AN ABSTRACT OF THE THESIS OF. Marcie L. Dodge for the degree of Master of Science in Nutrition and Food Management

Transcription

1 AN ABSTRACT OF THE THESIS OF Marcie L. Dodge for the degree of Master of Science in Nutrition and Food Management presented on April Title: The Effect of Selenium on the Fatty Acid Profiles of Human Breast Milk in Chinese Women. Abstract approved: Rosetrfary Wander Numerous dietary factors have been shown to influence the fatty acid profiles (FAP) in breast milk from lactating women. However, few studies have evaluated the effect of trace minerals on milk FAP. Consequently, the purpose of this study was to determine the effect of selenium status on the FAP in breast milk. Subjects were lactating women from three different regions in China; Xichang (n=21), an area where selenium intakes are among the lowest in the world, Beijing (n=20), where there are adequate selenium intakes, and Enshi (n^lq), where selenium intakes are among the highest in the world. Plasma and milk samples were obtained from women at birth of their baby and within 10 months postpartum and analyzed for selenium content, glutathione peroxidase (Gpx) activity and FAP. Plasma and breast milk selenium levels were significantly lower in the Xichang women and significantly higher in the Enshi women when compared to Beijing women. Despite the fact that the highest level of plasma selenium was measured in the samples from Enshi, the Gpx activity was greatest in the samples from Beijing; there was no effect of time of sampling on these samples. In breast milk, on the other hand, all the samples obtained at birth had similar activity of Gpx. The samples taken later, however, followed the same trend as plasma with the samples obtained from the women in Beijing having the highest activity. FAP indicated a significant difference in the amount of unsaturated fatty acids in both the plasma and milk for the Beijing women, when compared to the women from Xichang and Enshi. In particular, there were higher levels of linoleic acid, 18:2(n-6), in the plasma and milk of the women whose selenium intake was adequate.

2 Copyright by Marcie L. Dodge April 25, 1997 All Rights Reserved

3 The Effect of Selenium on the Fatty Acid Profiles of Human Breast Milk in Chinese Women by Marcie L. Dodge A THESIS submitted to Oregon State University in partial fulfillment of the requirements for the degree of Master of Science Completed April 25, 1997 Commencement June 1997

4 Master of Science thesis of Marcie L. Dodge presented on April 25 r 1997 APPROVED: Major Professor, representing Nutrition and Food Management ir «-i* it a «r Chair of Department of Nutrition and Food Management Dean of Graduare School I understand that my thesis will become part of the permanent collection of Oregon State University libraries. My signature below authorizes release of my thesis to any reader upon request. Marcie L. Dodge, Author NJ

5 ACKNOWLEDGMENT I would like to thank the laboratory of Dr. Phil Whanger from the Department of Agricultural Chemistry at Oregon State University for their assistance and contribution to my reserach project. Dr. Whanger provided me the opportunity to take part in an on-going research project, and provided the samples for my study. In particular I wish to thank Judy Butler for her assistance in analyzing and interpreting the data, and Pengchen Ha, for the cultural information she supplied. 1 am also grateful for the assistance from Dr. Dave Thomas, in the Department of Statistics at OSU. Most importantly, 1 wish to thank Dr. Rosemary Wander, my major professor, for the use of her laboratory, for her assistance and her mentorship. A special thanks to Researcher Shi-Hua Du who helped me obtain the samples from China, and assisted in the analysis and the interpretion of the data in our laboratory in the Department of Nutrition and Food Management.

6 TABLE OF CONTENTS I. Introduction ] Selenium 1 Function of selenium in plasma 1 Concentration of selenium in plasma 1 Concentration of selenium in breast milk 6 Fatty Acids 8 Content of fatty acids in breast milk 8 Content of plasma fatty acids during pregnancy 1 1 Plasma fatty acid profile and selenium concentration 12 Importance of long-chain fatty acids to infant development 13 Specific Aim 14 II. Methods and Materials 15 Overview 15 Subjects 15 Samples 16 Analytical Analyses 17 Statistical Analysis 19 III. Results 21 Dietary information 21 Demographics 21 Selenium Status 22 Selenium and protein concentrations and Gpx activities in plasma and breast milk 22 Fatty Acid Profiles of plasma and breast milk 27 IV. Discussion 43 Bibliography 49 Page

7 TABLE OF CONTENTS continued Appendices 55 Appendix A: Complete Fatty Acid Profile of Plasma Samples 56 Appendix B: Complete Fatty Acid Profile of Breast milk Samples 58 Appendix C: Plasma and Breast milk Selenium, Gpx and Protein Averages. 63 Appendix D: Detailed description of analytical analyses 65 Appendix E: Raw data 76

8 LIST OF FIGURES Figure Page 1 Plasma selenium concentration and Glutathione peroxidase activity Scatter plot of plasma selenium vs. plasma glutathione peroxidase activity Milk selenium concentration and Glutathione peroxidase activity Plasma polyunsaturated fatty acids (1) Plasma polyunsaturated fatty acids (2) Plasma monounsaturated fatty acids Plasma saturated fatty acids Milk polyunsaturated fatty acids (1) Milk polyunsaturated fatty acids (2) Milk polyunsaturated fatty acids (3) Milk monounsaturated fatty acids Milk saturated fatty acids Plasma Gpx vs. linoleic acid Milk Gpx vs linoliec acid, early samples Milk Gpx vs. linoleic acid, late samples 42

9 LIST OF TABLES Table Page 1. Average age of the women and collection time of the plasma and milk samples for each location at both of the collection times Correlations of selenium and Gpx activity 38 ^. Correlations of Gpx activity and fatty acids 40

10 THE EFFECT OF SELENIUM ON THE FATTY ACH) PROFILE OF HUMAN BREAST MILK IN CHINESE WOMEN I. INTRODUCTION Selenium Function of selenium in plasma Selenium is an essential trace element important in human nutrition. It is usually associated with glutathione peroxidase (Gpx), selenoprotein P and albumin, the three major selenium containing proteins in plasma. The majority of the selenium is associated with selenoprotein P (Deagan et al. 1993) when dietary selenium intake is adequate. However, with elevated selenium intake, the majority is associated with albumin (Deagan et al. 1993) About 20% of plasma selenium is associated with the selenoprotein glutathione peroxidase (Gpx) (Deagan et al. 1993). Gpx appears to be the primary enzymatic role of selenium. It protects cells from oxidative damage (Zachara et al. 1986, Thompson et al. 1980). Gpx protects against oxidative injury by reducing hydrogen peroxide and lipid hydroperoxide to alcohol, thus removing a reactive oxygen species from cells and plasma (Diplock 1994). Concentration of selenium in plasma The current RDA for selenium for adults is between ng/day. Associated with this dietary intake of selenium are whole blood levels that range from 155 to 278

11 2 ng/ml (Whanger et al. 1988). The maximum safe level of intake for an extended period of time is less than or equal to ng/day (Whanger 1989). Deficiencies may occur at levels below 17 ng/day (Whanger 1989). The level of selenium in plasma depends primarily on dietary intake. Geographical areas with low selenium content in the soil have low levels in the food supply. Populations in these areas have low dietary intakes of selenium resulting in low levels of selenium in blood (Levander 1987). The selenium content in the soil in the People's Republic of China varies dramatically among regions resulting in intakes that range from extremely low to extremely high. Estimates of intake are from 7 to 38,000 ig/day (Levander 1987). In cities such as Shanghai and Beijing, selenium intakes are adequate and average 110 Hg/day, and plasma concentrations average 96.2 ng/ml. Low selenium areas in China run from the northeast to the southwest in a long belt. Within this belt there are small zones or "safety islands" where selenium intakes are adequate (Jiang and Xu 1988). Most of this area is rural. The inhabitants in this area often consume only foods that are grown locally, therefore their intakes of selenium reflect the soil levels of selenium. Seasonal variation in plasma selenium concentrations occur, due to the fact that field workers consume more food during the planting and harvesting seasons, when their energy requirements increase due to hard labor. Individual variations are explained in part by variable consumption of imported wheat or other selenium-rich foods (Li et al. 1988). The Sichuan Province is an area characterized by low intakes of selenium. Before addition of selenium to salt, the intakes averaged 11 (ig/day and plasma selenium concentrations averaged 17.4 ng/ml (Xia et al. 1992).

12 3 There are also areas of China where the selenium intake is high. This can result from several factors. In some areas of China, high levels of selenium are found in the soil and crops grown here become concentrated in selenium. A form of coal known as stony coal is often used as a fiiel source in China. It has a high selenium content. When burned, the smoke may contain volatilized selenium which can be inhaled by the people. This coal is also used to cook and dry foods and "smoke" the fields to fertilize the land on which the crops are grown (Whanger 1989). All of these factors contribute to extremely high intakes of selenium for certain population groups. For instance, people living in the Hubei Province in central China tend to have high intakes. They average 759 (ig/day and plasma concentrations average 494 ng/ml (Xia et al. 1992). Low plasma selenium concentrations, although not as extreme as those seen in China, have been measured in individuals from Finland and New Zealand. In these two countries the daily intakes average (ig/day and plasma concentrations average 60 ng/ml. In the United States, intakes are generally adequate and range from Hg/day. Plasma concentrations average 200 ng/ml. An exception to this is in South Dakota and nearby areas where plasma concentrations of 400 ng/ml of selenium have been reported (Whanger et al. 1988). Gpx activity is often used as a measure of selenium status, although at high intakes it is less than satisfactory as an indicator. In humans, concentrations of selenium in blood are linearly correlated with Gpx activity when selenium intake is low; however, adequate or greater selenium intakes show similar Gpx activity in blood, demonstrating that the amount of selenium associated with Gpx can become saturated (Whanger et al. 1988,

13 4 Thompson et al. 1980, Mannan and Picciano 1987). At elevated levels of selenium intake, selenium is located in proteins other than Gpx (Xia et al. 1992) Pregnancy has been shown to influence measurements of selenium status. Both lower Gpx activity and lower selenium content have been found in the plasma of pregnant women when compared to nonpregnant women (Alaejos and Romero 1995, Perrone et al. 1994, Levander et al. 1987, Lee et al. 1995, Zachara et al. 1986). For instance, in a healthy Polish population, Zachara et al. (1986) reported that plasma levels of selenium and glutathione peroxidase activity were 42% and 20% lower, respectively, in pregnant women than in nonpregnant women. However, another study demonstrated that Gpx activity gradually increased during pregnancy even though whole blood and plasma selenium levels decreased (Butler et al. 1982). The selenium concentrations in erythrocytes of pregnant and nonpregnant women are similar, suggesting that plasma levels are more sensitive to selenium intakes than erythrocytes (Alaejos and Romero 1995, Zachara et al. 1986, Mannan and Picciano 1987). Lactating women also have lower plasma selenium levels than nonlactating women (Levander et al. 1987). Selenium concentrations in blood may also be influenced by the chemical form in which it is ingested. McGuire et al. (1993) and Kumpulainen et al. (1985) showed that a supplement of organic selenomethionine was more bioavailable than an inorganic selenite supplement. Selenomethionine and selenium-enriched yeast supplements prevent the decline of plasma selenium and Gpx activity in lactating mothers (McGuire et al. 1993). High selenium wheat bread also increased plasma, whole blood and erythrocyte selenium levels, as well as increasing the Gpx activity in these cells (Thompson et al. 1985).

14 5 Supplementation with selenomethionine was shown to reult in higher plasma levels of selenium in a population of pregnant and lactating women in New Zealand than was found in unsupplemented pregnant and lactating women (Butler et al. 1995). The dietary protein level may also affect the bioavailability of selenium. At low selenium intakes, a low protein diet results in higher selenium concentrations in the blood when compared to a high protein diet. This may mask a potential selenium deficiency. On the other hand, a higher protein intake helps to prevent selenium deficiency symptoms, even on a very low selenium diet (Yang 1984). Low selenium intakes have been associated with several diseases. Keshan disease, an endemic cardiomyopathy found in China, is a disease that is geographically restricted to rural mountainous areas scattered in a belt that extends from the northeast to the southwest of the country (Levander 1987). Symptoms of the disease can be classified as acute, subacute, chronic and latent, and often involve multifocal necrosis and fibrous replacement of the myocardium. Those persons most susceptible are multiparous women and children (Levander 1987). Kashin-Beck disease is also found in China and low selenium intakes appear to be one of the factors involved. It is an osteoarthropathy, a disabling polyarticular degenerative disease whose onset typically occurs during the first or second decade of life. It can cause failure of proper bone development and enlargement of the joints (Levander 1987). High intakes of selenium can lead to health problems as well. Selenosis may occur if intakes of selenium are above 750 ng/day (Yang et al. 1983). The disease is often characterized by loss of hair and nails, which become brittle and easily broken. The skin

15 6 may incur lesions, the incidence of tooth decay increases, and in extreme cases, the nervous system may be affected (Yang et al. 1983). Selenosis can be remedied by reducing the selenium intake in the diet, and frill recovery is possible. However, symptoms of the nervous system need a longer recovery time (Yang et al. 1983). Concentration ofselenhtm in breast milk Selenium is found in breast milk but at a lower concentration than that measured in plasma. Most of the selenium in breast milk is bound to proteins. At least nine selenium containing proteins have been identified in human breast milk, one of them being Gpx (Milner et al. 1987). Gpx activity has been measured in breast milk as well as plasma (Milner et al. 1987, Mannan and Picciano 1987). Gpx accounts for only 15-30% of the total selenium in milk and the function of the other selenoproteins is still unknown (Milner et al. 1987). Several selenoproteins in breast milk may be isozymes of Gpx (Debski et al. 1988). It is believed that selenomethionine can be incorporated into general body proteins in place of methionine, whether or not this also occurs in breast milk remains to be determined (Milner et al. 1987). In addition to its relationship with proteins in breast milk, about 5% of the selenium is associated with lipids. This association may be with the outer membrane proteins surrounding the fat globule (Milner et al. 1987). The concentration of trace minerals is typically low in breast milk when compared to other body fluids. Selenium is no exception. Mannan and Picciano (1987) reported its concentration was seven times lower in breast milk than in plasma, which is an even

16 7 greater reduction in concentration than that of other trace minerals found in breast milk (Picciano 1985). As with plasma, the primary factor determining selenium concentrations in milk is thought to be dietary intake. The effect of dietary selenium on its concentration in breast milk can be approximated by an equation derived from data collected from 18 countries (Alaejos and Romero 1995). It is as follows: log(ng Se in milk/l) = log(ng Se intake/day) As plasma selenium concentration and Gpx activity increase, both milk selenium concentration and Gpx activity increase (Alaejos and Romero 1995, Mannan and Picciano 1987). Supplementing lactating women with selenium increases maternal serum selenium and corresponding increases in breast milk selenium are seen (Kumpulainen et al. 1985, Butler et al. 1995). In North America, the average level of selenium in breast milk is (ig/l (Mannan and Picciano 1987). Selenium levels in breast milk from Chinese women were reported at 3.8 ig/l from a low selenium area, 20.0 f.tg/l from Beijing, and 28.3 ig/l from a high selenium area (Yang 1984). Other dietary factors also influence the concentration of selenium in breast milk. A vegetarian diet by the mother increases the concentration of selenium and Gpx activity in breast milk, even when dietary intakes of selenium are similar (Debski et al. 1988). It has been suggested (Debski et al. 1988) that the higher selenium concentration in breast milk is associated with the higher content of linoleic acid that accompanies a vegetarian diet. Geographical location and diet are not the only factors involved in the selenium content of breast milk. It can also be affected by the time postpartum and the moment of

17 8 feeding (Jensen and Neville 1985). Selenium levels are higher in colostrum then drop off and tend to plateau at about one month postpartum (Perrone et al. 1994). Typically concentrations are 20 fig/l at one month, and 15 ag/l at three and six months postpartum (Levander 1987). Hind milk selenium concentrations are also slightly higher than fore milk (Jensen and Neville 1985, Mannan and Picciano 1987, Smith et al. 1982). Mothers who have given birth to preterm infants have higher levels of long-chain polyunsaturated acids in their breast milk than mothers who give birth to term infants, and a similar trend is noted in the Gpx activity in the milk (Ellis et al. 1990). This relationship suggests that the enzyme may have an association with the fatty acids in the breast milk, possibly playing an antioxidant role (Ellis et al. 1990). Fatty Acids Content of fatty acids in breast milk: The total amount of lipids found in mature human milk ranges from 3% to 5% by weight or 40% to 50% of the total kilocalories and remains fairly constant. However, small variations occur as a function of the stage of lactation. Mature milk is 50% of the energy from lipid while colostrum may be as low as 20% to 30% (Jensen 1989). Although the total lipid content of mature breast milk remains fairly constant, the fatty acid profile can be influenced by several factors with the major ones being the stage of lactation (time postpartum), the maternal nutritional status, and the maternal diet. During the first six weeks of lactation, lauric (12:0) and myristic (14:0) acids tend to

18 9 increase slightly while the the long-chain polyunsaturated fatty acids, with the exception to linoleic acid, decrease over time (Jensen 1989). A mother who is on a low calorie diet or one who is undernourished will produce milk with the fatty acid composition resembling depot fat (Worthington-Roberts and Williams 1993). Overall, the most influential factor affecting individual fatty acids is the maternal diet. The major saturated fatty acids in breast milk are 12:0, 14:0, 16:0 (palmitic acid) and 18:0 (stearic acid). Globally, saturated fatty acids in human breast milk range from a low level of about 39% of the total fatty acids found in Spanish women to a high level of 51% found in Finnish women. Most countries have concentrations between 41% to 48% of the total fatty acids (Koletzko et al. 1992). The concentration present is influenced by the maternal diet. High intakes of dietary saturated fats increase their concentrations in breast milk (Jensen 1989). Women consuming a high carbohydrate diet have higher concentrations of saturated fatty acids in their breast milk due to an increased mammary synthesis of these fatty acids (Jensen 1989). It was recently found that the saturated fatty acid concentration in breast milk of selenium-deficient women from New Zealand was 14% higher than that of selenium-supplemented lactating women (Butler et al. 1995). The concentration of monounsaturated fatty acids in mature breast milk ranges from 37%) to 41% of the total fatty acid content. The major contributor is oleic acid, 18:l(n-9)c. Traces of the trans-fatty acid, 18:l(n-9)t ) can also be detected in the milk of women who consume partially hydrogenated food fats (Jensen and Neville 1985). The concentration of polyunsaturated fatty acids in mature breast milk is 6-24% of the total fatty acids (Koletzko et al. 1992). The major polyunsaturated fatty acid in breast

19 10 milk is linoleic acid, 18:2(n-6), ranging from 6-16% of the total fatty acids (Bitman et al. 1983). The diet of the lactating mother can influence the levels of polyunsaturated fatty acids as well, and extreme variations in levels of linoleic acid have been observed. A high corn oil diet in one subject was shown to increase the 18:2(n-6) content of milk from 8% to 42% (Insulletal. 1959). However, it is the concentration of 22:6(n-3) (docosahexanoic acid, DHA), 20:5(n-3) (eicosapentanoic acid, EPA), and 20:4(n-6) (arachadonic acid, AA) that has been the primary concern of researchers in recent years. These fatty acids also respond to maternal dietary manipulations. In mature breast milk DHA typically ranges from 0.05% to 0.59% in the United States and in Europe. Higher concentrations, ranging from 0.9% to 2.8%, are found in women who consume large quantities of seafood (Koletzko et al. 1992, Chulei et al. 1995). EPA concentrations range from 0.04% to 0.61% in Europe, and again, higher concentrations, up to 1.1%, can be found in the breast milk from women who consume high levels of seafood (Koletzko et al. 1992, Chulei et al. 1995). AA levels range from 0.3% to 1.22%. Animal products, as well as seafood, may increase AA levels in human breast milk (Chulei et al. 1995). Although diets can influence the fatty acid composition of breast milk, when comparing European and African women, Koletzko et al. (1992) found similar AA:DHA ratios, 2.7:1 and 2.4:1 respectively. These ratios are similar to the ratio of these fatty acids found in neonatal brain lipids (1992). This suggests that the milk's ratio of (n-6) to (n-3) polyunsaturated fatty acids may be regulated by metabolic processes that maintain it at a relatively constant value in the infant's diet (Koletzko et al. 1992).

20 11 Content of plasma fatty acids during pregnancy During pregnancy, the fatty acid profile of plasma lipids of the mother may show an abnormal pattern. Deficiencies of both the (n-6) and the (n-3) polyunsaturated acids may occur (Holman et al. 1991). They appear to be marginally deficient in essential fatty acids (EFA). It is speculated that this is because of the increased need for essential fatty acids of the growing tissues. Holman et al. (1991) found that lactating women showed less of a recovery from the deficiencies than non-lactating women, but neither approached the normal profiles by six weeks postpartum (Holman et al. 1991). Measurement of the plasma fatty acid content and ratios of fatty acids can be used to determine EFA deficiencies. The EFA status index is defined as the ratio between the sum of the (n-3) and (n-6) fatty acids and the sum of the (n-7) and (n-9) fatty acids (Hornstra et al. 1992). In general, if the supply of (n-3) and (n-6) fatty acids is sufficient, the desaturation of oleic acid to the long-chain polyunsaturated fatty acids of the (n-9) family is limited, leading to a higher EFA status (Hornstra et al. 1992). Therefore, higher levels of Mead acid, 20:3(n-9), in the plasma would indicate a poor EFA status. Since various fatty acids compete for the same enzymic desaturation-elongation system, if amounts of linoleic and a-linolenic acid are insufficient to occupy the delta-6-desaturase, this allows oleic acid to be converted to Mead acid (Al et al. 1990). A value of 0.2 for the ratio of Mead acid to arachadonic acid in human serum phospholipids is considered the upper limit of normal (Holman et al. 1979).

21 12 Plasma fatty acid profile and selenium concentration There have been a few studies that have looked at the relationship between plasma fatty acid profiles and plasma selenium concentrations. Crespo et al. (1995) found that rats supplemented with moderate levels of selenium had a larger concentration of the polyunsaturated fatty acids, mainly in adipose tissue, than those rats fed either no selenium supplement or a high level selenium supplement. A study conducted in a healthy human population from Spain found serum selenium was directly related to percent essential fatty acids (linoleic and linolenic acids) and the sum of the (n-6) polyunsaturated fatty acids and inversely related to the sum of the percent saturated fatty acids in plasma phospholipids (Cabre et al. 1992). Selenium was also a predictor of the unsaturation index, calculated by this equation (Cabre et al. 1992) : UI = S(Fatty acid percent value X number of double bonds) The researchers noted that the population in this area had a lower plasma selenium status (61.6 ng/ml) than people from other Western countries. A similar relationship was seen in a healthy population from Finland whose selenium intakes were similar to the subjects in Spain. These subjects also demonstrated a positive correlation between seaim selenium and plasma long-chain polyunsaturated fatty acids (Miettinen et al. 1983). However, this relationship was not seen in a Dutch population where serum selenium concentrations were higher (Kok et al. 1987). Cabre et al. (1992) hypothesized that only subjects with marginal selenium status demonstrate a relationship between serum selenium and the unsaturation of plasma phospholipids. Dotson et al. (1991) have also suggested that the amount of selenium in breast milk relates to the amount of individual fatty acids in the

22 13 breast milk. Recently, it has been shown that women from New Zealand supplemented with selenium had higher levels of polyunsaturated and monounsaturated fatty acids in breast milk than the amount measured in a similar group of women not given a supplement (Butler et al. 1995). In addition, the amount of saturated fatty acids was less. To our knowledge, no one has investigated whether the same situation prevails in breast milk samples obtained from women who habitually consume a higher intake of selenium. Importance of long-chain fatty acids to infant development Although the amounts of DHA and AA in breast milk are relatively small, comprising on average 0.2% and 0.5% of the total lipid content respectively, they provide an ample supply of these fatty acids for the infant throughout lactation (Innis et al. 1994). These fatty acids are highly concentrated in the brain and retinal tissues and accumulate markedly in the late fetal and early neonatal life (Nettleson 1993, Martinez 1992, Innis et al. 1994). DHA accumulates in brain tissue and continues to increase after birth. The levels of two (n-6) fatty acids, AA and adrenic acid (22:4n-6), also increase in the brain tissue after birth, corresponding to the occurrence of increased myelination of neurons. Dietary levels of (n-3) fatty acids influence the retinal function and visual development in human infants (Carlson et al. 1994, Neuringer et al. 1994). Infants fed breast milk have higher levels of long chain (n-3) and (n-6) fatty acids in tissues than do formula fed infants, and this corresponds to better developed visual functions (Carlson et al. 1994, Makrides et al. 1993). It is thought that AA is needed for infant growth. Infants in one trial were given a marine oil supplement, which was high in both DHA and

23 14 EPA. A decline was seen in circulating levels of AA and poorer growth rates were observed (Carlson et al. 1994). In the second trial, infants were fed a marine oil with DHA but less EPA than before, and no decline of AA levels were observed. It is apparent that a proper balance between DHA, EPA and AA is needed for neonatal development. Specific Aim The purpose of this study was to determine if breast milk fatty acid profiles differ in lactating women whose habitual diets differ markedly in selenium content. In particular, we hypothesized that lactating women who live in Xichang, a region of China in which the traditional diet is characterized by a low intake of dietary selenium, would have a breast milk fatty acid profile that contained fewer (n-6) fatty acids than would be found in the breast milk of women who live in Beijing, a region in which the traditional diet is characterized by a moderate intake of selenium, which in turn would have a lower content of (n-6) fatty acids than the breast milk obtained from women who reside in Enshi, in which the traditional diet is characterized by a high intake of dietary selenium.

24 15 IT, Materials and Methods Overview Samples of breast milk and plasma were obtained from 60 lactating Chinese women from three geographic regions. Selenium status of the subjects was determined by measuring plasma and breast milk selenium concentrations and glutathione peroxidase activity. Fatty acid profiles were measured in plasma and breast milk. The samples from this study were obtained through the assistance of Dr. Yiming Xia of the Chinese Academy of Preventative Medicine in Beijing. Dr. Xia has collaborated extensively with Dr. Phil Whanger from the Department of Agricultural Chemistry, Oregon State University, on other human selenium studies. Subjects Twenty-one of the women were from an area in central China near the village of Xichang in Sichuan Province. This area is associated with low levels of selenium in the diet. Estimated average daily intakes were 11 (ig/day before supplementation of salt with selenium. The estimated daily intake is now about 30 ng/day. Twenty of the women were from a rural area near the city of Beijing in northeast China. The people in this area have adequate intakes of selenium; they average 110 (ig/day. Nineteen women were from two areas in eastern central China, in and around the village of Enshi in the Hubei Province

25 16 Here there are high dietary intakes of selenium, estimated to average 759 (ig/day. Approximately one half of the women from each location provided samples shortly after the birth of their infants (termed early samples). The other one half of the subjects gave later postpartum samples (later samples). This means that the two samples were not from the same woman. All of the subjects were lifelong residents of China. They were selected based on similar lifestyles, incomes and diets. The subjects from Xichang and Enshi were farmers, while the subjects from the rural area near Beijing were clerks. Income levels from all three groups are similar. This study was approved by the Oregon State University Institutional Review Board and by the Chinese Academy of Preventative Medicine in Beijing. Samples Blood was collected from the women into Vacutainers tubes containing 0.10 ml of 15% EDTA from the antecubital arm vein by trained medical personnel. Plasma was prepared at the site of the collection by Dr. Yiming Xia's laboratory group at the Chinese Academy of Preventative Medicine in Beijing. It was frozen and held at -20 C. This sample was used for the measurement of selenium concentrations, Gpx activities and fatty acid profiles. Twenty-five milliliter breast milk samples were collected by the subjects by hand expression. Early milk samples were obtained within 1-7 days from parturition Later

26 17 milk samples were obtained 3-10 months postpartum. All of the milk samples were taken at the beginning of the nursing period. These samples were obtained on the same day as the blood samples. The milk was aliquoted. One fraction was frozen immediately and stored at C and used for the determination of selenium concentration and Gpx activity. The other fraction was extracted, as discussed below, and the extract frozen. All the samples were held at -20 o C until transported to Oregon. They were transported in dry ice in an insulated container to Oregon State University where they were stored in a -80 o C freezer until analyses were performed. Analytical analyses Selenium and Gpx activity in plasma and milk was determined by routine procedures as described in Beilstein and Whanger (1983). Selenium concentrations were determined using a semiautomated fluorometric method (Brown and Watkinson 1977) with modifications (Beilstein and Whanger 1983). In this method samples are digested with nitric acid and perchloric acid, converting Se to the +6 oxidation state. Hydrochloric acid was added and the sample was briefly heated to reduce the selenium to the +4 oxidation state. The acidity was adjusted to ph 2 - ph 3 and the selenium complexed to 2,3-diaminonapthalene (DAN) in a water bath. After extraction with cyclohexane, fluorescence was measured with a 325 nm filter for excitation and a 556 nm filter for emission. Peak heights were measured in mm and selenium content calculated from the standard curve in ng/ml of milk or plasma.

27 Activity of Gpx was assayed by a coupled enzyme procedure using 25 mm t-butyl hydroperoxide as a substrate. The following reaction describes the chemistry that occurs: 18 (Gpx) (GSSG-R) 2GSH + H 1 0, -» GSSG + H,0 ^ 2 GSH /\ NADPH NADP GSH is maintained at a constant concentration by exogenous glutathione reductase (GSSG-R) and NADPH which immediately converts any glutathione (GSSG) produced back to the reduced form, GSH. The rate of GSSG formation is measured by following the decrease in the absorbance of the reaction mixture at 340 nm as nicotinamide adenine dinucleotide phosphate (NADPH) is converted to NADP (Paglia and Valentine 1967) To run the assay, 25 il of plasma diluted with 75 (il of water or 100 (.il of milk was placed in a cuvette, followed by 0.8 ml of freshly prepared reaction mixture. The reaction mixture contained the following: M phosphate buffer (ph 7.0), 4.5 mm EDTA, 4.7 mm sodium azide, 2.8 nmoles NADPH, 49.9 nmoles reduced glutathione, and 0.67 units glutathione reductase. The reaction was initiated by adding 0.1 ml of freshly prepared 25 mm t-butyl hydroperoxide solution. Gpx activity was measured spectrophotometrically at 340 nm using a Shimadzu UV-VIS Spectrophotometer UV- 160A. Activity was expressed as nmoles NADPH oxidized per minute per ml of sample or as nmoles NADPH oxidized per minute per mg of protein. Protein concentrations were determined by methods described by Lowry et al. (1951).

28 19 Lipids were extracted from the breast milk while still in China and plasma samples were extracted at Oregon State University with chloroform/methanol (Folch et al. 1957). The fatty acid profiles of the breast milk and plasma were measured by gas chromatography (GC) as described by Song and Wander (1991). Fatty acid results are presented as the weight percent of total fatty acids measured. Because total milk fat concentrations change during nursing and from feeding to feeding, milk sampling conditions affect fatty acid concentrations; however, the relative percent contributions of major and minor fatty acids in human milk are unaffected by changing milk fat contents (Koletzko et al. 1992). Therefore, weight percent data are used for comparisons of fatty acid values in the study. Statistical analysis The means and standard errors for all variables were calculated. A 2-way ANOVA was performed to determine statistically significant effects with the two factors being time at which the samples were taken (early or later milk) and geographic location (Xichang, a low selenium area; Beijing, an adequate selenium area; and Enshi, a high selenium area). If there were no interactions between time and location, the variables were analyzed by observing main effect means. However, if interactions between time and location occurred, cell means were analyzed. To check whether or not assumptions were obeyed, we looked at the plot of the residuals to determine distribution and homogeneity of the data. A natural log transformation was performed on the selenium

29 20 and Gpx activity values to obtain homogeneity of variance. Relationships between the appropriate variables were evaluated using a linear regression model and Pearson correlation coefficients (Snedecor and Cochran 1989). Data were analyzed using SAS 6.11 (SAS Institute Inc 1985) and was considered statistically different if p < 0.05.

30 RESULTS Dietary Information Since specific dietary information from our subjects was unavailable, the dietary intake was generalized for the women based on regional food habits (Junshi et al. 1990). When compared to a Western diet, a typical Chinese diet includes a higher proportion of cereals and vegetables, and a lower proportion of meat, poultry, eggs and milk. The diet has a high intake of dietary fiber, and a low intake of protein, fat, calcium, retinol and riboflavin. Rice is a staple dietary component, especially for the farmers in Xichang, Sichuan Province and Enshi, Hubei Province. Rural Beijing subjects eat a little more wheat products, fruits, milk products and meat, but less dark vegetables than the farmers. The farmers often raise swine in the Sichuan Province and pork fat is easily obtained, as is also true in the Hubei Province. Demographics The average age of the women and collection time of the samples are given in Table 1. As access to Xichang and Enshi is difficult, little control could be exercised by the faculty from the Chinese Academy of Preventative Medicine on when the samples were obtained. Consequently, the samples were broadly grouped into two times, termed early and late.

31 22 Table 1: Average age of the women and collection time of the plasma and milk samples for each location at both of the collection times. Age Sample Collection Times Region Earlv Late Earlv Late years days poslpartum Xichang (n=21) 24.5 ± ± ± ±25.3 Beijing (n=20) 28.2 ± ± ± ±8.4 Enshi (n=19) 25.4± ± ± ±65.6 Selenium Status Selenium and protein concentrations and Gpx activities in plasma and breast milk There were significant differences in the selenium concentration and glutathione peroxidase activity in both plasma and breast milk. In the plasma, there were no interactions between time and location for selenium levels and glutathione peroxidase activity. Consequently, the statistical model used to analyze the data included only the main effects of time and location. Plasma selenium did not change as a function of time but increased dramatically from the areas with low selenium, Xichang, compared to that with adequate selenium intake, Beijing, and to the area with high selenium, Enshi (Figure 1). Selenium levels were 33.3 ±41.5 mol/l, ± 42.5 mol/l, and ± 43.7 mol/l, respectively. These values were each significantly different from one another (p =

32 ). The concentration of selenium measured in the women from Xichang was significantly lower than that measured in the women from Beijing (p = ) and Enshi (p = ); the concentration measured in the women from Beijing was significantly lower than that measured in the women from Enshi (p = ). The plasma Gpx activity also varied as a function of geographic location (p = ) but not time. Plasma Gpx activity was ±8.1, ±8.3, and ± 8.5 nmols NADPH ox/minml at Xichang, Beijing, and Enshi, respectively. The activity measured in the samples from Xichang was significantly lower than that measured in those from Beijing (p = ) and Enshi (p = ); the activity of the samples obtained from Beijing was significantly greater than that of the samples obtained from Enshi (p = 0.003). The relationship between the activity of Gpx and the plasma selenium concentration from all the samples is given in a scatter plot (Figure 2). This plot clearly shows the increased activity of the samples obtained from Beijing, compared to the other two locations. In contrast to the selenium and Gpx activity in plasma, in the breast milk there was a significant interaction for these two dependent variables between time and location (p = 0.007, and p = 0.02 respectively). Consequently, the statistical model used in evaluating these data used cell means rather than main effect means (Figure 3). For milk selenium concentrations, the interaction occurred because the early milk samples had a higher selenium concentration than did the later milk samples in Xichang and Beijing but in Enshi, the early milk samples had slightly lower concentration of selenium than the later milk samples. Because the selenium concentration of the samples from Enshi was so

33 Plasma Selenium Concentration and Glutathione Peroxidase Activity Plasma Sc 2 Plasma Gpx < z Xichang Beijing F.nslii Figure I: Plasma selenium concentration and Gpx activity from three locations in China. Values represent mean ± SE. Different letters above the values indicate significant differences at p < 0.05 when comparing values measured in samples obtained from the three locations. Plasma Selenium vs Plasma Gpx 100 < z Plasma Sclcnimn (rnol/ Figure 2: Scatter plot of plasma selenium vs. plasma glutathione peroxidase activity for all subjects (n = 60).

34 Milk Selenium Concentration and Glutathione Peroxidase activity O Xichang Beijing F.nshi Figure 3: Milk selenium concentration and Gpx activity from three locations in China. Values represent mean ± STD. Different letters above the values indicate significant differences at p < 0.05 when comparing values measured in samples obtained from the three locations. much larger than the values measured in the samples from Xichang and Beijing, the interaction may be of relative unimportance in interpreting the data. Consequently, the main effect of location was evaluated in addition to comparing cell means for each location at each of the time points. At the early time, the selenium concentration was 27.0 ± 6.2 mol/l in samples from Xichang, 32.5 ± 3.9 mol/l in samples from Beijing, and ± 20.0 mol/l in samples from Enshi. The samples from Enshi had a significantly higher selenium concentration than the samples from Xichang (p = ) and Beijing (p = ). The selenium concentrations from Xichang and Beijing were statistically equivalent. At the later time, the selenium concentration was 9.7 ± 1.6 mol/l in samples

35 26 from Xichang, 17.7 ± 2.0 mol/l in samples from Beijing, and ± 20.1 mol/l in samples from Enshi. The samples from Enshi had a significantly higher selenium concentration than both Xichang and Beijing (p = ), and the samples from Xichang and Beijing were statistically equivalent. With respect only to location, the selenium concentration in the breast milk samples from Xichang was 18.2 ± 8.0 mol/l, a value that was significantly lower (p = 0.003) than that measured in the samples from Beijing (25.1 ± 8.1 mol/l) or Enshi (111.6 ± 8.4 mol/l, p = ); the value measured in the samples from Beijing was significantly lower (p = ) than that measured in those from Enshi. There was a significant interaction (p = 0.02) between time and location for the activity of Gpx in the breast milk samples. In the samples obtained at the early time, despite the fact that the selenium concentrations differed, there was no significant difference in the Gpx activity at the three locations. The measured values were 69.7 ± 6.0 in the samples from Xichang, 79.0 ± 6.5 from Beijing, and 68.3 ± 4.9 nmols NADPHox/min-ml from Enshi. At the later time, on the other hand, the activity measured in the samples from Xichang (39.3 ± 5.9) was significantly lower than that in the samples from Beijing (66.8 ± 4.5, p = 0.001), and Enshi (60.7 ± 5.5, p = 0.01). The activity measured in the samples from Beijing and Enshi were statistically equivalent. The protein concentration of the blood and breast milk was also determined. There was no interaction between time and location on the protein concentration of plasma, so main effect means are discussed. The protein content of the plasma samples obtained from the three locations differed significantly (p = ). The protein concentration of the plasma samples from Xichang was 81.8 ± 2.9 g/l, from Beijing ± 3.0 g/l, and from

36 27 Enshi ± 3.0 g/l. The samples from Xichang were significantly lower than those from Beijing (p = ) and Enshi (p = ). The samples from Beijing and Enshi were statistically equivalent. For the protein concentration of breast milk there was an interaction between time and location (p = ). This occurred because the protein concentration of the early samples from Enshi was significantly lower than those from Beijing (p = ) or Xichang (p = ), while the protein concentration in the later samples from all three locations was statistically equivalent. The early and late protein concentrations were 3.1 ± 0.2 and 1.7 ± 0.2 g/dl in samples from Xichang, 3.2 ± 0.2 and 1.7 ±0.1 g/dl in samples from Beijing, and 1.9 ± 0.1 and 1.6 ± 0.1 g/dl in samples from Enshi. The plasma and milk Gpx levels were also expressed relative to the protein concentration but the data indicated the same trends as when expressed relative to the volume (data not shown). Fatty Acid Profiles of plasma and breast milk The complete fatty acid profiles for the plasma and milk samples are given in Appendices A and B. However, selected fatty acids are discussed. To evaluate changes in the fatty acid profiles due to sampling time and to location, composites of three categories of fatty acids were compared: the sum of the saturated fatty acids ( SFA), ^g sum of the monounsaturated fatty acids ( MUFA) and the sum of the polyunsaturated fatty acids (XPUFA). In plasma few interactions were observed between time and location so main effect means are discussed.

37 28 There were significant differences in the J^PUFA (Figure 4) found in the plasma as a function of location (p = ): 35.8 ± 1.0% in samples from Xichang, 45.5 ± 1.0% from Beijing, and 36.9 ± 1.0% from Enshi. The women from Beijing had significantly higher concentrations of PUFA than the women from Xichang (p = ) and Enshi (p = ) but the concentrations measured in the samples from Xichang and Enshi were statistically equivalent. Differences were also seen in the plasma concentration of ^MUFA (p = , Figure 6): 30.4 ± 0.7% in samples from Xichang, 20.8 ± 0.7% from Beijing, and 30.4 ± 0.7% from Enshi. The women from Beijing had significantly lower concentrations of MUFA in their plasma, when compared to Xichang (p = ), and Enshi (p = ), and the concentrations of ^MUFA in the samples from Xichang and Enshi were statistically equivalent. There were no significant differences between the three locations in terms of the amount of X^SFA (Figure 7) found in the plasma. The major contributor to the differences seen in the concentration of PUFA is linoleic acid. Linoleic acid makes up approximately 70% of the PUFA in plasma. Its content was 24.1 ± 0.9% in samples from Xichang, 32.9 ± 0.9% from Beijing, and 26.1 ± 0.9% from Enshi (Figure 4). Beijing subjects had significantly higher concentrations of linoleic acid than those from Xichang (p = ) or Enshi (p = ), and the concentrations of linoleic acid in samples from Xichang and Enshi were statistically equivalent. Other important polyunsaturated fatty acids include arachadonic acid (AA), linolenic acid, eicosapentanoic acid (EPA), docosahexanoic acid (DHA), and Mead acid. The concentration of AA (Figure 4) was significantly lower in the samples from Enshi than

38 29 Plasma Polyunsaturated Pally Acids b <io - XicliBng Mcijing Xichang Beijing f.nshi Figures 4 and 5: The concentration of selected polyunsaturated fatty acids from plasma. Individual fatty acids or composites of fatty acids with different letters above them indicate significant differences at p < 0.05.

39 30 Plasma Monounsaluralcd Fally Acids Plasma Saturntcd Fatly Acids Xichang Doling Enshi Figures 6 and 7: The concentration of selected monounsaturated and saturated fatty acids from plasma. Individual fatty acids or composites of fatty acids with different letters above them indicate significant differences at p <, 0.05.

40 31 the samples from Xichang (p = 0.006) or the samples from Beijing (p = ). There were no significant differences in the concentration of linolenic acid from all three locations (Figure 5). The concentration of EPA and DHA from the three locations differed significantly (p = 0.02 and , respectively). Samples from Enshi had significantly higher concentrations of EPA than those from Beijing (p = ) and Xichang (p = 0.02), yet significantly lower concentrations of DHA than Beijing (p = ) and Xichang (p = 0.005). The concentration of Mead acid also differed among the three locations (p = 0.006): it was significantly higher in the samples from Xichang and Enshi compared to those from Beijing (p = and 0.04, respectively). The concentration in the samples from Enshi and Xichang were statistically equivalent. Oleic acid (Figure 6) is the primary contributor to the ^MUFA and comprises about 75% of the total monounsaturates and differed significantly among the three location (p = ). The concentration of oleic acid was 23.1 ± 0.56% in samples from Xichang, 15.8 ± 0.6% from Beijing, and 22.0 ± 0.6% from Enshi. The samples from the women from Beijing had significantly lower concentrations than those from the women from Xichang (p = ) and Enshi (p = ). Oleic acid concentrations were statistically equivalent in samples from Xichang and Enshi. Although there were no significant differences in the concentration of X^FA (Figure 7) among the three locations, the two major fatty acids from this group 16:0 and 18:0, differed (p = and , respectively). The concentration of 16:0 was significantly higher in the samples from Xichang compared to Beijing (p = 0.04) and Enshi (p = 0.001). The concentration in the samples from Beijing and Enshi were statistically

41 32 equivalent. The concentration of 18:0 was significantly higher in the samples from Enshi compared to those from Beijing (p = ) and Xichang (p = 0.002). The concentration in the samples from Xichang and Beijing were statistically equivalent. The effect of time on the plasma fatty acid profile was not as marked as the effect of location. As a function of time, the concentration of MUFA from the early plasma samples was higher than the later plasma samples (p = 0.009), while the concentration of PUFA showed the opposite trend, with lower concentrations in the early samples, and higher concentrations in the later samples (p = 0.01). In the breast milk samples (Figures 8-12) interactions were observed in the J^PUFA analysis and many of the fatty acids that compose this fraction. Therefore individual cell means are discussed. For women from all three locations, the concentration of PUFA in the breast milk was higher in the samples collected at the later time (Figure 8) but the increase was more pronounced in the samples from Beijing, thus causing the interaction. In the early milk samples, the concentration of PUFA was 12.5 ± 1.0% in samples from Xichang, 24.3 ± 0.7% from Beijing, and 13.9 ± 0.9% from Enshi. Beijing women had a significantly higher concentration of milk PUFA than the women from Xichang (p = ) or from Enshi (p = ). In the later milk samples, the concentration of PUFA was 13.6 ± 1.0%, 30.0 ± 1.4%, and 14.0 ± 1.3%, respectively. Again, the Beijing women had a significantly higher level than Xichang women (p = ) or Enshi women (p = ). In both the early and later milk samples, the concentrations of PUFA from the Xichang and Enshi women were statistically equivalent. As is the case in plasma, in breast milk linoleic acid is the major contributor to

42 Milk Polvunsaluratcd ('any Acids I StftiPUFAd) ^ S«nPUFAC) [Zl IB:2<*(I) lb:2nj(2) Xichau? Xiciim^ Beijing. a*-" " i Jb ^ T b Figures 8, 9 and 10: The concentration of selected polyunsaturated fatty acids from breast milk. Individual fatty acids or composites of fatty acids with different letters above them indicate significant differences at p < The (1) and (2) following the name of the fatty acid means early and late samples, respectively.

43 34 Milk Monounsaturated I'nttv Acids Xichang Dcijing Enshi Milk SaiuratctI l : attv Acids < 20 ~ Xichang Beijing Unshi Figures 11 and 12: The concentration of selected monounsaturated and saturated fatty acids from breast milk. Individual fatty acids or composites of fatty acids with different letters above them indicate significant differences at p < The (1) and (2) following the name of the fatty acid means early and late samples, respectively.

44 35 ^TPUFA (Figure 8). With respect to time, the concentration of linoleic acid increased in the samples from all three locations. In the early milk samples, the linoleic acid concentration was 8.8 ± 0.8% from Xichang, 19.1 ± 0.7% from Beijing, and 10.0 ± 0.6% from Enshi. In the later milk samples, the linoleic acid concentration was 10.5 ± 1.1% from Xichang, 26.1 ± 1.1% from Beijing, and 11.1 ± 1.1% from Enshi. At both time points, the Beijing women had significantly higher concentrations of linoleic acid in their breast milk than did the women from Xichang (p = ) or Enshi (p = ) and the concentrations of linoleic acid were statistically equivalent in the samples from Enshi and Xichang. Individual polyunsaturated fatty acids of importance include AA, linolenic acid, EPA, and DHA. At all three locations, the AA concentration decreased as a function of time. In the early milk samples, the AA concentration (Figure 9) followed the same pattern as linoleic acid concentration in breast milk, i.e. the concentration of the samples from Beijing was significantly higher than those from Xichang (p = 0.03) or Enshi (p = ) and those from Xichang were significantly higher than those from Enshi (p = 0.007). In the later milk samples, the AA concentration in samples from Enshi was significantly lower than samples from Xichang (p = 0.02) or from Beijing (p = ). The AA content in samples from Xichang and Beijing were statistically equivalent Linolenic acid (Figure 9) concentration in the early milk samples from Enshi was significantly greater than that in the samples from Xichang (p = ) or from Beijing (p = 0.01). Linolenic acid concentrations in early samples from Xichang and Beijing were statistically equivalent. Linolenic acid in the later milk samples from Beijing was

45 36 significantly higher than its concentration in samples from Xichang (p = 0.005) or Enshi (p = 0.02), while the linolenic acid content from Xichang and Enshi was statistically equivalent. Both EPA and DHA (Figure 10) concentrations decreased at all locations as a function of time, but the effect was more marked in Beijing, thus causing an interaction. In early milk, the EPA content in samples from Beijing was significantly higher than in samples from Xichang (p = 0.006) and statistically equivalent to its content in samples from Enshi. The EPA content in the samples from Xichang and Enshi were also statistically equivalent. In the later milk samples, the EPA content was statistically equivalent at all three locations. The DHA content in early milk samples from Enshi was significantly lower than the DHA content in samples from Xichang (p = ) or Beijing (p == ). The DHA content in samples from Xichang and Beijing were statistically equivalent. In later milk samples, the DHA content in samples from Beijing was significantly higher than in samples from Enshi (p = 0.008) and the DHA content in samples from Xichang was statistically equivalent to samples from Beijing and Enshi. The concentration of Mead acid in breast milk was barely discernible in most samples; however, enough was present in some of the late samples from Enshi that these samples tended to contain more than the samples from the other two locations. There was no interaction between time and location in the analysis of the XS>FA (Figure 12) and the MUFA (Figure 11) and most of the fatty acids that make up these composites. There were also no significant differences with respect to time. For the SFA, milk content was 46.6 ± 1.6% in samples from Xichang, 36.2 ± 1.6% from Beijing, and 42.5 ± 1.7% from Enshi. Milk concentration of SFA was significantly lower in the

46 37 samples from Beijing than samples from Xichang (p = ) or Enshi (p = 0.009). The concentration of ^SiFA j n sam p es f rom Enshi and from Xichang were statistically equivalent. The largest contributors to the SFA of breast milk are 16:0 and 18:0. There were no significant differences in the concentration of 16:0 with respect to location but there were in the concentration of 18:0. In addition, there was an interaction between time and location in the concentration of 18:0 in breast milk (p = 0.01). In the early milk samples the concentration of 18:0 was significantly higher in the samples from Xichang than in those from Beijing (p = ) or Enshi (p = ). The concentration in the samples from Beijing and Enshi were statistically equivalent. In the later milk samples the concentration of 18.0 in the samples from Beijing were significantly lower than that in the samples from Xichang (p = ) or Enshi (p = ). The concentration in the samples from Xichang and Enshi were statistically equivalent. There were significant differences in the breast milk concentration of MUFA (p = 0.003). The MUFA concentration in milk samples was 38.0 ± 1.4% from Xichang, 35.1 ± 1.4% from Beijing, and 42.3 ± 1.4% from Enshi. The women from Enshi had significantly higher concentrations of ^MLTFA t j ian ^ women from Xichang (p = 0.03) and Beijing (p = ). The concentration of MUFA was statistically equivalent in samples from Xichang and Beijing. The main contributor to the MIJFA was 0 e j c ^jj However, there were no statistically significant differences in the concentration of oleic acid in the samples obtained from any of the locations. Pearson correlation coefficients were calculated to explore relationships between the plasma and breast milk selenium concentrations and Gpx activities (Table 2). The

47 38 plasma selenium concentrations and Gpx activities showed strong positive correlations with breast milk selenium concentrations and Gpx activites. Note: When the Pearson correlation coefficients were calculated, the negative sign was removed from Gpx activity to make the discussion clearer. The correlation between plasma Gpx activity and concentration of selected fatty acids in the plasma was also evaluated (Table 3). The concentrations of oleic acid and the ^IVTUFA were negatively correlated with the activity of Gpx while the concentrations of linoleic acid and ^PUFA were positively correlated to the activity. The concentration of Mead acid in plasma also increased as the activity of Gpx decreased. Table 2: Pearson correlation coefficients between plasma and breast milk concentration of selenium and Gpx activity (n = 60). Breast Milk Selenium Gpx Plasma Selenium 0.84 a 0.15 Gpx : a p ^ 0.00 Correlation coefficients were also calculated for the concentration of breast milk fatty acids with both plasma and breast milk Gpx activities (Table 3). The concentration of SFA in breast milk was negatively correlated with plasma Gpx activity, but had no correlation with breast milk Gpx activity. The concentrations of oleic acid and MUFA

48 39 were negatively correlated with breast milk Gpx activity but had no correlation with plasma Gpx activity. The concentration of PIJFA was positively correlated with both plasma and breast milk Gpx activities. The concentrations of linoleic acid and EPA were positively correlated with plasma Gpx activity. Breast milk Gpx activity was positively correlated with AA and DHA. To further evaluate the relationship between plasma and milk linoleic acid concentration and Gpx, the data were fit to linear regression models. In contrast to what was done with the data concerning the Pearson correlation coefficients, the negative sign on Gpx activity was retained. For all samples, there was a significant positive linear relationship between plasma Gpx activity and the concentration of plasma linoleic acid (Figure 13, r 2 = 0.28, p = ). In the early milk samples, there was no significant relationship between milk Gpx activity and the concentration of milk linoleic acid (Figure 14, r 2 = 0.04, p = 0.31). However, in later milk samples, there was a significant positive linear relationship between milk Gpx activity and milk linoleic acid (Figure 15, r 2 = 0.21, p = 0.01).

49 Table 3: Pearson correlation coefficients between plasma and breast milk glutathione peroxidase activity and select fatty acids. Data from all subjects (n = 60) 40 Plasma Fatty Acid Gpx ISFA :ln XMUFA :2n c 18:3n :4n :3n :5n :5n :6n IPUFA 0.48 Breast milk Fatty Acid Plasma Gpx Milk Gpx ESFA -0.40'' :ln a ^MUFA a 18:2n c :3n :4n b 20:3n :5n a :5n :6n b ^PUFA 0.62 c 0.25 a a p ^ b p^ c p ^ 0.001

50 Plasma Gpi (nmol NAOPH oxjm.n ml) Figure 13: Linear regression model of plasma Gpx activity and the concentration of plasma linoleic acid (n=60).

51 42 20 X = Xichang = Oeii'ng = Ensh, o Early samples Mfl* Gp«(nmol NADPH on/nun ml) 30 T = Bc. '"g Laic samples Milk Gpx {nmol NADPH OK/min ml) Figure 14 and 15: Linear regression model of milk Gpx activity and the concentration of milk linoleic acid in early (n=29) and late (n=3 1) samples

52 43 IV. DISCUSSION A unique aspect to this study was the naturally occuring variable intake of the nutrient selenium. The populations selected from China provided the opportunity to examine the effect of three different levels of selenium intakes without depleting or supplementing the subjects. The plasma selenium concentrations determined in this study confirmed our knowledge about the selenium intakes of the people living in the three regions in China. As expected, the women from Xichang had low plasma selenium concentrations, which put them at risk for selenium deficiency. Beijing subjects had adequate concentrations of plasma selenium and Enshi subjects had very high plasma selenium concentrations, perhaps an indication of potential selenium toxicity. In breast milk, many nutrients are maintained in relatively adequate amounts during lactation, regardless of maternal dietary intake. However, selenium is a trace element and for these nutrients the maternal intake is usually reflected in the concentration found in breast milk (Jensen and Neville 1985). Thus it is not surprising that the breast milk selenium concentrations are positively correlated with plasma selenium concentrations. The concentration of selenium that we measured in breast milk was higher relative to plasma concentrations than previously reported. While others have shown breast milk selenium concentrations to be seven times lower than plasma concentrations (Mannan and Picciano 1987), we found that in Xichang, Beijing and Enshi, breast milk samples were approximately 2, 5 and 6 times lower, respectively, than plasma concentrations. In early and later breast milk samples from Enshi the concentration of selenium were 21 2 ± 4.9

53 44 and 7.7 ± 1.3 ng/ml (27.0 ± 6.2 and 9.7 ± 1.6 mol/l). The later breast milk samples have a selenium concentration that is somewhat lower than the concentration of selenium reported in breast milk from United States (15 ng/ml, Levander et. al. 1987) and New Zealand (13.4 ng/ml, Dolamore et al. 1992), and comparable to concentrations in milk from Finland (7.2 ng/ml, Kumpulainen et al. 1985), where selenium intake is also known to be low. Low breast milk selenium concentrations and low plasma selenium concentrations may indicate a potential for selenium deficiency in the women and their infants from Xichang. The other measure of selenium status is glutathione peroxidase activity. The Gpx activities measured were consistent with other studies that demonstrate a positive correlation between plasma selenium levels and Gpx activity at low selenim intakes (Whanger et al. 1988). Sunde et al. (1988) showed that the regulation of Gpx and its mrna levels are dependent on selenium concentrations in plasma. Low levels of selenium lead to a decrease in the Gpx expression, thus a lower level of activity. Peak Gpx activity was measured in subjects with adequate plasma selenium levels, indicating saturation of the Gpx enzyme and Gpx activity declined as plasma selenium levels became excessive. Thus, high intakes of selenium decrease Gpx activity The women's selenium status, however, was not necessarily reflected in the activity of Gpx in breast milk. The enzymatic activity was adequate and statistically equivalent at all locations in the early milk samples, even though breast milk selenium concentrations varied markedly. On the other hand, in the late samples, the activity of Gpx in breast milk showed the same pattern that it did in plasma: low when the selenium

54 45 intake was marginal or excessive but appropriate when the selenium intake was adequate. This may suggest that Gpx plays an important role in breast milk, particularly right after birth, and mechanisms exist to maintain its adequate activity even if selenium intake is low. The data suggests that the activity of Gpx in both plasma and breast milk plays a role in determining their respective fatty acid profiles. The protective enzymatic action of Gpx prevents the oxidation of unsaturated fatty acids. Linoleic acid is one of the most abundant polyunsaturated fatty acids in both plasma and breast milk and the activity of Gpx is effective in maintaining high concentrations of this essential fatty acid. Linoleic acid is easily oxidized since much of it is found in triglycerides and free fatty acid pools (Crawford 1993). This hypothesis is supported by the positive correlation between plasma Gpx activity and the plasma concentration of linoleic acid and between breast milk Gpx activity and linoleic acid in the later milk samples. The early breast milk samples do not demonstrate this relationship because it appears that milk Gpx activity is maintained at high levels right after birth, irrespective of selenium concentrations. The range of concentrations of other polyunsaturated fatty acids, i.e. arachadonic acid and docasahexanoic acid, was smaller among the three groups of women than was that of linoleic acid. This may be due to the fact that these fatty acids are preferentially incorporated into cell membranes where they are less susceptible to oxidation (Crawford 1993). Crawford et at. (1993) calculated an average ratio of arachadonic acid to docasahexanoic acid in human breast milk of about 2:1. The women in our study had breast milk samples with AA:DHA ratios ranging from 2:1 (Xichang) to 2.3:1 (Enshi), demonstrating that these fatty acids were relatively unaffected by selenium intakes.

55 45 intake was marginal or excessive but appropriate when the selenium intake was adequate. This may suggest that Gpx plays an important role in breast milk, particularly right after birth, and mechanisms exist to maintain its adequate activity even if selenium intake is low. The data suggests that the activity of Gpx in both plasma and breast milk plays a role in determining their respective fatty acid profiles. The protective enzymatic action of Gpx prevents the oxidation of unsaturated fatty acids. Linoleic acid is one of the most abundant polyunsaturated fatty acids in both plasma and breast milk and the activity of Gpx is effective in maintaining high concentrations of this essential fatty acid. Linoleic acid is easily oxidized since much of it is found in triglycerides and free fatty acid pools (Crawford 1993). This hypothesis is supported by the positive correlation between plasma Gpx activity and the plasma concentration of linoleic acid and between breast milk Gpx activity and linoleic acid in the later milk samples. The early breast milk samples do not demonstrate this relationship because it appears that milk Gpx activity is maintained at high levels right after birth, irrespective of selenium concentrations. The range of concentrations of other polyunsaturated fatty acids, i.e. arachadonic acid and docasahexanoic acid, was smaller among the three groups of women than was that of linoleic acid. This may be due to the fact that these fatty acids are preferentially incorporated into cell membranes where they are less susceptible to oxidation (Crawford 1993). Crawford et al. (1993) calculated an average ratio of arachadonic acid to docasahexanoic acid in human breast milk of about 2:1. The women in our study had breast milk samples with AA:DHA ratios ranging from 2:1 (Xichang) to 2.3:1 (Enshi), demonstrating that these fatty acids were relatively unaffected by selenium intakes.

56 46 When there is an increase in PUFA in plasma or breast milk, there must be a compensatory decrease in a different fatty acid composite. In the plasma samples from Beijing, an increase in PUFA corresponds to a decrease in ^MUFA. In the breast milk fatty acids, the samples from Xichang and Enshi had a lower concentration of ^PUFA than the samples from Beijing. This corresponded to higher X^FA an( j MUFA concentrations in the samples from Xichang and Enshi when compared to Beijing, in particular, a higher concentration of XlSFA i n samples from Xichang, and a higher concentration of ^TMUFA in samples from Enshi. A limitation to this study is the limited knowledge that we have about the diets of these three groups of women. In particular, the dietary intake of polyunsaturated fatty acids, especially linoleic acid, is not known. Consequently, it is impossible to determine to what extent the very high concentration of linoleic acid measured in the breast milk is due to a high dietary intake of linoleic acid. The amount measured in the samples from Beijing is extremely high when compared to the amounts measured in samples from 24 European and African countries (Koletzko et al. 1992), yet very consistent with the amounts found in five different regions in China (Chulei et al. 1995). There are several factors that support the rationale that the high levels of linoleic acid found in the Beijing women's breast milk are not a consequence of high dietary intakes. Plasma concentrations of linoleic acid would also reflect dietary intake. The plasma concentration of linoleic acid measured in samples from each of the three areas do not differ as markedly as in the breast milk, and are similar to values that have been previously reported in humans (Wander et al. 1996) thus suggesting that the differences in linoleic acid are not induced by diet. If the

57 47 women from Xichang and Enshi were consuming a low fat diet, synthesis of saturated fatty acids would occur. Thus, the plasma levels of saturated fatty acids should be lower in Beijing compared to Enshi and Xichang. The plasma level of saturated fatty acids were statistically equivalent. The high level of monounsaturated fatty acids in the plasma samples from Xichang and Enshi may be the result of their consuming diets that contain greater amounts of MUFA than do the women from Beijing. It is not known if this occurs. However, no foods that contain large amounts of MUFA were cited by Junshi et al (1990) as food sources in those two areas. Collectively, this information argues against the differences in the plasma and breast milk concentrations of linoleic acid existing because of differences in intake. Another interesting outcome of this study was the observation that in the plasma samples, a decrease in Gpx was associated with an increase in Mead acid, a fatty acid whose presence may indicate an essential fatty acid deficiency. As mentioned earlier, Mead acid is formed when there are not enough essential fatty acids present to occupy the desaturase enzymes. These enzymes turn their actions towards the (n-9) fatty acids and produce Mead acid. Although there was no measurable amount of Mead acid in the breast milk samples, the plasma samples from both Xichang and Enshi had trace amounts. This could indicate a potential low essential fatty acid status in these women. The role of Gpx in breast milk may be to protect the lipids from further peroxidation by lipid peroxides or to guard against the mechanisms they may trigger, i.e. transcription factors such as nuclear factor kappab (NF-KB) and activator protein 1 (AP-

58 48 1) (Winyard et al. 1994). The fact that we have observed increased concentrations of linoleic acid in plasma and breast milk of women with appropriate selenium nutriture suggests that lipid peroxidation may be less than in women with compromised selenium status. The breast milk for their infants may be more protected from lipid oxidation. There may be clinical relevance to these data. Studies in New Zealand, an area where selenium intakes are also known to be low, have explored the relationship between selenium and Gpx levels and the health of premature infants. Premature infants are at high risk for oxidative diseases such as bronchopulmonary dysplasia and retinopathy of prematurity (Inder et al. 1996). Since glutathione peroxidase removes hydrogen peroxide and lipid hydroperoxides, two reactive oxygen species, one may speculate that the infants from New Zealand with low selenium status are at a higher risk than infants with appropriate selenium intake (Sluis et al. 1992). In conclusion, it appears that the health of both the mother and the infant may be at risk when selenium intakes are not appropriate. Indicators of health status, such as Gpx activity and lipid profiles may be negatively affected when selenium intakes are too low or too high. The hypothesis predicted a positive linear relationship between selenium intake and the concentration of (n-6) fatty acids. The results from this study indicate that this relationship is appropriate at low and adequate selenium intakes, but the relationship does not exist at very high selenium intakes. Instead, the concentration of (n-6) fatty acids appears to have a stronger relationship with the Gpx activity. The findings are limited due to the fact that our subjects were from very specific areas in China. If adequate selenium intakes lead to healthier status indicators in other populations needs to be investigated.

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65 APPENDICES 55

66 56 APPENDIX A Complete Fatty Acid Profile of Plasma Samples

67 57 Plasma lime 1 time 2 time locp loc 1 loc 2 loc 3 pl-2 P 1-3 P 2-3 IA 14:0 0.5<i ± * ± * * : i ±0.0! ± ± ± : * * * * » ± ± ± « J :0 0 28* * ± ± ± : ± ± * * i : ± * ± ± ± : ± * O96 * * ± SFA ± * * * ± I6:ln7 2 10* * *0.19 l.6o± *0.20 oooi :1 n * ± * * » :ln9c 2I.3<5± * * ± * I8:ln7 2 07* * ± * *006 OOOI.2 oooi 03 20:ln * * * ± S± :ln * * OI3i Oil 0 loi 0 II 0 82 i :1 1 93* * * * * MUFA ± ± * i ± I8:2II6C 26.25* * ± ± ± * ± ± i * I8:4n3 0.02* * i i * :2n ± ± ± i J :3n6 1.46* ± * i :3n * * Oil ± ± * :4n6 6.19* * * ± * :5ii * * ± * ± :5n * * ± ,29 ± ± :6ni 1.85* * ± * * OOOI 05 PUFA 37 92* * ± * * n * ± * * * n *0 II 3.43* * * * n-s/n * * * * * PI 62.75* * * ± *

68 58 APPENDIX B Complete Fatty Acid Profiles of Breast Milk Samples

69 59 MILK limt 1 lime 2 time p loc 1 loc 2 loci P 1-2 P 1-3 P i OIW J62» i O OOOOOiOOO i OOOOOi MO i i i 0 01.«4S i <HII i i i i i OOlMiOOO O5S t OI , uo: t <M l i i MILK tin* I lime 2 liltk > \*c ) l<«2 IUC3 f.t-2,.1-3 p3-3 14: t i * I6:ln to to t n9t J i IS;ln9c * i I8.1n * * * O9 ] : In? I.4IJ2 IO.U * * ! t :ln * * : * * * MILK time 1 lintel wnep loc 1 loc 3 It*: 3 pl-2 pl-3 pj-3 I8.2n6i *0 0I :21.6c * i i > * * »0.il :4, t OO0 > > JI * * n *0.02 0,3028* t J0 3n * * > * O Sn * * ( rO O.OI * , > } t t O0

70 60 R Pi S 8 5 S

71 MILK TIME LOC ] LOC 2 LOC 3 p 1-7 pl-3 p2-3 LOCP TIME 1 TIME 2 T1MEP Inleraction M:l ± ± ± ± ± ± ± ± I6:ln ± ± ± ± ± ± ± ± I8:ln9i ± ± ± ± ± ± ± ± :ln9c ± ± ± ± ± ± ± ± I8:ln ± ± ± ± ± ± ± ± ln * ± ± ± ± ± ± :ln ± ± ± ± = ± ± ± : ± ± ± i ± ± ± ± IMV?A i i ± J 1 II = ± ± ± ON

72 62

73 63 APPENDIX C Plasma and Breast Milk Selenium, Gpx and Protein averages

74 PLASMA lime 1 j lime 2 lime p LOC 1 LOC 2 LOC 3 Pl-2 P 1-3 P2-3 Se (ng/ml) ± ± * ± ± ± '.0001*.0001* Gpx (ng/mm ml) ± ± " -104.j8± ± ± '.0001*.0035* protein (g/dl) ± ± ± ± ± Gpx (ng/min mg) ± * ± ± ± MILK Se (ng/ml) 43.2I2± ± * ± ± ± *.0001* Gpx (ng/min ml) ± ± ' ± = ± *.0195*.1956* proiein (g/dl) ± ± = ± * Gpx (ng/min mg) ± ± ± ± ± PLASMA th^i Tmic > \.IK i :.iy. : M t -1 p ; > INTER/iCTlON Se truyml) ntsd, roi?ll!1. 119^ I* JO- la!b3. 3:R* ii 4i:. 13 ai *M "i. u \y 0001 oooi- 0001* OJTO- Gpx Ifttfrnm ml) t *'N.69 1?». IW3H...> -2INO)I n: JJ4 0J.S11 ooor 0001* 003 V 3037* Loctoon 1 Locaoon I Loc»oon 3 Timt I Ttmt i MILK tune 1 Qjnt 1 urn* I cmel tm\tl ome! 7in«P LocP lt\ief»ctjoo Loe 1-2 Loc 1-1 Loc M Ux 1- Loc t- Loe I- S* tftg/ml) M Dot ;j6»«in HO:J < is J J7? 1JS J- 0001* 007*' Ooxtn^/min mi) -o9 6*) > log l«l oil -OOS76 t.» -oa:9j. *oj *1 C«J«* 001 i- 0:34* om 3831

75 65 APPENDIX D Analytical Analyses

76 66 PROCEDURE FOR SEMI-AUTOMATED SELENIUM ANALYSIS: Reference: Brown, MW and Watkinson JH. (1977) Principle: Selenium concentrations are determined by digesting the samples with nitric and perchloric acids, then adding hydrogen chloride, which converts selenium to the (+4) oxidation state. 1. Digestion of the sample: The first step in the assay is to digest the tissue sample, to release all of the selenium from the organic compounds. Weigh and label a 50 ml erlenmeyer flask for each sample to be digested. To the flask, add 1 ml of either plasma or breastmilk to be analyzed. Re-weigh the flask to determine sample weight. Under the fume hood, wearing goggles and a lab coat, add 10 ml cone. HNO, and 3 ml cone. HCI0 4 Place the flasks on the hot plates under the other fume hood with fan on, temperature set at level "4". Watch for white fumes to appear, about 30 min, and let fume for 15 min on the hot plate. Remove and let cool. Add 1 ml of cone. HCI, place back on the hot plate, and again let fume for 15 min. After the flasks have cooled, they are ready for the next step. 2. Titration: Chelate other contaminating metals in the sample by adding 15 ml of M EDTA. Use 4 drops of the selenium indicator (cresol red and brom cresol green) in each sample, then titrate the sample to pm 2-3, with 5 N NH 4 OH, so the color changes from red to yellow. If too green or purple, add 0.1 N HCI to get the correct color. 3. Calculation: Weigh the final volume of the flask and record, fill cuvettes with sample and run thru the automated Fluorimeteric system, run standards before and after all of the samples, and one standard every 20 samples or so. With a ruler, measure the peak heights of the recordings in millimeters, and determine the concentration of the ng of selenium per volume of sample according to the standard curve. 4. Standard preparation: Use selenium standard stock 1000 fig/ml. from NBS liver standard. Weigh out 1.0 g into a 1 liter flask containing 100 ml of concentrated HNO3. Dissolve and dilute to volume with deionized water. Working standards: (10, 20, 30 ng/ml) Add 1, 2, 3 mis to 100 ml volumetric flasks and dilute to volume with deionized water. Calculate the standards first, should be within ± range specified by NBS on the bottle, then calculate unknowns. Typical range for selenium concentrations ng/ml.

77 67 GLUTATHIONE PEROX1DASE PROCEDURE - FOR PLASMA AND MILK Reference: Paglia and Valentine. (1967) Whanger et al (1977). Principle: Assay determines the activity of Glutathione peroxidase by using a coupled enzyme procedure. 1. Sample preparation: Spin thawed plasma samples to precipitate the fibrinogen. Use a tabletop centrifuge, fliptop tubes, and spin at 14 RPMs for 5-10 minutes. Spin the thawed milk samples with the centrifuge in the cold room, so as to allow the fat to rise to the top of the tube. Scrape offthe fat layer with a small metal spatula before removing sample. 2. Reagent preparation: Reaction mixture for 10 assays tertiary-butyl hydroxide: 125ul + 50ml H mg NAJDPH (reduced form, Sigma) mg reduced glutathione 8.0 ml phosphate-edta-azide buffer 26.8 ul glutathione reductase Keep in 30 o C bath til use. 3. Calibrate the spectrophotometer and program accordingly. 4. Using the microcuvette holder, prepare the samples, six at a time. Initially run two blanks, holding 100 pi of water. The samples should total 100 il and dilutions with water may be made. For example, 25 pi sample of plasma + 75 pi of water. There is no dilution for the milk, and 100 pi is used. To each cuvette, add 800 pi of reaction mixture, then 100 pi of T-butyl. Cover the cuvettes with parafilm and invert to mix three times. Place the cuvettes in the spectrophotometer and run machine. After completion of the readings, remove samples and repeat with next samples. TO RUN SPECTROPHOTOMETER MACHINE IN OUR LAB: 1. At menu, select mode #9. Attachments 2. Then select program #5. CPS Kinetics 3. Change parameters: nin = wavelength 2. ±2.0 A = absorbance range 3. Lag time = 0 cycle T = 30 (time interval) N = 7 (total time) 4. Factor (multiplication factor = extinction coefficient of NADPH) 5. Upper (start point) 6. Yes (data print) 7. No (Reagent blank) 8. No (Gain x 10) 9. No (Cell balnk)

78 68 Be consistent with temperature, use 30 C for all runs. 5. Calculations: For nmols NADPH oxidized per minute per ml sample Aabsorbance ("unknown) - Aabsorbance (blank) x 1.0 ml (total volume') x mmol ml of sample 6.22 A m) 1 (extinction coefficient of NADPH) Typical absorbance values range from 0 to -500.

79 69 FATTY ACID EXTRACTIONS Reference: Song and Wander (1991), Bligh and Dyer (1959) Principle: Using chloroform/methanol, the fatty acids were extracted from the samples, and their quantities were measured by gas chromatography. Plasma 1. Prepare the internal standards for each sample. The internal standard used for the plasma samples is 17:0 ( g/ml). Heat up the hot water bath under the hood, and bring the external standard to room temperature before use. Pipette 15 ^1 of standard into a 100x10 mm screw top test tube, and dry down the sample using N2 gas for 2-3 mins. 2. Add 200 il of vortexed plasma sample to the test tube, 800 (il of 0.8% saline solution, and 3.75 ml of 1:2 chloroform/methanol. At this stage the test tube is placed on the mechanized shaker for at least 1 hour. The samples can shake on the shaker for 2-3 hours. 3. Spin the test tubes in the upstairs centrifuge at 3000 rpm for 10 min. at 16 C, speed high and max. Remove supernatent and put into 120x10 mm size tube. Resuspend the precipitate, with 1 ml of water and 3.75 ml of chloroform/methanol and vortex. Spin again as above. Remove supernatent and add to previous test tube. 4. To the test tube with supernatent, add 2.5 ml of water and 2.5 ml of chloroform. Vortex and spin mixture as above. 5. Aspirate the top layer from the test tube, and using a disposable pipette, remove the bottom layer in the test tube (get all of it) and place in a clean test tube. The fatty acids are now extracted into the chloroform, and can remain stable for 2 weeks in the refrigerator. 6. Methylation: Be sure that the blower under the hood has the water bath full and turned on. Add 200 \i\ of benzene and 1ml of boron trichloride (Supelco, Inc.) Must be at room temperature and add it under the hood to the sample. Put the test tube in a heating block at95 o Cfor90min. 7. Cool test tube to room temperature, add 5 ml hexane (HPLC grade) and vortex thoroughly for 2 minutes. 8. Centrifuge at 1500 rpm for 10 minutes, remove top hexane layer. Add 5 ml of hexane to supernatent and vortex again for 2 minutes.

80 9. Centrifuge again, remove top layer of hexane, add it to previous amount. To this test tube, add g of solid Na2S04, mix for 45 sec. on vortex. Transfer the extract to a 100x10mm test tube and evaporate under hood in bath. 10. Reconstitute sample with 250 nl of iso-octane (HPLC grade) and transfer to an Eppendorf size tube. Can be stored in refrigerator til injection. 11. Injection: Turn on the computer connected to the GC. Turn the knobs on the "air" and "Nitrogen" tanks. For the "Helium" tank, adjust psi to 40. On the GC, turn aux gas knob all the way on, to light flame, push "sig 1" then "FID ignition" want it to read between In the computer program, enter the date and time. Specify method, METHOD3.M, then load method. Edit file, give a file name for each run, always use V3 for the drive. Name the RAWDATA and the INTERGRATION the same. QUIT the program and push RUN METHOD. 13. Now you are ready to inject 2 (il of the sample, push the start button as soon as the syringe is emptied. The syringe must be rinsed 20 times with octane between each use. 14. Each run will go for about 30 minutes; run a standard sample as well. The standard contains all the fatty acids and their elution times. The unknown samples can be determined by comparing the chromatograms. The standard for the plasma fatty acid is COMP30, inject 2 \i\, and use these elution times to determine the unknown samples fatty acid profiles. 70 Breast milk The milk samples were partially prepared for the fatty acid analysis by the researchers in China before being delivered to Oregon. The treatment of the breast milk was as follows: Two ml of milk are added to 14 ml of chloroform/methanol (1:1 mixture containing 71 mg/l BHT), homogenize by sonication, add 4 ml water, mix with a vortex, centrifuge at 1100 x g for 15 minutes and remove the bottom phase. This is the chloroform phase that was sent to Oregon. Unfortunately, the samples were stored in plastic screw top vials, and there was some leakage of many of the samples, and samples were unclearly labelled. I only know the region and the time point of these samples, not the individual subject numbers. 1. To complete the preparation for analysis, I first added 500 (il of a standard to the test tubes. Samples 22-39, used the internal standard 17:0. I ran out of that standard,

81 so I used 23:0 (lomg/ml) for samples 1-21, For samples 1-10, the standard had not been completely dissolved in the solution, so the peaks were a bit diluted. 2. After drying down the standard with nitrogen gas under the hood, the contents of the plastic vials were poured into the test tube. These samples were also dried down under the hood with nitrogen gas. 3. Add 0.2 ml of benzene and 1.0 ml of room temperature boron trichloride, cap tightly, and put into 95 0 C heat block for 90 minutes. 4. Remove from heat block and let cool for 5 minutes. Then add 5 ml of distilled water, and 5 ml of hexane to the test tube. Using the multi-tube vortexer, vortex for 1 minute. Centrifuge the test tubes at 3000 rpm for 10 min at 16 0 C. Transfer the top hexane layer to a 125x16mm test tube. 5. Add 5 ml of hexane to first tube again, vortex and centrifuge as above. Remove hexane layer and add to previous layer. Add about 0.3 g solid Na 2 SO, ) to each test tube and vortex for 45 seconds. Transfer the extracts to a 100x10 mm test, tube and dry down samples under the hood with nitrogen gas. Reconstitute the samples in 1.0 ml of iso-octane. This sample is generally too concentrated to obtain accurate peaks from the GC, so the samples were diluted before injection. Dilution 1:100 il original + 1 ml iso-octane All the samples were run at this concentration; however, some samples were too dilute at this concentration so they were rerun at a more concentrated solution. I looked for the appearance of the fatty acid 20:5n3 (area % at least 50) to determine if it was concentrated enough. RERUN 1 If the absolute sum of the fatty acids was less than 1000, the samples were run at no dilution Subjects: 7, 8, 2h, 3a Sum less than 10,000, samples diluted 250 nl original ^1 of iso-octane Subjects: 4, 6, 11, 17, 2b, 2c, 2d, 2e, 3i, 69 Sum less than 30,000, samples diluted 100 nl original ^1 of iso-octane. Subjects: 1, 9, 10, 14, 16, 19, 20, 2i, 3h, 52, 53, 54, 56, 57, 59, 61, 67, 68 RERUN 2 Try more concentrted 250 il original il iso-octane for; Subjects: 20, 2i, 56, 59 Try no dilutions for: Subjects: 11, 17,2b, 2d The GC program that was run for the milk samples had a lower temperature than the 71

82 plasma, so we could obtain the short chain fatty acids, the method selected was MJLK3.M. For the first thirty minutes the temperature was 150 C, instead of at 170 C. Then the temperature increased 2 C every minute to 200 C, then 3 C every minute to 220 C. Additional notes about the fatty acid extracts: Samples 3 a and 2h were empty vials. I rinsed the vials with 2 ml of chloroform, and continued with the procedure for methylation. Samples 40, 48, 49, 5 I were tainted with ink from their labels, but I continued the methylation treatment same as the other samples. 72

83 73 Reference: Lowry et al. (1951) LOWRY PROTEIN DETERMINATION Principle: There is an initial interaction between the protein and cupric ion in an alkali solution, and then a reduction of the phosphotungstic and phosphomolybdic acids to tungsten blue and molybdenum blue by the Cu-protein complex and by the tryosine and tryptophan of the protein. The latter two amino acids give color in the absence of the Cu ion, while the rest of the protein gives color in the presence of the Cu ion. About three quarters of the color is dependent on the Cu ion. The color is not proportional to concentration of the protein. Reagents: I made a 2% Na 2 C03 dissolved in 0.1 N NaOH with 4.0g of NaOH in 1 liter of distilled water, and dissolving 20.Og of Na 2 C03 ' n t' 16 solution. Already prepared were: 2.7% sodium potassium tartrate in water r/ocuso,, 5H 2 0 in water IN Folin & Ciocalteu's phenol reagent, before use, dilute l:l with water from 2N reagent, make fresh each day. Standards: BSA solution To prepare the standard BSA, weigh out 1.5 g of BSA and gently layer it on top of 50 ml of distilled water in a 100 ml beaker, let it sink to dissolve, do not shake or stir. After dissolved, filter through Whatman paper #l. This is a solution of 300mg/ml. Put 3.3 ml of 30 mg/ml solution into a 100 ml volumetric flask, fill with distilled water to volume. You now have the 1 mg/ml stock solution Absorbance of BSA at 280nm is 0.66 for 1.0 mg/ml concentration of the stock = abs280nm/0.66 x I Procedure: 1. Make the 6 standards in 10 x 1.5cm test tubes: 2. Prepare seaim samples: 1 me/ml stock in ^1 distilled water in il

84 Dilute serum 50 times with water. Put 50ul of diluted serum into a test tube, add 450ul of distilled water. For milk, no initial dilution is made. Pipette 5ul of milk into test tube, then add 495ul of distilled water. 3. Place all of the test tubes to be analyzed in a test tube rack 4. Prepare 4.9ml of 2% Na^Oj, 0.05ml of sodium potassium tartrate, and 0.05 ml of 1% CuS0 4 mixture for each sample, be sure to add CuS0 4 last. 5. Add 5ml of the prepared mixture at timed intervals in each of the samples and mix thoroughly on a Vortex mixer. Start the stopwatch when adding reagent to tube 1, wait 15sec before adding reagent to tube 2. Keep the same timing between tubes, it is critical. At the end of 10 min, add 0.5ml phenol reagent to the first tuve, mix on vortexer, 15sec later add reagent to the second tube, ect. this way each tube has a 10 minute incubation under room temp before the phenol reagent is added. After the last of phenol to the tubes, begin timing a 30 min incubation. 6. After incubation, read the absorbance of the samples at 750nm using the spectrophotometer. See following progam. 7. Calculate ug of protein in each sample using the standard curve. P = Ps(ug) x D x 0.1 (g/100ml) Vs (ul) P = serum protein concentration (g/100ml) Ps = ug of protein in standard Vs = ul of diluted serum used D = serum diluted factor, 50 To calculate the standard curve, you must use the PHOTOMETRIC program on the spectrophotometer. Read the BSA stock solution at 280nm. Calculate the correct concentration of the solution by using this equation: X x 100= _ug/ul STATISTICS The data collected for each subject was put into tabular form, using QuattroPro 6.02 spreadsheets. In order to analyze the data and to look for differences between variables,

85 the statistical program SAS 6.11 and SAS/INSIGHT was used. After grouping the different variables, the distribution of the data was analyzed. In order to conduct a statistical analysis, it is important that the data fits several assumptions (1) that the distribution is normal and (2) that there is normal variance. For the selenium and the glutathione peroxidase activity, taking the natural log of the results allowed the data to better fit the assumptions. Without the (log Y) function, the plot of the residuals vs. the predicted did not demonstrate a normal variance between groups of subjects. Box plots of the location vs. the response variable can indicate the outliers in the data set, however, an ANOVA analysis is robust enough to be able to deal with these outliers. By using SAS/INSIGHT, an exploratory analysis of the data was possible. From that information, several statistical programs were inputted into SAS libname temp 'b:'; 75 data temp, (filename); infile 'b:\(filename).txt' firstobs=2; input sub time loc varl var2...var''n"; run; proc print; run; OPTION: if time = ' 1' then delete; '2' proc corr; run; proc glm; proc glm; classes time loc; class loc; model varl var2...var"n" = time loc time*loc; model varl var2...var"n Ismeans loc/stderr pdiff; Ismeans loc/stderr pdiff; Ismeans time/stdert pdiff, run; run; proc reg; proc means n mean stderr; by time; classes time; model var 1 = var 2; var varl var2...var"n" run; run loc

86 76 APPENDIX E Raw Data

87 Individual plasma fatty acids (area %), ' 1 Oiect ' I ibo In) : '80 3 '"91 '3 in9<: 'e".;! Q C 3 7 "51 6 2n6< 20 0 '8 3n3c 20 in9 ' 8 4..J ; i c?o ;n6 20 J/iS i 16029! I c ' ' ' o; W Ol S I ! ! I I ! ! S l ii l : U C ) ! ! i ' '2; Ol I ' 3 L ! i : 41 o: 0! i ; : Oi Ol o ' ' ' 3' ' ^ ' 9"'\ IS Ol 0! i ' : ol l 28226! i 53206!! 7 Ol : Ol Oi Ol * , i «9316! i i 25043! ! i l l l ol l l ol ! ol l ' ! Ol I ! ' l JO Ol l ' C l ' l l o l i i i Ol l l ! w l o i 3981; ol ' 54997! ! ; Ol 01 l 59711! Ol C« l J ol ! l o l o Ol i I ol S ! l l ; Ol Ol * ol S47S5I S l 2862S Oi l I ol i 1530: S9I Ol l C i c I942< 1 7 U566I r _ o l i i I7» c Ol ' c ' i 3365( i SUM 52 1 c Ol i I c i i 3446 l ' c ) I l «i c c Ol ) ! SUSJ ) c SI ! f ) c l l\ ! e ) 31 C Ol i 33891

88 : Individual plasma fatty acids (area %) n9 i 20 4n InTl 230 I!0 5n3c i 240!?4 1, 2?" 5.0 ' OlMefi : SUM i 170 ; SAt ; wuf A i >Uf A R6 -O n6">) p,! U96I ! S ; 591? r ! i ? T 61 d i ! ) : Ol ! J4J Ol ,0 70 ' f o i90?e , O8748I 5 7B S II ! ! IS I o: ? o ! ! ! loo l? O ? ! O I ! SS 56 5 S( ; I looi I Ol i ! ( , ? i OI ! 11! 55 i i : : ' ' ! i J44I ' ; ; ! ' ' I c , ; ! I i S ! I i I ! ; ', ! I093< > ( > : i i l Si ^ J5I2 12O56I S322I ' ' ! ! : Jl ! ! , ; 1001 S3 0266! ! ' Q4663 l < ' : ! ! ! ' l i I 0! ! ( i ! ! f 0' ! " Si , ! looi : , i 'C i ' i '30472; ' 2669: '001 4?37Si X) 3583! 41,932, : : ; ' Si : i 0564i i 31643: ' i : ; S1563, CU ' : i ? iqo ; ! : , Ji , 0 13W5 C 2i8i; ! i 36314, : i ; ' "To 5! 00

89 Individual breast milk fatty acids (area %) 2a ;i6ln7! 180, H lfi9l 18 ln9cl '8 I8;n6l'ia?n6cl in9' 18 4n n6 20 3"n6r'HO 2l }" l l '< Oi I?'}, '"_pl '0 '44 0 IS66I O45! : oi ? Ol ' l ! ! ! Mill I I r ]" I 502Tp l O9r '7181 l pi pi ' '51 I ) " l 0061 i ' ' I3 ;4i67. 0! ' l l 0437! ' "J*: ' ?46i ' ^ ' '81 i ' ' 0! 0! ' '. ' O63; ' ' " 0 "" 01 ' l ' Ol ' "' I7SI ? ' ' Q ' 10" Ql ', I6a7. _0i ! _ 0 01 ' ] ~ i 60'. ~0J_"" oto '434. oi ' ' ~IL L J^ ' '3651 '"" J ; 0 29" ol i I 7766! ' ! ' _ Q 3178! 0 '532; :03_7I '2 151[ Oi ~5 " "51' ' ! ;3I ? ll " I ' 5756 ' ! ! 'l I30l l 242?! i 2064' O049i i ;II 0' ' ?'! ' ' ^S' i 4942! 79 2& ' »706l 3 967' ll 64i , 7 486r 70i6i " ' 3369 Ol" '323l 40' ! ' 3578' 31 08: '9 023' ' '315! ~ i'2 si ' ' ' JL2J ' isiii 305;? 0' 17?Oii " Oi 0 ' ' i??' 4?. 0 77" ' "0; 0, ' 566" 6 304? ' : 0 3': : 3435i ' 067« , " : 6 304?: ; ' ! ' , " 01 44, 0! ' Ol Oi ' 3187! 7 ' , }02'e- 3 0 ' ' ' 0049! 0 0 ' 4? ' 0: 01 "5' 0 '658. ' 6622' , ! ^: 19 'Ji '938! ' 260'. 2i _o_ ' j Oi '22" ' ' 842" 59035' ' 0 0, ? "ot"" 0 ' ' , Ol? ' 4i?a ; 2 716i 0! 0, ' " Oi ""0 ' 368' ' Oi ! Oi ? ' " ' 05017! " ! " " Ol 0 5" ? ' 0941! ' " " ! i ' 073? ! or J ? O5O "31 ' ! ? " ??8 6?l? " i " '677' 0 Ol '605" Ol Oi ! ? ! I695' ; ! ' ! ! ! 01 ' 1 08«l ! ! 0 5" ' : ! ' ?! Ol ' ? ! " 7 l?07l i ' "! 0 0! I0 4i4 0? " , 0 246? ' III " O5t I ' 0 0! ' ! 10569! " l I ' 6657! 0875" ^1

90 Individual breast milk fatty acids (area %) SuOioct ln n3<l :5n n3 I Olheu ISUM j 170 ISA! IMUFA IPUFA n6 i -.3 n&ro PI ' H 0 2[ 0 U O ! II l lool i d l J sr er J ! ' ' : 39 28] li L 0.92 H ! OI 2! ' ' IU : ] , ! !» ' ! ' ] ! I « ] lool ! , I9 429J li IB ) mi ) ! ] ] ' 2a ] : 2b lool , 2c ! * ! « ! !8 4a , !! ] OL , X i C Ol < ] ! ! , , S I! ! a ! S fll ! ] Ol f I : ! ! ! 100 I37i: : ! Hll ! U I ) I II <! : < S « S 9 970ll ! ! » U49! ! l t I ! l ! : 1 37l< I O; lool ' O 91! I ] ! c