Cardiovasc Drugs Ther (2014) 28:459–468 DOI 10.1007/s10557-014-6542-9
ORIGINAL ARTICLE
Extreme Urinary Betaine Losses in Type 2 Diabetes Combined with Bezafibrate Treatment are Associated with Losses of Dimethylglycine and Choline but not with Increased Losses of Other Osmolytes Michael Lever & Christopher J. McEntyre & Peter M. George & Sandy Slow & Jane L. Elmslie & Helen Lunt & Stephen T. Chambers & Amber Parry-Strong & Jeremy D. Krebs
Published online: 25 July 2014 # Springer Science+Business Media New York 2014
Abstract Purpose Betaine deficiency is a probable cardiovascular risk factor and a cause of elevated homocysteine. Urinary betaine excretion is increased by fibrate treatment, and is also often elevated in diabetes. Does fibrate further increase betaine excretion in diabetes, and does it affect the plasma concentrations and excretions of related metabolites and of other osmolytes? Methods Samples from a previous study of type 2 diabetes were selected if participants were taking bezafibrate (n=32). These samples were compared with participants matched for age and gender and not on a fibrate (comparator group, n=64). Betaine, related metabolites, and osmolytes were measured in plasma and urine samples from these 96 participants. Results Median urinary betaine excretion in those on bezafibrate was 5-fold higher than in the comparator group (p300 mg/ day) [4]. Also, about 30 % of patients with diabetes have an abnormally high urinary betaine excretion [3], and this latter finding has been recently confirmed in a larger population [5]. Betaine deficiency disturbs one-carbon metabolism, and for example is associated with an elevated plasma homocysteine [6–8], which can therefore be a marker of betaine deficiency. It is not clear how frequently elevated plasma homocysteine is
460
the result of betaine deficiency, but if it is common this could explain some of the negative results of lowering homocysteine by treatment with B-vitamins, since B-vitamin treatment will not correct a betaine deficiency. Since betaine is an osmolyte, its deficiency compromises cell volume regulation, yet we have previously found that elevations in betaine excretion and elevations in the excretion of the renal osmolyte sorbitol correlate positively in diabetes [9]. It is not uncommon and sometimes recommended to prescribe fibrate drugs for treating severe hypertriglyceridemia in diabetes [10–13], with increasing numbers of prescriptions in North America [14], and one question is whether the already-elevated betaine excretion is further increased by this treatment. We have shown that, in healthy volunteers, treatment with fenofibrate increases betaine excretion and that the larger the baseline excretion of betaine, the larger the increase on fenofibrate treatment [15]. Thus it could be anticipated that in diabetes, when baseline betaine excretion is often greatly elevated, fibrate treatment could lead to very large losses of betaine. In view of our observation with sorbitol, another important question is whether there would be also significant losses of other osmolytes or of other onecarbon metabolites. Additionally, we would like to know whether the diabetes and fibrate induced urinary losses of betaine share a mechanism, and whether the mechanism or mechanisms are related to one-carbon metabolism or to osmoregulation. The specimens collected during the DEWL (Diabetes Excess Weight Loss) study [16] provided an opportunity to illuminate some of these questions: the DEWL cohort was a group of overweight and obese participants with type 2 diabetes, 32 of whom were being treated with bezafibrate. Elevated blood lipid is a strong risk factor for cardiovascular disease. Statins are effective and extensively used drugs for lowering blood lipids, but 65–75 % of the cardiovascular disease risk persists after statin therapy [17–19]. Fibrates have been promoted as an adjunct therapy, especially in hypertriglyceridaemia, which is common in type 2 diabetes [12, 13]; however, they can cause apparently unfavourable changes in renal function markers such as plasma creatinine, urea and homocysteine, although these are reversible and there are no changes in the glomerular filtration rate [20]. There is no compelling evidence to suggest that fibrates reduce cardiovascular mortality, though at least one member of this therapeutic family has a beneficial effect on microvascular outcomes in type 2 diabetes and recently it has been confirmed that fibrates reduce risk in the sub-population with atherogenic dyslipidaemia [21]; however, this is also a population that is likely to be betaine deficient [3, 7]. It is possible that the effect of fibrates on betaine, and consequentially homocysteine, metabolism contributes to the lack of uniformly positive outcomes of fibrate trials. We asked whether fibrate therapy also affects the plasma concentrations and urinary excretions of betaine-related metabolites, and of other
Cardiovasc Drugs Ther (2014) 28:459–468
osmolytes. If the effect is predominantly on betaine, this could be expected to attenuate the cardiovascular benefits from fibrates and therefore raise the possibility that concurrent dietary betaine supplementation would be synergistic with fibrate treatment, and justify further studies to evaluate the benefit of this therapeutic combination. A number of recent reports have associated the betaine analog, trimethylamine-N-oxide (TMAO), with cardiovascular events [22–25]. Elevated plasma TMAO has been previously associated with renal failure [26, 27]. Therefore we also measured this component of plasma and urine to assess whether it also is elevated in participants receiving fibrate therapy. Because fibrate therapy, and changes in betaine, its metabolite N,N-dimethylglycine [28] and TMAO, have all been associated with abnormal renal function, a cardiovascular risk factor, we also examined the relationship of osmolytes and one-carbon metabolites with markers of renal function in this study.
Methods Participants, Sample Collection and Selection of the Comparator Group Samples from the DEWL study were archived at >−80 C, then used for the current study. The DEWL study compared prescriptions of hypocaloric high protein and high carbohydrate diets in overweight and obese (BMI >27 kg/m2 ) subjects with type 2 diabetes, aged 30–75 years [16]. Exclusion criteria were current weight-reducing medications, a weight loss of >5 % in the past 3 months, or a psychiatric or eating disorder. Participants were also excluded if their glycated haemoglobin (HbA1c) was >9.5 % (80 mmol/mol) or they had had renal disease (estimated glomerular filtration rate 30 mg/mmol based on a 24-h urine collection), abnormal liver enzymes, heart failure, known active malignancy or myocardial infarction in the preceding 6 months. A total of 419 participants were recruited, but urine samples were only available on 312 of these. The original DEWL study compared plasma and urine collected at four time points (0, 6, 12 and 24 months). No significant differences or even trends (p0.9); only one participant (also taking a statin and bezafibrate) was being treated concurrently with niacin.
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Cardiovasc Drugs Ther (2014) 28:459–468
Table 1 Mass transitions and instrument settings for mass spectrometry Analytes: Betaine Choline N,N-Dimethylglycine Acetylcarnitine
DP decoupling potential, CE collision energy, CXP collision cell exit potential
Carnitine Trimethylamine-N-oxide Glycerophosphorylcholine Taurine Internal standards: D9-Betaine D9-Choline D3-N,N-Dimethylglycine D3-Carnitine D9-Trimethylamine-Noxide D4-Taurine
Mass transition
DP (eV)
CE (eV)
CXP (eV)
Internal standard
118.2 → 59.0 104.1 → 60.2 104.1 → 58.1 204.3 → 85.2
56 16 51 81
27 25 21 31
4 4 4 8
D9-Betaine D9-Choline D3-Dimethylglycine D3-Carnitine
162.1 → 85.0 76.1 → 58.1 258.2 → 104.0 124.1 → 80
71 16 66 −55
31 27 23 −30
16 10 8 −13
D3-Carnitine D9-TMAO D9-Betaine D4-Taurine
127.2 → 68.0 113.2 → 69.0 107.2 → 61.0 165.2 → 85.0 85.2 → 66.0
61 61 46 61 91
27 27 29 29 29
4 2 4 6 2
128.1 → 80
−55
−30
−13
Differences Between Bezafibrate Participants and the Comparator Group The median urinary betaine excretion in the participants taking bezafibrate was 5-fold more than in the comparator group not on fibrates, p
ORIGINAL ARTICLE
Extreme Urinary Betaine Losses in Type 2 Diabetes Combined with Bezafibrate Treatment are Associated with Losses of Dimethylglycine and Choline but not with Increased Losses of Other Osmolytes Michael Lever & Christopher J. McEntyre & Peter M. George & Sandy Slow & Jane L. Elmslie & Helen Lunt & Stephen T. Chambers & Amber Parry-Strong & Jeremy D. Krebs
Published online: 25 July 2014 # Springer Science+Business Media New York 2014
Abstract Purpose Betaine deficiency is a probable cardiovascular risk factor and a cause of elevated homocysteine. Urinary betaine excretion is increased by fibrate treatment, and is also often elevated in diabetes. Does fibrate further increase betaine excretion in diabetes, and does it affect the plasma concentrations and excretions of related metabolites and of other osmolytes? Methods Samples from a previous study of type 2 diabetes were selected if participants were taking bezafibrate (n=32). These samples were compared with participants matched for age and gender and not on a fibrate (comparator group, n=64). Betaine, related metabolites, and osmolytes were measured in plasma and urine samples from these 96 participants. Results Median urinary betaine excretion in those on bezafibrate was 5-fold higher than in the comparator group (p300 mg/ day) [4]. Also, about 30 % of patients with diabetes have an abnormally high urinary betaine excretion [3], and this latter finding has been recently confirmed in a larger population [5]. Betaine deficiency disturbs one-carbon metabolism, and for example is associated with an elevated plasma homocysteine [6–8], which can therefore be a marker of betaine deficiency. It is not clear how frequently elevated plasma homocysteine is
460
the result of betaine deficiency, but if it is common this could explain some of the negative results of lowering homocysteine by treatment with B-vitamins, since B-vitamin treatment will not correct a betaine deficiency. Since betaine is an osmolyte, its deficiency compromises cell volume regulation, yet we have previously found that elevations in betaine excretion and elevations in the excretion of the renal osmolyte sorbitol correlate positively in diabetes [9]. It is not uncommon and sometimes recommended to prescribe fibrate drugs for treating severe hypertriglyceridemia in diabetes [10–13], with increasing numbers of prescriptions in North America [14], and one question is whether the already-elevated betaine excretion is further increased by this treatment. We have shown that, in healthy volunteers, treatment with fenofibrate increases betaine excretion and that the larger the baseline excretion of betaine, the larger the increase on fenofibrate treatment [15]. Thus it could be anticipated that in diabetes, when baseline betaine excretion is often greatly elevated, fibrate treatment could lead to very large losses of betaine. In view of our observation with sorbitol, another important question is whether there would be also significant losses of other osmolytes or of other onecarbon metabolites. Additionally, we would like to know whether the diabetes and fibrate induced urinary losses of betaine share a mechanism, and whether the mechanism or mechanisms are related to one-carbon metabolism or to osmoregulation. The specimens collected during the DEWL (Diabetes Excess Weight Loss) study [16] provided an opportunity to illuminate some of these questions: the DEWL cohort was a group of overweight and obese participants with type 2 diabetes, 32 of whom were being treated with bezafibrate. Elevated blood lipid is a strong risk factor for cardiovascular disease. Statins are effective and extensively used drugs for lowering blood lipids, but 65–75 % of the cardiovascular disease risk persists after statin therapy [17–19]. Fibrates have been promoted as an adjunct therapy, especially in hypertriglyceridaemia, which is common in type 2 diabetes [12, 13]; however, they can cause apparently unfavourable changes in renal function markers such as plasma creatinine, urea and homocysteine, although these are reversible and there are no changes in the glomerular filtration rate [20]. There is no compelling evidence to suggest that fibrates reduce cardiovascular mortality, though at least one member of this therapeutic family has a beneficial effect on microvascular outcomes in type 2 diabetes and recently it has been confirmed that fibrates reduce risk in the sub-population with atherogenic dyslipidaemia [21]; however, this is also a population that is likely to be betaine deficient [3, 7]. It is possible that the effect of fibrates on betaine, and consequentially homocysteine, metabolism contributes to the lack of uniformly positive outcomes of fibrate trials. We asked whether fibrate therapy also affects the plasma concentrations and urinary excretions of betaine-related metabolites, and of other
Cardiovasc Drugs Ther (2014) 28:459–468
osmolytes. If the effect is predominantly on betaine, this could be expected to attenuate the cardiovascular benefits from fibrates and therefore raise the possibility that concurrent dietary betaine supplementation would be synergistic with fibrate treatment, and justify further studies to evaluate the benefit of this therapeutic combination. A number of recent reports have associated the betaine analog, trimethylamine-N-oxide (TMAO), with cardiovascular events [22–25]. Elevated plasma TMAO has been previously associated with renal failure [26, 27]. Therefore we also measured this component of plasma and urine to assess whether it also is elevated in participants receiving fibrate therapy. Because fibrate therapy, and changes in betaine, its metabolite N,N-dimethylglycine [28] and TMAO, have all been associated with abnormal renal function, a cardiovascular risk factor, we also examined the relationship of osmolytes and one-carbon metabolites with markers of renal function in this study.
Methods Participants, Sample Collection and Selection of the Comparator Group Samples from the DEWL study were archived at >−80 C, then used for the current study. The DEWL study compared prescriptions of hypocaloric high protein and high carbohydrate diets in overweight and obese (BMI >27 kg/m2 ) subjects with type 2 diabetes, aged 30–75 years [16]. Exclusion criteria were current weight-reducing medications, a weight loss of >5 % in the past 3 months, or a psychiatric or eating disorder. Participants were also excluded if their glycated haemoglobin (HbA1c) was >9.5 % (80 mmol/mol) or they had had renal disease (estimated glomerular filtration rate 30 mg/mmol based on a 24-h urine collection), abnormal liver enzymes, heart failure, known active malignancy or myocardial infarction in the preceding 6 months. A total of 419 participants were recruited, but urine samples were only available on 312 of these. The original DEWL study compared plasma and urine collected at four time points (0, 6, 12 and 24 months). No significant differences or even trends (p0.9); only one participant (also taking a statin and bezafibrate) was being treated concurrently with niacin.
462
Cardiovasc Drugs Ther (2014) 28:459–468
Table 1 Mass transitions and instrument settings for mass spectrometry Analytes: Betaine Choline N,N-Dimethylglycine Acetylcarnitine
DP decoupling potential, CE collision energy, CXP collision cell exit potential
Carnitine Trimethylamine-N-oxide Glycerophosphorylcholine Taurine Internal standards: D9-Betaine D9-Choline D3-N,N-Dimethylglycine D3-Carnitine D9-Trimethylamine-Noxide D4-Taurine
Mass transition
DP (eV)
CE (eV)
CXP (eV)
Internal standard
118.2 → 59.0 104.1 → 60.2 104.1 → 58.1 204.3 → 85.2
56 16 51 81
27 25 21 31
4 4 4 8
D9-Betaine D9-Choline D3-Dimethylglycine D3-Carnitine
162.1 → 85.0 76.1 → 58.1 258.2 → 104.0 124.1 → 80
71 16 66 −55
31 27 23 −30
16 10 8 −13
D3-Carnitine D9-TMAO D9-Betaine D4-Taurine
127.2 → 68.0 113.2 → 69.0 107.2 → 61.0 165.2 → 85.0 85.2 → 66.0
61 61 46 61 91
27 27 29 29 29
4 2 4 6 2
128.1 → 80
−55
−30
−13
Differences Between Bezafibrate Participants and the Comparator Group The median urinary betaine excretion in the participants taking bezafibrate was 5-fold more than in the comparator group not on fibrates, p
