# Comparison of osmolality and refractometric readings of Hispaniolan Amazon parrot (Amazona ventralis) urine.

Abstract: To evaluate the relationship between osmolality and specific gravity of urine samples from clinically normal adult parrots and to determine a formula to convert urine specific gravity (USG) measured on a reference scale to a more accurate USG value for an avian species, urine samples were collected opportunistically from a colony of Hispaniolan Amazon parrots (Amazona ventralis). Samples were analyzed by using a veterinary refractometer, and specific gravity was measured on both canine and feline scales. Osmolality was measured by vapor pressure osmometry. Specific gravity and osmolality measurements were highly correlated (r = 0.96). The linear relationship between refractivity measurements on a reference scale and osmolality was determined. An equation was calculated to allow specific gravity results from a medical refractometer to be converted to specific gravity values of Hispaniolan Amazon parrots: [USG.sub.HAp] = 0.201 + 0.798([USG.sub.ref]). Use of the reference-canine scale to approximate the osmolality of parrot urine leads to an overestimation of the true osmolality of the sample. In addition, this error increases as the concentration of urine increases. Compared with the human-canine scale, the feline scale provides a closer approximation to urine osmolality of Hispaniolan Amazon parrots but still results in overestimation of osmolality.Key words: osmolality, refractometry, specific gravity, urine, avian, Hispaniolan Amazon parrots, Amazona ventralis

Introduction

Osmolality, a measure of the number of dissolved particles in a liquid (osmoles per kg of water), is considered to be the gold standard for measuring urine concentration. However, measurement of osmolality can be expensive, and the necessary equipment is generally unavailable to the practitioner. Refractometers are used frequently in veterinary medicine to allow practitioners to have a rapid and inexpensive assessment of urine osmolality. A refractometer uses a sample's refractive index, the degree to which it bends light, which depends on the concentration and type of solutes in the sample, to provide insight into other properties. In clinical medicine, refractometry is used routinely to estimate urine specific gravity (USG) and plasma protein concentration. The method is easy, fast, and inexpensive.

Medical refractometers designed for human samples use a reference scale based on human urine refractivity and USG. Although canine urine has the same refractivity as human urine, other mammalian species are documented to have differences in relative refractivity of urine, likely due to differences in the solute composition of each species' urine. Because of these differences, species-specific scales for USG have been developed for mammalian species, including cats and guinea pigs. (1-3) These species-specific scales allow for more accurate estimation of USG based on the refractivity of the sample. A similar scale or conversion factor has not been published for any avian species.

Measurement of USG is an essential component of urinalysis. The USG correlates strongly with osmolality and is used clinically as a marker of renal concentrating ability. Various studies report urate-free USG values for avian species between 1.005 and 1.020. (4,5) However, in most cases, the specific gravity scale that was used to obtain these values is not identified, thus reducing the clinical value of this information. A previous study in racing pigeons (Columba livia) established normal urine osmolality for this species (6) but did not correlate the results with refractometric USG values. Another study reported urine specific gravity values and osmolality in pigeons with salmonellae-induced polyuria, (7) but those values were not compared. Establishing the range of normal urine osmolality values and the relationship between osmolality and refractometric USG in various avian species would enhance the ability of veterinarians to accurately diagnose and monitor renal disease in those species.

The specific objective of this study was to evaluate the relationship between osmolality and specific gravity of urine samples from clinically normal adult Hispaniolan Amazon parrots (Amazona ventralis) and to determine a formula to convert USG measured on a reference scale, [USG.sub.ref], to a more accurate USG value for this avian species

Materials and Methods

Nineteen adult Hispaniolan Amazon parrots housed individually or in pairs as part of a research colony at the University of Tennessee, College of Veterinary Medicine (Knoxville, TN, USA) were used for this study. All the birds had access to fresh food and water throughout the collection periods. The birds were fed a commercial pelleted diet (Lafeber Premium Daily Diet Pellets for Parrots, Lafeber Company, Cornell, IL, USA) and were not known to have any significant health concerns at the time of the study. These parrots were not being used for any other studies during the time of sample collection. Examinations and laboratory evaluations were not performed on any of the birds nor were the urine samples collected identified to have come from specific individuals.

The bottom of each cage was lined with wax paper. To ensure that the samples were fresh, the cages were visually monitored from the time the paper was put in place until all the samples were collected. By using 1-mL syringes, at least 0.1 mL of urine was collected from each of the observed droppings. Only urine samples that were not visibly contaminated with feces were collected. The collected urine was centrifuged for 5 minutes to precipitate urates and other insoluble material. The supernatant was removed and stored for at least 1 hour at room temperature before analysis.

Samples were analyzed by using a refractometer (Reichert VET 360 Temperature Compensated Hand-Held Refractometer, Reichert Inc, Depew, NY, USA) that had previously been calibrated with distilled water. For each sample, 1 drop of urine was placed on the prism of the refractometer. Specific gravity of each sample was visually determined by a single observer (V.L.G.) by using both the canine and the feline scales. This test was performed in duplicate on each sample, and the mean value was used for statistical analysis. Measurement error was assumed to have zero mean with magnitude less than one-half the measurement resolution (5 x [10.sup.-4]). Twenty samples that ranged in specific gravity were selected, and the osmolalities of these samples were determined with a vapor pressure osmometer (Wescor 5500XRS, Wescor Inc, Logan, UT, USA). The osmometer was double calibrated by a medical technologist (UTCVM Clinical Pathology Department). For each sample, 10 [micro]L of urine was transferred by pipette onto a disk of filter paper, which was loaded into the osmometer. Three osmolality readings were determined for each sample.

Osmolality values were determined by using the mean value observed from 3 repeated measurements for each of the 20 samples. The difference between each measurement and its corresponding osmolality value was then used to estimate the measurement variance. Correlation was determined by using the Pearson correlation coefficient, r. Linear regression was performed by using Deming regression. Coefficient of determination, [R.sup.2], was used to characterize the linear regression performance. Calculations were performed by using MATLAB v2008b (MathWorks, Natick, MA, USA). The equation used to obtain the urine specific gravity of Hispaniolan Amazon parrots, [USG.sub.HAp], based on refractivity measured with a reference scale, [USG.sub.ref], is:

[USG.sub.HAp] = [[alpha].sup.*] + [[beta].sup.*] ([USG.sub.ref]). (1)

Two separate, but similar formulas are used to obtain estimates of [[alpha].sup.*] and [[beta].sup.*] in the above equation. The first formula defines the linear relationship between osmolality, [phi], and refractivity as measured on a reference scale:

[phi] = [alpha] + [beta]([USG.sub.ref] - 1). (2)

Because error is present in both refractivity and osmolality measurements, Deming regression was used to estimate the [alpha] and [beta] values from Eq (2)

The second formula defines a similar relationship between osmolality and USG for any animal species, in this case Hispaniolan Amazon parrots:

[MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII], (3)

The [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] values above were experimentally determined by Dossin et al (8) as -25(39/88) and 36 646 (34 318/38 974), respectively

Results

Refractometer and osmometer measurements of the Hispaniolan Amazon parrot urine were highly correlated (r = 0.96). By assuming a uniform distribution with range of 5 x [10.sup.-4], the refractometer measurement error variance was estimated as 2.08 x [10.sup.-8](by definition, [[sigma].sup.2] = [span.sup.2]/12). In stark contrast, the osmolality measurement error variance was found to be 183 (mOsm/kg) (2). Consequently, the error ratio used for Deming regression was (183/2.08 x [10.sup.-8 ]). Deming regression yielded the following estimates of [alpha] and [beta] ([R.sup.2] = 0.96) for Eq. 1, which describes the linear relationship between refractivity measured on a reference scale, [USG.sub.ref], and osmolality, [phi]:

[phi] = (-57) + 25 749([USG.sub.ref] - 1). (4)

A conversion equation was determined to translate specific gravity measured by using a reference (human-canine) refractometer scale to specific gravity values for Hispaniolan Amazon parrot urine. By noting that Eq. (2) and Eq. (3) both describe the same osmolality values, the righth- and sides of each equation can be set equal the other:

[MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII].

A conversion equation in the form of Eq. (1) can then be established by solving for [USG.sub.HAp] :

[MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII], (5)

where

[MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII]

and

([beta]/[??]) = [[beta].sup.*].

Substituting the [alpha] and [beta] estimates from Eq. (4) and [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] as described by Dossin et al (8) yields:

[USG.sub.HAp] = 0.201 + 0.798([USG.sub.ref]). (6)

The need for an appropriate conversion equation is illustrated in Figures 1 through 3, which depict the osmolality that would be computed by using 3 separate specific gravity scales. Depicted in Figures 1 and 2 is how use of a canine or feline specific gravity scale, respectively, on a refractometer would overestimate the osmolality of Hispaniolan Amazon parrot urine. The osmolality that would be estimated when using Eq. (4), which mathematically represents the Hispaniolan Amazon parrot scale, is depicted in Figure 3.

Although use of the above conversion factor is recommended when osmolality needs to be calculated exactly for urine samples, avian practitioners may need a faster estimate in practice. An approximate specific gravity for Hispaniolan Amazon parrot urine can be calculated by using the following equation:

[USG.sub.HAp] = [USG.sub.fel] + 0.003. (7)

Discussion

As is illustrated in Figure 1, use of the canine scale to approximate the osmolality of Hispaniolan Amazon parrot urine leads to an overestimation of the true osmolality of the sample. In addition, this error increases as the concentration of urine increases. Based on these data, we concluded that the feline scale provides a closer approximation to Hispaniolan Amazon parrot urine osmolality when compared with the human-canine scale, but it still causes an overestimation of osmolality, as seen in Figure 2.

The human-canine scale on handheld refractometers provides measurable values from which the osmolality of Hispaniolan Amazon parrot urine can be calculated. We have also provided a conversion factor from which the specific gravity of Hispaniolan Amazon parrot urine can be calculated based on the specific gravity measurement on a human-canine scale. Equation 6 should be used when urine is examined when using a medical refractometer or when using the canine scale on a veterinary refractometer.

For practitioners with a veterinary refractometer, a simplified equation (Eq. 7) has been provided that will give a rapid estimation of the USGHAp; however, this simplified formula does not fully account for the proportional bias: as the concentration of the urine increases, the error in this estimation also increases. Despite the slightly increased error incurred by this method, Eq. 7 provides a busy clinician with rapid mental calculation of [USG.sub.HAp] without the use of a calculator. However, this formula can only be used in conjunction with a veterinary refractometer with a feline USG scale.

Collection of avian urine samples presents unique challenges, including the increased potential of contamination with feces due to the shared cloacal opening. Although we collected samples that were not grossly contaminated with feces, microscopic contamination was not ruled out. Centrifugation allowed for removal of any non-soluble material. We were not able to collect urine with higher specific gravity than 1.038 on the human-canine scale and, therefore, cannot comment on whether the human/canine scale would be an accurate scale for more concentrated samples. More research is needed to determine if this scale would be appropriate for use in other avian species.

Acknowledgments: We thank the Tennessee Valley Exotic Bird Club for their support of the study. We also thank Karl Snyder, medical technologist at the University of Tennessee, College of Veterinary Medicine, for his technical assistance throughout this project.

References

(1.) Rubini ME, Wolf AV. Refractometric determination of total solids and water of serum and urine. J Biol Chem. 1957;225(2):869-876.

(2.) George JW. The usefulness and limitations of handheld refractometers in veterinary laboratory medicine: an historical and technical review. Vet Clin Pathol. 2001;30(4):201-210.

(3.) Wolf AV, Brown MG, Prentis PG. Concentrative properties of aqueous solutions: conversion tables.

In: Weast RC, ed. The CRC Handbook of Chemistry and Physics. 70th ed. Boca Raton, FL: CRC Press; 1990:D252-D253.

(4.) Styles DK, Phalen DN. Clinical avian urology. Semin Avian Exot Pet Med. 1998;7(2):104-113.

(5.) Sturkie PD. Kidneys, extrarenal salt excretion, and urine. In: Sturkie PD, ed. Avian Physiology. New York, NY: Springer-Verlag; 1986:359-382.

(6.) Halsema WB, Alberts H, de Bruijne JJ, Lumeij JT. Collection and analysis of urine from racing pigeons (Columba livia domestica). Avian Pathol. 1988; 17(1):221-225.

(7.) Gevaert D, Nelis J, Verhaeghe B. Plasma chemistry and urine analysis in Salmonella-induced polyuria in racing pigeons (Columba livia). Avian Pathol. 1991;20(3):379-386.

(8.) Dossin O, Germain C, Braun JP. Comparison of the techniques of evaluation of urine dilution/concentration in the dog. J Vet Med A Physiol Pathol Clin Med. 2003;50(6):322-325.

A. Paige Brock, DVM, Dipl ACZM, Vanessa L. Grunkemeyer, DVM, Dipl ABVP (Avian), Michael M. Fry, DVM, MS, Dipl ACVP, James S. Hall, PhD, PE, and Joseph W. Bartges, DVM, PhD, Dipl ACVIM, Dipl ACVN

From the Department of Small Animal Clinical Sciences, College of Veterinary Medicine, University of Florida, 2015 SW 16th Ave, Gainesville, FL 32608, USA (Brock); Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, 1060 William Moore Dr, Raleigh, NC 27607, USA (Grunkemeyer); Department of Biomedical and Diagnostic Sciences, College of Veterinary Medicine, University of Tennessee, 2407 River Dr, Knoxville, TN 37996, USA (Fry); Hidden Solutions, LLC, 7862 W Irlo Bronson Memorial Hwy, PMB-139, Kissimmee, FL 34747, USA (Hall); and Department of Small Animal Clinical Sciences, College of Veterinary Medicine, University of Tennessee, 2407 River Dr, Knoxville, TN 37996, USA (Bartges).

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Author: | Brock, A. Paige; Grunkemeyer, Vanessa L.; Fry, Michael M.; Hall, James S.; Bartges, Joseph W. |
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Publication: | Journal of Avian Medicine and Surgery |

Article Type: | Report |

Date: | Dec 1, 2013 |

Words: | 2434 |

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