SPECIAL COMMUNICATION
Metabolite levels in human skeletal muscle and adipose tissue studied with microdialysis at low perfusion flow

H. Rosdahl1,2, K. Hamrin1, U. Ungerstedt1, and J. Henriksson1

1 Department of Physiology and Pharmacology, Karolinska Institutet, and 2 Department of Human Biology, Stockholm University College of Physical Education and Sports, S-11486 Stockholm, Sweden

    ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References

To identify a perfusion flow at which the interstitial fluid completely equilibrates with the microdialysis perfusion fluid, a protocol with successively lower perfusion flows was used. A colloid was included in the perfusion fluid to make sampling possible at the lowest perfusion flows. At 0.16 µl/min, the measured metabolites had reached a complete equilibration in both tissues, and the measured concentrations of glucose, glycerol, and urea were in good agreement with expected tissue-specific levels. The glucose concentration in adipose tissue (4.98 ± 0.14 mM) was equal to that of plasma (5.07 ± 0.07 mM), whereas the concentration in muscle (4.41 ± 0.11 mM) was lower than in plasma and adipose tissue (P < 0.001). The concentration of lactate was higher (P < 0.001) in muscle (2.39 ± 0.22 mM) than in adipose tissue (1.30 ± 0.12 mM), whereas the glycerol concentration in adipose tissue (233 ± 19.7 µM) was higher (P < 0.001) than in muscle (40.8 ± 3.0 µM) and in plasma (68.7 ± 3.97 µM). The concentration of urea was equal in the two tissues. Overall, the study indicates that microdialysis at a low perfusion flow may be a tool to continuously monitor tissue interstitial concentrations.

dextran; glucose; lactate; glycerol; urea

    INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References

MICRODIALYSIS is an in vivo sampling technique used for studies of tissue-specific events in many species, including humans. Substances in the interstitial space diffuse across a semipermeable membrane to a perfusion fluid, which is pumped through the microdialysis catheter. The degree of equilibration between the interstitial and perfusion fluids (recovery) is mainly dependent on the perfusion flow, the size of the dialysis membrane, and the diffusivity of substances in the tissue. Most of the microdialysis experiments reported in the literature have been performed with an incomplete recovery. With this approach, the changes measured represent relative changes of an unknown fraction of the interstitial concentration. The microdialysis catheters may be calibrated by determining the recovery. This can be performed by perfusing with different concentrations of the substance of interest (no net flux method; see Ref. 13) or by extrapolation to zero flow (8). These techniques, however, both take several hours to perform, and, in addition, neither technique can be used to detect changes in recovery over time. In other studies, an internal standard has been used to calculate the recovery (7, 14, 16, 17, 23). An alternative approach that does not involve any recalculation of the data is to ensure a complete equilibration (100% recovery) by the combination of a large dialysis membrane and a low perfusion flow. With the use of this combination, a high recovery, but not a complete equilibration, has previously been reported in human adipose tissue and skeletal muscle (3, 7, 16).

The aim of the present study was to use a successively lower perfusion flow to identify a perfusion flow at which the interstitial fluid in human skeletal muscle and adipose tissue completely equilibrates with the microdialysis perfusion fluid with respect to glucose, lactate, glycerol, and urea.

    METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References

Subjects. Results are presented for 18 healthy male subjects. Mean age, height, weight, and body mass index were 24.9 ± 0.9 yr (range 19-32), 1.80 ± 0.02 m (range 1.70-1.98), 79.7 ± 2.6 kg (range 65-98), and 24.0 ± 0.56 (range 20.1-29.9). Abdominal skin and subcutaneous tissue thickness, as measured by a skinfold caliper, was 21.6 ± 2.22 mm (caliper values). Before the start of investigation, a detailed description of the study was given to the subjects, who gave their informed consent. The study was approved by the ethical committee of the Karolinska Institute.

Experimental design. Initially, 14 subjects participated in experiments with different perfusion flow rates (1.33, 0.66, 0.33, and 0.16 µl/min) with dextran added to the perfusion fluid (results are presented in Figs. 1A-4A). Subjects 1-8 had two additional catheters inserted (one in adipose tissue and one in skeletal muscle), perfused at a constant flow rate (0.33 µl/min) during the whole experiment (results are presented in Figs. 1B-4B). Subjects 9-14 underwent an additional experiment on a separate day with different perfusion flow rates (1.33, 0.66, 0.33, and 0.16 µl/min) without dextran added to the perfusion fluid (results are presented in METHODS, Comparison of experiments with or without dextran in the perfusion solution). The perfusion fluid used in these subjects was furthermore supplemented with 50 mM ethanol to compare the basal blood flow in skeletal muscle and adipose tissue. Five subjects (15-19) participated in experiments with different perfusion flow rates (0.33, 0.16, and 0.075 µl/min) with dextran added to the perfusion fluid (results are presented in Figs. 1C-4C). In the protocols with different perfusion flows, each flow rate was monitored for 2 h, except at 0.075 µl/min, which was monitored for 4 h.

Procedure. The participants were asked to eat a regular breakfast at 6:00-6:30 AM and to arrive at 8:00 AM at the laboratory. The investigation was performed with the subject in the supine position in a room kept at 24-25°C. An indwelling polyethylene catheter was placed in an antecubital vein and kept patent by a slow infusion of saline. After local anesthesia with 0.3 ml of 1% Carbocain (Astra, Södertälje, Sweden) to the skin and muscle fascia, the microdialysis catheters were inserted as previously described (21). Briefly, microdialysis catheters were inserted bilaterally into the quadriceps femoris muscles (vastus lateralis) at 25-30% of the distance from the superior margin of the patella to the anterior superior iliac spine. In the periumbilical subcutaneous adipose tissue, the microdialysis catheters were inserted 70-80 mm lateral to the umbilicus and were then introduced 45-50 mm in a medial direction (in parallel to the skin). A distance of 30 mm was kept between two microdialysis catheters if they were placed in the same tissue. Microdialysis samples were collected in 20-, 30-, or 60-min fractions as shown in Figs. 1-4. In all protocols, venous blood samples were obtained at -5 and 45 min and 1.45, 2.45, 3.45, 4.45, 5.45, 6.45, and 7.45 h.

Insertion of microdialysis catheters and reasons for exclusion. In all subjects, two microdialysis catheters were placed in the right periumbilical subcutaneous adipose tissue, and two were inserted bilaterally into the right quadriceps femoris muscle (vastus lateralis). In subjects 1-8, one additional catheter was inserted in the left periumbilical subcutaneous adipose tissue, and one catheter was inserted in the left quadriceps femoris muscle. From the total of 116 microdialysis catheters inserted, the results from a total of 20 microdialysis catheters were disregarded (12 adipose tissue and 8 muscle).

Reasons for exclusion of results from adipose tissue catheters. The results of four catheters were excluded due to a rupture of the dialysis membrane during the insertion procedure. The results of five catheters (two of these being in the same subject) were excluded due to uncertainties regarding their position in the tissue. The abnormally low glucose and high lactate concentrations in the microdialysis samples indicated that these catheters were placed too close to the skin (10). One catheter was excluded due to the lack of results from a corresponding muscle catheter, and two catheters were excluded due to the lack of results from corresponding adipose tissue catheters perfused with dextran.

Reasons for exclusion of results from skeletal muscle catheters. One catheter was excluded due to a rupture of the dialysis membrane during the insertion procedure. Two catheters were excluded since they displayed a continuous decrease in the glucose concentration in the microdialysis samples combined with a continuous increase in the lactate concentration. One catheter was excluded due to abnormally high glucose combined with a low lactate concentration in the microdialysis samples. Two catheters were excluded due to the lack of results from the corresponding adipose tissue catheters, and two catheters were excluded due to the lack of results from a corresponding muscle catheter perfused with dextran.

Microdialysis equipment and perfusion fluid. The microdialysis technique and the microdialysis catheters have previously been described in detail (21). Briefly, in the present study, the catheters consisted of a 30-mm-long dialysis membrane (polyamide, 20,000 mol wt cutoff) with an outer diameter of 0.62 mm (CMA 60; CMA Microdialysis, Stockholm, Sweden). The microdialysis catheters were connected to syringes (1 ml Plastipak) placed in a microinfusion pump (CMA 100; CMA Microdialysis). The perfusion fluid was a modified Krebs-Henseleit bicarbonate buffer (KHB) described previously (21) or KHB supplemented with 40 g/l Dextran 70. In five subjects in the protocol with perfusion flows of 1.33-0.16 µl/min with dextran and in the protocol with perfusion flows of 1.33-0.16 µl/min without dextran, the perfusion fluid was supplemented with 50 mM ethanol.

Microdialysis sample volumes. To prevent evaporation, the microdialysis samples were collected in capped microvials (CMA Microdialysis). The amount of fluid that had evaporated from microvials (n = 10) containing 5 µl of the perfusion fluid was determined after 0.25, 0.5, 1, 2, 5, 10, and 20 h storage at 22-23°C. No significant evaporation was detected between 0 and 2 h. After 5, 10, and 20 h, the evaporation was detectable, being 0.03 ± 0.006, 0.23 ± 0.013, and 0.44 ± 0.018 µl, respectively. During the experiments, the amount of fluid collected in each sample was determined by weighing the sample vial before and immediately after collection. The collected sample volumes (per 30-min fractions) at perfusion flows of 1.33, 0.66, 0.33, and 0.16 µl/min with dextran were from catheters placed in adipose tissue (n = 22): 41.7 ± 0.11, 20.9 ± 0.09, 10.3 ± 0.06, and 5.2 ± 0.07 µl, respectively. The corresponding values from catheters placed in skeletal muscle (n = 23) were 41.9 ± 0.19, 20.9 ± 0.12, 10.5 ± 0.11, and 5.3 ± 0.09. From the catheters perfused at 0.33 µl/min during the entire experiment, the collected sample volumes (per 20 min) were 7.0 ± 0.07 (adipose tissue, n = 7) and 7.1 ± 0.16 (skeletal muscle, n = 7) µl, respectively. The average coefficients of variation in sample volume as calculated from 24 samples/catheter were 5.03 ± 0.5% (adipose tissue) and 4.95 ± 0.4% (skeletal muscle). The collected sample volumes at 0.33 and 0.16 µl/min (per 30 min) and at 0.075 µl/min (per 60 min; protocol with perfusion flow of 0.33-0.075 µl/min with dextran) were from catheters placed in adipose tissue (n = 9): 11.0 ± 0.16, 5.3 ± 0.10, and 5.2 ± 0.17 µl, respectively. The corresponding values from catheters placed in skeletal muscle (n = 10) were 11.3 ± 0.32, 5.5 ± 0.18, and 5.3 ± 0.34 µl.

Analyses. The concentrations of glucose, lactate, glycerol, and urea in the microdialysis samples were analyzed immediately after collection using ordinary enzymatic methods on a CMA 600 Microdialysis Analyzer. A high-precision pipetting device handles the sample (0.2-0.5 µl) and reagent volumes (14.5-14.8 µl). For glucose, lactate, and glycerol, the rate of formation of the colored substance quinoneimine is measured in a filter photometer at 546 nm. For urea, the rate of utilization of NADH is measured at 365 nm. The instrument performs a kinetic measurement by extracting the maximal absorbance change per second from the generated absorbance versus time curve. The complete measurement cycle takes between 60 and 120 s, depending on the substance being measured. Reagents used were obtained from CMA Microdialysis. At all analysis occasions, we included standards every 2nd h to check stability during the analysis (8 h) and the day-to-day variation. The ethanol concentration in the microdialysis samples was determined with the previously described (21) modification of the procedure described by Bernt and Gutmann (1). Samples were read fluorometrically against a standard curve before and after the addition of enzyme (alcohol dehydrogenase). Blood samples (5 ml) for analyses of glucose, lactate, and glycerol were transferred to heparinized tubes, and blood samples (2 ml) for analyses of lactate were transferred to tubes with 2 ml perchloric acid. Samples in heparinized tubes were kept on ice, and samples with perchloric acid were kept at room temperature for 15 min before they were centrifuged at 3,000 revolutions/min for 8 min. The supernatant was removed and stored at -80°C. All plasma samples were analyzed using colorimetric methods on a Cobas Mira-s clinical analyzer (Roche Diagnostics, Basel, Switzerland). Plasma glucose and urea were analyzed using the Unimate 7 and Unimate 5 reagent kits, respectively (Roche Diagnostics). Plasma glycerol and lactate were analyzed using Randox reagent kits GY 105 and LC 2389, respectively (Randox Laboratories, Crumlin, UK). In all analyses, standards were included to secure the stability over time of the Cobas Mira-s analyzer. To verify that the concentrations in plasma could be directly compared with those of the dialysates, 24 microdialysis samples from adipose tissue and skeletal muscle (8 at 1.33 µl/min and 4 at 0.66 µl/min from each tissue) were analyzed both on the CMA 600 Microdialysis Analyzer and on the Cobas Mira-s clinical analyzer. In relation to the results obtained with the CMA 600 analyzer (100%), the results (mean ± SD) of the Cobas Mira-s analyzer were 98.5 ± 3.9% [not significant (NS)] for glucose, 100.0 ± 4.4% (NS) for lactate, 101.7 ± 3.6% (P < 0.05) for glycerol, and 97.0 ± 3.4% (P < 0.001) for urea.

Calculations and statistics. Results are presented as means ± SE. When results were achieved for two catheters in a given tissue, the mean of these was calculated and constituted the value for that subject. Values given in the text are mean values of the last three samples from each perfusion flow period. These values were also used in the statistical calculations. To evaluate the significance of differences between skeletal muscle, adipose tissue, or plasma, a one-way ANOVA with repeated measures design followed by a Newman-Keuls multiple comparison test was used. Student's t-test for paired comparisons was used to evaluate if a change in the perfusion flow from 0.33 to 0.16 µl/min resulted in a significant change in the concentration of a substance in dialysate, to evaluate the difference between the plasma and muscle glycerol concentration, and to compare results from the protocol with perfusion flows of 1.33-0.16 µl/min with dextran with the protocol with perfusion flows of 1.33-0.16 µl/min without dextran.

Comparison of experiments with or without dextran in the perfusion solution. To study if the metabolite concentrations in the microdialysis samples were changed when dextran was used to counteract the loss of perfusion fluid, five subjects underwent an additional experiment without dextran added to the perfusion fluid (protocol with perfusion flows of 1.33-0.16 µl/min without dextran). The results of the experiments without dextran are given only for perfusion flow 0.33 µl/min. This perfusion flow resulted in a large fluid loss (per 30 min: 2.5 µl in adipose tissue and 5 µl in skeletal muscle) but still yielded enough volume for the analyses of metabolites. No statistical difference was found between experiments with and without dextran. The number of subjects studied was, however, too low in relation to the differences in mean values. The differences, which, with the present number of subjects, would be found statistically significant with 80% power, are given in the parentheses below. Concentrations in samples from skeletal muscle in the experiments, with and without dextran, respectively, were 3.91 ± 0.24 and 4.11 ± 0.25 mM (0.74) for glucose, 2.82 ± 0.35 and 2.78 ± 0.31 mM (0.98) for lactate, 38.1 ± 1.90 and 32.4 ± 9.6 µM (20.5) for glycerol, and 5.21 ± 0.47 and 4.99 ± 0.37 mM (1.25) for urea. The corresponding results for adipose tissue were 3.86 ± 0.28 and 4.46 ± 0.36 mM (0.95) for glucose, 1.49 ± 0.22 and 1.57 ± 0.24 mM (0.69) for lactate, 257.2 ± 16.9 and 255.9 ± 13.2 µM (45.1) for glycerol, and 4.93 ± 0.41 and 4.81 ± 0.32 mM (1.1) for urea.

    RESULTS
Top
Abstract
Introduction
Methods
Results
Discussion
References

Glucose concentration in venous plasma and in microdialysis samples from adipose tissue and skeletal muscle. With each decrease in the perfusion flow, the concentration of glucose was significantly increased in samples from both tissues (Fig. 1A). At the two highest perfusion flows (1.33 and 0.66 µl/min), the glucose concentration in samples from adipose tissue was significantly lower than in samples from skeletal muscle. This difference was abolished at 0.33 µl/min, and, at 0.16 µl/min, the glucose concentration in samples from adipose tissue (4.98 ± 0.14 mM) was higher (P < 0.001) than in samples from skeletal muscle (4.41 ± 0.11 mM) and equal to the concentration in venous plasma (5.07 ± 0.07 mM). The increased glucose concentration in muscle and adipose tissue samples, recorded during decreased perfusion flow, has only to a small extent been influenced by a gradual change over time (Fig. 1B). The concentration in the samples from skeletal muscle followed the same trend as the plasma levels, i.e., a small gradual decrease. This pattern was not apparent in adipose tissue, mainly due to a low initial and gradually improving recovery in three subjects. To test if a complete equilibration had been obtained at 0.16 µl/min, five additional experiments were included in which the perfusion flow was decreased further from 0.16 to 0.075 µl/min. These results generally support that a complete equilibration for glucose had been obtained at 0.16 µl/min. Thus no further increase was seen when the perfusion flow was decreased to 0.075 µl/min, although, in samples from adipose tissue, there was a tendency (P < 0.1) toward a higher glucose concentration at 0.075 compared with 0.16 µl/min. In addition, the differences in the glucose concentrations in samples from skeletal muscle (4.12 ± 0.31 mM), adipose tissue (5.29 ± 0.17 mM), and plasma (4.98 ± 0.10 mM) noted at 0.16 µl/min persisted at 0.075 µl/min. Although the data at different perfusion flows indicate that a complete equilibration for glucose in adipose tissue is achieved at 0.16 µl/min, a lower flow may be necessary for individuals with a large skinfold thickness. As illustrated in Fig. 5, skinfold thickness is negatively correlated with recovery for glucose at 1.33, 0.66, and 0.33 µl/min.


View larger version (19K):
[in this window]
[in a new window]
 
Fig. 1.   Glucose concentration (mean ± SE) in venous plasma and in microdialysis samples from skeletal muscle and subcutaneous adipose tissue. A: results from experiments with microdialysis catheters (22 in adipose tissue and 23 in skeletal muscle) perfused at successively lower perfusion flow (1.33-0.16 µl/min) in 13 subjects. B: results from experiments with 7 microdialysis catheters perfused at 0.33 µl/min for 8 h in 7 subjects. C: results from experiments with microdialysis catheters (9 in adipose tissue and 10 in skeletal muscle) perfused at successively lower perfusion flow (0.33-0.075 µl/min) in 5 subjects. When results were achieved for two catheters in a given tissue, the mean of these was calculated and constituted the value for that subject.

Lactate concentration in venous plasma and in microdialysis samples from adipose tissue and skeletal muscle. The concentrations of lactate in samples from skeletal muscle and adipose tissue (Fig. 2, A-C) were not significantly changed when the perfusion flow was decreased from 0.33 to 0.16 µl/min (skeletal muscle: 2.28 ± 0.20 to 2.39 ± 0.22 mM, NS; adipose tissue: 1.22 ± 0.13 to 1.30 ± 0.12 mM, NS). The lactate concentration in samples from skeletal muscle was significantly higher than in samples from adipose tissue and plasma (P < 0.001). In addition, the lactate concentration in samples from adipose tissue was significantly higher (P < 0.001) than in venous plasma (0.52 ± 0.06 mM). These differences were also found in the experiments with perfusion flows of 0.33, 0.16, and 0.075 µl/min (Fig. 2C). The lactate concentrations in control catheters perfused at 0.33 µl/min for 8 h (Fig. 2B) showed no major systematic change over time in either tissue.


View larger version (21K):
[in this window]
[in a new window]
 
Fig. 2.   Lactate concentration (mean ± SE) in venous plasma and in microdialysis samples from skeletal muscle and subcutaneous adipose tissue. A: results from experiments with microdialysis catheters (22 in adipose tissue and 23 in skeletal muscle) perfused at successively lower perfusion flow (1.33-0.16 µl/min) in 13 subjects. B: results from experiments with 7 microdialysis catheters perfused at 0.33 µl/min for 8 h in 7 subjects. C: results from experiments with microdialysis catheters (9 in adipose tissue and 10 in skeletal muscle) perfused at successively lower perfusion flow (0.33-0.075 µl/min) in 5 subjects. When results were achieved for two catheters in a given tissue, the mean of these was calculated and constituted the value for that subject.

Glycerol concentration in venous plasma and in microdialysis samples from adipose tissue and skeletal muscle. The concentration of glycerol in microdialysis samples from skeletal muscle was initially unexpectedly high and then decreased gradually for 90 min (Fig. 3, A-C). Thereafter, the glycerol levels were unchanged at perfusion flows of 0.66 µl/min and below (0.66 µl/min: 39.0 ± 4.3 µM; 0.33 µl/min: 40.8 ± 3.3 µM; 0.16 µl/min: 40.8 ± 3.0 µM, NS). In samples from adipose tissue, a relatively constant level was achieved when decreasing the perfusion flow from 0.33 to 0.16 µl/min (219.9 ± 13.7 to 232.8 ± 19.7 µM, NS; Fig. 3A). In the control catheters in adipose tissue perfused at 0.33 µl/min for 8 h (Fig. 3B), there was a gradual decrease during the last 5 h of the experiment. This decrease may have masked a possible increase when the perfusion flow was lowered from 0.33 to 0.16 µl/min. In addition, there was a marked gradual increase during the first 3 h (Fig. 3, B and C), which may have influenced results at the two highest perfusion flows (Fig. 3A). Although not completely identical, a similar pattern of an initial increase with a subsequent decrease was also noted for the glycerol concentration in venous plasma. At a corresponding time, the concentration of glycerol in venous plasma (68.7 ± 3.97 µM) was significantly higher (P < 0.001, t-test) than in samples from skeletal muscle achieved at 0.16 µl/min (40.8 ± 3.04 µM). In samples from adipose tissue achieved at 0.16 µl/min, the concentration of glycerol was 3.4-fold higher than the glycerol level in plasma at the corresponding time.


View larger version (24K):
[in this window]
[in a new window]
 
Fig. 3.   Glycerol concentration (mean ± SE) in venous plasma and in microdialysis samples from skeletal muscle and subcutaneous adipose tissue. A: results from experiments with microdialysis catheters (22 in adipose tissue and 23 in skeletal muscle) perfused at successively lower perfusion flow (1.33-0.16 µl/min) in 13 subjects. B: results from experiments with 7 microdialysis catheters perfused at 0.33 µl/min for 8 h in 7 subjects. C: results from experiments with microdialysis catheters (9 in adipose tissue and 10 in skeletal muscle) perfused at successively lower perfusion flow (0.33-0.075 µl/min) in 5 subjects. When results were achieved for two catheters in a given tissue, the mean of these was calculated and constituted the value for that subject.

Urea concentration in venous plasma and in microdialysis samples from adipose tissue and skeletal muscle. The concentration of urea (Fig. 4A) in samples from skeletal muscle was almost completely equilibrated at 0.66 µl/min (4.4 ± 0.29 mM). A complete equilibration was reached at 0.33 µl/min (4.6 ± 0.28 mM) and 0.16 µl/min (4.6 ± 0.26 mM). In contrast, the concentration of urea in samples from adipose tissue increased markedly when the perfusion flow was changed from 0.66 to 0.33 µl/min (0.66 µl/min: 3.5 ± 0.29 mM, 0.33 µl/min: 4.4 ± 0.28 mM) and was slightly increased to 4.7 ± 0.27 mM when perfusion flow was decreased from 0.33 to 0.16 µl/min. The results of the experiments, including a change in perfusion flow from 0.33 to 0.16 and 0.075 µl/min, support that a complete equilibration may be achieved in both tissues at 0.33 µl/min. However, the results from the experiments performed with a perfusion flow of 0.33 µl/min for 8 h indicate that the equilibration at this perfusion flow may be slightly incomplete in adipose tissue (Fig. 4B). As was discussed above for glucose, these experiments included subjects with a large skinfold thickness, in whom the equilibration was decreased. As illustrated in Fig. 6, skinfold thickness is also negatively correlated with the recovery for urea at 1.33 and 0.66 µl/min but, unlike glucose (Fig. 5), not at 0.33 µl/min. The concentration of urea in plasma at 7-8 h (4.2 ± 0.24 mM) was significantly lower than the plateau levels both in skeletal muscle (4.6 ± 0.26) and in adipose tissue (4.6 ± 0.27) perfused at 0.16 µl/min (P < 0.001).


View larger version (19K):
[in this window]
[in a new window]
 
Fig. 4.   Urea concentration (mean ± SE) in venous plasma and in microdialysis samples from skeletal muscle and subcutaneous adipose tissue. A: results from experiments with microdialysis catheters (22 in adipose tissue and 23 in skeletal muscle) perfused at successively lower perfusion flow (1.33-0.16 µl/min) in 13 subjects. B: results from experiments with 7 microdialysis catheters perfused at 0.33 µl/min for 8 h in 7 subjects. C: results from experiments with microdialysis catheters (9 in adipose tissue and 10 in skeletal muscle) perfused at successively lower perfusion flow (0.33-0.075 µl/min) in 5 subjects. When results were achieved for two catheters in a given tissue, the mean of these was calculated and constituted the value for that subject.


View larger version (24K):
[in this window]
[in a new window]
 
Fig. 5.   Relationship between glucose concentration in microdialysis samples and skinfold thickness. Results are from 21 microdialysis catheters perfused at different perfusion flow in 12 subjects. Correlation coefficient, significance, and slope were at 1.33 µl/min: 0.830, P < 0.001, -2.22 ± 0.34; 0.66 µl/min: 0.694, P < 0.001, -2.26 ± 0.54; 0.33 µl/min: 0.608, P < 0.01, -1.68 ± 0.50; 0.16 µl/min: 0.247, NS, -0.35 ± 0.32.


View larger version (23K):
[in this window]
[in a new window]
 
Fig. 6.   Relationship between the urea concentration in microdialysis samples and skinfold thickness. Results are from 21 microdialysis catheters perfused at different perfusion flow in 12 subjects. Correlation coefficient, significance, and slope were at 1.33 µl/min: 0.704, P < 0.001, -2.15 ± 0.50; 0.66 µl/min: 0.544, P < 0.05, -1.42 ± 0.50; 0.33 µl/min: 0.209, not significant (NS), -0.30 ± 0.33; 0.16 µl/min: 0.315, NS, -0.33 ± 0.23.

Ethanol outflow-to-inflow ratio in adipose tissue and skeletal muscle. In the four subjects in whom the microdialysis perfusion solution was supplemented with 50 mM ethanol, the ethanol outflow-to-inflow ratios in skeletal muscle (7 catheters) were 0.071 ± 0.011 at 1.33 µl/min and 0.010 ± 0.003 at 0.66 µl/min. Values in adipose tissue (7 catheters) were 0.390 ± 0.063 at 1.33 µl/min, 0.187 ± 0.055 at 0.66 µl/min, and 0.036 ± 0.017 at 0.33 µl/min.

Variation between two microdialysis catheters inserted in the same tissue. In the eight subjects in whom a complete set of results was obtained from two catheters in each tissue, it was found that the methodological variability generally was greater in adipose tissue than in skeletal muscle. It was also found that the variability for glucose and urea was reduced when the perfusion flow was lowered (Table 1).

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Relative difference between the substance concentrations obtained in two microdialysis catheters inserted in the same tissue

    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

In the present study, very low perfusion flows were used to achieve a complete equilibration between the interstitial fluid and the microdialysis perfusion fluid. To make sampling at these low perfusion flows possible, without a substantial loss of fluid to the tissue, we have previously found that a colloid needs to be included in the perfusion fluid (20). If no colloid is added to the perfusion fluid at a perfusion flow of 0.16 µl/min, one-half of the sample volume is lost in adipose tissue and almost all of the sample in skeletal muscle. It may be expected that the addition of a colloid to the perfusion fluid would significantly decrease metabolite concentrations in the microdialysis samples by increasing the effective perfusion flow through the microdialysis catheter. On the other hand, the colloid may also increase the dialysate concentration by preventing a dilution of the surrounding interstitial space. In the present study, these opposing effects might have been in balance, since the metabolite concentrations were not significantly different when perfusing with or without dextran, although the sample volumes in the latter situation were markedly smaller. However, since the present results could only exclude that no major differences exist, more detailed studies are needed on this issue.

Another unresolved issue is the cause for the differences in methodological variability calculated from the substance concentrations in adjacent microdialysis catheters inserted in the same tissue. In the present study, we observed that the variability for glucose and urea was markedly lower than for lactate and glycerol in both tissues. This may indicate that the methodological variability is higher for substances that, to some extent, are produced in the tissue than for those being delivered to the tissue by the circulation. Furthermore, we found that the variability for glucose and urea was reduced when the perfusion flow was lowered. We propose that this may be due a lowered removal of these substances from the tissue by the microdialysis catheter. A lowered removal will make the measurements less sensitive to unavoidable differences in the microdialysis recovery (which is mainly determined by the supply of substances from the tissue to the microdialysis catheter). Our data on glucose, lactate, and urea indicate that these differences in recovery between catheters are higher in adipose tissue than in skeletal muscle, since the methodological variation was higher for these substances in adipose tissue.

To identify a perfusion flow at which the interstitial fluid completely equilibrates with the microdialysis perfusion fluid, we used a protocol with successively lower perfusion flows. A complete equilibration was defined as having occurred when the dialysate concentration was constant in spite of a further lowering of the perfusion flow. In addition, experiments with catheters perfused at 0.33 µl/min during the entire study period were performed to study if possible changes over time could have influenced the protocol with successively lower perfusion flow. With the exception of a lack of steady state in skeletal muscle during the 60- to 90-min period immediately after catheter insertion, the results did not show any signs of systematic changes that could have influenced the results at the lowest perfusion flows.

Our results indicate that a complete equilibration for glucose is generally achieved at a perfusion flow of 0.16 µl/min both in adipose tissue and in skeletal muscle. A lower perfusion flow in adipose tissue may, however, be necessary for individuals with a large skinfold thickness. At complete equilibration, the concentration of glucose in samples from adipose tissue was found to be the same as in venous plasma. On the other hand, the glucose concentration in samples from skeletal muscle was 0.6 mM lower than the concentration in venous plasma and than in samples from adipose tissue. The observed difference between adipose tissue and skeletal muscle is interesting in the light of investigations (5), which indicate a higher resting glucose uptake in skeletal muscle compared with adipose tissue. Our definition of when the true interstitial concentration of a substance is recorded (defined as the dialysate concentration, which remains constant in spite of a lowering of the perfusion flow) has not been used in practice previously. The most frequently used method to determine the true interstitial concentration of glucose has been based on perfusion with different glucose concentrations (the no net flux method). Results obtained with this method in human adipose tissue, with one exception (15), support the concept that the interstitial concentration of glucose in adipose tissue is the same as in venous plasma (2, 9, 13). Compared with the present study, results obtained with the no net flux method in skeletal muscle have shown a similar (17, 18) or a larger (15) difference between the estimated interstitial concentration and the concentration in venous plasma. With the same type of microdialysis catheters and perfusion pump as in the present study, a recent investigation at a perfusion flow of 0.3 µl/min (16) found glucose concentrations in skeletal muscle and adipose tissue equal to those in the present study obtained at 0.16 µl/min. Based on the diffusion of an internal standard, these authors found the recovery of glucose to be incomplete (90% in adipose tissue and 95% in muscle) at the perfusion flow that was used. An incomplete recovery at 0.3 µl/min is in agreement with the present results (0.33 µl/min: 79% in adipose tissue and 90% in muscle). In contrast to the referred results, the present data were obtained at complete equilibration. It is therefore puzzling that the values obtained were nearly identical.

The concentration of lactate from the microdialysis samples in the present study was not significantly changed when the perfusion flow was decreased from 0.33 to 0.16 µl/min. With our definition of when a complete equilibration is achieved, this indicates that the true interstitial concentration of lactate may be recorded at a perfusion flow of 0.33 µl/min. The fact that the perfusion flow, at which a complete equilibration occurs, is higher than that for glucose is consistent with the higher diffusivity of the lactate molecule. We found the interstitial concentration of lactate in adipose tissue to be significantly higher than the plasma level and significantly lower than the level in skeletal muscle. The concentration of 1.3 mM found in adipose tissue agrees well (10, 11, 25) or is slightly lower than (24) previously reported values determined with the microdialysis no net flux method. The values recorded with microdialysis are, however, higher than values (0.6 mM) measured in a vein draining subcutaneous abdominal adipose tissue (5). In skeletal muscle, we found the interstitial concentration of lactate to be 2.4 mM. In a previous microdialysis investigation performed at 0.3 µl/min, a value of 1.9 mM was found (7). On the other hand, a value of 0.8 mM was found with the no net flux method in another human study (17). The latter study agrees well with literature data on the concentration of lactate in human muscle biopsies, which, under resting conditions, are of the order of 0.5-0.7 mmol/kg wet wt (19, 22). Further studies are required to resolve the uncertainty regarding the interstitial concentration of lactate in human skeletal muscle and adipose tissue.

In the present study, a complete equilibration for glycerol was achieved at a perfusion flow of 0.66 µl/min in skeletal muscle and at a flow of 0.33 µl/min or possibly 0.16 µl/min in adipose tissue. We found the interstitial concentration of glycerol in adipose tissue to be 233 µM, which was significantly higher than the levels in plasma and skeletal muscle. Previously reported values, as determined by the microdialysis no net flux method, range from 185 to 350 µM (7, 12, 24, 25), and values obtained from a vein draining subcutaneous adipose tissue range from 140 to 312 µM (4-6, 24). Thus the present results are within the range of the previously reported values, but we acknowledge the fact that the different studies can not be directly compared, since the degree of lipolytic activation is difficult to control. The interstitial concentration of glycerol in skeletal muscle was found to be around 40 µM and was significantly lower than the concentration in venous plasma. This is at odds with previous microdialysis investigations that have shown values that are higher (7) or remarkably higher (15) than the concentration in venous plasma. An observation made in the present study, which could provide an explanation for the discrepancy, is that the glycerol concentration in the microdialysis samples was very high initially and required 90-120 min to become stable.

The interstitial concentration of urea in human adipose tissue and skeletal muscle has not been determined with microdialysis previously. Consistent with its smaller molecular size, urea was found to equilibrate more rapidly than glucose. This was especially evident in skeletal muscle in which a near complete equilibration was found at a perfusion flow of 0.66 µl/min. In adipose tissue, the equilibration of urea was clearly slower than in muscle, but, at the lowest perfusion flow, the concentrations were equal in the two tissues. This finding is consistent with the fact that urea is not metabolized in either tissue. At complete equilibration, the concentrations in the two tissues were found to be slightly higher than the concentration in venous plasma but were equal to the concentration in plasma water. The most correct comparison between microdialysis and plasma samples is made when the plasma sample is obtained from a vein draining the tissue studied and is recalculated into plasma water. Because the plasma concentrations of glucose, lactate, and glycerol in the present study were not obtained in a vein close to the tissue, these values have not been recalculated into concentrations in plasma water. For urea, however, we have included the concentration in plasma water, since the concentration in the antecubital vein is expected to be equal to the concentration in a vein draining adipose tissue or skeletal muscle. The slower equilibration in adipose tissue than in skeletal muscle was also evident for glucose, which, like urea, is transported to the tissue from the circulation. The lower ethanol clearance found in adipose tissue than in skeletal muscle is consistent with the hypothesis that a lower blood flow is one factor explaining the slower degree of equilibration of these substances in adipose tissue than in skeletal muscle. Interestingly, skinfold thickness was found to negatively correlate with the recovery of glucose and urea in adipose tissue, indicating that the reduced blood flow with increasing adipose tissue mass (10) might impair the microdialysis recovery.

We believe that the main advantage of using a perfusion flow that results in a complete equilibration is that this approach directly yields the interstitial concentration and that no recalculation from a prior calibration or internal standard is required. An additional advantage is that a low perfusion flow minimizes the amount of substances removed from the tissue. A limitation with this approach is, however, that the small volume of collected sample may result in a low time resolution. It is therefore crucial that the analytical methods used are sensitive and that no perfusion fluid is lost to the tissue. In the present study, the analysis of four metabolites required a collected sample volume of 3 µl. At a perfusion flow of 0.16 µl/min, this corresponds to a time resolution of ~20 min, which often is enough in metabolic studies in humans. A better time resolution could, if necessary, be achieved by pooling samples from two or more microdialysis catheters.

In conclusion, we have shown that it is possible to find a perfusion flow at which the interstitial fluid in human skeletal muscle and adipose tissue completely equilibrates with the microdialysis perfusion fluid. At complete equilibration, the measured concentrations of glucose, glycerol, and urea were in good agreement with expected tissue-specific levels. Overall, the study indicates that microdialysis at a low perfusion flow may be a tool for the continuous monitoring of tissue interstitial concentrations.

    ACKNOWLEDGEMENTS

We thank CMA Microdialysis, Stockholm, Sweden, for good collaboration and generous support.

    FOOTNOTES

This work was supported in part by grants from the Swedish Medical Research Council (Project 07917) and the Swedish National Center for Research in Sports. Financial support was also given by the Swedish Society for Medical Research and the Research Foundations of the Karolinska Institute, Novo Nordisk, Magn Bergvall, Clas Groschinskys Minnesfond, Lars Hiertas Minne, and Fredrik and Ingrid Thuring.

Address for reprint requests: H. Rosdahl, Dept. of Physiology and Pharmacology, Karolinska Institutet, PO Box 5626, S-11486, Stockholm, Sweden.

Received 2 July 1997; accepted in final form 28 January 1998.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

1.   Bernt, E., and I. Gutmann. Determination with alcohol dehydrogenase and NAD. In: Methods of Enzymatic Analysis, edited by H. U. Bergmeyer. Weinheim, Germany: Verlag Chemie, 1974, p. 1499-1505.

2.   Bolinder, J., E. Hagström, U. Ungerstedt, and P. Arner. Microdialysis of subcutaneous adipose tissue in vivo for continuous glucose monitoring in man. Scand. J. Clin. Lab. Invest. 49: 465-474, 1989[Medline].

3.   Bolinder, J., U. Ungerstedt, and P. Arner. Long-term continuous glucose monitoring with microdialysis in ambulatory insulin-dependent diabetic patients. Lancet 342: 1080-1085, 1993[Medline].

4.   Coppack, S. W., R. D. Evans, R. M. Fisher, K. N. Frayn, G. F. Gibbons, S. M. Humphreys, M. L. Kirk, J. L. Potts, and T. D. R. Hockaday. Adipose tissue metabolism in obesity: lipase action in vivo before and after a mixed meal. Metabolism 41: 264-272, 1992[Medline].

5.   Coppack, S. W., K. N. Frayn, S. M. Humphreys, P. L. Whyte, and T. D. R. Hockaday. Arteriovenous differences across human adipose and forearm tissues after overnight fast. Metabolism 39: 384-390, 1990[Medline].

6.   Frayn, K. N., S. W. Coppack, S. M. Humphreys, and P. L. Whyte. Metabolic characteristics of human adipose tissue in vivo. Clin. Sci. (Colch.) 76: 509-516, 1989[Medline].

7.   Hagström-Toft, E., S. Enoksson, E. Moberg, J. Bolinder, and P. Arner. Absolute concentrations of glycerol and lactate in human skeletal muscle, adipose tissue, and blood. Am. J. Physiol. 273 (Endocrinol. Metab. 36): E584-E592, 1997[Abstract/Free Full Text].

8.   Jacobson, I., M. Sandberg, and A. Hamberger. Mass transfer in brain dialysis devices---a new method for the estimation of extracellular amino acids concentration. J. Neurosci. Methods 15: 263-268, 1985[Medline].

9.   Jansson, P.-A., J. Fowelin, U. Smith, and P. Lönnroth. Characterization by microdialysis of intercellular glucose level in subcutaneous tissue in humans. Am. J. Physiol. 255 (Endocrinol. Metab. 18): E218-E220, 1988[Abstract/Free Full Text].

10.   Jansson, P.-A., A. Larsson, U. Smith, and P. Lönnroth. Lactate release from the subcutaneous tissue in lean and obese men. J. Clin. Invest. 93: 240-246, 1994[Medline].

11.   Jansson, P.-A., U. Smith, and P. Lönnroth. Evidence for lactate production by human adipose tissue in vivo. Diabetologia 33: 253-256, 1990[Medline].

12.   Jansson, P.-A., U. Smith, and P. Lönnroth. Interstitial glycerol concentration measured by microdialysis in two subcutaneous regions in humans. Am. J. Physiol. 258 (Endocrinol. Metab. 21): E918-E922, 1990[Abstract/Free Full Text].

13.   Lönnroth, P., P.-A. Jansson, and U. Smith. A microdialysis method allowing characterization of intercellular water space in humans. Am. J. Physiol. 253 (Endocrinol. Metab. 16): E228-E231, 1987[Abstract/Free Full Text].

14.   Lönnroth, P., and L. Strindberg. Validation of the "internal reference technique" for calibrating microdialysis catheters in situ. Acta Physiol. Scand. 153: 375-380, 1995[Medline].

15.   Maggs, D. G., R. Jacob, F. Rife, R. Lange, P. Leone, M. J. During, W. V. Tamborlane, and R. S. Sherwin. Interstitial fluid concentrations of glycerol, glucose, and amino acids in human quadricep muscle and adipose tissue. J. Clin. Invest. 96: 370-377, 1995[Medline].

16.   Moberg, E., E. Hagström-Toft, P. Arner, and J. Bolinder. Protracted glucose fall in subcutaneous adipose tissue and skeletal muscle compared with blood during insulin-induced hypoglycaemia. Diabetologia 40: 1320-1326, 1997[Medline].

17.   Müller, M., A. Holmäng, O. K. Andersson, H.-G. Eichler, and P. Lönnroth. Measurement of interstitial muscle glucose and lactate concentrations during an oral glucose tolerance test. Am. J. Physiol. 271 (Endocrinol. Metab. 34): E1003-E1007, 1996[Abstract/Free Full Text].

18.   Müller, M., R. Schmid, M. Nieszpaur-Los, A. Fassolt, P. Lönnroth, P. Fasching, and H.-G. Eichler. Key metabolite kinetics in human skeletal muscle during ischaemia and reperfusion: measurement by microdialysis. Eur. J. Clin. Invest. 25: 601-607, 1995[Medline].

19.   Nordheim, K., and N. K. Völlestad. Glycogen and lactate metabolism during low-intensity exercise in man. Acta Physiol. Scand. 139: 475-484, 1990[Medline].

20.   Rosdahl, H., U. Ungerstedt, and J. Henriksson. Microdialysis in human skeletal muscle and adipose tissue at low flow rates is possible if dextran-70 is added to prevent loss of perfusion fluid. Acta Physiol. Scand. 159: 261-262, 1997[Medline].

21.   Rosdahl, H., U. Ungerstedt, L. Jorfeldt, and J. Henriksson. Interstitial glucose and lactate balance in human skeletal muscle and adipose tissue studied by microdialysis. J. Physiol. (Lond.) 471: 637-657, 1993[Abstract].

22.   Sahlin, K., A. Katz, and J. Henriksson. Redox state and lactate accumulation in human skeletal muscle during dynamic exercise. Biochem. J. 245: 551-556, 1987[Medline].

23.   Scheller, D., and J. Kolb. The internal reference technique in microdialysis: a practical approach to monitoring dialysis efficiency and to calculating tissue concentration from dialysate samples. J. Neurosci. Methods 40: 31-38, 1991[Medline].

24.   Simonsen, L., J. Bülow, and J. Madsen. Adipose tissue metabolism in humans determined by vein catheterization and microdialysis techniques. Am. J. Physiol. 266 (Endocrinol. Metab. 29): E357-E365, 1994[Abstract/Free Full Text].

25.   Stallknecht, B., L. Simonsen, J. Bülow, J. Vinten, and H. Galbo. Effect of training on epinephrine-stimulated lipolysis determined by microdialysis in human adipose tissue. Am. J. Physiol. 269 (Endocrinol. Metab. 32): E1059-E1066, 1995[Abstract/Free Full Text].


AJP Endocrinol Metab 274(5):E936-E945
0193-1849/98 $5.00 Copyright © 1998 the American Physiological Society