a simple and improved method for extraction of phospholipids from hemoglobin solutions
TRANSCRIPT
Artificial Cells, Blood Substitutes, and Biotechnology, 36: 19–33, 2008Copyright © Informa Healthcare USA, Inc.ISSN: 1073-1199 print / 1532-4184 onlineDOI: 10.1080/10731190701857751
A Simple and Improved Method for Extractionof Phospholipids from Hemoglobin Solutions
Kun-Ping YanCollege of Life Science, Northwest University, Xi’an, P. R. China
Jing HaoShaanxi Lifegen Co., Ltd., Xi’an, P. R. China
Ning Dan and Chao ChenCollege of Life Science, Northwest University, Xi’an, P. R. China
Abstract: This study introduced a liquid-liquid extraction method designed forcomplete recovery of phospholipids from protein-rich samples, such as hemoglobin(Hb) solutions. In order to minimize protein denaturation and maximize lipid extractionfrom protein-rich samples, isopropanol was used as the major organic extractionsolvent. In a wide range of the volume ratio of isopropanol to Hb solution at lowtemperatures, such extraction system resulted in limited protein precipitation and didnot cause heme pigment contamination. The efficiency of phospholipid extraction fromHb solutions was pH-dependent, with the lowest extraction yield around neural pHand the highest extraction yield around pH 5.0. At pH 5.0, salt with concentrationsup to 400 mmol/L in the samples did not increase extraction efficiency. Comparedto other available methods, this method is simpler, needs significantly less organicsolvent and, most importantly, consumed much less Hb, which is very expensive tomake for the purpose of production of therapeutic products, hemoglobin-based oxygencarriers (HBOCs). This method is suitable not only for Hb solutions but also for otherprotein-rich biological samples, such as erythrocytes, plasma and liver.
Keywords: hemoglobin, hemoglobin-based oxygen carriers, phospholipids, lipidextraction
This study was supported in part by the National Scientific Research Program “863Program” (Grant No: 2004AA205030) China.
Address correspondence to Dr. Ning Dan or Dr. Chao Chen, National Engineering Re-search Center for Miniaturized Detection System, Northwest University, Xi’an, China,710069. E-mail: [email protected] or [email protected].
19
Art
if C
ells
Blo
od S
ubst
it Im
mob
il B
iote
chno
l Dow
nloa
ded
from
info
rmah
ealth
care
.com
by
The
Uni
vers
ity o
f M
anch
este
r on
11/
04/1
4Fo
r pe
rson
al u
se o
nly.
20 K.-P. Yan et al.
INTRODUCTION
Hemoglobin-based oxygen carriers (HBOCs) are hemoglobin-derived,potential biological therapeutics, which have been under active commercialdevelopment for more than twenty years [1]. Due to the unique oxygendelivering properties of hemoglobin, reduction or supplement of bloodtransfusion is a major therapeutic application of HBOC products in clinicaltrial studies for various clinical indications. Using HBOCs as red blood cellsubstitutes presents a unique challenge in their manufacturing process due tothe large or even enormous dosage amount required in order to achieve theirintended therapeutic effects. For example, a dosage of up to 20 units (10 literscontaining 1,000 g of hemoglobin) of PolyHeme, a HBOC product, has beenused in clinical trials [2]. Therefore, manufacturing of HBOC products holdsvery high quality standards requiring not only ultra-pure hemoglobin but alsovery low level of potential harmful contaminants to ensure product safety.
Currently, HBOCs have been manufactured from hemoglobin purified fromhuman, bovine or porcine blood [3–4]. Contamination of cellular membranephospholipids and associated biologically active molecules is a major concernin product safety. Lipid microparticles formed from vascular and circulatingcellular membrane components may exert a wide range of physical and patho-logical impacts, contributing to hemostatic and inflammatory responses, cellsurvival and atherothrombosis [5]. It is well known that phosphatidylserine(PS), a major component of the inner layer of cellular plasma membrane,promotes assembly and catalytic activity of various coagulation factors [6].PS accessible on cell surface induces apoptosis [7]. Stroma-contaminatedhemoglobin solution activated the alternative pathway of complement, andcaused thrombocytopenia, leukopenia, and disseminated intravascular coagu-lation associated with multiorgan dysfunctions in animals [8–9]. Therefore,effective techniques have been applied in hemoglobin purification processes toensure only less than tolerable trace amounts of phospholipids, especially PS,present in purified material and final products.
Accordingly, for quality control and quality assurance (QC/QA), completeextraction of phospholipids from hemoglobin or HBOC solutions is an impor-tant step in analysis of trace amounts of phospholipids in tested samples. Forthis purpose, various kinds of lipid extraction techniques, both in liquid-liquidformat [10–11] and in solid format [12], have been tested or developed. Inliquid-liquid extraction format, several widely used methods for extraction oflipids, such as the Folch’s method [13] or its modified version described byBligh and Dyer [14], have been tested for use in extraction of lipids from ery-throcytes [10] and hemoglobin solutions [11]. However, these methods, whichwere originally designed for extraction of lipids from lipid-rich solid tissuessuch as brain by using strong extraction power organic solvent, chloroform,have been shown to be problematic for erythrocytes and hemoglobin solu-tions [10–11], suffering from low recoveries, significant recovery variationsand heme pigment contamination, which may interfere with lipid analysis,
Art
if C
ells
Blo
od S
ubst
it Im
mob
il B
iote
chno
l Dow
nloa
ded
from
info
rmah
ealth
care
.com
by
The
Uni
vers
ity o
f M
anch
este
r on
11/
04/1
4Fo
r pe
rson
al u
se o
nly.
Extraction of Phospholipids from Hemoglobin Solutions 21
presumably due to the fast and severe protein-denaturing effect of the organicsolvent so that protein-associated lipids are trapped in aggregated proteins de-natured to become unextractable. For improvements, Rose and Oklander [10]introduced an extraction system with less powerful organic solvents to extractphospholipids from red blood cells. A direct comparison study showed thatthis extraction system significantly improved phospholipid recoveries by up to20% and had only trace amounts of heme pigment contamination compared tothe Folch’s method. However, liquid-liquid extraction systems often involve ex-traction and multiple washing steps, rendering them labor intensive, costly andenvironmentally harmful due to consuming large volumes of expensive hazardorganic solvents, such as chloroform. In addition, a large volume (30 mL to50 mL) of ultra-pure Hb or HBOC solutions is required for each extraction toobtain a reasonable amount of phospholipids for subsequent quantitative anal-ysis. Since Hb or HBOC solutions are made through a very expensive process,it is desirable to consume as less amounts of samples as possible in QC/QAprocess. In order to avoid these disadvantages, a solid-phase extraction systemusing styrene-divinylbenzene (SDB) macroreticular resins has been applied inextraction of phospholipids from erythrocytes and hemoglobin solutions [12].This method is relatively simple and requires relatively less organic solvents.However, it still consumes a large volume of ultra-pure Hb solutions (30 mLfor 8% Hb) and bears a high cost on the extraction materials. Here we present aliquid-liquid extraction method for extraction of phospholipids from untra-pureHb or HBOC solutions through modification and optimization of the proceduredescribed by Rose and Oklander [10]. Compared to the parent method andother available methods, the new extraction method described in this paper issimpler, much less expensive, significantly less environmentally harmful andmore effective in extracting the major phospholipids from Hb or HBOC so-lutions. It only requires 10–15 mL of 8% Hb solutions for each extraction tomeet related regulatory QC/QA requirements.
MATERIALS AND METHODS
Soybean L-α-phosphatidylinositol (PI), bovine brain 3-sn-phosphatidyl-L-serine (PS), sphingomyelin (SM) and 3-sn-phosphatidylethanolamine (PE),phosphatidylcholine (PC), were purchased from Sigma-Aldrich. All solventswere obtained from Tedia or Fisher (HPLC grade). Centrifuge 5804 R (Eppen-dorf, Germany), rotary evaporator RE-52A (Yarong, Shanghai, China).
Purification of Porcine Hemoglobin
Pig blood was freshly collected from a slaughterhouse and stored at 4◦C witha citric acid/sodium citrate buffer as the anticoagulant. Erythrocytes were thor-oughly washed and lysed. Membrane-associated lipids and Hb were separatedthrough filtration to obtain stroma-free Hb solution. Subsequent pasteurization
Art
if C
ells
Blo
od S
ubst
it Im
mob
il B
iote
chno
l Dow
nloa
ded
from
info
rmah
ealth
care
.com
by
The
Uni
vers
ity o
f M
anch
este
r on
11/
04/1
4Fo
r pe
rson
al u
se o
nly.
22 K.-P. Yan et al.
treatment of the Hb solution significantly reduced macromolecule impurityand the risk of viral contamination. Further purification steps were carried outto obtain Hb with purity larger than 99.5%. For the purpose of this study, ahemoglobin solution contaminated with a significant amount of erythrocytemembrane phosphlipids, called Stroma-contaminated Hb (SC-Hb) was inten-tionally prepared by lysing erythrocytes under a sub-optimal condition duringthe purification process described above.
Extraction of Phospholipids from Hemoglobin Solutions
pH of a Hb solution was adjusted to an appropriate level with 0.5 mol/Lphosphatic acid or 0.5 mol/L trisodium phosphate. With mixing, a certainvolume of isopropanol was slowly added to a clean glass test tube containing5.0 mL of a Hb solution. After 1 hour with occasional shaking of the testtube to mix the sample, chloroform was added at a volume half of that of theisopropanol previously added. The sample was mixed and allowed to stand foranother hour to further extract lipids. During the process Hb became denaturedand precipitated. All extraction solvents were pre-chilled at 4◦C. The abovesteps were all carried out on ice. It is crucial that the volume ratio of isopropanolto chloroform is kept in a narrow vicinity of 2:1 in the final stage of extractionso that a mono-phase can be maintained in the testing sample in the final stageof extraction. After the extraction, the sample was centrifuged at 10,000 × g for20 min at 4◦C. The supernatant was transferred to a clean 50 mL evaporatingflask. The test tube was then washed three times with a small amount ofchloroform. After each wash, the test tube was centrifuged and the supernatantwas transferred to the evaporating flask. After rotating the flask under a vacuumin a water-bath at 25◦ to evaporate the solvent, the dried material was redissolvedin a small amount of chloroform and stored at 4◦ or −20◦.
Phospholipid Analysis by HPLC
HPLC analysis was conducted with a Shimadzu HPLC system (Shimadzu-10Avp, Japan), which was composed of a SCL-10Avp system controller, anLC-10ATvp double pump system, a DGU-12A degasser, and a CTO-10ASvpcolumn oven. Combined with a guard column (10 × 4.6 mm I.D.), a stainless-steel Hypersil silica analytical column (250 mm × 4.6 mm I.D.) with 5 µmparticle size and 120 A pore size (Thermo Electron Corp., USA) was used.Separation of phospholipids on the column was achieved by an isocratic elutionsystem with a mobile phase of acetonitrile-methanol-water-trifluoroacetic acid(100:25:1.7:2.5, v/v) at a flow-rate of 1.0 mL/min and with the column oventemperature set at 40◦C. Detection was performed with an Alltech 2000ESevaporative light scattering detector (ELSD, Alltech, USA) with the drift tubetemperature set at 80◦C and gas (N2) flow rate set at 2.0 l/min.
Art
if C
ells
Blo
od S
ubst
it Im
mob
il B
iote
chno
l Dow
nloa
ded
from
info
rmah
ealth
care
.com
by
The
Uni
vers
ity o
f M
anch
este
r on
11/
04/1
4Fo
r pe
rson
al u
se o
nly.
Extraction of Phospholipids from Hemoglobin Solutions 23
Recovery of Phospolipid Extraction from Hb Solutions
Each of four phospholipid standards (20 µg PS, PE or PC and 30 µg SM) wasadded to five clean test tubes and then dried down with a gentle flow of N2 gas.5.0 mL of 8% pure Hb solution was added. The samples were sonicated for30 min on ice. The phospholipids were extracted from the solution first with15 mL isoproponal and then further with 7.5 mL chloroform according tothe extraction procedure described above. A calibration curve based on thelogarithm of the area of the detector response peak to a phospholipid standardversus the logarithm of the known analyzed amount of the phospholipidwas used for the calculation of extraction recovery of the correspondingphospholipid. The mean of correlation coefficients of all calibration curveswas at least 0.99. The concentration of an extracted phospholipid wascalculated based on the calibration curve of the corresponding phospholipid.The percentage recovery of a phospholipid was calculated by:
Recovery % = µg (calculated extracted amount)
µg (spiked weighted amount)× 100
RESULTS
HPLC Analysis of Stroma-contaminated Hb Solutions
HPLC analysis showed that the ultra-pure Hb solution obtained through our nor-mal purification procedure contained almost no detectable signals (Figure 1A),while stroma contaminated Hb (SC-Hb) solution obtained through a purifi-cation procedure in an undesirable purification condition as described in theMaterials and Methods section yielded four well-resolved major peaks, whichwere verified to be PS, PE, PC and SM, respectively, using correspondingphospholipid standards spiked in the samples (Figure 1B). The amount of PIrecovered from SC-Hb was minimal. Furthermore, the relative percentage ofeach phospholipid extracted from SC-Hb solution was compatible to that of thecorresponding phosphlipid extracted from porcine erythrocytes, indicating thecontaminated phospolipids in the SC-Hb solution was mainly originated fromerythrocyte membrane as shown in Table 1. In order to better reflect the real
Table 1. Comparison of relative percentages of major phospholipids extracted fromporcine erythrocytes and SC-Hb solution
PS (%) PE (%) PC (%) SM (%)
Erythrocytes 25.08 31.48 37.41 6.02SC-Hb solution 26.38 34.95 33.36 5.29
Art
if C
ells
Blo
od S
ubst
it Im
mob
il B
iote
chno
l Dow
nloa
ded
from
info
rmah
ealth
care
.com
by
The
Uni
vers
ity o
f M
anch
este
r on
11/
04/1
4Fo
r pe
rson
al u
se o
nly.
24 K.-P. Yan et al.
Figure 1. Separation of phospholipids extracted from in porcine Hb solution (panel A)and SC-Hb solution (panel B) by HPLC on a silica gel column with a mobile phaseof mixture of acetonitrile:methanol:water:trifluoroacetic acid (100:25:1.7:2.5, v/v) andan ELSD for signal detection. SF: solvent front; PS: phosphatidylserine, PE: phos-phatidylethanolamine; PC: phosphatidylcholine; SM: sphingomyelin; 1, 2: unknownsubstances.
world scenario, we used the SC-Hb solution in most of our experiments in thisstudy, instead of our normal Hb solution spiked with standard phospholipids.
Effect of the Relative Amount of Organic Extraction Solventon the Physical Status of Denatured Hb
Our data showed that the relative volume of isopropanol used at room tem-perature in extraction of phospholipids from Hb solutions affected the extentof pigment contamination and physical status of precipitated Hb, as shown inTable 2. At room temperature, with the increase of the relative volume ratioof isopropanol to the Hb solution (the volume ratio of isopropanol to Hb solu-tion = 1, 2,. . . . . .12), the physical state of precipitated hemoglobin underwentsignificant changes from tight packed clumps with significant pigment in the
Art
if C
ells
Blo
od S
ubst
it Im
mob
il B
iote
chno
l Dow
nloa
ded
from
info
rmah
ealth
care
.com
by
The
Uni
vers
ity o
f M
anch
este
r on
11/
04/1
4Fo
r pe
rson
al u
se o
nly.
Extraction of Phospholipids from Hemoglobin Solutions 25
Table 2. The effect of the relative amount of isopropanol on the physical status of Hbsolution at room temperature
Volume ratio of Isopropanol to Hb solution
1 2 4 8
Solutionpigmentation∗
5+ 3+ No No
Status ofprecipitates
Hard clumps Particles Fine particles Very fine flocculent
Color ofprecipitates
brown Light brown Light red Light red
∗Arbitrary scales.
solution, to loosely packed clumps with slight pigment in the solution, to finelydivided particles in a colorless solution, and then to an even more finely dividedflocculent form without color in the solution (Table 2). However, at a lowertemperature, such as on ice, a small proportion of isopropanol would result infinely divided particles of precipitated hemoglobin with no pigment contami-nation in the solution (data not shown). Therefore, when a small proportion ofisopropanol (with the volume ratio of isopropanol to Hb solution ≤2) is used,it is important to carry out the extracting step on ice to ensure that there are nocontaminated heme pigments and that hemoglobin is precipitated as flocculentfine particles so that hemoglobin molecules are in close contact with a largeexcess of the solvent to achieve maximum extraction of phospholipids.
Effect of Relative Volume of the Organic Solvent on the Efficiencyof Phospholipid Extraction
In keeping the ratio of isopropanol and chloroform at 2:1 (v/v), varying thevolume ratio of isopropanol to the SC-Hb solution did not affect the yields ofPC, PE and SM in a wide range of the volume ratios (2:1 to 8:1) of isopropanolto the SC-Hb solution when the extraction was carried out on ice, as shown inFigure 2. If the volume ratio of isopropanol to the SC-Hb solution was lowerthan 2:1, phospholipid extraction efficiency became significantly reduced forall types of phospholipids, presumably due to insufficient extraction capacityof the limited amount of the organic solvents. On the other hand, when thevolume ratio of isopropanol to the SC-Hb solution was larger than 6:1, the effi-ciency of PS extraction became significantly lower. This finding has importantimplications. Since PS is a potent toxin, underestimation of PS concentrationsin Hb solutions due to significant low efficiency of PS extraction may compro-mise product safety. Therefore, we chose a volume ratio (such as 3:1) at thelower end of the effective volume ratio range in our subsequent studies to use asmaller but effective amount of the organic solvent in the extraction process. In
Art
if C
ells
Blo
od S
ubst
it Im
mob
il B
iote
chno
l Dow
nloa
ded
from
info
rmah
ealth
care
.com
by
The
Uni
vers
ity o
f M
anch
este
r on
11/
04/1
4Fo
r pe
rson
al u
se o
nly.
26 K.-P. Yan et al.
Figure 2. The effect of relative volumes of the organic extraction solvent on the ef-ficiency of phospholipid extraction. Increasing volume of isopropanol were added to5.0 mL of SC-Hb solution resulting in the volume ratio of isopropanol to SC-Hb so-lution from 1:1 to 8:1 as shown 1 to 8 in the x axis to extract phospholipids for 1 hron ice before addition of chloroform at 2:1 volume ratio of isopropanol to chloroformfor further extraction. The extracted phospholipids were quantified by HPLC analysis.Symbols: �:PE; �:PC; �:PS; ×: SM.
contrast, the volume ratio of isopropanol to erythrocyte samples in the parentmethod of this study was 5.5:1 [10], which was at the upper end of the effectivevolume ratio range as determined in this study, and therefore consumed muchmore organic solvent (Figure 2).
Effect of pH on the Efficiency of Phospholipid Extraction
Our study found that the efficiency of this method for extraction of PE, PCand PS from the SC-Hb solution was pH-dependent. As shown in Figure 3 as atypical set of results, significantly larger amounts of these phospholipids wereextracted from the SC-Hb solution at acidic and basic pH than at neutral pHwith an optimal pH value around 5.0. Compared to the neutral pH condition,the optimal pH condition (pH 5.0) yielded about 50%, 70% and 140% more PS,PC and PE, respectively. The extraction efficiencies of these phospholipids atbasic conditions (around pH 9.0) were 15% to 30% higher than that at neutralpH. Hb and phospholipids are all amphophilic. The isoelectric point of Hb isaround 7.0. Therefore, the above data indicates that ion-ion interactions mayplay a significant role in binding of these phospholipids to Hb. For instance,at acidic pH, the head groups of phospholipids bear relatively less negativecharges due to their acidic groups becoming less deprotonized, presumably
Art
if C
ells
Blo
od S
ubst
it Im
mob
il B
iote
chno
l Dow
nloa
ded
from
info
rmah
ealth
care
.com
by
The
Uni
vers
ity o
f M
anch
este
r on
11/
04/1
4Fo
r pe
rson
al u
se o
nly.
Extraction of Phospholipids from Hemoglobin Solutions 27
leading to a weaker interaction between phospholipids and Hb. However, theextraction efficiency for SM was not significantly affected by pH.
It has been reported that human erythrocyte membrane binds Hb in a pH-dependent fashion in various experiment systems, such as erythrocyte ghostcells [15–16], phospholipid monolayer films [17] and inside-out vesicles [18].The binding sites are predominantly at the inner half of the membrane andlargely at two transmembrane glycoproteins, band 3 and glycophorin, with asmall portion of the binding directly through phospholipids [16–18]. However,the interaction between Hb and membrane components was stronger at acidicpH (pH = 4.0), much weaker at basic pH [15, 17]. The reason for the dis-crepancy between our results and these studies is not clear. It is possible thatat acidic pH, increased tendency of autooxidation of Hb may result in con-formation changes or even mild denaturation of related proteins, resulting inexposure of some of the hydrophobic patches of their molecular structures toincrease hydrophobic interactions between membrane lipid and the proteins.In our mild extraction condition, such interaction would be disrupted to releaselipids from Hb. Such disrupting force would not be available in the experimentconditions in the studies cited above.
Figure 3. The effect of pH on the efficiency of phospholipid extraction. 10 mL ofisopropanol was added to 5 mL SC-Hb solution with pH value ranging between 4.0 to10.0. (equivalent to a volume ratio of isopropanol to the SC-Hb solution at 2:1) to extractphospholipids for 1 hr before addition of 5 mL chloroform (equivalent to a volume ratioof isopropanol to chloroform at 2:1) for further extraction. The extracted phospholipidswere quantified by HPLC analysis. Symbols: �:PE; �:PC; �:PS; ×: SM.
Art
if C
ells
Blo
od S
ubst
it Im
mob
il B
iote
chno
l Dow
nloa
ded
from
info
rmah
ealth
care
.com
by
The
Uni
vers
ity o
f M
anch
este
r on
11/
04/1
4Fo
r pe
rson
al u
se o
nly.
28 K.-P. Yan et al.
Effect of Salt Concentrations Hb Solutions on the Efficiencyof Phospholipid Extraction
Since ion-ion interaction may be an important mode for binding of phospholidsto Hb, a further attempt was made to understand how strong this interactioncould be through investigation of the effect of salt concentrations in the SC-Hbsolution on the extraction efficiency. Our data demonstrated that concentrationsof NaCl ranging 10 mmol/L to 300 mmol/L in the SC-Hb solution did notsignificantly affect the yields of all the major phospholipids at pH 5.0. However,further increase of NaCl concentrations resulted in some degree of loss ofextraction efficiency (Figure 4).
Recovery of Phospholipids from Hb Solutions at the OptimumExtraction Condition
Based on the results of the above experiments, we set the optimum extractioncondition as Hb solution (pH 5.0):isopropanol:chloroform =1:3:1.5 performedon ice. Next we used standard phospholipids to study the recovery of PS,
Figure 4. The effect of salt concentration in Hb solutions on the efficiency of phospho-lipid extraction. 10 mL of isopropanol was added to 5 mL SC-Hb solution (equivalentto volume ratio of isopropanol to SC-Hb solution at 2:1) with pH value at 5.0 andcontaining varying amounts of NaCl to extract phospholipids for 1 hr before additionof 5 mL chloroform (equivalent to volume ratio of isopropanol to chloroform at 2:1)for further extraction. The extracted phospholipids were quantified by HPLC analysis.Symbols: �:PE; �:PC; �:PS; ×: SM.
Art
if C
ells
Blo
od S
ubst
it Im
mob
il B
iote
chno
l Dow
nloa
ded
from
info
rmah
ealth
care
.com
by
The
Uni
vers
ity o
f M
anch
este
r on
11/
04/1
4Fo
r pe
rson
al u
se o
nly.
Extraction of Phospholipids from Hemoglobin Solutions 29
PE, PC and SM under the optimum extraction condition by spiking standardphospholipids into normal Hb solutions and extracting phospholipds under theoptimum extraction condition. The recoveries of PE, PC and SM were about100%, as shown in Table 3. The recovery of PS was about 93%. Similar resultshave been reported using the parent method [11].
DISCUSSION
In biological samples processed for lipid extraction, phospholipids may bepresent as membrane vesicle (particles) or in a form associated with proteins.The proportion of the lipids in these two forms in a sample depends on thenature of the biological material and the properties of proteins in the sample. Insample prepared from lipid-rich tissues such as that of brain, most of the lipidsare membrane-associated, while in protein-rich solutions, such as plasma andhemoglobin solution, protein-associated portion of lipids could be significant.Since hemoglobin solutions made for manufacturing HBOC products maycontain trace amounts of contaminated phospholipids, a significant amount ofthe lipids may be protein-associated. In fact, it has been reported that about15% of phospholipids spiked in a hemoglobin solution was not extractableby a solid-phase extraction method [12], presumably due to association of thelipids with hemoglobin. Such a “phospholipid masking effect” of Hb solutionnwould result in underestimating of the phospholipid concentrations in HBOCproducts and constitute a significant concern in analysis of trace amounts ofcontaminated phospholipid in Hb solutions from a product safety point of view.
Due to the protein-rich nature of hemoglobin solutions and the require-ment of extraction of trace amounts of contaminated phospholipids from suchsolutions for QC/QA purposes, several important factors have to be consideredin the design of an effective extraction method using a liquid-liquid extractionapproach.
First is the choice of an organic extraction solvent system. Different or-ganic solvent systems have been established to extract lipids from tissue sam-ples. The Folch’s method [13] and related modified versions, such as the Bligh& Dyer’s [14] and Christiansen’s [19], are most widely used. These systemsusing a chloroform/methanol mixture as their organic extraction solvent are
Table 3. Recoveries of standard phospholipids spiked in Hb solution andextracted at the optimum extraction condition (n = 5)
Phospholipids Recovery (%) SD (%)
PS 93 1.3PE 103 1.1PC 109 1.9SM 102 3.5
Art
if C
ells
Blo
od S
ubst
it Im
mob
il B
iote
chno
l Dow
nloa
ded
from
info
rmah
ealth
care
.com
by
The
Uni
vers
ity o
f M
anch
este
r on
11/
04/1
4Fo
r pe
rson
al u
se o
nly.
30 K.-P. Yan et al.
particularly effective for use in lipid-rich tissues, such as brain tissue. How-ever, they have been shown to suffer from significant loss in efficiency in lipidextraction from protein-rich samples, such as red blood cells, heart and liver[10,19–21]. A significant part of the loss may be contributed to the ineffi-ciency of the strong organic solvents in extraction of protein-associated lipidsin protein-rich samples due to strong protein-denaturing nature of the organicsolvents, leading to protein-associated lipids to be too tightly trapped in heavilyprecipitated proteins (hard clumps) to be extractable. Milder organic solventshave been shown to yield better results in protein-rich samples. A method us-ing n-butanol as the extracting organic solvent is reported to be more effectivethan the Bligh & Dyer’s method for in liver samples [21]. The method usingisopropanol introduced by Rose and Oklander [10] yielded better recoveriesfor cholesterol (up to 19% higher) and phospholipids and had less heme pig-ment contamination extracted from red blood cells than those obtained by theFolch’s method extraction. It is also reported that nearly 100% recoveries ofphospholipids from Hb solutions by this method [11]. Our experiment in thisstudy further demonstrated that isopropanol was so mild in denaturing proteinsthat the finely divided flocculent form of hemoglobin precipit could be achievedduring the extracting phase with a wide range of the volume ratio of isopropanolto hemoglobin solution. This ensures maximum exposure of protein-associatedphospholipids to the organic solvent and increase the extraction efficiency.Therefore, isopropanol would be a suitable organic solvent of choice for ex-traction of lipids from hemoglobin solutions or other protein-rich biologicalsamples.
In liquid-liquid extraction, lipid extraction by an organic solvent systemand subsequent washing of the extract by an aqueous system are the two mainsteps. Depending on the samples for lipid extraction and the composition ofthe organic solvent system, the extracted samples exist as either monophasic orbiphasic (consisting of an organic phase and aqueous phase) after the extraction.The soluble material in monophasic samples or in the organic phase of biphasicsamples are either directly processed for recovery of lipids or undergo furtherwashing by an aqueous system to remove contaminants in the aqueous-organicinterface prior to being further processed.
It has been shown that phase separation and washing of organic extractin a lipid extraction process are the main steps resulting in loss (up to 30%)of lipid recoveries, largely because there is always a certain amount of lipidsin an emulsified form or similar status in the aqueous-organic interface, whichwill be carried away from the samples during further sample processing steps.[13, 19, 20]. The main purpose of elimination of contaminants from the lipidextract is to reduce interference of these substances to assays of the extractedlipids. For example, phosphate compounds, such as ATP, may interfere withquantitative detection of unknown phospholipids [11]. For detection of knownphospholipids as in Hb solutions, such contamination would not be a concern.Therefore, a monophasic method with no washing steps would be appropriate
Art
if C
ells
Blo
od S
ubst
it Im
mob
il B
iote
chno
l Dow
nloa
ded
from
info
rmah
ealth
care
.com
by
The
Uni
vers
ity o
f M
anch
este
r on
11/
04/1
4Fo
r pe
rson
al u
se o
nly.
Extraction of Phospholipids from Hemoglobin Solutions 31
for a high degree of recovery of lipids from Hb solutions. The Rose & Oklan-der’s isopropanol/chloroform system yields a monophasic extract [10]. There-fore, we adopted this system in this study, but omitting the washing steps.
It has been shown [20] that biphasic extraction systems, either chloro-form/methanol (Folch’s, Bligh and Dyer’s) or n-butanol system (Daae andBremer’s), yielded lower recoveries of more hydrophilic lysoPC and lysoPEthan those of PC and PE from rat heart homogenates, indicating that the hy-drophilic force is more significant in lysoPC and lysoPE than PC and PE so thatthese lyso-phospholids tend to be washed away from the samples along withassociated proteins. In fact, it has been known that increasing salt concentra-tions in the samples or washing solution in biphasic extraction systems couldsignificantly increase the recovery of phospholipids [13], presumably throughweakening the hydrophilic interactions between phospholipids and proteins.Such an interaction mechanism becomes more evident by the finding that pHplays a significant role in recoveries of acidic phospholipids. For example, theFolch’s method using 0.9%NaCl as the washing solution yielded almost norecovery of lysophosphatidic acid from rat liver mitochondria while a very sig-nificant recovery of such lipid was obtained when a washing solution containing0.01 NHCI was used instead [22].
Our data clearly showed that recoveries of phsopholipids from Hb solutionare pH-dependent. An acidic pH favored better extraction of PC, PE, PS fromHb solutions in our monophasic non-wash extraction system (Figure 3). Atthe optimum pH, salt concentration up to 300 mmol/L in the SC-Hb solutionseemed not to significantly affect the recoveries of phospholipids, especially PSand SM (Figure 4). Furthermore, complete recovery of spiked phospholipidsin Hb solutions was obtainable at the optimal condition. These data suggestthat phospholipids do interact with hemoglobin proteins through weak ionicinteractions.
In this study, we developed a phospholipid extraction method based onthe organic solvent system introduced by Rose and Oklander [10], which hasbeen demonstrated to be effective in extraction of lipids from erythrocytes andmild in denaturing of proteins, such as Hb (Figure 2). Optimized pH condition(pH 5.0) resulted in significant increase in extraction of PS, PE and PC from Hbsolutions (Figure 3) and from porcine erythrocytes (data not shown) comparedto the condition used in its parent method. Extraction at low temperaturesminimized heme pigment contamination at lower volume ratio of the organicextraction solvent to Hb solution. Monophasic extraction without washingsteps further decreased loss of recoveries. Compared to the parent method,the established method in this study was simpler and used 3/4 less organicsolvents. Combined with an improved phospholipid analytic system (data to bepublished), this method consumed at least 2/3 less Hb solution (used less than10 mL instead of 30 mL or more) compared to other commonly used methods[10–11]. Since ultra-pure Hb and HBOCs are very expensive to make, such asavings in QC/QA process would be significant. Apart from Hb solutions, this
Art
if C
ells
Blo
od S
ubst
it Im
mob
il B
iote
chno
l Dow
nloa
ded
from
info
rmah
ealth
care
.com
by
The
Uni
vers
ity o
f M
anch
este
r on
11/
04/1
4Fo
r pe
rson
al u
se o
nly.
32 K.-P. Yan et al.
method may have a wide application for other protein-rich biological samples,such as erythrocytes, plasma, heart and liver.
REFERENCES
1. Winslow, R.M. (2006). Current status of oxygen carriers (“blood substitutes”):2006. Vox. Sang. 91: 102–110.
2. Johnson, J.L., Moore, E.E., Offner, P.J., Partrick, D.A., Tamura, D.Y., Zallen, G,Silliman, C.C. (2001). Resuscitation with a blood substitute abrogates pathologicpostinjury neutrophil cytotoxic function. J. Trauma. 50: 449–455.
3. Stowell, C. P. (2005). What happened to blood substitutes? Transfus. Clin. Biol.12: 374–379.
4. Wu, B.P., Wang, F., Lou, C., Chen, C. and Dan, N. Studies of porcine hemoglobinand glutaraldehyde-polymerized porcine hemoglobin as hemoglobin-based oxygencarriers. To be published.
5. Diamant, M, Tushuizen, M.E., Sturk, A., Nieuwland, R. (2004). Cellular micropar-ticles: new players in the field of vascular disease? Eur. J. Clin. Invest. 34: 392–401.
6. Zwaal, R.F., Comfurius, P. and Bevers, E.M. (1998). Lipid-protein interactions inblood coagulation. Biochim. Biophys. Acta. 1376: 433–453.
7. Wu, Y., Tibrewal, N. and Birge, R.B. (2006). Phosphatidylserine recognition byphagocytes: a view to a kill. Trends Cell Biol. 16: 189–197.
8. Feola, M., Simoni, J., Dobke, M., Canizaro, P.C. (1988). Complement activationand the toxicity of stroma-free hemoglobin solutions in primates. Circ Shock. 25:275–290.
9. Feola, M., Simoni, J., Canizaro, P.C., Tran, R., Raschbaum, G., Behal, F.J. (1988).Toxicity of polymerized hemoglobin solutions. Surg. Gynecol. Obstet. 166: 211–222.
10. Rose, H.G., Oklander, M. (1965). Improved procedure for the extraction of lipidsfrom human erythrocytes. J. Lipid Res. 6: 428–431.
11. Biessels, P.T., Berbers, G.A., Broeders, G.C., Landsvater, R., Huisman, H.G.,Bleeker, W.K., Bakker, J.C. (1992). Detection of erythrocyte membrane compo-nents in hemoglobin-based blood substitutes. Clin. Chim. Acta. 212: 113–122.
12. Horne, T., Holt-Larkin, S. (1997). Solid-phase extraction of phospholipids fromhemoglobin solutions using Empore styrene-divinylbenzene disks. J. Chromatogr.B. 695: 259–267.
13. Folch, J., Lees, M., Sloane Stanley, G.H. (1957). A simple method for the isolationand purification of total lipides from animal tissues. J. Biol. Chem. 226: 497–509.
14. Bligh, E.G. and Dyer, W.J. (1959). A rapid method of total lipid extraction andpurification. Can. J. Biochem. Physiol. 37: 911–917.
15. Dodge, J.T., Mitchell, C., Hanahan, D.J. (1963). The preparation and chemicalcharacteristics of hemoglobin-free ghosts of human erythrocytes. Arch. Biochem.Biophys. 100: 119–130.
16. Shaklai, N., Yguerabide, J., Ranney, H.M. (1977). Classification and localizationof hemoglobin binding sites on the red blood cell membrane. Biochem. 16: 5593–5597.
Art
if C
ells
Blo
od S
ubst
it Im
mob
il B
iote
chno
l Dow
nloa
ded
from
info
rmah
ealth
care
.com
by
The
Uni
vers
ity o
f M
anch
este
r on
11/
04/1
4Fo
r pe
rson
al u
se o
nly.
Extraction of Phospholipids from Hemoglobin Solutions 33
17. Szundi, I., Szelenyi, J.G., Breuer, J.H., Berczi, A. (1980). Interactions ofhaemoglobin with erythrocyte membrane phospholipids in monomolecular lipidlayers. Biochim. Biophys. Acta. 595: 41–46.
18. Rauenbuehler, P.B., Cordes, K.A. and Salhany, J.M. (1982). Identification of thehemoglobin binding sites on the inner surface of the erythrocyte membrane. BiochimBiophys Acta. 692: 361–370.
19. Christiansen, K. (1975). Lipid extraction procedure for in vitro studies of glyceridesynthesis with labeled fatty acids. Anal. Biochem. 66: 93–99.
20. Kolarovic, L., Fournier, N.C. (1986). A comparison of extraction methods for theisolation of phospholipids from biological sources. Anal. Biochem. 156: 244–250.
21. Daae, L.N., Bremer, J. (1970). The acylation of glycerophosphate in rat liver. Anew assay procedure for glycerophosphate acylation, studies on its subcellular andsubmitochondrial localization and determination of the reaction products. Biochim.Biophys. Acta. 210: 92–104.
22. Bjerve, K.S., Daae, L.N.W., Bremer, J. (1974). The selective loss of lysophos-pholipids in some commonly used lipid-extraction procedures. Anal. Biochem. 58:238–245.
Art
if C
ells
Blo
od S
ubst
it Im
mob
il B
iote
chno
l Dow
nloa
ded
from
info
rmah
ealth
care
.com
by
The
Uni
vers
ity o
f M
anch
este
r on
11/
04/1
4Fo
r pe
rson
al u
se o
nly.