Analysis Of Urinary Polyamine Measurements In Breast Cancer Patients

The purpose of this study is to create a standard operating procedure (SOP) for the analysis of urinary polyamine measurements in breast cancer patients as a measurement of response to therapy. The preliminary tests conducted were carried out to observe the polyamine levels; to distinguish if there is a significant difference in the polyamine levels between the healthy volunteers and the breast cancer patients. The findings within are not solid, accurate data, the analysis was conducted to obtain statistical information to predict if the SOP data perhaps alludes to supporting published literature.

2.0 Polyamines, what are they?
Polyamine are aliphatic, organic molecules with three or four amine groups attached (Larqu?? et al., 2007; Soda, 2011). These low molecular weight polycations are ubiquitous and promiscuous molecules, their function depends on the electric charge. Polyamines react electrostatically with negatively charged molecules such as DNA, RNA, and proteins, modulating their function and transport in cells (Igarashi and Kashiwagi, 2010; Igarashi and Kashiwagi, 2000; Liu et al., 2013; Larqu?? et al., 2007).
The natural 'free' polyamines; putrescine, spermidine and spermine are commonly produced in almost all mammalian cells (H??kkinen et al., 2013; Soda, 2011). Polyamines can exist as both free and acetylated forms in biological fluids, they are excreted by means of oxidative reactions and are present in metabolic form (Larqu?? et al., 2007) in human urine.

Figure 1: Chemical structures of free polyamines

Figure 1 is a pictorial of polyamine structural information taken from the journal by Minois et al., (2011).

The amino acids; arginine, ornithine and methionine are responsible for polyamine synthesis (see Figure 2). Putrescine is synthesised from the enzymatic decarboxylation of L-ornithine, catalysed by ornithine decarboxylase (ODC) and subsequently spermidine is synthesised from putrescine. Spermine is synthesised from spermidine and the synthesis of both these polyamines are synthesised from arginine. (Igarashi and Kashiwagi, 2010; Soda, 2011).

Figure 2: Synthesis and interconversion of polyamines

Figure 2 is a pictorial describing the synthesis of polyamines obtained from Larqu?? et al., (2007).

The increased activity of enzymes such as ODC are said to correlate to the increased levels of polyamine synthesis linked to cell growth and cancer, the enhanced levels are therefore easily detected in each cancer patient's urine but reduced by therapy (Paik et al., 2008; Soda, 2011).
Polyamines are mainly associated with multifunctional roles in cell growth and proliferation (Soda, 2011; Paik et al., 2008; Moinarda et al., 2005; Minois et al., 2011) by increasing transcriptional activation at gene promoters including cell adhesion via hormone dependent, and independent transcription factors; polyamines are critical for cell survival. As regulators, it is proposed that polyamines may affect onset and progression (Schipper et al., 2000) of multiple diseases such as Alzheimer's (Minois et al., 2011) and cancer; hence why they are a target for therapeutic intervention for chemotherapeutics; or studied to ideally create a diagnostic dipstick style test for early prognosis or indicative of relapse and recurrence.

This study will focus on its relation to cancer with particular interest in the levels in breast cancer patients. Carcinomas and age affects polyamine concentrations, as they naturally decline with age and as the concentrations are elevated in cancer tissues. They therefore may be implicated as a potential diagnostic biomarker for many malignancies (Bachrach, 2010). Intratumour polyamine content and tumour recurrence in breast cancer patients has been demonstrated in published literature, with high levels observed. Existing literature was reviewed in order to obtain an understanding of how polyamines levels correlate with tumour growth.

The aim of the preliminary experiments was to support or oppose the trends in current literature, posing such questions as:
1. Does the data support the hypothesis that polyamine levels continually decline with age?
2. Is there a significant difference in polyamine levels between the healthy controls and the breast cancer patients? Does these findings refute the data concluded by (Lipton et al., 1975)?
3. Do polyamine levels increase after surgery?
4. Are all polyamines detectable at various concentrations in each patient undergoing treatment for breast cancer?
5. Are there significant differences in polyamine levels in the follow up clinic samples?

3.0 Urinary Analysis
Urine is an excreted waste product; a watery solution containing salts, urea and uric acid that is produced by the kidneys. In healthy, hydrated individuals you would expect the urine to be transparent however the colour and turbidity of urine samples correlate with the health and diet of each individual. In unhealthy or poorly nourished individuals, their urines samples can range from pale yellow-amber- dark yellow in colour. Many post theatre urine samples collected in this study were blue, this is due to a dye used when conducting a sentinel lymph node biopsy (SLNB). The dye highlights the flow of the lymph fluid to help determine the extent of cancer in the body.
Urine is an easily accessible, non-invasive body fluid and it is therefore patient friendly in collection. In this study, all samples were collected without preservatives and an aliquot of each sample was taken for the separate analysis of creatinine. The samples were stored at -80??C. An advantage of urinary analysis is that high sample volumes obtainable and subsequently this achieves greater accuracy through the capacity of numerous repetition tests (I.H.T, 2008). The preparation of biological samples is critical to the integrity and quality of the method, as polyamines are difficult to quantify, the preparation is long and laborious in order to enhance their sensitivity for detection.

3.1 Creatinine
Creatinine is a waste product resulting from the nonenzymatic conversion of creatine to phosphocreatine, and excreted by the kidneys. It is a useful tool for the normalisation of polyamines and other molecules to nmoles per mg creatinine (analysis 5??g creatinine/ 200??L) and thus creating a baseline reference specific to each individual. However, creatinine levels are dependent on numerous exogenous and endogenous factors such as age, gender, food consumption etc.
Measurement of urinary polyamines will be an independent marker in response to therapy and potential importance as an early indicator of relapse and disease recurrence.

4.0 Liquid Chromatography- Mass Spectrometry (LC-MS)
The analytical methods used to quantify polyamines is usually based on chromatographic separation and commonly done by Liquid Chromatography- Mass Spectrometry (LC-MS). LC-MS is a hugely popular hyphenated analytical technique which is used for quantitative analysis by chromatographically separating compounds based on polarity. The mass spectrometer in this case acts as a detector but the instrument itself is highly sensitive and selective, obtaining molecular masses and structural information from mass spectra.
In liquid chromatography a C18 column was used, this acts as the stationary phase. The samples were analysed by an auto sampler, loaded onto the column and the column was washed by a liquid mobile phase consisting of methanol, acetonitrile (these solutions were also used in sample preparation) and deionised water. The retention rate of the samples are based on affinity for the column, separation based on the differences in partitioning behaviour between the stationary phase and the sample; the samples are purified as they pass through the column.

For specification; the chromatographic process can be fractionated via gradient elution. Dependent on the volume of samples and standards requiring analysis, the process can be time-consuming and it is therefore preferential to run the analysis overnight as each sample analysis takes approximately seven minutes to run, as the auto sampler must equilibrate between samples.

4.1 Electrospray Ionisation
Electrospray ionisation (ESI) revolutionised LC-MS as a powerful analytical technique. The coupling success lies with the interface, developmental issues arose due to finding a means to transform the liquid effluent from the liquid chromatograph (LC) to gas phase ions for analysis in the mass spectrometer (MS). The development of ESI meant that the eluent from the LC is passed to the MS, heat is applied to evaporate the solvent; nebulised into a spray of gaseous ions. Nebulisation is conducted under vacuum in the MS chamber and passed on to the mass analyser, the gas phase ions are separated based on their mass to charge ratio (m/z+) using single reaction monitoring (SRM) mode.

The MS Triple Quadrupole instrument consists in two quadrupoles made from four parallel carbon steel rods arrange in a square, the quadrupoles are separated by a collision cell. The analyte ions are directed down the centre of the quadrupoles, voltage is applied to the rods to generate an electromagnetic field. The first quadrupole selects the first ion which is fragmented in the collision cell, accelerating the ions in the presence of a collision gas, nitrogen (N+) and onto the second quadrupole for analysis to determine the m/z+ ratio of the ions. The MS acts as a dual detector, sensitive and selective for quantitative analysis.

5.0 Ethics
The study of polyamines is of great importance as there has been little or no advancement in this area of cancer research over the past thirty years. One of the main factors hindering this research is discrepancies in published literature as there is large variability and inconsistencies in the data including variation within, and between studies. It is due to these errors that no power calculations are obtainable presently. Based on the evidence available in literature, few standard deviations are concluded and therefore authorisation for a large 'proof-of concept' study has been granted in order to achieve this information. The study falls between a pilot and full study due to the number of participants, the large pilot study will use one hundred patients with primary breast cancer so that estimates can be made with a reasonable degree of precision.
However, in this project the analysis conducted was to ultimately obtain a tried and tested standard operating procedure (SOP) for the large pilot study.

5.1 Study Approval
The process from application to approval is lengthy, requiring meticulous attention to detail as numerous documents are submitted for ethical approval. The Principal Investigator (PI) must inform and report every major or minor change to the governing regulatory bodies via the Integrated Research Application (IRAS) form and each amendment was version controlled. The governing bodies include; Research Ethics Committee (REC), North of Scotland Research Ethics Committee (NosREC), National Health Service Research and Development (NHS R&D) and the National Research Ethics Service (NRES). Each governing body comprises of various members with differing levels of academia, professions and specialist experts. If after any changes are made after approval, authorisation is sought from the Research Governing Manager (RGM) prior to submitting to the REC.
All NHS organisations require anyone who does not have a contractual relationship with the NHS, they must instead have an honorary research contract for permission to conduct research using NHS resources.
The IRAS form includes all information related to the study such as site specific information (SSI), research and development (R&D) and all approved documents by the REC (REC application, protocol, letter from statistician, consent forms etc.).

5.2 Consenting
The research nurse involved in the study provided information about the aims and objectives of the study prior to asking for participation and consent. All participation and information collected was anonymised, under the strictest confidence. All information is securely stored electronically is password protected.
To be eligible for the study each patient had to be between 18-80 years old.
Once verbal consent was achieved, the participant was provided with three consent forms; one for the patient, one for hospital records and one for the PI. After written consent was obtained from each patient, they were given four universal tubes and asked to provide four urine samples: a before theatre and after theatre sample and two follow up clinic samples.

6.0 Preliminary tests
In the ten week research study involved the collection and storage of biological samples from breast cancer patients, and the principle aim was to develop an SOP for polyamine analysis. During the development process, the available protocol was optimised, changes were made, tested and validated.
Controls were gathered consisting of ten individuals and broken down into two subset groups to provide intra-assay variation. The groups consisted of females; five controls aged between 20-25 years (mean age 21.5) and the final five controls were of age 50+ years old; as this control group is appropriately age matched to the patients. The logic behind gathering the control groups was to determine if the data can support the understanding that polyamine levels naturally deplete with age and if there is a significant difference in polyamine levels between the age-matched controls and the patients. The large pilot study will involve a different control group comprising of ten individuals taken from the 'Orange Juice Study'.
For data analysis, 24 patient samples were analysed against the ten healthy controls and a set of polyamine standards.
Seven polyamines were measure in the analysis; free polyamines; Putrescine, Spermine and Spermidine, the acetyls; N-acetylspermine, N1-acetylspermidine, N8-acetylspermidine and the diacetyl; N1, N12 'diacetylspermine.
The initial analysis was to determine competency in the laboratory, however the quality of data produced was hindered by inexperience which ultimately impacted the experiments leading to inaccuracies; poor linearity, precision, contamination, one major factor was to obtain reproducibility between replicates and reduce analytical errors.

Figure 3: The importance of good laboratory practice (GLP) in sample analysis, comparing N1,N12-Diacetyl Spermidine and N8-Acteyl Spermidine

As one can see from Figure 3, there is large variability, poor precision and reproducibility in the samples on the left but on the right, it is clearly a stark contrast with better linerarity and reproducibility.
Preliminary tests will be discussed further and in greater detail under method development.

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