J Neurol Res

Journal of Neurology Research, ISSN 1923-2845 print, 1923-2853 online, Open Access

Article copyright, the authors; Journal compilation copyright, J Neurol Res and Elmer Press Inc

Journal website https://www.neurores.org

Original Article

Volume 10, Number 6, December 2020, pages 209-219

Ceramide Dynamics and Prognostic Value in Acute and Subacute Ischemic Stroke: Preliminary Findings in a Clinical Cohort

Figure 1. Box plots showing difference in ceramide levels and ratios between two time points. Coral box plots represent circulating plasma ceramide levels (a) or ratios (b) at time point 1 (< 12 h from the symptom onset), and teal box plots represent ceramide levels or ratios at time point 2 (> 24 h from the symptom onset). The line in box plots represents the median, interquartile range and the whiskers correspond to minimum and maximum values. Wilcoxon signed rank test results are displayed above each comparison. ns: not significant, P ≥ 0.05. *0.01≤ P <0.05. **0.001≤ P <0.01. *** P < 0.001.

Figure 2. Box plots showing difference in levels of ceramides and ceramide ratios between two time points stratified by prior statin exposure. Light blue and orange box plots represent circulating plasma ceramide levels (a, c) or ratios (b, d) at time point 1, and dark blue and orange box plots represent ceramide levels or ratios at time point 2. Panels (a) and (b) show data for statin-naive participants; panels (c) and (d) show data for participants with prior statin exposure. The line in box plots represents the median, interquartile range and the whiskers correspond to minimum and maximum values. Wilcoxon signed rank test results are displayed above each comparison. ns: not significant, P ≥ 0.05. *0.01≤ P <0.05. **0.001≤ P <0.01. *** P < 0.001.

**Table 1.** Demographic, Clinical and Laboratory Characteristics of the Study Participants
	N(%) or median (range)	P value^b
LKW: last known well; BMI; body mass index; NIHSS: National Institutes of Health Stroke Scale; BP: blood pressure; IV-tPA: intravenous tissue plasminogen activator; ESUS: embolic stroke of undetermined source; LDL: low-density lipoprotein; HDL: high-density lipoprotein; T1: time point 1; T2: time point 2. ^aStatin-naive: participants were not on statin therapy prior to admission. ^bFor categorical variables, P values are from Fisher’s exact test; for continuous, from Wilcoxon rank sum test. ^cOther mechanisms: dissection (n = 1), hypotension (n = 1), transient ischemic attack (n = 1), small vessel occlusion (n = 1).
Demographic characteristics
Female	12 (52%)	7 (54%)	5 (50%)	1.000
Age, years	76 (45 - 96)	76 (45 - 96)	77 (53 - 92)	0.7093
BMI, kg/m²	25.6 (20.5 - 46.6)	28.9 (20.5 - 46.6)	24.7 (20.9 - 38.4)	0.2036
Risk factors
Hypertension	20 (87%)	11 (85%)	9 (90%)	1.000
Diabetes mellitus	7 (30%)	3 (23%)	4 (40%)	0.6500
Hyperlipidemia	19 (83%)	9 (69%)	10 (100%)	0.1045
Obstructive sleep apnea	6 (26%)	2 (15%)	4 (40%)	0.3413
Cardiac disease	15 (65%)	8 (62%)	7 (70%)	1.000
Prior stroke	8 (35%)	3 (23%)	5 (50%)	0.2213
Deep vein thrombosis	3 (13%)	2 (15%)	1 (10%)	1.000
Medications prior to admission
Antiplatelet therapy	13 (57%)	7 (54%)	6 (60%)	1.000
Anticoagulation	4 (17%)	1 (8%)	3 (30%)	0.2806
Acute ischemic event characteristics
NIHSS score	5 (0 - 27)	8 (0 - 27)	4 (0 - 22)	0.4541
Infarct volume, cm³	1.4 (0.0 - 36.5)	2.3 (0.04 - 36.5)	1.2 (0.0 - 25.5)	0.6418
Systolic BP, mm Hg	158 (98 - 190)	149 (98 - 190)	160 (136 - 175)	0.4949
Diastolic BP, mm Hg	89 (47 - 127)	89 (47 - 127)	87 (64 - 109)	0.8768
IV-tPA administered	10 (44%)	7 (54%)	3 (30%)	0.4015
Embolectomy	5 (22%)	4 (31%)	1 (10%)	0.3394
Modified Rankin scale	2 (0 - 6)	2 (0 - 6)	1.5 (0 - 3)	0.6784
Mechanism of the acute ischemic event
Cardioembolic	8 (35%)	6 (46%)	2 (20%)	0.3788
Large vessel occlusion	4 (17%)	2 (15%)	2 (20%)	1.000
ESUS	7 (31%)	2 (15%)	5 (50%)	0.1688
Other^c	4 (17%)	3 (23%)	1 (10%)	0.6036
Laboratory results
Total cholesterol, mg/dL	161 (110 - 244)	177(111 - 244)	146 (110 - 240)	0.0694
LDL, mg/dL	79 (55 - 162)	120 (55 - 125)	70 (55 - 162)	0.0158^a
HDL, mg/dL	45 (20 - 76)	40 (20 - 67)	51 (29 - 76)	0.2758
Triglycerides, mg/dL	114 (59 - 355)	132 (59 - 355)	88 (70 - 171)	0.9474
Hemoglobin A1c, %	5.7 (5.1 - 9.3)	5.5 (5.1 - 9.3)	5.9 (5.2 - 7.1)	0.0964
Ejection fraction, %	60 (14 - 73)	64 (14 - 73)	55 (20 - 60)	0.0742
Time from LKW to blood sample acquisition
T1, hours	8.5 ( 3.8 - 11.5)	8.8 (3.8 - 11.0)	8.4 (5.5 - 11.5)	0.8515
T2, hours	44.5 (24.3 - 151)	43.5 (29.0 - 183)	46.9 (24.3 - 151)	0.8768

Table 1. Demographic, Clinical and Laboratory Characteristics of the Study Participants

N(%) or median (range)

P value^b

All (n = 23)

Statin-naive (n = 13)^a

Statin-exposed (n = 10)

LKW: last known well; BMI; body mass index; NIHSS: National Institutes of Health Stroke Scale; BP: blood pressure; IV-tPA: intravenous tissue plasminogen activator; ESUS: embolic stroke of undetermined source; LDL: low-density lipoprotein; HDL: high-density lipoprotein; T1: time point 1; T2: time point 2. ^aStatin-naive: participants were not on statin therapy prior to admission. ^bFor categorical variables, P values are from Fisher’s exact test; for continuous, from Wilcoxon rank sum test. ^cOther mechanisms: dissection (n = 1), hypotension (n = 1), transient ischemic attack (n = 1), small vessel occlusion (n = 1).

Demographic characteristics

Female

12 (52%)

7 (54%)

5 (50%)

1.000

Age, years

76 (45 - 96)

77 (53 - 92)

0.7093

BMI, kg/m²

25.6 (20.5 - 46.6)

28.9 (20.5 - 46.6)

24.7 (20.9 - 38.4)

0.2036

Risk factors

Hypertension

20 (87%)

11 (85%)

9 (90%)

1.000

Diabetes mellitus

7 (30%)

3 (23%)

4 (40%)

0.6500

Hyperlipidemia

19 (83%)

9 (69%)

10 (100%)

0.1045

Obstructive sleep apnea

6 (26%)

2 (15%)

4 (40%)

0.3413

Cardiac disease

15 (65%)

8 (62%)

7 (70%)

1.000

Prior stroke

8 (35%)

3 (23%)

5 (50%)

0.2213

Deep vein thrombosis

3 (13%)

2 (15%)

1 (10%)

1.000

Medications prior to admission

Antiplatelet therapy

13 (57%)

7 (54%)

6 (60%)

1.000

Anticoagulation

4 (17%)

1 (8%)

3 (30%)

0.2806

Acute ischemic event characteristics

NIHSS score

5 (0 - 27)

8 (0 - 27)

4 (0 - 22)

0.4541

Infarct volume, cm³

1.4 (0.0 - 36.5)

2.3 (0.04 - 36.5)

1.2 (0.0 - 25.5)

0.6418

Systolic BP, mm Hg

158 (98 - 190)

149 (98 - 190)

160 (136 - 175)

0.4949

Diastolic BP, mm Hg

89 (47 - 127)

87 (64 - 109)

0.8768

IV-tPA administered

10 (44%)

7 (54%)

3 (30%)

0.4015

Embolectomy

5 (22%)

4 (31%)

1 (10%)

0.3394

Modified Rankin scale

2 (0 - 6)

1.5 (0 - 3)

0.6784

Mechanism of the acute ischemic event

Cardioembolic

8 (35%)

6 (46%)

2 (20%)

0.3788

Large vessel occlusion

4 (17%)

2 (15%)

2 (20%)

1.000

ESUS

7 (31%)

2 (15%)

5 (50%)

0.1688

Other^c

4 (17%)

3 (23%)

1 (10%)

0.6036

Laboratory results

Total cholesterol, mg/dL

161 (110 - 244)

177(111 - 244)

146 (110 - 240)

0.0694

LDL, mg/dL

79 (55 - 162)

120 (55 - 125)

70 (55 - 162)

0.0158^a

HDL, mg/dL

45 (20 - 76)

40 (20 - 67)

51 (29 - 76)

0.2758

Triglycerides, mg/dL

114 (59 - 355)

132 (59 - 355)

88 (70 - 171)

0.9474

Hemoglobin A1c, %

5.7 (5.1 - 9.3)

5.5 (5.1 - 9.3)

5.9 (5.2 - 7.1)

0.0964

Ejection fraction, %

60 (14 - 73)

64 (14 - 73)

55 (20 - 60)

0.0742

Time from LKW to blood sample acquisition

T1, hours

8.5 ( 3.8 - 11.5)

8.8 (3.8 - 11.0)

8.4 (5.5 - 11.5)

0.8515

T2, hours

44.5 (24.3 - 151)

43.5 (29.0 - 183)

46.9 (24.3 - 151)

0.8768

**Table 2.** Median Difference in Levels of Circulating Ceramides Between the Two Time Points
	Time point 1^a	Time point 2	Median difference (95% CI)^b	P value^c
CI: confidence interval; C: ceramide. ^aData shown for time point 1 and 2 are median (range). ^bMedian difference and 95% confidence intervals are from smoothed empirical likelihood quantiles. ^cP values shown are from Wilcoxon signed rank test, P < 0.05 was considered statistically significant.
All study participants (n = 23)
C(16:0), µmol	0.33 (0.18 - 0.70)	0.32 (0.20 - 0.48)	-0.01 (-0.02, 0.01)	0.5235
C(18:0), µmol	0.14 (0.04 - 0.33)	0.14 (0.05 - 0.30)	0.02 (0.00, 0.03)	0.0243
C(20:0), µmol	0.10 (0.05 - 0.25)	0.10 (0.05 - 0.17)	-0.01 (-0.01, 0.00)	0.0562
C(22:0), µmol	0.87 (0.63 - 2.38)	0.81 (0.54 - 1.41)	-0.10 (-0.14, -0.06)	< 0.0001
C(24:0), µmol	2.63 (1.70 - 6.64)	2.56 (1.47 - 3.95)	-0.34 (-0.49, -0.14)	0.0002
C(24:1), µmol	1.05 (0.48 - 2.73)	1.04 (0.55 - 2.02)	-0.02 (-0.09, 0.05)	0.4591
Total C, µmol	5.32 (3.62 - 13.03)	5.23 (3.22 - 8.16)	-0.39 (-0.69, -0.14)	0.0012
C(16:0)/(24:0)	0.12 (0.07 - 0.21)	0.12 (0.09 - 0.20)	0.01 (0.00, 0.02)	0.0160
C(18:0)/(24:0)	0.05 (0.01 - 0.11)	0.06 (0.02 - 0.12)	0.01 (0.007, 0.012)	< 0.0001
C(24:1)/(24:0)	0.39 (0.15 - 0.76)	0.44 (0.19 - 0.74)	0.04 (0.03, 0.05)	0.0013
C(24:0)/C(24:1)	2.53 (1.32 - 6.57)	2.28 (1.35 - 5.15)	-0.25 (-0.42, -0.14)	< 0.0001
Ceramide score	8 (0 - 12)	9 (1 - 12)	1 (0, 2)	0.0543
Statin-naive participants (n = 13)
C(16:0), µmol	0.36 (0.19 - 0.70)	0.36 (0.20 - 0.48)	0.00 (-0.03, 0.02)	0.6836
C(18:0), µmol	0.15 (0.05 - 0.33)	0.15 (0.07 - 0.30)	0.02 (0.003, 0.04)	0.0303
C(20:0), µmol	0.10 (0.05 - 0.25)	0.11 (0.05 - 0.17)	0.00 (-0.01, 0.01)	0.6537
C(22:0), µmol	0.93 (0.63 - 2.38)	0.89 (0.56 - 1.41)	-0.07 (-0.12, -0.02)	0.0247
C(24:0), µmol	3.26 (1.70 - 6.64)	3.00 (1.80 - 3.95)	-0.21 (-0.51, 0.02)	0.0803
C(24:1), µmol	1.05 (0.48 - 2.73)	1.26 (0.55 - 2.02)	0.00 (-0.08, 0.07)	0.9597
Total C, µmol	5.70 (3.62, 13.03)	5.52 (3.53, 8.16)	-0.23 (-0.62, 0.07)	0.0942
C(16:0)/(24:0)	0.12 (0.07 - 0.21)	0.12 (0.09 - 0.19)	0.00 (-0.004, 0.01)	0.3320
C(18:0)/(24:0)	0.05 (0.02 - 0.11)	0.06 (0.03 - 0.12)	0.01 (0.01, 0.02)	0.0020
C(24:1)/(24:0)	0.36 (0.22 - 0.76)	0.44 (0.25 - 0.74)	0.03 (0.01, 0.05)	0.0234
C(24:0)/C(24:1)	2.78 (1.32 - 4.63)	2.29 (1.35 - 4.02)	-0.25 (-0.47, -0.09)	0.0034
Ceramide score	8 (1 - 12)	10 (2 - 12)	1 (0, 2)	0.0508
Statin-exposed participants (n = 10)
C(16:0), µmol	0.31 (0.18 - 0.41)	0.31 (0.22 - 0.39)	-0.01 (-0.06, 0.05)	0.6738
C(18:0), µmol	0.14 (0.04 - 0.17)	0.12 (0.05 - 0.20)	0.01 (-0.02, 0.02)	0.4922
C(20:0), µmol	0.11 (0.06 - 0.14)	0.08 (0.06 - 0.13)	-0.01 (-0.024, -0.004)	0.0194
C(22:0), µmol	0.80 (0.67 - 1.19)	0.61 (0.54 - 0.94)	-0.14 (-0.21, -0.09)	0.0003
C(24:0), µmol	2.48 (1.88- 4.43)	2.22 (1.47 - 3.71)	-0.42 (-0.57, -0.27)	0.0039
C(24:1), µmol	1.04 (0.52 - 1.74)	0.90 (0.62 - 1.56)	-0.06 (-0.23, 0.06)	0.3398
Total C, µmol	4.88 (4.03 - 8.04)	4.04 (3.22 - 6.76)	-0.65 (-1.28, -0.09)	0.0098
C(16:0)/(24:0)	0.13 (0.07 - 0.17)	0.14 (0.09 - 0.20)	0.02 (0.002, 0.05)	0.0469
C(18:0)/(24:0)	0.06 (0.01 - 0.07)	0.06 (0.02 - 0.10)	0.01 (0.00, 0.02)	0.0547
C(24:1)/(24:0)	0.40 (0.15 - 0.67)	0.45 (0.19 - 0.62)	0.04 (0.00, 0.08)	0.0859
C(24:0)/C(24:1)	2.52 (1.49 - 6.60)	2.21 (1.61 - 5.15)	-0.30 (-0.76, -0.04)	0.0371
Ceramide score	8 (0 - 10)	7 (1 - 10)	1 (-3, 3)	0.6328

Table 2. Median Difference in Levels of Circulating Ceramides Between the Two Time Points

Time point 1^a

Time point 2

Median difference (95% CI)^b

P value^c

CI: confidence interval; C: ceramide. ^aData shown for time point 1 and 2 are median (range). ^bMedian difference and 95% confidence intervals are from smoothed empirical likelihood quantiles. ^cP values shown are from Wilcoxon signed rank test, P < 0.05 was considered statistically significant.

All study participants (n = 23)

C(16:0), µmol

0.33 (0.18 - 0.70)

0.32 (0.20 - 0.48)

-0.01 (-0.02, 0.01)

0.5235

C(18:0), µmol

0.14 (0.04 - 0.33)

0.14 (0.05 - 0.30)

0.02 (0.00, 0.03)

0.0243

C(20:0), µmol

0.10 (0.05 - 0.25)

0.10 (0.05 - 0.17)

-0.01 (-0.01, 0.00)

0.0562

C(22:0), µmol

0.87 (0.63 - 2.38)

0.81 (0.54 - 1.41)

-0.10 (-0.14, -0.06)

< 0.0001

C(24:0), µmol

2.63 (1.70 - 6.64)

2.56 (1.47 - 3.95)

-0.34 (-0.49, -0.14)

0.0002

C(24:1), µmol

1.05 (0.48 - 2.73)

1.04 (0.55 - 2.02)

-0.02 (-0.09, 0.05)

0.4591

Total C, µmol

5.32 (3.62 - 13.03)

5.23 (3.22 - 8.16)

-0.39 (-0.69, -0.14)

0.0012

C(16:0)/(24:0)

0.12 (0.07 - 0.21)

0.12 (0.09 - 0.20)

0.01 (0.00, 0.02)

0.0160

C(18:0)/(24:0)

0.05 (0.01 - 0.11)

0.06 (0.02 - 0.12)

0.01 (0.007, 0.012)

< 0.0001

C(24:1)/(24:0)

0.39 (0.15 - 0.76)

0.44 (0.19 - 0.74)

0.04 (0.03, 0.05)

0.0013

C(24:0)/C(24:1)

2.53 (1.32 - 6.57)

2.28 (1.35 - 5.15)

-0.25 (-0.42, -0.14)

< 0.0001

Ceramide score

8 (0 - 12)

9 (1 - 12)

1 (0, 2)

0.0543

Statin-naive participants (n = 13)

C(16:0), µmol

0.36 (0.19 - 0.70)

0.36 (0.20 - 0.48)

0.00 (-0.03, 0.02)

0.6836

C(18:0), µmol

0.15 (0.05 - 0.33)

0.15 (0.07 - 0.30)

0.02 (0.003, 0.04)

0.0303

C(20:0), µmol

0.10 (0.05 - 0.25)

0.11 (0.05 - 0.17)

0.00 (-0.01, 0.01)

0.6537

C(22:0), µmol

0.93 (0.63 - 2.38)

0.89 (0.56 - 1.41)

-0.07 (-0.12, -0.02)

0.0247

C(24:0), µmol

3.26 (1.70 - 6.64)

3.00 (1.80 - 3.95)

-0.21 (-0.51, 0.02)

0.0803

C(24:1), µmol

1.05 (0.48 - 2.73)

1.26 (0.55 - 2.02)

0.00 (-0.08, 0.07)

0.9597

Total C, µmol

5.70 (3.62, 13.03)

5.52 (3.53, 8.16)

-0.23 (-0.62, 0.07)

0.0942

C(16:0)/(24:0)

0.12 (0.07 - 0.21)

0.12 (0.09 - 0.19)

0.00 (-0.004, 0.01)

0.3320

C(18:0)/(24:0)

0.05 (0.02 - 0.11)

0.06 (0.03 - 0.12)

0.01 (0.01, 0.02)

0.0020

C(24:1)/(24:0)

0.36 (0.22 - 0.76)

0.44 (0.25 - 0.74)

0.03 (0.01, 0.05)

0.0234

C(24:0)/C(24:1)

2.78 (1.32 - 4.63)

2.29 (1.35 - 4.02)

-0.25 (-0.47, -0.09)

0.0034

Ceramide score

8 (1 - 12)

10 (2 - 12)

1 (0, 2)

0.0508

Statin-exposed participants (n = 10)

C(16:0), µmol

0.31 (0.18 - 0.41)

0.31 (0.22 - 0.39)

-0.01 (-0.06, 0.05)

0.6738

C(18:0), µmol

0.14 (0.04 - 0.17)

0.12 (0.05 - 0.20)

0.01 (-0.02, 0.02)

0.4922

C(20:0), µmol

0.11 (0.06 - 0.14)

0.08 (0.06 - 0.13)

-0.01 (-0.024, -0.004)

0.0194

C(22:0), µmol

0.80 (0.67 - 1.19)

0.61 (0.54 - 0.94)

-0.14 (-0.21, -0.09)

0.0003

C(24:0), µmol

2.48 (1.88- 4.43)

2.22 (1.47 - 3.71)

-0.42 (-0.57, -0.27)

0.0039

C(24:1), µmol

1.04 (0.52 - 1.74)

0.90 (0.62 - 1.56)

-0.06 (-0.23, 0.06)

0.3398

Total C, µmol

4.88 (4.03 - 8.04)

4.04 (3.22 - 6.76)

-0.65 (-1.28, -0.09)

0.0098

C(16:0)/(24:0)

0.13 (0.07 - 0.17)

0.14 (0.09 - 0.20)

0.02 (0.002, 0.05)

0.0469

C(18:0)/(24:0)

0.06 (0.01 - 0.07)

0.06 (0.02 - 0.10)

0.01 (0.00, 0.02)

0.0547

C(24:1)/(24:0)

0.40 (0.15 - 0.67)

0.45 (0.19 - 0.62)

0.04 (0.00, 0.08)

0.0859

C(24:0)/C(24:1)

2.52 (1.49 - 6.60)

2.21 (1.61 - 5.15)

-0.30 (-0.76, -0.04)

0.0371

Ceramide score

8 (0 - 10)

7 (1 - 10)

1 (-3, 3)

0.6328

**Table 3.** Sensitivity and Specificity of Proposed Ceramide Cut-Off Values for Prediction of Risk and Severity of Stroke
	Time point 1 (LKW< 12 h)	Time point 2 (LKW > 24 h)
C: ceramide; AUROC: area under the receiver operating curve; LKW: last known well; NIHSS: National Institutes of Health Stroke Scale
A. Plasma ceramide levels and the risk of stroke
C(16:0)	0.68	87%	0%	0.70	86%	67%
C(22:0)	0.66	100%	0%	0.74	73%	67%
C(24:0)	0.42	64%	33%	0.50	54%	33%
B. Plasma ceramide levels and the severity of stroke (NIHSS ≥ 6)
C(16:0)	0.51	78%	31%	0.63	56%	23%
C(22:0)	0.74	78%	15%	0.76	44%	23%
C(24:0)	0.78	33%	15%	0.74	33%	31%

Table 3. Sensitivity and Specificity of Proposed Ceramide Cut-Off Values for Prediction of Risk and Severity of Stroke

Time point 1 (LKW< 12 h)

Time point 2 (LKW > 24 h)

AUROC

Sensitivity

Specificity

AUROC

Sensitivity

Specificity

C: ceramide; AUROC: area under the receiver operating curve; LKW: last known well; NIHSS: National Institutes of Health Stroke Scale

A. Plasma ceramide levels and the risk of stroke

C(16:0)

0.68

87%

0.70

86%

67%

C(22:0)

0.66

100%

0.74

73%

67%

C(24:0)

0.42

64%

33%

0.50

54%

33%

B. Plasma ceramide levels and the severity of stroke (NIHSS ≥ 6)

C(16:0)

0.51

78%

31%

0.63

56%

23%

C(22:0)

0.74

78%

15%

0.76

44%

23%

C(24:0)

0.78

33%

15%

0.74

33%

31%