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Abstract. – OBJECTIVE: Very low-calorie di-
ets (VLCDs, < 800 kcal day-1) and Ketogenic diet
(KD) are generally used as part of integrated in-
tervention, medical monitoring and a program of
lifestyle modification, to improve a multitude of
clinical states. The effect of three different very
low calories KD (VLCKD), with (VLCKD1) or with-
out (VLCKD2,3) synthetic amino acid replacement
of the 50% protein intake, were analyzed after
weight loss.
PATIENTS AND METHODS: The clinical study
used a cross-over randomized double-blind place-
bo-controlled trial. Obese subjects, who were eli-
gible for the study, were randomly (R) divided in-
to three groups: one intervention group (IG) and
two control groups (CG1 and CG2). We compre-
hensively analyzed body composition, serum
metabolites, superoxide dismutase (SOD1), nu-
clear factor kappa-light-chain-enhancer of acti-
vated B cells (NfKB), Chemokine (C-C Motif) Lig-
and 2 (CCL2) gene expression.
RE S U LTS : Af ter VLDKD s a sig nif ica nt de-
creased in BMI was observed. TBF (kg) signifi-
cantly decrease after VLCKD1and VLCKD3. After
VLCKD2, a reduction of waist circumference (p=
0.02), FM L2-L5 (p< 0.05) was observed. After
VLCKD1reduction of IMAT (p= 0.00), LDL-C (p=
0.00) and HDL-C (p= 0.00) were observed. No
significant changes of GH, ESR, and fibrinogen
were highlighted. CRP (p= 0.02) reduced signifi-
cantly after VLCKD3. Significant modulation of
SOD1 expression (p= 0.009), CRP and decrease
of glucose levels (p= 0.03) were obtained after
VLCKD3.
European Review for Medical and Pharmacological Sciences
Effects of very-low-calorie diet on
body composition, metabolic state, and
genes expression: a randomized
double-blind placebo-controlled trial
G. MERRA1, S. GRATTERI2, A. DE LORENZO3, S. BARRUCCO4, M.A. PERRONE5,6,
E. AVOLIO7, S. BERNARDINI5, M. MARCHETTI8, L. DI RENZO4
1Emergency Department, “A. Gemelli” General Hospital Foundation, Catholic University of the
Sacred Heart, School of Medicine, Rome, Italy
2Department of Surgery and Medical Science, University “Magna Graecia”, Catanzaro, Italy
3Columbia University, New York, NY, USA
4Department of Biomedicine and Prevention, Section of Clinical Nutrition and Nutrigenomic,
University of Rome “Tor Vergata”, Rome, Italy
5Division of Clinical Biochemisty and Clinical Molecular Biology, University of Rome “Tor Vergata”,
Rome, Italy
6Division of Cardiology, University of Rome “Tor Vergata”, Rome, Italy
7Comparative Neuroanatomy Laboratory, University of Calabria, Cosenza, Italy
8Department of Surgical Sciences, “Umberto I” General Hospital, “Sapienza” University, Rome, Italy
Corresponding Author: Giuseppe Merra, MD; e-mail: giuseppe.merra@policlinicogemelli.it 329
CON CL USION S: This is the first study that
analyzes comprehensively body composition,
metabolic profile, and inflammation and oxida-
tive stress genes expression after VLCKD. Our
results show the efficacy of VLCKD with syn-
thetic aminoacidic protein replacement, for the
reduction of cardiovascular risk, without the
development of sarcopenia and activation of in-
flammatory and oxidative processes.
Key Words:
Obesity, Ketogenic diet, Inflammation, Glucose,
Lipid profile.
Introduction
The effects of diet on metabolic pathways re-
lated to obesity, diabetes, cardiovascular dis-
eases, and other chronic non-communicable dis-
eases (CNCD) are currently under investigation.
The primary determinant of weight loss is energy
deficit, and several dietary strategies available,
divided between low calories diet (LCD, 800
kcal day-1) and very-low-calorie diets (VLCDs,
< 800 kcal day-1)1can arise this goal.
VLCDs are generally used as part of an inte-
grated intervention that includes medical moni-
toring and a program of lifestyle modification,
and they are considered safe and effective at the
2017; 21: 329-345
G. Merra, S. Gratteri, A. De Lorenzo, S. Barrucco, M.A. Perrone, et al.
termination of their relationships with quality of
loss, regarding total body fat (TBF) mass loss
and maintenance of total body lean (TBL) mass,
metabolic states, inflammatory and oxidative
stress profile, remains of extreme importance.
In the present study, we wanted to check the
criterion of efficacy and safety in the short term
of VLCKD. We asked whether body composi-
tion, lipid and glucose profiles, inflammatory and
oxidative stress responses could be modulated in
a different manner by three different dietary
treatments (DTs). We conducted a randomized
double-blind placebo-controlled trial, and we
comprehensively analyzed body composition,
serum metabolites, superoxide dismutase
(SO D 1 ) , n uclear fac t o r k a ppa-light - c h a in-
enhancer of activated B cells (NfKB),
Chemokine (C-C Motif) Ligand 2 (CCL2) gene
expression in obese subjects during weight loss
with different DTs.
Patients and Methods
Clinical Study Design and Participants
The clinical study used a crossover random-
ized double-blind trial with placebo, between
October 2015 and April 2016. Subjects were con-
secutively recruited within a program of a routine
medical check-up at the Section of Clinical Nu-
trition and Nutrigenomic, University of Rome
“Tor Vergata”.
Eligibility criteria for the study were as fol-
lows: age between 18 and 65 years, BMI ≥25
kg/m2, the percentage of body fat (PBF) ≥25%
for male, and ≥30% for female. On the other
hand, exclusion criteria were as follows: preg-
nancy, breastfeeding, type 1 diabetes, heart fail-
ure, endocrine disorders, liver dysfunction, liver,
kidney, autoimmune, viral chronic (Hepatitis C,
B, HIV), neurologic disorders, and neoplastic
diseases; corticosteroid and chronic inflammato-
ry therapy; participating in another diet trial.
Subjects, who were eligible for the study, were
randomly (R) divided into three groups: one in-
tervention group (IG) and two control groups
(CG1 and CG2) were utilized. A simple random-
ization was carried out and was determined by an
external contract research organization and coor-
dinated with the Section of Clinical Nutrition and
Nutrigenomic, at the University of Rome “Tor
Vergata”, independently of the investigators. For
each group 20 subjects were allocated.
The study was conducted in double blind.
condition that it is used for highly selected pa-
tients, under careful medical supervision2.
VLCDs includes the very-low-carbohydrate
and high-fat ketogenic diet (VLCKD), and the
therapeutic use of this dietary treatment (DT) has
been extensively studied for the amelioration of a
multitude of clinical states, to manage obesity,
diabetes, epilepsy, seizure disorders, and malig-
nancies of the central nervous system3-7. VLCKD
is becoming an elective choice to promote weight
loss, especially in the case of severe obesity and
its metabolic complications, because this dietary
regimen seems to be more effective than the tra-
ditional calorie restriction. The VLCKD creates a
distinctive, but not well defined, cellular, molecu-
lar, and integrated metabolic state.
The effect of the ketogenic diet (KD) and VL-
CKD on lipid metabolism and the mechanisms
through which it can promote weight loss re-
mains controversial8.
According to Ellenbroek et al9after long-term
treatment, KD lead to increasing of plasma mark-
ers associated with dyslipidemia and inflamma-
tion, such as cholesterol, triglycerides, leptin,
monocyte chemotactic protein-1, IL-1beta, and
IL-6 without weight loss. However, different pa-
pers referred opposite effects. It has been demon-
strated that inflammation and thermal nocicep-
tion was significantly attenuated by KD treat-
ment10, probably because the KD decreases reac-
tive oxygen species (ROS) production by in-
creasing the expression and activity of mitochon-
drial uncoupling proteins11.
Garbow et al12 reported that, in C57BL/6J mice,
a very low-carbohydrate, low-protein, and high-fat
ketogenic diet determines a reduction, up to the
suppression, of the expression of inflammatory cy-
tokines and chemokines, as well as the production
of reactive species oxy-hydrogen (ROS).
Mutations in the gene encoding the enzyme
Cu/Zn superoxide dismutase 1 (SOD1) were the
first mutation identified to be associated with fa-
milial amyotrophic lateral sclerosis (ALS). It has
been demonstrated that a ketogenic diet in the
G93A-SOD1 transgenic mice model of ALS pro-
motes ATP synthesis and neuroprotection7.
In the context of tumor hypoxia and angiogen-
esis, animals fed ad libitum with KD significant-
ly reduced the activation of Nuclear Factor of
Kappa light polypeptide gene enhancer in β-cells
(NF-κB), blood glucose, and increased blood β-
hydroxybutyrate levels (βHB) levels13.
Although low-carbohydrate ketogenic diets are
effective for weight control, comprehensive de-
330
The study had no. 3 DTs conducted in three
arms: (1) a VLCKD1, in which 50% of protein in-
take is replaced with synthetic amino acids; (2) a
VLCKD2wit h place bo; (3) a VLC KD3with
placebo.
At arm no. 1 for three weeks (wks), the group
IG received the VLCKD1, the group CG1 re-
ceived the VLCKD2, and the group CG2 received
the VLCKD3. After 3 wks of washout period, to
avoid additive effects on treatments to follow, the
DT for each group was reversed (arm no. 2). Af-
ter 3 wks of washout period, the DT for each
group was reversed again (arm no. 3).
At the Baseline (T0) and at the end of each
arm (T1), all the subjects were evaluated for their
health and nutritional status, by anthropometric,
body composition, biochemical evaluation. Fur-
thermore, the genomic profile was evaluated for
every participant.
It was asked to the subjects not to change their
lifestyle habits. Any adverse effect has been
properly signed.
The first outcome was the evaluation of body
composition changes after DTs, evaluated by an-
thropometry and Dual X-ray Absorptiometry
(DXA). The second outcome was the evaluation
of metabolic profile by blood analysis. The third
outcome was the evaluation of nutrigenomic pro-
file by transcriptomic analysis.
The participants received no financial compen-
sation or gifts. All measurements were performed
at the Section of Clinical Nutrition and Nutrige-
nomic, Department of Biomedicine and Preven-
tion of the University of Rome “Tor Vergata”.
All subjects gave informed consent to partici-
pate in the interventional study, which was per-
formed, in accordance with principles of the De-
claration of Helsinki. All procedures followed
were in accordance with the ethical standards of
the responsible committee on human experimen-
tation (Ethics Committee “Centro, Calabria Re-
gion” 30.11.02.2016).
Clinical trial registration: the study has been reg-
istered by ClinicalTrials.gov Id: NCT01890070.
Sample Size
The minimum sample size was calculated with
respect to a two-tailed one-sample Student’s t-
test, considering as (1) insulin level to be detect-
ed between the two DTs | | ≥15 µU/mL − 1, (2)
SD of the paired differences SD = 15 µU/mL − 1,
(3) type I error probability α= 0.05 and power 1
−β= 0.90. The result was a minimum sample
size of 10 per group.
Dietary Treatment
We selected three different VLCKD, in which
the daily kcal amount was calculated subtracting
to the estimated basal metabolism 1000 kcal/day.
We considered a diet as ketogenic, when a num-
ber of carbohydrates were < 50 g/day.
The VLCKD1aimed at a daily energy intake
of 450-500 kcal per day for female, with 35-45%
of calories from proteins (corresponding to 1.2
g/kg of ideal body weight), 45-50% from fat (<
10% of calories from saturated fat), and 15%
from carbohydrates (< 20 g). The VLCKD1for
male aimed at a daily energy intake of 650-700
kcal, with 50-55% of calories from proteins (cor-
responding to 1.5 g/kg of ideal body weight), 35-
40% from fat (< 10% of calories from saturated
fat), and 10% of calories from carbohydrates (<
20 g). Both VLCKD1provided an intake of 20
mg of fiber per day. The half of the amount of
daily p r o t e i n was reached using syntheti c
aminoacid supplementation (SAS), contained:
whey protein (13.42/bag), carbohydrate
(0.03/bag), fat (0.15/bag), isoleucine (0.31/bag),
ornithine alpha-ketoglutarate (0.25/bag), L-cit-
rulline (0.25/bag), taurine, (0.25/bag), L-trypto-
phan (0.05/bag), potassium citrate (0.45/bag), for
a total of 64 kCal (268 KJ) (Macresces, Italfar-
macia s r l , R o m e , I t a ly). The pow d e r o f
aminoacid was dissolved in water and drunk at
breakfast and lunch or dinner.
The VLCKD2aimed at daily energy intake of
450-500 kcal for female with 20-30% of calories
from proteins (corresponding to 0.9 g/kg of ideal
body weight), 45-50% from fat (< 10% of calo-
ries from saturated fat) and 20-25% of calories
from carbohydrates (< 35 g; > 80% from simple
sugars). The VLCKD2for male aimed at a daily
energy intake of 650-700 kcal with 45-50% of
calories from proteins (corresponding to 1.1 g/kg
of ideal body weight), 35-40% fat (< 10% of
calories from saturated fat) and 20-25% of calo-
ries from carbohydrates (< 35 g; > 80% from
simple sugars). Both treatments provided an in-
take of 20 mg of fiber per day.
The VLCKD3aimed at an energy intake of
450-500 kcal per day for female with 25-35% of
calories from proteins (corresponding to 0.9 g/kg
of ideal body weight), 45-50% from fat (< 10%
of calories from saturated fat) and 20-25% of
calories from carbohydrates (< 30 g; > 35% from
complex sugars). The VLCKD3for male aimed at
a daily energy intake of 650-700 kcal with 45-
50% of calories from proteins (corresponding to
1.1 g/kg of ideal body weight), 35-40% fat (<
331
Effects of very-low-calorie diet on body composition, metabolic state, and genes expression
332
10% of calories from saturated fat) and 15-20%
of calories from carbohydrates (< 30 g; > 35%
from complex sugars). Both treatments provided
an intake of 20 mg of fiber per day.
The CG1 and CG2 received VLCKD2and VL-
CKD3respectively, with the placebo represented
by inert material (flour type 00). The powder of
placebo was dissolved in water and drunk at
breakfast and lunch or dinner.
In all DTs, a capsule of multivitamin, proper
integration of mineral salts and an alkalizing
product were prescribed. The correct administra-
tion of diet was evaluated by urinary keto-stick.
Anthropometric Measurements
After a 12-hour overnight fast, all subjects un-
derwent anthropometric evaluation. All the indi-
viduals were instructed to take off their clothes
and shoes before undergoing the measurements.
Waist and hip circumferences were taken us-
ing a flexible steel metric tape to the nearest 0.5
cm. Hip circumference was measured according
to International Society for the Advancement of
Kin anthropometry protocol taken at the greatest
posterior protuberance of the buttocks. Waist cir-
cumference was measured just above the iliac
crest to the nearest 0.1 kg, using a balance scale
(Invernizzi, Rome, Italy). Height (m) was mea-
sured using a stadiometer to the nearest 0.1 cm
(Invernizzi, Rome, Italy). BMI was calculated us-
ing the formula: BMI = body weight /height2
(kg/m2).
Dual X-ray Absorptiometry
To assess body composition analysis given the
possibility to measure total body fat (TBF) and
total body lean (TBL), DXA (i-DXA, GE Med-
ical Systems, Milwaukee, WI, USA).
The technique combined a total body scanner,
an X-ray source, an internal wheel to calibrate
the bone mineral compartment, and an external
lucite/aluminum phantom to calibrate soft mass.
Calibration and verification of the reproducibility
of the data were daily performed. The subjects
have received instructions before attending to the
medical views. Individuals were asked to remove
all clothing except for undergarments including
shoes, socks, and metal items before beginning
DXA examination in the supine position, with
the scan from the head and moving in a rectilin-
ear pattern down the body to the feet. The aver-
age measurement time was 20 min. The effective
radiation dose from this procedure is about 0.01
mSv. The coefficient of variation (coefficient of
variation = 100 × SD/mean) intra and inter-sub-
jects ranged from 1% to 5%. The coefficient of
variation for bone measurements is less than 1%;
coefficient of variation on this instrument for five
subjects scanned six times over a nine months
period were 2.2% for TBF, and 1.1% for TBL.
Total body fat percentage (PBF) was calculat-
ed as TBF mass divided by total mass of all tis-
sues, considering also the total body bone (TBB),
as the follow:
PBF = (TBF + TBL + TBB) × 100. (1)
Equations used for the percentage estimation
of fat mass for region and tissue parameters were
the following:
Region (%) = [TBF (kg) / (TBF (kg) + TBL (kg)
+ BCM (kg)] × 100 (2)
Tissue (%) = [TBF (kg) / (TBF (kg) + TBL
(kg)] × 100 (3)
where BCM represents the Bone Mineral Con-
tent.
Appendicular Skeletal Muscle Mass Index
(ASMMI)
ASMMI = (Legs Muscle Mass (kg) + Arms
Muscle Mass (kg)/Height (m2)
(Men < 7.59 kg/m2, Women < 5.47
kg/m2). (4)
Intermuscular Adipose Tissue (IMAT) was cal-
culated according to Bauer et al14 with the fol-
lowing formulas:
Log (IMAT) = [-2.21 + (0.12 × fat) + (-0.0013
× fat2)] for women (5)
Log (IMAT) = [-2.05 + (0.12 × fat) + (-0.0013
× fat2)] for men (6)
Body fat mass (FM) relative to the lumbar area
was calculated taking into account the area be-
tween the lumbar spine 2 (L2) and lumbar spine
5 (L5).
Hand Grip Strength Analysis
For the strength evaluation it was used an
electronic dynamometer (DynEx, Akern, Flo-
rence, Italy). The subjects were given complete
G. Merra, S. Gratteri, A. De Lorenzo, S. Barrucco, M.A. Perrone, et al.
instructions on the testing procedure, as specify
by Shechtman et al15. T he participant s p er-
formed all grip strength tests in the seated posi-
tion. The subjects were seated in a chair without
arm rests, with feet on the floor, hips as far back
in the chair as possible, and the hips and knees
positioned at approximately 90 degrees. The
shoulder of the tested extremity was adducted
and neutrally rotated, the elbow flexed at 90 de-
grees, the forearm in neutral position and the
wrist between 0 and 30 degrees of dorsiflexion
and between 0 and 15 degrees of ulnar devia-
tion. Subjects were instructed to maintain their
position during the grip strength test. Three rep-
etitions were execute d consecutively by the
right hand and only then by the left hand. There
was a 30-second rest period between each of the
three repeated trials and a two-minute rest peri-
od between each hand.
Biochemical Analyses
Blood tests were performed at each time of
evaluation, after a 12-hour overnight fast. Blood
samples (10 mL) were collected into sterile tubes
containing EDTA (Vacutainer®). All materials
were immediately placed on ice and plasma was
separated by centrifugation at 1600 × g for 10
min at 4°C.
La borat ory tes t inclu ded com plete b lood
count, Hemoglobin (HB) and Hematocrit blood
testing (HBT), fasting glucose, Total cholesterol
(TC), HDL-cholesterol (HDL-C), LDL-choles-
terol (LDL-C), triglycerides (Tg), fibrinogen,
Erythrocyte Sedimentation Rate (ESR), C-reac-
tive protein (CRP), Insuline (I), insulin growth
factor-1 (IGF-1) and growth Hormone (GH) lev-
els were recorded at baseline, and at the end of
each arms. All clinical chemistry analyses, ex-
cept glucose, serum lipid, CRP, and triglycerides
analysis, were carried out with an ADVIA®1800
Chemistry System (Siemens Healthcare, Munich,
Germany).
Plasma glucose concentrations were measured
using the glucose oxidase method with an auto-
mated glucose analyzer (COBAS INTEGRA
400, Roche Diagnostics, Indianapolis, IN, USA),
Serum lipid profile components were determined
by standard enzymatic colorimetric techniques
(Roche143 Modular P800, Roche Diagnostics,
Indianapolis, IN, USA). Serum CRP was mea-
sured by a high-sensitivity sandwich enzyme im-
munoassay from Immundiagnostik (Immundiag-
nostik AG, Bensheim, Germany). Serum triglyc-
erides were measured on the Beckman Synchron
LX 20 (LX 20; Bec kman C oulte r, B rea, CA,
USA) automated system by a coupled enzymatic
method that produces a red-coloured complex.
All tests were performed using the same lot of
reagents or assay plates to minimize variability
due to differences in reagent lots.
Lipid Accumulation Product (LAP) is an in-
dex, calculated through waist circumference
(WC) and triglyceride (TG) ratio according to the
formula:
LAP = [wais t cir cumf eren ce (cm) – 58] ×
triglycerides (mmol/l)16,17. (7)
Inflammatory indices Platelet Lymphocyte ra-
tio (PLR) and Neutrophils Lymphocytes ratio
(NLR) were evaluated during the study18.
Analyses were carried out at the accredited
Clinical Chemical Laboratories of the “Universi-
ty Hospital Tor Vergata” of Rome, Italy.
Sample Collection, RNA Extraction,
and Reverse Transcription
A blood sample was collected and stabilized in
Tempus Blood RNA Tubes (Applied Biosystems,
Foster City, CA, USA), and stored at -20°C until
RNA extraction. The total RNA of each collected
sample was purified using the Stabilized Blood
to Ct Nucleic Acid Preparation Kit for qPCR
(Life Technologies, Carlsbad, CA, USA).
Aliquots of total RNA were quantified and as-
sessed for quality by spectrophotometry (Nan-
odrop, Wilmington, DE, USA) and agarose gel
electrophoresis. Reverse transcription of each
sample of RNA was performed with High Capac-
ity RNA-to-cDNA Kit (Applied Biosystems,
Foster City, CA, USA).
Quantitative Real Time PCR and
Data Analysis
Real-time PCR was performed and analyzed
using Taqman Gene Expression Assay primer-
probe sets. We analyzed the following genes: su-
peroxide dismutase 1 (SOD1) (Hs00533490_m1),
Peroxisome prolifer ator activated rec eptor-γ
(PPAR-γ) (Hs00234592_m1), Nuclear factor kap-
pa-ligh t-chain-enhanc er of activated B cells
(NfKB) (Hs00765730_m1), Chemokine (C-C Mo-
tif) Ligand 2 (CCL2) gene (Hs00234140_m1).
Each qRT-PCR experiment was performed in trip-
licate and repeated at least twice, according to
manufacturer’s instruction (Applied Biosystems,
Foster City, CA, USA).
333
Effects of very-low-calorie diet on body composition, metabolic state, and genes expression
334
Comparative threshold (Ct) cycle was used to
determine gene expression level relative to the
calibrator from controls. The Ct value for each
gene was normalized using the formula:
∆Ct = Ct (gene) – Ct (Housekeeping Gene). (8)
The housekeeping gene used for this analysis
was Actin β(Hs01060665_g1) (Applied Biosys-
tems, Foster City, CA, USA).
Statistical Analysis
A paired t-test or a non-parametric Wilcoxon
test were performed to evaluate differences at
baseline and after a nutritional intervention.
The differences between parameter at baseline
and after diet were calculated as the follow:
∆% = [(Z-W)/W] × 100 (9)
where ∆% is the percentage variation of each
parameter, calculated as the ratio of absolute
variation to the base value.
The null hypothesis was rejected at the 0.05
level of probability.
Results
Of the sixty-five subjects enrolled from Octo-
ber 2015 to April 2016, five of them did not meet
the inclusion criteria, therefore sixty participants
resulted eligible for the study. Two subjects in-
cluded in IG of arm 1, and one included in GC2
declined to participate after one week; two sub-
jects included in IG of arm 2 declined to partici-
pate after two weeks, and one after two weeks of
the arm3.
Fifty-four patients completed the study, with a
mean age of 44.60 ± 15.06 years. The population
was represented by the 70% female and 30%
male.
At baseline (T0), the mean of BMI was 31.31
± 3.32 kg/m2. According to BMI the 50% of the
population was overweight and the 50% was
obese. All the subjects were obese according to
TBF percentage, estimated by DXA (> 30% for
female, > 25% for male).
At baseline only 10% of study population had
metabolic syndrome according to international
diabetes federation (IDF). After all the DT no
subject had the inclusion criteria for metabolic
syndrome diagnosis19.
Comparison of body composition parameters
after 3 wks of each DTs are shown in Table I-II.
All groups had a significant decreased in BMI:
after VLCKD1the ∆% of BMI was = -4.72% (p=
0.00), after VLCKD2the ∆% of BMI was = -
6.1% (p= 0.00) and after VLCKD3the ∆% of
BMI was = -7.84% (p= 0.00).
After VLCKD1treatment a significant de-
crease of Android Fat Percentage (AFP) of tissue
(∆% = -1.8%, p= 0.01), TBF (kg) (∆% = -7.8%,
p= 0.00), and tissue TBF percentage (∆% = -
0.9% p= 0.03) were highlighted.
IMAT value decreased in all diet treatments,
but only in VLCKD1a significant reduction was
observed (p= 0.00).
After VLCKD2, there was a significant reduc-
tion of waist circumference (p= 0.02), accord-
ingly with the result of FM L2-L5 (p< 0.05).
Conversely, to the other two DTs, no significant
changes were observed for hip circumferences
and fat mass parameters.
VLCKD3determined a significant decrease of
TBF (kg) (∆% = -7.9%, p= 0.00), and Fat Mass
L2-L5 (FM L2-L5) (p< 0.05).
VLCKD1determined a significant reduction of
TBL ma s s ( k g ) ( ∆% = - 4 . 7 % , p= 0.01).
VLCKD2treatment did not change any lean mass
parameters. After VLCKD3there was a signifi-
cant lowering of TBL mass (kg) (∆% = -7.8%, p
= 0.00).
Contrary to the two other treatments, VLCKD3
determined a significant decrease of ASMMI (p
= 0.00). However, submaximal resistance time
(at 70%), measured by handgrip, was significant-
ly reduced only after VLCKD1treatment (p=
0.01).
Comparison of blood parameters after 3 wks
of each DTs are shown in Table III and IV.
Blood tests underlined a significant reduction
for White Blood Cells (p= 0.00), neutrophils (p
= 0.01) and lymphocytes (p= 0.00) in VLCKD1
treatment. Even after VLCKD2it was underlined
a significant decrease of lymphocytes (p= 0.01),
but no other changes in blood tests were ob-
served.
No changes were observed in RBC, hemoglo-
bin, and hematocrit values.
PL R i ndex in crease d s ignificantl y ( ∆%=
+17. 3%, p= 0.00) on ly after VLCKD1. Red
Blood cells number (RBC ) sig nificantly in-
creased after VLCKD3treatment (p= 0.03).
After VLCKD1, it was noticed a significative
lowering of lipid profile values like TC (∆%=-
16.1%, p= 0.00), LDL-C (∆% = -21.4%, p=
G. Merra, S. Gratteri, A. De Lorenzo, S. Barrucco, M.A. Perrone, et al.
335
Effects of very-low-calorie diet on body composition, metabolic state, and genes expression
VLCKD1VLCKD2VLCKD3
T0 T1 T0 T1 T0 T1
Mean ± SD Mean ± SD Mean ± SD Mean ± SD Mean ± SD Mean ± SD
(min-max) (min-max) p(min-max) (min-max) p(min-max) (min-max) p
Systolic pressure (mm/Hg) 121.50 ± 15.28 120.00 ± 15.09 0.59 117.50 ± 9.57 117.50 ± 15.00 1.00 123.33 ± 10.33 124.17 ± 8.01 0.77
(100.00-150.00) (100.00-150.00) (110.00-130.00) (100.00-130.00) (110.00-140.00) (110.00-130.00)
Diastolic pressure (mm/Hg) 80.00 ± 9.43 74.00 ± 9.37 0.06 72.50 ± 5.00 70.00 ± 8.16 0.32a79.17 ± 8.01 75.00 ± 5.48 0.18a
(70.00-100.00) (60.00-90.00) (70.00-80.00) (60.00-80.00) (70.00-90.00) (70.00-80.00)
Heart Rate (bpm) 72.30 ± 5.72 71.00 ± 6.27 0.36 67.25 ± 10.87 71.00 ± 10.13 0.15 69.17 ± 6.46 72.50 ± 10.27 0.22
(64.00-81.00) (62.00-79.00) (54.00-78.00) (62.00-84.00) (60.00-74.00) (60.00-86.00)
BMI 30.10 ± 4.16 28.68 ± 4.20 0.00 30.98 ± 3.64 29.09 ± 3.31 0.00 29.99 ± 2.35 27.64 ± 2.58 0.00
(24.42-37.00) (23.38-36.30) (27.16-34.80) (25.46-32.48) (27.99-34.30) (25.60-32.20)
WC (cm) 89.75 ± 9.96 85.30 ± 8.75 0.00 94.65 ± 5.06 90.88 ± 3.66 0.02 86.47 ± 4.07 82.33 ± 3.87
(78.00-109.00) (75.00-101.50) (90.50-102.00) (88.00-96.00) (82.30-93.00) (78.00-89.00) 0.00
AFP (%) Tissue 45.10 ± 6.12 43.30 ± 7.32 0.02 41.50 ± 9.15 38.75 ± 12.95 0.14a49.33 ± 3.50 48.33 ± 5.13 0.35
(38.00-59.00) (33.00-57.00) (35.00-55.00) (30.00-58.00) (45.00-55.00) (41.00-55.00)
GFP (%) Tissue 43.10 ± 6.77 42.60 ± 7.09 0.38 35.50 ± 7.94 36.25 ± 10.90 0.70 48.67 ± 2.58 48.67 ± 3.20 1.00
(29.00-50.00) (30.00-53.00) (27.00-46.00) (27.00-52.00) (45.00-52.00) (45.00-54.00)
TBF (%) Tissue 40.70 ± 6.48 39.80 ± 7.02 0.03 35.00 ± 8.68 33.00 ± 9.38 0.07a45.00 ± 2.76 45.33 ± 3.72 0.61
(32.00-51.00) (31.00-50.00) (30.00-48.00) (27.00-47.00) (40.00-48.00) (39.00-50.00)
TBF (%) Region 39.20 ± 6.03 8.40 ± 6.883 0.15 33.50 ± 8.39 31.50 ± 9.04 0.07a43.83 ± 2.71 43.17 ± 2.79 0.17
(31.00-49.00) (30.00-48.00) (28.00-46.00) (26.00-45.00) (39.00-47.00) (38.00-46.00)
TBF (Kg) 31.69 ± 5.28 29.23 ± 5.25 0.00 30.13 ± 4.80 26.62 ± 5.02 0.07a33.81 ± 4.40 31.12 ± 4.18 0.00
(22.28-43.31) (19.19-39.89) (27.35-37.30) (23.75-34.14) (26.82-39.26) (23.76-35.71)
TBL (Kg) 47.60 ± 12.30 45.32 ± 11.59 0.01a57.93 ± 11.35 55.86 ± 11.17 0.07a40.86 ± 3.26 37.67 ± 3.94 0.00
(37.51-68.83) (33.21-63.93) (41.00-65.34) (39.21-62.92) (36.45-45.67) (32.19-43.85)
ASMMI 7.96 ± 1.60 7.95 ± 2.35 1.00 10.38 ± 1.93 9.17 ± 0.98 0.28 7.31 ± 0.75 6.57 ± 0.96 0.00
(6.30-11.34) (5.64-12.86) (8.54-12.92) (8.18-10.52) (6.42-8.55) (5.50-8.17)
IMAT 1.38 ± 0.26 1.25 ± 0.26 0.00 1.13 ± 0.59 0.43 ± 0.68 0.07a1.42 ± 0.20 0.94 ± 0.52 0.09
(0.83-1.73) (0.68-1.66) (0.26-1.58) (0.07-1.45) (1.08-1.64) (0.30-1.52)
FM L2-L5 3.87 ± 1.42 3.49 ± 1.05 0.06 4.24 ± 0.91 3.43 ± 0.72 0.00 3.82 ± 0.61 3.47 ± 0.68 0.04
(2.20-6.88) (1.80-5.53) (3.26-5.45) (2.66-4.41) (2.97-4.54) (2.51-4.53)
Submaximal Strenght (kg) 27.72 ± 11.57 29.47 ± 11.72 0.26a37.15 ± 10.55 37.78 ± 15.69 0.84 22.07 ± 4.71 23.35 ± 5.56 0.39
(17.30-51.70) (17.80-49.20) (23.60-46.10) (15.70-49.70) (17.60-28.50) (16.40-31.00)
Submaximal resistance 20.32 ± 8.33 13.93 ± 6.14 0.01 16.03 ± 9.81 14.70 ± 5.69 0.82 15.28 ± 9.99 16.70 ± 3.87 0.60a
(70%) sec (9.50-33.90) (7.30-28.60) (5.80-29.40) (9.10-22.50) (7.50-29.30) (13.00-23.00)
Table I. Anthropometric measurements and body composition parameters at before and after dietary treatment.
All parameters were evaluated before and after three different dietary treatments. All results were expressed as mean ± standard deviation (SD) followed by minimum and maxi-
mum. Statistical significance were attributed to results with p< 0.05 after parametric test (Student t-test) or non-parametric test (a)(Wilcoxon-Mann-Whitney). Body Mass Index
(BMI); Waist Circumference (WC); Android Fat Percentage (AFP); Gynoid Fat Percentage (GFP); Total Body Fat (TBF); Total Body Lean (TBL); Appendicular Skeletal Muscle
Mass Index (ASSMI); Inter Muscular Adipose Tissue (IMAT); Fat Mass L2-L5 (FM L2-L5).
336
0.00) and HDL-C (∆% = -10.6%, p= 0.00). After
VLCKD3we observe a significant decrease of
glycemia (∆% = -14.6%, p= 0.00).
LAP decreased significantly only after VL-
CKD1(∆%= -32.4%, p= 0.004). GH, ESR and
fibrinogen have not undergone any significant
changes after the DTs; instead, IGF-1 reduced
significantly after VLCKD2(∆% = -36.9%, p=
0.01), as well as CRP (∆% = -30.1%, p= 0.02).
Gene expression analysis shown a significant
decrease of SOD1 gene only after VLCDK3(p=
0.009). No significant changes were observed
for CCL2, NFkB in any dietary treatment (Table
V-VI).
Discussion
To our knowledge, this is the first study to ana-
lyze comprehensively body composition, to as-
sess the loss of muscle mass and abdominal fat,
metabolic profile and the regulation of certain
genes of inflammation and oxidative stress after
VLCKD.
Several studies 20,21 demonstrated the efficacy
of ketogenic diet on weight loss, maybe due to
lower energy intake, satiety protein-induced and
low-carbohydrates consumption.
Weight loss induced by VLCKD depends on
different variables like loss appetite, through sati-
G. Merra, S. Gratteri, A. De Lorenzo, S. Barrucco, M.A. Perrone, et al.
All parameters were compared between the three different dietary treatments. All results were expressed as mean ± standard devi-
ation (SD) followed by minimum and maximum. Statistical significance were attributed to results with p< 0.05 after parametric
test (Student t-test) or non-parametric test (a)(Wilcoxon-Mann-Whitney). Body Mass Index (BMI); Waist Circumference (WC);
Android Fat Percentage (AFP); Gynoid Fat Percentage (GFP); Total Body Fat (TBF); Total Body Lean (TBL); Appendicular
Skeletal Muscle Mass Index (ASSMI); Inter Muscular Adipose Tissue (IMAT); Fat Mass L2-L5 (FM L2-L5).
Mean ± Standard Deviation p
VLCKD1VLCKD1VLCKD2
vs. vs. vs.
VLCKD1VLCKD2VLCKD3VLCKD2VLCKD3VLCKD3
Systolic pressure (mm/Hg) 1.50 ± 8.51 0.00 ± 16.33 -0.83 ± 6.65 0.73a0.49a0.91
(-15.00-20.00) (-20.00-20.00) (-10.00-10.00)
Diastolic pressure (mm/Hg) 6.00 ± 8.76 2.50 ± 5.00 4.17 ± 8.01 0.45a0.49a0.91a
(-10.00-20.00) (0.00-10.00) (0.00-20.00)
Heart Rate (bpm) 1.30 ± 4.30 -3.75 ± 3.86 -3.33 ± 5.79 0.06 0.09 0.90
(-3.00-11.00) (-8.00-0.00) (-12.00-4.00)
BMI 1.42 ± 0.95 1.90 ± 0.36 2.34 ± 0.37 0.35 0.04 0.10
(0.00-2.55) (1.52-2.32) (1.75-2.80)
WC (cm) 4.45 ± 1.74 3.78 ± 1.66 4.13 ± 1.43 0.52 0.71 0.72
(2.00-7.50) (2.50-6.00) (1.80-6.00)
AFP (%) Tissue 1.80 ± 2.10 2.75 ± 3.86 1.00 ± 2.37 0.30a0.49 0.26a
(-2.00-5.00) (-3.00-5.00) (-3.00-4.00)
GFP (%) Tissue 0.50 ± 1.72 -0.75 ± 3.59 0.00 ± 1.41 0.38 0.56 0.65
(-3.00-3.00) (-6.00-2.00) (-2.00-2.00)
TBF (%) Tissue 0.90 ± 1.10 2.00 ± 0.82 -0.33 ± 1.51 0.08a0.12a0.02a
(-2.00-2.00) (1.00-3.00) (-2.00-1.00)
TBF (%) Region 0.80 ± 1.62 2.00 ± 0.82 0.67 ± 1.03 0.19 0.86 0.06
(-3.00-3.00) (1.00-3.00) (-1.00-2.00)
TBF (Kg) 2.46 ± 1.09 3.51 ± 0.26 2.69 ± 0.92 0.09 0.68 0.13
(0.65-4.14) (3.17-3.79) (1.34-3.71)
TBL (Kg) 2.28 ± 1.49 2.06 ± 0.72 3.19 ± 1.16 0.79 0.22 0.12
(0.70-4.90) (1.20-2.84) (1.82-4.92)
ASMMI 0.00 ± 1.70 1.21 ± 1.85 0.75 ± 0.32 0.45a0.04a0.26a
(-4.78-1.09) (0.24-3.99) (0.38-1.14)
IMAT 0.12 ± 0.07 0.70 ± 0.62 0.48 ± 0.56 0.01 0.26a0.17a
(0.03-0.25) (0.13-1.24) (0.07-1.22)
FM L2-L5 0.38 ± 0.57 0.81 ± 0.18 0.35 ± 0.31 0.17 0.90 0.03
(-0.35-1.35) (0.60-1.05) (-0.06-0.71)
Submaximal Strenght (kg) -1.75 ± 4.00 -0.63 ± 5.79 -1.28 ± 3.34 0.68 0.81 0.82
(-8.60-3.20) (-4.80-7.90) (-6.50-3.70)
Submaximal resistance sec 6.39 ± 6.16 1.33 ± 10.82 -1.42 ± 10.12 0.54a0.12a0.69
(70%) (0.90-17.60) (-8.20-14.70) (-10.60-16.20)
Table II. Anthropometric measurements and body composition parameters comparison between dietary treatments.
337
Effects of very-low-calorie diet on body composition, metabolic state, and genes expression
Table continued
VLCKD1VLCKD2VLCKD3
T0 T1 T0 T1 T0 T1
Mean ± SD Mean ± SD Mean ± SD Mean ± SD Mean ± SD Mean ± SD
(min-max) (min-max) p(min-max) (min-max) p(min-max) (min-max) p
RBC ( 106/µL) 4.99 ± 0.42 5.06 ± 0.42 0.06 5.52 ± 0.33 5.14 ± 0.12 0.06 4.67 ± 0.27 4.98 ± 0.23 0.03a
(4.40-5.62) (4.55-5.72) (5.22-5.99) (5.03-5.30) (4.31-4.89) (4.71-5.38)
HB (g/µL) 14.16 ± 1.85 14.33 ± 1.84 0.09 15.63 ± 1.42 15.10 ± 1.10 0.06 13.08 ± 1.06 13.93 ± 1.04 0.07a
(11.30-17.40) (11.50-17.70) (13.80-17.10) (13.60-16.10) (11.30-14.10) (12.00-15.10)
HCT (%) 43.19 ± 4.71 43.63 ± 4.87 0.098 47.73 ± 4.48 44.98 ± 2.35 0.12 40.38 ± 2.91 42.82 ± 3.06 0.03a
(36.00-51.70) (36.50-52.70) (42.00-52.30) (41.80-47.30) (36.60-43.70) (38.10-47.70)
MCV (fl) 86.60 ± 5.82 86.11 ± 5.42 0.31a86.38 ± 4.32 87.63 ± 5.14 0.19 86.57 ± 5.51 86.13 ± 5.56 0.23a
(73.80-92.00) (74.00-92.10) (80.50-90.90) (81.50-94.00) (75.60-90.50) (74.90-89.60)
MCH (pg) 28.41 ± 2.69 28.27 ± 2.41 0.37a28.25 ± 1.34 29.40 ± 2.05 0.06 28.07 ± 2.41 28.05 ± 2.37 0.92
(23.20-31.00) (23.30-30.90) (26.40-29.60) (26.50-31.20) (23.30-30.00) (23.60-30.60)
MCHC (g/dl) 32.73 ± 1.12 32.79 ± 0.99 0.78 32.75 ± 0.13 33.55 ± 1.05 0.25 32.40 ± 0.98 32.57 ± 1.03 0.44
(30.60-34.20) (31.20-34.10) (32.60-32.90) (32.50-35.00) (30.90-33.50) (31.50-34.10)
RDW-CV (%) 13.59 ± 1.14 13.48 ± 1.45 0.56 13.18 ± 0.93 13.40 ± 0.80 0.55 13.45 ± 1.41 13.43 ± 1.32 0.93
(12.20-15.10) (11.50-15.90) (12.20-14.40) (12.60-14.50) (12.00-15.50) (11.80-15.60)
PLT (× 103/µL) 301.80 ± 67.98 287.40 ± 55.78 0.15 260.25 ± 69.16 254.50 ± 59.65 0.53 316.00 ± 44.47 295.50 ± 49.36 0.06
(199.00-423.00) (188.00-372.00) (197.00-358.00) (191.00-335.00) (262.00-391.00) (244.00-386.00)
WBC (× 103/µL) 6.52 ± 1.23 5.35 ± 1.15 0.00 6.62 ± 1.03 5.68 ± 0.73 0.07 6.73 ± 1.39 6.01 ± 1.30 0.17
(4.44-8.87) (3.39-7.79) (5.61-8.05) (4.90-6.66) (4.49-8.18) (4.59-7.64)
NEUTR (× 103/µL) 3.57 ± 1.02 2.95 ± 0.97 0.01 4.04 ± 0.79 3.16 ± 0.75 0.01 4.46 ± 2.38 3.33 ± 1.11 0.15
(1.99-5.75) (1.43-5.08) (3.08-4.94) (2.29-3.80) (1.93-8.90) (1.86-4.87)
LYMP (× 103/µL) 2.34 ± 0.30 1.89 ± 0.26 0.00 2.03 ± 0.30 1.96 ± 0.50 0.80 2.31 ± 0.35 2.09 ± 0.27 0.14
(1.97-2.79) (1.46-2.36) (1.70-2.42) (1.52-2.67) (1.92-2.76) (1.77-2.55)
MON (× 103/µL) 0.42 ± 0.14 0.35 ± 0.13 0.23 0.40 ± 0.08 0.36 ± 0.13 0.52 0.40 ± 0.07 0.42 ± 0.10 0.92a
(0.28-0.73) (0.16-0.55) (0.28-0.48) (0.22-0.54) (0.29-0.49) (0.33-0.60)
EOS (× 103/µL) 0.16 ± 0.08 0.14 ± 0.08 0.11 0.14 ± 0.08 0.19 ± 0.26 0.71a0.19 ± 0.11 0.15 ± 0.11 0.11
(0.08-0.35) (0.06-0.28) (0.07-0.24) (0.04-0.58) (0.09-0.34) (0.05-0.35)
BAS (× 103/µL) 0.03 ± 0.02 0.03 ± 0.02 0.36 0.02 ± 0.01 0.02 ± 0.01 1.00 0.04 ± 0.02 0.03 ± 0.02 0.20
(0.01-0.06) (0.01-0.05) (0.01-0.03) (0.01-0.03) (0.01-0.07) (0.01-0.06)
NEUTR (%) 54.01 ± 6.61 54.05 ± 6.90 0.96 60.70 ± 5.39 55.50 ± 11.04 0.19 55.33 ± 7.75 54.25 ± 8.03 0.58
(44.80-64.80) (42.20-65.20) (54.80-67.70) (40.80-67.60) (42.90-65.50) (40.60-65.30)
LYMP (%) 36.60 ± 5.45 36.26 ± 6.55 0.66 30.88 ± 3.44 34.70 ± 9.20 0.31 35.22 ± 6.56 35.80 ± 7.27 0.73
(26.80-44.40) (26.40-46.60) (26.60-34.80) (27.30-47.60) (27.70-45.70) (25.50-48.10)
MON (%) 6.39 ± 1.69 6.46 ± 1.76 0.93 6.15 ± 1.59 6.43 ± 2.54 0.68 6.03 ± 0.82 6.98 ± 1.16 0.20
(4.20-9.70) (3.30-9.50) (4.40-7.50) (4.00-9.60) (4.60-6.90) (5.80-8.30)
EOS (%) 2.56 ± 1.15 2.76 ± 1.64 0.67 2.00 ± 0.99 3.05 ± 3.80 0.72a 2.88 ± 1.51 2.43 ± 1.35 0.30
(1.30-4.70) (0.90-5.50) (1.10-3.00) (0.70-8.70) (1.50-4.70) (0.70-4.60)
Table III. Blood tests before and after dietary treatment.
338
G. Merra, S. Gratteri, A. De Lorenzo, S. Barrucco, M.A. Perrone, et al.
VLCKD1VLCKD2VLCKD3
T0 T1 T0 T1 T0 T1
Mean ± SD Mean ± SD Mean ± SD Mean ± SD Mean ± SD Mean ± SD
(min-max) (min-max) p(min-max) (min-max) p(min-max) (min-max) p
BAS (%) 0.44 ± 0.18 0.47 ± 0.28 0.72 0.28 ± 0.10 0.33 ± 0.15 0.50 0.53 ± 0.34 0.53 ± 0.34 1.00
(0.20-0.70) (0.20-1.00) (0.20-0.40) (0.20-0.50) (0.20-1.10) (0.20-1.20)
ESR (mm/h) 23.10 ± 14.65 24.00 ± 10.58 0.78 17.25 ± 14.97 12.75 ± 9.74 0.27 33.83 ± 8.08 30.67 ± 7.50 0.14
(5.00-58.00) (7.00-40.00) (5.00-39.00) (6.00-27.00) (26.00-46.00) (20.00-41.00)
Fibrinogen (mg/dL) 356.39 ± 89.32 394.14 ± 102.91 0.09 307.10 ± 63.25 298.73 ± 52.44 0.78 383.68 ± 77.87 438.35 ± 76.66 0.05
(258.00-541.30) (272.20-541.30) (257.80-399.00) (231.90-353.00) (285.50-485.20) (329.10-553.80)
Tg (mg/dL) 98.40 ± 40.93 81.80 ± 23.62 0.22 99.25 ± 24.28 77.25 ± 15.31 0.21 97.17 ± 41.14 92.00 ± 21.78 0.68
(56.00-174.00) (37.00-110.00) (71.00-120.00) (58.00-91.00) (47.00-141.00) (63.00-113.00)
TC (mg/dL) 198.70 ± 31.08 166.70 ± 28.94 0.00 187.75 ± 17.78 159.75 ± 13.38 0.12 207.00 ± 49.27 177.83 ± 37.49 0.05
(162.00-273.00) (132.00-225.00) (173.00-212.00) (146.00-178.00) (160.00-293.00) (138.00-233.00)
LDL-C (mg/dL) 127.00 ± 24.88 99.80 ± 22.96 0.00 131.25 ± 25.49 102.75 ± 4.99 0.12 125.83 ± 43.17 108.50 ± 42.50 0.16
(87.00-176.00) (57.00-124.00) (109.00-167.00) (99.00-110.00) (68.00-187.00) (48.00-178.00)
HDL-C (mg/dL) 62.00 ± 15.25 55.40 ± 14.45 0.00 46.75 ± 6.55 44.50 ± 4.04 0.65 66.50 ± 15.06 58.50 ± 18.29 0.02
(39.00-88.00) (38.00-79.00) (41.00-55.00) (41.00-50.00) (49.00-89.00) (33.00-87.00)
CRP (mg/dL) 1.67 ± 2.84 3.14 ± 4.13 0.40a2.76 ± 1.72 1.93 ± 1.60 0.02 1.26 ± 2.00 1.79 ± 3.32 0.66a
(0.10-8.94) (0.10-11.84) (0.85-5.35) (0.10-4.09) (0.10-4.23) (0.10-6.77)
IGF-1 (ng/mL) 159.54 ± 67.06 129.81 ± 61.43 0.12 225.50 ± 84.35 142.18 ± 56.79 0.01 142.17 ± 27.13 103.87 ± 51.55 0.10
(65.40-297.00) (42.40-279.00) (165.00-349.00) (95.70-223.00) (101.00-185.00) (41.60-185.00)
GH (ng/mL) 1.12 ± 1.12 2.73 ± 2.64 0.11a2.29 ± 2.01 1.42 ± 0.88 0.43 1.24 ± 1.01 2.40 ± 2.61 0.28
(0.06-3.72) (0.10-6.11) (0.49-4.87) (0.25-2.32) (0.11-2.50) (0.20-7.51)
AIP -0.14 ± 0.25 -0.19 ± 0.21 0.38 0.16 ± 0.28 -0.13 ± 0.09 0.07 -0.23 ± 0.23 -0.16 ± 0.21 0.45
(-0.48-0.29) (-0.62--0.01) (-0.11-0.56) (-0.25--0.04) (-0.57-0.04) (-0.45-0.07)
Glycemia (mg/dL) 87.13 ± 11.70 78.95 ± 9.80 0.08 99.76 ± 12.05 81.99 ± 6.50 0.07 88.02 ± 7.53 75.20 ± 15.62 0.00a
(73.00-108.00) (58.00-90.00) (91.00-118.00) (72.00-87.00) (77.00-98.00) (45.00-93.00)
PLR 132.15 ± 39.54 155.01 ± 36.41 0.00 127.97 ± 25.50 134.60 ± 38.26 0.60 139.47 ± 32.62 144.30 ± 37.51 0.75a
(74.81-203.37) (94.47-204.73) (95.63-148.82) (93.26-173.58) (112.68-203.65) (119.61-218.08)
NLR 1.53 ± 0.44 1.57 ± 0.48 0.51 2.00 ± 0.41 1.73 ± 0.68 0.18 1.97 ± 1.20 1.61 ± 0.56 0.28
(1.01-2.42) (0.91-2.47) (1.58-2.55) (0.86-2.47) (0.94-4.30) (0.84-2.56)
LAP 34.57 ± 22.50 23.35 ± 8.07 0.04a 36.00 ± 13.04 28.63 ± 10.24 0.49 30.66 ± 11.99 24.37 ± 4.48 0.16
(12.65-86.51) (7.11-31.73) (22.14-49.75) (17.04-41.42) (14.11-43.98) (19.10-29.67)
Table III (Continued).Blood tests before and after dietary treatment.
All parameters were evaluated before and after three different dietary treatments. All results were expressed as mean ± standard deviation (SD) followed by minimum and maxi-
mum. Statistical significance were attributed to results with p< 0.05 after parametric test (Student t-test) or non-parametric test (a)(Wilcoxon-Mann-Whitney). Results with statis-
tical significance were reported in bold. Red Blood Cells (RBC); Hemoglobin (HB); Hematocrit blood testing (HCT); Mean Corpuscular Volume (MCV); Mean Corpuscular He-
moglobin (MCH); Red cell distribution width (RDW); Platelets (PLT); White Blood Cells (WBC); Neutrophils (NEUTR); Lymphocytes (LYMP); Monocytes (MON); Eosinophils
(EOS); Erythrocytes Sedimentation Rate (ESR); Triglycerides (Tg); Total Cholesterol (TC); Low Density Lipoprotein Cholesterol (LDL-C); High Density Lipoprotein Cholesterol
(HDL-C); C-Reactive Protein (CRP); Insulin Growth Factor-1 (IGF-1); Atherogenic Index of Plasma (AIP); Platelets/Lymphocytes Ratio (PLR); Neutrophils/Lymphocytes Ratio
(NLR); Lipid Accumulation Product (LAP).
339
Effects of very-low-calorie diet on body composition, metabolic state, and genes expression
Mean ± Standard Deviation p
VLCKD1VLCKD1VLCKD2
vs. vs. vs.
VLCKD1VLCKD2VLCKD3VLCKD2VLCKD3VLCKD3
RBW (× 106/µL) -0.07 ± 0.11 0.39 ± 0.26 -0.31 ± 0.24 0.00 0.02 0.00
(-0.20-0.17) (0.09-0.69) (-0.58/-0.03)
HB (g/µL) -0.17 ± 0.29 0.53 ± 0.36 -0.85 ± 0.73 0.00 0.02 0.01
(-0.60-0.30) (0.20-1.00) (-1.70-0.00)
HCT (%) -0.44 ± 0.75 2.75 ± 2.58 -2.43 ± 1.80 0.00a 0.04a 0.01
(-1.10-1.50) (0.20-6.30) (-4.60/-0.30)
MCV (fl) 0.49 ± 1.01 -1.25 ± 1.48 0.43 ± 1.00 0.02 0.91 0.06
(-0.50-2.50) (-3.10-0.50) (-1.10-1.80)
MCH (pg) 0.14 ± 0.53 -1.15 ± 0.79 0.02 ± 0.41 0.00 0.64 0.01
(-0.60-1.00) (-1.90--0.10) (-0.60-0.50)
MCHC (g/dl) -0.06 ± 0.65 -0.80 ± 1.12 -0.17 ± 0.49 0.14 0.74 0.25
(-0.90-0.90) (-2.30-0.40) (-0.60-0.60)
RDW-CV (%) 0.11 ± 0.57 -0.23 ± 0.67 0.02 ± 0.43 0.36 0.74 0.50
(-0.80-0.80) (-1.20-0.20) (-0.50-0.60)
PLT (× 103/µL) 14.40 ± 28.69 5.75 ± 16.21 20.50 ± 20.25 0.59 1.00a 0.48a
(-38.00-77.00) (-16.00-23.00) (5.00-60.00)
WBC (× 103/µL) 1.17 ± 0.85 0.94 ± 0.69 0.72 ± 1.12 0.64 0.37a0.76a
(-0.33-2.45) (0.00-1.53) (-0.10-2.70)
NEUTR (× 103/µL) 0.62 ± 0.56 0.88 ± 0.25 1.13 ± 1.60 0.40 0.37 0.77
(-0.27-1.47) (0.57-1.14) (-0.33-4.03)
LYMP (× 103/µL) 0.45 ± 0.24 0.08 ± 0.54 0.23 ± 0.31 0.09 0.13 0.59
(0.10-0.93) (-0.72-0.49) (-0.16-0.77)
MON (× 103/µL) 0.07 ± 0.17 0.04 ± 0.11 -0.02 ± 0.15 0.77 0.33 0.53
(-0.18-0.44) (-0.12-0.12) (-0.26-0.16)
EOS (× 103/µL) 0.02 ± 0.07 -0.06 ± 0.19 0.05 ± 0.06 0.24a0.79a0.61a
(-0.17-0.07) (-0.34-0.05) (-0.01-0.13)
BAS (× 103/µL) 0.01 ± 0.02 0.00 ± 0.01 0.01 ± 0.01 0.58 0.96a0.35a
(-0.02-0.03) (-0.01-0.01) (-0.01-0.01)
NEUTR (%) -0.04 ± 2.72 5.20 ± 6.11 1.08 ± 4.45 0.04 0.54 0.25
(-4.00-3.90) (0.10-14.00) (-6.10-7.70)
LYMP (%) 0.34 ± 2.38 -3.83 ± 6.20 -0.58 ± 3.85 0.08 0.56 0.33
(-3.20-4.10) (-12.80-1.10) (-5.80-5.00)
MON (%) -0.07 ± 2.42 -0.28 ± 1.22 -0.95 ± 1.57 0.84a0.44 0.48a
(-3.60-4.10) (-2.10-0.40) (-3.40-1.10)
EOS (%) -0.20 ± 1.42 -1.05 ± 3.11 0.45 ± 0.95 1.00a0.56a0.91a
(-4.00-0.70) (-5.70-0.90) (-0.50-2.10)
BAS (%) -0.03 ± 0.25 -0.05 ± 0.13 0.00 ± 0.14 0.89 0.80 0.59
(-0.50-0.30) (-0.20-0.10) (-0.20-0.20)
ESR (mm/h) -0.90 ± 9.83 4.50 ± 6.61 3.17 ± 4.36 0.34 0.36 0.71
(-19.00-20.00) (-2.00-12.00) (-3.00-9.00)
Fibrinogen (mg/dL) -37.75 ± 63.91 8.37 ± 55.58 -118.62 ± 214.39 0.23 0.64a0.17a
(-155.90-43.90) (-56.80-74.50) (-553.80-10.00)
Tg (mg/dL) 16.60 ± 39.95 22.00 ± 27.35 5.17 ± 29.06 0.81 0.55 0.39
(-31.00-101.00) (-17.00-47.00) (-39.00-33.00)
TC (mg/dL) 32.00 ± 22.49 28.00 ± 25.56 29.17 ± 28.22 0.73a0.83 0.76a
(3.00-76.00) (12.00-66.00) (1.00-82.00)
LDL-C (mg/dL) 27.20 ± 21.88 28.50 ± 26.06 17.33 ± 25.91 0.93 0.43 0.52
(0.00-57.00) (10.00-67.00) (-15.00-61.00)
HDL-C (mg/dL) 6.60 ± 5.40 2.25 ± 9.07 8.00 ± 5.97 0.28 0.64 0.26
(1.00-16.00) (-9.00-13.00) (2.00-16.00)
CRP (mg/dL) -1.47 ± 4.05 0.37 ± 2.29 -0.53 ± 1.35 0.18a0.73a0.11a
(-8.72-4.14) (-4.09-2.24) (-2.54-0.41)
IGF-1 (ng/mL) 29.73 ± 54.36 83.33 ± 28.46 38.30 ± 46.95 0.19a0.75 0.07a
(-32.00-134.60) (68.00-126.00) (-4.00-110.40)
GH (ng/mL) -1.61 ± 2.95 0.87 ± 1.90 -1.16 ± 2.34 0.15 1.00a0.17a
(-4.73-2.99) (-1.40-3.07) (-5.72-0.69)
Table IV. Blood tests comparison between dietary treatments.
Table Continued
340
ety protein-induced21,22, hunger control by hor-
mones23 increased lipolysis and reduction of lipo-
genesis24,25, raise of gluconeogenesis metabolic
costs and proteins thermic effect26,27, lowering of
respiratory quotient, increased metabolic effi-
ciency for fats consumption28,29.
There have been no previous studies to investi-
gate the VLCKD effects of abdominal fat distrib-
ution, lean mass reduction on inflammatory sta-
tus.
Sarcopenia, the loss of muscle mass leading to
muscle weakness, limited mobility, and increased
susceptibility to injury. It is defined, as a condi-
tion that involves a loss of type II muscle fibers,
a decline in total muscle area, the reduction of
muscle capillarization, shortening velocity and
declining strength and/or physical performance30.
DXA-derived total TBF, TBL and ASMMI
measures can reflect both the percentage of total
body fat (PBF), than muscle mass and muscle
strength, providing a reliable measure for assess-
ment of sarcopenia risk also induced by unbal-
anced diets31.
High protein intake, typical of KD treatments,
determines an increase in protein synthesis, be-
cause of the augmented systemic amino acid
availability32, which in turn is a muscle protein
sy nthes is stim ulant 3 3. Alth ough i t has been
proven that high protein intake prevents the loss
of muscle mass and promotes the reduction of
body fat during reduced caloric intake period34,35,
VLCKD1(p= 0.01) and VLCKD3(p= 0.00) de-
termined a significant reduction of TBL mass.
However, the VLCKD1seems to protect better
than VLCKD2and VLCKD3against the risk of
sarcopenia, either because no consistent reduc-
tion of ASMMI was observed, either because the
strength and muscular endurance do not change.
VLCKD2treatment did not change any lean mass
parameters, showing that the protein with biolog-
ical value is essential during weight lost. Further-
more, ASMMI was lowered after VLCKD3then
VLCKD1treatment (p= 0.04).
According to previous data36 and our results to
prevent and manage the risk of muscle mass loss,
ASMMI reduction and muscular strength, VL-
CKD1would s eem to be the e lective choice
among DTs.
In our study measurement of body composi-
tion revealed that obese subjects fed the VLCKD1
and VLCKD2for 3 wks, significantly reduced
BMI (p= 0.04), where BMI reduction was higher
in VLCKD1. Matching the three DTs, it was no-
ticed a reduction of TBF among them, but only
VLCKD2compared to VLCKD3, determined a
significant reduction of tissue TBF (p= 0.04).
G. Merra, S. Gratteri, A. De Lorenzo, S. Barrucco, M.A. Perrone, et al.
All parameters were compared between the three different dietary treatments. All results were expressed as mean ± standard
deviation (SD) followed by minimum and maximum. Statistical significance were attributed to results with p< 0.05 after
parametric test (Student t-test) or non-parametric test (a)(Wilcoxon-Mann-Whitney). Results with statistical significance
were reported in bold. Red Blood Cells (RBC); Hemoglobin (HB); Hematocrit blood testing (HCT); Mean Corpuscular Vol-
ume (MCV); Mean Corpuscular Hemoglobin (MCH); Red cell distribution width (RDW); Platelets (PLT); White Blood
Cells (WBC); Neutrophils (NEUTR); Lymphocytes (LYMP); Monocytes (MON); Eosinophils (EOS); Erythrocytes Sedi-
menta-tion Rate (ESR); Triglycerides (Tg); Total Cholesterol (TC); Low Density Lipoprotein Cholesterol (LDL-C); High
Density Lipoprotein Cholesterol (HDL-C); C-Reactive Protein (CRP); Insulin Growth Factor-1 (IGF-1); Growth Hormone
(GH); Atherogenic Index of Plasma (AIP); Plate-lets/Lymphocytes Ratio (PLR); Neutrophils/Lymphocytes Ratio (NLR);
Lipid Accumulation Product (LAP).
Mean ± Standard Deviation p
VLCKD1VLCKD1VLCKD2
vs. vs. vs.
VLCKD1VLCKD2VLCKD3VLCKD2VLCKD3VLCKD3
AIP 0.05 ± 0.18 0.29 ± 0.21 -0.07 ± 0.21 0.05 0.24 0.03
(-0.24-0.36) (0.14-0.60) (-0.41-0.12)
Glycemia (mg/dL) 8.18 ± 11.74 17.77 ± 10.32 12.82 ± 10.15 0.26 0.69 0.54
(-5.00-38.00) (3.00-36.00) (3.00-33.00)
PLR -22.86 ± 18.08 -6.63 ± 22.73 -4.84 ± 17.88 0.18 0.07 0.89
(-49.15/-0.29) (-25.64-26.23) (-36.06-12.67)
NLR -0.03 ± 0.15 0.27 ± 0.31 0.36 ± 0.73 0.03 0.11 0.82
(-0.30-0.11) (0.07-0.72) (-0.36-1.74)
LAP 11.22 ± 18.64 7.36 ± 18.73 6.29 ± 9.21 0.84a1.00a0.91
(-4.61-56.40) (-19.28-24.53) (-6.22-17.63)
Table IV (Continued).Blood tests comparison between dietary treatments.
IMAT increase seems to determine the rise of
insulin resistance and the risk of type 2 diabetes
in obese subjects, depending on the release of in-
flammatory cytokines within skeletal muscle37.
IMAT may also compromise physical perfor-
mance and muscle function. Moreover, the re-
duction of IMAT was associated with the im-
provements in lipid profile38.
Af ter VLCKD1a significant reduc tion o f
IMAT was observed (p= 0.00). Furthermore,
IMAT was significantly lower in VLCKD1than in
VLCKD2(p= 0.01). Our data highlighted the
possibility of reducing the IMAT by VLCKD1,
with improvement of myosteatosis, and conse-
quently decreasing the risk of cardiometabolic
diseases associated with obesity.
Regarding fat mass and its distribution, after
VLCKD1a significant decrease of AFP of tissue
(p= 0.01) was highlighted. The reduction of fat
mass in android level only observed with VL-
CKD1, associated with the decrease in waist cir-
cumference; support the hypothesis that this DT
is a positive factor to reduce the cardiometabolic
risk. On the contrary, the reduction observed af-
ter VLC K D 2a n d V L C K D 3were limited to
FML2-L5 (p< 0.05).
The effect of prolonged KD feeding on ob/ob
mice was associated with normalization of fast-
ing glycemia, reduction of reduced insulin and
lipid levels in the absence of weight loss. More-
over, it produces a significant increase in lipid
oxidative genes and reduction expression of lipid
synthetic genes, but no change in expression of
inflammatory markers39.
According to a previous study40, no differences
were observed between the VLCKD2and VL-
CKD3regarding lipid profiles. However, LAP,
that reflects lipid accumulation and is an effec-
tive predictive index for metabolic syndrome and
insulin resistance16,17 (p= 0.004), LDL-C (p=
0.00) and HDL-C (p= 0.00) significantly de-
creased after VLCKD1.
It has been reported41 that a KD induces a se-
vere reduction of IGF-I concentration in rats. In-
terestingly, we observed a significant reduction
of IGF-1 levels only after VLCKD2(p= 0.01).
Different studies4 2 reported that the use of
whey proteins could increase anabolic hormones
(i.e. insulin and GH). Furthermore, whey pro-
teins are potential functional food component,
with the ability to generate satiety signals, which
take part to the body weight regulation.
Muscle protein synthesis is the main metabolic
mechanism, and whey proteins are the perfect
341
Effects of very-low-calorie diet on body composition, metabolic state, and genes expression
VLCKD1VLCKD2VLCKD3
T0 T1 T0 T1 T0 T1
Mean ± SD Mean ± SD Mean ± SD Mean ± SD Mean ± SD Mean ± SD
(min-max) (min-max) p(min-max) (min-max) p(min-max) (min-max) p
SOD1 6.76 ± 1.09 5.93 ± 1.35 0.114 6.57 ± 0.64 6.08 ± 1.59 0.417 6.89 ± 1.36 5.41 ± 1.24 0.009
(5.63-9.45) (3.46-7.75) (5.72-7.27) (4.01-7.50) (5.63-9.45) (3.53-7.01)
CCL2 12.14 ± 2.13 12.14 ± 2.13 0.515 9.57 ± 5.44 11.92 ± 1.93 0.469 11.60 ± 4.17 13.19 ± 1.08 0.566
(7.36-14.85) (7.36-14.85) (4.94-15.54) (9.44-13.88) (4.31-14.85) (12.16-14.90)
NFKB 7.66 ± 2.75 6.92 ± 1.38 0.469 9.06 ± 3.76 6.25 ± 1.86 0.339 6.73 ± 1.57 6.81 ± 2.16 0.938
(4.68-14.30) (4.55-8.34) (5.40-14.30) (4.54-8.91) (4.68-8.38) (4.06-9.30)
Table V. Gene expression ∆Ct before and after each dietary treatment.
All genes were evaluated before and after VLCDK1, VLCDK2, VLCDK3dietary treatment. All results were expressed as mean ± standard deviation (SD) followed by minimum
and maximum. Statistical significance were attributed to results with p< 0.05 after parametric test (Student t-test) or non-parametric test (a)(Wilcoxon-Mann-Whitney). Results
with statistical significance were reported in bold. Superoxide Dismutase-1 (SOD-1); Chemokine (C-C Motif) Ligand 2 (CCL2); Nuclear factor kappa-light-chain-enhancer of ac-
tivated B cells (NfKB).
342
substrate for this goal24. Furthermore, whey pro-
teins seem to cause a reduction of glycemia. This
effect is probably mediated by incretins24. Inter-
estingly, it was observed a significant reduction
in blood glucose only after the VLCKD3; after
VLCKD1a decrease of glycemia value was ob-
served, even if without statistical significance.
GH secretion increases with low glycemia, but
according to our results, we do not observe sig-
nificant variations in the levels of this hormone,
proving that the chosen DTs do not raise GH lev-
els.
Moreover, RBC, HB and HBT were signifi-
cantly different among all three DTs (p< 0.05).
In our study, ESR and fibrinogen were not signif-
icantly different among the DTs. NLR was sig-
nificantly different between VLCKD1and VL-
CKD2(p= 0.03). Notably, CRP values were sig-
nificantly different between VLCKD3, respect to
the two other DTs (VLCKD1vs. VLCKD3,p=
0.02; VLCKD2vs. VLCKD3,p= 0.01).
The data confirm that despite the loss of mus-
cle mass observed, the reduction of inflammatory
parameters could depend on the reduction of fat
in the abdominal region. Our data are in accor-
dance with the previous results43, demonstrated
that calorie and carbohydrates restrictions ex-
ceeded the possible oxidative stress induced by
high fat and ketosis. In fact, Nazarewicz et al43
demonstrated that after a short-term ketogenic di-
et , total ant ioxi dative stat us, ur ic acid, and
sulfhydryl content were significantly increased,
without any alteration of malondialdehyde, or su-
peroxide dismutase or catalase activity.
Looking to genes expression of inflammatory
pathway, we have obtained no significant results
after the three DT, despite the significant reduc-
tion in BMI resulting in weight loss.
Jeong et al44demonstrated that the KD inhibit-
ed kainic acid (KA)-induced seizures, decreasing
neuroinflammation via the TNF-αand PPARγ
activation-mediated NF-κB-dependent COX-2
signaling pathway, and providing a novel thera-
peutic approach for Parkinson’s disease, stroke,
and Alzheimer’s disease.
NF-κB modultaes the response to hypoxia45,
and it is activated by various stimuli intro and ex-
tra-cellular, promoting a wide range of biological
functions. NF-κB and it is linked to various sig-
nal transduction pathways and to transcriptional
activation events that mediate inflammation, cell
proliferation, cell migration, apoptosis, and an-
giogenesis46.
Chemokine (CC Motif) Ligand 2 (CCL2) is
one of the several genes of cytokine cluster, lo-
cated on the q arm of chromosome 17, involved
in immunoregulation and flamers processes, with
chemo tactic activity for monocytes and b a-
sophils, but not for neutrophils or eosinophils.
We expected to observe a change in the levels
of expression of, CCL2, and NF-κB genes due to
the loss of body fat mass. However, the lack of
modulation observed in the expression of CCL2,
and NF-κB after the three DTs, could be ex-
plained by the fact that reductions of both fat
mass and muscle mass were obtained. The loss of
lean body mass does not allow to observe the
benefits of weight loss, in terms of reduction of
the expression of inflammatory genes.
Interestingly, the present study is the first, to
our knowledge, to d emons trate a significant
modulation of SOD1 mRNA after VLCKD3(p=
G. Merra, S. Gratteri, A. De Lorenzo, S. Barrucco, M.A. Perrone, et al.
Mean ± Standard Deviation p
VLCKD1VLCKD1VLCKD2
vs. vs. vs.
VLCKD1VLCKD2VLCKD3VLCKD2VLCKD3VLCKD3
SOD1 -0.83 ± 1.16 -0.49 ± 1.04 -1.48 ± 0.87 0.62 0.26 0.14
(-2.52-0.66) (-1.72-0.51) (-2.44/-0.29)
CCL2 2.51 ± 6.46 2.36 ± 5.70 1.32 ± 9.77 0.97 0.77 0.86
(-8.18-12.91) (-3.37-7.93) (-13.42-14.90)
NFKB -1.44 ± 3.96 -2.81 ± 4.95 0.08 ± 2.50 0.59 0.42 0.25
(-9.75-3.54) (-9.77-1.08) (-3.24-3.72)
Table VI. Gene expression ∆Ct comparison between dietary treatments.
All genes were compared between the three different dietary treatments. All results were expressed as mean ± standard devia-
tion (SD) followed by minimum and maximum. Statistical significance were attributed to results with p< 0.05 after parametric
test (Student t-test) or non-parametric test (a)(Wilcoxon-Mann-Whitney). Results with statistical significance were reported in
bold. Superoxide Dismutase-1 (SOD-1); Chemokine (C-C Motif) Ligand 2 (CCL2); Nuclear factor kappa-light-chain-enhancer
of activated B cells (NfKB).
0.009), concurrently with the reduction of CRP
and the decrease of glucose levels (p= 0.03).
SOD1 gene encodes an enzyme localized in the
cytoplasm, which plays an essential role in the
inactivation of free radicals generated by the
process of cellular respiration. In particular, this
enzyme binds to the copper molecules and zinc
for dismutase oxygen molecules charges whose
accumulation within the cells would be toxic47.
We hypothesized that the reduction of inflam-
mation and glycemia (p= 0.03) will depend on a
better response to oxidative stress induced by
SOD1 expression, during weight loss, despite the
reduction of muscle mass.
This study has a few potential limitations.
Firstly, a limit of the study was due to the small
number of participants, although it is acceptable
for genomic studies48. More data are needed on a
larger population. Secondly, the follow-up was
short: our results need to be confirmed in larger
long-term clinical studies. Thirdly, the study
lacked in some evaluation of biomarkers, such as
ketogenic bodies to verify the real status of keto-
sis; c r e a t i n uria to control kidn e y d a m age;
adipocytokines to evaluate the inflammatory sta-
tus related to the expression of studied genes49,
and ghrelin or leptin to check hunger and sati-
ety50.
More studies are needed to clarify changes in
adipose tissue distribution, activity and to estab-
lish the modification of early markers of inflam-
matory status during VLCKD.
Conclusions
The increasingly widespread use of VLDKD,
sometimes also self-prescribed, raises the impor-
tant issue of the risk assessment, even in the short
term, that the use of these prescriptions can lead
to individuals. Our work has wanted to check the
criterion of efficacy and safety in the short term.
Our results show the efficacy of a short-term
VLCKD with 50% of protein replaced by syn-
thetic aminoacidic, able to ensure weight loss,
ectopic fat reduction, as demonstrated by IMAT
and AFP decreases. Moreover, our results con-
firm the possibility of reducing cardiometabolic
risk, without committing the possibility of devel-
oping sarcopenia and activation of inflammatory
and oxidative processes. Results observed in this
exploratory study support the scientific evidence
regarding the important clinical implications in
selecting a dietary treatment, according to of
quality, efficacy and safety indicators.
Although the present data do not allow the
conclusion that VLCKD has a prolonged protec-
tive value for the metabolic consequences of obe-
sity, it seems reasonable to conclude that these
results show the favorable acute effects on some
risk factors, such as glycemia, inflammatory
markers, and overexpression of oxidative stress
related genes.
Further studies are needed to increase knowl-
edge of therapeutic mechanisms and ensure its
efficacy and safety in the long term.
––––––––––––––––––––
Acknowledgements
We are indebted to all the subjects who volunteered in the
clinical trial. We also thank Doctor Paola Gualtieri for statis-
tical analysis of data, Doctor Giorgia Cioccoloni for techni-
cal research assistance and the entire medical team from the
clinical research unit for their technical assistance in con-
ducting the clinical aspects of this study. This study was
supported by grants from Ministero Politiche Agricole Ali-
mentari e Forestali (D.M.: 2017188 03/24/2011).
–––––––––––––––––-––––
Conflict of Interest
The Authors declare that there are no conflicts of interest.
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