Hyaluronic acid-lipid binding.

BACKGROUND: Phospholipid (PL)-hyaluronic acid (HA) interactions are relevant to aging-associated vitreous humor liquefaction, therapies for dry eye disease, skin-care products and synovial joint lubrication. Phosphatidyl choline-HA interactions have been well characterized. However, other major lipids found in tears, vitreous humor and synovial joints have not. The purpose of this study was to bridge this gap of knowledge. METHODS: HA (1600 kDa) at 5 mg/mL, was mixed with various lipids ranging in concentration from 0.1 to 10 mg/mL in D2O. HA-PL binding was measured from the decrease in HA proton resonance intensity with binding using a nuclear magnetic resonance spectrometer. RESULTS: Cholesterol weakly bound to HA, followed by monoglyceride and palmitoyl palmitate < phosphatidyl choline, phosphatidic acid and sphingomyelin. The maximum amount of PL bound was 14 ± 1 µmoles inferring a 1 to 1 molar ratio of bound PL to HA dimer. Monoglyceride and palmitoyl palmitate required two to three times more lipid to achieve 100% bound HA compared to PL. CONCLUSIONS: Physiological levels of HA, phosphatidyl choline and sphingomyelin would result in only 4% of the hydrophobic hydrogens of HA to be bound. HA-PL binding interactions could be important for therapeutic use of HA in eye drops in future studies to treat dry eye and to trap PL entering the VH to keep them from forming light scattering micelles. HA-lipid binding may also be relevant to the therapeutic effects of topical skin-care products. Both head group and hydrocarbon chain moieties influence HA-lipid interactions.

In-vitro and ex-situ regional mass spectral analysis of phospholipids and glucose in the vitreous humor from diabetic and non-diabetic human donors.

The causes of vitreous humor (VH) liquefaction remain unclear. Diabetes accelerates this process and other ocular diseases. The weakening of the blood-retina barrier observed with diabetes could enhance the rate of transfer of relatively small molecules such as glucose (Glu) and phospholipids (PLs) from the retina to the VH. Glucose and PLs have been detected previously in VH but their regional distributions are not known. The mapping of Glu and PLs in VHs from subjects with and without diabetes could reveal the roles of these molecules in VH liquefaction. Diabetic and non-diabetic human eyes were acquired from the Kentucky Lions Eye Bank and frozen immediately. Each VH was removed and halved along the sagittal plane. One half was stamped on a matrix assisted laser desorption ionization (MALDI) plate. Either p-Nitroanaline (26 mg/mL MeOH:CHCl3) or 2,5-dihydroxybenzoic acid (20 mg/mL H2O:acetonitrile) was used as matrix. Glu and PLs were extracted from the remaining sections and analyzed. Data were acquired using a MALDI-mass spectrometer. The levels of Glu and PLs were significantly greater in VH from diabetics (VHd) compared with VH from non-diabetics (VHnd). VHds showed the highest relative levels of PLs in the posterior VH, followed by the anterior and central regions. Throughout the entire VH, the most abundant PLs were phosphatidylcholines followed by sphingomyelins. For Glu, the relative intensities were ~3 times higher in the posterior region of VHd (12 ± 1.3) compared with VHnd (6.5 ± 0.7) VHs. Regional studies showed that relative to the posterior VHd, the Glu levels were lower in the anterior (8.1 ± 1.0) and central (6.7 ± 0.8) regions. For the VHnds, the values for the central and anterior regions were 5.9 ± 1.2 and 4.7 ± 0.9, respectively. PLs and Glu are most abundant in the posterior region relative to the central and anterior zones of VHs. This trend was observed in VHd and VHnd, but PLs and Glu levels were significantly higher in VHds. These results support the possibility that higher levels of Glu and PLs accelerate VH liquefaction in diabetic patients. As liquefaction begins in the posterior region, the higher abundance of PLs and Glu in this zone also suggests that they may play a role in liquefaction. The specific molecular interactions affected by Glu and PLs in the collagen/hyaluronan/water network need to be examined.

Regional distribution of phospholipids in porcine vitreous humor.

This project explores the regional phospholipid distribution in porcine vitreous humor, retina, and lens. Matrix-assisted laser desorption mass spectrometry has been used previously to image lipids, proteins, and other metabolites in retinas and lenses. However, the regional composition of phospholipids in vitreous humors is not known. To address this issue, we have applied this mass spectral method to explore the regional phospholipid distribution in porcine vitreous humor both ex-situ and in-vitro. To establish the possible source(s) of phospholipids in the vitreous humor, compositional studies of the lens and retina were also pursued. Due to the overall low levels of phospholipids in vitreous humor, it was necessary to optimize the experimental approaches for ex-situ and in-vitro studies. The sensitivity observed in the spectra of methanol extracts from the lens and retina was higher than that for methanol:chloroform extracts, but the compositional trends were the same. A fourfold improvement in sensitivity was observed in the analysis of vitreous humor extracts obtained with the Bligh and Dyer protocol relative to the other two extraction methods. For ex-situ studies, the 'stamp method' with para-nitroaniline as the matrix was chosen. Throughout the vitreous humor, phosphatidylcholines were the most abundant phospholipids. In-vitro results showed higher relative levels of phospholipids compared to the 'stamp' method. However, more details in the regional phospholipid distribution were provided by the ex-situ approach. Both in-vitro and ex-situ results indicated higher levels of phospholipids in the posterior vitreous region, followed by the anterior and central regions. The posterior region contained more unsaturated species whereas more saturated phospholipids were detected in the anterior region. The observed trends suggest that the phospholipids detected in the posterior vitreous humor migrate from the retina and associated vasculature while those present in the anterior regions are likely to derive from the lens. Not all species found in the lens were observed in the vitreous humor. For example, whereas cholesterol was present in lens extracts, it was not detected in the vitreous humor. Overall, the higher relative abundance of unsaturated species in the posterior vitreous humor and also present in the retina suggests that these species may be able to disrupt the water-collagen-hyaluronan network and contribute to vitreous liquefaction.