Relationship between paraoxonase 1 activity and high density lipoprotein concentration during naturally occurring babesiosis in dogs

Paraoxonase 1 (PON1) is a negative acute phase protein bound to high density lipoproteins (HDL) and during the acute phase response (APR) protects HDL from peroxidation. The aim of this study was to assess the relationship between PON1 and HDL in canine babesiosis, a disease characterized by oxidative damages and by an APR. PON1, HDL and C-reactive protein (CRP), were measured in blood collected from 15 controls and 29 dogs with babesiosis sampled at admission, and on days 1 and 7 after treatment. At admission, PON1 and HDL were significantly lower in affected dogs. HDL concentration increased at day 1 while PON1 increased and CRP decreased at day 7. This suggests that the decrease of PON1 at admission is in part due to an increased consumption, the decreased HDL may depend on lipid peroxidation and its rapid increase after treatment may depend on the antioxidant activity of PON1.     

Paraoxonase (PON) 1, 2 and 3 Expression in Granulosa Cells and PON1 Activity in Follicular Fluid of Dairy Cows

Normal metabolic activity in ovarian follicles may result in oxidative stress and damage to oocytes. The aim of this study was to evaluate expression of the natural anti‐oxidants paraoxonase (PON) 1, 2 and 3 in granulosa cells and PON1 activity in follicular fluid (FF) and plasma of dairy cows. For the first experiment, ovaries were collected from cows at slaughter, after which follicles were dissected and classified as oestrogen active (EAF) or atretic (ATF). Expression of PON1, PON2 and PON3 mRNA was evaluated in granulosa cells, and activity of PON1 was measured in FF. PON1 mRNA was undetectable in granulosa cells, PON2 mRNA expression was not different between follicle types, and PON3 mRNA tended to be higher in EAF (p = 0.11). The activity of PON1 in FF was higher (p = 0.01) for EAF (82.6 ± 8.0 kU/L) than ATF (53.9 ± 6.8 kU/L), as were high‐density lipoproteins (HDL), low‐density lipoproteins (LDL) and total cholesterol concentrations. In the second experiment, we aimed to compare plasma and FF PON1 activity in early lactation Holstein cows (n = 15) with pre‐ovulatory EAF. Activity of PON1 was twofold higher (p < 0.0001) in plasma (122.5 ± 11.1 kU/L) than in FF (61.4 ± 5.2 kU/L). Plasma concentrations were also higher (p < 0.0001) for HDL, LDL and total cholesterol when compared to FF. In conclusion, FF concentrations of PON1, HDL, LDL and total cholesterol were higher in healthy oestrogen active bovine follicles than in atretic follicles. PON1 was not expressed by granulosa cells indicating that high PON1 activity in bovine FF is apparently derived by transfer from blood in association with HDL.     

Exogenous paraoxonase‐1 during oocyte maturation improves bovine embryo development in vitro

Paraoxonase‐1 (PON1) is an enzyme found in serum and follicular fluid that protects cell membrane and circulating lipids against oxidative damage. The aims of this study were to measure the direct effects of recombinant PON1 (rPON1) on bovine oocyte maturation at the molecular level (gene expression) and to measure the carry‐over effects of PON1 on pre‐implantation embryo development in vitro. COCs were submitted to IVM with the addition of 0.0, 0.02, 0.04 and 0.08 mg ml^?1 of rPON1, corresponding to an average PON1 arylesterase enzyme activity of 2.2 ± 0.4, 15.5 ± 1.5, 30.2 ± 3.0 and 57.9 ± 5.0 U ml^?1, respectively. The results indicated that addition of rPON1 during IVM improved embryo development in a dose‐dependent manner as D7 embryo development was 22.2%, 29.4%, 32.2% and 37.0% for the treatment groups, respectively (p = 0.02). In conclusion, addition of PON1 enzyme during IVM exerted dose‐related positive effects on embryo development rates to blastocysts.     

Serum paraoxonase 1 and butyrylcholinesterase in dogs with hyperadrenocorticism

The aim of this study was to evaluate serum activities of paraoxonase 1 (PON1) using three substrates; (1) 4-nitrophenylacetate (PON1n), (2) phenylacetate (PON1p), and (3) 5-thiobutyl butyrolactonase (PON1t), and butyrylcholinesterase (BChE) in dogs with hyperadrenocorticism (HAC). Serum activities of PON1 and BChE were higher in dogs with HAC than healthy dogs. There were strong positive correlations between PON1 activity measured with the three different substrates. This study demonstrated increased serum PON1 and BChE activities in dogs with HAC that could be attributed to the direct effect of glucocorticoids and lipid mobilisation.     

Bovine apolipoprotein E in plasma: increase of ApoE concentration induced by fasting and distribution in lipoprotein fractions

Apolipoprotein E (apoE) is a protein constituent of lipoproteins, and acts as a receptor-binding ligand. Although the existence of bovine apoE in lipoprotein fractions has already been reported, quantitative studies on the changes of apoE in plasma and lipoprotein fractions are lacking. In the present study, an increase of a 38 kDa protein in the very low-density lipoprotein (VLDL) fraction obtained from fasted calves was detected. This 38 kDa protein was identified as bovine apoE by determination of the N-terminal amino acid sequence. Bovine apoE was purified and an enzyme-linked immunosorbent assay (ELISA) was developed. Using this system, the effect of fasting on the concentration of apoE in plasma and the distribution of apoE in lipoprotein fractions were investigated. After 3 days of fasting, the concentration of plasma apoE increased significantly (p<0.05) by 280 %, and was returned to the basal level by 3 days of refeeding. The lipoprotein fractions obtained from before and after fasting was separated by ultracentrifugation. ApoE was significantly increased in VLDL, low-density lipoprotein (LDL) and non-lipoprotein fractions by fasting (p<0.05). On the other hand, in high-density lipoprotein (HDL) fractions obtained from both before and after fasting, the level of apoE was very low compared to the other fractions. These results suggested that bovine apoE contents in triglyceride-rich lipoproteins are modulated by nutritional treatment and closely associated with triglyceride-rich lipoprotein metabolism.     

Effect of high‐density lipoprotein on oocyte maturation and bovine embryo development in vitro

High‐density lipoprotein (HDL) is the main lipoprotein in the follicular fluid, and it has anti‐inflammatory, antioxidant and cryoprotectant properties. The anti‐inflammatory potential and antioxidant potential are derived from its lipid composition, especially the apolipoprotein AI (ApoAI) and paraoxonase 1 (PON1). The aim of this study was to evaluate the effect of HDL during in vitro maturation (IVM) on oocyte maturation and early bovine embryo development. For this, cumulus–oocyte complexes (COCs) were obtained from bovine ovaries collected at a local slaughterhouse. COCs (n = 2,250) were allocated into three groups (n = 50 COCs/group) according to the addition of HDL protein (HDL‐P) during IVM for 22 hr: 0 (control), 50 and 150 mg/dl. After IVM, COCs were inseminated (in vitro fertilization) and cultivated for 7 days. Total cholesterol concentration, total protein, triglycerides and ApoAI concentrations on IVM medium increased proportionally to HDL‐P addition. However, PON1 activity was not detected in any treatment. The addition of HDL‐P did not affect nuclear maturation rate, endogenous reactive oxygen species and glutathione levels in COCs (p > 0.05). The highest HDL‐P concentration (150 mg/dl) decreased cleavage and blastocyst rate (p < 0.05). Moreover, the HDL‐P 150 mg/dl group had lower cellular count/blastocyst than the 50 mg/dl group (p < 0.05). However, the addition of HDL‐P did not affect relative gene expression of evaluated genes. In conclusion, the complex HDL/ApoAI obtained from human plasma, in the absence of PON1 activity during in vitro oocyte maturation, decreased initial embryo development.     

Intrafollicular paraoxonase 1 activity and the steroidogenic potential of the first post‐partum dominant follicle in dairy cows

Cows experiencing high levels of inflammation and specific metabolic conditions tend to have slower follicular growth and lower serum and follicular concentrations of oestradiol (E2). Paraoxonase1 (PON1) activity decreases during inflammatory processes. Therefore, the aim of this study was to evaluate the association between serum and intrafollicular (FF) PON1 activity and the serum and intrafollicular levels of E2 and progesterone (P4), as well as the mRNA expression of genes related to steroidogenesis, metabolism and inflammation in the first post‐partum dominant follicle of Holstein cows. No correlation was found between PON1 activity, the expression of the analysed genes and levels of follicular E2 and P4, except for a negative correlation between serum E2 and follicular PO1 activity. Also, no correlation was found between serum and follicular PON1 during the first post‐partum follicular wave.     

Composition and electrophoretic mobility of plasma lipoproteins of dromedary camels (Camelus dromedarius)

OBJECTIVE: To determine the lipid composition and electrophoretic pattern of plasma lipoproteins in samples obtained from healthy 1-humped camels (Camelus dromedarius). ANIMALS: 34 healthy camels raised under similar farming and dietary conditions. PROCEDURES: Plasma samples were subjected to density-gradient ultracentrifugation for separation of plasma lipoproteins, including very-low-density lipoproteins (VLDL), low-density lipoproteins (LDL), and high-density lipoproteins (HDL). Purity of the separation was assessed by use of polyacrylamide gel disk electrophoresis. Concentrations of triglycerides, cholesterol, and phospholipids were measured in each lipoprotein fraction, and lipoprotein electrophoretic patterns were determined in plasma samples. RESULTS: Phospholipid was the major constituent of VLDL (mean +/- SD concentration, 10.62 +/- 1.2 mg/dL), LDL (24.66 +/- 3.12 mg/dL), and HDL (38.08 +/- 0.76 mg/dL). Low-density lipoprotein, VLDL, and HDL were important plasma lipoprotein carriers for cholesterol (67.94 +/- 9.51%), triglyceride (55.83 +/- 7.81%), and phospholipid (51.91 +/- 1.55%), respectively. On the basis of electrophoresis results, relative percentages of alpha- and beta-lipoproteins were 31.72 +/- 4.88% and 68.3 +/- 4.68%, respectively. CONCLUSIONS AND CLINICAL RELEVANCE: The lipoprotein profile in 1-humped camels differed substantially from that of other ruminants. Results may be useful in the evaluation of metabolic disorders in camels.     

Paraoxonase 1 (PON1) activity in serum,follicular fluid and seminal plasma of sheep

This work aimed to describe the activity of paraoxonase 1 (PON1) in serum, follicular fluid and seminal plasma of sheep. Average serum PON1 activity was 286.8 ± 96.2 U/ml in females and 237.6 ± 18.9 U/ml in males. There was a positive correlation between PON1 activity in serum and follicular fluid in females, being twice higher in serum than in follicular fluid (148.8 ± 15.7 U/ml). PON1 activity in males’ serum was 10‐fold higher than in seminal plasma (21.18 ± 14.2 U/ml), and there was no correlation between PON1 activity in both compartments. Finally, this work suggests that PON1 activity of in sheep is higher compared to other mammalian species, and there is an association between PON1 in serum and follicular fluid only.     

Serum paraoxonase type-1 activity in pigs: Assay validation and evolution after an induced experimental inflammation

Paraoxonase 1 (PON1) is a serum enzyme synthesised and secreted primarily by the liver. It possesses anti-inflammatory properties limiting the production of pro-inflammatory mediators. The objectives of this study were to validate three spectrophotometric assays for the quantification of PON1 activity in pig serum, and to determine if PON1 activity in porcine behaves as a negative acute phase protein (APP), decreasing in inflammatory conditions. An analytical validation using three different substrates – 5-thiobutil butyrolactone (TBBL), phenylacetate (PA) and 4-(p)-nitrophenyl acetate (pNA) – was performed. In addition, inflammation was experimentally induced in five pigs by subcutaneous injection of turpentine oil, while five control pigs were left untreated. The treated pigs showed significant increases in CRP and decreases in albumin, indicating an inflammatory condition. The three substrates used would be suitable for PON1 activity measurements in serum samples, since they offer adequate precision (coefficients of variation < 10%), sensitivity (0.01, 0.15, 0.02 U/mL for TBBL, pNA and PA respectively) and accuracy (r = 0.99). In addition, PON1 behaves as a negative APP in pigs since a significant decrease (P < 0.05) in its activity after 72 h of the induction of the inflammation was observed with all substrates.     

Decreased apolipoprotein C-III concentration in the high-density lipoprotein fraction from calves inoculated with Pasteurella haemolytica and bovine herpes virus-1

Lipoprotein lipid and apoprotein concentrations are known to be altered during the acute-phase response. We have previously shown that the serum activity of lecithin:cholesterol acyltransferase (LCAT) and concentration of cholesteryl esters, both constituents of the high-density lipoprotein (HDL) fraction, are reduced in calves inoculated with Pasteurella haemolytica and bovine herpes virus-1, the two major pathogens for calf pneumonia. The concentration of apolipoprotein C-III (apoC-III), a low molecular mass protein component distributed mainly in the HDL fraction, was therefore examined in bacteria- and virus-inoculated calves. An enzyme-linked immunosorbent assay demonstrated that it was decreased by inoculations of Pasteurella haemolytica and bovine herpes virus-1. The decrease was detected as early as 1 day after inoculation in both groups. A decreased serum apoC-III concentration was also observed by immunoblot analysis. It was detected in the HDL fractions from the bacteria- and virus-inoculated calves, and HDL apoC-III concentrations in the inoculated calves were decreased compared with controls. These results, coupled with the previous findings on LCAT activity and the cholesteryl ester concentration, indicate that a decreased HDL concentration is one of the early events occurring during the acute-phase response evoked by infections with Pasteurella haemolytica and bovine herpes virus-1.     

Serum butyrylcholinesterase and paraoxonase 1 in a canine model of endotoxemia: effects of choline administration

Butyrylcholinesterase (BChE) and paraoxonase 1 (PON1) are two serum enzymes synthesized by the liver that are related with inflammation. The main objectives of this study were to determine changes in serum BChE and PON1 by using a canine model of endotoxemia, and to evaluate whether choline alters BChE and PON1 activities during inflammation. For this purpose, a total of 20 mongrel dogs were divided into four groups: control, choline (C), lipopolysaccharide (LPS), and LPS+C. Dogs in the control group were injected with 0.9% NaCl (0.2 ml/kg, i.v.). Dogs in C and LPS+C groups received choline chloride (20 mg/kg, i.v., three times with 4 h intervals). Endotoxin was injected (0.02 mg/kg, i.v., once) to the dogs of LPS and LPS+C groups. Statistically significant decreases in BChE and PON1 activities in LPS group were detected 24 and 48 h post injection, respectively. No statistically significant changes in BChE and PON1 activities at different times were detected in control, C, or LPS+C groups. In conclusion, the data obtained in present study revealed a decrease in serum BChE and PON1 activities in dogs during experimentally induced endotoxemia and that choline administration attenuates these changes.     

Progesterone production by cultured luteal cells in the presence of bovine low- and high-density lipoproteins purified by heparin affinity chromatography.

The objectives of this study were to separate plasma lipoprotein particles based on the presence (low-density lipoproteins; LDL) or absence of apolipoprotein B (high-density lipoproteins; HDL) and to compare the abilities of bovine LDL and HDL to stimulate progesterone production by bovine luteal cells in culture. Plasma lipoproteins were isolated by ultracentrifugation and separated into LDL and HDL by heparin affinity chromatography. Luteal cultures were treated with LDL or HDL cholesterol for 48 h on d 3 of the culture (d 0 = day of dissociation). Progesterone production by luteal cells increased in a dose-dependent manner with increasing concentrations of either LDL or HDL cholesterol. There were no differences in the ability of LDL or HDL cholesterol to stimulate luteal cells to produce progesterone. Because LDL and HDL were equally potent, and experiment was designed to investigate the ability of modified LDL or reconstituted particles without apolipoproteins to stimulate progesterone production. Stimulation of luteal cell progesterone production by lysine-modified LDL was 70% of unmodified LDL. Progesterone production in the presence of phosphatidylcholine liposomes or BSA containing cholesterol was 50 and 108% of that obtained with HDL or LDL. Evidence indicated that apolipoprotein-free particles that contained free cholesterol but not cholesterol esters stimulated luteal cells to produce progesterone.     

Serum paraoxonase activity in dairy cows during pregnancy

Preparturient dairy cows are at high risk of metabolic and reproductive disorders and oxidative stress is considered to be involved in these events. We investigated the serum paraoxonase activity in dairy cows during pregnancy and alterations in lipid and lipoprotein patterns in this period. The relation between paraoxonase activity and HDL-cholesterol concentration was also compared. The study was carried out on 76 pregnant lactating and 26 pregnant dry Holstein dairy cows. The serum paraoxonase activity was determined by the method of hydrolysing of paraoxon, while triglyceride, cholesterol and HDL-cholesterol concentrations were measured by the enzymatic kit methods. A significantly higher serum triglyceride concentration (P<0.001) was observed in dry cows compared to lactating cows. The total cholesterol and HDL-cholesterol concentrations were significantly lower (P<0.001) in dry cows than in lactating ones. In dry cows, paraoxonase activity was significantly lower than in those lactating (P<0.001). There was no significant difference in paraoxonase/HDL-cholesterol ratio between the investigated groups. It seems that the lower HDL concentration could be one of the causes of reduced paraoxonase activity considering the role of HDL as a carrier of most paraoxonase molecules in the blood. A decreased serum paraoxonase activity could diminish the effectiveness and total capacity of the whole antioxidative system during prepartum period in dairy cattle.     

Lipid status, paraoxonase-1 activity and metabolic parameters in serum of heifers and lactating cows related to oxidative stress

The objective of this study was to investigate serum lipids, metabolic parameters and activity of the anti-oxidative enzyme paraoxonase-1 (PON1). The study was conducted on non-pregnant heifers with optimal health status and on healthy dairy cows in the period of intensive lactation, assuming that the energy and metabolic demands during lactation reduce anti-oxidative protection. Total cholesterol and HDL-cholesterol concentrations were significantly higher (P < 0.05) in lactating cows than in heifers. Bilirubin concentration and γ-glutamyltransferase (GGT) activity were also significantly higher in lactating cows (P < 0.05), indicating increased hepatic efforts of cows to meet energy requirements for lactation. Significantly lower PON1 activity and PON1/HDL ratio in lactating cows compared to heifers (P < 0.05) showed that metabolic efforts during pregnancy, parturition and lactation influence PON1 activity due to oxidative stress. Concurrent increase in total and HDL-cholesterol during lactation indicated that the HDL particle is a major carrier of cholesterol in cows.     

Semen traits and seminal plasma biochemical parameters in white leghorn layer breeders

This study was designed to determine the semen quality and seminal plasma biochemical parameters in White Leghorn layer breeders in the early phase of maturity. Individual ejaculates from 25 males were analysed for the determination of volume, sperm concentration, dead sperm percentage (DS) and sperm motility. Seminal plasma was separated and analysed for total oxidant status (TOS), total antioxidant capacity (TAC), paraoxonase (PON1), arylesterase, ceruloplasmin, triiodothyronine, thyroxine, aspartate aminotransferase (AST) and alanine aminotransferase (ALT). Our findings showed a significant (p < 0.05) positive correlation between TOS and DS in layer breeders. The values of TAC were significantly negatively correlated with TOS and DS, while positively correlated with PON1. Conversely, AST showed significant negative correlation with motility and DS. Significantly, negative correlation was also observed between ALT and sperm concentration. In conclusion, these parameters provide some valuable basal data that may help better understanding the semen quality characteristics of White Leghorn layer breeders.     

The effect of Eimeria acervulina infection on plasma lipids and lipoproteins in young broiler chicks

In young broiler chicks inoculated with 2 x 10(6) sporulated oocysts of Eimeria acervulina per bird, total plasma lipids were significantly depressed compared with controls in the first week after inoculation. The lowest level observed was at 5 days post-inoculation (d.p.i.), at which time the chick host is known to experience malabsorption in the chick host (Ruff and Wilkins, 1980). Analysis of plasma components of infected chicks at 4 and 7 d.p.i. showed that triglycerides, total cholesterol, free fatty acids, pigments and total protein were significantly decreased compared with controls. At 7 d.p.i., reduction of total cholesterol reflected mainly reduction in high density lipoprotein (HDL) cholesterol. However, the ratio of HDL cholesterol/total plasma cholesterol was not significantly different from the control ratio. Density gradient ultracentrifugation of chick plasma separated lipoproteins into three main fractions: portomicrons plus very low density lipoproteins (PM + VLDL), low density lipoproteins (LDL) and HDL. These fractions were analyzed for lipid content. Infection with E. acervulina caused (1) significant reduction in the triglyceride and cholesterol contents of the PM + VLDL fraction at 3 and 5 d.p.i., (2) significant reduction of LDL cholesterol at 9 d.p.i. and LDL phospholipid at 5-9 d.p.i., and (3) significant reduction of HDL cholesterol at 3-9 d.p.i. and HDL phospholipid at 5-9 d.p.i. Starvation of uninfected chicks for 48 h caused significant reduction in plasma triglycerides and phospholipids, but an increase in total cholesterol. Density gradient ultracentrifugation showed that the changes in these components reflected mainly reduction of the lipids in the PM + VLDL fraction. The LDL fractions, however, appeared more intense than those of the controls and contained more cholesterol and phospholipids. These results suggest that changes at 3 and 5 d.p.i. in the plasma lipoprotein pattern of chicks infected with E. acervulina most closely resemble changes seen in chicks starved for 48 h as far as PM + VLDL fraction is concerned. However, changes seen from 7 to 9 d.p.i. involve the LDL and HDL fractions and may reflect alterations in lipid and/or lipoprotein synthesis in the liver and intestine.     

香猪对氧磷酯酶基因家族的克隆及序列分析

采用RT-PCR技术,从香猪的肝脏总RNA中克隆了3个对氧磷酯酶(paraoxonase,PON)基因:PON1、PON2、PON3,其cDNA分别长1164、1212和1080 bp,各包含1个完整的开放阅读框,分别编码355、354、354个氨基酸组成的前体,其N-端的15、23、23个氨基酸为信号肽。香猪PON1基因中有1个碱基变异,不改变编码的氨基酸,为无义突变;香猪PON2基因中有2个碱基改变,导致成熟肽发生Q234R、V269I替换;香猪PON3基因中有1个碱基变化,使成熟肽出现R127H替换。经三维结构预测分析,这3个氨基酸位点的变化将影响蛋白质内部区域的微环境,影响对氧磷酯酶的功能,可能与香猪的抗逆性有一定的联系。     

Influence of Body Condition on Serum Metabolic Indicators of Lipid Mobilization and Oxidative Stress in Dairy Cows During the Transition Period

The objectives of this study were to examine the influence of body condition of cows on metabolic and antioxidative status, as well as to investigate the relationship between metabolic indicators of lipid mobilization and oxidative stress during transition period. The study was conducted on 24 Holstein‐Friesian dairy cows divided into 2 groups according to their body condition score (BCS) as optimal (n = 12; BCS from 3.25 to 3.75) or adipose (n = 12; BCS ≥4). Metabolic status (glucose, triglycerides, total cholesterol, HDL cholesterol, NEFA and BHB), paraoxonase‐1 (PON1) and apolipoprotein A‐I (ApoA‐I) were analysed in sera taken on days ?30, ?10, ?2, 0, 5, 12, 19, 26 and 60 relative to parturition. Adipose cows had significantly higher glucose concentration at parturition being significantly decreased after parturition on days 12 and 19. Total cholesterol and HDL‐C concentrations were the lowest at parturition and significantly higher on days 26 and 60 after parturition in both groups of cows. Both investigated groups had significantly higher NEFA concentration from parturition until day 19 after parturition, indicating energy deficit and an increased lipid mobilization after calving. There were no significant differences in BHB concentration during transition period in both groups. No significant differences were found in PON1 activity and ApoA‐I concentration during transition period in both groups of cows. However, in adipose cows, although not significantly different, PON1 was decreased from calving until day 19 after parturition indicating a disturbance in antioxidative status in adipose cows. PON1 significantly positively correlated with total cholesterol and HDL‐C concentrations and negatively with NEFA indicating a strong relationship of PON1 with lipid metabolism. Significant positive correlation between NEFA and BHB in both groups of cows points out on energy deficit during transition period that cows tend to overcome by lipid mobilization providing alternative source of energy needed for parturition and lactation.     

Isolation of the fifth component of the bovine complement system

Bovine C5 has been isolated from fresh bovine serum by a five-step procedure: polyethylene glycol precipitation, sequential ion-exchange chromatography on DEAE-Sephacel and CM-Sephadex, hydroxylapatite chromatography, and affinity chromatography. The purified C5 was a protein of apparent molecular weight 202,000 +/- 9,000 composed of two chains: an alpha-chain of molecular weight 127,000 +/- 5,000 and a beta-chain of molecular weight 74,000 +/- 2,000. The alpha-chain was cleaved by Sepharose-CVF.Bb (a cobra venom factor (CVF)-induced C3/C5 alternative pathway convertase) in the absence of any C3 or C3b. The monocarboxylic acid form of K-76, a sesquiterpene compound isolated from the culture filtrates of Stachybotris complementi, inhibited the alternative pathway of bovine serum, and the inhibited hemolytic activity was restored, in a dose dependent manner, by bovine C5. This provided the basis for a C5 functional assay throughout the purification procedure. The purified C5 showed species specificity and was functionally distinct from bovine C3.