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Purpose: To investigate the hypothesis that methotrexate causes demyelination due to a deficiency in S-adenosylmethionine (SAM) during the treatment of acute lymphoblastic leukemia (ALL)., Patients and Methods: Twenty-four patients treated on the Medical Research Council United Kingdom ALL trial no. 11 (MRC UKALL XI) were studied. The trial randomized patients at the presymptomatic CNS treatment (PCNS) phase to receive (1) intrathecal methotrexate and cranial radiotherapy (CRTX); (2) high-dose intravenous methotrexate with folinic acid rescue and continuing intrathecal methotrexate (HDMTX); and (3) continuing intrathecal methotrexate alone (ITMTX). Serial CSF samples were collected throughout treatment and concentrations of 5-methyltetrahydrofolate (MTF), methionine (MET), SAM, and myelin basic protein (MBP) were measured. The results were grouped into treatment milestones and compared with an age-matched reference population., Results: There was a highly significant effect of both treatment milestones and trial arm on the metabolite and MBP concentrations. CSF MTF reached a nadir during the induction phase of treatment, while SAM and MET reached their nadir during the consolidation phase. CSF MBP mirrored SAM concentration and there was a significant inverse relationship between the two. MTF, SAM, and MBP returned to normal values by the end of treatment, while MET was increased significantly. The effect of treatment was decremental across the ITMTX, HDMTX, and CRTX groups., Conclusion: Treatment of ALL causes marked abnormalities in the single-carbon transfer pathway and subclinical demyelination. Methotrexate is one cause of this. Whether these abnormalities contribute to the late cognitive deficits requires further study.

A method for the simultaneous determination of the antifolates methotrexate and 7-hydroxymethotrexate as well as the folates 5-methyltetrahydrofolic acid and folinic acid (5-formyltetrahydrofolic acid) in serum and cerebrospinal fluid (CSF) is described. High-performance liquid chromatography with gradient elution and dual detection (ultraviolet absorption and fluorescence) was used to separate and quantitate the analytes. Serum samples containing high levels of the substances of interest and CSF samples were injected directly onto the HPLC column. For determination of low concentrations, serum samples were subjected to a solid-phase extraction method for clean-up and concentration purposes. The determination limits were 10 ng/ml for both antifolates, 100 ng/ml for folinic acid, and 0.1 ng/ml for the physiologically occurring methylated folate which is about 1/100 the serum concentration in healthy children. The suitability of the method for pharmacokinetic monitoring of high-dose methotrexate therapy combined with leucovorin rescue administered to children with acute lymphoblastic leukemia was demonstrated. Minimum values of the serum folate during treatment ranged from 0.2 to 3.1 ng/ml. Even those very low concentrations could be reliably measured.

Osteosarcoma patients free of CNS metastases are at risk for acquiring leukoencephalopathy after receiving multiple courses of high dose intravenous methotrexate followed by oral leucovorin rescue (MTX-LV). A prospective study of the adequacy of CNS rescue of MTX biochemical toxicity by oral leucovorin was undertaken in newly diagnosed neurologically normal osteosarcoma patients. Prior to surgical resection of the primary tumor, ten patients received 4 weekly courses of MTX-LV. During the fourth weekly MTX-LV treatment, 0 and 72 hr serum and CSF determinations of MTX, 5-methyl-tetrahydrofolate (5-MTHF) and LV were made. No CSF MTX was detectable at 0 hr in any patient, but a significant elevation in CSF MTX occurred in 9/9 patients at 72 hr (mean 47.2 +/- 31.8 ng/ml or 1.04 +/- 0.7 X 10(-7) M). There was no significant change in mean CSF 5-MTHF over 72 hr despite a rise in serum 5-MTHF. MTX exceeded 5-MTHF in 6/9 patients in CSF, whereas only 3/8 patients had higher MTX in the serum at 72 hr. No acute systemic or neurotoxicity was seen. The failure of oral leucovorin to consistently elevate CSF 5-MTHF levels at 72 hr in the context of significant levels of CSF MTX may result in intermittent CNS folate deficiency. The clinical and pathological syndrome of leukoencephalopathy may be related to this phenomenon and may evolve after repeated MTX-LV treatments.

Serum and cerebrospinal fluid (CSF) concentrations of citrovorum factor (CF) and 5-methyltetrahydrofolic acid (5-MTHFA) were measured after i.v. infusion of leucovorin (50 or 100 mg/sq m) in seven patients undergoing treatment for meningeal carcinomatosis by intra-Ommaya reservoir injection of methotrexate (MTX). Serum CF levels rapidly rose after leucovorin administration as did 5-MTHFA, its conversion product. A small amount of CF entered the CSF, but peak CSF 5-MTHFA increased about 10-fold. The concentration X time (C X t) of additional 5-MTHFA in the CSF was greater [114.4 +/- 36.1 (S.E.) microgram/ml X min] after 100-mg/sq m doses of leucovorin than after 50 mg/sq m [14.2 +/- 4.3 micrograms/ml X min] (p less than 0.05). The CSF MTX concentration exceeded CSF 5-MTHFA by 2 to 3 logs throughout the 48 hr of observation, while serum 5-MTHFA and CF exceeded serum MTX by 0.5 to 2 logs. This study demonstrates that leucovorin administered i.v. to patients receiving intra-Ommaya MTX does not increase CSF concentrations of "rescue" folate above those of CSF MTX and are unlikely to interfere with MTX action against meningeal tumor. On the other hand, i.v. leucovorin does permit serum "rescue" folate to operate, thus reducing the systemic toxicity that may follow intraventricular administration of MTX.

In the first part of this study the availability of folinic acid (FA) and its main active circulating metabolite, 5-methyltetrahydrofolate (5-MTHF), were studied in plasma and cerebrospinal fluid (CSF) from normal subjects after i.v. administration of 100 and 250 mg of FA. 5-MTHF rapidly appeared in plasma, the maximum value being reached at the first observation time point (1 h). FA was eliminated in plasma more slowly than 5-MTHF. Between the two doses, there was no evidence of modification in pharmacokinetic parameters (terminal half-life, clearance) for either FA or 5-MTHF in plasma and CSF; 5-MTHF was the only product detectable in CSF. Considering FA plus 5-MTHF together, the AUC (area under the curve) ratios between CSF and plasma were close to 1%. 5-MTHF was cleared very slowly from CSF (t 1/2 = 85 h). This finding suggested possible accumulation of 5-MTHF in CSF during repeated administration of FA combined with medium or high dose MTX. In the second part of the study, dealing with a group of eight children treated by such protocols, an increase in CSF 5-MTHF was detected from cycle to cycle in five (r = 0.91, P less than 0.01) with a maximum at 5 x 10(-8) M. This progressive accumulation of 5-MTHF in CSF may have a negative effect on the local action of MTX and should be taken into account for therapeutic strategies designed for the management of meningeal leukaemia.

A high-performance liquid chromatographic method for the determination of N5-methyltetrahydrofolic acid, leucovorin, methotrexate and 7-hydroxymethotrexate in plasma and liquor samples is presented. Gradient elution is used to increase the sensitivity. Four sample preparation methods were compared with respect to the stability of the injectable sample. Samples can be pretreated with a simple deproteinization method. For enhanced selectivity a solid-phase extraction procedure is described.