PORPHYRIN BIOSYNTHESIS AND ENZYMIC STUDIES IN ERYTHROCYTES FROM NORMALS AND PORPHYRIC HUMANS

Abstract 1. 1. Studies on porphyrin biosynthesis from exogenus ALA, at various time intervals as well as direct enzyme measurements (aminolevulimc acid dehydratase (ALA-D); porphobilinogenase (PBG ase) and deaminase were carried out in hemolysates of human erythrocytes from healthy controls and patients with lead poisoning (Pb), acute intermittent porphyria (AIP), porphyria cutanea tarda (PCT), erythropoietic protoporphyria (EPP), variegate porphyria (VP) and congenital erythropoietic porphyria (CEP). 2. 2. Inhibited ALA-D in Pb, reduced PBGase and deaminase in AIP, lower uroporphyrinogen decarboxylase in PCT, and diminished isomerase in CEP, were confirmed. In addition, ALA-D was found, reduced in AIP, unchanged in PCT and increased in EPP, VP and CEP. PBGase and deaminase were, on the other hand, increased in Pb and PCT, unchanged in VP and diminished in EPP and CEP. 3. 3. Total porphyrin biosynthesis is a function of time; compared to normals, is lower in CEP and AIP, but higher in PCT. 4. 4. The porphyrin profile changes along the time; uroporphyrin increases at longer intervals while that of coproporphyrin concomitantly diminished. A significance enhancement of octacarboxylic porphyrins was observed during the entire duration of the incubation in PCT hemolysates. In CEP the main porphyrin was always uroporphyrin I. 5. 5. Studies on both total porphyrins formed and their distribution were performed in hemolysates from cases of non-hereditary and hereditary PCT and AIP, before and after therapy.

The porphyrias, either inherited or acquired are a group of diseases characterised by a combination of enhanced synthesis, accumulation and excretion of porphyrins and/or their precursors, S-aminolevulinic acid (ALA) and porphobilinogen (PBG), due to an enzymic disorder in porphyrin metabolism.
Besides their distinctive clinical syndromes, their unique and typical biochemical patterns of excretion are very useful tools for distinguishing each porphyria. However, we are aware that the sole study of the excretion profiles in urine and feces cannot always provide enough information to establish a differential diagnostic nor on the site of abnormal synthesis, that is why it is often necessary and advisable to determine the specific enzyme deficiency.
It has been already observed by others and in our laboratory (Doss, 1973;Schermuly & Doss, 1976;Viljoen et al., 1976;Miyagi et al., 1976;Batlle et al., 1979a,b), that information about the underlying enzymic defect and inheritance in many porphyrias can also be obtained by carrying out in oitro studies on porphyrin biosynthesis from ALA and PBG by erythrocytes, under different experimental conditions as well as by direct enzyme measurements.

MATERIALS AND METHODS
Blood samples were obtained and hemolysates prepared following Batlle et al. (1978).
Incubation conditions were those reported by Batlle er ul. (1979b). Incubation time varied between 2 and 8 hr. Fractionation, identification and quantitative spectrophotometric determination of the porphyrinogens formed & well as other methods and matdriais not specified here were those alreadv described bv Llambias & Batlle (197 1 a) and Batlle et al. (1979a). Total rate of biosynthesis is expressed as nmol of porphyrin per ml of RBC. ALA-D was assayed by the method of Batlle et al. (1967). PBGase and deaminase were measured as described by Batlle et rrl. (1978).
Enzyme units; one unit of enzyme activity is defined as the amount of enzyme which catalyses the formation of I nmol of product in 60 min under the standard incubation conditions, and enzymic activities are expressed as units per ml of RBC.
We wiil only repoit here, results on porphyrin biosynthesis from exogenous ALA at various time inter-

RESULTS AND DISCUSSION
vals, by human erythrocytes hemolysates from nor-Enzyme determinations mals and patients with lead poisoning (Pb), acute intermittent porphyria (AIP), porphyria cutanea tarda Findings obtained are presented in Fig. 1 ._. Fig. 1. Enzyme determinations. Every measurement was carried out by duplicates and in many patients repeated periodically. n = number of cases.
cantly inhibited, but it was consistently found here, that both PBGase and deaminase activities were 15-20?; increased. These results are not easy to explain because it has been demonstrated that in vitro, pmolar to mmolar concentrations of lead, depending on the tissue, inhibited PBG consumption and porphyrin formation by these enzymes (Llambias & Bathe, 1971b;Piper & Tephly, 1974;Tephly, 1979;Sancovich et al., 1976). However it is interesting to add, that PBG urinary levels in these patients were normal; probably, accumulation of lead in tissues was not high enough to in oiuo inhibit conversion of PBG to uro~rphyrinogen, leading to its accumulation and its finally increased excretion in urine. It therefore appears that depending on whether or not PBG is excreted in excess in urine, we could expect enzymic activity levels of erythrocytes PBGase and deaminase be lower or higher than normals.
It was found that in AIP, not only PBGase and deaminase were reduced to half but also ALA-D activity was 2530% diminished. In PCI' either inherited or acquired, as an average ALA-D was not modified, however values obtained were within a great range of variability, while PBGase and deaminase were slightly augmented; in some cases where ALA-S was determined, this activity was 25-507; increased.
Curiously in EPP, VP and CEP, ALA-D was again about 30, 20 and SOP: respectively, above normal; ALA-D has also been found to be enhanced in blood and liver of a bull with CEP (Batlle et al., 1979a), moreover, we and others have observed that in both patients and animals with CEP, ALA-S was 2-4 times increased in blood and bone marrow (Moore et al., 1978;Bathe et al., 1979a); therefore it could be, that in the last four types of porphyria the observed enhancement in ALA-D activity is in fact a reflex of an excess of substrate. PBGase was unchanged in EPP and VP but around 60% reduced in CEP, while deaminase was 20% lower in EPP and CEP. These findings would suggest that at least in these two CEP patients a decreased activity of isomerase might be the primary enzymic defect, in agreement with the hypothesis of Levin (1968). However it has been demonstrate that in some CEP human and animal cases, both decreased activity of isomerase and increased activity of deaminase are co-existent (Moore et al., 1978;BatIle er al., 1979a), supporting the view that CEP is phenotypically homogeneous in most respects but genetically heterogeneous, that is, that the inborn error in porphyrin biosynthesis may vary among different types of CEP (Hofstad et al., 1973). In general we would say that meaningful conclusions can only be drawn by studying individual

Biosynthetic activity
Total porphyrin biosynthesis. Although porphyrias might be p~m~ily liver or bone marrow disturbances, studies on porphyrin biosynthesis from the precursors. ALA  be of significance in the interpretation of porphyria excretion patterns and advance information about the specific enzymic defect and inheritance. They have also the advantage that synthesis cannot go beyond protoporphyrin, thus making easier the interpretation of porphyrin patterns. As we are here reporting results obtained using ALA as substrate, lead poisoning cases will be excluded from now on.
In Fig. 2, total porphyrin formed by hemolysates from healthy volunteers and different porphyrics at 2, 4 and 8 hr is illustrated. It must be noted that longer incubations, up to 24 hr were also performed, however the longer the time the greater the proportion of isomers I found; therefore it is not possible to make valid inferences from those experiments.
As it is well known, in general, total porphyrin biosynthesis is a function of time (Doss, 1976;Batlle et al., 1979ab). In PCT, porphyrin formation is significantly increased as compared to controls. So far, these findings are in agreement with an augmented urinary excretion of porphyrins and would suggest that the activity of one or more of the enzymes after ALA-S might be elevated. It was found, in fact, that PBGase and deaminase were slightly higher than normal ( Fig. 1) and Doss (1976) postulated that the activity of deaminase can be increased in PCT. However, they do not explain, yet, the typical constellation of porphyrins excreted in PCT; an analysis of the distribution of porphyrins is therefore compulsory.
As expected, at shorter intervals, porphyrin synthesis in AIP was reduced to 45-50x (Doss, 1973;Batlle et al., 1979b), this might be due to the known enzymic defect of deaminase, but we should also take into account the diminished activity of ALA-D here found; which all together well agree with the abnormal excessive quantities of PBG and ALA excreted in AIP urine, and can be of diagnostic usefulness.
On the other hand, no significant differences are apparent between total porphyrins formed in EPP, VP and CEP and in controls, so the determination of their porphyrin biosynthesis pattern is necessary to get information as to the type of porphyrins and isomers formed.
Total porphyrin biosynthesis in PCT and AIP. We considered it interesting to determine if there were  any changes in the porphyrin biosynthesis by hemolisates from hereditary PCT and AIP patients, before and after therapy. As shown in Fig. 3, Part B, the answer to the first question was clearly negative. On the other hand, (Part A), although in both hereditary and non-hereditary PCT synthesis was 1.3 to 2 times as high as in controls. There was consistently a clear cut difference between one and the other, being greater in the latter, which was correlated with a higher increase in PBGase and deaminase activities, confirming previous results (Bathe et al., 1979b). ~isrribution of bjosynthetic ~rphyri~s. The pattern corresponding to porphyrins synthesized by hemolysates of controls and porphyrics from ALA, after 2, 4 and 8 hr of incubations is shown in Fig. 4. In general the proportion of highly carboxylated porphyrins, mainly Uro, increases with time, while the percentage of less carboxylated, as Copro, concomitantly decreases.
It has been confirmed that in PCT porphyrin distribution is different from normal and quite distinctive, after 2 hr there is an increased synthesis of Uro and heptacarboxylic porphyrins as well as reduced percentages of hexa and Coproporphyrins, although their absoiute con~ntrations were within normal values. These results are clearly indicative of a defect in the activity of Uroporphyrinogen decarboxylase, which was observed in both symptomatic PCT patients and relatives with latent PCT (Doss, 1976;Batlle et al., 1979b) and can also be of diagnostic si~ifican~.
ft is interesting that, at shorter time intervals the distribution of porphyrins in AIP is quite similar in patients and controls (Doss% 19'73) except that the percentage of Copro is lower, and that of Uro III slightly higher; we must remember that the absolute amount of total porphyrins was 45-50% lower, which would be compatible with the demonstrated defect in deaminase and also ALA-D activity.
A different pattern, quite similar to that seen in PCT, was found for the distribution of porphyrin in VP. In these instances we do know that the qualitative analysis of the porphyrins excreted in both urine and feces is of great importance for a differential diagnostic. As we have observed that in VP, ALA-D, PBGase and deaminase seem not to be modified, it could be interesting to measure the levels of uroporphyrinogen decarboxylase, as the proportion of highly carboxylated porphyrins is so high.
In CEP, the general picture is not much different from controls, but uroporphyrinogen I is the dominant isomer. Decarboxylation is apparently not affected, but the lower proportion of Uro III, seems to reflect a defect at the level of isomerase, which is in agreement with both the typical constelIation of porphyrins excreted in CEP, and enzymic determinations. However as is has already been discussed, CEP, a rare disease, might be genetically expressed in different types. Nevertheless, these kind of studies are useful for distinguishing the nature of the inborn error.

Porphyrin biosynthetic patterns in cases of hereditary and non-hereditary PCT and AiP, before and after
therapy. It was shown (Fig. 2), that there were differences between the total amount of porphyrins formed by hemolysates from patients with non-hereditary and hereditary PCT'. We wanted to know if those differ-ences were only quantitative or if there was also any qualitative change. In addition, the distribution of biosynthetic porphyrins was further studied in PCT and AIP patients before and after therapy.
As we can see in Fig. 5, the pattern produced by hereditary PCT is somehow different from that found in non-hereditary PCT. After 4 and 8 hr incubation the percentage of 8-carboxylic porphyrins in the former is greater than in the latter, as if the first decarboxylation step were more inhibited here, although after 2 hr, the effect was apparently opposite.
As far as treatment is concerned, it did not modify the picture in PCT; however, in one AIP patient, after folic acid therapy (Wider de Xifra et al., 1979), the initial pattern, which was anyhow unusual before therapy, showed a tendency to normal, but only after 4 hr in~bation; more recently, these findings were confirmed with another AIP patient subjected to the same treatment. Nevertheless, we have not enough experimental data to claim that the analysis of the biosynthetic pattern can be useful in following the course of the therapy in AIP, and further studies are indeed necessary to support or not these results. Moreover as stated above, in many cases it is not always possible to arrive at valid conclusions from mean values, and in these instances it is highly advisable to carry out complete individual studies.