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Investigations of tissue folates in normal and malignant tissues.

The folates present in liver, gut and tumour tissue were examined before and after autolysis. Before autolysis 10-formylfolate tetraglutamate (10-CHOFA(glu)4), 5-methyltetrahydrofolate triglutamate (5-CH3THF(glu)3) and possibly tetrahydrofolate polyglutamate(s) (THF(glu)n) were detected. Liver contained all 3 species whereas no 5-CH3THF(glu)3 was present in the tumours; gut showed an intermediate situation. After autolysis the predominant monoglutamates formed were 5-CH3THF in the liver, 10-formylfolates in the gut and possibly tetrahydrofolate (THF) in the tumour extracts. These differences illustrate changes in tissue folates with the proliferation rate of the tissue and suggest an explanation for the methionine auxotrophy of Walker 256 carcinosarcoma cells.

Folate-dependent reactions are centrally involved in the biosynthetic pathways critical for cell replication and there are a number of reports in the literature of the differing patterns of folate co-enzymes exhibited by rapidly dividing tissues compared to resting tissues. These include studies on neoplasms (Sotobayashi et al., 1966) and on normal proliferating tissue (Barbiroli et al., 1975). Overall, the results show a shift from 5methyltetrahydrofolate (5-CH3THF) to formylfolates in the rapidly-dividing tissue. The major naturally-occurring tissue folates are polyglutamate conjugates (Whitehead, 1971) and changes in the glutamyl chain length have also been suggested to coincide with an increase in cellular proliferation. Krumdiek et al. (1976) described changes in the ratio of monoglutamyl-topolyglutamyl folate in the uteri of rats at different stages of the oestrus cycle and suggested that the increase in free folate (i.e. monoglutamate derivatives) at proestrus may be connected with the increase in uterine cell growth at this stage. Whitehead (1973) and Marchetti et al. (1980) reported a shift from high chain length forms to mono-and triglutamate forms in regenerating rat liver but Lavoie et al. (1975) were unable to confirm this finding.
Studies of the folate-dependent enzymes in liver and a number of hepatomas have shown that the activity of the majority of these enzymes is lower in the hepatomas (Lepage et al., 1972). Thymidylate synthetase is a notable exception, its activity being considerably greater (Jackson & Niethammer, 1979).
Other studies indicate that the folate-requiring enzymes involved directly or indirectly with nucleic acid synthesis show maximal activity in dividing cells (Rosenblatt & Erbe, 1973;Johnson et al., 1978;Rode et al., 1979;Rowe et al., 1979). These enzymatic changes would be expected to result in a displacement in the equilibrium between the various coenzymes.
In this investigation the folates present in liver (a slow-growing tissue), gut (a rapidly-growing normal tissue) and tumour tissue were compared. Two approaches were used; (a) the tissues were allowed to autolyse and the resulting monoglutamates studied; (b) direct analysis of the folates present in the different tissues was performed.

Tissue autolysis experiments
Normal and tumour-bearing male rats (Chester Beatty Wistars) in groups of 3, were given p.o. a mixture of 2yuCi 2-[14C]and 5yuCi 3', 5', 7,-[3H) folic acid (10OIgkg-' body wt.) and maintained in cages designed to prevent coprophagy with free access to food and water. Forty-eight hours after administration of the labelled folic acid the animals were killed and the livers, whole gut tissue or tumours removed, chilled and homogenised in 0.25 M-sucrose/0.05 M sodium phosphate buffer, pH 7.0 containing 2.0% (w/v) sodium ascorbate. The homogenates were centrifuged at 4°C for h at 100,000 g and the resulting cytosol fraction was used in the subsequent incubations.
Samples of cytosol (5-10 ml) were allowed to autolyse at 37°C for various times (0-2 h). The reaction was stopped by heating to 100°C for 5 min., cooling and centrifuging to remove precipitated protein. The supernatant was then sequentially chromatographed on Sephadex G15 and DEAE-cellulose.
Identification of tissue folates Normal and tumour-bearing rats (Chester Beatty or WAB/NOT Wistars) were given p.o. a mixture of 2 yiCi 2-[14C] and 5 jCi 3 ',5',7,9-[3H]-folic acid (80jigkg-1 body wt.) and kept in cages as above. At various times (2h-11 days) after administration, rats were killed in pairs and the tissues under investigation removed and extracted in hot 0.05M sodium phosphate buffer, pH 7.0, containing 2% (w/v) sodium ascorbate as described by Barford et al. (1977). Analysis of tissue extracts was carried out using sequential Sephadex G15 and DEAE-cellulose chromatography. Isolated fractions were examined by HPLC.

Animals
Chester Beatty Wistar rats were supplied by Mr B. Mitchley, Chester Beatty Research Institute, Institute of Cancer Research London. Tumours were initiated by s.c. injections of 2 x 106 Walker 256 carcinosarcoma ascitic cells into the right flank and the animals used one week later.
WAB/NOT Wistar rats were supplied by Dr M.V. Pimm, University of Nottingham. Animals received s.c. trocar implantation of fragments of the MC103B sarcoma (Pimm et al., 1980) on the right flank. The tumour mass was allowed to grow for 2-3 weeks before experimentation.
Determination of radioactivity Column effluents were counted as described by Connor et al. (1979).
Column chromatography Sephadex G1 5 gel filtration and DEAE-cellulose chromatography (using linear gradients of 0-1.2 M NaCI in 0.05 M sodium phosphate buffer, pH 7.0) were performed as described by Barford et al. (1977). In certain cases fractions from Sephadex GI5 columns were collected into 1 ml sodium ascorbate (2% w/v) to prevent oxidation of labile folates.
High performance liquid chromatography (HPLC) HPLC was carried out on a Partisil anion-exchange column (M9, 10/50 SAX, 10mm x 50cm) using 2 LDC constametric pumps controlled by a LDC gradient master. Folate markers were detected by their U.V. absorbance at 280 nm using a LDC spectro monitor (Model III). Prior to loading onto HPLC (sample loop injection, 2 ml) samples were filtered (0.22 jim Millipore) and adjusted to pH 4.5. Column elution was affected over 1 h with a flow rate of 1 ml min-1 using a linear Na2SO4 gradient (0-0.5 M) in 0.05 M sodium phosphate buffer (pH. 4.5) containing dithiothreitol (5 mg%0). On completion of gradient time (1 h), elution was continued at maximum salt concentration for an additional hour. Where appropriate, fractions (2 ml) were collected and the total radioactivity in each one determined. The retention times of various folate derivatives under these conditions are given in Table I. Unlike the other columns, this method also separates folate polyglutamates differing in the number of glutamate residues.  (1959) and 10-CHOTHF by the method of Rowe (1971). Authentic folate polyglutamate derivatives were synthesised in this laboratory as follows: (iii) 5-CH3THF(glu). (n=3 and 4) from the corresponding FA(glu). according to Blair & Saunders (1970).

Results
Tlissue autolysis experiments (a) Liver. Sephadex G 15 chromatography separated the radioactivity in the autolysed liver samples into 3 dual-labelled peaks eluting at tube numbers 11 (Peak I), 20 (Peak II) and 34 (Peak III). These are the elution positions of folate polyglutamates, formylfolates (lO-CHOFA + 10-CHOTHF) and 5-CH3THF respectively. The identity of peaks II and III was confirmed by cochromatography with authentic markers on DEAE-cellulose. As the incubation time increased the proportion of radioactivity present as folate polyglutamates fell and that as 5-CH3THF increased until, at the end of the experiment, 5-CH3THF was the dominant species representing >70% of the radioactivity (Table II). (b) Gut: Sephadex G15 chromatography of autolysed gut extracts showed the presence of peaks II and III only. No polyglutamates were detected at any time. The presence of formyl folates and 5-CH3THF was again confirmed by rechromatography of peaks II and III on DEAEcellulose. In this case there was little change with time, equilibrium between the folates being established rapidly. In contrast to the liver, formyl folates were the major monoglutamates formed (Table III). Some breakdown of labile folates to scission products is indicated by the excess [3H] associated with peak II (probably paminobenzoyl-L-glutamate) and the excess ["4C] associated with peak III (possibility a pterin). These single-labelled peaks separated on rechromatography on DEAE-cellulose.

Identification of tissuefolates Sephadex
Gi 5 chromatography showed polyglutamate synthesis to be complete 10 h after the administration of folic acid. DEAE-cellulose chromatography of the folate polyglutamate peak separated on G15 showed the presence of 3 duallabelled species (A, B and C). Folate polyglutamates A and B were incompletely resolved and eluted between 0.4 M and 0.6 M NaCl while polyglutamate C eluted between 0.8-1.00 M NaCl. In most cases variable amounts of single-labelled species including pterin, were present indicating that some breakdown occurred during handling.
Folate polyglutamate A (the earliest eluting species) co-chromatographed on DEAE-cellulose with the authentic 10-CHOFA(glu)4 and corresponds to the species isolated from rat liver and identified as such by Connor & Blair (1980). Folate polyglutamate B eluted from DEAEcellulose in the region of the 5-CH3THF derivatives  and co-chromatographed with 5-CH3THF(glu)3 on HPLC.
Folate polyglutamate C exhibited marked lability and was not detected unless precautions were taken to prevent breakdown during chromatography. Rechromatography on HPLC was not possible because of its instability. It failed to chromatograph with 5-CH3THF(flu)4 or FA(glu)4 and no change was observed following NaBH4 treatment suggesting that folate polyglutamate C is not a 5,10-CH2THF derivative. The considerable lability of folate polyglutamate C and the detection of pterin, an oxidation product of THF (Blair & Pearson, 1974) suggest that it may be a THF polyglutamate. This is supported by Shin et al. (1972) who identified a THF polyglutamate in rat liver; a species exhibiting very similar chromatographic properties on DEAE-cellulose to folate polyglutamate C. Tissue distribution of folate polyglutamates: All 3 species were present in liver up to 11 days after the administration of labelled folic acid. A and B were present in approximately equal amounts whereas C was the minor species. However the levels of B 5-CH3THF(glu)3 were reduced in the intestinal extract at 24 h and B was absent from both types of tumour tissue at this time.
At earlier time periods (2-6 h) labelled folate monoglutamates were present in the tissue extracts. Folic acid and 5-CH3THF were identified in all 3 tissues; 10-CHOTHF was present in gut and tumour extracts but was not detected in liver.

Discussion
These studies again show differing patterns of folate coenzymes in different tissues. During the autolysis experiments conjugase released from the lysosomes hydrolyses the folate polyglutamates to monoglutamates and an equilibrium is set up determined by the balance of folate-metabolising enzymes in that tissue. The rapid destruction of the polyglutamates in gut and tumour extracts presumably reflects greater lysosome fragility since all 3 tissues were shown to contain principally polyglutamate after 10 h when conjugase was rapidly inactivated by heat. The pattern of monoglutamates at equilibrium was markedly different in the 3 tissues examined. The 5-CH3THF:10-CHO folate ratio of 4.7:1 for the liver was reversed in the gut to 1:3.9 and the major monoglutamate formed in tumour tissue was a very labile species possibly THF. There is also evidence of a greater amount of this species in the gut than the liver. The folate polyglutamates identified by direct analysis were l0-CHOFA(glu)4, 5-CH3THF(glu)3 and possibly THF(glu)n. These observations agree with the isolation of 10-CHOFA(glu)4 from rat liver (Connor & Blair, 1980) and the detection of 5-CH3THF polyglutamates (Shin et al., 1972;Hillman et al., 1977) and THF(glu)4 (Wittwer & Wagner, 1980) by others. Again there was a difference in the balance of these folates in the 3 tissues studied. In the tumour tissue and to a lesser extent the gut, there was a shift away from 5-CH3THF(glu)3 in favour of 10-CHOFA(glu)4. Some of this is presumably 10-CHOTHF(glu)4 in vivo since identification of 10-CHOFA(glu)4 has been attributed to the oxidation of 10-CHOTHF(glu)4 during extraction and analysis (Connor & Blair, 1980). This shift away from 5-methyl forms of folate in favour of formyl folates or THF derivatives in the tumour and gut is consistent with the results of autolysis experiments and with the detection of 10-CHOTHF at early time periods in gut and tumour but not liver. It also explains the increased levels of 10-CHOTHF in the plasma of humans suffering from malignant disease (Ratanasthien et al., 1977) and in the urine of rats bearing the Walker 256 carcinosarcoma (Barford & Blair, 1978). These changes may reflect the differing requirement for purine and pyrimidine synthesis in the tissues examined. As these tissues have different proliferation rates these changes in folate coenzyme distribution may be related to tissue proliferation.
The Walker 256 carcinosarcoma in cell culture shows methionine auxotrophy (Halpern et al., 1974) which cannot be explained by the lack of methionine synthetase (Hoffman & Erbe, 1976). In these experiments no 5-CH3THF derivatives were found in Walker 256 carcinosarcoma extracts except shortly after administration of FA. This offers an explanation of the methionine auxotrophy since no methionine can be synthesised in the absence of the appropriate coenzyme and all folate entering the tumour appears to be channelled away from 5-CH3THF.