Stability of 8-Aminolevulinic Acid and Porphobilinogen in Urine Under Varying Conditions Irene Bossenmaier

and Ruth Cardinal

A study was made on the stability of 8-aminolevulinic acid and porphobilinogen in urine. Data are presentedshowing that both substancescan be preserved in the same sample by adjusting the pH to 6-7 and storing at -20#{176} for periods up to 1 month, or at 4#{176} for periods up to 2 weeks. As previously shown (1), these conditions are also satisfactory for preservation of coproporphyrin. of the porphyrin precursors 8-aminolevulinic acid (ALA) and porphobilinogen (PBG) in human urine is of importance from the standpoint of analysis in the clinical laboratory. The qualitative Ehrlich reaction for PBG may change from strongly positive to negative in a few hours if the urine is kept at room temperature. This may result either from the formation of an inhibitor of the reaction and/or simple disappearance of PBG through conversion to porphyrin, porphobilin, or other unidentified substances. If due to ‘the inhibitor as previously described (2), the same amount of PBG will be found as initially determined when the resin method of Mauzerall and Granick (3) is used. When analysis of the freshly voided urine is not possible, as with 24-hr. collections, it is important to know the optimum conditions under which the urine can be preserved. Haeger-Aronsen (4) stated that PBG is labile and should be determined as soon as possible, but if delayed, the urine should be maintained at pH 7-9 at 40 in the dark. ALA, on the other hand, was found to keep for at least 20 days at pH 4-7 at 4#{176}. According to these criteria, two samples would have to be stored-one at the former and one at the latter pH range. Henry (5) stated that ALA in urine is stable if the urine is kept

1THE

From

QUESTION

time University

OF

of

STABILITY

Minnesota

Medical

Unit,

Northwestern

Hospital,

Minneapolis,

Research

Fund.

Minn.

55407. Supported in part by the John H. and Mary Briggs The authors are grateful to Professor C. J. Watson

the course Received

Porphyria

for his advice and encouragement of this study and preparation of the manuscript. for publication Mar. 21, 1968; accepted for publication Apr. 15, 1968. 610

during

Vol. 14, No. 7, 1968

PORPHYRIN

PRECURSORS’

611

STABILITY

below pH 7.0, and that PBG must also be kept at an acidic pH; in fact, lie believes the pH for the latter should be maintained below 1.0. This conclusion is based on previous work by Cookson and Rimington (6), which referred purely to PBG in 0.5 N HC1, not in urine. It was previously shown by Schwartz et al. (1) that coproporphyrin is better preserved in urine of pH > 6.0, preferably 7-9. From these various observations it is evident that if determination of all three substances is to be carried out on the same urine sample, e.g., a 24-hr. collection, it would be best to maintain a neutral pH. The present study was undertaken to provide more definite information about keeping the urine specimen prior to determination of these compounds.

Materials and Methods Urine samples from 5 patients with acute intermittent porphyria were used in the study, their native concentrations of PBG- and ALA being recorded as initial values ill the tables. The quantitative method Table

I.

EFFECT

OF

pH, TEMPERATURE, IN 24-HR.

URINE

AND TIME

ON

PBG

AND

ALA

ConnEcTioNs Conditions PBG

E xperiment

dat,

p11

Temp.

EXPERIMENT

(CC)

1*

94

11)

1

20-23 4 20-23 4

72 94 sO 75

IS 19 19 IS

7

20-23

35

Initial 1 day

7

7

4 clays

4

55

16 18

1

4

11 60

IS IS

7

4

76

17

71

20

7 1

‘

iSdays

ALA

(mg/day)

(mg/day)

20-23

EXPERIMENT

2*

Initial

7 days

7

-20

70

20

21 days

7

-20

72

15

56 days *

ment

Experiments

1 was started

7 1 and

2 were

on Aug.

performed

on

urine

23, 1965, and Experiment

-20 samples

from

2 on Sept.

65 the same 10, 1965.

18 patient

(P.K.).

Experi-

612

BOSSENMAIER

Table

2.

EFFEc’

is Conditions p11

ph,

TEMPERTcRE,

URINE

COLLECTIONS

(mF

24-flit.

& CARDINAL

4 days

Temp.

(‘C)

AL.4

i’BG

3

EXPERIMENT

4 4

3.6 9.0

7

4

1) 1

4 -20

5 7 9

-20 -2() -20

10.4 10.4 2.7 10.7 10.4 1(1.1

IS

ALA

I’BG

7.5

8.6 8.6

8.2 7.6

9.9 9.9

8.2 6.9

9.0 8.8

7.4 6.0

6.() 7.1

6.9 4.9

7.5 8.5 8.8 8.2

1.4 11.5

t

8.5

10.7 10.7 10.0

8.5 8.0

8.2 9.3

8.5 7.4

7.9

8.5

7.5

7.4 8.1) 8.2 7.9

11.2 11.0

92.1)

94.4

39.1)

94.4

39.() 35.5

72.8 96.1) 1)3.2 90.0

39(1

40.0

50.8 97.6

37.6 38.4

94.4 91.2

Performed

on Jan.

8, 1967. Initial 7, 1967.

Initial

values: values:

8.2

4 (.mc)t

4

Not determined. Performed omi Feb.

f 3.3

4

t

t

8.6 8.5

31) .8

*

ALA

6.1)

42.0

-20 -20 -20 -20

I’BG

8.2 9.0

70.0

1 5 7 9

23 days .4LA

1.9 8.2

86.8

#{182}1

ALA

(J(.)*

40.1) 42.2 40.1)

4

AND

I)Y)

14 days

83.2 9S2 94.1)

4

MG.,

Chemistry

8.4

EXPERIMENT

1 5 7

ON PI3U

\Nl) TIME

(\Am.rEs days

PBG

1 i

Clinical

PBG, PBG,

f

39.2 41.0 38.4

42.0 37.1) 35.4 40.4 41.0 37.0 39.4

t 94.8 1)9.6 90.0

38.4 12.9; ALA, 100.4;

42.6

89.2 94.8 94.8

73.6 85.2 86.8

t 92.4 94.4 86.0

41 .2 42.4 26.1) 30.6 4L6 41.8 35.0

38.9

7.6.

ALA

37.0.

of Mauzerall and (lraiiick (3) was employed to determine the ALA and PBG. Urine pH was measured with a Beckman J)H meter, adjustments being made with hydrochloric acid, acetic acid, or sodium carbonate to provide the range which was studied. lii Experiment 1 (Table 1), an aliquot of a 24-hr. urine collection was unprotected from light and left at room temperature. A duplicate portion was kept in the dark at 4#{176}. In Experiment 2 (Table 1), the urine was kept in the dark at -20#{176}. In Experiments 3 and 4 (Table 2) and Experiments 5 and 6 (Table 3), separate tul)es of urine at various pH levels were stored as in Experiment 2, with their duplicate test samples kept at 4#{176} under similar conditions. All tubes were stoppered, and one of each was taken out at the specified times for the determinations. The initial controls in Experiments 1-6 were run 011 the same day that the 24-hr. urine collection was complete(l. In Experiment 7 (Table 4) the control was a freshly voided sample; the determinations were run as indicated, the tubes being stored at 4#{176} in the dark. No appreciable change in pH was observed in any of the stored samples. Duplicate determinations of ALA and 1’BG were analyzed Ofl various samples, as noted in Experiments 5 and 6 (Table 3).

Vol. 14, No. 7, 1968

Table

PORPHYRIN

3.

EFFECT

Conditions pH

OF

24-us.

IN

pH,

TEMPER.\TLTRE,

URINE

(‘C)

I’BG

ALA

PBG

.4LA

-20

7

127 127

-20

23.9 23.6

-

-

5

4

127

24.2

7

4

-

-

-20

7

-

-20

4

-

7

4

-

Duplicate

PBG: *

determinations

PBG

14 days PBG

ALA

28 days

ALA

PBG

ALA

5 (.ic)

23.0 22.6

122 122

23.0 23.3

123 120

23.5 23.8

120 121

24.9 24.7

117 117

23.3

122

23.0

122 123

22.0 22.2

120

22.0

122

23.2

120 121

20.4 20.4

123 123

22.1) 22.2

116

22.5

114 114

23.5 23.S

108 102

24.2 24.8

122

22.4

115

21.8

116

22.7

116

21.4

108

15.7

187

6

62.0

22.8

23.7

94

(.K.)t

194 195

62.2 62.4

191 189

60.2 60.8

189 187

62.4 60.9

189

60.2 60.8

193 192

60.6 60.7

19() 190

61.6 60.2

193 191

60.0 51)5

192 190

58.3 57.0

187 190

S2.7 52.7

187

62.6

191

62.2

185

61.8

166

62.8

153

58.4

-

191

59.3

188

60.2

190

55.7

176

56.4

175

46.4

have

been

performed

-

5

ALA

.\NI)

123 123

193

-

PB(

7 lays

ALA

EXPERIMENT

5

(IN

IN MG./DY)

days

PBG

EXPERIMENT

5

TIME

(V.LcEs

4

1 day

613

STABILITY

AND

COlLECTIONS

6 hr.

Temp.

PRECURSORS’

wheim

127 and 127 are values for duplicate determinations. Performed on Nov. 20, 1967. Initial values: PBG,

two

values

127 and

are

given:

Experiment

127 (duplicates);

ALA,

23.6

5, and

23.8.

t

Performed

Table

oh

Dec.

7, 1967.

Initial

7 (S.M.L.):

4. EXPERIMENT

IN A FRESH

values:

EFFECT

URINE

PBG,

6.2 9.0 Performed

(‘C)

24, 1967. Initial

%N1) TIME

MG./100

PBU,

amid 60.8.

(IN

PBG

.NI)

ALA

MI..) 24 hr. PBG

ALA

6.8 6.8 values:

IN

61.0

hr.

PBG

4 4 on July

(VALUES

4 Temp.

195; ALA,

phi, TEMPERATURE,

oF

SPECIMEN

Conditions p11

198 and

1.6 1.6 7.6; ALA,

6.5 6.7

ALA

1.7 1.6

1.6.

Results As SCQII from the results iii Experiments 1, 3, and 4, IB( not stable at 1)11 1.0, under aiy of the C011(litiOlLS studied. At pH 5, 7, and I) (Experiments 3 and 4) alIt! pH 5 all(l 7 (Experinieiits 5 and 6) the -20#{176} temperature was better for preserving PBG over the longer period of time, although 4#{176} was adequate for at least 1 week, during which time

614

BOSSENMAIER

& CARDINAL

Clinical

Chemistry

only inconsequential diminution was observed. Slight decrease of PBG was noted after 4 and 24 hr. in a single freshly passed urine sample (Experiment 7) kept at 4#{176}, either at the native pH of 6.2 or after addition of Na2CO3 to pH 9.0. The ALA appeared quite stable under all conditions studied with only moderate decline in a few instances-most noticeable in the 28-day, 4#{176} values at pH 7 and 9 (Table 2) and at pH 7 (Table 3). The slight increase of ALA seen in Table 2 may have resulted from resin batch differences which were not completely controlled during these determinations. Attention had been drawn to this possibility by Williams and Few (7) on the basis of a personal communication by Rimington.

References I.

2.

3.

4.

5. 6.

7.

Schwartz, S., Zieve, L., and Watson, C. J., An improved method for the determination of urinary coproporphyrin and an evaluation of factors influencing the analysis. J. Lab. GUn. Med. 37, 843 (1951). Watson, C. .1.,Bossenmaier, I., and Cardinal, R., Acute intermittent porphyria: Urinary porphobilinogen and other Ehrlich reactors in diagnosis. J. Am. Med. Assoc. 175, 1087 (1961). Mauzerall, D., and Granick, S., The occurrence and determination of #{246}-aminolevulinic acid and porphobilinogen in urine. J. Biol. Chem. 219, 435 (1956). Haeger-Aronsen, B., Studies on urinary excretion of a.aminoiaevuhic acid and other heme precursors in head workers and lead-intoxicated rabbits. Scand. J. Clin. Lab. Invest. 47 (Suppl.), 26 (1960). Henry, R. J., Clinical Chemistry: Principles and Technics. Hoeber, New York, 1964, p. 828. Cookson, G. H., and Rimington, C., Porphobilinogen. Biochem. J. 57, 476 (1954). Williams, M. K., and Few, J. D., A simplified procedure for tile determination of urinary -aminolaevuiinic acid. Brit. J. md. Mcd. 24, 294 (1967).