Determination Of Alkaline Phosphatase Research Articles

Impact of Dried Thyme Leaf Meal on Production Performance, Egg Quality and Blood Parameters of Laying Hens.

The use of commercially extracted phytogenic compounds to maintain poultry health and productivity in the absence of in-feed antibiotics is prohibitively costly in developing countries. The goal of the study is to determine the effect of dietary supplementation with Thymus schimperi leaf meal (TLM) on production performance, egg quality and haemato-biochemical parameters of Bovan brown layers. A total of 96 laying hens at 25 weeks of age were randomly assigned to 4 treatments with 6 replications each. The treatments include the control (standard commercial laying diet), TLM1.5 (control+1.5% TLM), TLM2.5 (control+2.5% TLM) and TLM3.5 (control+3.5% TLM). Egg production, feed intake and feed conversion ratio were recorded for each replicate. Two eggs per replication were used to measure internal and external egg quality traits on a monthly basis. At the end of the trial, blood samples were collected from 2 birds/replicate for the determination of albumin, uric acid, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, luteinizing hormone, prolactin and progesterone. All blood parameters were within the normal ranges of the breed. Egg production, feed conversion ratio, internal egg quality traits and external egg quality traits of hens fed diets containing 2.5% TLM were significantly higher than the control. Furthermore, diets containing 2.5% TLM led to a significantly reduced feed conversion ratio compared to all other dietary treatments. In conclusion, 2.5% TML is recommended to improve egg production and egg quality without adverse effect on hen health.

The release behavior and in vitro osteogenesis of quercetin-loaded bioactive glass/hyaluronic acid/sodium alginate nanocomposite paste

Injectable pastes based on bioactive compounds and natural polymers are of interest in non-invasive bone surgeries. Several quantities of quercetin (100, 150, and 200 μM) were added to a sol-gel derived mesoporous bioactive glass. Injectable pastes based on quercetin-loaded bioactive glass, sodium alginate, and hyaluronic acid were prepared. Aggregated nanoparticles of bioactive glass and quercetin-loaded bioactive glass with mesoporous morphologies were confirmed by TEM and BET techniques. The quercetin release study was assessed in phosphate-buffered solution medium over 200 h and the obtained data were fitted by different eqs. A sustained release of quercetin was found, in which a better regression coefficient was achieved using Weibull equation. Human-derived mesenchymal stem cells were utilized to determine alkaline phosphatase activity and bone-related protein expression by western blotting and real-time PCR evaluations. Quercetin-loaded pastes increased the levels of alkaline phosphatase activity and the expression of Collagen-1, Osteopontin, Osteocalcin, and Runx2 proteins in a concentration-dependent manner. Due to the mesoporous architecture and high specific surface area of bioactive glass, the paste made of these particles and sodium alginate/hyaluronic acid macromolecules is appropriate matrix for quercetin release, resulting in promoted osteogenesis. The further in vivo studies can support the osteogenesis capacity of the quercetin-loaded paste.

Toward the point-of-care testing of alkaline phosphatase in human serum

This work addresses challenges of simple but reliable determination of enzyme alkaline phosphatase (ALP) in μL-sized serum samples. The single-tube assay and plate-based immunoassay were developed by using a commercial glucose test strip (GTS) to transduce analytical signal from the ALP + glucose-6-phosphate (G6P) reaction. The glucose released from G6P contributed to the flow of anodic charge Q20s through GTS. The interferences from serum matrix including those from native glucose were eliminated by devising a signal ΔQ20s = Q20s (total) – Q20s (matrix). The ΔQ20s was proportional to ALP activity within a linear range of 7.4–720 IU L−1 for assay (R2, 0.995) and 10–300 IU L−1 for immunoassay (R2, 0.983), which far exceeded a normal clinical range for ALP in adult human serum (30–150 IU L−1). Both methods required no auxiliary enzymes or labels, and were accurate (93–99 % signal recovery) and precise (RSD ≤10%). The assay was a simpler option because it required only one 20-min incubation step to determine ALP. The immunoassay involved three steps but it was still less laborious (by ∼70%) than a traditional enzyme-linked immunosorbent assay (ELISA) of ALP due to the substitution of ELISA detection antibody with GTS. The GTS-G6P-based methods bring ALP testing closer to the point-of-care locations, which is important considering the growing role of ALP as a prophylactic, therapeutic, and anti-inflammatory agent, and a marker of human diseases.

Simple hydrated electron-induced electrochemiluminescence determination of alkaline phosphatase with novel time-resolved internal standard strategy at boron doped diamond electrodes

Hydrated electrons were generated at boron doped diamond electrodes in fully aqueous solutions during cathodic pulse polarization and utilized for the electrochemiluminescence (ECL) determination of alkaline phosphatase (ALP) by adapting one of the most common fluorescent methods. In this approach, highly fluorescent 4-methylumbelliferyl (MU) is enzymatically generated from non-fluorescent 4-methylumbelliferyl phosphate (MUP) substrate and chemically excited in the presence of oxidizing radicals and reducing hydrated electrons. A novel time-resolved internal standard strategy is presented to counter matrix effects to certain degree and to lower signal variation among concurrent measurements. In this unique approach, the cathodic ECL signals of MU and Tb(III)-chelate were separated on the basis of their exceptionally different excited state lifetimes. This enabled more precise determination of ALP by simply measuring the ratio of ECL emissions during and after cathodic excitation pulse with simple two-electrode, single photomultiplier tube setup. The ratiometric ECL signals in the absence and presence of 1.0 % serum (v/v) were interchangeable whereas ratiometric signal recovery in the presence of 10.0 % serum was ∼85 %.

Detection of alkaline phosphatase activity with a CsPbBr3/Y6 heterojunction-based photoelectrochemical sensor.

An ultra-sensitive photoelectrochemical (PEC) sensor based on perovskite composite was developed for the determination of alkaline phosphatase (ALP) in human serum. In contrast toCsPbBr3 or Y6 that generated anodic current, the heterojunction of CsPbBr3/Y6 promoted photocarriers to separate and generated cathodic photocurrent. Ascorbic acid (AA) was produced by ALP hydrolyzing L-ascorbic acid 2-phosphate trisodium salt (AAP), which can combine with the holes on the photoelectrode surface, accelerating the transmission of photogenerated carriers, leading to enhanced photocurrent intensity. Thus, the enhancement of PEC current was linked to ALP activity. The PEC sensor exhibits good sensitivity for detection of ALP owing to the unique photoelectric properties of the CsPbBr3/Y6 heterojunction. The detection limit of the sensor was 0.012 U·L-1 with a linear dynamic range of 0.02-2000 U·L-1. Therefore, this PEC sensing platform shows great potential for the development of different PEC sensors.

Engineering a microparticle-loaded rough membrane for guided bone regeneration modulating osteoblast response without inducing inflammation

Guided bone regeneration (GBR) is a widely used procedure that prevents the fast in-growth of soft tissues into bone defect. Among the different types of membranes, the use of collagen membranes is the gold standard. However, these membranes are implanted in tissue location where a severe acute inflammation will occur and can be negatively affected. The aim of this study was to develop a collagen-based membrane for GBR that incorporated alginate-hydroxyapatite microparticles. Membranes were manufactured using collagen type I and gelatin and alginate-hydroxyapatite microparticles. Membranes were assessed in terms of topography by scanning electron microscopy and confocal microscopy; stability by swelling after an overnight incubation in saline and enzymatic degradation against collagenase and mechanical properties by tensile tests. Furthermore, the biological response was assessed with SaOs-2 cells and THP-1 macrophages to determine alkaline phosphatase activity and inflammatory cytokine release. Our results showed that the incorporation of different percentages of these microparticles could induce changes in the surface topography. When the biological response was analyzed, either membranes were not cytotoxic to THP-1 macrophages or to SaOs-2 cells and they did not induce the release of pro-inflammatory cytokines. However, the different surface topographies did not induce changes in the macrophage morphology and the release of pro- and anti-inflammatory cytokines, suggesting that the effect of surface roughness on macrophage behavior could be dependent on other factors such as substrate stiffness and composition. Collagen-gelatin membranes with embedded alginate-hydroxyapatite microparticles increased ALP activity, suggesting a positive effect of them on bone regeneration, remaining unaffected the release of pro- and anti-inflammatory cytokines.

Hemolysis in blood samples

Hemolysis is one of the interfering factors that can limit the results of various biochemical tests, and this study aimed to assess the interference of hemolysis in the reliability of biochemical tests conducted in clinical-surgical procedures in the veterinary hospital routine. The methodology was retrospective, based on the analysis of records of animals treated at the Veterinary Hospital Unit (SUHVU) of the Federal University of Fronteira, Campus Realeza (Paraná) from January of 2020 to December of 2022. The tests conducted in the Veterinary Clinical Analysis laboratory included serum determination of total proteins, albumin, alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), gamma-glutamyl transferase (GGT), creatinine and urea. Based on the biochemical results, animals were divided into three groups according to the degree of hemolysis observed in the serum: mild hemolysis (HD); moderate hemolysis (HM) and intense hemolysis (HI). The control group (GC) was determined based on reference values specific to the species. Data were tabulated in spreadsheets and analyzed using Pearson's correlation test (p). Mild hemolysis was predominant and, in dogs, did not affect the analyzed biochemical values in comparison to the reference averages for the species, regardless of the degree. Isolated, most measurements increased. In cats, changes were more significant concerning the group average, with ALP showing the greatest variation, not consistent with the elevation of hemolysis. None of the parameters had a strong correlation of interference between hemolyses, only moderate negative and positive correlations in ALP and urea.

Nanozyme Rich in Oxygen Vacancies Derived from Mn-Based Metal-Organic Gel for the Determination of Alkaline Phosphatase.

Vacancy engineering as an effective strategy has been widely employed to regulate the enzyme-mimic activity of nanomaterials by adjusting the surface, electronic structure, and creating more active sites. Herein, we purposed a facile and simple method to acquire transition metal manganese oxide rich in oxygen vacancies (OVs-Mn2O3-400) by pyrolyzing the precursor of the Mn(II)-based metal-organic gel directly. The as-prepared OVs-Mn2O3-400 exhibited superior oxidase-like activity as oxygen vacancies participated in the generation of O2•-. Besides, steady state kinetic constant (Km) and catalytic kinetic constant (Ea) suggested that OVs-Mn2O3-400 had a stronger affinity toward 3,3',5,5'-tetramethylbenzidine and possessed prominent catalytic performance. By taking 2-phospho-l-ascorbic acid as the substrate, which can be converted into reducing substance ascorbic acid in the presence of alkaline phosphatase (ALP), OVs-Mn2O3-400 can be applied as an efficient nanozyme for ALP colorimetric analysis without the help of destructive H2O2. The colorimetric sensor established by OVs-Mn2O3-400 for ALP detection showed a good linearity from 0.1 to 12 U/L and a lower limit of detection of 0.054 U/L. Our work paves the way for designing enhanced enzyme-like activity nanozymes, which is of significance in biosensing.

One-Step Ultrasonic Preparation of Stable Bovine Serum Albumin-Perovskite for Fluorescence Analysis of L-Ascorbic Acid and Alkaline Phosphatase.

Halide lead perovskite has attracted increased attention due to its excellent optical properties. However, the poor stability of the halide lead perovskite nanocrystals has been a major obstacle to their application in biosensing. Here, we proposed a method to synthesize CsPbBr3/BSA NCs perovskite using bovine serum albumin (BSA) as a zwitterion ligand. Then, a fluorescent sensor for alkaline phosphatase determination based on CsPbBr3/BSA NCs was successfully built via the interaction of L-ascorbic acid (AA) with BSA on the perovskite surface. Under optimal conditions, the sensor showed a linear concentration range from 50 to 500 μM with a detection limit of 28 μM (signal-to-noise ratio of 3) for AA, and demonstrated a linear concentration range from 40 to 500 U/L with a detection limit of 15.5 U/L (signal-to-noise ratio of 3) for alkaline phosphatase (ALP). In addition, the proposed fluorescent biosensor exhibited good selectivity and recovery in the determination of ALP in human serum. This strategy offers an innovative way for enhancing the water stability of lead halide perovskite and promoting their application in biosensing areas.

Evaluation of the effects of radiation doses from computed tomography on the biochemical parameters of the hepatorenal organs of albino rats

Background Aims: Computed tomography (CT) uses X-rays which are ionizing radiation. Interaction of ionizing radiation with the body may result in harmful effects on the organism depending on the radiation dose. This study aimed to evaluate the impact of different doses of CT radiation on the hepatorenal organs of male albino rats using biochemical parameters as the pathological response of the organs to radiation. Materials and Methods: Thirty healthy male Wistar albino rats weighing 180–200 g were assigned into five groups of six rats each. Rats in groups B, C, D, and E underwent noncontrast helical total body CT irradiation and received varying doses of CT radiation while group A received sham irradiation and served as control. At 72 h postirradiation, blood was collected using conventional methods, and serum was harvested for the determination of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total bilirubin (TB), total protein (TP), albumin (Alb), urea, uric acid, and creatinine. Results: The mean serum levels of TP in the control (group A) and in the irradiated groups B, C, D, and E were 8.21 ± 0.34, 6.08 ± 0.30, 5.86 ± 0.42, 5.22 ± 0.30, and 4.72 ± 0.16, respectively. The mean values of TP in the irradiated groups decreased significantly in a dose-dependent manner compared to the control. The mean serum levels of ALP in the group A and in the irradiated groups B, C, D, and E were 75.20 ± 3.14, 111.60 ± 1.60, 117.60 ± 1.12, 124.40 ± 2.48, and 133.60 ± 2.46, respectively. The mean serum levels of AST in the group A and in the irradiated groups B, C, D, and E were 33.80 ± 0.58, 54.40 ± 2.29, 52.60 ± 1.78, 58.60 ± 2.64, and 63.60 ± 0.81, respectively. The mean serum levels of ALT in the group A and in the irradiated groups B, C, D, and E were 21.20 ± 0.86, 36.00 ± 0.84, 40.40 ± 1.17, 37.40 ± 0.93, and 41.60 ± 1.03, respectively. The mean serum levels of TB in the group A and in the irradiated groups B, C, D, and E were 2.78 ± 0.28, 3.70 ± 0.18, 4.77 ± 0.40, 5.88 ± 0.11, and 6.34 ± 0.04, respectively. The mean serum levels of Alb in the group A and in the irradiated groups B, C, D, and E were 3.68 ± 0.16, 5.96 ± 0.21, 6.65 ± 0.26, 8.15 ± 0.23, and 8.91 ± 0.17, respectively. The mean serum levels of urea in the group A and in the irradiated groups B, C, D, and E were 42.40 ± 0.17, 54.40 ± 1.33, 56.00 ± 1.76, 54.60 ± 1.72, and 57.80 ± 1.39, respectively. The mean serum levels of uric acid in the group A and in the irradiated groups B, C, D, and E were 5.65 ± 0.36, 8.32 ± 0.46, 8.87 ± 1.11, 9.13 ± 0.29, and 9.91 ± 0.29, respectively. The mean serum levels of creatinine in the group A and in the irradiated groups B, C, D, and E were 2.05 ± 0.13, 4.44 ± 0.32, 5.81 ± 1.17, 6.31 ± 0.03, and 6.63 ± 0.40, respectively. The mean values of ALP, AST, ALT, TB, Alb, urea, uric acid, and creatinine increased significantly in the irradiated groups compared to the control group. Conclusion: From the lowest to the highest doses of CT irradiation in the study, alterations in the biochemical parameters were evident and significant. Moreover, this suggests a possible deleterious biological effect of CT radiation on the hepatorenal organs probably due to the effects of radiation-induced free radicals which adversely affected the hepatorenal organs.

Colorimetric and Fluorescent Determination of Alkaline Phosphatase and Ascorbic Acid based upon the Inner Filter Effect using up-Conversion Nanoparticles

A novel fluorescence and colorimetric dual probe based upon the inner filter effect (IFE) is reported to selectively and sensitively determine alkaline phosphatase (ALP). First, up-conversion nanoparticles (UCNPs) were prepared via a facile hydrothermal method and characterized by transmission electron microscopy (TEM), X-ray diffractometry (XRD), Fourier transform infrared spectroscopy (FT-IR), and zeta potential. Under near infrared (NIR) excitation at 980 nm, the prepared nanoparticles emit green light at 541 nm. The absorption band of p-phenylenediamine (PPD) overlaps the emission of UCNPs, which effectively quenches the fluorescence of UCNPs. However, ascorbic acid (AA) generated from the reaction of ascorbic acid 2-phosphate (AAP) and ALP impedes the oxidation of PPD and the fluorescence is restored. Based on this change, a “turn-on” sensor was used to determine AA and ALP with the limits of detection of 2.49 μM and 0.018 U/L, respectively. The constructed assay was applied to determine ALP in serum and the recoveries were from 93.3% to 101.3% with a relative standard deviation from 1.1% to 3.3%. A facile and efficient approach is reported for the determination ALP and offers a theoretical basis for the related diseases.

A Strategy for the Determination of Alkaline Phosphatase Based on the Self-Triggered Degradation of Metal-Organic Frameworks by Phosphate.

Alkaline phosphatase (ALP) is widely present in the human body and is an important biomarker. Numerous ALP detection studies have been carried out, and ascorbic acid (AA) is often used as the reducing component in the sensors to monitor ALP levels since it can be produced from ascorbic acid 2-phosphate (AA2P) hydrolysis in the presence of ALP. However, it is well-known that AA is a strong reducing agent and can be easily oxidized. The disproportion between oxidized AA and reduced AA reactions results in the generation of AA free radicals with single electrons that may lead to inaccurate results in assays. To solve this problem, we synthesized a core-shell metal-organic framework sensor (PATP-Au@ZIF-8 NP) and used it as a sensitive and accurate ALP detection sensor with self-triggered control of phosphate ions (Pi) to avoid the potential inaccuracy of the method that uses AA as the reducing component. By establishing a physical shell on the surface of the gold nanoparticles (Au NPs), the sensor not only can eliminate the random assembly of metal nanoparticles caused by plasma exposure but also can generate self-triggering of Pi caused by ALP. Pi can decompose ZIF-8 through coordination with Zn2+ and thus can destroy the ZIF-8 shell structure of the prepared PAZ NPs. Au NPs are released and then become aggregated, in turn causing the SERS "hot spot" area to increase. The enhancement of the SERS signals was found to be directly associated with the level of Pi released from ALP-triggered hydrolysis. The response of the strategy was linear at ALP concentrations ranging from 0.1 to 150 mU/mL (r = 0.996) with a detection limit of 0.03 mU/mL. Lastly, the developed strategy was employed in the evaluation of ALP inhibitors, and the possibility to implement the developed SERS strategy for rapid and selective analysis of ALP in human serum was demonstrated.

Spectrophotometric Determination of Alkaline Phosphatase in Serum by A Copper Prussian Blue Analog as A Novel Polyphenol Oxidase-Like Nanozyme

Nanomaterials with natural enzyme-like activities have attracted widespread interest and hence their development is significant. Here, a copper Prussian blue analogue named Cu-HCF was prepared and its polyphenol oxidase (PPO)-like activity was investigated for the first time. Cu-HCF nanozyme showed nearly 7-fold higher substrate affinity than natural PPO and was more stable under alkaline conditions, high temperature, high salt concentrations, and long-time storage. The catalytic activity of Cu-HCF nanozyme was reduced by pyrophosphate (PPi). Alkaline phosphatase (ALP) hydrolyzed PPi to orthophosphate (Pi) and hence the PPO-like activity of Cu-HCF nanozyme was restored. A method was therefore developed for selective determination of the ALP activity. The linear range was from 1 to 50 mU/mL with a limit of detection (LOD) of 0.35 mU/mL. This approach was validated in human serum samples, showing its potential in diagnostic applications.

Maneuvering the Peroxidase-Like Activity of Palladium-Based Nanozymes by Alloying with Oxophilic Bismuth for Biosensing.

Engineering the catalytic performance of nanozymes is of vital importance for their broad applications in biological analysis, cancer treatment, and environmental management. Herein, a strategy to boost the peroxidase-like activity of Pd-based nanozymes with oxophilic metallic bismuth (Bi) is demonstrated, which is based on the incorporation of oxophilic Bi in the Pd-based alloy nanocrystals (NCs). To synthesize PdBi alloy NCs, a seed-mediated method is employed with the assistance of underpotential deposition (UPD) of Bi on Pd. The strong interaction of Bi atoms with Pd surfaces favors the formation of alloy structures with controllable shapes and excellent monodispersity. More importantly, the PdBi NCs show excellent peroxidase-like activities compared with pristine Pd NCs. The structure-function correlations for the PdBi nanozymes are elucidated, and an indirect colorimetric method based on cascade reactions to determine alkaline phosphatase (ALP) is established. This method has good linear range, low detection limit, excellent selectivity, and anti-interference. Collectively, this work not only provides new insights for the design of high-efficiency nanozymes, expands the colorimetric sensing platform based on enzyme cascade reactions, but also represents a new example for UPD-directed synthesis of alloy NCs.

PSVIII-1 Hy-D Increases Ca, P and Serum Metabolites Related to Growth in Grazing Animals Receiving a P Deficient Supplement

Abstract The aim with this trial was to evaluate the effects of 25-OH Vit D3 in serum metabolites of growing grazing Nellore young bulls during the rainy season with two different supplementary P levels. Two independent and sequential trials were performed: first one using a 250 g/kg CP supplement containing 8 g/kg of P, and the second one using the same 25% CP supplement, with no addition of P. In each trial, eighteen young bulls of 385 kg BW and 18 months of age were randomly assigned to the two treatments (n=9 animals/treatment): addition or not (control) of 25-OH Vit D3 (ROVIMIX Hy-D 1.25%, Hy-D) to the supplement. Animals were placed in a paddock of B. brizantha, cv. Marandú, equipped with electronic feeders that provided individual access (animal/treatment). On day 31 of each trial, blood samples were taken for determination of 25-OH Vit D3, IGF-1, Ca, P, Alkaline Phosphatase (ALP), and osteocalcin (OCa). In each trial, data were analyzed according to a complete randomized design. In the first trial (8 g/kg of P in supplement) the use of Hy-D (Control vs Hy-D) increased serum concentrations of 25-OH Vit D3 (25.8 vs 30.0 ng/mL, P< 0.03); not affecting the others variables. In the second trial, when no P was not added to the supplement, the use of Hy-D (Control vs Hy-D) increased serum concentrations of 25-OH Vit D3 (25.4 vs 44.7 ng/mL, P< 0.01); Ca (10.8 vs 11.3 mg/dL, P< 0.07); P (7.4 vs 8.3 mg/dL, P< 0.08); ALP (205 vs 263 U/L, P< 0.04); IGF-1 (256 vs 327 ng/mL, P< 0.07); and did not affect OCa (P< 0.27). These results showed that positive effects of Hy-D on metabolism could be dependent of dietary P level, with Hy-D being able to maintain the levels of Ca, P and serum metabolites related to growth when no P was supplied in the supplement.

Correlative study on the effect of toxic paint chemicals on the hepatorenal of paint factory workers in Enugu, Nigeria

Objective: Some chemical components of paint have been classified as toxigenic and carcinogenic to human health. The liver and kidney function markers have been found useful in assessing toxic effect of substances. This study assessed the liver and kidney function parameters of paint factory workers in Enugu, Nigeria. Methods: One hundred and forty apparently healthy men, aged (21-40 years), were recruited for this cross-sectional study. Eighty paint factory workers (PFW) as test subjects and sixty non-paint factory workers (NPFW) as controls. Blood samples were collected from participants for the determination of serum electrolytes, urea, creatinine, alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), conjugated and total bilirubin using standard methods. Data were analysed using SPSS computer software version 22. Results: The results showed that mean serum level of AST, ALP, Sodium and Chloride were significantly higher in PFW compared to NPFW (P< 0.05). Workers use of personal protective equipment were hand gloves (20%), goggles (7.5%), safety boots (52.5%), dust masks (20%), factory gown (36.25%). Self-reported occupational health problems amongst the PFW was also evaluated, and headache had the highest percentage (68.75%), followed by eye irritation (60%), skin irritation (56.25%), dizziness (23.75%), sleep disorder (20%), anxiety (12.5%), memory loss (0%). Conclusion: This study shows that occupational exposure of humans to volatile organic compounds and heavy metals in paints may have long term deleterious effects on liver, kidney or brain function in paint factory workers.

Silver ion-regulated ratiometric fluorescence assay for alkaline phosphatase detection based on carbon dots and o-phenylenediamine

In this work, a novel silver ion (Ag+)-regulated ratiometric fluorescence method for the effective and sensitive determination of alkaline phosphatase (ALP) was established based on carbon dots (CDs) and o-phenylenediamine (OPD). OPD can be oxidized by Ag+ to generate fluorescent 2, 3-diaminophenazine (DAP). Thus, based on inner-filter effect (IFE) or/and fluorescence resonance energy transfer (FRET) between CDs and DAP, the CDs-Ag+-OPD system can generate dual-emission at 454 nm and 570 nm respectively when excited at 360 nm. The introduction of ascorbic acid (AA) can react with Ag+ to produce dehydroascorbic acid (DHAA), which inhibits the generation of DAP, resulting in the fluorescence decrease at 570 nm and fluorescence recovery of CDs at 454 nm. Meanwhile, DHAA can react with OPD to generate quoxaline (QX), which emits strong blue fluorescence at 440 nm, further inhibiting the IFE or/and FRET between CDs and DAP. An obvious ratiometric fluorescence response was observed with the increase of the concentration of AA introduced. Due to the fact that AA can be generated by the enzyme catalysis reaction between ALP and 2-phospho-l-ascorbic acid (AAP), the CDs-Ag+-OPD ratiometric system was applied to the determination of ALP successfully. The ratiometric fluorescence value of F454/F570 increases with increasing ALP concentration, with a linear range of 0.2 to 40 U/L and detection limit of 0.1 U/L. In addition, the CDs-Ag+-OPD ratiometric system was successfully applied to the detection of ALP in human serum samples.

Holder Pasteurization: Comparison of Water-Bath and Dry-Tempering Devices.

BackgroundHuman milk (HM) for premature infants is frequently Holder pasteurized (heated at 62.5 ± 0.5°C for 30 min) despite its detrimental effects on heat-sensitive milk components. This tolerated compromise ensures HM's microbial safety while less detrimental methods like short-time HM treatments (HTST) are still being evaluated. Dry-tempering devices (DT-HoP) were recently introduced in clinical practice due to hygienic concerns about water-based Holder pasteurizers (WB-HoP). Evidence on the impact of such dry-tempering devices on HM quality is lacking. The aim of this study was to compare protein retention rates after DT-HoP, WB-HoP and HTST.MethodsWe colorimetrically determined alkaline phosphatase activity (ALP), concentrations of secretory immunoglobulin A (sIgA), and lactoferrin (LF) before and after DT-HoP, WB-HoP and HTST.ResultsALP was below the detection limit after HoP, but retained 52.8 ± 13% activity after HTST (p < 0.01). Secretory IgA (WB-HoP = 73.2 ± 13.5% vs. DT-HoP = 57 ± 14%, p = 0.0018) and LF retention (WB-HoP=47 ± 40% vs. DT-HoP=25 ± 8%, p = 0.07) differed between the two HoP modes. Again, retention was better maintained after HTST compared to HoP (80.4 ± 23% sIgA and 70 ± 42% LF concentration, all p < 0.01).ConclusionDry-tempering milk lowers even further the quality of HM when performing HoP compared to water-bath pasteurization, while HTST warrants continued evaluation for clinical application.

Diagnosis of asymptomatic bone metastases from breast cancer.

e13042 Background: The purpose of this study was to analyze the prognostic value of blood levels of fibrinogen (F) in bone metastases from breast cancer (BC). Methods: The study included 160 patients with nodular BC pT2-3N0-1M0-1, including 48 patients in a prospective group (PG) and 112 patients in a retrospective group (RG). All patients underwent initial clinical and laboratory examinations, including determination of alkaline phosphatase (AP) activity (Cobas Integra 4000 plus, Switzerland) and parameters of coagulation hemostasis (STA Compact, France). Bone scintigraphy was performed for complaints of bone pain and/or in cases of elevated AP activity, regardless of SXCT data. Results were considered statistically significant at p &lt; 0.05. Results: Patients in PG were divided into two subgroups depending on F levels: in 31 (64.6%) patients F = 3.67±0.5 g/L, insignificantly exceeding the norm (2.8±0.3 g/L); in 17 (35.4%) patients F = 6.9±0.3 g/L, being 1.9 and 2.5 times (p &lt; 0.05) higher than in subgroup 1 and the norm, respectively. AP activity exceeded the norm by 26% and 40%, respectively (p &lt; 0.1). Bone metastases were detected before treatment in 24 (21.4%) women of RG with pain syndrome; all these patients showed increased blood levels of F, on average up to 7.86±0.8 g/L, similarly to patients in subgroup 2 of PG. F levels in other 88 (78.6%) patients in RG were 3.4±0.6 g/L. The nature and severity of AP changes in RG patients and in both PG subgroups were similar. Initially elevated F levels in 17 PG patients remained unchanged after antitumor treatment (6.2±0.6 g/L). AP activity values were stable after treatment being only 36% and 28% (p &lt; 0.1) higher than the norm, respectively. None of the patients with high F levels complained about bone pain. Unscheduled bone scintigraphy was appointed to 17 patients of PG without clinical symptoms or x-ray (SXCT) signs of pathology but with initially high F which did not decline after treatment. Bone scintigraphy showed metastases to the spine and pelvic bones in 12 of 17 (70.5%) patients 1 month after treatment, subsequently confirmed by MRI results. Conclusions: Initial levels of F within 6.9±0.6 g/L which do not decline after the special treatment allow predicting unfavorable BC course and the presence of metastases to the spine and/or pelvic bones without clinical signs of progression. The proposed method allows diagnosis of advanced tumor process in the absence of clinical signs of the disease with an accuracy of 87.8%, and allows timely corrections to the treatment plan.

One-Step Determination of Alkaline Phosphatase in Human Serum Based on Manganese (IV) Dioxide/Manganese (II)-Mediated Nuclear Magnetic Resonance (NMR) Relaxation

Alkaline phosphatase (ALP) is a significant biomarker of diseases, such as osteoblastic bone cancer, hepatitis, and myeloma, and hence its determination is essential and important. MnO2/Mn2+ conversion-based magnetic relaxation sensing was developed to directly determine ALP in blood. In the presence of ALP, 2-phospho-L-ascorbic acid trisodium salt was catalyzed to produce ascorbic acid (AA), which reduced MnO2 to Mn2+, causing transverse relaxation time (T2) decay. A linear relationship of ALP from 5 mU·L−1 to 500 mU·L−1 with T2 signal and a low limit of detection (LOD) of 3.32 mU·L−1 were obtained using the optimized conditions of 200 µM of ascorbic acid-phosphate (AAP), 10 µg·mL−1 of MnO2, 37 °C, pH 7.4, and a 30 min reaction time. When directly measuring ALP in serum with the proposed method, the recoveries were from 99.2% to 101.3%, in accordance with values obtained by a commercial assay. In conclusion, a sensitive one-step method for ALP in complex samples was established which has potential for clinical applications.