The spectrum characteristics of unconjugated bilirubin and transition metal ions in NaOH solution

Yang Fang, Zheng Wenjie, Bai Yan, Feng Dexiong, Ouyang Jianming
Department of Chemistry,Jinan University, Guangzhou 510632, China

Received Nov. 7, 2002; Supported by the Natural Science Foundation of Guangdong Province, China (970630)

AbstractIt was discovered by our team a novel fluorescence characteristic of unconjugated bilirubin in NaOH solution. Fluorescence and UV-visible spectra characteristics of unconjugated bilirubin and transition metal ions Zn2+, Cu2+ in NaOH solution were further investigated. The results indicated that zinc ion impacted a distinct enhancement and cuprum ion quenching on the fluorescence of bilirubin. The fluorescence quenching could be reduced under the condition of high concentration of zinc ion, which was used to develop the fluorimetry of bilirubin in high concentration of zinc ion, and to determine bilirubin in Chinese medicine. The UV-visible spectrum also showed that zinc ion was prior to cuprum ion to interact with bilirubin in Bilirubin-Zn-Cu (BR-Zn-Cu) system.

Bilirubin (BR for short) and biliverdin (BV for short) are important bile pigments. In mammals, BR is deacidized to BV by BV reductase. BR is both the internal toxin and antioxidant in human body and has positive effects to the regeneration of hepatic cells. BR is also the main ingredient in cow bezoar which is a famous Chinese medicine. Together with its complexes, BR is closely related to the formation of gallstone[1-2]. As a result, much attention has been paid to the study of the interactions between BR and metal ions [3-11]
But many researches on BR were performed in organic solvents. As to the fluorescence of BR, it was rarely studied because of its low fluorescence intensity in aqueous solution and organic solvents. In 1996, J. R. Ferraro[1] studied the coordination between Cu2+ and BR in NaOH by FT-IR and EPR.
The fluorescence of BR had been studied by our team and it was discovered that unconjugated BR had a novel fluorescence peak in NaOH solution at room temperature[12]. Its excitation wavelength was 464nm and emission wavelength was 524nm, both of which would not shift with the change of the concentration of NaOH, illumination, and heat treatment of BR. But the fluorescence intensity would change greatly. The fluorescence characteristics of BR were quite similar to that of BV, and the excitation wavelength of BV was 465nm, the emission wavelength being 525nm. The interaction between various metal ions and BV led to three kinds of changes in the fluorescence spectrum of BV: Zn2+ and Cd2+ had a distinct fluorescence enhancement on BV; Cu2+ and Hg2+ caused fluorescence quenching; Li, Ba2+, Mn2+, Fe3+, Co2+, Ni2+, Pb2+, Bi3+, La3+, Ce3+ and Zr4+ had no effects on the fluorescence of BV. Therefore Zn2+ and Cu2+ were chosen to be studied in this paper. So were the fluorescence and UV-visible spectra of BR-M (Zn2+, Cu2+) system in NaOH solution at room temperature. The fluorimetry of BR in high concentration of zinc ion was also developed because the Chinese medicine cow bezoar included a quantity of metal complexes of BR which were active constituents in cow bezoar. This method made Zn2+ as a good factor and reduced the disturbance from other metal ions, and it could be applied to the determination of BR in Chinese medicines.

2.1 Instrumentation

Throughout the experiments a Shimadu (Kyoto Japan) UV260 spectrum recording meter was used. The fluorescence experiments were carried out with a 970-MC fluorescence spectrometer (The Third Analytical Instrument Factory of Shanghai, China).
2.2 Reagents
Bilirubin was obtained from Sigma Chemical Company. Zn (Ac) , Cu (Ac) and NaOH were all made in China and analytically pure. Double distilled water was used for the preparation of all solutions. An accurate quantity of BR was dissolved into 1.2mol× L-1 NaOH to form the aqueous alkaline solution of BR, which was called fresh BR solution.
2.3 Heat treatment of BR solution
Fresh BR solution was heated at a constant temperature and the temperature was controlled at 75±0.1°C, then it was cooled by water to room temperature.
2.4 Adding Zn2+, Cu2+
After heat treatment of fresh BR solution, Zn (Ac) and Cu (Ac) solutions were added with different ratios. Then fluorescence intensity was examined atex=464nm andem=524nm. As to the UV-visible spectrum, the blank solution was NaOH solution at the corresponding concentration.

3.1 Fluorescence of Zn2+, Cu2+ and BR

Table 1 was fluorescence intensities of BR and different concentration of Zn2+. The concentration of BR was 5mol× L-1 in this table. It could be figured out that the fluorescence intensities were greatly enhanced when Zn2+ was added to NaOH solution of BR. And the changes of fluorescence intensities were not so great when the ratio of BR and Zn2+ was varied from 1:1 to 1:10000. So Zn2+ had a distinct fluorescence enhancement to BR and the ratio of coordination between BR and Zn2+ was 1:1.

Tab. 1 Fluorescence intensities at
different concentration ratio
Tab. 2 Influences of Zn2+, Cu2+ on fluorescence intensity of BR
BR : Zn2+  
1:0 14.1
1:1 25.7
1:2 26.0
1:4 26.9
1:5 27.5
1:7 28.3
1:8 27.5
1:10 28.4
1:100 29.6
1:1000 28.1
1:10000 29.9
BR=5 BR=20
BR: cZn2+=1:10000 29.9 88.6
BR: cCu2+=1:10 5.3 9.3
Zn2+: Cu2+BR =10000:10:1 23.7 70.7
Zn2+BR: Cu2+=10000:1:10 24.0 71.6
Cu2+BR: Zn2+=10:1:10000 25.6 71.6

Table 2 showed the fluorescence intensities of BR-Zn-Cu system. BR was in unit ofmol× L-1. The ratio of three substances was the same, i.e., Zn2+Cu2+BR =10000:10:1. But the sequences of being mixed together were different. The ratio sequences listed in Table 2 were the mixing sequences. Table 2 indicated that fluorescence quenching of BR caused by Cu2+ could be reduced under the condition of high concentration of Zn2+, which was not influenced by the mixing sequences of the three substances.

3.2 UV-visible spectra of Zn2+, Cu2+ and BR
The color of fresh aqueous alkaline solution of BR was orange. It had three absorption peaks (see Fig. 1a) respectively at 421.4nm, 329.6nm, 224.4nm. The peak 421.4nm was related to →* transition of two pairs of pyrrole rings; the peak 329.6nm was contributed to n→* transition; and the narrow high peak 224.4nm was due to →* transition.

Fig.1 UV-visible Spectra of BR, BR-Zn and BR-Cu
a. fresh BR; b. BRZn2+=1:1; c. BRCu2+=1:1

The orange color would change into dark green when Zn2+ was added to the fresh BR solution. With the increasing of concentration of Zn2+, blue shift would happen at the peak 421.4nm. Fig. 1b was BRZn2+=1:1. When 1a was compared with 1b, the peak at 421.4nm in 1a shifted to 407.2nm in 1b, and the intensity reduced greatly. This peak would disappear if the concentration of Zn2+ was increased. And yet red shift happened at the peak 329.6nm and the intensity increased gradually. This peak shifted to 335.0nm in fig. 1b. This demonstrated that BR was oxidized to BV and complex BV-Zn was formed after the interaction between Zn2+ and BR.
When Cu2+ was added to the fresh BR solution, the orange color also changed into dark green. Fig. 1c was BRCu2+=1:1. When 1a was compared with 1c, the peak 421.4nm in 1a shifted to 395.0nm in 1c and the intensity increased. The peak 329.6nm in 1a also shifted to 290.0nm in 1c, but the intensity reduced greatly. And it would disappear when the concentration of Cu2+ was ten times as much as that of BR. This also demonstrated that Cu2+ and BR were coordinated and consequently complex BR-Cu was formed. Because of the oxidation of Cu2+, complex BV-Cu was also formed.
The UV-visible spectra of BR, Zn2+ and Cu2+ in the same solution were further studied. The ratio and the mixing sequences in Table 3 were the same as that in Table 2.
It could be seen from the figures in Table 3 that the peak 386.2nm in Table 3a was very weak and blue-shifted obviously if compared with the peak of fresh BR at 421.4nm, which was similar to the spectrum of BR and Zn2+. The concentration of Zn2+ was 1000 times as much as that of Cu2+. Although the peak 386.2nm was weak, it did not disappear for it was related to the influence of Cu2+. So in this system Zn2+ and Cu2+ both interacted with BR, and the interaction of Zn2+ was more distinctive because the concentration of Zn2+ was higher than that of Cu2+
The peak 340.2nm (intensity being 0.094) in 3a was quite similar to the peak 338.8nm (intensity being 0.099) in 3b. And the peak 386.2nm in 3a had disappeared in 3b. It was similar to the spectrum of BR and Zn2+. So in this system Zn2+ first interacted with BR and it was Zn2+ that produced the main effect.
The intensities of two peaks, 397.0nm and 357.4nm in 3c, were both 0.103. Because Cu2+ first coordinated with BR, the peak 421.4nm of fresh BR blue-shifted to 397.0nm and its intensity increased. However, when Zn2+ was added, Zn2+ competed with Cu2+ to interact with BR, which caused the peak 329.6nm of fresh BR to red-shift to 357.4nm and the intensity also increased.
A conclusion could be drawn from the above result that the high concentration of Zn2+ was prior to Cu2+ to interact with BR in NaOH solution.

Tab. 3 UV-visible Spectra of BR-Zn-Cu system

absorption peak (intensity)
  Zn2+Cu2+BR=10000:10:1 386.2nm (0.060) 340.2nm (0.094) 224.6nm (0.760)
  Zn2+BRCu2+=10000:1:10 —— 338.0nm (0.099) 224.6nm (0.764)
  Cu2+BRZn2+=10:1:10000 397.0nm (0.103) 357.4nm (0.103) 224.4nm (0.770)

3.3 Fluorimetry of bilirubin
The fluorescence quenching of bilirubin by cuprum ion could be reduced under the condition of high concentration of zinc ion, which was utilized to develop the fluorimetry of bilirubin in the presence of zinc ion. After the heat treatment of fresh BR solution, Zn2+ was mixed into it. The concentration of Zn2+ in the mixed solution was 0.1mol× L-1. Fluorescence intensities of different concentration of BR were recorded and the regression equation was F=1.56+3.65, where F was the fluorescence intensity and was the concentration of BR in unit ofmol×L-1, the regression coefficient being R=0.998. The linear range of bilirubin was 0.15-50.00mol× L-1, and the detection limit was 0.077mol×L-1
The recovery of two Chinese medicines detoxicating tablet of cow bezoar and zhongsheng wan were determined. First, the medicines were peeled and ground to powder. Second, absolute ethyl alcohol was added to the powder. After being thoroughly mixed and slightly heated, the mixture was centrifuged and the solution was poured out which was yellow. Third, 1.2mol× L-1 NaOH was added to the remaining solid substance to extract BR. Then the mixture was centrifuged for the second time and the deposited matter was gotten rid of. After the heat treatment of NaOH solution which contained BR now, Zn2+ was added to it and the concentration of Zn2+ in mixed solution was 0.1 mol× L-1. Finally, the mixed solution was equally separated into two. One was determined by fluorescence directly; the other was added in an accurate quantity of BR, which was also after heat treatment, and then the fluorescence was determined, recovery being computed. Since BR could not be dissolved in absolute ethyl alcohol, the quantity of BR would not lose during the procedure. The average recovery ratio of detoxicating tablet of cow bezoar was 94.60% (RSD = 1.48%, n=8) and the average recovery ratio of zhongsheng wan was 96.65% (RSD = 1.37%, n=6).
Fluorimetry of BR was presented in the condition of high concentration of Zn2+. This method made Zn2+ as a good factor and reduced the disturbance caused by the fluorescence quenching of Cu2+. It was a convenient and accurate method to determine the concentration of BR in medicine.

The mechanism of fluorescence enhancement of BR caused by Zn2+ was approached as follows: Intramolecular hydrogen bond and intermolecular hydrogen bond could be easily formed due to the ligand atoms and coordination groups in BR molecule. And intramolecular hydrogen bond often led to fluorescence quenching. In the aqueous alkaline solution of BR, ionization happened on both the proton of carboxylic group and the proton of nitrogen atom in pyrrole. So intramolecular hydrogen bond in BR molecule had been broken to some degree. When Zn2+ was added to it, Zn2+ also broke intramolecular hydrogen bond and strengthened the flatness of conjugate system. Therefore, conjugatebond was lengthened in favor ofp ® p* transition. On the other hand, when BR did not coordinate with metal ions, it had more degree of freedom and was easily to deform. If the deformation happened to molecules in excited state, energy band crossing would happen between ground state and excited state, and so would the multi-phonon process of nonradiation transition. The coordination both broke the intramolecular hydrogen bond and strengthened the rigidity structure of molecule. As a result the deformation was difficult to happen, and the nonradiation transition was greatly reduced, thus fluorescence enhancement happened.