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Development and Validation of a Method for Simultaneous Separation and Quantification of 5 Different Sugars in Canine Urine Jorg M

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Development and Validation of a Method for Simultaneous Separation and Quantification of 5 Different Sugars in Canine Urine Jorg M . I ! Development and validation of a method for simultaneous separation and quantification of 5 different sugars 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, xylose, sucrose, and lactulose 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 arabinose 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 disaccharides, 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 sugar 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. monosaccharides (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 monosaccharide 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.
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