Decarboxylated-5-adenosylmethionine Excretion: A Biochemical Marker … · ylase; OFMO,...
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[CANCER RESEARCH 47, 890-895, February 1, 1987] Decarboxylated-5-adenosylmethionine Excretion: A Biochemical Marker of Ornithine Decarboxylase Inhibition by a-Difluoromethylornithine Klaus D. Haegele,' Ted A. W. Splinter, J. C. Romijn, Paul J. Schechter, and A. Sjoerdsma Merrell Dow Research Institute, 67084 Strasbourg Cedex, France [K. D. H., P. J. S.J; Department of Oncology, University Hospital Dykzigt, 3015 GD Rotterdam The Netherlands [T. A. W. S., J. C. RJ; and Merrell Dow Research Institute, Cincinnati, Ohio 45215 [A. S.J ABSTRACT In an attempt to define a biochemical marker of ornithine decarbox- ylase inhibition in humans, a-difluoromethylornithine hydrochloride (DFMO), an irreversible ornithine decarboxylase inhibitor, was infused i.v. in seven cancer patients over 10-day courses at doses of 10-90 g/day and 24-h urinary excretion of polyamines and decarboxylated-5-adeno- sylmethionine was determined before, during, and after treatment. DFMO produces marked increases in urinary decarboxylated-5-adeno- sylmethionine excretion, up to 84 times pretreatment values. This re sponse appears to be time dependent, requiring several days to reach a maximum and lasting at least 4-5 days after stopping DFMO. In contrast, urinary excretion of the polyamines putrescine, cadaverine, spermidine, /Vi-monoacetylspermidine, /Vg-monoacetylspermidine, and spermine, were not consistently altered by DFMO. We conclude that urinary excretion of decarboxylated-S-adenosylmethionine represents a valid bio chemical indicator of ornithine decarboxylase inhibition in humans, whereas urinary polyamines are of no value. INTRODUCTION The regulatory role of the polyamines in cell growth, cell proliferation, and cell replication is well established (1-6). The first step in the biosynthesis of the polyamines is the formation of putrescine from L-ornithine, a reaction catalyzed by ODC2 (7, 8). Spermidine and spermine are then formed by sequential aminopropylation of putrescine and spermidine, respectively. This aminopropyl moiety originates from dc-SAM, biosyn thesis of spermidine and spermine being catalyzed by the en zymes spermidine synthase and spermine synthase, respectively. Formation of dc-SAM itself is regulated by the enzyme SAM- DC, which catalyzes the formation of dc-SAM from S-adeno- sylmethionine (9-13). DFMO is a specific, enzyme-activated, irreversible inhibitor of ODC (14) which causes, as a consequence of ODC inhibition, a marked depletion of putrescine and spermidine but not of spermine in normal organs (15) and tumors (16). In addition, DFMO administration to normal or tumor-bearing rats has been reported to reduce 24-h urinary excretion of some polya mines (17). Furthermore, as a consequence of the negative regulatory role of spermidine on SAM-DC activity (12), DFMO produces an increase of SAM-DC activity and a dramatic (e.g., up to 450-fold) increase of dc-SAM in tissues (18-22). MDL 72.175, another irreversible ODC-inhibitor, resulted in a 4- to 5-fold increase of the 24-h urinary excretion of dc-SAM after its administration in the drinking water to male rats (23). Since DFMO is currently on clinical trials (24-31) for evaluation in human malignant disease and for therapy of parasitic infections, it is of primary importance to discover a convenient method to Received 6/10/86; revised 10/15/86; accepted 10/23/86. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1To whom requests for reprints should be addressed, at Merrell Dow Research Institute, 16, rue d'Ankara, 67084 Strasbourg Cedex, France. J The abbreviations used are: ODC, L-ornithine decarboxylase; dc-SAM, de- carboxytated-5-adenosylmethionine; SAM-DC, S-adenosylmethionine decarbox ylase; OFMO, DL-a-difluoromethylornithine monohydrate hydrochloride (eflor- nithine hydrochloride); dc-SAE, decarboxy-S-adenosylethionine. ascertain and monitor ODC inhibition in man. We therefore determined the 24-h urinary excretion of the polyamines and dc-SAM in patients prior to, during, and following continuous i.v. infusion of large doses of DFMO. MATERIALS AND METHODS Cancer Patients and Dosing Schedule Seven patients (five males, two females) with advanced cancer (Table 1) received DFMO as a 24-h i.v. infusion during a 10-day period. All subjects, with the exception of patient 2, had been previously treated with a variety of chemotherapies. The interval between the last course of chemotherapy and DFMO treatment was at least 6 weeks in all cases. Patients 1-3 received 10 g of DFMO on days 1 and 2, 20 g on days 3 and 4, increasing by 10 g increments every third day up to 50 g on days 9 and 10. Patients 4-5 received 50-90 g at 10-g ascending- dose intervals every third day. Patient 6 received a constant i.v. infusion dose of 50 g for 10 days and a 25 g dose for a further 10 days, approximately 2 months later. Patient 7 was infused with 10 g/day for 10 days and then 25 g/day for 10 days, approximately 2 months later. Patients 6 and 7 were added to the original study in an attempt to evaluate the time effects of DFMO administration on biochemical markers of ODC inhibition. Daily i.v. doses up to 64 g/m2 have been reported in cancer therapy (32) and currently recommended i.v. doses for susceptible protozoal infections are approximately 30 g/day (33). Sampling Schedule Twenty-four-h urine was collected during the 3 days prior to DFMO administration, during each 10-day treatment course, and at least 1 day, in some cases up to 5 days, postinfusion. Urines were collected into 2-liter containers containing 25 ml of 6 N HC1, total volume was measured, and 50-ml aliquots were taken and kept at -20°C until analysis for DFMO, the polyamines, and dc-SAM. During the ascend ing-dose study, plasma samples were obtained always on the second day of each dosage level for analysis of steady-state concentrations of DFMO. Analytical Methods DFMO. The assay of DFMO in biological samples has been de scribed (34). After addition of 20% trichloroacetic acid and centrifuga- tion, the supernatant was injected via a Kont n m APE-100 injector onto the column of a Kontron Liquimat II amino acid analyzer. Postcolumn derivatization was accomplished with o-phthaldialdehyde-mercaptoeth- anol to yield a fluorecent derivative which was detected with an Aminco spectrophotometer. Polyamines. The method used to determine putrescine, spermidine, spermine, AVmonoacetylspermidine, JV-g-monoacetylspermidine, and cadaverine, a polyamine arising via decarboxylation of lysine, was based on gradient elution high-performance liquid chromatography and postcolumn derivatization with o-phthaldialdehyde-mercaptoethanol (20, 35). dc-SAM. Adenine and its derivatives are known to react with 2- chloroacetaldehyde to form highly fluorescent tricyclic derivatives (36). This reaction was utilized to give a sensitive and specific method for measuring dc-SAM in urine (23) and plasma samples. As internal standard, dc-SAE was used throughout the work-up. This work-up consisted of absorption of dc-SAM and dc-SAE on a Dowex cation exchange column, elution with l N and 3 N HC1 to elute undesired products, and finally 6 N HC1 to elute dc-SAM and dc-SAE. To this 890 on June 18, 2021. © 1987 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from
Transcript of Decarboxylated-5-adenosylmethionine Excretion: A Biochemical Marker … · ylase; OFMO,...
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[CANCER RESEARCH 47, 890-895, February 1, 1987]
Decarboxylated-5-adenosylmethionine Excretion: A Biochemical Marker ofOrnithine Decarboxylase Inhibition by a-DifluoromethylornithineKlaus D. Haegele,' Ted A. W. Splinter, J. C. Romijn, Paul J. Schechter, and A. Sjoerdsma
Merrell Dow Research Institute, 67084 Strasbourg Cedex, France [K. D. H., P. J. S.J; Department of Oncology, University Hospital Dykzigt, 3015 GD Rotterdam TheNetherlands [T. A. W. S., J. C. RJ; and Merrell Dow Research Institute, Cincinnati, Ohio 45215 [A. S.J
ABSTRACT
In an attempt to define a biochemical marker of ornithine decarbox-ylase inhibition in humans, a-difluoromethylornithine hydrochloride(DFMO), an irreversible ornithine decarboxylase inhibitor, was infusedi.v. in seven cancer patients over 10-day courses at doses of 10-90 g/dayand 24-h urinary excretion of polyamines and decarboxylated-5-adeno-sylmethionine was determined before, during, and after treatment.DFMO produces marked increases in urinary decarboxylated-5-adeno-sylmethionine excretion, up to 84 times pretreatment values. This response appears to be time dependent, requiring several days to reach amaximum and lasting at least 4-5 days after stopping DFMO. In contrast,urinary excretion of the polyamines putrescine, cadaverine, spermidine,/Vi-monoacetylspermidine, /Vg-monoacetylspermidine, and spermine,were not consistently altered by DFMO. We conclude that urinaryexcretion of decarboxylated-S-adenosylmethionine represents a valid biochemical indicator of ornithine decarboxylase inhibition in humans,whereas urinary polyamines are of no value.
INTRODUCTION
The regulatory role of the polyamines in cell growth, cellproliferation, and cell replication is well established (1-6). Thefirst step in the biosynthesis of the polyamines is the formationof putrescine from L-ornithine, a reaction catalyzed by ODC2
(7, 8). Spermidine and spermine are then formed by sequentialaminopropylation of putrescine and spermidine, respectively.This aminopropyl moiety originates from dc-SAM, biosynthesis of spermidine and spermine being catalyzed by the enzymes spermidine synthase and spermine synthase, respectively.Formation of dc-SAM itself is regulated by the enzyme SAM-DC, which catalyzes the formation of dc-SAM from S-adeno-sylmethionine (9-13).
DFMO is a specific, enzyme-activated, irreversible inhibitorof ODC (14) which causes, as a consequence of ODC inhibition,a marked depletion of putrescine and spermidine but not ofspermine in normal organs (15) and tumors (16). In addition,DFMO administration to normal or tumor-bearing rats hasbeen reported to reduce 24-h urinary excretion of some polyamines (17). Furthermore, as a consequence of the negativeregulatory role of spermidine on SAM-DC activity (12), DFMOproduces an increase of SAM-DC activity and a dramatic (e.g.,up to 450-fold) increase of dc-SAM in tissues (18-22). MDL72.175, another irreversible ODC-inhibitor, resulted in a 4- to5-fold increase of the 24-h urinary excretion of dc-SAM afterits administration in the drinking water to male rats (23). SinceDFMO is currently on clinical trials (24-31) for evaluation inhuman malignant disease and for therapy of parasitic infections,it is of primary importance to discover a convenient method to
Received 6/10/86; revised 10/15/86; accepted 10/23/86.The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1To whom requests for reprints should be addressed, at Merrell Dow ResearchInstitute, 16, rue d'Ankara, 67084 Strasbourg Cedex, France.
J The abbreviations used are: ODC, L-ornithine decarboxylase; dc-SAM, de-carboxytated-5-adenosylmethionine; SAM-DC, S-adenosylmethionine decarboxylase; OFMO, DL-a-difluoromethylornithine monohydrate hydrochloride (eflor-nithine hydrochloride); dc-SAE, decarboxy-S-adenosylethionine.
ascertain and monitor ODC inhibition in man. We thereforedetermined the 24-h urinary excretion of the polyamines anddc-SAM in patients prior to, during, and following continuousi.v. infusion of large doses of DFMO.
MATERIALS AND METHODS
Cancer Patients and Dosing Schedule
Seven patients (five males, two females) with advanced cancer (Table1) received DFMO as a 24-h i.v. infusion during a 10-day period. Allsubjects, with the exception of patient 2, had been previously treatedwith a variety of chemotherapies. The interval between the last courseof chemotherapy and DFMO treatment was at least 6 weeks in allcases. Patients 1-3 received 10 g of DFMO on days 1 and 2, 20 g ondays 3 and 4, increasing by 10 g increments every third day up to 50 gon days 9 and 10. Patients 4-5 received 50-90 g at 10-g ascending-dose intervals every third day. Patient 6 received a constant i.v. infusiondose of 50 g for 10 days and a 25 g dose for a further 10 days,approximately 2 months later. Patient 7 was infused with 10 g/day for10 days and then 25 g/day for 10 days, approximately 2 months later.Patients 6 and 7 were added to the original study in an attempt toevaluate the time effects of DFMO administration on biochemicalmarkers of ODC inhibition. Daily i.v. doses up to 64 g/m2 have been
reported in cancer therapy (32) and currently recommended i.v. dosesfor susceptible protozoal infections are approximately 30 g/day (33).
Sampling Schedule
Twenty-four-h urine was collected during the 3 days prior to DFMOadministration, during each 10-day treatment course, and at least 1day, in some cases up to 5 days, postinfusion. Urines were collectedinto 2-liter containers containing 25 ml of 6 N HC1, total volume wasmeasured, and 50-ml aliquots were taken and kept at -20°C until
analysis for DFMO, the polyamines, and dc-SAM. During the ascending-dose study, plasma samples were obtained always on the secondday of each dosage level for analysis of steady-state concentrations ofDFMO.
Analytical Methods
DFMO. The assay of DFMO in biological samples has been described (34). After addition of 20% trichloroacetic acid and centrifuga-tion, the supernatant was injected via a Kont n m APE-100 injector ontothe column of a Kontron Liquimat II amino acid analyzer. Postcolumnderivatization was accomplished with o-phthaldialdehyde-mercaptoeth-anol to yield a fluorecent derivative which was detected with an Amincospectrophotometer.
Polyamines. The method used to determine putrescine, spermidine,spermine, AVmonoacetylspermidine, JV-g-monoacetylspermidine, andcadaverine, a polyamine arising via decarboxylation of lysine, was basedon gradient elution high-performance liquid chromatography andpostcolumn derivatization with o-phthaldialdehyde-mercaptoethanol(20, 35).
dc-SAM. Adenine and its derivatives are known to react with 2-chloroacetaldehyde to form highly fluorescent tricyclic derivatives (36).This reaction was utilized to give a sensitive and specific method formeasuring dc-SAM in urine (23) and plasma samples. As internalstandard, dc-SAE was used throughout the work-up. This work-upconsisted of absorption of dc-SAM and dc-SAE on a Dowex cationexchange column, elution with l N and 3 N HC1 to elute undesiredproducts, and finally 6 N HC1 to elute dc-SAM and dc-SAE. To this
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solution was then added an aqueous solution of 2-chloroacetaldehyde,after adjustment of the pH to 3-4 with sodium acetate. The reactionmixture was incubated at 40°Covernight. An aliquot of this mixture
was then injected onto an Altex Ultrosphere column and Chromatographie separation achieved as described (20,23). Detection was accomplished using a Perkin-Elmer LS-4 spectrofluorometer.
Tolerance
Blood pressure, heart rate, and body temperature were monitoreddaily in each patient during the study. In addition, prior to treatment,daily during treatment and weekly for 4 weeks after treatment, bloodwas analyzed for hemoglobin concentration, leucocyte and plateletcounts, and hematocrit. Serum analysis included glucose, uric acid,urea nitrogen, creatinine, electrolytes, calcium, alkaline phophatase,albumin, bilirubin, inorganic phosphates, and total protein content.Twenty lour h urines were analyzed for sodium, potassium, magne
sium, phosphate, and creatinine excretion. Furthermore, patients werequestioned daily as to subjective complaints. For treatment-related sideeffects, the WHO grading system (37) was used.
RESULTS
Daily urinary excretion of dc-SAM, DFMO, and creatinineare tabulated in Table 2 for the dosage regimen of 10-50 g ofDFMO/day, in Table 3 for the dosage regimen of 50-90 g ofDFMO/day, and in Table 4 for the two patients receiving either50 g of DFMO/day for 10 days followed by 25 g for 10 days or10 g for 10 days followed by 25 g for 10 days. A graphicaldemonstration of the 24-h urinary excretion of dc-SAM is
Table 1 Patient characteristics
Patient1234First
courseSecondcourse56First
courseSecondcourse7First
courseSecondcourseWeight(kg)78.551.548.094.057.084.079.565.061.0SexMMFMMFMAge(yr)64597058595260Creatinineclearanceat
entry(ml/min)87567710079106107877936Creatinineclearanceatend(ml/min)nd6080919396951108819TypeofcancerColonColonGastricHyperne-phromaGastricOvarianProstatic
presented in Figs. 1-3. Pretreatment 24-h urinary excretion ofdc-SAM was 203 ±134 (SD) nmol/24 h (as measured for sixpatients and three pretreatment urine collections each). Thesevalues compare favorably with the 24-h urinary excretion of dc-SAM in healthy volunteers, where a range of 150-275 nmol/24 h had been found. An increase of the 24-h urinary excretionof dc-SAM was noticeable as early as the first day of treatmentwith DFMO in some patients, but usually occurred only 2-4days after DFMO infusions had commenced, regardless of theadministered dose. Compared to the respective pretreatmentvalues, dc-SAM excretion was increased up to 16-fold in patient1, 19-fold in patient 2, and 84-fold in patient 3, for the threepatients on the 10-50 g/day dosage regimen. For the twopatients (4 and 5) receiving from 50-90 g/day of eflornithinehydrochloride, the maximum increase seen in the 24-h urinaryexcretion of dc-SAM was 42-fold in patient 4 and 37-fold inpatient 5. Patient 6 showed a maximum rise of 17-fold followingthe constant 50 g/day infusion and a 12-fold increase followingthe second treatment course with 25 g/day. Patient 7 showedan 11-fold increase at both the 10 and 20 g/day dosage regimen.
The 24-h urinary excretion of dc-SAM continued to be elevated for at least 4-5 days after stopping DFMO treatment(the longest time posttreatment monitored) at which time littleor no DFMO could be found in urine. Except for a few 24-hurine collections, daily creatinine excretion remained relativelyconstant, suggesting complete 24-h urinary collections. In addition, relatively constant values of plasma creatinine concentrations (values not shown) indicate unchanged creatinine clearance values for each individual patient over the study period(Table 1), with one exception; Patient 7 had a drasticallyreduced creatinine clearance value of 36 ml/min at the start ofthe second treatment course as compared to a mean value of82 ml/min during the first treatment course 2 months earlier.This value further decreased during the second study periodand reached 19 ml/min during the last treatment day (day 66)(Table 1). The reduced kidney function is attributed to anoverall deterioration in the disease state of this patient duringthe study and, perhaps, to treatment with cisplatin on day 63of study.
Total urinary recovery of unchanged drug over the entirestudy period was 73.7 ±9.3% for all dosage regimens, a valuecomparable to the urinary recovery obtained when much smallerdoses of DFMO were given as single i.v. injections to normal
Table 2 Urinary excretion of dc-SAM, DFMO, and creatinine in patients 1-3
Day-3-2-112345678910111213141516DFMO(8)000101020203030404050SO000000dc-SAM(nmol/24h)13320015530846964710201866218124061360258015172648Patient
1DFMO(%
administered)0.00.00.094.987.075.171.289.492.686.538.970.851.63.3Creatinine
dc-SAM(mmol/24 h) (nmol/24h)10.22
1252181472363014095739171584229288231365012188Patient
2DFMO(%
administered)0.00.00.042.843.234.642.060.156.346.087.963.863.69.0Creatinine(mmol/24h)7.576.007.4110.359.7410.006.396.408.158.607.31dc-SAM(nmol/24h)1021031051731553051668299044443911699783517714867984953952978409Patient
3DFMO(%
administered)0.00.00.061.574.376.183.676.268.165.377.172.767.217.31.30.5
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URINARY dc-SAM EXCRETION AS BIOCHEMICAL MARKER
Table 3 Urinary excretion of dc-SAM, DFMO, and creatinine in patients 4 and 5
Patient4DFMODFMODay-3-2-1I234567891011\213143435363738(g)00050506060707080809090000009090900dc-SAM(
mimi Mh)1311751061352866431,0682,0592,6414,9564,8425,4275,7273,8375255679732,3842,082(%admin
istered)00081.668.682.383.880.171.071.074.171.875.733.9074.059.052.122.7Creatinine(mraol/24h)14.8814.0013.2315.7214.8513.6813.2014.0013.7212.4612.0813.5012.9013.0011.559.597.9617.6413.27dc-SAM((nmol/24
h)2385579751,3343,5384,3124,7805,8365,6444,436i¡12,966°4,2792,896Patient
5DFMO;%
administered)00065.976.768.173.5110.289.6121.390.784.763.1i¡18.0°0.40.2Creatinine(mmol/24h)10.2314.466.9010.436.939.6910.2610.449.989.5011.807.591¡18.80°9.327.80*
Forty-eight h urine collection.Table
4Urinary excretion of dc-SAM, DFMO, and creatinine in patients 6 and7Patient
6DFMODay-3-2-11234567891011121314155657585960616263646566(g)0005050505050505050505000000025252525252525252525dc-SAM(nmol/24
h)336249670410515102924722330391928285053504483984204907967044778887512021727230331993450329729394805DFMO(%admin
istered)0.00.00.077.251.670.0118.080.499.455.671.464.479.614.80.00.00.066.885.272.374.877.475.469.083.077.895.0Creatinine(mmol/24h)14.258.7523.8714.4312.2911.2510.0113.5212.5017.4210.388.529.0013.8512.0011.2110.1911.5011.6511.2511.769.4018.62DFMO(g)0001010101010101010101000000025252525252525252525Patient
7dc-SAM(nmol/24
h)201226274451320628621117312667011911247521861976117567556639120418725042085811601206144611023501642DFMO(%admin
istered)00082.665.667.845.077.067.833.2104.199.577.822.93.21.90.60.40.030.060.180.770.857.657.277.649.216.288.0Creatinine(mmol/24h)10.2310.7310.0015.0810.129.078.259.0810.506.6510.4012.889.3010.3711.1111.7915.0411.7614.5012.408.408.267.387.745.405.004.261.107.13
volunteers (28). Renal clearance values of DFMO, calculatedfrom steady-state plasma concentrations and urinary excretion,were also in the same range (46-116 ml/min) as reportedpreviously for normal volunteers (50-97 ml/min) with theexception of patient 7 on the second treatment course with avalue of 21 ml/min at the beginning of treatment and 13 ml/min at the end of this treatment period, mirroring similarlydeclining values of creatinine clearance.
Tables 5 and 6 list the 24-h urinary excretion in patients 1-5 of the polyamines putrescine, cadaverine, spermidine, andspermine and of the two monoacetylated metabolites of spermidine, N,- and yVg-acetylspermidine.
As can be seen from the tables, 24-h urinary excretion of allmeasured polyamines prior to DFMO treatment varied considerably in the same individual and among the individuals. Duringtreatment, no consistent changes were observed in any polya-mine in any patient, with the exception of patient 3 (Table 5)where increased cadaverine, and perhaps putrescine, excretionwere seen during DFMO administration, i.e., days 1-10. Analysis of urinary polyamine concentrations in selected samples,before and during treatment, from patients 6 and 7 also did notshow consistent changes in any polyamine.
Loss of appetite, slight nausea (grade 1), and tiredness werereported in a majority of the subjects during treatment. One
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URINARY dc-SAM EXCRETION AS BIOCHEMICAL MARKER
£
S
<Wiu•o
" p•oE
2 *S i
« l
PATIENT No. I
5O
10
-2 O 2 4 6 8 IO 12 14 38 40 42
DAYS
-I—I Fif¡.i- Twenty-four h urinary excretion of dc-SAM as a function of treatmentwith DFMO. Patients 4 and 5.
PATIENT No 7
-2 4 6DAYS
10 12 14
Fig. I. Twenty-four h urinary excretion of dc-SAM as a function of treatmentwith DFMO. Patients 1-3.
subject (patient 6) developed a urinary tract infection with fever1 day following the first DFMO treatment course. In twopatients (patients 3 and 6), both heavily pretreated, leucopenia(grade 1 in patient 3 and grades 3 and 1 for the two dosageschedules in patient 6) and thrombocytopenia (grade 3 andgrades 4 and 3, respectively) developed towards the end oftreatment. These hematological abnormalities recovered spontaneously and completely by 14 days posttreatment. Thrombophlebitis developed in patient 2 at the site of infusion. Severalseizure episodes occurred in patient 7 on the last day of thesecond treatment course. A computed tomography scan of thebrain was found to be normal.
DISCUSSION
The administration of the ODC inhibitor, DFMO, is associated with a multifold increase in the urinary excretion of dc-SAM in humans. This increase appears to be time dependent,with several days required for maximum response. The likelybiochemical mechanism of this increase centers around theability of DFMO to decrease intracellular putrescine and sper-midine concentrations. The enzyme catalyzing the formationof dc-SAM, 5-adenosylmethionine decarboxylase, has beenshown to be under strict negative control by spermidine concentrations in cultured cells (12). Under normal circumstancesdc-SAM does not accumulate because of its rapid utilization inthe aminopropylation reaction to form spermidine and sperm-ine. Decreases in polyamine concentrations increase markedly
3 . PATIENT No 6
mi-r-r-n-TTI-2
O 2 « 69-,ri
r- |_nn
/. , Õ1 1 1 i 1 I H 11IO12 14 56 58 60 62 64 66
DAYS
i 25« IO
Fig. 3. Twenty-four h urinary excretion of dc-SAM as a function of treatmentwith DFMO. Patients 6 and 7.
the activity of the enzyme catalyzing the formation of dc-SAMat the same time decreasing the utilization of dc-SAM as anaminopropyl group donor. These combined effects enormouslyincrease the concentrations of dc-SAM, as demonstrated incultured mammalian cells (18) and in vivo in rat tissues (19).
In contrast to the increases observed in urinary excretion of
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Table 5 Twenty-four h urinary excretion of polyamines (¡anal)
Patient1Day
Put"-3
11.8-228.1-1
14.6118.2224.6318.1417.1521.267.5715.985.89
13.1109.81116.7121314ISCad4.58.65.310.110.26.77.08.54.08.73.37.47.110.3N,-AS3.17.63.83.53.92.32.33.51.22.61.01.71.64.0N.-AS1.63.61.93.22.71.91.92.60.91.80.81.51.14.1Spd0.40.50.40.80.5
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URINARY dc-SAM EXCRETION AS BIOCHEMICAL MARKER
relatively low but increase markedly during a treatment coursewith an inhibitor of ODC, such as DFMO. In contrast, urinarypolyamine excretion appears to be of no value in monitoringODC inhibition.
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1. .Ianin-. J., Pösö,H., and Raina, A. Polyamines in rapid growth and cancer.Biochem. Biophys. Acta, 473: 241-293, 1978.
2. Raina, A., and Jänne,J. Physiology of the natural polyamines putrescine,spermidine and spermine. Med. Biol., 53:121-147, 1975.
3. Snyder, S. H., and Russell, D. H. Polyamine synthesis in rapidly growingtissue. Fed. Proc., 29: 1575-1582, 1970.
4. Russell. D. H., and Snyder, S. H. Amine synthesis in rapidly growing tissues.Ornithine decarboxylase activity in regenerating rat liver, chick embryo andvarious tumours. Proc. Nati. Acad. Sci. USA, 60: 1420-1427,1968.
5. Williams-Ashman. H. G., and Canellakis, Z. N. Polyamines in mammalianbiology and médecine.Perspect. Biol. Med., 22: 421-453, 1979.
6. Heby, O. Role of polyamines in the control of cell proliferation and differentiation. Differentiation. 19: 1-20, 1981.
7. Pegg, A. E., and Williams-Ashman, H. G. Biosynthesis of putrescine in theprostate gland of the rat. Biochem. J., 108: 533-539. 1968.
8. Pegg. A. E.. and Williams-Ashman, H. G. Biosynthesis of putrescine. In: D.R. Morris and L. J. Marlon (eds.), Polyamines in Biology and Medicine, pp.3-42. New York: Marcel Dekker, 1981.
9. Pegg, A. E., and Williams-Ashman, H. G. Aminopropyl-group transfers inpolyamine biosynthesis, ¡n:D. R. Morris and L. J. Marlon (eds.), Polyaminesin Biology and Medicine, pp. 43-73. New York: Marcel Dekker. 1981.
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