The Importance of Chiral Metabolomics

Chronic kidney disease (CKD) is a highly-prevalent, global health problem; for example, in Japan, it is estimated that about 10 percent of the population have CKD. The number of patients with worsening kidney functions, eventually requiring costly kidney replacement therapy or transplantation, is increasing. In addition, the risk of life-threatening cardiovascular diseases increases with the progression of CKD stages. Preventing CKD patients from progressing to end-stage kidney disease is therefore critical, but unfortunately there are no effective methods to predict the progression of CKD. Currently, prediction relies on kidney functions estimated from serum creatinine and some additional information, such as proteinuria, but these are insufficient. Naturally, nephrologists are earnestly searching for better biomarkers.

Could amino acids, those vital components of human bodies, help provide the answer? The levels of amino acids (which comprise 20 percent of the body) are influenced by the functions of many organs, including the kidneys; kidneys regulate the body’s amino acid balances via reabsorption. Scientists have been studying amino acids ever since their discovery – for more than a hundred years. But because people only detected L-forms in nature, D-amino acids were regarded as unnatural and have not been studied vigorously. The presence of D-amino acids started to be reported sporadically, including in the blood of patients with kidney diseases. Some studies also indicated the physiological roles of D-amino acids in bodies (for example, D-serine is also known as a neurotransmitter of NMDA receptors in neurons) but once again these reports were sporadic – mainly because of the measurement challenge; typically, the amount of D-amino acids in human bodies are present at trace levels, and the chemically similar nature of amino acid enantiomers makes it difficult to separate them and measure them simultaneously. And because reliable methods to measure D-amino acids are lacking, their functions and presence in tissues have remained a mystery.

It is only recently that methods have been devised to measure D-amino acids precisely via a metabolomic approach. Kenji Hamase of Kyushu University in Japan and his colleagues went to great lengths to develop a metabolomic platform – based on micro-2D-HPLC – that can detect whole sets of chiral amino acids from human samples with precision. In the first dimension of HPLC, 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F)-labelled amino acids are separated by reverse-phase separation. Then, the fraction of each NBD-derived amino acid is automatically transferred to the enantioselective (chiral-selective) column for chiral separation. The 2D-HPLC system is powerful enough to detect all amino acid enantiomers from clinical samples ranging from around 1 fmol to 100 pmol – quantitatively.

Our research group from Osaka and Kyushu University searched for prognostic biomarkers of CKD by using such chiral amino acid metabolic profiling. Our study revealed that D-amino acids, particularly D-serine and D-asparagine, were robustly associated with the progression of CKD to end-stage kidney disease. The risk of progression to ESKD was elevated from two- to four-fold in those with higher levels. What is more interesting is that this trend is only seen in D-amino acids, and not in L-amino acids. The fact that just a trace portion of amino acids have a stronger relationship with disease processes and prognosis strongly supports the importance of chiral separations.

A D-amino acid test could provide a powerful tool for clinicians, helping them identify high-risk CKD patients for intensive care. The development of a device suitable for clinical use – designed to increase throughput – is currently under way. Another important direction for the future will be undertaking further detailed research to study the physiology and metabolism of D-amino acids, both of which are poorly understood, so that we can enrich our understanding of kidney diseases. Through chiral metabolomics, I believe that the mysterious world of D-amino acids will turn out to be a fruitful one for clinicians.