Tissue and plasma globotriaosylsphingosine could be a biomarker for assessing enzyme replacement therapy for Fabry disease

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Abstract

Fabry disease is a genetic disease caused by a deficiency of α-galactosidase A (GLA), which leads to systemic accumulation of glycolipids, predominantly globotriaosylceramide (Gb3). With the introduction and spread of enzyme replacement therapy (ERT) with recombinant GLAs for this disease, a useful biomarker for assessing the response to ERT is strongly required. We measured the tissue level of lyso-globotriaosylsphingosine (lyso-Gb3) in Fabry mice by means of high performance liquid chromatography, and compared it with the Gb3 level. The results revealed a marked increase in the lyso-Gb3 level in most tissues of Fabry mice, and which decreased after the administration of a recombinant GLA as in the case of Gb3, which is usually used as a biomarker of Fabry disease. The response was more impressive for lyso-Gb3 compared with for Gb3, especially in kidney tissues, in which a defect significantly influences the morbidity and mortality in patients with this disease. The plasma level of lyso-Gb3 also decreased after the injection of the enzyme, and it was well related to the degradation of tissue lyso-Gb3. Thus, lyso-Gb3 is expected to be a useful new biomarker for assessing the response to ERT for Fabry disease.

Research highlights

► A marked increase in the lyso-Gb3 level was found in most tissues of Fabry mice. ► The lyso-Gb3 was decreased after an administration of GLA, especially in the kidneys. ► Tissue and plasma lyso-Gb3 is a useful new biomarker for assessing ERT for Fabry disease.

Introduction

Fabry disease (MIM 3010500) is an X-linked genetic disease resulting from deficient activity of α-galactosidase A (GLA, MIM 300644, EC 3.2.1.22). The enzymatic defect leads to the accumulation of glycolipids, predominantly globotriaosylceramide (Gb3), primarily in the kidneys and cardiovascular system, and it results in systemic manifestations, including renal, cardiac and vascular diseases [1].

So far, two different recombinant GLAs, agalsidase beta (Fabrazyme®; Genzyme Therapeutics, Cambridge, MA) and agalsidase alfa (Replegal®; Shire HGT, Cambridge, MA), have been available for enzyme replacement therapy (ERT) for Fabry disease, and their efficacy has been discussed mainly from the clinical aspect [2], [3], [4], [5], [6], [7], [8], [9]. However, there have been few reports assessing ERT from the biochemical aspect, because there are few biomarkers reflecting the disease. The level of Gb3 accumulated in tissues and body fluids would be one biomarker candidate, and it has been measured for diagnosis and monitoring of the response to ERT in Fabry disease [10], [11], [12]. But recent analysis revealed that Gb3, which itself does not exhibit any toxicity, was not necessarily an ideal biomarker [13]. Not only for assessing the effects of presently available therapeutics but also for developing new ones, a useful biomarker for this purpose is strongly required. Recently, there have been some reports that the plasma level of globotriaosylsphingosine (lyso-Gb3) was apparently increased in patients with Fabry disease and was likely to be well related to the clinical condition of Fabry patients receiving ERT [14], [15]. Furthermore, Sanchez-Niño et al. revealed that lyso-Gb3 induced the release of secondary mediators of injury in glomerular podocytes, which may be deeply related to the renal disorder in this disease [16].

In this study, we measured the levels of lyso-Gb3 in various tissues and plasma of Fabry mice and examined the effect of repeated administration of a recombinant GLA on cleavage of the lyso-Gb3 to determine whether tissue and plasma lyso-Gb3 could be a useful biomarker for assessing the response to ERT or not, especially in the kidneys, which are the most important organs of which a defect influences the prognosis of the disease.

Section snippets

Materials

Lyso-Gb3 and Gb3 were obtained from Sigma Chemical Co. (St. Louis, MO). A recombinant human GLA generated in Chinese hamster ovary cells, agalsidase beta, was obtained from Genzyme Japan Co. (Tokyo, Japan). o-Phthalaldehyde (OPA) was purchased from Nacalai Tesque (Kyoto, Japan). All other chemicals used were of analytical grade.

Animals

Fabry mice (GLA knock-out mice donated by Kulkarni and Ohshima et al.) [17], [18] and wild-type C57BL/6 mice were used in this experiment. All animal experiments were

Determination of lyso-Gb3 levels in mouse tissues and plasma

The lyso-Gb3 levels in the kidneys, heart, liver, lungs, spleen, brain and plasma were determined by means of HPLC. Peak identification was performed by comparison of the retention times with that of authentic lyso-Gb3. The lyso-Gb3 level was determined from the calibration curve obtained by plotting the lyso-Gb3 peak area against the concentration, which was linear over the range of 0.1–10 nmol/g for tissues and of 2–500 nmol/l for plasma.

Fig. 1 shows HPLC-chromatograms of lyso-Gb3 in the kidney

Discussion

The results of newborn screening performed in Taiwan [22] and Italy [23] revealed that the incidence of Fabry disease is unexpectedly high, being 1 in 1250–4000 male live births, and the clinical importance of this disease is increasing more and more.

With the introduction and spread of ERT, a useful biomarker for monitoring the response to ERT has been required. In particular, as a renal disorder is one of the most clinically significant features of Fabry disease, and constitutes an important

Acknowledgments

We thank Drs. Ashok B. Kulkarni (NIH) and Toshio Oshima (Waseda University) for providing us with the Fabry mice. We also thank Ms. Yoshiko Tanabe for typing the manuscript. This work was supported by the Program for Research on Intractable Diseases of Health and Labor Science Research Grants (HS); the Program for Promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation (ID: 09-15, HS); the JAPS Asia/Africa Scientific Platform Program (HS); the

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    These authors equally contributed to this work.

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