The stability of albumin-bilirubin complex was investigated depending on pH of solution. It was shown that the stability of complex increases in presence of Mn(II) ions. It was also investigated the paramagnetic composition of gallstones by the electron spin resonance (ESR) method. It turned out that all investigated gallstones contain a free bilirubin radical-the stable product of its radical oxidation. Accordingly the paramagnetic composition gallstones could be divided on three main types: cholesterol, brown pigment and black pigment stones. ESR spectra of cholesterol stones is singlet with g=2.003 and splitting between components 1.0 mT. At the same time the brown gallstones, besides aforementioned signal contain the ESR spectrum which is characteristics for Mn(II) ion complexes with inorganic compounds and, finally, in the black pigment stones it was found out Fe(III) and Cu(II) complexes with organic compounds and a singlet of bilirubin free radical. It is supposed that crystallization centers of gallstones could be the polymer network of bilirubin radical polymerization in complex with different metal ions earlier discovered in gallstones.
Deep Dive into Ternary complexes of albumin-Mn(II)-bilirubin and Electron Spin Resonance studies of gallstones.
The stability of albumin-bilirubin complex was investigated depending on pH of solution. It was shown that the stability of complex increases in presence of Mn(II) ions. It was also investigated the paramagnetic composition of gallstones by the electron spin resonance (ESR) method. It turned out that all investigated gallstones contain a free bilirubin radical-the stable product of its radical oxidation. Accordingly the paramagnetic composition gallstones could be divided on three main types: cholesterol, brown pigment and black pigment stones. ESR spectra of cholesterol stones is singlet with g=2.003 and splitting between components 1.0 mT. At the same time the brown gallstones, besides aforementioned signal contain the ESR spectrum which is characteristics for Mn(II) ion complexes with inorganic compounds and, finally, in the black pigment stones it was found out Fe(III) and Cu(II) complexes with organic compounds and a singlet of bilirubin free radical. It is supposed that crystalli
Serum albumin is a main carrier of bilirubin in blood which is a toxic product of hemoglobin decomposition.
Marx [1] supposed that in the bilirubin binding with albumin the bivalent metal ions Fe +2 , Cu +2 , Mn +2 , etc. could take part as the cation-bridges.
It is known that at aggressive and chronic hepatitis, along with the increase of nonconjugated bilirubin concentration in blood, the increase of divalent manganese -Mn(II) ions concentration takes also place. Investigations carried out with radioactive manganese ions confirmed that the increase of nonconjugated bilirubin concentration correlated with Mn(II) concentration [2][3][4]. We have investigated the stability of albumin-Mn(II) complex depending on pH of a solution [5]. The analysis of data shows that the binding energy of Mn(II) ions with serum albumin is comparable with the energy of weak hydrogen bonding~(1-1.5) kcal/mole at pH=7. 4. This indicates that the investigated complex decays easily with the varying pH of solution. The adsorption of Mn(II) ions on serum albumin depends linearly on the ionic strength of solution (Fig. 1) The increase of ionic strength of the solution decreases the pK value, but after reaching the I=0.1 value, it becomes a constant. It could be explained by a partial neutralization of charge by sodium ions in the binding centers of Mn(II) with albumin [5]. The investigation data on stability of albumin-bilirubin and albumin-Mn(II)-bilirubin complexes depending on pH of solution are shown in (Fig. 2). In the case of ternary complexes the binding constant value increases. The measurements carried out by the ESR method did not show the presence of Mn(II) ions in blood what is possibly related with their small concentration. Therefore it was decided to study the gallstones by the ESR method, as natural blood filter where it is possible the accumulation of compounds and metal ions complexes insoluble in bile.
Measurements of ESR spectra of brown pigment stones showed the presence of a high concentration of manganese ion complexes (Fig. 3 b) in them, although in cholesterol and black pigment stones they were not observed (Fig. 3 a,c).
During last years it has been carried out the intensive investigations of gallstones etiology but up to present time the chemical nature of the crystallization centers is not known, as well as the role of paramagnetic metal ions in their formation [6][7][8][9][10].
Investigations carried out by Liu X.-T. and Hu I. [11] showed that in the creation of gallstones the free bilirubin radicals should participate, which, accordingly authors’ opinion, appear at interaction of metabolic free radicals, presented in living organisms, with molecules of indirect bilirubin, followed by the radical polymerization of bilirubin and formation of complexes with metal ions. On the participation of free bilirubin radicals in formation of gallstones points out also Blazovich [12]. The formation of polymeric network of bilirubin salts in gallstones was observed still in works Okubo et al. [13]. By the method of infrared spectroscopy and equilibrium swelling authors showed that polymeric network insolvable in bile is particularly strongly developed in black pigment stones.
As it was shown by ESR method, a singlet with parameters g=2.003 and ΔH=1.0 mT is observed in all investigated gallstones. This ESR signal is attributed to a free radical of the indirect bilirubin. Bilirubin is a photosensitizer. Under irradiation by visible light it takes place the photoisomerization and photooxidation of bilirubin [14][15][16][17][18][19][20]. As it was shown in work [5] at irradiation of its powder by blue ( m =450 nm) or green ( m =500 nm) light, it appears the singlet ESR signal with parameters g=2.003 and ΔH=1.0 mT (Fig. 4). The ESR signal with the same parameters appears in the bilirubin chloroform solution at aforementioned light irradiation. In (Fig. 5) it is presented a curve of accumulation of the ESR signal of free bilirubin radical at influence of light.
The ESR spectrum of brown pigment stones consists of six intensive components with splitting between them ΔH=8,7 mT and a singlet with g=2.003 and ΔH=1.0 mT (Fig. 3) characteristic for a free stable bilirubin radical. The ESR spectrum of black pigment stones besides a singlet from free bilirubin radical, consists of also from a wide structurized signal with g =2.05, g =2.37 and A =18.6 mT, and also an asymmetric singlet with g=4.19 (Fig. 4).
Crystal serum albumin of “Serva” firm was used without its further purification. Bilirubin of “Alfa Aesar” firm was purified on chromatographic column (L100/400 “Chemapol” Praque) [5].
The purity of preparate from free radical products of oxidation was checked by help of ESR method.
In experiments it was used chromatically pure NaCl, MnCl, Tris (2-amino-2-hydroxymethyl-1,3propanediol),pure for analysis from “Reanal” Hungary firm. The thin layer of bilirubin powder (30 mg) was irradiated by blue and green lamps “O
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