The Origin of Some Enzymes in Tear Fluid, Determined by Comparative Investigation with Two Collection Methods IT. .J. \-AK HAERISGES* ,\;etherln?uEs
Ophthalmic Wilhelaknn
(Receiaed
19 Xeptember
AND E. GLASIW
Research Institute ad University Eye Clittiic, Amsterdam. The Nether1an.ti.r
Gnsthuis,
1975. nrd in ,revised form
30
October 1975, London)
Comparative investigations of human tear fluid. collected in capillaries as IT-e11 on filter paper strips, revealed that the enzymes lactate dehydrogenase (LDH), malste dehylrogenaw (RIDH), pyruvate kinase (PI high concentration in tear fluid allowed its concentration to lrv measured with greate,qt ;tccuracv. Total’ a-p1 and P-hex concentrations were assayed ilr tear Fluid \vit,h it tiuor.inlet,rlc. method, slightly modified from the procedures of Johnson, Del Monte, Cotlier and Desnivk (1975) and Singer, Cotlier and Krimmer (1973). In the blank det,erminations t’he suhst,ratv was incubated under the same conditions a,s ill the ~JS:IV but the enzvme solution WIG, added just after the alkaline buffer solution as described originally by Mead, Smit,h ;I,lri Williams (195.5). The solutions of t,he substrates : l-rr~eth!;lur~~l~ellifer~l-~-~~-~~~l~~~~o~~~ -cteos~-13-r,-glucop~ranositle I’or rwnoside for z-gal ant1 4-n~ethylunlhellifer~l-;2-aceta,llitl p-hex. mere freshly prepared before each assay. The stock sol&ion of 1 mmol -k-rnethvlumbelliferone standard was prepared. b\~ dissolving the co~~~pc~u~lcl~II boiling water :IIIII was stored in dark at 37°C.
ICDH
GOT
PI,, G-6-PDH
ALD
I SDH
2
4
6
I GLDH
HK
0
/
GPT
8
IO
2
4
6
8
IO
ORIGIN
OF
ENZYXES
IN
TEARS
269
3. Results The concentrations of the enzymes in tear fluid were compared with the LDH concentration. The results are given as scattergrams in Fig. 1 and show the relationship between the concentration of LDH and the other enzymes, using both methods of tear collection. The regression equations and correlation coefficients defining these relat.ionships are given in Table I. TABLE
Qrrantitative
I
relationships between enzyme comentmtions* in tearJlu,id the LDH concentrations. used CISCI reference
md
Enzyme.
LDH MDH 1% ICDH G(,T G-6.1’DH A I,D SDH (:Ll)H Hli (:l’l a-pal p-bcs
regression
= = = = = = = = = = = = z
line vs. LDH
LDH 0.61LDH 0.54LDH 0.025LDH 0.019LDH 0.018LDH O.OlSLDH 0.0077LDH 0.0075LDH 0.0060LDH 0.0036LDH O.OOOlSLDH+ 0.0142LDH
Correlation? coefficient
ill
&38nt+S.E.M.
capillary
sample
230 * 26 (42) 0.9743 (9) 245 +122 (3) 0%37* (9) 235 * 38 +28 (3) + 9.1 0.Y623 (9) 15.6 -j= 6.7 (3) + 6.4 0.YY23 (9) 11.0 k 1.0 (3) + 4.7 O.Y2@ (9) 5.6 & 2.7 (3) + 3.3 0.Y633 (9) 10.3 -& 3.3 (3) + 1.4 0.9773 (9) 3.0 * 2.0 (3) + 1.3 0.9753 (9) 5.0 + 0 (3) + 12.2l 0.Y523 (9) 17.3 & 1.7 (3) $- 7.81 0.9623 (9) 9.6 -& 2.0 (3) 1G362 0.78g3 (20) 2.061 0.08 (8) + 66.8” 0%37” (26) 72.5 f 2.7 (11) +102
* E:nzyme concentrations are given in U/l tear fluid. t ?Tumber of determinations is indicated in brackets. Significance is indicated thus 1 2’ < 0.05; 2 P < 0.01, J P < 0.001,
“33“ I *.. t237 (92) 822 Zl85 (6) 902 *200 (‘3) 70.5 * 13.0 (6) 61.0 5 10.2 (6) 49.0 i 10.5 (6) 28.3 f 5.9 (6) 30.0 + 12.0 (6) 20.1 & 4.8 (6) 23.5 $ 6.3 (6) 18.0 & 4.3 (6) 2.74$ 0.21 (12) 133 & 12 (15)
3 ’ ’ 2 z 2 ’ n.s. * n.s. ll.S. 2 :I
n.s. not significant.
A significant positive correlation can be seen of the values of LDH with those of the other metabolic enzymes and with the lysosomal enzymes ct-gal and P-hex (P -: 0.001, except for PK, where P < 0.01). For a-gal, P-hex, HK and GPT the regression line meets the ordinate at a point above the origin, indicating substantial values for these enzymes in absenceof LDH. These values (the intercept constants in the equations) are significant for cr-gal and &hex (P < 0.01) and for HK and GPT (P < 0.05) ; for the other enzymes the intercept constants are not significant (P > O-05). For a,11the enzymes lower values were found in the capillary samplesthan in the samples obtained by collection on filter paper. In Table I the significance of the difference between the mean capillary values and the mean filter paper values is given for each enzyme. It is significant for LDH and p-hex (P < O*OOl),a-gal, ICDH. GOT and G-6-PDH (P < 0.01) and MDH, PK, ALD and GLDH (P < 0.05). Only in the case of SDH, HK and GPT are the differences not significant.
The slope of the regression lines represents the increase in cnzyrnc (x)rlcelltriltiorl ItI relation to LDH. In Pig. 2 the regression coefficientx of t)hc equatiorls in Tat& I arc compared with the ratio’s of the corresponding enzvntes ant1 J,I)H in human corntv1.l qjithelium and lacrimal gland tissue. The enzyme pattern of tear fluid rc!semtjlt~~ t,ha.t of cornea1 epithelium and shows less correspondence wit,11 tShat of lacrimal glmd tissw.
(b) I or
l GOT l ICDH
_
GPTO
GDtH .HK
l ALD 0
.
G-6PDH
OOOl jLlllllill( 001
[enzyme]
Ratio of [LDHI
01
IO
I” tear fluid
FIG. 3. Enzyme pattern of cornral epithclium (a) and lacrimal gland tissue (I)). cmnpared with tlut 01 tear fluid. For the ratio of enzyme concentration/LDH concentration in tear iiuitl t,he regression CO. efficients of Table I have been used. The values of enzyme conccntration/LYH wncrntration fw w1~1~~a1 cpithelium have been calculated from R&n, Hmnighausm, Hildebrandt, and Maier (1971) ;rwl 1;~ lacrimal gland tissue from Reim (1964).
4. Discussion The results of this study make clear, that the ~nethotl of collection ma!- ati& markedly the composition of the tear fluid obtained. Tears obtainecl with filter paper strips usually show a considerable increase in the amount of enzymes, whereas many of these enzymes scarcely can be detected in tear fluid collected in capillaries.
ORIGIN
OF
ENZYMES
IN
TEAliS
2il
It seems most likely that some trauma to the conjunctival and cornea1 epithelia is caused by their mechanical irritation with the filter paper strips and that’ this is sufficient to liberate many enzymes, possibly as a consequence of lysis of clesquamatt4 epithelial cells. These conclusions agree with those of Kah&n and Ottovay (1975), who found a M-fold higher activity for LDH in tears of rabbits than in hunlan tears, which prol)nhly reflects the much higher concentration of LDH in rabbit cornea1 epithelinm sincc~ thr LDH level in the tear glands did not differ significantly in rabllits ~1~1 tlulllnns.
1’111~main source of the lysosomal enzymes, rx-gal and P-hex, is the lacrimal gland. since, r&her high values are found in tear fluid collected under conditions whertl t)htb cpithelial cells remain intact and containing very low levels of LDH. Epithelial cells III~~ act as second source for cc-gal and ,&hex in tear fluid. as indicated by the higher va,lutls found in samples collected on filter paper st’rips. HK and GPT are present at :I significant level in tear fluid when LDH is absent suggesting t,hat these enzymes lllil\‘ also Itr present as secretion products of the lacrimal gland. ( ‘t nnl)ariaon of the enzyme pattern in tear fluid with the values available in the, litrraturc concerning cornea1 and lacrimal gland tissue, rc>veal that the cornea1 enz?mcA patt.c‘rtl has more in common with the former t,han with the enzvme pattern of t,tlcL I;l.criln;~l gland itself. ‘l’ll(~ lack of good correlation of lysozyme and amylase with LDH in tears. collect,tlcl wit11 fiber paper strips, was established in earlier st#udies (van Haeringen and Glasills. I97~1~). Vrom later studies on the relevant &sues it, appcarcstl that the former CIIZ~IIIC~ arc’ l)r(~,~t~ntonly in the lacrimal gland and not, in conjunctiva or cornea (van HaCringtbll (‘1, al.. 1975). \\-Jl(‘rt’as the production of lysozpme, amylase. cc-gal. P-hex, HK and GPT t)v tlrta lacri111a1gland now seems certain, the question of the ccallular origin is not yet cledidrcl. ITsing in~munofluorescent techniques, Kraus and Mest,ecky (1971) found in 1lutt1a11 h&ma1 gland sections that lysozyme was contained only in cert)ain cells of thus striattxtl clucts, which refutes our arguments for an acinar origin (van Haeringen ant1 (Zlsius. 1974c). Amylase may be assumed to origiriatc from zymogen gram&s in tllcb ~11s of a(:ini, analogous to those Peen in the human parotid gland (Kmus and Mestccki-. 197 I). The enzymes cr.-gal and P-hex are probably sinlilar to P-glucuronidasc. whic.11 Kiihnt~l (1968) found to be present in the basal portion of t)he acinar cells and in the rpithclium of the ducts, by means of enzyme histochemical investigations of’ hunlan tear gland section*?. It would appear from this study that LDH is a very sensitive measure for epitheliwl damage, of t’he surface of the eye; it is possible therefore that the analysis of tpars. nollt~ctc4 in capillaries, can indicate biomicroscopicall>- non-visible cornea1 damage.
The authors wish to thank Dr F. Ensink for useful discussions and for crit,icallv reading the tnanuscript and Mr A. F. Blijleve for the illustrat.ions. REFERENCES
/
Hseringen, N. J. van, Ensink, F. and Glasius, E. (1975). Amylase in human tear fluid: origin and characteristics, compared with salivary and urinary amylases. Exp. Eye Res. 21, 395-403. Haeringen, N. J. van and Glasius, E. (1974a). Lactate dehydrogenase in tear fluid. Exp. Eye Kes. l&345-9.
S. J. VAN
272
HAEBINGEN
ANI)
E. (II,ASII!S
Haeringen, N. J. van and Glasius, E. (1974b). Enzymes of energy producing metabolism in hutuarl tear fluid. Exp. Eye Res. l&407-9. Haeringen, N. J. van and Glasius, E. (1974c). Enzymat,ic studies in lacrimal secretion. E,r.//. l’:yr Res. 19, 135-9. Halberg, G. P. and Berens, C. (1961). Standardized Schirmer tear test kit. Amez. .I. (+hthtr/no/.
51, 840-2. Johnson, D. L., Del Monte, M. A.. Cotlier, E. and Desnink, K. tJ. (1975). Fabry disease: diagnosis by cr-galactosidase activities in tears. Clin. Chim. Acts 63, X1-90. Kah&n, I. L. and Ottovay, E. (1975). Lactat,e dehydrogenasr of t,cars and c*ornral epithclium. Exp. Eye Res. 20, 129-33. Kraus, F. W. and Mestecky, J. (1971). Immunohistochemical localisation of amylase. I~soz,vrr~c~ and immunoglobulins in the human parotid gland. drchs Ornl Biol. 16, 7X1-9. Kiihnel, W. (1968). Vergleichende histologische, histochemisrhe und elektronenmikroskopiwh~~ Untersuchungen an Triinendriisen: VI. Menschliche TCnendriise. Z. %dljiisch. 89, 55lb-7?. Jlead, J. A. R., Smith, J. N. and Williams. R. T. (1955). Studies in detoxication. The biosynthesis of the glucuronides of umbelliferone and 4-methylumbelliferone and their use in fluorimetri~~ determination of B-glucuronidase. Biochem. J. 61,569-74. Reim, M. (1964). Die Enzyme des energielieferenden Stoffwec nels in der Xinendriw vo11 Kaninchen. Albrecht van Orrxefes Arch. Ophthalmol. 167, 398409. Reim, M.. Henninghausen, U., Hildebrandt, D. and Maier. R. (1971). Enzyme activit,ws 111 thcb cornea epithelium and endothelium of different speries. fphthrrl. Res. 2, 171-82. Singer. J. D., Cotlier, E. and Krimmer, R. (1973). Hexonaminidase A in tears and saliva for rapid identification of Tay-Sachs disease and it,s carriers. Laacet ii, Ill (j--19. Thompson, R. and Gallardo. E. (1936). The concent,ration of lysoeyme in the wars in il(‘uTt’ ~IIII chronic conjunctivitis. With a note on the source of the lysozyme in tears. .dmer. d. O~hthrr/naol.
19. 684-S.
Ophthalmic Wilhelaknn
(Receiaed
19 Xeptember
AND E. GLASIW
Research Institute ad University Eye Clittiic, Amsterdam. The Nether1an.ti.r
Gnsthuis,
1975. nrd in ,revised form
30
October 1975, London)
Comparative investigations of human tear fluid. collected in capillaries as IT-e11 on filter paper strips, revealed that the enzymes lactate dehydrogenase (LDH), malste dehylrogenaw (RIDH), pyruvate kinase (PI high concentration in tear fluid allowed its concentration to lrv measured with greate,qt ;tccuracv. Total’ a-p1 and P-hex concentrations were assayed ilr tear Fluid \vit,h it tiuor.inlet,rlc. method, slightly modified from the procedures of Johnson, Del Monte, Cotlier and Desnivk (1975) and Singer, Cotlier and Krimmer (1973). In the blank det,erminations t’he suhst,ratv was incubated under the same conditions a,s ill the ~JS:IV but the enzvme solution WIG, added just after the alkaline buffer solution as described originally by Mead, Smit,h ;I,lri Williams (195.5). The solutions of t,he substrates : l-rr~eth!;lur~~l~ellifer~l-~-~~-~~~l~~~~o~~~ -cteos~-13-r,-glucop~ranositle I’or rwnoside for z-gal ant1 4-n~ethylunlhellifer~l-;2-aceta,llitl p-hex. mere freshly prepared before each assay. The stock sol&ion of 1 mmol -k-rnethvlumbelliferone standard was prepared. b\~ dissolving the co~~~pc~u~lcl~II boiling water :IIIII was stored in dark at 37°C.
ICDH
GOT
PI,, G-6-PDH
ALD
I SDH
2
4
6
I GLDH
HK
0
/
GPT
8
IO
2
4
6
8
IO
ORIGIN
OF
ENZYXES
IN
TEARS
269
3. Results The concentrations of the enzymes in tear fluid were compared with the LDH concentration. The results are given as scattergrams in Fig. 1 and show the relationship between the concentration of LDH and the other enzymes, using both methods of tear collection. The regression equations and correlation coefficients defining these relat.ionships are given in Table I. TABLE
Qrrantitative
I
relationships between enzyme comentmtions* in tearJlu,id the LDH concentrations. used CISCI reference
md
Enzyme.
LDH MDH 1% ICDH G(,T G-6.1’DH A I,D SDH (:Ll)H Hli (:l’l a-pal p-bcs
regression
= = = = = = = = = = = = z
line vs. LDH
LDH 0.61LDH 0.54LDH 0.025LDH 0.019LDH 0.018LDH O.OlSLDH 0.0077LDH 0.0075LDH 0.0060LDH 0.0036LDH O.OOOlSLDH+ 0.0142LDH
Correlation? coefficient
ill
&38nt+S.E.M.
capillary
sample
230 * 26 (42) 0.9743 (9) 245 +122 (3) 0%37* (9) 235 * 38 +28 (3) + 9.1 0.Y623 (9) 15.6 -j= 6.7 (3) + 6.4 0.YY23 (9) 11.0 k 1.0 (3) + 4.7 O.Y2@ (9) 5.6 & 2.7 (3) + 3.3 0.Y633 (9) 10.3 -& 3.3 (3) + 1.4 0.9773 (9) 3.0 * 2.0 (3) + 1.3 0.9753 (9) 5.0 + 0 (3) + 12.2l 0.Y523 (9) 17.3 & 1.7 (3) $- 7.81 0.9623 (9) 9.6 -& 2.0 (3) 1G362 0.78g3 (20) 2.061 0.08 (8) + 66.8” 0%37” (26) 72.5 f 2.7 (11) +102
* E:nzyme concentrations are given in U/l tear fluid. t ?Tumber of determinations is indicated in brackets. Significance is indicated thus 1 2’ < 0.05; 2 P < 0.01, J P < 0.001,
“33“ I *.. t237 (92) 822 Zl85 (6) 902 *200 (‘3) 70.5 * 13.0 (6) 61.0 5 10.2 (6) 49.0 i 10.5 (6) 28.3 f 5.9 (6) 30.0 + 12.0 (6) 20.1 & 4.8 (6) 23.5 $ 6.3 (6) 18.0 & 4.3 (6) 2.74$ 0.21 (12) 133 & 12 (15)
3 ’ ’ 2 z 2 ’ n.s. * n.s. ll.S. 2 :I
n.s. not significant.
A significant positive correlation can be seen of the values of LDH with those of the other metabolic enzymes and with the lysosomal enzymes ct-gal and P-hex (P -: 0.001, except for PK, where P < 0.01). For a-gal, P-hex, HK and GPT the regression line meets the ordinate at a point above the origin, indicating substantial values for these enzymes in absenceof LDH. These values (the intercept constants in the equations) are significant for cr-gal and &hex (P < 0.01) and for HK and GPT (P < 0.05) ; for the other enzymes the intercept constants are not significant (P > O-05). For a,11the enzymes lower values were found in the capillary samplesthan in the samples obtained by collection on filter paper. In Table I the significance of the difference between the mean capillary values and the mean filter paper values is given for each enzyme. It is significant for LDH and p-hex (P < O*OOl),a-gal, ICDH. GOT and G-6-PDH (P < 0.01) and MDH, PK, ALD and GLDH (P < 0.05). Only in the case of SDH, HK and GPT are the differences not significant.
The slope of the regression lines represents the increase in cnzyrnc (x)rlcelltriltiorl ItI relation to LDH. In Pig. 2 the regression coefficientx of t)hc equatiorls in Tat& I arc compared with the ratio’s of the corresponding enzvntes ant1 J,I)H in human corntv1.l qjithelium and lacrimal gland tissue. The enzyme pattern of tear fluid rc!semtjlt~~ t,ha.t of cornea1 epithelium and shows less correspondence wit,11 tShat of lacrimal glmd tissw.
(b) I or
l GOT l ICDH
_
GPTO
GDtH .HK
l ALD 0
.
G-6PDH
OOOl jLlllllill( 001
[enzyme]
Ratio of [LDHI
01
IO
I” tear fluid
FIG. 3. Enzyme pattern of cornral epithclium (a) and lacrimal gland tissue (I)). cmnpared with tlut 01 tear fluid. For the ratio of enzyme concentration/LDH concentration in tear iiuitl t,he regression CO. efficients of Table I have been used. The values of enzyme conccntration/LYH wncrntration fw w1~1~~a1 cpithelium have been calculated from R&n, Hmnighausm, Hildebrandt, and Maier (1971) ;rwl 1;~ lacrimal gland tissue from Reim (1964).
4. Discussion The results of this study make clear, that the ~nethotl of collection ma!- ati& markedly the composition of the tear fluid obtained. Tears obtainecl with filter paper strips usually show a considerable increase in the amount of enzymes, whereas many of these enzymes scarcely can be detected in tear fluid collected in capillaries.
ORIGIN
OF
ENZYMES
IN
TEAliS
2il
It seems most likely that some trauma to the conjunctival and cornea1 epithelia is caused by their mechanical irritation with the filter paper strips and that’ this is sufficient to liberate many enzymes, possibly as a consequence of lysis of clesquamatt4 epithelial cells. These conclusions agree with those of Kah&n and Ottovay (1975), who found a M-fold higher activity for LDH in tears of rabbits than in hunlan tears, which prol)nhly reflects the much higher concentration of LDH in rabbit cornea1 epithelinm sincc~ thr LDH level in the tear glands did not differ significantly in rabllits ~1~1 tlulllnns.
1’111~main source of the lysosomal enzymes, rx-gal and P-hex, is the lacrimal gland. since, r&her high values are found in tear fluid collected under conditions whertl t)htb cpithelial cells remain intact and containing very low levels of LDH. Epithelial cells III~~ act as second source for cc-gal and ,&hex in tear fluid. as indicated by the higher va,lutls found in samples collected on filter paper st’rips. HK and GPT are present at :I significant level in tear fluid when LDH is absent suggesting t,hat these enzymes lllil\‘ also Itr present as secretion products of the lacrimal gland. ( ‘t nnl)ariaon of the enzyme pattern in tear fluid with the values available in the, litrraturc concerning cornea1 and lacrimal gland tissue, rc>veal that the cornea1 enz?mcA patt.c‘rtl has more in common with the former t,han with the enzvme pattern of t,tlcL I;l.criln;~l gland itself. ‘l’ll(~ lack of good correlation of lysozyme and amylase with LDH in tears. collect,tlcl wit11 fiber paper strips, was established in earlier st#udies (van Haeringen and Glasills. I97~1~). Vrom later studies on the relevant &sues it, appcarcstl that the former CIIZ~IIIC~ arc’ l)r(~,~t~ntonly in the lacrimal gland and not, in conjunctiva or cornea (van HaCringtbll (‘1, al.. 1975). \\-Jl(‘rt’as the production of lysozpme, amylase. cc-gal. P-hex, HK and GPT t)v tlrta lacri111a1gland now seems certain, the question of the ccallular origin is not yet cledidrcl. ITsing in~munofluorescent techniques, Kraus and Mest,ecky (1971) found in 1lutt1a11 h&ma1 gland sections that lysozyme was contained only in cert)ain cells of thus striattxtl clucts, which refutes our arguments for an acinar origin (van Haeringen ant1 (Zlsius. 1974c). Amylase may be assumed to origiriatc from zymogen gram&s in tllcb ~11s of a(:ini, analogous to those Peen in the human parotid gland (Kmus and Mestccki-. 197 I). The enzymes cr.-gal and P-hex are probably sinlilar to P-glucuronidasc. whic.11 Kiihnt~l (1968) found to be present in the basal portion of t)he acinar cells and in the rpithclium of the ducts, by means of enzyme histochemical investigations of’ hunlan tear gland section*?. It would appear from this study that LDH is a very sensitive measure for epitheliwl damage, of t’he surface of the eye; it is possible therefore that the analysis of tpars. nollt~ctc4 in capillaries, can indicate biomicroscopicall>- non-visible cornea1 damage.
The authors wish to thank Dr F. Ensink for useful discussions and for crit,icallv reading the tnanuscript and Mr A. F. Blijleve for the illustrat.ions. REFERENCES
/
Hseringen, N. J. van, Ensink, F. and Glasius, E. (1975). Amylase in human tear fluid: origin and characteristics, compared with salivary and urinary amylases. Exp. Eye Res. 21, 395-403. Haeringen, N. J. van and Glasius, E. (1974a). Lactate dehydrogenase in tear fluid. Exp. Eye Kes. l&345-9.
S. J. VAN
272
HAEBINGEN
ANI)
E. (II,ASII!S
Haeringen, N. J. van and Glasius, E. (1974b). Enzymes of energy producing metabolism in hutuarl tear fluid. Exp. Eye Res. l&407-9. Haeringen, N. J. van and Glasius, E. (1974c). Enzymat,ic studies in lacrimal secretion. E,r.//. l’:yr Res. 19, 135-9. Halberg, G. P. and Berens, C. (1961). Standardized Schirmer tear test kit. Amez. .I. (+hthtr/no/.
51, 840-2. Johnson, D. L., Del Monte, M. A.. Cotlier, E. and Desnink, K. tJ. (1975). Fabry disease: diagnosis by cr-galactosidase activities in tears. Clin. Chim. Acts 63, X1-90. Kah&n, I. L. and Ottovay, E. (1975). Lactat,e dehydrogenasr of t,cars and c*ornral epithclium. Exp. Eye Res. 20, 129-33. Kraus, F. W. and Mestecky, J. (1971). Immunohistochemical localisation of amylase. I~soz,vrr~c~ and immunoglobulins in the human parotid gland. drchs Ornl Biol. 16, 7X1-9. Kiihnel, W. (1968). Vergleichende histologische, histochemisrhe und elektronenmikroskopiwh~~ Untersuchungen an Triinendriisen: VI. Menschliche TCnendriise. Z. %dljiisch. 89, 55lb-7?. Jlead, J. A. R., Smith, J. N. and Williams. R. T. (1955). Studies in detoxication. The biosynthesis of the glucuronides of umbelliferone and 4-methylumbelliferone and their use in fluorimetri~~ determination of B-glucuronidase. Biochem. J. 61,569-74. Reim, M. (1964). Die Enzyme des energielieferenden Stoffwec nels in der Xinendriw vo11 Kaninchen. Albrecht van Orrxefes Arch. Ophthalmol. 167, 398409. Reim, M.. Henninghausen, U., Hildebrandt, D. and Maier. R. (1971). Enzyme activit,ws 111 thcb cornea epithelium and endothelium of different speries. fphthrrl. Res. 2, 171-82. Singer. J. D., Cotlier, E. and Krimmer, R. (1973). Hexonaminidase A in tears and saliva for rapid identification of Tay-Sachs disease and it,s carriers. Laacet ii, Ill (j--19. Thompson, R. and Gallardo. E. (1936). The concent,ration of lysoeyme in the wars in il(‘uTt’ ~IIII chronic conjunctivitis. With a note on the source of the lysozyme in tears. .dmer. d. O~hthrr/naol.
19. 684-S.
