Features of Hair Research for Biological and Forensic Chemical Research

Pavlova Al'bina Zakharovna Doctor of Medicine Researcher, Russian Center for Forensic Medical Examination 101000, Russia, Moscow, Polikarpova str., 12 himija@rc-sme.ru Kalekin Roman Anatol'evich Doctor of Pharmacy Researcher, Russian Center for Forensic Medical Examination 101000, Russia, Moscow, Polikarpova str., 12 himija@rc-sme.ru Orlova Alevtina Mikhailovna PhD in Pharmacy Researcher, Russian Center for Forensic Medical Examination 101000, Russia, Moscow, Polikarpova str., 12 himija@rc-sme.ru Volkova Alla Andreevna PhD in Pharmacy Researcher, Russian Center for Forensic Medical Examination 101000, Russia, Moscow, Polikarpova str., 12 himija@rc-sme.ru

Larev Zakhar Vasil'evich Senior Lecturer, Chuvash State University 101000, Russia, Cheboksary, Moskovsky Ave., 45 himija@rc-sme.ru

IntroductionHair is one of the metabolically active and informative tissues, and in terms of the speed of metabolic processes it occupies the second place after the bone marrow.

One of the defining features of hair is its chemical structure: they belong to the family of homologous tissues of epidermal origin, solid alpha-keratins; the main part is protein.

In the hair, there is a root immersed in the skin and a rod above the surface of the skin. In the root, in its lower part, the so-called matrix, there is an area of active cell division and hair growth, which surrounds the hair follicle, through which the hair is nourished and innervated. In its development, the hair goes through the phases of active growth – anagen and rest – catagen and telogen. The active phase lasts on average 1000 days. In this phase, the cells of the hair papilla are activated, DNA and RNA synthesis increases in them, melanin is actively produced in the melanocytes of the matrix. By the end of the anagen – metanagen phase, the cells of the matrix and the inner root vagina begin to keratinize and the hair enters the catagen phase. This phase lasts about 100 days and it persists until the appearance of a hair above the surface of the skin. The obsolete hair is pushed out by the growing hair – the telogen phase. New hair reaches the surface of the skin at the 3rd week. The daily lengthening of the hair on the head averages 0.37 mm / day, during the month – 1.5–3.2 cm. When forcibly separating the hair, the configuration of its root and the pigment content depend on the phase of hair growth. In the faces of fair–haired (fair–haired, light-haired, dark-haired) - blondes, the amount of pigment is less than in dark-haired (black) - brunettes, where the hair is completely filled with black pigment. Depending on the growth phase and the initial color of the hair, the quantitative content of pigment in the bulb and in the hair shaft changes.

Recently, in connection with the development of science and the improvement of technical equipment, the study of hair has entered into widespread practice in order to establish the causes of the disease, prescribe treatment, study the environment; put forward the need to study a wide range of metabolic, trophic, mental and other diseases. It turned out that the first symptoms and signs of the disease can manifest in the hair: its color, shape, structure, growth pattern, content and composition of enzymes, hereditary factors and others. To identify these changes, serological, enzymatic, morphological, elemental, genetic and other components of the hair are studied. The complete composition of the elements throughout the hair can be as unique for each individual as fingerprints.

In forensic investigative practice, hair is often removed from the scene of an accident for investigation, forensic doctors are involved to resolve issues that have arisen. Due to the peculiarity of the structure and function of hair (they do not enter into feedback with the body and, as it were, are an archive of human metabolic processes), their importance increases. For their work, forensic experts have developed methods of seizure, sequence and technique of conducting research.

However, there are no such developments for clinicians and toxicologists, even forensic chemists. The condition of hair removed from diseases and poisoning is often studied without observing the rules for their removal, studying the morphological structure (length, presence of a core, filling the hair thickness with a toxicant, indicating magnification of the microscope, and others); taking into account the stage of hair growth, the nature of mechanical damage. The diagnosis is often made on the basis of the presence of "black pigment", "fusiform bulb", "flask-like bloating". All this leads to an incorrect interpretation of the results of the hair study

For forensic doctors, criminologists, anthropologists, hair is an object for identifying a person, race, determining the causes of poisoning, elemental composition, and others ^[1].

Given the current relevance of treatment with biologically active additives in the clinic, it is necessary to clarify the quantitative value of the body's need for individual elements. It is known, for example, the content of commonly used elements for the treatment of cancer: selenium, lithium and others.

Hair as an object of research is very interesting from the point of view of narcology and forensic toxicology. Hair analysis for the purpose of determining narcotic, psychotropic, potent poisons and medicines is one of the most dynamically developing areas of forensic chemistry, both in terms of developing methodological approaches to analysis and in terms of interpreting the results ^[2,3]. Forensic chemical and chemical-toxicological analysis of hair is considered a standard method for detecting chronic alcohol consumption and psychoactive substances, since information about their use is stored in the hair indefinitely for a long time. The most promising methods are chromato-mass spectrometry and tandem mass spectrometry ^[4,5].

No less promising is the study of the types of ?-polypeptides of hair keratin in humans and animals by electrophoretic analysis. It has been proved that 7 anodic ?-polypeptide fractions are determined in humans and higher primates. When examining the hair of representatives of different races, it was noted that in approximately 6% of cases, an atypical phoregram of polypeptides, different from the usual ones, was detected in Caucasians. Analysis of hair and nails in the families of these individuals showed hereditary transmission of these traits as dominant. In the hair of Negroids and Mongoloids, this feature was found much less often. Foreign studies using isoelectrofocusing in acidic PAAG have shown the possibility of using this method in humans to solve the issue of individual hair ownership.

The analysis of the enzymes of the hair root showed the presence of phosphoglucomutase, erythrocyte acid phosphatase, esterase D, fogsfogluconate dehydrogenase in them, inherited genetically. The hair bulb contains an enzyme system that catalyzes the metabolism of a carcinogen – benzopyrene. The activity of epoxide hydrotase and aryl carbohydrate hydroxylase was recorded.

Hair is a biological material in which functional groups of proteins bind to toxic and psychotropic substances. The ability of these substances to dissolve in fats is a critical factor in determining the level of transfer from the bloodstream through the cell membrane to the growing root bulb. There are other ways in which it is possible to receive the drug observed in the hair itself. The drugs may be in the secreted sweat glands and sebaceous gland, which is located in your own skin. The mouth of the sebaceous gland reaches the upper edge of the external root vagina and washes the hair. Sweat glands are located in the subcutaneous fat, the mouth of which comes to the surface of the skin. The substances obtained from the surface of the hair are mainly sebaceous secretions. Their chromatographic fractions consist mainly of free fatty acids and neutral fats – esters, glycerin, paraffin and others. Since these two secretions wash the hair shaft as it grows, this has its effect on foreign inclusions that have entered the structure of the hair. It is advisable to wash off these overlays on the surface of the hair with ethanol, which removes them more actively than benzene, ether, chloroform ^[6-10].

The study of hair for elemental composition, toxic and psychoactive substances from a clinical point of view is fraught with difficulties, such as the lack of the concept of "norm", especially in children. In addition, there is no sequence (algorithm) of withdrawal, construction of research methods and conclusions, which can lead to erroneous conclusions ^[11,12].

Relevance

Currently, despite the large number of studies conducted by forensic medical experts and clinicians, there is no complete, targeted program for the study of hair for elemental composition, psychoactive and toxic substances, enzyme system, in which methods of hair research would be announced not only for forensic, but also for clinical, environmental practice. Forensic doctors have developed methods and techniques for conducting such studies. However, there are no such developments for clinicians, toxicologists. Removal and examination of hair to study the morphological structure is carried out without following the rules (length, description of the structure, indication of magnification of the microscope, characteristics of inclusions and others). The lack of widely available data on the characteristics of hair; by determining the content of foreign substances introduced into the body, gives rise to contradictory and even erroneous data. Standardization of methods would make it possible, for example, to use photon activation to determine the structure of the hair throughout its entire length ^[13,15].

In our practice, diseases of children with unclear etiology, accompanied by alopecia, changes in nervous status, gastrointestinal tract and others were analyzed

The purpose of this study was to develop tactics for removing hair as samples, as well as from the scene of the incident and, the sequence of studying the structure of the hair, to establish the presence of damage, signs of poisoning with toxic substances – chemical elements and their compounds, psychoactive and toxic substances. To this end, we turned to our work experience and literature data.

Materials and methods of researchThe study material was the hair of children with suspected thallium poisoning, presented to us in the form of photographs.

From the descriptive part of the morphological study of the hair, it is known: "the hair roots are thinned; fusiform, the presence of black pigment, there is no hair bag. Pathology"; "Fluffy hair. Mostly normal. Some of them have a thinned root without a bag. There is a small deposit of pigment in a single hair. Partial pathology (new hair)"; "Fluffy hair. Accumulation of pigment. Pathology".

The number of patients with suspected thallium poisoning is 46, aged 9 months to 12 years. The presented hair of the mothers was not studied by us in view of the inexpediency. We studied photographs of 56 hairs in various growth phases, and hair rods. It is known that thallium poisoning is difficult to diagnose. Morphologically, this is manifested by inclusions of black-purple color in the bulb and the hair shaft in the phase of hair growth – anagen. The microstructure of the hair also depends on the color of the hair. We took these factors into account and divided the hair presented into:

1) hair in the anagen phase: with signs of poisoning (fusiform);

2) in the anagen phase (without signs of poisoning);

3) in the phases of catagen and telogen;

4) hair rods.

Of the 46 samples presented, we established pathology in 10 patients according to their morphological structure.

The second direction of our research was to study the hair for the establishment of surfactant – pregabalin in them by special preliminary preparation of the object for the study. Experimental studies were conducted with native hair by exposing them to the toxicant pregabalin.

The results of our own researchIt is known that when forcibly separated in the anagen phase, the hair has a diverse configuration, more often in the form of a hook, and is surrounded by vaginal membranes, contains a pigment that concentrates in the matrix zone.

In the 10 samples studied by us (1-4 hairs), the bulb is represented by a fusiform shape, without vaginal membranes, a black pigment that passes to the hair shaft.  The length of the bulb exceeds the thickness by 3-4 times. This shape of the bulb is inherent in a viable hair, separated not by force. For example, when a toxicant is exposed to the cells connecting the hair shaft with the vaginal membranes in the anagen phase. The toxicant, in particular thallium, entering the hair structure, simultaneously, usually occupies the entire thickness of the hair and, as it grows, moves up the shaft, leaving behind the natural color of the hair structure, which is especially visible in blondes. With intermittent intake, inclusions of black and purple color should alternate with the normal structure of the hair. At the same time, it should be noted that such a high continuous arrangement of pigment on the rod is also characteristic of the black pigmented hair of brunettes. The color scheme of the hair is described as black, whereas thallium forms purplish-black inclusions.

In 20 patients, 32 hairs in the anagen stage were examined. The bulbs contain a black pigment, the amount of which is not the same for different hair – from not significant to significant, passing to the core. This pigment creates the overall background of the hair. In the photos of the hair, probably of blondes, the background of the hair is lighter, and individual formations similar to clusters of pigment and longitudinal striation were visible in them. The described formations in the form of a "black pigment" are also the natural state of the hair. This is evidenced by the intermittent accumulation of pigment in the thickness of the hair and at the root.

Thus, some objects have signs similar to the effects of a toxicant, but it is not possible to assert that they are left by thallium, since the black color of the inclusions depends on the natural state of the hair, the location of inclusions in the thickness of the hair is not characteristic of thallium.

Aging hair (catagen phase, rest) – 11 patients, 14 hairs were examined. The pathology was discovered by researchers in one hair. Such hair is characterized by a flask-like shape of bulbs with remnants of the vaginal membranes surrounding the hair in the form of a "corolla". Pigment accumulations are not characteristic of thallium. These formations are clusters of pigment, or longitudinal striation. Such hair does not carry information about thallium poisoning after the end of the active phase.

Obsolete hair in the phase of telogen, rest – 2, 2 examined. Pathology was identified in one case.

In the resting phase, the hair is not associated with the metabolic process of the body and the toxicant does not enter the hair in these phases.

Pathology of the rod – 3, 6 hairs were examined. Pathology was found in one patient. However, the injuries presented are of a mechanical nature and are not associated with any intoxication. "Flask-like swellings" do not indicate poisoning, they can be formed due to mechanical damage to the hair. The results are presented in table 1.

Table 1. Results of hair research in different growth phases

Table 1. Results of hair research in different growth phases

Note: in the numerator - the number of patients, in the denominator – the number of detected pathology. Recently, there have been reports of the use of hair by toxicologists to determine the psychoactive substances that caused poisoning.

Thus, cocaine was detected in the peripheral and root sections of the hair by tandem mass spectrometry. However, the authors do not provide data on what stage of growth the hair was at and the time of the study. Simultaneous establishment of a toxicant in different parts of the hair indicates a prolonged, intermittent intake, or incorrect withdrawal. The use of the entire length of the hair during mechanical grinding reduces the sensitivity of the study. For a more accurate determination of the presence and quantity of toxicants, it is advisable to establish at least an approximate beginning of its receipt in the anamnesis and take certain areas of hair. To increase sensitivity, it is advisable to expose the internal structure of the hair by destroying its integrity. Of the presented methods, the most objective is the exposure of the internal structure of the hair by crushing it.

Experimental studies were carried out on the hair in order to prove the presence of the toxicant pregabalin in them in compliance with the algorithm of hair removal and the preparation of samples of objects using a technique that does not destroy the integrity of the hair. Based on our practical experience and experimental data, we have proposed a methodological approach to the removal and examination of hair to determine foreign inclusions from the structure of the hair.

Hair analysis can be an addition to the analysis of more conventional biological material during autopsy of a corpse – blood, urine, bile and internal organs, poisoning with heavy metal salts, investigation of causes of death from overdose, chronic drug use.

Hair samples are taken in accordance with the orders regulating the rules for taking biological material for research: Ministry of Health and Social Development of the Russian Federation dated 12.05.2010, No. 346n "On approval of the Procedure for organizing and conducting forensic examinations in state forensic institutions of the Russian Federation" (Registered with the Ministry of Justice of the Russian Federation on 10.08.2010 No. 18111), Ministry of Health of the Russian Federation dated 18.12.2015 No. 933n (ed. dated 25.03.2019) "On the procedure for conducting a medical examination for intoxication (alcoholic, narcotic or other toxic)" (Registered with the Ministry of Justice of Russia on 11.03.2016 No. 41390) and the Ministry of Health and Social Development of the Russian Federation No. 40 dated 27.01.2006 "On the organization of chemical and toxicological studies in the analytical diagnosis of the presence of alcohol, narcotic drugs, psychotropic substances in the human body and other toxic substances."

Hair is selected from different parts of the human body - hair from the head (frontal, temporal, occipital, parietal areas) for comparative research – in cases of murder or suspicion of them, traffic accidents and others. It is advisable to take hair from the armpits. The hair is cleaner there. Hair for toxicological examination is removed together with bulbs and vaginal membranes, dried and placed in separate paper envelopes, or special plastic bags, which are marked, sealed and sealed with the seal of the appropriate unit. Hair should be taken taking into account the time of the onset of intoxication and the rate of hair growth.

Collection of postmortem hair samples includes: drying; decontamination in order to remove surface impurities. Flushing can be carried out according to any method adopted in a particular laboratory. It is necessary to control the flushes.

Currently, the method of high-resolution atomic absorption spectroscopy with electrothermal atomization using a dispenser attachment for the study of liquid samples is used for detection and quantitative determination during elemental analysis. When using atomic absorption and emission absorption methods, the appropriate methodology should be applied. Sample preparation for elemental composition: 100 mg of hair is filled with 3 ml of concentrated nitric acid, 2 ml of hydrogen peroxide. The mixture is run into a microwave system and a decomposition program is started.

If poisoning with narcotic drugs, psychotropic substances and medications is suspected, hair weighing at least 300 mg is taken, the suspension is divided into two equal parts: working and control samples. It is necessary to take a sample of at least 100 mg for the study.

The stages of sample analysis take place without the participation of an operator under the control of the AAC contr AA 700 software. Calibration of the content of elements is carried out according to water standards (Figure 1.).

Figure 1. Thallium calibration graph

Figure 1. Waist calibration graph After establishing the quantitative determination of elements in a biological object, it is necessary to correlate this amount to toxic or lethal doses.

For these purposes, it is recommended to use the scientific and practical sources presented in the list of references.

It is recommended to take into account the following features when conducting a forensic chemical and chemical-toxicological examination of hair samples and interpreting the results of the study: the higher the lipophilicity of the toxicant and the longer its half-life from the body, the more the substance accumulates in the structure of the hair. Amphoteric, highly hydrophilic substances accumulate in small quantities.

The degree of accumulation of toxic substances in the hair depends on their acid-base properties and the presence of pigment (eumelanin and pheomelanin). Substances of acidic nature are evenly distributed, both in pigmented and non-pigmented hair, and substances of basic nature are mainly in hair with a high content of melanin (red, light, black, brown).

Hair with a predominant pheomelanin content (natural redheads, blondes) is more difficult to undergo all types of hydrolysis, therefore it is possible to obtain underestimated results when quantifying the toxicant.

The staining procedure does not lead to an increase in the matrix effect and does not affect the possibility of identification and quantification of toxic substances.

It is recommended to use sample preparation by enzymatic hydrolysis by proteases for retrospective and segmentation analysis of hair. The possibility of retrospective (according to the results of the study, 24 weeks after the last intake) detection of toxicants with the properties of weak acids and a high half-life from the body in quantities comparable to their amount with regular intake (example of phenobarbital) has been established. Such toxicants accumulate mainly in the root segment, that is, as the hair grows.

Retrospective detection of drugs of a basic nature and a low half-life from the body is possible 6 weeks after the last intake, the results of quantitative assessment may be lower than with regular intake (for example, for the presence of diphenhydramine hydrochloride, tropicamide, gamma-aminobutyric acid derivatives). Toxicants with these properties accumulate to a greater extent in the terminal segment, that is, during the short period of time while it was circulating in the blood (blood plasma).

The impossibility of detecting toxicants with a short half-life and highly hydrophilic in the hair with their single intake was determined.

The determination of substances in hair samples from corpses and living persons can be considered a sign of systematic (chronic) use of these compounds. The length of a strand of hair 1.5-2 cm corresponds to about a one-month period of time.

Algorithm of morphological examination of hair into elements: Hair removal.

Hair for morphological examination is removed from five areas of the head of at least 20 hairs. In cases of hair loss (alopecia), loose hair should be collected. Along the edge of the preserved hair, without making much effort, as if stroking, collect the fallen hair, and also pull out a few hairs with force. Note the length of the hair and the time of withdrawal. For control, remove the hair from the intact part of the head, put everything in separate bags. Packages should be labeled and sealed.

Macroscopic examination. A preliminary examination should establish the macroscopic color of the hair in the bundle, that is, on the head, as well as individual hair. In the bundle, the hair is designated as blond, light brown, light brown, dark brown, black, red, gray. Individual hairs are designated as yellow, white, brown, black, gray. According to the investigative data, establish the approximate time of receipt of the toxicant.

M icroscopic examination. At least 15-20 hairs from the alopecia focus should be examined, and the structure and features of each hair should be described according to the scheme used by forensic experts of the physical evidence research department. Thallium is characterized by compact clusters of purplish-black color in the structure of the hair, located throughout the thickness of the hair. With simultaneous admission, it can be located in the bulb or on the rod, depending on the prescription of poisoning. With chronic inclusion in the form of a continuous string throughout the bulb and rod.

ConclusionIn the microphotographs of hair submitted for the study with suspected poisoning of children with thallium, there are no signs indicating the effects of thallium.

This is evidenced by the absence of purple-black inclusions in the description of the hair. In addition, from the information provided by specialists, thallium was not detected by spectral analysis of leading laboratories.

The described lesions in the form of inclusions of "black color", "flask-like formations" and "thickening of the core" are characteristic of normal hair and are not signs of exposure to any toxicant.

The morphological shape of some hair indicates the effect of a toxic agent on the hair, which affected their structure as a result of which their loss occurred (alopecia). Probably, the damaging factor acted on the desmosomes of the cuticle of the rod and the vaginal membranes of the hair, and it seemed to come out of its own hair bed. The shape of such hair is fusiform. Such agents can be elements with variable valence, such as boron, hafnium, indium, strontium, as well as hydrazine-fluorinated chemicals and others

The hair carries information about the entry of an intoxicant into the body in the phase of active growth – anagen. The presence of such formations in the telogen phase indicates the receipt of a toxicant in the past, about three months ago. The catagen phase does not carry information. Taking into account the phase of hair growth and the time of the start of the toxicant intake, it is possible to more accurately determine the location of its detection on the hair. This is important when using point spectral analysis.