Table 1.

Biomarkers meriting further evaluation for use with BioSCIM.^a

Biomarkerb	Example	Physiological role or origin	Limitations/advantages
Isoprostanes (IsoPs)	The IsoPs represent the archetype biomarker for the original conceptualization of BioSCIM. They are not discussed in this paper because they have already been covered in depth (Daughton, 2012b).	Broad class of prostaglandin-like free-radical catalyzed oxidation products from certain polyunsaturated fatty acids (in contrast to the cyclooxygenase-formation of the analogous prostaglandins). A well-known example is 15-F2t-IsoP (8-isoPGF2α).	IsoPs serve as time-integrative biomarkers of systemic oxidative stress. They are extensively excreted in urine and display excellent chemical stability. Extensive array of analytical methodology as well as clinical studies already exist. But only preliminary studies relevant to BioSCIM have been published. IsoPs merit more study.
Desmosines	Comprise two non-natural amino acids, each substituted with four lysine residues. Desmosine (DES: 1,2,3,5-tetrasubstituted pyridinium amino acid) and isodesmosine (IDES: 1,3,4,5-tetrasubstituted pyridinium amino acid).	Serve as cross-links conferring elasticity to the protein elastin. Occur only in elastin, which is essential for flexibility of connective tissues. Only a finite supply in body, as synthesis is repressed after growth ceases. Extensively excreted in urine only upon damage by disease or injury to elastin, as well as from normal aging.	Excretion from normal aging process may obscure accelerated release as a result of disease. Excretion trends downward as elastin supply is depleted in advanced disease states. Unusual biomarker in that the body can only excrete a fixed amount over a lifetime. Very chemically stable. Excretion may be independent of drug therapy. Significant, extraneous dietary sources not known. Considerable published research.
Bone Turnover Markers (BTMs)	The principal urinary BTMs are NTX and CTX [“N-terminal telopeptide crosslinks” (aminoterminal collagen crosslinks) and “C-terminal telopeptide crosslinks” (carboxyterminal collagen crosslinks), respectively] along with the cross-linking pyridinolines [pyridinoline (PYD) and deoxypyridinoline (DPD, or D-PYR)].	Both of these classes are integral to the physical, cross-linked structure and stability of type I collagen, which is the major constituent of the organic matrix of bone. They are released during bone resorption (breakdown), which is a natural process required for maintaining bone health and strength but whose balance is disrupted by a number of diseases. This leads to elevated urinary BTM levels.	Substantial intra-individual variation in diurnal excretion. It is therefore unclear if collective population-wide BioSCIM data would reveal meaningful trends. Resorption can be exacerbated by a number of drug therapies. Excreted levels may not be influenced by diet. But unknown is whether BTMs in raw or digested foods might be released to sewage and therefore confound BioSCIM data.
Pterins	Neopterin is a member of this class of biochemicals that share a 4-keto-2-amino pteridine ring.	Neopterin is a catabolic by-product of cyclic guanosine triphosphate and is generated primarily as a result of activation of the cell-mediated immune system. Produced in large quantities as a result of a wide spectrum of infectious diseases and some diseases caused by—or associated with—excessive inflammation.	Occurs as a natural product in some foods, such as tomatoes, spinach, and beets, which, if entering sewers, could confound excreted levels resulting from endogenous production. Large intra-individual natural daily variance in excretion. Susceptible to photolysis. Chemically stable in urine. Excreted in substantial quantities. Serves to integrate the collective stress from a large number of adverse conditions.
mtDAMPs	“Mitochondrial-derived damage-associated molecular patterns” are members of larger classes called “alarmin” or “danger signal” molecules. One in particular is fMLP, the tri-peptide N-formylmethionine-leucylphenylalanine.	fMLP is a chemotactic peptide released from mitochondria in response to localized necrosis or serious systemic inflammation. It attracts neutrophils (via surface receptors) and stimulates oxidative bursts. fMLP is an integral part of the innate immune system. It serves as a mimic for the same peptide sequence released by pathogenic bacteria.	fMLP is known to occur in agricultural and house dusts. But it is not yet known if it also occurs in sewage other than via urine (e.g., such as from dusts or bacterial lysis). An exogenous source could confound its utility as a BioSCIM marker.
Polyamines	The aliphatic polyamine with most potential as a biomarker of disease is N1,N12-diacetylspermine (DAS or DiAcSpm).	The aliphatic polyamines (PAs), such as putrescine, spermidine, and spermine are essential for regulatory and control functions for all mammalian cells. DAS is a PA catabolite that is normally well regulated and extensively excreted in urine. It becomes dramatically upregulated with the onset and progression of a number of different cancers. Significantly, DAS exhibits little intra-individual or inter-individual daily variation among healthy individuals.	Diacetyl-PAs are probably exclusively endogenous biomarkers (of cancer, kidney disease, and diabetes, among others). They originate in sewer predominantly from urine, as they are not known to have meaningful dietary sources. PAs are very chemically stable with respect to heat and pH, indicating that they may persist in sewage. DAS should be examined as an alternative to creatinine for the normalization of biomarker levels.c
Nerve growth factor receptor	The extracellular domain of the transmembrane p75 neurotrophin receptor is called p75ECD.	p75ECD is enzymatically cleaved from the p75 neurotrophin receptor during neuronal development, injury, and certain neurological diseases (especially amyotrophic lateral sclerosis). It is extensively excreted in urine but only at very low, constant rates in healthy individuals	p75ECD is systemically shed not just during neuronal injury or disease, but also during pregnancy and the first month of life. It is therefore not just a marker for disease. Furthermore, the incidence of neurological disease in a population might be too low for detection of signal over background. This, however, could make p75ECD an alternative for creatinine in estimation of population size or for biomarker data normalization.c
Vitamin D-binding protein (VDBP)	A 58-kDa glycoprotein (also known as Gc-globulin) binds with vitamin D metabolites, serving to transport, protect, and recycle them in circulation. Also serves as a scavenger (along with gelsolin) of toxic levels of free actin.	Present at very high levels in the serum of healthy individuals (around 0.4 mg/mL) but correspondingly very low urinary levels. Urinary levels become elevated by several orders of magnitude during development and manifestation of a variety of kidney diseases.	The relative urinary levels between healthy and diseased states is much larger than for most biomarkers. This would greatly enhance the odds of successful VDBP detection in sewage.
Monocyte chemoattractant protein-1 (MCP-1)	A 13-kDa cytokine, also known as CC-chemokine ligand 2 (CCL2) or small inducible cytokine A2. Renowned as the most potent chemotactic factor for monocytes.	Becomes over-expressed with renal disease (e.g., diabetic nephropathy). Even though urinary levels of MCP-1 for healthy and diseased states do not differ as much compared with VDBP, they still change by over an order of magnitude.	Increased urinary levels of MCP-1 can be confounded by acute kidney damage, such as that caused by normal physiological responses to healthy activities, such as strenuous, sustained exercise.
Gelsolin	Together with VDBP, gelsolin is a ubiquitous, systemic protein that is one of the more important scavengers of actin.	Plasma levels in healthy individuals range from 200 to 300 mg/L. Key role in regulating disassembly of actin filament released from cellular injury and which can reach toxic levels. Can therefore become depleted after cellular injury, acting like a negative acute-phase protein. Gelsolin itself is cleaved by caspase-3, releasing the terminal (or truncated) fragment, t-gelsolin (or tGelsolin).	Most data on urinary levels involve fragments such as t-gelsolin. Little data reported on urinary levels of parent gelsolin. High plasma levels of gelsolin can therefore become translated into low gelsolin fragment levels in the urine, and vice-versa. As with other negative APPs, gelsolin can also be up-regulated in some disease states, so its plasma levels may not be easily interpreted as reflecting health or stress.

^aSee Supplementary materials for narrative discussions (and supporting references) regarding each biomarker (excluding the isoprostanes).

^bIndividual biomarker or class of related biomarkers.

^cFor further background, see section: Are there biomarker alternatives to creatinine for estimating population size or for data normalization?