and muscle membrane permeability in selenium deficiency (Chapter 15).
However, a rise in plasma MMA may precede the important clinical sign of cobalt deficiency, loss of appetite (see Chapter 10). By studying relationships between markers of depletion or deficiency and dysfunction, the interpreta- tion of each one can be clarified (Mills, 1987). It is becoming apparent that animals have internal biochemical systems for recognizing low mineral status and correcting it; mRNAs for serum ferritin – the storage protein – are switched off in iron deficiency and those for glutathione peroxidase (GPX) switched off in selenium deficiency, while those for cholecystokinin are induced in zinc deficiency (see appropriate chapters). The use of mRNAs for routine diagnosis is likely to remain prohibitively expensive, but their experi- mental use could resolve some long-standing arguments by letting the animal decide how mineral-deficient it is.
When using the mineral composition of animal tissues and fluids to indicate the quality of mineral nutrition, it is important to avoid the confounding effects of physiological and developmental changes. The normal newborn animal often has ‘abnormal’ tissue or blood mineral composition when judged inappropriately by adult standards (see Chapter 11 for liver and blood copper). Most blood mineral concentrations change abruptly and briefly around parturition, and sampling at this time is only appropriate for acute disorders of calcium and magnesium metabolism (see Chapters 4 and 6).
The choice of tissue or fluid for analysis varies with the mineral under investigation. Blood, urine, saliva and hair have obvious advantages because of their accessibility without harm to the animal.
Fig. 3.2. Relationship between growth rate and plasma or serum zinc in experimentally deprived lambs (White, 1996).
Physiological and developmental changes
Whole blood, blood serum or plasma is widely sampled and serum is usually chosen for analysis, because it avoids the cost and possible analytical complications of adding an anticoagulant, gives a more stable (i.e. haemolysis- free) form for transportation and usually reflects the status of the transport pool of the element, i.e. one step nearer to dysfunction than storage. The use of whole blood or erythrocytes brings complications and new possibilities into the assessment of mineral status. Minerals in their functional units are often incorporated into the immature erythrocyte prior to release into the bloodstream, and the mineral content of the younger erythrocytes alone accurately reflects recent mineral nutrition. Once released, the mineral and functional status of the erythrocyte may remain unchanged, despite marked fluctuations in the mineral status of the diet or tissues. Thus erythrocyte enzymes indicative of copper and selenium status (copper–zinc superoxide dismutase (CuZnSOD) and GPX, respectively) normally show a delay of about 3 weeks before indicating a rise in copper or selenium provision, with the arrival of sufficient recently enriched cells to increase mean values for the entire erythrocyte population. This may be an advantage if animals have been treated or inadvertently gained access to a new mineral supply (during handling) prior to blood sampling, since erythrocyte enzyme activities in the subsequent blood sample would not have been influenced. However, it could be disadvantageous in masking a recent downturn in copper or selenium supply. In extremely deficient animals, erythrocytes may be released in which the enzyme lacks its functional mineral (apoenzyme). Assay of enzyme protein by enzyme-linked immunosorbent assay (ELISA) or the extent of in vitro reconstitution may offer new insights into the severity of depletion in such circumstances (see Chapter 16). Where the normal function of an element is expressed by the erythrocyte, e.g. stability of the membrane and/or resistance to oxidative stress, as in copper, phosphorus and selenium deficiencies, functional status may be indicated by in vitrohaemolysis tests or staining for Heinz bodies; however, such tests would obviously be non- specific.
Analysis of saliva provides diagnostic information on several minerals, and samples are readily procured, using an empty 20 ml syringe fitted with a semirigid polypropylene probe and using a ‘trombone slide’ gag to open the mouth. The composition of the saliva is particularly sensitive to a dietary deficiency of sodium; as the concentration of sodium declines, that of potassium rises and the Na : K ratio in parotid saliva can decrease tenfold or more, providing a means of detecting sodium deficiency in the animal (Morris, 1980). Preparatory mouthwashes can reduce contamination from feed and digesta without disturbing the Na : K ratio. The analysis of Na : K in muzzle secretions can also indicate sodium status (Kumar and Singh, 1981).
The zinc content of saliva has been suggested as a sensitive indicator of zinc status in humans (Henkin et al., 1975), although its diagnostic value is not Blood
Secretions
entirely supported by other investigations (Johnson et al., 1978; Greger and Sickles, 1979) and it has yet to be critically evaluated in domestic livestock.
Subnormal urinary sodium output points to a dietary deficiency of sodium, and high urinary fluoride levels suggest fluorine toxicity. However, high uri- nary fluorine may reflect high previous intakes of the mineral, because uri- nary excretion from the skeletal fluoride stores continues for some time after high intakes have ceased. Interpretation of urine analysis is improved by also measuring indices of dilution (e.g. s.g. or creatinine).
Concentrations of minerals in faeces are influenced by diet and animal factors, but, after prolonged deprivation, they can contribute to a diagnosis (e.g. faecal phosphorus).
Of the body tissues, liver and bone have proved especially useful in anticipat- ing mineral disabilities in livestock, because they are storage organs for certain minerals and because simple sampling techniques by aspiration biopsy or trephine are available. For instance, subnormal concentrations of iron, copper and cobalt (or vitamin B12) in the liver are suggestive of possible dietary deficiencies of these elements. Subnormal concentrations of calcium and phosphorus in the bones can suggest deficiencies of calcium, phos- phorus or vitamin D, and high fluoride levels in bone indicate excess intakes of fluorine. Results may, however, vary widely from bone to bone, with the age of animal and with the mode of expression (fresh, dried, defatted or ashed bone basis), making interpretation difficult (see Chapter 5 in parti- cular). With heterogeneous organs, such as the kidney, results can be affected by the area sampled; for example, selenium concentrations are two- to five- fold higher in the cortex than in the medulla (Millar and Meads, 1988), making precise description of and adherence to sampling protocols essential.
Relationships between mineral concentrations in storage organs and ill health can, however, be poor because stores may be exhausted before any dysfunc- tion or disorder arises (Fig. 3.1).
The analysis of minerals in body appendages, such as hair, hoof, fleece or feathers, for diagnostic purposes has had a chequered history (Combs, 1987).
Relationships to other indices of status, both biochemical and performance, have rarely been sufficiently precise. There are a number of reasons:
exogenous surface contamination; the presence of skin secretions (e.g. suint in wool); failure to standardize on a dry-weight basis; and variable time periods over which the sample has accumulated. The value of analysing material from body appendages can clearly be increased by the rigorous washing and drying of samples. Historical profiles can be obtained by dividing hair and core samples of hoof into proximal, medial and distal
Urine and faeces
Tissues
Appendages
portions. Alternatively, the sampling of regrowth from a recently shaved site (e.g. a liver biopsy site) can give a measure of mineral status to compare with contemporary diet, blood or tissue samples.
Advances in assay procedures for enzymes and hormones have greatly increased the range and sensitivity of the diagnostic techniques now available. Serum assays for vitamin B12 rather than cobalt, triiodothyronine (T3) rather than protein-bound iodine, ceruloplasmin rather than copper and GPX rather than selenium now provide alternative indicators of the dietary and body status of these elements. These newer assays brought with them fresh problems of standardization, which were only slowly recognized, and their use still does not invariably improve the accuracy of diagnosis. The diagnostic strength and limitations of these and similar estimations are considered later, in the chapters dealing with individual elements and their interrelations. Secondary changes in serum enzymes, arising from tissue changes or damage due to mineral deficiencies or toxicities, also have diagnostic value, but caution is still necessary.
Chronic exposure to mineral excesses leads to a sequence of biochemical changes which is, in some respects, a mirror image of events during depriva- tion (compare Fig. 3.3 with Fig. 3.1). Firstly, there is accretionat storage sites;
secondly, levels in transport pools may rise; thirdly, dysfunction may be manifested by the accumulation of abnormal metabolites or constituents in the blood, tissues or excreta; and, fourthly, clinical signs of disorder become visible. For example, marginal increases in plasma copper and a rise in serum aspartate aminotransferase (AST) precede the haemolytic crisis in chronic copper poisoning in sheep; the latter is indicative of hepatic dysfunction.
However, increases in AST also occur during the development of white muscle disease, caused by selenium deficiency, because the enzyme can leak from damaged muscle as well as liver. Assays of glutamate dehydrogenase (found only in liver) and creatine kinase (found only in muscle) are of greater value than AST in distinguishing the underlying site of dysfunction. The whole sequence in Fig. 3.3 can become telescoped during acute toxicity.
There will be similar marginal bands of uncertainty when assessing mineral excesses, as when deprivation is assessed.
Successful procedures for the prevention and control of all mineral deficiencies (and many mineral toxicities) have been developed. The procedure of choice varies greatly with different elements, climatic environ- Functional forms and indices