Spurious Elevation of Multiple Urine Amino Acids by Ion-Exchange Chromatography in Patients with Prolidase Deficiency

Abstract

The enzyme prolidase cleaves dipeptides where the C-terminal amino acid corresponds to proline or hydroxyproline. As a consequence, a deficiency of this enzyme leads to accumulation of these dipeptides, which correspondingly are found to be elevated in urine. In fact, the absence of dipeptiduria is sufficient to rule out a diagnosis of prolidase deficiency. However, given the fact that these dipeptides elute at the same position as more common amino acids, the analyzer's software will instead call an elevation of these corresponding amino acids. Thus, an elevation of glycylproline, aspartylproline, glutamylproline, threonylproline and serylproline, valylproline, leucylproline, isoleucylproline, alanylproline, phenylalanylproline, and lysylproline will instead be interpreted as an elevation of leucine, citrulline, methionine, isoleucine, beta-aminoisobutyric acid, gamma-aminobutyric acid, ethanolamine, tyrosine, histidine, and anserine/carnosine, respectively. This particular profile of elevated amino acids, however, can easily be overlooked. We hope that the recognition of this characteristic pattern of falsely elevated urinary amino acids will aid in the recognition of prolidase deficiency.

Clinical Genetics of Prolidase Deficiency: An Updated Review

“…Analysis of urinary amino acids in all tested patients revealed a massive excretion of imidodipeptides such as proline-glycine or proline-hydroxyproline [4,7,11]. Imidopeptiduria is therefore an essential biochemical marker for the diagnosis, the excretion of imidodipeptides being negligible in a healthy person (Figure 1d) [15,19].…”

“…Elevated ninhydrin-positive peaks are secondarily identified after hydrolysis of the urine sample, followed by a second quantitative analysis of the profiles of proline and hydroxyproline. Other diagnostic approaches to detect the urinary imidopeptides are exchange chromatography, thin-layer chromatography, capillary electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) [7,11]. Imidopeptiduria can be detected early during the newborn period, even in an asymptomatic person [4,20].…”

“…Due to a severely reduced prolidase activity in PD, a large amount of proline remains in the form of imidodipeptides X-Proline and X-Hydroxypyroline, which are excreted in the urine [11]. Thus, the hallmark of PD is a massive imidopeptiduria associated with elevated proline or hydroxyproline containing dipeptides in plasma [3,6,[11][12][13]. The confirmation of PD diagnosis relies on the measurement of the cellular prolidase activity and on the identification of PEPD gene variant [4,12,14,15].…”

Structural analysis of new compound heterozygous variants in PEPD gene identified in a patient with Prolidase Deficiency diagnosed by exome sequencing

“…In order to confirm the diagnosis of PD, total hydroxyproline was quantified in the patient´s urine sample by high performance liquid chromatography. Before quantification, the urine samples were exposed to acid hydrolysis (using 6 N HCl), which gives rise to a marked increase in hydroxyproline (Ferreira and Wang, 2015;Ferreira and Cusmano-Ozog, 2017).…”

Prolidase deficiency diagnosed by whole exome sequencing in a child with pulmonary capillaritis

“…Based on this result, a diagnosis of prolidase deficiency was suspected. The diagnosis was confirmed with urine amino acid analysis [4], which showed imidodipeptiduria.…”