Endogenous substrates

FIGURE 3 3 The structure of the UGTl A1 endogenous substrate, bilirubin. 

2 GlycoUpids Sulfated, glucuronic acid-containing glycolipids (cera-mides) are present in the peripheral nerves and cauda equina (Chou et ah, 1991). 

There are many endogenous substrates, of widely different chemical structure, that are metabolized through oxidative, peroxidative, and reductive changes introduced by P450 enzymes. These include saturated and unsaturated fatty acids, eicosanoids, sterols and steroids, bile acids, vitamin D derivatives, retinoids, and uroporphyrinogens (Tables 9.4 and 9.5). 

TABLE 9.4. Substrates and Functions of Human CYP Gene Families 

Family Number of Subfamilies Number of Genes Substrates and Functions 

CYP1 2 3 Foreign chemicals, arachidonic acid, eicosanoids 

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Biotransformation reactions can be classified as phase 1 and phase 11. In phase 1 reactions, dmgs are converted to product by processes of functionalization, including oxidation, reduction, dealkylation, and hydrolysis. Phase 11 or synthetic reactions involve coupling the dmg or its polar metaboHte to endogenous substrates and include methylation, acetylation, and glucuronidation (Table 1). 

Sulfotransferases catalyze the transfer of sulfate from PAPS to wide-range xenobiotics that possess hydroxyl groups. Steroid alcohols are among the endogenous substrates. The sulfotransferases exist in different forms. 

All cytochrome P450s are hemoproteins and generally have a wide substrate specificity, acting on many exogenous and endogenous substrates. They represent the most versatile biocatalyst known. 

Capiello, M., Guiliani, L., Pacifici, G. M., Distribution of UDP-glucuronosyl transferase and its endogenous substrate uridine 5-diphosphoglucuronic acid in human tissues, Pur. J. Clin. Pharmacol. 1991, 41, 345-350. 

Unsaturated fatty acids are probably the most abundant oxidizable endogenous substrates. In the past it was erroneously believed that unsaturated fatty acids are just products of lipid peroxidation. Now, it has been shown that they have dietary origin. Family of unsaturated fatty acids includes linoleic (Ci8), arachidonic (C2o), docosahexaenoic (C22), and other fatty acids containing two, three, four, five, or six double bonds. Some acids can be in vivo converted into others for example, linoleic acid can be metabolized to linolenic and eicosa-trienoic acids [78]. 

Figure 8.13 Typical reaction trace of a coupled enzyme assay. The indicator reaction in many coupled assays will often show a demonstrable change after the addition of the sample but before the addition of the substrate for the test enzyme. This blank reaction may be due to the presence of endogenous substrates in the sample and its rate (B) must be measured in order to be able to calculate the activity of the test enzyme (T —B) from the total rate of reaction (7) which results from adding the substrate.

The primary objective of integration plot analysis is to analyze the data on influx of the test substrate from the circulating blood to the retina (i.e., blood-to-retina direction) across the BRB after intravenous administration of the test substrate. The advantage of this approach is that it allows reliable determination of the retinal uptake (i.e., clearance) of the test substrate which has a slow permeability across the BRB [28], On the other hand, due to the intravenous injection, interference by endogenous substrates and plasma-protein binding of the test substrate can produce an unseemingly low estimate of the retinal uptake. 

Shfe, C.W., Wang, E., Hunter, R., Wang, S., Burgess, C., Liotta, D.C., and Merrill, A.H.Jr., 1989, Free sphingosine formation from endogenous substrates by a liver plasma membrane system with a divalent cation dependence and a neutral pH optimum. J. Biol. Chem. 264 10371-10377. 

Summary of Brain P450s with Primarily Endogenous Substrates... 

Martinez C, Agundez JA, Gervasini G, Martin R, Benitez J. 1997. Tryptamine a possible endogenous substrate for CYP2D6. Pharmacogenetics 7 85-93. 

If Km for an endogenous substrate is known, then it establishes an approximate value for the endogenous concentration of that substrate. This is because it would not make sense kinetically or metabolically for a substrate to be present at concentrations significantly below or above its Km value. In the former case, the majority of enzyme protein molecules, synthesized by the cell at significant metabolic cost, would be redundant, and in the latter case, the cell would be unable to respond immediately to fluctuations in substrate concentration because enzymes would approach saturation and v would thus vary only marginally with fluctuations in [S]. 

Both xenobiotics and endogenous substrates are primarily metabolized by a super-family of enzymes, named cytochrome F450s (CYP450s,... 

Saghatelian A, Trauger SA, Want EJ, Hawkins EG, Siuzdak G, Cravatt BF, Assignment of Endogenous Substrates to Enzymes by Global Metabolite Profiling, Biochemistry 43 14332—14339, 2004. 

Correction for endogenous substrate present in enzyme preparations is difficult. Measuring the endogenous rate (obtained by omitting the substrate involved) and subtracting this from the overall rate is generally incorrect (R5). Due to the nonlinearity of the substrate saturation curve, the endogenous rate will be more important in the control mixture than... 

Enzyme induction properties Rifampin has enzyme induction properties that can enhance the metabolism of endogenous substrates including adrenal hormones, thyroid hormones, and vitamin D. Rifampin and isoniazid have been reported to alter vitamin D metabolism. In some cases, reduced levels of circulating 25-hydroxy vitamin D and 1,25-dihydroxy vitamin D have been accompanied by reduced serum calcium and phosphate, and elevated parathyroid hormone. 

Zhu, J. X., Doyle, H. A., Mamula, M. J., and Aswad, D. W. (2006). Protein repair in the brain, proteomic analysis of endogenous substrates for protein L-isoaspartyl methyltransferase in mouse brain. ]. Biol. Chem. 28,33802-33813. 

West. S.B. and Lu, A.Y. (1972) Reconstituted liver microsomal enzyme system that hydroxylates drugs, other foreign compounds and endogenous substrates. V. Competition between cytochromes P-450 and P-448 for reductase in 3,4-benzpyrene hydro-xylation. Archives of Biochemistry and Biophysics, 153, 298-303. 

Sulfotransferases (SULTs) are important for the metabolism of a number of drugs, neurotransmitters, and hormones, especially the steroid hormones. The cosubstrate for these reactions is 3 -phosphoadenosine 5 -phosphosulfate (PAPS) (Fig. 4.1). Like the aforementioned enzymes, sulfate conjugation typically renders the compound inactive and more water soluble. However, this process can also result in the activation of certain compounds, such as the antihypertensive minoxidil and several of the steroid hormones. Seven SULT isoforms identified in humans, including SULTs lAl to 1A3, possess activity toward phenolic substrates such as dopamine, estradiol, and acetaminophen. SULTIBI possesses activity toward such endogenous substrates as dopamine and triiodothyronine. SULTIEI has substantial activity toward steroid hormones, especially estradiol and dehydroepiandrosterone, and toward the anti-... 

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