- Email: [email protected]
Effect of Stimulus on the Chemical Composition of Human Tears
E F F E C T O F STIMULUS ON T H E C H E M I C A L C O M P O S I T I O N O F H U M A N TEARS W I L L I A M H. F R E Y II, P H . D . , D E N I S E D E S O T A - J O H N S O N , AND C A R R I E H O F F M A N , B.S.
B.A.,
St. Paul, Minnesota AND JOHNT. MCCALL,
PH.D.
Rochester, Minnesota
We examined the effect of the nature of the stimulus on the chemical composition of human tears. The protein concentration of emotional tears from women exceeded that of irritant-induced tears by 24% (P<.01). Polyacrylamide disk gel electrophoresis disclosed no significant difference between the distribution of positively or negatively charged proteins of irritant-induced and emotional tears. Manganese concentrations in tears (30 ng/ml) exceeded serum concentrations from the same subjects by 30-fold. These manganese concentrations in tears were considerably less than previously reported values. We found no differences for the concentrations of protein or manganese in human tears between the sexes.
The effect of the nature of the stimulus on the chemical composition of human tears has not been adequately investigat ed. The first chemical analysis of tears was published by Fourcroy and Vauquelin 1 in 1791. Since that time, the physical and chemical properties of tears resulting from a variety of causes have been exam ined.2"11 Most of these studies were per formed on tears induced by a variety of ocular irritants. 2 Tapaszto 12 reported unusually high concentrations of man-
From the Department of Psychiatry, St. PaulRamsey Medical Center, St. Paul, Minnesota (Dr. Frey, Ms. DeSota-Johnson, and Ms. Hoffman), and the Metals and Toxicology Laboratory, Mayo Medical Center, Rochester, Minnesota (Dr. McCall). This work was supported by grant No. 8241 from the St. Paul-Ramsey Hospital Medical Education and Re search Foundation (Dr. Frey). Reprint requests to William H. Frey II, Ph.D., Department of Psychiatry, St. Paul-Ramsey Medical Center, 640 Jackson St., St. Paul, MN 55101.
ganese for human tears. The manganese concentration in the tears of men (0.48± 0.04 mg/100 ml) was 24 times greater than that in the tears of women and 5,000 to 10,000 times greater than that normally found in human serum. 1213 This suggests a possible difference be tween the tears of men and women and an unusual ability of the lacrimal or acces sory glands to concentrate manganese. The protein composition of tears is complex. Measurements of total protein concentration range from as low as 1.36 mg/ml 14 to as high as 59 mg/ml. 15 Josephson and Weiner 16 presented evi dence that the protein concentration is inversely related to the weight of tears produced. They suggested that the pro tein being measured is that found in the conjunctival sac fluid at low tear flow and that this is diluted by a lacrimal secretion of low protein concentration. Their data did not indicate any further effect of
AMERICAN JOURNAL OF OPHTHALMOLOGY 92:559-567, 1981
559
560
AMERICAN JOURNAL OF OPHTHALMOLOGY
increasing tear volume on protein con centration after 35 μΐ of tears was collect ed. Despite this and other problems, a number of investigators have reported values ranging from 3 to 8 mg/ml.12,17"19 The only comparisons of irritantinduced tears and emotional tears were published by Brunish in 195620 and 1957.18 Brunish observed that even though the volume of tears excreted dur ing emotional circumstances was greater than that obtained by using ocular irri tants, the protein concentration of emo tional tears (about 6 mg/ml) exceeded that of irritant-induced tears (about 4 mg/ml). He further reported that the protein dis tribution among lysozyme, globulin, and albumin fractions, obtained with paper electrophoresis, was different for the two types of tears. 18 The percentage of total protein appearing in the albumin fraction was at least twice as great in emotional as in irritant-induced tears. This finding was disputed in 1959 by Krause 19 who compared the electrophoretic distribution of irritant-induced tears with those of patients with epiphora. Krause did not, however, examine emo tional tears. Some years later, Botelho 21 reported that, "The ratio between the proteins albumin and globulin in reflex (irritant) tears is about the same as in serum, but psychogenic (emotional) tears may contain less albumin." However, she gave no data to support this conclusion. Many other studies of human tears have reported these three major protein frac tions.2'18,19,22"24 Using paper electrophore sis, Caselli and Schumacher 26 observed five different peaks. An even larger num ber of protein fractions have been distin guished with agarose 12 or polyacrylamide disk gel26 electrophoresis. We conducted a study to determine whether the nature of tear-inducing stim ulus affects the chemical composition of the tears.
OCTOBER, 1981
S U B J E C T S AND M E T H O D S
Tear donors—We collected tears from healthy male and female volunteers rang ing in age from 18 to 60 years (average age, 28 years). Approximately 80% of the subjects were female. Some subjects wore spectacles or hard or soft contact lenses, but the lenses were not worn during the tear collection procedure. Tear collection—We asked all the sub jects to wash their faces with soap and water before participating in the study. (None of the subjects wore cosmetics during the collection procedure.) We told the subjects to avoid rubbing their eyes because rubbing could cause some dam age to the corneal epithelium and alter the chemical composition of the tears. 27 Release of epithelial cell components into the tears was assessed by measuring lactate dehydrogenase 27,28 and adenosine triphosphatase 29 activities. We detected no activity in four samples of irritantinduced and emotional tears selected at random, indicating that epithelial cell damage was not a problem. Also, centrifugation of tear samples to remove any cells did not significantly alter the manga nese concentration. The average volume of emotional tear samples, 250 μΐ (range, 30 to 1,230 μΐ), was twice as great as that of the irritant-induced tear samples, 110 μΐ (range, 30 to 642 μΐ). There was no correlation between the volume of tears collected and the protein or manganese concentration of irritant-induced or emo tional tears. We collected irritant induced tears by exposing the subjects to the vapors of freshly cut onions for about three min utes. The tears were placed in preweighed 12 X 75-mm glass test tubes held just below the lower eyelid. This method is preferable to collecting tears directly from the surface of the eye, which often results in damage to the
VOL. 92, NO. 4
CHEMICAL COMPOSITION OF HUMAN TEARS
corneal epithelium and release of cellular material into the tears. 27 Emotional tears were collected within one week of collecting the irritantinduced tears. The subjects watched sad movies about two hours in length and collected their own tears into preweighed glass test tubes held just below the lower eyelid. The emotional status of the subject immediately before and after lacrimation was assessed by the Profile of Mood States. The Profile of Mood States is a self-rated scale consisting of 65 adjectives and was designed to assess feelings, af fect, and mood and their changes under therapeutic intervention or experimental manipulation. The Profile of Mood States has been extensively evaluated and nor mative samples for subjects are avail able. 30 We asked the subjects to evaluate their moods before lacrimation retrospec tively, that is, after the lacrimation proc ess was complete. Once collected, the tears were weighed, frozen immediately, and stored at —20 C until analyzed. To determine if evaporation of the tear samples while they were at room temperature altered their protein concentration, we prepared a mock tear solution of albumin and lysozyme and left 20-, 50-, 100-, and 200-μ1 aliquots exposed to air at room tempera ture for two hours. No change in protein concentration was detected after the twohour period. Similar samples were frozen for one, seven, 14, and 30 days and then tested for protein concentration. Varying periods of storage at — 20 C had no effect on the protein concentration. To deter mine whether tears could be contaminat ed by protein or manganese picked up during brief exposure to the skin below the eyelid, 0.9% sodium chloride was allowed to drip down the face, and then collected and analyzed. We detected no protein in the saline samples, and
561
five saline samples contained an average of only 1.6 ng/ml of manganese. Saline washes of the lower eyelid also contained negligible concentration of protein and manganese. Tear protein concentration—We deter mined the protein concentration of human tear samples by the method of Bradford, 31 using bovine serum albumin (E280, 1% = 6.6) as the standard. 32 Nor mally, we assayed 3 μΐ of tears from each sample in triplicate. Tear protein electrophoresis—We ana lyzed the tear samples (3 μΐ) by polyacrylamide disk gel electrophoresis on 5.5 X 100-mm, 7.5% polyacrylamide gels. The electrophoresis was conducted at p H 8.9, using 3 mamps per gel, for nega tively charged proteins and at p H 3.6 for positively charged proteins. The gels were fixed in 10% trichloroacetic acid for 30 minutes and stained with Coomassie brillant blue R. After unbound stain was removed, we scanned the gels at 570 nm with a spectrophotometer. Tear manganese concentration—The manganese concentrations of the tear samples were determined with an atomic absorption spectrophotometer with a HGA-500 graphite furnace. Blood sam ples (10 ml) were collected and centrifuged at 1,700 g for ten minutes. The serum was stored at —20 C in sealed Teflon cups until analyzed. The tear sam ples were collected directly into Teflon cups or were transferred into the cups after collection in glass tubes. We usually diluted the tear samples 20-fold with glass-distilled deionized water containing a manganese concentration of less than 0.3 ng/ml. All glass and Teflon was washed with 10% nitric acid and glassdistilled deionized water before use. Tears collected in washed glass tubes were not significantly different from those collected in washed Teflon cups with respect to manganese concentration.
562
AMERICAN JOURNAL OF OPHTHALMOLOGY RESULTS
Protein concentration of human tears—The effect of stimulus on the pro tein concentration of human tears is shown in Table 1. When all of the tear samples from women were analyzed, the protein concentration of the emotional tears (42 subjects) exceeded that of the irritant-induced tears (61 subjects) by 21% (P < .001). To eliminate the influence of differ ences between individuals, we compared the protein concentration of emotional tears to the protein concentration of irritant-induced tears obtained from the same subject on a subsequent day. Paired samples from 25 women also showed that the protein concentration of emotional tears was 24% greater than that of irritant-induced tears (P < .01) (Table 1). We found a higher protein concentra tion for emotional tears from men as well (P < .05), although we obtained paired samples from only four subjects. There was no significant difference between the protein concentration of tears from men and women when the same stimulus was
OCTOBER, 1981
used. The protein concentration of the emotional tears exceeded that of the irritant-induced tears in 23 of the 29 subjects examined. The protein concentrations of irritantinduced and emotional tears from five women who wore hard contact lenses and five women who wore soft contact lenses were not significantly different from the results for the other subjects. The sub jects did not wear contact lenses when the tears were collected. Of the 25 paired samples from women examined (Table 1), six were from subjects who wore specta cles. The tear protein concentration of those who wore spectacles did not differ significantly from that of those who did not. The variation in tear protein concen tration was as great between different samples obtained from the same subject on different occasions as between sam ples obtained from different subjects. Electrophorettc distribution of human tear proteins—Polyacrylamide disk gel electrophoresis distinguished approxi mately eight different negatively'charged protein peaks (Fig. 1) and eight different positively charged protein peaks (Fig. 2)
TABLE 1 P R O T E I N CONCENTRATIONS IN TEARS F R O M W O M E N AND MEN
Tears Irritant-induced No. of samples Protein (mg/ml) Emotional No. of samples Protein (mg/ml) P (Student's t-test)
Women
Men
Combined
61 5.40 ± 0.17
16 5.75 ± 0.37
77 5.47 ± 0.16
42 6.54 ± 0.25 <.001
5 6.18 ± 0.67 Not significant
47 6.50 ± 0.23 <.001
25 5.36 ± 0.24
4 5.22 ± 0.66
29 5.34 ± 0.22
25 6.62 ± 0.30 <.01
4 6.01 ± 0.83 <.05
29 6.53 ± 0.28 <.001
P A I R E D SAMPLES*
Irritant-induced No. of samples Protein (mg/ml) Emotional No. of samples Protein (mg/ml) P (Student's t-test)
*Emotional and irritant-induced tears were obtained from each subject.
VOL. 92, NO. 4
CHEMICAL COMPOSITION OF HUMAN TEARS
Fig. 1 (Frey and associates). Electrophoresis of the negatively charged proteins of human emotional (dashed line) and irritant-induced (solid line) tears. The subject was a 33-year-old woman.
in both emotional and irritant-induced tears. Each peak probably contained more than one protein species. To quantitate the distribution of protein among the various charged species, we divided the negative proteins into four groups (α, β, 7, δ) and the positive proteins into three groups (α, β, 7) on the basis of their electrophoretic mobility (Figs. 1 and 2). Table 2 shows the distribution of positive proteins among these groups for emotion al and irritant-induced tears from men and women, and Table 3 the distribution of negative proteins. Our results for 20 paired samples from women and three
563
Fig. 2 (Frey and associates). Electrophoresis of the positively charged proteins of human emotional (dashed line) and irritant-induced (solid line) tears. The subject was a 33-year-old woman.
paired samples from men showed no sig nificant difference between the electro phoretic distribution of positive tear pro teins from emotional and irritant-induced tears. Also the distribution of positive tear proteins in tears from men was not significantly different from that in tears from women (Table 2). The electrophoretic distribution of negative proteins from emotional and irritant-induced tears was also remark ably similar. There was no significant dif-
TABLE 2 PERCENTAGE OF POSITIVELY CHARGED PROTEINS IN ELECTROPHORETIC FRACTIONS OF HUMAN TEARS
Proteins a protein (%) Emotional Irritant-induced ß protein (%) Emotional Irritant-induced y protein (%) Emotional Irritant-induced
Women (20 samples*)
Men (3 samples*)
Combined (23 samples*)
47.8 ± 1.9 49.3 ± 1.7
53.6 ± 4.1 52.0 ± 5.9
48.6 ± 1.7 49.7 ± 1.6
29.4 ± 2.3 27.4 ± 2.1
26.0 ± 7.8 27.4 ± 8.5
28.9 ± 2.1 27.4 ± 1.9
22.8 ± 0.9 23.3 ± 0.9
20.5 ± 3.9 20.6 ± 2.8
22.5 ± 0.9 23.0 ± 0.8
*All samples were paired, that is, emotional and irritant-induced tears were obtained from each subject.
564
AMERICAN JOURNAL OF OPHTHALMOLOGY
OCTOBER, 1981
TABLE 3 PERCENTAGE O F NEGATIVELY CHARGED PROTEINS IN E L E C T R O P H O R E T I C FRACTIONS O F HUMAN TEARS
Proteins a protein (%) Emotional Irritant-induced ß protein (%) Emotional Irritant-induced 7 protein (%) Emotional Irritant-induced δ protein (%) Emotional Irritant-induced
Women (20 samples*)
Men (3 samples*)
Combined (23 samples*)
36.0 ± 1.5 37.1 ± 1.5
36.5 ± 8.5 43.4 ± 5.5
36.0 ± 1.5 37.9 ± 1.4
10.5 ± 1.1 8.6 ± 0.5
14.3 ± 3.5 10.5 ± 3.7
11.0 ± 1.0 8.8 ± 0.6
7.3 ± 1.0 6.3 ± 0.9
9.8 ± 0.8 6.3 ± 1.2
7.6 ± 0.8 6.3 ± 0.8
46.2 ± 2.3 48.0 ± 2.0
39.5 ± 10.2 39.9 ± 10.1
45.4 ± 2.3 47.0 ± 2.0
*All samples were paired, that is, emotional and irritant-induced tears were obtained from each subject.
ference between emotional and irritantinduced tears from men and women (Table 3). Although we observed no sig nificant difference between the δ (albu min) fraction of emotional and irritantinduced tears (Table 3), we occasionally found samples with small differences (Fig. 1). However, these differences were not of the magnitude reported by Brunish, 18 and the percentage of protein in the δ fraction of emotional tears did not ex ceed that of irritant-induced tears in most pairs examined (Table 3). The distribu tion of negative proteins of the irritantinduced and emotional tears from men did not differ significantly from those from women. Emotional tear samples ob tained from the left and right eyes of three different subjects did not show any significant difference between the elec trophoretic distribution of tear proteins. Manganese concentration of human tears—The manganese concentration of emotional tears (25.2 ± 6 . 6 ng/ml) was not significantly different from that of irritant-induced tears (34.2 ± 8 . 8 ng/ml) obtained from the same healthy female subjects (Table 4). The 15 subjects ranged
in age from 19 to 56 years (average age, 28 ± 2 years). The manganese concentra tion of emotional tears from women ex ceeded that of serum (0.98 ± 0.09 ng/ml) obtained from the same subjects by about 30-fold (Table 4). The manganese concen tration of irritant-induced tears from men (37.8 ± 4 . 8 ng/ml) was not significantly different from that of irritant-induced tears from women (34.2 ± 8.8 ng/ml), contrary to the earlier report by Tapaszto 12 (Table 4). The 14 male subjects ranged in age from 19 to 40 years (average age, 28 ± 2 years). There was no signifi cant correlation of manganese concentra tion with age for male or female subjects. The manganese concentrations of irritant-
TABLE 4 MANGANESE
CONCENTRATIONS IN HUMAN AND SERUM
TEARS
Manganese Concentration (ng/ml)
Women (No. = 15)
Men (No. = 14)
Emotional tears Irritant-induced tears Serum
25.2 ± 6.6 34.2 ± 8.8 0.98 ± 0.09
37.8 ± 4.8
VOL. 92, NO. 4
CHEMICAL COMPOSITION OF HUMAN TEARS
induced tears from men and women re ported by Tapaszto 12 were approximately 127-fold higher and sixfold higher, re spectively, than those presented here. DISCUSSION
The increased protein concentration of emotional tears compared to irritantinduced tears demonstrated that the lacrimal and accessory glands do excrete different tears in response to different stimuli. This finding agrees qualitatively with the previous report by Brunish. 18 However, we do not know the sample size and method of protein determination Brunish employed. Furthermore, Brun ish included tears from some subjects with idiopathic excessive tearing (per haps epiphora) along with emotional tears for some of his studies because his results indicated that they were similar. He re ferred to these as normal tears and found they had a markedly greater albumin content than irritant-induced tears. 18 This finding was later disputed by Krause 19 who found no difference between the albumin content of irritant-induced tears and those of patients with epiphora, al though he did not examine emotional tears. Our results clearly showed that there was no difference between the percent age of proteins in irritant-induced and emotional tears which migrate as "al bumin" or any other charged species, contrary to the report by Brunish. 18 We compared irritant-induced tears with emotional tears obtained from the same subject in order to avoid problems with individual differences, something Brun ish did not do. With our polyacrylamide electrophoresis system, we were able to distinguish many more protein species than Brunish could with paper electro phoresis in 1957. However, neutral pro teins (zero net charge) and proteins with molecular weights over 150,000 would probably not be easily observed with our
565
electrophoresis system. Our failure to observe differences in the electrophoretic patterns of irritant-induced and emotion al tears does not prove that differences do not exist in individual proteins. Differ ences in individual proteins not constitut ing a high percentage of total protein may not be observed by this method. The manganese concentration of hu man tears exceeded that of serum by approximately 30-fold. This suggested that the lacrimal or accessory glands con centrate manganese from the serum (or some other source) and excrete it in the tears. The mechanism by which this pro cess occurs is unknown, as is the reason for its occurrence. The possibility that a por tion of the manganese found in tears is derived from damaged cells of the corneal epithelium cannot be eliminated, al though we were unable to detect any adenosine triphosphatase or lactate dehydrogenase activity (from damaged cells) in our tear samples. The concentration we found in irritant-induced tears from men (37.8 ng/ml) was less than 1% of the value previously reported by Tapaszto. 12 Tapaszto gave no information regarding the stimulus for tear secretion or the method of tear collection he used. Also, we, unlike Tapaszto, observed no differ ence between the manganese concentra tions of tears from women and men. The high values for tear manganese concen tration reported by Tapaszto led us to suspect that sample contamination may have been a problem. The ability to shed tears in response to emotional stress (psychogenic lacrimation) is a physiologic function that only developed with the evolution of the human being. 33 Psychogenic lacrimation is not only a phylogenetically late devel opment, but also it occurs late ontogenetically, because the human infant does not normally exhibit tears with its crying until it is several days, or even weeks, old. 22 This occurs despite a functional
566
AMERICAN JOURNAL OF OPHTHALMOLOGY
lacrimal system that secretes reflex tears in response to ocular irritation. There fore, it appears the human infant must wait for further development of the cen tral nervous system before it becomes capable of psychogenic lacrimation. 33 Psychogenic lacrimation is not inhibited by surface anaesthesia, paralysis of the fifth nerve, or section of the cervical sympa thetic nerve as is irritant-induced tear ing. 34 The purpose of psychogenic lacrima tion is unknown, although a number of theories regarding its function have been proposed. Through further investigations of psychogenic tears, we may learn more about the purpose of psychogenic lacri mation, the body's specific exocrine re sponse to emotional stress. REFERENCES 1. Fourcroy, A. F . , and Vauquelin: The chemical examination of tears and of the nasal secretion. Ann. Chimie 10:113, 1791. 2. Altaian, P. L. : Physical properties and chemi cal composition of tears. Man. In Dittmer, D. S. (ed.): Blood and Other Body Fluids. Washington, D.C., Federation of American Societies of Experi mental Biology, 1961, pp. 488-490. 3. Holly, F. J., Patten, J. T., and Dohlman, C. H. : Surface activity determination of aqueous tear components in dry eye patients and normals. Exp. Eye Res. 24:479, 1977. 4. Sorensen, T., and Jenson, F. T. : Methodologi cal aspects of tear flow determination by means of a radioactive tracer. Acta Ophthalmol. 55:726, 1977. 5. Norn, M. S.: Outflow of tears and its influence on tear secretion and break up time (B.U.T.). Acta Ophthalmol. 55:674, 1977. 6. Carney, L. G., and Hill, R. M.: Human tear pH. Diurnal variations. Arch. Ophthalmol. 94:821, 1976. 7. Winder, A. F., Ruben, M., and Sheraidah, G. A. K. : Tear calcium levels and contact lens wear. Br. J. Ophthalmol. 61:539, 1977. 8. Horowitz, G., Lammers, J. W., Sussman, S., and Botelho, S. Y. : Calcium in tears, lacrimal gland fluid, and fluid from the other orbital glands. Invest. Ophthalmol. 15:994, 1976. 9. van Haerington, N. J., and Glasius, E.: Collec tion method dependant concentrations of some me tabolites in human tear fluid, with special reference to glucose in hyperglycaemic conditions. Albrecht von Graefes Arch. Klin. Exp. Ophthalmol. 202:1, 1977.
OCTOBER, 1981
10. Hill, R. M., and Terry, J. E.: Human tear cholesterol levels. Arch. Ophthalmol. 36:155, 1976. 11. Zubareva, T. V., and Kiseleva, Z. M.: Catacholamine content of the lacrimal fluid of healthy people and glaucoma patients. Ophthalmologica 175:339, 1977. 12. Tapaszto, I.: Pathophysiology of human tears. Ophthalmol. Clin. 13:119, 1973. 13. Versieck, J., and Cornells, R. : Normal levels of trace elements in human blood plasma or serum. Anal. Chim. Acta 116:217, 1980. 14. Junnola, K. : Über die Eigenschaften der menschlichen Tränenflussigkeit, thesis, Helsinki, Helsingin, Liikekirjapaino Oy, 1952, pp. 1-90. 15. Rennert, O. M., Kaiser, D . , Sollberger, H., and Joller-Jemelka, S.: Antiprotease activity in tears and nasal secretions. Humangenetik 23:73, 1974. 16. Josephson, A. S., and Weiner, R. S.: Studies of the proteins of lacrimal secretions. J. Immunol. 100:1080, 1968. 17. Allansmith, M.I Immunology of tears. Int. Ophthalmol. Clin. 13:47, 1973. 18. Brunish, R.: The protein components of human tears. Arch. Ophthalmol. 57:554, 1957. 19. Krause, U.: A paper electrophoretic study of human tear proteins. Acta Ophthalmol. 53 (suppl.):l, 1959. 20. Brunish, R. : The protein components of human tears, abstract. Am. J. Ophthalmol. 41:540, 1956. 21. Botelho, S. Y. : Tears and the lacrimal gland. Sei. Am. 211:78, 1964. 22. Allerhand, J., Karelitz, S., Penbharkkul, S., Ramos, A., and Isenberg, H. D.: Electrophoresis and immunoelectrophoresis of neonatal tears. J. Pediatr. 62:85, 1963. 23. Erickson, E. F . , Hatlen, R., and Berg, M.: Industrial tear study. Filter-paper electrophoresis of tears, with results of an industrial study from 1,000 specimens sent by mail. Am. J. Ophthalmol. 47:499, 1959. 24. McEwen, W. K., Kimura, S. J., and Feeney, M. L. : Filter-paper electrophoresis of tears. III. Human tears and their high molecular weight com ponents. Am. J. Ophthalmol. 45:67, 1958. 25. Caselli, P., and Schumacher, H.: Über den nachweis des tränenlysozyms mittels mikroelektrophorese. Klin. Monatsbl. Augenheilkd. 124:148, 1954. 26. Sapse, A. T., Bonavida, B., Stone, W., Jr., and Sercarz, E. E.: Proteins in human tears. I. Immunoelectrophoretic patterns. Arch. Ophthalmol. 81:815, 1969. 27. van Haeringen, N. J., and Glasius, E.: The origin of some enzymes in tear fluid, determined by comparative investigation with two collection meth ods. Exp. Eye Res. 22:267, 1976. 28. Goldberg, E.: Amino acid corr.fjsition and properties of crystalline lactate dehydrogenase X from mouse testes. J. Biol. Chem. 247:2044, 1972. 29. Warren, G. B., Toon, P . A . , Birdsall, N. J. M., Lee, A. G., and Metcalfe, J. C : Reconsti-
VOL. 92, NO. 4
CHEMICAL COMPOSITION OF HUMAN TEARS
tution of a calcium pump using defined membrane components. Proc. Natl. Acad. Sei. 71:622, 1974. 30. Guy, W.: ECDEU Assessment Manual for Psychopharmacology, rev. ed. DHEW No. (ADM) 7&-338, Rockville, Maryland, 1976, pp. 529-531. 31. Bradford, M. M.: A rapid and sensitive meth od for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248, 1976.
567
32. Tanford, C , and Roberts, G. L.: Phenolic hydroxyl ionization in proteins (I) bovine serum albumin. J. Am. Chem. Soc. 74:2509, 1952. 33. Walsh F. B., and Hoyt F. W.: Clinical NeuroOphthalmology. Baltimore, Williams and Wilkins Co., 1969, vol. 1, pp. 551-557. 34. Mutch, J. R. : The lacrimation reflex. Br. J. Ophthalmol. 28:317, 1944.
O P H T H A L M I C MINIATURE
But it is not ignoble to feel that the fuller life which a sad experience has brought us is worth our own personal share of pain. Surely it is not possible to feel otherwise, any more than it would be possible for a man with cataract to regret the painful process by which his dim blurred sight of men as trees walking had been exchanged for clear outline and effulgent day. George Eliot, Adam Bede
B.A.,
St. Paul, Minnesota AND JOHNT. MCCALL,
PH.D.
Rochester, Minnesota
We examined the effect of the nature of the stimulus on the chemical composition of human tears. The protein concentration of emotional tears from women exceeded that of irritant-induced tears by 24% (P<.01). Polyacrylamide disk gel electrophoresis disclosed no significant difference between the distribution of positively or negatively charged proteins of irritant-induced and emotional tears. Manganese concentrations in tears (30 ng/ml) exceeded serum concentrations from the same subjects by 30-fold. These manganese concentrations in tears were considerably less than previously reported values. We found no differences for the concentrations of protein or manganese in human tears between the sexes.
The effect of the nature of the stimulus on the chemical composition of human tears has not been adequately investigat ed. The first chemical analysis of tears was published by Fourcroy and Vauquelin 1 in 1791. Since that time, the physical and chemical properties of tears resulting from a variety of causes have been exam ined.2"11 Most of these studies were per formed on tears induced by a variety of ocular irritants. 2 Tapaszto 12 reported unusually high concentrations of man-
From the Department of Psychiatry, St. PaulRamsey Medical Center, St. Paul, Minnesota (Dr. Frey, Ms. DeSota-Johnson, and Ms. Hoffman), and the Metals and Toxicology Laboratory, Mayo Medical Center, Rochester, Minnesota (Dr. McCall). This work was supported by grant No. 8241 from the St. Paul-Ramsey Hospital Medical Education and Re search Foundation (Dr. Frey). Reprint requests to William H. Frey II, Ph.D., Department of Psychiatry, St. Paul-Ramsey Medical Center, 640 Jackson St., St. Paul, MN 55101.
ganese for human tears. The manganese concentration in the tears of men (0.48± 0.04 mg/100 ml) was 24 times greater than that in the tears of women and 5,000 to 10,000 times greater than that normally found in human serum. 1213 This suggests a possible difference be tween the tears of men and women and an unusual ability of the lacrimal or acces sory glands to concentrate manganese. The protein composition of tears is complex. Measurements of total protein concentration range from as low as 1.36 mg/ml 14 to as high as 59 mg/ml. 15 Josephson and Weiner 16 presented evi dence that the protein concentration is inversely related to the weight of tears produced. They suggested that the pro tein being measured is that found in the conjunctival sac fluid at low tear flow and that this is diluted by a lacrimal secretion of low protein concentration. Their data did not indicate any further effect of
AMERICAN JOURNAL OF OPHTHALMOLOGY 92:559-567, 1981
559
560
AMERICAN JOURNAL OF OPHTHALMOLOGY
increasing tear volume on protein con centration after 35 μΐ of tears was collect ed. Despite this and other problems, a number of investigators have reported values ranging from 3 to 8 mg/ml.12,17"19 The only comparisons of irritantinduced tears and emotional tears were published by Brunish in 195620 and 1957.18 Brunish observed that even though the volume of tears excreted dur ing emotional circumstances was greater than that obtained by using ocular irri tants, the protein concentration of emo tional tears (about 6 mg/ml) exceeded that of irritant-induced tears (about 4 mg/ml). He further reported that the protein dis tribution among lysozyme, globulin, and albumin fractions, obtained with paper electrophoresis, was different for the two types of tears. 18 The percentage of total protein appearing in the albumin fraction was at least twice as great in emotional as in irritant-induced tears. This finding was disputed in 1959 by Krause 19 who compared the electrophoretic distribution of irritant-induced tears with those of patients with epiphora. Krause did not, however, examine emo tional tears. Some years later, Botelho 21 reported that, "The ratio between the proteins albumin and globulin in reflex (irritant) tears is about the same as in serum, but psychogenic (emotional) tears may contain less albumin." However, she gave no data to support this conclusion. Many other studies of human tears have reported these three major protein frac tions.2'18,19,22"24 Using paper electrophore sis, Caselli and Schumacher 26 observed five different peaks. An even larger num ber of protein fractions have been distin guished with agarose 12 or polyacrylamide disk gel26 electrophoresis. We conducted a study to determine whether the nature of tear-inducing stim ulus affects the chemical composition of the tears.
OCTOBER, 1981
S U B J E C T S AND M E T H O D S
Tear donors—We collected tears from healthy male and female volunteers rang ing in age from 18 to 60 years (average age, 28 years). Approximately 80% of the subjects were female. Some subjects wore spectacles or hard or soft contact lenses, but the lenses were not worn during the tear collection procedure. Tear collection—We asked all the sub jects to wash their faces with soap and water before participating in the study. (None of the subjects wore cosmetics during the collection procedure.) We told the subjects to avoid rubbing their eyes because rubbing could cause some dam age to the corneal epithelium and alter the chemical composition of the tears. 27 Release of epithelial cell components into the tears was assessed by measuring lactate dehydrogenase 27,28 and adenosine triphosphatase 29 activities. We detected no activity in four samples of irritantinduced and emotional tears selected at random, indicating that epithelial cell damage was not a problem. Also, centrifugation of tear samples to remove any cells did not significantly alter the manga nese concentration. The average volume of emotional tear samples, 250 μΐ (range, 30 to 1,230 μΐ), was twice as great as that of the irritant-induced tear samples, 110 μΐ (range, 30 to 642 μΐ). There was no correlation between the volume of tears collected and the protein or manganese concentration of irritant-induced or emo tional tears. We collected irritant induced tears by exposing the subjects to the vapors of freshly cut onions for about three min utes. The tears were placed in preweighed 12 X 75-mm glass test tubes held just below the lower eyelid. This method is preferable to collecting tears directly from the surface of the eye, which often results in damage to the
VOL. 92, NO. 4
CHEMICAL COMPOSITION OF HUMAN TEARS
corneal epithelium and release of cellular material into the tears. 27 Emotional tears were collected within one week of collecting the irritantinduced tears. The subjects watched sad movies about two hours in length and collected their own tears into preweighed glass test tubes held just below the lower eyelid. The emotional status of the subject immediately before and after lacrimation was assessed by the Profile of Mood States. The Profile of Mood States is a self-rated scale consisting of 65 adjectives and was designed to assess feelings, af fect, and mood and their changes under therapeutic intervention or experimental manipulation. The Profile of Mood States has been extensively evaluated and nor mative samples for subjects are avail able. 30 We asked the subjects to evaluate their moods before lacrimation retrospec tively, that is, after the lacrimation proc ess was complete. Once collected, the tears were weighed, frozen immediately, and stored at —20 C until analyzed. To determine if evaporation of the tear samples while they were at room temperature altered their protein concentration, we prepared a mock tear solution of albumin and lysozyme and left 20-, 50-, 100-, and 200-μ1 aliquots exposed to air at room tempera ture for two hours. No change in protein concentration was detected after the twohour period. Similar samples were frozen for one, seven, 14, and 30 days and then tested for protein concentration. Varying periods of storage at — 20 C had no effect on the protein concentration. To deter mine whether tears could be contaminat ed by protein or manganese picked up during brief exposure to the skin below the eyelid, 0.9% sodium chloride was allowed to drip down the face, and then collected and analyzed. We detected no protein in the saline samples, and
561
five saline samples contained an average of only 1.6 ng/ml of manganese. Saline washes of the lower eyelid also contained negligible concentration of protein and manganese. Tear protein concentration—We deter mined the protein concentration of human tear samples by the method of Bradford, 31 using bovine serum albumin (E280, 1% = 6.6) as the standard. 32 Nor mally, we assayed 3 μΐ of tears from each sample in triplicate. Tear protein electrophoresis—We ana lyzed the tear samples (3 μΐ) by polyacrylamide disk gel electrophoresis on 5.5 X 100-mm, 7.5% polyacrylamide gels. The electrophoresis was conducted at p H 8.9, using 3 mamps per gel, for nega tively charged proteins and at p H 3.6 for positively charged proteins. The gels were fixed in 10% trichloroacetic acid for 30 minutes and stained with Coomassie brillant blue R. After unbound stain was removed, we scanned the gels at 570 nm with a spectrophotometer. Tear manganese concentration—The manganese concentrations of the tear samples were determined with an atomic absorption spectrophotometer with a HGA-500 graphite furnace. Blood sam ples (10 ml) were collected and centrifuged at 1,700 g for ten minutes. The serum was stored at —20 C in sealed Teflon cups until analyzed. The tear sam ples were collected directly into Teflon cups or were transferred into the cups after collection in glass tubes. We usually diluted the tear samples 20-fold with glass-distilled deionized water containing a manganese concentration of less than 0.3 ng/ml. All glass and Teflon was washed with 10% nitric acid and glassdistilled deionized water before use. Tears collected in washed glass tubes were not significantly different from those collected in washed Teflon cups with respect to manganese concentration.
562
AMERICAN JOURNAL OF OPHTHALMOLOGY RESULTS
Protein concentration of human tears—The effect of stimulus on the pro tein concentration of human tears is shown in Table 1. When all of the tear samples from women were analyzed, the protein concentration of the emotional tears (42 subjects) exceeded that of the irritant-induced tears (61 subjects) by 21% (P < .001). To eliminate the influence of differ ences between individuals, we compared the protein concentration of emotional tears to the protein concentration of irritant-induced tears obtained from the same subject on a subsequent day. Paired samples from 25 women also showed that the protein concentration of emotional tears was 24% greater than that of irritant-induced tears (P < .01) (Table 1). We found a higher protein concentra tion for emotional tears from men as well (P < .05), although we obtained paired samples from only four subjects. There was no significant difference between the protein concentration of tears from men and women when the same stimulus was
OCTOBER, 1981
used. The protein concentration of the emotional tears exceeded that of the irritant-induced tears in 23 of the 29 subjects examined. The protein concentrations of irritantinduced and emotional tears from five women who wore hard contact lenses and five women who wore soft contact lenses were not significantly different from the results for the other subjects. The sub jects did not wear contact lenses when the tears were collected. Of the 25 paired samples from women examined (Table 1), six were from subjects who wore specta cles. The tear protein concentration of those who wore spectacles did not differ significantly from that of those who did not. The variation in tear protein concen tration was as great between different samples obtained from the same subject on different occasions as between sam ples obtained from different subjects. Electrophorettc distribution of human tear proteins—Polyacrylamide disk gel electrophoresis distinguished approxi mately eight different negatively'charged protein peaks (Fig. 1) and eight different positively charged protein peaks (Fig. 2)
TABLE 1 P R O T E I N CONCENTRATIONS IN TEARS F R O M W O M E N AND MEN
Tears Irritant-induced No. of samples Protein (mg/ml) Emotional No. of samples Protein (mg/ml) P (Student's t-test)
Women
Men
Combined
61 5.40 ± 0.17
16 5.75 ± 0.37
77 5.47 ± 0.16
42 6.54 ± 0.25 <.001
5 6.18 ± 0.67 Not significant
47 6.50 ± 0.23 <.001
25 5.36 ± 0.24
4 5.22 ± 0.66
29 5.34 ± 0.22
25 6.62 ± 0.30 <.01
4 6.01 ± 0.83 <.05
29 6.53 ± 0.28 <.001
P A I R E D SAMPLES*
Irritant-induced No. of samples Protein (mg/ml) Emotional No. of samples Protein (mg/ml) P (Student's t-test)
*Emotional and irritant-induced tears were obtained from each subject.
VOL. 92, NO. 4
CHEMICAL COMPOSITION OF HUMAN TEARS
Fig. 1 (Frey and associates). Electrophoresis of the negatively charged proteins of human emotional (dashed line) and irritant-induced (solid line) tears. The subject was a 33-year-old woman.
in both emotional and irritant-induced tears. Each peak probably contained more than one protein species. To quantitate the distribution of protein among the various charged species, we divided the negative proteins into four groups (α, β, 7, δ) and the positive proteins into three groups (α, β, 7) on the basis of their electrophoretic mobility (Figs. 1 and 2). Table 2 shows the distribution of positive proteins among these groups for emotion al and irritant-induced tears from men and women, and Table 3 the distribution of negative proteins. Our results for 20 paired samples from women and three
563
Fig. 2 (Frey and associates). Electrophoresis of the positively charged proteins of human emotional (dashed line) and irritant-induced (solid line) tears. The subject was a 33-year-old woman.
paired samples from men showed no sig nificant difference between the electro phoretic distribution of positive tear pro teins from emotional and irritant-induced tears. Also the distribution of positive tear proteins in tears from men was not significantly different from that in tears from women (Table 2). The electrophoretic distribution of negative proteins from emotional and irritant-induced tears was also remark ably similar. There was no significant dif-
TABLE 2 PERCENTAGE OF POSITIVELY CHARGED PROTEINS IN ELECTROPHORETIC FRACTIONS OF HUMAN TEARS
Proteins a protein (%) Emotional Irritant-induced ß protein (%) Emotional Irritant-induced y protein (%) Emotional Irritant-induced
Women (20 samples*)
Men (3 samples*)
Combined (23 samples*)
47.8 ± 1.9 49.3 ± 1.7
53.6 ± 4.1 52.0 ± 5.9
48.6 ± 1.7 49.7 ± 1.6
29.4 ± 2.3 27.4 ± 2.1
26.0 ± 7.8 27.4 ± 8.5
28.9 ± 2.1 27.4 ± 1.9
22.8 ± 0.9 23.3 ± 0.9
20.5 ± 3.9 20.6 ± 2.8
22.5 ± 0.9 23.0 ± 0.8
*All samples were paired, that is, emotional and irritant-induced tears were obtained from each subject.
564
AMERICAN JOURNAL OF OPHTHALMOLOGY
OCTOBER, 1981
TABLE 3 PERCENTAGE O F NEGATIVELY CHARGED PROTEINS IN E L E C T R O P H O R E T I C FRACTIONS O F HUMAN TEARS
Proteins a protein (%) Emotional Irritant-induced ß protein (%) Emotional Irritant-induced 7 protein (%) Emotional Irritant-induced δ protein (%) Emotional Irritant-induced
Women (20 samples*)
Men (3 samples*)
Combined (23 samples*)
36.0 ± 1.5 37.1 ± 1.5
36.5 ± 8.5 43.4 ± 5.5
36.0 ± 1.5 37.9 ± 1.4
10.5 ± 1.1 8.6 ± 0.5
14.3 ± 3.5 10.5 ± 3.7
11.0 ± 1.0 8.8 ± 0.6
7.3 ± 1.0 6.3 ± 0.9
9.8 ± 0.8 6.3 ± 1.2
7.6 ± 0.8 6.3 ± 0.8
46.2 ± 2.3 48.0 ± 2.0
39.5 ± 10.2 39.9 ± 10.1
45.4 ± 2.3 47.0 ± 2.0
*All samples were paired, that is, emotional and irritant-induced tears were obtained from each subject.
ference between emotional and irritantinduced tears from men and women (Table 3). Although we observed no sig nificant difference between the δ (albu min) fraction of emotional and irritantinduced tears (Table 3), we occasionally found samples with small differences (Fig. 1). However, these differences were not of the magnitude reported by Brunish, 18 and the percentage of protein in the δ fraction of emotional tears did not ex ceed that of irritant-induced tears in most pairs examined (Table 3). The distribu tion of negative proteins of the irritantinduced and emotional tears from men did not differ significantly from those from women. Emotional tear samples ob tained from the left and right eyes of three different subjects did not show any significant difference between the elec trophoretic distribution of tear proteins. Manganese concentration of human tears—The manganese concentration of emotional tears (25.2 ± 6 . 6 ng/ml) was not significantly different from that of irritant-induced tears (34.2 ± 8 . 8 ng/ml) obtained from the same healthy female subjects (Table 4). The 15 subjects ranged
in age from 19 to 56 years (average age, 28 ± 2 years). The manganese concentra tion of emotional tears from women ex ceeded that of serum (0.98 ± 0.09 ng/ml) obtained from the same subjects by about 30-fold (Table 4). The manganese concen tration of irritant-induced tears from men (37.8 ± 4 . 8 ng/ml) was not significantly different from that of irritant-induced tears from women (34.2 ± 8.8 ng/ml), contrary to the earlier report by Tapaszto 12 (Table 4). The 14 male subjects ranged in age from 19 to 40 years (average age, 28 ± 2 years). There was no signifi cant correlation of manganese concentra tion with age for male or female subjects. The manganese concentrations of irritant-
TABLE 4 MANGANESE
CONCENTRATIONS IN HUMAN AND SERUM
TEARS
Manganese Concentration (ng/ml)
Women (No. = 15)
Men (No. = 14)
Emotional tears Irritant-induced tears Serum
25.2 ± 6.6 34.2 ± 8.8 0.98 ± 0.09
37.8 ± 4.8
VOL. 92, NO. 4
CHEMICAL COMPOSITION OF HUMAN TEARS
induced tears from men and women re ported by Tapaszto 12 were approximately 127-fold higher and sixfold higher, re spectively, than those presented here. DISCUSSION
The increased protein concentration of emotional tears compared to irritantinduced tears demonstrated that the lacrimal and accessory glands do excrete different tears in response to different stimuli. This finding agrees qualitatively with the previous report by Brunish. 18 However, we do not know the sample size and method of protein determination Brunish employed. Furthermore, Brun ish included tears from some subjects with idiopathic excessive tearing (per haps epiphora) along with emotional tears for some of his studies because his results indicated that they were similar. He re ferred to these as normal tears and found they had a markedly greater albumin content than irritant-induced tears. 18 This finding was later disputed by Krause 19 who found no difference between the albumin content of irritant-induced tears and those of patients with epiphora, al though he did not examine emotional tears. Our results clearly showed that there was no difference between the percent age of proteins in irritant-induced and emotional tears which migrate as "al bumin" or any other charged species, contrary to the report by Brunish. 18 We compared irritant-induced tears with emotional tears obtained from the same subject in order to avoid problems with individual differences, something Brun ish did not do. With our polyacrylamide electrophoresis system, we were able to distinguish many more protein species than Brunish could with paper electro phoresis in 1957. However, neutral pro teins (zero net charge) and proteins with molecular weights over 150,000 would probably not be easily observed with our
565
electrophoresis system. Our failure to observe differences in the electrophoretic patterns of irritant-induced and emotion al tears does not prove that differences do not exist in individual proteins. Differ ences in individual proteins not constitut ing a high percentage of total protein may not be observed by this method. The manganese concentration of hu man tears exceeded that of serum by approximately 30-fold. This suggested that the lacrimal or accessory glands con centrate manganese from the serum (or some other source) and excrete it in the tears. The mechanism by which this pro cess occurs is unknown, as is the reason for its occurrence. The possibility that a por tion of the manganese found in tears is derived from damaged cells of the corneal epithelium cannot be eliminated, al though we were unable to detect any adenosine triphosphatase or lactate dehydrogenase activity (from damaged cells) in our tear samples. The concentration we found in irritant-induced tears from men (37.8 ng/ml) was less than 1% of the value previously reported by Tapaszto. 12 Tapaszto gave no information regarding the stimulus for tear secretion or the method of tear collection he used. Also, we, unlike Tapaszto, observed no differ ence between the manganese concentra tions of tears from women and men. The high values for tear manganese concen tration reported by Tapaszto led us to suspect that sample contamination may have been a problem. The ability to shed tears in response to emotional stress (psychogenic lacrimation) is a physiologic function that only developed with the evolution of the human being. 33 Psychogenic lacrimation is not only a phylogenetically late devel opment, but also it occurs late ontogenetically, because the human infant does not normally exhibit tears with its crying until it is several days, or even weeks, old. 22 This occurs despite a functional
566
AMERICAN JOURNAL OF OPHTHALMOLOGY
lacrimal system that secretes reflex tears in response to ocular irritation. There fore, it appears the human infant must wait for further development of the cen tral nervous system before it becomes capable of psychogenic lacrimation. 33 Psychogenic lacrimation is not inhibited by surface anaesthesia, paralysis of the fifth nerve, or section of the cervical sympa thetic nerve as is irritant-induced tear ing. 34 The purpose of psychogenic lacrima tion is unknown, although a number of theories regarding its function have been proposed. Through further investigations of psychogenic tears, we may learn more about the purpose of psychogenic lacri mation, the body's specific exocrine re sponse to emotional stress. REFERENCES 1. Fourcroy, A. F . , and Vauquelin: The chemical examination of tears and of the nasal secretion. Ann. Chimie 10:113, 1791. 2. Altaian, P. L. : Physical properties and chemi cal composition of tears. Man. In Dittmer, D. S. (ed.): Blood and Other Body Fluids. Washington, D.C., Federation of American Societies of Experi mental Biology, 1961, pp. 488-490. 3. Holly, F. J., Patten, J. T., and Dohlman, C. H. : Surface activity determination of aqueous tear components in dry eye patients and normals. Exp. Eye Res. 24:479, 1977. 4. Sorensen, T., and Jenson, F. T. : Methodologi cal aspects of tear flow determination by means of a radioactive tracer. Acta Ophthalmol. 55:726, 1977. 5. Norn, M. S.: Outflow of tears and its influence on tear secretion and break up time (B.U.T.). Acta Ophthalmol. 55:674, 1977. 6. Carney, L. G., and Hill, R. M.: Human tear pH. Diurnal variations. Arch. Ophthalmol. 94:821, 1976. 7. Winder, A. F., Ruben, M., and Sheraidah, G. A. K. : Tear calcium levels and contact lens wear. Br. J. Ophthalmol. 61:539, 1977. 8. Horowitz, G., Lammers, J. W., Sussman, S., and Botelho, S. Y. : Calcium in tears, lacrimal gland fluid, and fluid from the other orbital glands. Invest. Ophthalmol. 15:994, 1976. 9. van Haerington, N. J., and Glasius, E.: Collec tion method dependant concentrations of some me tabolites in human tear fluid, with special reference to glucose in hyperglycaemic conditions. Albrecht von Graefes Arch. Klin. Exp. Ophthalmol. 202:1, 1977.
OCTOBER, 1981
10. Hill, R. M., and Terry, J. E.: Human tear cholesterol levels. Arch. Ophthalmol. 36:155, 1976. 11. Zubareva, T. V., and Kiseleva, Z. M.: Catacholamine content of the lacrimal fluid of healthy people and glaucoma patients. Ophthalmologica 175:339, 1977. 12. Tapaszto, I.: Pathophysiology of human tears. Ophthalmol. Clin. 13:119, 1973. 13. Versieck, J., and Cornells, R. : Normal levels of trace elements in human blood plasma or serum. Anal. Chim. Acta 116:217, 1980. 14. Junnola, K. : Über die Eigenschaften der menschlichen Tränenflussigkeit, thesis, Helsinki, Helsingin, Liikekirjapaino Oy, 1952, pp. 1-90. 15. Rennert, O. M., Kaiser, D . , Sollberger, H., and Joller-Jemelka, S.: Antiprotease activity in tears and nasal secretions. Humangenetik 23:73, 1974. 16. Josephson, A. S., and Weiner, R. S.: Studies of the proteins of lacrimal secretions. J. Immunol. 100:1080, 1968. 17. Allansmith, M.I Immunology of tears. Int. Ophthalmol. Clin. 13:47, 1973. 18. Brunish, R.: The protein components of human tears. Arch. Ophthalmol. 57:554, 1957. 19. Krause, U.: A paper electrophoretic study of human tear proteins. Acta Ophthalmol. 53 (suppl.):l, 1959. 20. Brunish, R. : The protein components of human tears, abstract. Am. J. Ophthalmol. 41:540, 1956. 21. Botelho, S. Y. : Tears and the lacrimal gland. Sei. Am. 211:78, 1964. 22. Allerhand, J., Karelitz, S., Penbharkkul, S., Ramos, A., and Isenberg, H. D.: Electrophoresis and immunoelectrophoresis of neonatal tears. J. Pediatr. 62:85, 1963. 23. Erickson, E. F . , Hatlen, R., and Berg, M.: Industrial tear study. Filter-paper electrophoresis of tears, with results of an industrial study from 1,000 specimens sent by mail. Am. J. Ophthalmol. 47:499, 1959. 24. McEwen, W. K., Kimura, S. J., and Feeney, M. L. : Filter-paper electrophoresis of tears. III. Human tears and their high molecular weight com ponents. Am. J. Ophthalmol. 45:67, 1958. 25. Caselli, P., and Schumacher, H.: Über den nachweis des tränenlysozyms mittels mikroelektrophorese. Klin. Monatsbl. Augenheilkd. 124:148, 1954. 26. Sapse, A. T., Bonavida, B., Stone, W., Jr., and Sercarz, E. E.: Proteins in human tears. I. Immunoelectrophoretic patterns. Arch. Ophthalmol. 81:815, 1969. 27. van Haeringen, N. J., and Glasius, E.: The origin of some enzymes in tear fluid, determined by comparative investigation with two collection meth ods. Exp. Eye Res. 22:267, 1976. 28. Goldberg, E.: Amino acid corr.fjsition and properties of crystalline lactate dehydrogenase X from mouse testes. J. Biol. Chem. 247:2044, 1972. 29. Warren, G. B., Toon, P . A . , Birdsall, N. J. M., Lee, A. G., and Metcalfe, J. C : Reconsti-
VOL. 92, NO. 4
CHEMICAL COMPOSITION OF HUMAN TEARS
tution of a calcium pump using defined membrane components. Proc. Natl. Acad. Sei. 71:622, 1974. 30. Guy, W.: ECDEU Assessment Manual for Psychopharmacology, rev. ed. DHEW No. (ADM) 7&-338, Rockville, Maryland, 1976, pp. 529-531. 31. Bradford, M. M.: A rapid and sensitive meth od for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248, 1976.
567
32. Tanford, C , and Roberts, G. L.: Phenolic hydroxyl ionization in proteins (I) bovine serum albumin. J. Am. Chem. Soc. 74:2509, 1952. 33. Walsh F. B., and Hoyt F. W.: Clinical NeuroOphthalmology. Baltimore, Williams and Wilkins Co., 1969, vol. 1, pp. 551-557. 34. Mutch, J. R. : The lacrimation reflex. Br. J. Ophthalmol. 28:317, 1944.
O P H T H A L M I C MINIATURE
But it is not ignoble to feel that the fuller life which a sad experience has brought us is worth our own personal share of pain. Surely it is not possible to feel otherwise, any more than it would be possible for a man with cataract to regret the painful process by which his dim blurred sight of men as trees walking had been exchanged for clear outline and effulgent day. George Eliot, Adam Bede