Table 3.
Concentrations of RBC folate forms and total folate stratified by non-methyl folate concentrations in conventionally prepared WB-Lys and RBC-Lys samples stored at −70°C for up to 2 y
| Concentration1, nmol/L | Relative change compared to baseline2, % | |||
|---|---|---|---|---|
| Storage time (mo) | WB-Lys3 | RBC-Lys4 | WB-Lys3 | RBC-Lys4 |
| Samples with low non-methyl folate concentration | ||||
| 5-MethylTHF5 | ||||
| Baseline6 | 840±261 | 860±283 | n/a | n/a |
| 3 | 853±262 | 835±271 | 1.59 (1.01, 2.17) | −2.75 (−3.36, −2.14) |
| 6 | 842±259 | 827±270 | 0.36 (−0.55, 1.28) | −3.64 (−4.33, −2.96) |
| 12 | 835±259 | 835±271 | −0.57 (−1.04, −0.10) | −2.82 (−3.26, −2.39) |
| 24 | 818±255 | 817±266 | −2.69 (−3.14, −2.23) | −4.89 (−5.46, −4.31) |
| Non-methyl folate7 | ||||
| Baseline6 | 25.9±7.76 | 26.8±9.35 | n/a | n/a |
| 3 | 26.1±8.41 | 22.0±8.42 | 0.93 (−2.38, 4.24) | −17.2 (−21.7, −12.7) |
| 6 | 24.0±8.25 | 24.5± 8.94 | −7.61 (−11.1, −4.14) | −8.35 (−11.3, −5.39) |
| 12 | 24.1±8.17 | 23.9±8.42 | −7.39 (−9.85, −4.94) | −10.2 (−13.2, −7.06) |
| 24 | 22.4±7.85 | 22.9±8.35 | −13.9 (−17.1, −10.7) | −14.5 (−17.4, −11.5) |
| tFOLExcluding MeFox8 | ||||
| Baseline6 | 868±266 | 889±289 | n/a | n/a |
| 3 | 880±268 | 860±276 | 1.52 (0.96, 2.09) | −3.24 (−3.78, −2.69) |
| 6 | 868±265 | 855±276 | 0.11 (−0.70, 0.92) | −3.81 (−4.42, −3.21) |
| 12 | 861±264 | 861±277 | −0.75 (−1.21, −0.29) | −3.08 (−3.51, −2.65) |
| 24 | 842±260 | 843±272 | −3.04 (−3.49, −2.58) | −5.20 (−5.71, −4.69) |
| MeFox9 | ||||
| Baseline6 | 149±43.2 | 175±53.0 | n/a | n/a |
| 3 | 161±47.1 | 177±53.4 | 8.81 (7.63, 9.99) | 1.74 (0.08, 3.39) |
| 6 | 164±48.6 | 188±55.4 | 10.6 (9.36, 11.9) | 7.89 (6.95, 8.83) |
| 12 | 164±47.8 | 188±56.0 | 10.1 (8.99, 11.3) | 7.79 (7.01, 8.58) |
| 24 | 159±48.0 | 189±54.9 | 6.68 (4.76, 8.59) | 8.23 (7.39, 9.06) |
| Samples with high non-methyl folate concentration | ||||
| 5-MethylTHF5 | ||||
| Baseline6 | 788±252 | 769±238 | n/a | n/a |
| 3 | 786±251 | 742±236 | −0.31 (−1.45, 0.83) | −3.74 (−5.49, −2.00) |
| 6 | 780±252 | 741±237 | −1.23 (−2.71, 0.25) | −3.86 (−5.53, −2.19) |
| 12 | 777±246 | 746±237 | −1.42( −2.49, −0.34) | −3.23 (−4.63, −1.83) |
| 24 | 757±244 | 724±230 | −4.08 (−4.96, −3.19) | −6.05 (−7.53, −4.56) |
| Non-methyl folate7 | ||||
| Baseline6 | 175±111 | 169±105 | n/a | n/a |
| 3 | 173±114 | 158±99.8 | −2.38 (−5.62, 0.85) | −7.17 (−8.92, −5.41) |
| 6 | 174±115 | 160±99.3 | −1.91 (−4.12, 0.29) | −4.55 (−6.31, −2.80) |
| 12 | 170±108 | 161±100 | −2.38 (−4.44, −0.32) | −5.18 (−7.13, −3.23) |
| 24 | 164±114 | 163±103 | −8.89 (−14.8, −3.01) | −4.50 (−6.30, −2.70) |
| tFOLExcluding MeFox8 | ||||
| Baseline6 | 966±188 | 941±169 | n/a | n/a |
| 3 | 961±187 | 902±172 | −0.46 (−1.84, 0.91) | −4.24 (−5.83, −2.65) |
| 6 | 957±185 | 905±174 | −0.92 (−1.92, 0.07) | −3.94 (−5.51, −2.38) |
| 12 | 950±184 | 910±171 | −1.64 (−2.53, −0.75) | −3.33 (−4.70, −1.96) |
| 24 | 924±182 | 891±162 | −4.37 (−5.91, −2.83) | −5.31 (−6.32, −4.30) |
| MeFox9 | ||||
| Baseline6 | 140±49.7 | 159±56.5 | n/a | n/a |
| 3 | 151±52.8 | 162±63.5 | 8.01 (6.00, 10.0) | 1.11 (−3.40, 6.62) |
| 6 | 154±52.1 | 169±62.7 | 10.6 (7.09, 14.2) | 6.36 (4.02, 8.69) |
| 12 | 154±51.8 | 170±63.5 | 10.7 (7.05, 14.2) | 6.42 (4.22, 8.62) |
| 24 | 148±49.0 | 170±62.3 | 6.38 (0.88, 11.9) | 6.76 (5.58, 7.94) |
Concentrations are reported as mean±SD for low non-methyl folate samples (n=49) and high non-methyl folate samples (n=11); each sample was analyzed in 4 replicates per storage time point
Calculated as time point x minus baseline divided by baseline (expressed as percent) and then averaged across all samples; 95% CI is shown in parenthesis
WB-Lys, whole blood lysate; WB-Lys samples were conventionally prepared by 1/11 dilution with 1% ascorbic acid, then frozen; prior to analysis by HPLC-MS/MS, WB-Lys aliquots were incubated for 4 h at 37°C (in duplicate over 2 days for n=4 results per sample)
RBC-Lys, red blood cell lysate; RBC-Lys samples were washed RBC diluted ½ with saline prior to 1/11 dilution with 1% ascorbic acid, then frozen; prior to analysis by HPLC-MS/MS, RBC-Lys aliquots were treated with exoGGH enzyme (5 μg/mL of lysate) for 30 min at room temperature (in duplicate over 2 days for n=4 results per sample)
5-MethylTHF, 5-methyltetrahydrofolate; major circulating folate form
Baseline aliquots of WB-Lysate and RBC-Lysate were stored at −70°C immediately after preparation for up to 1 week prior to analysis by LC-MS/MS.
Non-methyl folate; sum of minor folate forms (5-formyltetrahydrofolate, tetrahydrofolate, and 5,10-methenyltetrahydrofolate)
tFOL, total folate; sum of biologically active folate forms (5-methylTHF and non-methyl folate)
MeFox is a pyrazino-s-triazine derivative of 4α-hydroxy-5-methylTHF (an oxidation product of 5-methylTHF)