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J. Dairy Sci. 2007. 90:3308-3313. doi:10.3168/jds.2006-891
© 2007 American Dairy Science Association ®
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Concentrations of 17ß-Estradiol in Holstein Whole Milk

D. A. Pape-Zambito, A. L. Magliaro and R. S. Kensinger1

Department of Dairy and Animal Science, The Pennsylvania State University, University Park 16802

1 Corresponding author: rsk7{at}psu.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 ACKNOWLEDGEMENTS
 REFERENCES
 
Some individuals have expressed concern about estrogens in food because of their potential to promote growth of estrogen-sensitive human cancer cells. Researchers have reported concentrations of estrogen in milk but few whole milk samples have been analyzed. Because estrogen associates with the fat phase of milk, the analysis of whole milk is an important consideration. The objectives of this study, therefore, were to quantify 17ß-estradiol (E2) in whole milk from dairy cows and to determine whether E2 concentrations in milk from cows in the second half of pregnancy were greater than that in milk from cows in the first half of pregnancy or in nonpregnant cows. Milk samples and weights were collected during a single morning milking from 206 Holstein cows. Triplicate samples were collected and 2 samples were analyzed for fat, protein, lactose, and somatic cell counts (SCC); 1 sample was homogenized and analyzed for E2. The homogenized whole milk (3 mL) was extracted twice with ethyl acetate and once with methanol. The extract was reconstituted in benzene:methanol (9:1, vol/vol) and run over a Sephadex LH-20 column to separate E2 from cholesterol and estrone before quantification using radioimmunoassay. Cows were classified as not pregnant (NP, n = 138), early pregnant (EP, 1 to 140 d pregnant, n = 47), or midpregnant (MP, 141 to 210 d pregnant, n = 21) at the time of milk sampling based on herd health records. Mean E2 concentration in whole milk was 1.4 ± 0.2 pg/mL and ranged from nondetectable to 22.9 pg/mL. Milk E2 concentrations averaged 1.3, 0.9, and 3.0 pg/mL for NP, EP, and MP cows, respectively. Milk E2 concentrations for MP cows were greater and differed from those of NP and EP cows. Milk composition was normal for a Holstein herd in that log SCC values and percentages of fat, protein, and lactose averaged 4.9, 3.5, 3.1, and 4.8, respectively. Estradiol concentration was significantly correlated (r = 0.20) with percentage fat in milk. Mean milk yield was 18.9 ± 0.6 kg for the morning milking. The mean E2 mass accumulated in the morning milk was 23.2 ± 3.4 ng/cow. Likewise, using the overall mean concentration for E2 in milk, the mean E2 mass in 237 mL (8 fluid ounces) of raw whole milk was 330 pg. The quantity of E2 in whole milk, therefore, is low and is unlikely to pose a health risk for humans.

Key Words: 17ß-estradiol • whole milk • pregnancy


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 ACKNOWLEDGEMENTS
 REFERENCES
 
Milk is a valuable nutrient source for humans, but recent reports have questioned its safety because of the presence of steroid hormones in milk. Estrogens are present in milk (Monk et al., 1975; Erb et al., 1977a,b) and if present at high concentrations, could affect biological processes within humans who consume milk. Hartmann et al. (1998) reported that dairy products might account for up to 60% of true estrogens (compared with phytoestrogens) in a typical German diet. It has been hypothesized that long-term exposure to estrogens increases the risk of breast cancer (Yue et al., 2003) and others have specifically stated that the estrogens in milk may be responsible for increased cancer risk (Li et al., 2003; Qin et al., 2004). Consequently, individuals with estrogen-sensitive cancers are concerned with the potential biological consequences of estrogens in dairy products.

Several researchers have quantified estrogens in skim milk (Wolford and Argoudelis, 1979; Abeyawardene et al., 1984; Lopez et al., 2002), but few have quantified 17ß-estradiol (E2) in whole milk. This distinction is important because E2 is a lipophilic molecule and is at a greater concentration in the fat fraction of milk (Schwalm and Tucker, 1978; Wolford and Argoudelis, 1979). Monk et al. (1975) reported that E2 concentrations in whole milk from cyclic (n = 6) and pregnant (n = 13) cows averaged 37 and 64 pg/mL, respectively. Erb et al. (1977a, b) reported a mean whole milk E2 concentration of 13 pg/mL in 6 nonpregnant cows that were between 3 and 25 DIM. Wolford and Argoudelis (1979) quantified E2 from 2 to 4 raw and commercial whole milk samples with E2 averaging 12.3 and 6.7 pg/mL, respectively. These studies were valuable and gave estimates of E2 in whole milk but the number of cows or samples used were small.

Previous research has also shown that E2 concentrations in plasma increase throughout pregnancy in the cow (Robertson and King, 1979; Patel et al., 1999) and are correlated with milk E2 concentrations (Batra et al., 1980; Abeyawardene et al., 1984). It is less clear whether milk E2 concentrations also increased throughout pregnancy. Given the reported concerns about estrogens in milk and because genetic variability and stage of pregnancy may lead to differences in E2 concentration, it is important to analyze E2 concentration in whole milk from a large number of cows.

The objectives of this study were to analyze E2 concentrations in milk from a large number of cows (>200) and to determine whether the E2 concentrations in milk from cows in the second half of pregnancy are greater than those in milk from cows in the first half of pregnancy or in nonpregnant cows.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 CONCLUSIONS
 ACKNOWLEDGEMENTS
 REFERENCES
 
Sample Collection
Triplicate milk samples were collected during a single November morning milking (0500 h) of 206 Holstein cows in the Pennsylvania State University dairy herd in a milking parlor equipped with the AFIfarm system (S.A.E. Afikim, Kibbutz Afikim, Israel; US Distributor: Germania Dairy Automation, Waunakee, WI). Duplicate samples were sent to DHIA for fat, protein, lactose, and SCC analyses. The remaining milk sample (9 mL) was homogenized using a Polytron homogenizer with a PTA probe on setting 6 for 8 s before storage at –20°C.

Milk weights and reproductive status for each cow were obtained from farm records. Herd reproduction was conventionally managed with a 60-d voluntary waiting period before breeding was attempted. Cows were given a 60-d dry period. All cows sampled were >5 DIM. Based upon the date of sampling, cows were categorized as not pregnant (NP; n = 138) or in the first or second half of pregnancy. Cows in the first half of pregnancy were designated as early pregnant (EP; 1 to 140 d pregnant, n = 47), whereas cows in the second half of pregnancy were designated as midpregnant (MP; 141 to 210 d pregnant, n = 21). Sixty-nine cows calved in the 3-mo period before our sampling day, which contributed to a larger number of NP cows relative to EP or LP cows.

E2 Extraction from Whole Milk
Solvent extraction procedures were adapted from Monk et al. (1975) and Wolford and Argoudelis (1979). Homogenized milk samples were thawed in a warm water bath and vortexed for 10 s before aliquoting 3 mL into a 50-mL screw cap glass extraction tube. Milk samples were extracted with 9 mL of ethyl acetate (HPLC grade, J. T. Baker, Phillipsburg, NJ). The mixture was vortexed for 30 s and then placed on an orbital shaker for 15 min. The vortex and shaking steps were repeated before incubation at –20°C for 2 h. The resultant liquid organic layer was transferred to a glass test tube and dried under N2 at 50°C. The ethyl acetate extraction was then repeated. After freezing, the organic layer from the second ethyl acetate extraction was transferred to a corresponding sample tube and dried again. Warm (50°C) methanol (2 mL, HPLC grade, Burdick and Jackson, Muskegon, MI) was added to the extract, and the mixture was incubated at 50°C for 1 h with thorough mixing at 0, 15, 30, 45, and 60 min. The mixture was subsequently incubated at –20°C for 1 h and then centrifuged at 1,370 x g for 30 min at 4°C to precipitate triglycerides. The supernatant solution containing the steroid hormone fraction was quantitatively transferred to a clean glass test tube and dried under N2 at 50°C.

The estrogen-containing extract was reconstituted in benzene:methanol (9:1, vol/vol; benzene from Sigma-Aldrich, St. Louis, MO). Column chromatography was used to separate E2 from residual cholesterol and other steroids (Mikhail et al., 1970). A Sephadex LH-20 column (GE Healthcare, Piscataway, NJ) was packed to a height of 2.5 cm in glass columns with an internal diameter of 1 cm. Steroids were eluted with benzene:methanol (9:1, vol/vol) as described by Kensinger et al. (1986). The E2 elution pattern was verified with 2, 4, 6, 7-3H-E2 (GE Healthcare; cat no. TRK322), and the estrone (E1) elution pattern was verified using crystalline estrone followed by RIA (Diagnostic Systems Laboratory, Webster, TX; cat no. DSL-8700). Estrone was eluted between 1.5 and 4.5 mL, and E2 was eluted between 4.7 and 8.3 mL at 25°C. Other experiments in our lab have verified the E1 elution pattern using 2, 4, 6, 7-3H-E1 (GE Healthcare; cat no. TRK321). In addition, we have confirmed the absence of cholesterol in the E2 fraction when this methodology is used. The E2 fraction was dried under N2 at 50°C.

E2 Quantification by RIA
The dried E2 fraction from each milk sample was reconstituted in 125 µL of pooled wether sera before quantification using an RIA specific for E2 (cat. no. 07-138106; MP Biomedicals, Irvine, CA). The immunogen used to generate the antiserum in rabbits was 6-keto-estradiol-17ß-6-oxime-BSA. The cross-reactivity of the antiserum with other steroids is as follows: estrone, 20%, estriol, 1.5%, 17{alpha}-estradiol, 0.68%, and <0.01% with cholesterol, progestins, and androgens. Samples were run in duplicate according to the manufacturer’s instructions.

Internal standards were run for each set of samples extracted to quantify percentage E2 recovery from whole milk. Tritiated E2 (0.01 µCi) was added to pooled homogenized whole milk samples. After the final reconstitution step in pooled wether sera, the internal standards (125 µL) were pipetted into 7-mL scintillation vials with 5 mL of Ecolite scintillation fluid (ICN, Costa Mesa, CA) and counted with a Beckman LS 6500 scintillation counter (Beckman Coulter, Fullerton, CA). Control pooled milk and plasma samples were also run in the RIA with each set of unknown samples to determine inter- and intraassay variation. Estradiol was analyzed in 4 assays with cows at all stages of pregnancy represented in each assay. The inter- and intraassay coefficients of variation were each 6.8%.

The E2 concentrations were corrected for recovery of 3H-E2. Fifty-six percent of the samples repeatedly predicted below the limit of quantification (LOQ) of the RIA (0.7 pg of E2/mL of milk). Low concentrations of E2 were detected in these samples, but the predicted value was less than the lowest concentration standard from the standard curve. Any milk sample that predicted below the LOQ was assigned the LOQ value of 0.7 pg of E2/mL of milk. The data presented, therefore, are conservative estimates of E2 concentrations in raw whole milk. Estradiol mass was calculated by multiplying E2 concentration by the volume of milk produced at that milking. Milk volume (mL) was calculated using the following conversion factors beginning with pounds of milk: 1 gallon of milk/8.8 pounds of milk; 3.78 L of milk/1 gallon of milk; 1,000 mL of milk/1 L of milk.

Statistical Analyses
Version 8.2 of the SAS software was used for statistical analyses (SAS Institute, Cary, NC); PROC GLM was used to determine if E2 concentrations were affected by reproductive status. Reproductive status was a fixed effect in the following model: E2 concentration, E2 mass, percentage fat, percentage protein, percentage lactose, log SCC, milk yield = reproductive status. The least squares means option was used to determine differences between reproduction categories. The MANOVA option within PROC GLM was used to determine partial correlations between E2 concentrations, milk components, and milk yield after adjusting for reproductive status.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 CONCLUSIONS
 ACKNOWLEDGEMENTS
 REFERENCES
 
Reported E2 concentrations were corrected for the mean recovery of E2 from raw whole milk, which was 59.4 ± 2.1% (mean ± SD). The E2 concentration in raw whole milk averaged 1.4 ± 0.2 pg/mL of milk (mean ± SEM) and ranged from nondetectable to 22.9 pg/mL of milk. Estradiol concentrations were 1.3 ± 0.2, 0.9 ± 0.3, and 3.0 ± 0.4 pg/mL milk for NP, EP, and MP cows, respectively. The concentration of E2 did not differ between NP and EP cows; however, E2 in whole milk of MP cows was greater compared with that in NP and EP cows (P < 0.001).

Figure 1Go shows a scatter plot of E2 concentrations in milk vs. days pregnant for the 68 pregnant cows. Milk E2 increased as days pregnant increased. The best-fit equation to describe this relationship was a second-order polynomial (r = 0.61, P < 0.01).


Figure 1
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  		Figure 1. Concentrations of 17ß-estradiol in whole milk from individual cows (n = 68) vs. days of pregnancy (d 1 to 210). The best-fit equation to describe the relationship, a second-order polynomial (y = 0.0001x2 – 0.0163x + 1.1005; r = 0.61, P < 0.01), is also shown.

 
Milk yield for the morning milking averaged 18.9 ± 0.6 kg overall and was lower (P < 0.05) for MP cows compared with NP and EP cows, which did not differ (Table 1Go). Milk fat averaged 3.5 ± 0.1% overall and 3.6, 3.3, and 3.6% in NP, EP, and MP cows, respectively. Total E2 mass accumulated in the morning milking averaged 23.2 ± 3.4 ng/cow and was greater (P < 0.05) in MP cows than in NP or EP cows. Estradiol concentrations in whole milk were positively correlated with percentage milk fat and negatively correlated with milk yield (Table 2Go).


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  		Table 1. Milk composition, milk yield, and mass of 17ß-estradiol (E2) accumulated in milk for cows grouped by reproductive status
 

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  		Table 2. Partial correlation coefficients among milk 17ß-estradiol (E2) concentrations and milk components, E2 mass, and milk yield (MY)1
 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
CONCLUSIONS
 ACKNOWLEDGEMENTS
 REFERENCES
 
The concentrations of E2 in raw whole milk were greater in cows >141 d pregnant compared with non-pregnant cows and cows <141 d pregnant, and E2 concentrations tended to increase during pregnancy (Figure 1Go). In contrast, Monk et al. (1975) did not show increased E2 concentrations in milk during pregnancy. Our results, however, were not surprising because milk E2 is correlated with plasma E2 (Batra et al., 1980; Abeyawardene et al., 1984) and plasma E2 concentrations increase throughout pregnancy in cows (Robertson and King, 1979; Patel et al., 1999). If E2 levels in plasma and milk are positively correlated and plasma concentrations increase throughout pregnancy, one would expect E2 in milk to increase throughout pregnancy.

Mean E2 concentrations in the present study were lower than those reported by others (Monk et al., 1975; Erb et al., 1977a; Wolford and Argoudelis, 1979). This could be attributed to differences in sample handling (i.e., homogenization), extraction and isolation methodologies, physiological states of the cows when the samples were collected, genetics, season during sample collection, purity of the E2 standard used, or the fact that different antibodies were used for detection of E2. Monk et al. (1975) discussed these factors and suggested that within-study comparisons are more useful than comparisons of absolute concentrations across studies. Plasma E2 concentrations steadily increase during pregnancy (Patel et al., 1999); however, plasma E2 concentrations greatly increase 20 to 30 d prepartum because of increased placental conversion of progesterone to E2 (Henricks et al., 1972; Patel et al., 1999). Estradiol concentrations in mammary secretions are greatest several days around parturition (Monk et al., 1975) coincident with the increase in plasma E2. Erb et al. (1977b) reported a mean E2 concentration of 13 pg/mL of milk in cows between 3 and 25 DIM. The values reported in the present study were representative of cows in various reproductive states (i.e., noncycling, follicular, luteal, and various stages of pregnancy) although all cows were >5 DIM. Milk samples collected during the first 25 DIM would likely have greater E2 concentrations given the carryover effects of the recent calving. That the 138 NP cows in our study averaged 123 DIM on the milk sampling day may explain, in part, why our values were lower than those of Erb et al. (1977b). Wolford and Argoudelis (1979) did not report the reproductive status or DIM for the 2 to 4 cows sampled for their E2 analyses. Their average milk E2 concentration of 12.3 pg/mL, however, is consistent with the range presented in this study. Monk et al. (1975) reported a mean milk E2 concentration of 63 pg/mL in 13 pregnant cows. Procedural differences including the antibody used may be one reason that our estimates of milk E2 concentration were lower. We adjusted for recovery of tritiated E2 standard from milk samples and showed that increasing the extracted milk volume led to greater E2 recovery (parallelism). We also measured 158% recovery of E2 added to whole milk in added-mass verification tests of our assay. The 158% is not perfect but it was deemed reasonable given the extensive sample manipulation throughout the assay procedure.

The extraction and column chromatography procedures used in the present study led to the separation of E2 from other whole milk components and potentially cross-reactive compounds such as cholesterol. The presence of structurally related compounds in the RIA could lead to inflated E2 concentrations. The average cholesterol concentration in raw milk is 144 ± 38 µg/mL (Bachman et al., 1976). Although the E2 antibody in the RIA cross-reacted <0.01% with cholesterol, cholesterol could, at µg/mL concentrations, interfere with anti-body-based detection of E2 in milk. If the antibody had significant cross-reactivity with cholesterol and measures were not taken to separate E2 from cholesterol, erroneously high concentrations of E2 would be reported.

The relatively low E2 concentrations reported in the present study were verified by repeating this experiment at a later date with a smaller number of randomly selected cows (n = 19; NP = 8, EP = 11). Estradiol concentrations in milk averaged 0.9 pg/mL in the replicated study with values ranging from nondetectable to 3.0 pg/mL. In retrospect, we would expect the mean value to be somewhat lower than the mean for the data set with 206 cows (1.4 pg/mL of milk) because the smaller study did not contain any MP cows.

Estradiol concentrations in whole milk were correlated with milk fat percentage (Table 2Go). This is not surprising because E2 is found in greater concentrations in the fat fraction of milk (Schwalm and Tucker, 1978; Wolford and Argoudelis, 1979; our own unpublished results). Previous research also showed that progesterone was significantly correlated (r = 0.98) with the fat percentage of milk (Ginther et al., 1976). Progesterone is less polar than E2, which could explain why its correlation with milk fat is greater than that observed between E2 and milk fat in the present study. Skim milk was used by others to measure relative changes in E2 concentrations associated with the estrous cycle (Abeyawardene et al., 1984; Scholey et al., 2005). Estradiol concentrations measured in skim milk, however, do not reflect E2 in whole milk, because a large proportion of the E2 associated with the fat would be removed before analysis. The r-value for the correlation between E2 and percentage milk fat in the current study was significant but small (0.20), suggesting that the concentration of E2 in milk is determined by multiple factors. Plasma E2 concentrations in the mammary blood vessels influence the concentrations in milk (Monk et al., 1975). However, genetics, integrity of tight junctions, and the presence of a transport protein such as albumin may also influence E2 concentration in milk. It is possible that mastitis could influence milk E2 concentrations if the integrity of tight junctions within the mammary gland was compromised, allowing increased concentrations of albumin (a nonspecific carrier of estradiol) into the alveolar lumen. Mastitis, therefore, might increase milk E2 concentration in infected glands relative to non-infected glands. The accumulation of E2 in milk is probably a complex process and is not well defined.

Fat percentage was greater in the milk of NP cows compared with EP cows and some may find this result surprising. We evaluated milk composition, however, in relation to reproductive status and reproductive status was confounded with DIM. Some of the NP cows were in an advanced stage of lactation and may have contributed to the greater milk fat percentages observed in NP cows compared with EP cows.

Although the E2 concentration in the milk of cows >141 d pregnant was greater than that in nonpregnant cows or cows <141 d pregnant, the concentrations in raw whole milk were low. The mass of E2 in 237 mL (8 fluid oz.) of raw whole milk would average 330 pg. This is a conservative estimate of E2 mass in milk because 56% of the analyzed samples repeatedly predicted below the LOQ of the RIA but were assigned the value for the LOQ (0.7 pg/mL of milk). Guidelines provided by the USDA suggest consuming 3 servings of dairy foods per day (www.mypyramid.gov). Based on the raw whole milk data in the present study, if a person consumed 24 fluid oz. (or 711 mL) of whole milk per day, then he or she would ingest slightly less than 1 ng of E2/d. This analysis assumes that pasteurization and homogenization do not affect concentrations of E2 in milk (Pape-Zambito et al., 2006) and does not account for a lower percentage of fat in milk that is sold commercially. Reported 24-h production rates of E2 in humans range from 400 ng/d in prepubertal girls (<8 yr old; Joint FAO/WHO Expert Committee on Food Additives, 2000) to 37.8 mg/d in late-pregnant women (Hoffmann and Evers, 1986). Three servings of whole milk would contain 0.25% of the E2 produced daily in the body of a prepubertal girl. Toxicologists often use the term "no observable effect level" to define the level at which a chemical has no observed biological effect. Whole milk consumption would have to be >215,000 L/d (56,800 gallons/d) to observe the bioactivity of E2 derived from whole milk based upon the "no observable effect level" of 300 µg of E2/d as reported by the World Health Organization (Joint FAO/WHO Expert Committee on Food Additives, 2000). These calculations also do not take into account the potential for lower E2 concentrations in reduced-fat milk, fecal excretion, and the extensive intestinal and hepatic metabolism of orally ingested estrogens (Ruoff and Dziuk, 1994). Based upon the results of our analyses, E2 concentrations in milk are low and unlikely to pose a health risk for humans.


   CONCLUSIONS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
ACKNOWLEDGEMENTS
 REFERENCES
 
Concentrations of 17ß-estradiol in cows >141 d pregnant were greater than those in nonpregnant cows or cows <141 d pregnant. 17ß-Estradiol concentrations were correlated with milk fat. Concentrations of E2 in raw whole milk are negligible relative to literature values for 24-h production rates of E2 in humans. Therefore, the minute E2 concentrations in milk are unlikely to pose a health risk for humans.


   ACKNOWLEDGEMENTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 ACKNOWLEDGEMENTS
REFERENCES
 
This work was supported by Hatch funds as well as funds from USDA Special grant no. 2003-34163-13404. The authors also thank Yolanda Michetti and Jessica Montresor-Lopez for help in collecting the data, as well as the excellent comments from 2 reviewers.

Received for publication December 23, 2006. Accepted for publication February 24, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 ACKNOWLEDGEMENTS
 REFERENCES
 


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D. A. Pape-Zambito, A. L. Magliaro, and R. S. Kensinger
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