HPLC analysis of closed, open, and reflex eye tear proteins

T Sitaramamma; S Shivaji; Gullapalli N Rao

ORIGINAL ARTICLE

Year : 1998 | Volume : 46 | Issue : 4 | Page : 239-245

HPLC analysis of closed, open, and reflex eye tear proteins

T Sitaramamma¹, S Shivaji², Gullapalli N Rao¹
¹ L.V. Prasad Eye Institute, Hyderabad, India
² Centre for Cellular and Molecular Biology, Hyderabad, India

Correspondence Address:
T Sitaramamma
L.V. Prasad Eye Institute, Road No. 2, Banjara Hills, Hyderabad - 500 034
India

Source of Support: None, Conflict of Interest: None

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PMID: 10218308

Abstract

Changes in the closed, open and reflex eye tear proteins of normal subjects were compared and analysed. Tear proteins were resolved by high-performance liquid chromatography (HPLC) utilising both gel filtration (P-300 SW) and reverse-phase (C-18) columns and the HPLC fractions were further analysed by sodium dodecyl sulphate - polyacrylamide gel electrophoresis (SDS-PAGE) under reducing and non-reducing conditions. The protein composition of the closed-eye tear was significantly different from that of the open and reflex-eye tear. Secretory IgA (sIgA) was the predominant protein in closed eye tears constituting 49% of the total protein compared to 11% in reflex tears, whereas lysozyme was the predominant protein (53%) in reflex tears. Levels of lactoferrin, lipocalin and lysozyme were relatively constant in both open and reflex tears. HPLC profiles of the closed-eye tears, upon continuous stimulation of lacrimal glands indicated that sIgA was significantly reduced whereas lactoferrin, lipocalin, and lysozyme were significantly increased. These results indicate that the tear composition upon waking attains that of the open eye within 4 to 5 minutes, and upon continuous stimulation this reflects the reflex-eye tear composition. It also indicates that mechanisms responsible for changes in concentration of constitutive and regulated tear protein with stimulus can be studied successfully using non-invasive methods to collect human tears.

Keywords: Tears, proteins, high-performance liquid chromatography

How to cite this article:
Sitaramamma T, Shivaji S, Rao GN. HPLC analysis of closed, open, and reflex eye tear proteins. Indian J Ophthalmol 1998;46:239-45

How to cite this URL:
Sitaramamma T, Shivaji S, Rao GN. HPLC analysis of closed, open, and reflex eye tear proteins. Indian J Ophthalmol [serial online] 1998 [cited 2023 Sep 26];46:239-45. Available from: https://journals.lww.com/ijo/pages/default.aspx/text.asp?1998/46/4/239/24172

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The protein constituents of human tears maintain the surface integrity of the cornea and conjunctiva, protect the eye from microbial invasion, maintain the stability of the tear film and also act as a lubricant between the eye and eyelids.[1][2][3] Because of these important attributes it is absolutely essential to characterise tear proteins from normal individuals since such studies could provide baseline information regarding the number of proteins, the type of protein and their quantitative differences, if any, depending on whether the tears were collected from an open eye, stimulated eye or a closed eye. With this in view a number of studies have been directed towards identification of proteins present in reflex, open and closed-eye tears.[4][5][6] These studies which were based on sodium dodecye sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) clearly revealed that the predominant proteins in reflex and open-eye tears were lactoferrin, lipocalin (previously designated as tear-specific pre-albumin), and lysozyme whereas in closed-eye tears the levels of lactoferrin, lipocalin and lysozyme were quantitatively less and secretory IgA (sIgA) was increased along with an increase in plasma-derived proteins.[4],[7]

Attempts have also been made to quantify the tear proteins from open and reflex tears using high-performance liquid chromatography (HPLC) and quantifying the individual proteins based on the peak areas.[8],[9] However, as yet protein components of closed-eye tears have not been quantified due to diminished and stagnant flow of tears and because activation of complement and recruitment of polymorphonuclear (PMN) cells is considered a sub-clinical inflammatory state. Moreover, although changes in human open-eye tear protein levels with progressively increasing stimulus has been monitored, such studies on closed-eye tears collected immediately upon eye opening after 8 hours of sleep have not been done.

Thus the present investigation was undertaken to quantify the proteins of closed eye tears and to compare this with open and reflex-eye tears; and to evaluate changes in the closed-eye tear composition upon continuous stimulation.

Materials And Methods

Collection of tear samples

This study was approved by the institutional human experimentation committee and tears were collected with the consent of the participants. Tear samples were collected from healthy female and male volunteers (aged 20-40 years) according to the method of Sack et al.[4] None of the donors wore contact lenses. Closed-eye tears were collected immediately upon waking using a calibrated fire-polished disposable glass microcapillary tube of 10 μl capacity. The collection was undertaken under dim light and precautions were taken to prevent blinking of the eye by manually holding the eyelid. By this procedure 2-3 μl of tears could be collected from each eye. If during the collection reflex tearing occurred the sample was not included in the study. Tears collected without any stimulation are referred to as open-eye tears and 3-5 μl tears were collected from each eye by holding open the lower eyelid. Tears were collected from the temporal meniscus at the lower lid margin. Care was taken to avoid touching the lid margins or the corneal surface. Reflex tears were usually collected by holding open the upper eyelid so as to prevent blinking and to induce heavy tearing. Reflex tears were collected at a flow rate always greater than 50 μl per minute. Closed, open and reflex-eye tears were either pooled separately or analysed individually.

Handling of tears

Immediately after collection tear samples were centrifuged at 4° C for 10 min at 5,000 rpm and used as such or stored as aliquots at -70° C for future use.

HPLC of tear proteins

The tear proteins were resolved by HPLC using a Hewlett-Packard 1090 system consisting of two pumps, a Rheodynes injector fitted with a 250 μl loop, a photodiode array detector, an integrator (model 3392A) and a data system controller (model HP 85-B). Samples were analysed on a gel filtration column (P-300 SW from Waters, Milford, MA, USA) capable of separating proteins in the range of 10,000 to 5,00,000 daltons according to their mol. wt. [8,9] The column was equilibrated with the running buffer of 0.1 M PO₄ buffer (pH 5.0) containing 0.5 M NaCl prior to loading samples from various tear types. For all tear types the amount of total protein loaded onto the column was kept constant. HPLC was performed at a constant flow rate of 0.5 ml/min and the eluted proteins were monitored at 280 ran by an online UV detector and fractions corresponding to a particular protein peak were collected separately. The HPLC protein profiles of the tears were automatically analysed for peak areas by the integrator. Each HPLC run lasted for 30 min.

Attempts were also made to separate the tear proteins on a Waters C₁₈ reverse-phase column (46 by 250 nm; DuPont, Wilmington, DE) which separates proteins based on their hydrophobicity. The pore size of the column was 10 μm. The elution buffer used was a linear-gradient of 0 to 100% acetonitrile in 0.1% trifluoroacetic acid and the flow rate was 1 ml/min. The run lasted for 60 minutes. The eluted proteins were monitored at 280 nm and the protein fractions corresponding to each peak were collected separately and stored at -20° C for future use.

Protein estimation

Protein concentration of the tears and the HPLC fractions were measured using the Bradford's method.[10]

SDS-PAGE

SDS-PAGE was performed under reducing and non-reducing conditions according to Laemmli,[11] on a 10% polyacrylamide gel using a mini-gel apparatus. After the completion of electrophoresis the gels were stained with 0.08% Coomassiee Brilliant Blue R-250 in methanol-acetic acid-water (50:7:43, vol by vol) and destained in the above solvent mixture without the dye.

Results

Preliminary studies indicated that closed, open and reflex-eye tears following HPLC on a gel filtration column (P-300 SW) resolved into 4 distinct peaks (designated as P1 to P4 based on their increasing retention times) when 0.1 M phosphate buffer (pH 5.0) containing 0.5 M NaCl was used as the running buffer [Figure - 1]. Significant difference was observed in peak 1 of closed and reflex-eye tears.

Proteins present in human tears and the 4 individual protein peaks resolved by HPLC were further separated by SDS-PAGE [Figure - 2]. The reflex, open and closed-eye tears resolved into 7 distinct proteins designated 1 to 7 of mol. wt. 85, 76, 66, 50, 25, 16, and 13 kDa. Proteins 1, 3, 4, 5, 6 and 7 were present in open, reflex and closed-eye tears whereas protein 2 of mol. wt. 76 kDa was present only in closed-eye tears [Figure:2a]. Based on their mol. wts. and data from earlier literature[8],[9] 6 out of the 7 proteins, namely proteins 1, 2, 3, 5, 6, and 7 were identified as lactoferrin, sIgA heavy chain, albumin, sIgA light chain, lipocalin and lysozyme respectively. SDS-PAGE analysis of the individual HPLC peaks of reflex tears indicated lactoferrin in peak 2, lipocalin in peak 3 and lysozyme in peak 4. Peak 1 following SDS-PAGE did not reveal the presence of any protein under reducing [Figure - 2]b and non-reducing conditions. However, peak 1 of closed-eye tears following SDS-PAGE under non-reducing conditions showed a slow moving band corresponding to sIgA ([Figure:2c], lane 1) and 3 bands corresponding to sIgA heavy chain, albumin and sIgA light chain ([Figure - 2]c, lane 2) after SDS-PAGE under reducing conditions.

Quantifying proteins in reflex and closed-eye tears

HPLC protein profiles of reflex and closed-eye tears were analysed for the purpose of quantifying each of the 4 different types of proteins present in tears. In these experiments the protein concentration of reflex and closed-eye tears which were analysed by HPLC was kept constant so that the area under each peak (which reflects the amount of protein) could be taken as a measure of the protein concentration directly or it could be converted to a percentage of the total area so as to obtain its relative amount in the tear type. [Table - 1] shows the relative amounts of the 4 proteins present in reflex and closed-eye tears and the results indicate a significant difference in the amounts of all the 4 proteins in reflex and closed-eye tears. Further, it was also observed that sIgA (~49%) was the predominant protein in closed-eye tears whereas lysozyme (~53%) was predominant in reflex tears. Closed-eye environment is referred to as a sub-clinical inflammatory state and is known to consist mainly of sIgA. It also shows a marked rise in albumin (67 kDa) content [Figure:2a]. However, using the gel-filtration column the 67 kDa protein was not visible. Hence, a reverse-phase column was also used to resolve the proteins of the closed-eye tears and reflex tears [Figure - 3]. The tears resolved into 5 peaks which were designated as C1 to C5 and each peak was analysed separately by SDS-PAGE. Protein recovery was negligible in peaks C1, C2 and C5 in reflex tears [Figure:4a] and C1 and C2 in closed-eye tears. In reflex tears following SDS-PAGE under reducing conditions proteins were not detected in peaks C1, C2 ([Figure - 4]a, lanes 3 and 4) and C5 (not shown) whereas peak C3 (lane 5) was a mixture of 2 proteins, lactoferrin (85 kDa) and lipocalin (16 kDa) [Figure - 4]a. The protein in peak C4 (lane 6) had a mol. wt. of 13 kDa and was identified as lysozyme. However, under non-reducing conditions in closed-eye tears [Figure:4b] proteins were absent in peaks C1 and C2 only. Peak C3 (lane 1) contained a high mol. wt. protein which did not enter the 10% gel and was identified as sIgA (380 kDa), lactoferrin (85 kDa) and lipocalin (16 kDa) and peak C4 (lane 2) contained lysozyme (13 kDa). Albumin (67 kDa) which is absent in reflex-eye tears but present in closed-eye tears was detected in peak C5 (lane 3).

Changes in closed and open-eye tear composition following continuous stimulation

Closed-eye tears were collected immediately after eye opening and following dilution due to continuous stimulation and analysed for protein content. They were also subjected to HPLC to ascertain the changes in tear proteins [Table - 2]. The protein content of the closed-eye tears was about 15μg/μl when collected immediately after eye opening but following dilution due to stimulation the protein content decreased to 12, 9, 6 and 4μg/μl of tears collected after the second, third, fourth and the fifth collection. The entire collection process lasted 7 minutes. The HPLC results indicated a significant decrease in sIgA with continuous stimulation and concomitantly an increase in the levels of lysozyme, lactoferrin and lipocalin. Similar studies were also carried out with open-eye tears. In these experiments, 5μl of open-eye tears were collected from each eye and immediately afterwards reflex tears were collected from the same eye by inducing tearing which was achieved by holding up the upper eyelid. Ample care was taken to collect the same volume of reflex tears from all the subjects. Analysis of the open-eye tears and reflex tears by HPLC and subsequent quantification indicated that both the tear types contain sIgA, lactoferrin, lipocalin and lysozyme but following stimulation the concentration of sIgA decreased significantly in reflex tears [Table - 3] but the concentration of lactoferrin, lipocalin and lysozyme were not significantly different.

Discussion

Earlier studies have indicated that the predominant proteins in reflex and open-eye tears are lactoferrin, lipocalin and lysozyme whereas the closed-eye tear is characterised by an increase in sIgA, albumin and decrease in lactoferrin, lipocalin and lysozyme.[4] The present study confirms the above observations and provides additional data based on HPLC and SDS-PAGE which clearly demonstrate a quantitative increase in albumin, sIgA heavy chain and sIgA light chain in closed-eye tears. HPLC of closed-eye tears using a gel-filtration column resolved the proteins into 4 peaks which following electrophoresis were identified as containing sIgA and albumin in peak 1 ([Figure - 2]c, lane 1 and 2), lactoferrin, lipocalin and lysozyme in peaks 2, 3 and 4 respectively. The inability to resolve albumin by gel filtration may be due to the fact that albumin probably aggregates with the sIgA fraction (peak 1) as shown in [Figure - 2]c, lane 2. Hence attempts were made to obtain a better resolution of the tear proteins by reverse-phase HPLC. By this method it was indeed possible to resolve albumin as a distinct separate peak. Lysozyme also resolved into a single separate peak but sIgA, lactoferrin and lipocalin were not resolved, indicating that these three proteins may have the same degree of hydrophobicity. Thus, in order to quantify the albumin present in tears it would be necessary to resolve the proteins by reverse-phase HPLC. The present results confirm earlier studies with respect to HPLC of open and reflex tear proteins using a gel-filtration column and also provide for the first time the HPLC profile of closed-eye tears.

Based on SDS-PAGE and Western blot analysis, Sack et al[4] had indicated that sIgA and albumin were predominant in closed-eye tears. The present study confirms this observation and demonstrates that in closed-eye tears sIgA plus albumin constitute 49% of the total protein compared to 11% in reflex tears. The closed-eye environment which exhibits complement activation and recruitment of PMN cells closely resembles a sub-clinical inflammatory state. Thus it is possible that the dramatic increase in sIgA and albumin upon eye closure may have a vital role in protecting the ocular surface along with the changes that occur during the sub-clinical inflammation.

Previous studies[5],[6],[12] had indicated differences between stimulated and non-stimulated human tears with respect to the quantities of the various proteins present in tears. It was observed that certain proteins like immunoglobulins decreased progressively with stimulation while others like albumin and transferrin decreased immediately on stimulation and showed no further change on prolonged stimulation. Further the concentration of lactoferrin, lipocalin, lysozyme, and peroxidase were relatively constant both in the non-stimulated and stimulated tears. The present study also demonstrates a decrease in sIgA concentration following stimulation of open eye and no significant change in the levels of lactoferrin, lipocalin and lysozyme [Table - 3]. The decrease in sIgA is due to the fact that it is a constitutively secreted protein and its secretion is therefore dependent on the rate of synthesis of the protein and not on the rate of secretion.[6] Further, lactoferrin, lipocalin, and lysozyme are the main lacrimal gland proteins and their secretion is known to increase with increase in stimulation[13],[14] and thus their concentrations are maintained. Though much is known about the tear proteins of open eye prior to and after stimulation nothing is known as to changes, if any, that occur immediately following eye opening (closed-eye tears) and stimulation. This study shows a significant decrease in sIgA and increase in levels of lactoferrin, lipocalin and lysozyme [Table - 2] such that by 4 to 5 minutes the protein profile closely resembles that of open-eye tears. Thus continuous stimulation changes the protein composition of closed-eye tears to the open-eye type which subsequently on prolonged stimulation show changes described above for stimulated tears (reflex tears).

Acknowledgment

This project was supported by the Hyderabad Eye Research Foundation, Hyderabad, India.

References

1.	van Haeringen NJ. Clinical biochemistry of tears. Surv Ophthalmol 1981;26:84-96. [PUBMED]
2.	Bron AJ, Seal DV. The defences of the ocular surface. Trans Ophthalmol Soc UK 1986;105:18-25. [PUBMED]
3.	Holly FJ, Lemp MA. Tear physiology and dry eyes. Surv Ophthalmol 1977;22:69-87. [PUBMED]
4.	Sack RA, Tan KO, Tan A. Diurnal tear cycle. Evidence for a nocturnal inflammatory constitutive tear fluid. Invest Ophthalmol Vis Sci 1992;33:626-40. [PUBMED]
5.	Fullard RJ, Snyder AC. Protein levels in non-stimulated and stimulated tears of normal human subjects. Invest Ophthalmol Vis Sci 1990;31:1119-26.
6.	Fullard RJ, Tucker DL. Changes in human tear protein levels with progressively increasing stimulus. Invest Ophthalmol Vis Sci 1992;32:2290-301.
7.	Fukuda M, Fullard RJ, Willcox MDP, Baleriola-Lucas C, Bestawros F, Sweeney D, et al. Fibronection in the tear film. Invest Ophthalmol Vis Sci 1966;32:459-67.
8.	Boonstra A, Kijlstra A. Separation of human tear proteins by high performance liquid chromatography. Curr Eye Res 1976;3:1461-69.
9.	Boonstra A, Breebaart AC, Brinkman CJJ, Luyendijk L, Kuizenga A, Kijlstra A. Factors influencing the qualitative determination of tear proteins by high performance liquid chromatography. Curr Eye Res 1988;7:893-901.
10.	Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilising the principle of protein-dye binding. Anal Biochem 1976;72:248-54. [PUBMED] [FULLTEXT]
11.	Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage. Nature 1970;227:680-85. [PUBMED]
12.	Fullard RJ. Identification of proteins in small tear volumes with and without size exclusion HPLC fractionation. Curr Eye Res 1988;7:163-79. [PUBMED]
13.	Dartt DA. Signal transduction and control of lacrimal gland protein secretion. A review. Curr Eye Res 1989;8:619-36. [PUBMED]
14.	Burgess TL, Kelly RB. Constitutive and regulated secretion of proteins. Annu Rev Cell Biol 1987;3:243-93. [PUBMED] [FULLTEXT]