Microchim Acta (2007) 159: 223–228 DOI 10.1007/s00604-006-0699-2 Printed in The Netherlands
Original Paper Microdialysis sampling and chemiluminescence detection for in vivo and real-time study of the lead metabolism in rabbit blood Xiao Fang Hou, Zhu Jun Zhang , Yan Zhao, and Jie Ma School of Chemistry & Materials Science, Shaanxi Normal University, Xi’an 710062, Shaanxi, China Received May 29, 2006; accepted September 11, 2006; published online December 28, 2006 # Springer-Verlag 2006
Abstract. The chemiluminescence (CL) system K2MnO4-luminol is shown to be useful for the determination of lead(II). The method is based on the catalytic effect of Pb(II) on the CL reaction. The linear range was 3103 –9101 mg L1 (r ¼ 0.9971) and the relative standard deviation (R.S.D.) for 5102 mg L1 Pb(II) measurement was 0.7% (n ¼ 11). The detection limit was 3 104 mg L1 (3) Pb(II). Based on this, an in vivo, on-line, real-time analytical system for monitoring the metabolism of free lead(II) in rabbit blood was developed. A microdialysis probe, implanted in the vein of a rabbit, was perfused with perfusate at a flow rate of 5 mL min1 . The concentration of free lead(II) in the dialysate was determined on-line with a flow-injection CL system. This system included microdialysis sampling, on-line separation and chemiluminescence detection. The concentration-time curve of lead(II) was in accordance with the one-compartmental open model, T1=2 (elimination half-life), Tmax (peak time) and Cmax (peak concentration) were 37.77 min, 85.20 min and 0.137 mg L1 , respectively. Key words: Microdialysis; chemiluminescence; lead(II); metabolism.
Lead is a ubiquitous environmental pollutant, which causes great damage to human health [1]. Environment lead enters the human body mainly from the Author for correspondence. E-mail:
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digestive tract and the respiratory tract. Lead absorbed by the duodenum and lung alveoli will be distributed throughout the body following the blood circulation. Most of the blood lead (90%) that binds with red blood cells (RBC) becomes indiffusible lead and balances with plasma blood. However, the plasma blood is composed of lead that binds with protein in plasma and diffusible lead. Although the latter is very low, they are active centers in the lead metabolism of the body. They can combine with quite small biomolecules and go through capillary vessels freely. When they penetrate the pericellular membrane and enter the central nervous, immune, endocrine and cardiovascular system and the liver, kidney and essential cells of other organs, this will cause great and irreversible harm to the human body [2]. Therefore, in vivo and real-time monitoring of the metabolism of diffusible lead(II) in blood is a great importance. A number of existing analytical methods for the determination of blood lead(II) have been published, such as atomic absorption spectrometry [3, 4], isotope dilution inductively coupled plasma mass spectrometry [5], and capillary zone electrophoresis [6]. Regrettably, hardly any of these methods reported have ever been applied to in vivo and real-time monitoring of the lead metabolism of blood. Chemiluminescence (CL) is an attractive detection method for analytical determination because of the very low detection limits and wide linear working range
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that can be achieved while using relatively simple instrumentation [7]. Microdialysis (MD) can reflect the bioprocess without necessarily depleting the process under investigation [8]. An analytical system for monitoring the metabolism of free lead(II) in vivo and in real-time in rabbit blood was developed in this study. This system includes microdialysis sampling, on-line separation and chemiluminescence detection. A few reports on the chemiluminescence of Mn(VI) with flow-injection analysis have been reported. Xu et al. were the first in 2001 to propose that lead(II) could enhance the chemiluminescence signal of K2MnO4 and luminol [9]. In 2004 Du et al. also found that alkaline earth metal ions could enhance the chemiluminescence system of K2MnO4-luminol [10]. However, in their study, K2MnO4 was made indirectly following the reaction of K2MnO4 and luminol, which leads to a strong background and affects the detection limit. In order to lower the background and increase the signal to noise ratio, K2MnO4 was synthesized and applied directly to the analytical system of K2MnO4luminol-Pb for determination of Pb(II) in their paper.
Experimental Reagents Stock standard solution of lead(II) (1.0 103 mg L1 ) was prepared by dissolving 1.598 g Pb(NO3)2 in 2 mL of 10% HNO3 and diluting to 1000 mL with Ringer’s solution (147.1 mM NaCl, 4.0 mM KCl, 2.3 mM CaCl2, pH 7.4) [11]. Testing standard solutions of lead nitrate were prepared by appropriate dilution of the stock solution with Ringer’s solution. A stock solution of luminol (1.0 102 M) was prepared by dissolving 1.7772 g of luminol in 1 L of 0.5 M NaOH solution. Work solutions of luminol (5 104 M) were prepared from the stock solution of luminol by appropriate dilution in KOH (pH ¼ 11). Heparin sodium injection (12,500 IU) was purchased from Biochemical Pharmaceutical Co. (Jiangsu, China). Prior to use, the heparin sodium injection was diluted to 5000 IU using physiological salt solution. Potassium manganate (K2MnO4), a green substance, was obtained after about 25 min when melting manganese(IV) oxide (2.175 g) with excess potassium hydroxide. In order to avoid interference with the CL reaction with luminol, oxidizing agents such as potassium nitrate (KNO3) or potassium perchlorate (KClO4) should not be added. Potassium manganate is soluble in strong alkali (pH > 13) only, otherwise it will disproportionate into potassium permanganate (KMnO4) and manganese(IV) oxide (MnO2). So after cooling, 80 mL 2 mol L1 KOH is required to dissolve the solid potassium manganate. Subsequently, a centrifuge with 3000 rpm is needed to filter solid manganese(IV) oxide (MnO2). The concentration of K2MnO4 is identified by titration with oxalate. The final result of 0.027 mol L1 was the average value of three measurements. K2MnO4 (5 104 M) was diluted with potassium hydroxide (pH ¼ 13) from the original solution during the time of the experiment.
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Fig. 1. Schematic diagram of the study of the metabolism of free lead(II) in rabbit blood using flow-injection CL detection with online microdialysis sampling. SP Microdialysis syringe pump; TCIII Model TCI-II syringe micropump controller system; M microdialysis probe; R1 water carrier; R2 luminol solution (in KOH); R3 K2MnO4 solution; P peristaltic pump; V injection valve; F flow cell; PMT photomultiplier tube; HV negative high-voltage supply Apparatus The microdialysis sampling system was composed of a TCI-II model microinjection pump (Beijing Silugao High Technology Development Co. Ltd., Beijing, China, http:==www.medone.cn=co.asp?id¼473), a 5 mL non-metallic gas-tight syringe filled with Ringer’s solution and home-made microdialysis probes (0.15 mm i.d., 0.25 mm o.d., 2 cm membrane length, exclusion limit 10,000 Da) the material of which was obtained from Xi’an Kangpei New Technology Co. (Xi’an, China). The microdialysis manifold is shown schematically in Fig. 1. CL measurements were performed using an IFFM-D type flow injection CL analyzer (Xi’an REMEX Electronic Science-Techno, Ltd., Xi’an, China, http:==remex.instrument.com.cn). The emitted CL was collected with a CR-105 photomultiplier tube (operated at 700V, Hammamatsu, Tokyo, Japan) of the CL analyzer. Data acquisition and treatment were performed with IFFM-D software running under Windows XP. The flow system (Fig. 1) consisted of two peristaltic pumps, which delivered each reagent solution at an equally slow rate (2.5 mL min1 ) through flow tubes, and a six-way injection valve with a 50 mL loop, through which the sample solution was injected into the carrier stream. The flow cell was made by coiling 30 cm of colorless glass tube (1 mm i.d. and 2 mm o.d.) into a spiral disk shape with a diameter of 2 cm and placing it close to the window of the photomultiplier tube. PTFE tubing of 0.25 mm i.d. and 0.8 mm o.d. was used for all connections. A model SHH W21 402 super thermostat water bath (YuYao XinBo Instrument Co. Ltd, YuYao, China, http:==www.cnxinbo. com) was used. Procedures Establishment of the CL system of lead determination Flow lines were inserted into luminol solution, K2MnO4 solution, and water carrier, respectively. With the injection valve in the load position, the pump was started at a constant speed of 2.5 mL min1 on each flow line to wash the whole flow system until a stable baseline was recorded. The 50 mL lead nitrate solution was injected into the carrier stream (water) through the injection valve. This stream was merged with the mixture of luminol and K2MnO4 solution in the flow cell, producing CL emission. The CL emission intensities vs. lead nitrate concentrations were used for the calibration. Optimization of microdialysis system In this paper, the dialytic efficiency of the microdialysis probe was required to assess the free concentration of lead(II) in the rabbit blood. Therefore, the dialytic efficiency of the probe must be investigated prior to using the microdialysis probe. It is often influenced
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by various factors, such as temperature, perfusion flow rate, probe membrane length type of analyte, and sample composition (ionic strength, pH etc.) [12]. When the probe and analyte were fixed, temperature and perfusion flow rate became two important factors determining the dialytic efficiency of the microdialysis probe [13]. Calibration experiments of dialytic efficiency can be carried out in vivo and in vitro. The purpose of our experiment was to obtain the concentration-time curve of lead(II) in blood, not the concentration; Moreover, our previous research revealed that the dialytic efficiency obtained in vitro was almost the same as the one obtained in vivo using no net flux method [14], so we chose the in vitro calibration. The relative dialytic efficiency RDE was estimated according to the following equation: RDE ¼ Cout =Cin where Cout was the lead(II) concentration in microdialysate, and Cin was the lead(II) concentration surrounding the probe or the free lead(II) in blood. Prior to use, the probe was immersed in perfusion medium at 37 C for 24 h. In these experiments, aqueous standards were used instead of real samples from test animals. The aqueous standards were prepared by diluting stock standards with Ringer’s solution, so that the aqueous standards had the same osmotic pressure and pH as real samples from test animals. Calibration experiments of dialytic efficiency were carried out by placing a 2 cm probe successively into three 50 mL lead nitrate solutions (0.8, 1.0, 1.2 mg L1 ) that were maintained at 37 C in a thermostated water bath. The probe was perfused with Ringer’s solution at a speed of 5 mL min1 .
Determination of free lead(II) in rabbit blood Male specific pathogen-free rabbit (1.5 kg) was purchased from the center of feeding rabbits in Xi’an. The rabbit had free access to food and water until 12 h prior to being used in the experiment, at which time only the food was removed. The rabbit was fixed on a wooden dissecting plate (home-made) without anesthetization. About 20 min before the experiments, the rabbit was given an intravenous injection of 5000 IU of heparin sodium injection via the edge vein of the test rabbit’s ear. The sterilized microdialysis probe was inserted into the edge vein of the test rabbit’s other ear. Then the microdialysis probe was connected with the pump and valve and perfused with the perfusion solution at a flow rate of 5 mL min1 . Pb(NO3)2 (0.75 g) was administered orally. Dialysates of about 50 mL were injected at 30, 50, 65, 85, 105, 150, 195, 230 min following the lead nitrate administered. The concentrations of free lead(II) in rabbit blood dialysates were determined from the calibration graphs. The concentrations of free lead(II) in rabbit blood were calculated from the concentrations of lead(II) in the dialysates by the following equation: Cin ¼ Cout=RDE. The metabolism parameters of lead nitrate were obtained by treating the observed data utilizing the pharmacokinetic calculation software ‘DAS ver1.0’ (China Pharmaceutical University, P.R. China).
Fig. 2. Effect of luminol pH on the CL system (CPb2þ 0.1 mg L1 , CK2 MnO4 0.2 mM, Cluminol 0.5 mM)
KOH and NaOH were introduced into the CL system. The effect of the pH of KOH and NaOH in the range of 10.5–13 was investigated for 0.1 mg L1 lead(II). The results showed that KOH promoted a greater CL intensity than NaOH. Moreover, maximum CL emission was observed at pH 11, as shown in Fig. 2. So this pH was chosen for subsequent experiments. The effect of luminol concentration was examined in the range of 3.0105 –7.0104 mol L1 . The dependence of the CL intensity on the concentration of luminol was investigated for 0.1 mg L1 lead(II). It was found that the highest CL intensity was obtained when the concentration of luminol was 5104 mol L1 . Effect of K2MnO4 concentration on the CL intensity The effect of K2MnO4 concentration was investigated from 3105 to 5104 mol L1 (Fig. 3). The result showed that the CL intensity increased with increasing K2MnO4 concentration. However, while the K2MnO4 concentration was higher than 1104 mol L1 , the
Results and discussions Conditions of the CL detection system Effect of pH and concentration of luminol on the CL intensity Preliminary assays showed that the CL emission of luminol evolved in the basic medium [15]. Therefore,
Fig. 3. Effect of K2MnO4 concentration on the CL system (CPb2þ 0.1 mg L1 , Cluminol 0.5 mM, pH 11)
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CL intensity declined slowly. Therefore, 1 104 mol L1 K2MnO4 was chosen as the most suitable concentration for further studies. Effect of flow rate on the CL intensity The flow rate is an important factor in the flow injection CL system. Therefore, an optimum flow rate is necessary to collect the maximum emitted light in the flow cell to deliver the solutions. The effect of the flow rate on the CL emission was tested in the range of 0.3–3.0 mL min1 . The result showed that the highest CL intensity was achieved when the flow rate of each line was fed at 2.5 mL min1 . Hence, a flow rate of 2.5 mL min1 was selected in the present study.
Fig. 4. Effect of perfusate flow rate on the recovery rate and determination time
Interference study The influence of foreign species was studied by analyzing a solution of 5.0102 mg L1 lead(II) to which increasing amounts of potential interfering species were added. The tolerable limit of a foreign species was taken as a relative error not greater than 5%. The tolerable ratio of foreign ions to 5.0102 mg L1 lead(II) was 1000 for NH4(I), SO4(II), Br(I), F(I), I(I), Br(I), Cl(I); 200 for As(III), Sn(II), Al(III), Zn(II), PO4(III), CO3(II); 100 for ascorbic acid, Ba(II), Ca(II), Mg(II); 50 for Fe(II), Fe(III), Ag(I), Mn(II); equal concentrations of Ni(II) and Co(II) interfered with the determination of Pb(II) ion. Analytical characteristics of the CL system for lead(II) Under the optimum conditions described above, the calibration graph of concentrations of lead(II) vs. CL peak height was linear in the range of 3103 –9101 mg L1 (I ¼ 3.2154 [Pb(II)]103 (mg L1 ) þ 59.071, r ¼ 0.9971, n ¼ 10), and the relative standard deviation (R.S.D.) for 5102 mg L1 Pb(II) was 0.7% (n ¼ 11). The detection limit (3), defined as three times the S.D. for the reagent blank signal, was 3104 mg L1 . Dialytic efficiency calibration of microdialysis probe In order to make full use of the microdialysis probe for the experiment, its dialytic efficiency for lead nitrate in different flow rates of perfusate and under various temperatures must be calibrated first.
Effect of perfusate flow rate on the dialytic efficiency of the microdialysis probe The effect of perfusate flow rate on the dialytic efficiency of the microdialysis probe was examined in the range of 1–10 mL min1 at 37 C (Fig. 4). It was seen that the dialytic efficiency of the probe decreased with the increment of perfusate flow rate, and a lower flow rate of the perfusate through the probe generated a higher dialytic efficiency, but the time for one determination would be longer. Combined with the observed results, 5 mL min1 gave the dialytic efficiency of 10.3 1.43% (n ¼ 3) and was chosen as optimum dialysis flow rate.
Effect of temperature on the dialytic efficiency of the microdialysis probe Microdialysis is a dynamic sampling method based on analyte diffusion across a semipermeable membrane driven by a concentration gradient. Diffusion processes are temperature-dependent. So it is necessary to study the temperature effect. The effect of temperature on the dialytic efficiency of the microdialysis probe was investigated at 25 and 37 C with a perfusate flow rate of 5 mL min1 , respectively. The results showed that a higher temperature produced a higher dialytic efficiency (Fig. 5). Since the dialytic efficiency of the probe varies in different temperature, the dialysis system must be controlled strictly at 37 C in order to obtain and maintain an accurate dialytic efficiency in a physiologically normal temperature (37 C).
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Table 1. Estimated metabolic parameters following oral Pb(NO3)2 administration
Fig. 5. Effect of temperature on the recovery rate
Parameters
Estimated
Ka Ke AIC T1=2, ka (min) T1=2, ke (min) Vd=F (L Kg1 ) CL=F (L h1 Kg1 ) Cmax (mg L1 ) Tmax (min) AUC0–3.83 (mg=L h) AUC0–1 (mg=L h)
1.299 1.101 7.665 31.98 37.8 9115.276 10036.386 137.000 85.200 273.787 281.818
AUC Area under the concentration-time curve; Tmax peak time; Cmax peak concentration; CL clearance; F bioavailability; AIC Akaike’s information criterion; T1=2, ka absorption half-life; T1=2, ke elimination half-life; Ke elimination rate constant; Ka absorption rate constant; Vd apparent volume of distribution.
Fig. 6. Concentration of lead(II) in the rabbit blood after oral administration
Characteristics of lead(II) metabolism in rabbit blood The concentrations of free lead(II) vs. time in rabbit blood are shown in Fig. 6. Based on these data, the metabolism parameters of lead(II) in rabbit blood were performed utilizing the pharmacokinetics calculation software ‘DAS ver1.0’. The areas under the plasma concentration-time curves (AUC0-T) were calculated using the trapezoid method. The AUC0-1 were calculated based on the trapezoidal rule and extrapolated to time infinity by addition of AUCT-1. Half-life (T1=2) values were calculated using the equation: T1=2, Ke ¼ 0.693= for elimination half-life; T1=2, Ka ¼ 0.693= for distribution half-life, where and are the distribution and elimination rate constants, respectively. The clearance (CL) was calculated as: CL ¼ Vd. The apparent volume of distribution (Vd) was calculated as: Vd ¼ dose (mg)=C (mg L1 ). Based on the pharmacokinetic calculation, the metabolism of lead(II) was fitted to a one-compartmental open model (‘‘One-compartment open model’’ means
that after administration, a drug may spread into all of the accessible regions instantly. In that case the body is considered a homogenous container for the drug, and the disposition kinetics of the drug can be described as a one-compartment open model.). T1=2 (elimination half-life), Tmax (peak time) and Cmax (peak concentration) were 37.77 min, 85.20 min and 0.137 mg L1 , respectively. Other metabolism parameters of free lead(II) in rabbit blood are shown in Table 1. Conclusion In this paper we developed in vivo, on-line, real-time monitoring of the metabolism of free lead(II) by combining FIA-CL with microdialysis sampling techniques. The concentration of free lead(II) in rabbit blood increased slowly and reached a maximum concentration of 0.137 mg L1 after 85.20 min following Pb(NO3)2 administration, and the disposition of lead(II) in rabbit blood conformed to a one-compartmental open model. The present results confirm that microdialysis sampling followed by flow injection chemiluminescence detection represents a viable approach to the measurement of free lead(II) in rabbit blood. Moreover, compared to other methods, relatively simple instruments and cheap reagents as well as automation of the analytical process makes this technique suitable for common application. Acknowledgements. This study was supported by the Natural Science Foundation of China (No. 30470886).
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