Measurement of Glass Transition Temperatures of Freeze-Concentrated Solutes by Differential Scanning Calorimetry

Thermal analysis of aqueous solutions in which the solute does not crystallize immediately upon freezing was carried out to define the effects of experimental parameters on thermograms in the glass transition region. The intensity of enthalpy relaxations in the glass transition region is related to both the rate of cooling and the rate of heating through the glass transition region—slow cooling or slow heating increases the extent of structural relaxation in the glassy state and increases the intensity of the endotherm. Plots of the logarithm of heating rate versus l /Tg are linear, and activation enthalpies for structural relaxation are in the range of 210–350 kJ/mol. For polymeric solutes, both the activation enthalpies for structural relaxation and the heat capacity change accompanying the glass transition increase with increasing molecular weight of the solute. Molecular weight dependence of the observed midpoint of the glass transition agrees with the Fox–Flory relationship. Results are compared and contrasted with glass transitions in solid polymers and with the glass transition of hyperquenched water. Practical implications for characterization of formulations intended for freeze-drying are discussed.     

Higher genetic susceptibility to inflammation in mild disease activity of systemic lupus erythematosus

In order to test the hypothesis that stratification of Mexican Modification of the Systemic Lupus Erythematosus Disease Activity Index (MEX-SLEDAI) simplifies the genetic study of SLE, we evaluated the genetic susceptibility to inflammation and defects in clearance of immune complexes among SLE patients in Taiwan. SLE phenotypes were stratified according to the MEX-SLEDAI scores into two subgroups (≤10 and >10), and then according to renal disorder and neurological disorder, aiming to minimize any loss of power associated with disease heterogeneity. Upon stratification, IL1-β polymorphism and LTA were significantly associated with SLE within the MEX-SLEDAI ≤10 subgroup. When SLE patients were classified into two subgroups with or without renal disorder to stratify the genetic study, we could find that the stratification with renal disorder could partially confirm the hypothesis that stratification of MEX-SLEDAI score simplifies the genetic study of complex diseases such as SLE. So we concluded that in the mild disease state of SLE, stratification of disease phenotypes, especially IL1-β and LTA, according to MEX-SLEDAI scores could reveal new associations between candidate genes and disease activity index of SLE. Li-Jen Tsai and Sheng-Hsiung Hsiao have contributed equally to this study.     

Pharmacodynamics and Biophasic Drug Levels of Methionine Enkephalin

Methionine enkephalin (Met-E) is a naturally occurring pentapeptide. It appears to mediate pain perception by blocking CNS pathways. Using rabbits, a log dose relationship was obtained between Met-E-induced dilation of the pupil (mydriasis) and constant intravenous infusion dose rates (14, 20, 28, 37, 56, 73, and 129 µg/min/kg). Steady-state dilation is reached within 9 min. Dose-effect curves (DEC) were fitted by a linear regression analysis of the log dose versus percentage dilation plots. Fitted DEC were used to determine temporal profiles for the relative biophasic drug level.     

Effects of oral arginine and glutamine on radiation-induced injury in the rat

BACKGROUND: Exposure of the abdominal region to ionizing radiation is associated with serious untoward symptoms of intestinal dysfunction and some reports indicate that nutrient supplements may reduce these adverse effects. This study was designed to investigate the possible beneficial effects of oral arginine or glutamine supplementation on the radiation-induced tissue injury. MATERIALS AND METHODS: Rats were given one of three feeding regimens: standard diet and water (control group), diet and water containing 2% arginine (arginine group), diet and water containing 2% glutamine (glutamine group) for 3 days prior to radiation. All rats were then subjected to a single does of 1100 cGy to the abdomen. Several serum biochemical parameters and the histologic alterations in different segments of gastrointestinal tract and liver were measured 4 days after irradiation. RESULTS: All the arginine-fed rats developed diarrhea on Day 4 postirradiation, compared to 71% incidence in control rats and 86% in glutamine-fed rats. Serum levels of aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) in the arginine group were markedly higher than those in other groups. On histological examination, radiation caused more serious damage to various segments of intestine in the arginine-fed rats compared to rats on other feeding regimens. CONCLUSION: These observations seriously question the beneficial effects of arginine and glutamine supplementations on radiation-induced tissue injury.     

Involvement of superoxide anion in the pathogenesis of simple mechanical intestinal obstruction

BACKGROUND: Mechanism underlying the pathogenesis of mechanical intestinal obstruction has been suggested to be closely associated with bowel inflammatory response in which reactive oxygen metabolites might play an important role. This study was designed to examine the involvement of superoxide anion in the obstruction-induced intestinal injury. MATERIALS AND METHODS: Rats were randomly aasigned to four groups: sham, obstruction, obstruction with polyethylene glycol (PEG), and obstruction with polyethylene glycol-superoxide dismutase (PEG-SOD) groups. A ligation at the ileum 20 cm proximal to the cecum was created under anesthesia. The superoxide anion production and the pathological manifestations in the obstructed intestine were measured after 24 h of ligation. RESULTS: There were significant intestinal shortening, distension, fluid accumulation and mucosal damage in the segment proximal to the ligation site. Pronounced generation of superoxide anion was found in the obstructed intestinal segment. Supplement of SOD, a superoxide free radicals scavenger, ameliorated obstruction-induced bowel distension, fluid accumulation and mucosal damage. CONCLUSION: These data suggest superoxide anion is one of the important mediators in the pathophysiologic changes of simple mechanical intestinal obstruction.     

Periodic alterations of jejunal mucosa morphology following free microvascular transfer for pharyngoesophageal reconstruction.

BACKGROUND: Free jejunal flap reconstruction is the treatment of choice for patients after pharyngoesophagectomy. It remains unclear as to how the transplanted jejunal mucosal damage proceeds after the warm ischaemia. The current study aims to assess the relationship between the duration of ischaemia and the damage of jejunal mucosa. PATIENTS AND METHODS: From May 2002 to February 2003, 15 free jejunal flaps in 15 patients were transplanted to the cervical area for the reconstruction after pharyngoesophagectomy. Biopsy specimens were taken from the monitor loop at the time of pedicle ligation, 10 min after reperfusion, every day for 10 days, 14th day, 28th day, and 40th day after operation. Mucosal injury was assessed based on an accepted three-point scale which evaluates oedema, inflammation, mucosal necrosis or exfoliation, shortening of villi, and increase of goblet cells. FINDINGS: All 15 jejunal flaps survived. The mean ischaemia time was 68.7+/-5.2 min (range: 37-116). Serious injury to the mucosa was observed at 10 min after reperfusion, and gradually recovered until the 8th day, when it became normal in all flaps. The degree of damage was not found to be correlated with the length of ischaemia (less than 116 min). Severe ischaemia/reperfusion-induced mucosal damage occurs immediately following reperfusion and gradually recovers with time. The severity of the damage is not related linearly to the ischaemia time within 2h. The mucosa recovers gradually from the 8th day and returns to normal at the 28th day.     

An Improved Microscope Stage for Direct Observation of Freezing and Freeze Drying

A microscope stage for observation of freezing and freeze drying is described. The stage uses thermoelectric (Peltier) heaters configured in two stages, with circulating fluid as a heat sink on the high temperature side. Lowest attainable sample temperature is about –47°C. Principal advantages of this system are closed-loop control of stage temperature, rapid response to changes in temperature set point, and improved documentation of experiments by use of a video recorder system with a character generator which allows display of sample identity and temperature. Accuracy of measuring the sample temperature in the field of view was validated by comparing observed values of eutectic melting with published values for a series of solutes with eutectic temperatures in the range from –2°C to –32°C. Good agreement was obtained throughout this range.     

Measurement of Glass Transition Temperatures in Freeze Concentrated Solutions of Non-Electrolytes by Electrical Thermal Analysis

The electrical resistance (R) of frozen aqueous solutions was measured as a function of temperature in order to determine whether this technique can be applied for determination of glass transition temperatures of maximally freeze concentrated solutions (T_g) of non-electrolytes which do not crystallize during freezing. Electrical thermal analysis (ETA) thermograms of frozen solutions containing the solute alone show a gradual change in slope over the temperature range of interest, with no inflection point which corresponds to T_g. However, addition of low levels (about 0.1%) of electrolyte changes the shape of the thermogram into a biexponential function where the intersection of the two linear portions of the log (R) vs. T plot corresponds to the glass transition region. The total change in log (R) over the temperature range studied increases as the ionic radius of the reporter ion increases. The sharpest inflection points in the log (R) vs T curves, and the best correlation with DSC results, were obtained with ammonium salts. T_g values measured by ETA were compared with values measured by DSC. DSC thermograms of solutes with and without electrolyte (0.1%) show that the electrolyte decreases T_g by about 0.5 to 1.0°C. However, T_g values measured by ETA are somewhat higher than those measured by DSC, and difference between the two methods seems to increase as T_g decreases. T_g as measured by ETA is less heating rate dependent than DSC analysis, and ETA is a more sensitive method than DSC at low solute concentrations and at low heating rates. Results of electrical thermal analysis of frozen solutions are compared and contrasted with the electrical resistance vs. temperature behavior of polymer-electrolytes. ETA appears to be a useful complementary technique to DSC for characterizing formulations intended for freeze drying.     

Electrolyte-Induced Changes in Glass Transition Temperatures of Freeze-Concentrated Solutes

Addition of electrolytes to solutions of non-crystallizing solutes can cause a significant decrease in the glass transition temperature (T_g) of the maximally freeze-concentrated solution. For example, addition of 2% sodium chloride to 10% solutions of dextran, PVP, lactose, and sucrose causes a decrease in T_g of 14° to 18°C. Sodium phosphate has a smaller effect on T_g, and is unusual in that 1% to 2% sodium phosphate in 10% PVP causes a second glass transition to be observed in the low-temperature thermogram, indicating a phase separation in the freeze concentrate. Comparison of DSC thermograms of fast-frozen solutions of sucrose with and without added sodium chloride shows that electrolyte-induced reduction of T_gis not caused by a direct plasticizing effect of the electrolyte on the freeze concentrate. Measurement of unfrozen water content as a function of temperature by a pulsed nmr method shows that the most likely mechanism for electrolyte-induced changes in T_g is by increasing the quantity of unfrozen water in the freeze concentrate, where the unfrozen water acts as a plasticizer and decreases T_g. The correlation time (_c) of water in the freeze concentrate is in the range of 10^–7 to 10^–8 seconds. The results underscore the importance of minimizing the amount of added salts to formulations in-tended for freeze drying.