Raw Milk Versus Conventional Milk Intake and Its Effect on Nutrient Intake, Obesity and Central Adiposity

Raw Milk Versus Conventional Milk Intake and its Effect on Nutrient Intake, Obesity and Central Adiposity

THESIS

Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in the Graduate School of The Ohio State University

Alyssa Marie Mark, BS

Graduate Program in Human Ecology

The Ohio State University

2011

Master's Examination Committee:

Professor Lydia Medeiros, Advisor

Professor Martha Belury

Adjunct Assistant Professor Carolyn Gunther

Copyrighted by

Alyssa Marie Mark

2011

Abstract

Although the overwhelming majority of people consume conventional milk, recently there has been a growing trend towards the consumption of raw milk. While there have been studies focusing on the food safety hazards associated with raw milk, there has been little research focusing on the nutrient contribution from raw milk consumption.

Therefore, the purpose of this research was to investigate how raw milk consumption affects the nutritional status of the individuals and their overall health. To examine differences in the consumption of calcium, vitamin D, fat and caloric intake, four-day food records were collected and analyzed using Nutrition Data System for Research.

Obesity was examined by calculating subjects’ Body Mass Index (BMI); central adiposity was examined using waist circumference (WC) and sagittal abdominal diameter

(SAD). Milk samples were obtained from subjects and analyzed for fat content and composition by gas chromatography. In total 57 subjects (age=41.9±14.9), both conventional milk (n=35) and raw milk consumers (n=22) were recruited. Results showed there were no differences in total calorie intake but raw milk consumers had higher total fat (46.9±1.88g/1000kcal/day vs 40.2g±1.21g/1000kcal/day, P=0.003) and saturated fat intake (19.6±1.29g/1000kcal/day vs 14.6±0.65g/1000kcal/day, P≤0.001). In addition, WC (89.4±2.69cm vs 98.2±3.29cm, P=0.06) and SAD (20.0±0.64cm vs

22.3±0.80cm, P=0.05) were smaller in raw-milk consuming subjects, but BMI was similar. The raw milk samples had higher total fat (0.05±0.003g/ml vs 0.02±0.002g/ml, ii p≤0.001), palmitic acid (34.5±0.67% vs 30.9±0.73%, P=0.001), α-linolenic acid

(1.0±1.0% vs 0.5±0.02%, P≤0.001), and c9t11-CLA (0.68±0.04% vs 0.60±02%, P=0.05) content; however, when percentages were calculated in grams per serving, raw milk was higher in all fatty acid content than conventional milk. Overall, the study has contradictory outcomes regarding nutrition related risk factors associated with raw milk consumption; raw milk consumers have a greater risk due to fat consumption but lower risk due to reduced central adiposity.

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Acknowledgements

There are so many individuals that deserve an enormous amount of gratitude for all of their help with this project. First and foremost, I would like to thank my advisor, Dr. Lydia

Medeiros for all of her guidance, patience and support over the past two years and for giving me the freedom to develop a project that I truly loved. I also would like to thank Janet Buffer for immense amount of guidance and support. Her constant open door and IRB assistance have been invaluable. I would also like to thank Lesley Fisher and Jessica Castronova-Brooks for their tremendous amount of work with data collection, entry and food record revisions. I have been very blessed to have such a strong, supportive research team.

I would also like to thank to my other data collectors, Erin Powell, David Ashman, and

Shelby Stuphen for their many hours of reviewing food records and dealing with subjects. Also, I cannot adequately express my gratitude to Rachel Cole for all her help analyzing the milk samples. Without her training and step by step instructions, I would still be in the lab.

I would also like to thank Dr. Martha Belury and Dr. Carolyn Gunther for not only serving on my committee, but also providing me with their expertise on this project.

In addition, I am grateful to my fellow classmates, especially Laura, Sam and Brian, for helping to keep me sane and motivated over the past year. Lastly, to my parents and Greg for their understanding, constant love and support during my thesis work and time at Ohio State. I have been so lucky to have so many people support me during this entire process.

Vita

June 2005 ...... Bloom Carroll High School

May 2009 ...... Foods and Nutrition, Ashland University

2009 to present ...... Graduate Associate, Department of Human

Nutrition, The Ohio State University

Fields of Study

Major Field: Human Ecology

Area of Emphasis: Human Nutrition

Table of Contents

Abstract ...... ii

Acknowledgments...... iv

Vita...... v

List of Tables ...... ix

List of Figures ...... x

Chapter 1: Introduction ...... 1

1.1: Overview ...... 1

1.2: Study Design ...... 3

1.3: Research Questions, Objectives and Hypotheses ...... 3

Chapter 2: Review of Literature ...... 5

2.1: Raw Milk ...... 5

2.1.1: The Raw Milk Trend ...... 5

2.1.2: Raw Milk Opposition ...... 6

2.1.3: Raw Milk Proponenets and the Pasteurization/Homogenization Debate ...... 7

2.2: Vitamin D and Calcium ...... 10

2.2.1: Vitamin D ...... 10

2.2.2: Calcium ...... 12

2.3: Dietary Fat ...... 11

2.3.1: Overview of Dietary Fat ...... 12

2.3.2: Milk Fat...... 13

2.4: Obesity and Central Adiposity ...... 15

2.4.1: Obesity and Dairy Consumption ...... 15

2.4.2: Central Adiposity and Dairy Consumption ...... 17

2.5: Concluding Remarks...... 18

Chapter 3: Raw milk versus coventional milk intake and its effect on nutrient intake, obesity, and central adiposity ...... 20

3.1: Introduction ...... 20

3.2: Materials and Methods ...... 22

3.2.1: Study Design and Subjects...... 22

3.2.2: Food Record ...... 23

3.2.3: Survey ...... 23

3.2.4: Body Mass Index ...... 24

3.2.5: Central Adiposity ...... 24

3.2.6: Lipid Extraction ...... 25

3.2.7: Lipid Methylation ...... 26

3.2.8: Gas Chromatography ...... 26

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3.2.9: Data Analysis ...... 27

3.3: Results ...... 27

3.4: Discussion ...... 37

Chapter 4: Epilogue ...... 45

4.1: Study Conclusion and Major Findings ...... 45

4.2: Limitations of Study ...... 45

4.3: Future Directions for Study ...... 46

References ...... 47

Appendix A: Recruitment Flyer ...... 53

Appendix B: Press Release ...... 54

Appendix C: Research Match Recruitment ...... 55

Appendix D: Phone Call Script ...... 58

Appendix E: Email Script ...... 60

Appendix F: Consent Form ...... 61

Appendix G: Food Record ...... 65

Appendix H: Protocol for Food Reviewer ...... 85

Appendix I: Milk Survey ...... 86

Appendix J: Health Assessment Form ...... 95

Appendix K: Measurement Protocol ...... 96

Appendix L: Supplement Used by Subjects ...... 98

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List of Tables

Table 3.1. Personal characteristics of subjects ...... 28

Table 3.2. Nutrient intake for consumption of raw milk and conventional milk adjusted for overall calorie consumption ...... 29

Table 3.3. BMI, self reported physical activity and sun exposure among subjects ...... 32

Table 3.4. Body composition measures of obesity and central adiposity by gender ...... 33

Table 3.5 Milk fat in subjects’ samples ...... 35

List of Figures

Figure 3.1. Overall dietary fat consumption for raw and conventional milk subjects per 1000 calories consumed per day ...... 31

Figure 3.2. Body composition measures of subjects by age ...... 34

Figure 3.3. Average fatty acid composition of raw and conventional milk ...... 36

Chapter 1

Introduction

1.1 Overview

Milk has long been considered a staple of diets around the world. In the US, pasteurized milk has been predominately consumed, but recently there has been an increase in the consumption of raw or unpasteurized milk (1, 2). The trend of drinking raw milk as been increasing, primarily because raw milk consumers believe that there are health benefits that are lost during the processing of conventional milk, primarily microbes and enzymes (3). Although this perception exists, raw milk is not recommended by health experts due to the potential for foodborne illness and other infectious diseases (1, 2). The confirmed presence of pathogenic bacteria includes Listeria monocytogenes, Salmonella spp., Campylobacter jejuni, and E. coli

O157:H7, along with many others in milk (1, 2).

Although raw milk and conventional milk are very similar in calcium, protein and carbohydrate composition, there are marked differences in other nutrients that could have a dramatic effect on human health (4). The amount of vitamin D and fat vary between raw and conventional milk. Due to the prevalence of rickets in the early 20th century, milk was fortified with vitamin D to help combat the disease (5). Milk naturally contains very little vitamin D, around 50-85 IU per quart, meaning that a person would need to consume about 8 quarts of milk per day to obtain the 400 IU recommendation (6). By law, fortification will increase the vitamin

D content of pasteurized milk to the recommended 400 IU per quart (5). Due to the lack of

1 vitamin D fortification in raw milk, it is possible that people who consume the unfortified milk will lack sufficient daily vitamin D intake to optimally absorb the calcium present in the milk, placing them at an increased risk for bone diseases such as rickets and osteoporosis (7).

Conversely, the fat content is much higher in raw milk than in conventional milk (8).

Unlike conventional milk production, the fat in raw milk is not removed during processing. As a result, the total fat content is greater, although the actual percentage is highly variable based on breed and feed (8). The percentage of fat in raw milk can be as high as 6%, while conventional milk can be purchased with 3.25% (whole), 2%, 1% and less than 0.5% (skim or nonfat) (8, 9).

More importantly milk fat is composed of 65 to 75% saturated fat, often called ―bad fat,‖ because it is known to increase low density lipoproteins and triglyceride levels, both of which are contributing factors to cardiovascular disease and metabolic syndrome (10). Theoretically this increased saturated fat intake could potentially place raw milk consumers at an increased risk for cardiovascular disease and metabolic syndrome. However, milk is one of the main sources of dietary conjugated linoleic acid (CLA), which has been shown to reduce central adiposity, plasma lipids and total cholesterol (8, 11). Milk from pasture-fed cows contains higher levels of CLA than grain-fed animals, meaning that raw milk consumers may have a greater CLA intake, thus lower central adiposity than their conventional milk counterparts (8, 10).

In addition to the health impacts of specific fatty acids, a higher total fat content can also have a dramatic effect on weight since fat contributes nine kilocalories per gram; thus, raw milk tends to have higher caloric content than conventional milk (7). If the diet is not adjusted to account for the extra calories consumed in the milk, it could lead to weight gain and an increase in body mass index (BMI).

Although there has been a multitude of information on raw milk pertaining to foodborne illnesses, there has been little research outside of food safety. While the overall nutrient

2 composition of pasteurized milk has been well documented (11), there has been little to no research about the nutrient intake of those consuming raw milk and how it may affect their overall health. With the trend to drink raw milk on the rise, it is vital for health care professionals to understand how the compositional differences between raw and pasteurized milk may affect the consumers’ diet and overall health, beyond just foodborne illnesses.

1.2 Study Design

The study was cross sectional in design and descriptive in nature. We aimed to identify selected nutrient intake of raw milk consumers compared with conventional milk consumers. We focused specifically on calcium, vitamin D and fat intake. The impact of nutrient intake on obesity and central adiposity was also examined. Subjects were recruited through fliers, advertisements, and press releases through state extension service list serves. During the initial contact, prescreening questions ensured that the participants met the study inclusion criteria of: 1) eighteen years old or over, 2) consumed milk or dairy products, and 3) the only person in the household participating in the study. Four day food records and milk sample tubes were collected from participants and height, weight, waist circumference and sagittal abdominal diameter were measured.

1.3 Research Question, Objectives and Hypotheses

The purpose of the study was to examine the nutrient intake of those consuming raw milk when compared to those consuming pasteurized milk and to determine if the nutrient intake, from total diet and milk, had any influence on obesity and central adiposity.

The study was guided by the following objectives:

1) Identify if there is any difference in calcium or vitamin D intake between those

consuming raw milk and those consuming conventional milk.

2) Identify if there is any difference in dietary fat or calorie intake between those

consuming raw milk and those consuming conventional milk.

3) Determine if there is any difference in obesity and central adiposity between those

consuming raw milk and those consuming conventional milk.

4) Determine if there is any difference in the fatty acid content and composition

between the raw and conventional milk consumed.

Based overall perception of raw milk and conventional milk consumers, it is hypothesized that raw milk consumers would be more health conscious and would consume more milk than conventional milk consumers because they would believe more strongly in the health benefits of milk, particularly raw milk. Thus, raw milk consumers will have greater calcium intake, but lower vitamin D intake due to the lack of fortification of the milk products. In additional, raw milk consumers will have greater dietary fat intake because the raw milk is higher in total fat content; however, they will have a lower total calorie intake because they will compensate for the extra calories from milk in another part of the diet. Because we believe that raw milk consumers will be more health conscious, it is hypothesized that they will have a lower incidence of central adiposity, through both WC and SAD measurements, and obesity. Also, it is hypothesized that the raw milk will have a greater total fat and CLA content than the conventional milk.

Chapter 2

Review of Literature

2.1. Raw Milk

2.1.1 The Raw Milk Trend

Although conventional, pasteurized milk has been the predominate source of dairy consumed in the United States over the past century, there has been a recent increase in the number of individuals consuming raw milk (1, 2). Raw milk is defined as milk that has not been processed in any way, which includes pasteurization, homogenization, fortification of vitamins A and D and standardization of fat content. It is estimated that nearly 3.5% of the United States population now consumes raw milk and the percentage is continuing to grow (13), with an estimated 100,000 Californians consuming raw milk (14).

Raw milk consumers can be categorized into two groups based on their choices for drinking raw milk: urban and non-urban consumers. Anywhere from 35 to 60% of non-urban, dairy farmers consume raw milk and primarily choose to do so because of tradition and convenience (15). Although a large proportion of dairy farmers consume raw milk, the trend is primarily growing among the urban consumers. This subset of consumers chooses raw milk because they feel that milk has health benefits that are lost during the processing, primarily during pasteurization (2). Social activist organizations, such as the Weston A. Price Foundation, have

5 emerged over the past two decades advocating for the consumption and legality of raw milk for the American public (16).

Regardless of the growing trend, there are many governmental regulations surrounding raw milk sales due to food safety concerns. Raw milk interstate sale was banned by the federal government in 1987 and many states have followed suit in making their own regulations (1).

Twenty six states have outlawed the direct sale of raw milk and many other states have placed regulations restricting the amount or location of sale (1). Although there are laws in place, many individuals have developed ways around the regulations. In states that have outlawed the direct sale of raw milk, such as Ohio, the herdshare program has gained momentum. In a herdshare program, individuals pay farmers to ―purchase‖ a part of their herd (1). Since there is no regulation on milk consumption from the animals that you own, it is a legal solution to the problem of obtaining raw milk. However, this is still a controversial solution to many government officials, particularly the United States Department of Agriculture (USDA) (14).

Consequently, many herdshare managers have adopted tight legal contracts with their shareholders to ensure both parties protection (14).

2.1.2 Raw Milk Opposition

In addition to the regulations about raw milk sales, countless health professionals discourage the consumption of raw milk. Currently the US Food and Drug Administration

(FDA), the USDA, the Center for Disease Control (CDC), American Medical Association (AMA) and many others advocate against the consumption of raw milk due to the risk of contracting a foodborne illness (2). Milk is an optimal growth medium for many strains of bacteria, including

Listeria monocytogenes, Salmonella spp, Campylobacter jejuni, and E. coli O157:H7, all of which have been linked to serious human illnesses (2). Pasteurization kills pathogenic and many of the nonpathogenic bacteria naturally occurring in the milk, making it safe for human

6 consumption. Although there are ―on farm‖ practices that can reduce the likelihood of contamination, these have not proven effective enough to eliminate disease onset; pasteurization is still deemed the best way to protect consumers (1).

The process of pasteurization is heating milk (or other food products) at a specified temperature and time to destroy pathogens present in the milk. Pasteurization emerged in the

1910’s as one solution to the milk contamination issue that was killing hundreds of urban children each year (17). Interestingly, the other proposed solution was certification of raw milk, which included inspection of the animals, workers and equipment at the dairies as well as the milk to ensure that it had a low bacterial count and was free of pathogens (17). Eventually pasteurization became the preferred method, and in many instances, is the only legal solution to eliminating milk contamination.

Currently, the FDA’s Pasteurized Milk Ordinance (2007 revision) states that pasteurization is the ―only practical, commercial measure, which if properly applied to all milk, will destroy all milkborne disease organisms‖ (18). The FDA requires that every particle of milk or milk product is heated to a specific temperature for a specific period of time. This ranges from

63°C for 30 seconds up to 100°C for 0.01 second, also called ultra high temperature (UHT) pasteurization, and cooled immediately (18). The most common form of pasteurization in the dairy industry is 72°C for 15 seconds. Although it has not completely eliminated milkborne outbreaks, it has significantly reduced diseases associated with contaminated milk consumption

(18).

2.1.3 Raw Milk Proponents and the Pasteurization/Homogenization Debate

Raw milk advocates claim, however, that pasteurization actually causes more harm than good (16). Their belief is that milk in its natural state has many protective components that naturally kill and hinder pathogens from harming the consumer, making pasteurization

7 unnecessary (16). Raw milk proponents purport pasteurization is not only superfluous, but can decrease the nutrient content and destroy beneficial bacteria and enzymes, vital for human health

(16). Enzymes, such as amaylase, lactase, catalase and lipase are destroyed during the pasteurization process, reducing the natural health properties of milk (19). In addition it is believed that vitamins are decreased during the pasteurization process, further impacting the nutritional content of the milk (19).

Due to the hypothesized loss of the nutrients, beneficial bacteria, and enzymes, raw milk supporters link pasteurized milk with various common diseases such as allergies, colic, Chron’s disease and ulcerative colitis, osteoporosis, arthritis, heart disease and cancer (16). Growth problems in children and tooth decay are also postulated to be linked with pasteurized milk consumption by raw milk advocates (16). A limited number of studies from Europe have shown that children who were raised on farms with exposure to raw ―farm‖ milk had lower prevalence of asthma and allergies (20, 21, 22). One study (23) found that raw milk consumption by farm children decreased sensitivity to skin prick tests by 70%, and others have shown similar reductions. Study outcomes are in conflict on whether this protection is due to the combined effect of the farm lifestyle or raw milk independently (21, 22). Others hypothesize that it is other aspects of raw milk, apart from lack of pasteurization, that cause the reduction in allergies and asthma. A review by Wjst (2006) suggests that ubiquitous, exogenous vitamin D exposure can induce allergies in those already susceptible individuals, as shown in newborns that are supplemented vitamin D and often exhibit allergenic responses later in life (24). Nevertheless, there is little to no scientific research to validate the claims that raw milk may have added protective effects against the development of osteoporosis, arthritis, heart disease, Chron’s and ulcerative colitis, or cancer (25).

While most of the legal focus of raw milk consumption centers on the process of pasteurization, raw milk advocates strongly protest the process of homogenization as well (20).

Homogenization is the process of separating the large fat globules into many smaller, more uniform fat particles, often by forcing pressurized milk through a small valve (26). This process keeps the fat suspended in the liquid matrix, rather than separating to the top; it also increases the shelf life (27). The reduction in fat globule size improves the digestibility of milk in adults, especially those with impaired intestinal function (26).

Homogenization became a common practice in the 1930’s, which coincided with the increase of cardiovascular disease in the US (26). For this reason some people link homogenization with an increase risk of heart disease (28). It has been postulated that bovine xanthine oxidase, an enzyme found in the fat membrane, is atherosclerotic and is released during the homogenization process (29). Many scientists disagree with this hypothesis due to gastric acid denaturization of the enzyme, and because there is yet to be sufficient evidence that this enzyme has any harmful effects in humans (26).

Homogenization has also been speculated to induce allergic reactions in some individuals

(28). In animal studies, homogenization appears to cause hypersensitivity to milk, and this has been replicated in a few human studies (30). One biological explanation for this phenomenon is that homogenization increases the surface area on the fat globule where the allergenic proteins can reside; conversely, in untreated milk, the proteins are located on the interior of the micelle

(28). However, others argue that the allergenic protein exposure is sufficient in untreated milk to stimulate an allergic response (26) and some studies have not shown any difference in allergic response between homogenized and unhomogenized milk (31). There has yet to be strong scientific evidence to show proof that homogenization has any deleterious effect on human health

(26).

Although proponents of raw milk strongly believe in the health benefits associated with

―real milk,‖ they are quick to explain that this does not encompass the entirety of milk produced in the US. Raw milk advocacy groups propose that milk from pasture-fed, humanely-raised animals is the only safe form of raw milk. The contention is that milk that is produced on large confinement, feedlot farms is not safe for consumption prior to pasteurization due to unsanitary conditions and unethical treatment of the animals. Furthermore, the Weston A. Price Foundation does not support the consumption of low fat or skim raw milk. They assert that the inherent protective features of the milk reside in the butterfat. The Campaign for Raw Milk succinctly explains their position, ―Real milk, that is, raw whole milk from grass-fed animals (fed pasture, hay and silage) produced under clean conditions and promptly refrigerated, contains many antimicrobial and immune-supporting components; but this protective system in raw milk can be overwhelmed and the milk contaminated in situations conducive to filth and disease. Know your farmer!‖ (17).

2.2 Vitamin D and Calcium

2.2.1 Vitamin D

Vitamin D is an essential, fat soluble vitamin that plays many roles in the human body, but its role in calcium absorption and bone health has been the most well recognized function (7,

32). Conventional milk is fortified to contain 400 IU of vitamin D per quart, which is sufficient to prevent the onset of rickets in children (6). This means that to meet the Recommended Dietary

Allowance (RDA) for vitamin D, one would need to consume 4 cups of milk per day. Raw and conventional milk alike naturally contain only 50 to 85 IU per quart without fortification meaning that one would need to consume 32 cups of unfortified milk per day (6) to meet the RDA for vitamin D. Furthermore, fortified milk provides 65-86% of vitamin D intake from food each day

10 for the average American (32). This suggests that it could be rather difficult to obtain adequate dietary vitamin D if consuming little or no fortified milk.

Vitamin D is distinctive from other vitamins because it can be synthesized by the human body. Ultraviolet light can change 7-dehydrocholestrol located in the skin to pre-vitamin D (33).

This pre-vitamin D can then go through enzymatic processes in the liver (to 25- hydroxycholcalciferol) and then the kidney to become the active form of vitamin D (1,25- dihydroxycholcalciferol) (33). Only fifteen minutes of sun exposure on the hands and face during the summer months in Ohio is enough to produce the recommended 400 IU per day (34). Since there are few dietary sources of vitamin D, especially outside fortified foods, this provides a considerable amount of daily vitamin D. Unfortunately, the amount of cutaneous production has been decreasing over the past decade (34). This is in part to the concern over skin cancer leading to increased sunscreen use (which blocks the UVB rays, thus lowering the conversion to pre- vitamin D) and limited the total sun exposure, meaning a reduction in endogenous vitamin D synthesis (34).

Although vitamin D can be obtained both through dietary intake and cutaneous production, 25 to 57% of the American population is considered vitamin D deficient and even more are classified as insufficient (35). This number is alarming since vitamin D deficiency has been linked to an increase prevalence of hypertension, diabetes, the metabolic syndrome, left ventricular hypertrophy, congestive heart failure and chronic vascular inflammation (36). It has also been associated with an increase in the premature deaths of individuals from breast, colon, ovarian and prostate cancer (37). Because of this, any decrease in the consumption of fortified milk and other dairy products, or sun exposure could have serious effects on human health.

2.2.2 Calcium

Calcium is an essential mineral that represents 1.5 to 2% of total body weight (7). It is vital for bone and teeth development and nerve transmission (7). Calcium’s beneficial effects on bone development has been long documented, and current dietary recommendations for calcium intake are based on optimal bone development and maintenance throughout the lifecycle (7). The primary source of calcium in the average American diet is through the consumption of milk and dairy products, which contain approximately 300 mg per serving (7).

There is evidence that dairy consumption, especially low fat dairy consumption, has an inverse relationship with hypertension (38). This attribute of dairy has been primarily linked with dietary calcium intake (38). Because of this, dairy has become an important component of the Dietary Approaches to Stop Hypertension (DASH) diet, with studies suggesting that the diet is less effective without the addition of low-fat dairy products (39). However the current data is conflicting as a few studies have shown no effect between calcium supplementation and changes in blood pressure, both systolic and diastolic (40).

2.3 Dietary Fat

2.3.1 Overview of Dietary Fat

While dietary fat often receives a bad reputation for its higher caloric contribution and tendency to aid in the accumulation of excess body fat, it is a vital part of the human diet (7). Fat is responsible for energy, cell membrane structure, insulation of body heat, protection of organs and nerves, as well as aiding in the absorption of fat soluble vitamins, such as vitamin D (7). In fact, fat should comprise 30% of the total calories in the diet per day, with less than 10% of calories being saturated fat (7). Dietary fat can be divided into three distinctive classes, based on the hydrocarbon structure and resulting physiological effects. Saturated fatty acids are

12 hydrocarbon chains that contain no double bonds. Saturated fats are often linked with increase in cholesterol, particularly low density lipoprotein (LDL) which is a risk factor for cardiovascular disease. Monounsaturated fats are hydrocarbon chains that contain one double bond in the structure. Monounsaturated fats have received a considerable amount of scientific focus over the past decade for their cardiovascular protective function (41). Similar to monounsaturated fats, polyunsaturated fatty acids are hydrocarbon chains that contain more than one double bond.

Polyunsaturated fats have also been extensively studied for their proposed cardiovascular protective properties, in particular omega-3, a group of polyunsaturated fatty acids that contain a double bond at the n3 location (7).

2.3.2 Milk Fat

Milk and dairy products are a major source of fat in the average American diet, with dairy products contributing 12% of daily dietary fat (42) and 10% of palmitic (16:0) and stearic acid (18:0) intake alone (38). Whole milk contains approximately 33g of fat per liter, consisting of approximately 70% saturated fatty acids, 25% monounsaturated fats and 5% polyunsaturated fats (10, 11, 43). Saturated fats in milk consist primarily of palmitic acid (16:0) and myristic acid

(14:0) (11). The most abundant unsaturated fat, and virtually the only monounsaturated fat found in milk is oleic acid (18:1n9), accounting for 25% of milk fat (11, 44). Linoleic (18:2) and α- linolenic (18:3) acids are the most commonly found polyunsaturated fatty acids in milk, but they can be elongated and desaturated into longer chain polyundsaturated fats (11). The specific fatty acid composition can change, but this average composition can have profound effects on overall health, especially related to obesity and cardiovascular disease (11).

Although the vitamin D content of raw milk is lower than conventional milk, the fat content is considerably higher. Conventional milk fat is standardized to whole (3.25%), 2% milk fat, 1% milk fat and nonfat/skim (less than 0.5%) (12). Since raw milk does not go through any

13 processing, the milk fat content is not standardized into one of these percentages. Thus, the fat content of the milk is dependent solely on the fat production of the dairy cow. This in itself is highly variable (8). The fat percentage and composition can vary based on the breed of the cow as well as the feed given to the cow during lactation (8). Jersey dairy cows have the highest milk fat percentage as well as the highest saturated fat content while Dual Purpose Belgian Blue dairy cows have the lowest percentage of milk fat and saturated fat production (10). Milk from

Holstein dairy cows, which are one of the predominant dairy breeds in Ohio, contains an overall lower fat percentage, lower saturated fat content as well as higher monounsaturated fat and polyunsaturated fat content (8). Because of this, the breed of animal that the milk comes from can greatly impact the fat content of the milk. This is not nearly as important in conventional milk, not only because the milk fat is standardized, but also because the milk from different breeds and farms are often pooled together before processing and packaging (18).

Milk consumption and particularly milk fat intake have been researched for their connection to various health outcomes, primarily cardiovascular disease, obesity and metabolic syndrome. Palmitic and myristic fatty acids, which are large constituents of milk fat, are known to increase total cholesterol, specifically LDL, as well as insulin resistance (10, 38, 44), thus leading to an increase risk of cardiovascular disease and type 2 diabetes mellitus. However, the short and medium chain fatty acids as well as oleic acid have been found to have many various health benefits (1). These counteract the effects of palmitic and myristic acids to improve insulin sensitivity (11, 38, 44). Overall, dairy fat intake has not been linked to an increase in obesity- related diseases, and in some cases may have protective effects against these chronic disorders

(38, 44).

Conjugated linoleic acid (CLA), which is a group of polyunsaturated fatty acids that are isomers of linoleic acid (18:2), predominately found in milk, and has been recently garnering

14 much focus due to potential health benefits. (45). The most commonly found natural forms are the c9t11-CLA, t7c9-CLA, 11,13-CLA, 8,10-CLA and t10c12-CLA and these fatty acids are the forms primarily found in beef and dairy products (45). CLA has been associated in animal studies with decreased prevalence of diabetes, cancer and atherosclerosis (45). One study in particular showed that rats fed CLA had reductions in fasting blood glucose and insulinemia, triglyceridemia, and free fatty acid levels that were similar to outcomes of rats fed a known antidiabetic drug (46). However, few clinical studies have shown any significant effect of CLA on the antidiabetic, anticarcenogenic and antiatherosclerotic properties exhibited in animal studies

(38). One study demonstrated that CLA supplementation of 6g/day decreased fasting blood glucose and BMI, but it also significantly raised LDL levels (48). In addition, it appears that these benefits are dependent on the specific CLA isomers. While one study found that a diet of many CLA isomers reduced total cholesterol, non-HDL cholesterol and early aortic atherosclerosis (49), another found no effect when the animal was fed a diet that contained only the t9c11-CLA isomer (50).

2.4 Obesity and Central Adiposity

2.4.1 Obesity and Dairy Consumption

Obesity, which is categorized as a Body Mass Index (BMI) greater than 30 kg/m2, has attracted much attention over the past decade. Recent reports have shown that the current prevalence of obesity is 33.8% in 2008 in the United States and preliminary data has suggested that this number is continuing to rise (51). Because of this statistic there has been an increase in research related to weight gain and obesity. This has included research on milk and dairy products and the role that they may play in obesity. A review by Pfeuffer (2006) indicated that

15 there is an inverse relationship between dairy consumption and body weight in both children and adults (44); however, not all studies support this relationship (52).

Calcium is often indicated as the primary nutrient associated with the observed inverse relationship between dairy products and BMI (38). Calcium has been shown to increase fecal fat excretion (38, 44), resulting in less fat absorption and caloric contribution from calcium containing foods. Researchers hypothesized that this is due to calcium binding to free fatty acids, primarily palmitic acid, resulting in an indigestible insoluble soap (38, 44). Calcium may also play a role in regulating energy metabolism by decreasing fatty acid synthesis and increasing lipolysis through reducing 1,25 (OH)-vitamin D activity and intracellular calcium influx (44). In addition reductions in 1,25(OH)-vitamin D has also been shown to up-regulate uncoupling protein 2 (UCP2) increasing thermogenesis and energy expenditure (53).

There has been some indication that vitamin D deficiency may increase body weight and prevalence of obesity (54). A study by Shahar showed an inverse relationship between serum

25(OH)-vitamin D and BMI and a positive relationship between serum vitamin D and weight loss during a two year intervention (55). This relationship was independent of calcium consumption, although dietary calcium intake also aided in weight loss during the study (55). In addition, it has also been reported that each 100mg increase in calcium was associated with a 3% reduction in the prevalence of obesity, although magnesium, also found in dairy products, had a greater effect

(56). This outcome is not equal among all dairy products either. Beydoun (2008) found that yogurt was inversely associated with BMI, while cheese was positively associated and there was no association with fluid milk (56).

Many studies have demonstrated that CLA reduces the accumulation of adipose tissue

(45). In animal studies, CLA was found to reduce adipose tissue mass by 50% (57). Some studies have shown that CLA can have similar, albeit less drastic, changes in humans (38). CLA

16 supplementation in diabetic women showed a significant decrease in BMI and weight, as well as a moderate decrease in body fat (48). Other studies have also shown that CLA supplementation can reduce body fat and body weight, although more studies are needed to elucidate dose and specific CLA isomer effect (58, 59, 60).

2.4.2 Central Adiposity and Dairy Consumption

Central adiposity, sometimes referred to as central obesity, is the amount of body fat that is located in the abdomen of individuals (13). These measurements are often used to examine an individuals’ risk of obesity related diseases (61). It has been well documented that excess body fat located in the midsection, or android adiposity, greatly increases the risk of developing cardiovascular disease and type 2 diabetes mellitus as the fat is viscerally located, or around vital organs (61). Measurements of central adiposity have been shown to have a better correlation to risks of obesity-related diseases than BMI alone (62).

There are many measurements used to analyze central adiposity, but those of interest in public health are waist circumference (WC) and sagittal abdominal diameter (SAD). Waist circumference is simply the distance around the abdomen, measured in inches or centimeters.

The definition of waist varies by studies but the National Health and Nutrition Examination

Survey (NHANES) defines the waist at the top of the iliac crest (13). A WC greater than 88 cm in women and 102 in men places the individual at an increased risk of developing heart disease, although the definition of waist for these reference points is not clear (13). While WC is one of the more common measurements of central adiposity, SAD has been shown to be a better predictor of risk of obesity-related diseases, such as glucose intolerance, insulin resistance, and cardiovascular disease (61). The SAD is the ―abdominal height‖, or the distance between the back and the top of the stomach at the iliac crest. The SAD directly correlates to the amount of

17 android adiposity and recent research suggests that the cutoff of SAD for cardiometabolic risk is

22 cm in men and 20 cm in women (63).

Many cross-sectional and prospective studies have shown an inverse association between higher dairy consumption (both low and regular fat) and reduced central adiposity (64). For example, high fat dairy products had an inverse association with central adiposity (56). However, results are contradictory. A study has linked low-fat dairy consumption to a positive relationship with central adiposity (as defined by WC) (56). In addition dairy consumption among men increased the prevalence of central adiposity by 5%, although this was not true for women (56).

The same study found a significant inverse relationship between overall dairy consumption and central adiposity. Most of this effect was attributed to the calcium and magnesium consumption from the dairy products (56). Another study also showed an inverse relationship between dairy consumption and central adiposity (as defined by SAD); however, this was only observed in those identified as under-reporters of food intake (65). In the same study, dairy fat had an inverse association with central adiposity, but only in under-reporters as well. Under-reporters are those subjects that report a lower dietary intake than their actual intake, which impacts the total energy and nutrient intake, thus potentially affecting the true association between diet and investigated outcomes (65). Conversely, an inverse relationship was observed between calcium intake and

SAD in all respondents (65). Although extensively studied, the data is still conflicting on the relationship between dairy consumption and central adiposity.

2.5 Concluding Remarks

In summary, raw milk consumption has been growing in popularity, although little is known about its effect on nutrient intake and health. Raw milk has a considerably lower content of vitamin D; dietary fat, on the other hand, is much higher in raw milk. Although milk appears

18 to reduce body weight and body fat, the studies have been done primarily on low-fat dairy products, which raw milk would not qualify. In conclusion, there needs to be research conducted to specifically address raw milk intake and to determine if it differs from conventional milk intake of total calories, calcium, vitamin D and dietary fat.

Chapter 3

Raw Milk versus Conventional Milk Intake and its Effect on Nutrient Intake, Obesity and Central Adiposity

3.1 Introduction

Although conventional, pasteurized milk has been predominately consumed in the United

States, recently there has been an increase in the consumption of raw or unpasteurized milk (1, 2).

This trend has been increasing primarily because raw milk consumers perceive that there are health benefits that are lost during the processing of conventional milk (17). Although such perceptions have yet to be validated, there are inherent differences that exist between raw and conventional milk (4). Specifically, the amount of vitamin D and fat vary between raw and conventional milk, regardless of pasteurization.

Milk naturally contains very little vitamin D, only around 50 to 85 IU per quart but fortification in conventional milk processing will increase the vitamin D content to the recommended 400 IU per quart (5). Conversely, the fat content is much higher in raw milk than in conventional milk (11). Unlike conventional milk production, the fat in raw milk is not removed during processing; as a result, the total fat content can be greater, although this actual percentage is highly variable (8). The percentage of fat in raw milk can be as high as 6% milk fat, while conventional milk can be purchased with 3.25% milk fat (whole), 2% milk fat, 1% milk fat and less than 0.5% milk fat (skim or nonfat milk) (8, 9). The higher total fat content of the

20 milk can also have a dramatic effect on weight since it contains more calories than conventional milk (7).

Consequently, the type and quantity of milk consumed may impact health outcomes, such as obesity and central adiposity. Although dairy consumption has been shown to have an inverse relationship between consumption and body weight, these studies have focused solely on conventional milk and dairy products made from conventional milk (38, 44). Since raw milk’s fat and calorie content is significantly higher than conventional milk, if the consumer’s diet is not adjusted to account for these extra calories in the milk, it could lead to weight gain, thus affecting body mass index (BMI) and obesity, and central adiposity. Conversely, conjugated linolenic acid (CLA) which may be higher in grass-fed, raw milk has been shown to reduce central adiposity in participants (45).

The objectives of this study were to determine if there were any differences in calorie, total fat, vitamin D and calcium intake from the diet as well as differences in obesity and central adiposity between raw and conventional milk consumers. Milk samples from raw and conventional milk consumers was also examined to determine the potential differences in the fat content and specific fatty acid composition of the milk.

We hypothesized that raw milk consumers would consume more milk because they believe strongly in the health benefits of the raw milk, thus consume more calcium; however, they would have a lower vitamin D intake due to the lack of fortification of the milk. In addition, raw milk consumers would consume more fat and particularly saturated fat, but would have a lower total calorie intake by compensating for the additional fat in other parts of the diet. We also hypothesized that raw milk consumers would have a lower rate of obesity and central adiposity from having an overall healthier lifestyle.

By elucidating any potential differences between raw and conventional milk consumers, health care professionals can address the nutritional recommendations, for those individuals consuming raw milk. With this information dietitians can also tailor dietary plans for raw milk consumers based on the nutrient composition of the raw milk.

3.2 Materials and Methods

3.2.1 Study Design and Subjects

Subjects were recruited through fliers, advertisements, and press releases through state extension service list serves. During the initial contact, prescreening questions ensured that the participants met the study inclusion criteria of: 1) eighteen years old or over, 2) consumed milk or dairy products, and 3) the only person in the household participating in the study. The prescreening questions also determined whether the subjects were conventional or raw milk consumers. After meeting the criteria and agreeing to participate, subjects were asked to attend an initial meeting with the researchers, from October to February. Subjects were given a packet and asked to complete a four-day food record as well as fill out a survey about their food safety and lifestyle practices. The participants were also provided with two 50 ml sample tubes to collect samples of milk, one sample from the milk as they typically consumed and one from the milk as it was received from the source, either from the store or herdshare to determine if consumers removed any of the fat prior to consumption. Subjects were then asked to attend a second meeting with the researchers at least four days later in which their height, weight, waist circumference, and sagittal abdominal diameter were measured (specific methods follow) and food record, surveys and samples tubes were collected. Due to difficulty traveling in the winter months, the January and February participants were mailed an explanatory cover letter, food record and sample tubes prior to their scheduled study date and completed the survey and

22 measurements at the study location. Subjects received up to $50.00 cash as compensation for participating in the study ($15 for completing survey, $15 for completing food record, $10 for participating in food safety focus group, $5 for health assessment, and $5 for providing the milk sample). All study procedures were reviewed by the Ohio State University Institutional Review

Board (IRB) for Social and Behavioral human studies research under protocol # 2009B0301.

3.2.2 Food Record

The four non-consecutive day food record (Appendix G) was used to analyze total nutrient intake. The participants were instructed to record all food, beverage and supplements that they consumed for four days including at least one weekend day. The food records were then reviewed with the participant by the researchers using a multi-pass approach to ensure completeness of the record (Appendix H) (13) The food record was entered into and analyzed using the Nutrient Data System for Research (NDSR) (version 2006, Minneapolis, Minnesota) to determine average nutrient intake over the four days analyzed. Raw milk was not available in the system; Mexican Pima Indian leche fresco de vaca (Code # 25321) was used as a substitute for raw milk.

3.2.3 Survey

Survey questions (Appendix I) were used to assess cutaneous vitamin D production, daily physical activity and weight loss supplement intake. The survey was reviewed with subjects by researchers to ensure completeness; however, to preserve the voluntary nature of the study as per

IRB protocol, the subjects were not required to answer all questions if they felt uncomfortable with certain questions. The data was entered into Access (Microsoft 2007, Redman, WA) and analyzed using Statistical Package for Social Sciences (SPSS, IBM, version 19, New York, NY).

3.2.4 Body Mass Index

Participants’ height was measured to the nearest tenth centimeter (cm) using a stadiometer. Participants’ weight was measured to the nearest tenth kilogram (kg) using electronic or balance scale. The measurements were entered into Access (Microsoft 2007,

Redman, WA), height (in cm) was converted to meters and BMI calculation (BMI = kg/m²) was conducted in Excel (Microsoft 2007, Redman, WA). In this study overweight was operationally defined as BMIs of 25kg/m2 to 29.9kg/m2, obesity was operationally defined 30kg/m2 or more.

3.2.5 Central Adiposity

Central adiposity was assessed using both waist circumference (WC) and sagittal abdominal diameter (SAD). The WC method was adapted from the NHANES method (12). The participants were asked to lift any clothing that would restrict direct access to the skin. The researcher then placed the linen tape measure at the top of the right iliac crest and proceeded to place the tape around the horizontal plane, ensuring that the tape was parallel to the floor. The measurement was recorded to the nearest half centimeter. The process was repeated twice and the average of the measurements was calculated. The method for the SAD was adapted from

Kahn (1996). The subjects were asked to lie on a flat surface with their knees bent and any clothing covering direct access to the skin was lifted. The top of the iliac crest was located and a small mark was made. The bottom arm of the Holtain-Kahn Abdominal Caliper (Holtain Ltd.,

U.K.) was placed under the back so that the upper arm edge ran across the abdomen at the mark at the iliac crest (66). The subject was asked to inhale and exhale; as the subject exhaled the upper arm was lowered to rest on the subject’s abdomen. The measurement along the vertical ruler was recorded at the end of exhalation to the nearest tenth cm. The process was repeated an additional two times and the average of the measurement was calculated.

3.2.6 Lipid extraction

After collection, milk samples were stored at -80⁰ C to prevent fatty acid oxidation.

Samples were then thawed and mixed on a vortex mixer to ensure equal distribution of fat in the milk sample. After vortexing, 1 ml of the milk was added to 2 ml of methanol (HPLC, Fisher

Scientific, Hampton, NH) with butylated hydroxytoluene (BHT) (Fisher Scientific, Hampton,

NH). Samples were homogenized at setting 3 (out of 10) for ten seconds to ensure complete mixture of milk and then poured into a 20 ml glass vial. An additional 1 ml of methanol with

BHT was added to the homogenate tube and immediately poured into the glass vial. The glass vial was vortexed for 20 seconds. Next, 1 ml of chloroform (HPLC grade 99.8%, Fisher

Scientific, Hampton, NH) was added to the homogenate tube and quickly poured into the glass vial containing the milk with methanol. An additional 5 ml of chloroform were added to the glass vial and samples were incubated for 2 days at -20⁰ C, and vortexed once per day for 10 seconds during the incubation period.

After incubation, samples were removed from the freezer and 1.5ml of 0.88% potassium chloride (KCl) (Fisher Scientific, Hampton, NH) was added to each vial. The samples were vortexed and then centrifuged at 1000 x g for 15 minutes at 4⁰C. Glass vials (20 ml) were then weighed and their weight recorded. After completion of the centrifugation, the bottom phase containing chloroform and fatty acids was transferred to the new glass vial. An additional 6 ml of chloroform was added to the original glass vial containing the top phase. The samples were then vortexed for 10 seconds and centrifuged at 1000 x g for 15 minutes at 4⁰ C. The lower phase was transferred into the glass vials containing the bottom phase from the previous run. After the entire bottom phase was removed, 1 ml of 0.88% KCl was added to the glass vial containing the bottom phase, vortexed and centrifuged at 1000 x g for 15 minutes at 4⁰ C. The top KCl layer was then removed from the bottom chloroform layer. A new glass vial was weighed, with the

25 weight recorded, and the bottom chloroform layer was transferred to the new glass vial and stored at -20⁰ C.

3.2.7 Lipid methylation

The previously extracted samples were removed from the -20⁰ C freezer and were dried under a steady stream of N₂ (UHP 99.99999%, OSU Stores; Columbus, Ohio) until completely dry (about 90 minutes). Samples were then weighed and the weight recorded. Methanol (0.4ml,

HPLC, Fisher Chemical 1L; Fisher Scientific) with 1,1,3,3 tetramethylguanidine (99%, Fisher

Scientific, Hampton, NH) was added to each sample. The samples were vortexed and then heated in a 100⁰ C water bath for 20 minutes. After removal from water bath, 1 ml of hexane (HPLC grade, Fisher Scientific, Hampton, NH) was added to each sample followed by 0.5 ml of 0.88%

KCl. Tubes were inverted to mix and then centrifuged at 1000 x g for 10 minutes at 4⁰ C. The top hexane layer was then transferred to new 10 ml glass vials. An additional 1 ml of hexane was added to the original glass vial and centrifuged at 1000 x g for 10 minutes at 4⁰ C. The top hexane layer was added to the glass vial containing the previous hexane layer. These vials were then dried under a steady stream of N₂ until dry (about 30 minutes). Once completely dry, 2 ml of hexane was added to the vials and vortexed. The sample was then transferred into a gas chromatograph vial and stored at -20⁰ C until fatty acid analysis.

3.2.8 Gas chromatography

After methylation, analysis of fatty acid methyl esters was completed by gas chromatography using a 30-m Omegawax™ 320 fused silica capillary column (Supelco,

Bellefonte, PA). Oven temperature started at 175 °C and increased at a rate of 3°C/min until reaching 220°C. Samples were initially run at a 100 to 1 split (sample that was allowed to leave through a vent); reruns were run at a 3 to 1 split. Flow rate of carrier gas helium was 30mL/min.

Retention times of samples were compared to standards of fatty acid methyl esters (Matreya,

LLC, Pleasant Gap, PA, Supelco, Bellefonte, PA and Nu-Check Prep Inc, Elysian, MN). Fatty acids are reported as percent of total fatty acids in milk samples and gm fatty acid per 8 oz of milk.

3.2.9 Data Analysis

At the conclusion of the study, data was analyzed using SPSS. An alpha level of 0.05 was used to declare significance. Descriptive statistics were used to demographically describe the raw milk and conventional milk consumers. To achieve the goals of all objectives means were calculated as dependent variables for analysis of variance (ANOVA).

3.3 Results

Fifty-nine individuals were recruited from a pilot study and participated in the initial part of the study. Two subjects dropped from the study before data collection (raw n=1, conventional n=1) due to time conflicts. In total there were 57 subjects (raw n=22, conventional n=35). There were no differences between the milk consumers in gender and age (Table 3.1), but raw milk consumers were more likely to be Caucasian (P=0.04), more highly educated (P=0.02) and earned higher income (P=0.004) than conventional milk consumers (Table 3.1).

Table 3.1 Personal characteristics of subjects.

Variables Raw milk Conventional milk N % n % Type of milk consumed 22 38.6 35 61.4

Gender a Male 8 36.4 15 42.9 Female 14 63.6 20 57.1

Age b 18-35 years 5 22.7 15 42.9 35-49 years 11 50.0 7 20.0 ≥ 50 years 5 22.7 13 37.1

Race/ethnicity c Caucasian 22 100.0 28 80.0 Asian 0 0.0 1 2.9 African American 0 0.0 4 11.4 Bi/Multi-racial 0 0.0 1 2.9

Self-reported highest level of education d High School 1 4.5 6 17.1 Technical school or some 1 4.5 10 28.6 college College graduate 10 45.5 9 25.7 Postgraduate or professional 10 45.5 10 28.6

Self-reported household income e $45K or less 7 31.8 21 60.0 $45K-$54K 2 9.1 5 14.3 $55K-$64K 2 9.1 2 5.7 $75K-$84K 1 4.5 1 2.9 $85K-$99K 5 22.7 1 2.9 $100K or more 5 22.7 3 8.6 a ANOVA within row F=0.01, P=0.63 b ANOVA within row F≤ 0.01, P=0.99 c ANOVA within row F=4.43, P=0.04 d ANOVA within row F=6.17, P=0.02; raw milk ≥ conventional milk e K = thousands of United States Dollars ($); ANOVA within row F=9.29, P=0.004; raw milk ≥ conventional

While the food choices differed between the groups of consumers, raw milk and conventional milk consumers had similar total caloric intake (Table 3.2). They also had similar 28

calcium and vitamin D intake (Table 3.2); however when supplements were removed from

dietary analysis (Appendix L), vitamin D was significantly (P ≥ 0.000) less in raw milk

consumers. Cutaneous vitamin D production was assessed through self-reported sun exposure,

and there was no significant difference between the groups (Table 3.3). The study was also

conducted during the winter months in Ohio (October thru February) so there would be less

cutaneous vitamin D production in both groups than during other months.

Table 3.2. Nutrient intake for consumption of raw milk and conventional milk adjusted for overall calorie consumption. Nutrient Type of milk consumed a With supplements b Without supplements c

Raw milk Conventional milk Raw milk Conventional milk

Total calories 2681.9 ± 218.31 2308.6 ± 105.54 2673.1 ± 219.52 2308.6 ± 105.64 (kcals/day) d Vitamin D 6.0 ± 2.18 5.0 ± 1.14 1.5 ± 0.19*** 3.8 ± 0.26 (mcg/1000kcal/day) e Calcium 563.8 ± 49.49 537.7 ± 35.24 539.3 ± 41.21 529.4 ± 29.69 (mg/1000kcal/day) f a Mean ± SEM b Nutrients provided by dietary supplements included in data c Nutrients provided by dietary supplements excluded from data d Average total caloric intake over four days (three weekdays and one weekend). ANOVA within row with supplement use, F=2.93, P=0.09. ANOVA within row and without supplement use, F=2.77, P=0.10. e Average vitamin D intake over four days (three weekdays and one weekend day). ANOVA within row and with supplement use, F=0.18, P=0.67. ANOVA within row and without supplement F=24.13, P≤0.001, raw milk ≤ conventional milk f Average calcium intake over four days (three weekdays and one weekend day). ANOVA within row and with supplement F=0.011, P=0.919; ANOVA within row and without supplement F=0.40, P=0.84 *** P≤ 0.001

Although there was no difference in total calories, calcium and vitamin D between the

groups, raw-milk consuming subjects had significantly higher total fat and saturated fat intake

than conventional milk consumers (Figure 3.1). Monounsaturated and polyunsaturated fats were 29 not different between the groups. In addition, carbohydrate intake (mean ± SEM) was less in raw milk consumers (104.40 ± 4.3) than in conventional consumers (122.55 ± 3.20; P = 0.001) and there was no difference in protein intake (data not shown).

50 **

20 *** gm/1000 kcals/day gm/1000 10

0 Total SFA MUFA PUFA Type of fat (mean ± SEM) Raw Conventional

Figure 3.1. Overall dietary fat consumption for raw and conventional milk subjects per 1000 calories consumed per day. Total fat ANOVA within group, F=9.83, P=0.003. Saturated fat (SFA) ANOVA within group F=15.31, P≤0.001. Mono unsaturated fat (MUFA) ANOVA within group F=2.93, P=0.09. Polyunsaturated fat (PUFA) ANOVA within group F=1.23, P=0.27. **P≤0.01, ***P≤0.001

The BMI (mean ± SEM) was similar between raw (25.8 ± 0.90) and conventional milk groups (27.5 ± 1.02; P=0.26), which classify both milk consumers as overweight, although the majority of people within each group were classified as healthy (Table 3.3). Conversely, WC was moderately lower in raw milk (89.2 ± 2.69) than conventional (98.2 ± 3.29; P=0.06), and SAD was significantly lower in the raw milk consumers (20.0 ± 0.64 vs 21.4 ± 0.57, P=0.05).

Table 3.3. BMI, self-reported physical activity and sun exposure among subjects.

Variables Raw milk Conventional milk n % n % Type of milk consumed 22 38.6 35 61.4

Body mass index (BMI) category a Healthy b 12 54.5 15 42.9 Overweight c 6 27.3 10 28.6 Obese d 4 18.2 10 28.6

Self-reported physical activity level e Sedentary f 0 0.0 7 20.0 Low active g 8 36.4 10 28.6 Active h 11 50.0 14 40.0 Very active i 3 13.6 4 11.4

Self-reported sun exposure j Less than 15 minutes per day 7 31.2 11 31.4 15 to 30 minutes per day 7 31.2 10 28.6 31 minutes to 1 hour per day 6 27.3 12 34.3 Greater than 1 hour per day 2 9.1 2 5.7 a BMI is defined as weight for height (kg/m2); ANOVA within row F=1.298, P = 0.259 b Healthy = BMI of 18.5 to 24.9 c Overweight = BMI of 25 to 29.9 d Obese = BMI ≥ 30 e ANOVA within row F = 2.176, P = 0.146 f Sedentary = little or no exercise in the week g Low active = exercise/sports for 30 minutes 1 to 3 days a week h Active = exercise/sports for 30 minutes 3 to 5 days a week I Very active = exercise/sports for 30 minutes 6 to 7 days a week j Sun exposure on hands and face. ANOVA within row F = 0.001, P = 0.98

However, when gender (Table 3.4) and age (Figure 3.2) were accounted for, there was no significant difference between the consumer groups. Although not significantly different, raw milk consumers had mean WC and SAD measurement below the measurements for increased risk of metabolic syndrome when examined by gender, while conventional milk consumers had measurements higher than the current recommendation in both genders (Table 3.4). Physical activity was also assessed between groups by self-reported physical activity level, and there were no significant differences between the groups (Table 3.3).

Table 3.4. Body composition measures of obesity and central adiposity by gender.

Body composition Type of milk consumed a measurement Raw milk Conventional milk Male Female Male Female

Body mass index (kg/m2) b 25.4 ± 1.21 26.6 ± 1.35 27.0 ± 1.54 28.1 ± 1.23 Waist circumference (cm) c 94.4 ± 4.23 86.5 ± 3.33 103.9 ± 4.23 94.0 ± 4.68 Sagittal abdominal diameter 20.8 ± 0.89 19.6 ± 0.86 22.0 ± 1.85 21.5 ± 1.14 (cm) d a Mean ± SEM b BMI is defined as weight for height (kg/m2); ANOVA within row and sex. ANOVA within row and male F=0.61, P=0.44. ANOVA within row and female F=0.61, P=0.44. c Waist circumference is defined as the horizontal distance around the abdomen at the iliac crest. ANOVA within row and male, F=2.01, P=0.16. ANOVA within row and female F=1.43, P=0.24 d Sagittal abdominal diameter is defined distance from the back to top of the stomach at the iliac crest. ANOVA within row and male F=3.19, P=0.09. ANOVA within row and female F=1.47, P=0.23

A 40

) ) 2 20 10

BMI (kg/m BMI 0 18 - 34 35-49 ≥ 50 Age (years)

120 B 100 80 60 40 20 (cm) 0

18 - 34 35-49 ≥ 50 Waist circumference Waist Age (years)

C 30 20

Diameter (cm)Diameter 18 - 34 35-49 ≥ 50 Sagittal Sagittal Abdominal Age (years) Raw Conventional

Figure 3.2. Body composition measurements of subjects by age (mean ± SEM). Age categories were defined by young adulthood (18-34); middle adulthood (35-49) and older adulthood (≥ 50). A) The Body Mass Index (BMI), as defined by a kg/m2 of subjects. ANOVA within milk consumption and ages 18-34 F=0.105, P=0.75. ANOVA within milk consumption and ages 35- 49, F=0.06, P=0.80. ANOVA within milk consumption and ages ≥ 50, F=2.71, P=0.12 B) Waist circumference is defined as the horizontal distance around the abdomen at the iliac crest. ANOVA within milk consumption and ages 18-34 F=0.46, P=0.50. ANOVA within milk consumption and ages 35-49, F=0.69, P=0.42. ANOVA within milk consumption and ages ≥ 50, F=4.2, P=0.06 C) Sagittal abdominal diameter is defined distance from the back to top of the stomach at the iliac crest. ANOVA within milk consumption and ages 18-34 F=0.78, P=0.39. ANOVA within milk consumption and ages 35-49, F=1.27, P=0.28. ANOVA within milk consumption and ages ≥ 50, F=0.50, P=0.49. 34

In addition to raw milk consumers having a higher dietary intake of total fat and saturated fat, the raw milk itself had considerably more total fat, both in the milk as it was consumed and as it was received by the subjects (Table 3.5). The raw milk was approximately 5% milk fat, compared to the 2% milk fat of the mean conventional milk. This would be the equivalent to 11 g of fat per 8 oz serving of fluid raw milk, compared to 3.5 g/8 oz service of milk fat in conventional milk. The caloric impact would be approximately 180 kcals per serving of raw milk and 120 kcal per serving of conventional milk. The amount of milk consumed did not differ between the groups, accounting for 15.8% of total grams consumed in the raw milk group and

14.6 % in the conventional milk group.

Table 3.5. Milk fat in subject samples. Sample provided Type of milk consumed a Raw milk Conventional milk

As consumed (g/mL)b 0.05 ± 0.003 ** 0.02 ± 0.002 As received (g/mL)c 0.05 ± 0.003 ** 0.02 ± 0.002 a Mean ± SEM c As consumed is defined as the milk the subject consumed; fat and skim may have been separated; ANOVA within row F=72.369, P≤0.001. Raw milk ≥ conventional milk d As received is defined as milk before any subject manipulation; fat is intact F=55.310, P≤0.001. Raw milk ≥ conventional milk ***P≤0.001

The fatty acid composition also differed between the milks. Raw milk was higher in palmitic, α-linolenic and c9t11-CLA and conventional milk was higher in linoleic acid (Figure

3.3A). Although, when the percentages were converted into grams per 8 oz serving, raw milk was significantly higher in each of the fatty acids selected for review in this study (myristic acid

P≤0.001; palmitic acid P≤0.001; stearic acid P≤0.001; oleic acid P≤0.001; linoleic acid P≤0.001;

α-linolenic P≤0.001; c9t11-CLA≤0.001; t10c12-CLA P=0.003) (Figure 3.3B). 35

40 *** A 35 30 25 20 15 10 5 *** ***

% oftotal % fat milk 0 Myristic Palmitic Stearic acid Oleic acid Linoleic acid α-Linolenic c9t10-CLA t10c12-CLA acid (14:0) acid (16:0) (18:0) (18:1n9) (18:2n6) acid (18:2n6) (18:2n6) (18:3n3) Fatty acid (mean ± SEM) 5 B *** 4 3 *** 2 *** ***

gms fat fat gms peroz 8 1 *** *** *** ** 0 Myristic Palmitic Stearic Oleic acid Linoleic α-Linolenic c9t11 -CLA t10c12 -CLA acid acid acid acid acid

Fatty acid (mean ± SEM) Raw Conventional

Figure 3.3 Average fatty acid composition of raw and conventional milk. A) As consumed is defined as the milk the subject consumed; fat and skim may have been separated. ANOVA within group and myristic acid F=3.70, P=0.06. ANOVA within group and palmitic acid F=11.90, P=0.001. ANOVA within group and stearic acid F=0.52, P=0.48. ANOVA within group and oleic acid F = 0.36, P = 0.55. ANOVA within group and linoleic acid F=42.78, P≤0.001. ANOVA within group and α-linolenic acid F=44.94, P≤0.001. ANOVA within group and t9c10-CLA F=4.22, P=0.05. ANOVA within t10c12-CLA F=3.26, P=0.08 B) Fatty acid grams of fat in 8 oz serving of milk. ANOVA within group and myristic acid F=79.32, P≤0.001. ANOVA within group and palmitic acid F=92.10, P≤0.001. ANOVA within group and stearic acid F=101.71, P≤0.000. ANOVA within group and oleic acid, F=69.00, P≤0.001. ANOVA within group and linoleic acid F=22.43, P≤0.000. ANOVA within group and α-linolenic F=85.56, P≤0.001. ANOVA within group and c9t10-CLA, F=85.67, P≤0.001. ANOVA within group and t10c12-CLA F=10.97, P=0.003 * P ≤ 0.05 **P ≤ 0.01 ***P ≤ 0.001

3.4 Discussion

In this study raw milk consumers in Ohio were more likely to be Caucasian, highly educated and high-income (Table 3.1). This conflicts with a study published in 1997 that investigated the demographics of raw milk consumers in California and found that they were more likely to be young, Hispanic, low-educated, and low-income men (3). This difference may be due to the time and geographic differences between the studies. To our knowledge these two studies appear to be the only two studies examining demographics of raw milk consumers; more research nationwide will need to be conducted to accurately determine the general demographic characteristics of raw milk consumers in the United States.

There were no differences in caloric, calcium and vitamin D intake from the food record data (Table 3.2). However, when supplements were removed, raw milk consumers had significantly lower vitamin D intake. Since the majority of dietary vitamin D intake in Americans comes from fortified dairy products, raw milk consumers have fewer opportunities to get adequate vitamin D from whole food sources (7). To meet their daily needs, they may rely on supplements, primarily during the winter months. While raw milk consumers took significantly more supplements (P=0.05), it is plausible that vitamin D supplement use may be higher during the winter months, due to the lower endogenous vitamin D production. Although high vitamin D and calcium intakes have been linked to various favorable health outcomes (4, 33), since there is little difference between groups, it is unlikely that these nutrients will have an impact on any health disparities observed in the subjects.

Although total caloric intake was similar between groups, raw milk consumers had much higher total fat intake than conventional consumers (Figure 3.1). To keep the caloric intake similar to the conventional milk consuming group, it appeared that raw milk consumers compensated total carbohydrate intake for the additional fat intake, as they had less carbohydrate

37 consumption than conventional milk subjects when total energy intake was accounted for. In particular, the raw milk consuming subjects had much higher saturated fat intake, while mono- and polyunsaturated fats were similar between groups. The Weston A. Price Foundation, a vocal proponent of raw milk, promotes a high fat and especially high saturated fat diet (17). Coconut oil intake in particular is strongly encouraged (17), which consists of 92% saturated fat (7). On the other hand, health care professionals support a low-saturated fat, high-monounsaturated fat and polyunsaturated fat diet with the consumption of olive and canola oils (7). The current national recommendation is for total fat intake to be less than 30% of total caloric intake, and more importantly, for total saturated fat to be less than 10% of total calories (7). Previous studies have showed that an even further decrease in total fat to 25% of calories and saturated fat to 6% can lower total cholesterol by 9% (67). Myristic and palmitic acids are predominately the saturated fats that have been shown to raise total cholesterol (11, 44), which is a contributing factor to cardiovascular disease and also increased glucose intolerance (11, 44). Biochemical tests will need to be examined to determine if the high fat and saturated fat intake observed among raw milk consumers has any effect on lipid levels and glucose intolerance.

Although BMI was similar between groups, raw milk consumers’ WC was moderately smaller and SAD was significantly smaller than conventional milk consumers. This difference was attenuated when age (Figure 3.2) and gender (Table 3.4) were examined. However, the mean

WC and SAD for raw milk consumers based on gender placed them below the risk threshold for developing obesity-related diseases, while conventional milk consumers were above the threshold points in both WC and SAD. The lack of significance within these measurements was most likely due to the small sample size. More studies with a larger sample size will be needed to unequivocally determine if there is a cause and effect relationship between raw milk consumption and central adiposity.

Although there were differences in central adiposity between the groups, there was no difference in the prevalence of obesity; on average, both raw and conventional milk consumers were classified as overweight. With BMI being similar, but central adiposity lower in raw milk subjects, it suggests that raw milk consumers may have greater lean body mass than conventional milk consumers. More lean body mass would increase weight, thus raising BMI, without affecting central adiposity. Increases in lean body mass are often attributed to increase in physical activity; however, there was no difference in self-reported physical activity between groups (13). The lack of difference in physical activity measures may have been an artifact of season or the sensitivity of the measurement tool used in this study. Another possibility is that body fat in raw milk consumers may be less centrally located. A higher prevalence of gynoid adiposity (as opposed to central adiposity) may be possible. Although having similar body fat, gynoid adiposity is considered more favorable than central adiposity when evaluating risk factors for metabolic syndrome (63). Skinfold measurements or direct measures of body fat percentage

(bioelectric impedance analysis, underwater weight, BodPod or iDEXA) may be useful to more accurately investigate this phenomenon.

The observation that the milk samples from raw milk subjects had a greater milk fat content in their milk was consistent with the observation that they consumed more overall dietary fat. The mean fat percentage of the raw milk was approximately 5%, while conventional milk was approximately 2% (Table 3.5). This would translate into an average of 11 g of fat per cup of raw milk compared to 3.7 g of fat per cup of conventional milk. The total grams of fat in raw milk are considerably higher than the average milk fat of 33g/L (8 g/8oz) in whole milk; conversely the conventional milk is considerably lower than this average (43).

Although the carbohydrate and protein levels may slightly fluctuate, most of the variability in caloric content in milk is due to the fat content (11). Based on the total fat content

39 in this study, raw milk would have approximately 180 calories per cup while conventional milk would have 120 calories per cup. The raw milk consuming subjects may have been aware of the differences in caloric content of the milk and compensated appropriately, since total daily calorie intake was not different between the groups. In addition, the daily consumption of fluid milk was similar between raw and conventional milk consumers (accounting for 11% and 8% of total caloric intake, respectively). Thus, although the caloric content of the milk was higher, the added caloric contribution of the milk in the diets of the raw milk subjects did not appear to increase body weight.

The higher fat content of the milk could be due to a variety of factors. Firstly, the lack of processing means that the fat is often not removed prior to consumption in raw milk. When examining the milk as it was consumed by the subjects and as it was received (either from the store or herdshare), there was no difference in the milk samples between the two in both groups of subjects, suggesting that milk fat was not skimmed by the subjects. Raw milk advocates propose that the inherent, protective benefits of the milk are found in the butterfat (17), so fat content remained intact. Thus the fat percentage of raw milk would be solely dependent on the fat production of the animal the milk came from. In addition, the fat content itself is highly variable (8). The fat production is dependent upon the breed of cow, feed and season of the year

(8). Jersey cows, which are one of the supported breeds by raw milk advocates, have a much higher total fat production and higher saturated fat content compared to other bovine dairy breeds

(10). Holstein cows, which are commonly used in large dairy operations (8), have a much lower overall fat production and higher mono- and polyunsaturated fatty acid composition (8). With this considered, it is not surprising that raw milk had a much higher overall fat content than conventional milk.

In addition to the total fat content varying based on the breed of cow, feed and season, the specific fatty acid composition of the milk can change based on these same factors. As shown in

Figure 3.3, raw milk contained significantly more palmitic acid than conventional milk, which is the single highest fatty acid in both milks. Palmitic acid accounted for 4.1 ± 0.24 g of total milk fat in raw milk and 1.3 ± 0.18 g in conventional milk. In combination with myristic and stearic acid, long-chain saturated fatty acids accounted for nearly 55% of the total fatty acids found in the raw milk, which is similar to previous studies which show milk naturally containing approximately 60% saturated fat (2, 11). The higher fat content of the raw milk, in addition to the higher total dietary saturated fat intake may place raw milk consumers at an even higher risk for increased total cholesterol and glucose intolerance than people consuming conventional milk.

Oleic acid, a monounsaturated fatty acid, is the highest unsaturated fat in milk, accounting for nearly 2.4 ± 0.05 g of fat in raw milk and 0.80 ± 0.11 g in conventional milk in this study. On average, oleic acid accounts for 25% of total milk fat (Haug), but it only represented 18% of fat in the raw milk samples in this study. Conversely, the conventional milk was closer to the average with oleic acid accounting for 26% of total milk fat. Similar to other milk fatty acids, if there was a difference in the breed selection of the dairy cows, it could have an effect on the fatty acid profile of the milk.

While the oleic acid percentages were not significantly different between groups, when calculated into overall grams per serving, raw milk had significantly more oleic acid than conventional milk. This effect is most likely due to the higher total fat of raw milk. Thus, the actual fatty acid content of the milk may be more important factor to consider than the overall difference in percentage of composition, as it better represents actual intake from milk. The higher oleic acid intake from the milk may prove beneficial in raw milk consumers, as oleic acid has been shown to lower total cholesterol, LDL and triglycerides (41). It is plausible that the

41 higher oleic acid content of milk may be able to counteract the plasma lipid raising effects of the saturated fat in milk.

Raw milk had a greater content of the polyunsaturated fatty acids, although the percentage of linoleic acid was greater in conventional milk. Raw milk contained 0.22 ± 0.01 g compared to the 0.11 ± 0.02 g per 8 oz glass of conventional milk (P ≥ 0.000); however, both of these are lower than the reported average content of 1.2g/l (0.29 g/8oz) (43). Similarly raw milk contained 0.12 ± 0.01 g of α-linolenic acid while conventional milk contained only 0.02 ± 0.002 g

(P ≥ 0.000) and both are lower than the average content of 0.75 g/l (0.18 g/ 8oz) (43). Linoleic and α-linolenic acids have opposing biological functions, and recent research suggests that a low omega-6: omega-3 ratio is optimum for overall health (11). Raw milk had a nearly 1:1 ratio while conventional milk had a 5:1 ratio, suggesting that raw milk consumers may have better health outcomes related to a more favorable polyunsaturated fatty acid ratio from their milk.

Conjugated linoleic acid (CLA), primarily the c9t11 isomer, was higher in the raw milk than conventional milk, although the total contribution was small in both types of milk. On average, it represented only 0.6% of total milk fat (11), and in this study it represented 0.67% in raw and 0.60% in conventional milk(P≤0.05). This corresponded to 0.08 ± 0.005 g of CLA in the raw milk samples and 0.02 ± 0.003 g (P≥0.000) in the conventional milk samples. This difference in CLA content was not surprising as it has been previously shown that milk from pasture-fed cows have twice the CLA content as grain fed cows (8). CLA content also varies by season, with the highest concentration during the summer months and the lowest concentration during the winter months (8). With data collection occurring in this study during the winter months, it is plausible that the CLA content was more similar between the milks than it would have been if data collection occurred at a different time of the year. This suggests that the

42 contrast in CLA content may be even greater during the summer months, with raw milk consumers having a considerably higher CLA intake than conventional milk consumers.

Many animal studies have shown that CLA can have a pronounced effect on many metabolic factors. Of importance, CLA has been linked with a decrease in total body fat mass and

BMI. Most of these effects have been shown in animal studies (57, 68), but it has been shown to have some effect in humans (48, 45). However, these effects have been shown in studies where

CLA was supplemented in high doses. One study showed that body fat mass was reduced when a supplement of 3.4 g of CLA was taken per day (58), but another study showed no significant weight or fat mass reduction when supplemented with 3.0 g per day (69). To achieve the 3.4 g of

CLA per day that has been associated with fat mass reduction from raw milk it would require approximately 42 servings of milk per day, which would provide 7560 kcals from milk alone.

Although not directly studied, it seems improbable that adequate CLA can be obtained through dietary measures alone. Thus, CLA may not necessarily account for the effect on reduced central adiposity of the raw milk subjects observed in this study.

Between the high total dietary intake and intake from milk, the raw milk subjects consumed considerably more total fat and saturated fat than the conventional milk consumers.

This is in contrast to the current recommendation for fat consumption by various health professionals in the US (7). High saturated fat intake has been linked with an increase in total cholesterol and glucose intolerance which are a risk factor associated with cardiovascular disease and type 2 diabetes mellitus (11, 44). Conversely, raw milk consumers had lower overall central adiposity which is below the cutoff points for the risk factors of cardiovascular disease and diabetes. With this conflicting data more research needs to be conducted on the biochemical/metabolic implications of consuming raw milk. Blood analyses may be more useful

43 in determining true risk factors for nutrition-related chronic disorders, such as cardiovascular disease and type 2 diabetes mellitus.

When all factors are considered, there is an inconsistent response for the possible effects of raw milk consumption on the health status of individuals. While the raw milk subjects had a considerably higher total fat and saturated fat intake which are linked to an increased cardiovascular disease and type 2 diabetes mellitus, they also had reduced central adiposity and increased CLA intake, theoretically placing them at a lower risk for developing obesity-related diseases. When considering dietary milk intake alone, it appears that raw milk consumption may be more harmful than conventional milk. However, when looking at actual body composition it appears that that the raw milk consumption may be beneficial. It is possible that this effect may be due to the raw milk lifestyle rather than the milk itself. Since the majority of raw milk consumers strongly believe in the health benefits associated with milk (17), they may be more inclined to follow an overall healthier lifestyle. In addition, more investigation is needed to determine the health status of raw milk consumers, particularly the serum lipids and glucose tolerance. More data is needed before tailoring nutrition messages specifically to raw milk consumers.

Chapter 4

Epilogue

4.1 Study Conclusions and Major Findings

This study demonstrated that the main dietary difference between raw and conventional milk consumers was in total fat and saturated fat intake from both the overall diet and the milk consumed. In addition, central adiposity was smaller in raw milk consumers, although there was no difference in BMI. Thus our study offers conflicting data regarding the potential health impact of raw milk consumption. The high saturated fat intake theoretically places consumers at a higher risk of obesity-related diseases, but reduced central adiposity places them at a decreased risk of the same disorders. It remains unclear if raw milk is beneficial or harmful regarding nutritional health status.

4.2 Limitations of the Study

One of the main limitations of the study was the small sample size. Since this was a pilot study, it was anticipated that sample size would be lower. However, to be able to explain the average raw milk consumer’s nutrient intake and body composition, a larger, more geographically diverse study needs to be conducted. The subjects were also a convenience sample of those who volunteered to participate. Because of this there may have been differences between the individuals who participated and those who chose not to. We cannot generalize the results of this study to anyone besides the study subjects. Another limitation was that physical activity was not directly measured. The physical activity plays an important role in obesity and lean body mass,

45 so self-reporting physical activity may not have accurately captured the impact this may have played on obesity and central adiposity. The use of food records may have also been a limitation.

Some subjects may have altered their eating habits, so the record did not capture true nutrient intake. However, there is no gold standard measurement of nutrient intake and we felt this was the best method for collecting information in this study.

4.3 Future Directions for Study

Firstly, an additional study with a larger and more geographically-diverse sample, randomly selected, should be conducted from this pilot study to be able to more accurately assess any differences in diet, physical activity and other lifestyle factors between raw and conventional milk consumers.

Since the data was inconsistent regarding risk factors for obesity-related diseases, future research should investigate the biochemical markers, such as serum lipid levels, glucose tolerance and insulin sensitivity. Present data can only speculate on the impact that the high fat and saturated fat diet may play on internal markers of cardiovascular disease and type 2 diabetes mellitus. A more direct measure of cardiovascular and diabetic risk is needed.

In addition, fatty acid composition of conventional and raw milk should be compared to grass-fed, pasteurized milk to determine if the fatty acid profile is affected by the lack of pasteurization or the type of feed given to the cows. Further examination of the breed of cows in each milk sample is also necessary to be able to limit any confounders that may have been introduced from the different breeds of dairy cows.

Lastly, scientific investigation is needed to validate or refute the nutrient claims made by raw milk advocates. There is much anecdotal information about raw milk aiding in lactose intolerance, digestive disorders, autism, cancer and heart disease, and other disorders and diseases; however, there is no scientific literatures to either support or counter these claims.

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Appendix A: Recruitment Flyer

Do you drink MILK?

We Need You For a Milk and Dairy Products Focus Group and Health Assessment Study

Researchers with the Department of Human Nutrition at Ohio State University are seeking people who drink milk or eat dairy products to participate in a focus group discussion and health assessment regarding safety and the nutrition quality of milk.

Participants will meet twice for up to 2 hours to on each study day:  Complete a brief (approximately 30 minute) survey  Participate in a moderated discussion group with 5 to 10 others  Participant in a short health assessment  Complete a dietary assessment  Provide us with a sample of your milk for nutrient analysis

When:  To be announced – please call for listing of dates and times

Where  Ohio State University Campus, Campbell Hall  OARDC – OSU Wooster Campus – Food Animal Health Program

Time Involved:  About half a day

Compensation:  Up to $50 cash  Refreshments

How do you sign up? Please call Janet Buffer, M.S., R.D., Project Coordinator at 614 247-8388 Lydia C. Medeiros, Ph.D., R.D., Project Director at 614-292-2699

Appendix B: Press Release

Volunteers Needed for Milk Study in Ohio

More people are choosing to drink raw milk these days, and Ohio State University researchers are hoping to find out why. We are seeking 60 participants who drink raw milk who either live on a farm or in a town or city, and 30 participants who drink pasteurized milk and live on a farm. The farm can be either a dairy farm or a non-dairy production farm. Participants must be 18 years or older. Volunteers will be asked to meet with researchers to complete a written survey, take part in a health assessment, supply a four-day dietary intake record, provide a sample of milk they normally drink, and take part in a 1.5-hour-long focus group session. They will be paid $50 for their time and trouble. To participate in the study or to learn more about locations and dates, contact Lydia Medeiros at 614 292-2699 or Janet Buffer at 614 247-8388.

Appendix C: Research Match Recruitment

ResearchMatch Recruitment Message for This Protocol

The message placed in the text box below, under photo of the girl and the dandelion and under the email greeting that says: ―A research team with The Ohio State University in

Columbus, OH, believes you might be good match for the following study‖ is the message that the volunteers will receive about this protocol. This initial email will be routed to those

ResearchMatch volunteers who fit your inclusion criteria; ResearchMatch will provide hyperlinks at the close of the message that will allow volunteers to respond yes or no to the invitation to release their contact information to this study. The recruitment language the research team enters into this form SHOULD NOT include identifiable contact information such as email address or phone numbers. This will help ensure that volunteers respond through the ResearchMatch quick links provided in the email message they receive regarding this study. (Researchers: Feel free to enlarge the text box and move the text below it to the next page if needed.)

Volunteers will see the message. They are asked to click ―yes‖ or ―no‖ in response to the recruitment message. A ―yes‖ response will release their contact information to the researcher. If volunteers click ―yes‖ they are again reminded that their contact information will be released. If the volunteer clicks ―no‖ or ignores the message, the researcher will not receive any information about the potential volunteer.

Sender: [email protected]

Message Subject: ResearchMatch – you may be a good match for this research study!

This window contains the contents of the email that you will be sending out to your selected volunteers. Please proofread and confirm the information you are sending is valid and correct. If you need to make changes, simply click on 'close preview'. If you want to submit this email message, click on 'contact volunteers'.

A research team with The Ohio State University in Columbus, OH, believes you might be good match for the following study:

Enhancement of Educational and Extension communications for the Prevention of Milkborne

Diseases.

DO YOU DRINK MILK? We need you for a milk and dairy products focus group and health assessment

study. We are seeking people who drink milk or eat dairy products to participate in a focus group discussion and a health assessment regarding the safety and nutrition quality of raw and pasteurized

milk. Study participants will:

 Meet twice for up to 2 hours on each study day;  Participate in a moderated discussion group with 5-10 other participants;  Participate in a short health assessment;  Complete a food record;  Provide a sample of milk;  And complete a brief survey.

Participants will be compensated up to $50 in cash.

There are 2 research sites, OSU-Columbus Campus and OSU Wooster Campus. If you are interested in this study and having the research team contact you directly, please select the "Yes, I'm interested" link below. By clicking the "Yes, I'm interested" link, your contact information will be released to the research team. If you select the "No, thanks." link or do not respond to this study message, your contact information will not be released to the research team. Yes, I'm interested! No, thanks. 56

You are receiving this email message since you have registered in the ResearchMatch registry. Should you wish to edit your profile or remove your contact information from this registry, please login here. ResearchMatch Disclaimer ResearchMatch is a free and secure tool that helps match willing volunteers with eligible researchers and their studies at institutions across the country. ResearchMatch is only providing a tool that allows you to be contacted by researchers about their studies. ResearchMatch therefore does not endorse any research, research institution, or study. Any recruitment message that you may receive about a study does not mean that ResearchMatch has reviewed the study or recommends that you consider participating in this study.

Appendix D: Phone Call Script

Enhancement of Educational and Extension Communications for the Prevention of Milkborne Diseases

Hello, I am Dr. Lydia Medeiros [Janet Buffer, Alyssa Mark] and we are conducting a focus group and health assessment study with people who drink milk or eat dairy products. Thank you for your interest in participating in the study. First I need to ask you some questions to be sure that you meet our requirements for participation in a research study.

Are you 18 years or older?

If yes, continue If no, thank the potential subject for their interest but explain that only people 18 years or older are eligible to participate.

Do you drink milk or eat dairy products?

If yes, continue If no, thank the potential subject for their interest but explain that only people who drink milk or eat dairy products are eligible to participate.

Do you drink unpasteurized milk?

If yes, ask ―would you be interested in taking part in a focus group with other people who consume raw milk as well as complete a food record?”. Provide information regarding the scheduled focus group and health assessment study location. Ask for contact information in order to send directions and a map to the focus group location. (State their personal information will not be linked in any way to the research data collected

If no, ask ―would you be interested in taking part in a focus group with other people who consume raw milk as well as complete a food record? Provide information regarding the scheduled focus group and health assessment study location. Ask for contact information in order to send directions and a map to the focus group location. State that their personal information will not be linked in any way to the research data collected.

Is anyone else in your household also participating in this study?

If yes, explain that only one person per household is eligible to participate in the study. Thank them for their interest and that you hope they will be interested in other studies in the future.

If no, tell them that they are eligible to participate in the study and proceed with the script.

Are you willing to provide us with a sample of the milk you drink so we can analyze it for vitamin D and fat content?

If no, thank them anyway. We still welcome them to participate in the focus group and health assessment parts of the study.

If yes, tell them you will provide them with a container at the focus group to fill with milk they typically drink and bring back to the health assessment.

Explain they will be paid up to $50 and that the focus group and health assessment will last up to 2 hours. The 4-day dietary record will take about 30 minutes, but up to 1 hour per day to complete for 4 hours total.

Summarize what the potential participate has agreed to at this point based on the information above. (this way both you and s/he are clear) If they are going to participate in the physical assessment explain what will be involved. They will be asked to lie on a table with their shirt lifted above their waist and their slacks or shorts rolled down below their waist. Suggest they bring a pair of elastic waist shorts with them. Height will involve the removal of their shoes.

Send location and parking information to recruited subject.

Appendix E: Email Script

Hello,

I received your contact information through (Research Match, your email) that you may be interested in participating in our study.

To provide you with a little more information about the study itself, the purpose of the study is to examine the differences between raw and pasteurized milk consumers, focusing on decision making models, dietary intake, health assessment and fatty acid composition of the milk consumed. As a participant you would be asked to: 1. Attend an hour to 2 hour focus group with other like milk consumers (either pasteurized or raw). 2. Complete a 4 day food record (that will be mailed to you ahead of time) 3. Complete an opinion survey (distributed after the focus group) 4. Provide us with a sample of the milk in your household (sample tubes will be mailed to you ahead of time) 5. Attend a health assessment appointment where height, weight, waist circumference and sagittal abdominal diameter are measured that will take no more than an hour

Overall, the focus group and assessment will be conducted the same day, lasting no more than 4 hours.

To make sure that you meet all of our criteria and that we get you into the correct group, can you please answer the questions below for me. 1. Do you drink milk or consume dairy products? 2. Do you consume raw or pasteurized milk? 3. Is anyone else in your household participating in the study? 4. Do you live on a farm? 5. Would you be willing to supply us with a sample of the milk in your household? 6. In what town do you currently reside?

After all of this information if you are still interested, just answer the questions and let me know what session works best for you and we will get you set up. Also, please let me know if you have any questions.

Again, thanks for your interest and I look forward to hearing from you soon! Alyssa Mark Graduate Research Associate Department of Human Nutrition The Ohio State University Campbell 319 60

Appendix F: Consent Form

The Ohio State University Consent to Participate in Research

Study Title: Enhancement of Educational And Extension Communications for the Prevention of Milkborne Diseases Researcher: Dr. Lydia Medeiros

Sponsor: USDA, National Integrated Food Safety Initiative This is a consent form for research participation. It contains important information about this study and what to expect if you decide to participate.

Your participation is voluntary. Please consider the information carefully. Feel free to ask questions before making your decision whether or not to participate. If you decide to participate, you will be asked to sign this form and will receive a copy of the form.

Purpose:

The purpose of the study is to examine the nutrient intake of those consuming raw milk when compared to those consuming pasteurized milk and to determine if any differences in nutrient intake has any influence on obesity and central adiposity. By raw milk we mean any milk or dairy product that was not pasteurized or homogenized before consumption. By conventional milk we mean any milk or dairy product that has been pasteurized, homogenized and fat content standardized.

Procedures/Tasks:

Upon arrival, you will be taken to a private room where data collection will begin. Your 4-day food record will be reviewed to ensure completeness. Anthropmetric measurements will be gathered, these include our height, weight, waist circumference and sagittal abdominal diameter. Measurement of your waist and sagittal diameter will require you to lift your shirt above your waist and to loosen the waist of your slacks or shorts. All data collection will be conducted by employees of The Ohio State University.

Duration:

This session should last approximately 2 hours. You may leave the study at any time. If you decide to stop participating in the study, there will be no penalty to you, and you will not lose any benefits to which you are otherwise entitled. Your decision will not affect your future relationship with The Ohio State University.

Risks and Benefits:

The information gained from this study will help health professionals understand any nutritional differences between raw and conventional milk consumers. The result may be improved information that helps us tailor nutrition interventions and diet plans to raw milk consumers.

It is not possible to identify all potential risks in research procedures, but the researchers have taken reasonable safeguards to minimize any known and potential, but unknown, risks. It is not anticipated that any of the questions asked will lead to emotional distress; however, if you are uncomfortable for any reason, you have the option to leave the discussion group at any time without recourse

Confidentiality:

Efforts will be made to keep your study-related information confidential. However, there may be circumstances where this information must be released. For example, personal information regarding your participation in this study may be disclosed if required by state law. Also, your records may be reviewed by the following groups (as applicable to the research):  Office for Human Research Protections or other federal, state, or international regulatory agencies;  The Ohio State University Institutional Review Board or Office of Responsible Research Practices; There will be no identifying information that will link your name or any other personally identifiable information to your survey, food –record, raw milk sample (if one is obtained) and anthropometric measurements.

Incentives:

You will be paid $15 for the completion of the food record, $15 for the completion of the milk survey, $5 for a milk sample and $5 for your participation in the physical assessment to compensate you for your time and participation.

Participant Rights:

You may refuse to participate in this study without penalty or loss of benefits to which you are otherwise entitled. If you are a student or employee at Ohio State, your decision will not affect your grades or employment status.

If you choose to participate in the study, you may discontinue participation at any time without penalty or loss of benefits. By signing this form, you do not give up any personal legal rights you may have as a participant in this study.

An Institutional Review Board responsible for human subjects research at The Ohio State University reviewed this research project and found it to be acceptable, according to applicable state and federal regulations and University policies designed to protect the rights and welfare of participants in research.

Contacts and Questions: For questions, concerns, or complaints about the study you may contact Dr. Lydia Medeiros at 614 292-2699 or [email protected]

For questions about your rights as a participant in this study or to discuss other study-related concerns or complaints with someone who is not part of the research team, you may contact Ms. Sandra Meadows in the Office of Responsible Research Practices at 1-800-678-6251.

If you are injured as a result of participating in this study or for questions about a study-related injury, you may contact Dr. Lydia Medeiros at 614 292-2699 or [email protected]

Signing the consent form

I have read (or someone has read to me) this form and I am aware that I am being asked to participate in a research study. I have had the opportunity to ask questions and have had them answered to my satisfaction. I voluntarily agree to participate in this study.

I am not giving up any legal rights by signing this form. I will be given a copy of this form.

Printed name of subject Signature of subject

AM/PM Date and time

Printed name of person authorized to Signature of person authorized to consent for consent for subject (when applicable) subject (when applicable)

AM/PM Relationship to the subject Date and time

Investigator/Research Staff

I have explained the research to the participant or his/her representative before requesting the signature(s) above. There are no blanks in this document. A copy of this form has been given to the participant or his/her representative.

Printed name of person obtaining consent Signature of person obtaining consent

AM/PM

Appendix G: Food Record

ID #______

Food Record Sheet Instructions

 Record each meal and snack right after you eat it for 4 days (3 weekdays and 1 weekend) in the same week.  List the time and under the corresponding meal. You do NOT have to consume food at every meal listed or use every line provided.  Write each food, beverage, or supplement on a separate line, skipping a line in between each food. If there are multiple ingredients in a homemade food then list each ingredient on a separate line, making sure to skip lines between each ingredient.  Fully describe each food, beverage and supplement. List name brands, if applicable, and the main ingredients in homemade foods  Write down the amounts of the foods, beverages and supplements. For foods, measure using cups, spoons, inches or the food label. For beverages use fluid ounces.  Use as many sheets as necessary, making sure to date each page.  If you have any questions please call Janet Buffer at 614-247-8388.

Sample Food Record

ID#_NFP 01______

Day: _ Monday______Date: 07_/_29_/2010

Time Breakfast Food or Beverage Amount 8:30 Multigrain Cheerios 1 cup

Milk, skim 4 oz

Orange juice, Minutemaid Low Pulp + Calcium 8 oz.

10:45 SNACK Water, tap 24 oz.

Carrots, raw ½ cup

Celery, raw ½ cup

11:00am Green Tea, Lipton (brewed) 16 oz

Sample Food Record

ID#_NFP01______

Day: _____Monday______Date: _0 7_/_29_/2010

Time LUNCH Food or Beverage Amount 12:20 Turkey Sandwich

Whole wheat bread, Wonder 2 slices

Turkey, ** deli roasted

Lettuce, romaine 1 oz

Mustard, Heinz 1 tsp.

Apple, small 1

SNACK

Sample Food Record

ID#_NFP01______

Day: ___Monday ______Date: _07_ /29/2010

Time DINNER Food or Beverage Amount Chicken breast, grilled 6 oz

Broccoli, frozen, steamed ¾ cup

Potato, baked, ate the skin 1 medium

Margarine, Parkay 1 tbsp.

Pop, Diet Coke 12 oz can

9:30 SNACK Vanilla Ice Cream, Kroger Deluxe 1 cup

Chocolate syrup, Herseys 1 tsbp

Sprinkles, chocolate 1 ½ tsp.

Food Record

ID#______