Effect of sodium polyacrylate on the hydrolysis of octacalcium phosphate

Octacalcium phosphate (OCP) hydrolysis into hydroxyapatite (HA) has been investigated in aqueous solutions at different concentrations of sodium polyacrylate (NaPA). In the absence of the polyelectrolyte, OCP undergoes a complete transformation into HA in 48 h. The hydrolysis is inhibited by the polymer, which is significantly adsorbed on the crystals, up to about 22 wt.%. A polymer concentration of 10(-2) mM is sufficient to cause a partial inhibition of OCP to HA transformation, which is completely hindered at higher concentrations. The small platelet-like crystals in the TEM images of partially converted OCP can display electron diffraction patterns characteristic either of OCP single crystals or of polycrystalline HA, whereas the much bigger plate-like crystals exhibit diffraction patterns characteristic of OCP single crystals. The polyelectrolyte adsorption on OCP crystals is accompanied by an increase of their mean length and by a significant reduction of the coherence length of the perfect crystalline domains along the c-axis direction. It is suggested that the carboxylate-rich polyelectrolyte is adsorbed on the hydrated layer of the OCP (100) face, thus inhibiting its in situ hydrolysis into HA.     

Proteome analysis of human serum proteins adsorbed onto different titanium surfaces used in dental implants

Titanium dental implants are commonly used due to their biocompatibility and biochemical properties; blasted acid-etched Ti is used more frequently than smooth Ti surfaces. In this study, physico-chemical characterisation revealed important differences in roughness, chemical composition and hydrophilicity, but no differences were found in cellular in vitro studies (proliferation and mineralization). However, the deposition of proteins onto the implant surface might affect in vivo osseointegration. To test that hypothesis, protein layers formed on discs of both surface type after incubation with human serum were analysed. Using mass spectrometry (LC/MS/MS), 218 proteins were identified, 30 of which were associated with bone metabolism. Interestingly, Apo E, antithrombin and protein C adsorbed mostly onto blasted and acid-etched Ti, whereas the proteins of the complement system (C3) were found predominantly on smooth Ti surfaces. These results suggest that physico-chemical characteristics could be responsible for the differences observed in the adsorbed protein layer.     

Sensitive detection of C-reactive protein in serum by immunoprecipitation–microchip capillary gel electrophoresis

Sepsis represents a significant cause of mortality in intensive care units. Early diagnosis of sepsis is essential to increase the survival rate of patients. Among others, C-reactive protein (CRP) is commonly used as a sepsis marker. In this work we introduce immune precipitation combined with microchip capillary gel electrophoresis (IP–MCGE) for the detection and quantification of CRP in serum samples. First high-abundance proteins (HSA, IgG) are removed from serum samples using affinity spin cartridges, and then the remaining proteins are labeled with a fluorescence dye and incubated with an anti-CRP antibody, and the antigen/antibody complex is precipitated with protein G-coated magnetic beads. After precipitation the complex is eluted from the beads and loaded onto the MCGE system. CRP could be reliably detected and quantified, with a detection limit of 25 ng/μl in serum samples and 126 pg/μl in matrix-free samples. The overall sensitivity (LOQ = 75 ng/μl, R² = 0.9668) of the method is lower than that of some specially developed methods (e.g., immune radiometric assay) but is comparable to those of clinically accepted ELISA methods. The straightforward sample preparation (not prone to mistakes), reduced sample and reagent volumes (including the antibodies), and high throughput (10 samples/3 h) are advantages and therefore IP–MCGE bears potential for point-of-care diagnosis.     

A bone substitute with high affinity for vitamin D‐binding protein―relationship with niche of osteoclasts

The biological activity of osteoblasts and osteoclasts is regulated not only by hormones but also by local growth factors, which are expressed in neighbouring cells or included in bone matrix. Previously, we developed hydroxyapatite (HA) composed of rod‐shaped particles using applied hydrothermal methods (HHA), and it revealed mild biodegradability and potent osteoclast homing activity. Here, we compared serum proteins adsorbed to HHA with those adsorbed to conventional HA composed of globular‐shaped particles (CHA). The two ceramics adsorbed serum albumin and γ‐globulin to similar extents, but affinity for γ‐globulin was much greater than that to serum albumin. The chemotactic activity for macrophages of serum proteins adsorbed to HHA was significantly higher than that of serum proteins adsorbed to CHA. Quantitative proteomic analysis of adsorbed serum proteins revealed preferential binding of vitamin D‐binding protein (DBP) and complements C3 and C4B with HHA. When implanted with the femur of 8‐week‐old rats, HHA contained significantly larger amount of DBP than CHA. The biological activity of DBP was analysed and it was found that the chemotactic activity for macrophages was weak. However, DBP‐macrophage activating factor, which is generated by the digestion of sugar chains of DBP, stimulated osteoclastogenesis. These results confirm that the microstructure of hydroxyapatite largely affects the affinity for serum proteins, and suggest that DBP preferentially adsorbed to HA composed of rod‐shaped particles influences its potent osteoclast homing activity and local bone metabolism.     

Determination of aconitine,hypaconitine and mesaconitine in urine using hollow fiber liquid-phase microextraction combined with high-performance liquid chromatography

Hollow fiber liquid-phase microextraction (HF-LPME) coupled with high-performance liquid chromatography was used to simultaneously determine three Aconitum alkaloids, including aconitine (AC), hypaconitine (HA) and mesaconitine (MA) in human urine sample. Analytes were extracted from 5 mL urine sample containing 1.0 mmol/L NaOH into 1-octanol membrane phase impregnated in the pores of hollow fiber wall, and then back extracted into acidified aqueous solution in the lumen of the hollow fiber. After extraction, 10 μL of the acceptor phase was analyzed directly by HPLC. In this method, some important extraction parameters, such as organic solvent, extraction time, stirring rate, pH of donor phase and acceptor phase, temperature, and the volume of acceptor phase were optimized. This method provided 98- to 288-fold enrichment factors within 60 min of extraction and good repeatability with RSDs of 0.99–7.22%. The calibration curves were linear over the ranges of 16.0–128.0 μg/L for AC, 11.0–88.0 μg/L for HA and 8.1–64.8 μg/L for MA in human urine sample, with correlation coefficients of 0.9949, 0.9969 and 0.9904, respectively. Limits of detection were from 0.7 to 1.5 μg/L, and recoveries from spiked urine sample varied from 84.4% to 106.2% for AC, 77.3% to 85.6% for HA and 90.1% to 100.8% for MA.     

Influence of bacteriocin in the control of Escherichia coli infection of broiler chickens in Nigeria

One hundred broiler chickens were divided into four groups. Group A was untreated and challenged with E. coli 02:KH6; group B was infected and treated with bacteriocin in their drinking water; group C was infected and treated by administration of Lactobacillus plantarum F1 culture in skimmed milk, and group D acted as a control. Their weight, feed intake, clinical signs, microscopic lesions, E. coli re-isolation and serum biochemistry were compared. Groups B and C showed fewer symptoms and gross lesions than those from group A, while body weight and feed intakes were similar to the control group. E. coli 02:KH6 was re-isolated in 60% of the livers from group A, compared to 8 and 12% of the livers from group B and C respectively. There was significant increase in the levels of total protein (3.78 ± 0.05 g/100 ml) and globulins (2.53 ± 0.03 g/100 ml) in group A compared to groups B and C. The cholesterol levels in group A (112.56 ± 0.15 mg/100 ml) were significantly reduced compared to those of groups B and C which had 127.25 ± 0.14 and 128.73 ± 0.13 mg/100 ml respectively. The study provides evidence of the potential therapeutic efficacy of bacteriocin in the control of an experimental E. coli infection in broiler chickens.     

Discrepancy between radioimmunoassay and high performance liquid chromatography tandem-mass spectrometry for the analysis of androstenedione

The discrepancy of results for the quantification of androstenedione in human serum between a radioimmunoassay (RIA) method and high performance liquid chromatography tandem-mass spectrometry (LC–MS/MS) was investigated. RIA overestimated concentrations compared to LC–MS/MS on 59 clinical samples (RIA = 1.79 × LC–MS/MS + 0.94). RIA kit and LC–MS/MS calibrants were also determined by both methods. The RIA performed with improved accuracy on the calibrants (RIA = 1.35 × LC–MS/MS − 0.28). Lipid, protein, electrolyte content, and pH of the two sets of calibrants were further investigated. The RIA calibrants contained little lipid material, while the LC–MS/MS calibrant material contained the same levels expected in normal serum/plasma. The pH and sex hormone binding globulin (SHBG) values were different between the RIA calibrants and the LC–MS/MS calibrant material (SHBG, 31 ± 2 and 38 ± 2 nmol/l; pH, 8.27 ± 0.18 and 8.66 ± 0.03, respectively). No correlation was observed between androstenedione RIA and LC–MS/MS discrepancy and lipid or protein. LC–MS/MS sample preparation was tested for the removal of protein-bound material and recovery determined (99–108%). The corresponding RIA results overestimated androstenedione by 52–174% compared to LC–MS/MS. The results here demonstrate that LC–MS/MS is the more accurate method.     

Quantitative targeted absolute proteomics of human blood-brain barrier transporters and receptors

We have obtained, for the first time, a quantitative protein expression profile of membrane transporters and receptors in human brain microvessels, that is, the blood-brain barrier (BBB). Brain microvessels were isolated from brain cortexes of seven males (16-77 years old) and protein expression of 114 membrane proteins was determined by means of a liquid chromatography-tandem mass spectrometric quantification method using recently established in-silico peptide selection criteria. Among drug transporters, breast cancer resistance protein showed the most abundant protein expression (8.14 fmol/μg protein), and its expression level was 1.85-fold greater in humans than in mice. By contrast, the expression level of P-glycoprotein in humans (6.06 fmol/μg protein) was 2.33-fold smaller than that of mdr1a in mice. The organic anion transporters reported in rodent BBB, that is, multidrug resistance-associated protein, organic anion transporter and organic anion-transporting polypeptide family members, were under limit of quantification in humans, except multidrug resistance-associated protein 4 (0.195 fmol/μg protein). Among detected transporters and receptors for endogenous substances, the glucose transporter 1 level was similar to that of mouse, while the L-type amino acid transporter 1 level was fivefold smaller than that of mouse. These findings should be useful for understanding human BBB function and its differences from that in mouse.     

Cementum protein 1‐derived peptide (CEMP 1‐p1) modulates hydroxyapatite crystal formation in vitro

A cementum protein 1‐derived peptide (CEMP1‐p1) consisting of 20 amino acids from the CEMP1's N‐terminus region: MGTSSTDSQQAGHRRCSTSN, and its role on the mineralization process in a cell‐free system, was characterized. CEMP1‐p1's physicochemical properties, crystal formation, and hydroxyapatite (HA) nucleation assays were performed. Crystals induced by CEMP1‐p1 were analyzed by scanning electron microscopy, Fourier‐transform infrared spectroscopy‐attenuated total reflectance (FTIR‐ATR), X‐ray diffraction (XRD), high resolution transmission electron microscopy (HRTEM), and atomic force microscopy. The results indicate that CEMP1‐p1 lacks secondary structure, forms nanospheres that organize into three‐dimensional structures, possesses affinity to HA, and induces its nucleation. CEMP1‐p1 promotes the formation of spherical structures composed by densely packed prism‐like crystals, which revealed a Ca/P ratio of 1.56, corresponding to HA. FTIR‐ATR showed predominant spectrum peaks that correspond and are characteristic of HA and octacalcium phosphate (OCP). Analysis by XRD indicates that the crystals show planes with a preferential crystalline orientation for HA and for OCP. HRTEM showed interplanar distances that correspond to crystalline planes of HA and OCP. Crystals are composed by superimposed lamellae, which exhibit epitaxial growth, and each layer of the crystals is structured by nanocrystals. This study reveals that CEMP1‐p1 regulates HA crystal formation, somehow mimicking the in vivo process of mineralized tissues bioformation.     

Production of a soluble single-chain variable fragment antibody against okadaic acid and exploration of its specific binding

Okadaic acid is a lipophilic marine algal toxin commonly responsible for diarrhetic shellfish poisoning (DSP). Outbreaks of DSP have been increasing and are of worldwide public health concern; therefore, there is a growing demand for more rapid, reliable, and economical analytical methods for the detection of this toxin. In this study, anti-okadaic acid single-chain variable fragment (scFv) genes were prepared by cloning heavy and light chain genes from hybridoma cells, followed by fusion of the chains via a linker peptide. An scFv–pLIP6/GN recombinant plasmid was constructed and transformed into Escherichia coli for expression, and the target scFv was identified with IC–CLEIA (chemiluminescent enzyme immunoassay). The IC₁₅ was 0.012 ± 0.02 μg/L, and the IC₅₀ was 0.25 ± 0.03 μg/L. The three-dimensional structure of the scFv was simulated with computer modeling, and okadaic acid was docked to the scFv model to obtain a putative structure of the binding complex. Two predicted critical amino acids, Ser32 and Thr187, were then mutated to verify this theoretical model. Both mutants exhibited significant loss of binding activity. These results help us to understand this specific scFv–antigen binding mechanism and provide guidance for affinity maturation of the antibody in vitro. The high-affinity scFv developed here also has potential for okadaic acid toxin detection.     

Simultaneous quantitation of perfluoroalkyl acids in human serum and breast milk using on-line sample preparation by HPLC column switching coupled to ESI-MS/MS

A high throughput analytical method using a column switching high-performance liquid chromatography combined with isotope dilution tandem mass spectrometry (column switching-HPLC–MS/MS) was developed to simultaneously quantitate the concentrations of 7 perfluoroalkyl acids (PFAAs) in serum and 3 PFAAs in breast milk samples. The sample preparation includes addition of the isotope-labelled internal standard solution to breast milk and serum, enzymatic hydrolysis and filtration of milk samples, precipitation of proteins and analysis by column switching-HPLC–MS/MS. The limits of quantitation ranged from 0.1 to 0.4 μg/l for serum and 0.02 to 0.15 μg/l for breast milk samples. The method accuracies ranged between 73.2% and 100.2% for the different analytes at two concentrations in PFAAs spiked samples. The validity of the method was confirmed by analysing 20 serum and 20 breast milk samples.     

Synthesis and evaluation of thiophenyl derivatives as inhibitors of alkaline phosphatase

Pathological calcifications induced by deposition of basic phosphate crystals or hydroxyapatite (HA) on soft tissues are a large family of diseases comprising of ankylosing spondylitis (AS), end-stage osteoarthritis (OA) and vascular calcification. High activity of tissue non-specific alkaline phosphatase (TNAP) is a hallmark of pathological calcifications induced by HA deposition. The use of TNAP inhibitor is a possible therapeutic option to address calcific diseases produced by HA deposition on soft tissues. We report the synthesis of a series of thiopheno-imidazo[2,1-b]thiazole derivatives which were evaluated as potential inhibitors of TNAP displaying a large range of IC₅₀ at pH 10.4 (from 42 ± 13 μM to more than 800 μM).     

Analysis by LC/ESI-MS of iophenoxic acid derivatives and evaluation as markers of oral baits to deliver pharmaceuticals to wildlife

Iophenoxic acid and its derivatives (methyl, ethyl, and propyl) are organic chemicals used as markers in baiting campaigns to deliver vaccines, pharmaceuticals, contraceptives or poisons to wildlife. In this study we develop a method of detection of IPA derivatives by LC/ESI-MS (using butyl-IPA as internal standard) obtaining a limit of detection and quantification in wild boar (Sus scrofa) serum of 0.037 μg/ml and 0.123 μg/ml, respectively. The average recovery of IPA derivatives was 88% at levels >0.2 μg/ml, with coefficients of variation <15%. Wild boars in captivity were orally treated with 5 mg/kg b.w. (three adults) or 15 mg/kg b.w (two piglets and three adults) of methyl-, ethyl- and propyl-IPA and the serum levels of these were monitored during 18 months after dosing. Ethyl- and propyl-IPA were detected up to 18 months after a single oral dose in wild boar, especially at 15 mg/kg. Methyl-IPA was detected until 9 months after dosing. Half-lives of methyl-, ethyl- and propyl-IPA were (mean ± SD) 41 ± 5, 183 ± 85 and 165 ± 45 days, respectively. One control piglet not exposed to IPA, but housed in the same facility than treated animals showed detectable IPA levels in serum. Piglets born from mothers exposed to marked baits also showed detectable IPA levels in serum. The high persistence of Et- and Pr-IPA must be considered in the field trials, because the presence of the product at low levels in one animal may not reflect a real ingestion of the marked bait.     

Aluminium and human breast diseases

The human breast is exposed to aluminium from many sources including diet and personal care products, but dermal application of aluminium-based antiperspirant salts provides a local long-term source of exposure. Recent measurements have shown that aluminium is present in both tissue and fat of the human breast but at levels which vary both between breasts and between tissue samples from the same breast. We have recently found increased levels of aluminium in noninvasively collected nipple aspirate fluids taken from breast cancer patients (mean 268 ± 28 μg/l) compared with control healthy subjects (mean 131 ± 10 μg/l) providing evidence of raised aluminium levels in the breast microenvironment when cancer is present. The measurement of higher levels of aluminium in type I human breast cyst fluids (median 150 μg/l) compared with human serum (median 6 μg/l) or human milk (median 25 μg/l) warrants further investigation into any possible role of aluminium in development of this benign breast disease. Emerging evidence for aluminium in several breast structures now requires biomarkers of aluminium action in order to ascertain whether the presence of aluminium has any biological impact. To this end, we report raised levels of proteins that modulate iron homeostasis (ferritin, transferrin) in parallel with raised aluminium in nipple aspirate fluids in vivo, and we report overexpression of mRNA for several S100 calcium binding proteins following long-term exposure of MCF-7 human breast cancer cells in vitro to aluminium chlorhydrate.     

Is there any association of apolipoprotein E gene polymorphism with obesity status and lipid profiles? Tehran Lipid and Glucose Study (TLGS)

Aims

Considering the key role played by the apolipoprotein E (Apo E) gene in the regulation of lipid metabolism and obesity, the current study has evaluate the association between abdominal obesity and Apo E gene polymorphism in a population of Tehran.

Materials and methods

A cross-sectional study was performed on 345 men and 498 women, aged 19–86 years, selected from among participants of the Tehran Lipid and Glucose Study. The RFLP-PCR technique was employed to investigate polymorphism in the gene fragments. Based on the national survey of risk factors for non-communicable diseases of Iran, waist circumference (WC) cut off was set at 89 cm for men and 91 cm for women. The risk effect of obesity related variables and lipid profiles in two groups of WC were examined by logistic regression. For body mass index (BMI), waist to hip ratio (WHR), high-density lipoprotein-cholesterol (HDL-C), triglyceride (TG), fasting blood sugar (FBS), total cholesterol (TC), low-density lipoprotein-cholesterol (LDL-C), and blood pressure (BP), the standard risk cut-offs were applied.

Results

Frequencies of E2, E3, and E4 alleles were 9.7, 73, and 14.6%, respectively. The presence of the E3 allele was significantly associated with higher TG level in subjects with high WC, while, the presence of E4 allele decreased the plasma HDL-C (E2:52.1 ± 13.1 vs., E3:48.9 ± 11.2 vs., E4:44.6 ± 10.6 mg/dl, p < 0.05), HDL-C2 (E2:20.4 ± 9.2 vs., E3:19.1 ± 8.8 vs., E4:16.3 ± 7.9 mg/dl, p < 0.05), and HDL-C3 (E2:32.1 ± 7.4 vs., E3:30.3 ± 6.2 vs., E4:28.3 ± 6.1 mg/dl, p < 0.05) in normal WC subjects. The presence of the E3 carrier increased the risk of having higher plasma TG, compared with the E2 carrier (95% CI OR = 1.91, 1.02–3.57; p = 0.04).

Conclusion

According to the results of this study, the E3 carrier, caused an approximately 90% increase in the levels of TG in the group with abdominal obesity.     

Fecal estradiol-17β and testosterone in prepubertal domestic cats

The aim of this article was to describe the time course of prepubertal sexual steroids in domestic cats. Fourteen newborn kittens were followed up until puberty (physical, behavioral, and hormonal changes). Fecal testosterone [T; males] and E estradiol 17-β [E2; females] concentrations were analyzed by repeated measures ANOVA and two consecutive time windows (TWs) were used to compare changes in both male (postnatal weeks 1–4 vs. 5–14) and females (postnatal weeks 1–5 vs. 6–13). Puberty was achieved 14.3 ± 0.3 and 13.3 ± 0.4 weeks after birth in male and female cats, respectively. In both genders, during TW-1 fecal steroids concentrations were similar (males) or even higher (females) to that previously described for mature cats. Fecal T (P < 0.01) and E2 (P < 0.01) varied throughout the weeks. Differences were found when hormonal concentrations of TW-1 were compared with those of TW-2 both for male (61.4 ± 7.9 vs. 16.9 ± 2.2 ng/g; P < 0.01) and female (78.2 ± 12.5 vs. 11.2 ± 4.0 ng/g; P < 0.01) cats. It is concluded that in domestic cats there is a sexual steroid surge during the first 4 and 5 postnatal weeks in male and female animals, respectively.     

Lipopolysaccharide aggregates in native agarose gels detected by reversible negative staining with imidazole and zinc salts

We investigated the use of imidazole and zinc salts for the detection of lipopolysaccharide (LPS) aggregates separated by native agarose gel electrophoresis (NAGE). As a result, a new staining procedure was established by which as little as 1.5 μg of Escherichia coli O55:B5 LPS aggregates was detected by means of inducing a clear, transparent pattern, contrasted against an opaque background. E. coli O55:B5 LPS preparations treated with nucleases and proteinase K proved that the reverse-stained LPS pattern is not related to any potential artifacts caused by unrelated biomolecules (e.g., nucleic acids, proteins). After this, we showed that the procedure is applicable to two-dimensional LPS separation using NAGE/SDS-PAGE, while at the same time confirming that real polydisperse LPS aggregates are represented by the stained profile. Also, we demonstrated the general applicability of this stain to the detection of different NAGE-separated LPS aggregates (e.g., from E. coli 026:B6, E. coli 0111:B4, Salmonella minnesota Re595). Finally, using lysozyme as a model protein, we found that imidazole–zinc may be combined with Coomassie brilliant blue R-250 into a double-staining process to enable the use of NAGE for investigating the interaction of cationic proteins and LPS aggregates and protein or LPS concentration effects on protein–LPS binding.     

Influences of a diet supplemented with linseed oil and antioxidants on quality of equine semen after cooling and cryopreservation during winter

Seasonal changes in the reproductive physiology of stallions contribute to a decrease in the quality of frozen-thawed semen during late winter. Changes in the lipid composition of the sperm plasma membrane may contribute to this phenomenon. In the present study, we have, therefore, investigated the effects of adding linseed oil (LO) in combination with antioxidants to the diet of breeding stallions on the motility and membrane integrity of cooled–stored and cryopreserved semen. Starting in November, the diet of LO stallions (n = 6) but not control (C) stallions (n = 5) was supplemented with LO (100 mL once daily) plus an antioxidant (Myostem Protect; Audevard, Clichy, France) for a total of 84 days. Before (November) and at the end of this period (February), ejaculates were processed for cryopreservation (n = 3 ejaculates per stallion) and cooled shipping at 5 °C. Frozen-thawed and cooled–shipped semen was sent to the laboratory for computer-assisted semen analysis of total motility, progressive motility, and velocity parameters (average path velocity [VAP], curved line velocity [VCL], and straight-line velocity [VSL]) and evaluation of membrane integrity. The quality of frozen-thawed semen decreased (P < 0.05) from November (e.g., total motility LO 69 ± 3% and C 67 ± 3%) to February (total motility: LO 55 ± 4% and C 59 ± 3%) independent of treatment (P > 0.05). A decrease in the velocity parameters VAP, VCL, and VSL was more pronounced in LO stallions than in C stallions (e.g., VSL: November LO 67 ± 1 μm/s, C 64 ± 2 μm/s; February LO 59 ± 2 μm/s, C 63 ± 2 μm/s; interaction month by treatment, P < 0.05). In cooled–stored semen, total motility, progressive motility, and membrane integrity were lower in February than in November (P < 0.001 for all parameters). Supplementation of the diet with LO and antioxidants attenuated this decrease (e.g., Day 1 of cooled storage = 24 hours after semen collection: total motility in November LO 88 ± 1% and C 87 ± 3%; in February LO 83 ± 2% and C 73 ± 11%; interaction month by treatment: P < 0.05). Velocity parameters VAP, VCL, and VSL were significantly lower in February than in November (P < 0.001), but this decrease was not affected by treatment. In summary, dietary supplementation of stallions with LO plus antioxidants attenuated a decline in motility and membrane integrity of cooled–stored stallion semen during winter. This may improve the fertility of cooled–shipped semen. In contrast, the treatment did not counteract the decrease in quality of frozen-thawed semen that occurs in late winter.     

11β-Hydroxysteroid dehydrogenase activity in acromegalic patients with normal or impaired carbohydrate metabolism

The 11β-hydroxysteroid dehydrogenase isoenzymes (11β-HSD) catalyse the interconversion of cortisol (F) and cortisone (E). Earlier studies demonstrated that growth hormone (GH) and insulin resistance may exert opposite effects on the conversion of E to F by 11β-HSD type 1. Therefore, in the present study we determined F and E concentrations in 562 plasma samples obtained from acromegalic patients during an active phase (76 patients) and after cure of the disease (68 patients). In addition, we examined whether type 2 diabetes mellitus or impaired glucose tolerance, which are frequently associated with active acromegaly could influence plasma F and E levels in these patients. We found that plasma F concentrations were similar in patients with active acromegaly and in those who were cured with pituitary surgery, irradiation and/or medical therapy (mean ± S.E., 12.4 ± 0.3 and 12.7 ± 0.4 μg/dl, respectively). However, plasma E levels were significantly higher in patients with active compared to those with cured acromegaly (2.8 ± 0.1 and 2.2 ± 0.1 μg/dl, respectively; p < 0.001), resulting in a lower F/E ratio in patients with active disease (4.6 ± 0.1 vs. 5.9 ± 0.2 in the cured group of patients; p < 0.001). When the effect of altered carbohydrate homeostasis on plasma F and E was analysed, the results indicated significantly lower plasma E levels and higher F/E ratios in active acromegalic patients with type 2 diabetes mellitus or impaired glucose tolerance compared to those with normal carbohydrate metabolism (E, 2.5 ± 0.1 and 3.0 ± 0.1 μg/dl, respectively; F/E, 5.1 ± 0.2 and 4.4 ± 0.1; p < 0.001), whereas plasma F concentrations were similar in these two groups (12.1 ± 0.4 and 12.6 ± 0.3 μg/dl, respectively). These findings indicate that disease activity exerts a significant impact on 11β-HSD in acromegalic patients, which is further modified with altered carbohydrate homeostasis, frequently present in patients with active disease.     

Absolute quantification of DcR3 and GDF15 from human serum by LC‐ESI MS

Biomarkers are widely used in clinical diagnosis, prognosis and therapy monitoring. Here, we developed a protocol for the efficient and selective enrichment of small and low concentrated biomarkers from human serum, involving a 95% effective depletion of high‐abundant serum proteins by partial denaturation and enrichment of low‐abundant biomarkers by size exclusion chromatography. The recovery of low‐abundance biomarkers was above 97%. Using this protocol, we quantified the tumour markers DcR3 and growth/differentiation factor (GDF)15 from 100 μl human serum by isotope dilution mass spectrometry, using ¹⁵N metabolically labelled and concatamerized fingerprint peptides for the both proteins. Analysis of three different fingerprint peptides for each protein by liquid chromatography electrospray ionization mass spectrometry resulted in comparable concentrations in three healthy human serum samples (DcR3: 27.23 ± 2.49 fmol/ml; GDF15: 98.11 ± 0.49 fmol/ml). In contrast, serum levels were significantly elevated in tumour patients for DcR3 (116.94 ± 57.37 fmol/ml) and GDF15 (164.44 ± 79.31 fmol/ml). Obtained data were in good agreement with ELISA and qPCR measurements, as well as with literature data. In summary, our protocol allows the reliable quantification of biomarkers, shows a higher resolution at low biomarker concentrations than antibody‐based strategies, and offers the possibility of multiplexing. Our proof‐of‐principle studies in patient sera encourage the future analysis of the prognostic value of DcR3 and GDF15 for colon cancer patients in larger patient cohorts.