Vitamin D kinetics in nonpregnant and pregnant women after a single oral dose of trideuterated vitamin D3.

The plasma pool of the hormone 1,25-dihydroxyvitamin D (1,25(OH)2D) is increased throughout most of human pregnancy. Mechanisms behind this adaptation are unclear, in part due to limited data on vitamin D kinetics during pregnancy. Stable isotopes make it possible to study vitamin D kinetics in vulnerable study populations like pregnant women. We conducted a pilot study of vitamin D kinetics in nonpregnant and pregnant women. We evaluated a clinical protocol and developed analytical methods to assess the serum appearance and disappearance of trideuterated vitamin D3 (d3-vitamin D3) and trideuterated 25-hydroxyvitamin D3 (d3-25(OH)D3) after a single oral dose of 25 µg of [6,19,19-2H]-vitamin D3 (d3-vitamin D3). Blood was collected at baseline and 2, 4, 6, 24, 168, 264, and 456 hours post-dosing. We then described the serum kinetic profiles of d3-vitamin D3 and d3-25(OH)D3 in nonpregnant and pregnant women. Serum kinetic profiles of d3-vitamin D3 and d3-25(OH)D3 followed a time course in line with previous pharmacokinetic studies. There was marked variability between participants in the area under the concentration-time curve (AUC) of d3-25(OH)D3 over the 20-day study period. This AUC of d3-25(OH)D3 was positively correlated with the serum vitamin D binding protein (DBP) concentration, which was higher in pregnant compared with nonpregnant women. The mean serum half-life of 25(OH)D3 was longer but not significantly different in pregnant women (18.8 days) compared with nonpregnant women (13.6 days). Our pilot study demonstrated that a single oral dose of 25 µg of d3-vitamin D3 can be used to study vitamin D kinetics. Serum DBP concentration is an important predictor of vitamin D kinetics, and more research is needed to fully understand the significance of elevated DBP concentration during pregnancy.

Inhalation of 2% Hydrogen Improves Survival Rate and Attenuates Shedding of Vascular Endothelial Glycocalyx in Rats with Heat Stroke.

ABSTRACT: Heat stroke is characterized by excessive oxidative stress and inflammatory responses, both of which are implicated in vascular endothelial glycocalyx shedding and heat-stroke mortality. Although molecular hydrogen has antioxidation and anti-inflammatory potency, its effect on the vascular endothelial glycocalyx in heat stroke has not been examined. Therefore, the aim of this study was to investigate the influence of hydrogen inhalation on the survival and thickness of the vascular endothelial glycocalyx of rats subjected to heat stroke. Altogether, 98 Wistar rats were assigned to the experiments. A heat-controlled chamber set at 40°C temperature and 60% humidity was used to induce heat stroke. After preparation, the anesthetized rats that underwent the heating process were subjected to an hour of stabilization in which 0%, 2%, or 4% hydrogen gas was inhaled and maintained until the experiment ended. In addition to survival rate assessments, blood samples and left ventricles were collected to evaluate the thickness of the vascular endothelial glycocalyx and relevant biomarkers. The results showed that 2% hydrogen gas significantly improved survival in the heat-stroked rats and partially preserved the thickness of the endothelial glycocalyx. In addition, serum levels of endotoxin, syndecan-1, malondialdehyde, and tumor necrosis factor-α decreased, whereas superoxide dismutase levels increased, indicating that inhalation of 2% hydrogen attenuated the damage to the vascular endothelial glycocalyx through its antioxidative and anti-inflammatory effects.

Estimation of radiation dose from ingested tritium in humans by administration of deuterium-labelled compounds and food.

Radiation doses from organically bound tritium (OBT) in foods have been a major concern near nuclear facilities. The current dose coefficient for OBT is calculated using a standard model from the International Commission on Radiological Protection, in which some biokinetic values are not based on human metabolic data. Here, the biokinetics of ingested OBT, and radiation doses from them, were estimated by administering labelled compounds and foods to volunteers, using a deuterium (D) tracer as a substitute for tritium. After the administration of D-labelled glucose, alanine, palmitic acid, or soybean, the D/H ratios in urine were measured for up to 119 days, and the biokinetic parameter values were determined for OBT metabolism. The slow degradation rates of OBT could not be obtained, in many volunteers administered glucose and alanine. The estimated committed effective dose for 1 Bq of tritium in palmitic acid varied from 3.2 × 10-11 to 3.5 × 10-10 Sv Bq-1 among volunteers and, for those administered soybean, it varied from 1.9 × 10-11 to 1.8 × 10-10 Sv Bq-1. These results suggest that OBT, present in some ingested ingredients, gives higher doses than the current dose coefficient value of 4.2 × 10-11 Sv Bq-1.

DMAPT­D6 induces death­receptor­mediated apoptosis to inhibit glioblastoma cell oncogenesis via induction of DNA damage through accumulation of intracellular ROS.

Glioblastoma (GBM) is an aggressive malignancy with a high rate of tumor recurrence after treatment with conventional therapies. Parthenolide (PTL), a sesquiterpene lactone extracted from the herb Tanacetum parthenium or feverfew, possesses anticancer properties against a wide variety of solid tumors. In the present study, a series of PTL derivatives were synthesized and screened. An inhibitor, dimethylaminoparthenolide (DMAPT)­D6, a derivative of the PTL prodrug DMAPT in which the hydrogen of the dimethylamino group is substituted for the isotope deuterium, induced significant cytotoxicity in GBM cells in vitro and induced cell cycle arrest at the S­phase in a dose­dependent manner. Furthermore, mechanistic investigation indicated that through increasing the levels of intracellular accumulation of reactive oxygen species (ROS), DMAPT­D6 triggered DNA damage and finally death receptor­mediated extrinsic apoptosis in GBM cells, suggesting that DNA damage induced by DMAPT­D6 initiated caspase­dependent apoptosis to remove damaged GBM cells. Taken together, these data suggested that ROS accumulation following treatment with DMAPT­D6 results in DNA damage, and thus, death­receptor­mediated apoptosis, highlighting the potential of DMAPT­D6 as a novel therapeutic agent for the treatment of GBM.

Human Milk Intake of Thai Breastfed Infants During the First 6 Months Using the Dose-to-Mother Deuterium Dilution Method.

BACKGROUND: Data on infant human milk intakes in low- and middle-income countries are limited, although the deuterium oxide dose-to-mother method (DTM) is an accurate tool for its estimation. OBJECTIVE: We assessed human milk intakes of Thai infants during the first 6 months comparing exclusive breastfeeding (EBF), predominant breastfeeding (PBF), and partial breastfeeding (PartBF). METHODS: One hundred and ten healthy Thai lactating mother-infant pairs were followed from birth. Human milk intakes were determined at 6 weeks, 3 months, and 6 months using the DTM method. Daily energy and protein intakes were assessed using 3-day nonconsecutive 24-hour recalls. Sociodemographic characteristics were collected using a questionnaire. To compare breastfeeding practices, one-way analysis of variance with Tukey post hoc test assessed normally distributed data, while the Kruskal-Wallis test with Mann-Whitney U test was used for not normally distributed data. RESULTS: No difference existed in human milk intakes during the first 6 months between EBF (743-776 g/d) and PBF (748-862 g/d). Human milk intakes of PartBF infants were significantly lower compared to other infants. Human milk intake was sufficient to meet infant energy and protein requirements during the first 3 months for EBF and PBF groups. Infant formula largely replaced human milk during the first 3 months; other solid foods were also introduced among the PartBF infants. CONCLUSIONS: Human milk intakes were comparable between EBF and PBF infants during the first 6 months and provided adequate energy and protein to meet requirements. Infant formula largely replaced breast milk among PartBF infants, although other foods were also given early. This study was registered at clinicaltrials.gov as NCT04020640.

Site-specifically deuterated essential lipids as new drugs against neuronal, retinal and vascular degeneration.

An original approach to drug discovery and development is now in clinical and preclinical trials. The approach is based on the 'kinetic isotope effect' (i.e., the effect of isotopic substitution on chemical reaction rates). By replacing selective hydrogen atoms with deuterium in essential and conditionally essential lipids, a novel class of potent drugs is being created that prevents cellular and vascular oxidative damage causing diverse pathologies, such as neurodegeneration, atherosclerosis and macular degeneration. This review describes the molecular mechanisms underlying the new treatment modalities in these diseases and the encouraging results of ongoing studies for candidate drugs. Also, the possible extension of this new drug platform to treatment of nonoxidative diseases by deuterium-reinforced amino acids and nucleobases is briefly discussed.

Neuroprotective Effects of Deuterium-Depleted Water (DDW) Against H2O2-Induced Oxidative Stress in Differentiated PC12 Cells Through the PI3K/Akt Signaling Pathway.

Oxidative stress plays an important role in the pathogenesis of neurodegenerative diseases, including Alzheimer's disease and Parkinson's disease. Induction of endogenous antioxidants to act against oxidative stress-mediated neuronal damage seems to be a reasonable strategy for delaying the progression of such diseases. In this study, we investigated the neuroprotective effect of deuterium-depleted water (DDW) against H2O2-induced oxidative stress in differentiated PC12 cells and the possible signaling pathways involved. The differentiated PC12 cell line was pretreated with DDW containing different concentrations (50-100 ppm) of deuterium and then treated with H2O2 to induce oxidative stress and neurotoxicity. We assessed cell survival, reactive oxygen species (ROS) generation, TUNEL assay, catalase (CAT), copper and zinc-containing superoxide dismutase (CuZn-SOD) and superoxide dismutase (SOD) activity and performed Western blot analysis to investigate the neuroprotective effect of DDW. The results indicated that DDW could attenuate H2O2-induced apoptosis, reduce ROS formation, and increase CAT, CuZn-SOD and SOD activity in H2O2-treated PC12 cells. Western blot analysis revealed that DDW treatment significantly increased the expression of p-Akt, Bcl-2 and GSK-3ß. However, the protective effect of DDW on cell survival and the DDW-mediated increases in p-Akt, Bcl-2 and GSK-3ß were abolished by pretreatment with the phosphatidylinositol-3-kinase (PI3K) inhibitor LY294002. In summary, DDW may protect differentiated PC12 cells against H2O2-induced oxidative stress through the PI3K/Akt signaling pathway.

The International Atomic Energy Agency International Doubly Labelled Water Database: Aims, Scope and Procedures.

BACKGROUND: The doubly labelled water (DLW) method is an isotope-based technique that quantifies total energy expenditure (TEE) over periods of 1-3 weeks from the differential elimination of stable isotopes of oxygen and hydrogen. The method was invented in the 1950s, but limited ability to measure low isotope enrichments combined with the high cost of isotopes meant it only became feasible to use in humans in the 1980s. It is still relatively expensive to use, and alone small samples are unable to tackle some of the important questions surrounding energy balance such as how have expenditures changed over time and how do expenditures differ with age, between sexes and in different environments? SUMMARY: By combining information across studies, answers to such questions may be possible. The International Atomic Energy Agency (IAEA) DLW database was established to pool DLW data across multiple studies. It was initiated by the main labs currently using the method and is hosted by the IAEA. At present, the database contains 6,621 measures of TEE by DLW from individuals in 23 countries, along with various additional data on the study participants. Key Messages: The IAEA DLW database is a key resource enabling future studies of energy demands.

Strengthened Data Systems to Mitigate the Double Burden of Malnutrition: The Role of Stable Isotope Technique-Derived Nutrition Indicators.

Indicators reflecting the double burden of malnutrition are rarely measured in nutrition surveys and are needed to strengthen national data systems. Indicators such as body composition reflect both metabolic response to undernutrition and obesity risk and nutrition-related noncommunicable diseases. Stable isotope techniques (SITs) provide accurate data on body composition, exclusive breastfeeding and vitamin A status that are otherwise problematic with routine methods. Integration of SIT-derived nutrition indicators in data systems could improve the design and evaluation of programmes focused on obesity prevention, food fortification and infant and young child feeding practices. The Working Group at the symposium considered "how SIT-derived nutrition indicators may be integrated into surveillance systems to strengthen data availability and capacity at national and regional levels". Practical considerations for the use of SITs include cost, sample size, rigorous training and logistics. It was concluded that SITs are best suited, at present, for use in sub-samples of population surveys and for validating tools that can be scaled-up more easily in population surveys. In the long term, SITs could be applied to larger surveys following potential innovations in more affordable, hand-held devices for analysis of stable isotope enrichment in the field and simpler specimen collection protocols.

An exploration of the methods to determine the protein-specific synthesis and breakdown rates in vivo in humans.

The present study explores the methods to determine human in vivo protein-specific myofibrillar and collagenous connective tissue protein fractional synthesis and breakdown rates. We found that in human myofibrillar proteins, the protein-bound tracer disappearance method to determine the protein fractional breakdown rate (FBR) (via 2 H2 O ingestion, endogenous labeling of 2 H-alanine that is incorporated into proteins, and FBR quantified by its disappearance from these proteins) has a comparable intrasubject reproducibility (range: 0.09-53.5%) as the established direct-essential amino acid, here L-ring-13 C6 -phenylalanine, incorporation method to determine the muscle protein fractional synthesis rate (FSR) (range: 2.8-56.2%). Further, the determination of the protein breakdown in a protein structure with complex post-translational processing and maturation, exemplified by human tendon tissue, was not achieved in this experimentation, but more investigation is encouraged to reveal the possibility. Finally, we found that muscle protein FBR measured with an essential amino acid tracer prelabeling is inappropriate presumably because of significant and prolonged intracellular recycling, which also may become a significant limitation for determination of the myofibrillar FSR when repeated infusion trials are completed in the same participants.

Analysis of the Complicated Nonlinear Pharmacokinetics of Orally Administered Telmisartan in Rats Using a Stable Isotope-IV Method.

This study aimed to kinetically analyze the nonlinear absorption and systemic exposure of telmisartan (TEL) after oral administration to rats by using a stable isotope-IV method. Rats were orally administered different dose of TEL, followed by the intravenous injection of 0.005 mg/kg of deuterium-labeled TEL (TEL-d3). Assuming that TEL-d3 shows same pharmacokinetic properties with TEL, systemic clearance (CLtot), oral bioavailability (Foral), and intestinal and hepatic availability (Fa*Fg, Fh) of TEL were calculated in each individual rat. AUCpo of TEL increased disproportionately with dose and showed a sigmoid-type relation, indicating the involvement of multi-nonlinear processes in oral absorption of TEL. Fa*Fg of TEL increased with dose at the low-dose range while decreased at the high-dose range. In contrast, Fh increased and CLtot decreased significantly in the middle range (2 to 6 mg/kg). As main factors of nonlinearity, saturations of solubility, efflux transport in the intestine, and the hepatic uptake of TEL were indicated. In conclusion, this study demonstrated a high possibility of a stable isotope-IV method to characterize complicated pharmacokinetic properties of oral drugs in animals, which can help to consider the future risks in their clinical use.

Influence of Deuterium-Depleted Water on the Isotope D/H Composition of Liver Tissue and Morphological Development of Rats at Different Periods of Ontogenesis

Background: This study aimed to evaluate the reaction of organism of laboratory animals on deuterium-depleted drinking diet. To assess the cell energy metabolism, the effect of a liquid medium with different deuterium contents on isolated liver mitochondria of random bred rats and Wistar rats was studied. Methods: This experimental study on the effect of deuterium-depleted drinking water (DDW) on 16-week-old male Wistar rats lasted for four weeks. Energy metabolism of mitochondria was examined through the production of hydrogen peroxide using an Amplex® Red Hydrogen Peroxide/Peroxidase Assay Kit. Results: Modification of isotope (deuterium-protium [D/H]) composition of rats' blood and organ tissues with DDW (-705‰), introduced into rats' diet within four weeks, led to the formation of isotope D/H gradient between blood plasma and organ tissues and affected isotope D/H exchange reactions on the adaptive processes. The isolated liver mitochondria from the random bred rats consumed DDW presented a maximum increase in H2O2 production during the incubation in DDW medium. This increased level of H2O2 production was higher in the isolated liver mitochondria of the rats consuming natural deuterium content drinking water (-24‰). Conclusion: The obtained results indicate the possibility of nutritional correction of isotope D/H metabolism in blood by means of products with modified isotope composition, as well as the prospects of using isotope exchange reactions in case of imbalance in function of the body's defense systems in different generations of animals.

Measuring Human Lipid Metabolism Using Deuterium Labeling: In Vivo and In Vitro Protocols.

Stable isotopes are powerful tools for tracing the metabolic fate of molecules in the human body. In this chapter, we focus on the use of deuterium (2H), a stable isotope of hydrogen, in the study of human lipid metabolism within the liver in vivo in humans and in vitro using hepatocyte cellular models. The measurement of de novo lipogenesis (DNL) will be focussed on, as the synthesis of fatty acids, specifically palmitate, has been gathering momentum as being implicated in cellular dysfunction, which may be involved in the development of non-alcoholic fatty liver disease (NAFLD). Therefore, this chapter focusses specifically on the use of 2H2O (heavy water) to measure hepatic DNL.

Lack of deuterium isotope effects in the antidepressant effects of (R)-ketamine in a chronic social defeat stress model.

RATIONALE: (R,S)-ketamine, an N-methyl-D-aspartate receptor (NMDAR) antagonist, exhibits rapid and long-lasting antidepressant effects and anti-suicidal ideation in treatment-resistant patients with depression. However, the precise mechanisms underlying the antidepressant actions of (R,S)-ketamine are unknown. Although the previous report demonstrated the deuterium isotope effects in the antidepressant actions of (R,S)-ketamine, the deuterium isotope effects in the antidepressant actions of (R)-ketamine, which is more potent than (S)-ketamine, are unknown. METHODS: We examined whether deuterium substitution at the C6 position could affect antidepressant effects of (R)-ketamine in a chronic social defeat stress (CSDS) model. RESULTS: Pharmacokinetic studies showed that levels of (2R,6R)-d1-hydroxynorketamine [(2R,6R)-d1-HNK], a final metabolite of (R)-d2-ketamine, in the plasma and brain after administration of (R)-d2-ketamine (10 mg/kg) were lower than those of (2R,6R)-HNK from (R)-ketamine (10 mg/kg), indicating deuterium isotope effects in the production of (2R,6R)-HNK. In contrast, levels of (R)-ketamine and its metabolite (R)-norketamine in the plasma and brain were the same for both compounds. In a CSDS model, both (R)-ketamine (10 mg/kg) and (R)-d2-ketamine (10 mg/kg) showed rapid and long-lasting (7 days) antidepressant effects, indicating no deuterium isotope effect in the antidepressant effects of (R)-ketamine. CONCLUSIONS: The present study suggests that deuterium substitution of hydrogen at the C6 position slows the metabolism from (R)-ketamine to (2R,6R)-HNK in mice. In contrast, we did not find the deuterium isotope effects in terms of the rapid and long-lasting antidepressant effects of (R)-ketamine in a CSDS model. Therefore, it is unlikely that (2R,6R)-HNK is essential for antidepressant effects of (R)-ketamine.

Validating the use of bioimpedance spectroscopy for assessment of fluid status in children.

BACKGROUND: Bioimpedance spectroscopy (BIS) with a whole-body model to distinguish excess fluid from major body tissue hydration can provide objective assessment of fluid status. BIS is integrated into the Body Composition Monitor (BCM) and is validated in adults, but not children. This study aimed to (1) assess agreement between BCM-measured total body water (TBW) and a gold standard technique in healthy children, (2) compare TBW_BCM with TBW from Urea Kinetic Modelling (UKM) in haemodialysis children and (3) investigate systematic deviation from zero in measured excess fluid in healthy children across paediatric age range. METHODS: TBW_BCM and excess fluid was determined from standard wrist-to-ankle BCM measurement. TBW_D2O was determined from deuterium concentration decline in serial urine samples over 5 days in healthy children. UKM was used to measure body water in children receiving haemodialysis. Agreement between methods was analysed using paired t test and Bland-Altman method comparison. RESULTS: In 61 healthy children (6-14 years, 32 male), mean TBW_BCM and TBW_D2O were 21.1 ± 5.6 and 20.5 ± 5.8 L respectively. There was good agreement between TBW_BCM and TBW_D2O (R2 = 0.97). In six haemodialysis children (4-13 years, 4 male), 45 concomitant measurements over 8 months showed good TBW_BCM and TBW_UKM agreement (mean difference - 0.4 L, 2SD = ± 3.0 L). In 634 healthy children (2-17 years, 300 male), BCM-measured overhydration was - 0.1 ± 0.7 L (10-90th percentile - 0.8 to + 0.6 L). There was no correlation between age and OH (p = 0.28). CONCLUSIONS: These results suggest BCM can be used in children as young as 2 years to measure normally hydrated weight and assess fluid status.

Stable isotope compounds - production, detection, and application.

Stable isotopes are used in wide fields of application from natural tracers in biology, geology and archeology through studies of metabolic fluxes to their application as tracers in quantitative proteomics and structural biology. We review the use of stable isotopes of biogenic elements (H, C, N, O, S, Mg, Se) with the emphasis on hydrogen and its heavy isotope deuterium. We will discuss the limitations of enriching various compounds in stable isotopes when produced in living organisms. Finally, we overview methods for measuring stable isotopes, focusing on methods for detection in single cells in situ and their exploitation in modern biotechnologies.

Stable Isotope Kinetic Study of ApoM (Apolipoprotein M).

OBJECTIVE: ApoM (apolipoprotein M) binds primarily to high-density lipoprotein before to be exchanged with apoB (apolipoprotein B)-containing lipoproteins. Low-density lipoprotein (LDL) receptor-mediated clearance of apoB-containing particles could influence plasma apoM kinetics and decrease its antiatherogenic properties. In humans, we aimed to describe the interaction of apoM kinetics with other components of lipid metabolism to better define its potential benefit on atherosclerosis. APPROACH AND RESULTS: Fourteen male subjects received a primed infusion of 2H3-leucine for 14 hours, and analyses were performed by liquid chromatography-tandem mass spectrometry from the hourly plasma samples. Fractional catabolic rates and production rates within lipoproteins were calculated using compartmental models. ApoM was found not only in high-density lipoprotein (59%) and LDL (4%) but also in a non-lipoprotein-related compartment (37%). The apoM distribution was heterogeneous within LDL and non-lipoprotein-related compartments according to plasma triglycerides (r=0.86; P<0.001). The relationships between sphingosine-1-phosphate and apoM were confirmed in all compartments (r range, 0.55-0.89; P<0.05). ApoM fractional catabolic rates and production rates were 0.16±0.07 pool/d and 0.14±0.06 mg/kg per day in high-density lipoprotein and 0.56±0.10 pool/d and 0.03±0.01 mg/kg per day in LDL, respectively. Fractional catabolic rates of LDL-apoM and LDL-apoB100 were correlated (r=0.55; P=0.042). Significant correlations were found between triglycerides and production rates of LDL-apoM (r=0.73; P<0.004). CONCLUSIONS: In humans, LDL kinetics play a key role in apoM turnover. Plasma triglycerides act on both apoM and sphingosine-1-phosphate distributions between lipoproteins. These results confirmed that apoM could be bound to high-density lipoprotein after secretion and then quickly exchanged with a non-lipoprotein-related compartment and to LDL to be slowly catabolized.

Altering Metabolic Profiles of Drugs by Precision Deuteration 2: Discovery of a Deuterated Analog of Ivacaftor with Differentiated Pharmacokinetics for Clinical Development.

Ivacaftor is currently used for the treatment of cystic fibrosis as both monotherapy (Kalydeco; Vertex Pharmaceuticals, Boston, MA) and combination therapy with lumacaftor (Orkambi; Vertex Pharmaceuticals). Each therapy targets specific patient populations: Kalydeco treats patients carrying one of nine gating mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) protein, whereas Orkambi treats patients homozygous for the F508del CFTR mutation. In this study, we explored the pharmacological and metabolic effects of precision deuteration chemistry on ivacaftor by synthesizing two novel deuterated ivacaftor analogs, CTP-656 (d9-ivacaftor) and d18-ivacaftor. Ivacaftor is administered twice daily and is extensively converted in humans to major metabolites M1 and M6; therefore, the corresponding deuterated metabolites were also prepared. Both CTP-656 and d18-ivacaftor showed in vitro pharmacologic potency similar to that in ivacaftor, and the deuterated M1 and M6 metabolites showed pharmacology equivalent to that in the corresponding metabolites of ivacaftor, which is consistent with the findings of previous studies of deuterated compounds. However, CTP-656 exhibited markedly enhanced stability when tested in vitro. The deuterium isotope effects for CTP-656 metabolism (DV = 3.8, DV/K = 2.2) were notably large for a cytochrome P450-mediated oxidation. The pharmacokinetic (PK) profile of CTP-656 and d18-ivacaftor were assessed in six healthy volunteers in a single-dose crossover study, which provided the basis for advancing CTP-656 in development. The overall PK profile, including the 15.9-hour half-life for CTP-656, suggests that CTP-656 may be dosed once daily, thereby enhancing patient adherence. Together, these data continue to validate deuterium substitution as a viable approach for creating novel therapeutic agents with properties potentially differentiated from existing drugs.

Human CD62Ldim neutrophils identified as a separate subset by proteome profiling and in vivo pulse-chase labeling.

During acute inflammation, 3 neutrophil subsets are found in the blood: neutrophils with a conventional segmented nucleus, neutrophils with a banded nucleus, and T-cell-suppressing CD62Ldim neutrophils with a high number of nuclear lobes. In this study, we compared the in vivo kinetics and proteomes of banded, mature, and hypersegmented neutrophils to determine whether these cell types represent truly different neutrophil subsets or reflect changes induced by lipopolysaccharide (LPS) activation. Using in vivo pulse-chase labeling of neutrophil DNA with 6,6-2H2-glucose, we found that 2H-labeled banded neutrophils appeared much earlier in blood than labeled CD62Ldim and segmented neutrophils, which shared similar label kinetics. Comparison of the proteomes by cluster analysis revealed that CD62Ldim neutrophils were clearly separate from conventional segmented neutrophils despite having similar kinetics in peripheral blood. Interestingly, the conventional segmented cells were more related at a proteome level to banded cells despite a 2-day difference in maturation time. The differences between CD62Ldim and mature neutrophils are unlikely to have been a direct result of LPS-induced activation, because of the extremely low transcriptional capacity of CD62Ldim neutrophils and the fact that neutrophils do not directly respond to the low dose of LPS used in the study (2 ng/kg body weight). Therefore, we propose CD62Ldim neutrophils are a truly separate neutrophil subset that is recruited to the bloodstream in response to acute inflammation. This trial was registered at www.clinicaltrials.gov as #NCT01766414.

Complexity of Stomach-Brain Interaction Induced by Molecular Hydrogen in Parkinson's Disease Model Mice.

Molecular hydrogen (H2), as a new medical gas, has protective effects in neurological disorders including Parkinson's disease (PD). In our previous report, the neuroprotective effect of drinking water with saturated H2 (H2 water) in PD mice might be due to stomach-brain interaction via release of gastric hormone, ghrelin. In the present study, we assessed the effect of H2-induced ghrelin more precisely. To confirm the contribution of ghrelin in H2 water-drinking PD model mice, ghrelin-knock out (KO) mice were used. Despite the speculation, the effect of H2 water was still observed in ghrelin-KO PD model mice. To further check the involvement of ghrelin, possible contribution of ghrelin-induced vagal afferent effect was tested by performing subdiaphragmatic vagotomy before treating with H2 water and administration of MPTP (1-methyl- 4-phenyl-1,2,3,6-tetrahydropyridine). The protective effect of H2 water was still observed in the vagotomized mice in substantia nigra, suggesting that stimulation of vagal afferent nerves is not involved in H2-induced neuroprotection. Other neuroprotective substitutes in ghrelin-KO mice were speculated because H2-induced neuroprotection was not cancelled by ghrelin receptor antagonist, D-Lys3 GHRP-6, in ghrelin-KO PD model mice, unlike in wild-type PD model mice. Our results indicate that ghrelin may not be the only factor for H2-induced neuroprotection and other factors can substitute the role of ghrelin when ghrelin is absent, raising intriguing options of research for H2-responsive factors.