Application of ion-pair high-performance liquid chromatography to detection of the atypical coproporphyrin isomers II and IV in human faeces.

A highly selective and sensitive method has been developed for the detection of small amounts of the atypical isomers II and IV of coproporphyrin in human faeces. This method combines liquid-liquid extraction and solid-phase sampling techniques using talc and C18-modified silica gel as the sorbents. Simultaneous separation of the four coproporphyrin isomers I-IV was achieved by isocratic ion-pair high-performance liquid chromatography. Stool samples of healthy subjects (n = 12) contained 1.1 +/- 0.4% (mean +/- S.D.) isomer II and 2.2 +/- 0.9% isomer IV of total coproporphyrins. A somewhat higher content of isomer II (2.7%) and isomer IV (5.4%) was found in faeces of a patient suffering from porphyria variegata.

The isomer ratios of urinary coproporphyrins I--IV are pH-dependent.

The percentage of porphyrinogens as related to total porphyrin excretion was determined in the urine of healthy subjects. Acidic urines (pH 5.0 to 5.9) contained 62.9 +/- 10.7% (means +/- s, N = 11) porphyrinogens, whereas in neutral urines (pH 6.0 to 7.2) a somewhat lower percentage (51.2 +/- 15.3%, N = 11) was detected. However, there was no significant difference between the mean porphyrinogen contents of acidic and neutral urines. Evidence was found for a previously unreported pH-dependent influence on the isomer ratios of urinary coproporphyrins I and III. Acidic urines (N = 18) from healthy subjects showed significantly higher percentages of isomer I (27.1 +/- 6.4%), isomer II (2.7 +/- 1.1%), and isomer IV (5.0 +/- 1.3%) as compared to respective values from neutral urines (22.2 +/- 5.1% isomer I, 0.6 +/- 0.6% isomer II, and 1.5 +/- 1.3% isomer IV; N = 16, p less than 0.001). Conversely, the percentage of isomer III was markedly lower in acidic urines than in neutral urines (65.1 +/- 7.9% vs. 75.9 +/- 5.4%; p less than 0.001). The same relationship was confirmed in an individual subject by analysis of a series of urines (N = 13) with pH values ranging from 5.4 to 7.3. These results point to the possibility that the atypical coproporphyrin isomers II and IV are predominantly formed by an increased isomerization rate of coproporphyrinogens under acidic intravesical conditions.

Coproporphyrin isomers II and IV are normal constituents of human urine.

We describe for the first time the detection of small amounts of the atypical coproporphyrin isomers II and IV in urine from healthy subjects. Efficient sample preparation procedures together with a highly selective isocratic ion-pair high-performance liquid chromatographic method enabled the determination of these unexpected compounds. Formation of the atypical coproporphyrin isomers was shown to result mostly from non-enzymatic isomerization at the coproporphyrinogen level within the human body, whereas only traces were formed in vitro during collection and storage of the urine samples under the conditions applied.