INDIAN JOURNAL o F PEDIATRICS r
~
Vol. 44
February, 1977
SCREENING F O R METABOLIC DISORDERS AMINOACIDOPATHIES*
No. 349
IN C H I L D R E N -
O . S . REDDI
Hyderabad It has become imperative to screen newborn children for aminoacidopathies in view of the accumulation of aminoacids in serum and consequent cause of mental retardation. T h e latest technology to screen these disorders is available in this country. I t is necessary that large scale screening is undertaken in the newborn so as to determine the component of genetic morbidity. Secondly, it would also contribute to the discovery of new aminoacidopathies. Garrod (1908) was the first to introduce the concept of inborn errors of metabolism in the beginning of the 20th century At that time only two inborn errors of metabolism were known, namely, alcaptonuria and cystinuria. O v e r 60 inborn errors of aminoacid metabolism are now recognisable. Some are associated with disease and some are not. The steady i m p r o v e m e n t of diagnostic precision and the broadening of the disease spectrum have promoted more concern for the role and t r e a t m e n t of inborn errors of aminoacid metabolism. Theoretical principles were first applied to the treatment of *From the Department University, Hyderabad-7.
of
Genetics,
Osmania
phenylketonuria (PKU) in the early 1950's. An element of success awakened interest in the value of early diagnosis in these rare patients with inborn errors a m o n g the thousands of normal births, who could be benefitted from specific diagnosis and treatment. Almost all inborn errors of metabolism are recessives defined in the strict Garrodian sense. In recessive disorders an enzyme or a defect in the peptide hormone such as growth hormone should be sought. In dominant disorders abnormality in ' a non-enzymatic protein is more likely. Theoretically, change in the specificity of enzyme might be the result of mutation, so that it acts on a substrate it ordinarily would not touch. T h e enzymatic disorder resulting from such a mutation might behave as a d o m i n a n t ( K i r k m a n 1970). T w o dominant disorders whose biochemical bases are now known are due to increased activity of phosphoribos~,l phosphate synthetase (PRPP) (Becker st al. 1973 a, b) and hyperlipoproteinaemia type II (McKusick 1975). I n bacteria, noninducible mutations involve a change in repressor such that its affinity for an inducer substance is lost and repression is main-
26
VoL. 44, No. 349
INDIAN J O U R N A L OF PEDIATRICS
tained. Again, the enzyme deficiency would be expected to behave as a dominant. In over 140 of the nearly 500 certain recesslves, an enzyme deficiency has been demonstrated.
Frequency of hereditary aminoacidopathies Too little
is known about the real
frequency. Considerable variation in frequency between geographic regions and ethnic groups occurs. Table 1 indicates approximate frequencies. The incidence of certain aminoacldopathies among newborn infants in a Massacheusetts hospital is shown in Table 2.
Table 1. Approximatefrequency of some hereditary aminoacidopathies (Sc*iver et al. 1978).
Phenotype
Apparent frequency per 100,000 live births
Region or Population
Argininosuccinicaciduria
0.4
N.E.N. America
Branched-chain keto aciduria (s)
0.3
General
Cystlnuria
7
General
Fanconi syndrome
0.4
N.E.N. America
Hartnup disease
4
General
Histidinaemia
4
General
Iminoglycinuria
5
General
Hyperlysinaemia
0.4
N.E.N. America
Hypermethioninaemia (with homocystlnuria)
0.4
General
Hyperprolinaemia
2
General
Phenylketonuria
8
General
Hyperphenylalaninaemia
3
(PKU rare in Ashkenazi Jews)
Tyrosinaemia (persistent}
< 0.1 30
General Isolate of French Canadians in Quebec
REDDI--SCREENING FOR METABOLIC
T a b l e 2.
27
D I S O R D E R S IN C H I L D R E N
Incidenceof aminoaddopathies among newborn znfants in Massacheusett~ (Levy 1974).
Number
Incidence
Estimated frequency of heterozygotes (per 1000)
D isor d er
Total screened
Phenylketonuria
981,361
67
1: 15,000
16
Cystinuria
332,143
21
1: 16,000
16
Hartnup disease
332,143
18
1 : 18,000
15
Histidinaemia
332,143
18
1: 18,000
15
Argininosuccinic acidaemia
332,143
5
1: 70,000
8
Galactosaemia
550,000
5
1:110,000
6
Maple-syrup-urine disease
842,004
5
1:170,000
5
Cystathionaemia
332,143
3
1:110,000
6
Homocystinuria
449,615
3
I: 150,000
5
Hyperglycaemia (non-ketotic)
332,143
2
1 : 170,000
5
Propionic acidaemia
332,143
i
< 1:300,000
3
Hyperlysinaemia
332,143
1
< 1:300,000
3
Hyperonnithaemia
332,143
1
< 1:300,000
3
Fanconi syndrome
332,143
1
< 1:300,000
3
Why screening is essential
mutation rate at the relevant gene locus.
T h e effect of relaxed selection on the frequency of mutant alleles in the future is also of concern ( W H O Technical report 1972). I f complete ascertainment is achieved now it will be possible to monitor its frequency over future generations. In this way, we may also be able to detect changes in frequency which reflect variation in the
From these data we may detect environmental factors influencing the mutation rate. This obvious and pertinent application as well as the long range perspective suggest why screening and surveillance of amino acid disorders in the population has grown in relevance.
28
INDIAN ] O U R N A L OF PEDIATRICS
T h e W H O (Technical R e p o r t 1968) has issued two instructive technical reports which discuss the practice and impact of screening for hereditary metabolic diseases including disorders of amino acid metabolism. These reports recommend that pilot studies be carried out to gather information. Test Procedures
The screening procedure has the primary objective (Wilson and Jungner 1968) to provide an opportunity for early detection o f such individuals for prospective appropriate action. Screening for aminoacid diseases can be done at four primary levels (Scriver I965): (a) T h e genetic materiaI ( b ) T h e gene product modified by the mutation (c) Cellular function or metabolic equilibrium which is controlled by the gene product (d) The disease caused by the perturbation
VOL. 44, No. 349 are responsible for the amount of protein in the cell. However, there is no firm evidence at the present time to indicate the presence of regulatory genes influencing aminoacid metabolism in human cells. Nonetheless, the amount of enzyme activity at some stage of aminoacid metabolism may be altered if the mutation affects the structure of the enzyme in such a way that its turnover in the ceil has been altered. One can visualise that the degradation rate, in the presence of a constant rate of enzyme synthesis, could be increased or decreased so that the amount of enzyme would be diminished or augmented respectively. The amount of enzyme in the cell could also be affected if the final form of enzyme reflects genes acting in sequence.
Screening at the first level is not practical at present although mapping of the human genome by in vitro cell hybridization techniques can be anticipated (Ruddle et al. 1972). The pedigree analysis will yield eventually useful information for genetic counselling under certain circumstances. T h e present discussion is limited to the remaining there levels of screening.
For instance, if protein formed under the influence of one gene is modified by the addition of side groups through the action of a second enzyme acting on the first, then mutation at the gene locus responsible for the second enzyme could prevent synthesis of the final form of the first enzyme. This could be reflected by a diminished quantity of the first enzyme. Alternatively, if the final form of the first enzyme is comprised of sub-units, or if it is a multi-enzyme complex, then mutation affecting synthesis or structure of one of the components of the final enzyme complex could alter the amount of the intact enzyme.
Screening for the gene product (enzyme phenot~,pe) T h e gene product whether it is an enzyme or other cellular protein will usually be altered in some way when the mutation is expressed so that either the primary structure of the protein or the amount of protein is affected. It has been customary to think that regulatory genes
Enzyme screening requires either that enzyme be present in an active soluble form in the body fluids or that the tissue source of an enzyme is available to simple and effective biopsy. T h e test requiring biopsy of tissues is not compatible with the principles of mass screening though it is useful in the investigation of a single patient or a pedigree.
of cellular function.
RIeIDDI -- s C R E E N I N G
t'OR METABOLILI DISOllDERS
29
IN GI-IILDREN
Screenine for metabolic imtm/a~wc (chemicql phenoO'f~e)
cultured amniotic fluid c~,lIs can be dime in a few cases also.
If'the gene product (enzyme) regulates a metabolic pathway or transport fimction, alteration in the function of the enzymes or transport site m a y cause imbalance between a precursor and product stage of the pathway or transfer and this will declare itself either as an accumulation of the substrate or a deficiency of the product of reaction catalysed by the mutant enzyme. Further accumulation or depletion of metabolites may lead to secondary or tertiary level of imbalance which may be identified by the appropriate screening test. For example, phenylalnine hydroxylase deficiency (gene product level) causes accumulation of phenylalanine and its metabolite phenylpyruvic acid (metabolite level). The phenylalanine accumulation secondarily causes serotonin and catecholomine depletion and inhibition of melanin synthesis.
Diagnosis of amznoacidopathtes
Aminoacidopathies have been classified by Dent and Walshe (1954) as high threshold (prerenal) and low threshold (renal) conditions which affect only the membrane transport of an aminoacid and are most appropriately detected by urine screening where large changes in concentration will occur. Dent (1951, 1957) argued that for the general purpose of diagnosis, the urine is the ideal fluid to mirror the state of aminoacid metabolism.
Clinical diseaJe (disease phenoO~e) It is not desirable to screen for the presence of disease since it may be too late to help the patient. The objective of screening is to prevent pathological change or at least to recognise the disease at its incipient stage. Prenatal diagnosis by
Diagnosis may be arrived at rationally by the application of investigative methods. The differential diagnosis of the disorder of the aminoacid metabolism is extensive. The procedure for investigating a patient with a suspected anfinoacidopathy is discussed below. The problem of diagnosis may arise at any time in the patient's lifetime such as an emergency or incidentally during the course of other investigations.
Ch~,ical aids 1. Historr.--(a)
Environment, including toxic drugs, foods which may cause excretion or accumulation of amino acids o1" their byproducts.
(b) Family consanguinity, origin of parents, health in bibs, parents and collateral branches. The proband clinical signs.
may provide helpful
2.
Odour of the urine can be diagnostic of aminoacidopathies (Gone 1968) such as, a musty odour, maple syrup odour, Oast-house odour, fish or cabbage like odour, fishy odour, cheesy odom.
3.
Colourof urine. It darkens on exposure to air, light and alkalis in alcaptonuria and is indigo blue in the blue diaper syndrome.
4.
Hair r
Hair colour is diagnostic in some aminoacidopathies (Rook 1969). The pigmentation is diluted in P K U and the Chediak-Higashi syndrome. Unusually fair hair is seen in patients with Oast-house urine disease, methionine malabsorption and homo-
,~0
INDIAN JOURNAL OF PEDIATRICS
cystinuria. In cystinosis the hair is straw-like and the pigment is yellow. 5.
Eyes. Useful hints are seen in the eyes in certain conditions (Cogan 1966). Cystine crystals are found in the cornea in three different types of cystinosis (infantile, nephropathic and adolescent nephropathic) whereas retinopathy is found only in the infantile type. Scleral pigmentation occurs in alcaptonuria and depigmentation of the liver is seen in two forms of albinism ('generalised', autosomal recessive, and 'ocular' X linked).
Downward lenticular dislocation occurs in homocystinuria due to cvstathionine synthase deficiency. A Kayser-Fleiseher ring in \u disease, cataracts in galactosaemia due to deficient galactokinase or uridyltransferase activity may signal primary disease accompanied by a generalised di sturbance of aminoacid metabolism. Chemical tests for screening A series of simple chemical tests can yield considerable information. For example, an infant in the second half of his first year of life, with pale hair, polyuria and a recent history of weight loss, can be
given a tentative diagnosis of the Fanconi syndrome if the urine contains glucose, a cyanide-nitroprusside positive substance and a heat soluble, low molecular weight protein. Should inspection of the patient's eyes with the-~- 40 diopter lens of the ophlhalmoscope under lateral illunination reveal crystals in the cornea, the diagnosis of cystinosis would be highly probable and the preliminary work-up would have taken ,only 10 minules.
VoL. 44, No. 349 Methodology. Perry et al. (1968), Berry tt al. (1958), Buist (1968), Snyderman (1971), Tocci (1967), O'Brien (1965) and Seriver etal. (1973) provide usefid screening tests. One dimensional partition chromatography (Eft-on el al. 1964) and electro-chromotography (Efron 1959) are methods preferred for further investigations. It should be noted lhat sugar chromatography should always be done on the urine sample that shows a positive test. A negative ferric chloride test does not exclude hyper-phenylalaniemia in the newborn because the conversion of phenylalanine to phenylpyruvic acid may be impaired at this age.
A positive 2, 4-dinitrophenyl-hydrazine (DNPH) test may only indicate ketonuria and the acetone test will be positive. But ketosis may occur in several diseases of aminoacid metabolism, and if the 3,4DNPH test remains positive after heating the urine to get rid of acetone, then the possibility of an aminoacidopathy should be investigated. In this situation gas chromatograph:/ of the urine may reveal one of the unusual organic acids.
Techniques Over the past few years wide scale screening of patients with mental retardation or disease of unknown origin have demonstrated many new inborn errors of metabolism. Most of these diseases are inherited in a recessive manner. They are usually caused by the absence or inactivity of a specific enzyme, required for normal metabolic activity. The biochemical disturbance produced by the missing enzyme may be severe enough to cause serious symptoms in the newborn period or be so minor
N E D D I - - S C R E E N I N G FOR M E T A B O L I C DISORDERS IN C H I L D R E N
that no clinical symptom can be attributed to it. It is now apparent, moreover, that many of these diseases have varying clinical manifestations and may occur in patients who appear quite normal at the time of examination. Patients suffering fi'om homocystinuria may vary from the abnormal phenotype of a mentally retarded patient with the picture of Marfan's syndrome to an adult of normal appearance and intelligence. Nonetheless, so far as can be judged by present knowledge, both these patients seem to be equally at risk in terms of developing severe visual disturbance through dislocation o f | h e lenses and early death through an abnormal clotting mechanism or a dissecting aneurysm of the aorta. It has been recently shown that very high plasma levels of phenylalanine m a y occur in early life and yet produce no clinical signs of phenylketonuria. It seems likely that several different types of phenylketonuria may exist. For the most part it is not known why apparently identical disturbances should have such widely varying clinical manifestationsThe clinical and biochemical abnormalities of these groups of diseases are not usually detectable until after birth, presumably because prenatally, accumulation of any abnormal metabolites is cleared through the mother circulation. In view of the fact that many of these diseases cause severe damage, it is important to detect affected patients in early life in order to develop further effective remedies and to study the effect of such therapy on the subsequent course of the disease. It is equally important that methods should be available to detect heterozygotes or asymptomatic homozygotes in order that the natural history of such metabolic
31
diseases can be elucidated and appropriate therapeutic measures and genetic counselling provided for such patients. At present it is not practical that all potential patients can be subjected to the detailed biochemical tests necessary to establish a precise diagnosis. This is particularly true since there is no knowledge of the true incidence of many metabolic disorders in the normal population. More or less, by the selection of patients whose symptoms suggest the possibility of underlying metabolic disease, and the use of a set of simple chemical tests, it is often possible to detect patients who require full biochemical evaluation and whose symptoms may be cured by appropriate therapy. A list of the conditions which appear to be most commonly associated with metabolic abnormalities is shown in Table 3. A number of clinical tests recommended to screen aminoacidopathies are shown in T a b l e 4. All these tests with slight modifications have been found extremely useful in our daily routine investigations (Reddi 1976). In addition to simple clinical tests, the quantitative estimation of aminoacids from urine and serum samples of the patients is extremely important as confirmatory tests. For this purpose both the high voltage electrophoresis followed by chromatography (Efron 19591 and only chromotography (Sherma and Zweig 1971) techniques have been found to be most useful. In this laboratory we have been carrying on extensive screening of aminoacidopathies with the above technology
32
INDINJOURNALOF PEDIATRICS T a b l e 3.
VOL. 44, No. 349
Conditions in which metabolic errors should be suggested ( Buist 1968) Type
Mental retardation Psychiatric illness Failure to thrive or dwarfing Unknown diagnosis Unexplained deaths at any age Relatives of patients with known metabolic diseases Food intolerance and distaste for protein
Renal calculi Cataracts and dislocated lenses Bone disease of uncertain origin Liver disease of uncertain origin in speech disorders
Marfan's syndrome Speech disorders TESTS Hair coiour Diluted pigment Absence of pigment Unsually fair hair Straw-like, yellow pigment /~.t'es Cysline crystals in cornea Scleral pigmentation Downward lenticular dislocation Kayser-Fleischer ring Cataracts
Cause Many possible causes
Fructosemia Disorders of urea cycle Dissacharidase deficiencies Isovalerie acidaemia Cystinuria Xanthinuria Disorders of uric acid metabolism Diabetes Homocystinuria Galactosaemia Mucopolysaccharidoses Fanconi syndrome Galactosaemia Tyrosinosis Mycopolysaccharidoses Glycogen storage disease Homocystinuria Histidinaemia
Phenylketonuria Chediak-Higashi syndrome Albinism Oast-house urine disease Methionine malabsorption Homocystinuria Cystinuria Cystinosis (infantile nephropathic adolescent Adult non-nephropathic) Alcaptonuria Homocystinuria Wilson's disease Galactosaemia (Contd.)
R E D D I ~ S C R E E N I N G FOR METABOLIC DISORDERS IN CHILDREN
35
Table 3 - - (Contd.)
Odour of urine Odour
Compound
DiSSEIS~
Musty or mousy Maple syrup sweetish caramel, curry Sour berry reminiscent of a brewery, cabbage like
Phenylacetic acid Branched chain ketoacids aketobutyric acid
Methionlne malabsorption
Sulphurous
Hydrogen sulphide
Cystinuria
Phenylketonurla Maple syrup urine disease
Homocystinuria Sweaty feet or cheesy
Isovalerie acid
lsovaleric acidaemia
Cheesy, sweaty feet
Butyric acid, hexanoic acid
Green acyldehydrogenase deficiency
Oast- house, hop-llke
ahydroxybutyric acid (and perhaps other substances)
Oast-house urine syndrome
Fishy or cabbage like
~ketobutyrate or eketo e-methiobutyrate
Hypermethioninaemia
Powerful fishy
Trimethylaminuria
Cabbage like, fishy
Tyrosinaemia
Colour of urine Darkens on standing from surface down on exposure to air, light and alkali
Alcaptonuria
Indigo blue Crystals Cystine crystals
Blue-diaper syndrome
Tyrosine crystals
Liver failure
Orotic acid crystals
Hyperammaninaemia
Cystinuria (s)
(OCT deficiency)
Turbidity Add 20% ( W ) sulphosaHcylic acid V Protein clears with heat-No clearing with heat--
Tubular disease Glomerular (with or without) disease
34 INDIANJOURNALOF PEDIATRICS Table 4. Tests 1. Benedict's
VOL. 44, No. 349
Clinical tests fot aminoacidopathiees Positive
Glucose, in Fanconi syndrome (s) Homogentisic acid, in alcaptonuria p-hydroxyphenylpyruvate, in tyrosinaemia
Remarks
False positive 1. Glucose in diabetes mellitus, renal glycosuria, renal tubu, lar dystrophies, cystinosisLowe's syndrome, vitamin D resistant rickets (Dent type II) 2. Fructose in fructosaemia (aldolase deficiency). Essential fructosuria 3. Galactose in galactosaemia and variants, galactokinase deficiency 4. Lactose in lactase deficiency congenital or acquired 5. Xylulose in pentosuria 6. Phenols in phenylketonuria, tyrosinosis, tyrosine transaminase deficiency
2.
Ferric chloride reaction --phenylpyruvate (blue green), in phenylketonuria and variants --p-hydroxyphenylpyruvate (transient blue green), m tyrosinaemia --imidazolpyruvate (grey-green), in bistidinaemia --a ketobutvrate (purple-red-brown), methionine malabsorption --homogentisic acid (transient blue-green), in alcaptonuria --Pyruvic acid (yellow), in hyper alaninemia --xanthurenic acid (green-brown), in xanthurenic aciduria - - e ketoisovalerlc acid (blue)maple syrup urine disease --~ ketone isocapric acid (yellow), maple syrup urine disease - - e ketomethylvaleric acid (blue-green), maple syrup urine disease
(Contd,
REDDI--SCREENING
F O R M E T A B O L I C DISORDERS IN C H I L D R E N
Table 4.
Test
35
(continued)
Positive
Remarks ii
Other causes
--Lactic acidosis (grey) --Pyruvic acidaemia (yellow) --Pyridoxine disorders, kynurinase deficieny (immediate brown) --Aceto acetic acid (brown red) --Melanin, isonicotine acid hydrazide (grey precipitate) --P.A.S., salicylates (purple) --Phenols, phenothiazines (purple brown) --Vanilic acid (red/brown-mauve) -Conjugated billirubin (blue green) . Cyanide-nitroprusside test --Cystine, in cystinuria (s) (classical and variants) --I-Iomocystine, in homocystinuria (cystathionlne synthasedeficiency) --Cystein~-homocysteine disulfide, in cystinuria and homocystinuria --P-mercaptolactate-cysteine disulfide, in defect of its metabolism --Penicillamine-cysteine disulfide, in treatment of cystinuria --Glutathione, in glutathionlnaemia Urine should not be acidic lest false negative results . Dinitrophenyl hydrozine (ketoacids) --Phenylpyruvic acid in phenylketonuria --Branched chain ketoacids in maple syrup urine disease --P-hydroxyphenyl in tyrosinosis --Pyruvic acid in tyrosyluria --Imidazole pyruvic acid in histidinemia - - a ketobutyric acid in methlonine malabsorption (Oast-House syndrome)
36
INDIAN JOURNAL OF
PEDIATRICS
VOL. 44, No. 349 Table
Test 5.
(continued)
4.
Positive
Remarks
Nitrosonpathol test (Tyxosine metabolites) - - O r a n g e red colour in tyrosine transaminase deficiency --Tyrosinosis (tyrosinemia) - - T r a n s i e n t tyrosinosis of newborn --Tyrosiluria of scurvy
6.
Isatin test for proline
7.
Ninhydrin test for excessive amino aciduria
which useful.
has
been
found
to be
highly
Urine microscopy Microscopic examination of the urine also gives an insight into cellular pathology and metabolic disorders. The test should be performed on fresh urine samples otherwise the renal cells may be destroyed. Metabolic diseases which may be associated with microscopic abnormalities are shown in Table 5. These tests except urine microscopy for cellular abnormaiities and Ehrlics' aldehyde for porphobilinogen can be performed on stored urine; samples may be kept in the refrigerator for a week. But if the tests are to be delayed for longer periods, the specimen should be frozen. It is our policy to perform the tests initially on random urine samples. When an abnormal result is found a further sample is obtained for repeat testing. When the second test is abnormal the results are confirmed by more refined techniques on accurately timed samples.
References
Becker, M.A., Messer, L.T., Seegmillen, J.E. (1973a). Gout with purine overproduction due to increased phosphoribosyl phosphate synthetase activity. Am. 07. Med. 55, 232. Becket, M.A., Kostel, P.H., Messer, L.T. and Seegmillen, I.E. (1973 b). Human phosphoribosyl phosphate synthetase. Increased enzyme specific activity in gout, and excessive purine synthesis. Proc. Nat. Acad. Sci 70, 2749. Berry, H.K., Leonard C., Peters, H., Granger~ M. and Chunekanrai, N. (1968). Detection of metabolic dimrders. Chromatographic procedures and interpretation of resuls. Clin. Chem. 14, 1033. Buist, N R.M. ~1968). Set of simple side-room urine tests for detection of inborn errors of metabolism. Brit. Med. 07. 2, 745. Cogan, D.G. (1966), Occular correlates of inborn metabolic defects. Can. Med. Assoc. ,7. 95, 1055. Cone, T.E Jr. (1968). I Jiagnosls and treatment: Some diseases, syndromes and conditions associated with an unusual odour. Pedmtrics, 41,993. Dent, C.E. (1951). Recent advances in clinical pathology In Dyke, S.C , Ed., 2nd edit. Biakistan, Phila. 238-258. Dent, C E. (1957). Clinical applications of amino acid chromatography. Scand.,~. Clin. and Lab. Inves . (supp. 32) 10, 122.
REDDI--SCREENING
FOR
METABOIIC
DISORDERS
IN
CHILDREN
~
~D r
c~
~o
c~ ~0
~D
.2 O
>-.
~ r
O
.,.~
f:L
~
~
o
r
o~
c~
~
r
~9
Q9
~ ~9
a~ O
~9 r
o,3
~
c~ ~9
38
INDIAN JOURNAL OF PEDIATRICS
VOL, 44,
N o . 349
Dent, C.E. and Walshe, J.M. (1954). Aminoacid metabolism. Brit. Med. Bull 10,247. Efron, M.L. (1959). Two-way separation of amino acids and other ninhvdrin reading substances by highvoltage electrophoresis followed by paper chromatography. Biochem. j . 72, 69i. Efron, M.L., Young, D., Moser, H.W. and Mac Cready, R.A. ~1964). A simple chromatographic screening test for the detecion of disorders of amino acid metabohsm. A technique using white blood or ~.lrine collected on filter paper. New Eng. J. Med. 270,
Ruddle, F., Riccuti, F., McMorria, F.A., Tischfield, J., Greegan, R., Dalington, G. and Chen, T. (1972). Somatic cell genetic assignment of peptidase C and the Rh linkage group to chromosome A-1 in man. Science, 176, 1429.
1376. Garrod, A.E.
Sharma, J. and Zweig Gunter (1971). Paper Chromatography. Vol. II. Academic Press, New York and London. Synderman, S.E. (197l). Diagnosis of metabolic disease. Pediat. Clio. N.A. 18, 199. Tocci, P.M. (1967). The biochemical diagnosis of metabolic disorders by urinanalysis and paper chromatography. In Aminoacid Metabolism and Genetic Variation (Ed. W.L. Nyhan) Mc Graw-Hill Book Pub., 161, 490. Udenfriend, S., Stein, S., Hohlen, P., Diagman, W., Leingruber, W. and Weegele, M. (1972). Fluorescamine. A reagent for assay of amino acids, peptides, proteins and primary amines on the picomole range. Science, 178, 871. Wilson, J.M.G. andJungner, G. (1968). Principles and practice of screening for disease. Public Health paper 34, World Health Organization, Geneva. World Health Organization, Technical Report series 40, Screening for inborn errors of metabolism. Geneva, 1968. World Health Organization Technical Report Series, 497. Genetic Disorders, Prevention, Treatment and Rehabilitation. Geneva. 1972.
0908).
The
Croonien lectures.
Lancet ii, 73, 142. Kirkman, H.N (1970). Dominant mutations. Biochemical basis of phenotype. In F.CI. Fraser and V.A. McKusick Eds: Congential malformations Excerpta Medica. Amsterdam. Levy, H.L. (1973). Incidence ofaminoacidopathles among newborn infants in Massachusetts. Bull. Pub. Hlth. Massachusetts Dept. of Public Health McKusick, V. (1975). Mendelian inheritance in man. The John's Hopkins University Press. Baltimore and London. O'Brien, D. (1965). Rare inborn errors of metabolism in children with mental retardation. Part B. Technical procedures, Children's Burea publ. No. 4291965, U.~ ). Dept. U.S. Govt. Printing O~ice, Washington I).C., P. 70-100. Perry, T.L., Hansen, S. and MacDougall, L. (1966) Urinary screening tests in the prevention of mental deficiency. Caoad. Med. Assoc. 07.95, 89. Reddi, O.S. (1976). Unpublished. Rook, A. (1969). Hair colour in clinical diagnosis. Indian. 07. Med. So. 7, 415.
Scrlver, C.R. (1965). Screening newborns for hereditary metabolic disease. Ped. Clin. N. Ame.r 12, 807, Striver, C.R., Clow, C.L. and Lamm, P. (1973). On tlae screening, diagnosis and investigations of hereditary aminoacidpoathies. Clio. Bio~hem. 6, 1t2, 188.
~
Vol. 44
February, 1977
SCREENING F O R METABOLIC DISORDERS AMINOACIDOPATHIES*
No. 349
IN C H I L D R E N -
O . S . REDDI
Hyderabad It has become imperative to screen newborn children for aminoacidopathies in view of the accumulation of aminoacids in serum and consequent cause of mental retardation. T h e latest technology to screen these disorders is available in this country. I t is necessary that large scale screening is undertaken in the newborn so as to determine the component of genetic morbidity. Secondly, it would also contribute to the discovery of new aminoacidopathies. Garrod (1908) was the first to introduce the concept of inborn errors of metabolism in the beginning of the 20th century At that time only two inborn errors of metabolism were known, namely, alcaptonuria and cystinuria. O v e r 60 inborn errors of aminoacid metabolism are now recognisable. Some are associated with disease and some are not. The steady i m p r o v e m e n t of diagnostic precision and the broadening of the disease spectrum have promoted more concern for the role and t r e a t m e n t of inborn errors of aminoacid metabolism. Theoretical principles were first applied to the treatment of *From the Department University, Hyderabad-7.
of
Genetics,
Osmania
phenylketonuria (PKU) in the early 1950's. An element of success awakened interest in the value of early diagnosis in these rare patients with inborn errors a m o n g the thousands of normal births, who could be benefitted from specific diagnosis and treatment. Almost all inborn errors of metabolism are recessives defined in the strict Garrodian sense. In recessive disorders an enzyme or a defect in the peptide hormone such as growth hormone should be sought. In dominant disorders abnormality in ' a non-enzymatic protein is more likely. Theoretically, change in the specificity of enzyme might be the result of mutation, so that it acts on a substrate it ordinarily would not touch. T h e enzymatic disorder resulting from such a mutation might behave as a d o m i n a n t ( K i r k m a n 1970). T w o dominant disorders whose biochemical bases are now known are due to increased activity of phosphoribos~,l phosphate synthetase (PRPP) (Becker st al. 1973 a, b) and hyperlipoproteinaemia type II (McKusick 1975). I n bacteria, noninducible mutations involve a change in repressor such that its affinity for an inducer substance is lost and repression is main-
26
VoL. 44, No. 349
INDIAN J O U R N A L OF PEDIATRICS
tained. Again, the enzyme deficiency would be expected to behave as a dominant. In over 140 of the nearly 500 certain recesslves, an enzyme deficiency has been demonstrated.
Frequency of hereditary aminoacidopathies Too little
is known about the real
frequency. Considerable variation in frequency between geographic regions and ethnic groups occurs. Table 1 indicates approximate frequencies. The incidence of certain aminoacldopathies among newborn infants in a Massacheusetts hospital is shown in Table 2.
Table 1. Approximatefrequency of some hereditary aminoacidopathies (Sc*iver et al. 1978).
Phenotype
Apparent frequency per 100,000 live births
Region or Population
Argininosuccinicaciduria
0.4
N.E.N. America
Branched-chain keto aciduria (s)
0.3
General
Cystlnuria
7
General
Fanconi syndrome
0.4
N.E.N. America
Hartnup disease
4
General
Histidinaemia
4
General
Iminoglycinuria
5
General
Hyperlysinaemia
0.4
N.E.N. America
Hypermethioninaemia (with homocystlnuria)
0.4
General
Hyperprolinaemia
2
General
Phenylketonuria
8
General
Hyperphenylalaninaemia
3
(PKU rare in Ashkenazi Jews)
Tyrosinaemia (persistent}
< 0.1 30
General Isolate of French Canadians in Quebec
REDDI--SCREENING FOR METABOLIC
T a b l e 2.
27
D I S O R D E R S IN C H I L D R E N
Incidenceof aminoaddopathies among newborn znfants in Massacheusett~ (Levy 1974).
Number
Incidence
Estimated frequency of heterozygotes (per 1000)
D isor d er
Total screened
Phenylketonuria
981,361
67
1: 15,000
16
Cystinuria
332,143
21
1: 16,000
16
Hartnup disease
332,143
18
1 : 18,000
15
Histidinaemia
332,143
18
1: 18,000
15
Argininosuccinic acidaemia
332,143
5
1: 70,000
8
Galactosaemia
550,000
5
1:110,000
6
Maple-syrup-urine disease
842,004
5
1:170,000
5
Cystathionaemia
332,143
3
1:110,000
6
Homocystinuria
449,615
3
I: 150,000
5
Hyperglycaemia (non-ketotic)
332,143
2
1 : 170,000
5
Propionic acidaemia
332,143
i
< 1:300,000
3
Hyperlysinaemia
332,143
1
< 1:300,000
3
Hyperonnithaemia
332,143
1
< 1:300,000
3
Fanconi syndrome
332,143
1
< 1:300,000
3
Why screening is essential
mutation rate at the relevant gene locus.
T h e effect of relaxed selection on the frequency of mutant alleles in the future is also of concern ( W H O Technical report 1972). I f complete ascertainment is achieved now it will be possible to monitor its frequency over future generations. In this way, we may also be able to detect changes in frequency which reflect variation in the
From these data we may detect environmental factors influencing the mutation rate. This obvious and pertinent application as well as the long range perspective suggest why screening and surveillance of amino acid disorders in the population has grown in relevance.
28
INDIAN ] O U R N A L OF PEDIATRICS
T h e W H O (Technical R e p o r t 1968) has issued two instructive technical reports which discuss the practice and impact of screening for hereditary metabolic diseases including disorders of amino acid metabolism. These reports recommend that pilot studies be carried out to gather information. Test Procedures
The screening procedure has the primary objective (Wilson and Jungner 1968) to provide an opportunity for early detection o f such individuals for prospective appropriate action. Screening for aminoacid diseases can be done at four primary levels (Scriver I965): (a) T h e genetic materiaI ( b ) T h e gene product modified by the mutation (c) Cellular function or metabolic equilibrium which is controlled by the gene product (d) The disease caused by the perturbation
VOL. 44, No. 349 are responsible for the amount of protein in the cell. However, there is no firm evidence at the present time to indicate the presence of regulatory genes influencing aminoacid metabolism in human cells. Nonetheless, the amount of enzyme activity at some stage of aminoacid metabolism may be altered if the mutation affects the structure of the enzyme in such a way that its turnover in the ceil has been altered. One can visualise that the degradation rate, in the presence of a constant rate of enzyme synthesis, could be increased or decreased so that the amount of enzyme would be diminished or augmented respectively. The amount of enzyme in the cell could also be affected if the final form of enzyme reflects genes acting in sequence.
Screening at the first level is not practical at present although mapping of the human genome by in vitro cell hybridization techniques can be anticipated (Ruddle et al. 1972). The pedigree analysis will yield eventually useful information for genetic counselling under certain circumstances. T h e present discussion is limited to the remaining there levels of screening.
For instance, if protein formed under the influence of one gene is modified by the addition of side groups through the action of a second enzyme acting on the first, then mutation at the gene locus responsible for the second enzyme could prevent synthesis of the final form of the first enzyme. This could be reflected by a diminished quantity of the first enzyme. Alternatively, if the final form of the first enzyme is comprised of sub-units, or if it is a multi-enzyme complex, then mutation affecting synthesis or structure of one of the components of the final enzyme complex could alter the amount of the intact enzyme.
Screening for the gene product (enzyme phenot~,pe) T h e gene product whether it is an enzyme or other cellular protein will usually be altered in some way when the mutation is expressed so that either the primary structure of the protein or the amount of protein is affected. It has been customary to think that regulatory genes
Enzyme screening requires either that enzyme be present in an active soluble form in the body fluids or that the tissue source of an enzyme is available to simple and effective biopsy. T h e test requiring biopsy of tissues is not compatible with the principles of mass screening though it is useful in the investigation of a single patient or a pedigree.
of cellular function.
RIeIDDI -- s C R E E N I N G
t'OR METABOLILI DISOllDERS
29
IN GI-IILDREN
Screenine for metabolic imtm/a~wc (chemicql phenoO'f~e)
cultured amniotic fluid c~,lIs can be dime in a few cases also.
If'the gene product (enzyme) regulates a metabolic pathway or transport fimction, alteration in the function of the enzymes or transport site m a y cause imbalance between a precursor and product stage of the pathway or transfer and this will declare itself either as an accumulation of the substrate or a deficiency of the product of reaction catalysed by the mutant enzyme. Further accumulation or depletion of metabolites may lead to secondary or tertiary level of imbalance which may be identified by the appropriate screening test. For example, phenylalnine hydroxylase deficiency (gene product level) causes accumulation of phenylalanine and its metabolite phenylpyruvic acid (metabolite level). The phenylalanine accumulation secondarily causes serotonin and catecholomine depletion and inhibition of melanin synthesis.
Diagnosis of amznoacidopathtes
Aminoacidopathies have been classified by Dent and Walshe (1954) as high threshold (prerenal) and low threshold (renal) conditions which affect only the membrane transport of an aminoacid and are most appropriately detected by urine screening where large changes in concentration will occur. Dent (1951, 1957) argued that for the general purpose of diagnosis, the urine is the ideal fluid to mirror the state of aminoacid metabolism.
Clinical diseaJe (disease phenoO~e) It is not desirable to screen for the presence of disease since it may be too late to help the patient. The objective of screening is to prevent pathological change or at least to recognise the disease at its incipient stage. Prenatal diagnosis by
Diagnosis may be arrived at rationally by the application of investigative methods. The differential diagnosis of the disorder of the aminoacid metabolism is extensive. The procedure for investigating a patient with a suspected anfinoacidopathy is discussed below. The problem of diagnosis may arise at any time in the patient's lifetime such as an emergency or incidentally during the course of other investigations.
Ch~,ical aids 1. Historr.--(a)
Environment, including toxic drugs, foods which may cause excretion or accumulation of amino acids o1" their byproducts.
(b) Family consanguinity, origin of parents, health in bibs, parents and collateral branches. The proband clinical signs.
may provide helpful
2.
Odour of the urine can be diagnostic of aminoacidopathies (Gone 1968) such as, a musty odour, maple syrup odour, Oast-house odour, fish or cabbage like odour, fishy odour, cheesy odom.
3.
Colourof urine. It darkens on exposure to air, light and alkalis in alcaptonuria and is indigo blue in the blue diaper syndrome.
4.
Hair r
Hair colour is diagnostic in some aminoacidopathies (Rook 1969). The pigmentation is diluted in P K U and the Chediak-Higashi syndrome. Unusually fair hair is seen in patients with Oast-house urine disease, methionine malabsorption and homo-
,~0
INDIAN JOURNAL OF PEDIATRICS
cystinuria. In cystinosis the hair is straw-like and the pigment is yellow. 5.
Eyes. Useful hints are seen in the eyes in certain conditions (Cogan 1966). Cystine crystals are found in the cornea in three different types of cystinosis (infantile, nephropathic and adolescent nephropathic) whereas retinopathy is found only in the infantile type. Scleral pigmentation occurs in alcaptonuria and depigmentation of the liver is seen in two forms of albinism ('generalised', autosomal recessive, and 'ocular' X linked).
Downward lenticular dislocation occurs in homocystinuria due to cvstathionine synthase deficiency. A Kayser-Fleiseher ring in \u disease, cataracts in galactosaemia due to deficient galactokinase or uridyltransferase activity may signal primary disease accompanied by a generalised di sturbance of aminoacid metabolism. Chemical tests for screening A series of simple chemical tests can yield considerable information. For example, an infant in the second half of his first year of life, with pale hair, polyuria and a recent history of weight loss, can be
given a tentative diagnosis of the Fanconi syndrome if the urine contains glucose, a cyanide-nitroprusside positive substance and a heat soluble, low molecular weight protein. Should inspection of the patient's eyes with the-~- 40 diopter lens of the ophlhalmoscope under lateral illunination reveal crystals in the cornea, the diagnosis of cystinosis would be highly probable and the preliminary work-up would have taken ,only 10 minules.
VoL. 44, No. 349 Methodology. Perry et al. (1968), Berry tt al. (1958), Buist (1968), Snyderman (1971), Tocci (1967), O'Brien (1965) and Seriver etal. (1973) provide usefid screening tests. One dimensional partition chromatography (Eft-on el al. 1964) and electro-chromotography (Efron 1959) are methods preferred for further investigations. It should be noted lhat sugar chromatography should always be done on the urine sample that shows a positive test. A negative ferric chloride test does not exclude hyper-phenylalaniemia in the newborn because the conversion of phenylalanine to phenylpyruvic acid may be impaired at this age.
A positive 2, 4-dinitrophenyl-hydrazine (DNPH) test may only indicate ketonuria and the acetone test will be positive. But ketosis may occur in several diseases of aminoacid metabolism, and if the 3,4DNPH test remains positive after heating the urine to get rid of acetone, then the possibility of an aminoacidopathy should be investigated. In this situation gas chromatograph:/ of the urine may reveal one of the unusual organic acids.
Techniques Over the past few years wide scale screening of patients with mental retardation or disease of unknown origin have demonstrated many new inborn errors of metabolism. Most of these diseases are inherited in a recessive manner. They are usually caused by the absence or inactivity of a specific enzyme, required for normal metabolic activity. The biochemical disturbance produced by the missing enzyme may be severe enough to cause serious symptoms in the newborn period or be so minor
N E D D I - - S C R E E N I N G FOR M E T A B O L I C DISORDERS IN C H I L D R E N
that no clinical symptom can be attributed to it. It is now apparent, moreover, that many of these diseases have varying clinical manifestations and may occur in patients who appear quite normal at the time of examination. Patients suffering fi'om homocystinuria may vary from the abnormal phenotype of a mentally retarded patient with the picture of Marfan's syndrome to an adult of normal appearance and intelligence. Nonetheless, so far as can be judged by present knowledge, both these patients seem to be equally at risk in terms of developing severe visual disturbance through dislocation o f | h e lenses and early death through an abnormal clotting mechanism or a dissecting aneurysm of the aorta. It has been recently shown that very high plasma levels of phenylalanine m a y occur in early life and yet produce no clinical signs of phenylketonuria. It seems likely that several different types of phenylketonuria may exist. For the most part it is not known why apparently identical disturbances should have such widely varying clinical manifestationsThe clinical and biochemical abnormalities of these groups of diseases are not usually detectable until after birth, presumably because prenatally, accumulation of any abnormal metabolites is cleared through the mother circulation. In view of the fact that many of these diseases cause severe damage, it is important to detect affected patients in early life in order to develop further effective remedies and to study the effect of such therapy on the subsequent course of the disease. It is equally important that methods should be available to detect heterozygotes or asymptomatic homozygotes in order that the natural history of such metabolic
31
diseases can be elucidated and appropriate therapeutic measures and genetic counselling provided for such patients. At present it is not practical that all potential patients can be subjected to the detailed biochemical tests necessary to establish a precise diagnosis. This is particularly true since there is no knowledge of the true incidence of many metabolic disorders in the normal population. More or less, by the selection of patients whose symptoms suggest the possibility of underlying metabolic disease, and the use of a set of simple chemical tests, it is often possible to detect patients who require full biochemical evaluation and whose symptoms may be cured by appropriate therapy. A list of the conditions which appear to be most commonly associated with metabolic abnormalities is shown in Table 3. A number of clinical tests recommended to screen aminoacidopathies are shown in T a b l e 4. All these tests with slight modifications have been found extremely useful in our daily routine investigations (Reddi 1976). In addition to simple clinical tests, the quantitative estimation of aminoacids from urine and serum samples of the patients is extremely important as confirmatory tests. For this purpose both the high voltage electrophoresis followed by chromatography (Efron 19591 and only chromotography (Sherma and Zweig 1971) techniques have been found to be most useful. In this laboratory we have been carrying on extensive screening of aminoacidopathies with the above technology
32
INDINJOURNALOF PEDIATRICS T a b l e 3.
VOL. 44, No. 349
Conditions in which metabolic errors should be suggested ( Buist 1968) Type
Mental retardation Psychiatric illness Failure to thrive or dwarfing Unknown diagnosis Unexplained deaths at any age Relatives of patients with known metabolic diseases Food intolerance and distaste for protein
Renal calculi Cataracts and dislocated lenses Bone disease of uncertain origin Liver disease of uncertain origin in speech disorders
Marfan's syndrome Speech disorders TESTS Hair coiour Diluted pigment Absence of pigment Unsually fair hair Straw-like, yellow pigment /~.t'es Cysline crystals in cornea Scleral pigmentation Downward lenticular dislocation Kayser-Fleischer ring Cataracts
Cause Many possible causes
Fructosemia Disorders of urea cycle Dissacharidase deficiencies Isovalerie acidaemia Cystinuria Xanthinuria Disorders of uric acid metabolism Diabetes Homocystinuria Galactosaemia Mucopolysaccharidoses Fanconi syndrome Galactosaemia Tyrosinosis Mycopolysaccharidoses Glycogen storage disease Homocystinuria Histidinaemia
Phenylketonuria Chediak-Higashi syndrome Albinism Oast-house urine disease Methionine malabsorption Homocystinuria Cystinuria Cystinosis (infantile nephropathic adolescent Adult non-nephropathic) Alcaptonuria Homocystinuria Wilson's disease Galactosaemia (Contd.)
R E D D I ~ S C R E E N I N G FOR METABOLIC DISORDERS IN CHILDREN
35
Table 3 - - (Contd.)
Odour of urine Odour
Compound
DiSSEIS~
Musty or mousy Maple syrup sweetish caramel, curry Sour berry reminiscent of a brewery, cabbage like
Phenylacetic acid Branched chain ketoacids aketobutyric acid
Methionlne malabsorption
Sulphurous
Hydrogen sulphide
Cystinuria
Phenylketonurla Maple syrup urine disease
Homocystinuria Sweaty feet or cheesy
Isovalerie acid
lsovaleric acidaemia
Cheesy, sweaty feet
Butyric acid, hexanoic acid
Green acyldehydrogenase deficiency
Oast- house, hop-llke
ahydroxybutyric acid (and perhaps other substances)
Oast-house urine syndrome
Fishy or cabbage like
~ketobutyrate or eketo e-methiobutyrate
Hypermethioninaemia
Powerful fishy
Trimethylaminuria
Cabbage like, fishy
Tyrosinaemia
Colour of urine Darkens on standing from surface down on exposure to air, light and alkali
Alcaptonuria
Indigo blue Crystals Cystine crystals
Blue-diaper syndrome
Tyrosine crystals
Liver failure
Orotic acid crystals
Hyperammaninaemia
Cystinuria (s)
(OCT deficiency)
Turbidity Add 20% ( W ) sulphosaHcylic acid V Protein clears with heat-No clearing with heat--
Tubular disease Glomerular (with or without) disease
34 INDIANJOURNALOF PEDIATRICS Table 4. Tests 1. Benedict's
VOL. 44, No. 349
Clinical tests fot aminoacidopathiees Positive
Glucose, in Fanconi syndrome (s) Homogentisic acid, in alcaptonuria p-hydroxyphenylpyruvate, in tyrosinaemia
Remarks
False positive 1. Glucose in diabetes mellitus, renal glycosuria, renal tubu, lar dystrophies, cystinosisLowe's syndrome, vitamin D resistant rickets (Dent type II) 2. Fructose in fructosaemia (aldolase deficiency). Essential fructosuria 3. Galactose in galactosaemia and variants, galactokinase deficiency 4. Lactose in lactase deficiency congenital or acquired 5. Xylulose in pentosuria 6. Phenols in phenylketonuria, tyrosinosis, tyrosine transaminase deficiency
2.
Ferric chloride reaction --phenylpyruvate (blue green), in phenylketonuria and variants --p-hydroxyphenylpyruvate (transient blue green), m tyrosinaemia --imidazolpyruvate (grey-green), in bistidinaemia --a ketobutvrate (purple-red-brown), methionine malabsorption --homogentisic acid (transient blue-green), in alcaptonuria --Pyruvic acid (yellow), in hyper alaninemia --xanthurenic acid (green-brown), in xanthurenic aciduria - - e ketoisovalerlc acid (blue)maple syrup urine disease --~ ketone isocapric acid (yellow), maple syrup urine disease - - e ketomethylvaleric acid (blue-green), maple syrup urine disease
(Contd,
REDDI--SCREENING
F O R M E T A B O L I C DISORDERS IN C H I L D R E N
Table 4.
Test
35
(continued)
Positive
Remarks ii
Other causes
--Lactic acidosis (grey) --Pyruvic acidaemia (yellow) --Pyridoxine disorders, kynurinase deficieny (immediate brown) --Aceto acetic acid (brown red) --Melanin, isonicotine acid hydrazide (grey precipitate) --P.A.S., salicylates (purple) --Phenols, phenothiazines (purple brown) --Vanilic acid (red/brown-mauve) -Conjugated billirubin (blue green) . Cyanide-nitroprusside test --Cystine, in cystinuria (s) (classical and variants) --I-Iomocystine, in homocystinuria (cystathionlne synthasedeficiency) --Cystein~-homocysteine disulfide, in cystinuria and homocystinuria --P-mercaptolactate-cysteine disulfide, in defect of its metabolism --Penicillamine-cysteine disulfide, in treatment of cystinuria --Glutathione, in glutathionlnaemia Urine should not be acidic lest false negative results . Dinitrophenyl hydrozine (ketoacids) --Phenylpyruvic acid in phenylketonuria --Branched chain ketoacids in maple syrup urine disease --P-hydroxyphenyl in tyrosinosis --Pyruvic acid in tyrosyluria --Imidazole pyruvic acid in histidinemia - - a ketobutyric acid in methlonine malabsorption (Oast-House syndrome)
36
INDIAN JOURNAL OF
PEDIATRICS
VOL. 44, No. 349 Table
Test 5.
(continued)
4.
Positive
Remarks
Nitrosonpathol test (Tyxosine metabolites) - - O r a n g e red colour in tyrosine transaminase deficiency --Tyrosinosis (tyrosinemia) - - T r a n s i e n t tyrosinosis of newborn --Tyrosiluria of scurvy
6.
Isatin test for proline
7.
Ninhydrin test for excessive amino aciduria
which useful.
has
been
found
to be
highly
Urine microscopy Microscopic examination of the urine also gives an insight into cellular pathology and metabolic disorders. The test should be performed on fresh urine samples otherwise the renal cells may be destroyed. Metabolic diseases which may be associated with microscopic abnormalities are shown in Table 5. These tests except urine microscopy for cellular abnormaiities and Ehrlics' aldehyde for porphobilinogen can be performed on stored urine; samples may be kept in the refrigerator for a week. But if the tests are to be delayed for longer periods, the specimen should be frozen. It is our policy to perform the tests initially on random urine samples. When an abnormal result is found a further sample is obtained for repeat testing. When the second test is abnormal the results are confirmed by more refined techniques on accurately timed samples.
References
Becker, M.A., Messer, L.T., Seegmillen, J.E. (1973a). Gout with purine overproduction due to increased phosphoribosyl phosphate synthetase activity. Am. 07. Med. 55, 232. Becket, M.A., Kostel, P.H., Messer, L.T. and Seegmillen, I.E. (1973 b). Human phosphoribosyl phosphate synthetase. Increased enzyme specific activity in gout, and excessive purine synthesis. Proc. Nat. Acad. Sci 70, 2749. Berry, H.K., Leonard C., Peters, H., Granger~ M. and Chunekanrai, N. (1968). Detection of metabolic dimrders. Chromatographic procedures and interpretation of resuls. Clin. Chem. 14, 1033. Buist, N R.M. ~1968). Set of simple side-room urine tests for detection of inborn errors of metabolism. Brit. Med. 07. 2, 745. Cogan, D.G. (1966), Occular correlates of inborn metabolic defects. Can. Med. Assoc. ,7. 95, 1055. Cone, T.E Jr. (1968). I Jiagnosls and treatment: Some diseases, syndromes and conditions associated with an unusual odour. Pedmtrics, 41,993. Dent, C.E. (1951). Recent advances in clinical pathology In Dyke, S.C , Ed., 2nd edit. Biakistan, Phila. 238-258. Dent, C E. (1957). Clinical applications of amino acid chromatography. Scand.,~. Clin. and Lab. Inves . (supp. 32) 10, 122.
REDDI--SCREENING
FOR
METABOIIC
DISORDERS
IN
CHILDREN
~
~D r
c~
~o
c~ ~0
~D
.2 O
>-.
~ r
O
.,.~
f:L
~
~
o
r
o~
c~
~
r
~9
Q9
~ ~9
a~ O
~9 r
o,3
~
c~ ~9
38
INDIAN JOURNAL OF PEDIATRICS
VOL, 44,
N o . 349
Dent, C.E. and Walshe, J.M. (1954). Aminoacid metabolism. Brit. Med. Bull 10,247. Efron, M.L. (1959). Two-way separation of amino acids and other ninhvdrin reading substances by highvoltage electrophoresis followed by paper chromatography. Biochem. j . 72, 69i. Efron, M.L., Young, D., Moser, H.W. and Mac Cready, R.A. ~1964). A simple chromatographic screening test for the detecion of disorders of amino acid metabohsm. A technique using white blood or ~.lrine collected on filter paper. New Eng. J. Med. 270,
Ruddle, F., Riccuti, F., McMorria, F.A., Tischfield, J., Greegan, R., Dalington, G. and Chen, T. (1972). Somatic cell genetic assignment of peptidase C and the Rh linkage group to chromosome A-1 in man. Science, 176, 1429.
1376. Garrod, A.E.
Sharma, J. and Zweig Gunter (1971). Paper Chromatography. Vol. II. Academic Press, New York and London. Synderman, S.E. (197l). Diagnosis of metabolic disease. Pediat. Clio. N.A. 18, 199. Tocci, P.M. (1967). The biochemical diagnosis of metabolic disorders by urinanalysis and paper chromatography. In Aminoacid Metabolism and Genetic Variation (Ed. W.L. Nyhan) Mc Graw-Hill Book Pub., 161, 490. Udenfriend, S., Stein, S., Hohlen, P., Diagman, W., Leingruber, W. and Weegele, M. (1972). Fluorescamine. A reagent for assay of amino acids, peptides, proteins and primary amines on the picomole range. Science, 178, 871. Wilson, J.M.G. andJungner, G. (1968). Principles and practice of screening for disease. Public Health paper 34, World Health Organization, Geneva. World Health Organization, Technical Report series 40, Screening for inborn errors of metabolism. Geneva, 1968. World Health Organization Technical Report Series, 497. Genetic Disorders, Prevention, Treatment and Rehabilitation. Geneva. 1972.
0908).
The
Croonien lectures.
Lancet ii, 73, 142. Kirkman, H.N (1970). Dominant mutations. Biochemical basis of phenotype. In F.CI. Fraser and V.A. McKusick Eds: Congential malformations Excerpta Medica. Amsterdam. Levy, H.L. (1973). Incidence ofaminoacidopathles among newborn infants in Massachusetts. Bull. Pub. Hlth. Massachusetts Dept. of Public Health McKusick, V. (1975). Mendelian inheritance in man. The John's Hopkins University Press. Baltimore and London. O'Brien, D. (1965). Rare inborn errors of metabolism in children with mental retardation. Part B. Technical procedures, Children's Burea publ. No. 4291965, U.~ ). Dept. U.S. Govt. Printing O~ice, Washington I).C., P. 70-100. Perry, T.L., Hansen, S. and MacDougall, L. (1966) Urinary screening tests in the prevention of mental deficiency. Caoad. Med. Assoc. 07.95, 89. Reddi, O.S. (1976). Unpublished. Rook, A. (1969). Hair colour in clinical diagnosis. Indian. 07. Med. So. 7, 415.
Scrlver, C.R. (1965). Screening newborns for hereditary metabolic disease. Ped. Clin. N. Ame.r 12, 807, Striver, C.R., Clow, C.L. and Lamm, P. (1973). On tlae screening, diagnosis and investigations of hereditary aminoacidpoathies. Clio. Bio~hem. 6, 1t2, 188.
