bims-supasi Biomed News
on Sulfation pathways and signalling
Issue of 2022‒08‒14
seventeen papers selected by
Jonathan Wolf Mueller
University of Birmingham

  1. Biomacromolecules. 2022 Aug 12.
      Sulfated glycosaminoglycans (sGAGs) such as heparan sulfate (HS) are structurally diverse linear polysaccharides that are involved in many biological processes and have gained interest as antiviral compounds. Their recognition is driven by a complex orchestra of structural parameters that are still under intense investigation. One distinct characteristic is the incorporation of sulfation patterns including highly sulfated and non-sulfated sequences that provide variations in flexibility and conformation, which in turn impact the biological function of sGAGs. However, these distinct features have not yet been fully realized in the synthetic preparation of sGAG mimetics. Here, we present the synthesis of three groups of sulfated glycomacromolecules as sGAG mimetics: (i) globally sulfated glycooligomers, (ii) glycooligomers with sequence-defined sulfation patterns, and (iii) a globally sulfated glycooligomer-oligo-L-proline hybrid structure. The complete synthesis, including chemical sulfation, was conducted on solid support, enabled by the introduction of a commercially available photocleavable linker allowing for the preservation of sensitive sulfates during cleavage of the products. Structures were obtained in good purity and with high degrees of sulfation demonstrating the wide applicability of this methodology to prepare tailor-made sulfated glycomacromolecules and similar sGAG mimetics. Structures were tested for their anticoagulant properties showing activity similar to their natural HS counterpart and significantly lower than HP.
  2. Methods Mol Biol. 2022 ;2531 163-184
      Proteoglycans are heavily glycosylated proteins, covalently linked to one or more glycosaminoglycan (GAG) chains, abundantly expressed in the extracellular matrix (ECM). Among GAGs, chondroitin sulfate (CS) and dermatan sulfate (DS) play an essential role at the ECM level; however, the composition of the hybrid CS/DS as well as the distribution of the sulfate groups along the chain were also shown to influence biological activities in brain. The elevated structural diversity of CS/DS motifs, in which sulfation may occur at GalNAc and/or IdoA/GlcA in various combinations, requires the development of specific high performance analytical methods for reliable elucidation. Due to its sensitivity, reproducibility, and efficiency, capillary zone electrophoresis (CZE) for separation of CS/DS oligosaccharides coupled to electrospray ionization mass spectrometry (ESI-MS) for their structure determination contributed an essential progress to this field.In the present chapter, two powerful methods based on CZE for separation and ESI-MS for identification and structural analysis of CS/DS are presented. The first part is devoted to offline CZE-ESI-MS based on fraction collection, screening by negative ion mode nanoESI, and fragmentation analysis in tandem MS using collision-induced dissociation (CID) at low ion acceleration energies. In the second part of the chapter, a strategy for online CZE-ESI-MS in normal polarity and negative mode ESI followed by tandem MS in real-time data-dependent acquisition mode for CS/DS separation, screening, and fragmentation is described in detail. The latter method entails the in-laboratory manufacturing of a simple yet sturdy interface for the online CZE coupling to ESI-MS and the optimization of the coupled system for total analysis of regularly sulfated and irregularly, i.e., under- and oversulfated CS/DS domains.
    Keywords:  Capillary zone electrophoresis; Chondroitin/dermatan sulfate; Electrospray ionization mass spectrometry; Glycosaminoglycans; Structural analysis
  3. Bioorg Chem. 2022 Aug 01. pii: S0045-2068(22)00476-X. [Epub ahead of print]128 106070
      Sulfation is a common modification of glycans and glycoproteins. Sulfated N-glycans have been identified in various glycoproteins and implicated for biological functions, but in vitro synthesis of structurally well-defined full length sulfated N-glycans remains to be described. We report here the first in vitro enzymatic sulfation of biantennary complex type N-glycans by recombinant human CHST2 (GlcNAc-6-O-sulfotransferase 1, GlcNAc6ST-1). We found that the sulfotransferase showed high antennary preference and could selectively sulfate the GlcNAc moiety located on the Manα1,3Man arm of the biantennary N-glycan. The glycan chain was further elongated by bacterial β1,4 galactosyltransferase from Neiserria meningitidis and human β1,4 galactosyltransferase IV(B4GALT4), which led to the formation of different sulfated N-glycans. Using rituximab as a model IgG antibody, we further demonstrated that the sulfated N-glycans could be efficiently transferred to an intact antibody by using a chemoenzymatic Fc glycan remodeling method, providing homogeneous sulfated glycoforms of antibodies. Preliminary binding analysis indicated that sulfation did not affect the apparent affinity of the antibody for FcγIIIa receptor.
    Keywords:  Chemoenzymatic synthesis; Glycoforms; Sulfated N-glycans; Sulfated antibody; Sulfation; Sulfotransferase
  4. Int J Mol Sci. 2022 Aug 08. pii: 8821. [Epub ahead of print]23(15):
      More than half of the hospital-associated infections worldwide are related to the adhesion of bacteria cells to biomedical devices and implants. To prevent these infections, it is crucial to modify biomaterial surfaces to develop the antibacterial property. In this study, chitosan (CS) and chondroitin sulfate (ChS) were chosen as antibacterial coating materials on polylactic acid (PLA) surfaces. Plasma-treated PLA surfaces were coated with CS either direct coating method or the carbodiimide coupling method. As a next step for the combined saccharide coating, CS grafted samples were immersed in ChS solution, which resulted in the polyelectrolyte complex (PEC) formation. Also in this experiment, to test the drug loading and releasing efficiency of the thin film coatings, CS grafted samples were immersed into lomefloxacin-containing ChS solution. The successful modifications were confirmed by elemental composition analysis (XPS), surface topography images (SEM), and hydrophilicity change (contact angle measurements). The carbodiimide coupling resulted in higher CS grafting on the PLA surface. The coatings with the PEC formation between CS-ChS showed improved activity against the bacteria strains than the separate coatings. Moreover, these interactions increased the lomefloxacin amount adhered to the film coatings and extended the drug release profile. Finally, the zone of inhibition test confirmed that the CS-ChS coating showed a contact killing mechanism while drug-loaded films have a dual killing mechanism, which includes contact, and release killing.
    Keywords:  antibacterial activity; biocompatibility; chitosan; chondroitin sulfate; contact killing; polyelectrolyte complex; surface functionalization
  5. Molecules. 2022 Aug 07. pii: 5026. [Epub ahead of print]27(15):
      Optosensing chitosan-based membranes have been applied for the detection of heavy metals, especially in drinking water. The novelty of this study is based on the use of sulphated polysaccharides, in such optosensing membranes, aiming at an improved analytical performance. The sulphated polysaccharides, such as ulvan, fucoidan and chondroitin sulfate, were extracted from by-products and wastes of marine-related activities. The membranes were developed for the analysis of aluminum. The variation in the visible absorbance of the sensor membranes after the contact between the chromophore and the aluminum cation was studied. The membranes containing sulphated polysaccharides showed improved signals when compared to the chitosan-only membrane. As for the detection limits for the membranes containing ulvan, fucoidan and chondroitin sulfate, 0.17 mg L-1, 0.21 mg L-1 and 0.36 mg L-1 were obtained, respectively. The values were much lower than that obtained for the chitosan-only membrane, 0.52 mg L-1, which shows the improvement obtained from the sulphated polysaccharides. The results were obtained with the presence of CTAB in analysis solution, which forms a ternary complex with the aluminum cation and the chromophore. This resulted in an hyperchromic and batochromic shift in the absorption band. When in the presence of this surfactant, the membranes showed lower detection limits and higher selectivity.
    Keywords:  CTAB; aluminum cation; biopolymers; membranes; optosensors; sulphated polysaccharides; surfactant
  6. Growth Horm IGF Res. 2022 Jul 27. pii: S1096-6374(22)00053-3. [Epub ahead of print]66 101496
      OBJECTIVE: Neurosteroids (NSs) are a distinct hormone group and, they are known for their contribution into the status of mood and cognitive functions. Whether they are also involved in the mood disturbances and cognition in acromegaly is not known. Herein we aimed to evaluate the relation of mood status and cognitive functions with the NS levels in cases with acromegaly.DESIGN: A total of 33 cases with acromegaly composed the acromegaly group (AG) and, 30 age and gender-matched cases without acromegaly composed the control group (CG). The levels of Allopregnanolone (AP), pregnenolone (PRG), 24S-hydroxycholesterol (24OHC), dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate (DHEAS), androsterone (ADT), GH and IGF-1 were measured in each group. Beck Depression Inventory (BDI) was used to assess depressive symptoms, whereas an extensive neuropsychological assessment with several neurocognitive tests were carried out for each subject by an experienced psychologist.
    RESULTS: Cases with acromegaly had lower 24OHC and DHEA levels (p = 0.002 and p = 0.007, respectively) in comparison to CG. Of the cognitive functions time to complete 1 s Series was significantly higher and, the scores on Switching Verbal Fluency Test, Boston Naming Test (BNT)-semantic and BNT-phonological, the highest learning point of Oktem Verbal Memory Processes Test (VMPT) were significantly lower in cases with acromegaly in comparison to those in controls (p = 0.004, p = 0.01, p < 0.001, p = 0.02 and p = 0.05, respectively). KAS-perseveration errors were higher in CG (p = 0.03). In AG the levels of AP were negatively correlated with the scores on Months backward Test (MBT), Animal Naming Test, Construction, BNT-spontaneous and positively correlated with BNT-incorrect answers; PRG was positively correlated with VMPT-retention scores, ADT was negatively correlated with MBT and 3 s Series scores, DHEAS was positively correlated with VMPT-the highest learning point whereas it was negatively correlated with MBT scores. Additionally, the scores on BDI were positively correlated with DHEA levels in AG.
    CONCLUSION: Cognitive changes may be encountered in acromegaly and, neurosteroids may contribute to the changes in certain cognitive functions.
    Keywords:  Acromegaly; Cognition; Depression; Neurosteroids
  7. Elife. 2022 Aug 09. pii: e73818. [Epub ahead of print]11
      Secreted molecules called morphogens govern tissue patterning in a concentration-dependent manner. However, it is still unclear how reproducible patterning can be achieved with diffusing molecules, especially when that patterning concerns differentiation of thin tissues. Wnt is a morphogen that organizes cardiac development. Wnt6 patterns cardiogenic mesoderm to induce differentiation of a thin tissue, the pericardium, in Xenopus. In this study, we revealed that a Wnt receptor, frizzled-7, is expressed in a Wnt-dependent manner. With a combination of experiments and mathematical modeling, this receptor-feedback appears essential to shape a steep gradient of Wnt signaling. In addition, computer simulation revealed that this feedback imparts robustness against variations of Wnt ligand production and allows the system to reach a steady state quickly. We also found that a Wnt antagonist sFRP1, which is expressed on the opposite side of the Wnt source, accumulates on N-acetyl-rich heparan sulfate (HS). N-acetyl-rich HS concentration is high between the sources of Wnt and sFRP1, achieving local inhibition of Wnt signaling via restriction of sFRP1 spreading. These integrated regulatory systems restrict the Wnt signaling range and ensure reproducible patterning of the thin pericardium.
    Keywords:  Wnt signal; Xenopus; developmental biology; frizzled; gene regulatory circuit; heart; morphogen; xenopus
  8. Foods. 2022 Aug 06. pii: 2360. [Epub ahead of print]11(15):
      Sulfated polysaccharides from red algae have a variety of biological activities, especially antitumor activities. Matrix metalloproteinase-9 (MMP-9) is a proteolytic metalloenzyme that degrades the central part of the extracellular matrix (ECM) and promotes tumor metastasis. In this research, we have investigated the influence and mechanism of GNP (sulfated polysaccharide from Gelidium crinale) on tumor metastasis and MMP-9 expression of human fibrosarcoma (HT1080) cells. The results inflected that the concentration of GNP below 100 μg/mL has no toxicity to HT1080 cells, but showed excellent activity in inhibiting cells migration and invasion. In addition, GNP effectively inhibits the mRNA of MMP-9 and reduces its expression and activity by regulating nuclear factor-kappa B (NF-κB), mitogen-activated protein kinases (MAPK) and mTOR/PI3K/Akt signaling pathways. GNP has great potential as MMP-9 inhibitor and could be developed as a functional food or drug to prevent tumor metastasis.
    Keywords:  Gelidium crinale; MMP-9; invasion; migration; sulfated polysaccharides
  9. Protein J. 2022 Aug 13.
      Heparan sulfate (HS) as a mediator is usually involved in both inflammation and fibrosis. Besides, pre-fibrils are the intermediates of amyloid fibrils that usually cause cell death and tissue damage, like the amyloid-β in Alzheimer's disease, α-synuclein in Parkinson disease and islet amyloid polypeptide in type II diabetes mellitus. However, the related study was involved rarely in breast. Therefore, the combing technologies including hematoxylin-eosin staining and thioflavin S staining were used to investigate the influence of HS on breast amyloidosis. To further study the toxicity of the pre-fibrils formed by β-casein on the HC11 cells and the breast mammary gland, the combing technologies including pentamer formyl thiophene acetic acid fluorescence analysis, MTT assay, Annexin V/PI staining and hematoxylin-eosin staining were performed. The results demonstrated that HS, acted as an endogenous molecule, induced the inflammation and amyloid fibril formation at the same time, and there was a close relationship between inflammation and fibrosis of breast. In addition, the pre-fibrils formed by β-casein were toxic because they induced the death and apoptosis of HC11 cells, as well as the inflammation of mammary gland of rats. Therefore, the early examination and identify of the pre-fibrils in the breast were worth considering to prevent the disease development, and it was interesting to explore the HS mimetics to impair the breast amyloidosis and attenuate the inflammatory response in the future.
    Keywords:  fibrosis; heparan sulfate; inflammation; pre-fibrils; toxicity; β-casein
  10. Curr Res Struct Biol. 2022 ;4 246-255
      The temporary or permanent chemical modification of biomolecules is a crucial aspect in the physiology of all living species. However, while some modules are well characterised also in insects, others did not receive the same attention. This holds true for sulfo-conjugation that is catalysed by cytosolic sulfotransferases (SULT), a central component of the metabolism of endogenous low molecular weight molecules and xenobiotics. In particular, limited information is available about the functional roles of the mosquito predicted enzymes annotated as SULTs in genomic databases. The herein described research is the first example of a biochemical and structural study of a SULT of a mosquito species, in general, and of the malaria vector Anopheles gambiae in particular. We confirmed that the AGAP001425 transcript displays a peculiar expression pattern that is suggestive of a possible involvement in modulating the mosquito reproductive tissues physiology, a fact that could raise attention on the enzyme as a potential target for insect-containment strategies. The crystal structures of the enzyme in alternative ligand-bound states revealed elements distinguishing AgSULT-001425 from other characterized SULTs, including a peculiar conformational plasticity of a discrete region that shields the catalytic cleft and that could play a main role in the dynamics of the reaction and in the substrate selectivity of the enzyme. Along with further in vitro biochemical studies, our structural investigations could provide a framework for the discovery of small-molecule inhibitors to assess the effect of interfering with AgSULT-001425-mediated catalysis at the organismal level.
    Keywords:  Crystal structure; Cytosolic sulfotransferase; Differential mRNA expression; Mosquito; Reproductive system; SULT
  11. Int J Mol Sci. 2022 Aug 03. pii: 8641. [Epub ahead of print]23(15):
      Increasing evidence has revealed that plant secretory peptides are involved in the long-distance signaling pathways that help to regulate plant development and signal stress responses. In this study, we purified small peptides from soybean (Glycine max) xylem sap via o-chlorophenol extraction and conducted an in-depth peptidomic analysis using a mass spectrometry (MS) and bioinformatics approach. We successfully identified 14 post-translationally modified peptide groups belonging to the peptide families CEP (C-terminally encoded peptides), CLE (CLAVATA3/embryo surrounding region-related), PSY (plant peptides containing tyrosine sulfation), and XAP (xylem sap-associated peptides). Quantitative PCR (qPCR) analysis showed unique tissue expression patterns among the peptide-encoding genes. Further qPCR analysis of some of the peptide-encoding genes showed differential stress-response profiles toward various abiotic stress factors. Targeted MS-based quantification of the nitrogen deficiency-responsive peptides, GmXAP6a and GmCEP-XSP1, demonstrated upregulation of peptide translocation in xylem sap under nitrogen-deficiency stress. Quantitative proteomic analysis of GmCEP-XSP1 overexpression in hairy soybean roots revealed that GmCEP-XSP1 significantly impacts stress response-related proteins. This study provides new insights that root-to-shoot peptide signaling plays important roles in regulating plant stress-response mechanisms.
    Keywords:  C-terminally encoded peptide; CLV3/ESR-related; long-distance; peptide signaling; peptidomic; post-translational modification; root-to-shoot; sulfated peptide; xylem sap
  12. Food Res Int. 2022 Sep;pii: S0963-9969(22)00673-1. [Epub ahead of print]159 111615
      Our previous study found dietary mannogluconic acid (MA) and fucogalactan sulfate (FS) from Laminaria japonica have distinct structure characterization and potential hypolipidemic effects in vitro. Herein, we compared the benefits of MA and FS on hyperlipidemia. The result showed only FS treatment decreased body weight and serum cholesterol levels. Compared with MA, FS was more effective in mitigating hepatic fat accumulation, promoting GSH-Px activity, reducing the MDA formation, and lowering the level of TNF-α in liver. Gut microbiota and metabolism analysis revealed that FS increased the relative abundance of beneficial bacteria and boosted the level of short chain fatty acids. Particularly, taurine and 3α,7α,12α-trihydroxy-24-oxo-5-β-cholestanoyl CoA were upregulated by FS, which might attribute to the increased Oscillibacter and thus affect the enterohepatic circulation of bile acids and serum TC level. Therefore, FS with more branches and sulfate ester groups could be a good lipid-lowering dietary supplement.
    Keywords:  Fucogalactan sulfate (FS); Gut microbiota; Hyperlipidemia; Mannogluconic acid (MA); Metabolome
  13. Toxicol Sci. 2022 Aug 09. pii: kfac082. [Epub ahead of print]
      Larval zebrafish is emerging as a new model organism for studying drug-induced liver injury (DILI) with superiorities in visual assessment, genetic engineering as well as high throughput. Metabolic bioactivation to form reactive intermediates is a common event that triggers DILI. This study first addressed the correlation between acetaminophen metabolism and hepatotoxicity in zebrafish larvae (3 days post-fertilization) and demonstrated the occurrence of cytochrome P450 enzymes-mediated APAP bioactivation at early developmental stage through characterizing the dose-effect (0-1.6 mg/mL) and the time-course (0-72 h) of liver injury and metabolism in the AB strain and LiPan transgenic line Tg(lfabp10a: DsRed; elaA: egfp) expressing liver-specific fluorescent protein. APAP caused multi-organ developmental retardation and elicited dose- and time-dependent hepatotoxicity. Liver imaging revealed significant changes earlier than histological and biochemical measurements. APAP bioactivation in larval zebrafish was first confirmed by the detection of the glutathione conjugate of the reactive intermediate NAPQI (NAPQI-GSH) and subsequent mercapturate derivatives NAPQI-cysteine and NAPQI-N-acetylcysteine after even short (0.5-hour post exposure) or low (0.2 mg/mL) APAP exposure. APAP overdose impaired metabolic function, in particular sulfation, while facilitated GSH depletion and APAP sulfate excretion. Meanwhile, APAP displayed triphasic accumulation in the larvae, agreeing with fluctuating metabolic capabilities with sulfation dominating the early larval developmental stage. Most importantly, the dose-response effects and time-course of APAP accumulation and metabolism agree well with those of the liver injury development. Overall, larval zebrafish has developed mammalian-like metabolic function, enabling it an ideal model organism for high throughput screening hepatotoxicity and mechanistic study of bioactivation-based DILI.
    Keywords:  acetaminophen; bioactivation; drug-induced liver injury; liver imaging; metabolic capacity; zebrafish model
  14. Arch Microbiol. 2022 Aug 11. 204(9): 551
      The enzymes are biological macromolecules that biocatalyze certain biochemical reactions without undergoing any modification or degradation at the end of the reaction. In this work, we constructed a recombinant novel Raoultella sp. NX-TZ-3-15 strain that produces heparinase with a maltose binding tag to enhance its production and activity. Additionally, MBP-heparinase was purified and its enzymatic capabilities are investigated to determine its industrial application. Moreover, the recombinant plasmid encoding the MBP-heparinase fusion protein was effectively generated and purified to a high purity. According to SDS-PAGE analysis, the MBP-heparinase has a molecular weight of around 70 kDa and the majority of it being soluble with a maximum activity of 5386 U/L. It has also been noted that the three ions of Ca2 + , Co2 + , and Mg2 + can have an effect on heparinase activities, with Mg2 + being the most noticeable, increasing by about 85%, while Cu2 + , Fe2 + , Zn2 + having an inhibitory effect on heparinase activities. Further investigations on the mechanistic action, structural features, and genomes of Raoultella sp. NX-TZ-3-15 heparinase synthesis are required for industrial-scale manufacturing.
    Keywords:  Heparinase; MBP tag; Raoultella sp. NX-TZ-3-15; Ultra-low-weight heparin
  15. mSphere. 2022 Aug 09. e0029922
      Entamoeba histolytica, a protozoan parasite, causes amoebiasis, which is a global public health problem. The major route of infection is oral ingestion of cysts, the only form that is able to transmit to a new host. Cysts are produced by cell differentiation from proliferative trophozoites in a process termed "encystation." During encystation, cell morphology is markedly changed; motile amoeboid cells become rounded, nonmotile cells. Concomitantly, cell components change and significant fluctuations of metabolites occur. Cholesteryl sulfate (CS) is a crucial metabolite for encystation. However, its precise role remains uncertain. To address this issue, we used in vitro culture of Entamoeba invadens as the model system for the E. histolytica encystation study and identified serum-free culture conditions with CS supplementation at concentrations similar to intracellular CS concentrations during natural encystation. Using this culture system, we show that CS exerts pleiotropic effects during Entamoeba encystation, affecting cell rounding and development of membrane impermeability. CS dose dependently induced and maintained encysting cells as spherical maturing cysts with almost no phagocytosis activity. Consequently, the percentage of mature cysts was increased. CS treatment also caused time- and dose-dependent development of membrane impermeability in encysting cells via induction of de novo synthesis of dihydroceramides containing very long N-acyl chains (≥26 carbons). These results indicate that CS-mediated morphological and physiological changes are necessary for the formation of mature cysts and the maintenance of the Entamoeba life cycle. Our findings also reveal important morphological aspects of the process of dormancy and the control of membrane structure. IMPORTANCE Entamoeba histolytica causes a parasitic infectious disease, amoebiasis. Amoebiasis is a global public health problem with a high occurrence of infection and inadequate clinical options. The parasite alternates its form between a proliferative trophozoite and a dormant cyst that enables the parasite to adapt to new environments. The transition stage in which trophozoites differentiate into cysts is termed "encystation." Cholesteryl sulfate is essential for encystation; however, its precise role remains to be determined. Here, we show that cholesteryl sulfate is a multifunctional metabolite exerting pleiotropic roles during Entamoeba encystation, including the rounding of cells and the development of membrane impermeability. Such morphological and physiological changes are required for Entamoeba to produce cysts that are transmissible to a new host, which is essential for maintenance of the Entamoeba life cycle. Our findings are therefore relevant not only to Entamoeba biology but also to general cell and lipid biology.
    Keywords:  amoebiasis; dormancy; infectious disease; lipid metabolism; membrane property control; morphogenesis; parasitology
  16. Phytochemistry. 2022 Aug 06. pii: S0031-9422(22)00279-5. [Epub ahead of print] 113363
      Tumor protein or cellular tumor antigen p53, is considered a critical transcriptional regulation factor, which can suppress the growth of tumor cells by activating other functional genes. The current study appraised the p53 activation pathways, which could be used as an alternative therapeutic strategy for the treatment of hepatocellular and ductal carcinoma. Algal polysaccharides have been used as emerging sources of bioactive natural pharmacophores. A sulfated galactofucan characterized as [→1)-O-4-sulfonato-α-fucopyranose-(3 → 1)-α-fucopyranose-(3→] as the main branch with [→1)-6-O-acetyl-β-galactopyranose-(4→] as side chain isolated from marine macroalga Turbinaria ornata exhibited prospective apoptosis on HepG2 (hepatocellular carcinoma) and MCF7 (ductal carcinoma) cells. Annexin V-fluorescein isothiocyanate-propidium iodide study displayed higher early apoptosis in MCF7 and HepG2 cell lines (56 and 24.2%, respectively) treated with TOP-3 (at IC50 concentration) than those administered with standard camptothecin. Upregulation of the p53 gene expression was perceived in TOP-3 treated HepG2 and MCF7 cells.
    Keywords:  Apoptotic effect; Sargassaceae; Sulfated galactofucan; Turbinaria ornata; p53 gene expression
  17. J Anal Toxicol. 2022 Aug 12. pii: bkac058. [Epub ahead of print]
      Although kombucha is a popular fermented beverage, the presence of alcohol markers has not been well studied despite being potential indicators of unintentional impairment. Ethyl glucuronide (EtG) and ethyl sulfate (EtS) were measured in oral fluid and urine collected after consumption of regular or hard kombucha. Participants drank within 20 min and provided all urine voids for 12 h, the first urine voids on days 2 and 3, and oral fluid specimens at fixed time points for 48 h. Screening employed liquid chromatography-tandem mass spectrometry (LC-MS-MS; EtS, 25 ng/mL cutoff [oral]; 100 ng/mL cutoff [urine]; EtG, 500 ng/mL cutoff [urine] and immunoassay (IA; EtG, 500 ng/mL cutoff[urine]). After consuming regular kombucha (n = 12 participants), EtS was not detected in oral fluid but both markers were detected by LC-MS-MS in urine specimens within the first 5 voids from 83% of participants with median (range) concentrations of 240 (100-3,700) ng/mL for EtS and 830 (530-2,200) ng/mL for EtG. Neither marker was positive by IA nor LC-MS-MS after day 1. After consuming hard kombucha (n = 7 participants), 2 (2.8%) of the 70 collected oral fluid specimens tested positive for EtS 3 h after consumption; however, 21 (30%) had EtS levels above the limit of detection (LOD, 10 ng/mL) after 0.5-8 h. Both markers were detected in urine specimens from all participants with median (range) concentrations of 3,381 (559-70,250) ng/mL for EtS and 763 (104-12,864) ng/mL For EtG. Urine specimens were negative for EtG and EtS by the end of the 48-hour study.
    Keywords:  Kombucha; LC–MS-MS; alcohol markers; ethyl glucuronide; ethyl sulfate