Quantitative evaluation of sebum lipid components with nuclear magnetic resonance

Abstract

A NMR spectroscopic method is described that enables the quantitation of specific lipid classes and components, independent of fatty acid composition. We demonstrate this method for measuring cholesterol, squalene, and pools of sterol esters, wax esters (WEs), and triglyceride (TG) components in sebum and meibum. When 600 MHz NMR equipment is used in conjunction with highly sensitive cryogenically cooled probes, this method has adequate sensitivity, and for some applications, advantages over commonly used HPLC-evaporative light-scattering detection and mass spectrometry-based approaches. This method is shown to be useful for preclinical and clinical monitoring of the efficacy of sebum-reducing agents in animals and humans. In Syrian hamsters, 3% topical flutamide and 20 mg/kg oral isotretinoin reduced sterol esters by 18.7% and 30.0%, respectively, and reduced WEs by 32.9% and 31.8%, respectively, as measured in a punch biopsy of the ear. In a 72 patient clinical methodology study, the assay delivered reproducible and noninvasive measurements of WEs, cholesteryl esters, TGs, and squalene from Sebutape: skin blots. The quantitative results of sebum analysis obtained by the NMR method correlate well with those obtained with HPLC-based approaches. This approach may be broadly applicable to cases in which fatty acid-independent quantification of lipid classes is desired. Mammalian skin is composed of three primary layers: the stratum corneum, the epidermis, and the dermis. The outer layer of the skin, the stratum corneum, primarily functions as a barrier to the external environment, preventing water loss and the invasion of microorganisms. Sebum secreted to the stratum corneum from the sebaceous glands is a key component of the skin surface and has various demonstrated and postulated functions (1). Sebum itself is a complex mixture of lipids and is produced by the sebaceous glands. At maturation, the acinar cells of the sebaceous glands lyse and release sebum into the lumenal duct, from which the sebum is secreted. Cholesterol, sterol esters, wax esters (WEs), and triglycerides (TGs) are the primary lipids found in the sebum of many mammals, and squalene is also a major component of human sebum. In humans, the predominant sterol esters are cholesteryl esters (CEs), whereas in male Syrian hamster (a common sebum model), they are chiefly fatty acid esters of a single predominant noncholesterol sterol (2). The identity of the sterol constituting these esters in this species has not been identified, and herein we will refer to these as hamster sterol esters (HSEs). WEs are unique to sebum in that they are not synthesized by other cells in the body. Sebum composition can be modified from its native state by the action of xenobiots, which can greatly increase the complexity of its biochemical makeup; for example, bacterial hydrolytic enzymes are known to break down TGs to produce free fatty acids (3).A number of methods to isolate and qualitatively and/ or quantitatively analyze the lipid components of sebum and/...

Beyond acne: Current aspects of sebaceous gland biology and function

“…The identification of the composing species as intact molecules is a suitable analytical approach to find variations in the sebum composition [9,53,54]. Information regarding preferential arrangement of fatty acids in complex lipids can be collected and contribute to the understanding of the role of alterations of sebum composition in the pathologies of the PSU [10].…”

Comprehensive analysis of the major lipid classes in sebum by rapid resolution high-performance liquid chromatography and electrospray mass spectrometry

“…However, information on their arrangement in the entire lipid was indefi nite. In contrast, sebum can be analyzed according to lipid classes using thin-layer chromatography (TLC) ( 12,13 ) and nuclear magnetic resonance (NMR) ( 14 ). Both TLC and NMR methods are advantageous because they are semi quantitative in terms of their ability to quantify the expression of the lipid families.…”

A physiologically based in silico kinetic model predicting plasma cholesterol concentrations in humans

“…Human sebum secretion rates from forehead skin are reported to lie between 1.3 and 3.3 mg/10 cm 2 /3 h ( 42 ). NMR studies indicate that the molar fractions of CE and squalene in sebum are 0.03 and 0.28, respectively, with the other constituents being triglycerides, fatty acids, and wax esters ( 43 ). With a total body area of approximately 2 m 2 , taking into account the molar weights of the different constituents and assuming uniform secretion rates, an upper bound for the total CE and squalene fl ux may be as high as 2.5 mmol/man/day.…”