In a previous article (CLI October 2021 “Diagnosis of acute hepatic porphyria”), we discussed in detail the biochemistry and the diagnosis of the different types of porphyria. Here, we give a brief recap before going on to discuss a recent paper by Gallagher et al. that examined the effects of different specimen storage and handling conditions on the degradation of the photo-labile analytes, notably porphobilinogen and delta-aminolevulinic acid.

Why analysis of porphyrins and their precursors is necessary

Porphyrins and their precursors form part of the heme biosynthesis pathway and pathogenic inherited mutations of the enzymes involved result in altered activity and accumulation of the precursor to toxic levels, causing a group of eight diseases collectively known as porphyria [1]. The accumulation of porphyrins in the body, which result in acute neurovisceral symptoms, skin lesions, or both. The acute hepatic porphyrias (AHPs) include acute intermittent porphyria (AIP), variegate porphyria (VP), hereditary coproporphyria (HCP) and the incredibly rare delta-aminolevulinic acid dehydratase deficiency porphyria (ADP). In acute porphyria, the mutant enzyme has enough residual function to allow heme synthesis to occur, but is deficient enough to cause a build-up of the porphyrin precursors. This can be exacerbated by up-regulation of delta-aminolevulinic acid synthase (ALAS), which is usually the rate-limiting step in the pathway, by certain therapeutic drugs as well as barbiturates and alcohol. Symptoms of AHP are caused by the accumulation of delta-aminolevulinic acid (ALA) and porphobilinogen (PBG), and can include abdominal pain (reported by 92% of patients), nausea, urine colour change, muscle weakness, tiredness, among others. However, although most patients only experience a few attacks in their lifetime, some patients have recurring symptoms that result in long-term complications. Management of the disease is possible, but requires laboratory testing of the different porphyrins and their precursors to obtain a definitive diagnosis.

Laboratory testing of porphyrins

A recommended testing algorithm has been developed by Woolf et al. [2]. In general, testing a random (i.e. non-timed) urine sample for PBG forms the first-line investigation and should be done without delay. In an acute attack, PBG concentrations will be grossly elevated, reaching 10 to 150 times the upper limit of the reference interval and a diagnosis of porphyria is clear. However, patients with AIP tend to have persistently high urine PBG levels and so interpretation is less straightforward if they are presenting with new symptoms. Additionally, if PBG is not elevated but a strong clinical suspicion of porphyria remains, then urine porphyrins should be analysed to prevent a missed diagnosis of VP or HCP, where PBG levels are less elevated and return to normal more quickly. Second-line or confirmatory testing should be done to identify the exact type of porphyria. This requires analysis of the original urine sample, EDTA whole blood and feces at a specialist porphyria lab.

Qualitative methods of PBG testing are simple to do but are no longer recommended because of low sensitivity and poor specificity. Quantitative analysis can be rapid and cost-effective with in-house tests using ion-exchange spin columns, as detailed by Roshal et al. [3] and Mauerzall & Granick [4], using Ehrlich’s reagent and spectrophotometric detection at 553nm.

Sample stability and implications for sample storage and handling

Porphyrins are photo-labile because of their structure and it is known that samples should be protected from light. Additionally, it is known that stability differs according to the type of porphyrin, as stability increases with the increasing number of carboxylic acid side chains. Current guidelines state that that light-protected urine and whole blood porphyrins are stable for 4 days at 4°C and 2 days at room temperature. However, as there is little empirical evidence on porphyrin degradation (and some of the published data is contradictory), Gallagher et al. decided to an investigation of the effects of light exposure and storage temperature on the stability of urinary ALA and porphobilinogen, total urine porphyrin (TUP) and plasma porphyrin [5]. The study tested the effects of the following on analyte stability. In all experiments, the 10 different pools of samples covered a range of initial analyte concentrations.

1. Exposure to light and storage temperature on PBG concentration of 10 urine pools over 7 days.
2. Exposure to light and storage temperature on ALA concentration of 10 urine pools over 7 days.
3. Exposure to light and storage temperature on TUP concentration of 10 urine pools over 7 days.
4. Exposure to light and storage temperature on plasma porphyrin concentration of 10 urine pools over 7 days.
5. Exposure to light on urinary PBG, ALA, TUP and plasma porphyrin for samples stored at room temperature for 6 hours.

The conditions tested were: (1) dark, fridge temperature; (2) dark, room temperature; and (3) light, room temperature. It is not possible to completely unpick the effects of light exposure and temperature, as the fourth combination of light and fridge temperature was – naturally – not tested. However, in general, the results showed that the best conditions for preservation of urine PBG were light-protected and fridge temperature, whereas the conditions that resulted in greatest loss of PBG were those of light exposure and room temperature. Interestingly, the concentration of TUP in urine samples stored in the dark at room temperature increased by 24 and 48 hours.

Analysis of urine porphobilinogen is usually sufficient to diagnose acute hepatic porphyria (NB samples must be protected from light!) (Adobe Stock.com)

The results led the authors to propose that for the best sample storage and handling: “after collection, samples should be stored in a fridge at 4°C and sent to the porphyrin laboratory as soon as possible, either by guaranteed overnight or next day delivery to be received within 24 hours if sent at ambient temperature or alternatively appropriately cooled.”

The authors also suggest that the samples should be accompanied by documentation that details date and time of sample collection and postage, as well as storage temperature and amount of light exposure before posting. This would then allow data to be interpreted appropriately. The key points being:

1. PBG concentrations are so high during an acute attack that analyte stability is adequate for a diagnosis for samples kept light-protected at room temperature for several days.
2. Delay in transit times may cause false-negative results for PBG concentrations in latent inactive or asymptomatic porphyria patients, and should also be considered in the interpretation of results in known AIP patients.
3. Delay in transit times may cause falsely raised concentrations of TUP.
4. If samples have been exposed to light for more than 4 hours, repeat samples should be requested.

This study does not, perhaps, make major changes to the basic urine sample storage and handling recommendations, but it does now mean that these recommendations are based on firm evidence. Where it is most useful is in guiding decisions about which results are reliable and which need to be treated with caution when storage and handling conditions have been less than ideal, which is becoming increasingly common with delays and irregularities in the postal service in the UK.

References
1. Ramanujam VS, Anderson KE. Porphyria diagnostics-part 1: a brief overview of the porphyrias. Curr Protoc Hum Genet 2015;86:17.20.1–17.20.26 (https://pmc.ncbi.nlm.nih.gov/articles/PMC4640448/).
2. Woolf J, Marsden JT, Degg T et al. Best practice guidelines on first-line laboratory testing for porphyria. Ann Clin Biochem 2017;54(2):188–198 (https://journals.sagepub.com/doi/10.1177/0004563216667965).
3. Roshal M, Turgeon J, Rainey PM. Rapid quantitative method using spin columns to measure porphobilinogen in urine. Clin Chem 2008;54(2):429–431 (https://academic.oup.com/clinchem/article/54/2/429/5628734).
4. Mauzerall D, Granick S. The occurrence and determination of delta-amino-levulinic acid and porphobilinogen in urine. J Biol Chem 1956 219(1):435–446 ( https://www.jbc.org/article/S0021-9258(18)65809-0/pdf ).
5. Gallagher CJ, Bentley LA, Challenger R et al. Stability of porphyrins and porphyrin precursors in urine and plasma samples: implications for sample handling and storage. J Clin Pathol 2024;77(8):574–578 (https://jcp.bmj.com/content/77/8/574.long).

Structure of heme. Porphyrias are diseases of the heme synthesis pathway (Adobe Stock.com)