We found that choline deficiency in humans was associated with significant damage to DNA and with apoptosis in peripheral lymphocytes. This association had not previously been shown in humans.
Caspase-3 is a critical effector protein in apoptosis, and it exists within the cytosol as an inactive dimer (37
). Cleavage within a linker segment is required for activation, which occurs only during the terminal execution cascade of signals mediating apoptosis (37
). Thus, activated caspase-3 is an extremely specific marker for apoptosis. We observed that activated caspase-3 increased in the lymphocytes of subjects who developed organ dysfunction when fed a choline-deficient diet (). TUNEL labeling is often cited as a specific biomarker for apoptosis because the technique identifies cells with single-strand nicks in DNA. However, the assay also detects nonapoptotic events that result in DNA damage (38
). Our data are consistent with this finding, because our TUNEL assay values appear to be intermediate between activated caspase-3 values, which increased only in subjects who developed organ dysfunction when fed a choline-deficient diet, and COMET tail moment values, which increased in all subjects fed a choline-deficient diet. The COMET assay measures double-strand breaks in DNA (34
) as well as DNA modifications such as abasic sites (AP sites) (40
). This assay is different from both the TUNEL assay, which detects single- and double-strand breaks in DNA, and the activated caspase-3 assay, which detects specific activation of the apoptotic signaling cascade that is responsible for cell execution (41
). Whether DNA lesions detected by the COMET assay go on to become gene mutations depends on whether they are correctly repaired without resulting in permanent genetic alterations (40
As noted earlier, in animal models, choline deficiency increased leakage of reactive oxygen species from mitochondria (8
) and increased DNA adduct formation and DNA damage in liver (14
). In the current study, we observed that all subjects, as compared with the values when they were fed the control diet, had twice as much lymphocyte DNA damage (COMET assay) when they were fed the choline-deficient diet, even if choline was not sufficiently depleted to lead to liver or muscle dysfunction (). No evidence exists of a mechanism for choline interactions with DNA that is particular to lymphocytes, rather than to other tissues, and thus it is likely that damage to DNA is not organ specific. Perhaps damage to DNA is the earliest functional effect of choline deficiency and occurs before intracellular concentrations of choline or phosphatidylcholine decrease enough to induce apoptosis and leakage of enzymes from liver and muscle or fatty liver. If so, increased DNA damage as assessed by COMET assays could be the earliest marker for identification of a tissue as choline-deficient.
We suggest that choline deficiency–induced apoptosis shares a mechanism with other measures of organ dysfunction associated with choline deficiency—ie, diminished synthesis of phosphatidylcholine. Fatty liver occurs in choline deficiency because phosphatidylcholine is not available for secretion of VLDL from liver (42
). Muscle dysfunction in choline deficiency likely occurs because lower membrane phosphatidylcholine concentrations make myocytes fragile (4
). We previously reported that membrane phosphatidylcholine concentrations may be the critical variable for choline deficiency–mediated induction of apoptotic cascades (9
Choline and folate metabolisms are interrelated (1
), and it has been suggested that folate status can modify susceptibility to choline deficiency (44
). In the current study, the changes in plasma folate were modest, although they did occur in the direction we expected. It is possible that the experimental period in the current study—which was as short as 2 wk and as long as 6 wk—was not long enough for the dietary changes (from 400 to 100 or 768 DFE) to reach a plateau in plasma folate; it has been reported that 6–14 wk is required for that outcome (45
). It could have been predicted that humans ingesting a choline-deficient diet containing 100 DFE/d would have decreased plasma folate concentrations because this folate intake is less than that estimated for humans eating an ad libitum diet (≈200 DFE/d from naturally occurring food folate, in addition to the contribution from foods fortified with folic acid) (46
). We present folate data to show that our subjects did not become frankly folate deficient (plasma folate concentrations were well above those indicative of folate deficiency; 36
). Supplementary folic acid mitigated some but not all of the effects of the choline-deficient diet on increasing TUNEL labeling but did not mitigate choline deficiency–associated changes in activated caspase-3 or in COMET tail moment. The restoration to the diet of choline, without restoration of folate, returned COMET and activated caspase-3 values to normal. Thus, choline deficiency and not diminished folate status was responsible for the changes that we report here.
That choline deficiency induces significant DNA damage in humans is an important observation. In rodent models, prolonged (ie, 1 y) choline deficiency results in the development of liver cancers (47
) and in greater sensitivity to chemical carcinogens (48
). These conditions could be the result of mutations due to DNA damage. Similarly, choline deficiency–induced apoptosis is of clinical significance, because liver damage is a common side effect of prolonged parenteral nutrition (16
). At present, there is no definitive clinical test that can be used to identify persons who are choline deficient. Plasma choline, betaine, and phosphatidylcholine concentrations decreased in humans fed a choline-deficient diet, but they plateau after falling 30%–50% (2
). Urinary betaine and choline concentrations also decreased in humans consuming a choline-deficient diet, and we used that measure as a compliance marker in this study. Lower concentrations of choline metabolites are necessary but not sufficient to predict who will develop organ dysfunction (4
). In the current study, we identified a number of biomarkers that can be used in conjunction with plasma choline metabolites to make a diagnosis of choline deficiency in humans. COMET tail moment may be the most sensitive measure, and dose-response studies should be performed to ascertain the exact dietary intake at which COMET values become abnormal. Activated caspase-3 measurements in lymphocytes correlated best with liver and muscle dysfunction that was associated with choline deficiency. Thus, this assessment has clinical value for physicians who might use it as an indicator for ordering studies of liver or muscle function in patients suspected of being choline deficient. The TUNEL assay was influenced by folate status as well as by choline status, and thus it is not as specific a biomarker for choline deficiency as are the other assays.
The human dietary requirement for an essential nutrient is defined as the amount that prevents organ dysfunction. The Institute of Medicine Dietary Reference Intake Panel defined the human requirement for choline according to the amount of dietary intake needed to prevent liver dysfunction (ie, elevated serum transaminase concentrations; 26
). Because apoptosis and DNA damage in lymphocytes are measures of significantly abnormal cell function, we suggest that these measures in a clinically accessible tissue could be used to define the human dietary requirement for choline.