Trace Mineral Analysis of Hair Samples of Dogs and Cats: A Review of Research, Clinical Applications, and Analytical Procedures

INTRODUCTION

This “white paper” is provided to pet owners, breeders, and veterinarians as an educational resource about the clinical uses of and supporting research for hair analysis in dogs and cats. It will survey the history and uses of hair analysis, detail the strengths and limitations of hair as a sample for health screening, review the substantial body of medical and veterinary literature on the subject, and explain the technical and quality assurance components of accurate laboratory testing and reporting. This document provides referenced support for the hair testing provided by PetTest.

HAIR AS A MEDIUM FOR ANALYSIS

For some pet owners and breeders, the words “hair analysis” conjure up images of testing adolescents or job applicants for drug use. However, long before parents and employers began to use hair analysis in this way, the test had earned the respect of clinicians as a cost-efficient, easy-to-use methodology for getting a representative picture of what is happening inside the human or animal body. Indeed, the first recorded use of hair analysis for these purposes occurred over 200 years ago when it was used to measure arsenic in the body. [i] Hair analysis continues to provide forensic evidence in the courtroom [ii] and historical insight for researchers. Hair has been examined by investigators looking for the causes of Napolean’s death (arsenic, it seems) and the erratic behavior of President Andrew Jackson (possible mercury and lead poisoning). Hair has been admitted as evidence in criminal trials to place defendants at crime scenes or exonerate them. Because of the stability of hair and its deposits of chemical compounds from the bloodstream, hair resists the decay that occurs within days and even hours with other diagnostic tissues, such as blood and urine, when they are not correctly preserved. The cases of Napoleon [iii] and Andrew Jackson [iv] are excellent testimony to the remarkable durability of this tissue. Historical research, forensic studies, and screening for illegal substances are not the only effective applications of hair analysis. However, these uses underscore the important benefits of screening for compounds based on their deposition in hair. • Hair is easily collected and maintains its structural integrity over long periods of time • Hair is composed of minerals and other compounds circulating in the blood and, thus, reflects the cumulative deposit of these minerals and compounds • Because of its growth patterns, hair can offer a more extensive history of the body’s metabolism than other specimens. Hair can pinpoint toxic burden or mineral deficiencies even if the present levels in blood or urine show no indication of them As modern laboratory techniques have evolved, it has become possible to determine smaller and smaller concentrations of minerals and compounds in the hair. These technical improvements have allowed clinicians to detect not only gross abnormalities indicative of obvious pathology but also more subtle indications of imbalances that can accumulate over time and lead to the development of disease in the future. As such, hair analysis as a health screen now offers an important predictive benefit that blood and urine cannot provide. Numerous scientific studies have supported the utility and reliability of hair analysis. Reports by independent international agencies, including the Global Environmental Monitoring System and the International Atomic Energy Agency, have also provided published support for hair analysis as a reliable means for determining trace minerals levels in humans. In a sweeping review of more than 250 research reports, the U. S. Environmental Protection Agency described hair as “a meaningful and representative tissue” for laboratory measurement of selected nutrients and toxins when specimens are prepared and analyzed properly. [v] In recent years, a growing body of literature has focused on the uses of hair analysis in cats, dogs, and horses. This research builds upon a much larger body of research on livestock and wildlife-related to the absorption of nutrients. [vi] In their 2002 study of racehorses, a Japanese research team, headed by Ryuji Asano of Nihon University, observed similar levels of minerals in the hair of humans and horses (including arsenic, aluminum, lead, cadmium, mercury, selenium, phosphorus, sodium, potassium, calcium, magnesium, iron, copper, zinc, magnesium, nickel and manganese). The research team offered a wide-ranging endorsement of hair analysis in nutritional monitoring: The levels of trace elements in mane hair of horses can be used to assess diseases, metabolic disorders, and nutritional status because sampling and storage of hair are easier than those for other biological materials. [vii] In one of several recent studies of minerals in dog and cat hair, John A. Lowe and Julian Wiseman emphasized the value of hair analysis in determining the bioavailability of zinc from different sources: In the adult dog, which is normally considered indifferent to a wide variations in Zn [zinc] supply, Zn deposited in hair, which combines the effects of Zn on cell differentiation (hair growth) and the concentration of zinc in hair (reflecting an increase in Zn storage), should be considered as a sensitive assay for the determination of RB [relative bioavailability] of Zn from different dietary sources under controlled conditions. [viii]

HAIR AS A SPECIMEN

Scores of researchers in human and animal studies have remarked on the diagnostic advantages of hair. Hair is an extremely stable and sensitive specimen. Mineral concentrations in hair can be several times higher in hair than in blood or urine – for some, as much as 200 times higher. Along with technical advances to detect smaller concentrations, this means that hair is more sensitive as a reflection of tissue levels, and laboratory analysis can yield greater precision. Hair also provides a wider window of information about mineral status, reflecting the cumulative body stores of many minerals over time more accurately than blood or urine. Ingested minerals and compounds circulate in the blood until they are deposited or eliminated, and blood levels reflect the acute status of such toxic and nutritional minerals as mercury and zinc for only a few hours. As these analytes are processed for excretion, they enter the urine, which then shows mineral levels over about 24 hours, reflecting the clearance of unabsorbed and mobilized compounds. Hair, on the other hand, stores these minerals in much the same way as do organs and solid tissues, such as bone, kidney, liver, and brain. Each specimen type has its advantages, and they are often used as complementary components of a comprehensive diagnostic evaluation of mineral status. There are also limitations to each sample type: no tissue provides complete representative results for every one of the minerals found in the body. Blood is the standard for measuring acute environmental exposure to toxics and current mineral availability. Urine is superior for analyzing the clearance of toxins and depletion of nutrient minerals in response to detoxification therapies. Hair, in contrast, averages out these short-term variations and offers a better indication of the mean content of the body over a longer period. Writing for the Environmental Protection Agency’s Biological Monitoring of Toxic Trace Elements, researcher D. W. Jenkins summarized the differences between different tissue types: milk, urine, saliva, and sweat measure the component that is absorbed but excreted. The blood measures the component absorbed and temporarily in circulation before excretion and storage… The hair, nails, and teeth are tissues in which trace metals are sequestered and stored. [ix] Since these specimen types reflect mineral status differently, the correlations to be found among hair, blood, and urine measures are not necessarily direct. Minerals are deposited in different tissues at different rates and in different ways, and any comparison of data from analysis of different types of specimen analysis should bear these differences in mind. The variations reflect differences in time-span and sources, and each specimen should be evaluated based on its applications in specific situations. As a rule, hair analysis reveals nothing definite about acute excesses or deficiencies or the rate of elimination; it does, however, provide important information about longer-term excesses and deficiencies stored in tissue that could have a cumulative effect on health and provide indications of the early development of diseases that may be prevented or diminished in their severity with timely and appropriate interventions.

HAIR AS A HEALTH SCREEN IN HUMANS AND LABORATORY ANIMALS

A large body of research using humans and laboratory animals has correlated the results of hair analysis to detect both deficiency and excess of trace minerals with diseases. Results of hair analysis have been repeatedly associated with long-term exposure to toxins and with detoxification ability, and, furthermore, they have been linked to detrimental effects on health and development. Hair analysis also provides information about chronic mineral deficiencies and malabsorption patterns that develop to impact health over time. PetTest has posted on its web site () a detailed survey of research, Published Studies Which Document a Correlation Between Hair Chemical Status and the Chemical Status of Other Body Tissues and Body Fluids. This survey includes many of the toxic and essential minerals measured on the Health Screen for cats and dogs and analyzed in the accompanying Interpretation or “Bob’s Page.” In 2001 Bob Smith also co-authored a laboratory “white paper” survey of over 190 studies related to hair. [x] The volume of human and laboratory animal studies and correlations is impressive and even intimidating, including such highlights as these – • Toxic exposures in hair test correlated with exposures to aluminum in radiographers, [xi] patients receiving an antimony-containing drug, [xii] arsenic contamination of groundwater, [xiii] cadmium in people living near a battery factory, [xiv] chromium in tannery workers, [xv] lead from motor traffic in roadside vendors, [xvi] mercury in fish consumed by residents of a nearby fishing village [xvii] and nickel in people living near contaminated soils. [xviii] • Nutrient mineral levels in hair correlated with calcium in the aorta, [xix] copper in plasma among those with lactovegetarian diet, [xx] markers for diagnosing iron deficiency, [xxi] magnesium in serum and urine, [xxii] selenium in serum and hair among people with selenium deficiency [xxiii] and low intake of bioavailable zinc and plasma levels. [xxiv] • Nutrient and toxic mineral levels in hair correlated with common diseases, including increased death rates from breast and lung cancer (cadmium and mercury), [xxv] risk of coronary heart disease (calcium), [xxvi] gestational diabetes (chromium), [xxvii] tumor malignancy (copper), [xxviii] ADHD (lead), [xxix] myocardial infarction (calcium/magnesium ratio, magnesium, copper, strontium and barium), [xxx] male fertility problems (mercury), [xxxi] breast cancer (selenium) [xxxii] and non-insulin dependent diabetes (zinc). [xxxiii]

HAIR AS A HEALTH SCREEN IN ANIMALS

Livestock and Wildlife: The majority of studies using larger animals have focused on the bioavailability of nutrients from different feed sources for livestock and feed patterns among wildlife. While these studies do not address the concerns of owners and breeders of smaller companion animals directly, they do illustrate the use of hair analysis by researchers in the fields of agriculture, nutritional science, veterinary medicine, environmental sciences, and wildlife biology. A few examples will illustrate these kinds of research efforts: • Studies of two populations of rhesus monkeys (free-ranging and caged) and nutritional status for calcium, magnesium and phosphorus6 and copper, iron, manganese and zinc to delineate differences attributable to diet and environment. [xxxiv] • Correlations between plasma, hair, and hoof samples for zinc, copper, and selenium and signs of clinical deficiencies in Bavarian horses. [xxxv] • Determination of the superior bioavailability of zinc from the chelate over zinc from inorganic salt for pigs and correlations of levels in serum, bone, and hair, as well as physical measurements. [xxxvi] • Correlations of hair levels of zinc, iron, and sodium in white-tailed doe deer to grazing areas. [xxxvii] Dogs and Cats: There is a significantly smaller number of published studies using hair analysis with dogs and cats – and consequently fewer sources for reference values to determine whether they may differ significantly from humans. However, hair analysis is an accepted method for the analysis of toxic exposures and steadily emerging as a useful tool in nutritional analysis as well. Together with the results of research in humans, laboratory animals, livestock, and wildlife, they make up a substantial body of work that can provide important data for interpretation of health screens for dogs and cats. • Similarity of hair mineral values in humans and other animals has been observed in studies of racing horses,7 of fox terriers, schnauzers and mini-schnauzers (for magnesium, calcium, iron, barium, copper, zinc, cadmium, nickel, aluminum, manganese, chromium, titanium and vanadium). [xxxviii] • Effects of toxic exposure to mercury and correlation with age and food source for cats and dogs in Japan, [xxxix] the toxicity for autopsied cats in Sweden of methylmercury in contaminated fish and methylmercury hydroxide added to fish, [xl] confirming low-level dietary inorganic arsenic exposure or poisoning in dogs, [xli] whole-body retention of methylmercury in adult cats, [xlii] correlation between hair lead levels in dogs and cats and distance from source of contamination. [xliii] • Depletion of nutrient minerals zinc, copper and manganese in female dogs treated for lead poisoning by EDTA chelation, [xliv] determining the relative bioavailability of three dietary zinc sources in adult Beagles,8 and hair, serum and various organs of iron, zinc and copper in dogs with induced exocrine pancreatic insufficiency, [xlv] corroborating zinc-responsive dermatosis in dogs with serum and hair analysis. [xlvi]

RELIABLE LABORATORY ANALYSIS: COLLECTION, PREPARATION, AND PROCESSING

Like any laboratory methodology, the quality of hair analysis is directly related to its components. The sample must be collected from a part of the body proximate to where the hair is most representative of tissue composition. The hair should be protected from excessive external contamination before and during collection, as well as washed adequately before analysis to remove contaminants. Finally, the sample must be processed according to the best laboratory practices in a clean environment. Owners and breeders need to select a Health Screen performed by a laboratory that observes high-quality standards at every step of collection, preparation, and processing of samples. Difficulties with hair analysis in past studies and controversy over its utility have arisen primarily from laboratory failure to observe these strictures. This may seem obvious, especially considering the unambiguous statement by the Environmental Protection Agency about reliability of hair analysis,5 but the most widely circulated critiques of hair analysis in humans [xlvii] and animals [xlviii] have been based on the authors’ comparison of labs using vastly different methods and their failure to understand the implications of these strictures fully. PetTest has selected one of the world’s top-rank trace mineral laboratories to perform Health Screen analysis and maintains a close working relationship with the laboratory, including regular monitoring of licensing and proficiency performance and on-site inspections, to ensure continued compliance with the best standards of laboratory practice. Collection: Avoid any hair treatment or shampooing with cosmetic products before collection (if necessary, use a product without additives, such as Johnson & Johnson No More Tears™). Clean hair should be clipped from the brisket (the chest in between the front legs) close to the skin, using clean scissors or very clean clippers. Hair should be collected from 8 to 10 places for a representative sample. The sample, which should include hair no longer than one inch closest to the skin, should amount to at least .25 gram of hair (If the animal’s hair is longer than one inch, keep only the one inch of hair closest to the skin should be kept for the sample.) The sample should be shipped in a clean plastic bag to protect against contamination during transit. Preparation: The lab uses a modified version of the standardized sample preparation method published by the International Atomic Energy Agency (IAEA/RL/50, Vienna). This method [xlix] consists of cutting the hair into .3 cm pieces and mixing to obtain a representative sample, washing the hair three times with Triton X-100 to remove external contamination, and rinsing with acetone and de-ionized water twice. The sample is then digested using trace metal-free nitric acid and temperature-controlled microwave digestion. This method has been demonstrated to remove external contamination yet retain volatile minerals that can be cooked off using other methods such as open beaker digestion. This method of washing hair prior to analysis has also been recommended for animal hair studies.38 Processing: Although such older methods as plasma-coupled atomic emission spectrometry (AES) are still used in many commercial labs (including those surveyed in recent critiques of hair analysis47, 48) the top-ranked trace mineral labs now rely on the far more sensitive and comprehensive technique for multi-analyte measurement of trace minerals, Inductively Coupled Mass Spectroscopy (ICP-MS), which has shown a high degree of concordance with certified values. [l] This method can detect minerals in hair in tiny concentrations, as little as one part per trillion, that may be undetected by AES. The laboratory is licensed as a clinical laboratory according to standards established by the federal Clinical Laboratory Improvement Amendments and the state health departments of New York, Florida, and Maryland. It has scored consistently high on the CLIA-mandated College of American Pathologists, Le Centre de Toxicologie du Quebec, and New York Department of Health proficiency testing programs, and performs inter-laboratory comparisons with other labs that use ICP-MS. The lab has comparable results to other laboratories using ICP-MS, as demonstrated by our successful participation in the comparison program for hair analysis by ICP-MS offered by Le Centre de Toxicologie du Quebec. In addition to proficiency testing programs, trace mineral labs should have extensive Quality Control processes that ensure precision and reliability, which include calibration verification and monitoring standards; preparation blanks; laboratory controls and reference materials (low, medium, and high controls); spiked samples and duplicate analyses. The lab where these health screens are processed uses a state-of-the-art laboratory clean room specially designed for trace mineral analysis, which features metal-free walls, floors, and ceilings, ultra-pure water, and HEPA filtration systems.

CONCLUSIONS

Collected, prepared, and processed correctly, samples of hair from cats and dogs can guide owners and breeders in helping their animals enjoy longer and healthier lives. Based on literature sources, human analogs, and clinical observations, PetTest has created working reference range values to evaluate levels of toxic and nutritional minerals in the hair of cats and dogs. Hair analysis is a Health Screen for gauging an individual animal’s mineral status to identify: • Signs of disease that could develop if not treated preventively • Deficiencies in diet that can be corrected by selection of more absorbable nutrients and supplements to provide optimal nutrition • Presence of toxic burden that could be causing idiopathic symptoms, contribute to development of disease or interfere with reasonable physical and neurological development • Indications of sub-optimal breeding potential • Response to treatment and dietary changes In some cases, Health Screen results will suggest follow-up examination by a veterinarian or further laboratory analysis of other tissue samples to confirm suspected imbalances and sub-clinical illnesses. PetTest in no way intends to misrepresent the clinical application potential of its Health Screens. Invasive or potentially harmful interventions should never be based on the results of hair analysis alone.