was from low socioeconomic state, use of mobiles, computers and microwaves was negligible. Moreover prenatal and postnatal exposure to all indoor electrically charged objects were not detected as significant environmental factors in the present study. This work defined the risk of environmental especially continuous pre and postnatal exposure to high voltage power lines and living in pollutant regions through the parents or children as well as the previously described risk factors of ALL for the first time in low socioeconomic status Iranian population.

A sensitive one-step method for quantitative detection of α-amylase in serum and urine using a personal glucose meter.

Assays of α-amylase (AMS) activity in serum and urine constitute the important indicator for the diagnosis of acute pancreatitis, mumps, renal disease and abdominal disorders. Since these diseases confer a heavy financial burden on the health care system, AMS detection in point-of-care is fundamental. Here, a one-step assay for direct determination of the AMS activity was developed using a portable personal glucose meter (PGM). In this assay, maltopentaose was used as a substrate for sensitive detection of AMS with the assistance of α-glucosidase. In the presence of AMS, maltopentaose can be readily hydrolyzed to form maltotriose and maltose quickly. With the enzymatic hydrolysis of α-glucosidase, maltotriose and maltose can be turned into glucose rapidly, which can be quantitatively measured using a portable PGM. This assay did not require any cumbersome and time consuming operations, such as surface modification, synthesis of invertase conjugate, washing and centrifugation. Detection of AMS can be achieved using only a one-step mixture, and the limit of detection was 20 U L(-1) which was lower than the clinical cutoff for AMS. More importantly, this sensitive and selective assay was also used for the detection of AMS in human serum/urine samples. The results showed that the recovery of AMS from human serum/urine samples was 91-107%. The rapid and easy-to-operate assay may have potential application in the fields of point-of-care (POC) clinical diagnosis, particularly in rural and remote areas where lab equipment may be limited.

Development of a novel, hemolysis-resistant reagent for assessment of α-amylase in biological fluids.

BACKGROUND: Although the assessment of α-amylase is an essential part of the diagnostic workout of several pancreatic and extra-pancreatic disorders, its enzymatic activity is significantly reduced in the presence of cell-free hemoglobin such as in samples with spurious hemolysis, due to chemical and spectrophotometric interference. We developed a new reagent that provides reliable results on hemolyzed biological specimens. METHODS: All tests were performed on Beckman Coulter AU5822. Intra-assay imprecision was assessed on three serum samples with low, intermediate and high α-amylase concentration. Linearity was assessed by serially diluting two samples with low and high values of α-amylase. The comparison with commercial reagent was performed on 40 serum samples. Hemolysis studies were carried out by mechanically hemolysis of 20 lithium-heparin samples. RESULTS: The intra-assay imprecision was comprised between 1.3% and 2.2%. The linearity was excellent (r=0.998), and highly significant agreement was observed with the commercial assay (r=1.00; mean bias -3.8%). Although a significant correlation between non-hemolyzed and hemolyzed specimens was found with both assays (p<0.001), a much greater agreement was observed with the experimental method (r=0.997 vs. 0.818). No measurement exceeded the total allowable error with the experimental assay, whereas the threshold was exceeded in 85% of samples using the commercial method. CONCLUSIONS: The clinical applications of the experimental reagent include α-amylase assessment in hemolyzed samples, in urine and other biological fluids contaminated with lysed erythrocytes, or in patients under frequent transfusions and hemoglobin-based blood substitutes therapy. The formulation of this reagent could be adapted for other clinical chemistry or immunochemistry assays.

Effect of clonidine in mice injected with Tityus discrepans scorpion venom.

A study was conducted to assess the effect of clonidine (α(2)-adrenoceptor selective agonist) on glycemia, serum and urine α-amylase, blood urea nitrogen (BUN), serum creatinine, white blood cell count, kidney histology and zymogen granule content in pancreatic acini, in mice under the effect of Tityus discrepans (Td) scorpion venom. BALB/c male mice (20 ± 2 g, n = 7-11) were intraperitoneally (ip) injected with a sublethal dose (1 µg/g) of Td venom, and were treated (ip) with 0.1 µg/g of clonidine (Catapresan(®)) or 0.9% NaCl 30 min after the venom injection, and then every 2 h. Six hours later, mice were anesthetized with diethylether and urine and blood samples were withdrawn by cystocentesis and cardiocentesis, respectively. Tissue samples were obtained and fixed immediately in buffered formalin (2%, pH 7.4) and then processed for stain H&E. Td venom did not cause hyperglycemia by itself. However, clonidine induced hyperglycemia, which was synergized by Td venom. Although the venom did not produce hyperamylasemia, clonidine significantly diminished serum α-amylase activity in envenomed mice. Td venom did not significantly increase urinary α-amylase activity, which was unaffected by clonidine. Morphometric analysis using microphotographs of pancreata from mice injected with Td venom showed a reduced zymogen granule content as judged by the acidophilic bidimensional area of acini. This effect was significantly reduced by clonidine. Kidney samples showed histological changes which were partially affected by the drug. Clonidine reduced the increase in BUN and serum creatinine concentration in envenomed mice. Td venom produced neutrophilia and lymphopenia, which were clonidine-resistant at the assayed dose. These results suggest that α(2)-adrenoceptor selective agonists would be able to reduce some scorpion venom-induced renal and pancreatic disturbances, possibly through the inhibition of neurotransmitter release from presynaptic cholinergic and noradrenergic terminals, as well as from adrenal medulla.

[Clinical manifestation of acute pancreatitis in children with caustic ingestion injury - the role of oxidative stress].

For a 10 years period (1996-2005) 66 children with severe caustic injuries of the esophagus and stomach were admitted at the Department of Pediatric Surgery. Subject of this article are 17 children with clinical, laboratory and intraoperative proven acute pancreatitis. The patients were admitted at the clinic 12 hours to 12 days after the ingestion of the corrosive agent. Fifteen of them underwent surgery and different surgical procedures were performed - gastric resection, transhiatal esophagectomy, gastrectomy, gastrostomy. In all patients were found elevated levels of alpha-amilase in blood serum and urine as well as elevated CRP in blood serum. Clinically manifested acute pancreatitis was diagnosed on ultrasound studies and laparotomy. The newest theories about the genesis of acute pancreatitis emphasize on the role of oxidative stress. Experimental models suggest that burn trauma (thermal or chemical) cause critical increase of free oxygen radicals and lipid peroxydation products in the tissue of the damaged organ and the bloodstream. The local tissue damage leads to release of inflammatory mediators which enter the bloodstream and cause distant organs damage of - lung, liver, kidneys and pancreas. In this preliminary report the authors discuss the pathogenesis of acute pancreatitis in children with acute corrosive ingestion injury of the esophagus and stomach. We call this phenomenon " caustic " oxidative stress. This is the first scientific report on this topic in the reviewed literature.

Diagnostic value of pancreatic elastase-1 in human acute pancreatitis.

The diagnosis of acute pancreatitis (AP) is usually confirmed by a significant increase of the serum amylase and/or lipase level. However, serum pancreatic elastase-1 (pEla-1) was found to be a more sensitive diagnostic marker in AP, when assayed by the radioimmunoassay procedure. We analyzed the serum concentration of pEla-1, measured by the ELISA technique in 46 patients with AP and in a control group of 12 healthy volunteers. On admission (day 1) we found significantly higher pEla-1 levels in patients with AP than in the controls. During the following days, the concentration of pEla-1 rapidly decreased to nearly undetectable values on the 3rd day. There was no significant difference between patients with mild and severe AP nor those of different etiology. We suggest that pEla-1 has little diagnostic value and does not provide additional information to that of the less expensive and more widely available serum amylase and lipase.

Tripsinógeno 2 urinario como marcador bioquímico de pancreatitis aguda / Urinary trypsinogen-2 as biochemical marker of acute pancreatitis

Se ha evaluado la detección de tripsinógeno 2 urinario mediante tiras reactivas frente a la determinación de la actividad catalítica de a-amilasa plasmática y urinaria como método diagnóstico, de pancreatitis aguda. Se estudiaron 34 pacientes consospecha clínica de pancreatitis aguda. A todos ellos se les determinaron las actividades catalíticas a-amilasa plasmática y urinaria mediante el reactivo a-Amylae EPS (Roche diagnostics) en el autoanalizador Synchron CX7 (Beckman) y el tripsinógeno 2 urinario mediante tiras Dipsticks`` (Medix Biochemica). Para confirmar el diagnóstico se revisaron las historias clínicas de los pacientes. La prueba más sensible fue la aamilasa plasmática y la más específica la a-amilasa urinaria aunque ninguna de ellas fue significativamente mejor que la detección de tripsinógeno 2 en orina mediante tiras reactivas (Dipsticks). Por ello la utilización de dichas tiras para el diagnóstico de pancreatitis aguda está plenamente justificado cuando no se pueda determinar la a-amilasa de forma urgente (AU)

Determination of alpha-amylase using 4-O-beta-D-galactopyranosylmaltotetraose (Gal-G4) as a substrate.

A new substrate, 4-O-beta-D-galactopyranosylmaltotetraose (Gal-G4) is applied for the determination of alpha-amylase in serum and urine in a coupled assay with alpha-glucosidase (EC 3.2.1.20), glucokinase (EC 2.7.1.2) and glucose-6-phosphate dehydrogenase (EC 1.1.1.49) as auxiliary enzymes. Gal-G4 having a 4-position of the non-reducing-end glucose residue modified by a beta-galactopyranose group is resistant for degradation by alpha-glucosidase as auxiliary enzyme. Moreover, this substrate is hydrolyzed at just one position by alpha-amylase in serum and urine. More than 99% of the products generated from Gal-G4 by alpha-amylase are identified 4-O-beta-D-galactopyranosylmaltose (Gal-G2), maltose, respectively. Glucose and maltose do not interfere the value of alpha-amylase activity at least up to 0.056 mmol/l (1 g/dl) glucose and 0.027 mmol/l (1 g/dl) maltose, respectively. We are now carrying out this work under the authority of The Enzyme committee of Japanese Society of Clinical Chemistry (JSCC) as a standard method for determination of alpha-amylase in clinical chemistry.

Determinants of a normal (versus impaired) oral glucose tolerance after combined pancreas-kidney transplantation in IDDM patients.

After successful pancreas transplantation, insulin-dependent diabetic patients are characterized by a normal or at worst impaired oral glucose tolerance (World Health Organisation criteria). It is not known which pathophysiological mechanisms cause the difference between normal and impaired oral glucose tolerance. Therefore, we studied 41 patients after successful combined pancreas-kidney transplantation using stimulation in the fasting state with oral glucose (75 g), intravenous glucose (0.33 g/kg) and glucagon bolus injection (1 mg i.v.). Glucose (glucose oxidase), insulin and C-peptide (immunoassay) were measured. Repeated-measures analysis of variance and multiple regression analysis were used to analyse the results which showed: 28 patients had a normal, and 13 patients had an impaired oral glucose tolerance. Impaired oral glucose tolerance was associated with a greatly reduced early phase insulin secretory response (insulin p < 0.0001; C-peptide p = 0.037). Age (p = 0.65), body mass index (p = 0.94), immunosuppressive therapy (cyclosporin A p = 0.84; predniso(lo)ne p = 0.91; azathioprine p = 0.60) and additional clinical parameters were not different. Reduced insulin secretory responses in patients with impaired oral glucose tolerance were also found with intravenous glucose or glucagon stimulations. Exocrine secretion (alpha-amylase in 24-h urine collections) also demonstrated reduced pancreatic function in these patients (-46%; p = 0.04). Multiple regression analysis showed a significant correlation of 120-min glucose with ischaemia time (p = 0.003) and the number of HLA-DR mismatches (p = 0.026), but not with HLA-AB-mismatches (p = 0.084). In conclusion, the pathophysiological basis of impaired oral glucose tolerance after pancreas transplantation is a reduced insulin secretory capacity. Transplant damage is most likely caused by perioperative influences (ischaemia) and by the extent of rejection damage related, for example, to DR-mis-matches.

alpha-Amylase and isoamylase levels in renal transplant recipients compared to uremic patients.

Hyperamylasemia is a common finding in chronic renal failure (CRF) patients. The present study was designed to evaluate the frequency, the type, and the hyperamylasemia levels in renal transplant recipients (RTR) compared to patients with renal failure with or without replacement of renal function. One hundred and forty-one subjects [42 with varying degree of renal insufficiency (group A), 74 on hemodialysis (group B), and 25 RTR (group C)] and 47 normal individuals were studied. Total serum alpha-amylase (Ta) as well as pancreatic (Pa) and salivary (Sa) types of serum isoamylases were elevated in all groups when compared to the levels found in normal subjects. A remarkable proportion of patients belonging to groups A and B had Ta as well as Pa levels over three times the upper normal limits. On the contrary, no RTR had such increased levels of both Ta and isoamylases. A statistically significant correlation was found between Ta, Pa, and Sa and serum creatinine in RTR. However, no statistically significant correlation was found between urine amylases and serum creatinine or between urine and serum levels in all amylases in this group. In conclusion, serum amylase levels are increased in RTR. However, no subject in this group had amylase and isoamylase values more than three times the upper normal limits, which was a common finding in the other groups of patients.

Urinary alpha-amylase and serum macroamylase activities in dogs with proteinuria.

Activities of urinary alpha-amylase and serum macroamylase; concentrations of serum creatinine, immunocomplexes, and urinary protein; and patterns of proteinuria were determined in 35 dogs with proteinuria. Urinary alpha-amylase activity ranged from 37 to 4,031 U/L. Macroamylasemia was detected in 77.14% of dogs and the percentage of alpha-amylase precipitated ranged from 4.68 to 61.63. Serum alpha-amylase activity after immunoglobulin precipitation ranged from 654 to 6,390 U/L in 51.42% of the dogs; the values were higher than the reference limits. Concentrations of serum creatinine and immunocomplexes were higher than reference limits for 25.71 and 60% of dogs, respectively. Urinary protein concentrations ranged from 0.1 to 8.9 g/L. All the patterns of proteinuria were represented. Linear regression indicated correlations between urinary alpha-amylase activities, serum creatinine concentrations (P < 0.01), and concentration of immunocomplexes (P < 0.05). Mann-Whitney test indicated significantly higher urinary alpha-amylase activity (P < 0.01) and percentage of alpha-amylase precipitated (P < 0.05) in dogs with renal insufficiency.

The activity of granulocyte alpha-amylase in acute appendicitis.

The activity of alpha-amylase was measured in isolated granulocytes, serum and urine of 35 patients with acute appendicitis. The measurements were performed before operation and on the 7th day after operation. Slightly increased activity of alpha-amylase was found in the serum and urine of 15 patients. On the 7th day after operation the activity of this enzyme reached normal value. The activity of granulocyte alpha-amylase was elevated in 22 patients. In 2 of them the increased activity still maintained on the 7th day after operation. Positive correlation between the serum and granulocyte alpha-amylase activities was found. These observations allow to conclude that granulocytes are the source of increased alpha-amylase activity in the serum of patients with acute appendicitis.