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Xylose absorption test using D-xylose-14C
Clin. Biochem. 5, 246-250 (1972)
XYLOSE ABSORPTION TEST USING D-XYLOSE-'~C
D. M. LYSTER, E. K. MINCEY, R. T. MORRISON, A. BOGOCH and F. PING
Divisio~ of Nuclear Medici~e, Department of Pathology, Vancouver Genera.! Hospital, Vancouver, British Colu~bia, Canada. (Received June 16, 197Z)
SUMMARY
1. D-xylose-'4C was used to measure oral xylose absorption in the investigation of malabsorption syndromes. The concentration of D-xylose-"C present in the 1 and 2 hour serum samples and the quantity of D-xylose and D-xylose-"C excreted in the urine in 5 hours were used as indices of xylose absorption. 2. The radioactive technique makes the test technically simple to perform on both serum and urine, analyses specifically for xylose, is not affected by other reducing substances present in the sample and does not require the collection of a fasting urine sample.
INTRODUCTION
The oral xylose absorption test (chemical assay) is a valuable screening test for determining the presence of malabsorption, although it does not measure dietary carbohydrate absorption because xylose is a five carbon monosaccharide. Xylose is passively absorbed in the proximal small intestine and excreted unchanged in the urine. The xylose absorption test may be abnormal in patients with massive bacterial overgrowth in the proximal small intestine and in disease states where there is a significant loss of the functional integrity of the jejunum (1). However, there are some conditions that may lead to erroneous urinary excretion results, thereby invalidating the test. Among these are inadequate hydration, Correspondence: Dr. D. M. Lyster.
XYLOSE ABSORPTION TEST
247
advanced age, or renal disease (2, 3). The determination of the blood xylose level therefore, is i m p o r t a n t to help differentiate diminished intestinal absorption from impaired renal clearance (4). It is also necessary that patients drink sufficient fluid for adequate hydration and that allowance be made for decreased u r i n a r y output in the elderly. The following s t u d y d e m o n s t r a t e s the use of xylose-'~C in screening for intestinal malabsorption by measuring the u r i n a r y output and serum content of xylose following an oral dose. The results of the xylose u r i n a r y excretion by radiometric assay are compared with those obtained using chemical means.
MATERIALS AND METHODS
The procedure for the xylose tolerance test used in the present investigation was essentially as reported in the literature (5). The patient was given orally 25g of xylose containing 10 tzCi of xylose-'4C dissolved in 250 ml of w a t e r followed by 250 ml of w a t e r one hour later. The xylose was labelled uniformly and is metabolized identically to non radioactive xylose. All urine was collected for a period of 5 hours from the time of administration of the xylose and chemical analysis for reducing sugars carried out using alkaline potassium ferricyanide (autoanalyses). Additionally, chemical analysis for glucose was carried out on a fasting urine sample from each patient since the presence of glucose in the urine will interfere with the test (5). Blood samples were taken at 1 hour and 2 hours a f t e r administration of the oral dose and 1 ml serum samples of each were obtained for counting. A 1 ml aliquot of the 5 hour urine collection was also taken for counting. The samples were added to 14 ml of a commercially available fluor (Unogel, Schwarz-Mann, Becton, Dickinson and Company, Orangeburg, N.Y.) and counted in a Liquimat 220 liquid scintillation counter (Picker Nuclear Corporation (Intertech) North Haven, Conn.). A quench correction curve was constructed using the isotope channels ratio technique. S t a n d a r d curves were prepared f o r serum and urine using non-radioactive serum or urine solutions and 14C-Toluene (Solution S t a n d a r d Model 188270 A m e r s h a m / S e a r l e , Des Plaines, Illinois). Increasing amounts of urine or serum were used for quenching and a constant volume of 1 ml was maintained, when necessary, with distilled water. The xylose -'~C was checked for radio-chemical p u r i t y before use by thin layer c h r o m a t o g r a p h y using two solvent systems: n-butanol: ethanol: water (5:3:1) and ethylacetate: pyridine : w a t e r (2:1:2). Radiochemical purity was always g r e a t e r than 995~. It was then dispensed in 10 /zci aliquots and frozen f o r later use.
L Y S T E R et al.
248
CALCULATIONS
Urine Volume of urine in 5 hours Amount of xylose given Activity given (determined from appropriate aliquot) administered to patient Activity in 1 ml of urine Therefore total activity Amount of xylose excreted
Blood Activity in 1 ml of serum Therefore, amount of xylose in 100 ml serum
=
= = = =
a mls bg
c cpm d cpm d cpm x a mls d cpm x a mls x b g c cpm e cpm e cpm x 100 x b g e cpm
Counts per minute were converted to a fraction of the administered dose absorbed and then normalized to 25g. The results were expressed as mg ~ in serum and as g r a m s of xylose in the 5 hour urine collection.
RESULTS AND DISCUSSIONS
It can be seen from Table 1 t h a t the total a m o u n t of u r i n a r y xylose as calculated by chemical and radioisotopic means compare very well. The chemical analysis determines total reducing substances present in the urine (xylose, glucose, etc.) and this may account for the difference. Abnormal urine and serum xylose values by both chemical and radioisotopic analyses were obtained for patients J.K., M.B., and A.K. P a t i e n t J.K. had an irritable bowel syndrome. P a t i e n t M.B. had undiagnosed abdominal pain, and patient A.K. had a gluten induced e n t e r o p a t h y with malabsorption of folic acid, iron, vitamin D and fat. Abnormal radioactive xylose blood values were obtained for patient W.C. although the 5 hour urine value was normal. He was found to have gluten induced enteropathy. This indicates t h a t 5 hour urine values m a y be misleading in certain instances and t h a t blood xylose levels should be determined as well. Caution m u s t be used when using blood or urine values alone. Results obtained by the usual chemical method were not reported for patient A.K. because of interference due to glycosuria which thus invalidated the clinical analysis. Since m e a s u r e m e n t of radioactive xylose-'~C is not affected by other sugars present in the urine, glycosuria is not a factor in per-
XYLOSE ABSORPTION TEST
249
TABLE 1 ABSORPTION OF XYLOSE CONCENTRATION IN PLASMA AND U R I N E
Patient
Urinary Excretion Total excretion in gms 5 hours after oral administration Chemical Method
Radioisotopic Method
Plasma Concentration Radioisotopic Method 1 hr. after/2 hrs. after administration
E.L.S. V.M.
6.2 5.6
5.9 5.1
49.2 40.6
27.7 55.8
J.K.
2.2
2.0
--"
12.2
S.M. W.C.
5.8 3.6
5.7 4.6
32.7 4.0
28.4 13.8
E.S.
6.8
10.0
72.8
54.1
M.W.
5.5
5.4
29.7
31.5
M.D.
8.3
7.4
48.7
73.5
4.0 3.3
__a
__a
--"
5.5
M.B. A.K.
3.0
__b
Diagnosis
Chron's Disease Chronic Diarrhea Irritable bowel syndrome Gastric stasis Gluten induced enteropathy Ulcerative colitis Chronic Diarrhea Chronic Diarrhea Abdominal Pain Gluten induced enteropathy
" ) no blood samples obtained results not reported due to glycosuria h
_ _
NORMAL VALUES Plasma - - 1 hour 29 to 72 mg/100 ml 2 hour 15 to 76 rag/100 ml Urine -- 5 hour 4.1g to 9.3g (total excretion)
f o r m a n c e of the r a d i o a c t i v e test. I n a d d i t i o n to e l i m i n a t i n g the c o n c e r n a b o u t the n o n s p e c i f i c i t y of the c h e m i c a l r e a c t i o n f o r xylose, the r a d i o a c t i v e m e t h o d does n o t r e q u i r e e l a b o r a t e p r o c e d u r a l steps or special o r g a n i c or c a u s t i c solvents. Once t h e s t a n d a r d s f o r s e r u m a n d u r i n e h a v e been made, t h e p r o c e d u r e m e r e l y involves a d d i n g 1 ml of p a t i e n t s e r u m or u r i n e to t h e s c i n t i l l a t i o n solution a n d a f t e r d a r k a d a p t i n g , c o u n t i n g the vials. No c h e m i c a l e x t r a c t i o n s are n e c e s s a r y and t h e scintillation cocktail r e a d i l y a c c e p t s I ml of a q u e o u s media. T h e isotope c h a n n e l s r a t i o t e c h n i q u e w a s c h o s e n f o r q u e n c h c o r r e c t i o n b e c a u s e of the p o s s i b i l i t y of h a v i n g both color a n d c h e m i c a l q u e n c h i n g in t h e s a m p l e s a n d s t a n d a r d s ( 8 ) . A f e w p a t i e n t s c o m p l a i n e d of a b d o m i n a l d i s c o m f o r t a n d d i a r r h o e a w h e n g i v e n the 25g of xylose, b u t this m i n o r side e f f e c t applies to the c h e m i c a l m e t h o d as well. T h i s f i n d i n g h a s also been r e p o r t e d b y o t h e r w o r k e r s w h o
250
LYSTER et aL
e l i m i n a t e d the p r o b l e m b y u s i n g 5g of xylose (9). T h e xylose-'*C is of h i g h specific a c t i v i t y , a n d t h e use of s m a l l e r q u a n t i t i e s of c a r r i e r xylose w o u l d n o t be a l i m i t i n g f a c t o r . O u r r a d i o m e t r i c a s s a y c u r r e n t l y uses 10 g m of xylose a n d we h a v e n o t o b s e r v e d this side e f f e c t to date. I t a p p e a r s t h a t t h e use of x y l o s e - " C o f f e r s c e r t a i n a d v a n t a g e s in t h e p e r f o r m a n c e of the xylese a b s o r p t i o n test. I t is t e c h n i c a l l y simple, n o t a f f e c t e d b y g l y c o s u r i a , p e r m i t s e v a l u a t i o n of b o t h u r i n e a n d s e r u m , a n a l y s e s s p e c i f i c a l l y f o r xylose a n d does not r e q u i r e collection a n d a n a l y s i s of a f a s t i n g u r i n e sample.
REFERENCES 1. WILSON, F. A., and DIETSCHY, J. M., Progress in Gastroenterology, Differential Diagnostic Approach to Clinical Problems of Malabsorption, Gastroenterology, 61, 911-931 (1971). 2. FINLAY, J. M., HOGARTH, J., and WIGHTMAN, K. J. R., A Clinical Evaluation of the D-Xylose Tolerance Test. Ann. Intern. Med., 61, 411-422 (1964). 3. KENDALL, M. J., the Influence of Age on the Xylose Absorption Test, Gut, 11, 498-501 (1970). $. HELMER, O. M., and FOUTS, P. J., Gastro-intestinal Studies II The Excretion of Xylose in Pernicious Anemia, J. Clin. Invest., 16, 343-349 (1937). 5. Clinical Chemistry Principles and Techniques, Ed. by Henry R. J., Hoeber Medical Division, Harper and Row,1967, p. 664. 6. HOEKINS, J. I., Pediatric Xylose Absorption Test; Measurements in Blood Preferable to Measurements in Urine, Clin. Chem. 16, 749-752 (1970). 7. BENSON, J. A., Jr., CULVER, P. J., RAGLAND, S., JONES, C. M., DRUMMEY, G. D., and BOUGAS, E., The D-Xylose Absorption Test in Malabsorption Syndromes, New Eng. J. Med., 256, 335-339 (1957). 8. NOUJAIM, A. A., EDISS, C., and WIEBE, L., Precision of some Quench Correction Methods in Liquid Scintillation Counting, Organic Scintillators and Liquid Scintillation Counting, 705-717 (1971). Acadamic Press Inc., New York. 9. HARRIS, A. L., Determination of D-XYLOSE in Urine for the D-XYLOSE Absorption Test, Clin. Chem., 15, 65-71 (1969).
XYLOSE ABSORPTION TEST USING D-XYLOSE-'~C
D. M. LYSTER, E. K. MINCEY, R. T. MORRISON, A. BOGOCH and F. PING
Divisio~ of Nuclear Medici~e, Department of Pathology, Vancouver Genera.! Hospital, Vancouver, British Colu~bia, Canada. (Received June 16, 197Z)
SUMMARY
1. D-xylose-'4C was used to measure oral xylose absorption in the investigation of malabsorption syndromes. The concentration of D-xylose-"C present in the 1 and 2 hour serum samples and the quantity of D-xylose and D-xylose-"C excreted in the urine in 5 hours were used as indices of xylose absorption. 2. The radioactive technique makes the test technically simple to perform on both serum and urine, analyses specifically for xylose, is not affected by other reducing substances present in the sample and does not require the collection of a fasting urine sample.
INTRODUCTION
The oral xylose absorption test (chemical assay) is a valuable screening test for determining the presence of malabsorption, although it does not measure dietary carbohydrate absorption because xylose is a five carbon monosaccharide. Xylose is passively absorbed in the proximal small intestine and excreted unchanged in the urine. The xylose absorption test may be abnormal in patients with massive bacterial overgrowth in the proximal small intestine and in disease states where there is a significant loss of the functional integrity of the jejunum (1). However, there are some conditions that may lead to erroneous urinary excretion results, thereby invalidating the test. Among these are inadequate hydration, Correspondence: Dr. D. M. Lyster.
XYLOSE ABSORPTION TEST
247
advanced age, or renal disease (2, 3). The determination of the blood xylose level therefore, is i m p o r t a n t to help differentiate diminished intestinal absorption from impaired renal clearance (4). It is also necessary that patients drink sufficient fluid for adequate hydration and that allowance be made for decreased u r i n a r y output in the elderly. The following s t u d y d e m o n s t r a t e s the use of xylose-'~C in screening for intestinal malabsorption by measuring the u r i n a r y output and serum content of xylose following an oral dose. The results of the xylose u r i n a r y excretion by radiometric assay are compared with those obtained using chemical means.
MATERIALS AND METHODS
The procedure for the xylose tolerance test used in the present investigation was essentially as reported in the literature (5). The patient was given orally 25g of xylose containing 10 tzCi of xylose-'4C dissolved in 250 ml of w a t e r followed by 250 ml of w a t e r one hour later. The xylose was labelled uniformly and is metabolized identically to non radioactive xylose. All urine was collected for a period of 5 hours from the time of administration of the xylose and chemical analysis for reducing sugars carried out using alkaline potassium ferricyanide (autoanalyses). Additionally, chemical analysis for glucose was carried out on a fasting urine sample from each patient since the presence of glucose in the urine will interfere with the test (5). Blood samples were taken at 1 hour and 2 hours a f t e r administration of the oral dose and 1 ml serum samples of each were obtained for counting. A 1 ml aliquot of the 5 hour urine collection was also taken for counting. The samples were added to 14 ml of a commercially available fluor (Unogel, Schwarz-Mann, Becton, Dickinson and Company, Orangeburg, N.Y.) and counted in a Liquimat 220 liquid scintillation counter (Picker Nuclear Corporation (Intertech) North Haven, Conn.). A quench correction curve was constructed using the isotope channels ratio technique. S t a n d a r d curves were prepared f o r serum and urine using non-radioactive serum or urine solutions and 14C-Toluene (Solution S t a n d a r d Model 188270 A m e r s h a m / S e a r l e , Des Plaines, Illinois). Increasing amounts of urine or serum were used for quenching and a constant volume of 1 ml was maintained, when necessary, with distilled water. The xylose -'~C was checked for radio-chemical p u r i t y before use by thin layer c h r o m a t o g r a p h y using two solvent systems: n-butanol: ethanol: water (5:3:1) and ethylacetate: pyridine : w a t e r (2:1:2). Radiochemical purity was always g r e a t e r than 995~. It was then dispensed in 10 /zci aliquots and frozen f o r later use.
L Y S T E R et al.
248
CALCULATIONS
Urine Volume of urine in 5 hours Amount of xylose given Activity given (determined from appropriate aliquot) administered to patient Activity in 1 ml of urine Therefore total activity Amount of xylose excreted
Blood Activity in 1 ml of serum Therefore, amount of xylose in 100 ml serum
=
= = = =
a mls bg
c cpm d cpm d cpm x a mls d cpm x a mls x b g c cpm e cpm e cpm x 100 x b g e cpm
Counts per minute were converted to a fraction of the administered dose absorbed and then normalized to 25g. The results were expressed as mg ~ in serum and as g r a m s of xylose in the 5 hour urine collection.
RESULTS AND DISCUSSIONS
It can be seen from Table 1 t h a t the total a m o u n t of u r i n a r y xylose as calculated by chemical and radioisotopic means compare very well. The chemical analysis determines total reducing substances present in the urine (xylose, glucose, etc.) and this may account for the difference. Abnormal urine and serum xylose values by both chemical and radioisotopic analyses were obtained for patients J.K., M.B., and A.K. P a t i e n t J.K. had an irritable bowel syndrome. P a t i e n t M.B. had undiagnosed abdominal pain, and patient A.K. had a gluten induced e n t e r o p a t h y with malabsorption of folic acid, iron, vitamin D and fat. Abnormal radioactive xylose blood values were obtained for patient W.C. although the 5 hour urine value was normal. He was found to have gluten induced enteropathy. This indicates t h a t 5 hour urine values m a y be misleading in certain instances and t h a t blood xylose levels should be determined as well. Caution m u s t be used when using blood or urine values alone. Results obtained by the usual chemical method were not reported for patient A.K. because of interference due to glycosuria which thus invalidated the clinical analysis. Since m e a s u r e m e n t of radioactive xylose-'~C is not affected by other sugars present in the urine, glycosuria is not a factor in per-
XYLOSE ABSORPTION TEST
249
TABLE 1 ABSORPTION OF XYLOSE CONCENTRATION IN PLASMA AND U R I N E
Patient
Urinary Excretion Total excretion in gms 5 hours after oral administration Chemical Method
Radioisotopic Method
Plasma Concentration Radioisotopic Method 1 hr. after/2 hrs. after administration
E.L.S. V.M.
6.2 5.6
5.9 5.1
49.2 40.6
27.7 55.8
J.K.
2.2
2.0
--"
12.2
S.M. W.C.
5.8 3.6
5.7 4.6
32.7 4.0
28.4 13.8
E.S.
6.8
10.0
72.8
54.1
M.W.
5.5
5.4
29.7
31.5
M.D.
8.3
7.4
48.7
73.5
4.0 3.3
__a
__a
--"
5.5
M.B. A.K.
3.0
__b
Diagnosis
Chron's Disease Chronic Diarrhea Irritable bowel syndrome Gastric stasis Gluten induced enteropathy Ulcerative colitis Chronic Diarrhea Chronic Diarrhea Abdominal Pain Gluten induced enteropathy
" ) no blood samples obtained results not reported due to glycosuria h
_ _
NORMAL VALUES Plasma - - 1 hour 29 to 72 mg/100 ml 2 hour 15 to 76 rag/100 ml Urine -- 5 hour 4.1g to 9.3g (total excretion)
f o r m a n c e of the r a d i o a c t i v e test. I n a d d i t i o n to e l i m i n a t i n g the c o n c e r n a b o u t the n o n s p e c i f i c i t y of the c h e m i c a l r e a c t i o n f o r xylose, the r a d i o a c t i v e m e t h o d does n o t r e q u i r e e l a b o r a t e p r o c e d u r a l steps or special o r g a n i c or c a u s t i c solvents. Once t h e s t a n d a r d s f o r s e r u m a n d u r i n e h a v e been made, t h e p r o c e d u r e m e r e l y involves a d d i n g 1 ml of p a t i e n t s e r u m or u r i n e to t h e s c i n t i l l a t i o n solution a n d a f t e r d a r k a d a p t i n g , c o u n t i n g the vials. No c h e m i c a l e x t r a c t i o n s are n e c e s s a r y and t h e scintillation cocktail r e a d i l y a c c e p t s I ml of a q u e o u s media. T h e isotope c h a n n e l s r a t i o t e c h n i q u e w a s c h o s e n f o r q u e n c h c o r r e c t i o n b e c a u s e of the p o s s i b i l i t y of h a v i n g both color a n d c h e m i c a l q u e n c h i n g in t h e s a m p l e s a n d s t a n d a r d s ( 8 ) . A f e w p a t i e n t s c o m p l a i n e d of a b d o m i n a l d i s c o m f o r t a n d d i a r r h o e a w h e n g i v e n the 25g of xylose, b u t this m i n o r side e f f e c t applies to the c h e m i c a l m e t h o d as well. T h i s f i n d i n g h a s also been r e p o r t e d b y o t h e r w o r k e r s w h o
250
LYSTER et aL
e l i m i n a t e d the p r o b l e m b y u s i n g 5g of xylose (9). T h e xylose-'*C is of h i g h specific a c t i v i t y , a n d t h e use of s m a l l e r q u a n t i t i e s of c a r r i e r xylose w o u l d n o t be a l i m i t i n g f a c t o r . O u r r a d i o m e t r i c a s s a y c u r r e n t l y uses 10 g m of xylose a n d we h a v e n o t o b s e r v e d this side e f f e c t to date. I t a p p e a r s t h a t t h e use of x y l o s e - " C o f f e r s c e r t a i n a d v a n t a g e s in t h e p e r f o r m a n c e of the xylese a b s o r p t i o n test. I t is t e c h n i c a l l y simple, n o t a f f e c t e d b y g l y c o s u r i a , p e r m i t s e v a l u a t i o n of b o t h u r i n e a n d s e r u m , a n a l y s e s s p e c i f i c a l l y f o r xylose a n d does not r e q u i r e collection a n d a n a l y s i s of a f a s t i n g u r i n e sample.
REFERENCES 1. WILSON, F. A., and DIETSCHY, J. M., Progress in Gastroenterology, Differential Diagnostic Approach to Clinical Problems of Malabsorption, Gastroenterology, 61, 911-931 (1971). 2. FINLAY, J. M., HOGARTH, J., and WIGHTMAN, K. J. R., A Clinical Evaluation of the D-Xylose Tolerance Test. Ann. Intern. Med., 61, 411-422 (1964). 3. KENDALL, M. J., the Influence of Age on the Xylose Absorption Test, Gut, 11, 498-501 (1970). $. HELMER, O. M., and FOUTS, P. J., Gastro-intestinal Studies II The Excretion of Xylose in Pernicious Anemia, J. Clin. Invest., 16, 343-349 (1937). 5. Clinical Chemistry Principles and Techniques, Ed. by Henry R. J., Hoeber Medical Division, Harper and Row,1967, p. 664. 6. HOEKINS, J. I., Pediatric Xylose Absorption Test; Measurements in Blood Preferable to Measurements in Urine, Clin. Chem. 16, 749-752 (1970). 7. BENSON, J. A., Jr., CULVER, P. J., RAGLAND, S., JONES, C. M., DRUMMEY, G. D., and BOUGAS, E., The D-Xylose Absorption Test in Malabsorption Syndromes, New Eng. J. Med., 256, 335-339 (1957). 8. NOUJAIM, A. A., EDISS, C., and WIEBE, L., Precision of some Quench Correction Methods in Liquid Scintillation Counting, Organic Scintillators and Liquid Scintillation Counting, 705-717 (1971). Acadamic Press Inc., New York. 9. HARRIS, A. L., Determination of D-XYLOSE in Urine for the D-XYLOSE Absorption Test, Clin. Chem., 15, 65-71 (1969).