Clin. Biochem. 9 (1) 19-21 (1976)

A Sensitive Automated Method for the Determination of Serum and Urine Xylose J. L. B R A I D W O O D

a n d M. B I N N S

SMITH

D e p a r t m e n t of Medical B i o c h e m i s t r y , St. J o s e p h ' s Hospital, L o n d o n , O n t a r i o a n d D i v i s i o n of Clinical B i o c h e m i s t r y , U n i v e r s i t y of W e s t e r n O n t a r i o (Accepted August

20, 1 9 7 5 )

CLBIA, 9, (1) 19-21 (1976) Clin. Biochem. lEATZNGBATH

Braidwood J. L. and Binns Smith M. Department of Medical Biochemistry, St. Joseph's Hospital, Lo*tdo~h Ontario and Division of Clinical Biochemistry, University of Western Ontario. A S E N S I T I V E AUTOMATED METHOD FOR THE D E T E R M I N A T I O N OF SERUM AND URINE XYLOSE A simple automated method allowing the rapid and precise assay of serum and urine xylose is described, even after small oral doses of xylose. With the same manifold but different reagents, the system may also be used for the assay of glucose.

A N ULTRA-MICRO M E T H O D FOR THE ASSAY OF SERUM XYLOSE using o-toluidine has already been described "~. However, in studies on carbohydrate metabolism, oral doses of glucose in oral glucose tolerance tests was supplemented with a small, 5 g dose of xylose '~ and during the early part of the study, it was found that the precision of assay in serum was poor due to the low concentration of xylose achieved and the need to use technologists not specially trained in ultra-micro techniques, a factor accentuated by the necessity to perform large numbers of assays. Correspondingly a sensitive and precise Autoanalyser method utilising the principles of the earlier method ''~ and a m o d i f i c a t i o n of a m a n i f o l d a l r e a d y described for a u t o m a t e d glucose assay c~ was evolved. would allow the assay of glucose on the same equipF u r t h e r m o r e , slight m o d i f i c a t i o n of the system ment. MATERIALS AND METHODS Equipment A u toanalyser

An Autoanalyser manifold prepared as shown in Fig. 1 was used for the o-toluidine reaction. However, a Carlo Erba peristaltic pump was used so that the size of tygon manifold tubing could not be used on an autoanalyser pump II without modification. Fig. 1 shows the appropriate sizes of tubing for both the Technicon Pump II and Carlo Erba pump. Correspondence: Dr. M. Binns Smith, Department of Medical Biochemistry, St. Joseph's Hospital, London, Ontario.

Black/Black ~Orange/Nhi ~A~ ~B~ • ~

SN{PLER II

~/ASII P~ECEPTACLE ~

WASTE DEBtrBBLER

SHC

\

~

Purpie/~hlte (Purple~ o-tolufdtne Blue (Grey) Air C,r~P~ (yp11.~ ~)

Nater Green (Yellow)" Waste

I J

I

COLORI TER h

15ram TI~ULARH FLOWCELL [ ] RECORDER 480 nm, I I

[

Fig. 1 - - Autoanalyeer manifold for ~ylose determb~ations. The tubing colour code in parenthesis is that used on the Carlo Erba Peristaltic Pump. ( A ) is an 0.015 inch I. P. Pulse eupp~'essor and (B) is an 0.005 inch I. D. Pulse suppressor. A~todih~ter

A Micromedic automatic pipettor was used to prepare the sample-glucose oxidase and sample-saline dilutions. Reagents

1. One "regular" vial "Glucostat" (Worthington Biochemical Corp.) was dissolved in 50 ml phosphate buffer, pH 7.0. This is prepared fresh. 2. Phosphate buffer, 0.1 Molar, pH 7.0. To 100 ml 0.1 M solution of disodium hydrogen phosphate add 0.1 M solution of potassium dihydrogen phosphate to adjust the pH to 7.0. 3. Stock xylose standard. 100 mg/100 ml in water. Prepare fresh. 4. Working xylose standards, 5, 10, 20, 30, 40, and 50 mg/100 ml Aqueous dilutions are prepared from the, stock. 5. Stock glucose standard, 1,000 mg/100 ml in water. Prepare fresh. 6. Working glucose standard, 300 mg/100 ml is prepared from the stock. 7. Ort~o-toluidine reagent, "Diagnostest". (Dow Diagnostics.) This consists of 12% W / V ortho-toluidine in 55% V/V acetic acid.

20

BRAIDWOOD and BINNS SMITH

Method

All urine samples are prediluted with 20 volumes of distilled water.

Table 1. Comparison of Manual Ultra-micro and Autoanalyser Xylose Methods for Serum.

(a) Xylose Assay

Dose of Xylose

0.1 ml sample (serum or diluted urine) and 0.5 ml glucose oxidase reagent are added to an Autoanalyser cup; the xylose standards and the 300 mg/100 ml glucose standard are treated similarly. After capping and incubating at 37 ° for three hours, the samples are assayed on the Autoanalyser using the o-toluidine reagent; the sampling rate used is 60/hour with a sample/wash ratio of 2:1 and colorimeter filter at 480 nm.

Method of Assay...

(b) Glucose Assay Glucose is assayed in the same way as xylose except that saline is substituted for glucose oxidase reagent, and a colorimeter filter of 660 nm wavelength is used. RESULTS 1. S E R U M B L A N K F a s t i n g .serum usually showed the presence of "xylose" whose measured c o n c e n t r a t i o n is usually low and c e r t a i n l y has never exceeded 1.5 m g / 1 0 0 ml serum. This "xylose" appears to be due to t u r b i d i t y of the l"eagent/serum m i x t u r e , and its value is deducted from the xylose c o n c e n t r a t i o n s assayed a f t e r the a d m i n i s t r a t i o n of xylose.

2. S P E C I F I C I T Y O F ASSAY (a) E f f e c t o f Glucc~e on Xylose A s s a y A d d i n g v a r y i n g q u a n t i t i e s of glucose to xylose cont a i n i n g s e r u m or u r i n e has shown t h a t c o n c e n t r a t i o n s of glucose up to 400 m g / 1 0 0 ml has no effect on the xylose c o n t e n t provided the glucose oxidase r e a g e n t and i n c u b a t i o n conditions are s a t i s f a c t o r y . (b) E f f e c t o f X y l o s e on G l u c o s e C o n t e n t '

Addition of xylose up to a c o n c e n t r a t i o n of 100 mg./100ml, had no influence on the glucose c o n t e n t of serum. F u r t h e r m o r e , u r i n e with a xylose load showed no detectable glucose. (c) P r e c i s i o n

R e p r o d u c i b i l i t y was assessed in two ways. (i) Paired Observations Sera collected a f t e r both 5 g and 25 g oral loads of D-xylose were assayed in duplicate and the 95% confidence limits for the m e a n were calculated u s i n g the tehniques of Snedecor with corrections for small n u m b e r s of o b s e r v a t i o n s as described by H e n r y ") . The results are shown in T a b l e 1. (ii) Long Term Stability Pools of serum and u r i n e c o n t a i n i n g no xylose were "spiked" with the compound, aliquoted and frozen. One aliquot of each assayed each week for e i g h t weeks: the means for the 8 o b s e r v a t i o n s were 52.9

Range of Observed Values Mean . . . . . . . . . . . . . S.D . . . . . . . . . . . . . . . . Coefficient of Variation Number of observat ions 95% Confidence Limits

5g

5g

25 g

1Vanual ultrarr icro

Autoanalyzer

Autoanalyser

4.4 - - 17.9 rag/100 n:l 7.4 mg/100 n:l 4- 1.57 mg/le0 rrl ± 21.2%

4.O I 16.0 mg/lO0 ml 8.8 mg/lO0 rrl ± 0.84 mg/lO0 rrl + 9.6%

14.5 - - 97.5 rag/100 n:l 48.6 rag/100 wl ± 1.27 rag/100 ml ± 2.6%

50

42

2'2

7.4 ± 3 . l

8.8 ± 1.7

48.6 ± 2.63

mg/100 ml and 1.30 g/5 hours with coefficients of v a r i a t i o n s of ± 2.9% and -- 2.3% for s e r u m and u r i n e respectively. (d) R e c o v e r i e s Xylose added to u r i n e with c o n c e n t r a t i o n s v a r y i n g between 0.1 and 1.5 g/100 ml showed recoveries v a r y i n g f r o m 97.4 to 102%. Ten d i f f e r e n t sera were "spiked" with xylose to a c o n c e n t r a t i o n of 10 mg/100 ml, an upper r a n g e conc e n t r a t i o n often seen a f t e r a 5 g. xylose oral dose; recoveries varied between 90 and 105%.

3. C O M P A R I S O N O F A U T O M A T E D W I T H ULTRA-MICRO MANUAL METHOD Samples of both s e r u m and u r i n e were obtained from normal donors who were given 5 g or 25 g xylose and each sample was assayed by both the A u t o a n a l y s e r technique and the e a r l i e r u l t r a - m i c r o method '') . The c o m p a r a t i v e results for both biological fluids from i n d i v i d u a l s given a 5 g or 25 g dose of xylose are shown in Table 2. I t will be noted that, in all instances, t h e r e was no s t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e between the results in all cases, u s i n g the P a i r e d S t u d e n t ' s "t" Test'~k Table 3 shows the r e g r e s s i o n equations and correlaticn coefficients r e l a t i n g the a u t o a n a l y s e r and m a n u a l methodg, for both 5 and 25 g doses of xylose in both s e r u m and urine. The correlation coefficient is close to 1.0 except in the case of s e r u m a f t e r a 5 g dose of xylose; t h i s is due to the i n f e r i o r precision shown by the m a n u a l u l t r a - m i c r o technique at very low c o n c e n t r a t i o n s .

DISCUSSION The adequate precision exhibited by the m a n u a l u l t r a - m i c r o t e c h n i q u e ' " when a s s a y i n g small n u m b e r s of samples u s i n g high oral doses of xylose (25 g) and the consequent relatively high s e r u m c o n c e n t r a t i o n achieved was not f o u n d when large n u m b e r s of samples with low s e r u m xylose c o n c e n t r a t i o n s needed analysis, u s i n g technologists u n f a m i l i a r with u l t r a micro techniques.

A S S A Y OF S E R U M XYLOSE

21,

Table 2. Statistical significance of difference between results from the manual ultra-micro and autoanalyser methods (using paired student's " t " test) i 1 both serum and urine after both 5 g and 25g doses of xylose. Oral Dose of Xylose

5g

Biological Fluid

Serum, Mg/100 MI

Range of Observations . . . . . . . . . . . . . . . . . . . . Means (Manual/AA) . . . . . . . . . . . . . . . . . . . . . . Degrees of Freedom . . . . . . . . . . . . . . . . . . . . . . . "t". .................................... P ......................................

2.0 - - 15.0 7.6/7.8 40 0.911 0.2 < P < 0.5

Table 3. Correlation between manual ultra-micro assay (y) and autoanalyser assay (x). All regression coefficients (b) are statistically significant (p 0.1 in all cases).

Dose of Xylose 5g.

25 g.

Regression Equation, Yffibx+a

Fluid Analysed

Correlation Coefficient

Serum

0.881

Y = 0.87X + 0.81

Urine

0.982

Y = 1.15X - 0.12

Serum

0.994

Y = 1.03X - 2.15

Urine

0.984 "

Y = 0.93X + 0.75

However, the a u t o a n a l y s e r system provided adequate precision and good c o r r e l a t i o n w i t h the m a n u a l method. The volume of blood used f o r the a u t o m a t e d method still r e m a i n s r e l a t i v e l y small and provides no serious problem to the patient. In the clinical use of xylose a b s o r p t i o n f o r the diagnosis of m a l a b s o r p t i o n syndrome, t h e r e would be much m e r i t in using a 5 g r a t h e r t h a n a 25 g dose of the compound since the f o r m e r avoids the f r e q u e n t a b d o m i n a l cramps, explosive d i a r r h o e a and a r t e f a c t u a l l y f l a t a b s o r p t i o n curves which are seen w i t h the latter'~L H o w e v e r , the a u t h o r s are not c u r r e n t l y a w a r e of any published data s h o w i n g n o r m a l responses to the 5 g xylose dose in serum, or w h e t h e r the m e a s u r e m e n t of s e r u m values p r o v i d e s a d e q u a t e resolution of n o r m a l i n d i v i d u a l s f r o m those w i t h m a l a b s o r p t i o n syndrome, a m a t t e r of considerable c o n t r o v e r s y even w i t h a 25 g xylose dose "~. T h u s u n d e r p r e s e n t circumstances, it is p r o b a b l y wise to assay the 5 h o u r u r i n a r y e x c r e t i o n of xylose w h e r e the lower dose is a d m i n i s t e r e d , since a d e q u a t e n o r m a l values are available f o r t h a t m e a s u r e m e n t ' 6 k N e v e r t h e l e s s , the aut h o r s feel t h a t t h e m e t h o d described is precise enough

25 g

Urine, G/5 Hours 0.45 - - 1.07 0.74/0.75 4 0.343 P > 0.5

Serum, Mg/100 MI 14.5 - - 84.0 35.3/36.3 14 1.919 0.1 > P > 0.05

Urine, G/5 Hours 3.6 - - 9.2 6.7/6.4 6 1.682 0.2 > P > 0.1

to allow a d e q u a t e s e r u m assay on the "low" dose and it would be of i n t e r e s t to obtain data f r o m a g r o u p of n o r m a l volunteers. Those w o r k e r s i n t e n d i n g to set up the a u t o a n a l y s e r method described here should take p a r t i c u l a r care to select the c o r r e c t m a n i f o l d t u b i n g ( F i g . 1). A l t h o u g h no data is presented here, this l a b o r a t o r y has found the use of the same m a n i f o l d f o r glucose a s s : y to b~ p e r f e c t l y s a t i s f a c t o r y . Fina!ly, the a u t h o r s would like to point out t h a t this c o m m u n i c a t i o n allows the o p p o r t u n i t y to correct a published e r r o r ' " w r o n g l y d e s c r i b i n g the o r i g i n a l m a n u a l u l t r a - m i c r o t e c h n i q u e ''~ as an automated method. ACKNOWLEDGMENTS The a u t h o r s wish to t h a n k the R i c h a r d and J e a n I v e y F u n d f o r a donation to aid the p u r c h a s e of equipment. Mrs. N i j o l a Gryn provided technical a s s i s t a n c e d u r i n g development. REFERENCES

1. Binns Smith, M. and Braidwood, J. L. (1971). Clin. Biochem., 4, pages 118-122. 2. Tevaarwerk, G. J. M., and Binns Smith, M. (1974). Unpublished observations. 3. "Automated Glucose Determination". Technical Data Sheet No. 21, Dow Diagnostics. 4. Henry, R. J., (1964). Clinical Chemist~T, Principles and Technics, Medical Division of Harper and Row, First Edition, page 124, New York, Hoeber. 5. Woolf, C. M. (1968). Principles o[ Biametry, page 70, Princeton, New Jersey, D. Van Nostrand Co. 6. Bouchier, I.A.D. (1969). Clinical Investigation of Gastro-intestinal FunctimL pages 86 - - 8~, Oxford and Edinburgh, Blackwell Scientific Publications. 7. Pileggi, V. J. and Szustkiewicz, C. P. (1974). Clinical Chemistry, Principles ~nd Technics, edited by R. J. Henry, D. C. Cannon and J. W. Winkelman, 2nd edition, page 1310, New York, Harper and Row. *

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A sensitive automated method for the determination of serum and urine xylose.

Clin. Biochem. 9 (1) 19-21 (1976) A Sensitive Automated Method for the Determination of Serum and Urine Xylose J. L. B R A I D W O O D a n d M. B I...
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