European Journal of
Europ. J. Pediat. 126, 45--52 (1977)
Pediatrics 9 by Springer-Verlag 1977
Quantitative Determination of Lactose, Maltose, and Sucrose in Urine Klaus M. D6rner University of Kiel Medical School, Department of Pediatrics (Prof. H.-R. Wiedemann), Frfbelstr. 15--17, D-2300 Kiel, Federal Republic of Germany
Abstract. A simple method for determination of lactose, maltose and sucrose in urine is described. The principle of the method consists of enzymatic splitting of these disaccharides and specific measurement the resulting glucose by the Beckman Glucose-Analyzer. The precision and accuracy of the method have been evaluated and its clinical application is briefly discussed.
Key words: Lactose - Maltose - Sucrose - Glucose - Urine.
Introduction
While the nonspecific tests for sugar in urine of adults have been replaced by the use of glucose-specific teststrips, the urine of children is often also examined by Nylanders test (Klinisches Labor, 12th ed.) or comparable methods to detect genetic metabolic defects such as galactosemia, fructosuria and pentosuria. As Nylanders test gives false-positive results with high concentrations of creatinine and protein, positive tests have to be verified by polarimetry on the drug-free urine. This is followed by qualitative two-dimensional thinlayer chromatography (TLC) of the sugars on silica-gel plates. Using this procedure, unusually high sucrose and fructose excretion was detected in a three year-old boy with as yet unclassified hypoglycemia, and idiopathic hypertrophic cardiomyopathy. Quantitative TLC is laborious and only moderately reproducible, and so a very simple quantitative enzymatic test for sucrose in urine was developed. Using this test, lactose and maltose can be measured in the urine of infants and patients with disturbances of intestinal absorbtion. Furthermore, the method is applicable to food analysis.
Material and Methods
a) Instrumentation The determination of glucose was performed by the glucose-analyzer supplied by Beckman Instruments, Munich. The same firm supplied the glucose oxidase reagent.
46
K.M.D6rner
b) Chemicals Buffers 1) Phosphate 0.4 mmol/1, pH 7.5 2) Acetate 0.1 mmol/l, pH 6.6 3) Acetate 0.1 mmol/1, pH 4.6. These buffer solutions are stable for 1 year at 4~ Enzymes 1) fl-galactosidase (EC 3.2.1.23); Boehringer cat. number 15079, 150U/ml 2) a-glucosidase (EC 3.2.1.20); Boehringer cat. number 15018, 100U/ml 3) fl-fructosidase (EC 3.2.1.26); Boehringer cat. number 15067, 20rag dry powder specific activity 150 U/mg in a solution of 1 ml buffer 3). Stock solutions of disaccharides 1) 5 g/dl lactose (lactose-monohydrate Merck 7660) 2) 5 g/dl maltose (maltose-monohydrate 99% Merck 5912) 3) 5 g/dl sucrose (Merck 7674). These sugars were dried for 24h before weighing at 45~ in vacuo over P205. Calibration of the glucose-analyzer was performed with aqueous standard solutions (Boehringer 15726). All other reagents were of analytical grade and were obtained from E. Merck, Darmstadt.
c) Principle of Method By the action of pure disaccharidases, glucose is liberated from lactose, maltose and sucrose and measured by a specific enzymatic process. The initial concentrations of disaccharides are calculated from these results.
d) Assay First, the glucose concentration (Co) of the untreated urine is determined by the method recommended by Beckman's: 10~tl sample are injected into the analyzer and the result is read off to 3 digits. Then 1 ml urine, 480 pl buffer and 20 lal enzyme solution are mixed, and after incubation at 37~C, the glucose concentration is determined once more. Determination of lactose: Buffer 1 and enzyme solution 1, incubation-time 60min. Determination of maltose: Buffer 2 and enzyme solution 2, incubation-time 120 min. Determination of sucrose: Buffer 3 and enzyme solution 3, incubation-time 60rain. The concentrations of the disaccharides in the original urine are calculated according to Table 1. If the concentration of glucose is below 10 mg/dl, the instrument does not give a stable reading. In these cases, the greatest value was taken as the result. A higher sensitivity might be achieved by using a sample volume of 100 ~tl instead of 10 ~tl. In the glucose-analyzer, samples Table 1. Calculation of disaccharide concentration in urine Clactose
= 3c G - - 2 %
Csucrose
= 3c F - - 2 %
Cmaltose
= 1.5 (c M ~ C F )
Co = glucose CM= glucose c F = glucose c G = glucose
concentration concentratiorl concentration concentration
without the addition of buffer and enzyme after a-glucosidase treatment after fl-fructosidase treatment after fl-galactosidase treatment
Quantitative Determination of Lactose, Maltose and Sucrose in Urine
47
are mixed with 1 ml buffered glucose oxidase solution for polarographic glucose determination (see "discussion"). As the buffer content of the assay mixture could possibly influence the glucose determination, buffered solutions of glucose were prepared as controls (see "buffer solutions"). These showed no deviations from the expected values. Screening of urines for disaccharides was done after acid hydrolysis in the following way. 1 ml urine was boiled with 5 drops of 1 n HC1 for 92min. 5 drops of the hydrolysate were mixed with 10 drops water and a "Clinitest" tablet. A greenish colour denoted a positive reaction.
Results
Linearity A n incubation time was chosen (see " m e t h o d " ) to ensure that the splitting o f disaccharides was nearly quantitative up to concentrations o f 200 m g / d l (Figs. 1--3). If the expected concentrations do not exceed 100mg/dl, an incubation time o f 30 min m a y be sufficient. It should b e noted, however, that glucosidase activity of the commercial enzyme solution varies f r o m lot to lot. It m a y be necessary to use incubation periods longer than 2 h for maltose and sucrose concentrations over 150 m g / d l . In a small n u m b e r o f cases, the urine was f o u n d to have a p H greater than 7.5. By dilution with water (1 : 1), or by adjusting p H o f the urine, a more suitable p H o f the reaction mixture was achieved.
Recovery The results for the recovery o f disaccharides added to urine are shown graphically in Figures 1--3 and the percentage recoveries given in Table 2. F o r clarity the
c [mg/dl] Glucose 150-
100-
160
'
2(50
' 300 c "Lactose" [mg/cll]
Fig. 1. Recovery of lactose added to urine. The blank value for the urine has been subtracted
48
K.M. D6rner c t
lmo~t~ C~r.om
~
p
150"
100"
50"
0
'
160
'
260
~ c[mg/dl] >
"Maltose"
Fig. 2. Recovery of maltose from urine. The blank value for the urine has been subtracted
C
[mg/dl] Glucose 150-
4~'>/6~
100-
50-
0
0
160
260
,
360 c
>
Soccharose [mg,4~]
Fig. 3. Recovery of sucrose from urine. The blank value for the urine has been subtracted
Quantitative Determination of Lactose, Maltose and Sucrose in Urine Table 2. The percentage recovery of lactose, maltose and sucrose from urine
49
Disaccharide added (mg/dl)
Disaccharide recovery (percent) Lactose
Maltose
Sucrose
33.6 66.7 133 167 333 667
81.8% 94.6% 103.3% 108.4% 98.7% 104.1%
95.8% 95.8% 96.4% 100.6% 90.8%
95.8% 99.1% 99.0% 96.0% 99.4% 95.8%
Table 3. Simultaneous recovery rates for lactose, maltose and sucrose added to urine Experiment
Added: Lactose (mg/dl) Maltose (mg/dl) Sucrose (mg/dl)
1
2
3
4
190.0 47.0 200.0
142.5 94.0 150.0
95.1 141.0 75.0
47.4 188.0 50.0
96.6% 91.3% 109.2%
99.5% 95.0% 110.9%
100.7% 93.8% 129.2%
Percentage recovery: Lactose 94.9% Maltose 73.4% Sucrose 105.7%
p r i m a r y glucose concentrations (co) have been substracted. Up to 150 m g / d l , the range o f clinical importance, there is g o o d recovery provided that c o is lower than 500 m g / d l . The recovery o f disaccharide mixtures f r o m urine is shown in Table 3. The results are g o o d except for one lactose and one maltose value.
Precision All assays show g o o d precision (Table 4). The coefficients o f variation lie between 2.9 and 4.4% (n = 10). The pipetting error leads to a greater deviation than the enzymatic reaction. The coefficient o f variation of the glucose determination itself is k n o w n to be 1.5--2%.
Analysis of Urines of Newborn and Infants In a survey on.lactose, maltose and sucrose in urine f r o m newborns and infants, all urines sent for analysis to the clinical l a b o r a t o r y over a two m o n t h period were screened for glucose after acid hydrolysis. 19 urines f r o m 18 children were thus selected for further-examination o f their glucose and disaccharide content. The results are shown in Table 5. While maltose was only f o u n d once at a
50
K.M. D6rner
Table 4. Precision of disaccharide determinations in 10 separate assays. The urine, stock solution of sugar, buffer and enzyme were pipetted separately for each assay Disaccharide
Mean value (mg/dl)
s.d.
CV %
Lactose Maltose Sucrose
70 68 83
+ 2.0 +_3.0 +_3.0
2.9 4.4 3.6
Table 5. Results from urines of newborns and infants with positive clinitest results after acid hydrolysis (n.d. = not detectable) Case
Glucose mg/dl
Lactose mg/dl
Sucrose mg/dl
Maltose mg/dl
D. W. R. U. H. B. D. B. J. O. B. G. T. B. 1 B. 2 O. M. K. S.
5 6 15 28 n,d. 17 15 12 16 18 14 13 9 n.d. 15 12 3 6 17
11 12 24 28 21 23 30 152 88 33 59 13 48 18 96 135 21 n.d. 35
n.d. 6 6 10 27 2 n.d. 6 n.d. 62 59 13 6 9 39 33 n.d. 18 5
n.d. 3 3 3 n.d. 3.5 n.d. 1.5 n.d. n.d. n.d. 12.5 3 1.5 n.d. n.d. 3 n.d. n.d.
c o n c e n t r a t i o n higher t h a n 5 m g / d l , lactose was often f o u n d above this level. This increase in lactose was n o t always a c c o m p a n i e d by a high sucrose c o n c e n t r a t i o n . If the calculated maltose, lactose a n d sucrose c o n c e n t r a t i o n s were below 1.5 m g / d l , 3 m g / d l a n d 3 m g / d l respectively, "n.d." (= n o t detectable) has been entered in the table.
Discussion A paediatric l a b o r a t o r y m u s t be p r e p a r e d for the a p p e a r a n c e of several carbohydrates i n u r i n e ( T a b l e 6). T h e q u a n t i t a t i v e d e t e r m i n a t i o n o f m o n o s a c c h a r i d e s is easily achieved b y c o m m e r c i a l test kits for glucose, galactose a n d the pentoses.
Quantitative Determination of Lactose, Maltose and Sucrose in Urine Table 6. List of the most important carbohydrates which can be found in urine (* Appearance is pathological or iatrogenic). Jolley et al. (1970)
51
Arabinose Ribose Xylose Xylulose* Fructose Galactose Glucose Mannose Sorbose Fucose (desoxygalactose) Desoxyglucose* Galacturonic acid Sorbitol* Lactose Maltose Melibiose Sucrose Inulin*
To measure the biologically important disaccharides (lactose, maltose and sucrose) by the method described above, two preliminaries must be fulfilled: 1. The glucose determination must not be disturbed by the other sugars shown in Table 6. 2. The disaccharidases used should not show side-effects.
Specificity of Glucose Determination The well known test kits for glucose use the oxidation offl-glucose with glucose oxidase foltowed by an indicator reaction such as "Perid" or "PAP". As opposed to this widely used method, the Beckman glucose-analyzer measures the oxygen consumption polarographically and by differentiation evaluates the rate of consumption, a kinetic measurement. Thus the specificity of determination is only dependent on the purity of the glucose oxidase, which must be free of oxygen consuming enzymes such as uricase, xanthinoxidase etc., and on its cross-reaction with other substrates. It is worth noting that this method has recently been selected as the recommended method for routine analysis by an expert committee of pathologists and clinical chemists (Passey, 1977). We agree with the literature that desoxyglucose shows a cross-reaction of 25% compared to glucose, whereas the disturbance by mannose and xylose, each with a cross-reaction of 1%, is normally of no importance. Lactose and maltose at concentrations of 2 g/dl result in slow consumption of oxygen. This is without practical importance, as lactose and maltose do not occur at such high concentrations in urine, and as the reaction to measure glucose is rapid. Glucose oxidase reacts only with/?-glucose. Under our reaction conditions, the equilibrium of mutarotation is achieved.
52
K.M. D6rner
Impurities and Specificity of Disaccharidases The purity of the disaccharidases is one of the limiting factors in the method described in this paper. The supplier claims that contaminating activities are lower than 0.01%. We did not check this, as impurities even ten times higher would not be expected to affect the results. O f all the biologically relevant sugars, fl-galactosidase only splits lactose, a-Glucosidase splits maltose and sucrose. Other potential substrates such as maltotriose have not yet been detected in urine. fl-Fructosidase reacts with sucrose, with raffinose (cross-reaction approx. 25%) and possibly with inulin (Bergmeyer, 1974).
Clinical Significance High concentrations of disaccharides in urine may be caused by disturbances of intestinal absorbtion (Bickel, 1960). Disaccharides which enter the blood are excreted in the urine u n c h a n g e d as only the brush border of the small intestine contains disaccharidases. This frequently happens in coeliac disease, in gastroenteritis of viral or bacterial origin and in cystic fibrosis (Lebenthal, 1975). Furthermore, very premature infants often show high lactose levels in their urine as lactase activity develops late in the prenatal period. Very occasionally, congenital sucrase/isomaltase deficiency or lactase deficiency is the cause of high sucrose or lactose excretion. As the laboratories of childrens' hospitals are often faced with high disaccharide concentrations in urine, the simple assays described here are a useful step prior to the laborious estimation of disaccharidase activity in duodenal biopsies.
References Bergmeyer, H. U. (Ed.): Methoden der enzymatischen Analyse. H. U. Bergmeyer, E. Bernt: Saccharose, pp. 1221--1224; G. Kurz, K. Wallenfels: Lactose und andere fl-D-Galactoside, pp. 1225--1229; I. Gutmann: Maltose, pp. 1230--1233.3. edition. Weinheim: Verlag Chemie 1974 Bickel, H.: Die nicht-diabetischen Melliturien des Kindes. Mod. Probl. Paediat. 6, 313--336 (1960) Jolley, R. L., Warren, K. S., Scott, C. D., Jainchill, J. L., Freemann, M. L.: Carbohydrates in normal urine and blood serum as determined by high-resulution column chromatography. J. Clin. Path. 53, 793--802 (1970) Suppl. Klinisches Labor 12th edition, p. 341. Darmstadt: E. Merck, 1974 (no author given) Lebenthal, E.: Small Intestinal Disaccharidase Deficiencies. Ped. Clin. North America 22, 757--766 (1975) Passey, R. B., Gillum, R. L., Fuller, J. B., Urry, F. M., Giles, M. L.: Evaluation and comparison of 10 glucose methods and the reference method recommended in the proposed product class standard (1974). Clin. Chem. 23, 131--139 (1977)
Received March 31, 1977
Europ. J. Pediat. 126, 45--52 (1977)
Pediatrics 9 by Springer-Verlag 1977
Quantitative Determination of Lactose, Maltose, and Sucrose in Urine Klaus M. D6rner University of Kiel Medical School, Department of Pediatrics (Prof. H.-R. Wiedemann), Frfbelstr. 15--17, D-2300 Kiel, Federal Republic of Germany
Abstract. A simple method for determination of lactose, maltose and sucrose in urine is described. The principle of the method consists of enzymatic splitting of these disaccharides and specific measurement the resulting glucose by the Beckman Glucose-Analyzer. The precision and accuracy of the method have been evaluated and its clinical application is briefly discussed.
Key words: Lactose - Maltose - Sucrose - Glucose - Urine.
Introduction
While the nonspecific tests for sugar in urine of adults have been replaced by the use of glucose-specific teststrips, the urine of children is often also examined by Nylanders test (Klinisches Labor, 12th ed.) or comparable methods to detect genetic metabolic defects such as galactosemia, fructosuria and pentosuria. As Nylanders test gives false-positive results with high concentrations of creatinine and protein, positive tests have to be verified by polarimetry on the drug-free urine. This is followed by qualitative two-dimensional thinlayer chromatography (TLC) of the sugars on silica-gel plates. Using this procedure, unusually high sucrose and fructose excretion was detected in a three year-old boy with as yet unclassified hypoglycemia, and idiopathic hypertrophic cardiomyopathy. Quantitative TLC is laborious and only moderately reproducible, and so a very simple quantitative enzymatic test for sucrose in urine was developed. Using this test, lactose and maltose can be measured in the urine of infants and patients with disturbances of intestinal absorbtion. Furthermore, the method is applicable to food analysis.
Material and Methods
a) Instrumentation The determination of glucose was performed by the glucose-analyzer supplied by Beckman Instruments, Munich. The same firm supplied the glucose oxidase reagent.
46
K.M.D6rner
b) Chemicals Buffers 1) Phosphate 0.4 mmol/1, pH 7.5 2) Acetate 0.1 mmol/l, pH 6.6 3) Acetate 0.1 mmol/1, pH 4.6. These buffer solutions are stable for 1 year at 4~ Enzymes 1) fl-galactosidase (EC 3.2.1.23); Boehringer cat. number 15079, 150U/ml 2) a-glucosidase (EC 3.2.1.20); Boehringer cat. number 15018, 100U/ml 3) fl-fructosidase (EC 3.2.1.26); Boehringer cat. number 15067, 20rag dry powder specific activity 150 U/mg in a solution of 1 ml buffer 3). Stock solutions of disaccharides 1) 5 g/dl lactose (lactose-monohydrate Merck 7660) 2) 5 g/dl maltose (maltose-monohydrate 99% Merck 5912) 3) 5 g/dl sucrose (Merck 7674). These sugars were dried for 24h before weighing at 45~ in vacuo over P205. Calibration of the glucose-analyzer was performed with aqueous standard solutions (Boehringer 15726). All other reagents were of analytical grade and were obtained from E. Merck, Darmstadt.
c) Principle of Method By the action of pure disaccharidases, glucose is liberated from lactose, maltose and sucrose and measured by a specific enzymatic process. The initial concentrations of disaccharides are calculated from these results.
d) Assay First, the glucose concentration (Co) of the untreated urine is determined by the method recommended by Beckman's: 10~tl sample are injected into the analyzer and the result is read off to 3 digits. Then 1 ml urine, 480 pl buffer and 20 lal enzyme solution are mixed, and after incubation at 37~C, the glucose concentration is determined once more. Determination of lactose: Buffer 1 and enzyme solution 1, incubation-time 60min. Determination of maltose: Buffer 2 and enzyme solution 2, incubation-time 120 min. Determination of sucrose: Buffer 3 and enzyme solution 3, incubation-time 60rain. The concentrations of the disaccharides in the original urine are calculated according to Table 1. If the concentration of glucose is below 10 mg/dl, the instrument does not give a stable reading. In these cases, the greatest value was taken as the result. A higher sensitivity might be achieved by using a sample volume of 100 ~tl instead of 10 ~tl. In the glucose-analyzer, samples Table 1. Calculation of disaccharide concentration in urine Clactose
= 3c G - - 2 %
Csucrose
= 3c F - - 2 %
Cmaltose
= 1.5 (c M ~ C F )
Co = glucose CM= glucose c F = glucose c G = glucose
concentration concentratiorl concentration concentration
without the addition of buffer and enzyme after a-glucosidase treatment after fl-fructosidase treatment after fl-galactosidase treatment
Quantitative Determination of Lactose, Maltose and Sucrose in Urine
47
are mixed with 1 ml buffered glucose oxidase solution for polarographic glucose determination (see "discussion"). As the buffer content of the assay mixture could possibly influence the glucose determination, buffered solutions of glucose were prepared as controls (see "buffer solutions"). These showed no deviations from the expected values. Screening of urines for disaccharides was done after acid hydrolysis in the following way. 1 ml urine was boiled with 5 drops of 1 n HC1 for 92min. 5 drops of the hydrolysate were mixed with 10 drops water and a "Clinitest" tablet. A greenish colour denoted a positive reaction.
Results
Linearity A n incubation time was chosen (see " m e t h o d " ) to ensure that the splitting o f disaccharides was nearly quantitative up to concentrations o f 200 m g / d l (Figs. 1--3). If the expected concentrations do not exceed 100mg/dl, an incubation time o f 30 min m a y be sufficient. It should b e noted, however, that glucosidase activity of the commercial enzyme solution varies f r o m lot to lot. It m a y be necessary to use incubation periods longer than 2 h for maltose and sucrose concentrations over 150 m g / d l . In a small n u m b e r o f cases, the urine was f o u n d to have a p H greater than 7.5. By dilution with water (1 : 1), or by adjusting p H o f the urine, a more suitable p H o f the reaction mixture was achieved.
Recovery The results for the recovery o f disaccharides added to urine are shown graphically in Figures 1--3 and the percentage recoveries given in Table 2. F o r clarity the
c [mg/dl] Glucose 150-
100-
160
'
2(50
' 300 c "Lactose" [mg/cll]
Fig. 1. Recovery of lactose added to urine. The blank value for the urine has been subtracted
48
K.M. D6rner c t
lmo~t~ C~r.om
~
p
150"
100"
50"
0
'
160
'
260
~ c[mg/dl] >
"Maltose"
Fig. 2. Recovery of maltose from urine. The blank value for the urine has been subtracted
C
[mg/dl] Glucose 150-
4~'>/6~
100-
50-
0
0
160
260
,
360 c
>
Soccharose [mg,4~]
Fig. 3. Recovery of sucrose from urine. The blank value for the urine has been subtracted
Quantitative Determination of Lactose, Maltose and Sucrose in Urine Table 2. The percentage recovery of lactose, maltose and sucrose from urine
49
Disaccharide added (mg/dl)
Disaccharide recovery (percent) Lactose
Maltose
Sucrose
33.6 66.7 133 167 333 667
81.8% 94.6% 103.3% 108.4% 98.7% 104.1%
95.8% 95.8% 96.4% 100.6% 90.8%
95.8% 99.1% 99.0% 96.0% 99.4% 95.8%
Table 3. Simultaneous recovery rates for lactose, maltose and sucrose added to urine Experiment
Added: Lactose (mg/dl) Maltose (mg/dl) Sucrose (mg/dl)
1
2
3
4
190.0 47.0 200.0
142.5 94.0 150.0
95.1 141.0 75.0
47.4 188.0 50.0
96.6% 91.3% 109.2%
99.5% 95.0% 110.9%
100.7% 93.8% 129.2%
Percentage recovery: Lactose 94.9% Maltose 73.4% Sucrose 105.7%
p r i m a r y glucose concentrations (co) have been substracted. Up to 150 m g / d l , the range o f clinical importance, there is g o o d recovery provided that c o is lower than 500 m g / d l . The recovery o f disaccharide mixtures f r o m urine is shown in Table 3. The results are g o o d except for one lactose and one maltose value.
Precision All assays show g o o d precision (Table 4). The coefficients o f variation lie between 2.9 and 4.4% (n = 10). The pipetting error leads to a greater deviation than the enzymatic reaction. The coefficient o f variation of the glucose determination itself is k n o w n to be 1.5--2%.
Analysis of Urines of Newborn and Infants In a survey on.lactose, maltose and sucrose in urine f r o m newborns and infants, all urines sent for analysis to the clinical l a b o r a t o r y over a two m o n t h period were screened for glucose after acid hydrolysis. 19 urines f r o m 18 children were thus selected for further-examination o f their glucose and disaccharide content. The results are shown in Table 5. While maltose was only f o u n d once at a
50
K.M. D6rner
Table 4. Precision of disaccharide determinations in 10 separate assays. The urine, stock solution of sugar, buffer and enzyme were pipetted separately for each assay Disaccharide
Mean value (mg/dl)
s.d.
CV %
Lactose Maltose Sucrose
70 68 83
+ 2.0 +_3.0 +_3.0
2.9 4.4 3.6
Table 5. Results from urines of newborns and infants with positive clinitest results after acid hydrolysis (n.d. = not detectable) Case
Glucose mg/dl
Lactose mg/dl
Sucrose mg/dl
Maltose mg/dl
D. W. R. U. H. B. D. B. J. O. B. G. T. B. 1 B. 2 O. M. K. S.
5 6 15 28 n,d. 17 15 12 16 18 14 13 9 n.d. 15 12 3 6 17
11 12 24 28 21 23 30 152 88 33 59 13 48 18 96 135 21 n.d. 35
n.d. 6 6 10 27 2 n.d. 6 n.d. 62 59 13 6 9 39 33 n.d. 18 5
n.d. 3 3 3 n.d. 3.5 n.d. 1.5 n.d. n.d. n.d. 12.5 3 1.5 n.d. n.d. 3 n.d. n.d.
c o n c e n t r a t i o n higher t h a n 5 m g / d l , lactose was often f o u n d above this level. This increase in lactose was n o t always a c c o m p a n i e d by a high sucrose c o n c e n t r a t i o n . If the calculated maltose, lactose a n d sucrose c o n c e n t r a t i o n s were below 1.5 m g / d l , 3 m g / d l a n d 3 m g / d l respectively, "n.d." (= n o t detectable) has been entered in the table.
Discussion A paediatric l a b o r a t o r y m u s t be p r e p a r e d for the a p p e a r a n c e of several carbohydrates i n u r i n e ( T a b l e 6). T h e q u a n t i t a t i v e d e t e r m i n a t i o n o f m o n o s a c c h a r i d e s is easily achieved b y c o m m e r c i a l test kits for glucose, galactose a n d the pentoses.
Quantitative Determination of Lactose, Maltose and Sucrose in Urine Table 6. List of the most important carbohydrates which can be found in urine (* Appearance is pathological or iatrogenic). Jolley et al. (1970)
51
Arabinose Ribose Xylose Xylulose* Fructose Galactose Glucose Mannose Sorbose Fucose (desoxygalactose) Desoxyglucose* Galacturonic acid Sorbitol* Lactose Maltose Melibiose Sucrose Inulin*
To measure the biologically important disaccharides (lactose, maltose and sucrose) by the method described above, two preliminaries must be fulfilled: 1. The glucose determination must not be disturbed by the other sugars shown in Table 6. 2. The disaccharidases used should not show side-effects.
Specificity of Glucose Determination The well known test kits for glucose use the oxidation offl-glucose with glucose oxidase foltowed by an indicator reaction such as "Perid" or "PAP". As opposed to this widely used method, the Beckman glucose-analyzer measures the oxygen consumption polarographically and by differentiation evaluates the rate of consumption, a kinetic measurement. Thus the specificity of determination is only dependent on the purity of the glucose oxidase, which must be free of oxygen consuming enzymes such as uricase, xanthinoxidase etc., and on its cross-reaction with other substrates. It is worth noting that this method has recently been selected as the recommended method for routine analysis by an expert committee of pathologists and clinical chemists (Passey, 1977). We agree with the literature that desoxyglucose shows a cross-reaction of 25% compared to glucose, whereas the disturbance by mannose and xylose, each with a cross-reaction of 1%, is normally of no importance. Lactose and maltose at concentrations of 2 g/dl result in slow consumption of oxygen. This is without practical importance, as lactose and maltose do not occur at such high concentrations in urine, and as the reaction to measure glucose is rapid. Glucose oxidase reacts only with/?-glucose. Under our reaction conditions, the equilibrium of mutarotation is achieved.
52
K.M. D6rner
Impurities and Specificity of Disaccharidases The purity of the disaccharidases is one of the limiting factors in the method described in this paper. The supplier claims that contaminating activities are lower than 0.01%. We did not check this, as impurities even ten times higher would not be expected to affect the results. O f all the biologically relevant sugars, fl-galactosidase only splits lactose, a-Glucosidase splits maltose and sucrose. Other potential substrates such as maltotriose have not yet been detected in urine. fl-Fructosidase reacts with sucrose, with raffinose (cross-reaction approx. 25%) and possibly with inulin (Bergmeyer, 1974).
Clinical Significance High concentrations of disaccharides in urine may be caused by disturbances of intestinal absorbtion (Bickel, 1960). Disaccharides which enter the blood are excreted in the urine u n c h a n g e d as only the brush border of the small intestine contains disaccharidases. This frequently happens in coeliac disease, in gastroenteritis of viral or bacterial origin and in cystic fibrosis (Lebenthal, 1975). Furthermore, very premature infants often show high lactose levels in their urine as lactase activity develops late in the prenatal period. Very occasionally, congenital sucrase/isomaltase deficiency or lactase deficiency is the cause of high sucrose or lactose excretion. As the laboratories of childrens' hospitals are often faced with high disaccharide concentrations in urine, the simple assays described here are a useful step prior to the laborious estimation of disaccharidase activity in duodenal biopsies.
References Bergmeyer, H. U. (Ed.): Methoden der enzymatischen Analyse. H. U. Bergmeyer, E. Bernt: Saccharose, pp. 1221--1224; G. Kurz, K. Wallenfels: Lactose und andere fl-D-Galactoside, pp. 1225--1229; I. Gutmann: Maltose, pp. 1230--1233.3. edition. Weinheim: Verlag Chemie 1974 Bickel, H.: Die nicht-diabetischen Melliturien des Kindes. Mod. Probl. Paediat. 6, 313--336 (1960) Jolley, R. L., Warren, K. S., Scott, C. D., Jainchill, J. L., Freemann, M. L.: Carbohydrates in normal urine and blood serum as determined by high-resulution column chromatography. J. Clin. Path. 53, 793--802 (1970) Suppl. Klinisches Labor 12th edition, p. 341. Darmstadt: E. Merck, 1974 (no author given) Lebenthal, E.: Small Intestinal Disaccharidase Deficiencies. Ped. Clin. North America 22, 757--766 (1975) Passey, R. B., Gillum, R. L., Fuller, J. B., Urry, F. M., Giles, M. L.: Evaluation and comparison of 10 glucose methods and the reference method recommended in the proposed product class standard (1974). Clin. Chem. 23, 131--139 (1977)
Received March 31, 1977
