Serum Antibodies to N-Glycolylneuraminic Acid Are Elevated in Duchenne Muscular Dystrophy and Correlate with Increased Disease Pathology in Cmah-/-mdx Mice.

Humans cannot synthesize the common mammalian sialic acid N-glycolylneuraminic acid (Neu5Gc) because of an inactivating deletion in the cytidine-5'-monophospho-(CMP)-N-acetylneuraminic acid hydroxylase (CMAH) gene responsible for its synthesis. Human Neu5Gc deficiency can lead to development of anti-Neu5Gc serum antibodies, the levels of which can be affected by Neu5Gc-containing diets and by disease. Metabolic incorporation of dietary Neu5Gc into human tissues in the face of circulating antibodies against Neu5Gc-bearing glycans is thought to exacerbate inflammation-driven diseases like cancer and atherosclerosis. Probing of sera with sialoglycan arrays indicated that patients with Duchenne muscular dystrophy (DMD) had a threefold increase in overall anti-Neu5Gc antibody titer compared with age-matched controls. These antibodies recognized a broad spectrum of Neu5Gc-containing glycans. Human-like inactivation of the Cmah gene in mice is known to modulate severity in a variety of mouse models of human disease, including the X chromosome-linked muscular dystrophy (mdx) model for DMD. Cmah-/-mdx mice can be induced to develop anti-Neu5Gc-glycan antibodies as humans do. The presence of anti-Neu5Gc antibodies, in concert with induced Neu5Gc expression, correlated with increased severity of disease pathology in Cmah-/-mdx mice, including increased muscle fibrosis, expression of inflammatory markers in the heart, and decreased survival. These studies suggest that patients with DMD who harbor anti-Neu5Gc serum antibodies might exacerbate disease severity when they ingest Neu5Gc-rich foods, like red meats.

Association between Neu5Gc carbohydrate and serum antibodies against it provides the molecular link to cancer: French NutriNet-Santé study.

BACKGROUND: High consumption of red and processed meat is commonly associated with increased cancer risk, particularly colorectal cancer. Antibodies against the red meat-derived carbohydrate N-glycolylneuraminic acid (Neu5Gc) exacerbate cancer in "human-like" mice. Human anti-Neu5Gc IgG and red meat are both independently proposed to increase cancer risk, yet how diet affects these antibodies is largely unknown. METHODS: We used world global data to demonstrate that colorectal cancer incidence and mortality are associated with increased national meat consumption. In a well-defined large cohort, we used glycomics to measure daily Neu5Gc intake from red meat and dairy, and investigated serum as well as affinity-purified anti-Neu5Gc antibodies. Based on 24-h dietary records, daily Neu5Gc intake was calculated for 19,621 subjects aged ≥ 18 years of the NutriNet-Santé study. Serum and affinity-purified anti-Neu5Gc antibodies were evaluated by ELISA and glycan microarrays in representative 120 individuals, each with at least eighteen 24-h dietary records (aged 45-60, Q1-Q4; aged > 60, Q1 and Q4; 10 men/women per quartile). RESULTS: We found that high-Neu5Gc diet, gender, and age affect the specificity, levels, and repertoires of anti-Neu5Gc IgG immune responses, but not their affinity. Men consumed more Neu5Gc than women, mostly from red meat (p = 0.0015), and exhibited higher overall serum anti-Neu5Gc IgG levels by ELISA (3.94 ng/µl versus 2.22 ng/µl, respectively; p = 0.039). Detailed glycan microarray analysis against 56 different glycans revealed high Neu5Gc-specificity with increased anti-Neu5Gc IgG and altered repertoires, associated with higher consumption of Neu5Gc from red meat and cow dairy. Affinity purification of serum anti-Neu5Gc antibodies revealed increased levels and biased array repertoire patterns, without an increase in antibody affinity, in individuals consuming higher Neu5Gc levels. Furthermore, in a high-meat diet, antibody diversity patterns on glycan microarrays shifted towards Neu5Gcα3-linked glycans, increasing the α3/α6-glycans ratio score. CONCLUSIONS: We found a clear link between the levels and repertoire of serum anti-Neu5Gc IgG and Neu5Gc intake from red meat and dairy. These precise rational methodologies allowed to develop a Gcemic index to simplify the assessment of Neu5Gc in foods that could potentially be adapted for dietary recommendations to reduce cancer risk.

Are there specific antibodies against Neu5Gc epitopes in the blood of healthy individuals?

Strong discrepancies in published data on the levels and epitope specificities of antibodies against the xenogenic N-glycolyl forms of sialoglycans (Hanganutziu-Deicher Neu5Gcɑ2-3Galß1-4Glc and related antigens) in healthy donors prompted us to carry out a systematic study in this area using the printed glycan array and other methods. This article summarizes and discusses our published and previously unpublished data, as well as publicly available data from the Consortium for Functional Glycomics. As a result, we conclude that (1) the level of antibodies referred to as anti-Neu5Gc in healthy individuals is low; (2) there are antibodies that seem to interact with Neu5Gc-containing epitopes, but in fact they recognize internal fragments of Neu5Gc-containing glycans (without sialic acids), which served as antigens in the assays used and; (3) a population capable of interacting specifically with Neu5Gc (it does not bind the corresponding NAc analogs) does exist, but it binds the monosaccharide Neu5Gc better than the entire glycans containing it. In other words, in healthy donors, there are populations of antibodies capable of binding the Neu5Gc monosaccharide or the inner core -Galß1-4Glc, but very few true anti-Neu5Gcɑ2-3Galß1-4Glc antibodies, i.e., antibodies capable of specifically recognizing the entire trisaccharide.

Detection of N-glycolylneuraminic acid biomarkers in sera from patients with ovarian cancer using an engineered N-glycolylneuraminic acid-specific lectin SubB2M.

N-glycolylneuraminic acid (Neu5Gc)-containing glycans are a prominent form of aberrant glycosylation found in human tumor cells and have been proposed as cancer biomarkers. The B subunit of the subtilase cytotoxin (SubB) produced by Shiga toxigenic Escherichia coli recognises Neu5Gc containing glycans. We have previously engineered this lectin, SubB2M, for greater specificity and enhanced recognition of Neu5Gc-containing glycans. Here we further explore the utility of SubB2M to detect Neu5Gc tumor biomarkers in sera from patients with ovarian cancer. Using surface plasmon resonance (SPR) we show that SubB2M can detect the established ovarian cancer biomarker, CA125, in a highly sensitive and specific fashion in the context of human serum. These studies established conditions for screening serum samples from patients with ovarian cancer for Neu5Gc glycans. We found that serum from patients with all stages of ovarian cancer had significantly elevated mean levels of Neu5Gc glycans compared to normal controls. Serum from patients with late stage disease (stages IIIC, IV) had uniformly elevated levels of Neu5Gc glycans. Detection of Neu5Gc-glycans using SubB2M has the potential to be used as a diagnostic ovarian cancer biomarker, as well as a tool for monitoring treatment and disease progression in late stage disease.

Quantitative determination and confirmatory analysis of N-acetylneuraminic and N-glycolylneuraminic acids in serum and urine by solid-phase extraction on-line coupled to liquid chromatography-tandem mass spectrometry.

N-acetylneuraminic acid (Neu5Ac) and N-acetylglycolylneuraminic acid (Neu5Gc), two acylated derivatives of 9-C carboxylated monosaccharides, are involved in a number of biological processes as modulators of glycoconjugates. A partially automated method is here presented for determination of these sialic acids in the two most important biofluids for clinical analysis: serum and urine. For this purpose, a solid-phase extraction (SPE) workstation was on-line connected to an LC-MS/MS triple quadrupole mass detector. Hydrolysis to release sialic acids bound to glycoconjugates and derivatization were the two steps implemented as sample preparation prior to SPE-LC-MS/MS analysis. Following thorough optimization of the SPE and LC-MS/MS conditions, the analytical method was validated using the standard addition approach to assess the presence of matrix effects. The proposed method affords detection limits of 0.03ng/mL and 0.04ng/mL for Neu5Ac and Neu5Gc, respectively. The precision (expressed as relative standard deviation) was 1.7 and 4.6% for within-day variability, and 4.8 and 7.2% for between-days variability. Accuracy, estimated using spiked (between 1 and 50ng/mL) and non-spiked samples of both biofluids, ranged from 95.2 to 99.6%. The method was applied to human serum and urine of healthy volunteers, thus showing its suitability for application in both clinical and research laboratories.

Are there two functionally distinguished Neu5Gc pools with respect to rouleau formation on the bovine red blood cell?

Bovine red blood cells (RBCs) do not exhibit any aggregation tendency in autologous plasma and, therefore, all bovine rouleaux obtained in vitro are regarded as artificial. The present study reports the bovine RBC rouleau formation by either bovine or human fibrinogen and Ca2+ ions. The phenomenon was induced through two-step cell incubation: in 0.9% NaCl and 1% bovine albumin at 37 degrees C for 30 min followed by 20 hrs incubation at 30 degrees C in the fresh solution supplemented with 10 mM glucose. Its mechanism is unknown. During the incubation the number of N-glycolylneuraminic acid molecules per cell decreased from 48.1 to 44.9 amoles, which accounted for 7%. The treatment of RBCs with V. cholerae sialidase under the same conditions resulted in a 94% drop in the Neu5Gc quantity and did not induce the rouleau formation in the same fibrinogen preparation. The preliminary results rise a question whether the bulk of sialic acid is required in the aggregation of bovine erythrocytes under static conditions. Only a minor pool of Neu5Gc seems to be responsible for suppression of the phenomenon.

Determination of sialic acids in the serum of cancer patients by capillary electrophoresis.

A novel method for the determination of N-acetylneuraminic acid (NANA) and N-glycolylneuraminic acid (NGNA) was developed by using high-performance capillary electrophoresis (HPCE) with UV detection at 195 nm. NANA and NGNA were separated directly and analyzed without pre- or postcolumn derivation. The detection limit of NANA is 9.6 x 10(-6) mol L(-1) and for mass 3.879 x 10(-14) mol (39 fmol). This method was applied for the determination of NANA in 30 normal human and 72 cancer patients. The results demonstrated that NANA in the sera of cancer patients increased significantly as compared with the normal human (P < 0.001). The new method is simple and sensitive, and is suitable for basic research and clinical application to malignant tumors.

Importance of high-performance liquid chromatographic analysis of serum N-acylneuraminic acids in evaluating surgical treatment in patients with early endometrial cancer.

The objectives of this study were the quantification of the two major sialic acid (Sia) forms - N-acetylneuraminic (Neu5Ac) and N-glycolylneuraminic acids (Neu5Gc) - in serum before and after surgical treatment of early endometrial cancer and the relation of their levels with the progress of surgical therapy. The major Sia forms were liberated from sera glycoconjugates by mild acid hydrolysis, separated as per-O-benzoylated derivatives by a highly sensitive reversed-phase HPLC method and detected at 231 nm. Total Sia content in sera of healthy women was not related to age and body weight. Neu5Ac was identified as the major Sia in sera from both cancer patients, healthy individuals as well as in tissue specimens (> or = 94% of total Sia). In patients with endometrial cancer the total Sia level before surgical treatment (709.5 +/- 306.5 mg/l) was significantly higher (p < or = 0.0001) than that of the control group (213.5 +/- 88.7 mg/l). The elevation in Sia level was exclusively due to Neu5Ac. Following surgical therapy, serum Neu5Ac levels (699.4 +/- 305.6 mg/l) were significantly decreased (305.9 +/- 114.5 mg/l). In one case, where Neu5Ac level was increased 15 days and eight months after surgery (1.8 and 2.5 times as compared to control, respectively), a metastasis not detected during surgery was recorded. The obtained results suggest that Neu5Ac level in serum may be used as a tumor marker in evaluating the suitability of surgical treatment in early endometrial cancer.

Analysis of N-acetyl and N-glycolylneuraminic acid in rat serum and tissues with Walker 256 carcinoma by high-performance liquid chromatography.

Serum and tissue specimens from healthy Wistar rats and from rats with Walker 256 carcinoma were analysed for N-acetyl and N-glycolylneuraminic acid by high performance liquid chromatography (HPLC) as per-O-benzoylated derivatives. Both neuraminic acids were identified, while N-acetylneuraminic acid was the predominant sialic acid. Samples from rats with generalized metastasis showed a significant increase (45-80%) of total sialic acids. This phenomenon in serum is caused by the overproduction of sialic acids, as a result of synthesis of both types of neuraminic acids to a similar molar ratio. The increase of sialic acids in rat bones with metastatic cancer is mainly because of increased N-acetylneuraminic acid synthesis. These results suggest that the molecular mechanisms responsible for cancer metastasis in different tissues may be closely associated with increased synthesis of dominating neuraminic acid.

Screening of N-acylneuraminic acids in serum and tissue specimens of mouse C57BI with Lewis' lung cancer by high-performance liquid chromatography.

Serum and tissue specimens from healthy C57BI mice and from mice with Lewis' lung cancer after metastasis were analyzed for N-acetyl- and N-glycolylneuraminic acid by high-performance liquid chromatography. Both neuraminic acids were present, while N-glycolylneuraminic acid was the predominant sialic acid in all tissues. Samples from mice with metastatic cancer showed a significant increase (67-200%) of total sialic acids mainly as a result of increased N-glycolylneuraminic acid synthesis. These results suggest that cancer metastasis in various tissues is closely associated with increased synthesis of the predominant neuraminic acid and may help to understand the underlying mechanisms of tumor development.

Implication of altered levels of plasma alpha(1)-acid glycoprotein and its derived sialic acid on plasma protein binding of trimethoprim in pigs in physiological and pathological states.

In growing pigs (0 to 158 days after birth), pregnant sows (0 to 90 days), and in pigs suffering from respiratory disease (Actinobacillus pleuropneumoniae), the plasma levels of alpha(1)-acid glycoprotein (AGP) and its derived sialic acid were determined, as was the meaning of their altered levels on the plasma protein binding of trimethoprim. The AGP level was very high immediately after birth (more than 10,000 mu g/ml), decreased markedly during 2 weeks after birth (about 700 mu g/ml) and thereafter stayed at a constant level (about 400 mu g/ml). Pregnant sows had a low AGP level with a narrow variation throughout pregnancy (about 190 to 260 mu g/ml). Pigs infected with A. pleuropneumoniae showed an increased AGP level (mean value; 732 mu g/ml) with a wide variation (range: 170-1,840 mu g/ml). N-acetylneuraminic acid (NANA) and N-glycolylneuraminic acid (NGNA) were sialic acid subtypes detected in porcine plasma. In growing pigs, the time course of changes in NANA concentrations was consistent with that of AGP, whereas that of NGNA was different, implying that NANA is a sialic acid subtype derived from porcine AGP, in contrast to NGNA. The relationship between AGP and NANA levels in growing pigs could be expressed by the following equation: NANA=0.14 x AGP + 159 mu g/ml, whereas that in pigs with respiratory disease could be expressed by NANA=0.O67 x AGP + 357 mu g/ml, indicating a low fraction of NANA in AGP in diseased pigs. The regression lines between the AGP level and the plasma protein binding of trimethoprim < or = 2,000 mu g of AGP/ml were similar as follows: binding(%)=0.O23 x GP + 34 in growing pigs and binding(%)=0.O22 x GP+29 in diseased pigs, implying a minor role of sialic acid residues in the binding of basic drugs to AGP. In conclusion, the wide change in plasma AGP levels in diseased pigs as well as during the initial growth phase can alter the plasma protein binding of basic drugs such as trimethoprim, probably leading to a change in drug disposition. The low sialylation of AGP in diseased pigs may not have a great influence on the binding of basic drugs to AGP, implying the quantitative importance of AGP.

Analysis of the sialic acid-binding activity of the transmissible gastroenteritis virus.

Porcine transmissible gastroenteritis virus (TGEV) has been shown to agglutinate erythrocytes using alpha 2,3-linked sialic acid on the cell surface as binding site. The hemagglutinating activity requires the pretreatment of virus with neuraminidase. We obtained evidence that TGEV recognizes not only N-acetylneuraminic acid but also N-glycoloylneuraminic acid, a sialic acid present on many porcine cells.

A hydrolysis method using microwaves: determination of N-acetyl- and N-glycolylneuraminic acids in biological systems by fluorometric high-performance liquid chromatography.

A simple, rapid, and extremely sensitive method for determining N-acetyl- and N-glycolylneuraminic acids in serum and in submandibular, sublingual, and parotid glands using high-performance liquid chromatography with a fluorometric detector is described. The neuraminic acids contained in the samples are released in the presence of 2 M CH3COOH by means of microwave hydrolysis (only 10 min required) and are subsequently converted using 1,2-diamino-4,5-methylenedioxybenzene, a fluorogenic reagent for alpha-ketoacids, into highly fluorescent derivatives. In order to optimize the release of sialic acids and to minimize the effect of destruction, the following analytical variables were investigated: temperature, time, and concentration of the acid. Within 12 min after derivatization, the compounds were separated on a reversed-phase column by means of isocratic elution using a mobile phase of water (pH 3 with H3PO4)-methanol-acetonitrile (86:6:8, v/v). Fluorescence detection was performed at an excitation wavelength of 373 nm and an emission wavelength of 448 nm.

Comparison of the N-glycoloylneuraminic and N-acetylneuraminic acid content of platelets and their precursors using high performance anion exchange chromatography.

N-Acetylneuraminic acid (Neu5Ac) and N-glycoloylneuraminic acid (Neu5Gc) are distributed widely in nature. Using a Carbopac PA-1 anion exchange column, we have determined the ratios of Neu5Ac and Neu5Gc in hydrolysates of platelets and their precursors: a rat promegakaryoblastic (RPM) cell line and a human megakaryoblastic leukemia cell line (MEG-01). The ratio of Neu5Gc:Neu5Ac in cultured RPM cells is 16:1, whereas in platelet rich plasma and cultured MEG-01 cells it is 1:38 and 1:28, respectively. The nature of these sialic acids from RPM cells was verified using thin layer chromatography and liquid secondary ion mass spectrometry. The relevance of increased Neu5Gc levels in early stages of development is discussed.

N-glycolylneuraminic acid and N-acetylneuraminic acid define feline blood group A and B antigens.

Blood group incompatibility causes transfusion reactions and neonatal isoerythrolysis in cats. We investigated the molecular nature of the blood group antigens from cats that had blood type A, B, and AB erythrocytes. Naturally occurring anti-type B antibodies, Triticum vulgaris lectin, monoclonal antibody (MoAb) 32-27, and MoAb R-24 were used in agglutination tests, Western blots, and thin-layer chromatography (TLC) enzyme immunostaining. Type A erythrocytes had NeuGc-NeuGc-Galactose-Glucose-Ceramide ([NeuGc]2GD3) where NeuGc represents N-glycolylneuraminic acid, and NeuAc-NeuGc-GD3, where NeuAc represents N-acetylneuraminic acid, and may have [NeuGc]2 disialylparagloboside and NeuAc-NeuGc-disialylparagloboside. Type B erythrocytes only had [NeuAc]2GD3. Type AB erythrocytes had [NeuGc]2GD3, NeuAc-NeuGc-GD3, and [NeuAc]2GD3. Blood group antigens were also found on a 50-Kd membrane protein. We conclude that type B erythrocytes are characterized by [NeuAc]2GD3 as the only form of this ganglioside and the presence of NeuAc on a 50-Kd membrane protein. NeuGc is the major determinant of the A antigen; specifically, [NeuGc]2GD3 is the major glycolipid form. The A antigen is also present on a 50-Kd membrane protein.

Diethylnitrosamine-induced metastasizing hepatocellular carcinomas in New Zealand white rabbits. A tumor model for clinical investigations.

The aim of this study was to produce large liver tumors reliably, and to diagnose the tumors during development. Therefore, New Zealand white rabbits were treated with N-nitrosodiethylamine orally three times per week by gavage and were examined by clinical-chemical assay at regular intervals during the average treatment period of 14 months. The total cumulative dose was 1200 mg N-nitrosodiethylamine over 14 months. After a short treatment period the initial dose of 3 mg/kg had to be reduced to 1.5 mg/kg. In all 11 treated animals (100%) liver tumors were seen at the end of the study. Four control animals did not show any neoplastic changes. Clinical parameters investigated were for an assessment of liver function, total protein, urea, creatinine, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, albumin and neuraminic acid as well as some serum electrolytes. The in vivo diagnosis of liver tumors based on changes in these parameters proved to be relatively unreliable. The liver enzyme tests and urea concentration only yielded significant changes when the liver tumors were very large. Changes in neuraminic acid levels were the most reliable indicator for the presence of a liver tumor in this animal model. In the 11 treated animals, serum values of this marker increased towards the end of the study by an average of 300 mg/dl. The induced tumors were mainly hepatocellular carcinomas. Only in 1 animal was a hepatocellular adenoma found. Further primary tumors diagnosed were six adenomas in the kidneys and two uterus adenomas, as well as nasal cavity tumors (two papillomas, one carcinoma, one adenoma and one adenocarcinoma). In 70% of the treated rabbits the hepatocellular carcinomas had metastasized to the lungs.

Determination of mono-O-acetylated N-acetylneuraminic acids in human and rat sera by fluorometric high-performance liquid chromatography.

A simple, rapid, and highly sensitive fluorometric high-performance liquid chromatographic method for the determination of N-acetylneuraminic acid and its mono-O-acetyl derivatives in human and rat sera is described. The neuraminic acids, released by hydrolysis of serum in 2 M acetic acid, are converted with 1,2-diamino-4,5-methylenedioxybenzene, a fluorogenic reagent for alpha-keto acids, to highly fluorescent derivatives without the occurrence of O-acetyl migration and de-O-acetylation. The derivatives are separated isocratically within 25 min by reversed-phase chromatography using a TSK gel ODS-120T column. The limits of detection are 57-192 fmol in a 10-microliters injection volume at a signal-to-noise ratio of 3. This sensitivity permits precise determination of the neuraminic acids in 5 microliters of human and rat sera.

Fluorometric high-performance liquid chromatography of N-acetyl- and N-glycolylneuraminic acids and its application to their microdetermination in human and animal sera, glycoproteins, and glycolipids.

A simple, rapid, and highly sensitive high-performance liquid chromatographic method is described for the determination of N-acetyl- and N-glycolylneuraminic acids in human and animal sera, glycoproteins, and glycolipids. The neuraminic acids, released by acid hydrolysis of these biological samples, are converted in dilute sulfuric acid with 1,2-diamino-4,5-methylene-dioxybenzene, a fluorogenic reagent for alpha-keto acids, to highly fluorescent derivatives. The derivatives are separated within 12 min on a reversed-phase column (Radial-Pak cartridge C18) with an isocratic elution and detected fluorometrically. The detection limits are 25 fmol (7.7 pg) for N-acetylneuraminic acid and 23 fmol (7.5 pg) for N-glycolylneuraminic acid in a 10-microliter injection volume at a signal-to-noise ratio of 2. This method permits precise determination of the neuraminic acids in 5 microliter of human and animal sera or in 0.25-2.5 micrograms of glycoproteins and glycolipids.

[Value of neuraminic acid as a clinical parameter in 42 patients with small-cell bronchial cancer]. / Der Wert der Neuraminsäure als klinischer Parameter bei 42 Patienten mit kleinzelligem Bronchialkarzinom.

The serum concentrations of neuraminic acid, CEA, gamma-Gt, 5-nucleotidase, haptoglobin, and alkaline phosphatase were determined before therapy and three and six months after the initiation of therapy in 42 patients with small cell bronchial carcinomas. Before therapy, a significantly increased serum concentration as compared to normal values was found for neuraminic acid in 97% of patients (43/44), for CEA in 54.7% (23/42), for gamma-Gt in 19% (8/42), for 5-nucleotidase in 11.9% (5/42), for haptoglobin in 36.3% (14/44), and for alkaline phosphatase in 9.5% of all patients (4/42). Three and six months later, these laboratory investigations did not give any valuable hint with respect to therapy results, with the exception of neuraminic acid (p less than 0.05). Prior to therapy, the concentrations of neuraminic acid were considerably increased in patients (mean = 3.16 +/- 0.47 mumol/ml) with regard to normal values (mean = 1.90 +/- 0.14 mumol/ml). Within the total group of 42 patients suffering from small cell bronchial carcinomas, there was no significant difference between the serum concentrations of neuraminic acid of eleven patients with localized tumors (mean = 3.12 +/- 0.53) and those of 31 advanced tumor patients (mean = 3.16 +/- 0.45) (p less than 0.05). During the six months' treatment period, the concentration of neuraminic acid was valuable as clinical parameter in 36 patients, i.e. 85% of all cases (0.001 less than p less than 0.01). It was shown that the serum concentration of neuraminic acid indicated a regression of the disease in 23 patients (p less than 0.001), a progression in 8 patients (0.02 less than p less than 0.05), and a regression with subsequent progression of the tumor in 5 patients (0.001 less than p less than 0.01).