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The Spectrophotometric Sulfo-Phospho-Vanillin Assessment of Total Lipids in Human Meibomian Gland Secretions

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Lipids

Abstract

Human meibomian gland secretions (meibum) are the major lipid component of the human preocular tear film. The predominant lipid classes found in meibum include waxes (WE), cholesteryl esters (CE), and varying amounts of cholesterol (Chl). The classical sulfo-phospho-vanillin assay (SPVA), adapted for a microplate reader, was used to quantitate lipids in meibum. To account for varying reactivities of different lipids in SPVA, a model meibomian lipid mixture (MMx) that approximated the WE/CE/Chl composition of meibum was developed and used to quantitate meibomian lipids. The overall SPV responses of MMx and meibum were found to be close, with similar intermediate and final reaction products for both. Saturated WE that had not been expected to be reactive were found to be SPV-positive. A reaction mechanism for these compounds in SPVA which involves the formation of alkenyl ethers is proposed and discussed. Tested proteins were non-reactive in SPVA. Thus, by comparing the results of gravimetric analyses of meibum samples with the results of a properly calibrated SPVA, it was estimated that the SPV-reactive lipid content of dry meibum in tested samples was about 78 % (w/w). The SPV method can also be adopted for analyzing other types of complex lipids secretions, such as sebum, as well as whole lipid extracts from other lipid-enriched organs and tissues, if proper standards are chosen.

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Abbreviations

BO:

Behenyl oleate

BS:

Behenyl stearate

BSA:

Bovine serum albumin

Chl:

Cholesterol

CE:

Cholesteryl ester

Chl-O:

Cholesteryl oleate

Chl-S:

Cholesteryl stearate

FA:

Fatty acid

HD:

Heptadecane

LYZ:

Lysozyme

MMx:

Model meibum lipid mixture

MUC:

Mucin

OAHFA:

(O-Acyl)-omega-hydroxy fatty acid

1-OD:

1-Octadecanol

SPVA:

Sulfo-phospho-vanillin assay

STA:

Stearic acid

SO:

Stearyl oleate

TAG:

Triacylglycerol

TO:

Triolein

TS:

Tristearin

UV-Vis:

Ultraviolet-visible

WE:

Wax ester

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Acknowledgments

This project was supported by the National Institutes of Health grants R01EY019480, 3R01EY019480-01A1S1, a core grant 5P30EY020799-03, and an unrestricted grant from Research to Prevent Blindness (New York, NY).

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Authors and Affiliations

  1. Department of Ophthalmology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX, 75390-9057, USA

    Anne McMahon, Hua Lu & Igor A. Butovich

  2. The Graduate School of Biomedical Sciences, University of Texas Southwestern Medical Center, Dallas, TX, 75390-9057, USA

    Igor A. Butovich

Authors
  1. Anne McMahon
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  2. Hua Lu
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  3. Igor A. Butovich
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Correspondence to Igor A. Butovich.

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McMahon, A., Lu, H. & Butovich, I.A. The Spectrophotometric Sulfo-Phospho-Vanillin Assessment of Total Lipids in Human Meibomian Gland Secretions. Lipids 48, 513–525 (2013). https://doi.org/10.1007/s11745-013-3755-9

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  • DOI: https://doi.org/10.1007/s11745-013-3755-9

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