Biomarker Metabolomics for Diagnosing Tuberculosis J.

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1 Biomarker Metabolomics for Diagnosing Tuberculosis1

2 Joey R. Bacus Jr.2,*

3 1Paper presented during the lecture session on Metabolomics for Health in BIOCH 150:

4 Advances in Biochemistry, May 10, 2017, RT Skills Lab 2, College of Medical Radiation

5 Technology, De La Salle Health Sciences Institute, Dasmarinas, Cavite 4114,

6 Philippines.

7 2Department of Chemistry and Biochemistry, College of Humanities and Sciences, De

8 La Salle Health Sciences Institute, Dasmarinas, Cavite 4114.

9 *Corresponding author; email address: joeyrb@dlshsi.edu.ph; bacusjoey@gmail.com

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10 INTRODUCTION

11 Tuberculosis, although as far ancient as 15,000 to 35,000 years ago upon its first

12 emergence to it’s previously thought eradication since the discovery of antibiotics, is

13 currently reemerging in several countries often marked as a public health crisis with

14 some strains exhibiting resistance to several antibiotic drugs. Over one third of the

15 estimated nine million people worldwide who has been infected by tuberculosis (TB) are

16 not diagnosed, treated and reported to national health authorities. The countries of

17 Cambodia, China, Viet Nam and even the Philippines account for 93% of the estimated

18 regional TB statistics of 1.6 million cases with 110 000 deaths each year (WHO, 2016).

19 Significant improvements and advancements for, at least, TB diagnosis must be

20 utilized to better address the problem at hand and prevent further destructive

21 implications of prolonged exposure to the pathogen. With such in mind, metabolomics-

22 based approach through biomarker discovery can be a potential alternative to better

23 understand the bacterium cellular components, host-pathogen interactions and pathway

24 derivation for the elevated or reduced metabolites that correlates with the said infection.

25 Yet, the study of metabolomics is quite extensive requiring specialized instrumentation,

26 various statistical analyses and metabolite integration to established or hypothetical

27 derived metabolic pathways.

28 This review aims to present metabolomics and its application to tuberculosis

29 diagnosis to the readers by briefly discussing the overview regarding the concepts

30 behind metabolomics, tuberculosis, biomarker research and role in tuberculosis

31 diagnosis, the potential of two dimensional GC-TOFMS for metabolites identification

32 and quantification, statistical data elimination using principal component analysis and
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33 provide relevant articles for the current progress of the tuberculosis markers in both

34 urine and sputum sample media. The integration of the findings between several

35 researches to both the sample media may strengthen the possibility of a given set of

36 reproducible/repeatable metabolite markers to be a good indicator of active-tuberculosis

37 disease and may even discover missing metabolites that may complete a previously

38 hypothesized metabolic pathway that a given pathogen may undergo.

39 METABOLOMICS OVERVIEW

40 Metabolomics is the study of the identification and quantification of the

41 metabolites in a given organism, cell or tissue. Such metabolites are low-molecular

42 weight compounds (less than 1500 Daltons) which collectively represent the complex

43 enzymatic pathways of the biochemical, physiological and pathophysiological networks

44 occurring within the cell at a particular time forming a metabolic profile called

45 metabolome. The given compounds may be produced as the starting, intermediate or

46 end product of an organism’s metabolic process. A metabolomics study can utilize

47 either targeted analysis or nontargeted analysis approaches towards analyzing the

48 quantitative and qualitative properties of the given compounds. Targeted analysis

49 measures specific known metabolites present within the sample unlike the nontargered

50 analysis (i.e. metabolic profiling analysis) which completely surveys the metabolic

51 compounds (Tang, 2011).

52 BIOMARKER METABOLOMICS FOR DISEASE DIAGNOSIS

53 By analyzing the outcome of various factors or alterations within established

54 metabolic pathways, metabolomics can provide a comprehensive analysis, called

55 metabolic fingerprinting, of the all measurable metabolites to examine the actual

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56 physiological state of an organism during an illness. Such exploits the concept of time

57 varying metabolites to keep track of the disease progression processes and

58 host/pathogen adaptation. With each tissue having distinct metabolome variations, one

59 can also deduce the effects of the disease to specific organs. Such compounds upon

60 careful and extensive evaluation may be classified as a biomarker metabolite which can

61 correlate to a particular disease, infection or any factors that may significantly affect its

62 metabolism. By profiling diseases through a metabolomics approach, established

63 biomarkers may be used for diagnosis, prognosis, and therapeutic response monitoring

64 the drug efficiency and effectiveness during drug development (Weiner, 2012).

65 TUBERCULOSIS AND METABOLOMICS-APPROACH DIAGNOSIS

66 With the development of metabolomics, the diagnosis of bacterial infections

67 which requires prolonged incubation process can be shortened significantly thus easily

68 avoiding serious complications to patients within the given period. Tuberculosis, in

69 particular, can be diagnosed by culture-related procedures which requires around 21

70 days of culturing on agar due to its slow division rate of 12- to 24- hour period

71 (Sakamoto, 2012).

72 Tuberculosis (TB) is known as a potential deadly highly contagious disease

73 caused by the intracellular bacterial pathogen Mycobacterium tuberculosis. Typically,

74 the bacterium initially infects the lungs causing pulmonary tuberculosis but may

75 eventually spread throughout the body upon entry into the lymph nodes found within the

76 chest causing extrapulmonary tuberculosis. The transmission stage of the disease

77 occurs from droplet nuclei or airborne particles with infective material released orally
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78 (such as coughing or speaking) by infected individuals with pulmonary tuberculosis

79 (World Health Organization, 2016).

80 ANALYTICAL PLATFORM UTILIZING 2D GCxGC-TOFMS

81 There are multiple spectroscopic instruments that can be utilized in

82 metabolomics research including NMR, GC-MS and LC-MS (Wishart, 2013). The study

83 of Luier and Loots (2016) utilizes two dimensional gas chromatography (GCxGC)

84 coupled with time-of-flight mass spectrometer (TOFMS) where samples are identified

85 and quantified through separation using a system of primary and secondary capillary

86 columns, modulator and a detector which ultimately interpret the results and evaluate

87 such using its stored MS database to determine the sample metabolites/constituents.

88 The modulator placed between the two GC columns functions to trap, isolate and

89 reinject the eluates of the first column towards the second dimension or second column

90 for further separation.

91 The initial data from the first column obtained (primary dimension eluting peak)

92 combined with the final data obtained through the second column forms the GC x GC

93 chromatogram featuring the three dimensional plots of retention time vs signal plus the

94 additional dimension of 2D retention time (Shimadzu, 2012). Data processing of mass

95 spectral deconvolution, peak alignment and identification can be performed using

96 ChromaTOF software (Luier and Loots, 2016).

97 ADVANTAGES OF USING GC x GC-TOFMS

98 The extensive number of metabolites that are produced by an individual cannot

99 fully be detected and identified using one instrument/ technique and rather requires

100 multiple analytical techniques such as GC × GC–TOFMS (Ralston-Hooper et al., 2011).

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101 The GC × GC has the advantage of increasing the resolution or the degree of

102 differentiation between the chromatogram peaks and sensitivity due to further

103 separation of the fraction from the first column to the second through the modulation

104 process, allowing trace level detection and separating coelluted metabolites. Time-of-

105 flight mass spectroscopy (TOFMS) separates and individually detects the ionized

106 fragmented metabolites based on its ionic weight. GC x GC-TOFMS combines the

107 improved chromatographic resolution of the GC x GC and the analytical resolving power

108 of the TOFMS (Dekeirsschieter, 2012) creating a powerful instrument for separating the

109 several metabolites within the given sample.

110 STATISTICAL ANALYSIS USING PRINCIPAL COMPONENET ANALYSIS (PCA)

111 In a typical metabolomics study, an average of hundreds of metabolites are

112 measured but only few compounds are examined upon determining its significance in its

113 variations between two sample groups say TB-negative healthy control and TB- positive

114 patient groups.

115 Multi-statistical approach using various statistical tests like PCA, PLS-DA, effect

116 size and t-test are often utilized altogether to potentially compensate each method’s

117 flaws thus eliminating false-positive compounds. One particularly interesting method is

118 the principal component analysis (PCA) which functions to reduce the high dimensional

119 data through projection into a small dimensional subspace, usually a 2D graph

120 representation that captures as much variance of the original data as possible. PCA

121 designates the first principal component axis as the axis with the greatest variance and

122 the second principal component axis as the second largest variance that is orthogonal
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123 (perpendicular) to the first axis. Such comprises the linear combination of all metabolites

124 providing the groundwork for the visualization of the correlated data.

125 Unrelated variables to tuberculosis metabolomics with large variance may

126 obscure important metabolites with relatively small variance using PCA. Such important

127 metabolites may be detected by Pareto scaling the variables through dividing such by

128 the square root of its variance which ultimately reduces high variance entirely. When

129 combined with clustering analysis, the projected data in the PCA graph seen in Figure 1

130 will be clustered based on distance function where the distance between the data within

131 the same group are smaller compared to the distance to other clusters (Luier and Loot,

132 2016; Blekherman et al., 2011). PCA can be quantified using the modeling power

133 defined as the standard deviation per variable. A modeling power of 1 shows a

134 completely relevant variable (Wold, 1987) and can be distinguished between the control

135 and infected patients.

136 METABOLOMICS FOR TB URINE BIOMARKERS IDENTIFICATION

137 In Luier and Loots (2016), urine metabolic biomarkers selected through a multi-

138 statistical approach were used in detecting active tuberculosis (TB). Samples were

139 obtained from 30 TB-negative healthy control population and 46 culture-confirmed

140 active TB-positive patients. The samples were analyzed using GC x GC- TOFMS.

141 Overall, twelve compounds are identified to be a potential metabolite markers passing

142 the parameters set by the study from related literature. The PCA among other statistical

143 approach requires a modeling power > 0.5 lifted from Brereton (2013) as a cut-off

144 metabolite biomarker but ideally the PCA value should be closer to one for such to be

145 completely relevant (Wold, 1987). A total of 88 metabolites are within the said range but
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146 has been reduced to twelve upon further elimination from other statistical test to remove

147 the remaining 76 insignificant data.

148 Considering that Kamfar (2013) and Serra (2013) recognized twenty-five and

149 twenty TB urine metabolite markers respectively together with Luier and Loots (2016) of

150 twelve compounds, selection of urine metabolites usually are relatively few upon

151 statistical analysis completion. Additionally, Kamfar (2013) determined ribitol, the same

152 compound identified by Luier and Loots, to be one of two biomarkers for the M.

153 tuberculosis metabolism suggesting that the compound may arise from the bacterial cell

154 wall repeats, ribitol phosphate repeats. The remaining metabolites identified from all

155 papers are determined as markers for altered host metabolism.

156 The given papers all addressed the variations in urine sampling volume and

157 concentration through the utilization of creatinine as the internal standard. Due to its

158 relatively diverse presence in most cells, the normalization process of the detected

159 endogenous metabolite concentrations are expressed as ratios relative to the

160 individual’s creatinine level (Warrak et al., 2009) thus may provide relative consistency

161 across all samples regardless of the variations in terms of the extent of an individual’s

162 metabolic rate.

163 Since potential biomarker amino acids and certain lipids can cause significant

164 alterations under specific storage conditions yielding false results, Rotter et al. (2017)

165 recommends urine storage temperatures of at least -20C and to minimize the number of

166 freeze and thaw cycles to prevent its degradation for metabolomics research.
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167 METABOLOMICS FOR TB SPUTUM BIOMARKERS

168 Although sputum obtained from patients with HIV associated TB was proved

169 inconclusive by Kampfer (2013), research groups are still experimenting on the

170 reliability of sputum samples for the diagnosis of tuberculosis. Since conventional

171 methods utilizing microscopic examination of stained sputum smear on glass slide

172 (Arslan et al., 2010) aside from culture-related procedures involves sputum, such

173 samples may provide a more direct and sensitive diagnosis. Currently, little to no

174 dependable biomarkers has yet been identified (Schoeman et al., 2012).

175 Due to the uneven consistency and its varying viscosity among sputum samples,

176 reproducible results are difficult to obtain. In lieu of such, Schoeman et al (2012)

177 compared four sputum pre-extraction preparation methods (sputolysin, NALC-NaOH,

178 NaOH) to identify the most optimum procedure for sputum metabolomics research. The

179 samples were read using GCxGC-TOFMS and analyzed using PCA and PLS-DA. The

180 study concluded ethanol homogenization method to have the best extraction efficiency

181 and repeatability by which majority of the selected markers were elevated amounts of

182 several carbohydrates compounds in active-TB patients. Although NALC-NaOH has a

183 relatively poor detected limit, it work best for cell isolation method as such has been

184 able to differentiate TB infected sputum samples from control. The method also even

185 identified tuberculostearic acid (TBSA), a well known almost universal M. tuberculosis

186 biomarker, and other associated bacterial fatty acids.

187 Preez and Loots (2013) further studied sputum biomarkers adapting the ethanol

188 homogenization pre-extraction from the work of Schoeman et al (2012). Sputum

189 samples from 95 patients composing of sixty-one identified as TB-negative and thirty-
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190 four proven TB positive were subjected to the same instrumentation and minor

191 statistical data analysis alterations. With the twenty-two metabolites selected, thirteen

192 compounds were correlated to M. tuberculosis bacterial cell wall components including

193 fatty acids, mycolic acids and carbohydrates and seven markers to be of the host

194 response. Although TBSA was detected, its concentration was insufficiently elevated to

195 be considered a differentiating marker for patient sputum. Unlike Schoeman et al

196 (2012), the researches detected elevated levels of citramalic acid which signify the

197 presence of the citramalate cycle metabolic pathway being utilized by the pathogen as

198 seen in Figure 2. Such may be derived from the elevated glyoxylate and propionyl-CoA

199 levels in the glyoxylate cycle, a metabolic pathway bypassing the decarboxylation steps

200 to produce glucose from acetyl-CoA upon the host’s response to deprive the pathogen

201 of food sources. Preez and Loots (2013) identified such compound to be in vivo growth

202 M. tuberculosis markers.

203 RECOMMENDATIONS

204 Future reviews can highlight other statistical methods namely the multivariate

205 partial least squares-discriminant analysis (PLS-DA) and univariate unpaired t-test and

206 effect size biostatistical analysis not mentioned due to the limitations set within the

207 paper. Appreciating the essence of the multi-statistical approach to a qualitative and

208 quantitative identification of biomarkers requires such statistical tests. Also, other

209 spectroscopic instruments may be reviewed as the paper only focuses on GCxGC-

210 TOFMS. Additionally, future publications release after the review should be

211 incorporated, if not replace, outdated information provided.

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212 CONCLUDING REMARKS

213 In summary, metabolomics can provide the basis for alternative methods in

214 tuberculosis diagnosis as conventional methods of culture-related practices tend to be

215 slow and tedious. Alterations from the metabolite concentrations of infected patients

216 may provide insightful ideas for deriving or supporting hypothetical pathways involved in

217 the host-pathogen interaction. The selection for the differentiating marker metabolites

218 greatly depends on the quality of separation and the comprehensive statistical analysis

219 employing the multivariate approach to eliminate insignificant metabolite concentrations

220 with insufficient elevation levels. Two dimensional chromatography such as GCxGC-

221 TOFMS may provide better separation compared to single instrument/technique. Urine

222 and sputum sample mediums, although both noninvasive in nature, differs on sample

223 preparation. Yet, the metabolites selected from both mediums shows certain similarities

224 in terms of its classification: M. tuberculosis components, M. tuberculosis growth

225 markers and host response markers. The study may provide the groundwork towards

226 better understanding the overview of biomarker discovery through multistatistical

227 elimination of instrumental data analysis for tuberculosis characterization and host

228 metabolic interaction in terms of metabolomics approach.

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291 APPENDIX

292
293 Figure 1. Example of a PCA graph retrieved from Loots and Luier (2016) showing two
294 non overlapping clusters signifying a good separation between the groups
295

296
297 Figure 2. Citramate cycle upregulation upon M. tuberculosis forced conversion of
298 glucose from acetyl coa using glyoxylate cycle (retrieved from Preez and Loots,
299 2013)