Kinetic simulation method of C-reactive protein in beagle dogs during acute inflammation

Abstract

The half-life (t_1/2) of C-reactive protein (CRP) and its ability to stimulate weak inflammatory responses were investigated in beagle dogs. Four beagle dogs were administered 20 mg/kg indomethacin and blood was collected from the cephalic vein pre-dosing and at 24, 48, 72, 96, 144, 192, 240, 312, and 360 h post-administration. The serum concentrations of CRP were measured by enzyme-linked immunosorbent assay. The serum t_1/2 was calculated using the equation 0.693/elimination rate constant. The serum concentration of CRP beyond 192 h post-administration declined to levels in the normal range. The t_1/2 was 148.3 h, which is considered to be the essential t_1/2 of CRP. The simulation of CRP serum concentrations at arbitrary times using the elimination rate constant obtained in this study became possible.

Keywords

Beagle dogs CRP elimination rate constant simulation

Introduction

C-reactive protein (CRP) is a typical acute-phase protein produced by the liver in response to inflammation in mammals, including canines, and serum concentrations of CRP increase during acute inflammation.^1–4 CRP is a useful inflammatory marker in veterinary clinical medicine and non-clinical studies.^5–7 It has been reported that the half-life of CRP is 161 h in beagle dogs following oral administration of 60 mg/kg indomethacin.⁸ Vomiting and blood in the feces were observed in beagle dogs administered high dose of indomethacin, which are considered to be indicators of the induction of severe acute inflammation.⁸ The serum CRP concentration reflects the status of inflammation, and high concentrations are maintained during severe inflammation.⁹ As a result, the calculation of the extended half-life of CRP is thought to be intrinsically problematic. In this study, the half-life of CRP in beagle dogs following administration of a low dose of indomethacin was estimated in an effort to avoid discrepancies in half-life affected by the degree of inflammatory stimulation.

Materials and methods

Animal experiment

Four beagle dogs (body weight from 11 to 15 kg) were used in this study. Indomethacin (ICN Biomedicals, Inc., OH, USA) was suspended at 20 mg/mL in 0.5% carboxymethyl cellulose (Wako Pure Chemical Industries, Ltd., Osaka, Japan). Indomethacin was orally administered at a dose of 1 mL/kg after fasting for 18 h. Blood was collected from the cephalic vein pre-administration and at 24, 48, 72, 96, 144, 192, 240, 312, and 360 h post-administration. Serum was stored at −80°C until analysis. This animal experiment was approved by the Institutional Animal Care and Use Committee of Azabu University (approval no. 1606201).

Measurement of CRP

Serum CRP concentrations were measured by enzyme-linked immunosorbent assay according to the procedure of Yamamoto et al.¹⁰

Analysis of kinetic parameters

The maximum serum concentrations (C_max) of CRP were used for analysis. The linear slope of serum CRP concentration versus time was plotted on a log-linear regression.¹¹ The elimination rate constant (K) was calculated using a minimum of three measured serum concentrations. Half-life (t_1/2) was calculated from the formula

Slope = \frac{\log C^{A} - \log C^{B}}{Time (A) - Time (B)}

K (h^{- 1}) = (- 2.303) \times slope

t_{1 / 2} (h) = 0.693 / K

where K is the elimination rate constant, C^A is the serum concentration at Time (A), and C^B is serum concentration at Time (B)

Results

The change in serum concentrations and kinetic parameters of CRP are shown in Figure 1 and Table 1, respectively. Administration of 20 mg/kg indomethacin produced a mean CRP C_max that was one-seventh that observed following administration of 60 mg/kg indomethacin.⁸ This result indicated that the degree of inflammatory stimulation in this study was weak. The serum concentrations of CRP exhibited biphasic alterations following the peak concentration. The first phase (α-phase) of CRP was from peak concentration to 192 h and the terminal phase (β-phase) was from 192 and 360 h after administration of indomethacin. The mean t_1/2 of the α- and β-phases was 148.3 and 510.3 h, respectively. The mean K of the α- and β-phases was 0.0049 and 0.0022 h⁻¹, respectively.

Figure 1.

Serum concentrations of C-reactive protein (CRP) in beagle dogs after administration of indomethacin (Dose: 20mg/kg). Each point was represented mean ± standard deviation (n=4).

Table 1.

Kinetic parameters of C-reactive protein in beagle dogs administered with indomethacin (dose: 20 mg/kg).

C_max (µg/mL)	C_max/pre	α-phase		β-phase
		α (h⁻¹)	t_1/2α (h)	β (h⁻¹)	t_1/2β (h)
21.8 ± 5.7	8.4 ± 5.1	0.0049 ± 0.0010	148.3 ± 35.4	0.0022 ± 0.0022	510.3 ± 295.0

Each data were represented mean ± standard deviation (n = 4).

Discussion

The elimination rate of CRP in beagle dogs was estimated following induction of acute inflammation by weak immune stimulation. The serum concentration of CRP in healthy beagle dogs is reported to range from 8.0 to 10.0 µg/mL.^12,13 The serum concentrations of CRP beyond 192 h post-administration were found to be approximately 10 µg/mL. These results suggest that the serum concentration of CRP beyond 192 h declined to a normal concentration range. The t_1/2 of the β-phase was considered to be within the normal range and not consistent with the overall t_1/2 of CRP. Conversely, the t_1/2 of α-phase was very similar to the t_1/2 in beagle dogs observed following administration of 60 mg/kg indomethacin. Thus, the t_1/2 of the α-phase was considered to be the essential t_1/2 of CRP in beagle dogs. The t_1/2 of CRP in mice and humans is reported to be 4 and 18 h, respectively.^14,15 These parameters were calculated based on changes in serum concentrations following intravenous injection. Generally, CRP decreased in two phases—a distribution phase and an elimination phase—after intravenous injection, with the t_1/2 of the distribution phase being shorter than that of the elimination phase.^16,17 Previously, serum concentrations of CRP in mice and humans were measured until 72 and 96 h, respectively.^14,15 Thus, the t_1/2 of CRP in mice and humans was calculated using shorter periods than in this study of beagle dogs. As a consequence, it was presumed that the t_1/2 of CRP reported for mice and humans reflects the distribution phase, which accounts for the observed differences in CRP t_1/2 between beagle dogs and mice or humans.

In a previous study, the simulation of serum CRP concentrations during acute inflammation was performed from 96 to 192 h.⁸ However, the half-life in the terminal elimination phase was calculated using serum concentrations from 96 h onward in beagle dogs with weak inflammatory stimulation. Accordingly, optimal serum concentrations after 96 h can be estimated using the following formula

C^{1} = C^{2} \times \exp (- 0.0049 t)

where C¹ is the simulated serum concentration at the optimal time, C² is the serum concentration at 96 h after inflammatory stimulation, exp is the base of natural logarithm, and an elimination rate constant used is 0.0049, obtained in this study.

Thus, if the actual serum concentration of CRP indicated the stimulation of high concentrations, the recovery from acute inflammation is presumed to be caused by delayed effects, such as postoperative complications and suture-related complications.

In conclusion, the t_1/2 of CRP was estimated to be 148.3 h. Moreover, using the elimination rate obtained in this study, it is possible to simulate the serum concentrations of CRP at optimal times after 96 h of inflammatory stimulation during acute inflammation, such as during a surgical operation. The comparison between the actual measured value and simulated values is useful in determining the status of acute inflammation. Thus, this kinetic simulation method of plasma CRP concentrations is expected to be useful for non-clinical and clinical studies.

Footnotes

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

References

Yamamoto

Shida

Miyaji

et al . (1993) Changes in serum C-reactive protein levels in dogs with various disorders and surgical traumas. Veterinary Research Communications 17: 85–93.

Burton

Honor

Mackenzie

et al . (1994) C-reactive protein concentration in dogs with inflammatory leukograms. American Journal of Veterinary Research 55: 613–618.

Sheahan

Bell

Mellanby

et al . (2010) Acute phase protein concentrations in dogs with nasal disease. Veterinary Record 168: 865–899.

Chase

McLauchlan

Eckersall

et al . (2012) Acute phase protein levels in dogs with mast cell tumours and sarcomas. Veterinary Record 170: 648.

Cerón

Eckersall

Martínez-Subiela

(2005) Acute phase proteins in dogs and cats: Current knowledge and future perspective. Veterinary Clinical Pathology 34: 85–99.

Nielsen

Toft

Eckersall

et al . (2007) Serum C-reactive protein concentration as an indicator of remission status in dogs with multicentric lymphoma. Journal of Veterinary Internal Medicine 21: 1231–1236.

Kjelgaard-Hansen

Strom

Mikkelsen

et al . (2013) Canine serum C-reactive protein as a quantitative marker of the inflammatory stimulus of aseptic soft tissue surgery. Veterinary Clinical Pathology 42: 342–345.

Kuribayashi

Seita

Momotani

et al . (2015) Elimination half-lives of acute phase proteins in rats and beagle dogs during acute inflammation. Inflammation 38: 1401–1405.

Otabe

Ito

Sugimoto

et al . (2000) C-reactive protein (CRP) measurement in canine serum following experimentally-induced acute gastric mucosal injury. Laboratory Animals 34: 434–438.

10.

Yamamoto

Shida

Okimura

et al . (1994) Determination of C-reactive protein in serum and plasma from healthy dogs and dogs with pneumonia by ELISA and slide reversed passive latex agglutination test. Veterinary Quarterly 16: 74–77.

11.

Veilleux-Lemieux

Castel

Carrier

et al . (2013) Pharmacokinetics of ketamine and xylazine in young and old Sprague-Dawley rats. Journal of the American Association for Laboratory Animal Science 52: 567–570.

12.

Otabe

Sugimoto

Jinbo

et al . (1998) Physiological levels of C-reactive protein in normal canine sera. Veterinary Research Communications 22: 77–85.

13.

Kuribayashi

Shimada

Matsumoto

et al . (2003) Determination of serum C-reactive protein (CRP) in healthy beagle dogs of various ages and pregnant beagle dogs. Experimental Animals 52: 387–390.

14.

Baltz

Rowe

Pepys

(1985) In vivo turnover studies of C-reactive protein. Clinical and Experimental Immunology 59: 243–250.

15.

Vigushin

Pepys

Hawkins

(1993) Metabolic and scintigraphic studies of radioiodinated human C-reactive protein in health and disease. Journal of Clinical Investigation 91: 1351–1357.

16.

Lee

DeVito

Vercelli

et al . (2017) Ex vivo antibacterial activity of levofloxacin against Escherichia coli and its pharmacokinetic profile following intravenous and oral administrations in broilers. Research in Veterinary Science 112: 26–33.

17.

Scoble

Owens

Jr Puttagunta

et al . (2015) Pharmacokinetics, safety, and tolerability of a single 500-mg or 1000-mg intravenous dose of Dalbavancin in healthy Japanese subjects. Clinical Drug Investigation 35: 785–793.