The binding of bilirubin to albumin. A study using spin-labelled bilirubin.

Binding between human serum albumin and a spin-labelled derivative of bilirubin was investigated by circular dichroism, fluorescence quenching, electron spin resonance and visible spectroscopy. The orders of magnitude of the binding constants obtained by flurorescence quenching and electron spin resonance spectroscopies were 10(7) and 10(3) 1 . mol-1, respectively. These data suggest that most spin-labelled bilirubin interacts with human serum albumin at the side not holding the spin-labelled side-arm. CD measurements showed the presence of at least two sites, associated with opposite Cotton effects. It is worthy of note that the Cotton sign of the first site is inverted with respect to the corresponding one of bilirubin. CD measurements on mixed systems (spin-labelled bilirubin/human serum albumin/bilirubin) were also performed. The decomposition of the ternary curves shows that the rotatory power of bilirubin bound to human serum albumin is higher in the ternary system than in the binary (bilirubin/human serum albumin). The corresponding CD measurements for the binding between spin-labelled bilirubin and bovine serum albumin are also reported and discussed.

Observations on the chemical structure and cytotoxic activity of marycin, a hematoporphyrin derivative.

This paper describes the preparation, chemical structure and cytotoxic activity of marycin, a hematoporphyrin derivative. Marycin has been prepared by condensing hematoporphyrin dimethyl ester in the presence of p-toluenesulfonic acid and reducing the product with lithium aluminum hydride. The product appeared to be pure by thin-layer chromatography (TLC) and high-performance liquid chormatography (HPLC). The product, analyzed by UV-visible absorbance and fluorescence spectra, appears to be related to the parent hematoporphyrin compound. The product was also analyzed by NMR and Mass spectra: a dimeric structure can be assigned to marycin: this appears to have an oxide bridge between C2-chains of two porphyrin units and hydroxyl groups instead of carboxyls. Marycin was screened for cytotoxic activity against ZR-75, MCF-7, HT-29, K-562, human tumor cell lines and the MRC-9 human embryonic cell line. Marycin decreases the growth index, measured in the radiometric assay, as 14CO2 production. The cytotoxic activity was dose-dependent and is attributable to the pure compound, marycin. Marycin is active at low doses but the activity varies with the cell line studied. The compound had low toxicity versus MRC-9 normal cell line. The compound is active without light activation. How marycin acts is a matter of speculation. Marycin is highly liposoluble and would be expected to have high toxicity for tumors.

Interaction of bilirubin with small unilamellar vesicles of dipalmitoylphosphatidylcholine.

Interaction of bilirubin with phospholipid bilayers was studied at physiological pH above and below the gel-liquid crystalline phase transition of small unilamellar vesicles of dipalmitoylphosphatidylcholine. Chromatographic, calorimetric and 1H-NMR evidences strongly suggest that dianion form of bilirubin binds to the polar heads of the phosphatidylcholines protruding from the outer leaflet of the vesicles, whilst acid bilirubin, which is insoluble in water, is hydrophobically inserted into the lipophilic region of the bilayers. The surface-bound bilirubin is promptly removed from vesicles, whilst the acid form hydrophobically inserted into the vesicles is firmly bound to the membrane in the gel state. This pool of bilirubin could perturb the chemico-physical properties of the membrane (i.e., fluidity, phase transition, etc. ...) thus contributing to perturbation of the biological properties of living cells.

Anti-inflammatory activity of Maytenus senegalensis root extracts and of maytenoic acid.

Maytenus senegalensis (Lam.) Excell (Celastraceae) root extracts were investigated for their topical anti-inflammatory properties by measuring the inhibition of the Croton oil-induced ear oedema in mice. The highest anti-inflammatory activity was detected in the chloroform extract, which reduced the oedematous response with a potency similar to that of the NSAID reference drug indomethacin (ID(50)=84 and 93 microg/cm(2), respectively). Fractionation of the chloroform and of the hexane extracts led to the isolation of maytenoic acid (1), which exhibited a dose-dependent antiphlogistic effect (ID(50)=0.11 micromol/cm(2)) twice that of indomethacin (ID(50)=0.26 micromol/cm(2)) and only three times lower than that of hydrocortisone (ID(50)=0.04 micromol/cm(2)).

Aloeresin I, an anti-inflammatory 5-methylchromone from cape aloe.

A new diglucoside having a 5-methylchromone moiety was isolated from a commercial sample of Cape aloe, the dried exudate from Aloe ferox Miller, and named aloeresin I. Its structure was established as 1 on the basis of spectral and chemical evidence. Aloeresin I (1) (1 micromol/cm2) reduces in vivo the oedematous response (39 %) induced by Croton oil in the mouse ear with the same potency as aloesin, one of the most abundant Cape aloe constituents, and to a higher extent than aloeresin H (2). Indomethacin (0.3 micromol/cm2), the reference anti-inflammatory compound, provokes 61 % oedema inhibition.

Evidence for an NIH shift as the origin of the apparently anomalous distribution of deuterium in estragole from Artemisia dracunculus.

Feeding experiments of [2-(2)H]- and [4'-(2)H]phenylalanine in Artemisia dracunculus validate the hypothesis that the marked difference in deuterium content at the natural abundance level between the aromatic carbons of estragole (1) is due to an NIH shift during the hydroxylation of the benzene ring.

pH dependence of the water solubility of bilirubin photo-derivatives and its relevance to phototherapy.

Deoxygenated chloroform solutions of bilirubin were irradiated with visible light and continuously extracted with aqueous solutions at different pHs in the range of 7.20-8.20. The aqueous solutions became yellow rapidly and progressively: the higher their pH, the more intense their coloration. The water soluble E-isomers of bilirubin may not represent the only photoproducts transferred into water. It clearly appears from visible absorption measurements that the photopigment formed in our experiments can be partitioned from chloroform into water at pH as low as 7.2-7.4. Although the water solubility of the photopigment cannot be exactly calculated from experimental data, a direct relationship between water partitioning and water solubility can be reasonably assumed. The fact that the water solubility of the photopigment sharply increases in inverse proportion to the hydrogen ion concentration can be of great relevance to the treatment of jaundiced infants with phototherapy.

Drugs affecting porphyrin and lipid metabolism in rats: effects exerted by allylisopropylacetamide and related molecules.

Allylisopropylacetamide (AIA), a drug known to cause lesions in porphyrin and lipid metabolism, and drugs with structure related to AIA [propylisopropylacetamide (PIA) and 2-isopropyl-4-hydroxyvaleric acid] were injected subcutaneously into rats. Measurements were taken of the effect of these compounds on the levels of 5-aminolevulinic acid synthetase (ALA synthetase) and hepatic porphyrins and on the values of hepatic and plasmatic triglycerides and plasma free fatty acids. To a different degree, both AIA and PIA increase the activity of ALA synthetase, and also increase the levels of hepatic porphyrins and hepatic and plasmatic triglycerides, while they both initially lower the levels of plasmatic free fatty acids (FFAs). The administration of 2-isopropyl-4-hydroxyvaleric acid has no effect on the parameters studied. The findings seem to suggest that the activity affecting porphyrin and lipid metabolism is connected in both cases with the presence of the amide function.

Biliverdin as an electron transfer catalyst for superoxide ion in aqueous medium.

Stopped flow experiments gave evidence of the formation of a biliverdin-superoxide complex and/or a biliverdin radical anion by reaction of aqueous O2- with biliverdin. Such transient species are likely intermediates both in the bleaching of biliverdin, during exposure to the aerobic xanthine oxidase reaction, and in the reduction of ferricytochrome c under the same conditions.

An evaluation of bilirubin kinetics with respect to the diagnosis of Gilbert's syndrome.

1. The kinetics of the plasma disappearance of bilirubin (2 mg/kg intravenously) were studied in 106 patients with Gilbert's syndrome and in 13 normal subjects. 2. All patients had significant decreases in hepatic bilirubin clearance and transfer rates from plasma to liver, resulting in increased values for plasma retention at 4 h. The calculated value for unconugated bilirubin production was normal in 40% of patients and increased in the remainder. 3. In 29 of the Gilbert's patients their bromosulphthalein kinetics were studied 1 week before the bilirubin test. These results were essentially normal and it was concluded that the hepatic clearance mechanisms for bilirubin and bromosulphthalein are different. 4. In 10 patients the bilirubin transport maximum (Tm) was found to be low whereas the relative storage capacity (S) was normal. Phenobarbitone treatment in four patients resulted in an increase in Tm, and S decreased in two patients and remained unchanged in the other two. 5. These results support the hypothesis that there are several variants of Gilbert's syndrome and that the bilirubin tolerance test is a useful diagnostic test.