Microfiltration sampling in rats and in cows: toward a portable device for continuous glucocorticoid hormone sampling.

To monitor temporal patterns of glucocorticoids hormones in living animals, most often blood samples are collected. Blood sampling is invasive and subjects may find it--in particular--unpleasant when multiple samples are collected. We have developed a microfiltration collection device (MCD) sampling continuously, pulse-free, over a selected period of time, with minimum invasiveness as the device is inserted with only one venipuncture. The MCD consists of a hollow fiber membrane (probe), capillary collection coil and flow creator. Three biocompatible hollow fiber membranes were assessed on flow rate in rats, by placing the probe intraperitoneally, subcutaneously, or intravascularly and with or without heparin coating. The probe made from polyethylene coated with ethylene vinyl alcohol-heparin conveyed the best results and had the most benefit of the heparin coating. Consequently this probe was built into a collection device and tested in cows, sampling blood microfiltrate. Cortisol (protein-bound and -free) could be monitored in cows over a period of 7 hours. This device has several major advantages compared to manual blood collection: minor stress is induced by the application of the device; it has a low weight and can therefore be used in freely active subjects being in their own surroundings. The device can be sterilized and manufactured as a disposable tool, and the filled MCD can be shipped by regular mail to a specialized laboratory facility for analysis.

In vitro sampling and storage of proteins with an ultrafiltration collection device (UCD) and analysis with absorbance spectrometry and SELDI-TOF-MS.

Frequent in vivo sampling of blood proteins is often stressful, making it difficult to obtain more than a few samples. As a result, only limited time-profiles can be made. We have developed an ultrafiltration collection device (UCD) for continuous sampling. The UCD consists of a hollow fiber, a coil and a flow creator. Hollow fiber membranes are often hydrophobic and this can result in adsorption of protein and/or peptides, leading to clogged membranes. Adsorption was tested with a hydrophobic and hydrophilic peptide and two biocompatible hollow fibers made from different materials. The hollow fiber made from poly(ethylene) coated with ethylenevinyl alcohol gave near 100% recovery for both peptides. This was in contrast to the poly(sulfone) hollow fiber when sampling the hydrophobic peptide. Filling the coil with various peptide concentrations gave good recovery and insignificant diffusion even after storage for 6 d at 37 degrees C. Continuous pulse-free sampling was tested by vacuum. An average flow rate of 423 +/- 50 nl min(-1) over a period of 4 d was created using S-Monovette. The flow rate gradually declined during this period by <5% every consecutive day. In addition, we also examined a complex sample-serum in the poly(ethylene) hollow fiber. Serum and ultrafiltrate were spotted onto a protein chip and analyzed by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS). Six proteins out of 64 were found to be significantly different between serum and the ultrafiltrate (p < 0.05). The UCD has the potential to be used for in vivo real-time monitoring.

Modeling cerebral arteriovenous lactate kinetics after intravenous lactate infusion in the rat.

Venous-arterial lactate differences across the brain during lactate infusion in rats were studied, and the fate of lactate was described with a mathematical model that includes both cerebral and extracerebral kinetics. Ultrafiltration was used to sample continuously and simultaneously arterial and venous blood. Subsequent application of flow injection analysis and biosensors allowed the measurement of glucose and lactate concentrations every minute. Because of the high temporal resolution, arteriovenous lactate kinetics could be modeled in individual experiments. The existence of both a cerebral lactate sink and a lactate exchangeable compartment, representing approximately 24% of brain volume, was thus modeled.

The potential of biosensor technology in clinical monitoring and experimental research.

Glucose or lactate biosensors are very useful for monitoring metabolism. Continuous monitoring of glucose is for example very important in diabetic patients. The measurement of lactate, a marker for oxygen deficiency, is used in the intensive care unit to monitor the patients' condition. In our laboratory we have developed two types of on-line biosensors to measure in vivo glucose and lactate: a sandwich-type biosensor and, very recently, a miniaturized flow-through biosensor. These biosensors are not placed in the body itself, but are connected to implanted microdialysis or ultrafiltration probes. Both types of biosensors are based on the oxidation of substrate using glucose oxidase or lactate oxidase and electrochemical detection. In the sandwich-type sensor, the enzymes are physically immobilized between two cellulose nitrate filters, and operate with ferrocene as a mediator. In the miniaturized biosensor, with an internal volume of 10-20 nanolitres, the enzymes are immobilized on the electrode via in situ encapsulation in poly(1,3-phenylenediamine). In this review we shall explain the working of these biosensors, and describe their application in clinical monitoring and experimental research.

Quantitative on-line monitoring of cellular glucose and lactate metabolism in vitro with slow perfusion.

An on-line in vitro perfusion technique is described that allows the continuous quantification of cellular glucose metabolism in vitro. Using biosensor technology, we measure glucose and lactate metabolism at a minute-to-minute time resolution for periods up to several days. The application of our perfusion-detection technique for in vitro monitoring is demonstrated in a wide variety of cells, including primary neuronal and astroglia cultures, yeast cells, and human lymphocytes. The method shows that variations in oxygen delivery or exposure to a noncompetitive pseudosubstrate (here 2-deoxyglucose) affects normal glucose metabolism. The innovative advantage of the present system is that, in contrast to other devices including a recently described system, metabolism per cell can be quantified. The potential of in vitro on-line monitoring is discussed for application in studying normal and abnormal metabolism, toxic and nontoxic drug effects, and human tissue biopsies.

Minimally invasive technique based on ultraslow ultrafiltration to collect and store time profiles of analytes.

A device is described to collect and store continuously time profiles of analytes over periods of 24 h suitable to sample freely moving individuals (humans and animals). The device consists of a hollow fiber ultrafiltration probe, a long capillary and a nonmechanical unit (a disposable medical syringe) driven by vacuum to withdraw fluid. The principle is that at low rates (< or = 100 nL/min), sample fluid is collected through the ultrafiltration probe into the capillary. A time resolution of less than 5 min over a 24-h collection and storage period was achieved for lactate and glucose. To illustrate an in vivo application, devices were fixed under the wing of freely moving broiler chickens, with subcutaneous or intravenous probe placements. The device can be produced as a disposable, and it may become applied for ex vivo and in vitro monitoring.

Utilization of in vivo ultrafiltration in biomedical research and clinical applications.

Ultrafiltration (UF) is a filtrate selection method with a wide range of biomedical and clinical applications, including detoxification of blood in hemodialysis and peritoneal dialysis. New is, however, the use of UF as a convenient in vivo sampling method that, for example, has been used in diabetics. Ultrafiltration avoids complicated and time-consuming recovery calculations that are necessary when using in vivo microdialysis, as recoveries of low molecular weight molecules are near 100%. The subcutaneously or intravenously placed UF probes have been studied for off-line sample analysis and for continuous on-line monitoring, in a wide variety of species, including dogs, rats, pigs and humans. This review discusses the potential of in vivo UF as a continuous tissue sampling technique in clinical research areas, and in several major biomedical applications including glucose and lactate monitoring and drug kinetic studies.

Evidence for a lactate pool in the rat brain that is not used as an energy supply under normoglycemic conditions.

Lactate derived from glucose can serve as an energy source in the brain. However, it is not certain how much lactate, directly taken from the blood circulation, may replace glucose as an energy source. This study aimed to estimate the uptake, release, and utilization of lactate entering the brain from the blood circulation. The change in cerebral venous-arterial glucose and lactate differences after lactate infusions in the anesthetized rat were measured. Ultrafiltration probes were placed in the aorta and in the jugular vein, and connected to a flow injection analysis system with biosensors for glucose and lactate. Measurements were taken every minute. Lactate efflux was observed at baseline, whereas an influx of lactate was seen during lactate infusion. Immediately after the infusion there was a net efflux of lactate from the brain. The results suggest that the majority of lactate moving into the brain is not used as an energy substrate, and that lactate does not replace glucose as an energy source. Instead, the authors propose the concept of a lactate pool in the brain that can be filled and emptied in accordance with the blood lactate concentration, but which is not used as an energy supply for cerebral metabolism.

Quantitative on-line monitoring of hippocampus glucose and lactate metabolism in organotypic cultures using biosensor technology.

Quantitative glucose and lactate metabolism was assessed in continuously perfused organotypic hippocampal slices under control conditions and during exposure to glutamate and drugs that interfere with aerobic and anaerobic metabolism. On-line detection was possible with a system based on slow perfusion rates, a half-open (medium/air interface) tissue chamber and a flow injection analytic system equipped with biosensors for glucose and lactate. Under basal conditions about 50% of consumed glucose was converted to lactate in hippocampal slice cultures. Using medium containing lactate (5 mm) instead of glucose (5 mm) significant lactate uptake was observed, but this uptake was less than the net uptake of lactate equivalents in glucose-containing medium. Glucose deprivation experiments suggested lactate efflux from glycogen stores. The effects of drugs compromising or stimulating energy metabolism, i.e. 2-deoxyglucose, 3-nitropropionic acid, alpha-cyano-4-hydroxycinnamate, l-glutamate, d-asparate, ouabain and monensin, were tested in this flow system. The data show that maintaining Na+ and K+ gradients consumed much of the energy but do not support the hypothesis that l-glutamate stimulates glycolysis in hippocampal slice cultures.

A lightweight measuring device for the continuous in vivo monitoring of glucose by means of ultraslow microdialysis in combination with a miniaturised flow-through biosensor.

BACKGROUND: Tight regulation of blood glucose levels from patients suffering from diabetes mellitus can significantly reduce the complications associated with this disease. For this reason, elaborate research efforts have been devoted to the development of a glucose sensor for the continuous in vivo monitoring of glucose. Although the use of microdialysis as a sampling interface between the body and the biosensor is widely accepted, a major drawback of conventional microdialysis is the limited in vivo recovery. Here, ultraslow microdialysis is proposed in order to obtain (near) quantitative in vivo recoveries. To avoid, however, unacceptable long delay times, the need for a small and low dead volume measuring device was recognised. METHODS: A portable lightweight measuring device for continuous in vivo monitoring of glucose in subcutaneous tissue is presented. The measuring device consists of a miniaturised flow-through biosensor, connected to a microdialysis probe and a semi-vacuum pump. The biosensor is based on the amperometric detection of hydrogen peroxide after conversion of glucose by immobilised glucose oxidase. A portable potentiostat equipped with data logging is used for detection and registration. RESULTS: The device was validated for its accuracy, precision, linearity, sensitivity, selectivity and stability during ex vivo and in vivo experiments. The linearity was found to be up to 30 mmol/l with a limit of detection of 0.05 mmol/l. The precision, depending on the biosensor tested was found to be 2-4%. No contribution to the signal could be observed from several tested electroactive species. The accuracy was found to be well in accordance with the criteria set for methods of Self Monitoring of Blood Glucose for patients with diabetes mellitus. The biosensors could be used for up to 3 days in the continuous mode. In vivo monitoring of glucose in dialysate of subcutaneous sampled tissue during glucose tolerance tests in healthy volunteers demonstrated the potential of this measuring device. CONCLUSIONS: A portable lightweight measuring device is presented which can measure continuously glucose in vivo without excessive calibration steps. The performance characteristics determined justify the application of this measuring device for the in vivo monitoring of glucose in subcutaneous sampled interstitium of diabetic patients.