Autor:innen:
F. Gauß (Greifswald, DE)
K. Budde (Greifswald, DE)
A. Henning (Greifswald, DE)
A. Hafke (Göttingen, DE)
R. Krätzner (Göttingen, DE)
M. Nauck (Greifswald, DE)
A. Petersmann (Oldenburg, DE)
Aims
Multiple studies show the potential of amino acids (AA) to serve as biomarkers in prognosis and diagnosis of various diseases, e.g. diabetes. 1H-NMR-platforms are increasingly used in metabolic profiling studies due to their inherent advantages like robust and reproducible measurements. Still, NMR methods are not used in clinical settings.
In order to evaluate the use of NMR methods for AA measurement in research and routine patient care, we compared levels of AA measured on a 1H-NMR platform with those measured on a well established HPLC platform.
Materials and Methods
Eleven AA were measured in 90 EDTA-plasma samples from patients of the University Medicine Greifswald. Measurements were conducted on a HPLC platform (Biochrom 30+ system, Biochrom, Cambridge, UK) and on a 600 MHz 1H-NMR platform (Bruker AVANCE-III, Bruker Biospin, Ettlingen, Germany). All measurements were performed in duplicates. For evaluation of precision of the two methods, coefficients of variation (CV%) were calculated from duplicates and Passing-Bablok regression analysis was conducted. For the comparison of NMR and HPLC measurements, we determined Pearson correlation coefficients and performed Passing-Bablok regression.
Results
CV based on duplicates were lower on average for HPLC than for NMR measurements: median CV were 2.79% (interquartile range IR: 2.75% – 3.25%) and 10.22% (IR: 6.73% – 13.77%) for HPLC and NMR, respectively. Further, Passing-Bablok analysis revealed no or only small bias between the first and second measurement with very few exceptions for NMR-measured AA. The comparison of NMR and HPLC measured levels showed a median correlation coefficient of r=0.82 (IR 0.76 – 0.91). Highest correlation was found for phenylalanine (r=0.99), tyrosine (r= 0.98), glycine (r= 0.92) and alanine (r = 0.89). In Passing-Bablok regression, no or only very small bias was found for phenylalanine and alanine.
Discussion
In the comparison of AA levels measured on a NMR and a HPLC platform, two AA, namely phenylalanine and alanine, were highly comparable in terms of high correlation and no or only small bias. Thus, NMR methods have the potential to be applied in clinical context. In research context, NMR measurements of AA, which correlate well with HPLC levels, may be used in profiling studies, e.g. in the epidemiological field, to detect significant associations to clinical outcomes despite some bias between the exact AA levels.
Generally, precision of NMR measurements and correlation between HPLC and NMR measured levels were higher for those AA with the highest measured concentration levels. Still, also AA with low concentrations, such as tyrosine, showed a good performance, which indicates, that improvement of quantification algorithms or measurement time may lead to a higher analytical quality of NMR methods.