Accepted Manuscript

Urine volatile organic compounds as biomarkers for minimal change type nephrotic
syndrome

Desheng Liu, Nana Zhao, Mingao Wang, Xin Pi, Yue Feng, Yue Wang, Hongshuang
Tong, Lin Zhu, Changsong Wang, Enyou Li

PII: S0006-291X(17)32572-X
DOI: 10.1016/j.bbrc.2017.12.164
Reference: YBBRC 39164

To appear in: Biochemical and Biophysical Research Communications

Received Date: 19 December 2017

Accepted Date: 29 December 2017

Please cite this article as: D. Liu, N. Zhao, M. Wang, X. Pi, Y. Feng, Y. Wang, H. Tong, L. Zhu, C. Wang,
E. Li, Urine volatile organic compounds as biomarkers for minimal change type nephrotic syndrome,
Biochemical and Biophysical Research Communications (2018), doi: 10.1016/j.bbrc.2017.12.164.

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 ACCEPTED MANUSCRIPT

Urine Volatile Organic Compounds as Biomarkers for Minimal Change type Nephrotic

Syndrome

Desheng Liu2, Nana Zhao2, Mingao Wang3, Xin Pi2, Yue Feng1, Yue Wang1，Hongshuang Tong1, Lin

Zhu3, Changsong Wang1*, Enyou Li2*

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1
Department of Critical Care Medicine, the Third Affiliated Hospital of Harbin Medical University,

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Harbin, China.

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Department of Anesthesiology, the First Affiliated Hospital of Harbin Medical University, Harbin,

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China

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Department of Nephrology, the First Affiliated Hospital of Harbin Medical University, Harbin, China
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*Corresponding authors:

Changsong Wang. E-mail: changshongwangicu@163.com

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Tel: 86-45185718862 Fax number: 86-458685555725

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Department of Critical Care Medicine, the Third Affiliated Hospital of Harbin Medical University,
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Harbin, China.

Enyou Li. E-mail: lienyou1543@163.com

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Tel: 86-45185555566 Fax number: 86-458685555725

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Department of Anesthesiology, the First Affiliated Hospital of Harbin Medical University, Harbin,
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China.

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Desheng Liu. E-mail: liudeshengmz@sina.com

Nana Zhao. E-mail: 767721743Q@qq.com

Mingao Wang. E-mail: 1029031687@qq.com

Xin Pi. E-mail: 758819438@qq.com

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Yue Feng. E-mail: 1175635758@qq.com

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Yue Wang. E-mail: 1206743386@qq.com

Hongshuang Tong. E-mail: 623521637@qq.com

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Lin Zhu. E-mail: 370774491@qq.com

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Changsong Wang. E-mail: changsongwang@aliyun.com
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Abstract

Urinary volatile organic compounds (VOCs) profiling has recently received considerable attention

because it can be obtained noninvasively and conveniently while it can be successfully used in a

variety of diseases and can provide unique biomarkers. The aim of current study was to investigate

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potential biomarkers between minimal change type nephrotic syndrome (MCNS) and normal. Urinary

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samples were collected from 38 minimal change type nephrotic syndrome patients and 15 healthy

controls. Solid phase microextraction (SPME) and chromatography– mass spectrometry (GC-MS) were

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used to analysis the urinary metabolites. To deal with the final data, the statistical methods principal

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component analysis (PCA) and orthogonal partial least-squares discriminant analysis (OPLSDA) were
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performed. Six specific VOC biomarkers were present at abnormal levels in the urine of MCNS

patients. These VOCs included trans-2,2-dimethyl-4-decene; pyrrole; carbamic acid, monoammonium

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salt; 1-butyne, 3,3-dimethyl-; diisopropylamine; and 4-heptanone. These biomarkers may be useful as a
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new diagnostic method and for monitoring the prognosis for MCNS patients.
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Keywords Minimal change type nephrotic syndrome, volatile organic compounds, Solid-Phase
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Microextraction, gas chromatography-mass spectrometry, diagnostic method

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List of abbreviations
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VOCs Volatile organic compounds

MCNS Minimal change type nephrotic syndrome

SPME Solid phase microextraction

GC-MS Chromatography– mass spectrometry

PCA Principal component analysis

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OPLSDA Orthogonal partial least-squares discriminant analysis

MCD Minimal change disease

FSGS Focal segmental glomerulosclerosis

OPLSDA Orthogonal partial least-squares discriminant analysis

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VIP Variable importance in the projection

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SD Standard deviation

SCR Serum creatinine

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ALB Plasma albumin

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Introduction

Minimal change type nephrotic syndrome (MCNS) is the most common cause of nephrotic
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syndrome in children, accounting for 70% and 25% of primary nephrotic syndromes in children and
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adults, respectively[1,2]. The disease has higher incidence in boys and is inversely proportional to age,
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with the incidence decreasing after puberty [3,4].

MCNS is usually triggered by immunogenic stimuli, such as viral infection, vaccination or

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allergens [5]. The pathological types and clinical features of nephrotic syndrome determine its
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prognosis and treatment. MCNS is a pathological diagnosis, often having typical clinical
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manifestations of nephrotic syndrome, i.e., massive proteinuria, hypoalbuminemia, edema, and

hyperlipidemia. Most people are sensitive to glucocorticoid therapy for MCNS, and approximately

60% of patients will relapse [1]. To reduce recurrence, a combination of glucocorticoids and cytotoxic

agent therapy has been adopted in China and abroad in recent years.

MCNS diagnosis mainly relies on clinical manifestations and laboratory test results, the

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confirmation of which also relies on the pathological diagnosis of renal biopsy specimens. It is

necessary for children who are glucocorticoid resistant or dependent and adults with MCNS to confirm

their diagnosis and to guide the treatment and prognosis via renal biopsy. However, the technology is

costly, invasive and prone to cause complications; it is absolutely contraindicated in cases of significant

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bleeding tendency, severe hypertension, solitary kidney, small kidney, psychosis or non-cooperative

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patients [6,7]. In addition, if a sufficient tissue specimen is not available, the pathological diagnosis of

renal biopsy may not be able to provide an accurate diagnosis [8]. Therefore, renal biopsy has

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limitations, and multiple punctures are not applicable, factors that restrict its clinical applications and

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the feasibility of patient follow-up. Thus, it is necessary to identify a new diagnostic technique that is
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precise, non-invasive, and able to continuously monitor the disease progression and the response to

therapy to replace renal biopsy.

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Recently, a growing number of researchers have become dedicated to the research of urinary
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metabonomics, including VOC profiling and urinary non-volatile metabolic profiles. In particular,
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urinary levels of nucleosides as biomarkers of cancers have already been applied to the identification of

cases of leukemia [9], hepatocellular carcinoma [10], colorectal cancer [11], and breast cancer [12],
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which are not associated with patient age or gender. In addition, Vanessa Pérez et al. [13] proposed that
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urinary peptide profiling, as a type of urinary metabonomics, could differentiate minimal change
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disease (MCD) and primary focal segmental glomerulosclerosis (FSGS), which are also not associated

with patient age or gender.

Notably, urinary VOC profiling has recently received considerable attention because it is an

ideal source of biomarkers and can be obtained noninvasively and conveniently. In fact, urinary VOC

profiling can be successfully used in a variety of diseases and can provide unique biomarkers. Wu et al.

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[14].confirmed that the pattern of urinary VOCs in patients with hepatocellular carcinoma was different

from those of healthy controls. Jobu et al. [15] demonstrated that urinary VOC metabolites in bladder

cancer patients were more sensitive than urine cytology. CL Silva et al. [16] concluded that different

urinary volatile metabolomic profiles for healthy people and cancer patients were successfully

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recognized. Therefore, based on the above, we postulated that kidney disease can affect the

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composition and content of urinary VOCs. However, it is still unknown whether urinary VOC profiling

can be used to diagnose MCNS.

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We assumed that, compared with healthy subjects, MCNS patients might produce potential

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urinary VOCs. Our main goals of this study involved focusing on urinary VOC profiling of MCNS to
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improve the diagnosis of MCNS patients.

Statistical Analysis
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Normalized data were exported to SIMCA-p 11.5 platform for principal component analysis (PCA) and
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orthogonal partial least-squares discriminant analysis (OPLSDA) after being performed total area
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normalization. In order to guard against overfitting, we applied the default seven-round

cross-validation in the SIMCA-P software, and performed permutation tests using 100 iterations to
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further validate the supervised model. In addition, the nonparametric Kruskal–Wallis rank sum test was
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performed for each metabolite. The potential metabolic biomarkers were selected based on variable
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importance in the projection (VIP) values calculated from the OPLSDA model of 1.2. In this study, the

P value was set at 0.05.

Material and Methods

Human Subjects

The present experiments were conducted in accordance with the Declaration of Helsinki. The

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protocol in this study was approved by the Ethics Committee at the First Affiliated Hospital of Harbin

Medical University (No.201314), and written informed consent was obtained from patients prior to

study enrollment. This study was conducted between Dec. 2013 and Oct. 2014 at the Department of

Anesthesiology at the First Affiliated Hospital of Harbin Medical University.

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Included in this study were men between 16 and 65, and women between 20 and 61 years of age

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identified as ASA I and II individuals and scheduled for renal biopsy. In addition to the group of kidney

disease patients, this study also examined healthy volunteers, whose inclusion criterion were the

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absence of a history of kidney diseases and urinary infection, renal function were normal at the same

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time. This study involved a total of 53 individuals, including 38 individuals with MCNS and 15 healthy
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volunteers. The demographic characteristics are summarized in Table 1.

As detailed in Table 1, the normal control group involved 15 patients. The 38 MsPGN patients
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who were selected included 21 males and 17 females. The mean age of the MsPGN patients was 33 y,
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with a standard deviation (SD) of 13.8 y, and 15 of these patients were smokers.
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Urine Collection.

Portions of midstream urine samples of patients without breakfast were collected severally in the
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morning before analysis. All samples were analyzed within 1h after they were collected.
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Solid-Phase Microextraction(SPME).
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A manual SPME holder with carboxen/ polydimethylsiloxane (CAR/PDMS) fibers of 75um

thickness was purchased from Supelco (Bellefonte, USA). The SPME fiber was inserted into the vial

and exposed to the gaseous sample for 20 min at 40 . Subsequently, the desorption of volatiles

occurred in the hot GC injector at 200 for 2 min.

Gas Chromatography-Mass Spectrometry (GC/MS) Analysis.

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Analysis was performed on a GC/MS (Shimadzu GC-MS QP 2010, Shimadzu, Japan) equipped

with a DB-5MS (length 30 m *ID 0.250*film thickness 0.25 um; Agilent Technologies, USA) plot

column. Injections were performed in the splitless mode. The temperature of injector was 200 . The

flow rate of the helium (99.999%) carrier gas was kept constant at 2 ml min-1. The column temperature

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was held at 40 for 1 min to concentrate the hydrocarbons at the head of the column and then

min-1 to 200 min-1 to 230

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increased by 5 for 1 min, then ramped 15 . The MS analyses were

performed in full-scan mode, using a scan range from 35–350 amu. The ion source was maintained at

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230 , and an ionization energy of 70 eV was used for each measurement.

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Extraction and Pretreatment of the GC/MS Raw Data.
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Raw GC/MS data were converted into CDF format (NetCDF) files using Shimadzu GCMS

Postrun Analysis software and subsequently processed using the XCMS toolbox (http://metlin.scripps.
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edu/download/). The XCMS parameters consisted of the default settings with the following exceptions:
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xcmsSet (fwhm=8, snthresh=6, max=200); retcor (method=‘‘linear,’’ family=‘‘gaussian,’’

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plottype=‘‘mdevden’’); and a bandwidth of eight for first grouping command and four for the second

grouping command. The data set of the aligned mass ions was exported from XCMS and could be
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further processed using Microsoft Excel to normalize the data prior to multivariate analyses.
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Results
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Additionally, the VOCs in the exhaled breath of 38 patients with MCNS and 15 healthy subjects

were analyzed using GC-MS. Between the patients with MCNS and the healthy subjects, we obtained

two-dimensional PCA score plots using 215 parameters, with good separation tendency (Figure 1). A

PLS-DA score plot was then used to separate these two groups with three components (R2X=0.362,

R2Y=0.837, and Q2=0.697) (Figure 2). A validation plot obtained from 100 permutation tests revealed

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that all R2 and Q2 values calculated from the permutated data were lower than the original values

(Figure 3). All VIP values of the examined factors in either the PLS-DA model or OSC-PLS-DA model

were calculated. The distinct metabolic biomarkers were selected based on the standard of a VIP value

greater than 1.2 using the NIST 11 database with a similarity threshold of 75% (Tables 2).

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Discussion

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In a previous study, Solid-Phase MicroExtraction (SPME), a powerful, reliable and selective

technique, was applied to the analysis of urinary VOCs and was combined with GC-MS [16,17]. Based

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on this advantage, our study elucidated potential biomarkers of urinary metabolomics in MCNS

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patients using SPME-GC-MS and multivariate data analysis.
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VOCs are emitted by several biological fluids (e.g., urine, blood) and tissues (e.g., lungs, bowels).

Urine is a promising source of volatile markers due to the kidneys’ function of preconcentration. Indeed,
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approximately 230 types of urinary volatiles have been detected and identified in human urine [17-20] .
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These volatiles include pyrroles, ketones, hydrocarbons, aldehydes, terpenes, furans, and heterocyclic
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and sulfur compounds. Currently, a large number of trials have primarily focused on the biomarkers of

urinary metabolomics (e.g., VOCs) for many diseases, such as detecting urinary tract infections [21]
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and colorectal cancer [22], monitoring lung cancer development [23], differentiating pathological types
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of lung cancer [24], and discriminating tuberculosis patients from healthy subjects [25]. A previous
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study differentiated glomerular kidney disease patients and healthy subjects by urinary peptides [26].

However, thus far, relatively few studies have investigated the urinary VOCs in MCNS.

In our study, different urinary volatile profiles for healthy human and MCNS patients were

recognized by SPME-GC-MS. These VOCs included trans-2, 2-dimethyl-4-decene; pyrrole; carbamic

acid, monoammonium salt; 1-butyne, 3,3-dimethyl-; diisopropylamine; and 4-heptanone. Compared

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with healthy subjects, these specific VOC biomarkers were present at abnormal levels in the urine of

MCNS patients.

Our results revealed that 4-heptanone, a type of ketone, exhibited a decreasing concentration trend

in MCNS patients. In fact, there were relatively abundant ketones in human urine [18,20,27] . Human

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breath, blood and urine contained a major ketone, acetone (2-propanone). Ketones can originate from

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exogenous sources and the decarboxylation of oxo-acids[18]. Perbellini et al. confirmed that

2-heptanone was proposed to be one of the n-heptane metabolites[28]. It is mainly formed in

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hepatocytes from acetoacetate during the decarboxylation of excess acetyl–CoA in healthy humans[29].

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A previous study demonstrated that 4-heptanone levels were increased in the blood and breath of
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end-stage renal disease patients[30]. In addition, Hans Gunther Wahl et al. found that 4-heptanone was

significantly elevated in the plasma of hemodialysis patients compared with controls[31]. Therefore,
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we speculate that any impairment of kidney function may reduce the filtration of 4-heptanone, which is
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consistent with the study of H. M. LIEBICH [32]. The decreasing concentration trend of 4-heptanone
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detected in the urine of the MCNS patients suggests that it is difficult for MCNS patients to filter

4-heptanone through the kidney’s glomerulus, which results in 4-heptanone remaining in blood and
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breath at increased levels. However, the mechanism of this phenomenon in MCNS patients is unclear
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and demands additional studies.

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One heterocyclic compound, pyrrole, also exhibited a decreasing concentration trend in MCNS

patients. It is still unclear how pyrroles are produced in the human body and appear in the urine.

Jackson et al. found that endogenous pyrroles can form in the central nervous system by the

metabolism of amino sugars and N-acetylneuraminic acid [33]. Additionally, endogenous pyrroles are

by-products in the synthesis process of porphyrin and bile pigment and are oxidation products of

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hemopyrrole and bilirubin [34]. Urine pyrroles can also be provided exogenously in beverages and

meat [35]. Hence, in our study, to avoid the influence of exogenous factors on urinary VOCs, the

middle portions of the early morning fasting urine of normal subjects and MCNS patients, both of

whom were subjected to more than eight hours of fasting, were used. However, it is still unclear which

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endogenous factors gave rise to the reduced urinary VOCs in the MCNS patients.

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Whatever the interaction mechanism, the significant decrease in pyrrole concentration indicates

that pyrrole is a potential biomarker for distinguishing the differences between normal subjects and

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MCNS patients.

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There was a decrease in urinary excretion of trans-2,2-Dimethyl-4-Decene, which is an olefin, in
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the MCNS groups. We found no reports on trans-2,2-Dimethyl-4-Decene in urinary samples. However,

many previous studies have demonstrated isoprene, which is a by-product of cholesterol

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biosynthesis[36-38] and is also a olefin. Isoprene is the major hydrocarbon present in exhaled breath
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and blood [39,40]. We postulated that the origin of trans-2,2-Dimethyl-4-Decene is similar to that of
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isoprene. In addition, squalene, another olefin, is a cholesterol precursor and can form some VOCs by

peroxidation [41]. The cause of MCNS is unknown, but the disease may be preceded by viral infection,
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allergic reactions, or recent immunizations [1]. Therefore, it may be speculated that MCNS could be
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the result of peroxidation due to the above-stated possible causes. Further,

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trans-2,2-Dimethyl-4-Decene was consumed in the MCNS patients. Hence, volatile

trans-2,2-Dimethyl-4-Decene biomarkers are also a potential biomarker to distinguish the differences

between normal subjects and MCNS.

Three other decreased biomarkers, Carbamic acid, monoammonium salt; 1-Butyne, 3, 3-dimethyl-;

and Diisopropylamine, were not found in the literature as biomarkers in urinary samples. Further

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investigation and identification of potential biomarkers is needed.

Compared with healthy subjects, the unique VOC metabolites are present at reduced

concentrations in the body of MCNS patients, suggesting that MCNS patients have a unique VOC

profile. This profile may be useful as a diagnostic assay and for monitoring the prognosis in MCNS

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patients.

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Funding This work was supported by grants from the National Natural Science Foundation of China

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(No.81402462).

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Authors' Contributions EL and CSW have provided the conception and design. DSL and DW have
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been involved in drafting the manuscript. NNZ, MAW, XP, YF, YW, HST and LZ performed samples

collection and data analysis. All authors have read and given final approval of the version to be
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published.
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Compliance with ethical standards

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Competing interests All authors declare that they have no conflict of interest.

Ethical approval Authors state that they have obtained appropriate institutional review board approval
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or have followed the principles outlined in the Declaration of Helsinki for all human or animal
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experimental investigations. In addition, for investigations involving human subjects, informed consent
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has been obtained from the participants involved.

References

1 Eddy AA, Symons JM, Nephrotic syndrome in childhood, Lancet. 362 (2003) 629-639.

12
 ACCEPTED MANUSCRIPT

2 Nakayama M, Katafuchi R, Yanase T, et al., Steroid responsiveness and frequency of relapse in

adult-onset minimal change nephrotic syndrome, American journal of kidney diseases : the official

journal of the National Kidney Foundation. 39 (2002) 503-512.

3 Tarshish P, Tobin JN, Bernstein J,et al., Prognostic significance of the early course of minimal

PT
change nephrotic syndrome: Report of the international study of kidney disease in children, Journal of

RI
the American Society of Nephrology : JASN. 8 (1997) 769-776.

4 Nephrotic syndrome in children: Prediction of histopathology from clinical and laboratory

SC
characteristics at time of diagnosis. A report of the international study of kidney disease in children,

U
Kidney international. 13 (1978) 159-165.
AN
5 Mathieson PW: Minimal change nephropathy and focal segmental glomerulosclerosis, Seminars

in immunopathology. 29 (2007) 415-426.

M

6 Corapi KM, Chen JL, Balk EM, et al., Bleeding complications of native kidney biopsy: A
D

systematic review and meta-analysis, American journal of kidney diseases : the official journal of the
TE

National Kidney Foundation. 60 (2012) 62-73.

7 Eiro M, Katoh T, Watanabe T, Risk factors for bleeding complications in percutaneous renal
EP

biopsy, Clinical and experimental nephrology. 9 (2005) 40-45.

C

8 Fogo AB, Approach to renal biopsy, American journal of kidney diseases : the official journal of
AC

the National Kidney Foundation. 42 (2003) 826-836.

9 Zambonin CG, Palmisano F, Electrospray ionization mass spectrometry of

5-methyl-2'-deoxycytidine and its determination in urine by liquid chromatography/electrospray

ionization tandem mass spectrometry, Rapid communications in mass spectrometry : RCM. 13 (1999)

2160-2165.

13
 ACCEPTED MANUSCRIPT

10 Yang J, Xu G, Zheng Y, et al., Diagnosis of liver cancer using hplc-based metabonomics avoiding

false-positive result from hepatitis and hepatocirrhosis diseases, Journal of chromatography B,

Analytical technologies in the biomedical and life sciences. 813 (2004) 59-65.

11 Zheng YF, Yang J, Zhao XJ, et al., Urinary nucleosides as biological markers for patients with

PT
colorectal cancer, World journal of gastroenterology : WJG. 11 (2005) 3871-3876.

RI
12 Zheng YF, Kong HW, Xiong JH, et al., Clinical significance and prognostic value of urinary

nucleosides in breast cancer patients, Clinical biochemistry. 38 (2005) 24-30.

SC
13 Perez V, Ibernon M, Lopez D, et al., Urinary peptide profiling to differentiate between minimal

U
change disease and focal segmental glomerulosclerosis, PloS one. 9 (2014) e87731.
AN
14 Wu H, Xue R, Dong L, et al., Metabolomic profiling of human urine in hepatocellular carcinoma

patients using gas chromatography/mass spectrometry, Analytica chimica acta. 648 (2009) 98-104.
M

15 Jobu K, Sun C, Yoshioka S, et al., Metabolomics study on the biochemical profiles of odor
D

elements in urine of human with bladder cancer, Biological & pharmaceutical bulletin. 35 (2012)
TE

639-642.

16 Silva CL, Passos M, Camara JS, Investigation of urinary volatile organic metabolites as potential
EP

cancer biomarkers by solid-phase microextraction in combination with gas chromatography-mass

C

spectrometry, British journal of cancer. 105 (2011) 1894-1904.

AC

17 Smith S, Burden H, Persad R, et al., A comparative study of the analysis of human urine

headspace using gas chromatography-mass spectrometry, Journal of breath research. 2 (2008) 037022.

18 Mills GA, Walker V, Headspace solid-phase microextraction profiling of volatile compounds in

urine: Application to metabolic investigations, Journal of chromatography B, Biomedical sciences and

applications. 753 (2001) 259-268.

14
 ACCEPTED MANUSCRIPT

19 Statheropoulos M, Sianos E, Agapiou A, et al., Preliminary investigation of using volatile organic

compounds from human expired air, blood and urine for locating entrapped people in earthquakes,

Journal of chromatography B, Analytical technologies in the biomedical and life sciences. 822 (2005)

112-117.

PT
20 Wahl HG, Hoffmann A, Luft D, et al., Analysis of volatile organic compounds in human urine by

RI
headspace gas chromatography-mass spectrometry with a multipurpose sampler, Journal of

chromatography A. 847 (1999) 117-125.

SC
21 Perbellini L, Brugnone F, Cocheo V, et al., Identification of the n-heptane metabolites in rat and

U
human urine, Archives of toxicology. 58 (1986) 229-234.
AN
22 Arasaradnam RP, McFarlane MJ, Ryan-Fisher C, et al., Detection of colorectal cancer (crc) by

urinary volatile organic compound analysis, PLoS ONE. 9 (2014) e108750.

M

23 Yuan J-M, Butler LM, Gao Y-T, et al., Urinary metabolites of a polycyclic aromatic hydrocarbon
D

and volatile organic compounds in relation to lung cancer development in lifelong never smokers in the
TE

shanghai cohort study, Carcinogenesis. 35 (2014) 339-345.

24 Hanai Y, Shimono K, Matsumura K, et al., Urinary volatile compounds as biomarkers for lung
EP

cancer, Bioscience, biotechnology, and biochemistry. 76 (2012) 679-684.

C

25 Banday KM, Pasikanti KK, Chan EC, et al., Use of urine volatile organic compounds to
AC

discriminate tuberculosis patients from healthy subjects, Analytical chemistry. 83 (2011) 5526-5534.

26 Navarro-Munoz M, Ibernon M, Bonet J, et al., Uromodulin and alpha(1)-antitrypsin urinary

peptide analysis to differentiate glomerular kidney diseases, Kidney & blood pressure research. 35

(2012) 314-325.

15
 ACCEPTED MANUSCRIPT

27 Mochalski P, Krapf K, Ager C, et al., Temporal profiling of human urine vocs and its potential

role under the ruins of collapsed buildings, Toxicology mechanisms and methods. 22 (2012) 502-511.

28 Guernion N, Ratcliffe NM, Spencer-Phillips PT, et al., Identifying bacteria in human urine:

Current practice and the potential for rapid, near-patient diagnosis by sensing volatile organic

PT
compounds, Clinical chemistry and laboratory medicine : CCLM / FESCC. 39 (2001) 893-906.

RI
29 Schwarz K, Pizzini A, Arendacka B, et al., Breath acetone-aspects of normal physiology related to

age and gender as determined in a ptr-ms study, Journal of breath research. 3 (2009) 027003.

SC
30 Mochalski P, King J, Haas M, et al., Blood and breath profiles of volatile organic compounds in

U
patients with end-stage renal disease, BMC nephrology. 15 (2014) 43.
AN
31 Wahl HG, Hong Q, Hildenbrand S, Liebich H: 4-heptanone is a metabolite of the plasticizer

di(2-ethylhexyl) phthalate (dehp) in haemodialysis patients, Nephrology, dialysis, transplantation

M

official publication of the European Dialysis and Transplant Association - European Renal Association.
D

19 (2004) 2576-2583.
TE

32 Liebich HM, Woll J, Volatile substances in blood serum: Profile analysis and quantitative

determination, Journal of chromatography. 142 (1977) 505-516.

EP

33 Jackson JA RH, Neathery SS, Mayer K, Urine pyrrolles revisited, Journal of Orthomolecular
C

Medicine. 15 (2000) 47–48.

AC

34 Jackson JA RH, Neathery SS, Riordan NH, Urinary pyrrole in health and disease, Journal of

Orthomolecular Medicine. 12 (1997) 96–98.

35 GA B, Fenaroli’s handbook of flavor ingredients, Boca Raton, London, New York Washington DC,

CRC Press. (2005).

16
 ACCEPTED MANUSCRIPT

36 Hyspler R CS, Zadak Z, Gasparic J, Breath isoprene as a measure of the depression in cholesterol

synthesis in intensive care patients, Atherosclerosis Supplements. 2 (2001) 102.

37 Zadak Z HR, Crhova S, Gasparic J, et al., Isoprene in expired air as a marker of cholesterol

synthesis for the statin therapy monitoring, Atherosclerosis. 144 (1999) 206.

PT
38 DeMaster EG NH, Isoprene, an endogenous constituent of human alveolar air with a diurnal

RI
pattern of excretion, Life Sci. 22 (1978) 91–97.

39 King J, Koc H, Unterkofler K, et al., Physiological modeling of isoprene dynamics in exhaled

SC
breath, Journal of theoretical biology. 267 (2010) 626-637.

U
40 Miekisch W, Schubert JK, Vagts DA, et al., Analysis of volatile disease markers in blood, Clinical
AN
chemistry. 47 (2001) 1053-1060.

41 Stein RA, Mead JF, Small hydrocarbons formed by the peroxidation of squalene, Chemistry and
M

physics of lipids. 46 (1988) 117-120.

D
TE
C EP

Table 1 Demographic characteristics of the study subjects

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Table 2 Related metabolites that exist at abnormal levels in the urine from MCNS patients.

Figure legends

Figure. 1 PCA score plot: (6 components, R2X=0.781, Q2=0.553)

Figure. 2 OPLSDA score plot: (2 components, R2X=0.362, R2Y=0.837, Q2=0.697)

Figure. 3 PLSDA validation plot intercepts: R2=(0.0, 0.148); Q2=(0.0,-0.263).

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Table 1
Normal MCNS
subjects（n） 15 38
age 35(7.6) 33(13.8)
male 7 21
female 8 17
smokers（n） 3 15
SCR(umol/L) 60.6(8.4) 73.0(29.5)

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ALB(g) 47.9(4.7) 22.1(7.3)
24h urine protein (g) 0.06(0.04) 3.8(2.1)

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Table 2
Potential biomarker RT p-value FC VIP
4-Decene, 2,2-dimethyl-, (E)- 843.41 0.002069 2.663267 1.5469
Pyrrole 189.02 0.000022 2.925238 3.55659
Carbamic acid, monoammonium salt 71.66 0.012750 2.281669 9.64736
1-Butyne, 3,3-dimethyl- 195.17 0.017817 1.515409 3.13792
Diisopropylamine 89.68 0.018791 1.678709 1.27765
4-Heptanone 333.48 0.000128 2.394785 4.87288

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Highlights

1、Compared with healthy subjects, the unique VOC metabolites are present at

reduced concentrations in the body of MCNS patients, suggesting that MCNS patients

have a unique VOC profile.

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2、These biomarkers may be useful as a new diagnostic method and for monitoring the

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prognosis for MCNS patients.

3、These biomarkers may be useful as a new diagnostic method and for monitoring the

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