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EuropeanReviewforMedical and Pharmacological Sciences 2013;17:2555-2565
Body composition phenotype:
Italian Mediterranean Diet and C677T
MTHFRgenepolymorphisminteraction
1,5 1 1 1 1
L. DI RENZO , M. RIZZO , L. IACOPINO , F. SARLO , E. DOMINO ,
2 3 4 1,5
F. JACOANGELI , C. COLICA , D. SERGI , A. DE LORENZO
1
Division of Clinical Nutrition and Nutrigenomic, Department of Biomedicine and Prevention, University
of RomeTorVergata,Rome,Italy
2
Department of Internal Medicine, UOSD Service of Clinical Nutrition, Parenteral Therapy and
Anorexia Nervosa, University Tor Vergata Rome, Rome, Italy
3
CNR, ISN UOS of Pharmacology, Department of Pharmacology, University Magna Graecia,
Roccelletta di Borgia, Catanzaro, Italy
4
Department of Clinical Medicine, UOC of Cardiology, University of Rome Tor Vergata, Tor Vergata
Hospital, Rome, Italy
5
I.N.Di.M., National Institute for Mediterranean Diet and Nutrigenomic, Amantea, Italy
Abstract. – OBJECTIVES: Strategies to im- Introduction
proveweightmaintenancearefocusedonconsid-
ering the genetic makeup and its interaction with A correct life style is necessary to preserve
dietary intake, with the aim to identify vulnerable health status and wellbeing. The Mediterranean-
individuals that will benefit from a variety of more like dietary pattern is known to be associated
personalized dietary recommendations. The aim
of the study was to examine the impact of the with health beneficial effects and lower incidence
C677T MTHFR gene Polymorphism on body com- of various chronic diseases, as atherosclerosis,
position changes induced by a balanced 1
hypocaloric Italian Mediterannean diet (IMD). cardiovascular diseases, and cancer . The Italian
SUBJECTS AND METHODS: Participation in Mediterranean Diet (IMD), typical diet rich in
the study included a complete screening of an- fruit, vegetables, legumes, whole grains, fish and
thropometry and body composition by Dual-en- low-fat diary products, all coming from mediter-
ergy X-ray absorptiometry (DXA), and a genotyp- ranean place, is related to better management of
ing for the C677T MTHFR polymorphism. body weight and cardiovascular risk factors2,3. In
70 Italian Caucasian obese were enrolled and particular, we demonstrated the effectiveness of
56 of them completed the screening at baseline IMD, the so called Nicotera Diet, in reducing fat
and12weeksafterthenutritional intervention.
RESULTS:T(+) carriers had a higher content of mass and preserving lean mass in healthy and ill
Total Body Fat (TBFat), and Lean (TBLean), re- 4-6
flecting on higher weight and BMI, thanT(-) carri- subjects . Indeed, there are data suggesting the
ers. After IMD, the 28.6% and 71.4% of total sub- existence of significant gene-environment inter-
jects decreased weight and TBFat (Kg), respec- action effects that make some individuals more
tively. The relative changes were: delta % = susceptible to body weight gain or loss than oth-
−9.09±3.85 for weight; delta % = −15.79±8.51 for ers because of genetic differences, when exposed
TBFat; delta % = −3.80±5.60 forTBLean.The 5.3% to an obesogenic environment7. Moreover, body
of subjects who reached the end point of inter- composition changes could be partially explained
vention, and the 8.9% who reducedTBFat (%) be- by genetic factors or by the interaction between
low the cut-off of preobesity, were T(-) carriers. A 8
loss of TBLean (Kg) was observed in the 5.1% genes and environment .
and23.5%ofT(-)andT(+)carriers. Acommon polymorphism of methylenetetrahy-
CONCLUSIONS: MTHFR genetic variations drofolate reductase (MTHFR), the C677T muta-
analysis would be an innovative tool for the nu- tion, results in a reduced specific MTHFR activity
tritional assessment, in order to predict the ther- (~34% residual activity in T677T, ~71% residual
apeutic response of obese subjects, in terms of activity in C677T relative to C677C). MTHFR by
fat and lean mass loss.
catalyzing the conversion of 5, 10 methylenete-
Key Words: trahydrofolate to 5-methyltetrahydrofolate, is a piv-
Lean Body Mass, Body Composition, Diet, Human otal enzyme in folate metabolism and regulates the
Genetics, Genetic Susceptibility. proportional usage of one-carbon units between
Corresponding Author: Antonino De Lorenzo, M.D., Ph.D.; e-mail: delorenzo@uniroma2.it 2555
L. Di Renzo, M. Rizzo, L. Iacopino, F. Sarlo, E. Domino, F. Jacoangeli, C. Colica, D. Sergi, A. De Lorenzo
methylation reactions and nucleic acid synthesis. A they entered a baseline period .After baseline mea-
direct consequence of MTHFR deficiency is hy- surements subjects were entered the intervention
perhomocysteinemia due to the lack of 5-methyl- period and were followed at 4-week intervals. Fi-
tetrahydrofolate, the necessary methyl donator nal assessment was made at 12 weeks.
for homocysteine (tHcy) to be transformed into
methionine. Recent meta-analyses showed an Subjects
overall significantly higher cardiovascular dis- Seventy Caucasian Italian obese subjects were
ease risk in people with, compared to those with- consecutively recruited during three months from
9
out, C677T MTHFRpolymorphism . March 2009 by medical and nutritional staff of
Recently, the MTHFR gene polymorphisms the Obesity Centre of Nicotera Hospital (VV,
were found to be associated with BMI-defined Italy). Subjects having serious chronic dis-
obesity and lean mass10, sustaining a linkage of eases/conditions that may have potential influ-
body mass index (BMI) and lean mass to chro- ence on endocrine and metabolism were exclud-
mosome 1p36, where the MTHFR gene is ed. Participation in the study included a complete
located11. It has been also demonstrated an asso- medical history to gather informations about
ciation of C677T MTHFR polymorphism with health status, current medications history, includ-
BMI and central adiposity indices in healthy ing supplements of vitamins and minerals, social
postmenopausal women. Moreover, epidemiolog- habits, like alcohol drinking and smoking, food
ical studies found that tHcy was correlated with habits, physical activity and family history for
12,13
lean mass and BMI . chronic diseases, a complete screening of anthro-
However, BMI alone may not be accurate pometry and body composition, and a genotyp-
enough for assessing the extent of excessive fat ing for the C677T MTHFR polymorphism.
accumulation and, thus, the obesity related dis- The study group consisted of 56 subjects, 37
ease risk, since body weight is composed of Total females (age: 46.54 ± 12.32) and 19 males (age:
Body Lean (TBLean), Total Body Fat (TBFat), 43.84 ± 14.10), who completed the screening at
and bone mass. Dual energy X-ray absorptiome- baseline and 12 weeks after the nutrtional inter-
try (DXA) can measure body composition with vention. The subjects were categorized in BMI-
high precision and provides more reliable and subgroups according to World Health Organiza-
14,15 17
precise measure for obesity . tion (WHO) criteria . Moreover, gender, age and
Furthermore, we have shown a significant re- TBFat (%) cutoff points were also used to classi-
14,18
lationship between the Normal Weight Obese fy the total population .
phenotype, a status of TBFat accumulation ac- Amultidisciplinary team of dieticians and nu-
companied by TBLean deficiency, and posses- tritionists met with each patient, and provided an
sion of wild type and heterozygous genotypes of educational session for nutrition and meal-plan-
16
C677T MTHFR enzyme polymorphism . These ning guidance. A balanced hypocaloric IMD for
results jointly imply that MTHFR may play a 12 weeks was assigned to all eligible subjects. A
role in variation of TBLean. loss of -10% of weight or TBFat (%) below the
14
Upto now, there has been no study investigat- cut-off of obesity were considered the end-point
ing the relationship between MTHFR gene poly- of the intervention, and a treatment success.
morphism and the lost of TBLean after an A statement of informed consent was signed
hypocaloric IMD. by all participants in accordance with principles
Due to recent lines of evidence supporting the of the Declaration of Helsinki. The study was ap-
functional and genetic relevance of MTHFR and proved by the Ethical Commission of the Univer-
variation in TBLean and TBFat, this study was de- sity of Rome “Tor Vergata”, Italy. None of the
signed to determine whether a balanced subjects was receiving drug treatments at the
hypocaloric IMD differentially affected body com- time of the assessment.
position and TBLean of carrier T(+) or non carrier Nutritional status assessment was conducted at
T(-) of C677T MTHFRgenepolymorphism. the Obesity Centre of Nicotera Hospital (VV, Italy).
Genetic analysis was performed at the University of
Subjects and Methods RomeTorVergata,HumanNutritionUnit.
Study Design Physical Activity (PA) Assessment
Weusedprospective, cohort design with repeat- Leisure time and PA habits were monitored us-
29
ed measures. After screening and subject selection ing a validated questionnaire . Participants were
2556
Body composition phenotype
asked to maintain their usual exercise habits dur- retable/Animal (V/A) protein ratio as close to 1.5
ing 3 months of follow-up after the beginning of as possible, monounsatured/satured fatty acid ratio
the nutritional intervention. close to 2, polyunsatured/satured fatty acid ratio
close to 0.4-1, and low nutritional indices above
Dietary Assessment mentioned4. The MAI index value was attended
Usual dietary intakes over the past 12 months around 720. The IMD-based meal plan for each
were collected by a semiquantitative food-fre- subject was obtained by a dietetic software pack-
quency questionnaire that included 127 food age (Dietosystem, DS Medica SRL, Milan, Italy).
items and three portion-size pictures for 17
items. The alimentary diary and nutrient intake Anthropometric Measurements
were analysed using diet analyser software IN- After a 12-h overnight fast, all subjects under-
DALI. Daily and weekly food intake in g calcu- went anthropometric evaluation. Anthropometric
lated from food intake frequency and portion parameters of all the participants were measured
sizes. Dietary intake of macronutrients (lipids, according to standard methods (body weight,
22
proteins and carbohydrates), folate, vitamin B6 height, waist and hip circumferences) . Subjects
and vitamin B12 was estimated. The dietary pat- were instructed to take off their clothes and shoes
tern of each subject was evaluated by nutritional before performing all the measurements. Body
indices: vegetable to animal protein ratio (V/A), weight (kg) was measured to the nearest 0.1 kg,
Cholesterol/Satured fat Index (CSI), Atherogenic using a balance scale (Invernizzi, Rome, Italy).
Index (AI), Thrombogenic Index (TI); the Height (cm) was measured using a stadiometry to
4,20
MediterraneanAdeguacy Index (MAI) . the nearest 0.1 cm (Invernizzi, Rome, Italy). The
waist and hip circumferences were measured
Nutritional Intervention with a flexible steel metric tape to the nearest 0.5
The Italian Mediterranean Diet (IMD), the so cm. Waist circumference was measured at the
called Nicotera Diet, was used3,4,20. Total daily horizontal plane that corresponds with the nar-
energy content of the diet was determined on an rowest point between the crest iliac and the bot-
individual basis (max 2625 Kcal/day; min 2195 tom rib. Hip circumference was measured at the
Kcal/day), taking into account resting metabolic largest point when observed on a horizontal
rate (RMR), calculated using De Lorenzo et al. plane. BMI was calculated using the formula:
2
(2001) prediction equation for the Italian popula- BMI=bodyweight(kg)/height(m) .
21
tion, and level of PA .
The macronutrient’s composition of the dietary Dual-Energy X-ray Absorptiometry (DXA)
regimen was as follows: carbohydrates, 55% to Body composition analysis was assessed by
60%; proteins, 15% to 20% (of which about 60% DXA(i-DXA,GEMedicalSystems, Milwaukee,
was comprised of vegetable proteins); total fat, WI, USA), according to the previously described
less than 30% (saturated fat < 10%; poliunsaturat- procedure, for evaluating soft tissues, i.e. TBFat
ed fatty acids (PUFA) 6-10%: 5-6% of n-6 PUFA and TBLean23,24. The subjects were instructed not
and 1-2% of n-3 PUFA; monounsaturated fatty to exercise within 24 h from the test. The sub-
acids (MUFA), about 15%; trans fatty acids < jects were given complete instructions on the
1%; cholesterol consumption of 100 mg/day), testing procedure. They wore a standard cotton t-
sodium chloride less than 5 g and 30 g of fibers, shirt, shorts and socks. They laid supin on the
per die. No alcoholic beverages were allowed. DXA, without moving while the DXA scan
The daily intake of fruit and vegetables was more recorded their results. The average measurement
than 400 g. Extra-virgin olive oil was consumed time was 20 min. The effective radiation dose
daily in the amount of 20-25 g. The daily intake from this procedure is about 0.01 mSv. The coef-
of carbohydrates was mainly derived from wheat ficient of variation (CV% = 100 × s.d./mean) in-
(pasta and bread), other cereals and legumes (at tra- and intersubjects ranged from 1 to 5%. The
least 3 times/week). The weekly frequency of coefficient of variation for bone mass measure-
consumption of animal foods was 3-4 times for ments is < 1%; coefficient on this instrument for
fish, 1-2 for meat, 1 for eggs, 1 for cheese. No five subjects scanned six times over a 9-month
change in total energy intake (Kcal/day) was re- period were 2.2% for TBFat and 1.1% for
quired during the experimental time. TBLean. Subjects were classified as obese by us-
The composition of the diet in terms of foods ing gender and age Percentage Body Fat (PBF)
14
and food combinations was planned to obtainVeg- cutoff points .
2557
L. Di Renzo, M. Rizzo, L. Iacopino, F. Sarlo, E. Domino, F. Jacoangeli, C. Colica, D. Sergi, A. De Lorenzo
Analysis of Blood Samples parisons among genotype groups were performed
Blood samples (10 mL) were collected into using independent t test and non parametric
®
sterile tubes containing EDTA (Vacutainer ), All Mann Whitney test. Possible interactions be-
materials were immediately placed on ice and tween the C677T MTHFR polymorphism and
plasma was separated by centrifugation at 1600 x gender were investigated with the general linear
g for 10 min at 4°C. method. A paired t test or a non parametric
Homocysteineconcentration was determined by Wilcoxon test were performed to evaluate differ-
a fully automated HPLC method, using reversed- ences before and after IMD nutritional interven-
phase separation and fluorescence detection, with tion. All tests were considered significant at p ≤
reagents provided by the same company. 0.05. Statistical analysis was performed using a
Analyses were carried out by the accredited computer software package (SPSS for Windows,
Clinical Chemical Laboratories of the “Tor Ver- version 13.0; SPSS Inc., Chicago, IL, USA).
gata” Polyclinic (PTV) of Rome, Italy.
MTHFRGenotypeAnalysis Results
DNA was isolated from peripheral leukocytes
by using the Gentra DNA isolation kit (Gentra Dietary and Physical Assessment
Systems Inc, Minneapolis, MN, USA). Accord- Of all recruited subjects, 56 (F=39, M=17)
ing to a previously described procedure, genotyp- completed the study and their results were eligi-
ing for the MTHFR point polymorphism C677T ble for data analysis. According to WHO criteria
was performed by polymerase chain reaction am- of obesity, based on TBFat (%) evaluation, all
plification with the primers 5’TGAAGGA- subjects were obese at the starting point, before
GAAGGTGTCTGCGGGA3’ (sense) and 5’AG- the nutritional intervention. Dietary assessment is
GACGGTGCGGTGAGAGTG3’ (antisense). reported in Table I. A comparison of macronutri-
Thirty cycles (95°C for 45 s, 64°C for 30 s, and ents, micronutrients, and nutritional indices be-
72°C for 30 s) were used to amplify the 198-base tween baseline and IMD standard meal is high-
pair (bp) product. Because the C-to-T transition lighted. PA assessment, at baseline both in men
at nucleotide 677 produces a HinfI digestion site, and women, is shown in Table II. The level of PA
the amplified product derived from the mutant was classified into three categories (sedentary,
gene was cleaved into 175-bp and 23-bp frag- moderate and vigorous) based on the time spent
ments by HinfI, which leaves the wild-type gene on life activity or programmed physical exercise.
unaffected. After electrophoresis through a 6%- According to PA questionnaire, any subjects
polyacrylamide gel, the digestion products were spent time for vigorous PA, the most spent 1-2
visualized by staining with ethidium bromide. time/week for moderate PA, with a sedentary be-
havior ranging from 10 to 16 h/day.
Statistical Analysis
Data are presented as group means (or medi- Genotyping Assessment
an) ± standard deviation (SD), percentage, or < ∆ The T allele frequency was 19.6%, and the
%>(∆%). ∆% expresses the relative change, in CC, CT, and TT genotype frequencies were
percent, of a parameter respect to baseline. For 69.6% (n=39), 21.4% (n = 12), and 8.9% (n =
the calculation we used the following formula: 5), respectively. The T allele frequency in our
sample was similar to that in other white popula-
tions. The MTHFR genotypes were similarly dis-
[(Value at week 12) – (Value at baseline)] tributed in men and women (in men, CC: 63.2%,
∆%=–––––––––––––––––––––––––––––––––––––––––––––––––––––– CT: 21.1%, TT: 15.7%, and in women, CC: 73%,
(Value at baseline) *100 CT: 21.6%, TT: 5.4%; p = ns). The study popula-
tion was divided in 2 subgroups, i.e. T(+) carriers
Data were analyzed to check assumptions and T(-) non-carriers of the C677T MTHFR gene
about the distribution of the measured variables. polymorphism.
Three genotype groups were first considered to
check differences in considered variables be- Effects of IMD on Anthropometric and
tween groups. Because a dominant or recessive Body Composition Parameters
effect existed, analysis was repeated comparing Anthropometric parameters, laboratory parame-
carriers T(+) vs. non-carriers T(-) groups. Com- ters and body composition characteristics by
2558
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