Victor Herbert

A Palatable Diet for Producing Experimental Folate Deficiency in Man

F OR many years, it has been believed that folate deficiency on a nutritional basis could not be produced in man because of folate synthesis by the bacterial flora of the intestine.’ Indeed, no hematologic abnormalities were noted in human volunteer subjects after subsisting on a synthetic diet2 of vitamin free casein, Crisco,#{174} dextrimaltose, a mineral mixture, cod liver oil concentrate and a supplementary vitamm mixture containing no folic acid for eighteen months. This diet contained 14 to 17 g. of folate activity per day as measured by S. lactis R.’ Iii 1961 an abstract3 appeared which stated: “A study has been made of four patients fed on an artificial liquid diett which appears to provide a complete replacement of all known dietary requirements except folic acid. Estimation has shown that its folic acid content is less than 0.5 sg./kg. (Fleming, A., personal communication). On such a diet, individuals showed an abnormal excretion of FIG1u after 4-5 weeks. A further patient fed on the same diet with the addition of foods containing 2-5

Formiminoglutamicaciduria in Humans with Megaloblastic Anemia: Diminution by Methionine or Glycine.∗

Summary Five patients with megaloblastic anemia due to Vit. B12 and/or folic acid deficiency who excreted abnormally large quantities of formiminoglutamic acid (FGA) after a 20 g oral L-histidine · HCl load were studied. FGA excretion was markedly reduced in all 4 subjects given 20 g DL-methionine and in both subjects given 20 g glycine together with L-histidine · HCl. Of 3 subjects given 20 g DL-serine, one showed no reduction in FGA excretion and 2 showed moderate reduction.

Evidence Against Preferential Intestinal Absorption of Physiologic Quantities of Liver-Bound Vitamin B12 by Patients with Pernicious Anemia

I N 1959 it was reported’ that the fecal excretion of radiovitamin B,2 bound in pig liver (liver-bound vitamin B12) by patients with pernicious anemia was much less than that of free radiovitamin B12. These findings suggested either preferential absorption of the liver-bound vitamin B12 or a nonspecific delayed intestinal passage (i.e., fecal excretion) of this material. Subsequent experiments,2 which suggested the presence of intrinsic factor in bile, raised a third possibility-that the preferential absorption of liver-bound vitamm B15 may have been caused by the presence of variable quantities of intrinsic factor in the preparation. On the contrary, enhanced absorption of liver-bound radiovitamin B12 could not be demonstrated in either gastrectomized rats3 or in patients with intestinal malabsorption.4 Other studies5 indicate that coenzyme vitamin B, represents the majority of the cobamide in human liver, and that cyanocobal-

Studies suggesting the presence of intrinsic factor in bile.

Summary Four lines of evidence are presented suggesting that rat and human bile may contain intrinsic factor: 1. Human, and to a lesser extent rat, bile enhances Co60-B12 uptake by rat liver homogenate. 2. Some rat bile specimens enhance Co60-B12 uptake by everted sacs of rat small intestine. 3. Some rat bile specimens appear to substitute for rat intrinsic factor in a gastrectomized rat. 4. In Ouchterlony agar double diffusion analysis a reaction of identity was formed by human bile and intrinsic factor concentrates from hog, rat, and human sources to antibody induced by injection of purified hog intrinsic factor concentrate. If these findings in fact indicate that bile contains intrinsic factor, it is possible that an enterohepatic circulation of intrinsic factor exists as a further phase of its activity with relation to Vit. B12. The authors are indebted to Rebecca Fisher, Barbara Bean, and Laurie Dancy for technical assistance; to Drs. Jack Remington and Matthew D. Scharff for advice on immunologic technique; and to Dr. Shu Chu Shen who performed the rat gastrectomy.

Releasing Factor and Endogenous Vitamin B12

Summary Experiments were performed for the purpose of grossly identifying the substance responsible for the release, by rat intestinal segment extracts and human organ extracts, of vitamin B12 bound to rat stomach extract or normal human gastric juice. It has been found that the active principle in the proximal area and mid-ileum of the rat small intestine is dialyzable and that its distribution parallels closely that of endogenous vitamin B12. Non-radioactive vitamin B12 has been found to be very active in the release of radioactive vitamin B12 bound to rat or human intrinsic factor concentrate. This appears to be due to its ability to equilibrate with the bound form. In a complex equilibrium system, endogenous vitamin B12 appears to account for substantially all of the activity displayed by the proximal area and mid-ileum of the rat small intestine, and by mucosa homogenates from beef small intestine and various human organ extracts. Vitamin B12 bound to hog intrinsic factor on the other hand shows little tendency to equilibrate with unbound vitamin B12.