. I !

Development and validation of a method for simultaneous separation and quantification of 5 different in canine urine Jorg M. Steiner, David A. Williams, Erik M. Moeller Abstract

The objective of this project was to develop and validate a method for concurrent separation and quantification of methylglucose, rhamnose, , , and in canine urine by using high pressure anion exchange liquid chromatography and pulsed amperometric detection. The method was validated by evaluating dilutional parallelism, spiking recovery, intra-assay variability, and inter-assay variability. Observed to expected ratios for 3 urine samples, and all sugars, ranged from 77.6%7c to 106.9%7c for a 1:2 dilution, 85.2%7c to 121.4%7c for a 1:4 dilution, and 91.6%7c to 163.7%7c for a 1:8 dilution. Observed to expected ratios for spiking recovery of 3 urine samples, all sugars, and 5 different spiking solutions, ranged from 85.5% to 116.7 %7c (mean ± SD, 100.5 ± 6.0%). The intra-assay coefficients of variation were 1.6%7c, 3.4%7c, and 4.7%7c for methylglucose; 1.6%, 2.0%7c, and 3.6%7c for rhamnose; 2.7%7c, 1.4%, and 1.1% for xylose; 9.8%7c, 3.4%7c, and 4.0% for sucrose; and 3.2%7c, 3.3%, and 3.3%7c for lactulose. Inter-assay coefficients of variation were 3.2%7c, 5.7%7c, and 4.2%7c for methylglucose; 4.3%, 5.4%, and 6.4% for rhamnose; 3.3%7G, 5.0%7, and 4.2% for xylose; 9.4%, 9.9%/c, and 9.4%7c for sucrose; and 6.1%7c, 4.9%7c, and 2.7%7c for lactulose. In conclusion, a method for simultaneous separation and quantification of 5 sugars in canine urine was established and found to be linear, accurate, precise, and reproducible. This method may prove useful in the simultaneous evaluation of gastric permeability, small intestinal perme- ability, and small intestinal mucosal function in dogs with gastrointestinal disorders. Resume

Cette e'tude flit e'aliseie afin de detvelopper et de valider iu;c itne'thode permettant la sLparationi ct la qulanitification tidt et/u/liylgluicose, doi rham- niose, dot xylose, doi slucrose et doi lacttulose danis de l'trinie de clienl par onule mentltode de chromatographie d'e'chnange d'aniionis enz phtase liqilide soIus hlalute pressionl et onlle dttectioni ainpirotniitriqtue ptolsec. La tnethode ftot zvalidie ent k'7aloianit les resiiltats de diluitions ent paralec, le taiux de recou zvreiien t d 'clantillonis contaminie's, la zvariabilitc ijutra-e`prevze et in ter-`preuvz7e. Les ratios des resiultats obten uis zersuts les r'tsIltats attentdius potur 3 (chanttillotns d'iurinie, et polor tolis les stucres, zariaienit de 77,67c n 106,9% pooir utne diluitioni 1:2, de 85,2%7c n 121,4%7c polur oniie diluitioni 1:4, et de 91,6%, a' 163,7%7c potir oniie diluitiont de 1:8. Pouir totls les stucres, les ratios des resioltats obtenuiis zversuis les rtstul- tats atteniduis poll1r 3 eclaniitillonis d'lurinie conita,nints azvec 5 sollutionis cottanitinantes onit varie de 85,5% t4 116,7% (mtioyientne ± rcart-type: 100,5 ± 6,0%). Les coefficientts de zvariationl initra-jprectze itaienit de 1,6%7c, 3,4%7c et 4,7%7c pooir le mtnthylgltocose; de 1,6%c, 2,0% et 3,6%7c polur le rhamn11ose; 2,7%7c, 1,4%G et 1,1%7c polor le xylose; 9,8%7c, 3,4%c et 4,0% pouir le suicrose; et 3,2%c, 3,3% et 3,3% pouIr le lacttolose. Les coefficienit de variationl initer-&prelovie ttaient de 3,2%c, 5,7% et 4,2% pooir le inttlylgltucose; 4,3%, 5,4% et 6,4% pooar le rhamn1nose; 3,3%, 5,0% et 4,2% poolr le xylose; 9,4%, 9,9% et 9,4% pooir le suicrose; et 6,1%, 4,9% et 2,7%7c potor le lacttolose. Une mentltode polur la st'para- tionl et la qluanitificationi si,ntultantec de cinlq slucres danis l'torinie de c/Penl ftot mise ati pointt et s'est averee linieaire, precise, exacte et repro- dolctible. Cette ;ncthlode poiorrait s'avz7rer lutile polur lvz7altuationi simtultanie' de la permnabilit6 gastriqtue, de la perinmabilite' do petit initestint et de la foniction tie la ;nitoqlueiose dot petit initestini chez des cIliens avec des de'sordres gastro-inltestitnalux. (Tratdl(it p7ar tioctciu Scrgc MeAssier)

of a wide range of relatively small molecules can traverse the intes- tinal mucosa. The integrity of the barrier function of the gastroin-

The intestinal mucosa serves as a route of entry for essential testinal tract has been evaluated by permeability testing in human nutrients into the body. It also serves as a barrier against uncon- beings; laboratory animals, such as rabbits and rats; and in cats and trolled entry of molecules that are potentially harmful to the body dogs (2-6). In 1930, McCane and Madders were the first to evaluate (1). In normal animals, the barrier is not complete and trace amounts intestinal permeability using rhamnose, xylose, and as

Gastrointestinal Laboratory, Department of Small Animal Medicine and Surgery, College of Veterinary Medicine, Texas A&M University, College Station, Texas 77843-4474 USA. Address correspondence and reprint requests to Dr. Jorg M. Steiner, telephone: 979-862-4046; fax: 979-458-4015; e-mail: jsteiner@cmm. tamu.ed u. This material was preseinted as a research abstract at the 1999 ACVIM Forum in Chicago, Illinois, USA. Received December 3, 1999. Accepted April 18, 2000.

164 iv

+5mV

Figure 1. Voltammogram. This figure shows the plot of a voltammogram using a mobile phase of 167 mM NaOH at the flow-cell level. The potential sweeps from +600 mV to -600 mV, and back to +600 mV. One complete sweep Is shown In this figure. Note the transient decrease in current at approximately +5 mV.

marker molecules (2). Since then, the use of many different marker Currently, gastrointestinal permeability and function testing is not molecules, including 51Cr-EDTA, polyethylene glycol, and mono- and being routinely performed in small animal practice, but is mostly , has been evaluated, and well over 1200 reports on this reserved for research settings. This may be because commercial topic have been published (4,7-10). veterinary laboratories currently do not offer analysis of In general, the gastrointestinal mucosa is believed to have 2 types concentrations in urine samples. A further limitation is that of aqueous pores that allow non-carrier-mediated uptake of small most investigators have used different testing protocols, including molecules. The smaller pores are hypothesized to be located within different methodologies for the quantification of the marker the cell walls and are believed to have a maximum radius of molecules in urine samples, making it difficult for the clinician approximately 0.4 nm, thereby allowing permeation by small mol- to choose from the many different protocols published in the ecules (approximate molecular mass (MM) 150-185 Da), such as literature. (2,11). The overall frequency of these transcellu- The long-term goal of this research is to establish a standardized lar pores is believed to be quite high and mostly dependent on gastrointestinal permeability and function testing protocol that the total surface area of the intestinal mucosa. Small intestinal dis- concurrently evaluates gastric and intestinal permeability and ease is often associated with a decrease in this surface area, leading intestinal absorptive function, and is available to veterinary clini- to a decrease in intestinal permeability to markers cians regardless of the location of their practice. A first step is to (12). The larger pores, with a maximum radius ranging from establish and validate a method for concurrent separation and approximately 0.5 nm to 0.8 nm, allow permeation of larger mole- quantification of 5 different sugars: 3-o-methyl-D-glucopyranose cules, such as 51Cr-EDTA and disaccharides (approximate (methylglucose), L-rhamnose (rhamnose), D(+)xylose (xylose), MM 340 Da). These pores are believed to be located paracellularly sucrose, and lactulose. It should be pointed out that the concurrent in the area of the tight junctions (2,12,13). The overall frequency of separation of several different sugars, including the ones used in this these pores is much lower and is largely dependent on mucosal protocol, has been reported using methods similar to those used here integrity (10,13). In many small intestinal disorders, tight junc- (4). The validation of a protocol for the concurrent quantification of tions become "leaky," leading to increased permeation of disac- methylglucose, rhamnose, xylose, and lactulose, but not sucrose, in charide markers (12). There is, as yet, no direct evidence to support canine urine has also been reported (4). However, this is the first the existence of these different types of pores. Other theories have report about the complete validation of a protocol for the concurrent been suggested to explain clinical observations of differential per- quantification of all 5 sugars in canine urine. meability to molecules of different sizes. Recently, sucrose has been used as a specific marker molecule to evaluate the permeability of the gastric mucosa (5,14-16). The uri- nary recovery of other monosaccharides, such as methylglucose and The sugars were separated using an NaOH gradient on a metal-free xylose, that are transported across the intestinal mucosa by spe- high-pressure liquid chromatography system (Autosampler 717+ cialized carriers, has also been used to evaluate small intestinal and pump 625; Waters Corporation, Milford, Massachusetts, USA) absorptive capacity, thereby concurrently evaluating another aspect at a flow rate of 1 mL/min. Three different concentrations of NaOH of small intestinal mucosal function (4,17-19). were used to create the NaOH gradient and were kept in containers

The Canadian Journal of Veterinary Research 165 0.2

0.16

z 0.12 0 z 0.08

0.04- MR / XS L |

0.0 1 10I 15 I1 I I

0 5 10 15 20 25 30 35 40 45 minutes Figure 2. NaOH gradient and approximate elution times. This figure shows the flnal NaOH gradient used to resolve the 5 sugars. It also shows the approx- imate elution times of the 5 sugars (M - methylglucose; R - rhamnose; X - xylose; S - sucrose; L- lactulose).

(ultra-ware HPLC-reservoir; Kontes Glass Company, Vineland, Standard curves for the 5 sugars were prepared by using standards New Jersey, USA) under a helium cap. An anion exchange col- of 100 mg/L, 50 mg/L, 25 mg/L, 12.5 mg/L, and 6.25 mg/L for umn (Carbopac PA10 analytical and guard columns; Dionex methylglucose, rhamnose, xylose, and sucrose, respectively, and Corporation, Sunnyvale, California, USA) was used for separa- 50 mg/L, 25 mg/L, 12.5 mg/L, 6.25 mg/L, and 3.125 mg/L for tion of the sugars. A post-column pump (Duros CC-30-S-PK; Eldex lactulose, using the area under the curve of the peaks in the chro- Laboratories, Napa, California, USA), adding 0.5 M NaOH at a matogram and a linear curve fit (Millenium 32 chromatography man- flow rate of 0.5 mL/min, was used to narrow the NaOH gradient at ager; Waters Corporation). the detector level. Sugars were quantified by pulsed amperometric Urine samples, used for validation, were collected from dogs after detection (PAD) (Electrochemical detector 464; Waters Corporation). being enrolled in different gastrointestinal permeability studies First, the approximate optimal settings for the PAD detector were and mixed to result in different concentrations of the 5 sugars. All determined by voltammograms sweeping from +600 mV to urine samples were filtered through a 0.4 p,m pore size syringe fil- -600 mV and back to +600 mV, using a mixture of mobile phases ter and diluted 1:100 with filtered deionized water containing 0.1 g/L that would lead to NaOH concentrations of 167 mM and 183 mM at NaN3. the flow-cell level. The voltammograms were prepared by run- The linearity of the assay was determined by evaluating dilutional ning a constant mobile phase through the flow cell and applying a parallelism of 3 different urine samples measured neat and diluted constantly changing potential across the working electrode. The 1:2, 1:4, and 1:8 in filtered deionized water containing 0.1 g/L resulting current was charted on a plotter and the optimal setting for NaN3. Results were expressed as observed to expected (O/E) ratios. El was set at the potential that led to a small decrease in current as Accuracy of the assay was determined by evaluation of spiking the potential swept back from -600 mV to +600 mV (20). Values for recovery of 3 different urine samples with solutions containing E2 and E3 were calculated by using E2 = E1 + 600 mV and E3 = El 50 mg/L each of methylglucose, rhamnose, xylose, and sucrose, and - 600 mV. 25 mg/L of lactulose or dilutions of this solution of 1:2, 1:4, 1:8, and An NaOH-gradient was established that would allow sepa- 1:16. Results were also expressed as O/E ratios. ration of a sugar solution containing 100 mg/L 3-o-methyl-D- The precision of the assay was determined by evaluating intra- glucopyranose (M-4879, Sigma Chemical Company, St. Louis, assay variability by measuring 3 different urine samples 10 times Missouri, USA), L-rhamnose (R-3875, Sigma Chemical Company), within the same assay run and calculating coefficients of varia- D(+)xylose (X-1500, Sigma Chemical Company), sucrose (S-7903, tion for all 5 sugars and all 3 samples. Sigma Chemical Company), and lactulose (L-7877, Sigma Chemical Finally, the reproducibility of the assay was determined by eval- Company) in deionized water. uating inter-assay variability by measuring 3 different urine samples

166 The Canadian Journal of Veterinary Research auto-scaled chromatogram

N on 800.00- rn 0 CIIA C) N N C c .c .,6 c --4 N X 600.00- (U Iu t'l cr (U cr, Lr 0 .1, r-0 C) a 0SC; a- :un E 4 0 ct X 400.00 b-0 E IE u 200.00- E

-OIlk C}.- A - 1111 I T1111' lI ,I I I I I II I I, I I I I I I I,I I I II I I 0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00

Figure 3. Typical chromatogram. This figure shows a typical chromatogram for separation of a standard containing 50 mg/L methylglucose, rhamnose, xylose, and sucrose, and 25 mg/L lactulose.

in 10 consecutive assay runs and calculating coefficients of variation Table I. Dilutional parallelism of 3 different urine samples 5 sugars and 3 for all all samples. Dilution O/E ratios (%) 1:2 1:4 1:8 minimum 77.6 85.2 91.6 maximum 106.9 121.4 163.7 A voltammogram is shown in Figure 1. This representative meana 94.0 ± 8.5 100.0 ± 9.5 108.1 ± 17.8 voltammogram was recorded using a mobile phase of 167 mM a Data in this row are expressed as mean ± standard deviation NaOH. The voltammogram shows a transient decrease in current at These data are the O/E ratios for all 5 sugars and all 3 urine +5 mV. A similar voltammogram was performed approximately samples (total data points: 15) using a mobile phase of 183 mM NaOH and the decrease of current was noted at approximately +26 mV. Therefore, El was set between these 2 values at +10 mV, with E2 at +610 mV, and E3 at -590 mV. Table II. Observed to expected ratios for spiking recovery of 3 urine An optimal NaOH gradient for separation of the 5 sugars was samples, 5 sugars, and 5 spiking solutions ranged from 85.5% to established. The gradient was mixed using 3 mobile phases. Mobile 116.7 % (mean ± SD: 100.5 ± 6.0%). The mean intra-assay coefficient phase A was 100% filtered and deionized water, mobile phase B was of variation for intra-assay variability of 5 sugars in 3 samples 20 mM NaOH, and mobile phase C was 500 mM NaOH, both in fil- was 3.0% (SD 1.3%; Table III). Finally, the mean coefficient of vari- tered, deionized water. The final gradient ran as follows: 0-15 min, ation for inter-assay variability of 5 sugars in 3 samples was 5.6% 0.004 M NaOH (80% A, 20% B); 15-16 min, continuously increasing (SD 2.3%; Table IV). to 0.02 M NaOH (100% B); 16-17.5 min, continuously increasing to 0.078 M NaOH (88% B, 12% C); 17.5-24 min, 0.078 M NaOH; 24-27 min, continuously increasing to 0.198 M NaOH (63% B, 37% C); 27-37 min, 0.198 M NaOH; 37-39 min, continuously decreasing The use of a mixture of monosaccharides and disaccharides for to 0.02 M NaOH; 39-42 min, continuously decreasing to 0.004 M intestinal permeability and mucosal function testing in the dog NaOH; and 42-45 min, 0.004 M NaOH (Figure 2). Approximate elu- has been reported previously (4). However, the addition of a specific tion times were 14.5 min for methylglucose, 15.8 min for rhamnose, marker molecule for gastric permeability testing has not been 22.7 min for xylose, 23.8 min for sucrose, and 30.1 min for lactulose reported. (Figure 2). Figure 3 shows a typical chromatogram for separation of Different methods, such as anion exchange, cation exchange, or a standard solution containing 50 mg/L each for methylglucose, thin layer chromatography have all been used to separate mono- and rhamnose, xylose, and sucrose, and 25 mg/L for lactulose. disaccharides (11,18,21). We chose anion exchange chromatography Table I shows the results for dilutional parallelism of 3 different because of its relative simplicity compared to thin layer chro- urine samples. Results for the spiking recoveries are shown in matography and because it had been used successfully by other

167 Table I. Spiking recovery of 3 different urine samples O/E ratios Sugar addeda (%) 50b (25)c mg/L 25 (12.5) mg/L 12.5 (6.25) mg/L 6.25 (3.13) mg/L 3.13 (1.56) mg/L minimum 87.8 85.5 89.3 89.7 96.6 maximum 116.1 104.0 111.3 110.0 116.7 meand 102.3 ± 6.4 97.5 ± 5.7 99.9 ± 5.8 99.6 ± 5.5 103.0 ± 5.5 a methylglucose (M), rhamnose (R), xylose (X), sucrose (S), lactulose (L) b this first number represents the amount of M, R, X, and S added in mg/L c the number in parentheses indicates the amount of L added in mg/L d data in this row are expressed as mean ± standard deviation These data are the O/E ratios for all 5 sugars and all 3 urine samples (total data points: 15)

Table Ill. lntra*assay variability of the concentration of 5 sugars in 3 urine samples repeated 10 times within the same run Sample 1 Sample 2 Sample 3 Sugar meana (mg/L) CVb(%) mean (mg/L) CV(%) mean (mg/L) CV(%) methylglucose 38.5 1.7 73.4 3.4 84.5 4.7 rhamnose 11.4 3.3 59.2 2.0 87.5 3.6 xylose 49.5 0.8 75.0 1.4 110.7 1.1 sucrose 30.4 4.7 81.6 3.4 33.4 4.0 lactulose 6.1 4.4 29.6 3.3 12.5 3.3 a mean concentration of sugar in that urine sample from the 10 measurements within that same run b the coefficients of variation for each sugar (CV = standard deviation/mean)

Table IV. Inter-assay variability of the concentration of 5 sugars in 3 urine samples repeated 10 times in consecutive assay runs Sample 1 Sample 2 Sample 3 Sugars meana (mg/L) CVb (%) mean (mg/L) CV (%) mean (mg/L) CV (%) methylglucose 39.2 3.2 56.5 5.7 35.7 4.2 rhamnose 20.5 4.3 49.7 5.4 10.3 6.4 xylose 70.5 3.3 64.3 5.0 46.9 4.2 sucrose 30.7 9.4 73.5 9.9 25.6 9.4 lactulose 8.3 6.1 26.7 4.9 6.1 2.7 a mean concentration of sugar in that urine sample from the 10 consecutive assay runs b the coefficients of variation for each sugar (CV = standard deviation/mean) investigators (4,11). Most simple , such as mono- tine analysis (22). Some investigators have used refractive index and disaccharides, have a pK between 12 and 14. While these mol- detection for sugar analysis in urine samples, but this method is not ecules are neutral at any pH below the pK, they lose hydrogen very sensitive and is used mostly for sugar analysis in food (23,24). ions and become negatively charged at a higher pH. Therefore, in Colorimetric assays, if available at all, are labor intensive, because order to be able to separate carbohydrates by anion exchange chro- each sugar has to be analyzed by using a separate assay, and are, matography, the mobile phase must be basic. At the same time, the therefore, not useful for routine analysis (3,25,26). Pulsed amper- hydroxyl groups of NaOH carry a negative charge and compete with ometric detection was chosen because it is very sensitive, easy to per- the sugar anions for the positively charged groups on the anion form, and economically feasible (4,27-29). Detection is based on the exchange beads. When a low concentration of NaOH, such as 4 mM, redox potential of the sugars to be analyzed. A potential across the is chosen for the mobile phase, the sugars can be separated. flow cell is established between the gold working electrode and the However, the separation of all 5 sugars takes several hours, making reference electrode. If oxidizable molecules flow through the cell, the such an approach impractical for routine analysis. On the other hand, electrons lost from that molecule lead to the flow of a current. when a high concentration of NaOH, such as 200 mM, is chosen the This current is amplified and analyzed by use of a computer software total run time decreases, but the sugars can no longer be resolved. package. However, carbohydrates are not the only molecules that Therefore, a NaOH gradient is needed to optimally resolve the can be oxidized. Sodium hydroxide can also be oxidized; therefore, 5 sugars. the gradient necessary to separate the sugars causes large shifts in Many methods for quantification of the sugars have been the base line. In order to decrease these base line shifts, post-column described. Mass spectrometry is a highly sensitive and reliable addition of a highly concentrated NaOH was used. So, the range of method, but is very expensive and, therefore, not suitable for rou- NaOH concentrations over the run of the gradient at the detector

168 The Canadian Journal of Veterinary Research level was changed from a range of 4 to 198 mM (equating to an (4,11,27,28). Use of this column necessitated the establishment of a almost 50-fold increase in NaOH concentration at the detector specific protocol for PAD detection, the use of 3 rather than 2 mobile level without post-column addition) to a range of 169 (4 mM NaOH phases, and the establishment of a more complex gradient at 1 mL/min plus 500 mM at 0.5 mL/min) to 299 mM (198 mM (4,11,27,28). It is hoped that this work will ultimately lead to the NaOH at 1 mL/min plus 500 mM at 0.5 mL/min), equating to a less adoption of a uniform protocol used for gastrointestinal permeability than 2-fold increase in NaOH concentration after post-column and mucosal function testing in dogs. addition. Dilutional parallelism for the 5 sugars in 3 different urine samples showed O/E ratios between 77.6% and 106.9% for a 1:2 dilution, 85.2% and 121.4% for a 1:4 dilution, and 91.6% and 163.7% for a 1. Hollander D. The intestinal permeability barrier. A hypothesis 1:8 dilution. The O/E ratios for the 1:2 and the 1:4 dilutions suggest as to its regulation and involvement in Crohn's disease. Scand sufficient linearity of the assay for clinical use. For the 1:8 dilution, J Gastroenterol 1992;27:721-726. the 163.7% is an outlier caused by the low expected concentration of 2. Travis S, Menzies I. Intestinal permeability: Functional assess- lactulose (3.6 mg/L). It is not surprising that the assay loses linearity ment and significance. Clin Sci 1992;82:471-488. in the extreme areas of the standard curves. The next highest O/E 3. Hall EJ, Batt RM. Differential sugar absorption for the assessment ratio for the 1:8 dilution was 112.2%, which is acceptable. Observed of canine intestinal permeability: the /mannitol test to expected ratios for all 5 sugars, all 5 spiking solutions added, and in gluten-sensitive enteropathy of Irish setters. Res Vet Sci all 3 urine samples ranged between 85.5% and 116.7% (mean ± 1991;51:83-87. SD, 100.5 ± 6.0%, 75 data points). These results would suggest that 4. Sorensen SH, Proud FJ, Adam A, Rutgers HC, Batt RM. A the assay is sufficiently accurate for clinical use. Coefficients of novel HPLC method for the simultaneous quantification of variation for intra-assay variability for all 5 sugars and all 3 urine monosaccharides and disaccharides used in tests of intestinal samples were between 0.8% and 4.7%. Those values would suggest function and permeability. Clin Chim Acta 1993;221:115-125. that the assay is sufficiently precise for clinical use. Finally, coeffi- 5. Meddings JB, Gibbons I. Discrimination of site-specific cients of variation for inter-assay variability for methylglucose, alterations in gastrointestinal permeability in the rat. rhamnose, xylose, and lactulose for all 3 urine samples were Gastroenterology 1998;114:83-92. would suggest that the assay between 2.7% and 6.4%. These values 6. Papasouliotis K, Gruffydd-Jones TJ, Sparkes AH, Cripps PJ, of variation is sufficiently reproducible for clinical use. Coefficients Millard WG. Lactulose and mannitol as probe markers for in for inter-assay variability for sucrose were slightly higher at 9.4%, vivo assessment of passive intestinal permeability in healthy cats. 9.9%, and 9.4% for the 3 urine samples, respectively. However, Am J Vet Res 1993;54:840-844. these values would still allow the conclusion that the reproducibility 7. Van Nieuwenhoven MA, Geerling BJ, Deutz NEP, Brouns F, of this assay for quantification of sucrose in canine urine is sufficient Brummer RJM. The sensitivity of the lactulose/rhamnose gut for clinical use. test. Eur J Clin Invest 1999;29:160-165. In summary, a protocol for the simultaneous separation and permeability Klein PD. quantification of methylglucose, rhamnose, xylose, sucrose, and lac- 8. Irving CS, Lifschitz CH, Marks LM, Buford LN, as tulose in canine urine by high pressure anion exchange liquid Polyethylene glycol polymers of low molecular weight chromatography and pulsed amperometric detection was described. probes of intestinal permeability. I. Innovations in analysis The method is sufficiently linear, accurate, precise, and repro- and quantitation. J Lab Clin Med 1986;107:290-298. ducible for clinical use. Further studies are needed to describe the 9. Marks SL, Williams DA. Time course of gastrointestinal tract per- kinetics of urinary excretion of these 5 sugars after oral adminis- meability to chromium 51-labeled ethylenediaminetetraacetate tration in order to select the optimal sampling time of urine samples, in healthy dogs. Am J Vet Res 1998;59:1113-1115. to evaluate the influence of the different marker molecules on the 10. Hall EJ, Batt RM, Brown A. Assessment of canine intestinal urinary recovery of concurrently used markers, to describe a con- permeability, using 51Cr-labeled ethylenediaminetetraacetate. trol range of this intestinal permeability and mucosal function Am J Vet Res 1989;50:2069-2074. testing protocol described, and to evaluate changes of intestinal per- 11. Kynaston JA, Fleming SC, Laker MF, Pearson AD. Simultaneous meability and mucosal function in dogs with different gastroin- quantification of mannitol, 3-O-methylglucose, and lactulose in testinal and systemic disorders. All of these studies are currently urine by HPLC with pulsed electrochemical detection, for use underway. in studies of intestinal permeability. Clin Chem 1993;39:453-456. As indicated before, both ion exchange high pressure liquid 12. Hall EJ. Clinical laboratory evaluation of small intestinal func- chromatography and pulsed amperometric detection have been tion. Vet Clin North Am Small Anim Pract 1999;29:441-469. used by other investigators to quantify various mono- and disac- 13. Maxton DG, Bjarnason I, Reyonlds AP, Catt SD, Peters TJ, charide markers in urine samples (4,11,27,28). However, this is Menzies IS. Lactulose 51Cr-labeled ethylenediaminetetraacetate, the first report about validation of a protocol to simultaneously quan- L-rhamnose and polyethyleneglycol 400 as probe markers for tify methylglucose, rhamnose, xylose, sucrose, and lactulose in assessment in vivo of human intestinal permeability. Clin Sci urine from any species. We chose to use different equipment than 1986;71 :71-80. had been described by other investigators (4,11,27,28). We also 14. Meddings JB, Kirk D, Olson ME. Non-invasive detection of chose a different column, in order to consistently resolve sucrose canine NSAID-gastropathy. Am J Vet Res 1995;56:977-981.

The Canadian Journal of Veterinary Research 169 15. Meddings JB, Sutherland LR, Byles NI, Wallace JL. Sucrose: A variability of markers and marker ratios in healthy subjects. novel permeability marker for gastroduodenal disease. Scand J Gastroenterol 1993;28:274-280. Gastroenterology 1993;104:1619-1626. 23. Miki K, Butler R, Moore D, Davidson G. Rapid and simultane- 16. Sutherland LR, Verhoef M, Wallace JL, Van Rosendaal G, ous quantification of rhamnose, mannitol, and lactulose in Crutcher R, Meddings JB. A simple, non-invasive marker of gas- urine by HPLC for estimating intestinal permeability in pediatric tric damage: sucrose permeability. Lancet 1994;343:998-1000. practice. Clin Chem 1996;42:71-75. 17. Heyman M, Desjeux JF, Grasset E, Dumontier AM, Lestradet H. 24. Quigg J, Brydon G, Ferguson A, Simpson J. Evaluation of canine Relationship between transport of D-xylose and other mono- small intestinal permeability using the lactulose/rhamnose saccharides in jejunal mucosa of children. Gastroenterology urinary excretion test. Res Vet Sci 1993;55:326-332. 1981;80:758-762. 25. Wood AJJ. Abnormal intestinal permeability: an aetiological fac- 18. Jenkins AP, Menzies IS, Nukajam WS, Creamer B. The effect of tor in chronic psychiatric disorder? Br J Psychiatry 1987;150: ingested lactulose on absorption of L-rhamnose, D-xylose, and 853-856. 3-o-methyl-D- in subjects with ileostomies. Scand 26. Deitch EA. Intestinal permeability is increased in burn patients J Gastroenterol 1994;29:820-825. shortly after injury. Surgery 1990;107(4):411-416. 19. Cherbut C, Meirieu 0, Ruckebusch Y. Effect of diet on intestinal 27. Fleming SC, Kapembwa MS, Kaker MF, Levin GE, Griffin GE. xylose absorption in dogs. Dig Dis Sci 1986;31(4):385-391. Rapid and simultaneous determination of lactulose and mannitol 20. Andrews RW, King RM. Selection of potentials for pulsed in urine, by HPLC with pulsed amperometric detection, for amperometric detection of carbohydrates at gold electrodes. Anal use in studies of intestinal permeability. Clin Chem 1990;36: Chem 1990;52:2130-2134. 797-799. 21. Willems D, Cadranel S, Jacobs W. Measurement of urinary 28. Sanghi SK, Kok WT, Koomen GCM, Hoek FJ. Determination of sugars by HPLC in the estimation of intestinal permeability: inert sugars in urine by liquid chromatography with pulsed Evaluation in pediatric clinical practice. Clin Chem 1993;39: amperometric detection. Anal Chim Acta 1993;273:443-447. 888-890. 29. Morris TH, Sorensen SH, Turkington J, Batt RM. Diarrhoea 22. Blomquist L, Bark T, Hedenborg G, Svenberg T, Norman A. and increased intestinal permeability in laboratory beagles Comparison between the lactulose/mannitol and 51Cr- associated with proximal small intestinal bacterial overgrowth. ethylenediaminetetraacetic acid/14C-mannitol methods for Lab Anim 1994;28:313-319. intestinal permeability. Frequency distribution pattern and

170 The Canadian Journal of Veterinary Research