Leukocyte Enzyme Research Articles

2-Arylalkyl-substituted anthracenones as inhibitors of 12-lipoxygenase enzymes. 2. Structure–activity relationships of the linker chain

A series of 2-arylalkyl-substituted anthracenones were tested as inhibitors of three types of 12-lipoxygenase isoforms in epidermal homogenate of mice, bovine platelets and porcine leukocytes. Their inhibitory activities were compared with those to inhibit the 5-lipoxygenase enzyme in bovine leukocytes. The compounds were synthesised by Marschalk, Wittig or Horner–Emmons reaction at the anthracenedione stage and then reduced to the anthracenones. Structure–activity relationship for the chain linking the anthracenone nucleus and the phenyl ring terminus was investigated. The 2-phenylethyl analogues were among the most potent inhibitors, and 3,4-dimethoxy-substituted 10f was identified as a selective inhibitor of the 12-LO enzymes over 5-LO. Selectivity for 12-LO isoforms was observed with an increase in the overall lipophilicity of the inhibitors. However, none of the linker chains of the 2-substituted anthracenones provided inhibitors that were able to discriminate between the 12-LO isoforms.

Association between myeloperoxidase levels and risk of coronary artery disease.

Myeloperoxidase (MPO), a leukocyte enzyme that promotes oxidation of lipoproteins in atheroma, has been proposed as a possible mediator of atherosclerosis. To determine the association between MPO levels and prevalence of coronary artery disease (CAD). Case-control study conducted from July to September 2000 in a US tertiary care referral center, including 158 patients with established CAD (cases) and 175 patients without angiographically significant CAD (controls). Association of MPO levels per milligram of neutrophil protein (leukocyte-MPO) and MPO levels per milliliter of blood (blood-MPO) with CAD risk. Leukocyte- and blood-MPO levels were both significantly greater in patients with CAD than in controls (P<.001). In multivariable models adjusting for traditional cardiovascular risk factors, Framingham risk score, and white blood cell counts, MPO levels were significantly associated with presence of CAD, with an OR of 11.9 (95% CI, 5.5-25.5) for the highest vs lowest quartiles of leukocyte-MPO and an OR of 20.4 (95% CI, 8.9-47.2) for the highest vs lowest quartiles of blood-MPO. Elevated levels of leukocyte- and blood-MPO are associated with the presence of CAD. These findings support a potential role for MPO as an inflammatory marker in CAD and may have implications for atherosclerosis diagnosis and risk assessment.

Degenerative Disorders of the Central Nervous System

Degenerative Disorders of the Central Nervous System

Induction of NAD(P)H oxidase by oxidized low-density lipoprotein in human endothelial cells: antioxidative potential of hydroxymethylglutaryl coenzyme A reductase inhibitor therapy.

Elevated oxidative stress and superoxide anion formation in vascular cells could promote conversion of LDL to atherogenic oxidized LDL (oxLDL), contributing to endothelial dysfunction and atherosclerosis. As a major source of vascular superoxide anion formation, an endothelial NAD(P)H oxidase, similar to the leukocyte enzyme, has been identified. To elucidate functional differences between NAD(P)H oxidases of endothelial cells and leukocytes, DNA sequences of endothelial NAD(P)H oxidase subunits were determined. Gp91phox cDNA sequence showed no difference between the 2 cell types. Endothelial p67phox cDNA sequence revealed 2 known polymorphisms, which do not affect NAD(P)H oxidase function. Next, we analyzed relative mRNA expression of NAD(P)H subunits in human umbilical vein endothelial cells (HUVECs) and leukocytes using a common cRNA standard in competitive reverse transcription-polymerase chain reaction. NAD(P)H oxidase subunits p22phox and p47phox are expressed at a similar level in both cell types, whereas p67phox (2.5%) and gp91phox (1.1%) are expressed at a much lower level in endothelial cells than in leukocytes. Differences of gp91phox expression in leukocytes and HUVECs correlate with differences in superoxide release. Gp91phox mRNA and endothelial superoxide anion formation are induced in response to oxLDL in HUVECs. Furthermore, a lower gp91phox mRNA expression was found in internal mammary artery biopsy samples of patients with coronary artery disease treated with HMG-CoA reductase inhibitors before coronary bypass surgery. We conclude that oxLDL induces proatherosclerotic NAD(P)H oxidase expression and superoxide anion formation in human endothelial cells and an antioxidative potential of HMG-CoA reductase inhibition via reduction of vascular NAD(P)H oxidase expression.

Innate immune mechanisms in the pathogenesis of systemic lupus erythematosus (SLE)

Immune imbalance in SLE increases the susceptibility to infectious diseases. The aim of this study was to analyze several mechanisms related to non-specific immunity in this autoimmune disorder. We studied in vivo CD11b expression, phagocytosis, and chemotaxis in polymorphonuclear cells (PMN) from SLE patients. All tests were also performed under hrIL-8 stimulating conditions and analyzed by flow cytometry. Intracellular leucocyte (monocytes and PMN) enzyme activity was evaluated using specific substrates for cathepsin B and D, collagenase, and oxidative burst by flow cytoenzymology. An exaggerated in vivo CD11b expression was observed on PMN from SLE patients without noticeably in vitro effect upon hrIL-8. Similarly both, phagocytosis and chemotaxis were diminished and showed no response to hrIL-8 stimulation. The opposite was found in PMN from controls. Intracellular enzyme activity was comparable between groups as far as cathepsin B and D are concerned. A tendency of decreased oxidative-burst induction was noted in monocytes and PMN from SLE patients, whereas collagenase activity was found clearly increased in both leucocyte subpopulations. Our results may represent a deficient ability of the innate immune mechanisms for the clearance of infectious agents, immune complexes, satisfactory resolution of inflammatory processes and tissue repair in SLE.

2-Arylalkyl-substituted anthracenones as inhibitors of 12-lipoxygenase enzymes. 1. Structure–activity relationships of the terminal aryl ring

A series of 2-arylmethyl-substituted anthracenones were synthesized and tested as inhibitors of three types of 12-lipoxygenase isoforms in epidermal homogenate of mice, bovine platelets and porcine leucocytes. Their inhibitory activities were compared with those to inhibit the 5-lipoxygenase enzyme in bovine leucocytes. Structure–activity relationships are described with particular emphasis on modifications of the terminal aryl nucleus. The ability of the compounds to selectively inhibit the 12-lipoxygenase enzymes was dependent on a high overall lipophilicity of the inhibitor, whereas compounds with decreased lipophilicity were also inhibitors of the 5-LO enzyme. Among the more lipophilic inhibitors, the unsubstituted 2-phenylmethyl analogue 6a as well as the carboxylic acid ester 6q appeared to be selective inhibitors of platelet-type 12-LO isoform.

Neutrophil antiserum response to decrease in proteolytic activity in loaded rat muscle.

The leukocytes that are found in skeletal muscles after intense muscular activity have been shown to infiltrate areas of injury in skeletal muscle tissue. We believe that leukocyte enzymes, which appear in the tissue after the degranulation or destruction of leukocytes, could play a role in tissue enzyme activities. Neutrophil proteinases were investigated. Histological data provided evidence of rat muscular tissue infiltration by leukocytes after intense physical loading. To reduce the influx of leukocytes in rat muscles, a rabbit antiserum against rat peritoneal leukocytes was injected into rats after muscle loading. This resulted in a reduction in muscle cytosol proteolytic activity as compared to control animals (who received saline injections). The levels of proteolytic activities of media conditioned by the soleus muscle isolated from antiserum-treated rats were also reduced. These data provide evidence that the increase in proteolytic activity observed in rat skeletal muscles after physical loading is partially induced by neutrophil proteinases.

The role of the antioxidative defense system in papulopustular acne.

Acne vulgaris frequently occurs in the second decade of life. The pathogenesis of the disease is multifactorial. In this study, we aimed to investigate the role of reactive oxygen species in inflammation of acne by determining the activity of antioxidant defense enzymes in leukocytes. Fifty-two patients with papulopustular type acne vulgaris and 36 healthy controls were enrolled. The severity of the disease was examined by the Global Acne Grading System, and the activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and catalase (CAT) enzymes as well as the level of thiobarbituric acid reactive substance (TBARS) were detected in leukocytes. The activities of SOD and GSH-Px were significantly decreased in the acne group. CAT activity and TBARS level were higher in patients than controls. Only a poor correlation was detected between GSH-Px activity and severity of the disease. Antioxidative defense enzymes are impaired in papulopustular acne, and drugs with antioxidative effects might be valuable in treatment.

Capsaicin protects against ethanol-induced oxidative injury in the gastric mucosa of rats

In this study, we investigated the protective effects of capsaicin on gastric mucosal oxidative damage induced by ethanol. Sprague Dawley rats intragastrically received 0.5–10mg/kg, BW capsaicin or vehicle; 30 min later gastric lesions were induced by intragastric administration of absolute ethanol. Lipid peroxidation was estimated by measuring thiobarbituric acid reactive substances in gastric mucosa. Myeloperoxidase activity, a marker enzyme of polymorphonuclear leukocytes for tissue inflammation, was also measured in the gastric mucosa. The expression level of cyclooxygenase-2, which increases in inflammatory region, was determined by Western blot analysis. Capsaicin significantly suppressed gastric haemorrhagic erosions induced by ethanol. Capsaicin inhibited lipid peroxidation and myeloperoxidase activity in ethanol-induced gastric mucosal lesion in a dose-dependent manner. Capsaicin also inhibited the expression of cyclooxygenase-2 in the gastric mucosal lesion. The gastroprotective activity of capsaicin on the ethanol-induced oxidative damage may be important for chemoprevention.

Innate immune response mechanisms in non-insulin dependent diabetes mellitus patients assessed by flow cytoenzymology

It is well known that infections in patients with diabetes mellitus are more severe, although there is controversy for increased susceptibility to them. Non-specific immune response mechanisms could be related to defense and/or susceptibility to pathogens. The aim of this study was to investigate the activity of several enzymes involved in the primary host defense mechanisms in non-insulin dependent diabetes mellitus (NIDDM). Twenty NIDDM females with a mean HbA 1c level of 8.19% were included. No patient had clinical evidence of infection. As controls 20 healthy females were studied. The enzymes tested were dipeptidyl-peptidase I (DPP-I), cathepsin B and D, NADPH oxidase and superoxide dismutase (oxidative burst) and collagenase. Isolated leukocytes were incubated with the specific substrates in pyrogen free conditions. The intracellular enzyme activity was analyzed by flow cytometry. Collagenase enzymatic activity was similar in the three leukocyte subpopulations studied. Oxidative burst induction in monocytes was comparable between both groups. Enzyme activity of cathepsin B and D in all cell subsets, oxidative burst in PMN cells, and DPP-I in lymphocytes and monocytes from patients, was higher than those from healthy females ( P<0.05). Overall, our findings demonstrate an enhanced functional status of several intracellular leukocyte enzymes in NIDDM. Furthermore, the increased oxidative burst induction and the consequent production of free radicals, may contribute to vascular complications. Other mechanisms — either from the non-specific or specific immune response — deserve investigation to establish if diabetic patients are more susceptible to infectious diseases.

Effect of collection, transport, processing and storage of blood specimens on the activity of lysosomal enzymes in plasma and leukocytes.

This study was designed to evaluate the effect of different conditions of collection, transport and storage on the quality of blood samples from normal individuals in terms of the activity of the enzymes ss-glucuronidase, total hexosaminidase, hexosaminidase A, arylsulfatase A and ss-galactosidase. The enzyme activities were not affected by the different materials used for collection (plastic syringes or vacuum glass tubes). In the evaluation of different heparin concentrations (10% heparin, 5% heparin, and heparinized syringe) in the syringes, it was observed that higher doses resulted in an increase of at least 1-fold in the activities of ss-galactosidase, total hexosaminidase and hexosaminidase A in leukocytes, and ss-glucuronidase in plasma. When the effects of time and means of transportation were studied, samples that had been kept at room temperature showed higher deterioration with time (72 and 96 h) before processing, and in this case it was impossible to isolate leukocytes from most samples. Comparison of heparin and acid citrate-dextrose (ACD) as anticoagulants revealed that ss-glucuronidase and hexosaminidase activities in plasma reached levels near the lower normal limits when ACD was used. In conclusion, we observed that heparin should be used as the preferable anticoagulant when measuring these lysosomal enzyme activities, and we recommend that, when transport time is more than 24 h, samples should be shipped by air in a styrofoam box containing wet ice.

A gp91phox containing NADPH oxidase selectively expressed in endothelial cells is a major source of oxygen radical generation in the arterial wall.

Reactive oxygen species (ROS) play an important role in regulating vascular tone and intracellular signaling; the enzymes producing ROS in the vascular wall are, however, poorly characterized. We investigated whether a functionally active NADPH oxidase similar to the leukocyte enzyme, ie, containing the subunits p22phox and gp91phox, is expressed in endothelial cells (ECs) and smooth muscle cells (SMCs). Phorbol 12-myristate 13-acetate (PMA), a stimulus for leukocyte NADPH oxidase, increased ROS generation in cultured ECs and endothelium-intact rat aortic segments, but not in SMCs or endothelium-denuded arteries. NADPH enhanced chemiluminescence in all preparations. p22phox mRNA and protein was detected in ECs and SMCs, whereas the expression of gp91phox was confined to ECs. Endothelial gp91phox was identical to the leukocyte form as determined by sequence analysis. In contrast, mitogenic oxidase-1 (mox1) was expressed in SMCs, but not in ECs. To determine the functional relevance of gp91phox expression, experiments were performed in aortic segments from wild-type, gp91phox(-/-), and endothelial NO synthase (eNOS)(-/-) mice. PMA-induced ROS generation was comparable in aortae from wild-type and eNOS(-/-) mice, but was attenuated in segments from gp91phox(-/-) mice. Endothelium-dependent relaxation was greater in aortae from gp91phox(-/-) than from wild-type mice. The ROS scavenger tiron increased endothelium-dependent relaxation in segments from wild-type, but not from gp91phox(-/-) mice. These data demonstrate that ECs, in contrast to SMCs, express a gp91phox-containing leukocyte-type NADPH oxidase. This enzyme is a major source for arterial ROS generation and affects the bioavailability of endothelium-derived NO.

Mucopolysaccharidosis type I: clinical and biochemical study

Of 1240 outpatients referred to the Human Genetics Clinic between 1997 and 1998, 248 (20%) had inborn errors of metabolism, 36 (14%) of which were diagnosed as mucopolysaccharidoses. Parental consanguinity was present in 82% of these patients. Deficiency of alpha-L-iduronidase (IDUA) enzyme in leukocytes and increased urinary mucopolysaccharides excretion were detected in 17 patients. The urinary spot test for glucosaminoglycans was inconclusive in 4 of the 17 cases. Results showed a correlation between the biochemical enzyme activity in leukocytes, the amount of excreted mucopolysaccharides and the subtype and course of mucopolysaccharidosis type I. We conclude that estimation of IDUA enzyme activity in leukocytes can differentiate between clinically overlapping cases of MPS I and MPS II and given the clinical manifestations of MPS I is a definitive and unequivocal method of diagnosis while the urinary spot test is inconclusive.

Characterization of β-galactosidase in leukocytes and fibroblasts of GM1 gangliosidosis heterozygotes compared to normal subjects

Objectives: Characterization of β-galactosidase in leukocytes and fibroblasts of heterozygotes for GM1 type I. Design and methods: Leukocyte and fibroblast β-galactosidase activity was determined fluorimetrically using 4-methylumbelliferyl-β-D-galactoside as an artificial substrate. Optimum pH, Km, Vmax and thermostability of the enzyme at 42 °C were determined. Results: The leukocyte and fibroblast enzyme of heterozygotes have an optimum pH of 4.0 and 4.2, respectively. In normal subjects, the optimum pH was 4.2 in both cells, according to previous studies. The Km of the enzyme of heterozygotes was determined to be 0.65 mM in leukocytes and 0.59 mM in fibroblasts. The Vmax was determined in 167.21 nmol/h/mg of protein in heterozygotes leukocytes and 541.2 nmol/h/mg of protein in heterozygotes fibroblasts compared to 291.7 and 1768.1 nmol/h/mg of protein in controls leukocytes and fibroblasts, respectivelly. When leukocyte and fibroblast heterozygote β-galactosidase was preincubated at 42 °C, after 80 min the residual activity was determined to be 25 to 30% of the initial activity. These results are similar to the control group. Conclusions: We have found significant differences between the two groups in some investigated parameters. Both fibroblasts and leukocytes showed a virtually similar level of reliability as source of enzyme for the detection of heterozygotes.

Deacylation of Lipopolysaccharide in Whole Escherichia coli during Destruction by Cellular and Extracellular Components of a Rabbit Peritoneal Inflammatory Exudate

Deacylation of purified lipopolysaccharides (LPS) markedly reduces its toxicity toward mammals. However, the biological significance of LPS deacylation during infection of the mammalian host is uncertain, particularly because the ability of acyloxyacyl hydrolase, the leukocyte enzyme that deacylates purified LPS, to attack LPS residing in the bacterial cell envelope has not been established. We recently showed that the cellular and extracellular components of a rabbit sterile inflammatory exudate are capable of extensive and selective removal of secondary acyl chains from purified LPS. We now report that LPS as a constituent of the bacterial envelope is also subject to deacylation in the same inflammatory setting. Using Escherichia coli LCD25, a strain that exclusively incorporates radiolabeled acetate into fatty acids, we quantitated LPS deacylation as the loss of radiolabeled secondary (laurate and myristate) and primary fatty acids (3-hydroxymyristate) from the LPS backbone. Isolated mononuclear cells and neutrophils removed 50% and 20-30%, respectively, of the secondary acyl chains of the LPS of ingested whole bacteria. When bacteria were killed extracellularly during incubation with ascitic fluid, no LPS deacylation occurred. In this setting, the addition of neutrophils had no effect, but addition of mononuclear cells resulted in removal of >40% of the secondary acyl chains by 20 h. Deacylation of LPS was always restricted to the secondary acyl chains. Thus, in an inflammatory exudate, primarily in mononuclear phagocytes, the LPS in whole bacteria undergoes substantial and selective acyloxyacyl hydrolase-like deacylation, both after phagocytosis of intact bacteria and after uptake of LPS shed from extracellularly killed bacteria. This study demonstrates for the first time that the destruction of Gram-negative bacteria by a mammalian host is not restricted to degradation of phospholipids, protein, and RNA, but also includes extensive deacylation of the envelope LPS.

Increased intracellular calcium and decreased activities of leucocyte Na+,K+-ATPase and Ca2+-ATPase in asthma

The present study was carried out to determine the intracellular free calcium concentration ([Ca(2+)](i)) and the activity of its regulatory enzymes (Na(+),K(+)-ATPase and Ca(2+)-ATPase) in leucocytes. Levels of plasma lysophosphatidylcholine (LPC) were also measured. Then the relationship between these parameters and the clinical severity of asthma and bronchial reactivity was studied. Patients with asthma were divided into three groups: acute asthma (subjects in acute exacerbation), uncontrolled asthma (subjects currently symptomatic) and stable asthma (subjects currently asymptomatic). A group of normal subjects was also studied. Spirometry, specific airway conductance and bronchial reactivity measurements were carried out. The following biochemical parameters were studied in venous blood: leucocyte [Ca(2+)](i), Na(+), K(+)-ATPase and Ca(2+)-ATPase activities, and plasma LPC. Leucocyte [Ca(2+)](i) was increased and the activities of Na(+),K(+)-ATPase and Ca(2+)-ATPase were decreased in patients with asthma. Plasma levels of LPC were also increased. These changes were observed to be greatest among asthmatics in acute exacerbation of asthma, and lesser in magnitude in patients with less severe asthma. The activities of both ATPases were found to have a significant positive correlation, and [Ca(2+)](i) and the levels of plasma LPC a significant negative correlation, with predicted forced expiratory volume in 1 s (FEV(1)). No significant correlation was observed between the biochemical parameters and bronchial reactivity. It is concluded that intracellular calcium homoeostasis is abnormal in asthma; specifically, the activities of Na(+),K(+)-ATPase and Ca(2+)-ATPase are decreased. These abnormalities may modulate the clinical severity of asthma.

Changes in Superoxide Dismutase Activities and Concentrations and Myeloperoxidase Activities in Leukocytes from Patients with Diabetes Mellitus

To investigate whether the two free-radical scavengers, Cu, Zn- and Mn-superoxide dismutase (SOD), are changed in leukocytes of diabetic patients, and the alteration of these enzymes correlates with the diabetic state, we measured the activity and concentration of these enzymes in leukocytes from diabetic patients. Both Cu, Zn-SOD and Mn-SOD activities in neutrophils and lymphocytes were significantly lower in patients with non–insulin-dependent diabetes mellitus than in healthy controls. The concentrations of these enzymes in leukocytes from diabetic patients, however, did not differ from those in controls. Cu, Zn-SOD and Mn-SOD activities in neutrophils inversely correlated with HbA 1c concentrations. Myeloperoxidase activity in leukocytes was significantly reduced in NIDDM patients. These findings suggest that changes in these enzymes may affect the susceptibility to infection and immunocompetence of patients with diabetes.

Diagnostic value of redox leukocyte enzymes in postmenopausal osteopenia

Activities of redox enzymes of peripheral blood leukocytes is studied in patients with postmenopausal osteopenia. Neutrophil myeloperoxidase (MP), alkaline phosphatase (AP) and lymphocyte succinate dehydrogenase (SDH), and succinate.cytochrome C oxyreductase (SCOR) activities were measured by the cytochemical method. Densitometric examinations of 49 postmenopausal women without risk of secondary osteoporosis revealed osteoporosis in 15 (31%)) and osteopenia in 24 (49%)); in 10 (20%o) mineral compactness of bones was normal. AP and SDH activities were increased and SCOR and MP activities normal in patients with osteopenia. Changes in the activities of AL and SDH in subjects with different duration of the postmenopausal period indicate that the main factor affecting the activities of these enzymes was osteopenia but not the duration of postmenopause or age. Significant correlations between enzyme values and mineral compactness of the spine were the most numerous during the first three years of postmenopause, and therefore changes in leukocyte enzyme activities are apparently not caused by inflammations associated with chronic macroand microfractures of the vertebrae but by osteopenia. The detected specific correlation between enzyme activities and localization of the pathological process in certain segments of the skeleton permits us to propose that leukocyte AL and SDH values may serve as criteria for optimal choice of skeletal area for subsequent densitometry or x-ray examination.

Biochemical studies on leukocyte and fibroblast human β-galactosidase

Objectives: Some biochemical characteristics of the human leukocyte and fibroblast β-galactosidase were studied. Design and Methods: Leukocyte and fibroblast enzyme activity was determined fluorometricaly using 4-methylumbelliferyl-β-D-galactoside as artificial substrate. Optimum pH, K m, V max and thermostability of the enzyme at 42 °C were determined. Results: The leukocyte and fibroblast enzyme has an optimum pH at 4.2, which is in agreement with the lysosomal origin of the enzyme. The K m of the enzyme was 0.62 in leukocytes and 0.67 in fibroblasts, and V max was 289.9 nmol/h/mg of protein and 1779.2 nmol/h/mg of protein in the two tissues, respectively. When fibroblast or leukocyte β-galactosidase was pre-incubated at 42 °C, it did not retain its activity because the residual activity after 80 minutes of pre-incubation at this temperature was lower than 30% of the initial activity both in leukocytes and fibroblasts. Conclusions: This was the first study of K m, V max and thermostability of β-galactosidase performed on leukocytes and provided data for a better characterization of the enzyme β-galactosidase, allowing the improvement of the analytical conditions.

Acyloxyacyl hydrolase activity of neutrophil leukocytes in normal early postpartum dairy cows and in cows with retained placenta

Acyloxyacyl hydrolase (AOAH) is an enzyme of bovine polymorphonuclear neutrophil leukocytes (PMN) that is capable of detoxifying endotoxin (25). The activity of AOAH in PMN isolated from the blood was investigated in dairy cows that expelled the fetal membranes normally (Group NFM) and in cows with retained fetal membranes (Group RFM) to obtain better insight into the role of the AOAH enzyme of neutrophils in endotoxin-related diseases, which occur frequently in dairy cows during the early postpartum period, especially in RFM cows. Twenty early postpartum dairy cows were used in the study: 13 NFM cows and 7 RFM cows. In the RFM cows, the percentage of PMN in blood (29 ± 4%) was significantly (P < 0.05) lower than in NFM cows (43 ± 4%). The average AOAH activity in RFM cows (mean ± SEM = 89 ± 13 pmol fatty acid/ 10 7 PMN / h) was lower than in NFM cows (107 ± 6 pmol fatty acid / 10 7 PMN / h), but the difference in neutrophil AOAH activity between the 2 groups was not significant. There was also a higher percentage of immature neutrophils in isolated leukocyte suspensions from RFM cows (22 ± 8%) than from NFM cows (15 ± 4%), so that impairment of AOAH activity in early postpartum cows could be explained, in part, by immaturity of the neutrophils. These results suggest that the decreased AOAH activity of PMN could play a role in the pathogenesis of endotoxin-related diseases in dairy cows during the early postpartum period.