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Amylase in human tear fluid: Origin and characteristics, compared with salivary and urinary amylases
hp.
Eye Res. (1975) 21, 395-403
Amylase in Human Tear Fluid: Origin and Characteristics, Compared with Salivary and Urinary Amylases* 3. J. VAN HAERINGEN,
F. EMINR AND E. GLASIUS
Xetherlards Ophthalmic Research Iwtitute and lbkversity Eye Chic, Tl’il~helrni~v~nGnsthuis. Eerste Helmersstmot 104, Amsterdam, The Netherlards (Received 12
d!!ny
1975,
London)
In the tissues, coming in contact with the tear fluid, amylase activity is only demonstrable in lacrimal gland tissue and this is in accord with the concept that the lacrimal gland is the source of tear amylase. Isoenzymes of amylase in human tear fluid were separated with agarelectrophoresie and demonstrated with an iodine-starch method. The isoenzyme pattern of tear fluid amylase was different from that of urine and saliva. The influence of pH in t,he range (i-8 and of the ionic activators Ca2+ and Cl- also indicated different behaviour of t,he amylases.
1. Introduction In previous work (van Haeringen and Glasius, 1974a, b) we confirmed the presence of an amylase in human tear fluid. The common amylases, produced by the pancreas and the salivary glands and also those produced by the milk gland and Miiller’s epithelium of ovary and testis, have all been observed to consist of a number of isoenzyrnes (Merritt, Rivas, Bixler and Newell. 1973; Fridhandler. Berk. Montgomer! and Wong, 1974). It was the purpose of this study to investigate the origin of the amylase in tear fluid and to compare the isoenzyme pattern, the influence of pH and of the ionic activators Ca2+ and Cl- on this enzyme, with that of urinary and salivary anlylases.
2. Methods and Materials Avuy1a.wclctiuity (I) The saccharogenic amylase assay of Bernfeld (1951) as described by Rick and Stegbauer (1973), was used for activity measurement in specimen of tear fluid, urine and serum, subjected to electrophoresis. Units of amylase activity were expressed as micromoles of malt,ose liberated per minute. Amylase actiaitg (II)
For measurement of low amylase activity in small tear specimens, as occurring in t,he experiments with non-optimal ionic concentrations and pH, Bernfeld’s method did not seem satisfactory. In this method soluble Zulkowski starch is digested under standard conditions and the reducing cleavage products (mainly maltose) are estimated by reactiou with 3,5dinitrosalicylic acid (DNS) and spectrophotometry at 540 nm. The differencespect)rum for DNS in reduced and non-reacted form showed, however, in our hands an absorption-maximum at 470 nm [Fig. l(a)], U se of this wavelength instead of 540 nm enhances the sensitivity of the amylase assay appreciably. This confirms the results of ,Jamieson, Pruitt and Caldwell (1969) and refutes the criticism of them by Wesley-Hadzija and Pigon (1972). * Reprints requests to: Dr N. J. van Hawingen, Wilhelmina Gasthuis, Eerste Helmersstraat 104, Amsterdam, The Netherlands.
N. J. VAN
3%
HAERlS(:ES.
P. ENSISK
AND
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A standard curve for 470 nm with m&ose as reducing agent is given in Fig. I( 11j. I ‘1) tr) absorption values of about 0.3 the curve remains linear, therefore dilution of the samples was chosen so that with a maximal incubation-time of 15 min and a temperature of ?.?‘C’ the resulting absorption did not greatly exceed 0.3. Apart from the change in \vn\-elctlgttt. a micromodification was used : 0.10 1111 dilution of tear fluid, saliva or concentratetl uritw in the appropriate buffer was incubated with O-10 ml 19/h Zulkowsky starch in \v;lt,er. Ttrr reaction was stopped by addition of 0.20 1111 DNS solution of the following compositic~r~ : 100 mg DNS, 20 ml 1 xl-NaOH and 30 g Na-Ii tartrate made up to IIU ml with I\ ;rt,er. Reduction of DNS by the reaction products is completed by heating in il nat.el,-hat-It at. 100°C for 5 min. After addition of 3.60 ml \rater, the absorption ws tllrasurerl at 4741tltti against a blank cont,aining starch and D?jS.
J 400
500 Wavelength
600
0
20 Maltose
(nm)
40
60
concentration
tKl
I00
(nmol)
FIG. 1. Amylase assay, method II:
difference-spect,ra of reduced DNS against non-reacted DNS show an absorbance maximum at 470 nm (a). The curves represent two points from the standard curve fat 470 nm (b), for which the procedure is used as indicated in Materials and Methods, replacing the sample dilution by 0.10 ml of a solution containing varying amounts of a known maltose standard. hIalt,ose is given in final concentrations, i.e. after reaction with DNS and dilution with water.
Prepration
of aprose plates with amylase- and lysosylne-substmtes
The lysoplates of Bonavida and Sapse (1968) were modified by addition of insoluble Remazol brilliant blue-starch (RBB-starch, “Amylose Azure”, B grade Calbiochem) according to Ceska (1971) and Spiekerman, Perry, Hightower and Hall (1974). A suspension of 0.25% RBB-starch and 1% agarose (L’industrie Biologique Francaise) in phosphate buffer (67 mM, pH 6.3), containing ‘&XI mM-NaCl, was heated for 5 min in a boiling water bath, cooled to 65°C and then O.O5o/oXicrococcus lysodeicticus (Lysozyme substrate, Difco) was added as a thick suspension in phosphate buffer. This mixture wins immediately pipetted on to glass plates of 1.5 mm thickness. Pieces of the gel were stored in 5 cm Petri-dishes in a refrigerator. Tissue assay technique for amylase ad
lysozyme
The tissue to be studied was first rinsed with saline, blotted with filter paper, and then placed in pieces of about 2 mg wet weight on a filter paper disk of 5 mm diameter 071 the surface of the blue colored and opalescent agarose gel. For tear fluid 2 ~1 was applied on the filter paper. The agarose plates were observed for areas of lysis after 24 hr at 37°C. Lysozyme and amyla,se, if present together, produced two superimposed lysed areas: amylase a colorless area on the blue background of undigested RBB-starch and lysozyrue a transparent area on the opalescent’ background of non-lysed JI. lysodeicticus. The enzyme activity was stopped by treatment with 5: h acetic acid for 5 min. After drying, the plates could be stored.
AMYLASE
IN
TEAR
FLUID
397
The validity of this technique has been checked using filter paper disks soaked with solutions of known concentrations of lysozyme (Boehringer) and amylase (saliva dilutions). The observed diameters of the lytic zones, plotted against the amylase and lysozyme concentrations on a logarithmic scale, gave straight lines. Byar electroph~oresis. This was carried out in a Wieme apparatus. The agar blocks were made of ly/, purified agar (Oxoid Ltd.) in 0.1 M-Tris-HCl buffer, pH 8.2 and the same buffer was used in the electrode chambers. Gels were made of glass slides measuring 80~ 80 mm, covered with 10 ml 12/o agar solution in 0.05 nil-Tris-HCI buffer, pH 8.2. Three lo-mm slots were placed at the anodal side of the gel in order to cope with the marked endosmosis. After suction of about 10 ~1 of buffer solution from the slots into a 10 x 10 mm filt)er paper, the slots were filled with the samples. The agar plat,es in the rlectrophoresis cell \yere immersed in petroleum ether (b.p. 30-50°C): which ww kept c,ooled at ahout 10°C. With this set-up a volt)age of 200 V (40 nrA per slide) was ;tpplirtl (luring 60 min. Amylase activity was demonstrated after 60 min incubation of the gels at 37°C in a 1“i,, &arch solution in buffer of the following composition: Na-acetate 30 JIGI, Ka -veronal 30 IIIM, CaCl, 20 mM and HCI to pH 6.9. This incubation was followed by a brief water rinse and the soluble starch, penetrated into t,he agar surface, was stained with iodine solution (0.01 &I-iodine in O-02SKI). The amylase isoenzyme patterns hecarne visible immediately as colorless bands against a purplk background of non-digest,ed starch. The patterns were recorded by contact printing on photographic paper. On these zymograms (lark bands of amylase activity are visible against a light background. Tuor,flu.icZ.This was collected with glass capillaries. I!,ir~. This was, if necessary, concentrated in collodion tubes (Sartorius SJI 133110)at ;L pressure differential of 500 mmHg. S’ali~a. This was diluted wit’h water or buffer solution for enzvme assay or with \%-arn1 I’), agar solution for agar electrophoresis.
3. Results In order to investigate the origin of amylase in tear fluid. t,issue of human lacrimal gland, cornea and conjunctiva was compared with tear fluid in respect, of the ability of producing amylolytic activity on agarose plates. Also lysozyme, an antibacterial enzyme present in high concentration in tear fluid, was determined in the same assay. Significant lysis of the RBB-starch and of M. lysodeict~icus occurred with lacrimal pl&d tissue and tear fluid. whereas a much lower degree of lpsis was fcJUnd with cornea1 and conjunctival tissue (Table I). No cletectahle hydrolysis of RBB starch even coulcl be found with conjunctival tissue. For the demonstration of isoenzyme patterns of amylase. in three in&idual atnylase activities were determined in specimens of tear fluid, saliva and urinr (Tahle II) and samples were applied on agar slides, containing per person about equal amounts of amylase activity, and after electrophoresis the zymograms were ohtainecl as shown in Fig. 2. In the pattern of tear fluid three bands can be distinguished. and in subject 3 even five. In saliva three bands are visible, but the third (anodal) band, lying between the position of the second and third bands of tear fluid, is less intensive and not always clearly dist~inpuishable in the photographs. The faint cathodal band in tear fluid of’ subject 3 is of the same intensity as a 100 times more diluted saliva sample [Fig. 2(d)]. The pattern of urinary amylase isoenzymes shows three bands for subject 2 and 3. the third (anodal) handin Fig. 2(b) is, however. not clearly distinguishablein the photocrraph. In subject 1 only two isoenzymes are detectable in the urine. ?-
398
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The amylase activity in tear fluid, saliva and urine from one individual (subject 2) was measured between pH 6.0 and 8-0, at intervals of 0.2 pH units. in 25 I~lM-pbOSpha~~~ buffer and in the same buffer containing 50 111M-N&1 with and without 2 I~M-F:DTA. to study the effects of the activators C’a2-+and Cl-. The pH-activity-curvrs for the amylase in 1: 10 diluted tear fluid, 1: 400 diluted saliva and net 1 : 5 diluted urine, are presented in Fig, 3. The activities in Fig. 3 are given as :i470-\~alues. but at&ute TABLE
Comprative
I
determinations of umylwse and 1ysozy~n.r iyh teur@uicl. lacrimal gland, cowhea nlzd cowjunctira
Diameter of lytic zone (mm) AmyI&% Lysozymr
9 10 (5) (-3
Tear fluid Lacrimal gland C’0Hlea
Cornea1 epithelium Conjunctiva
26 “8 12 12 9
(5) had only lysis beneath the 5 mm-filter
paper disk.
activities can be calculated from the standard curve and the dilution factor. The activity in 5 mM-Tris-HCl pH 7.0, was used as control in computing the curves from results obtained with samples of different amyIase activities.
TABLE
Values of amylase activity Subject Tear
1 2 4
625 945 4000
II
in tear Jluid, urine a,nd saliva
Amylase u/I Urine
2.50 1600 250
Saliva
150 x 102 80 x IO” 200 ‘4 lo’
Comparison of curves A and C in Fig. 3 shows that EDTA in low concentration inhibits all three amylases considerably; for the salivary and urinary enzymes the pH of maximum activity thereby shifts to lower values, in contrast to the amylase from tear fluid where it remains constant at about pH 7.0. The pH-optimum for the urinary enzyme lies, in the presence of Caaf and Cl- (curve A), at somewhat higher pH-values than for the other amylases. Presence of 50 mM-Cl- in the phosphate buffers without EDTA exerts in these experiments the greatest effect on the salivary amylase.
AMYLASE
IN
TElZR
FLUID
399
(a)
(d) FIG. 2. $mylase isoenzymes in tear fluid (TF), saliva (YA) and urine (UR) of three individuals. Samples contaiuinq respectively 6,5 and 24 mu of amylase activity for subject 1,2 and 3, were applied a8 follows: (a) subject 1: 10 ~1 TF, 9 ~1 SA diluted 1: 200 and 9 ~1 UR concentrat,ed : 3; (1~)subject 2: 5 yl TF, 6 ~1 SA diluted 1: 100 and 3 ~1 UR; (c) subject 3: 6 ~1 TF, 6 ~1 SA diluted 1:50 and 6 ~1 UR concentrated x 16; (tl) subject 3: 6 PI TF, 6 ~1 SA diluted 1:5000 and 6 ~1 OR concentrated :r 16.
o6
7
8
PH PIG. 3. pH-activity curves for the amylases in tear fluid, saliva and urine of subject 2. Curves A show the results of incubation in 25 m-phosphate buffer containing 50 mM-NaC1, curves B 25 mM-phosphate buffer only, and curves C 25 mx-phosphate buffer containing 50 mM-Na’aci and 2 mu-EDTA. (a) 1: 10 diluted tear fluid; incubation time 15 min. (b) 1: 400 diluted saliva; incubation time 5 min. (c) I:50 dilution of x 10 concentrated urine; incubation time 15 min.
4. Discussion In human tear fluid amylase was detected for the first time by Mylius (1961) with a histochemical method. The glycogen-dissolving capacity of tears was demonstrated on sections of tissues with a high content of glycogen. Liotet (1967) and van Haeringen and Glasius (1974a, b) demonstrated the presence of anlylase in human tear fluid with an amyloklastic method. In considering the origin of enzymes (and other constituents) of tear fluid, the different sources of this fluid have to be taken into account. The tears present in the conjunctival sac consist of a mixture of fluids, secreted, according to Jones (1966) by: basic secretors (goblet cells, crypts of Henle and glands of Manz), lacrimal secretors (accessory lacrimal glands of Krause- and of Wolfring and occasional glands), oil secretors (Meibomian glands, glands of Zeis and of Mall) and reflex secretors (main lacrimal gland and accessory gland). According to Ehlers, Kessing and Norn (1972) the amounts of conjunctival mucous secretion and lacrimal gland fluid are produced in a ratio of 1: 1060. Reflex stimulation by handling of Schirmer paper or capillaries will increase the production of tears and of course even change that ratio in favour of the lacrimal gland fluid. Prom this point of view it may be concluded that practically all the tear fluid. collected in our experiments is lacrimal gland fluid.
AMYLASE
IN
TEAR
FLUID
401
Thompson and Gallardo (1936) already proved, by comparative investigation of the lysozyme contents of lacrimal gland and conjunctiva in dogs, that the lysozyme of tears originated in the lacrimal gland. The substantial amount of amylase, found together with lysozyme in lacrimal gland tissue, as compared with the much smaller amounts of amylase and lysozyme in cornea1 and conjunctival tissue, therefore may provide the answer to the problem of the tissue origin of amylase in tear fluid. If cornea and conjunctiva contributed to the amylase activity of tear fluid, only lower activities would be expected during lacrimation as a result of the dilution of cornea1 and conjunctival secretions by lacrimal gland fluid. However, earlier studieh (van Haeringen and Glasius, 197413)revealed t’hat. during stimulated tear production. fluctuations in amylase activity of tear fluid occurred in a range of 50 up to 26@4;, of the initial value. The small amounts of amylase and lysozyme found in cornea and conjunctiva, as compared with lacrimal gland tissue and tear fluid, suggests the reverse situation: the epithelium of the tissues coming into contact with the tear fluid is contaminated hv penetration of these enzymes into the thin mucous layer covering the outer cells and into the intercellular spaces. This means that amylase and lysozyme can be found as normal constituents of cornea1 and conjunctival tissue. Indeed. amylase activit) could he demonstrated in bovine cornea extract by Jonadet (1964) and Thompson and Gallardo (1936) found small amounts of lysozyme in the conjunctiva of dogs, and in the experiments of Fraqois and Rabaey (1956,1963) and Kamerau and Ott (1961) the presence of lysozyme in extracts of bovine cornea1 epithelium can be concluded from the appearance of an unknown fraction in the electrophoretic pattern at bhe ca.thodal side of gamma globulin. The activities of amylase in tear fluid from different persons show a considerable variation; values of 200-3000 u/l were found with the iodine starch method (van Haeringen and Glasius, 1974a, b). In our later st,udies. however. Bernfeld’s methott was ljreferred. because in this method the reaction products are estimated directly and the amylase activity can be measured in a larger scale. In the present st,ud!activity values of 625-4000 u/l were found (Table II). As it was an aim of our study to compare the amylase isoenzyme pattern of tear fluid with those of other body fluids, we matched in agar electrophoresis the amount of salivary and urinary amylase with that in the applied sample of tear fluid. As a consequence. t,he patterns of subject 3 show bands of higher density, because thtb amount of amylase applied from subject 3 (24 mu) is here 4-5 times more than front subject 1 and 2 (6 and 5 mu resp.). Possibly for this reason the two additional faint bandx are visible in the tear fluid pattern of subject 3, besides the three bands occurring in all patterns. Figure 2(d) gives an impression of the relative activity of t,he fa.int most cathodical band in tear fluid, as it apl’ears in equal densit,p as the hand in ii 1 : ,500Odiluted saliva sample. The itioenzytne pattern of tear fluid is clearly different from that of saliva in respect of the migration of the anodal bands and their relative intensities. In serum. the pancreas is the main source of amylase. which because of its molecular weight of about) 48 000 can be freely excreted by the kidney. Therefore the isoenzyme pattern of urine corresponds to that of serum and only minute amounts of salivary amylase may bt f’oun11 in 20-fold concentrat.ed urine (Merritt et al.. 1973). The three different’ iso enzymes in tear fluid, present in the three subjects under investigation. do not corrclspond to the urinary amylase isoenzymes, which indicates that amylasr in t.eai fluicl does not originate from the blood, but that it is locally synthesized.
10”
N. 3. VAN
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(:I,ASlI‘h
The relative migration of amylase isoenzymes in saliva and tear fluid suggests r 1r;l.t the observed isoenzymes differ in degree of glycosidation or deamination (Kelhbr. Kauffman. Allan and Williams. 1971). No conclusion can be drawn as to I\-Mher these isoenzymes are the result of post-tmnscriptional and post-secretional motlificatiolls of a single gene product (Karn. Shulkin, Merritt and Newell. 1973) or that’ gvltrt’ie polymorphism plays a role. The isoenzpme variation as occurred in the l)at,ttlrll OI urine of the three individuals in this study msy have a genetic I)xsis (Merritt clt ill.. 1973). In view of these differences in the isoenzvnic patterns, it is not surprising that’ the pH-activity curves, in which for each amilase the properties of the isoc~nzymt~~ww summaked. differ as well. (‘a2+ is the main activator of amylases (Hsiu, Fischer ant1 Stein? 1964) ; bintling of C’a2+bv EDTA indeed inhibits all three amylases. while relatively high concentrations of c’l- do not restore the original activity (Fi,.0 3. curves C). Although phoxphatr~ can bind C’a2i- -and t.herefore no extra C’a”+ was added in the buffers--there was in our experiments no risk that the phosphate buffer affected the Cla-amylase complex in the sample dilutions because the binding of C’a2+ by the enzyme is extremelS st,rong (Stein, Hsiu and Fischer, 1964). For the same reason it is doubtful whether the amylase activity in diluted saliva samples is as dependent on relatively high (‘a’)+concentrations as claimed by Mermall. Hanhilla and Reeves (1973). Their results 011 the activation of salivary amylase by C’a2r and other cat,ions are, moreover, cliticult to interpret because no indication is given regarding pH-control. The presence of low concentrations of EDTA was saGsfactory for removal of ( ‘ill) ! from the enzyme. although the incubat’ion with starch had to follow the dilllt,ion in the phosphate-EDTA buffer as soon as possible. because amylasc is more suscc:pt,iI)lc to tlenaturation in the absence of C’a2’m(Hsiu et al.. 1964). The effects of Cl- on the activity and the pH-optimum of the Ca-amylase conll)ltfx a,re marked for the enzyme in salira [Fig. 3(b)] ; for the amylases in tear fluitl an(l urine the effects are less clear, because in curves B the W-content of tears anal urine may play a role in the activation: Levitzki and Steer (1974) showed that aniorls like C!l- can already. in concentrations below 5 111~.have a maximal effect on hog pancreas amylase. For the same reason the high EaC’l concentrations, often intlicatclc-I t’oor optimal amglase assay, do not seem necessary (cf. Ceska. 1971 and Spiekerman ct. al.. 1974). t!onaitlering the effects of C!l- on the pH-activity curves. it has aIso to be kept in mind that the ionic strength in the buffer solutions containing NaCJI is sorur~~hat higher than in those containing only phosphate and EDTA, which map ha\-c ;ltl additional effect on the amylases. Inhibition by EDTA does not affect the pH-optimum for the amylaae in human tear fluid, in contrast to that for salivary and urinary amylases; therefore it cannot, in general be concluded that the pH-optimum of the amylases depends on the degree ol activation of the enzyme. The physiological role of amylase in tear fluid is not yet clear. As suggested IJY Liotet (1969), tear amylase could play a part in the glycogen metabolism of the cornea1 epithelium, liberating glucose necessary for the nutrition of cornea1 cells. Amylase could also be involved in other metabolic processes of glycogen such as the formation of mucous material in epithelial cells (Pei and Rhodin, 1971). Adverse reactions after frequent administration of EDTA-containing therapeut,ics to the eye do not seem to have been recognized so far, but are a possibility to consider.
A>IYLASE
IN
TEAR
403
FLUID
REFERENCES Bcrnfeld, I’. (1951). Enzymes of starch degradation and synthesis. Advnn. Enzymol. 12, 379. Bonavida, B. and Lapse, A. T. (1968). Human tear lysozyme: II. Quantitat,ive determination u%h standard Schirmer strips. Amer. J. Ophthdnd. 66, 70. C’rska. M. (1971). A new approach for quantitative and semi-quantitative determinations ot enzymat’ic activities with simple laboratory equipment. DeterGon of amylase. Cli71. (‘hint. A& 33, 135. mucows Ehlers, N., Kessing, S. V. and Norn, N. S. (1972). Q uantit,ative amounts of conjwctival secretion and t,ears. Actn Ophthnlmol. 50, 210. Francois. J. and Rabaey, M. (1956). Les proteines de l‘epith6lium oornben. ,4/~n. Owl. 189, 445. Francois. J. and Rabaoy. M. (1963). Immunoelectrophoresis of the proteins of the cornea1 epithelium. F:xp. Eye Res. 2, 196. Fridhandler. I,.. Berk. J. E.. &Montgomery, li. A. and Wang, D. (1974). Column-chromatographit, studiw of isoamylases in human serum, urine, and milk. Clin. (‘hug)?. 20, 547. Hawingen, N. J. van and Glasius. E. (1974a). Enzymes of energy produring metabolism in humall t’ear fluid. &xl). Byr Res. 18, 407. Hwringcn, N. ,J. van a,nd Glasius, E. (197413). Enzymatic studies in lac~rimal secretion. /:‘.rlj. K,//r KfS. 19, 135. Hsiu, ,J., Fischer. E. H. and St,ein, E. A. (1964). Alpha-amylases as calrium-metalloenz~nl~s. II. (Calcium and the catalytic act#ivity. Biochemistry 3, 61. Jamieson, A. D.. Pruitt, K. M. and Caldwell, R. C. (1969). i\n improwd amplase assay. .I. Jkw1.
RP.u.48, 4M. .lonadet.. &I. (1964). Xutivit&s b&ta-glucuronidasique et amylasiqne au nivrau de In c.orn@e. KF/.. PdhoZ. Pomp. Ifyg. Gen. 67, 453. .Jones. 1,. T. (1966). The lacrimal secretory system and its treatment. dnler. .I. Ophthnlmol. 62,47. Karn. R’. (‘.. Shulkin. J. D., Merrit,t. A. D. and Sewell, K. C. (1973). Evidence for post-transcriptional modification of human salivary amylase (Aim,v-,) isozymes. Biochrw. Genet. 10, S41. I
4X67. I,witzki. :Z. and Seer. M. L. (1974). The allostwic activation of mammalian z-am,&se t)y chloride. Ew. J. Biochem. 41, 171. Liotet. S. (1967). Pouvoir amylasique des larmes humaincs. dr~n. Owl. 200, 526. 2lermall. H. L., Hanhila, M. 0. and Reeves, IV. ;\I. (1973). Effects of cations on the activation ot salivary amylaae: I. Nat, I(+, Ca2f and Mg2+. J. Dent. Krs. 52, 114X. Slerritt’. A. D., Rivas, 31. L., Biller, D. and Sewell. R’. (1973). Salivary and pancreatic amylaw: clrctrophoretic characterizations and genetic studies. Amer. J. Hum. (&net. 25, 510. Mylius. .I. E. (1961). Amylase in t,ears? A histochemical study. dctrr I’rctlrol. Nicrobiol. Svr,rt/. (Suppl.) 148, 143. I’cti. Y. F. and Rhodin. J. A. S. (1971). Electronmicroscopic study of thr development of thp n1ouse cornea1 epit,helium. Incest. Ophthdmol. 10, 824. Rick. 12’. and Steghauer. H. P. (1973). a-Amylase. 3Iessung der redurerendrn Gruppen. ,Ilethotlw rlrr rn:ymtrtis&n. Analyse (Ed. Bergmeyer. H. I:.). Band I. p. 918. Verlag Chemie, iVeinheirn. Spiekcrman, A. 11.. Perry. P., Hightower, S. C. and Hall, F. F. (1974). (:hromogenic-substratr method for demonstrating multiple forms of alpha-amylase after elertrophoresis. L’litl. (!hiw.
~-l&Z.20 ,t”‘P4. Sitein. E. A., Hsiu, tJ. and Fischer, E. H. (1964). -ilpha-amylases as calcium-metalloenzymes. 1. Preparation of calcium-free apoamylases by chelation and electrodialysis. Biochemistry 3, 5fi. Thompson. R. and Ciallardo, E. (1936). The concentration of lysozyme in the tears in acute and cahronic conjumtivitis. Wit,h a note on the source of the lysozyme in tears. Amer. J. Ophthnlmol.
19, 684. Wesley-Hadzija, B. and Pigon, H. (1972). Effect of diet in West Africa activity. ilrc11. OrnZ BioZ. 17, 141.5.
on himIan salivary
amylaw
Eye Res. (1975) 21, 395-403
Amylase in Human Tear Fluid: Origin and Characteristics, Compared with Salivary and Urinary Amylases* 3. J. VAN HAERINGEN,
F. EMINR AND E. GLASIUS
Xetherlards Ophthalmic Research Iwtitute and lbkversity Eye Chic, Tl’il~helrni~v~nGnsthuis. Eerste Helmersstmot 104, Amsterdam, The Netherlards (Received 12
d!!ny
1975,
London)
In the tissues, coming in contact with the tear fluid, amylase activity is only demonstrable in lacrimal gland tissue and this is in accord with the concept that the lacrimal gland is the source of tear amylase. Isoenzymes of amylase in human tear fluid were separated with agarelectrophoresie and demonstrated with an iodine-starch method. The isoenzyme pattern of tear fluid amylase was different from that of urine and saliva. The influence of pH in t,he range (i-8 and of the ionic activators Ca2+ and Cl- also indicated different behaviour of t,he amylases.
1. Introduction In previous work (van Haeringen and Glasius, 1974a, b) we confirmed the presence of an amylase in human tear fluid. The common amylases, produced by the pancreas and the salivary glands and also those produced by the milk gland and Miiller’s epithelium of ovary and testis, have all been observed to consist of a number of isoenzyrnes (Merritt, Rivas, Bixler and Newell. 1973; Fridhandler. Berk. Montgomer! and Wong, 1974). It was the purpose of this study to investigate the origin of the amylase in tear fluid and to compare the isoenzyme pattern, the influence of pH and of the ionic activators Ca2+ and Cl- on this enzyme, with that of urinary and salivary anlylases.
2. Methods and Materials Avuy1a.wclctiuity (I) The saccharogenic amylase assay of Bernfeld (1951) as described by Rick and Stegbauer (1973), was used for activity measurement in specimen of tear fluid, urine and serum, subjected to electrophoresis. Units of amylase activity were expressed as micromoles of malt,ose liberated per minute. Amylase actiaitg (II)
For measurement of low amylase activity in small tear specimens, as occurring in t,he experiments with non-optimal ionic concentrations and pH, Bernfeld’s method did not seem satisfactory. In this method soluble Zulkowski starch is digested under standard conditions and the reducing cleavage products (mainly maltose) are estimated by reactiou with 3,5dinitrosalicylic acid (DNS) and spectrophotometry at 540 nm. The differencespect)rum for DNS in reduced and non-reacted form showed, however, in our hands an absorption-maximum at 470 nm [Fig. l(a)], U se of this wavelength instead of 540 nm enhances the sensitivity of the amylase assay appreciably. This confirms the results of ,Jamieson, Pruitt and Caldwell (1969) and refutes the criticism of them by Wesley-Hadzija and Pigon (1972). * Reprints requests to: Dr N. J. van Hawingen, Wilhelmina Gasthuis, Eerste Helmersstraat 104, Amsterdam, The Netherlands.
N. J. VAN
3%
HAERlS(:ES.
P. ENSISK
AND
E. (iLAS1L.S
A standard curve for 470 nm with m&ose as reducing agent is given in Fig. I( 11j. I ‘1) tr) absorption values of about 0.3 the curve remains linear, therefore dilution of the samples was chosen so that with a maximal incubation-time of 15 min and a temperature of ?.?‘C’ the resulting absorption did not greatly exceed 0.3. Apart from the change in \vn\-elctlgttt. a micromodification was used : 0.10 1111 dilution of tear fluid, saliva or concentratetl uritw in the appropriate buffer was incubated with O-10 ml 19/h Zulkowsky starch in \v;lt,er. Ttrr reaction was stopped by addition of 0.20 1111 DNS solution of the following compositic~r~ : 100 mg DNS, 20 ml 1 xl-NaOH and 30 g Na-Ii tartrate made up to IIU ml with I\ ;rt,er. Reduction of DNS by the reaction products is completed by heating in il nat.el,-hat-It at. 100°C for 5 min. After addition of 3.60 ml \rater, the absorption ws tllrasurerl at 4741tltti against a blank cont,aining starch and D?jS.
J 400
500 Wavelength
600
0
20 Maltose
(nm)
40
60
concentration
tKl
I00
(nmol)
FIG. 1. Amylase assay, method II:
difference-spect,ra of reduced DNS against non-reacted DNS show an absorbance maximum at 470 nm (a). The curves represent two points from the standard curve fat 470 nm (b), for which the procedure is used as indicated in Materials and Methods, replacing the sample dilution by 0.10 ml of a solution containing varying amounts of a known maltose standard. hIalt,ose is given in final concentrations, i.e. after reaction with DNS and dilution with water.
Prepration
of aprose plates with amylase- and lysosylne-substmtes
The lysoplates of Bonavida and Sapse (1968) were modified by addition of insoluble Remazol brilliant blue-starch (RBB-starch, “Amylose Azure”, B grade Calbiochem) according to Ceska (1971) and Spiekerman, Perry, Hightower and Hall (1974). A suspension of 0.25% RBB-starch and 1% agarose (L’industrie Biologique Francaise) in phosphate buffer (67 mM, pH 6.3), containing ‘&XI mM-NaCl, was heated for 5 min in a boiling water bath, cooled to 65°C and then O.O5o/oXicrococcus lysodeicticus (Lysozyme substrate, Difco) was added as a thick suspension in phosphate buffer. This mixture wins immediately pipetted on to glass plates of 1.5 mm thickness. Pieces of the gel were stored in 5 cm Petri-dishes in a refrigerator. Tissue assay technique for amylase ad
lysozyme
The tissue to be studied was first rinsed with saline, blotted with filter paper, and then placed in pieces of about 2 mg wet weight on a filter paper disk of 5 mm diameter 071 the surface of the blue colored and opalescent agarose gel. For tear fluid 2 ~1 was applied on the filter paper. The agarose plates were observed for areas of lysis after 24 hr at 37°C. Lysozyme and amyla,se, if present together, produced two superimposed lysed areas: amylase a colorless area on the blue background of undigested RBB-starch and lysozyrue a transparent area on the opalescent’ background of non-lysed JI. lysodeicticus. The enzyme activity was stopped by treatment with 5: h acetic acid for 5 min. After drying, the plates could be stored.
AMYLASE
IN
TEAR
FLUID
397
The validity of this technique has been checked using filter paper disks soaked with solutions of known concentrations of lysozyme (Boehringer) and amylase (saliva dilutions). The observed diameters of the lytic zones, plotted against the amylase and lysozyme concentrations on a logarithmic scale, gave straight lines. Byar electroph~oresis. This was carried out in a Wieme apparatus. The agar blocks were made of ly/, purified agar (Oxoid Ltd.) in 0.1 M-Tris-HCl buffer, pH 8.2 and the same buffer was used in the electrode chambers. Gels were made of glass slides measuring 80~ 80 mm, covered with 10 ml 12/o agar solution in 0.05 nil-Tris-HCI buffer, pH 8.2. Three lo-mm slots were placed at the anodal side of the gel in order to cope with the marked endosmosis. After suction of about 10 ~1 of buffer solution from the slots into a 10 x 10 mm filt)er paper, the slots were filled with the samples. The agar plat,es in the rlectrophoresis cell \yere immersed in petroleum ether (b.p. 30-50°C): which ww kept c,ooled at ahout 10°C. With this set-up a volt)age of 200 V (40 nrA per slide) was ;tpplirtl (luring 60 min. Amylase activity was demonstrated after 60 min incubation of the gels at 37°C in a 1“i,, &arch solution in buffer of the following composition: Na-acetate 30 JIGI, Ka -veronal 30 IIIM, CaCl, 20 mM and HCI to pH 6.9. This incubation was followed by a brief water rinse and the soluble starch, penetrated into t,he agar surface, was stained with iodine solution (0.01 &I-iodine in O-02SKI). The amylase isoenzyme patterns hecarne visible immediately as colorless bands against a purplk background of non-digest,ed starch. The patterns were recorded by contact printing on photographic paper. On these zymograms (lark bands of amylase activity are visible against a light background. Tuor,flu.icZ.This was collected with glass capillaries. I!,ir~. This was, if necessary, concentrated in collodion tubes (Sartorius SJI 133110)at ;L pressure differential of 500 mmHg. S’ali~a. This was diluted wit’h water or buffer solution for enzvme assay or with \%-arn1 I’), agar solution for agar electrophoresis.
3. Results In order to investigate the origin of amylase in tear fluid. t,issue of human lacrimal gland, cornea and conjunctiva was compared with tear fluid in respect, of the ability of producing amylolytic activity on agarose plates. Also lysozyme, an antibacterial enzyme present in high concentration in tear fluid, was determined in the same assay. Significant lysis of the RBB-starch and of M. lysodeict~icus occurred with lacrimal pl&d tissue and tear fluid. whereas a much lower degree of lpsis was fcJUnd with cornea1 and conjunctival tissue (Table I). No cletectahle hydrolysis of RBB starch even coulcl be found with conjunctival tissue. For the demonstration of isoenzyme patterns of amylase. in three in&idual atnylase activities were determined in specimens of tear fluid, saliva and urinr (Tahle II) and samples were applied on agar slides, containing per person about equal amounts of amylase activity, and after electrophoresis the zymograms were ohtainecl as shown in Fig. 2. In the pattern of tear fluid three bands can be distinguished. and in subject 3 even five. In saliva three bands are visible, but the third (anodal) band, lying between the position of the second and third bands of tear fluid, is less intensive and not always clearly dist~inpuishable in the photographs. The faint cathodal band in tear fluid of’ subject 3 is of the same intensity as a 100 times more diluted saliva sample [Fig. 2(d)]. The pattern of urinary amylase isoenzymes shows three bands for subject 2 and 3. the third (anodal) handin Fig. 2(b) is, however. not clearly distinguishablein the photocrraph. In subject 1 only two isoenzymes are detectable in the urine. ?-
398
K.
HAE:RlS~:Eh-,
F. ESSTliK
ASI)
E. C:LAS;ll’s
The amylase activity in tear fluid, saliva and urine from one individual (subject 2) was measured between pH 6.0 and 8-0, at intervals of 0.2 pH units. in 25 I~lM-pbOSpha~~~ buffer and in the same buffer containing 50 111M-N&1 with and without 2 I~M-F:DTA. to study the effects of the activators C’a2-+and Cl-. The pH-activity-curvrs for the amylase in 1: 10 diluted tear fluid, 1: 400 diluted saliva and net 1 : 5 diluted urine, are presented in Fig, 3. The activities in Fig. 3 are given as :i470-\~alues. but at&ute TABLE
Comprative
I
determinations of umylwse and 1ysozy~n.r iyh teur@uicl. lacrimal gland, cowhea nlzd cowjunctira
Diameter of lytic zone (mm) AmyI&% Lysozymr
9 10 (5) (-3
Tear fluid Lacrimal gland C’0Hlea
Cornea1 epithelium Conjunctiva
26 “8 12 12 9
(5) had only lysis beneath the 5 mm-filter
paper disk.
activities can be calculated from the standard curve and the dilution factor. The activity in 5 mM-Tris-HCl pH 7.0, was used as control in computing the curves from results obtained with samples of different amyIase activities.
TABLE
Values of amylase activity Subject Tear
1 2 4
625 945 4000
II
in tear Jluid, urine a,nd saliva
Amylase u/I Urine
2.50 1600 250
Saliva
150 x 102 80 x IO” 200 ‘4 lo’
Comparison of curves A and C in Fig. 3 shows that EDTA in low concentration inhibits all three amylases considerably; for the salivary and urinary enzymes the pH of maximum activity thereby shifts to lower values, in contrast to the amylase from tear fluid where it remains constant at about pH 7.0. The pH-optimum for the urinary enzyme lies, in the presence of Caaf and Cl- (curve A), at somewhat higher pH-values than for the other amylases. Presence of 50 mM-Cl- in the phosphate buffers without EDTA exerts in these experiments the greatest effect on the salivary amylase.
AMYLASE
IN
TElZR
FLUID
399
(a)
(d) FIG. 2. $mylase isoenzymes in tear fluid (TF), saliva (YA) and urine (UR) of three individuals. Samples contaiuinq respectively 6,5 and 24 mu of amylase activity for subject 1,2 and 3, were applied a8 follows: (a) subject 1: 10 ~1 TF, 9 ~1 SA diluted 1: 200 and 9 ~1 UR concentrat,ed : 3; (1~)subject 2: 5 yl TF, 6 ~1 SA diluted 1: 100 and 3 ~1 UR; (c) subject 3: 6 ~1 TF, 6 ~1 SA diluted 1:50 and 6 ~1 UR concentrated x 16; (tl) subject 3: 6 PI TF, 6 ~1 SA diluted 1:5000 and 6 ~1 OR concentrated :r 16.
o6
7
8
PH PIG. 3. pH-activity curves for the amylases in tear fluid, saliva and urine of subject 2. Curves A show the results of incubation in 25 m-phosphate buffer containing 50 mM-NaC1, curves B 25 mM-phosphate buffer only, and curves C 25 mx-phosphate buffer containing 50 mM-Na’aci and 2 mu-EDTA. (a) 1: 10 diluted tear fluid; incubation time 15 min. (b) 1: 400 diluted saliva; incubation time 5 min. (c) I:50 dilution of x 10 concentrated urine; incubation time 15 min.
4. Discussion In human tear fluid amylase was detected for the first time by Mylius (1961) with a histochemical method. The glycogen-dissolving capacity of tears was demonstrated on sections of tissues with a high content of glycogen. Liotet (1967) and van Haeringen and Glasius (1974a, b) demonstrated the presence of anlylase in human tear fluid with an amyloklastic method. In considering the origin of enzymes (and other constituents) of tear fluid, the different sources of this fluid have to be taken into account. The tears present in the conjunctival sac consist of a mixture of fluids, secreted, according to Jones (1966) by: basic secretors (goblet cells, crypts of Henle and glands of Manz), lacrimal secretors (accessory lacrimal glands of Krause- and of Wolfring and occasional glands), oil secretors (Meibomian glands, glands of Zeis and of Mall) and reflex secretors (main lacrimal gland and accessory gland). According to Ehlers, Kessing and Norn (1972) the amounts of conjunctival mucous secretion and lacrimal gland fluid are produced in a ratio of 1: 1060. Reflex stimulation by handling of Schirmer paper or capillaries will increase the production of tears and of course even change that ratio in favour of the lacrimal gland fluid. Prom this point of view it may be concluded that practically all the tear fluid. collected in our experiments is lacrimal gland fluid.
AMYLASE
IN
TEAR
FLUID
401
Thompson and Gallardo (1936) already proved, by comparative investigation of the lysozyme contents of lacrimal gland and conjunctiva in dogs, that the lysozyme of tears originated in the lacrimal gland. The substantial amount of amylase, found together with lysozyme in lacrimal gland tissue, as compared with the much smaller amounts of amylase and lysozyme in cornea1 and conjunctival tissue, therefore may provide the answer to the problem of the tissue origin of amylase in tear fluid. If cornea and conjunctiva contributed to the amylase activity of tear fluid, only lower activities would be expected during lacrimation as a result of the dilution of cornea1 and conjunctival secretions by lacrimal gland fluid. However, earlier studieh (van Haeringen and Glasius, 197413)revealed t’hat. during stimulated tear production. fluctuations in amylase activity of tear fluid occurred in a range of 50 up to 26@4;, of the initial value. The small amounts of amylase and lysozyme found in cornea and conjunctiva, as compared with lacrimal gland tissue and tear fluid, suggests the reverse situation: the epithelium of the tissues coming into contact with the tear fluid is contaminated hv penetration of these enzymes into the thin mucous layer covering the outer cells and into the intercellular spaces. This means that amylase and lysozyme can be found as normal constituents of cornea1 and conjunctival tissue. Indeed. amylase activit) could he demonstrated in bovine cornea extract by Jonadet (1964) and Thompson and Gallardo (1936) found small amounts of lysozyme in the conjunctiva of dogs, and in the experiments of Fraqois and Rabaey (1956,1963) and Kamerau and Ott (1961) the presence of lysozyme in extracts of bovine cornea1 epithelium can be concluded from the appearance of an unknown fraction in the electrophoretic pattern at bhe ca.thodal side of gamma globulin. The activities of amylase in tear fluid from different persons show a considerable variation; values of 200-3000 u/l were found with the iodine starch method (van Haeringen and Glasius, 1974a, b). In our later st,udies. however. Bernfeld’s methott was ljreferred. because in this method the reaction products are estimated directly and the amylase activity can be measured in a larger scale. In the present st,ud!activity values of 625-4000 u/l were found (Table II). As it was an aim of our study to compare the amylase isoenzyme pattern of tear fluid with those of other body fluids, we matched in agar electrophoresis the amount of salivary and urinary amylase with that in the applied sample of tear fluid. As a consequence. t,he patterns of subject 3 show bands of higher density, because thtb amount of amylase applied from subject 3 (24 mu) is here 4-5 times more than front subject 1 and 2 (6 and 5 mu resp.). Possibly for this reason the two additional faint bandx are visible in the tear fluid pattern of subject 3, besides the three bands occurring in all patterns. Figure 2(d) gives an impression of the relative activity of t,he fa.int most cathodical band in tear fluid, as it apl’ears in equal densit,p as the hand in ii 1 : ,500Odiluted saliva sample. The itioenzytne pattern of tear fluid is clearly different from that of saliva in respect of the migration of the anodal bands and their relative intensities. In serum. the pancreas is the main source of amylase. which because of its molecular weight of about) 48 000 can be freely excreted by the kidney. Therefore the isoenzyme pattern of urine corresponds to that of serum and only minute amounts of salivary amylase may bt f’oun11 in 20-fold concentrat.ed urine (Merritt et al.. 1973). The three different’ iso enzymes in tear fluid, present in the three subjects under investigation. do not corrclspond to the urinary amylase isoenzymes, which indicates that amylasr in t.eai fluicl does not originate from the blood, but that it is locally synthesized.
10”
N. 3. VAN
HAERIS(:ES,
Fi. EKSISK
Ah-l)
IL
(:I,ASlI‘h
The relative migration of amylase isoenzymes in saliva and tear fluid suggests r 1r;l.t the observed isoenzymes differ in degree of glycosidation or deamination (Kelhbr. Kauffman. Allan and Williams. 1971). No conclusion can be drawn as to I\-Mher these isoenzymes are the result of post-tmnscriptional and post-secretional motlificatiolls of a single gene product (Karn. Shulkin, Merritt and Newell. 1973) or that’ gvltrt’ie polymorphism plays a role. The isoenzpme variation as occurred in the l)at,ttlrll OI urine of the three individuals in this study msy have a genetic I)xsis (Merritt clt ill.. 1973). In view of these differences in the isoenzvnic patterns, it is not surprising that’ the pH-activity curves, in which for each amilase the properties of the isoc~nzymt~~ww summaked. differ as well. (‘a2+ is the main activator of amylases (Hsiu, Fischer ant1 Stein? 1964) ; bintling of C’a2+bv EDTA indeed inhibits all three amylases. while relatively high concentrations of c’l- do not restore the original activity (Fi,.0 3. curves C). Although phoxphatr~ can bind C’a2i- -and t.herefore no extra C’a”+ was added in the buffers--there was in our experiments no risk that the phosphate buffer affected the Cla-amylase complex in the sample dilutions because the binding of C’a2+ by the enzyme is extremelS st,rong (Stein, Hsiu and Fischer, 1964). For the same reason it is doubtful whether the amylase activity in diluted saliva samples is as dependent on relatively high (‘a’)+concentrations as claimed by Mermall. Hanhilla and Reeves (1973). Their results 011 the activation of salivary amylase by C’a2r and other cat,ions are, moreover, cliticult to interpret because no indication is given regarding pH-control. The presence of low concentrations of EDTA was saGsfactory for removal of ( ‘ill) ! from the enzyme. although the incubat’ion with starch had to follow the dilllt,ion in the phosphate-EDTA buffer as soon as possible. because amylasc is more suscc:pt,iI)lc to tlenaturation in the absence of C’a2’m(Hsiu et al.. 1964). The effects of Cl- on the activity and the pH-optimum of the Ca-amylase conll)ltfx a,re marked for the enzyme in salira [Fig. 3(b)] ; for the amylases in tear fluitl an(l urine the effects are less clear, because in curves B the W-content of tears anal urine may play a role in the activation: Levitzki and Steer (1974) showed that aniorls like C!l- can already. in concentrations below 5 111~.have a maximal effect on hog pancreas amylase. For the same reason the high EaC’l concentrations, often intlicatclc-I t’oor optimal amglase assay, do not seem necessary (cf. Ceska. 1971 and Spiekerman ct. al.. 1974). t!onaitlering the effects of C!l- on the pH-activity curves. it has aIso to be kept in mind that the ionic strength in the buffer solutions containing NaCJI is sorur~~hat higher than in those containing only phosphate and EDTA, which map ha\-c ;ltl additional effect on the amylases. Inhibition by EDTA does not affect the pH-optimum for the amylaae in human tear fluid, in contrast to that for salivary and urinary amylases; therefore it cannot, in general be concluded that the pH-optimum of the amylases depends on the degree ol activation of the enzyme. The physiological role of amylase in tear fluid is not yet clear. As suggested IJY Liotet (1969), tear amylase could play a part in the glycogen metabolism of the cornea1 epithelium, liberating glucose necessary for the nutrition of cornea1 cells. Amylase could also be involved in other metabolic processes of glycogen such as the formation of mucous material in epithelial cells (Pei and Rhodin, 1971). Adverse reactions after frequent administration of EDTA-containing therapeut,ics to the eye do not seem to have been recognized so far, but are a possibility to consider.
A>IYLASE
IN
TEAR
403
FLUID
REFERENCES Bcrnfeld, I’. (1951). Enzymes of starch degradation and synthesis. Advnn. Enzymol. 12, 379. Bonavida, B. and Lapse, A. T. (1968). Human tear lysozyme: II. Quantitat,ive determination u%h standard Schirmer strips. Amer. J. Ophthdnd. 66, 70. C’rska. M. (1971). A new approach for quantitative and semi-quantitative determinations ot enzymat’ic activities with simple laboratory equipment. DeterGon of amylase. Cli71. (‘hint. A& 33, 135. mucows Ehlers, N., Kessing, S. V. and Norn, N. S. (1972). Q uantit,ative amounts of conjwctival secretion and t,ears. Actn Ophthnlmol. 50, 210. Francois. J. and Rabaey, M. (1956). Les proteines de l‘epith6lium oornben. ,4/~n. Owl. 189, 445. Francois. J. and Rabaoy. M. (1963). Immunoelectrophoresis of the proteins of the cornea1 epithelium. F:xp. Eye Res. 2, 196. Fridhandler. I,.. Berk. J. E.. &Montgomery, li. A. and Wang, D. (1974). Column-chromatographit, studiw of isoamylases in human serum, urine, and milk. Clin. (‘hug)?. 20, 547. Hawingen, N. J. van and Glasius. E. (1974a). Enzymes of energy produring metabolism in humall t’ear fluid. &xl). Byr Res. 18, 407. Hwringcn, N. ,J. van a,nd Glasius, E. (197413). Enzymatic studies in lac~rimal secretion. /:‘.rlj. K,//r KfS. 19, 135. Hsiu, ,J., Fischer. E. H. and St,ein, E. A. (1964). Alpha-amylases as calrium-metalloenz~nl~s. II. (Calcium and the catalytic act#ivity. Biochemistry 3, 61. Jamieson, A. D.. Pruitt, K. M. and Caldwell, R. C. (1969). i\n improwd amplase assay. .I. Jkw1.
RP.u.48, 4M. .lonadet.. &I. (1964). Xutivit&s b&ta-glucuronidasique et amylasiqne au nivrau de In c.orn@e. KF/.. PdhoZ. Pomp. Ifyg. Gen. 67, 453. .Jones. 1,. T. (1966). The lacrimal secretory system and its treatment. dnler. .I. Ophthnlmol. 62,47. Karn. R’. (‘.. Shulkin. J. D., Merrit,t. A. D. and Sewell, K. C. (1973). Evidence for post-transcriptional modification of human salivary amylase (Aim,v-,) isozymes. Biochrw. Genet. 10, S41. I
4X67. I,witzki. :Z. and Seer. M. L. (1974). The allostwic activation of mammalian z-am,&se t)y chloride. Ew. J. Biochem. 41, 171. Liotet. S. (1967). Pouvoir amylasique des larmes humaincs. dr~n. Owl. 200, 526. 2lermall. H. L., Hanhila, M. 0. and Reeves, IV. ;\I. (1973). Effects of cations on the activation ot salivary amylaae: I. Nat, I(+, Ca2f and Mg2+. J. Dent. Krs. 52, 114X. Slerritt’. A. D., Rivas, 31. L., Biller, D. and Sewell. R’. (1973). Salivary and pancreatic amylaw: clrctrophoretic characterizations and genetic studies. Amer. J. Hum. (&net. 25, 510. Mylius. .I. E. (1961). Amylase in t,ears? A histochemical study. dctrr I’rctlrol. Nicrobiol. Svr,rt/. (Suppl.) 148, 143. I’cti. Y. F. and Rhodin. J. A. S. (1971). Electronmicroscopic study of thr development of thp n1ouse cornea1 epit,helium. Incest. Ophthdmol. 10, 824. Rick. 12’. and Steghauer. H. P. (1973). a-Amylase. 3Iessung der redurerendrn Gruppen. ,Ilethotlw rlrr rn:ymtrtis&n. Analyse (Ed. Bergmeyer. H. I:.). Band I. p. 918. Verlag Chemie, iVeinheirn. Spiekcrman, A. 11.. Perry. P., Hightower, S. C. and Hall, F. F. (1974). (:hromogenic-substratr method for demonstrating multiple forms of alpha-amylase after elertrophoresis. L’litl. (!hiw.
~-l&Z.20 ,t”‘P4. Sitein. E. A., Hsiu, tJ. and Fischer, E. H. (1964). -ilpha-amylases as calcium-metalloenzymes. 1. Preparation of calcium-free apoamylases by chelation and electrodialysis. Biochemistry 3, 5fi. Thompson. R. and Ciallardo, E. (1936). The concentration of lysozyme in the tears in acute and cahronic conjumtivitis. Wit,h a note on the source of the lysozyme in tears. Amer. J. Ophthnlmol.
19, 684. Wesley-Hadzija, B. and Pigon, H. (1972). Effect of diet in West Africa activity. ilrc11. OrnZ BioZ. 17, 141.5.
on himIan salivary
amylaw