Reliability and accuracy of determining minerals and electrolytes in goat urine using a dried filter paper method

Abstract

The lack of falcilities for veterinary services, such as analytical laboratories, which are distant from the field and at immense distances are aggravated 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 till' concentration of creatinine has recently become generally accepted. A single sample obviates 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, inexpensive 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 vitro, using urine (collected from water-deprived goats) dried on filter paper and manipulated in various 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 filter 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, 595. 597, 598 for Schleicher&Schuell (very high quality). There were few 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 linear/standardised 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 (R1 >0.75) between the 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, Mg/Cr ratio, K/Cr ratio are stable for the 10 days, the Cl/Cr ratio, Ca/Cr ratio, and Na/Cr 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 Cl/Cr ratio and Ca/Cr 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 dilution and/or the volume impregnated onto the filter paper below 0.525 ml on Whatman® number 6 filter paper. An experiment with goats on the relationship between the influence of water deprivation on minerallelectrulvte to creatinine ratio over a period of time demonstrated that concentrations and excretion of electrolytes vary from animal to animal, but the mineral/electrolyte to creatinine ratios by DFPM hardly vary, even if the goat is deprived of water. 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 libitum. Interclass correlation agreement for the two methods was R1 >0.75. On the basis of the results, the method is robust for use as a urine sample preservation and transportation method for the determination of mineral/electrolyte to creatinine ratio with an added advantage of not needing either preservative or refrigeration.