Single hollow fiber microconcentrator - Analytical Chemistry (ACS

Mar 1, 1974 - Secretion of proteins by sertoli cell enriched cultures: Effects of follicle stimulating hormone, dibutyryl camp and testosterone and co...
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Table I. Relative Sensitivity Factors as Determined by Magnetic Peak Switching. RSF = apparent/true relative to Bey Sample

Bovine liver No. 1, day 1 Bovine liver No. 1, day 1 Bovine liver No. 1, day 2 Bovine liver No. 2, day 3 Bovine liver No. 1, day 4

“Mn

66Fe

2.50

1.56

1.50

1.57

4.60

0.679

1.80

2.40

1.57

1.45

1.58

4.81

0.664

1.99

2.25

1.42

1.55

1.72

4.28

0.580

1.84

2.34

1.57

1.51

1.56

4.85

0.479

2.04

2.45

1.56

1.57

1.68

5.32

0.597

2.31

Cert. wt %

Found, w t yo

C P Ti V Cr Mn co

0.15 0.053 (0.01) 0.024 0.13 0.36 0.26 0.34 0.028 ( < O .005) 0.011 0.30 0.022 0.012 (0 .003)

0.313 0.047 0.012 0.021 0.187 0.429 0.269 0.345 0.030 0.001 0.014 0.263 0.023 0.011 0.003

cu

As

Zr Nb MO

Sn W Pb a

75As

ZOBPb

80%

puter interface might be used to drive the MPS in a peak to peak elemental search.

Table 11. Determination of 15 Elements in NBS No. 461 by Magnetic Peak Switching SSMS Using a Nickel Internal Standard. Element

SSZn

W U ’

CONCLUSIONS

The ability to bring unequivocally any mass in the spark source spectrum onto the collector slit quickly and unambiguously has led to greater use in our laboratory of the higher precision of peak switching techniques. By comparison, for electrostatic peak switching, “reliable peak identification . . . requires close interaction and decision making by the instrument operator” (8). For our problems, the MPS has provided a solution which is both convenient and inexpensive (total cost including Hall probe is approximately $750). Received for review May 22, 1973. Accepted October 29, 1973. This research was supported by grants from EPA and LEAA.

wNoNi+lat 0.40 (wt %) internal standard.

Single Hollow Fiber Microconcentrator Rashid A. Zeineh Department of Microbiology, Chicago Medical School and the University of Health Sciences, Chicago, 111

Beverly J. Fiorella and Elie P. Nijm School of Associated Medical Sciences, University of Illinois, Chicago. 111.

George Dunea Departments of Nephrology and Hypertension, Cook County Hospital and the .Wektoen institute for Medical Research, and the University of .Yea/th Sciences, Chicago, 111.

The determination and study of various substances in body fluids frequently requires preliminary concentration of the specimen. This requirement is essential when only small volumes are available, ( e . g . , cerebrospinal fluid), or when concentrations are low ( e . g . , proteins in normal urine). An ideal concentrator should be efficient,, easy to operate, cause no denaturation or drying, and allow easy recovery of the concentrate. Concentrating systems currently in use include freeze drying, concentration against a n osmotic gradient, pervaporation in air, precipitation, and suction ultrafiltration (1-5). None of these methods satisfy all the requirements for an ideal concentrator. In this report we describe a single hollow fiber microconcentrator which is simple, efficient, easy to operate, reusable, and which represents a significant improvement over earlier models. (1) G. Schneider and G. Wallenius, Scan. J. Clin. Lab. Invest.. 3, 140 (1951 ) .

EXPERIMENTAL

Apparatus. The microconcentrator consists of a single hollow fiber of microtubular semipermeable membrane with an outlet and inlet (Figure 1); a test tube cap with three implanted separate tubular steel outlets; a mini-clamp; connecting tubing; and a Plexiglas stand (Figures 2 and 3). The hollow fiber used in this laboratory is 6 inches long, 0.7-mm inside diameter and has a priming volume of 5 microliters. The tubular membrane is made of acrylic material, has a maximal pore size of 25 Angstroms and is impermeable to substances with molecular weights over 10,000. The ultrafiltration rate by suction is 15 ml per hour.

(2) T. J. Greenwalt, C. J. Van Oss, and E. A . Steane. Arner. J , Clin. Pathol., 49, 472 (1968). (3) J. N. Cummings, J. Neurol. Neurosurg. Psychiat., 16, 152 (1953). (4) F. Miyasato and V. E. Pollak, J . Lab. Clin. Med., 67, 1036 (1966). (5) R. A. Zeineh and 8. J. Fiorella, Amer. J. Med. Techno/, 36, 1 (1970).

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Figure 1. Single hollow fiber microconcentrator consisting of a microtubular semipermeable membrane with inlet and outlet adaptors

AMPLE

CONCENTRATE

-- - __-_--ULTRAFILTRATE

Figure 2. Schematic representation of single hollow fiber microconcentrator operated by suction

Figure 3. Assembled system with Plexiglas stand

Assembly a n d Operation of Apparatus. The hollow fiber is connected to two of the outlets in the cap and is carefully lowered into the test tube. The cap is firmly fitted on the test tube (Figure 2) and is placed on a special stand (Figure 3). Another test tube (the sample reservoir) is placed into the upper well of the Plexiglas stand and is connected to a piece of tubing through the steel inlet to the inlet of the hollow fiber. The outlet from the hollow fiber is connected through the steel outlet in the cap to a piece of connecting tubing and a mini-clamp is placed on this tubing. The third and larger steel outlet in the cap is attached to vacuum (Figure 2 ) . The hollow fiber is primed by injecting distilled water with a syringe through the sample outlet. This drives out trapped air bubbles which would slow down concentration by forming a dead space and also retard the inflow of the sample to be concentrated. Priming is stopped when about 0.3 cm3 of water has been intro478

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duced into the sample reservoir; the outlet tubing is then clamped and the syringe disconnected. The integrity of the assembled concentrator may be tested for leakage by concentrating 1 cm3 of hemoglobin: The hemoglobin solution is put into the sample reservoir and vacuum is applied for 1 minute. Oozing clear drops of ultrafiltrate are observed immediately on the outside of the membrane, in the test tube. If leakage is present, red drops of hemoglobin solution are observed outside the fiber and replacement by another fiber is then needed. If there is no leakage, the system is emptied and flushed, the sample is placed into the reservoir, and concentration is started. At the end of concentration, 0.2-1 ml distilled water, buffer, or saline is used to rinse the walls of the sample reservoir and the inlet tubing. After the system is rinsed, more rinsing fluid is added to the reservoir to prevent drying and vacuum is stopped. A hematocrit capillary tubing is connected to the outlet tubing. The clamp is released and the concentrate flows by gravity into the tubing. When the desired volume has been collected in the hematocrit tubing, the outlet tubing is clamped and the hematocrit tubing is removed. For washing the system, the sample outlet is opened and water is allowed to flush through from the reservoir. When the system has been washed out, the apparatus is ready for another run. Materials and Methods. The concentrating device described was obtained from Biomed Instruments; a Shandon Immunoelectrophoresis system on agar plate was purchased. Normal serum, urine from 10 normal subjects and from 15 patients with proteinuria, and cerebrospinal fluid from ten patients were collected. Behring polyvalent antisera (lot No. 13223/A5), Behring monospecific albumin antisera (lot No. 1208 AB/L @), gamma-globulin (lot No. 1506 M / L 68). Hyland monospecific orosomucoid goat antisera (lot Xo. 8219 TOOA 1) and Pentex anti-transferrin rabbit antisera (lot No. 35641) were purchased. Experiments were done to evaluate the capability of the single hollow fiber device to concentrate small volumes and very dilute solutions. These experiments included protein recovery, degree of concentration, and protein alteration or denaturation during the process of ultrafiltration. To determine whether denaturation occurred during the ultrafiltration process, 0.2 rnl of serum was diluted 50-fold with saline and then concentrated to a final volume of 0.02 ml. The immunoelectrophoretic pattern was then determined on agar gel plate and compared to that of the original native serum. Similarly, native serum was diluted with normal urine and the above process repeated in order to demonstrate that the urinary proteins remain unchanged during the concentration process. To determine protein recovery, individual proteins were measured in native serum, in serum that was diluted with saline and then concentrated, and in serum which was diluted with normal urine and then concentrated. Individual proteins were determined in triplicate by the Mancini technique of immunoradial diffusion (6) and the total protein by the standard biuret method (7) and by a modification of this method (8). Since one practical application of this concentrator is to obtain a high protein content in a small volume of a few microliters, 1, 3, 8, and 10 ml of 1, 0.1, and 0.01 gram 70albumin in saline solution was concentrated to a final volume of 0.2, 0.1 and 0.2 ml. The concentrate was then reconstituted to original volume by adding saline and the albumin content was determined in duplicate by immunoradial diffusion (6) and by ultraviolet absorption a t 280 nm wave length. Then, protein recovery and degree of concentration of albumin in the retrieved concentrate was calculated. The device was evaluated for clinical use by concentrating urine and cerebrospinal fluid and applying the concentrate to immunoelectrophoresis, electrophoresis, radioimmunoassay of Australia antigen and the Ouchterlony radial immunodiffusion systems. Ten milliliters of urine from each of the 10 proteinuric and 8 hepatitis patients were concentrated to 0.2 ml. Urine from normal subjects was similarly concentrated and the results were compared with those of other workers (9). Specimens of cerebrospinal fluid, 0.36-3.00 ml, were concentrated to a final volume of 0.02 ml and then applied to immunoelectrophoresis and radial immunodiffusion systems. (6) G. Mancini, A. 0. Carbonara, and J . F. Hermans. Immunochemistry, 3, 235 (1 965). (7) A . Hiller, R. L. Grelf, and W. W . Beckman, J . BioI. Chem.. 176, 1421 (1948) (8) J. Savory, P. H. Pin, and F. W . Sunderman Jr., Ciin. Chem., 14, 1 160 (1 968) (9) J. Poortmans and R. W. Jeanloz. J . Ciin. invest.. 47, 386 (1968).

RESULTS The immunoelectrophoretic pattern of native serum against monospecific or polyvalent antisera showed no recognizahle difference in five different sera after dilution with saline and reconcentration or after dilution with normal urine and reconcentration. The normal urine used for dilution when Concentrated 50-fold revealed no precipitin arcs upon routine immunoelectrophoresis (Figure 4, B and C). The results of immunoradial diffusion determinations of individual serum proteins averaged 5.3 gram % albumin, 76.0 mg % orosomucoid, 390 mg % transferrin, and 850 mg % globulin in native sera and 5.2 gram % of albumin, 75.2 mg % orosomucoid, 386 mg % transferrin and 838 mg % globulin in diluted and concentrated sera. Thus, the concentrations of these individual proteins were essentially equal in the native sera and in the two diluted then reconcentrated sera. These experiments show that protein was wholly retained and that little if any was lost during concentration. The protein recovery was 85% for 0.02 ml and 98% for 0.1 ml of .collected concentrate, Table I. Similarly, the electrophoresis experiments revealed identical arcs, indicating that the antigenicity and apparent electrophoretic mobility were unchanged. Clinical Applications The results of immunoelectrophoresis on proteinuric and normal nrines are shown in Figure 4, A and B. Precipitin lines are seen in proteinuric urines which were concentrated 50-fold, hut no lines are seen in the 50-fold concentrated normal urines. Immunoelectrophoresis of cerebrospinal fluid us. p l y valent antisera resulted in well defined precipitin arcs (Figure 5). Study of individual urinary proteins by immunoradial diffusion in ten apparently normal subjects showed that our values agree with the usually reported normal values. Total protein averaged 6.6 mg %, and individual proteins averaged albumin 2.3 mg %, alpha-1 acidic glycoprotein 0.4 mg %, transferrin 0.29 mg %, and gamma globulin 0.45 mg %. Similarly the proportions of individual proteins in the urine of ten patients with renal disease were not in disagreement with present accepted ranges of albumin when compared to total protein. Determinations of individual proteins in the cerebrospinal fluid of eight patients with neurological disease were possible after concentrating a volume of 1.0 ml to a final volume of 0.54.1. The eight urines from hepatitis patients were all found positive of the Australia antigen after concentration. DISCUSSION The microconcentrator described here consists of a single microtube of semipermeable membrane which allows high solute flux rates. Ultrafiltration may he achieved solely by gravity (hydrostatic pressure) but is greatly enhanced by suction or by positive pressure. The sweeping effect of a continuous inflow of the sample sweeps down the proteins toward the outlet leading to a higher degree of concentration (5). The methods previously used for concentrating proteins from dilute solutions include: freeze drying, which is slow and costly and needs prior dialysis ( I ) ; osmotic concentration by dialysis us. concentrated polymer solutions such as PW, which produces a low yield and may denature some proteins (I)-e.g., haptoglobin looses its perioxidase and immunological activity after concentration by bag dialysis us. polyethylene glycol-40,000 mol wt (Carbowax, Union Carbide); pervaporation in air using a tubular membrane is a very slow method (4); precipitation techniques which

Figure 4 A. Immunoelectrophoresis of Eehring standardized serum (upper well) and 50-fold concentrated proteinuric urine (lower well) vs. Behring polyvalent antisera. B. lmmunoeiectrophoresis of proteinuric urine concentrated 50 times, vs. polyvalent antisera (lower well). In the upper well, normal urine concentrated 50-fold revealed undetectable levels: C. Immunoelectrophoresis of 50-fold concentrated proteinuric urine (lower well) and SO-fold concentrated normal urine against anti-albumin antisera (upper well)

Figure 5. Immunoelectrophoresis of cerebrospinal fluid from two different patients result in denaturation of some proteins (2, 9); and SUCtion ultrafiltration using semipermeable membranes is slow and utilizes apparatus which is more difficult to assemble and operate (5). The microconcentrator described here does not have the above disadvantages. No denaturation can be demonstrated and the immunological properties and apparent electrophoretic mobility of the proteins are retained. Concentration with this device is very fast; a protein solution of l ml volume'can be concentrated to a final volume of 0.02 ml with 85% recovery in 4 minutes. A comparison with other microconcentrators is shown in Table I. The speed of concentration increases by increasing the suction force and by using longer fiber or multiple fibers. A single hollow fiber, 52 inches long and coiled in a wider cylinder, 3%-inch inside diameter and 2% inches high, has the ultrafiltration rate of 115 ml/hr when operated by 25inch Hg suction. A bundle of ten 52-inch fibers has the rate of 1 l./hr. The speed also depends on the protein content of the sample-eg., 1cm3 of serum needs 10-15 minutes to be concentrated to 0.1 ml while the same volume of normal urine needs only 4 minutes. The hollow fiber should remain wet because drying for over 15 minutes causes irreversible damage. The tube containing the fiber is filled with distilled water and stored. The fiber can be used as often as 30 times. If the pores are clogged by pro-

ANALYTICAL CHEMISTRY, VOL. 46, NO. 3. MARCH 1974

*

479

Table I. Efficiency of Single Hollow Fiber Concentrator Relative to Others in Current Use.a Starting volume, ml

Final volume, ml

Time

Protein recovery, yo

Folds of concentration

10

0.25

3-4 hr

60

24

Reference

Kabat/Mayer (10)

10

0.1

6-8 hr

45

45

Grogan et al,

3

0.02

lhr

42

63

Zeineh et al. ( 5 )

0.2

lhr

65

32

Windish et al.

(11)

10

(12)

5

8

0.1

3hr

40

32

Barrows (13) and Whitaker

3

0.1

This report

0.02 0.02

98 85

30

3 1

6 min 6 min 4 min

128

85

43

et al. (14)

Type of concentrator

Bag dialysis us. osmotic solution (polyethylene glycol) Bag dialysis us. osmotic (PVP) Thin channel suction ultrafiltration Membrane funnel ultrafiltration by centrifugation Dialysis us. dextran solution under vacuum Single hollow fiber suction ultrafiltration

The single hollow fiber is faster and gives higher degrees of concentration. Other concentrators have large surface area that results in lower protein recovery.

--#

COLUMN

Figure 6. Schematic representation of using the single hollow fiber concentrator in line with column eluate

longed or repeated use, the fiber could be regenerated by reverse ultrafiltration using pressure outside the fiber or suction connected to the sample outlet. Prior concentration of various types of body fluids is required before chemical analysis or routine electrophoresis can be done. The concentrator described here is particularly useful in the study of cerebrospinal fluid, where only small amounts of the fluid may be available. It is also useful in the study of total and individual proteins in normal urine where available concentrators fail to meet the limit of sensitivity of the present analytical methods. Other potential uses of this concentrator are in line with column chromatography (Figure 6) where the column eluate is concentrated with complete retention of column resolution; in line with the AutoAnalyzer system (Figure 7 ) in order to increase sensitivity by increasing the concentration such as in analysis of urinary proteins and enzymes; and to obtain protein free ultrafiltration from small volumes of serum as in the determination of free amino acids by electrophoresis or unbound iron by flame photometry. The versatility and uniqueness of this single hollow fiber device is brought out by the following advantages: high efficiency in terms of being fast, simple, and with high degree of concentration and protein recovery; no manipulation of the membrane is needed during concentration or concentrate collection; the fiber is flexible and allows easy handling for quick assembly; and the minimal priming volume allows complete protein recovery by rinsing with minimal degree of redilution of the concentrate. Received for review January 19, 1973. Accepted October 18, 1973. The single hollow fiber system was devised by R. A. Zeineh and is marketed by Biomed Instruments Inc.

(10) E. A. Kabat and M. M. Mayer, "Experimentai Immunochemistry," 2nd ed.. Charles C Thomas. Springfield, Ill.. 1971, Chapter 43, p

728.

Figure 7. The single hollow fiber concentration incorporation in AutoAnalyzer in order to increase sensitivity through continuous flow concentration 480

ANALYTICAL CHEMISTRY, VOL. 46, NO. 3, MARCH 1974

(11) (12) (13) (14)

C. H . Grogan and E. J. Roboz, J . Lab. Ciin. Med., 45, 495 (1955) R. M .Windish and M. M.Bracken, Ciin. Chem., 16, 416 (1970). S. Burrows, Clin. Chem.. 11, 1068 (1965). J. N. Whitaker and H. A Lernrni, Tech. Buii. Regis? Med. Techno/.. 36, 91 (1966)