Redox state of human serum albumin as a post-discharge prognostic marker in patients hospitalized for heart failure.

BACKGROUND: Heart failure (HF) is a global health concern, and oxidative stress has been implicated in its progression. The redox state of human serum albumin, a systemic oxidative biomarker, holds promise as a prognostic marker in HF. This study aimed to investigate the association between the fraction of human mercaptalbumin (fHMA), an indicator of human serum albumin's redox state, and adverse events in HF within a prospective single-hospital-based cohort. METHODS: We enrolled patients hospitalized for HF and measured fHMA using high-performance liquid chromatography at discharge. The primary endpoint was the composite of HF rehospitalization and all-cause death within one year after discharge. RESULTS: A total of 221 participants (median age:79 years; 35 % female) were included in the study. Over the course of one year, 26.1 % of the patients experienced HF readmission, while 13.1 % died. The low fHMA group divided by median of fHMA (<57.6 %) showed higher composite outcome rates (41.4 % for the low fHMA vs. 24.6 % for the high fHMA, p = 0.0114). Multivariate analysis, accounting for seven potential confounders, identified low fHMA (adjusted HR: 1.79 [1.03-3.11]) and lower hemoglobin as independent predictors of HF prognosis. CONCLUSIONS: The findings in this study provide the first evidence that fHMA is a potential novel prognostic biomarker in patients with HF.

Effect of albumin infusion on oxidative albumin modification and albumin binding capacity in chronic liver failure.

Oxidative albumin modification and impaired albumin binding function have been described both in chronic liver failure and for therapeutic albumin solutions. The aim of the present study was to evaluate the effect of albumin infusion on redox state and binding function of circulating albumin. We studied 20 patients with cirrhosis who routinely received albumin infusions for prevention of post-paracentesis circulatory dysfunction or treatment of hepatorenal syndrome. We measured albumin fractions by redox state of cysteine-34 and albumin binding properties using dansylsarcosine as site II ligand. Therapeutic albumin solutions showed high contents of human nonmercaptalbumin-1 and human nonmercaptalbumin-2, exceeding the respective values in our patients with decompensated cirrhosis. An initial protocol for the first nine patients sampled at baseline, 24 h and 48 h after albumin infusion revealed no significant changes of oxidized albumin species or albumin binding properties. However, a modified protocol for the remaining 11 patients sampled at baseline, <1 h after and 24 h after albumin infusion revealed short-lived changes of oxidized albumin species while no changes in albumin binding properties were observed. In conclusion, therapeutic albumin infusion transiently changed albumin redox state but did not improve binding function of circulating albumin in chronic liver failure.

Albumin of People with Diabetes Mellitus Is More Reduced at Low HbA1c.

Oxidative stress is involved in the development, progression, and complications of diabetes mellitus (DM). Oxidative modification of human serum albumin's cysteine-34 is a marker for oxidative stress-related pathological conditions. We aimed to evaluate the redox state of albumin in patients with DM to investigate possible correlations with age, diabetes duration, and disease control status. Plasma aliquots were collected from 52 participants (26 type 1 and 26 type 2 DM). Patients were divided into two groups according to their glycated hemoglobin levels less than or equal to and greater than 58 mmol/L. Albumin redox state was assessed with high-performance liquid chromatography by fractionating it into human mercaptalbumin (HMA) and human nonmercaptalbumin 1 and 2 (HNA1 and HNA2). Albumin redox fractions were differently related to the age of study participants. In age-matched T1DM and T2DM groups, the albumin redox state was essentially the same. Irreversibly oxidized HNA2 was positively correlated with diabetes duration, especially in the T1DM group. HNA was increased in people with an increased HbA1c (>58 mmol/mol). Our results support the hypothesis that oxidative stress plays a crucial role in DM pathogenesis and emphasize the importance of diabetes control on systemic oxidative burden.

A Methodological Basis for Estimating Human Mercaptalbumin in Serum and Plasma Using a Thiol-Binding Resin.

Human serum albumin is categorized into human mercaptalbumin (HMA) and human non-mercaptalbumin (HNA), according to the redox state of the cysteine residue at position 34. The ratio of HMA to total albumin (%HMA) is a novel biomarker of oxidative stress as well as protein nutritional status, but measuring %HMA normally requires an expensive analyzer such as HPLC and LC-MS, and can hardly be conducted in many clinical sites. To address this issue, we aimed to develop a methodological basis for estimating %HMA without these analyzers. An analytical method was investigated consisting of three steps, i.e., 1) removal of HMA from serum or plasma by using a thiol-binding resin (i.e., thereby obtaining a HNA fraction), 2) determination of both total albumin and HNA concentrations by a colorimetric assay or ELISA, and 3) calculation of %HMA. Proof-of-concept experiments, using serum and plasma samples of 4 adult volunteers, showed that the estimated value of %HMA obtained by this analytical method was significantly correlated with the theoretical value of %HMA determined by HPLC. The subsequent validation experiment, using 86 serum samples of pregnant women in the Japanese participants of SMILE Iwamizawa, also confirmed the significant association between the estimated and theoretical values of %HMA. This analytical method can be a basis to determine %HMA without using HPLC or LC-MS, contributing to the universalization of %HMA measurement as a clinical test.

Albumin redox state of maintenance haemodialysis patients is positively altered after treatment.

BACKGROUND AND AIM: Maintenance haemodialysis patients have increased morbidity and mortality which is mainly driven by an elevated inflammation level due to the uraemic milieu. A major part of this increased inflammation level is the degree of oxidative stress which can be assessed by albumin redox state (ARS). Aim of this study was to evaluate how the ARS is affected by a haemodialysis treatment and different dialyzer properties. METHODS: ARS was determined before and after haemodialysis treatment by fractionating it into reduced human mercaptalbumin (HMA), reversibly oxidized human non-mercaptalbumin 1 (HNA-1), and irreversibly oxidized human non-mercaptalbumin 2 (HNA-2) by high-performance liquid chromatography. In healthy individuals, albumin circulates in the following proportions: HMA 70-80%, HNA-1 20-30% and HNA-2 2-5%. High flux (FX 100 [Fresenius Medical Care], BG 1.8 [Toray], Xevonta Hi 18 [B. Braun], CTA-2000 [Kawasumi]) and low flux FX10 [Fresenius Medical Care] dialyzers were used. RESULTS: 58 patients (59% male, median age 68 years, median time on haemodialysis 32 month) were included in the study. Before haemodialysis treatment, HMA (median 55.9%, IQR 50.1-61.2%) was substantially lower than in healthy individuals. Accordingly, oxidized albumin fractions were above the level of healthy individuals (median HNA-1 38.5%, IQR 33.3-43.2%; median HNA-2 5.8%, IQR 5.1-6.7%). Before haemodialysis treatment HMA was significantly higher in patients usually treated with high flux membranes (p < 0.01). After haemodialysis treatment there was a significant increase of HMA and a decrease of HNA-1 and HNA-2 (p < 0.01). These effects were more pronounced in patients treated with high flux dialyzers (p < 0.01). There were no differences of ARS alteration with regard to the dialyzer´s sterilization mode or the presence of diabetes. CONCLUSION: The study confirms that the ARS is positively altered by haemodialysis and shows for the first time that this effect depends on dialyzer properties.

Redox State of Human Serum Albumin in Multiple Sclerosis: A Pilot Study.

Like in many other pathologies, oxidative stress is involved in the development of neurodegenerative disorders. Human serum albumin (HSA) is the main protein in different body fluids including cerebrospinal fluid (CSF). By its redox state in terms of cysteine-34, albumin serves as marker for oxidative burden. We aimed to evaluate the redox state of HSA in patients with multiple sclerosis in serum and CSF in comparison to controls to identify possible correlations with disease activity and severity. Samples were stored at -70 °C until analysis by HPLC for the determination of albumin redox state in terms of the fractions of human mercaptalbumin (HMA), human nonmercaptalbumin1 (HNA1), and human nonmercaptalbumin2 (HNA2). Albumin in CSF showed significantly higher fractions of the reduced form HMA and decreased HNA1 and HNA2. There was no difference between albumin redox states in serum of patients and controls. In CSF of patients HNA2 showed a trend to higher fractions compared to controls. Albumin redox state in serum was associated with physical disability in remission while albumin redox state in CSF was related to disease activity. Thus, albumin redox state in serum and CSF of patients in relation to disease condition merits further investigation.

Association of Redox State of Human Serum Albumin with Severity in Patients with Heart Failure.

Oxidative stress plays a crucial role in the progression of heart failure (HF). We surveyed the fraction of human mercaptalbumin [f (HMA) ], an indicator of the redox state of human serum albumin (HSA), in patients with HF and examined whether f (HMA) is associated with the severity of HF.We enrolled consecutive elderly patients hospitalized for acute HF or exacerbation of HF. The redox state of HSA was measured by the high-performance liquid chromatography with postcolumn bromocresol green method using serum samples collected close to discharge. First, the distribution of f (HMA) in HF was compared to that in community-dwelling elderly individuals (n = 125; median age, 80 years) as a control group analyzed in a previous study. Overall, 133 patients (median age, 81 years; 75 men) were included. Patients with HF showed a lower level of f (HMA) than those of the control group (55.0% [IQR 47.7-61.3] versus 66.3% [IQR 62.8-70.0], P < 0.001]. Multiple regression analysis showed a negative correlation between f (HMA) and log-transformed B-type natriuretic peptide (standardized beta = -0.19).Patients with HF showed lower f (HMA) than those in the control group. Additionally, f (HMA) was related to HF independently with log-transformed B-type natriuretic peptide in the multivariate regression analysis, suggesting that f (HMA) is a biomarker that reflects the redox state in HF patients.

Redox state as assessed using the measurement of human non-mercaptalbumin in embryo culture media is associated with successful embryo development in human in vitro fertilization.

The role of oxidative stress in the pathogenesis of various diseases has been attracting attention. We speculated as to whether the redox state of treatment solutions used for various diseases may play a role in treatment success. In the current study, we focused on the human embryo culture medium used for in vitro fertilization (IVF). A total of 173 oocytes from a total of 91 patients treated with IVF were enrolled. The redox state was assessed by measuring the levels of human non-mercaptalbumin (HNA). We analyzed factors related to blastocyst formation on day 5 or 6 after insemination. We also developed a random forest (RF) model for the prediction of blastocyst formation. The variable importance in the predictive model was assessed using the mean decrease in the Gini impurity. Blastocyst formation was observed in 41.04% (71/173) of the oocytes and was associated with a lower %HNA in the culture medium, a younger patient age, and the fertilization method (standard IVF or intracytoplasmic sperm injection). The RF model developed using these factors and 70% of the samples (training set, n = 121) was validated in the remaining testing set (n = 52) and produced an area under the curve of 0.761, where the %HNA in the culture medium was the most important variable for predicting blastocyst formation. In conclusion, lower levels of oxidative stress in embryo culture media were associated with the success of IVF treatment. The redox state of treatment solutions should be considered to support treatment success.

A simple colorimetric assay to determine the concentration and proportion of human mercaptalbumin.

Objectives: Human serum albumin can take on two forms, mercaptalbumin (HMA) or non-mercaptalbumin (HNA), depending on the redox status of its Cys34. The ratio of HMA and HNA is considered to be a novel biomarker of oxidative stress. While HPLC and mass spectrometry are established methods to measure HMA and HNA, a simple colorimetric assay was applied to measure this biomarker. Design and methods: Michler's Hydrol (4,4'-Bis(dimethylamino)benzhydrol) is a blue dye with a maximum absorption at 612 nm, and its absorption decreases when it reacts with a thiol group. Concentrations of HMA in serum samples from 36 healthy subjects were measured based on absorption changes of Michler's Hydrol. The proportion of HMA (HMA%) in total albumin was also obtained by dividing the HMA concentration by total albumin concentration, which was obtained by a bromocresol purple (BCP) assay. The proportion of HNA (HNA%) was obtained by subtracting HMA% from 100%. Results: HMA concentrations obtained by Michler's Hydrol assay were highly correlated (r2 = 0.97) with reference values obtained by HPLC (HMA%) and BCP assay (total albumin). The HNA% obtained by Michler's Hydrol and BCP assays combined also gave a good correlation (r2 = 0.96) and a small deviation (average 2.4%) with respect to HPLC as a reference method. Conclusions: A colorimetric assay using Michler's Hydrol was optimized for a 96-well plate format so that it can be easily performed in a standard laboratory setting. This assay gives HMA concentrations and HNA proportions comparable to HPLC.

Cystine supplementation sustains plasma mercaptalbumin levels in rats fed low-protein diets more effectively than methionine.

We recently reported that dietary cystine maintained plasma mercaptalbumin levels in rats fed low-protein diets. The present study aimed to compare the influence of low-protein diets supplemented with cystine and methionine, which is another sulfur amino acid, on plasma mercaptalbumin levels in rats. Male Sprague-Dawley rats were fed a 20% soy protein isolate diet (control group), 5% soy protein isolate diet (low-protein group) or 5% soy protein isolate diet supplemented with either methionine (low-protein + Met group) or cystine (low-protein + Cyss group) for 1 week. The percentage of mercaptalbumin within total plasma albumin of the low-protein + Met group was significantly lower than that of the control and low-protein + Cyss groups. No significant differences in the mRNA levels of tumor necrosis factor-α, interleukin-6, interleukin-1ß, and cyclooxygenase 2 in blood cells were observed between the low-protein + Met and low-protein + Cyss groups. Treatment with buthionine-(S,R)-sulfoximine, an inhibitor of glutathione synthesis, did not influence the percentage of mercaptalbumin within total plasma albumin in rats fed the low-protein diet supplemented with cystine. These results suggest that supplementation with cystine may be more effective than that with methionine to maintain plasma mercaptalbumin levels in rats with protein malnutrition. Cystine might regulate plasma mercaptalbumin levels via the glutathione-independent pathway.

New colorimetric method with bromocresol purple for estimating the redox state of human serum albumin.

Oxidative damage results in protein modification and is observed in many diseases, such as heart failure and renal insufficiency. Human serum albumin is an index of oxidative change and is conventionally measured using high-performance liquid chromatography (HPLC). Although this method is more sensitive than the colorimetric method, it is time-consuming for clinical practice and the sera must be stored at -80°C before analysis. To overcome these limitations, in the present study we developed a new reagent for a more rapid and convenient quantification of oxidative stress, involving determination of the ratio of human nonmercaptalbumin to total albumin using a colorimetric method with bromocresol purple. The clinical utility of the developed reagent was confirmed by demonstrating the consistently higher oxidative stress levels in dialysis patients than in healthy control subjects, matching the results of the conventional HPLC method. This novel approach could be a valuable tool for immediate estimation of the state of oxidative stress during the course of disease and treatment, and could aid clinical treatment decisions.

A More Oxidized Plasma Albumin Redox State and Lower Plasma HDL Particle Number Reflect Low-Protein Diet Ingestion in Adult Rats.

BACKGROUND: Plasma albumin (ALB) redox state reflects protein nutritional status, but how it differs from other protein nutrition biomarkers remains to be fully elucidated. OBJECTIVE: This study aimed to delineate the characteristics of plasma ALB redox state as a protein nutrition biomarker. METHODS: Adult male Wistar rats were maintained on an AIN-93 M [14% casein, control (CT)] diet or an AIN-93 M-based 5% casein [low protein (LP)] diet ad libitum for 4 wk. Plasma samples were repeatedly obtained from the same rats at weeks 0-4, ALB redox state was determined by HPLC, and the concentrations of conventional protein nutrition biomarkers, ALB and transthyretin (TTR), were compared between the groups by Student t test. Body mass, relative muscle masses, plasma proteome, and plasma lipids at week 4 were also compared. RESULTS: Plasma ALB redox state shifted to a more oxidized state in the LP diet group compared with the CT diet group at weeks 1-4. The LP diet group also showed significantly lower plasma ALB concentrations at weeks 1 and 2 (13% and 11% lower, respectively) and significantly lower TTR concentration at week 1 (21% lower) compared with the CT diet group, but these concentrations did not differ significantly at weeks 3 and 4. After 4 wk, body mass and relative soleus and gastrocnemius muscle masses did not differ, but the relative plantaris muscle mass tended to be 4% lower (1.75 compared with 1.68 g/kg body mass) in the LP diet group compared with the CT group (P = 0.06). The LP diet group also had a significantly lower HDL particle number than the CT group (30% lower). CONCLUSIONS: A more oxidized plasma ALB redox state and lower plasma HDL particle number reflect LP diet ingestion in adult rats, which did not exhibit changes of plasma ALB and TTR concentrations.

Redox state of albumin affects its lipid mediator binding characteristics.

Depending on its redox status, albumin is known to exist as two forms: reduced albumin or human mercaptalbumin (HMA); and oxidised albumin or human nonmercaptalbumin (HNA). The ratio of HNA to HMA is reportedly elevated in several diseases. Since lipid mediators, such as eicosanoids and lysophospholipids, are typically bound to albumin, we examined the possible preferences of lipid mediators for HNA or HMA. We observed that DHA-derived and EPA-derived eicosanoids preferred to be bound to HMA, while the levels of lysophospholipid mediators, such as lysophosphatidic acids and sphingosine 1-phosphate, were higher in the HNA fraction. Considering the bioactivities reported in previous basic studies, these results suggest that proatherosclerotic lipid mediators might generally prefer HNA, while antiatherosclerotic ones might prefer HMA. Oxidative stress affects the redox status of albumin, which might modulate the dynamism of lipid mediators. This pathway might be partly involved in the association between oxidation and atherosclerosis.

A Method for Stabilizing the Proportion of the Reduced Form of Albumin During Cell-Free and Concentrated Ascites Reinfusion Therapy in Patients with Malignant Ascites.

Cell-free and concentrated ascites reinfusion therapy (CART) is used to treat malignant ascites. However, the qualities of albumin in malignant ascites, such as antioxidative activity, may decrease owing to oxidative stress caused by cancer cells and inflammatory reactions. We investigated the fraction percentages of mercaptalbumin (HMA%, reduced form) and non-mercaptalbumin (HNA%, oxidized form) in malignant ascites from 21 patients who received CART and compared the HMA% in the malignant ascites and human serum albumin (HSA) preparations. HMA% of albumin in malignant ascites (22.5%) was significantly lower than that in HSA preparation (42.2%). To ensure a high HMA%, we added L-cysteine to the paracentesis-treated ascites followed by dialysis 1 h later. As a result, the HMA% of albumin in malignant ascites was increased to 59.1%. Our results suggest that using this method in CART will improve patient's albumin quality.

Plasma Albumin Redox State Is Responsive to the Amino Acid Balance of Dietary Proteins in Rats Fed a Low Protein Diet.

We recently reported that plasma albumin redox state, which correlates with albumin synthesis rate, could be associated with the quality of dietary protein. Aiming to elucidate the association between them, plasma albumin redox state was investigated in rats fed various kinds of AIN-93G-based low protein diets. Plasma albumin redox state was shifted to a more oxidized state in rats fed 3% casein (CN) diet than those fed 3% whey protein or 3% wheat gluten diet, while supplementing 3% CN diet with cystine reversed it to a more reduced state, indicating that cystine would complement the shortage of cysteine in CN, thereby increasing albumin synthesis rate. Supplementation with glutathione, a cysteine-containing antioxidative tripeptide, normalized hepatic glutathione redox state modulated by ingestion of 3% CN diet, but it only reversed the oxidized shift of plasma albumin redox state to an extent similar to cystine alone or the constituting amino acid mixture of glutathione (i.e., glutamic acid, cystine, and glycine), indicating that glutathione would primarily serve as a source of cysteine rather than exert its antioxidative activity. Plasma albumin would thus be influenced by amino acid balance in dietary proteins, and it could be useful as a biomarker that contributes to prevention of protein under-nutriton, caused by not only insufficient protein intake but also ingestion of poor-quality protein.

Increased Ratio of Non-mercaptalbumin-1 Among Total Plasma Albumin Demonstrates Potential Protein Undernutrition in Adult Rats.

The redox state of plasma albumin shifts in response to dietary protein intake in growing rats, and the shift is more sensitive than that of plasma albumin level, a classical marker of protein nutritional status. While it has been suggested that plasma albumin redox state could be useful as a novel marker of protein nutritional status, the above animal model is highly sensitive to dietary protein intake and the observation may not be extrapolated widely to humans. This study aimed to investigate whether albumin redox state also reflects protein nutritional status in adult rats, which have a lower dietary protein requirement and are less responsive to protein intake. Male adult rats were placed on AIN-93M diet (14% casein), or AIN-93M-based low protein diets (10 or 5% casein) ad libitum for 24 weeks. Whereas there was no significant difference in body weight between the groups at the end of the experimental period, the 5% casein diet group had the smallest gastrocnemius muscle weight among the groups, which was significantly lower than that of the 10% casein diet group. Plasma albumin level was also lower in the 5% casein diet group compared with the other groups, but the differences were limited and inconsistent during the experimental period. Among the albumin redox isoforms such as mercaptalbumin, non-mercaptalbumin-1, and non-mercaptalbumin-2, the ratio of non-mercaptalbumin-1 among total albumin was significantly higher in the 5% casein diet group, and the increase remained constant throughout the experimental period. Increased non-mercaptalbumin-1 ratio would thus demonstrate the presence of potential protein undernutrition in adult rats, as manifested only by a decreased gain in a specific type of skeletal muscle; non-mercaptalbumin-1 among total albumin ratio could be useful as a robust marker of protein nutritional status, contributing to prevention of protein undernutrition-related diseases such as frailty and sarcopenia.

Dietary cystine is important to maintain plasma mercaptalbumin levels in rats fed low-protein diets.

The oxidized/reduced state of plasma albumin in rats is influenced by the quantity of dietary protein. However, the effects of the protein quality on the oxidized/reduced state of plasma albumin are not clear. We hypothesized that the quality of dietary protein might modulate the oxidized/reduced state of plasma albumin. The aim of the present study was to examine whether the amino acid composition of dietary protein modulates the oxidized/reduced state of plasma albumin in rats. Male Sprague-Dawley rats were fed low-protein diets containing 5% casein (CA), 5% egg white (EW), or 6% wheat gluten (WG) for 2 weeks. The plasma albumin concentration gradually decreased in rats fed each diet; however, there was no significant difference among the groups. In rats fed the 5% CA diet, the percentage of mercaptalbumin within the total plasma albumin was significantly lower than in those fed the EW or WG diet. Compared with EW or WG, CA contains lower amounts of glycine and cystine. In rats fed a 5% CA diet supplemented with cystine, the percentage of mercaptalbumin was significantly higher than that in rats fed a 5% CA diet supplemented with glycine. The expression of hepatic eukaryotic initiation factor 4E-binding protein 1 was significantly lower in rats fed the cystine-supplemented diet than in those fed the glycine-supplemented diet. These results suggest that dietary protein with a high cystine content maintains plasma mercaptalbumin levels in rats fed low-protein diets.

A simple, rapid and validated high-performance liquid chromatography method suitable for clinical measurements of human mercaptalbumin and non-mercaptalbumin.

Background Human mercaptalbumin and human non-mercaptalbumin have been reported as markers for various pathological conditions, such as kidney and liver diseases. These markers play important roles in redox regulations throughout the body. Despite the recognition of these markers in various pathophysiologic conditions, the measurements of human mercaptalbumin and non-mercaptalbumin have not been popular because of the technical complexity and long measurement time of conventional methods. Methods Based on previous reports, we explored the optimal analytical conditions for a high-performance liquid chromatography method using an anion-exchange column packed with a hydrophilic polyvinyl alcohol gel. The method was then validated using performance tests as well as measurements of various patients' serum samples. Results We successfully established a reliable high-performance liquid chromatography method with an analytical time of only 12 min per test. The repeatability (within-day variability) and reproducibility (day-to-day variability) were 0.30% and 0.27% (CV), respectively. A very good correlation was obtained with the results of the conventional method. Conclusions A practical method for the clinical measurement of human mercaptalbumin and non-mercaptalbumin was established. This high-performance liquid chromatography method is expected to be a powerful tool enabling the expansion of clinical usefulness and ensuring the elucidation of the roles of albumin in redox reactions throughout the human body.

A revised method for determination of serum mercaptalbumin and non-mercaptalbumin by high-performance liquid chromatography coupled with postcolumn bromocresol green reaction.

BACKGROUND: Previously, high-performance liquid chromatography (HPLC) equipped with ultraviolet or fluorescence detectors has been used for separation of human mercaptalbumin (HMA) and human non-mercaptalbumin (HNA). However, it is difficult to perform reliable chromatographic analysis due to peak interference of such serum compounds as uric acid and bilirubin. The aim of this study is to explore a selective and simple analytical method for the determination of HMA and HNA. METHOD: HMA and HNA in serum sample were separated by HPLC and reacted with bromocresol green using a postcolumn reaction scheme. RESULTS: A complete separation of HMA and HNA is achieved in less than 30 min by using weak anion exchange columns and isocratic elution. Within-run and between-day precisions at albumin concentration of 45 g/L were 4.2 and 1.7% for HMA and 4.5 and 4.6% for HNA, respectively. There was no interference in HMA and HNA peaks when bilirubin-, haemoglobin- or chyle-spiked pooled serum samples were analysed. CONCLUSION: Our method is reliable and not labour-intensive and, therefore, might be applicable for clinical and epidemiological studies.

L-Cysteine conjugated poly L-lactide nanoparticles containing 5-fluorouracil: formulation, characterization, release and uptake by tissues in vivo.

CONTEXT: Targeted delivery of drugs is still a therapeutic challenge and numerous methods have been reported for the same. OBJECTIVE: In this study, emphasis was placed on developing nanoparticles loaded with 5-fluorouracil (FU) and modifying the surface of the nanoparticles by conjugation with amino acid, to improve the distribution of 5-FU in the lungs. METHODS: An emulsion solvent evaporation technique was used to formulate nanoparticles of FU using Poly L-lactide and Pluronic F-68. The nanoparticles were conjugated with L-Cysteine using EDC as the activator of COOH group and were evaluated for product yield, particle size, surface morphology, amount of conjugation by Ellman's method and in vitro drug release study. RESULTS AND CONCLUSION: The results indicated 60-65% yield with an average particle size of 242.7 ± 37.11 nm for the cysteine conjugated nanoparticle (CNP) formulation and more than 70% conjugation of cysteine. The cumulative percentage of drug released over a period of 24 h was found to be 58%. An increase in distribution of the delivery system in lungs (11.4% ID after 1 h) in mice was found indicating the role of L-Cysteine in the transport mechanism to the lungs. In vivo kinetic studies in rats revealed higher circulation time of CNP as compared to pure FU solution. The study helps in designing a colloidal delivery system for increased distribution of drugs to the lungs and may be helpful in delivery of drugs in conditions like non-small cell lung carcinomas.