<clinical_study>
  <!-- This xml conforms to an XML Schema at:
    https://clinicaltrials.gov/ct2/html/images/info/public.xsd -->
  <required_header>
    <download_date>ClinicalTrials.gov processed this data on September 20, 2023</download_date>
    <link_text>Link to the current ClinicalTrials.gov record.</link_text>
    <url>https://clinicaltrials.gov/ct2/show/NCT03305042</url>
  </required_header>
  <id_info>
    <org_study_id>2015-1482</org_study_id>
    <nct_id>NCT03305042</nct_id>
  </id_info>
  <brief_title>Vitamin A Liver Reserves and Serum Markers of Vitamin A in US Adults at Time of Death</brief_title>
  <official_title>Vitamin A Liver Reserves and Serum Markers of Vitamin A in US Adults at Time of Death</official_title>
  <sponsors>
    <lead_sponsor>
      <agency>University of Wisconsin, Madison</agency>
      <agency_class>Other</agency_class>
    </lead_sponsor>
    <collaborator>
      <agency>National Institute on Deafness and Other Communication Disorders (NIDCD)</agency>
      <agency_class>NIH</agency_class>
    </collaborator>
  </sponsors>
  <source>University of Wisconsin, Madison</source>
  <oversight_info>
    <has_dmc>No</has_dmc>
    <is_fda_regulated_drug>No</is_fda_regulated_drug>
    <is_fda_regulated_device>No</is_fda_regulated_device>
  </oversight_info>
  <brief_summary>
    <textblock>
      Minimal human data exist on actual liver vitamin A compared with blood biomarkers. One blood&#xD;
      biomarker, the percent of total serum retinol (vitamin A) in the form of retinyl esters, has&#xD;
      been suggested to diagnose hypervitaminosis A with cutoffs of 5% and 10%. In this study,&#xD;
      investigators aim to compare total liver vitamin A reserves with the percent total serum&#xD;
      retinol as retinyl esters to evaluate hypervitaminosis A using autopsy samples from US&#xD;
      adults. Investigators also evaluate the sensitivity (the ability of the biomarker to&#xD;
      correctly identify those with deficiency) and specificity (the ability of the biomarker to&#xD;
      correctly identify those without deficiency) of serum retinol to determine vitamin A&#xD;
      deficiency, variation of liver vitamin A concentration among lobes, and liver alpha retinyl&#xD;
      ester concentrations, a cleavage product of alpha-carotene, a vitamin A precursor. To conduct&#xD;
      the study, matched serum and liver samples were procured from 27 US adult cadavers (from&#xD;
      donors age 49-101 years) and their vitamin A biomarkers were analyzed.&#xD;
    </textblock>
  </brief_summary>
  <detailed_description>
    <textblock>
      Minimal human data exist on actual liver vitamin A compared with blood biomarkers. One blood&#xD;
      biomarker, the percent of total serum retinol (vitamin A) in the form of retinyl esters, has&#xD;
      been suggested to diagnose hypervitaminosis A with cutoffs of 5% and 10%. In this study,&#xD;
      investigators aim to compare total liver vitamin A reserves with the percent total serum&#xD;
      retinol as retinyl esters to evaluate hypervitaminosis A using autopsy samples from US&#xD;
      adults. Investigators also evaluate the sensitivity (the ability of the biomarker to&#xD;
      correctly identify those with deficiency) and specificity (the ability of the biomarker to&#xD;
      correctly identify those without deficiency) of serum retinol to determine vitamin A&#xD;
      deficiency, variation of liver vitamin A concentration among lobes, and liver alpha retinyl&#xD;
      ester concentrations, a cleavage product of alpha-carotene, a vitamin A precursor.&#xD;
&#xD;
      To conduct the study, matched serum and liver samples were procured from 27 US adult cadavers&#xD;
      (from donors age 49-101 years) and their vitamin A biomarkers were analyzed. Matched human&#xD;
      liver and serum samples were collected from 27 cadavers, 13 male and 14 female, and acquired&#xD;
      as a consecutive sample through a request to the National Disease Research Interchange&#xD;
      service. De-identified clinical history summaries were also obtained. The donor criteria were&#xD;
      as follows: no restrictions on race or sex, no history of chemotherapy and/or radiation, no&#xD;
      history of prolonged endotracheal intubation or ventilation, no evidence of sepsis or&#xD;
      infectious disease (negative serology panel), and age ranges of 21-54 y, 55-74 y, and &gt;75 y&#xD;
      (target collection was 5 male and 5 female donors per age range to provide a non-homogenous&#xD;
      sample). After 10 individuals were acquired in each age range (i.e., 55-74 y and &gt;75 y),&#xD;
      samples were no longer requested. All tissues and sera were procured within 16 h post-mortem&#xD;
      from September 2013 through August 2016. The procurement protocol numbers were DWEN1 002 022,&#xD;
      002 023, and 002 024 for frozen liver, embedded liver, and serum, respectively. Serum samples&#xD;
      were isolated via gel separation and then frozen; primary liver samples were snap frozen in&#xD;
      liquid N2; a second liver sample from each donor was embedded in optimal cutting temperature&#xD;
      compound (OCT) and snap frozen in a cryomold. Two livers were procured almost whole and&#xD;
      evaluated for VA distribution within and among lobes. Samples were shipped on dry ice to&#xD;
      Madison, WI, and stored in a -80°C freezer.&#xD;
&#xD;
      OCT-embedded liver samples were prepared as 5-µm frozen sections using a cryostat and stained&#xD;
      using routine hematoxylin and eosin and Masson's trichrome protocols and reagents (Newcomer&#xD;
      Supply, Middleton, WI). Images were captured using a Nikon E-600 bright field microscope&#xD;
      (Melville, NY) and Olympus DP-71 digital microscopy camera (Center Valley, PA). Liver samples&#xD;
      (1 g) for VA and carotenoid analyses were weighed and then ground with sodium sulfate (4-5 g)&#xD;
      in a mortar and pestle using published methods with minor modifications. Purified C-23&#xD;
      beta-apo-carotenol was added to determine extraction efficiencies. Samples were extracted&#xD;
      repeatedly with dichloromethane and filtered through WhatmanTM #1 (GE Healthcare Life&#xD;
      Sciences, Pittsburgh, PA) into 50-mL volumetric flasks. An aliquot (5 mL) was dried under&#xD;
      nitrogen, re-dissolved in 300 µL methanol:dichloroethane (75:25, volume:volume) and an&#xD;
      aliquot of 1 µL was injected into a Waters Acquity H-Class ultra-pressure liquid&#xD;
      chromatograph (UPLC®) system equipped with a photodiode array detector (Milford, MA).&#xD;
      Aliquots were further saponified for quantification of total alpha-retinol as described below&#xD;
      for the lobe analyses. For the liver lobe sample evaluations, 1 g samples were extracted in&#xD;
      triplicate from each available lobe. A 5-mL aliquot of the extract was saponified with 400 mL&#xD;
      potassium hydroxide:water (50:50, weight:volume) at 45°C for 1 h. The reaction was quenched&#xD;
      with 0.5 mL water and extracted three times with 0.5 mL hexanes. The hexane layers were&#xD;
      pooled and dried under nitrogen, redissolved in 150 µL methanol:dichloroethane (75:25,&#xD;
      volume:volume), and an aliquot of 1 µL was injected into the UPLC® system.&#xD;
&#xD;
      Serum samples (0.5 mL) were aliquoted into test tubes for retinol and RE determination; 1.25&#xD;
      X volume of ethanol was added to denature proteins. The internal standard was&#xD;
      C23-β-apo-carotenol. Samples were extracted three times with 0.75 mL hexanes; the supernatant&#xD;
      fractions were pooled and dried under nitrogen and reconstituted in 100 µL methanol:&#xD;
      dichloroethane (75:25, volume:volume). Two µL was injected into the UPLC® under the&#xD;
      conditions described below. Serum C-reactive protein (CRP) and alpha1-acid glycoprotein (AGP)&#xD;
      were analyzed to evaluate inflammation using ELISA kits (CRP: Cayman Chemical Company; AGP:&#xD;
      Abcam). Lycopene and alpha- and beta-carotene concentrations were determined by routine HPLC&#xD;
      analysis.&#xD;
&#xD;
      Ultra-high Performance Liquid Chromatograph (UPLC) method A Waters Acquity UPLC HSS C18 1.8&#xD;
      µm VanGuard pre-column was used in conjunction with a Waters Acquity UPLC® HSS C18 column&#xD;
      (1.8 µm, 2.1 x 150 mm). The method utilized two solvent mixes programmed for a 29-min&#xD;
      gradient. Solvent A was acetonitrile-water-isopropanol (70:25:5, volume:volume:volume) with&#xD;
      10 mmol/L ammonium acetate and solvent B was methanol-isopropanol (75:25, volume:volume). The&#xD;
      column temperature was set to 32°C and flow rate was 0.4 mL/min. The gradient began by&#xD;
      holding 100% solvent A for 7 min, followed by a 4-min linear transition to 5% A, and another&#xD;
      transition to 1% A over 12 min. The solvent gradient was then reversed to 100% A in 2 min and&#xD;
      equilibrated for 4 min. Chromatograms were generated at 311 nm for α-retinol and α-REs, 325&#xD;
      nm for retinol and REs, and 450 nm for carotenoids. alpha-REs, derived almost exclusively&#xD;
      from beta-carotene in the diet, were quantified as a proxy for long-term vegetable exposure.&#xD;
      The concentrations were calculated using standard curves derived from authentic HPLC-purified&#xD;
      standards of α-retinol and retinol.&#xD;
    </textblock>
  </detailed_description>
  <overall_status>Completed</overall_status>
  <start_date type="Actual">February 1, 2012</start_date>
  <completion_date type="Actual">January 31, 2017</completion_date>
  <primary_completion_date type="Actual">January 31, 2017</primary_completion_date>
  <study_type>Observational</study_type>
  <has_expanded_access>No</has_expanded_access>
  <study_design_info>
    <observational_model>Other</observational_model>
    <time_perspective>Cross-Sectional</time_perspective>
  </study_design_info>
  <primary_outcome>
    <measure>Serum retinyl esters, nmol/L</measure>
    <time_frame>Enrollment</time_frame>
    <description>Concentration of retinyl esters in subjects' serum</description>
  </primary_outcome>
  <primary_outcome>
    <measure>Liver vitamin A reserves, µmol/g</measure>
    <time_frame>Enrollment</time_frame>
    <description>Concentration of vitamin A in subjects' liver samples</description>
  </primary_outcome>
  <secondary_outcome>
    <measure>Serum retinol, µmol/L</measure>
    <time_frame>Enrollment</time_frame>
    <description>Concentration of retinol in subjects' serum</description>
  </secondary_outcome>
  <secondary_outcome>
    <measure>Liver alpha-retinyl ester, nmol/g</measure>
    <time_frame>Enrollment</time_frame>
    <description>Concentration of alpha-retinyl esters in subjects' liver samples</description>
  </secondary_outcome>
  <number_of_groups>3</number_of_groups>
  <enrollment type="Actual">27</enrollment>
  <condition>Vitamin A Deficiency</condition>
  <condition>Vitamin A Toxicity</condition>
  <arm_group>
    <arm_group_label>21-54 y</arm_group_label>
    <description>Ages 21-54 y</description>
  </arm_group>
  <arm_group>
    <arm_group_label>55-74 y</arm_group_label>
    <description>Ages 55-74 y</description>
  </arm_group>
  <arm_group>
    <arm_group_label>&gt;75 y</arm_group_label>
    <description>Age &gt;75 y</description>
  </arm_group>
  <biospec_retention>Samples Without DNA</biospec_retention>
  <biospec_descr>
    <textblock>
      Serum and liver samples&#xD;
    </textblock>
  </biospec_descr>
  <eligibility>
    <study_pop>
      <textblock>
        US adult cadavers from the the National Disease Research Interchange service age 21 years&#xD;
        and older, death from any cause.&#xD;
      </textblock>
    </study_pop>
    <sampling_method>Non-Probability Sample</sampling_method>
    <criteria>
      <textblock>
        Inclusion Criteria:&#xD;
&#xD;
          -  Human liver and serum samples were acquired as a consecutive sample through a request&#xD;
             to the National Disease Research Interchange service, based on sex and age range&#xD;
             targets (5 male and 5 female per age group; age groups: 21-54 ; 55-74; 75 and above).&#xD;
&#xD;
        Exclusion Criteria:&#xD;
&#xD;
          -  None&#xD;
      </textblock>
    </criteria>
    <gender>All</gender>
    <minimum_age>21 Years</minimum_age>
    <maximum_age>N/A</maximum_age>
    <healthy_volunteers>Accepts Healthy Volunteers</healthy_volunteers>
  </eligibility>
  <location>
    <facility>
      <name>University of Wisconsin - Madison</name>
      <address>
        <city>Madison</city>
        <state>Wisconsin</state>
        <zip>53706</zip>
        <country>United States</country>
      </address>
    </facility>
  </location>
  <location_countries>
    <country>United States</country>
  </location_countries>
  <verification_date>October 2017</verification_date>
  <study_first_submitted>September 29, 2017</study_first_submitted>
  <study_first_submitted_qc>October 3, 2017</study_first_submitted_qc>
  <study_first_posted type="Actual">October 9, 2017</study_first_posted>
  <last_update_submitted>October 13, 2017</last_update_submitted>
  <last_update_submitted_qc>October 13, 2017</last_update_submitted_qc>
  <last_update_posted type="Actual">October 17, 2017</last_update_posted>
  <responsible_party>
    <responsible_party_type>Sponsor</responsible_party_type>
  </responsible_party>
  <keyword>aging</keyword>
  <keyword>alpha-retinol</keyword>
  <keyword>hypervitaminosis A</keyword>
  <keyword>vitamin A biomarkers</keyword>
  <keyword>serum retinol</keyword>
  <condition_browse>
    <!-- CAUTION:  The following MeSH terms are assigned with an imperfect algorithm            -->
    <mesh_term>Night Blindness</mesh_term>
    <mesh_term>Vitamin A Deficiency</mesh_term>
  </condition_browse>
  <patient_data>
    <sharing_ipd>No</sharing_ipd>
  </patient_data>
  <!-- Results have not yet been posted for this study                                          -->
</clinical_study>


download date: ClinicalTrials.gov processed this data on September 20, 2023
link text: Link to the current ClinicalTrials.gov record.
url: https://clinicaltrials.gov/ct2/show/NCT03305042
org study id: 2015-1482
nct id: NCT03305042
lead sponsor: 
collaborator: 
has dmc: No
is fda regulated drug: No
is fda regulated device: No
Minimal human data exist on actual liver vitamin A compared with blood biomarkers. One blood
      biomarker, the percent of total serum retinol (vitamin A) in the form of retinyl esters, has
      been suggested to diagnose hypervitaminosis A with cutoffs of 5% and 10%. In this study,
      investigators aim to compare total liver vitamin A reserves with the percent total serum
      retinol as retinyl esters to evaluate hypervitaminosis A using autopsy samples from US
      adults. Investigators also evaluate the sensitivity (the ability of the biomarker to
      correctly identify those with deficiency) and specificity (the ability of the biomarker to
      correctly identify those without deficiency) of serum retinol to determine vitamin A
      deficiency, variation of liver vitamin A concentration among lobes, and liver alpha retinyl
      ester concentrations, a cleavage product of alpha-carotene, a vitamin A precursor. 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.
Minimal human data exist on actual liver vitamin A compared with blood biomarkers. One blood
      biomarker, the percent of total serum retinol (vitamin A) in the form of retinyl esters, has
      been suggested to diagnose hypervitaminosis A with cutoffs of 5% and 10%. In this study,
      investigators aim to compare total liver vitamin A reserves with the percent total serum
      retinol as retinyl esters to evaluate hypervitaminosis A using autopsy samples from US
      adults. Investigators also evaluate the sensitivity (the ability of the biomarker to
      correctly identify those with deficiency) and specificity (the ability of the biomarker to
      correctly identify those without deficiency) of serum retinol to determine vitamin A
      deficiency, variation of liver vitamin A concentration among lobes, and liver alpha retinyl
      ester concentrations, a cleavage product of alpha-carotene, a vitamin A precursor.

      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.
observational model: Other
time perspective: Cross-Sectional
measure: Serum retinyl esters, nmol/L
time frame: Enrollment
description: Concentration of retinyl esters in subjects' serum
measure: Liver vitamin A reserves, µmol/g
time frame: Enrollment
description: Concentration of vitamin A in subjects' liver samples
measure: Serum retinol, µmol/L
time frame: Enrollment
description: Concentration of retinol in subjects' serum
measure: Liver alpha-retinyl ester, nmol/g
time frame: Enrollment
description: Concentration of alpha-retinyl esters in subjects' liver samples
arm group label: 21-54 y
description: Ages 21-54 y
arm group label: 55-74 y
description: Ages 55-74 y
arm group label: >75 y
description: Age >75 y
Serum and liver samples
study pop: 
sampling method: Non-Probability Sample
criteria: 
gender: All
minimum age: 21 Years
maximum age: N/A
healthy volunteers: Accepts Healthy Volunteers
facility: 
country: United States
responsible party type: Sponsor
mesh term: Night Blindness
mesh term: Vitamin A Deficiency
sharing ipd: No