The aflatoxin-lysine adduct quantified by high-performance liquid chromatography from human serum albumin samples

Aflatoxin B1 (AFB1) exposure from the diet is a major risk factor for the development of liver cancer in people living in regions of China and Africa. Rapid methods to assess the exposure status of these individuals to genotoxic damage imparted by AFB1 will be very important for cancer prevention strategies. Serum albumin is a readily accessible target protein for AFB1 and we report here the development of an accurate and sensitive method to quantitate the major AFB1 serum albumin adduct, aflatoxin-lysine, from less than 100 microliters of serum by combined immunoaffinity chromatography/high-performance liquid chromatography (IAC/HPLC) with fluorescence detection. For this method, serum is digested with Pronase and the adducts are purified by monoclonal antibody IAC and quantified by HPLC. Analysis of human serum samples obtained from an exposed population revealed a highly significant correlation coefficient (up to 0.82 for male samples) between aflatoxin-lysine adduct levels and AFB1 consumption. These data suggest that aflatoxin-lysine is an excellent molecular dosimeter for exposure assessment. To determine whether the liver is the sole site of aflatoxin-albumin adduct formation, preliminary experiments with isolated perfused rat liver were done. These data showed that AFB1 metabolites covalently react not only with albumin in the hepatocyte, but also with circulating proteins in the perfusate. This suggests that a reactive aflatoxin metabolite secreted by the liver may form serum albumin adducts in circulating blood. Taken together, the analysis of aflatoxin-lysine could prove a very useful tool for epidemiological studies.     

Production and characterization of monoclonal antibody against human serum albumin

Hybridomas secreting monoclonal antibodies (MAbs) producing stable, specific and high affinity against human serum albumin (HSA) have been established. The aim of the present study was the production of MAbs that will be potentially used in designing immunoassay methods especially immunochromatography assay kit for screening of microalbuminuria (MAU) in the early detection of diabetic and nondiabetic nephropathy. The hybridomas were obtained by fusion of spleen cells from immunized mice with mouse myeloma cell line (SP2). After limiting dilutions three clones producing antibodies were designed as EMRC1-3, which displayed different pattern of fine specificity for HSA and low cross reaction with other proteins as elucidated by inhibition enzyme-linked immunosorbent assay (ELISA). These clones were found to be of immunoglobulin G (IgG) class with k light chain. Subclass determination showed that all three MAbs secreted IgG1 type of antibody. The results of affinity purification for the two selected clones (EMRC1 and EMRC3) displayed high affinity with no cross reactivity with any of the related protein molecules. The stable hybridomas secreting anti-HSA were expanded in 50-mL flasks for large-scale production of the required antibodies. The standard curves were constructed with a sensitivity of 10 pg per well covering up to 100 ng per well. The high binding activity to HSA antigen and having no cross reactivity with other related molecules illustrated the potential application of these antibodies as an immunodiagnostic reagent in designing an immunochromatography assay kit for screening of MAU in diabetic and nondiabetic patients.     

Identification of the major serum albumin adduct formed by 4-aminobiphenyl in vivo in rats

Serum albumin was isolated from rats at 27 h after administration of the carcinogen [2,2'-3H]-4-aminobiphenyl. Pronase digestion of the purified albumin yielded a mixture of radiolabeled materials which was resolved into 5 major components by reverse-phase liquid chromatography. From detailed UV, 1H-NMR, and mass spectral analyses, four of these were determined to be 4-aminobiphenyl, 4'-hydroxy-4-acetylaminobiphenyl, and two other metabolites, all of which are presumed to be non-covalently associated with the serum albumin. The fifth component, however, resulted from covalent bond formation and was identified as a tetrapeptide containing 3-tryptophanyl-4-acetylaminobiphenyl, the amino acid sequence of which was H2N-ala-trp-ala-val. Since rat serum albumin contains only a single tryptophan residue in a hydrophobic drug binding site, its high selectivity for carcinogen binding suggests a unique role for this protein in the detoxification and/or transport of ultimate carcinogenic metabolites.     

Interaction of human serum albumin with anticancer agents in vitro.

The influence of human serum albumin (HA) on the biological effects of 13 chemotherapeutic agents was studied in vitro in the human leukaemia cell line MOLT-3. On the basis of changes in biological activity influenced by HA, these drugs may be divided into three types. Type I agents include cis-diamminedichloroplatinum (II), 4''-(9-acridinylamino)methanesulphon-m-anisidide, neocarzinostatin, nitrogen mustard, adriamycin, daunorubicin and mitomycin C--drugs whose biological activities are reduced in the presence of HA. The biological activities of Type II drugs (cytosine arabinoside, fluorouracil and actinomycin D) are not influenced by HA. The biological activities of Type III drugs (bleomycin, vincristine and vinblastine) are increased in the presence of HA. These results indicate that serum HA interferes in vitro with certain anticancer agents in terms of biological activity and, probably, clinical effectiveness. HA-drug interaction may be a major factor governing the pharmacology of Type I anticancer agents in man.     

Reactive oxygen species damaged human serum albumin in patients with hepatocellular carcinoma

The role of reactive oxygen species (ROS) damaged human serum albumin (HSA) in hepatocellular carcinoma (HCC) has been investigated in the present study. HSA was modified by hydroxyl radical. Modification occurred in HSA was characterized by physico-chemical techniques. ROS modified HSA was found to be highly immunogenic in rabbits. The binding characteristics of circulating antibodies in HCC patients against native and ROS-modified HSA were assessed. HCC patients (n = 31) were examined by direct binding ELISA and their results were compared with healthy age-matched controls (n = 22). High degree of specific binding by 77.4% of HCC sera towards ROS-HSA, in comparison to its native analogue (p < 0.05) was observed. Competitive ELISA reiterates the direct binding results. Gel retardation assay further substantiated the enhanced recognition of ROS-HSA by circulating antibodies in HCC patients. The increase in total serum protein carbonyl levels in the HCC patients was largely due to an increase in oxidized albumin. Purified HSA of HCC patients (HCC-HSA) contained higher levels of carbonyls than HSA of normal subjects (normal-HSA) (p < 0.01). HCC-HSA was conformationally altered, with more exposure of its hydrophobic regions. Collectively, the oxidation of plasma proteins, especially HSA, might enhance oxidative stress in HCC patients.     

Technetium-99m human serum albumin in the differentiation of cerebrovascular and neoplastic lesions

Technetium-99m pertechnetate and technetium-99m human serum albumin have been used within a 24-hr interval to evaluate the vascularity of abnormalities detected on routine technetium-99m pertechnetate brain scans. Human serum albumin tagged with technetium-99m has provided superior resolution of vascular lesions on static images but offered no advantage over technetium-99m pertechnetate for early static imaging of neoplastic lesions. The use of technetium-99m human serum albumin in the resolution of suspicious areas observed on routine technetium-99m pertechnetate static scans is advised because it (1) more accurately detects vascular lesions, (2) serves better to indicate their total vascularity, and (3) will differentiate vascular lesions from neoplastic lesions.     

血清白蛋白检测对胰腺癌高剂量少分次放疗的预后评估

目的:评估血清白蛋白检测在预测胰腺癌高剂量少分次放疗预后的价值.方法:收集未经治疗的胰腺癌Ⅲ期患者,采用螺旋断层放疗设备治疗,按照高剂量少分次放疗剂量模式,放疗前行血清白蛋白检测.通过随访获取患者生存时间,评估生存时间采用Kaplan-Meier法,生存差异的比较采用Log-rank检验,相关分析采用Spearman相关分析法.结果:97例患者中位总生存时间12个月.血清白蛋白高值组中位生存时间15个月,低值组中位生存时间9个月,生存分析显示血清白蛋白水平与总生存时间在统计学上有关(P=0.007),放疗前血清白蛋白水平与放疗前减黄(r=-0.24,P=0.018)呈负相关,与肿瘤部位(r=0.279,P=0.006)呈正相关.结论:胰腺癌高剂量少分次放疗前血清白蛋白检测在预测患者总生存时间方面具有重要价值.     

Anti-tumor activity of fenretinide complexed with human serum albumin in lung cancer xenograft mouse model

Sufficient knowledge regarding cellular and molecular basis of lung cancer progression and metastasis would help in the development of novel and effective strategies for the treatment of lung cancer. 4HPR is a synthetic retinoid with potential anti-tumor activity but is still limited because of its poor bioavailability. The use of albumin as a complexing agent for a hydrophobic drug is expected to improve the water solubility and consequently their bioavailability.This study investigated the antitumor activity of a novel complex between albumin and 4-HPR in a mouse model of human lung cancer and focuses on role and mechanism of Cav-1 mainly involved in regulating cancer and Acsvl3 mainly connected with tumor growth.Their expressions were assayed by immunohistochemistry and qRT-PCR, to demonstrate the reduction of the tumor growth following the drug treatment. Our results showed a high antitumor activity of 4HPR-HSA by reduction of the volume of tumor mass and the presence of a high level of apoptotic cell by TUNEL assay. The downregulation of Cav-1 and Acsvl3 suggested a reduction of tumor growth.In conclusion, we demonstrated the great potential of 4HPR-HSA in the treatment of lung cancer. More data about the mechanism of drug delivery the 4HPR-HSA are necessary.     

In vitro and in vivo antitumor activity of methotrexate conjugated to human serum albumin in human cancer cells.

To avoid systemic toxicity of the cytotoxic drug methotrexate (MTX) and to improve tumor selectivity, MTX was bound to human serum albumin (HSA) as a drug carrier. To understand more about the mechanism of action of MTX conjugated to HSA (MTX-HSA), the uptake of MTX-HSA into the cell was determined as well as the effect of MTX-HSA on thymidylate synthase (TS), cell cycle distribution, and cell proliferation. Different uptake kinetics were observed for [(3)H]MTX and [(3)H]MTX-HSA. However, similar uptake kinetics were measured for (125)I-HSA and (125)I-MTX-HSA (2.1 and 1.8 pmol/10(7) cells/h when cells were treated with 10 micro M (125)I-HSA and (125)I-MTX-HSA, respectively), suggesting that MTX-HSA enters the cells by albumin-mediated endocytosis. We observed no effect of MTX-HSA on TS when folate receptor-expressing KB cells were treated for 4 h (IC(50), >50 micro M). However, 24 h after incubation, MTX-HSA inhibited TS with an IC(50) of 6.9 micro M. In addition, we found that MTX-HSA had a delayed effect on the cell cycle compared with MTX and that this effect could be inhibited with the lysosomal inhibitor methylamine, suggesting that MTX-HSA activity is dependent on lysosomal processes. The proliferation of different wild-type and MTX-resistant tumor cell lines was inhibited at IC(50) concentrations between 2 and 78 micro M, respectively. MTX-HSA accumulates in vivo in the tumor tissue. Local concentrations of 18-29 micro M were measured, which are effective antiproliferative concentrations as determined in vitro. We also investigated the antitumor activity of MTX-HSA in vivo in different human tumor xenografts grown s.c. in nude mice. Fourteen tumors from eight different tissues were tested. Nine of 14 tumors (64%) showed a clear response with tumor inhibition, stasis, or regression; 5 of 14 (36%) gave a moderate response with tumor growth delay or no response. In conclusion, MTX-HSA is effectively taken up by the cells via albumin receptor- or folate receptor-mediated endocytosis and time-dependently released as an active compound into the cytosol to exert an inhibiting effect on TS and to induce cell cycle alterations. In vivo, effective concentrations of MTX-HSA were reached in tumor tissue to exhibit antitumor activity.     

In vitro targeted killing of human endothelial cells by co-incubation of human serum and NGR peptide conjugated human albumin protein bearing alpha (1-3) galactose epitopes

The NGR/alpha1,3Gal-HSA peptide was designed to specifically target CD13 positive cells and induce cell lysis. NGR is the targeting component of the peptide in that it binds the CD13 isoform (aminopeptidase) that is expressed in tumor vessels. Galactose alpha1,3-galactose terminal carbohydrate epitope (alpha1,3Gal) induces a strong antibody reaction in human and Old World Monkeys and in vivo, this reaction leads to organ rejection. The human serum albumin (HSA) bearing alpha1,3Gal epitope was therefore used to lyse cells. In the present study, we were able to demonstrate that NGR/alpha1,3Gal-HSA binds CD13 positive human umbilical vein endothelial cells (HUVEC). We also found by live/dead fluorescent staining that NGR/alpha1,3Gal-HSA was able to induce lysis of HUVECs upon incubation with human serum. Therefore, by conjugating NGR to HSA bearing alpha1,3Gal epitopes, we are able to specifically target and lyse cells expressing CD13. This strategy may be potentially useful in tumor anti-angiogenesis therapy.     

Effect of pH and human serum albumin on the cytotoxicity of a glucuronide prodrug of 9-aminocamptothecin

Purpose 9-aminocamptothecin glucuronide (9ACG) is a prodrug of 9-aminocamptothecin (9AC) that displays potent antitumor activity against human tumor xenografts in nude mice. Camptothecins exist in a pH dependent equilibrium between active lactone and inactive carboxy forms that can be altered by binding to human serum albumin (HSA). Here we investigated the influence of pH and HSA on the lactone-carboxy equilibrium, HSA binding, and cytotoxicity of 9ACG. Methods Microfiltration and HPLC were used to measure the influence of pH on lactone to carboxy conversion and HSA binding of 9ACG as compared to other camptothecins. In vitro cytotoxicity of drugs was determined against EJ human bladder carcinoma cells and CL1-5 human lung cancer cells. Results The rate of lactone to carboxy conversion was similar for 9ACG and 9AC. Decreasing the pH from 7.6 to 6.0 increased the equilibrium levels of the lactone forms of the drugs from 20 to almost 95% of total drug. HSA moderately diminished the amount of free 9ACG lactone but did not change the ratio of 9ACG lactone to 9ACG carboxy. Consistent with the effect of pH on lactone levels, lowering the pH of EJ human bladder carcinoma cells from 7.6 to 6.8 decreased the IC₅₀ of 9ACG from 480 to 98 nM and 9AC from 33 to 12 nM. Activation of 9ACG by human β-glucuronidase anchored on the surface of EJ cells further decreased its IC₅₀ value to 26 nM. Although HSA significantly decreased the cytotoxicity of 9AC and 9ACG, activation of 9ACG at cancer cells with an antibody-β-glucuronidase immunoconjugate produced greater cytotoxicity than 9AC. Conclusions Acidification and targeted delivery of β-glucuronidase can enhance 9ACG cytotoxicity even in the presence of HSA.     

A structural-functional assessment of serum albumin in cancerous diseases

HSA structure was studied in healthy subjects and cancer patients using infrared spectroscopy. No significant differences were observed although the intensity of valent C-H fluctuation lines in alkyl groups of HSA was slightly increased in cancer patients. Gas-liquid chromatography established changes in the profile of polyunsaturated fatty acids binding to HSA in cancer patients. The data obtained suggest inhibition of omega-6 pathway of fatty acid metabolism and activation of omega-3 pathway. Marked binding of POL products to serum albumin was found nonspecific and was considered evidence of protective function of that protein.     

Correlation of immune complexes in disseminated neuroblastoma with serum antibody to bovine serum albumin

To evaluate the relationship between tumor burden and circulating immune complexes (IC) in children with neuroblastoma (NBL), we studied sera collected at intervals from patients with disseminated (Stage III or IV) NBL. Sera from 10 of 12 patients contained IC by the Raji cell assay at some time during the first 9 to 11 months of the study. Higher IC levels were observed in sera of female patients. Fluid-phase C1q binding tests detected IC in only 16% of sera. IC measurements by either assay did not correlate with tumor burden. However, serum IC levels, as measured by the Raji cell assay, correlated significantly with serum antibody to bovine serum albumin (BSA) (rs = 0.54; p less than 0.001, rs = r as determined by Spearman rank correlation test). Measurement of anti-BSA antibodies in sera from the 12 patients, tested serially for circulating IC, and from five additional patients revealed that these had significantly higher anti-BSA activity than was found in sera from 13 age-matched controls. Sera from females also had relatively high levels of anti-BSA. Levels of antibody to bovine gamma-globulin and casein were not abnormal. Three sera with high IC levels (greater than 800 micrograms equivalents of heat-aggregated IgG) and relatively low anti-BSA activity appeared to contain "hidden" antibodies to BSA. These were demonstrated by measuring the increase in the ability of sera to bind 125I-BSA after they had been briefly acidified and then neutralized in the presence of the labeled BSA. The possible relevance of these results to the pathophysiology of NBL is discussed in light of earlier work that reported that serum IC levels correlate with the stage of the disease in NBL.     

Spectroscopic interaction of a coumarin derivative with bovine serum albumin

Abstract The absorption and fluorescence spectra of the 7-diethylamino-4-methyl coumarin (DAMC) in ethanol-water (1:9 v/v) solution at varying pH values were investigated. The interaction between DAMC and bovine serum albumin (BSA) was investigated by fluorescence spectroscopy. The Stern-Volmer quenching constant, the quenching rate constant of the bimolecular reaction (kq), the binding constant, and number of binding sites are mentioned but not calculated in the paper. Moreover, in a preliminary pharmacological study, DAMC not only remarkably increased cellular apoptosis in a concentration-dependent manner but also clearly induced A549 cell cycle arrest. Thus, these coumarin derivatives merit investigation as novel potential antitumor agents with further structural modification to produce an optimal lead compound and elucidate the detailed pharmacological mechanism.     

Vinblastine-C4 alkyl maleoyl and amino acid maleoyl derivatives: III. Experimental antitumor activities of lactosaminated serum albumin conjugates

Vinblastine-C4 acyl derivatives were synthesized by linking alkyl maleoyl or amino acid maleoyl compounds through an ester linkage at C4 position of the Vindoline moiety of Vinblastine. To target these derivatives selectively to hepatoma, conjugates were prepared with a neoglycoprotein i.e. lactosaminated human albumin, as a specific carrier. The method of preparation of lactosaminated albumin and of coupling to Vinca alkaloid derivatives is described and the mechanism of addition of the protein to the derivative is discussed. The experimental antitumor activity of these conjugates has been screened against the experimental P-388 Leukemia. The activity of the best conjugate has been evaluated in several human tumor xenografts. Further, the therapeutic potential of this type of conjugate has been demonstrated in a model of hepatoma xenograft developed in our laboratory, the HepG2 carcinoma.