Conversion of S-phenylsulfonylcysteine residues to mixed disulfides at pH 4.0: utility in protein thiol blocking and in protein-S-nitrosothiol detection.

A three step protocol for protein S-nitrosothiol conversion to fluorescent mixed disulfides with purified proteins, referred to as the thiosulfonate switch, is explored which involves: (1) thiol blocking at pH 4.0 using S-phenylsulfonylcysteine (SPSC); (2) trapping of protein S-nitrosothiols as their S-phenylsulfonylcysteines employing sodium benzenesulfinate; and (3) tagging the protein thiosulfonate with a fluorescent rhodamine based probe bearing a reactive thiol (Rhod-SH), which forms a mixed disulfide between the probe and the formerly S-nitrosated cysteine residue. S-Nitrosated bovine serum albumin and the S-nitrosated C-terminally truncated form of AdhR-SH (alcohol dehydrogenase regulator) designated as AdhR*-SNO were selectively labelled by the thiosulfonate switch both individually and in protein mixtures containing free thiols. This protocol features the facile reaction of thiols with S-phenylsulfonylcysteines forming mixed disulfides at mild acidic pH (pH = 4.0) in both the initial blocking step as well as in the conversion of protein-S-sulfonylcysteines to form stable fluorescent disulfides. Labelling was monitored by TOF-MS and gel electrophoresis. Proteolysis and peptide analysis of the resulting digest identified the cysteine residues containing mixed disulfides bearing the fluorescent probe, Rhod-SH.

Oxidative damage of retinal rod outer segment membranes and the role of vitamin E.

Highly purified bovine rod outer segment membranes show loss of structural integrity under an air atmosphere. Obvious ultrastructural changes are preceded by increases in absorbance below 400 nm. These changes are inhibited by Ar or N2 atmospheres and appear to be due primarily to oxidative damage to the polyunsaturated fatty acids of the membrane lipids. Loss of polyunsaturated fatty acids, formation of malonaldehyde and fluorescent products characteristic of lipid oxidation accompany the spectral alterations. The elevated ultraviolet absorbance can largely be removed from the membranes by gentle extraction of the lipids using phospholipase C and hexane without changing the visible absorbance of rhodopsin. We have found a large seasonal variation in the endogenous level of alpha-tocopherol (vitamin E) in the bovine rod outer segment preparations. For much of the year we find that the rod outer segment membranes contain higher levels of alpha-tocopherol than have been previously reported in biological membranes. Rod outer segments which are low in endogenous tocopherol can be protected from oxygen damage by adding exogenous tocopherol. The rod outer segments are extremely susceptible to oxygen damage due to the unusually high content of polyunsaturated fatty acids in the membrane lipids. The presence of tocopherol inhibits oxygen damage but does not eliminate it. The tocopherol in the rod outer segments is consumed in air, thus complete protection from peroxidation in vitro requires an inert atmosphere as well as high levels of tocopherol. This work suggests that extensive precautions against oxidative degradation should also be employed in studies of other membrane systems where important deleterious effects of oxygen may be less obvious.

Increased glutathione-S-transferase activity in antioxidant-deficient rats.

Rats fed a diet deficient in vitamin E and selenium show an increased activity of glutathione-S-transferase (EC 2.5.1.18) in all tissues tested, with the possible exception of the retina. Glutathione-S-transferases are detoxifying enzymes that are induced by a variety of electrophilic drugs or toxins. Therefore, the induction of glutathione-S-transferase in vitamine E- and selenium-deficient rats indicates that substrates for the enzyme probably increase in vivo with dietary antioxidant deficiency. These substrates are likely to be lipid peroxides and/or other lipid peroxidation products.

Sidechain and backbone requirements for anti-invasive activity of laminin peptide 11.

The structure of laminin peptide 11 (CDPGYIGSR-NH2) contains valuable information for the design of mimetic compounds with anti-invasive and anti-metastatic properties. An alanine scan replacement experiment identified Tyr5, Ile6 and Arg9 residues as contributing significantly to anti-invasive activity. Circular dichroism spectra and NMR alphaH chemical shift values both supported the existence of populations of nonrandom coil solution structures for the analogs tested. A D-Ala4 for Gly4 substituted analog completely lost activity, while an L-Ala4 for Gly4 substituted analog retained half the activity of the parent peptide. These results complement our previous findings with D/L alanine substitutions at the Gly7 position, and together they suggest an 'S'-shaped backbone as likely for the active peptide conformation. NMR-constrained molecular modeling supported a direct involvement of the Tyr5 and Ile6 sidechains in conferring bioactivity, and indicated that the Tyr5 sidechain was buried in the Ala2 for Asp2 substitution. Based on the fact that the peptide 11 sequence derives from the disulfide bonded c-loop of an LE-repeat, we synthesized the cyclic CDPGYIGSRC-NH2 peptide. This analog exhibited good anti-invasive and anti-metastatic activity. NMR modeling experiments suggested that the trans-proline cyclic peptide, would favor an 'S'-shaped backbone conformation. Full retro-inverso analogs of peptide 11 were shown to have anti-invasive activity inferior to that of peptide 11. This weak bioactivity was probed using NMR-constrained molecular dynamics, and revealed potential conformations which limited the ability of the required sidechains to mimic the positions of those in the native peptide conformations.

Binding and activation of rod outer segment phosphodiesterase and guanosine triphosphate binding protein by disc membranes: influence of reassociation method and divalent cations.

Attempts to optimize the recovery of light-stimulated phosphodiesterase activity following reassociation of the hypotonically extractable proteins derived from retinal rod segments with hypotonically stripped disc membranes lead to the following observations: the best reassociations were obtained by mixing proteins and stripped disc membranes under hypotonic conditions and slowly increasing the salt concentration; the binding of G-protein and phosphodiesterase to stripped disc membrane occurs in less than 5 minutes and the recovery of light-stimulated phosphodiesterase activation in response to subsaturating stimulus levels requires 2-3 h to plateau. Stripped disc membranes and proteins were reassociated in 'isotonic' buffers containing KCl/NaCl, KCl/NaCl plus Mg2+, or KCl/NaCl plus Ca2+. Large fractional rhodopsin bleaches produced nearly identical light-stimulated phosphodiesterase activities in each of these samples and in the control rod outer segment membranes. Rod outer segment membranes and reassociated stripped disc membrane samples containing divalent cations showed similar phosphodiesterase activities in response to low fractional rhodopsin bleaches (e.g. less than or equal to 0.1%), however, samples devoid of divalent cations during reassociation required rhodopsin bleaches up to 10-fold larger to elicit comparable phosphodiesterase activities. These results suggest that not all phosphodiesterase and/or G-protein molecules bound to the disc membrane surface are equivalent with regard to their efficiency of activation by bleached rhodopsin and that divalent cations can modulate the distribution of G-protein and/or phosphodiesterase between these populations.

Isoelectric focusing studies of bacteriorhodopsin.

Purified bacteriorhodopsin (BR) samples show a minimum of four isoelectric forms in immobilized pH gradient isoelectric focusing gels. The bands occur as doublets with isoelectric points (pI) centered at 5.20 (principal species) and 5.60. In typical preparations additional bands may be observed at 4.90, 5.07 and 5.50. Purple membrane (PM) was proteolyzed with papain to calibrate the pI shift produced by changing the number of charges on the protein. Asp-242 is removed during the first cleavage between residues 239 and 240 resulting in the loss of a single negative charge and a shift of the principal doublet by +0.35 pH units to pI 5.55. The second papain cleavage occurs between residues 231 and 232 which removes Glu-232, -234 and -237 and shifts the pI by +0.60 pH units to pI 6.10. The +0.60 pH shift upon the second papain cleavage is consistent with the loss of two negative charges and is supported by prior evidence that at least one of the three glutamate residues lost during the second proteolysis step is protonated and neutral in the intact protein. The native and proteolyzed products of BR retain the characteristic 550 nm absorption maxima for solubilized BR. A model for the structural origin of the pI heterogeneity of BR species in proteolyzed PM is presented.

Characterization of complex formation between Gi2 and octyl glucoside solubilized neutrophil N-formyl peptide chemoattractant receptor by sedimentation velocity.

The reversible formation of complexes between N-formyl peptide chemoattractant receptor (FPR) and Gi2 protein was analyzed by velocity sedimentation in linear sucrose density gradients. FPR complexed with heterotrimeric Gi2, sediments at different rate than uncomplexed FPR and the two forms have apparent sedimentation coefficients of 7S and 4S, respectively. The biochemical variables important for the reconstitution of the 7S complex from the 4S receptor and Gi2 were studied. The formation of 7S was saturable with Gi2 and addition of excess Gi did not cause oligomerization. The reconstituted 7S complex was stable under a variety of conditions including octyl glucoside concentrations below and above the critical micellar concentration. The optimum pH for the reconstitution is between 7 and 9, where the 4S and 7S species sedimented reproducibly, at distinct positions in the gradient. Below pH 6 both the 4S and the 7S species appear to undergo denaturation and form precipitates. Magnesium ions have no significant effect on the sedimentation of either forms of FPR. Reconstitution was stable up to a NaCl concentration of 0.2 M. At 1 M NaCl reconstitution was inhibited and at 3 M salt FPR aggregated. Since guanine nucleotides GTP, GTP gamma S, GDP beta S selectively dissociated the 7S complex in a concentration-dependent manner and adenine nucleotides had no effect, we conclude that the FPR-Gi2 system displays a vacant guanyl nucleotide binding site, the hallmark of a functional guanine nucleotide exchange complex. Moreover, our results indicate that the reconstitution of FPR-Gi2 complexes is reproducible at physiologically relevant conditions, shows selectivity, specificity, and biochemically functional properties consistent with a specific and functional interaction between solubilized FPR and G protein.

Disaturated and dipolyunsaturated phospholipids in the bovine retinal rod outer segment disk membrane.

Thin-layer chromatography was used to separate the major phospholipid headgroup classes of the rod outer segment disk membrane into subfractions which differ markedly in fatty acid composition. At least 18% of the rod outer segment phosphatidylcholine must contain two saturated fatty acids. Furthermore, two unsaturated fatty acids are found in at least 43% of the phosphatidylserine, 24% of the phosphatidylcholine, and 24% of the phosphatidylethanolamine. The unsaturated acids are predominantly polyunsaturated in all cases. A similar separation, but with less resolution, was achieved with silicic acid column chromatography. The temperature dependence of the polarization of the fluorescence of trans-parinaric acid (9,11,13,15-all-trans-octadecatetraenoic acid) showed that the thermal behavior of aqueous dispersions of the phosphatidylcholine subfractions was consistent with their fatty acid compositions.

Effects of vitamin E and selenium deficiency on the fatty acid composition of rat retinal tissues.

The fatty acid composition of retinal tissues was measured in rats maintained for 26--32 weeks on each of the following diets: a purified basal diet deficient in alpha-tocopherol and selenium, an identical control diet supplemented with alpha-tocopherol and selenium, and a commerical laboratory rat chow. Dietary deficiencies of antioxidant nutrients were found to cause a large decrease in total polyunsaturated fatty acids in the retinal pigment epithelium, a small decrease in the retinal rod outer segments, but no change in the whole retina or liver when compared to tissues from animals fed the vitamin E- and selenium-supplemented control diet. The polyunsaturated fatty acid content which we have observed for the retinal pigment epithelium from rats fed commerical lab chow is similar to that which we observed for bovine retinal pigment epithelium. Our results indicate that changes in fatty acid composition are not generalized to all tissues in severely antioxidant-deficient animals, but that changes do occur in some tissues, such as the retinal pigment epithelium, which appears to be particularly sensitive to in vivo lipid peroxidation.

4-Hydroxyhexenal: a lipid peroxidation product derived from oxidized docosahexaenoic acid.

4-Hydroxynonenal and 4-hydroxyhexenal are cytotoxic aldehydic products of lipid peroxidation with high biological activity. Peroxidation of n - 6 fatty acids produces 4-hydroxynonenal, but the origin of 4-hydroxyhexenal has been uncertain. We now present evidence that 4-hydroxyhexenal is generated by oxidation of docosahexaenoic acid, the most abundant n-3 fatty acid in tissues.

Phosphorylation reactions in bovine rod outer segments studied by 32P-labelling of intact retina.

The protein phosphorylation pattern in the intact bovine retina has been investigated by labelling with 32P-phosphate under incubation conditions that preserve the electrical photoresponse of the photoreceptor cells. The phosphorylation of rod outer segment proteins was analysed after isolation of outer segments from the labelled retina. The global influence of light, Ca2+ and the phosphodiesterase inhibitor, isobutylmethylxanthine, on protein phosphorylation in rod outer segments was analysed. A 12 kDa protein is the most prominent phosphorylated species in the intact bovine retina. Its phosphorylation is increased by light and/or Ca2+. Evidence is presented that this strongly phosphorylated protein is not located in the outer segment, and we suggest that it may be a synaptic protein. Retinal rod outer segment membrane proteins with apparent molecular weights of 245, 226, 125, 110, 50, 46, 38 and 20 all show light-stimulated phosphorylation. Lowering the extracellular Ca2+ levels results in a decrease of the phosphorylation level of some of these proteins, viz. at 125, 50, 38 and probably at 20 kDa. Such proteins, whose phosphorylation level is influenced both by light and by elevated Ca2+, are candidates for mediators of phototransduction. The phosphorylated species at 245, 226, 110, 50 and 20 kDa are enriched in rod outer segment plasma membrane preparations. These protein species could participate in the light-regulated modulation of the Na+-conductance of the plasma membrane.

Two-dimensional crystallization of bovine rhodopsin.

Bovine rhodopsin has been clustered into two-dimensional crystals in highly purified native rod disk membranes and studied with negative staining and transmission electron microscopy. The lattice is P2(1) with dimensions of 8.3 X 7.9 nm and interaxis angles of 86 +/- 3 degrees. 110 images of ordered areas were digitized and aligned with computer-correlation methods to calculate an average image with diffraction to the fourth order. The images were computer-filtered and reconstructed to approx. 2 nm resolution. When crystals appeared they covered 20-40% of the surface of the preparation and, since rhodopsin is at least 95% of the protein, there is no doubt that the crystals were due to rhodopsin. There appear to be two rhodopsin dimers per unit cell. Each rhodopsin molecules takes up about 7.5 nm2 of membrane area and is estimated to be associated with about 12 lipids on each side of the membrane. The membrane area found for bovine rhodopsin supports the rhodopsin origin of rarely seen but more highly ordered two-dimensional crystals found in detergent-treated frog rod membranes (Corless, J.M., McCaslin, D.R. and Scott, B.L. (1982) Proc. Natl. Acad. Sci. USA 79, 1116-1120). Furthermore, the rhodopsin membrane area is close to that of bacteriorhodopsin and is consistent with a seven transmembrane helix structure proposed for rhodopsin (for references see Dratz, E.A. and Hargrave, D.A. (1983) Trends Biochem. Sci. 8, 128-131). Crystallization was accomplished by lowering the pH to 5.5 near the isoelectric point of rhodopsin, raising the salt concentration of 2 M (NH4)2SO4, adding 5% glucose and 0.02% Hibitane (Ayerst), a cationic amphipathic antiseptic that favored crystal growth.

A carboxyl-terminal tail peptide of neutrophil chemotactic receptor disrupts its physical complex with G protein.

The binding of G protein to the N-formyl peptide receptor of human neutrophils was investigated with site-specific synthetic peptides. Peptide CT336(322) (322RALTEDSTQTSDTAT336) from the carboxyl-terminal tail region of the receptor competed with the receptor for binding to bovine Gi protein. The peptide competition was assayed by dissociation of a GTP-sensitive, rapidly sedimenting (7S) form of receptor-G protein complex as analyzed by velocity sedimentation on linear sucrose density gradients. An IC50 of 590 microM was determined for CT336(322) peptide. A control peptide, with the reverse sequence, rCT322(336) (336TATDSTQTSDETLAR322), did not perturb the sedimentation of the reconstituted receptor-G protein complex up to the highest tested concentration, 3 mM. Other peptides tested, corresponding to central portions of the predicted intracellular loop regions CII140(127) (127VLHPVWTQNHRTVS140) and CIII239(227) (227KIHKQGLIKSSRP239) of the receptor, failed to dissociate the reconstituted receptor-G protein complex. Control peptides from the extracellular region EII184(170) (170KTGTVACTFNFSPWT184) and an unrelated sequence matching a portion of neutrophil cytochrome b, CYT306(296) (296KVVITKVVTHPFKTIE306), were also ineffective. Our results suggest that the cytoplasmic tail of the formyl chemotactic peptide receptor is involved in its coupling to the signal-transducing G protein.

Immunocytochemical detection of lipid peroxidation in phagosomes of human neutrophils: correlation with expression of flavocytochrome b.

Oxidants generated by the NADPH oxidase of activated neutrophils can react with a number of tissue targets to form toxic metabolites such as 4-hydroxynonenal (4-HNE). 4-HNE is a lipid peroxidation product generated by free radical attack on omega-6 polyunsaturated fatty acids and is a marker for membrane lipid peroxidation. In this study, we examined the accumulation of 4-HNE-protein adducts in phagosomes of neutrophils obtained from a male patient with homozygous X-linked, flavocytochrome b-deficient chronic granulomatous disease (CGD), his heterozygous mother, and his normal father. Specific polyclonal antibodies recognizing 4-HNE-protein adducts and gp91-phox (flavocytochrome b large subunit) were prepared and used to immunocytochemically detect these antigens in cryofixed, molecular distillation-dried neutrophils. No 4-HNE-protein adducts were detected in flavocytochrome b-deficit cells from the homozygous patient or from the heterozygous CGD carrier. However, in gp91-phox-positive cells from both the normal and heterozygous CGD carrier, significant 4-HNE-protein adduct labeling was observed, primarily in the phagosomes. When data from single- and double-labeled cells were combined, the frequency distribution of the labels in phagosomes supported this observation, showing that neutrophils from the heterozygous CGD carrier were 71% 4-HNE-protein adduct-positive and 56% gp91-phox-positive, while cells from the normal father were > 97% positive for both 4-HNE-protein adducts and gp91-phox. These results confirmed the nitroblue tetrazolium tests of 100%, 60 +/- 2%, and 0% positive for the father's, mother's, and son's cells, respectively, and demonstrated that 4-HNE-protein adduct antibodies are useful and accurate probes of the occurrence of lipid peroxidation in vivo. We conclude that 4-HNE and resulting 4-HNE-protein adducts are generated as a result of NADPH oxidase activity in the phagosomes of human neutrophils and that these lipid peroxidation products may contribute to microbial killing and/or damage of neutrophil phagolysosomal proteins.

Identification of transmembrane tryptic peptides of rhodopsin using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

The application of mass spectrometry for determining the topography of integral membrane proteins has focused primarily on the mass determination of fragments that do not reside in the lipid bilayer. In this work, we present the accurate mass determination of transmembrane tryptic peptides of bovine rhodopsin using matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The ability to determine the accurate mass of hydrophobic transmembrane peptides will facilitate the mapping of ligand binding sites in membrane receptors. It will also augment the determination of membrane spanning regions from integral membrane proteins digested in lipid bilayers. Affinity-purified rhodopsin in detergent and rhodopsin in retinal rod membranes were digested with trypsin. Tryptic peptides were separated using reverse-phase, high-performance liquid chromatography at 55 degrees C with the detergent octyl-beta-glucoside in the mobile phase. Four of the six transmembrane tryptic peptides of rhodopsin were identified, ranging in mass from 3,260 Da to 6,528 Da. The identities of the peptides were confirmed by Edman microsequencing. In addition, heterogeneity in the glycosylation of the N-terminal tryptic peptide of rhodopsin was identified by MALDI MS, without modifying the carbohydrate prior to analysis.

Actin surface structure revealed by antibody imprints: evaluation of phage-display analysis of anti-actin antibodies.

Phage-display peptide library analysis of an anti-F actin polyclonal antibody identified 12 amino acid residues of actin that appear, in its X-ray crystal structure, to be grouped together in a surface accessible conformational epitope. Phage epitope mapping was carried out by isolating immune complexes containing members of the J404 nonapeptide phage-display library formed in diluted antiserum and isolated on a protein A affinity matrix. Immunoreactive clones were grown as plaques, replica plated onto nitrocellulose, and labeled with anti-actin immune serum. One hundred and forty-four positively staining clones identified in this way were sequenced. Of these, 54 displayed peptides with sequence similarities. When the most abundantly selected sequence, KQTWQQLWD, was produced as a synthetic peptide and derivatized to ovalbumin, the complex was strongly recognized by the antiserum on Western blots and inhibited the binding of the antibody to immobilized F-actin by 60%. A scrambled version of this sequence WQDK WLQTQ, when coupled to ovalbumin, was not recognized by the antiserum and minimally inhibited binding of antiserum to immobilized F-actin by 10%. KQTWQQLWD contained four residues that corresponded, in frame, to a highly conserved six residue region of the chicken beta-actin sequence 351TFQQMW356 (identical residues are shown in bold). Examination of the rabbit skeletal muscle X-ray crystal structure suggested that within a 15 A radius of W356, nine additional residues were arranged on the actin surface in such a way that they could be mimicked by several of the selected phage sequences with root-mean-square deviation fits of 2.1-2.5 A. We conclude that phage-display analysis can provide information about the relative location of amino acids on the surfaces of proteins using antibody imprints of the protein surface structure.

Detection of phospholipid peroxides in biological samples.

Peroxidation of membrane lipids has been hypothesized to play a key role in various types of tissue degeneration and pathology. Lipid peroxides are formed when oxygen reacts with an unsaturated fatty acid chain. Virtually all of the unsaturated fatty acids in biological systems are bound by ester linkages in phospholipids or triglycerides. Phospholipid and triglyceride peroxides are primary products of lipid peroxidation and have rarely been measured. Most of the commonly used methods for detection of lipid peroxidation are based on detection of malondialdehyde or other chemical species that are derived from oxidized fatty acids. This review presents an overview of recently developed methods aimed at identifying and measuring oxidized phospholipids and triglycerides which are direct evidence of the occurrence of lipid peroxidation in vivo.

Relative reactivity of lysine and other peptide-bound amino acids to oxidation by hypochlorite.

Antibacterial and inflammatory responses of neutrophils and macrophages produce hypochlorite as a major oxidant. Numerous side chains of amino acids found in extracellular proteins can be modified by hypochlorite, including His, Arg, Tyr, Lys, Trp, and Met. We studied the relative reactivity of each of these amino acid residues in short N-blocked peptides, where other residues in the peptide were highly resistant to hypochlorite attack. Hypochlorite treatment led to modified peptides in each case, which were detected by changes in retention on reversed-phase HPLC. A distinct single product, consuming two equivalents of hypochlorite per equivalent of peptide, was obtained from the Lys-containing peptides. UV spectroscopy, nuclear magnetic resonance (NMR), and electrospray/mass spectroscopy identified this product as the dichloramine at the epsilon-amino group of the Lys side chain. The dichloramine at Lys did not decompose to form a detectable amount of carbonyl reactive with dinitrophenylhydrazine. The dichloramine at Lys did however quantitatively revert back to Lys during HCl digestion of the tetrapeptide for amino acid analysis, with simultaneous modification of the adjacent Phe residue. The formation of the dichloramine at Lys was not blocked by peptides or acetylated amino acids that contained Tyr, His, or Arg. In contrast, the presence of equimolar Met-containing peptide, or N-Acetyl-Trp, both inhibited the formation of the dichloramine at Lys. Thus, Met and Trp side chains of proteins might be able to protect Lys from chloramine formation under some circumstances, but this interpretation must consider that Met and Trp are typically found in relatively inaccessible hydrophobic sites, whereas lysine is typically exposed on the protein surface. The hierarchy of amino acid reactivities examined here will aid in the prediction of residues in biological samples most likely to be modified by hypochlorite.

Mg2+-ATP induces filament growth from retinal rod outer segments with disrupted plasma membranes.

Mg2+-ATP produces a large decrease in near-IR light scattering when added to suspensions of rod outer segments (ROS) when the plasma membranes have been disrupted by a gentle dialysis procedure. When this process is studied by light microscopy with video-enhanced image contrast, the Mg2+-ATP-dependent signal is seen to be associated with the formation of filaments which extend only from those ROS lacking plasma membranes. Both the IR light scattering signal and filament growth are inhibited by vanadate and DCCD but not by colchicine, colcemid or cytochalasins.

Human adipose alpha-tocopherol and gamma-tocopherol kinetics during and after 1 y of alpha-tocopherol supplementation.

Alpha-tocopherol and gamma-tocopherol were monitored in human adipose by using needle biopsies in four subjects during a 1-y supplementation trial with 800 mg all-rac-alpha-tocopherol/d, and for 1 additional year after cessation of supplement. Some increase in adipose alpha-tocopherol (per milligram adipose cholesterol) and a more consistent decrease in gamma-tocopherol were observed during the supplementation period. The alpha-tocopherol/gamma-tocopherol ratio rose consistently during supplementation and fell only gradually after the supplement was stopped. We estimate that > or = 2 y are required for the alpha-tocopherol/gamma-tocopherol ratio to reach a new steady state after a change in alpha-tocopherol intake. In a cross-sectional measurement in five subjects who reported long-term use of alpha-tocopherol supplements (> or = 250 mg/d), and in five other subjects who reported no supplement use, the adipose alpha-tocopherol/gamma-tocopherol ratio clearly discriminated between the two groups (P < 0.002). This ratio may be of value in ranking individuals according to long-term alpha-tocopherol intake.