The deuterated water dosing protocol consisted of 1 loading day (day 7) and 2 maintenance days (days 8 and 9) to achieve and maintain 0.6% body water deuterium enrichment on days 8 and 9. On day 7 participants arrived at the laboratory overnight fasted at 6:00 pm and consumed a total of 6 mL·kgBM-1 of deuterated water (70 atom %; Cambridge Isotope Laboratories Inc, Tewksbury, MA, USA) in 8 doses spaced 1.5 hours apart. Participants were provided with breakfast after the first dose and remained in the laboratory until the 4th dose was consumed (12:30 pm ) and then returned home to consume the remaining 5 doses (ending at 6:30 pm ). Thereafter, participants consumed a daily maintenance dose of 0.54 mL·kgBM-1 70% deuterated water upon waking on days 8 and 9.
Deuterated water
Manufactured by Cambridge Isotopes
Sourced in United States
Deuterated water, also known as heavy water, is a stable isotope of water where the hydrogen atoms are replaced with the heavier isotope of hydrogen, deuterium. It is used as a solvent and tracer in various scientific and research applications.
Automatically generated - may contain errors
Lab products found in correlation
Formic acid, by Merck Group (2 mentions)
Basic alumina, by Thermo Fisher Scientific (1 mentions)
Acetone, by Thermo Fisher Scientific (1 mentions)
Hexane, by Thermo Fisher Scientific (1 mentions)
Methanol, by Thermo Fisher Scientific (1 mentions)
Silica gel 60 f254 coated aluminum backed tlc sheets, by Merck Group (1 mentions)
Sodium hydroxide (naoh), by Thermo Fisher Scientific (1 mentions)
Sodium hydrogen carbonate, by Merck Group (1 mentions)
Silica gel p60, by Silicycle (1 mentions)
Ethyl acetate, by Merck Group (1 mentions)
Tmsp d4, by Cambridge Isotopes (1 mentions)
Varian inova nmr spectrometer, by Agilent Technologies (1 mentions)
Nmr suite 7, by Chenomx (1 mentions)
Methanol, by Beijing Chemical Works (1 mentions)
Echinacoside, by Merck Group (1 mentions)
Sodium 3 trimethlysilyl 2 2 3 3 d4 propionate tsp d4, by Cambridge Isotopes (1 mentions)
β glucose, by Merck Group (1 mentions)
Methanol d4, by Cambridge Isotopes (1 mentions)
Sucrose, by Merck Group (1 mentions)
Deuterated chloroform, by Cambridge Isotopes (1 mentions)
12 protocols using deuterated water
1
Deuterated Water Dosing for Body Composition
2
Purification and Characterization of NR Chloride
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NR chloride (beta form) was a gift from ChromaDex Company. Sodium dithionate was purchased from VWR, sodium hydrogen carbonate was purchased from Aldrich, silica gel (P60, 40–63 μm, 60 Å) was purchased from SiliCycle, and basic alumina (50–200 μm, 60 Å, pH 8) was purchased from Acros. Methanol (99.9%, Certified ACS, Fisher), acetone (99.8% Certified ACS, Fisher), sodium hydroxide (certified ACS, Fisher Chemical), hexanes (≥98.5%, GR ACS), ethyl acetate (EtOAc, >99.9%, certified ACS) and silica gel 60 F254 Coated Aluminum-Backed TLC Sheets were purchased from EMD Millipore (Billerica, MA, USA). Deuterated water and dimethyl sulfoxide (DMSO-d6, D, 99.9%) were purchased from Cambridge Isotope Laboratories, Inc.
3
NMR-based Metabolic Profiling Protocol
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The lyophilized metabolic extracts were suspended in 0.6 ml of deuterated water (Cambridge Isotope Laboratories Inc., Tewksbury, MA, USA) containing phosphate buffer (100 mM) and TSP (3-3-(trimethylsilyl)-2,2,3,3-tetradeuteropropionic acid or TMSP-d4, Cambridge Isotope Laboratories) (0.5 mM) and inserted into a 5 mm NMR tube for immediate acquisition. Spectra were acquired on a Varian Inova NMR spectrometer (Agilent Technologies Inc., Santa Clara, CA, USA), equipped with a triple resonance probe and operating at a resonant frequency of 1H at 500 MHz and a constant temperature of 25°C. Two hundred and fifty-six scans were performed with delays of 1.5 seconds, a reading window of 16 ppm, and an acquisition time of 4 seconds. Phase and baseline corrections, as well as metabolic identification and quantification, were performed in Chenomx NMR Suite 7.1 (Chenomx Inc., Edmonton, Canada). In total, 78 1H-NMR spectra were analyzed. Metabolite set enrichment analyses were performed with the platform MetaboAnalyst (33 (link)) (http://www.metaboanalyst.ca ).
4
Preparation of Isotope-Labeled Compounds
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methanol-d4 (CD3OD, deuterium abundance, 99.8 atom % D), deuterated water (D2O, deuterium abundance, 99.8 atom % D), and sodium 3-trimethlysilyl [2,2,3,3-d4] propionate (TSP-d4) were obtained from Cambridge Isotope Laboratories (Andover, MA, USA). Analytical grade methanol was purchased from Beijing Chemical Works (Beijing, China).
Authentic compounds, including echinacoside, acteoside, and mannitol, were purified from CD in our laboratory previously14 , and sucrose, β-galacose, along with β-glucose were supplied by Sigma–Aldrich (St. Louis, MO, USA). The purities of all references were determined to be more than 98% by HPLC–UV and 1H NMR analyses.
Authentic compounds, including echinacoside, acteoside, and mannitol, were purified from CD in our laboratory previously14 , and sucrose, β-galacose, along with β-glucose were supplied by Sigma–Aldrich (St. Louis, MO, USA). The purities of all references were determined to be more than 98% by HPLC–UV and 1H NMR analyses.
5
Extraction and Characterization of Krill Oil
6
Synthesis of Cysteine Derivatives
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l -Cystine (99%), l -cystine
dimethyl ester dihydrochloride (≥95%),l -cystine dibenzyl
ester ditosylate,l -cysteine methyl ester hydrochloride (98%), l -cysteine (97%), l -homocystine (≥98%), triethylamine
(≥99%),l -N,N′-diboc-l -cystine (l -Cys-Boc), S-methyl-l -cysteine, ditert-butyl dicarbonate (≥99%),
hydrochloric acid (37%), sodium hydroxide (reagent grade), methanol
(reagent grade), ethanol (reagent grade), isopropanol (reagent grade), tert-butanol (reagent grade), hexane (reagent grade), dichloromethane
(reagent grade), ethyl acetate(reagent grade), 4-(dimethylamino)pyridine
(DMAP) (≥99%), N,N′-dicyclohexylcarbodiimide
(DCC) (≥99%), and Grubb’s second generation catalyst
were obtained from Sigma-Aldrich (MO, USA) and used without further
purification. Deuterated chloroform (CDCl3; 99.8 atom %
D) and deuterated water (D2O; 99.994 atom % D) were obtained
from Cambridge Isotope Laboratories (MA, USA). hydrochloric acid in
ethyl acetate andl -allylglycine hydrogen chloride salt were
obtained from TCI (OR, USA) and used without purification. Aqueous
solutions were prepared using deionized water (18.2 MΩ) purified
with a Direct-Q 3 Millipore purification system.
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dimethyl ester dihydrochloride (≥95%),
ester ditosylate,
(≥99%),
hydrochloric acid (37%), sodium hydroxide (reagent grade), methanol
(reagent grade), ethanol (reagent grade), isopropanol (reagent grade), tert-butanol (reagent grade), hexane (reagent grade), dichloromethane
(reagent grade), ethyl acetate(reagent grade), 4-(dimethylamino)pyridine
(DMAP) (≥99%), N,N′-dicyclohexylcarbodiimide
(DCC) (≥99%), and Grubb’s second generation catalyst
were obtained from Sigma-Aldrich (MO, USA) and used without further
purification. Deuterated chloroform (CDCl3; 99.8 atom %
D) and deuterated water (D2O; 99.994 atom % D) were obtained
from Cambridge Isotope Laboratories (MA, USA). hydrochloric acid in
ethyl acetate and
obtained from TCI (OR, USA) and used without purification. Aqueous
solutions were prepared using deionized water (18.2 MΩ) purified
with a Direct-Q 3 Millipore purification system.
7
Purification and Characterization of Enzymes
8
Deuterated Water Assessment of DNL
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A background blood sample was drawn in the afternoon before intake of the deuterated water. Together with the evening meal, participants were given 2.86 g/kg body weight of deuterated water (70% 2H2O, Cambridge Isotope laboratories) in two servings. A blood sample for DNL analysis was drawn at fasting in the morning, 16 h after the first serving of deuterated water and in the postprandial state, and 4 h after the breakfast meal. The DNL was analyzed via enrichment in VLDL-TG of deuterated water [28 (link),29 (link)].
9
Deuterium Incorporation for Lipid Synthesis
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A background blood sample was drawn in the afternoon before intake of the deuterated water in 18 healthy overweight/obese participants. Together with the evening meal, participants were given 2.86 g/kg body weight deuterated water (70% 2H2O, Cambridge Isotope laboratories) in two servings. A blood sample for DNL analysis was drawn at fasting in the morning, 16 h after the first serving of deuterated water. The DNL was analyzed from enrichment in VLDL-TG of deuterated water10 (link),32 (link). Two participants were excluded from the analysis of DNL because of TG levels higher than 4 mmol/L, influencing the reliability of the deuterated water measurement.
10
Synthesis and Characterization of Functional Hydrogels
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Unless otherwise noted, compounds and solvents were purchased from commercial suppliers and used without further purification. 1-Vinylimidazole and 2-(2-chloroethoxy)ethanol were purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). 2,2′-Azobis(2-methylpropionitrile) (AIBN), N,N′-methylene bis(acrylamide) (BIS), acrylamide (AAm), and anhydrous ethanol were purchased from FUJIFILM Wako Pure Chemical Co., Ltd. (Osaka, Japan). Acetic acid and diethyl ether were purchased from Junsei Chemical Co., Ltd. (Tokyo, Japan). 4-Cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanol and 2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone were purchased from Sigma-Aldrich Co., Ltd. (St. Louis, MO, USA). Deuterated water was purchased from Cambridge Isotope Laboratories Inc. (Tewksbury, MA, USA). Graphite (≥99% carbon basis) was gifted from Nippon Graphite Industries Co., Ltd. (Shiga, Japan). Dialysis membranes were purchased from Repligen Co., Ltd. (Spectra/Por 3, MWCO: 3.5 kDa, Waltham, MA, USA). An elastic carbon-coated copper transmission electron microscopy (TEM) grid was purchased from Okenshoji Co., Ltd. (ELS-C10, Tokyo, Japan).
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