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A miniaturized rapid paper chromatographic procedure for quality control of technetium-99m sestamibi.

A miniaturized rapid paper chromatographic technique (MRPC) for quality control of a technetium-99m sestamibi preparation was developed and compared with the manufacturer's and Hung et al. techniques. The MRPC system involves the use of 6.0x0.5cm Whatman 3MM paper strip developed in ethyl acetate. The procedure was completed within 3min while that of the manufacturer and Hung techniques took 30-35 and 4min respectively. The Rf range of 99mTc-sestamibi using MRPC was 0.55-0.75 while that of the other two techniques was 0.9-1.0. The results indicate that MRPC can be used to separate 99mTc-sestamibi from any 99mTc contaminant that migrates with the solvent front. The MRPC is a fast and effective chromatographic technique for routine quality control testing of 99mTc-sestamibi preparation.

Chromatography, Paper↗

Serial blood sampling from the portal and hepatic vein in conscious unrestrained miniature pigs.

A technique of implantation of portal and hepatic venous catheters in miniature pigs is described in detail. To overcome early patency loss, Swan-Ganz balloon catheters were used. Portal as well as hepatic venous catheters could be held open to withdraw blood and to apply fluids over a period of at least 50 days. The collected hepatic venous blood represents mixed hepatic outflow. The method is recommended to study absorption of drugs and substrates from the gut and their metabolism in the liver of conscious unrestrained experimental animals.

Animals↗

Liver blood flow measured by indocyanine green in conscious unrestrained miniature pigs.

For studies in physiology and biochemistry of the liver the measurement of hepatic blood flow is a fundamental precondition. Therefore, the least invasive method, the measurement of liver blood flow by indocyanine green (ICG) dye elimination, was re-evaluated for the miniature pig, an animal model of increasing interest. The accuracy of spectrophotometric determination of ICG was enhanced by addition of 5% of desoxycholic acid. It was shown that ICG was eliminated exclusively by the liver. An extraction rate of 30%-50% was found in mini-pigs, but an insufficient rate of 5%-10% in pigs of German landrace. A liver blood flow ranging from 70 to 160 ml/100 g X min-1 was measured. These values are in good accordance with datas from simultaneous estimation of blood flow by 133Xenon washout. As shown for a starvation/refeeding cycle, liver blood flow measurement by ICG combined with permanently implanted catheters is a practicable tool for studies of the metabolic and pharmacologic fate of substances resorbed from the gut.

Animals↗

Effect of pancreatic secretions upon ileal disaccharidase activities of neonatal miniature pigs.

Mechanisms by which pancreatic secretions influence disaccharidase activities in the distal small intestine have been investigated in 1-week-old miniature pigs. Using a combination of biochemical, cytochemical and morphological techniques it has been found that the decrease in lactase specific activity is due solely to a reduction in villus surface area. By contrast, the increased sucrase-isomaltase activities, which occur despite the reduction in villus surface area, are due entirely to increased enzyme expression during enterocyte differentiation.

Animals↗

Glucoregulatory function of glucagon in hypo-, eu- and hyperthyroid miniature pigs.

The glucoregulatory function of glucagon was investigated in hypo-, eu- and hyperthyroid miniature pigs. Infusion glucagon, (3 ng x kg body weight-1.min-1) transiently increased blood glucose (p less than 0.01) and hepatic glucose production (p less than 0.01) in euthyroidism, but was without effect in hyperthyroidism. Infusing glucagon plus somatostatin (2 ng x kg body weight-1.min-1 and 0.2 microgram x kg body weight-1.min-1) transiently increased blood glucose (delta 3.0 to 4.3 mmol/l) and hepatic glucose production (delta 3.3 to 7.7 mumol x kg body weight-1.min-1) in all thyroid states, the effect was less pronounced in hyperthyroid pigs. By contrast, hypoglucagonaemia (74 to 107 pg/ml) at basal insulin (28 to 35 microU/ml) provoked hypoglycaemia (1.4 to 2.2 mmol/l) and a fall in glucose production (delta 4.7 to 8.3 mumol x kg body weight-1.min-1), which was independent of the thyroid state; the effect was most pronounced in hyperthyroidism (p less than 0.01). Hepatic glycogen content, arterial gluconeogenic precursor concentrations as well as the glycaemic response (delta 0.60 mmol/l) to alanine infusion (23 mumol x kg body weight-1.min-1) were all unaffected by hyperthyroidism. We conclude that moderate experimental hyperthyroidism reduces glucagon action due to reduced glycogen mobilisation. This may in part result from increased insulin sensitivity.

Animals↗

Two-way communication for programming and measurement in a miniature implantable stimulator.

Implantable stimulators are needed for chronic electrical stimulation of nerves and muscles in experimental studies. The device described exploits the versatility of current microcontrollers for stimulation and communication in a miniature implant. Their standard outputs can provide the required selectable constant-current sources. In this device, pre-programmed stimulation paradigms were selected by transcutaneous light pulses. The potential of a programmable integrated circuit (PIC) was thus exploited. Implantable devices must be biocompatible. A novel encapsulation method that require no specialised equipment and that used two classical encapsulants, silicone and Teflon was developed. It was tested for implantation periods of up to four weeks. A novel way to estimate electrode impedance in awake animals is also presented. It was thus possible to follow the evolution of the nerve-electrode interface and, if necessary, to adjust the stimulation parameters. In practice, the electrode voltage at the end of a known constant-current pulse was measured by the PIC. The binary coded value was then indicated to the user as a series of muscle twitches that represented the binary value of the impedance measurement. This neurostimulator has been successfully tested in vitro and in vivo. Thresholds and impedance values were chronically monitored following implantation of a self-sizing spiral cuff electrode. Impedance variations in the first weeks could reflect morphological changes usually observed after the implantation of such electrodes.

Animals↗

Cholesterol transport and uptake in miniature swine fed vegetable and animal fats and proteins. 1. Plasma lipoproteins and LDL clearance.

In a 2 X 2 factorial arrangement, miniature pigs were fed four diets containing vegetable protein/fat (soybean) and animal protein (egg white)/fat (beef tallow) to demonstrate the effects of protein and fat source on total plasma cholesterol, lipoprotein distribution, low density lipoprotein (LDL) composition, and plasma clearance of LDL-cholesterol and protein. Beef tallow consumption resulted in greater plasma cholesterol concentration, decreased LDL-cholesterol concentration, and a lower LDL-cholesterol to LDL-protein ratio than did consumption of soybean oil. High density lipoprotein (HDL)-cholesterol concentration was increased by beef tallow consumption. Cholesterol percentage by weight in LDL was significantly greater in pigs consuming soybean oil than those consuming beef tallow. Percentages by weight of protein, triglyceride and phospholipid in LDL were not significantly different in any group. Dietary protein source had no significant effect on total plasma cholesterol concentration, lipoprotein concentration or LDL composition. Egg white consumption decreased fractional catabolic rate and irreversible loss of LDL-cholesterol and LDL-protein when compared with consumption of soy protein. Dietary fat source had no consistent effect on LDL clearance from plasma. Dietary fat and protein seemed to influence lipoprotein metabolism by different mechanisms. Fat source altered lipoprotein concentration and LDL composition, whereas protein source affected the removal rate of LDL from plasma.

Animals↗

Cholesterol transport and uptake in miniature swine fed vegetable and animal fats and proteins. 2. LDL uptake and cholesterol distribution in tissues.

In a 2 X 2 factorial arrangement, miniature pigs were fed four diets containing vegetable protein/fat (soybean) and animal protein (egg white)/fat (beef tallow) to demonstrate the effects of protein and fat source on tissue cholesterol concentrations, uptake of intact low density lipoproteins (LDL) and free cholesterol exchange from LDL to tissues. Soybean oil feeding, compared with beef tallow feeding, resulted in greater concentrations of cholesterol in aorta, heart, and large and small intestines. Similar trends were seen in liver, adipose tissue and skeletal muscle. Dietary protein source had little or no effect on tissue cholesterol concentrations. Uptake of intact LDL, as measured by using [14C]sucrose-LDL, tended to be greater in pigs fed soybean oil or soy protein. Net exchange of free cholesterol from LDL, as measured with [3H]cholesterol, tended to be greater when vegetable products were fed. Relative contributions of whole tissues to total uptake by either mechanism were not influenced by diet. Mechanisms in addition to uptake of cholesterol from LDL seem to be involved in the greater accumulation of tissue cholesterol resulting from polyunsaturated fat feeding.

Animals↗

Immunological state of adult germfree miniature Minnesota pigs.

Fundamental hematological and immunological data were obtained on sexually mature germfree miniature pigs fed, after the milk diet period, with cereal-type diet sterilized by gamma-radiation, and were compared with data of control conventional animals. Germfree adult pigs had a lower count of peripheral blood leukocytes with a lower percentage of neutrophil granulocytes and without any younger forms, a lower total serum protein level with a negligible amount of gamma and alpha 2 globulin fractions and a higher serum albumin and beta globulin level. In the mesenteric lymph nodes and in spleen, surface IgA-bearing cells predominated over surface IgG-bearing cells. Also a large amount of IgA-containing cells was found in the intestinal lamina propria, where the IgG cells were present in a negligible amount. IgM cells were the most frequent surface isotype in peripheral blood. The count of blood T lymphocytes was more than doubled.

Animals↗

Lysosomal enzyme activities in polymorphonuclear leukocytes, macrophages, serum, and spleen of conventional, germ-free, and antigen-free Minnesota miniature swine.

The activities of lysozyme, myeloperoxidase, and elastase were lower in PMNs and AMs from GF and AF Minnesota miniature piglets than in the leukocytes from their CONV counterparts. In the spleen and serum of gnotobiotic piglets only the levels of lysozyme were slightly reduced. Substantially depressed activities of these LEs were found also in PMNs from precolostral piglets in comparison with PMNs from their CONV mother. The bisassociation of GF piglets with Enterococcus liquefaciens and Escherichia coli caused an increase of LE activities in their AMs, spleens, and sera. Fewer LEs were released after phygocytic stimulation with zymosan from PMNs of GF, AF, and precolostral piglets than from PMNs of CONV animals of the same age. These data suggest that the antigenic-microbial stimulation is important for the development of normal lysosomal enzyme activities in PMNs and AMs from gnotobiotic animals.

Animals↗

Feasibility of laparoscopic cholecystectomy with miniaturized instrumentation in 50 consecutive cases.

A new technique utilizing miniaturized (2 mm) instrumentation to perform laparoscopic cholecystectomy (LC) is introduced. The safety and efficacy of this mini-LC were assessed. Fifty consecutive mini-LCs were performed using one 10-mm port and three 2-mm ports (cumulative port size 16 mm). A 2-mm fiberoptic video-laparoscope was placed in the mid-epigastrium through a 2-mm port. A 10-mm umbilical port was then placed under direct visualization, allowing access for standard laparoscopic instruments. Two additional 2-mm ports were placed in the right upper quadrant allowing insertion of grasping instruments. Parameters reviewed included total operative time (OT), postoperative length of stay (LOS), anatomic pathology, complications, and rate of conversion to conventional LC and open cholecystectomy. Results were compared to those of 50 consecutive conventional LCs using two 10-mm and two 5-mm ports (cumulative port size 30 mm). The OT for the mini-LC and conventional LC were 88 +/- 5.9 and 78 +/- 5 minutes (mean +/- SD), respectively, (p = NS), and postoperative LOS for the mini-LC and conventional LC were 1.5 +/- 0. 2 and 1.8 +/- 0.4 days (mean +/- SD), respectively, (p = NS). Of the 50 mini-LC cases, 5 required conversion to conventional LC. One cystic duct leak was detected and successfully treated conservatively; no common bile duct injuries occurred; and no patients required conversion to open cholecystectomy. This study demonstrates the safety and efficacy of minilaparoscopic instruments for the performance of cholecystectomy. The data reveal that this new technique is comparable to conventional LC.

Adult↗

Miniature surface-plasmon resonance immunosensors--rapid and repetitive procedure.

Miniaturized immunosensors based on surface-plasmon resonance (SPR) have been developed by using the sensor recently developed by Texas Instruments. By using this sensor for human immunoglobulin G (IgG) rapid and repetitive measurements could be performed by alternate injection of sample and glycine-HCl buffer. A sensor for human serum albumin (HSA) could also be developed similarly. One difficulty in immunosensor development is dissociation of the strongly bound antigen-antibody complex. A "regenerable" immunosensor has also been developed for this reason.

Antigen-Antibody Reactions↗

Miniaturized solid-phase extraction as a sample preparation technique for the determination of phthalates in water.

Miniaturized solid-phase extraction (SPE) has been developed and successfully employed for the determination of organic species in water samples by liquid chromatography (LC). The method is based on the concept of a microscale extraction technique using a fused-silica capillary column for gas chromatography (GC), so-called in-tube solid-phase microextraction (SPME). The extraction conditions, such as the extraction time and flow-rate for the extraction and desorption process, were investigated as well as the effect of the internal structure of the extraction capillary on the efficiency. By inserting a stainless steel wire into the extraction capillary to reduce the internal volume of the capillary with the same surface area of the coating, an improved extraction and pre-concentration effects were obtained. Further pre-concentration was accomplished by the extraction device with a novel fiber-in-tube configuration. The direct coupling of the extraction method with a LC system has made it possible to determine low levels of phthalates in water samples without high consumption of organic solvents. The system developed must have potential applications for the analysis of environmental and biological samples in aqueous sample matrices.

Capillary Action↗

On-line coupling of miniaturized solid-phase extraction and microcolumn liquid-phase separations.

On-line coupling of miniaturized solid-phase extraction methods and microcolumn separation techniques are reviewed. A conventional solid-phase micro-extraction device and fiber-in-tube solid-phase extraction was employed as the sample preparation method. In the fiber-in-tube technique, a novel fibrous polymeric material was introduced as the extraction medium for the microscale sample preparation step which could be directly coupled with microcolumn liquid-phase separation systems, such as microcolumn liquid chromatography, capillary electrophoresis, and capillary electrochromatography. The applications of these hyphenated systems, consisting of the sample preparation and separation processes, are also reviewed.

Capillary Action↗

Miniaturized dynamic liquid-liquid extraction of organophosphate triesters from blood plasma using the hollow fibre-based XT-tube extractor.

A miniaturized liquid-liquid extractor for bioanalytical sample preparation is described. The extractor consists of a polypropylene hollow fibre mounted inside polytetrafluoroethylene (PTFE) tubing by means of a cross (X) connector and a tee (T) connector. All parts are commercially available, inexpensive, and easily assembled. The aqueous sample, injected into a carrier flow, is pumped along the outside of the fibre and the organic phase, which also wets the pores of the hollow fibre wall, is pumped inside. Eight organophosphate triester (OPE) plasticisers/flame retardants were extracted from 50 microL spiked blood plasma that had been mixed with 50 microL formic acid to denature plasma proteins. The organic phase was a mixture of hexane and methyl tert-butyl ether (MTBE). A high concentration of formic acid in the sample and of MTBE in the organic phase had positive effects on the recovery of some OPE. When investigating the recovery as a function of extraction time it was found that the extraction reached a maximum after 10 min, at a flow-rate of 15 microL min(-1). Recoveries varied between 40 and 80% with RSD around 4% for most compounds. The whole 150- microL extract was injected into a GC-MS system equipped with a programmed-temperature vaporization (PTV) injector. With the MS in selected-ion monitoring (SIM) mode, the LOD for triphenyl phosphate and 2-ethylhexyl diphenyl phosphate were 0.3 and 0.2 ng mL(-1), respectively. More than 40 plasma extractions were performed with the same fibre without any detectable change in extraction efficiency.

Animals↗

A miniaturized active sampler for the assessment of personal exposure to nitrogen dioxide.

A personalized, miniaturized air sampling system was evaluated to estimate the daily exposure of pediatric asthmatics to nitrogen dioxide (NO2). The lightweight device (170 g) uses a sampling pump connected to a solid sorbent tube containing triethanolamine (TEA)-impregnated molecular sieve. The pump is powered by a 9 V battery and samples air over a 24 h period at a collection rate of 0.100 L/min. After exposure, the solid sorbent is removed from the tubes for spectrophotometric analysis (Griess Assay). The lower detection limit of the overall method for NO2 is 11 microg/m3. The linearity, precision and accuracy of the sampler was evaluated. Different NO2 concentrations generated in the laboratory (range: 50 to 340 microg/m3) were simultaneously measured by the TEA tube samplers and colocated continuous chemiluminescent NO(x) analyzers (reference method). The coefficient of determination for the laboratory test derived from ordinary linear regression (OLR) was r2 = 0.99 (y(OLR) = 0.94 x -4.58) and the precision 3.6%. Further, ambient NO2 concentrations in the field (range: 10-120 microg/m3) were verified with continuous chemiluminescent monitors next to the active samplers. Re-weighted least squares analysis (RLS) based on the least median squares procedure (LMS) resulted in a correlation of r2 = 0.68 for a field comparison in Riverside, CA (y(RLS) = 1.01 x -0.94) and r2 = 0.92 in Los Angeles, CA (y(RLS) = 1.31 x -7.12). The precision of the TEA tube devices was 7.4% (at 20-60 microg/m3 NO2) under outdoor conditions. Data show that the performance of this small active sampling system was satisfactory for measuring environmental concentrations of NO2 under laboratory and field conditions. It is useful for personal monitoring of NO2 in environmental epidemiology studies where daily measurements are desired.

Air Pollutants↗

Miniaturized total analysis systems for biological analysis.

This review article discusses and documents the basic philosophies, concepts and current advances in the field of micro-TAS development, with special emphasis on applications in the arena of biosciences. After a brief overview of miniaturization theory and fabrication techniques, areas of microfluidic component development, detection protocols, biochemical assays, and integrated biological analyses are addressed.

Biochemical Phenomena↗