Appropriate Methods of Diagnosing Mineral Deficiencies Jeffery O. Hall, D.V.M., Ph.D., Diplomat A.B.V.T. Associate Professor and Head of Diagnostic Toxicology Utah Veterinary Diagnostic Laboratory Department of Animal, Dairy, and Veterinary Sciences College of Agriculture Utah State University Introduction responsive correction of some other clinical condition. Many minerals have been proven in research studies to be essential for optimal growth, An individual mineral may have multiple physiologic function, and productivity in means of measurement for identification of ruminants. Historically, testing for these deficiencies, but most have one that is more minerals has been performed on diets and/or specific than the others. For example, dietary dietary components to ensure adequate concentrations may or may not be reflective of concentrations of specific minerals in the diet. the amount of bioavailable minerals. Or, an However, general mineral analysis does not individual tissue concentration may or may not identify the chemical forms of the minerals, reflect functionally available mineral which can dramatically alter their bioavailability concentrations at the target or functional site. and utilization. The age of the animal being tested also is Although not possible for some of the important for proper interpretation of mineral minerals, the most specific means of diagnosing status. For example, feti accumulate some a mineral deficiency is by testing an animal for minerals at different rates during gestation, unique functional deficits or deficiencies of necessitating adequate aging of the fetus for specific mineral containing compounds or interpretation. In addition, some minerals, for enzymes. This type of testing is often which little is provided in milk, accumulate at impractical from a field perspective, due to higher concentrations during gestation in order to individual test costs or rigorous sample handling provide the neonate with adequate body reserves requirements. But, when possible, this type of for survival until it begins grazing. This is testing eliminates the need to know the specific especially prevalent with copper, iron, selenium, molecular characteristics of a dietary mineral and and zinc. Thus, the normal range for these the potential of interactions with antagonistic minerals would be higher in early neonates than minerals. For minerals that do not have in an adult animal. identified physiologic indices for which testing can be performed, direct quantification from When individual animals are tested, the prior animal tissues or serum may provide a reliable health status must be considered in interpreting indication of adequacy or deficiency. mineral concentration of tissues. Disease states can shift mineral from tissues to serum or serum Mineral deficiencies can be suggestively to tissues. For example, diarrhea can result in diagnosed by the development of clinical disease significant loss of sodium, potassium, and or by post-mortem identification of tissue calcium from the body. Acidosis will cause lesions. However, proof of deficiencies often electrolyte shifts between tissues and circulating requires analytical verification since most do not blood. It is known that infectious disease, stress, have very unique clinical signs or lesions. In fever, endocrine dysfunction, and trauma can some instances, circumstantial proof of a alter both tissue and circulating serum/blood deficiency can be provided by positive response concentrations of certain minerals and to supplementation of the suspect mineral. But, electrolytes. Thus, multiple animal evaluations positive response may have nothing to do with are much more reflective of mineral status within the supplementation and may be just a time a group than testing individual animals that are ill or have died from other disease conditions. 21 2005 Mid-South Ruminant Nutrition Conference This paper is directed at the animal testing Post-Mortem Animal Sampling side of diagnosing mineral deficiencies and provides a summarization of the most commonly A variety of post-mortem animal samples utilized tissues and fluids that are used for are available that can be analyzed for mineral diagnosing specific mineral deficiencies in content. The most common tissue analyzed for animals. mineral content is liver, as it is the primary storage organ for many of the essential minerals. Live Animal Sampling In addition, bone is used as the primary storage organ for calcium, phosphorous, and magnesium. A variety of samples are available from live Other post-mortem samples that can be animals that can be analyzed for mineral content. beneficial in diagnosing mineral deficiencies The most common samples from live animals are include urine and ocular fluid. serum and whole blood. These samples are adequate for measurement of several minerals; Post mortem samples should be stored frozen but it must be recognized that some disease until analyzed to prevent tissue degradation. If states, as well as feeding times, can result in samples are to be analyzed within 1-2 days, they serum fluctuations. Other common samples can be stored under refrigerated conditions. from live animals include liver biopsies, urine, and milk. But since milk mineral content can Calcium vary through lactation, vary across lactations, and be affected by disease; it is not typically Analysis for calcium deficiency falls into two used to identify deficiencies. distinct classes. The first of which is metabolic calcium deficiency, often referred to as milk Serum should be separated from the red/white fever. The second is due to a true nutritional blood cell clot within 1 to 2 hours of collection. deficiency which is associated with long term If the serum sets on the clot for long periods of dietary calcium deficits. time, minerals that have higher intracellular content than serum can leach into the serum and Analysis for metabolic calcium deficiency is falsely increase the serum content. Minerals for aimed at detection of low systemic or circulating which this commonly occurs include potassium calcium content. In live animals, testing is and zinc. In addition, hemolysis from both performed on serum to determine circulating natural disease and due to collection technique calcium content. However, in dead animals can result in increased serum concentrations of testing is more difficult, as serum collected post- iron, manganese, potassium, selenium, and zinc. mortem will not accurately reflect true serum calcium content prior to death. But, circulating The best type of tube for collecting serum or serum calcium content can be approximated whole blood is royal blue-top vaccutainer tubes, from analysis of ocular fluid, with a vitreous to as they are trace-metal free. Typical red-top clot serum ratio of approximately 0.54 (McLaughlin tubes will give abnormal results for zinc content and McLaughlin, 1987). The Utah Veterinary as a zinc containing lubricant is used on the Diagnostic Laboratory has been able to confirm rubber stoppers. For minerals other than zinc, and disprove the potential of clinical serum samples from the typical red-top clot hypocalcemia in numerous post-mortem cases tubes are adequate. via vitreous fluid analysis. Samples should be appropriately stored for True, nutritional calcium deficiency is preservation. Liver biopsies, urine, and serum associated with weak, poor doing animals that can be stored frozen long term or refrigerated if have swollen joints, lameness, weak bones, and a analysis is to be completed within 1-2 weeks. propensity for broken bones (Puls, 1994). Whole blood and milk should be refrigerated, but Analytical verification of calcium deficiency not frozen, as cell lysis or coagulation of solids requires analysis of bone, since approximately will result. 98-99% of the body calcium content is in bone and serum concentrations are maintained by both diet and turnover of bone matrix. The bone analysis should be performed as fat-free, dry 22 2005 Mid-South Ruminant Nutrition Conference weight to remove the age variability of moisture animals to have normal serum concentrations. and fat content. At the Utah Veterinary Diagnostic Laboratory, it is commonly recommended that 10% of a herd Cobalt or a minimum of 10-15 animals be tested in order to have a higher probability of diagnosing a copper deficiency via serum quantification. Cobalt deficiency is associated with Even with a severe deficiency in a herd, low deficiency of Vitamin B12 (cobalamin) in serum copper concentrations may be seen in as ruminants. Deficiency is associated with few as 20% of the individuals tested. Herds that decreased feed intake, poor feed conversion, may be classified as marginally deficient based reduced growth, weight loss, hepatic lipidosis, on liver testing may have predominantly normal anemia, immunosuppression, and impaired serum copper concentrations. Thus, serum reproductive function (Graham, 1991; Puls, copper analysis should be viewed as a screening 1994). Cobalt deficiency can also lead to method only. Another factor that can influence subnormal copper retention in the liver. diagnosis of copper deficiency in serum is the presence of high serum molybdenum. As the Tissue and serum concentrations of cobalt are copper-sulfur-molybdenum complex that forms generally quite small, as the B12 is produced in is not physiologically available for tissue use, the rumen by the microflora. Since cobalt normal serum copper content in the presence of concentrations may not truly reflect the B12 high serum molybdenum