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(Investigative Ophthalmology and Visual Science. 2002;43:2622-2625.)
© 2002 by The Association for Research in Vision and Ophthalmology, Inc.

Substance P and Its Metabolites in Normal Human Tears

Masakazu Yamada1, Masaro Ogata1, Masataka Kawai1, Yukihiko Mashima1 and Teruo Nishida2

1 From the Department of Ophthalmology, Keio University School of Medicine, Tokyo, Japan; and the 2 Department of Ophthalmology, Yamaguchi University School of Medicine, Yamaguchi, Japan.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
PURPOSE. To determine amounts and biochemical characteristics of substance P-like immunoreactivity (SPLI) in tears of normal human subjects.

METHODS. Forty-three healthy subjects (16 males and 27 females; age range, 17–80 years) participated. Ten microliters of unstimulated tears were collected with a micropipette from one eye of all subjects. Tear samples were partially purified by C-18 cartridges. SPLI concentrations in purified samples were measured by enzyme immunoassay (EIA). For biochemical characterization of SPLI, tear extracts were fractionated by high-performance liquid chromatography (HPLC). Each fraction then was subjected to EIA. To determine the metabolism of substance P in tears, synthetic substance P was incubated in medium containing pooled tears and then analyzed by HPLC with the detector set at a 210-nm wavelength.

RESULTS. The SPLI concentration in normal human tears was 306.0 ± 96.5 pg/mL (mean ± SD; range, 148–555 pg/mL). SPLI did not significantly vary by age or gender. HPLC analysis indicated that SPLI in tears consisted of five different substances and that substance P was converted to several fragments, including SP8-11 by enzymes present in tears.

CONCLUSIONS. Substance P, a normal component of human tears, presumably is released from the nerve endings in the ocular surface and converted to fragments by degradative enzymes in tears.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
The cornea is innervated by trigeminal sensory nerve fibers that contain several neuropeptides, including substance P and calcitonin gene-related peptide.1 2 3 When these neuropeptides are depleted with capsaicin, wound healing in the corneal epithelium is delayed.4 Substance P, both alone and in combination with other factors, such as insulin and insulin-like growth factor-1, promotes migration5 6 7 and proliferation8 9 of corneal epithelial cells. Recently, topically applied substance-P–derived peptide (FGLM peptide) combined with insulin-like growth factor-1 was reported to be effective in treating neurotrophic keratopathy.10 11 These findings suggest that release of axonally transported substance P may be involved in the mechanisms by which trigeminal sensory nerves exert trophic effects on the cornea.

Substance P recently has been detected in human tears.12 13 14 Although the role and the source of substance P in tears remains to be established, several studies have suggested that concentrations of substance P in tears differ in association with certain disorders of the ocular surface. Fujishima et al.13 reported that tears of patients with allergic conjunctivitis and vernal keratoconjunctivitis showed a significant excess of substance P compared with tears from normal control subjects. We recently reported that substance P concentrations in tears from eyes with unilateral corneal hypesthesia were decreased compared with tears from contralateral healthy eyes.14 Concentrations of substance P in tears of patients with diabetic keratopathy also were lower than those in normal control subjects (our unpublished data, 2001). Therefore, to some extent, concentrations of substance P in tears appear likely to reflect neuropeptide contents in ocular tissues.

When applied to the eye, substance P produces miosis15 16 and may enhance intraocular inflammation and conjunctival hyperemia.17 Obviously, substance P present in tears of normal quiescent eyes does not provoke such a response. Degradative enzymes, such as enkephalinase, angiotensin-converting enzyme, and trypsin, are thought to cleave substance P released from nerve endings.18 Therefore, substance P sometimes is considered a precursor of a variety of closely related peptides exhibiting different actions. For example, SP1-9, which contains the N-terminal portion of substance P, is involved in the mechanism of neurogenic inflammation in allergic disorders,19 whereas C-terminal SP8-11 (FGLM peptide) influences the epithelial cell migration in the cornea.11 However, little is known about the metabolites of substance P in the cornea or in tears.

This study was undertaken to determine concentrations and biochemical characteristics of substance P and its related peptides in tears of normal human subjects.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Subjects
Forty-three subjects (16 males and 27 females; age range, 17–80 years) participated in the study. Each participant underwent a thorough initial eye examination, including a slit lamp evaluation, Schirmer testing, and a cotton-thread test. All subjects had more than 10 mm of Schirmer strip wetting, and more than 15 mm of cotton-thread wetting. Normal corneal sensation (55 mm or greater) was confirmed by using a Cochet-Bonnet esthesiometer. Except for eight elderly subjects with age-related cataract, all subjects were free of any ocular disease. Subjects who wore contact lenses, had diabetes mellitus, or had a history of any ocular surgery were excluded from study. The principles of the World Medical Association’s Declaration of Helsinki were followed. Each subject received a full explanation of all procedures and provided informed consent for participation before the experiment. Approval for this investigation was granted by the Committee for the Protection of Human Subjects at Keio University School of Medicine.

Substance P–like Immunoreactivity Assay
Ten microliters of unstimulated tears were collected with a micropipette (Drummond Scientific, Broomall, PA) from one eye of all subjects. Tear samples were placed into chilled test tubes containing a 40-µL aprotinin-EDTA mixture (500 kallikrein inhibitor unit/mL aprotinin and 1.2 mg/mL EDTA) and stored at -80°C until assay.

Samples were diluted fivefold with 4% acetic acid and loaded onto C-18 cartridges (Sep-Pak; Waters, Milford, MA). After samples were washed with 3 mL of 4% acetic acid, immunoreactive substance P in tears was eluted with 3 mL ethanol-water-acetic acid (90:10:0.04, vol/vol/vol).20 A standard substance P solution (100 pg/mL; Sigma, St. Louis, MO) was used to estimate the extent of the recovery rate during this partial purification step.

The eluate was concentrated by evaporation and then reconstituted in 50 µL phosphate buffer. Immunoreactive substance P in purified samples was measured using an enzyme immunoassay (EIA) kit (Cayman Chemical, Ann Arbor, MI). The antiserum used in the assay, which was specific to the C-terminal portion of substance P, reacted with SP2-11, SP4-11, and SP5-11, but not with SP8-11 or SP1-9. The final results are expressed as picograms per milliliter.

High Performance Liquid Chromatography
An HPLC system was used that consisted of a solvent delivery system (LC-10AD), a programmable detector (UV-VIS) set at 210 nm, and a chromatography workstation (C-R6A; Shimadzu, Tokyo, Japan). Elution was performed with a 77:23 mixture of 0.1 M Tris-phosphate (pH 2.8) and acetonitrile in a chromatography column (ODS-2; Whatman, Clifton, NJ) at a rate of 0.5 mL/min.21 Synthetic substance P, SP2-11, SP4-11, SP5-11, and SP8-11 (Sigma) were used for the identification of peaks.

Three 100-µL samples of pooled tears (each was collected from both eyes of five subjects) mixed with 400 µL aprotinin-EDTA were used for HPLC-EIA. The samples were purified by a C-18 cartridge, as just described. The purified samples, reconstituted in 10 µL HPLC solvent, were fractionated by HPLC (fraction size, 0.25 mL). Each fraction was concentrated by evaporation under nitrogen gas and then reconstituted in 50 µL EIA buffer. These residues were subjected to EIA.

To determine metabolism of substance P by degradative enzymes in human tears, three 50-µL samples of pooled tears (each was collected from one eye of five subjects) were used. Ten micrograms of synthetic substance P (Sigma) was incubated for 60 minutes at 37°C in 0.5 mL DMEM/Ham’s F-12 containing 50 µL pooled human tears. Substance P (10 µg) incubated in 0.5 mL DMEM/Ham’s F-12 alone served as the negative control. The fluids were purified by C-18 cartridge and then analyzed by HPLC with UV detection at 210 nm.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
SPLI Concentration in Human Tears
The mean SPLI concentration in normal human tears was 306.0 ± 96.5 pg/mL (mean ± SD; range, 148–555 pg/mL; Table 1 ). SPLI concentrations in tears were not significantly associated with age or gender (Fig. 1) .


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  		Table 1. Substance P–like Immunoreactivity in Normal Human Tears

 


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  		Figure 1. SPLI in tears was not significantly associated with the subject’s age.

 
To test reproducibility of the measurement, tear samples were collected twice on different days in 10 subjects (6 men and 4 women; age range, 24–27 years). The mean SPLI concentration in tears collected on the first day was 316.6 ± 99.3 pg/mL, whereas that on the second day was 299.5 ± 95.3 pg/mL, which was not significantly different.

Tear samples were collected from both eyes in 10 subjects (5 men and 5 women; age range; 24–27 years). Tears were collected from the right eye first and then from the left. The mean SPLI concentration in tears from right eyes was 267.1 ± 48.7 pg/mL, and in tears from left eyes was 272.7 ± 70.2 pg/mL, which was not significantly different.

HPLC-EIA of SPLI
HPLC-EIA indicated that SPLI in tears consisted of five different substances. A representative chromatogram is shown in Figure 2 . The first peak, with a retention time shorter than that for SP8-11, was assumed to be SP-sulfoxide.13 18 No peak for SP8-11 was seen, possibly because the antibody used in the EIA system did not react with SP8-11. The second to the fifth peaks of HPLC-EIA comigrated with authentic substance P, SP2-11, SP5-11, and SP4-11, respectively. Substance P was the major SPLI in tears, although such fragments as SP2-11, SP5-11, and SP4-11 were identified.



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  		Figure 2. HPLC-EIA revealed that SPLI in tears consisted of five different substances. The first peak (6), with a shorter retention time than that of standard SP8-11 (1), is SP-sulfoxide. The second to fifth peaks in the HPLC-EIA comigrated with authentic substance P (2), SP2-11 (3), SP5-11 (4), and SP4-11 (5).

 
Metabolism of Substance P by Degradative Enzymes in Human Tears
HPLC analysis revealed that six peaks were present after substance P was incubated in a medium containing human tears. A representative chromatogram is shown in Figure 3 . The peaks were identified as SP sulfoxide, SP8-11, substance P, SP2-11, SP5-11, and SP4-11 (peaks 1–6, respectively). When substance P was incubated in medium alone, two small peaks (SP-sulfoxide and SP8-11) were identified in addition to substance P (Fig. 4) .



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  		Figure 3. Chromatogram obtained after 10 µg substance P was incubated in medium containing human tears. Six peaks were identified as SP-sulfoxide (1), SP8-11 (2), substance P (3), SP2-11 (4), SP5-11 (5), and SP4-11 (6). SP8-11 was the major metabolite of substance P.

 


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  		Figure 4. Chromatogram obtained after 10 µg of substance P was incubated in medium alone (negative control). Besides the large peak of substance P (3), two small peaks can be identified as SP-sulfoxide (1) and SP8-11 (2).

 

   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
Our results showed substance P to be a component of tears obtained from normal human eyes. The mean SPLI concentration in normal human tears was 306.0 ± 96.5 pg/mL (range, 148–555). Although there was considerable variation between individuals, concentrations of SPLI in tears was not significantly associated with age or gender. Reproducibility of measurements in a subject for different days or eyes was reasonably good.

The concentration of SPLI in tears in the present study was higher than previously reported by Varnell et al.12 and Fujishima et al.13 Although all three studies used EIA to quantitate the concentration of SPLI, differences in the purification procedure and the antibody used in the assay may account for the discrepancy. Fehder et al.20 have reported that proteins in samples influence measurements of SPLI, and partial purification is therefore required to measure SPLI in samples containing differing amounts of various proteins.

HPLC-EIA analysis indicated that SPLI in tears consisted of at least five different substances. After SP-sulfoxide, a nonenzymatic oxidative product of substance P, and substance P itself, the third to the fifth peaks of HPLC-EIA comigrated with authentic SP2-11, SP5-11, and SP4-11, respectively. As mentioned in the Methods section, the antiserum used in the assay was specific for the C-terminal portion of substance P. N-terminal fragments of substance P or C-terminal fragments smaller than SP5-11 were not detected by the assay. Accordingly, synthetic substance P was incubated in a medium containing human tears, to determine metabolism of substance P by degradative enzymes in tears. HPLC detected the six peaks mentioned earlier. SP8-11 was the major metabolite of substance P after incubation with human tears. These results suggest that SP8-11 is present in normal tears. Our results also suggest that several enzymes in tears take part in degrading substance P, although nonenzymatic degradation and nonenzymatic oxidation occur to some extent. Candidate degradative enzymes include trypsin, dipeptidyl aminopeptidase IV, postproline-cleaving enzyme, and enkephalinase.18

Enzymatic degradation of substance P released from nerve endings in tears appears to be biologically advantageous. The miotic effect of substance P would be avoided or attenuated by the degrading process.22

N-terminal fragments of substance P including SP1-9, which is involved in the mechanism of allergic inflammation,19 were not identified in normal human tears in the present study. It is of interest to know whether N-terminal fragments of substance P are present in tears of patients with severe allergic conjunctivitis, because elevated levels of substance P have been reported in tears of such patients.13 Nakamura et al.23 have shown that FGLM peptide, a sequence of four amino acids in the C-terminal of substance P, is the minimum sequence necessary to produce synergistic effects of substance P and insulin-like growth factor-1 on corneal epithelial wound healing. Notably, all metabolites of substance P detected in this study retained this minimum sequence. Therefore, degradation of substance P in tears may have a beneficial role, permitting maintenance of the ocular surface by exerting trophic effects of substance P while avoiding undesirable effects.


   Footnotes
 
Submitted for publication October 29, 2001; revised March 14, 2002; accepted March 22, 2002.

Commercial relationships policy: N.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Corresponding author: Masakazu Yamada, Department of Ophthalmology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan; yamadam{at}sc.itc.keio.ac.jp.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

  1. Tervo, T, Tervo, K, Eranko, L, Vannas, A, Eranko, O, Cuello, AC. (1983) Substance P immunoreaction and acetylcholinesterase activity in the cornea and Gasserian ganglion Ophthalmic Res 15,280-288[Medline][Order article via Infotrieve]
  2. Lehtosalo, JL. (1984) Substance P-like immunoreactive trigeminal ganglion cells supplying the cornea Histochemistry 80,273-283[Medline][Order article via Infotrieve]
  3. Jones, MA, Marfurt, CE. (1998) Peptidergic innervation of the rat cornea Exp Eye Res 66,421-435[Medline][Order article via Infotrieve]
  4. Gallar, J, Pozp, MA, Rebollo, I, Belmonte, C. (1990) Effects of capsaicin on corneal wound healing Invest Ophthalmol Vis Sci 31,1968-1974[Abstract/Free Full Text]
  5. Nishida, T, Nakamura, M, Ofuji, K, Reid, TW, Mannis, MJ, Murphy, CJ. (1996) Synergistic effects of substance P with insulin-like growth factor-1 on epithelial migration of the cornea J Cell Physiol 169,159-166[Medline][Order article via Infotrieve]
  6. Nakamura, M, Ofuji, K, Chikama, T, Nishida, T. (1997) Combined effects of substance P and insulin-like growth factor-1 on corneal epithelial wound closure of rabbit in vivo Curr Eye Res 16,275-278[Medline][Order article via Infotrieve]
  7. Nakamura, M, Chikama, T, Nishida, T. (1998) Up-regulation of integrin alpha 5 expression by combination of substance P and insulin-like growth factor-1 in rabbit corneal epithelial cells Biochem Biophys Res Commun 246,777-782[Medline][Order article via Infotrieve]
  8. Reid, TW, Murphy, CJ, Iwahashi, CK, Foster, BA, Mannis, MJ. (1993) Stimulation of epithelial cell growth by the neuropeptide substance P J Cell Biochem 52,476-485[Medline][Order article via Infotrieve]
  9. Garcia-Hirschfeld, J, Lopez-Briones, LG, Belmonte, C. (1994) Neurotrophic influences on corneal epithelial cells Exp Eye Res 59,597-605[Medline][Order article via Infotrieve]
  10. Brown, SM, Lamberts, DW, Reid, TW, Nishida, T, Murphy, CJ. (1997) Neurotrophic and anhidrotic keratopathy treated with substance P and insulin-like growth factor 1 Arch Ophthalmol 115,926-927[Medline][Order article via Infotrieve]
  11. Chikama, T, Fukuda, K, Morishige, N, Nishida, T. (1998) Treatment of neurotrophic keratopathy with substance-P-derived peptide (FGLM) and insulin-like growth factor I Lancet 351,1783-1784[Medline][Order article via Infotrieve]
  12. Varnell, RJ, Freeman, JY, Maitchouk, D, Beuerman, RW, Gebhardt, BM. (1997) Detection of substance P in human tears by laser desorption mass spectrometry and immunoassay Curr Eye Res 16,960-963[Medline][Order article via Infotrieve]
  13. Fujishima, H, Takeyama, M, Takeuchi, T, Saito, I. (1997) Elevated levels of substance P in tears of patients with allergic conjunctivitis and vernal keratoconjunctivitis Clin Exp Allergy 22,372-378
  14. Yamada, M, Ogata, M, Kawai, M, Mashima, M. (2000) Decreased substance P concentrations in tears from patients with corneal hypesthesia Am J Ophthalmol 129,671-672[Medline][Order article via Infotrieve]
  15. Anderson, JA, Hernandez, E, Duzman, E, Malfroy, B. (1990) Recombinant enkephalinase effectively inhibits substance P-induced miosis in the rabbit eye cup model Curr Eye Res 9,543-547[Medline][Order article via Infotrieve]
  16. Alessandri, M, Fusco, BM., Maggi, CA, Fanciulacci, M. (1991) In vivo pupillary constrictor effects of substance P in man Life Sci 48,2301-2308[Medline][Order article via Infotrieve]
  17. Tsuji, F, Hamada, M, Shirasawa, E. (1998) Tachykinins as enhancers of prostaglandin E2-induced intraocular inflammation Ocular Immunol Inflamm 6,19-25[Medline][Order article via Infotrieve]
  18. Rissler, K. (1995) Sample preparation, high-performance liquid chromatographic separation and determination of substance P-related peptides J Chromatogr B 665,233-270[Medline][Order article via Infotrieve]
  19. Iwamoto, I, Yamazaki, H, Tomioka, H, Yoshida, S. (1991) Inhibitory effect of tranilast on substance P-induced plasma extravasation in rat skin Immunopharmacol Immunotoxicol 13,65-71[Medline][Order article via Infotrieve]
  20. Fehder, WP, Ho, W-Z, Campbell, DE, et al (1998) Development and evaluation of a chromatographic procedure for partial purification of substance P with quantitation by an enzyme immunoassay Clin Diagn Lab Immunol 5,303-307[Abstract/Free Full Text]
  21. Rissler, K, Cramer, H. (1990) High-performance liquid chromatographic separation of tachykinins using tris-phosphate as the buffer component of the mobile phase J Chromatogr 533,179-186[Medline][Order article via Infotrieve]
  22. Nishida, T. (1997) Neurotrophic keratopathy: studies on substance P and the clinical significance of corneal sensation J Jpn Ophthalmol Soc 101,948-974
  23. Nakamura, M, Chikama, T, Nishida, T. (1999) Synergic effect with Phe-Gly-Leu-Met-NH2 of the C-terminal of substance P and insulin-like growth factor-1 on epithelial wound healing of rabbit cornea Br J Pharmacol 127,489-497[Medline][Order article via Infotrieve]



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