A Depletion-Repletion Folate Bioassay Based on Growth and Tissue Folate Concentrations of Rats

Abstract

To improve standardization of a folate bioassay, folate-depleted rats were repleted with a folate-free amino acid-based diet supplemented with 29 levels of folic acid. Growth was the main response variable and body tissue folate concentrations were also assessed. Because a positive correlation was observed between low levels of dietary folic acid and growth and little or no correlation was observed between high levels and growth, six regression models with a steep slope for low levels and a shallow or zero slope for high levels of dietary folic acid were evaluated. The model referred to as the "two-phase regression" or "change-point" model best described the relationship. Depleted rats needed 674 +/- 71 nmol folic acid/kg diet to reach their full growth potential. This value is biologically sensible, and this regression model is well established in the statistical literature. The change-point model is highly recommended to characterize the growth response, because growth is a functional response and, in the range of 226 to 680 nmol folic acid/kg, this response is linear, which is an additional advantage. Linear responses are easier to interpret because of complicated issues of interpretation and confidence intervals with nonlinearities. Linear regressions described serum and liver folate responses, whereas exponentials described whole blood and carcass folate responses. Depleted rats needed 5920 and 5780 nmol folic acid/kg diet to maximize their whole blood and carcass folates, respectively.

Folate Deficiency Induces Genomic Uracil Misincorporation and Hypomethylation But Does Not Increase DNA Point Mutations

“…Parallel to the observed decrease in plasma folate, we detected an increase in plasma homocysteine (60% increase at 8 months, P < .001). Folate deficiency caused a moderate but not significant growth retardation of 14% in wild-type mice ( P =.17) and 5.3% in Ung −/− mice ( P = .50), which correlates with previous studies (see Supplementary Figure 1 and Table 1 online at at www.gastrojournal.org)31.…”

High-dose folic acid supplementation in rats: effects on gestation and the methionine cycle

“…When general nutritional status was studied, we observed that supplementation with folic acid did not affect normal growth in rats. This is in accordance with other studies in which folate-enriched diets did not improve growth response (Clifford et al 1993;Alonso-Aperte, 1997). As expected, pregnant rats had a higher FER when compared with virgin rats, but folic acid level in the diet did not influence FER.…”

“…Both diets were adjusted to rat requirements (National Research Council, 1995), and were based on the pure amino acid diet (170 g amino acid/kg, Dyets, Bethlehem, PA, USA) described by Walzem & Clifford (1988). This is the most reliable system for studying the exclusive effect of dietary folic acid, without confounding factors, as demonstrated in several previous studies (Walzem & Clifford, 1988;Clifford et al 1989Clifford et al , 1993Varela-Moreiras & Selhub, 1992;Varela-Moreiras et al 1995b;Alonso-Aperte & Varela-Moreiras, 1996).…”

Experimental Folate and Vitamin B12 Deficiency Does Not Alter Bone Quality in Rats

“…However, Clifford et al and Birn et al have shown that the absence of dietary folate and B 12 readily reduces tissue levels of both vitamins in most nonbony organs, such as liver, kidney, heart, lungs, and others, within 4-8 wk. (41)(42)(43)(44) Based on these data, there is no reason to believe that the time of intervention (12 wk) was insufficient in this study. However, Clifford et al and Birn et al did not measure bone levels of folate and B 12 .…”