Clinical Trial Details
— Status: Not yet recruiting
Administrative data
| NCT number |
NCT01502826 |
| Other study ID # |
CJ7CTW-002 |
| Secondary ID |
|
| Status |
Not yet recruiting |
| Phase |
N/A
|
| First received |
November 24, 2011 |
| Last updated |
January 18, 2012 |
| Start date |
February 2012 |
| Est. completion date |
December 2013 |
Study information
| Verified date |
January 2012 |
| Source |
Universita di Verona |
| Contact |
Claudio Maffeis, Professor |
| Phone |
+ 39 045 4932011 |
| Email |
claudio.maffeis[@]univr.it |
| Is FDA regulated |
No |
| Health authority |
Italy: Ethics Committee |
| Study type |
Observational
|
Clinical Trial Summary
Childhood obesity is increasing at a fast pace, together with its complications. The aim of
the present study is to assess several candidate triggering agents, mechanisms and
intermediate phenotypes of atherosclerosis during the post-prandial phase in the obese
insulin-resistant child/adolescent.
Description:
Some of earlier outlines of the relationships between enlarged fat mass and insulin
resistance have not been proven experimentally in man. For instance, most of the
relationships shown in humans between cytokines and insulin sensitivity are correlational
observations.
In the obese child, fatty liver is quite prevalent and is more closely associated than other
fat depots to insulin resistance, dyslipidemia, high blood pressure and to high levels of
inflammation markers. The entire picture, therefore, is consistent with an accelerated drive
to atherosclerosis in the obese child/adolescent. Indeed, several reports have demonstrated
that intermediate phenotypes of atherosclerosis can be detected in the obese child.
Furthermore, the post-prandial phase is thought to play a role of its own in atherogenesis.
The investigators hypothesize that in the obese insulin-resistant child mechanisms of
atherogenesis, such as hyperlipemia, oxidant stress and sub-inflammation, are activated in
the post-prandial phase resulting in impairments of vascular function.
Parameters under study:
1. Candidate triggering agents
- Post-prandial glucose
- Post-prandial hyperlipemia
- Post-prandial small dense LDL
- Post-prandial insulin
- Candidate mechanisms
- Complement activation
- C3
2. Inflammation
- TNF-alpha
- Interleukin-6 (IL-6)
- Interleukin-10 (IL-10)
- C-reactive protein (CRP)
- Oxidant Stress
- Urinary iso-PGF2-alpha
3. Intermediate Phenotypes
- Carotid artery intima-media thickness (IMT) (only at baseline)
- Endothelial Function (flow-mediated vasodilation) (FMD)
- Arterial Stiffness (pulse-wave velocity) (PWV)
Experimental protocol Subjects will arrive at the Division of Endocrinology and Metabolic
Diseases of the Verona City Hospital at 0800 h after an overnight fast. Weight, height,
waist circumference and blood pressure will be measured. During the entire study subjects
will be lying in bed in a quiet, temperature controlled (22 °C) room. A Teflon venous
catheter will be inserted into an antecubital vein for blood sampling and will be kept
patent with a normal saline slow-drip infusion. Baseline state will be considered attained
after at least 20 minutes of rest in bed following vein puncture.
Subjects must have been on no drugs known to interfere with vascular function for at least 2
weeks and will be asked to refrain from caffeine containing beverages for at least 24 hours
before study. Systolic blood pressure, diastolic blood pressure, mean arterial pressure and
heart rate will be monitored at time intervals with Cardiocap II (Datex, Finland) throughout
the study.
Carotid-femoral PWV will be assessed using Complior (Colson, Garges les Genosse, France) .
Carotid artery IMT will be assessed by high resolution US color-doppler scan. Then,
flow-mediated endothelium-dependent vasodilation of the nondominant common femoral artery
will be assessed by high resolution echo-doppler (Esaote Biomedica AU6, Genova, Italy), with
a 10 MHz linear vascular probe with axial resolution of 0.01 mm. Baseline diameter and blood
flow velocity, a proxy of shear stress, will be assessed at least in triplicate. After this,
a sphygmomanometric cuff will be put under the knee and inflated 50 mm Hg higher than
systolic blood pressure and maintained for 5'. Vessel diameter and flow velocity will be
measured at time 0.5', 2', 4', 6', and 8' after deflating the cuff, according to
investigators' well established protocol . Under these conditions, when the cuff is deflated
postischemic hyperemia will take place in the tissues which are distal to the
sphygmomanometric cuff. This hyperemia will induced an increase in blood flow velocity of
the common femoral artery, which is located proximally to the cuff. The increase in flow
velocity, and the ensuing increase in shear stress, elicit an endothelium-dependent
vasodilation. It has been shown that this dilation is NO-dependent. A global quantitative
index of flow-dependent vasodilation will be obtained by calculating the area under the
curve of change in vessel diameter as a function of time (8 min of observation), expressed
both as absolute values and as percent changes over the baseline vessel diameter. In
investigators' hands, the between-day coefficient of variation of this technique (n=15,
covering a wide range of vascular response) is 23.0±3.8%. Endothelium-independent
vasodilation will not be evaluated because glyceryl-trinitrate administration for research
purpose is deemed ethically unjustifiable in children by the local Institutional Ethical
Committee.
In investigators' current experience (n=42 severely obese children), average FMD after an
overnight fast is 1.40±0.91% per 8 minutes (mean±SD), thereby leaving substantial room to
show dynamic changes. In these severely obese children these investigators found no
correlation between FMD and BMI. An ad hoc mechanical US probe holder will be employed in
the present paired studies, with the goal to help increasing reproducibility of the
technique. Furthermore, a specific automatic contour tracking technique for the automatic
calculation of the changes in arterial diameter will be used to minimize the operator
dependency of this method.
Baseline (-10 min and 0') blood samples will be collected to measure glucose, insulin,
C-peptide, standard lipid profile (total cholesterol, HDL-cholesterol, and triglycerides),
oxidized-LDL, adiponectin, leptin, C3, TNF-alpha, IL-6, IL-10, and CRP by micro-methods to
limit blood loss. A separate sample will be collected to measure LDL and HDL subfractions.
Baseline urines will be collected to measure iso-PGF2-alpha, a product derived from
nonenzymatic lipid peroxidation, catalyzed by oxygen free radicals on cell membranes and LDL
particles (43).
At time 0', subjects will ingest a mixed liquid meal (Ensure Plus, 1.5 kcal/cc) of known
caloric content (300 kcal per m2 of body surface area) and composition (53.8% carbohydrates,
29.5% lipids, 16.7% proteins) over 10 minutes. Glucose, insulin and C-peptide will be
measured at +15', +30', +45', +60', +90', +120', +150', +180', +210', +240' and +300'.
Standard lipids and PWV will be measured at +60', +120', +180', +240' and +300'.
Adiponectin, leptin, C3, TNF-alpha, IL-6, IL-10, CRP and LDL and HDL subfractions will be
measured at +180' and +300'. At time +180' assessment of FMD will be repeated. At the end of
the meal test urines will be collected to assess 8-iso-PGF2-alpha.
Blood samples will be quickly spun at 1500 g at +4 °C, plasma/serum will be collected and
stored at -80° C. Urine samples will be stored at -80° C. Total blood loss will be kept
below 100 cc.
Analytical Methods Except for LDL/HDL subfraction assessment and urine PGF2alpha, methods
specifically optimized to use tiny amounts of plasma/serum will be used to minimize blood
loss. Glucose will be measured at bedside with a Yellow Spring Glucose Analyzer by the
glucose oxidase method. C-peptide and insulin will be measured by in-house ELISA
micro-methods. Total and HDL cholesterol and total triglycerides will be assessed by
standard in-house enzymatic micro-methods. Adiponectin, leptin, C3, TNF-alpha, IL-6, IL-10,
CRP will be assessed by in-house immunometric micro methods. 8-Iso-PGF2alpha will be
assessed by an immunometric assay after extraction from urines.
LDL/HDL subfractions will be measured in the laboratory of prof. Alberto Zambon, at
University of Padua School of Medicine. Briefly, after creating a discontinuous salt density
gradient in an ultracentrifuge tube, the samples will be centrifuged at 65,000 rpm for 90
min at 10°C in a Sorvall TV-865B vertical rotor. Thirty-eight 0.45-ml fractions are then
collected from the bottom of the centrifuge tube. Cholesterol is measured in each fraction.
The relative flotation rate (Rf), which characterizes LDL peak buoyancy and is a measure of
LDL size/density, is obtained by dividing the fraction number containing the LDL cholesterol
peak by the total number of fractions collected.
Assessment of beta-cell function
Beta-cell function will be evaluated by analyzing the glucose and C-peptide curves during
the mixed meal test, according to the general strategy proposed by several laboratories with
some slight modifications. Briefly, insulin secretion is the sum of three components:
1. basal (postabsorptive) insulin secretion rates;
2. insulin secretion in response to the rate of increase in plasma glucose ("dynamic" or
"derivative" secretion component);
3. insulin secretion in response to the actual glucose levels above the postabsorptive
glucose concentration ("static" or "proportional" secretion component).
A complete description of the modeling strategy can be found in investigators' earlier
publications. Parameters will be estimated by implementing this minimal model of C-peptide
secretion in the SAAM 1.1.2 software (SAAM Institute, Seattle, WA). Numerical values of the
unknown parameters will be estimated by using nonlinear least squares.
Weights are chosen optimally, i.e., equal to the inverse of the variance of the measurement
errors, which are assumed to be additive, uncorrelated, with zero mean, and a constant
coefficient of variation (CV) of 8%.
This analysis will provide the hormonal scenario in which the postprandial atherogenetic
processes take place.
Statistical analysis Data will be summarized as means±SEM. The size of the sample (n=20 in
each group) has been selected with the aim of having 80% chance of attaining statistical
significance for differences of 20-40% (the minimum detectable difference varies from
variable to variable under study). The general estimating equation procedure (GEE) will be
used to run 2-way ANOVA for repeated measures and to adjust for covariates, if needed.
Gaussian distribution will be tested in all variables, and, if statistically significant
deviations from normality are found, logarithmic (or other) transformation will be applied.
Correlations will be sought by the Spearman's rank correlation coefficient. Statistical
significance will be declared at p<0.05. Statistical analyses will be performed using SPSS
12.0 (or higher) for MacOS X.
Reaching the goals of the present research project will:
1. enlarge the knowledge on the relationship between childhood obesity, inflammation,
insulin resistance and vascular dysfunction;
2. elucidate whether postprandial hyperlipemia, postprandial inflammation and/or
postprandial oxidant stress are associated to intermediate phenotypes of
atherosclerosis in the obese child/adolescent;
3. establish potential novel targets of prevention of obesity complications in the
child/adolescent and potentially indicate novel tools for the treatment of obesity
associated disturbances.
