AHP is an autosomal dominant genetic disorder caused by the defect of the enzymes during heme biosynthesis pathway12. Due to the partial deficiency of these enzymes, excess amounts of 5-aminolevulinic acid (ALA) and PBG accumulate and cause dysfunction of the autonomic system and neuropathy13. Common presentations of AHP include abdominal pain (85–95%), constipation (48–84%), extremity pain (50–52%), nausea and vomiting (43–88%), tachycardia (28–80%), and hypertension (36–54%)3. Bullous skin lesions may be present during an attack of VP or HCP14. In severe cases, seizures, hallucination and respiratory distress might also present and patients could require intensive care15. In our study, 100% of patients complained of abdominal pain, which was much higher than that reported in previous studies. This might be due to a potential missed diagnosis of AHP patients with other symptoms caused by the poor awareness of physicians in our hospital.
The key issue in AHP management is to suspect the diagnosis16, and the elevation of urinary PBG is the main diagnostic criteria for AHP14. However, the urine PBG detection is only ordered and performed when AHP is suspected by physicians. In our study, we found that 50% of the patients were diagnosed by endocrinologist, which was rare in the published articles whose authors usually were from the neurology, general surgery or emergency departments. This finding highlights the importance of experienced physicians for AHP diagnosis. However, as a rare disease, awareness of AHP in China is limited8. According to a previous investigation carried out in a tertiary hospital in China, the misdiagnosis rate of AHP was 70% in the hospital9. Further, case reports are barely reported from secondary or communal hospitals. Thus, a simple way for AHP screening is needed to help physicians notice the possibility of AHP.
Here, for the first time, we reported the important role of the urinary urobilinogen/serum total bilirubin ratio in the screening of AHP. Compared to the abdominal pain patients of other causes, the AHP patients showed a significantly higher urinary urobilinogen level and urinary urobilinogen/serum total bilirubin ratio. Urobilinogen is a product of bilirubin metabolism by anaerobic bacteria in the intestine. Up to 20% of the urobilinogen produced daily are reabsorbed from the intestine and undergo enterohepatic recirculation. The majority of the reabsorbed urobilinogen is taken up by the liver and then re-excreted into bile, while a small amount is excreted in the urine and being detected as urinary urobilinogen17. In healthy people, the urinary urobilinogen in urinalysis is negative since the amount of urobilinogen is too low to be detected. However, in certain diseases, such as hemolytic anemia, hepatic jaundice, and biliary disease, the serum bilirubin level is greatly elevated and leads to the excessive production of urobilinogen. Thus, a positive result shows in the urinalysis18,19. However, our study demonstrated that most of the AHP patients showed normal serum bilirubin levels, consistent with the literature9,15,20−22. Therefore, we believe that the elevated urinary urobilinogen level in AHP patients was a false-positive result caused by the urinary PBG, which was proved by Watson-Schwartz test.
The Watson-Schwartz test has been a widely used method for urinary PBG detection for more than 80 years. Ehrlich's reagent in the first step reacts with PBG and forms a red condensation product. However, PBG is not the only substance that can react with Ehrlich's reagent. Urobilinogen, a substance being routinely examined in urinalysis, can also react with Ehrlich's reagent and form a red product23. As such, in the second step of Watson-Schwartz test, chloroform is added to the solution to distinguish the PBG-Ehrlich compound from the urobilinogen-Ehrlich complex. When chloroform is added, the red PBG-Ehrlich compound is located in the aqueous phase while the red urobilinogen-Ehrlich complex is located in the chloroform phase16.
At present, the dipstick method is the most widely used method of urinalysis. The Ehrlich's reagent pad on the dipsticks is used for detection of urinary urobilinogen. Following the manufacturer’s instructions or using an automated dipstick reader, we can obtain semiquantitative results of urobilinogen. However, a false-positive result can occur due to urinary PBG since it is difficult to use chloroform to distinguish the red product on the dipstick. To avoid this, we used serum total bilirubin, which is a routine examinations and generally performed in patients with abdominal pain, for calibration. In the AHP patients in this study, the urinary urobilinogen increased greatly (caused by PBG) while the serum total bilirubin was normal or slightly elevated. This led to a great increase in the urinary urobilinogen/serum total bilirubin ratio. Via ROC curve analysis, we found that the cutoff point for AHP diagnosis was 3.22. So in patients with typical clinical symptoms such as abdominal pain, the AHP diagnosis should be considered when the urinary urobilinogen/serum total bilirubin ratio was above 3.22 and further investigation of urinary PBG and ALA should be carried out to confirm the diagnosis.
The potential clinical use of the urinary urobilinogen/serum total bilirubin ratio suggests the importance of urinalysis in the diagnosis of AHP. Dark urine is very common in AHP patients and sometimes becomes the first clue for AHP in the intensive care unit5,24. However, in the emergency department, dark urine color is often overlooked both by patients and physicians. In our study, all the urine specimens showed amber color during urinalysis, but only two patients mentioned a change of urine color in their complaints. In addition, a menstrual period is a common predisposing factor of an acute attack, but urinalysis is often avoided both by the physicians and female patients during their periods. Therefore, we strongly suggest that all patients with abdominal pain undergo urinalysis, and the urinary urobilinogen/serum total bilirubin ratio should be calculated for a diagnosis of potential AHP.
Our study has several limitations. Since AHP is a rare disease, we only enrolled 12 patients from one single center in this study. Due to the limited sample size, there might be variations in the cut-off point. In this retrospective study, although we enrolled patients whose diagnosis of diseases other than AHP were confirmed by postoperative pathological results, typical imaging and laboratory findings, no urinary PBG detection was done to fully rule out the possibility of AHP in the control groups. Future multi-center studies with larger sample sizes are needed.