Development of a particle packed bed model for homogeneity evaluation of liquid chromatography column.

Particle packed columns used for liquid chromatography (LC) can suppress a column internal band broadening (hereinafter referred to as band broadening) by packing monodisperse particles homogenously. However, a quantitative evaluation for the effects of particle shape and packed state on band broadening needs to be more investigated. In this study, we fabricated a model of particle packed bed using microfluid LC columns that have pillar array structure prepared by microfabrication technology, evaluating how structural factors inside of a column affect its band broadening. At first, microfluid LC columns was prepared using Si-quartz glass (Si-Q column) for the optimization of LC measurement system. Through the evaluation, it showed 11.6 times higher pressure tolerance compared to that of PDMS-soda lime glass (PDMS-g column). Then, an optimized LC measurement system was constructed using a microfluidic LC column made of Si-Q column, and it was confirmed that the measurement error was small enough and the LC measurement could be performed with high repeatability. Additionally, the effect of a distribution of structural size on band broadening was evaluated. It was confirmed that wide distribution of the structural size provided large band broadening in actual measurements. Comparing two columns having different structural log-normal distributions of 0 and 0.22 showed approximately 1.8 times difference in both real LC measurement. Lastly, the relationship between packed state and band broadening was evaluated. As packed state, we employed void arrangement and structural arrangement in the columns. Different location arrangements of 50 and 100 µm pillar sizes afforded different band broadening. Well-homogenized array showed approximately two times worse band broadening compared to that of delocalized array. Based on these results, the developed packed bed of particles model was able to evaluate the relation between structural factors and band broadening.

A Boy with Reset Osmostat Who Developed Chronic Hyponatremia due to Hypothalamic Injury Caused by a Giant Arachnoid Cyst.

Reset osmostat (RO) is classified as type C among the four subtypes of the syndrome of inappropriate secretion of antidiuretic hormone based on antidiuretic hormone (ADH) secretion. It is characterized by a lower plasma osmolality threshold for ADH excretion when plasma sodium concentration is reduced. We report the case of a boy with RO and a giant arachnoid cyst (AC). The patient had been suspected of having AC since the fetal period, and a giant AC in the prepontine cistern was confirmed by brain magnetic resonance imaging 7 days after birth. During the neonatal period, there were no abnormalities in the general condition or blood tests, and he was discharged from the NICU at the 27 after birth. He was born with a -2 standard deviation short stature and mild mental retardation. When he was 6 years old, he was diagnosed with infectious impetigo and had hyponatremia of 121 mmol/L. Investigations revealed normal adrenal and thyroid functions, plasma hyposmolality, high urinary sodium, and high urinary osmolality. The 5% hypertonic saline and water load tests confirmed that ADH was secreted under low sodium and osmolality conditions, and the ability to concentrate urine and excrete a standard water load; therefore, RO was diagnosed. In addition, an anterior pituitary hormone secretion stimulation test was performed, which confirmed growth hormone secretion deficiency and gonadotropin hyperreactivity. Hyponatremia was untreated, but fluid restriction and salt loading were started at age 12 because of the risk of growth obstacles. The diagnosis of RO is important from the viewpoint of clinical hyponatremia treatment options.

Selective Separation of Thyroid-Hormone-Receptor-Binding Substances Using Molecularly Imprinted Polymers.

To date, an identification protocol for endocrine disruptors that bind to the thyroid hormone receptor (TR) has not been established. A method for screening and identifying TR-binding substances is highly required due to the existence of unknown TR-binding substances from the environment. Here, we conceived a chromatographic method using a molecularly imprinted polymer (MIP) to create a novel screening protocol for the endocrine disruptors. A receptor-imitating MIP was prepared using N-acetylthyroxine (AcetylT4) and 4-vinylpyridine as a pseudo-template and a functional monomer, respectively, based on the existing molecular recognition mechanism of the TR. The receptor-imitating MIP provided molecular recognition ability for all the TR-binding substances that were employed in this study. The prepared MIPs were packed into a high-performance liquid chromatography column for the simultaneous analysis of TR-binding and non-binding substances. The former was strongly retained, while the latter was not. The presence or absence of TR-binding/non-binding activity resulted in successful dichotomous separation. Additionally, the surface imprinting technique was applied to improve the separation performance of the MIP packing material. MIP-coated uniformly sized silica-based particles of 5 µm were successfully prepared, and the MIP-coated silica column enabled more efficient dichotomous separation of TR-binding and non-binding substances.

Rare de novo inversion-duplication case with pure 3qter duplication syndrome including an overlap of the dup(3q) critical region: A case report.

We report here a very rare case of de novo inversion-duplication chromosomal abnormality with a pure 3qter duplication syndrome. Interestingly, the 3q duplication includes an overlap of the syndromes critical region. Although there have only been 9 cases of this syndrome reported in the past, our patient had more severe neurological abnormalities than anticipated. In this regard, we have gathered the 3q chromosomal duplication abnormalities known to cause pure 3q duplication syndrome to date as a reference for comparisons and we discuss the particulars of our case.

Biallelic TBCD Mutations Cause Early-Onset Neurodegenerative Encephalopathy.

We describe four families with affected siblings showing unique clinical features: early-onset (before 1 year of age) progressive diffuse brain atrophy with regression, postnatal microcephaly, postnatal growth retardation, muscle weakness/atrophy, and respiratory failure. By whole-exome sequencing, we identified biallelic TBCD mutations in eight affected individuals from the four families. TBCD encodes TBCD (tubulin folding co-factor D), which is one of five tubulin-specific chaperones playing a pivotal role in microtubule assembly in all cells. A total of seven mutations were found: five missense mutations, one nonsense, and one splice site mutation resulting in a frameshift. In vitro cell experiments revealed the impaired binding between most mutant TBCD proteins and ARL2, TBCE, and ß-tubulin. The in vivo experiments using olfactory projection neurons in Drosophila melanogaster indicated that the TBCD mutations caused loss of function. The wide range of clinical severity seen in this neurodegenerative encephalopathy may result from the residual function of mutant TBCD proteins. Furthermore, the autopsied brain from one deceased individual showed characteristic neurodegenerative findings: cactus and somatic sprout formations in the residual Purkinje cells in the cerebellum, which are also seen in some diseases associated with mitochondrial impairment. Defects of microtubule formation caused by TBCD mutations may underlie the pathomechanism of this neurodegenerative encephalopathy.

Urinary neutrophil gelatinase-associated lipocalin is an early predictor of acute kidney injury in premature infants.

Urinary neutrophil gelatinase-associated lipocalin (uNGAL) is produced in response to tubular epithelial injury and is a biomarker of tubulointerstitial injury. The aim of the present study was to examine whether acute kidney injury (AKI) could be predicted by measuring uNGAL in very low-birth weight (VLBW) infants. Forty VLBW infants with birthweight below 1,500 g were enrolled in the present study. uNGAL and serum creatinine (sCre) were measured daily from postnatal days 0 to 8. Infants with sCre ≥1.2 mg/dl were diagnosed with AKI. The relationship of uNGAL with sCre was measured on the day after uNGAL measurement (next-day sCre) was examined. The results showed that 16 infants had sCre ≥1.2 mg/dl in this period. Logistic regression analysis revealed that uNGAL on postnatal days 2, 3, 4, 5 and 6 was correlated with next-day sCre (P<0.05). uNGAL corrected by urinary Cre (uCre) (uNGAL/uCre) was only correlated with an increase in next-day sCre on postnatal days 5 and 6 (P<0.05). For the logistic analysis, subjects with high and low uNGAL levels based on the median value for each day, uNGAL on postnatal days 2, 3 and 6 in the high uNGAL group was correlated with an increase in next-day sCre. Thus, AKI may be predicted by measuring uNGAL in VLBW infants. This measurement was non-invasive, and is potentially useful for the evaluation of renal function in VLBW infants.

Safety of lipid emulsion in very low-birthweight infants according to cytokine level.

BACKGROUND: The aim of this study was to verify whether lipid emulsion treatment aggravates infection and inflammation in very low-birthweight (VLBW) infants. STUDY DESIGN: Very low-birthweight (<1500 g) infants born at <32 weeks gestational age between October 2013 and October 2014 at Dokkyo Medical University Hospital (Mibu, Tochigi, Japan) were treated with or without i.v. nutrition with a lipid emulsion. Infants were excluded who had congenital abnormalities, could not receive i.v. nutrition because of poor general condition, or on physician decision. Lipid emulsion with purified soybean oil was initiated at 0.5 g/kg/day on postnatal day 1. The dose was increased to 1 g/kg/day, and then to 1.5 g/kg/day (maximum dose). Blood tests were performed before (day 1) and after (day 8) initiation of lipid emulsion treatment. Interleukin (IL)-6, IL-8, monocyte chemotactic protein 1 (MCP-1), tumor necrosis factor-α (TNF-α), C-reactive protein (CRP), total bilirubin (T-Bil), direct bilirubin (D-Bil) and insulin were measured. Changes in respiratory condition, amount of oxygen used, and phototherapy duration were investigated. RESULTS: A total of 17 treated and 15 untreated VLBW infants were enrolled. IL-6, IL-8, MCP-1, TNF-α, CRP, T-Bil, D-Bil and insulin on days 1 and 8; respirator or surfactant use; amount of oxygen used; and phototherapy duration were not significantly different between the two groups. CONCLUSIONS: Lipid emulsion treatment did not increase inflammatory cytokine levels or aggravate respiratory disorders. Lipid emulsions, if proven safe, could be used to treat VLBW infants soon after birth, which may prevent extrauterine growth restriction and improve intellectual development prognosis.

Trace level determination of polycyclic aromatic hydrocarbons in river water with automated pretreatment HPLC.

A novel on-line pretreatment pump-injection HPLC system for polycyclic aromatic hydrocarbons is proposed. We report novel pump-injection HPLC-based on-line SPE with a specially designed pretreatment column for the determination of trace amounts of chemical substances in surface water. Polycyclic aromatic hydrocarbons are well known for strong carcinogenicity and thus a severe concentration control is required for drinking water and/or river water, which is the main resource of tap water. We found it possible to detect ng/L levels of polycyclic aromatic hydrocarbons by using pump-injection column switching HPLC with fluorescence detection. To avoid the phenomenon, in which polycyclic aromatic hydrocarbons can be often adsorbed on the surface of flow lines of HPLC by their highly hydrophobicity especially resin-made parts in sample delivery pump, we employed "autodilution" device that provides reliable recovery and repeatability. Additionally, real water samples were collected and then the spiked polycyclic aromatic hydrocarbons were determined at ng/L levels.

Hypophosphatemia in small for gestational age extremely low birth weight infants receiving parenteral nutrition in the first week after birth.

OBJECTIVES: To investigate the risk of hypophosphatemia and hypercalcemia in small for gestational age (SGA) extremely low birth weight infants (ELBWI) receiving parenteral nutrition. METHODS: A retrospective review of 58 ELBWI was conducted. Serum calcium (Ca) and phosphate (PO4) concentrations on days 1 and 8 after birth were examined for associations with body measurements and nutritional factors in the 1st week of life. RESULTS: Lower birth weight standard deviation (SD) scores were correlated with hypophosphatemia and hypercalcemia in SGA ELBWI on day 8. Higher parenteral amino acid (AA) administration was correlated with hypophosphatemia on day 8. SGA ELBWI exhibited lower serum PO4 concentrations compared to appropriate for gestational age (AGA) ELBWI on day 8. CONCLUSIONS: This is the 1st study to report that parenteral nutrition, in the first 7 days after birth for the treatment of SGA ELBWI, was correlated with hypophosphatemia and hypercalcemia. It is important to determine an ideal nutrition protocol for treatment of SGA ELBWI.

Determination of bisphenol A with effective pretreatment medium using automated column-switching HPLC with fluorescence detection.

A practical way for reducing contaminants, such as humic acids, and solving column-clogging problem in environmental water analysis with liquid chromatography is proposed. Detection interference by contamination is one of the most important issues of the environmental analyses. Moreover, due to the recent smaller diameter and fine particle size of an analytical column for HPLC system, a column-clogging problem is another practical difficulty as well. We found it possible to solve these problems by employing column-switching HPLC, which consists of a pretreatment column containing surface-modified polymer particles and flow changeover valves for cleaning the remaining matrices in the pretreatment column prior to analysis. This method was successfully applied to actual HPLC-fluorescence detection of bisphenol A. Limit of detection (LOD) in real sample was <0.7 ng/L. Repeatability was around 1.4% and recovery was around 97% or more. A particular pressure increase was not observed in 150 repeated analyses of real river water samples.

Retention properties of macroporous spongy monolith and its application for concentration of polyaromatic hydrocarbons.

We report the fundamental chromatographic retention properties of spongy monolith that was previously reported as a novel separation medium for the effective pretreatment of environmental pollutants. According to the detailed examination on liquid chromatographic evaluations using hydrophobic, hydrophilic, and polyaromatic compounds as solutes, the spongy monolith consisted of poly(ethylene-co-vinyl acetate) and having macroporous co-continuous structures showed the selective retention ability for polyaromatic hydrocarbons (PAHs), especially coplanar compounds, whereas lower selectivity was observed for typical hydrophobic and hydrophilic compounds compared with the commonly used C18 medium. Moreover, we demonstrate the effective preconcentration of benzo[a]pyrene using a spongy monolith as a pretreatment column of an online column switching HPLC system.

Thyroxine for transient hypothyroxinemia and cerebral palsy in extremely preterm infants.

BACKGROUND: The relationship of thyroxine supplementation for transient hypothyroxinemia of prematurity to the incidence of cerebral palsy (CP) in infants <28 weeks of gestation is unclear. METHODS: The incidence of CP at a corrected age of 18 months was compared between infants born in a 3-year period in which routine measurement of free T4 (FT4) in the blood was not performed (first period, n= 54), and those born in a later 3-year period in which FT4 was measured (second period, n= 60; mainly at 7 days old), and in which l-thyroxine 5-10 µg/kg per day (mean, 9 µg/kg/day) was administered for FT4 levels <0.8 ng/dL. Incidence of CP at 3 years of age was also compared between the same groups. RESULTS: Background clinical factors between the two groups were comparable except for prenatal steroid administration, which was reduced in the second period. Incidence of CP at a corrected age of 18 months was significantly lower in the second period (3.3%) than in the first period (16.6%). Incidence of CP at 3 years of age was also significantly lower in the second period. Multiple logistic regression analysis using factors except thyroxine supplementation, for the total of 114 infants from both groups, found no perinatal factors related to the development of CP at a corrected age of 18 months. CONCLUSIONS: Thyroxine supplementation for transient hypothyroxinemia of prematurity may reduce the incidence of CP in extremely preterm infants. Large-scale randomized controlled trials are essential to determine the effects of thyroxine supplementation in reducing the incidence of CP among extremely preterm infants.

The IG-DMR and the MEG3-DMR at human chromosome 14q32.2: hierarchical interaction and distinct functional properties as imprinting control centers.

Human chromosome 14q32.2 harbors the germline-derived primary DLK1-MEG3 intergenic differentially methylated region (IG-DMR) and the postfertilization-derived secondary MEG3-DMR, together with multiple imprinted genes. Although previous studies in cases with microdeletions and epimutations affecting both DMRs and paternal/maternal uniparental disomy 14-like phenotypes argue for a critical regulatory function of the two DMRs for the 14q32.2 imprinted region, the precise role of the individual DMR remains to be clarified. We studied an infant with upd(14)pat body and placental phenotypes and a heterozygous microdeletion involving the IG-DMR alone (patient 1) and a neonate with upd(14)pat body, but no placental phenotype and a heterozygous microdeletion involving the MEG3-DMR alone (patient 2). The results generated from the analysis of these two patients imply that the IG-DMR and the MEG3-DMR function as imprinting control centers in the placenta and the body, respectively, with a hierarchical interaction for the methylation pattern in the body governed by the IG-DMR. To our knowledge, this is the first study demonstrating an essential long-range imprinting regulatory function for the secondary DMR.

MR imaging appearance of laryngeal atresia (congenital high airway obstruction syndrome): unique course in a fetus.

Congenital high airway obstruction syndrome (CHAOS) is a rare life-threatening syndrome. Most cases are diagnosed prenatally by US. We report a fetus with this syndrome that showed a unique course revealed on MRI. Ultrasonography at 22 weeks demonstrated that the fetus had ascites and bilaterally enlarged hyperechoic lungs. Congenital infection, congenital cystic adenomatoid malformation or CHAOS was suspected. Subsequent MRI performed at 24 weeks demonstrated bilaterally enlarged high-signal lungs, dilated bronchi, massive ascites, subcutaneous oedema and polyhydramnios. MRI confirmed the diagnosis of CHAOS. A second MRI at 35 weeks showed that the bilateral lung enlargement, ascites, oedema and polyhydramnios had resolved, but that the appearance of the airway was unchanged. The infant was delivered by caesarean section at 38 weeks of gestation and immediate tracheostomy was performed. This spontaneous regression was explained by a tracheo-oesophageal fistula that may have decreased the intrathoracic pressure.

Fully automated liquid chromatography-mass spectrometry determination of 17beta-estradiol in river water.

Surface modified molecularly imprinted polymers (SM-MIPs) for 17beta-estradiol (E2), utilizing 6-ketoecradiol as a pseudo template were prepared. MIPs for E2 were synthesized using 4-vinyl pyridine and ethylene dimethacrylate as a functional monomer and cross-linking agent, respectively. MIPs selectively retained E2 and provided excellent chromatographic resolution from interfering compounds inherent in river water sample matrices. Therefore, freshly prepared MIPs were applied to quantitative mass spectrometric (negative electrospray ionization mode) detection of low levels of E2 in river water sample. In order to pre-concentrate the target compound for HPLC analysis, column switching was coupled with a pretreatment column packed with the MIPs. The repeatability of actual determinations of river water sample, in which background E2 was not detected, spiked with 50 ng/L of E2 was 2.2% RSD with a detection limit and qualification limit of 1.8 and 5.4 ng/L, respectively. Surface modification of MIP particlefs packed in the pretreatment column provided selective affinity and on-line concentration of low levels of E2 while simultaneously eliminating sample matrix interference, resulting in a significant increase in sensitivity and reproducibility for liquid chromatography-mass spectrometry analysis of E2 in river water sample.

Degeneration of monoaminergic fibers in the aged micrencephalic rat.

Age-related changes in the monoaminergic neuron systems in the brains of methylazoxymethanol acetate (MAM)-induced micrencephalic rats were studied. Neurochemical analysis revealed high levels of serotonin, norepinephrine and associated metabolites in several brain areas of MAM-treated rats. In particular, serotonin levels in the frontal cortex, cingulate cortex and hippocampus of 12-month-old (12 M) MAM-treated rats were significantly higher than in corresponding age-matched controls. Immunohistochemical analysis demonstrated numerous aberrant serotonin-immunoreactive fibers and small numbers of aberrant tyrosine hydroxylase-immunoreactive fibers in the septum, caudate putamen, thalamus, cerebral cortex, hippocampus and midbrain tegmentum of 12 M MAM-treated rats. Aberrant monoaminergic fibers characterized by swollen varicosities and thickening of intervaricose segments were common compared to 12 M control rats. In the cortex and hippocampus of 12 M MAM-treated rats, aberrant fibers were observed near cortical heterotopic tissue. These results indicate early onset of age-related degeneration of monoaminergic fibers in micrencephalic rats. Aged MAM-treated rats may thus offer a good model for studying age-related monoaminergic changes in the cortical heterotopic tissue of human cortical malformations.

Silica monolithic membrane as separation medium. Summable property of different membranes for high-performance liquid chromatographic separation.

We studied an applicability of a silica monolithic membrane as separation medium for high-performance liquid chromatography (HPLC). We prepared porous monolithic silica membranes having a three-dimensional network structure to cut and shape into a membrane separation medium. We evaluated chromatographic properties of a variety of solutes using a column containing the membranes with HPLC to elucidate summable property of the membrane separation media. In addition, we made brief study on separation of HbA1c in whole blood with the stacked" membranes having different surface characteristics in one column, which is a membrane column. We confirmed that the membrane column was able to separate HbA1c from other matrix in whole blood to some extent, and it also had an excellent ability for hydrophobic and ion exchange adsorption.

Novel surface modified molecularly imprinted polymer focused on the removal of interference in environmental water samples for chromatographic determination.

Uniformly sized molecularly imprinted polymers (MIPs) for bisphenol A (BPA) with surface modification and immobilized intervals of functional monomers afforded by utilizing 4,4'-methylenebisphenol as a pseudo component have been prepared. MIPs for BPA were prepared using 4-vinyl pyridine immobilized in the most effective interval and ethylene glycol dimethacrylate as a functional monomer and cross-linking agent, respectively. Prepared MIPs showed significant selectivity for BPA retention and removal performance for interference in actual samples as the HPLC stationary phase compared to those of ordinary MIPs. These MIPs were employed as pretreatment media of column switching HPLC and the HPLC system provided a detection limit of 0.36 ppt when electrochemical detection was used. Actual samples, including Suwannee River natural organic matter (NOM), were applied and BPA was detected in the NOM even if widely used UV detection was employed.