The estimation of total sugar, lactic acid and pH in stool has been described as ..... After lactose, glucose, or galactose tolerance tests (2 g/kg) in children.
Z. Kinderheilk. 115, 141--153 (1973) 9 by Springer-Verlag 1973
Sugars, Lactic Acid and pH in Feces of Children. A Useful Diagnostical Approach for Gastrointestinal Disorders ? Jtirgen Schaub and Michael Lentze Universit~ts-Kinderklinik (Director: Prof. Dr. K. Betke), Miinchen Received May 23, 1973
Abstract. The estimation of total sugar, lactic acid and pH and sugar chromatography of the stool was done in 168 normal children and children suffering from malabsorption syndromes and diarrhea. No significant difference of the parameters between normals and patients could be established. Key words: Feces - - Total sugar - - Lactic acid - - pit - - Malabsorption syndrome - - Diarrhea. Zusammenfassung. Im Stuhl yon 135 gesunden Frfihgeborenen, Neugeborenen, S~nglingen und Kleinkindern und im Stuhl yon 28 Kindern mit Diarrhoe wurden bestimmt: Gesamtzuckergehalt, Milchs~urekonzentration, pit und diinnschichtchromatografisch die Zuckerspektren. Es konnte gezeigt werden, dab weder die Altersgruppen untereinander noch die gesunden Kinder gegenfiber den Kindern mit Diarrhoe signifikante Unterschiede in einem der untersuchten Parameter boten. Es wird gefolgert, dal~ die Diagnose einer Zuckermalabsorption aus diesen Parametern nicht zu stellcn ist.
Introduction The estimation of total sugar, lactic acid and p H in stool has been described as valuable for the diagnosis of congenital and acquired glucose galactose malabsorption, hereditary or acquired disaccharidase deficiency and other malabsorption syndromes [1, 22, 27, 37, 42, 43]. Different methods were applied in these investigations and contradictory results were obtained. There are few k n o w n reports on the excretion of sugars and lactic acid in the stools and the p i t in stools of normal full-term babies, prematures, infants and children [6, 10, 13, 15, 39]. While the digestion and the splitting of the carbohydrates ingested with the food are subjected to various mechanisms b o t h in the small and in the large intestine, the origin of the sugars excreted in the stool has n o t been fully elucidated. Furthermore, the mechanism leading to proliferation of bacteria in the small intestine of infants with diarrhea and the simultaneously high content of carbohydrates has not been satisfactorily explained.
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I n t h e p r e s e n t s t u d y we e x a m i n e d t h e e x c r e t i o n of t o t a l sugar, lactic acid a n d p H a n d t h e different sugar s p e c t r a of stools from 140 n o r m a l subjects aged 1 d a y to 2 y e a r s a n d of 28 children suffering from a c u t e a n d chronic diarrhea. Materials and Methods
Materials Sucrose (2-[a-D-glucopyranosyl-] D-fructofuranoside), maltose (4-[a-D-glucopyranosyl-] D-glucose) monohydrate, lactose (4-[fl-D-galactopyranosyl-]D-glucose), D(+)glueose, D(--)fructose, D(§ L(~-)arabinose, D(--)ribose, D(§ xylose, D(A-)mannose, L(-?)rhamnose, special indicator paper pH 5.4--7.0 and pH 3.8--5.4, anthrone, thin-layer chromatography plates (Silica gel F 254 precoated, layer thickness 0.25 ram) and naphthoresoreine were obtained from Merck AG, Darmstadt, Germany. Laetate-dehydrogenase was obtained from Boehringer & Soehne, Mannheim, Germany. Palatinose (6-0-[a-D-glucopyranosyl]-D-fruetose) [44] was a gift of Dr. Leonhi,user, Boehringer & Soehne, Tutzing, Germany. Isomaltose (6-0-[a-D-glueopyranosyl]-glucose) was obtained from Baker Co., U.S.A., L-fucose from Fluka AG, Buchs, Switzerland. Filters for bacteria (Milipore HAWP 02500 and filter unit Milipore Swinney 25) were obtained from Milipore, U.S.A.
Methods Fresh stool specimens were collected in plastic boxes and immediately frozen and stored until analysis was performed. pH o/the stool. Assay was performed on the thawed stool specimen with the special pI-I paper (Merck), p i t 3.8--5.4 and 5.4---7.0 after the method of Auricchio et al. [4]. This method shows good correlation in comparison with the pH assay using the glass-electrode pH meter. There was no difference between the p i t readings of fresh stools and readings of stools after they had been frozen and then thawed. Estimation of the pH of meeonium hampered the correct pH reading with p i t paper because of its green color. Lactate Assay. Aliquot volumes of the deep-frozen stool were prepared after the method of Weijers et al. [45] and the lactic acid was measured by the lactate dehydrogenase method, according to Auricchio et al. [4]. Thin-Layer Chromatographic Identification o/Stool Sugars. The water content of stools of normal children is quite different from those of children with diarrhea and the results axe therefore not comparable to those obtained with fresh stool specimens. For this reason the deep-frozen stool was lyophilized and then diluted 1:10 (v/w) with water. This suspension corresponded with a normal fresh stool with an average water content of 80% [2]. The suspension was centrifuged at 5000 r/rain. 5--10 ~zlof the supenmtant was spotted onto silica gel plates 10 X 20cm. Ascending thin-layer chromatography was carried out by using 0.016 M boric acid in isopropanol-n-butanol-water (50: 30: 20). To avoid the "phenomenon of the edge" the tanks were lined with fat-free paper which was then saturated with the solvent [38]. The plates were dried with warm air after which a second run followed in the same direction. Identification o/the Sugar Spots. The sugar spots were stained with naphthoresorcine reagent and then developed 10 rain at 90~ [25]. The following standard
Sugars, Lactic Acid and pH in Feces of Children
143
solutions of 100 mg% in isopropanol- water (1 : l v/w) were used for identification of the different sugars: xylose, glucose, galaetose, fructose, sucrose, maltose, lactose, palatinose, rhamnose, arabinose, ribose, isomaltose, mannose and fucose. Total Sugars in Stool. The total sugar content was measured using the anthrone method [34]. The stool suspension mentioned above has been filtered germfree using millipore filters. The procedure is necessary because a third of the dry weight of the stool consists of living and dead bacteria [35]. The cell walls of the bacteria contain oligosaccharides and polysaccharides which are measured by the anthrone method. Results The stool specimens of 168 children were analyzed; 43 of t h e m were p r e m a t u r e babies, 97 full-term newborns and infants and 28 children with acute and chronic diarrhea. All subjects were between 1 d a y and 2 years old. Fig. 1 shows the total sugar excretion of normal newborn and prem a t u r e babies. The curves shown in the figure are the ranges of all values. The shaded area represents the excretion of total sugar in stools of prem a t u r e babies, starting at the third d a y because t h e y do not excrete stool in the first days of life. The excretion of total sugar of full-term newborn babies is high in the first 8 days of life, then drops sharply and decreases gradually in an exponential curve. Prematures show the same p a t t e r n except t h a t the sharp drop begins 8 days later. The excretion of total sugar in stools was followed until the 7th week, the average duration of their s t a y in the hospital. g
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newborns, and infants (n = 140). The areas represent the ranges of all measurements Fig. 2 shows a survey of the normal values for total sugar, lactic acid and p H of stool in children of different ages. The total sugar excretion decreases and the range of the three parameters varies considerably. I n Fig. 3 the mean values of total sugar, lactic acid and p H in stools of newborns in the first 7 days are shown. From the first to the second day the excretion of sugar shows a decreasing tendency diametrically opposite to the excretion of lactate which reaches a peak at the second day and drops sharply at the third day. From the third day onwards, the increase of sugar is followed b y an increase of lactate. The p H shows no correlation with the lactic acid concentration. The lactic acid concentration in the stool of prematures is desereased significantly (p < 0.05) in comparison the concentration in newborns (Fig. 4). The lactate content in the stool of newborn babies with diarrhea is not significantly increased compared with normal prematures and newborns. There is no significant difference between the amount of sugar in the stool ofprematures, newborns and newborns suffering from diarrhea. The p H of the stool of newborns with diarrhea is not significantly decreased in comparison with normals. Fig. 5 shows the three parameters in normal infants and children, and infants and children with diarrhea. The lactate content in the stool of
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normal infants is increased significantly (p < 0.05) compared with those with diarrhea. Sugar excretion and pH do not vary significantly in either group. Table 1 shows the result of the thin-layer chromatographic identification of sugars and the different sugar spectra in stools of prematures, newborns, infants and children, and children with diarrhea. We found sugars in the stools of all premature babies, 95% of newborns, infants and children and 97% of children with diarrhea. The sugars excreted in the largest quantities in prematures are the monosaccharides (galactose with 81%, glucose 48%, and fructose 23%) and disaccharides (lactose 18%, palatinose 16%). Newborns, infants and children show the same proportion with the exception that glucose, with 750//0,is the single sugar excreted in the largest quantities. The sugar spectra of children suffering from diarrhea show almost no difference from those of normals. It is remarkable, however, that the percentage of fucose excretion in children with diarrhea is greater than in normals. 10"
146
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Table 1. Excretion of mono- a n d disaccharides in stools of prematures, newborns, infants, children a n d children suffering from acute and chronic diarrhea Prematures
Newborns, young infants, infants
Children with diarrhea
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148
J. Schaub and M. Lentze
Even the excretion of the different sugars estimated semiquantitatively by thin-layer chromatography shows no difference in the two groups. After lactose, glucose, or galactose tolerance tests (2 g/kg) in children with malabsorption syndromes, the excretion of total sugars and lactic ~cid in feces is not changed. Nor does the chromatographic separation show either an increase or a different spectrum of sugars. In order to explain the origin of the different sugars in the stool, the sugar spectra of different stools were compared with the respective spectra of the formula given to each child. The sugar content of the foodstuff was analyzed chromatographically and plotted against the sugar excretion in the stool (Fig. 6). I t was demonstrated that sugars identified in stool closely correspond with those in the food. The high percentage of glucose and galactose in the stool demonstrates the hydrolytic power in the small and large intestine. No difference is shown in the sugar spectra between normals and cases with diarrhea. Discussion Quite different data exist in the literature concerning the total sugar content, lactic acid concentration and pH of feces in normal children and children with diarrhea [3, 4, 10, 11, 18, 21, 25, 37, 43]. Sugars excreted in the feces depend on three mechanisms: the absorption rate of the mucosa, the time of passage and the number and species of intestinal bacteria. The activities of a- and fl-glycosidases in the brush border region of the microvilli of the intestinal mucosa show a maximum in full-term newborns [5]. The absorption of monosaccharides per active transport or diffusion is 90.2% in normal subjects [29]. The colonization in the intestine after birth reaches a maximum at the fourth and fifth day with an exponential increase of number of bacterial [!9]. Likewise, the intestinal flora of breastfed babies, consisting 90% of B. bifidum, reaches its peak between the fifth and the sixth day [12]. These two observations correspond with our curve of sugar excretion rate plotted as a function of time (Fig. 1). After the sixth day the sugar excretion decreases in newborns in an approximately reciprocal exponential curve to the above-mentioned curve of colonization. I t can be assumed that the excretion of sugars correlates with the number of bacteria. In Fig. 1, the daily range of values varies considerably. In particular, there is some confusion over the quantities of sugar concentrations in stools of normal children and those with diarrhea. Some authors find no excretion of sugars in feces of normal controls [3, 4, 11] while others find variable concentrations. Gryboski reports that
Sugars, Lactic Acid and pH in Feces of Children
149
1--3-day-old newborns excrete 40--500 mg sugar/100 g stool, and 1--12month-old infants 20--800 rag/100 g stool [15]. Hooft et al. describe an excretion of more than 50 mg sugar/100 g stool as pathologic [18], other authors define fecal sugar over 250 mg/100 g stool as pathologic [21, 25, 37]. Our normal average value of 2.5 g/100 g stool is higher and correlates with those of Ford et al. [13] and Davidson et al. [10]. These wide differences reported in the literature can be attributed to the various methods and their inherent errors: the copper-reduction method [13], the modified copper-reduction method, i.e. the elinitest method [21], the anthrone method [41], high-voltage electrophoresis [15], paper and thinlayer chromatography [11, 22, 32, 36] and enzymatic methods [10, 27]. These methods in themselves explain the confusing results. For example, in reduction as well as in the clinitest method, other reducing products besides carbohydrates are measured, whereas sucrose gives no positive result. With the anthrone method all carbohydrates even oligosaccharides and polysaccharides of both the food and the bacteria are measured. The most constant and comparable results are obtained with the last four methods described. The reports on the thin-layer chromatographic separation of sugars in feces of normal children and those with diarrhea differ. Durand et al. found no sugars in 20 normal controls with chromatography [il], whereas we and others found various monosaccharides, disaccharides and oligosaccharides [6, 10, 32] when using this method. We found no difference in the sugar spectra and in the quantitative excretion of sugars between normals and children suffering from diarrhea, which confirmed these observations (Figs. 4 and 5, Table 1). Holzel found no lactose in the stools of two siblings with laetase deficiency and attributed this to quick hydrolysis of lactose by intestinal flora [17]. Kaijser et al. found only small amounts of sugar in the feces of a case of glucose galactose malabsorption [20] and Carredu et al. found reducing substances rarely after disaccharide tolerance tests in children suffering from enteritis [7]. A possible explanation of the origin of the sugars excreted with the feces is demonstrated in Fig. 6. Their origin may be attributed to the sugars ingested with the food, with the reservation that intestinal bacteria are capable of splitting and synthesizing carbohydrates. The amount of sugars found in the feces may depend on the quantity of bacteria and the rate of hydrolysis. The number of bacteria in the small intestine of normal subjects ranges from l0 s to 105/ml of intestinal fluid [14, 40]. Coello-Ramirez et al. reported that all children with diarrhea had bacterial proliferation; a linear increase in the bacterial counts in duodenal aspirates (10 s to 10 s) was observed with increasing severity of ca,rbohydrate intolerance [9]. The rate of bacterial proliferation
150
J. Schaub and M, Lentze
depends upon the quantity of the unabsorbed carbohydrates metabolized to organic fatty acids [45] and to CO 2 [29]. The mechanism leading to increased proliferation of bacteria by a high percentage of unabsorbed carbohydrates remains to be elucidated. The mechanism of osmotic diarrhea is discussed and attributed either to the decreased split of disaccharides in hereditary or acquired disaccharidase deficiency [45] or to the decreased absorption of monosaccharides in glucose galaetose malabsorption [33]. The time of passage of the unabsorbed carbohydrates may be diminished in these cases and they may be excreted in higher quantities or metabolized by bacterial hydrolysis. This interpretation is in accordance with the observation that the absorption of glucose and the net flux of water is altered in diarrhea; the absorption of glucose and the net flux of water and salt depend upon the proportion os glucose absorbed [42]. The pentose fucose contained both in oligosaceharides [6] and in N-containing carbohydrates of the human breast-milk [16] is frequently excreted in the stool. The high excretion rate of galaetose in feces of prematures correlates with the observations of Auricchio et al. [5], who found a low level of fl-glycosidases in prematures. Lactose which cannot be split completely is hydrolyzed by the intestinal bacteria to glucose and galactose. Whereas some authors found no lactic acid in the feces of normal children, e.g. Auricchio et al. [3, 4] and Weijers et al. [45], our findings (Figs. 2 and 3) correspond with those of Clarke et al. [8] and Hooft et al. [18], who maintain that lactic acid is a normal constituent of the stool. Tomaszewski et al. [39] even assert that a lactic acid content of 300 mg lactate/100 g stool is normal for prematures and newborns. Our investigation shows no difference between the quantity of lactic acid in stools of normal children and those with diarrhea (Figs. 4 and 5). This is in keeping with the findings of McMichael et al. [32] and Clarke et al. [8]. Lactate is produced in the intestine from various sugars, e.g. arabinose, xylose, rhamnose, glucose, sucrose, maltose and lactose [31]. It makes up about of 80% of the hydrolyzing product of hexoses by B. bifidum in breast-fed babies [36]. The interdependence between sugar content and lactic acid concentration in feces of newborns shown in Fig. 3 correlates with the results of other authors [3, 4]. No statistical correlation exists between lactic acid concentration and total sugar excretion of feces in prematures, newborns, infants, children and children with diarrhea. Similarly contradictory results have been published on the p i t in stools of normal children and those suffering from diarrhea. Some authors assume that a low fecal pH, others that a pH value under 6.0 indicates sugar malabsorption in the form of hereditary or acquired disaceharidase deficiency or glucose galactose malabsorption [1, 25, 26, 28, 41, 45]. We found no significant difference in the pH of stools of normal children and
Sugars, Lactic Acid and pH in Feces of Children
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children with diarrhea (Figs. 4 and 5). These observations confirm those of other authors [10, 32]. Because the carbohydrates ingested with the food are subjected to multifarious metabolic influences, it is n o t possible to deduce the t y p e of sugar malabsorption, e.g. hereditary or acquired disaccharidase deficiency and hereditary or acquired glucose galactose malabsorption, from the determination of fecal carbohydrates, lactic acids, and pH, even in the case of a sugar loading test.
References 1. Abraham, J. M., Levin, B., Oberholzer, V. G., Russel, A.: Glucose-galactose malabsorption. Arch. Dis. Childh. 42, 592 (1967) 2. Andersen, D. It.: Celiac syndrome; determination of fat in faeces; reliability of 2 chemical methods and of microscopic estimate; excretion of faeces and of fecal fat in normal children. Amer. J. Dis Child. 69, 141 (1945) 3. Auricehio, S., Dahlquist, A., Miirset, G., Prader, A.: Isomaltose intolerance causing decreased ability to utilize dietary starch. J. Pedlar. 62, 165 (1963) 4. Auricchio, S., Prader, A., Mfirset, G., Witt, G. : Saccharoseintoleranz: Durchfall infolge hereditEren Mangcls an intestinaler Saccharaseaktivit~t. ttelv, paediat. Acta 16, 483 (1961) 5. Auricchio, S., Rubino, A., Miirset, G. : Intestinal glycosidase activities in the human embryo, fetus, and newborn. Pediatrics 85, 944 (1965) 6. Baar, S., Bickel, It.: Oligosaccharides in infants faeces. J. elin. Path. 17, 273 (1964) 7. Carredu, P., Giovannini, ]~., Cevini, G. : Voriibergchende Disaccharidintoleranz bei den Ern~hrungsstSrungen des S~uglingsalters. Helv. paediat. Acta 18, 97 (1963) 8. Clarke, A. D., Podmore, D. A. : The enzymatic determination of lactic acid in faeces in galaetosidase deficiency. Clin. chim. Acta 13, 725 (1966) 9. Coello-Ramirez, P., Lifshitz, F.: Enteric microflora and carbohydrate intolerance in infants with diarrhea. Pediatrics 49, 233 (1972) 10. Davidson, A. G. F., Mullinger, M.: Rcxlucing substances in neonatal stools detected by Clinitest. Pediatrics 46, 632 (1970) 11. Durand, P., Martino, A. M., Lamedica, G. 1~. : Diagnosis of carbohydrate intolerance diarrheas by stool chromatography. Lancet 1961 II, 374 12. Feldheim, G., Schmidt, E. F., Haenel, H. : ~t~oerdie Besiedlung des l~eeoniums. Zbl. Bakt., I. Abt. Orig. 177, 62 (1960) 13. Ford, J. D., Haworth, J. C. : The fecal excretion of sugars in children. J. Pedlar. 63, 988 (1963) 14. Gorbach, S. L., Nahas, L., Lerner, P. I., Weisntein, L.: Studies on intestinal microflora: I. Effects of diet, age, and periodic sampling on numbers of fecal microorganisms in man. Gastroenterology 53, 845 (1967) 15. Gryboski, J. D., Zillis, J., 5~a, O. It. : A study of fecal sugars by high voltage electrophoresis. Gastroenterology 47, 26 (1964) 16. Gy6rgy, P.: Biochemical aspects (the uniquness of human milk). Amer. J. olin. Nutr. 24, 970 (1971) 17. ttolzel, A., Sehwarz, V., Suteliffe, K. W. : Defective lactose absorption causing malnutrition in infancy. Lancet 1959 I, 1126
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18. Hooft, C., Devos, E., Kriekemans, J., van Damme, J. : Malabsorption and diabetes melfitus in children. Helv. paediat. Acta 23, 478 (1968) 19. Ichihashi, Y. : Colonization of bacteria in man. J. Jap. med. Ass. 67, 702 (1972) 20. Kaijser, K., 0ckermann, P. A. : Diagnostic problems in glucose galactose realabsorption. Acta paediat, seand. 59, 214 (1970) 21. Kerry, K. R., Anderson, C. M.: A ward test for sugar in faeces. Lancet 1964 I, 981 22. Kraffczyk, F., Helger, R., Bremer, H. J. : Thin layer chromatographic screening tests for carbohydrate anomalies in plasma, urine and faeces. Clin. chim. Acta 4~, 303 (1972) 23. Lentze, M., Schaub, J., Harms, K. : A simple and rapid thin-layer chromatographic method for the assay of intestinal disaccharidases. Clin. chim. Acta 83, 301 (1971) 24. Lentze, M. : DiinnschichtchromatographischeUntersuchungen zur Diagnose yon kindlichen Malabsorptionssyndromen.Med. Dissertation, Mfinchen 1973, in press 25. Lifshitz, F., Coello-Ramirez, P., Contreras-Gutierrez, M. L.: The response of infants to carbohydrate oral loads after recovery from diarrhea. J. Pediat. 79, 612 (1971) 26. Lifshitz, F., Coello-Ramirez, P., Gutierrez-Topete, G., Cornado-Cornet, M. C. : Carbohydrate intolerance in infants with diarrhea. J. Pediat. 79, 760 (1971) 27. Lindquist, B.: Die intestinale Monosaccharid-Malabsorption. Msehr. Kinderheilk. 117, 18 (1969) 28. Lindquist, B., Meeuwisse, G. W. : Chronic diarrhea caused by monosaccharidmalabsorption. Acta paediat, scand. 51, 674 (1962) 29. Linneweh, F. : Isotopen-Anwendungbei der Erforschung erblieher Stoffwechselkrankheiten. Mschr. Kinderheilk. 118, 191 (1970) 30. Malpress, 1~. H., Hytten, F. E. : The oligosaccharides in human milk. Biochem. J. 6S, 708 (1958) 31. Mayer, J. B., Tewes, G., Pinta, U., Dittmann, J.: Zum Stoffwechsel des Bact. bifidum. VIII. Mitteiinng. Typenbestimmung unserer St~mme. Z. Kinderheilk. 94, 141 (1965) 32. McMichael, It. B., Webb, J., Dawson, A. M.: Lactase deficiency in adults: A cause of functional diarrhea. Lancet 1965 I, 717 33. Meeuwisse, G. W., Melin, K. : Glucose galactose malabsorption. Acta paediat. stand., Suppl. 188, 3 (1969) 34. Roe, J. : The determination of sugar in blood and spinal fluid with anthrone reagent. J. biol. Chem. 212, 335 (1955) 35. Rosebury, T. : Microorganisms indigenous to man, pp. 435. New York: McGraw Hill 1962 36. Sinterhauf, K., Mayer, J. B., Dittmann, J. : Zum Stoffwechsel des Bact. bifidum. IX. Mitteilung. Abbau yon Kohlenhydraten durch Bact. bifidum. Z. Kinderheilk. 97, 146 (1966) 37. Soeparto, P., Stobo, E. A., Walker-Smith, J. A. : Role of chemical examination of the stool in diagnosis of sugar malabsorption in children. Arch. Dis. Childh. 47, 56 (1972) 38. Stahl, E. : Dfinnschichtehromatographie. Berlin-Heidelberg-NewYork: Springer 1967 39. Tomaszewski, L., Hofmann, H., Brzozowska, I., Zalewski, T. : Lactic acid excretion with stools by premature babies, newborns, infants, and healthy children. Pediat. pol. 46, 13 (1971) 40. Tabaqchali, S., Booth, C. C. : Relationship of the intestinal bacterial flora to absorption. Brit. med. Bull. 23, 285 (1967)
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