Tubular reabsorption of free amino acids in infants with scurvy

CLINICA CHIMICA ACTA

TUBULAR

409

REABSORPTION

OF FREE

AMINO

ACIDS

IN

INFANTS

WITH

SCURVY

J. BRODEHL

Univwsitiits (Received

AND W. P. KAAS Iiindevklinik

November

Bonn

(It’.

Crvmany)

10, 1969)

SUMMARY

Clearance

studies

were performed

in 3 infants

with florid scurvy

in order to

determine the transport of free amino acids in this state of vitamin deficiency. The following results were obtained: I. Glomerular filtration rate and effective renal plasma flow were slightly reduced in two infants, and were normal in the third one. 2. Tubular reabsorption of phosphate was also slightly reduced in two infants, which was considered to be due to high phosphate intake rather than to direct impairment of tubular phosphate transport or secondary hyperparathyroidism. 3. The serum concentrations of some free amino acids (threonine, glycine, lysine, histidine and arginine) were below normal, in florid scurvy, the other amino acids were within the normal range. 4. In all three scorbutic infants the tubular reabsorption of almost all free amino acids was completely normal, as proved by urinary excretion rates, endogenous clearance rates and percentage tubular reabsorption. Only the tubular transport of glycine was slightly impaired in two infants. The significance of this finding, however, is questionable. 5. It is concluded that obviously, vitamin C deficiency does not necessarily produce renal hyperaminoaciduria.

INTR9DUCTION

In 1950,Jonxis and Wadman’ reported that vitamin C deficiency in infancy was accompanied by an increased excretion of amino acids. A few years later this finding was confirmed in other cases of scurvy by the same group of investigators213. The mechanism of hyperaminoaciduria in scurvy was found to be of renal origin, since the plasma concentrations of amino acids remained within the normal ranged. Since then, vitamin C deficiency has been listed as one of the many causes which can produce renal hyperaminoaciduria. There are, however, only very few additional reports with contradictory results concerning the excretion of amino acids in scorbutic humans5-’ and animalsR-“. Clin.

Chim.

Acta,

28 (1970)

409-416

BRODEHL, KAAS

410

Therefore, in order to clarify the degree and type of hyperaminoaciduria in vitamin C deficiency, clearance studies were performed in three scorbutic infants, and their urinary excretion, endogenous clearance rates and tubular reabsorption of free amino acids were determined. As will be shown, no generalized impairment of tubular reabsorption of amino acids could be demonstrated in these infants with scurvy. It is concluded that obviously, vitamin C deficiency does not necessarily produce renal hyperaminoaciduria. METHODS AND MATERIALS

The renal clearance studies were performed in 3 infants aged (1 to 12 months, with scurvy. Some of their biographical and biochemical data are listed in Table I. Two of these infants TABLE

were normally

developed,

one infant (H.L.) was mentally

1

SOME BIOGRAPHICALAND BIOCHEMICALDATA OF 3 INF4NTS WITH sCUK\‘Y, 1N ACID

and

CLEARANCE

STUDIES

WERE

WHOM

? ‘HE

AMINO

PERFORMED l3.E.

m

Sex

Birth weight (kg) Age* (months) Weight* (kg) Body surface area* (m*) Serum calcium (mgO;,) Serum phosphorus (mgqh) Alk. phosphatase (mu/ml) ______ * At date of clearance study.

‘t.3 9 7.6 0.39 9.8 4.0 56

M.P.

H.I..

f 3.0 10 74 0.36

f 1.8 IZ 6.9 o.34 9.7 4.5 27

1O.b

6.1 48

physically retarded due to cerebellar hypoplasia with amaurosis. When they were admitted to the hospital they suffered from severe pain in the legs, gingival bleeding and hematomas and typical scorbutic changes of the skeletal system on x-ray. They all had the same history of feeding difficulties and received almost exclusively cow’s milk with carbohydrates without additional fruit juice, vegetables or vitamin C supplementations. All of them had received vitamin D sufficiently and none of them showed clinical, biochemical or roentgenological signs of rickets (Table I). The clearance studies were performed before the treatment with vitamin C was started. In two infants (M.P. and H.L.) the clearance test was repeated after 3 respectively 4 weeks of vitamin C treatment, when all clinical signs of florid scurvy had disappeared. The procedures of the short term clearance test were the same as described elsewherelz. The infants were fasted overnight. Glomerular filtration rate was measured with inulin. Amino acids were determined in serum and urine by exchange chromatography, using the Technicon Auto-Analyzer@ as described elsewhere13. Twelve children aged 2 to 13 years served as a control group whose data were obtained by exactly the same methods as used in this study12. The mean age of this control group was higher than that of the scorbutic infants, but normal values of a group having exactly the same age were not available to us. The children’s group of our previous study was preferred for comparison to the infant’s group, whose ages Clin. Chiwz. Acta,

ZX (1970) 409-416

TUBULAR

REABSORPTION

OF AMINO ACIDS IN SCURVY

4’1

were between 16 days and 4 months and who were known to show physiological hyperaminoaciduria (see also’ j). Inulin was determined in the serum and urine by a micro adaption of the resorcinol method15, $-aminohippurate (PAH) by the method of Smithlo. Phosphate in the serum and urine was determined by the method of Fiske and SubbaRow17. RESULTS

The results of short term clearance studies in three infants with scurvy are listed in Table II. Inulin clearance was slightly reduced in two infants (M.P. and H.L.) and normal in the third one. After treatment with vitamin C the clearance values TARLE

II

THE VALUES OF IXULIN CLEARANCE,PAW-CLEARANCE, TRANSPORT

OF

PHOSPHATE

IN

3 INFANTS

WITH

FILTRATION

PRACTIOS,

ASD

TUBULAR

SCURVY

The clearance studies mere repeated in two infants (M.P. and H.L.) after 3 respectively treatment with vitamin C. All values are corrected to surface area of adults (1.73 m2). H.L.

1W.P.

D.E.

after

befovr

ml/min/r.73 ml/min/r.73 4’ G/min/r.73 9/, mglminlr.73

Gin CPAH

I’F CP

TRP Tmp __. Ci* CPAH I’F Cp TKP Tmp P AA

= = = = = =

m2 m2

113 4.53 24.8 19.3 82.9 5.37

m2 m2

4 weeks of

83 94 395 429 21.1 22.0 16.6 13.5 82.5 83.8 2.3r 4.48 _______~_

befove

after

80 320 25.0 27.5

91 3’5 28.9

65.3

88.1

11.0

__

inulin clearance PAH clearance filtration fraction phosphate clearance percentage tubular phosphate reabsorption maximal tubular phosphate reabsorption

n

mg %

F vitaminC

- deficiency

(n=3)

V-7 EI

1t s

normal

children

in = 12 )

30,

20

r.0

I

0

0 Asp

Thr

Ser

Pro

G/y

Ala

Vol

CyS

bkt

3/w

Leu

ryr

Phe

Orn

Lys

His

Are

Fig. I. Mean concentrations of free amino acids in the serum of 3 infants with florid scurvy (black columns) in comparison to normal values (x + SD) of 12 children (= white columns). The decreases of glycine, lysine and histidine are statistically significant (p < 0.025). Aspartic acid, threonine, and arginine, determined only in two infants with scurvy, are also beyond the normal range (x + 2 SD). For individual values see Table III. Clin. Chim. Acta,

28 (1970)

409-416

HRODEHL,

412 TABLE

KAAS

III

VALUES OF SERUMSCONCENTRATI~S ,ug/min/r.73 m2) 0F FREE .uwivo *cIIx

(PAAexpressetl

in mguo ) IK 3 INFAXTS wm xuwY

AXU OF URISARY

EXCRRTION

RATES

(I’,\,\\

The data mere obtained under clearance study. In t\vo infants (M.1’. and H.L.) the clearance study was rcpea after 3 resp. 4 weeks of treatment with vitamin C. The mean \-alues of the swum concentrations in the inf: with florid scurvy are shown in Fig. I The control T-alues are derived from I2 children’“.

.4sp Thr Scr l’ro c;ly .4la

0.33

0.37

0.33

I.09

I.IS

I.jI

\‘a1 Cys Met Ileu LCU Tyr

I.87 0.88

~

0.21

0.04

I.73 I.27

O.IY 0.1-j

2.Oj I.65 2.42

0.38 0.25 0.32

7.8 20.4 3x.2 0.0 1rS.X

6.3

6.Y

IO.3 IO.9

12.9 4.3

4.Y 8.2

0.d.

n.d.

13 1.q 5.7

10.X

21 .q

IO.0

12.h

17. I

LI.7 2.4

I.3

I.1

I.45

T.hj

2.81

I.SI

2.12

0.2.+

I.32 0.18 0.60 0.98

0.94 0.07 0.30 0.74

1.x7 0.86

I.Oj

0.16

0.20 0.56 0.97

I.73 0.90 O.I9 0.46 0.84

2.10

0.30 0.43 o.xj

0.24

0.05

0.58

0.08

I.19

0.17

0.8.5

0.65

1.10

0.72

0.74

0.83

O.IS

7.4 23.1

2.5 7.6

2.3 ‘3.7

0.80 0.70 1.25 0.84

0.8s 0.62 7.33 0.75

o.i5 0.49 I.79

I.22 0.5’ 1.1.3

0.66 O.SI 1.6-j

I.24

O.j2

1.00

0.95

1.08

0.09 0.1 I 0.29 0.21 0.27

‘5.’ -I.-l 27.j X4.4 i.4

6.3 2.5 13.0 56.7 2 .5

5.7 2.3 13.7 so.3

0.80

0.78 O.hI I.91 I.24 I.49

0.9b

I(,.0

n.tl. 3.7

0.91

not determined

37.5

4.6 4.6

I.37 ~

:

OS.+

2.5 3.8

I.IO

I.84

~

hO.0

9.x 7-4

1.80

I.45 I.34 2.73

n.d. = not detectable.

5’).H ‘4.5

n.tl.

I.SO

I.35 I.I9 I.78

1’he Orn LVS H-is :Zrg

16.1

23.Y 5.0 40.3 IO.1

I.2j

I.68 1.48 I.51

2.3.5

i.3 ‘7 4 3 1’1

‘7.5 38.9 9. 2 57.2 LO.0

Ij.1

23.’

due to technical

1..3 ~~~

4.”

0.5 3.2 3 f, JO..+

1.S 14.5

41.6 I .o

4.3 31.7 39.1 IO.7

0.0

22.3

I IO.2 .VY

difficulties.

VALUES OF ENDOGENOUS CLEARAP~CE RATES OF FREE AMIXO ACIDS (CAA expressed in ml/min/1.73 m2) AKII ov PERCEKTAGE TUBULAR REABSORPTION OF FREE AMINO ACIDS (“” TAA) IN 3 INFANTS WITH SCURVY III two infants (M.P. and H.L.) the clearance study was repeated The values of the Dercentaee tubular reabsorDtion in the florid Fig. 2. The control;alues a& derived from r2children12.

Amino ads

ASP Thr SCr Pro Gly Ala Val CYS Met TlCU Leu Tyr Phe Orn Lys His Arg

Endogenous D.E.

clpavance vales ml/wzin/r.73

hf. P.

H.L.

I.9 3.0 0.0

7.7 I.3 1.6 0.4

2.1 1.8 2.2 0.6

x.7 I.5

3.4 0.6

4.3

2.4

0.5 0.8

0.8

0.1

0.2

0.4 0.2 0.3

2.7

0.4 0.7 0.3 0.3 I.3

I.2

I.9

0.7

0.8

0.6

0.4

0.5

2.2

1.0

7.6 0.3

0.4

_ 0.7 0.8

10.0

0.7

I.0

2.1 0.0

I.2 ~

6.0 0.9

4.’ I.3

4.2

0.9 I.2

0.7 0.5

0.2

2.4 0.1 0.8

0.8

0.8

0.9 0.2 0.5 0.2

btafort 97.9 98.4 97.3 100

92.3 9x.7

0.1 0.2

99.5 99.3 99.4 99.3 97.6

0.3 0.5

I.5

0.7 2.4 3.0

2.0

I.0

2.6

0.5 1.9

1.5 0.4

0.3 0.1

0.8

0.3 I.3

1.2

4.1

8.0

4.0

0.4 2.6 0.1

9.5 0.3

1Zf.P.

I.4 0.4

0.3 0.1 0.2 0.8

1.1

C treatm arc show

mz Contvols S.D. 3.6

0.2 0.j

after 3 resp. 4 weeks, of vitamin scorbutic state of the 3 infants

9x.3 99.4 98.1 91.1 99.4

9x.0 98.4 9X.1 99.6 95.9 99.3

97.x 9X.1 97.7 99.3 95.5 99.2

97.3

9x.7

IO”

91.X gs.9

95.5 98.5

99.8 99.5 99.2 99.7 99.6 98.6

99.8 99.6

98.9 98.8

99.2 99.5

99.8 99.8 98.7

99.8 99.4 97.9

99.2 97.4 96.8

99,1 99.5 98.7 90.9 99.7

99.2 99.5 99.2 95.7 99.5

98.7

97.2 99.4 97.9 95.6 9X.8

99.6 98.4 90.0

99.8 ‘j9.3 9cj 3 99.7 99.6 98.3 9x.s ‘j9.7 9Cj.0 92.2 99.x

TtJRlJLAR REABSORPTION

increased

slightly.

phate clearance

OF AMINO ACIDS IN SCC’RVY

The same applies for the PAH

was elevated

in one infant

(H.L.),

413 clearance.

The endogenous

and in another

infant

phos-

(M.P.)

the

rate of maximal tubular phosphate reabsorption (Tmr) was reduced, After treatment with vitamin C these values returned to normal in both infants. The values of the serum concentrations (PAA expressed in mgy,i,,) and urinary excretion rates (IJnaV in pglminlI.73 m2) of free amino acids are given in Table III. For comparison, the mean values of 12 normal childrenI are added. The mean values A

D.E.

9 months JOJZ

so ,I 05

Asp

-

I f 3 SD normd children

”

Thhr

SET

Pro

G/y

Alo

Fig. z. The values of percentage scurvy (A, @, q) in comparison inciividual =m_lulues see Table IV.

tubular reabsorption of free amino acids in 3 infants with florid to values in normal children (_r t 2 SD == hatched area). For

of the serum concentrations of the three infants in the florid state of scurvv are compared with the normal values in Fig. I. The rates of endogenous clearance (C.4, in ~~l/~nilliI.7~ m2) and percentage tubular reabsorption (11, TA~x) of free amino acids are listed in Table IV. The values of the percentage tubular reabsorption in the scorbutic infants in comparison with the normal values are shown in Fig. 2. The serum concentrations of some amino acids were lower in the infants with florid scurvy than in normals (Table III, Fig. I). This was especially valid for threonine, glycine, lysine, histidine and arginine, in which the differences were statistically significant ($ < o.ozg). The concentration of aspartic acid was higher in two scorbutic infants than in normals, all other amino acids, including tyrosine, were within the normal range. The rates of urinary excretion were within the normal range for almost all free amino acids measured (Table III). There were only very few exceptions : the excretion of glycine was somewhat higher in one infant (D.E.) than in the control group. The value, however, was still in the normal range of younger infants, whose mean value for glycine excretion is C OiYV = I1cj.o i- ~~.4~,g~rnin~r.~3 m2 (ref. 12). The excretion of valine was elevated in infant I-I. L. This, however, cannot be related to vitamin C Uin.

Chim.

.4cta,

AS (1970) py-.+rf>

BRODEHL,

414

KAAS

deficiency, since it remained on the same level in the control clearance examination after vitamin C treatment. Endogenous clearance rates were normal for almost all amino acids measured (Table IV) with the same few exceptions: the clearance of glycine in D.E. was higher than in the control group, but was still within the normal range of younger infants, whose mean

value is Ccl, = 7.4 _C 3.2 ml/min/r.73

valine was elevated

in H.L. before and after treatment

amino acids had normal clearance rates. The percentage tubular reabsorption,

m2 (ref. rz). The clearance with vitamin

the most sensitive

of

C. All other free

parameter

of tubular

function, was within the normal range for almost all free amino acids measured in the scorbutic infants (Table IV and Fig. 2). Only glycine showed a slightly less complete reabsorption than normal in two infants (D.E. and H.L.). Their values, however, were again within the normal range of younger infants, whose mean value for percentage tubular reabsorption of glycine is 87.6 + 6.415 (ref. 12). Therefore it is doubtful whether reduced glycine reabsorption is really significant. The reabsorption of valine in infant H.L. was somewhat lower before and after treatment with vitamin C. The reabsorbtion infants.

of all other

amino acids was completely

normal

in all three scorbutic

DISCUSSIOiX

Our findings demonstrate that there is no real impairment of kidney functions and, especially, of tubular reabsorption in florid scurvy. Glomerular filtration rate (Gin) and effective renal plasma flow (C PA”) were only slightly and nonsignificantly reduced in two infants, and normal in the third one. The tubular reabsorption of phosphate was also low in two infants and became normal after treatment with vitamin C. Since there was no hypophosphatemia (Table I), it is more likely, that the reduced phosphate reabsorption was due to the high phosphate intake from cow’s milk feedingIS, rather than to secondary hyperparathyroidism responsible for phosphate diabetes in ricketslg, or to a direct tubular impairment of phosphate reabsorption produced by vitamin C deficiency. The tubular reabsorption of free amino acids was almost completely normal in all three infants with scurvy. The only exception seemed to be the transport of glytine, which was slightly reduced in two of the three infants. Their percentage tubular glycine reabsorption was lower than in the control group. As already pointed out, the mean age of the control group used for comparison was higher than that of the scorbutic infants studied, and the values of the glycine reabsorption in the two infants were still within the normal range of younger infants. Therefore it is questionable, whether or not the observed reductions are significant. They are certainly not very spectacular. After treatment with vitamin C, the value of glycine clearance and glycine reabsorption became normal in H.L. This might indicate that glycine reabsorption had indeed been slightly impaired by vitamin C deficiency in this infant. Our findings are in contrast to those of Jonxis and coworkerslpd. In their early studies, these authors described increased excretion of cc-amino nitrogen and generalized hyperaminoaciduria in a total of 4 infants with scurvy. The hyperaminoaciduria was to a considerable degree due to increased excretion of bound amino acids, as proved by hydrolysis of the urine and its subsequent analysis. Furthermore, in one of

TUBULAR

REABSORPTION

OF

AMINO

ACIDS

IN

4’5

SCtTRVY

the reported infants the hyperaminoaciduria treatment with vitamin C2, but even increased

did not disappear after appropriate during the first 60 days of treatment.

Therefore it seems questionable, whether in those cases the reported increases of urinary amino acids were exclusively caused by vitamin C deficiency, and whether the physiological variability of amino acid excretion in infancy could be correctly interpreted at that pioneering time of amino acid investigations. Further reports gave contradictory results : Dustin and Bigwood6 reported of a T-day-old(!) infant who was diagnosed to have vitamin C deficiency and relative hyperaminoaciduria. Chadwick and coworkers5 reported of an elderly scorbutic patient with abnormal amounts of amino acids in the urine, which disappeared after vitamin C treatment. Hodges and coworkers20 mentioned increased excretion of hydroxyproline in experimental human scurvy. Efron et al.‘, studying the influence of vitamin C deficiency in a patient with congenital hydroxyprolinemia, did not find increased excretion of any free amino acid other than hydroxyproline. The latter finding was thought to be due to increased turnover of collagen rather than to renal impairment in the state of vitamin C deficiency. Robinson and \J(:arbutonZ1 did not detect increased amounts of tyrosine or tyrosine derivatives either in 5 adults and one child with florid scurvy. In scorbutic guinea pigs, hyperaminoaciduria was observed which was mainly due to increased excretion of glycine 9t11. In a recent study, Barnes d al.” suggested that scurvy in guinea pigs was associated

with some degree of general aminoaciduria,

which offsets the decreased excretion of free amino acids to be found in scorbutic guinea pigs as a consequence of inanition due to vitamin C deficiency. These authors observed also an increased excretion of free glycine and histidine in scorbutic guinea pigs, which was only partly attributed to renal defect and partly to the anomalous excretory response of those amino acids to inanition. Free and bound hydroxyproline were found to be excreted in lower amounts by scorbutic guinea pigs than by the controls. The same observation was reported by Martin and coworkerP. Our knowledge of the biochemical action of vitamin C is very limited (see ,,). Vitamin C is known to be involved in the hydroxylation of proline to hydroxyproline within the protocollagen molecule 23-28. Furthermore it acts as a cofactor of the p-hydroxyphenylpyruvic acid oxidase of the liver, which is characterized by slow maturation in prematurity and early infancy 28--34.Loading with protein and tyrosine, therefore, produces hypertyrosinemia and tyrosyluria in premature and young infants and in patients with scurvy31933J-37. These metabolic defects due to vitamin C deficiency seem to be of much greater clinical significance than the slight tubular defect, if any. The tubular dysfunction in scurvy does not stand any comparison with the generalized tubular defect of amino acid reabsorption regularly encountered in rickets due to vitamin D deficiencv.

ACKNOWLEDGEMENTS

The authors gratefully acknowledge the technical assistance of Mrs. G. Servos, Miss A. Jakel, Miss C. Kern and Miss M. Kowalski, R. N. The investigations were supported by grants of the Deutsche Forschungsgemeinschaft. Bad Godesberg, Germany. Clip.

Chim.

Acta, 28 (1970)

409-~1h

416

BRODEHL,

KAAS

REFERENCES J. H. I’. JONXIS AND S. Ii. WADMAN, d%faandschv. IiindP~gcnc~sk., 81 (1950) 251. J. H. P. JONXIS AND T. H. J. HUISMAK, Pediatvics, I4 (1954) 238. T. H. J. HUISMAK ANIJ J. H. 1). JONXIS, Avch. Diseases Childhood, 32 (1957) 77. T. H. J. HUEMAX, Prdiatvics, 14 (1954) 245. J. M. CHADWICK, J. S. FAWCETT ASD F. (G. WARHURTON, .4nwv. J. (Ilin. AVb., 7 (1959) 7’1. J. P. DUSTIN ASD E. J. BIGWOOD, P?C~c.A’b’utv. sm., 12 (1953) 293. &I. L. EFRON, E. nr. BIXHY, T. L). Ii. ~IoCKADAY, 1,.H. SMITH AxI) 1:.AIESMORER. ~i0ChZm. Biophys. Acta, 165 (1968) 238. 8 M. J. BARKES, H. J, CONSTABLE AND E. KODICEK, Biochiun.Biophys. Arta, 1x4 (1969) 358. 9 E. hI. GADDIS, L. J. FISHER, C. IX. MILLER AKD L. I:.ROSENBERG, Pvoc. Sot. Exptl.Riol. Mrd., 1zo(Ig65) 185. IO G. R. MARTIN, S. E. MERGESHAGEK AND U. J, I'ROKOP, A’atuv?,191 (1961) IO&. 17 (1968) 386. II S. J. SAUNDERS ANI) R. E. KIRSCH, Metabolism, 12 J. BRODEHL AND K. GELLISSEK, Pediatrics, .+z (1968) 395. 13 J, BRODEHL, _-1. JXKEL ASD W. HAGGE, Trchnicon Sywzp., Frankfwt /96-j, 'I‘echnicotl GtnbH, Frankfurt, 1966, p. 473. I-1 F. SERINI, H. MCNAMARA, N. SHIRUYA, ?C:.KRETCHMER AXL) H. I,.BARNHTT, Prdiatvics, 15 I 2 3 4 5 6 7

15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37

(1955) 575. J. H. ROE, J. H. EPSTEIK AICD r\'. f'.GOLDSTEIK, J.Bzol. Chew., 178 (19~9) 839. H. W. SMITH, Przncipl~s of Henal Physiology, Oxford liniv.r-'ress, New York, 19.57. C. H. FISKE 4xD x7-. SCBBAROW, ,I.Rid. Chenz., 66 (1925) 3j’j. D. 11.THOMPSOK AND H. H. HIATT, ,I.cli?z.~?n!est., 36 (1957) 566. 11.FRASER, S. 'iv,KOOH 4xD C. R. SCRIVER, Pcdiat. Res., I (1967) 42.5. R. E. HODGES, E. hl. B.~KER, J. HOOD, H. E. SAURERLLCH ANI) S. C. MARCH, Aww. J. C/in. N&v., 22 (1969) 535. Ii. ROHINSON API‘DF. G. \VARBUTOZ, Katurc, LIZ (1966) 1605. \V. E. I?',. /JW5f., 2s (1949) 8Oh.