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Variegate porphyria: diagnostic value of fluorometric scanning of plasma porphyrins
ELSEVIER
Clinica Chimica Acta 238 (1995) 163-168
Variegate porphyria: diagnostic value of fluorometric scanning of plasma porphyrins V. Da Silva, S. Simonin, J.C. Deybach, H. Puy, Y. Nordmann* Cenrre Fran@
des Porphyries, INSERM
U.409. Hdpital Louis Mourier, 92701 Colombes cedex, France
Received 22 November 1994; revision received 3 April 1995; accepted IO April 1995
Variegate porphyria (VP) is a dominantly inherited acute hepatic porphyria characterized by a 50% decrease in activity of protoporphyrinogen oxidase (PO) which catalyses the last step of heme biosynthesis. In VP families, most of the gene carriers are asymptomatic but at risk of developing acute attacks if subjected to precipitating factors. Recognition of the carrier status is the first step of an efficient preventive care. This could be achieved by measurement of PO activity which is a sensitive and specific but tedious method. A specific plasma fluorometric emission at 626 nm has been shown in VP patients. Here we show that this simple and inexpensive method is specific but poorly sensitive, especially in detection of asymptomatic carriers. We conclude that this procedure should not replace PO aetivity measurement in VP family studies. Keywords:
Variegate
porphyria;
Protoporphyrinogen
oxidase;
Heme biosynthesis;
Fluorometric scanning
1. Introdluction Variegate porphyria (VP) is an autosomal dominant disorder with low penetrance and variable clinical expression classified as one of the acute hepatic porphyrias [ 11. VP patients and asymptomatic carriers have a 50% decrease in the activity of protoporphyrinogen oxidase (PO) (EC 1.3.3.4); PO catalyses the penultimate step in -_ Corresponding author.
??
0 1995 Elsevier Science B.V. All rights reserved 0009-898V95609.50 SSDI 0009-8981(95)06085-R
164
V. Da Silva et al. / Clinica Chimiea Acta 238 (1995) 163-168
heme biosynthesis, i.e. the conversion of protoporphyrinogen to protoporphyrin within the mitochondria [2,3]. Acute attacks in VP patients include abdominal, neurological and psychiatric symptoms. Another common feature is the development of photosensitive cutaneous lesions on exposed areas [ 1). During acute attacks, the urinary abnormalities are an increased excretion of porphyrin precursors 6-aminolevulinic acid (ALA) and porphobilinogen (PBG). In the faeces, there is an increased excretion of protoporphyrin and, to a lesser extent, of coproporphyrin. This typical porphyrin overexcretion profile in the faeces is found not only during acute attacks but also sometimes between them. Another biochemical feature is the presence in VP patients’ plasma of a specific porphyrin compound which has been described as showing a fluorometric emission at 626 nm when excitation wavelength is set at 405 nm [4]. For other types of porphyrias, the maximum emission peak is consistently located at different wavelengths [4,5]. As in other acute porphyrias, prevention of acute attacks is based on the recognition of latent cases and asymptomatic carriers in VP families. Asymptomatic carriers are rarely detected by measurement of urinary ALA and PBG and/or faecal porphyrins, which are most often within the normal range. However, patients and carriers always exhibit the same 50% defect in PO activity in nucleated cells [2]. The measurement of PO activity is tedious and usually requires specialized laboratories [2,6,7]. A rapid screening method based on plasma fluorescence emission spectroscopy has been proposed [4,5,8,9]. In a recent study, this qualitative fluorescence emission scanning was shown to be an easy, specific and sensitive tool for the detection of asymptomatic VP gene carriers [5], but the sensitivity was calculated on statistical expected frequencies of asymptomatic carriers in first and second-degree relatives and assuming a 100% specificity of the test. The aim of the present study was to evaluate the clinical relevance of this screening method in samples from symptomatic and asymptomatic VP subjects previously recognized by measurement of PO activity in circulating lymphocytes.
2. Materials and methods 2.1. Subjects Lymphocytes and plasma samples from 88 subjects were studied: 32 unrelated patients (18-65 years old) with overt VP in which diagnosis has been established by characteristic clinical symptoms, porphyrin excretion abnormalities and a documented 50% decreased PO activity in lymphocytes, and 56 VP relatives, identified as asymptomatic carriers according to their clear-cut half PO activity in lymphocytes, 20 of whom were children (< 18 years old). 2.2. Chemicals Potassium phosphates, metallic sodium, mercury, orthophosphoric acid, potassium hydroxide, ethyl alcohol, were from Prolabo (France). Tris, EDTA, Triton X100, Tween 20, glutathione (GSH) were from Sigma (France). Ficoll was from Eurobio (France). Bio-Rad Protein Assay was from Bio-Rad Laboratories (Germany). Free protoporphyrin was from Porphyrin Products, Logan, UT (USA).
V. Da Silva et al. / Clinica Chimica Acta 238 (1995) 163-168
165
2.3. Fluorometric scanning of plasma porphyrins
Plasma (100 ~1) was diluted with 900 ~1 of 250 mmol/l phosphate buffer (pH 6.7) in a fluorometric microcuvette. After stirring, the fluorescence emission spectrum was recorded in a Shimadzu-RF 540 spectrofluorometer fitted with a red sensitive photomultiplier (excitation at 405 nm, scan from 570 to 750 nm). In these conditions, the observed sensitivity was 5 nmol/l. 2.4. PO activity in lymphocytes The assay follows the kinetic development of protoporphyrin fluorescence from as previously described [2] with slight non-fluorescent protoporphyrinogen, modifications. Briefly, lymphocytes from 10 ml venous blood were isolated by the Ficoll method and storeld at -80°C. The lymphocyte pellet was resuspended in 400 ~1 of 150 mmol/l Tris-HCl (pH 8.7) 1 mmol/l EDTA, 1% Tween 20, and sonicated for three 10-s periods. After centrifugation at 10 000 x g, the protein concentration in the supernatant w,as measured with the Coomassie Blue reagent (Bio-Rad Protein Assay); 50 ~1 of the lymphocyte lysate were transferred to at least three fluorometric microcuvettes held at 4°C. Protoporphyrin (1.2 mmol/l in 80:20 v/v KOH-0.01 mol/l ethyl alcohol,) was reduced to protoporphyrinogen with 3% sodium amalgam in a dim light, under a nitrogen flow. After a 2-fold dilution with 300 mmol/l Tris-HCl (pH 7.0), 2 mmol/l EDTA, 2% Tween 20, 50 mmohl GSH, the substrate solution was adjusted to a pH value between 8.0 and 9.0 by addition of 17% phosphoric acid; 5 ~1 of this solution were immediately added to each microcuvette containing the enzymatic source. After stirring, the microcuvettes were placed at 37°C in the dark. Enzymatic reaction was stopped by addition of 1 ml of 100 mm0111Tris-HCl (pH 8.7) with 1 mmol/l EDTA, 0.1% Triton X-100, 2 mmol/l GSH at timed intervals (5, 15, 25... min). For each microcuvette the fluorescence emission was immediately measured at 635 nm with an excitation wavelength of 410 nm. The rate of non-enzymatic oxidation of protoporphyrinogen was estimated in a similar assay, performed with a tissue blank containing heat-inactivated enzyme. The amount of the non-enzymatic protoporphyrin formed in 20 min (AFB) was subtracted from the enzymatic value (AFE). To convert the fluorescence values to nanomoles of protoporphyrin formed, a reference standard was prepared as described: 10 ~1 of the 1.2 mmol/l protoporphyrin solution was diluted 120-fold with KOH-ethyl alcohol; 1 ml of 100 mmol/l Tris-HCl (pH 8.7), 1 mmol/l EDTA, 0.1% Triton X-100, 2 mmol/l GSH was added to 50 ~1 of this solution in a microcuvette. The fluorescence emission (FSTD) was immediately measured in the same conditions. The enzymatic activity expressed as nanomoles of protoporphyrin formed per hour per milligram of protein at 37°C was calculated as follows: (AFE .- AFB) x 3 x standard final cont. FSTD x mg protein in incubation The between-batch reproducibility of the enzymatic assay ‘was 10% (5 different subjects in 4 different assays). Student’s unpaired t-test was used to compare data from controls and VP subjects.
166
V. Da Silva et al. /Clinica
Chimica Acta 238 (1995) 163-168
61
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51
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*%‘A** ttfft: . . . .
41
i-
3
;
$31
I
:!
2 !
.f:*. t
II .. . ‘t:’ .;.
:
0’
P
.... ::
..;:.:.z . .............
5v.t .
AC
C
Fig. 1. Protoporphyrinogen oxidase activity (PO), nmol protoporphyrinih per mg protein. P: patients (n = 32). mean= 2.5 (S.D. = 0.4); AC: asymptomatic carriers (n = 56), mean = 2.6 (SD. = 0.3); C: controls (n = SO), mean = 4.4 (SD. = 0.4).
3.Results 3.1. Enzymatic assay
PO activity in lymphocytes was significantly decreased both in asymptomatic carriers and VP subjects (-50% P c 0.005) compared with controls (Fig. 1). 3.2. Plasma jluorometric assay An illustration of the specific plasma fluorescence emission spectrum is shown in Fig. 2 and the results of the fluorometric screening in plasma are detailed in Table 1. In our experimental procedure, all VP patients had a fluorescence emission peak at 630 nm. In the same conditions of excitation (405 nm) the emission peak in other types of porphyrias was located at different wavelengths (620 nm in porphyria cutanea tarda, acute intermittent porphyria, hereditary coproporphyria and 635 nm in erythropoietic protoporphyria; data not shown). Moreover, the fluorescence peak at 630 nm was found in only 50% of the adults and in none of children who were VP asymptomatic carriers. 4. Discussion Lymphocyte PC activity determined by fluorometric measurement of protoporphyrinogen oxidation was the reference method for the diagnosis of VP patients and
V. Da Silva et al. / Clinica
Chimica
Acta 238 (1995)
163-168
167
630 s
_. -.~ _ _.-._-
.--750
570
Fig.
2.
nm
Fluorometric screening of plasma porphyrins (excitation 405 nm). 1: VP patient; 2: control.
asymptomatic carriers [2]. The high sensitivity and specificity of this method was confirmed in this study: we clearly distinguished VP subjects from controls and no overlapping area appeared between these groups. Both in asymptomatic carriers of VP and in VP patients, the PO activities were found to be significantly decreased by approximately 50%. The screening method by fluorometric emission scanning has been proposed as a simple, rapid and inexpensive test to detect VP [4,5,8,9]. The purpose of our study
Table 1 Fluorometric screening results in patients and carriers of variegate porphyria Total number of specimens
Controls Asymptomatic carriers Adults Children Patients
Number of specimens o/ of specimens with with emission peak at emission peak at 630 nm 630 nm
50
0
0
36 20 32
18 0 32
50 0 100
168
V. Da Silva et al. / Clinica Chimica Acta 238 (1995) 163-168
was to evaluate its value as a diagnostic tool, not only for VP patients as previously described [4] but also for asymptomatic carriers of VP. Our results clearly showed the indications and limits of the screening method: none of the normal control plasma samples exhibited the characteristic emission peak at 630 nm; on the contrary, all the plasma samples from VP patients showed this peak suggesting that the fluorometric screening is a specific and sensitive marker in symptomatic VP subjects. However, only 50% of the plasma samples from adult asymptomatic carriers and none from child carriers exhibited a fluorescence peak. Therefore, there is a large lack of sensitivity of the test in detecting latent VP carriers; this underlines the limit of the procedure since the detection of asymptomatic carriers in VP families is the first and most important step in the prevention of acute attacks. In conclusion, plasma fluorescence characteristics should be used with great caution. If all symptomatic VP subjects are found positive, only 50% of asymptomatic carriers can be detected by this method. All family studies to detect gene carriers both in adults and children should use the measurement of lymphocyte PO activity, which is the reference method. Acknowledgement This work has been done with grants from INSERM (U. 409) and UER X. Bichat (University Paris 7). The authors would like to thank Mrs Catherine Guyomard for her help in preparing the manuscript.
References ItI Kappas A, Sassa S, Galbraith RA, Nordmann Y. The porphyrias. In: Striver CR, Beaudet AL, Sly WS, Valle D, eds. The metabolic basis of inherited diseases. 6th edn. Vol. I. McGraw Hill, New York, 1989;1305-1366. 121Deybach JC, de Vemeuil H, Nordmann Y. The inherited enzymatic defect in porphyria variegata. Hum Genet 1981;58:425-428. [31 Deybach JC, Da Silva V, Grandchamp B, Nordmann Y. The mitochondrial location of protoporphyrinogen oxidase. Eur J Biochem 1985;149:431-435. [41 Poh-Fitzpatrick MB. A plasma porphyrin fluorescence marker for variegate porphyria. Arch Dermatol 1980;116:543-547. ISI Long C, Smyth SJ, Woolf J et al. Detection of latent variegate porphyria by fluorescence emission spectroscopy of plasma. Br J Dennatol 1993;129:9-13. 161 Brenner DA, Bloomer JR. A fluorometric assay for measurement of protoporphyrinogen oxidase activity in mammalian tissue. Clin Chim Acta 1980;100:259-266. 171 Jacobs NJ, Jacobs JM. Assay for enzymatic protoporphyrinogen oxidation, a late step in heme synthesis. Enzyme 1982;28:206-219. 181 Henriquez de Salamanca R, Sepulveda P, Moran MJ, Santos JL, Fontanellas A, Hemandez A. Clinical utility of fluorometric scanning of plasma porphyrins for the diagnosis and typing of porphyrias. Clin Exp Dermatol 1993;18:128-130. 191 Martasek P. Jirsa M, Kordac V. Porphyrinogens in plasma. Arch Dermatol 1982;118:624-625.
Clinica Chimica Acta 238 (1995) 163-168
Variegate porphyria: diagnostic value of fluorometric scanning of plasma porphyrins V. Da Silva, S. Simonin, J.C. Deybach, H. Puy, Y. Nordmann* Cenrre Fran@
des Porphyries, INSERM
U.409. Hdpital Louis Mourier, 92701 Colombes cedex, France
Received 22 November 1994; revision received 3 April 1995; accepted IO April 1995
Variegate porphyria (VP) is a dominantly inherited acute hepatic porphyria characterized by a 50% decrease in activity of protoporphyrinogen oxidase (PO) which catalyses the last step of heme biosynthesis. In VP families, most of the gene carriers are asymptomatic but at risk of developing acute attacks if subjected to precipitating factors. Recognition of the carrier status is the first step of an efficient preventive care. This could be achieved by measurement of PO activity which is a sensitive and specific but tedious method. A specific plasma fluorometric emission at 626 nm has been shown in VP patients. Here we show that this simple and inexpensive method is specific but poorly sensitive, especially in detection of asymptomatic carriers. We conclude that this procedure should not replace PO aetivity measurement in VP family studies. Keywords:
Variegate
porphyria;
Protoporphyrinogen
oxidase;
Heme biosynthesis;
Fluorometric scanning
1. Introdluction Variegate porphyria (VP) is an autosomal dominant disorder with low penetrance and variable clinical expression classified as one of the acute hepatic porphyrias [ 11. VP patients and asymptomatic carriers have a 50% decrease in the activity of protoporphyrinogen oxidase (PO) (EC 1.3.3.4); PO catalyses the penultimate step in -_ Corresponding author.
??
0 1995 Elsevier Science B.V. All rights reserved 0009-898V95609.50 SSDI 0009-8981(95)06085-R
164
V. Da Silva et al. / Clinica Chimiea Acta 238 (1995) 163-168
heme biosynthesis, i.e. the conversion of protoporphyrinogen to protoporphyrin within the mitochondria [2,3]. Acute attacks in VP patients include abdominal, neurological and psychiatric symptoms. Another common feature is the development of photosensitive cutaneous lesions on exposed areas [ 1). During acute attacks, the urinary abnormalities are an increased excretion of porphyrin precursors 6-aminolevulinic acid (ALA) and porphobilinogen (PBG). In the faeces, there is an increased excretion of protoporphyrin and, to a lesser extent, of coproporphyrin. This typical porphyrin overexcretion profile in the faeces is found not only during acute attacks but also sometimes between them. Another biochemical feature is the presence in VP patients’ plasma of a specific porphyrin compound which has been described as showing a fluorometric emission at 626 nm when excitation wavelength is set at 405 nm [4]. For other types of porphyrias, the maximum emission peak is consistently located at different wavelengths [4,5]. As in other acute porphyrias, prevention of acute attacks is based on the recognition of latent cases and asymptomatic carriers in VP families. Asymptomatic carriers are rarely detected by measurement of urinary ALA and PBG and/or faecal porphyrins, which are most often within the normal range. However, patients and carriers always exhibit the same 50% defect in PO activity in nucleated cells [2]. The measurement of PO activity is tedious and usually requires specialized laboratories [2,6,7]. A rapid screening method based on plasma fluorescence emission spectroscopy has been proposed [4,5,8,9]. In a recent study, this qualitative fluorescence emission scanning was shown to be an easy, specific and sensitive tool for the detection of asymptomatic VP gene carriers [5], but the sensitivity was calculated on statistical expected frequencies of asymptomatic carriers in first and second-degree relatives and assuming a 100% specificity of the test. The aim of the present study was to evaluate the clinical relevance of this screening method in samples from symptomatic and asymptomatic VP subjects previously recognized by measurement of PO activity in circulating lymphocytes.
2. Materials and methods 2.1. Subjects Lymphocytes and plasma samples from 88 subjects were studied: 32 unrelated patients (18-65 years old) with overt VP in which diagnosis has been established by characteristic clinical symptoms, porphyrin excretion abnormalities and a documented 50% decreased PO activity in lymphocytes, and 56 VP relatives, identified as asymptomatic carriers according to their clear-cut half PO activity in lymphocytes, 20 of whom were children (< 18 years old). 2.2. Chemicals Potassium phosphates, metallic sodium, mercury, orthophosphoric acid, potassium hydroxide, ethyl alcohol, were from Prolabo (France). Tris, EDTA, Triton X100, Tween 20, glutathione (GSH) were from Sigma (France). Ficoll was from Eurobio (France). Bio-Rad Protein Assay was from Bio-Rad Laboratories (Germany). Free protoporphyrin was from Porphyrin Products, Logan, UT (USA).
V. Da Silva et al. / Clinica Chimica Acta 238 (1995) 163-168
165
2.3. Fluorometric scanning of plasma porphyrins
Plasma (100 ~1) was diluted with 900 ~1 of 250 mmol/l phosphate buffer (pH 6.7) in a fluorometric microcuvette. After stirring, the fluorescence emission spectrum was recorded in a Shimadzu-RF 540 spectrofluorometer fitted with a red sensitive photomultiplier (excitation at 405 nm, scan from 570 to 750 nm). In these conditions, the observed sensitivity was 5 nmol/l. 2.4. PO activity in lymphocytes The assay follows the kinetic development of protoporphyrin fluorescence from as previously described [2] with slight non-fluorescent protoporphyrinogen, modifications. Briefly, lymphocytes from 10 ml venous blood were isolated by the Ficoll method and storeld at -80°C. The lymphocyte pellet was resuspended in 400 ~1 of 150 mmol/l Tris-HCl (pH 8.7) 1 mmol/l EDTA, 1% Tween 20, and sonicated for three 10-s periods. After centrifugation at 10 000 x g, the protein concentration in the supernatant w,as measured with the Coomassie Blue reagent (Bio-Rad Protein Assay); 50 ~1 of the lymphocyte lysate were transferred to at least three fluorometric microcuvettes held at 4°C. Protoporphyrin (1.2 mmol/l in 80:20 v/v KOH-0.01 mol/l ethyl alcohol,) was reduced to protoporphyrinogen with 3% sodium amalgam in a dim light, under a nitrogen flow. After a 2-fold dilution with 300 mmol/l Tris-HCl (pH 7.0), 2 mmol/l EDTA, 2% Tween 20, 50 mmohl GSH, the substrate solution was adjusted to a pH value between 8.0 and 9.0 by addition of 17% phosphoric acid; 5 ~1 of this solution were immediately added to each microcuvette containing the enzymatic source. After stirring, the microcuvettes were placed at 37°C in the dark. Enzymatic reaction was stopped by addition of 1 ml of 100 mm0111Tris-HCl (pH 8.7) with 1 mmol/l EDTA, 0.1% Triton X-100, 2 mmol/l GSH at timed intervals (5, 15, 25... min). For each microcuvette the fluorescence emission was immediately measured at 635 nm with an excitation wavelength of 410 nm. The rate of non-enzymatic oxidation of protoporphyrinogen was estimated in a similar assay, performed with a tissue blank containing heat-inactivated enzyme. The amount of the non-enzymatic protoporphyrin formed in 20 min (AFB) was subtracted from the enzymatic value (AFE). To convert the fluorescence values to nanomoles of protoporphyrin formed, a reference standard was prepared as described: 10 ~1 of the 1.2 mmol/l protoporphyrin solution was diluted 120-fold with KOH-ethyl alcohol; 1 ml of 100 mmol/l Tris-HCl (pH 8.7), 1 mmol/l EDTA, 0.1% Triton X-100, 2 mmol/l GSH was added to 50 ~1 of this solution in a microcuvette. The fluorescence emission (FSTD) was immediately measured in the same conditions. The enzymatic activity expressed as nanomoles of protoporphyrin formed per hour per milligram of protein at 37°C was calculated as follows: (AFE .- AFB) x 3 x standard final cont. FSTD x mg protein in incubation The between-batch reproducibility of the enzymatic assay ‘was 10% (5 different subjects in 4 different assays). Student’s unpaired t-test was used to compare data from controls and VP subjects.
166
V. Da Silva et al. /Clinica
Chimica Acta 238 (1995) 163-168
61
..
!!!. . ..*.*. ....
51
>
*%‘A** ttfft: . . . .
41
i-
3
;
$31
I
:!
2 !
.f:*. t
II .. . ‘t:’ .;.
:
0’
P
.... ::
..;:.:.z . .............
5v.t .
AC
C
Fig. 1. Protoporphyrinogen oxidase activity (PO), nmol protoporphyrinih per mg protein. P: patients (n = 32). mean= 2.5 (S.D. = 0.4); AC: asymptomatic carriers (n = 56), mean = 2.6 (SD. = 0.3); C: controls (n = SO), mean = 4.4 (SD. = 0.4).
3.Results 3.1. Enzymatic assay
PO activity in lymphocytes was significantly decreased both in asymptomatic carriers and VP subjects (-50% P c 0.005) compared with controls (Fig. 1). 3.2. Plasma jluorometric assay An illustration of the specific plasma fluorescence emission spectrum is shown in Fig. 2 and the results of the fluorometric screening in plasma are detailed in Table 1. In our experimental procedure, all VP patients had a fluorescence emission peak at 630 nm. In the same conditions of excitation (405 nm) the emission peak in other types of porphyrias was located at different wavelengths (620 nm in porphyria cutanea tarda, acute intermittent porphyria, hereditary coproporphyria and 635 nm in erythropoietic protoporphyria; data not shown). Moreover, the fluorescence peak at 630 nm was found in only 50% of the adults and in none of children who were VP asymptomatic carriers. 4. Discussion Lymphocyte PC activity determined by fluorometric measurement of protoporphyrinogen oxidation was the reference method for the diagnosis of VP patients and
V. Da Silva et al. / Clinica
Chimica
Acta 238 (1995)
163-168
167
630 s
_. -.~ _ _.-._-
.--750
570
Fig.
2.
nm
Fluorometric screening of plasma porphyrins (excitation 405 nm). 1: VP patient; 2: control.
asymptomatic carriers [2]. The high sensitivity and specificity of this method was confirmed in this study: we clearly distinguished VP subjects from controls and no overlapping area appeared between these groups. Both in asymptomatic carriers of VP and in VP patients, the PO activities were found to be significantly decreased by approximately 50%. The screening method by fluorometric emission scanning has been proposed as a simple, rapid and inexpensive test to detect VP [4,5,8,9]. The purpose of our study
Table 1 Fluorometric screening results in patients and carriers of variegate porphyria Total number of specimens
Controls Asymptomatic carriers Adults Children Patients
Number of specimens o/ of specimens with with emission peak at emission peak at 630 nm 630 nm
50
0
0
36 20 32
18 0 32
50 0 100
168
V. Da Silva et al. / Clinica Chimica Acta 238 (1995) 163-168
was to evaluate its value as a diagnostic tool, not only for VP patients as previously described [4] but also for asymptomatic carriers of VP. Our results clearly showed the indications and limits of the screening method: none of the normal control plasma samples exhibited the characteristic emission peak at 630 nm; on the contrary, all the plasma samples from VP patients showed this peak suggesting that the fluorometric screening is a specific and sensitive marker in symptomatic VP subjects. However, only 50% of the plasma samples from adult asymptomatic carriers and none from child carriers exhibited a fluorescence peak. Therefore, there is a large lack of sensitivity of the test in detecting latent VP carriers; this underlines the limit of the procedure since the detection of asymptomatic carriers in VP families is the first and most important step in the prevention of acute attacks. In conclusion, plasma fluorescence characteristics should be used with great caution. If all symptomatic VP subjects are found positive, only 50% of asymptomatic carriers can be detected by this method. All family studies to detect gene carriers both in adults and children should use the measurement of lymphocyte PO activity, which is the reference method. Acknowledgement This work has been done with grants from INSERM (U. 409) and UER X. Bichat (University Paris 7). The authors would like to thank Mrs Catherine Guyomard for her help in preparing the manuscript.
References ItI Kappas A, Sassa S, Galbraith RA, Nordmann Y. The porphyrias. In: Striver CR, Beaudet AL, Sly WS, Valle D, eds. The metabolic basis of inherited diseases. 6th edn. Vol. I. McGraw Hill, New York, 1989;1305-1366. 121Deybach JC, de Vemeuil H, Nordmann Y. The inherited enzymatic defect in porphyria variegata. Hum Genet 1981;58:425-428. [31 Deybach JC, Da Silva V, Grandchamp B, Nordmann Y. The mitochondrial location of protoporphyrinogen oxidase. Eur J Biochem 1985;149:431-435. [41 Poh-Fitzpatrick MB. A plasma porphyrin fluorescence marker for variegate porphyria. Arch Dermatol 1980;116:543-547. ISI Long C, Smyth SJ, Woolf J et al. Detection of latent variegate porphyria by fluorescence emission spectroscopy of plasma. Br J Dennatol 1993;129:9-13. 161 Brenner DA, Bloomer JR. A fluorometric assay for measurement of protoporphyrinogen oxidase activity in mammalian tissue. Clin Chim Acta 1980;100:259-266. 171 Jacobs NJ, Jacobs JM. Assay for enzymatic protoporphyrinogen oxidation, a late step in heme synthesis. Enzyme 1982;28:206-219. 181 Henriquez de Salamanca R, Sepulveda P, Moran MJ, Santos JL, Fontanellas A, Hemandez A. Clinical utility of fluorometric scanning of plasma porphyrins for the diagnosis and typing of porphyrias. Clin Exp Dermatol 1993;18:128-130. 191 Martasek P. Jirsa M, Kordac V. Porphyrinogens in plasma. Arch Dermatol 1982;118:624-625.