Anal. Chem. 1998, 70, 526-533
Dialysis with Semipermeable Membranes as an Efficient Lipid Removal Method in the Analysis of Bioaccumulative Chemicals Bo Strandberg,* Per-Anders Bergqvist, and Christoffer Rappe
Institute of Environmental Chemistry, Umeå University, S-901 87 Umeå, Sweden
Herein is a procedure described using a semipermeable membrane (SPM) for enrichment of organic chemicals from lipid-containing samples. Dialysis with SPMs in an organic solvent can separate organochlorine contaminants such as non-, mono-, and di-o-PCBs, PCDDs, PCDFs, PCDTs, PCNs, pesticides, and PAHs from lipids. The method is nondestructive and more than 20 g of lipid can be dialyzed in a single membrane with acceptable recoveries of the internal standards, nearly independent of amount and type of lipid dialyzed. The lipid removal process shows good consistency between subsamples, and the lipid content can be reduced by 90-99%, depending on species and amount lipid. Neither triglycerides nor phospholipids were obtained in the dialysate fraction. The utility and reliability of the method is displayed by good precision for 72 PCBs and 27 organochlorine pesticides in the concentration range 0.0550 µg/sample for triplicate subsamples, by the consistency in PCDD/F levels compared to a classic analytical procedure, and by the analysis of the above listed chemicals in ∼200 biological samples of a wide variety of types. This technique can also be used as an enrichment device for contaminants when huge amounts of lipids are extracted for toxicological studies. Moreover, it is possible to use SPM to cleanup other samples from molecules with relatively high masses, e.g. sediments, soil, compost, and tar materials. The use of semipermeable membranes (SPMs) as nondestructive dialytic enrichment devices for the separation of organic contaminants from fish lipid was first demonstrated by Huckins et al.1 Further, Meadows et al.2 reported conditions for maximizing recoveries of some organochlorines while minimizing the residual amount of lipids coeluting with the analytes through the membranes into the dialytic solvent, so-called lipid carry-over. Transfer of analytes through the SPM is governed by a high concentration gradient.1 Other variables that are likely to affect analyte recoveries are SPM area in contact with the dialytic solvent, the ratio of dialytic solvent to sample volume as well as osmotic effects.2 SPM pore size is ∼10 Å,1 which allows smaller (1) Huckins, J.; Tubergen, M.; Lebo, J.; Gale, R.; Schwartz, T. J. Off. Anal. Chem. 1990, 2, 290-293. (2) Meadows, J.; Tillitt, D.; Huckins, J.; Schroeder, D. Chemosphere 1993, 11, 1993-2006.
526 Analytical Chemistry, Vol. 70, No. 3, February 1, 1998
molecules through but restricts molecules bigger than this size, e.g., triglycerides and phospholipids, to penetrate the film. Huckins et al.1 reported a relatively slow diffusion rate through the SPM for mirex (cross-sectional diameter 9.2 Å) and also suggested mirex to be the operational size limit for this particular setup. In environmental studies, extraction of large samples of biological tissue is often required to achieve low detection limits in residue analyses. Similarly, in toxicological studies and bioassays,3 large amounts of sample must be extracted to achieve sufficient amounts of contaminants. This often generates large amounts of lipids after extraction and thus stresses the importance of a high-capacity step for lipid removal in the sample cleanup procedure. For biological samples, several different methods have been used to remove lipids, including destructive methods such as sulfuric acid or sodium hydroxide treatment and nondestructive methods such as partitioning procedures,4,5 gel permeation chromatography (GPC)6,7 and supercritical fluid extraction (SFE).8 However, the destructive methods will degrade some organochlorine pollutants,2 while nondestructive methods can be used without destruction of these labile substances. The problem is that the majority of nondestructive methods can only remove limited amounts of lipids or are costly to perform. As the sample size of environmental samples, especially biological organisms, usually is limited, it is desirable to analyze all of the target contaminants in the same sample, using a multiresidue analytical method.9,10 A high-capacity nondestructive, and nondiscriminating, lipid removal procedure is therefore urgently needed. The objective of this paper is to describe a simple, efficient, and versatile lipid removal procedure, without advanced equipment or excessive solvent usage. The lipid removal process was studied in detail using a batch of salmon roe lipid, and important (3) Tillitt, D.; Giesy, J.; Ankley, G. Environ. Sci. Technol. 1990, 25, 87-92. (4) Jensen, S.; Jansson, B. Ambio 1976, 5, 257-260. (5) Jensen, S.; Athanasiadou, M.; Bergman, A° . Organohalogen Compd. 1992, 8, 79-80. (6) Stalling, D.; Tindle, R.; Johnson, J. J. Off. Anal. Chem. 1972, 55, 32-38. (7) Norstrom, R.; Simon, M.; Mulvihill, M. Int. J. Environ. Anal. Chem. 1986, 23, 267-287. (8) Nam, K.; Kapila, S.; Yanders, A.; Puri, R. Chemosphere 1990, 20, 873-880. (9) Bergqvist, P.-A.; Bandh, C.; Broman, D.; Ishaq, R.; Lundgren, K.; Na¨f, C.; Pettersen, H.; Rappe, C.; Rolff, C.; Strandberg, B.; Zebu ¨ hr, Y.; Zook, D. Organohalogen Compd. 1992, 9, 17-20. (10) Jansson, B.; Andersson, R.; Asplund, L.; Litze′n, K.; Nylund, K.; Sellstro ¨m, U.; Uvemo, U.; Wahlberg, C.; Wideqvist, U.; Odsjo¨, T.; Olsson, M. Environ. Toxicol. Chem. 1993, 12, 1163-1174. S0003-2700(97)00647-1 CCC: $15.00
© 1998 American Chemical Society Published on Web 01/06/1998
Table 1. Environmental Samples Successfully Analyzed for a Multitude of Organic Chemicals Using SPM in the Lipid Detachment Process; in addition, Number of Samples with Range of Lipid Weight Dialyzed, Obtained Lipid Carry-Over, and Recoveries for the Internal Standards Listed in Table 3, for Each Sample Type sample type
no. of samples
amt lipid dialyzed (g)
abiotic sediment sediment trap material
17 5
0.02-0.1 0.06-0.3
fish species lamprey (Lampetra fluviatilis) perch (Perca fluviatilis) lesser sand eel (Ammodytes tobianus) cod (Gadus morhua) flounder (Platichthys flesus) eelport (Zoarces viviparus) round goby (Neogobius melanostomus) stickleback (Gasterosteus aculeatus) sand eel (Hyperoplus lanceolatus) herring (Clupea harengus) pike perch (Stizostedion lucioperca) fourhorn sculpin (Oncocottus quadricornis) sea trout (Salmo trutta) whitefish (Coregonus lavaretus) whitefish roe
2 11 1 1 3 1 1 4 1 16 1 14 3 7 1
17-23 0.8-1.1 18 10 9.9-14 9.5 15 3.5-7.4 6.9 0.7-6.9 6.2 1.5-5.6 1.6-3.1 0.3-14 3.7
0.9-2.1 0.9-1.9 3.4 4.1 2.1-2.7 8.1 3.2 4.6-6.6 3.2 2.4-8.0 3.5 2.6-8.6 2.1-6.1 3.8-12 2.6
55-80 50-99 59-102 60-65 43-114 65-75 74-85 42-103 65-102 48-106 62-81 41-106 54-92 61-89 45-84
3
6.9-10
1.4-2.2
54-115
3 3
1.2-1.6 0.8-1.0
9.6-13 15-16
55-110 51-111
4 5
0.4-4.0 1.2-2.3
3.9-19 7.8-10
39-109 50-95
12 17 2 1 17 18 9
0.02-2.5 0.1-4.1 1.1-1.2 3.2 0.6-5.5 0.4-2.0 0.6-5.1
6.2-25 4.4-25 9.9-12 4.8 1.9-6.7 4.4-11 4.7-9.9
43-102 34-120 43-100 53-60 42-96 49-113 47-99
marine mammal species porpoise (Phocoena phocoena) bird species black cormorant (Phalacrocorax carbo sinensis) muscle tissue liver tissue white-tailed sea eagle (Haliaeetus albicilla) muscle tissue liver tissue other marine species phytoplankton zooplankton mussels (Mytilus trossulus) crabs (Carcinus meanas) amphipods (Monoporeia affinis) isopods (Saduria entomon) mysis (Mysis sp.)
SPM parameters such as dialysis time, volume dialytic solvent, membrane wash procedures, etc. were optimized. We show the utility and repeatability of the SPM method for a number of polychlorinated biphenyl (PCB) congeners and pesticides by analysis of triplicate seal blubber and liver tissue samples and by the parallel analysis of polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs) in chicken egg and sediment using both the SPM procedure and a traditional procedure. We evaluate the experiments in regard to internal standard recoveries and lipid carry-over and also discuss the experiences gained from the analysis of a multitude of organic compounds, in more than 200 biological samples. EXPERIMENTAL SECTION Materials. The SPMs are made from a single lot of a “layflat” polyethylene dialysis tube, 26 mm wide and 80 µm thick (EST, St Joseph, MO). The glass funnels used for the dialysis procedure were 300-500 mm × 45 mm i.d., with a Teflon stopcock placed at the bottom. A TEW (QS) impulse heat sealer purchased from Mo¨llerstro¨m (Gothenburg, Sweden) was used to seal the tubes. All adsorbents, Florisil (deactivated 1.2% w/w, Merck), silica gel (activated 550 °C, Merck), and acidic and basic alumina (BioRad Laboratories), were methanol and dichloromethane rinsed
lipid carry-over (%) 12-24 16-23
recovery range (%) 35-109 46-110
before use. All solvents were of glass distilled quality (Burdick and Jackson). The 13C-labeled internal standards and the PCDD/F native mix were obtained from Cambridge Isotope Laboratories (Andover, MA). A synthetic reference standard containing pesticides and PCBs was obtained from Dr. Ehrenstorfer (Augsburg, Germany). Samples. Ringed seal (Phoca hispida) blubber (3 g) and liver (4 g) tissues, originating from the Baltic Sea, were obtained from the Swedish Museum of Natural History. Six chicken eggs were collected from an adjacent outdoor breeding farm, and dry sediment (2 g) from the Baltic Sea was obtained from the Swedish Environmental Protection Agency. Approximately 6 kg of salmon roe was sampled from salmon (Salmo salar) from the Umea River, Stornorrfors, Sweden. Furthermore, ∼200 biological samples were dialyzed and analyzed and are presented in Table 1. These included 22 different aquatic species, one fish roe, muscle and liver tissue from two bird species, and sediment and sediment trap material, collected in the Gulf of Bothnia or the Gulf of Gdansk in the Baltic Sea. Sample Extraction and Pretreatment. The sediment sample was Soxhlet extracted with toluene for 24 h. The chicken egg and seal tissue samples were ground with sodium sulfate in a highAnalytical Chemistry, Vol. 70, No. 3, February 1, 1998
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Table 2. A Dialysis Process Evaluated for 2, 7, 14, and 20 g of Salmon Roe Lipida lipid weight 2g transfer volume (mL) add solvent from start (period 1) (mL) add solvent for period 2 (mL) add solvent for period 3 (mL)
2 40 70 94
solvent pool outside to inside the membrane after periods 1, 2, and 3 (%)
81, 86, 92
7g 7 140 207 276 84, 92, 94
14 g 14 280 395 523 85, 82, 84
20 g 20 400 584 781 85, 92, 94
a Dialysis time was 16, 24, and 24 h for periods 1-3, respectively. The given dialytic solvent volumes should give the ratio ∼1:10 of volume inside to outside the SPM at the start of each dialysis period with compensation for dialytic solvent entering the film (osmosis). Maintaining a positive contaminant flux, a solvent pool outside to inside the membrane of 80% is advised. Obtained solvent pools in the end of each period is also displayed.
Table 3. Recovery Mean Values (%) for 13C-Labeled Internal Standards Spiked Prior to the Dialysis Process for Triplicate Seal Blubber and Liver Tissue Samples recovery (%) internal standard
blubber (n ) 3)a
liver (n ) 3)a
[13C]PCB 80 [13C]PCB 153 [13C]γ-HCH [13C]-p,p′-DDT [13C]hexachlorobenzene [13C]dieldrin
106 95 79 102 73 81
95 97 68 96 69 90
a
n, number of samples.
capacity blender, packed into a glass column (60-mm i.d.) and extracted with acetone/n-hexane (2.5:1, v/v) and n-hexane/diethyl ether (9:1, v/v), as described elsewhere.11 The samples included in Table 1 were homogenized and extracted wet in Soxhlet (Dean-Stark) extractor with toluene (24 h) and a mixture of n-hexane and acetone (59/41, v/v 24 h).12 The solvent was evaporated and the residue weights were determined gravimetrically. Seal extracts were spiked with 13C-labeled PCBs and organochlorine pesticides (Table 3) and split into triplicate subsamples. The egg and sediment extracts were spiked with 13C-labeled PCDD/Fs and split into duplicates. The biological samples (Table 1) were spiked with 30 different 13C-labeled (PCDDs, PCDFs, PCBs, pesticides) and deuterated (PAHs) internal standards (IS). The salmon roe lipid extract (0.4 kg) was divided into portions of 2, 7, 14, and 20 g. SPM Dialysis Procedure. SPMs were cut into pieces of appropriate length (350-550 mm), soaked in a beaker with cyclopentane for 24 h, rinsed, air-dried for 10 min, and heat-sealed in one end. The following dialysis procedure was then used (see Table 2): Lipid extracts of the samples were redissolved in cyclopentane (∼1 mL/g of lipid) and were quantitatively transferred to the SPM. If solubility difficulties occurred, some dichloromethane was added (11) Falandysz, J.; Strandberg, L.; Bergqvist, P.-A.; Kulp, S.-E.; Strandberg, B.; Rappe, C. Environ. Sci. Technol. 1996, 30, 3266-3274. (12) Bavel, B.; Na¨f, C.; Bergqvist, P.-A.; Broman, D.; Lundgren, K.; Papakosta, O.; Rolff, C.; Strandberg, B.; Zebu ¨ hr, Y.; Zook, D.; Rappe, C. Mar. Pollut. Bull. 1995, 32, 210-218.
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to improve solvation of the lipids, giving a final dichloromethane concentration of maximum 5%. The film was placed in the dialysis funnel (described under Materials), the open end was folded over the edge and fixed by piece of elastic, dialytic solvent was added (∼10 mL/mL of sample extract), and the device was covered with aluminum foil. The dialysis time varied depending on the sample type and the aim of the experiment. The sediment, chicken egg, and seal samples were dialyzed for 40 h, and the biological samples included in Table 1 were dialyzed for 64 h according to Table 2. The SPM procedure for the salmon roe lipid was varied in order to evaluate different lipid removal process variables (next section). The dialytic solvent was replaced by draining the funnel, opening the Teflon stopcock, and then refilling it with fresh solvent. Following dialysis, the dialysates were pooled for each sample and the residue weights were determined gravimetrically after complete solvent removal. From the salmon roe extracts, an aliquot was taken (2 g), prior to lipid weight determination, for lipid class measurement. Sample Cleanup. The seal and salmon roe samples were cleaned using a method developed by Norstrom et al.13 The method uses Florisil to fractionate PCBs and chlorinated pesticides according to their polarity. The egg and sediment samples were cleaned using the procedure of Smith et al.,14 with some minor modifications. Briefly, lipid removal and target isolation were achieved by using a multilayer column packed with silica, and acid- and base-modified silica, and then an activated carbon column. The PCDD/Fs are retained on the carbon adsorbent and are subsequently recovered by reverse elution with toluene. The samples were finally fractionated on a silica column followed by an acidic or basic aluminum oxide column. All other biological samples (Table 1) were cleaned, enriched, and fractionated for a multitude of target compounds as described elsewhere.9,11,15 Prior to the GC/MS analyses, tetradecane (keeper) and 13Clabeled recovery standards were added, and the solvent was evaporated. Analysis. Analyses were accomplished by gas chromatography/low-resolution mass spectrometry (GC/LRMS) or high(13) Norstrom, R.; Simon, M.; Muir, D. G.; Schweinsburg, R. Environ. Sci. Technol. 1988, 22, 1063-1071. (14) Smith, L.; Stalling, D.; Johnson, J. Anal. Chem. 1984, 56, 1830-1842. (15) Bavel, v. B. Doctor Thesis, Umeå University, Umeå, Sweden, 1995.
resolution mass spectrometry (GC/HRMS) using selected ion recording (SIR). The GC/LRMS instrument, a Fisons GC8000/MD800, was used for congener specific PCBs (Cl3-Cl10) and organochlorine pesticide analysis. Splitless injection at 250 °C was used as sample introduction technique with helium as carrier gas. The GC oven was equipped with a nonpolar fused-silica column (PTE-5, 60 m × 0.32 mm, 0.25-µm film thickness, Supelco, Bellefonte, PA) and was temperature programmed as follows: 180 °C (2 min hold), 15 °C/min increase to 205 °C, and 2 °C/min increase to 300 °C. The temperature of the GC/MS interface was 270 °C, and the ion source was at 250 °C. The SIR descriptor included the most intense, or two most intense, ions of the molecular ion isotopic clusters of the 13C-labeled and native compounds, respectively. PCB congeners identification was performed according to Schwartz and Stalling,16 and Bavel et al.12 Every PCB congener was quantified using the relative response factor (RRF) to a native PCB congener with the same degree of chlorination. The octachlorochlordane components MC4, MC5, MC7, and U82 and the nonachlordane component MC6 (Miyazaki et al.,17 Dearth and Hites18) were identified by retention time comparison with a technical chlordane mixture or the data reported by Buser et al.19 The octachlordane components were quantified using RRFs to synthetic cis-chlordane, and MC6 was quantified using synthetic trans-nonachlor. The other pesticides were quantified using a reference standard, which contains all targets. The GC/HRMS instrument, a Hewlett-Packard 5890 GC coupled to a VG Analytical 70S, was used for PCDD/F analysis. The MS was operated in the SIR mode with electron impact ionization (35 eV). The mass selection was performed by accelerating voltage (7000 V initially) switching. The SIR descriptor included the two most intense ions of the molecular ion isotopic clusters of the 13C-labeled and native compounds. All compounds were quantified to a native mix containing all of the 2,3,7,8-PCDD/ Fs. The lipid classes determination of the salmon roe included triglycerides, phospholipids, sterols, sterol esters, and a group of unknown highly polar compounds. The analysis was performed by normal-phase high-performance liquid chromatography with evaporative light-scattering detection. The separation was achived using a diol column, from Merck, and a ternary mobile-phase system, i.e., hexane, 2-propanol, THF/water.20 RESULTS AND DISCUSSION SPM Dialysis Procedure. Meadows et al.2 compared cyclopentane, n-hexane, and a mixture of n-hexane/dichloromethane (80:20 v/v) as dialytic solvent and reported that both cyclopentane and n-hexane effected high analyte recovery with low or moderate lipid carry-over. Of these solvents, cyclopentane is more expensive but less harmful.21 Considering the toxicological aspects, we selected cyclopentane as dialytic solvent. (16) Schwartz, T.; Stalling, D. Arch. Environ. Contam. Toxicol. 1991, 20, 183199. (17) Miyazaki, T.; Yamagishi, T.; Matsumoto, M. Arch. Environ. Contam. Toxicol. 1985, 14, 475-483. (18) Dearth, M.; Hites, R. Environ. Sci. Technol. 1991, 25, 245-254. (19) Buser, H.; Mu ¨ ller, M.; Rappe, C. Environ. Sci. Technol. 1992, 26, 15331540. (20) Nordbeck, J. Personal communication. Marine Research Center, Ho¨rnefors, Sweden.
Figure 1. Characterization dialysis process using percentage lipid carry-over (% LC), osmosis volume, ratio dialysate vs retentate volume, and type of membrane wash. Triplicate salmon roe lipid samples were utilized in all cases: (A) % LC vs dialysis time; (B) % LC vs amount of lipids; (C) osmosis volume vs dialysis time; (D) osmosis volume vs amount of lipids; (E) % LC and osmosis volume for two dialytic solvent volume ratios (1:10 and 1:30, inside:outside SPM) after dialysis of 2 g of lipids; (F) comparison of two membrane wash procedures, viz., traditional soaking and Soxhlet extraction in regard to % LC after 64 h, osmosis volume after 16 h, and SPM residues released after 64 h dialysis of 20 g lipid.
Variables affecting contaminant recovery are solvent volume ratio outside to inside the SPM and number of dialytic solvent replacement.2 To achieve minimal solvent use and acceptable recoveries, a ratio of ∼1:10 at the start of each dialysis period is used. During the dialysis process some dialytic solvent enters the film (osmosis), and thus additional solvent must be added at the start of the next dialysis periods to maintain a sufficient solvent pool (∼80%2) outside the SPM at the end of each dialysis period; cf. Table 2. The osmosis effect will depend on the sample type and room temperature.2 To characterize the SPM procedure, salmon roe lipid was dialyzed to investigate parameters such as percent lipid carryover (% LC), osmosis volume, and dialysate vs retentate ratio. Further, two membrane prewash procedures were also compared. The results from this study are presented in Figure 1. All samples were processed in triplicates with a good repeatability, RSD (p ) 0.05) 2-4%. (21) Berglind, R. Personal communication. National Defence Research Establishment, FOA 4, Umeå, Sweden.
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Table 4. Evaluation of Type and Amount of Lipid Carry-Over (LC) for Four Lipid Classes and a Group of Unknown Residues during Dialysis of 1.8 g of Salmon Roe Lipid for 112 ha lipid classes
lipid content (%) weight from start (mg) LC after 112-h dialysis mg % lipid class composition of LC (%)
triglycerides
phospholipids
sterols
sterol esters
unknowns
45 815
25 453
11 199
7 127
12 216
ndb 0 0
nd 0 0
39 20 38
3.2 2.5 3
61 28 59
a Data show percent lipid composition from start with weight (mg) for each lipid class, LC in weight (mg) and percent (%) after 112-h dialysis for each lipid class, and the percentage composition of the LC. b nd, not detected.
Figure 1A shows the % LC for 1.8- and 20-g salmon roe lipid after 16, 40, 64, and 112 h of dialysis. The % LC increases over time for both samples although the increase levels off as the dialysis time increase. The small sample seems, however, to penetrate the membrane to a greater extent compared to the large sample (see discussion below). The effect of sample size on % LC is also illustrated by Figure 1B (88 h dialysis time). The osmosis volume also increases with increased sample size; cf. Figure 1C. In fact, there is an almost linear relationship between amount lipid dialyzed and osmosis volume; cf. Figure 1D. The influence of two different dialysate vs retentate volume ratios on osmosis and % LC for 2 g of salmon roe lipid is displayed in Figure 1E. The larger ratio apparently increases both osmosis volume and % LC, probably due to an enhanced flux through the membrane. A perfect wash procedure with minimal wash solvent before SPM use would remove all polymer residues so that none would be released during dialysis, without changes in dialytic performance. Figure 1F shows the differences between SPMs washed by either soaking for 24 h in cyclopentane or Soxhlet extraction with cyclopentane for 16 h. The membranes were tested for the dialysis of 20 g of salmon roe lipids together with additional blanks for measurement of released SPM residue, respectively. The Soxhlet-washed membrane released considerably less polymeric material (0.5 vs 1.4 mg), but exhibited an increased LC (2 vs 15%) and osmosis volume (17 vs 52 mL, after 16 h). Thus, the Soxhlet wash removes polymeric residues more effectively, with less solvent consumption, but the SPM performance changed dramatically and negatively affects % LC and osmosis. Therefore we have used the soaking procedure throughout this study. The percent recovery of the environmental pollutants will also depend on the number of solvent changes. Replacement of the dialytic solvent increases the chemical potential across the dialysis membrane and leads to a high initial transfer rate of chemicals through the membrane, but the transfer rate is rapidly reduced as equilibrium is approached. Using the dialysis variables described herein, most chemicals reach equilibrium after 12-20 h under room-temperature conditions (20-25 °C). One solvent replacement is sufficient to achieve a recovery of >80% for the pollutants covered in this paper, and thus, a third dialysis period will only yield small recovery improvements at the expense of additional solvent usage and lipid carry-over. However, there are 530 Analytical Chemistry, Vol. 70, No. 3, February 1, 1998
transfer rate differences among the pollutants due to differences in physical properties such as polarity, and molecular size and shape.1 Reduced diffusion rates for some of the chemicals, e.g., mirex, might necessitate a third dialysis period. To overcome such difficulties, we recommend the use of labeled internal standards covering the chemical spectrum of interest. Characterization of the Lipids Penetrating the SPM. We have characterized the salmon roe lipids that are penetrating the SPM membrane. Four different lipid classes were measured, viz. triglycerides, phospholipids, sterols, and sterol esters. Further, a group of unknowns were also detected and quantified. Table 4 shows the percentage composition of the original salmon roe lipid and the composition of the penetrating lipids. The residues found in the dialysate after 112 h of dialysis mainly consist of polar unknowns (59%), sterols (
