RELIABILITY AND ACCURACY OF DETERMINING MINERALS AND ELECTROLYTES IN GOAT URINE USING A DRIED FILTER PAPER METHOD P. J. D. C. Bagasse ©© UUnniivveerrssiittyy ooff PPrreettoorriiaa Paulo Bagasse - MSc. Dissertation of DFPM RELIABILITY AND ACCURACY OF DETERMINING lVlINERALS AND ELECTROLYTES IN GOAT URINE USING A DRIED FILTER PAPER METHOD Submitted in partial fulfilment of the requirements for the degree MSc (Master of Science) in the Department of Companion Animal Medicine, Faculty of Veterinary Science, University of Pretoria Paulo Bagasse - MSc. Dissertation of DFPM Summary .... Samevattillg .. Acknowledgements . XIII Index of Tables . xv Index of Figures . Index of Tables in Appendix . XXIII Glossary of Abbreviations . XXVI l. BACKGROUND . 2. LITERA1'LJRE REVIE"V... . '" . 2.2. Minerals/Electrolytes Fractional Excretion " . 2.3. Analytes . 2.3.1. Creatinine . 2.3.1.1. Metabolism and function '" '" '" '" . 2.3.1.2. Sample stability . 2.3.1.3. Analytical method '" '" '" '" '" . 2.3.2. Calcium . 2.3.2.1. Metabolism and function . 2.3.2.2. Calcium imbalance . 2.3.2.3. Analytical method . 2.3.3. Inorganic phosphorus/phosphate . 2.3.3. 1.Metabolism and function . 2.3.3.2. Phosphorus imbalance . 2.3.3.3. Analytical method . 2.3.4. Magnesium . 2.3.4.1. Metabolism and function . 2.3.4.2. Magnesium imbalance '" '" '" .., . 2.3.4.3. Analytical method . Paulo Bagasse - MSc. Dissertation of DFPM 2.3.5.2. Chloride imbalance . 2 3.5.3. Analytical method . 2.3.6 Sodium and Potassium . 2.3.6. 1. Metabolism and function . 2.3.6.2. Sodium and Potassium imbalance ,. 2.3.6.3. Analytical method . 2.4. Filter Paper Method '" '" ." '" ,. 2.5. Assessing The Minerals Using a Diluent.. . 7. MATERIALS AND METHODS '" . 7.1. Procedures Common To Both Phases . 7.l.l. Solutions, Materials, and Formulae '" '" , . a) Artificial c) Calculations Formulas . d) Filter Paper . e) Procedures in General . 7.1.2. Analytical Methods " " . 7.1.3. Statistical Methods '" '" . 7.2. Experimental Design and Model System.............................................. 45 7.2.1. PHASE I (in vitro) '" 46 7.2.1. 1. Testing different types of filter paper...... 46 7.2\.\ I. ModeL................................... 46 72. I. 1.2. Experimental Design and Procedures... . . 46 72.1 I 3. StatisticaIMethod.................................... 47 7.2.1.2. Testing the diluent " .'" '" .... 47 7.2.1.2.1. ModeL........................................................................ 47 7.2. 1.2.2. Experimental Design and Procedures. .................................... 47 7.2.1.2.3. Statistical Methods ,'" ,.................. 48 7.2.1.3. Detel'luination of the reproducibility of the method , '" .. 48 7.2.1.3.1. Model , '" '" ,.. ,. 48 7.2. I.3.2. Experimental Design and Procedures. .................................. 48 7.2.1.33. Statistical Methods '" 49 7.2.1.4. Testing the effect of temperature and storage time on the method......... 49 7.2. I -\..I. iVlodeL........................................................................ 50 7.2. IA.2. Experimental Design and Procedures 1... ................................ 50 7.2. 1.4.3. Experimental Design and Procedures 2. ............................. 51 7.2. 1.4.4 . Statistical Methods. ............................................... .......... 51 7.2.1.5. Testing the effect of different volumes of impregnation and different urine dilutions on the method '" '" '" .,. 51 7.2.1.5.1. Model......................................................................... 52 7.2.1.5.2. Experimental Design and Procedures................................. .... 52 7.2.1.5.3. Statistical Methods.......................................................... 53 7.2.2. PHASE II (in vitro and in vivo)... 53 7.2.2.1. Testing the effect of differences in water intake on mineral/electrolyte to creatinine ratio in goat urine by using the method......................... ... 53 7.2.2.1.1. Model......................................................................... 53 7.2.2.1.2. Experimental Design and Procedures.................................. 54 7.2.2.1.3. Statistical Methods '" '" '" 55 8. RESULTS . 8.1. PHASE I . 8. I.I. Testing different types of filter paper. . 8.1.2. Testing the diluent . 8. 1.3. Determination of the reproducibility of the method . 8 14 Testing the eflect of temperature and storage time on the method . 8.\.5 Testing the eflect of different volumes of impregnation and diflerent urine dilutions on the method........... .. . 8.2. TABLES . 8.3 FIGU RES . 8.4.TAB LES . 8.5. FIGURES " '" , . 8.6. TAB LES . 8.7.\. Testing the eflect of different water intake on mineral/electrolyte to creatinine ratio in goat urine by using the method , , 84 8.8. TAB LES ............................................................................. 86 9. DISCUSSiON................................................................................. 92 9. I. Testing different type of filter paper. ............................................. 92 9.2. Testing the diluent. ................................................................. 93 9.3. Determination of the reproducibility of the method............................ 95 9.4. Testing the effect of temperature and storage time on the method.. ......... 96 9.5. Testing the effect of different volumes of impregnation and different urine dilutions on the method........................... 97 9.6. Testing the effect of different water intake on mineral/electrolyte to creatinine ratio in goat urine by using the method............................ 98 10.CONCLUSiON.............................................................................. 100 II. APPEN 0IX. ....................... ...................................... ........... 101 12.REFERENCES '" .., '" '" '" '" '" 115 Paulo Bagasse - MSc. Dissertation of DFPM The lack of facilities for veterinary services, such as analytical laboratories, which are distant from the Held and at immense distances are aggravatted by the constraint of transporting and preserving the samples. A method where a certain amount of the urine sample is absorbed onto filter paper, but dried in the field and then sent to the laboratory (Dried Filter Paper Method, DFPM), has been used in human medicine, but never applied in the veterinary field. The practice of expressing various urinary minerals/electrolytes relative to the concentration of creatinine has recently become generally accepted. A single sample ob"iatt:s the need for collection of urine over a 24-hour period. Fractional excretion (FE) of minerals and electrolytes (which is the mineral or electrolyte to creatinine ratio), is a simple, ine"pensive measurement and a reasonable indicator of the renal clearance of minerals/electrolytes, using a single urine sample. The investigation was divided into two phases, The first was conducted entirely in vitro, using simulated urine (artificial and goat urine) dried on filter paper, manipulated in various ways under laboratory conditions, and the second phase was conducted in vivo and in \'itro, using urine (collected from water-deprived goats) dried on filter paper and manipulated invarious ways under laboratory conditions. Determination of mineral/electrolyte to creatinine ratios using the dried filter paper method consisted of Impregnating a specific filter paper with a specific volume of artificial/goat urine, diluted in a specific eluent, and then analysed for the analytes (creatinine, phosphate, chloride, magnesium, calcium, sodium, and potassium). Due to the wide range of filter papers, which could have been used for the trial, it was necessary to run an experiment to identify the best for the purpose. The experiment used tilter papers from :2 different brands (Whatman® and Scheicher & Schuel filter papers). The following filter paper were compared: Whatman® no 2, 3, 5, 6, 42, 43, 44, and 542 and 860, 593, 59). 597, 598 for Schleicher & Schuell (very high quality). There were few Paulo Bagasse - MSc. Dissertation of DFPM significant differences. Whatman filter paper number 6, was chosen, because of the constant and uniform mineral/electrolyte to creatinine ratios. The very high variability of urine mineral/electrolyte concentrations in ruminants, and the limited linearity range of routine analytical equipment, constrains the routine use of laboratory analysers in urine mineral and electrolyte assessment. One of the approaches is to use a diluent, with a particular mineral/electrolyte concentration near to the lower end of the linearlstandardised range. Therefore, "mixing" a small volume of urine with the diluent will result in a final mineral/electrolyte concentration, that falls into a linear and controlled/standard range for the particular mineral and electrolyte. A diluent was tested and the results for analytes show a high interclass correlation (R >0.75) between the 1 expected and the calculated values of this ratio. The stability of mineral/electrolyte to creatinine ratio (B/Cr) in artificial and goat urine specimens using the dried filter paper method stored at 2 different temperatures during ten days was also evaluated experimently. While it has been found that P/Cr ratio, MglCr ratio, K/Cr ratio are stable for the 10 days, the Cl/Cr ratio, CaiCr ratio, and NaiCr were found to be less stable during the same period of time. The average results do not differ significantly from the control in either the artificial urine or the goat urine. Experiments were conducted to evaluate the reproducibility of artificial urine and artificial urine diluted 1:5 to simulate reproducibility of mineral/electrolyte to creatinine ratios with higher and lower concentrations, respectively. The results indicate a relatively good reproducibility of the method, because the variation, as measured by standard deviation, is small relative to the mean, except for CVCr ratio and CaiCr ratio, where the results presented showed a relatively low reproducibility. In theory, since ratios can be obtained, and should be unchanged by taking measurements at different dilutions even if the amount of specimen is unknown, there should be no need to absorb a fixed amount of urine onto the filter paper when urine is collected, using this method. However, there appear to be limits to this in reality, dilution of urine below a 1:10 Paulo Bagasse - MSc. Dissertation of DFPM dilution and/or the volume impregnated onto the filter paper below 0.525 ml on Whatman® number 6 filter paper. An experiment \vith goats on the relationship between the influence of water deprivation on mineral/electrulyte to creatinine ratio over a period of time demonstrated that concentrations and excretion of electrolytes vary from animal to animal, but the mineral/electrdl,,!\:' to creatinine ratios by DFPM hardly vary, even if the goat is deprived of \Vater. Using goat urine to determe mineral/electrolyte to creatinine ratio with the dried filter paper method gives high interclass correlation for mineral/electrolyte to creatinine ratio between the control (fresh urine sample, preserved in freezer) and the dried filter paper method on goats given water ad lihitulll. Interclass correlation agreement for the two methods was R, >0.75. On the basis ,,)1' the results, the method is robust for use as a urine sample preservation and transportatiu;: 11lL'lhodfor the determination of mineral/electrolyte to creatinine ratio with an added ad\ antage of not needing either preservative or refrigeration. Die tekort aan fasiliteite vir veeartsenykundige dienste, soos analitiese laboratoria, wat dikwels groot afstande van die boerderye gelee is, word vererger deur die beperkings wat deur die vervoer en bewaring van monsters teweeg gebring word. 'n Metode waar 'n hoeveelheid uriene monster op filtreerpapier geplaas en gedroog word en dan na die laboratorium gestuur word vir die ontleding van sekere stowwe (Droe Filtreer Papier Metode, DFPM), is al in menslike geneeskunde gebruik maar nog nooit in die veeartsenykundige bedryf nie. Die gebruik waar die urinere mineraal of elektroliet konsentrasie in verhouding tot die konsentrasie van urinere kreatinien uitgedruk word, word nou nogal algemeen. Dit laat die gebruik van 'n enkele uriene monster toe wat baie voordelig is in vergelyking met die versameling van 'n 24-uur monster. Fraksionele uitskeiding (FE) van mineraale en elektroliete, synde die mineraallelektroliet tot kreatinien verhouding, is 'n eenvoudige, ekonomiese meetinstrument asook 'n redelike indikator van die renale klaring van minerale en elektroliete, met die gebruik van 'n enkele uriene monster. Hierdie ondersoek is in twee fases uitgevoer. Eerstens, 'n uitsluitelike in vitro fase met die gebruik van gesimuleerde, (kunsmatige) uriene, op filtreerpapier gedroog en dan op verskye wyses, onder laboratorium omstandighede, gemanipuleer. Die tweede fase was beide in vitro sowel as in vivo en het die versamel van uriene van bokke wat aan verskye vlakke van waterweerhouding onderwerp was, en die verdere manipulasie daarvan in die laboratorium behels. Die bepaling van urinere mineraal/elektroliet tot kreatinien verhoudings met die gebruik van die gedroogde filtreerpapiermetode behels die toediening van 'n sekere volume uriene (kunsmatig en bok) op 'n gespesifiseerde tipe filtreerpapier, uitweek met 'n spesifieke oplosmiddel en dan die bepaling van mineral, elektroliet (fosfaat, chloride, magnesium, kalsium, kalium en natrium) en kreatinien konsentrasies.