Pancreatic Extracts Research Articles

Diabetes mellitus and pernicious anemia: interrelated therapeutic triumphs discovered shortly after William Osler’s death

William Osler died on December 29, 1919, at the age of 70. Less than 1 year later, Frederick Grant Banting began a research project at the University of Toronto to find a treatment for diabetes mellitus. John James Rickard Macleod, director of physiology, gave him space, funding, and supplies. Charles Herbert Best, an undergraduate medical student, joined Banting. In 1921, Banting and Best isolated and purified insulin from pancreatic extracts of dogs. James Bertram Collip, a biochemist, helped in the purification process. The first American patient was treated with Toronto insulin in May 1922. Banting and Macleod were awarded the Nobel Prize in 1923 “for the discovery of insulin.” George Richards Minot, a young hematologist in Boston, had an obsessive interest in the effect of diet on anemia. In October 1921, Minot developed weight loss and was diagnosed with severe diabetes mellitus. By January 1923, the pioneering diabetologist, Elliott Proctor Joslin, began to treat Minot with insulin. Minot’s condition improved and he returned to work. In 1926, Minot and William Parry Murphy amazed the medical world when they eradicated anemia in 45 pernicious anemia patients by feeding them a half-pound of beef liver daily. Minot shared the 1934 Nobel Prize with Murphy and George Hoyt Whipple “for their discoveries concerning liver therapy in cases of anemia.” Minot remained on insulin the rest of his life. Osler described the clinical findings and blood picture of pernicious anemia nearly a half century before Minot but his observations were largely ignored. Osler had an intriguing connection to Banting. Had he lived, Osler would have been ecstatic over these two monumental therapeutic breakthroughs.

The Applicability of a Checklist for the Diagnosis and Treatment of Exocrine Pancreatic Insufficiency: Results of the Italian Exocrine Pancreatic Insufficiency Registry.

To evaluate a rapid checklist capable of identifying exocrine pancreatic insufficiency in outpatients. Prospective observational study of a multicenter cohort. One hundred and two patients were enrolled; 61.8% of the patients had medically-treated benign or malignant pancreatic disease, and 38.2% had a pancreatic resection. Visual examination of the feces was evaluated in 84 patients and it was related to steatorrhea in 51 patients (50.0%). Receiver operating characteristic curves were evaluated for each symptom or clinical sign and four of them (ie, increase in daily bowel movements, number of bowel movements, fatty stools, >10% weight loss) had a satisfactory area under the curve. At multivariate analysis, fatty stools and >10% weight loss entered into this analysis having an area under the curve of 0.916 (95% confidence interval, 0.851-0.981). At 1 month and at one year of follow-up, the pancreatic enzyme replacement therapy administered showed that pancreatic extracts were able to significantly improve the increase in daily bowel movements, the number of bowel movements, fatty and bulky stools and >10% weight loss. Both fatty stools and >10% weight loss were able to clinically evaluate steatorrhea, and their improvement was sufficient to evaluate substitution therapy.

Effect of thyroid hormones on rat exocrine pancreas morphology and function

AimThe influence of thyroid hormones on exocrine pancreas function is poorly understood, and limited to the postnatal development period. Here, we evaluated the effects of hypo- and hyperthyroidism on the morphology and enzyme content of this tissue. Main methodsTo induce hypothyroidism male Wistar rats were subjected to a thyroidectomy (Tx) or sham operated (SO). After 40 days, some of the Tx and SO rats were treated with T3 for 7 days. Following euthanization, the pancreas was removed and evaluated for morphology, as well as amylase, lipase and trypsin content, using histological and immunoreactive techniques analyses, respectively. Serum amylase levels were also evaluated. Key findingsThe pancreatic acinar cells of Tx rats were smaller, exhibited reduced Haematoxyllin stained areas, and contained lower amylase and lipase levels, indicative of low cell activity. Tx rats also presented higher collagen levels, and high trypsin content in pancreatic extracts. Interestingly, T3 administration reversed the observed acinar cell alterations and restored pancreatic enzyme content, by augmenting amylase and lipase and attenuating trypsin levels, but failed to change collagen content. Increased levels of lipase and decreased trypsin were also observed in T3-treated SO rats. SignificanceThyroid hormones play an important role in acinar cell morphology and function. In the hypothyroid state there is a decrease in pancreatic enzyme levels that is restored with T3 treatment. In addition to participating in insulin sensitivity and glycemic control, THs also modulate enzyme expression and activity in the exocrine pancreas, consequently, delivering metabolic substrates to specific organs and tissues.

Pancreatic Extracts for the Treatment of Diabetes (1889-1914): Acomatol.

Historical review on the early development of organotherapy for diabetes [pancreatic extracts (PE)] and its relationship with the social and political circumstances. The diagnosis of diabetes relied only in the presence of glycosuria and cardinal symptoms. Blood glucose determinations were not regularly available, requiring large volumes for sampling. Micromethods for glycemia were developed just in the last years of the investigated period. Hypoglycemia remains undiscovered. Isolation and purification of PE were difficult tasks due to the unknown chemical structure of the antidiabetic hormone. (1) Berliner Medizinhistoriches Museum der Charité (Humboldt University). (2) GeDenKort Charité-Wissenschaft in Verantwortung. (3) Geheim Staatsarchiv Preuβischer Kulturbesitz. (4) Archival Collections, University of Toronto: Thomas Fisher Rare Book Library. Academy of Medicine Collection, F. G. Banting Papers, C. H. Best Papers, J. J. R. Macleod Papers. (5) National Library of Medicine: Pubmed search for the topic of history of insulin. History of Medicine-on syllabus archive. (6) Selected books: The Discovery of Insulin (M. Bliss); Diabetes, Its Medical and Cultural History (D. von Engelhardt); Brown-Séquard (M. J. Aminoff); Diabetes: The Biography (R. Tattersall); The Endocrine Organs (E. Schäfer); The Internal Secretions (E. Gley); Health, race and German politics between national unification and Nazism, 1870-1945 (P. Weindling). Demonstration that diabetes is a pancreatic disease. The outstanding progress of medical physiology led to the birth of endocrinology and the key concepts of homeostasis. Experimental scientists designed new procedures for complete pancreatectomy and elaboration of PE containing the antidiabetic principle. Organotherapy achieved complete success in the treatment of myxedema and partial success in the treatment of experimental and clinical diabetes. The organotherapy of diabetes was an obliged step to facilitate the identification of the antidiabetic hormone. Organotherapy of diabetes was a paradigm for the integration of basic and applied knowledge about hormone action and development of endocrine pharmacology.

Biosimilars: A novel perspective in diabetes therapy

Diabetes mellitus, an endocrine disorder, has a wider reach among most of the world population. The incidence of diabetes is high not only among adults but wider-age groups are also becoming susceptible to this disease because of modified food habits and lifestyle changes that are alien to the physiological system. The control of blood glucose level would be the prime focus of all the therapeutic targets, which is achieved through drugs, modified lifestyle, and paleo-based diets. To find a solution to these problems, earlier humans have revolutionized the science with the discovery of insulin from the porcine pancreatic crude extract. Later, developments have been made with artificial recombinant insulin and even insulin analogs that would mimic the physiological basal insulin in controlling the blood sugar levels. Various factors such as cost and logistics for quality delivery to the end-user at various corners of the world have impeded the reach of the original product. Hence, biosimilar insulins that are original insulin analogs were designed to execute similar physiological functions. In the current situation, the use of biosimilars has been approved in various clinical conditions that are very promising in its functions. In the present review, the various developmental phases of biosimilar preparations and the regulations enforced ensuring a quality product in the market through the Food and Drug Administration and the European Medicines Agency have been discussed.

Experiences of First Insulin-Treated Patients (1922-1923).

Historical description of first insulin trials just after its discovery. The review includes first initiatives of insulin treatment. The probability of other trials, not reported to the Insulin Committee of the University of Toronto and conducted in the years 1922 and 1923, is quite low. (1) Archival Collections, University of Toronto: Insulin Discovery and Early Developments of Insulin (University of Toronto Libraries digital special collection, with a particular section entitled "From a Patient's Point of View" containing letters, patient charts, newspaper clippings, and photographs). (2) Thomas Fisher Rare Book Library: Academy of Medicine Collection, F. G. Banting Papers, C. H. Best Papers, J. B.Collip Papers, W. R. Feasby Papers, E. Hugues Papers, J. J. R. Macleod Papers. (3) National Library of Medicine: PubMed search for the topic of history of insulin, History of Medicine-on syllabus archive. (4) Selected Journals for History of Medicine: Bulletin of the History of Medicine, Journal of the History of Medicine and Allied Sciences, Medical History. (5) Selected books: The Discovery of Insulin (M. Bliss); Diabetes, Its Medical and Cultural History (D. von Engelhardt); H. C. Hagedorn and Danish Insulin (T.Deckert), Continuing Quest (W. A. Tomkins). This historical review shows the quick progress from impure pancreatic extract to the selective isoelectric precipitation of the hormone, which made possible the introduction of insulin in the clinic. The coordination between the Departments of Physiology (Connaught Laboratories) and Medicine (Toronto General Hospital) was essential for the discovery and implementation of insulin therapy. The Insulin Committee was decisive for the negotiation with the pharmaceutical industry, the purification, grand-scale production, patents' achievement, and provision of licenses to expert clinicians and prestigious health centers. At the end of the year 1923, insulin treatment was already extended to Europe (mainly Scandinavia, Great Britain, and Spain). Insulin discovery and treatment changed the clinical spectrum of diabetes.

Dogs and November: What Do They Have in Common?

November has dual importance in the world of skin and wound care: it is a time to raise awareness about diabetes mellitus (DM; November is National Diabetes Month and November 14 is World Diabetes Day) and pressure injuries (PIs; Thursday, November 21 is World Wide PI Prevention Day). Dogs were used for key research discoveries in both. Early work on the discovery of insulin started with a German scientist, Dr Paul Langerhans.1 He identified two types of pancreatic cells, including the Langerhans islet cells. Six years later, two other German investigators, Oskar Minkowski (a physiologist and pathologist) and Joseph von Mering (a physician) removed a dog’s pancreas, resulting in an elevated blood glucose and metabolic changes similar to the physiologic changes in a person with diabetes. In 1916, a Romanian physician from Bucharest, Nicolae Constantin Paulescu, isolated and injected aqueous pancreatic extract into a diabetic dog with a normalizing effect on blood sugar levels. Most scientists and clinicians are familiar with the work of Sir Frederick G. Banting (a Canadian orthopedic surgeon), Charles H. Best (his chemistry skills assistant), James B. Collip (who worked on the pancreas extract to purify it), and John James Richard Macleod (expert in carbohydrate metabolism), who completed further dog experiments between 1921 and 1923 that completed the knowledge translation cycle. They injected insulin into a young boy dying with type 1 DM at the Toronto General Hospital and saved his life! The rest is history; in 1923, Banting and Macleod received the Nobel Prize for the discovery of insulin.1 November 14, Dr Banting’s birthday, is World Diabetes Day. Can you do your part to identify persons with a high-risk foot and prevent foot ulcers and lower limb amputation? In this issue, a validated General Foot Screen is presented to help clinicians in their battle against DM. This screening tool can help detect persons with a high-risk foot who may not know they have diabetes. This is especially important because only 11.6% of adults with prediabetes know they are at risk; approximately 25% of persons with prediabetes will develop type 2 DM in 3 to 5 years, with up to 70% of persons with prediabetes developing DM during their lifetime.2 In contrast with DM, PIs have been observed clinically over several centuries. The classic work by Dr Michael Kosiak3 on dog thighs (1959) is often credited as breakthrough research into the etiology of pressure as the cause of PI. Each year, on the third Thursday of November, the global community pauses to raise awareness of PI. The National Pressure Ulcer Advisory Panel has many resources, including media materials, posters, buttons, drafts of proclamations, and clinical imagery that you can use to formulate a plan for prevention, educate the public, and celebrate this event at your insitution.4 Another key November PI event will be the launch of the third edition of the PI Clinical Guideline.5 The guidelines are the result of extensive collaboration of many healthcare professionals from numerous associations and stakeholders around the world who have synthesized current evidence into one document. If you cannot be in California for this landmark conference, be sure to go to the National Pressure Ulcer Advisory Panel website and download the guideline information once it is released. Fortunately, the use of dogs in scientific research has decreased since the start of the century, commensurate with the increase in humane laws implemented around the world to protect man’s best friend.6 That said, we wish to congratulate the human researchers, clinicians, educators, and funders who continue to work together to provide the evidence and knowledge that results in improved patient outcomes.Elizabeth A. Ayello, PhD, MS, BSN, RN, CWON, ETN, MAPWCA, FAANR. Gary Sibbald, MD, DSc (Hons), MEd, BSc, FRCPC (Med Derm), FAAD, MAPWCA, JM

Targeting nNOS ameliorates the severe neuropathic pain due to chronic pancreatitis

BackgroundPain due to pancreatic cancer/PCa or chronic pancreatitis/CP, is notoriously resistant to the strongest pain medications. Here, we aimed at deciphering the specific molecular mediators of pain at surgical-stage pancreatic disease and to discover novel translational targets. MethodsWe performed a systematic, quantitative analysis of the neurotransmitter/neuroenzmye profile within intrapancreatic nerves of CP and PCa patients. Ex vivo neuronal cultures treated with human pancreatic extracts, conditional genetically engineered knockout mouse models of PCa and CP, and the cerulein-induced CP model were employed to explore the therapeutic potential of the identified targets. FindingsWe identified a unique enrichment of neuronal nitric-oxide-synthase (nNOS) in the pancreatic nerves of CP patients with increasing pain severity. Employment of ex vivo neuronal cultures treated with pancreatic tissue extracts of CP patients, and brain-derived-neurotrophic-factor-deficient (BDNF+/−) mice revealed neuronal enrichment of nNOS to be a consequence of BDNF loss in the progressively destroyed pancreatic tissue. Mechanistically, nNOS upregulation in sensory neurons was induced by tryptase secreted from perineural mast cells. In a head-to-head comparison of several genetically induced, painless mouse models of PCa (KPC, KC mice) or CP (Ptf1a-Cre;Atg5fl/fl) against the hypersecretion/cerulein-induced, painful CP mouse model, we show that a similar nNOS enrichment is present in the painful cerulein-CP model, but absent in painless genetic models. Consequently, mice afflicted with painful cerulein-induced CP could be significantly relieved upon treatment with the specific nNOS inhibitor NPLA. InterpretationWe propose nNOS inhibition as a novel strategy to treat the unbearable pain in CP. FundDeutsche Forschungsgemeinschaft/DFG (DE2428/3-1 and 3-2).

Characterization of all the lipolytic activities in pancreatin and comparison with porcine and human pancreatic juices

Porcine pancreatic extracts (PPE), also named pancreatin, are commonly used as a global source of pancreatic enzymes for enzyme replacement therapy in patients with exocrine pancreatic insufficiency. They are considered as a good substitute of human pancreatic enzymes and they have become a material of choice for in vitro models of digestion. Nevertheless, while the global PPE contents in lipase, protease and amylase activities are well characterized, little is known about individual enzymes. Here we characterized the lipase, phospholipase, cholesterol esterase and galactolipase activities of PPE and compared them with those of porcine (PPJ) and human (HPJ) pancreatic juices. The phospholipase to lipase activity ratio was similar in PPJ and HPJ, but was 4-fold lower in PPE. The galactolipase and cholesterol esterase activities were found at lower levels in PPJ compared to HPJ, and they were further reduced in PPE. The enzymes known to display these activities in HPJ, pancreatic lipase-related protein 2 (PLRP2) and carboxylester hydrolase/bile salt-stimulated lipase (CEH/BSSL), were identified in PPJ using gel filtration experiments, SDS-PAGE and LC-MS/MS analysis. The galactolipase and cholesterol esterase activities of PPE indicated that PLRP2 and CEH/BSSL are still present at low levels in this enzyme preparation, but they were not detected by mass spectrometry. Besides differences between porcine and human enzymes, the lower levels of phospholipase, galactolipase and cholesterol esterase activities in PPE are probably due to some proteolysis occurring during the production process. In conclusion, PPE do not provide a full substitution of the lipolytic enzymes present in HPJ.

Protective effects of urocortin 2 against caerulein-induced acute pancreatitis.

Because little is known about the role of corticotropin-releasing factor (CRF) agonists in regulating responses in pancreatitis, we evaluated the effects of urocortin 2 (UCN2) and stressin1 in caerulein-induced acute pancreatitis (AP) model in rats. Male rats were pretreated with UCN2 or stressin1 for 30 min followed by induction of AP with supraphysiologic doses of caerulein. Serum amylase and lipase activity, pancreatic tissue necrosis, immune cell infiltrate, nuclear factor (NF)-κB activity, trypsin levels, and intracellular Ca2+ ([Ca2+]i) were ascertained. UCN2, but not stressin1 attenuated the severity of AP in rats. UCN2, but not stressin1, reduced serum amylase and lipase activity, cell necrosis and inflammatory cell infiltration in AP. NF-κB activity in pancreatic nuclear extracts increased in AP and UCN2 treatment reduced caerulein-induced increases in NF-κB activity by 42%. UCN2 treatment prevented caerulein-induced degradation of IκB-α in the cytosolic fraction as well as increased levels of p65 subunit of NF-κB in the cytosolic fraction. Pancreatic UCN2 levels decreased in AP compared with saline. UCN2 evoked [Ca2+]i responses in primary acinar cells and abolished caerulein-evoked [Ca2+]i responses at 0.1nM, and decreased by ~50% at 1.0nM caerulein. UCN2 stimulation resulted in redistribution of a portion of F-actin from the apical to the basolateral pole. UCN2 prevented the massive redistribution of F-actin observed with supraphysiologic doses of caerulein. UCN2, but not stressin1 attenuated severity of an experimental pancreatitis model. The protective effects of UCN2, including anti-inflammatory and anti-necrotic effects involve activation of the CRF2 receptor, [Ca2+]i signaling, and inhibition of NF-κB activity.

Short communication: Endophytic actinobacteria isolated from ginger (Zingiber officinale) and its potential as a pancreatic lipase inhibitor and its toxicity

Abstract. Rahayu S, Fitri L, Ismail YS. 2019. Short communication: Endophytic actinobacteria isolated from ginger (Zingiber officinale) and its potential as a pancreatic lipase inhibitor and its toxicity. Biodiversitas 20: 1312-1317. Endophytic actinobacteria from ginger (Zingiber officinale Rosc.) is a bacterium that is capable of producing secondary metabolites that are the same as their hosts. This study aims to look at the potential of endophytic actinobacteria from ginger as a pancreatic lipase inhibitor and its toxicity. Endophytic actinobacteria were isolated, purified, then tested for pancreatic lipase inhibitors and their toxicity using the BSLT method (Brine Shrimp Lethality Test) and phytochemical tested on ethanol extract of selected isolates. Seven endophytic actinobacterial isolates were isolated from the ginger rhizome. The isolates had different morphological diversity based on colony and microscopic observations and 5 isolates had pancreatic lipase inhibitor activity. The highest inhibitors were found in AJ4 isolates (89.9%), compared with pancreatic lipase inhibitors crude extracts of ginger (68.9%) and orlistat (88.1%) as positive controls. The LC50 value of AJ4 isolates was 653,381 ppm and the value of LT50 was 17,569 hours. AJ4 isolates contain terpenoids, phenols, tannins, flavonoids, alkaloids, and saponins. This research data is considered as new information about the potential of endophytic actinobacteria from ginger as pancreatic lipase inhibitors and their toxicity.

Bariatric/Metabolic Surgery for Diabetes: Lessons From the Past and Present.

One of the most important breakthroughs in diabetes was announced in 1921 by a surgeon-led team working on the gastrointestinal tract. Frederick Banting, a general surgeon in Canada, partnered with a physiologist, John Macleod, and others to purify insulin from pancreatic extracts and demonstrated that this gut hormone could treat diabetes. Eighty years later, more than 30 gut-derived hormones have been described (1), many of which have also been shown to influence glucose as well as lipid metabolism, appetite control, and energy expenditure. And these are just the ones we know about. Despite its gastrointestinal origins, research into the mechanisms of insulin’s action and its use for the treatment of diabetes became the domain of endocrinologists, who to this day, it may be argued, have otherwise ignored the largest endocrine organ in the body to focus on the five or six hypothalamic-pituitary axes. As a result of these efforts, diabetes is now understood to be a chronic disease that manifests when too little insulin is secreted by pancreatic islets in response to the body’s needs: an absolute deficiency in the case of type 1 diabetes and a relative insulin deficiency in type 2 diabetes. It is frustrating that, despite nearly 100 years of research since insulin’s discovery, the specific causes of insulin resistance and impaired insulin secretion that lead to diabetes remain largely elusive. Enter the surgeons again. Starting in the latter half of the last century, obesity became an increasingly prevalent medical problem in the U.S. and other developed nations. As obesity rates have risen, so has our understanding of the importance of excess fat storage in expression of …

Lipolysis-Permeation Setup for Simultaneous Study of Digestion and Absorption in Vitro.

Lipid-based formulations (LBFs) are a delivery strategy to enhance intestinal absorption of poorly water-soluble drugs. LBF performance is typically evaluated by in vitro lipolysis studies, but these do not accurately predict the in vivo performance. One possible reason is the absence of an absorptive membrane driving sink conditions in the serosal compartment. To explore the impact of absorption under sink conditions on the performance evaluation, we developed a lipolysis-permeation setup that allows simultaneous investigation of intestinal digestion of an LBF and drug absorption. The setup consists of two chambers, an upper one for digestion (luminal), and a lower, receiving one (serosal), separated by a Caco-2 monolayer. Digestions were performed with immobilized lipase, instead of the pancreatic extract typically used during lipolysis, since the latter has proven incompatible with Caco-2 cells. Danazol-loaded LBFs were used to develop the setup, and fenofibrate-loaded LBFs were used to establish an in vitro in vivo correlation. As in regular lipolysis studies, our setup allows for the evaluation of (i) the extent of digestion and (ii) drug distribution in different phases present during lipolysis of drug-loaded LBFs (i.e., oil, aqueous, and solid phase). In addition, our setup can determine drug permeation across Caco-2 monolayers and hence, the absorptive flux of the compound. The presence of the absorptive monolayer and sink conditions tended to reduce aqueous drug concentrations and supersaturation in the digestion chamber. The drug transfer across the Caco-2 membrane accurately reflected in vivo drug exposure upon administration of three different LBFs loaded with fenofibrate, where the traditional lipolysis setup failed to predict in vivo performance. As the new setup reflects the dynamic processes occurring in the gastrointestinal tract, it is a valuable tool that can be used in the development of LBFs prior to in vivo studies.

Emerging Peptide Science in Canada

Emerging Peptide Science in Canada

In vitro digestion of galactolipids from chloroplast-rich fraction (CRF) of postharvest, pea vine field residue (haulm) and spinach leaves.

The removal of intact chloroplasts from their cell wall confinement offers a novel way to obtain lipophilic nutrients from green biomass, especially carotenoids and galactolipids. These latter are the main membrane lipids in plants and they represent a major source of the essential α-linolenic acid (18:3; ALA). Nevertheless, knowledge on their digestion is still limited. We have developed a physical method of recovering a chloroplast-rich fraction (CRF) from green biomass and tested its digestibility in vitro under simulated gastrointestinal conditions. Using a two-step static model, CRF from both spinach leaves and postharvest, pea vine field residue (haulm) were first exposed to enzymes from rabbit gastric extracts and then either to pancreatic enzymes from human pancreatic juice (HPJ) or to porcine pancreatic extracts (PPE). The lipolysis of monogalactosyldiacylglycerol (MGDG) and digalactosyl diacylglycerol (DGDG) was monitored by thin layer chromatography and gas chromatography of fatty acid methyl esters. For both CRF preparations, MGDG and DGDG were converted to monogalactosylmonoacylglycerol (MGMG) and digalactosylmonoacylglycerol (DGMG), respectively, during the intestinal phase and ALA was the main fatty acid released. Galactolipids were more effectively hydrolysed by HPJ than by PPE, and PPE showed a higher activity on MGDG than on DGDG. These findings may be explained by the higher levels of galactolipase activity in HPJ compared to PPE, which mainly results from pancreatic lipase-related protein 2. Thus, we showed that CRF galactolipids are well digested by pancreatic enzymes and represent an interesting vehicle for ALA supplementation in human diet.

NMR-based metabolic profiling of urine, serum, fecal, and pancreatic tissue samples from the Ptf1a-Cre; LSL-KrasG12D transgenic mouse model of pancreatic cancer.

Pancreatic cancer is the third leading cause of cancer deaths in the United States with more than 53,000 expected to be diagnosed with the disease in 2018. The median survival time after diagnosis is four to six months. The poor survival statistics are due in part to the fact that pancreatic cancer is typically asymptomatic until it reaches advanced stages of the disease. Although surgical resection provides the best chance of survival, pancreatic cancer is rarely detected when surgery is still possible due, in part, to lack of effective biomarkers for early detection. The goal of the research reported here was to determine if it was possible to identify metabolic biomarkers for detection of pre-cancerous pancreatic intraepithelial neoplasia (PanIN) that precede pancreatic adenocarcinoma. The transgenic Ptf1a-Cre; LSL-KrasG12D mouse strain was used as a model of pancreatic cancer progression. Nuclear magnetic resonance (NMR) spectroscopy was employed to compare metabolic profiles of urine, sera, fecal extracts, and pancreatic tissue extracts collected from control and study mice aged 5, 11, and 15 months, including 47 mice with tumors. We were able to identify the following potential biomarkers: decreased 3-indoxylsulfate, benzoate and citrate in urine, decreased glucose, choline, and lactate in blood, and decreased phenylalanine and benzoate and increased acetoin in fecal extracts. Potential biomarkers were validated by p-values, PLS-DA VIP scores, and accuracies based on area under ROC curve analyses. Essentially, all of the metabolic profiling changes could be explained as being associated with the consequences of bicarbonate wasting caused by a complete substitution of the normal pancreatic acinar tissue by tissue entirely composed of PanIN. Given the nature of the mouse model used here, our results indicate that it may be possible to use NMR-based metabolic profiling to identify biomarkers for detection of precancerous PanIN that immediately precede pancreatic cancer.

Effects of Renshenjian decoction on pancreatic metabonomic profiles in insulin resistance rats based on ¹H-NMR metabonomics

Pancreas metabonomic profiles of the type 2 diabetic rats' induced by streptozotocin(STZ) and high-sugar, high fat diet on the treatment of Renshenjian decoction(RSJD) after 8 weeks were investigated.In this study, 48 Rats were randomly divided into four groups: normal control (NC), Pathological model (PM), Renshenjian decoction(RSJD 3.76 g·kg⁻¹) and glimepiride control (GC 0.04 mg·kg⁻¹). They are induced insulin resistance model of type 2 diabetes mellitus by streptozotocin(STZ) after 4 weeks' high-sugar, high fat diet except for NC. After sucessful modeling, they are given intragastric administration respectively with same amount of saline, RSJD and glimepiride in 4 weeks. At the end of the 8th week, the pancreatic tissue of rats in each group was collected, and the ¹H-NMR spectrum was collected after being treated by certain method, and analyzed by principal component analysis (PCA). Compared with NC's rats, we found PM's a significant elevation in the level of leucine/isoleucine, valine, lactic acid, creatine but reduction in the level of inose and less obvious changes in the level of creatine, cholic acid, taurine in pancreatic extract. After having been recieved RSJD, reduction level in leucine/isoleucine, valine, alanine, creatine, choline, taurine are also found in pancreatic extract of RSJD's rats, together with the increase of creatinine and tryptophan levels. The results showed that RSJD could regulate the level of amino acids in pancreas of IR rats, promoting a recovery in the process of metabolism. It's helpful to simulate the metabolic changes of IR rats via ¹H-NMR for a further understanding to study the mechanism how RSJD treat IR rats.

Caco-2 Cell Conditions Enabling Studies of Drug Absorption from Digestible Lipid-Based Formulations

PurposeTo identify conditions allowing the use of cell-based models for studies of drug absorption during in vitro lipolysis of lipid-based formulations (LBFs).MethodsCaco-2 was selected as the cell-based model system. Monolayer integrity was evaluated by measuring mannitol permeability after incubating Caco-2 cells in the presence of components available during lipolysis. Pure excipients and formulations representing the lipid formulation classification system (LFCS) were evaluated before and after digestion. Porcine mucin was evaluated for its capacity to protect the cell monolayer.ResultsMost undigested formulations were compatible with the cells (II-LC, IIIB-LC, and IV) although some needed mucin to protect against damaging effects (II-MC, IIIB-MC, I-LC, and IIIA-LC). The pancreatic extract commonly used in digestion studies was incompatible with the cells but the Caco-2 monolayers could withstand immobilized recombinant lipase. Upon digestion, long chain formulations caused more damage to Caco-2 cells than their undigested counterparts whereas medium chain formulations showed better tolerability after digestion.ConclusionsMost LBFs and components thereof (undigested and digested) are compatible with Caco-2 cells. Pancreatic enzyme is not tolerated by the cells but immobilized lipase can be used in combination with the cell monolayer. Mucin is beneficial for critical formulations and digestion products.

РАЗРАБОТКА КОМПОНЕНТНОГО СОСТАВА ПИТАТЕЛЬНОЙ СРЕДЫ НА ОСНОВЕ ТВОРОЖНОЙ СЫВОРОТКИ ДЛЯ КУЛЬТИВИРОВАНИЯ ШТАММА BIFIDOBACTERIUM LONGUM B379M

In the theoretical part of this paper, we discuss the role of natural intestinal microflora - in particular, bifidobacteria - in maintaining the homeostasis of the human body. We also explain the importance of the development of culture media for growing these important bacteria. In the experimental part, we present a step wise development of a culture medium composition for growing the Bifidobacterium longum B379M strain. Milk whey, a secondary raw material of the dairy industry containing numerous nutrients, seems to be a suitable basis for such a medium. The use of milk whey for these purposes also solves the problem of its rational utilization in the production of cottage cheese. The constituent components of the culture under development were selected taking into account the nutritional needs of the bacteria. The optimal amounts of these components were determined by comparing the logarithms of CFU concentrations in 1 mL of culture samples following a 24- hour incubation at 37°C and their active acidity values, which are considered to be indicative of the acid-forming ability of the bifidobacterium at its various cultivation stages. Pancreatic casein hydrolysate, peptone and yeast extract were used as sources of nitrogen; whereas inulin, lactose and glucose supplied the carbon requirement. A particular attention is paid to the creation of a neutral medium, which is necessary for bifidobacteria, by adding sodium citrate, ascorbic acid and a solution of phosphate buffer. The medium was also neutralized during the cultivation process using a sodium bicarbonate solution. As a result, the cell yield on the developed culture medium exceeded 109 CFU/cm3, which is sufficient for laboratory studies.

Insulin-From its Discovery to the Industrial Synthesis of Modern Insulin Analogues.

After the discovery of insulin as a drug for diabetes, the pharmaceutical companies were faced with the challenge to meet the demand for insulin with the highest possible degree of purity in the required quantities from animal sources. The observation of an immune reaction of patients to insulin from animal pancreatic extracts made the availability of human insulin of highest priority. Only the enzyme-catalyzed semisynthesis at the C-terminus of the insulin B-chain led to a commercial process, but it depended on porcine insulin and was aggravated by supply concerns. The advent of rDNA technology allowed the commercial preparation of human insulin by biosynthesis in virtually unlimited quantities. An increased chemical diversity was only envisaged through chemical synthesis, which was simplified by advances in solid-phase peptide synthesis and chemical ligation. Single-chain insulin precursors are now being synthesized that should enable fast screening of insulin analogues for improved biophysical, biological, and thus promising new therapeutic properties, as well as for the industrial manufacture of insulin analogues not accessible by biosynthesis.