SBOG- 20 Anos: Novos Horizontes para Ciência e Tecnologia em Óleos e Gorduras

Florianópolis Novembro 2013

ASPECTOS QUÍMICOS E NUTRICIONAIS DOS ÁCIDOS

GRAXOS CONJUGADOS

Jorge Mancini-Filho

Departamento de Alimentos e Nutição Experimental

Faculdade de Ciências Farmacêuticas

Universidade de São Paulo

E-mail:jmancini@usp.br

Structures of isomers t-10, c-12 CLA; c-9, t-11 CLA and of c-9, c-12 linoleic acid. Adapted from Parisza et al. 2001

A romã (Punica granatum, L.)

OH

O

Ácido Punícico

OH

O

OH

O

Ácido punícico

Ácido α-linolênico

OH

O

OH

O

Ácido punícico

Ácido α-linolênico

Punicic acid

α-linolenic acid

Structures of positional and geometric isomers of conjugated fatty acids [Adapted figure from Tran et al. (2010)].

Analytical methods for detection of CLAs

1) Preparation of lipids methyl esters containing trans fatty acids;

2) Gas chromatography with flame ionization detector (FID) • Polar columns of 50m or 100m, covered with 100% of cyanopropyl polysiloxane. Eg.: CP-Sil 88; SP 2380 and SP 2560; 3)High performance liquid cromatography – Ag-HPLC

• Column Ag-HPLC – Chom Spher Lipids Varian – Chrompack International;

4) Gas cromatography associated with mass spectrometer; 5) Nuclear Magnetic Resonance Spectroscopy.

CG: Chromatogram sigma standard 18919 by CLA01method.

RMN: Commercial CLAs

Metabolic ways of vacenic acid biosynthesis. Adapted from Bauman and Griinari (2001)

Rumen

Linoleic acid Cis-9, cis-12 C 18:2

Cis-9, trans-11 C18:2 (CLA)

Trans-11 C18:1 (Vacenic acid)

C18:0 (Estearic acid)

Tissue

Linoleic acid Cis-9, cis-12 C18:2

Cis-9, trans 11 C18:2 (CLA) 9 -Desaturase

Trans-11 C18:1 (Vacenic acid)

C18:0 cis-9 C18:1 9 -Desaturase

FORMAÇÃO DE CLAs

Ocorrem naturalmente na gordura do leite e na carne de animais

ruminantes. (BENJAMIM e SPENER, 2009)

Rúmen

C18:2 cis9, cis12

(Ácido linoléico)

C18:2 cis9, trans11

(CLA)

C18:1 trans11

(Ácido vacênico)

C18:0

(Ácido esteárico)

C18:2 cis9, cis12

(Ácido linoléico)

C18:2 cis9, trans11

(CLA)

C18:1 trans11

(Ácido vacênico)

∆9 dessaturase

∆9 dessaturase

C18:0 (Ác. esteárico)

C18:1 cis9 (Ac. Oléico)

Tecido Mamário

Graphic of CLA content in bovine cuts.

g/1

00

g fa

t

Muscle Fat cover

• Produced by isomerization of oils rich in linoleic acid

CLAs FORMATION

Linoleic acid

NAGAO and YANAGITA, 2008

• CLNA

It has been shown that the synthetic CLNAs have several physiological functions that are important and unique, with anti-obesity and anticarcinogenic effects (KOBA et al, 2007).

α- or γ-linolenic acid CLNAs Alkaline

Isomerization

Anti-carcinogen: • Membrane fluidity • Cytotoxicity • Eicosanoid synthesis • Immune system

CLA

Anti-atherogen: • LDL/HDL • Total cholesterol • LDL

Physiological effects of conjugated linoleic acids (CLA) Fett/Lipid 100 (1998), Nr. 6, S. 190-210

Body fat Muscle growth

CLAS supplementation effects

CLAs

Anti-carcinogenic (PARIZA et al, 1985; PARK, 2009)

Corporal Composition

(PARK; PARIZA et al, 2007)

Atherosclerosis (MITCHELL; McLEOD, 2008)

Diabetes (ZHOU et al, 2008)

Anti-inflammatory (REYNOLDS; ROCHE, 2010)

Antioxidant (FAGALI; CATALÁ, 2008;

SANTOS-ZAGO et al, 2008)

Elevated triglycerides (DINIZ et al, 2008)

Liver damages (WENDELL et al, 2008)

Positive Negative Inconclusive

CLA has been widely used by athletes as a dietary supplement because of its supposed effect on increasing the fat utilization by the body and thus promote weight loss and decreases adipose tissue.

In addition to reducing body fat, CLA could be useful for diabetics to help prevent hyperglycemia

03/29/2007 ANVISA published the

resolution No. 833 RE that determines the

seizure of all batches of the product

Conjugated Linoleic Acid (CLA). No company

in Brazil has ANVISA authorization to

manufacture, import or sell this product.

Possible CLA mechanisms of action:

1-Induction of lipolysis by catecholamines, promoting selective reduction of visceral fat and abdominal fat indirectly; 2-Increase in the activity of hormone-sensitive lipase and thus lipolysis in adipocytes, accompanied by increased fatty acid oxidation in both skeletal muscle and adipose tissue; 3-Increased activity of carnitine palmitoyl-transferase; and thermogenic effect, possibly related to the induction in gene expression of uncoupling proteins (UCPs); 4 In addition, CLA has an affinity receptor-binding peroxisomal proliferation and activation (PPARs) are transcription factors that control beta-oxidation, the process of transport of fatty acids and differentiation of adipocytes.

Some undesirable effects related to the use of CLA were found in both studies with humans and in animals and include: increased insulin resistance, elevated blood glucose and fasting insulin, increased lipid peroxidation and reduced HDL-cholesterol in individuals with metabolic syndrome. Factors that could contribute to the development of future chronic diseases, among them obesity.

CLAs SUPLEMENTATION EFFECTS

Resolution n° 833, March 28, 2007: ANVISA prohibited CLAs commercialization.

Controversial Results

(A) Redução do radical DPPH• pelos óleos da semente de romã (PSO), de linhaça (OL) e de soja (OS) e (B) comparação do efeito na redução do radical de 1mg dos óleos e do BHT.

B

A

PSO OL OS BHT

0

20

40

60

80

100

ab

a

c

% d

e r

ed

ução

do

rad

ical

ENSAIO IN VIVO

56 animais (n=8)

Grupo controle – Água

1% Ac. Linoleico

2% Ac. Linoleico

4% Ac. Linoleico

1% de FFA – CLA

2% de FFA – CLA

4% de FFA – CLA

Ração comercial - Nuvilab CR-1, descontaminada e irradiada a 12KGy. Duração do Ensaio – 40 dias.

isômeros cis9, trans11 e trans10, cis12

(FFA – CLA)

Óleo de Soja (OS)

IN VIVO ASSAY- TBARS

FFA-CLAs e OS supplementation effect in the lipid peroxidation in serum (a), liver tissue (b), brain (c) and muscle (d)) - mol TBARS / mg protein. Mean and standard deviation (n = 7-8). p <0.05.

TBARS in the muscle for the groups treated with CLAs. (RAHMAN et al, 2009).

Serum Liver

Brain Muscle

Antioxidants enzymes activities

CAT - Figado

Contr

ole

LNA 1

%

LNA 2

%

LNA 4

%

CLN

A 1

%

CLN

A 2

%

CLN

A 4

%

0

500

1000

1500

U /

mg

de p

rote

ína

GPx - Fígado

Contr

ole

LNA 1

%

LNA 2

%

LNA 4

%

CLN

A 1

%

CLN

A 2

%

CLN

A 4

%

0.0

0.5

1.0

1.5

U /

mg

de

pro

teín

a

OL (source of LNA) and PSO (CLNA source) supplementation effect on the antioxidant enzymes (CAT, SOD and GPx) in liver of rats.

SOD - Fígado

Con

trol

e

LNA 1

%

LNA 2

%

LNA 4

%

CLN

A 1

%

CLN

A 2

%

CLN

A 4

%

0

50

100

150

a aab

bcbc

cc

U /

mg

de p

rote

ína

LIVER

Liver

Liver

Lipidic Profile - Gastrocnêmius

Formula Name Control FFA -

CLA

1%

FFA - CLA

2%

FFA - CLA

4%

Saturated 34,89 35,54 35,20 35,46

Totals Monounsaturated 19,42 28,91 28,41 21,98

Polyunsaturated 31,65 34,55 35,24 33,64

18 : 2

(9c11t)

Conjugated

Linoleic Acid

0,95

0,00a

1,70 0,00b 4,04 0,00c

18 : 2

(10t12c)

Conjugated

Linoleic Acid

0,91

0,00a

1,48 0,00b 3,07 0,00c

Different lowercase letters indicate differences (P <0.01).

• CLNAs metabolism

CLNAs are well absorbed by the body and is metabolized to CLAs (KOBA et al, 2007; Yuan et al, 2009).

Tsuzuki et al. (2004): CLNA (α-eleostearic acid – 9c,11t,13t-18:3) is partially saturated in the position Δ13 and converted to 9c,11t-CLA; the reaction is carried out by the enzyme Δ13-saturase which is NADPH-dependent.

Resumo dos Componentes da Via Inflamatória TLR: receptor Toll-like; TNF: fator de necrose tumoral; IL-1: interleucina-1; IL-6: interleucina-6; CCL2: quimiocina CCL2; CXCL8: interleucina-8. Fonte: Extraído de Medzhitov (2010).

Mecanismos de ação dos CLAs no processo inflamatório LPS = lipopolissacarídio; TLR4 = receptor toll-like 4; CD14 = cluster de diferenciação 14; TIRAP = proteína adaptadora do

receptor toll-interleucina 1; TOLLIP = proteína toll-interativa; MyD88 = fator de diferenciação mielóide 88; IRAK = quinase

associada ao receptor de interleucina 1; TRAF6 = fator 6 associado ao receptor do fator de necrose tumoral

citoplasmático.; NF-B= fator nuclear kappa B; IB= inibidor de kappa B; IKK = quinase de IB, PPAR- = receptor gama

ativado por proliferador de peroxissomo; COX 2 = cicloxigenase 2; CLA = ácido linoléico conjugado. Setas verdes indicam a ação positiva dos CLAs e setas vermelhas indicam a ação inibitória destes ácidos graxos conjugados. Fonte: Extraído de Reynolds, Roche (2010).

Sctelma – Safflower oil

From: R$ 109,00

By: R$ 89,10

SAFFLOWER (Cartamus) It is an oleaginous plant and its seeds have high oil content (35 to 40%) of excellent quality for human consumption. Safflower oil has high levels of linoleic acid (70%) and oleic (20%). One of the most important characteristics of this oil is the presence of low cholesterol content, which is a substance harmful to human body. The use of safflower oil has produced an increase in lean body mass, decreased fat accumulation and has an important role in muscle growth. There is evidence that safflower oil inhibits the mechanism that causes our body to accumulate fat and causes it to use our reserves of fat for energy. Supplementation with Safflower oil produces many effects in sedentary people and those who perform physical activities, but research suggests that safflower oil is more effective in reducing the fat of those who perform physical activities, be they aerobic or anaerobic (weight training).

SOY BEAN It is an oilseed plant with oil content of approximately 20%. Its fatty acid composition is: Oleic 22.3%; Linoleic 54.5%; Alpha Linolenic 8%

CONCLUSION Given the structural characteristics of conjugated fatty acids and their presence in plants and animals, and also many works without a consistent biochemical evidences, becomes necessary to perform most biological studies to have more information on their influence in the animal organism.

Referências Bibliográficas ARAO, K.; YOTSUMOTO, H.; HAN, S.Y.; NAGAO, K.; YANAGITA, T. The 9cis, 11trans,13cis isomer of conjugated linolenic acid reduces apolipoprotein B100 secretion and triacylglycerol synthesis in HepG2 cells. Bioscience, Biotechnology, and Biochemistry, v. 68, n. 12, p. 2643-2645, 2004.

BLOIS, M.S. Antioxidant determinations by the use of a stable free radical. Nature. v. 118, p. 119-1200,1958.

BRAND-WILLIANS,W.; CUVELIER, M.E.; BERSET, C. Use of free radical method evaluate antioxidant activity. Lebensm, wiss. u.-Technology., v. 28, n. 1,p. 25-30,1995.

CARVALHO, E.B.T; MELO,I.L.P.; MANCINI-FILHO, J. Chemical and physiological aspects of isomers of conjugated fatty acids. Ciência e Tecnologia de Alimentos, Campinas, v.30, p. 295-307, 2010.

CHEN, J.; CAO, Y.; GAO, H.; YANG, L.; CHEN, Z.Y. Isomerization of conjugated linolenic acids during methylation. Chemistry and Physics of Lipids, v. 150, p. 136-142, 2007.

DHAR, P.; BHATTACHARYYA, D.; BHATTACHARYYA, D.K.; GHOSH, S. Dietary Comparison of Conjugated Linolenic Acid (9 cis, 11 trans, 13 trans) and a-Tocopherol Effects on Blood Lipids and Lipid Peroxidation in Alloxan-Induced Diabetes Mellitus in Rats. Lipids, v. 41, n. 1, p. 49-54, 2006.

JARDINI, F.A.; MANCINI FILHO, J. Composição centesimal e perfil dos ácidos graxos de romã (Punica granatum, L.) cultivada no Brasil. Higiene Alimentar, v. 21, n. 148, p. 81-85, 2007.

MANCINI FILHO, J.; TAKEMOTO, E.; AUED-PIMENTEL, S. Parâmetros de identidade e qualidade de óleos e gorduras. In: ALMEIDA-MURADIAN, L. B.; PENTEADO, M. D. V. C. Vigilância Sanitária: tópicos sobre legislação de alimentos. Rio de Janeiro, RJ: Guanabara Koogan, 2007. cap. 6, p. 81-107.

MÉNDEZ, E. et al. Validation of the rancimat test for the assessment of the relative stability of fish oils. Journal American Oil Chemistry Society., v. 73, n. 8, p. 1033-1037, 1996.

SCHUBERT, S.Y.; LANSKY, E.P.; NEEMAN, I. Antioxidant and eicosanoid enzyme inhibition properties of pomegrante seed oil and fermented juice flavonoids. Journal of Ethnopharmacology, v. 66, p. 11-17, 1999.

ZOCK, P.L. Health problems associated with saturated and trans fatty acids intake. In: WILLIAMS, C.; BUTTRISS, J. Improving the fat content of foods. Boca Raton: CRC Press, 2006. cap. 1, p. 3-24.

CYTED

Mecanismos de ação dos CLAs no processo inflamatório LPS = lipopolissacarídio; TLR4 = receptor toll-like 4; CD14 = cluster de diferenciação 14; TIRAP = proteína adaptadora do receptor toll-interleucina 1; TOLLIP = proteína

toll-interativa; MyD88 = fator de diferenciação mielóide 88; IRAK = quinase associada ao receptor de interleucina 1; TRAF6 = fator 6 associado ao receptor do fator de

necrose tumoral citoplasmático.; NF-B= fator nuclear kappa B; IB= inibidor de kappa B; IKK = quinase de IB, PPAR- = receptor gama ativado por proliferador de

peroxissomo; COX 2 = cicloxigenase 2; CLA = ácido linoléico conjugado. Setas verdes indicam a ação positiva dos CLAs e setas vermelhas indicam a ação inibitória

destes ácidos graxos conjugados. Fonte: Extraído de Reynolds, Roche (2010).

Possible mechanisms of action of conjugated linoleic acid (CLA) related to body composition in adipocytes and skeletal muscle. MOURÃO, D. M. et al. Revista de Nutrição, vol. 18 (3) 2005.