Hair mineral analysis has been used in forensics for decades. Today, hair analysis is gaining widespread recognition as an analytical tool for receiving information about mineral patterns and drug abuse. Hair analysis is a valid analytical technique that provides important answers to puzzling historical questions, including Beethoven's habits. Nearly 170 years ago, an admirer (grieving the death of Ludwig van Beethoven snipped a lock of the great composer's hair for a keepsake and kept it in a locket. It is this strand of hair that is expected to provide key answers. Did the deaf composer use drugs? Was he suffering from syphilis? Did he die of arsenic poisoning or was his health affected by mineral deficiencies? Today's sophisticated analytical methods can provide these and other answers, and explanations are often unexpectedly simple. For instance, during Beethoven's time, mercurial drugs were used to treat syphilis and arsenic was used to kill rodents. In minute doses, arsenic was deliberately taken to increase virility and physical strength. Other toxins such as lead were ingested by drinking lead-containing water, causing, a host of neurological and behavioral problems such as Beethoven's feared moodiness and ill-tempered conduct. The 582 strands of hair recently auctioned off at Sotheby's are expected to provide important information regarding Beethoven's biochemical makeup and its link to behavior. 

Researchers William Walsh and Ronald Isaacson have been studying the relationship between body chemistry and behavior for decades. They have published an Impressive amount of data, including the relations between toxic elements and hyperactivity. They recognized that heavy metal exposure is higher in people prone to violent behavior and that a specific pattern of toxic exposure and mineral deficiencies is seen among death-row inmates. The researchers also noticed what they considered a "genius pattern," characterized by extraordinarily high levels of copper and sodium but low zinc levels in hair. Individuals with this type of hair mineral pattern are often highly intelligent and a bit eccentric, Walsh said. The scientists documented that hair mineral analysis is a valid test of body mineral concentration when used appropriately. "Hair is a dairy of what is going on in your body," Isaacson said. After decades of studying chemicals in hair an associating mineral patterns with behavior, the researchers opened the HRI Pfeiffer Treatment Center seven years ago. It aims at treating biochemical problems, and a strand of hair often reveals the cause of psychiatric ailments that did not respond to other more conventional treatment. 

Hair analysis has been utilized for decades, and one of the principal factors in hair analysis is the accessibility of hair to the external environment. Contamination can occur from air, water, perspiration, shampoos, dyes and other hair preparations, however, washing techniques utilized by laboratories alleviate these problems. Hair analysis is a ideal complement to serum and urine as a diagnostic tool. Hair is collected without trauma, an important point when it comes to the toxic screening of children or the frail. I many cases when mineral deficiencies have been noted, the individuals inadequate mineral status may not be solely due to a mineral deficient diet, but also be compounded by digestive problems that cause inadequate mineral absorption. 

A review of over 1400 articles indicate that hair is the prime tissue to be utilized when analyzing for heavy metal and other trace mineral concentrations. Most notable results have been obtained on heavy metal pollutants such as lead, arsenic, cadmium and mercury. Scientists in the United States, Canada, Germany, Japan and Sweden have all shown that elemental concentrations in hair provide a relatively permanent record of exposure an that there is good correlation between concentrations in human hair and certain organs. 

Chattopadyay of Dalhousie University reported during the Second Human Hair Symposium in Atlanta, Georgia, that concentrations of lead in hair were lowest in rural population groups, higher in urban groups and highest in individuals who live close to lead smelters. 

Trace Minerals International, Inc. of Boulder, Colorado compared the toxic content of hair in American, German and Mexican children and found that concentrations were highest in Mexican children, lower in American and lowest in German children. 

Petering and a coworker at the University of Cincinnati College of Medicine have fed heavy metals to animals in measured quantities and monitored hair concentrations in an effort to correlate exposure with concentration. 

Harry Shwachman of the Children's Hospital Medical Center in Boston along with Kopito of the Massachusetts Institute of Technology have shown that children with cystic fibrosis have as much as five times the normal concentration of sodium in their hair, but only about ten percent of the normal concentrations of tightly-bound calcium. 

Shwachman and Kopito have also found low concentrations of sodium and potassium in the hair of patients with celiac disease ( disorder in the digestion and utilization of fat) and that there is generally three to four times as much sodium and potassium in the hair of healthy individuals. This is in spite of the fact that the analysis of sodium and potassium in hair is not considered one of the stronger points of hair analysis, due to instrumentation limitations and difficulties with sample preparation. 

Shwachman and Kopito also demonstrated that hair from victims of phenylketonuria (PKU) contains below-normal concentrations of magnesium and calcium; and that hair from victims of kwashiorkor, a severe protein-calorie malnutrition disease, has markedly increased concentrations of zinc. 

Prasad of Wayne State University has shown that marginal zinc-deficiencies in the diet can be identified by below normal hair zinc concentrations. 

Hambidge of the University of Colorado Medical Center has confirmed this work and suggested that the problem may exist because diets of people with low-income generally provide little zinc. Hambidge has tested children in Denver's Head Start Program and found that both their hair and blood serum contain significantly lower concentrations of zinc than specimens from children of middle-income families. The researcher picked six children with the lowest hair zinc concentrations for further testing and found that taste perception was impaired in five. Zinc supplementation restored taste perception and increased zinc concentrations in both blood and hair. 

Both Hambidge and Walter Mertz of the U.S. Department of Agriculture in Beltsville, Maryland, have individually demonstrated the below-normal hair concentrations of chromium in victims of juvenile onset diabetes. 

Sheard and Carter of the Clinical Chemistry Division of the Center for Disease Control collected data from over 21,000 individuals and developed a standardization technique for measuring chromium in hair. 

Gordus of the University of Michigan reported that the hair of students with high academic marks contained substantially more copper and less iodine, lead and cadmium than the hair of students with low marks. 

Robert Pihl and colleagues of McGill University in Montreal, Canada, report that based on hair mineral results, they can distinguish with 98% accuracy normal children and those with learning disabilities. 

Barlow of the University of Aston in Birmingham, England, and Kapel of the University of Leeds. have observed a relationship between trace element profiles in hair and four different abnormalities. Hair mineral evaluations of three sets of identical twins revealed similar patterns even when one of the siblings lived in a different environment, suggesting that the metabolic pathways of minerals and trace elements are as similar as has been found with other diagnostic procedures. 

Brain and hair tissues of Alzheimer patients have been found to contain substantially-elevated aluminum concentrations and researchers at the National Institute of Health in Bethseda, Maryland, and at the Elizabeth Hospitals in Washington, D.C. noticed an improvement in symptoms after hair aluminum decreased. 

Elemental concentrations in organs are not identical, but certain elements are more densely concentrated in specific organs a better understanding of the biochemical pathway and pathophysiology of elements improves the diagnostic and therapeutic a limitations of trace element analysis. Hair mineral analysis evaluates tissue storage and is in many cases the best choice when chronic exposures and deficiencies are suspected. As was stated by M. Laker, "HMA is an excellent, simple, and accurate test to establish mineral and trace element concentrations. Since the structure of hair remains unchanged, the minerals and trace elements are fixed, whether a sample is tested now or in a few years time. The levels are not subject to change." 

Sampling hair is a simple procedure, but test results greatly depend on using the correct procedure. Coloring, bleaching and perming hair permanently alters the structure and negatively influences the analysis, causing falsely-elevated test results. For example, black hair coloring agents may contain manganese, lead and or other elements, while red dyes may result in elevated copper or iron levels. Bleaching agents drastically change the hair shaft and cause falsely elevated calcium and magnesium results. Generally, a report that shows overall high levels is not a reflection of toxicity, but indicates chemical treatment of hair. Certain shampoos, colors, and conditioners contain relatively large amounts of certain trace elements which penetrate the hair and cannot be removed by a laboratory's washing procedure. Some laboratories have choosen not to wash hair samples prior to analysis, a highly- questionable practice that leads to falsely-elevated results. Clinicians interested in hair analysis must investigate laboratories and ask pertinent information about sample preparation. It is only logical that test results of unwashed hair exposed to sweat would provide falsely elevated sodium results. 

Note: If hair has been subjected to multiple treatment (bleaching, coloring, perming) and the patient is not willing to wait until sufficient untreated new growth is available, it is best to use pubic hair or nails as a sample material. 

Procedures to follow:  

1. Hair does not require washing before collection since each sample will be washed meticulously at the laboratory with deionized soap. Surface residues, such as spray, conditioner, and dust are removed during this process. 

2. To cut a sample, use a hair dresser's shaving blade when collecting very short hair. Use normal scissors for hair of 5 cm (2 inches) or longer. 

3. Pin hair up at the back of the head. 

4. Carefully cut several strands of hair. Cut narrow strands to avoid visible gaps. 

5. Cut off ends if hair is very long; only keep strand at a length of up to 10 cm (about 6 inches) for analysis. 

6. Repeat procedure higher up, until enough hair has been obtained. 

7. Write all patient information and needed details on sample envelope. 

How to collect Beard and Pubic Hair:  

Both facial and pubic hair should be cut as close to the skin as possible. Pubic hair from female patients should be collected from the upper part of the pubic hair line. Axillary hair is unsuitable for analysis. 

Samples for Re-testing or Control:  

It is advisable that the sample be collected from the approximately the same area. For example, if the sample has been collected from the nape for the first analysis, it is advisable to collect again from the nape. 

Laboratory Preparation:  

Any sample that reaches the laboratory is first numbered and recorded. After that, the hair sample is washed in a special solution and rinsed several times with deionized water. After the sample has been dried, it is weighed and dissolved in nitric acid. The solution obtained is then diluted with de-ionized water and tested with control solutions by atomic emission spectrophotometry (ICPAE) or mass spectrometry (MS). The emission occurs at approximately 10,000 degrees Celsius, and the accuracy of the analysis is monitored after five to ten tests by comparing test results of known standard solutions. Test results are immediately entered and analyzed by computer, and all reports are checked and approved by competent personnel. 

Nail analysis has been used in forensics for the evaluation of severe arsenic poisoning when hair loss prohibited hair mineral analysis. Nail analysis is also used when untreated hair is not available in sufficient quantity. Finger or toenails may be used as a testing material. but nails must be free of varnish or polish. A minimum of 200 mg of nails Is needed for analysis. 

Reference Values  

1. Definition of Normal  

In medicine. the definition "normal" has several meanings. It used to distinguish a "normal" or healthy person from the abnormal or unhealthy individual, and in this context, we refer to "normal" iron levels as values that reflect good health, in fact "normal" iron concentrations can be found in the presence of disease. In the absence of disease or disease symptoms, a person is medically and legally considered normal or healthy however, a person without symptoms of disease does not necessarily enjoy even optimal health. In the laboratory, "normal" is used to describe a set of laboratory results that is based on statistics. For many analytes such as serum iron or aluminum, whole blood lead or urine mercury, reference levels have been established by the Center for Disease Control (CDC). For other analytes less known in conventional medicine, including the important blood chromium or urine nickel, reference values have not been standardized. This applies for most elemental reference ranges in hair, with arsenic being one exception. In some cases, instrument sensitivity and detection limits dictate that in spite of existing reference ranges, a new set of values needs to be established. For instance, while ICP-MS provides detection limits in the PPB range traditional ICP-AES measurement of blood lead concentrations are difficult due to instrumentation limitations. However, the experienced analytical chemist can statistically and mathematically correct some of these problems by carefully evaluating the known concentrations of standard solutions. Thus, the establishment of a specific set of reference values that is based on existing laboratory conditions and variables provides a valid rationale for establishing "laboratory-specific" reference values. While the "normal" range of such "laboratory-specific" reference value deviates from official or otherwise used "normal" ranges, analytical data that is outside that range reflects abnormal conditions 

When no reference values are officially available or recommended by the CDC or other government agencies, the laboratory has the responsibility to establish reference ranges. Since 1994, All laboratories engaged in testing human samples have to provide licensing inspectors with sufficient statistical data that evidences the statistical and mathematical basis which led to the establishment of a specific set of reference values, The only difference is that 'established' reference ranges have demonstrated to define "normal" for a longer period of time, and more clinical data is available to support them. 

2. Statistical Basis:  

For the establishment of reference ranges, a Gaussian distribution is generally used, although additional statistical and mathematical measurements are taken into account. 

3. Detection Limits  

The detection limit is defined as the concentration of an element or compound that produces an instrumental signal that is acceptably greater in intensity than the instrumental noise (or background) level. The instruments used at TNU are the inductively-coupled plasma mass spectrometer (ICP-MS) and the inductively-coupled plasma atomic emission spectrophotometer (ICP-AES). The ICP-MS is a state-of-the-art instrument designed to provide quantitative measurements in the parts-per-trillion range. The ICP-AES does not provide such low detection limits, but is more than sufficient for many applications and better suitable to test elements that are present in higher concentrations. 

Bibliography:  

Blaurock-Busch E.; Mineralstoffe und Spurenelemente und deren Bedeutung in der Haar Mineralstoff Analvtik. Biol Arb und Forschkr, 1984. 

Blaurock-Busch E.; Confirming the Biochemical Identity of Identical Twins using Mineral Analysis. 

Versieck J., Cornelis R.; Trace Elements in Human Plasma or Serum. CRC Press 1989. 

Kaplan A.L. Pesce A.J.; Clinical Chemistry, 2nd ed. Mosby Co. 

Thomas L.; Labor und Diagnose. 4.ed. Med Verlagsges Marburg, 1982. 

Valkovic V.; Human Hair Vol I & II. CRC Press 1988.