Citric acid analysis

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The citrate anion exists in all living cells. It is not only an important intermediate in the tricarboxylic acid cycle, but also a key component of fatty acid, cholesterol and hormone synthesis, photorespiration, the glyoxylate cycle and nitrogen metabolism. Due to this metabolic significance, abnormal citrate levels have been linked to the characteristics of several diseases. Recently, citrate has been selected as a marker for the discrimination of prostate cancer as a result of the highly specialized anatomical function of the prostate gland.

The main function of the prostate gland is to store and secrete a clear fluid (pH 7.3) that constitutes about 50% of the volume of the seminal fluid that, along with spermatozoa, constitutes the semen.  Prostate cancer (PCa) is the most common non-skin related male cancer type in the world, affecting one in ten men in the UK. It is classified as adenocarcinoma and also considered as a malignant disease.

Prostate secretory epithelial cells have the specialised function and capability of accumulating and secreting extraordinarily high levels of citrate . The diminishedability of neoplastic epithelial cells, mainly in the peripheral zone of the prostate is a consistent factor in their development of malignancy and the subsequent dramatic decrease in prostatic citrate concentration. The analysis of citrate levels is  striking as the disease progresses. Prostate cancer screening is an attempt to detect unsuspected cancers in their earliest stages. Screening test may lead to more specific follow-up diagnostic tests and if required cancer treatments. As PCa is a slow-growing cancer, the chances to identify the disease in early stage are high. The most commonly used screening procedure today are the Prostate Specific Antigen (PSA) blood test and the Digital Rectal Examination (DRE) and, if required, biopsy involving identification of prostate epithelial cell morphology, using the well established Gleason-score.

A major problem involved in prostate cancer (PCa) diagnosis is the absence of sensitive, accurate, and preferably non-invasive diagnostic procedures. The current diagnostic and screening procedures are considered to be highly inaccurate and/or possess the possibility of increasing the risk of developing prostate cancer. Therefore, there is a need for a uniform, well established non-invasive screening procedure. Moreover, procedures are needed that allow early detection, staging, location, and estimated volume of the malignancy; preferably, a mapping of the prostate for follow-up of progression and regression of the malignancy. Citrate level tests from prostate or seminal fluid samples may overcome the risk and inaccuracy of current screening procedures.

One main drawback of citrate level measurement in the prostate fluid is that obtaining the sample requires a biopsy. Prostate fluid, as the main source (about 50%), gives rise to seminal fluid citrate level of about 10 - 40 mM. The high citrate content of seminal fluid is consistent with the use as a buffer to maintain the pH of semen (7.2 – 8.0).  Its role may be complex, but importantly the citrate level in seminal fluid is proportionate to that in the prostate fluid. Therefore, citrate level measurements from seminal fluid samples could aid the detection of prostate malignancy.

We have set out to develop a robust, responsive, single component luminescent citrate sensor that can used to measure citrate levels in prostate or seminal fluid samples. This assay may be of general utility and significance to the analytical and life sciences. Based on ratiometric luminescent methodology established by the Parker group and FScan Ltd. a  single component citrate sensor has been developed for use in clinical samples of prostatic origin.  The analystical  method based on this sensor utilises ratiometric detection, by plotting changes in the ratio of up to 4 different wavelengths as a function of citric acid concentration. Therefore, following establishment of a suitable calibration curve and appropriate sample dilution this provides a fast method for the determination of citrate in various fluids.

The precision of the measurement, which is an inherent feature of such a ratiometric assay, allows less than 1% variance in the measured intensity ratio for a given citrate concentration. It is also worth mentioning that one analysis in a typical sample volume of 100 μL (diluted x100 with commercially available buffer solution) requires 0.5 μg of compound with simple and fast sample preparation and a fast (3 min) acquisition time. Since there is always biological autofluorescence present in such spectroscopic measurements using bio-flids, causing interference in the form of a sloping baseline, time gated measurements are preferred for precise calibration. This can been achieved using a our own brand spectrophotometer incorporating a time gating device. It is worth noting here that this relatively low budget yet versatile instrument may be successfully used for numerous related spectroscopic applications, including the proprietary lactate and urate determinations (for details please see the dedicated instrumentation section of this portfolio). Using this device, the short-lived auto-fluorescence can be successfully eliminated. Using the built in detection software with its  pre-programed calibration curve, citrate concentrations of clinical fluid samples have been successfully differentiated. As this compound and the associated analytical method has been shown to be successful in the rapid determination of citrate concentration in a wide variety of biological samples (plasma, seminal and prostate fluid and urine), with confirmation of the accuracy of the analysis by comparison with a commercially available enzymatic kit (Megazyme™ citric acid assay kit) with an average error of ± 5 mM (Fig. 1).  Current commercially available citric acid assays are based on similar enzymatic methods has not only been found to be sensitive to pH and transparency of the sample, but more importantly to certain interferents, such as protein, fatty acids, and pyruvate, making the analysis rather elaborate and time consuming. Considering all associated issues, a single component, fast response citrate sensor is the only way forward to establish a user friendly and accurate method of measuring the citrate concentration of low volume clinical samples.

In order to demonstrate the potential application of this sensor and the methodology associated with it, we carried out analysis of several various bio-fluid samples with unknown citrate levels. To co-validate and simultaneously compare/highlight its beneficial properties we also evaluated these samples using the above detailed commercially available enzymatic citric acid kit (Fig. 1). The typical analysis time of or Eu-based sensor was established at an average 3 min using our  own brand prototype time-gated spectrophotometer and 50 μL total sample volume (0.5 μL actual bio-fluid content) compared to up to 30 min acquisition time, 3 mL total sample volume required using the enzyme based kit. Not only the assay time and the required sample volume have been significantly reduced, but more importantly the experimental error associated with the method has also been improved.

    

Figure 1. Comparison of results regarding citrate concentrations in various biofluid samples using CS05 Eu(III) based sensor compound and a commercially available enzymatic citric acid assay kit.

In summary, a simple, rapid, non-invasive luminescence assay has been developed for citrate and the new analytical method has been shown to be successful in the rapid analysis of both seminal and prostate fluids. The method employs ratiometric detection, using either low-cost home-built equipment or devices commonly found in high throughput screening laboratories. These extensive encouraging results suggest this ‘single component’ citrate assay to be applicable for low volume samples for the detection and staging of prostate adenocarcinoma. The fast response, short acquisition time, good photophysical and chemical properties of the probe used each support its use to determine citrate concentration in low volume clinical samples, overcoming the inaccuracy, hazard and elaborate nature associated with current screening processes.

                          For more detailed information on our Citrate assay please visit our 'Literature' section.

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