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Automated enzymatic assays for the determination of intestinal permeability probes in urine. 1. Lactulose and lactose
79
Clinica Chimica Acta, 187 (1990) 79-88 Elsevier
CCA 04657
Automated enzymatic assays for the determination of intestinal permeability probes in urine. 1. Lactulose and lactose Christine A. Northrop, Peter G. Lunn and Ronald H. Behrens Dunn Nutritional (Received
5 November
Laboratory,
Downham’s Lane, Cambridge (UK)
1988; revision received 29 September
Key words; Intestinal
permeability;
Lactulose;
1989; accepted
Lactose;
Automated
16 October
enzyme
1989)
assay
Summary Lactulose is becoming the disaccharide of choice in the dual sugar assessment of passive permeability of the small intestinal mucosa. However its more widespread use is hampered by current analytical methods which are tedious and time consuming. An automated spectrophotometric technique for the assay of this sugar in urine is presented in which lactulose is linked by a series of enzyme reactions to the equimolar production of NADPH. In addition to lactulose, the procedure also gives accurate values for lactose, glucose and fructose in the urine sample. The assay has been shown to be highly specific for lactulose and lactose and was not affected by high concentrations of other sugars or other urinary constituents. Within assay and between assay precision were similar with the coefficient of variation for both sugars in the range 0.4-1.6s. The technique represents a significant improvement in time, simplicity and precision on existing methods of analysis.
Introduction The measurement of passive intestinal permeability using the dual sugar technique is becoming accepted as a useful noninvasive method for assessing mucosal integrity in the small bowel [l]. Clear differences in the uptake of either mono- or disaccharides, or both, have been demonstrated in a number of illnesses in which pathological alterations in the mucosa of the small intestine are known to occur. Abnormal passive uptake of sugars has been observed in coeliac disease [2,3],
Correspondence to: C.A. Northrop, Cambridge CB4 lXJ, UK.
0009-8981/90/$03.50
Dunn
Nutritional
0 1990 Elsevier Science Publishers
Laboratory,
Downham’s
B.V. (Biomedical
Division)
Lane,
Milton
Road,
80
Crohn’s disease [4,5], cow’s milk protein intolerance [6,7] and acute and chronic diarrhoea [8,9]. In addition, several studies have reported a good correlation between dual sugar permeability data and morphological assessments of mucosal damage made on biopsy material [7,10,11]. The literature however has become confused by the range of different mono- and disaccharide sugars which have been used by different research groups and one of the major causes of this disparity lies in the tedious and difficult techniques which have been used to measure the concentrations of the probe molecules in the urine. Most methods so far described have involved chromatographic separation of the sugars, either on paper or thin layer plates [12,13], or by using gas liquid or high pressure liquid chromatography columns often following sugar derivatisation [ 14-161. Clearly such procedures are slow and limit the number of samples which can be processed. Moreover, the number of steps involved makes such techniques prone to operator error. In this communication and an earlier one [17], we describe rapid, automated enzyme assays for a disaccharide, lactulose, and a monosaccharide, mannitol, both of which have been frequently used in permeability assessment. In a recent study these two sugars have been shown to fulfill all the criteria required for their use in measuring passive permeability in the small intestine [18]. Materials and methods Theory The basic principle of the assay was first described as a manual Behrens et al. [19], but this has since been modified and automated. The principle reactions are: lactulose
P-galactosidase
) fructose
+ galactose,
lactose
/3-galactosidase ) glucose + galactose,
fructose
+ ATP - hexokinase fructose-6-phosphate
glucose + ATP B fructose-6-phosphate glucose-6-phosphate
glucose-6-phosphate
by
(11 (la) + ADP, + ADP,
phosphogluco- ) glucose-6-phosphate, isomerase + NADP
method
g1ucose~6~phosphate ) gluconate-6-phosphate dehydrogenase
(2) (2a) (3) + NADPH. (4)
Thus the amount of NADPH, at 340 nm is directly proportional
obtained by measuring the increase in absorption to lactulose concentration if this is the only sugar
81
TABLE
I
Programme
parameters
for the assays of lactulose
alpha 1 Units
2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
Calculation factor Standard 1 Standard 2 Standard 3 Limit Temperature (O C) Type of Analysis Wavelength (nm) Sample volume (PI) Diluent volume (~1) Reagent volume (pl) incubation time (sf Start reagent volume @I) Time of first reading(s) Time interval (s) Number of readings Blanking mode Printout mode
and lactose
on the Cobas-Bio
+ Lactose
+ Lactulose
w/l
m/l 0
250 500 1000 I 000 40 4 340 20 30 300 0 0 0.5 60 6 1 1
0 250 500 1000 1000 40 6 340 0 0 0 10 5 0.5 60 6 5 1
present. However, urine, particularly of ill patients may well contain other sugars, such as glucose, fructose and lactose, which would interfere with the assay. Any lactose in the sample would also be hydrolysed by /I-galactosidase to produce glucose (reaction (la)) which would be phosphorylated by reaction (2a), and subsequently take part in the final reaction to generate NADPH. Similarly, any free glucose in the urine would enter the system at reaction (2a), and free fructose at reaction (3) and both would increase the amount of NADPH. In the method described below these interfering factors are taken into account and accurate measurements of the urinary concentrations of lactose, glucose and fructose as well as lactulose are obtained during the procedure (Table I). Reagents were obtained and prepared as described previously [19] except that the /3-galactosidase (EC 3.2.1.23) was dissolved in the triethanolamine/MgSO, buffer and not ammonium sulphate. Using the Cobas-Bio (Roche UK, Welwyn Garden City) technique, only 300 ~1 of the buffer-enzyme cocktail is required per assay.
Protocol Two Cobas-Bio sample cups were set up for each urine. Into the first were pipetted 50 ~1 of sample, 50 ~1 of TE/MgSO, buffer and 25 ~1 of P-galactosidase solution. The mixture was incubated at 37°C for 1.5-2 h to hydrolyse both lactulose and lactose to their constituent monosaccharides. A further 50 ~1 of sample, followed by 7.5 ~1 of TE/MgSO, buffer, but no P-galactosidase was added to the second sample cup which was used to measure free urinary glucose and
82
NO
free mono-
main
isomerase
saccharides
reagent -/---+
P-galacto/
sample
P-galacto-\ sidase
\
\ free
L
monosaccharides
+ hydrolysis
products
main ------+A reagent
isomerase
4
4
glucose+
fructose+
lactose
lactulose
Fig. 1. Flow diagram of lactose and lactulose assay.
fructose. This second sample should also be incubated at 37°C for 1.5-2 h, but in practice this has been found to be an unnecessary precaution. Following incubation, sample cups were placed in the Cobas-Bio, with the cocktail in the main reagent reservoir and phosphoglucoseisomerase (PGI) in the start reagent cup. The analysis was performed in two stages, using the programme settings shown. The first step was performed before PGI was added so the increase in NADPH observed was proportional to the glucose concentration. In samples not treated with /3-galactosidase, this was a measure of free urinary glucose, but in the /I-galactosidase treated samples, it was a measure of both free glucose and glucose released by hydrolysis of lactose. Thus a subtraction of the free from the combined value gave the lactose concentration. The second stage of the reaction was then performed after addition of PGI. In samples not exposed to /?-galactosidase, the further increase in NADPH is a measure of free fructose whereas in the P-galactosidase incubated aliquot, combined free fructose and fructose hydrolysed from lactulose is measured. Subtraction of the free from the combined gives the lactulose concentrations in the original sample. The protocol is summarised in Fig. 1.
83
/
I
1200
400 Lactuloseilactose Fig. 2. Relationship
between
urinary
2000
concentration
concentrations of lactose 340 nm.
(mgll)
or lactulose
and change
in absorbance
at
Results
The change in absorbance (340 nm) with rising concentrations of lactose and Iactulose up to 6.84 mmol/f (2~0 mg/l) is shown in Fig. 2. However, production of NADPH was found to increase linearly with lactose concentrations up to 14.6 mmol/l (5000 mg/l) whereas lactulose gave a linear response up to 8.76 mmol/l (3 000 mg/l). The assay is capable of measuring down to 0.03 mmol/l (10 mg/l) but in practice, 0.073 mmol/l (25 mg/l) is taken as the lowest level of accurate assessment for both sugars. Following a standard permeability test dose of 2 g Iactuiose/lO kg body weight (up to a maximum of 10 g), the concentration of this sugar in urine generally lies in the range 0.15-2.9 mmol/l(50-1000 mg/l). Table II shows the inter and intra-assays reproducibility of the method over this range.
TABLE
II
The inter- and intra-assay
precision
at different
concentrations
Sugar cone
Within
assay
Between assay
Values are expressed
of lactose
and iactulose
CV (%)
(mmol/l)
(mg/B
lactose
lactulose
0.37 0.73 1.46 2.92
125 250 500 1000
1.14 0.83 0.52 1.22
1.47 1.21 0.89 0.66
0.37 0.73 1.46 2.92
125 250 500 loo0
1.03 0.89 0.97 0.60
1.59 1.03 0.94 0.40
as the CV for 15 determinations.
84 TABLE
III
Mean recoveries
of different
concentrations
of lactose
Lactose
and lactulose
added
to five normal
urines
Lactulose
Cone mmol/l
mS/l
0.37 0.73 1.46 2.92
125 250 500 1000
% Recovery
+ SE
99.1 k 2.4 102.7 + 2.2 105.1 * 3.3 102.9i4.2
Overall
Cone mmol/l
mS/l
0.37 0.73 1.46 2.92
125 250 500 1000
% Recovery
95.4 i 98.6 k 98.7 k 96.9+
102.5 +0.8
* SE
2.0 1.9 2.6 2.8
97.4 f 0.6
More important however is assay performance in the presence of other sugars and in urine itself. Table III shows the mean recoveries from five different urines to which lactose and lactulose had been added to give four different concentrations. Recovery of lactose can be seen to be complete throughout whereas lactulose recovery at 97.4% was only slightly less than complete. The variation in recovery rates from the different urines were however very constant and similar for both sugars. It therefore seems unlikely that urine contains any substances which seriously inhibit the sequence of reactions. In some patients, e.g. breast-fed infants with diarrhoea and lactase deficiency, considerable amounts of lactose may appear in the urine and consequently it was felt necessary to investigate the effect of high concentrations of lactose on the lactulose estimation. Table IV shows this effect to be minimal, lactulose values were essentially unaffected by lactose concentrations up to 2.92 mmol/l (1000 mg/l). Above this concentration, lactulose recovery was constant at concentrations up to 1.46 mmol/l(500 mg/l), but the 2.92 mmol/l (1000 mg/l) estimate was only 92.4% of expected. In practice this problem is easily overcome by prior dilution of the urine sample. To assess specificity, a range of common sugars, made up at 29.2 mmol/l were tested in the standard assay procedure, and the results are shown in Table V.
TABLE
IV
Determination Actual cone
of lactulose
lactulose
in the presence
of different
Observed lactulose % of actual value
of lactose
cone as 2.92 mmol/l
lactose
5.34 mmol/l
mmol/l
mS/l
1.46 mmol/l
0.37 0.73 1.46 2.92
125 250 500 1000
103.8 100.1 102.7 100.1
99.0 96.5 100.4 96.5
101.3 96.5 99.7 92.4
101.7
98.1
97.5
Mean
lactose
concentrations
lactose
85
TABLE
V
Specificity of lactose and assayed for both lactose lactose/lactulose. Sugar
lactulose assays Solutions containing 29.2 mmol/l and lactulose. Results are shown as a percentage
Lactose
Arabinose
0
Fructose Galactose Glucose Lactose
3.2 0
Lactulose Maltose Mannitol Raffinose Rhamnose Sorbose Sucrose Trehalose Turanose Xylose
ww (100) 5.8 0.16 0 0 0 0 0 5.5 0.48 0.28
assay
Lactulose
of various sugars were activity compared to
assay
0 (TOO) 0 0.3 0.6 (100) 0 0 0 0 0 0 0.26 0.03 0.08
Clearly the level of interference is extremely low furthermore, some of the apparent interference may be due to impurities in the sugars as purchased. Lactulose from Sigma, for example, is only guaranteed to be 90% pure, with lactose as the most likely contaminant. Thus, the apparent 5.8% cross-reaction of lactulose with the lactose measurement is probably a measure of the lactose content of the preparation. Discussion The determination of lactose and lactulose using specific enzymatic procedures has a number of advantages over previously used assay techniques which invariably required chromatographic separation. Although recent advances in gas-liquid chromatography and high pressure liquid chromatography have resulted in improved accuracy, easier sample preparation and faster flow rates, the highly reproducible automated analysis of 25 samples each 20 min makes the enzyme technique far faster and less tedious than previous methods. Moreover, the assay has been shown to be highly specific for lactose and lactulose and the presence of other sugars does not interfere with the determination. Although the method can be performed manually, use of the Cobas-Bio centrifugal analyser not only increases the speed of the assay, it also increases versatility. For example, increasing the sample volume from 20 to 60 ~1 immediately gives a three-fold increase in sensitivity, whilst lowering it to 5 ~1 extends the linear range of the assay to 29.2 mmol/l (10000 mg/l). The method described is also an improvement on that of Behrens et al. [19] in that accurate measurements of glucose
86
and fructose are obtained in addition to lactose and lactulose. The estimation of lactose has proven to be particularly important in permeability studies of breast-fed Gambian infants with diarrhoea and partial secondary lactase deficiency [9,20]. Using the assay as described, several thousand urines have been assayed for lactulose and lactose. The results, combined with mannitol concentrations measured as described in an earlier paper [17], have been used to assess small bowel permeability in lean, obese and fasting adults [21], malnourished Gambian infants with diarrhoea 191, Bangladeshi children with ascariasis [22] and in rats infected with the nematode parasite ~~pp~s~~~n~~~~lu~~ ~~~s~l~~~sis [23,24]. The technique was also used in a pharmacokinetic study of lactulose in man [lg]. References 1 Menzies IS. Transmucosal passage of inert molecules in health and disease. In: Intestinal and secretion. Falk Symposium 36. London MTP Press, 1983;527-543. 2 Cobden I, Rothwell R, Axon ATR. Intestinal permeability and screening 1980;21:512-518.
absorption
tests for coeliac disease. Gut
3 Wheeler PC, Menzies IS, Creamer B. Effect of hyperosomolar stimuli and coeliac disease on the permeability of the human gastrointestinal tract. Clin Sci Mol Med 1978;54:495-501. 4 Pearson ADJ, Eastham L, Laker MF, Craft AW, Nelson R. Intestinal permeability in children with Crohn’s disease and coeliac disease. Br Med J 1982;285:20,21. 5 Nathavitharana KA, Lloyd DR, Raafat F. Brown GA, NcNeish AS. Urinary mannitol: lactulose excretion ratios and jejunal mucosal structure. Arch Dis Child 1988;43:1054-1059, 6 DuPont C. Barau E, Dehennin L, Molkhu P. Intestinal permeability to sugars: results in celiac disease and in cow’s milk sensitive enteropathy. Path01 Biol 1987;35:1179-J182. 7 Hamilton I, Hill A, Bose B. Bouchier AD, Forsyth JS. Small intestinal perm~bility in pediatric clinical practice. J Pediatr Gastroenterol Nutr 1987;6:697-701. 8 Noone C, Menzies IS, Banatvala JE, Scopes JW. Intestinal permeability and lactose hydrolysis in human rotaviral gastroenteritis assessed simultaneously by non-invasive differential sugar permeation. Eur J Chn Invest 1986;16:217-225. 9 Behrens RH, Lunn PG. Northrop CA, Hanlon PW, Neale G. Factors affecting the integrity of the intestinal mucosa of Gambian children, Am J Clin Nutr 1987;45:1433-1441. 10 Ford RPK, Menzies IS, Philips AD, Walker-Smith JA, Turner NW. Intestinal sugar permeability: relationship to diarrhoeal disease and small bowel morphology. J Pediatr Gastroenterol Nutr 1985;4:568-574. 11 Juby LD, Dixon MF, Axon ATR. Abnormal intestinal permeability and jejunal morphometry. 3 Ciin Path01 1987;40:714-718, 12 Menzies IS. Quantitative estimation of sugars in blood and urine by paper chromatography using direct densitometry. J Chromatogr 1973;81:109-127. 13 Menzies IS, Mount NJ, Wheeler MJ. Quantitative estimation of clinically important monosaccharides in plasma by rapid thin layer chromatography. Ann Clin Biochem 1978;15:65-76. 14 Laker MF. Estimation of disaccharides in plasma and urine by gas-liquid chromatography. J Chromatogr 1979;I63:9-18. 15 Laker MF, Mount JN. Mannitol estimation in biological fluids by gas-liquid chromatography of trimethylsiiyl derivatives. Clin Chem 1980;26:441-443. 16 Delahunty T, Hollander D. Liquid chromatographic method for estimating urinary sugars: applicability to studies of intestinal permeability. Clin Chem 1986;32:7542,1543. 17 Lunn PG. Northrup CA, Northrup AJ. Automated enzymatic assays for the determination of intestinal permeability probes in urine. 2. Mannitol Clin Chim Acta 19~9;1~3:I63-17~. 18 Elia M, Behrens R, Northrop C, Wraigbt P, Neale G. Evaluation of mannitol, lactulose and s’Cr-labelledEDTA as markers of intestinal permeability in man. Clin Sci 1987;73:197-204.
87 19 Behrens RH, Docherty H, Elia M, Neale G. A simple enzymatic method for the assay of urinary lactulose. Clin Chim Acta 1984;137:361-367. 20 Northrop CA. Lunn PG, Downes RM. Intestinal permeability studies in Gambian infants. In: Kager PA, Polderman AM, ed. Proceedings of the XIIth international congress for tropical medicine and malaria. Amsterdam: Excerpta Medica, 1988;214. 21 Elia M. Goren A, Behrens R, Barber RW, Neale G. Effect of total starvation and very low calorie diets on intestinal permeability in man. Clin Sci 1987;73:205-210. 22 Northrop CA, Lunn PG, Wainwright M, Evans J. Plasma albumin concentrations and intestinal permeability in Bangladeshi children infected with Ascaris lumbricoides. Trans R Sot Trop Med Hyg 1987;81:811-815. 23 Lunn PG. Northrop CA, Behrens RH, Martin J. Wainwright M. Protein losing enteropathy associated with N~ppostrongy/w brasiliensis infection and its impact on albumin homeostasis in rats fed two levels of dietary protein. Clin Sci 1986:70:469-475. 24 Lunn PG. Northrop CA, Wainwright M. Hypoalbuminemia in energy malnourished rats infected with Nippostrongyh brasiliensis (Nematoda). J Nutr 1988;118:121-127.
Clinica Chimica Acta, 187 (1990) 79-88 Elsevier
CCA 04657
Automated enzymatic assays for the determination of intestinal permeability probes in urine. 1. Lactulose and lactose Christine A. Northrop, Peter G. Lunn and Ronald H. Behrens Dunn Nutritional (Received
5 November
Laboratory,
Downham’s Lane, Cambridge (UK)
1988; revision received 29 September
Key words; Intestinal
permeability;
Lactulose;
1989; accepted
Lactose;
Automated
16 October
enzyme
1989)
assay
Summary Lactulose is becoming the disaccharide of choice in the dual sugar assessment of passive permeability of the small intestinal mucosa. However its more widespread use is hampered by current analytical methods which are tedious and time consuming. An automated spectrophotometric technique for the assay of this sugar in urine is presented in which lactulose is linked by a series of enzyme reactions to the equimolar production of NADPH. In addition to lactulose, the procedure also gives accurate values for lactose, glucose and fructose in the urine sample. The assay has been shown to be highly specific for lactulose and lactose and was not affected by high concentrations of other sugars or other urinary constituents. Within assay and between assay precision were similar with the coefficient of variation for both sugars in the range 0.4-1.6s. The technique represents a significant improvement in time, simplicity and precision on existing methods of analysis.
Introduction The measurement of passive intestinal permeability using the dual sugar technique is becoming accepted as a useful noninvasive method for assessing mucosal integrity in the small bowel [l]. Clear differences in the uptake of either mono- or disaccharides, or both, have been demonstrated in a number of illnesses in which pathological alterations in the mucosa of the small intestine are known to occur. Abnormal passive uptake of sugars has been observed in coeliac disease [2,3],
Correspondence to: C.A. Northrop, Cambridge CB4 lXJ, UK.
0009-8981/90/$03.50
Dunn
Nutritional
0 1990 Elsevier Science Publishers
Laboratory,
Downham’s
B.V. (Biomedical
Division)
Lane,
Milton
Road,
80
Crohn’s disease [4,5], cow’s milk protein intolerance [6,7] and acute and chronic diarrhoea [8,9]. In addition, several studies have reported a good correlation between dual sugar permeability data and morphological assessments of mucosal damage made on biopsy material [7,10,11]. The literature however has become confused by the range of different mono- and disaccharide sugars which have been used by different research groups and one of the major causes of this disparity lies in the tedious and difficult techniques which have been used to measure the concentrations of the probe molecules in the urine. Most methods so far described have involved chromatographic separation of the sugars, either on paper or thin layer plates [12,13], or by using gas liquid or high pressure liquid chromatography columns often following sugar derivatisation [ 14-161. Clearly such procedures are slow and limit the number of samples which can be processed. Moreover, the number of steps involved makes such techniques prone to operator error. In this communication and an earlier one [17], we describe rapid, automated enzyme assays for a disaccharide, lactulose, and a monosaccharide, mannitol, both of which have been frequently used in permeability assessment. In a recent study these two sugars have been shown to fulfill all the criteria required for their use in measuring passive permeability in the small intestine [18]. Materials and methods Theory The basic principle of the assay was first described as a manual Behrens et al. [19], but this has since been modified and automated. The principle reactions are: lactulose
P-galactosidase
) fructose
+ galactose,
lactose
/3-galactosidase ) glucose + galactose,
fructose
+ ATP - hexokinase fructose-6-phosphate
glucose + ATP B fructose-6-phosphate glucose-6-phosphate
glucose-6-phosphate
by
(11 (la) + ADP, + ADP,
phosphogluco- ) glucose-6-phosphate, isomerase + NADP
method
g1ucose~6~phosphate ) gluconate-6-phosphate dehydrogenase
(2) (2a) (3) + NADPH. (4)
Thus the amount of NADPH, at 340 nm is directly proportional
obtained by measuring the increase in absorption to lactulose concentration if this is the only sugar
81
TABLE
I
Programme
parameters
for the assays of lactulose
alpha 1 Units
2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
Calculation factor Standard 1 Standard 2 Standard 3 Limit Temperature (O C) Type of Analysis Wavelength (nm) Sample volume (PI) Diluent volume (~1) Reagent volume (pl) incubation time (sf Start reagent volume @I) Time of first reading(s) Time interval (s) Number of readings Blanking mode Printout mode
and lactose
on the Cobas-Bio
+ Lactose
+ Lactulose
w/l
m/l 0
250 500 1000 I 000 40 4 340 20 30 300 0 0 0.5 60 6 1 1
0 250 500 1000 1000 40 6 340 0 0 0 10 5 0.5 60 6 5 1
present. However, urine, particularly of ill patients may well contain other sugars, such as glucose, fructose and lactose, which would interfere with the assay. Any lactose in the sample would also be hydrolysed by /I-galactosidase to produce glucose (reaction (la)) which would be phosphorylated by reaction (2a), and subsequently take part in the final reaction to generate NADPH. Similarly, any free glucose in the urine would enter the system at reaction (2a), and free fructose at reaction (3) and both would increase the amount of NADPH. In the method described below these interfering factors are taken into account and accurate measurements of the urinary concentrations of lactose, glucose and fructose as well as lactulose are obtained during the procedure (Table I). Reagents were obtained and prepared as described previously [19] except that the /3-galactosidase (EC 3.2.1.23) was dissolved in the triethanolamine/MgSO, buffer and not ammonium sulphate. Using the Cobas-Bio (Roche UK, Welwyn Garden City) technique, only 300 ~1 of the buffer-enzyme cocktail is required per assay.
Protocol Two Cobas-Bio sample cups were set up for each urine. Into the first were pipetted 50 ~1 of sample, 50 ~1 of TE/MgSO, buffer and 25 ~1 of P-galactosidase solution. The mixture was incubated at 37°C for 1.5-2 h to hydrolyse both lactulose and lactose to their constituent monosaccharides. A further 50 ~1 of sample, followed by 7.5 ~1 of TE/MgSO, buffer, but no P-galactosidase was added to the second sample cup which was used to measure free urinary glucose and
82
NO
free mono-
main
isomerase
saccharides
reagent -/---+
P-galacto/
sample
P-galacto-\ sidase
\
\ free
L
monosaccharides
+ hydrolysis
products
main ------+A reagent
isomerase
4
4
glucose+
fructose+
lactose
lactulose
Fig. 1. Flow diagram of lactose and lactulose assay.
fructose. This second sample should also be incubated at 37°C for 1.5-2 h, but in practice this has been found to be an unnecessary precaution. Following incubation, sample cups were placed in the Cobas-Bio, with the cocktail in the main reagent reservoir and phosphoglucoseisomerase (PGI) in the start reagent cup. The analysis was performed in two stages, using the programme settings shown. The first step was performed before PGI was added so the increase in NADPH observed was proportional to the glucose concentration. In samples not treated with /3-galactosidase, this was a measure of free urinary glucose, but in the /I-galactosidase treated samples, it was a measure of both free glucose and glucose released by hydrolysis of lactose. Thus a subtraction of the free from the combined value gave the lactose concentration. The second stage of the reaction was then performed after addition of PGI. In samples not exposed to /?-galactosidase, the further increase in NADPH is a measure of free fructose whereas in the P-galactosidase incubated aliquot, combined free fructose and fructose hydrolysed from lactulose is measured. Subtraction of the free from the combined gives the lactulose concentrations in the original sample. The protocol is summarised in Fig. 1.
83
/
I
1200
400 Lactuloseilactose Fig. 2. Relationship
between
urinary
2000
concentration
concentrations of lactose 340 nm.
(mgll)
or lactulose
and change
in absorbance
at
Results
The change in absorbance (340 nm) with rising concentrations of lactose and Iactulose up to 6.84 mmol/f (2~0 mg/l) is shown in Fig. 2. However, production of NADPH was found to increase linearly with lactose concentrations up to 14.6 mmol/l (5000 mg/l) whereas lactulose gave a linear response up to 8.76 mmol/l (3 000 mg/l). The assay is capable of measuring down to 0.03 mmol/l (10 mg/l) but in practice, 0.073 mmol/l (25 mg/l) is taken as the lowest level of accurate assessment for both sugars. Following a standard permeability test dose of 2 g Iactuiose/lO kg body weight (up to a maximum of 10 g), the concentration of this sugar in urine generally lies in the range 0.15-2.9 mmol/l(50-1000 mg/l). Table II shows the inter and intra-assays reproducibility of the method over this range.
TABLE
II
The inter- and intra-assay
precision
at different
concentrations
Sugar cone
Within
assay
Between assay
Values are expressed
of lactose
and iactulose
CV (%)
(mmol/l)
(mg/B
lactose
lactulose
0.37 0.73 1.46 2.92
125 250 500 1000
1.14 0.83 0.52 1.22
1.47 1.21 0.89 0.66
0.37 0.73 1.46 2.92
125 250 500 loo0
1.03 0.89 0.97 0.60
1.59 1.03 0.94 0.40
as the CV for 15 determinations.
84 TABLE
III
Mean recoveries
of different
concentrations
of lactose
Lactose
and lactulose
added
to five normal
urines
Lactulose
Cone mmol/l
mS/l
0.37 0.73 1.46 2.92
125 250 500 1000
% Recovery
+ SE
99.1 k 2.4 102.7 + 2.2 105.1 * 3.3 102.9i4.2
Overall
Cone mmol/l
mS/l
0.37 0.73 1.46 2.92
125 250 500 1000
% Recovery
95.4 i 98.6 k 98.7 k 96.9+
102.5 +0.8
* SE
2.0 1.9 2.6 2.8
97.4 f 0.6
More important however is assay performance in the presence of other sugars and in urine itself. Table III shows the mean recoveries from five different urines to which lactose and lactulose had been added to give four different concentrations. Recovery of lactose can be seen to be complete throughout whereas lactulose recovery at 97.4% was only slightly less than complete. The variation in recovery rates from the different urines were however very constant and similar for both sugars. It therefore seems unlikely that urine contains any substances which seriously inhibit the sequence of reactions. In some patients, e.g. breast-fed infants with diarrhoea and lactase deficiency, considerable amounts of lactose may appear in the urine and consequently it was felt necessary to investigate the effect of high concentrations of lactose on the lactulose estimation. Table IV shows this effect to be minimal, lactulose values were essentially unaffected by lactose concentrations up to 2.92 mmol/l (1000 mg/l). Above this concentration, lactulose recovery was constant at concentrations up to 1.46 mmol/l(500 mg/l), but the 2.92 mmol/l (1000 mg/l) estimate was only 92.4% of expected. In practice this problem is easily overcome by prior dilution of the urine sample. To assess specificity, a range of common sugars, made up at 29.2 mmol/l were tested in the standard assay procedure, and the results are shown in Table V.
TABLE
IV
Determination Actual cone
of lactulose
lactulose
in the presence
of different
Observed lactulose % of actual value
of lactose
cone as 2.92 mmol/l
lactose
5.34 mmol/l
mmol/l
mS/l
1.46 mmol/l
0.37 0.73 1.46 2.92
125 250 500 1000
103.8 100.1 102.7 100.1
99.0 96.5 100.4 96.5
101.3 96.5 99.7 92.4
101.7
98.1
97.5
Mean
lactose
concentrations
lactose
85
TABLE
V
Specificity of lactose and assayed for both lactose lactose/lactulose. Sugar
lactulose assays Solutions containing 29.2 mmol/l and lactulose. Results are shown as a percentage
Lactose
Arabinose
0
Fructose Galactose Glucose Lactose
3.2 0
Lactulose Maltose Mannitol Raffinose Rhamnose Sorbose Sucrose Trehalose Turanose Xylose
ww (100) 5.8 0.16 0 0 0 0 0 5.5 0.48 0.28
assay
Lactulose
of various sugars were activity compared to
assay
0 (TOO) 0 0.3 0.6 (100) 0 0 0 0 0 0 0.26 0.03 0.08
Clearly the level of interference is extremely low furthermore, some of the apparent interference may be due to impurities in the sugars as purchased. Lactulose from Sigma, for example, is only guaranteed to be 90% pure, with lactose as the most likely contaminant. Thus, the apparent 5.8% cross-reaction of lactulose with the lactose measurement is probably a measure of the lactose content of the preparation. Discussion The determination of lactose and lactulose using specific enzymatic procedures has a number of advantages over previously used assay techniques which invariably required chromatographic separation. Although recent advances in gas-liquid chromatography and high pressure liquid chromatography have resulted in improved accuracy, easier sample preparation and faster flow rates, the highly reproducible automated analysis of 25 samples each 20 min makes the enzyme technique far faster and less tedious than previous methods. Moreover, the assay has been shown to be highly specific for lactose and lactulose and the presence of other sugars does not interfere with the determination. Although the method can be performed manually, use of the Cobas-Bio centrifugal analyser not only increases the speed of the assay, it also increases versatility. For example, increasing the sample volume from 20 to 60 ~1 immediately gives a three-fold increase in sensitivity, whilst lowering it to 5 ~1 extends the linear range of the assay to 29.2 mmol/l (10000 mg/l). The method described is also an improvement on that of Behrens et al. [19] in that accurate measurements of glucose
86
and fructose are obtained in addition to lactose and lactulose. The estimation of lactose has proven to be particularly important in permeability studies of breast-fed Gambian infants with diarrhoea and partial secondary lactase deficiency [9,20]. Using the assay as described, several thousand urines have been assayed for lactulose and lactose. The results, combined with mannitol concentrations measured as described in an earlier paper [17], have been used to assess small bowel permeability in lean, obese and fasting adults [21], malnourished Gambian infants with diarrhoea 191, Bangladeshi children with ascariasis [22] and in rats infected with the nematode parasite ~~pp~s~~~n~~~~lu~~ ~~~s~l~~~sis [23,24]. The technique was also used in a pharmacokinetic study of lactulose in man [lg]. References 1 Menzies IS. Transmucosal passage of inert molecules in health and disease. In: Intestinal and secretion. Falk Symposium 36. London MTP Press, 1983;527-543. 2 Cobden I, Rothwell R, Axon ATR. Intestinal permeability and screening 1980;21:512-518.
absorption
tests for coeliac disease. Gut
3 Wheeler PC, Menzies IS, Creamer B. Effect of hyperosomolar stimuli and coeliac disease on the permeability of the human gastrointestinal tract. Clin Sci Mol Med 1978;54:495-501. 4 Pearson ADJ, Eastham L, Laker MF, Craft AW, Nelson R. Intestinal permeability in children with Crohn’s disease and coeliac disease. Br Med J 1982;285:20,21. 5 Nathavitharana KA, Lloyd DR, Raafat F. Brown GA, NcNeish AS. Urinary mannitol: lactulose excretion ratios and jejunal mucosal structure. Arch Dis Child 1988;43:1054-1059, 6 DuPont C. Barau E, Dehennin L, Molkhu P. Intestinal permeability to sugars: results in celiac disease and in cow’s milk sensitive enteropathy. Path01 Biol 1987;35:1179-J182. 7 Hamilton I, Hill A, Bose B. Bouchier AD, Forsyth JS. Small intestinal perm~bility in pediatric clinical practice. J Pediatr Gastroenterol Nutr 1987;6:697-701. 8 Noone C, Menzies IS, Banatvala JE, Scopes JW. Intestinal permeability and lactose hydrolysis in human rotaviral gastroenteritis assessed simultaneously by non-invasive differential sugar permeation. Eur J Chn Invest 1986;16:217-225. 9 Behrens RH, Lunn PG. Northrop CA, Hanlon PW, Neale G. Factors affecting the integrity of the intestinal mucosa of Gambian children, Am J Clin Nutr 1987;45:1433-1441. 10 Ford RPK, Menzies IS, Philips AD, Walker-Smith JA, Turner NW. Intestinal sugar permeability: relationship to diarrhoeal disease and small bowel morphology. J Pediatr Gastroenterol Nutr 1985;4:568-574. 11 Juby LD, Dixon MF, Axon ATR. Abnormal intestinal permeability and jejunal morphometry. 3 Ciin Path01 1987;40:714-718, 12 Menzies IS. Quantitative estimation of sugars in blood and urine by paper chromatography using direct densitometry. J Chromatogr 1973;81:109-127. 13 Menzies IS, Mount NJ, Wheeler MJ. Quantitative estimation of clinically important monosaccharides in plasma by rapid thin layer chromatography. Ann Clin Biochem 1978;15:65-76. 14 Laker MF. Estimation of disaccharides in plasma and urine by gas-liquid chromatography. J Chromatogr 1979;I63:9-18. 15 Laker MF, Mount JN. Mannitol estimation in biological fluids by gas-liquid chromatography of trimethylsiiyl derivatives. Clin Chem 1980;26:441-443. 16 Delahunty T, Hollander D. Liquid chromatographic method for estimating urinary sugars: applicability to studies of intestinal permeability. Clin Chem 1986;32:7542,1543. 17 Lunn PG. Northrup CA, Northrup AJ. Automated enzymatic assays for the determination of intestinal permeability probes in urine. 2. Mannitol Clin Chim Acta 19~9;1~3:I63-17~. 18 Elia M, Behrens R, Northrop C, Wraigbt P, Neale G. Evaluation of mannitol, lactulose and s’Cr-labelledEDTA as markers of intestinal permeability in man. Clin Sci 1987;73:197-204.
87 19 Behrens RH, Docherty H, Elia M, Neale G. A simple enzymatic method for the assay of urinary lactulose. Clin Chim Acta 1984;137:361-367. 20 Northrop CA. Lunn PG, Downes RM. Intestinal permeability studies in Gambian infants. In: Kager PA, Polderman AM, ed. Proceedings of the XIIth international congress for tropical medicine and malaria. Amsterdam: Excerpta Medica, 1988;214. 21 Elia M. Goren A, Behrens R, Barber RW, Neale G. Effect of total starvation and very low calorie diets on intestinal permeability in man. Clin Sci 1987;73:205-210. 22 Northrop CA, Lunn PG, Wainwright M, Evans J. Plasma albumin concentrations and intestinal permeability in Bangladeshi children infected with Ascaris lumbricoides. Trans R Sot Trop Med Hyg 1987;81:811-815. 23 Lunn PG. Northrop CA, Behrens RH, Martin J. Wainwright M. Protein losing enteropathy associated with N~ppostrongy/w brasiliensis infection and its impact on albumin homeostasis in rats fed two levels of dietary protein. Clin Sci 1986:70:469-475. 24 Lunn PG. Northrop CA, Wainwright M. Hypoalbuminemia in energy malnourished rats infected with Nippostrongyh brasiliensis (Nematoda). J Nutr 1988;118:121-127.