ARTERIOSCLEROSIS PROGNOSTICATION PROCEDURE AND THERAPEUTIC METHOD

Abstract: A procedure for rating the potential of an individual for developing demonstrable coronary artery disease associated with VLDLC toxicity, comprising determining quantitatively from a sample of said individual's serum the level therein of each of the components PF and RF, calculating from these component levels the value of the ratio PF/RF, comparing this value with a rating standard for said potential.

Description: ARTERIOSCLEROSIS PROGNOSTICATION PROCEDURE AND THERAPEUTIC METHOD This invention concerns a procedure for prognosticating the susceptibility of a subject to developing arteriosclerosis, particularly atherosclerosis, through the destructive effect of very low density lipoproteins (VLDL, d<1.006 g/ml) on vascular wall cells, in which procedure essentially only established chemical analytical techniques are employed and onerous biological procedures are avoided. The invention also concerns a therapeutic method for reducing such susceptibility. The references 1-41 are incorporated herein by reference. The firmly established correlation of blood lipoprotein levels with atherosclerosis and its sequel coronary heart disease (1,2,3), shows such lipoproteins to be intimately involved in the etiology of the disease process. Despite these correlations however, lipoprotein measurements per se do not accurately delineate subjects of varying potential for developing such disease. Many individuals do not develop atherosclerosis despite an unfavorable ratio (4,5) of risk lipoproteins [very low density lipoprotein (VLDL) (2,6,7,8,9) and low density lipoprotein (LDL) (1)], to protective lipoproteins [high, density lipoprotein (HDL) (3)], and many do develop atherosclerosis despite a favorable ratio of risk to protective lipoproteins. In a study involving 21 known risk factors in 431 consecutive patients who had undergone cardiac catheterization, Holmes et al (10) concluded that despite a highly significant correlation between current risk factors and the presence of documented coronary artery disease, these discriminators are often not diagnostically useful -when applied to individual S ITUTE SH££T patients. A response-to-injury hypothesis of atherosclerosis dates back over 100 years to the work of Virchow (11) . However, the specific factors and mechanism of endothelial injury have continued to elude investigators. In 1982, elevated levels of VLDL were shown to injure aortic endothelial cells in vitro (12, 13). Subsequently, and as part of the present invention, human serum was found to contain an identifiable substance, designated toxicity preventing activity (TxPA) , which prevents this observed injury caused by elevated VLDL levels (14) . In two clinical studies (14, 15) , an atherogenic index which incorporated TxPA as the principal protective component of the protective factor PF was found to classify individuals with angiographically demonstrable coronary artery disease with a much higher accuracy ( 93%) than had been found using lipoprotein values alone. This index in its most generally applicable form is equal to the numerical ratio of quantified PF to quantified RF wherein the TxPA identified in detail below is a protective component of the PF and the RF identified in detail below is a risk factor. This Atherogenic Index PF/RF provides a highly accurate classification of individuals with cholesterol levels throughout the normal and pathological range. It is noted that the terminology such as VLDL and VLDLC are interchangeab1e herein. The present invention is defined therefore as the procedure for rating the potential of an individual for developing demonstrable coronary artery disease associated with VLDLC toxicity r comprising determining quantitatively from a sample of said individual's serum the level therein of each of the PF and RF components, and calculating from these component levels the value of PF/RF, and comparing this value with a rating standard for said potential. The TxPA which is the principal protective factor is identified as albumin material forming part of the plasma protein fraction of the serum and has an isoelectric point of approximately 5.6 as determined by isoelectric focusing. The quantity of albumin in this 5.6 pi peak was determined by the Bromcresol Green binding assay (ref. Doumas, BJ, Watson, W. , and Biggs, H.G., Albumin Standards and the measurement of Albumin with Bromcresol Green, Clin. Chem. Acta ϋ:87, 1971). It is noted that this analytical technique very accurately and dramatically shows peaks at approximately 5.6 pH for serums having substantial TxPA levels. In this regard, the therapeutically ineffective portion of the albumin material focuses mainly at a pH of about 4.8. This analytical technique is highly preferred for quantifying TxPA levels, however, other methods and combinations of these methods such as adsorption chromatography, polarization chromatography, electrophoresis, reverse phase chromatography, affinity chromatography (particularly cibacron blue absorption) isoelectric focusing in other supporting media, indicator dye binding, ion exchange chromatography, high performance liquid chromatography, ligand binding and the like known for blood fractionation find utility to varying degrees in carrying out the present procedure. The RF or risk factor represents those materials which have been recognized in the field as contributing to cell toxicity and mainly comprises the very low density lipoprotein cholesterol LDLC but also generally includes all of the lipoprotein cholesterol material which is not high density (HDLC) , and also includes total triglyceride, especially for non-fasting subjects. Also, the protective factor or numerator of the ratio can be supplemented by other known protective factors such as HDLC as shown in the exemplary ratios given below. The Atherogenic Inde'x therefore, stated as PF/RF SUBSTITUTE SH££T is actually a generic index and contains within its meaning various other ratios. These ratios include, for example, TxPA x HDLC. TxPA x HDLC. RF non-HDLC (LDLC + VLDLC) TxPA x HDLC , and TxPA/Total Triglyceride (TG) . Total Cholesterol(TC) The quantification of the HDLC, and non-HDLC of the serum samples is done by well established procedures such as described by Allain, C.A, Poon, C.S. and Richmond, W. in Enzymatic Determination Of Total Serum Cholesterol, Clin. Chem. Acta .31:87, 1971, and by Warnick, G.R. and Albers, J.J. in A Comprehensive Evaluation Of Heparin Managamese Precipitation: A procedure for estimating high density lipoprotein cholesterol, J. Lipid Res. 19:65, 1978). The rating standard against which an individual•s atherogenic index is compared for assessment of his -* vascular disease potential, comprises a numerical scale having a sufficient range to include very high to very low potential. The numerical data for this standard were developed from the sera of a number of subjects who represented the full range of very high to very low potential by correlating their vascular condition to their TxPA. The procedures for developing these data are given, below. Endothelial cells were obtained from procine thoracic aortas following 40 minutes collagenase (CLS II, Worthington, Freehold, NJ) digestion (12) . Cells were plated in 96-well tissue culture cluster plates with flat bottom wells (Costar, Cambridge, MA) in 200^1 of Medium 199 (16) with Earle's Salts, 2.2 g/1 NaHC0 3 , 2.5 μ g/ml Amphotericin B, 50 g/ml gentamicin sulfate and 17% fetal bovine serum. All tissue culture media and supplements were obtained from Grand Island Biological Co., Grand Island, NY. Cultures were maintained at 37°C in a humidified atmosphere (95% relative humidity) of 5% C0 2 in air in a Forma Scientific Model 3028 C0 2 Incubator (Marietta, OH) . After 18 hours the cultures were rinsed 3 times in Medium 199 and changed to the test medium which consisted of 20 υl of sample, 30 /L of Medium 199 and 50 y± of a mixture of diabetic rat VLDL (17, 18) (0.90 mg triglyceride/ml) and [methyl- 3 H] thymidine, (1.0// Ci/ml, ICN Radiochemicals, Irvine, CA) in M 199. After 3 days cells were harvested on glass fiber filter paper using a MASH II Cell Harvester (Microbiological Associates, Inc., Walkersville, MD) . The VLDL preparation is as follows: Preparation of VLDL Blood obtained from streptozotocin-induced diabetic rates is allowed to clot for 1 h at 37°C and then centrifuged at 300 x g for 10 in at 4°C to prepare serum (supernatant) . The serum is then centrifuged in a SW-41 rotor in a Beckman L5-75B ultracentrifuge (Smith Kline-Beckman, Philadelphia, PA) at 175,000 x g for 18 h at 4°C. The VLDL (d < 1.006 g/ml) fraction and the d > 1.006 infranatant are separated using a tube slicer. The VLDL fraction is diluted with cold 0.15 M NaCl and given a wash spin (175,000 x g) at 4°C. The serum and serum fractions are electrophoresed in 0.8% agarose gel and stained with oil red 0 to determine the electrophoretic mobility of the lipoproteins (Noble, R.P. Electrophoretic separation of plasma lipoproteins in agarose gel, J. Lipid Res. 9:693, 1968) . When a quantitative analysis of TxPA was required, the sample was serially diluted with Medium 199 to obtain concentrations of 1, 2, 3, 4, 6, 8, 12 and 16% (50 l) . Fifty jj± of the mixture of the mixture of diabetic rat VLDL and [methyl- 3 H] thymidine in Medium 199 was then added to each well and the cells cultured for 3 more days. Cells were harvested as described above (14) . U STITUTE SHZZT A power regression (y = ax " , y = cpm, x = % sample) provided the best fit of the data and was used to calculate values of a and b for each sample. A single standard serum which had a value of approximately 5 100 TxPA units/ml in a previous study (15) was used as the standard and given the value of 100 units/ml. Horizontal shifts of the sample regression lines from the standard regression line were then calculated at 12% serum for each of the samples using the a and b values 10 obtained from their individual regression analysis. This analysis provided a quantitative measure of TxPA in the individual samples. By isoelectric focusing, the standard serum having the 100 TxPA units/ml was found to contain 14 mg/ml of 15 proteins in the 5.6 albumin peak. From this serum analysis the following rating standard was evolved. Cell Based x 14 = Direct Albumin Atherogenic Index Atherogenic Index ,mα.HDLC/dl x mo:. 5.6 Alb/dl 20 * > mg.non-HDLC/dl 0 « 0 10 140 15 210 20 280 25 30 420 40 560 60 840 80 1120 For this standard, it has been found for an 30. experimental clinical population of angiographed subjects that the values of up to about 280 represent a high potential for atherosclerosis as evidenced by greater than about 15% cross-sectional reduction of normal coronary artery diameter, while values above 35 about 280 demonstrate less than 15% arterial narrowing. It is noted that this data is viable for greater than about 90% of the subjects tested. Clinical population studies of this type are made, for example, as follows: The subject population consists of 29 men (age 43 + 8 yr) who have undergone coronary angiography using the Judkins technique. Left ventriculograms are obtained in the 30° right anterior oblique projection and selective coronary angiograms are performed in multiple projections. Subjects undergoing catheterization have wither abnormal exercise tests, significant arrhythmis, valvular disease or left bundle block. Angiographic data are reviewed independently by two cardiologists and interpreted without knowledge of laboratory tests results. Individuals with 15% or less narrowing are considered normal while those • having greater than 15% narrowing are classified as having coronary artery disease. None of the subjects have electrocardiographic evidence of a previous myocardial infarction and less than 10% are suspected of definite angina. No subject has hypertension that required any medication other than hydrochlorothiazide and none have diabetes mellitus. Total serum cholesterol and HDLC are measured on fasting serum samples using an ASA-100 bichromatic analyzer (Abbott Laboratories, North Chicago, ILL.) with BMC Autoflor cholesterol reagents (Cat. No. 148393, Biodynamics/bmc, Indianapolis, IN) . Triglycerides are enzymatically measured using the Stat-Pack Enzymatic Triglyceride (Car. No. 869262, Calbiochem-Behring, LaJolla, CA) procedure. The following procedures detail the identification and properties of TxPA. Dialysis One ml aliquots of serum were dialyzed for 48 hr at room temperature (RT) against either 10%, 20% or 30% ethanol, 5% or 10% dimethyl sulfoxide, 10% glycerol, 10% dextrose, 8% sucrose, 1% N,N-dimethylformamide or 0.1% 2-mercaptoethanol in 0.05 M HEPES, pH 7.4. Dialysis was then continued for 4 hr against M 199 alone. The E samples were then immediately tested for TxPA as described above. In a second set of experiments, serum was dialyzed 48 hr at RT against the antioxidants dithiothreitol, 2-mercaptoethanol and EDTA in 0.05 M HEPES. Duplicates were then transferred to M 199 and dialysis continued for 4 hr. Samples were then immediately tested for TxPA. Precipitation In order to characterize the TxPA activity, tests for precipitability in trichloroacetic acid (TCA) (19) and (NH ) 2 Sθ4 (19) were conducted. Two ml of serum were diluted with 8 ml of 0.15 M NaCl and solid TCA added to obtain a 0.15 M solution. Alternately, solid (NH 4 ) Sθ4 was added to solutions of 1 ml of serum in 4 ml of 0.15 M NaCl to obtain 1.0 - 4.0 M solutions of (NH 4 ) 2 S0 4 . Both the TCA and (^H 4 ) 2 Sθ4 precipitations were stirred overnight at 4°C and centrifuged at 5,000 x g for 10 min at 4°C. The pellets from the (NH 4 ) 2 S0 4 precipitation were resuspended in 5 ml of 0.15 M NaCl and reprecipitated at the same molarity of (NH 4 ) 2 S0 4 as the original precipitation. After centrifugation, the pellets from the TCA precipitation and the second (NH 4 ) 2 S0 4 precipitation were dissolved in 0.15 M NaCl (2 and 1 ml respectively) . The resulting solutions and supernatants from the TCA precipitation and the second (NH 4 ) 2 S0 4 precipitation were dialyzed overnight at 4°C against 3 changes of 0.05 M NaN-2-hydroxyethyl- piperazine-N'-2-ethanesulfonate (HEPES) (Sigma Chemical Co., St. Louis, MO), pH 7.4. TCA, (NH 4 ) 2 S0 4 and NaCl were obtained from Fisher Scientific Co. (Fairlawn, NJ) . Cohn Fractions Cohn Fractions I, II, ΣV l f IV 4 , V and VI of human serum (19) were purchased from Sigma Chemical Co., dissolved in M 199 (30 mg/ml) and tested quantitatively for TxPA activity. Column Chromatography Samples were subjected to gel filtration on a 1.5 x 100 cm column of Sephadex G-100. All columns, gels and the UV monitor were obtained from Pharmacia, Uppsala, Sweden. Timed fractions (approximately 8 ml) were collected on a Buchler Fraction Collector (Fort Lee, NJ) by elution with 0.05 M HEPES buffer, pH 7.4, 0.1 M NaCl, 0.4 m M ethylenediaminetetraacetic acid, disodium salt (EDTA) (Fisher) , and 50 ^g/ml gentamicin sulfate. Samples were also separated by ion exchange chromatography on diethylaminoethyl (DEAE) Sephacel columns (1.5 x 30 cm). Columns were eluted with a linear gradient of NaCl generated from 100 ml of 0.05 M HEPES, pH 7.4, 0.4 m M EDTA plus 50 g/ml gentamicin sulfate and 200 ml of 0.4 M NaCl in the same buffer. Both DEAE Sephacel and Sephadex G-100 columns were eluted at RT by gravity feed. Isoelectric Focusing Isoelectric focusing was performed in an LKB 8100- 2, 440 ml column at 4°C. A linear gradient of 5% to 50% sucrose (Sigma) was prepared using an LKB gradient maker. The sample and 0.04 m M EDTA were included in the gradient. Ampholines (1%) from Pharmacia, LKB (Bromma, Sweden) or BioRad (Richmond, CA) yielded comparable results. The cathode solution at the top of the gradient was 0.25 M NaOH and the anode solution was 0.15 M H23P0 4 in 60% sucrose (20). The sample was electrophoresed at 30 w for 18 hours using a Pharmacia 2000/300 constant power supply. The column was eluted by pumping H 2 0 into the top and absorbance was monitored at 280 " X . The pH of each fraction was measured at RT. Radial I munodiffusion Radial immunodiffusion (RID) was carried out on selected isoelectric focusing fractions using albumin EE LC-Partigen RID and apolipoprotein A M-Partigen RID Plates (Calbiochem, La Jolla, CA) . RESULTS Serum which had been stored for 3 years at -20°C or -70°C or at 4°C for 6 months had TxPA levels comparable to those in freshly drawn serum from the same individual. Incubation of serum for 2 days at room temperature or 1 hour at 60°C did not significantly change TxPA levels. TxPA was completely destroyed in 3 minutes at temperatures above 80°C (data not shown) . Dialysis of serum against deionized water for 48 hours resulted in a complete loss of TxPA. Various substances which included ethanol, dimethylsulfoxide, glycerol, HEPES, dextrose, sucrose, N,N- dimethylformamide and 2-mercaptoethanol were tested for their ability to prevent this loss of activity. Only serum dialyzed against 2-mercaptoethanol was found to ' retain activity (data not shown) . The reducing agents, 2-mercaptoethanol and dithiothreitol, and the chelator, EDTA, were then tested at various concentrations. Dialysis against either 0.4 or 0.04 M EDTA yielded the highest recovery of activity (Table 1) . A concentration of 0.4 M EDTA was used to prevent loss of activity in subsequent experiments. Precipitation of TxPA by established techniques was used to investigate the nature of the activity and as an early purification step. TxPA precipitated in 0.15 M TCA and was reconstituted with a good (85%) recovery of activity (Table 2) . Activity was also found to precipitate above 3.0 M ammonium sulfate with a 46-49% recovery of activity (Table 2) . In an attempt to find a commercially available source of starting material which had undergone some enrichment of TxPA activity, the Cohn fractions of human serum were tested (Table 3) . Cohn fraction I proved to be toxic to the endothelial cells and could not be tested. Cohn fractions II, IV ! and VI did not contain TxPA. Only Cohn fractions IV 4 and V demonstrated activity. Cohn fraction V which had been treated to remove fatty acids contained less TxPA activity than Cohn fraction V. In order to further purify and characterize TxPA, both human serum and Cohn fraction V were subjected to gel filtration and ion exchange chromatography. Toxicity preventing activity in serum eluted from the Sephadex G-100 column in fractions 22 to 32 (Fig 1) . TxPA from Cohn fraction V eluted in a similar position (data not shown) . TxPA activity from Cohn fraction V, eluted from DEAE Sephacel under the predominant albumin peak (Fig 2) . Thus, TxPA co-eluted with albumin on both gel filtration and ion exchange. On ion exchange, however, TxPA was narrower than the total albumin peak suggesting that TxPA was either a subcomponent of albumin or a separate component co-purifying with albumin on these two chromotographic columns. Isoelectric focusing was used to resolve this question. TxPA in Cohn fraction V (Fig 3) was found to focus with an isoelectric point of pH 5.6. Column fractions were tested on cells for TxPA both before and after adjusting the pH of the fractions to 7.4 with similar results. TxPA activity was never found to be associated with albumin focusing below pH 5.2. The presence of albumin was measured by radial immunodiffusion and Bromcresol Green binding on the TxPA containing peak. By both of these techniques total albumin and TxPA were found to coincide (Fig 3) . Three sera from individuals with high and three sera from individuals with low levels of TxPA were subjected to isoelectric focusing to determine if there was a relationship between the absorbance in the pH 5.6 sϋ B srrrυτE SHEET region and the amount of TxPA. Two representative sera are shown in Figure 4 A and B. The serum focused in Figure 4A has 260 units/ml and also has a major protein peak around pi 5.6 which corresponds to the peak of 3 H- thymidine incorporation. The serum in Figure 4B has less than 10 units/ml of TxPA and likewise has only minor peaks in the pi 5.6 region with no detectable incorporation of 3 H-thymidine above base line. The volume of serum applied to each column was identical (2 ml) and column fractions were tested on endothelial cell cultures from the same pool of cells. Two other sera with high and two other sera with low levels of TxPA were tested with similar results. DISCUSSION TxPA was found to be extremely stable while in serum, however, activity was progressively lost during dialysis or multiple purification steps. Inclusion of chelators such as EDTA, or antioxidants and reducing agents such as H 2 or N 2 purge, dithiothreitol, 2- mercaptoethenol, or the like, for example, in Table 1, in the purification procedure results in recoverable TxPA activity through purification steps beyond Cohn fractionation. Complete loss of activity usually occurs upon extensive purification. Human albumin is known to contain groups such as SH which should be maintained in reduced form for maximum TxPA activity. The TxPA has been shown to copurify with albumin in a number of isolation procedures as described below, however,, the isoelectric focusing readily distinguishes the TxPA. TxPA precipitates in 0.15 M TCA and above 3.0 M (NH 4 ) 2 S0 4 , conditions which have also been shown to precipitate albumin (19) . Most of the TxPA activity was found to be associated with Cohn fraction V which • routinely contains 96-99% albumin. ' Cohn fraction IV 4 which contained significant TxPA activity also contains albumin (19) and has a substantial absorbance peak in the PI 5.6 region (data not shown). Fractionation of serum or Cohn fraction V by gel filtration or ion-exchange chromatography resulted in a single peak of TxPA which co-eluted with the albumin peak. TxPA eluted as a much narrower peak than total albumin on ion-exchange columns (Fig 2) , suggesting that TxPA was either a subcomponent of albumin, a small molecule bound to a species of albumin or a separate protein which co-eluted with albumin. TxPA was separated from the bulk of albumin by isoelectric focusing (Fig 3) . The 280 absorbance peak at pH 5.6 contained all of the TxPA. RID and Bromeresol Green detectable albumin and 3 H-thymidine incorporation also coincided with the 280^, absorbance peak at pH 5.6. When the pi 5.6 peak was pooled and chromatographed on DEAE Sephacel, only one protein peak was found, further indicating the purity of this fraction. Albumin exists as two major isoelectric forms in plasma, at pi 4.8 and 5.6 (25,26). Removal of fatty acids either by defatting before focusing or by gradually defatting during the focusing process itself decreases the pi 4.8 peak and increases the pi 5.6 peak (26). The pi 4.8 peak contains 2.5 - 10 moles of fatty acid per mole of albumin while the pi 5.6 peak contains 0 - 1.3 moles of fatty acid per mole of albumin (26) . Since the binding of one charge to albumin would shift the pi only 0.07 units (27), the shift from 5.6 to 4.8 is likely to be due to a conformational change which alters the pKa of several ionizable groups. Toxicity preventing activity is thus identified as the pi 5.6 component of albumin (Fig 3) . A prominent 280 absorbance peak was present in sera with high levels of TxPA (Fig 4A) . TxPA as determined by 3 H-thymidine incorporation in the presence UTE S H ST of toxic levels of VLDL also corresponded with this peak. Serum from individuals with low levels of TxPA had minor peaks in absorbance around pH 5.6 and had no increase in 3 H-thymidine in this area over background (Fig 4B) . The critical factors, as determined in the clinical studies (14,15), appear to be the level of pi 5.6 albumin and the non-fasting maximum level of VLDL as expressed by the VLDLC + LDLC to HDLC ratio. These two components accurately delineate the extent of in vitro injury. In vivo endothelial cell injury is proposed to occur during periods of elevated VLDL levels. Maximum VLDL levels occur several hours after means and are more variable than fasting levels (28) . The VLDLC + LDLC/HDLC ratio correlates with peak VLDL levels (29) and may help to explain the in vivo data in the light of the in vitro results. Although the actual factors involved in endothelial injury are VLDL and pi 5.6 albumin (determined by the in vitro cell assay) , the ratio of VLDLC + LDLC/HDLC empirically substitutes for maximal VLDL levels in the atherogenic index ' equation. Since LDLC contributes the majority of the VLDLC + LDLC component in normolipemic individuals and since LDLC and HDLC are relatively constant throughout the diurnal cycle, they can be readily measured and used to determine an individual's susceptability to atherosclerosis. TABLE 1 The Effect of Antioxidants on TxPA Toxicit Preventin Activit UBSTITUTE SHBZΎ TABLE 2 Precipitation of TxPA from Serum TABLE 3 TxPA Content of Cohn Fractions SUBSΠTUTE S ZT FIGURE LEGENDS Figure 1. Elution profile of TxPA from Sephadex G-100. V 0 represents the void volume (blue dextran) and V j - the total volume of the column. Two ml of human serum were applied to the column and absorbance at 28 o measured (—) . Eluted fractions (20.^1) were then incubated with primary cultures of porcine aortic endothelial cells for 3 days and subsequent incorporation of 3 H-thymidine (see METHODS) was plotted for each fraction (•...*). Figure 2. Elution profile of Cohn fraction V from a DEAE Sephacel column. Cohn fraction V (1 gm) in 2 ml 0.05 M HEPES, pH 7.4 and 0.4 m M EDTA was applied to the column and absorbance at 280^ measured (—) . Twenty microliters of each fraction were incubated with primary cultures of porcine aortic endothelial cells " and 3 H- thymidine measured (#...#). Figure 3. Isoelectric focusing of Cohn fraction V. Cohn fraction V (1 gm) was electrophoresised on a pH 4-6 gradient for 18 hr and absorbance at 280 X (—) was measured during elution. The pH of the fractions was measured at RT (—) . Albumin (x—x) was measured on 5 vl of the fractions by radia immunodiffusion. TxPA was determined by incubating aliquots of the fractions with endothelial cells and measuring 3 H-thymidine incorporation (#...#). Figure 4A. Isoelectric focusing of human serum containing 260 units/ml of TxPA. Serum (2 ml) was mixed in a pH 4-6 gradient and electrophoresised for 18 hr. Absorbance at 280 (—)_, pH, (—) and TxPA as determined by 3 H-thymidine incorporation (»...#) were measured as described in Fig 3. Figure 4B. Isoelectric focusing of human serum containing less than 10 units/ml of TxPA. Conditions were identical to those in Fig 4A. SU8STΓΠJT£ SHEET REFERENCES 1. Kannel, W. , Castelli, W. , Gordon, T. and McNamara, P.M., Serum cholesterol, lipoproteins and the risk of coronary heart disease: The Framingham Study, Ann. Int. Med. , 74 (1971) 1. 2. Carlson, L.S. and Bottiger, L.E., Ischaemic heart disease in relation to fasting values of plasma triglycerides and cholesterol, Lancet, i (1972) 856. 3. 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Claims: I Claim: 1. The procedure for rating the potential of an individual for developing demonstrable coronary artery disease associated with VLDLC toxicity, comprising determining quantitatively from a sample of said individual's serum the level therein of each of the components PF and RF, calculating from these component levels the value of the ratio PF/RF, and comparing this value with a rating standard for said potential. 2. The procedure of Claim 1 wherein the PF component contains the TxPA and the quantitation of its level is obtained by isoelectric focusing. 3. The procedure of Claim 1 wherein the TxPA of the Pf component is held in a non-oxidizing environment prior to and during its quantitation. 4. The procedure of Claim 3 wherein the said environment includes an antioxidant material, a reducing agent, a chelating agent, or any mixture thereof. 5. The procedure of Claim 1 wherein the PF component comprises the total of TxPA x HDLC. 6. The procedure of Claim 5 wherein the RF component comprises VLDLC, or LDLC, or non-HDLC (LDLC + VLDLC) , or total cholesterol, or total TG either alone or additive with any or all of VLDLC, LDLC or total cholesterol. 7. The therapeutic process for protecting against or alleviating the toxic effects of VLDL on human vascular wall cells, comprising providing an albumin material having an isoelectric point of about 5.6 and a substantial level of TxPA activity, providing a transport system containing said material for admixing the same with an individual's blood stream, and employing said system to elevate the TxPA level in said blood stream to provide an atherogenic index of at least about 280. SUBSTITUTE: SHZET 8. The process of Claim 7 wherein the TxPA level is monitored and regulated by determining quantitatively from a sample of said individual*s serum the level therein of each of the components PF and RF, calculating from these component levels the value of the ratio PF/RF comparing this value with a rating standard. 9. The process of Claim 7 wherein said transport system is selected form (a) an injectable aqueous solution of said albumin material, (b) an orally administrable encapsulation of an aqueous solution of or the solid albumin material, (c) a nasal spray of an aqueous solution of said albumin material, or (d) a suppository containing an aqueous solution of or the solid albumin material. UB ITUTE HE

Inventors: ARBOGAST BRADLEY W, ARBOGAST, BRADLEY, W.
Applicants: UNIV TENNESSEE EAST
Application year: 1989
Main classification: A61B5
IPC codes: A61B5, G01N33