Table 2.
Concentrations of RBC folate forms and total folate in conventionally prepared WB-Lys and RBC-Lys samples stored at −70°C for ≤2 y
| Concentration1, nmol/L | Relative difference2, % | Relative change compared to baseline3, % | ||||
|---|---|---|---|---|---|---|
| Storage time (mo) | WB-Lys4 | RBC-Lys5 | WB-Lys + exoGGH6 |
RBC-Lys vs WB-Lys | WB-Lys | RBC-Lys |
| 5-MethylTHF7 | ||||||
| Baseline | 831±258 | 843±276 | 867±268 | 1.22 (−0.58 to 3.02) | n/a | n/a |
| 3 | 840±259 | 818±265 | n.m.8 | −2.88 (−4.88 to −0.88) | 1.24 (0.70 to 1.78) | −2.93 (−3.50 to −2.36) |
| 6 | 830±257 | 812±265 | n.m. | −2.58 (−4.23 to −0.94) | 0.07 (−0.73 to 0.87) | −3.68 (−4.30 to −3.07) |
| 12 | 825±256 | 818±266 | n.m. | −0.97 (−2.82 to 0.87) | −0.72 (−1.15 to −0.30) | −2.90 (−3.32 to −2.48) |
| 24 | 807±252 | 800±261 | n.m. | −0.95 (−3.02 to 1.11) | −2.94 (−3.36 to −2.52) | −5.10 (−5.63 to −4.56) |
| Non-methyl folate9 | ||||||
| Baseline | 53.3±74.4 | 52.9±70.7 | 53.0±75.6 | 2.27 (−1.91 to 6.44) | n/a | n/a |
| 3 | 53.0±74.5 | 46.9±67.4 | n.m. | −14.8 (−17.8 to −11.8) | 0.32 (−2.43 to 3.08) | −15.3 (−19.1 to −11.5) |
| 6 | 51.5±75.5 | 49.5±67.5 | n.m. | 1.49 (−2.90 to 5.89) | −6.60 (−9.47 to −3.67) | −7.66 (−10.1 to −5.21) |
| 12 | 50.9±72.7 | 49.0±67.8 | n.m. | −1.25 (−4.64 to 2.14) | −6.47 (−8.55 to −4.39) | −9.23 (−11.8 to −6.67) |
| 24 | 48.4±72.9 | 48.5±69.6 | n.m. | 2.82 (−1.41 to 7.04) | −13.0 (−15.8 to −10.2) | −12.6 (−15.2 to −10.0) |
| tFOLExcluding MeFox10 | ||||||
| Baseline | 886±255 | 899±271 | 922±263 | 1.37 (−0.43 to 3.18) | n/a | n/a |
| 3 | 895±255 | 867±259 | n.m. | −3.15 (−5.13 to −1.17) | 1.16 (0.61 to 1.70) | −3.42 (−3.9 to −2.91) |
| 6 | 884±253 | 864±260 | n.m. | −2.44 (−4.12 to −0.75) | −0.08 (−0.76 to 0.60) | −3.83 (−4.38 to −3.29) |
| 12 | 878±253 | 870±260 | n.m. | −0.88 (−2.68 to 0.92) | −0.92 (−1.32 to −0.51) | −3.12 (−3.53 to −2.72) |
| 24 | 857±248 | 851±255 | n.m. | −0.58 (−2.63 to 1.48) | −3.28 (−3.74 to −2.82) | −5.22 (−5.67 to −4.77) |
| MeFox11 | ||||||
| Baseline | 147±44.1 | 172±53.5 | 165±50.8 | 16.8 (14.8 to 18.8) | n/a | n/a |
| 3 | 160±47.9 | 175±55.1 | n.m. | 9.26 (6.94 to 11.6) | 8.66 (7.65 to 9.67) | 1.62 (0.10 to 3.14) |
| 6 | 163±48.9 | 185±56.7 | n.m. | 13.9 (11.2 to 16.5) | 10.6 (9.46 to 11.8) | 7.61 (6.75 to 8.47) |
| 12 | 162±48.2 | 185±57.3 | n.m. | 14.1 (11.7 to 16.5) | 10.2 (9.14 to 11.3) | 7.54 (6.81 to 8.28) |
| 24 | 157±47.9 | 185±56.2 | n.m. | 18.2 (15.7 to 21.9) | 6.60 (4.84 to 8.40) | 7.96 (7.24 to 8.68) |
Concentrations are reported as mean±SD for n=60 samples (4 results per sample and storage time point)
Calculated as RBC-Lys minus WB-Lys divided by WB-Lys (expressed as percent) and then averaged across all 60 samples; 95% CI is shown in parenthesis
Calculated as time point x minus baseline divided by baseline (expressed as percent) and then averaged across all 60 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)
exoGGH, exogenous gamma-glutamyl hydrolase; WB-Lys samples were conventionally prepared by 1/11 dilution with 1% ascorbic acid, then frozen; prior to analysis by HPLC-MS/MS, exoGGH was added to WB-Lys aliquots (5 μg enzyme/mL sample added to the internal standard mixture), and samples were incubated for 4 h at 37°C (in duplicate over 2 days for n=4 results per sample)
5-MethylTHF, 5-methyltetrahydrofolate; major circulating folate form
n.m., not measured
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)