Newborn screening for galactosemia and other galactose metabolic defects

Newborn screening for galactosemia and other galactose metabolic defects

T H E J O U R N A L OF PEDIATRIC S J U N E 1978 Volume 92 Number 6 I MEDICAL PROGRESS Newborn screening for galactosemia and other galactose met...

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T H E J O U R N A L OF

PEDIATRIC S J U N E 1978

Volume 92

Number 6

I

MEDICAL PROGRESS

Newborn screening for galactosemia and other galactose metabolic defects Harvey L. Levy, M.D.,* and Gerhard Hammersen, M.D.,** B o s t o n , M a s s .

W I T H THE INTRODUCTION of the Guthrie assay in

1962 as a screening test for phenylketonuria, newborn screening for metabolic disorders has become widely accepted? Though PKU has been the major focus in screening programs and in many is the only disorder sought, testing for other disorders is becoming more common2 Among the latter, galactosemia (severe transferase deficiency) is particularly suitable for routine newborn screening because clinical complications m a y develop rapidly in early infancy and are entirely preventable or reversible with prompt diagnosis and the institution of a relatively simple diet? However, screening for galactosemia and other disorders of galactose metabolism is more complicated than that for PKU. Not only is there uncertainty regarding which of the several available tests is best, but the technical difficulties are magnified by the From the State Laboratory Institute, Massachusetts Department o f Public Health, the Joseph P. Kennedy Jr. Laboratories of the Neurology Service, Massachusetts General Hospital, and the Department of Neurology, Harvard Medical School. Supported by USPHS H S M H A M C H Grant 01-H000111-07, National Institute of Health Grant N S 05096, and by Deutsche Forsehungsgemeinschaft Grant HA 855/1. *Reprint address: State Laboratory Institute, 305 South St.,

Boston, MA 02130. **Dr. Hammersen complied these data while on a leave of absencefrom the University Children's Hospital, Heidelberg (Director Prof. Dr. H. Bickel).

0022-3476/78/0692-0871500.70/0 9 1978 The C. V. Mosby Co.

necessity for rapid detection if neonatal death or irreversible damage is to be prevented, 4 Consequently, a review and analysis of screening for galactosemia is timely and important. Abbreviations used phenylketonuria PKU: galactose-l-phosphate uridyltransferase transferase: uridine diphosphate galactose-4-epimerase epimerase: uridine diphosphate glucose UDPG: uridine diphosphate galactose UDPGal: galactose-l-phosphate gal-l-P: reduced nicotinamide adenine dinucleotide NADPH: phosphate GENETIC DISORDERS METABOLISM

OF GALACTOSE

Three separate inborn errors of galactose metabolism are known to occur in man (Fig. 1). The one described first and most frequently recognized is a defect in transferase (enzyme 2), commonly designated galactosemia. Gatactose accumulates in the blood and urine of affected individuals, gal-l-P is present in erythrocytes and other cells and galactitol is found in urine, ocular lenses, and other body tissues? In its classical form wherein there is essentially no detectable transferase activity, galactosemia is characterized by failure to thrive, jaundice, hepatomegaly, and, often, death. Sepsis due to Escherichia coli seems to be particularly frequent among galactosemic neonates and is usually the cause of death? If the infant survives

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Levy and Hammersen

t T ~ GALACTOSE~ ATP i

The Journal of Pediatrics June 1978

O3.

GALACTO~.E-I-P+ U D ~ G ~ U ~ ADP

.....::'.:::::::......

~.....

..... ::::: .....

4,)

I~ GLUCOSE-6-P

.... i; .....

..;::: ........... "........ .....

~. . . . . . .

UTP

GW_COSE-I-P

y

~

....... ::21 m.. ,.~1110"2:

GALACTITOL

lol +

EX~Os "~ r- H E PP

IMONOPHOSPHATE I\ / SHUNT L

l,- }~

-~~I_~COLYSIS~

Fig. 1. Pathway of galactose metabolism. The enzymes involved are as follows: one, galactokinase; two, galactose-1phosphate uridyltransferase; three, uridine diphosphate galactose-4-epimerase;four, phosphoglucomutase. In addition, five, uridine diphosphate galactose (or glucose) pyrophosphorylase could have some role, albeit limited, in bypassing the transferase reaction to convert galactose-l-phosphate to UDPGal. In galactosemia or galactokinase deficiency, six, aldose reductase is active in forming galactitol from galactose. The known human disorders are in enzymes one, two and three. but remains untreated, the disease results in mental retardation, cirrhosis, and cataracts. Individuals with transferase partially deficient in activity and abnormal in electrophoretic mobility have also been described? This is most commonly due to the compound heterozygous state for the Duarte and galactosemia variants. 7 Clinical findings range from none in the various Duarte variant states to most or all of the signs of classical galactosemia in the Rennes 8 and Indiana variantsY In the so-called Negro variant there appears to be residual liver metabolism of galactose but no detectable transferase activity in erythrocytes. TM Blacks with this form of galactosemia may have milder clinical disease than whites with classical galactosemia. In a second disorder of galactose metabolism, galactokinase (enzyme 1) deficiency (Fig. 1), galactose accumulates in blood and urine and galactitol accumulates in urine and presumably ocular lenses but gal-l-P is not found." The only clinical sign of this disease is cataracts developing early in life. Most recently, epimerase, a third enzyme (enzyme 3) involved in galactose metabolism (Fig. 1), was discovered to be deficient in erythrocytes and leukocytes from clinically normal individuals but with normal activity in liver, activated lymphocytes and cultured skin fibroblasts. '-~ Blood from these individuals contains slightly increased concentrations of galactose and markedly increased concentrations of gal-l-P. Epimerase activity in tissues may account for the apparent lack of clinical effects in this disorder. Treatment for the clinically significant deficiencies of transferase and galactokinase consists of a diet as free of lactose as possible, by the exclusion of milk and milk products. 13 If begun within the first four or five days of life, the clinical problems are a!most always prevented. As in all inherited disorders, genetic counseling is an

important aspect of management. Since the inheritance pattern of each is autosomal recessive, there is a 25% risk of recurrence for a given defect in any sibling of an affected infant. The parents should be so advised for the purposes of family planning. Prenatal diagnosis of galactosemia is possible?" The carrier states can also be identified by appropriate quantitative assays of enzyme activity in erythrocytes~~ This service should be offered to all members of a family in which galactosemia or galactokinase deficiency has occurred, particularly to those of child-bearing age or younger. SCREENING

TESTS

These tests and their characteristics ate listed in Table I.

Galactoseassays. Inhibition assay. Shortly after the introduction of the Guthrie test for PKU, a screening test for galactose elevations in blood which utilized the dried filter paper b)ood specimen obtained for PKU screening was introduced. '~ Discs from these blood specimens are incubated in an agar gel that has been inoculated with a transferasedeficient mutant strain of E. coll. Inhibition of bacterial growth is noted around those discs that contain galactose. Growth inhibition because of increased concentrations of valine may also occur. A serious disadvantage of this auxotrophic assay is, that with time, the mutant bacteria tend to lose their sensitivity to galactose. Paigen assay. Paigen and Pacholec TM recently developed a relatively stable bacterial assay for galactose that employs an epimerase-deficient strain of E. coli which, in the presence of galactose, resists destruction by C21 bacteriophage. Discs of dried filter paper blood specimens that contain galactose are surrounded by bacterial growth that is proportional to the galactose concentration. The assay also responds to gaM-P if the bacterial cells produce

Volume 92 Number 6

Newborn screening for galactosemia

873

Table I. Newborn blood screening tests for galactosemia and other inborn errors of galactose metabolism

Test*

Principle

Inhibition assaf 5

Bacterial assay

Paigen 16

Bacterial assay

Galactose dehydrogenase TM

Automated spectrophotometric Enzyme assay

Beutler19

Compound(s) tested Galactose Lactose Valine Galactose Gal-I-P Lactose Galactose Transferase

Advantages All galactose disorders; hypervalinemia detection

Disadvantages Instability

All galactose disorders; responds May be partially inhibited by to gal-l-P; technically simple antibiotics All galactose disorders Enzyme detection

Technically more difficult than bacterial assay Limited to galactosemia; many false positives

*The test names are as commonlyused. The reference for each is given. phosphatase (W. H. Murphey, personal communication) or if alkaline phosphatase is incorporated into the medium. 17 Since growth is more readily visualized than inhibition, the Paigen assay can be scanned with greater reliability compared to the inhibition assay. Furthermore, considering the response to gal-l-P, the Paigen assay has greater sensitivity and may be used with umbilical cord blood. Other galactose tests. A number of chemical tests for the detection of galactose exist. An automated spectrophotometric assay has been developed for use on blood eluted from filter paper discs. TM This tends to be technically more difficult and more expensive than a bacterial assay. Galactose can be demonstrated in liquid urine by either a dipstick method (Cralactostix, Ames Co., Elkhart, IN) or a nonspecific test for reducing substance (Clinitest, Ames Co.) However, the collection of liquid urine specimens and the transportation of specimens to a central laboratory is not an efficient method of routine neonatal screening. Enzyme assay. An ingenious screening procedure for galactosemia, based on testing for enzyme activity rather than for metabolite increase was introduced in 1966 by Beutler and Baluda. 19 This enzyme spot screening test is performed on blood eluted from a disc of filter paper; transferase activity is detected by the fluorescence of NADPH. Though this test has been a valuable asset in genetic screening, there are certain difficulties. The most serious of these is the frequency with which specimens that have little or no activity are encountered. In most instances, this seems to be due to loss of activity following exposure of the specimen to the heat and humidity of summer~ ~~ Another cause may be a benign low activity transferase variant. In order to identify the infant with galactosemia, screening programs using this assay must request a repeat specimen from each infant with this

apparently positive finding. The frequent requests for repeat Specimens result in anxiety among parents and physicians and severely compromise the effectiveness of this assay. RECOMMENDATIONS SCREENING

FOR NEWBORN

The Paigen assay for galactose and gal-l-P is the most effective single test currently available for the detection of galactosemia and other inborn errors of galactose metabolism in the newborn infant. Since it is a metabolite assay, infants with galactokinase deficiency as well as those with galactosemia can be detected. Frequent requests for repeat specimens are avoided since few neonates have substantially increased concentrations of galactose or gal1-P. By responding to gal-l-P the Paigen assay will identify the infant with galactosemia who has ingested little or no milk and may not have accumulated large amounts of gaiactose. In Massachusetts, the Paigen assay with added alkaline phosphatase (for gal-l-P responsiveness) is performed on filter paper specimens of umbilical cord blood as well as on newborn filter paper blood specimens. Thus, gal-l-P in umbilical cord blood, a characteristic finding in galactosemia, 3 can be detected and the information given to the attending physician earlier than is possible with the testing of blood from newborn infants, This may he crifical in the care of an infant who might otherwise die with galactosemia and secondary neonatal sepsis. The infant with galactosemia who might be missed by umbilical cord b l o o d testing or the infant with increased galactose or gal-l-P due to another galactose metabolic disorder or to nongalactosemic liver disease would likely be detected via the newborn blood test, If ametabolite assay is used for routine screening it is important that the Beutler test for transferase be available

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The Journal of Pediatrics June t978

Table II. R o u t i n e screening a m o n g n e w b o r n infants for g a l a c t o s e m i a a n d o t h e r i n b o r n errors o f galact0se m e t a b o l i s m *

No. with disorder~ Methodt North America California Connecticut Maine Massachusetts Minnesota New York Ohio Oregon Quebec Rhode Island Europe Austria Belgium Czechoslovakia Germany (North) Germany (West) Ireland Switzerland Asia Japan New Zealand

Newborn tested

Enzyme Enzyme Galactose Enzyme Galactose Enzyme Enzyme Enzyme Enzyme or Galactose or both Galactose Galactose

236,944 313,367 16,085 326,261 582,800 51,000 365,000 595,927

Galactose Cralactose Galactose Galactose Galactose Galactose Enzyme or Galactose or both

840,112 106,511 132,392 119,024 300,355 144,843

Enzyme or Galactose Enzyme

376,670 224,000 11,408

Kinase

Total

Transferase

[

Epimerase

3 4 1 2 10 1 8 5

1

5

673,846 160,784 389,760 5,967,089

I

5 1 0 19 0 3 4 7 3 11

6

2

3 7 97 (1:62,000)

*Data were obtained as follows: references 28 and 29; personal communication from G.N. Donnell and P. Pepe (California), J. S. Tucker (Connecticut), L. J. Damsky (Minnesota), C. C. Croft (Ohio), W. H. Murphey (Oregon), C. Laberge (Quebec), O. Thalhammer (Austria), R. Gitzelmann (Switzerland). H. Naruse (Japan), A. O. Veale and I. C. T. Lyon (New Zealand). The most recent data for New York are those through 1972.'~" tEnzyme refers to the spot screening test for transferase of Beutler and Baluda19; galactose refers to an assay for galactose, either the bacterial method of GuthrieTM or of Paigen and PacholecTM or the chemical method of G-renierand LabergeTM or of Weidemann.3~ :~Disorders of galactose metabolism referred to are as follows: kinase is galactokinase deficiency; transferase is galactose-l-phosphate uridyl transferase deficiency; epimerase is uridine diphosphate galactose-4-epimerase deficiency.

so t h a t infants with galactose elevations due to transferase deficiency c a n be differentiated from those with galactose accumulations caused by o t h e r factors. Thus, w h e n a galactose increase is discovered, the i n f a n t with galactosemia can be presumptively identified a n d the necessary measures, including i m m e d i a t e physician contact a n d confirmatory testing, b e instituted. Blood for screening should b e o b t a i n e d n o later t h a n the third d a y o f life in order to avoid the possible fatal delay in the identification o f galactosemia. CONFIRMATORY

TESTING

N e o n a t a l screening serves only to separate the vast majority of infants w h o h a v e n o r m a l findings f r o m the few w h o have a b n o r m a l test results. Most a m o n g the latter will have a n a b n o r m a l result because of factors

other t h a n a significant disorder (e.g., artifact, t r a n s i e n t abnormality). Only a n occasional i n f a n t will h a v e a primary or secondary disorder o f galactose m e t a b o l i s m . Confirmatory tests m u s t b e available so t h a t a precise metabolic diagnosis c a n b e m a d e or excluded. This i n f o r m a t i o n is essential for the p r o p e r u n d e r s t a n d i n g a n d t r e a t m e n t of any condition. Since the transferase-deficient states are the most severe clinically a n d are the most frequently e n c o u n t e r e d disorders of galactose metabolism, the i m p o r t a n t confirmatory tests are those which allow for a q u a n t i t a t i v e m e a s u r e o f erythrocyte transferase activity. These include the U D P G c o n s u m p t i o n assay ~1 a n d a radioactive assay. 2~- Starch gel electrophoresis for transferase mobility is also essential. '-'3 W i t h these tests, the presence or absence o f transferase can be ascertained, the a m o u n t of any residual transferase

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activity can be measured, and a determination of whether or not a variant of transferase is present can be made. The availability of additional tests will depend upon the focus and resources ofthe screening laboratory. Assays for galactok!nase~ and epimerase ~2 may be useful and detailed investigations of affected individuals may necessitate studies of cultured skin fibroblasts? ~ Methods for quantifying galactose and related metabolites, such as gas-liquid chromatography, ~6 may be of value. In most screening laboratories it is not feasible to employ such relatively elaborate testing methods. Consequently, a referral system for this testing should be developed; transfer of specimens long distances is suitable since galactose and its metabolites as well as enzymes in the galactose metabolic system are quite stable. Collaborative arrangements should be established before screening is begun. COSTS Newborn screening for galactosemia, when added to other screening tests such as PKU performed on filter paper blood specimens, costs approximately 35r per test if 50,000 to 100,000 tests are performed per year in a single laboratory. Approximately 70% of this cost is for personnel (one technician per 50,000 tests) and the remainder is for material, clerical services, laboratory overhead (one room), and equipment (small incubator). The costs of testing by a galactose assay or by the Beutler enzyme spot test are similar though material costs for the latter may be slightly higher due to the relatively high cost of the co-substrate gal-l-P. These expenses do not include the costs of confirmatory testing. This type Of screening (as well as all other types of newborn screening) should always be conducted in laboratories receiving at least 50,000 specimens per year. 27 The necessary experience in performance and interpretation as well as in cost efficiency can be realized only in laboratories of this size or larger. INCIDENCE In Table II are listed those routine newborn screening programs that include or have included screening for galactosemia and (perhaps) other disorders of galactose metabolism. Among the 19 programs, six have used an enzyme assay for transferase exclusively, nine have used only a galactose assay, and four have used either type of assay or a combination of both. The enzyme assay has be'en more popular among programs in the United States, whereas in Europe a galactose assay has been used in virtually every program. The incidence o f galactosemia, reported from newborn Screening, is about 1:62,000. The highest reported frequencies are from Hamburg (1:30,000) and Austria

Newborn screening for galactosemia

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(1:39,000). In Massachusetts, we previously reported an incidence of 1: i87,000. 2~ However, we now believe that cases of galactosemia may have been overlooked, resulthag in a falsely low incidence.:" Since 1972, when we added the Paigen assay to newborn blood testing and later to umbilical cord blood testing, we have detected ten cases among 401,823 screened infants for an incidence of i :40,000. It may be that few, if any, of the reported statistics reflect the true frequency of galactosemia. The problems of interpreting the test result or in obtaining repeat specimens could result in missed cases. In this regard, it is interesting to compare the incidence of galactosemia as determined by galactose assay only and that determined exclusively by the Beutler enzyme test for transferase. Among 2,477,530 infants tested by a galactose assay only, 48 cases of galactosemia have been reported, a frequency of 1:52,000. Among 2,278,259 infants tested by the Beutler assay only, 30 cases of galactosemia have been reported, a frequency Of 1:76,000. Thus, the reported frequency of galactosemia detected by programs using a galactose assay is higher compared to those using the spot enzyme test. Though this difference may be indicative of a greater effectiveness of a galactose assay it could also be due to a higher gene frequency in Europe inasmuch as most of the infants screened by a galactose assay have been in Europe and most of those screened by the enzyme assay have been in the United States. Only one clinically significant transferase variant has been identified on the basis of routine neonatal screening, 6 though it is probable that other infants, believed to have classical galactosemia, may also represent variant states. Nevertheless, it appears that the incidence of clinically significant transferase variants is considerably lower than that of classical galactosemia. Conversely, the incidence of benign transferase variants, such as the Duarte variant, is quite high 7 and such infants may be incorrectly recorded as cases of galactosemia unless gel electrophoresis or other confirmatory methods are employed, The data from screening programs that employ only the Beutler enzyme spot screening test for tranSferase are necessarily limited to galactosemia and other transferase deficiencies. However, in programs employing a galactose assay infants with galactokinase deficiency or epimerase deficiency have also been identified (Table II). Judged from screening data, infants with these deficiencies seem to be far less frequently encountered than those With galactosemia. On the other hand, this lower reported incidence could be due partially to an artifact of screenhag, in that the blood galactose concentration in newborn infants with galactokinase or epimerase deficiency may

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not be increased as markedly as in those with galactosemia? ~ SUMMARY Routine newborn screening is necessary for the effective diagnosis of galactosemia The criticism that routine screening for galactosemia is unnecessary is based on the misconception that the diagnosis will almost always be made clinically, since the disease produces clinical manifestations in the infant. Actually most newborn infants, detected by routine screening, were not suspected to have galactosemia, even when ill. Furthermore. some infants with clinically important transferase variants are not ill as neonates. In Massachusetts. a black infant who probably has the Negro variant form of galactosemia and an infant with the Rennes variant of galactosemia ~ were clinically normal at five days of age when detected by routine screening. Complications of galactosemia can occur in both variants if treatment is not given. In addition, galactokinase deficiency, which results in cataracts but not in acute illness? 2 cannot be detected on a clinical basis until the cataracts have become apparent, when it is too late for preventive therapy. The full impact of galactosemla on the n e w b o r n infant has become evident only since routine n e w b o r n screening has been initiated. It is now apparent that death associated with bacterial sepsis may occur in about 30% of those with untreated classical galactosemia. 4 In most of these deaths diagnosis and treatment were delayed until the second week of life. Consequently, screening for galactosemia should be performed on a blood specimen that is obtained no later than the third or fourth day of life and is delivered promptly to the screening laboratory. There should be sufficient medical back-up available to the laboratory so that the attending physician can be quickly contacted by telephone when an abnormality suggesting galactosemia is encountered. Further procedures should follow: (1) Urine should be tested for reducing substance. (2) If urinary reducing substance is present, galactosemia shou M be presumed; milk feedings should be discontinued; and blood and urine specimens should be sent to a laboratory for confirmatory testing. (3) If the infant is ill, bacterial cultures should be obtained and treatment for sepsis initiated. (4) If galactosemia is confirmed, treatment should be continued. Otherwise, treatment can be discontinued. Galactosemia screening should be routine for all newborn infants. It is a disorder with definite and severe complications, but one in which the complications can be prevented with simple and inexpensive treatment. W h e n a test for galactose and gaM-P, such as the Paigen assay, is also applied to the newborn blood specimen that is,

The Journal o f Pediatrics June 1978

submitted for P K U screening, galactosemia can be detected efficiently and effectively. The screening for galactosemia in Massachusetts has been performed with extraordinary diligence by Mrs. Sally Houghton and Mrs. Jane Carr. Dr. Robert A. MacCready was responsible for the introduction of this procedure to the program and Dr. Viviau E. Shih has been instrumental in its evaluations. REFERENCES

1. Committee for the Study of Inborn Errors of Metabolism: Genetic screening, Washington, 1975, National Academy of Sciences. 2. Levy HL: Genetic screening, in Harris H, and Hirshhorn K, editors: Advances in human genetics, vol 4, New York, 1973, plenum Publishing Corp., p 1. 3. Donnell GN, Koch R, and Bergren WR: Observations on results of management of galactosemia patients, in Hsia DYY, editor: Galactosemia, Springfield, Ill, 1969, Charles C Thomas, Publisher, p 247. 4. Levy HL, Sepe SJ, Shih VE, Vawter GF, and Klein JO: Sepsis due to Escherichia eoli in neonates with galactosemia, N Engl J Med, 297:825, 1977. 5. Segal S: Disorders ofgalactose metabolism, in Stanbury JB, Wyngaarden JB, .and Fredrickson DS, editors: The metabolic basis of inherited disease, ed 4, New York, 1978, McGraw-Hill Book Company, p 160. 6. Hammersen G, Houghton S, and Levy HL: Rennes-like variant of galactosemia: Clinical and biochemical studies, J PEDIATR87:~0~ 1975. 7. Levy HL, Sepe SJ, Walton DS, Shih VE, Hammersen G, Houghton S, and Beutler E: Galactose-l-phosphate uridyl transferase deficiency due to Duarte/galactosemia combined variation: Clinical and biochemical studies, J PEDIATR 92:390, 1978. 8. Sehapira F, and Kaplan JC: Electrophoretic abnormality of galactose-l-phosphate uridyl transferase in galactosemia, Biochem Biophys Res Commun 35:451, 1969. 9. Chacko CM, Christian JC, and Nadler HL: Unstable galactose-l-phosphate uridyl transferase: A new variant of galactosemia, J PEDIATR78:454, 1971. 10. Baker L, Mellman WJ, Tedesco TA, and Segal S: Galactosemia: Symptomatic and asymptomatic homozygotes in one Negro sibship, J PEDIATR68:551, 1966. 11. Gitzelmann R: Hereditary galactokinase deficiency, a newly recognized cause of juvenile cataracts, Pediatr Res 1:14, 1967. 12. Gitzelmann R, Steinmann B, Mitchell B, and Haigis E: Uridine diphosphate galactose 4-epimerase deficiency IV. Report of eight cases in three families, Helv Paediatr Acta 31:44, 1976. 13. Donnell GN, Bergren WR, and Koch R: Abnormal galactose metabolism in man, in Mclsaac WM, Claghorn J, and Farrell G, editors: Congenital mental retardation, Austin, 1969, University of Texas Press, p 87. 14. Ng WG, Donnell GN, Bergren WR, Alfi O, and Golbus MS: Prenatal diagnosis of galactosemia, Clin Chim Acta 74:227, 1977. 15. Guthrie R: Routine screening for inborn errors in the newborn: "inhibition assays," instant bacteria and multiple tests, in Proc Intern Copenhagen Cong Scient Study of Mental Retard, Vol 2, Copenhagen, 1964, p 495.

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Newborn screening for galactosemia

16. Paigen K, and Pacholec F: A new method of screening neonates for galactosemia, J Lab Clin Med (in press). 17. Gitzelmann R: Frfiherfassung von Anomalien im Galaktosestoffwechsel Methoden und Resultate, Mchr Kinderheilk 124:654, 19=/6. 18. Grenier A, and Laberge C: Rapid method for screening for galactosemia and galactokinase deficiency by measuring galactose in whole blood spotted on paper, Clin Chem 19:463, 1973. 19. Beutler E, and Baluda MC: A simple spot screening test for galactosemia, J Lab Clin Med 68:137, 1966. 20. Shih, VE, Levy HL, Karolkewicz V, Houghton S, Efron ML, Isselbacher KJ, Beutler E, and MacCready RA: Galactosemia screening of newborns in Massachusetts, N Engl J Med 284:753, 1971. 21. Beutler E, and Baluda MC: Improved method for measuring galactose-l-phosphate uridyl transferase activity of erythrocytes, Clin Chim Acta 13:369, 1966. 22. Ng WG, Bergren WR, and Donnell GN: An improved procedure for the assay of hemolysate galactose-l-phosphate uridyl transferase activity by the use of 1'C-labelled galactose-l-phosphate, Clin Chim Acta 15:489, 1967. 23. Ng WG, Bergren WR, Fields M, and Donnell GN: An improved electrophoretic procedure for galactose-t-phosphate uridyl transferase: demonstration of multiple activity bands with the Duarte variant, Biochem Biophys Res Commun 37:354, 1969.

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24. Beufler E, and Matsumoto F: A rapid simplified assay for galactokinase activity in whole blood, J Lab Clin Med 82:818, 1973. 25. Hammersen G, Mandell R, and Levy HL: Galactose-1phosphate uridyl transferase in fibroblasts: isozymes in normal and variant states, Ann Hum Genet 39:147, 1975. 26. Sweeley CC, Bentley R, Makita M, and Wells WW: Gasliquid chromatography of trimethylsilyl derivatives of sugars and related substances, J Am Chem Soc 85:2497, 1963. 27. Brandon GR: Regionalization of public health metabolic laboratories, Public Health Lab 34:56, 1976. 28. Thalhammer O: Frequency of inborn errors of metabolism, especially PKU, in some representative newborn screening centers around the world. A collaborative study, Humangenetik 30:273, 1975. 29. Kelly S, Burns J, and Desjardins L: Incidence of galactosemia at birth in New York state, Am J Epidemiol 99:8, 1974. 30. Weidemarm VG: Screening-test zum Nachweis einer Galactos~tmie, Z Klin Chem Klin Biochem 9:527, 1971. 31. Levy HL, and Shih VE: Galact0semia in Massachusetts, N Engl J Med 287:723, 1972. 32. Thalhammer O, Gitzehnann R, and Pantlitschko M: Hypergalactosem!a and galactosuria due to galactokinase deficiency in a newborn, Pediatrics 42:441, 1968.

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Most of the provisions of the Copyright Act of 1976 became effective on January 1, 1978. Therefore, all manuscripts must be accompanied by the following statement, signed by each author: "The undersigned author(s) transfers all copyright ownership of the manuscript entitled (title of article) to The C. V. Mosby Company in the event the work is published. The author(s) warrants that the article is original, is not under consideration by another journat, and has not been previously published." Authors will be consulted, when possible, regarding republication of their material.