WO2000032806A1

WO2000032806A1 - Method for the quantitative determination of proteinase inhibitors

Info

Publication number: WO2000032806A1
Application number: PCT/US1999/028703
Authority: WO
Inventors: Cindy L. Bristow
Original assignee: Bristow Cindy L
Priority date: 1998-12-02
Filing date: 1999-12-02
Publication date: 2000-06-08
Also published as: AU2587800A

Abstract

A method is provided for the quantitative determinations of functionally active concentrations of proteinase inhibitors, such as α1 PI and α2M, in the serum or plasma of humans and animals.

Description

METHOD FOR THE QUANTITATIVE DETERMINATION OF PROTEINASE INHIBITORS

RELATED APPLICATIONS

This application claims priority under provisional application, 60/110,580, filed

12/2/98, the entire contents of which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

1) Field of the Invention

This invention relates to a method for the quantitative determination of the specific

activity of proteinase inhibitors. In one aspect, the invention is directed to the quantitative

determination of α, Proteinase Inhibitor in human serum or plasma.

2) Background Art

Emerging evidence suggests a mnctional link between proteinases and cell signaling.

As would be predicted, activation and inactivation of proteinase-activated receptors are

selective, e.g. , the thrombin-activated receptor can be inactivated by cathepsin G or neutrophil

elastase. Examination of the influence of proteinase inhibitors on proteinase-activated

receptors is complicated by the variance in affinities, concentrations , and species of proteinase

inhibitors represented in serum. The proteinase inhibitor in serum exhibiting the greatest

concentration is α, proteinase inhibitor (α,PI, α antitrypsin), and the proteinase inhibitor

encompassing the broadest spectrum is a₂ macroglobulin (α₂M).

In the acute phase of inflammation, quantitative levels of ,PI have been reported to

significantly increase, as do proteolytic fragmentation and proteinase complexation, both of

which can diminish the functional capacity of ,PI. The functional capacity of o^PI during the acute phase has not previously been examined. Further, in some situations, the association

of α₂M with neutrophil elastase in plasma is competitively favored, and in this case elastase-

mediated proteolysis of low-molecular-weight peptides and cytokines can persist. The relative

concentrations of elastolytic proteinases, the inhibitor α,PI, and the substrate-restricting o^M form a tightly regulated mechanism for discreet targeting of elastase activity.

It has been previously observed using serially diluted serum that the residual

uriinhibited enzymatic activity of exogenously added elastase exhibits bimodal regulation. The

bimodal behavior of serum was demonstrated to result from the dual activities of ,PI and

α₂M; however, these investigators did not attempt to derive a numerical value for quantitating

the active fraction of α,PI. While elastase is completely inhibited by α,PI, association of

elastase with α₂M excludes its activity except toward low molecular substrates. When the

concentration of α₂M exceeds that of elastase, catalytic activity if unaffected by adding α,PI

since α₂M is not replaced by α,PI in these complexes. When the concentration of elastase

exceeds that of α₂M in this scenario, elastase is available for complexing with added ,PI

resulting in a decrease in catalytic activity. Physiologic concentrations of the common

phenotypes of ocjPI have been approximated as 20-53 μM and that of α₂M as 1.56-4.96 μM

so that as serum is diluted and incubated with a constant concentration of elastase, the

contribution from α₂M in elastase protection becomes negligible by this method of detection,

and the contribution from c^PI is detected as increased inhibition. On the other hand, as

serum becomes excessively dilute, the contribution from a_λ PI also becomes negligible to

detection resulting in decreased inhibition. Therefore, a serum dilution exists at which

minimum catalytic activity can be measured by exploiting the properties of unequal serum

concentration and unequal outcomes of complexes between elastase and α,PI and α₂M. The maximum reduction in catalytic activity is a measure of the functionally active concentration of α,PI in competition with α₂M for elastolytic enzymes. The relationship between reduction

in catalytic activity and the precise quantitation of functional α,PI in competition with α₂M has

not previously been examined.

Proteinase inhibition is only one of the diverse biologic activities of ,PI and α₂M

including alteration of the cellular effects of polymorphonuclear neutrophils, found that α,PI decreases antigen-driven, PHA and, Con A, but not PWM, lymphocyte responsiveness. In

fact, inhibition of DNA synthesis and proliferation by α,PI has been demonstrated in erythroid

progenitor cells and lymphocytes. It has been reported that α,PI deficient serum mediates

enhancement of lymphocyte response to PHA and increases zymosan activation of monouclear

cells and PMN. The ability to measure the functional capacity of proteinase inhibitors in

serum is paramount to determining the interrelationship between proteinase inhibitors and

immune responsiveness in pathology. Association rates previously derived using isolated

proteinases and inhibitors suggested the feasibility for measuring these activities in serum.

Quantitative determination of serum α,PI has traditionally been performed

nephelometrically; however, antigenically quantitated levels may not be representative of

functional capacity. It has previously been observed that α,PI in serum exhibits bimodal

behavior as the result of various concentrations of proteinase inhibitors, specifically cwnacro-

globulin (α₂M) and inter-a-trypsin inhibitor, which compete in binding to a panel of serine

proteinases . Consequently . it has not previously been possible to assign a numerical value for

the specific activity of these competing proteinase inhibitors in serum. By applying known

constants representing the association of proteinase inhibitors with porcine pancreatic elastase

(PPE), the theoretical relationship between the functional and antigenic values for α,PI and oc₂M has been empirically derived allowing, for the first time, the calculation of their specific

activities in serum. The serum concentration of α,PI was found to be highly correlated with

residual uninhibited PPE catalytic activity in healthy individuals, but not in individuals

exhibiting fragmented or complexed α,PI. Using these techniques, both the antigenic and mnctional levels of α,PI were determined in sera from subjects with insulin-dependent diabetes

mellitus (IDDM) who has been clinically diagnosed as having either periodontal disease or gingival health. Determination of quantitative levels by antigen-capmre suggests that the

IDDM subjects with periodontitis manifest dramatically increased levels of fragmented serum

α,PI compared with their orally healthy counterparts or normal controls.

The following abbreviations are employed in the specifications and amended claims:

α,PI - α, proteinase inhibitor (α,-antitrypsin)

α₂M - α₂ Macroglobulin

HNE - human neutrophil elastase

led - inter- -trypsin inhibitor

APE - porcine pancreatic elastase

PBS - 0.01M phosphate, 0.15M NaCl, pH 7.2

TBS - 0.05 M Tris, 0.15M NaCl, pH 7.8

Sa_qNA - succinyl-L-Ala-L-Ala-L Ala p-Nitro-anilide.

EDTA - ethylenediaminotetraacetic acid

ACD - acetate-citrate-dextrose

IDDM- insulin-dependent diabetes anellites

SUMMARY OF THE INVENTION In its broad aspect, the present invention relates to a method for the quantitative

determination of proteinase inhibitors. The method comprises the steps of:

a) obtaining a sample of blood from a subject; b) separating the blood into a fluid containing serum or plasma; c) preparing a first plurality of serial dilutions of the fluid decreasing

concentrations; d) incubating the dilutions with varying concentrations of PPE and monitoring the

catalytic activity which decreases linearly in relation to the dilutions of the fluid

to a minimum point after which the catalytic activity increases linearly in

relation to this dilution of the fluid;

e) by means of regression analysis calculating the coordinates of the intersection

of two linear lines formed by the fluid concentration and residual activity; and

f) calculating the functionally active proteinase inhibitor by computer-fit least

squares regression analysis and comparing with a standard curve.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1A - IC depict competitive assays for ,PI (black squares) and α₂M (black circles).

Fig. 2 A - 2F show serum concentration vs. catalytic activity from which residual catalytic

activity is calculated.

Fig. 3 depicts the relationship between residual catalytic activity and antigenically quantitated

levels of α,PI.

Fig. 4 A - 4C shows a comparison of HNE and PPE in measuring serum ,PI.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The stoichiometric relationship between α₂M with any proteinase and interactive proteinase inhibitor can therefore be used to derive the precise equation to calculate the specific activity and concentration of the active concentrations of the proteinase inhibitor in

serum or other complex body fluids. The active concentrations of proteinase inhibitors can

be determined by the present invention without interference by the presence of substrates,

buffering pH , temperatures or optical detection device - the active concentration of proteinase inhibitors can be determined in blood corrected with no additive (serum), or into tubes

containing the anticoagulants heparin, EDTA or ACD or any other anticoagulant.

The method of the present invention is effective for the determination of the active

concentration of a proteinase inhibitor in complex body fluids using a wide variety of

proteinases. For example, serine proteinases can be employed to quantitate serine proteinase

inhibitors, aspartyl proteinases to quantitate aspartyl proteinase inhibitors, cystine proteinases

to quantitate cystine proteinase inhibitors, metalloproteinase to quantitate metalloproteinase

inhibitors, and the like.

The method is useful regardless of whether the proteinase is reversibly or irreversibly

bound by the proteinase inhibitor of interest or whether the inhibitor is synthetic, trans genie

or exogenously induced to expression. Also, the method is effective whether the proteinase

inhibitor is bacterial, viral or parasitic or associated with a specific organ or cell type. The

proteinase inhibitor can be introcellular, involved in coagulation, fibrinolysis, or complement

inactivation and still be determinable by the present method.

In practice, the method of this invention is a useful diagnostic tool for evaluating the

medical condition in man and animals. Such condition can include but are not limited to,

arthritis, atherosdersosis, diabetes, asthma, systemic lupus erythematosis; conditions which

are of lymphoid origin, such or agammoglobulinemia, hypo-gammaglobulinemia, hypergammaglobulinemia, NK cells, T-lymphocytes, B-lymphocytes, thymocytes, bone

marrow or null cells; conditions which are age-related illnesses, or anaphylatic; and conditions

which involve a malignant illness, including but not limited to, lymphoma, leukemia, or

tumors of any origin primary or secondary. The condition can be an autoimmune illness, or infection of bacterial, viral, or other parasitic origin. It can be a damyclinating disease or a degenerative disease of any tissue; or

the condition can be genetic, hemolytic anemia, or cardiovascular; or related to a toxin or

toxoid including cholera, pertussis, diphtheria, tetanus or E. coli.

The method of the present invention is also useful where the condition is related to a

poison, such as stings, bites, ingested poisons, or those which contact the skin. The condition

can be murosal including gastrointestinal disorders, pulmonary, a granulomatous, one that is

inflammatory, an immune disorder, or an unknown condition. The condition can be one

which is secondary to any pathologic process, such as those mentioned above.

The method can be used as a therapeutic tool for many medical conditions such as

organ transplantation, transfusions, inducing immune tolerance, immunization, vaccination,

inducing immune suppression or activations and for replacement therapy.

The following reagents were employed in the examples:

Porcine pancreatic elastase, type 1(EC 3.4.21.36, lot No. 16H8045, Sigma, St. Louis,

MO) or human neutrophil elastase (EC 3.4.21.37, lot No. #EH9602a, Athens Research and

Technology, Inc. , Athens, GA) were active-site titrated in 0.05 M Tris, 0.15 M NaCl, pH 7.8, (TBS) using the substrate N-t-boc-L-alanine /7-nitrophenyl ester (lot No. 46F-0330, Sigma),

and the steady state was determined by optical absorbence at 410 ran in a Vmax kinetic microplate reader (Molecular Devices, Palo Alto, CA) for 3 min with 6-s intervals (30

measurements) at 20°C. The functionally active fractions of preparations of α,PI (Sigma

A6150, lot No. 82H9323) and α₂M (generous gift of Dr. Hanne Grøn, Duke University

Medical Center, Durham, NC) were determined. One mole of PPE was found to saturate

12.12 mol α,PI and 1.10 mol α₂M, suggesting that oc,PI and α₂M were 8 and 91 % active,

respectively. Concentrations of HNE, PPE, α,PI, and α₂M throughout represent the

functionally active concentrations. Residual catalytic activity refers to uninhibited PPE in the

presence of competing α,PI and α₂M.

The following examples are illustrative of the inventions:

Example 1

Serum or Plasma Collection Blood was collected with informed consent from healthy volunteers using Vacutainer

tubes (Becton Dickinson, Rutherford, N.J.) Containing either heparin,

ethylenediaminetetraacetic acid (EDTA, K₃), acetate-citrate-dextrose (ACD, Solution A), or

no additive. Serum was prepared from blood collected in tubes with no additive by allowing

blood to clot for 1 h at 2°C and 2h at 20°C followed by centrifugation at 200g for 10 min.

Serum was stored at -70°C, thawed at 37 °C, and maintained at 2°C for no longer than 3 days before discarding. Two subjects, Nos. 2 and 6, were asked to participate based on known

αjPI deficiency. Sera from patients with documented insulin-dependent diabetes mellitus

(IDDM) were collected in the School of Dentistry during routine examination.

Example 2

Quantitation of ct.PI by ELISA

Wells of microtiter plate (Nunc, Denmark) were coated overnight at 20 °C with the

immunoglobulin fraction of chicken anti-α,PI (lot No. 18823680, O.E.M. Concepts, Toms

River, NJ) at a concentration of 0.5 μg/ml in 0.02 M carbonate-bicarbonate buffer, pH 9.4.

Microtiter plates were washed once in 0.01 M sodium phosphate buffer, 0.15 M NaCl, pH 7.2

(PBS), containing 0.0 % Tween-20 (PBS-Tween) and blocked for 60 min at 20°C with 5 %

fish gelatin (Norland, N. Brunswick, NJ) in PBS-Tween. After washing once, wells were

incubated for 60 min at 20 °C with serum serially diluted twofold beginning with a 1/1000

dilution in a 100-μl volume of 5% fish gelatin, PBS-Tween. After washing three times, wells

were incubated for 60 min at 20°C with polyclonal rabbit anti-human ,PI (lot No. 0180,

Boehringer Mannheim, Indianapolis, IN) at a concentration of 1/4000 in 5 % fish gelatin, PBS-

Tween. After washing three times, wells were incubated for 60 min at 20 °C with horseradish

peroxidase-coupled goat anti-rabbit immunoglobulin (lot No. 025H-4831, Sigma) at a

concentration of 1/4000 in 5% fish gelatin, PBS-Tween. After being washed five times, the

ELISA was developed using the substrate orthophenylenediamine HCI (0.4 mg/ml in 0.05 M

citrate buffer, PH 5.0, containing 0.025 % H₂0₂). The initial slopes were determined at 490

nm in a Vmax kinetic microplate reader (Molecular Devices) and concentrations from at least

four sequential dilutions of a single serum sample were calculated based on standard curves

of α,PI (1.6-200nM). Standard deviations were less than 10% of the mean suggesting little interference in the ELISA from serum contaminants such as complement.

To detect the fraction of serum α,PI in complex with HNE, polyclonal rabbit anti-

human neutrophil elastase (anti-HNE, lot No. 8K3185, Biodesign, Kennebunkport, ME) at a

concentration of 1.1000 in 5% fish gelatin, PBS-Tween, was used in the detection step.

Quantitation was based on a standard curve of preformed equimolar complexes (1.6-200 pM)

between α,PI and HNE (Athens Research & Technology).

Example 3

Residual Catalytic Activity

It has been previously demonstrated that a serum dilution exists at which minimum

catalytic activity can be measured by exploiting the properties of unequal serum concentration

and unequal outcomes of proteinase complexes with α,PI and ₂M. The maximum reduction

in catalytic activity of elastase was used to determine the functionally active concentration of

α,PI.

A PPE standard curve was prepared in each microtiter plate, and units of activity were

determined where one unit is defined as the amount of PPE that hydrolyzes lμM of the

elastase substrate, succinyl-L-Ala-L-Ala-L-Ala -nitroanilide (SA₃NA, Sigma), per minute at

20 °C at pH 7.8. Serum was serially diluted twofold in wells containing 50 μl TBS. Final

serum concentrations for each sample ranged from 0.05 to 20% . PPE was added to wells in

a 10-μl volume at an estimated concentration of 50 U and incubated for 2 min at 37 °C

followed by 25μl SA₃NA in TBS with a final concentration of 0.6 mM in 0.06% dimethyl

sulfoxide (Me₂SO). The maximum (100%) PPE activity was determined in each microtiter

plate by incubating in TBS lacking serum. Optical absorbency (ΔmOD₄₀₅/min) was monitored

at 405 nm for 15 min with 6-s intervals (151 measurements) in a Vmax kinetic mincroplate reader (Molecular Devices) at 20°C. Activity was calculated using the initial 30

measurements by regression analysis (r² > 0.98) and expressed as units of activity (μM/min)

based on a final volume of 85 μl having a path length of 0.18 cm. Activity was determined

for each of 8 or 16 different serum concentrations, and the serum concentration demonstrating the greatest reduction in activity was determined by regression analysis performed for these concentrations. The uninhibited PPE activity at this serum concentration is represented as a

fraction of the total activity residual catalytic activity (μM)

residual catalytic activity (μM)

uninhibited PPE activity (uM/mi ) [1]

= maximum PPE activity (μM/min)

HNE was substituted for PPE in one set of measurements. For comparison, exogenous α,PI

or α₂M were added to sera prior to measuring activity.

The validity of determining the mnctional concentration of serum c^PI using residual

catalytic activity was established by comparing the same measurements using purified

preparations of α,PI, α₂M, PPE, and HNE. Catalytic activity for a constant concentration of

active-site titrated PPE (0.2 μM) was measured after incubation with varied concentrations

of functionally determined α₂M (0.0008-0.1 μM) either before (Ganrot Assay) or after

(competition assay) addition of excess functionally determined α,PI (lμM). In these

experiments, the theoretical fraction of PPE bound to each concentration of α₂M was

calculated assuming 1 : 1 stoichiometry as theoretical molecules PPE/molecule α₂M

= aM (uM). [2]

PPE (0.2μM) where ₂M and PPE represent the active concentrations. The fraction of PPE actually bound

to α₂M was empirically determined in the presence of various concentrations of α₂M and excess α,PI, and this value was calculated as molecules PPE/molecule o^M

= residual catalytic activity [3] theoretical molecules PPE/molecule c^M

The fraction of PPE bound to ,PI was calculated from the competition assay as molecules

PPE/molecule α,PI

= 1 - molecules PPE/molecule α₂M. [4]

Western Blot Analysis

Electrophoresis on 0.75-mm gels composed of 12% total polyacrylamide was

performed using standard SDS-polyacrylamide gel electrophoresis buffers in reducing

conditions after boiling samples 5 min, Proteins were transferred to Immobilon (Millipore

Corp., Bedford, MA) by electrophoresis of proteins in 0.025 M Tris, 0.193 M glycine, 20%

MeOH, and blocked in 3 % nonfat, dried milk. For detection of α,PI, blots were incubated

with rabbit anti-α,PI (0.7 μg/ml, Boehringer Mannheim). Binding was detected by incubation

of blots with horseradish peroxidase conjugated with goat anti-rabbit immunoglobulin (1/1000 ,

Sigma). After being washed extensively, substrate consisting of 0.3 mg/ml 3,3'-

diaminobenzidine in 20 mM Tris, pH 7.4, 0.3 M NaCl, 0.03% H₂0₂ was added.

Example 4

Determination of α,PI Antigen Concentration in Serum

A sandwich ELISA was developed for quantitating α,PI in serum, as well as the

proportion α,PI complexed with HNE. Incubation of untreated serum or plasma with

immobilized antibodies in a microtiter plate can initiate complement activation and aggregation

of proteins resulting in unreliable values. Because chicken antibodies have been shown to lack the capacity to activate human complement, serum α,PI was first captured with chicken anti-

human α,PI. It was observed that sensitivity and consistency were improved when values were based on estimates of Vmax as opposed to the traditional endpoint method (data not shown).

Since gender differences in α,PI quantitative levels have been reported, healthy adult males

and healthy adult females were recruited for the study (Table 1) below. Siblings having

known α,PI deficiency were recruited for the study. Genotype analysis by PCR was performed by Dr. R. Farber, UNC Hospitals, to confirm that these individuals were

homozygous for the deficient Pi_^allele. Because serum concentrations of α,PI are routinely

measured using nephelometric methods, the antigenically quantitated level a ,PI in serum

from Subject No. 6 was confirmed by nephelometry (Dr. J. Katzmann, Mayo Clinic,

Rochester, MN). Serum concentrations of α,PI were found to be 3.27+/= 1.3μM by the

ELISA method and 4.18 μM by nephelometry. By ELISA, healthy individuals demonstrated

a wide range of serum levels of α,PI (10-84 μM) with a minor fraction (0.3-0.4%) in complex

with HNE. The individuals with known α,PI deficiency demonstrated roughly 10% normal

α,PI levels (3-5 μM) and a proportionally increased fraction (3%) was HNE complexed.

r IIΠI.I.IUIU

1 M 22 305 = US 012 O1590 0Q90 39.45 Sδ7

2 M 47 4Λ = 08 OU 09433 O0439 33? 2M

3 46 2X0 = 3Λ 09 t ?l?^B 0138 ma- 3 57

4 M 45 3_LS = 5.1 aΛT 2190 O0083 208Z ■un

5 M 27 37.6 = 10 —L 01403 0085 soββ 3.86

6 F 43 03 i 1-3 0.22 0U390 0 QSS3 2.45 1.79

7 F 39 100 = is 07 03284 O0132 037 iβδ a^» F 44 03 = . •tu 05319 0.0131 058 1.70

9 F 43 200 = ZS ml 02128 0.0032 2Z03- 3Λ1

10 F 44 814 = 13.4 I L 01152 O0061 75.12 A.28

11 F 37 40.1 = 5.0 014 02S97 0.0034 13.72 3.50

12 F 45 405 = 15 JLJL 01656 0.0060 3039 •l.ω

13 F 31 209 = 2.6 &0, 01978 0.0074 2SJ2 L34

14 F 49 37.0 = 12.1 r . 0.1573 0.0073 4034 IL20

18* M 41 282 fl = 42.1 T 2240 0Q19 19Λ9 :i J5

" Mean z standard deviation measured in ELISA by eanturiπo; -ri b anti-αiPI and detecting with anti-αjPI.

* Measured in ELISA by capturing with ano-o,?! sod detecting -^χi~ anti-HNE.

* Not done.

* Measured as described in Fig, 2.

* Measured as described in Fig. 4 using Eq. {61. ' Measured as described in Fig. 5 using Eq. (7].

*^■' Sample collected from Subject No. 6 at a dlfferent time point.

* This IDDM periadontall d sease subject is included w demenstrate the diaerepaney between βtFI quanπtaώn* by V,^UUΛ.^Λ- npresemaoσn in Fig. 4.

Example 5

Determination of Residual Catalytic Activity

The covalent nature of the interaction of PPE within c^M has been shown to be at a site

not inhibitory for the catalytic site of PPE, and this property allows c^M-complexed PPE to

retain discretionary cleavage of low-molecular- weight substrates such as SA₃NA. In contrast,

incubation of PPE with c^PI results in stoichiometrically decreased PPE activity. The rate of

association of PPE to c^M (4.4 x 10⁶ M"¹ s"¹) is 44-fold greater than that of PPE to α,PI (1 x

10³ M^'1 s^"1), and this suggests that in the presence of sufficient competing concentrations of

α₂M, PPE should not be inhibited by ,PI. This principle forms the basis for the Ganrot

Assay which allows determination of α₂M activity by first saturating tM in the presence of

a 2: 1 molar excess of PPE:α₂M after which the noncomplexed proteinase is neutralized by

incubation with 10: 1 molar excess ,PI:α₂M. The stoichiometry of PPE binding to ocjPI is 1: 1. Although each molecule of α₂M has the capacity to bind two proteinase molecules, it has been mechanistically documented that excessively great proteinase concentrations are necessary to achieve a 2: 1 stoichiometry.

Hypothetically, the difference in the residual uninhibited catalytic activity in the Ganrot

assay and the residual activity in the competitive assay represents the concentration of

competing o^PI as long as all other contaminating proteins (e.g., Ial) are without influence.

To establish the validity for measuring elastase inhibitory capacity of o^PI in the presence of competing α₂M, the molecular ratios at which cCjPI and α₂M demonstrate competitively

equivalent elastase binding capacity were examined using isolated proteins. PPE was

incubated with a constant concentration of cc,PI in the presence of varying concentrations of

competing α₂M (competition assay). These values were compared with those obtained using

the Ganrot assay in which PPE was incubated with varying concentrations of ₂M and

subsequently incubated with a constant concentration of ctjPI. As expected, residual

uninhibited catalytic activity was diminished in the competition assay in comparison to activity

using the Ganrot assay (Fig. 1A). In the Ganrot assay, a 2: 1 ratio of PPE (0.2 μM) to α₂M

(0.1 μM) resulted in 1.1 molecules PPE bound to 1 molecule α₂M (Fig. IB), and this result

is consistent with previous mechanistic smdies demonstrating binary α₂M:proteinase

complexes. As the ratio of α₂M to PPE approached 60: 1, two molecules of PPE were

associated with one molecule α M. In contrast, in the competition assay, fewer than 0.6

molecules of PPE were associated with one molecule α₂M as the ration of PPE to α₂M

approached 60: 1. The protection of PPE by α₂M in the competition assay was relatively

constant when the ration of ₂M (0.0031-0.1 μM) to ,PI (lμM) was between 1 : 100 and 1 :320

(Fig. IC). When the concentration of ₂M fell below these values, the protection of PPE by α₂M decreased in proportion. At the intersection of the two regression lines, the

concentrations of PPE, α₂M, and ocjPI are 0.31, 0.003, and 1.0 μM, respectively.

Reliability of Residual Catalytic Activity as a Measure of Serum c.ιPI

Serum dilutions were incubated with varying concentrations of PPE, and catalytic activity was monitored (Fig. 2-2F). Catalytic activity was found to decrease linearly in

relation to the dilution of serum to a minimum point, after which the catalytic activity

increased linearly in relation to the dilution of serum. Regression analysis was used to

calculate the coordinates of the intersection of these two lines, the maximum reduction in catalytic activity. As expected, the abscissa (serum concentration) and ordinate (residual

catalytic activity) at the intersection were found to increase in collinear manner in the presence

of increasing PPE. In other words, increased serum concentration (and 0C]PI concentration)

was necessary to achieve maximum inhibition using increased PPE; likewise, increased PPE

resulted in increased residual PPE activity at the point of maximum inhibition. Since a

collinear relation exists between the concentration of PPE and the values of the coordinates

at the intersection, residual catalytic activity at the serum dilution demonstrating maximum

inhibition can be reliably estimated as the fraction of total PPE activity (0.2008 +/= 0.0138

for Subject No. 11). Variation in multiple determinations of residual catalytic activity for a

single serum sample was < 1 % using consistent concentrations of active-site titrated PPE (near

50 U). Values for some subjects in this study were calculated using sera collected on more

than one occasion, and variation in residual catalytic activity observed to occur between

different sera collections (1-13 %) was interpreted as true variations in residual catalytic

activity.

As expected, when exogenous ,PI (2.7 μM) was added to serum, residual catalytic activity decreased representing an increased concentration of ,PI. When exogenous α₂M (2.7

μM) was added to serum, residual catalytic activity increased representing increased protection

of PPE. In this experiment, the concentration of cc₂M was added in great excess to

demonstrate its effect on the distribution of PPE activity. Since the physiologic concentration of α₂M is 1.56-4.96 μM , and proteinase-complexed o^M is rapidly cleared (half-life 2-4 min) , fluctuations in serum concentrations of α₂M would not be expected to significantly affect measurements of serum iPI. Results suggest that approximately 50% of the exogenous α,PI

(1.50 +/= 0.44 μM) and 80% the exogenous α₂M (2.23 +/= 0.45 μM) were inactivated

upon addition probably as a result of proteo lysis. However, these results support the

hypothesis that residual PPE catalytic activity is primarily determined by the functionally

active fractions of c^PI and α₂M.

Comparison of Residual Catalytic Activity in Serum and Plasma

Evidence has suggested that heparin decreases the rate of α,PI inhibition of HNE, but

not Ppe. The residual catalytic activities were compared in a single individual when blood was

collected with no additive (serum) or into tubes containing the anticoagulants heparin, EDTA

or ACD . The residual catalytic activity for plasma collected in ACD (0.276) was greater than

that for plasma collected in heparin or EDTA or for serum (0.2373 =/+ 0.0018). These

results suggest that serum or plasma can yield equivalent residual catalytic activities using this

method.

Serum from a patient (No . 15) previously determined to have a history of infection with

periodontopathogenic bacteria exhibited higher than normal quantitative levels (283 μM), but

low normal functional levels of ,PI (20μM). When serum from this patient was examined

by Western blot, proteolytic fragmentation was observed. Serum from Subject No. 11 also exhibited evidence of fragmentation compared with that of Subject Nos. 6 and 13. These

results suggest that increased antigenic levels of α,PI as determined by ELISA or nephelometry

might not be representative of the functionally active αjPI.

Determination of the Relationship between Residual Catalytic Activity and the Functional Concentrations of Serum o^PI and α₇M

It was found that residual catalytic activity decreased in direct relation to increased α,PI

concentration in healthy individuals (Fig. 3). Based on these individuals, the relationship between residual catalytic activity and functionally active α,PI was determined using computer-

fit least-squares regression analysis to be

log(x) = log(y)

-0.5

where x represents the functional concentration of α,PI (μM), and y represents residual

catalytic activity of PPE (μM). This equation is equivalent to

log [PPE] Log [α,PI] = log (0.316)

which corresponds to a ratio of PPE:α,PI equivalent to 0.316: 1. This ration is virtually

identical tot he ratios for PPE: ₂M:α,PI determined using isolated proteins in Fig. 1 which

were 0.31:0.003: 1.0.

The stoichiometric relationship between PPE, α,PI AND α₂M demonstrated in Fig. 1

suggests that α₂M might also be estimated using concentrations of PPE and ,PI. Since two

molecules of PPE were shown to associate with ₂M when the concentration of PPE exceeded

60-fold α₂M, the serum concentration of α₂M was estimated using the serum concentrations

at which PPE and α₂M have 1 : 1 stoichiometry as log[α₂M]

[uninhibited PPE activity/maximum PPE activity] = log log [serum concentration]

The results of Fig. 1 suggest the concentration of α₂M might also be calculated using α,PI using the ratio 0.003 : 1.0 (or 1:333) as

log[α,Pr] Log[α₂M] = log[333]

Based on Eqs. [7] and [8], the average ₂M concentration of these individuals was 3.32 +/ =

1.27 μM and 3.25 +/= 1.20 μM, respectively, and these values are within the previously

reported range, 1.56-4.96 μM determined by antigen capture. However, because α₂M has

broad specificity, proteinases that do not compete for and are not eliminated by α₍PI retain

their capacity to interact with and diminish α₂M, and this effect escapes detection by the

methods described here. This suggests that measuring α₂M by competition with ,PI might

vary as a result of serum proteinases other than elastase. Further examination of the behavior

of α₂M by comparison using ELIS or the Ganrot assay is needed for verification of the

measurements described here.

Comparison of HNE with PPE as a Measure of Serum of o^PI

Although α₂M inhibition of proteinases including PPE involves covalent proteinase

interaction with a thiol ester, the noncovalent complex between HNE and α₂M has been shown

to be unique in lacking a thiol ester bond. The rate of association of HNE for α,PI (6.5 x 10⁷

M^s^'1). During the acute phase, c^PI increases 2- to 4-fold, and this is consistent with the

critical ratio for 0C[PI:a₂M during control of HNE. In the absence of influence by other serum

inhibitors, measurement of residual catalytic activity with HNE or PPE should yield identical

values for α,PI concentration. Residual catalytic activity of a single serum sample was compared using PPE or HNE as described. Based on the relationship described in Fig. 3 , total c^PI concentration was determined to be 22.25 μM. To more easily compare molar relationships, serum concentrations were converted to represent α,PI concentrations (FIG.

4A). In parallel, residual catalytic activity of the same serum sample was measured using HNE. As expected, HNE was found to exhibit bimodal activity when varying concentrations of serum were incubated with a constant concentration of active-site titrated HNE (Fig. 4B).

Based on total serum α,PI as determined using PPE (22.25 μM), the serum concentrations

between 10"¹ and 10"³ were converted to represent alP concentrations (Fig. 4C). At the serum

concentration (0.0107) displaying minimum catalytic activity, the HNE concentration was

0.2227 μM and the c^PI concentration was 0.2388 μM a ratio of 0.93:1. Then the total

concentration of α,PI can be calculated from the serum concentration as

0.238 uM α.PI = 22.24 μM 0.0107

Since this value is virtually identical with the concentration of α,PI determined using PPE, it

can be concluded that the assay is highly specific and that the empirically derived relationship

accurately represents the theoretical relationship. Further, the consistency in these

measurements suggests that serum inhibitors other than α,PI and α₂M do not contribute

significantly to inhibition of PPE or HNE.

Determination of Quantitative and Functional Levels of Serum c^PI in an IDDM Population

It has been previously reported that patients with insulin-dependent diabetes mellitus

have greater or lower (36-38) c^PI values compared with the normal population. Applying

the methods developed here, the ,PI quantity and function in a population of IDDM patients

with and without periodontal disease were determined. Because IDDM patients are known to have altered glaciation of secreted proteins, patients demonstrating aberrant glycosylated hemoglobin were eliminated from the study. The results clearly demonstrate that there is a significant quantitative difference between the IDDM and the normal population. However, when IDDM patients are dichotomized based on evidence of periodontal disease, it becomes

apparent that IDDM patients without periodontal disease have normal levels of α,PI, whereas those with periodontal disease have significantly greater than normal α,PI. In comparison, when the elastase inhibitory capacity in these patients was determined, there was a less dramatic, although statistically significant, difference between subjects suggesting an

underlying pathology perhaps of bacterial origin unrelated to periodontitis . These data suggest

that although the IDDM patients manifesting periodontal disease have increased levels of

antigenically determined c^PI, a significant proportion my be inactivated. Further, these data

demonstrate an attendant systemic manifestation associated with periodontal disease in IDDM

patients.

It is evident from the foregoing that the present inventions provides a reproducible,

inexpensive and expedient method for determinations of the functionally active concentrations

of o^PI and α₂M in serum or plasma.

Although the invention has been illustrated by the preceding disclosures, it is not to be

considered as being limited to the examples disclosed therein, but rather, the invention is

directed to the generic area or hereinbefore disclosed. Various modifications and embodiments

thereof may be made without departing from the spirit and scope thereof.

Claims

WHAT IS CLAIMED IS:

1. A method for the quantitative determination of a proteinase inhibitor in serum or

plasma which comprises the steps of:

a) obtaining a sample of blood from a subject;

b) separating the blood into a fluid containing serum or plasma; c) preparing a first plurality of serial dilutions of the fluid of decreasing

concentrations;

d) incubating the dilutions with varying concentrations of PPE and monitoring the

to a minimum point, after which the catalytic activity increase linearly in

relation to the dilution of the fluid;

of two linear lines formed by the fluid concentration and residual catalytic

activity; and

squares regression analysis and comparing with a standard curve.

2. The method of claim 1 wherein the proteinase inhibitor in α, _PI.

3. The method of claim 1 wherein the proteinase inhibitor is α₂M.

4. The method of claim 1 wherein the subject is human.

5. The method of claim 1 wherein the subject is animal.

6. The method of claim 1 wherein the fluid is blood serum.

7. The method of claim 1 wherein the fluid is blood plasma.

8. A method for the evaluation of noninflammation condition in a subject which comprises determining the quantity of functionally active proteinase inhibitor in the subject by the method of claim 1.

9. The method of claim 8 wherein the condition is insulin - dependent diabetes mellitus.

10. The method of claim 9 wherein the subject has a periodontal disease.

11. The method of claim 8 wherein the condition is an age related illness. '

12. The method of claim 8 wherein the condition is HIV infection.

13. The method of claim 8 wherein the condition is HIV infection.

14. The method of claim 8 wherein the condition is arthritis.

15. The method of claim 8 wherein the condition is atherosclerosis.

16. The method of claim 8 wherein the condition is asthma.

17. The method of claim 8 wherein the condition is systemic lupus erythematosis.

18. The method of claim 8 wherein the condition is of lymphoid origin.

19. The method of claim 8 wherein the condition is a malignant illness.

20. The method of claim 8 wherem the condition is an infection of bacterial, viral or other

parasitic origin.