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.

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

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

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

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

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).

Cmax (µg/mL)	Cmax/pre	α-phase		β-phase
		α (h−1)	t1/2α (h)	β (h−1)	t1/2β (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

$$C^1=C^2\times \exp \left(-0.0049\mathrm{t}\right)$$

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.