Biochemical studies of experimental porphyria. II. The influence of porphyrinogenic substances in mice treated with low concentrations of griseofulvin

Our previous study showed that a 0.1% concentration of griseofulvin (GF) in feed induced an abnormality of porphyrin metabolism in some dd-K strain mice. To investigate the kind of changes in porphyrin metabolism that would be produced by other chemicals compared to the administration of 0.1% GF alone, estrogen, ethyl alcohol-iron mixture and PCB was given to dd-K strain mice. The numbers of mice with high liver protoporphyrin levels were increased in the group treated with both 0.1% GF and ethyl alcohol-iron mixture, in comparison to the levels in the 0,1% GF alone and the ethyl alcohol-iron mixture alone groups. A slight elevation in the liver protoporphyrin level was noted in only one mouse of the ethyl alcohol-iron mixture group. The remaining mice showed normal levels. From this finding, it was presumed that the administration of ethyl alcohol-iron mixture has the potential to intensify the activity of 0.1% GF alone. An elevation of the liver coproporphyrin and protoporphyrin levels was seen in the group treated with both 0.1 % GF and estrogen, as compared to the 0.1% GF alone and the estrogen alone groups. In those treated with estrogen alone, liver porphyrin levels were within normal limits except for one mouse, which showed an increase of liver protoporphyrin. An elevation of liver coproporphyrin and protoporphyrin was seen in 8 of 12 mice treated with both PCB and 0.1% GF, while there was no change in the PCB alone group. From these findings, it was inferred that the mechanism of abnormal porphyrin metabolism due to estrogen and PCB combined with 0.1% GF differs from the mechanism of abnormality due to 0.1% GF alone. It appears that the addition of some chemicals to a 0.1% GF feed enhances the action of the 0.1% GF alone.     

The synergistic effect of isonicotinic acid hydrazide (INH) and griseofulvin (GF) on porphyrin metabolism

Abnormal porphyrin metabolism can be induced by several chemicals. To investigate the synergistic effect on porphyrinopathy of isonicotinic acid hydrazide (INH) with a low concentration of griseofulvin (GF), the two chemicals were given to mice simultaneously. INH was added to drinking water at a concentration of 0.05%. GF was mixed with feed at a concentration of 0.1%. The mice (dd-y strain) were divided into four groups. Those in group A were fed normally. Group B received only 0.1% GF, group C received only 0.05% INH, and group D received both 0.1% GF and 0.05% INH. The treatment was continued for 13 to 30 weeks. After treatment, the mice were anesthetized and sacrificed. The liver and whole blood were taken for analysis of porphyrins. The results revealed a slight elevation of erythrocytic porphyrins in the groups treated with 0.1% GF or 0.05% INH alone and remarkable abnormalities in the hepatic and erythrocytic porphyrin levels of the group simultaneously treated with both chemicals. These results show that INH itself may have a small potential for the induction of porphyric abnormalities, and that the administration of INH with a low concentration of GF induces marked porphyrinopathy in dd-y strain mice.     

Experimental Murine Protoporphyria Induced by Griseofulvin (GF): The Relationship between Hepatic Porphyrin Levels and Liver Function Test Values in Mice Treated with GF

To investigate the hepatic abnormalities accompanying experimental protoporphyria due to griseofulvin (GF), liver function test values and porphyrin levels in mice were assayed at days 2, 4, 8, and 16 after starting the administration of 0.5% GF feed. Furthermore, in an attempt to elucidate the harmful effects of GF on liver functions, the above mentioned assay was also performed after the feed was discontinued in mice given 0.5% GF feed for 16 days. The hepatic protoporphyrin (PP) level had already risen by day 2, but the erythrocytic PP level was within normal limits at that time. Hepatic PP levels increased gradually, followed by an increase in erythrocytic PP levels. The variation in liver function test values roughly paralleled the porphyrin levels. Over the time span of the response to GF, the variations in the serum glutamate oxaloacetate transaminase (S-GOT) levels, serum glutamate pyruvate transaminase (S-GPT) levels, and leucine amino peptidase (LAP) levels resembled those in hepatic PP. On the other hand, the changes in alkaline phosphatase (ALP) levels paralleled those of the erythrocytic PP levels. Erythrocytic and fecal protoporphyrin levels decreased to the normal level one month after the discontinuation of GF administration, but the hepatic protoporphyrin level still was 53.6 times higher than the normal level two months after switching to normal feed. The values of liver function tests had returned to within the normal range after one month. By the fourth day after the administration of GF, a brown pigmented material could be observed around the hepatocytes and the Glisson sheath; the amount of this material increased day by day. Its amount gradually decreased after cessation of GF. These results suggested that the hepatic abnormalities were not induced by the deposition of PP in the liver, but by the direct toxicity of GF. This means that GF-induced protoporphyria mice are not suitable for investigating porphyric damage to the liver because of the strong hepatic intoxication by GF. However, they may be useful for studies of the removal of deposits of porphyrin granules from the liver.     

Porphyrin concentrations in various human tissues

Abstract We measured the concentrations of total porphyrins and their metabolites (uro-, hepta-, hexa-, penta-, copro- and protoporphyrin) in various human tissues: liver, erythrocytes, skin, adipose tissue, and mammary gland. The porphyrin concentrations varied within minor limits, e.g., 3.1 ±2.3 nmol porphyrins/g liver and 0.50±0.10 nmol/g erythrocytes. No significant differences were detectable in other tissues in comparison with liver. In all tissues, the predominant metabolite was protoporphyrin, followed by coproporphyrin, whereas only low concentrations of higher carboxylatcd porphyrins such as uroporphyrin were detectable. It is concluded that porphyrin metabolism and its regulation is similar in all human tissues, perhaps with some small differences in the erythrocytes.     

The influence of ursodesoxycholic acid (URSO) on griseofulvin (GF)-induced protoporphyria

To investigate the influence of ursodesoxycholic acid (URSO) on griseofulvin (GF)-induced protoporphyria mice, analysis of hepatic, erythrocytic, and fecal porphyrin levels and histopathological examinations were performed in dd-Y strain mice treated with 0.5% GF and/or 0.5% URSO. We observed no difference of hepatic and fecal porphyrin levels between the GF group and GF with URSO group, although an elevation of erythrocytic porphyrin levels was seen in the GF with URSO group. However, remarkable hepatic atrophy revealed in the GF with URSO group. Furthermore, a strong emission of red fluorescence was observed in the liver under long wave ultraviolet. Histopathologically, many focal necrosis was found in the liver specimen treated with GF and URSO. We expected that URSO might facilitate the excretion of porphyrin from bile to feces because of suppression of transfer from serum to erythrocyte like cholic acid (CA). But, the action of URSO appears to be different from that of CA. We consider that the 0.5% concentration of URSO plays a role in the cytotoxic effect to the liver.     

In vivo porphyrin production by P. acnes in untreated acne patients and its modulation by acne treatment

Propionibacterium acnes is often discussed as a contributing pathogenic factor in the aetiology of acne lesions. The aim of this study was to test which porphyrin patterns are synthesized by P. acnes in vivo in untreated acne patients and during standard acne regimens. These photosensitive compounds are potential targets for photo-dynamic therapy of acne and need to be better characterized in the skin. Using high-performance liquid chromatography coproporphyrin III was the main porphyrin identified in all patients. Coproporphyrin I and protoporphyrin were found at considerably lower concentrations. When the porphyrin concentration of individual patients receiving isotretinoin was analysed repeatedly over time, clinical improvement was associated with lowered levels of porphyrins. Statistical analysis demonstrated a significant reduction in the porphyrin fractions only in the isotretinoin group which was associated with clinical improvement 2 months after starting therapy.     

Transient porphyrinemia in a liver transplant recipient

We report a case of acquired porphyrinemia in a liver transplant recipient. Thrombosis of the portal vein and hepatic artery led to massive necrosis of the patient's first donor liver, necessitating its removal. After an 18-hour anhepatic period, a second donor liver was transplanted. During this interval, the patient was warmed with an infrared heat lamp that emitted bright visible light. Following the anhepatic period, a photodistributed cutaneous erythema was noted. The level of total serum porphyrins during the anhepatic period was significantly elevated at 1.548 mumol/L (normal, less than 0.018 mumol/L). Fractionation showed that the levels of the hydrophobic porphyrins, coproporphyrin and protoporphyrin, were elevated, while the levels of the hydrophilic porphyrins remained normal. Following the successful second liver transplantation, the patient's porphyrin levels declined. This is the first reported case of acquired porphyrinemia in a liver transplant recipient.     

Interdependence between degree of porphyrin excess and disease severity in congenital erythropoietic porphyria (Günther’s disease)

Various clinical and biochemical observations point to a relationship between degree of disease expression and metabolic disturbance in autosomal recessive congenital erythropoietic porphyria (Günther’s disease). Although the clinical manifestations have been well described since Günther’s fundamental observations, an interdependence between disease severity and porphyrin excess has yet to be elucidated. We investigated porphyrin metabolism in nine Indian patients suffering from the characteristic clinical symptoms: skin photosensitivity, red-colored urine as a sign of extremely elevated porphyrinuria and mild to severe hemolytic anemia. Porphyrins in urine, feces and blood were analysed by HPTLC and HPLC in conjunction with spectrophotometry and spectrofluorometry. Uroporphyrinogen III synthase activities in red blood cells were determined using a coupled-enzyme assay. Biochemical studies revealed varying degrees of porphyrinuria with total urinary porphyrins between 23 and 102 μmol/24 h (normal <0.2 μmol/24 h) and uroporphyrin predominance. Urinary and fecal coproporphyrin isomer I were markedly elevated to 87– 97% and 81–93% (normal <31%, <75%), respectively. Overproduction of porphyrins led to a considerable porphyrinemia with mainly copro- and protoporphyrin. A hitherto undescribed fecal porphyrin pattern with increased protoporphyrin levels was found in three patients. This atypical finding was probably related to severe hemolysis since protoporphyrin can be excreted only via the liver with bile in the feces. High porphyrin levels in urine, feces and blood were associated with worse cutaneous symptoms. Activities of uroporphyrinogen III synthase in red blood cell lysates were decreased to between 9% and 30% of controls. Patients showed increased porphobilinogen deaminase activities, up to 190% of control. Deficiency of uroporphyrinogen III synthase activity was reflected by inversion of the relationship between and isomer III leading to dominance of isomer I. Elevation of porphobilinogen deaminase activities is related to hemolysis and, additionally, to regulatory compensation for the enzyme deficiency. Variations in both the severity of photosensitivity and the enhancement of porphyrin production and excretion indicate the molecular heterogeneity of this disease. These findings suggest a close relationship between the metabolic disturbance reflected by porphyrin excess and the severity of disease expression. Received: 12 July 1996     

Effects of KC-400 (polychlorinated biphenyls) on porphyrin metabolism--liver and blood porphyrin analyses in rats treated with KC-400

To investigate the influence of polychlorinated biphenyls (PCB) on porphyrin metabolism, Wistar rats were orally administered KC-400. The mean value of liver/body weight ratio in normal rats was 3.86%, with a range of 3.44% to 5.22%. Their mean blood protoporphyrin level was 23.3 μg/dl packed cell volume (p.c.v.), with a range of 11.8 to 64.4 μg/dl p.c.v., and their mean liver protoporphyrin level was 0.17 μg/gm wet weight, with a range of 0.03 to 0.40 μg/gm wet weight. A feed containing KC-400 (140–160 ppm) was given for 2 to 20 weeks to 20 rats. The mean value of liver/body weight ratio was 5.00%, with a range of 3.80% to 6.16%. The mean erythrocyte protoporphyrin level was 47.4 μg/dl p.c.v., with a range of 19.7 to 83.2 μg/dl p.c.v., and the mean liver protoporphyrin level was 0.36 μg/gm wet weight, with a range of 0.04 to 0.67 μg/gm wet weight. The liver coproporphyrin level showed abnormalities in 2 of 20 rats fed the feed containing KC-400, with values of 0.71 and 9.89 μg/gm wet weight, respectively. After administration of KC-400 feed for 43 weeks, the mean value of blood coproporphyrin level rose to 32.1 μg/dl p.c.v., with a range of 11.4 to 54.6 μg/dl p.c.v. The mean erythrocyte protoporphyrin level was 74.3 μg/dl p.c.v., with a range of 49.5 to 102.1 μg/dl p.c.v. The mean liver coproporphyrin level was elevated to 5.15 μg/gm wet weight, with a range of 0.45 to 9.48 μg/gm wet weight, and the mean liver protoporphyrin level was 0.65 μg/gm wet weight, with a range of 0.43 to 9.88 μg/gm wet weight. Thus, there was an increase in both liver porphyrin and coproporphyrin following KC-400 feeding. Liver cells in the rats with abnormal porphyrin levels exhibited a red fluorescence. From these results, we can deduce that KC-400 may induce abnormalities in porphyrin metabolism, when it is fed orally for a long period of time. These abnormalities in porphyrin metabolism are limited to the liver only.     

Relationship between N-Acetyltransferase (NAT) Activity and Liver Protoporphyrin Level in Experimental Porphyria

The results of our previous studies demonstrated that isonicotinic acid hydrazide (INH) can aggravate griseofulvin (GF)-induced protoporphyria in mice. To elucidate this phenomenon, we studied the relationship between liver protoporphyrin (PP) levels and N-acetyltransferase (NAT) activity, which is known to be a major catabolic enzyme of INH metabolism in the liver. The results revealed a significant correlation between liver PP levels and NAT activity in the mice fed 0.1% GF and 0.05% INH. In this group, mice with high NAT activity developed severe protoporphyria. There was no correlation, however, between liver PP levels and NAT activity in the mice fed GF alone or INH alone. The result suggested that INH metabolites formed by NAT enhance the hepatotoxicity of GF in mice.     

Biochemical study of fecal porphyrin in porphyria cutanea tarda

Fecal, urinary and erythrocyte porphyrin analyses using the solvent extraction method were performed in 26 patients with porphyria cutanea tarda (PCT) and 144 normal controls. The levels of fecal uroporphyrin (UP) and coproporphyrin (CP) were markedly increased in the PCT group, especially in comparison with the protoporphyrin (PP) level. The values of the UP/PP and CP/PP ratios in the feces were also elevated over those in the control group. It appears that fecal porphyrin excretion is basically similar to urinary porphyrin excretion in PCT. The analysis of fecal porphyrins and the observation of fecal UP/PP and CP/PP ratios may be helpful in the biochemical diagnosis of PCT. In particular, the elevation of CP/PP ratio is characteristic of PCT.     

Urinary porphyrin excretion measurements in healthy neonates.

The objective of this study was to establish normal reference ranges for porphyrins in healthy neonates. There is little information about urinary porphyrin excretion in this age group. This knowledge may provide an early diagnostic tool for detecting subtle alterations or latent forms in disorders of heme biosynthesis. Fifty healthy neonates were selected from the Department of Obstetrics. Total urinary porphyrins in random specimens were analysed by a spectrofluorometry method. The measurement of porphyrin fractions was made by fluorometric high-performance liquid chromatography (HPLC). The results were adjusted to urinary creatinine excretion to correct any imprecision and interindividual variation in body mass. The urinary total porphyrin had a median value of 331.50 (nmol/L). A statistically significant relationship between total porphyrin (nmol/L) and creatinine (mmol/L) was found (p < 0.01). The porphyrin/creatinine ratio showed a median value of 56.30 nmol/mmol creatinine. The study of individual porphyrins revealed that coproporphyrin and uroporphyrin were the major porphyrins excreted in neonates (coproporphyrin represents 81.98% and uroporphyrin 16.64% of total porphyrin); in both cases, isomer I was predominant with median values of 22.36 and 6.25 nmol/mmol creatinine, respectively. No significant relationships were found between porphyrin excretion and sex, gestational age, weight, or height. Our data provide the reference limits for porphyrins in neonates as a diagnostic guideline for evaluation of subtle alterations in heme biosynthesis.     

Urinary porphyrin analyses in patients with porphyria cutanea tarda

Urinary porphyrin analyses were carried out in five patients with porphyria cutanea tarda and in a control group of 44 individuals. Quantitative analysis revealed that the mean value of coproporphyrin was 67.3 μg/l (range 13.1–189.1 μg/l) in 44 normal individuals and the mean value of uroporphyrin was 9.7 μg/l (range 5.0 to 13.7 μg/l) in 11 of 44 controls, but not detectable in the remainder. Four of the five patients with porphyria cutanea tarda, however, had markedly elevated coproporphyrin and uroporphyrin levels. Urinary porphyrin pattern analysis was carried out using one dimentional thin layer chromatography. The pattern in controls revealed that the mean value of coproporphyrin was 85.5%, while the other fractions consituted less than 6.0%. In the porphyria cutanea tarda group, however, hepta-carboxyl porphyrin and uroporphyrin were predominant in two; all five fractions appeared in roughly equal amounts in one; and in the remaining two, coproporphyrin was decreased to a value of only about 50%. A patient treated by phlebotomy was also evaluated by this method. His urinary porphyrin pattern showed a shift from uroporphyrin to a high concentration of coproporphyrin during and after the treatment. With this method, it is very simple to obtain data for urinary porphyrin pattern analysis, and many samples can be analyzed simultaneously. The profile can aid in the diagnosis of porphyria cutanea tarda, especially when the patients show low levels of urinary porphyrins quantitatively.     

Latent porphyria cutanea tarda

A total of 142 subjects have been examined; of these 49 healthy relatives of patients with manifest porphyria cutanea tarda (PCT) (group 1), 48 subjects with melanodermal skin changes characteristic of PCT abd with anamnesis aggravated for alcoholism (group 2), and 45 patients with chronic liver diseases (group 3). None of the examinees has developed photosensitization symptoms. The findings have been compared to the results of examinations of 24 normal subjects and of 145 patients with manifest PCT. Minimal abnormalities of porphyrin metabolism have been detected in 43 subjects (30.2%). In group 1 subjects these abnormalities presented as increased levels of uroporphyrin and fecal coproporphyrin, in Groups 2 and 3 as secondary coproporphyrinuria and a symptomatic rise of fecal protoporphyrin level. Latent PCT has been diagnosed in 18 patients (12.7%). In latent PCT the total porphyrin excretion with the urine has been 10-fold lower than in manifest PCT, not exceeding 1000 nmol/day; in has been associated with a relative elevation of uroporphyrin level (up to 42-65% of the total porphyrin content). Increased coproporphyrin concentrations have been recorded, with coproporphyrin share making up over 60% of the total amount. It is possible that the minimal shifts of porphyrin metabolism anticipate the development of the biochemical syndrome of latent PCT. The author suggests criteria for the early diagnosis of the latent forms of the disease. He considers that the examinees should be referred to a group at risk of developing manifest PCT.     

Effect of oral contraceptives on urinary porphyrins

Several studies have reported on the appearance of cutaneous porphyria in people treated with estrogen. This study was undertaken to investigate the effects of oral contraceptives (OCs) on levels of porphyrins in urine in nonporphyric women. 30 women aged 21-40 and on OCs were observed as the study group, and 10 women using other contraceptive methods were observed as the control group. Urinary porphyrins were measured by the Remington method. Levels of urinary coproporphyrin and of urinary uroporphyrin were higher in the study group than in the control group. These differences, however, are not statistically significant. To study the effect of duration of OC treatment, women who had taken OCs for 3-6 months and women who had taken OCs for longer than 6 months were observed, and the means of urinary coproporphyrin and uroporphyrin measured. Again, the differences were found to be insignificant, although slightly higher values of urinary coproporphyrin were obtained for women who had been on OCs for a longer period. These results compare favorably with others reported in the published literature. It can be concluded that OC treatment has no significant bearing on porphyrin metabolism.     

Influence of pH on porphyrin production in Propionibacterium acnes

Summary Propionibacterium acnes was grown on Eagle's medium for 4–15 days at pH 5.3–7.2 The porphyrin production was measured both by direct fluorometry and by high pressure liquid chromatography (HPLC). The greatest concentration of porphyrins was produced at pH 6.1. Protoporphyrin was the dominant porphyrin species present in the bacteria in all samples. The relative amount of coproporphyrin was greatest at pH 6.7 after 4 days of incubation. In human skin there are local variations in the pH; therefore our findings may be of importance for porphyrin production in acne.     

Five cases of porphyria cutanea tarda with mild cutaneous changes: evaluation of the efficacy of phlebotomy by the pattern analysis of urinary porphyrins

Five cases of porphyria cutanea tarda (PCT) with mild cutaneous changes are reported. Acute episodes of photosensitivity were not seen in all patients. Laboratory examinations showed a predominance of uroporphyrin (UP) excretion in the urine in 4 out of 5 cases. One case showed a relatively low urinary porphyrin level even though abnormality of the porphyrin pattern was present. Four cases showing a predominance of UP excretion in the urine revealed a high level of serum coproporphyrin (CP) as well. Histopathologically, all cases showed a deposition of PAS positive materials surrounding the small blood vessels in the upper dermis, in addition to the deposition of IgG materials visualized by the immunofluorescent technique. Phlebotomy was performed fifteen times in one case. The urinary porphyrin level was gradually reduced to normal levels and the urinary porphyrin pattern shifted from a predominance of UP and hepta-carboxyl porphyrin to a predominance of CP excretion. Analysis of the urinary porphyrin profile is useful as a laboratory tool for diagnosis or observation of the course of PCT, as well as for the quantitative analysis of urinary porphyrins.     

ERYTHROPOIETIC PROTOPORPHYRIA

Summary.— A case of erythropoietic protoporphyria is described. To our knowledge this is the second to be reported in Jugoslavia. The disease started at 3 years of age, with skin photosensitivity. burning and itching of the face and hands. Symptoms and signs were present almost throughout the year, with only short periods of freedom. In recent years the disease had tended to decrease in severity, but lately she has developed some bullous haemorrhagic lesions on the feet.
Increased values of RBC protoporphyrin, and to a lesser extent of coproporphyrin, were found. Increased values of faecal protoporphyrin and coproporphyrin were also found. Urinary porphyrins were within normal limits.     

Urinary porphyrin excretion in a human population highly exposed to hexachlorobenzene

BACKGROUND: Data from an epidemic reported in Turkey (1955-1959) is the only information about the relationship between hexachlorobenzene (HCB) intake and porphyria cutanea tarda in humans. No information is available on the HCB threshold exposure level to induce porphyria cutanea tarda. OBJECTIVES: To study HCB serum levels and urinary porphyrin excretion in the inhabitants of a village located near an organochlorine compound factory with high HCB concentrations in the air and to detect possible alterations in urinary porphyrin excretion and examine their relationship with HCB serum levels. DESIGN: Cross-sectional study. SETTING: Unit of Porphyrias of a tertiary care facility in Barcelona, Spain. PARTICIPANTS: Six hundred four inhabitants of the village who were older than 14 years provided serum and urine samples (185 participants were factory workers). MAIN OUTCOME MEASURES: Serum HCB was analyzed by gas chromatography coupled to electron capture detection. Quantification of urinary total porphyrins was performed by spectrofluorimetry. Porphyrin profile was determined by high-pressure liquid chromatography. RESULTS: Hexachlorobenzene was detected in all serum samples (mean, 39.8 ng/mL; range, 1.1-1616.0 ng/mL), and levels were higher in factory workers. Mean +/- SD level urinary total porphyrin average concentration was 98 +/- 69 nmol/L (range, 9-1009 nmol/L). Only the urine sample with the highest porphyrin concentration showed an increase of highly carboxylated porphyrins, with a typical profile of porphyria cutanea tarda. In the remaining 603 urine samples, coproporphyrin was the predominant fraction. CONCLUSION: The airborne exposure to and increased body burden of HCB in the Flix village population are not enough to trigger a significant alteration of the heme biosynthesis pathway.     

外用苯烯莫德对特应性皮炎样小鼠的治疗作用

目的：评价外用苯烯莫德对卡泊三醇（MC903）诱导的特应性皮炎小鼠模型的治疗作用。方法：36只BALB/c小鼠，6只作为空白对照，30只给予MC903诱导特应性皮炎后再随机分为基质组、0.5%苯烯莫德组、1.0%苯烯莫德组、0.1%糠酸莫米松组和0.1%他克莫司组，治疗前后测量皮肤厚度、经皮水丢失（TEWL）、皮损内CD4+T细胞、胸腺基质淋巴细胞生成素（TSLP）蛋白表达。结果：0.5%和1.0%苯烯莫德乳膏组皮损评分、耳部皮肤厚度、TEWL、皮损内CD4+T细胞及TSLP蛋白水平较基质组均显著降低（P<0.05），1.0%苯烯莫德组疗效优于0.5%苯烯莫德组和他克莫司组（P<0.05），与糠酸莫米松组差异无显著性（P>0.05）。结论：外用苯烯莫德乳膏可减轻特应性皮炎样小鼠局部炎症反应。