Suitability and safety of L-5-methyltetrahydrofolate as a folate source in infant formula: A randomized-controlled trial

L-5-methyltetrahydrofolate is the predominant folate form in human milk but is currently not approved as a folate source for infant and follow-on formula. We aimed to assess the suitability of L-5-methyltetrahydrofolate as a folate source for infants. Growth and tolerance in healthy term infants fed formulae containing equimolar doses of L-5-methyltetrahydrofolate (10.4 mu g/100 ml, n = 120, intervention group) or folic acid (10.0 mu g/100 ml, n = 120, control group) was assessed in a randomized, double-blind, parallel, controlled trial. A reference group of breastfed infants was followed. Both formulae were well accepted without differences in tolerance or occurrence of adverse events. The most common adverse events were common cold, poor weight gain or growth, rash, eczema, or dry skin and respiratory tract infection. Weight gain (the primary outcome) was equivalent in the two groups (95% CI -2.11;1.68 g/d). In line with this, there was only a small difference in absolute body weight adjusted for birth weight and sex at visit 4 (95% CI -235;135 g). Equivalence was also shown for gain in head circumference but not for recumbent length gain and increase in calorie intake. Given the nature of the test, this does not indicate an actual difference, and adjusted means at visit 4 were not significantly different for any of these parameters. Infants receiving formula containing L-5-methyltetrahydrofolate had lower mean plasma levels of unmetabolized folic acid (intervention: 0.73 nmol/L, control: 1.15 nmol/L, p<0.0001) and higher levels of red cell folate (intervention: 907.0 +/- 192.8 nmol/L, control: 839.4 +/- 142.4 nmol/L, p = 0.0095). We conclude that L-5-methyltetrahydrofolate is suitable for use in infant and follow-on formula, and there are no indications of untoward effects

The patterns of gO- and MCK-2-dependent virus spread differ.

<p>NIH3T3, NIH3T3-MCK2, and NIH3T3-gO cells were infected at an m.o.i. of 0.1 with the double mutant Δm74/131stop reconstituted in NIH3T3-gO cells (A-C). (A) Spread in culture was followed by staining cells for IE1 expression 3 and 6 days after infection. (B) Infection was enhanced by a centrifugation step at 2,000× g for 30 min at RT. Cells were stained for IE1 24 h after infection to show the comparable initial infection of NIH3T3, NIH3T3-gO, and NIH3T3-MCK-2 cells (left panel). Release of infectious virus was monitored in multistep growth curves (right panel). (C) The release of DNAse-protected viral DNA was followed by real-time PCR using supernatants from the growth curves under (B, right panel) plus an additional time point at 6 h post infection. The column labeled “in” shows the amount of DNAse-protected viral DNA in the Δm74/131stop inoculum used to infect the cells. (A–C) p.i., post infection.</p

MCK-2 associates with gH in a complex distinct from gH/gL/gO.

<p>(A) Supernatant virus from NIH3T3 cells infected with MCMV-gH-HA or MCMV-gO-HA was lysed in red. or non-red. sample buffer. gO-HA and gH-HA were detected with an anti-HA antibody. Monomeric gO-HA (black arrow) and gH/gL/gO (white arrow) are indicated by arrows. Two exposures of the Western blots showing non-red. extracts are depicted. As a control, gO-HA was precipitated from a lysate of MCMV-gO-HA infected cells with an anti-HA antibody and analyzed under non-red. conditions. (B) MEF were infected with MCMV-gO-HA, and three days after infection, cells were harvested and either directly analyzed for gO-HA expression (total cell extract) or after immunoprecipitation using an anti-HA antibody, an anti-gH antibody, an anti-MCK2 (2H9), or a mouse IgG control antibody. (A–B) The gO-HA specific protein band runs at about 70 kDa. The positions of the molecular weight markers (kDa) are indicated.</p

MCK-2 dependent infection of MEF.

<p>(A) MEF (left panel) and ANA-1 cells (right panel) were infected with MCMV mutants 131stopB and Δm74 and MEF additionally with Δm74/m74trans. The latter mutant was trans-complemented with gO by growth in NIH3T3-gO cells. Virus was either preincubated with anti-MCK2 rabbit antiserum or with an anti-pUL131A rabbit antiserum, which served as a control rabbit antiserum, at a dilution of 1∶10, or with medium as a mock control. Infection of cells was monitored by indirect immunofluorescence staining for IE1⁺ cells 6 h after infection. The percentage of IE1⁺ cells is expressed relative to the percentage of IE1⁺ cells of mock-treated infections. As indicated, both, Δm74 MCMV and Δm74/m74trans MCMV were significantly (Student's t-test) inhibited by the MCK-2 antiserum when compared to the control rabbit antiserum. The P values are indicated in the histograms. Shown are means +/−SEM of 3 to 4 independent experiments. (B) MEF were infected with a Δm74/131stop mutant either grown in NIH3T3-gO (gO-trans) or NIH3T3-MCK-2 (MCK-2 trans) cells in the presence or absence of energy depletion medium, bafilomycin A1, or NH₄Cl. Three hours after infection, cells were fixed and stained for IE1 expression. The percentage of IE1⁺ cells is expressed relative to the percentage of IE1⁺ cells of mock-treated infections. For all inhibitors, inhibition of the gO trans-complemented and of the MCK-2 trans-complemented mutant was significantly different (Student's t test). The P values are indicated in the histograms. Shown are means +/− SEM of 4 to 6 independent experiments.</p

Expression of full-length MCK-2 facilitates the infection of macrophages in vivo.

<p>(A) BALB/C mice were i.p. infected with 5×10⁵ PFU of wildtype or 131stopD MCMV. Six hours after infection PEC were harvested and CD11b⁺ F4/80⁺ macrophages analyzed for MCMV m06 expression by FACS analysis. The number of infected cells was significantly lower for the 131stop mutant. Shown are means +/−SD. Data were compiled from two independent experiments with altogether 9 mice analyzed per group. (B, C) Quantitation of infected F4/80⁺IE1⁺ liver-resident and recruited macrophages in situ. Two-color IHC analysis was performed on liver tissue sections derived from immunocompromised BALB/c mice on day 10 after intraplantar infection with viruses vpSM3fr, 131stopD, and wildtype virus. (B) Representative image of wildtype virus-infected liver tissue with red staining of the intranuclear viral protein IE1 for detecting infected cells and black staining of the marker antigen F4/80 (Ly71) for detecting macrophages. The red arrow exemplarily points to an enlarged hepatocyte nucleus containing the CMV-typical inclusion body that indicates productive infection. The black arrow exemplarily points to an uninfected F4/80⁺IE1⁻ macrophage. Black-and-red striped arrows highlight cases of infected F4/80⁺IE1⁺ macrophages. The bar marker represents 50 µm. (C) Quantitation of infected F4/80⁺IE1⁺ liver macrophages and statistical analysis. Symbols represent individual mice infected with the indicated viruses. Data represent numbers of F4/80⁺IE1⁺ macrophages counted per 10-mm² areas of liver tissue sections and normalized to the total numbers of infected IE1⁺ cells and F4/80⁺ macrophages present in the same sections to take account of differences in the levels of overall infection and macrophage recruitment. Mean values and SDs are indicated. (A–C) P values were calculated by unpaired, two-tailed Student's t test with Welch's correction not assuming equal variance. Differences are considered significant for P<0.05.</p

MCK-2 is a virion protein forming a high-molecular weight complex co-migrating with gH.

<p>(A–E) Lysates of cells, supernatant virus, or precipitated proteins were separated on SDS-polyacrylamide gels and analyzed by Western blot using the anti-MCK-2 antibody 5A5 to detect MCK-2 or the anti-HA antibody 3F10 to detect gH-HA. The positions of the molecular weight markers (kDa) are indicated. (A) Infected cells and supernatant virus from NIH3T3 cells infected with wildtype MCMV were harvested 5 days after infection and lysed in reducing sample buffer. (B) Gradient purified wildtype MCMV was lysed in reducing (red.) and non-red. sample buffer. The MCK-2 specific bands are indicated by arrows. (C) Supernatant virus from NIH3T3 cells infected with MCMV-gH-HA was either directly lysed in red. or non-red. sample buffer (extract) or proteins were precipitated from lysates with anti-gH or anti-MCK2 antibodies and then analyzed under non-red. conditions. Monomeric gH-HA (black arrow) and gH/gL/MCK-2 (white arrow) are indicated by arrows. (D) Supernatant virus from NIH3T3 cells infected with MCMV-gH-HA or MCMV-gH-HA/129stop was lysed in red. or non-red. sample buffer. The upper panel shows extracts prepared under red. and non-red. conditions and stained with an anti-MCK-2 antibody. The lower panel shows the same extracts stained with an anti-HA antibody to detect gH-HA. The gH/MCK-2 band formed under non-red. conditions is indicated by an arrow. The MCMV-gH-HA/129stop extracts showed an at least fivefold higher protein content. (E) NIH3T3 cells were infected with MCMV-gH-HA and supernatant virus harvested 5 days after infection. Lysates of supernatant virus were either directly analyzed for gH-HA expression (supernatant virus) or after immunoprecipitation using a mouse IgG control antibody, an anti-HA antibody, an anti-gH antibody, or an anti-MCK2 (2H9) antibody recognizing the m131 region of MCK-2. The gH-HA specific protein band is indicated.</p

Expression of full-length MCK-2 facilitates the infection of macrophages in vitro.

<p>(A) MEF, NIH3T3, MHEC-5T and TCMK-1 cells were plated in 96 well plates, infected with wildtype MCMV and 131stop mutants of MCMV at an m.o.i. of 0.5, and 6 h after infection stained for MCMV immediate early 1 (IE1) protein by indirect immunofluorescence. Each infection was done in triplicates and for each well IE1⁺ cells were counted and the means of these counts related to the mean of IE1⁺ MEF. The value for MEF was set to 100%. Shown are means +/− SEM of at least 4 independent experiments. Infection of TCMK-1 with m131stop mutants was significantly enhanced compared to wildtype infection. The P values (Student's t-test) are indicated in the histograms. (B) Immortalized macrophages ANA-1 (left and middle panel) and J774 (right panel) were infected in suspension with wildtype MCMV or MCMV-gH-HA carrying a wildtype MCK-2 or with the MCK-2 mutants m131stopB or D, MCMV-gH-HA/129stop, or pSM3fr BAC-derived virus. The numbers of infected macrophages were determined by intracellular FACS staining for IE1⁺ cells. All infections with MCK-2 mutants showed significantly lower numbers of infected macrophages than infections with wildtype virus. The P values (Student's t-test) are indicated in the histograms. Shown are means +/−SEM of 3 to 4 independent experiments. (C) Primary BMDM (left panel) and cells in the macrophage-enriched gate of PEC (PEC/M) (right panel) were infected with wildtype virus or the MCK-2 mutant 131stopD as described under (B). The numbers of infected cells were significantly lower for the 131stop mutant. The P values (Student's t-test) are indicated in the histograms. Shown are means +/−SEM of 3 independent experiments. (B and C) All virus preparations have been titrated on MEF and for each experiment MEF were infected in parallel to confirm that wildtype and mutant viruses infected comparable percentages of MEF.</p