-9-

observations suggest that at 12 weeks of gestation, wethylmalonic
acid is undetectable in the aaniotic fluid surrounding a normal
fetus and, probably, a fetus who is heterozygous for the
(recessive) gene (ref 24). Gooinan, et al. (ref 25) have
successfully diagnosed argininosuccinic aciduria in a 16 week
fetus. This involved detection of aryininosuccinic acid in the
amniotic fluid plus enzymatic studies on cultured amniotic fluid
cells. Norreally this compound is not detected in amniotic fluid
at 16 weeks of gestation. There are reports of other conditions
being diagnosed in the fetus by direct assay of amniotic fluid
(ref 26) as well.

The accuracy of diaynostis procedures probing amniotic fluid
for soluble constituents has been questioned because of the
possibility of contamination with maternal blood and because of
lack of information on the normal state at various tiges of
gestation. If the fluid component could be used for pre-natal
diagnostic purposes, the phenotype of the fetus could be detected
relatively capidly as compared to the time required to culture
sufficient arnniotic cells. Hereditary diseases which are
potentially amenable to diagnosis by analysis of the soluble
constituents of amniotic fluid are those in which the
accumulating metabolite is not cleared by the placenta but is
expected to appear in fetal urine. Defects in epithelial
transport, e.g, cystinuria and Hartnup disease, are exaaples of
such conditions. However, it is clear that this class of
metabolic errors is not the only one which might be detectable by
direct assay of amniotic fluid. The examples provided above
suggest that fetuses with overflow type metabolic errors say also
be detected.

The development of diagnostic and screening techniques
suitable for various inborn errors of setabolisa will require a
suitable coaputer based sethodoloyy for screening a large
selected sasple of subjects with the subsequent resolution of
data into classifications describing norgsal states and ranges as
well as specific correlations of GC/MS analysis abnoragalities
vith disease states. With a modest augmentation of existing
instrumentation facilities, ve can accomplish these analytical
tasks on increasing numbers of patients.

Be. SPECIFIC AIMS

a) We plan to use GC/SS to screen urine and plasma from
Norgal individuals of various ages, including premature and
newborn infants, in order to establish adequate control data
and to anderstand variations encountered.

b) We plan to use GC/MS in the diagnosis of inherited
metabolic abnormalities ani in the detection and study of
previously unrecognized metabolic disorders.
~10-

c) We plan to use GC/MS to study normal variation of
Clinically significant metabolites in amniotic fluid and then
to apply these techniques to pre-natal diaynosis of
hereditacy inborn errors of metabolisa.

d) In support of the above goals, we plan to augment our
existing GC/MS system and to more fully automate it for
screening larger numbers of patients throuyh improved
computer management. This would fit with current concepts of
regionalization of diagnostic facilities for genetic
disorders and would be appropriate for a Genetics Research
Center.
-11-

C. METHODS OF PROCEDURE

We use the following procedures to fractionate urine, blood,
and amniotic fluid in preparation for their analysis by GC/MS. in
the case of plasma, the protein is first precipitated (by the
addition of ethanol) and the supernatant liquid dried IN VACUO
and processed as for urine. Amniotic fluid is treated in the sane
way as urine.

URINE (pH = 1, internal standards added)

|

| *

{ ethyl acetate extraction
|

organic phase aqueous phase
\
(free acids) 9 wer mrt mtr tt rrr rrr
A 1 i |
(carbohydrates) (amino acids) } HCl hydrolysis
Cc B } and ethyl acetate
| extraction
|
| |
organic phase agueous phase
|
(hydrolyzed acids) (amino acids)
D E

The experimental procedure used for working with a fluid
sample is as follows. To an aliquot (2.5 ml.) of fluid is added
6N hydrochloric acid until the pH is 1. Two internal standards,
n-tetracosane and 2-amino octanoic acid are then added. Ethyl
acetate extraction isolates the free acids {fraction A) which are
then methylated and analyzed by GC/MS. An aliquot of the aqueous
phase (6.5 ml.) is concentrated to dryness, reacted with
n-butanol/hydrochloric acid followed by methylene chloride
containing trifluoroacetic anhydride. This procedure derivatizes
any amino acids (or water soluble amines) which are then
subjected to GC/MS analysis (fraction 6). Another aliquot (0.5
ml) of the aqueous phase can be derivatized (T&S) for the
detection of carbohydrates (Fraction C).

Concentrated hydrochloric acid (0.15 ml) is added to the
urine (1.5 al) after ethyl acetate extraction and the mixture
hydrolyzed for 4 hours under raflux. Ethyl acetate extraction
then separates the hydrolyzed acid fraction (D) which is
methylated and analyzed by GC/MS. A portion of the aqueous phase
(0.5 ml), From hydrolysis of the urine, is concentrated to
-~1l2-

dryness and derivatized and analyzed for amino acids (Fraction
BE).

The above scheme represents a general method for preparing
body fluids for analysis. The conditions required for gas
chromatographic separation vary with different classes of
compounds. For instance, uSing our method for chromatographing
derivatized free acids, amino acids, and carbohydrates, steroids
will not be detected. A different gas chromatography column and
derivatization procedure would be required. Large molecular
weight compounds (e.g., tripeptides and mucopolysaccharide
fragments) cannot be analyzed by this system without their
degradation to smaller unit molecules.

A suitable computer data system can Significantly ease the
tedious burden of analyzing the large amounts of data emanating
from GC/MS instrumentation. The computer assists in instrument
set-up and calibration, data collection and filing, and data
reduction and analysis. We have developed considerable experience
with such data systems, both for low and high resolution
instruments, since our tirst design was described in 1966 (ref
27, copy attached). Our most recent efforts have focussed on
facilitating user interaction with the data system and on
automating various aspects of data analysis (ref 3c). The
research proposed in this application entails the analysis of
increased numbers of body fluid samples, beyond the capacity of
the present system. We therefore propose to augment the current
system to handle the larger sample volume and at the same time to
extend the capabilities of the system to reduce GC/MS data.

Our present system is built around the NiH-subsidized ACHE
time-shared computer facility (IBM 360/50) with the real time
data interface being through an IbM 1800 computer. These machines
will disappear at the termination of the ACME grant in July 1973
and will be replaced by a fee-for-service IBM 370/158 system with
more limited real time support capabilities. With the transition
implied by the machine changeover, we have reevaluated the
approach to be taken in implementing our GC/MS support neecs
relative to the new requirements presently proposed, the
projected costs of alternative approaches, and the evolving
computer technology. Mini-computer capabilities have advanced
Significantly since the previous decision was made in 1965 to
implement the existing system on the ACME facility.

After examining the options of implementing a data system on
the new tima-shared facility or on a stand-alone mini syste, we
feel that a mini-system is the more advantayeous for our needs.
Such a system is the cheaper of the two based on projected costs
and is more responsive to real time needs in view of a smaller
set of demands on the mini-machine. There are limitations
inherent in mini-machines, however, in terms of memory size and
software/language support. The large central system (370/153 or
SUMEX iff approved) will act as a backup for the mini-system in
the relatively small numcer of instances where these
considerations are important in low resolution GC/M5s

gy

fr not
-13-

applications.

In preparation for the transition this July, we are
iaplementing a minimal data system on a PDP-11/20 computer. This
machine is well suited for "front end" data collection and filing
activities as well as simple analysis tasks. [t cannot
simultaneously support data acquisition and the more
sophisticated analysis activities contemplated in this projraa,
however, because of capacity limitations. [n addition, it lacks
the arithmetic speed desirable for these analysis procedures.
Since the proposed research program will entail a heavy duty
cycle of data collection throughout the day to analyze the
increased number of body fluid samples, data analysis and prograrm
development would have to take place during off hours. We propose
to solve this problem by acquiring a second machine to sapport
the PDP-11/20. This machine, tentatively selected as a PDP-11/45,
would provide for data analysis and program development support
and would be well suited to anticipated data analysis functions
because of its speed and extended arithmetic capabilities. We
have also considered a PDP-11/480 machine but feel the PDP-11/45
to be a better choice because of the relatively small cost
differential (approximately $10,000) in return for a factor of
2-3 in performance. The proposed system configuration is shown in
Figure 1. Oux choice of bigital Equipment Corporation (DEC)
machines is based on our existing hardware and software expertise
with this equipment. Our existing high resolution MS system
utilizes a similar PDP-11/20 machine as the data acquisition
computer as well.

The PDP-11/45 would have 24K words of memory, floating point
hardware and a programmable clock. The memory size is based on
projected needs for FORTRAN-based analysis programs. The two disk
drives (an existing fixed head drive, 262K words and a proposed
moving head drive, 1.24 words) provide needed space for system
software, programs (source and object files), and for spectral
data. It should be noted that a single GC/MS run contains some
700 spectra, each containing 500 12-bit spectral amplitude
measurements. Each such file therefore requires 350,000 words
(uncompressed) so that our proposed disk space would be quickly
consumed by several such files in residence. We can increase the
effective space available by compressing the data files to
eliminate insignificant measurements. Note that this requires at
least one data analysis pass over the full file, however. he
propose to augment this disk capacity in the second year by
adding an additional drive.

In general, data tiles will be stored on magnetic tape. Both
DEC tape and industry compatible tape drives are provided. The
former is required for system maintenance and is ideal for
relatively small private program files, data files, etc. The
industry compatible tape provides for large volume storage of raw
data during data acquisition and for archival storage.

This augmented data system will allow increased
sophistication in the analysis of GC/MS runs and in prescreenin,
-14-

GC profiles. In processing body fluid samples to establish norms
and to investigate specitic clinical abnormalities, full MS
analyses of the GC effluent will be required. This involves
extracting from the approxisately 700 spectra collected during
each run, the 100 or so representing the components of the body
fluid sample for identification. The raw spectra are contasinated
with background “column bleed" and some are composited with
adjacent constituent spectra unresolved by the GC.

We have begun to develop a solution to the problem of
effectively increasing the resolution of the GC by computer
analysis of the data. These programs will allow us to
automaticaly locate the body fluid constituent spectra and remove
the distortions caused by background and poor resolution. These
"“cleaned-up" spectra can then be analyzed by library search
techniques or first principles as necessary. By using a
disk-oriented matrix transposition algorithm developed for image
processing applications, we can rotate the entire array of 700
spectra by 500 mass sasples for each run. In this way we can gain
convenient access to the “mass chromatogranm® form of the data.
This forw of the data, displayed at a few selected masses, is
used in masS fragmentography described elsewhere in this
proposal. Sass chromatograms have the important property of
displaying auch higher resolution in localizing GC effluent
constituents. The automatic analysis techniques currently being
developed foc mass fragmentograms can be extended to this rore
general case. Thus by transposing the raw data to the sass
chromatogram domain, we can systematically analyze these data for
baselines, peak positions, and correct amplitudes thereby
deriving idealized mass spectra for the constituent materials.
These spectca are free from background contamination and
influences of adjacent unresolved GC peaks.

The results of this work can also lead to reliable
prescreening analyses of GC traces alone by having available a
detailed list of expected GC effluent positions and amplitudes
for the particular body fluid fraction under consideration. By
dynamically determining peak shape parameters for detected GC
Singlet peaks, interpretation of more complex peaks can be made
to determine if unexpected constituents or abnormal amounts of
expected constituents are present. If so, a more thorough GC/MS
analysis can be made. This typ2 of prescreening is valuable in
processing body fluid samples which may or may not be abnormal
and saves by committing the expensive mass spectrometer
instrumentation to analyzing only suspect samples. This, of
course, assanes that norms have been previously established by
processing body fluid samples in detail from a large population
of normal subjects.

The problem of spectrum iientification is addressed by using
rapid library search techniques for the identification of
previously encountered compounis. Those not in the library will
be identified by the manual interpretation of the spectrum using
other inforaation as available. These results will be
incorporated into the library to extend its domain of usefulness.

P-3/
~15-

This progressive compilation of a library of biomedically
relevant compounds will speed the throughput of the system. This
library will be freely shared with other investigators.
Eventually the extension of the library domain will be assisted
by adapting the computer programs under development in the
DENDRAL project (NIH RR-00612). These seek to emulate the
reasoning processes organic chemists employ in interpreting
spectra using fragmentation rules and other knowledge to infer
the correct molecular structure from among those possible.

The most appropriate target material for this developmental
effort is surely the metabolic output of NORMAL subjects under
controlled conditions of diet and other intakes. The eventual
application of this kind of analytical methodology to the
diagnosis of disease obviously depends on the establishment of
normal baselines, and much experience already tells us how
important the influence of nutrient and medication intake can be
in influencing the composition of urine, body fluids, and breath
(ref 18). Among the most attractive subjects for such a baseline
investigation are newborn infants already under close scrutiny in
the Premature Research Center and the newborn nurseries of the
Department of Pediatrics at this institution. Such patients are,
for valid medical reasons, under a degree of dietary control
difficult to match under any other circumstance. Many other
features of their physiological condition are being carefully
monitored for other purposes as well. The examination of their
urine and other effluents is therefore accompanied by the sost
economical context of other information and reguires the least
disturbance of these subjects.

Two obvious factors which could profoundly influence the
excretion of metabolites detected by GC/MS are maturity and diet.
We have already initiated a program for serial screening ot
urinary metabolite excretion in premature infants of various
gestational ages and a determination of changes in the pattern of
excretion of various vwetabolites as a function of age following
birth. A synopsis of this research is presented later in this
section. These studies are being performed on infants admitted to
the Center for Premature Infants and the Intensive Care Nursery
at Stanford, a source of some 500 premature infants per year. In
addition, in conjunction with an independent study on the effects
of both guality and quantity of oral protein intake on the
incidence and pathogenesis of late metabolic acidosis of
prematurity, we plan to measure the urinary excretion patterns of
various metabolites and thereby partially assess the effect of
diet on this screening method.

We shall use the analyses on blood and urine specimens fror
normal individuals in the final development of rapid, automated
identification of compounds described by mass spectrometry. By
compiling records of the gas chromatographic profiles and
associated computer-identified constituents of the body fluids of
normal individuals, we will establish the statistical norms and
expected variations for the component and levels present.
Quantitation is achieved by introduciny internal standards into

Sy oss
fors
we.
-16-

the body fluid fractions prior to analysis. We will seek to
minimize or at least stabilize the variations in measurement
results by assessing the effects of various aspects of diet and
medication. Normal blood and urine specimers will also allow us
to test the system's operational capacity for vapid and accurate
metabolite identification.

Given our ability to identify various constituents of urine
and plasma and to understand normal variation, we shall apply the
GC/MS system to pathology, making use of patients with already
identified metabolic defects for control purposes. The main
application will, of course, be diagnostic and patients with
suggestive clinical manifestations, such as psychomotor
retardation and progressive neurologic disease, as well as
suggestive pedigrees (e.y. affected offspring of consanguineous
parents or aultiplex sibships) will be investigated. Actually, we
have been studying limited numbers of such patients already.
These patients are seen relatively frequently at any university
hospital, and their presence in the various in-patient and
out-patient services of the Stanford Department of Pediatrics is
well documented.

The GC/MS system will be invaluable in diagnosing various
inborn errors of metabolism, especially those involving excretion
of various fatty and other organic acids. Some of these are
isovaleric acidemia (ref 15), methylmalonic acidemia (ref 28),
the recently reported inherited disorder of isoleucine retabolisa
causing accumulation of alpha-methylacetoacetic acid and
alpha-aethyl-beta-hydroxybutyric acid (ref 29), lactic acidemia
(ref 20), Refsum's disease (a iefect in the oxidation of phytanic
acid - ref 31), orotic aciduria (ref 32), and as illustrated
above, ornithine transcarbamylase deficiency (ref 19). We
recognize the potential of this methodology for defining
previously andescribed ("new") inborn errors of metabolism (e.g.,
ref 15).

In considering the strategy for applying GC/MS techniques to
problems of screening and characterizing inborn errors of
metabolisa, particular limitations in our present system in terms
of detection capabilities or throughput capacity must be
accounted for. For example, our present system is somewhat
limited for screening and diagnosing those conditions whose
phenotypes include the accumulation of amino acids in urine
and/or blood. We can detect all of the naturally occurring alpha
amino acids except hogocystine, cystine, and tryptophan. In
addition, the derivatization procedure being used, converts
aSparagine and glutamine respectively to aspartic acid and
glutamic acid. Thus the presence and quantity of asparagine and
glutamine cannot be separately measured from that of aspartic
acid and glutamic acid by current procedures. This situation
could be remedied by the use of a different GC column in order to
detect homocystine, cystine, and tryptophan. By using an
additional derivatization procedure, asparagine and glutamine
could be measured. This approach, while rigorous in its ability
to detect and quantitate amino acids, complicates the higher

P-33
-1)-

volume screening throughput. In effect, it would be necessary to
install and operate an additional gas chromatograph just for this
purpose because the temperature programming conditions for the
new column differ from those required for the column presently
used for acid and carbohydrate fractions. We are aware that an
amino acid analyzer, separate from the GC/MS system, offers a
practical solution to larger scale screening for metabolic errors
involving the accumulation of amino acids. This approach also has
the advantage of providing increased assurance that the clinical
analysis of amino acid accumulations can be performed at times
when the GC/AS system may be tied-up, under repair, or undergoing
engineering change. Such an analyzer, using ion exchange
chromatography, does not reguire derivatization of the specimen
and can detect all of the naturally occurring amino acids. We
therefore propose to acquire an amino acid analyzer to assist in
the screening of the increased number of sasples expected from
our work at Stanford and from collaborating researchers
elsewhere. In selected cases where the amino acid analyzer alone
cannot provide sufficient resolution and quantitative accuracy in
characterizing amino acid accusulations, the sore lengthy,
augmented GC/MS approach will be available.

Another area in which our existing equipment would limit
throughput is in the capacity of the single low resolution pass
spectometer. The present GC/MS system can process 1-Z specimens
per day (including all five derivatized fractions). This
throughput has sufficed for small sample research applications
but we anticipate that this load will at least be tripled when we
are actively studying normal variations and are screening
patients for metabolic errors. We could, ot course, eliminate the
bottleneck by buying more mass spectrometers. This would be quite
expensive. An alternative approach appears feasible.

By passing the samples submitted for screening through a
preliminary GC profile analysis (without MS), we feel it will be
possible to identify those deviating from established normal
limits and warranting a more rigorous GC/MS analysis. fhis will
allow more efficient use to be made of the MS instrument time.
For abnormal samples, the deviation, under MS analysis, may be
readily identified by library search techniques or ray require
more lengthy analysis procedures. These could require ditferent
chemical derivatization techniques or additional information such
aS high resolution mass spectrometry. This additional analysis
could consume days or weeks of effort. Thus it is important to
make efficient use of these analysis capabilities. The number of
samples eliminated in GC prescreening depends, of course, on the
nature of the samples submittei. Prescreening will be less useful
for the processing of samples which have been carefully selected
as Clinically suspect than on a general sampling of “normal"
individuals. It is in this latter case that we see a benefit to
developing the prescreening capability. For this purpose, we
propose to add to our laboratory a 4 column gas chromatograph.

The Department of Pediatrics has affiliations with the
Kaiser-Permanente Medical Center in Santa Clara, the Santa Clara

vy
-18-

County Hospital, and the Children's Hospital at Stanford. These
Clinical anits will funnel samples (urine, blood, amniotic fluid,
etc.) from suspect patients into the laboratory of this program.
We are already receiving samples from various physicians in
California (e.g., Fresno - see below) and from Dr. Jose #.
Garcia-Castro, Medical Geneticist of the University of Puerto
Rico Medical School (see below also). Arrangements are in process
to obtain samples from the newborn nurseries of the Los Angeles
County Hospital. The proposed augmentation of our instrumentation
with an amino acid analyzer and an additional gas chromatograph
will assist in being able to handle this anticipated specimen
load. With the experience afforded by our augmented screening and
diagnostic facilities, we will have the capabilities to develop
into a regional center for the screening and diagnosis of various
metabolic errors.

As an example of the application of these methods to
biomedical problems, we can use some recent studies we have
undertaken on the urine of a patient suffering from acute
lymphoblastic leukemia. The gas chromatographic profile (figure
2) of the amino acid fraction of his urine showed the presence of
an abnormal peak (A). The sass spectral analysis of this
chromatographic peak (Figure 3) identified this component as
beta-amino [SObutyric acid (BAIB) from a coaparison with a
literature spectrum of authentic material (ref 32a). The
literature on human urinary BAIB excretion is extensive and it
has been reviewed in reference 33 . Awapara (ref 34) noted
increased urinary BAIB excretion from patients receiving nitrogen
mustard therapy while gubini (ref 35) reported high levels of
BAIB excretion in people exposed to excessive radiation levels.
Wright and Fink (ref 36) found elevated urinary BAIB levels frorm
mongoloid children and non-agjongoloid mental defectives but these
conclusions were subsequently jJisputed (ref 37). Harris in
England (ref 38) and Calchi-Novata in Italy (ref 39) found that
6-10% of a sample population had elevated urine concentrations of
BAIB while DeGrouchy and Sutton (ref 40) in a study of families
of oriental extraction, and Yani, et al. (ref 41) with Japanese
families, found about 40% of their subjects excreted elevated
levels of BAIB. Gartler concluded that the variation underlying
BAIB excretion by New York fanilies (ref 42), Apache Indians and
Black Caribs of British Honduras (ref 35) was due to genetic
difference at one locus. Later studies showed that thymine was
probably a precursor of BAIB in man (ref 44).

At least two authors (refs 33 and 41) have questioned the
accuracy of the methods used for the detection and quantitation
of BAIB in urine. The early work (refs 32 and 51), including the
first isolation of BAIB trom urine (ref 38), was completed using
paper chromatography and reaction with ninhydrin to develop the
appropriate spot for detection and quantitation. As suggested
(refs 25 and 34) other compounds in urine migrate with
characteristics similar to BAIB thereby introducing an element of
imprecision into the published quantitation results. In those
instances where BAIB is present in only a small amount, or for
that matter in large amounts, it is always possible that another

s:
Prax
oY
-19-

metabolite could be present within the confines of the "BAIS
spot". Subsequent methods erployed for BAIB quantitation include
thin layer chromatography (ref 47 ) and paper electrophoresis
(ref 48). These methods sust also be considered as non-specific
for the estimation of BAILB in urine. Our own procedure, sust be
considered far superior to these other techniques for both the
detection and guantitation of BAIB in urine because it combines
the quantitative aspects of gas chromatography with the positive
identification of a mass spectrum. We have observed several
instances in which the gas chromatographic peak associated with
BAIB was shown by mass spectrometry to contain other components.
Using the technique of mass fragmentography (refs 3a and 3b), we
can quantitate for BAIB under these conditions. In this
technique, a characteristic fragment ion from the mass spectrum
of BAIB (figure 3, a/e=153 or 182, for example) is summed over
the total ion chromatogram to give an area corresponding to the
amount of BALB present. Using an internal standard and a
characteristic ion from its mass spectrum and knowing the
ionization efficiency of both BAIB and the internal standard,
quantitation can be achieved. This work is almost finished and a
paper describing a convenient way to quantitate BAIB in urine
will be prepared in the near future.

We have examined urine saaples from 18 patients suffering
from leukeaia and have observed BAIB in a total of five
individuals. The patient whose urine was used to record the
chromatograag shown in figure 2 was excreting 1.2 grams of BAIB
per day and following drug therapy this compound could no longer
be detected nor was it detected in a urine sample collected 3
months later. Awapara (ref 34) claimed that all leukemic patients
excrete varying amounts of BAIB which was not detected in normal
urine. Killmann, et al. (ref 49) noted that two out of three
patients with chronic granulocytic leukemia produced high levels
of BAIB but following drug therapy, the level returned to normal.
Lee, et al. (ref 50) examined the urine of 33 patients with
leukemia receiving specific drug therapy and failed to observe
BAIB excretion in any of these subjects. Our own observations, in
agreement with the literature (refs 45, 49, and 50), suggest that
during the disease cycle, leukemic patients may excrete large
amounts of BAIB but this ceases following chemotherapy. Schreier
(ref 51) studied the urinary excretion of BAIB in leukemic
subjects and noted extremely variable results with "massive
amounts" of BAIB present in the terminal stages of the disease.

As mentioned earlier in this section, we started pilot GC/a#S
studies of biological fluids by investigating the urinary
metabolic output ot premature children. So far we have studied
over 86 urine specimens from a total of 11 premature or "small
for gestational age" infants. Of this population, six infants
were closely studied for periods of between six and eight weeks
beginning at day 3 of life. Five of the six babies showed
clinical syaptoms of late metabolic acidosis (ref 52) and all
five showed the same abnormal metabolic profile of their acidic
fractions. All five children excreted excessive amounts of
p-hydroxryphenyllactic acid (I), together with smaller quantities

P-3k
~20-

of p-hydroxyphenylpyravic acid (II), and p~hydroxyphenylacetic
acid (III) (see figure WA and the structures shown below). After
reaching a maximum, the daily excretion of these abnormal
metabolites diminished until they were almost completely absent
when the child’s pH and weight gain had returned to normal values
(see figure 4-B). The sixth infant showed no clinical symptoms of
acidosis and excreted only minute amounts of I, II, and III
during the observation period. None of the six subjects showed
any increased level of urinary tyrosine excretion. A blood
sample, from a patient who excreted large urinary amounts of I,
II, and IIIf, showed no detectable quantity of these acids.

CO3H CO, Hl
t
CHOb! c=
l

=0
" Hy "
OH OH
r m

Ir

The heavy urinary content of I and II by premature infants
ingesting a relatively high protein diet (cow's milk) was
described in 1941 (ref 53). Classical cheaical isolation
procedures were used for the identification of I and II and
quantitation was achieved by two colorimetric assays. The
quantity of I was determined from the difference between the two
colorimetric assays, assuming that no p-hydroxyphenylacetic acid
(III) was present. Our finding of appreciable amounts of IIY in
the urine of these premature infants suggests that the
quantitation values given previously (ref 53) for the excretion
of (I) are incorrect. Figure 4 illustrates the gas
chromatographic trace from the analysis of the acid fraction (as
their methyl esters) present in the urine of one premature infant
while acidotic (A) and after recovery (8). The identification of
the cospounds on Figure 4 follow from their mass spectral
Signatures while quantitation was achieved from measuring the
area under the gas chromatographic peaks using tetracosane as an
internal standard.

C0,H
I

—

During the past six months we have commenced a preliminary
investigation of the use of GC/MS for analyzing body fluids from
both normal patients and those whose clinical history suggested
they had a high risk of suffering from a metabolic disorder. The
first two cases reflect some of the results we have obtained
while the third describes the preliminary work undertaken with
amniotic fluid from normal pregnancies being terminated for
medico-social reasons.
-21-

The first case involves a deceased child, whose parents were
first cousins and who was suspected of having a branched chain
amino aciduria as indicated by thin layer chromatography at
another institution (ref. 54). Preliminary screening by GC/MS of
the urine which was sent to us showed no abnormal levels of
leucine, isoleucine or valine, nor of their respective alpha-keto
acids. In order to verify this point we analyzed the amino acid
fraction using our recently developed mass fragmentography
technique (refs. 2, 3). A copy of the computer output for this
analysis is reproduced as Figure 5. Clearly there are no elevated
quantities of any of the branched chain amino acids, which we
know we can detect, and the cause of this child's death remains
unknown at this time. [It is possible that this child showed
intermittent excretion of branched chain amino acids (54). This
urine was sent to us by Dr. Jose M. Garcia-Castro, Puerto Rico
(see note on Collaborative Arrangements).

Recently a child died 33 hours after birth in Fresno,
California, with the classical signs of hypophosphatasia (ref
55). This genetic defect is marked by high phosphoethanolamine
(PEA) concentrations in urine of affected homozygotes and
unaffected heterozygotes. After derivatization (in this instance
the TMS ethecs of the water soluble carbohydrate fraction were
prepared) we were able to detect by GC/MS large concentrations of
ethanolamine and phosphoric acid but not PEA itself. The
derivatization procedure we used aost likely hydrolyzed PEA (ref
56). We were able to quantitate for this compound in the infant's
urine using an amino acid analyzer, and PEA excretion was
extremely high (over 2CQ times normal values for infants)
confirmjing the diagnosis. Wext we examined urine samples from the
child's parents, presumed heterozygotes, by GC/MS and by the
amino acid analyzer. Again, no PEA was detected by the former
method although the presence of ethanolamine and phosphoric acid
was demonstrated. We determined the following excretion levels of
PEA by amino acid analyzer:

Newborn infant: 94% micromoles per 100 wl. (Normal 0.21-0.33)
Father: 269 micromoles per 24 hours (normal 17-99)
Mother: 32 microaoles per 24 hours (normal 17-99)

It is of interest that in this family the affected infant
and his unaffected father both show subnormal serum alkaline
phosphatase activity. The sother, who did not excrete increased
amounts of PEA, was found to have normal activity of this enzyme
in her serum. The following table summarizes the serua
phosphatase activity measuresents:

Newborn infant: 0.2 units* (normal 2.8-6.7)
Pather: 0.7 units (normal 0.8-2.3)
Mother: 3.4 units (normal 0.8-2.3)

(* - 1 unit is that phosphatase activity which will liberate
1 millimole of p-nitrophenol per hour per liter of serun)
-22~

These findings are quite consistent with hypophosphatasia.
The heterozygous unaffected parents of children with
hypophosphatasia may or may not show biochemical evidence of the
carrier state.

The analysis of the metabolic content of amniotic fluid has
been pursued by many investigators with the aim of identifying
metabolic defects IN UTEKO. Historically the first studies on
amniotic fluid utilized paper chromatography (refs 57, 58, 59).
Amino acid quantitation in amniotic fluid was attempted by a
visual corgparison of paper chromatographic spots with standard
spots (refs 60, 61) and by spectrophotometrically measuring the
intensity of spots eluted from paper chromatograms. (ref 62). Ion
exchange chromatography was applied (refs 20, 21) for the
analysis of the free amino acids in amniotic fluid while gas
chromatography (refs 63, 64) and GC/MS (cef 65) were used for the
determination of the free acid content of amniotic fluid. On the
basis of their results, Levy and Montay Suggested (ref 21) that
agniotic fluid arises from at least 3 sources: maternal blood,
fetal blood, and fetal urine.

Our own endeavors in the field of amniotic fluid analysis
has involved a study to determine those constituents found in
normal amniotic fluid. Ten specimens have been analyzed. All of
these samples were obtained at Stanford, most at 14-16 weeks of
gestation for chromosore studies of the fetus, usually because of
increased maternal age. Following separation by centrifugation
and removal of the cells for culture, the amniotic fluid was
again centrituged (2900 rpm, International Clinical Centrifuye,
for 10 minutes) and the supernatant was stored at -20 degrees C
until analyzed by GC/MS. To date we have identified the following
acids in amniotic fluid: citric, nyristic, palmitic, Stearic,
Oleic and esters of phthalic acid (probably leached from the
plastic containers used to store the specimens). The agniotic
fluid from one patient containad salicylic acid (aspirin
ingestion) and from another caffeine (tea or coffee ingestion).
The following amino acids have been identified in these "normal"
amniotic fluid samples: alanine, threonine, serine, glycine,
valine, i-leucine, leucine, proline, methionine, aspartic acid,
phenylalanine, tyrosine, glutamic acid and lysine. In view of the
current interest in the pre-natal diagnosis of
methylmalonicaciduria, we can state that methylmalonic acid was
not detected in the acidic fraction of any of the "normal”
amniotic fluid specimens. We are continuing these baseline
studies of noraal amniotic fluid in order to eventually identify
abnormal accurmulations ot wetabolites in the at-risk pregnancy.
Variables which we will consider include gestational age,
diagnosis and status of the fetus, maternal plasma concentration
of the relevant substances and maternal plasma concentrations of
hormones and medications and their metabolites. We are presently
planning to investigate variation in amniotic fluid
pregnanetriol, 17-ketosteroids and some of the Specific androgens
which are elevated in the adrenogenital syndrome (ref 66).
-2 3-

At this tine we are not prepared to predict that GC/MS
analysis of amniotic fluid alone will provide the accurate
diagnostic information about the fetus required for a decision
for selective abortion. Heterozygous fetuses and contamination of
the amniotic fluid with maternal blood (the result of the
amniocentesis procedure) raise possibilities for false positive
diagnoses which may lead to abartion of unaffected fetuses if
cultured aaniotic fluid cells are not also studied. However,
studies relating quantity of the pertinent metabolite in amniotic
fluid to enzyme activity in the cultured cells should be helpful
in providing information for discriminating between heterozyyous
and homozygous fetuses by GC/MS. In other words, we will monitor
at-risk pregnancies with GC/MS and with biochemical studies of
cultured cells until we are confident we can predict the
homozygous state from GC/MS analysis alone. Maternal blood
contamination will be monitored by gross and microscopic
examination of amniotic fluid samples, and the behavior of
contaminated fluid on GC/SS analysis will be anticipated by
studying a series of amniotic fluid samples to which various
amounts of blood from a heterozygous individual (The mother in
the high cisk pregnancy is usually a heterozygote) have been
added.

D. SIGNIFICANCE

An accurate diagnosis is especially important for genetic
counseling purposes. The diagnosis allows reference to published
data on the mode of inheritance and, thus, expresses the
recurrence risk. Furthermore, accurate diagnosis of the
accumulated metabolite provides insight into the biochemical
pathogenesis and into therapeutic approaches to the control of
hereditary inborn errors of metabolism. The GC/MS system, with
its potential for automated and rapid identification of many
metabolites, provides the diagnostic accuracy necessary for a
clinical program. This system also provides the methodology for
detecting previously unrecognized inherited metabolic errors.

The methodology developed by this project will decrease the
time required for antenatal diagnosis of certain metabolic
disorders. The elapsed time until diagnosis is important because
legal, psychological and, perhaps, obstetrical considerations
have set a deadline of 20 weeks of gestation for selective
abortion. This deadline is sometimes not amet when amniotic cells
obtained for diagnostic purposes fail to divide sufficiently
rapidly in culture to provide adequate material for biochemical
testing.

The study of systen designs for automated GC/MS systems in
the clinical environment will pave the way for a prototype systen
which will sake more routinely available these powerful
analytical tools. Such tools will be important in the inevitable
regionalization of facilities for the screening, diagnosis and
study of hereditary inborn errors of metabolisa.

P-¥C
-24-
E. FACILITIES AVAILABLE

We will derive much of the clinically significant asaterial
for analysis from patients in the Premature Research Center and
the Clinical Research Center of the Department of Pediatrics.
Analyses will be performed in part on existing GC and MS
equipment in the Department of Genetics. We now have a two column
Varian Aerograph 2100 gas chromatograph used for specimen
prescreening and an older, less effective Varian Aeroygraph 1200
gas chromatograph connected to a Finnigan 1015 quadrupole mass
spectrometer. We have access to a Varian-MAT 711 high resolution
GC/MS system to assist in the identification of compounds not
readily identified by low resolution spectrometry alone. Also
available to the project are the electronics facilities and
software experience of the Instrumentation Research Laboratory of
the Deparctwent of Genetics. Assuming the approval of the SUMEX
resource proposal, we will have access to large scale computing
support for the later applications of artificial intelligence to
this research from the SUMEX PDP-10 machine.

F. COLLABORATIVE ARRANGEMENTS

This project involves an interdisciplinary collaboration
between Drs. J. Lederberg (Principal Investigator) and A.
Duffield (Associate Investigator) of the Department of Genetics,
Drs. N. Kretchmer and H. Cann (Associate Investigators) of the
Department of Pediatrics, and the Instrumentation Research
Laboratory (including Dr. &. C. Levinthal and Mr. T. c.
Rindfleisch). Dr. Jose S. Garcia-Castro, University of Puerto
Rico School.of Medicine has agreed to send us samples (urine and
blood) from selected patients. We will also receive samples from
collaborators at the Kaiser-Permanente Medical Center in Santa
Clara, the Santa Clara Valley Medical Center, the Children's
Hospital at Stanford, and the Los Angeles County Hospital.

This arrangement is a prototype of efforts to organize a
systematic network of support to physicians at outlying centers
for the mutual benefit of better care for their patients, and
providing pre-screened, high-yield material for scientific study.
Before this is formalized, we wish to build up practical
experience with collaborations where personal understanding
allows good communication about respective needs and flexibility
in meeting urgent requirements.
-25-

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-26-

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~27-

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26c) Nadlec, H. L., and MeSsina, A. M., In-Utero Detection of
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27) Reynolds, W.E., Bacon, V.A., Bridyes, J.C., Coburn, T.C.,
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28) Morrow, G., Schwartz, R.H., Hallock, J.A., and Barness,
L.A., “Prenatal Detection of Methylmalonic Acidemia," J.
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29) Daua, R. S., Seviver, C. R., Mamer, O. A., Delvin, E., Lamn,
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30) Haworth, J.C., Ford, J.D., and Younoszai, A.K., “Familial
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"9
-~2b-

32) Fallon, J.H., Smith, L.H., Graham, J.H., and Burnett, C.H.,
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-29-

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-30-

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