Development of an In Vivo Retrodialysis Calibration Method Using Stable Isotope Labeling to Monitor Metabolic Pathways in the Tumor Microenvironment via Microdialysis.

Microdialysis is a technique that utilizes a semipermeable membrane to sample analytes present within tissue interstitial fluid. Analyte-specific calibration is required for quantitative microdialysis, but these calibration methods are tedious, require significant technical skill, and often cannot be performed jointly with the experimental measurements. Here, we describe a method using retrodialysis with stable-isotope-labeled analytes that enables simultaneous calibration and quantification for in vivo tumor microdialysis. Isotope-labeled amino acids relevant to immuno-metabolism in the tumor microenvironment (tryptophan, kynurenine, glutamine, and glutamate) were added to the microdialysis perfusate, and microdialysis probes were inserted in subcutaneous CT26 and MC38 tumors in mice. The levels of both the endogenous and isotope-labeled amino acids in the perfusate outlet were quantified using LC-MS/MS. Plasma and tumor tissue samples were also collected from the same mice and amino acid levels quantified using LC-MS/MS. Amino acids which showed statistically significant differences between the CT26-bearing and MC38-bearing mice in tumor lysate (tryptophan, kynurenine, and glutamine) and plasma (glutamate) were not the same as those identified as significantly different in tumor interstitial fluid (kynurenine and glutamate), underscoring how microdialysis can provide unique and complementary insights into tumor and immune metabolism within the tumor microenvironment.

A prolonged run-in period of standard subcutaneous microdialysis ameliorates quality of interstitial glucose signal in patients after major cardiac surgery.

We evaluated a standard subcutaneous microdialysis technique for glucose monitoring in two critically ill patient populations and tested whether a prolonged run-in period improves the quality of the interstitial glucose signal. 20 surgical patients after major cardiac surgery (APACHE II score: 10.1 ± 3.2) and 10 medical patients with severe sepsis (APACHE II score: 31.1 ± 4.3) were included in this investigation. A microdialysis catheter was inserted in the subcutaneous adipose tissue of the abdominal region. Interstitial fluid and arterial blood were sampled in hourly intervals to analyse glucose concentrations. Subcutaneous adipose tissue glucose was prospectively calibrated to reference arterial blood either at hour 1 or at hour 6. Median absolute relative difference of glucose (MARD), calibrated at hour 6 (6.2 (2.6; 12.4) %) versus hour 1 (9.9 (4.2; 17.9) %) after catheter insertion indicated a significant improvement in signal quality in patients after major cardiac surgery (p < 0.001). Prolonged run-in period revealed no significant improvement in patients with severe sepsis, but the number of extreme deviations from the blood plasma values could be reduced. Improved concurrence of glucose readings via a 6-hour run-in period could only be achieved in patients after major cardiac surgery.

A protocol for studying the interaction between small-molecular drug and DNA using microdialysis sampling integrated with chemiluminescent detection.

It is of great significance to understand how drug molecules interact with DNA, which is one of the most important aspects of biological investigations in drug discovery at molecular level. Herein, with the model of ractopamine and calf thymus DNA (ct-DNA), a protocol using microdialysis (MD) sampling integrated with flow injection (FI)-chemiluminescent (CL) detection was developed for studying the interaction between small-molecular drug and DNA. After incubating ractopamine with ct-DNA, unbound ractopamine was on-line sampled using a MD probe, followed by being introduced into a FI-CL system for quantitation. The detected concentrations of unbound ractopamine were calibrated with the recovery of the MD probe, and then treated with Klotz analysis and Scatchard analysis to acquire the binding parameters. The MD probe exhibited a mean recovery of 27.3% for ractopamine sampling under the optimal conditions. The binding constants obtained by Klotz analysis and Scatchard analysis were 3.8 × 106 M-1 and 3.9 × 106 M-1, respectively, showing negligible difference. Ractopamine was estimated to have only one type of binding site on ct-DNA. The obtained results demonstrated that the protocol using on-line MD sampling integrated with FI-CL detection is a simple and reliable technique platform for studying the interaction between small-molecular drug and DNA.

Intraabdominal microdialysis - methodological challenges.

Microdialysis is used for in vivo sampling of extracellular molecules. The technique provides a continuous and dynamic view of concentrations of both endogenous released and exogenous administered substances. Microdialysis carries a low risk of complications and has proven to be a safe procedure in humans. The technique has been applied in several clinical areas, including gastrointestinal surgery. Microdialysis may be used for studies of tissue metabolism, and the technique is also a promising tool for pharmacological studies of drug penetration into abdominal organ tissue and the peritoneal cavity. The clinical significance of intraabdominal microdialysis in postoperative monitoring of surgical patients has yet to be proven. In this review, we introduce the microdialysis technique, and we present an overview of theoretical and practical considerations that should be taken into account when using microdialysis in intraabdominal clinical research.

Consensus statement from the 2014 International Microdialysis Forum.

Microdialysis enables the chemistry of the extracellular interstitial space to be monitored. Use of this technique in patients with acute brain injury has increased our understanding of the pathophysiology of several acute neurological disorders. In 2004, a consensus document on the clinical application of cerebral microdialysis was published. Since then, there have been significant advances in the clinical use of microdialysis in neurocritical care. The objective of this review is to report on the International Microdialysis Forum held in Cambridge, UK, in April 2014 and to produce a revised and updated consensus statement about its clinical use including technique, data interpretation, relationship with outcome, role in guiding therapy in neurocritical care and research applications.

Effect of Perfusion Fluids on Recovery of Inflammatory Mediators in Microdialysis.

Microdialysis is an excellent tool to assess tissue inflammation in patients, but in vitro systems to evaluate recovery of inflammatory mediators have not been standardized. We aimed to develop a reference plasma preparation and evaluate different perfusion fluids with respect to recovery of metabolic and inflammatory markers. The reference preparation was produced by incubation of human blood with lipopolysaccharide and cobra venom factor to generate cytokines and activate complement, respectively. Microdialysis with 100 kDa catheters was performed using different colloid and crystalloid perfusion fluids (hydroxyethyl starch (HES) 130/0.4, HES 200/0.5, hyperosmolar HES 200/0.5, albumin 200 g/l, T1 perfusion fluid and Ringer's acetate) compared to today's recommended dextran 60 solution. Recovery of glucose, glycerol and pyruvate was not significantly different between the perfusion fluids, whereas lactate had lower recovery in HES 200/0.5 and albumin perfusion fluids. Recovery rates for the inflammatory proteins in comparison with the concentration in the reference preparation differed substantially: IL-6 = 9%, IL-1ß = 18%, TNF = 0.3%, MCP-1 = 45%, IL-8 = 48%, MIG = 48%, IP-10 = 25%, C3a = 53% and C5a = 12%. IL-10 was not detectable in microdialysis dialysate. HES 130/0.4 and HES 200/0.5 yielded a recovery not significantly different from dextran 60. Hyperosmolar HES 200/0.5 and albumin showed significantly different pattern of recovery with increased concentration of MIG, IP-10, C3a and C5a and decreased concentration of IL-1ß, TNF, MCP-1 and IL-8 in comparison with dextran 60. In conclusion, microdialysis perfusion fluid dextran 60 can be replaced by the commonly used HES 130/0.4, whereas albumin might be used if specific immunological variables are in focus. The present reference plasma preparation is suitable for in vitro evaluation of microdialysis systems.

Role of microdialysis in pharmacokinetics and pharmacodynamics: current status and future directions.

Diagnostic and therapeutic decisions in medical practice are still generally based on blood concentrations of drugs and/or biomolecules despite the knowledge that biochemical events and pharmacological effects usually take place in tissue rather than in the bloodstream. Microdialysis is a semi-invasive technique that is able to measure concentrations of the free, active drug or endogenous compounds in almost all human tissues and organs. It is currently being used to monitor brain metabolic processes and quantify tissue biomarkers, and determine transdermal drug distribution and tissue pharmacokinetics, confirming its importance as a widely used sampling technique in clinical drug monitoring and drug development as well as therapy and disease follow-up, contributing to rationalizing drug dosing regimens and influencing the clinical decision-making process.

Cerebral microdialysis in traumatic brain injury and subarachnoid hemorrhage: state of the art.

Cerebral microdialysis (CMD) is a laboratory tool that provides on-line analysis of brain biochemistry via a thin, fenestrated, double-lumen dialysis catheter that is inserted into the interstitium of the brain. A solute is slowly infused into the catheter at a constant velocity. The fenestrated membranes at the tip of the catheter permit free diffusion of molecules between the brain interstitium and the perfusate, which is subsequently collected for laboratory analysis. The major molecules studied using this method are glucose, lactate, pyruvate, glutamate, and glycerol. The collected substances provide insight into the neurochemical features of secondary injury following traumatic brain injury (TBI) and subarachnoid hemorrhage (SAH) and valuable information about changes in brain metabolism within a short time frame. In this review, the authors detail the CMD technique and its associated markers and then describe pertinent findings from the literature about the clinical application of CMD in TBI and SAH.

Clinical applicability of dOFM devices for dermal sampling.

BACKGROUND: Sampling the dermal interstitial fluid (ISF) allows the pharmacokinetics and pharmacodynamics of dermatological drugs to be studied directly at their site of action. Dermal open-flow microperfusion (dOFM) is a recently developed technique that can provide minimally invasive, continuous, membrane-free (thus unfiltered) access to the dermal ISF. Herein, we evaluate the clinical applicability and reliability of novel wearable dOFM devices in a clinical setting. METHODS: Physicians inserted 141 membrane-free dOFM probes into the dermis of 17 healthy and psoriatic volunteers and sampled dermal ISF for 25 h by using wearable push-pull pumps. The tolerability, applicability, reproducibility, and reliability of multiple insertions and 25 h continuous sampling was assessed by pain scoring, physician feedback, ultrasound probe depth measurements, and 25 h-drift and variability of the sodium relative recovery. RESULTS: Insertion pain was moderate and decreased with each additional probe. Probe insertion was precise, although slightly deeper in lesional skin. The wearable push-pull pump enabled uninterrupted ISF sampling over 25 h with low variability. The relative recovery was drift-free and highly reproducible. CONCLUSION: dOFM sampling devices are tolerable and reliable for prolonged continuous dermal sampling in a multiprobe clinical setting. These devices should enable the study of a wide range of drugs and their biomarkers in the skin.

Desalting DNA by drop dialysis increases library size upon transformation.

It is often desirable to obtain gene libraries with the greatest possible number of variants. We tested two different methods for desalting the products of library ligation reactions (silica-based microcolumns and drop dialysis), and examined their effects on final library size. For both intramolecular and intermolecular ligation, desalting by drop dialysis yielded approximately 3-5 times more transformants than microcolumn purification.

Long-term calibration considerations during subcutaneous microdialysis sampling in mobile rats.

The level at which implanted sensors and sampling devices maintain their calibration is an important research area. In this work, microdialysis probes with identical geometry and different membranes, polycarbonate/polyether (PC) or polyethersulfone (PES), were used with internal standards (Vitamin B(12) (MW 1355), antipyrine (MW 188) and 2-deoxyglucose (2-DG, MW 164)) and endogenous glucose to investigate changes in their long-term calibration after implantation into the subcutaneous space of Sprague-Dawley rats. Histological analysis confirmed an inflammatory response to the microdialysis probes and the presence of a collagen capsule. The membrane extraction efficiency (percentage delivered to the tissue space) for antipyrine and 2-DG was not altered throughout the implant lifetime for either PC- or PES membranes. Yet, Vitamin B(12) extraction efficiency and collected glucose concentrations decreased during the implant lifetime. Antipyrine was administered i.v. and its concentrations obtained in both PC- and PES-membrane probes were significantly reduced between the implant day and seven (PC) or 10 (PES) days post-implantation suggesting that solute supply is critical for in vivo extraction efficiency. For the low molecular weight solutes such as antipyrine and glucose, localized delivery is not affected by the foreign body reaction, but recovery is significantly reduced. For Vitamin B(12), a larger solute, the fibrotic capsule formed around the probe significantly restricts diffusion from the implanted microdialysis probes.

TheClinical Research Tool: a high-performance microdialysis-based system for reliably measuring interstitial fluid glucose concentration.

BACKGROUND: A novel microdialysis-based continuous glucose monitoring system, the so-called Clinical Research Tool (CRT), is presented. The CRT was designed exclusively for investigational use to offer high analytical accuracy and reliability. The CRT was built to avoid signal artifacts due to catheter clogging, flow obstruction by air bubbles, and flow variation caused by inconstant pumping. For differentiation between physiological events and system artifacts, the sensor current, counter electrode and polarization voltage, battery voltage, sensor temperature, and flow rate are recorded at a rate of 1 Hz. METHOD: In vitro characterization with buffered glucose solutions (c(glucose) = 0 - 26 x 10(-3) mol liter(-1)) over 120 h yielded a mean absolute relative error (MARE) of 2.9 +/- 0.9% and a recorded mean flow rate of 330 +/- 48 nl/min with periodic flow rate variation amounting to 24 +/- 7%. The first 120 h in vivo testing was conducted with five type 1 diabetes subjects wearing two systems each. A mean flow rate of 350 +/- 59 nl/min and a periodic variation of 22 +/- 6% were recorded. RESULTS: Utilizing 3 blood glucose measurements per day and a physical lag time of 1980 s, retrospective calibration of the 10 in vivo experiments yielded a MARE value of 12.4 +/- 5.7. Clarke error grid analysis resulted in 81.0%, 16.6%, 0.8%, 1.6%, and 0% in regions A, B, C, D, and E, respectively. CONCLUSION: The CRT demonstrates exceptional reliability of system operation and very good measurement performance. The ability to differentiate between artifacts and physiological effects suggests the use of the CRT as a reference tool in clinical investigations.

Evaluation of in vivo and in vitro recovery rate of anatoxin-a through the microdialysis probe.

In vivo microdialysis is a versatile sampling technique commonly employed to observe changes in neurotransmitters levels that occur in response to different treatments, being these treatments administered through a microdialysis probe implanted into a specific brain region in living animals. In previous works we have used this technique to study the effects of the drug anatoxin-a, a nicotinic acetylcholine receptor agonist, on dopamine release in striatum. The aim of the present study was to assess the recovery of anatoxin-a through the microdialysis probe. This information allows knowing the exact amount of the drug crossing the microdialysis membrane, acting on extracellular tissue. High Performance Liquid Chromatography (HPLC) with Fluorescence Detection (FLD) has been used for the analysis of anatoxin-a. We observed that the recovery of anatoxin-a was about 0.5%. Under our experimental conditions, the results suggest that anatoxin-a can be used as an important tool in the study of neuronal nicotinic receptors by in vivo microdialysis technique and also show a reliable estimation of the anatoxin-a recovery through the microdialysis probe under both in vivo and in vitro conditions.

Clinical microdialysis in skin and soft tissues: an update.

Traditionally, plasma or serum drug concentrations have been used for the assessment of bioavailability and bioequivalence. Since in the majority of cases the site of drug action is in the tissue rather than the blood, the use of corresponding free, unbound concentrations in the tissue is a much more meaningful approach. This can become especially important for topical drug administrations, where locally active drug concentrations can significantly exceed free concentrations in plasma. The ability to measure these free concentrations at the site of drug action over time makes microdialysis a very valuable tool for the assessment of bioavailability and bioequivalence. This has been recognized by industry and regulatory authorities, resulting in a recommendation of the microdialysis technique as a tool for bioequivalence determination of topical dermatologic products. The aim of this article is to provide an updated review of the microdialysis technique, its applications in skin and soft tissues, and the resulting impact on clinical drug development.

Assessment of in vitro and in vivo recovery of sinomenine using microdialysis.

BACKGROUND/PURPOSE: For microdialysis studies in the skin, laboratory-made linear probes are often used. The application of the microdialysis technique to the investigation of pharmacokinetics and pharmacodynamics of drugs requires careful assessment of the linear probes' performance to ensure validity of the data obtained using this technique. The aim of this study was to establish and validate the microdialysis technique for investigation of the pharmacokinetics and pharmacodynamics of sinomenine. METHODS: Using different lengths of the dialysis membrance and different perfusion flow rates, a flow rate of 2 microL/min with 20-min sampling intervals was selected for the subsequent studies, based on the analysis of sinomenine from the microdialysis probe. In vitro recovery of sinomenine from the microdialysis probe was independent of concentration, stable over an 8-h period. Comparable in vitro recoveries were obtained by different established approaches including recovery estimation by gain, loss and the zero-net flux (ZNF) method. Recovery by loss was used to study the in vivo recovery of sinomenine from rat subcutaneous tissue. RESULTS: The performance of the microdialysis system was stable over an 8-h study, resulting in a mean in vitro recovery of 51.91+/-1.29%. Recovery obtained using the ZNF plot was 52.43%. In vivo recovery of sinomenine was 34.46+/-0.76% and was stable over the 7-h study period. CONCLUSION: The in vitro and in vivo performance of the microdialysis technique was established for the study of sinomenine. It would prove to be a useful and reliable tool to study the pharmacokinetics and pharmacodynamics of sinomenine. The data obtained in our study highlight the importance of a systematic examination of microdialysis linear probe validation.

Neurochemical monitoring using intracerebral microdialysis during systemic haemorrhage.

BACKGROUND: Intracerebral microdialysis is a sensitive tool to analyse tissue biochemistry, but the value of this technique to monitor cerebral metabolism during systemic haemorrhage is unknown. The present study was designed to assess changes of intracerebral microdialysis parameters both during systemic haemorrhage and after initiation of therapy. METHODS: Following approval of the Animal Investigational Committee, 18 healthy pigs underwent a penetrating liver trauma. Following haemodynamic decompensation, all animals received a hypertonic-hyperoncotic solution and either norepinephrine or arginine vasopressin, and bleeding was subsequently controlled. Extracellular cerebral concentrations of glucose (Glu), lactate (La), glycerol (Gly), and the lactate/pyruvate ratio (La/Py ratio) were assessed by microdialysis. Cerebral venous protein S-100B was determined. Haemodynamic data, blood gases, S-100B, and microdialysis variables were determined at baseline, at haemodynamic decompensation, and repeated after drug administration. RESULTS: Microdialysis measurements showed an increase of La, Gly, and La/Py ratio at BL Th compared to BL (mean +/- SEM; La 2.4 +/- 0.2 vs. 1.4 +/- 0.2 mmol x l(-1), p < 0.01; Gly 37 +/- 7 vs. 27 +/- 6 micromol x l(-1), n.s.; La/Py ratio 50 +/- 8 vs. 30 +/- 4, p < 0.01), followed by a further increase during the therapy phase (La 3.4 +/- 0.3 mmol x l(-1); Gly 69 +/- 10 micromol x l(-1); La/Py ratio 58 +/- 8; p < 0.001, respectively). Cerebral venous protein S-100B increased at decompensation and after therapy, but decreased close to baseline values after 90 min of therapy. CONCLUSIONS: In this model of systemic haemorrhage, changes of cerebral energy metabolism detected by intracerebral microdialysis indicated anaerobic glycolysis and degradation of cellular membranes throughout the study period.

Microdialysis sampling extraction efficiency of 2-deoxyglucose: role of macrophages in vitro and in vivo.

Macrophages are a class of inflammatory cells believed to direct the outcome of device biocompatibility. Despite their relevance to implanted in vivo devices, particularly implanted glucose sensors, few studies have attempted to elucidate how these cells affect device performance. Microdialysis sampling probes were used to determine glucose uptake alterations in the presence of resting and activated macrophages in vitro. Significant differences for 2-deoxyglucose (2-DG) relative recovery at 1.0 microL/min were observed between resting (74 +/- 7%, n = 18) and lipopolysaccharide (LPS) (1 microg/mL)-activated (56 +/- 6%, n = 18) macrophages in culture that had 2-DG spiked into the media (p < 0.005). To establish if in vitro characterization could be correlated to in vivo studies, microdialysis probes were implanted into the dorsal subcutis of male Sprague-Dawley rats for 0, 3, 5, and 7 days. An internal standard, 2-DG, was passed through the microdialysis probe during in vivo studies. No significant differences in 2-DG extraction efficiency from the probe into the tissue site were observed in vivo among microdialysis probes implanted into the subcutaneous space of Sprague-Dawley rats for either 3, 5, or 7 days vs probes implanted the day of sample collection. These results suggest that macrophage activation in vivo at implant sites is much lower than highly activated macrophages in vitro. It is important to note that these results do not rule out the potential for increased glucose metabolism at sensor implant sites.

Effect of cyclosporine a on glucose interstitial concentration in renal cortex and medulla from rats.

AIM: To standardize microdialysis in rat kidneys and address cyclosporine A (CsA) effects on renal cortex and medulla interstitial glucose. METHODS: Munich-Wistar rats were treated with vehicle or CsA (15 mg/kg/day) for 3 weeks. Glucose was assessed by spectrophotometry in dialysate samples from cortex, medulla and arterial plasma. Plasma insulin was measured by radioimmunoassay. Renal blood flow (RBF) was measured by Doppler ultrasound. Creatinine and urea were measured by spectrophotometry. RESULTS: CsA significantly increased the plasma levels of urea and creatinine (1.5 +/- 0.20 vs. 0.73 +/- 0.03 mg/dl in controls, p < 0.05). Medullary glucose in control was 44% lower than arterial glucose (56 +/- 6 vs. 101 +/- 8 mg/dl, p < 0.05). At the same time, CsA increased arterial (163 +/- 35 vs. 101 +/- 8 mg/dl in controls, p < 0.05) and medullary interstitial glucose (100 +/- 18 vs. 56 +/- 6 mg/dl in controls, p < 0.05), but did not affect cortical glucose (114 +/- 21 vs. 90 +/- 11 mg/dl in controls). These changes occurred in the presence of a decreased plasma insulin level (2.7 +/- 0.2 vs. 9.3 +/- 0.4 microU/ml in controls, p < 0.05). The increment in medullary glucose in CsA group occurred despite a reduction in RBF (4.6 +/- 0.8 vs. 6.5 +/- 1.0 ml/min/kidney in controls, p < 0.05). CONCLUSIONS: Microdialysis was an adequate tool to investigate in vivo regulation of renal glucose metabolism. Renal glucose uptake was dependent on medullary cells and CsA treatment induced diabetogenic effects on renal medulla in situ.

Normal values and differences between intraperitoneal and subcutaneous microdialysis in patients after non-complicated gastrointestinal surgery.

OBJECTIVE: Visceral ischemia is an early event in the development of shock and organ failure. Microdialysis has been presented as a promising method for detection of visceral hypoxia and ischemia. The aim of this study was to investigate differences in the metabolic response measured by microdialysis between intraperitoneal and subcutaneous locations and to estimate normal values of lactate/pyruvate ratio, glucose and glycerol. MATERIAL AND METHODS: Intraperitoneal and subcutaneous metabolic responses were compared regarding lacate/pyruvate ratio, glucose and glycerol, during 45 postoperative hours in 33 patients undergoing various non-complicated elective major gastrointestinal surgery. RESULTS: Intraperitoneal lactate/pyruvate ratio started around 15 and decreased over time, while subcutaneous levels were stable around 9. Glucose levels were higher intraperitoneally and increased rapidly during the first 9 h to 8.6 mM, while the subcutaneous levels increased during 21 h to 7.5 mM. Intraperitoneal glycerol levels were stable around 100 microM, while subcutaneous values started around 230 microM and then increased. CONCLUSIONS: In a non-complicated postoperative course the lactate/pyruvate ratio and glucose levels are higher intraperitoneally, suggesting a higher postoperative intraperitoneal metabolism. Glycerol levels are higher and increase subcutaneously, suggesting increased postoperative energy demand, particularly in the visceral organs, as being responsible for the lipolysis seen in the subcutaneous tissue.