SEARCH

SEARCH BY CITATION

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

This study aimed to assess the overall glucuronidation capacity of cats, using prototypic substrates identified for human UDP-glucuronosyltransferases (UGTs). To this end, Michaelis–Menten kinetics were established for the substrates using feline hepatic microsomal fractions, and results were compared with similar experiments carried out with dog liver microsomes. Cats are known for their low capacity of glucuronide formation, and UGT1A6 was found to be a pseudogene. However, functional studies with typical substrates were not performed and knowledge of the enzymology and genetics of other glucuronidation enzymes in felidae is lacking. The results of this study showed extremely low formation of naphthol-1-glucuronide (1.7 ± 0.4 nmol/mg protein/min), estradiol-17-glucuronide (<0.7 nmol/mg protein/min), and morphine-3-glucuronide (0.2 ± 0.03 nmol/mg protein/min), suggesting a lack of functional UGT1A6 and UGT2B7 homologues in the cat's liver. Dog liver microsomes were producing these glucuronides in much higher amounts. Glucuronide capacity was present for the substrates 17β-estradiol (estradiol-3-glucuronide, 2.9 ± 0.2 nmol/mg protein/min) and 4-methylumbelliferone (31.3 ± 3.3 nmol/mg protein/min), assuming that cats have functional homologue enzymes to at least the human UGT1A1 and probably other UGT1A isozymes. This implies that for new drugs, glucuronidation capacity has to be investigated on a substance-to-substance base. Knowledge of the glucuronidation rate of a drug provides the basis for pharmacokinetic modeling and as a result proper dosage regimens can be established to avoid undesirable drug toxicity in cats.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Cats and other felidae are known for their low capacity for glucuronide conjugation of drugs and toxins. This assumption is based on early data on the biotransformation of phenolic compounds in cats in comparison with other species (Capel et al., 1972a,b, 1974). Subsequently, acetaminophen (paracetamol, a phenolic derivative) is the most prominent example of a drug that is toxic for cats, when common dosage regimens established for dogs or humans are applied. This toxicity has been largely attributed to the fact that cats express an inactive pseudogene UGT1A6, hence being unable to glucuronidate acetaminophen resulting in an accumulation of paracetamol and its phase I metabolites in the liver followed by liver cell damage (Court & Greenblatt, 2000). However, little is known about the enzymology and genetics of other glucuronidation enzymes in felidae.

The formation of glucuronide conjugates is accomplished by a superfamily of enzymes, the UDP-glucuronosyltransferases (UGTs). The UGTs are membrane-bound enzymes that catalyze the conjugation of the glycosyl group of glucuronic acid to many lipophilic endogenous and exogenous substrates (Mackenzie et al., 2005). The resulting water soluble glucuronide conjugates are generally less active than the parent compound and can be excreted into the bile or urine. UGTs have a broad and overlapping substrate specificity albeit with often significant differences in affinity (Radominska-Pandya et al., 1999; Tukey & Strassburg, 2000). The human UGT proteins are classified into two families denoted UGT1 and UGT2, based on evolutionary divergence and homology in amino acid sequence. The UGT1 and UGT2 families are abundant in the liver although UGT activity is also found in extra-hepatic tissue such as the intestines and kidneys. Based on the preference for certain substrates and similarity in amino acid sequences, UGT1 is further subdivided into the subfamilies UGT1A and UGT1B, even as UGT2 is subdivided in UGT2A and UGT2B (Court & Greenblatt, 2000; Mackenzie et al., 2005; Dong et al., 2012). Two other UGT families, denoted UGT3 and UGT8, were also discovered, but they conjugate other glycosyl groups to their substrates and are considered unlikely to play a significant role in the detoxification of drugs or other xenobiotics (Mackenzie et al., 2005; Rowland et al., 2013). UGT1A and UGT2B are important isozymes in the human liver responsible for glucuronidation (Miners et al., 2004; Mackenzie et al., 2005; Ohno & Nakajin, 2009; Dong et al., 2012; Harbourt et al., 2012). Substrates for human hepatic UGTs are, among others, endogenous steroids, bile acids (Gall et al., 1999), bilirubin (Bosma et al., 1994; Senafi et al., 1994), and many exogenous substances such as drugs, alcohols, phenols, lipid soluble vitamins, amines, and thiols (Ebner & Burchell, 1993; Green & Tephly, 1996; Hashizume et al., 2008). Examples of drugs often used in veterinary medicine, which are substrates for human UGT2B, are various NSAIDs, opioids, and benzodiazepines (Miners & Mackenzie, 1991; Jin et al., 1993; Coffman et al., 1997; Stone et al., 2003; Kiang et al., 2005).

A similar classification of UGT enzymes of the cat based on substrate specificities and genetics has not been prepared. As mentioned above, Court and Greenblatt (2000) initiated the genetic research on UGTs in the cat and showed the inactive pseudogene of UGT1A6. Furthermore, they suggested a limited expression of other hepatic UGT1A isoforms, denoted as UGT1A1 and UGT1A02 (Court & Greenblatt, 1997, 2000). However, limited data have been published on the activity and substrate preference of these other feline UGTs.

Hence, it was the aim of the current study to assess the overall glucuronidation capacity of cats, using prototypic substrates, identified for human UGT isozymes. To this end, Michaelis–Menten kinetics were established for the substrates using feline hepatic microsomal fractions. Results were compared with similar experiments carried out with dog liver microsomes as this could allow a broader interpretation of the clinical relevance of the findings.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Drugs and chemicals

Alamethicin solution, 17β-estradiol water soluble, β-estradiol 3-β-D-glucuronide (E3G), β-estradiol 17-β-D-glucuronide (E17G), 11β-hydroxytestosterone, 1-naphthol, naphthylβ-D-glucuronide (N1G), magnesium chloride hexahydrate, 4-methylumbelliferone (4MU), 4-methylumbelliferyl-β-D-glucuronide hydrate (4MUG), testosterone, and uridine 5′-diphosphoglucuronic acid (UDPGA) were purchased from Sigma Chemical Co. Morphine 3-β-D-glucuronide (M3G) was purchased from Cerilliant Corporation. Morphine hydrochloride was purchased from BUFA B.V.

Tissue samples

Liver tissue was collected from adult healthy European Shorthair cats (n = 8, five males and three females, aged from 11 to 13 months) directly after euthanasia, and samples were immediately frozen in liquid nitrogen and stored at −70 °C. The cats had served as controls in a study for the development of FIV vaccines and had been sacrificed as cell donors. The same applies to the liver samples of Beagle dogs (n = 7, two males and five females, aged from 3.5 to 4.5 years) that had also served as controls in clinical trials. Animals were sacrificed with permission of the Animal Ethical Committee of the Utrecht University and performed according to the Dutch law on Animal Experiments.

Preparation of submitochondrial fractions

The isolation of submitochondrial fractions (commonly referred to as microsomes) containing predominantly microsomal proteins followed the procedure as described by Rutten et al. (1987) with minor modifications. In brief, cat and dog liver samples of approximately 10 g were obtained from adult healthy cats and dogs directly after euthanasia and were quickly frozen in liquid nitrogen and stored at −70 °C. The tissue samples were homogenized with 1.15% KCl, containing 0.1 mm EDTA at 4 °C. The homogenates were centrifuged at 9000 g for 25 min at 4 °C, and the supernatant obtained (S9-fraction) was centrifuged at 100 000 g for 1 h and 15 min at 4 °C. The microsomal pellet was resuspended in 1.15% KCl 0.05 m phosphate buffer, pH 7.4, containing 0.1 mm EDTA and 20% glycerol. The microsomes were then quickly frozen in liquid nitrogen and stored in Eppendorf-cups at −70 °C until use (Rutten et al., 1987).

The protein concentrations of the microsomal fractions were determined by the method of Bradford using bovine serum albumin (BSA) as a standard (Bradford, 1976), and data were expressed as nmol/mg protein/min. As a quality control for the enzymatic activity of the microsomal preparations, cytochrome P450 activity was measured using 6β-testosterone hydroxylation as described by Chauret et al. (Chauret et al., 1997).

Incubation protocols

All glucuronidation assays were performed with pooled microsomes according to standard protocols with only minor modifications (Miners et al., 1988; Furlan et al., 1999; King et al., 2000; Stone et al., 2003; Mano et al., 2004; Court, 2005). Details regarding the specific incubation conditions of each substrate are given in Table 1. Incubation mixtures contained 100 mm phosphate buffer (KH2PO4, pH 7.4), 5 mm MgCl2, alamethicin (50 μg/mg protein), and a concentration range of the selected substrates (1-naphthol, 17β-estradiol, morphine, 4MU, respectively) in a total volume of 500 μL. All substrates were dissolved in water, except for 4MU, which was dissolved in methanol resulting in a final concentration of 0.5% methanol. Pooled hepatic microsomal protein was added to obtain a protein concentration ranging from 0.1 to 0.5 mg/mL, and pre-incubations were performed for 5 min at 37 °C. The glucuronidation reactions were initiated by adding UDPGA with a final concentration of 5 mm, and samples remained at 37 °C in a heat block for the indicated time and were shaken regularly to ensure an equal temperature within the incubating mixtures. Reactions were terminated by addition of ice-cold acetonitrile, followed by a rapid-cooling step. Samples were centrifuged at 13 000 g for 5 min, and the supernatants were directly injected onto the HPLC column. HPLC conditions are given in Table 2. Assays were tested for linearity in incubation time and protein concentration by duplicate measurements in two independent experiments. All measurements for Km and Vmax determination were performed at least in duplicates, and the maximum activity measurements were performed in triplicate in three independent experiments. Blanks were obtained from incubations without UDPGA.

Table 1. Incubation conditions for the different substrates
SubstrateGlucuronide formedIncubation timeProtein concentrationSubstrate concentrationProcedure
  1. Incubation mixtures contained 100 mm phosphate buffer (KH2PO4, pH 7.4), 5 mm MgCl2, alamethicin (50 μg/mg protein), and different substrate concentrations in a total volume of 500 μL. Pooled hepatic microsomal protein was added, and pre-incubations were performed for 5 min at 37 °C. Thereafter, UDPGA (final concentration 5 mm) was added, and the reaction mixture was incubated at 37 °C in a heat block for the indicated time.

1-naphtholN1G4 min0.1 mg/mL750 μm

Miners et al., 1988;

Furlan et al., 1999;

17β-estradiol

E3G

E17G

15 min0.5 mg/mL3 mmCourt, 2005;
MorphineM3G90 min0.25 mg/mL5 mm

Miners et al., 1988;

King et al., 2000;

Stone et al., 2003;

4-methylumbelliferone4MUG4 min0.1 mg/mL750 μmMano et al., 2004
Table 2. HPLC conditions for the glucuronidation activities of different substrates with their stationary phase, mobile phase, and detection conditions
Glucuronidation activityStationary phaseMobile phaseDetection conditions
  1. *The HPLC system consisted of two Genkotek high-precision pumps (model 300), a Gynkotek autosampler and a Jasco FP-920 fluorescence detector. The HPLC system consisted of two Genkotek high-precision pumps (model 480), a Marathon autosampler and a Shimadzu SPD-10AVP UV-VIS-detector. The HPLC system consisted of two Genkotek high-precision pumps (model 480), a Marathon autosampler and a Merck-Hitachi F1050 fluorescence detector.

1-naphthol glucuronidationPhenomenex Synergi, polair (150 × 4.6 mm, 5 μm)*10 mm phosphate buffer, containing 20% acetonitrile (pH 2.7, adjusted with 85% ortho-phosphoracid H3PO4)Fluorometric emission: 330 nm,extinction: 290 nm
β-estradiol-3-glucuronidationPhenomenex Synergi Hydro, polair (250 × 4.6 mm, 4 μm)

A: 20 mm KH2PO4 containing 25% acetonitrile

B: 100% acetonitrile

10 min A, balance up to 65% B for 4 min, 65% B for 8 min and then to 100% A in 13 min

UV 280 nm
β-estradiol-17-glucuronidation
Morphine-3-glucuronidationPhenomenex Synergi Hydro, polair (250 × 4.6 mm, 4 μm)10 mm KH2PO4, containing 5% acetonitrile (pH 2.1)UV 220 nm
4-methylumbelliferyl glucuronidationPhenomenex Synergi, polair (250 × 4.6 mm, 4 μm)

A: 10 mm ammoniumacetate: acetonitrile (9:1)

B: 20 mm ammonium acetate: acetonitrile (6:4)

10 min A, linear from A to B in 10 min, 20 min B and then 20 min A

Fluorometric

emission: 365 nm,

extinction: 315 nm

Data analysis

Data of the glucuronide formation were fitted by nonlinear regression curve fitting analysis according to the Michaelis–Menten equation by means of GraphPad Prism 6.01 software (GraphPad Software, San Diego, CA, USA). Subsequently, apparent Km and Vmax were calculated with the same software. Michaelis–Menten enzyme kinetics could not always be applied to the data of the feline liver microsomes due to the low activity of glucuronide formation, which was beneath the limit of detection at lower substrate concentrations. Therefore, the maximum activity of glucuronide formation in the feline liver microsomes was measured at substrate concentrations at which the canine microsomal activity was saturated, which is in general assumed to be at a concentration exceeding five times the Km value. Data of these maximum observed activities were expressed as means ± SD of three independent experiments with samples performed in triplicate. Data of Vmax or the maximum activity, which was measured, were analyzed using an independent two-sample Student's t-test with P < 0.05 denoting a significant difference.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

As a quality control for the enzymatic activity of the microsomal fractions, the cytochrome P450 activity was tested by testosterone metabolism, and hydroxylated metabolites were analyzed by HPLC as described by Chauret et al. with minor modifications (1997). The testosterone hydroxylation (6β-OH TST) activity in the microsomes from cats was approximately one-third of the activity in the microsomes from dogs (data not shown).

In initial experiments, the linear range for the rate of product formation vs. time was established for the incubation period and protein content for each reaction (data not shown). All data were further obtained within the linear phase of product formation.

A fast formation of N1G was observed for the dog microsomes with a Vmax of 61.6 ± 1.3 nmol/mg protein/min (see Fig. 1a). The observed data were fitted according to the equation for Michaelis–Menten enzyme kinetics, and the calculated Km and Vmax are presented in Table 3. The formation of N1G in the microsomes prepared from cat livers was much lower, but above the limit of quantification of the used analytical method. The data could, however, not be fitted according to the Michaelis–Menten equation. The maximum rate of N1G formation that was measured when incubating the feline microsomes with 750 μm 1-naphthol (the substrate concentration at which the canine microsomal activity was approximately saturated) was 1.7 ± 0.4 nmol/mg protein/min.

Table 3. Michaelis–Menten kinetics for different formed glucuronides in pooled cat (n = 8) and dog (n = 7) liver microsomes. Data represent the mean ± SD of at least two independent experiments with samples performed in duplicate
Glucuronide formedHepatic human UGT isoformCatDog
Km (μM)Vmax (nmol/mg/min)Maximum activity measured (nmol/mg/min)Km (μM)Vmax (nmol/mg/min)
  1. Due to low glucuronidation activity in the feline liver microsomes, fitting was not possible for N1G, E17G, and M3G. Therefore, the maximum glucuronidation activity in cats was measured at the substrate concentration for achieving approximately the Vmax of dogs. E17G was below the LOD. These data represent three independent investigations with samples performed in triplicate. P < 0.05 denotes a significant difference (*) of Vmax between cats and dogs. †4MU is a substrate in the human liver for UGT1A1, 1A3, 1A6, 1A9, 2B7, 2B15, and 2B17.

N1GUGT1A61.7 ± 0.445.3 ± 4.461.6 ± 1.3*
E3GUGT1A1545.2 ± 163.62.9 ± 0.2 780.8 ± 108.510.2 ± 0.4*
E17GUGT2B7<0.7287.9 ± 61.825.1 ± 1.1*
M3GUGT2B70.2 ± 0.032045 ± 206.234.0 ± 1.5*
4MUGNonselective274.3 ± 83.831.3 ± 3.3 120.5 ± 26.889.6 ± 5.2*
image

Figure 1. Enzyme kinetics for the glucuronidation of different substrates in pooled cat (○) and dog (■) liver microsomes. (a) 1-naphthol; (b) 17β-estradiol; (c) morphine; (d) 4-methylumbelliferone. Data represent the mean ± SD of at least two independent experiments with samples performed in duplicate.

Download figure to PowerPoint

The formation of E3G from estradiol was observed in both the canine and feline liver microsomes and could be fitted according to the Michaelis–Menten equation. The graphs are shown in Fig. 1b, and the calculated Km and Vmax values are given in Table 3. Vmax for the canine and feline liver microsomes was 10.2 ± 0.4 nmol/mg protein/min and 2.9 ± 0.2 nmol/mg protein/min, respectively. The formation of E17G from 17β-estradiol was only observed in the canine microsomes, with a Vmax of 25.1 ± 1.1 nmol/mg protein/min, as in cats values were below the detection limit.

Morphine was glucuronidated to M3G in dogs with a Vmax of 34.0 ± 1.5 nmol/mg protein/min. Cats almost completely lacked the capacity to form M3G as they did only produce 0.2 ± 0.03 nmol/mg protein/min after incubation with 5 mm morphine, which was the substrate concentration to obtain approximately the Vmax in dog liver microsomes. The formation of M3G from morphine could be fitted according to the Michaelis–Menten equation for dogs and is shown in Fig. 1c and Table 3. A relative low affinity can be seen for morphine in dogs compared with the other tested substrates. For cats, fitting was not possible due to the low M3G activity of the feline microsomes.

4MU is the only substrate, which is glucuronidated substantially in cats, as a Vmax of 31.3 ± 3.3 nmol/mg protein/min was found. Dogs had a Vmax of 89.6 ± 5.2 nmol/mg protein/min for the same substrate, which is not even three times higher than in cats. The formation of 4MUG from 4MU in both cats and dogs could be fitted according to the Michaelis–Menten kinetics and is shown in Fig. 1d and Table 3. It can be observed that the affinity for 4MU is lower in cats than in dogs as the Km is twice that of dogs.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

In this study the in vitro glucuronidation activity was characterized in cat liver microsomes using typical substrates for human UGT1A and 2B isozymes, as available evidence in human indicates that UGT1A1, 1A3, 1A4, 1A6, 1A9, 2B7, and 2B15 are the enzymes of greatest importance in hepatic drug and xenobiotic metabolism (Ohno & Nakajin, 2009; Harbourt et al., 2012). As a control for the assay and for comparison between species, dog liver microsomes were subjected to the same typical substrates as the microsomes from the cats. The results of the current study showed that the overall assumption of cats having a general low glucuronidation capacity needs to be refined.

Probe substrates used for testing the activity of UGT1A enzymes were 1-naphthol, 17β-estradiol, and 4-methylumbelliferone (4MU). The feline liver microsomes hardly showed glucuronidation activity for the UGT1A6 probe substrate 1-naphthol, while in the canine liver microsomes, a rapid formation of N1G was observed. The low capacity of N1G formation in cats demonstrates that cat liver indeed lacks a functional UGT1A6 homologue. The small amount of product formed in feline liver microsomes likely resulted from the activity of other UGT isoforms that commonly show an overlap in substrate specificity, although with a much lower affinity. The formation of E3G from 17β-estradiol, which in human is predominantly catalyzed by UGT1A1 (Senafi et al., 1994; Soars et al., 2004), was slower in the feline microsomes in comparison with the canine microsomes with the maximum formation rate being approximately 28% of that of the canine microsomes. The substrate 4MU is predominantly glucuronidated by human liver UGT1A6 and 1A9, but is not as specific as the other tested substrates and can therefore be seen as a substrate to test overall glucuronidation capacity (Uchaipichat et al., 2004). The maximum rate of formation of 4MUG in the feline microsomes was approximately 35% from that of the canine liver microsomes.

For the functional characterization of the UGT2B enzyme family in feline liver microsomes, the typical substrates 17β-estradiol and morphine were used. The formation of estradiol glucuronides is not only catalyzed by UGT1A1 but also by UGT2B7 (Gall et al., 1999; Soars et al., 2004) in humans resulting in the E17G product. Feline liver microsomes only formed the glucuronide E3G, but E17G could not be detected, as mentioned above. The other probe substrate for UGT2B7 in human is morphine (Coffman et al., 1997), which was used for measuring the formation of M3G. Feline liver microsomes demonstrated a very limited capacity for the formation of M3G compared with the canine liver. This almost lacking capacity of M3G and E17G formation suggests a low expression or absence of an UGT2B7 homologue in the liver of cats.

As yet, the feline homologue to the human UGT1A6 had received most attention. Studies in the 1970s already showed that cats do not form glucuronide conjugates of certain phenolic compounds in vivo, and genetic analyses by Court and Greenblatt (2000) demonstrated that the feline UGT1A6 is a pseudogene that is not translated into a functional isozyme. However, the liver microsomal activity for glucuronidation of the typical UGT1A6 probe substrate 1-naphthol had never been studied in cats in detail. The very low capacity of N1G formation that was observed in the current study confirms previous investigations (Watkins & Klaassen, 1986) and further demonstrates that the cat's liver lacks a functional UGT1A6 homologue.

In contrast, formation of glucuronides was found in substantial amounts for the substrates 17β-estradiol and 4MU in the feline liver microsomes, demonstrating that cats have functional homologue enzymes to at least the human UGT1A1 and possibly other isoforms. Previous genetic analyses suggested a low expression of a feline homologue to human UGT1A1, while another feline UGT was named UGT1A02 that was predicted to be homologue to human UGT1A2, 3, 4, and 5 based on phylogenetic analysis (Court & Greenblatt, 2000). In this respect, it is noteworthy to mention that despite a high sequence identity, human UGT1A3 and UGT1A4 are functionally very different as a resultant of only one amino acid difference (Green et al., 1998; Kubota et al., 2007). Moreover, human UGT1A4 has no activity toward 4MU, while human UGT1A3 glucuronidates 4MU but with a very low efficiency (Uchaipichat et al., 2004). Therefore, it can be concluded that a homologue to the human UGT1A3 or another not yet identified UGT, which is specific for the cat, catalyzes the 4MUG formation in the feline liver.

The absent or negligible formation of the glucuronides E17G and M3G found in feline liver microsomes indicates a lack of a functional UGT2B7 homologue in the cat's liver. In vivo investigations did also not find M3G in the blood plasma of cats following the application of morphine, nor did they find M6G in all cats in substantial amounts (Taylor et al., 2001). Previously, M3G has been found only at very low concentrations in urine and feces after administration of morphine to cats, and the major conjugated metabolite was found as morphine-3-ethereal sulfate instead (Yeh et al., 1971). An indication that cat livers do not express a functional UGT2B7 homologue is also provided by the low glucuronidation capacity of chloramphenicol, a typical substrate for human UGT2B7, in cats (Watkins & Klaassen, 1986).

In this study, a comparison between cat and dog liver microsomes was prepared using liver specimen of Beagle dogs. This selection was prepared in consideration of the broad use of Beagles in drug research. However, the limitations of such an approach are obvious, as variations in glucuronidation capacity can be assumed comparable with those described for differences in cytochrome P450 activity across canine breeds (Martinez et al., 2013).

Our results suggest that the cat does not have functional homologues to the human UGT1A6 and UGT2B7, while glucuronidation capacity in the liver is present that may be catalyzed by UGTs that have functional similarities to the human UGT1A1 and possibly other UGT1A isozymes. Indeed, there are some examples of drugs that are well glucuronidated by cats: phenolphthalein (Pekanmaki & Salmi, 1961; Watkins & Klaassen, 1986), lorazepam (Schillings et al., 1975; Elliott, 1976; Ruelius, 1978), pradofloxacin (EMEA/V/C/099, 2007), ibuprofen (Magdalou et al., 1990), and telmisartan (Ebner et al., 2013) can be found as glucuronides in cats. Telmisartan and ibuprofen are even glucuronidated with a higher rate in feline hepatic microsomes than in other species. Although it is not known which UGT isozymes are involved in the metabolism of all these drugs, for the most recently investigated compound telmisartan in cats, it is known that in human hepatocytes, this drug is metabolized mainly by UGT1A3 (Yamada et al., 2011).

In conclusion, the present functional data indicate that the cat has a number of functional UGT1A enzymes that are homologues to the human UGT1A isoforms. Other glucuronidation enzymes in the cat remain to be characterized in more detail. This implies that for new drugs, glucuronidation capacity has to be investigated on a substance-to-substance base to provide the basis for PK-modeling to establish proper dosage regimens in cats and to avoid undesirable drug toxicity.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

The authors especially thank L.B. Chaigneau and J.L. de Nijs-Tjon for their technical assistance.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  • Bosma, P.J., Seppen, J., Goldhoorn, B., Bakker, C., Oude Elferink, R.P., Chowdhury, J.R., Chowdhury, N.R. & Jansen, P.L. (1994) Bilirubin UDP-glucuronosyltransferase 1 is the only relevant bilirubin glucuronidating isoform in man. Journal of Biological Chemistry, 269, 1796017964.
  • Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72, 248254.
  • Capel, I.D., French, M.R., Millburn, P., Smith, R.L. & Williams, R.T. (1972a) The fate of (14C)phenol in various species. Xenobiotica, 2, 2534.
  • Capel, I.D., French, M.R., Millburn, P., Smith, R.L. & Williams, R.T. (1972b) Species variations in the metabolism of phenol. Biochemical Journal, 127, 25P26P.
  • Capel, I.D., Millburn, P. & Williams, R.T. (1974) The conjugation of 1- and 2-naphthols and other phenols in the cat and pig. Xenobiotica, 4, 601615.
  • Chauret, N., Gauthier, A., Martin, J. & Nicoll-Griffith, D.A. (1997) In vitro comparison of cytochrome P450-mediated metabolic activities in human, dog, cat, and horse. Drug Metabolism and Disposition, 25, 11301136.
  • Coffman, B.L., Rios, G.R., King, C.D. & Tephly, T.R. (1997) Human UGT2B7 catalyzes morphine glucuronidation. Drug Metabolism and Disposition, 25, 14.
  • Court, M.H. (2005) Isoform-selective probe substrates for in vitro studies of human UDP-glucuronosyltransferases. Methods in Enzymology, 400, 104116.
  • Court, M.H. & Greenblatt, D.J. (1997) Molecular basis for deficient acetaminophen glucuronidation in cats. An interspecies comparison of enzyme kinetics in liver microsomes. Biochemical Pharmacology, 53, 10411047.
  • Court, M.H. & Greenblatt, D.J. (2000) Molecular genetic basis for deficient acetaminophen glucuronidation by cats: UGT1A6 is a pseudogene, and evidence for reduced diversity of expressed hepatic UGT1A isoforms. Pharmacogenetics, 10, 355369.
  • Dong, D., Ako, R., Hu, M. & Wu, B. (2012) Understanding substrate selectivity of human UDP-glucuronosyltransferases through QSAR modeling and analysis of homologous enzymes. Xenobiotica, 42, 808820.
  • Ebner, T. & Burchell, B. (1993) Substrate specificities of two stably expressed human liver UDP-glucuronosyltransferases of the UGT1 gene family. Drug Metabolism and Disposition, 21, 5055.
  • Ebner, T., Schanzle, G., Weber, W., Sent, U. & Elliott, J. (2013) In vitro glucuronidation of the angiotensin II receptor antagonist telmisartan in the cat: a comparison with other species. Journal of Veterinary Pharmacology and Therapeutics, 36, 154160.
  • Elliott, H.W. (1976) Metabolism of lorazepam. British Journal of Anaesthesia, 48, 10171023.
  • Furlan, V., Demirdjian, S., Bourdon, O., Magdalou, J. & Taburet, A.M. (1999) Glucuronidation of drugs by hepatic microsomes derived from healthy and cirrhotic human livers. Journal of Pharmacology and Experimental Therapeutics, 289, 11691175.
  • Gall, W.E., Zawada, G., Mojarrabi, B., Tephly, T.R., Green, M.D., Coffman, B.L., Mackenzie, P.I. & Radominska-Pandya, A. (1999) Differential glucuronidation of bile acids, androgens and estrogens by human UGT1A3 and 2B7. Journal of Steroid Biochemistry and Molecular Biology, 70, 101108.
  • Green, M.D. & Tephly, T.R. (1996) Glucuronidation of amines and hydroxylated xenobiotics and endobiotics catalyzed by expressed human UGT1.4 protein. Drug Metabolism and Disposition, 24, 356363.
  • Green, M.D., King, C.D., Mojarrabi, B., Mackenzie, P.I. & Tephly, T.R. (1998) Glucuronidation of amines and other xenobiotics catalyzed by expressed human UDP-glucuronosyltransferase 1A3. Drug Metabolism and Disposition, 26, 507512.
  • Harbourt, D.E., Fallon, J.K., Ito, S., Baba, T., Ritter, J.K., Glish, G.L. & Smith, P.C. (2012) Quantification of human uridine-diphosphate glucuronosyl transferase 1A isoforms in liver, intestine, and kidney using nanobore liquid chromatography-tandem mass spectrometry. Analytical Chemistry, 84, 98105.
  • Hashizume, T., Xu, Y., Mohutsky, M.A., Alberts, J., Hadden, C., Kalhorn, T.F., Isoherranen, N., Shuhart, M.C. & Thummel, K.E. (2008) Identification of human UDP-glucuronosyltransferases catalyzing hepatic 1alpha,25-dihydroxyvitamin D3 conjugation. Biochemical Pharmacology, 75, 12401250.
  • Jin, C., Miners, J.O., Lillywhite, K.J. & Mackenzie, P.I. (1993) Complementary deoxyribonucleic acid cloning and expression of a human liver uridine diphosphate-glucuronosyltransferase glucuronidating carboxylic acid-containing drugs. Journal of Pharmacology and Experimental Therapeutics, 264, 475479.
  • Kiang, T.K., Ensom, M.H. & Chang, T.K. (2005) UDP-glucuronosyltransferases and clinical drug-drug interactions. Pharmacology & therapeutics, 106, 97132.
  • King, C., Finley, B. & Franklin, R. (2000) The glucuronidation of morphine by dog liver microsomes: identification of morphine-6-O-glucuronide. Drug Metabolism and Disposition, 28, 661663.
  • Kubota, T., Lewis, B.C., Elliot, D.J., Mackenzie, P.I. & Miners, J.O. (2007) Critical roles of residues 36 and 40 in the phenol and tertiary amine aglycone substrate selectivities of UDP-glucuronosyltransferases 1A3 and 1A4. Molecular Pharmacology, 72, 10541062.
  • Mackenzie, P.I., Walter Bock, K., Burchell, B., Guillemette, C., Ikushiro, S., Iyanagi, T., Miners, J.O., Owens, I.S. & Nebert, D.W. (2005) Nomenclature update for the mammalian UDP glycosyltransferase (UGT) gene superfamily. Pharmacogenetics and Genomics, 15, 677685.
  • Magdalou, J., Chajes, V., Lafaurie, C. & Siest, G. (1990) Glucuronidation of 2-arylpropionic acids pirprofen, flurbiprofen, and ibuprofen by liver microsomes. Drug Metabolism and Disposition, 18, 692697.
  • Mano, Y., Usui, T. & Kamimura, H. (2004) Effects of beta-estradiol and propofol on the 4-methylumbelliferone glucuronidation in recombinant human UGT isozymes 1A1, 1A8 and 1A9. Biopharmaceutics & Drug Disposition, 25, 339344.
  • Martinez, M.N., Antonovic, L., Court, M., Dacasto, M., Fink-Gremmels, J., Kukanich, B., Locuson, C., Mealey, K., Myers, M.J. & Trepanier, L. (2013) Challenges in exploring the cytochrome P450 system as a source of variation in canine drug pharmacokinetics. Drug metabolism reviews, 45, 218230.
  • Miners, J.O. & Mackenzie, P.I. (1991) Drug glucuronidation in humans. Pharmacology & therapeutics, 51, 347369.
  • Miners, J.O., Lillywhite, K.J. & Birkett, D.J. (1988) In vitro evidence for the involvement of at least two forms of human liver UDP-glucuronosyltransferase in morphine 3-glucuronidation. Biochemical Pharmacology, 37, 28392845.
  • Miners, J.O., Smith, P.A., Sorich, M.J., McKinnon, R.A. & Mackenzie, P.I. (2004) Predicting human drug glucuronidation parameters: application of in vitro and in silico modeling approaches. Annual Review of Pharmacology and Toxicology, 44, 125.
  • Ohno, S. & Nakajin, S. (2009) Determination of mRNA expression of human UDP-glucuronosyltransferases and application for localization in various human tissues by real-time reverse transcriptase-polymerase chain reaction. Drug Metabolism and Disposition, 37, 3240.
  • Pekanmaki, K. & Salmi, H.A. (1961) The absorption and excretion of phenolphthalein and its glucuronide by the cat. Acta Pharmacologica et Toxicologica, 18, 133140.
  • Radominska-Pandya, A., Czernik, P.J., Little, J.M., Battaglia, E. & Mackenzie, P.I. (1999) Structural and functional studies of UDP-glucuronosyltransferases. Drug Metabolism Reviews, 31, 817899.
  • Rowland, A., Miners, J.O. & Mackenzie, P.I. (2013) The UDP-Glucuronosyltransferases: their role in drug metabolism and detoxification. The international journal of biochemistry & cell biology, 45, 11211132.
  • Ruelius, H.W. (1978) Comparative metabolism of lorazepam in man and four animal species. The Journal of Clinical Psychiatry, 39, 1115.
  • Rutten, A.A., Falke, H.E., Catsburg, J.F., Topp, R., Blaauboer, B.J., van Holsteijn, I., Doorn, L. & van Leeuwen, F.X. (1987) Interlaboratory comparison of total cytochrome P-450 and protein determinations in rat liver microsomes. Reinvestigation of assay conditions. Archives of Toxicology, 61, 2733.
  • Schillings, R.T., Sisenwine, S.F., Schwartz, M.H. & Ruelius, H.W. (1975) Lorazepam: glucuronide formation in the cat. Drug Metabolism and Disposition, 3, 8588.
  • Senafi, S.B., Clarke, D.J. & Burchell, B. (1994) Investigation of the substrate specificity of a cloned expressed human bilirubin UDP-glucuronosyltransferase: UDP-sugar specificity and involvement in steroid and xenobiotic glucuronidation. Biochemical Journal, 303(Pt 1), 233240.
  • Soars, M.G., Petullo, D.M., Eckstein, J.A., Kasper, S.C. & Wrighton, S.A. (2004) An assessment of udp-glucuronosyltransferase induction using primary human hepatocytes. Drug Metabolism and Disposition, 32, 140148.
  • Stone, A.N., Mackenzie, P.I., Galetin, A., Houston, J.B. & Miners, J.O. (2003) Isoform selectivity and kinetics of morphine 3- and 6-glucuronidation by human udp-glucuronosyltransferases: evidence for atypical glucuronidation kinetics by UGT2B7. Drug Metabolism and Disposition, 31, 10861089.
  • Taylor, P.M., Robertson, S.A., Dixon, M.J., Ruprah, M., Sear, J.W., Lascelles, B.D., Waters, C. & Bloomfield, M. (2001) Morphine, pethidine and buprenorphine disposition in the cat. Journal of Veterinary Pharmacology and Therapeutics, 24, 391398.
  • Tukey, R.H. & Strassburg, C.P. (2000) Human UDP-glucuronosyltransferases: metabolism, expression, and disease. Annual Review of Pharmacology and Toxicology, 40, 581616.
  • Uchaipichat, V., Mackenzie, P.I., Guo, X.H., Gardner-Stephen, D., Galetin, A., Houston, J.B. & Miners, J.O. (2004) Human udp-glucuronosyltransferases: isoform selectivity and kinetics of 4-methylumbelliferone and 1-naphthol glucuronidation, effects of organic solvents, and inhibition by diclofenac and probenecid. Drug Metabolism and Disposition, 32, 413423.
  • Watkins, J.B., 3rd & Klaassen, C.D. (1986) Xenobiotic biotransformation in livestock: comparison to other species commonly used in toxicity testing. Journal of animal science, 63, 933942.
  • Yamada, A., Maeda, K., Ishiguro, N., Tsuda, Y., Igarashi, T., Ebner, T., Roth, W., Ikushiro, S. & Sugiyama, Y. (2011) The impact of pharmacogenetics of metabolic enzymes and transporters on the pharmacokinetics of telmisartan in healthy volunteers. Pharmacogenetics and Genomics, 21, 523530.
  • Yeh, S.Y., Chernov, H.I. & Woods, L.A. (1971) Metabolism of morphine by cats. Journal of Pharmaceutical Sciences, 60, 469471.