The Biological Variability of Plasma Ceramides in Healthy Subjects

Abstract

Background

Ceramides are bioactive lipid species that mediate numerous cell-signaling events. Elevated plasma ceramides concentration constitutes a risk factor for several pathologies. Multiple studies have affirmed the plasma concentrations of 4 specific ceramides (Cer16:0, Cer18:0, Cer24:0, and Cer24:1) can predict cardiovascular disease risk. Furthermore, these ceramides can be altered by many lipid-lowering therapies. Understanding the biological variability within an individual, and within a population, will further inform the clinical use of plasma ceramides as a biomarker. In this study, we aimed to define the intra- and interbiological variability of ceramides in a healthy reference population in a weekly and monthly manner.

Methods

Fasting plasma from 24 healthy adults was collected daily (5 days), weekly (4 weeks), and monthly (7 months). Ceramide concentrations were measured with liquid chromatography–mass spectrometry (LC–MS). For analysis, we used random-effects regression models to estimate variance components.

Results

The analytical variability was smaller compared to the biological variability overall. The greatest variation reported was between-subject variation for all ceramide species. The critical difference-reference change value (RCV) for within-subject variations monthly were 0.07 mcmol/L (Cer16:0), 0.04 mcmol/L (Cer18:0), 1.09 mcmol/L (Cer24:0), and 0.27 mcmol/L (Cer24:1). The index of individuality (IOI) of ceramides were 0.82 (Cer16:0), 0.96 (Cer18:0), 1.06 (Cer24:0), and 0.89 (Cer24:1). The most consistent ceramide species was Cer18:0 with the lowest within- and between-subject critical differences in weekly and monthly measurements.

Conclusions

Overall, this study demonstrates that the variability of ceramide concentrations at different time points is minimal within individuals, allowing a single draw to be sufficient at least in a yearly time frame.

RCV, reference change value, atherosclerotic cardiovascular disease, sphingolipids

Impact Statement

With increasing available information of the role of ceramides in disease, it is important to establish biomarker parameters such as the biological variability between and within individuals. This study investigated ceramide biological variability in healthy individuals. Patients with conditions such as cardiovascular diseases, renal impairment, Type 2 diabetes mellitus, and nonalcoholic liver disease will benefit from a better understanding of ceramide variability and its potential use as a biomarker.

Introduction

Ceramides are bioactive lipid species that support the structure of the plasma membrane and mediate numerous cell-signaling events in eukaryotic cells. Ceramides are composed of a sphingosine base and an amide linked fatty acid chain varying in length. Ceramides are associated with cellular processes such as cell proliferation, apoptosis, migration, and growth. Under normal conditions, ceramide concentrations in the plasma membrane are very low (<1 mole %); however, on stress or stimuli, ceramide concentrations can increase up to 10-fold (1).

Elevations of plasma ceramides are associated with several cardiovascular disease risk factors such as renal impairment (2, 3) Type 2 diabetes mellitus, insulin resistance (4), hypertension (5), and nonalcoholic fatty liver disease (6). The primary emphasis has been on associations with atherosclerotic cardiovascular disease (ASCVD). Ceramide species N-palmitoyl-sphingosine (Cer16:0), N-stearoyl-sphingosine (Cer18:0), N-nervonoyl-sphingosine (Cer24:1), and N-lignoceroyl-sphingosine (Cer24:0) have been repeatedly identified as key biomarkers for predicting adverse ASCVD events (i.e., heart attacks, strokes) (7, 8). Associations between adverse cardiovascular events and ceramides has been repeatedly confirmed in cohorts with known ASCVD (2, 3, 7, 9–12) and in general population cohorts (13–17).

Given that ceramide concentrations are used to assess ASCVD risk, understanding the biological variability over time within an individual, and within a population of individuals, would inform clinical decision-making. Understanding the reference change value (RCV) or critical difference within-subjects reported will help determine the expected variation in plasma ceramides between serial measurements. Accordingly, this study was designed to define the intra- and interbiological variability of ceramides in a healthy reference population over a frequency of days, weeks, and months.

Methodology

Subject Characteristics

The participant characteristics are outlined in Table 1. The study cohort included 24 healthy adults, 10 males, and 14 females. Participants were matched for age and BMI between females and males. Individuals with diabetes or cardiovascular disease, prescription drug use for lipid lowering and/or hypertension, pregnancy, current smokers, blood pressure ≥130/85 mmHg, fasting glucose ≥100 mg/dL, triglycerides ≥150 mg/dL, HDL cholesterol <40 mg/dL (males) or <50 mg/dL (females), and/or waist circumference >101 cm (males) or >89 cm (females) were excluded. All participants provided written informed consent and the study was approved by the Mayo Clinic Institutional Review Board. The median (range) age and weight of participants was 30 (22–47) years and 71.8 (55.1–99.3) kg, respectively. BMI median and range were 25.04 (19.70–31.20). There was no significant difference in age between male (median 30.5, range 22–36) and female (median 30.0, range 24–47) participants (P = 0.95, Wilcoxon rank-sum test).

Table 1

Subject characteristics.

	Females (n = 14)1	Males (n = 10)1	P value
Age (years)	32 ± 2	31 ± 2	0.59
BMI (kg/m2)	24.9 ± 0.9	25.5 ± 0.7	0.68
Waist circumference (cm)	82.2 ± 1.4	89.9 ± 1.5	0.001*
Apolipoprotein A1 (mg/dL)	163.9 ± 7.9	142.2 ± 6.7	0.82
Apolipoprotein B (mg/dL)	76.0 4.9	81.3 ± 5.8	0.53
Total Cholesterol (mg/dL)	169.1 ± 9.6	170.9 ± 10.5	0.91
Triglycerides (mg/dL)	80.9 ± 10.1	91.4 ± 14.2	0.57
HDL-C (mg/dL)	62.2 ± 3.7	52.2 ± 3.5	0.09
LDL-C (mg/dL)	90.7 ± 7.0	100.4 ± 7.9	0.41
Cer16:0 (mcmol/L)	0.25 ± 0.05	0.25 ± 0.04	0.97
Cer18:0 (mcmol/L)	0.08 ± 0.05	0.07 ± 0.02	0.31
Cer24:0 (mcmol/L)	2.97 ± 0.54	3.34 ± 0.62	0.05*
Cer24:1 mcmol/L)	0.71 ± 0.14	0.74 ± 0.16	0.61

1

Values are means ± SEMs, unless otherwise noted, for subject characteristics.

Plasma Collections and Ceramide Analysis

Plasma-EDTA from fasting (≥8 h) participants was collected between 6 AM and 9 AM daily for 5 days. Fasting samples were drawn weekly for 4 weeks, and monthly for 7 months. Plasma aliquots were stored frozen at −80 °C for 6 years before testing. Specimens were allowed to thaw for one hour before preforming lipid extraction and underwent one previous freeze–thaw cycle. Plasma ceramides were quantitated using liquid chromatography–tandem mass spectrometry as previously described (3, 18). Briefly, plasma was diluted in ethyl acetate and isopropanol (20:80 v:v) with 0.1% formic acid before addition of deuterium-labeled internal standards (Avanti Polar Lipids, Alabaster, AL). To minimize analytical variation, samples were prepared, and internal standards were added using Perkin Elmer JANUS automated liquid handling system. Ceramides were separated using Thermo Scientific Dionex Ultimate 3000 Series, TLXII Liquid Chromatography System (column heated) and detected using Sciex API 6500 MS/MS (AB Sciex, Framingham, MA). Analytical variability was determined based on the variation of pooled plasma measured by 22 unique extractions over 11 weeks. The mean (SD) concentration was 0.21(0.01) mcmol/L for Cer(16:0), 0.06(0.01) mcmol/L for Cer(18:0), 2.96(0.15) mcmol/L for Cer(24:0), and 0.68(0.03) mcmol/L for Cer(24:1).

Calculations and Statistical Methods

Age was compared between males and females with a Wilcoxon rank-sum test. Average fasting ceramide concentration by gender was analyzed using linear regression with generalized estimating equations to account for within-subject correlation among the repeated observations, separately for daily, weekly, and monthly measurements. The coefficient of variation (CV) was determined based on each source of variability [between-subject, within-subject across months, and analytical (estimated with the 5 daily fasting specimens collected within the first week of month 1)] using the same methods as used in previous work by the group Donato et al. (18) and adapted from Khuseyinova, et al. (19). Briefly, we used random-effects regression models to estimate each variance component, and each estimate of variability was computed from these components. The reported estimates include the variance and SD, CV, and proportion of total variance (between-subject, within-subject across months, and analytical), along with the critical difference, intraclass correlation coefficient (ICC), and index of individuality (IOI) (18). The probability (Z) used in the critical difference calculation was 1.96. The within-subject critical difference is regarded as the RCV. All analyses were conducted using SAS v.9.4 (SAS Institute, Inc.). The risk score combined all ceramide measures into a whole integer between 0 and 12 and was derived from 352 participants with and without cardiovascular disease (2, 3).

Results

Participant characteristics are shown in Table 1. An average of 4.9 daily fasting samples were collected per individual during the first week (21 subjects had all 5 daily fasting samples, 3 participants had 4 daily fasting samples). An average of 3.7 fasting samples were drawn weekly over 1 month (17 participants had all 4 samples, 7 had 3 samples) and 5.2 fasting samples were drawn monthly over 7 months (16 participants had 6–7 samples, 5 had 5 samples, and 3 had <5 samples). No outliers were noted, and the results were approximately normally distributed.

The clinically established reference intervals for this ceramide method are as follows, Cer16:0, 0.19–0.36 mcmol/L, Cer18:0, 0.05–0.14, Cer24:1, 0.65–1.65 mcmol/L (3) In this study, ceramide concentrations ranged as follows, Cer16:0, 0.16–0.37 mcmol/L, Cer18:0, 0.03–0.14 mcmol/L, Cer24:0, 1.5–4.9 mcmol/L, and Cer24:1, 0.4–1.1 mcmol/L for all measurements combined (Fig. 1). There was a slight sex difference in the ceramide Cer24:0 species for daily measurements (P = 0.049), with males having slightly higher average ceramide (average difference 0.5 mcmol/L). None of the other ceramides showed a sex difference for the daily, weekly, or monthly measurements.

Fig. 1

Plasma ceramides were measured serially over 7 months in a cohort of healthy volunteers (n = 24). Concentrations are in mcmol/L (x axis), patient number (y axis), and error bars indicate range. (black = male, gray = female).

The average within-subject CV results from daily; weekly; and monthly analyses were 8.49%, 8.79%, and 8.34% [Cer16:0]; 18.10%, 18.63%, and 18.61% [Cer18:0]; 8.14%, 11.77%, and 11.41% [Cer24:0]; and 10.73%, 11.16%, and 12.67% [Cer24:1], respectively. Within-subject variation for all ceramide species ranged from <0.01 to 0.09 mcmol/L, showing negligible differences for all ceramide species at daily, weekly, and monthly measurements except for Cer24:0 (0.26–0.37 mcmol/L) (Table 2). Between-subject variability was observed to be higher for Cer24:0, however, the percentage CV was higher for Cer18:0. Overall between-subject variability results may discourage the use of population reference intervals. Monthly variability estimates are shown in Fig. 1. The IOI is a measure of the capability of detecting unusual individual results, and an IOI ≤0.6 may indicate low utility in the reference values for detecting unusual individual results (19). Generally, the IOI in the data was >0.6 for all measures. The ICC is a useful tool for understanding the correlation status of measurements within a variability source. ICC spans from 0–1, ICC closer to 1 indicates a higher correlation for a particular variability source. Our data set showed ICCs of 0.54–0.96, indicating positive correlation.

Table 2

Monthly biological variability of plasma ceramides.

Ceramide species	16:0	18:0	24:0	24:1
Samples, N	233	232	233	233
Subjects, N	24	24	24	24
Overall mean	0.25	0.07	3.16	0.72
Within-subject variation
Daily; SD (CV%)	0.02 (8.49)	0.01 (18.10)	0.26 (8.14)	0.08 (10.73)
Weekly; SD (CV%)	0.02 (8.79)	0.01 (18.63)	0.36 (11.77)	0.08 (11.16)
Monthly; SD (CV%)	0.02 (8.34)	0.01 (18.61)	0.37 (11.41)	0.09 (12.67)
Between-subject variation
Weekly; SD (CV%)	0.04 (16.83)	0.14 (20.61)	0.55 (18.09)	0.12 (17.53)
Monthly; SD (CV%)	0.04 (16.41)	0.02 (26.43)	0.50 (15.75)	0.14 (19.63)
Analytical variation
Weekly; SD (CV%)	0.01 (4.91)	0.005 (6.78)	0.15 (4.79)	0.03 (4.72)
Monthly; SD (CV%)	0.01 (4.77)	0.005 (6.66)	0.15 (4.58)	0.03 (4.76)
Critical difference—RCV
Weekly within subject (RCV, ICC)	0.07,0.74	0.04, 0.52	1.08,0.67	0.24,0.68
Monthly within subject (RCV, ICC)	0.07,0.74	0.04, 0.64	1.09,0.62	0.27,0.68
Analytical—weekly (RCV, ICC)	0.03,0.94	0.01, 0.94	0.41,0.95	0.10,0.95
Analytical—monthly (RCV, ICC)	0.03,0.94	0.01, 0.96	0.41, 0.95	0.10,0.96
IOI
Weekly	0.81	1.23	0.92	0.91
Monthly	0.82	0.96	1.06	0.89

Abbreviations: standard deviation (SD), coefficient of variation percentage (CV), critical difference (RCV), intraclass correlation coefficient (ICC), and index of individuality (IOI).

Discussion

These data provide index information to guide clinicians when interpreting plasma ceramides clinically and support the practice of serial measures to follow the trajectory of patients with cardiovascular disease. In aggregate, they suggest overall that values are reasonably stable over time. This suggests that when therapies are applied, the likelihood that most changes are attributable to the biological effects of the intervention is high. This is in contrast to analytes such as natriuretic peptides where conjoint analytical and biological variation is quite high, making interpretation of small changes problematic (20).

In this cohort, the RCV for all ceramides was lower than the difference of within-subject measurements. The percentage of variation shows the overall distribution, along with the %CV, the greatest variation reported comes from between-subject variation for all the ceramide species. The analytical variability was smaller compared to the biological variability. Overall, we found no sex differences in the daily, weekly, and monthly measurements of all ceramide species, except for Cer24:0 with males showing a slightly higher concentration. According to our measurements, the most consistent ceramide species was Cer16:0 with the lowest within- and between-subject CV% in the daily, weekly, and monthly measurements.

A potential limitation of the study was that ceramide concentrations were generally low. This is expected since the cohort was selected because the subjects were considered healthy. Thus, these data might not be representative of a clinical population. However, it is unclear how accurately one can measure variation over months in a diseased population, especially when the disease can at times be very overt (21). An additional limitation of the study was the storage of samples before ceramide measurement. In-house studies have established that freeze–thaw and 30 days frozen storage do not appreciably alter plasma ceramide measures, however, the potential influence of 6 years at −80 °C has not been evaluated.

The highest percentage of variation was seen from between-subject variation. Historically a high IOI >1.4 was said to favor the use of population-based reference intervals. However, more recent reports have disputed this convention and suggested that IOI has limited impact on the utility of population-based reference intervals (22) In any case, the IOI for ceramides measured was intermediate (>0.6 but <1.4).

The within-subject variation in our cohort showed minimal percentage of variation, this allows us to report a very low variability of all the species within-subjects. The within-subject critical difference results indicate that for a stable patient 95% of measured ceramide concentrations will be within 0.07 mcmol/L for Cer16:0, 0.04 mcmol/L for Cer18:0, and 0.24 mcmol/L for Cer24:1. These observations are particularly relevant given reports that aerobic exercise (23) and lipid-lowering pharmaceuticals such as simvastatin, rosuvastatin, and PCSK9 inhibitors reduce ceramide concentrations by >0.1 mcmol/L for Cer16:0, 0.05 for Cer18:0, and >1.00 mcmol/L for Cer24:1 (7, 23, 24, 25).

Overall, this study demonstrates that ceramide concentrations vary minimally within healthy individuals over time, allowing a single draw to be sufficient in stable patients for at least 1 year.

Nonstandard Abbreviations: Cer: ceramide; ASCVD: atherosclerotic cardiovascular disease; RCV: reference change value; ICC: intraclass correlation coefficient; IOI: index of individuality

Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 4 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; (c) final approval of the published article; and (d) agreement to be accountable for all aspects of the article thus ensuring that questions related to the accuracy or integrity of any part of the article are appropriately investigated and resolved.

Authors’ Disclosures or Potential Conflicts of Interest: Upon manuscript submission, all authors completed the author disclosure form. Disclosures and/or potential conflicts of interest:Employment or Leadership: None declared. Consultant or Advisory Role: A.S. Jaffe, Abbott, Siemens, Roche, Beckman-Coulter, Ortho Diagnostics, Radiometer, ET Healthcare, Sphingotec, Astellas, Amgen, Novartis, and Medscape. Stock Ownership: None declared. Honoraria: None declared. Research Funding: None declared. Expert Testimony: None declared. Patents: None declared. Other Remuneration: S.M. Jenkins does not receive any payments directly but works for a department that performs statistical analysis on a charge-out basis, and all funds are handled internally in the institution.

Role of Sponsor: No sponsor was declared.