Rapid concentration of urinary peptides and proteins

ANALYTICAL

BIOCHEMISTRY

123, 291-294 (1982)

Rapid Concentration

of Urinary Peptides

ULF-H~KANST~NMAN,KRISTINA Clinical

Laboratory,

Department

of Obstetrics SF-00290

and Proteins’

PESONEN, ANDMARJA-LISA and Gynecology, Helsinki Helsinki 29, Finland

University

HUHTALA Central

Hospital,

Received December 21, 1981 A method for rapid concentration and dialysis of urinary peptides and proteins is described. The method is based on the use of a cheap disposable dialyzer and a two-channel peristaltic pump. One to twenty liters of urine can be concentrated to 0.3-0.5 liter in 2-24 h. The recovery of peptides larger than 6000 daltons is 80-95%. The recovery of smaller peptides varies depending on the conditions used.

The urine of cancer patients contains proteins, which seem to be useful as tumor markers ( 1,2). Early studies on urinary peptides indicate that they may also display typical changes in cancer (3). These data suggested that it might be worthwhile to perform a systematic study on urinary peptides in cancer patients. A prerequisite for such a study was an efficient method for concentrating peptides from large volumes of urine. We describe a method that is simple, rapid, and permits dialysis and concentration of peptides and proteins in I to 20 liters of urine to less than 0.3-0.5 liter in 2 to 24 h. The method is based on the use of cheap disposable dialysis coils. MATERIALS

AND METHODS

Urine was obtained from healthy laboratory personnel and from pregnant women. Sodium bicarbonate (5 g/liter) and sodium azide (0.5 g/liter) were added to the collection vials as preservatives. Urine was filtered and stored at 4°C before dialysis. Dialysis and concentration were performed at 4°C using a disposable dialyzer intended for treatment of kidney patients. ’ This work was supported by grants from the Finnish Academy of Sciences, the Finnish Cancer Association, and the Finska Lakareslllskapet.

The following types were used: a Hospal CAP I, 1.06-m’ hollow fiber and a Cobe PPD, 1.3-m* parallel-plate dialyzer. Urine and dialysis fluid were pumped through the coil with a variable speed two-channel peristaltic pump. Urine contained in a large beaker or a bucket was fed through a 5-mm-i.d. tubing to the pump, which was connected to the dialyzer. From the dialyzer the urine was led back to the bucket. The transmembrane pressure in the dialyzer was adjusted with a clamp on the outlet tubing and monitored with a gauge (Fig. 1). The pump speed varied between 0.1 and 1 liter/min. The highest pressure used was 0.5 bar. The urine was dialyzed against 5-10 liters of distilled water, which was changed at OS- to 2-h intervals. The dialysis was monitored by measurement of the volume of urine, A*,,,, and the concentration of sodium. Recovery of proteins was determined by measuring the content of human chorionic gonadotropin (hCG)’ in the urine by radioimmunoassay (RIA) (4). Recovery of peptides was studied by determining the concentration of a urinary 7000-dalton peptide (5). * Abbreviations used: hCG, human chorionic gonadotropin; RIA, radioimmunoassay.

291

0~3-269~~82~1~291-04$02.~/0 Copyright 0 I?82 by Academic Press, Inc. All rights of reproduction in any form rwsved.

292

STENMAN.

PFSONEN.

CLamp w ii

t

il II LF Z-Channel

Urk!e

Dialysis fluid

FIG. 1. Equipment used for dialysis and concentration of urinary peptides and proteins, The dialyzers used have a surface area of 1.O to I .3 m*, an internal volume of 70 to 90 ml, and a membrane thickness of 11 to 13 m. The ultrafiltration rate is controlled by varying the transmembrane pressure in the dialyzer with the clamp on the urine outlet tubing.

AND HUHTALA

rate of loss is increased. The content of hCG is very constant until 15 h, but during the last 2 h there is a loss of hCG similar to the loss of uv-absorbing substances. This loss of uv-absorbing substances occurs when the volume of the dialyzed urine drops below 450-300 ml. This volume range is critical for the recovery of proteins; when the volume of the sample is reduced to less than 200 ml, the loss of protein is considerable. In gel-filtration experiments of unprocessed normal urine, the main uv-absorbing peak is caused by substances with a molecular mass of less than 1000 daltons. A very small uv absorbance is caused by peptides in the range lOOO- 10,000 daltons and by proteins. After dialysis the proteins form the main uv-absorbing peak. The ~n~entration of peptides larger than 2000 daitons has been increased, whereas that of smaller molecules has been reduced (Fig. 3).

Samples of urine taken before and during dialysis were also studied by gel filtration on a Sephadex G-50 column (1.6 X 80cm) in 0.1 mol/liter acetic acid, pH 3.0. The uv absorbance of the eluate at 214 nm was monitored with a Pharmacia l/2 I4 uv monitor. The column was calibrated using cytochrome c, aprotinin, and angiotensin II as molecular weight markers.

RESULTS

The progress of a dialysis experiment with low transmembra~e pressure is shown in Fig. 2. During the first part of dialysis the Naf is removed rapidly. The decrease in volume is slow in the beginning but becomes faster with decreasing salt concentration. The content of uv-absorbing substances also drops rapidly during the first hours; it levels off after 5 to 10 h, but during the last 2 h the

0

5

IO

15

FIG. 2. Change in volume and content of Na*, uvabsorbing substances, and hCG during analysis and concentration of urine. The pump speed was 100 ml/min and the transmembrane Dressure 0. I bar.

RAPID CONCENTRATION

A214 1:

i I

i

I:

6k I

13k I

Y

293

OF PEPTIDES

fk I

I

I‘ I

I'

200

150

100 Elutlon

volume

tmll

FIG. 3. Gel filtration on Sephadex G-50 of untreated urine (-f and a sample of the same urine after dialysis and 20-fold ~n~ntration (- - -). The column size was 1.6 X SO cm and the eluant acetic acid, 0.1 mol/liter, pH 3.0. The eluate was monitored for absorbance at 214 nm.

When dialysis is performed at a higher flow rate and a transmembrane pressure of 0.4 bar instead of 0.1 bar, the concentration rate is increased and the dialysis time shortened (Table 1). Under these ~nditions, the recovery of proteins and large peptides is about the same as with lower pressure. Smali organic substances are removed less efficiently, as evidenced by a four- to five fold

higher absorbance at 2 14 nm. In spite of this, salts (Na+) are removed fairly completely. The recovery of proteins and large peptides measured by specific assays was better than 80% as long as the final volume of the dialyzed urine was larger than 300-400 ml (Table 1). Further concentration caused a considerable drop in recovery. When 1 liter of urine was concentrated to 0.15 liter, the

TABLE

1

CHANG~I~VOL~MEANDCO~TE~OFN~', UV-ABSORBING SUBSTANCES, PEPTIDEDURINGDIALYSISATDIFFERENTTRANSMEMBRANEPRESSURES

Na’ Time (h) Pressure, 0.1 bar 0 5 10 Pressure, 0.4 bar 0 1.1 2.0

A2,4

Vol (I)

mm01

(o/o)

AU. 1

1.80 1.29 0.30

244 3.9 co. 1

flO0) (1.6) (<0.05)

524 18 7

1.80 1.00 0.31

196 4 0.1

(100)

458

(2.0) (0.05)

16

30

hCG, ANDTHE~~DALTON 7000-dalton peptide

hCG

X vol

IU

(%J

mg

(%I

(100) (3.4) (1.3)

21000 20500 17000

(100) (98)

14.0 12.8 11.3

(toot (91)

(100) (16.5)

24000 23000 20400

(100)

12.6 11.8 11.2

(100) (94) (89)

(%jo)

(6.5)

(80) (96) (85)

Note. Recovery of the different substances is also given as percentage of the initial values.

(81)

294

STENMAN,

PESONEN,

recovery was 50-70’S, and when 5 liters of urine was concentrated to 0.5 liter, the recovery was 90-95s. The capacity of the dialyzer decreased with time. Depending on the samples handled, a 50-70’S loss in concentration rate occurred after dialysis of 10 to 30 liters of urine. After this, the loss of performance was slow. DISCUSSION

The method described here permits rapid removal of electrolytes and low molecular weight substances from large volumes of urine. Proteins and large peptides are concentrated lo-20-fold with a recovery of 80 to 95%. The recovery is dependent on several factors, and it is possible to affect the recovery by changing the experimental conditions; e.g., when maximal recovery of peptides is important, it is advantageous to dialyze only until most of the electrolytes are removed and then to lyophilize. After lyophilization the peptides usually dissolve well, but some urinary proteins are poorly soluble. Therefore, a better recovery of proteins is achieved if the sample is concentrated to a volume compatible with the next fractionation step. If the next step is ion-exchange chromatography, the sample can be conveniently dialyzed against the proper buffer. Concentration to the smallest possible volume should be used only when necessary, e.g., prior to gel filtration. This volume is dependent on the dialyzer and the pressure used. In parallel-plate dialyzers, the internal volume increased with pressure from about 80 to 90 ml up to about 150 ml at 0.5 bar. In hollow-fiber dialyzers, the internal

AND

HUHTALA

volume, 80-90 ml, is not dependent on the pressure. The dialyzers used in this study represent the most common types in clinical use. Equivalent models are available from many manufacturers. In addition, there are small dialyzers available for pediatric use as well as larger ones. Most dialyzers used have a cuprophane membrane, with a thickness of 11 to 17 pm. In the CAP I dialyzer the thickness was 11 pm and in the Cobe PPD, 13.5 pm. The M, cut-off limits of these membranes are not given by the manufacturers, but a rough estimate can be obtained from the gel-filtration experiments (Fig. 3), which indicate that molecules larger than 2000 daltons are concentrated during dialysis, whereas smaller ones are eliminated. The price of dialyzers intended for clinical use is very favorable in comparison to the corresponding laboratory equipment. They also have a much larger surface area than most laboratory dialyzers. This limits their use to large-scale applications, but for this purpose they offer a very competitive alternative to conventional procedures. REFERENCES 1. Rudman, Nixon,

D., Chawla, R. K., Wadsworth, D. W., and Schwartz, M. (1978)

A. D., Trans.

Assoc. Amer. Physicians 90, 286-299. 2. Anderson, N. G., Anderson, N. L., and Tollaksen, S. L. (1979) Clin. Chem. 25, 1199-1210. 3. Skartyhski, B., and Sarnecka-Keller. M. (1962) Advan. Clin. Chem. 5, 107-l 34. 4. Stenman, U.-H., Tanner, P., Ranta, T., Schrijder, J., and Sepplll, M. (1982) 0bstef. Gynecol. 59,

375-377. 5. Huhtala, M.-L., Koistinen, Stenman, U.-H. (1981)

Biochem. 19, 705.

R., Sepplll, M., .I. Clin. Chem.

and

Clin.