Soluble lipofuscin in commercially-available human serum albumin solutions

Two studied commercial human serum albumin solutions had developed yellow colors during storage. These yellow materials were isolated and shown to be soluble lipofuscin. Aqueous solutions of this lipofuscin exhibited fluorescence spectra with 355 nm excitation and 432 nm emission maxima. After acid hydrolysis of this lipofuscin a nonhydrolysable lipid-melanin fraction was obtained. Ethanol-ether extraction yielded a lipid-containing solution. When evaporated and mixed with water, a solution-suspension was obtained that produced very similar fluorescence spectra to those described above, with 368 nm excitation and 432 nm emission maxima. The separated melanin component was not fluorescent. The isolated lipofuscin exhibited a weak electron paramagnetic resonance spectrum and its g-value has been found to be 2.0069 and its line width 9.8 G. The albumin solution contained approximately 0.23 g of melanin precipitate per 9.31 g of soluble lipofuscin isolated from 25 g of albumin. The deleterious cardiac, pulmonary, renal and clotting changes associated with the use of albumin solution might be due to this lipofuscin.     

Isolation and characterization of pteridines from heads of Drosophila melanogaster by a modified thin-layer chromatography procedure

An improved thin-layer chromatography technique is described for the separation of fluorescent compounds found in extracts of heads of Drosophila melanogaster. Eighteen to twenty fluorescent spots are resolved, two of which are xanthurenic acid and 3-hydroxykynurenine, and the remaining spots are presumably pteridines. Of these, nine have been identified and quantitated directly on the chromatograms with a fluorometer. One of the spots present on the chromatogram apparently has not been described previous to this work. Characteristics of this substance, termed quench spot, are presented, several of which indicate that it may be a pteridine or pteridine derivative.T. G. W. is a predoctoral trainee supported by Grant GM 1974 from the National Institute of General Medical Sciences, National Institutes of Health.The Oak Ridge National Laboratory is operated by Union Carbide Corporation for the U.S. Energy Research and Development Administration.     

The autofluorescent products of lipid peroxidation may not be lipofuscin-like

The fluorescent molecules of cellular age pigment granules (lipofuscin) are commonly thought to be end products of membrane lipid autoxidation. Lipofuscin fluorophores of the retinal pigment epithelium (RPE) appear to be derived from photoreceptor outer segment membranes. Experiments were therefore conducted to determine whether the in vitro oxidation of retinal homogenates would generate fluorophores similar to the naturally occurring lipofuscin fluorophores of the RPE. Neural retina and RPE-choroid homogenates from young (2-3 month old) albino rats were subjected to an iron-ascorbate-air pro-oxidant reaction medium, and compared to unoxidized control samples from young age-matched animals as well as senescent (24 month old) rats. In addition, neural retina and RPE-choroid homogenates from 3 month old albino rats were subjected to a 100% oxygen atmosphere to test whether the fluorescent products of autoxidation differ substantially from those generated in the pro-oxidant medium. The chloroform-soluble fluorophores of chloroform-methanol sample extracts were analyzed by corrected fluorescence spectroscopy and thin-layer chromatography (TLC). In vitro pro-oxidation of both the neural retina and the RPE from young rats produced blue-emitting fluorophores which differed from the orange- and yellow-emitting fluorophores extracted from the RPE of senescent rats. Corrected fluorescence spectroscopy of aged tissue extracts revealed vitamin A-related fluorescence (330 nm excitation maximum; 515 nm emission maximum) and a spectrally resolvable age-related fluorescence (420 nm excitation maximum; 600 nm emission maximum). Only the vitamin A-related fluorescence could be measured in the control of young samples.(ABSTRACT TRUNCATED AT 250 WORDS)     

Human plasma lipofuscin melanins formed from tryptophan metabolites

Incubation of human plasma with the tryptophan metabolites 3-hydroxy-DL-kynurenine (3HK) or 3-hydroxyanthranilic acid (3HAA) yields soluble brown pigments with the fluorescence spectrophotometric and paper chromatographic properties of plasma lipofuscin (PL). An alternative to the recognized tyrosine pathway of melanogenesis is therefore demonstrated in human plasma.     

Isolation of melanin from human plasma lipofuscin

Human plasma lipofuscin and its melanin component were isolated and quantified. Electron paramagnetic resonance, infrared, ultraviolet and visible spectra of this melanin exhibited absorption characteristics very similar to those of known melanins. The human plasma lipofuscin contained approximately 85% protein, 3% melanin, 0.4% lipid and 0.25% mucoprotein constituents and emitted yellow-green fluorescence in 366-nm light. The ethanol-ether lipid extract obtained after acid hydrolysis from the lipid-melanin fraction of this lipofuscin was also found to fluoresce in yellow-green color in 366-nm light and produced similar fluorescence excitation and emission spectra as those of the human plasma lipofuscin in water solution. The isolated melanin component was not fluorescent.     

Lipofuscin like compound in mango

Thin layer chromatographic separation of chloroform-methanol extracts of mango on silica gel revealed a fluorescent substance in mango peel and pulp. The compound had fluorescence spectrum similar to that of lipofuscin, the age pigment of animal tissues and was found to be water insoluble and stable to ultraviolet irradiation. The fluorescent material appeared to be a lipoprotein.     

Isolation of Biopterin-α-glucoside from Spirulina (Arthrospira) platensis and Its Physiologic Function

A fluorescent substance was isolated from the cyanobacterium with a yield of 4.5 mg per 10 g of dried Spirulina (Arthrospira) platensis cells by gentle extraction and ethanol fractionation followed by column chromatography. The fluorescent substance, which has absorption maxima at 256 nm and 362 nm (pH 8.4), was identified as biopterin-α-glucoside by spectrophotometry and nuclear magnetic resonance spectroscopy. Biopterin-α-glucoside prevented decolorization of the photosynthetic pigments, chlorophyll a, phycocyanin, and carotenoids in photosynthetic vesicles of Spirulina platensis cells, by ultraviolet irradiation. Received June 23, 1998; accepted September 10, 1998.     

Studies on rheomelanins. IV. The apparent occurrence in vivo of rheomelanins in human blood.

Paper chromatograms of the rheomelanins made earlier in this laboratory in human plasmas during incubation with each of the catecholamines or with L-dopa had yellow-green fluorescence in ultraviolet light of 366 nm (Hegedus & Altschule, 1970). In the present studies, a yellow-green fluorescent spot was found in each paper chromatogram. All of these spots were eluted and their excitation and emission spectra were recorded and compared to one another. The rheomelanins made from the catecholamines, L-dopa, catechol or from mixtures of these in human plasmas during incubation were chromatographed twice on paper with two different solvent systems. These artificial rheomelanins in vitro and the apparent in vivo rheomelanins present in plasmas moved together during the two chromatographies. These yellow-green fluorescent compounds were eluted as one spot after the second chromatography. This mixture produced high intensity excitation and emission spectra closely similar to the low intensity excitation and emission spectra of the apparent in vivo rheomelanins of unincubated human plasmas treated the same way without any chemical under nitrogen. The RF-values of the various rheomelanin spots after each chromatography were closely similar. These above results were also obtained with other solvent systems. It appears therefore that rheomelanins are formed in vivo in the human body.     

Detection of age-related fluorescent substances in rat tissues

Studies are reported on the detection of fluorescent substances and their relationship to the accumulation of the aging pigment and lactate dehydrogenase (LDH) isoenzyme composition in tissues of the rat. Comparison of fluorescent substances in nine tissues in 6- and 100-week-old rats by thin-layer chromatography showed that one substance in particular accumulated with age. This substance exhibited the typical excitation-fluorescence spectrum of lipofuscin pigments and its detection in tissues correlated closely with histologic analysis of the aging pigment. Accordingly, it was designated as an age-related fluorescent substance (ARFS). Because of the high intensity of its fluorescence and its relatively small mass, it appeared to be a constituent of aging pigment that was dissociated and extracted upon treatment of the tissue with chloroform-methanol. Histochemical analysis revealed the presence of both hemosiderin deposits and a golden brown pigment which was devoid of iron. These substances as well as the ARFS appeared to accumulate more in tissues which were classified generally as aerobic based on their LDH isoenzyme composition.     

Solid-phase purification and analysis of dicarboxylic porphyrins extracted from cultured tumor cells

We describe here a sensitive method for the purification and analysis of porphyrins present in hematoporphyrin derivative. Hematoporphyrin derivative is a solution containing a complex mixture of dicarboxylic porphyrins such as hematoporphyrin IX, monohydroxyethyl monovinyl deuteroporphyrin isomers, and protoporphyrin IX in addition to porphyrin aggregates of variable molecular sizes. This mixture is known for its ability to be selectively retained by tumor cells and for its cytotoxicity in the presence of light. In order to study the mechanisms of hematoporphyrin derivative uptake and its cellular metabolism, extraction methods are required that combine high recoveries with minimum changes of very labile components. Extraction with perchloric acid: methanol mixtures recovered only some 60% of the porphyrins taken up by tumor cells and artifactual fluorescent spots were seen on thin-layer chromatograms. Improved yields were obtained upon extraction with dimethyl sulfoxide or Triton X-100:4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (Hepes) buffer mixture, but the extracts were not suitable for reverse-phase thin-layer chromatography (RTLC). The procedure described here consists of extracting porphyrins from cultured tumor cells with a buffered detergent followed by sequential chromatography on DEAE-cellulose columns and on reverse-phase octadecylsilyl cartridges. Identification of the isolated free dicarboxylic porphyrins is conveniently done by RTLC.     

Actinomycin synthesis in Streptomyces antibioticus: enzymatic conversion of 3-hydroxyanthranilic acid to 4-methyl-3-hydroxyanthranilic acid.

A methyltransferase which utilizes 3-hydroxyanthranilic acid (HAA) as a substrate was identified in detergent-treated extracts of the bacterium Streptomyces antibioticus. The enzyme catalyzes the transfer of methyl groups from [14C]S-adenosylmethionine to HAA, but does not catalyze the methylation of 3-hydroxy-DL-kynurenine. Enzyme, substrate, time, and pH dependencies for the methyl transfer reaction were examined. Reaction products obtained from scaled-up reaction mixtures were fractionated by chromatography on Dowex 1, and the Dowex 1 fractions were examined by paper and thin-layer chromatography. One Dowex fraction was shown to contain a radioactive product with the chromatographic properties of 4-methyl-3-hydroxyanthranilic acid (MHA), a known intermediate in the biosynthesis of actinomycin. Available evidence indicates that the conversion of HAA to MHA is an early step in the biosynthesis of actinomycin by S. antibioticus and other actinomycin-producing streptomycetes.     

Preparation of Water and Ethanolic Extracts of Propolis and Evaluation of the Preparations

Propolis was extracted using water and various concentrations of ethanol as solvents. The extracts were investigated by measurement of absorption spectrum with a UV spectrophotometer, reversed phase-high pressure thin-layer chromatography and reversed phase-HPLC. Maximum absorption of all extracts was 290 nm, resembling flavonoid compounds, and the 80% ethanolic extract showed highest absorption at 290 nm. The most isosakuranetin, quercetin, and kaempferol were extracted from mixtures of propolis and 60% ethanol, while 70% ethanol extracted the most pinocembrin and sakuranetin, but 80% ethanol extracted more kaempferide, acacetin, and isorhamnetin from propolis. The 60 to 80% ethanolic extracts of propolis strongly inhibited microbial growth and 70 and 80% ethanolic extracts had the greatest antioxidant activity and 80% ethanolic extract strongly inhibited hyaluronidase activity.     

Elevated levels of plasma lipofuscins in patients with chronic renal failure

The fluorescence excitation and emission spectra observed in plasma from patients with chronic renal failure were reproduced by the generation of soluble lipofuscins in normal plasma samples by incubation with mixtures of L-dopa, dopamine, L-norepinephrine, L-epinephrine, 3-hydroxy-DL-kynurenine and 3-hydroxy-anthranilic acid for 24 h at 37 degrees C. Relative fluorescence intensity measurements consistently showed elevated plasma levels of the soluble lipofuscins in chronic renal failure: the means (n = 27) were 73.9 +/- 33.4 (SD) and 71.1 +/- 14.8 at emissions 413 nm and 445 nm respectively, in contrast to those of normal plasma samples (n = 11), 18.2 +/- 5.3 and 23.1 +/- 5.6. The maximum or shoulder at approximately 413 nm represents soluble lipofuscin that can be generated from 3-hydroxyanthranilic acid and the maximum or shoulder at approximately 445 nm represents soluble lipofuscins derived from the precursors listed above and probably from other related precursors. Gravimetric measurements also showed elevated levels of melanins in the plasma samples of patients with chronic renal failure: 2.72 +/- 0.38 mg/ml (n = 16), as compared to normal values: 1.70 +/- 0.10 mg/ml (n = 6). In individual patients haemodialysis reduced the fluorescence intensities to a range of 65-99% and the melanin levels to a range of 86-99% of the pre-dialysis values.     

Serum glycoconjugates in children with schizophrenia and conduct and adjustment disorders

The serum glycoproteins represented by the individual protein-bound carbohydrate components and glycosaminoglycans represented by the hexuronic acid contents were determined in the sera of black and Caucasian normal children and children with diagnoses of schizophrenia, conduct disorder, and adjustment disorder. There were no race-related or sex-related differences in glycoproteins and glycosaminoglycans in the sera of normal children. Although the serum glycosaminogltents were determined in the sera of black and Caucasian normal children and children with diagnoses of schizophrenia, conduct disorder, and adjustment disorder. There were no race-related or sex-related differences in glycoproteins and glycosaminoglycans in the sera of normal children. Although the serum glycosaminogltents were determined in the sera of black and Caucasian normal children and children with diagnoses of schizophrenia, conduct disorder, and adjustment disorder. There were no race-related or sex-related differences in glycoproteins and glycosaminoglycans in the sera of normal children. Although the serum glycosaminoglycans were significantly elevated in children with a diagnosis of schizophrenia, the levels were in normal range in children with conduct and adjustment disorders. All of the protein-bound carbohydrates were elevated in schizophrenic children. However, only arabinose and galactosamine were significantly elevated in children with a diagnosis of conduct disorder, while only galactosamine was elevated in children with adjustment disorder. The presence of arabinose in serum glycoprotein was confirmed by chemical ionization-mass spectrometry. The possible causes of the differential elevation of the glycoconjugates in psychiatric disorders in relation to the effect of stress and environment are discussed.     

The anemia of chronic renal failure: in vitro response of bone marrow to erythropoietin.

Bone marrow cells of patients with chronic renal failure were studied in short-term in vitro cultures to determine erythropietin responsiveness. Seven normals and fourtheen patients on hemodialysis were studied. Bone marrow cells of normal subjects and of patients with chronic renal failure responded similarly to erythropoietin. Total heme synthesis was significantly lower in cultures prepared with uremic serum than normal serum. We conclude that there is a substance in the serum of uremic patients which suppresses general heme synthesis and that this "uremic toxin" may be responsible, in part, for the clinically severe anemia seen in these patients.     

Fluorescent pigments in the newly isolated methylotrophs: Pseudomonas J16 and Methylomonas Pl1.

The pigments showing fluorescence maxima at 390, 366, 450-460 and 520 nm at excitation wavelength 254, 366 and 450 nm respectively, were detected in the cells and culture media of the obligate methylotroph Methylomonas Pl1 and facultative methylotroph Pseudomonas J26. The maximum at 520 nm is associated with the occurrence of a flavin pigment enabling growth of Lactobacillus casei E ATCC-7469 on the vitamin B2 deficient medium. The remaining fluorescence maxima are related to the prosthetic group of methanol dehydrogenase.     

Studies on rheomelanins. V. Hemolysis associated with the transformation of catechol into rheomelanin in human blood.

Incubation of 2 mg amounts of catechol in 5 ml samples of heparinated blood plasma from four subjects at 38 degrees C for 24 h produced plasma-soluble rheomelanins. These solutions had the brown color and the yellow-green fluorescence in ultraviolet light of 366 nm of other rheomelanins. Their differential ultraviolet and visible spectra showed a rheomelanin absorption maximum at 344 nm. Paper chromatograms of the rheomelanin-plasma solutions in 5% methanol-95% water showed elongated spots of rheomelanins with RF values of 0.82, on Whatman No. 1 paper. Using heparinated distilled water adjusted to pH 7.4 with sodium bicarbonate instead of human blood plasma gave markedly different findings from those obtained with the plasma rheomelanin solutions. Incubation of 4 mg amounts of catechol in 10 ml samples of heparinated whole blood from four subjects for 24, 32 and 48 at 38 degrees C produced rheomelanins as found in the plasma separated from the blood after incubation. The differential ultraviolet and visible spectra of these solutions revealed hemolysis caused by the catechol rheomelanins; this was more marked with longer incubations. The hemolysis was manifested by two absorption peaks at about 270 and 400 nm. Paper chromatography revealed the brown elongated spots of catechol rheomelanins with an RF value of 0.82. Other spots owing to the products of hemolysis were also present.     

Inhibition of normal rat macrophage functions by soluble tumor products

Summary The phagocytic and chemotactic activities of normal rat peritoneal macrophages were inhibited by sera from tumor-bearing rats (TBR) and 3 M KCl extracts of tumor mass. However, sera from Corynebacterium parvum- or Listeria monocytogenes-treated TBR did not inhibit phagocytosis. On the other hand, sera from C. parvum-treated, but not from L. monocytogenes-treated TBR still inhibited the chemotactic response of the normal macrophages. Furthermore, 3 M KCl extracts of tumors from C. parvum-treated TBR did not inhibit phagocytosis and chemotactic response of the same cells. Similar results were obtained with extracts of tumor masses from L. monocytogenes-treated rats. It is suggested that treatment with bacterial immunomodulators can influence the release from neoplastic cells of soluble products influencing normal macrophage functions.     

Characteristics of the composition of fluorescent lipopigments of the liver and brain in rats of various ages

Spectral characteristics of the lipid liver and brain extracts were studied in experiments on rats of different age (1, 3, 12, 24 months). The aim of the experiments was to study age peculiarities of the lipopigments composition by means of thin-layer and liquid chromatography. Different fractions of lipids which differ in spectral absorption and fluorescence were extracted. Proceeding from the results obtained certain conclusions may be derived: the processes of structural recombination of the whole lipid constituents are accompanied by the age accumulation of lipofuscin; the process of accumulation of lipofuscin is not monotonous and has pronounced age peculiarities; the formation of the lipid-pigment complex is tissue-specific.     

A new HPLC analytical method for o-hydroxyhippuric acid in uremic serum

o-Hydroxyhippuric acid (HHA) is formed in liver during the process of detoxication of salicylic acid which arises from either salicylic-containing dietary vegetables or hydrolysis of aspirin. Recently, HHA has also been shown as one of the so-called 'uremic toxins'. By derivatization of HHA with o-phthaldialdehyde (OPA), the resulting fluorescent product can easily be measured with the limit of measurement somewhere below 3 pmol on high performance liquid chromatography. Using the method described, an approximately 85% recovery of added HHA in human sera was obtained. This analytical system is now being employed to determine the concentration of HHA in human sera of uremic patients. Determination of HHA content in human uremic patients is very important, since uremia is associated with defective binding of many acidic drugs to serum protein(s) and HHA replaces these drugs by tightly binding to these proteins. This could potentially affect the therapeutic effectiveness of various pharmacologic agents.