Choline deficiency increases lymphocyte apoptosis and DNA damage in humans

Abstract

Background: Whereas deficiency of the essential nutrient choline is associated with DNA damage and apoptosis in cell and rodent models, it has not been shown in humans.

Objective: The objective was to ascertain whether lymphocytes from choline-deficient humans had greater DNA damage and apoptosis than did those from choline-sufficient humans.

Design: Fifty-one men and women aged 18-70 y were fed a diet containing the recommended adequate intake of choline (control) for 10 d. They then were fed a choline-deficient diet for up to 42 d before repletion with 138-550 mg choline/d. Blood was collected at the end of each phase, and peripheral lymphocytes were isolated. DNA damage and apoptosis were then assessed by activation of caspase-3, terminal deoxynucleotide transferase-mediated dUTP nick end-labeling, and single-cell gel electrophoresis (COMET) assays.

Results: All subjects fed the choline-deficient diet had lymphocyte DNA damage, as assessed by COMET assay, twice that found when they were fed the control diet. The subjects who developed organ dysfunction (liver or muscle) when fed the choline-deficient diet had significantly more apoptotic lymphocytes, as assessed by the activated caspase-3 assay, than when fed the control diet.

Conclusions: A choline-deficient diet increased DNA damage in humans. Subjects in whom these diets induced liver or muscle dysfunction also had higher rates of apoptosis in their peripheral lymphocytes than did subjects who did not develop organ dysfunction. Assessment of DNA damage and apoptosis in lymphocytes appears to be a clinically useful measure in humans (such as those receiving parenteral nutrition) in whom choline deficiency is suspected.

FIGURE 1

Overview of study design. The choline content of the baseline, depletion, and repletion phases is indicated. Subjects who developed organ dysfunction when fed the choline-deficient diet underwent repletion with graded increasing concentrations of choline for 10 d each until signs of organ dysfunction resolved [137.5 (25%), 275 (50%), 412.5 (75%), and finally 550 mg choline · 70 kg⁻¹ · d⁻¹ (100%)]. The baseline diet provided 400 dietary folate equivalents (DFE)/d. The choline-depletion and -repletion diets provided 100 DFE/d. On day 11, subjects were randomly assigned to receive placebo (100 DFE/d) or 400 μg folic acid supplement/d for the remainder of the study.

FIGURE 2.

Mean (±SE) urinary betaine and choline concentrations (n = 32–45/time point), from which dietary compliance was assessed. Subjects were treated as described in the legend for Figure 1. Urine samples (24-h) were collected at the end of the baseline (550 mg), depletion (Depl), and repletion (Repl) phases and on days 13 and 31 of the choline-deficient diet (D13CD and D31CD, respectively), and aliquots were analyzed for betaine () and choline (□) concentrations nmol/mg creatinine as described in Methods. *Significantly different from baseline, P < 0.01 (one-factor ANOVA and Dunnett's test).