Further studies of blood infectivity in an experimental model of transmissible spongiform encephalopathy, with an explanation of why blood components do not transmit Creutzfeldt-Jakob disease in humans

BACKGROUND: Solid evidence from experimentally infected animals and fragmentary evidence from naturally infected humans indicate that blood may contain low levels of the infectious agent of Creutzfeldt-Jakob disease (CJD), yet blood components have never been identified as a cause of CJD in humans. STUDY DESIGN AND METHODS: Blood components and plasma fractions were prepared from the pooled blood of mice that had earlier been infected with a mouse-adapted strain of human transmissible spongiform encephalopathy (TSE). Infectivity bioassays were conducted in healthy mice, and the brains of all assay animals dying during the course of the experiments were examined for the presence of proteinase-resistant protein. RESULTS: Infectivity in the blood during the preclinical phase of disease occurred in the buffy coat at infectious unit (IU) levels between 6 and 12 per mL and was either absent or present in only trace amounts in plasma and plasma fractions. Infectivity rose sharply at the onset of clinical signs to levels of approximately 100 IU per mL of buffy coat, 20 IU per mL of plasma, 2 IU per mL of cryoprecipitate, and less than 1 IU per mL of fractions IV and V. Plasma infectivity was not eliminated by either white cell-reduction filtration or high-speed centrifugation. Approximately seven times more plasma and five times more buffy coat were needed to transmit disease by the intravenous route than by the intracerebral route. CONCLUSION: Epidemiologic evidence of the absence in humans of disease transmission from plasma components can probably be explained by 1) the absence of significant plasma infectivity until the onset of symptomatic disease, and comparatively low levels of infectivity during the symptomatic stage of disease; 2) the reduction of infectivity during plasma processing; and 3) the need for at least five to seven times more infectious agent to transmit disease by the intravenous than intracerebral route. These and other factors probably also account for the absence of transmission after the administration of whole blood or blood components.     

Preparation of soluble infectious samples from scrapie-infected brain: a new tool to study the clearance of transmissible spongiform encephalopathy agents during plasma fractionation

BACKGROUND: Concern about the safety of blood, blood components, and plasma-derived products with respect to prions has increased since the report of two blood-related infections of variant Creutzfeldt-Jakob disease in the United Kingdom. Efforts were directed toward the development of procedures able to remove or inactivate prions from blood components or plasma-derived products with brain fractions of transmissible spongiform encephalopathy (TSE)-infected rodents as spiking materials. These spiking materials, however, are loaded with pathological prion protein (PrP(TSE)) aggregates that are likely not associated to blood infectivity. The presence of these aggregates may invalidate these studies. STUDY DESIGN AND METHODS: Brains from 263K scrapie-infected hamsters were suspended in 10 percent phosphate-buffered saline. After low-speed centrifugation, the supernatant was collected and ultracentrifuged at 220,000 x g at 25 degrees C for 30 minutes. The high-speed supernatants (S(HS)) and pellets were collected; the proteinase-resistant PrP(TSE) was measured by Western blot and infectivity by intracerebral inoculation into weanling hamsters. RESULTS: A substantial amount of prion infectivity (more than 10(5) LD(50) per mL of a 10% suspension of brain tissues) is present in the S(HS) fraction of 263K scrapie-infected hamster brains. Concomitantly, this fraction contains none or only traces of PrP(TSE) in its aggregate form. CONCLUSION: This study describes a simple and fast protocol to prepare infectious material from 263K scrapie-infected brains that is not contaminated with PrP(TSE) aggregates. This S(HS) fraction is likely to be the most relevant material for endogenous spiking of human blood in validation experiments aimed at demonstrating procedures to remove or inactivate TSE infectious agents.     

Improved method for analysis of glycosaminoglycans in glycosaminoglycan/protein mixtures: application in Cohn-Oncley fractions of human plasma

BACKGROUND: Glycosaminoglycans are found in human tissues including plasma. They encompass chondroitin sulphates, heparan sulphate/heparin, hyaluronic acid, and keratan sulphate. Glycosaminoglycans, in particular heparan sulphate and heparin, are strongly associated with plasma proteins, so that their purification results quite difficult. METHODS: In order to study the distribution of glycosaminoglycans in plasma subfractions, we developed a novel method that allows their identification even if they were still associated with proteins or peptides. Plasma was fractionated following the procedure of Cohn-Oncley, and each fraction was treated with proteases. After centrifugation, glycosaminoglycan/protein complexes in the supernatant were analysed using a modified cellulose acetate electrophoresis which allowed identification of glycosaminoglycans in mixtures of glycosaminoglycans/proteins. RESULTS: Chondroitin sulphate was recovered in cryoprecipitate and in all Cohn-Oncley fractions. Glycosaminoglycans belonging to the class of heparan sulphate/heparin, however, were recovered in the cryoprecipitate and in fractions I and IV-1, and, in smaller amount, in fraction II+III. CONCLUSIONS: Since the largest amount of plasma proteins is partitioned in Factions II+III and V, these results demonstrate that heparan sulphate/heparin are not randomly distributed in Cohn-Oncley fractions and are associated with certain plasma proteins. This association might play a role in the physiological function of heparan sulphate/heparin, regulating hemostasis and atherogenesis.     

Failure to transfer tuberculin sensitivity passively with plasma fractions containing alpha globulin

We are unable to confirm the observations reported by Cole and Favour that passive transfer of plasma fractions containing alpha globulin (IV + V or IV-10) from tuberculin-sensitive guinea pigs confers delayed sensitivity to tuberculoprotein upon normal animals. In guinea pigs with chronic cervical adenitis due to infection with group C hemolytic streptococci, injection of fraction IV + V and repeated skin tests with PPD induced indurated skin reactions of 9 to 10 mm., maximal at 24 hours, and, in one experiment, a positive tissue culture response to tuberculin. It is suggested that this reactivity was actively induced. Normal or infected recipients of fraction I + II + III from the plasma of tuberculin-sensitive donors manifested edematous responses to tuberculopolysaccharide, maximal at 4 to 6 hours, as reported. Skin tests with pneumococcal C polysaccharide revealed no evidence of passive transfer of C-reactive protein in fractions I + II + III or IV + V.     

Universal white blood cell reduction in Europe: has transmission of variant Creutzfeldt-Jakob disease been prevented?

Universal white blood cell (WBC) reduction was introduced in Europe to prevent transmission of variant Creutzfeldt-Jakob disease (vCJD) by transfusion. Findings from rodent models indicate that WBC reduction should not prevent vCJD transmission because the residual plasma infectivity suffices to infect transfusion recipients even under optimistic infectivity assumptions. Although infectivity in human blood may not partition in the manner in which it is distributed in rodents, prion-reduction filters remove the residual plasma infectivity in rodent models. Precautionary introduction of prion filtration in the UK--for patients without dietary exposure to bovine spongiform encephalopathy and in the absence of a reported case of vCJD transmission attributable to infectivity residing in plasma--is consistent with the (already in place for such subjects) precautionary importation to the UK of fresh frozen plasma from low-risk countries. Thus, implementation of prion filtration in the UK does not imply that universal WBC reduction--as currently practiced in Europe--does not abrogate transmission of vCJD. Because neither a human case of vCJD transmission through transfusion of WBC-reduced red blood cells nor a case of experimental bovine spongiform encephalopathy transmission by WBC-reduced transfusion to sheep has been reported, it cannot be concluded that ordinary WBC reduction is ineffective in preventing transfusion transmission in humans. Accordingly, universal WBC reduction for the prevention of vCJD in Europe should continue.     

Removal of exogenous (spiked) and endogenous prion infectivity from red cells with a new prototype of leukoreduction filter

BACKGROUND: Two recent probable cases of transmission of a variant of human Creutzfeldt-Jakob disease (vCJD) through blood transfusion suggest that the disease can be transmitted through transfusion of blood components from presymptomatic blood donors. In the absence of a preclinical screening test, removal of the infectious agent by processing is the only means by which risk to recipients of blood from donors with inapparent vCJD infections can be eliminated. STUDY DESIGN AND METHODS: In the endogenous infectivity study, a pool of 500 mL of whole blood was collected into CP2D anticoagulant from 263K-strain scrapie-infected hamsters, processed into 300 mL of red cells (RBCs), and then passed through a prion removal filter. Pre- and postfiltration samples were tested for PrP(sc) by Western blot and for infectivity by inoculation of healthy hamsters. In the exogenous (spiking) infectivity study, 30 mL of 10 percent (wt/vol) scrapie-infected brain homogenates was added to 270 mL of human RBCs and then filtered. Levels of PrP(sc) and infectivity were determined by Western blot and bioassay. RESULTS: In the endogenous infectivity study, the prefiltered RBCs transmitted disease to 6 of 43 animals, whereas the postfiltered RBCs did not transmit disease to any of 35 animals, and a barely visible prefiltration PrP(sc) Western blot signal was reduced below the level of detection in the postfiltration sample. In the exogenous (spike) study, infectivity was reduced by 3.7 log LD50 per mL, from 9.2 to 5.5 log LD50 per mL. CONCLUSION: The new filter was effective in removing both infectivity and PrP(sc) from RBCs. The use of this type of filter should reduce the risk of vCJD transmission through blood transfusion.     

Similar levels of infectivity in the blood of mice infected with human-derived vCJD and GSS strains of transmissible spongiform encephalopathy

BACKGROUND: The possible transmission of variant CJD (vCJD) through blood transfusion or use of plasma‐derived products prompted this study comparing infectivity in murine models of vCJD and Gerstmann‐Sträussler‐Scheinker (GSS) disease, a non‐vCJD form of transmissible spongiform encephalopathy (TSE). STUDY DESIGN AND METHODS: RIII/Fa/Dk (RIII) or Swiss‐Webster (Swiss) mice were inoculated intracerebrally (IC) with mouse‐adapted strains of vCJD or GSS (Fukuoka‐1) of similar infectivity. Groups of RIII mice were euthanized 17 weeks after inoculation (during the incubation period), and another 23 weeks after inoculation (when symptomatic). Blood was collected, separated into components, and inoculated into groups of healthy mice; brains and spleens from all mice were harvested and tested for the presence of PrPres by Western blot using 6H4 MoAb. RESULTS: Levels of 20‐30 infectious doses per mL were present in buffy coat and plasma during both the incubation and symptomatic stages of disease; PLT pellet infectivity was lower (10 ID/mL) and RBCs were not infectious. The disease was transmitted more efficiently by IV than IC inoculation of plasma, but there was no difference observed with inoculation of buffy coat. The incubation period was shorter after IC inoculation of GSS‐ than vCJD‐brain inocula. The amount of PrPres in spleens was similar for both TSE agents, but was slightly lower in brains of vCJD than GSS mice. CONCLUSION: Infectivity was detected in blood components of mice infected with a human‐derived strain of vCJD during both the preclinical and clinical phases of disease in a similarly low range of concentrations as in mice infected with a human‐derived nonvariant strain (GSS, Fukuoka‐1). Other measures of virulence, including brain infectivity titers, incubation periods, and the accumulation of PrPres in spleens and brains, were also comparable in both experimental models.     

A direct relationship between the partitioning of the pathogenic prion protein and transmissible spongiform encephalopathy infectivity during the purification of plasma proteins

BACKGROUND: Experimental evidence from rodent models indicates that blood can contain transmissible spongiform encephalopathy (TSE) infectivity, which suggests a potential risk for TSE transmission via proteins isolated from human plasma. Because methods that can reduce TSE infectivity typically are detrimental to protein function, infectivity must be removed to ensure the safety of these therapeutic proteins. Animal bioassays are conventionally used to detect infectivity, but the pathogenic form of the prion protein (PrP^Sc) can serve as a marker for TSE infectivity. STUDY DESIGN AND METHODS: Seven plasma protein‐purification steps were performed after the plasma intermediates were spiked with TSE‐infected material. Resulting fractions were analyzed for PrP^Sc by using a Western blot assay and for TSE infectivity by using an animal bioassay. Western blots were quantitated by an endpoint dilution analysis, and infectivity titers were calculated by the Spearman‐Kärber method. RESULTS: PrP^Sc partitioning paralleled TSE infectivity partitioning, regardless of the nature of the protein‐purification step. The detection ranges for PrP^Sc and infectivity were 0 to 5.3 log and 1.1 to 8.9 log median infectious dose per unit, respectively. Clearance of PrP^Sc and infectivity ranged from 1.0 to 6.0 log. CONCLUSION: Purification steps for isolating therapeutic proteins from human plasma showed the removal of both PrP^Sc and TSE infectivity. PrP^Sc partitioning coincided with infectivity partitioning, which showed a close relationship between PrP^Sc and TSE infectivity. By exploiting this association, the in vitro Western blot assay for PrP^Sc was valuable for estimating the partitioning of TSE infectivity during plasma protein purification.     

Comparison of nanofiltration efficacy in reducing infectivity of centrifuged versus ultracentrifuged 263K scrapie-infected brain homogenates in "spiked" albumin solutions

BACKGROUND: The safety of plasma‐derived products is of concern for possible transmission of variant Creutzfeldt‐Jakob disease. The absence of validated screening tests requires the use of procedures to remove or inactivate prions during the manufacture of plasma‐derived products to minimize the risk of transmission. These procedures need proper validation studies based on spiking human plasma or intermediate fractions of plasma fractionation with prions in a form as close as possible to that present in blood. STUDY DESIGN AND METHODS: Human albumin was spiked with low‐speed or high‐speed supernatants of 263K scrapie‐infected hamster brain homogenates. Spiked albumin was then passed through a cascade of filters from 100 nm down to 20 to 15 nm. Residual infectivity was measured by bioassay. RESULTS: The overall removal of infectivity spiked into albumin through serial nanofiltration steps was 4 to 5 logs using low‐speed supernatant and 2 to 3 logs with high‐speed supernatant. CONCLUSION: These findings confirm the utility of nanofiltration in removing infectivity from plasma (or other products) spiked with scrapie brain homogenate supernatants. However, efficiency is diminished using supernatants that have been ultracentrifuged to reduce aggregated forms of the infectious agent. Thus, filtration removal data based on experiments using “standard” low‐speed centrifugation supernatants might overestimate the amount of prion removal in plasma or urine‐derived therapeutic products.     

Variant Creutzfeldt–Jakob disease and its transmission by blood

Summary.  Variant Creutzfeldt–Jakob disease (vCJD) is a novel acquired human prion disease resulting from human exposure to the agent causing bovine spongiform encephalopathy (BSE). vCJD differs from all other human prion diseases in that the disease-associated form of the prion protein and infectivity are present in lymphoid tissues throughout the body. Lymphoid tissues and lymphocytes are implicated in the peripheral pathogenesis of prion diseases (where infectivity may be detected during the preclinical phase of the illness), giving rise to concerns that blood and blood products may also contain infectivity, thus representing a possible source of iatrogenic spread of vCJD. These concerns have been reinforced by the recent transmission of BSE in an experimental sheep model by blood transfusion from an infected animal in the preclinical phase of the illness. Studies in other animal models suggest that most infectivity in blood may be cell-associated, with lower levels in the plasma, and there is evidence to indicate that any infectivity present may be reduced during the process of plasma fractionation. At present, the attempts to detect disease-associated prion protein and infectivity in buffy coat from vCJD patients have been negative, but these studies have been limited in size and in the sensitivity of the detection systems employed. Further studies are required to develop more sensitive means of detection of disease-associated prion protein in blood; such techniques could also be employed for screening purposes, both individually and to help ascertain more precisely the likely numbers of future cases of vCJD.     

Effect of protease treatment on plasma infectivity in variant Creutzfeldt-Jakob disease mice

BACKGROUND: The emergence of variant Creutzfeldt-Jakob disease (vCJD) and of a probable transmission of the disease through blood transfusion from a presymptomatic case has underlined the need for a reliable, sensitive, and specific screening test. This study was initiated to explain why attempts to identify protease-resistant prion protein (PrPres) following treatment with proteinase K (PK) in blood or blood components have so far failed. STUDY DESIGN AND METHODS: RIII mice were inoculated intracerebrally (i.c.) with vCJD agent. As soon as some mice became ill, blood from all mice was collected, pooled, and separated into components. Aliquots of plasma were treated with either 100 and 500 microg per mL PK or left untreated. Samples were analyzed for total protein and for PrPres by Western blot with 6H4 antibodies. Infectivity in PK-treated and untreated samples was bioassayed by i.c. inoculation into healthy mice. RESULTS: Estimated infectivity in untreated control plasma was 20.6 IU per mL. Treatment of plasma with 100 or 500 microg per mL PK resulted in estimated infectivity levels of 8.4 and 5.2 IU per mL, respectively. Coomassie staining revealed substantial changes in the protein profile after PK treatment, with massive degradation of proteins at 500 microg per mL PK. No PrPres was detected in plasma samples by Western blotting. CONCLUSION: Infectivity in plasma of vCJD-infected mice showed a trend toward reduction following enzymatic treatment with increasing doses of PK, possibly because of activity against proteolysis-sensitive isoforms of abnormal prion protein. It is concluded that the use of PK in protocols for the detection of PrPres may decrease the sensitivity of blood-based assays.     

Four methods for determining total protein compared by using purified protein fractions from human serum

The proportional bias of four methods frequently used for determining low concentrations of protein was evaluated with human serum protein fractions (Cohn Fractions II, III, IV, and V). Each fraction was assigned a protein concentration value as determined by the biuret method, calibrated with purified human serum albumin monomer. All four methods (Coomassie Brilliant Blue dye-binding, the method of Lowry et al., ultraviolet absorption, and immunonephelometry gave acceptable results for Fraction V (albumin). The ultraviolet absorption and the Lowry methods overestimated the three globulin fractions (II, III, and IV), whereas the other two methods underestimated these fractions. The method of Lowry et al. gave the least proportional bias for the globulin fractions.     

Prions and blood products

The transmission of Creutzfeldt-Jakob disease (CJD) by human pituitary-derived growth hormone has led to concerns that blood products might also provide a route for the iatrogenic transmission of CJD. A number of actions have been implemented by regulatory authorities to address such concerns, and numerous studies have been undertaken to determine whether or not there is a risk of CJD being transmitted in this manner. To date, no excess risk has been identified, leading to a growing consensus that there is little or no risk of long established forms of CJD being transmitted to recipients of blood products. This opinion does not extend to new variant CJD (vCJD) which is found predominantly in the UK and is believed to have resulted from the transmission of bovine spongiform encephalopathy (BSE) to humans. Unlike that of CJD, the prevalence of vCJD is not known. In addition, the detection of abnormal prion protein in the tonsils of vCJD-infected individuals has led to speculation that blood infectivity may be greater than in patients with CJD. A number of precautionary measures have been taken to address the possibility that vCJD may be transmissible by blood products; however, further scientific advances are needed to enable this risk to be defined. A suitable screening test is required to identify any infected blood donors, particularly where cellular blood components are being derived from populations believed to be at risk from BSE infection. Recent experimental data suggest that process operations used in the manufacture of plasma products may be capable of removing prion agents to a significant extent. However, further work is required to confirm these observations and to determine whether or not all potential vCJD infectivity would be removed by these means.     

Long-term stability of human immunodeficiency virus viral load and infectivity in whole blood

BACKGROUND: We intended to evaluate the stability of human immunodeficiency virus (HIV) type 1 virions in whole blood and in culture medium. MATERIALS AND METHOD: EDTA whole-blood samples taken from 12 patients were left at room temperature for up to 7 days, and aliquots of a laboratory virus stock spiked in EDTA, in heparinized or in citrated whole blood, with or without the addition of Triton X-100, or spiked in culture medium were left at room temperature for up to 120 days before plasma was separated and frozen at -80 degrees C. Viral load was measured for all frozen plasma samples using different viral load assays. p24 antigen and infectivity were also measured in the spiked samples. RESULTS: The patient whole-blood samples did not show any decrease in viral load during this 7-day period. The spiked samples decayed by not more than 1 log after 120 days (about 4 months), with the fastest decay in medium. Virus infectivity decayed very slowly from 20,000 units mL-1 to undetectable amounts after 56 days. CONCLUSIONS: These results indicate that HIV-1 virions in whole blood possess a long-term stability in terms of viral load, p24 antigen level and infectivity, which is not sufficiently recognized by laboratory and health care workers.     

Advances in screening test development for transmissible spongiform encephalopathies

The blood of patients with transmissible spongiform encephalopathy or prion disease can no longer be considered free of infectivity. There have been two recent reports of highly probable transfusion-associated iatrogenic variant Creutzfeldt-Jakob disease infections, and there is supporting experimental evidence of scrapie transmission by the transfusion of blood from sheep with naturally occurring disease. In the absence of a preclinical diagnostic test for transmissible spongiform encephalopathy, the main precautionary measures undertaken by blood agencies employ donor exclusion criteria, ensuring that the number of any further iatrogenic cases will be small. The development of a sensitive, specific and reliable diagnostic test is urgently needed for early identification of infected individuals in order to ensure the safety of blood supplies. During the past 5 years, significant progress has been made in improving the sensitivity and specificity of tests using brain and lymphoreticular tissues to identify Creutzfeldt-Jakob disease-infected individuals. However, the quest for a blood test is still in its infancy and requires extensive further research.     

An overview of prion biology and the role of blood filtration in reducing the risk of transfusion-transmitted variant Creutzfeldt-Jakob disease

Prions are infectious proteins believed to be responsible for a variety of progressive and fatal neurodegenerative diseases, collectively referred to as transmissible spongiform encephalopathies (TSE). By 1996, it was recognized that ingestion of beef from cattle afflicted with a TSE known as bovine spongiform encephalopathy, could result in a devastating human TSE known as variant Creutzfeldt-Jakob disease (vCJD). Two recent reports of probable transfusion-transmitted vCJD have raised concerns about the safety of the blood supply. The relatively long asymptomatic latency of vCJD, as well as the lack of sensitive and specific antemortem tests, increase the risk that asymptomatic, infected individuals may become blood donors. To this point, donor deferral has been a strategy used to reduce this risk. Nevertheless, this strategy may be unreliable and, furthermore, may threaten blood availability. Leukoreduction has also been helpful in reducing cell-associated infectious prion, which has been reported to reduce up to 42% of the infectivity in blood. Proprietary prion affinity surface modifications have been developed and applied to filters, which exploit an understanding of the unique chemical characteristics of prion surfaces. These have been successfully adapted to existing high-efficiency blood filter matrices for the reduction of prions present in blood components for transfusion.     

Antithrombin III in fresh frozen plasma, cryoprecipitate, and cryoprecipitate-depleted plasma

Antithrombin III (AT III) is a plasma protein that inhibits several activated procoagulants. Hereditary disease or acquired conditions such as severe hepatic dysfunction, nephrotic syndrome and intravascular coagulation may be associated with reduced levels of AT III. Its replacement may be essential in controlling thrombosis. In order to determine the most effective form of replacement, we compared AT III biological activity and antigen levels in conventionally prepared fresh frozen plasma, cryoprecipitate and cryoprecipitate depleted plasma (CDP). Both the activity and antigen levels were comparable in all three products (approximately 100%) and AT III was not concentrated in cryoprecipitate. These results indicate that conventionally prepared CDP, fresh frozen plasma and cryoprecipitate contain equal quantities volume for volume of AT III. On this basis, all products are equally effective as therapy for AT III deficiency, but CDP and fresh frozen plasma are recommended as convenient sources of this factor.     

Three turbidimetric methods for determining total protein compared

We used human serum protein fractions to evaluate the sensitivity and bias of three turbidimetric methods for determining concentrations of proteins. Each fraction (Cohn Fractions II, III, IV, and V) was assigned a protein concentration value that was determined by the biuret method, which we calibrated with purified monomer of human serum albumin. All three turbidimetric methods (those involving sulfosalicylic acid/sodium sulfate, trichloroacetic acid, and alkaline benzethonium chloride) gave acceptable results for Fraction V with crystallized human serum albumin as the reference material, but there was bias by each of the three methods for the three globulin fractions. The method involving alkaline benzethonium chloride with measurement at 450 nm had the best sensitivity within the range of linearity and the most consistent bias among the three globulin fractions. These results define the dilemma for valid calibration of these methods for total serum protein in cerebrospinal fluid and urine.     

Evidence for the identity of the major apoprotein in low density and very low density lipoproteins in normal subjects and patients with familial hyperlipoproteinemia

The major apoprotein(s) from human plasma low density lipoproteins was isolated and compared with a major protein fraction (fraction I) from very low density lipoproteins (VLDL). Fraction I had been previously found to comprise approximately 40% of the total protein of VLDL. Fraction I from VLDL and apoLDL from normal subjects were indistinguishable in amino acid compositions and circular dichroic spectra. They yielded indistinguishable displacement curves of LDL-(125)I by radioimmunoassay and formed immunoprecipitin lines of complete identity. Fraction I from VLDL of normal subjects was compared with the fraction isolated from patients with familial types II, III, IV, and V hyperlipoproteinemia. There were no detectable differences between any of these fractions in amino acid compositions, circular dichroic spectra, and immunochemical properties. It was, therefore, concluded that short of peptide mapping or determination of amino acid sequence, fraction I from VLDL of each subject with familial hyperlipoproteinemia appears to be identical with fraction I and apoLDL from normal individuals.A new convenient method of preparation of soluble apoLDL, modified from a procedure previously described from this laboratory, is presented.     

Direct demonstration of an inhibitor of sodium, potassium dependent adenosine triphosphatase (Na+, K+-ATPase) in plasma from normotensive and hypertensive subjects

An endogenous inhibitor of Na+, K+-ATPase was extracted from human plasma and sera and concentrated by a novel reverse-phase octadecylsilane chromatography method. The active extracts (eluates) were dried and reconstituted in the minimum volume of the non-adsorbed fraction of the plasma from which they had been derived. Reconstituted eluates, non-adsorbed plasma fractions and native plasma samples were then tested for their ability to inhibit phosphate production in standard Na+, K+-ATPase incubation mixtures. In a pilot study 31 samples of pooled normal human sera were assayed. The eluates gave a significantly lower production of phosphate than the non-adsorbed fractions or the native sera (n = 31, p less than 0.0025). Further concentration of the eluates by repeated chromatography increased the inhibitory power of the eluate proportional to the concentration achieved, as quantified by ouabain dose-equivalents. In clinical studies, samples from 12 normotensive subjects and from 12 untreated patients with essential hypertension were tested. Significant inhibition of the ATPase by the eluates, as compared to the corresponding non-adsorbed fractions was seen for samples from both normotensive (p less than 0.05) and hypertensive (p less than 0.05) subjects. There was no significant difference in incidence or degree of inhibition between the normotensive and hypertensive groups. This study provides direct evidence for the presence of an endogenous inhibitor of Na+, K+-ATPase in human plasma.