Henning Nielsen

Covalent binding of peroxidized phospholipid to protein: III. Reaction of individual phospholipids with different proteins

Various peroxidized phospholipids were reacted with proteins under N2. In all cases, phospholipid is bound covalently to the proteins whose molecular size is increased. Both the amount of bound phospholipid and the increase in molecular size of the protein depends on the nature of the phospholipid. Ultraviolet (UV) absorption of the proteins is increased in qualitatively similar ways. Their difference spectra, which show a gradual increase in absorption from 400 nm toward shorter wavelength, differ from that of malonaldehyde-protein complexes. The various complexes of proteins and peroxidized phospholipids have similar fluorescence spectra showing two excitation maxima at 310–320 nm and at 340–350 nm, respectively, and emission maximum at ca. 400 nm. This is different from both fluorescence spectra of malonaldehyde-protein complexes and fluorescence spectra reported for proteins after reaction with peroxidized polyunsaturated fatty acids. Amino groups of the proteins are consumed in the reaction with peroxidized phospholipids. Blocking the amino groups decreases the binding of phospholipid considerably. Besides amino groups, other structures of the protein molecule react with the peroxidized phospholipids. The similar features of UV absorption, fluorescence, decrease of amino groups, and covalently bound phospholipid phosphorus of the various complexes suggest that they are formed by common type of reactions. The reactions seem to be different from those generally believed important between peroxidized lipid and protein. Important reacting species are compounds other than malonaldehyde.

Reaction of phenylhydrazine with erythrocytes: Cross-linking of spectrin by disulfide exchange with oxidized hemoglobin

Phenylhydrazine causes deleterious oxidations of components of erythrocytes. These reactions and their effects on the mechanical properties of rabbit erythrocytes are investigated to provide insight into the mechanisms leading to destruction of oxidatively damaged erythrocytes. After 1 hr of incubation with phenylhydrazine, precipitated denatured protein (Heinz body protein) amounts to 25-30% of membrane protein, but deformability of erythrocytes as measured by filtrability is unchanged. After 4 hr of incubation filtrability drops sharply. Polymerization of spectrin and covalent binding of hemoglobin to spectrin, but no peroxidation of membrane lipids is observed. Precipitated protein amounts to 85-95% of membrane protein. It contains Fe, porphyrin and globin peptide in the proportion 1:1:1. Heinz body protein precipitated when hemoglobin is incubated under similar conditions has 90% of its sulfhydryl groups oxidized and no other amino acids than cysteine are destroyed. Addition of this Heinz body protein to erythrocyte ghosts causes polymerization of spectrin. Incubation of tetrathionate, a specific cross-linking agent, causes filtrability to drop sharply after about 80 min. This effect is similar to that observed after 4 hr incubation with phenylhydrazine, and is accompanied by polymerization of spectrin and band 3. The results indicate that cross-linking of membrane proteins by disulfide exchange with precipitated hemoglobin may play a major role in decreasing deformability during incubation with phenylhydrazine.

A pivotal role of the human kidney in catabolism of HDL protein components apolipoprotein A-I and A-IV but not of A-II.

Renal handling of major HDL components was studied by analyzing urine from patients with Fanconi syndrome, a rare renal proximal tubular reabsorption failure, including dysfunction of the kidney HDL receptor, cubilin. A high urinary excretion of apolipoprotein A-I and A-IV corresponding to a major part of the metabolism of these proteins was measured. In contrast, no urinary excretion of apolipoprotein A-II which is more hydrophobic and tighter bound to HDL was found. Control urines displayed absence of the three apolipoproteins. Urinary excretion of phospholipids, triglycerides, cholesterol and cholesterol esters in patients was as low as in controls. In conclusion, these data indicate that the human kidney is a major site for filtered nascent apolipoprotein A-I and A-IV but not for HDL particles.

Accumulation of triglycerides in the proximal tubule of the kidney in diabetic coma

Aims: The present study was initiated by a very recent histochemical observation of lipid accumulation in the renal cortex of a woman who died in a diabetic coma. Two older reports of lipid accumulation in the kidneys of patients who died, most likely in a state of non‐regulated diabetes, supported this observation. We have examined whether lipid accumulation in the renal cortex is characteristic of diabetic coma and, if so, which type of lipid accumulates. Methods: Three groups were studied. Ten subjects who died in a diabetic coma, eight diabetics who died of known causes unrelated to diabetes, and seven normal control subjects without any diagnosed diabetes who died of known causes. All were subjected to histological examination for lipid accumulation in the renal cortex. Detailed analysis of cortex lipids was performed for two of the subjects who died in a diabetic coma and all diabetic controls as well as non‐diabetic control subjects. Results: All subjects who died in a diabetic coma showed vacuolar lesions staining strongly for lipid in the proximal tubules. Neither normal controls nor non‐coma diabetics showed these lesions. Compared with normal controls, renal cortex lipid was about tripled in the two analysed diabetic coma subjects due to 60–100‐fold increases of triglycerides. The non‐coma diabetics did not differ from the other controls with respect to triglycerides or other types of lipid, except that cholesteryl esters were elevated, though still a quantitatively minor component. Conclusion: Our findings strongly indicate that vacuolar lesions in the proximal tubules are characteristic of diabetic coma and that they are caused by accumulated triglycerides. Therefore, histological examination of renal cortex using a lipid stain may be a useful forensic tool in establishing diabetic coma as the cause of death.

Effect of genetic variation on the fatty acid-binding properties of human serum albumin and proalbumin

In the circulation, non-esterified fatty acids are transported by albumin which also facilitates their removal from donor cells and uptake into receptor cells. We have studied whether genetic variations in the albumin molecule can affect its in vivo fatty acid-binding properties. The fatty acids bound to 25 structurally different variants and to their wildtype counterparts, isolated from heterozygous carriers, were determined gas chromatographically. The variants were proalbumins, albumins with single amino acid substitutions and glycosylated or truncated albumins. In eight cases the total amount bound to the variants was diminished (0.4-0.8-fold), and in seven cases the load was increased to 1.3 or more of normal. Twenty-one fatty acids were quantitated, and for 19 alloalbumins significant deviations from normal were found. Usually, changes in total and individual fatty acid binding were of the same type, but several exceptions to this rule was found. The glycosylated albumin Casebrook showed the largest changes, the total load and the amount of bound palmitate was 8.6 and 14 times, respectively, the normal. The most pronounced changes and the majority of cases of increased binding were caused by molecular changes in domain III. Mutations in domain I, II and the propeptide resulted in smaller effects, if any, and these were often reductions in binding.

Reaction between peroxidized phospholipid and protein: I. Covalent binding of peroxidized cardiolipin to albumin

A system is described for study of the reaction of peroxidized cardiolipin (diphosphatidylglycerol) with albumin. Covalent binding of peroxidized cardiolipin to albumin occurs in this system as evidenced partly by a decrease in lipid-extractable P and partly by lipid P being inseparable from the albumin by gel filtration in the presence of a detergent (sodium deoxycholate) under conditions known to separate the lipid moiety and apoprotein of lipoproteins. Based on analyses of the decrease in lipid-extractable P, the average number of cardiolipin molecules bound covalently per molecule of albumin is about 5 when peroxidized cardiolipin (4 moles O2/mol cardiolipin) at about 10 times molar excess is allowed to react with albumin. However, the data of the gel filtration experiment indicate that the bound peroxidized cardiolipin molecules may not be evenly distributed on the albumin molecules. Therefore, the number of cardiolipin molecules bound per albumin molecule may actually vary over a range and be considerably higher for part of the albumin. The findings have been discussed in relation to peroxidation in vivo.

Structural characterization, stability and fatty acid-binding properties of two French genetic variants of human serum albumin.

Four bisalbuminemic, unrelated persons were found in Bretagne, France, and their variant and normal albumins were isolated by DEAE ion exchange chromatography, reduced, carboxymethylated and treated with CNBr. Comparative two-dimensional electrophoresis of the CNBr digests showed that three of the variants were modified in fragment CB4, whereas the fourth had an abnormal fragment CB1. These fragments were isolated, digested with trypsin and mapped by reverse-phase HPLC. Sequencing of altered tryptic peptides showed that the three variants modified in CB4 were caused by the same, previously unreported, amino acid substitution: Asp314-->Val (albumin Brest). The fourth, however, was a proalbumin variant with the change Arg-2-->Cys (albumin Ildut). Both amino acid substitutions can be explained by point mutations in the structural gene: GAT-->GTT (albumin Brest) and CGT-->TGT (albumin Ildut). The proalbumin Ildut is very unstable and already in vivo it is to a large extent cleaved posttranslationally to Arg-Albumin and normal albumin. Furthermore, we observed that during a lengthy isolation procedure the remaining proalbumin was changed to Arg-Albumin or proalbumin lacking Arg-6. In addition, part of normal albumin had lost Asp1. Gas chromatographic investigations using isolated proteins indicated that albumin Brest has improved in vivo fatty acid-binding properties, whereas the structural modification(s) of albumin Ildut does not affect fatty acid binding.

On the structure of rat tissue cardiolipins.

Abstract The object of the present investigation was to study the new structure(s) suggested for rat tissue cardiolipins. 1. 1. Conditions are given for a chromatographic procedure by which cardiolipin is isolated free of other phospholipids. 2. 2. Cardiolipins from rat liver, heart, kidney and ox heart were studied. 3. 3. The cation composition of the cardiolipins was similar with about 90% of the cation equivalents being calcium and about 10% magnesium. The cation: P equivalence ratio was approx. 1. 4. 4. The P content of the rat tissue cardiolipins, that were isolated by silicic acid column chromatography, was 3.59, 3.83 and 3.61% for liver-, heart- and kidney cardiolipin, respectively. After Chromatographic purification of their sodium form, the P content of the tissue cardiolipins was found to be very close to the theoretical value of 4.15%. 5. 5. The percentage of cardiolipin phosphorus rendered water-soluble by mild acid hydrolysis was shown to be deeply influenced by divalent metal ions bound to the cardiolipins. When the Ca/Mg form of the cardiolipins was subjected to mild acid hydrolysis, a minor part only of the cardiolipin phosphorus was rendered water-soluble, whereas approx. 80% of the cardiolipin phosphorus was rendered water-soluble when the divalent metal ions had been replaced by sodium prior to the hydrolysis. The divalent metal ions were shown to affect the partitioning of the phosphorus-containing product of hydrolysis between the water phase and the organic phase rather than the extent of hydrolysis. 6. 6. Spectral studies of the cardiolipins in the region 220–400 nm revealed only an absorbance characteristic of autoxidized lipid. 7. 7. The data obtained for rat tissue cardiolipins are consistent with the structure of beef-heart cardiolipin and synthetic cardiolipin.

Reaction between peroxidized phospholipid and protein: II. Molecular weight and phosphorus content of albumin after reaction with peroxidized cardiolipin.

Peroxidized cardiolipin (diphosphatidylglycerol) reacts covalently with albumin. Incubation of albumin with increasing amounts of peroxidized cardiolipin produces a gradual increase in molecular size. Incubation with a small amount of peroxidized cardiolipin (molar ratio of cardiolipin/albumin 21) produces a mixture of complexes that differs considerably with respect to the number of cardiolipin molecules bound per molecule of albumin. With larger amounts of peroxidized cardiolipin (molar ratios of cardiolipin/albumin 54 and 114), the complexes formed seem to be of a more uniform type since the numbers of cardiolipin molecules bound per molecule of albumin are similar. No polymerization occurs for reactions in which up to at least 15 moles of cardiolipin have become bound per mole of albumin, and 20–25 moles may be bound with only very little polymerization. Only when the ratio of peroxidized cardiolipin to albumin was increased to a high value of 314 did polymerization occur. The present findings show that extensive covalent binding of peroxidized cardiolipin to albumin can occur without intermolecular crosslinking of the protein.

Silica gel thin-layer chromatography of acidic phospholipids : I. Effect of metal ions of the adsorbent and phospholipid on the chromatographic mobility of cardiolipin and phosphatidylinositol

Abstract The monovalent and divalent salt forms of two acidic phospholipids (cardiolipin and phosphatidylinositol) have been subjected to thin-layer chromatography on two different adsorbents commonly used for the chromatography of lipids. It was found that in some thin-layer chromatographic systems, an acidic phospholipid displays different chromatographic behaviour when applied to the plate as its monovalent salt and when applied as its divalent salt. The results show that misleading results will often occur in the thin-layer chromatography of acidic phospholipids unless the ionic states of both the phospholipid and the adsorbent are properly appreciated.