URICA-II; a Longitudinal Study to Analyse the Correlation Between Urinary Carbonic Anhydrase (CAI), a Marker of Haemolysis, and Bilirubin

In newborns, intravascular hemolysis can range from mild, as part of the physiological turnover of red blood cells, to severe in cases such as jaundice or ABO incompatibility. Early biomarkers of haemolysis would improve neonatology practice by identifying at-risk patients, particularly if the assay is simple, rapid, non-invasive and quantitative. Our now-completed URICA trial on full-term male babies (Trial registration CPMS 19576) showed that the small cytoplasmic protein carbonic anhydrase I (CAI), found abundantly in red blood cells, was detected (by immunoassay and mass-spec), in 17 out of 26 urine samples collected once per recruited baby at the neonatology ward. CAI-positive samples were obtained from babies with levels of bilirubin that are rapidly rising or peaking above the threshold for phototherapy. CAI-negative urine was obtained when either bilirubin did not reach phototherapy threshold, or after it had recovered from its peak. On four occasions, the cause of CAI-positive urine was undetermined (https://doi.org/10.1093/jalm/jfaa051). Since CAI is normally absent from urine, a positive signal is indicative of intravascular hemolysis and confirms that CAI crossed the glomerular barrier. However, the quantitative power of urinary CAI to predict and estimate an impending haemolytic crisis requires a new longitudinal study, which is the objective of the URICA-II trial.

Clinical rationale: The health of neonates must be monitored regularly for one of many rapidly progressing and potentially life-threatening conditions that implicate intravascular hemolysis. These include sepsis (e.g. Gram-positive bacteria), protozoan infection (e.g. malaria), alloimmunity (ABO and Rhesus incompatibility), genetic traits (e.g. sickle cell, glucose-6-phosphate dehydrogenase [G6PD] deficiency) or electrolyte imbalance. Some of these conditions (e.g. RhD, G6PD, sickle trait) are particularly prevalent in minority ethnic groups [1-4]. In advanced healthcare facilities, tests for haemolysis involve taking blood samples for microscopy analyses (e.g. cell count and morphology) or biochemical assays (e.g. for unconjugated bilirubin or lactate dehydrogenase). Other resource-intensive tests are available to diagnose specific causes of haemolysis, such autoimmune haemolytic anaemia with Coombs test or G6PD deficiency by redox assays. These tests require trained personnel, advanced laboratory equipment and high standards of hygiene. Simpler, cheaper and faster tests on novel biomarkers of disease, adapted for point-of-care, minimal-laboratory conditions, would be medically and economically justified. This technology would also benefit the developing world, where hemolysis can be more common.

Scientific background: The soluble enzyme carbonic anhydrase I (CAI) is, after haemoglobin isoforms, the most abundant protein in the cytoplasm of red blood cells [5]. Plasma normally contains only trace amounts of CAI but levels will increase upon haemolysis [6]. Based on its small size (29 kDa and Stokes radius 25 Å [7-9] CAI is predicted to filter across the glomerular barrier and appear in urine [10]. Quantitative immuno-techniques can detect CAI even at 1:10,000 dilution. Urine from healthy adults is normally devoid of CAI, producing a very low background signal. Urinary CAI excretion is expected to be proportional to the extent of hemolysis, and therefore serve as a quantitative biomarker that can be assayed non-invasively.

Objectives of the (first) URICA trial: The objective of the now-completed URICA trial (URInary excretion of Carbonic Anhydrase; trial registration CPMS 19576) was to test for the presence of CA isoforms in the urine of full term neonates admitted to Evelina's Children Hospital. The cohort included controls who were not suspected of undergoing hemolysis (beyond the mild physiological form) as well as babies with ABO incompatibility, anaemia and undergoing treatment for jaundice. The URICA trial obtained ethical approval on May 12th, 2015 (REC: 15/NS/0042). The collection and analysis of samples was completed by June 2017. Measurements on urine comprised of western blotting for CAI (a sensitive but semi-quantitative method), ELISA for CAI (a quantitative method for determining CAI levels), mass-spec for obtaining the proteome of urine, plus measurements of total protein and creatinine (to assess evidence for renal failure as an exclusion criterion). In addition, clinical data were collected about gestation, and blood haemoglobin, serum or transcutaneous bilirubin and C-reactive protein at several time points, where possible, to test for anaemia, jaundice and infection, respectively. 26 babies were recruited.

Outcomes of the URICA trial: Western blotting showed no detectable CAI signal in 9 samples (indistinguishable from background-signal in adult urine) and a range of positive signals in 17 samples. These results were supported by ELISA measurements. Two samples had elevated protein/creatinine levels, possibly indicating renal damage yet these did not produce CAI-positive urine. Mass spec analysis showed that CAI level did not correlate with any other red blood cell protein, indicating strongly that the hemolysis had occurred inside vessels and not in the urinary tract (where it would also lead to signals from e.g. haemoglobin or anion exchanger 1). When compared to CAI-negative urine, the most over-abundant protein in CAI-positive samples was, in fact, CAI, arguing for the excellent resolving power of this biomarker. When considering the time point at which urine was collected, it is possible to explain the CAI signal relative to bilirubin measurements as follows: