2025/01/01 by Brianna D. Guarino, Adam S. Ptolemy, Michelle A. Baum +3 · 1 voice
Biochemistry, Genetics and Molecular Biology · Medicine · #Metabolism and Genetic Disorders #Neurological and metabolic disorders #Methemoglobinemia and Tumor Lysis Syndrome
paper · doi:10.1093/clinchem/hvae090
openalex publication_date 2025/01/01 · openalex created_date 2025/10/10 · openalex updated_date 2026/06/23
What are the potential causes of pink- or red-colored urine? What could cause an apparent increased urine protein result in the context of otherwise normal urinalysis? A 3-year-old female with no previous relevant medical history was referred to the nephrology clinic for evaluation of episodic red-brownish colored staining in her diaper and on the toilet seat. She was not prescribed any medications and had no exposure to hormonal creams, nor were there any obvious dietary factors. The patient initially presented to her primary care physician at 2½ years of age following the discovery of apparent blood on the toilet seat. Around this time, the child also visited the emergency room, where urinalysis was negative for blood, protein, or evidence of urinary infection. A renal-pelvic ultrasound was also unrevealing. Consultation with nephrology resulted in serial urinalyses, which were repeatedly negative for blood or protein, and urine microscopy was normal. She was referred to endocrinology and gynecology for concerns of precocious puberty and vaginal bleeding, respectively. Endocrine assessment revealed that the child was Tanner stage 1 and had an appropriate bone age. Gynecological examination was positive for mild vulvovaginitis but was otherwise normal. In the interim period, she exhibited further episodes of pink/red splattering on the toilet. The patient was then referred to urology. Despite no outward cause of the episodes, the urologist recommended cystoscopy if symptoms persisted past 1 year. Several months later, the child developed some periumbilical abdominal pain and was assessed by gastroenterology. At this visit, a family history (maternal great aunt) of acute intermittent porphyria was revealed. However, subsequent laboratory workup for acute intermittent porphyria, including urine porphobilinogen, coproporphyrins I and III, and uroporphyrin, was unremarkable. Approximately 1 year later, she developed proteinuria, which was noted on follow-up urinalysis screening by other providers (Table 1), triggering a referral back to nephrology. Despite persistently increased urine protein measurements over the following 5 months, other urine analyses, including albumin, β2- and α1-microglobulins, and protein electrophoresis, were all normal. A urine purine/pyrimidine panel was also negative, excluding the possibility of hyperuricosuria, a rare cause of orange-colored crystals in children’s urine. The patient’s serum albumin remained normal, and she had no edema. The nephrology service consulted the clinical laboratory regarding the ongoing apparent proteinuria without etiology that supported classic proteinuria. A chromogenic interference from the substance within the child’s urine causing artifactually increased urine total protein determinations was strongly suspected. Patient’s urine analyses. aUrinalysis performed using Bayer Multistix SG10 method. Patient’s urine analyses. aUrinalysis performed using Bayer Multistix SG10 method. Innocuous causes of red or red/brown-colored urine include certain foodstuffs, such as beetroot or other strongly pigmented foods or dyes. Certain medications, such as rifampin (1), propofol (2), and senna (3), can also have a similar effect on urine color. In this case, the child’s father denied any recent ingestion of beet or the use of therapeutics of any type. Pathological causes of red discoloration of urine include hematuria, menstrual bleeding, and rare hereditary porphyria (3). Although the patient did have one episode of borderline urinary red blood cell elevation at 4/hpf (reference interval: <3/hpf), all subsequent urinalyses were negative for hematuria. Additionally, her gynecological and endocrine examinations were not suggestive of precocious puberty, therefore excluding the possibility of menstrual bleeding. A porphyria workup in 2 separate urine collections were also negative, making a diagnosis of acute intermittent porphyria less likely. The cause of new-onset proteinuria was also exhaustively investigated. Urine protein concentrations ranged from 92 to 270 mg/dL (reference interval: <12 mg/dL) over a 5-month period, despite normal urine albumin determinations (Table 1). Notably, urine protein electrophoresis did not suggest proteinuria (urine total protein, <25 mg/dL) in a split specimen, which returned an increased total protein concentration in our laboratory-based urine total protein assay (total protein, 270 mg/dL). Urine protein determinations using the latter approach are based on the modified benzethonium chloride method (Cobas c503, Roche Diagnostics). Analytical interferences that have been described with this method include hemolysis, calcium-dobesilate, ascorbic acid, and phenylbutazone (4, 5). A lesser-known interferent is homogentisic acid, associated with the rare inborn error of metabolism alkaptonuria (6). In our case, the child was not receiving any medications or contrast imaging prior to any collections, and the specimens were free from hemolysis. Therefore, the possibility of homogentisic acid as the source of the interference was explored. Alkaptonuria (OMIM 203500) is a rare autosomal recessive disorder of tyrosine metabolism caused by pathogenic variants in the HGD gene, which encodes the enzyme homogentisate 1,2-dioxygenase, responsible for the conversion of homogentisic acid to maleylacetoacetic acid. Alkaptonuria results in the production and excretion of large quantities of homogentisic acid, which oxidizes to benzoquinone acetate and a dark-colored polymerization product. Since this disorder is not included in population newborn screening programs, alkaptonuria cases are detected with a combination of clinical symptoms and standard biochemical laboratory screening tests. The majority of alkaptonuria patients are diagnosed in early childhood, based on the observation of dark-colored urine or episodes of dark-colored staining/particulate matter in diapers. As a result of its low incidence (approximately 1 in 500 000 to 1 million), there is little awareness of this condition within the general medical community. Consequently, many patients with alkaptonuria undergo diagnostic odyssey with multiple referrals to specialists before eventually seeing a geneticist or having the appropriate biochemical screening test performed. Notably, our patient presented with pink/red-colored urine, which is atypical and only previously reported in a small number of cases. In one such case, a 12-year-old female was misdiagnosed with pyelonephritis following episodes of severe abdominal pain and episodes of pink/red-colored urine, which was later found to be due to alkaptonuria (7). In another reported case of alkaptonuria, the child was initially misdiagnosed at 2 years of age with urinary bleeding until he presented at age 6 with severe joint and abdominal pain, which represent early symptoms of the disorder (8). Thus, as with our case, the literature supports the assertion that pink- or red/brown-colored urine observed in rare cases of alkaptonuria (as opposed to the characteristic brown/black-colored urine typically seen) can delay diagnosis, particularly in pediatric patients. Difficulties with capturing urine specimens from a small child in an outpatient setting may also delay diagnosis. The clinical course in alkaptonuria reflects the progressive accumulation of homogentisic acid in the tissues, most notably in cartilage. Ochronosis, a pathological process defined by the accumulation of the dark pigment in connective tissue, leads to degeneration and arthritis, typically presenting after 30 years. On the suspicion of homogentisic acid being both the cause of the colored urine and the source of assay interference, it was recommended that the patient provide a fresh urine specimen for organic acid testing by gas chromatography-mass spectrometry. Marked excretion of homogentisic acid was revealed, and the child was referred to the metabolism service for follow-up. Molecular testing revealed 2 compound heterozygous variants (c.1111dup and c.16-1G > A) in the HGD gene, confirming the diagnosis. In this case, apparent proteinuria in the context of unremarkable alternative urine protein determinations was an important clue to the diagnosis. In the modified benzethonium chloride method, the primary reagent reacts with protein in the presence of alkali to generate a turbidimetric reaction. In the Roche version of the assay, positive interference by homogentisic acid is exhibited at concentrations in excess of 1.2 mmol/L. Homogentisic acid is extremely polar, and large quantities are excreted in alkaptonuria, typically in excess of 4 to 8 g per day. Notably in our patient, serial urine dipstick measurements (Siemens Multistix 10 SG) for protein using the tetrabromophenol blue methodology were all negative (Table 1). Similarly, one previous study demonstrated that the addition of 5 mg/mL of homogentisic acid to a negative protein urine specimen caused a positive interference (1 g/L) with the benzethonium chloride method but not on dipstick analysis (9). According to a recent survey from the College of American Pathologists, approximately 35% of all participating laboratories use the benzethonium chloride method for urine total protein (10). The presence of homogentisic acid is not known to interfere with the pyrogallol red method. This case provides an insight into the plight of patients with an undiagnosed rare disease, particularly in the context of atypical presenting symptoms. Since the majority of inborn errors of metabolism are rare (the vast majority of which are not screened for at birth), and are generally underrecognized, patients often undergo a diagnostic odyssey and are seen by multiple specialists. Although most of the complications of alkaptonuria present in adulthood and treatment is supportive, early identification in childhood could prevent unnecessary testing, specialty visits, and parental anxiety. Counseling for patients and families should include education about the need for future disease surveillance for cardiac, joint, thyroid, and renal involvement in adulthood. Nitisinone, a compound that blocks the metabolism of tyrosine upstream of homogentisic acid, is used for the treatment of alkaptonuria in adults in Europe and the United States. However, there is no consensus regarding its use in children and adolescents. This case was complicated by the appearance of the urine itself, which was not considered classic for this disorder. Rather, the possibility of an interfering compound producing unexpected or conflicting urine protein concentrations ultimately led to greater suspicion for this particular disorder, with the laboratory providing a key role in reaching the diagnosis. The presenting signs of alkaptonuria may not always be classic and can be a source of delayed diagnosis. A polymerization product of benzoquinone acetate is responsible for the colored urine in alkaptonuria. The diagnosis of alkaptonuria is possible through the detection of homogentisic acid in urine by gas chromatography-mass spectrometry. Homogentisic acid is a source of interference with the benzethonium chloride method of urine protein. Orthogonal testing is a useful approach to investigate potential sources of interference in chemistry analyses. Written consent was obtained, granting permission to publish this case report. The corresponding author takes full responsibility that all authors on this publication have met the following required criteria of eligibility for authorship: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; (c) final approval of the published article; and (d) agreement to be accountable for all aspects of the article thus ensuring that questions related to the accuracy or integrity of any part of the article are appropriately investigated and resolved. Nobody who qualifies for authorship has been omitted from the list. Brianna Guarino (Writing—original draft-Equal), Adam S. Ptolemy (Writing—review & editing-Equal), Michelle Baum (Investigation-Supporting, Writing—review & editing-Supporting), Melinda J. Palma (Writing—review & editing-Supporting), Mark Kellogg (Investigation-Supporting, Writing—review & editing-Supporting), and Roy Peake (Investigation-Supporting, Writing—original draft-Equal). Upon manuscript submission, all authors completed the author disclosure form. This project was funded by the Endowment Fund of the Department of Laboratory Medicine, Boston Children’s Hospital. M.D. Kellogg has received consulting fees from ProterixBio, Inc., Nexus Laboratories, Inc., and Roche Diagnostics and meeting and travel support from ADLM, Clinical & Laboratory Standards Institute, and The American Society for Clinical Laboratory Science. R.W.A. Peake is an associate editor for Clinical Chemistry, ADLM.