Formation and degradation of lipid droplets in human adipocytes and the expression of aldehyde oxidase (AOX)
Lipid droplet (LD) binding proteins in mammary glands and in adipocytes were previously compared and striking similar sets of these specific proteins demonstrated. Xanthine oxidoreductase (XOR) together with perilipins and the lactating mammary gland protein butyrophilin play an important role in the secretion process of LDs into milk ducts. In contrast, in adipose tissue and in adipocytes, mainly perilipins have been described. Moreover, XOR was reported in mouse adipose tissue and adipocyte culture cells as “novel regulator of adipogenesis”. This obvious coincidence of protein sets prompted us to revisit the formation of LDs in human-cultured adipocytes in more detail with special emphasis on the possibility of a LD association of XOR. We demonstrate by electron and immunoelectron microscopy new structural details on LD formation in adipocytes. Surprisingly, by immunological and proteomic analysis, we identify in contrast to previous data showing the enzyme XOR, predominantly the expression of aldehyde oxidase (AOX). AOX could be detected tightly linked to LDs when adipocytes were treated with starvation medium. In addition, the majority of cells show an enormous interconnected, tubulated mitochondria network. Here, we discuss that (1) XOR is involved—together with perilipins—in the secretion of LDs in alveolar epithelial cells of the lactating mammary gland and is important in the transcytosis pathway of capillary endothelial cells. (2) In cells, where LDs are not secreted, XOR cannot be detected at the protein level, whereas in contrast in these cases, AOX is often present. We detect AOX in adipocytes together with perilipins and find evidence that these proteins might direct LDs to mitochondria. Finally, we here report for the first time the exclusive and complementary localization of XOR and AOX in diverse cell types.
KeywordsLipid droplets Perilipins Mammary gland Xanthine oxidoreductase Adipocytes Aldehyde oxidase
Adipose triglyceride lipase
- BTN or BTN1a
Milk lipid globule membrane
Outer mitochondrial membrane
Peroxisome proliferator-activated receptor
Reactive oxygen species
Sodium dodecyl sulfate polyacrylamide gel electrophoresis
Small lipid droplet
In recent years, the origin, the formation and the accumulation of lipid droplets (LDs) in a variety of different cells have been investigated intensely in order to contribute insights to a general problem: obesity as an increasing worldwide major health concern in modern life (Sztalryd and Kimmel 2014). Of particular interest were proteins of the perilipin family bound to LDs (Wang and Sztalryd 2011). The LDs in milk are surrounded by a proteinaceous coat, i.e., the milk lipid globule membrane (MLGM), comprise members of the perilipin family (PLIN2-3), the lactation-specific transmembrane protein butyrophilin (BTN1A) and xanthine oxidoreductase (XOR) as major LD-binding proteins (see Franke et al. 1981; Jarasch et al. 1981; Heid and Keenan 2005). During lactation, the enzyme XOR is involved in the secretion of LDs from the mammary epithelial cells into the mammary gland ducts. This enzyme was also found and localized in capillary endothelial cells (Jarasch et al. 1981; Jarasch et al. 1986; Bruder et al. 1984). In general, XOR regulates cellular redox and detoxification processes, representing an evolutionary old oxidative defense mechanism (Vorbach et al. 2003; Vorbach et al. 2006). XOR is also reported to be responsible for the final two steps in purine metabolism by converting hypoxanthine to xanthine and xanthine to uric acid with concomitant generation of reactive oxygen species (ROS). Generated ROS products are considered responsible for antimicrobial activity and are involved in various pathological situations (McCord et al. 1985; Berry and Hare 2004; Martin et al. 2004; Battelli et al. 2019).
In LDs isolated from adipocytes, specific protein members of the perilipin family are well established as major LD surface-associated proteins in association with the intermediate filament protein vimentin (Franke et al. 1987; Greenberg et al. 1991; Blanchette-Mackie et al. 1995; Wolins et al. 2005; Heid et al. 2014). Using gene profiling methods and a novel algorithm, XOR was reported (Cheung et al. 2007) as “regulator of adipogenesis and PPARy activity”. These striking similarity of sets of LD-associated proteins found in lactating mammary gland cells and in adipocytes prompted us to revisit early adipocyte differentiation in more detail, with special focus on XOR expression.
Here, we show by electron and immunoelectron microscopy new structural details on early droplet formation in cultured human adipocytes. Further, we identify and demonstrate the expression of aldehyde oxidase (AOX) by proteomic and biochemical analysis. We present evidence that AOX is primarily not participating in LD formation and LD packaging during adipogenesis but is involved in lipolysis processes by nutrition deprivation. Implications and possible functions of XOR for packaging and secretion of LDs in the lactating mammary gland versus AOX directing LDs to mitochondria of adipocytes, assisting ordered release of fatty acids for β-oxidation, are discussed.
Material and methods
Antibodies and reagents
The generation of monoclonal (mab) and polyclonal (pab) antibodies (abs) against members of the perilipin family of LD-binding proteins was recently described (Heid et al. 2013). New abs against butyrophilin (BTN1A), xanthine oxidoreductase (XOR) and aldehyde oxidase (AOX) were raised accordingly. These abs are summarized in Table S1 of Supplementary material. Additional abs used for testing and for controls in this study are also listed or given in previous publications (Heid et al. 2013; Heid et al. 2014).
Human preadipose cells (Poietics from subcutaneous preadipocytes), growth media and adipocyte differentiation medium (ADM; also named adipocyte induction medium, AIM)—containing insulin, dexamethasone, indomethacin and isobutyl-methylxanthine—were obtained from Lonza (Basel, Switzerland; cat. # PT-5020, PT-8002 and PT-8202). Cell lysates were obtained as previously described (Heid et al. 2013).
Isolation of MLGM material was according to Franke et al. (Franke et al. 1981). For nitrogen cavitation, density gradient separation, SDS-PAGE, immunoblotting, immunofluorescence microscopy, immunoprecipitation and mass spectrometry analysis, see Heid et al. (2013).
Electron and immunoelectron microscopy
For electron microscopy (EM), cells were grown on cover slips and fixed as previously described using glutaraldehyde and OsO4—either simultaneously (Franke et al. 1969) or sequentially (Heid et al. 2013). For further details of immunoelectron microscopical techniques, see Franke et al. (1987).
Expression of lipid droplet–associated proteins in human mammary gland and in adipocytes
Selecting suitable antibodies for cultured adipocytes
In order to test the adipocyte cell system for proper performance of the various antibodies (abs) for correct localization, we used low passages of human preadipocytes (schematically shown in Fig. 1b). Several of the commercial available abs have been proven unspecific and unreliable, e.g., one ab reacted positive in immunoblotting with a band at 27 kD, within the reference lane and turned out to be triosephosphate isomerase (Escherichia coli). This protein is completely unrelated to XOR or AOX (see Fig. S5). In recent years, false findings by abs are spread all over the field and the specificity of abs became a matter of intensive debate (Baker 2015). It is evident that proper validation of abs, including eventual cross-reactivity and other drawbacks, should be supplied. For example, one report on xanthine oxidase inhibition by febuxostat is stating for used ab only “XO (1:100, Abcam)” and no product number and no further information are given (Yisireyili et al. 2017). Another example with a company statement not very useful on an antigen source (“Peptide internal sequence: considered to be commercially sensitive”) is given in Table S1. Therefore, we characterized available XOR abs in parallel to immunofluorescence microscopy (IFM) by immunoblotting and obtained some conflicting and/or negative results (Fig. S5). To overcome this problem, we obtained additional commercial abs and further also generated a series of novel abs (Table S1). We tested and selected 7 different abs specific for PLIN, with 12 different abs specific for XOR and 7 different abs specific for aldehyde oxidase (AOX). Using these abs, we demonstrate exemplarily a wide heterogeneity of LD labeling (Fig. S1).
Electron microscopic studies on the endogenous formation of LDs
EM studies of LDs of adipocyte conversion and OA uptake
IEM localization studies of adipocyte conversion and uptake of OA
Specific oxidases in adipocytes and mammary gland
AOX association with LDs in adipocytes
Linking lipid droplets, perilipins and oxidases to membranes of human cells
LD binding protein
Epithelial cells of lactating mammary gland
Discovery of a complementary localization of XOR and AOX
Localization of xanthine oxidoreductase and aldehyde oxidase in diverse cell types
XOR is present in:
XOR is absent from:
Alveolar epithelial cells of lactating mammary gland
Ductal epithelial cells of mammary gland
Capillary endothelial cells of
Myoepithelial cells of mammary and salivary gland
- Small and large intestine
- Heart and skeletal muscle
Endothelial cells of
- Large blood vessels (arteries and veins)
- Capillaries of brain (gray and white matter)
- Capillaries of testis
Epithelial cells of
- Choroid plexus of brain
- White adipose tissue
- Arterial wall
Fibroblasts, adipocytes, muscle cells, nerve cells and diverse blood cells
AOX is present in:
Visceral and subcutaneous fat depots
The report on XOR in adipose tissue and adipocytes and the postulated involvement of this enzyme in adipogenesis (Cheung et al. 2007) gave rise to revisit and extend our former investigations on the formation of LDs. We found strikingly similar sets of major LD binding proteins reported for MLGs from the lactating mammary gland and for adipocytes. XOR is tethering cytoplasmic LDs in the lactating mammary gland to the apical plasma membrane by interacting with the LD binding protein PLIN2 and the cytoplasmic part of the transmembrane, milk specific protein BTN (Mather and Keenan 1998; McManaman et al. 2002; Heid and Keenan 2005; Monks et al. 2016). We wondered what specific role such an enzyme might play in adipocytes, cells that are LD storage cells and not LD secretory cells.
We highlight our major findings
EM and IEM on the formation of LDs in adipocytes are extended and connections of LDs with mitochondria under starvation conditions are presented.
We generated novel and reliable abs for XOR and AOX suitable for new experiments.
We identified AOX expressed in adipocytes instead of XOR as previously reported.
We recognized an exclusive and complementary localization of XOR and AOX in diverse cell types and summarized all the data in tables and cartoons.
A hypothesis is presented where XOR together with PLIN2 is tethering LDs to the apical plasma membrane of lactating mammary gland cells and AOX together with PLIN1 is linking LDs with mitochondria in adipocytes under starvation condition.
Is XOR involved in adipogenesis?
XOR has been suggested as a novel regulator of adipogenesis (Cheung et al. 2007); however in this report, XOR was not shown at the protein expression level and the XOR-related enzyme AOX was not tested. Like XOR, AOX belongs to a family of molybdo-flavoproteins with an identity towards XOR of 49.7% on the protein level and with almost a similar molecular mass of 150 kD. The genes encoded have almost identical intron/exon organization and both enzymes have a similar spectrum of enzymatic reactions. Therefore, the oxidase activity of XOR activity measurement in the absence and presence of NAD+, claimed to distinguish activities between XO and XHD, could be prompted also by AOX and oxygen. Current data are based on microarray analysis, activity measurement, a HPLC assay for uric acid levels, working with PPARy agonists, which all can be influenced also by AOX, because both enzymes, XOR and AOX, are able to generate ROS (Kundu et al. 2007; Kundu et al. 2012). In addition, inhibitors like allopurinol and others, used for generating these data, can work on both enzymes and can create misinterpretations (see, e.g., Weidert et al. 2014; Williams et al. 2014). The evolution of AOX from XOR was by gene duplication and the enzymes are vicinally situated on the same chromosome (see, e.g., Krenitsky 1978). Therefore, the claimed cascade of factors that controls adipogenesis might hold true perhaps also for AOX and not exclusively for XOR. To date, many researchers believe that XOR is the principal enzyme responsible for oxidation of purine metabolites to uric acid and that hyperuricemia is highly correlated with increased adiposity, obesity and the metabolic syndrome (Berry and Hare 2004; Harrison 2002; Pacher et al. 2006). Still, the question remains: why could XOR not be shown directly at the protein level in adipocytes? Interestingly, Weigert et al. reported on AOX in high amounts in adipose tissue and mouse 3T3-L1 and demonstrated with fenofibrate, an agonist of PPAR-α, reduced AOX protein contents in differentiated 3T3-L1 cells (Weigert et al. 2008).
These different interpretations prompted us to revisit data on adipogenesis with special emphasis on these oxidases XOR and AOX.
Endogenously and exogenously derived LDs in adipocytes and validation of novel antibodies to XOR and AOX
Human preadipocytes were converted to adipocytes to test commercially available abs for XOR. We wanted to confirm the expression of XOR during adipogenesis in relation to the LD binding perlipins. We took an approach on LD formation and generated two different kinds of LDs (Fig. 1b; described by Heid et al. 2014). We obtained in cells internally generated, endogenous LDs derived from ER with monolayer membranes (Murphy and Vance 1999; Heid and Keenan 2005; Martin and Parton 2006; Wilfling et al. 2013; Choudhary et al. 2015). By addition of albumin-complexed OA to the culture medium, exogenous LDs with bilayer membranes from the uptake via an endocytosis process were additionally generated. By IFM, the huge variety and heterogeneity of resulting LDs by such approaches could be demonstrated (Fig. S1). We discovered using proper abs and by mass spectrometry that instead of the proclaimed XOR, the related enzyme AOX is expressed in adipocytes (see below).
EM findings on vimentin, layers of ER and the formation of LDs
In a next step, we extended our previous findings on the perilipin-vimentin cortex surrounding nascent LDs (Figs. 2,S2 and S4; see Heid et al. 2014). These structures of forming LDs are often encased by multiple layers of ER. With starting the conversion condition, occasionally some mitochondria seemed to be linked via vimentin intermediate filaments to LDs (Fig. S2b). In general, only a few, small mitochondria are seen in the cytoplasm during LD forming and the perilipin-vimentin cortex and ER layers are keeping the growing LDs separated from mitochondria (Figs. 2 and S3). We would like to emphasize that we and formerly others (Novikoff et al. 1980; Franke et al. 1987; Heid et al. 2014), so far, have not found by EM any evidence yet for LDs contacting microtubules. In support of our findings are results on the ablation of vimentin in steroidogenic tissues where the movement of cholesterol from cytosolic LDs to mitochondria is defected (Shen et al. 2012).
EM and OA uptake
When albumin-complexed OA was added to the conversion medium of adipocytes (cp. Fig. 1b), almost immediately many endocytotic vesicles and invaginations appear in the cytoplasm, especially near the plasma membrane (Fig. 3a). These endocytotic vesicles have bilayer membranes derived from the plasma membrane material, also seen in clusters of “vesicles-like structures” or of “rosette-like structures” (Fig. 3b). These structures were first described in rat adipose tissue (Williamson 1964) and in mouse 3T3-L1 cultured cells (Novikoff et al. 1980). The invaginations of the plasma membrane are not labeled by perilipin abs. Still in the process of endocytosis, these invaginations are likely be filled with OA cargo. As further consequence of OA supply and “ER decay,” the tight intermediate filament structures at the surface of LDs begin to loosen and occasionally, via these vimentin intermediate filaments, mitochondria can be seen linked to individual LDs (Fig. 3c). How bilayer membrane-surrounded, endocytosis-derived LDs can assemble and fuse with monolayer membrane-surrounded, ER-derived LDs, is still a matter of debate. One explanation might be the unique architecture of the “monolayer LDs”, which can give rise to “lipidic bridges” (Schuldiner and Bohnert 2017).
IEM localization of perilipins in adipocytes
IEM localization of PLIN1 upon adipocyte conversion is solely found at the surface of endogenously, ER-derived LDs (Fig. 4). These LDs are connected by a network of intermediate filaments. With OA uptake, smaller vesicle-like structures appear in the sections; many are seen in clusters. These are not labeled for PLIN1 but are cross-sectioned invaginations. In addition, possible association sites of mitochondria and LDs with positive labeling of PLIN1 can be detected (Fig. 4, black brackets). These IEM results support an involvement of PLIN1 in tethering mitochondria with LDs (see below for further arguments). IEM localization in adipocytes of PLIN2 is shown in Fig. 5. PLIN2-positive labeling of LDs is seen as a minor population of midsize droplets during adipocyte conversion in midst of PLIN1-positive, bigger LDs. LD anchored by intermediate filaments is clearly detectable. Clusters of very small, positively labeled droplets can be detected additionally (“sLD”). These seem to be derived from endocytosed lipid material by OA treatment.
AOX is expressed in adipocytes and links LDs and mitochondria under nutrition depletion
Despite testing a panel of abs against XOR with samples of adipocytes, we were not able to confirm the report on XOR on the protein level (Fig. S5). Additional experiments and having identified unambiguously AOX instead of XOR in adipocytes by mass spectrometry (MS) and immunoblotting using a broad spectrum of commercial available and newly generated, validated specific abs (Figs. 6, S6, and S7; Table S1), we wondered why the XOR related enzyme AOX so far was not tested but rather excluded in the work of many reports. So far, only one report describes AOX in high amounts in fat depots and mouse 3T3-L1 cells (Weigert et al. 2008). AOX is known for broad substrate specification used in many clinical studies on the metabolism of drugs and xenobiotics (Terao et al. 2016; Romao et al. 2017; Beedham 2019). Humans are characterized by a single AOX enzyme, while rodents express four isoenzymes (AOX1–4). Both enzymes AOX and XOR are able to generate besides ROS also nitric oxide (NO) (see Maia and Moura 2018; Bender and Schwarz 2018). Under normal culturing conditions of adipocytes, AOX localizes in the cytoplasm of cells (Fig. 6b–d). XOR is also localized soluble in epithelial cells of the mammary gland (Bruder et al. 1984). In lactating mammary gland epithelial cells, XOR is binding additionally the LD protein PLIN2 and supporting with LDs docking to the apical plasma membrane via BTN. XOR is clustering BTN within the apical membrane and regulates milk lipid secretion into milk ducts (Mather and Keenan 1998; Jeong et al. 2009). This clustering was shown also with mammary specific XOR knockout mice, where XOR was found to make the secretion of LDs highly efficient (Monks et al. 2016). Knowing that a distinct combination of perilipins and molybdo-flavoenzymes—PLIN2 and XOR—is responsible for delivering/transporting LDs to the apical plasma membrane in the lactating mammary gland, we speculate that such a similar tethering system might also exist within adipocytes under starvation condition (Fig. 7). LD trafficking to mitochondria in starved mouse embryonic fibroblasts cells (MEFs) was described recently (Rambold et al. 2015) and thus, we propose for adipocytes a combination of PLIN1 and AOX as a tether for such linkages (Figs. 4 and 7). Adipose triglyceride lipase (ATGL) might also be involved for direct, immediate release of fatty acids (FAs) at the LD-mitochondria interface (Smirnova et al. 2006; Granneman and Moore 2008; Haemmerle et al. 2011; Walch et al. 2015). This assumption awaits verification but some arguments in favor are given by a report with experiments using Saccharomyces cerevisiae (see below; Pu et al. 2011). Direct delivery of FAs from the storage organelle to the degradation/β-oxidation organelle would be a highly efficient way to prevent otherwise cytotoxic FA effects in the cytosol. Whether acyl-CoA synthetases (ACSLs) and carnitine palmitoyltransferases (CPTs) (Young et al. 2018; Coleman 2019) play a role as a docking station of outer mitochondria membranes (OMMs) remains to be established. The same holds true also for synaptosomal-associated protein 23 (SNAP23) reported for interaction of LDs and mitochondria (Jagerstrom et al. 2009; Strauss et al. 2016).
LD binding protein PLIN5 of muscle cells is a major player in recruiting LDs to mitochondria (Wang et al. 2011; Andersson et al. 2017; Pribasnig et al. 2018; Gemmink et al. 2018). AOX might be the possible oxidase partner participating together with PLIN5 in linking of LDs to mitochondria in myocytes, because XOR is localized only in capillary endothelial cells of heart and muscle tissue (Jarasch et al. 1981; Jarasch et al. 1986) and AOX is found during myogenesis in the mouse skeletal myoblast cell line C2C12 (Kamli et al. 2014). A summary of the combinations for perilipins and oxidases tethering LDs to specific membranes is given in Table 1.
Localization of XOR and AOX in cells and tissues
A surprising finding of our study is the recognition of a complimentary, mutually exclusive localization of the molybdo-flavoenzymes XOR and AOX in diverse cell types (Table 2). XOR expression has so far only been reported on the protein level solely in epithelial cells of lactating mammary gland and in capillary endothelial cells of small blood vessels (Jarasch et al. 1981; Jarasch et al. 1986; Bruder et al. 1984). For clinical aspects of endothelial XOR in reperfusion injury and inflammatory signal transduction, see Meneshian and Bulkley (2002). For therapeutic effects of XOR inhibitors in a comprehensive compilation of clinical studies, see Pacher et al. (2006). The many discrepancies currently found in the literature for XOR originate mainly from incorrect addressing and clear discrimination between the two enzymes XOR and AOX. With the knowledge of source and localization of the two enzymes by using proper antibodies, the measurements on purine metabolism—analyzing hypoxanthine and uric acid in blood and urine—have to be credited in most cases rather to AOX than to XOR.
Interestingly, Williams et al. (2014) requested that hundreds of studies using high doses of allopurinol claimed as XO inhibitors should be revisited. Of course, these corrections should be extended also for the localization and antibody-based publications on XOR and AOX. Starting with publicly available reports on the localization of the enzymes (Jarasch et al. 1986; Moriwaki et al. 2001; Weigert et al. 2008), we compared both enzymes (Table 2). Our results now highlight that XOR is not an ubiquitously and constitutively expressed metallo-flavoprotein as stated in most current XOR publications.
Other functions than housekeeping
XOR and AOX; both enzymes can activate lipid peroxidation. This process generates naturally ROS on a small level in the body. Thereby, polyunsaturated FAs of membranes are attacked and a chain reaction is started. When the antioxidant defense mechanism is overcome, great damage and destruction of membrane lipids could take place. ROS production can change membrane properties and alter membrane deformability.
XOR has been described as a housekeeping protein with enzymatic functions in purine catabolism and additionally in a variety of other roles. The enzyme has a protective and antimicrobial defense mechanism, can participate in wound healing through effects on ROS production and is supposed to be part of an innate immune system (Jarasch et al. 1986; Vorbach et al. 2002; Vorbach et al. 2003; Vorbach et al. 2006; Madigan et al. 2015). Mouse XOR gene knockout (Vorbach et al. 2002; Ohtsubo et al. 2004; Ohtsubo et al. 2009; Murakami et al. 2014) and mouse isoenzyme AOX4 knockout studies are beyond the scope of this manuscript and have to be discussed separately. Besides ROS, the generation of NO by the two enzymes, XOR and AOX, should be emphasized (Lundberg et al. 2011; Millar et al. 1998). With its localization in capillary endothelial cells, XOR is a major player and in a key position to control vasodilatation of small vessels by NO and inflammation events by ROS. Blood pressure lowering can also be achieved by allopurinol, an inhibitor of both enzymes, XOR and AOX. This drug is mostly prescribed in gout disease for the reduction of uric acid formation. Using a large clinical data collection of adults with hypertension, allopurinol-treated patients were associated with a significantly lower risk of both stroke and cardiac events than with non-exposed patients (MacIsaac et al. 2016).
In addition to these multiple activities and roles of XOR and AOX, we propose completely new functions, tethering of LDs to biomembranes in conjunction with specific perlipins (Table 1).
Pu et al. (2011) investigated the interaction between LDs and mitochondria in S. cerevisiae. Using a bimolecular fluorescence complementation assay, they identified an LD protein named Erg6 that can link to mitochondria. Erg6 is described as a regulator of membrane permeability and fluidity (Gaber et al. 1989). With additional Erg6 binding assays, Pu and coworkers came up with other associated LD proteins. Identified were proteins with sterol esterase activity (Yeh1), with triglyceride lipase activity (Tgl3, Tgl4) and with oxidoreductase activity (Yor246c). With lipases and the oxidoreductase binding on LDs and Erg6 linking to mitochondria, they find—at least viewed from functional aspects—a very similar set of proteins in S. cerevisiae as we now propose for human adipocytes. Since early in evolution an oxidoreductase enzyme seems to be important for docking and locally changing membrane properties for proper channeling of lipid products.
We greatly appreciate Werner W. Franke and Thomas W. Keenan for their continuous generous support and advice for more than 40 years. We especially thank Stefanie Winter, Caecilia Kuhn, Heiderose Schumacher and Edeltraut Noffz (Helmholtz Group for Cell Biology, DKFZ) for valuable help with biochemical characterizations and cell culture work. Special acknowledgement goes to Andrea Janicová (Department Biology, Friedrich-Alexander University of Erlangen-Nuremberg, Germany) for experiments in the framework of her Master Thesis at the Helmholtz Group for Cell Biology in Heidelberg. Martina Schnoelzer and Uwe Warnken (Functional Proteome Analysis, DKFZ) are gratefully acknowledged for proteomic analysis. We thank Ian Mather (University of Maryland, College Park, MD, USA) for his supply of one XOR antibody and Nicole B. (Heidelberg) for her generous supply of milk used for the isolation of milk lipid globule membranes.
Conceived and designed the experiments: HH. Performed the experiments: HH, RZ, YD and SR. Analyzed the data: HH and SR. Wrote the paper: HH and SR. Drawings and finalizing figures: HH and RZ.
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