A novel enzymatic method for isolation of plastic particles from human blood.

Microplastic particles have been detected in the human body. This study aimed to develop a blood digestion method that preserves microplastics during analysis. Acidic and alkaline reagents, commonly used for isolating plastic particles from organic materials, were tested on human blood samples and microplastics. Nitric acid, hydrochloric acid, potassium hydroxide, and sodium hydroxide were examined over time. Additionally, a pepsin-pancreatin combination was utilized for blood digestion. Light microscopy assessed digestion efficiency and particle count changes, while Raman microspectroscopy distinguished between plastic and cell debris. The acidic reagents were ineffective in removing the organic material, while alkaline reagents were effective without significant effects on microplastics. Blood digestion using pepsin and pancreatin demonstrated efficient digestion without negative consequences for the particles. While potassium hydroxide digestion is already established, novel use of the pepsin-pancreatin combination was introduced to digest human blood, indicating its potential for isolating plastic particles from tissue and human food.

In vitro Comparison of Pancreatic Enzyme Preparations Available in the Indian Market.

PURPOSE: Pancreatic enzyme replacement therapy (PERT) involves exogenous enzyme supplementation and is used in the treatment of pancreatic exocrine insufficiency. Clinical efficacy of PERT preparations is a function of physical properties and release kinetics that vary between commercially available products. In this study, we evaluated the physical properties, in vitro dissolution, and release kinetics of commercially available pancreatic enzyme preparations available in the Indian market. METHODS: Physical properties such as particle size distribution and water content of the capsules were measured by dynamic light scattering and Karl-Fischer titration method, respectively. An analytical procedure based on the European pharmacopoeia (EP) method was used to determine lipase activity, and a modified United States pharmacopoeia (USP)-based method was used for dissolution studies. Enzyme release was ascertained under gastroduodenal conditions in buffered media. RESULTS: Considerable variations in physical properties such as particle size and water content were observed between pancreatic enzyme preparations. Some preparations failed to meet the labeled lipase content as per USP standards (>90% label claim) and showed inconsistent release behavior (>5% relative standard deviation). CONCLUSION: Differences exist between pancreatic enzyme preparations in terms of physical properties, dissolution, and release behavior that can affect their clinical efficacy. The present study suggests, therefore, that these preparations should not be used interchangeably.

In vitro digestion of salmon: Influence of processing and intestinal conditions on macronutrients digestibility.

Salmon is the main dietary source of omega-3 lipids and contains high-biological value protein. However, processing techniques could affect macronutrient digestibility. Also, altered intestinal conditions, particularly given in pancreatic insufficiency, could threaten digestibility. This study tested both hypotheses by subjecting raw, marinated and microwave-cooked salmon to static in vitro digestion under healthy (pH 7, bile concentration 10 mM) and altered (pH 6, bile 1 or 10 mM) intestinal conditions with different pancreatin concentrations. In the standard conditions, proteolysis was not affected by processing, but lipolysis decreased in marinated (46%) and raw salmon (57%) compared to the cooked matrix (67%). In altered conditions, proteolysis and lipolysis decreased to different extents depending on the treatment. Overall, processing affected proteolysis the most (f-ratio = 5.86), while intestinal conditions were the major determinants of lipolysis (f-ratio = 58.01). This study could set the ground to establish dietary recommendations of salmon for specific population groups.

Evaluation of a molecular method for hepatitis E virus (HEV) detection in pancreatin of porcine origin.

Pancreatin is a combination of enzymes, principally amylase, lipase, and protease, used in the treatment of pancreatic endocrine insufficiency in humans. Pancreatin manufactured from imported porcine pancreas carries the risk of hepatitis E virus (HEV) contamination. About 1 % of the starting material for pancreatin manufacture is invariably constituted of the small intestine, which is known to be a major extrahepatic site of HEV replication in pigs. The aim of this study was to evaluate a method to detect and quantify HEV in pancreatin of porcine origin. Because HEV cannot be easily grown by conventional cell culture, an approach based on an established quantitative RT-PCR (RT-qPCR) was selected. This entailed the use of a non-HEV internal control to monitor RNA extraction efficacy and the production of HEV synthetic RNA as a reference to account for the efficacy of reverse-transcription. The method was evaluated by experiments in which HEV (from naturally infected pigs) was spiked in both the starting material (i.e., porcine pancreas homogenate for industrial production) and in the pancreatin itself. A laboratory protocol matching the industrial production workflow was set up and RT-qPCR experiments were carried out to evaluate the method's ability to detect HEV in pancreatin made from HEV-contaminated porcine tissues. The results showed that the method may be employed in two different strategies: to test the porcine pancreas homogenate (quantitative performance) or directly on pancreatin (qualitative assay). While the risk of HEV contamination in pancreatin may be low, it cannot be completely ruled out. Testing for HEV based on the precautionary principle ought to be the guiding rule.

Chromatographic Study of Azintamide in Bulk Powder and in Pharmaceutical Formulation in the Presence of Its Degradation Form.

Two specific, sensitive, and precise stability-indicating chromatographic methods were developed, optimized, and validated for the determination of Azintamide (AZ) in the presence of its degradation product. The first method was TLC combined with the densitometric determination of the separated bands. Separation was achieved using silica gel 60 F254 TLC plates and chloroform-acetone-glacial acetic acid (7.5 + 2.1 + 0.4, v/v/v) as the developing system. Good correlations were obtained between the integrated peak area of the studied drug and its corresponding concentrations in the linearity range. The second method used HPLC with UV diode-array detection, in which the proposed method was applied for the quantitative determination of AZ in the presence of its acidic degradation product and the quantitative determination of the acid-induced degradation product of AZ (AZ Deg) using pentoxifylline as the internal standard. The proposed components were separated on a reversed-phase C18 analytical column using acetonitrile-water (50 + 50, v/v). The flow rate was maintained at 0.55 mL/min and the detection wavelength was 260 nm. Linear regressions were obtained in the range of 1-30 and 0.3-16 µg/mL for AZ and AZ Deg, respectively. Different parameters affecting the suggested methods were optimized for maximum separation of the cited components. The suggested methods were validated in compliance with the International Conference on Harmonization guidelines and successfully applied for the determination of AZ in its pure powder form and in its pharmaceutical formulation. Both methods were also statistically compared with the reported method with no significant difference in performance observed.

Degree of hydrolysis and peptide profile of whey proteins using pancreatin / Grau de hidrólise e perfil peptídico das proteínas do soro de leite utilizando a pancreatina

In the present study, we used pancreatin for hydrolyzing whey proteins and evaluated the degree of hydrolysis (DH) and peptide size distribution. The following methods were used for DH determination: formol titration, soluble protein content, ortho-phthalaldehyde (OPA), and freezing point. Peptide size distribution was conducted by size-exclusion high-performance liquid chromatography (HPLC). The DH varied from 3.26% to 36.41%, and the highest yield was obtained by the soluble protein content method (mean of 35.85%). Formol titration was considered the most suitable method for assessing the DH, because it showed a marked increase with reaction time (from 1h-17.20% to 2h-24.86%). Significant positive correlation of strong intensity was observed between the following methods: formol titration and OPA (r=0.9616; p=0.0090), formol titration and freezing point (r=0.8784; p=0.0493), and OPA and freezing point (r=0.9515; p=0.0127). The highest contents of di- and tri-peptides and free amino acids were 9.07% and 8.22%, respectively. A significant positive correlation of strong intensity was also observed between the degree of hydrolysis and the fraction of medium-sized peptides by the formol titration (r =?0.9274; p=0.0232) and OPA (r=?0.8977; p=0.0386) methods.

En este trabajo, se utilizó una pancreatina para hidrolizar las proteínas del suero de leche, evaluándose el grado de hidrólisis (GH) y la distribución de tamaño de los péptidos. Los métodos de valoración con formol, determinación de proteínas solubles, ortoftalaldehído (OPA) y disminución del punto de congelación se emplearon para evaluar el GH. La distribución del tamaño de los péptidos se realizó por cromatografía líquida de exclusión molecular. El GH varió de 3,26% a 36,41% y los mejores resultados se obtuvieron con el método de determinación de proteínas solubles (media de 35,85%). La valoración con formol se consideró el método más adecuado para evaluar el GH, en función de los significativos incrementos con el tiempo de reacción (de 17,20% a 24,86%). Se observó una correlación positiva y de fuerte intensidad entre los siguientes métodos: valoración con formol y OPA (r=0,9616, p=0,0090); valoración con formol y disminución del punto de congelación (r=0,8784, p=0,0493); OPA con la disminución del punto de congelación (r=0,9515, p=0,0127). Los contenidos más altos de di-tripéptidos y de aminoácidos libres fueron de 9,07% y 8,22%, respectivamente. Una correlación positiva y de fuerte intensidad se verificó entre la fracción de péptidos medios y el GH evaluado con los métodos de la valoración con formol (r=?0,9274, p=0,0232) y OPA (r=?0,8977, p=0,0386).

Neste trabalho, uma pancreatina foi utilizada para hidrolisar as proteínas do soro de leite, avaliando-se o grau de hidrólise (GH) e a distribuição de tamanho dos peptídeos. Os métodos de titulação com formol, determinação de pro-teínas solúveis, ortoftalaldeído (OPA) e depressão do ponto de congelamento foram empregados para avaliar o GH. A distribuição do tamanho de peptídeos foi realizada por cromatografia líquida de exclusão molecular. O GH variou de 3,26% a 36,41% e os melhores resultados foram obtidos com o método de determinação de pro¬teínas solúveis (média de 35,85%). A titulação com formol foi considerada como o método mais adequado para avaliar o GH, em função dos significativos aumentos com o tempo de reação (de 17,20% a 24,86%). Observou-se uma correlação positiva e de forte intensidade entre os métodos de titulação com formol e OPA (r=0,9616, p=0,0090); titulação com formol e depressão do ponto de congelamento (r=0,8784, p=0,0493); OPA e depressão do ponto de congelamento (r=0,9515, p=0,0127). Os maiores teores de di-tripeptídeos e de aminoácidos livres foram de 9,07% e 8,22%, respectivamente. Correlação positiva e de forte intensidade foi verificada entre a fração de peptídeos médios e o GH avaliado pelos métodos da titulação com formol (r=?0,9274, p=0,0232) e OPA (r=?0,8977, p=0,0386).

Novel agents for enzymatic and fungal hydrolysis of stevioside / Novos agentes para a hidrólise enzimática e fúngica do esteviosídeo

A comparative study on the potential of some biological agents to perform the hydrolysis of stevioside was carried out, aiming at establishing an alternative methodology to achieve the aglycon steviol or its rearranged derivative isosteviol, in high yields to be used in the preparation of novel bioactive compounds. Hydrolysis reactions were performed by using filamentous fungi (Aspergillus niger, Rhizopus stolonifer and Rhizopus arrhizus), a yeast (Saccharomyces cerevisiae) and enzymes (pancreatin and lipases PL250 and VFL 8000). Pancreatin showed the best hydrolytic activity, furnishing isosteviol at 93.9% of yield, at pH 4.0, using toluene as a co-solvent. Steviol was produced using both pancreatin at pH 7.0 (20.2% yield) and A. niger atpH 7 (20.8% yield).

Um estudo comparativo do potencial de alguns agentes biológicos capazes de hidrolisar o esteviosídeo foi realizado,objetivando-se estabelecer uma metodologia alternativa para a obtenção da aglicona esteviol ou seu produto de rearranjo, isoesteviol, em rendimentos elevados que permitam o uso destas agliconas para o preparo de novos compostos bioativos. As reações de hidrólise foram realizadas usando fungosfilamentosos (Aspergillus niger, Rhizopus stolonifer e Rhizopus arrhizus), uma levedura (Saccharomyces cerevisiae) e enzimas(pancreatina, lipase PL250 e lipase VFL 8000). A pancreatina mostrou a melhor atividade hidrolítica dentre os sistemastestados, fornecendo isoesteviol com rendimento de 93,9% em pH 4,0, usando tolueno como co-solvente. Esteviol foi produzido tanto usando pancreatina em pH 7,0 (20,2% derendimento) quanto usando A. niger em pH 7,0 (20,8% de rendimento).

[Placental site trophoblastic tumor: case report and review of literature]. / Placental site trophoblastic tumor--Ein Fallbericht und Literaturübersicht.

A 32-year-old para 3 patient is presented having suffered from a silent uterine rupture on the occasion of her second and third Caesarean sections. After the third Caesarean section, there was light uterine bleeding whilst the patient breastfed for five months. Amenorrhoea then ensued due to oral contraception. Regular clinical and ultrasound checks revealed negative serum beta-HCG values and retrovesicular resistance, which increased in size after 18 months and became symptomatic. Because the patient's family was complete, abdominal hysterectomy without adnexectomy was performed. Histology revealed a placental site trophoblastic tumour (PSTT). This is a rare tumour with malignant potential, whose prognosis depends on the stage of the primary tumour, the period of time between the last pregnancy and onset of disease, the patient's age, and the rate of mitosis, and whose progress cannot be assessed using the WHO Prognostic Index Score for Gestational Trophoblastic Disease. In therapeutic terms, hysterectomy is recommended. Chemosensitivity is low and, due to the infrequency of the tumours, the most suitable chemotherapy scheme is unknown. In the case of metastasising or recurrent PSTT, the EP/EMA regime has proved to be most effective.

[Autoimmune pancreatitis: new concepts of an often overlooked disease]. / Autoimmune Pankreatitis: Aktuelle Konzepte einer häufig übersehenen Krankheit.

Autoimmune pancreatitis is characterized by a lympho-plasmacytic infiltrate centred around the pancreatic ducts along with venulitis; it can produce a mass-like fibroinflammatory lesion and often simulates pancreatic malignancy or chronic pancreatitis of other types. This may lead to unnecessary surgical interventions. Patients, who are usually over 40 years of age, show 1) mild unspecific abdominal pain, 2) increased serum immunoglobulins (specifically IgG4) and autoantibodies, and 3) diffuse or focal enlargement of the pancreas with pancreatic strictures and sometimes jaundice due to biliary obstruction (detectable by US, CT, MRI, ERCP and/or endoscopic ultrasound (EUS)). The diagnosis can be strongly supported by EUS- or US-guided biopsies showing typical histological changes and specific indirect immunohistochemistry with the patient's serum or a steroid trial showing often a dramatic decrease of pathological findings within weeks.

Variability in the composition of physiologic duodenogastric reflux.

Duodenogastric reflux has long been associated with various diseases of the foregut. Even though bile is often used as a marker, duodenogastric reflux consists of other components such as pancreatic juice and duodenal secretions. The aim of this study was to investigate the occurrence of duodenogastric reflux, its components, and the variability of its composition in normal subjects. Twenty healthy volunteers (7 men and 13 women) whose median age was 24 years underwent combined 24-hour bilirubin and gastric pH monitoring and intraluminal gastric aspiration. All probes were placed at 5 cm below the lower border of the lower esophageal sphincter. Aspiration was performed hourly and at any time when bilirubin and/or pH monitoring showed signs of duodenogastric reflux. Elastase and amylase were measured in the aspirate. All volunteers had episodes of physiologic duodenogastric reflux. A total of 70 episodes of duodenogastric reflux were registered with a median of three episodes (range 1 to 8) per subject. Most bile reflux occurred separately from pancreatic enzyme reflux. Pancreatic enzyme aspirate was significantly more often associated with a rise in pH in comparison to bile reflux (P <0.01). Duodenogastric reflux is a physiologic event with varying composition. Both bile and pancreatic enzyme reflux frequently occur separately. These findings could explain the disagreement regarding assessment and interpretation of duodenogastric reflux in the past. Thus monitoring of duodenogastric reflux requires more than the detection of just one component.

Indicators of inflammation and cellular damage in chronic asymptomatic or oligosymptomatic alcoholics: correlation with alteration of bilirubin and hepatic and pancreatic enzymes

Indicadores bioquimicos e hematimetricos de inflamacao e lesao celular foram correlacionados com bilirrubina e enzimas hepaticas e pancreatica em 30 alcoolistas cronicos do sexo masculino internados em hospital psiquiatrico para desintoxicacao e tratamento do alcoolismo. A aspartato aminotransferase, alanino aminotransferase, gamaglumiltransferase, fosfatase alcalina e bilirrubina total estavam alteradas em 90(por cento), 63(por cento), 87(por cento), 23(por cento) e 23(por cento) dos casos, respectivamente. Entre os indicadores de inflamacao (desidrogenase latica, alterada em 16 dos casos; alfa-1 globulina, 24(por cento), alfa-2 globulina, 88(por cento); contagem de leucocitos, 28(por cento) nenhum estava correlacionado com as alteracoes da bilirrubina e enzimas hepaticas...

Lesäo hepatica na pancreatite aguda experimental: influencia da reduçäo da reserva enzimatica do pancreas / Evaluation of hepatic lesion of experimental acute pancreatitis: influence of pancreatic enzyme reduction

A pancreatite aguda (PA) produz alteracoes morfologicas e funcionais no figado. A administraçäo de doses baixas de ceruleina diminui o conteudo enzimatico do pancreas. O objetivo do presente estudo foi estudar o efeito da reduçäo do conteudo enzimatico do pancreas na funçäo mitocondrial hepatica. Ratos machos Wistar foram submetidos a PA atraves de injeçäo retrograda intraductal de taurocolato de sodio a 5 por cento com e sem infusäo de ceruleina (0,133 ug/Kg/h) durante tres horas : Grupo I (GI): sem reducao enzimatica do pancreas, com induçäo de PA, GRUPO II (GII): com reduçäo enzimática do pancreas, com induçäo de PA, GRUPO III (GIII): com reduçäo enzimatica do pancreas, sem induçäo de PA, GRUPO IV (GIV): sem reduçäo enzimatica do pancreas, sem induçäo de PA (Controle). Após duas horas da inducao da PA os animais foram sacrificados e os figados retirados para avaliaçäo da funçäo mitocondrial hepatica, determinada polarograficamente com eletrodo de Clark, medindo-se o consumo de oxigenio na ausência de ADP (S4-Basal) e na presença de ADP (S-3 Ativado), utilizando-se succinato de potassio como substrato. Os conteudos de tripsina, amilase e proteinas totais no liquido ascitico foram determinados. Após duas horas da induçäo da PA observamos aumento significativo no estado 4 da respiraçäo (41 por cento) e diminuiçäo do RCR e da relaçäo ADP/O nos animais do GI (PA sem ceruleina) quando comparados com os animais do GII (PA com ceruleina) (p<0,05). O conteudo de amilase (A) e tripsina (T) no liquido ascitico mostraram-se diminuidos nos animais do GII (A=80 +- 10 U/ml, T= 9,75 +- 1,25 U/ml), quando comparados com os animais do GI que nao receberam ceruleina ( A= 231 +- 24 U/ml, T = 40,32 +- 5,19 U/ml) (p<0,001). Infusäo somente de ceruleina (GIII) näo alterou a funçäo mitocondrial hepatica. Estes achados sugerem que a reduçäo do conteudo enzimatico do pancreas através de infusäo de ceruleina atenua a disfunçäo mitocondrial hepatica na PA experimental evidenciada por desacoplamento da oxigenaçäo fosforilativa

[Possible decontamination of pancreatin with gamma rays and chemical agents]. / Möglichkeiten der Dekontamination von Pankreatin mit gamma-Strahlen und chemischen Mitteln.

Certain pre-conditions given, the microbial contamination of pancreatin in powder form can be reduced below international standard values. However, each of the methods whether physical or chemical, causes a variable loss of activity depending on the methods applied and the concentration of the decontaminating agent used. Therefore during preparation of this complex enzymatic compound, highly aseptic conditions must be kept in all stages of the process to avoid an increase of germal contamination. For a final toxicological assessment of the decontaminated products, further tests are required.

[In vitro studies of pancreatic enzyme substitution]. / In-vitro-Untersuchungen zur Pankreasenzymsubstitution.

In-vitro activity of 14 commercial pancreatin preparations, commonly used in the Federal Republic of Germany, were tested. All had been declared by their manufacturers to contain more than 6000 FIP (Fédération International Pharmaceutique) units of lipase and to be acid resistant. The declared lipase and amylase amounts were found to be present in 11 of the 14 preparations. Three of the 14 preparations, said to be acid resistant were found not to be so in buffer with falling pH values between 4.0 and 2.5, so that there occurred an, at times marked, loss of enzyme activity. Most noticeable was the poor solubility of most preparations at pH 6.6. Only three of the 14 liberated their total enzyme content within 60 minutes, as they should for theoretical reasons, based on the relatively short duodeno-cecal transit time.

Temporal adjustments in sympathoadrenal activity in rats with obesity-producing hypothalamic knife cuts.

Sympathoadrenal activity was assessed in adult rats with obesity-producing hypothalamic knife cuts prior to and after the onset of gross obesity by measuring urinary excretion of norepinephrine and epinephrine and by determining rates of norepinephrine turnover in selected organs. Urinary excretion of norepinephrine, as an index of overall sympathetic nervous system activity, was approximately doubled throughout the 4-week study in knife-cut rats, as was intake of the high-fat diet. Three days after knife-cut surgery (before the onset of gross obesity) rates of norepinephrine turnover (ng X organ-1 X hr-1) were 23-33% lower in three of the four organs examined than in the corresponding organs of control rats; rates of norepinephrine turnover were depressed in pancreas, interscapular brown adipose tissue, and abdominal white adipose tissue and unchanged in hearts. Four weeks after surgery when gross obesity was evident, rates of norepinephrine turnover were accelerated in heart (+82%) and pancreas (+63%), but remained low in interscapular brown adipose tissue (-27%) and abdominal white adipose tissue (-28%). Adrenal medullary activity, assessed by urinary excretion of epinephrine, was suppressed within the 1st day after knife-cut surgery and remained suppressed for several weeks. Brown adipose tissue and white adipose tissue appear to be selectively excluded from the generalized activation of the sympathetic nervous system in adult hyperphagic rats with obesity-producing hypothalamic knife cuts. Activation of the sympathetic nervous system was associated with reciprocal suppression of adrenal medullary responses in knife-cut rats.

[Radiation sterilization of pancreatin preparation].

The enzymatic activities (in detail, amylase-, lipase- and protease-activity) of gamma-irradiated pancreatin powder preparation decrease to 50-95% at doses range of 50 kR-8 MR. The dose-inactivation relation-ships vary depending on every different production lot. In the case of higher moisture content preparation as irradiation sample, amylase activities are more stable, the other side, protease activities are less stable. On the variation of these activities by gamma-irradiation, there are no difference between room temperature and lower temperature (-70 degrees C) as irradiation condition. The D10 values of contaminating bacteria and fungus (contamination number: about 1 X 10(3) cells/g and 9 X 10(2) spores/g) of a pancreatin preparation are 74 C/kg (280 kR) and 60 C/kg(230 kR). Then, these values are corresponded to 645 C/kg (2.5 MR) and 540 C/kg (2.1 MR) as perfect sterilization.

Pancreatic spasmolytic polypeptide (PSP): III. Pharmacology of a new porcine pancreatic polypeptide with spasmolytic and gastric acid secretion inhibitory effects.

Pancreatic spasmolytic Polypeptide (PSP) is a new porcine pancreatic polypeptide, which inhibits gastrointestinal motility and gastric acid secretion in laboratory animals after parenteral as well as oral administration. (1) PSP inhibits the amplitude of electrically stimulated contractions of the isolated guinea pig ileum. PSP's inhibitory effect is antagonized by phentolamine, but not by yohimbine. (2) PSP inhibits the motility of isolated guinea pig intestinal segments after intraluminal dosing. (3) PSP reduces intestinal motility in rabbits in vivo after intravenous and intraluminal administration, and in mice in vivo after subcutaneous injection. (4) PSP delays absorption of protein hydrolysate when it is administered orally in capsules to pigs and to pancreatectomized dogs. (5) PSP inhibits pentagastrin induced gastric acid secretion in rats after oral administration and in cats after subcutaneous and oral administration. The mechanism of action of PSP has so far not been finally elucidated. It seems likely that PSP interferes with endogenous acetylcholine release. Furthermore it might act by release of somatostatin from somatostatin cells in the gastrointestinal tract. It may have a direct or an indirect stimulant effect on alpha 2-receptors.