High fat and high cholesterol diet induces DPP-IV activity in intestinal lymph.

Recent studies have reported that dipeptidyl-peptitase IV (DPP-IV) is correlated with diabetic conditions and also with dyslipidemia caused by overnutrition, especially a high fat diet. However, the role of DPP-IV in diabetes during dyslipidemia has been unclear. We utilized a lymph fistula rat model to determine whether intestinal lymph, which absorbs dietary fats, is affected by a chronic high-fat and high-cholesterol diet (HFHC). HFHC diet rats showed significantly higher DPP-IV activity in intestinal lymph and plasma compared to rats receiving a normal chow diet. In addition, HFHC diet rats showed significantly increased DPP-IV mRNA expression in the intestine. However, DPP-IV mRNA in the lymphocytes isolated from intestinal lymph and mesenteric lymph nodes did not show significant differences from that in the normal diet rats. In conclusion, HFHC diets increased DPP-IV expression in intestinal lymph; these results indicate the applicability of a previously unrecognized role for DPP-IV in metabolic disorders, including diabetes.

Effect of intraperitoneal and intravenous administration of cholecystokinin-8 and apolipoprotein AIV on intestinal lymphatic CCK-8 and apo AIV concentration.

CCK and apolipoprotein AIV (apo AIV) are gastrointestinal satiety signals whose synthesis and secretion by the gut are stimulated by fat absorption. Intraperitoneally administered CCK-8 is more potent in suppressing food intake than a similar dose administered intravenously, but the reason for this disparity is unclear. In contrast, both intravenous and intraperitoneally administered apo AIV are equally as potent in inhibiting food intake. When we compared the lymphatic concentration of CCK-8 and apo AIV, we found that neither intraperitoneally nor intravenously administered CCK-8 or apo AIV altered lymphatic flow rate. Interestingly, intraperitoneal administration of CCK-8 produced a significantly higher lymphatic concentration at 15 min than did intravenous administration. Intraperitoneal injection of apo AIV also yielded a higher lymphatic concentration at 30 min than did intravenous administration. Intraperitoneal administration of CCK-8 and apo AIV also resulted in a much longer period of elevated CCK-8 and apo AIV peptide concentration in lymph than intravenous administration. Furthermore, enzymatic activity of dipeptidyl peptidase IV (DPPIV) and aminopeptidase was higher in plasma than in lymph during fasting, and so, satiation peptides, such as CCK-8 and apo AIV in the lymph, are protected from degradation by the significantly lower DPPIV and aminopeptidase activity levels in lymph than in plasma. Therefore, the higher potency of intraperitoneally administered CCK-8 compared with intravenously administered CCK-8 in inhibiting food intake may be explained by both its higher concentration in lymph and the prolonged duration of its presence in the lamina propria.

Rapid inactivation of a moth pheromone.

We have isolated, cloned, and expressed a male antennae-specific pheromone-degrading enzyme (PDE) [Antheraea polyphemus PDE (ApolPDE), formerly known as Sensillar Esterase] from the wild silkmoth, A. polyphemus, which seems essential for the rapid inactivation of pheromone during flight. The onset of enzymatic activity was detected at day 13 of the pupal stage with a peak at day 2 adult stage. De novo sequencing of ApolPDE, isolated from day 2 male antennae by multiple chromatographic steps, led to cDNA cloning. Purified recombinant ApolPDE, expressed by baculovirus, migrated with the same mobility as the native protein on both native polyacrylamide and isoelectric focusing gel electrophoresis. Concentration of ApolPDE (0.5 microM) in the sensillar lymph is approximately 20,000 lower than that of a pheromone-binding protein. Native and recombinant ApolPDE showed comparable kinetic parameters, with turnover number similar to that of carboxypeptidase and substrate specificity slightly lower than that of acetylcholinesterase. The rapid inactivation of pheromone, even faster than previously estimated, is kinetically compatible with the temporal resolution required for sustained odorant-mediated flight in moths.

Compartmentalization of the protease-antiprotease balance in early severe acute pancreatitis.

OBJECTIVE: To assess the balance between trypsin and protease inhibitors simultaneously in the systemic circulation and in the thoracic lymph and peritoneal exudate. METHODS: Twenty patients with early severe acute pancreatitis were studied. Enzymatically active and immunoreactive trypsin in conjunction with its major inhibitors were measured in the 3 compartments at the onset of end-organ failure(s). The molecular forms of trypsin were determined in the lymph and ascites by gel filtration chromatography to separate trypsinogen and free-and inhibitor-bound trypsin. RESULTS: Both enzymatically active trypsin and immunoreactive trypsin levels were highest in ascites and lymph compared with the systemic circulation. Intracompartmental alpha1- protease inhibitor gradient moved in the opposite direction, whereas alpha2 macroglobulin concentration was highest in ascites and lowest in the lymph. Although most of the enzymatically and immunoreactive material in ascites and lymph consisted of trypsin complexed with alpha2 macroglobulin and trypsinogen, respectively, free active trypsin was detected in more than 80% of the samples. CONCLUSIONS: In patients with early severe acute pancreatitis, there is a significant trypsinogen activation resulting in protease-antiprotease imbalance and thereby free enzymatically active trypsin in the 2 body fluid compartments in close vicinity to the inflammatory process. This may be involved in the pathophysiology of local and distant tissue damage.

Effects of intravenous apolipoprotein A-I/phosphatidylcholine discs on paraoxonase and platelet-activating factor acetylhydrolase in human plasma and tissue fluid.

We have previously shown that intravenous apolipoprotein (apo) A-I/phosphatidylcholine (apo A-I/PC) discs increase plasma high-density lipoprotein (HDL) concentration in humans. We have now studied the associated changes in two enzymes, paraoxonase (PON) and platelet-activating factor acetylhydrolase (PAF-AH) that are carried in whole or in part by HDLs, and are thought to influence atherogenesis by hydrolyzing oxidized phospholipids in lipoproteins. Apo A-I/PC discs (40 mg/kg over 4 h) were infused into eight healthy males. Although plasma apo A-I and HDL cholesterol increased on average by 178 and 158%, respectively, plasma total PON and total PAF-AH concentrations did not rise. By the end of the infusion, HDL-associated PAF-AH had increased by 0.56 +/- 0.14 microg/mL (mean +/- S.D., P < 0.01), and nonHDL-associated PAF-AH had decreased by 0.84 +/- 0.11 microg/mL (P < 0.05). These changes were accompanied by an increase in the HDL-associated PAF-AH/apo A-I ratio from 0.19 to 0.35 (P < 0.05), and by a decrease in the nonHDL-associated PAF-AH/apo B ratio from 2.1 to 1.4 (P < 0.05). No changes in PON or PAF-AH concentrations were detected in prenodal lymph (tissue fluid), collected continuously from the leg. Our results show that the total concentrations of PON and PAF-AH in plasma are uninfluenced by plasma HDL concentration. PAF-AH transfers readily between HDLs and LDLs in vivo, and its distribution between them is determined partly by their relative concentrations and partly by HDL composition.

Intestinal alkaline phosphatase release is not associated with chylomicron formation.

Intestinal alkaline phosphatase (IAP) is one of the major sources of alkaline phosphatase in circulation. It is secreted into the intestinal lumen, serum, and lymph. After the ingestion of lipid, lymphatic alkaline phosphatase secretion increases significantly. We have found that the nonabsorbable fat olestra is unable to stimulate lymphatic alkaline phosphatase secretion. We also found that the hydrophobic surfactant Pluronic L-81, which blocks chylomicron formation, fails to inhibit this increase in lymphatic alkaline phosphatase secretion. These results suggest that it is the lipid uptake into the mucosa and/or reesterification to form triacylglycerols, but not the formation of chylomicrons, that is necessary for the stimulation of the secretion of alkaline phosphatase into the lymph.

Phospholipase A(2)--derived neutral lipids from posthemorrhagic shock mesenteric lymph prime the neutrophil oxidative burst.

BACKGROUND: Our previous work identified posthemorrhagic shock mesenteric lymph (PHSML) lipids as key elements in polymorphonuclear neutrophil (PMN)--provoked acute lung injury. We hypothesize that gut phospholipase A(2) (PLA(2)) is responsible for the generation of proinflammatory lipids in PHSML that primes circulating PMNs for enhanced oxidative burst. METHODS: Mesenteric lymph was collected from rats (n = 5) before (preshock), during the induction of hemorrhagic shock (mean arterial pressure, 40 mm Hg x 30 minutes), and at resuscitation (shed blood + 2x lactated Ringer's solution). PLA(2) inhibition (quinacrine, 10 mg/kg, intravenously) was given before shock was induced. Extracted lipids were separated by normal phase high-pressure liquid chromatography and resuspended in albumin. PMNs were exposed to a 5% vol:vol concentration of eluted lipids and activated with N-formyl-methionyl-leucyl-phenylalanine (1 micromol/L). Superoxide production was assessed by cytochrome C reduction. RESULTS: High-pressure liquid chromatography--extracted neutral lipids of lymph collected before hemorrhagic shock did not prime the PMN oxidase, whereas isolated neutral lipids of postshock lymph primed PMNs 2.6- +/- 0.32-fold above baseline (P <.05). PLA(2) inhibition returned PHSML neutral lipid priming to baseline levels. CONCLUSIONS: PLA(2) inhibition before hemorrhagic shock abrogates the neutrophil priming effects of PHSML through reduction of the accumulation of proinflammatory neutral lipids. Identification of these PLA(2)-dependent lipids provides a mechanistic link that may have therapeutic implications for postshock acute lung injury.

Protein phosphatase 2Calpha expression in normal human tissues: an immunohistochemical study.

Protein phosphatase (PP2Calpha) is a member of the mammalian serine threonine-specific protein phosphatases family. We produced monoclonal antibodies against the recombinant PP2Calpha and evaluated the immunoreactivity of normal human tissues. The reactivity was strong in normal skin, the digestive tract, lung, kidney, breast, prostate, endocrine glands, and brain, while it was moderate in the ovary, testis, and liver. Epithelial cells revealed high levels of PP2Calpha expression, but stromal cells, including fibroblasts and endothelial cells, showed no or little PP2Calpha expression. Given the broad reactivity in endocrine and secreting epithelial cells, we propose that PP2Calpha expression might contribute to secretory cell function.

The role of phospholipase A2 in the pathogenesis of respiratory damage in hemorrhagic necrotizing pancreatitis--assessment of a new experimental model.

The new experimental model has been set as a standard with the purpose to explore and analyze the role of phospholipase A2 in predicting the severity of acute pancreatitis with specific interest for the onset of hemorrhagic necrotizing pancreatitis and pleuropulmonary complications. The experiments were performed on dogs (n = 25), and acute pancreatitis was induced with the injection of 10% sodium-taurocholate into pancreatic duct. Four experimental groups of animals were formed, so that 2 groups had lymph held by draining the thoracic duct. In the other two groups, the lymph had been put into system circulation, so that the nylon bag was set around the pancreas in order to prevent pancreatic juice to be spilt into abdominal cavity. The analyses of the levels of amylase, lipase, pancreatic amylase and phospholipase A2 in of serum, lymph and exudate were performed. The positive correlation was found between increased concentration of phospholipase A2 in serum and reduction of partial pressure of oxygen and pH of blood and the degree of severity of respiratory failure. The speed and intensity of development of the acute hemorrhagic necrotizing pancreatitis, as well as the severity of pulmonary complications might be estimated upon the of increase of the levels of phospholipase A2 in serum and lymph. This experimental model enables the studying of pathogenesis of acute pancreatitis and systemic complications.

Nutrients regulate diamine oxidase release from intestinal mucosa.

Diamine oxidase is continuously released from the intestinal mucosa and carried to the circulation by the lymphatics. The effect of nutrients on this release was examined. Rats were prepared with duodenal and intestinal lymph cannulas. Test mixtures of lipid emulsions containing triolein, oleic acid, or tricaprylin and solutions of carbohydrate and protein were infused into the duodenum. The enzyme release and triglyceride transport were determined and in some experiments were done in the presence and absence of Pluronic L-81, an inhibitor of chylomicron formation, and aminoguanidine, an inhibitor of diamine oxidase activity. The data indicate that nonlipid nutrients did not increase diamine oxidase activity in the intestinal lymph, but the mucosal tissue content was significantly reduced in the distal small intestine, particularly after protein infusion. Triglycerides and fatty acids increased diamine oxidase in the intestinal lymph, and the longer-chain triglyceride was more effective. Inhibition of triglyceride transport did not interfere with the enzyme release, and the inhibition of diamine oxidase activity had no significant effect on lipid absorption. According to our observations, only lipids increase intestinal lymph diamine oxidase. Nonfat nutrients appear to increase diamine oxidase in the intestinal lumen. Diamine oxidase is not directly required for lipid absorption.

Does hyperamylasemia in choledochal cyst indicate true pancreatitis? An experimental study.

Patients with choledochal cyst often have repeated attacks of abdominal pain accompanied by hyperamylasemia, and they may be diagnosed as having acute pancreatitis. However, the attacks generally tend to subside in a short period by conservative treatment, and evidence of pancreatitis is rarely observed at the time of surgery. Choledochal cyst is commonly associated with pancreatobiliary maljunction, and high concentrations of pancreatic enzymes in bile are usually observed. When the bile duct pressure increases due to obstructive cholangitis, pancreatic enzymes in bile may regurgitate into the blood stream. Cholangiovenous reflux of amylase might cause hyperamylasemia. In order to investigate the mechanism of hyperamylasemia by cholangiovenous reflux, canine pancreatic juice or bile from a patient with choledochal cyst was injected into the obstructed common bile duct in dogs. The pancreatic enzymes in bile could readily enter into the blood stream at the pressure level of 15 mmHg or more in the bile duct. The peak amylase level in the thoracic lymph was observed to be more than 4 times higher than that in the blood serum, and the lymph flow during 30 minutes increased significantly from 8.1 to 20.4 ml at the bile duct pressure level of 20 mmHg. The reflux of amylase in bile into the blood stream via both the hepatic vein and thoracic duct might result in hyperamylasemia in the patients with choledochal cyst.

Contribution of lymphatic myogenic activity and respiratory movements to pleural lymph flow.

In 11 anesthetized spontaneously breathing rabbits, we studied the contribution to total pleural lymph flow of myogenic activity of pleural lymphatics ("intrinsic mechanism") and the effect due to mechanical action of respiratory movements ("extrinsic mechanism"). Isoncotic saline solution (5 ml) containing 100 microCi of 125I-lactate dehydrogenase (LDH) was injected into right pleural space; in all but three control rabbits, injectate contained 1 mM amiloride in dimethyl sulfoxide to induce relaxation of smooth muscle tone. At 3 h, rabbits were killed and pleural fluid was collected and its volume measured. LDH radioactivity in pleural liquid and parietal pleural tissue was counted. In control rabbits, net pleural liquid flow (Jnet) at 3 h was -0.17 +/- 0.04 (SD) ml.kg-1.h-1; LDH concentration (C) and quantity (Q) decreased by 40.3 and 51.1% of initial value, respectively; total pleural lymphatic flow (Jl), calculated from LDH clearance, was 0.58 +/- 0.01 ml.kg-1.h-1. In amiloride-treated rabbits, Jnet was 0.01 +/- 0.1 ml.kg-1.h-1, C decreased by 34.4% and Q by 33.1%, and Jl averaged 0.39 +/- 0.02 ml.kg-1.h-1. C in parietal pleura, rich in lymphatics, was 13-fold higher in control than in amiloride-treated animals. The significant decrease of pleural lymphatic flow observed with amiloride (-40% relative to control) resulted from impairment of intrinsic mechanism, whereas, at comparable breathing frequencies, extrinsic mechanism remained unaltered. The direct effect of topical application of 1 mM amiloride was confirmed on exposed mesenteric collecting lymphatic ducts (data from 5 rats): amiloride reduced lymph flow by 40% by decreasing stroke volume without greatly affecting contraction rate of lymphatic walls.

[Enzyme liberation and activation of the kallikrein-kinin system in experimental pancreatitis. Studies of portal vein blood, pancreatic lymph and peritoneal effusion]. / Enzymfreisetzung und Aktivierung der Kallikrein-Kinin-Systeme bei experimenteller Pankreatitis. Untersuchungen in Pfortaderblut, Pankreaslymphe und Peritonealexsudat.

The clinical course of acute pancreatitis is strongly influenced by secondary cardiac, pulmonary and renal damage. The aim of the present study was to gather information about the compartment promoting the systemic damage. Therefore the activity of lipase, phospholipase A and plasma pro-kallikrein and the concentration of tissue kallikrein and kininogen were measured in portal venous blood, pancreatic lymph and peritoneal exudate. Anaesthetized pigs were subjected to fluid resuscitation to keep systemic haemodynamic parameters constant. The pancreas was isolated in situ. The pigs were randomly assigned to a control group (n = 9) or one of the two pancreatitis groups (n = 10 each). Pancreatitis was induced by i.a. infusion of free fatty acid (FFS) or retrograde infusion of 5% sodium taurocholate intraductally (NaT). In both pancreatitis groups the activity of lipase and phospholipase A increased. The most pronounced changes were seen in the peritoneal exudate (phospholipase A activity 40 min after induction: control 10.0 U/l, NaT 72.2 U/l). In both pancreatitis groups there was evidence for activation of the tissue kallikrein kinin system in the form of an increase in the kallikrein concentration and a decrease in the kininogen concentration. Again the changes were most pronounced in the peritoneal exudate (tissue kallikrein 40 min after induction: control 14.7 ng/ml, NaT 452 ng/ml).

[Comparative studies of diagnostically significant enzymes in plasma, udder lymph and milk of healthy cows and cows with udder diseases]. / Vergleichende Untersuchungen diagnostisch bedeutsamer Enzyme in Blutplasma, Euterlymphe und Milch von gesunden und euterkranken Kühen.

Different secretions (colostrum, milk, dry udder secretion) of every quarter, peripheral lymph from superficial lymph vessels of the mammary gland and blood from the V. epigastrica superficialis were obtained in 43 cows at different stages of lactation. In these samples the activity of 5 enzymes (LDH, NAG, AP, LAP, GGT) was determined. Levels of LDH and NAG were highest in blood plasma and udder lymph. Levels of LAP, AP and GGT were highest in milk increasing in this order. LDH, NAG, AP and LAP were correlated in both compartments. Changes of the functional state (dry or colostral period) and tissue disturbances of the mammary gland were accompanied by marked changes of enzyme activity in the secretions, but were without obvious influence on enzyme levels in blood plasma and udder lymph.

[Transferase contents in lymph and blood in fever reaction]. / Soderzhanie transferaz v limfe i krovi pri likhoradochnoi reaktsii.

The aim of the present investigation was the study of the content of alanine, asparagine-transaminases, gamma-glutamyl transferase and their isoenzymes, as well as leucine aminotransferase in the lymph of thoracic duct, hepatic and intestinal lymph and peripheral blood in dynamics of fever reaction of various duration in the experiments on rabbits. Irrespective of its duration, the fever was followed by a significant activation of the enzymes in the body fluids. However, in many-day fever reaction, a rise of enzymes level in the lymph was more prolonged than that in the blood. The above studies make it possible to assume that the released enzymes in fever reaction are primarily resorbed by lymphatic capillaries and their activity indices in the blood serum are largely evidenced by the transport function of the lymphatic system.

[Phosphatase activity in the lymph during a fever reaction]. / Aktivnost' fosfataz v limfe pri likhoradochnoi reaktsii.

Activity of acid and alkaline phosphatases, as well as isoenzymes of alkaline phosphatase in the lymph of the thoracic lymphatic duct, hepatic lymph and the peripheral blood have been studied on rabbits in the dynamics of the fever reaction of different duration. The fever reaction was followed by enzyme activity increase in all the body biologic fluids. However the degree of increase of their activity in the lymph was greater that that in the blood. Our data indicate that in the transport of phosphatases released from the tissues in the common circulation the essential role is played by the lymphatic system, the resorption and transport functions of which significantly characterise the dynamics and the level of their changes in the blood in fever reaction.

Active drainage of cardiac lymph in relation to reduction in size of myocardial infarction: an experimental study.

Active drainage of cardiac lymph using hyaluronidase was attempted in dogs. The results were satisfactory and the ischemic myocardium was salvaged. The infarct risk area (I/R) ratio decreased after drainage. Regional myocardial ischemia and infarction were provided by means of ligature of the left coronary artery for 120 and 240 minutes respectively. Cardiac lymph was collected by conventional procedures. Enzymes released from the myocardium increased significantly in the cardiac lymph. The volume of cardiac lymph gradually increased after ligature of the coronary artery. Administration of hyaluronidase further increased the cardiac lymph flow and significantly decreased the I/R ratio as determined by triphenyl tetrazolium chloride (TTC) and methylene blue staining. Drainage of the cardiac lymph salvaged the ischemic myocardium. Reduction of interstitial edema and augmentation of cardiac lymph flow with the hyaluronidase prevented the development of the infarction. This is the first documentation of the effect of active drainage of cardiac lymph on the development of infarction through observation of the I/R ratio.

[Activity of several carbohydrate exchange enzymes in the lymph during fever reaction]. / Aktivnost' nekotorykh fermentov uglevodnogo obmena v limfe pri likhoradochnoi reaktsii.

This work includes a comparative study of aldolase enzyme activity, LDG and its isoenzymes in thoracic duct lymph and blood serum of rabbits in fever reaction dynamics of various duration. Irrespective of its duration the fever was followed by a significant activation of investigated enzymes in the body liquids. However, in many day fever reaction a rise of LDG level in the lymph was more prolonged then that in the blood. The above studies make it possible to assume that the released enzymes in fever reaction are primarily resorbed by lymphatic capillaries and their activity indices in the blood serum are largely evidenced by the transport function of the lymphatic system.