How To Study The Effect Of Lcohol On The Body

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1 CFPA Cnning Fruit Producers Assoc. Submit to: Wiehhn Victor Tel: +27 (0) SAAPPA / SASPA / SAT Fruitgro Science Submit to: Louise Liebenberg Tel: +27 (0) /1 louise@fruitgro.co.z DFTS Dried Fruit Technicl Services Submit to: Dppie Smit Tel: +27 (0) dppies@dtd.co.z Winetech Submit to: Jn Booysen Tel: +27 (0) booysenj@winetech.co.z X Indicte (X) client(s) to whom this finl report is submitted. Replce ny of these with other relevnt clients if required. FINAL REPORT FOR 2011 PROGRAMME & PROJECT LEADER INFORMATION Progrmme leder Title, initils, surnme Dr DP vn Velden Prof MJ Kotze Project leder Present position Senior Lecturer Senior Lecturer & Resercher Address Dept of Pthology, US Dept of Pthology, US Tel. / Cell no Fx E-mil dpvv@sun.c.z mrith@sun.c.z PROJECT INFORMATION Project number Project title Project Keywords Industry progrmme N09/08/225 The effect of regulr lcohol consumption intercting with genetic risk fctors for crdiovsculr Wine Brndy Crdiovsculr helth CFPA Deciduous DFTS Winetech X Other Fruit kind(s) Grpes Strt dte 01/01/2009 (dd/mm/yyyy) End dte 31/12/2011 (dd/mm/yyyy) (Note: djust footer insert the project number no, resercher nd reserch institution)

2 Finl report 2 FINAL REPORT 1. Executive summry In this reserch project the effect of regulr lcohol consumption in the form of red wine or brndy ws investigted on the lipoprotein profile, oxidtive stress sttus nd inflmmtory mrkers in 37 volunteers. We ssessed the physiologicl effects in reltion to genetic risk fctors for crdiovsculr disese. Nutrition intervention my be more effective to lower crdiovsculr risk when the genetic bckground is tken into considertion for identifiction of gene-environment interctions. ORIGINALITY OF THE STUDY The effect of regulr moderte lcohol consumption wine vs brndy - on the lipoprotein profile, oxidtive stress nd inflmmtory mrkers ws studied for the first time in the South Africn popultion in the context of genetic risk fctors known to interct with lcohol. It is envisged tht this study will led to the development of guidelines for sfer drinking prctices in the locl popultion. It is importnt to determine whether moderte intke of wine nd brndy hve similr crdiovsculr protective properties. Ultimtely, sfe limits of wine nd brndy consumption will be determined bsed prtly on the genetic profile nd other lifestyle fctors studied. 2. Problem identifiction nd objectives The specific ims were s follows: 1. To compre the effect of moderte red wine consumption compred to brndy on the therogenic lipoprotein profile, oxidtive stress nd inflmmtory sttus. 2. To perform genetic screen of CVD risk fctors to determine individul muttion/llele frequencies in the study popultion 3. To correlte the presence of genetic risk fctors with relevnt biochemicl prmeters nd ssess gene expression in the presence nd bsence of known environmentl triggers 4. To determine the impct of muttions/functionl polymorphisms included in the genetic screen on the response to the intervention (smll smple size my be limiting fctor) 3. Workpln (mterils & methods) We recruited 37 helthy dult volunteers between the ges of 18 nd 70 yers with or without crdiovsculr risk fctors tht fulfilled the inclusion criteri. All specimens nd dt were obtined with informed consent nd re kept strictly confidentil. Blood pressure, body weight, height s well s wist nd hip circumferences were mesured following stndrdized procedures on first nd follow-up visits of prticipnts to clculte the body mss index (BMI) nd the wist/hip rtio (WHR). No mediction ws llowed t ny time during the whole study period. Study design After 2 week wshout period, 50% of the prticipnts consumed 250 / 175 ml red wine per dy x 28 dys, nd 50% consumed 50 / 40 ml per dy brndy x 28 dys respectively for men

3 Finl report 3 nd women. This intervention ws followed immeditely by cross-over period of wine or brndy consumption for 28 dys. Wine consumption per dy Mles: 13.5% lcohol = g lcohol per dy (13.5x 2.5 = 33.75g) Femles % lcohol = g lcohol per dy (13.5x1.75 = 23.63g) (or 0.35g lcohol/kg body mss for mles nd femles) Brndy consumption per dy Mles: 50 43% lcohol = 21.5 g lcohol per dy (43 x 0.5 = 21.5 g) Femles: 40 43% lcohol = 17.2 g lcohol per dy (43 x 0.4 = 17.2 g) (or 0.35g lcohol/kg body mss for mles nd femles) Fsting blood specimens were tken from the nterior cubitl vein: 50 ml per test dy: After the 2 week wshout period (Bseline) After the 28 dy red wine/brndy consumption period After the next 28 dys consumption of wine/brndy consumption cross-over period Biochemicl nlysis of serum/plsm Blood were obtined from n ntecubitl vein in the morning fter 12 hour overnight fst. Blood tests were performed ex vivo, i.e. the wine or brndy ws consumed by volunteers undergoing certin blood tests before nd fter ech period of wine / brndy consumption to observe the effect tht wine or brndy hd on the specified biologicl systems (plsm levels of different lipoprotein frctions, oxidtive nd inflmmtory sttus). Clinicl nd biochemicl prmeters included totl cholesterol, homocysteine, serum ferritin nd oxidtive stress mrkers. Cholesterol nd triglyceride levels were determined in plsm by enzymtic methods using the RA 1000 nlyzer, while the high density lipoprotein (HDL) levels were obtined fter precipittion of polipoprotein B contining lipoproteins. The cholesterol nd triglyceride contents of the HDL frction were mesured, while the low density lipoprotein cholesterol ws clculted using the Friedewld eqution. A RA 1000 utomted nlyzer ws used. High sensitive C-rective protein (ELISA test kit, HELICA) nd homocysteine (Arki nd Sko, 1987) concentrtions were nlyzed in the plsm of ll the prticipnts. CRP is n importnt immunomodultor in host defence while elevted levels of both CRP nd homocysteine hve been identified s risk fctors for crdiovsculr diseses s well. Biomrkers for oxidtive stress nd inflmmtion included lipid peroxidtion mrker, mlondildehyde (MDA) equivlents; reduced to oxidized glutthione rtios (GSH:GSSG); oxygen rdicl bsorbnce cpcity (ORAC); nitric oxide metbolic products; oxidized low density lipoproteins (Ox-LDL); C-rective protein (CRP) mong others. Full lipid profiles serum liver nd kidney function indictors were lso done on ll study prticipnts to exclude possible toxicity induced by the consumption of the vrious lcoholic beverges (red wine nd brndy). Totl phenolic cid content (TP) of plsm ws determined using the Folin-Cioclteu method modified by Serfini et l (1998) to void plsm protein interference. Plsm ntioxidnt cpcity ws determined using the Trolox equivlent ntioxidnt cpcity (Arendt et l. 2001). Sub smples of the plsm were used to determine the oxygen rdicl bsorbnce cpcity (ORAC) by the method of Co nd Prior (1998); the impct on the

4 Finl report 4 performnce of ntioxidnt enzymes including superoxide dismutse (Flohe nd Otting, 1984), ctlse (Aebi, 1984), glutthione peroxidse (Flohe nd Gunzler, 1984), the rtio of reduced to oxidised glutthione (GSH:GSSG) s n independent predictor of crotid intimmedi thickness (IMT) nd ultimtely the presence of erly therosclerosis (Ashfq et l., 2006) nd the effect on lipid peroxidtion by mesuring thiobrbituric cid recting substnces (Esterbuer et l., 1990) Genetic Studies Reltively common genetic vritions underlying dyslipidemi nd the development of CVD in the presence of certin lifestyle risk fctors were tested to identify individuls who my not benefit from drinking lcohol s consequence of gene-environment mismtches. DNA ws extrcted from whole blood for muttion screening using polymerse chin rection (PCR)- bsed methods. Ptients with monogenic CVD subtypes such s fmilil hypercholesterolemi (FH) nd fmilil defective polipoprotein B100 (FDB) were excluded from the study bsed on the lipid profile nd strong fmily history of erly-onset CHD. The genetic nlysis included screening for muttions / functionl polymorphisms the ApoE, MTHFR nd HFE genes known to influence CVD risk the effect of lcohol intke, using previously described methods (Kotze et l. 2003; Kotze nd Thirt 2003) with some modifiction to llow high-throughput nlysis. Direct DNA sequencing of PCR-mplified DNA ws performed to verify the results obtined with rel-time TqMn technology pplied in high-throughput genotyping. STATISTICAL ANALYSIS The sttisticl nlysis will be performed by Professor Mrtin Kidd, the chief sttisticin t the Centre for Sttisticl Consulttion, University of Stellenbosch. ETHICAL CONSIDERATIONS Prior to this study, study prticipnts were informed of the fct tht E4 llele of the Apo E gene included in the genetic nlysis does not only increse the risk of coronry hert disese in the presence of environmentl risk fctors such s smoking nd high-sturted ft diet, but lso increse the risk of Alzheimer s disese, with specific gene-lcohol effect documented in vrious studies. This hs to be considered before the results of the genetic testing cn be requested by the prticipnts nd/or provided by the ttending clinicin to prticipnts. 4. Results nd discussion Stte results obtined nd list ny benefits to the industry. Include short discussion if pplicble to your results. This finl discussion must cover ALL ccumulted results from the strt of the project, but plese limit it to essentil informtion. All the dt obtined during the study relted to the lipoprotein profile, oxidtive stress, inflmmtory mrkers nd genetic risk fctors hve been cptured in n electronic dtbse. The sttisticl nlysis hve been performed in four phses nd discussed fterwrds t project meetings rrnged with the sttisticin. Four members of the reserch tem Dr DP vn Velden, Dr Dee Blckhurst, Prof M Mrnewick, nd Prof MJ Kotze ttended these meetings nd provided their cdemic input. After the finl meeting held on the 13 th of Mrch dditionl genetic nlyses ws requested tht ws received on the 15 th of Mrch These results will be used towrds the completion of severl students involved in this study.

5 Finl report 5 Milestone Achievement 1. Recruitment 37 prticipnts completed the study 2. Biochemicl determintions Completed in 37 subjects 3. Genetic testing 8 SNPs performed 4. Sttisticl nlysis Completed Lipid Profile The most significnt finding relted to the response in the lipid profile ws the effect of lcohol consumption on HDL-cholesterol (good cholesterol) levels. Both the wine nd brndy (p= ) intervention resulted in significnt increse in HDL-cholesterol (Figure 1), known to hve crdiovsculr protective effect tretment; LS Mens Current effect: F(2, 66)=25.930, p= Verticl brs denote 0.95 confidence intervls b 1.6 c HDL bseline brndy wine tretment Figure1. Effect of lcohol consumption on HDL-cholesterol levels. The HDL-cholesterol rising effect vried significntly ccording to Apo E genotype (llele E4 p=0.027, llele E2 p=0.51). The HDL-cholesterol levels incresed with lcohol intke in both the presence nd bsence of the Apo E4 llele. In the group tht consumed brndy first the HDL-cholesterol levels incresed throughout the intervention period irrespective of Apo E genotype. However, in the group tht consumed wine first, it seems tht this lcohol effect ws suppressed in the Apo E4-positive study group. In the wine-first group without the Apo E4 llele HDL-cholesterol levels incresed while the brndy hd no further effect. Although HDL-cholesterol rising effect of lcohol ws slightly modified by genetic vrition in the MTHFR gene (p=0.048), the HDL-cholesterol rising effect ws confirmed in both muttionpositive nd negtive individuls with lcohol intke (Figure 2).

6 Finl report MTHFR 677 SNP PANEL*tretment; LS Mens Current effect: F(2, 66)=3.1632, p= Verticl brs denote 0.95 confidence intervls HDL bseline brndy wine tretment MTHFR 677 SNP PANEL WT MTHFR 677 SNP PANEL MU Figure 2. Effect of lcohol consumption on HDL-cholesterol levels in reltion to genetic vrition on the MTHFR gene. An importnt finding ws tht the sttisticlly significnt increse in totl cholesterol levels observed fter the lcohol intervention (p=0.034) (Figure 3) ws only found in individuls with the C282Y nd/or H63D muttions in the HFE gene (p=0.016) (Figure 3b). 6.4 tretment; LS Mens Current effect: F(2, 66)=3.5649, p= Verticl brs denote 0.95 confidence intervls CHOLESTEROL b b bseline brndy wine tretment Figure 3. Effect of lcohol consumption on totl cholesterol levels.

7 Finl report HFE*tretment; LS Mens Current effect: F(2, 66)=4.4227, p= Verticl brs denote 0.95 confidence intervls b CHOLESTEROL c cb cb cb bseline brndy wine tretment HFE MU HFE WT Figure 3b. Effect of lcohol consumption on totl cholesterol levels in reltion to genetic vrition on the HFE (muttions C282Y nd H63D) gene. An increse of triglyceride levels ws observed with lcohol intke, which ws only seen in HFE muttion-positive individuls (p=0.02) (Figure 4). This finding demonstrtes n importnt gene-environment interction nd my explin why some individuls in the generl popultion, but not ll, experience n increse in triglyceride levels with lcohol intke HFE*tretment; LS Mens Current effect: F(2, 66)=4.0338, p= Verticl brs denote 0.95 confidence intervls log10(triglyceride) b b b b bseline brndy wine tretment Figure 4. Effect of lcohol consumption on triglyceride levels in reltion to genetic vrition on the HFE (muttions C282Y nd H63D) gene. HFE MU HFE WT The results of the blood biomrkers indictive of ntioxidnt ctivity nd content, lipid peroxidtion nd redox sttus (GSH:GSSG) re shown in Tble 1. When considering the plsm ntioxidnt cpcity (ORAC) nd the level of totl polyphenols, no significnt chnges fter the wine or brndy interventions were detected when compred to the bseline. This could be expected s the plsm used ws fsting smples nd polyphenolic compounds hve reltive short hlf life (1-5 hrs) in the plsm. No significnt chnges in

8 Finl report 8 plsm mrkers for lipid peroxidtion (CDs, TBARS) when compring the different interventions with the bseline were detected in this study. The level of totl glutthione ws significntly (P=0.038) decresed fter the brndy intervention when compred to the bseline (Figure 5), while the wine intervention did not cuse ny significnt decrese nd showed similr vlues compred to the bseline. Tble 1: Antioxidnt sttus nd oxidtive stress prmeters Group Conjugted dienes (umol/l) Bseline 128.1± 18.8 Wine ± 19.0 Brndy ± 20.7 TBARS (umol/l) 0.89 ± ± ± 0.17 tgsh (µmol/l) 1006 ± ± ± 144b (P= GSSG (µmol/l) 18.8 ± ± 14.1b (P=0.019) 27.4 ± 23.1b (P=0.0004) GSH:GSSG rtio ORAC (µmol/l) 145 ± ± ± ± 63b (P=0.0009) 76 ± 90b (P=0.0002) 6147 ± ± ± ± 16 Totl polyphenols (mg/l) Vlues in columns re verges ± SD. Vlues followed by the sme letter indictes P>0.05 (non-significnce), while when letters differ indictes P<0.05 (significnce) tretment; LS Mens Current effect: F(2, 64)=7.0604, p= Verticl brs denote 0.95 confidence intervls GSH (µmol/l) b 880 bseline brndy wine tretment Figure 5. Effect of brndy nd wine interventions on totl glutthione levels in the blood compred to bseline levels Significntly incresed levels of oxidised glutthione (GSSG) were shown for both interventions when compred with the bseline (Figure 6), thus cusing significnt decrese in the rtio of GSH: GSSG (n indictor of oxidtive stress sttus).

9 Finl report tretment; LS Mens Current effect: F(2, 64)=7.0067, p= Verticl brs denote 0.95 confidence intervls 1.5 log10(gssg (µmol/l)) b bseline brndy wine tretment Figure 6. Effect of brndy nd wine interventions on oxidised glutthione levels in the blood compred to bseline levels. Whole blood GSH levels were mesured s it is n importnt circulting endogenous ntioxidnt. Reduced glutthione is powerful intrcellulr ntioxidnt tht plys vitl role in stbilizing vrious enzymes nd could lso be considered good mrker for tissue ntioxidnt cpcity [Wng & Jio, 2000; Vn Acker et l., 2000]. Severl clinicl conditions re ssocited with decrese in cellulr GSH levels tht my result in lowered cellulr redox potentil [Exner, et l., 2000]. A recent study by Cmpolo et l. [2007] lso suggested blood glutthione nlyses to be included in dietry supplementtion trils to ssess the thiol redox sttus especilly in chronic hert filure (CHF) ptients, s incresed free rdicl production in these ptients could result from bnormlities in intrcellulr GSH cycling, tht ws ssocited with incresed lipid peroxidtion (mesured s MDA) in CHF [Cmpolo et l., 2007]. In this study, it ws only the brndy consumption tht dversely ffected the level of glutthione nd not the red wine. No other increses in oxidtive stress prmeters could be shown fter brndy intervention. Previously, the bility of red wine to enhnce the red blood cell s reduced glutthione level (GSH) hs been reported (Urquig et l., 2010). References: Wng, S.Y.; Jio, H. Scvenging cpcity of berry crops on superoxide rdicls, hydrogen peroxide, hydroxyl rdicls nd singlet oxygen. J. Agric. Food Chem. 48: ; Vn Acker, F.; Schouten, O.; Henen, G.R.M.; Vn der Vijg, W.J.F.; Bst, A. Flvonoids cn replce R-tocopherol s ntioxidnt. Fed. Euro. Biochem. Soc. Lett. 473: ; Exner, R.; Wessner, B.; Mnhrt, N.; Roth, E. Therpeutic potentil of glutthione. Wien. Klin. Wochenschr. 112: ; Cmpolo, J.; De Mri, R.; Cruso.; Accinni, R.; Turzz, F.; Prolini, M.; Roubin, E.; De Chir, B.; Cighetti, G.; Frigerio, M.; Vitli, E.; Prodi, O. Blood glutthione s independent mrker of lipid peroxidtion in hert filure. Intl. J. Crdiol. 117:45-50; 2007.

10 Finl report 10 Urquig et l., 2010: Mediterrnen diet nd red wine protect ginst oxidtive dmge in young volunteers. Atheroclerosis: 21, Accumulted outputs List ALL the outputs from the strt of the project. The yer of ech output must lso be indicted. A set of stndrd operting procedures (SOPs) were developed for the SNPs nlysed, which formed prt of student trining: Yndisw Yko, registered PhD student. She ws ppointed nd reserch ssistnt for this project. The results of the study will be presented t n interntionl Winehelth Congress in Austrli during 2013 nd submitted for publiction in peer reviewed journl. Technology development, products nd ptents Indicte the commercil potentil of this project (intellectul property rights or commercil product(s)). The industry will benefit from the results of this study in the following res: The comprison of the effect of moderte red wine consumption to brndy on the therogenic lipoprotein profile, oxidtive stress nd inflmmtory sttus hs enbled us to document the effect of brndy in reltion to the well documented helth benefits of moderte wine consumption. The genetic screen of CVD risk fctors identified the proportion of individuls who my not benefit from drinking lcohol s consequence of gene-environment mismtches, bsed on the muttion/llele frequencies in the study popultion. With this informtion the presence of genetic risk fctors cn in future be correlted with relevnt biochemicl prmeters to compre gene expression in the presence nd bsence of known environmentl triggers. We were lso be ble to determine the impct of muttions/functionl polymorphisms included in the genetic screen on the response to the intervention to ultimtely develop guidelines for sfe drinking hbits. Humn resources development/trining Indicte the number nd level (e.g. MSc, PhD, post doc) of students/support personnel tht were trined s well s their cost to industry through this project. Add in more lines if necessry. Student level (BSc, MSc, PhD, Post doc) Cost to project (R) 1. Y Yko, PhD R (SOPs development, extrctions) 2. L Fisher, MSc R (ABI-lightcycler muttion screening 3. S Spgni, MSc R (THRIP project prticiption) 4. L Nutt, mtric R (dmin support, consent forms) 5.

11 Finl report 11 Publictions (populr, press releses, semi-scientific, scientific) Books: Vn Velden D P, Die Dokter s Vennoot Prote Uitgewers 2011 Articles: 1. vn Velden DP, Kotze MJ, Blckhurst D, Kidd M. Helth clims on the benefits of moderte lcohol consumption in reltion to genetic profiles. Journl of Wine Reserch, 2011, Vol 22, pp Vn Velden DP. Gesondheidsvoordele vn wyn is fhnklik vn interksie tussen gene en omgewing. Wynboer, Augustus 2010; Kotze MJ, vn Velden DP. Wr stn ons nou met lkohol en gesondheid? Wynboer, Oktober vn Velden DP vn der Merwe S, Fourie E, Blckhurst DM, Kidd M, Kotze MJ, Mnsvelt EPG. The influence of Mediterrnen-like diet with nd without red wine on ptients with the metbolic syndrome. S Afr J Enol Vitic. 2007;28 (1): Mnsvelt EPG, Fourie E, Blckhurst D, Kotze T, Stofberg H, vn der Merwe S. Kotze MJ, vn Velden DP. The influence of Mediterrnen Diet with nd without red wine on the hemosttic nd inflmmtory prmeters of subjects with the metbolic syndrome. S Afr J Enol Vitic. 2007;28 (1): Presenttions/ppers delivered 4 th Interntionl Wine nd Helth Conference, 3-6 October 2010, Friuli, Itly. Invited speker: Helth clims on the benefits of moderte lcohol consumption in reltion to genetic profiles

12 Finl report Totl cost summry of project Totl cost in rel terms for yer 1 Totl cost in rel terms for yer 2 Totl cost in rel terms for yer 3 Totl cost in rel terms for yer 4 Totl cost in rel terms for yer 5 TOTAL Yer CFPA Deciduous DFTS Winetech THRIP Other TOTAL