The overall purpose of this thesis was to develop analytical methods for the determination of drugs and melanin content of human hair for practical use in forensic and clinical toxicology. The thesis consists of five papers, two of which are controlled single dose studies where codeine or selegiline were administered to healthy volunteers. One patient study, looked at the concentrations of selegiline metabolites in both pigmented and senile white hairs as well as plasma. Another study involved forensic autopsy cases where the occurrence of drugs of abuse in post-mortem blood, urine and hair were compared. Finally, one in vitro study investigated the binding of [³H]-flunitrazepam to melanin. To our knowledge, the controlled dosage studies are the first to quantitatively determine the relationship between drug concentration in human hair and melanin content.

The results demonstrated that pigmentation was an important factor for the incorporation of codeine, methamphetamine, and amphetamine into human hair. The relationships could be described by exponential functions with correlations coefficients r²>0.8. We have shown that the pigmented portion of hair from grey-haired patients incorporated more methamphetamine and amphetamine than the non-pigmented portion. The mean pigmented/white-hair ratios were 3.7±1.9 and 3.0±1.2 for methamphetamine and amphetamine respectively. Segmental hair analysis showed decreasing drug-concentrations over three months as a sign of noncompliance or of instability of the drugs in hair owing to cosmetic treatment of hair or wear. In the controlled studies, we demonstrated that the drug concentration in hair was fairly constant up to one month. In the study on autopsy cases, we found that hair analysis revealed patterns of multi-drug use not found by analysis of a single blood sample. Also, in 6 of 19 cases of heroin overdose, no opiates could be detected in hair. This suggested "first" or only occasional use of heroin, which might have been a contributing factor to the overdose death, because of lack of tolerance. The results from the in vitro study showed that binding of flunitrazepam to eumelanin occurs by two mechanisms, a Langmuir-like binding and a diffusion limited binding. We propose that these are expressions of an initial binding to the melanin surface (surface binding) followed by the diffusion of drug molecules into the melanin granule (bulk binding).

Hair as a specimen for toxicological analysis has hitherto not been investigated in Sweden. This thesis address questions raised by international research on the incorporation of drugs into hair and its implications for clinical and forensic toxicology.

Melanin has been established as an important factor for incorporation and binding of certain drugs into human hair and methods that allow correction for this are presented. Together, the results from the various studies provide a framework for both future research and the start of drug analysis in hair for forensic and clinical applications in Sweden.

This thesis deals with studies of melanophores and melanosomes by means of a physical approach. Melanophores are pigment cells that give the black colour to many vertebrates, e.g. fishes, frogs, and reptiles. Covering large areas of the skin, these cells are approximately 0.1 mm in diameter, and they contain black pigment granules called melanosomes.

The geometry and the electric charge of isolated melanosomes were two physical properties that were studied. The electric charge was measured by electrophoresis and was found to be (-1.7 ± 0.2)·10^-16 Coulomb in average. The geometry of melanosomes was measured using Scanning Force Microscopy, SFM, and resulted in an ellipsoidal shape with an average major diameter of 840 ± 20 nm.

Under nervous and hormonal control, melanophores rearrange the intracellular melanosomes from a scattered distribution, called dispersed, to a state where all melanosomes are accumulated in the cell centre, called the aggregated state. In this way, melanophores change from black towards transparent. This gives an animal the ability to change not only between being pale or dark, but also between different colours by using melanophores to cover and uncover the colours of different types of pigment cells from underlying layers.

The volume of melanosomes was measured with SFM. This study resulted in a difference of 18% when individual melanosomes from aggregated and dispersed melanophores were measured separately.

Magnetic field exposure of melanophores has been reported to affect the aggregation. However, contradicting results are presented in the literature. To clarify the possible effect of magnetic fields on melanophores, experiments by aggregating fish melanophores under exposure to strong (8 and 14 Tesla), homogenous, static magnetic fields were carried out. Both the magnetic field-induced Lorentz force on the charged melanosomes and the reorientation of the cytoskeleton were considered as possible explanations of any effects. Whenfield experiments were compared to control experiments with zero field, no difference in aggregation levels were found. However, a more irregular speed of aggregation was seen in the 8 Tesla field than in the control experiments.

A theoretical model was developed to explain switch-like responses in biological systems. A switch-like response to a graded stimulation was sometimes seen in the case of melanophores but was shown not to have a very large so-called Hill coefficient. The model is simple in its approach. It may be applied to general phenomena and is based on the assumption of a simultaneous desorption of an activator (agonist; substrate molecule; ... ) and an inactivator (antagonist, inhibitor; ... ) caused by a collision or interaction between two effector molecules (e.g. receptors or enzymes).

Melanosomes are also found in the human body and have a remarkably capacity to bind other molecules. A well-established forensic application of this is to detect (illegal) drugs that have bound to melanosomes in hair shafts. So far this application is only qualitative. This thesis includes a characterisation of the binding of flunitrazepam to melanin. Flunitrazepam is the active substance of Rohypnol, which is a sedative that is illegal in several countries and sometimes called the "date-rape-drug".

Melanophores are excellent model systems for studies of cellular phenomena. Moreover, melanophores are also interesting in sensing aspects. The change from black to transparent is a highly visible response to substances in their surroundings and has previously been the measurand in melanophore-based biosensors. The physical approach of these studies of melanophores also had the objective of evaluating new biosensor solutions.