SPECIFICITY OF THE HUMAN INTESTINAL AND IMPLICATIONS FOR HEREDITARY INTOLERANCE

Arne Dahlqvist

J Clin Invest. 1962;41(3):463-470. https://doi.org/10.1172/JCI104499.

Research Article

Find the latest version: https://jci.me/104499/pdf Journal of Clinical Investigation Vol. 41, No. 3, 1962 SPECIFICITY OF THE HUMAN INTESTINAL DISACCHARIDASES AND IMPLICATIONS FOR HEREDITARY DISACCHARIDE INTOLERANCE * By ARNE DAHLQVIST (From the Department of Physiological Chemistry, University of Lund, Sweden) (Submitted for publication June 27, 1961; accepted October 27, 1961)

The existence of several different a- 1) Jejunal sample, from a 40 year old woman; resection has recently been demonstrated in extracts of pig of the middle part of the was indicated by an intestinal tumor. From an 8-cm piece of normal intestinal mucosa (1-7). These have intestine, located in the distal decimeters of the , varying specificity for with a-D-glu- 1.6 g of mucosa was scraped off with a glass slide. The copyranoside structure (e.g., , , iso- mucosa was homogenized for 1 minute in an Ultra-Tur- maltose, ), and thus the intestinal hy- rax homogenizer with 4.8 ml of 0.9 per cent NaCl, the drolysis of these is caused by a more com- tube being chilled with crushed ice during homogenization. previously After removal of nuclei and greater cell debris by cen- plicated mixture than had been trifugation in an ordinary laboratory centrifuge (Wifug, believed (8, 9). rotor no. 103-30A, 4,000 rpm) for 10 minutes, the opales- The 8-glucosidase and /3-galactosidase activities cent supernate, which contains the disaccharidases (22), of extracts of pig intestinal mucosa, in contrast, was used for analysis. 2) Ileal sample, from a 26 year seem to be exerted by one enzyme, intestinal lac- old woman suffering from chronic ulcerative ; re- operation of ileocecal anastomosis indicated by abdominal tase (10, 11). abscess. A 15-cm piece of the distal part of the It is not known whether the specificity of the was removed. The intestinal wall of this piece showed human intestinal disaccharidases parallels that of considerable induration, but the mucosa appeared macro- the pig enzymes. This question is of clinical im- scopically normal; 3.0 g of mucosa was obtained by portance, however, since several cases of hereditary scraping with a glass slide. The mucosa was homoge- nized as described above with 9.0 ml of 0.9 per cent inability to digest certain disaccharides have been NaCl. recently described, and are apparently caused by Assay of activities was performed by inherited deficiency of single intestinal disacchari- the methods previously described (23, 24) using the dases (12-19). Tris- oxidase reagent (24). One unit of disac- Since the human intestinal content, obtained by charidase activity causes 5 per cent in 2.0 ml a catheter during the digestion of a meal, contains of 0.028 M substrate, which is equal to the formation of 1 mg (23), in 60 minutes at 370 C and at essentially no disaccharidases (20, 21), this ma- optimum pH. terial cannot be used for the study of the specificity Assay of polysaecharidase activities was done as previ- of these enzymes. Therefore homogenates have ously described (25). One unit of or been prepared of human intestinal mucosa, ob- activity catalyzes an increase in reducing power corre- tained from pieces of small intestine cut out during sponding to 1 mg of disaccharide (monohydrate) in 60 minutes at 370 C, and may thus be compared with the surgical operations. These homogenates had unit for disaccharidase activity. powerful disaccharidase activities, and the speci- Assay of protein was performed by the method of ficity of the enzymes responsible for these activi- Lowry, Rosebrough, Farr and Randall (26) with the ties has been studied by heat inactivation. modified reagent B introduced by Eggstein and Kreutz (27). A standard curve was prepared with human serum by AB Kabi (Stockholm). AND METHODS albumin, kindly supplied MATERIALS Heat inactivation. When a solution of an enzyme is Homogenates of human intestinal mucosa. Two sam- heated at a sufficiently high temperature the enzyme is ples were obtained, one from the distal part of the inactivated, following the kinetics of a first-order reac- jejunum, and the other from the distal part of the ileum. tion. The temperature interval within which the velocity constant for the inactivation increases from zero (no * This investigation was supported by grants from the measurable inactivation) to very high values (complete Swedish Medical Research Council and the Royal Physi- inactivation within a few minutes) is usually less than 100. ographic Society at Lund, Sweden. Since the different glycosidases present in extracts of pig 463 464 ARNE DAHLQVIST

intestinal mucosa have widely different sensitivities to paper indicate that the human intestine contains disac- heat, fractionated heat inactivation provided a useful charidases which are essentially similar to the correspond- method for the separation of these enzymes (3, 28, 29). ing enzymes of the pig, although some differences are The rate of the heat inactivation at a certain tempera- found. The chromatographic separation of the pig en- ture is strongly dependent on the pH of the solution; zymes succeeded only with the use of a special tech- thus an appropriate buffer must be added. It is also nique-mutual displacement chromatography (29)- desirable to heat the enzyme solution to the desired in- which demanded rather large amounts of enzyme ma- activation temperature as quickly as possible and then to terial. The limited amount of enzyme available with hu- maintain this temperature within 0.10 during the ex- man preparations prohibited separation experiments with periment. The enzyme solution which is to be inacti- this method. Chromatographic separation of the human vated (usually a volume of 5 to 10 ml) was therefore disaccharidases will have to await the development of a first immersed in a preheating bath that had a tempera- suitable micromethod. ture 7° to 80 higher than the inactivation bath. Dur- RESULTS ing the preheating the tube was continously shaken, and the temperature of the enzyme solution was measured The homogenates had powerful disaccharidase with a thermometer having 0.10 gradations. At the activities. The relative rate of hydrolysis of dif- moment the desired inactivation temperature was reached (under these conditions this occurred in less than 1 min- ferent disaccharides was essentially the same as ute) the tube was transferred to the inactivation bath; at that reported earlier for preparations from the mu- the the same time a zero-time sample was taken for en- cosa of the adult pig (23, 25), except that the rela- zyme analysis and a stopwatch was started. Care was tive isomaltase activity of the human preparations taken that the inactivation temperature was never ex- was stronger and the activity somewhat ceeded during preheating. New samples for enzyme analy- sis (0.5 to 1 ml) were withdrawn at intervals, immedi- weaker than in the pig (Table I). ately blown down into tubes chilled with crushed ice, and The activity of the jejunal sample was so stored until analyzed. strong, but this activity was practically absent in The enzyme solutions for heat inactivation in the pres- the ileal sample, indicating that lactase in the hu- ent case were prepared by mixing 1 vol of homogenate man is localized in the proximal part of the in- (the j ejunal sample, which was strongest in disaccharidase activity, had first been diluted 1:2 with distilled water) testine as it is in the pig (25). This is also in ac- with 4 vol of 0.0125 M sodium phosphate buffer, pH 7.0. cordance with the finding that is absorbed Validity of separation by heat inactivation. The sepa- in the proximal part of he human small intestine ration of two enzymatic activities, present in the same (21). preparation, only on the basis of their different sensitivi- The cellobiase and gentiobiase activities in the ties to heat, does not conclusively prove that the two ac- (Table tivities are manifestations of different enzyme proteins; two samples paralleled the lactase activity heating the solution may affect activators or inhibitors, I), indicating that also in the human intestine breaking single bonds within the enzyme molecule may ,8-glucosides and /3-galactosides may be hydrolyzed alter the specificity of the enzyme, and so forth. In- by a single enzyme (10, 11). versely, the parallel heat inactivation of two different en- zymatic activities does not necessarily mean that these are caused by the same enzyme; two different enzymes TABLE I may very well have similar heat inactivation properties. Carbohydrase activities of the homogenates of human small The results of heat inactivation experiments therefore intestinal mucosa * must be compared with those of other separation meth- ods, such as fractionation with different precipitants, Carbohydrase a) Jejunal b) Ileal electrophoresis, ion-exchange chromatography, and so on. activity sample sample the existence of several different disac- By such methods unitsIg mucosa charidases in pig intestinal mucosa has been conclusively (total) 948 292 demonstrated and the specificity of the different enzymes I nvertase 360 40 elucidated (3-7, 10, 28-30). In this study the results of Isomaltase 272 69 Trehalase 31 17 the heat inactivation experiments agreed very well with Turanase 74 23 those of the other methods. Furtthermore, the heat in- Palatinase 59 12 activation analysis was found to be the most simple and Lactase 412 3.2 accurate method for the analysis of the mixture of disac- Cellobiase 78 0.6 Gentiobiase 2.4 0.0 charidases present in extracts of pig intestinal mucosa Amylase 1,328 960 (3, 29). Dextranase 32 8.8 The heat inactivation properties of disaccharidase preparations of human intestinal mucosa described in this * Protein content: a) 82 mg, b) 92 mg/g tissue. DISACCHARIDASES AND DISACCHARIDE INTOLERANCE 465

0--O Irvetose activity 0--O Malta&V activity concluded from their sensitivity to heat and their 0-0 Isomalfaso activity relation to the activity, maltase Ib, II, 5 and III in these samples correspond to maltase I, II, and III, respectively, in the pig intestinal mu- cosa (3, 4, 29). Thus there exists in the human intestine one further enzyme with maltase activity, maltase Ia. This new enzyme was inactivated parallel with the isomaltase activity (Figure 1), which may indicate that the maltase Ia and the isomaltase activities are produced by the same en-

0 2030 __ }at@ zyme, a hypothesis that, of course, needs further This ~a toi-IIII experimental proof. hypothetical enzyme , }asM e m was responsible for about half of the total maltase 0 20 40 60 0 20 40 60 0 20 40 60 0 20 40 60 MMU105 activity in the preparations of human mucosa FIG. 1. HEAT INACTIVATION OF THE HUMAN INTES- (Table II) and accounted for essentially all of the TINAL a-GLUCOSIDASES IN FOUR STEPS. The enzyme solu- isomaltase activity of the preparations. About tion contained 2 mg protein per ml in 0.01 M sodium phos- 15 to 20 per cent of the invertase activity also was phate buffer, pH 7.0. The original disaccharidase ac- tivities (before heating) were: invertase 7.9, maltase inactivated during heating at 450 C for 60 minutes (total) 21.4, and isomaltase 6.1 U per ml. (Figure 1). The inactivation of this fraction of the invertase activity proceeded, however, more The amylase activity, in contrast to the disac- slowly than the inactivation of the maltase Ia and charidases, was weaker in the mucosal samples isomaltase activities, and thus there seems to be than in intestinal contents (21), in harmony with no reason to ascribe any measurable invertase ac- the conception that this enzyme is mainly se- tivity to maltase Ia. creted from the pancreas (21, 25). It cannot be B. Invertase activity. The heat inactivation of stated whether the amylase present in the mucosal invertase at all temperatures was in accordance homogenates originates from the mucosa per se or with the kinetics of a first-order reaction, indi- from contaminating intestinal contents. cating that the human intestinal invertase activity, Dextranase which, like the disaccharidases, is like that of the pig (3, 4, 6, 28), is caused by a located in the intestinal mucosa (25), had activity single enzyme. The human maltase lb activity comparable with that in preparations from pig was inactivated simultaneously with the invertase mucosa. activity, and the invertase and maltase lb activity is probably caused by one enzyme, which seems to Heat inactivation of the disaccharidases have properties similar to the pig maltase I (in- A. Maltase activity. The course of the heat in- vertase) (6). With this assumption the maltase/ activation of the maltase activity of the homoge- invertase activity quotient for this enzyme was cal- nates did not follow first-order kinetics, indicating culated to be 1.0, which may be compared with that the maltase activity was caused by a mixture 0.7 for the pig enzyme (23). of several enzymes with different sensitivities to heat. By heating at temperatures increased step- TABLE_ II wise, the maltase activity could be divided into Fractions of the total maltase activity of the homogenates four different fractions Ib, II, produced by each of the different maltases of human (maltase Ia, and intestinal mucosa III 1), in order of increasing resistance to heat (Figure 1). The fraction of the total maltase a) Jejunal b) Heal Enzyme sample activity in the homogenates that was produced sample by each of these enzymes is seen in Table II. As %,of total maltase activity 1 By analogy with the pig enzymes (28, 29), naming the Maltase Ia 50 44 two most heat-labile human maltase Ia Maltase lb 34 16 fractions and lb Maltase II 9 20 seems more consistent than calling the human maltase Maltase III 7 20 fractions I-IV. 466 ARNE DAHLQVIST

O-0 Celloblase activity C. Isomaltase activity. The isomaltase activity % also seemed to be caused by a single enzyme in 0- Lactose octivity the human preparations, as concluded from the jA00 t 60 kinetics of the heat inactivation. Like the specific 1 isomaltase in the pig (31), which in this species 40 ,,8 accounts for the major part of the isomaltase ac- C 20 tivity (7, 23), the human isomaltase was inacti- vated with the most heat-sensitive disaccharidase a ,0 fraction (Figure 1). As concluded from the heat c 4 51 inactivation experiments, the human isomaltase t C to 20 30 40 50 60 mInutes and maltase Ia are caused by the same enzyme. FIG. 3. HEAT INACTIVATION OF THE HUMAN INTESTI- If this is the case, there is a difference in speci- NAL CELLOBIASE (,8-GLUCOSIDASE) AND LACTASE (a8-GA- ficity between the human and the pig isomaltase, LACTOSIDASE) ACTIVITIES AT 500 C. The enzyme solution contained 2 mg of protein per ml in 0.01 M sodium phos- for the specific isomaltase of the pig has no mal- phate buffer, pH 7.0., Cellobiase activity 1.8, and lactase tase activity (3, 7, 31). The maltase/isomaltase 7.5 U per ml. The cellobiase and lactase activites are in- activity quotient for the human enzyme was cal- activated slowly and parallel each other, indicating that culated to be 1.8. these activities may be produced by a single enzyme. Although the specific isomaltase in the pig ac- counts for the major part of the isomaltase activity activity comparable with that of the pig enzymes, (23), the pig maltase II and maltase III also pro- although this activity would have been too weak to duce weak isomaltase activity, caused by these be detected. enzymes per se (4, 30). The present experi- D. Trehalase activity. The human trehalase ac- ments do not rule out the possibility that the tivity seemed homogeneous in the heat inactiva- human maltase II and maltase III have isomaltase tion experiments (Figure 2). At 520 C the tre- halase activity was slowly inactivated, and thereby

0-0 Invertqsee ctivit completely distinguished from the isomaltase, in- _- Malteae activity G-0 lsomaitase activty vertase, maltase Ia, and maltase lb activities, o r,-ehafase activity which were rapidly inactivated; and also from the maltase II and maltase III activities, which were IZ not measurably affected (Figure 2). As in the pig (5), therefore, the trehalase in the human in- testine seems to be a specific enzyme, distinguished i from the other human intestinal a-glucosidases. IIt 1% IL E. Cellobiase and lactase activity. Both cellobi- I11 0 ase and lactase activities were inactivated in ac- Q 0 cordance with the kinetics of a first-order reac- k 46 tion, and the inactivation of these activities ran k parallel (Figure 3). With the human enzymes it was thus indicated that a single intestinal disac- I I 0 t0 20 30 40 50 60 m*Wft charidase may hydrolyze both /8-glucosides and FIG. 2. SEPARATION OF TREHALASE FROM THE OTHER ,8-galactosides, in accordance with previous re- HUMAN INTESTINAL a-GLUCOSIDASES BY HEATING AT 520 sults with the pig enzyme ( 10, 11 ). C. The enzyme solution contained 2 mg of protein per ml in 0.01 M sodium phosphate buffer, pH 7.0. The original Comparison of the human intestinal disaccharidases disaccharidase activities were: invertase 7.9, maltase with those of the pig (total) 21.4, isomaltase 6.1, and trehalase, 0.5 U per ml. The trehalase activity decreases slowly, following the The heat inactivation experiments indicate the kinetics of a first-order reaction. It is thereby clearly existence of at least six different disaccharidases distinguished from the isomaltase, invertase, maltase Ia, in the human small intestinal mucosa (Table III). and maltase Ib activities, which are rapidly inactivated, and from the maltase II and maltase III activities which The main difference from the pig enzymes (3, 10) are not measurably affected. seems to be that the human isomaltase probably DISACCHARIDASES AND DISACCHARIDE INTOLERANCE 467

TABLE III tration of the disaccharide they cannot utilize, but Specificity of human intestinal disaccharidases and their the administration of the corresponding mono- quantitative importance for the hydrolysis of different disaccharides as concluded from heat inactivation saccharides or other disaccharides is followed by experiments the normal blood response. In some cases the unhydrolyzed disaccharide has been demon- % of total activity against each strated in the urine or in the feces ( 12, 14-16, 18). Enzyme Substrate substrate Loss of weight, flatulence, abdominal pain, in- creased excretion of volatile fatty acids (formed Isomaltase = maltase Ia Maltose 00 by intestinal bacteria), vomiting, and renal acido- Invertase = maltase Ib fSucrose 100 sis have been reported, and the condition may be IMaltose 25 fatal. If the poorly tolerated disaccharide is ex- Maltase III Maltose 15 cluded from the diet and replaced by monosac- Maltase III Maltose 10 charides or other utilizable carbohydrates, the Trehalase Trehalose 100 symptoms disappear. Although enzymatic analy- sis of the intestinal mucosa of these patients has Lactase~ ~ ~ Lactose 100 Lactase fCellobiose 100 hitherto not been performed, there seems to be sufficient evidence for the conception that the dis- has considerable maltase activity, which the cor- ease is caused by inherited deficiency of single in- responding pig enzyme (specific isomaltase) has testinal disaccharidases. not (7, 31)). As a consequence, there exist in The enzyme deficiency is apparently compen- the human intestine not fewer than four different sated after the first years of life, but the mechanism enzymes with maltase activity, as compared with for this is unknown (18). three in the pig. A. Disaccharide intolerance to be expected from There appears to be a developmental difference deficiency of the different disaccharidases. Some in the intestinal disaccharidases of the two species. of the intestinal disaccharidases act on more than The intestinal lactase activity is strong at birth in one kind of substrate, and certain substrates are both species (11, 32). In the newborn pig, how- hydrolyzed by more than one intestinal enzyme. ever, the intestinal a-glucosidases are weak or With the assumptions made above about the speci- absent, and these enzymes ficity of the human intestinal disaccharidases, cer- develop gradually dur- tain ing growth (11, 33, 34). As a consequence, the predictions can be made about the disacchari- newborn pig cannot utilize sucrose or dase intolerance to be expected from the deficiency dietary mal- of of tose The normal is each the different intestinal disaccharidases. (35). child, however, able to intolerance utilize sucrose or maltose as well Sucrose can occur by itself, without equally as lac- for tose (17-19), and invertase has been demonstrated simultaneous intolerance other disaccharides. in the intestine of the newborn child and hu- Although the human intestinal invertase seems to the be identical man fetus as early as the third month of intra- with maltase Ib, and consequently the uterine life (36). The intestinal a-glucosidases of absence of invertase will entail the absence also of the maltase Ib activity, the other maltases will the human therefore appear to be already well de- be veloped at birth. strong enough to ensure sufficient hydrolysis of maltose. Isomaltose intolerance for corresponding DISCUSSION reasons also can occur as an isolated defect. Mal- Implications of disaccharidase specificity for he- tose intolerance cannot be caused by a single en- reditary disaccharide intolerance zyme deficiency but demands the simultaneous lack of four different enzymes. Maltose intol- Several cases have been recently described of erance will probably always be accompanied by in- inherited inability to utilize one or another disac- tolerance for sucrose and isomaltose. Trehalose charide (12-19). These patients suffer from mal- intolerance can occur separately, since intestinal nutrition and usually develop on peroral trehalase is a specific enzyme without action on ingestion of the disaccharide. No rise in blood the other disaccharides. can sugar is noted in these patients on peroral adminis- occur without simultaneous deficiency in a-glu- 468 ARNE DAHLQVIST cosidases, but will probably always be accompanied only case so far known in whom more than one by cellobiose intolerance. disaccharidase was shown to be absent. In this B. Nutritional importance of the different disac- patient isomaltose intolerance would also be ex- charides. The clinical consequences of intolerance pected, but no test with this disaccharide was de- for (inability to utilize) a disaccharide will be de- scribed. In fact, no case of intolerance for isomal- pendent on the amount of this disaccharide present tose has so far been described, although this de- in the diet. will give marked fect can be expected to cause clinical symptoms symptoms when the child begins to get a mixed (decreased ability to utilize starch). diet, since sucrose is one of the major carbohy- Trehalose intolerance has never been described, drate components in our food (37). In artificial and as an isolated defect will probably not cause feeding of the newborn child sucrose is commonly clinical symptoms. added, and may thus initiate the symptoms in an Lactose intolerance, which was first described invertase-deficient infant. Both maltose and iso- by Durand (12), has hitherto been described in 11 maltose intolerance will interfere with the utiliza- patients (12-16) 2. In several cases this defect ran tion of dietary starch (1, 2), which is the main a fatal course (12, 14-16), reflecting the nutri- source for these disaccharides and related oligo- tional importance of lactose for the young child. saccharides, formed by the action of amylase. In no case has it been stated that cellobiose intol- Trehalose intolerance will give scarcely any clini- erance is involved, as would be expected from the cal symptoms at all at any age, since trehalose is specificity studies. Sucrose, maltose, and starch present only in very small amounts in our food have been tolerated by these patients (13). (37). This seems especially remarkable, since The cases of disaccharide intolerance described intestinal trehalase has very marked specificity for above seem, with one exception, to have been its substrate (5). Lactose intolerance will cause caused by deficiency in a single intestinal disac- severe malnutrition in the newborn child, especially charidase. The findings concerning the speci- if the child is breast fed, since lactose constitutes ficity of the human intestinal disaccharidases agree practically all of the carbohydrate in milk, and well with the intolerance observed in these patients, accounts for nearly 50 per cent of its caloric value. so far as they have been investigated. In further Cellobiose intolerance is associated with lactase work the possibility of isolated isomaltase defi- deficiency. The cellobiase deficiency as such will ciency has to be kept in mind as a possible cause not, however, be of any clinical importance, since of failure in the utilization of starch. cellobiose is not present in our diet in any signifi- cant amount (37), and we possess no SUMMARY hydrolyzing cellulose to disaccharide. C. Comparison of theory with cases of disac- Human intestinal mucosa homogenates hydro- charide intolerance described. Prader, Auricchio lyze a number of different disaccharides. Heat and Murset (18) have described five patients with inactivation of the homogenates at varying tem- sucrose intolerance, all of whom had normal toler- peratures in 0.01 M sodium phosphate buffer, pH ac- ance for maltose and lactose. These patients prob- 7.0, has indicated that these disaccharidase ably suffered from isolated deficiency of intestinal tivities are accounted for by a mixture of at least invertase. Weijers and associates (17, 19) de- six separate enzymes-namely, five different a-glu- scribed three patients with sucrose intolerance. cosidases, four of which have maltase activity, and Two of these patients, like those of Prader and co- lactase, which hydrolyzes both f8-glucosides and workers, had normal tolerance for maltose and ,8-galactosides. The a-glucosidases separated are: lactose, while the third showed intolerance also maltase Ia (= isomaltase), maltase Ib (= inver- for maltose. The latter patient thus may be de- tase), maltase II, maltase III, and trehalase. ficient in all the four different intestinal disac- Hereditary deficiency of single intestinal disac- charidases responsible for maltase activity (isomal- charidases is known to appear in children. From = tase = maltase Ia, invertase maltase Ib, mal- 2 Weijers, van de Kamer, Dicke and Ijsseling (19) tase II and maltase III). The tolerance for lac- have observed five further cases of this defect, which tose was normal. This -patient seems to be the have not yet been described in detail. DISACCHARIDASES AND DISACCHARIDE INTOLERANCE 469 the specificity of the human intestinal disacchari- 13. Holzel, A., Schwarz, V., and Sutcliffe, K. W. De- dases, it may be expected that intolerance for iso- fective lactose absorption causing malnutrition in infancy. Lancet 1959, 1, 1126. maltose, sucrose, or trehalose can appear as an 14. Darling, S., Mortensen, O., and Sondergaard, G. isolated defect. Lactose intolerance will probably Lactosuria and aminoaciduria in infancy. A new always be associated with cellobiose intolerance; inborn error of ? Acta paediat. (Upp- and maltose intolerance, which demands the simul- sala) 1960, 49, 281. taneous absence of four separate enzymes, will 15. Durand, P. Intolerance au lactose. Insuffisante hy- drolyse intestinale du lactose. Pediatrie 1960, 15, probably always be accompanied by intolerance 407. for sucrose and isomaltose. 16. Jeune, M., Charrat, A., Cotte, J., Fournier, P., and As far as the disaccharidase-deficient patients Hermier, M. Sur un cas de lactosurie congenitale. described in the literature have been examined, Pediatrie 1960, 15, 411. their defects accord with this theory. 17. Weijers, H. A., van de Kamer, J. H., Mossel, D. A. A., and Dicke, W. K. 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A,