Development of a fluorescence and quencher-based FRET assay for detection of endogenous peptide:N-glycanase/NGLY1 activity.

Cytosolic peptide:N-glycanase (PNGase/NGLY1 in mammals) catalyzes deglycosylation of N-glycans on glycoproteins. A genetic disorder caused by mutations in the NGLY1 gene leads to NGLY1 deficiency with symptoms including motor deficits and neurological problems. Effective therapies have not been established, though, a recent study used the administration of an adeno-associated viral vector expressing human NGLY1 to dramatically rescue motor functions in young Ngly1-/- rats. Thus, early therapeutic intervention may improve symptoms arising from central nervous system dysfunction, and assay methods for measuring NGLY1 activity in biological samples are critical for early diagnostics. In this study, we established an assay system for plate-based detection of endogenous NGLY1 activity using a FRET-based probe. Using this method, we revealed significant changes in NGLY1 activity in rat brains during aging. This novel assay offers reliable disease diagnostics and provides valuable insights into the regulation of PNGase/NGLY1 activity in diverse organisms under different stress conditions.

Rat hepatocytes secrete free oligosaccharides.

We recently established a method for the isolation of serum-free oligosaccharides, and characterized various features of their structures. However, the precise mechanism for how these glycans are formed still remains unclarified. To further investigate the mechanism responsible for these serum glycans, here, we utilized rat primary hepatocytes to examine whether they are able to secrete free glycans. Our findings indicated that a diverse array of free oligosaccharides such as sialyl/neutral free N-glycans (FNGs), as well as sialyl lactose/LacNAc-type glycans, were secreted into the culture medium by primary hepatocytes. The structural features of these free glycans in the medium were similar to those isolated from the sera of the same rat. Further evidence suggested that an oligosaccharyltransferase is involved in the release of the serum-free N-glycans. Our results indicate that the liver is indeed secreting various types of free glycans directly into the serum.

ELISA-based highly sensitive assay system for the detection of endogenous NGLY1 activity.

Cytosolic peptide:N-glycanase (NGLY1, PNGase) is an enzyme that cleaves N-glycans from misfolded glycoproteins. In 2012, a human genetic disorder, NGLY1 deficiency, was first reported to be caused by mutations of the NGLY1 gene. Since then, there has been rapid progresses on NGLY1 biology, and gene therapy has been proposed as a promising therapeutic option for NGLY1 deficiency. While a plasma/urine biomarker has also been developed for this disease, detection of NGLY1 activity could be another viable option for early diagnosis of NGLY1 deficiency. Thus far, several in vitro and in cellulo NGLY1 assays have been reported, but those assay systems have several issues that must be addressed in order to develop an assay system compatible for routine clinical examination. Here, we show a facile, highly sensitive in vitro assay system that could be used to detect NGLY1 activity by utilizing its sequence editing function, i.e. conversion of glycosylated Asn into Asp, followed by a detection of newly generated epitope (HA)-tag by anti-HA antibody. Using this ELISA-based assay, we detected endogenous NGLY1 activity in as little as 2 µg of crude extract, which is the equivalent of 5 × 103 cells. Our system also detects NGLY1 activity from cells with compromised NGLY1 activity, such as iPS cells from patient samples. This assay system could be applied in future clinical examinations to achieve an early diagnosis of NGLY1 deficiency.

Conformational preference of 2-(4-methoxyphenyl)ethanol studied by supersonic jet spectroscopy: Intramolecular OH/π interaction.

A π-type hydrogen bonding between the OH group and the π electron is a crucial factor for the conformational preference of the molecular structure with a flexible group. However, the information on the effect of the substituent on the OH/π interaction is insufficient. The laser-induced fluorescence (LIF) excitation, the dispersed fluorescence (DF), the IR-UV hole-burning, and the IR dip spectra of jet-cooled 2-(4-methoxyphenyl)ethanol were measured for the first time. Almost all bands observed in the spectral region of 35 550-36 500 cm-1 in the LIF excitation spectrum were successfully assigned with the DF and the IR-UV hole-burning spectra coupled with the quantum chemical calculation at M06-2x/6-311G and MP2/6-311G levels. Five conformers were found in the LIF excitation spectrum. The most stable conformer was Ggπ, and the second most stable conformer was Ggπ' (the trans rotamer of the methoxy group for Ggπ). Ggπ and Ggπ' had the OH group directed toward the π electron system of the benzene ring. The OH stretching frequency of Ggπ/Ggπ' of MPE in the IR dip spectra was red-shifted against that of Ggπ of phenylethanol, indicating that the introduction of the methoxy group would enhance the intramolecular OH/π interaction. In addition, the torsional vibration between the benzene ring and the side chain (-CH2CH2OH) (mode 63) was observed in the DF spectra of the Ggπ-00 and Ggπ'-00 band excitation, but their intensities were rather different, resulting from the different orientation of the OH group for each conformer toward the π electron system. The methoxy group would increase the negative charge on the benzene ring and would enhance the intramolecular OH/π interaction through the electrostatic interaction.

Loss of peptide:N-glycanase causes proteasome dysfunction mediated by a sugar-recognizing ubiquitin ligase.

Mutations in the human peptide:N-glycanase gene (NGLY1), which encodes a cytosolic de-N-glycosylating enzyme, cause a congenital autosomal recessive disorder. In rodents, the loss of Ngly1 results in severe developmental delay or lethality, but the underlying mechanism remains unknown. In this study, we found that deletion of Fbxo6 (also known as Fbs2), which encodes a ubiquitin ligase subunit that recognizes glycoproteins, rescued the lethality-related defects in Ngly1-KO mice. In NGLY1-KO cells, FBS2 overexpression resulted in the substantial inhibition of proteasome activity, causing cytotoxicity. Nuclear factor, erythroid 2-like 1 (NFE2L1, also known as NRF1), an endoplasmic reticulum-associated transcriptional factor involved in expression of proteasome subunits, was also abnormally ubiquitinated by SCFFBS2 in NGLY1-KO cells, resulting in its retention in the cytosol. However, the cytotoxicity caused by FBS2 was restored by the overexpression of "glycan-less" NRF1 mutants, regardless of their transcriptional activity, or by the deletion of NRF1 in NGLY1-KO cells. We conclude that the proteasome dysfunction caused by the accumulation of N-glycoproteins, primarily NRF1, ubiquitinated by SCFFBS2 accounts for the pathogenesis resulting from NGLY1 deficiency.

Synthesis of azafluoranthenes by iridium-catalyzed [2 + 2 + 2] cycloaddition and evaluation of their fluorescence properties.

We report a method for the synthesis of azafluoranthenes under neutral reaction conditions in a highly atom-economical manner by the iridium-catalyzed [2 + 2 + 2] cycloaddition of 1,8-dialkynylnaphthalenes with nitriles. A variety of nitriles react with methyl- or phenyl-substituted 1,8-dialkynylnaphthalenes to give a wide range of azafluoranthenes. Azafluoranthenes bearing an amino group show intense fluorescence at around 500 nm. Comparison of the fluorescence properties of amine-substituted azafluoranthenes with related compounds revealed the importance of the amine moiety for obtaining a high fluorescence quantum yield. The choice of the solvent affected the emission maxima and the fluorescence quantum yield. Azafluoranthenes bearing pyrrolidine exhibited blue-shifted emission bands in a non-polar solvent and gave a fluorescence quantum yield of 0.76 in toluene. A Lippert-Mataga plot and computational studies provide insight into the origin of the fluorescence of azafluoranthenes. Furthermore, cellular experiments using human breast adenocarcinoma cells SK-BR-3 demonstrated the feasibility of using azafluoranthenes as fluorescent probes.

Effects of Two Electron-Donating and/or -Withdrawing Substituents on Two-Photon Absorption for Diphenylacetylene Derivatives.

Two-photon absorption for diphenylacetylene (DPA) derivatives with two substituents (-OMe and/or -NO2) at the 4,4'-position was investigated experimentally and theoretically. The two-photon absorption spectra and the two-photon absorption cross-sections σ(2) for DPA derivatives were obtained by optical-probing photoacoustic spectroscopy (OPPAS). The simulated two-photon absorption spectra of the DPA derivatives, obtained with the time-dependent density functional theory within the Tamm-Dancoff approximation, agreed well with the experimental ones. The mechanisms for enhancement of the σ(2) for centrosymmetric and non-centrosymmetric DPA derivatives were found to be different. The large σ(2) for centrosymmetric molecules (DPA-OMeOMe and DPA-NO2NO2) results from the magnitude of the transition dipole moment, while for non-centrosymmetric molecules (DPA-OMeNO2), it is enhanced by the smaller detuning energy. Information on two-photon absorption properties of DPA derivatives obtained in this study will be important for the molecular design of two-photon absorption materials.

A method for assaying peptide: N-glycanase/N-glycanase 1 activities in crude extracts using an N-glycosylated cyclopeptide.

Cytosolic peptide: N-glycanase (PNGase; NGLY1), an enzyme responsible for de-glycosylation of N-glycans on glycoproteins, is known to play pivotal roles in a variety of biological processes. In 2012, NGLY1 deficiency, a rare genetic disorder, was reported and since then, more than 100 patients have now been identified worldwide. Patients with this disease exhibit several common symptoms that are caused by the dysfunction of NGLY1. However, correlation between the severity of patient symptoms and the extent of the reduction in NGLY1 activity in these patients remains to be clarified, mainly due to the absence of a facile quantitative assay system for this enzyme, especially in a crude extract as an enzyme source. In this study, a quantitative, non-radioisotope (RI)-based assay method for measuring recombinant NGLY1 activity was established using a BODIPY-labeled asialoglycopeptide (BODIPY-ASGP) derived from hen eggs. With this assay, the activities of 27 recombinant NGLY1 mutants that are associated with the deficiency were examined. It was found that the activities of three (R469X, R458fs and H494fs) out of the 27 recombinant mutant proteins were 30-70% of the activities of wild-type NGLY1. We further developed a method for measuring endogenous NGLY1 activity in crude extracts derived from cultured cells, patients' fibroblasts, iPS cells or peripheral blood mononuclear cells (PBMCs), using a glycosylated cyclopeptide (GCP) that exhibited resistance to the endogenous proteases in the extract. Our methods will not only provide new insights into the molecular mechanism responsible for this disease but also promises to be applicable for its diagnosis.

Occurrence of free N-glycans with a single GlcNAc at the reducing termini in animal sera.

Recent studies demonstrated the occurrence of sialyl free N-glycans (FNGs) in sera from a variety of animals. Unlike the intracellular FNGs that mainly carry a single N-acetylglucosamine at their reducing termini (Gn1-type), these extracellular FNGs have an N,N'-diacetylchitobiose at their reducing termini (Gn2-type). The detailed mechanism for how they are formed, however, remains unclarified. In this study, we report on an improved method for isolating FNGs from sera and found that, not only sialyl FNGs, but also neutral FNGs are present in animal sera. Most of the neutral oligomannose-type FNGs were found to be Gn1-type. We also found that a small portion of sialyl FNGs were Gn1-type. The ratio of Gn1-type sialyl FNGs varies between species, and appears to be partially correlated with the distribution of lysosomal chitobiase activity. We also identified small sialylated glycans similar to milk oligosaccharides, such as sialyl lactose or sialyl N-acetyllactosamine in sera. Our results indicate that there are varieties of free oligosaccharides in sera and the mechanism responsible for their formation is more complicated than currently envisaged.

Ngly1 -/- rats develop neurodegenerative phenotypes and pathological abnormalities in their peripheral and central nervous systems.

N-glycanase 1 (NGLY1) deficiency, an autosomal recessive disease caused by mutations in the NGLY1 gene, is characterized by developmental delay, hypolacrima or alacrima, seizure, intellectual disability, movement disorders and other neurological phenotypes. Because of few animal models that recapitulate these clinical signatures, the mechanisms of the onset of the disease and its progression are poorly understood, and the development of therapies is hindered. In this study, we generated the systemic Ngly1-deficient rodent model, Ngly1-/- rats, which showed developmental delay, movement disorder, somatosensory impairment and scoliosis. These phenotypes in Ngly1-/- rats are consistent with symptoms in human patients. In accordance with the pivotal role played by NGLY1 in endoplasmic reticulum-associated degradation processes, cleaving N-glycans from misfolded glycoproteins in the cytosol before they can be degraded by the proteasome, loss of Ngly1 led to accumulation of cytoplasmic ubiquitinated proteins, a marker of misfolded proteins in the neurons of the central nervous system of Ngly1-/- rats. Histological analysis identified prominent pathological abnormalities, including necrotic lesions, mineralization, intra- and extracellular eosinophilic bodies, astrogliosis, microgliosis and significant loss of mature neurons in the thalamic lateral and the medial parts of the ventral posterior nucleus and ventral lateral nucleus of Ngly1-/- rats. Axonal degradation in the sciatic nerves was also observed, as in human subjects. Ngly1-/- rats, which mimic the symptoms of human patients, will be a useful animal model for preclinical testing of therapeutic options and understanding the detailed mechanisms of NGLY1 deficiency.

TMEM2 expression is downregulated as bladder cancer invades the muscle layer.

Cell surface hyaluronidase transmembrane protein 2 (TMEM2), which also serves as a reportedly functions in malignancy of several solid tumors. However, TMEM2 involvement in bladder cancer (BCa) is unknown. Therefore, we investigate potential changes in expression of TMEM2 during BCa invasion and over the course of the epithelial mesenchymal transition (EMT). Immunohistochemical analysis of 127 clinical specimens revealed that TMEM2 expression changed with pathological stage (pT) and infiltration pattern (INF) and was highest in pTa-pT1 of INFa tumors and significantly lower at stages from pTa-pT1 to pT2 or 3 in INFb or INFc. E-cadherin expression was highest in INFa and lowest in INFc, a pattern comparable to TMEM2 expression. TMEM2 protein expression analysis of BCa cell lines showed that muscle-invasive T24 and YTS-1 cells with low TMEM2 expression exhibited EMT phenotypes in vitro, in contrast to high TMEM2-expressing non-muscle invasive RT4 cells. EMT-induced non-muscle invasive RT4 cells also showed significantly decreased plasma membrane expression of TMEM2. Our data suggested TMEM2 expression is higher in non-invasive cancers, whereas invasive cancer cells are less likely to express TMEM2 during muscle-invasion and "partial EMT".

The ER-associated protease Ste24 prevents N-terminal signal peptide-independent translocation into the endoplasmic reticulum in Saccharomyces cerevisiae.

Soluble proteins destined for the secretory pathway contain an N-terminal signal peptide that induces their translocation into the endoplasmic reticulum (ER). The importance of N-terminal signal peptides for ER translocation has been extensively examined over the past few decades. However, in the budding yeast Saccharomyces cerevisiae, a few proteins devoid of a signal peptide are still translocated into the ER and then N-glycosyl-ated. Using signal peptide-truncated reporter proteins, here we report the detection of significant translocation of N-terminal signal peptide-truncated proteins in a yeast mutant strain (ste24Δ) that lacks the endopeptidase Ste24 at the ER membrane. Furthermore, several ER/cytosolic proteins, including Sec61, Sec66, and Sec72, were identified as being involved in the translocation process. On the basis of screening for 20 soluble proteins that may be N-glycosylated in the ER in the ste24Δ strain, we identified the transcription factor Rme1 as a protein that is partially N-glycosylated despite the lack of a signal peptide. These results clearly indicate that some proteins lacking a signal peptide can be translocated into the ER and that Ste24 typically suppresses this process.

A Substituent Effect on Two-Photon Absorption of Diphenylacetylene Derivatives with an Electron-Donating/Withdrawing Group.

Two-photon absorption for diphenylacetylene derivatives with an electron-donating (ED) or electron-withdrawing (EW) group (DPA-Rs) was investigated by high-sensitivity optical-probing photoacoustic spectroscopy. Two-photon absorption spectra and two-photon absorption cross sections σ(2) for DPA-Rs were successfully obtained. Two-photon absorption spectra of DPA-Rs with stronger ED or EW groups display more significant red-shifts and larger σ(2) values. Simulated two-photon absorption spectra, using time-dependent density functional theory within the Tamm-Dancoff approximation, compared well with the experimental spectra. Based on the three-state model, the substituent effect on the two-photon absorption for DPA-Rs was expected to manifest in the transition dipole moments and detuning energies. Information obtained from investigating the monosubstituent effect on two-photon absorption of DPA is critical for an improved understanding of two-photon absorption.

Excited States of Thio-2'-deoxyuridine Bearing an Extended π-Conjugated System: 3',5'-Di-O-acetyl-5-phenylethynyl-4-thio-2'-deoxyuridine.

A new thio-2'-deoxyuridine with an extended π-conjugated group was successfully synthesized: 3',5'-di-O-acetyl-5-phenylethynyl-4-thio-2'-deoxyuridine. The thio-2'-deoxyuridine derivative has a large red-shifted absorption band in the UVA region and also shows fluorescence, a rare photo-property among thionucleobases/thionucleosides. The triplet-triplet absorption spectrum and the rate constants (the intrinsic decay rate constant of the triplet state, the self-quenching rate constant, and the quenching rate constant of the triplet state by an oxygen molecule) of the thio-2'-deoxyuridine were obtained by transient absorption spectroscopy. The quantum yield of intersystem crossing and the quantum yield of singlet molecular oxygen formation (ϕΔ) under an oxygen atmosphere were also determined. The Ï•Δ value of the new thio-2'-deoxyuridine was found to be substantially higher than all reported values of other thio-2'-deoxyribonucleosides in low oxygen concentrations similar to cancer cell environments. The fluorescence quantum yield depended on the excitation wavelength, revealing certain photochemical reactions in the higher excited singlet states. However, when excited into the higher excited state with non-resonant two-photon absorption, the Ï•Δ of the thio-2'-deoxyuridine derivative was found to remain sufficiently large. These findings should be very useful for the development of thio-2'-deoxyribonucleoside-based pharmaceuticals as DNA-specific photosensitizers for photochemotherapy.

JF1/B6F1 Ngly1-/- mouse as an isogenic animal model of NGLY1 deficiency.

N-Glycanase 1 (NGLY1) deficiency is a congenital disorder caused by mutations in the NGLY1 gene. Because systemic Ngly1-/- mice with a C57BL/6 (B6) background are embryonically lethal, studies on the mechanism of NGLY1 deficiency using mice have been problematic. In this study, B6-Ngly1-/+ mice were crossed with Japanese wild mice-originated Japanese fancy mouse 1 (JF1) mice to produce viable F2 Ngly1-/- mice from (JF1×B6)F1 Ngly1-/+ mice. Systemic Ngly1-/- mice with a JF1 mouse background were also embryonically lethal. Hybrid F1 Ngly1-/- (JF1/B6F1) mice, however, showed developmental delay and motor dysfunction, similar to that in human patients. JF1/B6F1 Ngly1-/- mice showed increased levels of plasma and urinary aspartylglycosamine, a potential biomarker for NGLY1 deficiency. JF1/B6F1 Ngly1-/- mice are a useful isogenic animal model for the preclinical testing of therapeutic options and understanding the precise pathogenic mechanisms responsible for NGLY1 deficiency.

Free glycans derived from O-mannosylated glycoproteins suggest the presence of an O-glycoprotein degradation pathway in yeast.

In eukaryotic cells, unconjugated oligosaccharides that are structurally related to N-glycans (i.e. free N-glycans) are generated either from misfolded N-glycoproteins destined for the endoplasmic reticulum-associated degradation or from lipid-linked oligosaccharides, donor substrates for N-glycosylation of proteins. The mechanism responsible for the generation of free N-glycans is now well-understood, but the issue of whether other types of free glycans are present remains unclear. Here, we report on the accumulation of free, O-mannosylated glycans in budding yeast that were cultured in medium containing mannose as the carbon source. A structural analysis of these glycans revealed that their structures are identical to those of O-mannosyl glycans that are attached to glycoproteins. Deletion of the cyc8 gene, which encodes for a general transcription repressor, resulted in the accumulation of excessive amounts of free O-glycans, concomitant with a severe growth defect, a reduction in the level of an O-mannosylated protein, and compromised cell wall integrity. Our findings provide evidence in support of a regulated pathway for the degradation of O-glycoproteins in yeast and offer critical insights into the catabolic mechanisms that control the fate of O-glycosylated proteins.

Cytosolic N-GlcNAc proteins are formed by the action of endo-ß-N-acetylglucosaminidase.

NGLY1 is a widely conserved eukaryotic cytosolic deglycosylating enzyme involved in the endoplasmic reticulum-associated degradation (ERAD) process, which eliminates misfolded proteins through retrograde translocation and proteasomal degradation. A human genetic disorder called NGLY1-deficiency has been reported, indicating the functional importance of NGLY1 in humans. Evidence suggests that Ngly1-KO is embryonic lethal in mice, while additional deletion of the Engase gene, encoding another cytosolic deglycosylating enzyme (endo-ß-N-acetylglucosaminidase; ENGase), partially rescued lethality. Upon compromised Ngly1 activity, ENGase-mediated deglycosylation of misfolded glycoproteins may cause excess formation of N-GlcNAc proteins in the cytosol, leading to detrimental effects in the mice. Whether endogenous N-GlcNAc proteins are really formed in Ngly1-KO cells/animals or not remains unclarified. Here, comprehensive identification of O- and N-GlcNAc proteins was carried out using purified cytosol from wild type, Ngly1-KO, Engase-KO, and Ngly1/Engase double KO mouse embryonic fibroblasts. It was revealed that while there is no dramatic change in the level of O-GlcNAc proteins among cells examined, there was a vast increase of N-GlcNAc proteins in Ngly1-KO cells upon proteasome inhibition. Importantly, few N-GlcNAc proteins were observed in Engase-KO or Ngly1/Engase double-KO cells, clearly indicating that the cytosolic ENGase is responsible for the formation of N-GlcNAc proteins. The excess formation of N-GlcNAc proteins may at least in part account for the pathogenesis of NGLY1-deficiency.

Functional glycomics: Application to medical science and hepatology.

Glycomics refers to the comprehensive analysis of glycans. Recent progress in glycotechnology enables the determination of a variety of biological functions of glycans. Among different glycosylation patterns, certain types of aberrant glycosylation are linked to cancer and/or inflammation, and thus have biological importance. Glycotechnology has been applied to many fields of medical science, including hepatology. In particular, dramatic changes in glycosylation are observed in the progression of liver diseases. As the liver produces so many serum glycoproteins, changes in glycosylation of these proteins might provide useful disease biomarkers. Furthermore, many patients with genetic diseases of glycosylation who have liver dysfunction have been found as a result from whole genome sequencing, and various kinds of glycotherapy have been developed, especially in immunotherapy. In this review, we describe our basic knowledge of glycobiology and discuss the application of these data to medical science, especially hepatology.

Simultaneous Two-Photon Absorption of the Thioguanosine Analogue 2',3',5'-Tri-O-acetyl-6,8-dithioguanosine with Its Potential Application to Photodynamic Therapy.

2',3',5'-Tri-O-acetyl-6,8-dithioguanosine (taDTGuo) is an analogue of nucleosides and currently under investigation as a potential agent for photodynamic therapy (PDT). Excitation by simultaneous two-photon absorption of visible or near-infrared light would provide an efficient PDT for deep-seated tumors. The two-photon absorption spectrum of taDTGuo was obtained by optical-probing photoacoustic spectroscopy (OPPAS). A two-photon absorption band corresponding to the S5 ← S0 transition was observed at 556 nm, and the two-photon absorption cross-section σ(2) was determined to be 26 ± 3 GM, which was much larger than that of other nucleobases and nucleosides. Quantum chemical calculations suggested that the large σ(2) value of taDTGuo was responsible for large transition dipole moments and small detuning energy resulting from the thiocarbonyl group at 6- and 8-positions. This is the first report on two-photon absorption spectra and cross-sections of thionucleoside analogues, which could be used to develop a more specific PDT for cancers in deep regions.

Pre-dialysis serum creatinine as an independent predictor of responsiveness to zinc supplementation among patients on hemodialysis.

BACKGROUND: To investigate whether pre-dialysis level of serum creatinine (SCre) could indicate the responsiveness to zinc supplementation of patients on maintenance hemodialysis (MHD). METHODS: We retrospectively reviewed the results of our previous randomized study of 91 patients who had been on MHD and received zinc supplementation with either zinc acetate hydrate (ZAH; zinc, 50 mg/day) or polaprezinc (PPZ; zinc, 34 mg/day). A late response to zinc supplementation was defined as a serum zinc level of < 80 µg/dL three months after the study began. Patients were divided into two groups: late response (serum zinc level < 80 µg/dL) and early response (serum zinc level ≥ 80 µg/dL). Factors independently associated with a late response to zinc supplementation were determined using inverse probability of treatment weighting (IPTW) multivariate logistic analysis. RESULTS: Of 91 patients, 86 continued to receive zinc supplementation after three months. The mean pre-dialysis SCre level was 10.0 mg/dL. The number of patients with a late response and response to zinc supplementation was 32 and 54, respectively. There was a significant negative correlation between the pre-dialysis SCre and the Δserum zinc change for 3 months. (r = - 0.284, P = 0.008). IPTW multivariate analysis showed that a pre-dialysis SCre level ≥ 10.0 mg/dL (odds ratio, 3.71; 95% confidence interval; 1.24-11.1, P = 0.022) was an independent factor associated with a late response to zinc supplementation. CONCLUSIONS: Pre-dialysis SCre level was independently associated with responsiveness to zinc supplementation after three months in patients on MHD.