Extracellular Fluid

All women were omnivore without any flaxseed in their daily diet. The premenopausal women were investigated either in the follicular and the luteal phase in one menstrual cycle or during two consecutive luteal phases of two menstrual cycles. The luteinizing hormone (LH) peak was determined in urine samples and thereafter the first microdialysis investigation was performed within 5–10 days. Of the twenty premenopausal women eleven were controls and investigated either in one menstrual cycle or during two consecutive luteal phases without diet modifications whereas nine women added 25 g of freshly ground flaxseed/day to their diet after the first microdialysis session. The next microdialysis investigation was performed at the same day after the LH-peak as in the first un-exposed cycle i.e. if the first microdialysis was performed at day 5 after the LH-peak the next microdialysis investigation was also performed at day 5. The five healthy postmenopausal control women were investigated at one time point.
The exposed postmenopausal women had been treated for early breast cancer and tamoxifen 20 mg/day had been prescribed as an adjuvant therapy. Tamoxifen treatment to early breast cancer patients is a standard regimen in low risk patients in Sweden. All had had a normal breast examination and mammography on the contra-lateral side. Before the start of tamoxifen, microdialysis was performed in the healthy breast and in subcutaneous fat. After six weeks of tamoxifen treatment the women returned for the second microdialysis.
As plasma and serum represent different compartments compared with the extracellular space in tissues, and as they are collected with another technique they are not ideal controls for microdialysates. Therefore, microdialysis performed on abdominal subcutaneous fat was used as an internal control. Additionally, blood samples were collected at the start of each microdialysis investigation.
Prior to insertion of the microdialysis catheters 0.5 ml lidocain (10 mg/ml) was administrated intracutaneously. One microdialysis catheter was placed in abdominal subcutaneous fat and one in the upper lateral quadrant of the breast and directed towards the nipple as previously described [4] (link), [26] (link), [27] (link), [28] (link). At the next microdialysis investigation the catheters were inserted at the same locations as in the previous session. The premenopausal women and the postmenopausal controls were investigated in the left breast whereas the women treated for breast cancer were investigated in their healthy breast. A microdialysis catheter (CMA 71/Microdialysis AB, Solna, Sweden), which consists of a tubular dialysis membrane (20 mm long x 0.52 mm in diameter, 100,000 atomic mass cut-off) glued to the end of a double-lumen tube (80 mm long x 0.8 mm in diameter), was used. The catheters were inserted guided by a splitable introducer (CMA Microdialysis AB). The catheters were connected to a microinfusion pump (CMA 107, CMA/Microdialysis AB) and perfused with NaCl 154 mmol/L and hydroxyethyl starch 60 g/l (Voluven®, Fresenius Kabi, Uppsala, Sweden), at a perfusion rate of 0.5 µl/min. The solution entered the catheter through the outer tube and left it through the inner tube, from which it was collected. After a 30-min equilibration period, the outgoing perfusate containing extracellular proteins was collected and stored at −70°C for subsequent analysis.
Microdialysis is a sampling technique, which allows continuous sampling of the extracellular fluid by passive diffusion of molecules over a semi-permeable membrane. The recovery i.e. the amount of substances from the tissue that diffuse into the perfusion fluid depends on the membrane properties, the flow rate, tissue temperature, and the size of the compound of interest. Diffusion of low molecular substances over the dialysis membrane has been shown to be almost complete at low flow rates using a 30 mm long dialysis membrane [29] (link). However, for larger molecules the recovery over the membrane decreases and the measured levels in the microdialysis sample cannot be considered as the absolute concentrations in the tissue. The recovery of a certain substance may be measured in vitro by putting a microdialysis in a vial containing the compound of interest, perfuse the catheter and divide the concentration of the substance in the dialysate by the concentration in the vial. The in vitro recovery of VEGF was 8%, of angiogenin 31%, and of endostatin 33%. This in vitro recovery can only be an estimate of the in vivo recovery since other factors such as tissue pressure and temperature will affect the diffusion of substances in vivo. Therefore, all microdialysis values are given as original raw data without any re-calculations.

The hydrolytic degradation of the hydrogels was carried out in PBS, while the degradation was performed in simulated exudate solution in pseudo extracellular fluid (PECF) (0.68 g of NaCl, 0.229 g of KCl, 2.5 g of NaHCO₃, and 0.4 g of NaH₂PO₄) containing lysozyme (1 mg/mL). The dried disks were weighted, and each disk was then immersed in 5 mL of the degradation solution at 34 °C, under agitation at 180 rpm. After 24 h and 48 h, the disks were washed by immersion in DD water and dried. The dried disks were weighed, and the weight loss was calculated through Equation (3), where w₀ is the weight of the dried disks and w_24/48 are the weights of the sample, after 24 h or 48 h in the degradation solutions, respectively, after drying [38 (link)]. The assay was done in quintuplicate.
$$weight loss \left(\%\right)=\frac{{\mathit{w}}_{\mathbf{0}}-{\mathit{w}}_{\raisebox{1ex}{$\mathbf{24}$}\!\left/ \!\raisebox{-1ex}{$\mathbf{48}$}\right.}}{{\mathit{w}}_{\mathbf{0}}}\times \mathbf{100}$$