Lead-revealed lipid organization in human hair

Abstract

Human hair lipids form a complex mixture of composition close to that of sebum. Part of these lipids appears in an organized state that has been studied by diffraction techniques in the literature. Nevertheless, information on the structure of these lipids remains very scarce due to their low contribution to global hair diffraction pattern. Here we show that appropriate lead treatment considerably enhances organized lipid features observed by microbeam synchrotron radiation diffraction experiments. We attribute these features to the formation of lead soaps of free fatty acids. Specific orientation of hair “lipid crystals” in planes parallel to the hair axis is clearly demonstrated. Inclusion of these lipids in the bulk of the hair shaft is shown by diffraction experiments following removal of the cuticular outermost layer of hair. Moreover, microfluorescence and diffraction experiments are consistent with part of the lipids being present as calcium soaps in native hair. We therefore consider lead fixation as a powerful tool to evaluate the lipid organization in human hair for medical, environmental and archaeological purposes, including lead poisoning.

Introduction

Human hair is a keratinized fibre of 50–100 μm in diameter. Its section is divided into three main concentric structural zones. Discontinuous medulla cells form the 5–10 μm in diameter centre of the fibre. They are surrounded by the tightly packed 100-μm-long cells of cortex that represent an average of 90% of hair total mass. The 5–10 overlapping scales of cuticle, 5-μm-thick outermost layer of the hair strand, isolate the fibre from its environment.

Hair cortex consists of keratin-filled elongated cells (keratinocytes) that are separated by an intercellular lipido-proteinic matrix or cell membrane complex (CMC). These cells are mostly filled with keratin intermediate filaments embedded in an interfilamentous protein material. Keratins are fibrous proteins characterized by a succession of organized supramolecular assemblies of increasing size, at several scales from 1 nm to several micrometers. It is this intricate supramolecular organization that confers to hair and epidermis their important function of protective barrier against chemical, mechanical and physical aggressions.

Hot solvent extraction removes an average of 4 wt.% of lipids, with a large dispersion. These lipids can be divided in structural lipids, free internal lipids and surface lipids. Hair total lipid content is reported to depend on age, sex and origin of the subject [1]. Recent synchrotron infrared lipid mapping on Caucasian hairs shows that the global content is distributed between the medullary canal and the outer regions of the cuticle and the cortex [2]. Removable lipids form a complex mixture of composition similar to that of the fatty secretion of scalp sebaceous glands [3]. Sebum is reported to contain mainly triglycerides (41% wt.), free fatty acids (16%), wax esters (25%) and squalene (12%) [4], [5]. A rough estimation of available carboxylate content in the lipid fraction, derived from this composition, is of 30 μmol per gram of hair.

Structural lipids are found in the CMC and at the surface of cuticle scales (or epicuticle). Some of these lipids are covalently bound to hair cells and require a saponification step to be removed. They contain free fatty acids (mainly the C21 18-methyl-eicosanoic free fatty acid), ceramides and cholesterol sulfate as major components [6], [7], [8], but no phospholipids.

Part of lipids appears in an organized state and leads to fine rings in the hair diffraction pattern. A 4.5-nm ring and observable higher harmonics are reported to correspond to the lamellar periodicity of structured lipid bilayers in early publications [9], [10]. Two reflections around 0.41 and 0.37 nm are related to interatomic distances within these layers. Such organized lipids have been observed in skin stratum corneum [11]. Hair lamellar lipids would be present as lipid granules, or “lipid crystals” and not lipid layers of cell membranes [10], [12], [13]. Neither the composition nor the origin of these lipids has been elucidated so far.

Lipids are expected to play a very important role in water diffusion processes through hair and skin stratum corneum, which is of primary importance for pharmaceutical and medical applications. Furthermore, obtaining more information on the interaction between lead compounds and keratinized tissues finds a direct impact on the analysis of trace elements in human body in a medical (lead poisoning), environmental (heavy metal contamination) and archaeological context (preservation of ancient tissues in contact with metal objects). Recently, hair lipid diffraction rings have also been controversially suggested as a breast-cancer diagnosis tool [14], [15], [16].

In the present experiments, we follow the biocrystallography approach of the soaking heavy atom solution. Lead can induce a strong contrast of diffraction spots due to its high electronic density. We couple here diffraction and fluorescence microbeam techniques, using high-flux synchrotron X-ray beams to study the structural and elemental properties of these lipids. Third-generation synchrotron facilities allow work on single hair, through the strong enhancement of the hair diffraction pattern. We show here that the organized fraction of lipids in human hair appears to consist of fatty acid bilayers, partly under calcium soap form. Lead fixation on specific sites belonging to these lipids leads to a strongly enhanced diffraction pattern and shows a strong orientation of these soaps parallel to the hair axis.