Evaluation of rat intestinal lactase in vivo with 4-galactosylxylose

Clinica Chimica Acra. 210 (1992) 221-226 0 1992 Elsevier Science Publishers B.V. All rights reserved. 0009-8981/92/.$05.00

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CCA 05375

Short Communication

Evaluation of rat intestinal lactase in vivo with 4-galactosylxylose* Juan J. Aragbn”, Alfonso Fernindez-Mayoralasb, Jestis JimCnez-Barber0 b, Manuel Martin-Lomas b, Alfonso Rivera-Sagredob and Daniel Villanueva”?** ‘Departamento de Bioquimica de la U.A.M. and Instituro de Investigaciones BiomPdicas del C.S.I.C.. Facultad de Medicina de la Universidad Aurdnoma de Madrid, 28029-Madrid and hGrupo de Carbohidraros, InsMuto de Quimica Orgrinica General, C.S.I.C., Juan de la Cierva, 3, 28006-Madrid (Spain)

(Received 16 December 1991; revision received 25 June 1992; accepted 26 July 1992)

Key words: Intestinal lactase evaluation; Lactase deficiency; 4-Galactosylxylose

Introduction A deficiency of small intestinal lactase (EC 3.2.1.23) acting in adult type alactasia is the most frequent genetically based syndrome in man, affecting one-third to onehalf of the humans [l]. Its evaluation is important in pediatrics and gastroenterology. This can be carried out directly by mucosal sampling [2], or indirectly from blood glucose determination after a load of lactose [3], administration of lactose and ethanol and determination of galactose in blood [4], determination of 14C02 in the breath after ingestion of a small quantity of 14C-labelled lactose [5], measurement of either breath hydrogen [6,7] or urinary galactose [8] after lactose ingestion and determination of differential urinary excretion of disaccharides [9]. We have reported an entirely different, non-invasive evaluation procedure based on oral administration of 3-methyllactose followed by measurement of 3methylglucose in the urine [ 10,111. 3-Methyllactose proved to be an acceptable Correspondence lo: J.J. Aragbn, Departamento de Bioquimica, Facultad de Medicina de la Universidad Aut6noma de Madrid, Arzobispo Morcillo 4, 28029 Madrid, Spain. *Dedicated to the memory of Prof. Albert0 Sols. **On leave of absence from the Universidad de1 AtlBntico, Barranquilla, Colombia.

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substrate for intestinal lactase and, when administered orally to suckling rats, led to the urinary excretion of 3-methylglucose which could be determined by gas (GLC) or liquid (HPLC) chromatography. Nevertheless, the application of this method is restricted to hospitals having these analytical facilities and this may be a serious drawback for widespread diagnostic application. We now report on a new method based on the use of 4-galactosylxylose (4-0-&Dgalactopyranosyl-D-xylose), a disaccharide very similar to lactose, which we have synthesized and found to be a substrate of intestinal lactase yielding galactose and xylose. Xylose is passively absorbed from the small intestine [12] and although a small proportion may be metabolized [13], it is mainly eliminated in the urine in which it can be measured using a simple calorimetric procedure [ 141. Correlation of the data obtained by the method now reported with the levels of intestinal lactase activity has been assessed by evaluating, in the same animals, the normal decline in lactase activity that takes place after weaning [l]. Materials and Methods Intestinal lactase, free of lysosomal ~-galactosida~, was partially purified from sheep intestinal mucosa according to Schlegel-Haueter et al. [ 1S] and was used for the evaluation of kinetic parameters. The disaccharide 4-galactosylxylose has been previously synthesized following a multi-step procedure starting from tetra-o-acetyla-D-arabinopyranose [ 16,171. We have now prepared this compound in good yield from benzyl4-O-/3-D-xylopyranoside by acetalation to give the corresponding 2,3O-isopropylidene acetal and then glycosylation with 2,3,4,6-tetra-O-benzoyl-a-Dgalactopyranosyl bromide. A full account of this novel synthetic procedure has been reported [ 181. The hydrolysis of either 4-galactosylxylose or lactose was measured in a total volume of 60 ~1 containing 66 mM sodium maleate, pH 6.0, and a portion of the enzyme preparation. After 2 min at 37°C the reaction was initiated by addition of variable concentrations of substrate. After 15 min, 140 ~1 of 0.18 M Tris-acetate, pH 8.4, were added to the mixture and the reaction was stopped by boiling the mixture for 2 min. The liberated galactose was dete~ined using galactose dehydrogenase [ 191or chromato~aphically. Xylose was determined colo~metrical1y with phloroglucinol (141 or chromatographically. A Hewlett-Packard 5590 gas chromatograph equipped with a glass capillary column of 0.2 mm internal diameter and 25 m length filled with SE-30 was used for chromatographic evaluation of 3methylglucose, xylose and galactose. Trimethylsilyl derivatives of the sugars were prepared as in Dutton [20]. Details concerning administration of sugars to animals and concentration of sugar administered are described in the legends of Tables I and II. For the measurement of rat intestinal lactase, animals were sacriliced by a blow on the neck, small intestine was rapidly removed, cut open and the mucosa scraped off with a glass slide. The mucosa was homogenized in 10 vol. of 5 mM sodium maleate, pH 6.5 and lactase activity was determined in the homogenate as described above, except that the total volume was 200 ,ul and that 50 mM sodium maleate, pH 6.0, was used. The liberated galactose was determined with galactose dehydrogenase [19]. Protein was determined by the method of Lowry [21]. The institution’s guide

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for the care and use of laboratory animals was followed. All chemicals were obtained from Sigma (St. Louis, MO). ResuIts and Discussion The disaccharide 4-galactosylxylose was hydrolyzed using intestinal lactase, isolated as in [ 151. The V,,, was 25% of that of lactose and the Km was 370 mM versus 27 mM for lactose. A V,,, of 5% of that of lactose and a Km of 120 mM have been reported for 3-methyllactose [ 111; thus, lactase exhibits a higher V,.,,,,IK,,, ratio for 4-galactosylxylose. The synthetic disaccharide and D-xylose were administered orally to suckling rats, collecting urine after 5 h and analyzing it calorimetrically and chromatographically as described in Table I. These results show that xylose appeared in the

TABLE I Elimination of D-XylOSe (X) and 3-methylglucose (3MG) in the urine of suckling rats after oral administration of Cgalactosylxylose (4GX) and 3-methyllactose (3ML), respectively and intestinal lactase activity Rats

Sugar elimination in urine within 5 h

Oral sugars (mg) 4GX

3ML

X

3MG

Calorimetric determination (“/of X

18.2 18.2 18.2 18.2 18.2 18.2 _ _ _

18.2 18.2 18.2 18.2 18.2 18.2 _ _

9.0 9.0 9.0 _ _ _

9.0 9.0 9.0 _ -

I8 24 22 18 20 22 ._ _

determination

X

x (from 4GX)

x

3MG (from 3ML)

3MG

38 45 36 _ _

14 I4 12 IO 9 9 _ _ _

31 25 27 _ _ _

I7 21 I9 I8 I7 22 _ _ _

98 95 92 _ _ -

(from 4GX) Al A2 A3 BI B2 B3 Cl C2 C3 C4 C5 C6

Chromatographic (o/o)

Lactase activity (nmol/ minimg prot.)

- 69 58 22 60 73 36

A group of six suckling rats (Al-A3, Bl-B3) of the same litter and age (15 days) were fasted for 4 h in metabolic cages at 3O’C and orally administered sugars in 0.4 ml of water as indicated. 4GX and 3ML were given to the six rats above in separate experiments with 24 h interval; rats Al-A3 received 4GX before 3ML and vice versa for rats Bl-B3; 24 h after administering the second disaccharide, X was given to rats Al-A3 and 3MG to rats Bl-B3. Urine was collected by intermittent transabdominal bladder pressure during 5 h. Sugars eliminated during this time are indicated in the table as percentage of the amount administered. X was determined in the urine both calorimetrically and chromatographically. Intestinal lactase activity was determined postmortem in rats Cl-C6 that were of the same litter.

224 TABLE II Elimination of D-xylose (X) and 3-methylglucose (3MG) in the urine of adult rats after oral administration of 4-galactosylxylose (4GX) and 3-methyliactose (3ML). respectively and intestinal lactase activity Rats

Oral sugars fmg) 4GX

Al A2 A3 Bl B2 B3 DI D2 D3

36.4 36.4 36.4 36.4 36.4 36.4 -

3ML

36.4 36.4 36.4 36.4 36.4 36.4 -

Sugar elimination in urine within 20 h X

_18.2 18.2 18.2

3MG

-. 18.2 18.2 18.2

Calorimetric determination W)

Chromatographic ((%I)

X (from 4GX)

X

X (from 4GX)

X

3MG (from 3ML)

3MG

7.4 11.4 4.7 8.0 6.8 4.6 -

28.0 43.0 45.0

2.8 3.6 6.0 8.0 7.5 3.8 -

.,.I. ,.I.I 35 3I 22

4.3 2.7 1.8 0.6 3.0 2.5 -

-

determination

83 100 86

Lactase activity (nmoll min/mg prot.}

4.6 18*5 9.8 6.0 4.1 9.4 -

Rats Al-A3 and Bl-B3 were the same animals used in the experiments described in Table I except that they were now 40 days old. Other conditions were as described in Table I except that X and 3MG were given to rats D1 -D3 from the same litter in different experiments with 24 h interval and that urine was coflected during 20 h. After completing the experiment, rats Al--A3 and Bl -B3 were sacrificed and intestind lactase activity was determined.

urine shortly after the administration of the disaccharide, For comparison purposes, 3-methyllactose and 3-methylglucose were also administered orally to the same rats, urine was collected as above and analyzed for sugars chromatographically. The results are also given in Table I. The percentage of both 3-methylglucose and xylose derived from 3-methyllactose and 4-galactosylxylose, respectively, eliminated in the urine, was in the same range. The elimination of sugars was not signi~~antly affected by the order of administration of both disac~harid~s~ Nevertheless, the recovery of 3-methylglucose in the controls was higher than 90% of the administered dose whereas that of xylose averaged 28 to 40% depending on the determination procedure, the percentage being not signi~cantly higher in adult rats where the urine collection was extended to 20 h {see Table II). This agrees with data reported in normal humans, where no more than No/ of the xylose administered was recovered in the urine in 24 h [13,22]. The difference is attributable to non-absorption and/or metabolization. Xylose is not phosphorylated by either hexokinase or glucokinase 123,241, but as much as 15% of the orally given pentose has been shown to be recovered in the urine as D-threitol in man and glucose dehydrogenase or a more specific pentose dehydrogenase were proposed for the initiation of xylose metabolism [ 131.The levels of intestinal lactase activity determined in six more suckling rats of the same litter are included in Table I.

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To assess if the excretion of xylose after giving 4-galactosylxylose varies on the basis of lactase activity, an experiment similar to that described in Table I was carried out in the same animals (groups A and B) when they were 40 days old, i.e. after weaning. They were subsequently sacrificed to determine intestinal lactase activity. Table II shows that the urinary elimination of xylose derived from administered 4galactosylxylose decreased as compared to the values previously obtained in the suckling period, since xylose excretion in adult rats was 35-47% (depending on the determination procedure) of that observed in suckling rats. This reduction in xylose elimination was concomitant with an important decrease in the levels of intestinal lactase, that reflects its normal decline in the postweaned mammal [ 11.A substantial decrease in the elimination of 3-methylglucose derived from 3-methyllactose was observed as well, these experiments being, to our knowledge, the first evaluation in vivo of the decline of intestinal lactase detected in the same animals. The data presented in this paper show that xylose is eliminated in the urine as a result of the hydrolysis in vivo of 4-galactosylxylose by intestinal lactase and that this sugar can be easily determined calorimetrically. Values obtained by calorimetric determinations of xylose were somewhat higher than those obtained by chromatographic measurements, the difference being probably related to the higher specificity provided by the second method. However, semiquantitative evaluation can be simply and confidently carried out as indicated by the decrease of both excretion of xylose derived from 4-galactoxylxylose and intestinal lactase activity after weaning. Both 3-methyllactose and 4-galactosylxylose could be used for the noninvasive evaluation of intestinal lactase in vivo, however, the latter avoids the use of gas chromatography and therefore could be widely and routinely adopted. Application of the method reported herein to humans as a screening test for lactase de% ciency can be of particular interest in pediatrics, on account of its simple performance. Furthermore, the important proportion of xylose excreted in the urine, due to its poor metabolization, suggests that lower doses of disaccharide could be administered in comparison with other non-invasive tests [8,9]. Acknowledgements

This research was supported by the Direction General de Investigation Cientitica y Tecnica (Grants PB-87-0367 and PB-87-0132). We thank Mr. G. Corrales for excellent technical assistance, the Ministerio de Education y Ciencia and the Ministerio de Asuntos Exteriores for fellowships (to A.R.-S. and D.V., respectively). References Semenza G, Auricchio S. Small-intestinal disaccharidases. In: Striver CR, Beaudet AL, Sly WS, Valle D, eds. The metabolic basis of inherited disease, 6th edn. New York: McGraw-Hill, 1988;2975-2997. Dalqvist A. Assay of intestinal disaccharidases. Enzyme Biol Clin 1970;11:52-56. McGill DB, Newcomer AD. Comparison of venous and capillary blood samples in lactose intolerance testing. Gastroenterology 1967;53:371-374. Fischer W, Zapf J. Zur erworbenen lactose intoleranz. Klin Wochenschr 1965;43:1243-1246. Sasaki Y, Ilo M, Kameda H, Ueda H, Aoyagi T, Christopher NL, Bayless TM, Wagner HN.

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6 7

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19 20 21 22 23 24

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