EXCRETION OF FORMIMINOGLUTAMIC ACID IN RETICULOSIS AND CARCINOMA

454 treated with lymphocytes from a patient with hsemorrhagic vasculitis with intense involvement of the kidneys. These experiments demonstrated the role of immune lymphocytes in the pathogenesis of S.L.E. We assume that it is these cells that constitute the main link in the pathological process and produce the destruction of tissues while autoantibodies are only of secondary importance. These findings should be further investigated by examining the dynamics of the clinical course of the disease. The findings may be important for the evaluation of the intensity of the disease besides predominant involvement of some or other organs.

In order to assess abnormal levels we investigated six patients with untreated addisonian pernicious anasmia, seven with vitamin-B12 deficiency due to other causes; and fourteen with megaloblastic anaemia as a result of folic-acid deficiency. Fortyseven patients with diseases other than megaloblastic anasmia or malignancy were investigated in order to establish the range of normal (table i). Table 11 summarises the findings in fifty-two patients with malignant disease. At the time of investigation they had not received antibiotics, corticosteroids, or cytotoxic agents for a period of at least three weeks.

Conclusions

8-hour test.-The details of this test were as described by Chanarin and Bennett (1962). The patients were fasted overnight. 15 g. of L-histidine monochloride was given by mouth, and the urine was collected for 8 hours into 5 ml. of N hydrochloric acid. 12-hour test.-The conditions were the same as for the 8-hour test, but the urine collection was continued for a further 4 hours. This test was introduced because some patients had difficulty in voiding urine at the end of the 8-hour period. 24-hour test.-The details of histidine loading were as described by Tabor and Wyngarden (1958). Patients were not fasted; three separate doses of 5 g. histidine (one after each main meal) were given; and the urine was collected for 24 hours after the first dose of histidine.

Lymphocytes from patients with systemic lupus erythematosus and scleroderma destroyed fibroblasts of human embryo in tissue culture. A similar effect was observed with lymphocytes from patients with systemic lupus erythematosus in human kidney-cell cultures. Lymphocytes from a control group showed no such effect. It is suggested that these findings not only indicate the role of immune lymphocytes in the pathogenesis of systemic lupus erythematosus and scleroderma but may also have some diagnostic significance. Requests for reprints should be addressed to Dr. George J. SvetMoldavsky, Institute of Experimental and Clinical Oncology A.M.S., U.S.S.R., Kashirscoe Shosse 6, Moscow M-478. REFERENCES

Berg. O., Kallen, B. (1964) J. Neuropath. exp. Neurol. 23, 550. Govaerts, A. J. (1960) J. Immun. 85, 516. Koprowski, H., Fernandes, M. V. (1962) J. exp. Med. 116, 167. Perlmann, P., Broberger, O. (1963) ibid. 117, 717. Rosenau, W., Moon, H. D. (1961) J. natn. Cancer Inst. 27, 171. Tareev, E. M. (1965) Collagenoses. Moscow. Walker, R. G., Palmer, I. G. (1962) Blood, 20, 109.

OF THE UNIVERSITY DEPARTMENT OF CLINICAL

HÆMATOLOGY, INFIRMARY, MANCHESTER, 13

THE excretion of formiminoglutamic acid (FIGLU) in the urine after a loading dose of histidine has been regarded as evidence of folic-acid deficiency (Luhby et al. 1958). Raised excretion is also found in some patients with addisonian pernicious anxmia (Knowles and Prankerd 1962, Chanarin 1963, Villemil and McCracken 1963), liver disease (Knowles et al. 1963a), and skin disease (Knowles et al. 1963b). Raised FiGLU excretion in leukxmia was noted by Villemil and McCracken (1963), and in lymphoma by Rose (1965). We report here our evaluation of a FIGLU-excretion test " in hxmatological practice and its significance in malignant disease, using the term non-specifically to indicate the eventually fatal group of leukxmias, reticuloses, and myeloma in addition to carcinoma. "

Patients and Methods All

patients had been admitted to the wards of Manchester Royal Infirmary for the investigation and treatment of suspected blood-diseases. In most of them the diagnosis was established before the FIGLU test became evident later.

was

carried out, but in

investigated:

All urine samples were assayed by the method of Chanarin and Bennett (1962) which estimates the content of urocanic acid and FiGLU. No attempt was made to separate these two substances and the total was expressed as the equivalent amount of FiGLU in mg. per specimen. Serum-folate.-Serum was taken after an overnight fast and assayed with Lactobacillus casei as the test organism in Difco folic acid casei ’ medium. Ascorbic acid was added and conditions for the assay were as described by Waters and Mollin

some

Serum-vitamin B12 levels were assayed with Lactobacillus leichmannii using Dano Bl2 assay’ medium. Bone-marrow examination.-Aspirates were obtained from the sternum or the iliac crest. They were classified as showing megaloblastic hsemopoiesis if either megaloblastic erythroblasts or indubitable macrometamyelocytes were present.

Results

S. NOEYPATIMANOND M.B. Bangkok E. J. WATSON-WILLIAMS M. C. G. ISRAËLS M.B. Cantab., M.R.C.P. M.D., M.Sc. Manc., F.R.C.P.

only

were

(1963).

EXCRETION OF FORMIMINOGLUTAMIC ACID IN RETICULOSIS AND CARCINOMA

ROYAL

The FIGLU Test Three separate tests

it

Eight patients with intestinal malabsorption, as evidenced by increased excretion of fat and abnormal jejunal biopsy specimens, excreted 30-370 mg. FiGLU in the 8-hour test, 44-630 mg. in 12 hours, and 56-560 mg. in the 24-hour test. The specimen containing 30 mg. measured 110 ml. and a further 29 mg. FiGLU was excreted in the next 4 hours. The original collection was probably incomplete owing to difficulty in emptying the bladder. Serum-folate levels were from 0-6 mg. to 2.0 mg. per ml. Six patients with megaloblastic ansemia due to folic-acid deficiency from other causes excreted between 42 mg. and 880 mg. FIGLU in every specimen collected. The serum-folate level of five of these patients was less than 2-0 mg. per ml. but was 4-1 m per ml. in one (her serum-vitamin-B12 level was normal and there was a complete hxmatological response to 100 g. sodium folate intramuscularly daily for 5 days). Control Values Ten patients had

no hasmatological disorder, and seven with normal haemoglobin levels. The hxmophilia highest values of FIGLU excretion were 32 mg. in 8 hours, 35 mg. in 12 hours, and 40 mg. in 24 hours. We therefore accept 40 mg. as the upper limit of normal for FiGLU excretion in any of the three urine collections in hospital patients. By this definition excretion of FIGLU was abnormal in six out of eleven patients with primary

had

455 TABLE I-FIGLU EXCRETION IN MEGALOBLASTIC ANaeMIA AND OTHER CONDITIONS

in the 8-hour test, five out of nine in the 12-hour test, and four out of ten in the 24-hour test. In other patients without megaloblastic anaemia or malignancy there were also two abnormal 8-hour tests, two abnormal 12-hour tests, and two abnormal 24-hour tests

vitamin-B12 deficiency

(table III). Malignant Disease Of fifty-two patients with malignant disease, twentynine had FIGLU excretion measured in 8-hour, 12-hour,

patient gave an abnormal excretion in a single 8-hour test. Again there was no correlation with serum-folate levels. Two patients with Hodgkin’s disease had grossly megaloblastic erythropoiesis. Serum-folate levels were below 2 mfl-g. per ml., and both had very high values of FiGLU excretion. After therapeutic doses of sodium TABLE IV-SERUM-FOLATE LEVELS COMPARED WITH FIGLU EXCRETION IN MALIGNANT DISEASE

and 24-hour tests: twelve gave increased excretion in each test; nine gave consistent normal results; and eight gave increased excretion in one test and normal in at least one other test. There was no obvious reason for these inconsistent results and no correlation with serum-folate levels (table iv). For the purpose of further analysis FIGLU excretion was regarded as abnormal when more than 40 mg. was found in any specimen. Eleven patients had only 8-hour and 24-hour collections: six had only 12-hour and 24-hour collections; and fom had only 8-hour and 12-hour collections. In ten the results were abnormal, and in eleven they were normal. One TABLE II-FIGLU EXCRETION IN MALIGNANT DISEASE

folate

TABLE III-CONTROLS GIVING INCREASED FIGLU EXCRETION

(15 mg. intramuscularly per day for 4 days) erythropoiesis became normoblastic; FiGLU excretion was unchanged in one, and fell to normal levels in the other. One patient with chronic lymphatic leukxmia showed slight megaloblastic change in the bone-marrow which was associated with a low serum-folate level (0-8 m[tg. per ml.) and normal FiGLU excretion. In all other patients, erythropoiesis was normoblastic at the time of the investigation. Thus, out of fifty-two patients, thirty-one excreted abnormal amounts of FIGLU after a histidine load. Abnorwas found in at least some patients in all the disease groups (table v). It seemed to be commoner in solid tumours (sixteen out of twenty cases) than in patients with leukxmia, myeloma, or myeloid reticulosis (fifteen out of thirty-one). There was no relation with hxmoglobin level, number or size of tumours, or duration of disease before the test was carried out. A raised FIGLU excretion does, however, appear to suggest a worse prognosis. Twenty-five out of thirty-one patients with abnormal excretion have died, whereas in the same period only

mal excretion

456 TABLE V-FIGLU EXCRETION COMPARED WITH SERUM-FOLATE LEVEL IN VARIOUS MALIGNANT DISEASES

raised in persons with solid

tumours

than in leukasmic

patients. It is difficult to see why FiGLU excretion should be abnormal, although many patients with malignant disease show other evidence of folic-acid deficiency and megaloblastic haemopoiesis is not uncommon (Rose 1965). However, there is no correlation in our series with serumfolate levels, and when folic acid is given the FIGLU excre-

tion does

become normal. In some patients with malignant disease a disturbance of folic-acid metabolism not

probable, but it is clearly not the same as a true deficiency. Unless haemopoiesis is megaloblastic, these patients are unlikely to benefit from treatment with folic seems

acid. al. (1965) found low serum-folate levels in some with leuksemia, lymphoma, and other malignant patients but there was no correlation between folate levels tumours, and the extent of the disease. Hellman et al. (1964) found low serum-folate levels (less than 3 m[Lg. per ml.) in six of eighteen patients with carcinoma of the head and neck, but there was no difference in their response to anti-folate therapy. Hoogstraten et al. (1961) found low serum-folate levels in some patients with leukaemia, Hodgkin’s disease, and lymphosarcoma, but only in acute lymphoblastic leukaemia were these low levels related to resistance to antifolate treatment. Rao

four of twenty-one with normal results have died

(table vi). Effect of Folic acid on

FIGLU Excretion

In six patients with megaloblastic anaemia due to folicacid deficiency, the FIGLU excretion became normal shortly after they were given adequate treatment with folic acid. Two other patients had a pronounced reduction in FIGLU excretion after small doses of folic acid. No constant change was observed in four patients with malignant disease who were given folic acid (table vn). Discussion

Our results and those of Villemil and McCracken (1963) and Rose (1965) show that FiGLU excretion is often raised in malignant disease. In our series it was more commonly TABLE VI-FIGLU EXCRETION RELATED TO SURVIVAL IN MALIGNANT DISEASE

et

Despierres (1965) found that some patients with maligdisease excreted abnormally little folic acid in the

nant

urine after a standard oral dose. He believed that this finding might have some value in screening procedures for possible malignancy. Of forty-three patients with car-

cinoma, thirty-two gave a positive test as against only ten unexplained positives in fifty-one patients with chronic non-malignant disease. These findings are curiously similar to ours. We suggest that the FiGLU test might be useful where the diagnosis of malignant disease is being considered.

Summary

TABLE

VII-EFFECT

OF

TREATMENT WITH EXCRETION

FOLIC

ACID

ON

FIGLU

Fourteen patients with megaloblastic anaemia due to folic-acid deficiency excreted more than 40 mg. FiGLU in the urine after a loading dose of histidine. This figure was exceeded in five of forty-six patients with other diseases, but 40 mg. FIGLU was taken as the upper limit of normal after a histidine load. Thirty-one of fifty-two patients with various malignant diseases excreted abnormal amounts of FIGLU. There was no correlation between FIGLU excretion and haemoglobin level, clinical manifestations of the extent of disease, or serum-folate level. The finding of a raised FIGLU excretion seemed to be associated with a poor

prognosis. Requests

for

reprints

should be addressed to M. C. G. I. REFERENCES

Chanarin, I. (1963) Br. J. Hœmat. 9, 141. — Bennett, M. G. (1962) Br. med. J. i, 27. Despierres, G. (1965) Poumon Coeur, 21, 169. Hellman, S., Iannotti, A. T., Bertino, J. R. (1964) Cancer Res. 24, 105. Hoogstraten, B., Baker, H., Reizenstein, P. (1961) Blood, 18, 787. Knowles, J. P., Prankerd, T. A. J. (1962) Clin. Sci. 22, 233. Shaldon, S., Fleming, A. (1963a) ibid. 24, 39. — Shuster, S., Wells, G. C. (1963b) Lancet, i, 1138. Luhby, A. L., Cooperman, J. M., Teller, D. N., Donnenfield, A. M. (1958) J. clin. Invest. 37, 915. Rao, P. B. R., Lagerlöf, B., Einhorn, J., Reizenstein, P. G. (1965) Cancer Res. 25, 221. Rose, D. P. (1965) J. clin. Path. 18, 486. Tabor, H., Wyngarden, L. (1958) J. clin. Invest. 37, 824. Villemil, A., McCracken, B. H. (1963) Br. med. J. i, 717. Waters, A. H., Mollin, D. L. (1963) Br. J. Hœmat. 9, 319. —

*

Figures refer

to

8-hr., 12-hr., i.m.

or 24-hr. urine collections. Intramuscular.