C. E. Butterworth

A study of folate absorption and metabolism in man utilizing carbon-14—labeled polyglutamates synthesized by the solid phase method

The absorption and metabolism of synthetic polyglutamates of folic acid have been compared with free pteroylglutamic acid in four subjects having chronic lymphatic leukemia and one with Hodgkins granuloma. Pteroylpolyglutamates containing either three or seven glutamate residues were prepared by the solid-phase method permitting placement of carbon-14 labels in either the pteridine ring or in a selected glutamate unit of the gamma peptide chain. Complete dissociation was observed between biological folate activity and radioactivity of plasma after ingestion of pteroyltriglutamate labeled in the middle glutamate. This indicates cleavage to the monoglutamate form at the time of absorption from the intestine or very soon thereafter. A large portion of radioactivity liberated from the middle glutamate is recoverable as carbon dioxide in the exhaled air. Fecal losses of folate tended to be greater with increasing length of the poly-gamma-glutamyl chain. Higher blood levels and greater urinary losses of folate tended to occur after ingestion of mono- and triglutamates than with the heptaglutamate. Calculations based on radioactivity determinations in feces plus urinary folate losses, judged by either radioactivity or microbiological assays, indicated net retention of 37-67% of the dose irrespective of chain length ingested and major avenue of loss. During the peak of absorption the folate circulating in plasma was active for both Streptococcus fecalis and Lactobacillus casei and carried specific radioactivity which was virtually identical with that of the administered dose. This suggests that neither methylation, conjugation, nor displacement of nonradioactive folate occurred to any significant extent during the 1st 2 hr. The specific radioactivity of 24-hr urine specimens as measured with L. casei corresponded closely with that of the administered dose. Evidence exists that methylation of the radioactive folate may occur, but significant displacement of nonradioactive methylfolate was not observed under the conditions of this study. Since 50-75% of administered heptaglutamate appears to be absorbable in man, estimates of dietary intake should include this fraction as well as the free folate.

Jejunal Perfusion of Simple and Conjugated Folates in Man

The technique of human jejunal perfusion was used to study the process of digestion and absorption of conjugated folates in five healthy volunteers. The test solution of isotonic saline contained equimolar concentrations of purified [3H]pteroylmonoglutamate ([3H]PG-1) and of [14C]pteroylheptaglutamate ([14C]PG-7) which was labeled on the first glutamyl unit. Calculations were made of the luminal disappearance of each labeled folate, and degradation products of [14C]PG-7 were identified in luminal contents obtained 15 and 30 cm from the infusion site. The percentage of disappearance of [3H]PG-1 was 74.7% and of [14C]PG-7 was 52.6% (P less than 0.001)9 Column chromatography of intestinal aspirates demonstrated a spectrum of 14C-labeled folates corresponding to chain lengths from [14C]PG-1 to [14C]PG-7, with distal accumulation of derived [14C]PG-1. Intraluminal hydrolysis of [14C]PG-7 was excluded by finding the compound unchanged after in vitro incubation with intestinal juice obtained by saline perfusion or siphonage. These data indicate that there are separate rates for the luminal disappearance of PG-1 and PG-7, and show that the digestion of PG-7 requires its contact with the intestinal mucosa. The evidence suggests that progressive mucosal hydrolysis is integral to the process of PG-7 absorption in man.

Studies on the absorption and metabolism of folic acid: I. Folate absorption in the dog after exposure of isolated intestinal segments to synthetic pteroylpolyglutamates of various chain lengths

Folic acid absorption was studied in anesthetized dogs by determining the amount and chemical nature of folate in venous blood emerging from isolated intestinal segments containing free folic acid and/or pteroylpolyglutamates of a known chain length. Chromatographically pure test materials placed in the lumen were prepared by unambiguous solid phase synthetic methods. This synthetic procedure not only yields compounds of known structure, it also provides a means by which glutamic acid residues at any given position in the gamma glutamyl chain can be made radioactive. For example, teropterin (pteroyltriglutamate) was synthesized in such a way that (14)C was present only in the middle glutamic acid unit. Suitable placement of label permitted assessment of the extent of peptide cleavage. The action of plasma conjugase was inhibited by copper chloride. Plasma samples were analyzed by Lactobacillus casei and Streptococcus faecalis assay, by column chromatography, and by quantitative measurement of pteridine-bound radioactivity. It was observed that biologically active folate appeared in the mesenteric vein with either pteroylmono-, di-, tri-, penta-, or heptaglutamate in the lumen. Generally speaking the absorption rate appeared to be inversely related to the length of the gamma glutamyl side chain. Roughly twice as much folic acid appeared in the circulation from (3)H-labeled pteroylmonoglutamate as from (14)C-labeled pteroylpentaglutamate when equimolar amounts of each were placed simultaneously in a single intestinal segment. Pteroylmonoglutamate appeared to be the predominant form entering the blood from each of the precursors tested. However, evidence was obtained that pteroyldiglutamate may enter the mesenteric vein soon after placing pteroyldi-, or triglutamate in the lumen, but not with the higher polyglutamates. Comparison of radioactivity and biological activity patterns suggests little conversion, if any, to reduced or methylated forms during the first 30 min of passage through the intestinal mucosa. We conclude that both pteroylmonoglutamates and pteroyldiglutamates may across the intestinal mucosa of the dog, and that reduction and methylation are not essential to the absorption process.

Partial Purification and Some Properties of Pteroylpolyglutamate Hydrolase (Conjugase) from Chicken Pancreas

Summary A simple and rapid procedure for the purification of pteroylpolyglutamate hydrolase (conjugase) from chicken pancreas for the purpose of standardization of microbiological assays of folates has been developed. It yields a stable folate-free preparation in quantity. The purification steps included extraction of conjugase from crude lyophilized chicken pancreas in mer-captoethanol followed by DEAE-cellulose chromatography. The enzyme was further purified by absorption to alumina-C-γ-gel from which it was eluted with phosphate buffer, pH 6.5. The enzyme was concentrated by dialysis against 20 vol of 2 M sucrose, lyophilized, and vacuum-sealed. The purified conjugase exhibited two peaks on Sephadex G-75 chromatography corresponding to molecular weights of 50,000 and 25,000, respectively. The optimum