Gas chromatography/mass spectrometry analysis of very long chain fatty acids, docosahexaenoic acid, phytanic acid and plasmalogen for the screening of peroxisomal disorders

Very long chain fatty acids (VLCFAs) and docosahexaenoic acid (DHA), phytanic acid, and plasmalogens are usually measured individually. A novel method for the screening of peroxisomal disorders, using gas chromatography/mass spectrometry (GC/MS), was developed. Saturated and unsaturated fatty acids, including VLCFAs and DHA, phytanic acid, and plasmalogen were detected by a selected ion monitoring-electron impact method, using 100 microl of serum or plasma. Methyl-esterification and extraction could be done in one tube, and data were obtained within 4 h. All patients with Zellweger syndrome (ZS), X-linked adrenoleukodystrophy (ALD), isolated deficiency of peroxisomal beta-oxidation enzyme, and most ALD carriers showed increased VLCFA ratios, including C24:0/C22:0, C25:0/C22:0 and C26:0/C22:0. The ratio of DHA to palmitic acid (C16:0) and plasmalogen (measured as hexadecanal dimethyl acetal) to C16:0 in ZS patients was significantly lower than for the controls (P<0.001 for healthy high school students, P<0.05 for infants with other disorders). Plasmalogen was also decreased in patients with isolated deficiency of plasmalogen biosynthesis. Two of eight patients with ZS, two of four with RCDP, and all of three classical Refsum patients showed increased levels of phytanic acid. This method will simplify the screening for peroxisomal disorders.     

Globoid cells, glial nodules, and peculiar fibrillary changes in the cerebro-hepato-renal syndrome of Zellweger

In addition to a distinct malformation (pachymicrogyria, heterotaxic lamination of the cerebellar cortex, olivary dysplasia), unusual degenerative changes were found in the nervous system of 2 unrelated babies with the Zellweger syndrome. Cerebral clefts were present in 1 case. In both infants there was neuron loss and accumulation of glial nodules and globoid cells in the gray matter as well as degeneration of the white matter. There was fatty change in astrocytes and diffuse gliosis. Neurons in the column of Clarke and the lateral cuneate nucleus showed peculiar fibrillary changes. Cytoplasmic inclusion bodies were seen in the spinal ganglia. Swelling of cortical astrocytes was remarkable in the older infant. The combination of a rare malformation with the cell changes described here gives the syndrome a unique neuropathological profile.     

Plasma very long chain fatty acids in 3,000 peroxisome disease patients and 29,000 controls

The assay of plasma very long chain fatty acids (VLCFAs), developed in our laboratory in 1981, has become the most widely used procedure for the diagnosis of X-linked adrenoleukodystrophy (X-ALD) and other peroxisomal disorders. We present here our 17 years' experience with this assay. Three VLCFA parameters, the level of hexacosanoic acid (C26:0), the ratio of C26:0 to tetracosanoic acid (C24:0), and of C26:0 to docosanoic acid (C22:0), were measured in 1,097 males (hemizygotes) with X-ALD, 1,282 women heterozygous for this disorder, including 379 obligate heterozygotes, 797 patients with other peroxisomal disorders, and 29,600 control subjects. All X-ALD hemizygotes who had not previously received Lorenzo's oil or a diet with a high erucic acid content had increased VLCFA levels, but the application of a discriminant function based on all three measurements is required to avoid the serious consequences of a false-negative result. VLCFA levels are increased at day of birth, thus providing the potential for neonatal mass screening, are identical in the childhood and adult forms, and do not change with age. Eighty-five percent of obligate heterozygotes had abnormally high VLCFA levels, but a normal result does not exclude carrier status. VLCFA levels were increased in all patients homozygous for Zellweger syndrome, neonatal adrenoleukodystrophy, infantile Refsum's disease, and in patients with deficiencies of peroxisomal acyl-coenzyme A oxidase, bifunctional enzyme, and 3-oxoacyl-coenzyme A thiolase. In these patients the degree of VLCFA excess correlated with clinical severity. Ann Neurol 1999;45:100–110     

Unusual orthochromatic leukodystrophy with epitheloid cells (Norman-Gullotta): increase of very long chain fatty acids in brain discloses a peroxisomal disorder

Summary Very long chain fatty acids (VLCFA) were found to be markedly increased and phytanic acid was borderline above normal in formalin-fixed brain white matter of case with an unusual type of familial leukodystrophy with epitheloid cells as described previously by Gullotta et al. [Neuropädiatrie (1970) 2: 173–186]. Increased VLCFA in brain clearly demonstrate that the patient had suffered from a peroxisomal disease. This diagnosis is corroborated by ultrastructural findings in brain showing typical lamellar inclusions. The particular type of peroxisomal disorder present in case (heterozygote of X-linked adrenoleukodystrophy?) remains speculative.     

Presence of plasma branched-chain fatty acids in multineuronal degeneration,hepatosplenomegaly and adrenocortical insufficiency

We have previously reported a unique disorder in two brothers with multisystem neuronal degeneration, hepatosplenomegaly and adrenocortical deficiency. The clinical features were different from Refsum's disease. Biochemical analysis suggested that a metabolic defect of the ω6 polyenoic fatty acid pathway may be involved. In the present study, were have further identified by gas chromatography-mass spectrometry two branched-chain fatty acids, phytanate and pristanate, in these two patients' plasma. This small, but unequivocally elevated amount of branched-chain fatty acids was primarily localized in the triacylglycerols of plasma low density lipoprotein. Such branched-chain fatty acids were not detected in skin, liver and sural nerve samples. These two cases may represent an alternative metabolic error to that found in Refsum's disease leading to phytanate accumulation.     

Correlation of very long chain fatty acid accumulation and inflammatory disease progression in childhood X-ALD: implications for potential therapies

This study was designed to understand the role of inflammatory mediators involved in the neurobiology of childhood adrenoleukodystrophy (cALD) by comparing the differential expression of the inflammatory mediators with metabolite very long chain fatty acids that accumulate in this disease. Histopathological examinations indicated extensive demyelination and accumulation of infiltrates in perivascular cuffs in plaque area (PA) and inflammatory area (IA) compared to normal looking area (NLA) of the cALD brain and controls. The PA had excessive accumulation of cholesterol ester (25-30-fold), VLC fatty acids (8-12-fold), and exhaustive depletion of cholesterol (60-70%) and sphingomyelin (50-55%) in comparison to controls. The mRNA expression of cytokines (IL-1alpha, IL-2, IL-3, IL-6, TNF-alpha, and GM-CSF), chemokines (CCL2, -4, -7, -11, -16, -21, -22, CXCL1, CX3CL1, and SDF-2) and iNOS in IA was significantly increased compared to NLA of the cALD and controls determined by gene array, semiquantitative RT-PCR, and immunohistochemistry. These results indicate that accumulation of VLC fatty acid contents in membrane domains associated with signal transduction pathways may trigger the inflammatory process through activation of resident glial cells (microglia and astrocytes) resulting in loss of myelin and oligodendrocytes.     

Accumulation of very long chain fatty acids is common to 3 variants of adrenoleukodystrophy (ALD): “Classical” ALD,atypical ALD (female patient) and adrenomyeloneuropathy

Fatty acids of cholesterol esters were analyzed by gas chromatography in affected CNS white matter of 3 variants of ALD (“classical” ALD, atypical ALD (adult female) and AMN) and of 10 controls with myelin breakdown of an etiology other than ALD. In all 3 ALD variants a marked accumulation of very long chain fatty acids (VLFA) as compared to control material was observed. This was due to the accumulation mainly of saturated C₂₄–C₃₂ fatty acids, particularly of C_26:0, C_25:0 and, to a lesser extent, C_24:0 and C_27:0 fatty acids. Our results demonstrate for the first time an accumulation of VLFA in an adult female patient (atypical ALD), who probably is an ALD heterozygote rather than a variant of AMN, and confirm and extend earlier findings in classical ALD and AMN, respectively. It appears that ALD may be a single nosological entity with clinically and morphologically different variants sharing specific ultrastructural (accumulation of paired leaflets) and neurobiochemical (accumulation of VLFA) diagnostic markers.     

The Refsum disease marker phytanic acid, a branched chain fatty acid, affects Ca2+ homeostasis and mitochondria, and reduces cell viability in rat hippocampal astrocytes

The saturated branched chain fatty acid, phytanic acid, a degradation product of chlorophyll, accumulates in Refsum disease, an inherited peroxisomal disorder with neurological clinical features. To elucidate the pathogenic mechanism, we investigated the influence of phytanic acid on cellular physiology of rat hippocampal astrocytes. Phytanic acid (100 microM) induced an immediate transient increase in cytosolic Ca2+ concentration, followed by a plateau. The peak of this biphasic Ca2+ response was largely independent of extracellular Ca2+, indicating activation of cellular Ca2+ stores by phytanic acid. Phytanic acid depolarized mitochondria without causing in situ swelling of mitochondria. The slow decrease of mitochondrial potential is not consistent with fast and simultaneous opening of the mitochondrial permeability transition pore. However, phytanic acid induced substantial generation of reactive oxygen species. Phytanic acid caused astroglia cell death after a few hours of exposure. We suggest that the cytotoxic effect of phytanic acid seems to be due to a combined action on Ca2+ regulation, mitochondrial depolarization, and increased ROS generation in brain cells.     

Analysis of very long-chain fatty acids and plasmalogen in the erythrocyte membrane: a simple method for the detection of peroxisomal disorders and discrimination between adrenoleukodystrophy and Zellweger syndrome

We analyzed the sphingomyelin very long-chain fatty acids (VLCFAs) and phosphatidylethanolamine (PE) plasmalogen contents of the erythrocyte membrane in patients suffering from peroxisomal disorders. In a patient with Zellweger syndrome, both a decrease in the PE plasmalogen content and an increase in the sphingomyelin VLCFAs content of the erythrocyte membrane were noted. In patients with adrenoleukodystrophy, however, there was no decrease in PE plasmalogen, although the sphingomyelin VLCFAs content of the membrane was significantly increased in comparison with control values. Analyses of both sphingomyelin VLCFAs and PE plasmalogen were carried out simultaneously, using both the same process and the same sample.     

Two siblings of Leber's congenital amaurosis with an increase in very long chain fatty acid in blood: relationship between peroxisomal disorders and Leber's congenital amaurosis

We reported two siblings of Leber's congenital amaurosis associated with increased level of very long chain fatty acid (VLCFA) in blood. Case 1, a 3 1/2-year-old boy had congenital blindness, severe psychomotor retardation, hepatomegaly, profound hypotonia, loss of deep tendon reflexes, muscular atrophy and weakness, and non-convulsive status epilepticus characterized by a sudden respiratory failure, and also showed a flat electroretinogram, non-pigmentary retinal degeneration, severe atrophy of the brain stem and cerebellum, hepatic fibrosis, decreased motor and sensory conduction velocities and atlanto-axial instability. Sural nerve biopsy revealed severely decreased number of total myelinated fibers without remarkable demyelination or remyelination. Case 2, an elder sister of case 1, with pigmentary retinal degeneration, hepatomegaly and pericarditis had died at 3 months. Autopsy revealed hypomyelination and heterotopy of the cerebral white matter, hepatic fibrosis, renal microcysts and normal adrenal cytoarchitecture. In case 1, the level of VLCFA was increased twofold and sevenfold of controls in serum and in red cell membrane, respectively. Phytanic or trihydroxycholestanoic acid was not detected in the serum and bile. Normal shaped peroxisomes were definitely recognized in biopsied liver by means of electronmicroscopic histochemistry. From the above findings, these patients was thought to be a new variant of peroxisomal disorders relating to degradation of VLCFA, other than Zellweger syndrome, infantile Refsum disease and infantile adrenoleukodystrophy. It was concluded that peroxisomal functions should be studied in cases of Leber's congenital amaurosis.     

Sialic acid and fatty acid concentrations in lymphocytes, red blood cells and plasma from patients with multiple sclerosis

Lipids and constituents of lipids were isolated from peripheral blood lymphocytes (PBL), red blood cells (RBC), plasma and sera from 51 patients with multiple sclerosis (MS) and 51 controls, matched for age, sex and race. The amount of sialic acid released by incubation of intact PBL from MS patients was significantly lower (P less than 0.02) than that of the sialic acid from control lymphocytes. The amounts of sialic acid released by neuraminidase from intact RBC, on the other hand, did not differ significantly between the MS group and the control group. The concentrations of ganglioside sialic acid in PBL from MS and control groups did not vary significantly. Similarly the concentration of ganglioside sialic acid in RBC from MS patients was not significantly different from that in the controls. Analyses of the fatty acids isolated after alkaline methanolysis of the lipids from the PBL of MS patients and controls showed a small but significant decrease (P less than 0.01) in the relative percentage of linoleic acid in patients with MS. Determination of the ester-linked fatty acids in RBC lipids from patients with MS showed a significant decrease (P less than 0.001) in the relative percentage of linoleic acid and an increase (P less than 0.01) in palmitic plus palmitoleic acids compared to control values. The fatty acid composition of the plasma neutral lipids plus free fatty acids showed a very significant decrease (P much less than 0.001) in the relative percentage of linoleic acid, a small decrease (P less than 0.05) in arachidonic acid and significant increases in palmitic (P less than 0.001) and oleic acids (P less than 0.001) in MS, compared to controls. These results are suggestive of possible differences in metabolism of lipids between patients with MS and controls.     

Effect of exogenous fatty acids on lipid synthesis, marker-enzymes, and development of glial cells maintained in serum-free culture

Glial cells were isolated from the cerebra of 7-day-old rats and maintained in culture in a chemically defined medium that favours the development of oligodendrocytes. Acetate, butyrate, or albumin-bound hexanoate, octanoate, decanoate, laurate, myristate, palmitate, oleate, linoleate, or arachidonate was added to the culture medium. The incorporation of [3H]acetate into fatty acids and cholesterol and [35S]sulphate into sulphatide, and the activities of the oligodendrocyte marker enzymes 2',3'-cyclic-nucleotide 3'-phosphodiesterase and glycerol 3-phosphate dehydrogenase were measured. The composition of the glial cell population (the number of astrocytes and oligodendrocytes) in these cultures was studied by immunocytochemistry. Results show that 1) long-chain fatty acids depress the synthesis of fatty acids, cholesterol, and sulphatide; and 2) the presence of long-chain, in contrast to short-chain, fatty acids in the culture medium lowers the activities of 2',3'-cyclic-nucleotide 3'-phosphodiesterase and glycerol 3-phosphate dehydrogenase and decreases the number of oligodendrocytes. Our results suggest that long-chain fatty acids exert a negative influence on the development of oligodendrocytes in the culture system used.     

Elevated plasma beta-amyloid peptide Abeta(42) levels, incident dementia, and mortality in Down syndrome

BACKGROUND: Deposition of the beta-amyloid peptide Abeta(42) is thought to be an important initial step in the pathogenesis of Alzheimer disease (AD). Individuals with Down syndrome have increased levels of beta-amyloid peptides and an increased risk for AD. OBJECTIVE: To examine the relation of plasma levels of Abeta(42) and Abeta(40) to the risk of dementia in nondemented participants and all-cause mortality in adults with Down syndrome. DESIGN: Prospective, community-based longitudinal cohort study. SETTING: State and voluntary service providers in New York State. PARTICIPANTS: Adults with Down syndrome (N = 204). MAIN OUTCOME MEASURE: Plasma Abeta(42) and Abeta(40) levels were measured at initial examination. Participants were assessed for cognitive and functional abilities, behavioral/psychiatric conditions, and health and vital status at 14- to 18-month intervals for 4 cycles of data collection. RESULTS: Among participants who were nondemented at baseline, those in the middle and highest tertiles of plasma Abeta(42) levels were more than 2 times as likely to develop AD as those in the lowest tertile. Compared with participants without AD, participants with prevalent AD had higher levels of plasma Abeta(42) but not Abeta(40). Among all participants, those in the highest tertile of plasma Abeta(42) level at baseline were more than twice as likely to die during the study period as those in the lowest tertile, whereas there was no difference in risk of death between those in the middle and lowest tertiles of plasma Abeta(42) level. CONCLUSION: Elevations in plasma Abeta(42) peptide levels are associated with earlier onset of AD and increased risk of death.     

Influence of IFN-β1b (Betaferon) on cytokine mRNA profiles in blood mononuclear cells and plasma levels of soluble VCAM-1 in multiple sclerosis

Inflammatory cell infiltration within the central nervous system (CNS) and upregulation of both pro- and anti-inflammatory cytokines are characteristic for multiple sclerosis (MS). Treatment with interferon-β1b (IFN-β1b) reduces the number and severity of MS relapses. To examine whether treatment with IFN-β1b affects levels of cytokine mRNA expressing blood mononuclear cells (MNC) we employed in-sit hybridization with synthetic oligonucleotide probes to detect and enumerate IFN-γ, TNF-α, IL-10, TGF-β and perforin mRNA expressing cells in MS patients before treatment with IFN-β1b and during tretmetn for 3–6 weeks and for 3–6 monts. Numbers of blood MNC spontaneously expressing TNF-α and IL-10 mRNA were lower after 3–6 months of treatment, while numbers of IFN-γ, TGF-β and perforin mRNA expressing MNC were not affected by treatment. IFN-β1b had no influence on levels of MBP-reactive IFN-γ, TNF-α, TGF-β, IL-10 or perforin mRNA expressing blood MNC determined after 3–6 weeks 3–6 months of treatment. Parallel measurements of plasma concentrations of soluble vascular cell adhesion molecule-1 (sVCAM-1) revealed elevated levels after 3–6 weeks of treatment and these levels remained higher after 3–6 months of treatment. The results suggest that IFN-β1b treatment upregulates plasma levels of sVCAM-1, but has little effects on numbers of blood MNC expressing mRNA of the pro- and anti-inflammatory cytokines under study.     

Proton and 31-phosphorus neurospectroscopy in the study of membrane phospholipids and fatty acid intervention in schizophrenia, depression, chronic fatigue syndrome (myalgic encephalomyelitis) and dyslexia

Neurospectroscopy allows biochemical processes in the brain to be studied non-invasively. At magnetic field strengths of 1.5 T or higher, cerebral proton neurospectroscopy allows the ascertainment of values of myo-inositol, choline-containing compounds, creatine, glutamate, glutamine, and N-acetyl aspartate. At similar field strengths, cerebral 31-phosphorus neurospectroscopy allows the ascertainment of values of phosphomonoesters, inorganic phosphate, phosphodiesters, phosphocreatine, and the gamma, alpha and beta nucleotide triphosphate (mainly adenosine triphosphate) resonances. Since choline is a common polar head group at the Sn3 position of membrane phospholipid molecules, a raised level of free choline, as indexed by proton neurospectroscopy, can indicate relatively low anabolism of membrane phospholipid molecules. Furthermore, the choline peak includes phosphorylcholine and glycerophosphorylcholine and even ethanolamine. The phosphomonoesters peak measured using 31-phosphorus spectroscopy includes major contributions from phosphocholine, phosphoethanolamine and L-phosphoserine, which are important precursors of membrane phospholipids, while the phosphodiesters peak includes contributions from glycerophosphocholine and glycerophosphoethanolamine, which are important products of membrane phospholipid catabolism. Hence proton neurospectroscopy and 31-phosphorus neurospectroscopy can yield important information relating to the metabolism of cerebral membrane phospholipids. The application of these techniques to the investigation of membrane phospholipid metabolism in schizophrenia, depression, chronic fatigue syndrome (myalgic encephalomyelitis or M.E.) and dyslexia is described.     

Proton and 31-phosphorus neurospectroscopy in the study of membrane phospholipids and fatty acid intervention in schizophrenia,depression, chronic fatigue syndrome (myalgic encephalomyelitis) and dyslexia

Summary

Neurospectroscopy allows biochemical processes in the brain to be studied non-invasively. At magnetic field strengths of 1.5?T or higher, cerebral proton neurospectroscopy allows the ascertainment of values of myo-inositol, choline-containing compounds, creatine, glutamate, glutamine, and N-acetyl aspartate. At similar field strengths, cerebral 31-phosphorus neurospectroscopy allows the ascertainment of values of phosphomonoesters, inorganic phosphate, phosphodiesters, phosphocreatine, and the gamma, alpha and beta nucleotide triphosphate (mainly adenosine triphosphate) resonances. Since choline is a common polar head group at the Sn3 position of membrane phospholipid molecules, a raised level of free choline, as indexed by proton neurospectroscopy, can indicate relatively low anabolism of membrane phospholipid molecules. Furthermore, the choline peak includes phosphorylcholine and glycerophosphorylcholine and even ethanolamine. The phosphomonoesters peak measured using 31-phosphorus spectroscopy includes major contributions from phosphocholine, phosphoethanolamine and L-phosphoserine, which are important precursors of membrane phospholipids, while the phosphodiesters peak includes contributions from glycerophosphocholine and glycerophosphoethanolamine, which are important products of membrane phospholipid catabolism. Hence proton neurospectroscopy and 31-phosphorus neurospectroscopy can yield important information relating to the metabolism of cerebral membrane phospholipids. The application of these techniques to the investigation of membrane phospholipid metabolism in schizophrenia, depression, chronic fatigue syndrome (myalgic encephalomyelitis or M.E.) and dyslexia is described.     

Biosynthesis of prostanoids, tissue fatty acid composition and thrombotic parameters in rats fed diets enriched with docosahexaenoic (22:6n3) or eicosapentaenoic (20:5n3) acids

The objective of this experiment was to elucidate the effect(s) of eicosapentaenoic (20:5n3) vs docosahexaenoic (22:6n3) acid on prostaglandin biosynthesis and related thrombotic parameters. Diets were formulated to contain oils absent in (control) or enriched with either 20:5n3 (EPA) or 22:6n3 (DHA). The diets were fed to rats for three weeks and the following evaluated: 1) bleeding time; 2) blood viscosity; 3) platelet aggregation; 4) tissue fatty acids; 5) serum thromboxane (TXB₂), aortic prostacylin (6-keto) and 6) arachidonic acid conversion to eicosanoids by lung microsomes. There were no significant differences between treatments for bleeding time, red blood cell viscosity or platelet aggregation. In EPA fed rats 20:5n3 increased significantly in platelet and aorta phospholipids. In platelets and aorta 20:4n6 was slightly decreased in EPA and DHA animals. Platelet 22:6n3 levels were not altered by treatment, but 22:6n3 increased in the aorta of EPA and DHA fed rats. Similar changes were noted in lung and liver fatty acid composition. Serum TXB₂levels were significantly decreased only in the EPA vs control group. No differences were noted for aortic 6-keto levels or in the amount of hydroxy fatty acids, PGE, TXB₂or PGF₂ produced by lung microsomes. While fish oils have been shown to alter hematologic parameters in humans this study suggests that the rat is not similarly affected. Furthermore, it is evident that in the rat, 20:5n3 and not 22:6n3 is responsible for the alterations in platelet prostaglandin biosynthesis; however, these observations may not be directly applicable to other species.     

K1K2Pu, a recombinant t-PA/u-PA chimera with increased thrombolytic potency, consisting of amino acids 1 to 3 and 87 to 274 of human tissue-type plasminogen activator (t-PA) and amino acids 138 to 411 of human single chain urokinase-type plasminogen activator (scu-PA). Purification in centigram quantities and conditioning for use in man.

K1K2Pu, a recombinant t-PA/u-PA chimera with increased thrombolytic potency in animal models of venous and arterial thrombosis, which consists of amino acids 1 to 3 and 87 to 274 of human tissue-type plasminogen activator (t-PA) and amino acids 138 to 411 of human single chain urokinase-type plasminogen activator (scu-PA), was produced and conditioned for use in patients. Chinese hamster ovary cells were transfected with an expression plasmid containing the K1K2Pu cDNA, high producer cell lines were selected and scaled up in 800 cm2 roller bottles, and 350 ml conditioned cell culture medium was harvested 3 to 7 times at 2 to 5 day intervals. Batches of 21 +/- 4 liter (mean +/- SD, n = 28) containing 1.8 +/- 0.6 mg/l of K1K2Pu related antigen were purified by chromatography on Copper chelate-Sepharose and immunoadsorption on an insolubilized murine monoclonal antibody (MA-1C8). Yields were 8.6 +/- 3.4 mg K1K2Pu per batch with a specific activity of 83,000 +/- 44,000 IU/mg. The final material, obtained at a concentration of approximately 0.7 mg/ml, was dialyzed against 0.3 M NaCl, 0.02 M Tris-HCl buffer, pH 7.5, containing 0.01% Tween 80 and 10 KIU/ml aprotinin. It was homogeneous on SDS-PAGE, contained 6.5 +/- 6.9 percent two chain material and the contamination with murine monoclonal antibody was less than 0.1 percent. After filtration of pools of 3 to 5 selected batches on 0.22 microns Millipore filters the material was sterile and virus free by routine screening; it was obtained at a concentration of approximately 0.5 mg/ml with a specific activity of 110,000 +/- 16,000 IU/mg (mean +/- SD, n = 3) and an endotoxin content of 0.5 to 7 units/mg. Bolus injection at a dose of 1 mg/kg in mice did not produce weight loss within 8 days. Thus, this material appears to be suitable for the investigation on a pilot scale of the pharmacokinetic and thrombolytic properties of K1K2Pu in patients with thromboembolic disease.