Elevated plasma glucosylsphingosine in Gaucher disease: relation to phenotype, storage cell markers, and therapeutic response

Abstract

Gaucher disease, caused by a deficiency of the lysosomal enzyme glucocerebrosidase, leads to prominent glucosylceramide accumulation in lysosomes of tissue macrophages (Gaucher cells). Here we show glucosylsphingosine, the deacylated form of glucosylceramide, to be markedly increased in plasma of symptomatic nonneuronopathic (type 1) Gaucher patients (n = 64, median = 230.7 nM, range 15.6-1035.2 nM; normal (n = 28): median 1.3 nM, range 0.8-2.7 nM). The method developed for mass spectrometric quantification of plasma glucosylsphingosine is sensitive and robust. Plasma glucosylsphingosine levels correlate with established plasma markers of Gaucher cells, chitotriosidase (ρ = 0.66) and CCL18 (ρ = 0.40). Treatment of Gaucher disease patients by supplementing macrophages with mannose-receptor targeted recombinant glucocerebrosidase results in glucosylsphingosine reduction, similar to protein markers of Gaucher cells. Since macrophages prominently accumulate the lysoglycosphingolipid on glucocerebrosidase inactivation, Gaucher cells seem a major source of the elevated plasma glucosylsphingosine. Our findings show that plasma glucosylsphingosine can qualify as a biomarker for type 1 Gaucher disease, but that further investigations are warranted regarding its relationship with clinical manifestations of Gaucher disease.

Figure 1

Quantification of glucosylsphingosine in plasma of type 1 Gaucher type patients. (A) Fragmentation spectrum of glucosylsphingosine. Predominant product ions (m/z 282.2 and 264.2) generated by collision induced fragmentation of glucosylsphingosine (m/z 464.2) are indicated. (B) Diagnostic value of glucosylsphingosine in type 1 Gaucher disease. Glucosylsphingosine and glucosylceramide levels in plasma of type 1 Gaucher patients (n = 64 and n = 49, respectively) and controls (n = 28). Patients homozygous for the mutation N370S are indicated with open circles (n = 19). Lines represent the median value in each group.

Figure 2

Plasma glucosylsphingosine levels in relation to GBA genotype. Glycolipid concentration was determined in plasma samples collected before initiation of therapy. Controls (n = 28), N370S heterozygotes (n = 22), L444P heterozygotes (n = 12), N370S homozygotes (n = 19), and N370S/L444P compound heterozygotes (n = 18).

Figure 3

Relation between plasma glucosylsphingosine levels and established Gaucher biomarkers. Correlation between plasma glucosylsphingosine and chitotriosidase activity in type 1 Gaucher patients with wild-type CHIT genotype (n = 34; A) and carriers of the 24 bp duplication in the CHIT gene (n = 15; B). Correlation of plasma glucosylsphingosine level of type 1 Gaucher patients and cytokine CCL18 (n = 36; C) and macrophage inflammatory protein MIP-1β (n = 37; D).

Figure 4

Response to therapy. (A) Characteristic reduction in plasma glucosylsphingosine, chitotriosidase and CCL18 in 2 type 1 Gaucher patients after enzyme therapy. Reductions are expressed as percentage of amount at start of treatment. Patient with wild-type chitotriosidase genotype (left panel); patient homozygous for a 24 bp duplication in the chitotriosidase gene, lacking chitotriosidase activity (right panel). (B-D) Changes in plasma glucosylsphingosine (left panels) and chitotriosidase (right panels) levels in 3 siblings suffering from type 3 Gaucher disease after enzyme replacement therapy (ERT; variable dosage) in combination with miglustat treatment (SRT). Patients received different combinations of enzyme replacement therapy and treatment with miglustat (3 × 100 mg/kg/d), starting at a very young age. The individual treatment regimens are indicated as ERT and SRT.

Figure 5

Relation between plasma glucosylsphingosine levels and other sphingolipids. (A) Glucosylceramide; (B) Ganglioside GM3; (C) Ceramide; and (D) Globoside Gb3. (n = 49).

Figure 6

Accumulation of glucosylceramide and glucosylsphingosine in cultured macrophages afterinactivation of glucocerebrosidase. Macrophages were differentiated from peripheral blood monocytes obtained from 2 different donors. After 7 days of culture, glucocerebrosidase was irreversibly inactivated for 4 days with an excess of the suicide inhibitor MDW941. Cells were harvested at indicated time points, and glucosylsphingosine (A) and glucosylceramide content (B) determined.

Figure 7

Relation between plasma glucosylsphingosine and disease manifestations or bone marrow markers. (A) Plasma glucosylsphingosine and disease severity score (SSI; n = 46). (B) Plasma glucosylsphingosine and liver volume (n = 45). (C) Plasma glucosylsphingosine and skeletal disease (n = 48). (D) Plasma glucosylsphingosine and bone marrow fat fraction (n = 34). (E) Plasma glucosylsphingosine and osteocalcin (n = 24). (F) Plasma glucosylsphingosine and P1NP (n = 24).