Over the last two decades, organic minerals have been used as a partial replacement of trace mineral supply to improve certain areas of poultry productivity; this includes fertility in breeders, shell strength, and carcass and meat quality. Current research has identified future opportunities by replacing inorganic trace mineral sources with lower levels of the more available organic trace mineral forms, thereby minimizing the amount excreted and also optimizing trace mineral-associated performance traits.
Ensuring, via testing, that the organic trace mineral source used is presented in a form designed to optimize uptake and retention in the bird is essential to enable this new frontier of poultry mineral nutrition. Further, and perhaps more importantly, understanding the limitations of these tests is critical if one is to apply them successfully in the assessment of OTMs.
The chemistry of complexation, or chelation as it is commonly known, has created a great deal of confusion in the animal feed industry. Terms such as metal amino acid complexes, metal amino acid chelates, metal polysaccharide complexes and metal proteinates abound, yet official definitions remain vague and unhelpful.
Tests of varying scientific nature and credibility are widely claimed as having the ability to differentiate between “good and bad” organic mineral chelates.
Basic parameters that can be analyzed include: total mineral, amino acid profiling, the nitrogen-to-mineral ratio, the percentage of bound mineral, size or molecular weight, solubility and stability. In vitro tests designed to mimic bioavailability have also been developed. While some of the analyses in use have the ability to provide meaningful and valuable information on defined or individual products, no single test can be used to adequately compare and contrast all classes of OTM. Additionally, these techniques have as yet provided no information as to how the mineral sources react under conditions present in the animal’s gastrointestinal tract.
Accurate quantification of the total mineral content of OTMs is a routine quality control procedure used by all manufacturers. Cost comparisons between different products will consider this factor when calculating the relative value between OTMs. However, it is important to remember that the total mineral content gives no information regarding the bioavailability of individual products, and as such, is limited in terms of calculating the true relative value of a product.
Amino acid profiling
Amino acid profiling can be used in the case of amino- and protein-based products to determine the composition and type of bonding group used. With defined amino acid products, it can indicate if a carrier is in use and for more complex protein sources, the relative proportion of individual amino acids will enable one to quite easily distinguish between plant and animal protein sources.
Calculating the molar ratio of nitrogen to metal can be a useful way of assessing defined amino acid products such as glycine-based chelates, in which nitrogen plays a key role in mineral bonding. In general terms, if this ratio is 1-to-1 or greater, the product only contains enough glycine to bind the mineral as a charged complex (not a true chelate). If it is less than 1-to-1, it contains insufficient nitrogen to bind the metal. Obviously a ratio of nitrogen to mineral of 2-to-1 or greater is desirable in the case of simple amino acids such as glycine.
When assessing more complex products such as proteinates, the nitrogen-to-mineral ratio will not give a fully accurate reflection of the true potential for mineral bonding. Amino groups such as cysteine, histidine, aspartic and glutamic acids can bind metal atoms through their side chains via sulphur and oxygen atoms. As there is no involvement of nitrogen in this side chain bonding, an underestimation of the potential for binding can be made if one only considers the nitrogen-to-mineral ratio.
Products can also have their nitrogen contents artificially inflated through the addition of nitrogenous compounds such as urea, thus giving the false impression of a product with high nitrogen-to-mineral ratio.
Percent bound mineral
Typically, determination of the percentage bound mineral utilizes tests whereby aqueous solutions or suspensions of the OTM are filtered through a low molecular weight membrane before determination of the total mineral content. The material retained behind the filter is assumed to be bound to higher molecular weight components, while the mineral in the filtrate (solution) is assumed to be either unbound or bound to single amino acids or very low molecular weight peptides. Additional testing of the filtrate using an ion-specific electrode can indicate if the mineral in the filtrate is free or bound, but no interpretation of the strength of the bond between the ligand and the metal can be made. Importantly, this methodology is only applicable to copper-based OTMs. Filtration based techniques have, as yet, provided no information as to how the mineral sources react under the changing pH conditions present in the animal’s gastrointestinal tract.
It is often erroneously claimed that a metal complex or chelate must be soluble to be bioavailable. Many peer reviewed publications have shown that insoluble OTMs can potentially be more bioavailable than their soluble counterparts. Evaluation of OTM solubility is of little benefit unless one considers the effects of digestive processes and the changing pH environment present within the GI tract.
Numerous claims have been made as to the merits of comparing OTMs on the basis of size. In most instances the claims attempt to assert that a smaller size bonding group creates a more stable and more bioavailable OTM. In general, the perception is that peptide or proteinate based OTMs are too large to be effective, yet the molecular weight of OTMs is not a good gauge of bioavailability.
Correlating the molecular weight of a defined ligand, such as an amino acid or a peptide, with what’s known as its stability constant, indicates quite clearly that rather than size being of critical importance in generating a stable OTM, the type of ligand is of far greater significance.
Arguments concerning size persist in the industry, due mainly to misleading marketing literature.
In vitro lab-based assays, which attempt to assess bioavailability, have been developed over the last number of years. Most make use of cell culture-based assay systems in which transfer and uptake of mineral across cellular membranes is determined. This can be either by direct measurement of the mineral (free or bound) or indirectly through the measurement of cellular processes stimulated during or following mineral uptake.
One pitfall of the use of artificial membranes or isolated cell culture techniques is that they don’t possess the ability to accurately reflect the digestive processes found during GI transit of the OTM. As such they have limited value when comparing different products.
In general, when we talk about stability of OTMs, we are referring to the bond strength that exists between the bonding group and the mineral. The greater the bond strength, the more stable the product, although (as in the case of Ethylenediaminetetraacetic acid complexes and chelates), there is an upper limit beyond which bioavailability is negatively impacted upon.
Polarography using a controlled growth mercury electrode is a well-established physical chemistry tool although it is quite complicated for use as a standard testing procedure to assess the stability or bond strength of organic trace minerals.
One of the main drawbacks is that as a technique, polarography, can only be applied to test materials in solution and as such can only assess the soluble fraction of organic mineral products. Many of the OTMs on the market have insoluble fractions which can vary widely depending upon the product. Under the in vivo conditions of the GI tract, the initially insoluble fraction will be increasingly solubilised and it is recognised in the literature that metal solubility by itself is not a reliable indicator of chelate quality. Consequently, the results from polarographic tests alone should be interpreted with caution.
While simple tests based on total mineral, nitrogen to mineral ratio, percent mineral bound/free, solubility, stability and molecular weight are used by different manufacturers, they only provide limited information regarding individual products and may not be suitable for comparing different classes of product.
In reality, the most accurate assessment of OTMs is to assess the actual bioavailability of the product through animal feeding trials in which indices of mineral availability and animal performance are measured.
The development of a simple assay to compare different classes of OTM that is applicable under all conditions has yet to be developed. As such, the results from single in vitro tests alone should be interpreted with caution.