New Approach to Detect Prostate Cancer

Introduction 

The objective of our article is to share about the recent developments in the techniques for detection of prostate cancer, with special emphasis on the role of spectroscopy and chromatography. Conventional techniques of detection using prostate-specific antigen and various newly developed techniques of detection will be discussed in our article. We also included an evaluation of the techniques as well as possible areas of improvement with respect to the discipline of analytical chemistry. Ultimately, we hope that through our article, we can shed light on the detection of prostate cancer, a deadly disease which is the third most common cancer affecting Singaporean men [1]. 

Prostate Cancer

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The objective of our article is to share about the recent developments in the techniques for detection of prostate cancer, with special emphasis on the role of spectroscopy and chromatography. Conventional techniques of detection using prostate-specific antigen and various newly developed techniques of detection will be discussed in our article. We also included an evaluation of the techniques as well as possible areas of improvement with respect to the discipline of analytical chemistry. Ultimately, we hope that through our article, we can shed light on the detection of prostate cancer, a deadly disease which is the third most common cancer affecting Singaporean men [1]. 

Prostate cancer is a disease condition where cells in the prostate become cancerous. The prostate is a small gland that serves important function in the male reproductive system and is located beneath bladder and surrounds the urethra [2].

Prostate cancer is commonly diagnosed when the cancer has reached advanced stages because it shows no symptoms. However, the cancer has usually become fatal by then. If diagnosed early, patients have a very high survival rate[3]. As such, many detection techniques have been established. The most conventional method used clinically is a blood test for the Prostate Specific Antigen (PSA) level.

Conventional Prostate Specific Antigen Level Test

Prostate Specific Antigen (PSA) is a protein secreted solely by cells lining the prostate. This test measures the level of PSA in a man’s blood. The concentration of PSA increases greatly in men with prostate cancer [4].  As such, blood sample of patients are analysed for presence of elevated PSA concentrations in an effort to diagnose patients with prostate cancer.

Normal PSA Scores

• 15% of men with psa levels less than 4 ng/mldevelop prostate cancer.
• 31% of men with prostate cancer screening between 4 - 10 ng/ml develop prostate cancer
• 50% - 65% of men with psa scores over 10 ng/ml develop prostate cancer

An important part of the your results is finding both the:

1. Total amount of PSA in your blood.
2. Ratio of free vs bound PSA.

http://www.mens-hormonal-health.com/images/psa-test.jpg 

While this method is used worldwide, there remains controversies over the accuracy of the test because other conditions may also cause a rise in PSA level (prostatitis and benign prostatic hyperplasia) and the blood test also risk showing false positive and false negative results. In fact, only about 25 percent of men who have a prostate biopsy due to an elevated PSA level actually have prostate cancer. [5] Hence, many other detection techniques have been developed, such as analyzing the level of metabolites related to prostate cancer.

Digital Rectal Examination

To test asymptomatic men for prostate cancer. It assesses the size, shape and texture of the prostate via the rectum. This is usually done in conjunction with conventional Prostate Specific Antigen Level Test.

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Sarcosine

Metabolites is important in the onset of cancer typically because of its regulation of various signal transduction pathway. Metabolomics (Metabolic profiling) involves the total pool of metabolites in cells, body fluids (urine, serum, seminal fluid, etc.) or tissues from different patient groups to be subjected to comparative analysis.[6]

Urine is used often as a biological fluid for investigation due to easy collection, repeatability, rich metabolite composition, and the often-higher metabolite concentrations than blood plasma or serum. One such metabolite that was initially found to be linked to prostate cancer in urine samples is sarcosine. Eventually, the correlation was refuted [1, 2], but the importance of similar metabolites as biomarkers which are linked to prostate cancer remains. Moreover, the ultimate aim is not only to find these tumour markers that correlate to all cancers, but also to ensure that the analytical method is simple and accurate. This way, detection of cancer could be done with a simple blood test. Only then would it be possible to have such screening to proliferate within the population.


Hence, there has been research on spectroscopy techniques to detect prostate cancer. One can either extract the prostate via transurethral resection (a urological operation which visualizes the prostate through urethra) or by directly scanning on the subjects. Common spectroscopic techniques:

1. Magnetic Resonance (MR) Spectroscopy
• Especially suitable for metabolomics
• Requires little to no sample preparation
• Rapid, nondestructive, noninvasive, and highly reproducible
• Simple data set conversion provides a complete overview of a biological sample in one experiment
• Relatively high-throughput sampling
• New developments to improve sensitivity (cryo-cooled probe head technology)
• Still has lower sensitivity compared to MS methodologies.

2. Infrared Spectroscopy
3. Mass spectroscopy
• Coupled with forms of chromatography (liquid, gas or capillary electrophoresis) as the biomarkers of interest would have to be separated from other compounds in the samples first

Using these techniques, direct quantification of the metabolite sarcosine was possible but there were problems of:

1. Difficult application due to high instrumentation costs
2. Interferences from chemicals in the sample with similar mass (alanine) resulting in difficult separation and detection. [3]

Using spectroscopy analytic techniques, a new approach was thus discovered other than PSA level testing and digital rectal examination, using: (1) enzyme specificity to bind to the metabolite and (2) fluorescence. An enzyme sarcosine oxidase could act on sarcosine specifically to catalyze production of formic acid. This creates a pH change which could be measured by fluorimetric analysis.

Reaction Mechanism of enzymatic approach with fluoimetric analysis

In Burton et. al. [4], both techniques (LC-MS/MS and fluorescence) were compared and indeed the specificity of the enzyme had eliminated interferences (caused by alanine, which is of similar mass to sarcosine). This specificity would allow analysis of complex samples which are easier to be obtained such as urine samples. This methodology could be easily applied to other metabolites (since sarcosine is shown to be obsolete in detection of prostate cancer). For example, D-amino acid oxidases (DAOs) would similarly produce a keto acid. Moreover, the metabolite sarcosine could be in consistent ratio with another metabolite which could be tapped on for prostate cancer detection. An example is creatinine. [5] Even though LC-MS/MS technique is more sensitive, the new novel technique would be more practical as it is cheaper and faster due to the use of fluorimetric analysis.

Comparison plot of the fluorimetric and LC-MS/MS techniques used to analyze sarcosine concentrations in 10 urine samples. Correlation coefficient (R²) is 0.8466.

Nevertheless, chromatography techniques could also be modified to be practical. Below are 2 approaches using the Gas Chromatography (GC) and High Performance Liquid Chromatography (HPLC).Using Gas Chromatography (GC) ^[6]
Similar to LC, quantification of sarcosine using just GC-MS was possible, but sample preparation was tedious, and it was not direct for a complex derivatization step is needed.

Optimizing SPME and GC

A new approach through Solid Phase Microextraction (SPME) hopes to eliminate the time-consuming step of esterification/actylation, and to minimize the organic solvent used, involving only sorption and desorption. However, analysis of amino acids using GC would require carboxylic acid groups and amino group to be derivatized (chemically modified) to increase their volatility. In this study, they tested that the a few microliters of ethyl chloroformate could convert them to esters within 1 minute.

Derivatization of sarcosine using ethyl chloroformate

Interference between Alanine and Sarcosine

Initially, the transition from m/z=116 to m/z=44 was selected and collision energy and scan time were optimized based on it. However, separation of alanine and sarcosine in a human urine sample was no clear as it contains high concentration of alanine. Nevertheless observation from the full-scan mass spectra showed that alanine had a significantly lower ion current at m/z=189 compared to that of sarcosine. This is explained by the difference in energetics of unimolecular dissociations in the reacting species (sacrosine being more stabilized by the donor effect of methyl group on nitrogen atom), despite of identical reaction paths in gas phase. This is further supported by the fact that the ratio of m/z 189→m/z 116 was similar for both alanine and sarcosine.

Thereby, the transition (m/z 189→m/z 116) was chosen since the response factor for alanine is significantly smaller. Together with an appropriate GC oven temperature program, there was good separation between alanine and sarcosine. The discrimination is crucial in order for accurate prognosis since typical urine sample would have high concentration of alanine.

Using Liquid Chromatography (LC): Ion Exchange Liquid Chromatography (IELC) [7]

Overall Scheme of separation and detection of Sarcosine: IELC-Vis with post column derivatization with ninhydrin.

Separation of amino acids using IELC

Stationary phase of IELC is the cation exchanger in the sodium cycle. The mobile phase is in variable gradient of pH and ionic strength (by increasing the concentration of salt) with a flow rate of 0.5ml/min and initial pH=2.7. Owing to the amphiphilic properties of amino acids, they can be separated by IELC based on their isoelectric point (pI).

The mixture of 17 amino acids and sarcosine was well separated. The chromatogram showed a retention time of 32.93 minutes and that it elutes between serine (pI=5.76) and glutamic acid (pI=3.22). The sarcosine peak is also symmetrical and baseline separated. Highlighted in the blue line is the increase in gradient of buffers: 0-7 min (pH 2.7, 4.06 M), 7-20 min (pH 3, 4.24 M), 20-31 min (pH 4.25, 5.30 M), 31-90 min (pH 9.7, 8.96 M).

How organic and inorganic content can affect the elution time

Indeed, there was good separation of sarcosine from other amino acids in the urine samples. However, in biological samples, as there could be other organic and inorganic content which affects the retention time of sarcosine. In urine sample, that would meant salt (NaCl) and uric acid. Proline (Pro) and glycine (Gly) were also investigated as their pI is similar to that of sarcosine. Change in the compound's concentration was plotted with the respective retention time of sarcosine.

1. A linear increase in NaCl and glycine concentration caused a significant linear decrease in the retention time (RT) of sarcosine.
2. Adding of uric acid however increased the RT of sarcosine, but increasing concentration did not subsequently cause a significant shift of the RT.

Adding Proline also not only increased RT of sarcosine, higher concentrations further increased RT (but nonlinearly). The higher the concentration of nonpolar proline side chain, the higher the attraction of more polar sarcosine to the stationary phase, hence the longer the RT of sarcosine.

It was also observed that increasing concentration of these compounds did not affect the peak height of sarcosine significantly. Thus, we can correlate that peak area was probably similar and that the amount of sarcosine detected would not be affected, an important factor in diagnosis of prostate cancer.

Detector explains for the practical application of methodology

Instead of the usual mass spectrometer after chromatography, eluted compounds are goes through post-column derivatization by ninhydrin and are detected by photometric detector at 570 nm. This meant faster detection and less sample preparation. They validated this approach by showing that: 

• Measurable range of concentration of sarcosine is ideal.
• The concentration of sarcosine and the absorbance varies linearly. 

Hence, it can be concluded that IELC with post-column derivatization by ninhydrin and photometric detection is a robust and reliable method. Nevertheless, optimization of the photometric detection was followed to increase the sensitivity.

Conclusion

Burton’s technique of detecting prostate cancer using urine samples is more accurate and reliable than conventional PSA testing for PSA cannot 100% differentiate benign prostate conditions from active prostate cancer. Moreover, high-tech instrumentation is not required to detect metabolites, thus the technique could be less costly as it requires a simple chemical reaction and pre-existing diagnostic equipment found in most hospitals. It has been postulated to cost a tenth of a penny per sample as compared to $70—cost to get a PSA test.

Overall Scheme of separation and detection of Sarcosine: IELC-Vis with post column derivatization with ninhydrin.