Individual variations in human meibomian lipid composition

llxp.

Eye Res. (1978) 27, 289-300

Individual Variations in Human Meibomian Lipid Composition J. M. TIFFANY Nt@eld

Laboratory of Ophthalvvaolog,y, T rvlicersity of Oxfordz Walton. Street, Oxford OX.2 6dW’: F.K.

Samples of meibomian lipid from several human donors were analyzed by thin-layer chromatography. Great variations were seen between individual samples in the relative proportions of the different lipid classes present, although all contained the three principal classes of wax esters, cholesteryl esters and triglycerides. Appreciable amounts of hydrocarbons and free fatty acids were seen in some cases. but the fatty acid did not seem to be derived by hydrolysis from triglycerides as in skin surface lipid. Samples from individual donors were also hydrolysed and the fatty acids and fatty alcohols (minus cholesterol) analysed by gas-liquid chromatography. The results were broadly similar for all samples. The fatty acids were of both odd and even carbon numbers, and both straight and branched-chain isomers were present in most cases; t,he unsaturated chain content was low. A characteristic pattern of chain length distribut,ion was seen over the range C,-C,,. and a similar pattern was noted for alcohols, but about’ four carbon atoms per chain longer. The significance of these and earlier results is discussed. It is concluded that no single romposition exists for human meibomian oil, and that the results from pooled material from many individuals may be misleading as an indicator of ocular function. Key words: human meibomian lipids; individual compositional variations; lipid classes; fatty acids; fatty alcohols; TLC’: GLC: branched-chain isomers.

1. Introduction A number of studies have been made of the composition of human meibomian lipids. These have largely been qualitative investigations. 114thonly rough indications of the relative magnitudes of different, lipid classes,and, with one exception, no attempt to define the constituent fatty acids and alcohols. Thus Pes (1897) identified cholesterol. fatSty acids and “fat” ; Linton. (‘urnow and Riley ( 1961)reported that the major part, of the material was“neutral fats” and other unident.ified material, plus not more than lO’l& of phospholipid, but could not confirm the presenceof cholesterol. Ehlers (1965) employed rather more reliable analytical methods. and found both cholesterol and chol&er!;l esters, fatty acids, phospholipids ant1 21trace of triglycerides. Nico1aide.q (1965) identified both wax esters and cholesteryl esters among the neutral lipids, R!: well as traces of squalene and other hydrocarbon, fatty acids, and mono-, di- antI triglycerides. Keith (1967) compared normal meibomian lipid, sebum, and meibomian lipid from patients with seborrhoeic blepharo-kerato-conjunctivitis by thin-layer chromatography (TLC). He found the major component to be wax and cholesteryl esters, with small amounts of triglycerides, fatty acids and sterols, but found meibomian samples did not contain the large amounts of squalene and triglyceride+ present in sebum. No significant, difference was seetll)etween the normal and abnormal meibomian samples. Gory, Hinks, Burton and Shuster (1973) were the tist workers to give quantitative results of a TLC analysis on lipid from individual &JllOrS, in a comparative study of the meibomian gland secretion from control subjects ancl those with red eye associated with rosacea. The major component was mixed stcrol and wax mono-esters, with a smaller amount of diesters (not identified further). and still lessof triglycerides, fatty OUl4-4835/78/090289+

12 $01.00/O

!@I 197s Academic 289

Press Inc. (London)

Limited

‘l’ht> rlitwt striking feature 01’ thaw iyorts is tht: lack of agmmitwt 011 thtb iiatilw antI relati\-c ;miouiits of tht! tlifftmiit lil)itl classes in the secretic~n. The work reportttcl time wils intentlet to cstnl~lish wh(~tticr. the cot~sitlcml~le intliviclual tlif&rcticcs itr lil~i~l c:l&SS (10 exist,. illltl whether xxriiit,ioir in tmlltw: Clilsws is reflet:tSecI ii1 tllc: ovt~l’illl itC\-l ;tiicl ally1 chain wi~il~osititm of the: w!lll)lt:.

2. Materials and Methods S;mples \vere collected at the slit-lauip microscope froiti voluiiteers iii the trut~patielit c,lillic of Oxford Eye Hospital or from workers in this laboratory, all free frotil lid 01’ glai~(l disease. The lid mxrgins were swabbed wit,lr ether before collection to avoid coiltairrii~atioll of the sample bv skin lipids. The use of topical anaesthetics was generallv avoide(l. Li,gltt thuml) pressure‘was exerted on the eyelids. against the globe, and the sen;i-solid espresseci lipitl was scoopetl into a 2 mm chalaziou wvette, previously washed iu ether. The s;rtnple wan ~vashetl off into freshly distilletl ether in screwcappetl collectin g tubes. Samples 11(0t :iiiiilvsecl inrmedixtelv were drietl ul~tlrr ;I, SttWLlll of nitrogen n,iirl stored ;tt -21 j^C.

The rthereal ~~JhltioilS were tlried onto alurninium planchets rind weighetl. Coiwiderable variation was found in the amounts of lipid obtainable from different donors, in part because only light pressure was used during collection, and individuals differed iu their tolcranre t,o the procedure. Hydrolysis and gas-liquid chromatography of fatty acids au11 :&~~l~ols cwultl be done on even a small sample. but thiwlayer chromatography of lipitl classes could only he carried out ott samples great’er than about 0.5 nlg. In no case \VW CIIOII~~I material available from ;L single donor to permit a complete analysis of indivitlu:~l lipid classes, such as has been done t>Alhovine material (Baron and Blough, 1976).

Hexatle was shaken several times with concentratetl sulphuric acid, washed thoroughly with water, dried over calcium chloridta :LIIC~ distilled. All other solvents usetl were Anal;~l, gratle ant1 were dried with 3A nloleculirr sieves and redistilled before use. Dietlryl c:ther was stored over sodium. The handling processes associated with collecting, weighing and anwlvsing t,he meibomiau lipid samples involve frequent solut,ioii. tlryiug down ant1 redissolvingii~ fresh solvent,. ;n~l small impurities in the solvent may be concentrated and appear as substantial componetlts, apparently of meibomian origin. It is possible that the presence of xuchspurious compotleilts could account for apparent, variations in lipid composition between different SiilllplC2S. ‘rt) avoid t’his misinterpretation, a stringent, test of purity \\rits applied to all the solvellt,s usc(l. Approximately 18 nil of solvent was placed in one of the concentration tubes uwtl ii1 preparing samples for GM! (test tubes I\-ith capillary tips) and evaporated tlown in ;I. stream of high-purity nitrogen to al)out 10 ~1, which \vils injected directly into t,he C+IX. Solvents showing any appreciable irnpurit>r peaks even after this 1800-folcl concentration were re-purified before use.

pin single TLC’ syst’em is available which can adeqtltttely separate all the possible lipid Of these. the hardest to separate are the \VilX esters and cholesteryl esters, which :tre expected to be among the major constit,uent,s (Ait(lrews. 1970). A known amount of samplr ill ether solution was applietl as a streak on to 34 .<%I ,211 glass plates coatetl with .\l?lY’li ;\luntina E 0.25 mm thick. Par:illel lanes of stantlitrtl lipids and a 1iai7-nw tlii~rket. 1;1tt~ sc,ttte prcjteitt) were sctx~~e~l off. The atttounth (I f lipi(l itt these atttl the hiitt(ls frottt tltc. ;tltttttitt;i pl:ttc \vcre i~ssa~etI II!- the rltarrittg ntc*tltoci of .\l;trsh itn(l Weinstein (I%iti). ;q;~it~il t’itIil)r;Ltillll ~‘11I’ves for kllo\I’t1 stcltlthr(l~. 111 SOttIe t itst's. :L lJilllf1 \VilS llotf'tl ~tet\I'tY'tl tltc‘ II;IS (Bsttbr an(l c,holestervl ester l~attrls ott tlte alunritta jJl;tte which might contain eitlt~~r or lt1,1ti of these c,lasses. Tl;js battrl was tlivitletl ittto two. one llillf being measured 1tV the c.lr;trt.ittg i~+:it~ ;ttrtl the other half anwlvsed for cholcstervl c+tPr contettt hv the trtetllolI of %I;ltlii... iLlI< lLtl,l Boyle (1963). .A c,orrectiott \V~IS tltrtt ilt;ttle for tltis c,holester\.l rsttlt, itt PliLSSf?S,

Ilcil~otitiit ti lipid samples were dried under a stre;ttn of nitrogen iti screlr-capped test tttl,ry ;111cl\vere hydrolyzed by Itrating overnight at Wt”C with 3 r111of 1 S-KOH in Sj”,, et,l~;i tidal. .Xfter cooling, fatty alcoliols and cholest,erol were extrac.ted wit.11 3 X 1 ml of liesane (;t(ltlittF it little I\-ater to encourage separation into t,n-ct layers and rentrifuging brirf’tv to Iw:II< up interfacial material). The hexane extract, its washed with water and the wash&s ;~tfclrcl lttt(~k to the hydrolysis tube. This was acidified with 6 s-HCI and the free fatty acids ww cxstracted \vitlt 3 x 1 ml of hexane. Jlethvl esters of the free fatty acids were preparetl It\- )trJ;tting the drietl extract overnight at 70% with 0.3 1111of 14”; BF, in met8hnnol ant1 O: t tttl q)f benzene, in screw-rapped tubes wit.h Teflon-lined caps. Aft.er cooling, 1 1111of wtttrxtrtl ;2a,SU, solution was added and the methyl esters were extracted with 3 :’ 1 1111 of ht~;t tte and concentrated clo\vn under nitrogen. Approsituatel~ half the nteth,vlaltetl nt:ttcri;tl was taken and reduced by bubbling in hydrogen for 30 tttin in the presence of ,Y”:, [t;tll~~.(li~U~~/(!lttt.r~oa~l(Futterntan ant1 Andrew-s. 1%-C).

Tht* ;~lcol~ol fraction following hydrolysis contained both fatty alcohols and cholesterol? a,ttcl. if run directly ou GLC, the large cholesterol peak obscured fatty alcohol peaks in the C,,-C.,, region. Changing th e chromatographic conditions or column type altered the retetition t,itne of this peak, but no conditions were found under which it fell clearly between or lattar than other peaks. A novel TLC technique was therefore developed (to be described fully elsewhere) using silica gel G plates impregnated with urea and developed in trichlorrlethylene: ether (1: 1 v/v). On these plates, relative to their migration in the absence

‘9L’

J. iV. TIFFANY

~rf urea, cholesterol is little affected, but straight-chain fatty alcohols, aud branched-chaitt ;~lcohols eotttaittittg au unbranched region eight or more carbott atoms long (Truter, l%l), ;IPCretarded bv formation of inclusion cotupounds with urea. The result is a spot cotttaittitq the alcohols streaked upwards front the origin but, ttot overlapping the cholesterol spot. This streaked spot \vas scraped off the plate, the lipid extracted with chloroform: tnethattc~l (2 : 1 \y’v) and used for GLC of alcoltols. The extractiott process did ttot sigttificatttlv ;altet the reIati\-c proportions of peaks seett Ity GI,C prior to this step.

tzll ;ttlitl~~f3 were performed isothertttally in a Pye 104 itistrutttent w&h flame iottiz;ttiott deteot,or, 011 .i mm ‘,: 1.5 nt columus of 3”;, 01:-l or 11t’ib DEGS on 100-120 mesh Chrotttosorb IV support. Runs on the OVl colnnut were at, 190’ and 220°C. and ou DEW at I ‘it t ;rtt(l 19 t’C, with nitrogeit as eitrriet gas at 4( t tnljtttitt. lit some cases t~rintetliylsilyl ether derivatives were made of the fatty alcohol samples, but, generally no advatttage \\Tas foutttl over GLC directly on the free alcohols. Peaks were idetttified b> rcfereuce to standard mixtures, run utttler the same conditiotts, of straight- atl(l brattc:ltedchain fatty acid tttethvl esters (Applied Science Laboratories Irtc.), and the correspotttlittg alwho tttixt~ures prepared from these lty TAlH, reduot~iott. I’nsaturated aotttponettts werc~ identified by cotttparisott of the ~l~r(~ttt;lto~rattts of the normal and ltrdrogettat~ecl satttple~: a peak correspottding t’o an uttsaturated cltaitt tlisa,ppeared ant1 the relative peak area of its 5ittllrittfA ;malogue ittcreiisetl followitt,: Iiv(lt~c~peni~t~i,)ti.

As the tiutiiber of peaks iti each c~l~rc~ttii~t~ogr~tt~iwns large ilmlld the rutitiittg time altctnt 3 hr, it) \vas occasiottally foutttl that slight, changes itt eonditiotts durittg a GLC rutt alterecl peak retctttiott titttes enough to make it, clifhcult to decide which peaks represented straight chaitts at111which their branched isottters. This difficult,y catt readilr be retnor-ccl br a(ldittg ittt~crxtl stSn.tttlartls. but their use requires adtlitjiottal GLC runs to compensate for ol~scwetl peaks. attd there \VitS getterall\- too little material available for this to be tlone. .A sattrltlc ntrichetl itt The br~tttabetl-clta.ill isomers \vas obtained from the urea-TIX system previousI\ usetl for removal of cholesterol. hterial tttigratittg bepld the origin but less th;ttt tltc c~holesterol had lost, a litqe proportiotr of its straight c*haitts (iml a sma,lIer proportiotr of its brnttched cltaitts) by adduct fortttatiotr. rendering i~lentification of GLC pea,ks as straight or hrattched eltaitts simple. A sittril;~ I’ ettric~lttttettt, using partition of the alcohols betweett ltesatte attd a saturated tnethanoli~ urea solution, has heett described by Kicolai(lw it ~1 Ray (1965) atit1 used iti itletttitieatiott of hri-Lticlietl-~li;~iti fatty acids i ti I’rrkli.r, ~~~sfws~~ (Kicolaides, Apott and \Vottg, 197(i). Peak areas were tiieasuretl by platiititeter, and the carboit ttutitbers of utisaturatetl it tttl br~attchecl it(:ids tklt(I ;~lcoltols WC~E expressed itt terms of their equivalent8 chain lettgtlt (ECL) b,y interpolation front t,ltr carbott numbers of their ecpiivaletit~ stra,i~lit-c,ltailt tteighbours. The reliability of the hnlrolrsis and metlt~%tt~iotr procedures were tested using &ttdard fatty acitl ttiethvl ester mixtures. Relative peak itreits in the original stmtlmtl tnistures differed hy 110 ttiow thtt 5’!,,: over the wliole tmge of chain lettpths obset~vd iti ttreibotrtiatt lipitl, from those in tlie saute ttiistures followittg hvdrolysis att11 re-tiirtlt\-l;ttil,tl.

3. Results The results of the charring assay on four samples of meibomian lipid are shown in Table I. The response to charring of hvclrocarltotts other than qualene is poor. so that the actual percentage of this class is in some doubt. In more recent work it has been found preferable to-measure this class from total peak area on GLC relative to

HT32~4X

MEIBOMIAN

LIP1 I>S

%U

internal standards. Nicolaides (1965) suggeststhat the hydrocarbon content found in lipid samplesoften reflects the degree of care exercised by the operator. So far as possible. all sampleswere treated in an identical manner, yet they show considernl~le differences in their content of both hydrocarbons and sqnalene. Contamination of the samplt~by skin lipids seemsunlikely, in view of the cleansing of t.he lid margins prior to collection, and the fact that the sgualene content, is lo\\-. The origin of the hydrocarbon may in J)art he traffic or other industrial fumes in t#heair of t,he laborator\ ccmtairiinating glassware during handling of the specimens.The pattern of hvdrocarbon Jleaks on GJX did not correspond to either par&in wax or vaseline, but no conlJ)arisonwas made with fuel oil. Gnfortunately. no comparison can l)e made with t.Jlerwults of C’ory et al. (1973) as t,heir TJX technicluc involve(l l)urning hydrocarbc~n~ off thll platX.

S(lu:l,leneis \\ell-estaJ&shed as a comJ,onent of skin lipitls, but has not Jnevionsly been rcJlort,ed in meibomian lipid. The TLC separation reported 1)y Keith (1967) ditl not, slrow it dist,inct spot for squalene above that for coml tinetl wax esters and cholcst~erolestersas did sebumunder the sa,meconditions ; however, it is clear that the ester fraction of sebum had a different composition from that of meibomian secretion a.ntl a loser Rf. sothat the upJwr part of the meihomia,nest,erspot may in fact conce:rl sq”“lwC:.

Ver!- witlc variation is seenJ)et+reensamplesfor all IiJnd classes,and no consistent J>at,ternof comJ)osition can be tletected, except that all the satnJ)lescontain at least, the t,hree classeswax esters.cholesteryl estersand trigl!-ceritles. In view of the uniform a.titl raJ)itl liantllin p of t,he lipicl folloaing csJuYwion, it. ii utilikel,v that the great differences in free fatty acid content indicate 1iJJolysisof briglvceride, as is the case with skin sebum (Nicola,itlesand Wells, 1957’).Sebum 4~owsa reciprocal relationshiJ) l)etswwn triglvcerik and free fatty acid levels, indic~iting t8hatSthe fatty a&Is are tlcriwtl from triglycericle rather than from lipolysis of was estersor cholesteryl ester<. The present results do not show any significsnt relationshiJ) of this kind. If breakdown of triglycerides hacl taken place, one might also ex]Dect to seehigher percentages of tho nwno- and diglyceride classes.The low levels of phospholiJnd, and J)ossibly of cholesterol, mav reflect the small amount of cellular debris expressedwith the lipid by light sqeezing.

q

Acids

IO

12

q

Alcohols

14

16

I8 Carbon

20

22

24

26

28

30

32

number

FIG. 1. Distribution of straight-chain fatty acids and fatt,y alcohols of six individual samples WDK meibomian oil. The letters (a)-(f) rorrespond with the sample identifyin, w numbers Tables II and III. B, Acids: n, alcohols.

of human gircn in

were seen(Table II). The distribution of these straight chains is also shown graphically in Pig. 1. Many of these were accompaniedby a smaller iso- or ant&so- branched-chain peak of the same carbon number. On the 3% OV-1 column, the equivalent chain lengths (ECL) denoting Go-chains were 0.62-0.66, and for mnteiso-chainsO-70-0.75, relative to the next whole number (i.e. since branched-chain retention times are less than those of the Ft-isomer,iso-stearic acid would have ECL 17.65). ECLs in Table II are given only to the nearest tenth, so the monomethyl-branched ECL region is 0.6-04. In all cases,the pattern of straight chain lengths consistedof a relatively large proportion of short chains, a medium-chain region with few components of any size,

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a~ntla larger region of long and very long chains showing an appreciable content (of all chain Ien.&, but falling off to trace amounts at the upper end. In samples (a)--(tt) OF Fig. 1 t,hcsc ranges were approximately (‘&1,,, (‘13P(1,5 and (‘16-C3,j, rcdpectivcly. nlt~hough small variations were seen for these limits. Samples (e) and (f) showeci SOI~I(:what similar regions, but their limit’s were three to five chain length units greatt>r. Hydrogenation showed that very few unsaturated fatty acids were present except in ssmple (f), in marked contrast to the findings of Andrews (1970) for meibomian nnc.l Nicolaides (1974) for skin lipids, where a large amount of oleic acid was detectetl. The alcohols had a chain length range of about C12-C,,, and the pattern or’ rlistribution of straight chain lengths was roughly similar to that seen with the acids. but occurring about four carbon atoms later (Fig. 1). The distribution was much more variable in the C,,&, region for sample (e), and in sample (f) did not in fact show

HUMAS

XIEIBOMIAN

LIPIDS

L”3i

the four-carbon shift relative to the fatty acid pattern. However. the alcohols of sample (f) contained only 33% saturated straight chains and 28: b unsaturated chains. in addition to the very large figure of 27q’,0 unidentified material. Only the straight chains are represented in Fig. 1. As pointed out ]~y Nicolaides et al. (1976)? t’he retention times of polymethyl-substituted chains, with additional methyl bra,nches in positions other than iso- or anteiso-, may resemble those of skaight chains of lower ca,rbon number; although they may be present in small amount. uo such bra,nchetI chains I\-ere detected here as no mass-spectra were obtainetl on these samples. Nevertheless, any peaks not accounted for as straight chains or unsaturated analogues are assumed to be branched in an utqecified manner. In addition, no figures for the proportion of cholesterol are included among the alcohols. since cholesterol had been remowc] by the TLC-urea technique; all the higher figuws it1 the C’24-C,‘28region thewforf, in fact alcohols. an

firttv

4. Discussion Thcw olwervations tend to confirm the results of C’ory et al. (1973), in that no two sa.qlw are al&f. and profound differences in Iipit] class CfJlll~~flSitiO~l ma\- be seen between individuals with satisfactory tear film function. No ‘Xypical” cokposition can be (liscernerl, and “mean” values woult] bc meaningless even on a much large1 population of samples, since in terms of physical function of the meibomian secretion a synthetic mixture of the average composition might be less desirable than any one of several widely-differing natura.1 compositions. As iuentionetl earlier, it is remarkable that a number of t,he compositions pre\-iously rcportetl (Pes, 1897 ; Linton et a,l., 1961; Ehlers. 1965) apparently do not contain sonLe of thtb three principal classes of lipid, ant1 that there are wide disviz. wax esters. cholesteryl esters alld triglvceridw. ., cropallcics also in other classes. such as cholesterol ant1 l)ll~)s])l~(~lil~ids. Part of t,he diffiCU~t,V hS ill t.he tlf~lll~~lC~~~~~ll~f1 IlSfVl for the lipid5 : Pw (1 895) re]“‘rts “fat”. which ])rol):tl)ly inclutl~~s was esters although perhaps intentled to tlenote triglycerides: in ad(lition to triglyceride. It is (‘orv c+ al. (19i3) list b‘nIonowtc’rs” and “&esters” not clwr in the latter case u-hethrr “tliesters” arc tliglyceritles, or li,vdroxS-fatty Ads est,erifi(a(l in I)oth the hvdroxyl and carboxrl groups. as fount1 in set)um lw Nicolairlta (196.5). Xico]ait]w (197”4) refers t’o unpubl~shetl olwrvaticms tzhart h& nl(~d~o~nia~~ li])itl 111il!- coiitaill two as yet iincharacterizecl diwtcxrs (4 this t,v])t’. The ~wt~hotls of’ separation or iclcnt,ificat~ion of lipit cli~ssrs mav also introthw error. Thus l,int,on et, aI. (1961) usetl paper cluomiltogr~~])ll~ to qx;ate lipitl bantls. and tlctwt,tvl tht: llan(ls on the paper wit’11 Sudan 2. The prehetlctb 01’ c~hol~~st~erolwas testrti lgttf~ntlticnt~ of’ t#lie bnntls with ncet,ic and sul]lhiiric~ aci(ls (t,lie IJi(tl)frIli:lIill--I:urctt;~,rclt rtwkn). No cholesterol was tletrctwl. an(l this rcwlt also swmetl fo inclic:ltf~ that, no cholesteryl esters \verc present. Howvw. it has Iwn shown (( ‘ain ant1 1 Iibrriso11. 1950) that Sudan tlws only stain lipitls in the melted st;ite, nw~ t,hat li])itls 01 high iiielting point. such as cholesterol anti its hltturated esters. will riot stain at ~)~II tctnperature. Sirnila,rly. the silver nitrate tctst applied for fatty acids tna,y bc itw)nclasi\-e if the acids are nearlr all saturated. The taxtensive stutlies of Ehlers (1965) suffer from the same drawbacks. “Since no wa,x ester standartl was used, his itlentification of t#hemajor TLC spot of high Rf as cholesteryl ester is incomplete. Andrew-s (1970) (Jhtaillfd a TLC! spot of higher Rf than his major ester spot, referred to as hydrocarlJon, hut the spot did not reappear on rechromatogragll~~ of this region of the chronlatogram. ;Lntl was not further identifictl. It is pfJSdde that the ])hospho]ipids reported by

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several workers, including the present investigation, are in fact derived from membranes in cellular debris released with the lipid, and the manner of expression of the lipid from the glands may influence the apparent content of this and other lipid classes in tho secretion. In the absence of a test for phosphorus in addition to charring 1)~ sulphuric acid! the phospholipid figures presented here may include glycolipids or protein. ‘I’ht fatty acids and alc,ohols shown in Fig. 1 arc the straight-chain saturated ant1 unsaturated con~ponents only; although not as numerous as in the samples of Andrews (1970) or the bovine material of Haron and Blough (1976), appr~iable quantities of l,ranched chains are present. l’a~ble III shows the percentage \\‘eight ratios of I)lullcllccl t,o st,Gght chairls in the six samples in\:estigatecl here. These ratios are calculatcltl using al1 the available percentage figures and riot, only those appearing in ‘ra11lc 11, whia II rrcortls only components present as 1’,‘1, or nior’e of total weight. There is little uotrsistc~llcv I)ctween sa~nples, ljrtb, \vith the clsception of sanrl~l~~ -IO. t Ilt: r;l.ticbs For ncitls ant1 dcoliols in the same sample are roughly similar. The results of Illltlrcl\vs (1950) for the combined wax ant1 stcrol ester CliLSSW give a br~tllclietl/straigllt ratio of

38 39 40 43 AB DK

The arailable area.

0.19 0.32 0.15 0.17 0.40 0.40

(total branchctl-chain peak area)/(total GLC figures, including peaks oxcludcd from

ratio

0.13 0.25 0.30 0.17 0.36 0.31

straight-chain peak arc*&) \vr2s cnlculatecl using al1 T;~blc IL as m&ing up less than I’;;, of total pez~k

1.15 for both acids and alcohols. The function of the branched chains seems to be to reduce the melting point of the meibomian secretion to uear body temperature. If all chains were straight, the melting point would probably he some X-40°C higher, and the lipid would be unable to spread on the lid margills or tear film surface. Nicolaides (1965) separated skin surface wax esters lay urea adduct formation into predominantly straight-chain and branched-chain fractions, and found a branched/straight ratio of about 0.3. This is in good agreement with the values in Table III, and emphasizes a certain degree of biochemical similarity between meibomian and sebaceous glands. The similarity in branched/straight ratios for acids and alcohols reinforces the hypothesis formulated from work on sebum, that alcohols are derived from fatty acids by a mechanism involving the addition of two acetate units (Cunliffe, 1971; Nicolaides, 1974; O’Neill and Gershbein, 1976). The straight-chain patterns of acids and alcohols seen in Pig. 1 show roughly a four-carbon difference, and the results of Andrews (1970) also show a three or four-carbon difference in average chain lengths of alcohols and acids. In the bovine sample of Baron and Blough (1976) the alcohols are exclusively long-chain and branched, but the major components of both acids and alcohols are

HUMAN

MEIBOMIAIL

LIPIDS

299

and ante&o-C,,:,. The mechanismof synthesis of alcoholsin the cow may be entirely different from that in man, and more akin to the enzyme system in mouse preputial gland tumour (Snyder and Malone, 1970), which reduced fatty acids to alcoholsof the samechain length. There seemsto be no marked chain length preference in the conversion of fatty acids to alcoholsin man, although the present results do not rule out the possihility, referred to hy Andrews (1970), t,hat someselectivity for chain length exists in the enzymic coupling of alcohols and acids to produce wax esters. C’unl’usionin the past as to the composition of human meihomian lipid may have :~risenin part from inadequate analytical proceduws, l)ut also according to whet’her the sw~t~plcanalysed wa,sfront it single donor or pc)oled from several donors. Thr~ results j)resentet1alcove indicate that reliance on either wurce would be unwise. until a suffkicnt~lr large numl~cr of intLivitlua1 samples has lwt+n analysetl to define the normal range, an(l bhe functions of the intlivitlnal c.onlponwlts hiI,.\-f? heen deterniinrtl. arateiso-C,,,,

1 wish to thank Drs A. J. Bran ant1D. V. Kauffnlat~ for their help in collectiq lipid san$w, and for helpful discussions,and Jlrs JIarv Gales for her escellent technicd msist~m~c~e. The work wassupported by grants from tlw Jletlical ResearchCouncil and the Royal &w&v. A preliminary report (Jf this work U-Wpresentedat the 2nd Intern:ttionn~ Congre+ of k,ve Research,Jerusalem.1!)7(i. RRFERENC!ES dndrrws. .J. S. (1970). Human tear film lipids. I. Composition of the principal non-polar component. 6.z~. l
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Si~~olnitl~~z. N. (1965). Skin lipids. II. I,ipid class composition of samples from -\-nrions spcciw and ~~l~~it~~~~~i~~~lsites. .I. Am. Oil Cl~~~~i.~t~‘SOC. 42, 691~7Or’. Sic~olaitlw. S. ( 1974). Skin lipids: thrir biochemical uniquerwss. S&we 186, 19-26. Sic~olaitlw, N. and Rap, T. (1965). Skin lipids. III. Fatty chains in skin lipids. The use of T-v/,)ri.~ rvs, INO to different,iate between endogenous and csogenolls components in human skin surfaw lipid.

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S. and C\rells. G. <‘. (1957). On the biogenesis of thv free fatty acids in human skin fat. J. Inwest. llerm. 29. 4E-43. S.. Apon, J. M. B. and 11-ong. D. H. (1976). Further studies of the saturated methyl fatty acids of Verk ~uwosn lipid. Lipids 11, 78 L-90. .J. and Gershbein. L. I,. (1976). Analysis of fatty acid and alcoholic components of schceous lipid t,ypes. J. Chromnfoq. Sci. 14, 28-X.