A population-based urinary and plasma metabolomics study of environmental exposure to cadmium.

BACKGROUND: The application of metabolomics-based profiles in environmental epidemiological studies is a promising approach to refine the process of health risk assessment. We aimed to identify potential metabolomics-based profiles in urine and plasma for the detection of relatively low-level cadmium (Cd) exposure in large population-based studies. METHOD: We analyzed 123 urinary metabolites and 94 plasma metabolites detected in fasting urine and plasma samples collected from 1,412 men and 2,022 women involved in the Tsuruoka Metabolomics Cohort Study. Regression analysis was performed for urinary N-acetyl-beta-D-glucosaminidase (NAG), plasma, and urinary metabolites as dependent variables, and urinary Cd (U-Cd, quartile) as an independent variable. The multivariable regression model included age, gender, systolic blood pressure, smoking, rice intake, BMI, glycated hemoglobin, low-density lipoprotein cholesterol, alcohol consumption, physical activity, educational history, dietary energy intake, urinary Na/K ratio, and uric acid. Pathway-network analysis was carried out to visualize the metabolite networks linked to Cd exposure. RESULT: Urinary NAG was positively associated with U-Cd, but not at lower concentrations (Q2). Among urinary metabolites in the total population, 45 metabolites showed associations with U-Cd in the unadjusted and adjusted models after adjusting for the multiplicity of comparison with FDR. There were 12 urinary metabolites which showed consistent associations between Cd exposure from Q2 to Q4. Among plasma metabolites, six cations and one anion were positively associated with U-Cd, whereas alanine, creatinine, and isoleucine were negatively associated with U-Cd. Our results were robust by statistical adjustment of various confounders. Pathway-network analysis revealed metabolites and upstream regulator changes associated with mitochondria (ACACB, UCP2, and metabolites related to the TCA cycle). CONCLUSION: These results suggested that U-Cd was associated with metabolites related to upstream mitochondrial dysfunction in a dose-dependent manner. Our data will help develop environmental Cd exposure profiles for human populations.

Reliability of Time-Series Plasma Metabolome Data over 6 Years in a Large-Scale Cohort Study.

Studies examining long-term longitudinal metabolomic data and their reliability in large-scale populations are limited. Therefore, we aimed to evaluate the reliability of repeated measurements of plasma metabolites in a prospective cohort setting and to explore intra-individual concentration changes at three time points over a 6-year period. The study participants included 2999 individuals (1317 men and 1682 women) from the Tsuruoka Metabolomics Cohort Study, who participated in all three surveys-at baseline, 3 years, and 6 years. In each survey, 94 plasma metabolites were quantified for each individual and quality control (QC) sample. The coefficients of variation of QC, intraclass correlation coefficients, and change rates of QC were calculated for each metabolite, and their reliability was classified into three categories: excellent, fair to good, and poor. Seventy-six percent (71/94) of metabolites were classified as fair to good or better. Of the 39 metabolites grouped as excellent, 29 (74%) in men and 26 (67%) in women showed significant intra-individual changes over 6 years. Overall, our study demonstrated a high degree of reliability for repeated metabolome measurements. Many highly reliable metabolites showed significant changes over the 6-year period, suggesting that repeated longitudinal metabolome measurements are useful for epidemiological studies.

Metabolic profiling of charged metabolites in association with menopausal status in Japanese community-dwelling midlife women: Tsuruoka Metabolomic Cohort Study.

BACKGROUND: Emerging evidence has shown that charged metabolites, such as amino acids, may play an important role in the pathogenesis of various metabolic disorders, many of which women in the postmenopausal period are at high risk of developing. This study examined the metabolic profile of middle-aged Japanese women to investigate alterations in charged metabolites induced by menopausal transition. METHODS: The participants were 1193 female residents aged 40-60 at the baseline survey of the Tsuruoka Metabolomics Cohort Study. We investigated the cross-sectional association of menopausal status with 94 metabolomic biomarkers assayed in fasting plasma samples via capillary electrophoresis time-of-flight mass spectrometry using linear regression analysis. RESULTS: Among the participants, 529 were premenopausal, 132 were in menopausal transition (MT), and 532 were postmenopausal. Significant differences were found in age, blood pressure, glucose and lipid levels, and smoking and drinking habits among the three groups. The concentrations of 5 metabolites in the MT group and 15 metabolites in the postmenopausal group were significantly higher than those in the premenopausal group after adjusting for confounding factors. When classified into pathways, these metabolites were related to the tricarboxylic cycle, urea cycle, and homocysteine metabolism, some of which are linked to arteriosclerosis. CONCLUSION: Multiple charged metabolites were associated with women's menopausal status, showing a gradual increase as women shifted from pre-, to peri-, to postmenopause. These findings might reflect the early changes behind the increased risk of dyslipidemia, diabetes, cardiovascular disease, and osteoporosis in later life.

Reliability of urinary charged metabolite concentrations in a large-scale cohort study using capillary electrophoresis-mass spectrometry.

Currently, large-scale cohort studies for metabolome analysis have been launched globally. However, only a few studies have evaluated the reliability of urinary metabolome analysis. This study aimed to establish the reliability of urinary metabolomic profiling in cohort studies. In the Tsuruoka Metabolomics Cohort Study, 123 charged metabolites were identified and routinely quantified using capillary electrophoresis-mass spectrometry (CE-MS). We evaluated approximately 750 quality control (QC) samples and 6,720 participants' spot urine samples. We calculated inter- and intra-batch coefficients of variation in the QC and participant samples and technical intraclass correlation coefficients (ICC). A correlation of metabolite concentrations between spot and 24-h urine samples obtained from 32 sub-cohort participants was also evaluated. The coefficient of variation (CV) was less than 20% for 87 metabolites (70.7%) and 20-30% for 19 metabolites (15.4%) in the QC samples. There was less than 20% inter-batch CV for 106 metabolites (86.2%). Most urinary metabolites would have reliability for measurement. The 96 metabolites (78.0%) was above 0.75 for the estimated ICC, and those might be useful for epidemiological analysis. Among individuals, the Pearson correlation coefficient of 24-h and spot urine was more than 70% for 59 of the 99 metabolites. These results show that the profiling of charged metabolites using CE-MS in morning spot human urine is suitable for epidemiological metabolomics studies.

Correction: Charged metabolite biomarkers of food intake assessed via plasma metabolomics in a population-based observational study in Japan.

[This corrects the article DOI: 10.1371/journal.pone.0246456.].

Changes in the microcirculation in periodontal tissue due to experimental peri-implantitis.

OBJECTIVES: Peri-implantitis causes dislodgement of dental implants due to inflammation in the peri-implant tissue. The microcirculation in the periodontal tissue undergoes morphological and physiological changes due to inflammation. The immune mechanism of peri-implantitis differs from that of periodontitis. In this study, we examined the changes in the microcirculation in the peri-implant tissue with experimentally induced inflammation, using morphological and physiological techniques. METHODS: Six beagle dogs were used in the experiment. After extracting both mandibular premolars, three titanium screw implants were inserted on each side of the mandibular jaw. Dental floss was placed on the right side for 90 days in the study group but not in the control group. Microvascular resin cast models were created, and morphological changes were observed using scanning electron microscopy. Periodontal blood flow was measured using laser Doppler flowmetry. RESULTS: Ninety days after induction of inflammation, bone resorption was observed around the implant body. Osseointegration was impaired, and a gap at the implant-bone interface was observed. The resin cast models showed that inflamed gingival blood vessels had invaded the bone marrow through the resorbed apical margin of the alveolar bone. Analysis of the physiological data obtained using laser Doppler flowmetry showed a significant increase in blood flow around the implants with experimentally induced inflammation. CONCLUSIONS: Significant morphological and physiological changes occur in the gingival microcirculation of peri-implant tissue due to inflammation. Evaluating the vasculature and blood flow in the tissue surrounding the site of peri-implantitis may be helpful for pathologic analysis in clinical settings.

Charged metabolite biomarkers of food intake assessed via plasma metabolomics in a population-based observational study in Japan.

Food intake biomarkers can be critical tools that can be used to objectively assess dietary exposure for both epidemiological and clinical nutrition studies. While an accurate estimation of food intake is essential to unravel associations between the intake and specific health conditions, random and systematic errors affect self-reported assessments. This study aimed to clarify how habitual food intake influences the circulating plasma metabolome in a free-living Japanese regional population and to identify potential food intake biomarkers. To achieve this aim, we conducted a cross-sectional analysis as part of a large cohort study. From a baseline survey of the Tsuruoka Metabolome Cohort Study, 7,012 eligible male and female participants aged 40-69 years were chosen for this study. All data on patients' health status and dietary intake were assessed via a food frequency questionnaire, and plasma samples were obtained during an annual physical examination. Ninety-four charged plasma metabolites were measured using capillary electrophoresis mass spectrometry, by a non-targeted approach. Statistical analysis was performed using partial-least-square regression. A total of 21 plasma metabolites were likely to be associated with long-term food intake of nine food groups. In particular, the influential compounds in each food group were hydroxyproline for meat, trimethylamine-N-oxide for fish, choline for eggs, galactarate for dairy, cystine and betaine for soy products, threonate and galactarate for carotenoid-rich vegetables, proline betaine for fruits, quinate and trigonelline for coffee, and pipecolate for alcohol, and these were considered as prominent food intake markers in Japanese eating habits. A set of circulating plasma metabolites was identified as potential food intake biomarkers in the Japanese community-dwelling population. These results will open the way for the application of new reliable dietary assessment tools not by self-reported measurements but through objective quantification of biofluids.

Morphological study of human facial fascia and subcutaneous tissue structure by region through SEM observation.

Fascia of the facial area is contiguous between fat tissues of the subcutaneous and connective tissue layers and does not envelope the muscle surface like other parts of the human body. This structure is called the superficial musculoaponeurotic system (SMAS), which is accepted as an international anatomical terminology. This special structure is commonly used to pull facial muscles during plastic surgeries such as a face lift. Most reports regarding the facial subcutaneous tissue structure including SMAS are in the field of plastic surgery, and only a few studies from a morphological perspective has been reported. Since the facial fascia does not envelope the muscular surface layer which is different from the deep fascia found on the general skeletal muscle surface, a clear definition of this structure has not been established yet. The purpose of this study was to clearly identify the basic morphological structure of the subcutaneous tissue layer containing the SMAS three-dimensionally through a scanning electron microscope using dissected specimen rather than living subjects. Moreover, this study explores structural differences among seven aging facial areas; thereby further clarifying the properties of the structure and add clinical significance and considerations.

Severe kyphosis as a risk factor of umbilical trichophytosis in elderly people.

Objective: To determine whether severe kyphosis is one of the risk factors of umbilical trichophytosis in elderly people. Patients and Methods: Four cases with umbilical trichophytosis in the last 3 years and all 143 patients who visited to our clinic during from April 2018 to March 2019 were included in this study. The relationship between the condition of umbilical region and kyphosis was investigated. As regards the levels of senile kyphosis, simple visual classification of the kyphosis method was used (Level 0-4). Results: All four cases with umbilical trichophytosis were elderly women aged ≥80 years, had severe kyphosis, skin overlaps, filthy umbilical region, and lumps of dirt in the umbilical region and had nail tinea. Only two cases had umbilical trichophytosis among 143 patients who visited our clinic during the 1 year study period, with incidence is of 1.4%/year. About 65.7%, 60%, and 37.1% of 35 patients with kyphosis with forward-bent posture had skin overlap, moist, and filthy umbilical regions, respectively. However, these symptoms were not found in patients without severe kyphosis. Conclusion: Severe kyphosis with forward-bent posture could be one of the risk factors of umbilical trichophytosis in elderly people.

Study of morphological structure in human fetus Wharton's duct opening area.

OBJECTIVES: Not many morphological studies have been reported regarding the human salivary main duct, especially those based on observations of the fetal salivary main duct at various developmental stages. The relationships between fetal structural development and its function, functional disorders and diseases certainly have clinical significance. METHODS: In this study, we conducted a morphological observation of seven 5-9 month old human fetal sublingual caruncula, the common opening between the submandibular (Wharton's) duct and the sublingual (Bartholin's) duct. After seven specimens were removed and paraffin sections were prepared, HE and EVG staining were used for histological study of structural differences in the various developmental stages. Furthermore, anti-α-SMA staining was used to observe the distribution of the smooth muscle inside of the Wharton's duct wall. RESULTS: An abundance of elastic fibers were observed in the duct walls of both five and nine month old specimens with EVG staining. The smooth muscle fibers of five month old fetus were immature and were starting to distribute throughout the duct wall, whereas nine month old fetus' smooth muscle fibers were further developed and composed in layers. CONCLUSION: In this study, we confirmed that the Wharton's duct wall structure of nine month old fetuses was close to its adult structural formation, while the inner structure consisted of an ample distribution of elastic fibers and smooth muscle fibers. Based on the distribution pattern of the smooth muscle, we speculate that the Wharton's duct's opening area does not possess a sphincter-like function in the regulation of salivary flow.

Hyperactive mutation occurs adjacent to the essential glutamate 286 for transport in the yeast tryptophan permease Tat2.

In Saccharomyces cerevisiae, high-affinity tryptophan import is mediated by the plasma membrane permease Tat2. Herein, we identified hyperactive Tat2 mutations, I285V and I285T, which allowed the cells to grow at very low tryptophan concentrations (<4 µg/mL). The Km value of wild-type Tat2 for tryptophan appeared to be 24 µg/mL, whereas that of Tat2I285V and Tat2I285T was 17 and 11 µg/mL, respectively. Normalized values of Vmax/Km for Tat2I285V- and Tat2I285T-mediated tryptophan import were 2-fold higher than that for Tat2, suggesting that these mutations increase the affinity for tryptophan, and mediate transport at very low tryptophan concentrations. I285 resides adjacent to E286, a fully conserved residue among amino acid pemreases. According to a pKa prediction for E208 (pKa ∼8.3-11.7) of Escherichia coli AdiC antiporter, a structural homologue of Tat2, the E286 carboxyl chain of Tat2 could get loaded with a proton during tryptophan/H+ symport. Hence, I285V and I285T mutations might affect the buried residue environment of Tat2, thereby facilitating tryptophan import. Additionally, Tat2I285V and Tat2I285T levels increased rapidly, and were efficiently localized to the cell surface after transferring the cells to low tryptophan medium (0.5 µg/mL). Our findings provide a clue to gain insights into the property of high-affinity transport mechanisms, and offer a unique approach to improve the functionality of broad types of amino acid permeases.

Morphometric aspects of the facial and skeletal muscles in fetuses.

OBJECTIVES: There are few research reports providing a comparison of the muscle fiber morphometry between human fetuses and adults. Data on fetal and adult muscle fibers would be valuable in understanding muscle development and a variety of muscle diseases. This study investigated human muscle fiber growth to clarify the difference between the facial muscles and other skeletal muscles. METHODS: The materials were obtained from three male fetuses (6-month-old) and 11 Japanese male cadavers aged 43-86 years (average: 71.8). Human buccinator muscles (facial muscles), masseter and biceps brachii muscles (skeletal muscles) were resected. We counted the muscle fibers and measured their transverse area. We also calculated the number of muscle fibers per mm(2) (NMF) and the average transverse area of the muscle fibers (TAMFs). RESULTS: The average of the NMF of the buccinator, masseter and biceps brachii muscles in fetuses had, respectively, 19, 37, and 22 times as many fibers as those in adults. The average fetus/adult ratios of the TAMF of the buccinator, masseter and biceps brachii muscles were 4.0%, 2.4%, 4.1%, respectively. CONCLUSIONS: The average NMF for all kinds of muscles decreased after birth; however, the peak in life-span or decreases with the aging process tended to vary with the kind of muscles examined. The average TAMF for all kinds of muscles enlarged after birth. We considered that the enlargement of the TAMF was connected with the emergence of fetal movements and functional demands after birth.

Nerve innervations in the human adult and fetal parotid duct

The human adult parotid duct is the longest of all major salivary gland ducts, approximately 6-8 cm in length. Its unique structure extends over the masseter muscle, penetrates through the buccinator muscle and opens into the oral cavity. Salivary secretion is under basic control of the sympathetic and parasympathetic divisions of the autonomic nervous system. Scarce reporting on the parotid duct nerve distribution led us to this study; to investigate the nervous distribution in the human adult and fetal parotid ducts using an antibody against protein gene product 9.5 (PGP9.5), a molecular marker for nerve cells and fibers. In order to identify the nerve fibers distributed throughout the parotid duct and confirm them to be part of the autonomic nervous system, we stained adult parotid ducts with tyrosine hydroxylase (TH) and choline acetyltransferase (ChAT) for observation. PGP9.5 staining of the parotid duct’s inside wall where it traverses over the masseter prior to penetrating the buccinator revealed a dense concentration of nerve fibers in the area. Staining revealed both sympathetic and parasympathetic nerve fibers in the same area, with the majority of the sympathetic nerve fibers surrounding blood vessels. However, the section of the duct penetrating the buccinator showed less concentration of nerve fibers in both adult and fetal specimens. The difference in the nerve distribution of the parotid duct suggests its direct association with the salivary transport function of the duct. PGP9.5 expression in fetuses over five months of age further suggests that the nerve distribution in the human parotid duct is fully established at six months of gestation

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Study of human adult parotid duct in the area of penetration through buccinator muscle and their functional relationship as a sphincter.

The adult human parotid duct is roughly 6-8 cm long. From the parotid gland, parotid duct traverses through masseter muscle, penetrates through buccinator muscle, and opens into the oral cavity. This unique form of the parotid duct is likely correlated with the function of the duct, directly affected by the movement of the buccinator muscle during mastication and swallowing. Histological structure of the duct is known to be different in each region, and details of smooth muscle present in the parotid duct are mostly unclear. In this study, we conducted SEM and histological observations of the area where the parotid duct penetrates the buccinator muscle, and an observation of smooth muscle to investigate its existence using alpha-smooth muscle antibody. We confirmed the presence of an abundance of skeletal muscle bundles likely originating from the buccinator muscle under the epithelium of the parotid duct wall in the region where it penetrates the buccinator muscle. We also observed that some of the muscle fibers were completely attached to the epithelium. We observed a lack of smooth muscle in this region of the duct wall. From these results, we suggest that the area of the duct penetrating buccinator muscle plays a role in regulating the salivary passage through the contraction of the surrounding buccinator muscle fibers.

Morphological study of the fetal parotid duct and buccinator muscle and the relationship to salivary secretion.

The parotid glands secrete about 25% of all saliva produced. In the presence of a stimulus, the amount of saliva secreted from the parotid gland increases to 50%. A decrease in the amount of produced saliva due to aging and parotiditis results in a dry mouth. Therefore, the parotid duct is important to maintaining a healthy oral cavity. In human adults, the parotid duct, approximately 6-8-cm long, travels over the masseter muscle and penetrates the buccinator muscle to enter the oral cavity. Although there have been various studies regarding the parotid gland, only few suggest a functional role of the parotid duct, especially its area of penetration of the buccinator muscle. In this study, 34 fetal specimens ranging from 4 to 10 months of age at death were dissected for anatomical and histological examinations. The area of the parotid duct penetrating the buccinator muscle was fully formed in 5-month-old fetuses. This study found buccinator muscle fibers invading the parotid duct wall near its opening in 6-month-old fetuses and older. Our results support the claim that the buccinator muscle may act as a sphincter, playing a role in regulating and possibly preventing the reflux of salivary secretions into the parotid duct.

Morphological study of the parotid duct in human fetuses with special emphasis on the relationship between the buccinator muscle and the parotid duct.

Parotid glands secrete about 25% of all saliva produced in the salivary glands. In the presence of a stimulus, the amount of saliva secreted from the parotid gland increases to 50% (1). In human adults, the parotid duct, approximately 6-8 cm long, traverses the masseter muscle and penetrates through the buccinator muscle into the oral cavity. Although various studies have been conducted on the parotid gland, there are only few suggesting the functional roles of the parotid duct, especially of the area penetrating the buccinator muscle. In the present study, we observed parotid ducts of human fetuses to morphologically analyze the function of the buccinator muscles in the flux of parotid saliva. Thirty fetal specimens ranging from five to ten months of age were dissected for anatomical and histological examinations. The area of the parotid duct penetrating the buccinator muscle was fully formed in six-month-old fetuses. Furthermore, this study confirms the existence of thin buccinator muscle fibers underneath the epithelium of the parotid duct's distal portion. Results suggest that the buccinator muscle may play a major role in preventing the reflux of salivary secretions by assisting the contraction of the parotid duct.

Morphometric aspects of peripheral nerves in adults and the elderly.

There have been inconsistencies among reports of age-related differences in human peripheral nerves (PNs). For such studies, normal control values are necessary. Moreover, the diversity of methods employed makes it difficult to compare results. We used the same histological procedures and methods to measure 12 PNs: 8 in the cranial nerves, 2 motor nerves in the lower limb, and 2 nerves in the autonomic system. We performed a morphometric analysis of nerve fibers and estimated the change in the total number (TN) and average transverse area (ATA) of myelinated axons from adulthood to old age. The spinal nerves demonstrated notable age-related changes in TN and ATA. Most of the cranial nerves also demonstrated notable age-related changes in TN and ATA. However, some nerves demonstrated no such age-related changes and were affected more by other factors. With regards to the autonomic nerves, the lesser splanchnic nerve indicated age-related changes in TN, but the greater splanchnic nerve indicated no age-related changes in either TN or ATA. The autonomic nerves were affected not only by the aging process but also by the pathological changes to the peripheral tissues that they innervate.

In vitro apatite induction by osteopontin: interfacial energy for hydroxyapatite nucleation on osteopontin.

Noncollagenous phosphoproteins that interact with type I collagen are thought to nucleate the mineral phase to the collagen fibril network of mineralized tissues. We previously reported that a specific dentin phosphoprotein, phosphophoryn, crosslinked to an insoluble substrate such as type I collagen fibrils, was an effective nucleator of apatite. In this study, we investigated the capacity of another phosphoprotein in dentin, osteopontin, for apatite nucleation in vitro. Osteopontin purified from bovine milk was either absorbed to agarose beads or crosslinked to agarose beads. Each preparation was incubated at 37 degrees C in metastable solutions. Apatite was induced by osteopontin that was crosslinked to agarose beads, whereas osteopontin adsorbed to agarose beads failed to induce apatite crystal formation. Using classical nucleation theory, the interfacial energy for hydroxyapatite nucleation on osteopontin crosslinked to agarose beads was determined to be 94.7 ergs/cm(2). This value is larger than that for phosphophoryn, though it is similar to that for hydroxyapatite. The present study indicates that osteopontin, like phosphophoryn, has a high potential to induce apatite formation when it is covalently bound to some substrate in vitro, and suggests the possibility that osteopontin bound to type I collagen may induce apatite formation in vivo.