MAPLE SYRUP DISEASE

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Isolation

and Identifkation

of Organic

Acids

in the Urine

By John H. Menkes, M.D.

Divi,cion of Pediatric Neurology, Neurological Institute, Columbia-Presbyterian Medical Center

CH3

MAPLE

SYRUP

DISEASE

348

S

EVERAL years ago Menkes, Hurst and

Craig1 reported four cases of a familial

cerebro-degenerative disease, characterized

by the excretion of urine with a pleasant,

maple syrup-like odor. At that time we

were unable to determine the nature of the

substance responsible for the urinary odor.

Since then, six additional cases of the

syn-drome, occurring in three unrelated

fami-lies, have been brought to our attention.2

The disease, as it occurred in all these

chil-dren, was characterized by early onset of

spasticity and myoclonic seizures in previ-ously intact infants, with rapid progression

to decerebrate rigidity. After 1 or 2 days of

age, the unusual urinary odor became very

evident, and was present throughout life

with some slight variations in intensity.

Death ensued between 15 days and 20

months of age.

From one such case, previously reported

by Westall et al., we were fortunate to

ob-tam,

through the courtesy of these authors,

various samples of urine excreted during

the last months of the infant’s life. In these

specimens we were able to demonstrate the

presence of large amounts of

alpha-ketoiso-caproic acid,

(CH:CHCH2COCOOH)

the keto analogue of leucine, together with

lesser quantities of the keto acids of valine

(

alpha-ketoisovaleric acid), and isoleucine

(alpha-keto-beta-methyl-n-valeric acid). It

is these substances that may indirectly be

responsible for the unusual urinary odor.

(Accepted August 20, 1958; submitted July 22.)

This research was supported in part by a grant and Blindness, Public Health Service.

ADDRESS: 622 West 168th Street, New York, New York.

CHEMICAL STUDIES

Initial Observations on the Urine

of Maple Syrup Disease

The urine was a clear amber liquid, with

the characteristically pleasant, maple

syr-rup-like odor. To the usual qualitative

cx-aminations, there was no reaction for

al-bumin or sugar. Upon addition of ferric

chloride (10% in alcoholic solution), a

blue-black color was produced which persisted

for several hours before fading. Addition of

a 0.1% solution of

2,4-dinitrophenylhydra-zinc in 2 normal hydrochloric acid gave an

almost immediate dense yellow precipitate,

even when a 1 : 50 dilution of the urine was

tested.

In contrast to this, various specimens of

urine of normal infants produced no

im-mediate precipitate, and only after heating

the urine with the reagent for about 30

minutes at 80#{176}Cwas a small amount of

precipitate produced.

Isolation of the

2,4-Dinitrophenylhydrazones (DNPH)

Since the formation of a

dinitrophenyl-hydrazone is a fairly specific test for

car-bonyl compounds, further qualitative

cx-amination of the urine was directed towards

the isolation and identification of these

de-rivatives.

Two milliliters of urine were mixed with

2 ml of 0.1% 2,4-dinitrophenylhydrazine in

2 normal hydrochloric acid. The precipitate

formed after 30 minutes at room

tempera-ture was separated by centrifugation,

washed several times with cold water, and

dried. Its melting point range was between

from the National Institute of Neurological Diseases

(2)

TABLE I

CIII1OMATOGRAPIIY OF THE DNI’H i)EHIvATIvES

“Maple syrup” urine

Neutral 0.64

ketones 0.84

0.90

Keto 0.61

aci(is 0.77-0.85

0.91

ARTICLES

Re/a/ire

1”rac/ion I/f Magnitude

of Spot

Neutral

ketones

0.91

‘2+

,

Normal urine ket()

a(’)ds

0..56

0.59

0.9

1+

trace

1+

Descending chromatography. Solvent phase of etha-nol 10 parts; water 40 parts; n-hutanol 50 parts. Wlmt

1flH11 No. I paper. Spots identified by their intrinsic color.

‘rile overlap between the neutral and the acid I)NPII

is (lue to tile relative incompleteness of the extraction procedures employed.

1370 and 143#{176}C,and it represented a

miX-ture of the DNPH of various ketones an(l

keto acids, some of which were present in

normal urine. This was demonstrated by

extraction of the urinary acids with 10

so-dium carbonate, as described by Seligson and Shapiro, and precipitating any car-bonyl compound present in both the acid fraction and the acid-free residue as the

DNPH. Results of chromatography of these

compounds are shown in Table I.

Isolation of the

2,4-Dinitrophenylhydrazone

of Alpha-ketoisocaproic Acid

Since several attempts to isolate the keto

acids as such from the urine were unsuc-cessful, probably because of the relative

CdSC of decomposition of these substances,

the isoltioii of the DNPH from the keto

acids was undertaken instead.

The mixture of the DNPH of the keto

acids was dissolved in ethyl acetate, and subjected to an initial purification by pas-sage through an acid-washed (i.e., neutral)

alumina column prepared as directed by

Adkins.5 The DNPH derivatives were then

progressively eluted with ethyl acetate, ethyl alcohol and sodium carbonate, as de-scribeci by Datta.6 The greater portion of

the DNPH was eluted with 90% ethyl

alco-hol. The eluants were concentrated by

evaporation with a water pump, and

acidi-fled ‘ith 2 normal hydrochloric acid. The

precipitate was separated and

recrystal-lized from hot water.

The melting point of the DNPH thus

oh-trace tamed was 159 to 161#{176}C (uncorrected).

I+ About 15 mg of DNPH could he isolated + from 5 lTd of urine. The melting point of the

I+ DNPH of alpha-ketoisocaproic acid, as

re-4+ corded in the literature, is

162#{176}C(uncor-trace rectecl).7

Calculated for C2F1,0N,: C: 46.45

H: 4.55%

N: 18.06% Obtained : C: 45.60%

H: 4.58%

N: 17.67%

A mixed melting point with the DNPH

of authentic alpha-ketoisocaproic acidf

showed no depression.

To further prove the identity of the

com-pound, chromatography of the DNPH of

alpha-ketoisocaproic acid, as prepared from

the urine, parallel with the DNPH of

an-thentic alpha-ketoisocaproic acid was

per-formed (Table II).

Ultraviolet spectroscopy of the DNPH of

alpha-ketoisocaproic acid in alcohol

solu-tion showed the expected peaks at 260 and

370 mt.#{176}’#{176}

Conversion of Alpha-keto Acids to Alpha-amino Acids

In order to further confirm the identity of

the DNPH isolated from the urine, and at

the same time obtain some evidence as to

the nature of the other keto acids present,

a Analyses for carl)OI1, hydrogen and nitrogen

1)erfOrlfled by Schwarzkopf Micro-Analytical Labo-ratories, New York.

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‘2+

‘2+

0.81 trace

3+

‘2,4-dinitrophenyiliydrazine

HCI

conversion of the keto acids to their

respec-tive alpha-amino acids was accomplished

according to the following scheme:

1)

Cit3

(1) Cl13-C-C1I3-C-COOlI

-H

Aipha-ketoisocaproic Acid

(1113 II

(‘2) Ci13-C-CH2-C-COOH

II NH2

Leucine

This reaction sequence has been

de-scribed previously by Meister and

Abend-schein” and by Towers

et

al.12 The

alpha-amino acids thus produced were identified

by conventional methods of

chromatogra-phy.

The mixture of the DNPH of the keto

acids from 3 ml of urine was purified by

passage through acid washed alumina with

elution by 90% ethyl alcohol. The alcohol

was evaporated to dryness using a water

pump, and the residue was resuspended in

0.5 normal hydrochloric acid. Two

milli-grams of platinum oxide were added, and

the suspension was hydrogenated at 24#{176}C

and 40 lb/in.2 pressure for 16 hours. At the

end of that period all of the DNPH had

gone into solution. The platinum was

fil-tered off, and the solution was used for

chromatography without further

purifica-tion.

350 MAPLE SYRUP DISEASE

TABLE II

CHROMATOGRAPHY OF THE DNPH OF ALPHA-KETOI.SOCAPHOIC Acm

. Relalire Magnitude

Fraction R1

of Spot

DNPH of “maple syrup” urine (acidic and neutral 0.86 ketones) equivalent to 0.01 ml urine 0.91

I)NP1I of sup1)osed alpha-ketoisocaproic acid (10 g) 0.86

I)NPII of authentic alpha-ketoisoeaproic acid (10 g) 0.86

Descending chromatography. Solvent phase: ethanol 10 parts; water 40 parts; n-butanol 50 parts. Whatman No.

40 filter-paper. Spots identified by intrinsic color.

Chromatography of the Amino Acids

Chromatography of the mixture of the

amino acids produced by hydrogenolysis of

Cu3

---*CH3-C-CH2-C--COOH

II

NO N-N--/\

11

NO2

l)NPII II.,

PtO.

the DNPH of the alpha-keto acids was

mi-tially performed with phenol-water and

In-tidine-collidine-water as the solvent phases

according to the method of Dent.13

With these solvents, spots with Rf 0.75

and 0.42 to 0.49, respectively, were

pro-duced. In relationship to the markers

em-ployed, this corresponded to an area

occu-pied by leucine, isoleucine, nor-leucine and

valine. Repeat chromatography using

n-butanol-acetic acid solvent confirmed this

spot to be composed of leucine, with small

amounts of isoleucine and valine also being

present (Table III, A). Similar results were

obtained by the use of a n-amyl

alcohol-pyridine solvent mixture, as suggested by

Heyns and Walter’ (Table III, B).

From the above, the presence of valine

and isoleucine in the amino acid mixture,

and thus the respective keto acid analogues,

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alpha-keto-TABLE III

AIINo-AcID CHROMATOGRAPHY

Amino Acid

Soi vent A Solvent B

----Distance (cm)

---

Magnitude of Spot

-

-_________

R1

-

Magnitude of Spot

Nor-leucine (1’2.5 gamma) ‘26.’2 4+ 0.4 3+

Isoleucine (1’2.5gamma) ‘24.3 4+ 0.38 3+

Leucine(1’2..Sgamma) ‘2.5.4 4+ 0.36 3+

Valine (1’2 ..5 gamma) 19 ..5 3+ 0.‘29 3+

Norvaline (1’2.5 gamma) - - 0.3’2 3+

Amino acid mixture (equiv- ‘2.5.3 4+ 0.40 trace

alent to 0.03 ml “maple ‘24.0 1+ 0.37 3+

syrup” urine) 19.0 trace 0.Q9 trace

Solvent A: Alcoholic phase of a niixture of n-butanol 40 parts; glacial acetic acid 10 parts; water .50 parts. “Durchiauf” chromatography (17 hours) on Whatnian No. 40 paper. Spots identified by spraying with a 0.1%

solution of ninhydrin in acetone.

Solvent B: Pyridine 35 parts; n-amyl alcohol 35 parts; water 30 parts. Chromatography on Whatman No. 40 paper by descent. Spots identified by spraying with a 0.1% solution of ninhydrin in acetone.

beta-methyl-n-valerie acid in the original

“maple syrup” urine can be deduced.

When the partially purified DNPH of

alpha-ketoisocaproic acid (m.p. 158-160#{176}C)

was hydrogenated, the presence of leucine

together with a trace of valine was noted.

Starting with 2.9 mg of the DNPH,

approxi-mately 2 mg of the amino acid

hydrochlo-rides were obtained, showing the conver-sion to be about quantitative.

Identity of the “Maple Syrup” Odor

Various attempts were made to obtain a

pure sample of the urinary component

re-sponsible for the maple syrup-like odor,

but as yet this has not been successful. Pure

alpha-ketoisocaproic acid has a faint,

cheese-like odor, not at all like that of the

urine.

DISCUSSION

The presence of large quantities of keto

acids in the urine represents a distinctly

abnormal phenomenon. Only small

quanti-ties of pruvic and alpha-ketoglutaric acid have been reported to occur in urine of

normal subjects. Therefore, the presence of

several keto acids in association with a severe cerebro-degenerative disease

im-mediately suggests these two findings to be

causally related, and recalls the findings in

phenylketonuria. In the latter condition,

the urine has a peculiar “mousy” odor,

prob-ably caused by the presence of phenylacetic

acid. Phenylpyruvic acid is excreted in large

quantities, with the corresponding amino

acid, phenylalanmne, present in high levels

both in the blood and urine. In

phenyl-ketonuria the urinary phenylpyruvic acid is

due to the transamination by the kidney of

the excess amount of phenylalanine. In

maple syrup disease, several possibilities

present themselves to account for the

ac-cumulation of alpha-keto acids in the urine.

The first, a failure of the transamination

reaction, must be rejected on the basis of

evidence accumulated by Westall et a!.

These authors showed the presence of

dc-vated levels of valine, leucine and

isoleu-cine in both blood and urine of a child with

this condition, and also found normal

ac-tivity of transaniinase in tissues obtained at

necropsy.

The next postulate, a failure in excretion

or utilization of amino acids, is probably

excluded by the finding of a normal amino

acid pattern in infants during the initial

stages of the illness.1’

Thuis it would appear that the further

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Step I:

CH:,C11CH2COCOOH CH3CHCll3(’O-S-CoA

Cl!3 CH3

Alpha-ketoisocaproic acid Isovalerate

Step’2:

(H:,ClI(ll.(’(’oACH3CC1l(’OSCoA

Cl!3 Cli:

Isovalerate Beta-beta ‘-dillict li’1 acrvla t e

Step 3:

crotonase Oil

C113-C=CH-CO-S-CoA--- -CH3-C-CH2CO-S-CoA

Cl3 1130 Cl!3

Beth-beta’-dimetiiyl acrylate Beta-methyl-beta ‘-hydroxybutyrate

Valine and isoleucine undergo similar reactions, leading to the production of CH3-CH-CO-S-CoA from

OH CH3

valine, and CH,-CH-CH-CO-S-CoA from isoleucine.’5.16 (S-CoA here stands for coenzyme A.)

OH CH3

_______

352 MAPLE SYRUP DISEASE

in this condition. This degradation has, in

the case of leucine, been postulated to

oc-cur along the following pathway:15

a patient with maple syrup disease, a famil-ial cerebral degenerative condition.

Alpha-ketoisovaleric acid and alpha-ket

)-From this point on, however, the

degra-dative pathways of the branched-chain

amino acids diverge. Leucine is ultimately

converted to acetoacetate, while isoleucine

and valine enter the glucogenetic route. It

would, therefore, appear fairly likely that

the degradation of the branched chain

amino acids is interrupted anywhere

be-tween step 1 and step 3 above. Whether

this interruption is due to a congenitally

deficient enzyme necessary in the

degrada-tion of all three keto acids, or whether the

accumulation of alpha-ketoisocaproic acid

inhibits the degradation of the other keto

acids, one cannot as yet say. Umbarger and

Magasanik,hT however, reported that in

cul-tures of mutants of Escherichia coli,

alpha-keto-beta-methyl-n-valeric acid, the keto

analogue of isoleucine, appeared to inhibit

the transamination of alpha-ketoisovaleric

acid to valine.

Further studies now under way to

demon-strate the presence or absence of other

in-termediary metabolic products may

eluci-date the site of arrest.

SUMMARY

A mixture of alpha-keto acids, the most

important of which has been identified as

aipha-ketoisocaproic acid, was found to be

excreted in large quantities in the urine of

beta-methyl-n-valeric acid were present in

smaller amounts.

Their excretion would suggest an

im-pairment of the degradative metabolism of

the branched-chain amino acids as an

im-portant cause of the cerebral degeneration

in this condition.

ACKNOWLEDGEM ENT

The author wishes to express his

ap-preciation to Drs. Sidney Carter, H. H.

Merritt, Rustin McIntosh, Irwin B. Wilson,

and Sara Ginsburg for their assistance in

preparing this paper.

REFERENCES

1. Menkes,

J.,

Hurst, P., and Craig,

J.

:

Pro-gressive familial infantile cerebral dys-function. PEDIATRICS, 14 :462, 1954.

2. Dick,

J.

: Personal communication;

Mai-mon, A. C. : Personal communication.

3. Westall, R. C., Dancis,

J.,

Miller, S., and

Levitz, M. : Maple sugar urine disease.

Fed. Proc., 17:334, 1958.

4. Seligson, D., and Shapiro, B. : Alpha-keto acids in urine. Anal. Chem., 24:754, 1952.

5. Adkins, H. : Alumina chromatography of 2,4-dinitrophenyihydrazones.

J.

Am.

Chem. Soc., 71:3051, 1949.

6. Datta, S. P. : Chromatography of 2,4-dmnitrophenylhvdrazones on alumina.

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HANS H. Z1N55ER, M.D. 7. \Ieister, A. : Enzymatic preparation of

alpha-keto acids.

J.

Biol. Chem., 197:

309, 1952.

8. \Vaters, K. L. : Alpha-keto acids. (l#{236}em.

Rev., 41:585, 1947.

9. Braude, E. A., and Jones, E. R. H. : Studies in light absorption of

2,4-dinitrophenyl-hdrazones.

J.

Chem. Soc., 1945, p. 498.

10. Johnson, G. D. : The ultraviolet absorption

spectra of 2,4-dinitrophenylhvdrazones.

J.

Am. Chem. Soc., 75:2720, 1953.

1 1. \Ieister, A., and Abendschein, P. A. : Chro-matographv of alpha-keto acids. Anal.

Chem., 28:171, 1956.

12. Towers, C. H. N., Thompson,

J.

F., and

Steward, F. C. : Detection of keto acids in plants.

J.

Am Chem. Soc., 76:2392,

1954.

13. Dent, C. E. : Chromatography of 60 amino

acids. Biochem.

J.,

43:169, 1948.

I 4. Heyns, K., and Walter, W. : Papier

chro-matische Trennung der isomeren

Len-cine. Ztschr. phvsiol. Cheni., 287:15,

1951.

15. Coon, M.

J.,

Robinson, W. C., and

Bach-hawat, B. K. : Enzymatic studies on the

biological degradation of the branched

chain amino acids, in McElroy, W. D.,

and Glass, B.: Amino Acid Metabolism.

Baltimore, Johns Hopkins Univ. Press,

1955, p. 431.

16. Robinson, W. C., Nagle, R., Bachhawat,

B. K., Kupiecki, F. P., and Coon, M.

J.:

Role of coenzyme A in valine

metabo-lism.

J.

Biol. Chem., 224:1, 1957.

17. Umbarger, H. E., and Magasanik, B.:

Isoleucine and valine metabolism in E.

coli.

J.

Am. Chem. Soc., 74:4253, 1952.

THE URETEROVESICAL JUNCTION, John A. Hutch, M.D. Berkeley, California, Uni-versity of California Press, 1958, 178 pp., $7.50.

This little book contains an excellent sum-niary of many aspects of the problem of neuro-genie bladder with particular emphasis on the author’s operation for returning competence to

decompeiisated ureterovesical junctions. The

initial section describing the clinical course of

the patient with a traumatic neurogenic bladder is excellent 1)0th ifl detail and in tone. The book

is profusely illustrated both with

roentgeno-grams of excellent quality, drawings and

micro-scopic sections. On this scaffolding a series of

interesting discussions of the pathogenesis of various dysfunctions of the ureterovesical valve

and the details of the two procedures which the

author favors for correction are presented. The

case histories appended are interesting, and in sufficient detail to make them of considerable teaching value to students of this specialized

problem.

The section of the book discussing the

author’s own Pen1tio11 is lucid and, while his results, as might be expected, have been con-siderably l)etter than those of others in the

ap-plication of this procedure, shows remarkable

critical faculty . As he says in referring to an

alternate procedure, “the true indications for these operations cannot be discussed defini-tively until the procedures have had adequate

clinical application to determine their merits.

Only after many operations have been

per-formed and there has been adequate time for long-term evaluation will the true indications

be evident.” This certainly applies to Dr.

Hutch’s own procedure in the minds of most

practicmg urologists who have had the

oppor-tunity to utilize the operation.

The second section, dealing with mechanical

disabilities of the upper and lower urinary tract secondary to other congenital anomalies, is less complete and less lucid. It fails to give

ade-(mate enthusiasm or credit for developments

involving sacral rhizotomy, to the first pioneers

in the field, and to the extensive work with

substitute urinary reservoirs utilizing small in-testine which has aroused considerable interest

in the field at this time.

The make-up of the book is excellent, except

for minor awkward spacings on occasion, and it is embarrassing that Figure 24, entitled “The relationship of the lateral border of the trigone

to the saccule about the ureteral orifice,” and Figure 62, which would appear to be identical to Figure 24, is entitled, “An artist’s conception of how this ureteral vesical junction would have

looked if it had been possible to cvstoscope the

patient.”

The book should certainly be required

read-ing for those undertaking the care of paraplegic

patients, and has many points of stimulation

for the urologist doing a more general type of

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1959;23;348

Pediatrics

John H. Menkes