Abstract

BACKGROUND
Hyperbilirubinemia in jaundiced neonates is routinely assessed by use of total serum bilirubin. However, the unbound or free form (B(f)), not total bilirubin, crosses the blood-brain barrier and can be neurotoxic. Although the peroxidase-mediated oxidation of bilirubin can be used to measure plasma concentrations of B(f), this measurement is relatively complex and the assay is not routinely used. We describe a fluorescence sensor for quantifying B(f) in plasma.
METHODS
Our method uses a mutated fatty acid binding protein labeled with the fluorescent molecule acrylodan (BL22P1B11), whose fluorescence is quenched upon binding bilirubin. Another configuration (BL22P1B11-Rh) was developed that uses BL22P1B11 together with the fluorophore rhodamine B, which responds by a change in the ratio of its fluorescence.
RESULTS
The "B(f) probes" were calibrated with aqueous solutions of bilirubin and yielded similar bilirubin dissociation constants [K(d) = 16 (1.5) nmol/L]. We used the probes to determine B(f) concentrations in equilibrium with human serum albumin (HSA) and in human plasma samples supplemented with bilirubin. We obtained equivalent B(f) values in both systems, and the B(f) probe results were in agreement with the peroxidase assay. B(f) measurements revealed that bilirubin-HSA binding was well described by 2 sites with K(d) values of 15.4 (1) nmol/L and 748 (14) nmol/L. We measured B(f) concentrations in the range expected in jaundiced neonates with a mean CV of approximately 3%.
CONCLUSIONS
The BL22P1B11-Rh probe provides accurate plasma sample B(f) concentrations with a single measurement, in 1 min with either a handheld B(f) meter or a laboratory fluorometer.

Links

Publisher Full Text

Authors

Huber AH, Zhu B, Kwan T, Kampf JP, Hegyi T, Kleinfeld AM

Institution

FFA Sciences LLC, San Diego, CA, USA.

Source

Clinical chemistry 58:5 2012 May pg 869-76

MeSH

2-Naphthylamine
Adult
Animals
Bilirubin
Biosensing Techniques
Calibration
Fatty Acid-Binding Proteins
Fluorescent Dyes
Humans
Infant, Newborn
Mutation
Protein Binding
Rats
Rhodamines
Serum Albumin

Pub Type(s)

Journal Article
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

22395024