Rise in plasma trimethyllysine levels in humans after oral lysine load

Rise after

in plasma trimethyllysine oral lysine 2

C Vijayasarathy,

MSc;

Latifa

Khan-Siddiqui,

ABSTRACF marked

Oral and

load.

Urinary

after

the

load.

This

did

load.

plasma

SN Murthy,

not

register rise

a similar

rise.

in TML

or carnitine

synthesis,

urinary

lysine

e-N-Trimethyllysine

is a known

(TML)

in many

species

including

man.

(4) and

(5). The lysine,

as a free

three

amino

acid

e-N-methylated

-N-dimethyllysine,

lysines, and

precursor In rats,

of

is given

Materials

to

in plasma

and

urine

&N-monomethyl-

-N-trimethyllysine,

healthy

male

out. In the first experiment volunteers were given an

six

oral

load of 5 g lysine. In the second experiment, effect ofa nonprecursor amino acid, tryptophan, was tested by administering 5 g orally to six healthy male volunteers. The protocol was reviewed

772

carnitine

seen

after

produced also

trimethyllysine,

the

a lysine

increased

a 5 g tryptophan

is not a nonspecific

plasma

PhD

consequence

urinary

of

trimethyl-

by the human experimental ethics and informed consent was obtained lected for the study. The experimental

earlier

estimated

TML method trations

(7). Plasma

according

was isolated of Kakimoto ofTML

and

to the

methods

from

plasma

and Akazawa

in plasma,

samples

committee of the Institute from all the volunteers sedesign was similar to that urinary carnitine levels were

described and urine (5). Because from

at each time. For comparability, these subjects were also pooled.

neutral,

(7).

to the

oflow

the same

were pooled

of acidic,

earlier

according

concen-

two

subjects

urine

samples

from

and imidazole

amino

acids,

the

basic amino acids were selectively eluted from Dowex 50 x 8 column (8 X 1 cm Sigma Chemical Co, St Louis, MO) with 3 M ammonium hydroxide. This fraction was flash evaporated and the residue was dissolved in water and loaded on Dowex 1 x 8 (OH) column. TML was eluted with water and quantitated without derivatization in an automatic amino acid analyzer (model 1 19 CL, Beckman Instruments, Inc, Fullerton, CA), connected to a Hewlett-Packard integrator 3390 (Hewlett-Packard Co, Palo Alto, CA).

The diameter and

the column

ofthe

column

was packed

containing to a height

W3P resin was 6 mm of 22 cm.

The

buffer

and ninhydrin flow rates were 44 mL and 22 mL/h, respectively. Details of buffer changes are given in Table 1. Starting temperature was 40 #{176}C. After 42 mm the system was programmed for

adults.

were carried adult

urinary not

After removal were

and methods

Two experiments apparently

normal

load,

and

and

volunteers 3 to 24 h after

was

reported

meth-

first demonstrated and quantitatively determined in human urine by Kakimoto and Akazawa (5). Methylation of free lysine has been demonstrated in N crassa (6). In an earlier study (7) we observed a significant rise in plasma and urinary carnitine levels 3-6 h after an oral load of lysine. A similar increase was not observed after oral administration of other amino acids such as tryptophan and threonine. It was proposed that unlike the rat humans may have a direct nonpeptidyl pathway for methylation of lysine. This study examines the changes in plasma and urinary TML levels after an oral load of 5 g lysine or 5 g tryptophan

Plasma

from

S Bamji

carnitine

ylation of amino acids has been shown to be a posttranslational process and the amino acid derivatives so formed are not reincorporated into protein (1 , 2). TML has been identified in various proteins including histones (3) and myosin

male

(TML)

that the effect is specific for lysine Am J C/in Nuir 1987;46:772-7.

carnitine,

and Mahiab

to healthy

trimethyllysine

Introduction

carnitine

MSc;

of 5 g of lysine

in plasma

A similar

Carnitine

WORDS

lysine,

rise

in humans

Am J C/in

Nuir

I From the Department of Biochemistry, National Institute of Nutrition, Indian Council of Medical Research, Hyderabad, India. 2 Address reprint requests to Latifa Khan-Siddiqui, PhD, Department

ofBiochemistry, National Institute ofNutrition, Indian Council of Medical Research, Jamal Osmania P0, Hyderabad-500 007, India. Received June 2, 1986. Accepted for publication December 23, 1986.

1987;46:772-7.

Printed

in USA.

© 1987 American

Society

for Clinical

Nutrition

Downloaded from ajcn.nutrition.org by guest on August 12, 2015

KEY

TML

lysine

PhD;

administration

progressive

suggests acid load.

amino

levels

TRIMETHYLLYSINE TABLE 1 Buffer changes

LEVELS

IN HUMANS

resolution for the resolution

of trimethyllysine

Buffers

Time

AtoB BtoC C to LiOH LiOH to A

nmol. from

A-0.2 N lithium citrate 3.70; C-l.0 N lithium citrate lithium hydroxide (regenerating

pH 2.83; pH 3.75 alkali).

B-0.2 N lithium + 6% isopropanol;

a change to 64 #{176}C for the remainder of the system was done at 64 #{176}C with

mm. These conditions

of the buffer

The

sensitivity

ofthe

instrument is 10-100 could be detected and

levels as low as 2.5 nmol

quantitated.

start

To check the authenticity of TML fraction, TML thesized from a-N-acetyl lysine by a modification ofthe of Dc Lange et al (HP Broquist, personal communication)

66mm 100mm 195 mm 200 mm (regeneration)

*

ofTML.

However,

773

lysine and Reinecke

a-N-acetyl Vanderbilt citrate pH and 0.3 N

University,

lysine standard yield

(9).

salt were gifts from

Nashville,

TN.

after corrections 1 gives

Figure

TML

was synmethod (8).

HP Broquist,

was quantitated

with

were made for ninhydrin

the aminograms

showing

color

resolution

of

TML added to a standard mixture of amino acids in a protein hydrolysate and TML isolated from plasma and urine. Data on plasma and urinary TML were analyzed by log trans-

run. Equilibration A (Table 1) for

15

were found to give good and reproducible

formation

nary

A

followed

carnitine

were

by Student’s t test. analyzed by paired

Data on plasma t test (10).

and

un-

B .

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FIG 1 . Aminograms showing TML resolution: A, standard plus TML; C, TML isolated from plasma; and D, TML isolated

protein hydrolysate; from urine.

B, standard

protein

hydrolysate

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FIG I.(Continued)

Results

Neither changed

Changes in plasma oral administration in Figures

3. The

2 and

2. In healthy

and urinary of 5 g lysine

adult

men

mean

and

carnitine

or tryptophan

values

a significant

TML after are shown

are given increase

in Table

(Figs

carnitine significantly

3A, B, C, and

nor

TML

after

the

D; Table

in oral

plasma load

and of

urine

tryptophan

2).

Discussion

in plasma

levels occurred within 3 h oforal administration of 5 g lysine (Fig 14). The levels continued to be high up to 24 h. Urinary carnitine showed an increase within 3 h and continued to be high up to 24 h (Fig 2B). Plasma TML also showed an increase after the lysine load, the rise being significant after 6 h. The values con-

The plasma carnitine values observed before the lysine load are comparable to the values for adult Indian men reported by us earlier (7, 1 1). The higher plasma values observed in Indian men compared with Western men may be due to the smaller physique ofthe former (1 1). Plasma carnitine levels for apparently healthy men tend to be

tinued

inversely (1 1). The

carnitine

ofTML failed

to increase

up to 24 h (Fig

in urine to show

was markedly

a further

increase

2C).

The

concentration

higher

than

in plasma

after

the

lysine

2D). In fact urinary TML tended to decrease after load of lysine or tryptophan (Figs 2D and 3D).

load

but (Fig

an oral

related increase

after a lysine but this study confirms the rise in plasma

to their in plasma not

after

anthropometric

index

and

carnitine

urinary

a tryptophan

load

(wt/ht2)

observed

levels

in

our earlier observations (7). However, carnitine tended to be lower than in

TRIMETHYLLYSINE

LEVELS

IN

775

HUMANS

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2

B

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T

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FIG 2. Changes in plasma and urinary carnitine and TML after oral administration A, plasma carnitine; B, urinary carnitine; C, plasma TML; and D, urinary TML. subject at different times.

the earlier

are not

study.

clear.

in response

Reasons

for these

There

is marked

to a lysine

load.

quantitative

differences

interindividual

variation has also been after a lysine

Malnutrition

observed to affect the rise in plasma carnitine load (7). TML also shows a significant and progressive increase in plasma. Absence of a detectable rise in urinary TML after a lysine load may be due to the fact that human kidney has an efficient mechanism for converting TML to carnitine. Recent studies in rat show that TML can be

of5 g L-Lysine monohydrochloride: Each group of bars represents

one

taken up by the tissues and also reabsorbed by the kidney (12, 13). The fall in urinary TML appears to be a nonspecific effect of amino acid bolus load since it was seen in all the samples after the lysine or tryptophan load. The mechanism of rise in plasma TML after a lysine load is not clear. It is unlikely to be due to a greater protein turnover (synthesis and breakdown) since only a single amino acid was administered. Because a tryptophan load did not produce a similar change suggests that the effect is specific

for lysine

and

notjust

a nonspecific

consequence

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