LOS ÍNDICES PRONÓSTICOS EVALUADOS

These experiments were dedicated to explore the comparison of different methods for the quantitative analysis of nitrate and nitrite in biological samples (plasma and urine). As been discussed earlier in this chapter, there are various analytical methods based on different analytical principles, such as colorimetry, spectrophotometry, fluorescence, chemiluminescence, gas and liquid chromatography, electrophoresis and mass spectrometry (Tsikas, 2005a). The method for the assessment of nitrate and nitrite in humans depends on the availability of resources and equipment, technical and research expertise.

In this study, CL (with and without deproteination) method and the Griess method were used for quantifying concentrations of nitrate and nitrite in plasma and urine. These methods were used based on the availability of the machine in the laboratory (ozone based CL) and the commercially available of ‘ready to use’ kits for the Griess method. In addition, the GCMS method was used for the analysis of saliva samples in the third phase of this PhD project (details in Chapter 5).

In general, following comparison of the three techniques (CL, Griess and GCMS) used in measuring nitrate/nitrate in plasma, urine and saliva samples, the following conclusions have been made:

1) Even though CL is the best technique for measuring nitrate (Piknova et al., 2016), extra precaution is required when using this method. Injecting the samples into the purge vessel resulting in excessive bubbles even though an antifoaming agent was used, and this was exacerbated with for non-deproteinised due to the protein present in the samples. The bubbles should be monitored by adjusting the needle valve so that the bubbles did not get into the inlet tube since that will damage the machine. At maximum of 10 injections of samples were done before the nitrate reducing agent solution in the bubble base needed to be changed. The cell pressure should be constantly monitored at

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4 – 7 torr. The samples need to be deproteinised prior to the analysis, which may add considerable time and expense to the analysis. Due to tedious, time consuming and technically difficult, it can be suggested that CL method is only suitable to be used in research laboratory setting.

2) The Griess method is practical for analysing large numbers of samples where the fast, cheaper and reproducible measurement is a considerable advantage. However, the presence of protein in the sample, can interfere in the outcome. Therefore, performing protein removal prior to the Griess procedure might be beneficial into reducing inaccuracy in the determination of nitrate/nitrite concentrations in samples (Hunter et

al., 2013).

3) GCMS, although quite cumbersome to performed, is considered the most accurate technique of measurement nitrate/nitrite in biological samples (Romitelli et al., 2007). In the study reported in the Chapter 5 of this thesis, the saliva samples have to undergone several steps of derivatisation before being analysed in the GCMS machine. Drying down, heating, washing and filtration were the procedures done before the sample were placed into the injection port of the GC device. Due to the multiple derivatisation procedures prior to the measurement, time consuming and expensive equipment, it can be suggested that GCMS is more suitable to be used in the clinical and research laboratory setting and performed by experienced personnel.

Discrepancies and methodological problems on diverging values of nitrate/nitrite measured are depends on the analytical principles of the method (Tsikas, 2005b). Tsikas et al reported a relatively poor correlation (R= 0.8) between estimates of nitrate in human urine samples (n=33) when estimated using the Griess assay and by GCMS (Tsikas et al., 1997). This discrepancy may be due to the interference by free reduced thiols, proteins, and other plasma constituents in the Griess assay.

There are various methods of deproteination available for treating plasma samples prior to nitrate/nitrite analysis such as acetonitrile-chloroform, ultrafiltration, zinc sulfate, zinc sulfate-

sodium hydroxide and methanol-diethyl ether. The complete removal of protein is essential

because any remaining protein may cause turbidity and may lead to the inaccuracy in determination of nitrite and nitrate concentration. In this study, authors suggested that

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acetonitrile-chloroform is the most efficient method for protein precipitation (Romitelli et al.,

2007).

In the present PhD study, for nitrate analysis using CL method, there was no difference in the effects of MTH and ETH as deproteination agents prior to the measurement of nitrate concentrations in plasma samples (Figure 3.6). However, for nitrite analysis using CL method, nitrite concentrations were significantly higher after using the MTH than the ETH approach

(Figure 3.9). However, both deproteination methods resulted in lower plasma nitrite

concentrations than were observed when non-deproteinised samples were analysed. These results suggest that both ETH and MTH can be used as deproteination agents for nitrate measurements. Deproteination using ETH method is adviced for the measurement of nitrite concentrations as it produced less foaming and shorter protocol for the processing of the samples. While non-deproteinised samples had higher nitrite concentrations, the formation of foam makes the analyses cumbersome and technically difficult.

The comparison between the Griess method and the CL method showed relatively poor agreement for the measurement of nitrate in both plasma and urine. Both deproteination methods (ETH and MTH) showed a similar degree of under-estimation of plasma nitrate concentrations by the Griess compared with CL (Figure 3.7). Finally, the Griess method significantly over-estimated urinary nitrate concentrations (Figure 3.8).

For the analysis using Griess method, we can conclude this method performs poorly in plasma and urine samples for the measurement of nitrate concentrations and its results should be interpreted cautiously. In the case of proportional difference variability between measurements, i.e. constant coefficient of variation across the range of concentration, the effect on the B & A revealed that in all experiments performed, all unit difference has a widening trend of the agreement range with increasing concentrations on nitrite/nitrate in urine/plasma.