Our data provide clear evidence that metabolic profiling reflects human metabolism. We calculated correlation of all metabolite concentration pairs. Clustering results revealed that metabolites in related functional contexts are highly correlated. This is also consistent with similar conclusions of a mouse study based on 67 studied metabolites 
. This demonstrates that metabolic profiles are biologically and statistically meaningful.
We applied our metabolic profiling to investigate the impact of cigarette smoking. Significant changes were observed mainly for clusters of lipid metabolites. Moreover, the 23 biomarkers that we could identify are all lipids, consistent with the observation that cell membranes are affected or damaged due to the influence of tobacco smoking 
. The physiological importance of lipids is illustrated by the numerous diseases to which lipid abnormalities contribute, including atherosclerosis, diabetes, obesity, and Alzheimer's disease 
. Lipids are major components of biological membranes, which maintain the integrity of cells and allow the compartmentalization of the cytoplasm into specific organelles. Cigarette smoke, then, might affect or even damage cell membranes, thus influencing the concentrations of related metabolites, namely the biomarkers discovered in this study.
Glycerophospholipid metabolism and ether lipid metabolism share one small molecule, 1-acyl-glycerone 3-phosphate 
. In the ether lipid metabolism pathway, a unique biochemical reaction is catalyzed by the enzyme alkylglycerone phosphate synthase (alkyl-DHAP, EC 220.127.116.11) resulting in the formation of the ether bond by replacement of the sn
-1 fatty acid with a long chain fatty alcohol (). The following biosynthesis steps in both ether lipid and diacyl-phospholipids converge in a reaction catalyzed by acylglycerone phosphate reductase (EC 18.104.22.168), which is utilized in the synthesis of both ether lipids and diacylated phopholipids 
. Following similar synthesis steps in the ether lipid and glycerophospholipid pathways, acyl-alkyl-phosphatidylcholines and diacyl- phosphatidylcholines will be either intermediate or end products of the two pathways. A
ynthase is encoded by AGPS
, the gene in Homo sapiens
. Interestingly, in a human lung project 
, it was found that the AGPS
gene expression is highly increased in former and non-smokers relative to current smokers (Figure S6
). The upregulation of alkyl-DHAP seen in our metabolite profiles and independent in a gene expression study further corroborates its role in defects linked to smoking.
In smokers, reduced or lack of activity of the enzyme alkyl-DHAP may further regulate the ratio of acyl-alkyl- to diacyl- phosphatidylcholines in the ether lipid- and glycerophospholipid pathways.
Human newborns of nicotine-exposed pregnancies reveal growth retardation due to impairment of uteroplacental circulation as a result of the vasoconstricting effect of nicotine 
. Studies in the rat showed that mechanisms involving deterioration development of fetal alveolae and up regulation of lipid peroxidation by P450 enzymes 
. In this respect, our study provides a novel insight in that nicotine affects plasmalogen levels. Plasmalogen comprise a major portion of the phospholipids in the adult human central nervous system. Overall, it was shown that newborn plasmalogen levels are relatively low (7% of total phospholipid mass) 
. As the plasmalogens may influence the surface tension in alveolar surfactants 
, we hypothesize that this would be triggered as well by nicotine. Isolated (single gene defect) deficiency in human AGPS
gene function further indicate that this gene is embryonic essential and its inactivation leads to a lethal phenotype 
. This gene is also affected in other disorders of biogenesis, such as Zellweger Syndrome or Rhizomelic chodrodisplasia punctata type 3 
. Therefore, all factors that influence ether lipid balance, including nicotine as shown here, are of potential risk to human health.
Our metabolic profiling provides a snapshot of the complex human metabolome. More detailed profiles in combination with kinetic experiments for blood sample collection are necessary to draw a comprehensive map and will reflect physiological processes as responses to developmental, genetic or environmental factors 
The 198 detected metabolites are a large dataset in human blood samples, though much smaller in comparison to the human metabolomics database, which currently has a collection of about 2,500 metabolites 
. Previously identified biomarkers of ND metabolites 
, such as nicotine, cotinine and carbon monoxide, are not in our dataset. In addition to further technical improvements in metabolite detection sensitivity, samples from urine and other tissues are needed to enlarge our dataset.
Our study represents the first large screen of metabolites to study the influence of cigarette smoking on human blood serum. Albeit we are aware that the sample size of current smokers in this pilot study is small, our results are encouraging and we could show that the smokers are distinctly separated from former and non-smokers. In general, similar observations were obtained at an individual level, though with large variance. An interesting observation is that former smokers were found to be separated from non-smokers, suggesting that the influence of cigarette smoke in human blood remains for years. We note, however, that the group of former smokers is not well-defined in this study because the time when these individuals quit smoking is not documented. Damage to the cell membrane from smoking may be reversed over time due to the repair mechanisms in the human body 
The independent but consistent observation from our metabolic profile analysis and AGPS gene expression data may indicate that smoking affects the enzymatic activity of alkyl-DHAP and thus change the ratios of two types of metabolites. However, the overall fat metabolism is likely not be affected, as the BMI does not vary significantly between the groups of current, former and non-smokers (data not shown).
Our analyses suggest that small molecules that activate the enzyme alkyl-DHAP could be developed to treat plasmalogens deficiency disorders that are caused by nicotine consumption in smokers.