Received: 22 July 2011, Revised: 29 August 2011, Accepted: 1 September 2011 Published online in Wiley Online Library: 11 November 2011
(wileyonlinelibrary.com) DOI 10.1002/jat.1752
418
Effect of lipopolysaccharide on alterationof phospholipids and their fatty acidcomposition in spleen and thymus by in vitrometabolic labelingJayaraja Sabarirajan,a Panneerselvam Vijayaraj,a
*Correspondence to: V. Nachiappan, Department of Biochemistry, School of LifeSciences, Bharathidasan University, Tiruchirappalli 620024., Tamilnadu, India.E-mail: [email protected]
aBiomembrane Laboratory, Department of Biochemistry, School of Life Sciences,Bharathidasan University, Tiruchirappalli 620024, India
bBiochemistry Department, Indian Institute of Science, Bangalore 560012, India
INTRODUCTIONThe mammalian immune system is a cooperative venturebetween innate and acquired arms, offers an optimal envi-ronment for defense against the invasion of pathogens atany site in the body (Mebius and Kraal, 2005). The thymusis one of the primary central lymphoid organs and plays animportant role in cellular immunity by generating circulatingT lymphocytes (Hong, 2001). The spleen is a secondary lym-phoid organ that plays an essential role in the primarydefense against all types of antigens that emerge in the cir-culation and is a major site of antibody production (Swirskiet al., 2009; Bohnsack and Brown, 1986). Lack of the spleencauses an augmented susceptibility to systemic infectionsby encapsulated bacteria (Engelhardt et al., 2009).
Phospholipids (PLs) are major components of cellular mem-branes that take part in a series of metabolic events includingcellular permeability, regulation of proteins associated withthe membrane and regulation of intracellular signaling byproviding signaling molecules (Yamashita et al., 1997; Exton,1994). Alterations in phospholipid (PL) composition can leadto modifications in membrane integrity, permeability, cellinjury and intracellular membrane trafficking that eventuallycan lead to death (Stenger et al., 2009; Yorio and Frazier1990).
Fatty acids can trigger membrane enzymes, channels andtransporters by direct interaction with proteins, by altering the
interaction with the lipid bilayer or indirectly by metabolic con-version of arachidonic acid (AA) to oxygenated metabolites(Ordway et al., 1991).
Lipopolysaccharide (LPS) is an endotoxin and a powerfulactivator in the immune system (Niehaus and Lange, 2003).It consists of lipid A, an inner core region and the O-specificside chain. LPS is a potent inducer for acute respiratorydistress syndrome (ARDS). Our earlier study has shown multi-ple organ dysfunctions caused by LPS (Sabarirajan et al.,2010). The immunological effect of LPS on thymus andspleen is well established, but the role of phospholipidsduring endotoxemia is yet to be elucidated. Hence elucidat-ing the role of phospholipid may provide a better under-standing of the relationship between fatty acid alterationsand immune impairment. The present study clearly showsthe influence of LPS on spleen and thymus phospholipidand their fatty acid composition.
MATERIALSLipopolysaccharide (Escherichia coli, serotype 055:B5) andheptadecanoic acid were obtained from Sigma ChemicalCompany (St Louis, MO, USA). Radioactive work was carriedout at the Biochemistry Department, Indian Institute of Sci-ence, Bangalore. Silica gel 60F254 thin layer chromatography(TLC) plates were from Merck. All chemicals and solventswere purchased from Sigma unless specifically mentioned.
Animals
Healthy male albino Wistar rats (200–250 g body weight) wereobtained from the Indian Institute of Science, Bangalore andhoused at the Central Animal Facility, Bharathidasan University.The rats were fed on pellet diet (Sree Sai Durga Feeds & FoodsPvt. Ltd, Bangalore) and water ad libitum. Animals were kept un-der conventional housing conditions (22 �C, 55% humidity, and12 h day/night cycle). The acclimatization period was at least7 days before use. LPS was dissolved in saline (phosphate freemedium) and kept at 4 �C until use. All procedures were ap-proved and complied with the standards for the care and useof animal subjects as stated in the guidelines laid down by theInstitutional Animal Ethical Committee, Bharathidasan Univer-sity, Tiruchirappalli, Tamilnadu, India.
Preparation of Tissue Slices
Animals (n=6) were anesthetized using ketamine (50mg kg�1)intraperitoneally and killed by cervical dislocation; thymus andspleen were quickly excised and kept on ice, chopped into smallslices (0.7mm) using a tissue chopper. Tissue slices of 25mgwere used for in vitro lipid labeling experiments. The spleentissue of the animals were processed separately, whereasthymus tissues (n= 6) were pooled due to minimal in size(~100–150mg). Three independent experiments were carriedout for each analysis.
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In vitro [32P]orthophosphate Metabolic Labeling ofTissue Phospholipids
[32P]orthophosphate labeling is the best tool to study thephospholipid metabolism (Dawson, 1954; Marinetti et al.,1958). In this present study, spleen and thymus PL alterationwas studied by [32P]orthophosphate labeling in the presenceor absence of LPS. Alterations were monitored by three dif-ferent sets: (i) PLs were labeled along with the compound;(ii) tissues were pre-incubated with the compound followedby PL labeling; and (iii) tissues were labeled with [32P]ortho-phosphate and post-incubated with LPS.
The tissue slices (~25mg) were incubated at 37 �C with 5mCiof [32P]orthophosphate (5000Cimmol�1) in the presence or ab-sence of LPS in phosphate-free medium. The final volume of thereaction mixture was 100μl. The effective concentration of LPSon PLs were studied using different concentrations of LPS rang-ing from 0 to 100μg (0, 12.5, 25, 50 and 100μg) and the maxi-mum incorporation of [32P]orthophosphate was monitored byincubation of tissue slices with the presence or absence of LPSat different time points (0, 0.5, 1, 2 and 4 h). After incubation
unused [32P]orthophosphate was removed by washing withsaline and subjected to lipid extraction (Folch et al., 1957).For pre-incubation, tissue slices (spleen and thymus) were
pre-incubated in the presence or absence of LPS for 1 h at37 �C in phosphate-free medium followed by [32P]orthophos-phate labeling.In post labeling, tissue slices were first labeled with [32P]
and unused label was removed by washing with saline thenpost-incubated with the presence or absence of LPS for 1 hat 37 �C. The reaction was stopped by the addition of 2:1(v/v) chloroform–methanol mixture and lipids were extracted(Folch et al., 1957).
Extraction of Lipids
Lipids were extracted according to Folch et al. (1957). Briefly,chloroform–methanol (2:1, v/v) mixture was added to stop thereaction, vortexed well and centrifuged at 10 000 rpm for2min. The lower organic phase (containing lipids) was trans-ferred into a fresh tube. The entire organic layer was pooledand washed with acidified water (2% orthophosphoric acid).Centrifugation was performed and the organic layer was driedunder a vacuum concentrator. Then the lipids were resuspendedin a minimal volume of 2:1 (v/v) chloroform–methanol mixtureand loaded onto 2D-TLC.
Two-dimensional TLC and autoradiography
Lipids were separated by 2D-TLC using chloroform–methanol–ammonia (65:35:5, v/v) as the first-dimensional solventsystem and chloroform–methanol–acetone–acetic acid–water(50:10:20:15:5, v/v) as the second-dimensional solvent system. In-dividual PL were identified by comparing the Rf values of theunknowns with the Rf values of the standards. The TLC plates weresubjected to autoradiography; PL spots were scrapped andcounted with toluene-based scintillation cocktail using a liquidscintillation counter (Perkin Elmer, Life Sciences).
Fatty Acid Analysis
Fatty acid analysis was performed in altered PL of spleen andthymus which was subjected to LPS treatment in cold condi-tions. The PL spots were scraped from the TLC plate andextracted from silica gel with chloroform–methanol (2:1, v/v)mixture. To the dried lipid 100 μg heptadecanoic acid wasadded as an internal fatty acid standard and methylated withBF3-methanol (14%) to convert it into the correspondingmethyl esters. The fatty acid methyl esters were analyzedby GC/MS (Morrison and Smith, 1964).
Statistical Analysis
Statistical analysis was performed using one-way analysis of var-iance (ANOVA) and a least significant difference (LSD) post hoctest and sample-t test were used to compare individual means(SPSS for Windows 11.5; SPSS Inc., Chicago, IL, USA). The resultswere expressed as the means� SD of three independent exper-imental values and a statistical probability of P< 0.05 andP< 0.001 was considered to be significant.
RESULTS AND DISCUSSIONLPS is a potent stimulator of immune response and has a defi-nite impact on lymphoid organs like spleen and thymus. LPSenters the lipid layer of biological membranes and modifiesthe phospholipid (Rothfield and Romeo, 1971). PLs are impor-tant components of the cell membranes of all living species.They contribute to the physicochemical properties of the mem-brane and thus influence the conformation and function ofmembrane-bound proteins such as receptors, ion channels andtransporters, and also influence cell function by serving as pre-cursors for prostaglandins and other signaling molecules andmodulating gene expression through the transcription activa-tion (Gimenez et al., 2011). In PLs, biosynthesis of phosphatidyl-choline (PC) and phosphatidylethanolamine (PE) occurs fromdiacylglycerol (DAG) or conversion of PE to phosphatidylserine(PS) and PC to sphingomyelin (SM) (Van Den Bosch, 1974). How-ever very little is known about PL metabolism during endotoxe-mic conditions in spleen and thymus. In the current study, PLalteration was monitored by [32P]orthophosphate labeling inspleen and thymus in the presence or absence of LPS. The de-crease in PL observed may have been due either to decreasedsynthesis or increased degradation and was measured usingpre- and post-incubation studies with LPS.
LPS Alters the Phospholipids of Spleen and Thymus Tissues
In the present study, spleen and thymus were incubated at 37 �Cfor different time intervals (0–4 h) and the maximum reductionwas seen with 2 and 4 h for spleen and thymus, respectively. In-terestingly, LPS treatment in spleen and thymus (in vitro)showed a significant decrease in phospholipid contents andthe reductions were concentration-dependent (Fig. 1A). Inspleen the maximum reduction was observed in PC (~52%), fol-lowed by phosphatidylglycerol (PG; ~40%) with 50μg of LPStreatment, whereas in thymus PC alone showed ~65% reduction(Fig. 2A). Figures 1(B) and 2(B) depict time-dependent reductionof PL and maximun reduction was observed at 50μg concentra-tion after 2 and 4 h incubation periods with spleen and thymus.From the labeling experiments, we showed that 50 μg LPS has asignificant inhibitory effect on the PLs in spleen and a profoundeffect with major phospholipids, PC and PG, when comparedwith the control. All other PLs, such as PE, phosphatidic acid(PA), PS, phosphatidylinositol (PI) and lysophosphatidylcholine(LPC), did not show much alteration. In thymus LPS also showedsignificant (P< 0.001) reduction in PC and not much alteration inother PLs. Hence these concentrations and time points wereused for further studies.
The turnover of PC has been reported to increase instantlyafter the stimulation with lectins; a mitogen and transient ac-cumulation of LPC was observed (Shier et al., 1976). In thepresent study, LPS-treated spleen and thymus showed signif-icant (P< 0.001) depletion in PC but no accumulation of LPC.PC is an essential phospholipid in mammalian cells andtissues. It functions as a primary lipid in cellular membranesand is a precursor of signaling molecules. PC serves as a di-rect substrate for sphingomyelin synthesis via sphingomyleinsynthases (Li and Vance, 2008), a major source for secondmessengers (Li and Vance, 2008). PC has several known met-abolic pathways and plays a critical role in membrane struc-ture (Cui and Houweling, 2002; DeLong et al., 1999). Anextensive network of phospholipases, acyltransferases and
other metabolizing enzymes makes PC the source of aunique group of signaling mediators. PA, DAG, LPC and AAare signaling molecules generated from PC (Exton, 1994;Billah and Anthes, 1990). Alterations in PC homeostasis canoccur during many pathophysiological conditions (Whiteand McCubrey, 2001; Mattson, 2000). Perturbation of PC ho-meostasis in mammalian cells may lead to cell death. (Exton,1994; Billah and Anthes, 1990; Kiss, 1990; Kester et al., 1989).
PG is an anionic minor phospholipid, at ~2% in most ani-mal tissues, but is the second most abundant phospholipid(~10–15%) in lung surfactant (in a few species, it is replacedby another acidic lipid, PI). It is well established that the con-centration of PG increases during fetal development. PG is aprecursor for cardiolipin (CL), an important component of the in-ner mitochondrial membrane, and is found almost exclusively inthe inner mitochondrial membrane, where it is essential for theoptimal function of numerous enzymes that are involved in mito-chondrial energy metabolism (Schlame et al., 2000). The labelingstudies showed a marked impact on the PC and PG of spleen andPC of thymus upon LPS treatment.
To further understand the effect of LPS on PL metabolism weperformed pre-incubation and post-incubation studies to deter-mine the changes in PL synthesis and degradation. Figures 3(A)and 4(A) depict the pre-incubated labeling of spleen and thymustissues with LPS. Incorporation of [32P]orthophosphate in spleenwas significantly diminished in PC (~87%) followed by PG(~44%), whereas in thymus PC was significantly depleted (~70%)when compared with their respective controls. Post-incubationalso decreased the phospholipid content. Figures 3(B) and 4(B) de-pict the labeled phospholipid post-incubated with LPS. In spleen,significant (P< 0.001) reductions in PC (~57%), PG (~62%) andLPC (~74%) were observed. In thymus, reductions in PC (~76%)and LPC (~8%) were also observed. A profound inhibitory effectwas observed in PC and PG in spleen upon both pre- and post-incubation with LPS. Thymus also showed a significant (P< 0.001)decrease in the PC level upon both pre- and post-incubation withLPS. From these labeling results, it is clear that LPS inducessignificant alterations in PC and PG of spleen and PC of thymus.
In the present study, the pattern of PLs in the presence ofcompound was the focus. Reduction in PL level was observedduring both pre- and post-incubation treatments and the func-tion of PLs depended on their fatty acid (FA) composition. TheFA molecular species analysis of post-incubated spleen PC, PGand thymus PC did not show much alteration (data not shown),whereas both spleen and thymus PL showed significant changesin their FA composition when treated with LPS (Tables 1–3). Tocheck whether the alteration of PLs is attributed to FA alterationof the membrane, we analyzed the PL fatty acid profile usingGC/MS. It is generally accepted that biomembrane compositionis altered during nutritional, environmental or xenobiotic expo-sure (Yorio and Frazier, 1990).
Fatty Acid Composition of LPS-treated Spleen andThymus PLs
The FA species composition has a genetic component asevidenced by organ- and tissue-specific molecular speciespatterns (Ordway et al., 1991). Biological tissues seem to have adegree of freedom within which an oscillation in molecular spe-cies composition is allowed. Beyond certain threshold values,the organ may be incapable of functioning in an optimal way.
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Figure 1. Metabolic labeling of spleen tissue phospholipids. Spleen tissues (25mg) and 5 μCi of [32P]orthophosphate were incubated with differentconcentration (0–100μg) of lipopolysaccharide (LPS) for 4 h at 37 �C (A), treated with different time intervals (0–4 h) in the presence of saline/effectiveconcentration of LPS at 37 �C (B). Lipids were extracted and separated by two-dimensional silica TLC using chloroform–methanol–ammonia–water(70:30:2:3, v/v) as the first-dimension and chloroform–methanol–water (65:35:5, v/v) as the second-dimension solvent systems. Lipids were identifiedby comparison with known standards. The amounts of [32P]orthophosphate incorporated into lipids are presented as cpm per 25 mg of tissue. Valuesare the means of three separate experiments. Data are means� SD *P< 0.001 compared with control.
Figure 2. Metabolic labeling of thymus phospholipids. Thymus tissues (25 mg) and 5 μCi of [32P]orthophosphate were treated with different concen-trations (0–100 μg) of lipopolysaccharide (LPS) for 4 h at 37 �C and quantified (A). Tissues were incubated with different time intervals (0–4 h) in thepresence of saline/effective concentration of LPS at 37 �C (B). Lipids were extracted and separated by two-dimensional silica TLC using chloroform–methanol–ammonia–water (70:30:2:3, v/v) as the first-dimension and chloroform–methanol–water (65:35:5, v/v) as the second-dimension solvent sys-tems. Lipids were identified by comparison with known standard. The amounts of [32P]orthophosphate incorporated into lipids are presented as cpmper 25mg of tissue. Values are the means of three separate experiments. Data are means� SD; *P < 0.001 compared with control.
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Interestingly in spleen PC, arachidonic acid (C20:4) was in-creased ~5.37-fold with LPS (Table 1), whereas palmitic (~51%)and stearic acids (~100%) were decreased. The ratio betweenunsaturated fatty acids (UFA) and saturated fatty acids (SFA) ofspleen PC was 0.44 and 3.57, respectively for control and LPS-treated. FA molecular species of spleen PG showed ~5-foldincrease in UFA content, and ~20-fold increase in linoleic acidwhen compared with the control (Table 2), whereas palmitic(~34%) and stearic acids (~32%) were decreased. In LPS treat-ment, the UFA/SFA of spleen PG was 0.078 for control andincreased to 0.598 during LPS treatment.
It has been reported that SFA and UFA are also affected in lym-phocytes during both in vivo and in vitro conditions (Erickson,1986; Gurr, 1983; Meade and Mertin, 1978). The mitogenic stimula-tion of lymphocytes is related to a decrease in the proportions ofpalmitic (C16:0), stearic (C18:0), linoleic (C18:2) and arachidonic(C20:4) acids and an increase in the proportion of oleic acid(C16:1; Calder et al., 1994). Fascinatingly, in this present study, treat-ment with LPS showed amarked enhancement of C20:4 (5.37-fold)and a decrease in C18:0 and C16:0. Earlier reports showed that fattyacidmolecular species alteration of PC directly reflects the immunefunction via the fatty acid remodeling pathway (Gurr, 1983).
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Figure 3. Pre- and post-incubation of spleen tissue phospholipids withlipopolysaccharide (LPS). Spleen tissues (25mg) were pre-incubated for1 h at 37 �C with saline or with 50 μg of LPS followed by 2 h incubationwith 5μCi of [32P]orthophosphate. (A) Amount of [32P]orthophosphateincorporated into various phospholipids. [32P]Orthophosphate-labeledspleen tissues were post-incubated for 1 h at 37 �C either alone or to-gether with 50 μg LPS. (B) Amount of [32P]orthophosphate after post-incubation. Lipids were extracted and separated by two-dimensionalsilica TLC by chloroform–methanol–ammonia–water (70:30:2:3, v/v) asthe first-dimension and chloroform–methanol–water (65:35:5, v/v) asthe second-dimension solvent systems. Lipids were identified bycomparing with known standards. The amounts of [32P]orthophosphateincorporated into lipids are presented as cpm per 25mg of tissue after2 h of labeling. Values are the means of three separate experiments. Dataare means� SD; *P < 0.001 compared with control.
Figure 4. Pre- and post-incubation of thymus tissue phospholipids withlipopolysaccharide (LPS). Thymus tissues (25mg) were pre-incubated for1 h at 37 �C either with saline or together with 50 μg of LPS followed by4 h incubation with 5 μCi of [32P]orthophosphate. (A) Amount of [32P]or-thophosphate incorporated into various phospholipids after pre-incubation with LPS followed by [32P]-labeling of thymus tissues.[32P]Orthophosphate-labeled thymus tissues (25mg) were post-incubated for 1 h at 37 �C either alone or together with 50μg LPS.(B) Amount of [32P]orthophosphate after post-incubation. Lipids wereextracted and separated by two-dimensional silica TLC by chloroform–methanol–ammonia–water (70:30:2:3, v/v) as the first-dimension andchloroform–methanol–water (65:35:5, v/v) as the second-dimensionsolvent systems. Lipids were identified by comparison with knownstandards. The amounts of [32P]orthophosphate incorporated into lipidsare presented as cpm per 25mg of tissue after 4h of labeling. Valuesare the means of three separate experiments. Data are means� SD;*P < 0.001 compared with control.
Impact of LPS on spleen and thymus phospholipids
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Conjugated linoleic acid has been reported to modulate theimmune response, modify cytokine production and increaselymphocyte proliferation in in vitro animal studies (Yang andCook, 2003; Hayek et al., 1999; Turek et al., 1998; Chew et al.,1997). Importantly in this study, spleen PG had a marked enrich-ment in linoleic acid (~20-fold), which is a precursor for arachi-donic acid.
When thymus tissue was exposed to LPS, the FA molecularspecies of PC showed significant (P< 0.001) increase, especiallyin SFA (Table 3). Stearic acid (C18:0) was drastically increasedby ~32% during LPS exposure and palmitic acid (C16:0) was de-creased. Interestingly, upon treatment with LPS, thymus PCshowed a reduction in linoleic acid (C18:2) along with significantincrease in AA (~2.4-fold). The ratio of UFA/SFA decreased from0.302 to 0.159. These results clearly showed that LPS has astrong impact on thymus PC. Two polyunsaturated fatty acids(PUFAs), linoleic acid (18:2n-6) and a-linolenic (18:3n-3), whichcannot be synthesized de novo by human cells, are essentialfatty acids and there are a series of saturation, desaturationand elongations steps leading to the formation of long-chainPUFAs, arachidonic acid (20:4 n-6), which along with otherPUFAs (including some n-3 fatty acids) can serve as the
precursors for mediators of inflammation (prostaglandins,thromboxanes and leukotrienes; Siddiqui et al., 2004; Lee andAusten, 1986; Smith et al., 1985).It has been reported that SFA and UFA markedly affect lym-
phocyte function during both in vivo and in vitro conditions(Erickson, 1986; Gurr, 1983; Meade and Mertin, 1978). Studiescarried out with LPS in in vivo (Bennett et al., 1987; Erickson,1986; Erickson et al., 1980) and in vitro conditions (Pourbohloulet al., 1985; Buttke, 1984) have revealed that fatty acid alterationsand have a greater effect on T cells and cell-mediated immunitythan on B cells and humoral immunity. Alteration in fatty acidcomposition occurs during the pathophysiology of many dis-eases with major changes in PUFA content (Peet et al., 1998;Maes et al., 1996, 1997). Polyenoic acid content was increasedduring in vitro and in vivo stimulation. Thus the ratio of polyenoicto SFA in position 2 of rabbit lymphocyte PC increased duringin vitro stimulation from 0.538 to 0.926. It is reasonable to guessthat this increase in the content of polyenoic acids during
Spleen tissue was treated with 50μg lipopolysaccharide(LPS) in a 37 �C water bath for 2 h and lipids were extractedand resolved by 2D-TLC. The major spleen phospholipid PCwas extracted from silica plates. A 100 μg aliquot of heptade-canoic acid was added as the internal fatty acid standard andmethylated. The fatty acid methyl esters were analyzed byGC/MS. Each data point represents the mean of three inde-pendent experiments� SD.*P< 0.001, $P< 0.05 against control was considered to besignificant.
Table 3. Fatty acid molecular species analysis of thymusphosphatidylcholine
Thymus tissue was treated with 50 μg lipopolysaccharide(LPS) in a 37 �C water bath for 4 h in cold conditions andlipids were extracted and separated by 2D-TLC. The controland LPS-treated thymus PC was methylated by BF3–methanolfor the conversion of volatile fatty acid methyl esters(FAME). The FAME were analyzed by GC/MS. The fatty acidswere quantified using internal standard heptadecanoicmethyl ester. Each data point represents the mean of threeindependent experiments� SD.*P< 0.001, $P< 0.05 against control was considered to besignificant.
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stimulation leads to a higher fluidity of cellular membranes(Ferber et al., 1975).
The increased content of polyenoic acids in PLs appears to bethe outcome of redistribution of their fatty acid moieties ratherthan de novo synthesis of the whole molecule. A change in fattyacid composition helps adaptation in organisms. The FA chain
Table 2. Fatty acid molecular species analysis of spleenphosphatidylglycerol
The spleen phosphatidylglycerol was re-extracted from silicaplates and methylated by BF3–methanol for the conversionof volatile fatty acid methyl esters (FAME).The FAME were an-alyzed by GC/MS. Each data point represents the mean ofthree independent experiments� SD.*P< 0.001 against control was considered to be significant.
length and number of double bonds in fatty acids mainly regu-late the membrane fluidity (Ferber et al., 1975). The presentstudy shows the PC of spleen to increase the ratio betweenUFA, SFA from 0.441 to 3.566, whereas in thymus it is reversed(0.302–0.159). Increased unsaturated fatty acid in spleen mightbe due to the sensitivity of LPS and decrease in thymus mightbe due to adaptation of the organs.
In summary, to understand the spleen and thymus phospho-lipid metabolism during LPS endotoxemia in vitro, we studiedrat spleen and thymus tissue with [32P]orthophosphate labeling.For the first time we report here that LPS specifically alters themajor structural phospholipids, PC and PG, of spleen and PC ofthymus tissue. The fatty acid analysis showed marked alterationof UFA and SFA of spleen PC and PG and thymus PC. We believethat these in vitro lipid metabolic results could open up new vis-tas for exploring LPS-induced immune impairment in spleen andthymus, as well as the underlying mechanism.
Acknowledgement
The financial support fromBharathidasan University, Tiruchirappalli,India is gratefully acknowledged. We also acknowledge Depart-ment of Biochemistry, Indian Institute of Science, Bangalore,India for providing the radioactive material and also helpingus to conduct the radioactive study. JS is a senior research fel-low of Council of Scientific and Industrial Research, India.
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