Supplementary Figure 1: Sphingolipid Metabolism.
Enzymes in bold were analysed for gene expression in the VKC and
controls
Diagnosis of VKC and inclusion criteria for study recruitment:Patients with ocular itching, photophobia, presence of active
tarsal/limbal papillae, bulbar congestion, Horner Tranta’s dots or
superficial punctate keratitis were diagnosed as active VKC. The
quiescent stage was diagnosed based on inactive or flat-topped papillae,
a non -inflamed ocular surface with previous history of chronic itching.
All cases were included after a complete ophthalmic examination and
ruling out any other ocular or systemic morbidity or history of ocular
surgery. Tear specimen and conjunctival imprints were collected in cases
and controls, while blood sample was collected in a sub-set based on
consent of the study participant. All cases with active VKC and age
> 6 years were included in the study. Patients with history
of previous ocular surgery, ocular co-morbidity other than VKC, any
systemic disease other than allergic disorders; age < 6 years
and quiet eye at presentation were excluded from the study. On basis of
Bonini’s classification, patients with grade 1 and 2 (mild to moderate
form of seasonal VKC) were considered as non-refractory (NR-VKC) and
those with grade 3 and 4 (severe to very severe form of perennial VKC)
as refractory (R-VKC). Patients with refractory VKC were further
sub-grouped based on the duration and/ or use of topical steroid or
immunomodulator, (C) those who were on topical steroid/immunomodulator
<8 weeks; (D) those without steroid or immunomodulator, who
presented as fresh case with a repeat episode; (E) those who were on
steroid/immunomodulator > 8 weeks. Duration of 8 weeks was
considered so that the maximum effect of topical immunomodulator could
be assessed. All cases had active VKC at recruitment and were advised to
defer use of any topical on the day of collection. Non refractory and
fresh refractory cases (group D) were only on lubricants at presentation
whereas refractory (groups C and E) were on dual acting mast cell
stabilizer (Olopatadine 0.2% w/v, Alcon Laboratories, India) in
addition to lubricants and topical steroid / immunomodulator
(Fluoromethalone 0.1%w/v, Allergan India Private Limited/Tacrolimus
ointment 0.03% w/w, Aurolab, India).
LC-MS/MS analysis of sphingolipids in tear: The sphingolipid
standards were procured from Avanti Polar Lipids (Alabama, USA). Based
on calibration of the LC column and linearity check, the following
standards were finalized for the assay in pooled specimen of tear in
cases and controls LC-ESI-MS/MS experiments were performed using a
triple quadrupole tandem mass spectrometer (4000 Q-Trap, AB Sciex,
Foster City, CA, USA) coupled with high performance liquid
chromatography system (HPLC, Agilent Technologies, 1260 Infinity, Santa
Clara, CA, USA) that consisted of quaternary pump (G1311C),
multi-sampler (G7167A), HIBAR (30 x 2.1mm, 2µm) column compartment
(G1316A) with variable wavelength UV detector (G1314F) and online
degasser. All the parameters of tandem mass spectrometer and HPLC were
controlled by Analyst software, version 1.5.2 (AB Sciex, Foster City,
CA, USA) and Open LAB control panel software (Agilent Technologies, 1260
Infinity, Santa Clara, CA, USA), respectively.