MEIC and current efficiency were greater for SUP60 and SIT60 compared to SUP20 and SIT20. The vastus lateralis and medialis showed lower θp and higher Lf at SUP60 and SIT60, while for the rectus femoris, in SUP60 there were lower θp and higher Lf than in all positions. The vastus intermedius had a similar pattern to the other vastii, except for lack of difference in θp between SIT60 compared to SUP20 and SIT20. The TAC stiffness index was greater for SUP60. We concluded that NMES generate greater torque and current efficiency at 60° of knee flexion, compared to 20°. For these knee angles, lengthening the QF at the hip did not promote significant change. Each QF constituent demonstrated muscle physiology patterns according to hip and/or knee angles, even though a greater Lf and lower θp were predominant in SUP60 and SIT60. QF TAC index stiffened in more elongated positions, which probably contributed to enhanced force transmission and slightly higher torque in SUP60. Our findings may help exercise physiologist better understand the impact of hip and knee angles on designing more rational NMES stimulation strategies.Muscle-tendon unit length plays a crucial role in quadriceps femoris muscle (QF) physiological adaptation, but the influence of hip and knee angles during QF neuromuscular electrical stimulation (NMES) is poorly investigated. We investigated the effect of muscle length on maximum electrically induced contraction (MEIC) and current efficiency. We secondarily assessed the architecture of all QF constituents and their tendon-aponeurosis complex (TAC) displacement to calculate a stiffness index. This study was a randomized, repeated measure, blinded design with a sample of twenty healthy men aged 24.0 ± 4.6. The MEIC was assessed in four different positions: supine with knee flexion of 60° (SUP60); seated with knee flexion of 60° (SIT60); supine with knee flexion of 20° (SUP20), and seated with knee flexion of 20° (SIT20). The current efficiency (MEIC/maximum tolerated current amplitude) was calculated. Ultrasonography of the QF was performed at rest and during NMES to measure pennation angle (θwww.ClinicalTrials.gov, identifier NCT03822221. Neuromuscular electrical stimulation (NMES) has been applied to increase, or attenuate loss, in muscle strength and size . AdjustiThe length of the knee extensor quadriceps femoris muscle (QF), often targeted for NMES , dependsLf) is associated with the number of sarcomeres in series, which determines fiber/muscle shortening velocity and excursion (p) is related to the parallel number of sarcomeres within the fiber and determines the maximal capacity for force transmission efficiency  at rest and during NMES of the four QF constituents: RF, vastus lateralis (VL), vastus medialis (VM), and vastus intermedius (VI), and the QF TAC stiffness. We hypothesized that: (1) during NMES, greater knee extensor torque and current efficiency would be obtained with the hip at 0° and the knee at 60° of flexion; (2) at rest and during the plateau of the evoked contraction, θp would be lower and Lf would be greater when the knee is at 60°, although for RF, the hip at 0° would decrease the θp and increase the Lf even more; and (3) the TAC stiffness would be greater at more elongated positions compared to shortened positions, as predicted for other muscle groups (The primary aim of this study was to investigate the effect of quadriceps muscle length by manipulating hip (0° or 85°) and knee (60° or 20°) joint knee angles on MEIC and current efficiency by NMES. We secondarily investigated the muscle architecture  from the Research Ethics Committee of the University of Brasília/Faculty of Ceilândia following the Helsinki Declaration of 1975. The present study is reported according to the CONSORT .Participants were recruited through flyers distributed at the University and by verbal invitation. The inclusion criteria were: healthy males, aged 18–30 years, and physically active, but not engaged in systematic lower limb strengthening in the previous 6 months. The exclusion criteria were none responsiveness or discomfort with the NMES, considered as an MEIC lower than 40% of the MVC, and any musculoskeletal injury that could limit performance during the tests.Four positions were randomly tested during NMES: lying supine with knee flexion of 60° (SUP60); seated with knee flexion of 60° (SIT60); lying supine with knee flexion of 20° (SUP20); and seated with knee flexion of 20° (SIT20), where supine was 0° of hip extension, seated was 85° of hip flexion, and full knee extension = 0° . One resParticipants were blinded to the study aim and hypotheses to avoid having their expectations affecting the performance. However, neither the researcher nor the volunteers could be blinded to the positions during the assessment.Torque assessment), to verify if participants tolerated a current amplitude enough to generate a MEIC ≥ 40% of the MVC. The four positions were tested separately in each experimental session and consisted of eight MEIC during QF NMES. The timeline and content of the familiarization and experimental sessions are shown in This study involved five laboratory visits: a familiarization and four experimental sessions, 7 days of washout between each one, and conducted consistently between 09.00 AM and 4.00 PM. We instructed subjects not to ingest alcohol the previous 24 h or stimulants  the previous 6 h before each visit. The familiarization consisted of: anthropometry (body mass and height); motor point localization on the VL and VM ; and two2 applied over the motor points of the VL and VM  was connected to two isolated cables, each connected to a pair of self-adhesive electrodes of 25 cmL and VM . A biphaL and VM . The curL and VM . MoreoveL and VM .p and Lf) and the TAC stiffness of the four QF constituents  and the current efficiency. Secondary outcomes were the muscle architecture at rest and during NMES  were used to measure the knee extensor torque. The equipment axis was visually aligned with the knee flexion-extension axis. The knee and hip angles were determined using a goniometer, and the lever arm of the dynamometer transducer was firmly attached 2–3 cm above the lateral malleolus with a strap. A warm-up  was performed prior to data collection. Before recording the evoked torque during NMES, participants completed two MVCs of the right knee extensors separated by a 2 min rest. During each MVC, participants were encouraged verbally to perform maximally and received visual feedback of the torque produced.p analysis  was connected to an ultrasound system . With cine-loop ultrasound imaging, two recordings (from rest to MEIC plateau) were obtained of each QF constituent, and the best recording  was used for analysis. For all ultrasound imaging outcomes, three measurements were performed and averaged. The probe was positioned longitudinally to the muscle fiber and perpendicular to the skin at 50% (RF), 60% (VL), 75% (VM), and 80% (VI), from proximal to the distal, of the distance between the medial aspect of the anterior superior iliac spine and the patella base, as adapted from previous reports . These ranalysis . The Lf  studies . The θp ®, Porto Alegre/RS, Brazil) on the lateral aspect of the knee to correct displacement overestimation due to any knee joint angular rotation. Only the corrected values were used to calculate the stiffness of each constituent  collected with a sampling rate of 2,000 Hz per channel, A/D converter of 14 bits, common rejection mode of 110 db (at 60 Hz). For this, the data acquisition device was connected to the computerized dynamometer, and a high-definition camera was positioned to capture the ultrasound display. When the assessor started recording cine-loop ultrasound images prior to the MECs, a visual indicator appeared on the ultrasound screen, which enabled the synchronization of all data on a torque-time recording generated by the device  was applied to verify differences between positions for MVC, maximal evoked torque, current efficiency, current amplitude, and TAC stiffness index. A two-way ANOVA [“position”  by “condition” (2 levels: rest and MEIC)] was applied to verify changes in θp and Lf. When a significant difference was detected, a Tukey post hoc test was applied to identify the differences. Effect sizes  and statistical power were provided. For reliability of consecutive MEIC, a mean of multiple measurements, absolute-agreement, 2-way mixed-effects model was used for intra-class correlation (ICC) . Moreover, a single-measurement, absolute-agreement, 2-way mixed-effects model was used for the ICC of Lf, θp, and TAC displacement . Reliability was classified as: poor (<0.5), moderate (0.5–0.75), good (>0.75–0.9), and excellent (>0.9)  with the level of significance set at p = 0.05 and power (1−β) = 0.80 to detect a large effect (ηρ2 > 0.14). According to a previous study . Repeated measures one-way ANOVA with a within-subject of “t (>0.9) . Coefficus study , we estiTwenty-three men were evaluated for eligibility criteria, and twenty men were included for randomization and completed the study. No cases of skin burn, or injury caused by NMES, occurred. p of RF , VL , VM , and VI , and good reliability for the Lf of RF , VL , VM , and VI . For the TAC displacement, the ICC was good to excellent for RF , VL , VM , and VI .ICC was excellent for evoked torque at SUP60 , SIT60 , SUP20 , and SIT20 . We obtained good reliability for the θρ2 for MVC, MEIC , current amplitude, and current efficiency. There was a significant main effect for MVC (p < 0.001), MEIC (p < 0.001), and current efficiency (p < 0.001). SUP60 and SIT60 showed greater MVC (p < 0.001), absolute evoked torque (p < 0.001), and current efficiency (p < 0.01) than SUP20 and SIT20. For these variables, no differences were found when we compared positions with the same knee angle . For normalized MEIC, there was no main effect among positions (p = 0.14). Current amplitude presented a significant main effect among positions (p < 0.001). A lower current amplitude was found at SUP20 compared to SIT60 (p < 0.001), SIT20 (p < 0.03), and SUP60 (p < 0.001), and SIT60 was greater than SIT20 (P < 0.02). SUP60 did not differ from SIT60 and SIT20 (P = 0.45). All participants reported 8 out 10 of perceived discomfort that was considered the maximum tolerated current amplitude.p, and Lf of the QF constituents at rest and during MEIC and the ANOVA results (except for muscle thickness). p and Lf at rest and (as well as the main effect of position) during evoked contraction for the QF constituents, along with the post hoc significance.position and intensity for θp (p = 0.017). In comparison to rest, NMES promoted higher θp in SIT60, SUP20, and SIT20 (p < 0.001). During MEIC, SUP60 presented lower θp compared to SIT60, SUP20, and SIT20 (p < 0.001). There was no interaction of factors for Lf (p = 0.90), but position effect was significant (p < 0.001). SUP60 presented greater Lf (p < 0.001–0.002) than other positions.For RF , there wp (p = 0.095) or Lf (p = 0.79). Position factor was significant for both θp (p < 0.001) and Lf (p < 0.001). The post hoc analysis showed lower θp (p < 0.001–0.011) and greater Lf (p < 0.001–0.031) for SUP60 and SIT60 compared to SUP20 and SIT20.Considering VL , there wLf (p = 0.044). The post hoc showed that SIT60 (p < 0.001) and SIT20 (p < 0.001) were different from rest to contraction, but not SUP60 (p = 0.10) and SUP20 (p = 0.083). Moreover, comparing positions, Lf was significantly greater for most analyses of SUP60 and SIT60 compared to SUP20 and SIT20 (p < 0.001 –0.03), except SUP60 vs. SIT20 at rest (p = 0.25) and SIT60 vs. SUP20 during NMES (p = 0.077). There was no interaction for θp (p = 0.097), but there was a significant main effect of position (p < 0.001). SUP60 and SIT60 showed lower θp (p < 0.001) compared to SUP20 and SIT20.VM  demonstrp (p < 0.25) or Lf (p = 0.15), but position factor was significant for both θp (p < 00.1) and Lf (p < 0.001). Lf was greater at SUP60 and SIT60 than SUP20 and SIT20 (p < 0.001). However, for θp, SUP60 was greater than SUP20 (p < 0.001) and SIT20 (p < 0.001), but SIT60 was not .For VI , there wp < 0.001 ηρ2: 0.50, power: 0.99) for the TAC stiffness index (p < 0.001). SIT60 and SUP20 were not different (p = 0.99). For SIT20, the stiffness index was also lower than in SUP20 (p = 0.02) and SIT60 (p = 0.01).There was a significant main effect of position  The TAC stiffness index was greater in SUP60. These new findings may help exercise physiologists and clinicians to develop effective strategies when applying NMES by choosing a knee angle around 60° when the goal is to induce high MEIC of knee extension. Furthermore, the QF elongation at the hip may be chosen to provide greater mechanical stress to the muscle-tendon unit.The main findings of this study were: (1) MEIC and current efficiency were greater at 60° than 20° of knee flexion. At these knee angles, changing the hip angle  did not affect torque output; (2) the QF constituents showed lower θWe demonstrated that NMES produces greater MEIC of knee extension and current efficiency applied with the knee at 60° compared to 20° irrespective of hip angle. Our results are in agreement with previous reports that found higher MEIC at 60° of knee flexion compared to more extended positions (15° and 30°) . In addip and greater Lf when the knee was flexed at 60° compared to 20°. It is possible that the positions SUP60 and SIT60 placed the QF at a better physiologic architectural configuration for torque generation, i.e., allowed improved transmission of the muscle force to the tendon and ideal sarcomere/fiber length  than all other positions , indicatEarlier studies  found grp of the RF was not significant at SUP60  from the Research Ethics Committee of the University of Brasília/Faculty of Ceilândia. The participants provided their written informed consent to participate in this study.JC, RM, KS, MB, NB, and JD conceived and designed research. JC performed the experiments and drafted the manuscript. JC, NB, and JD analyzed the data. JC, RM, JG, IS, BB, KS, MB, NB, and JD interpreted the results of experiments, edited and revised the manuscript, and approved the final version of the manuscript. JC and IS prepared the Figures. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Canis lupus familiaris) sperm cryosurvival, spermatozoa were diluted in a standard freezing medium and cooled to 5°C. Then, more freezing medium was added to obtain 200 × 106 cells/mL, and 5% glycerol. Diluted spermatozoa were treated with melatonin , and packaged in 0.25 mL straws, which were further cooled to −5°C before freezing in liquid nitrogen. Thawing was carried out at 70°C for 5 s, and the progressive motility, viability, plasma membrane integrity, acrosome integrity, capacitation status, and plasma membrane fluidity of the spermatozoa (at 37°C) were assessed. Data were analyzed using ANOVA to detect differences between the melatonin doses. There were statistical differences (P < 0.05) in the percentage of sperm having hyper-fluid membranes, intact acrosome, capacitated acrosome-intact, and acrosome-reacted. The values for the high melatonin doses (0.002 and 0.0035 mol/L) were better than for the low melatonin doses (0.0 and 0.0005 mol/L). In conclusion, 0.002 and 0.0035 mol/L of melatonin improved the cryosurvival of sperm from male bulldogs.Cryopreservation compromises the capacity of sperm fertilizing due to a series of alterations in the structure and physiology of the sperm. The use of antioxidants, such as melatonin, added to freezing media, may help to reduce sperm cryoinjury. To test the effect of melatonin on Bulldog  sperm survival and quality after freezing. We found that adding melatonin to the freezing media improved sperm quality after thawing. Artificial insemination (AI) employing frozen-thawed spermatozoa represents a practical tool for dog breeding, especially for breeds such as the English Bulldog. Usually, males of this breed are unable to mate. Therefore, many litters are conceived by AI. Despite this, English bulldogs are a beloved breed in many countries, and their semen serves as a model to develop cryopreservation protocols for dog spermatozoa.Freeze–thawing spermatozoa irreversibly impair the structure and function of the sperm . During 1 and MT2, which have been identified in several animal species, including dogs  and oxidative stress (antioxidants) during cryopreservation. This study aimed to test the effect of pre-freeze cooling to −5°C and MLT on dog sperm cryosurvival.All experiments complied with the Institutional Subcommittee for Care of Animals in Experimentation from the National Autonomous University of Mexico . This work was carried out at the Unit of Multidisciplinary Research, Laboratory of Animal Reproduction, from the National Autonomous University of Mexico (Faculty of Superior Studies Cuautitlan).n = 8, two from each male) from 4 English bulldogs (2–6 years old), collected throughout June 2017, were employed in a preliminary experiment. Ejaculates  from 8 English bulldogs (3–7 years old), collected from June to November 2018, were employed in the first experiment.Ejaculates  once a week from each male. Immediately after collection, the semen was left for 30 min at approximately 23°C (room temperature) to adjust. Then, it was macroscopically assessed for volume, color, appearance, and presence of strange particles . Immediately after centrifugation and resuspension in a standard freezing medium (see: semen processing), a small sample (200 μL) of diluted spermatozoa was further diluted in phosphate-buffered solution  at 37°C in a water bath, mixed, left for 10 min to equilibrate, and microscopically assessed. Only ejaculates showing at least 80% progressive motility and viability (SYBR14/PI) were included. The microscopic assessment was carried out as follows:Progressive motility (percentage of cells showing forward movement) was subjectively assessed under light microscopy (Leica DMLS) using the 20× objective. A smear stained by eosin/nigrosine was employed to assess normal/abnormal spermatozoa  under liThe sperm plasma membrane integrity was assessed by fluorescent staining using SYBR14/propidium iodide (PI) (L7011 Invitrogen). Moreover, 50 μL of diluted spermatozoa  was added to 5 μL of SYBR14 (100 nmol/L) and mixed. Immediately after this, 5 μL of PI (12 μmol/L) was added and mixed again for 10 s. Finally, 5 μL of glutaraldehyde (0.4%) was promptly added to immobilize the spermatozoa . The perAcrosome integrity was assessed by fluorescent lectins. Diluted sperm were smeared on a slide and air-dried, and the cells were permeabilized in alcohol for 60 min. Then, 50 μL of PSA-FITC lectin  was spread on a slide that was left in the dark for 10 min. Immediately after, the slide was gently washed with distilled water and air-dried (Sperm capacitation status was assessed using the chlortetracycline (CTC) assay as follows: 100 μL of diluted sperm  was added to 100 μL of CTC solution (pH 7.8), mixed for 30 s, and 20 μL of glutaraldehyde (0.2%) was added to immobilize the sperm . CTC-staTo assess the sperm plasma membrane fluidity, a merocyanine 540 assay was carried out as follows: a stock solution of merocyanine (0.005 mol/L) in dimethyl sulfoxide  was prepared and stored at room temperature (23°C), protected from light until use. Then, a work solution of merocyanine (0.000004 mol/L) in PBS (495 μL PBS + 5 μL merocyanine (0.005 mol/L) in DMSO) was freshly prepared  containing 3% glycerol (v/v) (6 cells/mL. These procedures were performed at about 23°C (room temperature). The sperm concentration was estimated from a 1:200 dilution  with the aid of a Neubauer chamber using the 40x objective.The semen was centrifuged at 750 ol (v/v)  to obtai6 cells/mL and 5% glycerol. Then, the diluted spermatozoa were divided into aliquots and MLT was added as follows:Sperm diluted in EYT (3% glycerol) were cooled from room temperature to 5°C for approximately 2 h (0.15°C/min). Then, EYT medium containing 7% glycerol (v/v) was slowly added in three equal parts with 10 min periods between each to obtain a final concentration of 200 × 10Preliminary experiment: (i) 0.0, (ii) 0.001, and (iii) 0.002 mol/LFirst experiment: (i) 0.0, (ii) 0.0005, (iii) 0.002, and (iv) 0.0035 mol/LA mix of DMSO + PBS  was added to the control group (i.e. 0.0 mol/L of MLT) so that all treatments contained the same amount of DMSO (used to dissolve MLT). Then, the diluted spermatozoa were packaged in 0.5 mL (preliminary experiment) or 0.25 mL (first experiment) plastic straws, which were sealed with polyvinyl alcohol. The straws were placed in a special recipient containing a saline solution  at 5°C. Each straw was positioned inside a plastic cylinder with a diameter slightly larger than the straw. One end of these cylinders was sealed and fixed to the bottom of the recipient using plasticine, while the other end was opened and stood above water . In thisThe straws  were thawed at 37°C for 30 s (preliminary experiment) or 70°C for 5 s (first experiment) in a water bath. The content was immediately poured into dry tubes located in another water bath at 37°C. After 10 min, the sperm were assessed as described previously.To assess the effect of MLT on the gross measures of sperm quality, a preliminary experiment was carried out to test the effect of 0.0, 0.001, and 0.002 mol/L of MLT on the cryosurvival of spermatozoa from English bulldogs , two ejaculates per male). The semen was collected at the laboratory, assessed, and processed as previously mentioned. Each ejaculate was processed separately and subjected to all the treatments . The diluted spermatozoa were cooled to either +5°C or −5°C, and frozen–thawed as previously mentioned. The progressive motility, plasma membrane integrity, capacitation status, and acrosome integrity were assessed before and after cryopreservation. This experiment was replicated four times.In the first experiment, the effects of 0.0, 0.0005, 0.0020, and 0.0035 mol/L of MLT were tested on the cryosurvival of spermatozoa from English bulldogs , three ejaculates per male). The semen was collected at the laboratory, assessed, and processed as mentioned. Each ejaculate was processed separately and subjected to all treatments . The diluted spermatozoa were cooled to −5°C, and freeze–thawed as previously mentioned.The progressive motility, plasma membrane integrity, plasma membrane fluidity, capacitation status, and acrosome integrity were assessed before and after cryopreservation. This experiment was replicated eight times.s.e.m. Values were considered to be significant at P < 0.05.Sperm quality data from the fresh semen were analyzed using descriptive statistics only. Data regarding the sperm quality from the cooling treatments of the preliminary experiment (i.e. +5 vs −5°C) were analyzed using the paired T test; data from the MLT treatments and individual dogs were pooled for each cooling temperature. For the ANOVA of cooling temperatures x MLT doses, an individual dog was a fixed effect. The sperm quality data from the (i) cooling temperatures × MLT treatments (preliminary experiment), and (ii) MLT treatments (first experiment) were arcsine transformed to normalize the data before the ANOVA . The genP < 0.05) between the cooling temperatures in the percentages of non-capacitated, acrosome-intact spermatozoa : 17.9 ± 3.50% (+5°C) vs 30.8 ± 6.32% (−5°C); capacitated, acrosome-intact spermatozoa : 69.9 ± 2.53% (+5°C) vs 59.4 ± 4.71% (−5°); and acrosome-intact spermatozoa (PSA-FITC lectin): 58.9 ± 5.56% (+5°C) vs 72.8 ± 4.29%  in the percentages of sperm with hyper-fluid membranes (MC540 brilliant pattern) between the 0.0 and either 0.002 or 0.0035 mol/L of MLT  in the percentages of sperm with intact acrosomes between the low (0.0 and 0.0005 mol/L) and high (0.002 and 0.0035 mol/L) MLT treatments. The values of the high MLT doses were higher than those of the low MLT treatments  in the percentages of capacitated acrosome-intact spermatozoa  between the low and high MLT treatments. There were also significant differences (P < 0.05) in the percentages of acrosome-reacted spermatozoa  between the low and high MLT treatments (There were significant differences (eatments .Table 3P < 0.05) between some dogs (regardless of MLT treatment) in sperm motility: male 1 had the highest (61.5 ± 4.12%) and males 4, 6, and 8 the smallest . Also, there were significant differences (P < 0.05) in sperm plasma membrane integrity: males 1 and 2 displayed the highest  and male 8 the smallest (24.6 ± 4.37%) percentages  or smaller  values in some variables when high levels of MLT were added . In contIn the preliminary experiment, the effects of MLT  and pre-freeze cooling to −5°C were tested on the cryosurvival of sperm from male English bulldogs. MLT produced no differences in sperm cryosurvival. However, pre-freeze cooling to −5°C improved some sperm attributes. In a similar study, using a different breed of dogs (Belgian and German Shepherds), cooling reduced the percentage of hyper-fluid membranes in comparison to the usual cooling to +5°C . BalasubIn the first experiment, different doses of MLT were tested on the cryosurvival of sperm from male English bulldogs. In general, sperm cryosurvival was significantly better with high (0.002 and 0.0035 mol/L) than low (0.0 and 0.0005 mol/L) doses of MLT. In this experiment, some modifications to the freeze–thawing protocol  were introduced in addition to the different doses of MLT.vs frozen-thawed sperm) were similar to those reported by others: 11% vs 91%  of MLT significantly reduce the percentage of sperm with hyper-fluid membranes after freeze–thawing. However, similar results were obtained in dog sperm by the mere effect of pre-freeze cooling (The protective role of MLT in sperm preservation is based on its antioxidant and anti-apoptotic properties that may either improve or maintain sperm quality (MLT in high doses may reduce the potential of sperm fertilizing after cryopreservation. The sperm motility and viability are reduced due to ROS production being excessively neutralized and oxidative phosphorylation inhibited (In conclusion, 0.002 and 0.0035 mol/L of MLT was better than the other concentrations of MLT in terms of the quality of post-thawed dog spermatozoa.The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.J A M-R, F J C, and R M-D-A carried out semen collection, freeze–thawing experiments, collected data and drafted the paper. A A R and A M designed and supervised the work, analyzed data, drafted and corrected the paper.


Recently, metabolic syndrome (MetS) has been linked with HS.2A total of 470 outpatients from a university department from January 2016 to May 2020 were included , and alanine aminotransferase (ALAT) levels between Hurley stages. Association and mean difference (MD) between age and presence of MetS and distribution of continuous variables between sexes were analyzed with independent 3r = .59, P < .001 in females and r = .40, P = .034 in males) and , respectively, and leukocytes, neutrophils, and NLR in females only . In adjusted analysis, ALAT correlated with CRP in males , and neutrophils  and NLR  in females. There were no significant correlations among ALAT, ESR, and leukocytes in either sex.The hepatic status was investigated by analyzing ALP and ALAT. The median ALP and ALAT levels were 73 U/L (IQR 59‐89) and 21 U/L (IQR 16‐29), respectively. Unadjusted analysis showed that ALP levels correlated with markers of systemic inflammation in females; C‐reactive protein (CRP), erythrocyte sedimentation rate (ESR), leukocytes, neutrophils, and neutrophil‐lymphocyte ratio (NLR), whereas among males, ALP levels only correlated significantly with CRP. In males, ALAT levels were negatively correlated with CRP, ESR, leukocytes, neutrophils, and NLR, whereas in females, ALAT levels were positively correlated with CRP and ESR (Table P = .09) and , respectively, compared with those not receiving antibiotics.A total of 39 patients received tetra‐, lyme‐, or doxycycline, and nine patients received rifampicin at the time of visit. Although long‐term treatment with antibiotics is known to possibly affect hepatic function, we found no significant increase in ALP or ALAT levels ; in females  and males , even after adjustment . Only ALP levels correlated with the number of boils in the last month in females  but not in males , and for females even after adjustment .Mean levels of ALP and ALAT in patients with Hurley stages I, II, and III were 69, 79, and 94 U/L, P = .468), or when split by sex, with a prevalence for females of 24.8%, 36.4%, and 40.0% (P = .103) and 41.5%, 32.9%, and 30.0% for males (P = .515) in Hurley stages I, II, and III, respectively. However, there was a significant difference in the number of criteria met  between the Hurley stages in females (P = .005), but not in males (P = .79) where the association between MetS score and Hurley stage was modified by sex (P = .039). MetS score was correlated with HSS and disease duration in females  and , but not in males  and , respectively. HSS correlated with BMI in female patients  but not in males  , but not in males (P = .65).The prevalence of MetS  as were ALAT levels , but not for male patients. ALP levels correlated significantly with number of MetS criteria in females  but not in males , whereas ALAT was significantly correlated with MetS score for females  and males .ALP levels were significantly higher in female patients with MetS , P = .11. In the subgroup of patients with abnormally high ALP levels, inflammatory markers of CRP, ESR, leukocytes, neutrophils, and NLR were significantly higher compared with those below the reference level, with an MD of 8.4, 14.2, 1.4, 1.2, and 0.5, respectively . Moreover, this subgroup had significantly higher disease severity, with an MD in HSS of 14.3 (P < .001) including a larger fraction of patients with Hurley III , and presented with a larger mean MetS score  compared with those below the reference level.A total of 12.9% had ALP above the reference level of 105 U/L and 5.3% had ALAT above the reference level of 70 U/L for males and 45 U/L for females.4A large percentage of our female patients were obese, which associate with an abnormal cytokine production,Previous studies have shown that risk of type 2 diabetes and more advanced stages of liver disease increases when the number of MetS criteria increases,Even though we found no significant difference in the presence of MetS between females and males, the number of criteria was dependent on HS severity and female sex. Based on this association, including the correlation between ALP levels and the presence of MetS, we propose the use of ALP as a predictor for MetS in patients with HS. This test allows the clinician to quickly determine whether their patients with abnormal liver results possibly are in high risk for MetS and refer to appropriate care.Conceptualization: Simon Francis ThomsenInvestigation: Jesper Grønlund Holm, Astrid‐Helene Ravn Jørgensen, Yiqiu Yao, Hans Christian Ring, Simon Francis ThomsenFormal analysis: Valdemar Wendelboe Nielsen, Simon Francis ThomsenWriting ‐ original draft: Valdemar Wendelboe NielsenWriting ‐ review & editing: Valdemar Wendelboe Nielsen, Jesper Grønlund Holm, Astrid‐Helene Ravn Jørgensen, Yiqiu Yao, Hans Christian Ring, Simon Francis Thomsen.The corresponding author confirms that the manuscript is an honest, accurate, and transparent account of the study being reported; that no important aspects of the study have been omitted; and that any discrepancies from the study as planned have been explained.


Dental caries is known as a multimicrobial disease. Caries are very prevalent in numerous countries, and the incidence is higher in underdeveloped countries than in developed countries. Dental caries is a major public health problem, and it is the most prevalent health problem across the world, affecting 2.4 billion people. Natural mouthwashes can be beneficial in the prevention of dental caries and oral infections without the side effects of synthetic mouthwashes.  The aim of the present study was to investigate the antibacterial, antifungal, and cytotoxicity properties of sea salt from different areas of Iran. L. acidophilus, S. aureus, E. coli, S. mitis, S. mutans, S. salivarius, and C. albicans. The cytotoxic effect of salts was evaluated on human gingival fibroblasts by the MTT test.  Sea salts from different areas  of Iran were collected. In order to define the elemental and mineralogical features of different salt samples, X-ray powder diffraction (XRD) was employed. Different concentrations (0.19–50 mg/mL) of sea salt were used in the antimicrobial and antibiofilm tests. The antimicrobial  and antibiofilm (formation and degradation tests) effects were evaluated against −1 was as follows: S. salivarius (50), S. mutans (50), S. mitis (50), L. acidophilus (12.5 to >50), C. albicans (50), E. coli (12.5 to 25), and S. aureus (12.5 to 25), while MBC values were, S. mutans (>50), S. salivarius (>50), S. mitis (>50), L. acidophilus (50 to >50), C. albicans (>50), E. coli (50), and S. aureus (50). MTT results showed that more than 50% of cell viability depends on decreasing the salt concentration (<1.56 mg/ml).  The range of MIC values in mg mlC. albicans. Therefore, sea salts can be a suitable candidate for mouthwash. Sea salts had significant antimicrobial effects on cariogenic bacteria and  Streptococcus mutans, though it is not sufficient for caries development. In carious lesions, additional microbial species were also isolated such as Gram-negative bacteria lactobacilli. They have been related to the dental caries [Streptococcus mutans's capability to make extracellular polysaccharides (mainly glucans) is an essential factor for tooth caries [S. mutans which leads to caries and an acidic environment [Lactobacilli should be considered as “secondary invaders.” They are not capable of producing caries [Sanguinaria canadensis. It has an extensive range of action against several oral bacteria. It has been used in different oral care products due to its robust antibacterial usefulness. It was reported to be related with oral leukoplakia, therefore its application had been decreased. These complications necessitate more research for natural antibacterial materials which are specific for oral microorganisms and safe for humans [Dental caries is a multifactorial disease which is considered by local destruction of the tooth. Dental biofilm plays an important role in the progression of periodontal diseases and caries . Evidencl caries , 4–6. Thh caries , 3, 5. Tironment . The aciironment . Lactobag caries . Bacterig caries . Many efr humans , 9. Chemr humans . Saltwatr humans . Furtherr humans . There ir humans , 12, 13.r humans , 12, 13.Streptococcus salivarius, Streptococcus mutans, Streptococcus mitis, Candida albicans, Lactobacillus acidophilus, Staphylococcus aureus, and Escherichia coli, and human gingival fibroblast cells were gifted. Brain heart infusion (BHI) broth and agar and yeast peptone dextrose (YPD) broth and agar and crystal violet were provided from Merck . The MTT Kit was purchased from Bioidea (Iran). Dimethyl sulfoxide (DMSO) was purchased from Sigma‐Aldrich. Dulbecco's modified Eagle's medium (DMEM), trypsin, fetal bovine serum (FBS), phosphate‐buffered saline (PBS), antistreptomycin, and betaglycerol were bought from Gibco .Sea salts were collected in 2020 from Urmia (West Azerbaijan) , Qom , and Jarquyeh (Isfahan Province)  .α radiation and a secondary graphite monochromator at 40 kV and a current of 30 mA in a range from 2 to 70° 2θ/degree, with a speed of 3.0 2 θ/min). Patterns were recognized by X'Pert HighScore Plus with a PAN-ICSD database [Elemental characteristics of salt samples were characterized by X-ray powder diffraction (XRD)  with DMEM (serum-free) were added (100 µl/well). Then, the cultures were incubated (37°C) (moistened atmosphere of 5% CO2) for 24 h. In the next step, using the MTT solution (5 mg/ml), cell growth induction activity was assessed. MTT solutions were added to each well, and the plates were incubated (4 h at 37°C) (moistened atmosphere of 5% CO2). Dimethylsulfoxide (DMSO) (1000 µl) was used to change the well's medium. DMSO dissolves the dark blue crystals. The plates were read with an ELISA reader (EL X 808) after 10 min (room temperature) at 570 nm as the test wavelength and 630 nm as the reference wavelength. An MTT-based method was used to analyse cell mitochondrial activity after 24 h and 48 h of conditioning. Data were changed into percentages of viable cells [Human gingival fibroblasts were incubated in 96-well plates with different concentrations (0.19–50 mg/mL) of sea salt in the growth medium. Cell survival was analyzed in this test containing 3- 2,5-diphenyl tetrazolium bromide. 2 × 10le cells , 15. Thele cells , 15. TheStreptococcus mitis, Streptococcus mutans, Streptococcus salivarius, Lactobacillus acidophilus, Staphylococcus aureus, Escherichia coli, and Candida albicans. The bacterial strains were reactivated in brain heart infusion agar (BHI) medium  (48 h)). Then, a loopful of bacteria were suspended in BHI Broth medium (25 mL) . After incubation, the concentration of cells was determined (37°C for 24 h). In a spectrophotometer (at 625 nm) (absorbance of 0.18), a cell density equivalent of 1.0 × 108 CFU/mL was obtained [5 CFU/mL was used for the MIC assay [C. albicans was inoculated initially into sterilized yeast peptone dextrose (YPD) broth to form the C. albicans suspension. A concentration of 1.0 × 105 CFU/mL was used for the MIC assay .The bacterial and fungal strains used in this experiment were obtained . A conce(5% CO2) 8 h). TheμL) was injected into the wells of 96-well microtiter plates to assess MIC. In the next stage, sea salts (100 μL) were inserted into the first column of the wells at their primary concentration (50 mg/ml). Then, by transferring well content (100 μL) from the highest concentrated to the least concentrated, the sea salt solution was sequentially diluted (from 50 to 0.19 mg/mL) (1 : 1 v/v) [μL of the well contents of the last column were thrown away. At the final stage, bacterial and fungal inoculums were inserted (100 μL) (1.0 × 105 CFU/mL). The control groups were used as follows: (1) growth control ; (2) antimicrobial control (Chlorhexidine (CHX) 0.2%); and (3) sterility control (only sterile culture medium). The microplates were incubated  [BHI and YPD broth (100 : 1 v/v) . In the 8/ml of strains was streaked on BHI agar. By the DAD test, the antibacterial activity of sea salt solutions was measured. The plates of sea salt solutions were filled with 0.08 ml each of 2× MBC of each sea salt and 0.2% CHX (positive control). Then, plates were incubated (for 48 h) (at 37°C). The inhibition zone around the wells was measured and noted [The antimicrobial activity of sea salt solutions was determined by DAD using brain-heart infusion. Colonies of different strains grown on BHI agar were suspended in NaCl solution (145 mM). Then, they were adjusted to the McFarland 0.5 scale by a spectrophotometer. Sea salt solutions (400 ml) were mixed with BHI agar (40 ml) (at 45°C). Then, they poured on a set layer of BHI agar , 18. Thend noted , 18.2 5% for 48 h. Then, the broth was eliminated and the microplates were washed with PBS 3 times to remove nonadherent bacteria. Then, microplates were dried for 45 minutes at 60°C. Then, crystal violet (1% (w/v)) (100 μL) solution was added to each well. Then, microplates were incubated for 15 minutes. Using PBS, the microplates were washed. By adding ethanol (95%) (125 μL) to each well, biofilm formation was determined. With a microplate reader, the optical density of wells (OD) was measured in comparison to the control biofilm  at 590 nm [treatment and OD control refer to the absorbance at 570 nm in each well with and without the samples, respectively, after the addition of the dissolving solution.Biofilm growth was assessed by the crystal violet staining method. The strains were cultured in microplates with sucrose (1%) and sterile BHI agar. Microplates were cultured with 2×MBC of each sea salt solution, under anaerobic conditions at 37°C with COt 590 nm . The meaThe results were analyzed by Tukey post hoc test and one-way ANOVA to compare means among groups. Statistical evaluation becomes carried out with SPSS statistics model 20.The results for the mineral elements of the sea salt samples are found in To determine the possible effect of sea salt samples on cell growth, cells were incubated with different extracts (0.19 to 50 mg/ml) (for 24 and 48 h). Using samples, a significant reduction of viable cells was observed in a dose and time-dependent pattern. By determination of the optical density of vital cells after treatment with the samples for incubation of 24 and 48 h, the cell viability percentage for both cell lines was calculated and compared to the percentage of the control group. The cell viability was increased by decreasing the concentration of salt, and the 1.56 mg/ml was considered as more than 50 percentage of cell viability. Results are shown in −1 was as follows: S. salivarius (50); S. mutans (50); S. mitis (50); L. acidophilus (12.5 to >50); C. albicans (50); E. coli (12.5 to 25); and S. aureus (12.5 to 25)  .−1 were S. mutans (>50); S. salivarius (>50); S. mitis (>50); L. acidophilus (50 and > 50); C. albicans (>50); E. coli (50); and S. aureus (50)  .S. mutans (5 to 6 mm); S. salivarius (4 to 6 mm); S. mitis (4 to 6 mm); L. acidophilus (4 to 5 mm);C. albicans (4 to 5 mm); E. coli (3 to 4 mm); and S. aureus (4 to 5 mm)  as follows: to 5 mm) .The effect of samples on the prevention of biofilm formation was tested using the microdilution method. Biofilm formation rates of samples are reported in percentages in The effect of treatment with samples on biofilms which had already been formed was tested using the same method. In this case, the biofilm reduction rate in percentage was calculated . All salS. mutans only. The S. mutans' biofilm is commonly selected to assess the antibacterial activities of various materials. Sucrose and S. mutans are the main factors of the growth of biofilms [Some methods to control dental caries include the use of varnish, fluoride gel, professional tooth cleaning, antimicrobial agents, fissure sealant, fluoride mouthwashes, fluoride gels for personal use, fluoride supplements, oral hygiene, diet control, and noncariogenic sweeteners like xylitol. Natural products can also be used as coadjutant factors for caries control. Natural materials are a useful substitute to synthetic antimicrobials . Many babiofilms , 16. Mecbiofilms .Sea salt makes the environment alkaline, thereby accelerating the healing of the surgical wound . Sea salS. mutans (5 to 6 mm); S. salivarius (4 to 6 mm); S. mitis (4 to 6 mm); L. acidophilus (4 to 5 mm); C. albicans (4 to 5 mm); E. coli (3 to 4 mm); and S. aureus (4 to 5 mm). The best results were, respectively, related to Urmia, Qom, and Jarquyeh. In this study, we evaluate the effect of sea salts on the formation and degradation of microbial biofilm. Sample 2 (Qom) had the highest effect on the formation of biofilm, and sample 3 (Jarquyeh) had the lowest effect on the formation of biofilm. Sample 3 had the highest effect on the degradation of biofilm, and sample 2 had the lowest effect on the degradation of biofilm. MTT results showed that more than 50 percentage of cell viability depends on decreasing the salt concentration (≤1.56 mg/ml).In this study, the range of zones of microbial growth inhibition by sea salt samples was as follows: In this study, sea salts were collected from different regions in Iran. In this study, sea salts were effective against the tested microorganisms. Michel et al. assessed the usefulness of the application of sea salt rinse  on periodontitis in street children of Manila . HoweverAntimicrobial studies specifically demonstrate that sea salt solutions are potent plaque inhibitors that could be used as a mouthwash. The solutions blocked plaque formation and degraded biofilm formation. However, further long-term clinical trials are required to include the much needed standardization and certification of the mouthwash in order to overcome the drawbacks of the gold standard chlorhexidine.


Sarcopenia‐related parameters may have differential impacts on health‐related outcomes in older adults. We examined dose–response relationships of body composition, muscle strength, and physical performance with incident disability and mortality.2 for men and 5.7 kg/m2 for women; HGS: 28 kg for men and 18 kg for women; or UGS: 1.0 m/s for both sexes). Association shapes were examined using restricted cubic splines or fractional polynomial functions.This prospective study included 1765 Japanese residents  aged ≥65 years who participated in health check‐ups. Outcomes were incident disability and all‐cause mortality. Fat mass index (FMI) and skeletal muscle mass index (SMI), determined using segmental multi‐frequency bioelectrical impedance analysis, handgrip strength (HGS), and usual gait speed (UGS) were measured. We determined multivariate‐adjusted hazard ratios (HRs) for disability and mortality relative to sex‐specific reference values  was significantly associated with higher mortality risk in women, compared with median FMI. SMI did not impact disability in either sex and mortality in women, but showed a significant inverse dose–response relationship with mortality risk in men , independent of HGS and UGS. HGS and UGS showed a significant inverse dose–response relationship with disability in both sexes  and mortality in women , independent of body composition, but did not impact mortality in men.The median follow‐up was 5.3 years; 107 (12.7%) men and 123 (14.2%) women developed disability, and 101 (11.7%) men and 56 (6.2%) women died. FMI did not impact any outcome in men and disability in women, while an FMI ≤ 7.3 kg/mDisability risk was more dependent on muscle strength and physical performance in both sexes. Mortality risk in men was more dependent on muscle mass, and mortality risk in women was influenced by lower fat mass along with muscle strength and physical performance. Although improving muscle strength and physical performance should be the first target for health promotion, it is also necessary to pay attention to body composition to extend life expectancy in older adults. Sarcopenia was originally defined as an age‐related loss of skeletal muscle mass in older adults.These trends are attributable to the evidence showing that muscle strength and physical performance are more strongly associated with health‐related outcomes than muscle mass.Therefore, we examined the shapes of the associations of body composition, muscle strength, and physical performance, after accounting for the confounding effects of each on the other, with incident disability and all‐cause mortality among community‐dwelling older Japanese adults. Specifically, we aimed to clarify (i) whether body composition, muscle strength, and physical performance have differential impacts on incident disability and mortality and (ii) whether their impacts differ between sexes because of the previously established sex‐related differences in body composition, muscle strength, and physical performance.We used combined dataBody composition parameters were measured using direct segmental multi‐frequency bioelectrical impedance analysis To measure muscle strength, HGS was assessed using a Smedley‐type hand dynamometer .For physical performance measurements, UGS was measured over a distance of 5 m, with acceleration and deceleration phases of 3 m each.We ascertained the occurrence of disability and/or all deaths on 13 December 2017, in Kusatsu town and 31 December 2015, in Hatoyama town. All‐cause mortality was confirmed by checking local registries that have linked records with the Japanese National Vital Statistics System.Disability was identified in the participants using the nationally unified database of the long‐term care insurance (LTCI) system, enrolment in which is mandatory for all Japanese adults aged ≥ 40 years. The system provides formal care and support for eligible Japanese adults aged ≥ 65 years with physical and mental disabilities.2, calculated using creatinine concentration and equations developed for Japanese adultsThe covariates included baseline age, study area (Kusatsu or Hatoyama), year of first visit for health check‐up, alcohol consumption and tobacco smoking status , previous diagnosis of stroke, heart disease, cancer, hypertension , and diabetes , high total cholesterol , low total cholesterol (<160 mg/dL), hypoalbuminemia (<3.8 g/dL), anaemia (haemoglobin < 13.0 g/dL in men or <12.0 g/dL in women), and chronic kidney disease . Statistical significance was set at For primary analysis, we used the Cox proportional hazards model, with incident disability or mortality as the dependent variable and FMI, SMI, HGS, and UGS as independent variables. We then constructed two multivariate analytic models and examined multivariate‐adjusted hazard ratios (HRs) and 95% confidence intervals (CIs) in terms of FMI, SMI, HGS, and UGS for incident disability or mortality. Model 1 was adjusted for baseline age, study area, year of first visit for health check‐up, alcohol consumption and smoking status, hypertension, stroke, heart disease, diabetes, cancer, high total cholesterol, low total cholesterol, hypoalbuminemia, anaemia, chronic kidney disease, low activity, depressed mood, and cognitive impairment. In the analyses with FMI and SMI as independent variables, these two indices were mutually adjusted as were other covariates because they are mutual confounders and both have sex‐specific differences.2 for men and 5.7 kg/m2 for women; HGS: 28 kg for men and 18 kg for women; or UGS: 1.0 m/s for both sexesFurthermore, using fractional polynomial (FP) functions or restricted cubic spline (RCS), in accordance with the procedures of previous studies,In the secondary analyses of the possible influence of reverse causation on the association between each measure and disability or mortality, we performed sensitivity analyses using the same statistical approach, after excluding disability or death that occurred during the first year and the first 2 years of follow‐up.During a median (interquartile range) follow‐up of 5.3 (3.5–6.9) years, 107 (12.7%) men and 123 (14.2%) women presented with functional disabilities, with disability rates of 24.3 and 25.5 per 1000 person‐years, respectively. Of these, 9 (8.4%) men and 14 (11.4%) women presented with disabilities during the first year, and 22 (20.6%) men and 26 (21.1%) women presented with disabilities during the first 2 years. During a median follow‐up of 5.3 (4.4–7.5) years, 101 (11.7%) men and 56 (6.2%) women died, with mortality rates of 21.5 and 10.4 per 1000 person‐years, respectively. Of these, 2 (2.0%) men and 2 (3.6%) women died during the first year, and 6 (5.9%) men and 5 (8.9%) women died during the first 2 years.TablesTableTableTableTableTablesTableTableTablesFigureFigureFigureFigure2 (median) was significantly associated with higher mortality risk in Model 2 [HR (95% CI) of minimum value (2.1 kg/m2) compared with reference value (7.3 kg/m2): 3.56 (1.11–11.40)  Figure].FigureFigure2) and maximum (9.4 kg/m2) values compared with reference value (7.0 kg/m2): 2.45 (1.20–5.02) and 0.38 (0.17–0.82), respectively in model 1 ; 2.18 (1.07–4.48) and 0.43 (0.20–0.93), respectively in model 2 and physical performance (defined by UGS) than body composition in both sexes, while mortality risk was also influenced by body composition, independent of muscle strength and physical performance. In particular, mortality risk in men was more dependent on muscle mass than muscle strength and physical performance, and mortality risk in women was influenced by lower fat mass along with muscle strength and physical performance. Although improving muscle strength and physical performance (rather than only increasing muscle mass) should be the first target for health promotion, it is also necessary to pay attention to body composition to extend life expectancy in older adults.The authors declare that they have no competing interests.This study was supported by grants from the Tokyo Metropolitan Institute of Gerontology; Research Institute of Science and Technology for Society, the Japan Science and Technology Agency; Grants‐In‐Aid for Scientific Research (B) JP20390190, (B) JP21390212, (B) JP24390173, and (B) JP26310111 from the Ministry of Education, Culture, Sports, Science and Technology, Japan; the Japan Arteriosclerosis Prevention Fund for the Japan Arteriosclerosis Longitudinal Study (2001–2012); and the towns of Kusatsu and Hatoyama.Figure S1.Dose–response relationships of FMI with incident disability and mortality risks, excluding disabilities or deaths that occurred during the first year of follow‐upFigure S1a‐S1d show the relationships of FMI with disability  and mortality  risks in men. Figure S1e‐S1h show the relationships between FMI and disability  and mortality  risks in women. Figure S1a‐S1b were modeled using an FP function, and Figure S1c‐S1h were modeled using an RCS with three knots located at the 10th, 50th, and 90th percentiles of the distribution of the index. Model 1 was adjusted for baseline age, study area, year of first visit for health check‐up, drinking and smoking status, hypertension, stroke, heart disease, diabetes, cancer, high total cholesterol, low total cholesterol, hypoalbuminemia, anemia, chronic kidney disease, low activity, depressed mood, cognitive impairment, and SMI. Model 2 was adjusted for the variables in Model 1 plus HGS and UGS. The reference values for each model are the median FMI . The dashed lines indicate the 95% confidence intervals. AIC, Akaike's information criterion; FMI, fat mass index; FP, fractional polynomial; HGS, handgrip strength; HR, hazard ratio; RCS, restricted cubic spline; SMI, skeletal muscle mass index; UGS, usual gait speed.Click here for additional data file.Figure S2. Dose–response relationships of SMI with incident disability and mortality risks, excluding disabilities or deaths that occurred during the first year of follow‐upFigure S2a‐S2d show the relationships of SMI with disability  and mortality  risks in men. Figure S2e‐S2h show the relationships between SMI and disability  and mortality  risks in women. Figure S2a‐S2h were modeled using an FP function. Model 1 was adjusted for baseline age, study area, year of first visit for health check‐up, alcohol consumption and smoking status, hypertension, stroke, heart disease, diabetes, cancer, high total cholesterol, low total cholesterol, hypoalbuminemia, anemia, chronic kidney disease, low activity, depressed mood, cognitive impairment, and FMI. Model 2 was adjusted for the variables in Model 1 plus HGS and UGS. The reference values for each model are the cut‐off points for sarcopenia criteria defined by the Asian Working Group for Sarcopenia in 2019 . The dashed lines indicate the 95% confidence intervals. AIC, Akaike's information criterion; FMI, fat mass index; FP, fractional polynomial; HGS, handgrip strength; HR, hazard ratio; SMI, skeletal muscle mass index; UGS, usual gait speed.Click here for additional data file.Figure S3. Dose–response relationships of HGS with incident disability and mortality risks, excluding disabilities or deaths that occurred during the first year of follow‐upFigure S3a‐S3d show the relationships of HGS with disability  and mortality  risks in men. Figure S3e‐S3h show the relationships of HGS with disability  and mortality  risks in women. Figure S3a‐S3h were modeled using an FP function. Model 1 was adjusted for baseline age, study area, year of first visit for health check‐up, drinking and smoking status, hypertension, stroke, heart disease, diabetes, cancer, high total cholesterol, low total cholesterol, hypoalbuminemia, anemia, chronic kidney disease, low activity, depressed mood, and cognitive impairment. Model 2 was adjusted for the variables in Model 1 plus FMI and SMI. The reference values for each model are the cut‐off points for sarcopenia criteria defined by the Asian Working Group for Sarcopenia in 2019 . The dashed lines indicate the 95% confidence intervals. AIC, Akaike's information criterion; FMI, fat mass index; FP, fractional polynomial; HGS, handgrip strength; HR, hazard ratio; SMI, skeletal muscle mass index.Click here for additional data file.Figure S4. Dose–response relationships of UGS with incident disability and mortality risks, excluding disabilities or deaths that occurred during the first year of follow‐upFigure S4a‐S4d show the relationships of UGS with disability  and mortality  risks in men. Figure S4e‐S4h show the relationships of UGS with disability  and mortality  risks in women. Figure S4a‐S4h were modeled using an FP function. Model 1 was adjusted for baseline age, study area, year of first visit for health check‐up, drinking and smoking status, hypertension, stroke, heart disease, diabetes, cancer, high total cholesterol, low total cholesterol, hypoalbuminemia, anemia, chronic kidney disease, low activity, depressed mood, and cognitive impairment. Model 2 was adjusted for the variables in Model 1 plus FMI and SMI. The reference values for each model are the cut‐off points for sarcopenia criteria defined by the Asian Working Group for Sarcopenia in 2019 . The dashed lines indicate the 95% confidence intervals. AIC, Akaike's information criterion; FMI, fat mass index; FP, fractional polynomial; HR, hazard ratio; SMI, skeletal muscle mass index; UGS, usual gait speed.Click here for additional data file.Figure S5. Dose–response relationships of FMI with incident disability and mortality risks, excluding disabilities or deaths that occurred during the first two years of follow‐upFigure S5a‐S5d show the relationships of FMI with disability  and mortality  risks in men. Figure S5e‐S5h show the relationships of FMI with disability  and mortality  risks in women. Figure S5a‐S5b were modeled using an FP function, and Figure S5c‐S5h were modeled using an RCS with three knots located at the 10th, 50th, and 90th percentiles of the distribution of the index. Model 1 was adjusted for baseline age, study area, year of first visit for health check‐up, drinking and smoking status, hypertension, stroke, heart disease, diabetes, cancer, high total cholesterol, low total cholesterol, hypoalbuminemia, anemia, chronic kidney disease, low activity, depressed mood, cognitive impairment, and SMI. Model 2 was adjusted for the variables in Model 1 plus HGS and UGS. The reference values for each model are the median FMI . The dashed lines indicate the 95% confidence intervals. AIC, Akaike's information criterion; FMI, fat mass index; FP, fractional polynomial; HGS, handgrip strength; HR, hazard ratio; RCS, restricted cubic spline; SMI, skeletal muscle mass index; UGS, usual gait speed.Click here for additional data file.Figure S6. Dose–response relationships of SMI with incident disability and mortality risks, excluding disabilities or deaths that occurred during the first two years of follow‐upFigure S6a‐S6d show the relationships of SMI with disability  and mortality  risks in men. Figure S6e‐S6h show the relationships of SMI with disability  and mortality  risks in women. Figure S6a‐S6h were modeled using an FP function. Model 1 was adjusted for baseline age, study area, year of first visit for health check‐up, alcohol consumption and smoking status, hypertension, stroke, heart disease, diabetes, cancer, high total cholesterol, low total cholesterol, hypoalbuminemia, anemia, chronic kidney disease, low activity, depressed mood, cognitive impairment, and FMI. Model 2 was adjusted for the variables in Model 1 plus HGS and UGS. The reference values for each model are the cut‐off points for sarcopenia criteria defined by the Asian Working Group for Sarcopenia in 2019 . The dashed lines indicate the 95% confidence intervals. AIC, Akaike's information criterion; FMI, fat mass index; FP, fractional polynomial; HGS, handgrip strength; HR, hazard ratio; SMI, skeletal muscle mass index; UGS, usual gait speed.Click here for additional data file.Figure S7. Dose–response relationships of HGS with incident disability and mortality risks, excluding disabilities or deaths that occurred during the first two years of follow‐upFigure S7a‐S7d show the relationships of HGS with disability  and mortality  risks in men. Figure S7e‐S7h show the relationships of HGS with disability  and mortality  risks in women. Figure S7a‐S7h were modeled using an FP function. Model 1 was adjusted for baseline age, study area, year of first visit for health check‐up, drinking and smoking status, hypertension, stroke, heart disease, diabetes, cancer, high total cholesterol, low total cholesterol, hypoalbuminemia, anemia, chronic kidney disease, low activity, depressed mood, and cognitive impairment. Model 2 was adjusted for the variables in Model 1 plus FMI and SMI. The reference values for each model are the cut‐off points for sarcopenia criteria defined by the Asian Working Group for Sarcopenia in 2019 . The dashed lines indicate the 95% confidence intervals. AIC, Akaike's information criterion; FMI, fat mass index; FP, fractional polynomial; HGS, handgrip strength; HR, hazard ratio; SMI, skeletal muscle mass index.Click here for additional data file.Figure S8. Dose–response relationships of UGS with incident disability and mortality risks, excluding disabilities or deaths that occurred during the first two years of follow‐upFigure S8a‐S8d show the relationships of UGS with disability  and mortality  risks in men. Figure S8e‐S8h show the relationships of UGS with disability  and mortality  risks in women. Figure S8a‐S8h were modeled using an FP function. Model 1 was adjusted for baseline age, study area, year of first visit for health check‐up, drinking and smoking status, hypertension, stroke, heart disease, diabetes, cancer, high total cholesterol, low total cholesterol, hypoalbuminemia, anemia, chronic kidney disease, low activity, depressed mood, and cognitive impairment. Model 2 was adjusted for the variables in Model 1 plus FMI and SMI. The reference values for each model are the cut‐off points for sarcopenia criteria defined by the Asian Working Group for Sarcopenia in 2019 . The dashed lines indicate the 95% confidence intervals. AIC, Akaike's information criterion; FMI, fat mass index; FP, fractional polynomial; HR, hazard ratio; SMI, skeletal muscle mass index; UGS, usual gait speed.Click here for additional data file.


P < 0.05), and the higher values of them occurred in photoperiods ≥ 16 h, compared with 12L:12D (P > 0.05). Initial and bare stroma weight increased quadratically, while total large white follicle (LWF) number and weight increased linearly and quadratically, with increasing photoperiods (P < 0.05). The higher values of them occurred in 16L:8D and 18L:6D treatments as well as the higher total LWF weight also occurred in 20L:4D, compared with 12L:12D (P > 0.05). Besides, 16.93 and 16.93 h were the optimal photoperiods for bare stroma (follicles ≥ 2 mm in diameter removed) weight and total LWF weight, respectively, calculated from reliable regression equations (R2 ≥ 0.5071). Compared with 12L:12D, the higher levels of estradiol, progesterone, follicle-stimulating hormone (FSH) as well as the higher expressions of estrogen, luteinizing hormone (LH) and progesterone receptors were observed in ≥16 h photoperiods (P < 0.05), while the higher LH level and FSH receptor expression only occurred in 16L:8D and 18L:6D (P < 0.05). In the hypothalamus, higher mRNA expression of gonadotropin-releasing hormone occurred in 16L:8D and 18L:6D groups (P < 0.05). Meanwhile, gonadotropin-inhibitory hormone and prolactin increased in 20-hour photoperiod (P < 0.05), and the latter may be due to theup-regulation of vasoactive intestinal peptide expression (P < 0.05). To sum up, an appropriate photoperiod could improve the performance and reproductive organ and ovarian follicles development through reproductive hormones and their receptors, and 16.56 to 10.93 h is an adequate photoperiod for laying ducks.We investigated the effect of photoperiod on performance, ovarian morphology, reproductive hormones levels, and their receptors mRNA expressions in laying ducks. After adaption, 300 252-day-old Jinding laying ducks were randomly allocated to 5 groups, receiving 12L:12D, 14L:10D, 16L:8D, 18L:6D, or 20L:4D, respectively. Each treatment had 6 replicates of 10 birds each. The feeding trial lasted 8 wk. Egg production, egg mass, and ADFI increased linearly and quadratically with increasing photoperiods ( F3) will become the new largest follicle (F1) or the second-largest follicle (F2), following the maturation and ovulation of F1 and F2. Meanwhile, a small yellow follicle (SYF) will be chosen to enter the preovulatory follicle (POF) hierarchy. In poultry, photoperiod is one of the most significant environmental factors which impact reproductive activity, through regulating the development of reproductive organ and ovarian follicle  axis (GnRH) neurons activity. In fact, photoperiod was a crucial modulator for GnRH neurons activity (Dio2/Dio3 system) (GnIH)  axis . The uprPG) axis , while tactivity . In fowl system)  and be i) (GnIH)  or prola) (GnIH) . HoweverThere has been no consistent photoperiod protocol for laying ducks in practical production. Hence, more work is needed to evaluate the effect of photoperiod on the productive performance, reproductive organ and ovarian follicle development, and further explore the optimal photoperiod for laying ducks. Moreover, although the functions of these hormones have been extensively reported , the infAll experimental protocols were approved by the Animal Care and Use Committee of the Feed Research Institute of the Chinese Academy of Agricultural Sciences. After adaption, 300252-day-old Jinding laying ducks were randomly allocated to 5 treatments with a corn–soybean meal diet  for 8 wkBefore the formal experiment, the egg production of all replicates was adjusted to be approximately equal. Eggs were collected, and irregular  eggs were noted daily. Egg production, egg weight, and mortality were recorded every day by replicate. Feed consumption per replicate was weighted and recorded every 4 wk. Egg mass was calculated by multiplying average egg weight by egg production. Feed conversion ratio was obtained as grams of total feed consumption/total egg weight per replicate. ADFI and feed conversion ratio were calculated by replicate each 4 wk. Egg production and ADFI were adjusted by duck mortality in time.At the end of the experiment (310 d of age), 12 ducks from each group were randomly chosen (2 birds per replicate) and quickly killed by an overdose of anesthesia . Hypothalamus samples were collected, immersed in liquid nitrogen, and then stored at −80°C for subsequent measurement of mRNA expression for genes encoding GnRH, GnIH, Dio2, Dio3, and vasoactive intestinal peptide. In addition, the oviduct and ovary were collected and weighed. The initial stroma was weighted after the POF (>10 mm in diameter) being removed from the ovary, and the bare stroma was weighted after the SYF (6–10 mm in diameter) and LWF (2–5 mm in diameter) being removed from the initial stroma.ER), hormone receptor (FSHR), luteinizing hormone receptor (LHR), and progesterone receptor (PR).The preovulatory follicles, including F1, F2, F3, SYF, and LWF, were removed from the ovary, counted, weighed, and noted. Then, the granulosa layers of F1, F2, and F3 were divided, as per the description of FSH), luteinizing hormone (LH), and progesterone were measured using ELISA kits for ducks . The yield and integrity of RNA were determined using a NanoDrop 2000 spectrophotometer , and agarose−ethidium bromide electrophoresis. Expression quantification was conducted with a two-step reaction process, which contains reverse transcription and PCR using FastQuant Reverse Transcription kit , as per the description of t method . Primer P < 0.05. Data were showed as the mean and pooled SEM.All analyses were performed using SAS, version 9.2,. The replicate (1 room per replicate) was the experimental unit for the analysis of performance. For the other parameters, the mean of 2 ducks serving as the experimental unit for statistical analysis. The homogeneity of variances and normality of the data were evaluated first, which are the preconditions of ANOVA . The Sha1Xi + eij (linear regression), Yij = α + β1Xi + β2Xi2 + eij (quadratic regression).The PROC REG used in regression analysis and statistical models were as follows , Yij = α1 and β2 were regression coefficient; Xi was the studied factor effect as hour of light , and eij was the observational error for (ij)th observation.Yij was the response variable; α was the intercept (indicators with the 12 h of light); βP > 0.05, data not shown). Egg weight and feed conversion ratio were not affected by photoperiod during the whole period (P > 0.05). Increment in photoperiod linearly and quadratically increased egg production, egg mass, and ADFI (P < 0.05), during 1–4, 5–8 and 1–8 wk of the experiment. Compared with 12L:12D, the higher values of egg production, egg mass, and ADFI occurred in ≥16-hour photoperiods (P < 0.05), during 1–4, 5–8, and 1–8 wk of the trial.The effect of photoperiod on performance of laying ducks is presented in P > 0.05). Initial stroma weight, bare stroma weight, and percentage were quadratically affected in response to the increasing photoperiods (P < 0.05). Compared with 12L:12D, the higher initial and bare stroma weight occurred in 16L:8D and 18L:6D treatments (P < 0.05), while the higher bare stroma percentage occurred in 16L:8D (P < 0.05).As shown in P ˃ 0.05). Number of LWF and total LWF weight increased lineally and quadratically in response to the increasing photoperiods (P < 0.05). Compared with 12L:12D, the higher total LWF weight was observed in ≥16-hour photoperiods (P < 0.05), while the larger number of LWF occurred in 16L:8D and 18L:6D treatments (P < 0.05).The effect of photoperiod on the development of ovarian follicles, including F1, F2, F3, other POF, SYF, and LWF, is shown in P < 0.05). Meanwhile, the increment in photoperiod quadratically increased the levels of estradiol (12:00) and LH . Compared with 12L:12D, the higher levels of estradiol (18:00) were observed in ≥14-hour photoperiods (P < 0.05), and the higher values of estradiol (6:00), FSH (6:00), and progesterone (6:00 and 18:00) occurred in ≥16-hour photoperiods (P < 0.05). Meanwhile, 16L:8D and 18L:6D treatments had the higher values of estradiol (12:00) and LH .The changes of serum reproductive hormones (equidistant 4 time points) in response to photoperiods are detailed in P < 0.05).Real-time PCR analyses of ER, FSHR, LHR, and PR mRNA expressions in the granulosa cell layers of POF , SYF, and LWF are shown in P < 0.05); 16L:8D and 18L:6D treatments upregulated the mRNA expressions of ER (F3), FSHR , and LHR .Compared with 12L:12D, the higher mRNA expressions of ER (F1 and F2), LHR (F1), and PR (F1 and LWF) occurred in ≥16-hour photoperiods (The effect of photoperiod on the relative mRNA expressions of reproduction-related genes in the hypothalamus and serum level of prolactin is detailed in The potential benefits of photoperiod on poultry productive performance have been extensively reported, such as increasing egg mass of broiler breeders , laying 2 + 0.89907x – 2.70710, R2 = 0.5096, and 16.56 was the optimal photoperiod, obtaining from calculation. Therefore, a photoperiod of 16.56 h was supposed to be the most appropriate for the reproductive organ development of laying ducks.Laying performance of birds has a close link with the development of reproductive organs, including the oviduct, ovary, and ovarian stroma . The pho2 + 0.37788x – 2.01352, R2 = 0.5071. Based on this equation, the optimal photoperiod was calculated as 16.93 h/day. Taking the development of the reproductive organ and ovarian follicle into consideration, a 16.56- to 16.93-hour photoperiod could be appropriate for laying ducks.The response of reproductive organ development to photoperiod was always consistent with that of ovarian follicle development . In the The development of ovarian follicles and reproductive organs is mainly regulated by the HPG axis , throughThe higher values of these 4 hormones occurred in 16L:8D and 18L:6D treatments, and 3 of them were also observed in 20L:4D, which was consistent with the responses of ovarian morphology and laying performance to photoperiod. Thus, the positive effects of photoperiod on laying performance and reproductive organ and ovarian follicle development could be attributed to the increase in levels of reproductive hormones. The rise in serum FSH and LH probably led to the increase in the secretion of estradiol and progesterone, while the latter may stimulate the release of the former in turn. The biological effects of estradiol, FSH, LH, and progesterone on target cells can be carried out through their receptors . TherefoThe changes happened in serum levels of reproductive hormones can upstream trace to the effect of photoperiod on GnRH neurons in the hypothalamus . Hence, In conclusion, an increment in photoperiod improved productive performance of laying ducks in a linear and quadratic manner. Moreover, photoperiod quadratically promoted the development of reproductive organs and follicles, with 16.56 to 16.93 h as the optimal photoperiod. Besides, the higher levels of serum estradiol, FSH, LH, and progesterone accompanied with their receptors mRNA expressions were observed in ≥16-hour photoperiods, and the overall optimal values occurred in 16- to 18-hour photoperiod. These results could be ascribed to GnRH expression increasing in these 2 photoperiods and being suppressed by GnIH and prolactin when photoperiod reached 20 h. Thus, 16.56 to 16.93 h was the most suitable photoperiod for productive performance, ovarian morphology development, and reproductive hormone secretion of laying ducks.


Chronic kidney disease (CKD) has become a global public health problem. Accumulating evidence suggested that vitamins play important roles in the progression of CKD.A cross-sectional study was conducted to investigate the vitamin status of patients with CKD at stage 1–5. The serum concentrations of 9 vitamins, vitamin A, B1, B2, B6, B9, B12, C, D, and E were measured by electroanalytical method with a Multi-Vitamin Analyzer. Pearson correlation and multiple linear regression between serum level of vitamins were analyzed.The median levels of vitamin A, B1, B2, B6, B9, B12, C and E were within the reference ranges or on the borderline. Vitamin D deficiency was found in all patients. Weak correlation was found between vitamin A or vitamin D and estimated glomerular filtration rate (eGFR). The Pearson correlation coefficient were − 0.21766 and 0.19752, respectively. Hypertension, diabetes mellitus, and atherosclerosis were the major comorbidities.For the first time, the serum levels of 9 vitamins were measured simultaneously in patients with CKD at different stages. Vitamin D deficiency was found in all patients. Weak correlation between vitamin A or vitamin D and eGFR was found. Chronic kidney disease (CKD) refers to kidney damage and decreased renal function, which can be classified into 5 stages according to KDOQI guidelines . The gloOver the years, both basic research and clinical trials have improved our understanding of the interaction between disordered vitamins and CKD , 8. CKD Renal dysfunction of CKD patients is reportedly associated with a decrease in plasma vitamin C level. Moreover, decreased vitamin C may cause endothelial dysfunction via an increase in oxidative stress in non-diabetic chronic kidney disease patients . Hyperho2 and an albumin-creatinine ratio (ACR) of 30 mg/g or greater were retained [CKD was defined as abnormalities of kidney structure or function, present for more than 3 months, with implications for health (Not graded). The diagnostic thresholds were estimated glomerular filtration rate (eGFR) of less than 60 mL/min per 1.73 mretained , 20 CKD retained . The eGFFrom May 2018 to September 2019, consecutive patients with primary diagnosis of CKD and hospitalized at the Department of Nephrology, the First Medical Center of the General Hospital of People’s Liberation Army, were invited to participate the study. Exclusion criteria were (1) acute kidney injury (any of the following: increase in serum creatinine (sCr) by ≥ 0.3 mg/dl (≥ 26.5 µmol/l) within 48 h; or an increase in serum creatinine to ≥ 1.5 times baseline for 7 days; or a urine volume < 0.5 ml/kg/h for 6 h.);  (2) younThe study was approved by ethics committee of First Medical Center of Chinese People’s Liberation Army General Hospital and carried out in accordance with the declaration of Helsinki.2). Complete blood count, alkaline phosphatase (ALP), calcium, phosphorus, iron, albumin, creatinine, cystatin C, total cholesterol (CHOL), triglyceride (TG), high‑density lipoprotein (HDL), low‑density lipoprotein (LDL), homocysteine (Hcy) and other biochemical and clinical tests were performed as routine investigations. Renal biopsy was performed to investigate the possible etiology of CKD.Following our standardized protocols, demographic data and anthropometric measurements were collected by trained investigators. eGFR was calculated by the CKD Epidemiology Collaboration (CKD-EPI) equation . Body ma− 10 mol/L, vitamin B1: 3.4 × 10− 9 mol/L, vitamin B2: 4.3 × 10− 8 mol/L, vitamin B6: 1.5 × 10− 10 mol/L, vitamin B9: 2.3 × 10− 10 mol/L, vitamin B12: 1.7 × 10− 12 mol/L, vitamin C: 2.8 × 10− 6 mol/L, vitamin D: 3.8 × 10− 9 mol/L, vitamin E: 6.0 × 10− 6 mol/L.Serum concentrations of 9 vitamins, vitamin A, B1, B2, B6, B9, B12, C, D and E were measured by electroanalytical method with Multi-Vitamin Analyzer  following the manufacturer’s instructions. Briefly, 10ml venous blood sample was collected after 12-hour fasting and was centrifuged at 3000 rpm for 10 min quickly after collection. The separated serum sample were stored at 4 °C for testing. Vitamin E had to be tested within 24 h of sample collection, while other vitamins could be tested within 3 days. Sample preparation solution and vitamin standard solution for calibration were provided by the Tianjin Lanbiao Electronic Technology Development Co. Ltd, China. The detection limitation for different vitamins were vitamin A: 8.5 × 10The concentration of a specific vitamin in serum sample below the following reference value was defined as deficiency, vitamin A: 0.70 µM, vitamin B1: 80.3 nM, vitamin B2: 106 nM, vitamin B6: 20 nM, vitamin B9: 10 nM, vitamin B12: 150 pM, vitamin C: 11 µM, vitamin D: 50 nM, and vitamin E: 23 µM. Normal value of serum homocysteine is below 15 µM –31.p-values were two-tailed and those less than 0.05 were considered statistically significant.All statistical analyses were performed using SAS . Each variable was tested for normality before statistical analysis. Data were presented as median  or frequency (%). The difference between two groups were analyzed by the chi-squared test, Fisher’s Exact test, Kruskal-Wallis test or Student-Newman-Keuls test where appropriate. Pearson correlation coefficient was calculated to characterize the correlation between two variables. Multiple linear regression analysis was performed to examine the correlation between significant variables and eGFR. All In total, 759 CKD patients were included in this study. Based on their eGFR values, the participants were assigned to 5 different stages according to the classification criteria described in the p < 0.001). The major pathological type of CKD in this cohort was IgA nephropathy (40.6 %) followed by membranous nephropathy at a prevalence of 31.5 %.Based on the results of renal biopsy, the pathological classification of CKD patients was displayed in Table p < 0.001). The incidence rates of other comorbidities, such as liver disease (14.23 %), gastrointestinal diseases (11.86 %), respiratory diseases (10.93 %), cancer (5.80 %), and coronary heart disease (5.40 %) were relatively low, and no difference was found in different stages. More details of the common comorbidities in this cohort were shown in Table Hypertension was the most common comorbidity in this cohort. 437 (75.47 %) patients were suffering from hypertension. Its incidence increased along with the increase of CKD severity, ranging from 29 % of stage 1 patients to 87 % of stage 5 patients. 140 (18.44 %) and 81 (10.67 %) patients had diabetes mellitus (DM) and atherosclerosis, respectively. The incidence rates of hypertension, DM, and atherosclerosis were higher in advanced stages than in lower stages (p < 0.001). The changing of vitamin D level was associated with the severity of eGFR staging (p < 0.001), age (p = 0.004) and sex (p = 0.024). The concentration of vitamin A increased from stage 1 to stage 5. However, the changing of vitamin A level was only correlated with eGFR staging (p < 0.001) and sex (p = 0.002). No statistical difference was noticed in vitamin B9, C or E between different stages.The serum level of 9 vitamins were exhibited in Table p = 0.0307) was too small to be practically meaningful. Vitamin A was inversely associated with eGFR , whilst vitamin D  was positively associated  million CKD patients . The pre.9–125.0 Hypertension, DM and CVD are common coexisting chronic diseases of CKD in clinical setting, where one is not necessarily more central than the other. CKD is not only a common cause of hypertension but also a complication of uncontrolled hypertension . The prep < 0.001) between vitamin D and eGFR was identified, indicating that circulating vitamin D concentrations were lower in patients of advanced stages and with lower eGFR. Vitamin D deficiency may be related to a decrease in intake from food, supplement, insufficient sunlight exposure, or generation of vitamin D3 from 7-DHC [Vitamins, water or fat-soluble, are important for normal cellular function as well as the growth and development of human body. Due to this, vitamin deficiency may cause severe health problem. It was reported that vitamins played important roles in the prognosis of CKD. Hyperhomocysteinemia (hHcy) was highly prevalent and strongly associated with renal function deterioration in patients with CKD. Vitamins B9 and B12 regulate the metabolism of homocysteine and reduce the odds of CKD progression by 83 % . Vitaminom 7-DHC . Due to om 7-DHC . Since lom 7-DHC , 40 the Except for vitamin D, the serum levels of other vitamins were all within the reference range or on the borderline. Circulating vitamin A and its metabolites are essential for cellular maintenance, gene regulation, lipid metabolism, and inflammatory response . In the The limitations to our research include (1) absence of data about dietary intake, enteral feeds, vitamin supplements, and medications related to bone and mineral metabolism of the patients, which made it difficult to test etiologic hypotheses. (2) The presence of a vitamin in serum may neither be able to reflect the adequacy and content at the tissue or cellular level nor the efficiency of utilization at the tissue or cellular level. Patients may exhibit functional deficiency of a vitamin despite normal serum content. (3) This was a single-center study conducted in a tertiary hospital. Multi-center studies with a large sample size, particularly if outpatients are included, should be carried out in the future to alleviate the possible biases and confounding factors.To our knowledge, for the first time the status of 9 vitamins were assessed in a single center study at one time. Except for vitamin D, vitamin deficiency was not a serious problem in this cohort. Weak correlation of vitamin A or D with the severity of renal function was identified. Patients’ ages have been decreasing from those in previous reports; whether it is correlated to the elevated prevalence of comorbidities, such as hypertension and/or diabetes, requires further study.