Free-catch urine samples were collected from forty-one clinically normal domestic rabbits of various ages, breeds and both sexes. The Test γ GT Liquid-0018257640 was used for the in vitro quantitative determination of γ-Glutamyl-transferase (GGT) and reference intervals for γ-glutamyl transferase (γ-glutamyl transpeptidase, γ-GT, GGT) and GGT index (γ-glutamyl transferase to creatinine ratio) were established in fresh urine samples. Possible correlations of GGT and GGT index with sex and age were also explored. The stability of GGT after storage at +4°C for one week and -20°C for one month was investigated. The GGT and the GGT index reference intervals in fresh urine samples of healthy domestic rabbits were found to be 2.7–96.5 IU/l and 0.043–1.034, respectively. The urine GGT activity and the GGT index did not differ significantly between sexes in fresh urine samples. Nevertheless, a statistically significant difference was found in the GGT index with neutered status. Short-term storage at 4°C did not alter the enzyme stability, whereas, freezing did. Further investigations are needed to determine whether these parameters may be useful for early detection of renal tubular damage in rabbits, and in enabling better clinical management of affected animals.
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Rabbits are now the third most popular pet in the UK (after dogs and cats) with over 1.7 million kept, often as house pets rather than outdoor rabbits. Improvements in responsible management, husbandry and breeding practices and promotion of healthcare and welfare of these animals has lead to an extension of the life span with rabbits commonly now living up to nine or 11 years (Carey and Judge 2000). This expanded longevity is often accompanied by an increase in geriatric disorders, such as renal disease, and acute and chronic renal failure are considered common in older domestic rabbits (Klaphake and Paul-Murphy 2012). Clinical signs may be subtle in rabbits; therefore, early recognition is essential for immediate and more effective treatment.
The kidneys have a large functional reserve capacity. They receive almost 25% of the cardiac output and are a common target for toxic substances, including different chemicals and drugs. Kidneys are highly susceptible to damage due to their extractive and concentrating capabilities, through which these toxic substances are removed from the ultrafiltrate and accumulated in the tubular epithelium. Many toxicologic studies have suggested that the anatomic integrity of the kidney might be reflected by the output of urinary enzymes. γ -Glutamyl-transferase (GGT), also known as γ-glutamyl transpeptidase, is one of the numerous renal tubular enzymes which are excreted in the urine of many mammal species (Braun and others 1983, Bush 1991). GGT is considered a relatively stable enzyme that can be measured using the same equipment already used for serum GGT measurement in a clinical setting (Dierickx 1981). When a tubular injury occurs, renal tubular enzymes can be released into the nephronal lumen or into the urine. The urinary GGT activity was found to be four times higher than that of serum in rats (Malvoisin and others 1978), and about seven times higher in human beings (Orlowski and Szewczuk 1962). GGT urinary excretion has, therefore, been used for a long time in nephrotoxicity studies in human beings, laboratory rodents and dogs (Dierickx 1981, Stoykova and others 1983, De Schepper and others 1989, Grauer and others 1995, Whiting and Brown 1996, Clemo 1998). In most species, high GGT concentrations exist in the pancreas, mammary gland, small intestine and liver, but are found mainly in the kidney with GGT activity here ranging from 5% to 58% in the kidney cortex of horses, cows, sheep, swine and goats (Braun and others 1983). In the dog, GGT is a brush border enzyme of the proximal convoluted tubule (Brunker and others 2009). Pillion and others (1976) first demonstrated the existence in the rabbit of a urinary form of this enzyme of clear renal origin, while Shimada and others (1982) reported that GGT exhibits its highest activity in the straight proximal renal tubule. In human beings, raised serum GGT level is considered a sensitive indicator of hepatobiliary disease (Lum and Gambino 1972), but it lacks any specificity (Hardison 1979). Most research has been performed on liver GGT despite the fact that the kidney remains the most important source of this enzyme (Melillo 2007). GGT activity is high in rabbit kidney, but renal GGT does not reach the circulation because it is eliminated with the urine (Melillo 2007, Saunders and Davies 2005).
The diagnostic value and clinical significance, as a valid indication of renal disease, of many different urinary enzymes has been extensively investigated in many mammalian species, but not in domestic rabbits. Validation of a non-invasive screening test for detection of urinary enzymes, and GGT in particular, could be clinically useful as a possible indicator of early renal damage in rabbits. The purpose of this study was to determine a reference value for GGT excreted in fresh urine of clinically healthy domestic rabbits, and to explore the possible correlations with age and sex. The GGT index (urinary GGT/creatinine ratio) in fresh free-catch urine samples was also established. In addition, the GGT urinary activity was evaluated after sample storage for one week at +4°C, and for one month at -20°C, to determine the effect that storage at different temperatures could have on urinary enzymatic activity. After collection of a urine sample, analysis may be delayed. The laboratory may need batching of the samples, or these may need to be shipped to a different location, therefore, it is important to know whether storage may significantly affect the activity of the enzymes when the urine is kept refrigerated or frozen, before being analysed.
Materials and methods
Urine samples were collected from forty-one domestic rabbits presented to the Exotic Animal and Wildlife Service of The Royal (Dick) School of Veterinary Studies, University of Edinburgh, between March 2010 and March 2012. A full history, including medical history, was obtained from their owners, and all rabbits were considered healthy on the basis of an absence of any clinical signs, and a complete physical examination. Rabbits with any previously diagnosed disease, or those receiving any medications at the time of the study were not included. The study animals consisted of 13 sexually intact females, 13 neutered females, six sexually intact males and nine neutered males. Breeds included crossbreed (n = 7), dwarf lop (n = 19), lion-head (n = 2), lion-head cross lop (n = 2), French lop (n = 2), Netherland dwarf (n = 5), English cross lop (n = 3) and chinchilla (n = 1). The age ranged from three months to 11 years.
Urine samples were collected from spontaneous urination. Because bacteriology was not a requirement for the study, free catch from a clean plastic litter tray was considered the easiest way to obtain the samples without affecting the welfare of the animals. Samples were collected, between 8:30 and 16:30, by aspiration into a 2.5 ml or 5 ml sterile syringe which was then voided into standard plain sterile urine pots. The samples were sent to the Veterinary Pathology Unit of the University of Edinburgh and analysed within eight hours.
The urine samples were centrifuged at 1000 g for 5 minutes, and the supernatant separated. The Test γ GT Liquid-0018257640, generally intended for determination of GGT in human serum or plasma, was used for the quantitative in vitro determination of γ-glutamyl transferase (γ-GT) using the ILab 650 Chemistry System Analyser (Instrumentation Laboratories) within eight hours after sample collection. Rate analysis followed the Szazs/Persijn method (Bergmeyer 1983). Enzymatic activity was expressed in International Units per Liter (IU/l). Creatinine was measured on the same sample using a modified Jaffe test run on the automated clinical chemistry analyser. The urinary GGT activity (IU/l) was then divided by the urinary creatinine concentration (mg/dl) to obtain the urinary GGT index. Reference Value Advisor was used to calculate the reference intervals for urine GGT (IU/l) and GGT index in fresh urine samples (Geffre and others 2011). For nineteen of the rabbits, part of the fresh urine sample was stored for one week at +4°C. GGT activity was measured again after storage. For five samples, part of the freshly collected supernatant was also immediately stored at -20°C, and analysis repeated 30 days after collection.
All analyses were conducted by use of a commercially available software programme (Minitab 15; Minitab Ltd, Coventry, UK). Urine GGT activity was analysed for an effect of sex and neutered status, and any interaction between them by use of 2-way analysis of variance (ANOVA) test, after normality of residuals had been confirmed. Regression analysis was used for determination of the relationship between GGT (IU/l), GGT index and age in fresh and refrigerated urine samples, after normality of residuals had been confirmed. Enzyme stability at +4°C for one week and -20°C for one month was assessed by use of a paired t test, and correlation analysis was performed via a Pearson correlation test. For all analyses, a value of P < 0.05 was considered statistically significant.
The mean age of the rabbits was 2.8 ± 2.7 years (mean ± sd). The urine GGT reference range for fresh urine was 2.7–96.5 IU/l. The reference range for the GGT/creatinine ratio (GGT index) in fresh urine samples was found to be 0.043–1.034 (Table 1).
Urine GGT values did not differ significantly in fresh and refrigerated urine samples between sexes or neutered status (P > 0.150). Furthermore, there was no interaction between sexes and neutered status for GGT values in fresh and refrigerated urine samples (P > 0.698). The GGT index also did not differ significantly between sexes in fresh and refrigerated urine (P > 0.509). However, there was a statistically significant difference with neutered status (P = 0.025) compared with entire rabbits in the GGT index in fresh urine samples (mean GGT index 0.348 ± 0.38 and 0.158 ± 0.08).
No statistically significant relationship with age of rabbits and GGT values in fresh or refrigerated urine samples or GGT index in fresh or refrigerated urine samples was found (P > 0.088).
GGT activity and the GGT index were not significantly different after storage at 4°C for one week compared with that determined on fresh urine samples at day 0 (P = 0.410 and P = 0.331, respectively). While the resulting correlation between GGT (IU/l) activity in fresh and refrigerated urine was highly significant (P < 0.001, r = 0.901), this was not true for GGT index (P = 0.063, r = 0.435). By contrast, a decrease in enzyme activity and GGT index was significant (P = 0.012 and P = 0.046, respectively) after storage at -20°C for one month.
The present study has established for the first time a reference range for urine GGT activity in clinically normal domestic rabbits of 2.7–96.5 IU/l. To the authors' knowledge, values of this urinary enzyme have not been previously reported for domestic rabbits, but have been established for several other domestic species. In dogs, it is suggested that enzyme activity in a single, randomly collected urine sample could be an accurate representation of an animal enzyme's urine status (Brunker and others 2009). In sheep, reproducible GGT values between 48 IU/l and 373 IU/l have been reported, using the same method developed for the routine serum assay, despite urine pH and specific gravity variations (Van Den Berg 1990). In healthy horses, urine GGT activity has been found to be between 21 IU/l and 407 IU/l (Adams and others 1985), and unrelated to the serum GGT concentration. As in the present study, neither sex nor age of the horses influenced urinary enzyme activity whether expressed as absolute values or in relation to creatinine excretion (Brobst and others 1986). Serum GGT levels were not measured in the present study, so it is unclear as to how urinary levels might be related to serum levels in rabbits.
Brunker and others (2009) established a GGT index reference range for dogs, but values did not differ significantly between sexes (P = 0967), as found in the present study in rabbits. In dogs, the urinary GGT/creatinine ratio in spot urine samples appears to correlate with the 24-hour enzyme excretion (Gosset and others 1987). The quantity of GGT lost into the urine can be related to the amount of creatinine measured in the same sample because the latter is not reabsorbed by the tubules, and its excretion is relatively constant and stable. This will account for variation in urine flow rate at the time of sampling, and would minimise the variability in urinary enzyme excretion (Archer 2007). The potential diagnostic utility of the urine GGT index has been evaluated in an experimental canine model of aminoglycoside-induced nephrotoxicity, and the authors considered it unlikely that differences in age, gender or body weight could have a significant effect (Rivers and others 1996). A normal urine GGT index ratio of 0.14 ± 0.10 has been reported for adult dogs (Gosset and others 1987), whereas Grauer and others (1995) reported a value of 0.34 ± 0.53 (IU/l divided by mg/dl) in a gentamicin-induced nephotoxicity study of six-month-old male beagles, where a strong correlation was also found between 24-hour urinary enzyme level and spot enzyme/creatinine ratios. In sheep and horse urine, despite a significant variation in the measured GGT and creatinine values, the ratio of GGT/creatinine remains constant (Van Den Berg 1990, Adams and others 1985). GGT index has been shown to differ significantly in acidic and basic pH, but inactivation of GGT in urine occurs only at pH 4.7 (Brunker and others 2009). High urine pH has been reported to influence the activity of other urinary enzymes in pigeons (Wimsatt and others 2009), and this factor may be even more important in herbivorous species with alkaline urine. Simultaneous assessment of urine pH was not undertaken in this study, but the influence that this may have on urine enzyme activities in rabbits will need further evaluation.
Previous studies on the effects of storage on GGT activity in serum or plasma have demonstrated that activity was unchanged if stored for two weeks at +20°C, +4°C or -20°C in the cow, whereas in the horse, storage of serum for one month at -30°C caused 50% loss of activity (Braun and others 1983). Adams and others (1985) also found that GGT activity in equine urine decreased after storage, but this was greatest, most variable and significant at -20°C for 72 hours, whereas, greater stability was found at 4°C for 72 hours. In the rabbit, urine GGT activity is stable at +4° or at -20°C (Pillion and others 1976). GGT is also fairly stable in human and canine urine samples stored at room temperature (20°C) or at 4°C, regardless of centrifugation, whereas, the enzyme is labile at low temperature (Gosset and others 1987, Matteucci and others 1991). The results of the present study are in agreement with these previous reports. Short-term (one week) storage at +4°C did not significantly alter the GGT enzymatic activity compared with freshly collected urine. Freezing, however, appeared to destroy the activity of the enzyme, therefore limiting its practical use.
Assessment of renal function in many mammals is achieved by measuring blood urea nitrogen (BUN), serum creatinine and the urinary protein/creatinine ratio. In rabbits, BUN follows a circadian rhythm and depends largely on the protein intake and nutritional status of the animal. Rabbits have a reduced capacity to concentrate urea compared with other mammals; and intestinal absorption, activity of the caecal flora, liver function, GI haemorrhage, stress and hydration status can also influence serum BUN levels (Jenkins 2008). Creatinine is a product of protein catabolism that is freely filtered through the glomerulus and excreted at a constant rate (Melillo 2007). It is considered a more reliable indicator of kidney function compared with BUN levels, because it does not show the same variability due to external extrarenal factors. However, traditional clinical pathology indicators of nephrotoxicity, such as BUN, serum creatinine and urinalysis parameters, including total protein, albumin, electrolytes and sediment examination are considered relatively insensitive and non-specific indicators of modest renal dysfunction, as changes in these biomarkers tend to occur only after extensive damage has been sustained by the kidneys (Ferguson and others 2008). In renal disease, proteinuria seems to occur earlier than serum biochemical changes, although in rabbits, protein levels need to be evaluated along with urine specific gravity and sediment because healthy young rabbits may normally have small amounts of albumin in the urine, and adult rabbits may have trace proteinuria (Kraus and others 1984). Measuring the urinary protein/creatinine ratio may be a more useful test in clinical practice to quantify the proteinuria (Reusch and others 2009). There is, therefore, the need for more sensitive biomarkers as indicators of early kidney damage that could also prove more valuable in localising, anatomically, the kidney lesion. Several studies have revealed that evaluation of enzymuria may prove valuable in detecting early renal tubular injury in different species (Greco and others 1985, Rivers and others 1996, Da Silva Melo and others 2006). In human beings, measurement of tubular enzymuria is considered important as, according to Westhuyzen and others (2003), it can detect acute renal failure up to four days before alteration in GFR can even be detected. Urine, as a diagnostic medium, allows for non-invasive detection of biomarkers. With end-stage renal disease, in which loss of tubules is extreme, GGT concentration may increase substantially (Polito and others 1989). Whether GGT may be used as a biomarker of early renal tubular impairment and for prediction of developing azotaemia in rabbits requires further investigations as well as possible correlation with the glomerular filtration. Additional research is also warranted to evaluate the effect of urinary specific gravity and pH on urinary GGT excretion, and the relationship between urinary GGT/creatinine ratio and plasma creatinine concentration in rabbits. Future research in this area is also required to determine the sensitivity and specificity of this urinary index for identification of various causes of renal tubular damage in domestic rabbits. Despite the effort to ensure that the rabbits in this study were clinically healthy, it could still be possible that some of these animals had subclinical disease which may have affected the excretion of the urinary enzyme. Blood sampling for complete haematobiochemistry, in addition to history and physical examination, would be advisable in order to confirm health status, but it was not possible within the constraints of this study. Ideally, the quality of the urine should also be assessed, as the presence of red and white blood cells, faeces and bacteria may increase or decrease the urinary enzymatic activity (Clemo 1998). However, the reported reference range is applicable to other pet rabbits, and could be used for further studies to non-invasively identify rabbits with early renal tubular damage.
The authors would like to kindly thank Yvonne Crawford for performing the analysis of all urine samples; Livia Benato, Brigitte Lord and Wendy Bament for their help in sample collection.
Provenance: Not commissioned; externally peer reviewed
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