Plasma porphyrins

If the patient presents with acute burning pain, the examination of choice is erythrocytic protoporphyrins. If the free protoporphyrin is significantly elevated (more than 6 pmol/1), the diagnosis of erythropoietic protoporphyria is established. Plasma porphyrins are used in patients on chronic hemodialysis who suffer from skin blisters to differentiate between PCT and pseudoporphyria the latter does not show increased plasma porphyrins. 

Porphyrins in plasma are mainly used to distinguish between PCT and pseudoporphyria in patients with chronic hemodialysis. CEP patients also show characteristic elevations with dominance of I-isomers, especially of uroporphyrin and its decarboxylation products, in both plasma and erythrocytes. Plasma porphyrins may be used for follow-up of the patients. 

Figure 7.3.6 compares plasma porphyrin chromatograms from an individuals with PCT and an individual with CEP. 

Fig. 7.3.6a,b Plasma porphyrins from an individual with porphyria cutanea tarda (a) and from an individual with congenital erythropoietic porphyria (b)... 

Da Silva, V., Simonin, S., Deybach, J.C., Puy, H., Nordmann, Y. Variegate porphyria diagnostic value of fluometric scanning of plasma porphyrins. Clin. Chim. Acta 1995 238 163-168... 

Impaired glomerular function reduces the clearance of those water-soluble porphyrins normally excreted in the urine. Furthermore, these porphyrins are poorly cleared by dialysis and, as a consequence, plasma porphyrins are raised in end-stage renal failure. ... 

In PCT, excretion and plasma porphyrin concentrations return to normal during remissionj with the proportions of individual porphyrins in urine and feces remaining abnormal for longer than total porphyrin concentrations. Thus in a patient whose skin lesions have healed, and whose total urinary and fecal porphyrin concentrations are normal, determination of individual porphyrins may reveal the diagnosis. The plasma fluorescence scan in VP and fecal coproporphyrin-III excretion in HCP remain abnormal for many years after clinical remission. 

Fecal copro isomer I/Iilratio Plasma porphyrin fluorescence... 

Plasma porphyrins maybe determined by fluorescence emission spectroscopy of sahne-diluted plasma or depro-teinized extracts, or by HPLC. The first of these methods has the advantages of simplicity and including porphyrins that are bound covalently to plasma proteins and is detailed below. 

This method detects the fluorescent emission of plasma porphyrins when excited at 405 nm. It is described in detail on the Evolve site that accompanies this book. 

The plasma porphyrin concentration may be increased in conditions in which porphyrin excretion is impaired, such as renal failure and cholestasis. Although plasma porphyrin concentrations are usually higher in chronic renal failure with PCT than in renal failure alone, unequivocal diagnosis of PCT in this situation is best achieved by fecal porphyrin analysis or fractionation of plasma porphyrins by HPLC. ... 

Hindmarsh JT, Oiiveras L, Greenway DC. Plasma porphyrins in the porphyrias. Clin Chem 1999 45 1070-6. 

Especially useful in patients on chronic hemodialysis that is a precipitating factor of PCT, but also of the clinically similar disease pseudoporphyria with no increase in plasma porphyrins ... 

Plasma porphyrins and fluorescence under ultraviolet light are characteristic in catde. 

At another level, water-soluble polyphosphazenes are of interest as plasma extenders. In addition, specific polymers with pendent imidazolyl units have been studied as carrier macromolecules for heme and other iron porphyrins (structures and (44,45). (In structures M and the ellipse and Fe symbol represent heme, hemin, or a synthetic heme analog.)... 

Knowledge of the biochemistry of the porphyrins and of heme is basic to understanding the varied functions of hemoproteins (see below) in the body. The porphyrias are a group of diseases caused by abnormalities in the pathway of biosynthesis of the various porphyrins. Although porphyrias are not very prevalent, physicians must be aware of them. A much more prevalent clinical condition is jaundice, due to elevation of bilirubin in the plasma. This elevation is due to overproduction of bilirubin or to failure of its excretion and is seen in numerous diseases ranging from hemolytic anemias to viral hepatitis and to cancer of the pancreas. 

There are large numbers of naturally occurring representatives, especially of pyrrole that include the important polypyrroles (porphyrins and corrins), and the nitropyrrole antibiotics such as pyr-rolomycins and pyrroxamycin. Derivatives of furan have been used as fungicides and A-vinylpyr-rolidone is an important monomer for the production of blood plasma extenders and for cosmetic applications. On account of the similarity in the pathways for the aerobic degradation of monocyclic furan, thiophene, and pyrrole, all of them are considered here. Anaerobic degradation of furans is discussed in Part 2 of this chapter. 

The haem molecule would be incomplete without iron so this must be delivered to the progenitor red cells. Iron is toxic so it is carried in the plasma bound to a specific protein named transferrin (Tf). Uptake of iron is via a Tf receptor, of which there are approximately 300 000 per cell. The whole iron/Tf complex is taken into the cell by endocytosis where the iron is released and made available for incorporation into the porphyrin ring by ferrochelatase. 

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. 

Increased porphyrin levels in plasma can be shown by a specific fluorescence emission spectrum. 

Porphyrins are extracted from plasma or erythrocytes and separated according to their polarity on a reverse-phase system. Detection by their fluorescence is highly specific because both excitation and emission are at a relatively long wavelength. A high number of different isomers exist, and are especially detectable in pathological conditions. 

Table 7.3.6 Standards used in the HPLC determination of porphyrins in plasma and erythrocytes...

A 500-pl aliquot of sample, 100 pi trichloroacetic acid, and 400 pi HC1 30% are mixed by vortexing and then kept on ice for at least 1 h, centrifuged at 14,000 rpm (16,100 xg), and filtered (AcrodiscLC 13, Millipore) into brown HPLC autosampler tubes. Aliquots (20 pi) of standards and samples are injected into the HPLC. Samples with high concentrations of porphyrins should be diluted with 3 M HC11 2 initially. To measure the erythrocytes they are pretreated as follows 100 pi of 3 M acetic acid is pipetted into a tube, 50 pi of erythrocytes is added drop by drop under constant vortexing, followed by 1 min further mixing by vortex. This solution is further processed as described for plasma. 

Each laboratory should establish its own reference values. Those given here can thus only be interpreted as a guide. Porphyrins in plasma are rarely detectable in healthy individuals. Patients on chronic dialysis but without signs of PCT may show some measurable values, which are given here as reference values (nmol/1, mean 2SD) uroporphyrin <24.3 heptacarboxyporphpyrin <2.7 hexacarboxyporphyrin <1 pentacarboxyporphyrin < 1 coproporphyrin I < 1 coproporphyrin III < 1. 

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