Excess serum Na level in rats administered with high doses of (-)-epigallocatechin gallate-casein nanoparticles prepared with sodium caseinate.

(-)-Epigallocatechin gallate (EGCG)-incorporated casein nanoparticles benefit from excellent antioxidant, anti-inflammatory and anti-cancer activities due to their synergistic efficiency, but few studies have evaluated their safety. In this study, the EGCG-casein nanoparticles (EGCG-NPs) formulated using caseinate by ultrasonic treatment were evaluated for their subacute toxicity. The subacute toxicity test of EGCG-NPs through 28-day oral administration in rats did not exhibit adverse effect, with a no-observed-adverse-effect level (NOAEL) of at least 5.0 g per kg body weight (BW) per day, which was equivalent to 500 mg per kg BW EGCG per day. However, the serum Na level in females and males treated with 10.0 g per kg BW EGCG-NPs increased significantly as compared to the control rats (P < 0.05). Similar indications appeared in rats treated with 10.0 g per kg BW pure casein nanoparticles without EGCG, which indicated that high doses of caseinate nanoparticles result in an excess serum Na level. Therefore, we should consider the safety of the nanoparticle formulation of caseinate when it is used as a loading nutrient and a functional substance in foods.

HPLC with fluorescence detection for the bioanalysis and pharmacokinetic study of Doxorubicin and Prodigiosin loaded on eco-friendly casein nanomicelles in rat plasma.

A rapid, efficient, and sensitive liquid chromatographic assay hyphenated to fluorometric detector (HPLC-FLD) was developed and validated for the determination of doxorubicin (DXR) and prodigiosin (PDG) in rat plasma. The sample pre-treatment involves a protein precipitation with acetonitrile with satisfying extraction efficiency (98% and 85% for DXR and PDG, respectively). The chromatographic separation was accomplished using stationary phase: Agilent Zorbax Eclipse plus-C18 analytical column (250 × 4.6 mm, 5 µm) and gradient eluting mobile phase of ammonium acetate (pH = 3), acetonitrile and methanol with programmed fluorescence detection. As the proposed method has been validated, it was subsequently implemented to evaluate DXR and PDG loaded on novel eco-friendly Casein nano drug delivery system after intravenous injection in healthy rats. A comparative pharmacokinetics' study was carried out in rats for DXR in free form, DXR alone entrapped in the nanomicelle and DXR with PDG entrapped in the nano micelle. After testing the differences in pharmacokinetic parameters of the different formulations using ANOVA, the results showed insignificant differences among the tested parameters. This indicates that the presented nanomicelle delivery system has succeeded to incorporate PDG and DXR in a hydrophilic, safe, and potent formulation. This novel nanomicelle has negligible effect on the distribution and elimination of DXR.

Exercise Plus Presleep Protein Ingestion Increases Overnight Muscle Connective Tissue Protein Synthesis Rates in Healthy Older Men.

Protein ingestion and exercise stimulate myofibrillar protein synthesis rates. When combined, exercise further increases the postprandial rise in myofibrillar protein synthesis rates. It remains unclear whether protein ingestion with or without exercise also stimulates muscle connective tissue protein synthesis rates. The authors assessed the impact of presleep protein ingestion on overnight muscle connective tissue protein synthesis rates at rest and during recovery from resistance-type exercise in older men. Thirty-six healthy, older men were randomly assigned to ingest 40 g intrinsically L-[1-13C]-phenylalanine and L-[1-13C]-leucine-labeled casein protein (PRO, n = 12) or a nonprotein placebo (PLA, n = 12) before going to sleep. A third group performed a single bout of resistance-type exercise in the evening before ingesting 40 g intrinsically-labeled casein protein prior to sleep (EX+PRO, n = 12). Continuous intravenous infusions of L-[ring-2H5]-phenylalanine and L-[1-13C]-leucine were applied with blood and muscle tissue samples collected throughout overnight sleep. Presleep protein ingestion did not increase muscle connective tissue protein synthesis rates (0.049 ± 0.013 vs. 0.060 ± 0.024%/hr in PLA and PRO, respectively; p = .73). Exercise plus protein ingestion resulted in greater overnight muscle connective tissue protein synthesis rates (0.095 ± 0.022%/hr) when compared with PLA and PRO (p < .01). Exercise increased the incorporation of dietary protein-derived amino acids into muscle connective tissue protein (0.036 ± 0.013 vs. 0.054 ± 0.009 mole percent excess in PRO vs. EX+PRO, respectively; p < .01). In conclusion, resistance-type exercise plus presleep protein ingestion increases overnight muscle connective tissue protein synthesis rates in older men. Exercise enhances the utilization of dietary protein-derived amino acids as precursors for de novo muscle connective tissue protein synthesis during overnight sleep.

Predictors of a positive oral food challenge to cow's milk in children sensitized to cow's milk

INTRODUCTION AND OBJECTIVES: The diagnosis of IgE-mediated cow's milk allergy (CMA) is often based on clinical history and on specific IgE levels and/or skin-prick tests (SPT), both of which are sensitive but not specific. The gold standard, oral food challenge (OFC), is expensive and time-consuming and involves a risk of severe allergic reactions. This study aimed to determine the value of specific IgEs, ratios of specific IgEs for cow's milk and its components to total IgE, and wheal size on SPT for predicting a positive OFC for CMA. MATERIAL AND METHODS: We retrospectively studied 72 patients [median age, four years; age range 0.75-15 years] sensitized to cow's milk who underwent OFCs to milk. predictive variables between patients with positive and negative OFCs were compared. Receiver operator characteristic (ROC) curves were uses to assess variables' discriminatory capacity and Youden's index to determine the best cut-offs for predicting CMA. RESULTS: The OFC was positive in 39 (54%) patients. Wheal size on SPT and all specific IgEs and specific-to-total IgE ratios were significantly different between patients with positive OFCs and those with negative OFCs (p < 0.001). The variable with the greatest area under the ROC curve was casein-specific IgE (0.98), followed by β-lactoglobulin-specific IgE (0.923), casein-specific-to-total-IgE ratio (0.919), and α-lactalbumin-specific IgE (0.908). Casein-specific IgE ≥ 0.95kU/L yielded 88.9% sensitivity and 90.9% specificity. CONCLUSIONS: In our center, casein-specific IgE > 0.95kU/L can obviate an OFC to cow's milk for the diagnosis of CMA in patients sensitized to cow's milk with a compatible history

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Specific enrichment of phosphopeptides by using magnetic nanocomposites of type Fe3O4@graphene oxide and Fe3O4@C coated with self-assembled oligopeptides.

Iron(III-immobilized magnetic nano-composites (MNCs) were first fabricated using one-step aqueous self-assembly of oligopeptides (Glu-Pro-Ala-Lys-Ala-Lys-Ala-Lys; EPAK-VI) for the highly selective capture of phosphopeptides from complex biological samples. Under physiological conditions, EPAK-VI can readily self-organize into a robust and complete coating layer mainly composed of ß-sheets and ß-turns on the surface of Fe3O4@GO and Fe3O4@C MNCs. Tailored by the cyclic structure of proline, the Glu-Pro motifs of EPAK-VI are vertically erected on the surface and thus serve as an effective linker to chelate Fe3+ through carboxyl (COO-) group in the glutamic acid (E) residues. The ionic hydrogen bonds between the ε-amino groups and the surface negative charges coupled with intermolecular hydrogen bonds render the EPAK-VI coating on the MNCs insusceptible to repeated extreme washing conditions. The Fe3+-EPAK-VI coated MNCs exhibit high enrichment efficiency for ß-casein tryptic digest (0.05 fmol µL-1), excellent selectivity from mixed digests (ß-casein/bovine serum albumin, mass ratio 1:500), and high recovery rate (over 80%). Graphical abstractSchematic representation of the fabrication of Fe3+-immobilized MNCs for phosphopeptide enrichment.

Core-shell magnetic bimetallic MOF material for synergistic enrichment of phosphopeptides.

In proteomics, phosphorylation is an important process for protein post-translational modification (PTM), which greatly improves the diversity of proteomes. The PTM regulates almost all physiological and pathological processes such as signal transduction, cell division, proliferation, differentiation and metabolism. The abnormal expression of protein phosphorylation is also associated with cellular metabolic disorders and a range of diseases. However, in mass spectrometry-based phosphorylated peptideomics studies, phosphorylated peptide signals were inhibited by a high abundance of non-phosphorylated peptides; thus, highly selective enrichment was required. In this study, a newly designed material named Fe3O4@MIL(Fe/Ti) was synthesized using a layer-by-layer self-assembly technique that coats the surface of magnetic oxide nanospheres with bimetallic MOF of iron and titanium. The synergistic synthetic coating of the bimetallic MOF gives the material a large surface area and excellent hydrophilicity, which endow the nanoparticles with excellent phosphopeptide enrichment ability, high selectivity (ß-casein/BSA molar ratio 1:500), a low detection limit (3 fmol), high recovery rate (85%), strong binding capacity, size exclusion ability, and ideal batch-to-batch repeatability. For comparison, we used Fe3O4@MIL(Fe/Ti) and two single-metal MOF materials Fe3O4@MIL-100(Fe) and Fe3O4@MIL-125(Ti), to enrich α-casein in the middle. Thus, the iron-titanium bimetallic MOF can not only enrich all the phosphorylated peptides enriched by Fe3O4@MIL-100(Fe) and Fe3O4@MIL-125(Ti), but can also specifically enrich four phosphorylated peptides. Encouraged by the excellent results of characterization and standard protein enrichment, we used this material to analyze human serum and found that bimetallic materials can effectively enrich all four phosphorylated peptides and exclude high molecular proteins. These experimental results indicate that the novel bimetallic MOF is a good candidate to analyze protein phosphorylation in complex samples.

Amino Acid Availability of a Dairy and Vegetable Protein Blend Compared to Single Casein, Whey, Soy, and Pea Proteins: A Double-Blind, Cross-Over Trial.

Protein quality is important for patients needing medical nutrition, especially those dependent on tube feeding. A blend of dairy and vegetable proteins (35% whey, 25% casein, 20% soy, 20% pea; P4) developed to obtain a more balanced amino acid profile with higher chemical scores, was compared to its constituent single proteins. Fourteen healthy elderly subjects received P4, whey, casein, soy, and pea (18 g/360 mL bolus) on five separate visits. Blood samples were collected at baseline until 240 min after intake. Amino acid availability was calculated using incremental maximal concentration (iCmax) and area under the curve (iAUC). Availability for P4 as a sum of all amino acids was similar to casein (iCmax and iAUC) and whey (iCmax) and higher vs. soy (iCmax and iAUC) and pea (iCmax). Individual amino acid availability (iCmax and iAUC) showed different profiles reflecting the composition of the protein sources: availability of leucine and methionine was higher for P4 vs. soy and pea; availability of arginine was higher for P4 vs. casein and whey. Conclusions: The P4 amino acid profile was reflected in post-prandial plasma levels and may be regarded as more balanced compared to the constituent single proteins.

Aminoacidemia following ingestion of native whey protein, micellar casein, and a whey-casein blend in young men.

We examined the aminoacidemic, glycemic, and insulinemic responses following ingestion of 25 g of native whey protein, micellar casein, and a 1:1 blend of whey and casein in randomized order in young adult men. Blood samples were drawn at baseline and at regular intervals for 6 h following ingestion. Area under curve and peak plasma essential amino acid concentrations after the ingestion of the protein blend were similar to whey and greater compared with casein.

Immobilization of a Ce(IV)-substituted polyoxometalate on ethylenediamine-functionalized graphene oxide for selective extraction of phosphoproteins.

A sorbent for selective extraction of phosphoproteins was obtained by immobilization of a Ce(IV)-substituted polyoxometalate on ethylenediamine-functionalized graphene oxide (CeEGO). The resulting composites exhibit an adsorption capacity of 981 mg g-1 for ß-casein due to the synergistic effect of metal-affinity interaction between Ce(IV) and phosphate groups and π-stacking interaction between the polyoxometalate framework and the phosphate groups. The results of LC-MS and SDS-PAGE analysis show that the CeEGO composites can be applied to the extraction of phosphoproteins from protein mixture, and as little as 50 µg mL-1 of the phosphoprotein ß-casein can be detected by SDS-PAGE. It was also applied to the extraction of ß-casein from spiked biological samples such as drinking milk, whole blood and swine heart tissue extract. Graphical abstract An efficient sorbent is obtained by immobilization of a Ce(IV)-substituted polyoxometalate on ethylenediamine-functionalized graphene oxide (CeEGO). The resulting composites exhibit highly selective capture capacity towards phosphoproteins due to the synergistic effect of metal-affinity interaction between Ce(IV) and phosphate groups and π-stacking interaction between the polyoxometalate framework and the phosphate groups.

Metabolomic changes demonstrate reduced bioavailability of tyrosine and altered metabolism of tryptophan via the kynurenine pathway with ingestion of medical foods in phenylketonuria.

BACKGROUND: Deficiencies of the monoamine neurotransmitters, such as dopamine synthesized from Tyr and serotonin synthesized from Trp, are of concern in PKU. Our objective was to utilize metabolomics analysis to assess monoamine metabolites in subjects with PKU consuming amino acid medical foods (AA-MF) and glycomacropeptide medical foods (GMP-MF). METHODS: Subjects with PKU consumed a low-Phe diet combined with AA-MF or GMP-MF for 3weeks each in a randomized, controlled, crossover study. Metabolomic analysis was conducted by Metabolon, Inc. on plasma (n=18) and urine (n=9) samples. Catecholamines and 6-sulfatoxymelatonin were measured in 24-h urine samples. RESULTS: Intake of Tyr and Trp was ~50% higher with AA-MF, and AA-MF were consumed in larger quantities, less frequently during the day compared with GMP-MF. Performance on neuropsychological tests and concentrations of neurotransmitters derived from Tyr and Trp were not significantly different with AA-MF or GMP-MF. Plasma serotonin levels of gut origin were higher in subjects with variant compared with classical PKU, and with GMP-MF compared with AA-MF in subjects with variant PKU. Metabolomics analysis identified higher levels of microbiome-derived compounds synthesized from Tyr, such as phenol sulfate, and higher levels of compounds synthesized from Trp in the kynurenine pathway, such as quinolinic acid, with ingestion of AA-MF compared with GMP-MF. CONCLUSIONS: The Tyr from AA-MF is less bioavailable due, in part, to greater degradation by intestinal microbes compared with the Tyr from prebiotic GMP-MF. Research is needed to understand how metabolism of Trp via the kynurenine pathway and changes in the intestinal microbiota affect health for individuals with PKU. This trial is registered at www.clinicaltrials.gov as NCT01428258.

Glycomacropeptide in children with phenylketonuria: does its phenylalanine content affect blood phenylalanine control?

BACKGROUND: In phenylketonuria (PKU), there are no data available for children with respect to evaluating casein glycomacropeptide (CGMP) as an alternative to phenylalanine-free protein substitutes [Phe-free L-amino acid (AA)]. CGMP contains a residual amount of phenylalanine, which may alter blood phenylalanine control. METHODS: In a prospective 6-month pilot study, we investigated the effect on blood phenylalanine control of CGMP-amino acid (CGMP-AA) protein substitute in 22 PKU subjects (13 boys, nine girls), median age (range) 11 years (6-16 years). Twelve received CGMP-AA and nine received Phe-free L-AA, (1 CGMP-AA withdrawal). Subjects partially or wholly replaced Phe-free L-AA with CGMP-AA. If blood phenylalanine exceeded the target range, the CGMP-AA dose was reduced and replaced with Phe-free L-amino acids. The control group remained on Phe-free L-AAs. Phenylalanine, tyrosine and Phe : Tyr ratio concentrations were compared with the results for the previous year. RESULTS: In the CGMP-AA group, there was a significant increase in blood phenylalanine concentrations (pre-study, 275 µmol L-1 ; CGMP-AA, 317 µmol L-1 ; P = 0.02), a decrease in tyrosine concentrations (pre-study, 50 µmol L-1 ; CGMP-AA, 40 µmol L-1 ; P = 0.03) and an increase in Phe : Tyr ratios (pre-study, Phe : Tyr 4.9:1; CGMP-AA, Phe : Tyr 8:1; P = 0.02). In the control group there was a non-significant fall in phenylalanine concentrations (pre-study 325µmol/L: study 280µmol/L [p = 0.9], and no significant changes for tyrosine or phe/tyr ratios [p = 0.9]. Children taking the CGMP-AA found it more acceptable to L-AA. CONCLUSIONS: Blood phenylalanine control declined with CGMP-AA but, by titrating the dose of CGMP-AA, blood phenylalanine control remained within target range. The additional intake of phenylalanine may have contributed to the change in blood phenylalanine concentration. CGMP-AA use requires careful monitoring in children.

Impact of human milk pasteurization on gastric digestion in preterm infants: a randomized controlled trial.

BACKGROUND: Holder pasteurization has been reported to modify human milk composition and structure by inactivating bile salt-stimulated lipase (BSSL) and partially denaturing some of its proteins, potentially affecting its subsequent digestion. OBJECTIVE: We sought to determine the impact of human milk pasteurization on gastric digestion (particularly for proteins and lipids) in preterm infants who were fed their mothers' own milk either raw or pasteurized. DESIGN: In a randomized controlled trial, 12 hospitalized tube-fed preterm infants were their own control group in comparing the gastric digestion of raw human milk (RHM) with pasteurized human milk (PHM). Over a 6-d sequence, gastric aspirates were collected 2 times/d before and after RHM or PHM ingestion. The impact of milk pasteurization digestive kinetics and disintegration was tested with the use of a general linear mixed model. RESULTS: Despite inactivating BSSL, instantaneous lipolysis was not affected by pasteurization (mean ± SD at 90 min: 12.6% ± 4.7%; P > 0.05). Lipolysis occurred in milk before digestion and was higher for PHM than for RHM (mean ± SD: 3.2% ± 0.6% and 2.2% ± 0.8%, respectively; P < 0.001). Pasteurization enhanced the proteolysis of lactoferrin (P < 0.01) and reduced that of α-lactalbumin (only at 90 min) (P < 0.05). Strong emulsion destabilization was observed, with smaller aggregates and a higher specific surface for PHM (P < 0.05). Pasteurization did not affect gastric emptying (∼30-min half time) or pH (mean ± SD: 4.4 ± 0.8) at 90 min. CONCLUSIONS: Overall, pasteurization had no impact on the gastric digestion of lipids and some proteins from human milk but did affect lactoferrin and α-lactalbumin proteolysis and emulsion disintegration. Freeze-thawing and pasteurization increased the milk lipolysis before digestion but did not affect gastric lipolysis. Possible consequences on intestinal digestion and associated nutritional outcomes were not considered in this study. This trial was registered at clinicaltrials.gov as NCT02112331.

Resistance Exercise Augments Postprandial Overnight Muscle Protein Synthesis Rates.

INTRODUCTION: We have previously shown that protein ingestion before sleep increases overnight muscle protein synthesis rates. Whether prior exercise further augments the muscle protein synthetic response to presleep protein ingestion remains to be established. OBJECTIVE: This study aimed to assess whether resistance-type exercise performed in the evening increases the overnight muscle protein synthetic response to presleep protein ingestion. METHODS: Twenty-four healthy young men were randomly assigned to ingest 30 g intrinsically L-[1-C]-phenylalanine and L-[1-C]-leucine-labeled casein protein before going to sleep with (PRO + EX, n = 12) or without (PRO, n = 12) prior resistance-type exercise performed in the evening. Continuous intravenous L-[ring-H5]-phenylalanine, L-[1-C]-leucine, and L-[ring-H2]-tyrosine infusions were applied. Blood and muscle tissue samples were collected to assess whole-body protein balance, myofibrillar protein synthesis rates, and overnight incorporation of dietary protein-derived amino acids into de novo myofibrillar protein. RESULTS: A total of 57% ± 1% of the ingested protein-derived phenylalanine appeared in the circulation during overnight sleep. Overnight myofibrillar protein synthesis rates were 37% (0.055%·h ± 0.002%·h vs. 0.040%·h ± 0.003%·h, P < 0.001, based on L-[ring- H5]-phenylalanine) and 31% (0.073%·h ± 0.004%·h vs. 0.055%·h ± 0.006%·h, P = 0.024, based on L-[1-C]-leucine) higher in PRO + EX compared with PRO. Substantially more of the dietary protein-derived amino acids were incorporated into de novo myofibrillar protein during overnight sleep in PRO + EX compared with PRO (0.026 ± 0.003 vs. 0.015 ± 0.003 molar percent excess, P = 0.012). CONCLUSIONS: Resistance-type exercise performed in the evening augments the overnight muscle protein synthetic response to presleep protein ingestion and allows more of the ingested protein-derived amino acids to be used for de novo myofibrillar protein synthesis during overnight sleep.

Ingestion of Casein in a Milk Matrix Modulates Dietary Protein Digestion and Absorption Kinetics but Does Not Modulate Postprandial Muscle Protein Synthesis in Older Men.

BACKGROUND: The slow digestion and amino acid absorption kinetics of isolated micellar casein have been held responsible for its relatively lower postprandial muscle protein synthetic response compared with rapidly digested proteins such as isolated whey. However, casein is normally consumed within a milk matrix. We hypothesized that protein digestion and absorption kinetics and the subsequent muscle protein synthetic response after micellar casein ingestion are modulated by the milk matrix. OBJECTIVE: The aim of this study was to determine the impact of a milk matrix on casein protein digestion and absorption kinetics and postprandial muscle protein synthesis in older men. METHODS: In a parallel-group design, 32 healthy older men (aged 71 ± 1 y) received a primed continuous infusion of L-[ring-(2)H5]-phenylalanine, L-[ring-3,5-(2)H2]-tyrosine, and L-[1-(13)C]-leucine, and ingested 25 g intrinsically L-[1-(13)C]-phenylalanine and L-[1-(13)C]-leucine labeled casein dissolved in bovine milk serum (Cas+Serum) or water (Cas). Plasma samples and muscle biopsies were collected in the postabsorptive state and for 300 min in the postprandial period to examine whole-body and skeletal muscle protein metabolism. RESULTS: Casein ingestion increased plasma leucine and phenylalanine concentrations and L-[1-(13)C]-phenylalanine enrichments, with a more rapid rise after Cas vs. Cas+Serum. Nonetheless, dietary protein-derived phenylalanine availability did not differ between Cas+Serum (47 ± 2%, mean ± SEM) and Cas (46 ± 3%) when assessed over the 300-min postprandial period (P = 0.80). The milk matrix did not modulate postprandial myofibrillar protein synthesis rates from 0 to 120 min (0.038 ± 0.005 vs. 0.031 ± 0.007%/h) or from 120 to 300 min (0.052 ± 0.004 vs. 0.067 ± 0.005%/h) after Cas+Serum vs. Cas. Similarly, no treatment differences in muscle protein-bound L-[1-(13)C]-phenylalanine enrichments were observed at 120 min (0.003 ± 0.001 vs. 0.002 ± 0.001) or 300 min (0.015 ± 0.002 vs. 0.016 ± 0.002 mole percent excess) after Cas+Serum vs. Cas. CONCLUSIONS: Casein ingestion in a milk matrix delays protein digestion and absorption but does not modulate postprandial muscle protein synthesis when compared to the ingestion of micellar casein only in healthy older men. This trial was registered at Nederlands Trial Register as NTR4429.

High IgE levels to α-lactalbumin, ß-lactoglobulin and casein predict less successful cow's milk oral immunotherapy.

BACKGROUND: A new treatment option for persistent cow's milk allergy (CMA) is oral immunotherapy (OIT). Not all patients develop tolerance during therapy, and markers to identify those who will benefit from it are needed. The objective was to study the IgE and IgG4 antibody profiles to milk and milk proteins before and after OIT in relation to clinical outcome. METHODS: Seventy-six children (5-17 years) with challenge-verified CMA were subjected to a 6-month OIT protocol. The treatment aimed at reaching a maintenance dose of 200 ml CM (high dose = HD). Those who did not reach target were analysed as a low-dose (LD) group. Sera were characterized before and after OIT regarding serum levels of IgE and IgG4 to milk and five milk allergen components evaluated together with clinical CMA symptoms and outcome of OIT. RESULTS: Fifty-five (72%) patients reached the maintenance dose (HD) during therapy. High specific IgE levels towards the milk allergens α-lactalbumin (P = 0.048), ß-lactoglobulin (P = 0.006) and casein (P = 0.015) before OIT start were associated with lower maintenance dose reached. Patients who developed desensitization had a larger increase in IgG4 levels to α-lactalbumin (P = 0.034), ß-lactoglobulin (P = 0.010), casein (P = 0.047) and lactoferrin (P = 0.030) during treatment than those who failed. CONCLUSIONS: Component-resolved diagnostics before OIT can help to identify children with lower probability of a successful OIT outcome, as high IgE levels to α-lactalbumin, ß-lactoglobulin and casein are associated with lower maintenance dose reached. An increase in the IgG4 concentration to milk components during treatment indicated effective desensitization.

Mast cell activation syndrome as a significant comorbidity in sickle cell disease.

Some sickle cell anemia (SCA) patients suffer significantly worse phenotypes than others. Causes of such disparities are incompletely understood. Comorbid chronic inflammation likely is a factor. Recently, mast cell (MC) activation (creating an inflammatory state) was found to be a significant factor in sickle pathobiology and pain in a murine SCA model. Also, a new realm of relatively noncytoproliferative MC disease termed MC activation syndrome (MCAS) has been identified recently. MCAS has not previously been described in SCA. Some SCA patients experience pain patterns and other morbidities more congruent with MCAS than traditional SCA pathobiology (eg, vasoocclusion). Presented here are 32 poor-phenotype SCA patients who met MCAS diagnostic criteria; all improved with MCAS-targeted therapy. As hydroxyurea benefits some MCAS patients (particularly SCA-like pain), its benefit in SCA may be partly attributable to treatment of unrecognized MCAS. Further study will better characterize MCAS in SCA and identify optimal therapy.

Native chromatographic sample preparation of serum, plasma and cerebrospinal fluid does not comprise a risk for proteolytic biomarker loss.

We recently developed a native multidimensional chromatographic method for serum and plasma fractionation for proteomic biomarker search. This method has several advantages:parallelization and automation, high reproducibility and proteome coverage, flexible dynamic range with respect to molecular weight and sample amount, optional enzymatic and immunological analytics additional to mass spectrometry, retaining metabolites, and information on complex formation, modification, and fragmentation of constituents. Nevertheless, native conditions have the probable risk of proteome alteration and biomarker loss by intrinsic proteinases. Hence, we tried to quantify here intrinsic proteolytic activity in native samples and fractions from serum, plasma and cerebrospinal fluid, as well as the effectiveness of intrinsic anti-proteinases during sample handling and preparation under our fractionation conditions. Therefore, we used several quantitative measures: (1) total proportion of intrinsic protein and peptide fractions, (2) azocasein hydrolysis and (3) mass spectrometric protein coverage and peptide numbers. To 1: In all non-fractionated specimens, neither decrease of protein concentration or molecular weight nor increase of peptide concentration was found after variable clotting or pre-incubation time. To 2: No azocasein hydrolysis was seen in these samples when prepared within a few hours at room temperature. Trypsin, when added in concentrations not higher than 0.85 µg/mL (0.04 µM), even was completely inhibited. Moreover, in native 1-D fractions no proteinase activity could be observed. To 3: Mass spectrometry confirmed that neither protein coverage nor peptide numbers differ significantly in 1-D or 2-D fractions after variable incubation time. These results suggest that intrinsic, native proteinase inhibitors potentially protect the proteomes considered, enabling "top-down" proteomic approaches under native conditions with serum, plasma and cerebrospinal fluid.

Interaction between titanium and phosphoproteins revealed by chromatography column packed with titanium beads.

The biochemical mechanism behind the strong binding between titanium and living bone has not been fully elucidated, in spite of worldwide clinical application of this phenomenon. We hypothesized that one of the core mechanisms may reside in the interaction between certain proteins in the host tissues and the implanted titanium. To verify the interaction between titanium and proteins, we chose the technique of chromatography in that titanium spherical beads (45 µm) were packed into a column to obtain a bed volume of 16×50 mm, which was eluted with phosphate buffered saline (PBS) and a straight gradient system made by using PBS and 25 mM NaOH. Fetal calf serum, albumin, lysozyme, casein, phosvitin and dentin phosphoprotein (phosphophoryn) were applied to the column. Most part of albumin and lysozyme eluted with the breakthrough peak, indicating practically no affinity to titanium. Fetal bovine serum also eluted mostly as the breakthrough peak, but distinct retained peak was observed. On the other hand, α-casein, phosvitin and phosphophoryn exhibited a distinct retained peak separated from the breakthrough peak. We proposed that phosphate groups (phosphoserines) in the major phosphoproteins, α-casein, phosvitin and phosphophoryn may be involved in the binding of these proteins with titanium.

The safety of oral use of L-glutamine in middle-aged and elderly individuals.

OBJECTIVE: To evaluate the safety of nutraceutical oral administration of L-glutamine (L-Gln) in middle-aged and elderly individuals. METHODS: In this randomized, crossover, double-blind clinical study, 30 residents of a long-term-care institution, selected according to a modified SENIEUR protocol (Working Party of the EURAGE Concerted Action Programme on Ageing of the European Community), were studied. Fourteen subjects received orally 0.5 g kg(-1) d(-1) of L-Gln and 16 received calcium caseinate for 14 d, followed by a 5-d washout. Supplements were switched for the second 14-d trial. Laboratory tests for hepatic and renal functions and ammonemia were performed and the estimated glomerular filtration rate (eGFR) was calculated. RESULTS: Of the 30 subjects, 16 were men, mean age was 69+/-8.8 y, average weight was 61.8+/-14.2 kg, and mean serum albumin was 4.0+/-0.3g/dL. Neither adverse clinical effects nor clinically significant laboratory changes were noted during L-Gln supplementation. There was no difference in ammonemia between the groups. There were statistically but not clinically significant increases in plasma urea nitrogen and creatinine concentrations. There was no significant decrease in eGFR during calcium caseinate supplementation (-2.9%). The eGFR decreased significantly after L-Gln supplementation (-13.3%) but well below the 25% limit for biologic significance. CONCLUSION: Increases in serum urea nitrogen and creatinine and decrease in eGFR are probably due to difficulties by older kidneys in metabolizing the supplemented protein sources. Although not clinically significant, those alterations impose a rigorous control on the evaluation parameters of renal function during oral L-Gln supplementation, with doses of 0.5 g kg(-1) d(-1) in middle-aged and elderly individuals.

Early serum IGF-I response to oral protein supplements in elderly women with a recent hip fracture.

BACKGROUND & AIMS: In patients with recent hip fracture, reduced serum IGF-I in relation to protein undernutrition is frequent. Elevation of circulating IGF-I in response to a daily oral supplement of 20 g of casein was observed after 6 months. This study determined if the response to casein as compared to whey protein can be observed as early as after one week. METHODS: 45 women were randomized after recent hip fracture in 3 groups receiving a preparation of 20 g of casein, an isocaloric supplement of 20 g of whey protein or an isocaloric supplement of 15 g of whey protein combined with 5 g of essential amino acids (a.a.). RESULTS: A similar significant elevation of serum IGF-I was already observed after 7 days for casein (+37.3 microg/L), whey (+29.4) and for whey+a.a. (+34.3). From day 7-28, no further significant rise in IGF-I was recorded. CONCLUSION: After one week of protein supplementation, the percent increase of IGF-I was of similar magnitude to that previously observed after 6 months of protein supplementation. It suggests that in hip fracture patients, long-term effects of various protein preparations on IGF-I could be predicted from changes observed as early as 7 days after the onset of supplementation.