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N-nitrosodimethylamine, nitrate and nitrate-reducing microorganisms in human milk

J Uibu 1, O Tauts  2, A Levin 3, N Shimanovskaya 1 and R Matto 1

Institute of Experimental and Clinical Medicine 1, Tallinn Technical University 2 and Tallinn Children's Hospital 3, Estonia

Acta Pædiatr 1996;85:1140-2. Stockholm. ISSN 0803-5253.

    Of 54 milk samples from 54 healthy nursing women analysed for volatile N-nitrosamines, 42 appeared negative. Trace amounts (below the detection limit 0.5 µg l-11) of N-nitrosodimethylamine were detected in the milk of 10 mothers and two samples contained this compound at 1.1 and 1.2 µg l-1 respectively. Almost all samples investigated contained nitrate (mean 2.9 ± 2.3 mg l-1) and nitrate reducing microorganisms (mean 4.2 ± 1.0 log ml-1). The recent finding of N-nitrosodimethylamine in human milk gives evidence of the continuous endogenous formation of N-nitrosamines.

    Bacteria breastfeeding, dimethylnitrosamine, human milk, nitrates

    J Uibu. Department of Experimental Oncology, Institute of Experimental and Clinical Medicine. Hiiu 42, EE0016 Tallinn, Estonia
In recent decades breast milk has very often been investigated concerning the occurrence of the environmental chemical contaminants. There are at least three reasons for this: the growing pollution of the human environment, extension of our knowledge about the harmful effects of various chemicals and the concept that the occurrence of a chemical substance in breast milk describes well enough the situation in the organism as a whole.

Among the numerous studies on the undesirable chemical contaminants, the content of N-nitrosamines in human milk has been investigated only in limited cases (1-3). However, just N-nitroso compounds are continuously attracting a keen interest in experimental and epidemiological oncology as a large group of most potential chemical carcinogens, to which exposure may occur over a long time and which can be formed endo-genously.

In 51 milk samples from 13 nursing women in Philadelphia, US, seven samples contained N-nitrosodimethylamine at 0.3 – 1.1 µ g kg-1 (1). In a study carried out in Tokyo, 82% of 67 milk samples contained N-nitrosodimethylamine from trace amounts to 1.8 µg kg-1 (2). However, N-nitrosamines were not found in any samples in a human milk analysis in Germany (3).

In addition to the above-mentioned data on the actual detection of N-nitrosamines in mother's milk, indirect evidence exists that these harmful compounds are rather 'normal' constituents of breast milk: (i) presence of N-nitrosamines in the blood of healthy or diseased human subjects (4), (ii) endogenous formation of N-nitroso compounds, which is well demonstrated in many epidemiological studies by the N-nitrosoproline test (5), and (iii) nitrosation processes may take place in infected tissues (bacteria, parasites, viruses) via free radical of nitric oxide (5), Furthermore, there is a possible double role for bacteria concerning endogenous formation of N-nitrosamines — the production of nitrite from nitrate and the catalysis of the N-nitrosation reaction (6).

The above-mentioned circumstances determined the objective of this study – to investigate the occurrence of N-nitrosamines, nitrate and nitrate-reducing microorganisms in the milk of healthy nursing mothers.
Subjects and methods

Fifty four healthy nursing women 1.5 – 8 weeks post partum participated in this investigation as donors of breast milk. These volunteers stayed in the Tallinn Children's Hospital with their babies, who were undergoing medical treatment.

Collection of milk samples started in the morning and continued for 1-2 days until 150 ml of breast milk was accumulated. The claim, without any deficiency in the nutrition of infants, has been strongly observed during the collection of milk. Samples were taken in 50ml plastic tubes and stored at 4°C during collection. Subsequently milk samples were stored at -20°C before the chemical analyses. For a microbiological analysis, 5ml of breast milk was collected into a sterile test tube and stored at 4°C, but not for more than 4h, before the inoculation procedure.

N-nitrosamines were extracted from milk samples by steam distillation, extracted with dichloromethane and concentrated in a Kuderna-Danish evaporator. Quantitative measurements for volatile N-nitrosamines were carried out with a gas chromatograph PYE-104 interfaced to a Thermo Electron thermal energy analyzer, model TEA-502. The limit of detection and identification was 0.5 µg l-1 (7).

We determined the nitrate content of milk by reducing it to nitrite on a cadmium column and assaying the NO-2 by diazotization and coupling with a Griess reagent. The presence of nitrite in supernatant before reduction had been checked by a Griess reagent. The method of quantitative determination of nitrate reducing micro organisms on the basis of the most probable numbers method ("multiple tube dilution to extinction") was employed to study the total counts and the counts of nitrate-reducing microorganisms in nitrate broth medium (6).

Of 54 milk samples from 54 nursing women analysed for volatile N-nitrosamines by GC/TEA, 42 gave negative results. Trace amounts (below the detection limit 0.5 µg l-1) of N-nitrosodimethylamine were detected in milk of 10 mothers and only two samples contained this compound at 1.1 and 1.2 µg l-1, respectively. No other N-nitrosamines were detected.

Nitrite had not been found in any milk samples, but almost all of the 27 samples investigated contained nitrate with a few exceptions. Its concentration varied from below 0.5 to 6.1 mg l-1 NO-3, only one sample contained 11.3 mg l-1 NO-3. Mean concentration of nitrate was 2.9 ± 2.3 mg l-1 (mean ± SD).

All 31 milk samples investigated contained microorganisms, including nitrate-reducing microorganisms. Mean total counts of microorganisms and nitrate-reducing microorganisms were 4.5 ± 1.1 and 4.2 ± 1.0 log ml-1, respectively. It was observed that total count and count of nitrate-reducing microbes of the milk sample were very often the same.

We have not found any clear connection between the concentration of nitrate and the counts of nitrate-reducing microorganisms — the correlation coefficient was only 0.155.

Our results confirm the phenomenon that sometimes human milk may contain the potent chemical carcinogen N-nitrosodimethylamine. Provisionally it is possible only to suppose its origin and biological significance. The low amounts and rare occurrence of N-nitrosodimethylamine, together with the time-consuming analytical procedure, make it difficult to elucidate these acute problems.

Today we are not able to confirm or deny any possible negative effect of N-nitrosodimethylamine, sometimes found in human milk, on the health of an infant. This finding of a potent chemical carcinogen in breast milk requires very careful evaluation of the existing scientific data to continue research on the carcinogenic risk of this compound. Provided that risk is uncertain, clear benefits of breastfeeding do not allow us to recommend any change of breastfeeding policy, as carried out by the WHO (8). Actually, breastfeeding is an essential component of a truly baby-friendly environment based on the concept of the "psychological and biological umbilicus", which means continuous physical contact between the mother and newborn, breastfeeding immediately after birth, minimal contact with other care-givers or technological equipment, etc. (9). Also, it is necessary to note that bottle-fed infants are often exposed to N-nitrosamines derived from baby-bottle nipples and pacifiers. Thus, a highly contaminated nipple may cause a 5 kg infant who drinks 1 litre of formula per day to ingest approximately 10 µg of N-nitrosamines (10).

The presence of N-nitrosodimethylamine in human milk has subsequently raised a question about its endogenous formation and makes us re-examine the preconditions of this formation. The mean concentration of dimethylamine as a precursor compound in the blood of healthy volunteers is 0.91 mg l-1 (11), being almost three orders of magnitude larger than the N-nitrosodimethylamine content in some milk samples investigated by us. This dimethylamine concentration is evaluated as sufficient to support endogenous formation of N-nitrosodimethylamine (11). Results of studies carried out in recent years demonstrate that both bacteria and stimulated macrophages may increase the endogenous formation of N-nitroso compounds via nitro-oxide produced in infected and inflamed tissues (5). It seems plausible that just microorganisms, including nitrate-reducing bacteria, found in large amounts in milk, may provoke and maintain the synthesis of nitric oxide in breast tissues, mediated by macrophages and neutrophils.
Acknowledgments. The pioneering contribution of Professor P Bogovski and Professor J Kann to initiation of N-nitrosamines research in Estonia two decades ago is highly appreciated as a prerequisite for the recent study. Part of the work described here was supported by grant no. 97 from the Estonian Science Foundation. We thank the National Cancer Institute, Bethesda, MD, USA, for the TEA detector used in this work under contract no. NO1CP75975.
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