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Diagnostic Uses of Organic Acid Testing
by
William Shaw Ph.D.
Copyright 1996 by William Shaw Ph.D. No part of
this article may be reproduced in any form or by any means without permission
in writing by the author.
ORGANIC ACID TESTING AT
The Great Plains Laboratory, Inc.
We at The Great Plains Laboratory are proud of the service we provide
for organic acid testing. Our mission is to provide the best possible
service for the patients, physicians, and health providers who utilize
our services. The advantages you will obtain with testing from us are:
(1) Testing is supervised and results are reviewed by professionals who
are daily involved in the diagnosis and treatment of patients with metabolic
diseases, nutritional deficiencies, microbial dysbiosis due to intestinal
overgrowth of yeast or bacteria such as C. difficile, or metabolic sensitivity
to certain food additives such as adipic acid.
(2) Testing is continually updated to include screening for newly described
metabolic diseases that appear in the medical literature.
(3) Follow-up consultation and/or referrals to another appropriate local
medical facility are available if desired.
(4) Screening for over 60 different defined metabolic diseases is included.
All diseases tested are defined so that there is no doubt as to what diseases
are being tested. Some reference labs only test for 5 or 6 diseases or
don't even list what diseases are screened for. The reason that they don't
list the diseases they are testing is that often they don't know what
they're testing for.
(5) At The Great Plains Laboratory, gas chromatography/mass spectrometry(GC/MS),
an extremely powerful analytical tool, is used as the primary screen.
Some laboratories used gas chromatography or nonspecific color tests for
screening. GC/MS, if used at all, is only used for confirmatory tests
by many of these labs. Many genetic diseases might be missed by these
primitive technologies. The use of such methods could actually leave a
practitioner who utilizes them open to malpractice suits if a particular
genetic disease should have been detected but was missed.
(6) 48 hour turnaround time will be available for the vast majority of
samples. Some labs take a week, two weeks, or a month to get results back.
(7) All GC/MS results are archived on magnetic tape. Because of continuous
advances in this field, new diseases are being continuously discovered.
We will store all data for ten years on tape. At any time during the storage,
we can reanalyze the data for the existence of newly discovered genetic
diseases.
(8) Chart review and consultation are also available and may help to
establish a probable diagnosis in cases in which concentrations of abnormal
metabolites are not definitive.
(9) Quantitative results are available on many tests which help to distinguish
normal and abnormal results. Quantitative results are not generally available
from other reference labs or require an additional charge.
(10) A summary of all significant compounds and an evaluation of their
implications in over 60 different known diseases is provided. In addition,
new diseases may be diagnosed since our mass spectra library includes
over 2000 mass spectra and is continually updated.
(11) Sophisticated software on a state-of-the-art computer system allows
all organic acids in the urine to be rapidly identified and minimizes
the possibility of human error in analyzing extremely complex samples
such as urine which contains hundreds of different components.
OVERVIEW OF ORGANIC ACID TESTING
More than 50 phenotypically different organic acidemias are now known
since the oldest known disease, isovaleric acidemia, was described in
1966. An organic acid is any compound that generates protons at the prevailing
pH of human blood. Although some organic acidemias result in lowered blood
pH, other organic acidemias are associated with organic acids that are
relatively weak acids that do not typically cause acidosis. Organic acidemias
are disorders of intermediary metabolism that lead to the accumulation
of toxic compounds that derange multiple intracellular biochemical pathways
including glucose catabolism (glycolysis), glucose synthesis (gluconeogenesis),
amino acid and ammonia metabolism, purine and pyrimidine metabolism, and
fat metabolism. The accumulation of an organic acid in cells and fluids
(plasma, cerebrospinal fluid, or urine) leads to a disease called organic
acidemia or organic aciduria.
Organic acids are most commonly analyzed in urine because they are not
extensively reabsorbed in the kidney tubules after glomerular filtration.
Thus, organic acids in urine are often present at 100 times their concentration
in the blood serum and thus are more readily detected in urine. The number
of organic acids found in urine is enormous. Over 1000 different organic
acids have been detected in urine since this kind of testing started 25
years ago. The large number and complexity of dealing with such large
numbers of compounds led to the use of GC/MS to analyze these complex
body fluids. With GC/MS, organic acids are chromatographically separated
on the basis of their polarity and volatility and them bombarded by an
electron beam that fragments the eluting molecules in a pattern that is
characteristic of each organic acid. The patterns or spectra are stored
by a computer system and then are compared to known spectra that are compiled
in a spectral "library" that is stored on the hard disk of the
computer system. The computer system then compares an unknown spectrum
to all the spectra on the hard drive and prints out those with the best
fit. Since a single organic acid analysis generates over a thousand spectra
and each spectra may consist of 600 ions, the computer system must be
optimized to analyze the data in the most efficient and clinically relevant
manner.
Even before GC/MS is used to analyze a sample for organic acids, the
organic acids must be extracted from the urine sample and then be chemically
modified to allow the organic acids to be sufficiently volatile to be
analyzed by GC/MS. Bis-trimethysilyl-trifluoroacetamide (BSTFA) in combination
with 1% trimethylchlorosilane (TCMS) is reacted with alcohol, acid, and
sulfhydryl substituents on the organic acids to form trimethylsilyl ethers
and esters which are commonly termed TMS derivatives.
Clinical presentations of organic acidemias vary widely and may include
failure to thrive, mental and/or developmental retardation, hypo- or hyperglycemia,
encephalopathy, lethargy, hyperactivity, seizures, dermatitis, dysmorphic
facial features, microcephaly, macrocephaly, anemia and/or immune deficiency
with frequent infections, ketosis and/or lactic acidosis, hearing, speech,
or visual impairment, peripheral neuropathy, sudden cardiorespiratory
arrest, nausea and coma. Many organic acidemias are associated with slight
to marked increases in plasma ammonia. Some organic acidemias may be chronic
and present in the first few days of life. In others, such as medium chain
acyl dehydrogenase deficiency, a child might appear completely normal
until a potentially fatal episode of cardiorespiratory arrest.
The prognosis for patients with the organic acidemias varies widely but
drugs, dietary restrictions, vitamin and cofactor supplementation are
effective modes of long term therapy for most of these diseases. However,
delayed testing may lead to delayed or missed diagnoses or inadequate
treatment for these diseases. Some of these diseases may be lethal if
untreated. Symptoms may vary widely within an individual and different
individuals with the same organic acidemia may have markedly different
clinical symptoms. Some individuals with the organic acidemia, methylmalonic
acidemia, are symptom free while others may have mental and growth retardation,
psychosis and deafness.
As technology improves and our knowledge of intermediary metabolism improves,
new organic acidemias will be discovered and our skill in the diagnosis
and treatment of recognized diseases will improve.
WHEN TO COLLECT SAMPLES FOR ORGANIC ACIDS
The best time to collect samples for any genetic disease is prior to the
development of symptoms. However, newborn screening in many states is
very limited in scope. For example, screening in the state of Missouri
is limited to hypothyroidism, phenylketonuria, galactosemia, and sickle-cell
disease.
Phenylketonuria is a genetic disease involving altered metabolism of
the amino acid phenylalanine and can be detected by either organic acid
screening or by amino acid testing. The other three diseases would not
be detected by either organic acid screening or amino acid testing.
Metabolic diseases, missed by screening programs, are most readily detected
when the disease is most acute because lab results are most abnormal at
this time. In some organic acidemias, abnormal compounds cannot be detected
at all during well episodes.
Although there is a legitimate concern to reduce the cost of medical
expenses by reducing unneeded tests, organic acid testing is extremely
cost efficient since approximately
1000 different biologically important compounds may be tested on a single
sample. If a urine sample is not tested at the time of an acute episode,
invaluable information about the patient maybe lost, the diagnosis may
be missed, and the patient may suffer irreversible retardation and death
in the future at a much greater loss to the parents and society. My own
expectation for this service is that it should be somewhat "over-utilized"
and that there will be many more normal than abnormal results obtained.
COORDINATION OF ORGANIC ACID TESTING WITH OTHER
LAB TESTING
GLUCOSE: Many organic acidemias are associated with hypoglycemia
or hyperglycemia. Furthermore, blood sugar results may fluctuate rapidly
in the same individual because these compounds may inhibit both the enzymes
that synthesize glucose as well as those that break down glucose. Anomalous
lab results may occur including a decrease in glucose after glucose infusion.
Individuals with medium chain acyl dehydrogenase deficiency may present
in acute cardiorespiratory arrest with hypoglycemia, normoglycemia, or
hyperglycemia.
AMMONIA: Many, but not all, of the organic acidemias
are associated with blood ammonia values ranging from just below the upper
limit of normal to values several times the upper limit of normal. Children
with organic acidemias or amino acidemias often exhibit protein intolerance
and commonly develop dietary aversions to proteins such as meat, fish,
or milk products. Measurement of blood ammonia before and after a protein
meal may detect genetic defects in children between acute clinical episodes
but caution should be employed since fatal cardiorespiratory arrest may
occur with metabolic stress in certain of these diseases. Hyperammonemia
may occur in the absence of significant acidosis.
BUN: BUN which stands for blood urea nitrogen is the end
product of nitrogen metabolism in mammals. Since many of the organic acids
and amino acidemias result in impaired ammonia metabolism, the amount
of urea produced from ammonia will frequently be DECREASED in these diseases.
Although many clinicians are quick to note elevated BUN values, the significance
of low BUN values has not been fully appreciated.
ANION GAP: The anion gap is an indicator of the concentration
of organic acids or other anion drugs that may be present. An elevated
anion gap in the absence of drugs may indicate the presence of an organic
acidemia. However, some organic acids are such weak acids that they have
no significant effect on the anion gap. Thus a normal anion gap does not
rule out the presence of an organic acidemia.
ALBUMIN: Albumin binds organic acids and neutralizes their
toxic effect to some extent. A low-serum albumin is a significant risk
factor that results in a more serious clinical episode in patients with
organic acidemias. The administration of valproic acid (DEPAKENE) or salicylates
should be carefully evaluated in cases of suspected organic acidemias
since these drugs also bind to albumin and this diminishes the protective
effect of albumin in neutralizing toxic organic acids.
HEMATOPOIETIC FINDINGS: Certain organic acidemias such
as methylmalonic acidemia may result in megaloblastic anemia while others
such as propionic acidemia and biotinidase deficiency lead to immune deficiency
involving T and B lymphocytes and susceptibility to Candida infections.
A number of organic acidemias are associated with impaired granulopoiesis
or granulocytic function.
LACTIC ACID: Lactic acid may be elevated in a wide range
of diseases including the following: pyruvate dehydrogenase deficiency,
pyruvate carboxylase deficiency, glycogen storage disease type I, fructose
1,6-diphophatase deficiency, phosphoenol-pyruvate carboxykinase deficiency,
respiratory chain deficiencies and dihydrolipoyl dehydrogenase deficiency.
In addition to the above diseases in which lactic acid is the primary
abnormal organic acid, elevated lactic acid may also be excreted secondarily
as in methylmalonic acidemia due to the disruption of intermediary metabolism.
In addition, elevations of plasma and/or urine lactic acid may be due
to hypoxia due to septicemia, shock, or other causes.
Lactic acidosis is usually defined as a plasma lactic acid greater than
2.0 moles/L. Primary lactic acidosis is indicated if other organic acids
in urine are negative and other causes of hypoxia can be eliminated. Definitive
diagnosis of primary lactic acidosis requires provocation tests and/or
measurement of enzymes on tissue biopsies.
CPK: Extremely elevated CPK have been reported to occur
in cases of carnitine deficiency and in medium chain acyl dehydrogenase
deficiency.
URIC ACID: Uric acid is found in increased concentrations
in plasma or serum in gout and in conditions involving cellular destruction
such as leukemia, lymphoma, polycythemia vera, and during chemotherapy.
Elevations of uric acid in conditions in which massive cellular destruction
is not apparent are frequently due to organic acidemias. The exact reasons
for this elevation are not known but this elevation appears to be connected
to a disruption of cellular energy metabolism. Uric acid has been reported
as consistently elevated in medium chain acyl dehydrogenase deficiency,
glycogen storage diseases, Lesch Nyhan's syndrome, and in lead poisoning.
It also appears that the degree of elevation may be related to the severity
of clinical symptoms. Complaints of joint pains, arthritis, or pain in
the big toe in children may be due to uric acid deposits that have accumulated
from past bouts of undiagnosed organic acidemias.
TRIGLYCERIDES: Extremely elevated triglycerides (800 mg/dl
or more) may be associated with some organic acidemias.
URINALYSIS: High levels of the ketones beta-hydroxybutyrate,
acetoacetate, and acetone may be present in urine. These common products
of fat metabolism also will be detected by organic acid screening. However,
some organic acidemias are associated with low or normal ketones. Uric
acid crystals in the urine may also indicate the presence of an organic
acidemia.
STOOL ANALYSIS: Yeast overgrowth of the gastrointestinal
tract can be determined by stool analysis and is frequently associated
with elevated concentrations of certain organic acids that are produced
by yeast in the gastrointestinal tract and then absorbed into the body.
Summary of Lab Results that Indicate the Need
for Organic Acid Testing
Consider testing for organic acids and amino acids whenever any of the
following are present.
(1) Elevated glucose in a nondiabetic patient
(2) Low glucose
(3) Ammonia near the upper limit of normal or abnormally elevated
(4) Low BUN
(5) Elevated lactic acid
(6) Elevated serum uric acid
(7) Elevated anion gap (Some organic acidemias present with normal anion
gap)
(8) Presence of uric acid crystals in urine
(9) Elevated ketones (Some organic acidemias are also associated with
low or n o ketones)
(10) Severe anemias
(11) Immune deficiencies of unknown etiology
(12) CPK values that are several multiples of the upper limit of the normal
range
(13) Triglycerides that are 800 mg/dL or more
(14) Serum cholesterol greater than 250 mg/dL or less than 50 mg/dL.
(15) Low blood pH without a satisfactory clinical explanation.
Clinical symptoms that indicate the need for
organic acid testing
(1) The most important clinical finding is that there is not a good diagnosis
that explains severe, unusual or recurrent clinical symptoms.
(2) Frequent infections especially recurrent otitis media, thrush, yeast
infections and/or immunodeficiency
(3) SIDS, Near-miss SIDS or medical history of siblings with SIDS or near-miss
SIDS
(4) Lethargy, coma, Reye's syndrome
(5) Apneic spells or cardiorespiratory arrest
(6) Alopecia and/or severe dermatitis
(7) Physical and/or developmental retardation
(8) Failure to thrive
(9) Unexplained birth defects, microcephaly, macrocephaly, dysmorphic
features, cardiac defects
(10) Neurological symptoms including peripheral neuropathy,
(11) Hearing, speech, or visual impairment that has not been connected
to a defined disease.
(12) Marked dietary aversions to protein foods such as meat, fish, or
dairy products and/or history of nausea or vomiting after protein ingestion.
(13) Protracted unexplained periods of nausea and vomiting
(14) Juvenile arthritis and/or pain in the joints no confirmed as an autoimmune
disease
(15) Any child who seems to be much sicker than would normally be expected
(16) Peculiar body odor or urine odor or abnormally colored (black, blue,
red, green) urine
(17) Hepatomegaly
(18) Clotting disorder or disseminated intravascular coaguglation that
is not explained by the deficiency of known clotting factors or other
disease entities, lupus, etc.
(19) Psychosis, autism, or other severely abnormal behavior
(20) Severe protracted constipation and/or diarrhea not attributed to
any appropriate disease entity
(21) Emergency room admissions in which toxicology testing is usually
ordered in young children. Children with negative drug screens frequently
have metabolic diseases that can be detected by organic acid testing
DISEASES THAT CAN BE DETECTED
WITH ORGANIC ACID SCREENING
Diseases of Aromatic Amino Acid Metabolism
a) Phenylketonuria
b) Tyrosinemia
c) Tyrosinemia-hepatorenal form
d) Hawkinsinuria
e) Alcaptonuria
Diseases of Branched Chain Amino Acid Metabolism
a) Maple syrup urine disease
b) Isovaleric acidemia
c) Methylcrotonylglycinuria
d) Biotin responsive multiple carboxylase deficiency
e) Methylglutaconic aciduria
f) 3-hydroxy-3-methyl glutaric aciduria
g) 3-ketothiolase deficiency
h) Propionic acidemia
i) Methylmalonic acidemia
Diseases of Other Amino Acid Metabolism
a) Ketoadipic acidura
b) Glutaric aciduria type I
c) 5-oxoprolinuria
d) Ornithine transcarbamylase deficiency
e) Citrullinemia
f) Arginosuccinic aciduria
g) Arginemia
h) Canavan's disease
i) Methioninemia
Disease of Purine Metabolism - Lesch-Nyhan's syndrome
Disease of Pyrimidine Metabolism - Orotic aciduria
Diseases of Fatty Acid Oxidation
a) Short chain fatty acyl dehydrogenase (SCAD) deficiency
b) Medium chain fatty acyl dehydrogenase (MCAD) deficiency
c) Long chain fatty acyl dehydrogenase (LCAD) deficiency
d) Multiple acyl dehydrogenase (MAD) deficiency, also termed glutaric
acidemia type II
e) Long chain 3-hydroxyacyl dehydrogenase deficiency
Disease of Cholesterol Synthesis - Mevalonic acidemia
Miscellaneous
a) Glutathione synthetase deficiency
b) Fumarase deficiency
c) Succinic semialdehyde dehydrogenase deficiency with lactic acidosis
d) Malonyl CoA carboxylase deficiency
e) 2-Ketoadipic acidemia
f) Glutaric acidemia type I
g) Pyruvate dehydrogenase El and E2 subunit deficiency
h) Dihydrolipoyl dehydrogenase deficiency
i) Biotinidase deficiency
j) Alpha-ketoglutaric aciduria
k) Pyruvic dehydrogenase phosphatase deficiency
1) Primary hyperoxaluria type I and II
m) Oasthouse urine disease (methionine malabsorption syndrome)
n) Hartnup's disease
o) Fructose 1, 6 diphosphatase deficiency
p) Ethylmalonic-adipic acidemia
q) Neuroblastoma
r) Carcinoid syndrome
s) Pheochromacytoma
Diseases that are Associated with Lactic Acidemia and/or Pyruvic Acidemia
but No Characteristic Elevations of Other Organic Acids
a) Pyruvic carboxylase deficiency, neonatal and infantile forms
b) Phosphoenol pyruvic carboxykinase deficiency
c) NADH-Co Q oxidoreductase (Complex I) deficiency
d) Ubiquinol-cytochrome C reductase (Complex III) deficiency
e) Cytochrome oxidase (Complex IV) deficiency
f) Myoclonic epilepsy with ragged-red fibers (MERRF)
g) Mitochondrial encephalomyopathy lactic acidosis with strokelike episodes
(MELAS)
h) Kearns-Sayre syndromes
DISEASES NOT DETECTED
BY ORGANIC ACID TESTING
A scan of the list of diseases detected by organic acid testing reveals
that many diseases of amino acid testing can be detected by organic acid
screening. However, not all inborn errors of amino acid testing can be
detected by organic acid screening. PLASMA AND URINE AMINO ACID SCREENING
SHOULD ALSO BE DONE AT THE SAME TIME THAT ORGANIC ACIDS ARE REQUESTED.
Metabolic disturbances are most readily detected when clinical symptoms
are most severe.
Other metabolic diseases that require additional laboratory testing and
are not included in the organic acid screen are:
(1) Galactosemias
(2) Mucopolysaccharidoses such as Hurler's syndrome
(3) Mucolipidoses
(4) Sphingolipidoses such as Gaucher's disease
(5) Zellweger's syndrome
(6) Refsum's syndrome
(7) Adrenoleukodystrophy
(8) Leber congenital amaurosis
(9) Rhizomelic chondrodysplasia punctata
(10) Some disorders of amino acid metabolism
Testing for Zellweger's syndrome, Refsum's syndrome, adrenoleukodystrophy,
Leber congenital amaurosis, and rhizomelic chondrodysplasia punctata is
done by testing serum for phytanic acid, very long chain fatty acids,
and pipecolic acid. Such testing is not included in organic acid screening
of urine. However, some unusual non-pathognomonic organic acid compounds
are frequently detected in these peroxisomal disorders and are detected
with our current organic acid screen and will be reported with a suggestion
for follow-up testing.
Relationship between Amino Acid and Organic Acid Results
for the Diagnosis of Metabolic Disease
Oznand and Gascon (J.Child. Neurol. 6:288, 1992) have compiled an excellent
summary of the relations between amino acid changes and organic acidemia
which was used for the table below:
Increased Blood or Urine
Amino Acids in
Various Organic Acidemias.
Generalized Hyperaminoacidemia and Aminoaciduria
Isovaleric acidemia
3-Methylglutaconic aciduria with normal hydratase
Propionic acidemia
Holocarboxylase synthetase and biotinidase deficiencies
Short-chain acyl-CoA dehydrogenase deficiency
Pyruvate carboxylase deficiency, neonatal form
Glutathione synthetase deficiency (in red blood cells but not always true)
Elevated Branched-Chain Amino Acids in Blood
3-Methylcrotonyl-CoA carboxylase deficiency
Dihydrolipoyl dehydrogenase (E3) deficiency (periodic, during attacks)
Elevated Glycine
Isovaleric acidemia
Propionic acidemia
Methylmalonic acidemia
Glutaric aciduria type 1
d-Glyceric aciduria with defect in fructose metabolism
Succinic semialdehyde dehydrogenase deficiency (50%)
Elevated Alanine
Generally observed in disease with hyperlacticacidemia
Dihydrolipoyl dehydrogenase (E3) deficiency
Holocarboxylase synthetase and biotindase deficiencies
Phosphoenolpyruvate carboxykinase deficiency
Pyruvate carboxylase deficiency, neonatal form
Pyruvate carboxylase deficiency, infantile form
Pyruvate dehydrogenase El subunit deficiency, neonatal form
Pyruvate dehydrogenase El subunit deficiency, infantile form
Pyruvate dehydrogenase E2 subunit deficiency Pyruvate dehydrogenase phosphatase
deficiency
Pathognomonic Amino Acid Elevations
Pyruvate carboxylase deficiency, neonatal form: citrulline, lysine, proline
Multiple acyl-CoA dehydrogenase deficiency: sarcosine
Glutaric aciduria type 1: 2-aminoadipic acid and saccharopine
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