British Journal of Ophthalmology, 1984, 68, 316-320

Normal tear protein profiles and age-relatedchangesJ. I. McGILL,' G. M. LIAKOS,' N. GOULDING,2 AND D. V. SEAL'From the 'Southampton Eye Hospital, the 2Department of Microbiology, Southampton Medical School,and the 'Division of Communicable Diseases, Clinical Research Centre, Harrow, Middlesex

SUMMARY The specific and non-specific tear proteins have been analysed by means of the ELISAtechnique to establish the normal and age-related values. There is a linear and related decline oflysozyme and lactoferrin with age, and a similar but unrelated reduction in tear volume. IgA levelsgradually decline, while caeruloplasmin and IgG both increase after the fifth decade. The resultssuggest that tear IgG and caeruloplasmin are probably transudates from the serum, that IgA issecreted independently of tear volume, and that lysozyme and lactoferrin are secreted at the samesite but independently of tear volume.

The chemical composition ofhuman tears is complex,as they contain lipids, proteins, and other elementsdissolved in fluid secretions of the lacrimal gland. Theprotective properties of the protein fractions in healthand disease have long been appreciated, but onlyrecently has their enormous complexity becomeapparent. At least 60 components of the tear proteinfraction have been demonstrated,' among them thespecific and non-specific immunoglobulins. Theseform the first line of defence against an externalinfection and seem to be more effective than thesystemically produced antibodies.2 IgA is secreted intears,3 being locally produced in the lacrimal gland,and differs chemically and immunologicalty from itsserum counterparts.4The non-specific proteins include lactoferrin and

lysozyme secreted by the lacrimal acinar cells56 andknown to inhibit bacterial multiplication. Differentestimates of their concentration have been reported.Lactoferrin, one of the major tear proteins,' was firstidentified in tears in 1966,7 but little is known of howthe levels alter in disease, except that there is areduction in the dry eye patient. Similarly, caerulo-plasmin, a glycoprotein carrying copper, has beenidentified in tears by immunoelectrophoresis' and bythe use of monospecific antisera,3 but it has not yetbeen determined how age or ocular disease affectsthese levels. Furthermore, a combined or simult-aneous study of the specific and non-specific tearCorrespondence to James I. McGill, FRCS, Southampton EyeHospital, Wilton Avenuc, Southampton S09 4XW.

31(

proteins has not yet been carried out, either in thenormal eye or in various external eye diseases. Thus,there is a need to establish an accurate estimate ofthese protein concentrations. We have done this bymeans of the enzyme-linked immuno-assay (ELISA)method.9The purpose of our study has been to measure the

normal concentrations of the immunoglobulins IgAand IgG and the tear proteins lysozyme, lactoferrinand caeruloplasmin. Our findings have beencompared with those of other reports where differenttechniques were used. Secondly, we wished to estab-lish for the first time the normal concentration oflactoferrin and caeruloplasmin, and thirdlywe wishedto determine whether age-related changes occur inthe tear film protein components other than lysozyme.

Material and methods

Selection ofsubjects. Fifty-five subjects were selectedfor this study; their ages varied from 20 to 82, coveringthe different age groups equally. The majority ofthem had no ocular history, but a few cases withmacular degeneration were included in the older agegroups.

Collection of specimens. Tear fluid was collectedon Whatman no. 1 filter paper discs, 5mm in diameter,which were placed in the lower fornix of the un-anaesthetised eye."' The volume of tears collectedwas determined by weighing the paper discs in theirbottles before and after collection. Every effort was

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Normal tearprotein profiles and age-related changes

made to avoid injuring the ocular tissues, thusinducing excess lacrimation.Assay ofproteins. The concentrations of IgA, IgG,

lysozyme, lactoferrin, and caeruloplasmin in the tearfluid collected were estimated by ELISA' with teareluates collected as neat tears on filter paper discs towhich phosphate buffered saline was added. "'

Tear volume. This was estimated by the modifiedSchirmer's test " in the unanaesthetised eye bymeasuring the length of filter paper wetted over 5minutes which had been placed in the lower fornix. Amodified version of Schirmer's I test was used,whereby the Schirmer strip was moved to another sitewithin the lower fornix if wetting had not occurredafter 2 minutes.

Results

The mean concentrations of the tear proteins for eachdecade are shown in Table 1. The age-related changesTable I Mean normal values of tearprotein concentrationsfor each decade in mgldl

Age 20-29 30-39 40-49 50-59 60-69 70-18 eyes 18 eyes 18 eyes 18 eyes 17 eyes 19 eyes

Lactoferrin 136 139 118 90 114 87(26) (18) (24) (22) (32) (22)

Lysozyme 110 105 99 79 73 72(22) (18) (20) (16) (24) (28)

Caeruloplasmin 12 13 10 15 15 30(2) (2) (0-8) (4) (2) (14)

IgA 52 63 41 41 52 45(14) (14) (12) (12) (24) (14)

IgG 1-7 0-7 0-7 0-7 2-8 6-5(1-0) (0-2) (0-4) (0-2) (2-2) (22)

Numbers in parentheses are standard deviations.

60'

.5O.

0Cc

E3E.5_Z.2 20'

C 10.c0

(-

Mean

IgA

-2SDlimit

-25D

20 30 40 50 60Age

70 80

Fig. I Age-related changes ofthe immunoglobulins IgAand IgG (110 eyes).

of the specific immunoglobulins (Fig. 1) and non-specific antibacterial proteins are given (Fig. 2).Lysozyme and lactoferrin declined by a similaramount, 1 mg/dl (10 mg/l) for every year, linearly andprogressively with age. IgG and caeruloplasmin levelsremained static until an apparent increase after thefifth decade, while IgA levels only fell slightly withage. Tear flow, as measured by the modifiedSchirmer's test on the open eye, declined steadilyafter the age of 40 (Fig. 3), when there was thensignificant correlation (r= -0.8) between decreasedflow and increasing age.

-2SD

Lactoferrin

-2SDlimit

Caeruloplasmin

Fig. 2 Age-related changes oflysozyme,lactoferrin, and caeruloplasmin(110 eyes for each protein).

Mean

""IO, -2 SD............ .. ....-a"limit

v .

20 40 60 80 20 40Age

140-

_ 120'

c'6E 100'c

.@ 80'0a.

o; 60'0

0)-0 40-

0U- 20'

I 00----V- --I lmit

0 .

317

MeanIgG

t

60 80 20 40 60 80

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J. I. McGill, G. M. Liakos, N. Goulding, and D. V. Seal

30-

24-

18-ScNrmer(mm )

12

0 2o 4bAge (years)

240-

180-

Lactoferrin(mg/dI) 120-

60

0

*. S

0~~~

*z "S S@

00

0000 r1000

@0 0

00 0

0

:0 r-o0

000 0

so 0

O 60 1Ls 180Lysozyme ( mg/dli)

240

.-,----.-,-- Fig. 4 Showing linear relationship between lysozyme and60 80 lactoferrin concentration changes.

Fig. 3 Relationship between the modified Schirmer's testand age, showing that there is a linear decline in tearproduction in the normal subject after the age of4O.

The correlation of one protein with another illus-trates how the concentrations of one protein relate tothose of the other. A positive correlation signifiesthat the concentrations of the 2 behave similarly indifferent subjects, while a negative correlationsignifies that the concentrations alter in oppositedirections.There was a strong positive correlation between

lysozyme and lactoferrin (Fig. 4) and between IgGand caeruloplasmin (Fig. 5). Both lysozyme andlactoferrin showed a negative correlation to IgG andcaeruloplasmin, with IgA showing no significant cor-relation to any of the proteins (Table 2).

In relation to age, lysozyme and lactoferrin showeda negative correlation, as their concentrationsdeclined with age, IgG and caeruloplasmin a positiveone, and IgA no significant correlation. It thusemerged that lysozyme and lactoferrin formed onegroup of similarly behaving proteins (Fig. 4) and IgGand caeruloplasmin another (Fig. 5), but each groupbehaved in an opposite way to the other. IgA behavedindependently. No significant correlation could befound between tear volume collected on the discs andany of the proteins, except in the last 2 decades of life

(60-69, 70-79), when tear volume decreased and theconcentrations of caeruloplasmin (r= -0.33) and IgG(r=-0 3) increased.

Discussion

This work has established the concentrations ofvarious specific and non-specific tear proteins and,more importantly, has shown how these values insome instances alter quite appreciably with age.There is general agreement that IgA is the pre-

dominant tear immunoglobulin, but the reportedconcentrations have varied considerably in relationto the technique used. Our values of 40 mg/dl at theage of 50 are higher than those obtained by some-for example, 14 mg/dl, 2 20*6 mg/dl,'3 and 24-6 mg/dl 14

-but are comparable to others-e.g., 36-8 mg/dl. '5(SI conversion: mg/dlxlO=mg/l.) Only one grouphas measured the concentration at different ages andfound that IgA levels seem to be higher in youngerand older people.'4 Our findings are comparable,showing a small decrease in the 40-49 and the 50-59age groups, and then a small rise in the older groups

but still lower than the younger ones. Our method ofmeasuring IgA in tear eluates, as opposed to neattears, has proved satisfactory.Most reports agree that IgG tear levels are low.3 16

Some, though, have found it in equal quantities to

Table 2 Correlation coefficients between each tear protein measured

Age Lactoferrin Lysozyme Caeruloplasmin IgA

Lactoferrin -0-368* - - - -Lysozyme -0-417* 0-603* - - -

Caeruloplasmin 0-326 -0-234 -0-280 - -

IgA -0-121 0-098 0-082 0-104 -IgG 0-270 -0-271 -0-298 0-841 * 0-043

*Significant to p<0 01.

*\ e~00A

* 00

0 0

0

n=70 *0r--0776 000

318

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Normal tearprotein profiles and age-related changes

=25

°15

E*c 2).E0

.2 15

C-)

0

n=110(8 vc

0of b

r=+0 E*@0

* 0

0@* 0

00

6 x |0 16 32 48

IgG (mg/dl)Fig. 5 Showing relationship between IgGCcaeruloplasmin concentrations.

IgA,'2'" while others'7 have found theto be much higher at 79 mg/dl. Thistimes the concentration of IgA as msame authors. It is likely that these higiresult of using incompletely absorbedtechnique of detecting the specific imrso that there was significant cross-realbeing measured as IgG. Our mean agewas 2 mg/dl, but in some cases there wconcentration in the older group. Texplanation is increased concentrationtear flow. This is confirmed by the findconcentration increase in caeruloplasbe formed by hepatocytes. 8 A raticand caeruloplasmin thus has useparticularly in tear evaporation statesLysozyme, perhaps the most wide

tear protein, has been estimated by theplate " with hen egg lysozyme standarmean age-adjusted value of 100 mg/dl,lysozyme standard, which comparevalues of 170 mg/dl found with the lysoand 130 mg/dl by a less sensitive animmunoassay method.21 We foundlevels started falling after the age ofdecrease of 1 mg/dl per year of age,rate of decline as that for lactoferrreports exist as to the effect of age on 13some reporting no change,2' whilegradual decline with age. l 19 20 22 Thoscannot detect a decreasing concentratilikely to be using an insensitive metho

Lactoferrin levels were found to haadjusted value of 140 mg/dl, confirmiBroekuyse,23 and, just like lysozynsteadily with age at 1 mg/dl per year.

between lactoferrin and lysozyme in terms of con-centration and decline with age was impressive. Inspite of their different molecular weights (75 000 forlactoferrin and 15 000 for lysozyme), both are identi-fied within the same molecular range, ' have low serum

* * o levels324 and a similar production site, identified asthe acinar epithelial cells of both the main and the

alues out accessory lacrimal tissues.56 Both have antibacterial)ounds ) properties,2526 have a strong positive correlation to341 each other in terms oftheir concentration, and decline

at the same rate with age. It is therefore logical toexpect that a pathological reduction of tear secretion,

* such as in dry eyes, results in decreased levels notonly of lysozyme but also of lactoferrin. Other

6 4 8-0 workers have found decreased lysozyme levels in dryeyes 121 27 as well as decreased lactoferrin levels.28 It

and thus appears that the measurement of both lactoferrinand lysozyme will represent useful diagnostic tests indry eye investigations. This has already beensuggested in a recent study of tear lactoferrin.29

concentration The role and concentration of caeruloplasmin inis more than 3 tears has so far not been determined. We found aeasured by the mean age-adjusted value of 16 mg/dl, with a markedh values are the increase with age over 70 years, in a few cases. Asantisera in the with IgG, this apparent increase with advancing agenunoglobulins, might be due to increased concentration.ction, with IgA It was interesting that there was a significant-adjusted value positive correlation between IgG and caeruloplasmin,,as an increased suggesting a common source of origin, different from'he most likely that of lysozyme and lactoferrin, with which thereidue to reduced was a negative correlation. Both have probably beenling of a similar secreted from serum, either by the lacrimal gland or;min, known to via the conjunctiva, and suggests that the blood-tear) between IgG protein barrier is incomplete, allowing a small leakageful potential, of serum protein to occur.seen clinically. There are still a large number of tear proteins thatly investigated remain to be studied and quantified before the defen-lysozyme/agar sive mechanism of the tears against external infectionds. We found a is fully understood. We have estimated the concen-, using a human tration of 5 tear proteins and, by so doing, hope to bes with higher able to evaluate their significance in pathological)plate method'9 states. Expressing protein concentrations in ratiosd less accurate between each other magnifies changes that occur inthat lysozyme either of the proteins expressed, and offers a more40 at a rate of sensitive and accurate method of investigating path-with the same ological changes in various ocular disorders. Now

*in. Conflicting that these normal and age-adjusted values of the tearysozyme levels, proteins have been established, it will be possible toothers give a study their significance in various ocular disorders.

se workers whoion with age are)d.yve a mean age-ing the work ofne, it declines.The similarity

We thank the Research Committee of the Wessex Regional HealthAuthority (grant to N.J.G.) and the British National Committee forthe Prevention of Blindness (grant to J. M. and G. M. L.) for grants toenable this work to be carried out. We also thank Professor PeterWatt, Department of Microbiology, University of Southampton, formuch valuable advice as well as laboratory facilities.

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J. 1. McGill, G. M. Liakos, N. Goulding, and D. V. Seal

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age-related changes.Normal tear protein profiles and

J I McGill, G M Liakos, N Goulding and D V Seal

doi: 10.1136/bjo.68.5.3161984 68: 316-320 Br J Ophthalmol 

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