Key role of Levitt’s carbon monoxide breath test in revealing coexistent Gilbert syndrome and erythropoietic protoporphyria: A case report

Abstract

BACKGROUND

It is challenging to diagnose isolated hyperbilirubinemia with rare and complex etiologies under the constraints of traditional testing conditions. Herein, we present a rare case of coexisting Gilbert syndrome (GS) and erythropoietic protoporphyria (EPP), which has not been previously documented.

CASE SUMMARY

We present a rare case of coexisting GS and EPP in a 23-year-old Chinese male with a long history of jaundice and recently found splenomegaly. Serial non-specific hemolysis screening tests yielded inconsistent results, and investigations for common hemolytic etiologies were negative. However, Levitt’s carbon monoxide breath test, which measures erythrocyte lifespan (the gold-standard marker of hemolysis), demonstrated significant hemolysis, revealing a markedly shortened erythrocyte lifespan of 11 days (normal average 120 days). Genetic testing subsequently confirmed EPP with a homozygous ferrochelatase gene mutation and GS with a heterozygous uridine diphosphate glucuronosyl transferase 1A1 gene mutation.

CONCLUSION

The rapid, non-invasive Levitt’s carbon monoxide breath test resolved the diagnostic challenge posed by a rare and complex cause of hyperbilirubinemia.

Key Words: Isolated hyperbilirubinemia; Erythropoietic protoporphyria; Gilbert syndrome; Hemolysis; Levitt’s carbon monoxide breath test; Erythrocyte lifespan; Case report

Core Tip: This first-reported case of coexisting Gilbert syndrome and erythropoietic protoporphyria highlights Levitt’s carbon monoxide breath test as a pivotal non-invasive tool for diagnosing rare and complex causes of hyperbilirubinemia. The test confirmed severely shortened red blood cell lifespan (11 days), resolving diagnostic uncertainty after conventional tests failed, and guided definitive genetic diagnosis of dual uridine diphosphate glucuronosyl transferase 1A1 and ferrochelatase mutations.

INTRODUCTION

Isolated hyperbilirubinemia, characterized by elevated bilirubin without evidence of liver injury or cholestasis, arises from three mechanisms: (1) Increased bilirubin production (e.g., hemolysis); (2) Impaired hepatocellular uptake/conjugation (e.g., Gilbert syndrome (GS), the most common hereditary unconjugated hyperbilirubinemia); and (3) Reduced bilirubin secretion (e.g., Dubin-Johnson syndrome)[1]. While GS is common (prevalence: 6%-12%), erythropoietic protoporphyria (EPP), an inborn error of heme biosynthesis with protoporphyrin accumulation causing photosensitivity, hemolysis, and hepatopathy, is rare (1:75000-1:200000). Coexistence of these disorders is exceptionally rare and diagnostically complex. Traditional hemolysis screening [reticulocytosis, haptoglobin, lactate dehydrogenase (LDH)] lacks sensitivity/specificity[1,2], and etiological workups (e.g., flow cytometry, enzyme assays) may fail to identify rare causes. Levitt’s carbon monoxide (CO) breath test measures erythrocyte (RBC) lifespan - the gold-standard hemolysis marker - by measuring endogenous CO production from heme catabolism during hemoglobin degradation following RBC rupture[3-6]. We present a case where this test was pivotal in diagnosing concurrent EPP and GS after extensive negative investigations.

CASE PRESENTATION

Chief complaints

A 23-year-old Chinese male with a long-standing history of benign jaundice and a three-month history of splenomegaly was referred from a hematology clinic to our hospital’s gastroenterology department for further evaluation.

History of present illness

During each physical examination or medical visit, he had always been informed that it was constitutional jaundice and no treatment was required. Nevertheless, during a routine health check three months ago, an ultrasound surprisingly detected splenomegaly, which had never been present before. This discovery led to his referral to a hematology clinic for further assessment. The patient was uncertain whether any of his family members had a history of either jaundice or splenomegaly.

History of past illness

The patient had maintained overall good health since childhood, with the exception of mild yellowing of the eyes, which had persisted since it first appeared at the age of 7-8 years. He had no history of smoking, alcohol use, or drug abuse and enjoyed running.

Personal and family history

A telephone interview with the patient’s parents, who lived hundreds of kilometers away in a rural area, revealed that neither parent experienced photosensitivity, but the father had a history of “yellow eyes” since his youth.

Physical examination

In the clinic, the physical examination revealed mild icterus of the sclera (Figure 1A) and a spleen palpable just 1.0 cm below the left costal margin.

Figure 1 Diagnostic findings of the proband. A: Mild icterus of the sclera without facial injuring scar; B: A large number of fluorocytes in the peripheral blood.

Laboratory examinations

Primary laboratory findings showed mild normocytic anemia (hemoglobin 11.6 g/dL) with a significantly elevated reticulocyte count (10.5%). Additionally, there was isolated unconjugated hyperbilirubinemia with a total bilirubin of 75.4 μmol/L, direct bilirubin of 6.7 μmol/L, and indirect bilirubin of 68.7 μmol/L. Other parameters were within normal limits, including albumin (4.0 g/dL), globulin (2.0 g/dL), liver enzymes (alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, and gamma-glutamyl transferase), serum haptoglobin, and LDH levels. Given these findings, chronic hemolytic hyperbilirubinemia was suspected. However, all the subsequent investigations to identify the etiology of the hemolysis yielded negative results. These tests included blood smear analysis for abnormal red blood cell morphology (e.g., hereditary spherocytosis), hemoglobin electrophoresis for hemoglobinopathies (e.g., thalassemia and sickle cell anemia), glucose-6-phosphate dehydrogenase activity test for glucose-6-phosphate dehydrogenase deficiency, Coombs’ test for autoimmune hemolytic anemia, and flow cytometry for CD55 and CD59 for paroxysmal nocturnal hemoglobinuria. Other tests for non-hemolytic blood disorders also showed no significant abnormalities.

Further diagnostic work-up

Given the negative work-up, the patient was referred to our hospital’s gastroenterology department for further evaluation and management. According to the flowchart for the evaluation of isolated hyperbilirubinemia, it is essential to differentiate between hemolytic and non-hemolytic causes of unconjugated hyperbilirubinemia[1]. In this case, we first assessed the patient’s RBC lifespan, the gold standard marker for diagnosing hemolysis, using the simple, rapid, and inexpensive Levitt’s CO breath test, which is available only at a few hospitals in the city, including ours[2-4]. The basic principle of the test is that the lungs are the exclusive route for CO excretion from the body, and that exhaled endogenous CO originates predominantly (approximately 70%) from heme oxidation during hemoglobin degradation following RBC rupture. Accordingly, dividing the total CO capacity of hemoglobin by the daily amount of CO released yields an estimate of the mean RBC lifespan at the time of measurement. The test involves collecting a 200 mL sample of end-expiratory breath in the morning, then connecting the sample bag to the analyzer (ELS TESTER, Seekya Biotec Co. Ltd, Shenzhen, China) and inputting the same day’s hemoglobin data from a peripheral complete blood count. The machine automatically detects exhaled endogenous CO concentration and calculates the results using the pre-embedded Levitt’s formula, providing the outcome within 5 minutes. The patient’s RBC lifespan was found to be only 11 days (normal average: 120 days, range 75-175 days), which unequivocally confirmed the diagnosis of hemolysis[5,6].

Subsequently, drawing from our previous similar experiences, and considering the patient’s long history of benign jaundice, negative results from common hemolytic etiology tests, and the discordance between significant unconjugated hyperbilirubinemia and mild anemia, we chose to perform targeted diagnostic next-generation sequencing using a commercial sequencing panel (MyGenotics, Beijing, China)[7]. This panel covered 208 genes associated with hereditary erythroid disorders, as well as the gene encoding UDP-glucuronosyltransferase family 1 member A1 (UGT1A1). The results revealed a homozygous mutation in the ferrochelatase (FECH) gene (c.315-48T>C) combined with a heterozygous mutation in the UGT1A1 gene (c.-3279T>G). Congenital mutation in the UGT1A1 gene is known to cause the most common hereditary unconjugated hyperbilirubinemia, namely GS, while the FECH gene mutation is responsible for EPP, a rare inborn error of heme biosynthesis, characterized by the accumulation of photosensitive protoporphyrin in RBCs, hepatocytes, and vascular endothelium, which can lead to painful, non-blistering photodermatitis and/or hemolysis and hepatopathy in some cases. The genetic test indicated a comorbidity of GS and EPP. Genetic analysis of mailed blood samples showed that both parents carried the identical homozygous FECH mutation as the proband, with the same UGT1A1 mutation found in the father. The following was straightforward. A second round of medical history collection and physical examination was conducted. Although the patient denied any history of painful photosensitivity since childhood and a careful skin examination revealed no pigmentation or scarring in sun-exposed areas, further laboratory tests were consistent with EPP, including a large number of red-fluorescent RBCs in the peripheral blood smear under RBC fluorescence microscopy (Figure 1B).

Imaging examinations

No relevant imaging examinations have been performed.

FINAL DIAGNOSIS

The final diagnosis of the presented case was coexisted GS and EPP.

TREATMENT

The patient was advised to avoid sunlight.

OUTCOME AND FOLLOW-UP

The patient was scheduled for annual consultations.

DISCUSSION

The prevalence of GS in the population ranges as high as from 6% to 12%, while the estimated prevalence of EPP, despite being the most common form of porphyria in children, is reported to be as low as 1 in 75000 to 1 in 200000 individuals[8,9]. Consequently, the likelihood of having both GS and EPP together would be exceedingly rare. Furthermore, just a fraction of EPP patients, particularly in Asian populations, carry the c.315-48T>C low-expression allele without an additional severe FECH mutation in trans. Such patients, whether heterozygous or homozygous, tend to experience a later onset and mild symptoms, often with less severe or even absent photosensitivity[9-11]. Therefore, the case we encountered of a 23-year-old male is indeed exceptional. Although quantitative EPP measurement - a diagnostic gold standard - was unavailable in our clinical setting, the diagnosis was unequivocally established by integrating genetic test results with functional evidence of hemolysis from Levitt’s CO breath test. This case highlights the practical value of such a combinatorial diagnostic approach, particularly in settings where specialized assays are not readily accessible.

Despite its rarity, however, this case still falls within the category of chronic isolated hyperbilirubinemia, a common clinical laboratory finding in gastroenterology[1]. It is characterized by elevated serum bilirubin levels in the context of normal global liver function, with no significant biochemical markers indicating hepatocellular injury or cholestasis[1]. It is well known that chronic isolated hyperbilirubinemia arises from one or more of the following three bilirubin metabolism mechanisms: (1) Increased bilirubin production (common, due to various intra- or extra-hemolysis); (2) Decreased hepatocellular uptake and conjugation of unconjugated bilirubin (most common, due to hereditary abnormalities like GS and Crigler-Najjar syndrome, or occasional environmental interference such as rifampin and probenecid); and (3) Decreased hepatocellular secretion of conjugated bilirubin (rare, and so far only seen in Dubin-Johnson syndrome and Rotor syndrome)[1]. Meanwhile, a proven and effective algorithm for approaching chronic isolated hyperbilirubinemia has long been established (Figure 2)[1]. As shown in Figure 2, there are two sequential key steps in the algorithm: (1) Fractionating elevated serum bilirubin into indirect (unconjugated) or direct (conjugated, rare as described above); and (2) Evaluating for the presence or absence of hemolysis. Since more than 99% of isolated hyperbilirubinemia is indirect, and due to the potential errors in routine bilirubin fractionation tests, almost every individual should undergo hemolytic testing. However, it is not uncommon for a portion of patients, like in our case, to remain undiagnosed even after numerous tests. What complicates the issue is not the algorithm itself, but the traditional, commonly used hemolytic screening tests. Current hemolytic marker-based clinical laboratory tests - such as reticulocyte count, LDH, serum haptoglobin, plasma free hemoglobin - are non-specific, and far from satisfactory with respect to sensitivity and specificity. To compensate for this deficit, tests for common hemolytic etiologies are often conducted. Genetic tests sometimes are also employed. Undoubtedly, genetic testing is crucial for diagnosing some rare causes, such as in this case. However, it must be acknowledged that without a confirmed diagnosis of hemolysis, it is difficult for clinicians to routinely implement such costly tests.

Figure 2 The classical algorithm for the evaluation of an isolated hyperbilirubinemia. Incorporating the recently developed Levitt’s carbon monoxide breath test into the hemolysis evaluation step could provide substantial benefits. Adapted from Pratt[1]. LDH: Lactate dehydrogenase.

Hemolysis is defined as the accelerated destruction of RBCs, leading to their premature removal from circulation and a shortened lifespan[2]. This means that a shortened RBC lifespan is a fundamental characteristic of hemolysis and a gold-standard diagnostic indicator. However, RBC lifespan measurement is seldom conducted in basic research or clinical practice due to the cumbersome and protracted nature of classical RBC labeling methods (e.g., ⁵¹Cr, ¹⁵N-glycine, or biotin), which can take several weeks or even months. Based on the understanding that exhaled endogenous CO mainly comes from degraded hemoglobin following RBC destruction, established mainly by Coburn and Forman[12], Levitt’s team[3,4] developed a simple, rapid CO breath test to estimate RBC lifespan in 1992, which was later modified in 2003. Studies have confirmed that Levitt’s CO breath test yields results similar to those obtained with classical labeling methods[3,5,6]. This innovation greatly simplifies the diagnostic process for hemolysis. When it comes to isolated hyperbilirubinemia, regardless of the results from screening tests and other investigations, a normal RBC lifespan clearly rules out a hemolytic origin. Conversely, a shortened RBC lifespan strongly supports the contribution of hemolysis to hyperbilirubinemia, whether hemolysis is the sole cause or a contributing factor. The major limitation of Levitt’s CO breath test is that it is contraindicated in smokers (within 48 hours) or individuals exposed to significant environmental CO pollution.

Clearly, with Levitt’s CO breath test, the challenges encountered in the differential diagnosis of isolated hyperbilirubinemia are no longer focused on confirming or excluding hemolysis, but rather on determining whether hemolysis is the sole cause or one of the contributing factors, followed by the selection of appropriate diagnostic tests to investigate the underlying etiology. Our previous study found that significantly elevated bilirubin levels, along with mild RBC lifespan shortening or relatively higher hemoglobin concentrations, often indicated mild hemolysis in GS or a multifactorial cause of hyperbilirubinemia, such as hemolysis combined with GS, as observed in this case[7].

CONCLUSION

Levitt’s CO breath test, being rapid and simple, greatly facilitates the differential diagnosis of rare and complex causes of hyperbilirubinemia, as it can sensitively and specifically diagnose the presence or absence of hemolysis.