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Results

The combined treatment with EPA and lovastatin enhanced the regulatory effect on gene expression of HMGCoA reductase and LDL receptor in HepG2 cell line. Moreover, we detected a synergistic effect on the inhibition of cancer cell proliferation obtained by combination of EPA and Lovastatin.

Introduction

In the present study we investigated in vitro whether combined treatment with Eicosapentaenoic acid (EPA), belonging to omega-3 family, and lovastatin enhanced the regulatory effect on gene expression of HMGCoA reductase and LDL receptor in HepG2 cell line.

Cell culture conditions

HepG2, a cell line derived from human liver tissue with a well differentiated hepatocellular carcinoma, were obtained from the ICLC (IST, Genoa, Italy).

Lovastatin and EPA treatment

To examine the response to EPA, HepG2 cells were treated for 48 h with culture medium supplemented with various concentrations of EPA (1, 10, 25, 50 and 100 μM). Each experiment included a control without EPA and a control with the same amount of DMEM -BSA used for dissolving the fatty acid.

Lovastatin and EPA treatment

For the experiment testing the effect of combined treatment of Lovastatin and EPA on subconfluent HepG2 cells two sets of experiments were prepared. In the first set, HepG2 cells were incubated with 50 μM of EPA and increasing concentrations of Lovastatin (1,10, 25, 50 and 100 μM). The second set of experiments were performed by incubating HepG2 with Lovastatin at the concentration of 50 μM with increasing concentrations of EPA (1,10, 25, 50 and 100 μM) (data not shown). In these experimental conditions, HepG2 cells were allowed to growth for 48 h. Each experiment included an untreated control and a control with the equivalent concentration of solvent used for adding Lovastatin and EPA.

LDL receptor and HMG-CoA reductase gene expression

Analysis of gene expression was performed in HepG2 cells treated with 1, 10, 25, 50 and 100 μM of EPA and Lovastatin alone and with both compounds at established concentrations for 48 hours.

Abstract

Homocysteine (Hcy) has been recognized for the past five decades as a risk factor for atherosclerosis. However, the role of Hcy in the pathological changes associated with atherosclerosis as well as the pathological mechanisms triggered by Hcy accumulation is poorly understood. Due to the reversal of the physiological direction of the reaction catalyzed by S-adenosyl-L-homocysteine hydrolase Hcy accumulation leads to the synthesis of S-adenosyl-L-homocysteine (AdoHcy). AdoHcy is a strong product inhibitor of S-adenosyl-L-methionine (AdoMet)-dependent methyltransferases, and to date more than 50 AdoMet-dependent methyltransferases that methylate a broad spectrum of cellular compounds including nucleic acids, proteins and lipids have been identified. Phospholipid methylation is the major consumer of AdoMet, both in mammals and in yeast. AdoHcy accumulation induced either by Hcy supplementation or due to S-adenosyl-L-homocysteine hydrolase deficiency results in inhibition of phospholipid methylation in yeast. Moreover, yeast cells accumulating AdoHcy also massively accumulate triacylglycerols (TAG). Similarly, Hcy supplementation was shown to lead to increased TAG and sterol synthesis as well as to the induction of the unfolded protein response (UPR) in mammalian cells. In this review a model of deregulation of lipid metabolism in response to accumulation of AdoHcy in Hcy-associated pathology is proposed.

3. AdoHcy-Triggered Deregulation of Lipid Metabolism in Yeast

In yeast, the synthesis of PC from PE by the de novo phospholipid methylation pathway is particularly sensitive to AdoHcy accumulation [, ]. Both inhibition of S-adenosyl-L-homocysteine hydrolase and Hcy supplementation results in AdoHcy accumulation and inhibition of phospholipid methylation in yeast []. However, not only phospholipid methylation, but also a methylation-independent branch of lipid metabolism, namely, TAG synthesis, is affected by AdoHcy accumulation in yeast: yeast cells deficient in AdoHcy catabolism or supplemented with Hcy massively accumulate TAG []. Supporting the causal role of impaired phospholipid methylation in the deregulation of TAG metabolism in response to AdoHcy accumulation, it was found that yeast mutants that are deficient in the enzymatic activities required for methylation of PE to PC, cho2 and opi3, also accumulate TAG []. TAG is known to play an important role in buffering excess fatty acids []. Therefore, TAG accumulation under these conditions suggests accumulation of fatty acids and their redirection from phospholipid to TAG synthesis in methylation deficiency in yeast.

4. Phospholipid Methylation and Homocysteine: Impact on Lipid Metabolism in Mammals

AdoHcy inhibits phosphatidylethanolamine N-methyltransferase in vitro and in vivo also in mammals [, , ]. Similarly as in yeast, deficiency of phospholipid methylation in PEMT−/− knockout mice leads to a rapid decrease of the hepatic PC/PE ratio and accumulation of TAG in the liver, in the absence of choline supplementation []. However, TAG accumulation in the livers of these animals appears to be at least in part due to decreased TAG secretion from hepatocytes [].

4. Phospholipid Methylation and Homocysteine: Impact on Lipid Metabolism in Mammals

Elevated levels of Hcy are as well linked to deregulation of lipid metabolism in mammals. CBS−/− knockout mice exhibit severe hyperhomocysteinemia and accumulate AdoHcy in all tissues tested [, ]. These mutant animals show elevated TAG and nonesterified fatty acid levels in the liver and serum and develop hepatic steatosis [, ]. Another genetic disorder that results in moderately elevated Hcy levels, methylenetetrahydrofolate reductase (MTHFR) deficiency, leads to fatty liver development as well as to neuropathology and aortic lipid deposition in mouse models [, ]. Dietary-induced hyperhomocysteinemia in mice also causes fatty liver, further supporting the role of Hcy in deregulation of lipid metabolism in mammals []. In these mice as well as in the CBS−/− knockout mice lipid accumulates in liver rather than in serum [, ].

4. Curcumin Safety and Toxicity

In the systematic studies funded by the Prevention Division of the US National Cancer Institute (NCI) conducted in rats, dogs, or monkeys and at oral doses of curcumin up to 3,500 mg/kg-BW for up to 90 days, no adverse effects were observed []. In a preclinical study involving the administration of 2% dietary curcumin (approx. 1,200 mg/kg-BW) to rats for 14 days [] or in a study of 0.2% dietary curcumin (approx. 300 mg/kg-BW) administered to mice for 14 weeks, no toxicity was observed []. Furthermore, a reproductive toxicity study with the oral curcumin administration of up to 1,000 mg/kg-BW daily, no toxicity was observed in two successive generations in rats [].

4. Curcumin Safety and Toxicity

In a study performed in India, daily administration of 1,200–2,100 mg of oral curcumin to patients with rheumatoid arthritis for 2–6 weeks did not cause any toxicity []. In another study of high-dose oral curcumin by Cheng et al. in Taiwan, administration of 500, 1,000, 2,000, 4,000, and 8,000 mg of curcumin daily for three months to patients with preinvasive malignant or high risk premalignant conditions, no noticeable adverse effects were detected []. Lao et al. studied safety of curcumin in healthy volunteers using curcumin capsules (containing 75% curcumin, 23% demethoxycurcumin and 2% bisdemethoxycurcumin) with single escalating doses from 500 mg to 12,000 mg. Among 24 enrolled healthy subjects, seven developed adverse effects, including diarrhea, headaches, rashes and yellowish stools. All toxicities observed were of grade 1 and not related to the doses. In this study, the maximum tolerated dose could not be determined because more than 12,000 mg of curcumin is regarded as too bulky [].

4. Curcumin Safety and Toxicity

In a study performed in India, daily administration of 1,200–2,100 mg of oral curcumin to patients with rheumatoid arthritis for 2–6 weeks did not cause any toxicity []. In another study of high-dose oral curcumin by Cheng et al. in Taiwan, administration of 500, 1,000, 2,000, 4,000, and 8,000 mg of curcumin daily for three months to patients with preinvasive malignant or high risk premalignant conditions, no noticeable adverse effects were detected []. Lao et al. studied safety of curcumin in healthy volunteers using curcumin capsules (containing 75% curcumin, 23% demethoxycurcumin and 2% bisdemethoxycurcumin) with single escalating doses from 500 mg to 12,000 mg. Among 24 enrolled healthy subjects, seven developed adverse effects, including diarrhea, headaches, rashes and yellowish stools. All toxicities observed were of grade 1 and not related to the doses. In this study, the maximum tolerated dose could not be determined because more than 12,000 mg of curcumin is regarded as too bulky [].

4. Curcumin Safety and Toxicity

In a phase I clinical trial on oral curcumin in patients with advanced colorectal cancer in which the US NCI criteria were used to assess potential toxicity, curcumin was well tolerated at all dose levels up to 3,600 mg daily for up to four months []. Adverse effects related to curcumin consumption reported by patients in these studies were mainly gastrointestinal (nausea and diarrhoea). Two abnormalities were detected in blood tests in this trial, both possibly related to treatment: an increase in serum alkaline phosphatase level was observed in four patients (two NCI grade 1, and two grade 2); and three other patients had serum lactate dehydrogenase increases to >1.5 times the upper limit of normal. It is not clear whether these abnormal blood test results were related to the activity of the malignant disease in these patients or to treatment toxicity.

5.1. Animal Model Pharmacokinetics

Due to its poor systemic bioavailability after oral administration, many research groups have focused on ways to improve its bioavailability. Co-administration of oral curcumin with piperine, an alkaloid found in black pepper (Piper nigrum) and long pepper (Piper longa), appeared to increase serum concentrations of curcumin in rodents. In a study using high doses of oral curcumin (2,000 mg/kg) in rats, the investigators found that co-administration of piperine increased systemic bioavailability by as much as 154% []. The mechanism of this effect has not been elucidated yet.

Abstract

The peptidoglycan of Staphylococcus aureus is characterized by a high degree of crosslinking and almost completely lacks free carboxyl groups, due to amidation of the D-glutamic acid in the stem peptide. Amidation of peptidoglycan has been proposed to play a decisive role in polymerization of cell wall building blocks, correlating with the crosslinking of neighboring peptidoglycan stem peptides. Mutants with a reduced degree of amidation are less viable and show increased susceptibility to methicillin. We identified the enzymes catalyzing the formation of D-glutamine in position 2 of the stem peptide. We provide biochemical evidence that the reaction is catalyzed by a glutamine amidotransferase-like protein and a Mur ligase homologue, encoded by SA1707 and SA1708, respectively. Both proteins, for which we propose the designation GatD and MurT, are required for amidation and appear to form a physically stable bi-enzyme complex. To investigate the reaction in vitro we purified recombinant GatD and MurT His-tag fusion proteins and their potential substrates, i.e. UDP-MurNAc-pentapeptide, as well as the membrane-bound cell wall precursors lipid I, lipid II and lipid II-Gly5. In vitro amidation occurred with all bactoprenol-bound intermediates, suggesting that in vivo lipid II and/or lipid II-Gly5 may be substrates for GatD/MurT. Inactivation of the GatD active site abolished lipid II amidation. Both, murT and gatD are organized in an operon and are essential genes of S. aureus. BLAST analysis revealed the presence of homologous transcriptional units in a number of gram-positive pathogens, e.g. Mycobacterium tuberculosis, Streptococcus pneumonia and Clostridium perfringens, all known to have a D-iso-glutamine containing PG. A less negatively charged PG reduces susceptibility towards defensins and may play a general role in innate immune signaling.

Abstract

Compartmentalized redox faults are common to ageing diseases. Dietary constituents are catabolized to NAD(H) donating electrons producing proton-based bioenergy in coevolved, cross-species and cross-organ networks. Nicotinamide and NAD deficiency from poor diet or high expenditure causes pellagra, an ageing and dementing disorder with lost robustness to infection and stress. Nicotinamide and stress induce Nicotinamide-N-methyltransferase (NNMT) improving choline retention but consume methyl groups. High NNMT activity is linked to Parkinson's, cancers, and diseases of affluence. Optimising nicotinamide and choline/methyl group availability is important for brain development and increased during our evolution raising metabolic and methylome ceilings through dietary/metabolic symbiotic means but strict energy constraints remain and life-history tradeoffs are the rule. An optimal energy, NAD and methyl group supply, avoiding hypo and hyper-vitaminoses nicotinamide and choline, is important to healthy ageing and avoids utilising double-edged symbionts or uncontrolled autophagy or reversions to fermentation reactions in inflammatory and cancerous tissue that all redistribute NAD(P)(H), but incur high allostatic costs.

2. Pellagra

An NAD(H) deficiency state that illustrates the complexity of these coevolved energy networks was observed during the largely forgotten epidemics of pellagra caused by economically determined suboptimal foraging of nicotinamide and tryptophan (tryptophan is converted to NAD and serotonin) and dietary sources of methyl-groups. Such a dietary deficiency may still be important in sections of modern populations and intracellular and microenvironmental deficiency states can also occur from high NAD expenditure with NAD(H) ratios also affected by any disorder that causes hypoxia or hypoglycemia [, ].

Abstract

Two members of the Cryptococcus neoformans-gattii species complex, the etiologic agents of cryptococcosis, can be differentiated by biological, biochemical, serological and molecular typing techniques. Based on their differences in carbon and nitrogen utilization patterns, cost effective and very specific diagnostic tests using D-proline and canvanine-glycine-bromthymol blue (CGB) media have been formulated and are widely used for identification of the two species. However, these methods have yet to be tested for strains with confirmed molecular types to assess the degree of specificity for each molecular type in the two species. We collected global isolates of every major molecular type available and tested their patterns of nitrogen utilization. We confirmed specificity of the CGB test to be 100% regardless of molecular type while the D-proline test yielded 8–38% false negative results in three of the four C. gattii molecular types, VGI–VGIII. The utilization pattern of a new set of amino acids: D-alanine, L-tryptophan and L-phenylalanine, showed species specificity comparable to that of D-proline. We discovered that the transcription factor Gat1 (Are1) regulates the utilization of nitrogen differently between C. neoformans and C. gattii strains. Unlike in C. neoformans, expression of the genes encoding glycine decarboxylase complex in C. gatti was only partially suppressed by nitrogen catabolite repression in the presence of ammonium. GAT1 in C. neoformans controlled the induction of three of the four genes encoding the glycine decarboxylase complex when glycine was used as the sole nitrogen source while in C. gattii its regulation of these genes was less stringent. Moreover, while virulence of C. neoformans strains in mice was not affected by Gat1, the transcription factor positively influenced the virulence of C. gattii strain.

Discussion

The ability to utilize specific nitrogen and carbon sources is among the standard methods used for the identification of yeast species , as well as some filamentous fungi . Differentiation of the two species in Csc also has been effectively carried out by both nitrogen and carbon utilization tests , , , , . However, the efficacies of these biochemical tests were established long before molecular strain typing methods became available and the validity of these tests could have been compromised since the populations tested could contain strains of the same genotype. For example, previous studies have reported 100% and 95% specificity for D-proline utilization by C. gattii while we found a much lower specificity; only 80% of C. gattii strains yielded positive results in contrast to the 100% that were negative for C. neoformans. The CGB media, on the other hand, showed 100% specificity between the two species regardless of molecular type. Remarkably, the false negatives observed for D-proline utilization were found mainly among strains of the VGI and VGIII (5 negatives among 6 strains) molecular types. According to the phylogenetic analysis of global Csc strains based on multi gene sequence analyses, the VGI and VGIII molecular types formed a separate cluster with high bootstrap supports , . Our results underscore the biological variation between different molecular types within C. gattii.

Abstract

The multicomponent reaction (MCR) of aromatic aldehydes 1 and malononitrile (2) with active methylenes 5a–h in the presence of L-proline produced pyrans and thiopyrans 6a–h stereospecifically and in good yields. Moreover a novel MCR of ethyl propiolate (8) with 1 and 2 in the presence of L-proline to afford (R)-polysubstituted pyran is also reported. X-ray structures, e.e. and optical activity of the synthesized compounds indicated that L-proline as a catalyst is responsible for the observed enantioselectivity in the studied reactions.

2. Results and Discussion

First in an attempt to synthesize the chiral pyranopyrazoles 4, we have reacted benzaldehyde (1), malononitrile (2) and pyrazolon-5-one (3) with 10% mol L-proline as the only catalyst. In contrast to Muramulla et al’s. findings that for the same reaction using L-proline alone as a catalyst under the reported reaction conditions no product was obtained, in our case after the reaction mixture was refluxed in ethanol for 4 h, the pyranopyrazole 4 was isolated in 81% yield (). To initially test if L-proline has induced any enantioselectivity in this reaction, compound 4 was tested for optical activity and found to be optically active with a specific rotation of +247.02 ([α]D, 25 °C, c = 1, DMF).

3.2. General Experimental Procedure for the Synthesis of Pyrans 6a–h and Compound 7

A mixture of benzaldehyde (1, 0.01 mol), malononitrile (2, 0.01 mol) and 10% mol L-proline was stirred at r.t. for 2 min. then active methylenes 5a–h (0.01 mol) were added. The mixture was refluxed in ethanol (10 mL) for 4–6 h followed by TLC. The crude compounds formed were recrystallized from ethanol and further purified using column chromatography using 2:1 petroleum ether/ethyl acetate as an eluent.

2. Results and Discussion

Specific rotation measurements for some selected synthesized compounds revealed that these compounds are optically active, which supports the assumption that L-proline when used as a catalyst brings about enantioselectivity in such reactions. The specific rotation of some selected compounds is listed in .

2. Results and Discussion

In addition a novel synthesis of pyran 13 could be achieved by mixing benzaldehyde (1), and malononitrile (2) with ethyl propiolate (8) in ethanol and 10% L-proline as a catalyst. It is believed that initially L-proline (9) adds to ethyl propiolate (8) affording the enamine ester 10, while benzaldehyde (1) condenses with malononitrile (2) affording 2-benzylidenemalononitrile (11). This was followed by the addition of the electron rich β-carbon in the enamine ester to the electron poor π system in the benzylidine-malononitrile 11, affording an adduct. This adduct 12 is then hydrolyzed by H2O and cyclizes into 13 (). Compound 13 was also tested for optical activity and found to be optically active with a specific rotation of +272.0 ([α]D 25 °C, c = 1, DMF). The structure of 13 has been confirmed with certainty via X-ray crystal structure determination ().

Background

D-phenylglycine aminotransferase (D-PhgAT) of Pseudomonas stutzeri ST-201 catalyzes the reversible stereo-inverting transamination potentially useful in the application for synthesis of D-phenylglycine and D-4-hydroxyphenylglycine using L-glutamate as a low cost amino donor substrate in one single step. The enzyme is a relatively hydrophobic homodimeric intracellular protein difficult to express in the soluble functionally active form. Over-expression of the dpgA gene in E. coli resulted in the majority of the D-PhgAT aggregated into insoluble inclusion bodies that failed to be re-natured. Expression in Pichia pastoris was explored as an alternative route for high level production of the D-PhgAT.

Background

The D-phenylglycine aminotransferase (D-PhgAT) from a soil bacterium Pseudomonas stutzeri ST-201 [], catalyzes the reversible transamination specific for D-phenylglycine or D-4-hydroxyphenylglycine in which 2-oxoglutarate is an amino-group acceptor that are converted into benzoylformate or 4-hydroxybenzoylformate and L-glutamate (Figure ). Both D-phenylglycine and D-4-hydroxyphenylglycine are important side chain moieties in high demand for production of semisynthetic penicillin and cephalosporin antibiotics such as ampicillin, amoxicillin, cephalexine and cephadroxyl. Currently, D-phenylglycine and D-4-hydroxyphenylglycine are industrially produced by a process using two enzymes, the hydantoinase and carbamoylase []. However, the reaction rates are low since the solubilities of the substrates, (D,L) phenylhydantoin and (D,L)-4-hydroxyphenylhydantoin, are very poor. Alternatively, a single-step enzymatic synthesis using a D-amino acid aminotransferase is possible. But known D-amino acid aminotransferases have very low transamination activity towards D-phenylglycine [] and they accept only D-amino acids as the amino-group donors. Due to the high cost of D-amino acids (such as D-alanine, D-glutamic acid), their direct application as amino-group donors is impractical. Using an L-amino acid (such as L-alanine or L-glutamic acid) together with an amino acid racemase to convert it to the corresponding D-amino acid to serve as the amino-group donor for the D-amino acid aminotransferase will increase the production cost. Additionally, the racemase may interfere with the transamination reaction. By contrast, with the D-PhgAT, due to its characteristic “stereo-inverting” transamination activity, a low cost substrate L-glutamic acid can be used as an amino-group donor for the synthesis of enantiomerically pure D-phenylglycine or D-4-hydroxyphenylglycine in a single transamination step without the need for any amino acid racemase [].

Methods

Patients with type 2 diabetes (n = 34) and control subjects (n = 17) were recruited. We measured baseline fasting plasma and 4-hour post-methionine load (L-methionine, 0.1 mg/kg body weight) concentrations of homocysteine, betaine, and the betaine metabolite N,N-dimethylglycine. Baseline urine excretions of betaine, dimethylglycine and glucose were measured on morning urine samples as the ratio to urine creatinine. Statistical determinants of the post-methionine load increase in homocysteine were identified in multiple linear regression models.

Results

Plasma betaine concentrations and urinary betaine excretions were significantly (p < 0.001) more variable in the subjects with diabetes compared with the controls. Dimethylglycine excretion (p = 0.00014) and plasma dimethylglycine concentrations (p = 0.039) were also more variable. In diabetes, plasma betaine was a significant negative determinant (p < 0.001) of the post-methionine load increase in homocysteine. However, it was not conclusive that this was different from the relationship in the controls. In the patients with diabetes, a strong relationship was found between urinary betaine excretion and urinary glucose excretion (but not with plasma glucose).

Methionine load test study

The study was approved by the Canterbury Ethics Committee (New Zealand) and all subjects gave written informed consent. Patients with Type 2 diabetes were identified through the Canterbury retinal screening programme database and invited to participate. Controls were recruited by posting notices around the hospital campus. Thirty four patients (aged 51–75 yrs, 20 males) and 17 controls (aged 54–69 yrs, 10 male) were recruited. They were asked to fast for 16 hours and to withhold morning medications. Their height, weight, age, and medical history were recorded. Venous blood was drawn into EDTA tubes that were placed on ice immediately for determining fasting plasma betaine and homocysteine concentrations and a urine sample was collected. They were then given a methionine load of 0.1 g/kg body weight of L-methionine dissolved in 100 ml fruit juice. They also ate a standard snack consisting of a jam (jelly) sandwich to avoid hypoglycaemia. They took no further food or fluid until their return 4 hours later, when a second venous blood sample was collected. The betaine, dimethylglycine and homocysteine from the pre- and post- methionine load blood samples were measured in the same analytical batches.

Differences between subjects with and without diabetes

The post-methionine load plasma betaine and homocysteine concentrations were also significantly more variable in the subjects with diabetes (on log-transformed data, p < 0.001 for betaine and p = 0.0016 for N,N-dimethylglycine), as was the post-methionine change in plasma N,N-dimethylglycine (p = 0.013), but not the post-methionine change in plasma betaine (p = 0.7).

Methods

Patients with type 2 diabetes (n = 34) and control subjects (n = 17) were recruited. We measured baseline fasting plasma and 4-hour post-methionine load (L-methionine, 0.1 mg/kg body weight) concentrations of homocysteine, betaine, and the betaine metabolite N,N-dimethylglycine. Baseline urine excretions of betaine, dimethylglycine and glucose were measured on morning urine samples as the ratio to urine creatinine. Statistical determinants of the post-methionine load increase in homocysteine were identified in multiple linear regression models.

Methionine load test study

The study was approved by the Canterbury Ethics Committee (New Zealand) and all subjects gave written informed consent. Patients with Type 2 diabetes were identified through the Canterbury retinal screening programme database and invited to participate. Controls were recruited by posting notices around the hospital campus. Thirty four patients (aged 51–75 yrs, 20 males) and 17 controls (aged 54–69 yrs, 10 male) were recruited. They were asked to fast for 16 hours and to withhold morning medications. Their height, weight, age, and medical history were recorded. Venous blood was drawn into EDTA tubes that were placed on ice immediately for determining fasting plasma betaine and homocysteine concentrations and a urine sample was collected. They were then given a methionine load of 0.1 g/kg body weight of L-methionine dissolved in 100 ml fruit juice. They also ate a standard snack consisting of a jam (jelly) sandwich to avoid hypoglycaemia. They took no further food or fluid until their return 4 hours later, when a second venous blood sample was collected. The betaine, dimethylglycine and homocysteine from the pre- and post- methionine load blood samples were measured in the same analytical batches.

Glucose, betaine and homocysteine

Betaine excretion positively correlated with glucose excretion (log transformed data; r = +0.47, p = 0.005, n = 34) in patients with diabetes. Dimethylglycine excretion also correlated with glucose excretion (log transformed data; r = +0.53, p = 0.001, n = 34). There was an inconclusive correlation between plasma glucose and log-transformed betaine excretion (r = +0.33, p = 0.056, n = 34); the Spearman’s correlation coefficient was significant (rs = +0.37, p = 0.03). Neither fasting homocysteine, nor the post-methionine load increase in homocysteine, correlated with either plasma glucose or glucose excretion. Hemoglobin A1c did not strongly correlate with betaine excretion, but there was a significant positive correlation (r = +0.52, p = 0.003, n = 30) between hemoglobin A1c and the post-methionine load change in plasma dimethylglycine (measured as the difference in log(plasma dimethylglycine) before and after the load). Hemoglobin A1c did not correlate with either fasting homocysteine or the post-methionine load increase in homocysteine.

Methods

Patients with type 2 diabetes (n = 34) and control subjects (n = 17) were recruited. We measured baseline fasting plasma and 4-hour post-methionine load (L-methionine, 0.1 mg/kg body weight) concentrations of homocysteine, betaine, and the betaine metabolite N,N-dimethylglycine. Baseline urine excretions of betaine, dimethylglycine and glucose were measured on morning urine samples as the ratio to urine creatinine. Statistical determinants of the post-methionine load increase in homocysteine were identified in multiple linear regression models.

Methionine load test study

The study was approved by the Canterbury Ethics Committee (New Zealand) and all subjects gave written informed consent. Patients with Type 2 diabetes were identified through the Canterbury retinal screening programme database and invited to participate. Controls were recruited by posting notices around the hospital campus. Thirty four patients (aged 51–75 yrs, 20 males) and 17 controls (aged 54–69 yrs, 10 male) were recruited. They were asked to fast for 16 hours and to withhold morning medications. Their height, weight, age, and medical history were recorded. Venous blood was drawn into EDTA tubes that were placed on ice immediately for determining fasting plasma betaine and homocysteine concentrations and a urine sample was collected. They were then given a methionine load of 0.1 g/kg body weight of L-methionine dissolved in 100 ml fruit juice. They also ate a standard snack consisting of a jam (jelly) sandwich to avoid hypoglycaemia. They took no further food or fluid until their return 4 hours later, when a second venous blood sample was collected. The betaine, dimethylglycine and homocysteine from the pre- and post- methionine load blood samples were measured in the same analytical batches.

Predictors of the post-methionine load increase in homocysteine

In the subjects with diabetes, the statistically significant (p < 0.05) predictors of the post-methionine load increase in plasma homocysteine, identified by best subset regression, were fasting plasma homocysteine, either fasting or post-methionine load betaine, and (in most models) serum vitamin B12. Variables that did not enter any of 36 models with p < 0.1 included age, body mass index, red blood cell folate, hemoglobin A1c, plasma glucose concentrations, plasma N,N-dimethylglycine concentration (either fasting or post-methionine load), and the urinary excretions of glucose, betaine and N,N-dimethylglycine. Log-transformed variables were used where the raw data was not normally distributed (Kolmogorov-Smirnov test). The three strongest candidate predictors (all log-transformed) were included in an explicit multiple linear regression model (Figure ) which confirmed that the fasting plasma homocysteine was the strongest (positive) predictor, and the post-methionine load plasma betaine concentration was also a highly significant (p < 0.001) negative predictor. The serum vitamin B12 concentration also a positive predictor (p = 0.021). The model explained most of the variance in the post-methionine load increase in homocysteine (r2 = 0.69). When the fasting plasma betaine concentration was included in the model instead of the post-methionine concentration, a similar result was obtained (r2 = 0.56) but the fasting betaine concentration appears to be a weaker negative predictor (p = 0.013).

2. Results and Discussion

2-Octanol was successfully oxidized in nearly quantitative yield both by the composite and 30% aqueous H2O2 (). The application of UHP as an oxidant lowers the yield of the reaction to 69%. Moreover, when the reaction temperature was decreased to 70 °C, UHP exhibited low reactivity manifested only 6% yield of 2-octanone with comparison to 64% and 56% for aqueous H2O2 and the xerogel, respectively. Oxidation under microwave conditions gave very good results only when 30% H2O2 was applied.