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Peroxidized unsaturated fatty-acids show fluorescent properties in the absence of amino groups. Compounds formed from linolenic acid autooxidized for 5 days have been separated on thin-layer chromatograms with paired zero-time controls then located by their thiobarbituric acid and peroxidase reactivity. Spectrofluorimetric analysis of the eluted 5-day zones showed that two had ultraviolet fluorescence and a third visible fluorescence.

Biliverdin reacts with thiobarbituric acid to give a red chromogen with absorption peaks at 560, 530 and 465 nm in acid solution and 602, 566 and 482 nm in alkaline solution. The reaction in acid solution could interfere with lipid peroxidation studies in which malondialdehyde is measured at 532 nm. The TBA reaction in biological fluids is discussed together with its use as a possible measure of bile pigments.

1. The inhibitory (antioxidant) effect of serum on the autoxidation of tissue polyunsaturated fatty acids has been investigated. 2. Dialysis studies, DEAE-cellulose chromatography, gel filtration, immunoelectrophoresis and immunodiffusion experiments showed that this effect is the function of two serum protein fractions. 3. One fraction was identified as transferrin. The second fraction includes caeruloplasmin. 4. Studies with radioactively labelled α-tocopherol showed that serum tocopherol in the physiological concentration range contributes only marginally to the total antioxidant activity of serum. 5. These findings are discussed in the wider context of biological antioxidant protection.

The polymerization of malonyldialdehyde (MDA) formed after the acid hydrolysis of 1,1,3,3-tetraethoxypropane (TEP) and 1,1,3,3-tetramethoxypropane (TMP) has been studied using techniques of gas-liquid, thin-layer, and gel-filtration chromatography. The results show that, even while hydrolysis is still in progress, low-molecular-weight polymers of MDA begin to form. After hydrolysis at least 13 different polymers could be detected. Several of these had absorption peaks in both the visible and the ultraviolet region. With the thiobarbituric acid reagent (TBA) at room temperature the various polymers produced different colour reactions; but heating in 28% trichloroacetic acid leads to depolymerisation and the formation of the characteristic 532 nm MDA TBA coloured complex. MDA polymers could not be shown to account for any of the multiple TBA-reactive compounds produced by autoxidised linolenic acid. In experimental work designed to study the biological activity of MDA in its own right, the use of TEP or TMP as the source material and the strong tendency of MDA to polymerise must be taken into account.

An Auto-Analyzer system is described for the quantitative assay of urinary 4-hydroxy-3-methoxymandelic acid (‘vanilmandelic acid’, VMA) following manual solvent extraction at controlled pH. VMA is oxidised with peroxide, enzymically catalysed by peroxidase, and the coloured hydrazone formed from the oxidation product and added 2,4-dinitrophenylhydrazine is measured in alkali at 480 nm. A blank on each sample is run in the absence of peroxidase.

A simple technique is described by which normetadrenaline (NMA), metadrenaline (MA), 4-hydroxy-3-methoxymandelic acid (VMA) and 4-hydroxy-3-methoxy phenylglycol (HMPG) can be demonstrated and visually assessed on thin-layer chromatography plates. Patients showing an abnormal 24-h excretion patterns are further investigated by collecting sequential samples over a 24-h period. In these the concentration of the metabolites is related to diurnal variations in urinary osmolality.

A simple screening test for vitamin B 12 deficiency is described. It depends on the semi-quantitative visual assessment of urinary methylmalonic acid excretion. Chromatography is carried out on silica gel. Location with a Fast Blue B reagent increases sensitivity over existing methods, allowing detection of as litte as 0.1 mg/100 ml.

Summary The peroxidation of ox-brain phospholipid liposomes catalysed by oxidised and reduced iron and copper ions have been studied by the measurement of fluorescent non-polar MDA-amino complexes. The inhibitory activity of superoxide dismutase when added to the system implicated superoxide radicals as important intermediates in both reduced and oxidised metal-ion catalysed peroxidation of lipids.

The effect of calcium ions on the peroxidation of ox-brain phospholipid liposomes in different free-radical catalysing systems has been assessed. Using thiobarbituric acid-reactivity (TBA) as a measure of lipid peroxidation, calcium ions both inhibited and enhanced peroxidation in the different systems. Changing the composition of the ox-brain phospholipid liposome with synthetic non TBA-reactive phosphatidylcholine, significantly altered its susceptibility to peroxidation both in the presence and absence of calcium ions. The results are discussed with reference to the possibility that calcium ions induce conformational changes in membrane phospholipids. Susceptibility to peroxidation is then influenced by a complex interrelationship between the qualitative lipid composition of the membrane, the pro-oxidant catalyst and the presence of calcium or other active ions.

Thin-layer chromotographic techniques are described for the preparation and separation of water-soluble compounds formed by the autoxidation of pure linoleic and linolenic acids. Location of 15 main fractions with Schiffs reagent on fibreglass sheets was possible. These could be eluted for biological testing and chromogenic behaviour with thiobarbituric acid reagent. At least 7 bands from autoxidized linoleic acid and 9 from autoxidized linolenic acid had peak absorbance maxima at 532 nm identical to that characteristically given by malonyldialdehyde (MDA). Possible reactions and interpretations for the MDA test are discussed.

The specificity of the thiobarbituric acid reaction (TBA) has been investigated using techniques of high-performance thin-layer chromatography and spectrofluorimetry. It was found that malondialdehyde (MDA) derived from different lipid and nonlipid origins formed the same MDA-TBA complex. This complex could be separated from other TBA-reactive compounds by both chromatography and spectrofluorimetry. Normal human plasma and urine both formed an MDA-TBA complex along with other TBA-reactive compounds. In plasma this was associated mainly with phosphatidylcholine and appeared to be peroxidic in reaction. Urine, however, contained polar MDA-forming compounds probably resulting from the oxidation of 2-deoxyaldoses during the acid-heating stage of the TBA test.

A new colorimetric method for the assay of biliverdin in biological fluids is described. The method, based upon the reaction of biliverdin with barbituric acid, offers improved sensitivity and selectivity when compared to direct spectrophotometric measurements. Using this method biliverdinaemia was observed in two patients with obstructive jaundice of malignant origin.