Vitamin A Liver Reserves and Serum Markers of Vitamin A in US Adults at Time of Death
NCT ID: NCT03305042
Last Updated: 2017-10-17
Study Results
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Basic Information
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COMPLETED
27 participants
OBSERVATIONAL
2012-02-01
2017-01-31
Brief Summary
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Detailed Description
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To conduct the study, matched serum and liver samples were procured from 27 US adult cadavers (from donors age 49-101 years) and their vitamin A biomarkers were analyzed. Matched human liver and serum samples were collected from 27 cadavers, 13 male and 14 female, and acquired as a consecutive sample through a request to the National Disease Research Interchange service. De-identified clinical history summaries were also obtained. The donor criteria were as follows: no restrictions on race or sex, no history of chemotherapy and/or radiation, no history of prolonged endotracheal intubation or ventilation, no evidence of sepsis or infectious disease (negative serology panel), and age ranges of 21-54 y, 55-74 y, and \>75 y (target collection was 5 male and 5 female donors per age range to provide a non-homogenous sample). After 10 individuals were acquired in each age range (i.e., 55-74 y and \>75 y), samples were no longer requested. All tissues and sera were procured within 16 h post-mortem from September 2013 through August 2016. The procurement protocol numbers were DWEN1 002 022, 002 023, and 002 024 for frozen liver, embedded liver, and serum, respectively. Serum samples were isolated via gel separation and then frozen; primary liver samples were snap frozen in liquid N2; a second liver sample from each donor was embedded in optimal cutting temperature compound (OCT) and snap frozen in a cryomold. Two livers were procured almost whole and evaluated for VA distribution within and among lobes. Samples were shipped on dry ice to Madison, WI, and stored in a -80°C freezer.
OCT-embedded liver samples were prepared as 5-µm frozen sections using a cryostat and stained using routine hematoxylin and eosin and Masson's trichrome protocols and reagents (Newcomer Supply, Middleton, WI). Images were captured using a Nikon E-600 bright field microscope (Melville, NY) and Olympus DP-71 digital microscopy camera (Center Valley, PA). Liver samples (1 g) for VA and carotenoid analyses were weighed and then ground with sodium sulfate (4-5 g) in a mortar and pestle using published methods with minor modifications. Purified C-23 beta-apo-carotenol was added to determine extraction efficiencies. Samples were extracted repeatedly with dichloromethane and filtered through WhatmanTM #1 (GE Healthcare Life Sciences, Pittsburgh, PA) into 50-mL volumetric flasks. An aliquot (5 mL) was dried under nitrogen, re-dissolved in 300 µL methanol:dichloroethane (75:25, volume:volume) and an aliquot of 1 µL was injected into a Waters Acquity H-Class ultra-pressure liquid chromatograph (UPLC®) system equipped with a photodiode array detector (Milford, MA). Aliquots were further saponified for quantification of total alpha-retinol as described below for the lobe analyses. For the liver lobe sample evaluations, 1 g samples were extracted in triplicate from each available lobe. A 5-mL aliquot of the extract was saponified with 400 mL potassium hydroxide:water (50:50, weight:volume) at 45°C for 1 h. The reaction was quenched with 0.5 mL water and extracted three times with 0.5 mL hexanes. The hexane layers were pooled and dried under nitrogen, redissolved in 150 µL methanol:dichloroethane (75:25, volume:volume), and an aliquot of 1 µL was injected into the UPLC® system.
Serum samples (0.5 mL) were aliquoted into test tubes for retinol and RE determination; 1.25 X volume of ethanol was added to denature proteins. The internal standard was C23-β-apo-carotenol. Samples were extracted three times with 0.75 mL hexanes; the supernatant fractions were pooled and dried under nitrogen and reconstituted in 100 µL methanol: dichloroethane (75:25, volume:volume). Two µL was injected into the UPLC® under the conditions described below. Serum C-reactive protein (CRP) and alpha1-acid glycoprotein (AGP) were analyzed to evaluate inflammation using ELISA kits (CRP: Cayman Chemical Company; AGP: Abcam). Lycopene and alpha- and beta-carotene concentrations were determined by routine HPLC analysis.
Ultra-high Performance Liquid Chromatograph (UPLC) method A Waters Acquity UPLC HSS C18 1.8 µm VanGuard pre-column was used in conjunction with a Waters Acquity UPLC® HSS C18 column (1.8 µm, 2.1 x 150 mm). The method utilized two solvent mixes programmed for a 29-min gradient. Solvent A was acetonitrile-water-isopropanol (70:25:5, volume:volume:volume) with 10 mmol/L ammonium acetate and solvent B was methanol-isopropanol (75:25, volume:volume). The column temperature was set to 32°C and flow rate was 0.4 mL/min. The gradient began by holding 100% solvent A for 7 min, followed by a 4-min linear transition to 5% A, and another transition to 1% A over 12 min. The solvent gradient was then reversed to 100% A in 2 min and equilibrated for 4 min. Chromatograms were generated at 311 nm for α-retinol and α-REs, 325 nm for retinol and REs, and 450 nm for carotenoids. alpha-REs, derived almost exclusively from beta-carotene in the diet, were quantified as a proxy for long-term vegetable exposure. The concentrations were calculated using standard curves derived from authentic HPLC-purified standards of α-retinol and retinol.
Conditions
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Keywords
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Study Design
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OTHER
CROSS_SECTIONAL
Study Groups
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21-54 y
Ages 21-54 y
No interventions assigned to this group
55-74 y
Ages 55-74 y
No interventions assigned to this group
>75 y
Age \>75 y
No interventions assigned to this group
Eligibility Criteria
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Inclusion Criteria
Exclusion Criteria
21 Years
ALL
Yes
Sponsors
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National Institute on Deafness and Other Communication Disorders (NIDCD)
NIH
University of Wisconsin, Madison
OTHER
Responsible Party
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Locations
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University of Wisconsin - Madison
Madison, Wisconsin, United States
Countries
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References
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Olsen K, Suri DJ, Davis C, Sheftel J, Nishimoto K, Yamaoka Y, Toya Y, Welham NV, Tanumihardjo SA. Serum retinyl esters are positively correlated with analyzed total liver vitamin A reserves collected from US adults at time of death. Am J Clin Nutr. 2018 Nov 1;108(5):997-1005. doi: 10.1093/ajcn/nqy190.
Other Identifiers
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2015-1482
Identifier Type: -
Identifier Source: org_study_id