ABSTRACT

Objective: To measure the oestrogen concentrations in various supply of milk and milk products in Pune.

Background: Indian women consume milk every day. Breast and ovarian cancer are on rise in India. Oestrogen has been reported to be strongly associated with increased risk for breast cancer. The daily intake of milk could be a risk factor because of various attempts of hormone injections to cow/buffalo for increasing the milk production.

Methods: Milk and milk products being consumed in Pune were measured for oestrogen concentration using Brown’s method for extraction and CLIA for measurement.

Results: The median oestrogen concentration in any milk /milk product was 180 pg/gm which is far below the oncogenecity level of 320 pg/mL.

Conclusion: The daily consumption of dairy products is advocated to be not harmful because of its non-significant levels of oestrogen, on the contrary, a beneficial factor from health point of view in vegetarian Indian women as a supply of micronutrients scores out as safer.

Keywords: Oestrogen; Aetiology; Oncogencity

INTRODUCTION

Epidemiological and experimental studies have shown the role of oestrogens in the aetiology of breast cancer in humans probably acting through hormone-related pathways (1). Increased concentrations of circulating oestrogens have been found to be strongly associated with increased risk for breast cancer in postmenopausal women (2). It has also been hypothesized that oestrogen exposure is a major determinant of risk for breast cancer. Animal studies have shown that oestrogens promote mammary tumours and oophorectomy or anti-oestrogenic drug reverse the effect (3). Exposure to exogenous oestrogens from a variety of sources has become increasingly common, particularly from hormonal preparations for use as contraceptives or to cure the symptoms of the menopause (4). Hormone replacement therapy in older women containing oestrogens has become common (5). It has also been thought to be a risk factor for various clinical problems like obesity, pubertal disorders and various kinds of cancer like breast cancer and prostate cancer. Estrogen has been shown to reduce the serum levels of gonadotrophins in pre-pubertal children and affect ovulation in adult females (2). One potential dietary source is milk. Absolute quantities of total oestrogen was measured in a variety of commercial milks (whole, 2%, skim, and buttermilk) and found that the milk products contain considerable levels of oestrogen. The levels in skim milk were lower than that observed in whole milk, 2% milk, and buttermilk. Butter milk contained the lowest, while curd contained the highest, probably because estrogen is fat soluble hormone (1). Ganmaa et al. (4) pointed out that milk and dairy products represent the main source for animal estrogens adding up to 60–70% of all animal estrogens consumed. Modern genetically improved dairy cows continue to lactate throughout almost the entire pregnancy. Therefore, cow's milk contains large amounts of estrogens. So far women do not have any information on the estrogen content of the milk being consumed. Therefore, we examined concentrations of oestrogen in the various sources of milk and milk products being supplied in Pune city.

MATERIALS AND METHODS

Estimation of Estrogen in milk and milk products was carried out by Brown’s method (1955), modified in the laboratory (6). It was estimated by the following protocol:

The protocol was divided in two methods –

1. Manual extraction method
2. Instrumental analysis

1. Manual extraction method:

10 ml / gm of milk / milk product was taken in a glass bumper tube. 2ml of Conc. Hydrochloric Acid (HCl) (name of the company) was added in that and this mixture was mixed on vortex. The tube was then kept in boiling water for about 45 min – 1 Hr., which after removing was cooled at room temperature for about 10-15 min. and then cooled completely in ice. The hydrolysate was extracted with 40 ml Diethyl ether (Fischer Scientific) by keeping it on magnetic stirrer for about 15 min. The aqueous phase was discarded and the ether layer was extracted with 5-6 ml of 1 N Sodium hydroxide (NaOH) (name of the company). Lower alkaline layer was collected in which 800 mg Sodium Bicarbonate (NaHCO₃) (name of the company) was added and mixed thoroughly. This alkaline / neutralized solution was extracted with 20-25 ml diethyl ether (Fischer Scientific) by keeping it on magnetic stirrer for about 5 min. Organic ether layer was collected in a test tube and was evaporated to complete dryness. This was dissolved in measured assay buffer / diluent.

1. Instrumental analysis:

The estrogen content was estimated using CLIA technique on Architect (Abbott) Immunoassay analyzer.

The final estrogen value in the given sample was calculated per 100 ml / per 100 gm.

1. CLIA Technique:

Antibody coated micro-particles were reacted with the test sample. The Antibody bound estrogen reacts with fluorescence tagged estradiol antibody and the bound fraction having fluorescence was measured. Instrument was calibrated using internationally accepted calibrators and internal quality controls were used for quality assessment. The inter and intra assay variation was 5.3% and 3.4 % respectively

For all the samples, the milk and milk products were processed in such a way to replicate Indian scenario. Milk was boiled and cooled and then used without cream for sample processing.

All the milk cream was used for making homemade curds in which 1 gm of commercially available Govardhan curd was used as an inoculum. For all the homemade butter milk samples, water was added in all the homemade curds (curd: water -1:10 w/v) to make butter milk and mixed in a mixer. All the butter was removed and only lower butter milk was used for sample processing.

RESULTS

Milk from different sources of supply in Pune does not cross the limit of toxic level. Table 1 shows the oestrogen concentrations in various sources of milk supply in Pune. Milk oestrogen contents were below 320 pg/mL. Only the curds prepared from milk exhibited 424 and 400 pg/mL (Table 1 and 2). Krishnai milk contained 256 pg/mL and curd prepared from Amul and Chitale milk contained 424 pg/gm and 340 pg/gm respectively. However, the women consume not more than 150 mL every day. Commercially available milk powders also showed less than 320 pg/gm of oestrogen concentrations (Table 3).

DISCUSSION

Our results indicate that the milk supply in Pune contains about 180 pg/gm of estrogen in terms of any dairy product that is being consumed is far away from the level of oncogenicity. Curd is found to have maximum concentration (424 pg/gm) and the amount consumed is not more than 200 gm per day. Hence, milk or milk product intake may not be a causative factor for cancer (breast cancer) in Indian scenario.

Maruyama et al (1) reported that estrogens in milk were absorbed, and gonadotropin secretion was suppressed, followed by a decrease in testosterone secretion in male adults and children. Sexual maturation of prepubertal children could be affected by the ordinary intake of cow milk.

Hjartåker et al. (5) carried out a prospective cohort study in almost 50,000 Norwegian women examining childhood milk consumption and breast cancer incidence in later adult life. Milk intake during adult life was negatively correlated with breast cancer incidence. Subjects drinking more than 3 glasses of milk each day had a 44% lower incidence rate than women not drinking milk. There was no clear-cut association between various milk preparations and milk fat consumption. Combination of the data on childhood and adult life milk consumption revealed a significant negative trend for the incidence rate with increasing amount of milk consumed showing a protective effect of milk consumption. Gallus et al. (14) reported a large integrated network of hospital-based case-control-studies from Italy. The authors detected an inverse association between milk intake and colorectal, breast and ovarian cancer and concluded that dairy products do not appear as strong risk predictors for the carcinomas.

Moorman and Terry (16-17) strongly focused on epidemiological data from cohort or case-control-studies. They opined that most trials were unable to produce a clear-cut pattern of increased or diminished breast cancer risk. Therefore, they concluded that the epidemiological literature available did not contain strong evidence for an association between milk consumption and later breast cancer risk. Calcium and vitamin D contents of dairy products probably reduce breast cancer risk. Hence, our results stating that the oestrogen concentrations in milk and milk products do not exceed the undesirable limits and claim that consumption is safe as far as oncogenicity are concerned.

Limitation of the study is that we have not measured estrogen concentration season-wise.

Table 1: Maximum estrogen concentration was 256 pg/mL in Krishnai milk and the curd prepared from Amul milk and Chitale milk were 424 and 340 pg/gm respectively.

Table 3. The median estrogen concentrations are less than 320 pg/mL in all the samples.

CONCLUSIONS

Our data on milk and milk products indicate that estrogens in milk were below the so-called toxic levels. We also observed that the oestrogen concentrations in milk being consumed did not vary during the period. Therefore, consumption of milk does not lead to higher circulating oestrogen levels and is not responsible for oncogenicity. Milk for calcium and vitamin D contents need to be studied

REFERENCES

1. Maruyama K,Oshima T, Ohyama K; Exposure to exogenous estrogen through intake of commercial milk produced from pregnant cows. PediatrInt, 2010. 52: p. 33-38
2. Farlow DW, Xu X, Veenstra TD; Quantitative measurement endogenous estrogen metabolites, risk factors for development of breast cancer, in commercial milk products by LC-MS/MS. J Chromatogr B Analyt Technol Biomed Life Sci, 2009. 877: p. 1327-1334.
3. Ganmaa D, Wang PY, Qin LQ, Hoshi K, Sato A; Is milk responsible for male reproductive disorders? Med Hypotheses, 2001. 57: p. 510-514.
4. Hjartaker A, Laake P Lund E. Childhood and adult milk consumption and risk of premenopausal breast cancer in a cohort of 48, 844 women-the Norwegian women and cancer study. Int J cancer, 2001.93: p. 888-93.
5. Van der Pols JC, Bain C, Gunnell D, Smith GD, Frobisher C, Martin RM. Childhood dairy intake and adult cancer risk. 65-y follow-up of the Boyd Orr cohort. Am J Clin Nutr, 2007. 86: p. 1722-1729.
6. Brown JB. Oestrogen assays in gynaecology. Med J Aust. 1974 May 11; 1(19):756-757.
7. Qin LQ, Wang PY, Kaneko T, Hoshi K, Sato A; Estrogen: one of the risk factors in milk for prostate cancer. Med Hypotheses, 2004. 62: p. 133–142
8. Farlow DW, Xu X, Veenstra TD, Comparison of estrone and 17β-estradiol levels in commercial goat and cow milk. J Dairy Sci, 2012. 95(4): p. 1699-708.
9. Pape-Zambito DA, Magliaro AL, Kensinger RS, Concentrations of 17beta-estradiol in Holstein whole milk. J Dairy Sci, 2007. 90(7): p. 3308-13.
10. Mommers M, Schouten LJ, Goldbohm RA, van den Brandt PA, Dairy consumption and ovarian cancer risk in the Netherlands Cohort Study on Diet and Cancer. Br J Cancer, 2006. 94(1): p. 165-70.
11. Snow DR, Jimenez-Flores R, Ward RE, Cambell J, Young MJ, Nemere I, Hintze KJ, Dietary milk fat globule membrane reduces the incidence of aberrant crypt foci in Fischer-344 rats. J Agric Food Chem, 2010. 58(4): p. 2157-63.  
12. Politzer WM. Long-term clinical remission of a patient with chronic lymphocytic leukemia using alternative treatment option: cottonseed oil (gossypol). Phytomedicine, 2008. 15(8): p. 563-5.
13. Ganmaa D, Sato A. The possible role of female sex hormones in milk from pregnant cows in the development. Med Hypotheses, 2005.65(6): p. 1028-37.
14. Gallus S, L Scotti, E Negri, R Talamini, S Franceschi et al; Artificial sweeteners and cancer risk in a network of case–control studies. Ann Oncol. 2007. 8(1): p. 40-4.
15. Moorman PG and Terry PD. Consumption of Cow's Milk and Possible Risk of Breast Cancer. Breast Care (Basel), 2010. 5(1): p. 44-46.
16. Moorman PG1, Terry PD, Consumption of dairy products and the risk of breast cancer: a review of the literature. Am J ClinNutr, 2004.80(1): p. 5-14.
17. S Gallus, E. Negri, L Scotti,L Scotti1S Franceschi, M Montella, A Giocosa,L Dal Maso, C La Vecchia Artificial sweeteners and cancer risk in a network of case–control studies Annals of Oncology, 2007. 18: p. 40-44.

1. Maruyama K,Oshima T, Ohyama K; Exposure to exogenous estrogen through intake of commercial milk produced from pregnant cows. PediatrInt, 2010. 52: p. 33-38
2. Farlow DW, Xu X, Veenstra TD; Quantitative measurement endogenous estrogen metabolites, risk factors for development of breast cancer, in commercial milk products by LC-MS/MS. J Chromatogr B Analyt Technol Biomed Life Sci, 2009. 877: p. 1327-1334.
3. Ganmaa D, Wang PY, Qin LQ, Hoshi K, Sato A; Is milk responsible for male reproductive disorders? Med Hypotheses, 2001. 57: p. 510-514.
4. Hjartaker A, Laake P Lund E. Childhood and adult milk consumption and risk of premenopausal breast cancer in a cohort of 48, 844 women-the Norwegian women and cancer study. Int J cancer, 2001.93: p. 888-93.
5. Van der Pols JC, Bain C, Gunnell D, Smith GD, Frobisher C, Martin RM. Childhood dairy intake and adult cancer risk. 65-y follow-up of the Boyd Orr cohort. Am J Clin Nutr, 2007. 86: p. 1722-1729.
6. Brown JB. Oestrogen assays in gynaecology. Med J Aust. 1974 May 11; 1(19):756-757.
7. Qin LQ, Wang PY, Kaneko T, Hoshi K, Sato A; Estrogen: one of the risk factors in milk for prostate cancer. Med Hypotheses, 2004. 62: p. 133–142
8. Farlow DW, Xu X, Veenstra TD, Comparison of estrone and 17β-estradiol levels in commercial goat and cow milk. J Dairy Sci, 2012. 95(4): p. 1699-708.
9. Pape-Zambito DA, Magliaro AL, Kensinger RS, Concentrations of 17beta-estradiol in Holstein whole milk. J Dairy Sci, 2007. 90(7): p. 3308-13.
10. Mommers M, Schouten LJ, Goldbohm RA, van den Brandt PA, Dairy consumption and ovarian cancer risk in the Netherlands Cohort Study on Diet and Cancer. Br J Cancer, 2006. 94(1): p. 165-70.
11. Snow DR, Jimenez-Flores R, Ward RE, Cambell J, Young MJ, Nemere I, Hintze KJ, Dietary milk fat globule membrane reduces the incidence of aberrant crypt foci in Fischer-344 rats. J Agric Food Chem, 2010. 58(4): p. 2157-63.  
12. Politzer WM. Long-term clinical remission of a patient with chronic lymphocytic leukemia using alternative treatment option: cottonseed oil (gossypol). Phytomedicine, 2008. 15(8): p. 563-5.
13. Ganmaa D, Sato A. The possible role of female sex hormones in milk from pregnant cows in the development. Med Hypotheses, 2005.65(6): p. 1028-37.
14. Gallus S, L Scotti, E Negri, R Talamini, S Franceschi et al; Artificial sweeteners and cancer risk in a network of case–control studies. Ann Oncol. 2007. 8(1): p. 40-4.
15. Moorman PG and Terry PD. Consumption of Cow's Milk and Possible Risk of Breast Cancer. Breast Care (Basel), 2010. 5(1): p. 44-46.
16. Moorman PG1, Terry PD, Consumption of dairy products and the risk of breast cancer: a review of the literature. Am J ClinNutr, 2004.80(1): p. 5-14.
17. S Gallus, E. Negri, L Scotti,L Scotti1S Franceschi, M Montella, A Giocosa,L Dal Maso, C La Vecchia Artificial sweeteners and cancer risk in a network of case–control studies Annals of Oncology, 2007. 18: p. 40-44.