A Study of Racial Differences in Natriuretic Peptides Response to Glucose Challenge
Phase: NA
Conditions: Healthy, Natriuretic Peptides, Metabolic Syndrome
Sponsor: University of Alabama at Birmingham

January 20, 2021 
 
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PROTOCOL:  Racial Differences in the Natriuretic Peptide Response to Glucose Challenge 
 
Study Design, Setting, and Location 
 
Our study was a single-center, prospective, physiological clinical trial approved by the 
institutional review board at the University of Alabama at Birmingham (UAB).  Participants 
were recruited from the university campus and surrounding areas.  All participants signed 
informed consent.   
 
 
Sample Selection 
 
Prospective participants were first contacted by phone to determine eligibility prior to 
scheduling a screening visit at the clinical research unit (CRU).  Self-identified healthy black and 
white men and women were eligible to participate in the clinical trial based on the following 
criteria: between 18 and 40 years of age, blood pressure (BP) less than 140/90, estimated 
glomerular filtration rate >60 ml/min, and willingness to adhere to the study diet.  Exclusion 
criteria included the following: history of hypertension, history of cardiovascular, renal, or liver 
disease, history of diabetes or glucose-lowering medications, use of vasoactive or diuretic 
medications, anemia, abnormal serum sodium or potassium, pregnancy, elevated liver function 
tests defined as >3x the upper limit of normal, women taking hormonal birth control, history of 
smoking, body mass index (BMI) <18.5 kg/m2, or regular use of non-steroidal anti-inflammatory 
medications.   
 
Study Protocol 
 
For the screening visit, subjects were educated on the study protocol and directed to 
complete a questionnaire regarding medical and family history.  The study investigators further 
evaluated subjects who agreed to the study and signed consent.  Blood was drawn for a basic 
metabolic profile, liver enzymes, and complete blood count.  Additional samples were collected 
if consent was provided for DNA extraction and gene expressions for NPs.  The CRU nursing 
staff obtained height, weight, and BP (i.e., an average of three BP measurements) for a baseline.  
Female participants provided a urine sample to test for pregnancy.  Before completion of the 
screening visit, subjects met with the registered dietitian to discuss the study diet and were 
provided with a 3-day meal log to assess dietary habits.  
 
Upon review of screening labs, eligible subjects were enrolled in the study [stratified by 
race using permuted blocks for age and sex)] and invited back for food pick up and protocol (i.e., 
a high-carbohydrate challenge) visits.  Within seven days after the screening visit, participants 
visited the CRU metabolic kitchen for food pick-up.  Three days of standardized meals were 
provided to be consumed for the three days leading up to the protocol visit.  Three meals per day 
and snacks were provided based on participant preferences and by following macronutrient 
RDAs from the USDA 2005 Guidelines.  The study nutritionist calculated caloric needs.  In 
brief, each study participant was provided on an average 1,993 kilocalories worth of meals 
comprising 4,667 mg of sodium per day for three days.  The contribution of carbohydrate, fat, 
and protein in total caloric intake was 51%, 31%, and 18%, respectively.  Additional instructions 
were given to drink only water, to refrain from alcohol and caffeine use, and to consume only 

January 20, 2021 
 
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food provided by CRU metabolic kitchen for the remainder of the study.  Participants were also 
advised to avoid strenuous activities for 24 hours prior to the protocol visit.  For the protocol 
visit, participants fasted overnight for 10 hours, before reporting at 7:00 AM to the CRU.  Upon 
arrival, participants were assessed for compliance with the study diet and were positioned in the 
sitting position with the back-rest at 45 degrees and legs elevated.  Heart rate, oxygen saturation 
and BP (an average of three measurements using a Carescape V100 monitor) were obtained.  
Participants were given glucola, a 75mL, 75g glucose equivalent, and were provided only 
distilled water to drink as needed for the remainder of the protocol.  Blood pressure was 
measured every 30 minutes, and heart rate and oxygen saturation were measured every 60 min.  
Blood was drawn in 60 min intervals for 8 hours for biomarker profiling.  All the participants 
remained in fasting state (except distilled water) till the end of the study protocol. 
 
Statistical Analyses 
 
Our study was powered to detect the change in ANP levels in response to a high-
carbohydrate challenge among black individuals.  Based on a prior investigation among Whites, 
a high-carbohydrate challenge can reduce circulating plasma ANP levels up to 27%.  With an 
estimated standard deviation of 0.2 (a high estimate) and utilizing prespecified sample size of 30 
black individuals,  our study was powered to detect a change as small as 11% in ANP levels after 
a high-carbohydrate challenge, with β=0.8 and α=0.05.  We expected a similar effect size for 
change in ANP levels after a high-carbohydrate challenge among whites. 
 
All analyses were conducted using STATA, version 14.2 MP (StataCorp LP), and 
LIMMA R package.  Baseline characteristics of participants were compared between blacks and 
whites using Student’s t-test (normal distribution) or Mann-Whitney test (non-normal 
distribution) for continuous variables and Pearson χ2 test for categorical variables.  The normality 
of continuous variables was assessed using histogram and Q-Q plots (visual assessment) as well 
as Shapiro Wilk test (statistical assessment).  Plasma NP levels at baseline and after a high-
carbohydrate challenge were found to have a non-normal distribution and were log-transformed 
for the analyses.  Linear regression models were used to assess the racial differences in plasma 
NP levels.  Choosing white race as a reference, a relative percentage difference in plasma NP 
levels with 95% confidence interval (CI) in blacks was calculated using the following formula: 
(eβ – 1) x 100 (where β is the beta coefficient from linear regression).  Linear mixed-effect 
models excluding missing data (<1%) from 72 individuals were used to assess the effect of a 
high-carbohydrate challenge over time on plasma NP levels.  In these models, we assessed for 
repeated measurements of NP levels with fixed effects of race, time, and their multiplicative 
interaction term (race*time), accounting for the correlation among repeated measures in the same 
person.  Participants were treated as random effects in these models.  The percentage difference 
with 95% CI in plasma NP levels after 8 hours in overall as well as by race was calculated: [(eβ – 
1) x 100 multiplied by 8 hours (where β is the beta coefficient from linear regression)].  The 
models mentioned above were further assessed after adjusting for their baseline demographics 
[i.e., age, sex, body mass index (BMI), insulin].