[Determination of purity and impurities of ethylene glycol for industrial use by gas chromatography].

A new test method was established for the determination of purity and impurities of ethylene glycol (EG) by gas chromatography (GC) using a Rtx-624 column (30 m×0.32 mm×1.8 µm). The method determined not only the impurities in EG from the ethylene oxidation process, such as diethylene glycol, triethylene glycol and (1,3-dioxolan-2-yl)methanol, but also the impurities from the oxalate hydrogenation process, such as 1,2-butanediol, 1,4-butanediol, 1,2-hexanediol, and ethylene carbonate, etc. The method showed good repeatability and high detection sensitivity. The lowest LOD of the impurities reached to 0.0002% (mass fraction), and the recoveries of the impurities were in the range of 91.2%-105.4%. The proposed method can be applied in production control, product testing and market trade of ethylene glycol.

Vagal innervation and early postoperative ileus in mice.

INTRODUCTION: Postoperative ileus is characterized by infiltrates of leukocytes in the gut wall 24 h after surgery, which is subject to vagal modulation. We hypothesized that vagal modulation is irrelevant during earlier hours of postoperative ileus and aimed to determine whether afferent neuronal feedback to the central nervous system is altered by vagal innervation during this early period. METHODS: C57BL6 mice were laparotomized and received standardized small bowel manipulation to induce postoperative ileus. Subgroups were vagotomized 3-4 days prior to experiments while control animals were sham-operated. Three or 9 h later a 2-cm jejunal segment was harvested for multi-unit mesenteric afferent nerve recordings in vitro. Intestinal motility was monitored continuously and intestinal muscularis was stained for myeloperoxidase to determine infiltration of leukocytes. RESULTS: Peak amplitudes of intestinal motility and afferent nerve discharge at baseline were not different in all subgroups. Afferent discharge to 5-HT (500 µM) was virtually absent following vagotomy at 3 and 9 h of postoperative ileus (POI) compared to controls (p < 0.05). Maximum afferent nerve discharge to bradykinin and peak firing during maximum distension at 60 mmHg was not different in all subgroups while luminal distension from 10 to 30 mmHg was lower at 3 h of POI following vagotomy compared to controls (p < 0.05). The number of myeloperoxidase positive cells was similar at 3 h of POI in both subgroups; however, at 9 h of POI, ileus counts were increased to 713 ± 99 cells following vagotomy compared to 47 ± 6 cells per square millimeter in control animals. CONCLUSIONS: Vagal afferents mediate sensitivity to low-threshold distension and 5-HT during postoperative ileus but not to high-threshold distension and bradykinin. Vagal inhibition of the intestinal immune response is present at 9 h but not detectable earlier, i.e., at 3 h of postoperative ileus when spinal reflex inhibition may prevail.

Down-regulation of HSP60 expression by RNAi increases lipopolysaccharide- and cerulein-induced damages on isolated rat pancreatic tissues.

The objective of this study was to investigate the function of heat shock protein 60 (HSP60) on pancreatic tissues by applying HSP60 small interfering RNA (siRNA) to reduce HSP60 expression. Rat pancreas was isolated and pancreatic tissue snips were prepared, cultured, and stimulated with low and high concentrations of cerulein (10(-11) and 10(-5) mol/L) or lipopolysaccharide (LPS, 10 and 20 µg/mL). Before the stimulation and 1 and 4 h after the stimulation, the viability and the level of trypsinogen activation peptide (TAP) in the tissue fragments were determined and the levels of tumor necrosis factor-alpha (TNF-α) and interleukin 6 (IL-6) in the culture supernatants were measured. Real-time PCR and Western blotting were used to evaluate the HSP60 mRNA and protein expression. After the administration of siRNA to inhibit HSP60 expression in the isolated tissues, these injury parameters were measured and compared. The pancreatic tissues in the control (mock-interfering) group showed a decreased viability to varying degrees after being stimulated with cerulein or LPS, and the levels of TAP, TNF-α, and IL-6 increased significantly (p < 0.05) in the tissues and/or in the culture supernatant. The expressions of HSP60 mRNA and protein were raised moderately after stimulating 1 h with low concentrations of cerulein or LPS, but decreased with high concentrations of the toxicants. In particular, the expression of HSP60 protein was reduced significantly (p < 0.05) when the tissues were stimulated by the two toxicants for 4 h. In contrast, the tissue fragments in which HSP60 siRNA was applied showed much lower tissue viability (p < 0.01) and higher levels of TNF-a, IL-6, and TAP (p < 0.01) in the tissues or culture supernatant after stimulating with the toxicants at the same dose and for the same time duration as compared with those of the control groups (p < 0.05). The results indicated that both cerulein and LPS can induce injuries on isolated pancreatic tissues, but the induction effects are dependent on the duration of the stimulation and on the concentrations of the toxicants. HSP60 siRNA reduces HSP60 expression and worsens the cerulein- or LPS-induced injuries on isolated pancreatic tissues, suggesting that HSP60 has a protective effect on pancreatic tissues against these toxicants.

Role of the vagus nerve on the development of postoperative ileus.

INTRODUCTION: Postoperative ileus involves reflex inhibition of intestinal motility within hours after surgery and a subsequent intestinal inflammatory response that is characterized by efferent vagal modulation via acetylcholine receptors on intestinal macrophages. We aimed to characterize the role of vagal modulation of intestinal motility during the early hours after surgery. METHODS: C57BL6 mice underwent laparotomy and standardized small bowel manipulation to induce postoperative ileus. Subgroups were vagotomized 3-4 days prior to experiments or received pharmacological inhibition of the acetylcholine alpha7 subunit with the inhibitor alpha-bungarotoxin, while control animals were sham operated and remained otherwise untreated. Three hours later, a 2-cm jejunal segment was harvested with the mesentery attached. Mesenteric afferent nerve recordings were established in an organ bath generating a multiunit signal with subsequent computerized analysis. Intraluminal pressure was continuously recorded to assess intestinal motility. Afferent nerve responses were quantified at baseline and to chemical stimulation with bradykinin (0.5 microM) or serotonin (5-HT; 500 microM) and following mechanical stimulation by continuous ramp distension to 60 mmHg. RESULTS: Peak amplitudes of intestinal motility and afferent nerve discharge at baseline were not different following chronic vagotomy, alpha-bungarotoxin or sham operation. Maximum afferent discharge to 5-HT following alpha-bungarotoxin was comparable to sham controls, while the response was reduced in chronically vagotomized animals (p < 0.05). Maximum afferent nerve discharge to bradykinin and peak firing during maximum distension at 60 mmHg was similar in the different subgroups. At luminal distension from 10 to 30 mmHg, afferent discharge was lower in vagotomized animals compared to sham controls (p < 0.05) but unchanged after alpha-bungarotoxin. CONCLUSIONS: Sensitivity to low-threshold distension and 5-HT is mediated via vagal afferents during postoperative ileus, while sensitivity to high-threshold distension and bradykinin is independent of vagal afferent innervation. Early inhibition of intestinal motility at 3 h after onset of postoperative ileus does not appear to depend on vagal innervation.

Ascitic fluid and serum from rats with acute pancreatitis injure rat pancreatic tissues and alter the expression of heat shock protein 60.

Acute pancreatitis (AP) is an inflammatory process in which cytokines and chemokines are involved. After onset, extrapancreatic stimuli can induce the expression of cytokines in pancreatic acinar cells, thereby amplifying this inflammatory loop. To further determine the role and mechanism of irritating agents in the pathogenesis of AP, rat pancreatic tissues were stimulated with ascitic fluid (APa) and serum (APs) from rats with AP or with lipopolysaccharide (LPS). In addition, the alteration of heat shock protein 60 (HSP60) expression was evaluated. Rat pancreas was removed and meticulously snipped to fragments. The snips were cultured for up to 48 h. During this period, the tissue viability as well as amylase and TNF-alpha levels in the supernatant and the HSP60 expression in the pancreatic tissue before and after stimulation by APa, APs, and LPS were assayed time-dependently. At different time-points during the culture, the viability and the amylase activity in the pancreatic tissue remained largely stable. After stimulation with APa, APs, or LPS for 1 h, the pancreatic tissues showed some damage, and this was followed by a sharp decrease in the viability accompanied by increased levels of amylase and TNF-alpha in the culture medium 2 or 4 h after stimulation (p < 0.05). In contrast, both the HSP60 mRNA and protein levels had a relatively high expression in the freshly prepared tissue fragments (0 h). As the culturing period was extended, the expression of HSP60 mRNA decreased only slightly; at the same time, the HSP60 protein levels decreased over a prolonged culture time, significantly so from 12 through 48 h (p < 0.05). After stimulation with APs, APa, or LPS, both the expression of HSP60 mRNA and protein in the tissue fragments increased slightly at 1 h and decreased significantly thereafter at 2 and 4 h (p < 0.05). APa, APs, or LPS induce injuries on isolated pancreatic tissues, accompanied by an altered HSP60 expression pattern in a time-dependent manner.

Regulation of HSP60 and the role of MK2 in a new model of severe experimental pancreatitis.

The objective of this study was to investigate the role of MAPKAP kinase 2 (MK2) and heat shock protein (HSP) HSP60 in the pathogenesis of a new model of severe acute pancreatitis (AP). MK2 plays a significant role in the regulation of cytokines. It has been shown that induction and expression of several HSPs can protect against experimental pancreatitis. Interplay between both systems seems of high interest. Mice with a homozygous deletion of the MK2 gene were used. Severe AP was induced by combined intraperitoneal injections of cerulein with lipopolysaccharide (LPS). Severity of AP was assessed by biochemical markers and histology. The serum IL-6 and lung myeloperoxidase (MPO) levels were determined for assessing the extent of systemic inflammatory response. Expression of HSP25, HSP60, HSP70, and HSP90 was analyzed by Western blotting. Repeated injections of cerulein alone or cerulein plus LPS (Cer+LPS) resulted in local inflammatory responses in the pancreas and corresponding systemic inflammatory changes with pronounced severity in the Cer+LPS group. Compared with the C57Bl wild-type mice, the MK2-/- mice presented with significant milder pancreatitis and attenuated responses of serum amylase and trypsinogen activity. Furthermore, serum IL-6 was decreased as well as lung MPO activity. Injection of LPS alone displayed neither pancreatic inflammatory responses nor alterations of pancreatic enzyme activities but evidently elevated serum IL-6 levels and increased lung MPO activity. In contrast hereto, in the MK2-/- mice, these changes were much milder. Increased expression of HSP25 and HSP60 occurred after induction of AP. Especially, HSP60 was robustly elevated after Cer+LPS treatment, in both MK2-/- and wild-type mice. Thus the homozygous deletion of the MK2 gene ameliorates the severity of acute pancreatitis and accompanying systemic inflammatory reactions in a new model of severe acute pancreatitis. Our data support the hypothesis that MK2 participates in the multifactorial regulation of early inflammatory responses in AP, independently of the regulation of stress proteins like HSP25 and HSP60 and most likely due to its effect on cytokine regulation.