Microwave irradiation for a fast gas chromatography-mass spectrometric analysis of polysaccharide-based plasma volume expanders in human urine

In this contribution we tested the possibility to use microwave irradiation for the screening and confirmation pre-treatment steps of hydroxyethylstarch, with the aim to speed up gas chromatography-mass spectrometric procedures. Acid hydrolysis and derivatization processes were conducted in a temperature-controlled single beam microwave oven for organic synthesis. The kinetics of hydroxyethylstarch chemical hydrolysis and derivatization were investigated at different microwave power, incubation temperature and incubation time. The best hydrolysis conditions were found at a microwave power value of 1200 W (T 100°C) with an incubation time of 2 min; whereas the best derivatization conditions were found at a microwave power value of 1020 W (T 100°C) with an incubation time of 5 min. The effectiveness of this approach was evaluated by gas chromatography-mass spectrometry analyzing more than 20 different pools of blank urine samples spiked with hydroxyethylstarch at a concentration of 1 mg/mL. The results showed that the effect of microwave irradiation on the chemical hydrolysis process was very remarkable: the total sample preparation time can be shortened by 58 min compared to the reference method (2 min instead of 60 min). In addition to this, the time necessary for the derivatization process can also be drastically shortened with respect to the reference procedure (5 min instead of 30 min). The repeatability of the hydrolysis and derivatization recoveries, the limit of detection and the matrix interferences were comparable to the reference method accredited under the ISO 17025 guidelines and presently followed by the accredited sports anti-doping laboratory of Rome.     

Theoretical investigations on the functionalization of carbon nanotubes

In this paper, our recent work concerning theoretical studies on the functionalization of carbon nanotubes (CNTs) is reviewed. In particular, two different aspects of the functionalization process are taken into account. On the one hand, the chemical functionalization of the sidewall is exploited as a way to develop nanostructured gas sensing devices. On the other hand, we investigated the possibility of functionalizing the sidewall with transition metal complexes, thus extending the concepts of organometallic chemistry to CNTs. Calculations were performed by applying statical and dynamical (Car-Parrinello) density functional theory methods, as well as hybrid (quantum mechanics/molecular mechanics) schemes. The structural and electronic peculiarities of the CNT model under study, due, for example to the presence of defects, were found to play a crucial role in the modelization of the functionalization process. In most cases, the use of realistic models was essential to achieve a full agreement with experiments.     

Aryldiazenido derivatives: A new entry to the functionalization of Keggin polyoxometalates

A series of aryldiazenido polyoxomolybdates of the type (nBu₄N)₂[Mo₅O₁₃(OMe)₄(NNAr){Na(MeOH)}] (Ar = C₆F₅, 1; Ar = O₂N-o-C₆H₄, 2; Ar = O₂N-m-C₆H₄, 3; Ar = O₂N-p-C₆H₄, 4a; Ar = (O₂N)₂-o,p-C₆H₃, 5) have been obtained by controlled degradation of the parent compounds (nBu₄N)₃[Mo₆O₁₈(NNAr)] with NaOH in methanol. They have been characterized by elemental analysis and UV-Vis and IR spectroscopy. In addition, 4a has been characterized by ⁹⁵Mo NMR spectroscopy and the crystal structure of (nBu₄N)₂[Mo₅O₁₃(OMe)₄(NNC₆H₄-p-NO₂){Na(H₂O))]·H₂O (4b) has been determined by X-ray diffraction. The molecular structure of the anion of 4b features a lacunary Lindqvist-type anion [Mo₅O₁₃(OMe)₄(NNC₆H₄-p-NO₂)]³⁻ interacting with a sodium cation through the four terminal axial oxygen atoms. The 1:1 sodium complexes react with BaCl₂ and BiCl₃ to yield 2:1 complexes which have been isolated as (nBu₄N)₄[Ba{Mo₅O₁₃(OMe)₄(NNAr)}₂] (Ar = C₆F₅, 6; Ar = O₂N-p-C₆H₄, 7) and (nBu₄N)₃[Bi{Mo₅O₁₃(OMe)₄(NNAr)}₂] (Ar = C₆F₅, 8; Ar = O₂N-p-C₆H₄, 9). X-ray crystallography analysis of 9·Me₂CO has shown that the tetradentate [Mo₅O₁₃(OMe)₄(N₂C₆H₄-p-NO₂)]³⁻ anions provide a square-antiprismatic environment for Bi. In contrast, IR spectroscopy provides evidence for a square-prismatic environment of Ba in 6 and 7. In acetonitrile-methanol mixed solvent, [Mo₅O₁₃(OMe)₄(NNAr)]³⁻ and [PW₁₁O₃₉]⁷⁻, generated in situ by alkaline degradation of their respective parents, [Mo₆O₁₈(NNAr)]³⁻ and [PW₁₂O₄₀]³⁻, react together to give the Keggin-type diazenido compounds (nBu₄N)₄[PW₁₁O₃₉(MoNNAr)] (Ar = O₂N-o-C₆H₄, 10; Ar = O₂N-m-C₆H₄, 11; Ar = O₂N-p-C₆H₄, 12), which have been characterized by ³¹P and ¹⁸³W NMR spectroscopy.     

Laccase-mediated functionalization of chitosan by caffeic and gallic acids for modulating antioxidant and antimicrobial properties

A new strategy for the functionalization of chitosan with caffeic acid (CA) or gallic acid (GA) using laccase from Trametes versicolor is presented for the first time, yielding a product with modulated antioxidant and antimicrobial properties. UV-vis spectroscopy coupled to HPLC-SEC analysis and cyclic voltammetry kinetic studies showed that laccase catalyzes the oxidation of phenolic acids to electrophilic o-quinones, which undergo new oligomer/polymer-forming structures originated by C-C coupling between the benzene rings and C-O-C coupling involving phenolic side-chains. Furthermore, pH tunable reactions/interactions of the laccases oxidized o-quinones with nucleophilic amino groups of chitosan were determined with FTIR and ¹H NMR spectroscopy's. The highest antioxidant activity was found to be for chitosan modified with phenolic acids at pH 4.5, exhibit also an increased activity against Escherichia coli and Listeria monocytogenes compared to untreated chitosan.     

Plasma volume, osmolarity, total protein and electrolytes during treadmill running and cycle ergometer exercise

While haemoconcentration due to loss of plasma volume is well established during cycling, the existence of similar changes during running remains contentious. This study compared the changes in plasma volume and associated blood indices during 60 min of running and cycling at the same relative intensity (approximately 65% VO2max), with all changes referenced to blood indices obtained after 30 min seated at rest on a cycle ergometer. Plasma osmolarity increased similarly with both forms of exercise but was less than predicted for water loss alone, such that there was a net loss of sodium during exercise and of potassium postexercise, with essentially no loss of protein. Plasma volume decreased similarly (approximately 6.5%) in both exercise trials, but while that with cycling was initiated by exercise itself and was essentially maximal within 5 min, the reduction in plasma volume in the running trial was induced by adopting the upright posture and was complete before exercise began. These data would indicate that different mechanisms are responsible for the changes in plasma volume induced by running and cycling, while the similarity of change would suggest that there is a lower limit to any reduction in plasma volume, regardless of mechanism. Furthermore, the observation that the changes in plasma volume were complete before or early in exercise, would imply that oral water ingestion during prolonged exercise, which is essential for thermoregulation, may be more concerned with homeostasis of extravascular water rather than plasma volume.     

PEGylation of rhIL-1RA increased its solution stability at room temperature

Recombinant human interleukin-1 receptor antagonist (rhIL-1RA) is an important cytokine in the treatment of inflammatory diseases. However, it is instable in aqueous solution and prone to degrade without the addition of any excipient. Following the 30- or 60-day storage in 50 mM sodium acetate (pH 5.0) at room temperature, rhIL-1RA markedly degraded into three species (denoted as P1, P2 and P3 in this study), the bioactivities of which to a different extent was lost (from 9.72 × 10⁴ UI/mg to 3.07 × 10³ UI/mg for P1, 5.49 × 10³ UI/mg for P2, 1.09 × 10⁴ UI/mg for P3, respectively). To solve this problem, we prepared the mono-PEGylated rhIL-1RA with propionaldehyde mPEG (ALD-PEG, M_w 5000 Da). The conjugate showed more favorable stability than original protein, and remained homogeneous under the similar storage conditions. In addition, the activity of the conjugate was well retained (from 5.80 × 10⁴ UI/mg to 4.92 × 10⁴ UI/mg), compared to that of original protein. The results based on the combination analysis of CD, ion exchange chromatography and RP-HPLC, revealed that the stability improvement of rhIL-1RA majorly benefited from the PEG strands protection against the protein conformational changes occurred during the storage.     

Blood volume measurement in baboons: simultaneous estimation of red cell volume and plasma volume

A method is described for frequent sequential blood volume estimation in baboons using 32P for red cell volume measurements and 125I-albumin for simultaneous plasma volume measurements. Values for red cell, plasma, and total blood volumes are reported. Close correlations of the volumes to bodyweight were demonstrated. Circulatory half-lives of the isotopes, determined from disappearance curves, confirmed their suitability for serial measurements in these baboons.     

Athletes'' pseudoanaemia

To characterize the so-called pseudoanaemia of endurance-trained athletes, the plasma volume (PV), red cell volume (RCV) and total blood volume (TBV) of 12 male and 12 female athletes and 5 male and 5 female nonexercising controls were measured using 125I-labelled human serum albumin and 51Cr-labelled erythrocytes. The mean PV of the male athletes (52.8 ml.kg-1) was 37.5% higher than that of the controls (38.4 ml.kg-1), while the 18.1% increase measured in the female runners (51.5 ml.kg-1) over the controls (43.6 ml.kg-1) was a novel observation. Although the RCV was significantly greater (34.7%) in male athletes (32.6 ml.kg-1 vs 24.2 ml.kg-1 in the controls), a similar elevation (3.6%) was not found in the female athletes (25.9 ml.kg-1) compared to the sedentary women (22.8 ml.kg-1). This could have been due to iron-limited erythropoiesis because the RCV of the female athletes defined as clinically anaemic was markedly lower that of the nonanaemic women (P less than 0.05). The elevated plasma protein mass and concentration measured in the athletes partly accounted for their expanded PV. It was concluded that the decreased blood haemoglobin levels reported in the endurance athletes was largely a dilutional effect.     

PEGylated derivatives of cystamine as enhanced treatments for nephropathic cystinosis

The genetic disease, nephropathic cystinosis is characterized by lysosomal accumulation of the amino acid cystine. Crystallization of cystine in affected organs, if untreated, results in mortality of the affected individuals by their middle to late teens. The only approved treatment for cystinosis is administration of cysteamine. However, cysteamine is associated with an offending odor and taste and this, coupled to a rapid first pass metabolism and a 6 h dosing regimen, suggest a clear need to improve the therapy. A number of PEGylated derivatives of cystamine, the disulfide counterpart of cysteamine, have been synthesised and evaluated in cultured cystinotic fibroblasts for toxicity and efficacy. All of the tested compounds were non-cytotoxic and displayed a remarkable depletion of intralysosomal cystine.     

Small-protein Enrichment Assay Enables the Rapid,Unbiased Analysis of Over 100 Low Abundance Factors from Human Plasma

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Highlights

• •Development of a method for low-abundance small protein analysis in human plasma.
• •Deep proteome profiling demonstrated unbiased identification of diverse factors.
• •Method enabled identification of a novel human plasma protein C5ORF46.
• •Analysis of intermittent fasting response showed changes in iron regulation.

     

In situ PEGylation of recombinant hirudin on an anion exchange chromatography column

In this study, an integrated process was developed for successive solid-phase PEGylation of recombinant hirudin variant-2 (HV2) and separation of PEGylated HV2 species on an anion exchange chromatography column (so-called in situ PEGylation). The effects of different PEG sizes, ion exchange resins and reaction conditions on in situ PEGylation were investigated. The results showed that in situ PEGylation efficiently integrates the reaction, separation and purification into a single-unit operation using the same column. In situ PEGylation could improve the selectivity of PEGylation reactions by significantly reducing the formation of multi-PEG-HV2. The pore sizes and internal surface structures of different resins had a significant impact on the yield of mono-PEG-HV2. In contrast to liquid-phase PEGylation, the yield of mono-PEG-HV2 decreased as PEG size increased during the in situ PEGylation process, indicating that in situ PEGylation is a pore diffusion-controlled process. The in vitro and in vivo anticoagulant activities of mono-PEG-HV2 derived from in situ PEGylation were higher than those from liquid-phase PEGylation, indicating that in situ PEGylation could enhance the bioactivity retention of mono-PEG-HV2. The results of this study demonstrated that in situ PEGylation can be used as an effective approach for the development of PEGylated protein drugs.     

Conjugation of Multiple Copies of Polyethylene Glycol to Hemoglobin Facilitated Through Thiolation: Influence on Hemoglobin Structure and Function

PEGylation induced changes in molecular volume and solution properties of HbA have been implicated as potential modulators of its vasoconstrictive activity. However, our recent studies with PEGylated Hbs carrying two PEG chains/Hb, have demonstrated that the modulation of the vasoconstrictive activity of Hb is not a direct correlate of the molecular volume and solution properties of the PEGylated Hb and implicated a role for the surface charge and/or the pattern of surface decoration of Hb with PEG. HbA has now been modified by thiolation mediated maleimide chemistry based PEGylation that does not alter its surface charge and conjugates multiple copies of PEG5K chains. This protocol has been optimized to generate a PEGylated Hb, (SP-PEG5K)₆-Hb, that carries ~six PEG5K chains/Hb – HexaPEGylated Hb. PEGylation increased the O₂ affinity of Hb and desensitized the molecule for the influence of ionic strength, pH, and allosteric effectors, presumably a consequence of the hydrated PEG-shell generated around the protein. The total PEG mass in (SP-PEG5K)₆-Hb, its molecular volume, O₂ affinity and solution properties are similar to that of another PEGylated Hb, (SP-PEG20K)₂-Hb, that carries two PEG20K chains/Hb. However, (SP-PEG5K)₆-Hb exhibited significantly reduced vasoconstriction mediated response than (SP-PEG20K)₂-Hb. These results demonstrate that the enhanced molecular size and solution properties achieved through the conjugation of multiple copies of small PEG chains to Hb is more effective in decreasing its vasoconstrictive activity than that achieved through the conjugation of a comparable PEG mass using a small number of large PEG chains.     

Effect of blood volume on plasma volume shift during exercise

To assess the relationship between blood volume (BV) and the reduction in plasma volume (PV) during exercise in individual variations, we measured BV and changes in PV in thirteen male volunteers during treadmill exercise until exhaustion. The lactate threshold (LT), as a predictor of aerobic exercise capacity, was calculated from the exercise intensity at the point of plasma lactate concentration buildup to 4 mmol. The relationship of peak VO₂ with BV indicated a significant positive correlation. The strong positive relation between the shifts in PV and total PV, and resulted in a maintenance of the circulating BV.     

蛋白药物的聚乙二醇定点修饰策略与最佳位点

聚乙二醇修饰是一种改善蛋白质药物临床药效行之有效的方法。聚乙二醇修饰具有延长蛋白质药物在体内的半衰期、降低免疫原性和延缓蛋白酶降解、提高稳定性和溶解性等优点。而聚乙二醇的定点修饰由于能够获得均一性和高活性保留率的产物,并能提高产率,已经引起了广泛关注。综述了近年来聚乙二醇定点修饰蛋白质药物方面的研究进展,着重介绍了聚乙二醇定点修饰的策略及最佳修饰位点,并对聚乙二醇定点修饰技术的发展趋势进行了展望。     

A zeolite as a tool for successful refolding of PEGylated proteins and their reassembly with tertiary structures

In the present study, we demonstrated zeolites' potential contribution to establish a method for preparing successfully refolded and reassembled PEGylated protein nanoparticles without the use of protein denaturants through the proteins' reassembly process. At first, the PEGylated nanoparticles are disassembled into identical PEGylated protein subunits by means of protein denaturants, and then the denatured subunits are adsorbed to zeolites. After the complete removal of denaturants, high-molecular-weight poly(ethylene glycol) (PEG) molecules are added to a solution where the zeolites suspend. Consequently, the PEGylated proteins are gradually reassembled into nanoparticles because the subunits are desorbed from the zeolites by the steric hindrance of the added PEG molecules. The present study reveals that PEGylated encapsulin was reassembled and hollow encapsulin nanoparticles were obtained. The results clearly demonstrate the usefulness of zeolites as a tool for the successful refolding of PEGylated proteins and their reassembly with tertiary structures.     

Analysis of heterogeneity in nonspecific PEGylation reactions of biomolecules

The compositional heterogeneity associated with polymer conjugation reactions of biomolecules is analyzed for the particular case of nonspecific PEGylation reactions. It is shown that the distribution of the number of PEG moieties grafted to biomolecules such as proteins is a binomial‐type function of two parameters—the reaction efficiency as well as the number of binding sites per biomolecule. The nature of this distribution implies that uniform compositions are favored for increasing number of coupling sites per biomolecule as well as for increasing efficiency of the modification process. Therefore, the binomial distribution provides a rationale for the pronounced heterogeneity that is observed for PEGylated small enzyme systems even at high coupling efficiencies. For the particular case of PEGylated trypsin it is shown that the heterogeneity results in a broad distribution of deactivation times that is captured by a stretched exponential decay model. The presented analysis is expected to apply to general modification processes of compounds in which partial functionalization of a fixed number of reactive sites is achieved by means of a nonspecific coupling reaction. © 2012 Wiley Periodicals, Inc. Biopolymers 99: 427–435, 2013.     

Marathon running: physiological and chemical changes accompanying late-race functional deterioration

Twenty-one experienced runners were studied before, during and immediately after a marathon race to ascertain whether either depletion of energy substrate or rise in body temperature, or both, contribute to late-race slowing of running pace. Seven runners drank a glucose/electrolyte (GE) solution ad libitum (Na+ 21 mmol l-1, K+ 2.5 mmol l-1, Cl- 17 mmol l-1, PO4(2-) 6 mmol l-1, glucose 28 mmol l-1) throughout the race; 6 drank water and 8 drank the GE solution diluted 1:1 with water. Although average running speeds for the three groups were not significantly different during the first two-thirds (29 km) of the race, rectal temperature was significantly higher (P < 0.05) and reduction of plasma volume was greater (P < 0.05) in runners who replaced sweat losses with water. During the last one-third of the race, the average running pace of the water-replacement group slowed by 37.2%; the pace slowed by 27.9% in the 8 runners who replaced their sweat loss with GE diluted 1:1 with water (1/2 GE) and 18.2% in runners who replaced fluid loss with full-strength solution (GE). Eleven runners (5 in the water group, 4 in the 1/2 GE group and 2 in the GE group) lapsed into a walk/run/walk pace during the last 6 miles of the race. Ten of these had a rectal temperature of 39 degrees C or greater after 29 km of running, and plasma volume in these runners was reduced by more than 10%.(ABSTRACT TRUNCATED AT 250 WORDS)     

Extension Arm Facilitated PEGylation of Hemoglobin: Correlation of the Properties with the Extent of PEGylation

Human hemoglobin (Hb) conjugated with six copies of PEG-5K is nonhypertensive. The hexaPEGylated Hb exhibits molecular size homogeneity in spite of the chemical heterogeneity with respect to the sites of conjugation (Manjula et al., 2005). In the present study, Hb conjugated with an average of 4, 6, 8 and 10 copies of PEG-5K chains have been generated using the extension arm facilitated PEGylation protocol. Except for the tetraPEGylated Hb, all the other products exhibit molecular size homogeneity. The molecular, colligative and functional properties of PEG-Hb conjugates have been correlated with the extent of PEGylation. The results imply that six copies of PEG-5K chains are accommodated on Hb without significant crowding on the molecular surface. As more copies of PEG-5K chains are conjugated to form octa and deca PEGylated Hb, the PEG-chains conjugated appear to undergo transition from a mushroom (compact) to a brush-like conformation (extended conformation) with a concomitant decrease in the propensity of the molecule to transition from oxy to deoxy conformation in the presence of allosteric effectors. The viscosity and the colloidal osmotic pressure of Hb increase with the number of the PEG-chains conjugated in an exponential fashion. The composition of the PEGylated Hb generated appears to be controlled by (i) high reactivity of thiol groups of the extension arms on Hb with maleimide-PEG, (ii) increase in the viscosity of the reaction mixture as the level of PEGylation increases and (iii) increased resistance induced by the PEG-shell of PEGylated Hb to accommodate more PEG-chains as the level of PEGylation increases. Potential implications of extent of PEGylation on the oxygen delivery by PEG-Hb conjugate in vivo have been discussed.     

聚乙二醇定点修饰蛋白质药物的技术和临床研究进展

聚乙二醇(PEG)定点修饰蛋白药物是针对蛋白特定基团特定位点的修饰,相比于非定点随机修饰的特点是PEG修饰位点的单一与确定,避免了修饰异构体的干扰,能较好的保留药物体内外活性;修饰产物组成均一、性质稳定,便于质量控制,降低由修饰异构体引起的潜在的安全性风险,并很大程度上提高得率,降低成本。已有PEG定点修饰蛋白药物上市,还有部分处于临床试验阶段。本文综述了PEG定点修饰蛋白药物的技术研究与临床进展,包括PEG定点修饰剂、定点修饰方法、PEG定点修饰的上市和临床药物及面临的问题,并展望了PEG修饰技术未来的发展前景。     

Disulfide–bridging PEGylation during refolding for the more efficient production of modified proteins

Proteins that are modified by chemical conjugation require at least two separate purification processes. First the bulk protein is purified, and then after chemical conjugation, a second purification process is required to obtain the modified protein. In an effort to develop new enabling technologies to integrate bioprocessing and protein modification, we describe the use of disulfide‐bridging conjugation to conduct PEGylation during protein refolding. Preliminary experiments using a PEG‐mono‐sulfone reagent with partially unfolded leptin and unfolded RNAse T1 indicated that the cysteine thiols underwent disulfide‐bridging conjugation to give the PEGylated proteins. Interferon‐β1b (IFN‐β1b) was then expressed in E.coli as inclusion bodies and found to undergo disulfide bridging‐conjugation during refolding. The PEG‐IFN‐β1b was isolated by ion‐exchange chromatography and displayed in vitro biological activity. In the absence of the PEGylation reagent, IFN‐β1b refolding was less efficient and yielded protein aggregates. No PEGylation was observed if the cysteines on IFN‐β1b were first modified with iodoacetamide prior to refolding. Our results demonstrate that the simultaneous refolding and disulfide bridging PEGylation of proteins could be a useful strategy in the development of affordable modified protein therapeutics.