The concentration of high molecular weight compounds in interstitial tissue fluid: A study in patients with post-reconstructive leg oedema

Eur J VascEndovascSurg 13, 355-360 (1997)

The Concentration of High Molecular Weight Compounds in Interstitial Tissue Fluid: A Study in Patients with Post-reconstructive Leg Oedema R. H a a v e r s t a d .1, I. R o m s l o 2 and H. O. M y h r e 1

Departments of 1Surgery and 2Clinical Chemistry, University Hospital of Trondheim, N-7006 Trondheim, Norway Objectives: To study whether relative changes in the concentration of different proteins of the subcutaneous interstitial tissue fluid in patients with postreconstructive leg oedema could give information on the integrity of the capillary wall following vascular reconstruction. Materials: Sixteen patients developed ipsilateral leg oedema following femoropopliteal (n=14) or femorotibial (n=2) bypass surgery for intermittent claudication (n= 10) or critical lower limb ischaemia (n=6) caused by obliterating atherosclerosis. Methods: The leg volume increase was measured according to the formula of a truncated cone. The blister suction method was used for sampling of subcutaneous interstitial tissue fluid. The concentration of albumin, transferrin, immunoglobulin G and a2-macroglobulin of serum and blister fluid was measured with nephelometry. Results: Seven days postoperatively the mean leg volume increase was 32% (19.8%) in the operated leg compared to the contralateraI side (p
Introduction

Leg oedema is a common complication following reconstructive surgery for lower limb atherosclerosis. The oedema increases to a maximum at about 1 week postoperatively, and then declines gradually. 1The oedema fluid accumulates within the subcutaneous tissue, and the leg volume increase is in the order of 20% compared to the contralateral leg. 2'3 This leg oedema may concern the patient and delay rehabilitation following an otherwise successful operation. The microcirculatory changes responsible for this postreconstructive leg oedema formation are not fully understood. Impaired lymph drainage 4-~ and an increased transcapillary filtration~2have been suggested to be responsible. The oedema has also been explained as a manifestation of the reperfusion syndrome caused *Please address all correspondence to: Rune Haaverstad, Department of Surgery, University Hospital of Trondheim, N-7006 Trondheim, Norway.

by ischaemic damage to the t i s s u e . ~3'14 Deep venous thrombosis is uncommon after infrainguinal arterial reconstruction and is an unlikely explanation for the postoperative leg oedema formation. 15'16Because postreconstructive leg oedema is located subcutaneously, one would expect that pathophysiological mechanisms responsible for the oedema formation are mainly active in the subcutaneous tissue compartment. Since there seem to be altered albumin kinetics 17 as well as increased transcapillary filtration12in the operated limb following infrainguinal bypass surgery, changes in the capillary permeability seem likely. When studying the biochemical composition of subcutaneous interstitial tissue fluid of the lower limb following vascular reconstruction, the blister suction technique with large (8 mm) blisters is a preferred fluid sampling method. I8 However, little is known about the various protein concentrations in the subcutaneous interstitial tissue fluid of patients with postreconstructive leg oedema. By applying the blister suction technique, the intention of this study was to

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investigate whether relative changes in the concentration of compounds of different molecular weights occurred. This could give information on the integrity of the capillary wall following vascular reconstruction.

Material and Methods

Sixteen patients developed ipsilateral leg oedema following femoropopliteal (n = 14) or femorotibial (n =2) bypass surgery for intermittent claudication (n=10) or critical lower limb ischaemia (n = 6) caused by obliterating atherosclerosis. 19 The median age was 69 years (range 57-84 years) and there were 11 males and five females. The level of distal anastomosis was above the knee in 10 patients and below the knee in six. Autologous saphenous vein and PTFE graft (Goretex R) were each used as bypass graft in eight of the subjects. Three patients had mild type-2 diabetes mellitus of recent onset. No patients suffered from congestive heart failure, liver failure or renal disease. All patients were operated under epidural anaesthesia. To improve graft blood flow and as a prophylaxis against venous thrombosis, dextran 70 (Macrodex ~) was administered i.v.; 500 ml on the day of operation as well as 500 ml on the first postoperative day. 2° The ankle-brachial pressure index (ABPI) was measured pre- and postoperatively by the Doppler-technique. Leg volume increase was calculated 7 days postoperatively according to the formula of a truncated cone after measurement of the leg circumference 5-7 cm and 25-28 cm proximal to the medial malleolus. 21 The contralateral leg was used as a control. Postoperatively, air plethysmography (Stranden pletysmograph, STR-Teknikk, Oslo, Norway) or colourcoded duplex scanning (Vingmed CFM 750, Vingmed Sound, Horten, Norway) was used to exclude deep venous thrombosis. 22-25 Sampling of serum and subcutaneous interstitial tissue fluid was done 6-10 days (median 7 days) postoperatively. A blood sample was collected from a cubital vein at the same time as the sampling of interstitial fluid. Serum was isolated after coagulation and centrifugation.

Blister suction technique

The method was based on the principles described by Kiistala 26 with the modifications of Hellum et aI. 27 Blister suction devices were applied on the anteromedial side of the leg. The blister suction device Eur J Vasc Endovasc Surg Vol 13, April 1997

was made of a perspex block with either eight or 10 concentric holes, each 8 mm in diameter, into which a half-spherical blister could develop. By using a manually working pump (Stranden blister suction device, STR- Teknikk, Oslo, Norway) a controlled subatmospheric pressure of 200-220 mmHg was applied. 2s As soon as the blisters appeared, following 60-120 min of suction, the fluid volume of each blister (100-150 btl) was aspirated through a 27 gauge needle. No samples of blister fluid had to be discharged because of blood contamination. In five control legs and one operated leg no blisters appeared or too little sample fluid necessary for the biochemical analysis could be produced.

Protein measurements

Albumin (molecular weight 69 000), transferrin (77000), immunoglobulin G (160 000) and ~2-macroglobulin (725 000) of serum and blister fluid were measured with nephelometry (Behring nephelometer analyser). The accuracy of the assays (expressed as the coefficient of variation) was as follows: albumin: 2.9%; transferrin: 3.2 %; immunoglobulin G: 2.2%; %-macroglobulin: <5.0%. The recommended normal limits of serum concentrations given by the manufacturer and our own laboratory were as follows: albumin 35-55 g/l; transferrin 2.0-4.0 g/l; immunoglubulin G 7.0-15.0 g / 1; %-macroglobulin 1.1-3.2 g/1. Approximately 200 btl of each fluid sample was necessary for the protein measurements. All patient measurements took place at room-temperature (22-24°C). The patients were examined in the supine position following a resting period of at least 15-30 rain. Otherwise all patients had been mobilised from the first postoperative day and were included in a rehabilitation program following surgery. All subjects had given their informed consent for participation in the investigation.

Statistics

Statistical calculations on the relationship between leg oedema and clinical data (sex, limb ischaemia and level of distal anastomosis) were based on the Student's t-test for independent samples. Student's t-test for paired data were used for comparing changes in ABPI, leg volume and protein concentration. The results are given as mean (S.D.) and p<0.05 was accepted as the level of statistical significance.

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Table 1. Concentration of albumin, transferrin, immunoglobulin G and ~2-macroglobulinin serum (Cs) and blister fluid (CBr) as well as concentration ratio (CBL/Cs)in operated and contralateral limb. The values are given as mean (S.D.). Protein (MW)

Serum (n = 16) Cs

Albumin (69 000) Transferrin (77 000) Immunoglobulin G (160000) %-Macroglobulin (725 000)

Operatedleg (n = 15) CBL CBL/Cs

Control leg (n = 11) CBL CBL/Cs

30.9 (4.3)

1Z60 (3.80)

0.42 (0.12)

10.55 (3.08)

0.31 (0.15)

2.3 (0.5)

0.68 (0.19)

0.31 (0.08)

0.46 (0.29)

0,20 (0.12)

9.1 (1.7)

2.73 (0.80)

0.31 (0.10)

1.93 (0.63)

0.21 (0.05)

1.9 (0.5)

0.33 (0.17)

0.18 (0.08)

0.10 (0.07)

0.10 (0.13)*

* Median (range) = 0.05 (0.02-0.45). Results

Following vascular reconstruction, the ABPI increased from an average of 0.54 (0.14) preoperatively to 0.91 (0.11) postoperatively in the operated limb (p<0.01). All patients developed significant leg oedema on the operated side. Seven days postoperatively the m e a n leg volume increase was 32% (19.8%) in the operated leg compared to the contralateral side (p<0.01). The leg volume increase had no relation to sex, severity of preoperative limb ischaemia or the level of distal anastomosis. No patients developed generalised oedema or oedema in the contralateral control limb. Postoperatively, the mean serum concentration of albumin was 30.9g/1 (4.3g/1), transferrin 2.3g/1 (0.5 g/l), immunoglobulin G 9.1 g/1 (1.7 g/l) and %macroglobulin 1.9 g/1 (0.5 g/l). The concentration of the different proteins in serum and blister fluid, as well as the concentration ratio between the level in blister fluid and serum, are summarized in Table 1. Comparison of the protein levels in the operated vs. the control leg are shown in Fig. 1. In the blister fluid, each of the proteins had a significantly higher concentration in the operated leg compared to the control leg. This concentration difference was relatively larger for the proteins with the highest molecular weight, immunoglobulin G (p = 0.006) and %macroglobulin (p =0.002) (Fig. 1). However, the concentration difference had no relation to the increase of ABPI or leg volume in the various subjects. The protein concentration values in the patients with type2 diabetes did not differ from the rest of the patients.

Discussion

The leg oedema following vascular surgery is located in the subcutaneous tissue. 2'3 Normally there is a restricted capillary permeability for plasma proteins

which is expressed by a high capillary reflection coefficient.29-31 The mean serum values of transferrin, immunoglobulin G and %-macroglobulin were within the recommended normal range. The mean serum albumin concentration was a little below the lower normal limit, which is a c o m m o n observation following major general surgery as well as vascular surgery. 17'32-34However, the serum albumin concentration was far above the level which m a y cause generalised oedema. 33~5 For all four protein species investigated, the concentration in the blister fluid was higher in the operated than in the non-operated leg. The ratio between blister fluid and plasma increased by about 50% for albumin, transferrin and immunoglobulin G. However, for the larger ~2-macroglobulin the concentration ratio increased by nearly 100%. Because lymphatic drainage is non-selective, 31'36lymphatic stasis would consequently not be expected to cause any retention of high molecular weight proteins in the leg oedema. This difference between the protein species must therefore result either from a difference in the capillary membrane or in the interstitial matrix in the control leg vs. the operated leg. Assuming a steady state in both situations, there are two possible explanations for this observation. First, it could be due to a decreased selectivity of the capillary membrane, which w o u l d tend to raise the interstitial concentration of proteins with high molecular weight more than for those with low molecular weight. 29'3°'37 Second, the difference could also result if there was a relatively higher excluded volume for %-macroglobulin than for the other three protein species in the operated leg. However, the latter seems unlikely since increased interstitial volume and oedema w o u l d be expected to lower interstitial exclusion more for proteins with higher molecular weights, i.e. more for %-macroglobulin than for the three smaller proteins. 38 This should result in the smallest increase in the concentration for mRmacroglobulin. Thus, the most likely explanation for Eur J Vasc Endovasc Surg Vol 13, April 1997

358

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R. Haaverstad

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Fig. l. Blister fluid concentration of albumin (A), transferrin (B), immunoglobulin G (C) and a2-macroglobulin (D) in patients with postreconstructive leg oedema. The statistical calculations comparing the operated and contralateral control leg are based on measurements in those patients who developed blisters bilaterally (n = 10).

the observed difference in the concentration ratios between blister fluid and plasma would seem to be an increased capillary permeability in the operated leg. This supports the earlier observation of increased capillary filtration in limbs with post-reconstructive leg oedema. 12Furthermore, as the increased fluid content in the subcutaneous tissue compartment exceeds Eur J Vasc Endovasc Surg Vol 13, April 1997

the drainage capacity of the lymphatic system, a leg oedema develops and could thus be aggravated by a lymphatic disruption frequently caused by the surgical trauma. 11 The non-invasive blister suction technique is simple and more comfortable for patients than the wick technique, is The suction blisters occur at the

Interstitial Tissue Fluid

dermoepidermal junction between the basal cell membrane and the basement membrane without profound alterations in the viability of the epidermis or capillary wall permeability, as long as the suction pressure is kept at around 200 m l n H g . 26"39 Serial samplings and repeated measurements in a clinical routine may therefore be possible. The method is reliable for the investigation of interstitial fluid, is'4° as well as for dermatological 41 and pharmacological research. 42 The blister suction technique with 8 mm blisters also gives a sufficient sample volume. Approximately 200 ~tl of each sample fluid was needed for the chemical analysis. In six patients no blisters or too little blister sample volume were produced. It is easier to make blisters on the trunk than on the leg where the dermis is somewhat thicker. 2sA higher suction pressure might have solved the problem of blister formation in those patients who did not develop blister. The lack of correlation between protein concentration vs. the degree of leg oedema and ABPI might be due to the limited number of patients in the study. The selection of patients was relatively homogeneous with respect to age and the absence of major generalised disease except for lower limb atherosclerosis. Patients with diabetes mellitus may have a different pattern for regulation of the limb microcirculation compared to patients with lower limb atherosclerosis. However, the influence of diabetes on the microcirculation is outweighed by the effects of atherosclerosis when vascular disease becomes severe. 43 Since the lower limb atherosclerosis was advanced in all cases and the diabetes in three of our patients was of recent onset and of mild degree, they were included in the study. In conclusion, the blister suction technique is a simple method for the Clinical study of proteins of different molecular weight in the subcutaneous tissue of human legs with post-reconstructive leg oedema. Following vascular reconstruction for lower limb atherosclerosis, there is an increased concentration of plasma proteins in blister fluid from the operated limb. A relatively larger difference for the bigger molecules suggests increased capillary permeability to plasma proteins.

Acknowledgements The study was supported by grants from the Norwegian Council for Cardiovascular Diseases.

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Accepted 23 August 1996