Comparisons of mineral retention (‘comparative balance’) from two or more sources at two or more rates of intake have been used to assess the relative nutritional value of minerals in those sources from the ratio of the linear responses in retention. Partial body retention is commonly used; for example, haemoglobin responses have been used to assess the availability of iron (Saylor and Finch, 1953), bone growth to assess calcium sources (Lengemann et al., 1957) and increases in liver copper to compare copper sources for chicks and lambs (Pott et al., 1994). The results obtained can, however, vary, depending on the experimental conditions chosen (Suttle, 1983, 1985).
Comparisons of rates of urinary excretion and milk secretion between two mineral sources can also be used to assess relative availabilities, provided that the losses are linearly related to dose and there are no differences between sources in the partition of absorbed mineral prior to excretion or secretion.
Thus magnesium availability to sheep has been assessed from rates of urinary magnesium excretion. This ‘comparative loss’ approach has been modified by the use of double-radioisotope techniques, in which one source of the element is labelled with a radioisotope and then compared with another source labelled with a different radioisotope of the same element and given simultaneously. Lengemann (1969) compared iodate and iodide as sources for milk iodine (I) in lactating goats given sodium iodide (Na131I) and sodium iodate (Na125IO3) daily or as a single oral dose. It was concluded from the
125I :131I ratio of the milk that iodate had 0.86 of the value of iodide as a source of milk iodine. By a similar procedure, the iodine of 3,5-diiodosalicylic acid was claimed to be only 20% as available as that of sodium iodide (Aschbacher et al., 1966). Inferences can also be drawn about the value of different sources of minerals from the relative amounts that are required to ameliorate, prevent or potentiate signs of deficiency in the animal. Cantor et al. (1975) assessed the value of plant and animal feed sources of selenium for chicks from their ability to prevent exudative diathesis and raise activities of the seleno-enzyme, glutathione peroxidase. Using a chick growth assay, Odell et al. (1972) reported that zinc in wheat, fish-meal and non-fat milk was 59, 75 and 82% as available, respectively, as that in zinc carbonate.
Complications can arise when natural feeds are added to semipurified diets as the major source of a given mineral, because they provide variable amounts of other nutrients, which might also influence growth (Suttle, 1983).
The above techniques, whether radioactive or not, normally yield only qualitative assessments relative to a standard source, assumed to be highly, if not completely, available (e.g. ferrous sulphate (FeSO4) as an iron (Fe) source). However, repletion techniques can be modified to give quantitative data (Suttle, 1985). To obtain quantitative assessments of absorption, radio- isotopes have to be applied in special ways. Refinements were first intro- duced to differentiate between the unabsorbed dietary mineral fraction of the faeces and the fraction that has been absorbed and re-excreted by the gut,
Measures of relative availability
Measures of absolute availability
i.e. the endogenous faecal mineral (FE). The essential calculations (following a single parenteral injection of radioisotope, total collection of faeces and measurements of total excretion of both radioisotope and stable mineral) were first outlined by Comar (1956), using 45Ca; they were:
Endogenous
= Specific activity of faeces
3Total faecal Ca faecal Ca Specific activity of endogenous Ca
where specific activity is the ratio of radioisotopic to stable mineral concentra- tion (hence the term ‘radioisotopic dilution technique’). A crucial assumption has to be made, in that the specific activity of endogenous mineral is predicted from samples taken from an accessible pool with which the endogenous min- eral is supposedly in equilibrium (e.g. plasma or urine). Having calculated faecal endogenous Ca (FECa), calcium absorption coefficients are than derived as follows:
Ca absorption coefficient = Ca intake 2 (Faecal Ca 2FECa) Ca intake
The technique is sometimes modified by giving the radioisotope orally and intravenously, either sequentially to the same animal or simultaneously to matched groups. Where two isotopes of the same element are suitable (e.g.
45Ca and 47Ca), they can be given simultaneously by alternative routes to the same animal. Estimates of intake and faecal excretion may then be confined to the radioisotope and ‘intake’ is the dose of radioisotope given; this technique is useful for studies with elements which readily contaminate the average experimental environment, polluting both feed and faeces (e.g. iron, zinc and copper). The term ‘comparative radioisotope balance’ technique more accurately describes a further modification, in which retention of the radioisotope is measured in a whole-body monitor and route of excretion ignored. Thus Heth and Hoekstra (1965) gave 65Zn as the chloride or as a glycine complex to growing rats in the feed or by intramuscular injection.
Between 100 and 250 h after administration of the marker, the decline in radioactivity was a simple exponential function, with identical slopes for the two types of dosing (Fig. 2.4). By extrapolating these lines to zero time, it was estimated that 43% of the oral 65Zn was absorbed.
Yet another refinement of the radioisotope approach requires sophisticated compartmental analysis of changes in radioisotope activity in the bloodstream after a single dose, using computer programmes such as SAAM27. From the rate of dilution of radioisotope in the plasma caused by ‘influx’ of stable mineral by absorption and mobilization of body stores and ‘outflow’ by tissue uptake and endogenous loss, estimates can be made of the rate at which the stable mineral is entering the blood plasma pool from the gut (e.g. from phosphorus in herbage eaten by sheep (Grace, 1981)) and flowing to and from compartments (e.g. selenium in hay (Krishnamurti et al., 1997)).
Compartmental modelling
However, complexity is no guarantor of accuracy and, in the case of selenium, failure to recognize key ‘molecular’ compartments leads to results which cannot yet be trusted (see Chapter 15).
The most recent development in the assessment of availability has been the use of stable or non-radioactive isotopes as tracers (Turnlund, 1989).
Suitability depends on the existence of isotopes of the element of different natural abundance; the natural isotope ratio is then perturbed by dosing the animals with the least abundant isotope and the same dilution principles as those used for radioisotopes are applied (see Fairweather-Tait et al., 1989).
The stable isotope approach has the advantage of avoiding the hazards and safety constraints associated with radioisotopes; two disadvantages are the need for expensive capital equipment (e.g. inductively coupled ‘plasma’
generator plus mass spectrometer) and the slow (chromatographic) through- put of samples.
All isotopic tracer techniques have two further potential pitfalls, which may lead to false estimates of the value of a mineral source. First, they may fail to be representative of the mineral in the feed which they are supposed to trace or mimic; the lower the ‘true’ availability of the feed source, the greater is the Fig. 2.4. A comparison of retention curves for 65Zn given in the feed and by
intramuscular injection. Percentage absorption (A) of oral 65Zn is calculated by dividing Y2(the y intercept of the extrapolated retention curve for oral 65Zn) by Y1(the y intercept of the extrapolated retention curve for injected 65Zn) and multiplying by 100 (from Heth and Hoekstra, 1965).
Stable isotopes
Problems with isotopic markers
likelihood of underestimation. Secondly, the full potential of the mineral source may be masked by an animal capable of absorbing only what it needs; this applies to calcium, zinc and iron, for example (Suttle, 1985).
Accuracy of the tracer might theoretically be improved by growing the feed in isotope- (radioactive or stable) enriched nutrient solutions, a technique known as ‘intrinsic labelling’. However, intrinsic labelling is laborious and expensive and has yet to demonstrate marked differences when compared with extrinsic labelling for most trace elements, selenium, chromium and cadmium being the exceptions (Johnson et al., 1991). In vitro measurements of the completeness of labelling under conditions of simulated digestion might usefully and economically predict the accuracy of a potential tracer (Schwarz et al., 1982). When the tracer is given parenterally, the problem of poor representation is minimized, because absorption partitions the mineral into absorbed and non-absorbable forms and the former will generally be more readily labelled than the heterogeneous forms in the diet. Care must be taken not to administer so much tracer that the homeostatic control mecha- nisms are grossly disturbed; this problem is of particular concern in studies using stable isotopes, where sufficient tracer must be given to perturb the normal isotope distribution.
The problem of absorption according to need is theoretically overcome by measuring absorption with mineral intakes at or below need (AFRC, 1991).
However, need is determined by availability and ideal mineral intakes may therefore vary from source to source. There are a growing number of false estimates of availability in the literature, mostly involving chelated sources of trace elements (e.g. Mn, Chapter 14), in which the use of different ranges of intake for different mineral sources has led to animal influences being interpreted as hyperavailability of a particular source. The best approach is to assess each source at progressively lower intakes and note the maximal absorptive efficiency attained (AFRC, 1991). The term ‘absorbability’ should be used to describe values obtained by this type of approach and to distin- guish these proper assessments of the full potential of a mineral source from the sundry measurements covered in the past by the term ‘availability’.
The application of tracer and other techniques to the assessment of the value of mineral sources has led to a few major advances. In the experiments of Heth and Hoekstra (1965), a significant depression in zinc absorption was demonstrated when the calcium content of the diet was raised. In others, the calcium of milk was shown to be better absorbed by calves than the calcium of a hay and grain diet. Differences in the availability of various dietary forms of selenium are also well established. Cantor et al. (1975) found that, in most of the feeds of plant origin, the selenium was readily available (values ranging from 60% to 90%) to chicks, whereas selenium in animal products tested was less than 25% available. However, these isolated examples merely Problems introduced by the animal
Gaps in knowledge
illustrate how much remains to be learned of absorbability and its relation- ship to the chemical forms in which minerals occur. Why is fresh herbage less effective in promoting body copper stores in cattle and sheep than hay or dried herbage of equivalent total copper content (Hartmans and Bosman, 1970; Suttle, 1983), for example? Only sophisticated, physiologically and biochemically sound approaches to the use of tracers (e.g. Buckley, 1988) will provide the necessary information on these and other intriguing questions concerning mineral absorbability, arguably the most important determinant of mineral deficiency diseases. For a detailed text on the practical assessment of mineral availability, the reader should consult Ammerman et al. (1995).