Psoriatic plaques exhibit red autofluorescence that is due to protoporphyrin IX.

In evaluating the autofluorescence properties of normal and diseased skin we discovered that psoriatic plaques can emit a distinct red fluorescence when illuminated with UVA or blue light. Using a macrospectrofluorometer equipped with a 442 nm excitation laser, a sharp in vivo fluorescence emission peak around 635 nm could be demonstrated within the plaques of 34 of 75 (45%) patients with psoriasis. This peak was absent from normal appearing skin of psoriatic patients and also from the skin of 66 patients with other dermatologic diseases. A microspectrofluorometer coupled with the same excitation laser was used to obtain emission spectra of separated epidermal sheets and dermis from plaques demonstrating macroscopic red autofluorescence. An emission peak around 635 nm was observed in all three patients thus studied, but only on spectra obtained from the epidermis. Additional spectra of vertical microscopic sections of intact psoriatic skin from five other patients revealed that the peak originated from the stratum corneum. Emission spectra from other microlocations including the mid-epidermis and dermis of psoriatic and normal skin, as well as the stratum corneum of normal skin, failed to demonstrate a 635 nm peak. The excitation and emission fluorescence spectra of acid extracts of psoriatic scale from five patients were all similar to those of protoporphyrin IX in acid solution. High performance liquid chromatography identified the presence of protoporphyrin IX in the acid extracts from psoriatic scale of the same patients. We conclude that native psoriatic plaques can exhibit red autofluorescence that is due to elevated levels of protoporphyrin IX within scales.

A molecular view of the neurologic porphyrias.

At the present time, the use of molecular techniques for diagnosing the neurologic porphyrias is limited mainly to those laboratories doing research in the area. Because any of four genes may be involved, it is important to confirm the diagnosis in the index case before undertaking molecular investigation. With advances in methodology, it may eventually become possible to test for the many mutations that cause these disorders in a simple, cost-effective manner.

Environmental chemical exposures and disturbances of heme synthesis.

Porphyrias are relatively uncommon inherited or acquired disorders in which clinical manifestations are attributable to a disturbance of heme synthesis (porphyrin metabolism), usually in association with endogenous or exogenous stressors. Porphyrias are characterized by elevations of heme precursors in blood, urine, and/or stool. A number of chemicals, particularly metals and halogenated hydrocarbons, induce disturbances of heme synthesis in experimental animals. Certain chemicals have also been linked to porphyria or porphyrinuria in humans, generally involving chronic industrial exposures or environmental exposures much higher than those usually encountered. A noteworthy example is the Turkish epidemic of porphyria cutanea tarda produced by accidental ingestion of wheat treated with the fungicide hexachlorobenzene. Measurements of excreted heme precursors have the potential to serve as biological markers for harmful but preclinical effects of certain chemical exposures; this potential warrants further research and applied field studies. It has been hypothesized that several otherwise unexplained chemical-associated illnesses, such as multiple chemical sensitivity syndrome, may represent mild chronic cases of porphyria or other acquired abnormalities in heme synthesis. This review concludes that, although it is reasonable to consider such hypotheses, there is currently no convincing evidence that these illnesses are mediated by a disturbance of heme synthesis; it is premature or unfounded to base clinical management on such explanations unless laboratory data are diagnostic for porphyria. This review discusses the limitations of laboratory measures of heme synthesis, and diagnostic guidelines are provided to assist in evaluating the symptomatic individual suspected of having a porphyria.

Hereditary coproporphyria: exon screening by heteroduplex analysis detects three novel mutations in the coproporphyrinogen oxidase gene.

Hereditary coproporphyria is a dominantly inherited disorder of porphyrin metabolism caused by a partial deficiency of coproporphyrinogen oxidase, the sixth enzyme in the heme synthetic pathway. We investigated the molecular basis of hereditary coproporphyria in three unrelated patients, amplifying each exon of the coproporphyrinogen oxidase gene and performing heteroduplex analysis to look for mutations. Unique heteroduplex patterns were noted in exons 2, 3, and 6. Sequencing revealed different mutations in each patient: a G-->A point mutation encoding a glutamic acid to lysine substitution at codon 101 (E101K), a C-->T point mutation encoding a proline to serine substitution at codon 149 (P149S), and a one base-pair insertion in exon 6 (968insT). No other mutations were found on sequencing the remaining exons and their intron-exon junctions. The two point mutations affect amino acids that are conserved in all species studied to date. The one base-pair insertion in exon 6 is the first frameshift mutation to be described in the coproporphyrinogen oxidase gene. This study adds three new mutations to those that have been previously reported, and all have been restricted to single families. These results indicate that hereditary coproporphyria is a genetically heterogeneous disease.

Detection of a R173W mutation in the porphobilinogen deaminase gene in the Nova Scotian "foreign Protestant" population with acute intermittent porphyria: a founder effect.

OBJECTIVES: Acute intermittent porphyria (AIP) is caused by mutations in the porphobilinogen deaminase (PBGD) gene that disrupt the function of the enzyme. Many mutations that lead to decreased PBGD activity have been described. An Arg to Trp substitution at codon 173 (CGG-->TGG in exon 10) and designated R173W, which leads to a CRIM-negative phenotype, has been reported in Swedish, Finnish, Scottish, and South African kindreds, and in a Nova Scotian proband with fatal AIP. In this work, we investigated the presence of this mutation in a Nova Scotian patient population presenting with AIP. DESIGN AND METHODS: Single-strand conformation polymorphism analysis and DNA sequencing by TA cloning and Sanger's dideoxy chain termination method, were used to confirm the maternal transmission of this mutation to the proband. The mutation also eliminates an Ncil (also Mspl) endonuclease restriction site, which allows for detection of the mutant allele by polymerase chain reaction amplification and restriction enzyme digestion. RESULTS: The family of the Nova Scotian proband and four other AIP kindreds showed the presence of the same mutation. These five families are descendants of German, Swiss, and French immigrants historically known as the "Foreign Protestants," who were recruited to Nova Scotia in the 1750s. CONCLUSION: In all these families, descent from one couple that settled in Nova Scotia in 1751 has been identified by genealogy research, consistent with a founder effect within this population. This is the first identified mutation in PBGD causing AIP that has been linked to a founder effect in descendants of an immigrant population to North America, and which could be traced to such a distant background, similar to the South African variegate porphyria mutation.

Acute intermittent porphyria: laboratory diagnosis by molecular methods.

Acute intermittent porphyria is a neurologic disorder caused by a partial deficiency of porphobilinogen (PBG) deaminase, the third enzyme in the synthetic pathway for heme. The isolation and characterization of the gene for PBG deaminase has brought molecular techniques for diagnosing the disease within reach. Over 60 mutations causing acute intermittent porphyria have been found, most of which are confined to one or several families. Because no single mutation accounts for more than a fraction of cases, screening techniques for locating and identifying unknown mutations are very important. Once a mutation has been characterized, testing of family members is straightforward, and gene carriers can be identified or excluded with greater accuracy than is possible with conventional biochemical tests.

Heteroduplex analysis detects frameshift and point mutations in patients with acute intermittent porphyria.

We used heteroduplex analysis to screen for mutations in the porphobilinogen deaminase gene in 21 patients with acute intermittent porphyria (AIP). Unique banding patterns were investigated by direct sequencing of polymerase chain reaction products and, when indicated, sequencing of cloned DNA containing the exon of interest. Two frameshift mutations were found, a 2-bp deletion in exon 5 and a 1-bp insertion in exon 7. Both mutations generate a premature stop codon. Two point mutations, in exons 10 and 14, were also observed. The C-->T mutation in exon 10 codes for an Arg173 to Trp substitution, while a G-->A mutation in exon 14 changes Trp283 into a premature stop codon. This study extends the spectrum of mutations that cause AIP and demonstrates the utility of heteroduplex analysis as a screening technique.

Acute intermittent porphyria in a native North American family. Biochemical and molecular analysis.

A native North American family with acute intermittent porphyria was investigated by molecular methods to locate the causative mutation and identify carriers of the mutant allele. All 15 exons of the porphobilinogen deaminase gene were screened by single-strand conformation polymorphism analysis, and a unique banding pattern was observed in exon 14. Sequencing revealed a one base-pair insertion in this exon that shifts the reading frame of the mRNA, and generates a premature stop codon. Family members were tested for the mutation by amplification of exon 14 followed by digestion with the restriction enzyme NlaIII. The activity of erythrocyte porphobilinogen deaminase was measured in 36 family members. The results agreed with mutational analysis in 32 cases. However, four individuals who were not gene carriers had low enzyme activity, and in the absence of molecular genetic data would have been incorrectly diagnosed. This is the first study to identify the molecular basis of acute intermittent porphyria in native North Americans.

Molecular diagnosis of acute intermittent porphyria by analysis of DNA extracted from hair roots.

Analysis for mutations in the porphobilinogen deaminase gene offers a more definitive diagnosis of acute intermittent porphyria (AIP) than do conventional biochemical tests. We used single-strand conformation polymorphism analysis followed by direct sequencing to identify a new G-->A mutation at the last position of intron 7 in a patient with AIP. The mutation disrupts the invariant AG dinucleotide at the 3' splice acceptor site and therefore interferes with mRNA processing. To identify other individuals who inherited this mutation, we analyzed five hairs with intact roots collected by each participating family member and sent to us by mail. DNA was extracted from the hair roots and amplified by the polymerase chain reaction. The amplified products were digested with the restriction enzyme BsaJI to confirm the presence or absence of the mutation. All six family members who were known to have AIP tested positive, as did three members who had not been previously diagnosed. Hair roots provide a convenient, accessible, and economical alternative to blood as a source of DNA for molecular diagnostic testing.

Frameshift mutations in exons 9 and 10 of the porphobilinogen deaminase gene produce a crossreacting immunological material (CRIM)-negative form of acute intermittent porphyria.

Single-strand conformation polymorphism analysis was used to screen all 15 exons of the porphobilinogen deaminase gene from 13 patients with acute intermittent porphyria. Unique banding patterns in two amplified gene fragments, one containing exon 9 and another containing exon 10, were further investigated. Sequence analysis of cloned genomic DNA revealed a single base pair insertion in the middle of exon 9 in one patient and a single base pair deletion near the 3' end of exon 10 in two related patients. Both mutations change the reading frame of the mRNA transcript and predict proteins that are normal at their NH2-terminal ends but contain novel, unrelated sequences at their COOH-terminal ends and are prematurely terminated. Frameshift mutations in the porphobilinogen deaminase gene are uncommon; this is the first report of an insertion mutation causing acute intermittent porphyria.

Determining the optimal dose of Photofrin in miniswine atherosclerotic plaque.

The purpose of this study was to determine the lowest dose of Photofrin (P) that would produce a 3:1 or greater ratio between atherosclerotic (AS) and control arterial walls. Aortoiliac AS was created in 24 Yucatan miniswine by a combination of balloon endothelial injury and 2% cholesterol and 15% lard diet for 7 weeks. Arteriography was then performed to demonstrate AS lesions. Following this, swine were given intravenously P in one of the following single dosages: 2.5, 1.0 or 0.5 mg/kg. Twenty-four hours later, swine were sacrificed and aortoiliac and control carotid artery segments removed and photographed with ultraviolet light to differentiate fluorescent from nonfluorescent areas. Arterial specimens were submitted for histologic analysis and chemical extraction for determination of fluorescence using a spectrofluorometer. Tissue concentration (ng/g tissue) of P from AS vessels were: Group I, 130.4 +/- 82.7; Group II, 10.0 +/- 1.2; and Group III, 9.1 +/- 0.6, respectively (P < 0.05). Ratios of P concentration in AS: control vessels were: Group I, 8.1 +/- 13.7; Group II, 1.1 +/- 0.2; and Group III, 0.9 +/- 0.1, respectively (P < 0.05). These results demonstrated that a P dose of 2.5 mg/kg provided at least a 3:1 ratio between AS: control artery wall.

Electrophoresis underestimates the concentration of polyclonal immunoglobulins in serum.

The concentration of gamma-migrating immunoglobulins in blood serum was measured in 200 healthy blood donors by two methods, nephelometry and electrophoresis. Nephelometric values were calculated as IgG + IgM + 1/2 IgA because immunoglobulin G (IgG) and immunoglobulin M (IgM) migrate entirely within the gamma region and immunoglobulin A (IgA) is split between the gamma and beta regions. Electrophoretic values were calculated as % gamma x total protein. Gels were scanned with two different densitometers, providing two sets of electrophoretic data. In each case, the correlation between nephelometry and electrophoresis was very good (r = 0.94 and r = 0.96). However, electrophoresis consistently gave lower results than nephelometry (mean = 2.3 g/L and 4.8 g/L lower, respectively). The disparity between these methods was greater as the concentration of immunoglobulins increased. It is concluded that (1) electrophoresis gives a relative but not absolute value of immunoglobulin concentration in serum; (2) nephelometric and electrophoretic values cannot be used interchangeably; and (3) reference ranges for electrophoresis must take into account the densitometer used.

Quantitative fluorometric screening test for fecal porphyrins.

We describe a fluorometric method for screening and quantifying porphyrins in stool. A small sample of stool is extracted with concentrated HCI and is diluted 200-fold in 3 mol/L HCI before analysis. An excitation scan is done from 350 to 450 nm, monitoring emission at 603 nm. Total porphyrin is estimated at the isosbestic point for coproporphyrin and protoporphyrin (402.5 nm). Monitoring emission at 603 nm eliminates interference from chlorophyll, obviating the need for extraction with ether. The position of the excitation peak gives some indication of the nature of the porphyrins in the stool. The acid extract can be injected directly into an HPLC system for fractionation studies. Our method correlates well with the spectrophotometric method developed by Lockwood et al. (Clin Chem 1985;31:1163-7). However, in our method, the sample is easier to process and the assay has higher sensitivity than their assay. The reference interval for porphyrin in healthy individuals by the fluorometric method is less than 300 nmol/g dry weight. We can detect as little as 1 nmol of porphyrin per gram (dry weight) of stool. Results of the method vary linearly with stool porphyrin concentrations as great as 4000 nmol/g dry weight. The within-run imprecision of the method is 3%.

A unique bone-like variant of alkaline phosphatase.

The authors describe an unusual variant of alkaline phosphatase (ALP) discovered in a patient with a 90-fold increase in serum ALP. The variant ALP was indistinguishable from the bone isoenzyme when subjected to chemical inhibition, heat inactivation, lectin precipitation, and routine electrophoresis. However, treatment with neuraminidase produced a marked decrease in the ALP variant's electrophoretic mobility and a reduction in its molecular weight. A bone marrow biopsy revealed metastatic infiltration of the bone marrow by adenocarcinoma of the prostate accompanied by a remarkable amount of new bone formation, suggesting a bone origin for the unusual isoenzyme. The authors believe this to be the first report of an ALP variant with these properties.

An automated assay of glycogen phosphorylase in the direction of phosphorolysis.

We have developed a rapid, automated assay for glycogen phosphorylase that measures the degradative reaction. The substrate contains higher concentrations of phosphate and AMP than other methods, and the enzyme is preincubated with the substrate before adding phosphate to start the reaction. These modifications improve the activity and linearity of the assay. The new assay compares favourably with a standard phosphorylase assay in the synthetic direction and is linear to 500 U/L. We have used this assay to measure phosphorylase activity in human tissue samples and muscle biopsy specimens.