Urine porphyrins

Fig. 7.3.2 Characteristic color of a porphyrin urine (left urine on lower picture) and typical purple fluorescence (right urine on upper picture) both compared to a normal urine...

High levels of lead can affect heme metabohsm by combining with SH groups in enzymes such as fer-rochelatase and ALA dehydratase. This affects porphyrin metabolism. Elevated levels of protoporphyrin are found in red blood cells, and elevated levels of ALA and of coproporphyrin are found in urine. 

There are a large number of hereditary or acquired disturbances of porphyrin synthesis, known as porphyrias, some of which can cause severe clinical pictures. Several of these diseases lead to the excretion of heme precursors in feces or urine, giving them a dark red color. Accumulation of porphyrins in the skin can also occur, and exposure to light then causes disfiguring, poorly healing blisters. Neurological disturbances are also common in the porphyrias. 

Clinical samples often contain complex mixtures of different porphyrins. Porphyrin solubility in organic solvents is inversely proportional to the number of acid side chains. Indoles in the urine may also give a positive result. 

In most porphyrias, excess metabolites can be detected in urine. Less polar porphyrins (i.e., coproporphyrins and protoporphyrin) are detectable in feces as they are excreted by the bile. The apolar protoporphyrin is eventually only detectable in blood. Porphyrins can easily be detected and measured by their intense fluorescence in mineral acids. The excitation wavelength is around 404 nm, and emission at about 615 nm. ALA is derivatized to a pyrrole and both, ALA and PBG, are detected by dimethylaminobenzaldehyde (DMAB), as described by Mauzerall and Granick [7]. 

The types of test to be performed depend on the clinical situation. If the patient has an acute, severe disease and an acute porphyria is suspected, PBG should be assessed either qualitatively by a reliable screening test or quantitatively in a spot urine. Quantitative ALA can be added to uncover lead intoxication, which may be clinically undistinguishable from an acute porphyria. Furthermore, the extremely rare ALAD deficiency may show normal PBG. If increased values of PBG are present, urinary porphyrins can be determined to confirm the existence of porphyria. 

If the patient is actually asymptomatic, but has a family history of acute porphyria or prior symptoms suspicious of acute porphyria, hydroxymethylbilane synthase (HMBS) activity, plasma scanning, and fecal porphyrins should be measured. These tests will reveal AIP, P V, and HC. As a small percentage of AIP families exhibit normal HMBS activity, PBG in a urine sample can be added. PBG determination can also performed as a first choice, if an acute porphyria is suspected. But if normal, it does not exclude acute porphyrias in asymptomatic phases. Furthermore, the existence of an acute porphyria is only proved if the value exceeds at least five times the upper limit of normal. 

If a patient presents with blisters due to photosensitivity, a plasma scan and fecal porphyrins will not only reveal the presence of a porphyria, but will also enable the distinction between the three forms that may cause such symptoms porphyria cutanea tarda (PCT), PV, and HC. Urinary porphyrins and eventually quantitative ALA und PBG in urine may be added in questionable cases or for monitoring of therapy. 

Porphyrins and porphyrin precursors are assayed most often in a 24-h urine collected without additive. Alternatively, untimed urine samples may be used and excretion standardized to creatinine. The latter is especially recommended for children and in emergency situations. Alternative specimens for porphyrins are plasma, erythrocytes, and feces, depending on the medical indication. During collection and until arrival at the laboratory, specimens should be kept cold, preferably at about 4°C, and protected from light. Specimens in the laboratory are best kept frozen, as the metabolites in body fluids are stable at -20°C for at least 3 months. Some exceptions have been noted below. 

The first porphyrin intermediate of the biosynthetic pathway is uroporphyrinogen, which is stepwise decarboxylated by uroporphyrinogen decarboxylase to heptacarboxy-, hexacarboxy-, pentacarboxy-, and coproporphyrinogen. This latter compound proceeds, as indicated in Fig. 7.3.1, to protoporphyrinogen and protoporphyrin. The oxidized uroporphyrin and its decarboxylation products up to coproporphyrin are assayed in urine. Coproporphyrin and the further downstream intermediaries can be recovered from feces as described below. 

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. 

Gradient HPLC system identical to that used for urinary porphyrins. Protoporphyrin present only in feces but not in urine elutes last. If it is not eluted, the plateau phase can be prolonged for some minutes. 

Lim CK, Peters TJ (1984) Urine and faecal porphyrin profiles by reversed-phase high-performance liquid chromatography in the porphyrias. Clin Chim Acta 139 55-63 Minder El, Vuilleumier JP, Vonderschmitt DJ (1992) Prototype application of robot in the clinical laboratory enabling fully automated quantification of fecal porphyrins. Clin Chem 38 516-521... 

Urine from a patient with porphyria cutanea tarda (right) and from a patient with normal porphyrin excretion (left). 

The porphyrias. The human body does not use all of the porphobilinogen produced, and a small amount is normally excreted in the urine, principally as coproporpyrins (Fig. 16-5). In a number of hereditary and acquired conditions blood porphyrin levels are elevated and enhanced urinary excretion (porphy-... 

A gold(I)/tri(methoxyphenyl) phosphine complex incorporated into PVC membranes led to formulation of ISEs with fast (14 s) Nernstian response (10 2-10 6 M) to perchlorate, with excellent selectivity even over chlorate in both water and urine [28]. Chromium porphyrins in PVC gave ISEs with near-Nernstian response to salicylate (10 2-10 6 M) with UV spectra confirming binding of salicylate to the chromium atom [29]. 

It may be noted that although the human organism is capable of degrading metal-containing porphyrins such as heme, it has no means of degrading porphyrins containing no metal, for example coproporphyrins and uroporphyrins. These must be excreted as such. The latter two are water-soluble and are thus found in urine and to some extent in the feces. Protoporphyrins, however, are largely water-insoluble and are excreted in the feces. Finally, it may be mentioned that porphyrias make their appearance after puberty. This has been associated with the appearance of 5/3-steroid reductases, as discussed above. 

Which porphyrin is found in urine and/or feces if uroporphyrinogen III cosynthase is defective in a patient ... 

During an acute attack, a fresh urine sample which has been protected from light should be sent to a specialist laboratory to be tested for aminolaevulinic acid and porphobilinogen concentrations. If urinalysis confirms raised urinary excretion of aminolaevulinic acid and porphobilinogen, an analysis of faecal porphyrins can be used to identify the specific porphyria. In acute intermittent porphyria faecal porphyrin levels are generally normal. 

Essentially, all organ systems must be evaluated. Thus laboratory tests, should include complete blood count, liver and renal functional tests, and blood, nail and urine arsenic levels. Other biomarkers of arsenic exposure include nonerythrocyte porphyrin enzyme activities and urine transforming growth factor TNF-a, accompanied by induction of heme oxygenase, mitogen-activated protein kinases, the ubiquitin-dependent proteolytic pathway, and protein kinase C in various tissues. These tests are still being investigated in laboratories and their clinical usefulness remains to be proven (Chapell et al, 2001). 

Measure urine porphyrin and porphobilinogen before starting treatment. 

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