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

Abstract

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.

Keywords: Diabetes; Glucose; Lens; Liquefaction; Matrix assisted laser desorption ionization; Phospholipids; Retina; Vitreous humor.