Kateryna Goncharova

A piglet with surgically induced exocrine pancreatic insufficiency as an animal model of newborns to study fat digestion.

The maldigestion and malabsorption of fat in infants fed milk formula results due to the minimal production of pancreatic lipase. Thus, to investigate lipid digestion and absorption and mimic the situation in newborns, a young porcine exocrine pancreatic insufficient (EPI) model was adapted and validated in the present study. A total of thirteen EPI pigs, aged 8 weeks old, were randomised into three groups and fed either a milk-based formula or a milk-based formula supplemented with either bacterial or fungal lipase. Digestion and absorption of fat was directly correlated with the addition of lipases as demonstrated by a 30% increase in the coefficient of fat absorption. In comparison to the control group, a 40 and 25% reduction in total fat content and 26 and 45% reduction in n-3 and n-6 fatty acid (FA) content in the stool was observed for lipases 1 and 2, respectively. Improved fat absorption was reflected in the blood levels of lipid parameters. During the experiment, only a very slight gain in body weight was observed in EPI piglets, which can be explained by the absence of pancreatic protease and amylase in the gastrointestinal tract. This is similar to newborn babies that have reduced physiological function of exocrine pancreas. In conclusion, we postulate that the EPI pig model fed with infant formula mimics the growth and lipid digestion and absorption in human neonates and can be used to elucidate further importance of fat and FA in the development and growth of newborns, as well as for testing novel formula compositions.

Exogenous pancreatic-like enzymes are recovered in the gut and improve growth of exocrine pancreatic insufficient pigs

The exocrine pancreatic insufficient (EPI) pigs grow less due to different disturbances in feed digestion, absorption, and retention. Use of pancreatic-like enzymes of microbial origin in pigs may improve feed use and performance in slow-growing pigs. The aim was to study gut recovery and effectiveness of pancreatic-like enzymes of microbial origin supplementation on pig performance. Six male pigs 10 to 12 kg BW underwent pancreatic duct ligation surgery to induce total exocrine pancreatic insufficiency (EPI). Three cannulas to access the gastrointestinal tract content were installed in stomach, duodenum, and ileum in EPI pigs and in 3 control (healthy) pigs. One month after surgery, enzymes were given before feeding and digesta samples were collected for analyses. The BW of EPI pigs did not increase during 1 mo following surgery (11.7 vs. 11.6 kg BW); however, BW increased after 1 wk of enzyme supplementation (12.1 kg BW). Coefficient of fat and N absorption increased (P < 0.05) in EPI pigs after enzyme supplementation. Activity of amylase, lipase, and protease in chyme samples of EPI pigs was very low compared to controls. In EPI pigs after enzyme supplementation, amylase activity increased from 5.32 to 72.9 units/mL but remained lower than that of healthy pigs (162.7 units/mL). Lipase activity increased from 79.1 to 421.6 units/mL, which was similar to that of controls (507.3 units/mL). Proteolytic activity increased from 7.8 to 69.7 units/mL but still did not reach control pigs (164.3 units/mL). In conclusion, exogenous microbial enzymes mimic endogenous pancreatic enzymes being recovered along the lumen of the gastrointestinal tract. These enzymes might be a useful tool to stimulate growth of slower-growing pigs after the weaning period.

Impact of colostrum and plasma immunoglobulin intake on hippocampus structure during early postnatal development in pigs.

The first milk, colostrum, is an important source of nutrients and an exclusive source of immunoglobulins (Ig), essential for the growth and protection from infection of newborn pigs. Colostrum intake has also been shown to affect the vitality and behaviour of neonatal pigs. The objective of this study was to evaluate the effects of feeding colostrum and plasma immunoglobulin on brain development in neonatal pigs.

Effect of feeding colostrum versus exogenous immunoglobulin G on gastrointestinal structure and enteric nervous system in newborn pigs

Colostrum is an indispensable source of antibodies (IgG) protecting the newborn pig against infection. We studied the effect of feeding colostrum and purified IgG on early structure and development of the gastrointestinal tract (GIT). Newborn littermate pigs were fed either colostrum, an elemental diet (ED), or an ED supplemented with purified serum IgG (ED + IgG) for 24 h or then only ED up to 72 h. Afterwards, pigs were slaughtered. Colostrum-fed pigs or ED supplemented with IgG (ED + IgG) increased thickness (P < 0.001) of stomach mucosa and muscularis (P < 0.05) compared to the ED group not receiving IgG. Feeding an ED supplemented with IgG improved morphology of the GIT towards that of colostrum-fed piglets and indicates a beneficial effect of IgG on GIT development in neonatal pigs. Immunohistochemical studies indicate that ED feeding may influence the expression of nitric oxide synthase in jejunal myenteric (but not submucous) neurons of newborn pigs.

Experiments suggesting extra-digestive effects of enteral pancreatic amylase and its peptides on glucose homeostasis in a pig model

The studies presented were designed to highlight the impact of pancreatic enzymes on glycemic control and insulin response. Blood glucose and plasma insulin levels were monitored after intravenous, oral or direct gut glucose tolerance tests (GTT) in 6 pigs with an intact gastrointestinal tract and in 12 pigs following duodenal-jejunal bypass (DJB) surgery. In the intact pigs, pancreatic enzymes (Creon®) given orally 1 h prior to the GTT, lowered the blood glucose levels during the oral and meal GTT and reduced the plasma insulin response during the intravenous and meal GTT. In DJB pigs, blood glucose and plasma insulin levels were higher following glucose loading into the by-passed biliopancreatic limb as compared to that following glucose loading orally or into the common intestinal limb. Infusion of amylase or amylase peptides together with glucose into the biliopancreatic limb lowered blood glucose levels in DJB pigs. These preliminary data suggest new, extra-digestive, actions of enteral pancreatic enzymes – probably amylase or its peptides – on glucose homeostasis, with an reduction in net glucose absorption into the blood and in insulin response. This ability of digestive enzymes (amylase) to reduce post-prandial hyperglycaemia in an insulin-independent manner could aid in preventing the development of obesity and diabetes.

Importance of neonatal immunoglobulin transfer for hippocampal development and behaviour in the newborn pig

Neurological disorders are among the main clinical problems affecting preterm children and often result in the development of communication and learning disabilities later in life. Several factors are of importance for brain development, however the role of immunoglobulins (passive immunity transfer) has not yet been investigated. Piglets are born agammaglobulinemic, as a result of the lack of transfer of maternal immunoglobulins in utero, thus, they serve as an ideal model to mimic the condition of immunoglobulin deficiency in preterm infants. Thirty six, unsuckled newborn piglets were fed an infant formula or colostrum and supplemented orally or intravenously with either species-specific or foreign immunoglobulin and then compared to both newborn and sow-reared piglets. Two days after the piglets were born behavioural tests (novel recognition and olfactory discrimination of conspecifics scent) were performed, after which the piglets were sacrificed and blood, cerebrospinal fluid and hippocampi samples were collected for analyses. Both parameters of neuronal plasticity (neuronal maturation and synapse-associated proteins) and behavioural test parameters appeared to be improved by the appearance of species-specific porcine immunoglulin in the circulation and cerebrospinal fluid of the piglets. In conclusion, we postulate possible positive clinical effects following intravenous infusion of human immunoglobulin in terms of neuronal plasticity and cognitive function in preterm infants born with low blood immunoglobulin levels.

Oral uricase eliminates blood uric acid in the hyperuricemic pig model

An elevated level of serum uric acid—hyperuricemia, is strongly associated with the development of gout and chronic kidney disease (CKD) which is often accompanied by a significantly reduced glomerular filtration rate (GFR). In the present study, we investigated the extra-renal elimination of uric acid via the intestine in a healthy pig model and the effect of oral uricase therapy on plasma uric acid concentrations in pigs with induced hyperuricemia and CKD. The experiment was conducted on eleven, ten-week-old pigs (n = 11). The porcine model of CKD was developed by performing 9/10 nephrectomy surgery on eight pigs. A stable model of hyperuricemia was established in only five of the eight nephrectomized pigs by frequent injections of uric acid (UA) into the jugular vein. All pigs (three healthy pigs and five CKD pigs) were operated for implantation of jugular vein catheters and the three healthy pigs also had portal vein catheters inserted. Blood uric acid concentrations were measured spectrophotometrically, using the Uric Acid Assay Kit (BioAssay Systems, Hayward, USA). The piglets with CKD received orally administered uricase (treatment) and served as their own controls (without uricase supplementation). Oral uricase therapy significantly decreased plasma uric acid concentrations in pigs with CKD, whereas hyperuricemia was observed in the pigs whilst not being treated with uricase. Urinary uric acid excretion was similar during both the treatment and control periods during the first 8 h and 24 h after UA infusions in the CKD pigs. To demonstrate the elimination of UA via the intestine, the healthy pigs were infused with UA into the jugular vein. The blood collected from the jugular vein represents circulating UA concentrations and the blood collected from the portal vein represents the concentration of UA leaving the intestine. The final (after 2 h) concentration of UA was significantly lower in blood collected from the portal vein compared to that collected from the jugular vein (3.34 vs. 2.43 mg/dL, respectively, p = 0.024). The latter allows us to suggest that UA is eliminated from the blood via the gut tissue.

Is hyperoxaluria in a porcine model of Roux-en-Y gastric bypass (RYGB) associated with exocrine pancreatic insufficiency?

1Department of Epizootiology and Clinic of Infectious Diseases, University of Life Sciences, Lublin, Poland Head of the Department: prof. Stanislaw Winiarczyk, PhD 2Department of Preclinical Veterinary Sciences, University of Life Sciences, Lublin, Poland Head of the Department: prof. Jose L. Valverde Piedra, PhD 3Department of Animal Physiology, University of Life Sciences, Lublin, Poland Head of the Department: prof. Iwona Puzio, PhD 4Department of Biology, Lund University, Lund, Sweden Head of the Department: prof. Christer Lofstedt, PhD 5R&D, SGPlus, Malmo, Sweden Chief Executive Officer: prof. Stefan G Pierzynowski, PhD 6General Surgery Department of the District Specialist Hospital, Lublin, Poland Head of the Department: Jerzy Mackiewicz, MD, PhD 7Department of Vitreoretinal Surgery, Medical University of Lublin, Poland Head of the Department: Andrzej Chrościcki, MD, PhD 8Department and Clinic of Animal Internal Diseases, University of Life Sciences, Lublin, Poland Head of the Department: Jacek Madany, MD, PhD 9Department of Human Anatomy, Laboratory of Biostructure, Medical University of Lublin, Poland Head of the Department: prof. Ryszard Maciejewski, MD, PhD 10Institute of Rural Health, Lublin, Poland Head of the Institute: prof. Iwona Bojar, MD, PhD 11Department of Cytology, Bogomoletz Institute of Physiology, Kiev, Ukraine Head of Department: prof. Galyna Skibo, MD, PhD

Diet-induced changes in brain structure and behavior in old gerbils.

Background/Objectives:Aging is associated with many physiological alterations such as changes in metabolism, food intake and brain dysfunction. Possible ways to correct age-related brain dysfunction using dietary treatments still remains undeveloped. The aim of our research was to investigate whether long-term dietary treatment with 2-oxoglutarate (2-OX), which is involved in many regulatory pathways, together with pancreatic-like enzymes of microbial origin (PLEM), which ensure appropriate digestion and absorption of nutrients, affects age-related changes in the brain morphology and cognitive function in old Mongolian gerbils.Materials/methods:Experiment was comprised of two separate studies. Samples of the hippocampus were obtained from male Mongolian gerbils of different ages (n=63 in the first study, n=74 in the second study). Immunohistochemistry was used for visualization of the nestin/NeuN-positive neuronal progenitors. Changes in amount of neural cell adhesion molecules (NCAMs) were estimated using enzyme-linked immunosorbent assay. For assessment of cognitive and sensorimotor functions, the T-maze spontaneous alternation test and the adhesive removal test (ART) were used. The ultrastructure of the CA1 hippocampal area was visualized using transmission electron microscopy.Results:Long-term treatment with 2-OX+PLEM led to a significantly increased amount of nestin/NeuN-positive cells in the CA1 hippocampal area and positive changes in learning and sensorimotor functions. As for synaptic transmission, changes in the spatial distribution of synaptic vesicles, as well as the redistribution of NCAM forms, were observed in the hippocampal synapses of the old gerbils.Conclusions:Taken together, our data show that dietary supplementation with 2-OX+PLEM not only enhances the proliferation and differentiation of neuronal progenitors, but also improves age-related deficits in the morphological and functional state of the brain of old gerbils. Thus, suggesting that a 2-OX+PLEM-enriched diet could also improve brain functions that have deteriorated with age.

Novel potential of pancreatic-like enzymes of microbial origin in exocrine pancreatic insufficiency – study on a pig model

Introduction. The standard porcine-derived pancreatic enzyme replacement therapy (PERT) is a lifesaving treatment for patients with diseases causing exocrine pancreatic insufficiency (EPI). An attempt to replace PERT with microbial enzymes were undertaken. The aim was to highlight whether the mode of application, mixed with food or applied directly to the stomach, of pancreatic-like enzymes of microbial origin (PLEM) can affect their activity along the gastrointestinal tract. Materials and method. The activity of amylase, lipase and proteinase in the stomach, duodenum and ileum were tested in EPI pigs (n=6) after supplementation of PLEM, either orally – before and during feed consumption – or via the stomach – before and during feed consumption. Healthy pigs not treated with PLEM (n=3) served as controls. Activity of the enzymes measured in the chyme were obtained together with the digesta pH. Activity of the enzymatic residues in the stool samples was also checked. Results. The highest pancreatic enzyme activities were found in the duodenum of the healthy pigs (amylase 162,68 kU/ mL, lipase 507,34 kU/mL and protolitic (trypsin) activity 357,60 kU/mL). Nevertheless, the microbial enzymes remained also active along the entire length of the GIT – including stomach in EPI pigs, regardless of their route of administration. However, activity level was significantly lower. Discussion. Results indicate that the activity pattern of PLEM in the small intestine mimics the activity of the natural endogenous pancreatic enzymes in healthy pigs. The most physiological features of PLEM were observed when enzymes were offered orally. The magnitude of PLEM activity in the stomach of EPI pigs was essential and significantly higher than that measured in healthy pigs, thus being somewhat not physiological, and for health reasons of the patients should be further explored. Interestingly, specific trypsin-like activity was measured in all parts of the GIT after PLEM application. However, proteolytic activity of the experimental proteaze in in vitro studies did not exhibit trypsin-like activity.