Manuscript: Effects of Refined and Unrefined Corn Oil
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    Research project in the advanced nutrition course at SDSU
This was a research project in the Advanced Nutrition lab at San Diego State University. The students each had to research, design and budget a research project. Then, once a project was chosen, the students were participants in the study. This project is the completed manuscript that was prepared to be submitted to The American Journal of Clinical Nutrition. 
Effects of Refined andUnrefined Corn Oil on Blood Lipids, Oxidation and Glucose Metabolism
Amanda M. Hibshman and Mayumi Petrisko
San Diego State University
5500 Campanile Drive
San Diego, CA 92182
Funded by student lab fees for the Advanced Nutrition Lab
Keywords: Corn Oil, Cholesterol, LDL Cholesterol, Oxidized LDL, Olive Oil, Health Benefits


Many studies have looked at the effects of virgin olive oil and have compared it to its refined counterpart. Studies have concluded that the refined oil is nutritionally better, mainly due to its polyphenol content. The purpose of this study was to examine the benefits of corn oil and it was hypothesized that unrefined corn oil would have greater health benefits than refined. The study was a randomized, double-blind, cross-over design that consisted of two 2 week dietary intervention trials that were separated by a two week washout period. For one trial, participants consumed 40g of unrefined corn oil daily while participants consumed 40g of refined corn oil daily for the other trial. Antioxidants were avoided and participants diets were monitored by food journals every other day of the intervention period. Total cholesterol, LDL-C, HDL-C, OxLDL, triglycerides, glucose and insulin were evaluated. Significance was found in the TC, LDL and OxLDL results, showing that the refined oil had a more dramatic effect on lowering TC, LDL and OxLDL. Both the refined and unrefined corn oils had a beneficial effect. The unrefined corn oil was not significant, however the refined was significant. There was no significant effect on HDL, TG, glucose or insulin. This study demonstrates that the refining process does not diminish the health benefits of corn oil, like it does with olive oil.

The Mediterranean diet has been increasing in popularity because of its effects on preventing cardiovascular disease and myocardial infarctions because of its positive effects on serum cholesterol 1,2, 3. One of the main components of the Mediterranean diet is the wide use of olive oil, which is an oil that contains 74% monounsaturated fatty acids 4. Health benefits have been linked to the abundance of MUFA in Olive Oil, but many studies have also investigated the effects of the polyphenols in unrefined olive oil. The refining process diminishes the amount of polyphenols that are present in the olive oil 1,5.
Many studies have been conducted looking at the effects of refined olive oil against virgin olive oil. One study of healthy men found that unrefined olive oil decreased oxidized LDL and LDL more than the refined olive oil did 1. Another study, also looking at healthy men, consumed 25 ml daily of olive oil all with varying amounts of polyphenols (low, medium and high concentrations). The olive oil with the high and medium concentration of polyphenols (the less refined of the three) decreased oxidative damage, and the high polyphenol (the least refined) decreased LDL 5.
When olive oil is compared to corn oil, which has 24% MUFA and 58% PUFA 4, evidence is not conclusive. In one study of middle aged and elderly participants with elevated LDL levels, corn oil and olive oil were tested to see if there would be a significant change in blood lipid parameters. The study concluded that switching from saturated fat to either a MUFA or a PUFA was important, however there was no significance found between vegetable oils with differing concentrations of MUFA and PUFA 6. Another article compared corn oil to a mix of sunflower and olive oil. The corn oil proved to significantly decrease LDL, VLDL and TG, but had no effect on HDL 7.
There is such strong evidence that unrefined olive oil is a superior oil to its refined version 1,5. There are no studies that consider the effects of a different oil to its refined counterpart. Corn oil has been proven to beneficially influence serum cholesterol and LDL levels, but it is unknown if the benefits are greater in virgin corn oil compared to refined corn oil 8. This study will look to see if unrefined corn oil has greater health benefits tan refined corn oil. Blood lipids including TC, HDL-C, LDL-C, OxLDL and TG will be measure as well as blood glucose and insulin.
Unrefined corn oil is high in ubiquinone and vitamin E in the form of alpha- and gamma- tocopherols 8. Because of this, it is hypothesized that the unrefined corn oil will decrease OxLDL. The high MUFA content of both the refined and unrefined corn oils will cause an overall decrease TC, TG, LDL-C and raise HDL-C. It is expected to see more dramatic change in the unrefined oil. Glucose and insulin will be tested too, because the Mediterranean diet has been shown to improve glucose metabolism 3. It is expected that the unrefined corn oil will lower both serum insulin and serum glucose levels.
Research Design and Methods
Five male and 13 female subjects (ages 20-45 years, BMI: 18-40 kg/m 2) were recruited to participate in this study.  Prior to participation, all subjects were asked to complete a screening questionnaire to determine if they met the selection and exclusionary criteria. Exclusionary criteria included cigarette smoking, heart disease, metabolic disturbance diseases and chronic use of medications known to alter metabolism. Participants were asked to discontinue use of all vitamin and mineral supplements one week prior to and throughout the entire study.

Study Design
The study was a randomized, double-blind, cross-over design that consisted of two 2-week dietary intervention trials separated by a 2-week wash out period in between trials to allow for normalization. Subjects were instructed to maintain their normal diet and exercise regiments throughout the entire study and to avoid a high intake offoods containing antioxidants such as vegetables, legumes, fruit, tea, coffee, chocolate, wine, and beer. Dietary intake was monitored by completion of food records every other day during each trial period. Food records were analyzed using The Food Processor SQL (ESHA Research, Salem, OR USA).  During the two trial periods, each subject consumed two supplement shakes per day, separated by at least four hours. Each shake consisted of 20 grams of either refined or unrefined corn oil, 39 grams (2T) Hershey’s® chocolate syrup, and 240 ml non-fat milk.  Unrefined corn oil was provided by Spectrum (Boulder, CO USA) and refined corn oil was provided by ACH Food Companies, Inc (Memphis, TN USA). Prior to initiating the trials, subjects were instructed how to consume the supplement shakes and complete the food records. Height, weight, and body composition were also recorded. Body composition was measured by bioelectrical impedence using the InBody 520 (Biospace, Los Angeles, CA USA).
Biochemical Parameters
Fasting blood samples were obtained at the beginning and end of each trial. Subjects reported to the laboratory for blood draws between 7:30 am to 10:30 am after a 12-hour overnight fast. Subjects were instructed to refrain from consuming non-steroidal anti-inflammatory medications, caffeine, alcohol and from performing structured exercise for 24 hours prior to each blood collection. Subjects were weighed and sat quietly for 10 min before collection of the fasting blood samples. Blood samples were drawn by venipuncture from the antecubital region and collected into a Vacutainer SST tube with gel separator and clot activator. Blood was allowed to clot at room temperature for approximately 15 min and then centrifuged at 1,500 x g for 10 min at 2 to 8°C. Serum was separated and stored at  ~70°C for future analysis. Serum TG, TC and glucose was assessed enzymatically with a kit from Stanbio Laboratory (Boerne, TX). HDL-C was measured using a dextran sulfate precipitating technique followed by enzymatic cholesterol analysis using a kit from Stanbio Laboratory. LDL-C was calculated using the Friedewald equation 10.  Oxidized LDL was measured by ELISA using a kit from Mercodia (Uppsala, Sweden). Insulin was measured by ELISA using a kit from Calbiotech (Spring Valley, CA).

Descriptive data and biochemical data was expressed as means +/- SD. Data was analyzed using SPSS Version 16.0 and Microsoft Excel. A 2 (timepoint) x 2 (dietary trial) repeated measures ANOVA was used to compare serum lipids, glucose, insulin, oxidized-LDL cholesterol and body weight, followed by paired-comparison t-tests as post-hocanalyses.  Paired samples t-tests was used to compare dietary intake between trials. An alpha level of p <0.05 was selected as the criterion for statistical significance.

Out of the 18 recruited subjects, 10 women and 5 men participated in the study. The mean age was 24.7 +/- 6.40 years with a mean BMI of 24.2 +/- 8.03. During the first trial, one participant missed two shakes and one participant missed three shakes.
There was a significant decrease over time for total cholesterol, however there was no significant difference between oils. The mean total cholesterol with refined oil at baseline was 150.94mg/dL +/-33.53, and after two weeks it decreased to143.35mg/dL +/- 29.63 (P=0.029). The virgin oil had a mean of 147.92mg/dL +/-34.50 at baseline and 144.76mg/dL +/- 28.97 after two weeks, however it wasn’t statistically significant.  There was an effect on LDL over time, but not between trials. At baseline, there fined oil had a mean value of 78.10mg/dL  +/- 27.57 and a mean value of 71.14mg/dL +/- 26.96 after two weeks (P = 0.013). The virgin oil trial had a baseline value of 75.92mg/dL +/-28.86 and a mean value of 70.64mg/dL +/- 24.63.  Results summarized in figure 1.
OxLDLproduced a significant difference for overall time (P= 0.013), and there was a trend towards significance for the refined trial, with a decrease over time (P=0.102). The mean OxLDL at baseline was 62.55U/L +/- 14.07 for refined oil and 65.54U/L +/- 20.38 for unrefined oil. After two weeks, the refined oil decreased OxLDL to 56.17U/L +/- 21.55 and the unrefined oil decreased the OxLDLlevels to 64.58 U/L+/- 20.56. Significance was determined between oils, with the refined oil showing a more dramatic reduction in OxLDL (P=0.002). Results summarized in figure 2.
There was no significant difference over time or between trials in blood glucose, insulin or triglycerides over time or between oils. No significance was found over time or between trials in weight change. There was a trend towards significance over time for HDL, but not between trials (P = 0.104).
Dietary data of the participants was gathered and Kcal, protein, carbohydrate, fiber, total fat, saturated fat, cholesterol, vitamin C and vitamin E were analyzed.The means for each nutrient analyzed was compared for difference between trials, and significance was not determined for any nutrient and can be seen intable 1. There was a trend towards significance with the differences in fiber consumption, with a mean of 19.59g +/- 7.56 consumed during the refined trialand 18.03g +/- 9.41 consumed during the unrefined trial (P=0.0868).

This is the first study of its kind to examine the effects of unrefined corn oil to a refined corn oil. Results of this study do not support the original hypothesis that the unrefined corn oil will have a greater hypocholesterolemic effect than the refined oil. The refined oil had a more favorable influence on TC  and LDL than unrefined oil. This indicates that the refining process does not strip the nutrients from corn oil as it does from other oils such as olive oil.
Although there was a more dramatic decrease in TC, LDL and Ox LDL with the refined oil, there was still a decrease between baseline and post intervention for the unrefined oil. The mean HDL also increased from baseline to two weeks, however it was not significant.
Participants had the tendency to consumeless fiber during the unrefined trial and consumed more fiber during the refined oil trial. Because fiber can positively influence cholesterol levels, this could be the reason why the refined oil trial showed a more dramatic effect on lowering total cholesterol and LDL. The difference was not significant, but was approaching significance.
Antioxidants present in corn oil such as ubiqionone, alpha- and gamm-tocopherols could have been the main influence on the reduction in OxLDL. The diets of participants were analyzed for antioxidant consumption and their levels of vitamin C and E were not significantly different from each other.
 The corn oil that was used in the study was analyzed for its polyphenol content, and the results showed no phenols present in either the refined or unrefined oils. Studies that have looked into the benefits of refined vs. unrefined olive oil have found that the polyphenol content present in the unrefined oils had a positive effect on serum lipid levels and were protective antioxidants 1,5.
Insulin levels were also tested, however all values came back much lower than expected. Regular insulin levels in healthy individuals should be around 25μIU/ml, however the mean value was 1.6μIU/ml +/- 2. This could have been due to multiple freeze thaw cycles, which is cautioned against in the directions. Some insulin could have been destroyed through repeated freezing and thawing.
Future research should be conducted in the area of corn oil. A longer trial period of 3+ weeks would be desirable to see long term effects of a corn oil supplementation. Different dosages of less than 40g of oil a day should also be tried to see if benefits are just as significant in lower dosages. It can be argued that 40g is a high amount of raw oil to consume daily, however amounts of 30g-50g have been reported as usual in the typical Mediterranean diet 2.
Also, the benefits of refined corn oil should be compared to refined olive oil, to see if the PUFA content of corn oil has more benefits on blood lipids than the MUFA content of olive oil. If refined oil is used, then the polyphenol content wouldn’t be a contributing to the results. Other widely used oils should be investigated in comparison to olive oil, such as soybean oil and rapeseed oil.
The mean blood lipid levels of the participants were in the  healthy categories according to the National Cholesterol Education Program Guidelines. It is desirable to have cholesterol levels less than 200mg/dL and the mean for this study at baseline was150 mg/dL +/- 33.53 and 147.92 mg/dL+/- 34.50.  Optimal LDL levels are less than 100mg/dL and the mean for the baseline of this study were 78.10 mg/dL +/- 27.57 and 75.92 mg/dL +/-28.86. HDL levels above 60mg/dL are considered protective against heart disease, and the mean baseline HDL of participants was 56.71 mg/dL +/- 10.15and 56.75mg/dL +/- 11.06. Triglyceride levels under 150 are considered healthy, and the average TG levels of participants at baseline were 80.68 mg/dL +/-45.55 and 76.22mg/dL +/- 31.92 10.  A trial should be preformed on participants who have blood lipid levels in the unhealthy range.
In conclusion, the unexpected results were helpful at understanding the positive effects of corn oil. The refined and unrefined corn oil both lowered TC, LDL, TG and OxLDL and raised HDL. However, the refined oil lowered the TC, LDL and Ox LDL significantly more than the unrefined oil. This shows that the refining process for corn oil does not diminish the integrity of corn oil, it in fact enhances the health benefits. Future studies should be conducted in this area to determine just how great the benefits of corn oil are in comparison to other types of oils. 
Works Cited
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