Docosahexaenoic acid (DHA; 22:6n-3) is a critical constituent of the brain, but its metabolism has not been measured in the human brain in vivo. In monkeys, using positron emission tomography (PET), we first showed that intravenously injected [1-11 C]DHA mostly entered nonbrain organs, with ?0.5% entering the brain. Then, using PET and intravenous [1-11 C]DHA in 14 healthy adult humans, we quantitatively imaged regional rates of incorporation (K*) of DHA. We also imaged regional cerebral blood flow (rCBF) using PET and intravenous [15 O]water. Values of K* for DHA were higher in gray than white matter regions and correlated significantly with values of rCBF in 12 of 14 subjects despite evidence that rCBF does not directly influence K*. For the entire human brain, the net DHA incorporation rate J in , the product of K*, and the unesterified plasma DHA concentration equaled 3.8 6 1.7 mg/day. This net rate is equivalent to the net rate of DHA consumption by brain and, considering the reported amount of DHA in brain, indicates that the half-life of DHA in the human brain approximates 2.5 years. Thus, PET with [1-11 C]DHA can be used to quantify regional and global human brain DHA metabolism in relation to health and

In order to understand the role of the high levels of docosahexaenoic acid (DHA) in neuronal and retinal tissue, a study of the effect of membrane lipid composition on the visual pathway, a G protein-coupled system, was undertaken. The level of metarhodopsin II (MII) formation was determined to be a function of phospholipid acyl-chain unsaturation, with the highest levels seen in DHA-containing bilayers. Similarly, the rate of coupling of MII to the retinal G protein, Gt, to form a MII-Gt complex, was enhanced in DHA bilayers relative to less unsaturated phospholipids. Complex formation initiates the first stage of amplification in the visual pathway. The activation of the cGMP phosphodiesterase (PDE), the effector enzyme, represents the integrated pathway function. DHA-containing bilayers were found to support PDE levels comparable to those of the rod outer segment (ROS) disk membranes. Inclusion of 30 mol% cholesterol in the reconstituted bilayers had an inhibitory effect on each step in the visual pathway studied. Inclusion of cholesterol reduced MII formation and PDE activity and increased the lag time between the appearance of MII and the formation of the MII-Gt complex. However, signaling in DHA bilayers was far less affected by the addition of cholesterol than in bilayers containing less unsaturated phospholipids. These studies point up the importance of DHA acyl chains in promoting optimal function in G protein-coupled signaling pathways. The results reported here suggest that visual and cognitive deficits observed in n-3 deficiency may result from decreased efficiency in related neurotransmitter and visual signaling pathways in the absence of DHA.

We describe the characterization of degradation products responsible for color change in near UV-visible light-irradiated and heat-stressed monoclonal antibody (mAb) drug product in liquid formulation. The treated samples were characterized using reversed-phase HPLC and size-exclusion HPLC with absorption spectroscopy. Both methods showed color change was due to chromophores formed on the mAb but not associated with the formulation excipients in both light-irradiated and heat-stressed mAb samples. These chromophores were further located by a new peptide mapping methodology with a combination of mass spectrometry and absorption spectroscopy. Mass spectrometry identified the major tryptophan oxidation products as kynurenine (Kyn), N-formylkynurenine (NFK), and hydroxytryptophan (OH-Trp). The absorption spectra showed that each of the tryptophan oxidation products exhibited a distinct absorption band above 280 nm shifted to the longer wavelengths in the order of OH-Trp < NFK < Kyn. The Kyn-containing peptide was detected by absorption at 420 nm. No new absorption bands were observed for either methionine or histidine oxidation products. This confirmed that tryptophan oxidation products, but not methionine and histidine oxidation products, were responsible for the color change. It is worth noting that a new oxidation product with the loss of hydrogen (2 Da mass decrease) for Trp-107 of the heavy chain was identified in the heat-stressed mAb sample. This oxidized tryptophan residue exhibited a distinct absorption band at the maximum absorbance wavelength 335 nm, which is responsible for the color change to yellow. This study showed that the new peptide mapping methodology with a combination of mass spectrometry and absorption spectroscopy is useful to identify tryptophan oxidation products as chromophores responsible for color change in stressed mAb drug product.

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Bovine rhodopsin was reconstituted into mixtures of didocosahexaenoylphosphatidylcholine (di22:6-PC), dipalmitoylphosphatidylcholine (di16:0-PC), sn-1-palmitoyl-sn-2-docosahexaenoylphosphatidylcholine (16:0, 22:6-PC) and cholesterol. Rhodopsin denaturation was examined by using high-sensitivity differential scanning calorimetry. The unfolding temperature was increased at lower levels of lipid unsaturation, but the highest temperature was detected for native disk membranes: di22:6-PC < 16:0,22:6-PC < di16:0,18:1-PC < native disks. The incorporation of 30 mol% of cholesterol resulted in 2-4 degrees C increase of denaturation temperature in all reconstituted systems examined. From the analysis of van't Hoff's and calorimetric enthalpies, it was concluded that the presence of cholesterol in di22:6-PC-containing bilayers induces a level of cooperativity in rhodopsin unfolding. Fluorescence resonance energy transfer (FRET), using lipids labeled at the headgroup with pyrene (Py) as donors and rhodopsin retinal group as acceptor of fluorescence, was used to study rhodopsin association with lipids. Higher FRET efficiencies detected for di22:6-PE-Py, compared to di16:0-PE-Py, in mixed di22:6-PC-di16:0-PC-cholesterol bilayers, indicate preferential segregation of rhodopsin with polyunsaturated lipids. The effective range of the rhodopsin-lipid interactions facilitating cluster formation exceeds two adjacent lipid layers. In similar mixed bilayers containing no cholesterol, cluster formation was absent at temperatures above lipid phase transition, indicating a crucial role of cholesterol in microdomain formation.

ABP 215 is a biosimilar product to bevacizumab. Bevacizumab acts by binding to vascular endothelial growth factor A, inhibiting endothelial cell proliferation and new blood vessel formation, thereby leading to tumor vasculature normalization. The ABP 215 analytical similarity assessment was designed to assess the structural and functional similarity of ABP 215 and bevacizumab sourced from both the United States (US) and the European Union (EU). Similarity assessment was also made between the US- and EU-sourced bevacizumab to assess the similarity between the two products. The physicochemical properties and structural similarity of ABP 215 and bevacizumab were characterized using sensitive state-of-the-art analytical techniques capable of detecting small differences in product attributes. ABP 215 has the same amino acid sequence and exhibits similar post-translational modification profiles compared to bevacizumab. The functional similarity assessment employed orthogonal assays designed to interrogate all expected biological activities, including those known to affect the mechanisms of action for ABP 215 and bevacizumab. More than 20 batches of bevacizumab (US) and bevacizumab (EU), and 13 batches of ABP 215 representing unique drug substance lots were assessed for similarity. The large dataset allows meaningful comparisons and garners confidence in the overall conclusion for the analytical similarity assessment of ABP 215 to both US- and EU-sourced bevacizumab. The structural and purity attributes, and biological properties of ABP 215 are demonstrated to be highly similar to those of bevacizumab.

Interactions of hydrophobically-modified poly-(N-isopropylacrylamides) (HM PNIPAM) with phospholipid liposomes were studied as a function of the lipid type, the lipid bilayer fluidity, and the polymer conformation. Fluorescence experiments monitoring non-radiative energy transfer (NRET), between naphthalene attached to the HM PNIPAM and 1,6-diphenyl-1,3,5-hexatriene (DPH) incorporated into the lipid bilayer, confirmed the direct penetration of hydrophobic anchor groups linked to the polymer into the liposome hydrophobic core. Contraction of the polymer backbone above the lower critical solution temperature (LCST) resulted in a partial withdrawal of the anchor groups from the lipid bilayer. Analysis of polymer/lipid mixtures by centrifugation and quasi-elastic light scattering (QELS) revealed the polymer-induced fission of liposomes in the liquid-crystalline state, resulting in the formation of vesicles 150-230 nm in diameter. The process is reversible and upon transition of the bilayer into the gel state these vesicles are converted into larger aggregates. According to the results of gel-filtration experiments the HM PNIPAM is in dynamic exchange between the liquid-crystalline lipid bilayer and the water solution, while the binding to the bilayer in the gel state is more static in nature. The binding constant for mixture of HM PNIPAM with DMPC liposomes, evaluated from the centrifugation experiments, was found to be 120 M(-1).

The transition states of binary mixtures of dipalmitoyl-and dimyristoylphosphatidylcholines with sodium cholate at the reversible temperature-induced micellar-lamellar transformation were characterized by turbidimetry, electron microscopy, 31P NMR and differential scanning calorimetry. This transformation is triggered by the phospholipid acyl chain melting, and appears to include two structural pathways: (i) from discoidal mixed micelles to network-like structures composed of long interlaced rod-like micelles, then to multilayer membrane structures, and finally to multilamellar vesicles; and (ii) from discoidal micelles to membrane fragments and finally to unilamellar vesiCleS.