Quantification of Urine and Plasma Porphyrin Precursors Using LC-MS in Acute Hepatic Porphyrias: Improvement in Routine Diagnosis and in the Monitoring of Kidney Failure Patients.

BACKGROUND: The quantification of delta-aminolevulinic acid (ALA) and porphobilinogen (PBG) in urine are the first-line tests for diagnosis and monitoring of acute hepatic porphyrias (AHP). Ion-exchange chromatography (IEC), which is time- and staff-consuming and limited to urine, is still the preferred method in many specialized laboratories, despite the development of mass spectrometry-based methods. METHODS: We describe a new LC-MS method that allows for rapid and simple quantification of ALA and PBG in urine and plasma with an affordable instrument that was used to analyze 2260 urine samples and 309 blood samples collected in 2 years of routine activity. The results were compared to those obtained with IEC, and urine reference ranges and concentrations in asymptomatic carriers were determined. Plasma concentrations were measured in healthy subjects and subgroups of symptomatic and asymptomatic AHP carriers. RESULTS: In urine, the clinical decision limits were not impacted by the change of method despite discrepancies in low absolute concentrations, leading to lower normal values. Two-thirds of asymptomatic AHP carriers (with the exception of coproporphyria carriers) showed an increased urine PBG concentration. Urine and plasma levels showed a good correlation except in patients with kidney disease in whom the urine/plasma ratio was relatively low. CONCLUSION: We described an LC-MS based method for the routine diagnosis and monitoring of AHP that allows for the detection of more asymptomatic carriers than the historical method. Blood analysis appears to be particularly relevant for patients with kidney disease, where urine measurement underestimates the increase in ALA and PBG levels.

Porphyrin precursors and risk of primary liver cancer in acute intermittent porphyria: A case-control study of 188 patients.

Acute intermittent porphyria (AIP) is a rare hereditary metabolic disease characterized by acute attacks and accumulation of the porphyrin precursors 5-aminolevulinic acid (ALA) and porphobilinogen (PBG). Patients with AIP have a high risk of primary liver cancer (PLC). We aimed to assess the association between porphyrin precursor excretion and the risk for PLC in patients with AIP. We studied 48 patients with AIP who developed PLC between 1987 and 2015 and 140 age and sex matched controls with AIP but no PLC. Data on all available urinary PBG and ALA samples collected from 1975 until 1 year before PLC diagnosis were analyzed and compared between cases and controls using logistic regression. Porphyrin precursor excretion was higher in patients with PLC (PBG median 7.9 [IQR 4.4-21.9] mmol/mol creatinine) than in controls (3.8 [1.2-9.8]) (adjusted odds ratio 1.07, 95% confidence interval: 1.02-1.12). None of the 28 patients with all registered samples below the upper limit of normal (ULN) developed PLC, and only one of the 45 patients with all samples <2× ULN developed PLC. Among non-PLC controls, ALA and PBG levels decreased after age 50-60 while an increasing trend was observed after age 65 among those who developed PLC. Increased urinary porphyrin precursors are associated with a high risk of developing PLC. Patients with normal levels appear to have a low risk while high or increasing ALA and PBG after age 65 indicates high risk, which should be considered in surveillance decisions.

RNA interference therapy in acute hepatic porphyrias.

The acute hepatic porphyrias (AHPs) are inherited disorders of heme biosynthesis characterized by life-threatening acute neurovisceral attacks precipitated by factors that upregulate hepatic 5-aminolevulinic acid synthase 1 (ALAS1) activity. Induction of hepatic ALAS1 leads to the accumulation of porphyrin precursors, in particular 5-aminolevulinic acid (ALA), which is thought to be the neurotoxic mediator leading to acute attack symptoms such as severe abdominal pain and autonomic dysfunction. Patients may also develop debilitating chronic symptoms and long-term medical complications, including kidney disease and an increased risk of hepatocellular carcinoma. Exogenous heme is the historical treatment for attacks and exerts its therapeutic effect by inhibiting hepatic ALAS1 activity. The pathophysiology of acute attacks provided the rationale to develop an RNA interference therapeutic that suppresses hepatic ALAS1 expression. Givosiran is a subcutaneously administered N-acetylgalactosamine-conjugated small interfering RNA against ALAS1 that is taken up nearly exclusively by hepatocytes via the asialoglycoprotein receptor. Clinical trials established that the continuous suppression of hepatic ALAS1 mRNA via monthly givosiran administration effectively reduced urinary ALA and porphobilinogen levels and acute attack rates and improved quality of life. Common side effects include injection site reactions and increases in liver enzymes and creatinine. Givosiran was approved by the US Food and Drug Administration and European Medicines Agency in 2019 and 2020, respectively, for the treatment of patients with AHP. Although givosiran has the potential to decrease the risk of chronic complications, long-term data on the safety and effects of sustained ALAS1 suppression in patients with AHP are lacking.

AGA Clinical Practice Update on Diagnosis and Management of Acute Hepatic Porphyrias: Expert Review.

DESCRIPTION: The acute hepatic porphyrias (AHP) are rare, inborn errors of heme-metabolism and include acute intermittent porphyria, hereditary coproporphyria, variegate porphyria, and porphyria due to severe deficiency of 5-aminolevulinic acid dehydratase. Acute intermittent porphyria is the most common type of AHP, with an estimated prevalence of patients with symptoms of approximately 1 in 100,000. The major clinical presentation involves attacks of severe pain, usually abdominal and generalized, without peritoneal signs or abnormalities on cross-sectional imaging. Acute attacks occur mainly in women in their childbearing years. AHP should be considered in the evaluation of all patients, and especially women aged 15-50 years with recurrent severe abdominal pain not ascribable to common causes. The screening tests of choice include random urine porphobilinogen and δ-aminolevulinic acid corrected to creatinine. All patients with elevations in urinary porphobilinogen and/or δ-aminolevulinic acid should initially be presumed to have AHP. The cornerstones of management include discontinuation of porphyrinogenic drugs and chemicals, administration of oral or intravenous dextrose and intravenous hemin, and use of analgesics and antiemetics. Diagnosis of AHP type can be confirmed after initial treatment by genetic testing for pathogenic variants in HMBS, CPOX, PPOX, and ALAD genes. AHP is also associated with chronic symptoms and long-term risk of systemic arterial hypertension, chronic renal and liver disease, and hepatocellular carcinoma. Patients who have recurrent acute attacks (4 or more per year) should be considered for prophylactic therapy with intravenous hemin or subcutaneous givosiran. Liver transplantation is curative and reserved for patients with intractable symptoms who have failed other treatment options. METHODS: This expert review was commissioned and approved by the American Gastroenterological Association (AGA) Institute Clinical Practice Updates Committee (CPUC) and the AGA Governing Board to provide timely guidance on a topic of high clinical importance to the AGA membership, and underwent internal peer review by the CPUC and external peer review through standard procedures of Gastroenterology. These Best Practice Advice (BPA) statements were drawn from a review of the published literature and from expert opinion. Because systematic reviews were not performed, these BPA statements do not carry formal ratings of the quality of evidence or strength of the presented considerations. Best Practice Advice Statements BEST PRACTICE ADVICE 1: Women aged 15-50 years with unexplained, recurrent severe abdominal pain without a clear etiology after an initial workup should be considered for screening for an AHP. BEST PRACTICE ADVICE 2: Initial diagnosis of AHP should be made by biochemical testing measuring δ-aminolevulinic acid, porphobilinogen, and creatinine on a random urine sample. BEST PRACTICE ADVICE 3: Genetic testing should be used to confirm the diagnosis of AHP in patients with positive biochemical testing. BEST PRACTICE ADVICE 4: Acute attacks of AHP that are severe enough to require hospital admission should be treated with intravenous hemin, given daily, preferably into a high-flow central vein. BEST PRACTICE ADVICE 5: In addition to intravenous hemin, management of acute attacks of AHP should include pain control, antiemetics, management of systemic arterial hypertension, tachycardia, and hyponatremia, and hypomagnesemia, if present. BEST PRACTICE ADVICE 6: Patients should be counseled to avoid identifiable triggers that may precipitate acute attacks, such as alcohol and porphyrinogenic medications. BEST PRACTICE ADVICE 7: Prophylactic heme therapy or givosiran, administered in an outpatient setting, should be considered in patients with recurrent attacks (4 or more per year). BEST PRACTICE ADVICE 8: Liver transplantation for AHP should be limited to patients with intractable symptoms and significantly decreased quality of life who are refractory to pharmacotherapy. BEST PRACTICE ADVICE 9: Patients with AHP should be monitored annually for liver disease. BEST PRACTICE ADVICE 10: Patients with AHP, regardless of the severity of symptoms, should undergo surveillance for hepatocellular carcinoma, beginning at age 50 years, with liver ultrasound every 6 months. BEST PRACTICE ADVICE 11: Patients with AHP on treatment should undergo surveillance for chronic kidney disease annually with serum creatinine and estimated glomerular filtration rate. BEST PRACTICE ADVICE 12: Patients should be counseled on the chronic and long-term complications of AHP, including neuropathy, chronic kidney disease, hypertension, and hepatocellular carcinoma, and need for long-term monitoring.

Heterogeneous molecular behavior in liver tumors (HCC and CCA) of two patients with acute intermittent porphyria.

INTRODUCTION: Acute intermittent porphyria (AIP) is a very rare (orphan) metabolic disorder of porphyrin biosynthesis which is characterized by elevated plasma and urine levels of 5-aminolevulinic acid (5-ALA) and porphobilinogen (PBG). Patients with this disorder which is caused by a germline mutation of the hydroxymethylbilan-synthase (HMBS)-gene have a high risk of primary liver cancer which may be determined by disease activity. The exact mechanism of carcinogenesis of this rare tumor is unknown, however. MATERIALS AND METHODS: We analyzed paraffin-embedded formalin-fixed liver tumor and normal liver specimens of two female AIP patients treated at the Munich EPNET center. One patient had developed hepatocellular carcinoma (HCC), the other intrahepatic cholangiocarcinoma (CCA). Since biallelic inactivation of HMBS had been observed in one study, we used Sanger and next-generation sequencing with a 8 gene porphyria panel plus 6 potential modifier loci to search for mutations in DNA extractions. RESULTS: In the patient with the HCC, we found a second inactivating mutation in the HMBS gene in the tumor but not in the adjacent normal liver tissue. No mutation could be found in the liver tissues of the patient with CCA, however. CONCLUSIONS: Biallelic inactivation of HMBS or protoporphyrinogen-oxidase (PPOX), another enzyme of porphyrin biosynthesis, has been observed in patients with acute porphyrias and liver tumors. We could confirm this in our patient with HCC with a mutation in HMBS but not in the one with CCA. Since 5-ALA can be converted into carcinogenic substances such as 4,5-dioxovaleric acid (DOVA) or 3,6-dihydropyrazine-2,5-dipropanoic acid (= cyclic dimerization product of 5-ALA), local production of these metabolites in hepatic areas with complete loss of HMBS activity may contribute to liver carcinogenesis.

Evaluation of Metabolic Changes in Acute Intermittent Porphyria Patients by Targeted Metabolomics.

Acute intermittent porphyria (AIP) is an inherited rare hepatic disorder due to mutations within the hydroxymethylbilane gene. AIP patients with active disease overproduce aminolevulinic acid (ALA) and porphobilinogen (PBG) in the liver which are exported inducing severe neurological attacks. Different hepatic metabolic abnormalities have been described to be associated with this condition. The goal of this research was to explore the metabolome of symptomatic AIP patients by state-of-the art liquid chromatography-tandem mass spectrometry (LC-MS/MS). A case versus control study including 18 symptomatic AIP patients and 33 healthy controls was performed. Plasmatic levels of 51 metabolites and 16 ratios belonging to four metabolic pathways were determined. The results showed that the AIP patients presented significant changes in the two main areas of the metabolome under study: (a) the tryptophan/kynurenine pathway with an increase of tryptophan in plasma together with increase of the kynurenine/tryptophan ratio; and (b) changes in the tricarboxylic acid cycle (TCA) including increase of succinic acid and decrease of the fumaric acid/succinic acid ratio. We performed a complementary in vitro study adding ALA to hepatocytes media that showed some of the effects on the TCA cycle were parallel to those observed in vivo. Our study confirms in plasma previous results obtained in urine showing that AIP patients present a moderate increase of the kynurenine/tryptophan ratio possibly associated with inflammation. In addition, it also reports changes in the mitochondrial TCA cycle that, despite requiring further research, could be associated with an energy misbalance due to sustained overproduction of heme-precursors in the liver.

Mechanistic modelling of enzyme-restoration effects of new recombinant liver-targeted proteins in acute intermittent porphyria.

BACKGROUND AND PURPOSE: Acute intermittent porphyria (AIP) is a rare disease caused by a genetic mutation in the hepatic activity of the porphobilinogen-deaminase. We aimed to develop a mechanistic model of the enzymatic restoration effects of a novel therapy based on the administration of different formulations of recombinant human-PBGD (rhPBGD) linked to the ApoAI lipoprotein. This fusion protein circulates in blood, incorporating into HDL and penetrating hepatocytes. EXPERIMENTAL APPROACH: Single i.v. dose of different formulations of rhPBGD linked to ApoAI were administered to AIP mice in which a porphyric attack was triggered by i.p. phenobarbital. Data consist on 24 h urine excreted amounts of heme precursors, 5-aminolevulinic acid (ALA), PBG and total porphyrins that were analysed using non-linear mixed-effects analysis. KEY RESULTS: The mechanistic model successfully characterized over time the amounts excreted in urine of the three heme precursors for different formulations of rhPBGD and unravelled several mechanisms in the heme pathway, such as the regulation in ALA synthesis by heme. Treatment with rhPBGD formulations restored PBGD activity, increasing up to 51 times the value of the rate of tPOR formation estimated from baseline. Model-based simulations showed that several formulation prototypes provided efficient protective effects when administered up to 1 week prior to the occurrence of the AIP attack. CONCLUSION AND IMPLICATIONS: The model developed had excellent performance over a range of doses and formulation type. This mechanistic model warrants use beyond ApoAI-conjugates and represents a useful tool towards more efficient drug treatments of other enzymopenias as well as for acute intermittent porphyria.

Multibiomarker approach to assess the magnitude of occupational exposure and effects induced by a mixture of metals.

Metals and metalloids including lead (Pb), arsenic (As) and manganese (Mn) can occur as mixtures in occupational contexts, such as mines. These chemicals are all known to be neurotoxic and provoke changes in heme metabolism also known to induce neurotoxicity. The objective of this work was to propose a multi-biomarker (BM) methodology to screen subjects exposed to the mixture of Pb, As and Mn and assess the severity of their exposure/effects, in an individual basis. The urinary levels of the metals, dela-aminolevulinic acid (ALA) and porphyrins were determined in Portuguese miners and in a control group. The combination of Pb and As urinary levels had the highest capability to identify subjects occupationally exposed to this mixture in mines, as evaluated through Receiver Operating Characteristic (ROC) (A = 98.2%; p < 0.05), allowing that 94.2% of 86 studied subjects were properly identified and the generation of an equation indicating the odd of a subject be considered as exposed to the metal mixture. The combination of urinary ALA and porphyrins revealed to be best one to be applied in the assessment of subjects with high, intermediate, and low magnitudes of exposure/effects, with 95.7% of 46 miners classified correctly according to their severity sub-group and allowing to generate equations, which can be applied in new subjects. The proposed methodology showed a satisfactory performance, evaluating in an integrated manner the magnitude of exposure/effects of the exposed workers, may contributing to improve the control of their health.

Reassessment of the threshold of the blood lead level to increase urinary δ-aminolevulinic acid based on their relationship in recent lead workers in Japan.

OBJECTIVES: The present study investigated the quantitative relationship between blood lead (Pb-B) and urinary δ⁻aminolevulinic acid (ALA-U) in lead workers, and examined the Pb-B level that induces increases in ALA-U and the corresponding ALA-U. METHODS: We collected 10 562 data sets on Pb-B, ALA-U, age, and smoking habits from 808 workers (771 males and 37 females) who underwent multiple lead poisoning medical examinations at a lead-acid battery and lead smelting plant in Japan between 1995 and 2018. Females were excluded, and data collected in 169 subjects prior to engaging in lead work were used as the control. Pb-B and ALA-U levels were measured by graphite furnace atomic absorption spectrophotometry and high-performance liquid chromatography respectively. RESULTS: A significant dose-response relationship was observed between Pb-B and ALA-U based on Pb-B-classified observations of increases in ALA-U values and the prevalence of over-reference ALA-U as well as regression analyses independent of smoking habits. The results obtained revealed that the threshold of Pb-B to increase ALA-U was 25.1-35.0 µg/dL based on the significant elevation point of the prevalence of over-reference ALA-U and 16.2-22.3 µg/dL from a 3rd degree regression equation. CONCLUSIONS: We proposed a threshold of Pb-B to increase ALA-U of 20 µg/dL and a biologically acceptable value of ALA-U of 1 mg/L, corresponding to the threshold.

Protocolo de actuación en pacientes con sospecha de porfiria aguda / Protocol for patients with suspected acute porphyria

Las porfirias son errores congénitos del metabolismo de las porfirinas o ruta biosintética del hemo. El acúmulo de los precursores de las porfirinas, ácido delta aminolevulínico (ALA) y/o porfobilinógeno (PBG) es responsable de las crisis neuroviscerales de las porfirias agudas que cuando se expresan clínicamente se inician con intenso dolor abdominal. Durante las crisis la eliminación urinaria de PBG y ALA siempre es muy elevada. La excesiva concentración de PBG en orina es fácilmente identificable mediante el sencillo test de Hoesch. Un test negativo descarta crisis porfírica actual. El protocolo de actuación en pacientes con dolor abdominal agudo no filiado en los que el test de Hoesch positivo permite la sospecha de porfiria aguda se basa en los siguientes aspectos: valoración clínica inicial en el servicio de urgencias, supresión de los posibles factores desencadenantes, tratamiento específico de la crisis con hemina y/o sobrecarga de glucosa y tratamiento sintomático

Porphyrias are a group of congenital errors in porphyrin metabolism and in the heme biosynthetic pathway. Accumulation of porphyrin precursors (delta-aminolaevulinic acid and porphobilinogen) is responsible for the neurovisceral crises of acute porphyria, which, when expressed clinically, start with intense abdominal pain. During crises, the urinary elimination of porphobilinogen and delta-aminolaevulinic acid is always very high. Excessive porphobilinogen concentration in urine is easily identified using the simple Hoesch test. A negative test rules out a current porphyric crisis. The clinical protocol for patients with acute abdominal pain of unknown origin in whom a positive Hoesch test leads to the suspicion of acute porphyria is based on the following aspects: initial clinical assessment in the emergency department, suppression of potential triggers, specific treatment for the crisis with hemin and/or glucose overload and symptomatic treatment

Phase 3 Trial of RNAi Therapeutic Givosiran for Acute Intermittent Porphyria.

BACKGROUND: Up-regulation of hepatic delta-aminolevulinic acid synthase 1 (ALAS1), with resultant accumulation of delta-aminolevulinic acid (ALA) and porphobilinogen, is central to the pathogenesis of acute attacks and chronic symptoms in acute hepatic porphyria. Givosiran, an RNA interference therapy, inhibits ALAS1 expression. METHODS: In this double-blind, placebo-controlled, phase 3 trial, we randomly assigned symptomatic patients with acute hepatic porphyria to receive either subcutaneous givosiran (2.5 mg per kilogram of body weight) or placebo monthly for 6 months. The primary end point was the annualized rate of composite porphyria attacks among patients with acute intermittent porphyria, the most common subtype of acute hepatic porphyria. (Composite porphyria attacks resulted in hospitalization, an urgent health care visit, or intravenous administration of hemin at home.) Key secondary end points were levels of ALA and porphobilinogen and the annualized attack rate among patients with acute hepatic porphyria, along with hemin use and daily worst pain scores in patients with acute intermittent porphyria. RESULTS: A total of 94 patients underwent randomization (48 in the givosiran group and 46 in the placebo group). Among the 89 patients with acute intermittent porphyria, the mean annualized attack rate was 3.2 in the givosiran group and 12.5 in the placebo group, representing a 74% lower rate in the givosiran group (P<0.001); the results were similar among the 94 patients with acute hepatic porphyria. Among the patients with acute intermittent porphyria, givosiran led to lower levels of urinary ALA and porphobilinogen, fewer days of hemin use, and better daily scores for pain than placebo. Key adverse events that were observed more frequently in the givosiran group were elevations in serum aminotransferase levels, changes in serum creatinine levels and the estimated glomerular filtration rate, and injection-site reactions. CONCLUSIONS: Among patients with acute intermittent porphyria, those who received givosiran had a significantly lower rate of porphyria attacks and better results for multiple other disease manifestations than those who received placebo. The increased efficacy was accompanied by a higher frequency of hepatic and renal adverse events. (Funded by Alnylam Pharmaceuticals; ENVISION ClinicalTrials.gov number, NCT03338816.).

Uncontrolled Acute Intermittent Porphyria as a Cause of Spontaneous Abortion.

INTRODUCTION: The association of acute intermitetn porphyria (AIP) with pregnancy and as a cause of spontaneous abortion is rare. AIM: To show a case of AIP known before pregnancy in a patient who had a spontaneous abortion. CASE REPORT: A gynecologist examined 26-year-old patient in the 8th week of gestation, due to initial spontaneous abortion, abdominal pain, constipation, muscle weakness, vomiting and dark colour of urine. Her therapy was dydrogesterone. In consultation with an anesthesiologist, a short intravenous anesthesia, vacuum aspiration, and curettage were performed.During hospitalization, the patient ceased to take harmful drugs and she was given haemarginate, glucose and symptomatic drugs, and she recovered completely. CONCLUSION: Treatment of threatened spontaneous abortion in AIP remains the subject of dilemma and controversy, and future research is needed.

Pharmacokinetics and Pharmacodynamics of the Small Interfering Ribonucleic Acid, Givosiran, in Patients With Acute Hepatic Porphyria.

Givosiran is a small interfering ribonucleic acid agent that was recently approved in the United States for the treatment of acute hepatic porphyria (AHP). This phase I study evaluated the safety, pharmacokinetic, and pharmacodynamic profile of subcutaneously (SC) administered givosiran in patients with acute intermittent porphyria, the most common AHP type. Givosiran was rapidly absorbed from the SC injection site with peak plasma concentrations achieved within 0.5-5 hours followed by elimination with a short half-life of 4-10 hours. Plasma exposures of AS(N-1)3' givosiran, an active metabolite with equal potency as givosiran, was 35%-75%. Givosiran treatment resulted in a rapid and dose-dependent reduction in urinary aminolevulinic acid (ALA) and porphobilinogen (PBG) towards the upper limit of normal (ULN) in AHP patients. Greater and more sustained reductions in ALA and PBG were achieved with once monthly dosing compared with once quarterly dosing. After monthly dosing, trough ALA levels were reduced to below the ULN, approximately 95% reduction from baseline, at both the 2.5 and 5.0 mg/kg doses.

Manifestation of Systemic Toxicity in Rats after a Short-Time Inhalation of Lead Oxide Nanoparticles.

Outbred female rats were exposed to inhalation of lead oxide nanoparticle aerosol produced right then and there at a concentration of 1.30 ± 0.10 mg/m3 during 5 days for 4 h a day in a nose-only setup. A control group of rats were sham-exposed in parallel under similar conditions. Even this short-time exposure of a relatively low level was associated with nanoparticles retention demonstrable by transmission electron microscopy in the lungs and the olfactory brain. Some impairments were found in the organism's status in the exposed group, some of which might be considered lead-specific toxicological outcomes (in particular, increase in reticulocytes proportion, in δ-aminolevulinic acid (δ-ALA) urine excretion, and the arterial hypertension's development).

Metal environmental contamination within different human exposure context- specific and non-specific biomarkers.

Exposure to high levels of persistent pollutants, such as metal mixtures, is commonly encountered by the general population especially in industrialized countries. The aim of this work was to evaluate how metal pollution in contaminated areas is reflected in terms of biomarkers (BMs) of exposure and effect in human sub-populations living in distinct non-occupational environmental contexts. Thus, four Portuguese sub-populations living in different areas of Portugal were studied: i- the exposure of each member of these sub-populations to lead (Pb), manganese (Mn) and arsenic (As) was evaluated by determining metal levels in urine; ii- biochemical changes were assessed, establishing the levels of urinary metabolites of heme biosynthesis; iii- the ability of combinations of these BMs to predict the context of exposure of each subject was tested, as to develop a tool to identify adverse health effects in these environmentally exposed populations. Concerning the combinations of BMs, heme precursors in urine (delta-aminolevulinic acid and porphyrins), were predictive of contexts of environmental exposures, with 94.2% of the studied subjects correctly identified as to their sub-population origin. The use of non-specific BMs may affirm the exposure to Pb, Mn and As, also reflecting health effects induced by a chemical environmental mixture. Our studies affirm the difficulty in establishing a metal reference population.

Clinical Guide and Update on Porphyrias.

Physicians should be aware of porphyrias, which could be responsible for unexplained gastrointestinal, neurologic, or skin disorders. Despite their relative rarity and complexity, most porphyrias can be easily defined and diagnosed. They are caused by well-characterized enzyme defects in the complex heme biosynthetic pathway and are divided into categories of acute vs non-acute or hepatic vs erythropoietic porphyrias. Acute hepatic porphyrias (acute intermittent porphyria, variegate porphyria, hereditary coproporphyria, and aminolevulinic acid dehydratase deficient porphyria) manifest in attacks and are characterized by overproduction of porphyrin precursors, producing often serious abdominal, psychiatric, neurologic, or cardiovascular symptoms. Patients with variegate porphyria and hereditary coproporphyria can present with skin photosensitivity. Diagnosis relies on measurement of increased urinary 5-aminolevulinic acid (in patients with aminolevulinic acid dehydratase deficient porphyria) or increased 5-aminolevulinic acid and porphobilinogen (in patients with other acute porphyrias). Management of attacks requires intensive care, strict avoidance of porphyrinogenic drugs and other precipitating factors, caloric support, and often heme therapy. The non-acute porphyrias are porphyria cutanea tarda, erythropoietic protoporphyria, X-linked protoporphyria, and the rare congenital erythropoietic porphyria. They lead to the accumulation of porphyrins that cause skin photosensitivity and occasionally severe liver damage. Secondary elevated urinary or blood porphyrins can occur in patients without porphyria, for example, in liver diseases, or iron deficiency. Increases in porphyrin precursors and porphyrins are also found in patients with lead intoxication. Patients with porphyria cutanea tarda benefit from iron depletion, hydroxychloroquine therapy, and, if applicable, elimination of the hepatitis C virus. An α-melanocyte-stimulating hormone analogue can reduce sunlight sensitivity in patients with erythropoietic protoporphyria or X-linked protoporphyria. Strategies to address dysregulated or dysfunctional steps within the heme biosynthetic pathway are in development.

Computational disease model of phenobarbital-induced acute attacks in an acute intermittent porphyria mouse model.

INTRODUCTION: Acute intermittent porphyria (AIP) is characterized by hepatic over-production of the heme precursors when aminolevulinic acid (ALA)-synthase 1 is induced by endogenous or environmental factors. The aim of this study was to develop a semi-mechanistic computational model to characterize urine accumulation of heme precursors during acute attacks based on experimental pharmacodynamics data and support the development of new therapeutic strategies. METHODS: Male AIP mice received recurrent phenobarbital challenge starting on days 1, 9, 16 and 30. 24-h urine excretion of ALA, porphobilinogen (PBG) and porphyrins from challenges D1, D9 and D30 constituted the training data set to build the mechanistic model using the population approach. In a second study, porphyrin and porphyrin precursor excretion from challenge D16 were used as a validation data set. RESULTS: The computational model presented the following features: (i) urinary excretion of ALA, PBG and porphyrins was governed by unmeasured circulating heme precursor amounts, (ii) the circulating amounts of ALA and PBG were the precursors of circulating amounts of PBG and porphyrins, respectively, and (iii) the phenobarbital effect linearly increased the synthesis of circulating ALA and PBG levels. The model displayed good parameter precision (coefficient of variation below 32% in all parameters), and adequately described the experimental data. Finally, a theoretical hemin effect was implemented to illustrate the applicability of the model to dosage optimization in drug therapies. CONCLUSIONS: A semi-mechanistic disease model was successfully developed to describe the temporal evolution of urinary heme precursor excretion during recurrent biochemical-induced acute attacks in AIP mice. This model represents the first computational approach to explore and optimize current and new therapies.

Homozygous hydroxymethylbilane synthase knock-in mice provide pathogenic insights into the severe neurological impairments present in human homozygous dominant acute intermittent porphyria.

Acute intermittent porphyria (AIP) is an inborn error of heme biosynthesis due to the deficiency of hydroxymethylbilane synthase (HMBS) activity. Human AIP heterozygotes have episodic acute neurovisceral attacks that typically start after puberty, whereas patients with homozygous dominant AIP (HD-AIP) have early-onset chronic neurological impairment, including ataxia and psychomotor retardation. To investigate the dramatically different manifestations, knock-in mice with human HD-AIP missense mutations c.500G>A (p.Arg167Glu) or c.518_519GC>AG (p.Arg173Glu), designated R167Q or R173Q mice, respectively, were generated and compared with the previously established T1/T2 mice with ~30% residual HMBS activity and the heterozygous AIP phenotype. Homozygous R173Q mice were embryonic lethal, while R167Q homozygous mice (R167Q+/+) had ~5% of normal HMBS activity, constitutively elevated plasma and urinary 5-aminolevulinic acid (ALA) and porphobilinogen (PBG), profound early-onset ataxia, delayed motor development and markedly impaired rotarod performance. Central nervous system (CNS) histology was grossly intact, but CNS myelination was delayed and overall myelin volume was decreased. Heme concentrations in liver and brain were similar to those of T1/T2 mice. Notably, ALA and PBG concentrations in the cerebral spinal fluid and CNS regions were markedly elevated in R167Q+/+ mice compared with T1/T2 mice. When the T1/T2 mice were administered phenobarbital, ALA and PBG markedly accumulated in their liver and plasma, but not in the CNS, indicating that ALA and PBG do not readily cross the blood-brain barrier. Taken together, these studies suggest that the severe HD-AIP neurological phenotype results from decreased myelination and the accumulation of locally produced neurotoxic porphyrin precursors within the CNS.

Lifestyle factors including diet and biochemical biomarkers in acute intermittent porphyria: Results from a case-control study in northern Norway.

BACKGROUND: Lifestyle factors, including a low intake of carbohydrates, dieting, alcohol consumption, cigarette smoking and stress are some of the possible triggers of attacks in acute intermittent porphyria (AIP). The influence of lifestyle factors, including energy intake, diet and alcohol consumption on the biochemical disease activity in AIP and biochemical nutritional markers were examined. METHODS: A case-control study with 50 AIP cases and 50 controls matched for age, sex and place of residence was performed. Dietary intake was registered using a food diary in 46 matched pairs. Symptoms, alcohol intake, stress and other triggering factors of the last AIP attack were recorded on questionnaires. Porphyrin precursors, liver and kidney function markers, vitamins, diabetogenic hormones and other nutritional biomarkers were analyzed by routine methods. The Wilcoxon matched-pairs signed rank test was used to compare the cases vs. controls. The Spearman's rank correlation coefficient was used on the cases. RESULTS: Increasing total energy intake was negatively correlated with the biochemical disease activity. The intake of carbohydrates was lower than recommended, i.e., 40 and 39% of total energy intake in the AIP cases and controls, respectively. The plasma resistin level was significantly higher (p = .03) in the symptomatic than asymptomatic cases. Plasma insulin was lower in those with high porphobilinogen levels. The intake of sugar and candies were higher in the AIP cases with low U-delta aminolevulinic acid (ALA) levels (p = .04). Attacks were triggered by psychological stress (62%), physical strain (38%), food items (24%) and alcohol (32%) in the 34 symptomatic cases. Alcohol was used regularly by 88% of the cases (3.2 g ethanol/day) and 90% of the controls (6.3 g/day), but the intake was significantly lower in symptomatic than in asymptomatic cases (p = .045). CONCLUSION: A high intake of energy, sugar and candies and a higher insulin level were associated with a lower biochemical disease activity. The resistin level was higher in the symptomatic than the asymptomatic cases. AIP patients drink alcohol regularly, but the intake was significantly lower in the symptomatic cases. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT01617642.

Spectrophotometric photodynamic diagnosis of prostate cancer cells excreted in voided urine using 5-aminolevulinic acid.

To evaluate the feasibility of photodynamic diagnosis using 5-aminolevulinic acid (PDD-ALA) for detection of prostate cancer (PCa) cells in urine samples after prostate massage in patients who were suspected to have PCa. One hundred and eighty-nine patients with abnormal digital rectal examination and/or an elevated prostate-specific antigen (PSA) level who underwent initial prostate biopsy were recruited. After prostate massage, the first 60 mL of voided urine was collected. For PDD-ALA, 50 mL was used. The rest of collected urine was used for polymerase chain reaction (PCR) of PSA and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). After incubation for 2 h, the intensity was measured at 635 nm under a 405-nm wavelength excitation. The results of PDD-ALA were compared with those of an initial transrectal ultrasound (TRUS)-guided prostate biopsy. Overall, 126/189 (67%) samples that showed bands of both PSA and GAPDH on PCR in urine samples were analyzed. The area under the curve, sensitivity, and specificity of PDD-ALA were 0.74, 77, and 67%, respectively. The value of PDD-ALA was significantly higher in patients with Gleason scores of 6 (p = 0.03), 7 (p = 0.005), and 8-10 (p = 0.0002) than in those with negative biopsy results. In the multivariate analysis, high PSA density, abnormal findings on TRUS, and a high value of PDD-ALA were significant markers for prediction of positive biopsy results. PDD-ALA was useful to predict positive biopsy results in patients who underwent initial prostate biopsy with suspected PCa. This PCa-detection method has potential for clinical use.