Tohoku

J. exp.

Med.,

1977,

Determination

121,

of

173-178

Hydroxylysine

in

Urine

TOKUTARO SATO, TSUYOSHI SAITO, MASARU KOKUBUN, MASAAKIITO and KAORU YOSHINAGA

The Second Department of Internal Medicine, Tohoku University School of Medicine, Sendai 980

SATO, tion

of

T.,

SAITO,

A new

method

ml

for

titrated

to on

with

was

separated

by

preparative

was

cut by

and

sodium

assayed

with

pH

2.0

with

50 •~

4

NH4OH. the

2 (H+

with

by

chromotropic

1•~8

was

shown

acid

(formate to

form).

be

An

paper

formaldehyde

approximately

was

were

1.5

from

serine

and

the

of

110 ƒÊmoles

with from

the

NH4OH

threonine, hydroxy water

was

hydroxylysine

periodate

and

hydroxylysine per

from N

to

eluate

to

column

eluted

with

removing

rate

diluted to

eluate

liberated

after

Excretion

is described. hr

corresponding of

Determina 173-178•\•\

subjected

acids

the

aliquot

reagents,

(2),

24

then

especially The

and

for

Amino in

K.

121

hydroxylysine

and

acids,

water.

1977,

urinary

N HCl, form).

amino

YOSHINAGA,

collected

Hydroxylysine

other

and Med.,

urine

chromatography.

eluted

M.

exp. of

human

metaperiodate,

Dowex

adults

of

paper

ITO, J,

determination

Dowex N

from

was

oxidized

1.5

M.,

Tohoku

colorimetric

chromatography

lysine

Urine.

one-hundredth

and

column

KOKUBUN,

in

Approximately 25

T.,

Hydroxylysine

in

iodate urine

of

day. --•\urinary

hydroxylysine

Basement membranes belong to collagen family, criteria of which include the occurrence of hydroxylysine and hydroxyproline. The content of hydroxylysine in basement membranes is approximately 5 times as large as that of collagen fiber (Spiro 1967; Sato et al. 1975a), and the content of hydroxylysine in glomerular and tubular basement membranes from the diabetics is significantly larger than that from the nondiabetics (Beisswenger and Spiro 1973; Sato et al. 1975b). So, urinary excretion rate of hydroxylysine can be a good indicator in the studies of the basement membrane metabolism under pathological conditions as diabetes mellitus. Specific methods for the determination of certain amino acids such as hydroxy proline, threonine and serine are available, but the colorimetric method for hydroxylysine has not been reported. This communciation reports a method for determination of urinary excretion rate of hydroxylysine. MATERIALSAND METHODS Materials. the

collection

Received

Urine for

24

was hr.

collected The

for publication,

24

from hr

urine

October

individuals was

frozen

1, 1976. 173

and and

kept kept

at at

4•Ž -15•Ž

under until

toluene use.

during

174

T. Sato Separation

urinary

of

one-hundredth The form),

column

peptides ml

50%

After paper,

paper of

were

eluted

outline

Fig.

was was

1.

with

diluted

to

N

dryness

the

stored

to to with

1.5

of

The

applied

exhaustively

evaporated

An outline

which

was

corresponding

in

urine

25 a

ml

and

column

and

to

pH

cm)

remaining eluate

then

determination

thawed,

titrated

The The

vacuo,

and

was

(1.4•~11

water.

NH4OH. in

separation urine

of the procedure

for determination

at

10

centrifugation

standard

shown

washed

in

of

approximately

2.0

with

(H+

adsorbed amino

was

HOl.

50•~4

substances

residue

2 N

Dowex

containing the

of

and

on

acids

dissolved

and in

1.0

ethanol.

Fig. 1.

on

hr sample

was

was

of

24

urine

which

An

is shown

of

acidified

the

hydroxylysine.

hydroxylysine

et al.

to

mixture Fig.

2000

developed the of

position

leucine,

rpm in

for butanol: of

glycine

min acetic

hydroxylysine , threonine,

,

of hydroxylysine

100 ƒÊl acid:

of the water

was

cut

aspartic

supernatant (4:1:5)

according acid

was applied 12 hr . The

for to

and

in urine .

the

guide

strips

hydroxylysine

as

2.

Fig. 2.

Preparative

paper chromato

graphy of amino acid fractions from two cases (N.S. and S.H.). The paper was developed in butanol: acetic acid: water (4:1:5) for 12 hr, and amino acids were detected by ninhydrine. Leu, leucine; Tyr, tyrosine; Thr, threonine; Asp, aspartic acid; OHLy, hydroxylysine.

Hydroxylysine The glass

paper

wool

was

eluted

column,

Determination 1%

of

aqueous

reaction 400

mixture

the

was

The

The

through Aliquots

reagent

(O'Dea

absorbance

at

and 570

water, and

and

a

small the

at

was

eluate 2.0

ml)

column

read

cm)

a

of

for

0.1

30

ml

of

min.

The

Dowex 1•~8.

added

heated

through

with for

were

and

passed

water.

added

(0.3•~0.5

with

of

temperature

(0.2 ml)

1953)

was

ml

were

room

filtrate

Gibbons

nm

the in

(0.4

oxidized

of

175

dissolved

samples

and

passed form).

acid

dark.

with dryness,

metaperiodate

(formate

chromotropic

to

hydroxylysine.

sodium

mesh

exhaustively

evaporated

in Urine

200

with

30

2.0

min

at

ml

of

100•Ž

in

spectrophotometer.

RESULTS Periodate

and

acid

could

And

formaldehyde

which

be removed

Spectrograms

of

the

similar

Employing lysine

was The

followed standard

0.05 As

subjects

to

shown

acid

reagent.

the

color

excretion

an small

the

column

by

the

reaction

(Fig.

4).

Absorbance

curve

for

hydroxylysine

absorption

reaction,

the

at

560 is

1•~8

form).

in

Fig.

from

of

platen 5

after with

hydroxy 20

3.

min.

straight

line

Table

1,

approximately rate

daily

urinary

excretion

110 ƒÊmoles, was

corrected

ranging for

body

rates from surface,

of

hydroxylysine

42 and

to

of

239 ƒÊmoles. the

average

,ƒÊmoles/m2/day.

Fig.

3.

urine

nm.

oxidation

Fig.

reacted

shown

560 of

reached in

(formate

isolated at

shown

chromotropic

oxidation

are

course

nm

with

periodate

fraction

maximum acid

color

of Dowex by

hydroxylysine

with

chromotropic

brown

hydroxylysine

developed

and

intense

0.2 ƒÊmoles. in

were

by

front

spectrum the

develop

completely

hydroxylysine

showed

from

which

liberated

chroinotropic

Authentic

urinary

iodate,

Spectrograms

of the

reaction

mixtures

of chromotropic

acid

reaction.

adult The

was

68

176

T. Sato

Fig.

4.

Oxidation

using

1%

of

hydroxylysine

sodium

Fig. 5.

(0.2 ƒÊmole)

at

room

temperature

periodate.

Chromotropic

TALE

et al.

1.

acid

Urinary

reaction

of hydroxylysine,

excretion of hydroxylysine

DISCUSSION In the study

of the basement

a colorimetric

method

for basement

membrane

membrane

to determine or collagen.

metabolism,

hydroxylysine

it is advantageous

which

is a specific

to have amino

acid

Hydroxylysine In

this

periodate

study, to

Dowex

release

50•~4.

oxidation,

and

formaldehyde,

Threonine

so

paper

monosaccharide

these

amino

in Urine

polysaccharide,

were

and

serine

acids

were

177 which

separated can

by

also

column

release

separated

can

be

oxidized

chromatography

formaldehyde

from

by on

by

hydroxylysine

by

periodate preparative

chromatography. The

dark

periodate

to

oxidation

obviate

the

of

hydroxylysinee

occurrence

of

both

was

invariably

photochemical

carried

side

out

reactions

in

and

the

reagent

decomposition. Formaldehyde to

yield

But of

a

reacts

highly

chromotropic the

and lead

is

(1953)

acid

and

rapidly

periodate

Gibbons

chromotropic

compound,

acid

excess

with

colored

the

oxidized

together

with

succeeded

in

by the

on

heating

reaction

is

periodate,

and

inorganic

removing

in

strong

specific the

and

acid

formaldehyde.

complete

products

periodate

sulfuric

for

removal

is essential. iodate

as

O'Dea

precipitate

of

dithionate. In

this

of

lead

of

Dowex

study,

a small

dithionate

to

1•~8

smaller,

Chromotropic To

and

this

Absorption curve

and

color

this Urinary

rather about

wide 5

corresponds

absorbance

reaction

range

times to

is

approximately

form) order

in

was

to

water

reacting by

used

make

the

in

the

without

with reaction

mixture

between as

of

showed 0.05

an

to

amino

hydroxylysine ranging

that

out

color

reaction

sensitive of

variation, than

carried

In

using

urinary

between

stead

amount buffer.

pigments. chromotropic

subtracted.

line

as

rate of

larger

the

straight

(formate

iodate.

developed

was of

showed

excretion

and

a slight

samples spectrum

standard

Dowex 1•~8

was

developed

effect,

unoxidized

of

periodate

oxidation

acid

eliminate

acid

column

remove

acid

from

42

to

of

by

the

239 ƒÊmoles glycosides

glomerular

at of

560

nm.

The

hydroxylysine,

analyzer.

determined

O-hydroxylysyl

300 mg

a peak

02,ƒÊmoles

basement

method per

(Sato

day, et

al.

showed which 1976)

is and

membrane.

Acknowledgment

This work was supported

by a grant from the Ministry of Education (No. 167081).

References

1)

Beisswenger, P.L. & Spiro, R.G. (1973) Studies on the human glomerular basement membrane. Diabetes, 22, 180-193. 2) O'Dea, J.F. & Gibbons, R.A. (1953) The estimation of small amounts of carbohydrates and other substances with periodate. Biochem. J., 55, 580-586. 3) Sato, T., Munakata, H., Yoshinaga, K. & Yosizawa, Z. (1975a) Chemical composi tions of glomerular and tubular basement membranes of human kidney. Tohoku J. exp. Med., 115, 299-306. 4) Sato, T., Munakata, H., Yoshinaga, K. & Yosizawa, Z. (1975b) Comparison of the chemical composition of glomerular and tubular basement membranes obtained from human kidneys of diabetics and nondiabetics. Clin. Mini. Acta., 61, 145-150. 5) Sato, T., Saito, T. Yasuda, K., Kokubun, M., Yoshinaga, K. & Yosizawa, Z. (1976) Determination of 0-hydroxylysyl glycosides (OHLG) in urine. Tohoku J. exp. Med., 118, 81-87.

178

6)

T. Sato

et al.

Spiro, R.G. (1967) The structure of the disaccharide basement membrane. J. biol. Chem., 242, 4813-4823.

units of the

renal glomerular