SPECIAL COMMUNICATION
Evaluation of the microdialysis technique in the dog fat pad

Bente Stallknecht1,2, Joop Madsen1, Henrik Galbo1,2, and Jens Bülow3

1 Department of Medical Physiology, The Panum Institute, 2 The Copenhagen Muscle Research Centre, Rigshospitalet, and 3 Department of Clinical Physiology, Bispebjerg Hospital, DK-2200 Copenhagen N, Denmark.


    ABSTRACT
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Abstract
Introduction
METHODS
RESULTS
DISCUSSION
References

In the present study the microdialysis technique was evaluated in an isolated autoperfused dog fat pad. Concentrations of glucose, lactate, and glycerol were measured in interstitial fluid by microdialysis and simultaneously in arterial and adipose venous plasma. Adipose tissue blood flow was measured by both 133Xe washout and timed weighing of venous blood. Metabolite concentrations in adipose venous plasma calculated from interstitial and arterial metabolite concentrations and 133Xe washout were positively correlated with measured venous concentrations (glucose: r = 0.95, lactate: r = 0.92, glycerol: r = 0.81). Calculated and measured venous plasma concentrations did not differ for either glucose or lactate, but for glycerol, calculated concentration was on average 76% of measured concentration. Metabolite exchanges (Fick's principle) calculated from interstitial metabolite concentrations were positively correlated with measured exchanges only for lactate (r = 0.69). In conclusion, metabolite concentrations in adipose venous plasma can be calculated from microdialysis measurements with greater accuracy for glucose and lactate than for glycerol. The precision, however, is too low to allow calculation of metabolite exchange when arteriovenous metabolite differences are low.

adipose tissue; glucose; lactate; glycerol; blood flow


    INTRODUCTION
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Abstract
Introduction
METHODS
RESULTS
DISCUSSION
References

ADIPOSE TISSUE, especially from rats, has been extensively studied in vitro, but the tissue is difficult to study in vivo, especially in humans, because most depots do not have a vein that selectively drains the tissue and is easy to cannulate. Frayn et al. (7) described a technique that allows catheterization of a human subcutaneous abdominal vein draining mainly adipose tissue but to some degree also skin. The technique has given new insight into the regulation of lipid metabolism in humans, but its value is limited by the fact that it is only applicable to the subcutaneous abdominal adipose tissue depot.

Microdialysis is a technique by which interstitial water concentrations of various substances may be determined in, for example, adipose tissue in humans and animals (8, 9, 11, 15, 21-23). A small microdialysis tube is placed in the tissue of interest and perfused with a fluid that resembles the interstitial fluid. Water-soluble substances exchange over the microdialysis membrane, and the concentration change from the perfusate to the fluid collected after passage through the microdialysis probe, the dialysate, is an expression of the concentration difference between the perfusate and the interstitial fluid. The degree of equilibration between the microdialysis fluid and the interstitial fluid, the recovery, can be determined by various calibration techniques, e.g., no-net-flux or internal reference calibration. Thus the interstitial concentration of the substance can be determined (3, 8, 11, 15, 20-23).

Dialysate metabolite concentrations with or without correction for recovery have been interpreted as indicators of adipocyte metabolism (8). However, because the interstitial fluid is located between adipocytes and capillaries, interstitial metabolite concentrations reflect capillary permeability, arterial metabolite concentrations, and blood flow in addition to adipocyte metabolism. Accordingly, it must be important to take these factors into account when one wants to examine adipocyte metabolism. In previous studies we have done that by calculating metabolite concentrations in venous plasma drained from adipose tissue by use of "Fick's law of diffusion for a thin membrane" (21-23), as originally done by Jansson et al. (11). Metabolite exchange in adipose tissue could then be calculated by "Fick's principle."

Because of the many assumptions underlying such calculations, we compared venous metabolite concentrations calculated from interstitial metabolite concentrations with metabolite concentrations measured in one of the subcutaneous abdominal veins (21). That study showed good agreement between calculated and measured venous concentrations of glucose and glycerol, whereas calculated lactate concentrations were much higher than measured concentrations. Metabolite exchange measured by the two techniques varied in parallel, although absolute values differed significantly. However, the design in our previous study was not optimal, because it was not known exactly from which tissue the blood in the vein was drained.

The objective of the present study was therefore to evaluate the microdialysis technique during better-defined conditions. In an isolated autoperfused dog fat pad, which is drained by only one vein (18), we have investigated whether venous plasma concentrations and exchange of glucose, lactate, and glycerol can be calculated from interstitial metabolite concentrations determined by microdialysis.


    METHODS
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Abstract
Introduction
METHODS
RESULTS
DISCUSSION
References

Experimental animals. Eight female dogs weighing 14-34 kg, with right inguinal fat pads weighing 27-98 g after isolation, were examined after an overnight fast. They were fed standard dog chow. The experiments were performed according to national ethical guidelines for use of animals in research.

Operation. The dogs were premedicated with propionyl promazine (5 mg sc) and anesthetized with thiopental sodium (10 mg/kg) and pentobarbital sodium (20 mg/kg) supplemented intravenously during the experiment as necessary. They were intubated and ventilated with 50% O2-50% N2O. During the whole experiment, rectal temperature was kept at 37.5-38.5°C by use of a heating pad. A polyethylene catheter was inserted either in the left common femoral artery or in a carotid artery for blood sampling and monitoring of blood pressure and heart rate. The right subcutaneous inguinal fat pad was isolated from skin and muscle fascia. Medially the fat pad was isolated from the contralateral fat pad by cauterization, and laterally, cranially, and caudally from surrounding tissues by ligations. Care was taken not to damage the external pudendal artery, vein, and nerve.

Experimental protocol. Microdialysis fibers for determination of glucose, lactate, and glycerol were placed in the isolated fat pad, and 30 min before start of the experiment, 133Xe was injected in the fat pad. The fat pad was covered with gauze soaked in 40°C saline, and the gauze was frequently replaced. After placement of microdialysis fibers, a catheter was inserted into the external pudendal vein to allow collection of all blood from the fat pad, and the dog was anticoagulated with 5,000 IU of intravenous heparin. During the experiment all venous blood was collected in preweighed 10-ml test tubes on ice. At the beginning of the experiment and for every 5-10 ml of venous blood, an arterial blood sample was drawn. Microdialysate was sampled in 15-min periods during the experiment.

The first 15 min of the experiment was a control period. In the next 15 min epinephrine was infused in a foreleg vein. The amount infused was varied (0.05-0.2 nmol · kg body weight-1 · min-1) from experiment to experiment to obtain a wide range of metabolic conditions in the fat pads. This period was followed by a second 15-min control period (30 min in 1 dog). In five of the dogs, the metabolic conditions were also perturbed by infusion of adenosine (140 µg · kg body weight-1 · min-1) in a foreleg vein in an additional 15-min period. A bolus of [14C]lactate, [3H]glycerol, and 125I-labeled albumin was given in the external pudendal artery to determine the capillary permeability-surface area (PS) product for glycerol and lactate. Four biopsies were taken from each inguinal fat pad for determination of water, lipid, and dry matter content for calculation of the tissue-to-blood distribution coefficient (lambda ) for xenon and for detection of edema formation in the experimental tissue. In four dogs, biopsies of adipose tissue with a microdialysis fiber were also taken for histological examination. Finally, the right fat pad was taken out and weighed. At the end of the experiment, the dogs were killed by an intravenous bolus of saturated potassium chloride.

Microdialysis. Microdialysis was performed in principle as described by Lönnroth et al. (15). Dialysis fibers were obtained from artificial kidneys used for dialysis of humans with uremia, either Alwall GFE18 (glucose and lactate determination) or GFS+12 (glycerol determination) (Gambro AB, Lund, Sweden), both with a molecular mass cutoff at 5,000 kDa. A 4-cm-long dialysis fiber (0.20 mm ID, 0.22 mm OD) was glued at both ends to a nylon tube with a diameter of 0.50-0.63 mm. Dialysis fibers are coated with glycerol, and to wash out this glycerol, fibers used for glycerol determination were perfused at 10 µl/min with sodium chloride for 15 h before the experiment. Four fibers (2 for glucose and lactate and 2 for glycerol determinations) were placed in the inguinal fat pad at a distance of ~1 cm by use of 18-gauge cannulas. The fibers were perfused at a rate of 3 µl/min by use of a high-precision syringe pump (CMA/100, CMA/Microdialysis, Stockholm, Sweden). Calculated mean transit time was 25 s through each fiber. After insertion, fibers were perfused for >= 60 min before the experimental periods were begun.

The exchange over the microdialysis membrane in the present setup does not reach 100% equilibrium. Therefore, it is necessary to determine the relative recovery (RR) over the fibers. This was done by the internal reference calibration technique essentially as described by Scheller and Kolb (20). In the two fibers used for glucose and lactate determination, 6 nM D-[6-3H]glucose (sp act 1,850 GBq/mmol; NEN-Du Pont, Mechelen, Belgium) and 2.8 µM L-[U-14C]lactic acid, sodium salt (sp act 4.0 GBq/mmol; NEN-Du Pont) were added to the perfusate. In the two fibers used for glycerol determination, 1.5 µM [2-3H]glycerol (sp act 7.4 GBq/mmol; NEN-Du Pont) was added to the perfusate. Perfusate concentrations of glucose and lactate were 4 mM and 1 mM, respectively, during the whole experiment in fibers used for glucose and lactate determinations. In fibers used for glycerol determination, glycerol concentration was 0.025 mM during the whole experiment. Samples of perfusate (10 µl; quadruple determinations) and dialysate (10 µl; single determinations) were counted in a liquid scintillation counter (2200 CA Packard). RR was calculated for each microdialysis sample. It was assumed that RR from interstitial fluid to perfusate of unlabeled metabolite equals relative loss from perfusate to interstitial fluid of labeled metabolite: (C*<SUB>p</SUB> - C*<SUB>d</SUB>)/C*<SUB>p</SUB>, where C*<SUB>p</SUB> is count rate/min in 10 µl of perfusate, and C*<SUB>d</SUB> is count rate/min in 10 µl of dialysate. Interstitial concentrations of metabolites were calculated from individual RR values as (Cd - Cp)/RR + Cp, where Cd is dialysate concentration and Cp is perfusate concentration.

Blood flow. Adipose tissue blood flow (ATBF) was measured by weighing of venous outflow from the inguinal fat pad as well as by the local 133Xe washout method (12). About 3.7 MBq of 133Xe dissolved in 0.1-0.15 ml of isotonic saline were slowly injected between microdialysis fibers in the central part of the fat pad with a 25-gauge cannula. ATBF determined by the 133Xe washout method was calculated as k · lambda  · 100 (ml · 100 g-1 · min-1), where lambda  is the tissue-to-blood distribution coefficient for 133Xe at equilibrium, and k is the rate constant of the washout. lambda  was determined for each fat pad from four biopsies essentially as described (16).

Determination of capillary PS products for lactate and glycerol. The indicator diffusion method was used in principle as described by Crone (5). Two test substances, L-[U-14C]lactic acid-sodium salt (0.3 MBq; NEN-Du Pont) and [2-3H]glycerol (0.3 MBq; NEN-Du Pont), and one reference substance, 125I-labeled albumin (0.3 MBq; Institutt for energiteknikk, Kjeller, Norway), were mixed with isotonic sodium chloride in a 1-ml plastic syringe to give a final volume of 0.8 ml. The syringe was weighed, and ~0.5 ml was given as a bolus (1-2 s) in the external pudendal artery. The syringe was weighed again, and the rest of the injection fluid was kept for later scintillation counting. During and after injection, venous blood was sampled in preweighed 2.5-ml test tubes every 5 s for 150 s and then every 20 s for 200 s. Test tubes were immediately stoppered and weighed, and 2 ml of 0.6 M perchloric acid were added. The next day reference samples were prepared: 25 µl of 20% albumin and 2 ml of 0.6 M perchloric acid were added to each of 6 10-µl samples of the injection fluid. All samples were centrifuged for 30 min at 2,200 g, and the supernatants were decanted to scintillation vials. Pellets were rinsed with 1 ml of 0.33 M perchloric acid and centrifuged for 30 min at 2,200 g, and the new supernatants were added to the scintillation vials containing the previous supernatants. Supernatants were counted in a liquid scintillation counter (2200 CA Packard) and corrected for background, quenching, and cross-talk. Pellets were counted in an automatic gamma counter (1470 Wizard). Extraction values (E) were calculated for each sample as (Cref - Ctest)/Cref, where Cref and Ctest are dpm of the reference and test substances, respectively, normalized by their concentrations in the bolus. E was integrated from time 0 to the time when the lactate and glycerol tracer concentrations had decreased to 40% of their peak values (E40) (13). PS product was calculated as -Qwater · ln(1 - E40) (ml · 100 g-1 · min-1) (5), where Qwater is mean plasmawater flow during PS product determination. Qwater was calculated as Q · (1 - Hct) · 0.95 (ml · 100 g-1 · min-1), where Q is mean blood outflow from fat pad during PS product determination, and Hct is hematocrit determined in arterial blood.

Sampling. Blood was sampled into iced tubes, kept on ice, and centrifuged or precipitated with perchloric acid within 5 min. Blood for determination of glucose, lactate, glycerol, and triglyceride was stabilized with 2.5 mg potassium fluoride and 5.5 mg EDTA (dogs 1-3) or 20 IU heparin (dogs 4-8) per milliliter of blood. Plasma and microdialysate glucose and lactate determinations were done immediately, and all other blood and microdialysate samples were kept at -20°C until analysis. Respective blood or plasma concentrations will be described in Calculations.

Analyses. Blood for determination of glucose, lactate, and glycerol concentrations and plasma for determination of glycerol concentrations were precipitated with perchloric acid. Plasma and microdialysate glucose and lactate concentrations were determined by a YSI 2300 glucose/lactate analyzer (YSI, Yellow Springs, OH). Intra-assay coefficient of variation (CV) for 10 determinations was 1.6% for glucose (mean: 4.32 mM) and 1.5% for lactate (mean: 1.40 mM). Blood glucose (IL Test, Instrumentation Laboratory, Lexington, MA) (CV: 1.3%, mean: 5.01 mM) and plasma triglyceride (CV: 1.6%, mean: 0.64 mM) concentrations were determined by spectrophotometric analysis on a Monarch centrifugal analyzer (Instrumentation Laboratory, Warrington, Cheshire, UK). Blood lactate (CV: 1.2%, mean: 0.85 mM) and plasma, blood, and microdialysate glycerol (CV: 2.6%, mean: 0.104 mM) concentrations were determined by fluorometric analysis adapted to the Monarch centrifugal analyzer. Hematocrit was determined on heparinized blood.

Calculations. Concentrations in venous plasmawater (Cv,calc) were calculated by the principle described by Intaglietta and Johnson (10). The calculation is based on Fick's law of diffusion for a thin membrane: J = -P · S · (Cc - Ci), where J is the substrate flux, P is the membrane permeability of the substrate, S the membrane surface area, Cc is the concentration of substrate in the capillary plasmawater, and Ci is the substrate concentration in interstitial water. If this equation is integrated over the entire length of a capillary, the following expression is obtained: (Cv,calc - Ci)/(Ca,water - Ci) = e(-PS/Q,water), giving Cv,calc = (Ca,water - Ci) · e(-PS/Q,water) + Ci for the tissue uptake situation, or Cv,calc = (Ci - Ca,water) · [1 - e(-PS/Q,water)] + Ca,water for the tissue output situation. Ca,water is metabolite concentration in arterial plasmawater, and Qwater is the bloodwater flow in which the metabolite is distributed during one capillary transit time. For lactate and glycerol, Qwater was calculated as Q · (1 - Hct) · 0.95 (ml · 100 g-1 · min-1), where Q is blood flow determined by 133Xe washout, and Hct is hematocrit determined in arterial blood. Because near equilibrium exists between plasma and erythrocyte water for glucose (but not for lactate and glycerol) throughout the capillary, glucose Qwater was calculated as Q · (1 - Hct) · 0.95 + Q · Hct · 0.43 (ml · 100 g-1 · min-1); (0.43 is the distribution volume of glucose in erythrocytes as calculated from hematocrit and whole blood and plasma glucose concentrations in dogs 4-8). The mean PS products determined for lactate and glycerol in the present experiment (1.0 and 1.3 ml · 100 g-1 · min-1, respectively) were used in calculations. Glucose is a larger molecule than lactate and glycerol and should accordingly have a smaller PS product, so we estimated it to be 0.8 ml · 100 g-1 · min-1. The apparent PS products increased from ~0.5 to 1.5 ml · 100 g-1 · min-1 within the range of blood flow variations registered. Because this has little influence on calculated venous metabolite concentration (see DISCUSSION), for practical reasons a fixed PS product was used. When possible, Ci was the mean of values calculated from dialysates from two different probes. For every pair of simultaneously determined Ci values from two probes, the CV was calculated. Mean interfiber CV ± SD was 13 ± 11% for glucose (simultaneous values from 2 probes in 8 dogs 22 times), 17 ± 15% for lactate (simultaneous values from 2 probes in 8 dogs 22 times), and 18 ± 14% for glycerol (simultaneous values from 2 probes in 8 dogs 21 times).

For comparison between calculated and directly determined plasma concentrations, Cv,calc was expressed as metabolite concentration in plasma: Cv,calc,pl = Cv,calc · 0.95. For metabolites released from the fat pad (lactate and glycerol), Cv,calc,pl was also corrected for the transport of metabolites from plasma into erythrocytes during blood sampling to be comparable to the directly determined plasma concentrations: Cv,calc,pl = (Cv,calc · 0.95 - Ca,pl) · (1 - Hct)/(1 - Hct + Hct · D) + Ca,pl, where Ca,pl is metabolite concentration in arterial plasma, and D is distribution volume of metabolite in erythrocytes as calculated from hematocrit and whole blood and plasma metabolite concentrations in dogs 4-8. D was 0.42 for lactate and 0.75 for glycerol.

Glycerol in venous blood originates from arterial blood, intravascular lipolysis of triglycerides, and lipolysis in adipose tissue. With the assumptions that glycerol from intravascular lipolysis does not reach the microdialysis probe and, accordingly, is not contributing to Cv,calc (24), the measured concentrations in venous plasma (Cv,pl) and blood (Cv,blood) of glycerol were corrected for intravascular lipolysis as follows: Cv,pl,corrected = Cv,pl - (Ca,pl,TG - Cv,pl,TG) · (1 - Hct)/(Hct · 0.75 + 1 - Hct), and Cv,blood,corrected = Cv,blood - (Ca,pl,TG - Cv,pl,TG) · (1 - Hct), where Ca,pl,TG and Cv,pl,TG are triglyceride concentrations in arterial and venous plasma.

Exchange of metabolites in adipose tissue was determined by use of Fick's principle, with (Ca,blood - Cv,blood) · Q for the tissue uptake situation and (Cv,blood - Ca,blood) · Q for the tissue output situation. Ca,blood and Cv,blood are metabolite concentrations in arterial and venous whole blood, either directly measured or calculated from Ca,pl or Cv,calc,pl as (D · Hct + - Hct) · Cpl. Q is blood flow, either measured by timed weighing of venous blood outflow (used in determination of measured exchange) or estimated by 133Xe washout (used in determination of calculated exchange).

Before calculations, all parameters were integrated in 15-min periods (microdialysis sampling periods) to make direct comparison between calculated and measured values possible.

Statistics. The computer program SigmaStat for Windows version 1.0 (Jandel Scientific Software, San Rafael, CA) was used for statistical analysis. Data are presented as means ± SD. Calculated data are plotted against measured data. Pearson's correlation coefficient (r) and corresponding P value are presented if at least one of the data sets was normally distributed, and Spearman's r and corresponding P value are presented if data were not normally distributed. Linear regression was performed if assumptions for doing it were met, and then slope of regression line with 95% confidence interval, intercept with the y-axis, and corresponding P value are presented. A paired t-test was performed, if within-subject differences were normally distributed; otherwise Wilcoxon's signed rank test was performed. A P value of 0.05 in two-tailed testing was considered significant.


    RESULTS
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Abstract
Introduction
METHODS
RESULTS
DISCUSSION
References

Internal reference calibration. Including values from all dialysis fibers during all conditions, RR values were 0.22 ± 0.07 (n = 50) for glucose, 0.27 ± 0.08 (n = 50) for lactate, and 0.38 ± 0.14 (n = 50) for glycerol.

Effect of interventions. Depending on dose, epinephrine changed fat pad blood flow from -57% to +49% and glycerol output from -35% to +131%. Adenosine did not consistently influence blood flow.

Average concentrations of glucose, lactate, and glycerol. Average glucose, lactate, and glycerol concentrations in arterial and venous plasma and in fat pad interstitial fluid were calculated to include all measured values to give an impression of concentration differences. Glucose concentrations were 8.55 ± 2.11, 7.12 ± 1.02, and 5.29 ± 0.80 mM (n = 28) in arterial and venous plasma and in interstitial fluid, respectively. Corresponding values for lactate were 1.84 ± 1.31, 2.44 ± 2.21, and 3.61 ± 2.67 mM (n = 28) and for glycerol were 0.114 ± 0.057, 0.238 ± 0.121, and 0.286 ± 0.152 mM (n = 26).

Capillary PS product for lactate and glycerol. The PS products were 1.0 ± 0.5 ml · 100 g-1 · min-1 (n = 8) for lactate and 1.3 ± 0.6 ml · 100 g-1 · min-1 (n = 8) for glycerol.

Fat pad blood flow. The 133Xe tissue-to-blood distribution coefficient (lambda ) was 10.5 ± 1.0 ml/g (n = 8).

Concentrations of glucose, lactate, and glycerol in venous plasma from the fat pad calculated using microdialysis and blood flow determined by 133Xe washout. The venous plasma metabolite concentrations calculated from microdialysis were always positively correlated with the measured concentrations (Fig. 1). However, correlations were better for glucose and lactate (r = 0.95 and 0.92, respectively) than for glycerol (r = 0.81). The slope of the regression line did not differ from 1, and the intercept with the y-axis did not differ from 0 for glucose. For lactate, the slope of the regression line was close to 1, and the intercept with the y-axis was close to 0. Calculated and measured venous plasma concentrations did not differ for either glucose or lactate (glucose: 7.17 ± 1.10 vs. 7.12 ± 1.02 mM, P > 0.05, n = 28; lactate: 2.48 ± 1.79 vs. 2.44 ± 2.21 mM, P > 0.05, n = 28). Assumptions for performing linear regression were not fulfilled for glycerol, but calculated concentration was on average 76% of measured concentration (0.175 ± 0.088 vs. 0.230 ± 0.113 mM, P < 0.001, n = 26). In the plot of the difference between measured and calculated venous plasma concentrations vs. the mean of the two, it can be seen that for glucose and lactate the mean differences are close to zero, but for glycerol the mean difference is 0.055 mM. Variation around the mean is small relative to the venous plasma concentrations for glucose, larger for lactate, and even larger for glycerol.


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Fig. 1.   A: correlation between values of metabolite concentrations in venous plasma from fat pads calculated from microdialysis and 133Xenon measurements and those directly measured. Broken line, line of identity; solid line, regression line. B: difference between measured and calculated venous plasma metabolite concentration values plotted against the mean of the 2 values. Different symbols represent different dogs (1, ; 2, open circle ; 3, black-down-triangle ; 4, down-triangle; 5, ; 6, ; 7, black-diamond ; 8, star ). In all dogs, measurements were made in control periods and during infusion of epinephrine. In dogs 4-8, measurements were also made during infusion of adenosine.

Fat pad glucose uptake, lactate output, and glycerol output calculated using microdialysis and blood flow determined by 133Xe washout. Fat pad lactate and glycerol outputs calculated from microdialysis and 133Xe washout were correlated with those measured by Fick's principle (Fig. 2). For glycerol, however, the correlation was negative. Calculated and measured glucose uptakes tended to be positively correlated. Calculated mean glucose uptake was lower than measured mean glucose uptake (2.3 ± 1.3 vs. 3.5 ± 2.3 µmol · 100 g-1 · min-1, P < 0.05, n = 28); calculated mean lactate output was higher than measured mean lactate output (0.93 ± 0.93 vs. 0.37 ± 1.26 µmol · 100 g-1 · min-1, P < 0.01, n = 28); and calculated mean glycerol output was lower than measured mean glycerol output (0.14 ± 0.11 vs. 0.36 ± 0.18 µmol · 100 g-1 · min-1, P < 0.0001, n = 26).


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Fig. 2.   Correlation between metabolite exchange (arteriovenous or venoarterial whole blood metabolite differences multiplied by blood flows) in fat pads calculated from microdialysis and 133Xenon measurements and from direct measurements of blood concentrations and determination of blood flow by weighing, respectively. Broken line, line of identity; solid line, regression line. Different symbols represent different dogs (1, ; 2, open circle ; 3, black-down-triangle ; 4, down-triangle; 5, ; 6, ; 7, black-diamond ; 8, star ). In all dogs, measurements were made in control periods and during infusion of epinephrine. In dogs 4-8, measurements were also made during infusion of adenosine.

Fat pad water content. The water content of the fat pads used in the experiment did not differ from the water content of the contralateral fat pads (20 ± 8 vs. 20 ± 6%, P > 0.05, n = 7).

Histological evaluation of adipose tissue. In the examined samples, the adipose tissue around the microdialysis fibers showed no sign of cellular reaction or edema. Around some of the fibers there was an accumulation of red blood cells.


    DISCUSSION
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Abstract
Introduction
METHODS
RESULTS
DISCUSSION
References

The present experiments confirm that adipose venous metabolite concentrations can be determined by means of microdialysis for glucose and lactate but that the accuracy is not optimal for glycerol (Fig. 1). Moreover, output and uptake measurements based on microdialysis are associated with a high methodological variation when arteriovenous concentration differences are small (Fig. 2).

Four factors have to be known for metabolite concentrations in venous plasma from adipose tissue to be calculated by use of Fick's law of diffusion for a thin membrane: the interstitial metabolite concentration, the arterial plasmawater metabolite concentration, the bloodwater flow in which the metabolite is distributed during one capillary transit time, and the capillary PS product for the metabolite.

The interstitial metabolite concentrations may be determined by microdialysis, but metabolite recovery over the microdialysis membrane must be known for interstitial concentrations to be calculated from dialysate concentrations (15, 20). In the present study, recovery was determined by internal reference calibration. The microdialysis perfusate equilibrates only with the interstitial metabolites in the near vicinity (1-3 mm) of the probe (3). Therefore, microdialysis measurements represent only very small pieces of adipose tissue, and if interstitial metabolite concentrations are heterogeneous, comparisons between persons or between different sites of the body will be difficult. In the present study, mean interfiber CV values for interstitial concentrations were 13% for glucose, 17% for lactate, and 18% for glycerol. These figures reflect analytical errors in determination of dialysate metabolite concentrations (CV of 1-3%), errors in recovery determination, errors due to problems with dialysis fibers, e.g., glycerol liberation from or adherence to fiber wall, and true heterogeneity in metabolite concentrations in the tissue.

The arterial plasmawater metabolite concentration is easily determined in an arterial blood sample, but the bloodwater flow in which changes in concentration of the metabolite take place during one capillary transit time depends both on the blood flow and how fast the metabolite passes the erythrocyte membrane. Glucose exchange over the erythrocyte membrane is very fast in dogs (2), and thus changes in plasma glucose will be accompanied by parallel changes in erythrocytes during one capillary transit time (17). In contrast, lactate and glycerol exchanges are slow (4, 6), and changes in plasma lactate and glycerol will not result in concentration changes within erythrocytes during one capillary transit time.

Intravital microscopy of adipose tissue has shown that shunt vessels are present (17). Because the 133Xe washout technique measures capillary blood flow, this may imply that the total flow is underestimated by the 133Xe washout technique. The capillary blood flow (estimated by the 133Xe washout technique) is a more appropriate measure than total blood flow in microdialysis studies, as shunt vessels do not exchange metabolites. Because interstitial metabolite concentrations depend on local blood flow, the ideal method of measuring blood flow would in microdialysis studies be to measure capillary blood flow in exactly the same area in which metabolite concentrations are measured. It has been suggested that perfusion of microdialysis fibers with ethanol and measurement of the ratio between ethanol concentrations in outflow from and inflow to fibers make this possible (9). The drawback of the method is that many assumptions are needed to quantify the blood flow measurements (25).

We found the capillary PS products for lactate and glycerol to be 1.0 and 1.3 ml · 100 g-1 · min-1. Glucose is a larger molecule than lactate and glycerol and should, accordingly, have a smaller PS product, and we assumed it to be 0.8 ml · 100 g-1 · min-1. These PS products were used in calculations of venous metabolite concentrations. The PS products for sucrose and 51Cr-EDTA have earlier been determined to be 2 ml · 100 g-1 · min-1 in adipose tissue (14, 19). Glucose, lactate, and glycerol are smaller molecules than sucrose and 51Cr-EDTA and should, accordingly, have higher PS products. Some of the discrepancy between the studies could be due to the relatively low blood flow in the present study, if the capillary diffusion capacity is sensitive to blood flow (1) or if some capillaries are not perfused, causing the effective capillary surface area to be reduced in our preparation (17).

Correlations between calculated and measured metabolite concentrations of glucose, lactate, and glycerol in venous plasma were good, especially for glucose and lactate (Fig. 1). Furthermore, for glucose and lactate, calculated and measured venous plasma concentrations did not differ significantly, whereas, for glycerol, calculated venous concentration was on average 76% of measured concentration. In a human study, we have previously found that glucose concentrations in venous plasma calculated from microdialysis measurements were slightly lower than concentrations measured in plasma from an abdominal vein draining both the dialysed adipose tissue and skin (21). In contrast to the present study, in the human study we found calculated venous lactate concentrations to be much higher than measured concentrations (21). In our human study and in another study comparing the same techniques (24), calculated and measured venous glycerol concentrations were found not to differ significantly. In both studies, however, a rather large variation was present, and in both the present and the previous studies, calculated venous glycerol concentration was on average ~0.05 mM lower than the measured concentration.

The discrepancies between the present and the previous studies can partly be due to different PS products being used in calculations. In the present study, we found PS products for lactate and glycerol to be 1.0 and 1.3 ml · 100 g-1 · min-1, and we estimated the PS product for glucose to be 0.8 ml · 100 g-1 · min-1. In the previous studies, PS products were estimated to be 3 ml · 100 g-1 · min-1 for glycerol and lactate and 2 ml · 100 g-1 · min-1 for glucose. The calculated venous glucose concentration decreases with increasing PS product, and in the present study, an increase in PS product from 0.5 to 1.5 ml · 100 g-1 · min-1 would decrease calculated glucose concentrations 0.7 mM on average. Accordingly, calculated and measured venous glucose concentrations might not have differed in our human study (21) if we had used a lower PS product. Calculated venous lactate and glycerol concentrations increase with increasing PS product, but in the present study, the change in average concentrations with a change in PS product would be very little.

In the present study, the calculated venous glycerol concentration was lower than the measured concentration. Theoretically the reason could be that the measured venous glycerol concentration not only reflects lipolysis in the adipocytes but also intravascular lipolysis, whereas the venous glycerol concentration calculated from the interstitial concentration mainly reflects lipolysis in adipocytes (24). Although the intravascular lipolysis was negligible in the present study, we have corrected the measured venous glycerol concentration by subtracting the amount of glycerol formed by intravascular triglyceride breakdown. Another reason for the calculated venous glycerol concentration being lower than the measured one could be that the interstitial glycerol concentration from which the venous glycerol concentration was calculated was falsely too low. It might be that the trauma inherent in placement of microdialysis fibers interferes with local lipolysis by release of inhibiting substances. Minor trauma was indicated by an accumulation of red blood cells in some of the tissue samples we had histologically evaluated. Furthermore, if microdialysis recovery of glycerol was overestimated, a too low interstitial glycerol concentration would be calculated. Glycerol recovery would be overestimated if the [3H]glycerol used for calibration in the present study adheres to the microdialysis fiber wall. This will be interpreted as a loss from the perfusate and, accordingly, a falsely high microdialysis recovery.

Surprisingly, despite the good correlations between calculated and measured venous metabolite concentrations, metabolite exchange calculated from microdialysis and 133Xe washout was, nevertheless, badly correlated with metabolite exchange calculated from whole blood concentrations and directly measured blood flow (Fig. 2). Calculated and measured glucose uptakes tended to be positively correlated, calculated and measured lactate outputs were positively correlated, but calculated and measured glycerol outputs were negatively correlated. Measured arteriovenous plasma glucose and lactate differences were relatively small, means being 1.4 and 0.7 mM, respectively. Consequently, small errors in the absolute values of plasma glucose and lactate concentrations calculated from microdialysis had large influence on the calculated arteriovenous difference. Furthermore, when an erroneous arteriovenous difference is multiplied by an inaccurate blood flow estimated from 133Xe washout, the bad correlation between calculated and measured metabolite exchanges is not so surprising.

At first, the negative correlation for glycerol output was surprising too (Fig. 2). However, the large scatter around the line of identity for calculated vs. measured venous plasma glycerol concentrations (Fig. 1) explained the negative correlation: in the five dogs for which calculated values were lower than measured ones (Fig. 1), calculated venoarterial differences were very low, whereas measured differences were high. In consequence, glycerol output values from these dogs lie in the lower right part of Fig. 2. Conversely, in the two dogs for which calculated glycerol concentrations were higher than measured ones (Fig. 1), glycerol output values lie in the upper left part of Fig. 2. It appears that, even though they are small, errors inherent in the calculation of venous metabolite concentrations from microdialysis are too high to allow calculation of metabolite exchange, when arteriovenous metabolite concentration differences are low. Evidently, calculations based on mean concentrations determined for groups of subjects will be more reliable than calculations for individual subjects.

In conclusion, glucose and lactate concentrations in venous plasma from adipose tissue can be determined by means of microdialysis, but calculation of glycerol concentration should be done with caution. Moreover, the precision is too low to allow determination of metabolite exchange from individual microdialysis measurements when arteriovenous metabolite differences are low.


    ACKNOWLEDGEMENTS

We thank Lisbeth Kall, Birgitte Kiærskou, Regitze Kraunsøe, Inge Rasmussen, and Karen Klausen for skilled technical assistance and Dr. Dorthe Francis, Department of Pathology, Bispebjerg Hospital, for histological evaluation of samples of adipose tissue. We also acknowledge Drs. Per Sejrsen and Jørgen Vinten for valuable discussions of data.


    FOOTNOTES

This study was supported by grants from the Novo Nordisk Foundation and the Danish National Research Foundation (504-14).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests: B. Stallknecht, Dept. of Medical Physiology, The Panum Institute, Bldg. 12.4, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark.

Received 5 June 1998; accepted in final form 30 October 1998.


    REFERENCES
Top
Abstract
Introduction
METHODS
RESULTS
DISCUSSION
References

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