International Journal of Food Sciences and Nutrition, February 2011; 62(1): 5259

Effect of daily intake of yoghurt and bread enriched with biologically active substances on blood lipids and vitamin A in adolescents and young adults
HARALD K. WIDHALM1,5, KURT HERKNER2, INGRID KIEFER3, RUDOLF SEEMANN4, ANITA RIEDER3, MICHAEL KUNZE3, & KURT WIDHALM1
Division of Nutrition and Metabolism, Department of Pediatrics, Medical University of Vienna, Austria, Ludwig Boltzmann Institute, Department of Pediatrics, Medical University of Vienna, Austria, 3Institute of Social Medicine, Medical University of Vienna, Austria, 4Department of Cranio-Maxillofacial and Oral Surgery, Medical University of Vienna, Austria, and 5Center for Joints and Cartilage, Department of Traumatology, Medical University of Vienna, Austria
2 1

Abstract Objective: The aim was to investigate whether the daily intake of special nutrients, enriched with supplements from natural origins, has any effect on blood parameters. Design and subjects: In this double-blind placebo-controlled clinical trial, 80 healthy subjects (mean age 26.3 years) were statistically assigned to two groups. Group I had to eat two special yoghurt and bread products a day. The other probands represented the control group (II). Setting: Plasma concentrations of blood parameters were measured at the beginning and at the end of the study, and dietary intake was calculated. Results: In group I, total cholesterol decreased. This was due to a signicant drop of low-density lipoprotein-cholesterol from 106.0 to 99.0 mg/dl. A signicant reduction of the apolipoprotein B and an increase of vitamin A in group I were also observed. Conclusion: Regular intake of specially fortied food inuences parameters, especially lipids and lipoproteins.

Keywords: Nutrition, fortied food, young adolescents, blood lipids, vitamins

Introduction Food products enriched with nutritional supplements (vitamins, minerals, special fats) are increasingly being used to improve general nutrition and to achieve a higher level of health (Food and Drug Administration 1993). Due to their potential benecial health effects, these so-called functional foods are quickly gaining popularity. It has been claimed that many diseases can be prevented with the help of adequate nutritional support. For example, folic acidfortied grain products produced a drop in neural tube defects when administered to pregnant women (MRC Vitamin Research Group 1991). It was also shown that the incidence of cardiovascular diseases (Quinlivan et al. 2002) and cancer (Bailey et al. 2003) could have also been reduced by folic acid. As well as the above-mentioned studies, the most extensive study on the effects of folic acid fortication reported a 19% drop in the neural tube defects in the USA. Quinlivan and Gregory (2003) determined the estimated effect of folic acid fortication on the amount of folate consumed by persons in the USA. The study found that the increase in circulating folate concentrations was linearly related to folic acid intake over the range of 100 1,000 mg/day (P , 0.0001). Predicted increases in folic acid intake from fortied foods ranged from 215 to 240 mg/day. However,

Correspondence: Kurt Widhalm, Department for Nutrition and Metabolism, Medical University of Vienna, Waehringer Guertel 18-20, 1090 Vienna, Austria. Tel: 43 1 40 400/23 37. Fax: 43 1 40 400/23 38. E-mail: kurt.widhalm@meduniwien.ac.at
ISSN 0963-7486 print/ISSN 1465-3478 online q 2011 Informa UK, Ltd. DOI: 10.3109/09637486.2010.507186

Effect of daily intake on various parameters typical intakes of folic acid from fortied foods were more than twice the level originally predicted (Quinlivan and Gregory 2003). In addition to the benecial health effects of folic acid, other supplements have also been shown to have positive effects on health. The publication by Simon et al. (2001) highlighted a decrease of vascular disease incidence and mortality due to vitamin E when compared with ascorbic acid supplementation. Alongside the positive effects of folic acid mentioned earlier, other benecial health effects from vitamin E have been observed. Data by Bostick et al. (1993) found that vitamin E supplementation was associated with a lower risk of cancer, especially of the colon. Recently, a large prospective cohort study (88,758 women) has proved that a combination of folate and multivitamins (as supplements), similar to the supplementation used in the present study, is associated with a signicant reduction in the risk of colon cancer. Interestingly, however, the opposite effect was found with folate intake, where a direct inverse correlation was noted with respect to a reduction in cancer (Fuchs et al. 2002). Aside from these remarkable and scientically precious ndings, which have been achieved through the implementation of functional foods, the aim of this present study was to test functional foods fortied with vitamins, minerals, saturated and unsaturated fatty acids and bioactive substances and their ability to provoke positive changes in routine blood parameters and vitamin levels in healthy adolescents. Special interest should be given to any changes in lipid metabolism. Subjects (n 80) were compared on the basis of 38 blood parameters, which were randomly assigned to control and study groups, before and after a 2-month period. Subjects and methods Participants

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The cohort consisted of 46 healthy women aged between 20 and 36 (26.7 ^ SD 3.0 years), and 34 healthy men aged between 19 and 34 (26.7 ^ SD 3.8 years). All participants were recruited at the Medical University of Vienna. Members of the medical staff of the General Hospital of Vienna were given preference for participation in the study as their location allowed easy access to collect the weekly food package from the study center, which was based at the General Hospital of Vienna. Subjects were invited to a pre-study information lecture where they received detailed information about the study and what was expected of them. The mean initial body weight was 67.1 ^ 11.9 kg. Baseline data of the subjects is presented in Tables I and II.

Study design and duration This was a single-center, diet and placebo-controlled, randomized double-blind trial with duration of 2 months. At the beginning of the study, the subjects were required to complete a clinical test, including blood and urine tests, medical history, and a general questionnaire of close-ended questions regarding sociodemographic characteristics such as smoking, drinking, and socioeconomic backgrounds. Exclusion criteria included allergies concerning any special foods or intolerances, familial disorders of metabolism, diabetes mellitus type I/II, disorders of liver or kidney, viral infections, inuenza, and pregnancy. Furthermore, the subjects were advised not to take any drug or vitamin supplements during the 2-month study period.

Table I. Baseline demographic data and characteristics of 80 healthy subjects. Group I (verum)(n 40, 50%) Age (years) Body mass index (kg/m2) Total cholesterol (mg/dl) HDL-C (mg/dl) LDL-C (mg/dl) VLDL-C (mg/dl) Triglycerides (mg/dl) Lp(a) (mg/dl) Data shown as mean ^ standard deviation. 25.7 ^ 3.4 23 ^ 3.5 190.7 ^ 35.1 64.6 ^ 17.5 106 ^ 34.2 20.3 ^ 7.6 101.8 ^ 37.9 35.3 ^ 38.5 Group II (placebo) (n 40, 50%) 25.8 ^ 3.4 21.5 ^ 2.8 180.8 ^ 27 62.9 ^ 16.8 101.2 ^ 25.8 16.7 ^ 7.6 83.3 ^ 38.3 48.1 ^ 39.4 P value 1.000 0.253 0.986 1.000 1.000 0.242 0.221 1.000

Table II. Anthropometric data before and after 8 weeks study participation: comparison of group I (verum) and group II (placebo). Total Begin Weight (kg) BMI (kg/m2) 67.1 ^ 11.9 22.3 ^ 3.2 End 67.6 ^ 11.9 22.4 ^ 3.2 P value 0.060 0.301 Begin 69.6 ^ 12.9 23.0 ^ 3.5 Group I (verum) End 70.1 ^ 12.9 23.2 ^ 3.3 P value 0.455 0.455 Begin 64.5 ^ 10.3 21.5 ^ 2.8 Group II (placebo) End 65.1 ^ 10.4 21.6 ^ 2.9 P value 0.104 0.455

Data shown as mean ^ standard deviation. BMI: body mass index.

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H. K. Widhalm et al. subjects had to complete a questionnaire about their well-being and compliance. Body weights were recorded (indoor clothing without shoes). The body mass index was calculated and blood pressure was measured bilaterally. Laboratory analyses Blood samples were collected after a 12-h overnight fasting period at the beginning and the end (2 months later) of the study. Samples were then centrifuged at 48C for 10 min at 3,000 rpm. The plasma drawn was stored in tubes containing benzamidine (0.03%) at 48C until processed. Blood measurements included iron, glucose, complete blood count, creatinine, uric acid, total bilirubin, total glutamate-oxalacetatetransaminase, glutamate-pyruvate-transaminase, and g-glutamyltransaminase, by means of the usual methods. Lipid analysis included total cholesterol, triglycerides, high-density lipoprotein (HDL-C), lowdensity lipoprotein (LDL-C), very-low-density lipoprotein (VLDL-C), lipoprotein(a), and apolipoprotein B (apoB). Total cholesterol and triglycerides were measured after applying an enzymatic preparation on an autoanalyzer using special test kits, described by Allain et al. (1974). After the precipitation and removal by centrifuge of LDL-C and VLDL-C, HDL-C was separated and measured using colorimetry. LDL-C levels were calculated using the Friedewald formula (Friedewald et al. 1972), and lipoprotein(a) concentrations were determined using the immunological tarnish test, which assesses the quantitative in vitro values of lipoprotein(a) with clinical and chemical analyzers (Passing and Bavlok 1983, Bablok et al. 1988). Serum concentrations of apoB were measured by an immunoturbidimetric method. Vitamins Vitamins were measured by the high-performance liquid chromatography (HPLC) method, a process that simultaneously determines levels of vitamins in serum and whole blood. A determination of both vitamin A and E in serum with high-performance liquid chromatography was carried out in a reversed-phase column with a mobile phase of acetonitrile methanol HPLC water (48:48:4) and a ow-rate of 0.8 ml/min. The vitamins were monitored at 290 nm, as described by Arnaud et al. (1991) (100 ml injections/temperature: 358C). Vitamin B1 analysis was carried out using a uorescence detector at an excitation wavelength of 365 nm and an emission wavelength of 433 nm. The evaporated organic layer was reconstituted with a mobile phase of 10% tetrahydrofuran and a ow-rate of 0.5 ml/min (Bettendorff et al. 1986). Vitamin B2 analysis was performed at an excitation wavelength of 470 nm and an emission wavelength of

The study was approved by a local ethics committee (Medical Faculty, University of Vienna) and written consent was obtained from all subjects. After passing the physical examination and questionnaire, the participants were asked to record their eating habits for 3 days. Venous blood for testing was drawn after a 12-h overnight fast between 8:00 and 11:00 am in the morning in a sitting or supine position. According to the study design, numbers of subjects were statistically assigned to two groups: the supplemented group (group I), and the placebo control group (group II). Based on their assignment, the subjects were instructed to consume, as part of their regular meal, 2 x 150 g yoghurt and 2 x 80 g bread from the provided food package, everyday during the 2-month study period. Subjects and supervisors were blind as to group assignment and food packages were collected weekly. Ingredients of both supplemented and control products are presented in Table III. Subjects were instructed to store the yoghurt products in a refrigerator, and the bread products at a room temperature of lower than 188C. During the pickups,
Table III. Nutrient content of the study products of 300 g yoghurt and 180 g bread product: comparison of group I (verum) and group II (placebo). Group I (verum) products Water (g) Protein (g) Fat (g) Carbohydrate (g) Monosaccharides, disaccharides (g) Bread units Organic acids (g) Mineral nutrients (g) Energy (kcal) Energy (kJ) Dietary ber (g) Fatty acids Saturated(g) Monounsaturated (g) Polyunsaturated (g) Bioactive substances Phytosterols (mg) Carotinoids (mg) Bioavonoids (mg) Vitamins Vitamin C (mg) Vitamin D (mg) Vitamin E (mg) Total tocopherol (mg) a-Tocopherol (mg) Total tocotrienol (mg) Vitamin B1 (mg) Vitamin B2 (mg) Vitamin B6 (mg) Pantothen acid (mg) Folic acid (mg) Vitamin K1/3 (mg) Niacin (mg) Data given as absolute counts. 268.2 27.4 13.6 127.3 40.6 10.4 2.5 3.0 749.8 3134.0 9.6 3.2 2.5 5.7 165.3 1.52 346.3 75.5 0.06 9.3 13.2 8.2 4.9 0.5 0.9 0.5 1.9 215.5 43.8 1.6 Group II (placebo) products 286.8 26.3 5.2 133.7 40.5 11.1 2.7 3 696.6 2911.8 8.1 2.3 0.9 0.5 1.76 1.32 0 75 0.06 0.25 0.52 0.2 3.5 0.4 0.7 0.4 1.4 110.7 0.8 0.4

Effect of daily intake on various parameters 525 nm. The ow-rate was 1.5 ml/min with an injection volume of 100 ml and a pH of 2.9. The peak heights were plotted and integrated automatically, as described by Speek et al. (1982). Vitamin B6 analysis was carried out at an excitation wavelength of 300 nm and an emission wavelength of 400 nm. The ow-rate was 0.5 ml/min with an injection volume of 100 ml and a pH of 2.9 (Deitrick et al. 2001). Ascorbic acid levels were determined at a ow-rate of 0.75 ml/min with the mobile-phase of ammonium dihydrogenphosphate, monitored by 245 nm and an injection volume of 30 ml (Ruemelin et al. 1999). The HPLC analysis was performed isocratically by using a mobile phase of 20%. The peak heights were plotted and integrated automatically. Malonyldialdehyde levels in the urine were measured after extraction. Malonyldialdehyde was extracted from hydrolyzed urine using C18 solidphase extraction columns. The extract was analyzed by reversed-phase HPLC with electrochemical detection at a cell potential of 0.8 V (Peterson et al. 1991). Statistical analysis

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Analysis of variance models of the pre post differences of each parameter, using the baseline value and the group as explaining variables, were established. A P value under 5% was considered to indicate statistical signicance, given the high number of tests performed. A Bonferroni correction suggests that a signicant result should have a P value under 0.0013.

Results A total of 80 subjects consumed special study products as part of their regular meals over a 2-month period. Four subjects were excluded: one had gastrointestinal problems and three (two men and one woman) did not like the taste of the study products, and did not participate further in the study. Mean initial total cholesterol and LDL-C concentrations of all 80 subjects, 34 men and 46 women, were 185.7 mg/dl and 103.6 mg/dl, respectively. HDL-C mean levels were 63.7 mg/dl, the triglycerides had mean values of 92.5 mg/dl, which means that both were in the respective reference range. The coefcient for the baseline value was signicant in several of the models. Three parametersLDL-C, apoB and vitamin Ahad a P value under 5%. However, none of these mentioned parameters achieved statistical signicance under the Bonferroni-corrected level. At the end of the study period, total cholesterol decreased by 11.3 mg/dl from 190.7 ^ 35.1 mg/dl to 179.4 ^ 29.1 mg/dl, as well as LDL-C by 7.0 mg/dl from 106.0 ^ 34.2 to 99.0 ^ 27.4 mg/dl; the LDL-C values decreased signicantly in the supplemented group (group I). However, in group II a slight increase of total cholesterol by 1.3 mg/dl from 180.8 ^ 27.0 mg/dl to 182.1 ^ 28 mg/dl and of LDLC by 2.7 mg/dl from 101.2 ^ 25.8 mg/dl to 103.9 ^ 27.6 mg/dl was observed (Table IV). HDL-cholesterol decreased by 4.1 mg/dl without signicance from 64.6 ^ 17.5 mg/dl to 60.5 ^ 14.9 mg/dl in group I, as well as in group II by 2.5 mg/dl from 62.9 ^ 16.8 mg/dl to 60.4 ^ 11.4 mg/dl (not signicant). Triglycerides did not change substantially (101.8 ^ 37.9 mg/dl vs. 100.0 ^ 28.9 mg/dl) in group I; however, increased insignicantly by 6.3 mg/dl from 83.3 ^ 38.3 mg/dl to 89.6 ^ 42.1 mg/dl in group II. Vitamin A concentrations rose from 1.6 ^ 0.4 mg/l to 1.7 ^ 0.4 mg/l in group I (P , 0.05), whereas the values in group II decreased by 0.2 mg/l from 1.6 ^ 0.3 mg/l to 1.4 ^ 0.5 mg/l. The lipoprotein apoB dropped by 3.3 mg/dl from 71.4 ^ 17.3 mg/dl to 68.1 ^ 16.2 mg/dl in group I (P , 0.04), and increased by an insignicant amount of 1.4 mg/dl from 66.7 ^ 17.1 mg/dl to 68.1 ^ 17.7 mg/dl in group II.

Composition of study products (yoghurt & bread) The base of the study products was a supplement consisting of wheatgrass germs as well as a number of complex vitamins. These products contain so-called panvital nutrients, which include vitamins, minerals, and trace elements. Regarding their natural function, these products complement each other; they are able to evolve into a special density of nutrients. PMN is extracted from germ corn, biodynamically farmed and not genetically engineered. The effect of sunlight and dynamic water results in complex vital substances, which are getting concentrated to PMN. Yoghurt, either supplemented or unsupplemented, and special compounded bread, supplied by the producer, were distributed weekly and stored below 188C at the study center and at home by the subjects as instructed. The composition of the control and supplemented products are presented in Table III.

Food intake Three-day dietary diaries were obtained using a quantitative food frequency questionnaire. The subjects were asked to complete a questionnaire concerning their daily eating habits and were requested to document what they ate for the 3 days before the commencement of the study as well as for the last 3 days of the study period. The recall method crosschecked reported food intakes during those 3-day periods. The food intake records during those 3 days were similar for most subjects at the beginning and end of the study.

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H. K. Widhalm et al.

Table IV. Mean changes in blood lipid and vitamin concentrations and anthropometric data after 8 weeks study participation: comparison between group I (verum) and group II (placebo). Group I (verum) (n 40) Begin Total cholesterol (mg/dl) Triglycerides (mg/dl) HDL-C (mg/dl) LDL-C (mg/dl) VLDL-C (mg/dl) Lp(a) (mg/dl) ApoB (mg/dl) Vitamin A (mg/l) Ascorbic acid (mg/l) Body mass index (kg/m2) Weight (kg) 190.7 ^ 35.1 101.8 ^ 37.9 64.6 ^ 17.5 106 ^ 34.2 20.3 ^ 7.6 35.3 ^ 38.5 71.4 ^ 17.3 1.6 ^ 0.4 20.3 ^ 5.3 23 ^ 3.5 69.6 ^ 12.9 End 179.4 ^ 29.1 100 ^ 28.9 60.5 ^ 14.9 99 ^ 27.4 20.1 ^ 5.9 34.4 ^ 37.4 68.1 ^ 16.2 1.7 ^ 0.4 24.1 ^ 5.3 23.2 ^ 3.3 70.1 ^ 12.9 Group II (placebo) (n 40) Begin 180.8 ^ 27 83.3 ^ 38.3 62.9 ^ 16.8 101.2 ^ 25.8 16.7 ^ 7.6 48.1 ^ 39.4 66.7 ^ 17.1 1.6 ^ 0.3 19.6 ^ 3.3 21.5 ^ 2.8 64.5 ^ 10.3 End 182.1 ^ 28 89.6 ^ 42.1 60.4 ^ 11.4 103.9 ^ 27.6 17.9 ^ 8.4 46.3 ^ 35.8 68.1 ^ 17.7 1.4 ^ 0.5 20.7 ^ 6.7 21.6 ^ 2.9 65.1 ^ 10.4 P value 0.12 0.66 0.77 0.02 0.78 0.51 0.02 0.04 0.26 0.82 0.77

Data shown as mean ^ standard deviation. P , 0.05 is signicant.

The malonyldialdehyde concentration decreased by 0.1 mmol/l from 1.1 ^ 0.4 mmol/l to 1.0 ^ 0.5 mmol/l in group I, whereas no changes were seen in group II. The lp(a) levels dropped slightly in both groups, but were also insignicant. All the values of the parameters, which have been examined in this study, are listed in Table V.

Diet of subjects Dietary habits showed distinct changes, particularly an increased intake of calcium, magnesium, manganese and iron over both groups. An increase in calorie intake was seen in both groups. In the supplemented group (group I) there was an increase of 84 kcal/day from 2,040 kcal/day to

Table V. Mean changes of special parameters after 8 weeks study participation: comparison between group I (verum) and group II (placebo). Group I (verum) (n 40) Begin RBC (106/ml) MCHC (g/dl) MCV (f/l) MCH (pg) HCT (%) PLT (103/ml) Neutrophilic granulocytes (103/ml) Leukocytes (103/ml) Lymphocytes (103/ml) Monocytes (%) Eosinophilic granulocytes (103/ml) Basophilic granulocytes (103/ml) Reticulocytes (%%) Iron (mg/dl) Transferritin (mg/dl) Ferritin (ng/ml) Glucose (mg/dl) Kreatinin (mg/dl) Uric acid (mg/dl) Total bilirubin (mg/dl) Total protein (g/dl) GGT (U/l) GOT (U/l) GPT (U/l) Vitamin B2 (nmol/l) Vitamin B6 (mg/l) Vitamin C (mg/l) Vitamin E (mg/l) MDA (mmol/l) 4.9 ^ 0.5 34.5 ^ 0.9 86.7 ^ 4.3 29.9 ^ 1.6 42.3 ^ 3.9 248.5 ^ 49.0 2.9 ^ 0.9 5.5 ^ 1.2 2 ^ 0.5 8 ^ 1.9 0.2 ^ 0.1 0.03 ^ 0 10.3 ^ 3.7 119.5 ^ 38.2 291.4 ^ 43.3 40.4 ^ 30.8 78.3 ^ 6.5 0.9 ^ 0.1 4.9 ^ 1.4 0.7 ^ 0.5 7.6 ^ 0.5 12.2 ^ 4.5 9 ^ 2.1 12.1 ^ 5.7 191.2 ^ 33 13.2 ^ 5.9 20.3 ^ 5.3 9.5 ^ 5.9 1.1 ^ 0.4 End 4.9 ^ 0.4 34.2 ^ 1.0 87.4 ^ 4.3 29.9 ^ 1.6 42.6 ^ 3.6 251.6 ^ 48.1 3.2 ^ 1.5 5.9 ^ 1.7 2.1 ^ 0.6 7.9 ^ 1.9 0.2 ^ 0.1 0.03 ^ 0 10.3 ^ 3.5 126.1 ^ 37.8 302.7 ^ 56.3 41.8 ^ 31.4 81.1 ^ 7.8 0.9 ^ 0.1 4.7 ^ 1.3 0.8 ^ 0.6 7.6 ^ 0.4 12.2 ^ 4.8 9.2 ^ 2.0 15.5 ^ 5.8 179.8 ^ 19.6 14.6 ^ 5.2 24.1 ^ 3.3 11.5 ^ 6.5 1 ^ 0.5 Group II (placebo) (n 40) Begin 4.7 ^ 0.4 34.6 ^ 0.8 87.2 ^ 3.6 30.1 ^ 1.4 41 ^ 3.5 241.2 ^ 56.7 3 ^ 1.2 5.6 ^ 1.8 2 ^ 0.6 8.4 ^ 2.4 0.1 ^ 0.2 0.03 ^ 0 10 ^ 2.6 126.2 ^ 47.8 284.9 ^ 52.1 48.9 ^ 41.5 79.1 ^ 5.8 0.8 ^ 0.2 4.9 ^ 1.4 0.7 ^ 0.3 7.6 ^ 0.5 12.7 ^ 13.2 9.2 ^ 2.7 12.8 ^ 8.1 175.6 ^ 38.7 11.3 ^ 5.2 19.6 ^ 5.3 7.8 ^ 5.2 1 ^ 0.5 End 4.7 ^ 0.5 34.2 ^ 0.8 87.9 ^ 3.6 30.1 ^ 1.4 41.6 ^ 4.1 239.8 ^ 49.4 3.1 ^ 1.5 5.7 ^ 1.8 2 ^ 0.5 8.1 ^ 1.9 0.4 ^ 1.8 0.03 ^ 0 10.4 ^ 3.3 125.7 ^ 57.3 299.2 ^ 50.4 43.3 ^ 36.1 85.1 ^ 18.8 0.8 ^ 0.1 4.6 ^ 1.3 0.6 ^ 0.4 7.6 ^ 0.4 11.5 ^ 8.9 8.8 ^ 2.5 15 ^ 6.6 178.7 ^ 30.3 12.8 ^ 5.2 20.7 ^ 6.7 9.4 ^ 5.5 1 ^ 0.5 0.58 0.94 0.88 0.77 0.76 0.06 0.56 0.81 0.43 0.15 0.53 0.5 0.23 0.61 0.4 0.74 0.99 0.99 0.31 0.9 0.94 0.2 0.6 0.24 0.96 0.28 0.26 0.79 0.93 P value

Data shown as mean ^ standard deviation. RBC, red blood count; MCHC, mean corpuscular hemoglobin concentration; MCV, mean corpuscular volume; MCH, mean corpuscular hemoglobin; HCT, hematocrit; PLT, platelet; GGT, g-glutamyltransferase; GOT, glutamateoxalacetate-transaminase; GPT, glutamate-pyruvate-transaminase; MDA, malonyldialdehyde.

Effect of daily intake on various parameters

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Table VI. Mean changes in supply of special nutrients: 3 days before study and the last 3 days of participation in the study: comparison between group I (verum) and group II (placebo). Group I (verum) (n 40) Begin Energy supply (kcal/day) Protein supply (g/day) Fat supply (g/day) Fat supply: SAFA (g/day) Fat supply: MUFA (g/day) Fat supply: PUFA (g/day) Omega 3-fatty acids (g/day) Carbohydrate (g/day) Vitamin E (mg/day) Vitamin B1 (mg/day) Vitamin B2 (mg/day) Folic acid (mg/day) Vitamin C (mg/day) Calcium (mg/day) Magnesium (mg/day) Zinc (mg/day) Manganese (mg/day) Vegetables (g/day) Fruits (g/day) Alcohol (g/day) 2040.0 69.4 78.3 33.6 27.4 12.7 1.2 239.7 11.8 1.1 1.4 289.0 104.0 894.0 312.0 10.4 4.9 104.0 419.0 10.6 End 2124.0 76.0 78.3 33.1 26.1 14.0 1.6 262.6 16.5 1.2 1.8 454.0 151.1 989.0 342.0 11.3 5.8 88.0 312.0 6.6 Group II (placebo) (n 40) Begin 2075.0 73.1 80.5 33.9 26.4 13.6 1.3 247.2 13.2 1.1 1.4 288.0 111.0 877.0 307.0 10.3 4.9 120.0 361.0 6.1 End 2267.0 83.3 77.3 32.0 25.4 13.1 1.5 289.6 10.9 1.2 1.7 344.0 168.8 950.0 313.0 11.0 5.3 79.0 231.0 7.8 P value 0.05 0.81 1.000 0.57 1.000 1.000 1.000 0.79 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 0.59 1.000 1.000

Data shown as mean counts. SAFA, saturated fatty acids; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids.

2,124 kcal/day, and in the control group (group II) of 192 kcal/day from 2,075 kcal/day to 2,267 kcal/day. The protein intake also rose in both groups to about 10 g/day compared with initial values, whereas the fat intake did not change signicantly. In group I the supply of carbohydrates increased by 22.9 g/day from 239.7 g/day to 262.6 g/day, and in group II by 42.4 g/day from 247.2 g/day to 289.6 g/day. Whereas in group I a decrease in alcohol consumption was seen, 10.6 g/day vs. 6.6 g/day, there was a rise in group II from 6.1 g/day to 7.8 g/day. Furthermore, subjects from both groups consumed greater amounts of vitamin B1, vitamin B2, folic acid, vitamin C, calcium, magnesium, zinc and manganese. All values are listed in Table VI. The mean weight of all participants increased slightly from 67.1 to 67.6 kg (not signicant). The amount of weight gained was similar in both groups (Table II). During the study period, none of the subjects reported any serious side effects that could be related to the dietary products. Discussion The present study shows that college-age (19 36 years) women and men who are consuming products that are supplemented with a combination of monounsaturated and polyunsaturated fatty acids, tocopherol, bioavonoids, folic acid, and so forth are able to achieve signicant changes of certain blood parameters, especially a reduction of LDL-C, apoB, and an increase of vitamin A.

The fact, however, that a daily intake of yoghurt and bread supplemented with vitamins, within a normal range, leads to a rise of mean serum levels of ascorbic acid and vitamin B12 is of importance because such an effect might be associated with an improved cognitive function and work performance. In this regard, it has been shown by Duthie et al. (2002) in a longitudinal observational study based on brain aging and health (334 elderly individuals) that, in the elderly, blood concentrations of folate and vitamin B12 are positively associated with cognitive ability. Although mean total cholesterol and LDL-C levels were within normal ranges at the commencement of the study, a signicant decrease was seen in the supplemented group, after the 2-month duration of the study. In a similar study, Baro et al. (2003) investigated the effect of supplementing the diet with a mixture of n-3 polyunsaturated fatty acids, oleic acid, vitamin E, vitamin B6, and folic acid in a group of 15 men and 15 women with a mean age of 33.1 years. They focused on the risk factors associated with cardiovascular disease. The authors found a signicant decrease of 9.7 mg/dl in total cholesterol from 176.0 ^ 3.8 mg/dl to 166.3 ^ 4.2 mg/dl and a decrease in LDL-C concentration by 14.7 mg/dl from 90.9 ^ 4.6 mg/dl to 76.2 ^ 4.2 mg/dl, also reaching a level of signicance. However, HDL-C concentration increased insignicantly by 3.9 mg/dl from 63.8 ^ 2.7 mg/dl to 67.7 ^ 3.1 mg/dl, which is the main difference compared with our study results (Baro et al. 2003).

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H. K. Widhalm et al. Acknowledgements The study was planned by K.W., for the most part, and H.K.W., who was mainly responsible for the correct processing of the study. The laboratory analysis was carried out under observation of K.H. and evaluations of the 3-day dietary diaries and calculations were done by I.K. and M.K. H.K.W. contributed to the tables and the data content of the paper. Declarations of interests: The study was supported by a research grant provided by vis Vitalis-Salzburg. One position for a physician (H.W.) and for a technician (C.W.) has been supported by the company.

Yoghurts containing special bacteria (Streptococcus thermophilus) Causidow culture have also been shown to have a LDL-C-lowering effect in a group of subjects consuming 450 ml daily for 8 weeks (AgerholmLarsen et al. 2000b). A further study concluded that fermented yoghurt products promote up to a 4% decrease of total cholesterol and a 5% decrease in LDL-C in 70 healthy, overweight and obese individuals of 18 55 years age. However, it must be noted that an increase of brinogen was also documented, which cannot be regarded as a benecial effect (Agerholm-Larsen et al. 2000a). The effect of yoghurts, which contain special bacteria (Lactobacillus acidophilus) and fructo-oligosaccharides, on blood lipids has been studied by Schaafsma et al. (1998). They were able to show, in a double-blind crossover study, a signicant cholesterol-lowering effect of the fermented dairy products supplemented with fructo-oligosaccharides, as compared with traditional yoghurt with a fat content of 1% in 30 healthy men, aged between 33 and 64 years. A decrease in the LDL-C/HDL-C ratio could be interpreted as a decreased atherogenicity. Nonetheless, in that specic study, HDL-C remained unchanged (Schaafsma et al. 1998). This cholesterol-lowering effect has again been demonstrated in 32 patients who were suffering from mild to moderate primary hypercholesterolemia, aged between 36 and 65 years. A signicant mean reduction of 5.3% in total cholesterol (P , 0.05), 6.1% for LDL-C (P , 0.05) and no signicant change in HDL-C and triglycerides, achieved by fermented milk products, was seen (Bertolami et al. 1999). Interestingly, returning back to the present study, an increase of ascorbic acid and iron levels was shown, although neither study product contained these nutrients. Due to the fact that the nutritional supplement was a mixture of natural bioactive substances, it has been speculated that the supplement was able to increase the absorption of these substrates.

References
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Conclusion The results of the present study show that the daily intake of two special bread and yoghurt products, enriched with special biological active substances, leads to a demonstrable improvement of several important blood parameters. Thus, the risk for coronary vascular disease, and possibly cancer as well, can be reduced through an adequate nutritional diet, as has been seen previously. Nonetheless, supplementation is commonly practiced by younger, well-nourished individuals as well as the elderly. As such, further research with wellnourished individuals is needed to substantiate possible metabolic and functional benets.

Effect of daily intake on various parameters

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