Uroporphyrin chromatography

Increased porphyrins in clear fluid such as urine may be detected directly by their pink fluorescence if exposed to long ultraviolet (Fig. 7.3.2). The specificity of this screening assay may be improved if porphyrins are extracted by talcum [8]. These isolated porphyrins may be quantified using a spectrofluorimeter. As different porphyrias show specific excretion patterns, separation of the main porphyrins is desirable. The formerly used fractionated extraction enabled to separate the uroporphyrin fraction from the coproporphyrin fraction. In addition to uroporphyrin, the first fraction includes heptacarboxy- and part of hexacarboxyporphyrins, and in addition to coproporphyrin, the second fraction includes part of hexacarboxy- and pentacar-boxyporphyrins. Later on, thin-layer chromatography of methylester derivatives is used. 

Table 7.3.3 Example mean reference values for uroporphyrin, coproporphyrin I, and coproporphyrin III (these can only be interpreted as a guidance) collected by means of high-performance liquid chromatography (HPLC). The intermediary porphyrins were below the detection limit...

The extraction method for fecal porphyrins results in some interference with the chromatography caused by a proportion of the diethyl ether dissolvmg in the aqueous phase. As a result an extra peak elutes just before the uroporphyrin position. This peak contains any uroporphyrin in the sample, up to 50% of the heptacarboxylate porphyrins and smaller amounts of hexacarboxylate and pentacarboxylate porphyrins. 

Guo, R., and C. K. Lim. 1991. Determination of hydroxy and peroxy acid derivatives of uroporphyrin in the plasma of patients with congential erythropoietic porphyria by high-performance liquid chromatography. 7. Chromatography 550 603-7. 

As indicated in our earlier article, the direct polymerization of monopyrroles to porphyrins is of little synthetic value as far as Ae unsymmetrical natural pigments are concerned. However, this might provide a feasible approach at l( ast to the copro- and uroporphyrins if sufficiently sophisticated methods could be developed for their chromatographic separation on a preparative basis. This has already been achieved analytically by high performance liquid chromatography (hplc). ... 

Porphobilinogen when boiled in 0.5 N HCl for 20 minutes is converted to uroporphyrin to the extent of 40%. Cookson and Rimington isolated a crystalline octamethyl ester which had a m.p. of 255°. The major part of this material behaved as uroporophyrin III on paper chromatography, but at least one other isomer was present. To explain the formation of uroporphyrin III from porphobilinogen it may be necessary to assume some branched tri- or tetrapyrrylmethane as an intermediate, perhaps formed by a Corwin condensation vide infra). 

Uroporphyrinogen is an intermediate in the synthesis of haem. In certain types of porphyria there is increased conversion and excretion of uroporphyrins (porphyrins have acetate and propionate substituents). Elevated levels can be detected in faeces and urine by various screening tests (see porphyrins). If elevated urine levels of porphyrins are found, they can be identified as copro - or uroporphyrins by column or thin-layer chromatography. They can also be resolved by selective solvent extraction and estimated spectrophotometrically. 

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