WO1998005966A1 - Phospholipase a2 as a marker for the presence of pulmonary fat embolism - Google Patents

Abstract

A method of predicting the onset or severity of pulmonary fat embolism disorder in a patient, which comprises analyzing a body fluid of the patient for secretory phospholipase A2 concentration or activity; comparing the concentration or activity with a standard value selected from the group consisting of (1) a range of secretory phospholipase A2 concentrations or activities, respectively, for said body fluid in a control population and (2) a range of secretory phospholipase A2 concentrations or activities, respectively, of previously isolated samples of the body fluid of the patient; and relating a statistically significant higher concentration or activity, respectively, of secretory phospholipase A2 relative to the standard value as predictive of onset and severity of pulmonary fat embolism.

Description

PHOSPHOLIPASE A₂ AS A MARKER FOR THE PRESENCE OF PULMONARY FAT EMBOLISM

ACKNOWLEDGEMENTS This invention was supported in part by grant number HL20985 from the

National Institutes of Health. The U.S. Government has rights in this invention as a result of such support.

INTRODUCTION Technical Field

The present invention is in the field of diagnosis of incipient fat embolism syndrome in patients at risk for the same and, in particular, predicting the onset of acute chest syndrome in sickle cell disease patients and predicting the onset of multiple organ failure in patients having a trauma-related injury.

Background

Pulmonary fat embolism (PFE) has previously been diagnosed by clinical observation of symptoms after onset of the disorder has progressed sufficiently for the symptoms to come into existence. These symptoms include, for classic pulmonary fat embolism, pulmonary disease with hypoxia, mental status changes, and a fall in platelets or hemoglobin (Levy, review, Clin. Orthop. , 1990, 261 :281 ; Moylan et al., J. Trauma, 1976, 16:341 ; Peltier, J. Trauma, 1971, 11 :661). There is currently no recognized diagnostic or predictive assay for pulmonary fat embolism that is used either to assist in the diagnosis of existing disease, to predict the onset of the disease, or to predict the likely prognosis once disease has begun.

Among those particularly at risk for developing pulmonary fat embolism are trauma victims, especially those with long bone fractures (Levy 1990), as well as patients undergoing orthopedic or cardiac surgery. Prospective studies in trauma patients have revealed that although fat embolism occurs in the majority of trauma victims, "fat embolism syndrome" occurs in only about 10 percent of these same patients (McCarthy et al. , J. Trauma, 1973, 13:9; Fabian et al. , Crit. Care. Med. , 1990, 18:42). This suggests that "fat embolism syndrome" represents only _^ the most severe form of fat embolism and that, currently, pulmonary fat embolism is clinically unrecognized in most cases.

In addition to its association with trauma-related injuries, pulmonary fat embolism has been implicated in acute chest syndrome. Acute chest syndrome

(ACS) is the second most common cause of hospitalization and the leading cause of death in sickle cell disease (SCD) patients (Platt et al., N. Engl. J. Med., 1994, 330:1639; Vichinsky, Semin. Hemaϊol , 1991, 28:220; Sprinkle et al., Am. J. Pediatr. Hematol. Oncol., 1986, 8:105). A majority of SCD patients will experience at least one episode of ACS (Powars et al., Medicine, 1988, 67:66;

DeCeulaer et al., Eur. J. of Pediatr., 1985, 144:255). Repeated episodes can result in rapidly progressive chronic lung disease (Powars 1988). Despite its substantial morbidity and mortality, little is known about the etiology of ACS. Although generally attributed to infection and infarction (Sprinkle 1986; Powars 1993; Poncz et al., J. Pediatr. , 1985, 107:861; Castro et al. , Blood, 1994,

84:643; Charache et al. , Arch. Intern. Med. , 1979, 139:67), recent evidence suggests that pulmonary fat embolism may be related to many cases of severe ACS (Vichinsky et al., Blood, 1994 83:3107; Shapiro et al., Arch. Intern. Med. , 1984, 144: 181 ; Johnson et al. , Am. J. Hematol. , 1994, 46:354). Accordingly, a method for predicting the presence of pulmonary fat embolism at early stages, before the manifestation of symptoms, would be advantageous in the treatment and prevention of severe lung damage in patients at risk for developing pulmonary fat embolism. Similarly, a predictive assay that gives a reliable indication of the severity of existing pulmonary fat embolism would likewise be advantageous. The present inventors have now shown that an increased level of secretory phospholipase A₂ (sPLA₂) is associated with the presence of pulmonary fat embolism and that measurement of sPLA₂ is particularly useful for predicting the onset of ACS in SCD patients and for predicting the severity of the disease. The present inventors have also shown that an increased level of sPLA₂ activity correlates with postiηjury multiple organ failure (MOF).

Phospholipase A₂ (PLA₂) is an enzyme which cleaves phospholipids at the sn-2 position, generating free fatty acids and lysophospholipids. Type II secretory PLA₂ (sPLA₂) is found in low concentration in normal plasma (Kramer, Advances in Second Messenger and Phosphoprotein Research. , 1993, 28:81); however, its levels are increased in response to inflammation (Nevalainen, Clin. Chem., 1993, 39:2453; Oka et al., Biol. Chem. , 1991 , 266:9956; Pruzanski, Immunol. Today, 1991 , 12: 143; Dennis, Biol. Chem., 1994, 269: 13057), sepsis (Vadas 1984; Green et al. , Inflammation, 1991, 15:355; Vadas et al. , Can. J. Physiol. Pharmacol.,

1983, 61 :561 ; Vadas et al. , Crit. Care Med. , 1988, 16: 1) and arthritis (Green 1991 ; Pruzanski 1991). Elevated levels of sPLA₂ have been reported in cases of adult respiratory distress syndrome (ARDS) (Baur et al., Klin. Wochenschr, 1989, 67: 196; Rae et al., Lancet, 1994, 344: 1472; Vadas, J. Lab. Clin. Med., 1984, 104:873), and multi-organ dysfunction (Baur 1989; Fink, Crit. Care Med., 1993,

21 :957; Vadas et al. , Cήt. Care Med. , 1993, 21 : 1087). However, elevated sPLA₂ levels in these syndromes have not previously been known to precede or predict the onset of these or any disorders. Likewise, elevated sPLA₂ levels have not previously been associated with pulmonary fat embolism or with acute chest syndrome in sickle cell disease patients. Secretory PLA₂ is thought to be a mediator of inflammation in these conditions as it can hydrolyze arachidonic acid from the sn-2 position of phospholipids, thereby providing the essential substrate for a number of eicosanoids (Fink 1993). Secretory PLA₂ is also upregulated in response to proinflammatory cytokines such as tumor necrosis factor and interleukin-1 (Pruzanski 1991 ; Vadas 1993; Dennis 1994; Clark et al. , Biochem.

J., 1988, 250: 125; Vadas et al. , Infection and Immunity, 1992, 60:3928; Pfeilschifter et al., Biochem. Biophys. Res. Communi., 1989, 159:385). PLA₂ given intravenously or instilled intra-tracheally in animal models produces diffuse ARDS-like changes including diffuse alveolar edema and an inflammatory cell influx (Niewoehner et al. , J. Appl. Physiol. , 1989, 66:261; Edelson et al., Am.

Rev. Respir. Dis., 1991, 143: 1102), suggesting a role for PLA₂ in the pathophysiology of lung disease. This same lung injury can be prevented by pre- treatment with inhibitors of PLA₂ (Tighe et al., Intensive Care Med., 1987, 13:284; Koike et al., Surgery, 1992, 112: 173). In humans, sPLA₂ is increased in patients with ARDS and has been reported to correlate with outcome, severity of lung injury, and alveolar-arterial oxygen gradient (Baur 1989; Rae 1994; Schroder et al., Resuscitation, 1982, 10:79; Uhl et al., J. Trauma, 1990, 30: 1285). However, no correlation has been reported between the increase of sPLA₂ levels prior to onset of ARDS and diagnosis of ARDS.

A recent report has suggested pulmonary fat embolism as a distinct cause of severe acute chest syndrome in sickle cell disease patients (Vichinsky 1994). In SCD patients with ACS, those diagnosed as PFE positive by bronchoalveolar lavage experienced a different and more severe clinical course than those diagnosed as PFE negative. In a different study, sickle cell patients with ACS were shown to have elevated levels of both palmitic acid and oleic acid as well as total free fatty acids (Hassell et al. , Blood, 1994, 84:412A).

SUMMARY OF THE INVENTION The present invention provides a method and a kit for predicting the onset and severity of secretory PLA₂-related disorders including pulmonary fat embolism, multiple organ failure, and acute chest syndrome. The method can be described in several steps: A sample of body fluid is obtained from a patient and analyzed for the concentration or activity of secretory phospholipase A₂; the measured concentration or activity is then compared with a standard value, which may be a range of secretory phospholipase A₂ concentrations or activities, respectively, for the same body fluid in a control population. Alternatively, the standard value may be a secretory phospholipase A₂ concentration or activity of previously isolated samples of the same body fluid from the patient whose body fluid is being analyzed. A higher concentration or activity of secretory phospholipase A₂ relative to the standard value is related to the prediction of the onset and severity of various sPLA₂-related conditions.

In one embodiment, the current invention provides a method for predicting the onset and severity of secretory PLA₂-related disorders including pulmonary fat embolism, multiple organ failure, and acute chest syndrome by analyzing sequential samples of a body fluid of a patient to obtain a patient profile of secretory phospholipase A₂ concentration or activity over a period of time. These concentrations or activities, respectively, are compared with a standard profile of concentrations or activities, respectively, of secretory phospholipase A₂ for the same body fluid in a control population over a period of time. A higher concentration or activity of secretory phospholipase A₂ relative to the standard profile is related to the prediction or indication of the onset and severity of various sPLA₂-related conditions.

In another embodiment of the current invention, a kit is provided which contains an assay test kit for detecting secretory phospholipase A₂ and guidelines for predicting the onset or severity of various sPLA₂-related disorders, according to the method of this invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention now being generally described, the same will be better understood by reference to the following detailed description of specific embodiments and to the drawings that form part of this specification, wherein: Figure 1A depicts direct correspondence between secretory phospholipase A₂ (sPLA₂) activity as measured by a radio-assay and sPLA₂ protein concentration as measured by enzyme-linked immunosorbent assay (ELISA).

Figure IB depicts direct correspondence between sPLA₂ activity as measured by a fluorometric assay and sPLA₂ protein concentration as measured by ELISA. Figure 2 depicts sPLA₂ protein concentrations in various patient populations.

Figure 3 depicts profiles of sPLA₂ protein concentrations over twelve days from four patients admitted with vaso-occlusive crisis (VOC) who subsequently developed acute chest syndrome (ACS). Figure 4 depicts sPLA₂ concentrations in various patient groups having

ACS.

Figure 5 depicts profiles of sPLA₂ activity over time from patients with or without multiple organ failure.

DESCRIPTION OF SPECIFIC EMBODIMENTS

The present invention provides a method that is useful for the related purposes of predicting the onset of sPLA₂-related diseases such as pulmonary fat embolism (PFE), acute chest syndrome (ACS) and multiple organ failure (MOF), and of predicting the severity of this class of diseases. The method relates to the discovery by the present inventors of the association of PFE with an increased concentration of secretory phospholipase A₂ (sPLA₂) in a body fluid of the patient. Without wishing to be bound by theory, the inventors hypothesize that free fatty acids and lysophospholipids released by the hydrolysis of phosopholipids in the embolized fat directly cause acute lung injury (Styles et al. , 1996, Blood, 87:2573; Kuypers et al., 1995, Colloque INSERM, 234:501). The increase in sPLA₂ concentration or activity usually precedes the manifestation of symptoms currently used for diagnosis of PFE. By measuring the level of sPLA₂ in a patient, the physician can determine the presence of developing or existing PFE and can modify patient treatment accordingly.

The method of the present invention comprises analyzing a sample of a body fluid of a patient for sPLA₂ concentration or activity, comparing the concentration or activity, respectively, with a standard value selected from the group consisting of (1) a predetermined range of secretory phospholipase A₂ concentrations or activities, respectively, for the same body fluid in a control population and (2) a sPLA₂ concentration or activity, respectively, of a previously isolated sample of the same body fluid of the patient whose body fluid is being analyzed, and relating a higher concentration or activity of sPLA₂ relative to the standard value as an indication of the onset or severity of a sPLA₂-related disorder.

Currently known sPLA₂-related disorders or conditions include pulmonary fat embolism, acute chest syndrome, multiple organ failure, adult respiratory distress syndrome, sepsis and arthritis. It is understood, however, that the method of the invention can readily be applied to other sPLA₂-related disorders that may later become evident.

In a specific embodiment of the invention, the sPLA₂-related disorder is pulmonary fat embolism (PFE). Preferably, the method is used to predict onset of PFE in patients at risk of developing such embolism. Patients particularly at risk for developing PFE include those undergoing orthopedic or cardiac surgery, or any other surgery involving potential exposure of the circulating blood to bone marrow; trauma victims, particularly those suffering long bone fractures or other bone disturbance; blunt trauma victims; and sickle cell disease patients or patients with other disease states that might lead to the exposure of necrotic or apoptotic bone marrow to the blood circulation. The method of the present invention is not limited to use in these particular classes of patients, however, as this method can provide meaningful predictive information in any patient for whom development of PFE is a possibility, even if a remote one.

Prior to the current invention, diagnosis of PFE was made by evaluating body temperature, changes in mental status, a decrease in platelet and hemoglobin levels, and decreased blood oxygenation in patients showing respiratory distress.

The present invention advantageously provides predictive information at earlier stages and with greater certainty than previously available methods. sPLA, levels typically increase prior to the onset of certain symptoms of PFE, for example, respiratory distress, elevated body temperature, decreased blood oxygenation, or characteristic new white infiltrate visible on chest x-rays, by at least several hours to as much as several days, depending on the circumstances of the individual and the level of sPLA₂ observed to be present in the serum.

Analysis of sPLA₂ concentration or activity can be carried out on any body fluid, such as blood (or a blood fraction, especially serum or plasma), urine, sweat or saliva. Preferred samples for analysis are serum and plasma. The body fluid is obtained from a patient whose sPLA₂ levels are to be measured. It should be appreciated that methods and techniques for obtaining a patient's body fluids, especially serum, plasma, urine, sweat and saliva, are commonly practiced and well known to those of ordinary skill in the art. Concentration or activity of sPLA₂ in the body fluid assayed is compared with a standard value to determine the presence of PFE. The standard value is usually (1) a range of secretory phospholipase A₂ concentrations or activities, respectively, for the same body fluid in a control population or (2) a previously obtained sPLA₂ concentration or activity or a range of sPLA₂ concentrations or activities, respectively, of the same body fluid obtained from the same patient prior to isolating the sample being analyzed. It is apparent that comparison with both standard values (1) and (2) may be possible and may provide confirmatory evidence of the presence of PFE.

The first general standard value set out above, a range of secretory phospholipase A₂ concentrations or activities, for the body fluid in a control population, is typically obtained by using the same assay technique that will be used in the application of the method to the sample being tested in order to ensure the highest correlation. Sufficient measurements are made within the appropriate control population to produce a statistically significant range of control values to which a comparison will be made. It is appreciated that the appropriate control population will vary depending upon the particular patient being tested.

Preferably, the control population is selected such that its members approximately match the patient being tested with respect to any characteristic or condition, independent of PFE, known to affect the sPLA₂ level. The range of sPLA₂ concentration or activity determined from this population thus serves as a baseline value for sPLA₂ concentration or activity for the individual patient being tested. In many cases, the appropriate control population will consist of normal, healthy humans. In such a control population the concentration of sPLA₂ has been shown to be approximately 1-4 ng/ml in plasma or serum. A control population consisting of normal healthy humans will be appropriate particularly when the patient being tested is free of conditions unrelated to PFE that may contribute to an increase in sPLA₂ levels.

A number of conditions unrelated to PFE are known to cause an elevation in sPLA₂ levels. For example, SCD patients at baseline demonstrate a 2 to 3-fold _ elevated sPLA₂ level compared to normal controls (see Figure 2). For this reason, if the first general standard is used in the method of the present invention, the appropriate control population will consist of SCD patients without ACS or will take this variation of normal values into consideration. The reason for the higher level of sPLA₂ in SCD patients is not known. Elevated levels of sPLA₂ have also been reported in conditions such as ARDS, sepsis, multi-organ dysfunction, arthritis, and pneumonia. The levels reported vary widely with the particular condition. When the patient being tested is known to have a condition other than PFE that is associated with elevated sPLA, levels, the appropriate control population is preferably a population having the same condition.

The foregoing discussion is not to suggest, however, that an actual control population must be measured for every application of the method of the present invention. Once a clinically satisfactory standard is established, this predetermined standard range can be used for subsequent evaluations without additional testing of control populations. It is also possible to relate the sPLA₂ level for any patient to normal human controls by taking into consideration elevated sPLA₂ levels associated with other disorders. It will be apparent that to obtain the first general standard value set out above, the sPLA₂ concentration or activity for the appropriate control population can be determined in a number of ways. For example, it can be estimated from values in the relevant scientific or clinical literature, it can be constructed from a combination of measured values and estimated adjustment factors (i.e. adjustments for the presence or absence of a condition other than PFE affecting sPLA₂ concentration), or it may be actually measured. The first general standard value is particularly useful when it is likely that the patient being tested is in more advanced stages of PFE; for example, trauma victims from which no previous measurements of sPLA₂ were taken. The second general standard value set out as an alternative above is a previously obtained sPLA₂ concentration or activity or a range of sPLA₂ concentrations or activities from the same body fluid of the same patient to be tested. Measurements may be made on single or multiple samples of body fluid taken from the patient prior to the collection of the morbid sample. Typically the measurement of sPLA₂ concentration or activity in the previously isolated sample is taken using the same technique as that used in the test application. Preferably the previously measured sPLA₂ concentrations or activities are measured at times well before the onset of PFE and will fall within the predetermined range of values for the appropriate control population, although this is not essential to the application of the present invention. The second general standard is particularly useful when the nature of the medical condition of the patient to be tested is stable enough to allow monitoring in advance of conditions precipitating development of PFE, such as sickle cell patients with vaso-occlusive crisis, or patients who will later undergo orthopedic surgery, or trauma patients after injury but before onset of multiple organ failure.

When the first general standard value is used for comparison with the sPLA₂ value of the sample being analyzed, the threshold concentration or activity indicative of the onset of PFE can be determined by any appropriate statistical method. In general, the minimum concentration or activity indicative of onset of PFE is considered to be set by appropriate clinical trials, taking into account other simple conditional parameters. The concentration or activity set above the mean of the predetermined sPLA₂ concentration or activity range for the appropriate control population will indicate the threshold above which the onset of the syndrome is likely to occur with a particular level of certainty. It will be recognized by those familiar with statistics that the number of standard deviations used as a positive indication of PFE will be selected with an appropriate diagnosis goal in mind. A concentration or activity greater than one standard deviation from the mean may correlate with onset of the disease, particularly in combination with the presence of additional symptoms. A concentration or activity greater than two standard deviations from the mean generally indicates statistical significance and is predictive of onset of the disease. A concentration or activity value greater than three standard deviations is accordingly predictive of onset of the disease with a higher degree of certainty, and values greater than four standard deviations will be predictive of the disease with a still higher degree of certainty. It will also be recognized that concentration or activity levels falling outside the range observed . for the control population are statistically significant values. Preferably, a particular concentration or activity of sPLA₂ considered to reflect a positive indication of onset of a disorder is best selected by the attending physician and will vary depending on the condition of the patient as well as the presence of other, conventional indications of the onset of PFE.

As an example, in the case of ACS in sickle cell disease, a level above 100 ng/ml reflects a 75% probability of later onset of ACS. If sPLA₂ is above 100 ng/ml and the patient also has a fever, the probability of developing ACS rises to 95% . If the sPLA₂ is at or above 200ng/ml, the probability of later onset of ACS increases accordingly.

Similarly, when the second general standard value is used for correlation with the sPLA₂ value of the sample being analyzed, the threshold concentration or activity indicative of the onset of PFE can be determined by any appropriate statistical method. The concentration or activity set above the mean of the predetermined sPLA₂ concentration or activity range for the previously obtained samples will indicate the threshold above which the onset of the syndrome is likely to occur with a particular level of certainty, as described above for the first general standard value. If the second general standard value is taken as a single measurement of a single previously isolated sample, a positive assay may be confirmed by subsequent measurements that reflect continuing increase in sPLA₂ levels. In preferred embodiments this amount is a numerical threshold determined to be statistically significant based on comparison with a control population, as indicated above. Additional methods of statistical analysis are also available to determine if the individual patient's sPLA₂ concentration or activity is significantly different from standard values. Most preferably, a particular increased concentration or activity of sPLA₂ considered to reflect a positive result is best selected by the attending physician and will vary depending on the condition of the patient as well as the presence of other, conventional indications of the onset of

PFE.

It will be recognized by those skilled in clinical analysis that the method of the invention may be used in combination with other clinical indications observed by the attending physician to formulate a diagnosis. One specific embodiment of the present invention provides a method for predicting the onset and severity of acute chest syndrome in patients having sickle cell disease. In this embodiment, the method comprises analyzing an isolated body fluid of a patient having sickle cell disease for secretory phospholipase A₂ concentration or activity, comparing the measured concentration or activity with a standard value selected from the group consisting of (1) a range of secretory phospholipase A₂ concentrations or activities, respectively, for the same body fluid in persons with sickle cell disease and without acute chest syndrome and (2) a sPLA₂ concentration or activity or a range of secretory phospholipase A₂ concentration or activities, respectively, of previously isolated samples of the same body fluid of the same patient, and determining the statistical significance of a higher concentration or activity, respectively, of secretory phospholipase A₂ relative to the standard value as a predictor of onset and severity of acute chest syndrome.

Among patients with SCD, patients with ACS exhibit dramatically elevated levels of sPLA₂ (see Example 2). Similar elevations are not observed in patients with vaso-occlusive crisis alone, and the presence or absence of fever with pain crisis does not alter this result. Although sPLA₂ levels in patients with ACS show some overlap with sPLA₂ concentrations reported in acute respiratory distress syndrome (ARDS) patients, the levels found in ACS patients are frequently two to three times higher than those reported in ARDS patients (Vadas et al. , J. I tb. Clin. Med. , 1984, 104:873; Baur 1989). The sPLA₂ levels in ACS patients are up to 10-fold higher than those reported for pneumonia patients (Rintala et al., Clin.

Infect. Dis. , 1993, 17:864), clearly distinguishing ACS from pneumonia.

Secretory PLA₂ concentration in patients with ACS correlates with several measures of clinical severity in studies by the present inventors. Figure 4 shows a bar graph comparing sPLA₂ levels in ACS patients with three characteristic measurements of severity of ACS: hypoxia, increased alveolar-arterial O₂ gradient, and need for transfusion. Patients with hypoxia (PaO₂<70 mm Hg) display statistically significant increases in sPLA₂ concentration compared to patients without hypoxia (PaO₂ > 70 mm Hg, where PaO₂ equals partial pressure of oxygen _._ in arterial blood). Patients with increased alveolar-arterial O₂ gradient ([A- a]O₂> 30mm Hg) display statistically significant increase in sPLA₂ concentration compared to patients with normal alveolar-arterial O₂ gradient ([A-a]O₂ < 30 mm Hg). Patients who require transfusion (TXN +) display statistically significant increases in sPLA₂ concentration compared to patients who did not require transfusion (TXN -). In Figure 4, error bars indicate ± 1 SD and n= 15 for all three comparisons. For all three positive indications of severity of ACS, mean sPLA₂ levels are about 500 ng/ml, while none of the three negative indications of verity of ACS exhibit sPLA, concentrations equal to or greater than about 380 ng/ml. These data suggest that sPLA₂ levels equal to or greater than about 380 ng/ml in patients with ACS are predictive of increased severity of the disease and may be predictive of a need for transfusion. The predictive value of sPLA₂ with arterial-alveolar gradient is particularly relevant, as a recent report (Emre et al., J. Pediatr. , 1993, 123:272) indicates the alveolar-arterial gradient to be the strongest predictor of clinical severity in ACS.

In ACS patients followed over time, the increase in sPLA, coincides with the onset of ACS, and sPLA₂ levels decline as the patients improve. Figure 3 demonstrates sequential secretory phospholipase A₂ protein levels from four patients initially admitted with VOC who went on to develop ACS. Hospital day 0 is the day the clinical diagnosis of ACS was made. Hospital days before and after diagnosis of ACS are designated by negative and positive numbers, respectively, in Figure 3. In vaso-occlusive crisis patients followed over time, sPLA₂ levels are observed to increase in the 2-3 days prior to ACS. Application of the method of the present invention to patients undergoing vaso-occlusive crisis will beneficially result in earlier employment of therapies to prevent or lessen the consequences of ACS. The method of the present invention does not depend on or dictate any particular treatment of ACS.

Another specific embodiment of the present invention provides a method for predicting the onset of multiple organ failure. This method comprises obtaining and analyzing a body fluid of a patient for secretory phospholipase A₂ concentration or activity, comparing the measured concentration or activity with a standard value selected from the group consisting of (1) a range of secretory phospholipase A₂ concentrations or activities, respectively, for the same body fluid in a control population and (2) a single value or a range of secretory phospholipase

A₂ concentrations or activities, respectively, of previously isolated samples of the same body fluid of the same patient, and determining the statistical significance of a higher concentration or activity, respectively, of secretory phospholipase A₂ relative to the standard value as a predictor of onset of multiple organ failure. Preferably, the body fluid tested derives from a patient at risk for multiple organ failure. Patients particularly at risk are those suffering injury related to trauma, such as would result in a bone fracture or would otherwise result in bone marrow contacting circulating blood. Patients having a blunt- trauma related injury are also at risk for multiple organ failure. For this embodiment of the invention, it is appreciated that the control population in the first general standard value set will depend on the patient in question. For example, a patient at risk of multiple organ failure and having a trauma-related injury may be compared with a control population comprised of patients who also have a trauma-related injury, but who do not have multiple organ failure.

In one specific embodiment of the invention multiple sequential samples of a body fluid of a patient are isolated and analyzed to create a patient profile of sPLA₂ concentration or activity over a period of time. The profile may be used to chart changes in sPLA₂ levels in one patient over time. Elevation in sPLA₂ levels in the same patient are indicative of onset and severity of sPLA₂-related disorders. The patient profile can be compared with a standard profile, which is obtained by assembling the concentrations or activities of sPLA₂ for the same body fluid in a control population. Statistical significance can be determined for higher concentrations or activities of sPLA₂ in the patient profile compared to the standard profile, and a higher concentration or activity in the patient profile is related to an indication of onset of a sPLA₂-related disease.

For use in the method of the present invention, the concentration of sPLA₂ can be determined in any of a variety of ways that are well known in the art or that are later discovered. For example, a quantitative enzyme immunoassay or radioimmunoassay for laboratory testing may be used in the present invention. A preferred method utilizes a sandwich enzyme-linked immunosorbent assay (ELISA). In a typical assay using this technique, antibody is attached to a solid surface, such as a microtiter plate well, a test tube or a porous reagent strip (such as cellulose or glass fiber). The antibody-coated surface is then contacted with the sample and allowed to bind any antigen that is present in the sample. For use in the present invention, the antigen will typically be sPLA₂. The coated surface is washed free of unbound antigen and a second antibody is used to detect the presence of the bound antigen. The second antibody carries a label which is readily detectable, for example, by radiometric or calorimetric techniques. The amount of the second antibody bound is proportional to the concentration of antigen present in the sample.

Antibody production for use in an assay for sPLA₂ is conventional and is not described here in detail. Techniques for producing antibodies are well known in the literature and are exemplified by the publication Antibodies: A Laboratory

Manual (1988) eds. Harlow and Lane, Cold Spring Harbor Laboratories Press, and by U.S. Patent Nos. 4,381,292, 4,451,570, and 4,618,577. For an example of production of antibodies specific for sPLA₂, see Smith et al. (Smith et al., Br. J. Rheumatol., 1992, 31 :175). General steps involved in obtaining an antibody to sPLA₂ are as follows:

An animal is injected with a composition containing the antigen, usually purified sPLA₂ protein. The protein may by naturally occuring or recombinant. Multiple injections or the use of an adjuvant will ensure maximum stimulation of the animal's immune system and production of antibodies. If polyclonal antibodies are desired, they can be prepared by collecting blood from the immunized animal and separating the antibodies from other blood components by standard techniques. To obtain monoclonal antibodies, the spleen or lymphocytes from the immunized animal are removed and immortalized or used to prepare hybridomas by cell- fusion methods known to those skilled in the art. Antibodies secreted by the immortalized cells are screened to identify the clones that secrete antibodies of the desired specificity. For monoclonal anti-sPLA₂ antibodies, the antibodies must bind to sPLA₂. Cells producing antibodies of the desired specificity are selected, cloned, and grown to produce the desired monoclonal antibodies.

Antibody can be attached to a solid surface for use in an assay of the invention using known techniques for attaching protein material to solid support materials. The solid support can include plastic surfaces of test tubes or microtiter plates, polymeric beads, dip sticks, or filter materials. The attachment methods include non-specific adsorption of the protein to the support and covalent attachment of the protein, typically through a free amino group, to a chemically reactive group on the solid support, such as an activated carboxyl, hydroxyl, or aldehyde group. The concentration of sPLA₂ may also be determined indirectly by measuring sPLA, activity. sPLA₂ activity may be determined by any of a number of methods well known in the art. (Van den Bosch et al., Agents Actions 9:382, 1979.) In particular, sPLA₂ activity can be determined by measuring the breakdown of a substrate (phospholipid) of sPLA₂ or by measuring formation of the products (lysophospholipid and unesterified fatty acid). After incubation of the sample that contains phospholipase activity, the products are separated from the substrate and quantitated by well established lipid biochemistry techniques. Alternatively, the products of a sPLA₂ enzymatic reaction may have distinct characteristics from the substrate and can be measured without separation. The presence of a labeled fatty acyl group at the sn-2 position of the phospholipid substrate facilitates the measurement and increases substantially the sensitivity. The label attached to the fatty acid can be radioactive or a chromophore (light absorbing fluorescent or chemiluminescent compounds)(see Example 1). A direct linear correlation exists between sPLA₂ concentration as measured by ELISA and sPLA₂ enzyme activity as measured by radio-assay (see Figure 1A) or by fluorometric assay (see Figure IB and Example 1). For example, Figure 1A shows the correlation between (sPLA₂) activity as measured in arbitrary units (AU) by hydrolysis of radio-labeled phosphatidylethanolamine and sPLA₂ protein concentration in ng/ml as measured by enzyme-linked immunosorbent assay

(ELISA) in 26 plasma samples from sickle cell patients (r² = 0.953).

One embodiment of the current invention is a kit for diagnosing the potential onset of a sPLA₂-related disorder and the severity of a sPLA₂-related disorder. The kit includes assay materials for detecting sPLA₂ and guidelines for predicting the onset or severity of a secretory sPLA₂-related disorder according to the method of the current invention.

Assay materials include at least one labeled antibody specific for sPLA₂. The antibody is labeled with a means to make it readily detectable, such as with a radioactive, fluorescent or chemiluminescent moiety. Preferably, the antibody is provided on a suitable support or in a container for conducting the assay. Most preferably, the assay materials for detecting protein concentration are the components of an enzyme linked immunosorbent assay (ELISA) as described above.

In another embodiment, the kit comprises materials used in an assay capable of measuring the enzymic activity of sPLA₂. Assay materials in such case include a sufficient amount of fluorescing or radioactive substrate to allow monitoring of the accumulation of products of the enzymatic reaction or the depletion of substrate and a suitable container in which to conduct the assay.

The form of the guidelines includes but is not limited to written instructions and explanations, photographs, graphical depictions, diagrams, and charts. Such guidelines may be presented as written documentation or in electronic form such as on a computer or compact disk.

The invention now being generally described, the same will be better understood by reference to the following specific examples that are not to be considered limiting of the invention unless so specified and are provided primarily for the purpose of illustration of preferred embodiments.

EXAMPLES

Example 1 MEASUREMENT OF sPLA₂ ACTIVITY AND CONCENTRATION

Phospholipase A₂ activity was measured with l-acyl-2-[l-¹⁴C]linoleoyl-sn- glycero-3-phosphoethanolamine, prepared as described by Van den Bosch et al. , Biochem. Biophys. Ada., 1974, 348: 197, as substrate. Enzymatic activity was assayed by incubating 0.2 mM radioactive substrate (specific radioactivity 3000 dpm/nmol) in 0.2 M Tris/HCl (pH 8.5), 10 mM Ca²⁺ and 5 μl plasma in a final volume of 200 μl. After 30 minutes at 37 °C, reactions were stopped by extracting the liberated ¹⁴C-labeled fatty acid by a modified Dole-extraction procedure (Van den Bosch 1979) and the radioactivity was determined by liquid scintillation counting. Secretory phospholipase A₂ antigen levels in plasma were determined with a sandwich enzyme-linked immunosorbent assay (ELISA) that has been modified from Smith et al. 1992. Two different monoclonal antibodies against human sPLA₂ (provided for this study by and available from Dr. F.B.Taylor Jr. , Oklahoma Medical Research Foundation, Oklahoma City, OK) were used as coating and detecting antibodies, respectively. Microtiter plates were coated with the first antibody (lOOμl, 2.5 μg/ml) in phosphate-buffered saline (PBS) for 16 hours at 4°C. After washing, the wells were blocked with 150 μl PBS containing 30 mg/ml bovine serum albumin (BSA) for 30 minutes at room temperature.

Samples diluted in PBS with 0.1 mg/ml Tween 20 and 2 mg/ml gelatin (PTG) were incubated in the wells for 1 hour, and after washing the wells were incubated for 1 hour with the detecting antibody, which was biotinylated and diluted 1: 1000 in PTG. Thereafter the wells were incubated for 30 minutes with streptavidin- horseradish peroxidase conjugate, diluted 1 : 1000 in PTG. The plate was washed, and the whole complex was incubated with the chromogenic substrate 3,3', 5,5' tetramethyl benzidine (0.1 mg/ml), 30 μg/ml H₂O₂ in 0.1 M sodium acetate buffer pH 5.5 for 8 minutes. The reaction was stopped by adding an equal volume of 1 M H₂SO₄ to each well, and the absorbance was read at 490 nm in a microtiter plate reader (EAR 400, SLT-Labinstruments, Austria). Results were compared with those obtained with cultured medium from Hep G2 cells stimulated with human interleukin-6 (Crowl et al. , J. Biol. Chem., 1991 , 266:2647). The amount of sPLA₂ in this cultured medium was assessed by comparison with purified recombinant human sPLA₂ (provided by Dr. H.M. Verheij, Department of Enzymology and Protein Engineering, University of Utrecht, Utrecht, The

Netherlands). The lower limit of detection was about 1 ng/ml.

Secretory PLA₂ concentration as measured with ELISA in plasma was shown to have a linear correlation with sPLA₂ activity (r² =0.953), confirming that ._ the sPLA₂ found in the plasma is in an active form (Figure 1A). Virtually identical results were found when either plasma or serum was used. Hence, sPLA₂ concentration data was used to analyze the relationship between the presence of active sPLA₂ and ACS. To continuously measure sPLA₂ activity without the use of radioactive materials, we have developed a continuous fluorometric assay. The measurement is based on the selfquenching properties of 1 -palmitoy l-2-(N-4nitrobenzo-2-oxo- 1 , 3-diazole)aminododecanoy 1 phosphatidylethenolamine (NBD PE). Hydrolysis of the NBD PE at the sn-2 position will lead to an increase in fluorescence. This increase in fluorescence can be used to determine the initial rate of hydrolysis of the substrate and consequently the phospholipase A₂ activity in an unknown sample. To 2 ml of buffer (250 mM Tris pH 9.0, 2.0 mM Calcium Chloride) in a Perkin Elmer LS-5B Fluorometer, equipped with a thermostat and stirring device, 20 μl NBD PE (Avanti Polar Lipiods, Pelham, AL) was added to a final concentration of 3.5 μM. Due to die selfquenching properties of the NBD PE, a low fluorescence was recorded (Fmin). Addition of 30 μl, 0.1 IU/μl Bee Venom Phospholipase A₂ (Sigma, St. Louis, MO) leads to an increase in fluorescence to a maximal value (Fmax). The difference in fluorescence (Fmax-Fmin) is linearly related to the concentration of NBD PE in the buffer and was kept constant in all assays. The initial rate of NBE hydrolysis was linearly related to the amount of Bee Venom phospholipase A₂ added (not shown). The addition of 50 μl serum samples from patients with different amounts of sPLA₂ as measured by ELISA rendered a linear relation between the amount present in plasma and the initial rate of fluorescence increase (Figure IB). Thus, the three different assays render similar results and show that the sPLA₂ found in the serum of patients is active.

Example 2

DETERMINATION OF sPLA₂ LEVELS IN SICKLE CELL DISEASE PATIENTS sPLA₂ levels were determined in thirty-five SCD patients. Patients ranged in age from one to 20 years (mean = 11 years). All patients had diagnoses of SCD confirmed by standard electrophoresis and isoelectric focusing methods. There were 30 patients with hemoglobin SS, two with hemoglobin SC, and three with hemoglobin S-β thalassemia. Serum sPLA₂ levels were measured for 20 patients admitted for ACS and 10 patients admitted for vaso-occlusive crisis. The sPLA₂ level was also determined in eleven SCD patients during routine comprehensive health care visits in the sickle cell clinic. Four patients were tested during more than one hospitalization. Secretory PLA₂ levels in the plasma of nineteen normal (non-SCD) controls (hemoglobin AA) were also evaluated.

Acute chest syndrome was defined as the development of a new infiltrate on chest radiography in combination with fever, respiratory symptoms, or chest pain. Patients admitted with ACS were treated following a standard protocol which included hydration at one and one-quarter times maintenance, parenteral cefuroxime and oral erythromycin, arterial blood gas monitoring, and daily complete blood counts. Transfusion was used at the attending physician's discretion based on the patient's clinical course. Intravenous narcotics and non- steroidal anti-inflammatory medications were used to treat accompanying pain events.

Vaso-occlusive crisis was defined as an admission for pain which required parenteral narcotics and had no other cause for pain than SCD. Patients admitted with vaso-occlusive pain were treated following a protocol which included intravenous hydration, not to exceed one and one-half times maintenance, intravenous narcotics, and non-steroidal anti- inflammatory drugs. If fever developed, patients were evaluated with chest radiography and blood and urine cultures, and intravenous cefuroxime was administered. Clinical and laboratory data were collected on all hospitalized patients; these included history of preceding or accompanying pain, PaO₂ on room air arterial blood gas, transfusion history, and the presence or absence of fever. Arterial blood gas measurements were determined using an AVL 995 (AVL Scientific Corp, Roswell, GA). Alveolar-arterial oxygen gradient was calculated from room air arterial blood gas values according to the following formula: (A- a)PO₂= (713x FiO₂) - (PaCO₂x 1.2) - PaO₂. All sPLA₂ levels were measured using the method described in Example 1. Fifteen SCD patients had two or more sPLA₂ level determinations during a single hospital admission. In the patients that were followed with sequential sPLA₂ levels from before the onset of ACS through convalescence, the sample with the highest sPLA₂ value was used in the calculation of statistical significance. Statistical evaluation was performed using the unpaired t-test.

Mean sPLA₂ concentrations were significantly elevated in all three SCD patient groups studied (ACS, vaso-occlusive crisis, and steady state) compared to values obtained from control patients. Figure 2 graphically depicts sPLA₂ protein levels (in ng/ml) in sickle eel' natients with acute chest syndrome (ACS), sickle cell patients with vaso-occlusive crisis (VOC) and steady state sickle cell patients at the time of routine comprehensive health care visit (steady state) are shown. Twenty ACS patient samples, ten VOC patient samples and eleven steady state patient samples are represented. In addition, sPLA₂ levels from eleven non-SCD pneumonia patients are shown. Non-SCD pneumonia is a condition in which sPLA₂ has been reported to be elevated compared to healthy patients. sPLA₂ concentrations are presented in Table 1. Steady state SCD patients had a mean sPLA₂ level of 10.0 ±8.4 ng/ml (median = 9 ng/ml) which was three times higher than in the normal controls (mean = 3.1 + 1.1 ng/ml, median = 3.1 ng/ml). Sickle cell disease patients with vaso-occlusive crisis had a similar 3- to 5-fold elevation above normal controls. The mean sPLA₂ concentration of patients with vaso-occlusive crisis (mean = 23.7+40.5 ng/ml, median = 8.7 ng/ml) was not significantly different from SCD patients in the steady state. Four of the ten patients with vaso-occlusive crisis had fevers during hospital ization. Comparison of the febrile and afebrile groups did not reveal a significant difference in sPLA₂ concentration.

Acute chest syndrome patients had a mean sPLA₂ level of 336 ±209 ng/ml (median = 289 ng/ml) which was 100 times greater than normal controls and 35 times greater than in samples from SCD patients in the steady state. There was an increase in sPLA₂ concentration in 19 of 20 episodes of ACS. In 18 of the 20 ACS episodes there was a history of vaso-occlusive pain preceding or accompanying ACS. Both patients without a history of pain were under 3 years of age, and one of these was the only ACS patient without a significant elevation of sPLA₂ above baseline (12 ng/ml). sPLA₂ levels in ACS patients were also significantly elevated above non-SCD pneumonia patients (mean = 68.6 ±82.9 ng/ml, median = 38 ng/ml).

In the 15 SCD patients followed with serial sPLA₂ measurements, sPLA₂ levels paralleled the clinical course of the patient. Seven patients with vaso- occlusive crisis were followed with sequential sPLA₂ levels, and four of these went on to develop ACS. In all four of these patients, sPLA₂ levels rose abruptly with the development of ACS and then decreased as the patient clinically improved

(Figure 3). In the three patients who did not develop ACS, sPLA₂ levels remained low. The remaining eight patients were admitted at the time of ACS. Sequential evaluation of sPLA₂ concentration in these patients documented that sPLA₂ levels were highest with the onset of ACS and declined as the patient recovered.

Secretory PLA₂ levels were highest in patients with clinically more severe lung disease as measured by arterial blood gas results and the need for transfusion. ' Arterial blood gas measurements on room air were available on 15 ACS patients.

Comparisons of ACS patients with and without significant hypoxia (PaO₂ < 70 and > 70 mm Hg) and with and without increased alveolar-arterial O₂ gradients ( > 30 and < 30 mm Hg) revealed that sPLA₂ levels generally were predictive of clinical severity (Figure 4). Secretory PLA₂ concentrations were also compared in the transfused and untransfused patient groups. One patient from the transfused group was removed from the analysis because he was transfused secondary to aplastic crisis and not due to pulmonary disease. Also, one severely alloimmunized patient was removed from the analysis because, despite severe hypoxia, he could not be transfused secondary to a lack of compatible blood. Secretory PLA₂ levels were significantly higher in the group needing transfusion, again showing a relationship between sPLA₂ concentration and clinical severity (Figure 4).

The data above illustrate typical results found in the sickle cell patients. Based on these data we have developed a clinical study in which each sickle cell patient admitted for vas-occlusive crisis (VOC) to Children's Hospital Oakland is tested for sPLA₂ levels daily. To date we have analyzed over 3000 samples from 182 patients (ages 0.3 to 29 years, 52% male, 48% female). Our data confirm the conclusions as indicated in the examples above. All patients who develop ACS show dramatically increased levels of sPLA₂ activity in their serum, and the onset of ACS is preceded by an increase in sPLA₂. Based on these data, we are currently using a level of 100 ng/ml sPLA₂ in serum (which equals an activity of 1 AU in the fluorometric assay, as indicated in Figure IB) to predict the risk to develop ACS for a patient with VOC. Our data demonstrate that sPLA₂ levels above this threshold in the absence of fever translate into a risk of 75 % of developing ACS within 24 hours, while the presence of fever increases the risk to

95% . Example 3

DETERMINATION OF sPLA₂ ACTIVITY IN PATIENTS WITH MULTIPLE

ORGAN FAILURE

Multiple organ failure (MOF) is the most common cause of postinjury death in intensive care units. Despite intensive investigation, the pathogenesis of postinjury

MOF remains unclear. Various scoring systems have been developed to better define the population of patients who are susceptible to postinjury adult respiratory distress syndrome (ARDS) and MOF, syndromes in part characterized by capillary leakage and end organ damage. These scoring systems are largely descriptive and can be difficult to reproduce and implement clinically (Schuster, Chest, 1992,

102: 1861). An early and accurate prediction of the risk for developing MOF may reduce the mortality of trauma patients in the intensive care unit. We performed a study to characterize the activity of sPLA₂ in severely injured patients sequentially during their early hospital course and determine if the activity of sPLA₂ relates to the subsequent development of MOF. Seventeen patients were enrolled in this study to measure sPLA₂ levels. Six of the 17 patients developed MOF (35%). Figure 5 depicts the differential between sPLA₂ activities for the MOF and non- MOF patients as measured with the fluorescent assay described in Example 1. Within 36 h postinjury, MOF patients had a mean sPLA₂ activity of 2.4 ±0.97 AU, which was nearly threefold greater than the non-MOF patients (0.86±0.16

AU) with a p value of 0.051. In addition, sPLA₂ activity remained significantly higher in the MOF patients as compared to the non-MOF patients at 60 hours (2.7±0.74 vs. 0.97±0.18), 84 hours (1.87±0.29 vs. 1.05 ±0.23) and 132 hours postinjury (2.87 ±0.95 vs. 0.97 ±0.22). These data suggest that postinjury MOF develops secondary to systemic inflammation. The predictive value of sPLA₂ levels for MOF provide early warnings to pursue aggressive treatment early in the postinjury time period. Table 1

Secretory Phospholipase A₂ Levels in Sickle Cell Disease Patients Groups

MEAN (MEDIAN) RANGE P VALUE (ng/ml) (ng/ml)

Steady State SCD 10.0±8.4 (9) 1.1-28.5 < 0.05 (n= l l)

VOC (n = 10) 23.7±40.5 (8.7) 1.8-134.6 <0.05

ACS (n=20) 336 ±209 (289) 12-725 < .001

Pneumonia (n= ll) 68.6±82.9 (38) 6-267 <0.05 non SCD

P values are for differences between control and sickle cell patient groups; SCD = sickle cell disease; ACS = acute chest syndrome; VOC = vaso-occlusive crisis.

All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

The invention now being fully described, it will be apparent to one of ordinary skill in the art that many changes and modifications can be made thereto without departing from the spirit or scope of the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A method of diagnosing the potential onset of a secretory PLA₂-related disorder in a patient at risk for such disorder and the severity of a secretory PLA₂- related disorder in a patient having said disorder or said potential onset of said disorder, which comprises: analyzing a body fluid of said patient for secretory phospholipase A₂ concentration or activity; correlating said concentration with a standard value selected from the group consisting of

(1) a predetermined range of secretory phospholipase A₂ concentrations or activities, respectively, for said body fluid in a control population and

(2) a secretory phospholipase A₂ concentration or activity, respectively, of a previously isolated sample of said body fluid of said patient; and relating a higher concentration or activity, respectively, of secretory phospholipase A₂ relative to said standard value as an indication of potential onset or severity of secretory PLA₂-related disorder.

2. A method of predicting the onset or severity of a secretory PLA₂- related disorder in a patient, comprising: obtaining a sample of a body fluid of a patient; analyzing said sample for secretory phospholipase A₂ concentration or activity; comparing said concentration or activity, respectively, with a standard value selected from the group consisting of

(1) a range of secretory phospholipase A2 concentrations or activities, respectively, for said body fluid in a control population and

SUBSTITUTE SHEET (RULE 28) (2) a secretory phospholipase A₂ concentration or activity, respectively, of previously obtained sample of said body fluid of said patient; and relating said concentration or activity, respectively, of secretory phospholipase A₂ to said standard value, wherein said a statistically significant higher concentration is predictive of onset and severity of said secretory PLA₂- related disorder.

3. The method of claim 1 or 2, wherein said secretory PLA₂-related disorder is pulmonary fat embolism disorder, acute chest syndrome in a patient with sickle cell disease, or multiple organ failure.

4. The method of claim 1 or 2, wherein said patient is suffering from trauma.

5. The method of claim 1 or 2, wherein said at risk patient is at risk for pulmonary fat embolism resulting from cardiac surgery or orthopedic surgery.

6. The method of claim 1 or 2, wherein said body fluid is saliva, plasma, serum, urine, or sweat.

7. An article of manufacture comprising: an enzyme linked immunosorbent assay test kit for detecting secretory phospholipase A₂, and guidelines for predicting the onset or severity of secretory PLA₂- related disorder according to the method of claims 1-5.