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Urinary excretion of porphyrins, porphobilinogen and δ-aminolaevulinic acid following an attack of acute intermittent porphyria
  1. Joanne T Marsden1,
  2. David C Rees2
  1. 1Department of Clinical Biochemistry, King's College Hospital, London, UK
  2. 2Department of Haematological Medicine, King's College Hospital, London, UK
  1. Correspondence to Dr Joanne T Marsden, Department of Clinical Biochemistry, King's College Hospital NHS Foundation Trust, Denmark Hill, London SE5 9RS, UK; joannemarsden1{at}nhs.net

Abstract

Background and objectives The porphyrias are a group of rare, mainly inherited, diseases caused by a deficiency of one of the enzymes of the haem biosynthesis pathway. The biochemical hallmark of an acute attack is an increase in urine porphobilinogen (PBG), together with an increase in urinary excretion of δ-aminolaevulinic acid (ALA) and total urine porphyrins (TUP). In patients with acute intermittent porphyria (AIP) the concentrations of the porphyrin precursors are thought to remain elevated for many years following an acute attack, although this has not been well documented.

Methods We measured urine ALA, PBG and TUP excretion in 20 patients with AIP following an attack of acute porphyria over a time period of 3 months to 23 years after their last documented acute attack.

Results We showed that urinary concentrations of all metabolites remain elevated for many years. The urinary half life of TUP was 5.3 years, ALA 7.7 years and PBG 10.6 years. Even after 20 years, PBG concentrations remained elevated above the normal range.

Conclusions Our study highlights the difficulties of using urinary analysis for diagnosing recurrent attacks, and also raises important questions about the pathophysiology of the condition.

  • Metabolism
  • Laboratory Tests
  • Inherited Pathology

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Introduction

The porphyrias are a group of mainly inherited diseases affecting the haem synthesis pathway. There are four types of acute porphyrias: aminolaevulinic acid dehydratase (ALAD) deficiency porphyria, acute intermittent porphyria (AIP), hereditary coproporphyria and variegate porphyria; each is caused by a deficiency of one of the enzymes of the pathway shown in figure 1. The clinical presentation of the acute porphyrias is unpredictable although abdominal pain is nearly always present.1 There are other associated problems such as neuropathy, psychiatric symptoms and hypertension. Patients may complain of muscle pain and have constipation, diarrhoea and nausea.2

Figure 1

Haem biosynthesis pathway showing the enzymes and associated porphyrias.

Most individuals who inherit acute porphyria remain symptom free; there are no reliable estimates of clinical penetrance, although it is thought that 10–50% of patients may develop symptoms at some time in their life and women are more often affected than men.3 The symptoms are typically triggered by a combination of one or more of the following precipitants: exposure to an unsafe drug, substance misuse, stress, reduced calorie intake and infection. Women are affected by hormones, particularly progesterone that rises in the luteal phase of the menstrual cycle.4 ,5 Acute attacks are thought to occur when there is a functional deficiency of haem in the liver, which induces the activity of the first enzyme in the pathway, aminolaevulinic acid synthase (ALAS). This leads to the deficient enzyme becoming the rate-limiting step and a build up of precursors including δ-aminolaevulinic acid (ALA) and porphobilinogen (PBG).6 It has been suggested that ALA is a toxic precursor and is thought to cause most of the symptoms, at least partly by inducing neuropathy.7 There is specific treatment for an acute porphyria attack; patients are usually hospitalised and given treatment in the form of a glucose infusion or a course of intravenous human hemin (Panhaematin in the USA or Normosang in other countries). Adequate analgesia is also essential, which typically involves opiates (http://www.porphyria-europe.com).

The biochemical hallmark of an acute porphyria attack is the increase in urine excretion of PBG that increases to more than 10 times the normal reference range (0–1.5 µmol/mmol creatinine) during an attack.8 Following an attack, urine PBG excretion decreases slowly, particularly in attacks associated with AIP and it can be difficult to diagnose subsequent illness as reports suggest that concentrations can remain elevated for years in patients with AIP,911 although this is not well documented. Laboratories often also measure ALA and total urine porphyrins (TUP) excretion with urine PBG as an acute profile to aid in the monitoring of the disease.12 ,13

There are few reports on biochemical long term monitoring of patients following an acute attack; this report documents the extended time course of urine excretion of total porphyrins and precursors.

Design and methods

This study is a retrospective audit of urine porphyrin biochemistry in patients with AIP. Urine ALA, PBG and TUP excretion were measured in 20 patients (18 female and 2 male) with AIP confirmed by biochemical and genetic analysis. All patients were attending the Porphyria Clinic at King's College Hospital for routine follow-up of an acute attack of porphyria. Typically asymptomatic and latent patients attend the clinic on an annual basis, and samples were collected routinely at these appointments; patients were seen more frequently following an acute attack. The history was determined by examining hospital records and from the patient. Acute attacks were identified based on a history of acute abdominal pain with no other explanation, combined with biochemical abnormalities consistent with the diagnosis of AIP, as documented in hospital notes or letters and confirmed by the patient. The measurements were taken on urine samples collected either during an acute attack or at routine clinical appointments. The urine concentrations were plotted against the time since the last acute attack, which extended to more than 20 years (table 1).

Table 1

Minimum, maximum and median concentrations of urine PBG, ALA and TUP excretion from 0 to 72 months post-acute attack compared to latent and asymptomatic AIP patients

The audit also included the measurement of urine porphyrin metabolites in two groups of AIP patients: latent and asymptomatic. These patients were: (a) 20 patients with latent AIP (9 female and 11 male) who were biochemically normal and never had any symptoms; and (b) 8 patients with mild biochemical abnormalities and no reported symptoms at the time of sampling (7 female and 1 male). Prepubertal children were excluded from the study. None of the patients had abnormal kidney or liver function and were monitored annually as part of their follow-up.

The concentrations of urine ALA and PBG were measured using an ion-exchange chromatography method (Bio-Rad, Alpha Labs) and the results were expressed as a ratio to urine creatinine (Jaffe method, Siemens Diagnostics). The concentration of TUP was measured by spectrophotometric analysis.12 The interassay variations (as CVs) for the ion-exchange chromatography method were 4.2% for PBG (mean=75 µmol/L, n=12), 5.6% for ALA (mean=151 µmol/L, n=12) and 2.9% for TUP (mean=1070 nmol/L, n=12). The same assay was used throughout the study period.

All urine specimens (10–15 mL) were collected in 25 mL universal containers as patients attended the Porphyria Clinic at King's College Hospital. The urine was protected from light, transported to the laboratory and frozen immediately. Data analysis was carried out using SPSS predictive analytics software Version 21.0.

Results

Twenty patients were identified as having a definite acute attack with subsequent symptom free period and measurements of urine excretion of ALA, PBG and TUP on one or more occasions following the attack. Fourteen patients had only one attack, and biochemical results were available from the day of the attack to a maximum of 23 years; three patients reported two acute attacks before measurements were taken (3 months–17 years); and three patients had four or more acute attacks—the samples were collected only from the time when they were asymptomatic (3 months–1 year). The mean age at the time of this last acute attack was 28 years (range 19–34 years).

The time course for the excretion of the urine porphyrins and precursors is shown in figure 2. The data were expressed as a log-log plot and the best fit line is illustrated with 95% confidence limits. PBG is significantly elevated following an acute attack, with the lower 95% confidence limit remaining above the upper limit of normal for more than 10 years (figure 2A). Urinary ALA concentrations return to normal more quickly, with the lower 95% confidence limit approaching normal between 2 and 3 years (figure 2B), while urinary porphyrins return to normal most quickly (figure 2C). All three parameters remain elevated above the latent median for more than 6 years (table 1). PBG/ALA ratios were calculated for the time course from 0 to 23 years after the acute attack (figure 3) and show a similar pattern as the separate parameters with the ratio remaining elevated for up to 23 years after an acute attack.

Figure 2

Time course (log-log) and best-fit line with 95% confidence limits for (A) porphobilinogen (PBG, µmol/mmol creatinine), (B) aminolaevulinic acid (ALA, µmol/mmol creatinine) and (C) total urine porphyrins (nmol/mmol creatinine).

The approximate half-life of each marker was calculated using the median levels from 0 to 23 years for each urine marker using the formula: T1/2=(elapsed time×log2)/log (beginning amount/ending amount). The results showed that TUP decayed faster with a T1/2 5.3 years; ALA T1/2 was 7.7 years and PBG T1/2 10.6 years. Although these calculations are based on composite data from 20 patients, the data are fairly closely grouped around the regression line (figure 2), suggesting that these figures have some validity. Table 2 shows the urine concentrations of PBG, ALA and TUP for each patient from >7 days to >72 months after an acute attack. There is variation between the urinary concentrations for each patient, and a plot showing the mean percentage decrease in excretion of urine PBG, ALA and TUP over 72 months after an acute attack illustrates that the concentrations decrease incrementally over 36 months and then decrease more gradually over the rest of the time course (figure 3).

Table 2

Urine concentrations for PBG, ALA and TUP for the individual patients

Figure 3

Time course in years for urine PBG/ALA ratios in patients following an acute attack shown with the baseline ratio 0.3 for latent AIP patients.

Figure 4

Mean percentage decrease in the urine excretion of PBG, ALA and TUP from <7 days to 72 months after an acute attack. Access the article online to view this figure in colour.

Discussion

The data presented show that the concentrations of the porphyrin precursors ALA and PBG remained elevated for at least 20 years after an attack in some patients with AIP. This is the first time that the long term urine excretion of these porphyrins and precursors has been documented in patients with AIP. There is variability in the excretion rate of the individual porphyrins and precursors between patients and this highlights the need for urine monitoring after an acute attack. Other authors have followed the time course for shorter periods of time in AIP patients,14 ,15 and reported an overlap between the results obtained in the latent and in the acute phases, but did not specify the timescale. One group followed two patients with ALAD deficiency porphyria for 20 years and showed that the urine excretion of ALA and PBG also remained above the normal.16 It is not clear why urine excretion of porphyrin precursors remains elevated in patients for so long following an acute attack, despite the absence of obvious symptoms. This does not fit well with the model of acute induction of ALAS and toxic porphyrin precursors explaining the complications of AIP. It seems most likely that ALAS activity remains elevated for many years following an acute attack, although it is also possible that massive accumulation of ALA and PBG occurs in tissues during an acute attack and this is then excreted slowly over many years. This latter explanation is less likely, particularly as urinary excretion of ALA and PBG returns rapidly to normal following liver transplantation.17 Equally it is difficult to implicate either ALA or PBG directly in the symptomatology of AIP, in that the symptoms resolve in a few weeks but the marked biochemical abnormalities persist for more than 10 years. In vitro evidence from mouse studies suggests that ALA is a neurotoxin and might be responsible for many of the symptoms, although this study does not support this idea, unless it is proposed that the body adapts fairly quickly to elevated urine ALA and it is rapid changes in concentrations of ALA which cause complications.

The urine PBG/ALA excretion ratios for the AIP patients (latent and symptomatic) were in agreement with a report by previous authors11 with a ratio of ∼2.0 in AIP patients who were excreting elevated concentrations of the precursors and 0.3 in latent AIP carriers. The authors reported that the continuing increase in urine excretion of PBG in patients following an acute attack may be due to overloading the enzyme hydroxymethylbilane synthase and may result in selective accumulation of PBG, although our results show that elevated ALA concentrations also persist for many years.

The measurement of urine ALA is not essential for the diagnosis of acute porphyria. However there have been suggestions that it is neurotoxic and it is often measured with urine PBG. Urine ALA is also increased in hereditary tyrosinaemia and lead poisoning, the latter having similar symptomatology to acute porphyria.

We showed that the average half life for the urine metabolites was 8 years, with TUP concentrations falling at a faster rate than the urine precursors. This finding may be due to the prompt collection and storage of the urine samples, minimising the degradation of ALA and PBG in urine. However it is not clear why urinary concentrations of PBG continue to be higher than ALA over time. There have been suggestions by other authors11 ,18 that the increased excretion of ALA in urine may be a consequence of the upregulation of the enzyme ALAS1 to compensate for haem deficiency and a possible inhibitory effect of PBG on ALAD activity. The regression is based on average data from many patients and individual half-life excretion is variable, although for ALA and PBG the concentrations did not return to normal within the time course of the study. The percentage decrease in excretion of the porphyrins and precursors from the start of the acute attack is variable but the mean results confirm that there is a decline in excretion that begins to plateau 36 months after the acute attack. Similarly we have shown that the ratio did not return to normal and reached a plateau at 23 years post acute attack, with a PBG/ALA ratio of ∼1.4, similar to the asymptomatic AIP patients who had never had an acute attack (PBG/ALA ratio=1.3). PBG/ALA ratios in urine may be calculated as another measurement to monitor the time course of excretion of urine porphyrin metabolites following an acute porphyria attack.

Other studies also demonstrate that while urinary PBG measurement is very good at identifying a first attack, it is of very limited value in diagnosing subsequent attacks because the concentrations continue to be elevated.11 It is possible that subsequent attacks result in further increases in PBG above the already elevated baseline, although this is of very limited diagnostic value. The prediction of a new acute attack based on PBG concentrations may be valid if the attack occurs 1–2 years after the initial episode based on the biological variation of the metabolites.15 Of the three urinary measurements, TUP has the best potential as a diagnostic marker of recurrent attacks, returning to normal values within a few years, although in practice recurrent attacks typically occur more frequently.

The value of our study is limited by its retrospective nature, and the combination of results from 20 different patients. However, the pattern of urinary excretion with time is remarkably consistent across the different patients, and some clear patterns emerge. Our study highlights the difficulty of using urinary PBG as a diagnostic test following the first attack, and raises important questions about the pathophysiology of AIP. There is a need to develop a better understanding of the metabolic abnormalities involved, and to identify better biomarkers of acute symptoms.

Take-home messages

  • Urine concentrations of δ-aminolaevulinic acid (ALA) and porphobilinogen (PBG) remain significantly elevated for up to 20 years following an attack of acute intermittent porphyria (AIP).

  • Measurement of urine ALA and PBG is useful to diagnose a first attack of AIP, but not helpful to distinguish subsequent attacks from other conditions causing the same symptoms.

  • Better markers of recurrent acute attacks need to be developed.

  • It is not understood why urine porphyrin concentrations remain elevated for so long following an attack; the pathophysiology of the condition is still poorly understood.

References

Footnotes

  • Contributors Both authors contributed equally to the study design and data analysis, reviewing the manuscript and agreeing to the final version.

  • Competing interests None.

  • Provenance and peer review Not commissioned; externally peer reviewed.