http://informahealthcare.com/txm ISSN: 1537-6516 (print), 1537-6524 (electronic) Toxicol Mech Methods, 2014; 24(5): 342–346 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/15376516.2014.910851
RESEARCH ARTICLE
Toxicology Mechanisms and Methods Downloaded from informahealthcare.com by University of Toronto on 08/11/14 For personal use only.
Analysis of oxytocin in milk samples and intake pattern in different age groups of Indian population Manjari Mishra1,2, Shakir Ali2, and Mukul Das1 1
Department of Food, Drug and Chemical Toxicology, CSIR-Indian Institute of Toxicology Research (IITR), Lucknow, Uttar Pradesh, India and Department of Biochemistry, Jamia Hamdard, New Delhi, India
2
Abstract
Keywords
Background: Oxytocin (OT) injections have been indiscriminately used to milk cattle in dairy industries. There is no study available regarding surveillance of OT in market milk samples. Material and methods: OT from milk samples was extracted by precipitation with trichloroacetic acid and passed through the solid phase extraction column. OT was eluted and evaporated to dryness under a gentle stream of nitrogen. The residue was either dissolved in milli Q water or buffer for analysis through HPLC or EIA. The intake assessment of OT through milk was assessed through the Food Frequency Recall method employing a Food Frequency Questionnaire. On the basis of milk consumption and the values of OT in milk, the actual intake of OT was calculated. Results: In the present study, a total of 55 milk samples (39 milkman and 16 branded) were analyzed for occurrence of OT by EIA and UV-HPLC from different locations of Lucknow, Uttar Pradesh (India). OT contamination in milkman samples was found to be 21 pg/mL to 18.9 ng/mL with the mean value of 8.9 ng/mL. The average daily intake of OT in terms of mg/day/person was highest (2.3–2.4 mg/day/person) in 1–3-year age group. Conclusions: Since there is no prescribed level of OT in milk and the intake of OT through this commodity is quite high there is need to implement regulatory laws so that non-physiological OT exposure may not occur in children which may have deleterious effects.
Enzyme immunoassay, high-performance liquid chromatography, milk, oxytocin, SPE column
Introduction In India, oxytocin (OT) ampules, commercially known as pitocin or syntocinon, are indiscriminately used for enhanced milk production following intramuscular injections to cattle. The exogenous supplementation of OT to cattle get absorbed in blood and increases the plasma concentration by 4–5 folds with respect to physiological levels (Macuhova et al., 2004). It has been presumed that due to small size of OT (1 kd), it may cross the blood–milk barrier and reaches into the milk, as OT is known to cause changes in permeability at tight junctions of mammary gland (Nguyen & Neville, 1998). OT has been found to be stable in milk under normal conditions (Prakash et al., 2009) and its continuous exposure to cattle may increase the levels of OT in milk. Studies have shown that radioactive OT when given intraperitonially to dam rat on seven postpartum day, the radioactivity was detected in the plasma of neonates, indicating the potential transfer of OT into the milk (Takeda et al., 1986). OT is a schedule ‘‘H’’ Address for correspondence: Mukul Das, Department of Food, Drug and Chemical Toxicology, CSIR-Indian Institute of Toxicology Research (IITR), P.O. Box No. 80, Mahatma Gandhi Marg, Lucknow 226001, Uttar Pradesh, India. Tel: 091-522-2613786. Fax: 091-522-2628227. E-mail: [email protected], [email protected]
History Received 5 December 2013 Revised 28 March 2014 Accepted 28 March 2014 Published online 21 April 2014
drug in India which means that it cannot be bought or sold without a prescription, thus its use in dairy industries is not recommended (PCA Act, 1982). However, due to lack of detection and awareness, OT is being used for enhanced milk ejection in cattle. Being a hormone, small amount of OT is required to perform physiological function under specific periods of life, including pregnancy, labor and after delivery. However, nonphysiological exposure due to consumption of OT contaminated milk by humans may cause some unwanted effects on human health. The bioavailability of peptides for absorption is relatively low (0.5–1%) which may increase depending upon the conditions in gastrointestinal (GI) tract (Florence, 1980; Huck et al., 2006; Lundin et al., 1995). In a recent study, OT has been shown to be stable in milk under adverse conditions of temperature, pH and simulated gastric fluid (Mishra et al., 2013a) thereby suggesting that absorption may occur to elicit undesired effects. Hence, 0.5–1% of bioavailable OT through oral consumption of contaminated milk to neonates and children may affect the central nervous system, due to the leaky nature of infant blood brain barrier. Furthermore, recent studies have shown that oral OT exposure since early childhood may not show alteration in classical toxicological parameters but its target organ, ovary, is a matter of concern
DOI: 10.3109/15376516.2014.910851
as it enhances ovulation which may have unwanted consequences that needs to be explored further (Mishra et al., 2013b). Visualizing the overall scenario, surveillance of OT in milk samples is extremely necessary for assessing the exposure risk as no international guidelines are available for permissible levels of OT in milk. Since no method was available for detection of OT in milk, we developed the method using EIA and validated by LC-MS (Mishra et al., 2013a). The aim of the present study includes detection and quantification of OT in a variety of milk samples (milkman and branded) and assessment of likely intake of OT in Indian population. Toxicology Mechanisms and Methods Downloaded from informahealthcare.com by University of Toronto on 08/11/14 For personal use only.
Methods
OT in milk and likely intake in Indian population
343
Analysis of OT by EIA The analysis of the extracted material was carried out using a commercially available OT enzyme immunoassay kit and the protocol provided there in. Briefly, the extracted sample was dissolved in 1 ml of assay buffer and 100 ml samples were applied to each well of 96-well EIA plate (pre-coated with goat antibody). Subsequently, 50 ml of OT conjugate and 50 ml of OT antibody (supplied with kit) were added to the samples and incubated overnight at 4 C. Wells were washed three times with 300 ml of washing solution and then 200 ml of p-nitrophenylphosphate (pNpp) substrate was added to each well and incubated at room temperature for 1 h. A 50 ml of stop solution (tri-sodium phosphate solution) was added to each well and reading was taken at 405 nm.
Chemicals Standard OT was purchased from Sigma Chemicals Co. (St. Louis, MO). Solid phase extraction (SPE) columns and EIA kit for OT quantitation were procured from Waters (Milford, MA) and Assay Design (Ann Arbor, MI), respectively. All other chemicals and reagents used were of highest purity commercially available.
Analytical quality assurance The analytical quality assurance data for the HPLC and EIA method employed to quantify OT in milk showing linearity range; the limit of detection; the limit of quantification and percentage relative standard deviation (RSD) were generated earlier (Mishra et al., 2013a).
Procurement of milk samples
Surveillance for consumption pattern of milk
A total of 55 milk samples (39 samples from local milkman and 16 branded samples) were collected from different parts of Lucknow, India and after extraction, the dried forms were kept at 20 C till further analysis.
A limited household survey on milk consumption in 255 subjects from 150 families was conducted within 15 year of age. The subjects were subdivided into three age groups; 0.5–1 year (infant), 2–12 year (children) and 13–15 year (adolescent) as per Indian Council of Medical Research (ICMR) guidelines (ICMR, 2000).
Extraction of OT from milk OT from milk samples was extracted as described by Mishra et al. (2013a). In brief, milk sample (5 ml) was precipitated with 500 ml of 100% TCA (grams per volume) and stirred for 10 min. The precipitate was removed by centrifugation at 10 000 rpm for 15 min and supernatant was collected. The precipitate was re-suspended in 1 ml of 0.25% acetic acid and stirred for 15 min and again 100 ml of 100% TCA was added, stirred for 5 min and centrifuged. The supernatant was separated and pooled with previous supernatant. The pooled supernatant was passed through the SPE column. OT was eluted with 2 ml mixture of ethanol: 6N hydrochloric acid (1000:1:v/v) and evaporated to dryness under a gentle stream of nitrogen. The residue was either dissolved in milli Q water or buffer for analysis through HPLC or EIA. Analysis of OT by HPLC The analysis of OT was carried out by reverse phase HPLC (Waters, Vienna, Austria). The HPLC system consisted of injector fitted with a loop (20 ml), dual pump (model #510) and 4.6 250 mm SymmetryR C18 (5 mm) column (Waters; Vienna, Austria). Elute was monitored using a tunable absorbance detector (model #486) at wavelength 220 nm. Samples prepared in 100 ml mill Q water were passed through a filter (0.2 mm) and injected to HPLC. OT was separated using the solvent system consisting of 0.08 M phosphate buffer, pH 5 and acetonitrile (80:20, v/v) under isocratic condition at a flow rate of 1.5 ml min1. The total analysis time per injection was 20 min and the peak of OT was analyzed using millennium software.
Intake assessment of OT through milk The intake of OT through milk was assessed through the Food Frequency Recall method (Burdock, 1996) employing a Food Frequency Questionnaire, which searched for information of respondent’s name, age, gender; followed by the queries on the consumption and frequencies of consumption of specified milk. Quantities of milk consumed were recorded based on the standard stainless steel measuring vessels. On the basis of milk consumption and the values of OT in milk, the actual intake of OT was calculated.
Results Analytical performance The LOD, LOQ and % recovery of OT by EIA were 7.74, 10.3 pg/ml and 85–92%, respectively. Assay precision in terms of intra- and inter-assay coefficient of variations of OT spiked milk samples were below 14 (% RSD) (Mishra et al., 2013a). A linear HPLC response to OT standards was observed for a concentration range of 23.75–12 500 ng ml1. The LOD, LOQ and % recovery for OT by HPLC were 4.1, 9.8 ng/ml and 84–91%, respectively. The intra-day and inter-day repeatability was below 10% (Mishra et al., 2013a). Quantification of OT by EIA The quantitative analysis of OT in milk samples is summarized in Table 1. OT was detected in 100% of milk
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Toxicol Mech Methods, 2014; 24(5): 342–346
Table 1. Analysis of OT by EIA in milk samples from different locations of Lucknow, India. Sample type Milkman Branded Total
Positive samples in range (pg/ml) 5100
100–500
500–1000
1000–5000
45000
Range
Mean ± SE
90th Percentile
39 16 55
2 Nil 2
16 Nil 16
12 Nil 12
2 8 10
7 8 15
21–12 180 1320–18 923 21–18 923
2463 ± 694 8881 ± 1802 4336 ± 1506
11 358 17 129 14 334
Table 2. Analysis of OT by HPLC in milk from different locations of Lucknow, India.
Toxicology Mechanisms and Methods Downloaded from informahealthcare.com by University of Toronto on 08/11/14 For personal use only.
Sample type Milkman Branded Total
OT concentrations (pg/ml)
No. of samples analyzed
OT concentration (ng/ml)
No. of samples analyzed
Range
Mean ± SE
4 8 12
10.9–12.9 14.0–18.9 10.9–18.9
11.60 ± 0.462 16.14 ± 0.629 14.63 ± 0.777
samples (39 milkman and 16 branded), and concentrations ranged between 21 pg/ml and 18.9 ng/ml with a mean value of 4.3 ng/ml. In the case of samples collected from milkman, 46% had OT concentration below 500 pg/ml, other 31% samples were found in the range of 500–1000 pg/ml, while 5% samples were in the range of 1–5 ng/ml, remaining 18% exceed the value of 5 ng/ml. A range of 21 pg/ml to 12.1 ng/ml OT was observed in milk samples collected from milkman, with a mean and 90th percentile value of 2.5 and 11.4 ng/ml, respectively. In branded milk samples, not a single sample showed OT concentration within 1 ng/ml. A 50% samples had OT contamination in the range of 1–5 ng/ml while remaining 50% of the samples showed OT levels more than 5 ng/ml. The range of OT contamination was found to be 1.3–18.9 ng/ml with a mean and 90th percentile value of 8.9 and 17.1 ng/ml, respectively. Quantification of OT by HPLC The HPLC method having the LOQ of 9.8 ng/ml was employed to those positive samples (four milkman and eight branded) having the concentration of OT above 10 ng/ml for further confirmation. The quantitative analysis of OT by HPLC in milk samples is summarized in Table 2. OT contamination range in four samples collected from milkman was found to be 10.9–12.9 ng/ml with a mean value of 11.6 ng/ml whereas in branded milk samples (eight samples), the contamination range was 14.0–18.9 ng/ml having a mean value of 16.1 ng/ml. The results of HPLC analysis of 12 milk samples for OT levels were comparable to that analyzed by the EIA method with a % RSD ranging between 0.7% and 7.0% (Figure 1). Surveillance for consumption pattern The consumption pattern of milk in the surveyed population is shown in Table 3. Out of 255 subjects from 150 families, almost 98% subjects were milk consumers. The surveyed population contains 3% infant (0–1 year), 66% children (1–12 year) and 31% adolescent (13–15 year). In children group, 24% population belonged to 1–3 year, 16% were in the age
group of 4–6 year, 24% were in the age group of 7–9 year while maximum (36%) were in the age group between 10 and 12 years. There were relatively more females (n ¼ 136) than males (n ¼ 119) in this study. The average milk consumption was found to be highest (534.6–542.9 ml/day/person) in 1–3 year age group which is 1.3–1.9 fold higher than other groups while consumption based on 90th percentile was also maximum (900–1200 ml/day/person) in 1–3-year age group. Intake of oxytocin The intake of OT through milk in different age group populations is shown in Table 4. The average intake of OT through milk in 1–3-year age group showed a maximum value of 2.3–2.4 mg/day/person (1.3–1.9 fold higher than others) while intake based on the 90th percentile was also highest (3.9–5.2 mg/day/person) in the 1–3-year age group, which was 1.2–2.8 fold higher than the other age groups.
Discussion Milk is the main source of protein and essential dietary component in children, apart from being consumed by the population of all age groups. In dairy farming, exogenous OT is frequently administered to cattle for milk let down. During the past several years there has been concern regarding the use of exogenous OT in cattle in Asian countries for milking. However, no study regarding surveillance of OT hormone in the milk samples for human consumption is available. Hence, a detailed scientific investigation is warranted to elucidate the levels of OT in this commonly consumed commodity. Earlier, we have developed a method for quantitation of OT in milk (Mishra et al., 2013a) and have applied it for the first time to analyze the quantity of OT in milk samples. In the present study, it was observed that all the 55 milk samples were found to be contaminated with OT ranging from 21 pg/ml to 18.9 ng/ml. Based on the results of quantification of OT and the data of average actual consumption of milk in children, the average estimated daily intake of OT was found to be maximum in 1–3-year age group of children (2.3– 2.4 mg/day/person). When the data were calculated from 90th percentile value, the intake was also found to be highest in 1–3-year children. Though maximum intake of OT was in 1–3-year age group, nonetheless, intake was also observed up to 13–15-year group (1.2–1.7 mg/day/person) suggesting that children up to 15 year are continuously exposed to OT since birth. Since OT is stable against normal boiling conditions as well as in simulated gastric fluid assay (Mishra et al., 2013b; Prakash et al., 2009), there is likelihood of chronic exposure effect of OT through oral route. Studies of Noriega-Guerra et al. (1966) have shown that oral administration of OT as a
OT in milk and likely intake in Indian population
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Figure 1. Comparative analysis of OT in milk samples by EIA and HPLC.
Table 3. Consumption pattern of milk in population of different age groups.
Table 4. Intake pattern of OT through milk in population of different age groups.
Milk consumption (ml/day/person) Age groups (years) Infant (0–1) Male (0–1) Female Children (1–3) Male (1–3) Female (4–6) male (4–6) Female (7–9) Male (7–9) Female (10–13) Male (10–13) Female Adolescent (13–15) Male (13–15) Female
Intake of OT (mg/day/person)
No. of % of 90th subjects consumers Average Maximum Percentile 3 5
100 100
375.0 400.0 a
500 500
475 500 b
17 23 14 13 18 22 35 26
100 100 100 100 100 100 98 95
542.9 534.6c 382.0 405.8 345.6 344.3 420.7 372.3
1500 1000 750 1000 500 500 2000e 2000f
1200 900d 500 500 500 500 900 750
32 47
90 90
399.2 279.0
2000 1000
500 500
A total of 150 families having 255 subjects between 0.5 and 15 years of age were surveyed. Average consumption is derived from the questionnaire in (n) number of subjects as mentioned above. a Average consumption of milk in males is highest (1.4–1.6 fold) in 1–3-year age. b 90th Percentile consumption of milk in male is highest (1.3–2.5 fold) in 1–3-year age group. c Average consumption of milk in female is highest (1.3–1.9 fold) in 1–3-year age group. d 90th Percentile consumption of milk in female is highest (1.2–1.8 fold) in 1–3-year age group. e Maximum consumption of milk in male is highest (1–4 fold) in 10–13-year age group. f Maximum consumption of milk in female is highest (2–4 fold) in 10–13-year age group.
drug induces uterine contractibility. Buccal administration of OT to pregnant women at term was shown to induce uterine contractions along with increased plasma concentrations of OT (Noriega-Guerra et al., 1966). It has been reported earlier that the use of oral or transbuccal OT facilitate child birth (Dawood & Khan-Dawood, 1986; Dillon, 1958; Dillon et al., 1962; Elstein & Wright, 1963; Newman & Hon, 1963) while overdose may have a risk for fetal cardiac problems (Dillon et al., 1962; Maxwell, 1964). Furthermore, Takeda et al. (1986) showed that intraperitoneal administration of
Age group
Age (years), sex, body weight (kg)
Average
90th Percentile
Infant
(0–1) Male (8.9) (0–1) Female (9.1)
1.6 1.7
2.1 2.2
Children
(1–3) Male (14.8) (1–3) Female (11.9) (4–6) Male (19.4) (4–6) Female (18.4) (7–9) Male (27.3) (7–9) Female (25.4) (10–13) Male (35.4) (10–13) Female (35.6)
2.4a 2.3c 1.7 1.8 1.5 1.5 1.8 1.7
5.2b 3.9d 2.2 2.2 2.2 2.2 3.9 3.3
Adolescent
(13–15) Male (48.2) (13–15) Female (47.5)
1.7 1.2
2.2 2.2
OT is specifically banned under the Prevention of Cruelty to Animals Act, 1960. Body weight has been derived from the surveyed population, whose mean is indicated in parenthesis. Age group selection was based on ICMR guidelines (ICMR, 2000). a Average value in male is highest (1.3–1.6 fold) in 1–3-year age group. b 90th Percentile value in male is highest (1.3–2.8 fold) in 1–3-year age group. c Average value in female is highest (1.3–1.9 fold) in 1–3-year age group. d 90th Percentile value in female highest (1.2–1.8 fold) in 1–3-year age group.
radioactive OT to dam rat on seven postpartum days, the presence of radioactivity in the plasma of neonates proved the transfer of OT into the milk. The absorption of peptides is less due to low bioavailability (0.5–1%), which may be one of the reasons for irregular effects of oral supplementation of OT as a drug. However, if this small percentage of OT is bioavailable in GI tract through continuous consumption of milk contaminated with OT (21 pg/ml to 19 ng/ml), it may result in unwanted toxic manifestation as observed in the recent studies where OT caused enhanced ovulation (Mishra et al., 2013b). The intake assessment studies of OT in the present investigation provided evidences for exposure to OT as high as 5.2 mg/day/person. Though the tolerable dose of OT and safe level of daily intake has not been monitored through milk, nonetheless,
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Takeda et al. (1986) showed that the presence of OT in normal human breast milk ranges from 6.4 to 9.0 pg/ml. Considering 9 pg/ml concentration of OT as the normal value in milk, the presence of OT in milk sample was 273–987 fold higher as observed in the present study. In general, the level of OT in human blood ranges between 3 and 5 pg/ml (Amico & Seif, 1980; Leake et al., 1981). However, from this study children population shall be exposed to OT (2.1–5.2 mg/day/ person) through milk and considering 1% bioavailability, 21–52 ng/day/person of OT shall be absorbed through intestine, which shall enhance the serum level by 3 fold per day taking into account the blood volume as 5 l. Being a hormone, the small amount of OT is sufficient enough to perform physiological function in normal conditions while exposure of OT (through milk) at higher levels, which occurs since childhood, may be a cause of concern as OT enhances the process of ovulation under nonphysiological conditions (Mishra et al., 2013b).
Conclusion Present study shows the presence of OT in milk samples. Based on our survey data for OT intake, children are the main consumers of milk and continuous exposure of OT through milk may enhance the levels by three folds per day in human serum. Therefore, the daily intake of OT through milk since childhood is a matter of concern.
Acknowledgements We are grateful to the Director of our Institute for his keen interest in this present study. M.M. is thankful to Council of Scientific and Industrial Research (CSIR), New Delhi for the award of Senior Research Fellowship. Financial assistance of CSIR- INDEPTH Project- BSC-0111 is gratefully acknowledged. The manuscript is IITR communication # 3051.
Declaration of interest The authors declare that there is no conflict of interest.
References Amico JA, Seif SM. (1980). Oxytocin in human plasma; correlation with neurophysin and stimulation with estrogen. J Clin Endoc Metab 52: 88–99.
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Burdock GA. (1996). Encyclopedia of food and color additives. Vol. II. Boca Raton (FL): CRC Press Inc. Dawood MY, Khan-Dawood FS. (1986). Human ovarian oxytocin; its source and relationship to steroid hormones. Am J Obstet Gynecol 154:756–63. Dillon TF. (1958). Transbuccal administration of pitocin. Surg Forum 9: 679–82. Dillon TF, Douglas RG, Du Vigneaud V. (1962). Further observations of transbuccal administration of pitocin for induction and stimulation of labor. Obstet Gynecol 20:434–41. Elstein M, Wright HP. (1963). Transbuccal oxytocin in the induction of labour; a clinical evaluation. J Obstet Gyn Br Commun 70: 1005–09. Florence TM. (1980). Degradation of protein disulfide bonds in alkali. Biochem J 189:507–20. Huck CW, Pezzei V, Schmitz T, et al. (2006). Oral peptide delivery: are there remarkable effects on drugs through sulfhydryl conjugation. J Drug Target 14:117–25. ICMR. (2000). Nutrient requirements and recommended dietary allowances for Indians. A Report of the Expert Group of the Indian Council of Medical Research. New Delhi, India, NIN Hyderabad. Leake RD, Weitzman RE, Glatz TH, Fisher DA. (1981). Plasma oxytocin concentrations in men, nonpregnant women, and pregnant women before and during spontaneous labor. J Clin Endocrinol Metab 53: 730–3. Lundin DP, Lundin S, Olsson H, et al. (1995). Enhanced intestinal absorption of oxytocin peptide analogues in the absence of pancreatic juice in pigs. Pharmaceut Res 12:1478–82. Macuhova J, Tancin V, Bruckmaier RM. (2004). Effects of oxytocin administration on oxytocin release and milk ejection. J Dairy Sci 87: 1236–44. Maxwell AW. (1964). A comparison of buccal and intravenous oxytocin. J Obstet Gyn Br Commun 71:37–44. Mishra M, Ali S, Das M. (2013a). A novel method for the determination of oxytocin in milk by enzyme immune assay or high performance liquid chromatography; validation by liquid chromatography-mass spectrometry. Food Anal Methods 6:1308–19. Mishra M, Mishra V, Chaudhuri BP, et al. (2013b). Anomalies in ovary following oral exposure to oxytocin: mechanistic studies. Reprod Toxicol 40:24–34. Nguyen DA, Neville MC. (1998). Tight junction regulation in the mammary gland. J Mammary Gland Biol 3:233–46. Newman JW, Hon EH. (1963). Induction of labor with transbuccal oxytocic. Obstet Gynecol 21:3–12. Noriega-Guerra L, Aznar R, Arevalo N, et al. (1966). Disadvantages of administration of oxytocin by oral absorption. Am J Obstet Gynecol 96:849–56. PCA Act. (1982). The Prevention of Cruelty to Animals Act, 1960 (59 of 1960). As amended by central act 26 of 1982, pp. 1–19. Prakash BS, Paul V, Kliem H, et al. (2009). Determination of oxytocin in milk of cows administered oxytocin. Anal Chem Acta 636:111–15. Takeda S, Kuwabara Y, Mizuno M. (1986). Concentrations and origin of oxytocin in breast milk. Endocrinol Japon 33:821–6.
RESEARCH ARTICLE
Toxicology Mechanisms and Methods Downloaded from informahealthcare.com by University of Toronto on 08/11/14 For personal use only.
Analysis of oxytocin in milk samples and intake pattern in different age groups of Indian population Manjari Mishra1,2, Shakir Ali2, and Mukul Das1 1
Department of Food, Drug and Chemical Toxicology, CSIR-Indian Institute of Toxicology Research (IITR), Lucknow, Uttar Pradesh, India and Department of Biochemistry, Jamia Hamdard, New Delhi, India
2
Abstract
Keywords
Background: Oxytocin (OT) injections have been indiscriminately used to milk cattle in dairy industries. There is no study available regarding surveillance of OT in market milk samples. Material and methods: OT from milk samples was extracted by precipitation with trichloroacetic acid and passed through the solid phase extraction column. OT was eluted and evaporated to dryness under a gentle stream of nitrogen. The residue was either dissolved in milli Q water or buffer for analysis through HPLC or EIA. The intake assessment of OT through milk was assessed through the Food Frequency Recall method employing a Food Frequency Questionnaire. On the basis of milk consumption and the values of OT in milk, the actual intake of OT was calculated. Results: In the present study, a total of 55 milk samples (39 milkman and 16 branded) were analyzed for occurrence of OT by EIA and UV-HPLC from different locations of Lucknow, Uttar Pradesh (India). OT contamination in milkman samples was found to be 21 pg/mL to 18.9 ng/mL with the mean value of 8.9 ng/mL. The average daily intake of OT in terms of mg/day/person was highest (2.3–2.4 mg/day/person) in 1–3-year age group. Conclusions: Since there is no prescribed level of OT in milk and the intake of OT through this commodity is quite high there is need to implement regulatory laws so that non-physiological OT exposure may not occur in children which may have deleterious effects.
Enzyme immunoassay, high-performance liquid chromatography, milk, oxytocin, SPE column
Introduction In India, oxytocin (OT) ampules, commercially known as pitocin or syntocinon, are indiscriminately used for enhanced milk production following intramuscular injections to cattle. The exogenous supplementation of OT to cattle get absorbed in blood and increases the plasma concentration by 4–5 folds with respect to physiological levels (Macuhova et al., 2004). It has been presumed that due to small size of OT (1 kd), it may cross the blood–milk barrier and reaches into the milk, as OT is known to cause changes in permeability at tight junctions of mammary gland (Nguyen & Neville, 1998). OT has been found to be stable in milk under normal conditions (Prakash et al., 2009) and its continuous exposure to cattle may increase the levels of OT in milk. Studies have shown that radioactive OT when given intraperitonially to dam rat on seven postpartum day, the radioactivity was detected in the plasma of neonates, indicating the potential transfer of OT into the milk (Takeda et al., 1986). OT is a schedule ‘‘H’’ Address for correspondence: Mukul Das, Department of Food, Drug and Chemical Toxicology, CSIR-Indian Institute of Toxicology Research (IITR), P.O. Box No. 80, Mahatma Gandhi Marg, Lucknow 226001, Uttar Pradesh, India. Tel: 091-522-2613786. Fax: 091-522-2628227. E-mail: [email protected], [email protected]
History Received 5 December 2013 Revised 28 March 2014 Accepted 28 March 2014 Published online 21 April 2014
drug in India which means that it cannot be bought or sold without a prescription, thus its use in dairy industries is not recommended (PCA Act, 1982). However, due to lack of detection and awareness, OT is being used for enhanced milk ejection in cattle. Being a hormone, small amount of OT is required to perform physiological function under specific periods of life, including pregnancy, labor and after delivery. However, nonphysiological exposure due to consumption of OT contaminated milk by humans may cause some unwanted effects on human health. The bioavailability of peptides for absorption is relatively low (0.5–1%) which may increase depending upon the conditions in gastrointestinal (GI) tract (Florence, 1980; Huck et al., 2006; Lundin et al., 1995). In a recent study, OT has been shown to be stable in milk under adverse conditions of temperature, pH and simulated gastric fluid (Mishra et al., 2013a) thereby suggesting that absorption may occur to elicit undesired effects. Hence, 0.5–1% of bioavailable OT through oral consumption of contaminated milk to neonates and children may affect the central nervous system, due to the leaky nature of infant blood brain barrier. Furthermore, recent studies have shown that oral OT exposure since early childhood may not show alteration in classical toxicological parameters but its target organ, ovary, is a matter of concern
DOI: 10.3109/15376516.2014.910851
as it enhances ovulation which may have unwanted consequences that needs to be explored further (Mishra et al., 2013b). Visualizing the overall scenario, surveillance of OT in milk samples is extremely necessary for assessing the exposure risk as no international guidelines are available for permissible levels of OT in milk. Since no method was available for detection of OT in milk, we developed the method using EIA and validated by LC-MS (Mishra et al., 2013a). The aim of the present study includes detection and quantification of OT in a variety of milk samples (milkman and branded) and assessment of likely intake of OT in Indian population. Toxicology Mechanisms and Methods Downloaded from informahealthcare.com by University of Toronto on 08/11/14 For personal use only.
Methods
OT in milk and likely intake in Indian population
343
Analysis of OT by EIA The analysis of the extracted material was carried out using a commercially available OT enzyme immunoassay kit and the protocol provided there in. Briefly, the extracted sample was dissolved in 1 ml of assay buffer and 100 ml samples were applied to each well of 96-well EIA plate (pre-coated with goat antibody). Subsequently, 50 ml of OT conjugate and 50 ml of OT antibody (supplied with kit) were added to the samples and incubated overnight at 4 C. Wells were washed three times with 300 ml of washing solution and then 200 ml of p-nitrophenylphosphate (pNpp) substrate was added to each well and incubated at room temperature for 1 h. A 50 ml of stop solution (tri-sodium phosphate solution) was added to each well and reading was taken at 405 nm.
Chemicals Standard OT was purchased from Sigma Chemicals Co. (St. Louis, MO). Solid phase extraction (SPE) columns and EIA kit for OT quantitation were procured from Waters (Milford, MA) and Assay Design (Ann Arbor, MI), respectively. All other chemicals and reagents used were of highest purity commercially available.
Analytical quality assurance The analytical quality assurance data for the HPLC and EIA method employed to quantify OT in milk showing linearity range; the limit of detection; the limit of quantification and percentage relative standard deviation (RSD) were generated earlier (Mishra et al., 2013a).
Procurement of milk samples
Surveillance for consumption pattern of milk
A total of 55 milk samples (39 samples from local milkman and 16 branded samples) were collected from different parts of Lucknow, India and after extraction, the dried forms were kept at 20 C till further analysis.
A limited household survey on milk consumption in 255 subjects from 150 families was conducted within 15 year of age. The subjects were subdivided into three age groups; 0.5–1 year (infant), 2–12 year (children) and 13–15 year (adolescent) as per Indian Council of Medical Research (ICMR) guidelines (ICMR, 2000).
Extraction of OT from milk OT from milk samples was extracted as described by Mishra et al. (2013a). In brief, milk sample (5 ml) was precipitated with 500 ml of 100% TCA (grams per volume) and stirred for 10 min. The precipitate was removed by centrifugation at 10 000 rpm for 15 min and supernatant was collected. The precipitate was re-suspended in 1 ml of 0.25% acetic acid and stirred for 15 min and again 100 ml of 100% TCA was added, stirred for 5 min and centrifuged. The supernatant was separated and pooled with previous supernatant. The pooled supernatant was passed through the SPE column. OT was eluted with 2 ml mixture of ethanol: 6N hydrochloric acid (1000:1:v/v) and evaporated to dryness under a gentle stream of nitrogen. The residue was either dissolved in milli Q water or buffer for analysis through HPLC or EIA. Analysis of OT by HPLC The analysis of OT was carried out by reverse phase HPLC (Waters, Vienna, Austria). The HPLC system consisted of injector fitted with a loop (20 ml), dual pump (model #510) and 4.6 250 mm SymmetryR C18 (5 mm) column (Waters; Vienna, Austria). Elute was monitored using a tunable absorbance detector (model #486) at wavelength 220 nm. Samples prepared in 100 ml mill Q water were passed through a filter (0.2 mm) and injected to HPLC. OT was separated using the solvent system consisting of 0.08 M phosphate buffer, pH 5 and acetonitrile (80:20, v/v) under isocratic condition at a flow rate of 1.5 ml min1. The total analysis time per injection was 20 min and the peak of OT was analyzed using millennium software.
Intake assessment of OT through milk The intake of OT through milk was assessed through the Food Frequency Recall method (Burdock, 1996) employing a Food Frequency Questionnaire, which searched for information of respondent’s name, age, gender; followed by the queries on the consumption and frequencies of consumption of specified milk. Quantities of milk consumed were recorded based on the standard stainless steel measuring vessels. On the basis of milk consumption and the values of OT in milk, the actual intake of OT was calculated.
Results Analytical performance The LOD, LOQ and % recovery of OT by EIA were 7.74, 10.3 pg/ml and 85–92%, respectively. Assay precision in terms of intra- and inter-assay coefficient of variations of OT spiked milk samples were below 14 (% RSD) (Mishra et al., 2013a). A linear HPLC response to OT standards was observed for a concentration range of 23.75–12 500 ng ml1. The LOD, LOQ and % recovery for OT by HPLC were 4.1, 9.8 ng/ml and 84–91%, respectively. The intra-day and inter-day repeatability was below 10% (Mishra et al., 2013a). Quantification of OT by EIA The quantitative analysis of OT in milk samples is summarized in Table 1. OT was detected in 100% of milk
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Table 1. Analysis of OT by EIA in milk samples from different locations of Lucknow, India. Sample type Milkman Branded Total
Positive samples in range (pg/ml) 5100
100–500
500–1000
1000–5000
45000
Range
Mean ± SE
90th Percentile
39 16 55
2 Nil 2
16 Nil 16
12 Nil 12
2 8 10
7 8 15
21–12 180 1320–18 923 21–18 923
2463 ± 694 8881 ± 1802 4336 ± 1506
11 358 17 129 14 334
Table 2. Analysis of OT by HPLC in milk from different locations of Lucknow, India.
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Sample type Milkman Branded Total
OT concentrations (pg/ml)
No. of samples analyzed
OT concentration (ng/ml)
No. of samples analyzed
Range
Mean ± SE
4 8 12
10.9–12.9 14.0–18.9 10.9–18.9
11.60 ± 0.462 16.14 ± 0.629 14.63 ± 0.777
samples (39 milkman and 16 branded), and concentrations ranged between 21 pg/ml and 18.9 ng/ml with a mean value of 4.3 ng/ml. In the case of samples collected from milkman, 46% had OT concentration below 500 pg/ml, other 31% samples were found in the range of 500–1000 pg/ml, while 5% samples were in the range of 1–5 ng/ml, remaining 18% exceed the value of 5 ng/ml. A range of 21 pg/ml to 12.1 ng/ml OT was observed in milk samples collected from milkman, with a mean and 90th percentile value of 2.5 and 11.4 ng/ml, respectively. In branded milk samples, not a single sample showed OT concentration within 1 ng/ml. A 50% samples had OT contamination in the range of 1–5 ng/ml while remaining 50% of the samples showed OT levels more than 5 ng/ml. The range of OT contamination was found to be 1.3–18.9 ng/ml with a mean and 90th percentile value of 8.9 and 17.1 ng/ml, respectively. Quantification of OT by HPLC The HPLC method having the LOQ of 9.8 ng/ml was employed to those positive samples (four milkman and eight branded) having the concentration of OT above 10 ng/ml for further confirmation. The quantitative analysis of OT by HPLC in milk samples is summarized in Table 2. OT contamination range in four samples collected from milkman was found to be 10.9–12.9 ng/ml with a mean value of 11.6 ng/ml whereas in branded milk samples (eight samples), the contamination range was 14.0–18.9 ng/ml having a mean value of 16.1 ng/ml. The results of HPLC analysis of 12 milk samples for OT levels were comparable to that analyzed by the EIA method with a % RSD ranging between 0.7% and 7.0% (Figure 1). Surveillance for consumption pattern The consumption pattern of milk in the surveyed population is shown in Table 3. Out of 255 subjects from 150 families, almost 98% subjects were milk consumers. The surveyed population contains 3% infant (0–1 year), 66% children (1–12 year) and 31% adolescent (13–15 year). In children group, 24% population belonged to 1–3 year, 16% were in the age
group of 4–6 year, 24% were in the age group of 7–9 year while maximum (36%) were in the age group between 10 and 12 years. There were relatively more females (n ¼ 136) than males (n ¼ 119) in this study. The average milk consumption was found to be highest (534.6–542.9 ml/day/person) in 1–3 year age group which is 1.3–1.9 fold higher than other groups while consumption based on 90th percentile was also maximum (900–1200 ml/day/person) in 1–3-year age group. Intake of oxytocin The intake of OT through milk in different age group populations is shown in Table 4. The average intake of OT through milk in 1–3-year age group showed a maximum value of 2.3–2.4 mg/day/person (1.3–1.9 fold higher than others) while intake based on the 90th percentile was also highest (3.9–5.2 mg/day/person) in the 1–3-year age group, which was 1.2–2.8 fold higher than the other age groups.
Discussion Milk is the main source of protein and essential dietary component in children, apart from being consumed by the population of all age groups. In dairy farming, exogenous OT is frequently administered to cattle for milk let down. During the past several years there has been concern regarding the use of exogenous OT in cattle in Asian countries for milking. However, no study regarding surveillance of OT hormone in the milk samples for human consumption is available. Hence, a detailed scientific investigation is warranted to elucidate the levels of OT in this commonly consumed commodity. Earlier, we have developed a method for quantitation of OT in milk (Mishra et al., 2013a) and have applied it for the first time to analyze the quantity of OT in milk samples. In the present study, it was observed that all the 55 milk samples were found to be contaminated with OT ranging from 21 pg/ml to 18.9 ng/ml. Based on the results of quantification of OT and the data of average actual consumption of milk in children, the average estimated daily intake of OT was found to be maximum in 1–3-year age group of children (2.3– 2.4 mg/day/person). When the data were calculated from 90th percentile value, the intake was also found to be highest in 1–3-year children. Though maximum intake of OT was in 1–3-year age group, nonetheless, intake was also observed up to 13–15-year group (1.2–1.7 mg/day/person) suggesting that children up to 15 year are continuously exposed to OT since birth. Since OT is stable against normal boiling conditions as well as in simulated gastric fluid assay (Mishra et al., 2013b; Prakash et al., 2009), there is likelihood of chronic exposure effect of OT through oral route. Studies of Noriega-Guerra et al. (1966) have shown that oral administration of OT as a
OT in milk and likely intake in Indian population
DOI: 10.3109/15376516.2014.910851
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Figure 1. Comparative analysis of OT in milk samples by EIA and HPLC.
Table 3. Consumption pattern of milk in population of different age groups.
Table 4. Intake pattern of OT through milk in population of different age groups.
Milk consumption (ml/day/person) Age groups (years) Infant (0–1) Male (0–1) Female Children (1–3) Male (1–3) Female (4–6) male (4–6) Female (7–9) Male (7–9) Female (10–13) Male (10–13) Female Adolescent (13–15) Male (13–15) Female
Intake of OT (mg/day/person)
No. of % of 90th subjects consumers Average Maximum Percentile 3 5
100 100
375.0 400.0 a
500 500
475 500 b
17 23 14 13 18 22 35 26
100 100 100 100 100 100 98 95
542.9 534.6c 382.0 405.8 345.6 344.3 420.7 372.3
1500 1000 750 1000 500 500 2000e 2000f
1200 900d 500 500 500 500 900 750
32 47
90 90
399.2 279.0
2000 1000
500 500
A total of 150 families having 255 subjects between 0.5 and 15 years of age were surveyed. Average consumption is derived from the questionnaire in (n) number of subjects as mentioned above. a Average consumption of milk in males is highest (1.4–1.6 fold) in 1–3-year age. b 90th Percentile consumption of milk in male is highest (1.3–2.5 fold) in 1–3-year age group. c Average consumption of milk in female is highest (1.3–1.9 fold) in 1–3-year age group. d 90th Percentile consumption of milk in female is highest (1.2–1.8 fold) in 1–3-year age group. e Maximum consumption of milk in male is highest (1–4 fold) in 10–13-year age group. f Maximum consumption of milk in female is highest (2–4 fold) in 10–13-year age group.
drug induces uterine contractibility. Buccal administration of OT to pregnant women at term was shown to induce uterine contractions along with increased plasma concentrations of OT (Noriega-Guerra et al., 1966). It has been reported earlier that the use of oral or transbuccal OT facilitate child birth (Dawood & Khan-Dawood, 1986; Dillon, 1958; Dillon et al., 1962; Elstein & Wright, 1963; Newman & Hon, 1963) while overdose may have a risk for fetal cardiac problems (Dillon et al., 1962; Maxwell, 1964). Furthermore, Takeda et al. (1986) showed that intraperitoneal administration of
Age group
Age (years), sex, body weight (kg)
Average
90th Percentile
Infant
(0–1) Male (8.9) (0–1) Female (9.1)
1.6 1.7
2.1 2.2
Children
(1–3) Male (14.8) (1–3) Female (11.9) (4–6) Male (19.4) (4–6) Female (18.4) (7–9) Male (27.3) (7–9) Female (25.4) (10–13) Male (35.4) (10–13) Female (35.6)
2.4a 2.3c 1.7 1.8 1.5 1.5 1.8 1.7
5.2b 3.9d 2.2 2.2 2.2 2.2 3.9 3.3
Adolescent
(13–15) Male (48.2) (13–15) Female (47.5)
1.7 1.2
2.2 2.2
OT is specifically banned under the Prevention of Cruelty to Animals Act, 1960. Body weight has been derived from the surveyed population, whose mean is indicated in parenthesis. Age group selection was based on ICMR guidelines (ICMR, 2000). a Average value in male is highest (1.3–1.6 fold) in 1–3-year age group. b 90th Percentile value in male is highest (1.3–2.8 fold) in 1–3-year age group. c Average value in female is highest (1.3–1.9 fold) in 1–3-year age group. d 90th Percentile value in female highest (1.2–1.8 fold) in 1–3-year age group.
radioactive OT to dam rat on seven postpartum days, the presence of radioactivity in the plasma of neonates proved the transfer of OT into the milk. The absorption of peptides is less due to low bioavailability (0.5–1%), which may be one of the reasons for irregular effects of oral supplementation of OT as a drug. However, if this small percentage of OT is bioavailable in GI tract through continuous consumption of milk contaminated with OT (21 pg/ml to 19 ng/ml), it may result in unwanted toxic manifestation as observed in the recent studies where OT caused enhanced ovulation (Mishra et al., 2013b). The intake assessment studies of OT in the present investigation provided evidences for exposure to OT as high as 5.2 mg/day/person. Though the tolerable dose of OT and safe level of daily intake has not been monitored through milk, nonetheless,
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Takeda et al. (1986) showed that the presence of OT in normal human breast milk ranges from 6.4 to 9.0 pg/ml. Considering 9 pg/ml concentration of OT as the normal value in milk, the presence of OT in milk sample was 273–987 fold higher as observed in the present study. In general, the level of OT in human blood ranges between 3 and 5 pg/ml (Amico & Seif, 1980; Leake et al., 1981). However, from this study children population shall be exposed to OT (2.1–5.2 mg/day/ person) through milk and considering 1% bioavailability, 21–52 ng/day/person of OT shall be absorbed through intestine, which shall enhance the serum level by 3 fold per day taking into account the blood volume as 5 l. Being a hormone, the small amount of OT is sufficient enough to perform physiological function in normal conditions while exposure of OT (through milk) at higher levels, which occurs since childhood, may be a cause of concern as OT enhances the process of ovulation under nonphysiological conditions (Mishra et al., 2013b).
Conclusion Present study shows the presence of OT in milk samples. Based on our survey data for OT intake, children are the main consumers of milk and continuous exposure of OT through milk may enhance the levels by three folds per day in human serum. Therefore, the daily intake of OT through milk since childhood is a matter of concern.
Acknowledgements We are grateful to the Director of our Institute for his keen interest in this present study. M.M. is thankful to Council of Scientific and Industrial Research (CSIR), New Delhi for the award of Senior Research Fellowship. Financial assistance of CSIR- INDEPTH Project- BSC-0111 is gratefully acknowledged. The manuscript is IITR communication # 3051.
Declaration of interest The authors declare that there is no conflict of interest.
References Amico JA, Seif SM. (1980). Oxytocin in human plasma; correlation with neurophysin and stimulation with estrogen. J Clin Endoc Metab 52: 88–99.
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