• Determination of total porphyrines in urine

Chromatography

Determination of total porphyrines in urine

Oct 05 2011

General
Porphyrines are colored compounds occurring ubiquitous in the flora and fauna, for example in chlorophyll and vitamin B12. As the chemical matrix for heme, the color of the red blood cells, they are of essential importance in the transportation of blood oxygen in the metabolism of almost all organisms. The decomposition products of the blood color are responsible for the coloring or urine. With the column test developed by RECIPE® and subsequent absorption measurement using the SPECORD® 250 PLUS by Analytik Jena AG the total porphyrine content of the urine can be determined quickly and with ease. This test is used to verify metabolic illnesses, so-called porphyria, caused by enzyme defects or problems with porphyrine production.

Procedure
For the sample preparation 24h urine samples were taken and buffered with a 0.3% sodium hydrogen carbonate solution to a pH value of between 6 and 7. From the control and patient samples 1ml each were pipetted onto the prepared columns which were flushed with 10ml distilled water after run-out of the reagent. The cycle was discarded and then 2 x 1ml elution reagent each was pipetted onto the columns within 15 minutes. The eluate was then subjected to photometric analysis.

The samples were measured in cuvettes with a pathlength of 10mm and compared to water as a reference. The absorption measurements were initially carried out at fixed wavelengths and then across a defined wavelength range. The measuring parameters were set as follows:

For measurements at fixed wavelengths:

Correction: Reference Measurement mode: Wavelengths
Display: Absorbance   380nm and 430nm
Lamp replacement: 320nm Step width: 1nm
Slit: 1nm Integration time 0.2s

For measurements across a wavelength range

Correction: Reference Measurement mode: Scan
Display: Absorbance Range: 400 – 410nm
Lamp replacement: 320nm Step width: 1nm
Slit: 1nm Speed: 5nm/s

Results
Table 1 shows the measured extinction values of the urine samples and the control urine for both wavelengths. Figure 1 shows the corresponding absorption spectra across the respective wavelength range.

Table 1: Extinction values for defined wavelengths

Wavelength Control sample extinctions (level 1)      
Sample 1 Sample 2 Sample 3
380nm 0.0129 0.0107 0.0155 0.0144
430nm 0.0004 0.0002 0.0031 0.0017

 Fig. 1: Absorption spectra of the urine samples

Analysis
All spectra have a similar gradient and show a maximum extinction at 402nm, indicating the prevalent presence of copro-porphyrines (oxidized byproducts of heme biosynthesis).
Based on the maximum extinctions of the spectra (Fig. 1) and the specific extinctions detected at the two fixed wavelengths (Table 2), the total porphyrine concentration can be calculated as follows, shown here in the example of the control urine sample (level 1):
             (1)  Correction factor    F = 2 • Emax – (E380 + E430)
                                                      F = 2 • 0.0197 – (0.0129 + 0.0004) = 0.0261

             (2) Total porphyrines =       F • 4740 = 0.0261 • 4740 = 123.71µg/l

Here the factor 4740 represents an empirically determined value comprised of the average extinction coefficient of the porphyrines, the divisor constant and the dilution factor of the sample and the recovery rate.

The concentrations of all other samples were calculated analogously. The results for these are shown in table 2.

Table 2: Total porphyrine concentrations of the urine samples

  Control sample (level 1) Sample 1 Sample 2 Sample 3
Concentration [µg/L] 123.71 39.34 66.36 63.99
Control range 114 -172      
Average setpoint 143      

 With 123.71µg/l the total porphyrine content of the control sample is within the expected range. For the urine samples concentrations below the control urine were detected, but they were all within the normal range of < 150µg/l.
The sample preparation using the column test by RECIPE® followed by the measurement with the SPECORD® 250 PLUS offers a reliable and rapid method for the determination of the total porphyrine concentration in the urine as screening for the verification of porphyria.

More information:

Analytik Jena AG
Konrad-Zuse-Strasse 1
07745 Jena/ Germany
Tel.: +49/ 3641/ 77 70
Fax: +49/ 3641/ 77 9279
www.analytik-jena.com | info@analytik-jena.com


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