Biochemistry Laboratory Isolation and Characterization of Non-Saponifiable and Saponifiable Complex Lipids from Calf’s C alf’s Brain Dumo, Ephraim D., Emmanuel, Dave R.*, Ermino, Mia Joy DV., Espino, Audrey Joan . !olle"e o# $cience, %niversity o# $anto omas, Espana Blvd., Manila Abstract Lipids are biomolecules that are hydrophobic in nature, relatively insoluble in &ater and soluble in nonpolar solvents. Lipids can be broadly subdivided into t&o cate"ories' (on)$aponi#iable and $aponi#iable Lipids. (on)$aponi#iable (on)$aponi#iable lipids are those types o# lipids that cannot be hydrolyed, due to the absence o# an ester "roup. +n the other hand, saponi#iable lipids are those that can be easily h ydrolyed and those "enerally have ester lina"es. -n this eperiment, non)saponi#iable and saponi#iable comple lipids &ere isolated #rom cal#/s brain &ith the use o# the solubility in various chemical compositions o# the lipids. he comple lipids &ere o btained, namely' cholesterol, "lycerophosphatide, sphin"osine phosphatide and sphin"osine "lycoside. he isolated comple lipids &ere then characteried into various chemical color reaction tests in order to analye the composition o# each lipid. -solated comple lipids &ere then compared to the standards that &ere analyed and characteried also into di##erent chemical tests.
Introduction
Lipids Lipids are "roup "roup o# biolo biolo"ic "ical al molec molecul ules es that that conta contain in lon") lon")ch chain ain aliph aliphati atic c hydrocarbons hydrocarbons and their derivatives. Lipids may be "rouped into t&o main classes' (on) $aponi#iable lipids, lipids that cannot be broen up into smaller units since it does not react &ith &ater, &hich includes steroids and prosta"landins, &hile $aponi#iable lipids, those that may be converted into smaller molecules &hen hydrolysis occurs, and can then be #urther classi#ied into simple lipids &hich includes tri"lycerides and &aes and comple lipids that are phospho"lycerides and sphin"olipids. Basically, simple lipids contain 0ust t&o types o# components, &hereas comple lipids contain more than t&o. hre hree e stan standa dard rds s &ere &ere used used in the the epe eperi rime ment nt'' chol choles este tero rol, l, leci lecith thin in and and "alactoc "alactocereb erebros roside. ide. !holest !holestero eroll is placed placed under under nonsap nonsaponi# oni#iab iable le lipids lipids and can be 1|Page
Biochemistry Laboratory #urther classi#ied into the steroid cate"ory. Also, it is the precursor #or all the other important steroids o# mammalian metabolism and its chemical structure &ith the three si)membered rin"s and sin"le #ive)membered rin" #used to"ether servin" as the basic structure o# a steroid. 1!abatit, 23456 Lecithin or other&ise no&n as phosphatidyl choline, is placed under saponi#iable lipids and #urther classi#ied as a phospho"lyceride. -t is made #rom the "lycerol bacbone, t&o #atty acids, and a phosphoryl ester. 7urthermore, it contains t&o #atty acids and phosphoric acid that are esteri#ied to three hydroyl "roups o# "lycerol, &hich is the basis o# a phospho"lyceride. Lastly, "alactocerebroside, the last standard used, may also be #ound on the cell membranes o# the brain and is placed in saponi#iable lipids in the sphin"olipid "roup. $phin"olipids do not contain "lycerol but rather sphin"osine as its bacbone and it is a type o# cerebroside consistin" o# a ceramide &ith a "alactose residue at the 2)hydroyl moiety and the "alactose is cleaved by "alactosylceramidase . 1$&iter, 23336 he
lipids
that
&ere
etracted
in
the
eperiment
&ere
cholesterol,
"lycerophosphatides and sphin"osines. !holesterol is a steroid8 "lycerophosphatides is a phospho"lyceride, &hile sphin"osine phosphatides and sphin"osine "lycosides are sphin"olipids. -solation o# saponi#iable and non)saponi#iable lipids #rom cal#/s brain and various 9ualitative tests #or #urther characteriation &ere conducted to satis#y the main ob0ective o# ac9uirin" valid presumptions about the composition o# these comple lipids.
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Biochemistry Laboratory Methodolog
-n the isolation o# non)saponi#iable and saponi#iable lipids, cal#/ brain &as used in order to etract the three comple lipids that is needed #or the characteriin" into di##erent chemical test. 7or the isolation o# cholesterol, the isolated :;)<= " o# cal#/s brain &as triturated &ith := ml acetone usin" sand to aid in the disruption o# the cells. he miture &as trans#erred to a 2:; ml Erlenmeyer #las and &as added &ith ;= ml acetone. !or stopper &as used to cover the mouth o# the #las. he miture &as allo&ed to stand overni"ht in the re#ri"erator. he etract &as #iltered and the residue &as &ashed &ith 2; ml o# acetone. he residue &as saved #or the preparation o# "lycerophosphatides, sphin"osine phosphatides and sphin"osine "lycosides. he #iltrate &as evaporated to 2= ml over a steam bath inside the #ume hood, and the evaporatin" dish &as cooled over an ice bath. he crude cholesterol &as #iltered and collected. hen, the crude cholesterol &as recrystallied by dissolvin" it in a minimal volume o# :)< ml o# hot ethanol. the solution &as #iltered &hile it is hot and it &as allo&ed to cool. he recrystallied cholesterol &as collected by decantation and &as dissolve in a < ml o# methanol'chloro#orm miture &ith a ratio o# 2'<. -n the isolation o# "lycerophosphatide, the residue that &as le#t #rom the isolation o# cholesterol that &as done be#orehand &as trans#erred to a 2== ml beaer and it &as etracted &ith := ml heane or petroleum ether. he beaer &as covered &ith a &atch "lass. -t &as allo&ed to stand #or <= minutes &ith occasional stirrin". he product &as #iltered and the residue &as saved #or the isolation o# sphin"osine phosphatides and sphin"osine "lycosides. he etract &as concentrated over a steam bath inside the #ume hood. he concentrated etract &as poured into := ml o# acetone and &as stirred. 3|Page
Biochemistry Laboratory he precipitate &as #iltered and dissolved it at once in a < ml o# methanol'chloro#orm miture. Lastly #or the isolation o# sphin"osine phosphatide and sphin"osine "lycosides, the residue #rom the isolation o# "lycerophosphatide that &as done be#ore &as obtained and trans#erred to a 2== ml beaer and &as etracted &ith >= ml o# hot ethanol. he miture &as boiled #or ei"ht minutes over a &ater bath usin" a :;= ml beaer. he hot miture &as #iltered and the residue &as discarded. he #iltrate &as cooled and collected the resultin" precipitate by #iltration. he collected precipitate &as dissolved immediately in < ml o# methanol'chloro#orm miture. he isolated comple lipids yielded by the previous part o# this eperiment &ere utilied and &ere re9uired to proceed &ith the re9uirements o# this procedure. Alon" &ith the isolated comple lipids in test tubes prepared be#orehand, all isolates &ere #inally set #or #urther characteriation. -n Liebermann)Burchard test, =.; ml o# each lipid solution &as placed in separate small test tube. Acetic anhydride &as added ten drops to each test tube and &as "ently s&irled. !oncentrated sul#uric acid &as care#ully added #our drops do&n the side o# the tubes and &as mied &ell. hen, color produced &as noted. 7or the $alo&si test, about ten drops o# lipid solution &as placed in small test tubes. !oncentrated sul#uric acid &as care#ully added ten drops do&n the side o# each test tube and the t&o layers that &as #ormed &as not mied. he color o# interphase &as obtained.
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Biochemistry Laboratory he test o# phosphate necessitated the use o# crucible, on &hich 2.= ml o# the lipid &as mied &ith the #usion miture that is #ive times the bul o# the lipid solution. -t &as then i"nited over a #ree #lame until all or"anic matter is burned a&ay and the miture &as turned to a "rayish or colorless li9uid, or a &hite or "ray ash is obtained. he miture &as cooled and dissolved in a < ml o# &arm &ater. he contents o# the test tube &as trans#erred and acidi#ied &ith < M o# nitric acid. he solution &as heated under ?;o!. About < ml o# :.;@ o# ammonium molybdate &as added and &as heated. he color o# the precipitate and the solution &as observed. he raut/s test used ten drops o# lipid solution that &as put in a small test tube. he test tube &as put in a boilin" &ater bath in the #ume hood to evaporate o## the solvent #rom the lipid solution. he dried lipid &as suspended in ten drops o# distilled &ater. About 2; drops o# raut/s rea"ent &as added and the test tubes &ere heated 2): minutes. he color o# the solution and the precipitate #ormed &as noted. &o test tubes &ere prepared success#ully #or the succeedin" tests. he ninhydrin test re9uired ten drops lipid solution &as put in a small test tube. hen, #ive drops o# ninhydrin in ethanol &as added to the solution. he tube &as heated #or 2): minutes and the color o# the solution &as noted. Lastly #or Molisch test, about ten drops o# the lipid solution &as put in the small test tube. he test tube &as put in a boilin" &ater bath in the #ume hood to evaporate o## the solvent #rom the lipid solution. he lipid &as suspended in t&enty drops o# distilled &ater. &o drops o# Molisch rea"ent &as added in the solution and it &as mied &ell. he color o# the interphase &as noted a#ter this color reaction tests.
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Biochemistry Laboratory !esults and "iscussion #able $% Results o# isolation o# saponi#iable and non)saponi#iable comple lipids #rom
cal#/s brain Lipids Cholesterol &lcerophosphatide Sphingosine phosphatide and sphingosine
Appearance $li"htly turbid yello& solution urbid yello& solution !lear colorless solution
glcoside
-nitial process done #or this eperiment &as the isolation o# comple lipids. $ince lipids are amphipathic molecules, di##erent solvents &ere used in etractin" the di##erent lipids. rituration usin" sand and the addition o# acetone &as observed. he sand helped in the disruption o# the cells &hile the acetone provided a mild but rapid method o# dehydratin" the tissue. 7or the isolation o# cholesterol, acetone &as used to stabilie sterols lie cholesterol. Acetone etracts #ats, sterol esters, sterols and other simple lipids. he 3;@ ethanol &as used to obtain the crude product. -t &as then cooled over an ice bath to recrystallie. Recrystalliation &as done to avoid immediate precipitation. -n the isolation o# "lycerophosphatides, sphin"sosine phosphatides and sphin"osine "lycosides, methanol'chloro#orm 12'<6 &as used as the selective solvent to etract the "lycerophosphatides and sphin"olipids #rom the residue.
#able '% Results o# Liebermann)Burchard test
6|Page
Biochemistry Laboratory Standard Cholesterol Lecithin &alactocerebrosid e
Liebermann-Burchard test Appearance Sample Dar blue "reen Cholesterol solution Dar violet &lcerophosphatide solution !olorless solution Sphingosine glscoside ( Sphingosine phosphatide
Appearance Bro&nish oran"e solution Dar "reen solution
Li"ht "reen solution
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Biochemistry Laboratory 7or the Liebermann)Burchard est, it determines #or the presence o# cholesterol, &hich "ives a blue)"reen or rather deep "reen color. he addition o# acetic anhydride in its setup reacts &ith the hydroyl "roup o# cholesterol located at carbon)< then reacts &ith addition o# the conc. :$+>, thus "ivin" o## a deep color. he color is due to the hydroyl "roup 1)+6 o# cholesterol reactin" &ith the acetic anhydride, thus under"oin"
acetylation,
and concentrated sul#uric acid, &hich causes it to under"o sul#onation and +igure $% Liebermann)Burchard mechanism reaction in cholesterol
thereby
increasin"
con0u"ation
o#
the
unsaturation in the ad0acent #used rin" . o&ever, the chan"e in color taes time, #irst startin" out &ith a li"ht pin color then #inally deepenin" into dar "reen. he deeper the color, the more intense the concentration o# the solution is. he cholesterol standard depicted the ri"ht result &hich is the dar "reen solution. $ince Clycerophosphatides sho&ed dar "reen and $phin"osine "lycosides and $phin"osine phosphatides sho&ed a li"ht "reen, it can be stated that they do not contain cholesterol. o&ever, the cholesterol sample depicted bro&n oran"e solution instead o# the dar "reen product. ossible source o# error &as the cholesterol &as not isolated. #able ,% Result o# $alo&si test Sal)o*s)i test 8|Page
Biochemistry Laboratory Standard Cholesterol
Appearance Red interphase
Lecithin
Dar violet red interphase Li"ht pin interphase
&alactocerebrosid e
Sample Cholesterol
&lcerophosphatide Sphingosine glscoside ( Sphingosine phosphatide
Appearance Red interphase &ith clear solution Red interphase &ith yello& solution Bro&n interphase &ith clear solution
$alo&si est &as conducted to also sho& i# the sample contained cholesterol. -# the sample &as positive on this test, a yello& to bric)red color is #ormed. he cholesterol standard result
depicted
&hich
the
ri"ht
the
red
is
interphase. -t indicates #or a double bond in the cholesterol rin". hen the rea"ent used in
+igure '% Reaction o# !holesterol in $alo&si test
$alo&si test, concentrated sul#uric acid, is added to a cholesterol solution, a red interphase is visible. he red interphase is created by the reaction o# :$+> in the chloro#orm layer o# the solution. -n the presence o# the added acid, dehydration occurs #ormin"
a
bisteroid
&hich
"ives
a
red
color
interphase.
1arer,
235>6
Clycerophosphatides, $phin"osine phosphatide and sphin"osine "lycosides depicted a red)oran"e interphase. he cholesterol sample depicted a red interphase &ith clear soluton. ossible source o# error #or the other samples &as there &as still cholesterol included in the samples.
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Biochemistry Laboratory #able /% Result o# est #or hosphate
Standard Lecithin
#est for hosphate Appearance Sample Fello& solution &ith &lcerophosphatid yello& precipitate e
Appearance hite precipitate &ith clear solution
he test #or phosphate, &hich determines phospholipids, yello& precipitate sho&s the positive result #or the test. -# the sample &as positive on this test, the presence o# nitric acid causes the phosphorous to react &ith ammonium molybdate, #ormin" a yello& precipitate G &hich is ammonium phosphomolybdate. 1Luthra, :==56 Miin" #irst the lipid solutions &ith the #usion miture, &hich is composed o# potassium nitrate and sodium carbonate 1<'26, and then combustin" it under an open #lame is used to remove all the carbon components and to eep the phosphate in addition &ith the &ater. Addin" then
#able 0% Result o# raut/s test .raut’s test Standard Cholesterol
Lecithin
Appearance Bro&nish oran"e solution &ith blac precipitate Bro&nish oran"e solution &ith red
Sample Cholesterol
&lcerophosphatide
Appearance Bro&nish oran"e solution &ith blac precipitate Bro&nish oran"e solution &ith blac 10 | P a g e
Biochemistry Laboratory
&alactocerebrosid e
and blac precipitate Bro&nish oran"e solution &ith blac precipitate
and &hite precipitate Sphingosine glscoside ( Sphingosine phosphatide
Bro&nish oran"e solution &ith blac precipitate
raut/s est &as conducted to sho& i# the sample contained choline. his test is no&n to be the modi#ication o# Dra"endor##/s test. +nce the raut/s rea"ent &as eposed in the isolated lipid solution it "ives o## a red bric precipitate in the presence o# alaloids such as choline. he rea"ent is composed o# bismuth subnitrate &ith potassium iodide in
Standard Cholesterol Lecithin
Ninhdrin test Appearance Sample !olorless solution Cholesterol Violet red solution &lcerophosphatide
Appearance Dar purple solution Li"ht purple solution 11 | P a g e
Biochemistry Laboratory &alactocerebrosid e
!lear li"ht yello& solution
Sphingosine glscoside ( Sphingosine phosphatide
Dar purple solution
(inhydrin est ehibited i# the sample contained primary amines, rather it determines the presence o# #ree alpha amino acids. he ninhydrin test, &hich "ives o## a purple solution also no&n as the Ruhemann/s purple, ehibits a positive result. his test basically indicates the presence o# amino acids attached to lipids. -t is an oidative deamination and decarboylation reaction that is #ollo&ed by condensation. he $erine standard depicted the epected result o# violet li9uid. $ince $phin"osine phosphatides sho&ed a similar result to the standard, it can be said that it contained a primary amine. -# the sample &as positive on this test, the ninhydrin rea"ent decarboylates the primary amino "roup to #orm hydrindantin. -t &ould then condense to another molecule o# ninhydrin and ammonia to produce the Ruhemann/s purple. 1$ea"er H $labau"h, :==;6 he Clycerophosphatides and !holesterol samples sho&ed a dar and li"ht purple solution, respectively. -t can there#ore be stated that Clycerophosphatides does not contain primary amine. he !holesterol sample did not sho& the epected result. ossible source o# error &as the cholesterol &as not isolated.
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Biochemistry Laboratory +igure ,% Reaction o# (inhydrin test
Lastly, Molisch est &as conducted to sho& i# the sample contained a carbohydrate. he positive result #or this test is a red or violet rin" at the interphase. he principle behind this test is due to dehydration and condensation o# #ur#ural or ;) hydroymethyl#ur#ural &ith a)naphthol #ormin" violet rin". 1odd, 23436
#able 2% Result o# Molisch test Molisch test Standard Cholesterol Lecithin
Appearance urple interphase urple interphase
&alactocerebrosid e
urple interphase
Sample Cholesterol &lcerophosphatide Sphingosine glscoside ( Sphingosine phosphatide
Appearance (o interphase !loudy &hite interphase urple interphase
he "lycosidic bonds o# the lipids then under"o hydrolysis and are thus converted into monosaccharides and a"ain #ur#urals. +nce the Molisch rea"ent, composed o# I)naphthol dissolved in &ater, is added, it condenses the #ur#ural &ith its t&o phenols resultin" in a red or violet rin" at the interphase. -# the sample &as positive on this test, the sul#uric acid dehydrates the sample to #orm #ur#ural or ;)hydroymethyl #ur#ural. he #ur#urals &ill then react &ith the I)naphtol in Molisch/s rea"ent to produce the purple product. he I)naphtol serves as the condensation a"ent &hile sul#uric acid serves as the dehydratin" a"ent. he Calactoserebroside standard depicted the epected result o# purple interphase. $ince $phin"osine phosphatides sho&ed a similar result to the standard, it can be said that it contained a carbohydrate. he Clycerophosphatides sho&ed a cloudy &hite interphase. -t can there#ore be stated that 13 | P a g e
Biochemistry Laboratory Clycerophosphatides does contain a carbohydrate. he !holesterol sample ehibited an absence o# interphase. ossible source o# error &as the cholesterol &as not isolated.
+igure /% Mechanism o# Molisch test Conclusion As demonstrated by this eperiment, structural components o# the isolated comple lipid can be detected and validate &ith the utiliation o# the lipid miture and the "overnance o# the appropriate tests. (on)$aponi#iable lipids and $aponi#iable lipids &ere distin"uished by the process o# isolatin" comple lipids that yielded cholesterol &hich is a non)saponi#iable lipid and "lycerophosphatide and sphin"olipids &hich are considered as saponi#iable lipids.
%pon
completion o# a series o# tests, it &as ascertained that comple lipids o# the cal#/s brain constitutes by the presence o# cholesterol &hich that satis#ies by Liebermann)Burchard test, presence o# phospholipids that &as validated by the est #or hosphate, the presence o# choline by raut/s test, carria"e o# a #ree amino "roup by (inhydrin test and lastly Molisch test, that determines the presence o# a carbohydrate in the isolated comple lipids.
!eferences
!abatit, B. E. 123456. Biochemistry 9th Ed . Manila' %$ ress. 53)2:< Luthra, . 1:==56. Clinical Biochemistry . Basic concept o# biochemistry 14 | P a g e
Biochemistry Laboratory arer, $. 1235>6. McGraw-Hill Dictionary of Chemistry . %$A' McCra&)ill $ea"er, $. L. and $labau"h, $.R. 1:==;6. Organic and Biochemistry for Today. 5 th Ed .
%$A' homson Learnin" -nc.
$&iter, R.L. 123336. Eperimental Biochemistry. (e& For' .. 7reeman. 25?)255 odd, D. 12343 . E!"erimental Organic Chemistry . En"le&ood !li##s (.J.' rectice all. ?:)?4.
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