Metabolomic Signature in Sera of Multiple Sclerosis Patients during Pregnancy

Abstract

Multiple sclerosis (MuS) is an autoimmune disease of the central nervous system characterized by neuroinflammation, neurodegeneration, and degradation of the myelin sheath. Epidemiological studies have shown that the female gender is more susceptible than the male gender to MuS development, with a female-to-male ratio of 2:1. Despite this high onset, women have a better prognosis than men, and the frequency of the relapsing phase decreases during pregnancy, while it increases soon after birth. Therefore, it is interesting to investigate hormonal fluctuations during pregnancy and whether they correlate with metabolic signatures. To gain a deeper inside into the biochemical mechanism of such a multifactorial disease, we adopted targeted metabolomics approaches for the determination of many serum metabolites in 12 pregnant women affected by MuS by mass spectrometry analysis. Our data show a characteristic hormonal fluctuation for estrogens and progesterone, as expected. They also highlight other interesting hormonal alterations for cortisol, corticosterone, 11-deoxycortisol, 4-androstene-3,17-dione, testosterone, and 17α-hydroxyprogesterone. Furthermore, a negative correlation with progesterone levels was observed for amino acids and for acylcarnitines, while an imbalance of different sphingolipids pathways was found during pregnancy. In conclusion, these data are in agreement with the characteristic clinical signs of MuS patients during pregnancy and, if confirmed, they may add an important tessera in the complex mosaic of maternal neuroprotection.

Keywords: acylcarnitines; amino acids; biomarkers; ceramides; estrogens; mass spectrometry; metabolomics; multiple sclerosis; pregnancy; steroids.

Figure 1

(A) Scores scatter plot calculated on two components by using 42 observation of 12 MuS cases, divided into two time groups: during pregnancy and post-partum. The classification is based on each metabolite concentration, excluding steroids and estrogens. The Partial Least Squares Discriminant Analysis (PLS-DA) was used to classify sera from MuS patients in each time group as follow: purple diamonds: during pregnancy; dark red inverted triangles: post-partum obtained by R2Y = 0.865 and Q2(cum) = 0.758. (B) Scores scatter plot calculated on two components by using 42 observations divided into three different times of pregnancy and the post-partum period. classification is based on each metabolite concentration, excluding steroids and estrogens. The Partial PLS-DA was used to classify sera from MuS patients in each time group as follow: black boxes: first trimester of pregnancy; red dots: second trimester of pregnancy; blue diamonds: third trimester of pregnancy; green triangles: post-partum obtained by R2Y = 0.484 and Q2(cum) = 0.321.

Figure 2

(A,B) Concentrations of steroids and estrogens in each of the three trimesters of pregnancy and post-partum (P-P) as follow: black bars for the first trimester of pregnancy, red bars for the second trimester of pregnancy, blue bars for the third trimester of pregnancy, and green bars for the post-partum period. Data are mean values, and bars represent the corresponding standard deviations (SD). The significance, expressed in terms of p-value, is reported in Table 2 for each time comparison for the observed steroids and estrogens. CORT: cortisol, CCONE: corticosterone, 11DECOL: 11-deoxycortisol, ADIONE: 17-dione, TESTO: testosterone, 17-OHIP: 17α-hydroxyprogesterone, PROG: progesterone, E2: estradiol, E1: estrone.

Figure 3

Sphingolipid metabolic pathways and concentration levels of the related metabolites in each of the three trimesters of pregnancy and post partum as follow: black bars for the first trimester of pregnancy, red bars for the second trimester of pregnancy, blue bars for the third trimester of pregnancy, and green bars for the post-partum period. The de novo synthesis pathway is reported in Panel (A), the sphingomyelin pathway is represented in Panel (B), the catabolic pathway is schematized in Panel (C), and glucosylceramide synthesis is summarized in Panel (D). SPT: serinepalmitoyltransferase; 3-KSR: 3-ketosphinganine reductase; CS: ceramidesynthetase; DES: dihydroceramidedesaturase; Smase: sphingomyelinase; SMS: sphingomyelinsynthetase; Cdase: ceramidase; CS: ceramide synthase; SK: spingosine kinase; S1PP: spingosine-1-phosphate phosphatase; GCS: glycosylceramide synthase; Gcase: glycosylceramidase.

Figure 4

The heatmap displays data related to amino acids (AAs), free carnitine (C0), acylcarnitines (ACCs), succinyl acetone (SA), nucleosides, and lysophospholipids (LPCs) in the form of colored cells. It provides direct visualization of the relative levels of individual samples and time series. At the top of the image, the black bar indicates the first trimester of pregnancy (T1), the red bar indicates the second trimester of pregnancy (T2), the blue bar indicates the third trimester of pregnancy (T3), and the green bar indicates the post-partum period. The color code describes high serum levels (red) and low serum levels (blue) of each metabolite.

Figure 5

Panels (A–F) show the Rank correlations, performed by using MedCalc 7.6, between Progesterone (PROG), Sphingomyelin (d18:0/22:0) (SM), Sphinganine (Sa), Ceramide (d18:1/16:0) (C16Cer), Glucosylceramide (d18:1/16:0) (C16GlcCer), Tyrosine (TYR) and Free Carnitine (C0), respectively. The black squares indicate the first trimester of pregnancy, the red circles indicate the second trimester of pregnancy, the blue diamonds indicate the third trimester of pregnancy, and the green triangles indicate the post-partum period.