Blood glutathione redox status and global methylation of peripheral blood mononuclear cell DNA in Bangladeshi adults

Figures & data

Figure 1. Overview of one-carbon metabolism, transsulfuration pathway, and glutathione influx/efflux. Folate is delivered into the cell via a receptor-mediated [folate receptor alpha (FRα), beta (FRβ) or gamma (FRγ)] or carrier-mediated [solute carrier family 19, member 1 (SLC19A1), also known as RFC1] transport mechanism and is subsequently reduced to dihydrofolate (DHF) and tetrahydrofolate (THF), which then enters the one-carbon metabolic pathway. THF picks up a methyl group from serine and is converted to 5-methyl THF (5mTHF). The methyl group of 5mTHF can be transferred to Hcys via methionine synthetase (MTR), generating methionine (Met) and THF. Met is activated to form S-adenosylmethionine (SAM), which serves as the methyl donor for the methylation of CpG dinucleotides and many other substrates, yielding the methylated product and S-adenosylhomocysteine (SAH), which is hydrolyzed to regenerate Hcys. Hcys can be used to regenerate Met or be directed through the transsulfuration pathway via cystathionine-β-synthase (CBS). Glutathione (GSH), a product of the transsulfuration pathway, is a tripeptide comprised of glutamate (Glu), cysteine (Cys) and glycine (Gly). As the primary endogenous antioxidant, GSH donates an electron to reactive oxygen species (e.g., H₂O₂) with the enzyme glutathione peroxidase (GPx) and quickly reacts with another free radical GSH to form glutathione disulfide (GSSG). GSH can be regenerated from GSSG in a reaction catalyzed by glutathione reductase (GR). Under conditions of oxidative stress, CBS activity is upregulated to direct Hcys flux through the transsulfuration pathway for GSH production. Excess intracellular Hcys can also be exported extracellularly. GSH can be exported out of the cell and catabolized via the cell membrane enzyme γ-glutamyltransferase (GGT). GGT transfers the γ-glutamyl group to an amino acid, producing cysteinylglycine (Cys-Gly), which can be broken down to Cys and Gly via dipeptidase (DP). Cys is unstable extracellularly and rapidly oxidizes to cystine (CySS). The x_C⁻ antiporter can import CySS using a transmembrane Glu gradient, and the b⁰⁺ system can directly import CySS, which can be converted back to Cys to maintain the intracellular Cys pool.

Table 1. Descriptive characteristics for study sample (n = 320)

Baseline variables	Mean ± SD (range) or N(%)
Age (years)	43.2 ± 8.3 (30–63)
Male	159 (49.7)
BMI (kg/m^2)	20.3 ± 3.5 (13.8–35.3)
Underweight (BMI < 18.5 kg/m^2)	112 (35.1)
Ever cigarette smoking	123 (38.4)
Ever betel nut use	142 (44.4)
Television ownership	185 (57.8)
Water As (μg/L)	144.9 ± 122.5 (0.4–699.9)
Water As > 50 μg/L	226 (70.9)
Plasma folate (nmol/L)^a	12.6 ± 7.0 (2.4–60.6)
Folate deficiency (folate < 9.0 nmol/L)^a	100 (31.3)
Plasma Hcys (μmol/L)	11.2 ± 12 (3.0–165.4)
Hyperhomocystinemia (Hcys > 13 μmol/L)	56 (17.5)
DPM per μg DNA	149,123 ± 23,932 (61,398–215,666)
Blood GSH (μmol/L)	491.0 ± 170.6 (104.6–1072.1)
Plasma GSH (μmol/L)	2.6 ± 0.7 (1.0–5.8)
Blood GSSG (μmol/L)	37.3 ± 18.8 (9.6–121.8)
Plasma GSSG (μmol/L)	2.1 ± 0.6 (0.8–4.8)
Blood GSH Eh (mV)	−198.0 ± 13.0 (−227.9 to −144.4)
Plasma GSH Eh (mV)	−98.5 ± 7.2 (−118.0 to −73.4)
Urinary 8-oxo-dG/urinary Cr (μM)	16.0 ± 14.2 (0.9–89.6)
Blood SAM^b (μM)	1.29 ± 0.50 (0.44–3.38)
Blood SAH^b (μM)	0.31 ± 0.17 (0.10–1.37)

^a n = 319; ^bn = 312.

Table 2. Spearman correlation coefficients of blood and plasma glutathione redox variables with plasma Hcys and blood SAH (n = 320)

		Blood GSH	Blood GSSG	Blood Eh GSH	Plasma Eh GSH
Plasma Hcys	r P	0.22 (<0.0001)	−0.09 (0.10)	−0.27 (<0.0001)	0.07 (0.22)
Blood SAH^a	r P	0.22 (<0.0001)	−0.38 (<0.0001)	−0.42 (<0.0001)	0.02 (0.74)

^a n = 312.

Table 3. Unadjusted and adjusted regression coefficients for associations between predictors and [³H]-methyl incorporation of PBMC DNA (n = 320)

Predictor	Model	[^3H]-methyl incorporation
				B ± SE	P	R^2 %	ΔR^2 %
Blood GSH Eh	Unadjusted	303 ± 102	0.003	2.7
	Adjusted^a	271 ± 103	0.009	5.5	2.0
Blood GSH	Unadjusted	−13.0 ± 7.8	0.10	0.9
	Adjusted^a	−12.7 ± 8.0	0.11	4.2	0.7
Blood SAM^b	Unadjusted	−4799 ± 2698	0.08	1.0
	Adjusted^a	−4803 ± 2747	0.08	5.2	1.7

^a Adjusted for sex, age and well water As, n = 319; ^bn = 312.