Glycated albumin and diabetes mellitus

Background

Diabetes is a growing worldwide problem that is strongly associated with atherosclerosis. Screening and intervention for diabetes in the earliest stages are advocated for the prevention of diabetic complications and cardiovascular disease.

Scope of review

This review gives a background of and discusses the potential clinical utility of glycated albumin (GA) in diabetes.

Major conclusions

GA is a ketoamine formed via a non-enzymatic glycation reaction of serum albumin and it reflects mean glycemia over two to three weeks. GA can be used for patients with anemia or hemoglobinopathies for whom the clinically measured hemoglobin A1c level may be inaccurate. Because both serum and plasma samples can be used, GA can be analyzed from the same samples as common biological markers. GA is a useful marker for the screening of diabetes in a medical evaluation. It can be also used to determine the effectiveness of treatment before initiating or changing medications for diabetic patients. GA is potentially an atherogenic protein in the development of diabetic atherosclerosis.

General significance

GA measurement is useful as part of a routine examination to screen for both diabetes and atherosclerosis. This article is part of a Special Issue entitled Serum Albumin.     

High-throughput analytical gel filtration screening of integral membrane proteins for structural studies

Background

Structural studies of integral membrane proteins (IMPs) are often hampered by difficulties in producing stable homogenous samples for crystallization. To overcome this hurdle it has become common practice to screen large numbers of target proteins to find suitable candidates for crystallization. For such an approach to be effective, an efficient screening strategy is imperative. To this end, strategies have been developed that involve the use of green fluorescent protein (GFP) fusion constructs. However, these approaches suffer from two drawbacks; proteins with a translocated C-terminus cannot be tested and scale-up from analytical to preparative purification is often non-trivial and may require re-cloning.

Methods

Here we present a screening approach that prioritizes IMP targets based on three criteria: expression level, detergent solubilization yield and homogeneity as determined by high-throughput small-scale immobilized metal affinity chromatography (IMAC) and automated size-exclusion chromatography (SEC).

Results

To validate the strategy, we screened 48 prokaryotic IMPs in two different vectors and two Escherichia coli strains. A set of 11 proteins passed all preset quality control checkpoints and was subjected to crystallization trials. Four of these crystallized directly in initial sparse matrix screens, highlighting the robustness of the strategy.

Conclusions

We have developed a rapid and cost efficient screening strategy that can be used for all IMPs regardless of topology. The analytical steps have been designed to be a good mimic of preparative purification, which greatly facilitates scale-up.

General significance

The screening approach presented here is intended and expected to help drive forward structural biology of membrane proteins.     

Screening for Fabry disease in patients undergoing dialysis for chronic renal failure in Turkey: Identification of new case with novel mutation

Background

Chronic renal failure (CRF) is a serious complication of Fabry disease (FD). The aims of the present study were to determine the prevalence of unrecognized FD in Turkish hemodialysis population and to investigate the molecular background.

Method

Primarily, α-galactosidase A (α-Gal A) activity was investigated on DBS in 1136 patients of both sexes who underwent dialysis for CRF in Turkey. The disease was confirmed by analyzing enzyme activity in leukocyte and GLA gene sequencing in all patients in whom α-Gal A level was 40% of normal or less.

Results

Mean age of the patients (44.5% female, 52.5% male) was 56.46 ± 15.85 years. Enzyme activity was found low with DBS method in 12 patients (four males, eight females). Two men, but no women, were diagnosed with FD by enzymatic and molecular analysis. In consequence of genetic analysis of a case, a new mutation [hemizygote c.638C>T (p.P214S) missense mutation in exon 5] was identified, which was not described in literature. Family screening of cases identified six additional cases.

Conclusion

As a result of this initial screening study performed on hemodialysis patients for the first time with DBS method in Turkey, the prevalence of FD was detected as 0.17%. Although the prevalence seems to be low, screening studies are of great importance for detecting hidden cases as well as for identifying other effected family members.     

A high-throughput assay for the detection of Tyr-phosphorylated proteins in urine of bladder cancer patients

Background

Bladder cancer has the peculiarity of shedding neoplastic cells and their components in urine representing a valuable opportunity to detect diagnostic markers. Using a semi-quantitative method we previously demonstrated that the levels of Tyr-phosphorylated proteins (TPPs) are highly increased in bladder cancer tissues and that soluble TPPs can also be detected in patient's urine samples. Although the preliminary evaluation showed very promising specificity and sensitivity, insufficient accuracy and very low throughput of the method halted the diagnostic evaluation of the new marker. To overcome this problem we developed a quantitative methodology with high sensitivity and accuracy to measure TPPs in urine.

Methods

The Immobilized Metal Affinity Chromatography (IMAC) was miniaturized in a 96 well format. Luminescence, visible and infrared fluorescence antibody-based detection methods were comparatively evaluated.

Results

Due to their low abundance we evidenced that both phosphoprotein enrichment step and very sensitive detection methods are required to detect TPPs in urine samples. To pursue high throughput, reproducibility and cost containment, which are required for bladder cancer screening programs, we coupled the pre-analytical IMAC procedure with high sensitive detection phases (infrared fluorescence or chemiluminescence) in an automated platform.

Conclusions

A high throughput method for measuring with high sensitivity TPP levels in urine samples is now available for large clinical trial for the establishment of the diagnostic and predictive power of TPPs as bladder cancer marker.

General significance

The new assay represents the first quantitative and high throughput method for the measurement of TPPs in urine.     

Glycans as cancer biomarkers

Background

Non-invasive biomarkers, such as those from serum, are ideal for disease prognosis, staging and monitoring. In the past decade, our understanding of the importance of glycosylation changes with disease has evolved.

Scope of review

We describe potential biomarkers derived from serum glycoproteins for liver, pancreatic, prostate, ovarian, breast, lung and stomach cancers. Methods for glycan analysis have progressed and newly developed high-throughput platform technologies have enabled the analysis of large cohorts of samples in an efficient manner. We also describe this evolution and trends to follow in the future.

Major conclusions

Many convincing examples of aberrant glycans associated with cancer have come about from glycosylation analyses. Most studies have been carried out to identify changes in serum glycan profiles or through the isolation and identification of glycoproteins that contain these irregular glycan structures. In a majority of cancers the fucosylation and sialylation expression are found to be significantly modified. Therefore, these aberrations in glycan structures can be utilized as targets to improve existing cancer biomarkers.

General significance

The ability to distinguish differences in the glycosylation of proteins between cancer and control patients emphasizes glycobiology as a promising field for potential biomarker identification. Furthermore, the high-throughput and reproducible nature of the chromatography platform have highlighted extensive applications in biomarker discovery and allowed integration of glycomics with other -omics fields, such as proteomics and genomics, making systems glycobiology a reality. This article is part of a Special Issue entitled Glycoproteomics.     

Serum thioredoxin reductase levels increase in response to chemically induced acute liver injury

Background

Mammalian thioredoxin reductases (TrxR) are selenoproteins with important roles in antioxidant defense and redox regulation, principally linked to functions of their main substrates thioredoxins (Trx). All major forms of TrxR are intracellular while levels in serum are typically very low.

Methods

Serum TrxR levels were determined with immunoblotting using antibodies against mouse TrxR1 and total enzyme activity measurements were performed, with serum and tissue samples from mouse models of liver injury, as triggered by either thioacetamide (TAA) or carbon tetrachloride (CCl₄).

Results

TrxR levels in serum increased upon treatment and correlated closely with those of alanine aminotransferase (ALT), an often used serum biomarker for liver damage. In contrast, Trx1, glutathione reductase, superoxide dismutase or selenium-containing glutathione peroxidase levels in serum displayed much lower increases than TrxR or ALT.

Conclusions

Serum TrxR levels are robustly elevated in mouse models of chemically induced liver injury.

General significance

The exaggerated TrxR release to serum upon liver injury may reflect more complex events than a mere passive release of hepatic enzymes to the extracellular milieu. It can also not be disregarded that enzymatically active TrxR in serum could have yet unidentified physiological functions.     

Estrogen receptor alpha (ERα) promoter methylation status in tumor and serum DNA in Egyptian breast cancer patients

Background

Status of DNA methylation is one of the most common molecular alterations in human neoplasia. Because it is possible to detect these epigenetic alterations in the bloodstream of patients, we investigated the aberrant DNA methylation status of estrogen receptor alpha (ERα) in patient pretherapeutic sera and tissue.

Materials and methods

In this case control study the patient series consisted of 120 sporadic primary breast cancer cases and 100 patients with benign breast lesion. ER3, ER4, and ER5 primers were used for methylation-specific polymerase chain reaction (MSP) to analyze the CpG methylation of promoter region of ERα gene. Correlation between ER3, ER4, and ER5 methylation and clinicopathological characteristics of the patients was investigated.

Result

The methylation status of ER3, ER4 and ER5 was 65%, 26.7% and 61.7% in tissue respectively and 57.5%, 21.7% and 55.8% in serum respectively. The concordance between tumor and serum DNA methylation was 80%, 72% and 92% for ER3, ER4 and ER5 respectively.

Conclusions

This study demonstrated the potential utility of serum DNA methylation of ERα gene promoter as a non-invasive diagnostic and/or prognostic marker in patients with breast cancer.     

Validation of the organic electrochemical transistor for in vitro toxicology

Background

The gastrointestinal epithelium provides a physical and biochemical barrier to the passage of ions and small molecules; however this barrier may be breached by pathogens and toxins. The effect of individual pathogens/toxins on the intestinal epithelium has been well characterized: they disrupt barrier tissue in a variety of ways, such as by targeting tight junction proteins, or other elements of the junctions between adjacent cells. A variety of methods have been used to characterize disruption in barrier tissue, such as immunofluorescence, permeability assays and electrical measurements of epithelia resistance, but these methods remain time consuming, costly and ill-suited to diagnostics or high throughput toxicology.

Methods

The advent of organic electronics has created a unique opportunity to interface the worlds of electronics and biology, using devices such as the organic electrochemical transistor (OECT), whose low cost materials and potential for easy fabrication in high throughput formats represent a novel solution for assessing epithelial tissue integrity.

Results

In this study, OECTs were integrated with gastro-intestinal cell monolayers to study the integrity of the gastrointestinal epithelium, providing a very sensitive way to detect minute changes in ion flow across the cell layer due to inherent amplification by the transistor.

Major conclusions

We validate the OECT against traditional methods by monitoring the effect of toxic compounds on epithelial tissue. We show a systematic characterization of this novel method, alongside existing methods used to assess barrier tissue function.

General significance

The toxic compounds induce a dramatic disruption of barrier tissue, and the OECT measures this disruption with increased temporal resolution and greater or equal sensitivity when compared with existing methods. This article is part of a Special Issue entitled Organic Bioelectronics — Novel Applications in Biomedicine.     

A straightforward protocol for the preparation of high performance microarray displaying synthetic MUC1 glycopeptides

Background

Human serum MUC1 peptide fragments bearing aberrant O-glycans are secreted from columnar epithelial cell surfaces and known as clinically important serum biomarkers for the epithelial carcinoma when a specific monoclonal antibody can probe disease-relevant epitopes. Despite the growing importance of MUC1 glycopeptides as biomarkers, the precise epitopes of most anti-MUC1 monoclonal antibodies remains unclear.

Methods

A novel protocol for the fabrication of versatile microarray displaying peptide/glycopeptide library was investigated for the construction of highly sensitive and accurate epitope mapping assay of various anti-MUC1 antibodies.

Results

Selective imine-coupling between aminooxy-functionalized methacrylic copolymer with phosphorylcholine unit and synthetic MUC1 glycopeptides-capped by a ketone linker at N-terminus provided a facile and seamless protocol for the preparation of glycopeptides microarray platform. It was demonstrated that anti-KL-6 monoclonal antibody shows an extremely specific and strong binding affinity toward MUC1 fragments carrying sialyl T antigen (Neu5Acα2,3Galβ1,3GalNAcα1→) at Pro-Asp-Thr-Arg motif when compared with other seven anti-MUC1 monoclonal antibodies such as VU-3D1, VU-12E1, VU-11E2, Ma552, VU-3C6, SM3, and DF3. The present microarray also uncovered the occurrence of IgG autoantibodies in healthy human sera that bind specifically with sialyl T antigen attached at five potential O-glycosylation sites of MUC1 tandem repeats.

Conclusion

We established a straightforward strategy toward the standardized microarray platform allowing highly sensitive and accurate epitope mapping analysis by reducing the background noise due to nonspecific protein adsorption.

General significance

The present approach would greatly accelerate the discovery research of new class autoantibodies as well as the development of therapeutic mAbs reacting specifically with disease-relevant epitopes.     

Glucosylsphingosine Is a Highly Sensitive and Specific Biomarker for Primary Diagnostic and Follow-Up Monitoring in Gaucher Disease in a Non-Jewish,Caucasian Cohort of Gaucher Disease Patients

Background

Gaucher disease (GD) is the most common lysosomal storage disorder (LSD). Based on a deficient β-glucocerebrosidase it leads to an accumulation of glucosylceramide. Standard diagnostic procedures include measurement of enzyme activity, genetic testing as well as analysis of chitotriosidase and CCL18/PARC as biomarkers. Even though chitotriosidase is the most well-established biomarker in GD, it is not specific for GD. Furthermore, it may be false negative in a significant percentage of GD patients due to mutation. Additionally, chitotriosidase reflects the changes in the course of the disease belatedly. This further enhances the need for a reliable biomarker, especially for the monitoring of the disease and the impact of potential treatments.

Methodology

Here, we evaluated the sensitivity and specificity of the previously reported biomarker Glucosylsphingosine with regard to different control groups (healthy control vs. GD carriers vs. other LSDs).

Findings

Only GD patients displayed elevated levels of Glucosylsphingosine higher than 12 ng/ml whereas the comparison controls groups revealed concentrations below the pathological cut-off, verifying the specificity of Glucosylsphingosine as a biomarker for GD. In addition, we evaluated the biomarker before and during enzyme replacement therapy (ERT) in 19 patients, demonstrating a decrease in Glucosylsphingosine over time with the most pronounced reduction within the first 6 months of ERT. Furthermore, our data reveals a correlation between the medical consequence of specific mutations and Glucosylsphingosine.

Interpretation

In summary, Glucosylsphingosine is a very promising, reliable and specific biomarker for GD.     

Assessment of HER-2 gene overexpression in Isfahan province breast cancer patients using Real Time RT-PCR and immunohistochemistry

Purpose

Overexpression of proto-oncogene HER-2 is one of the main molecular markers of breast cancer involved in prognosis and diagnosis and also in trastuzumab therapy. Thus, a request for the evaluation of HER-2 status in breast cancer has been increasing. The aim of our study was assessment of HER-2 overexpression in malignant and benign breast cancer specimens by Real Time RT-PCR technique and comparison of its results with IHC outcomes.

Methods

Twenty benign and sixty malignant breast cancers in addition to fifteen normal breast tissue specimens were analyzed by Real Time RT-PCR method. Fresh tissue samples were disrupted by mortar and pestle. A syringe and a needle were used for complete homogenization of the tissues. The RNA was then isolated from the samples and converted to cDNA. A standard curve was initially plotted using BioEasy SYBR Green I and then all 95 specimens were studied by Real Time RT-PCR using 2^− ΔΔCt method.

Results

23.3% of 60 malignant specimens showed HER-2 overexpression, while all of the benign samples represented the normal expression level of HER-2 gene. The concordance rate between the results of Real Time RT-PCR and IHC was 86.6%.

Conclusion

Real Time RT-PCR method is an almost reliable technique and at least can be used as a complementary method for confirming IHC results. This is emanated from relatively high rate of concordance between outcomes of IHC test, as a routine method of detecting the HER-2 gene expression status, and Real Time RT-PCR technique.     

Honokiol: A non-adipogenic PPARγ agonist from nature

Background

Peroxisome proliferator-activated receptor gamma (PPARγ) agonists are clinically used to counteract hyperglycemia. However, so far experienced unwanted side effects, such as weight gain, promote the search for new PPARγ activators.

Methods

We used a combination of in silico, in vitro, cell-based and in vivo models to identify and validate natural products as promising leads for partial novel PPARγ agonists.

Results

The natural product honokiol from the traditional Chinese herbal drug Magnolia bark was in silico predicted to bind into the PPARγ ligand binding pocket as dimer. Honokiol indeed directly bound to purified PPARγ ligand-binding domain (LBD) and acted as partial agonist in a PPARγ-mediated luciferase reporter assay. Honokiol was then directly compared to the clinically used full agonist pioglitazone with regard to stimulation of glucose uptake in adipocytes as well as adipogenic differentiation in 3T3-L1 pre-adipocytes and mouse embryonic fibroblasts. While honokiol stimulated basal glucose uptake to a similar extent as pioglitazone, it did not induce adipogenesis in contrast to pioglitazone. In diabetic KKAy mice oral application of honokiol prevented hyperglycemia and suppressed weight gain.

Conclusion

We identified honokiol as a partial non-adipogenic PPARγ agonist in vitro which prevented hyperglycemia and weight gain in vivo.

General significance

This observed activity profile suggests honokiol as promising new pharmaceutical lead or dietary supplement to combat metabolic disease, and provides a molecular explanation for the use of Magnolia in traditional medicine.     

A rubber transfer gasket to improve the throughput of liquid–liquid extraction in 96-well plates: Application to vitamin D testing

Background

The unmitigated rise in demand for the assessment of vitamin D status has taxed the ability of clinical mass spectrometry laboratories to preserve turn-around times. We aimed to improve the throughput of liquid–liquid extraction of plasma/serum for the assay of 25-hydroxy vitamin D.

Methods

We designed and fabricated a flexible rubber gasket that seals two 96-well plates together to quantitatively transfer the contents of one plate to another. Using the transfer gasket and a dry-ice acetone bath to freeze the aqueous infranatant, we developed a novel liquid–liquid extraction workflow in a 96-well plate format. We applied the technology to the mass spectrometric quantification of 25-hydroxy vitamin D.

Results

Cross-contamination between wells was ≤0.13%. The interassay imprecision over 132 days of clinical implementation was less than 10%. The method compared favorably to a standard liquid–liquid extraction in glass tubes (Deming slope = 1.018, S_x|y = 0.022). The accuracy of the assay was 102–105% as assessed with the recently released control materials from NIST.

Conclusions

The development of a plate-sealing gasket permits the liquid–liquid extraction of clinical specimens in a moderate-throughput workflow and the reliable assay of vitamin D status. In the future, the gasket may also prove useful in other sample preparation techniques for HPLC or mass spectrometry.     

Genetic variants in TERT–CLPTM1L genetic region associated with several types of cancer: A meta-analysis

Background

TERT–CLPTM1L genomic region has been extensively associated with several types of cancer. However, results were inconsistent. We performed this meta-analysis to estimate the association between TERT (rs2736100, rs2736098), as well as CLPTM1L (rs402710, rs401681) polymorphisms and cancer risk.

Methods

Electronic search in PubMed, EMBASE and China National Knowledge Infrastructure (CNKI) was conducted to select the studies. Studies containing available genotype frequencies of those 4 polymorphisms were chosen, and overall odds ratio (OR) with 95% confidence interval (CI) was used to assess the association.

Results

The final meta-analysis included 16 published case–control studies. Increased cancer risk was found between TERT-rs2736100, as well as CLPTM1L-rs402710 and cancer susceptibility. In addition, we found an increased risk of cancer in both rs2736098 and rs401681 homozygous variant genetic model.

Conclusions

This meta-analysis suggested that TERT–CLPTM1L genomic region was associated with increased risk of cancer. To validate the association between these polymorphisms and cancer susceptibility, further studies with larger participants are needed.     

Ubiquitination and SUMOylation of annexin A1 and helicase activity

Background

While annexin A1 in nuclei is proposed to be involved in cell transformation, its functions remain poorly understood. Since annexin A1 has the consensus motif, ¹⁶⁰LKRD, for SUMOylation as well as Ks, acceptors for ubiquitination that regulates localization and functions of proteins, we investigated SUMOylation and ubiquitination of annexin A1.

Methods

SUMOylation and ubiquitination of bovine annexin A1 were biochemically tested in vitro by purified proteins, and were confirmed by cell experiments with L5178 lymphoma cells. Effects of the modifications on DNA helicase activity were measured by ssDNA binding activity and by dsDNA unwinding activity.

Results

SUMOylation of annexin A1 was catalyzed by Ubc9, while its ubiquitination was by Rad6-Rad 18. Ubiquitinated annexin A1 had higher affinity for damaged DNA, and promoted in vitro translesion DNA synthesis by Pol β. In mouse lymphoma L5178Y tk(+/−) cells, levels of SUMOylated annexin A1 decreased by DNA damaging agents, MMS or As³⁺, whereas those of ubiquitinated annexin A1 increased under the same conditions.

Conclusion

These observations suggest but do not necessarily prove that ubiquitinated annexin A1 in nuclei may be involved in DNA damage response, while SUMOylated annexin A1 functions in proliferation–differentiation.

Significance

Ubiquitination of annexin A1 may play an important role in mutagenesis, an initial step of cell transformation.     

Integrated Analysis of Gene Expression Profiles Associated with Response of Platinum/Paclitaxel-Based Treatment in Epithelial Ovarian Cancer

Purpose

This study aims to explore gene expression signatures and serum biomarkers to predict intrinsic chemoresistance in epithelial ovarian cancer (EOC).

Patients and Methods

Gene expression profiling data of 322 high-grade EOC cases between 2009 and 2010 in The Cancer Genome Atlas project (TCGA) were used to develop and validate gene expression signatures that could discriminate different responses to first-line platinum/paclitaxel-based treatments. A gene regulation network was then built to further identify hub genes responsible for differential gene expression between the complete response (CR) group and the progressive disease (PD) group. Further, to find more robust serum biomarkers for clinical application, we integrated our gene signatures and gene signatures reported previously to identify secretory protein-encoding genes by searching the DAVID database. In the end, gene-drug interaction network was constructed by searching Comparative Toxicogenomics Database (CTD) and literature.

Results

A 349-gene predictive model and an 18-gene model independent of key clinical features with high accuracy were developed for prediction of chemoresistance in EOC. Among them, ten important hub genes and six critical signaling pathways were identified to have important implications in chemotherapeutic response. Further, ten potential serum biomarkers were identified for predicting chemoresistance in EOC. Finally, we suggested some drugs for individualized treatment.

Conclusion

We have developed the predictive models and serum biomarkers for platinum/paclitaxel response and established the new approach to discover potential serum biomarkers from gene expression profiles. The potential drugs that target hub genes are also suggested.     

Discovery and Validation of New Potential Biomarkers for Early Detection of Colon Cancer

Background

Accurate detection of characteristic proteins secreted by colon cancer tumor cells in biological fluids could serve as a biomarker for the disease. The aim of the present study was to identify and validate new serum biomarkers and demonstrate their potential usefulness for early diagnosis of colon cancer.

Methods

The study was organized in three sequential phases: 1) biomarker discovery, 2) technical and biological validation, and 3) proof of concept to test the potential clinical use of selected biomarkers. A prioritized subset of the differentially-expressed genes between tissue types (50 colon mucosa from cancer-free individuals and 100 normal-tumor pairs from colon cancer patients) was validated and further tested in a series of serum samples from 80 colon cancer cases, 23 patients with adenoma and 77 cancer-free controls.

Results

In the discovery phase, 505 unique candidate biomarkers were identified, with highly significant results and high capacity to discriminate between the different tissue types. After a subsequent prioritization, all tested genes (N = 23) were successfully validated in tissue, and one of them, COL10A1, showed relevant differences in serum protein levels between controls, patients with adenoma (p = 0.0083) and colon cancer cases (p = 3.2e-6).

Conclusion

We present a sequential process for the identification and further validation of biomarkers for early detection of colon cancer that identifies COL10A1 protein levels in serum as a potential diagnostic candidate to detect both adenoma lesions and tumor.

Impact

The use of a cheap serum test for colon cancer screening should improve its participation rates and contribute to decrease the burden of this disease.     

High expression of miR-210 predicts poor survival in patients with breast cancer: A meta-analysis

Background

Many microRNAs (miRNAs) exhibit altered expression levels in cancers, and they may be considered as valuable prognostic biomarkers for cancers. Here we aimed to summarize the recent advances in miR-210 involvement in human breast cancer and analyze the predicting role of miR-210 for survival.

Methods

A meta-analysis was performed by searching PubMed, Cochrane Library, and Science Direct databases. Data were extracted from studies comparing survival in patients with breast cancer having higher expression of miR-210 with those having lower expression. Pooled hazard ratios (HRs) and 95% confidence intervals (CI) were calculated.

Results

A total of 511 cases of breast cancer were involved for this global meta-analysis. For post-operational survival, the HR of higher miR-210 expression in breast cancer tissue was 3.39 (95% CI: 2.04–5.63, P < 0.05), which could significantly predict poorer survival.

Conclusions

High expression of miR-210 might predict poor survival in patients with breast cancer.     

Albumin heterogeneity in low-abundance fluids. The case of urine and cerebro-spinal fluid

Background

Serum albumin is a micro-heterogeneous protein composed of at least 40 isoforms. Its heterogeneity is even more pronounced in biological fluids other than serum, the major being urine and cerebrospinal fluid. Modification ‘in situ’ and/or selectivity of biological barriers, such as in the kidney, determines the final composition of albumin and may help in definition of inflammatory states.

Scope of review

This review focuses on various aspects of albumin heterogeneity in low ‘abundance fluids’ and highlights the potential source of information in diseases.

Major conclusions

The electrical charge of the protein in urine and CSF is modified but with an opposite change and depending on clinical conditions.In normal urine, the bulk of albumin is more anionic than in serum for the presence of ten times more fatty acids that introduce equivalent anionic charges and modify hydrophobicity of the protein. At the same time, urinary albumin is more glycosylated compared to the serum homolog. Finally, albumin fragments can be detected in urine in patients with proteinuria.For albumin in CSF, we lack information relative to normal conditions since ethical problems do not allow normal CSF to be studied. In multiple sclerosis, the albumin charge in CSF is more cationic than in serum, this change possibly involving structural anomalies or small molecules bindings.

General significance

Massively fatty albumin could be toxic for tubular cells and be eliminated on this basis. Renal handling of glycosylated albumin can alter the normal equilibrium of filtration/reabsorption and trigger mechanisms leading to glomerulosclerosis and tubulo-interstitial fibrosis. This article is part of a Special Issue entitled Serum Albumin.     

Selective binding interactions of deramciclane to the genetic variants of human α1-acid glycoprotein

Background

α₁-Acid glycoprotein (AGP) plays a decisive role in the serum protein binding of several drugs.Genetic variants of AGP have different ligand binding properties. The binding of deramciclane (DER), a chiral anxiolytic agent, has been studied on A and F1/S genetic variants of AGP.

Methods

The effects of DER and reference drugs on the binding of specific fluorescent and circular dichroism (CD) probes of AGP were determined. Dicumarol (DIC) binding was measured by CD and equilibrium dialysis.

Results

DER effectively displaced probes bound to variant A, while it was less effective at displacing probes bound to variant F1/S. DER increased the binding and inverted the induced CD spectrum of DIC in the solution of variant F1/S. This phenomenon could not be brought about by the enantiomer of DER.

Conclusion

DER has high-affinity binding (K_a ≥ 2×10⁶ M^-1) to variant A, while its binding to the variant F1/S is about thirty times weaker. During simultaneous binding of DER and DIC to variant F1/S a ternary complex having about four times higher affinity is formed, in which the opposite chiral conformation of DIC is favored.

General significance

The binding interactions found prove that AGP can simultaneously accommodate different ligand molecules. Even weakly bound ligands can provoke unexpected allosteric protein binding interactions.