Clinical Science (1993) 85, 247-256 (Printed in Great Britain)
241
Techniques in Clinical Research
Assessment of adipose tissue metabolism in man: comparison
of Fick and microdialysis techniques
Peter ARNER and lens BULOW
Department of Medicine, Huddinge University Hospital, Huddinge, Sweden,
and *Department of Clinical Physiology, Bispebjerg Hospital, Copenhagen, Denmark
INTRODUCTION
Energy expenditure of different tissue types can
vary 100-fold, and the selection of fuels as well as
local energy expenditure can change substantially in
the same tissue. This has been elucidated for different tissue types in man primarily by the use of
arteriovenous catheterization techniques (Fick‘s
principle). A new technique, microdialysis, has
recently been introduced in clinical research. This
technique allows measurements of extracellular
water concentrations of the substances of interest
(e.g. metabolites, hormones, etc.). The aim of the
present paper is to review the microdialysis technique and the arteriovenous catheterization technique with special reference to the study of adipose
tissue metabolism in man.
MICRODIALYSIS
Direct measurement of the extracellular fluid has
several advantages. The concentration of the substance measured is that to which the cell is directly
exposed. The extracellular fluid is a low-protein
environment in which most drugs or hormones exist
in the ‘free’ active form, whereas protein binding
interferes with the measurement of the unbound
level of many substances in blood. In addition, the
distribution of a substance between the extracellular
space and blood may differ substantially depending
upon the local production and/or elimination, as
well as the physical properties of the substance
itself, such as lipophilicity, molecular charge, etc.
Several techniques exist to measure concentrations of active substances in the extracellular
water space. Some techniques involve the use of
dialysing sacs, cloths or wicks. However, these techniques are traumatic and most certainly alter the
normal capillary physiology, and, due to removal of
fluid volume, cannot be used to measure rapid
changes in the substance level in the extracellular
space. Less traumatic techniques use enzyme-coated
electrodes, whereby reactions between the substance
and the electrode are recorded as an electric current.
These methods do not consume fluid and enable
rapid measurements of substances in the water
space. However, it is difficult to calibrate the electrodes or to achieve reliable steady-state conditions,
and there are major problems with long-term stability. In addition, most electrodes allow measurements of only one particular substance, such as
glucose.
The microdialysis technique has several advantages over the above-mentioned techniques for
investigating the extracellular space. It is, generally
speaking, non-traumatic. Several substances can be
measured simultaneously. Reliable calibration of the
device is possible. Long-term measurements (i.e.
several days) can be made. Finally, and exclusively
to microdialysis, it is possible simultaneously to
deliver biologically active substances to the extracellular space and to measure the effects of the
substances on local metabolism.
Microdialysis was introduced over 20 years ago
by Delgado et al. [I] and was soon further developed by Ungerstedt and Pycock [2]. It was initially
mainly used for studies of the rat brain, although
other animal organs, such as skeletal muscle, liver,
heart and adrenals, have been investigated as well.
Recently, the microdialysis technique has been
introduced into clinical research on adipose tissue.
For many reasons this tissue is the first choice for
human investigations: it is easily available and can
be investigated without ethical problems. Furthermore, it can be used for long-term monitoring of
metabolism avoiding the problems of thrombosis
Key words: adipose tissue blood flow, arteriovenour, microdialysis.
Abbreviation: FFA. free fatty acid.
Correspondence: Dr lens Bulow, Department of Clinical Physiology, Birpebjerg Hospital. Bispebjerg Bakke 23, DK-2400 Copenhagen NV. Denmark.
248
P. Arner and J. Bulow
and infections associated with repeated blood
sampling.
1
PRINCIPLES OF MICRODIALYSIS
The microdialysis unit consists of a semipermeable membrane, which is a thin cylindrical
hollow fibre. It functions as an artificial vessel and
can deliver or remove substances to and from the
extracellular water space. It is coupled to a precision pump, and a neutral solvent (usually saline or
Ringer's solution), with or without biologically
active substances, is delivered to the cylinder at a
low perfusion speed (0.5-5 pl/min). The ingoing
solvent is called the perfusate. The solvent leaving
the cylinder, called the dialysate, is collected and
analysed for components collected from the extracellular space.
The type of substance that can be measured by
microdialysis or be delivered to the extracellular
space by microdialysis is determined by the permeability of the membrane. Usually membranes with a
molecular mass cut-off point between 3000 and
20000 Da are used. These membranes can easily
handle small molecules, such as glucose, lactate,
glycerol and adenosine, or small pharmacological
substances, such as adrenergic agents, prostaglandins and methylxanthines. In theory, it should be
possible to microdialyse large compounds, such as
proteins, by increasing the permeability of the membrane. However, when the molecular mass cut-off
point is too large (usually )lOOOOO Da) several
physical properties of the membrane are altered,
causing problems with diffusion, recovery and kinetics. Substances with a high degree of lipophilicity,
such as free fatty acids (FFAs), cannot be measured
with current microdialysis methods.
Two types of microdialysis devices have been
used for microdialysis of adipose tissue: (a) a dual
concentric tubing with a dialysis membrane at the
end; and (b) a transversally implanted dialysis
cylinder with separate input and exit sites. The
performance of the two probes is similar. The
former probe (Fig. 1) has to be manufactured with
special instruments [3], but is commercially available. The latter probe can be produced in the
laboratory without complicated equipment, but the
procedure is time-consuming.
For' many reasons it is most likely that the
microdialysis probes cause no or little harm to
adipose tissue. There is a transient rise in ATP in
the dialysate during the first 30 min after the
implantation of the probe, which probably reflects
the initial trauma [4]. However, histological studies
show no evidence of oedema or bleeding 12h after
the implantation of the probe [ S ] . Furthermore, the
recovery in uiuo of the metabolites, such as glucose,
is constant over several days after implantation [ 6 ] .
The major methodological problem with microdialysis is whether or not the measured concentration of a particular substance in the dialysate
0.
OO
O.
0 0
0
0.
O O
0
.O
3
Fig. I. Principles of microdialysis with a doublelumina probe. See
the text for further details.
reflects the actual level in the extracellular space of
adipose tissue. The experiments are usually conducted so that the recovery of a particular substance
is incomplete. Recovery is negatively correlated with
the flow velocity of the perfusion medium and
positively correlated with the length of the active
dialysis membrane. It is often necessary to use a
high perfusion speed (2.5-5 pl/min) in order to
obtain a sufficient dialysate volume which can be
used for analysis. It is difficult to construct a very
long membrane 0 3 0 mm) because such membranes
may fracture and/or cause tissue damage. Previously, the probe was calibrated in oitro, i.e. it was
placed in a solution with a known amount of the
substance to be determined and the concentration of
the substance in the dialysate was measured at the
currently used perfusion speed. This concentration
was divided by the actual concentration in the
incubation bath to obtain recovery. As discussed in
detail [7], this technique for determining recovery in
uitro seriously underestimates the true tissue level of
the substance to be measured because recovery in
uitro is higher than recovery in oiuo . The latter is
due to several factors, such as tissue resistance and
different physicochemical properties of the membrane and substance to be measured in biological
fluid as compared with baths in oitro .
The recovery in uivo problem has been solved in
two ways. Lonnroth et al. [S] have introduced a
calibration technique. Adipose tissue is microdialysed under steady-state conditions with solvents
containing different concentrations of the substance
measured and the concentration in the perfusate
that does not cause any change in the concentration
Aksessment of tissue metabolism
in the dialysate is estimated by linear regression
analysis (concentration,, = concentration,,, =
tissue concentration). One problem with this technique is that it is time-consuming (2-3 h) and therefore less suited to be used in connection with
subsequent experiments under non-steady-state conditions. In addition, the technique necessitates local
exposure of adipose tissue to high concentrations of
the substance, which may artificially alter tissue
behaviour in subsequent kinetic experiments.
Bolinder et al. [6] have constructed a long (30 mm)
dialysis membrane perfused at low speed (0.5 pl/
min). Using this technique it was possible to obtain
full recovery and to measure the change in the true
concentration of glucose in adipose tissue over time.
However, it was necessary to use a long collection
time (0.5-1.0 h) in order to obtain a suitable dialysis
volume. Therefore, the latter technique is unsuitable
for short-term kinetic experiments.
Another problem with the estimation of the true
tissue level of a substance using microdialysis
concerns tissue drainage of the substance, as discussed in detail in [7-91. When the tissue is perfused at a high speed, large amounts of substances
are removed from the extracellular space, although
recovery is low because large volumes of fluid per
unit of time pass through the microdialysis device
and remove molecules from the extracellular space.
This problem is of minor concern for substances
that are produced locally in adipose tissue, such as
glycerol, adenosine and lactate, but may cause analytical problems when the substance is one that is
only consumed by adipose tissue, such as glucose.
Some reports [lo] of changes in adipose tissue
glucose that were not paralleled by changes in
circulating glucose may be due to local drainage of
glucose around the microdialysis probe. The
drainage problem can be solved in different ways. It
is possible to add glucose in a low concentration to
the perfusion medium in order to avoid a large
concentration gradient between the tissue and the
microdialysis probe [S]. Alternatively, adipose tissue
can be dialysed at a very low speed (0.5 pl/min or
lower) so that insignificant amounts of glucose are
removed from the tissue by the perfusion solvent
PI.
Microdialysis is highly suitable for kinetic experiments. These studies can usually not be performed
at 100% recovery in vivo or be used in connection
with the time-consuming calibration method discussed above. On the other hand, the problem of
measuring the absolute level of a particular substance is of minor relevance in kinetic experiments.
Instead, the change in relation to the baseline level
is the major focus of the kinetic investigation.
However, the interpretation of kinetic experiments is
valid only when recovery in vivo is constant over a
large concentration interval of the substance in the
extracellular space [3]. Several reports have been
published which indicate that this recovery is constant. Thus, the ratio of blood glucose concentration
249
to adipose tissue glucose concentration was constant
in kinetic experiments when adipose tissue was
microdialysed at ~ 2 0 %recovery in vivo during
various types of glucose loads in healthy subjects
[l 13 or microdialysed at x 100% recovery in Type
1 diabetic patients who had large endogenous
swings in their glucose level [6]. It is also necessary
to consider the dead space in the outlet of the
dialysis tubing when rapid changes in the concentration of the substance are recorded. A slow perfusion rate and long sampling period will not detect
small rapid concentration changes in the extracellular space, in particular if the outlet of the dialysis
tubing is long.
As mentioned above, microdialysis can be used to
study the effect in situ of biologically active substances on adipose tissue metabolism. The tissue can
be exposed to a very high concentration of an agent
locally through the microdialysis device without
causing any systemic effect of the agent [12]. This is
probably due to the fact that the penetration distance from the probe is usually small (i.e. 1-2 mm),
as discussed in detail in [7].
An additional problem with concentration measurements concerns adipose tissue blood flow. The
concentration of a substance in the extracellular
water space depends largely upon two factors: (a)
production and utilization by the tissue cells; (b)
delivery and elimination through the blood vessels.
In this respect, it is of importance to know that only
the nutritive blood flow regulates the concentration
of the extracellular space. Blood which is shunted
through adipose tissue is of no or little importance
for the exchange between the two compartments.
Until recently, no methods have been available to
directly estimate the nutritive flow in the immediate
vicinity of the microdialysis probe. Attempts have
been made to combine a laser Doppler probe with a
microdialysis probe in order to determine nutritive
flow in brain microdialysis experiments [13]. The
method has, however, been questioned by its own
inventors [14]. An ethanol method has recently
been developed to study the nutritive flow [15]. The
ethanol is added to the dialysis solvent and its
escape from the perfusion medium to the surrounding extracellular space is measured. Changes in the
ratio of outgoing to ingoing ethanol concentration
reflect changes in nutritive flow. The method has
been thoroughly evaluated in rat skeletal muscle
[14]. Ethanol, in the concentration used (up to 1055
mmol/l), does not alter metabolism and has no
effect itself on blood flow. It does also not alter the
properties of the microdialysis membrane. The ethanol method has been modified to be used on
subcutaneous adipose tissue [16]. The methodological findings are similar to those with skeletal muscle.
The most effectiveethanol concentration (50mmol/l)
has no effect on local adipose tissue lipolysis or
blood flow. Although it is not possible to make true
quantitative evaluations of tissue perfusion with the
ethanol technique, the method appears to be a
250
P. Arner and J. Bulow
sensitive indicator of small variations in the nutritive blood flow to the tissue surrounding the microdialysis probe.
Owing to the small volumes involved and/or
incomplete recovery, the final concentration of a
substance to be measured in the dialysate with
analytical chemistry methods is low, often in the
picomolar or even femtomolar range. Consequently,
very sensitive detector systems have to be used to
measure substances in the dialysate. H.p.1.c. in combination with electrochemical detection or enzymic
assays, where the sensitivity has been enhanced by
luminescence, have proven useful. Radioimmunoassays, used to determine small polypeptides or
pharmacological drugs which can be recovered in
the dialysate, are probably too insensitive to be used
in combination with microdialysis experiments.
GLUCOSE METABOLISM
The glucose levels in subcutaneous adipose tissue
have been investigated in detail. Interstitial levels
identical or almost identical with those in blood
were determined with the calibration technique [8]
and with microdialysis at full recovery [6]. In
healthy non-obese or obese subjects and in Type 1
diabetic patients the kinetics of glucose in blood
and adipose tissue are very similar [4, 8, 10, 1 1 , 17,
181. During insulin infusions at a fixed blood glucose level (euglycaemic glucose clamp) the concentration decreases in the extracellular water space of
adipose tissue [191. After stopping the insulin infusion it returns to the initial level [19]. Because
nutritive blood flow does not change during this
condition (U. Johansson et al., unpublished work)
the change in adipose tissue glucose level during
insulin infusion probably reflects local glucose
uptake.
Subcutaneous adipose tissue has long since been a
target for continuous on-line measurements of glucose because it closely mirrors the blood glucose
level. The ultimate goal is to construct a closed-loop
insulin infusion system to diabetic patients using
continuous monitoring of the glucose level to adjust
the rate of insulin delivery from a pump. Microdialysis has recently been used for this purpose. By
combining a dialysis probe with an electrochemical
glucose sensor [20] it was possible to perform online measurements of the glucose level for up to 21 h
in diabetic patients and healthy subjects [l8].
Although many technical problems remain to be
solved, the combination of microdialysis for sampling glucose with an electrode to continously measure glucose has the potential to be used for closedloop insulin delivery in diabetic patients.
LACTATE METABOLISM
It has become increasingly evident that adipose
tissue is an important source of lactate production.
This question has been addressed using microdialy-
sis [21,22]. The steady-state levels of lactate are at
least 50% higher than in blood. When the water
space around the microdialysis probe is drained of
glucose (the major substrate for lactate) the concentration of lactate in adipose tissue decreases. The
kinetics for the increase in the lactate concentration
are not apparently different in blood or adipose
tissue during an oral glucose load. However, lactate
kinetics in adipose tissue are significantly altered
when the tissue is microdialysed with a catecholamine during the glucose load. Taken together, the
microdialysis data strongly favour the hypothesis of
active lactate production in adipose tissue.
ADENOSINE METABOLISM
The concentration of adenosine in the extracellular space of adipose tissue has been determined in
one study [23]. The level measured in the interstitial
space was high enough to tonically inhibit basal
lipolysis in vitro, thus allowing P-adrenergic lipolytic
effects of catecholamines to be revealed in vivo.
LlPOLYSlS
Lipolysis is a major metabolic event in adipose
tissue and has been an obvious target for microdialysis studies. The interstitial glycerol level in adipose
tissue reflects lipolysis, since glycerol, in contrast
with fatty acids, is metabolized by the tissue to an
insignificant extent. The steady-state levels of glycerol are 2-3 times higher in subcutaneous adipose
tissue than in blood [24,25] when determined with
the microdialysis calibration technique. When lipolysis is inhibited by an oral glucose load [25] or
stimulated by physical exercise [24] or mental stress
[26], the kinetics of glycerol in plasma and abdominal subcutaneous adipose tissue are similar, which
may suggest that changes in circulating glycerol
reflect lipolysis in adipose tissue. However, other
results indicate that the latter is not the case. Thus,
repeated intravenous injections of isoprenaline (lipolytic P-adrenoceptor agonist) to the rat caused an
entirely different kinetic response of glycerol in
subcutaneous adipose tissue as compared with
blood [12]. Since changes of the blood glycerol level
reflect production in different adipose depots, tissue
utilization and urinary output, it is evident that
several factors besides lipolysis in subcutaneous
adipose tissue can regulate the circulating glycerol
level. One such factor is regional variation in the
lipolytic rate, which has been repeatedly documented in vitro. Such variation is also found in vivo
with microdialysis. The steady-state glycerol level is
higher in abdominal than in femoral subcutaneous
adipose tissue [25]. During physical exercise or
mental stress the rise in the glycerol level is much
more marked in abdominal than in gluteal subcutaneous adipose tissue [24,27].
The data above suggest that abdominal subcutaneous adipose tissue is more lipolytically active than
Assessment of tissue metabolism
the peripheral subcutaneous adipose tissue. An
attempt has been made to directly measure the
lipolytic rate in uiuo in central versus peripheral
subcutaneous adipose tissue by combining the microdialysis method to measure glycerol with the Xe
clearance technique to measure blood flow [28].
Surprisingly, the rate of glycerol production per unit
of adipose tissue mass was similar in the two
adipose reigons and there was no apparent influence
of obesity on the results. However, some caution
should be exercised when these data are interpreted,
since their validity is largely dependent on the true
estimation of adipose tissue blood flow. When using
Xe clearance it is necessary to know the partition
coefficient for this gas between tissue and blood. A
fixed value is used [28], but this value may vary
between different adipose tissue depots as well as
between individuals, and it may also be influenced
by obesity, as discussed in detail below. Furthermore, blood flow measurement with Xe was performed in a different part of adipose tissue than that
immediately surrounding the microdialysis probe.
PHARMACOLOGICAL STUDIES
Human fat cells are equipped with a large
number of receptors which are coupled to lipolysis.
Agents that stimulate or block these receptors can
be added to the microdialysis perfusion medium and
their effect on lipolysis can be investigated after
exposure in situ.
So far the adrenergic regulation of lipolysis in
subcutaneous adipose tissue has been investigated in
some detail using microdialysis with solvents containing adrenoceptor agonists and antagonists in
various combinations. At rest, antilipolytic aadrenoceptors seem to regulate the lipolytic rate,
whereas during exercise lipolytic /?-adrenoceptors
are operating [24]. During mental stress lipolysis is
under the control of /?-adrenoceptors, whereas lactate formation by adipose tissue seems to be regulated by a-adrenoceptors [26] and evidence has
been found for increased glucose uptake in the
tissue during adrenaline infusion [29]. a- and /?adrenoceptor subtypes have also been investigated.
a,-adrenoceptors may play an important role in
regulating blood flow in addition to their antilipolytic effect [16] and are resistant to acute desensitization after catecholamine exposure [30]. The latter
is also true for a,-adrenoceptors, whereas the
/.?,-subtype is very sensitive to acute desensitization
~301.
The doseresponse relationship for catecholamine-induced lipolysis has been investigated in situ
[3 13. The dominant lipolytic receptor appears to be
the /?,-subtype. Marked lipolytic effects of noradrenaline were obtained when this hormone was
added to the perfusion medium in concentrations
that were 10-100 times lower than in blood. This
suggests that the concentration of catecholamines in
251
the extracellular space of human adipose tissue is
much lower than in plasma, which is in contrast
with microdialysis data obtained with rat adipose
tissue, where the catecholamine Concentration is
reported to be in the same range as that in blood
~321.
Finally, adipose tissue microdialysis has been
used for pharmacokinetic experiments. The distribution of caffeine between blood and the extracellular
fluid differed considerably after oral administration
C331.
FICK'S PRINCIPLE
Fick's principle, originally proposed as a means of
measuring blood flow [34], states that if arterial and
venous concentrations of a substance are constant
and blood flow is constant, then input = blood
flow x arterial concentration = output = blood
flow x venous concentration + tissue metabolism
of the substance.
Quantitative determination of uptake or output of
a substance from an organ is normally performed
by measurements of arteriovenous concentration
differences for the substances across the organ and
of blood flow through the organ. Under ideal
circumstances the organ is drained selectively by a
single vein, but this condition can seldom be fullfilled in studies of skeletal muscle and adipose
tissue, at least in man in uiuo. With regard to human
skeletal muscle metabolism, the so-called forearm
model has been widely applied, and it is normally
assumed that the blood taken from a deep forearm
vein is mainly derived from skeletal muscle [35,36].
During exercise and immediately after exercise this
assumption is probably correct, but during rest it
can be questioned. In a recent study [37] several
methodological problems of the forearm technique
has been emphasized, some of which are the mixing
of deep venous blood with blood derived from
superficial veins mainly draining skin and adipose
tissue, and blood drawn retrogradely during sampling. In addition to these problems the natural
fluctuations in resting forearm blood flow induce
another error if the blood samples are not taken
very slowly (over minutes) to obtain a representative
blood sample matching the mean blood flow. In this
context it is necessary to take the transit time of the
substance of interest through the system into consideration. It is shown that carbon dioxide can have
a transit time in the forearm from cellular production to appearance in the vein blood of more than
30 min [37].
Adipose tissue is not easily accessible for selective
venous catheterization in uiuo. Until recently, studies
of adipose tissue metabolism have been performed
either semi in uiuo in the dog or rabbit [38,39] or in
uiuo in the Syrian fat-tailed sheep [a]
or dog [41].
However, Frayn et al. [42] have developed a technique which allows the catheterization of a vein
draining the subcutaneous adipose tissue of the
P. Arner and J. Bulow
252
anterior abdominal wall in man. By this technique
adipose tissue metabolism can be studied quantitatively or semi-quantitatively during various conditions in uiuo when the measurements of arteriovenous concentration differences are combined with
measurement of the local adipose tissue blood flow.
However, since the blood in this very superficially
located vein (see Fig. 3) can only be partly derived
from adipose tissue and a significant fraction must
derive from skin (probably around 50% during
rest), the metabolite concentrations measured in this
vein can only give a picture of the metabolic events
taking place in adipose tissue to a certain degree. It
is difficult to estimate exactly the dilution effect in
man, but in experiments in dogs an attempt was
made to calculate the effect in the inguinal fat pad
preparation, and in these experiments it was concluded that it was of minor importance with regard
to estimates of lipolytic rate (glycerol output) during
stimulated lipolysis [41]. This is in accord with the
evidence presented in [42] that dilution with blood
deriving from skin may play a minor role in man as
well. With regard to glucose and lactate metabolism
the relative contributions of skin and adipose tissue
are difficult to estimate. First, the arteriovenous
concentration differences are very small for these
metabolites. Secondly, there are concentration differences to be taken into account in blood deriving
from subcutaneous adipose tissue and skin owing to
metabolic differences in the carbohydrate metabolism between the two tissue types [43]. In spite of
this unavoidable Achilles’ heel of the method, it has
provided new insight into the metabolism of human
abdominal subcutaneous tissue during various conditions. However, the catheterization technique is
not easy to use. In selected subjects with visible
veins the success rate is around 5 0 4 0 % . The major
problem is the low pressure in the vein, which leads
to very slow backflow in the catheterization cannula. This implies that the puncture of the vein may
not be recognized, and further advancement of the
cannula results in the formation of a haematoma. If
this happens, it is not possible to place a guide wire
in the vein even if the cannula tip is withdrawn to
the vessel lumen afterwards. In some cases the
puncture site may be moved proximal to the first
site with success. Another problem is that the low
flow and volume in the vein under some circumstances make it difficult to withdraw sufficient
amounts of blood. On the other hand, the vein is
not irreversibly damaged by catheterization. Thus, it
is possible to re-catheterize it in new experiments.
Similarly, when a catheter has been placed in a vein,
it is in such a stable position that it is possible to
perform experiments even during physical exercise.
H U M A N SUBCUTANEOUS ADIPOSE TISSUE
METABOLISM STUDIED BY FICK’S PRINCIPLE
The results of the investigations of human adipose
tissue metabolism performed with the catheterization technique have recently been reviewed by
Frayn [44]. In brief, it has been demonstrated that
abdominal subcutaneous adipose tissue takes up
glucose during fasting and that this uptake increases
after an oral glucose load. Similarly, lactate was
found to be produced even in the fasting state and
with an increasing rate after glucose intake. However, the lactate production could only account for
a minor fraction of the total glucose metabolism
[45]. It is estimated that less than 1 % of the
carbohydrate ingested in a mixed meal is converted
to lactate on a whole-body basis, but the lactate
production from adipose tissue can account for as
much as 30% of the glucose uptake [46]. It has
been shown that subcutaneous adipose tissue plays
a significant role in the metabolism of recently
ingested triacylglycerol. The results have given new
insight into the regulation of intracellular lipolysis
in adipocytes (via the hormone-sensitive lipase
systems) and the extracellular lipolysis of circulating
triacylglycerol (via the lipoprotein lipase bound to
the adipose tissue endothelium). During fasting
about 75 % of the glycerol output from subcutaneous adipose tissue is due to intracellular lipolysis,
whereas about 90% can be accounted for by intravascular lipolysis after a mixed meal [46]. The
regulation of lipoprotein lipase is abnormal in obesity [47]. The effects of insulin and of ethanol intake
on adipose tissue metabolism have been elucidated
[48,49], and adipose tissue has been shown to
participate in the metabolism of amino acids in a
manner qualitatively comparable with skeletal muscle [SO]. The technique has been applied in an
exercise study to elucidate the factors regulating
lipid mobilization from adipose tissue during exercise [Sl]. A decreasing rate of FFA re-esterification
during exercise was found, which is somewhat in
discord with previous studies [52,53] in which
evidence was presented for the hypothesis that FFA
re-esterification is a key process in the control of
FFA mobilization during exercise. The conclusion
in [Sl] that limitation of FFA release from adipose
tissue during exercise is a determinant of the muscle
utilization of FFA is questionable, since the concentration of circulating FFA increases about 10fold during prolonged exercise [54]. If FFA mobilization was limiting FFA utilization, an increase in
blood concentration could not take place. In addition, the catheterization technique cannot distinguish between FFA re-utilization by fat cells and
FFA retained in the extracellular space between fat
cells. Recent microdialysis data demonstrate a
marked capacity of the extracellular space to retain
lipids in adipose tissue [16].
Quantification of the results obtained by Fick’s
principle is crucially dependent upon the determination of the arteriovenous concentration differences and the blood flow. Measurements of arteriovenous concentration differences in subcutaneous
adipose tissue are difficult for many metabolites
253
Assessment of tissue metabolism
because they are very small. The reason is that the
blood supply to adipose tissue primarily is adjusted
to the lipolytic level in the tissue to enable rapid
mobilization of FFA and not to the aerobic demand
of the tissue [53]. This and the low oxygen consumption of adipose tissue imply a very low oxygen
extraction, and the oxygen saturation in the
superficial abdominal wall vein in man is very high
during rest and fasting (85-90 %), increasing to 9095% when vasodilatation is induced in the tissue,
e.g. after an oral glucose intake [46,55]. This
emphasizes that oxygen and carbon dioxide partial
pressures and metabolite concentrations have to be
measured in actual arterial blood instead of in
arterialized hand vein blood, since arterialization
always is more or less incomplete, which seriously
affects the calculations of uptake or output [37,45].
Another problem arises during non-steady state
conditions, since it may be necessary to take the
transit time of the metabolites through the tissue
into consideration in order to match venous blood
sampling correctly to a rapidly changing arterial
concentration [56].
Determination of local nutritive blood flow is also
subject to experimental difficulties. Adipose tissue
washout of ‘33Xe has been used as a reliable
method for three decades [57]. Several assumptions
have to be made in order to calculate the tissue
perfusion coefficient from the washout rate constant
of the radioactivity. The first assumption is that the
tissue is homogeneous with regard to composition
as well as to perfusion. The content of lipid, water
and protein in subcutaneous adipose tissue from the
anterior abdominal wall has been found to be rather
homogeneous, with a coefficient of variation of 6 %
between symmetrical biopsies [58]. In view of
this chemical homogeneity the modest difference
between symmetrical Xe depots, the coefficient of
variation is about 10% [59,60], can be taken as
evidence of a homogeneous perfusion pattern in
human subcutaneous abdominal adipose tissue. On
the other hand, whether a single Xe depot labelling
a tissue volume of about 1 ml is representative for
the whole tissue volume drained by the superficial
inferior epigastric vein can be questioned. It would
be preferable to label a larger tissue volume with
‘33Xe or 12’Xe. However, due to the increased local
radiation dose which is then given, this is not
practical [61]. This problem could be circumvented
by the use of the 99mTc-washouttechnique [62].
However, the major advantage of the Xe-washout
technique is that it allows studies for longer periods
(up to 24 h) [63] owing to the high solubility of Xe.
Tc has a low solubility in adipose tissue resulting in
a very fast washout rate from the isotope depot,
which normally is prohibitive for experiments of
longer duration than 1-2 h. A problem with the Xewashout technique is that it requires knowledge of
the specific tissue/blood partition coefficient in order
to allow calculation of tissue perfusion coefficients
from the washout rates. Otherwise, only relative
changes in blood flow can be obtained, and thus
only semi-quantitative changes in substrate flux can
be calculated. Traditionally a tissue/blood partition
coefficient of 10 for Xe in adipose tissue has been
applied [57]. However, in normal weight man this
value is too high (on average it has been found to
be 8 [58]) and the coefficient can vary considerably
from individual to individual. It is therefore necessary to estimate the partition coefficient individually,
preferably by chemical analysis, but this is seldom
practical. It was previously demonstrated [58] that
the local skinfold thickness correlates with the partition coefficient determined chemically. Thus, by
such additional anthropometric measurements it is
possible to obtain quantitative measurements of
local tissue perfusion enabling calculations of substrate fluxes with Fick’s principle.
QUANTIFICATION OF LOCAL TISSUE
METABOLISM BY MICRODIALYSIS
As pointed out above, microdialysis is very well
suited for measurements of qualitative changes in
local tissue metabolism, but only few attempts have
been made to quantify measurements of local substrate exchange [28,64]. One of the problems is
how to obtain a reliable measurement of local
nutritive blood flow in the environment of the
microdialysis fibre. Xe can be deposited in the close
vicinity of the fibre, thus giving an exact picture of
the washout rate in the tissue which is microdialysed. However, the injection may cause a local
oedema and disturb the extracellular space that is
surrounding the microdialysis probe.
Under the assumption that a reasonable estimate
of local tissue blood flow can be obtained, the next
problem is to recalculate the interstitial water concentrations to venous blood concentrations in order
to enable the use of Fick’s principle in the calculations of substrate exchange. Theoretically the relation between the interstitial water concentration and
the arterial and venous blood water concentrations
is given by the following equation:
(cv-ca)/(ci-ca) = 1 -e-PS’Q
which is the general equation for the Krogh tissue
cylinder model [65,66], and it can be generalized to
whole organs provided all capillaries are of similar
length, exhibit identical permeabilities, are homogeneously perfused. and the surrounding interstitial
space is homogeneous in composition.
In the tissue uptake situation c, can be calculated
as:
c, = (c,-ci)
x e-PSIQ
+ ci
and in the tissue output situation c, can be calculated as:
c, = (ci-c,)
x (l-e-PSIQ)
+
Ca
254
P. Arner and J. Bulow
In the above equations c,, c, and ci denote arterial,
venous and interstitial water concentrations, PS the
permeability surface area product and Q the plasma
water flow. The model implies that the exchange
over the capillary membrane is exponentially dependent on the concentration difference from the arterial to the venous end of the capillaries, a concept
that has been questioned experimentally, since the
permeability is higher at the venous end of a
capillary than at the arterial end [67]. Another
assumption is that the extracellular water space is a
well-mixed compartment, and that the substrate
concentration measured by microdialysis is the average concentration in this compartment. It appears
from the equations given above that it is necessary
to have an estimate of the plasma water flow
through the tissue. For small uncharged molecules
plasma water flow is normally calculated from
plasma flow by multiplication with the factor 0.94.
If the substance is negatively charged as, for example. lactate, the factor is 0.89, since a small potential
difference exists across the endothelial membrane
with the luminal side being negative (683. Thus, the
recalculations of ci concentrations to c, concentrations imply the same experimental dificulties as
do the measurements of local nutrititive blood flow.
Yet another problem to be taken into account is the
difference between plasma and whole blood concentrations [68]. The last experimental problem to
be taken into account when microdialysis data are
quantified is the timing of arterial blood sampling in
relation to the collection of the dialysate, a problem
which is particularly important during non-steadystate conditions. The perfusion rates used in microdialysis gives a low time resolution for this method,
usually greater than 10 min. Fig. 2 demonstrates the
time course of the interstitial glucose and lactate
concentrations in the crural subcutaneous adipose
tissue before, during and after 30 min of circulatory
arrest in six subjects, and it appears that the time
delay in the system applied [29,64] is approximately 10 min. Thus it is necessary to know this
delay exactly in order to be able to take the arterial
blood sample at the correct time in advance of the
microdialysis sample, and it may be an advantage to
use the average of several blood samples taken
during the period of microdialysate sampling.
Applying this method, Jansson et al. [28] measured glycerol production in two different subcutaneous locations, and glycerol and lactate production
and glucose uptake was measured in abdominal
subcutaneous adipose tissue during prolonged exercise [64].
DIRECT COMPARISON OF MICRODIALYSIS AND
CATHETERIZATION TECHNIQUES
Only a few experimental data have been published in which substrate fluxes in adipose tissue
have been measured simultaneously by microdialysis
T
t
d
Occlusion
Ib
$0
I
i lo
$0
Time (min)
Qo
7b
Fig. Z Subcutaneous interstitial glucose ( 0 )and lactate (m)
concentrations before, during and after circulatory arrest. Values are
means SD for six subjects.
and venous catheterization [55,64]. With regard to
glucose uptake, the results obtained by the two
methods are fairly equal, giving glucose uptakes in
the range 1-2 pmol min-'lOOg-' during fasting
and increasing during an oral glucose load. Differences of about 10% were found between the glucose
concentrations measured by the two methods [55]
with the calculated venous glucose concentration
being lower than the measured concentration. In
contrast, a great difference is found in the lactate
outputs. The lactate production calculated from
microdialysis data is several-fold higher than the
corresponding production found with the venous
catheterization method during an oral glucose load
[55, 641. Glycerol production rates are comparable
when it is taken into account that it can be
necessary to correct the total glycerol concentrations measured in the venous blood for glycerol
derived from lipolysis of circulating triacylglycerols
[46,47]. As regards glycerol concentrations measured by microdialysis, it is not necessary to
consider the blood level of glycerol, since the latter
concentration is 2-3 times lower than the concentration of glycerol in the extracellular space of
adipose tissue.
The quantification of substrate fluxes from microdialysis data is not only dependent on the flow
measurements but also on the estimates of the
permeability surface area products, defined as the
substrate flux across the capillary membrane divided
by the concentration difference between the blood
and the intercellular water space. For molecules in
the size range of glucose, lactate, and glycerol the
permeability surface area product is 2-3 ml
rnin-'lOOg-', and the value does not change
within the limits of the physiological flow variation
in adipose tissue [69]. Direct measurements of the
permeability surface area products for glucose, lactate and glycerol in adipose tissue have not been
published. Another explanation of the differences
Assessment of tissue metabolism
255
may be beneficial to combine the two techniques to
fully understand the turnover of substances that can
be both produced and re-utilized by adipose tissue
or be retained in the extracellular space of the tissue
(for example, adenosine, prostaglandins, fatty acids).
As regards measurements of true metabolic rates,
both methods are slightly hampered by current
problems with the determination of the true rate of
blood flow. The microdialysis method has some
theoretical advantages in comparison with the catheterization technique in the further development of
blood flow measurements, since microdialysis directly investigates the extracellular space and thereby
the nutritive flow. In conclusion, microdialysis and
venous catheterization each offer unique possibilities
to study human adipose tissue function in uiuo. With
these techniques it is for the first time possible to
study in detail transport of substances from the fat
cells to the bloodstream and vice versa, or to study
the regulation of metabolism and blood flow in the
intact tissue.
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