Abstract
Abstract
Purpose:
To determine the possibility of plasma citrate as a biomarker in patients with glaucoma.
Methods:
Twenty-one consecutive Caucasian patients with glaucoma and 21 sex- and age-matched controls were investigated. Plasma citrate, plasma creatinine, urine citrate, and urine creatinine were analyzed by ion chromatography. Mean (±standard deviation) concentrations and the calculated fractional citrate excretions were compared using the Mann–Whitney test. Sensitivity and specificity to detect glaucoma using plasma citrate levels were calculated.
Results:
The mean plasma citrate (104.8±23.2 vs. 128.2±31.1 μmol/L; P=0.01) concentrations were significantly lower among the patients with glaucoma, whereas the mean urine citrate concentrations (1.7±0.9 vs. 2.8±1.9 μmol/L; P=0.07) were slightly lower. Mean plasma and mean urine creatinine concentrations showed no significant differences (plasma creatinine: 63.0±16.7 vs. 63.4±15.5 μmol/L; P=0.72; urine creatinine: 9.6±5.1 vs. 11.5±8.4 μmol/L; P=0.67). The calculated fractional citrate excretions were also not different with 12.1% versus 13.6% (P=0.37). Setting the cut-off limit at 110 μmol/L, the plasma citrate level evaluation would have a sensitivity of 66.7% and a specificity of 71.4% to detect glaucoma.
Conclusion:
In this masked study, plasma citrate levels were significantly decreased in Caucasian patients with glaucoma giving the possibility to use them eventually as a biomarker. The kidney function was normal in both groups, leaving the etiology of this hypocitraemia yet unexplained.
Introduction
Molecular diagnostics rely on the analysis of biological processes at the molecular level by utilizing biomarkers. The FDA defines a biomarker as a characteristic, objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacological responses to therapeutic interventions. Biomarkers can change qualitatively (mutation/s) or quantitatively (expression level). Known mutations are involved in a negligible minority of patients with glaucoma. Therefore, searching for compounds with quantitative approaches and comparing their expression level might be more promising in terms of potential screening and diagnostic purposes in glaucoma.
So far, no biomarker is available for the detection of glaucoma. Accidentally, we found low levels of citrate in the plasma of a glaucoma patient. The aim of this study was therefore to verify the hypothesis that patients with glaucoma have lower plasma citrate levels, and to determine prospectively the plasma and urine citrate levels in patients with glaucoma and evaluate its possible use as a biomarker.
Methods
Study subjects
Twenty-one Caucasian patients with glaucoma referred to the Department of Ophthalmology of the University Hospital Basel, Switzerland, and 21 sex- and age-matched healthy persons were recruited. The study design was set up as a prospective, clinical, nonrandomized study, approved by the local ethics committee. Written informed consent was obtained from all participants before entry into the study. The study was designed and conducted in accordance with the tenets of the Declaration of Helsinki. The study was registered at ClinicalTrials.gov (NCT00804063) before its start.
In all patients and controls, best spectacle corrected visual acuity (BSCVA), automated Octopus program G2 perimetry visual field (VF), and intraocular pressure (IOP) were obtained. Gonioscopy and papillary morphology was determined and the dietary habits (eg, vegetarism and alcohol), systemic and topical medications, and tobacco consumption noted.
Patients or controls under the age of 18 years, with active ocular inflammatory disease, isolated ocular hypertension, severe kidney disease, intake of diuretics or drugs affecting kidney function, mitochondrial disease, or severe alcohol or drug abuse were excluded.
Glaucoma was defined as typical glaucomatous optic nerve head cupping, treated or nontreated IOP ≥21 mm Hg and confirmed on at least 2 VF showing typical glaucomatous visual field defects.
Citrate analysis
Twenty milliliters of fasting-blood was drawn in the morning (7 a.m.) as well as a urine sample taken at the same time. The fresh blood was immediately centrifuged (3,000 rpm) and the plasma and urine stored at −80°C until analysis. The anonymized probes were analyzed by ion chromatography in the central laboratories of the Bruderholzspital, Basel, as follows.
The measurement of citrate in urine was done on a Metrohm 761 Compact IC Ion chromatography system with a Metrosep A Supp4 column, using sodium carbonate/sodium hydrogen carbonate eluent with flow gradient, chemical suppression, and conductivity detection. The Metrohm Ion-Chromatography-System consisted of the 761 Compact IC Units; 813 Compact IC Sampler; Metrosep A supp 4; and Metrosep A supp 4/5 Guard. The details of the method are described in IC Application Work AW CH6-0829-12005- of Metrohm.
For plasma citrate determination, heparin plasma was diluted 1:1 with distilled water and centrifuged at 2,500 g through a Amicon Ultra Centrifugation filter 10K (Ref. UFC801024). The filtrate was analyzed according to urine protocol.
The standards and the samples, including QC samples, were injected by the 813 IC sample processor. Calculations were done by automatic integration with IC Net 2.3 Software using peak area integration for citrate. The CV for precision testing was below 5% and recovery 97%–105%. The laboratory is accredited according to IEC/ISO 17025 and ISO 15189.
Statistical analysis
The plasma and urine citrate and creatinine levels are expressed as the arithmetic mean±standard deviation. To determine kidney involvement, the fractional citrate excretion ratio was also calculated. The differences between patients with glaucoma and controls were evaluated statistically utilizing the 2-tailed Mann–Whitney test for independent samples. Differences with a first-order error of P<0.05 were considered as statistically significant.
Results
Twenty-one patients with glaucoma and 21 controls (12 women/group) were recruited in each group and their mean age was 67.2±10.9 and 67.2±10.9 years, respectively. Mean IOP in the right/left eye was 14.9±2.6/15.8±3.1 mm Hg, respectively, among patients with glaucoma and 13.5±2.5/14±2.6 mm Hg, respectively, among controls (P=0.01). The average VF mean defect in the glaucoma group in the right/left eye was 7.3±5.1/8.2±5.6 dB, respectively, versus 1.6±4.9/0.6±1.4 dB, respectively (P<0.0001). Cup/disc ratios in the glaucoma group in the right and left eyes were 0.68±0.16; 0.72±0.1 versus 0.35±0.17; 0.34±0.18 (P<0.00001).
No differences were found between the groups for BSCVA, refractive errors, diets, or tobacco consumption.
Twelve patients suffered from normal tension glaucoma (NTG), 8 were diagnosed with primary open angle glaucoma (POAG) and 1 patient had a pseudoexfoliation (PEX) glaucoma.
The mean plasma citrate concentration was 104.8±23.2 μmol/L and hence significantly lower among patients with glaucoma compared with the age- and gender-matched control patients (128.2±31.1 μmol/L; P=0.01 Mann–Whitney), whereas the mean plasma citrate/creatinine ratio among patients with glaucoma with 1.74±0.5 no significant difference to the control group revealed 2.12±0.7 (P=0.07 Mann–Whitney). The mean urine citrate concentrations in the same patients was in the glaucoma group 1.7±0.9 versus 2.8±1.9 μmol/L; (P=0.07 Mann–Whitney) and the mean urine citrate/creatinine ratios were 0.21±0.1 versus 0.29±0.2 (P=0.09 Mann–Whitney). The mean plasma and mean urine creatinine concentrations showed no significant differences (plasma creatinine glaucoma: 63.0±16.7 versus 63.4±15.5 μmol/L; P=0.72; urine creatinine glaucoma: 9.6±5.1 versus 11.5±8.4 μmol/L; P=0.67). The calculated fractional citrate excretions were also not different with 12.1% versus 13.6% (P=0.37 Mann–Whitney) (Fig. 1).
Significant lower citrate plasma concentrations were found in patients with glaucoma compared with nonglaucomatous controls, whereas no difference in plasma creatinine was found.
Utilizing a cut-off value at 110 μmol/L, the plasma citrate testing to detect glaucoma would have a sensitivity of 66.7%, a specificity of 71.4%, a positive predictive value of 70.0%, and a negative predictive value of 68.2%. Its likelihood ratio for a positive test (LR+) is then 2.34 and the likelihood ratio for a negative test (LR−) results in 0.47. 1
Discussion
The 3 keystones of glaucoma are typical optic nerve head cupping, glaucomatous visual field defects and often elevated IOP. Nevertheless, particularly in early stages it is sometime difficult for the specialist to diagnose glaucoma since IOP can be at normal levels. Therefore, it would be helpful to have additional parameters such as biomarkers for the early diagnosis of glaucoma. Some candidate genes involved in molecular pathways of stress response, apoptosis, DNA-repair, cell adhesion, tissue remodeling, transcription regulation, multi-drug resistance, and energy metabolism have been identified as possible biomarkers for glaucoma. 2 Nevertheless, the clinical relevance of these findings has yet to be investigated.
On protein levels, potential biomarkers described were, for example, altered concentrations of brain-derived neurotrophic factor in the tears NTG 3 or Alzheimer chemokines in the aqueous humor of patients with glaucoma. 4
Citrate is an organic trivalent anion. It is a major substrate for the generation of energy in most cells. It is produced in mitochondria and used in the Krebs' cycle. If not used for energy production, it is released into the cytoplasm through specific mitochondrial carriers. In addition to its metabolic function, it has a role as an important chelator of cations such as Ca, Zn, and Mg. Intracellular citrate levels indicate the energy status of a cell. By inhibition, high levels of intracellular citrate slow down glycolysis decreasing energy production. 5
In this study, citrate has been found to be significantly reduced in the plasma of patients with glaucoma, whereas the excretion of citrate and the relation between citrate and creatinine was not different between study groups. Therefore, it is very unlikely that a pathology of the kidneys may have influenced our results.
Theoretically, a lower plasma citrate concentration can be achieved by a lower citrate intake or production, a higher metabolism, an accumulation in tissues, or an increased excretion. Considering the known pathophysiological mechanisms for glaucoma, the decreased amount of citrate in the blood of patients with glaucoma might be explained, for example, by an increased usage as an antioxidant agent or by a dysfunction of mitochondria.
As a chelator, citrate has an important role as an antioxidant by eliminating Fe2+ ions that are involved in the formation of hydroxyl radicals from H2O2. 6 An increased plasma citrate concentration during thyrotoxicosis has been described as a protective response of the organism toward mobilization of the antioxidant system and therefore protecting the cells from free radicals. This process might be explained by a lowered activity of the aconitate hydratase, catalyzing the citrate conversion into isocitrate. 7 In glaucoma, decreased antioxidant activity has been found. 8 Thus, this might be an explanation, why and how hypothyroidism and glaucoma could be associated, 9 although this association has always controversially been discussed. 10
As described before, citrate is an important molecule in the energy production cycle in mitochondria. It is widely accepted that mitochondria might play a pivotal role in the pathophysiology of glaucoma. 11 Reduced mitochondrial activity might also lead to a decreased citrate production with resulting lower blood citrate levels.
The calculated sensitivity and specificity in this cohort is comparable with other biomarkers such as prostate specific antigen (PSA) for prostate cancer 12 and could proof citrate interesting as a biomarker for screening examination to detect glaucoma. The actual specificity of 71% would still make other additional exams necessary to confirm the diagnosis in practice.
Although the present pilot study might be interesting for future research, it has some important limitation: (1) While statistical results are significant, the samples size is relatively small. (2) All patients with glaucoma were Caucasians referred to a tertiary center due to glaucoma progression. The study cannot be applied to other ethnic groups. We cannot differentiate between nonprogressive and progressive glaucoma. (3) We could not differentiate between glaucoma subtypes yet. It would be interesting to investigate citrate levels in acute angle closure glaucoma and compare it to chronic progressive POAG or NTG. (4) There was no differentiation between early and late stage glaucoma.
Further studies are needed to describe the role of citrate in glaucoma and to investigate citrate levels in various ethnic groups, in glaucoma subtypes, and different glaucoma stages as well as the measurement of plasma citrate concentrations in a larger cohort in the normal/healthy population.
In conclusion, plasma citrate levels were reduced in patients with glaucoma, whereas the function of their kidneys and the excretion of citrate were normal. Plasma citrate might be a candidate as a potential biomarker for the screening and or diagnosis of glaucoma in the future.
Footnotes
Acknowledgment
The authors would like to thank Anja Kruse, M.D., Department of Nephrology and Hypertension, University Hospital Bern, Switzerland, for her helpful advice.
Author Disclosure Statement
The authors have no personal financial interests in any of the mentioned substances, companies, or competing companies related to the study.