A Methodological Basis for Estimating Human Mercaptalbumin in Serum and Plasma Using a Thiol-Binding Resin.

Human serum albumin is categorized into human mercaptalbumin (HMA) and human non-mercaptalbumin (HNA), according to the redox state of the cysteine residue at position 34. The ratio of HMA to total albumin (%HMA) is a novel biomarker of oxidative stress as well as protein nutritional status, but measuring %HMA normally requires an expensive analyzer such as HPLC and LC-MS, and can hardly be conducted in many clinical sites. To address this issue, we aimed to develop a methodological basis for estimating %HMA without these analyzers. An analytical method was investigated consisting of three steps, i.e., 1) removal of HMA from serum or plasma by using a thiol-binding resin (i.e., thereby obtaining a HNA fraction), 2) determination of both total albumin and HNA concentrations by a colorimetric assay or ELISA, and 3) calculation of %HMA. Proof-of-concept experiments, using serum and plasma samples of 4 adult volunteers, showed that the estimated value of %HMA obtained by this analytical method was significantly correlated with the theoretical value of %HMA determined by HPLC. The subsequent validation experiment, using 86 serum samples of pregnant women in the Japanese participants of SMILE Iwamizawa, also confirmed the significant association between the estimated and theoretical values of %HMA. This analytical method can be a basis to determine %HMA without using HPLC or LC-MS, contributing to the universalization of %HMA measurement as a clinical test.

Shear-Viscosity-Dependent Effect of a Gum-Based Thickening Product on the Safety of Swallowing in Older Patients with Severe Oropharyngeal Dysphagia.

Fluid thickening is a valid therapeutic strategy for patients with oropharyngeal dysphagia (OD). The main aim of this study was to determine the therapeutic effect of the xanthan-gum-based thickener Tsururinko Quickly (TQ, Morinaga Milk Co., Tokyo, Japan) in older patients with severe OD. A total of 85 patients (83.32 ± 6.75 y) with OD and a penetration-aspiration score (PAS) of n ≥ 3 were studied by videofluoroscopy while swallowing duplicate 10 mL boluses at <50 mPa·s, 100, 200, 400, 800, and 1600 mPa·s, to assess the safety and efficacy of swallowing and the biomechanics of a swallowing response at each viscosity level. At <50 mPa·s, only 16.25% patients swallowed safely, 45% had penetrations (PAS 3-5), and 38.75% had aspirations (PAS 6-8). Fluid thickening with TQ greatly increased the prevalence of patients with safe swallowing from 62.90% at 100 mPa·s to 95.24% at 1600 mPa·s in a shear-viscosity-dependent manner. The penetrations and aspirations were significantly reduced to 3.60% and 1.19%, respectively, at 1600 mPa·s. The threshold viscosity was 100 mPa·s and the increasing viscosity above 800 mPa·s did not further improve the therapeutic effect significantly. Increasing the shear viscosity significantly reduced the time to laryngeal vestibule closure (-16.70%), increased the time to upper oesophageal sphincter opening (+26.88%), and reduced the pharyngeal bolus velocity (-31.62%) without affecting the pharyngeal residue. TQ has a strong shear-viscosity-dependent effect on the safety of swallowing in older patients with severe OD without increasing the pharyngeal residue. The therapeutic range for TQ is 100-800 mPa·s, with 200 and 800 mPa·s being the optimal doses to cover the needs of older patients with OD.

A lower ratio of reduced to total albumin in serum is associated with protein nutritional status of pregnant women in Japan.

Protein-energy undernutrition is potentially prevalent among Japanese pregnant women, and biomarkers that objectively indicate the protein nutritional status during pregnancy may help in implementing appropriate protein supplementation to these women. We hypothesized that a serum parameter of pregnant women, the ratio of reduced to total albumin (reduced ALB ratio), would be associated with protein intake during pregnancy. The serum reduced ALB ratio of pregnant women was compared with protein intake and with gestation outcomes (gestation length and infant birth weight) in an observational study of 115 Japanese pregnant women. The serum reduced ALB ratio in the third trimester tended to be positively correlated with gestation length (P = .07). Infant birth weights tended to be different between protein intake tertiles (P = .09); the mean infant birth weight was higher in the third tertile compared with the first and second tertiles. The protein intake of pregnant women was significantly and positively correlated with the serum reduced ALB ratio in the second trimester. The serum reduced ALB ratio reflects protein nutritional status during pregnancy and may contribute to healthier gestation outcomes.

Associations between Maternal Diet, Human Milk Macronutrients, and Breast-Fed Infant Growth during the First Month of Life in the SMILE Iwamizawa in Japan.

Maternal diet may affect human milk macronutrients, but it remains to be elucidated whether this is also influential in infant growth. This study aimed to examine (1) how maternal diet influences human milk macronutrients, and (2) to what extent the variation in milk macronutrients affects infant growth during the first month of life. In 71 Japanese lactating women, maternal dietary information was collected from the brief-type self-administered diet history questionnaire, and anthropometry of mother-infant dyads was collected from medical records. Macronutrients in milk were analyzed by a Human Milk Analyzer. Maternal retinol intake was associated with the carbohydrate content in human milk at 1-month postpartum (standardized ß coefficient: 0.287; p = 0.038). Moreover, the energy content in human milk was associated with an increase in the weight standard deviation score based on the WHO growth standard at 1 month of age (standardized ß coefficient: 0.399; p = 0.046). Nevertheless, the milk macronutrient was not associated with the risk of infant growth abnormalities. In conclusion, a part of the maternal diet impacts macronutrient contents in human milk, but milk macronutrients have a limited effect on infant growth only within the normal growth curve during the first month of life.

Proposal for a Standard Protocol to Assess the Rheological Behavior of Thickening Products for Oropharyngeal Dysphagia.

Increasing shear viscosity (ShV) in thickening products (TP) is a valid therapeutic strategy for oropharyngeal dysphagia (OD). However, salivary amylase in the oral phase and shear rate in the pharyngeal phase of swallowing can change the viscosity of TPs when swallowed. This study aims to design and validate a rheological protocol to reproduce the oral and pharyngeal factors that affect the therapeutic effect of TPs and report the viscosity measurements in a standardized scientific and precise manner. We measured (a) the variability of the ShV measurements across several laboratories; (b) the in vitro and ex vivo properties of TPs and (c) the impact of the X-ray contrast Omnipaque, temperature and resting time on the rheological properties of TPs. A common protocol was applied in four international laboratories to assess five ShV values (100, 200, 400, 800 and 1600 mPa·s) for the xanthan-gum TP Tsururinko Quickly (TQ). The protocol included the dose (g/100 mL water), stirring procedure and standing time before measurement. Each value was characterized at the shear rate of 50 and 300 s-1 pre- and post-oral incubation in eight volunteers. The effect of temperature, standing time and Omnipaque was assessed. The main results of the study were: (a) The mean intra-laboratory variability on the ShV at all levels was very low: 0.85%. The mean inter-laboratory variability was higher: 9.3%; (b) The shear thinning of TQ at 300 s-1 was 75-80%. Increasing the temperature or standing time did not affect the ShV, and oral amylase caused a small decrease; (c) Omnipaque slightly decreased the dose of TP and hardly affected the amylase resistance or shear thinning. This study showed that different laboratories can obtain very accurate and similar ShV measurements using this protocol which uses scientific, universal SI units (mPa·s). Our protocol accurately reproduces oral and pharyngeal factors affecting the therapeutic effect of TPs. The addition of X-ray contrast did not produce significant changes.

Maternal Serum Albumin Redox State Is Associated with Infant Birth Weight in Japanese Pregnant Women.

BACKGROUND: Plasma albumin (ALB) reflects protein nutritional status in rats, but it is not clear whether it is associated with dietary protein insufficiency in pregnant women and/or their risk of low birth weight delivery. This study aimed to investigate whether maternal serum ALB redox state reflects maternal protein nutritional status and/or is associated with infant birth weights. METHODS: The relationship between the serum reduced ALB ratio and infant birth weight was examined in an observational study of 229 Japanese pregnant women. A rat model simulating fetal growth restriction, induced by protein-energy restriction, was used to elucidate the relationship between maternal nutritional status, maternal serum ALB redox state, and birth weight of the offspring. RESULTS: In the human study, serum reduced ALB ratio in the third trimester was significantly and positively correlated with infant birth weight. In the rat study, serum reduced ALB ratio and birth weight in the litter decreased as the degree of protein-energy restriction intensified, and a significant and positive correlation was observed between them in late pregnancy. CONCLUSIONS: Maternal serum reduced ALB ratio in the third trimester is positively associated with infant birth weight in Japanese pregnant women, which would be mediated by maternal protein nutritional status.

Serum Albumin Redox States: More Than Oxidative Stress Biomarker.

Serum albumin is the most abundant circulating protein in mammals including humans. It has three isoforms according to the redox state of the free cysteine residue at position 34, named as mercaptalbumin (reduced albumin), non-mercaptalbumin-1 and -2 (oxidized albumin), respectively. The serum albumin redox state has long been viewed as a biomarker of systemic oxidative stress, as the redox state shifts to a more oxidized state in response to the severity of the pathological condition in various diseases such as liver diseases and renal failures. However, recent ex vivo studies revealed oxidized albumin per se could aggravate the pathological conditions. Furthermore, the possibility of the serum albumin redox state as a sensitive protein nutrition biomarker has also been demonstrated in a series of animal studies. A paradigm shift is thus ongoing in the research field of the serum albumin. This article provides an updated overview of analytical techniques for serum albumin redox state and its association with human health, focusing on recent findings.

Receptor affinity, stability and binding mode of Shiga toxins are determinants of toxicity.

The closely related Shiga toxins, Shiga toxin 1 (Stx1) and Shiga toxin 2 (Stx2), can bind to Gb3 receptors. However, Stx2-producing enterohemorrhagic Escherichia coli (EHEC) strains are more commonly associated with serious human disease (viz., hemolytic-uremic syndrome) than Stx1-producing strains. To clarify the relationship between properties and toxicity of these toxins, we constructed and analyzed a hybrid holotoxin composed of Stx2A and Stx1B, designated as Stx2A1B, and a B subunit chimeric holotoxin composed of Stx2A and Stx2B (III V), designated as Stx2A2B (III V). The affinity of Stx2A1B to Gb3 was lower than that of Stx1, higher than that of Stx2 and identical to that of Stx2A2B (III V). On the other hand, the 50% lethal dose (LD(50)) for mice of Stx2A1B was lower than that of Stx1, but higher than that of Stx2. These results suggested that pathogenicity in mice was inversely related to the receptor affinity of the holotoxins. However, LD(50) of Stx2A1B was not identical to that of Stx2A2B (III V). Gel filtration analysis indicated that Stx2A2B (III V) was relatively less stable than Stx2A1B. Moreover, cross-linking experiments demonstrated that the modes of cell surface binding of Stx2A2B (III V) and Stx2A1B were different. These results indicated that the receptor affinity, stability and binding mode of Shiga toxins might be important determinants for toxicity in mice.

The serine 31 residue of the B subunit of Shiga toxin 2 is essential for secretion in enterohemorrhagic Escherichia coli.

Shiga toxins produced by enterohemorrhagic Escherichia coli (EHEC) include Shiga toxin 1 (Stx1) as well as Shiga toxin 2 (Stx2). Stx1 is cell associated, whereas Stx2 is localized to the culture supernatant. We have analyzed the secretion of Stx2 by generating histidine-tagged StxB (StxB-H). Although neither StxB1-H nor StxB2-H was secreted in StxB-H-overexpressed EHEC, StxB2-H-overexpressed EHEC showed inhibited Stx2 secretion. On the other hand, StxB1-H-overexpressed EHEC showed no alteration of Stx2 secretion. B-subunit chimeras of Stx1 and Stx2 were used to identify the specific residue of StxB2 that the Stx2 secretory system recognizes. Alteration of the serine 31 residue to an asparagine residue (S31N) in StxB2-H enabled the recovery of Stx2 secretion. On the other hand, alteration of the asparagine 32 residue to a serine residue (N32S) in StxB1-H caused the partial secretion of a point-mutated histidine-tagged B subunit in EHEC. Based on the evidence, it appeared possible that this residue might contain secretion-related information for Stx2 secretion. To investigate this hypothesis, we constructed an isogenic mutant EHEC (Stx1B subunit, N32S) strain and an isogenic mutant EHEC (Stx2B subunit, S31N) strain. Although the mutant Stx2 was cell associated in isogenic mutant EHEC, mutant Stx1 was not extracellular. However, when we used plasmids for the expression of the mutant holotoxins, the overexpressed mutant Stx1 was found in the supernatant fraction, and the overexpressed mutant Stx2 was found in the cell-associated fraction in mutant holotoxin gene-transformed EHEC. These results indicate that the serine 31 residue of the B subunit of Stx2 contains secretion-related information.

Effects of hypergravity environments on amphibian development, gene expression and apoptosis.

This study investigates how rearing under conditions of hypergravity affects amphibian development, Xotx2 and Xag1 gene expression and apoptosis. Uncleaved Xenopus laevis eggs 20 min after insemination, 2 cell stage embryos, and gastrula stage embryos were raised at 2G and 5G, while controls were raised in normal gravity. Apoptosis in brain and eye inner structures of hatching embryos was scored using the TUNEL staining method, and gene expression in tail-bud embryos was analyzed by whole-mount in situ hybridization. Results showed that: (1) 5G retarded the development of eggs and embryos and induced microcephaly and microphthalmia. (2) 5G suppressed the expression of the two genes, Xotx2 (involved in fore- and midbrain and eye development) and Xag1 (regulating cement gland formation). (3) Eggs and 2 cell stage embryos raised at 5G showed a greater extent of brain and eye apoptosis compared with controls, while those raised at 2G showed no significant difference. These findings suggest that high gravity suppresses certain gene functions and induces abnormal apoptosis in brain and eyes, resulting in developmental retardation and various morphological abnormalities.

Effects of high gravity on amphibian development.

In order to clarify the possible effects of high gravity environments on eggs and developing embryos, Rana rugosa and Xenopus laevis fertilized eggs and early embryos were raised in 2 G, 5 G, 7 G and 10 G up to the hatched tadpole stage. The results showed that: (1) High gravity significantly retarded the development of eggs and embryos beginning treatment before the blastula stage and induced various abnormalities, including two heads and microcephally suggesting that high gravity is apt to disrupt the animal-vegital axis. On the other hand, embryos beginning treatment after the gastrula stage showed a striking increase in the number of normal-appearing feeding tadpoles. (2) Autopsy revealed that brains, notochords and muscles were reduced in development and differentiation for embryos and tadpoles developed in high gravity. (3) It seems likely that the system for hydrogen peroxide detoxification develops abnormally in high gravity-treated embryos and tadpoles, which probably results in oxidative stress, leading to considerable cell damage.