Detection of Maple Syrup Urine Disease by a Bioassay James D. Linn, MS; S. M. Hussain Qadri, PhD, Diplomate ABMM, SM(AAM), FAAM; Sarvepalli B. Subramanyam, PhD; Pinar Ozand, MD, PhD From the Biological and Medical Research Department (Mr. Linn and Dr. Subramanyam), Department of Pathology and Laboratory Medicine (Dr. Qadri), and the Department of Pediatrics (Dr. Ozand), King Faisal Specialist Hospital and Research Centre, Riyadh. Address reprint requests and correspondence to: Dr. Qadri: Department of Pathology and Laboratory Medicine, King Faisal Specialist Hospital and Research Centre, P.O. Box 3354, Riyadh 11211, Saudi Arabia. Accepted for publication 21 March 1989. Judging from the number of clinical referrals, there may be an unusually large number of children born in Saudi Arabia suffering from maple syrup urine disease (MSUD). To assure early diagnosis and rapid treatment of these infants, we have developed a bioassay, using Bacillus subtilis, for screening high levels of leucine in the blood. Leucine overcame the inhibition of growth due to 4-aza-dl-leucine in a chemically defined medium. Addition of the redox indicator 2,3,5-triphenyl-2H-tetrazolium chloride enhanced the sensitivity and interpretation of the test. The test is simple, accurate, and economical for screening MSUD, and may have wide applicability in Saudi Arabia and elsewhere in the Middle East. JD Linn, SMH Qadri, SB Subramanyam, P Ozand, Detection of Maple Syrup Urine Disease by a Bioassay. 1989; 9(6): 579-583 Maple syrup urine disease (MSUD) is a hereditary metabolic disorder that results from a primary defect in oxidative decarboxylation of leucine, isoleucine, and valine, and is usually characterized by a maple syrup odor to the urine. MSUD is a genetic disease with devastating consequences in an undiagnosed infant and carries a much higher mortality than phenylketonuria (PKU). Although generally a rare disorder, its incidence varies in different parts of the world. 1-5 Thenumber of clinical referrals at this center and reports from Bahrain and Jeddah indicate that the incidence may be higher in Saudi Arabia and elsewhere in the Middle East than that reported in the U.S.A. and Europe. 6,7 MSUD is characterized by high levels of leucine, isoleucine, or yaline in blood, and this can form the diagnosis. Current methods of laboratory diagnosis include paper, thin-layer, or column chromatography, or complicated bioassays. 6-12 Unlike PKU assays, there are no commercial kits available for the screening and detection of MSUD, possibly because of its rare occurrence. Since early diagnosis accompanied by supportive therapy and dietary management can result in a good prognosis, there is need for an effective and simple screening procedure for the detection of MSUD. This has prompted us to develop a simple and reliable assay, and our findings are presented in this paper.
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Detection of Maple Syrup Urine Disease by a BioassayDetection of Maple Syrup Urine Disease by a Bioassay
James D. Linn, MS; S. M. Hussain Qadri, PhD, Diplomate ABMM, SM(AAM), FAAM;
Sarvepalli B. Subramanyam, PhD; Pinar Ozand, MD, PhD
From the Biological and Medical Research Department (Mr. Linn and Dr. Subramanyam), Department of Pathology and Laboratory Medicine (Dr. Qadri), and the Department of Pediatrics (Dr. Ozand), King Faisal Specialist Hospital and Research Centre, Riyadh.
Address reprint requests and correspondence to: Dr. Qadri: Department of Pathology and Laboratory Medicine, King Faisal Specialist Hospital and
Research Centre, P.O. Box 3354, Riyadh 11211, Saudi Arabia.
Accepted for publication 21 March 1989.
Judging from the number of clinical referrals, there may be an unusually large number of children born in Saudi
Arabia suffering from maple syrup urine disease (MSUD). To assure early diagnosis and rapid treatment of these
infants, we have developed a bioassay, using Bacillus subtilis, for screening high levels of leucine in the blood.
Leucine overcame the inhibition of growth due to 4-aza-dl-leucine in a chemically defined medium. Addition of the
redox indicator 2,3,5-triphenyl-2H-tetrazolium chloride enhanced the sensitivity and interpretation of the test. The
test is simple, accurate, and economical for screening MSUD, and may have wide applicability in Saudi Arabia and
elsewhere in the Middle East.
JD Linn, SMH Qadri, SB Subramanyam, P Ozand, Detection of Maple Syrup Urine Disease by a Bioassay. 1989;
9(6): 579-583
Maple syrup urine disease (MSUD) is a hereditary metabolic disorder that results from a primary defect in
oxidative decarboxylation of leucine, isoleucine, and valine, and is usually characterized by a maple syrup odor to
the urine. MSUD is a genetic disease with devastating consequences in an undiagnosed infant and carries a much
higher mortality than phenylketonuria (PKU). Although generally a rare disorder, its incidence varies in different
parts of the world. 1-5
Thenumber of clinical referrals at this center and reports from Bahrain and Jeddah indicate that
the incidence may be higher in Saudi Arabia and elsewhere in the Middle East than that reported in the U.S.A. and
Europe. 6,7
MSUD is characterized by high levels of leucine, isoleucine, or yaline in blood, and this can form the diagnosis.
Current methods of laboratory diagnosis include paper, thin-layer, or column chromatography, or complicated
bioassays. 6-12
Unlike PKU assays, there are no commercial kits available for the screening and detection of MSUD,
possibly because of its rare occurrence. Since early diagnosis accompanied by supportive therapy and dietary
management can result in a good prognosis, there is need for an effective and simple screening procedure for the
detection of MSUD. This has prompted us to develop a simple and reliable assay, and our findings are presented in
this paper.
Material and Methods
Fifty milliliters of PKU test agar without beta-2-thienylalanine (Difco Laboratories, Detroit, MI) was melted by
boiling and then cooled to 45 to 50°C in a water bath. 4-Aza-dl-leucine (Sigum Biochemical, St. Louis, MO) and
2,3,5-triphenyl-2H-tetrazolium chloride (TTC) (Kodak, Rochester, NY) were added to yield a final concentration of
1.0 mg/ml and 0.03 mg/ml, respectively. This was followed by addition of 0.1 ml of Bacillus sub-tilis spore
suspension (No. 2) (No. 981-52-0, Difco Laboratories) to the melted agar, mixed by shaking, poured into a 150-mm
petri dish, and allowed to solidify.
Control sample discs
A heparinized sample of normal blood was obtained from a volunteer and divided into 1.0-ml aliquots. L-
leucine (Kodak) was added to each aliquot to get a final concentration of 0.2,0.3, 0.5, 1.0, and 2.0 mM, and 25-μl
sample from each tube was spotted on Blood Test Form (No. 3240-30-8; Difco Laboratories). Blood Test Forms are
made of absorbent filter paper and have three circles on each to accommodate the control of test samples. The test
forms were wrapped in aluminum foil and autoclaved at 121°C for 3 minutes to fix the blood to the filter paper and
destroy any contaminating bacteria.
Patient sample discs
Patient specimens were collected by heel puncture 48 hours after first feeding, using standard technique, and
each circle of the test form was filled by single application of paper to a drop of blood. If the blood was collected in
heparinized vacutainer tubes, 25 μlwas applied to the circle on the test form. All forms were sterilized for 3 minutes
at 121°C.
Discs from the control and patient forms were then punched out with a 6-mm paper punch and then placed on
the agar surface using sterile forceps. The discs were gently pressed, the plate covered, inverted, and incubated at
37°C, and then examined for visible growth after 18 to 24 hours. Leucine concentration was estimated by comparing
the zone size of growth around the patient disc with those around standard discs.
High-performance liquid chromatography
Leucine concentration in the plasma of MSUD patients was also determined by using reversed-phase high-
performance liquid chromatography (HPLC). Plasma was separated from the clotted blood by centrifugation at 3000
rpm for 30 minutes in an Amicon MPS-1 Micropartition system. The amino acid standards were purchased from
Pierce Co. (Illinois) and contained 17 L-amino acids at a concentration of 2.5 mM, which were diluted to 0.5 mM.
Standard and patient samples (25 μl) were pipetted into 6 × 50 mm test tubes; the tubes were placed in reaction vials
and dried under vacuum on the PICO-TAG vacuum station (Waters Chromatography Division, Milford, MA). The
dried samples were extracted in phenol:water: phenylisothiocyanate: triethylamine (7:1:1:1, v/v) reagent and dried
again; 10 μlof tolune was added and subjected to the last drying cycle followed by addition of 100 μlof PICO-TAG
diluent. The samples were transferred to the WISP vials with a limited vacuum insert. Immediately after extraction,
10-μlsamples were injected into the HPLC system that consisted of a Waters Associates model 481
spectrophotometer (269-nm wavelength), model 510 HPLC pumps, and model 710 WISP for automatic injection
system (Waters Chromatography).
This system was controlled by a Waters 840 Data and Chromatography control station. A column heating
module and eluent stabilization system were also used. A uBondpak C18 reversed-phase PICO-TAG column (10-μm
particle size, 3.9 × 300 mm) was used in conjunction with a stainless steel guard column (3.9 × 20 mm) which was
packed with Waters Coracil C18 packing material. The temperature of the guard column was maintained at 40°C by
encasing it in a column heater (Waters) that was kept at 40° ± 1°C.
Peaks were identified with reference to the retention times of standard amino acids injected separately. The
peak areas of known concentrations of authentic amino acids were measured using the Waters 840 Data and
Chromatography control station. The accuracy of measurement was tested by analyzing the standard and treating it
as a control. Methionine sulfone was used as the internal standard to account for injection variables.
Results
The screening test for MSUD was based on the ability of 4-aza-dl-leucine to inhibit the growth of B. subtilis in
Detection of Maple Syrup Urine Disease by a Bioassay
Annals of Saudi Medicine, Vol 9 No. 6; 1989
a chemically defined medium, and reversal of inhibition by leucine. Since 1.0 mM leucine in blood is considered
positive for MSUD and less than 0.5 mM negative, the experiments were designed to determine the concentration of
4-aza-dl-leucine that yield small zones of growth with 0.3 mM leucine. Of the ten different concentrations varying
from 0.25 to 5.0 mg/m1 of 4-aza-dl-leucine, 1.0 mg/ml of the culture medium yielded no growth without leucine,
but there were 8 to 10 mm growth zones in the presence of 0.2 mM leucine (Figure 1). Using 1.0 mg/ml of 4-aza-dl-
leucine as the optimal concentration, we found the zones for growth varying from 8 to 25 mm in the presence of 0.2
to 2.0 mM leucine (Table 1).
Addition of the redox agent TTC was intended for ease of interpretation because the growth appears as a pink-
reddish zone. We found that TTC enhanced the inhibitory effect of the leucine analogue. This enabled us to reduce
the concentration of inhibitor by a factor of 5. Following trials with different concentrations of TTC, the optimal
concentration was found to be 0.03 mg/ml. Similarly it was found that the optimal incubation temperature was 37°C
for 18 to 24 hours. Too short an incubation (12 hours or less) increased the number of false negatives, and
incubation periods of over 30 hours caused more false positives. Total turnaround time for completion of the assay
was 24 hours. When used as a screening procedure, with 25 patient specimens and 7 controls on each assay plate,
the estimated cost was less than SR2.00 ($0.50) per test.
Table 1. Growth of Bacillus subtilis in the presence of leucine.
Leucine concentration (mM) Zone of growth
0.2 8-10 mm
0.3 9-11 mm
0.5 13-15 mm
1.0 16-19 mm
2.0 22-25 mm
During development of the test, a number of simulated specimens as well as confirmed cases of MSUD samples
were analyzed by both the bioassay and HPLC to determine the accuracy of the bioassay. Actual concentration of
leucine in the samples as well as predictive values were comparable with both methods (Table 2).
Table 2. Comparison of the bioassay and HPLC for the detection of MSUD.
Specimen
Bioassay HPLC Bioassay HPLC
Normal volunteer
HPLC = high-performance liquid chromatography.
Detection of Maple Syrup Urine Disease by a Bioassay
Annals of Saudi Medicine, Vol 9 No. 6; 1989
Figure 1. Maple syrup urine disease assay plate. Pink areas arounddifferent discs are
zones of growth. Leucine content in each disc was:1, none; 2,2.0 mM; 3,0.1 mM;
4,0.2 mM; 5,0.3 mM; 6, 0.5 mM; 6, 0.5 mM, 7,1.0 mM.
Discussion
MSUD was first reported by Menkes et al, 13
who found that four of six infants in a family died during the first
weeks of life with symptoms of vomiting, muscular hypertonicity, cerebral dysfunction, and a maple syrup odor in
the urine. Three years later, Westhall et al 14
described a similar case of an infant with brain damage, associated with
increased levels of leucine, isoleucine, and valine in blood and urine. Since then a number of studies have shown
that the disease is rare, with an incidence of 1:120,000 in Europe to 1:290,000 in the U.S. 2,3
Of the 872,660 infants
screened in Massachusetts, MSUD was detected in only three. 3 Using a similar incidence rate and assuming an
annual birth rate of 240,000, no more than one case per 1 to 2 years should be encountered in Saudi Arabia.
However, in less than two years we have confirmed MSUD in 14 infants in the Kingdom, indicating an incidence
that is several magnitudes higher than that in the U.S. and Europe. It is left to speculation how many infants have
died during the first week of life because MSUD is usually not considered in the differential diagnosis. We found
that 9 of 14 patients had progressed to irretrievable morbidity and brain damage by the time of their referral to the
Division of Inborn Errors of Metabolism at this center. If MSUD had been diagnosed during the first week of life,
with proper management these infants would have grown to be normal and healthy children.
The laboratory diagnosis of MSUD entails detection of high levels of leucine, isoleucine, or valine in blood or
urine. Commercial kits for rapid diagnosis or screening have not been developed because of the extremely low
incidence of the disorder in the West. Currently available methods consist of paper chromatography, thin-layer
chromatography, HPLC, or complicated microbiological assays that are not suitable for large-scale screening. These
tests are also beyond the scope and expertise of most institutions, except large medical centers and research
laboratories. This led us to the development of the method described in this paper. Essentially it is a modification of
the Guthrie test and utilizes methodology similar to that of the PKU inhibition assay. Since the development of this
procedure, we have screened more than 1200 infants in our diagnostic microbiology laboratory and the test is now
done routinely, along with PKU, on all infants born at this institution. Plans are under way to perform screening of
newborns at other hospitals in the Kingdom. We found ready acceptability of the assay procedure by our
technologists because the method is simple and uses familiar bacteriologic technique.
Acknowledgment
This work was supported in part by a grant from Shaikh Rafiq Al-Harir. We wish to thank Dr. Peter B. Herdson
for critical review and Miss Erlinda M. Umali for secretarial assistance in preparation of the manuscript.
Detection of Maple Syrup Urine Disease by a Bioassay
Annals of Saudi Medicine, Vol 9 No. 6; 1989
References
1. Naylor EW. Newborn screening for maple syrup urine disease (branched chain ketoaciduria). In: Bickel H, Guthrie R,
Hammersen G, eds. Neonatal screening for inborn errors of metabolism. Berlin: Springer-Verlag, 1980;19-28.
2. Editorial. Collective results of mass screening for inborn metabolic errors in eight European countries. Acta Paediatr Scand
1973;62:413.
3. Levy HL. Neonatal screening for inborn errors of amino acid metabolism. Clin Endocrinol Metabol 1974;3:153-66.
4. Auerbach VH, DiGeorge AM. Maple syrup urine disease. In: Hommes FA, Van den Berg CJ, eds. Inborn errors of
metabolism. London: Academic Press, 1973;337-52.
5. Marshall L, DiGeorge A. Maple syrup urine disease in Old Order Mennonites (abstract). Am J Hum Genet 1981;33:138A.
6. Mohammad AM. Maple syrup urine disease in Bahrain. Bahrain Med Bull 1985;7:114-7.
7. Hardy MJ, O'Connell JP. Maple syrup urine disease in two sibling Saudi Arabs. Saudi Med J 1987;8(3):250-2.
8. Bachhawat BK, Robinson WG, Coon MJ. Enzymatic carboxylation of beta-hydroxyisovaleryl coenzyme. Am J Biol Chem
1956;219:539.
9. Piez KA, Morris L. A modified procedure for the automatic analysis of amino acids. Anal Biochem 1960;1:187-201.
10. Berry HG, Schee C, Marks J. Microbiological test for leucine, valine, and isoleucine using urine sample dried on filter paper.
Clin Chem 1962;8:242-5.
11. DiGeorge AM, Rezvani I, Garibaldi LR, Schwartz M. Prospective study of maple-syrup-urine disease for the first four days
of life. N Engl J Med 1982;307(24):1492-5.
12. Guthrie R. Screening for "inborn errors of metabolism" in the newborn infant: a multiple test program. In: Bergsma D, ed.
Human Genetics (Birth Defects Original Article Series Vol. 4, No. 6). New York: The National Foundation, 1968;92-8.
13. Menkes JH, Hurst PL, Craig JM. New syndrome: progressive familial infantile cerebral dysfunction associated with an
unusual urinary substance. Pediatrics 1954;14:462-6.
14. Westall RG, Dancis J, Miller S. Maple sugar urine disease. Am J Dis Child 1957;94:571.
James D. Linn, MS; S. M. Hussain Qadri, PhD, Diplomate ABMM, SM(AAM), FAAM;
Sarvepalli B. Subramanyam, PhD; Pinar Ozand, MD, PhD
From the Biological and Medical Research Department (Mr. Linn and Dr. Subramanyam), Department of Pathology and Laboratory Medicine (Dr. Qadri), and the Department of Pediatrics (Dr. Ozand), King Faisal Specialist Hospital and Research Centre, Riyadh.
Address reprint requests and correspondence to: Dr. Qadri: Department of Pathology and Laboratory Medicine, King Faisal Specialist Hospital and
Research Centre, P.O. Box 3354, Riyadh 11211, Saudi Arabia.
Accepted for publication 21 March 1989.
Judging from the number of clinical referrals, there may be an unusually large number of children born in Saudi
Arabia suffering from maple syrup urine disease (MSUD). To assure early diagnosis and rapid treatment of these
infants, we have developed a bioassay, using Bacillus subtilis, for screening high levels of leucine in the blood.
Leucine overcame the inhibition of growth due to 4-aza-dl-leucine in a chemically defined medium. Addition of the
redox indicator 2,3,5-triphenyl-2H-tetrazolium chloride enhanced the sensitivity and interpretation of the test. The
test is simple, accurate, and economical for screening MSUD, and may have wide applicability in Saudi Arabia and
elsewhere in the Middle East.
JD Linn, SMH Qadri, SB Subramanyam, P Ozand, Detection of Maple Syrup Urine Disease by a Bioassay. 1989;
9(6): 579-583
Maple syrup urine disease (MSUD) is a hereditary metabolic disorder that results from a primary defect in
oxidative decarboxylation of leucine, isoleucine, and valine, and is usually characterized by a maple syrup odor to
the urine. MSUD is a genetic disease with devastating consequences in an undiagnosed infant and carries a much
higher mortality than phenylketonuria (PKU). Although generally a rare disorder, its incidence varies in different
parts of the world. 1-5
Thenumber of clinical referrals at this center and reports from Bahrain and Jeddah indicate that
the incidence may be higher in Saudi Arabia and elsewhere in the Middle East than that reported in the U.S.A. and
Europe. 6,7
MSUD is characterized by high levels of leucine, isoleucine, or yaline in blood, and this can form the diagnosis.
Current methods of laboratory diagnosis include paper, thin-layer, or column chromatography, or complicated
bioassays. 6-12
Unlike PKU assays, there are no commercial kits available for the screening and detection of MSUD,
possibly because of its rare occurrence. Since early diagnosis accompanied by supportive therapy and dietary
management can result in a good prognosis, there is need for an effective and simple screening procedure for the
detection of MSUD. This has prompted us to develop a simple and reliable assay, and our findings are presented in
this paper.
Material and Methods
Fifty milliliters of PKU test agar without beta-2-thienylalanine (Difco Laboratories, Detroit, MI) was melted by
boiling and then cooled to 45 to 50°C in a water bath. 4-Aza-dl-leucine (Sigum Biochemical, St. Louis, MO) and
2,3,5-triphenyl-2H-tetrazolium chloride (TTC) (Kodak, Rochester, NY) were added to yield a final concentration of
1.0 mg/ml and 0.03 mg/ml, respectively. This was followed by addition of 0.1 ml of Bacillus sub-tilis spore
suspension (No. 2) (No. 981-52-0, Difco Laboratories) to the melted agar, mixed by shaking, poured into a 150-mm
petri dish, and allowed to solidify.
Control sample discs
A heparinized sample of normal blood was obtained from a volunteer and divided into 1.0-ml aliquots. L-
leucine (Kodak) was added to each aliquot to get a final concentration of 0.2,0.3, 0.5, 1.0, and 2.0 mM, and 25-μl
sample from each tube was spotted on Blood Test Form (No. 3240-30-8; Difco Laboratories). Blood Test Forms are
made of absorbent filter paper and have three circles on each to accommodate the control of test samples. The test
forms were wrapped in aluminum foil and autoclaved at 121°C for 3 minutes to fix the blood to the filter paper and
destroy any contaminating bacteria.
Patient sample discs
Patient specimens were collected by heel puncture 48 hours after first feeding, using standard technique, and
each circle of the test form was filled by single application of paper to a drop of blood. If the blood was collected in
heparinized vacutainer tubes, 25 μlwas applied to the circle on the test form. All forms were sterilized for 3 minutes
at 121°C.
Discs from the control and patient forms were then punched out with a 6-mm paper punch and then placed on
the agar surface using sterile forceps. The discs were gently pressed, the plate covered, inverted, and incubated at
37°C, and then examined for visible growth after 18 to 24 hours. Leucine concentration was estimated by comparing
the zone size of growth around the patient disc with those around standard discs.
High-performance liquid chromatography
Leucine concentration in the plasma of MSUD patients was also determined by using reversed-phase high-
performance liquid chromatography (HPLC). Plasma was separated from the clotted blood by centrifugation at 3000
rpm for 30 minutes in an Amicon MPS-1 Micropartition system. The amino acid standards were purchased from
Pierce Co. (Illinois) and contained 17 L-amino acids at a concentration of 2.5 mM, which were diluted to 0.5 mM.
Standard and patient samples (25 μl) were pipetted into 6 × 50 mm test tubes; the tubes were placed in reaction vials
and dried under vacuum on the PICO-TAG vacuum station (Waters Chromatography Division, Milford, MA). The
dried samples were extracted in phenol:water: phenylisothiocyanate: triethylamine (7:1:1:1, v/v) reagent and dried
again; 10 μlof tolune was added and subjected to the last drying cycle followed by addition of 100 μlof PICO-TAG
diluent. The samples were transferred to the WISP vials with a limited vacuum insert. Immediately after extraction,
10-μlsamples were injected into the HPLC system that consisted of a Waters Associates model 481
spectrophotometer (269-nm wavelength), model 510 HPLC pumps, and model 710 WISP for automatic injection
system (Waters Chromatography).
This system was controlled by a Waters 840 Data and Chromatography control station. A column heating
module and eluent stabilization system were also used. A uBondpak C18 reversed-phase PICO-TAG column (10-μm
particle size, 3.9 × 300 mm) was used in conjunction with a stainless steel guard column (3.9 × 20 mm) which was
packed with Waters Coracil C18 packing material. The temperature of the guard column was maintained at 40°C by
encasing it in a column heater (Waters) that was kept at 40° ± 1°C.
Peaks were identified with reference to the retention times of standard amino acids injected separately. The
peak areas of known concentrations of authentic amino acids were measured using the Waters 840 Data and
Chromatography control station. The accuracy of measurement was tested by analyzing the standard and treating it
as a control. Methionine sulfone was used as the internal standard to account for injection variables.
Results
The screening test for MSUD was based on the ability of 4-aza-dl-leucine to inhibit the growth of B. subtilis in
Detection of Maple Syrup Urine Disease by a Bioassay
Annals of Saudi Medicine, Vol 9 No. 6; 1989
a chemically defined medium, and reversal of inhibition by leucine. Since 1.0 mM leucine in blood is considered
positive for MSUD and less than 0.5 mM negative, the experiments were designed to determine the concentration of
4-aza-dl-leucine that yield small zones of growth with 0.3 mM leucine. Of the ten different concentrations varying
from 0.25 to 5.0 mg/m1 of 4-aza-dl-leucine, 1.0 mg/ml of the culture medium yielded no growth without leucine,
but there were 8 to 10 mm growth zones in the presence of 0.2 mM leucine (Figure 1). Using 1.0 mg/ml of 4-aza-dl-
leucine as the optimal concentration, we found the zones for growth varying from 8 to 25 mm in the presence of 0.2
to 2.0 mM leucine (Table 1).
Addition of the redox agent TTC was intended for ease of interpretation because the growth appears as a pink-
reddish zone. We found that TTC enhanced the inhibitory effect of the leucine analogue. This enabled us to reduce
the concentration of inhibitor by a factor of 5. Following trials with different concentrations of TTC, the optimal
concentration was found to be 0.03 mg/ml. Similarly it was found that the optimal incubation temperature was 37°C
for 18 to 24 hours. Too short an incubation (12 hours or less) increased the number of false negatives, and
incubation periods of over 30 hours caused more false positives. Total turnaround time for completion of the assay
was 24 hours. When used as a screening procedure, with 25 patient specimens and 7 controls on each assay plate,
the estimated cost was less than SR2.00 ($0.50) per test.
Table 1. Growth of Bacillus subtilis in the presence of leucine.
Leucine concentration (mM) Zone of growth
0.2 8-10 mm
0.3 9-11 mm
0.5 13-15 mm
1.0 16-19 mm
2.0 22-25 mm
During development of the test, a number of simulated specimens as well as confirmed cases of MSUD samples
were analyzed by both the bioassay and HPLC to determine the accuracy of the bioassay. Actual concentration of
leucine in the samples as well as predictive values were comparable with both methods (Table 2).
Table 2. Comparison of the bioassay and HPLC for the detection of MSUD.
Specimen
Bioassay HPLC Bioassay HPLC
Normal volunteer
HPLC = high-performance liquid chromatography.
Detection of Maple Syrup Urine Disease by a Bioassay
Annals of Saudi Medicine, Vol 9 No. 6; 1989
Figure 1. Maple syrup urine disease assay plate. Pink areas arounddifferent discs are
zones of growth. Leucine content in each disc was:1, none; 2,2.0 mM; 3,0.1 mM;
4,0.2 mM; 5,0.3 mM; 6, 0.5 mM; 6, 0.5 mM, 7,1.0 mM.
Discussion
MSUD was first reported by Menkes et al, 13
who found that four of six infants in a family died during the first
weeks of life with symptoms of vomiting, muscular hypertonicity, cerebral dysfunction, and a maple syrup odor in
the urine. Three years later, Westhall et al 14
described a similar case of an infant with brain damage, associated with
increased levels of leucine, isoleucine, and valine in blood and urine. Since then a number of studies have shown
that the disease is rare, with an incidence of 1:120,000 in Europe to 1:290,000 in the U.S. 2,3
Of the 872,660 infants
screened in Massachusetts, MSUD was detected in only three. 3 Using a similar incidence rate and assuming an
annual birth rate of 240,000, no more than one case per 1 to 2 years should be encountered in Saudi Arabia.
However, in less than two years we have confirmed MSUD in 14 infants in the Kingdom, indicating an incidence
that is several magnitudes higher than that in the U.S. and Europe. It is left to speculation how many infants have
died during the first week of life because MSUD is usually not considered in the differential diagnosis. We found
that 9 of 14 patients had progressed to irretrievable morbidity and brain damage by the time of their referral to the
Division of Inborn Errors of Metabolism at this center. If MSUD had been diagnosed during the first week of life,
with proper management these infants would have grown to be normal and healthy children.
The laboratory diagnosis of MSUD entails detection of high levels of leucine, isoleucine, or valine in blood or
urine. Commercial kits for rapid diagnosis or screening have not been developed because of the extremely low
incidence of the disorder in the West. Currently available methods consist of paper chromatography, thin-layer
chromatography, HPLC, or complicated microbiological assays that are not suitable for large-scale screening. These
tests are also beyond the scope and expertise of most institutions, except large medical centers and research
laboratories. This led us to the development of the method described in this paper. Essentially it is a modification of
the Guthrie test and utilizes methodology similar to that of the PKU inhibition assay. Since the development of this
procedure, we have screened more than 1200 infants in our diagnostic microbiology laboratory and the test is now
done routinely, along with PKU, on all infants born at this institution. Plans are under way to perform screening of
newborns at other hospitals in the Kingdom. We found ready acceptability of the assay procedure by our
technologists because the method is simple and uses familiar bacteriologic technique.
Acknowledgment
This work was supported in part by a grant from Shaikh Rafiq Al-Harir. We wish to thank Dr. Peter B. Herdson
for critical review and Miss Erlinda M. Umali for secretarial assistance in preparation of the manuscript.
Detection of Maple Syrup Urine Disease by a Bioassay
Annals of Saudi Medicine, Vol 9 No. 6; 1989
References
1. Naylor EW. Newborn screening for maple syrup urine disease (branched chain ketoaciduria). In: Bickel H, Guthrie R,
Hammersen G, eds. Neonatal screening for inborn errors of metabolism. Berlin: Springer-Verlag, 1980;19-28.
2. Editorial. Collective results of mass screening for inborn metabolic errors in eight European countries. Acta Paediatr Scand
1973;62:413.
3. Levy HL. Neonatal screening for inborn errors of amino acid metabolism. Clin Endocrinol Metabol 1974;3:153-66.
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