Variations in erythrocyte uroporphyrinogen I synthetase activity in non porphyrias

119

Clinica Chimica Acta, 87 (1978) 119-125 @ Elsevier/North-Holland Biomedical Press

CGA 9472

VARIATIONS SYNTHETASE

M. BLUM,

IN ERYTHROCYTE UROPORPHYRINOGEN ACTIVITY IN NON PORPHYRIAS

C. KOEHL

* and J. ABECASSIS

Laboratoire d’rlnalyses Me’dicales, 67000 Strasbourg (France) (Received

January

I

31st,

Znstitut

de Chimie

Biologique,

11, rue Human,

1978)

Summary The activity of erythrocyte uroporphyrinogen I synthetase has been measured in various cases of non-porphyric affections. The results indicate a diminution of this activity in some of the studied cases of chronic renal insufficiency and chronic polyarthritis. This modulation in activity mitigates against its use as a diagnostic criterion of acute intermittent porphyria. On the other hand, an increase in urosynthetase activity has been noted in acute and especially chronic hepatic affections. This increase seems to be connected with the severity of the hepatic affection. The relationship is illustrated particularly in the case of viral hepatitis associated to an AIP, where the increasing activity of the urosynthetase masks for many weeks the congenital deficiency peculiar to this AIP. Our study thus indicates that the diagnosis of AIP based on the activity of the urosynthetase must take into account the pathological context in which the investigation is realised.

Introduction The importance of determining erythrocyte uroporphyrinogen I synthetase activity (Urosynthetase) in the diagnosis and identification of acute intermittent porphyria (AIP) was shown in 1972 by Strand and coworkers [1,2,3]. These studies have since been confirmed by numerous authors [4-131. However, Meyer et al. [14] have noted the existence of an overlapping zone between the values of a normal population and those obtained from a porphyric population. This zone is composed on the one hand by quite low activities among the non-porphyric population and on the other by normal activities in some cases of latent AIP.

* To

whom

correspondence

should

be addressed.

120

Therefore, it seemed necessary to check whether the low urosynthetase activity is exclusively specific for AIP. Moreover, we tried to find out if some pathological states would lead to the increase in activity, thus making the usual deficit of AIP. Material The first category of patients with hepatic affections was constituted by: a group of 10 cases of chronic alcoholic hepatitis (G I); a group of 14 cases of ethylic cirrhosis (G II); a group of 5 nonethylic cirrhosis cases (G III); a group including 4 cases of viral hepatitis and one case of acute hepatic steatosis (G IV). A less homogeneous category of patients suffering from affections with no nosological link between them was constituted by: a group of 3 cases of chronic lead poisoning (G V); a group of 7 cases of hyposideremic hypochrome anaemia (G VI); a group of 8 chronic renal insufficiency cases (G VII); a group of 7 chronic polyarthritis cases (G VIII); a group of 9 cases of epilepsy (G IX); a group of 6 neoplasic affections from various origins (G X). Furthermore, we have considered separately latent AIP with concurrent viral hepatitis. With the exception of this last case, family exploration of clinical and biological data enabled us to diagnose those patients with possible porphyria. Method The determination of erythrocyte urosynthetase activity was carried out according to a method described previously [14]. This method has been automated and is based on assay of the dosage of non-transformed porphobilinogen after the enzymatic reaction. All assays were performed twice on at least two different blood samples. The usual values obtained by this method were between 0.75 and 1.15 nmol of porphobilinogen/min/lOg erythrocytes. Results An elevation of the urosynthetase activities was observed in the following cases : All cases comprising the first group, with an average activity of 1.67 nmol/ with extreme values of 1.22 and 2.36 nmol/min/lOg min/lOg erythrocytes, erythrocytes. All cases of group II, with average activity of urosynthetase of 2.25 nmol/ min/lO’ erythrocytes and extreme values of 1.43 and 2.78 nmol/min/lOg erythrocytes. The five cases of the group III, presenting an average activity of 1.99 nmol/ min/lO’ erythrocytes, with extreme values of 1.27 and 2.90 nmol/min/109 erythrocytes. Table I presents the results obtained for cases of groups I, II and III. Table II indicates the variations of urosynthetase activity according to the degree of evolution of the cirrhosis group: we note that the average value obtained in the case of equilibrated cirrhosis (2.47 nmol/min/109 erythrocytes) is higher than the average value obtained for the group with non-equilibrated

121

TABLE

I

UROSYNTHETASE Case

No.

ACTIVITY Activity Group

OF

GROUPS

of erythrocyte I

I, II AND

III

urosynthetase Group

in nmol/min/109

II

Group

1

1.22

1.43

1.27

2

1.37

1.57

1.42

3

1.44

1.58

2.08

4

1.47

1.71

2.32 2.90

5

1.47

1.76

6

1.54

1.84

7

1.95

1.85

8

1.82

1.86

9

2.10

2.13

10

2.36

2.14

11

2.34

12

3.99

13

4.13

14

2.78

erythrocytes III

Average activity

1.67

+ 0.36

2.25

UPPER

LIMIT

OF

t 0.85

1.99

REFERENCE

f 0.66

VALUES

1.15

cirrhosis (1.86 nmol/min/lO’ erythrocytes). Two cases of group IV: one is a viral hepatitis of protracted form, the other is a gravidic hepatic steatosis. In both cases, the increase in the activity of the urosynthetase started at the icterus maximum, and proceeded to the clinical and biological improvement phase. We then noted a return to the usual values. A slight shift between the urosynthetase activity maximum and the bilirubin maximum values was observed (Fig. 1). Two of the three cases from group V, showing activities of 1.48 and 2.05,

TABLE

II

UROSYNTHETASE

ACTIVITY

IN

Activity

EQUILIBRATED

of erythrocyte

Equilibrated

cirrhosis

AND

urosynthetase

NON-EQUILIBRATED in nmol/min

Non-equilibrated

1.43

1.57

1.84

1.58

1.85

1.71

1.86

1.76

1.27

2.13

1.42

2.14

2.32

2.34 3.99

2.90

4.13 2.78 3.08 Average

value

1.86

f 0.53

2.47

? 0.92

lo9 cirrhosis

CIRRHOSIS erythrocytes

Days

Fig. 1. Evolution

of total bilimbin

ratio and urosynthetase

activity

in a

hepatitis

of protracted

form.

for the first case at separate times, and 1.82 nmol/min/lOg erythrocytes for the second case. The sideroblastic anaemia case of group VI whose enzymatic activity determined at a monthly interval was 2.52, then 2.48 nmol/min/lOg erythrocytes. And lastly an increase in the special case of viral hepatitis for a women patient suffering from AIP. The low urosynthetase activity, usually at 0.50 nmol/min/lOg erythrocytes for this patient, increased at the icterus maximum and was maintained for 3 months at normal values (0.97 to 1.06 nmol/min/lOg erythrocytes). The initial enzymatic activity was restored on recovery and a control value measured 6 months after the beginning of the viral hepatitis gave an enzymatic activity of 0.58 nmol/min/lOg erythrocytes. Fig. 2 shows the change of the total serum bilirubin compared with the variation in urosynthetase activity. We found a decrease in urosynthetase activity in the following cases: Six cases from group VII: the activities varied from 0.37 to 0.67 nmol/min/ lo9 erythrocytes. The two other cases showed normal urosynthetase activity (0.85 and 1.07 nmol/min/109 erythrocytes). Three cases from group VIII. In the first case the enzymatic activities determined at a month’s interval varied from 0.52 to 0.58 nmol/min/109 erythrocytes. For the second case the activity, initially at 0.72, decreased to 0.64 nmol/min/109 erythrocytes 5 months later. The third case showed an enzymatic activity of 0.43 nmol/min/109 erythrocytes. The four other cases showed normal activity. One case from group IX where the activity was at 0.65 and fell to 0.57 nmol/min/lOg erythrocytes one week after starting a barbituric treatment (phenylethylmalonyl urea). Two cases from group X: an operated langerhansian adenoma secreting

123

LLI

90 Days

Fig. 2. Evolution of total bilirubin ratio and urosynthetase activity in a case of latent AIP with concurrent viral hepatitis. -, total bilimbin in mg %; - - - - - -, urosynthetase activity in nmol/min/109 erythrocytes.

insulin with urosynthetase activity of 0.47 initially, changing to 0.44 nmol/ min/lO’ erythrocytes one month later; a case of neoplasic ascite of ovarian origin, for which the enzymatic activity was 0.61, falling to 0.58 nmol/min/109 erythrocytes five months later. Discussion The results presented illustrate diseases in which the urosynthetase activity may be increased or decreased. In the hepatic syndrome we constantly find high urosynthetase activity. This phenomenon seems to be definitive among chronic hepatic affections as well as among those connected with ethylism and non-ethylic cirrhosis. As indicated by the difference between the average values of equilibrated cirrhosis and nonequilibrated cirrhosis, the elevation of the urosynthetase activity seems to be proportional to the severity of the hepatic lesion. In acute hepatic disease the inconsistently observed increase in the urosynthetase activity seems to be transitory and reversible. These results suggest that non-porphyric syndromes, essentially of hepatic origin, may lead to an increase, sometimes of great magnitude, in erythrocyte urosynthetase activity. These hepatic diseases may be attended by high amounts of bilirubin and bile salts, liable to detach any fraction of the urosynthetase bound to erythrocyte membranes. This possibility, which would lead to a systematic elevation of urosynthetase activity, has, however, been questioned. We have shown the effects of increasing amounts of bilirubin and glycodeo-

124

xycholic acid on a normal erythrocyte pool: the activities of the urosynthetase remained the same as in a control sample. In the case of the AIP patient with viral hepatitis, the congenital deficiency of enzyme activity established prior to the infection was masked by the elevation of the urosynthetase activity observed during the viral infection. Among the other diseases with high urosynthetase activity, the result for the single sideroblastic anaemia is indicative, but not conclusive, of an elevation in the activity of the enzyme. On the other hand, the increased activities in two of three cases of lead poisoning seem to indicate that this increase may be frequent in lead intoxication. It follows from our work that the diagnosis of AIP based on the determination of erythrocyte urosynthetase activity must take into account the pathological context, particularly in cases of hepatic disease. Among the affections with decreasing activity no etiological common character could be noted. This diminution of the urosynthetase activity was not obtained systematically among the patients of groups VII, VIII and IX. The chronic renal insufficiency cases which have been studied show high plasmatic urea levels. Urea is known to inhibit the porphobilinogen coloration reaction with the Ehrlich reagent, and may thus give rise to an artefactual decrease of the urosynthetase activities. Treating erythrocytes with increasing levels of urea (from 1 to 4 g/l) did not alter the activity of the eythrocyte urosynthetase. The usual values zone was taken to include 95% of the normal population (mean + 2 standard deviations), those cases whose activities are between 0.60 and 0.70 nmol/min/lO’ erythrocytes would be part of the 5% of the cases considered as situated outside this range. However, the enzymatic activities lower than 0.60 nmol/min/109 erythrocytes cannot be explained in this way and raise the problem of the specificity of the diminution of the erythrocyte urosynthetase activity in AIP. Conclusion A diminuiton of the urosynthetase activity is observed in some diseases even though there is no nosological link between them. This modulation mitigates its use as a diagnostic criterion for AIP. An increase in urosynthetase activity is observed in some syndromes, especially hepatic affections. This modulation in activity may mask a possible deficiency of urosynthetase for an AIP. References Meyer,

U.A.,

Strand,

L..

Doss,

M.,

Redeker,

A.

Rees.

A.

and

Marver,

H.

(1972)

New

Eng.

J. Med.

286.

1277-

67,

1315-

1282 Strand,

L..

Felsher,

B.,

and

Marver,

H.

(1970)

Proc.

Natl.

Acad.

Sci.

U.S.A.

1320 Strand.

I.. Meyer,

Sassa.

S..

Sassa,

S..

U.S.A.

732-736

Bonkowsky.

H.L..

Koehl,

Felsher.

B.,

S..

Bickers,

D.R..

Granick,

71,

Med.

U.,

Granick.

S.,

Bickers,

Tschudy,

Redeker, D.R.,

D.P..

A.

Levere,

and

R.D.

Bradlow,

Weinbach.

Marver, and

H.L. E.C.,

H. (1972)‘J.

Kappas, and

A.

Kappas,

Ebert,

P.S.

Now.

Presse

CIin.

(1973)

and

Invest.

Enzyme

A.

(1974)

Doherty,

51,

16, Proc.

J.M.

C..

Granchamp,

Abecassis, B.,

J. and

Grelier.

Wertenschlag,

M. and

Nordmann.

J. (1975) Y.

(1975)

Now.

Med.

Presse

4, 2735

Med.

Natl.

(1975)

85.93-102 4, 1801-1802

2530-2536

326-333 Acad. J. Lab.

Sci. CIin.

125

9 Granchamp, B. (1975) Contribution P 1’6tude de I’UrosynthCtase, Paris VII 10 Meyer, U.A. and Schmid. R. (1973) Fed. Proc. 32, 1649-1655 11 Meyer, U.A. (1973) Enzyme 16,335-342

These de biochimie

12 13 14

53, 3542 32. 353-358 International Porphyrin Meeting on M. and Nawrocki, P., eds.). P. 155,

Van Eijk, H.J.. Wiltink, W.F. and Bos, G. (1974) Clin. Chim. Acta Mandel, P., Koehl. C. and Wertenschlag, J. (1974) Ann. Biol. Clin. Meyer, U.A. (1976) in Supplement to the Proceedings of the 1st Porphyrins in Human Diseases. Freiburg, May 14, 1975 (Doss, Karger. Base1

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