Structural Differences in Meibum from Donors after Hematopoietic Stem Cell Transplantations

Abstract

Purpose

Meibum is considered to be a key component of tears which serve to protect the eye and conformational changes in meibum have not been studied extensively within the population of patients that had hematopoietic stem cell transplantation (HSCT). The aim of this study was to determine possible lipid conformational changes in the meibum of patients that had HSCT.

Methods

Participants who had HSCT were randomly sampled for this prospective comparative study. Control participants did not have dry or had not undergone allogenic or autologous stem cell transplantation. Fourier transform infrared spectroscopy was used to measure meibum phase transition.

Results

Meibum was collected from both eyes of 36 donors without dry eye (M_c), and from 22 patients who had undergone HSCT (M_HSCT). There were no significant differences between the phase transition parameters based on gender or race. Significant differences (P < 0.0001), between the parameters for M_c compared with M_HSCT are: lipid order (% trans) at 33.4 °C increased from 40 (1) to 54 (2); cooperativity decreased from 7.9 (0.4) to 5.4 (0.3); the phase transition temperature (°C) increased 30.3 (0.4) to 34.2 (0.9); the magnitude of the phase transition (cm⁻¹) increased 4.0 (0.1) to 4.7 (0.5). (standard error of the mean).

Conclusions

Conformational and thermodynamic differences were observed between M_c and M_HSCT. The changes observed in the lipid conformation of meibum from patients receiving hematopoietic stem cell transplantations suggest that meibum composition changes after stem cell transplantation and clinicians should consider treating the meibomian glands to improve ocular surface.

INTRODUCTION

Autologous and Allogeneic Hematopoietic Stem Cell Transplantations (HSCT) have been important treatment options for various types of leukemias and lymphomas. Every year, there are over 20,000 hematopoietic stem cell transplantations performed within the United States and this number continues to grow annually.¹ Unfortunately, graft-versus-host disease (GvHD) is a common and potentially fatal complication despite improvements in immunosuppressive therapies and better human leukocyte antigen typing. The prevalence of GvHD in patients who receive HSCT is estimated to be between 30% and 70%.²

GvHD manifests when donor CD4+ and CD8+ T cells interpret the recipient’s histocompatibility antigens as foreign and mount an immunological reaction against them.³ Acute GvHD tends to occur within 100 days of the HSCT and can affect any part of the human body. However, the disease most commonly affects the skin, mouth, eye, gastrointestinal tract, liver, lung, joint and fascia, and genital tract.^3–5 These sites are then used to calculate a global score indicating the severity of disease.⁵ Chronic GvHD tends to occur 100 days after HSCT, and can be a continuation of acute GvHD, or can occur insidiously.³

Ocular derangements, including dry eye occur in 40% to 60% of patients with GvHD.^6–10 Typical disease presentation can range from meibomian gland disease (MGD) and scarring of the lacrimal gland to corneal erosions from chronic inflammation and aqueous deficiency dry eye (ADDE).¹⁰ Meibomian gland loss increases in patients with ocular GvHD and could serve as a predictor for the development of ocular GvHD.¹¹

Meibum becomes more ordered (stiffer) with age^12–15 and MGD.^16,17 Lipid order correlated with tear film stability and it is plausible that more ordered meibum could block the meibomian glands and form aggregated ‘islands’ of lipid on the tear film surface impeding the spreading of the tear film lipid layer which could contribute to tear film instability.¹⁵ In this study, meibomian lipid conformation with temperature was measured using Fourier transform infrared (FTIR) spectroscopy. Conformation is the arrangement of atoms in a molecule that differ by rotation about a single double bond and is important to the order (fluidity), strength of lipid-lipid interactions, and lipid structure in general.

The purpose of this study was to compare the physical properties of meibum from donors with HSCT (M_HSCT) to meibum from donors with MGD (M_MGD) and donors without dry eye (M_C). This study could help elucidate the relationships between tear lipid composition, structure and function to understand the pathogenesis of HSCT and also in future to see if changes in the Meibomian composition could serve as a predictor of ocular HSCT.

MATERIALS AND METHODS

In this prospective comparative study, we analyzed both eyes from all donors. Written informed consent was obtained from all donors and protocols and procedures were approved by the University of Louisville Institutional Review Board # 11.0319, August, 2016. All procedures were in accordance with the Declaration of Helsinki.

Human meibum samples were collected from participants recruited from the Kentucky Lions Eye Center and the James Graham Brown Cancer Center in Louisville, Kentucky. Participants were assigned to the cohort Cc when the patient’s Meibomian gland orifices showed no evidence of keratinization or plugging with turbid or thickened secretions and no dilated blood vessels were observed on the eyelid margin. The participants did not recall having dry eye symptoms. Participants were assigned to the cohort C_AHSCT if they had undergone AHSCT. Patients with GvHD were diagnosed by medical oncologists. Patients in C_HSCT underwent a full ophthalmic eye exam using slit lamp biomicroscopy. Tear film break up time was measured at the slit lamp after instillation of one fluorescein drop. The diagnosis of dry eye was based on the clinical examination results, including fluorescein stain uptake of the cornea or conjunctiva, irregular tear film, low tear meniscus, as well as symptoms. Symptoms that were considered positive included foreign body sensation, excessive tearing, excessive blinking, burning of eyes or blurry vision. The Schirmer’s test was performed on all patients by placing a standard strip in the lower conjunctival sac without anesthesia for 5 minutes. Meibomian gland orifices, eyelid changes at the mucocutaneous junction and expression of meibum by gentle pressure were all evaluated for diagnosis of MGD. The third cohort (C_MGD) consisted of patients that had not undergone AHSCT but had clinical MGD.

Meibum was collected and lipid phase transitions were measured as described previously.¹⁷ Curves were fit using Sigma plot 10 software (Systat Software, Inc., Chicago, IL, USA) and the confidence levels, were obtained from a critical value table of the Pearson product–moment correlation coefficient. Averages were compared using the Student’s t test. A value of P < 0.05 was considered statistically significant. Two of the phase transition parameters, the minimum and maximum vibrational frequency of the C-H symmetric stretch (ṽ_sym), correspond to the most ordered and disordered states of hydrocarbon chains, respectively. Another parameter was the phase transition temperature, which is the temperature at which half of the lipid molecules undergo a change from the gel to liquid crystalline phase. The relative cooperativity of the phase transition describes how the order of a lipid influences that of neighboring lipids. Broad phase transitions have a relatively smaller absolute value of the cooperativity. Lipid order was calculated at 33.4 °C the temperature at the surface of the eye and 36 °C the temperature of the eyelid. Data are reported as the mean plus or minus the standard error. Significance between groups was tested using the Student’s t test.

RESULTS

The participant donor demographics are listed in Table 1. The Cc contained more males and Hispanics than C_HSCT (Table 1). Their average visual acuity was 20/38 ± 6 and their average Schirmer score was 14 ± 1.6 mm.

Table 1.

Donor demographics and phase transition parameters.

				P
Parameter	Cc	CAHSCT	CMGDa	Cc verses CAHSCT	Cc verses CMGD	CMGD verses CAHSCT
Average Age (y)	35 (4)	45 (5)	66 (2)	> 0.05
Age Range (y)	13 to 88	13 to 81	8 to 87
Gender (% male)	74	44	65
Race (%)	C (75), B (5.6), A (11), H (5.6) ? (2.8)	C (84), B (12) A (4)	C (77), B (12.5), H (2.1) ? (8.3)
Tm	30.3 (0.4)	34.2 (0.9)	32.2 (0.6)	< 0.0001*	0.016*	> 0.05
Cooperativity (Hill coefficient)	7.9 (0.4)	5.4 (0.3)	9.0 (0.4)	< 0.0001*	> 0.05	< 0.0001*
Order 36.0 °C (% trans)	35 (1)	48 (2)	40 (2)	< 0.0001*	0.01*	0.01*
Order 33.4 °C (% trans)	40 (1)	54 (2)	44 (1)	< 0.0001*	0.017*	0.001*
Δ enthalpy (kcal/mol)	142 (6)	121 (7)	153 (7)	0.016*	> 0.05	0.0018*
Δ entropy (kcal.mol/degree)	0.48 (0.02)	0.39 (0.02)	0.49 (0.02)	0.0026*	> 0.05	0.003*
Magnitude (cm−1)	4.0 (0.1)	4.7 (0.5)	3.8 (0.6)	< 0.0001*	> 0.05	< 0.0001*
Minimum Frequency (cm−1)	2849.71 ( 0.06)	2849.33 (0.06)	2849.68 (0.06)	< 0.0001*	> 0.05	0.0006*
Maximum Frequency (cm−1)	2853.69 (0.09)	2854.00 (0.09)	2853.48 (0.08)	> 0.05	> 0.05	0.0004*
Δ Order 33.4 °C - 36.0 °C (% trans)	4.8 (0.2)	5.4 (0.2)	5.9 (0.3)	0.04*	0.001*	> 0.05
Number of Participants	36	22	49

(SEM), C = Caucasian; H = Hispanic; A = Asian; B = African American; X = unknown race; numbers are age. Meibum was collected only once from each donor.

^aFrom reference 17.

^*Significant difference, P < 0.05.

The frequency of the symmetric CH₂ stretching band near 2850 cm⁻¹ (ṽ_sym) was used to estimate the trans to gauche rotamer content of the hydrocarbon chains (Fig. 1 A and B),¹⁷ and it increased with an increase in temperature (Fig. 1C) concurrent with a decrease in intensity.^17,18 Lipid order was related to the phase transition temperature (Fig. 1D).

FIGURE 1.

A) Infrared spectra of the CH stretching region at 22 °C of a typical 36 year-old Caucasian adolescent female without dry eye (top), and a typical 31 year-old Caucasian adolescent female with dry eye and Graft-versus-host disease (bottom).

B) Schematic showing trans and gauche conformations in lipid hydrocarbon chains. The greater the number of trans rotamers, the tighter the lipids pack, the stronger the van der Wall’s forces, the greater the lipid order and higher the lipid phase transition temperature.

C) Typical lipid phase transitions of meibum from a 13 year-old Caucasian adolescent female without dry eye (●) and a 14 year-old Caucasian adolescent female with dry eye and Graft-versus-host disease (○). The CH₂ symmetric stretching frequency is related to lipid structural order. The higher the value for the frequency, the more disordered the lipid. (─) Curve fit of data using the sigmoidal equation 1 (Experimental Section).

D) Correlation between the lipid phase transition temperature and lipid order at 33.4 °C for human meibum. (●) Meibum from donors without dry eye. (o) Meibum from donors with dry eye and graft versus host disease. (★) Meibum from donors with meibomian gland dysfunction. Data are average ± the standard error of the mean.

Lipid phase transition parameters for human meibum are listed in Table 1. Key differences in the phase transition parameters are shown in Figure 2. Arrhenius plots used to calculate the ∆H and ∆S values from the lipid phase transitions were linear, with correlation coefficients greater than 0.998 (data not shown). There were no significant differences between the phase transition parameters and gender or race in any of the cohorts.

FIGURE 2.

Key lipid phase transition parameters from Table 1. All bars are statistically different from each other, P < 0.05 (Table 1). Data are average ± the standard error of the mean.

Due to the small sample size, there was no statistical difference, P > 0.4, between the phase transition parameters of M_GvHD and M_{w/o GvHD} or between M_GvHD classified as acute or chronic, although the trend in lipid order is worth noting M_{w/o GvHD} (49 % ± 6, n = 5) < acute M_GvHD (51 ± 7, n = 4) < chronic M_GvHD (53 ± 3, n = 8).

DISCUSSION

The current study was conducted as a step to elucidate meibum compositional, structural and functional relationships following hematopoietic stem cell transplantation. The major finding of the current study, one of the largest structural studies of meibum to date, was that the order or stiffness of M_HSCT was higher than that of any meibum measured to date and significantly much greater compared with M_C. It is plausible that more ordered meibum could block the meibomian glands that could cause inflammation and form aggregated ‘islands’ of lipid on the tear film surface impeding the spreading of the tear film lipid layer which could contribute to tear film instability.

Lipid order and dry eye

Lipid order is a measure of the structural ‘fluidity’ of the hydrocarbon chains of a lipid. More ordered lipids are solid like butter, whereas lipids that are more disordered are fluid and liquid like olive oil. There are more trans rotamers in the hydrocarbon chains of ordered lipids which result in hydrocarbon chains that are straight. The straight chains allow the lipids to pack closely together with increased Van der Waal’s interactions between the lipids. Disordered lipids have bends in their hydrocarbon chains so that they do not pack tightly together and Van der Waal’s interactions between the hydrocarbon chains are minimal (Figure 2B).³⁵ The lipid order of M_HSCT and M_MGD were significantly higher compared with the lipid order for M_c. The higher lipid order of M_HSCT and M_MGD compared with M_c suggests that a higher level of M_MGD and M_HSCT saturation compared with M_c could contribute to this observation.^14,29

With dry eye, M_MGD and M_HSCT were significantly more ordered (stiff) at the temperature of the eyelid (36 °C) compared with M_c. It has been speculated that a stiffer lipid would decrease the amount of meibum on the eyelid causing a decrease in the tear film thickness leading to the instability of the tear film and it has been suggested that at some point, “when the reservoir of meibum on the lid reaches a critically low amount, there will not be enough to cover the tear film.”³⁰ However, published data does not support this idea as on the lid margin there was 42% less M_c compared with M_MGD and meibum from donors with dry eye related to seborrhea and the tear film lipid layer is thicker with dry eye.³¹ Thus, more ordered stiffer meibum with dry eye is associated with more meibum on the lid surface and a thicker tear film lipid layer. This suggests that tear film stability is inversely related to tear film thickness or tear film stability is affected by the quality not the quantity of tear film lipid. It is interesting that babies and children, that have more stable tear films have much more fluid meibum compared with adults and people suffering from dry eye.¹⁵ It has been suggested, based on in vitro rheology that “a more fluid meibum from children and babies compared with meibum from adults reiterates higher stability in their films which spread better, resist deformation, and facilitates their ability to be quickly restored after blinking.”¹⁵ A similar rationale could be made for the increase in fluidity of meibum on the surface of tears at 36 °C, compared with meibum that is more stiff or ordered in the meibomian glands at 33.4 °C.

Between birth and adulthood¹⁵ and with dry eye, ^{16,17 and the current study} the lipid becomes significantly more ordered, an exacerbation of aging. A slight decrease in order as with age could result in a more stable tear film as discussed above, however, above a certain point, one may speculate that the lipid becomes too ordered, causing it to aggregate, and it does not spread on the tear film surface resulting in an unstable tear film. This could occur above a lipid order of 40 % trans at 36 °C. The elevated lipid order with dry eye may be more than circumstantial as C_MGD have elevated levels of lipid order and when their signs and symptoms of dry eye are ameliorated with treatment, their levels of lipid order were lower.³² Although lipid order could contribute to tear film instability with dry eye and is a good marker for it, other factors could contribute to tear film instability such as: protein and contaminants in the tear film^16,22,34 squalene levels,³³ which does not affect meibum lipid order,³⁵ sebum,²⁰ differences between the lipid composition of tears and meibum,^20,35–38 and cholesterylester content^39,40 which does not affect the order of waxes.¹⁷ The combination of elevated % trans rotamers and hydrocarbon chain melting temperature and decrease in cooperativity, as discussed below, suggests gross changes in the structure and properties of meibomian films at the air/tear surface in vivo. In particular, it suggests formation of discontinuous patchy tear film lipid layers which in turn will results in deteriorated spreading,⁴² decreased surface elasticity and attenuated capability to restore its structure between blinks. This topic has been reviewed.⁴³

Lipid phase transition temperature

As temperature increases, lipids go from an ordered state to a disordered state. The term ‘phase transition’ is used rather than melting, because at low temperatures, lipids are not completely ordered and are considered to be in a gel phase. At high temperatures, lipids are not completely disordered and are considered to be in a liquid crystalline phase. Thus, rather than use the term ‘melting’, biophysicists prefer to use the term ‘gel to liquid crystalline phase transition’ to describe the melting of lipids. The lipid phase transition temperature is the temperature in which half the lipids undergo a phase transition. This study shows that the phase transition temperature and lipid order at physiological temperatures, discussed in the section above, are directly related. With dry eye, M_MGD and M_HSCT had significantly higher phase transition temperatures compared with M_c. It has been proposed that if a therapy could be developed that decreases the lipid phase transition temperature, the meibum would be more fluid and flow out of the meibomian gland.²⁸

Lipid phase transition cooperativity

The relative cooperativity of the phase transition describes how the order of a lipid influences that of neighboring lipids. Broad phase transitions have a relatively smaller absolute value of the cooperativity. The cooperativity of the phase transition of M_HSCT was significantly lower than that for M_c. Cooperativity (Hill coefficient) is related to how the melting of one lipid influences the melting of another lipid and the cooperative unit size.²² The compositional differences between M_C and M_HSCT that contribute to lower cooperativity have yet to be determined. The homogeneity of the meibum lipid contributes to cooperativity as the phase transition cooperativity of pure waxes are orders of magnitude greater than that of meibum.^17,40 The phase transitions for mixtures of pure waxes are less cooperative than for pure waxes.^17,41 Unsaturation,¹⁴ contaminants and proteins could also lower the cooperativity.¹⁶ The lower cooperativity of the phase transition of M_HSCT compared with M_c indicates that M_HSCT is more heterogeneous, perhaps containing more contaminants and breakdown products compared with M_c.

Lipid phase transition magnitude

The magnitude of the phase transition was lower in M_c compared with M_HSCT. The magnitude of the phase transition is related to the number of trans rotamers that convert to gauche rotamers during the phase transition. Double bonds, hydrocarbon chain branching, hydroxyl moieties and shorter chain lengths could contribute to a smaller phase transition magnitude. As all of these factors could contribute to or be a marker of dry eye. Future studies to quantify the moieties responsible and to determine their effect on structure are warranted.

Differences in the phase transition parameters between M_MGD and M_AHSCT

There were significant differences between the phase transition parameters of M_MGD and M_HSCT. The phase transition cooperativity and change in enthalpy and entropy were higher and the magnitude lower in M_MGD compared with M_HSCT due to compositional differences. M_MGD is from donors with only MGD and no ADDE, whereas M_HSCT is mostly from donors with MGD and ADDE. As both M_HSCT and M_MGD are from donors with a MGD component, the differences between their phase transition parameters could be due to aqueous deficiency or systemic effects due to HSCT. A phase transition study of meibum from donors with only ADDE could help to determine the contribution of the aqueous deficient component.

A limitation of the current study is that the composition of the meibum was not measured so compositional-structural correlations could not be made. Compositional data on the samples used in this study are being calculated from nuclear magnetic resonance spectra and will be presented in another publication. Both compositional and structural differences could impact the function of meibum. The changes observed in the lipid conformation of meibum from patients receiving hematopoietic stem cell transplantations suggest that clinicians should consider treating the meibomian glands to ameliorate dry eye. Future studies into the meibomian composition could shed light into the pathophysiology of GVHD and could also potentially serve as an indicator for systemic response to anti-rejection treatment.