BIOCHEMICAL
12, 302-3 I:! (197.5)
MEDICINE
A Screening
Test
in Urine
for
Sulphur-Containing
Using
Auxotrophic
Escherichia
co/i
Metabolites Mutants
of
K12
M. G. PATRICK, R. C. H. COTTON, D. M. DANKS, AND D. GOLDSMITH Genetics Research Unit. ROJUI Childretl’s Hospitd Research Foundation. Pnrk~ille. Victoria, Australitr 3052, md Department of Genetics. Univer.sity of Melbourrw. ParC\~ille. Victor-kc. Australia 3052 Received
December
8. 1974
A simple method is described for testing samples of urine dried on blotting paper for a large range of sulphur containing compounds. including all those found in known inborn errors of metabolism. The test uses two auxotrophic strains of Escherichin co/i K 12, and can be semiquantitative if the method is carefully standardized to optimal conditions.
In studying human inborn errors of metabolism, it seems reasonable to anticipate a disease corresponding to each enzyme in intermediary metabolism. Consequently it is desirable to produce tests for abnormal levels of all possible metabolites in blood and/or urine. The use of separate tests for each metabolite is clearly impracticable, and tests for groups of compounds must be devised. The traditional approach has grouped compounds according to the possession of chemical properties which determine characteristic staining reactions-e.g., the use of ninhydrin for the recognition of (Yamino nitrogen groups. The compounds present in body fluids have been separated by chromatography or electrophoresis and revealed by staining. Chemicals having similar radicles may or may not be metabolically related. If bacteria and humans utilize very similar metabolic pathways for synthesis and degradation, respectively, it is possible to utilize the requirements of appropriate auxotrophic mutants to detect, and to quantitate, human metabolites which can act as growth factors (1). By using two sulphur requiring mutants (AT2035 and AB1875) of Escherichia co/i K12, it has been possible to detect a wide range of sulphur containing compounds derived from methionine and/or cysteine in man. Some of these compounds are easily detected by existing techniques, but others are not. Several known inborn errors of metabolism Copyright @ 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
SCREENING
TEST
FOR
SULPHUR-CONTAINING
METABOLITES
303
can be diagnosed by this method, and wider use of the test may identify new metabolic defects in this pathway. Certain factors which influence the sensitivity and reproducibility of the method have been investigated and an optimal technique has been defined. MATERIALS
AND
METHODS
Ofgutzisms. The original strains of the mutants used were obtained from Dr. A. Taylor, by courtesy of Professor A. J. Pittard. AT2035 (cys 1324) was believed to be blocked in conversion of sulphite to sulphide. and the results obtained confirmed this belief. The additional growth requirements of this mutant for tryptophan and histidine proved inconvenient and were corrected by transduction from the prototrophic strain AB259 using phage Pl and a standard method described by Lennox (2). The transduced substrain (AT203YMP2) showed comparable and satisfactory responses to the compounds to be detected and was used throughout this study. It is referred to as MP2. Several met A, B and C mutants were tested and strain AB1875 (met C56), blocked at cystathionine reductase, was chosen because of its satisfactory response to methionine and homocystine and some intermediates. Both the organisms chosen for further study give sharp-edged zones of growth under appropriate conditions-an important consideration when zone diameter is to be measured. Both strains are streptomycin resistant and AB1875 requires added thiamine. Bacteria were maintained on nutrient agar slopes and were subcultured monthly. Media. The minimal media and supplements used have been described (1). The minimal medium was solidified with 1% Ionagar (Oxoid). Streptomycin (50 mg/ml) and cycloheximide (500 mg/ml) were added. Nutrient broth contained 2.5% Nutrient Broth No. 2 (Oxoid) plus 0.5% yeast extract (Difco). Chrmicds. Chemicals were obtained commercially and were not further purified. All growth factors were autoclaved, except those which were unstable to heat. These were sterilized by filtration. Prepnmtion of Assay Plates. Liquid cuItures (10 ml in 50 ml flasks) were inoculated with a loop from maintenance cultures and shaken in a water bath, at 37”. For reasons described below, AB1875 was grown in minimal broth containing methionine 100 mg/ml for 20 hours, and AT2035 was grown in nutrient broth for 15 hours. Cells were centrifuged, washed twice in 5 ml of 56/2 buffer (3) and resuspended to approximately 1.5 x lo7 cells/ml. One milliliter of this suspension was added to 140 ml of minimal medium including agar, the growth factors (other than sulphur compounds) and triphenyltetrazolium chloride (0.4
304
PATRICK
ET
AL.
mg/ml). This volume was poured into rectangular perspex trays (25 x 18 cm) on a perfectly horizontal surface, to give an even layer 2 mm deep, and allowed to set. Testing Compounds as Growth Factors. All available compounds on the pathway (Fig. 1) were tested initially by applying 10 mg in the crystalline or powder form directly on the agar surface, and incubating for 40 hours. Compounds which did not promote growth were not tested further. Those which did promote growth were made up in solutions ranging from 0.05 to 5.0 pmoles/ml, applied to blotters and allowed to dry. Discs 5 mm in diameter were punched from the dried blotters and were applied to the surface of the agar. Zone diameters were measured with dividers after 40 hours incubation at 37°C. Dose-diameter curves were plotted. Mixtures of compounds, and of pure compounds added to pooled normal urine, were also tested. Testing of Urine Specimens. Blotting paper was soaked in urine and allowed to dry. Samples on blotters were stored in envelopes at 4°C until tested and were stable for very long periods (years). Discs 5 mm in diameter were applied to the agar, and zone diameters were measured after 40 hours incubation at 37°C. Every plate included discs of appropriate standards and of pooled normal urine. Discs were placed at least 2 cm away from the edges of the plate, and 3.5 cm apart. Plates were discarded if the zones around the standard discs deviated by more than 1.5 mm from the expected diameter or were of unusual appearance. Urine concentration was estimated by measurement of the a-amino nitrogen content using a spot test especially developed for the purpose (3). The color developed in a disc of dried urine-soaked blotter treated with ninhydrin was compared after 8 minutes with the color in discs of standards containing 10 to 500 mg/ml of an equimolar solution of alanine and glycine. This method showed no systematic error when compared with estimation of a-amino nitrogen in liquid samples and a sufficient degree of precision which was constant throughout the range of concentrations found in urine. zone diameter log,, (Y amino nitrogen concentration Bioautography. Plates were prepared as described already. Urine was subjected to HVE at pH 1.9 (3) or to chromatography, each sample being run in two adjacent lanes. After drying the lanes were cut out and one was stained with ninhydrin. The other was placed on the agar surface for 10 minutes and then removed, after which the plate was incubated as usual. Zones of growth were matched against the ninhydrin stained bands using added standard compounds for final identification.
SCREENING
TEST
,3 /
/
FOR SULPHUR-CONTAINING
METABOLITES
30s
P!ETRIONII?ZSULUIIOXIDE
RETHIOIJINE e
0 4 S-ADZNOSYL?ZSTHIONINE
-1 \ S-ADENOSYLHOXOCYSTEINE \ \\ 4 HOEIOCYSTEINEf--J \ 4 I AT 1875 CYSTATBIONIXE
?1.’ 1
.B
I
,a / SULPHIDE
/ /
CYSTEINE c----j
CYSTINE
/ I \L CYSTEINE SULPIIINATE
n
I
KP2 (?)
/
I
SULPEITE + I I
XYPOTAURINE
v.P2 (?)
YIN, CYSTEIC ACID
3-SULPSINYL PYRUVATE
I PROSPHO-ADENOSINE 5' SULPHATOPIiOSPHATE I ? I
SULPHITE
I ADZNINE 5' SULPHATOPHOSPZATE -. . .
. . . .c 1 SULPHATE
FIG. 1. Simplified pathway of metabolism of sulphur containing compounds in man (4) and bacteria (. .). Known enzyme defects in man are shown: A, homocystinuria. B. cystathionuria, C. sulphite oxidase deficiency. The names of the bacterial mutants are placed next to the step in which they are deficient.
306
PATRICK
ET
AL.
Urine Specimens. Urine samples were obtained day from 120 newborn babies
at varying times of
51 babies aged l-24 months 85 school boys aged 11-13 years 70 university students aged 19-24 years 165 pregnant women at varying stages of gestation 2050 mentally retarded children from the Children’s Training Centre, Kew 147 severely mentally retarded children from St. Nicholas Hospital, Carlton 195 children of low normal and borderline intelligence attending Special Schools in Victoria 636 patients from the Royal Children’s Hospital, Melbourne In addition urine samples were available from 15 patients with homocystinuria 3 patients with cystathioninuria, and 16 patients with cystinuria (homozygotes and heterozygotes) RESULTS Growth Factors. Table 1 shows the response of the two mutants to the more important compounds in sulphur amino acid metabolism. The responses were approximately those anticipated because of the proposed metabolic blocks in these strains. The results are expressed in 2 ways-the concentration of the compound yielding a 15 mm diameter zone, and the concentration necessary to be detectable when added to normal urine. (The cystine content of normal urine produces a sizeable zone on MP2.) Growth Response Curves. For all compounds tested in serial concentrations, the diameter of the zones of growth was found to be proportional to log,, concentration provided the zone diameter exceeded 10 mm. This ratio was chosen because radial diffusion from a point source is known to follow a logarithmic function. The slope of the straight lines obtained by plotting diameter against log,, concentration varied from one compound to another. The finite diameter of the disc caused distortion of the ratio at low diameters. Determination of Optimal Technique. The effects of numerous variables upon zone size and clarity were investigated. The depth of the medium was shown to alter zone diameter inversely (because of threedimensional diffusion of the growth factors). An even layer of agar is therefore essential and a shallow layer is desirable-2 mm proved to be the shallowest layer which could be poured evenly and reproducibly in the large trays used.
SCREENING
TEST
FOR SULPHUR-CONTAINING TABLE
GROWTH
RESPONSE
OF MUTANTS
METABOLITES
307
I TO SELECTED
Minimum concentration (~moleiml) detectable in urine
COMPOUNDS
Concentration (~moleiml) giving IS mm diameter zone
Compound
MP?
AB 1875
MP2
AB 1X7.5
L-Methionine o-Methionine L-Methionine sulphoxide L-Methionine sulphone S-Adenosyl methionine DL-Homocystine Homocysteic acid nL-Cystathionine I.-Cystine L-Cysteine D-Cystine Cysteic acid Cysteine sulphuric acid Thiosulphate (Na) Sulphite (Na) Sulphide (Na) Glutathione Taurine Penicillamine
0.5 + 0.6 + >5 0.1 + 0.3 0.5 +
0.3 + 0.4 + >S 0.3 + -
0.3 + 0.4 + >5 0.25 + 0.15 0.1
0.4 + 0.6 + 35 0.4 +
-
>5 0.4 0.15 >5 0.3
-. -
i -
-
+ >5 0.5
0.3 >5 0.8
+ -
+
-.
-
+ indicates growth factor, sensitivity not quantified. - indicates pure chemical did not promote growth.
The type of medium used to grow the inoculum and the duration of growth before preparing the medium proved to affect the zones very dramatically, especially with AB1875. Inocula in early exponential, late exponential and stationary phases of growth in both minima1 and nutrient broth were tested with both organisms. Several of these treatments gave reasonable responses with MP2, but cells at the stationary phase after growth in nutrient broth yielded the most distinct zones. Only cells at stationary phase after growth in minima1 broth gave satisfactory zones with AB 1875. Zone diameter was found to be inversely proportional to the concentration of bacteria in the medium. The clarity of the zones was influenced by washing the bacteria-two washes proved optimal. Allowing time for compounds to diffuse into the agar from the discs before incubation increased the zone size, but the clarity of the edges diminished. Addition of the redox dye triphenyltetrazolium aided the definition of the edge of the zone. Zones were distorted when discs were placed closer than 1.5 cm to the edges of the tray.
FIG. 2. Zones of growth of mutant AB1875 seen with urine samples from various patients. 1: normal urine + methionine (0.5 pmole/ml); 2 & 3: normal urine (adult); 4: normal urine (2 days old): 5-7: homocystinuria; 8: liver disease (advanced); 9: phenylketonuria (treated); 10: normal urine (9 days old); 11: liver failure; 12: patient with undiagnosed disease: 13: oculo-cerebra-renal syndrome (Lowe): 14: Wilson’s disease (hepatic); and 15:patient receivingparenteral nutrition.
SCREENING
TEST
FOR
SULPHUR-CONTAINING
METABOLITES
309
After these experiments methods were chosen to give zones of growth with very distinct margins and a diameter of approximately 12 mm around discs of normal urine. This size of zone combines optimal sensitivity and accuracy. Dose response is free of distortion by the diameter of the disc at this size and a modest increase in growth factor concentration causes an obvious increase in zone diameter. At larger diameters the logarithmic relationship means that each millimeter increase represents a larger increment in concentration. Normal urine samples Mutcrnt MP2. When the samples from normal babies and children were tested growth zones were obtained with diameters ranging from 8 to 24 mm. The zones had the annular appearance typically seen with cystine. which is known to be present in normal urine (Fig. 2). The distribution of zone diameters was skewed towards higher results. However, the ratio zone diameter: log,, (a-amino nitrogen concentration) proved to be normally distributed in all age groups. This ratio was therefore used to express all results. Results in the different groups of normal individuals were closely comparable except for the babies under 1 month (mostly under 1 week) who had significantly higher ratios than other groups (mean 7.62, SD 0.66), and pregnant women whose ratios were significantly lower (mean 6.85. SD 0.60). Combining all other subjects the mean ratio was 7.10 and the SD 0.62. Using 99% confidence limits as cut off points, ratios above 8.7 (or below 5.5) were defined as suggestive of abnormality. Mutant AB1875. This mutant showed no growth response to most urines, presumably because methionine is present in only small amounts and homocystine is absent from normal urine. The only normal urines which supported growth came from three babies less than 1 week of age. In fact only 34 of the 2569 samples tested contained growth factors, the remaining 31 samples coming from patients with diseases. Urine samples from patients
with known diseases
Cystinurin. Sixteen samples were available from persons homozygous or heterozygous for cystine-lysinuria. None supported growth of AB1875. All gave large growth zones of characteristic appearance on MP2 (Fig. 2). Ratios for three samples from proven homozygotes were 11.0, 11.3 and 13.4. The remaining samples were from proven or probable heterozygotes and yielded ratios of 8.8 to 11.7. Bioautography confirmed cystine as the principal growth factor present and revealed some cysteic acid. Honzocystinuria. Twenty-six samples from 15 patients were tested. Heavy growth zones were seen on AB1875 with samples from all un-
310
PATRICK
ET
AL.
treated cases. Results on samples from treated cases correlated well with the amount of homocystine detectable by HVE in the same sample. Bioautography showed growth over the methionine-homocystine area but separation of the compounds was inadequate to show two distinct zones. The samples from untreated cases also promoted growth on MP2, and the ratios obtained correlated quite well with the response of AB1875. The ratios ranged from 6.8 to 13.0, but were above the 99% confidence limit of normal in only 11 samples. Two untreated cases gave ratios below this limit. Cystathioninuria. All three urine samples failed to promote growth of AB1875, but produced large zones of striking appearance on MP2. The ratios were 11.3, 11.5 and 16.0. Bioautographs confirmed cystathionine as the growth factor. Other Diseases. Samples from patients with acute liver failure and with untreated Wilson’s disease promoted growth of both mutants, due to the presence of excess methionine and cystine. Glycinuria produced a low ratio in MP2 presumably because the glycine elevated the a-amino nitrogen level. Some patients with phenylketonuria showed generalized aminoaciduria at certain phases of treatment and samples obtained at these times supported growth of both mutants. Normal responses were seen with samples from children with erotic aciduria, histidinemia, arginino-succinic aciduria, cystinosis. oxalosis, tyrosinosis, untreated phenylketonuria and sarcosinemia. A large growth zone was obtained on MP2 (and no growth on AB1875) with a sample from a baby who died of degenerative brain disease. The morphological changes in the brain were similar to those described in a child with sulphite oxidase deficiency (4). There was insufficient urine to pursue the matter further and studies of the parents and healthy siblings were normal. DISCUSSION
Use of the two mutants described should provide good coverage of a wide range of sulphur containing compounds which may be excreted in urine in inborn errors of metabolism. It is unfortunate that 3-sulphinylpyruvate and hypotaurine could not be obtained for testing. MP2 responds to a very wide range of compounds, but its sensitivity in detecting abnormal metabolites is somewhat diminished by the level of cystine present in normal urine. AB1875 is conveniently specific in its response to methionine and homocystine. Expression of results as the ratio of zone diameter: log,, (o-amino nitrogen content) allows precise interpretation. The quality of the zone of growth with MP2 gives a clue
SCREENING
TEST
FOR
SULPHUR-CONTAINING
METABOLITES
31
I
to the compound present (Fig. 2) and bioautography provides confirmation. Standardization of the methodology is essential. and the optimal methods have been defined. Other methods are available for detection of many of the sulphur containing compounds. The cyanide nitroprusside test (5) will detect only cystine and homocystine. Comparative studies showed this test to be less sensitive than the bacterial method when applied to liquid urine samples and very much less sensitive for urine dried on blotter. Drying urine on blotters is extremely convenient for transport of samples to the laboratory in mass screening and it is therefore desirable to develop tests that can be applied to these samples. High voltage electrophoresis of urine on paper will detect the sulphur containing amino acids and the iodoplatinate stain is a very satisfactory method of confirming the sulphur content of ninhydrin stained bands (6). The presence of lysine, ornithine and arginine in addition to cystine aids the detection of cystinuria. Similar arguments can be advanced regarding chromatography on paper or thin-layers, although homocystine is less easily identified by these methods. A bacterial inhibition test applicable to the detection of increased levels of methionine in the blood is available (7). The argument for adding these bacterial tests in a laboratory which is already using one of the more general amino acid methods rests on its ability to detect some non-amino acid sulphur compounds. Sulphite oxidase deficiency is the only additional known disease which would be detected, but there would also be potential for recognizing new defects. The bacterial test may be more valuable to a laboratory which is depending upon bacterial inhibition methods for detecting inborn errors of metabolism. In addition, the test is sufficiently quantitative to be useful in monitoring treatment of diseases like homocystinuria. The next step is to put the test into use in a screening laboratory in order to appraise its practical value. The work capacity of the test seems satisfactory for this purpose.
ACKNOWLEDGMENTS Dr. Kathleen Hayes, Dr. D. Hagger (School Medical Service), Dr. D. B. Pitt (Kew Cottages Training Centre), and Dr. D. W. Maginn (St. Nicholas Hospital) organized the collection of the urine samples. Professor V. A. McKusick (Baltimore), Professor K. Visakorpi (Helsinki). the late Dr. B. Turner (Sydney) and Mr. H. Davies (R.C.H.) provided samples from patients with known inborn errors of metabolism. Dr. J. Camakaris and Professor A. J. Pittard assisted in the transduction of the mutants and provided other advice.
312
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ET
AL.
REFERENCES 1. 2. 3. 4. 5.
Cotton, R. G. H., Camakaris. J.. and Danks. D. M.. Biocltem. Med. 3, 326 (1970). Lennox, E. S., Virology 1, 190 (19.55). Tippett, P., Danks, D. M., and Dimech. L.. Auf. Puediat. J. 9, 297 (1973). Irreverre. F., Mudd, S. H.. Heizer, W. D., and Laster, L., Biochenz. Med. 1. 187 (1967). Fell. V., and Pollitt, R. J., in “Inherited Disorders of Sulphur Metabolism” (N. Carson and N. Raine, Ed.), S.S.I.E.M. Symposium No. 8, Churchill-Livingstone, 1971. 6. W&ken, B., Brown, D. A.. and Turner, B. Med. J. Aust. i, 1193 (1972). 7. Guthrie. R., Birth Defects Orig. Art. Series, IV(6). 92 (1968).
12, 302-3 I:! (197.5)
MEDICINE
A Screening
Test
in Urine
for
Sulphur-Containing
Using
Auxotrophic
Escherichia
co/i
Metabolites Mutants
of
K12
M. G. PATRICK, R. C. H. COTTON, D. M. DANKS, AND D. GOLDSMITH Genetics Research Unit. ROJUI Childretl’s Hospitd Research Foundation. Pnrk~ille. Victoria, Australitr 3052, md Department of Genetics. Univer.sity of Melbourrw. ParC\~ille. Victor-kc. Australia 3052 Received
December
8. 1974
A simple method is described for testing samples of urine dried on blotting paper for a large range of sulphur containing compounds. including all those found in known inborn errors of metabolism. The test uses two auxotrophic strains of Escherichin co/i K 12, and can be semiquantitative if the method is carefully standardized to optimal conditions.
In studying human inborn errors of metabolism, it seems reasonable to anticipate a disease corresponding to each enzyme in intermediary metabolism. Consequently it is desirable to produce tests for abnormal levels of all possible metabolites in blood and/or urine. The use of separate tests for each metabolite is clearly impracticable, and tests for groups of compounds must be devised. The traditional approach has grouped compounds according to the possession of chemical properties which determine characteristic staining reactions-e.g., the use of ninhydrin for the recognition of (Yamino nitrogen groups. The compounds present in body fluids have been separated by chromatography or electrophoresis and revealed by staining. Chemicals having similar radicles may or may not be metabolically related. If bacteria and humans utilize very similar metabolic pathways for synthesis and degradation, respectively, it is possible to utilize the requirements of appropriate auxotrophic mutants to detect, and to quantitate, human metabolites which can act as growth factors (1). By using two sulphur requiring mutants (AT2035 and AB1875) of Escherichia co/i K12, it has been possible to detect a wide range of sulphur containing compounds derived from methionine and/or cysteine in man. Some of these compounds are easily detected by existing techniques, but others are not. Several known inborn errors of metabolism Copyright @ 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
SCREENING
TEST
FOR
SULPHUR-CONTAINING
METABOLITES
303
can be diagnosed by this method, and wider use of the test may identify new metabolic defects in this pathway. Certain factors which influence the sensitivity and reproducibility of the method have been investigated and an optimal technique has been defined. MATERIALS
AND
METHODS
Ofgutzisms. The original strains of the mutants used were obtained from Dr. A. Taylor, by courtesy of Professor A. J. Pittard. AT2035 (cys 1324) was believed to be blocked in conversion of sulphite to sulphide. and the results obtained confirmed this belief. The additional growth requirements of this mutant for tryptophan and histidine proved inconvenient and were corrected by transduction from the prototrophic strain AB259 using phage Pl and a standard method described by Lennox (2). The transduced substrain (AT203YMP2) showed comparable and satisfactory responses to the compounds to be detected and was used throughout this study. It is referred to as MP2. Several met A, B and C mutants were tested and strain AB1875 (met C56), blocked at cystathionine reductase, was chosen because of its satisfactory response to methionine and homocystine and some intermediates. Both the organisms chosen for further study give sharp-edged zones of growth under appropriate conditions-an important consideration when zone diameter is to be measured. Both strains are streptomycin resistant and AB1875 requires added thiamine. Bacteria were maintained on nutrient agar slopes and were subcultured monthly. Media. The minimal media and supplements used have been described (1). The minimal medium was solidified with 1% Ionagar (Oxoid). Streptomycin (50 mg/ml) and cycloheximide (500 mg/ml) were added. Nutrient broth contained 2.5% Nutrient Broth No. 2 (Oxoid) plus 0.5% yeast extract (Difco). Chrmicds. Chemicals were obtained commercially and were not further purified. All growth factors were autoclaved, except those which were unstable to heat. These were sterilized by filtration. Prepnmtion of Assay Plates. Liquid cuItures (10 ml in 50 ml flasks) were inoculated with a loop from maintenance cultures and shaken in a water bath, at 37”. For reasons described below, AB1875 was grown in minimal broth containing methionine 100 mg/ml for 20 hours, and AT2035 was grown in nutrient broth for 15 hours. Cells were centrifuged, washed twice in 5 ml of 56/2 buffer (3) and resuspended to approximately 1.5 x lo7 cells/ml. One milliliter of this suspension was added to 140 ml of minimal medium including agar, the growth factors (other than sulphur compounds) and triphenyltetrazolium chloride (0.4
304
PATRICK
ET
AL.
mg/ml). This volume was poured into rectangular perspex trays (25 x 18 cm) on a perfectly horizontal surface, to give an even layer 2 mm deep, and allowed to set. Testing Compounds as Growth Factors. All available compounds on the pathway (Fig. 1) were tested initially by applying 10 mg in the crystalline or powder form directly on the agar surface, and incubating for 40 hours. Compounds which did not promote growth were not tested further. Those which did promote growth were made up in solutions ranging from 0.05 to 5.0 pmoles/ml, applied to blotters and allowed to dry. Discs 5 mm in diameter were punched from the dried blotters and were applied to the surface of the agar. Zone diameters were measured with dividers after 40 hours incubation at 37°C. Dose-diameter curves were plotted. Mixtures of compounds, and of pure compounds added to pooled normal urine, were also tested. Testing of Urine Specimens. Blotting paper was soaked in urine and allowed to dry. Samples on blotters were stored in envelopes at 4°C until tested and were stable for very long periods (years). Discs 5 mm in diameter were applied to the agar, and zone diameters were measured after 40 hours incubation at 37°C. Every plate included discs of appropriate standards and of pooled normal urine. Discs were placed at least 2 cm away from the edges of the plate, and 3.5 cm apart. Plates were discarded if the zones around the standard discs deviated by more than 1.5 mm from the expected diameter or were of unusual appearance. Urine concentration was estimated by measurement of the a-amino nitrogen content using a spot test especially developed for the purpose (3). The color developed in a disc of dried urine-soaked blotter treated with ninhydrin was compared after 8 minutes with the color in discs of standards containing 10 to 500 mg/ml of an equimolar solution of alanine and glycine. This method showed no systematic error when compared with estimation of a-amino nitrogen in liquid samples and a sufficient degree of precision which was constant throughout the range of concentrations found in urine. zone diameter log,, (Y amino nitrogen concentration Bioautography. Plates were prepared as described already. Urine was subjected to HVE at pH 1.9 (3) or to chromatography, each sample being run in two adjacent lanes. After drying the lanes were cut out and one was stained with ninhydrin. The other was placed on the agar surface for 10 minutes and then removed, after which the plate was incubated as usual. Zones of growth were matched against the ninhydrin stained bands using added standard compounds for final identification.
SCREENING
TEST
,3 /
/
FOR SULPHUR-CONTAINING
METABOLITES
30s
P!ETRIONII?ZSULUIIOXIDE
RETHIOIJINE e
0 4 S-ADZNOSYL?ZSTHIONINE
-1 \ S-ADENOSYLHOXOCYSTEINE \ \\ 4 HOEIOCYSTEINEf--J \ 4 I AT 1875 CYSTATBIONIXE
?1.’ 1
.B
I
,a / SULPHIDE
/ /
CYSTEINE c----j
CYSTINE
/ I \L CYSTEINE SULPIIINATE
n
I
KP2 (?)
/
I
SULPEITE + I I
XYPOTAURINE
v.P2 (?)
YIN, CYSTEIC ACID
3-SULPSINYL PYRUVATE
I PROSPHO-ADENOSINE 5' SULPHATOPIiOSPHATE I ? I
SULPHITE
I ADZNINE 5' SULPHATOPHOSPZATE -. . .
. . . .c 1 SULPHATE
FIG. 1. Simplified pathway of metabolism of sulphur containing compounds in man (4) and bacteria (. .). Known enzyme defects in man are shown: A, homocystinuria. B. cystathionuria, C. sulphite oxidase deficiency. The names of the bacterial mutants are placed next to the step in which they are deficient.
306
PATRICK
ET
AL.
Urine Specimens. Urine samples were obtained day from 120 newborn babies
at varying times of
51 babies aged l-24 months 85 school boys aged 11-13 years 70 university students aged 19-24 years 165 pregnant women at varying stages of gestation 2050 mentally retarded children from the Children’s Training Centre, Kew 147 severely mentally retarded children from St. Nicholas Hospital, Carlton 195 children of low normal and borderline intelligence attending Special Schools in Victoria 636 patients from the Royal Children’s Hospital, Melbourne In addition urine samples were available from 15 patients with homocystinuria 3 patients with cystathioninuria, and 16 patients with cystinuria (homozygotes and heterozygotes) RESULTS Growth Factors. Table 1 shows the response of the two mutants to the more important compounds in sulphur amino acid metabolism. The responses were approximately those anticipated because of the proposed metabolic blocks in these strains. The results are expressed in 2 ways-the concentration of the compound yielding a 15 mm diameter zone, and the concentration necessary to be detectable when added to normal urine. (The cystine content of normal urine produces a sizeable zone on MP2.) Growth Response Curves. For all compounds tested in serial concentrations, the diameter of the zones of growth was found to be proportional to log,, concentration provided the zone diameter exceeded 10 mm. This ratio was chosen because radial diffusion from a point source is known to follow a logarithmic function. The slope of the straight lines obtained by plotting diameter against log,, concentration varied from one compound to another. The finite diameter of the disc caused distortion of the ratio at low diameters. Determination of Optimal Technique. The effects of numerous variables upon zone size and clarity were investigated. The depth of the medium was shown to alter zone diameter inversely (because of threedimensional diffusion of the growth factors). An even layer of agar is therefore essential and a shallow layer is desirable-2 mm proved to be the shallowest layer which could be poured evenly and reproducibly in the large trays used.
SCREENING
TEST
FOR SULPHUR-CONTAINING TABLE
GROWTH
RESPONSE
OF MUTANTS
METABOLITES
307
I TO SELECTED
Minimum concentration (~moleiml) detectable in urine
COMPOUNDS
Concentration (~moleiml) giving IS mm diameter zone
Compound
MP?
AB 1875
MP2
AB 1X7.5
L-Methionine o-Methionine L-Methionine sulphoxide L-Methionine sulphone S-Adenosyl methionine DL-Homocystine Homocysteic acid nL-Cystathionine I.-Cystine L-Cysteine D-Cystine Cysteic acid Cysteine sulphuric acid Thiosulphate (Na) Sulphite (Na) Sulphide (Na) Glutathione Taurine Penicillamine
0.5 + 0.6 + >5 0.1 + 0.3 0.5 +
0.3 + 0.4 + >S 0.3 + -
0.3 + 0.4 + >5 0.25 + 0.15 0.1
0.4 + 0.6 + 35 0.4 +
-
>5 0.4 0.15 >5 0.3
-. -
i -
-
+ >5 0.5
0.3 >5 0.8
+ -
+
-.
-
+ indicates growth factor, sensitivity not quantified. - indicates pure chemical did not promote growth.
The type of medium used to grow the inoculum and the duration of growth before preparing the medium proved to affect the zones very dramatically, especially with AB1875. Inocula in early exponential, late exponential and stationary phases of growth in both minima1 and nutrient broth were tested with both organisms. Several of these treatments gave reasonable responses with MP2, but cells at the stationary phase after growth in nutrient broth yielded the most distinct zones. Only cells at stationary phase after growth in minima1 broth gave satisfactory zones with AB 1875. Zone diameter was found to be inversely proportional to the concentration of bacteria in the medium. The clarity of the zones was influenced by washing the bacteria-two washes proved optimal. Allowing time for compounds to diffuse into the agar from the discs before incubation increased the zone size, but the clarity of the edges diminished. Addition of the redox dye triphenyltetrazolium aided the definition of the edge of the zone. Zones were distorted when discs were placed closer than 1.5 cm to the edges of the tray.
FIG. 2. Zones of growth of mutant AB1875 seen with urine samples from various patients. 1: normal urine + methionine (0.5 pmole/ml); 2 & 3: normal urine (adult); 4: normal urine (2 days old): 5-7: homocystinuria; 8: liver disease (advanced); 9: phenylketonuria (treated); 10: normal urine (9 days old); 11: liver failure; 12: patient with undiagnosed disease: 13: oculo-cerebra-renal syndrome (Lowe): 14: Wilson’s disease (hepatic); and 15:patient receivingparenteral nutrition.
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After these experiments methods were chosen to give zones of growth with very distinct margins and a diameter of approximately 12 mm around discs of normal urine. This size of zone combines optimal sensitivity and accuracy. Dose response is free of distortion by the diameter of the disc at this size and a modest increase in growth factor concentration causes an obvious increase in zone diameter. At larger diameters the logarithmic relationship means that each millimeter increase represents a larger increment in concentration. Normal urine samples Mutcrnt MP2. When the samples from normal babies and children were tested growth zones were obtained with diameters ranging from 8 to 24 mm. The zones had the annular appearance typically seen with cystine. which is known to be present in normal urine (Fig. 2). The distribution of zone diameters was skewed towards higher results. However, the ratio zone diameter: log,, (a-amino nitrogen concentration) proved to be normally distributed in all age groups. This ratio was therefore used to express all results. Results in the different groups of normal individuals were closely comparable except for the babies under 1 month (mostly under 1 week) who had significantly higher ratios than other groups (mean 7.62, SD 0.66), and pregnant women whose ratios were significantly lower (mean 6.85. SD 0.60). Combining all other subjects the mean ratio was 7.10 and the SD 0.62. Using 99% confidence limits as cut off points, ratios above 8.7 (or below 5.5) were defined as suggestive of abnormality. Mutant AB1875. This mutant showed no growth response to most urines, presumably because methionine is present in only small amounts and homocystine is absent from normal urine. The only normal urines which supported growth came from three babies less than 1 week of age. In fact only 34 of the 2569 samples tested contained growth factors, the remaining 31 samples coming from patients with diseases. Urine samples from patients
with known diseases
Cystinurin. Sixteen samples were available from persons homozygous or heterozygous for cystine-lysinuria. None supported growth of AB1875. All gave large growth zones of characteristic appearance on MP2 (Fig. 2). Ratios for three samples from proven homozygotes were 11.0, 11.3 and 13.4. The remaining samples were from proven or probable heterozygotes and yielded ratios of 8.8 to 11.7. Bioautography confirmed cystine as the principal growth factor present and revealed some cysteic acid. Honzocystinuria. Twenty-six samples from 15 patients were tested. Heavy growth zones were seen on AB1875 with samples from all un-
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treated cases. Results on samples from treated cases correlated well with the amount of homocystine detectable by HVE in the same sample. Bioautography showed growth over the methionine-homocystine area but separation of the compounds was inadequate to show two distinct zones. The samples from untreated cases also promoted growth on MP2, and the ratios obtained correlated quite well with the response of AB1875. The ratios ranged from 6.8 to 13.0, but were above the 99% confidence limit of normal in only 11 samples. Two untreated cases gave ratios below this limit. Cystathioninuria. All three urine samples failed to promote growth of AB1875, but produced large zones of striking appearance on MP2. The ratios were 11.3, 11.5 and 16.0. Bioautographs confirmed cystathionine as the growth factor. Other Diseases. Samples from patients with acute liver failure and with untreated Wilson’s disease promoted growth of both mutants, due to the presence of excess methionine and cystine. Glycinuria produced a low ratio in MP2 presumably because the glycine elevated the a-amino nitrogen level. Some patients with phenylketonuria showed generalized aminoaciduria at certain phases of treatment and samples obtained at these times supported growth of both mutants. Normal responses were seen with samples from children with erotic aciduria, histidinemia, arginino-succinic aciduria, cystinosis. oxalosis, tyrosinosis, untreated phenylketonuria and sarcosinemia. A large growth zone was obtained on MP2 (and no growth on AB1875) with a sample from a baby who died of degenerative brain disease. The morphological changes in the brain were similar to those described in a child with sulphite oxidase deficiency (4). There was insufficient urine to pursue the matter further and studies of the parents and healthy siblings were normal. DISCUSSION
Use of the two mutants described should provide good coverage of a wide range of sulphur containing compounds which may be excreted in urine in inborn errors of metabolism. It is unfortunate that 3-sulphinylpyruvate and hypotaurine could not be obtained for testing. MP2 responds to a very wide range of compounds, but its sensitivity in detecting abnormal metabolites is somewhat diminished by the level of cystine present in normal urine. AB1875 is conveniently specific in its response to methionine and homocystine. Expression of results as the ratio of zone diameter: log,, (o-amino nitrogen content) allows precise interpretation. The quality of the zone of growth with MP2 gives a clue
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to the compound present (Fig. 2) and bioautography provides confirmation. Standardization of the methodology is essential. and the optimal methods have been defined. Other methods are available for detection of many of the sulphur containing compounds. The cyanide nitroprusside test (5) will detect only cystine and homocystine. Comparative studies showed this test to be less sensitive than the bacterial method when applied to liquid urine samples and very much less sensitive for urine dried on blotter. Drying urine on blotters is extremely convenient for transport of samples to the laboratory in mass screening and it is therefore desirable to develop tests that can be applied to these samples. High voltage electrophoresis of urine on paper will detect the sulphur containing amino acids and the iodoplatinate stain is a very satisfactory method of confirming the sulphur content of ninhydrin stained bands (6). The presence of lysine, ornithine and arginine in addition to cystine aids the detection of cystinuria. Similar arguments can be advanced regarding chromatography on paper or thin-layers, although homocystine is less easily identified by these methods. A bacterial inhibition test applicable to the detection of increased levels of methionine in the blood is available (7). The argument for adding these bacterial tests in a laboratory which is already using one of the more general amino acid methods rests on its ability to detect some non-amino acid sulphur compounds. Sulphite oxidase deficiency is the only additional known disease which would be detected, but there would also be potential for recognizing new defects. The bacterial test may be more valuable to a laboratory which is depending upon bacterial inhibition methods for detecting inborn errors of metabolism. In addition, the test is sufficiently quantitative to be useful in monitoring treatment of diseases like homocystinuria. The next step is to put the test into use in a screening laboratory in order to appraise its practical value. The work capacity of the test seems satisfactory for this purpose.
ACKNOWLEDGMENTS Dr. Kathleen Hayes, Dr. D. Hagger (School Medical Service), Dr. D. B. Pitt (Kew Cottages Training Centre), and Dr. D. W. Maginn (St. Nicholas Hospital) organized the collection of the urine samples. Professor V. A. McKusick (Baltimore), Professor K. Visakorpi (Helsinki). the late Dr. B. Turner (Sydney) and Mr. H. Davies (R.C.H.) provided samples from patients with known inborn errors of metabolism. Dr. J. Camakaris and Professor A. J. Pittard assisted in the transduction of the mutants and provided other advice.
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REFERENCES 1. 2. 3. 4. 5.
Cotton, R. G. H., Camakaris. J.. and Danks. D. M.. Biocltem. Med. 3, 326 (1970). Lennox, E. S., Virology 1, 190 (19.55). Tippett, P., Danks, D. M., and Dimech. L.. Auf. Puediat. J. 9, 297 (1973). Irreverre. F., Mudd, S. H.. Heizer, W. D., and Laster, L., Biochenz. Med. 1. 187 (1967). Fell. V., and Pollitt, R. J., in “Inherited Disorders of Sulphur Metabolism” (N. Carson and N. Raine, Ed.), S.S.I.E.M. Symposium No. 8, Churchill-Livingstone, 1971. 6. W&ken, B., Brown, D. A.. and Turner, B. Med. J. Aust. i, 1193 (1972). 7. Guthrie. R., Birth Defects Orig. Art. Series, IV(6). 92 (1968).
