BIOCHEMICAL MARKERS OR ENZYME CHANGES THAT MAY

PRESAGE THE PRESENCE OF CANCER

Final Report for Period

6/30/74 - 6/29/76

National Cancer Institute

Contract # NOl-CB-43902

Principal Investigator:    Joshua Lederberg,Ph.D.
Name/Address:         Stanford University
                    School of Medicine
Performance Site:       Department of Genetics
Organization:         Stanford University
                 Stanford, Ca. 94305


   Goal:  Our goal was to develop further the uses of gas-
liquidromatography and mass spectrometry instrumentation,
under computer management, for the study of derangements of human
biochemical metabolism due to neoplastic change; this was done for
the purpose of identifying new metabolites, or altered levels of
known metabolites, which might be diagnostic of cancer.

  Approach: (a) To screen urine by GC/MS from control subjects
and from individuals suffering from prostatic cancer, to determine
if polyamines are indicators of cancer;(b) the quantitation of uri-
nary protein amino acid and organic levels in cancer patients, and
quantitative intercomparison of these levels among patients and
controls; (c) the quantitation of urinary beta-aminoisobutyric acid
levels from cancer patients.

Progress:

A. Development of an Analytical Method for the Quantitation
of Urinary Polyamine Levels.

  Our intent in pursuing this study was to determine if the
polyamines can be used as markers for the early detection of pro-
static cancer. Prostatic cancer was chosen because the highest
concentration of polyamines in the human body can be found in the
male prostate gland. After the method had been developed, it was
applied to clinical analysis. We summarize in Table 1 the samples
available for study.

  A sensitive and specific method using mass fragmentography
for the analysis of the polyamines putrescine, cadaverine, spermi-
dine and spermine was developed, along with a method of synthesis
for their deuterated analogs. The procedure involves addition of a
known amount of a standard solution of deuterated analogs to the
urine, followed by overnight acid hydrolysis, butanol extraction
and ion exchange chromatography on a strongly cationic ion exchange
resin. These procedures are described in detail in Appendix I. The
polyamine extract is trifluoracetylated, and polyamine quantitation
is achieved by measuring peak height ratios of specific ions charac-
teristic of the trifluoroacetyl derivatives of the indigenous and
deuterated polyamines, m/e 126 and m/e 154 being characteristic of
the derivative of the indigenous material, and 128 and 156 being
characteristic of the derivative of the deuterated analog.


TABLE I.   Summary of Data Available for Polyamine Analysis

Lab. No.

229
238
239
302
324
329

Disease     Age

.   *
  Urine Volume

Ca. Prostate
Normal
Normal
Ca. Prostate
Hodgkins
Ca. Prostate
Grade II
Ca. Prostate
Grade I
BPH
BPH
BPH
BPH
BPH
BPH
BPH
BPH
i3PH
BPH
Ca Prostate
Grade II
BPH
Ca. Prostate
Grade II
Ca. Prostate
Grade II
Ca. Prostate
Grade II
Ca. Prostate
Grade III
Ca. Prostate
Grade III
Ca. Prostate
Grade III
Ca. Prostate
Grade I
Ca. Prostate
Grade III
Ca. Prostate
Breast Ca.
Ca. Prostate
Ca. Prostate
Ca. Prostate
Ca. Prostate
Control
Control
Control
Control
Control
Control
Control
Control
Control
Control

51
70
58
29
69

1100 cc
1410 cc (12 hr)
1840 (12 hr)
1610 cc
632 cc (12 hr)
1135 cc

330

61     2370 cc

336
337
340
343
344
345
347
348
349
351
354

56    1300 cc (12 hr)
61     1790 cc
64    1430 cc (12 hr)

62    990 cc (8 hr)
62     745 cc
71     1250 cc (16 hr)
71     372 cc (8 hr)
51    900 cc (8 hr)
51     590 cc (8 hr)
51     3220 cc

356~
362

52     620 cc (8 hr)
64     1425 cc

363

74

1550 cc

l-74

63    970 cc (12 hr)

2-74

64    720 cc (12 hr)

3-74

65     560 cc (12 hr)

4-75

56    440 cc (12 hr)

5-75

65     525 cc (12 hr)

6-75

54    620 cc (16 hr)

7-75
8-75
9-75
10-75
11-75
12-75
1
2
3
4
5
6
7
8
9
10

66    407 cc (12 hr)
50     1240 cc
50     695 cc (12 hr)
51     2140 cc
61     475 cc (12 hr)
?     1050 cc (12 hr)
39     837 cc
33     1760 cc
39     1460 cc
35     1758 cc
27     930 cc
26     1020 cc
41     1248 cc
43     2030 cc
35     1430 cc
?      1330 cc


  After injection of the samples into the GC/MS system, appro-
ximately one-half hour is required for analysis, followed by five
minutes for processing the analytical data. The concentrations of
indigenous polyamines are expressed in mg/lOO ml at the end of the
time required for processing. During the actual GC/MS runs, the
computer monitors the specific ions (126, 128, 154 and 156) charac-
teristic of the materials being analyzed, and at termination of the
runs, the total ion current (Fig. 1) is printed out at a CalComp
plotter.

  During processing of the analytical data, the contributions
of the individual specific ions to the total ion current are sepa-
rated and displayed on a T.V. Monitor along with background substrac-
tion (Fig. II), and the finding of the peak's maximum (Fig. III).

  Before the actual urine analysis, several quantitative mixtures
of pure deuterated polyamines and pure non-deuterated polyamines are
made up and derivatized. These mixtures are analyzed by GC/MS and
processed to determine the specific ion ratios for each polyamine.
These ratios are then plotted versus relative concentrations of the
nondeuterated and deuterated polyamines to establish calibration curves
for the individual polyamines. During the actual urine analysis, the
calibration curves are used to determine the relative concentration
of the non-deuterated indigenous materials, from the specific ion ratios
determined. The concentrations of the polyamines in the urine can then
be determined.

  We have examined the samples summarized in Table I using the
above procedure. Further methodological details and a presentation of
results are to be found in the attached preprint (Appendix I). Briefly,
we found, contrary to prev;ous reports, no significant differences
between diseased and normal patients in any of the polyamines analyzed.
In our opinion, based on this limited sample size, any further attempt
to detect or stage prostatic cancer using urinary polyamine levels is
worthless.  (See Appendix I for a review of the literature on this topic.

B. Screening of Urine for Metabolites Which Might Presage the
  Presence of Cancer.

  Each urine is fractionated as described in our original proposal
into an acidic + neutral fraction, an amino acid fraction and a sugar
fraction. We decided to divide the acidic + neutral fraction into equal
portions,one-half being methylated with diazomethane (as described in


TYPE OF CANCER
Bladder
Non-Hodgkin lymphomas
       C

Prostati
Leukemia
Breast
Lung
Pancreat
Colon

ic

# OF PATIENTS

3

the original proposal) and the second half is silylated (BSTFA). The
latter derivative was included in our analysis for two reasons. First,
diazomethane will cause complications in the derivatization of alpha-
keto acids and some heterocyclic systems.  This does not occur with sily-
lation and a large number of reference mass spectra of TMS derivatives
are available for the identification of unknown compounds from library
search routines.

  The acidic and neutral fraction was divided into two equal portions, one
of which was methylated with diazomethane (D-OME) while the other was sily-
lated with BSTFA + 1% TMCS (D-TMS).  The sugar fraction was derivatized to
the TMS derivative (S-TMS) with TRI-SIL-Z.  The amino acid fraction was also
divided into two equal portions with one portion silylated with BSTFA + 1%
TMCS (E-TMS) and the other converted to N-TFA-0-E-butyl derivative (E-TAB).
Details of the procedure have been presented in previous reports.

  Each of the six fractions of each urine was then analyzed by the GC/MS/
Computer system.  Each fraction yields about 600 complete mass spectra. These
spectra are processed by a computer program, called "CLEANUP", which is designed
to detect components and remove from the spectrum of each component interference
from background, column bleed and overlapping components. This procedure yields
spectra which are much more characteristic of the spectra of pure compounds than
are the raw data.  Each fraction may yield from 30-60 spectra representing a
GC/MS "profile" for each fraction for each patient.
  We have assembled libraries of mass spectra of known compounds by dividing
an available collection of over 3000 spectra of compounds of biological interest
into subclasses corresponding to the chemical fractions isolated in the above
procedure.  The appropriate library is searched for the spectrum of each compo-
nent detected by CLEANUP.  Spectra of components which were not matched to the
library are examined further in collaboration with the NIH supported DENDRAL
project for computer-assisted structure elucidation.

  Quantitative intercomparison of profiles to detect components present in
abnormal amounts is carried out by automated computer analysis of the GC/MS
profile by the HISLIB program.  This program is described in detail in
Appendix II.

  We have examined in detail the urinary profiles from the samples sum-
marized in Table II.

   Table II.


4

  Obviously, with this limited a sample size we depend on
detection of either a unique biochemical marker for cancer, or a
normal metabolite present in grossly abnormal amounts. The analytical
situation is further complicated by the fact that the sample we analyzed
were collected from persons under no dietary control and in various stages
of illness. Thus, there is no such thing as a "normal" profile of metabo-
lites. In short, all of the problems of drawing definitive answers from
urine analysis are present here , and further complicated by artifacts from
the collection procedures such as phthalates. We did, however, consider
an "average" profile of controls and patients as a basis for detection of
novel or abnormal amounts of corn onents
of techniques for comparing GC/M P    (see Appendix II for a description
                  profiles).
  We detected no novel biochemical markers in the group of patients

(Table II) or any subgroup which could be used to detect cancer. However,
we did note a remarkably high incidence of excretion of B-aminoisobutyric
acid (1) throughout the set of samples

            0
H2N - CH2 - CH - : - OH
              I

CH3

examined. The excretion of 1 is a highly complex problem involving
various disease states and genetic factors. The situation is made more
complex because not all of the samples analyzed displayed abnormal amounts
of L Whether this is due to stage of cancer, other illnesses or other
factors is unknown. The correlation is present, however, and in the next
section we describe work already done and more which might be done in the
future to clarify this problem.

C Q
. , uantitation of B-Aminoisobutyric acid (1).

   @-Aminoisobutyric acid occurs in significantly elevated amounts in
about 70% of the samples studied. It is most prevalent in urines from pa-
tients with leukemia (all 7), bladder ( in 7 of 8 samples) and lymphomas
(5 of 6 samples).

  The excretion of 1 has a genetic relationship. D-Beta-aminoisobutyric
acid (1) (BAIB) was first identified in normal urine by Crumpler and Dent
(1L and shown to be a catabolite of thymine (2,3). BAIB has also been
reported to be excreted in a variety of other conditions (4) including
cancer (4,5).


  The genetic basis for the excretion of BAIB in human urine was
first proposed by Harris (6) as a result of a study on a series of
families in London. Other studies have shown that in Caucasoid popula-
tions, the frequency of high excretors of BAIB is lower than 10% (6,7),
whereas in Oriental populations the frequency of high excretors is about
40% (8,9). As a result of family studies, a general hypothesis was put
forth, that the homozygote for a recessive allele is a high excretor,
and both the homozygote and heterozygote for a dominant allele are low
excretors. Experimental support for this hypothesis has been presented by
Yanai et al (9) who have shown that the distribution of BAIB concentration
in the urine of a Japanese population was bimodal, thereby classifying the
population into high and low excretors, the low excretor group being corn osed
of heterozygous and homozygous low excretors. Kakimoto and co-workers (8 P
isolated and purified the enzyme BAIB:pyruvate aminotransferase, which was
found to be present in mammalian livers. This enzyme metabolizes D-BAIB but
not the L-isomer. A later publication (10) showed that genetically determined
high excretion of BAIB was associated with reduced activity of BAIB:pyruvate
aminotransferase. The enzyme from human liver obtained at autopsy was isolated
and partially purified. The general character of human liver enzyme was found
to be similar to that of hog liver enzyme. The activity of human liver BAIB:
pyruvate aminotransferase was low in the high excretors, though not absent.

  This study showed as before that the distribution of BAIB in the urine
of a Japanese population was bimodal , classifying the population into the
high and low excretors, the low excretor group being composed of the hetero-
zygous and homozygous low excretors. The BAIB concentrations were higher in the
heterozygous low excretors than the homozygous low excretors, since the hetero-
zygous low excretors are expected to have a lower enzyme activity than that of
homozygous low excretors. Tariguchi and co-workers (10) suggested that if the
enzyme activity was determined with a larger number of liver specimens, dis-
tribution of the activity may be trimodal. In order to demonstrate this pheno-
menon, they tried to find a more readily available tissue or cell other than
liver, but found that human blood cells lacked detectable enzyme activity,
even when leukocytes from 20 ml of blood were used.

  Current methods of quantitation of BAIB involve either paper chromato-
graphy (ll), thin layer chromatography (lo), electrophoresis (8,9) or ion
exchange chromatography (13). These methods are, however, nonspecific for
the absolute and unambiguous identification of BAIB. A precise method of
quantitation would be highly desirable, especially when one is dealing with
low nanogram quantities of BAIB.

  The enzyme BAIB:pyruvate aminotransferase catalyzes the reversible
reaction

FH3

Y3

Y3

Y3

  &I-CH2FfH2 c=O        CiMH2     tn-clio
  1       I          I        I
  COOK      Ccm!          coot1        COOH

D-EAIR + Pyruvate <-->  L-al.ani.ne + methylmaloayl. senialdehyde


                          6

  Two methods have been used to measure enzyme activity. The first
involves determination of L-alanine formed in the reaction, using an
amino acid analyzer (8).  The second method involves the use of D-beta-
action ( -Me-14C) isobutyrate as substrate and measurement of radioactivity
in the deaminated product (10).  Both methods are non-specific, and the
products of the reaction have not been properly characterized.

                    REFERENCES

1.  H. R. Crumpler, C. E. Dent, H. Harris and R. G. Westall. Nature,
  167, 307 (1951).

2.  R. M. Fink, C. McGauchy, R. E. Cline and K. Fink. J. Biol. Chem. 218,
  1 (1956).
4. H. E. Sutton, in J. B. Stanbury and J. B. Wyngaarden (Eds.), Metabolic
   Basis of Inherited Disease, 1st Ed., McGraw Hill, New York, 1960, p. 792.

5.  H. R. Nielsen, K. Nyholm and K. E. Sjolin. Rev. Europ. Etud. Clin.
  Biol. 16, 444 (1971).

6. H. Harris.  Ann. Eugen. 18, 43 (1953).

7.  C. Calchi-Novati, R. Cepellini, I. Biancho, E. Silvestroni and H. Harris.
  Ann. Eugen. 18, 335 (1954).

8.  Y. Kakimoto, K. Taniguchi and I. Sano. 3. Biol. Chem. 244, 335 (1969).

9.  J. Yanai, Y. Kakimoto, T. Tsujio and I. Sano. Am. J. Human Genetics,
  21, 115 (1969).

10.  K. Taniguchi, T. Tsujio and Y. Kakimoto. Biochim. Biophys. Acta,
  279, 475 (1972).

11.  W. Sachietecatte, G. Maes, M. H. Faes and R. Bernaldelli. Clin. Chim.
  Acta, 11, 259 (1965).

12.  H. W. Goedde and Brunschede.  Clin. Chim. Acta, 11, 485 (1965).

13.  E. Solem, D. P. Agarwal and H. W. Goedde. Clin. Chim. Acta, 59, 203
  (1975).

  We have studied GC/MS methods for quantitation of BAIB (see Reference
1, below);an alternative method utilizing dentenium labelled BAIB would be
more precise.  Obviously considerable additional work remains to be done in
order to establish the significance of high incidence of excretion of BAIB
in cancer patients.  The question of genetic phenotype needs to be settled
for each patient before one can conclude whether or not high excretion is
normal.


7

Publications Relating to the Work

  1).  W. E. Pereira, R. E. Summons, W. E. Reynolds, T. C. Rindfleisch and
A. M. Duffield, "The Quantitation of B-Aminoisobutyric Acid in Urine by Mass
Chromatography," Clinica Chimica Acta, 9, 401 (1973).

  2). R.G. Dromey, B.G. Buchanan, D.H. Smith, J. Lederberg and
C. Djerassi, "Applications of Artificial Intelligence for Chemical
Inference, XIV. A General Method for Prediction of Molecular Ions in
Mass Spectra." J. Org. Chem. 9, 770 (1975).

  3). R.E. Carhart, S.M. Johnson, D.H. Smith, B.G. Buchanan, R.G.
Dromey, and J. Lederberg. "Networking and a Collaborative Research
Community: A Case Study Using the DENDRAL Programs," in Computer Net-
working and Chemistry, P. Lykos, Ed., American Chemical Society,
Washington, D.C. 1975.

  4). D.H. Smith, M. Achenbach, W.J. Yeager, P.J. Andersen, W.L.
Fitch, and T.C. Rindfleisch,"Quantitative Comparison of Combined Gas
Chromatographic /Mass Spectrometric Profiles of Complex Mixtures,"
Anal. Chem., 49-, 1623 (1977) (attached.)
  5). E.T. Everhart, T.C. Rindfleisch, W.L. Fitch, W.E. Pereira,
and A.M. Duffield, "Application of Computerized Stable Isotope Mass
Fragmentography to Analysis of Polyamines in Urines of Cancer Patients,"
submitted for publication (preprint attached).