MALDI-MS Redox Lipidomics Applied to Human Hair: A First Look.

Lipids are an amazingly diverse group of biomolecules with an array of biological functions including protecting and maintaining key properties and structure. Oxidative insult in the form of UV, hydrothermal, or other damage leads to compromised lipid function and can be linked to a wide range of consumer complaints. This proof-of-principle study applied and evaluated redox lipidomic approaches for the characterization and profiling of selected lipids and their oxidation products in human hair. It was observed that cholesterol and cholesterol derivatives regions appeared to be the most susceptible to oxidative damage and this leads to further experiments, including the systematic characterization of oxidative products, and correlation of modifications with damage protocol.

INTRODUCTION

For human hair and skin, lipids play an integral role in protecting and maintaining key properties and structure. Oxidative insult leads to compromised lipid function and is linked to a wide range of consumer complaints, including brittleness, splitting, dryness, and decreased luster.

Hair can contain up to 9% lipids by mass,[1] comprising two general types: sebaceous lipids, which contribute to waterproofing, lubrication, and sheen; and endogenous lipids, which are associated with chemical diffusion, cell cohesion, and physico-mechanical properties such as strength.

Redox lipidomics is a rapidly emerging sub-discipline of lipidomics and lipid chemistry. However, advances in this field have yet to be fully applied to the cosmetic sciences. This paper reports the preliminary development of redox lipidomic techniques for the characterization and profiling of selected lipids and their oxidation products in human hair.

MATERIALS AND METHODS

Light, straight brown virgin hair was sourced from De Meo Brothers, NY USA. Approximately 10 mg was divided into three regions: root, middle, and tip. The lipids were extracted by Soxhlet (8 h in chloroform/methanol 2:1, then 18 h in fresh chloroform/methanol 2:1 at 18 C), washed with chloroform, methanol, and then concentrated. A portion of the lipid extracts was treated with 9% hydrogen peroxide and 1% ammonium persulfate for an hour and then dried. The following MALDI matrices were trialed:[2] 9-Aminoacridine (9-AA) from Sigma-Aldrich at 10 mg/ml in 60:40 isopropanol:acetonitrile; saturated α-cyano-4-hydroxycinnamic acid (CHCA) from Bruker Daltonics in a 1:2 mixture of acetonitrile with 0.1% trifluoroacetic acid; and 2,5-dihydroxy benzoic acid (DHB) from Bruker Daltonics at 10 mg/ml in a 1:2 mixture of acetonitrile with 0.1% trifluoroacetic acid. Lipid analysis was performed on a Bruker Daltonics Ultraflex III MALDI-TOF/TOF. Matrix solutions and sample were mixed 1:1, and 1 μl was spotted on a polished steel target plate and allowed to dry. For calibration, Peptide Calibration Standard (Bruker Daltonics) and Lipid Standard, Mono-, Di-, and Tri-glyceride Mix (Sigma-Aldrich) were prepared according to the manufacturer's instructions. Samples were measured in the positive and negative reflectron modes.

RESULTS

An initial evaluation of varying matrices was performed to determine the optimal matrix for matrix-assisted laser desorption ionization (MALDI) mass spectrometric (MS) analysis. The lipid profiles of each of the matrices trialed appeared quite different (data not shown). A range of lipids were detected with both DHB and CHCA in both positive and negative modes. CHCA was selected as the matrix of choice on the basis of facilitating good lipid coverage across the full mass range of interest and the best resolution of the lipid/peptide standard used for calibration.

MALDI-MS/MS was utilized to characterize individual lipids. Several classes of internal hair lipids were identified including ceramides, diglycerides, and cholesteryl esters. An example of the structural assignment of a ceramide, N-(tetracosanoyl)-dihydroceramide, is shown in [Figure 1].

Figure 1

MS/MS spectrum of ceramide N-(tetracosanoyl)-dihydroceramide, with characteristic fragment ions highlighted

Peroxide treatment of the lipid extract revealed significant changes in the lipid profile [Figure 2]. From these profiles, the loss of characterized lipids can be observed, in addition to a wide range of new observed ions correlating to damage products. In negative mode, no discernable changes were seen in the lipid profiles. In positive mode the range of lipids found from m/z 700 to 1000 (including phosphocholines, di- and tri-glycerides) seemed to be primarily present in the root region with the exception of one phosphocholine seen in the tip region.

Figure 2

MS spectra of peroxide treated lipid extract of the root region of human hair in both positive and negative modes

DISCUSSION

This proof-of-principle study has demonstrated the validity and utility of redox lipidomic approaches for evaluating lipid damage in human hair.

While the small sample number utilized in this initial study made it hard to determine exact lipid differences between the three regions of the hair, peroxide treatment of extracted hair lipids revealed ion peak shifts and disappearances within the di- and tri-glyceride regions. The cholesterol and cholesterol derivatives regions appeared to be the most affected, indicating higher susceptibility to oxidative damage. This is consistent with hydroxyl radicals preferentially attacking electron rich aromatic ring structures.[3]

This preliminary evaluation has demonstrated that redox lipidomic-based approaches can be applied to the detection of molecular-level damage to important lipids in human hair, allowing pinpoint characterization of the damaging effects of treatments such as peroxide bleaching. The next key steps will include the systematic characterization of oxidative products, and correlation of specific modifications and relative abundances with damage protocol.

Significant potential exists for applying and expanding our approach to both fundamental and applied hair and skin research, as well as personal care product advances, though development of lipid damage tracking technologies.