METHODS OF MEASURING HORMONE LEVELS
IN ANIMAL PRODUCTS

by

R. Ferrando
Directeur du Laboratoire de nutrition et d'alimentation
Ecole nationale vétérinaire d'Alfort
94704 Maisons-Alfort (France)

1. INTRODUCTION

The question of the use in animal production of anabolic agents, including the natural steroid hormones and their derivatives, has been -- and continues to be -- badly stated, and therefore misunderstood. Those who brought the problem before the public had neither the information nor the technical competence, especially in biology, to speak with perfect objectivity.

Hormones are found naturally in many animal products (6, 7) and, as phytohormones, in many plants (3, 5). Only some of them raise public health problems, and these should be distinguished from the others. Indeed, a study of the practical and health aspects of the use of hormones in animal production should underlie all regulation of the matter, and therefore any decision on the measurement of hormone levels.

A considerable volume of documentation exists on the problem (6), which is as important for the developed as for the developing countries. Items 1 to 11 in the Bibliography of this paper indicate a number of general studies.

2. THE USE OF HORMONES IN ANIMAL PRODUCTION, AND ITS CONSEQUENCES

A reading of many popular, and even official, papers reveals that different types of anabolic agents, whatever their structure, are grouped together under the general name of hormones, among them those whose activity as oestrogens is emphasized and those which reinforce masculine traits.

Published material and research results make it possible to distinguish three types of anabolic agents.

The first type includes those not found in mammals and birds but obtained exclusively through organic synthesis: diethylstilboestrol (DES) or stilboestrol, dienoestrol, hexoestrol and their derivatives. These compounds, active when administered orally or as implants, are harmful for laboratory animals, whether given directly or as residues in the flesh and offal of treated animals. The use of these substances, a potential danger to human health, is prohibited in many countries, particularly since the methods of measuring DES levels in meat are unreliable, so that measurements must be made on the excreta.

The second type of anabolic agents consists of the natural steroids, normally present in all animals: testosterone, oestradiol, oestrone, progesterone. Secreted by the endocrine glands, these substances and their derivatives, although absorbed by the intestines, are deactivated in the liver. They produce measurable effects only when implanted.

The third type, found in the higher plants and moulds, consists of substances with a more or less clear oestrogenic and, at times, anabolic activity. Among these phytoestrogens, only zearalenone has been isolated for use in animal production: as zeranol, it gives good results, but has an effect on genital formation and the level of thyroxine in plasma (23). It should be added that Sharaf and Gomaa (21) attribute oestrogenic effects to Vitamins E, B₆, C and perhaps A, while Nelson et al. (20) have studied the same effects in O-p'-DDT.

This paper discusses only the consequences for the consumer of the use in animal production of the first two types: the synthetic agents such as DES and the natural steroids oestradiol, progesterone and testosterone.

First mention should be made of cases of cancer of the vagina and hypogonadism observed in the offspring of women treated with DES. Such observations (17, 18, 22) are verified by research based on the so-called relay methodology (13), based on the fact that methods of analysis for a residue and its metabolites fail to reflect practical realities, since they may lead to findings of residues without bioavailability or vice versa. Further, the individual biological evaluation of each metabolite fails to reflect the overall consumption of all of them. The relay, a farm animal, changes this situation.

In order to determine whether this was true of the first two types of anabolic agents, rats and mice of both sexes received (13) balanced diets containing 20% of the flesh or 6% of the liver of male and female calves implanted with DES (24 mg in each of two implants), oestradiol and progesterone (20 + 200 mg in two doses) or oestradiol + testosterone (20 + 200 mg in two doses). Animals were implanted at the beginning of the growth period and 60 days later, 38 days prior to slaughter.

Chemical measurement of residues showed DES levels of 30 g/kg in the flesh, but none in the liver. Levels of natural steroid hormones did not exceed levels in untreated animals.

Feeding rats and mice on the flesh of DES-implanted calves led to growth in both sexes, sterilized the females and sharply atrophied the testicles, seminal ducts and penis of the males (12, 15). Liver behaviour was less clear. Pokrovski et al. and Nesterin (2) obtained similar results with flesh. It was also observed that DES persisted for nearly 40 days in the environment and was found in alfalfa (5, 14).

Animals fed on the flesh and liver of calves implanted with oestradiol + progesterone or oestradiol + testosterone behaved in the same manner as controls fed on the flesh and liver of control calves. After 24 months, the percentage of tumors (primarily mammary adenomata, frequent in old Wistar rats) was the same in both groups.

Trenbolone acetate (TBA), a testosterone derivative, leaves little or no residue. Measured by radio-immunoassay, Gropp et al. (16) also studied their biological effects by the relay toxicity method, on Sprague-Dawley rats fed on the flesh of calves implanted with a mixture of 20 mg oestradiol-17β + 140 mg TBA, as well as with doses 10 and 25 times higher. The results of this study, which continued for 110 weeks and covered two generations, showed the absence of any unfavourable influence on growth, organ weight, reproduction, teratogenicity and cancerogenicity. Only females fed on the flesh of control calves to which TBA had been added in doses 25 times normal, had significantly lower growth(P< 0.05). The biochemical constants in the blood were the same in both groups.

These experiments show that compounds of natural steroid male and female hormones, and their derivatives, appear to be without danger for the consumer. They are useful in animal production, obviating a number of often costly and polluting therapeutic operations. On the contrary, the use of DES-type non-steroid anabolic agents has serious disadvantages. It is difficult to detect their residues in meat. Relay toxicity results show that analyses and biological behaviour fail to agree.

The indiscriminate prohibition of all types of anabolic agents may be leading to the use of DES and similar substances. Analyses of urine in which they can be identified most easily, have revealed them in 20% of cases. In the United States, McClung (19) estimates that 500 000 bovines are being treated illegally. In Europe, illegal use appears to be fairly general, but is tending to decrease. The use of DES and similar substances should thus be prohibited, while that of the steroids and their derivatives should be authorized under veterinary control. This was the view of many writers as early as 1967, and their conclusions were confirmed by the FAO/WHO Joint Committee of 1975 (2) and a recent international symposium (Warsaw, 27–30 April 1980) (9). Any new anabolic agent should be severely tested before it is distributed widely.

There is a need to develop practical, economic and rapid -- but very exact -- methods of detecting residues, particularly those originating in products chemically different from the natural steroid hormones and their derivatives.

3. METHODS OF MEASURING HORMONE LEVELS IN ANIMAL PRODUCTS

It should be clearly understood that this discussion concerns only measurement methods intended to detect residues remaining in flesh and viscera. These methods are, however, not very reliable, and other biological material must be examined to determine whether the animals have been treated. Specialists agree that there are still difficulties in testing for residues in the tissues of animals intended for use as food.

Many different methods have been proposed and discussed at the EEC level. While the trend is increasingly toward the use of radio-immunoassay, it is undeniable that the techniques are highly complex and still far from being perfected.

DES and similar substances should preferably be looked for in urine and faeces. Instructions to this effect were issued in the United States in 1979 and confirmed in 1980 (48).

Account should also be taken of the fact that no difference may be found between animals properly implanted with anabolic agents and untreated animals. This problem has received insufficient attention. The proceedings of the FAO/WHO Joint Meeting of March 1975 (2) contain reports emphasizing this lack of difference and the relatively high, but physiological, levels of testosterone and progesterone in the flesh of bulls and gestating cows, respectively. This being the case, how can the natural presence of these substances be distinguished from their presence due to the use of natural products?

Further, it should not be forgotten that very many animal feeds contain phytohormones which can cause modifications in the morphology and histology of certain organs.

After summarizing and stating the principles of the four main groups of useful techniques enumerated by Kroes et al. (39) and Richou-Bac and Pantaléon (42) -- histological, biological, physico-chemical and radioimmunoassay methods -- this paper will study in detail the techniques that can be applied in practice for routine control.

1. Histological methods (Kroes et al. (39)), used specially in young bovines, consist in the observation of modifications in the male and female genitalia. The prostate and the bulbo-urethral glands are particularly examined in male calves and lambs, while in females attention is focused on the glands of Bartholin, the teats, the vagina and the cervix. Modifications are obvious when DES and zeranol, as well as hexoestrol, have been used. In pigs the effects are less discernable. It is generally agreed that the histological method makes it possible to conclude that an oestrogen has been used at some earlier time. The use of natural steroid oestrogens, in combination with testosterone or progesterone, leads only to slight histological modifications. The phyto-oestrogens may also have some effect and lead to modifications; this is the case of Fusariuminfected maize.

2. The biological methods used are those of Astwood (26), which measures the increase in uterus weight of pre-puberal mice and rats, and of Allen and Doisy (25), based on modifications of the cytology of the vagina of adult mice after ovarectomy. The latter test, applied by Stob et al. (47) in 1954, is the most sensitive, but it is responsive only to compounds with oestrogenic activity. Martin (40) reports that the use of concentrated extracts placed in the vagina of mice also gives good results.

3. Physico-chemical methods, which make it possible to identify with precision the anabolic agent sought for, require a fairly long extraction, followed by thin-layer or gaseous-phase chromatography. Hans and Abraham (31) report that the latter techniques, combined with mass spectometry, can now be contemplated. Sensitivity is high. Further off is the possible use of liquid/liquid chromatography. Thin-layer chromatography is currently used, and Verbeke gives details in an EEC document (50), while Waldschmidt (52) and Schuller and Stephany (46) have described the method.

4. Radio-immunoassay, first described for the oestrogenic steroids by Jiang and Ryan (38), is based on competition between tritium-labelled and unlabelled antigens for the corresponding specific antibodies. This technique, increasingly used, is highly sensitive, making it possible to detect 0.05 to 0.005 ppb of DES and 0.1 ppb of TBA. It is, however, complex and long, although it is being improved. It has the same advantages as all uses of radioactive compounds. The technique has been well studied by Hoffmann (34) and Hoffmann and Karg (35).

Two new methods may also be mentioned: high-pressure liquid chromatography which, according to Richou-Bac and Pantaléon (42) does not appear to be sufficiently sensitive, and the so-called Elisa method, applied by Ruitenberg et al. (44) in the Netherlands to detect pig infection by Trichinella spiralis. This method, which uses an enzyme for labelling, followed by measurement with 450 nm spectrophotometry, needs further development, but it may be possible to automatize its use. Kroes, working at Bilthoven (Netherlands) is studying how it can be used to measure hormone levels.

Table I, completed from an original kindly furnished by Richou-Bac, indicates the sensitivity of all these new methods.

TABLE I. Sensitivity of techniques for testing
for “hormones” and anabolic agents
(ppb)

Figure A	Figure B
Source: Verbeke (50)	Source: Verbeke (49)

Figure C

URINE

(sample: 20 ml, diluted with 10 ml H₂O)
↓
Hydrolysis
↓
Ether extraction and washing of ether phase
↓
Silica gel column
↓
Eluate evaporated in N atmosphere
and collected in μl acetone
↓
HPTLC (bidim.)*
↓
Observation under UV 366 nm
(pink spot reveals presence of DES)

Source: French veterinary services.

* High-performance thin-layer chromatography (bi-dimensional).

In practice, the most urgent problem appears to be the detection of DES. Bories et al. (27) and other authors have shown that it is eliminated in the urine and faeces. It has also been demonstrated (30) that the saliva does not contain DES but that it does contain progesterone. DES cannot be detected in the flesh of animals treated three weeks before slaughter. It can be detected in the urine after one month by thin-layer chromatography and radio-immunoassay.

The bladder need only contain from 20 to 50 ml of urine for the thin-layer chromatographic method to be applied. The method, used in 1973 (29) to detect oestradiol residues, was modified and improved by Verbeke (50) in 1979. Vogt and Oehrle (51) have employed it for detecting steroid oestrogen residues and Stilbene in calves' urine. Ryan and Hoffmann (22) use it, together with radio-immunoassay, to study linked trenbolone residues.

For the latter compound, according to Richou-Bac and Pantaléon (42), Schuller and Stephany believe it possible to use thin-layer chromatography to attain a sensitivity level of 0.005 ppb, comparable to that for radioimmunoassay; this figure has not been included in Table I.

Figures A and B show, respectively, outlines of Verbeke's analytical methods for meat, fat and viscera and for urine (49, 50). Figure C shows the outline of a technique for urine used in the laboratories of the French veterinary services for the detection of DES.

Radio-immunoassay is based on competition between labelled and unlabelled antigens, i.e. a labelled hormone (indicated below by H*) and an unlabelled hormone (H), for the same antibody molecule. The diagram below shows the process of the reaction.

H* (Tritium-labelled hormone)

+

AB (Specific anti-body)

H* - AB (Localized labelled hormone)

+
        (Standard or
H* (biological

(Tritium-labelled hormone)

(Localized unlabelled hormone)

The levels of AB and H* being constant, any increase in H provokes a reduction in the labelled hormone localized in the specific antibody (H* - AB). The measurement, established by calculating the relationship

depends on the measurement of radioactivity levels between the localized labelled hormone and the free hormone.

Kroes et al. (39) and Richou-Bac and Pantaléon (42) give details of extraction and measurement techniques. Hoffmann and Laschuetza (37) have used the method to examine blood plasma and flesh for DES.

The hormone is rendered antigenic by localization on seric albumen. Labelling the hormone should in no case alter the molecule. The specificity of immunoassay depends on the intrinsic characteristics of the antibody and the efficiency of the hormone purification method. Many techniques exist for separating the free H*, but immunoprecipitation appears to be the method most commonly used. The immunological behaviour of the hormone present in its matrix should be identical with that of the reference standard.

Authors such as Exley et al. (28), Hoffmann and Karg (35) and Heinritzi (32) have used this method for the natural oestrogens, Abraham et al. (24) and Rombauts et al. (43) for DES, and Pottier et al. (41), Heitzman and Harwood (33) and Hoffmann and Oettel (36) for TBA.

It is not yet clear what techniques are the most exact and economical and are therefore practical for large-scale control.

The basic material which can be inspected also needs to be determined.

Flesh, fat and viscera cannot be used for DES, which can be identified only in the urine, with a sensitivity of about 6 ppb. Many analyses can be made by thin-layer chromatography and radio-immunoassay. The French control services use only the former, estimating the cost of one analysis at 25 FFr (= approx. US $5). The possibility of techniques applicable to flesh is under study, but in this regard the techniques speak of “chemical torture”. They concede that routine control of meat for all hormones appears to be difficult.

The same services also use urine for measuring natural hormones, applying Verbeke's technique (50). No results are obtained from flesh, even for natural hormones, over 3 weeks following treatment, levels being too low. Thus, as compared with untreated calves, thin-layer chromatography yields no information.

Tables 1 through 4 of ref. 50 show sensitivity levels for the various hormones, according to the solvent system used, as determined by Verbeke, and also the RF values and spot colours under UV fluorescence at 366 nm.

It can be concluded that routine control in flesh is very difficult in all cases, whatever the anabolic agent. Above all, the use of DES should be avoided, and for this purpose, using urine as the basic material, high performance bidimensional thin-layer chromatography is the method to be preferred.

REFERENCES

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