Dual-mode fluorescence and colorimetric smartphone-based sensing platform with oxidation-induced self-assembled nanoflowers for sarcosine detection.

BACKGROUND: Early prostatic cancer (PCa) diagnosis significantly improves the chances of successful treatment and enhances patient survival rates. Traditional enzyme cascade-based early cancer detection methods offer efficiency and signal amplification but are limited by cost, complexity, and enzyme dependency, affecting stability and practicality. Meanwhile, sarcosine (Sar) is commonly considered a biomarker for PCa development. It is essential to develop a Sar detection method based on cascade reactions, which should be efficient, low skill requirement, and suitable for on-site testing. RESULTS: To address this, our study introduces the synthesis of organic-inorganic self-assembled nanoflowers to optimize existing detection methods. The Sar oxidase (SOX)-inorganic hybrid nanoflowers (Cu3(PO4)2:Ce@SOX) possess inherent fluorescent properties and excellent peroxidase activity, coupled with efficient enzyme loading. Based on this, we have developed a dual-mode multi-enzyme cascade nanoplatform combining fluorescence and colorimetric methods for the detection of Sar. The encapsulation yield of Cu3(PO4)2:Ce@SOX reaches 84.5 %, exhibiting a remarkable enhancement in catalytic activity by 1.26-1.29 fold compared to free SOX. The present study employing a dual-signal mechanism encompasses 'turn-off' fluorescence signals ranging from 0.5 µM to 60 µM, with a detection limit of 0.226 µM, and 'turn-on' colorimetric signals ranging from 0.18 µM to 60 µM, with a detection limit of 0.120 µM. SIGNIFICANCE: Furthermore, our study developed an intelligent smartphone sensor system utilizing cotton swabs for real-time analysis of Sar without additional instruments. The nano-platform exhibits exceptional repeatability and stability, rendering it well-suited for detecting Sar in authentic human urine samples. This innovation allows for immediate analysis, offering valuable insights for portable and efficient biosensors applicable to Sar and other analytes.

Chemical modification of Arthrobacter sarcosine oxidase by N-methylisothiazolinone reduces reactivity toward oxygen.

N-Methylisothiazolinone (MIT) is a thiol group modifier and antimicrobial agent. Arthrobacter sarcosine oxidase (SoxA), a diagnostic enzyme for assaying creatinine, loses its activity upon the addition of MIT, and its inactivation mechanism remains unclear. In this study, SoxA was chemically modified using MIT (mo-SoxA), and its structural and chemical properties were characterized. Spectral analysis data, oxygen consumption rates, and reactions were compared between intact SoxA and mo-SoxA. These demonstrate that the oxidative half-reaction toward oxygen is inhibited by MIT modification. The oxidase activity of mo-SoxA was approximately 2.1% of that of intact SoxA, and its dehydrogenase activity was approximately 4.2 times higher. The C-to-S mutants revealed that cooperative modification of 2 specific cysteine residues caused a drastic change in the enzyme reaction mode. Based on the modeled tertiary structures, the putative entrance for oxygen uptake is predicted to be blocked by the chemical modification of the 2 cysteine residues.

Design of a H2 O2 -generating P450SPα fusion protein for high yield fatty acid conversion.

Sphingomonas paucimobilis' P450SPα (CYP152B1) is a good candidate as industrial biocatalyst. This enzyme is able to use hydrogen peroxide as unique cofactor to catalyze the fatty acids conversion to α-hydroxy fatty acids, thus avoiding the use of expensive electron-donor(s) and redox partner(s). Nevertheless, the toxicity of exogenous H2 O2 toward proteins and cells often results in the failure of the reaction scale-up when it is directly added as co-substrate. In order to bypass this problem, we designed a H2 O2 self-producing enzyme by fusing the P450SPα to the monomeric sarcosine oxidase (MSOX), as H2 O2 donor system, in a unique polypeptide chain, obtaining the P450SPα -polyG-MSOX fusion protein. The purified P450SPα -polyG-MSOX protein displayed high purity (A417 /A280  = 0.6) and H2 O2 -tolerance (kdecay  = 0.0021 ± 0.000055 min-1 ; ΔA417  = 0.018 ± 0.001) as well as good thermal stability (Tm : 59.3 ± 0.3°C and 63.2 ± 0.02°C for P450SPα and MSOX domains, respectively). The data show how the catalytic interplay between the two domains can be finely regulated by using 500 mM sarcosine as sacrificial substrate to generate H2 O2 . Indeed, the fusion protein resulted in a high conversion yield toward fat waste biomass-representative fatty acids, that is, lauric acid (TON = 6,800 compared to the isolated P450SPα TON = 2,307); myristic acid (TON = 6,750); and palmitic acid (TON = 1,962).

A Ti3 C2 TX /Pt-Pd based amperometric biosensor for sensitive cancer biomarker detection.

The detection of cancer biomarkers is of great significance for the early screening of cancer. Detecting the content of sarcosine in blood or urine has been considered to provide a basis for the diagnosis of prostate cancer. However, it still lacks simple, high-precision and wide-ranging sarcosine detection methods. In this work, a Ti3 C2 TX /Pt-Pd nanocomposite with high stability and excellent electrochemical performance has been synthesized by a facile one-step alcohol reduction and then used on a glassy carbon electrode (GCE) with sarcosine oxidase (SOx ) to form a sarcosine biosensor (GCE/Ti3 C2 TX /Pt-Pd/SOx ). The prominent electrocatalytic activity and biocompatibility of Ti3 C2 TX /Pt-Pd enable the SOx to be highly active and sensitive to sarcosine. Under the optimized conditions, the prepared biosensor has a wide linear detection range to sarcosine from 1 to 1000 µM with a low limit of detection of 0.16 µM (S/N = 3) and a sensitivity of 84.1 µA/mM cm2 . Besides, the reliable response in serum samples shows its potential in the early diagnosis of prostate cancer. More importantly, the successful construction and application of the amperometric biosensor based on Ti3 C2 TX /Pt-Pd will provide a meaningful reference for detecting other cancer biomarkers.

Using bimetallic Au/Cu nanoplatelets for construction of facile and label-free inner filter effect-based photoluminescence sensing platform for sarcosine detection.

Herein, we report a facile and label-free method for sensitive and specific determination of prostate cancer biomarker sarcosine via using photoluminescent bimetallic Au/Cu nanoplatelets (AuCu NPs) to construct an inner filter effect (IFE)-based photoluminescence (PL) sensing platform. The AuCu NPs were formed by the cysteine-induced co-reduction reaction, which displayed bright PL with an emission peak at 560 nm. Meanwhile, the Cu(I) doping caused a maximum 25-fold enhancement of quantum yield (QY), compared with the native Au(I) complexes, i.e., from 0.85 to 21.5%. By integrating the AuCu NPs with p-phenylenediamine (PPD) oxidation reaction, an IFE-based sensor for sarcosine detection was constructed. In this method, sarcosine is oxidized under the catalysis of sarcosine oxidase (SOx) to yield H2O2. The latter further oxidizes PPD to form 2,5-diamino-N,N'-bis(p-aminophenyl)-l,4-benzoquinone di-imine (PPDox) in the presence of horseradish peroxidase (HRP). The UV-vis absorption spectrum of the PPDox can overlap well with the excitation and emission spectra of the AuCu NPs, resulting in the efficient quenching of the AuCu NPs via the IFE effect. Therefore, this IFE-based AuCu NPs/SOx/PPD/HRP sensing platform can be used for highly sensitive and specific sensing of sarcosine. The sensing platform showed two linear regions of the PL intensity of the AuCu NPs versus the concentration of sarcosine in the range of 0.5-5 µM and 5-100 µM with a detection limit (LOD) of 0.12 µM (S/N = 3). Furthermore, this IFE-based sensing platform could be developed into a paper-based biosensor for simple, instrument-free, and visual detection of sarcosine.

Colorimetric paper-based sarcosine assay with improved sensitivity.

This manuscript reports on a simple paper-based bienzymatic colorimetric assay for sarcosine as an important urinary biomarker of prostate cancer. All required assay reagents are pre-deposited on hydrophilic filter paper spots surrounded by a hydrophobic barrier. Sarcosine in the sample solution is selectively oxidized in the presence of sarcosine oxidase (SOx), resulting in the formation of hydrogen peroxide, which is subsequently detected through the horseradish peroxidase (HRP)-catalyzed conversion of the colorless indicator 3,3',5,5'-tetramethylbenzidine (TMB) into its blue-colored oxidation product. By the modification of the paper with positively charged poly(allylamine hydrochloride) (PAH), a linear response to sarcosine between 0 and 10 µM and a significant lowering of the limit of detection (LOD) (0.6 µM) compared to the unmodified paper substrate (12.6 µM) has been achieved. The improvement of the LOD was attributed to the fact that the presence of the polymer limits the enzyme-driven colorimetric reaction to the surface of the paper substrate, resulting in stronger color development. In experiments in artificial urine matrix, the bicarbonate anion was identified as an inhibitor of the colorimetric reaction. This inhibition was successfully eliminated through on-device sample pH adjustments with pH-buffer components pre-deposited onto assay devices. The LOD for sarcosine achieved in artificial urine matrix (2.5 µM) is below the 5 µM threshold value for this urinary biomarker required for diagnostic purposes. Finally, good selectivity over all 20 natural amino acids and satisfactory long-term storage stability of reagent-modified paper substrates at - 20 °C for a period of 50 days were confirmed.

Specific Screening of Prostate Cancer Individuals Using an Enzyme-Assisted Substrate Sensing Platform Based on Hierarchical MOFs with Tunable Mesopore Size.

Rapid and specific identification of tumor metabolic markers is of great significance. Herein, a convenient, reliable and specific strategy was proposed to screen prostate cancer (PCa) individuals through indirectly quantifying sarcosine, an early indicator of PCa, in the clinical urine samples. The success roots in the rational design of a cascade response model, which takes integrated sarcosine oxidase (SOX) as a specific recognition unit and oxygen-sensitive molecule as a signal reporter. The newly developed hierarchical mesoporous Zr-based metal-organic frameworks with continuously tunable mesopore size ensure the synergetic work of the SOX and response unit spatially separated in their neighboring mesoporous and microporous domains, respectively. The large mesopore up to 12.1 nm not only greatly enhances the loading capacity of SOX but also spares enough space for the free diffusion of sarcosine. On this basis, the probe is competent to specifically check out the tiny concentration change of sarcosine in the urine sample between PCa patients and healthy humans. Such a concept of enzyme-assisted substrate sensing could be simply extended by altering the type of immobilized enzymes, hopefully setting a guideline for the rational design of multiple probes to quantify specific biomarkers in complex biological samples.

Iron doped graphitic carbon nitride with peroxidase like activity for colorimetric detection of sarcosine and hydrogen peroxide.

The successful synthesis is reported of Mn, Fe, Co, Ni, Cu-doped g-C3N4 nanoflakes via a simple one-step pyrolysis method, respectively. Among them, the Fe-doped g-C3N4 nanoflakes exhibited the highest peroxidase-like activity, which can be used for colorimetric detection of hydrogen peroxide (H2O2) and sarcosine (SA), within the detection ranges of 2-100 µM and 10-500 µM and detection limits of 1.8 µM and 8.6 µM, respectively. The catalytic mechanism of the Fe-doped g-C3N4 nanoflake was also explored and verified the generation of hydroxyl radical (•OH) through fluorescence method. It is believed that the Fe-doped g-C3N4 nanoflakes as enzyme mimics will greatly promote the practical applications in a variety of fields in the future including biomedical science, environmental governance, antibacterial agent, and bioimaging due to their extraordinary catalytic performance and stability. Graphical abstract.

Amperometric sarcosine biosensor based on hollow magnetic Pt-Fe3O4@C nanospheres.

Sarcosine is a recently identified biomarker for prostate cancer. However, the rapid detection methods for sarcosine are relatively lack because of the low concentration and the presence of complicated interfering substances in serum or urine. In this manuscript, hollow nanospheres of Fe3O4 was synthesized and used as carrier to disperse Pt (Pt) nanoparticles. In order to achieve excellent electron transfer ability, we use polyaniline to coat Pt-Fe3O4 nanoparticles, and pyrolyze the polyaniline to carbon (C). Thus, hollow magnetic Pt-Fe3O4@C nanocomposites with good electron transfer ability are formed. The Pt-Fe3O4@C nanocomposites have high catalytic activity and stability. The nanocomposites were immobilized on glassy carbon electrode (GCE) to construct a nonenzyme hydrogen peroxide (H2O2) sensor (Pt-Fe3O4@C/GCE). We further construct a sensitive sarcosine biosensor by immobilizing sarcosine oxidase (SOx) on the Pt-Fe3O4@C/GCE. The high catalytic activity and good biocompatibility of Pt-Fe3O4@C nanocomposites greatly retained the bioactivity of immobilized SOx, and the prepared sarcosine biosensor has good electrocatalytic performance towards sarcosine. It has a linear detection range between 0.5 and 60 µM with a limit of detection (LOD) of 0.43 µM (the signal to noise ratio is 3), and the sensitivity is 3.45 nA µM-1 (48.8 nA µM-1 cm-2), which has the potential to be used for rapid screening of prostate cancer.

An electrochemical sarcosine sensor based on biomimetic recognition.

A nonenzymatic electrochemical sensor is described for the prostate cancer biomarker sarcosine (Sar). Riboflavin was employed to mimic the active center of the enzyme sarcosine oxidase for constructing the biomimetic sensor. The use of riboflavon (Rf) avoids the disadvantages of an enzymatic sensor, such as high cost and poor stability. A glassy carbon electrode (GCE) was modified with a graphene-chitosan (GR) composite and further modified with gold-platinum bimetallic nanoparticles in a polypyrrole (PPy) matrix in order to enhance the catalytic activity of the enzyme mimic. Finally, Rf was electrodeposited on the surface of the AuPt-PPy/GR-modified GCE. Under optimized conditions, the GCE provided high sensitivity and selectivity for Sar at around 0.61 V. Response covers the 2.5-600 µM concentration range, and the detection limit is 0.68 µM. The method was successfully applied to the determination of Sar in spiked urine with 98.0%-103.2% recovery. Graphical abstract Schematic presentation of the fabrication of the Rf/AuPt-PPy/GR/GCE surface and the measurement principle by differential pulse voltammetry (DPV).

A Rapid Method for the Detection of Sarcosine Using SPIONs/Au/CS/SOX/NPs for Prostate Cancer Sensing.

BACKGROUND: Sarcosine is an amino acid that is formed by methylation of glycine and is present in trace amounts in the body. Increased sarcosine concentrations in blood plasma and urine are manifested in sarcosinemia and in some other diseases such as prostate cancer. For this purpose, sarcosine detection using the nanomedicine approach was proposed. In this study, we have prepared superparamagnetic iron oxide nanoparticles (SPIONs) with different modified surface area. Nanoparticles (NPs) were modified by chitosan (CS), and sarcosine oxidase (SOX). SPIONs without any modification were taken as controls. Methods and Results: The obtained NPs were characterized by physicochemical methods. The size of the NPs determined by the dynamic light scattering method was as follows: SPIONs/Au/NPs (100⁻300 nm), SPIONs/Au/CS/NPs (300⁻700 nm), and SPIONs/Au/CS/SOX/NPs (600⁻1500 nm). The amount of CS deposited on the NP surface was found to be 48 mg/mL for SPIONs/Au/CS/NPs and 39 mg/mL for SPIONs/Au/CS/SOX/NPs, and repeatability varied around 10%. Pseudo-peroxidase activity of NPs was verified using sarcosine, horseradish peroxidase (HRP) and 3,3',5,5'-tetramethylbenzidine (TMB) as a substrate. For TMB, all NPs tested evinced substantial pseudo-peroxidase activity at 650 nm. The concentration of SPIONs/Au/CS/SOX/NPs in the reaction mixture was optimized to 0⁻40 mg/mL. Trinder reaction for sarcosine detection was set up at 510 nm at an optimal reaction temperature of 37 °C and pH 8.0. The course of the reaction was linear for 150 min. The smallest amount of NPs that was able to detect sarcosine was 0.2 mg/well (200 µL of total volume) with the linear dependence y = 0.0011x - 0.0001 and the correlation coefficient r = 0.9992, relative standard deviation (RSD) 6.35%, limit of detection (LOD) 5 µM. The suggested method was further validated for artificial urine analysis (r = 0.99, RSD 21.35%, LOD 18 µM). The calculation between the detected and applied concentrations showed a high correlation coefficient (r = 0.99). NPs were tested for toxicity and no significant growth inhibition was observed in any model system (S. cerevisiae, S. aureus, E. coli). The hemolytic activity of the prepared NPs was similar to that of the phosphate buffered saline (PBS) control. The reaction system was further tested on real urine specimens. Conclusion: The proposed detection system allows the analysis of sarcosine at micromolar concentrations and to monitor changes in its levels as a potential prostate cancer marker. The whole system is suitable for low-cost miniaturization and point-of-care testing technology and diagnostic systems. This system is simple, inexpensive, and convenient for screening tests and telemedicine applications.

Construction and application of amperometric sarcosine biosensor based on SOxNPs/AuE for determination of prostate cancer.

An improved amperometric sarcosine biosensor was constructed based on covalent immobilization of sarcosine oxidase nanoparticles (SOxNPs) onto gold electrode (AuE). The SOxNPs/AuE was characterized by scanning electron microscopy (SEM), fourier transform infrared (FTIR) spectroscopy and electrochemical impedance spectroscopy (EIS) at different stages of its construction. The biosensor worked optimally within 2 s at a potential of 1.0 V, against Ag/AgCl, pH 6.5 and 35 °C. A linear relationship was observed between sarcosine concentration range, 0.1-100 µM and the biosensor response i.e. current in mA under optimum conditions. The biosensor offered a low detection limit of 0.01 µM and gratifying storage stability. The SOxNPs/AuE was unaffected by a number of serum substances at their physiological concentrations. The biosensor measured sarcosine level in sera collected from persons suffering from prostate cancer (mean13.5 µM, n = 8), which was significantly higher (p < 0.01) than those in apparently healthy persons (mean 2.2 µM, n = 8). The SOxNPs/Au electrode was reused 300- times during the span of 180 days, with only 10% loss in its initial activity while being stored dry at 4 °C.

Molecular assessment of glyphosate-degradation pathway via sarcosine intermediate in Lysinibacillus sphaericus.

The widespread use of glyphosate has permeated not only small- and large-scale agriculture, but also the fight against drug trafficking and illicit crops. Health, alimentary security, and the rights of peasant and indigenous communities have been compromised in countries with intensive use of glyphosate-based herbicides. In 2015, the International Agency for Research on Cancer classified this substance as probably carcinogenic to humans, leading to the suspension of aerial glyphosate spraying the same year in countries like Colombia, where glyphosate has been extensively used in illicit crop eradication. Notwithstanding, according to a study of the U.S. Geological Survey, traces of glyphosate and its main degradation product, AMPA, remain in soil year after year. This underscores the urgency and importance of assessing new technologies to degrade glyphosate present in soils and waterbodies without leaving persistent byproducts. The aim of this study was to evaluate Lysinibacillus sphaericus' glyphosate uptake as a carbon and phosphorous source by a sarcosine-mediated metabolic pathway that releases glycine as final degradation product. To accomplish this, molecular and analytic evidence were collected in vitro from sarcosine oxidase activity, a key enzyme of a degradation pathway which releases byproducts that are easy to incorporate into natural biosynthesis routes.

Fabrication of an amperometric sarcosine biosensor based on sarcosine oxidase/chitosan/CuNPs/c-MWCNT/Au electrode for detection of prostate cancer.

An amperometric sarcosine biosensor was fabricated based on covalent immobilization of sarcosine oxidase (SarOx) onto the nanocomposite of carboxylated multi-walled carbon nanotubes (cMWCNT)/chitosan (CHIT) and copper nanoparticles (CuNPs), electrodeposited on gold (Au) electrode. The SarOx/CHIT/CuNPs/c-MWCNT/Au electrode was characterized by scanning electron microscopy (SEM), electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). The enzyme electrode exhibited optimum current within 2 s at a potential of 0.2 V against Ag/AgCl, pH 7.0 and 35 °C. A linear relationship was obtained between sarcosine concentration in the range, 0.1-100 µM and current (mA) under optimum conditions. The biosensor exhibited a high sensitivity of 277.5 µA/µM/cm2, a low detection limit of 0.1 pM and excellent storage stability (180 days). The analytical recoveries of added sarcosine in sera at 0.5 µM and at 1.0 µM concentration were 95.5% and 97.30 respectively. The precision i.e. within and between-batch coefficients of variation (CVs) were 1.08% and 1.70% respectively. There was a good correlation (R2 = 0.99) between the level of sarcosine in sera as measured by the standard immuno kit method and the present biosensor. The biosensor measured sarcosine level in sera of prostate cancer patients, which was significantly higher than those of apparently healthy persons (p value <0.01).

Evidence for Pipecolate Oxidase in Mediating Protection Against Hydrogen Peroxide Stress.

Pipecolate, an intermediate of the lysine catabolic pathway, is oxidized to Δ1 -piperideine-6-carboxylate (P6C) by the flavoenzyme l-pipecolate oxidase (PIPOX). P6C spontaneously hydrolyzes to generate α-aminoadipate semialdehyde, which is then converted into α-aminoadipate acid by α-aminoadipatesemialdehyde dehydrogenase. l-pipecolate was previously reported to protect mammalian cells against oxidative stress. Here, we examined whether PIPOX is involved in the mechanism of pipecolate stress protection. Knockdown of PIPOX by small interference RNA abolished pipecolate protection against hydrogen peroxide-induced cell death in HEK293 cells suggesting a critical role for PIPOX. Subcellular fractionation analysis showed that PIPOX is localized in the mitochondria of HEK293 cells consistent with its role in lysine catabolism. Signaling pathways potentially involved in pipecolate protection were explored by treating cells with small molecule inhibitors. Inhibition of both mTORC1 and mTORC2 kinase complexes or inhibition of Akt kinase alone blocked pipecolate protection suggesting the involvement of these signaling pathways. Phosphorylation of the Akt downstream target, forkhead transcription factor O3 (FoxO3), was also significantly increased in cells treated with pipecolate, further implicating Akt in the protective mechanism and revealing FoxO3 inhibition as a potentially key step. The results presented here demonstrate that pipecolate metabolism can influence cell signaling during oxidative stress to promote cell survival and suggest that the mechanism of pipecolate protection parallels that of proline, which is also metabolized in the mitochondria. J. Cell. Biochem. 118: 1678-1688, 2017. © 2016 Wiley Periodicals, Inc.

Expression of sarcosine metabolism-related proteins in estrogen receptor negative breast cancer according to the androgen receptor and HER-2 status.

The aim of this study is to investigate the expression of sarcosine metabolism related proteins according to androgen receptor (AR) and HER-2 status in estrogen receptor (ER) negative breast cancer and to analyze its clinical implications. Tissue microarray was constructed for a total of 334 cases of ER negative breast cancer. Immunohistochemical stain was conducted for sarcosine metabolism related proteins such as glycine N-methyltransferase (GNMT), sarcosine dehydrogenase (SARDH), and l-pipecolic acid oxidase (PIPOX). There were 131 AR positive, 205 AR negative cases and 143 HER-2 positive, 193 HER-2 negative cases. When subdividing into four groups according to AR and HER-2 status, there were 55 AR(+)/HER-2(-) cases, 76 AR(+)/HER-2(+) cases, 67 AR(-)/HER-2(+) cases and 138 AR(-)/HER-2(-) cases. GNMT and PIPOX expression was highest in the AR(+)/HER-2(-) group while expressed lowest in the AR(-)/HER-2(-) group (P<0.001). Stromal PIPOX expression was highest in the AR(-)/HER-2(+) group and lowest in the AR(-)/HER-2(-) group (P=0.010). GNMT and PIPOX expression was higher in the AR positive group compared with those of AR negative group (P=0.001, and P<0.001, respectively), while tumoral and stromal PIPOX expression showed a significant association with HER-2 positivity (P=0.006, and P=0.005, respectively). AR positive group had the highest ratio of low sarcosine type while the AR negative group had the highest ratio of null type (P<0.001). In conclusion, ER negative breast cancer showed different expression of sarcosine metabolism related proteins according to AR and HER-2 status. GNMT and PIPOX expression was high in the AR positive group while tumoral and stromal PIPOX expression was high in the HER-2 positive group.

Strong Negative Interference by Calcium Dobesilate in Sarcosine Oxidase Assays for Serum Creatinine Involving the Trinder Reaction.

The vasoprotective drug calcium dobesilate is known to interfere with creatinine (Cr) quantifications in sarcosine oxidase enzymatic (SOE) assays. The aim of this study was to investigate this interference in 8 different commercially available assays and to determine its clinical significance. In in vitro experiments, interference was evaluated at 3 Cr levels. For this, Cr was quantified by SOE assays in pooled serum supplemented with calcium dobesilate at final concentrations of 0, 2, 4, 8, 16, 32, and 64 µg/mL. Percent bias was calculated relative to the drug-free specimen. For in vivo analyses, changes in serum concentrations of Cr, cystatin C (CysC; a renal function marker), and calcium dobesilate were monitored in healthy participants of group I before and after oral calcium dobesilate administration. In addition, variations in interference were also examined among different SOE assays using serum obtained from healthy participants of group II. Lastly, Cr levels from the 10 patients treated with calcium dobesilate were measured using 4 SOE assays and liquid chromatography-isotope dilution tandem mass spectrometry (LC-IDMS/MS) for comparison. Our in vitro analyses indicated that the presence of 8 µg/mL calcium dobesilate resulted in a -4.4% to -36.3% reduction in Cr serum concentration compared to drug-free serum for 8 SOE assays examined. In vivo, Cr values decreased relative to the baseline level with increasing drug concentration, with the lowest Cr levels obtained at 2 or 3 hours after drug administration in participants of group I. The observed Cr concentrations for participants in group II were reduced by -28.5% to -3.1% and -60.5% to -11.6% at 0 and 2 hours after administration related to baseline levels. The Cr values of 10 patients measured by Roche, Beckman, Maker, and Merit Choice SOE assays showed an average deviation of -20.0%, -22.4%, -14.2%, and -29.6%, respectively, compared to values obtained by LC-IDMS/MS. These results revealed a clinically significant negative interference with calcium dobesilate in all sarcosine oxidase-based Cr assays, but the degree of interference varied greatly among the assays examined. Thus, extra care should be taken in evaluating Cr quantification obtained by SOE assays in patients undergoing calcium dobesilate therapy.

Sarcosine oxidase composite screen-printed electrode for sarcosine determination in biological samples.

As the prostate cancer (PCa) progresses, sarcosine levels increase both in tumor cells and urine samples, suggesting that this metabolite measurements can help in the creation of non-invasive diagnostic methods for this disease. In this work, a biosensor device was developed for the quantification of sarcosine via electrochemical detection of H2O2 (at 0.6V) generated from the catalyzed oxidation of sarcosine. The detection was carried out after the modification of carbon screen printed electrodes (SPEs) by immobilization of sarcosine oxidase (SOX) on the electrode surface. The strategies used herein included the activation of the carbon films by an electrochemical step and the formation of an NHS/EDAC layer to bond the enzyme to the electrode, the use of metallic or semiconductor nanoparticles layer previously or during the enzyme immobilization. In order to improve the sensor stability and selectivity a polymeric layer with extra enzyme content was further added. The proposed methodology for the detection of sarcosine allowed obtaining a limit of detection (LOD) of 16nM, using a linear concentration range between 10 and 100nM. The biosensor was successfully applied to the analysis of sarcosine in urine samples.

Implications of differences in expression of sarcosine metabolism-related proteins according to the molecular subtype of breast cancer.

BACKGROUND: The goal of this study was to investigate the expression of sarcosine metabolism-related proteins, namely glycine N-methyltransferase (GNMT), sarcosine dehydrogenase (SARDH), and l-pipecolic acid oxidase (PIPOX), in the different breast cancer subtypes and to assess the implications of differences in expression pattern according to subtype. METHODS: We analyzed the expression of GNMT, SARDH, and PIPOX in a tissue microarray of 721 breast cancer cases using immunohistochemistry (IHC). We classified breast cancer cases into subtype luminal A, luminal B, HER-2, and triple negative breast cancer (TNBC) according to the status for the estrogen receptor (ER), progesterone receptor (PR), HER-2, and Ki-67. Sarcosine metabolism phenotype was stratified according to IHC results for GNMT, SARDH, and PIPOX: GNMT(+), SARDH and PIPOX(-) was classified as high sarcosine type; GNMT(-), SARDH or PIPOX(-) as low sarcosine type; GNMT(+), SARDH or PIPOX(+) as intermediate sarcosine type, and GNMT(-), SARDH and PIPOX(-) as null type. RESULTS: Expression of sarcosine metabolism-related proteins differed significantly according to breast cancer subtype (GNMT, p=0.005; SARDH, p=0.012; tumoral PIPOX, p=0.008; stromal PIPOX, p<0.001). These proteins were the most frequently expressed in HER-2 type tumors and the least in TNBC. Sarcosine metabolism phenotype also varied according to breast cancer subtype, with high sarcosine type the most common in HER-2, and null type the most common in TNBC (p=0.003). Univariate analysis revealed that GNMT expression (p=0.042), tumoral PIPOX negativity (p=0.039), and high sarcosine type (p=0.021) were associated with shorter disease-free survival (DFS). Multivariate analysis also revealed GNMT expression was an independent factor for shorter DFS (hazard ratio: 2.408, 95% CI: 1.154-5.024, p=0.019). CONCLUSION: Expressions of sarcosine metabolism-related proteins varied according to subtype of breast cancer, with HER-2 type tumors showing elevated expression of these proteins, and TNBC subtype showing decreased expression of these proteins. Expression of sarcosine metabolism-related proteins was also associated with breast cancer prognosis.

Expression of sarcosine metabolism-related proteins according to metastatic site in breast cancer.

We aimed to evaluate the expression of sarcosine metabolism-related proteins according to site of metastatic breast cancer, and the clinical implications. Immunohistochemical staining for glycine N-methyltransferase (GNMT), sarcosine dehydrogenase (SARDH), and l-pipecolic acid oxidase (PIPOX) was performed on tissue microarrays from 162 metastatic breast cancer (bone metastases = 47, brain metastases = 39, liver metastases = 24, and lung metastases = 52). Sarcosine metabolism-related proteins showed variable expression with regard to metastatic sites. GNMT was expressed in brain and lung metastases, but not in bone and liver metastases (P = 0.016). In view of the sarcosine metabolic phenotype, high sarcosine and intermediate type were only found in the brain and lung metastases, and low sarcosine type was observed more frequently in bone and lung metastases (P = 0.047). By univariate analysis, PIPOX positivity was correlated with shorter overall survival (OS) (P = 0.031). In lung metastases, PIPOX positivity (P = 0.019) and stromal PIPOX positivity (P = 0.001) were associated with shorter OS. In conclusion, in metastatic breast cancer, sarcosine metabolism-related proteins are differently expressed according to the metastatic site. Expression of GNMT and high sarcosine type are predominantly observed in brain and lung metastases.