Life Extension Magazine®

Issue: Nov 2010

Calcium

Role of dietary protein in the sarcopenia of aging.

Sarcopenia is a complex, multifactorial process facilitated by a combination of factors including the adoption of a more sedentary lifestyle and a less than optimal diet. Increasing evidence points to a blunted anabolic response after a mixed nutrient meal as a likely explanation for chronic age-related muscle loss. There is currently insufficient longer-term research with defined health outcomes to specify an optimal value for protein ingestion in elderly individuals. However, there is general agreement that moderately increasing daily protein intake beyond 0.8 g x kg(-1) x d(-1) may enhance muscle protein anabolism and provide a means of reducing the progressive loss of muscle mass with age. The beneficial effects of resistance exercise in aging populations are unequivocal. However, research has not identified a synergistic effect of protein supplementation and resistance exercise in aging populations. There is little evidence that links high protein intakes to increased risk for impaired kidney function in healthy individuals. However, renal function decreases with age, and high protein intake is contraindicated in individuals with renal disease. Assessment of renal function is recommended for older individuals before they adopt a higher-protein diet.

Am J Clin Nutr. 2008 May;87(5):1562S-1566S

Differential stimulation of muscle protein synthesis in elderly humans following isocaloric ingestion of amino acids or whey protein.

To counteract the debilitating progression of sarcopenia, a protein supplement should provide an energetically efficient anabolic stimulus. We quantified net muscle protein synthesis in healthy elderly individuals (65-79 yrs) following ingestion of an isocaloric intact whey protein supplement (WY; n=8) or an essential amino acid supplement (EAA; n=7). Femoral arterio-venous blood samples and vastus lateralis muscle biopsy samples were obtained during a primed, constant infusion of L-[ring-2H5]phenylalanine. Net phenylalanine uptake and mixed muscle fractional synthetic rate (FSR) were calculated during the post-absorptive period and for 3.5 h following ingestion of 15 g EAA or 15 g whey. After accounting for the residual increase in the intracellular phenylalanine pool, net post-prandial phenylalanine uptake was 53.4+/-9.7 mg phe leg-1 (EAA) and 21.7+/-4.6 mg phe leg-1 (WY), (P<0.05). Postabsorptive FSR values were 0.056+/-0.004% h-1 (EAA) and 0.049+/-0.006% h-1 (WY), (P>0.05). Both supplements stimulated FSR (P<0.05), but the increase was greatest in the EAA group with values of 0.088+/-0.011% h-1 (EAA) and 0.066+/-0.004% h-1 (WY), (P<0.05). While both EAA and WY supplements stimulated muscle protein synthesis, EAAs may provide a more energetically efficient nutritional supplement for elderly individuals.

Exp Gerontol. 2006 Feb;41(2):215-9

Amino acid ingestion improves muscle protein synthesis in the young and elderly.

We recently demonstrated that muscle protein synthesis was stimulated to a similar extent in young and elderly subjects during a 3-h amino acid infusion. We sought to determine if a more practical bolus oral ingestion would also produce a similar response in young (34 +/- 4 yr) and elderly (67 +/- 2 yr) individuals. Arteriovenous blood samples and muscle biopsies were obtained during a primed (2.0 micromol/kg) constant infusion (0.05 micromol.kg(-1).min(-1)) of L-[ring-2H5]phenylalanine. Muscle protein kinetics and mixed muscle fractional synthetic rate (FSR) were calculated before and after the bolus ingestion of 15 g of essential amino acids (EAA) in young (n = 6) and elderly (n = 7) subjects. After EAA ingestion, the rate of increase in femoral artery phenylalanine concentration was slower in elderly subjects but remained elevated for a longer period. EAA ingestion increased FSR in both age groups by approximately 0.04%/h (P < 0.05). However, muscle intracellular (IC) phenylalanine concentration remained significantly higher in elderly subjects at the completion of the study (young: 115.6 +/- 5.4 nmol/ml; elderly: 150.2 +/- 19.4 nmol/ml). Correction for the free phenylalanine retained in the muscle IC pool resulted in similar net phenylalanine uptake values in the young and elderly. EAA ingestion increased plasma insulin levels in young (6.1 +/- 1.2 to 21.3 +/- 3.1 microIU/ml) but not in elderly subjects (3.0 +/- 0.6 to 4.3 +/- 0.4 microIU/ml). Despite differences in the time course of plasma phenylalanine kinetics and a greater residual IC phenylalanine concentration, amino acid supplementation acutely stimulated muscle protein synthesis in both young and elderly individuals.

Am J Physiol Endocrinol Metab. 2004 Mar;286(3):E321-8

Increased protein requirements in elderly people: new data and retrospective reassessments.

Dietary protein requirements of elderly people were determined by short-term nitrogen-balance techniques and using calculations recommended by the 1985 Joint FAO/WHO/UNU Expert Consultation. Twelve men and women aged 56-80 y were randomly assigned to groups that consumed either 0.80 +/- 0.01 or 1.62 +/- 0.02 g protein.kg-1.d-1 (mean +/- SEM). Net nitrogen balance was negative for the lower-protein group (-4.6 +/- 3.4 mg N.kg-1.d-1) and positive for the higher-protein group (13.6 +/- 1.0 mg N.kg-1.d-1); the intake required for nitrogen equilibrium was estimated to be 1.00 g.kg-1.d-1. Nitrogen-balance data from three previous protein requirement studies in elderly people were recalculated by using the same balance formula and combined with the current study data to provide an overall weighted mean protein requirement estimate of 0.91 +/- 0.043 g.kg-1.d-1. Together, the current and retrospective nitrogen-balance data suggest that the mean protein requirement in elderly adults is considerably greater than the 0.60 g.kg-1.d-1 established by the 1985 Joint FAO/WHO/UNU Expert Consultation. A safe protein intake for elderly adults would be 1.0-1.25 g.kg-1.d-1 of high-quality protein.

Am J Clin Nutr. 1994 Oct;60(4):501-9

Amino acids and proteins in relation to the nutrition of elderly people.

In this short review some aspects of body protein and amino acid metabolism during ageing in human subjects have been explored. The picture that emerges is a progressive diminution of total body protein with ageing, due largely to a decline in the size of the skeletal muscle mass. These changes are accompanied by a shift in the overall pattern of whole body protein synthesis and breakdown, with muscle mass estimated to account for about 30% of whole body protein turnover in the young adult, as compared with a lower value of 20% or less in the elderly subject. The metabolic significance and possible functional implications of this alteration in the quantitative contribution by muscle to whole body amino acid and protein dynamics have been considered. The determination of requirements for individual essential amino acids and for total protein has been discussed, and it is evident that the data are limited and often contradictory. However, elderly individuals are more likely to be influenced by various biological, environmental and social factors, the effects of which would be generally to increase protein needs above those for younger adults. Thus, in practice, the protein needs of elderly people are probably higher than for the young. The decline in energy intake, together with its possible consequences for reduced dietary protein utilization, will also tend to increase the protein need of elderly subjects, compared with that for physically more active young adults. Until more data become available, it is recommended for food planning purposes that an appropriate protein allowance could be 12-14% of the total energy intake, for mixed protein sources characteristic of the diets of industrialized countries or the more affluent sectors of populations in developing countries. Energy intake should be at a level that meets the estimates proposed by FAO/WHO/UNU for older persons. Tentative recommendations are made herein that intakes of specific indispensable (essential) amino acids, per unit of protein need, should be similar to those for the young school-age child and they should be higher than those currently judged by international authorities (i.e. FAO/WHO/UNU) to be sufficient for maintenance of protein nutritional status in the adult. In view of (i) the increasing proportion of older individuals within technically advanced populations together with the need of this group for health care and (ii) the important role played by diet and food habits in health maintenance, and in the aetiology or progression of degenerative disease, it is vitally necessary to improve upon the current state of knowledge concerning protein and amino acid metabolism and nutrition during the later phases of our lives.

Age Ageing. 1990 Jul;19(4):S10-24

Dietary protein intake is associated with lean mass change in older, community-dwelling adults: the Health, Aging, and Body Composition (Health ABC) Study.

BACKGROUND: Dietary surveys suggest that many older, community-dwelling adults consume insufficient dietary protein, which may contribute to the age-related loss of lean mass (LM). OBJECTIVE: The objective of the study was to determine the association between dietary protein and changes in total LM and nonbone appendicular LM (aLM) in older, community-dwelling men and women. DESIGN: Dietary protein intake was assessed by using an interviewer-administered 108-item food-frequency questionnaire in men and women aged 70-79 y who were participating in the Health, Aging, and Body Composition study (n=2066). Changes in LM and aLM over 3 y were measured by using dual-energy X-ray absorptiometry. The association between protein intake and 3-y changes in LM and aLM was examined by using multiple linear regression analysis adjusted for potential confounders. RESULTS: After adjustment for potential confounders, energy-adjusted protein intake was associated with 3-y changes in LM [beta (SE): 8.76 (3.00), P=0.004] and aLM [beta (SE): 5.31 (1.64), P=0.001]. Participants in the highest quintile of protein intake lost approximately 40% less LM and aLM than did those in the lowest quintile of protein intake (x+/-SE: -0.501+/-0.106 kg compared with -0.883+/-0.104 kg for LM; -0.400+/-0.058 kg compared with -0.661+/-0.057 kg for aLM; P for trend<0.01). The associations were attenuated slightly after adjustment for change in fat mass, but the results remained significant. CONCLUSION: Dietary protein may be a modifiable risk factor for sarcopenia in older adults and should be studied further to determine its effects on preserving LM in this population.

Am J Clin Nutr. 2008 Jan;87(1):150-5

Protein turnover and requirements in the healthy and frail elderly.

There are as yet no definitive data that warrant the establishment of evidence-based dietary protein recommendations for the elderly. We reviewed the relevance of the new 2002 recommended protein intake of 0.80 g/kg body weight.d for adults to healthy and frail elderly persons. We found that data from published nitrogen balance studies indicate that, a higher protein intake of 1.0 - 1.3 g/k.d is required to maintain nitrogen balance in the healthy elderly, which may be explained by their lower energy intake and impaired insulin action during feeding compared with young persons. Although it needs to be confirmed, a decrease in efficiency of protein utilization with aging may also dictate a higher protein-intake recommendation. Measures of the dynamic aspects of protein metabolism done in the postabsorptive state have shown no change in whole body protein turnover per unit of active metabolic tissue with aging. However, the contribution of muscle protein to wholebody protein metabolism was significantly reduced in the elderly, and explained by their reduced muscle mass and lower rates of myofibrillar protein turnover. Consequently, the contribution of nonmuscle protein, especially that of visceral tissue whose rates of protein turnover are known to be more rapid was proportionally greater with aging. It is conceivable that higher protein consumption rates could compensate for the decrease in availability of muscle amino acids and spare the muscle mass. Despite a paucity of data on the frail elderly population, we present a rationale to justify a greater protein intake of at least equivalent to that of their healthy counterparts. We propose that higher protein intakes for the elderly, and especially the frail population, than those presently recommended may minimize the sarcopenia of aging and thereby protect against some of the health risks of aging.

J Nutr Health Aging. 2006 Jul-Aug;10(4):272-83

Protein requirement of elderly women: nitrogen balance responses to three levels of protein intake.

BACKGROUND: For elderly women, insufficient data exist to assess the accuracy of the assumed mean protein requirement of 0.6 g of protein x kg(-1) x day(-1), and the adequacy of the current Recommended Dietary Allowance (RDA) of 0.8 g of protein x kg(-1) x day(-1). The aims of this study were to assess the mean protein requirement and suggested safe and adequate protein intake (protein allowance) of elderly women using a shorter-term nitrogen balance protocol. METHODS: During three separate 18-day trials, 11 elderly women (age range, 70-81 years) were randomly fed eucaloric diets designed to provide either 0.50, 0.75, or 1.00 g of protein x kg(-1) x day(-1). Nitrogen balance was determined at Weeks 2 and 3 (Days 7-10 and 14-17, respectively) of each trial using data from total nitrogen analyses of duplicate food composites, 24-hour urine collections, and stool collections. The mean protein requirement was calculated using linear regression of individual women’s data from all three trials and inverse prediction. RESULTS: At protein intakes of 0.53 +/- 0.02, 0.76 +/- 0.02, or 1.06 +/- 0.05 g of protein x kg(-1) x day(-1), net nitrogen balances during Week 2 were -14.5 +/- 3.1, 3.8 +/- 2.5 and 23.4 +/- 3.3 mg of nitrogen x kg(-1) x day(-1), respectively, for these body weight- and body composition-stable women. At Week 3, the net nitrogen balances were -0.1 +/- 2.7, 8.5 +/- 3.6 and 42.0 +/- 3.0 mg of nitrogen x kg(-1) x day(-1). From Week 2 to Week 3, shifts to more positive nitrogen balances occurred due to decreases in urinary nitrogen excretion. The mean protein requirement at Week 2 was calculated to be 0.70 +/- 0.09 g of protein. kg(-1) x day(-1) (coefficient of variation [CV] = 13%) and at Week 3 was calculated to be 0.56 +/- 0.09 g of protein x kg(-1) x day(-1) (CV = 17%). From these data, an adequate protein allowance was estimated to be greater than the RDA at Week 2 (0.90 g of protein x kg(-1) x day [d](-1)), and not different than the RDA at Week 3 (0.76 g of protein x kg(-1) x d(-1)). CONCLUSIONS: The decrease over time in urinary nitrogen excretion from Week 2 to Week 3 suggests that these elderly women did not achieve a metabolic steady state during this shorter-term nitrogen balance study. Collectively, these data suggest that the total protein needs of elderly women are at or above the current RDA for protein. However, the results of this study indicate that shorter-term nitrogen balance protocols are insufficient to firmly establish the RDA for protein of elderly women, and further research is required using alternative criteria measures.

J Gerontol A Biol Sci Med Sci. 2001 Nov;56(11):M724-30

The protein digestibility-corrected amino acid score.

The protein digestibility-corrected amino acid score (PDCAAS) has been adopted by FAO/WHO as the preferred method for the measurement of the protein value in human nutrition. The method is based on comparison of the concentration of the first limiting essential amino acid in the test protein with the concentration of that amino acid in a reference (scoring) pattern. This scoring pattern is derived from the essential amino acid requirements of the preschool-age child. The chemical score obtained in this way is corrected for true fecal digestibility of the test protein. PDCAAS values higher than 100% are not accepted as such but are truncated to 100%. Although the principle of the PDCAAS method has been widely accepted, critical questions have been raised in the scientific community about a number of issues. These questions relate to 1) the validity of the preschool-age child amino acid requirement values, 2) the validity of correction for fecal instead of ileal digestibility and 3) the truncation of PDCAAS values to 100%. At the time of the adoption of the PDCAAS method, only a few studies had been performed on the amino acid requirements of the preschool-age child, and there is still a need for validation of the scoring pattern. Also, the scoring pattern does not include conditionally indispensable amino acids. These amino acids also contribute to the nutrition value of a protein. There is strong evidence that ileal, and not fecal, digestibility is the right parameter for correction of the amino acid score. The use of fecal digestibility overestimates the nutritional value of a protein, because amino acid nitrogen entering the colon is lost for protein synthesis in the body and is, at least in part, excreted in urine as ammonia. The truncation of PDCAAS values to 100% can be defended only for the limited number of situations in which the protein is to be used as the sole source of protein in the diet. For evaluation of the nutritional significance of proteins as part of mixed diets, the truncated value should not be used. In those cases, a more detailed evaluation of the contribution of the protein to the amino acid composition of the mixed diet is required. From such an evaluation, it appears that milk proteins are superior to plant proteins in cereal-based diets.

J Nutri. 2000 Jul;130(7):18655-75

Amino acid supplementation increases lean body mass, basal muscle protein synthesis, and insulin-like growth factor-I expression in older women.

CONTEXT: Inadequate dietary protein intake has been implicated in sarcopenia. OBJECTIVE AND DESIGN: The objectives of this study were to determine whether: 1) chronic essential amino acid (EAA) supplementation improves postabsorptive muscle protein fractional synthesis rate (FSR), lean body mass (LBM), and one-repetition maximum muscle strength, and androgen receptor and IGF-I muscle protein expression; and 2) the acute anabolic response to EAA ingestion is preserved after a 3-month supplementation period. Using a randomized, double-blinded, placebo-controlled design, older women (68 +/- 2 yr) were assigned to receive either placebo (n = 7), or 15 g EAA/d [supplemented treatment group (SUP)] (n = 7) for 3 months. Metabolic outcomes were assessed in association with stable isotope studies conducted at 0 and 3 months. SETTING: The study was performed at The University of Texas Medical Branch General Clinical Research Center. RESULTS: Ingestion of 7.5 g EAA acutely stimulated FSR in both groups at 0 months (P < 0.05). Basal FSR at 3 months was increased in SUP only. The magnitude of the acute response to EAA was unaltered after 3 months in SUP. LBM increased in SUP only (P < 0.05). One-repetition maximum strength remained unchanged in both groups. Basal IGF-I protein expression increased in SUP after 3 months (P = 0.05), with no changes in androgen receptor or total and phosphorylated Akt, mammalian target of rapamycin, S6 kinase, and 4E-binding protein.

CONCLUSIONS: EAA improved LBM and basal muscle protein synthesis in older individuals. The acute anabolic response to EAA supplementation is maintained over time and can improve LBM, possibly offsetting the debilitating effects of sarcopenia.

J Clin Endocrinol Metab. 2009 May;94(5):1630-7

Long-term metformin use is associated with decreased risk of breast cancer.

OBJECTIVE: To evaluate whether use of oral hypoglycemic agents is associated with an altered breast cancer risk in women. RESEARCH DESIGN AND METHODS: Using the U.K.-based General Practice Research Database, we conducted a nested case-control analysis among 22,621 female users of oral antidiabetes drugs with type 2 diabetes. We evaluated whether they had an altered risk of breast cancer in relation to use of various types of oral hypoglycemic agents. Case and control patients with a recorded diagnosis of type 2 diabetes were matched on age, calendar time, and general practice, and the multivariate conditional logistic regression analyses were further adjusted for use of oral antidiabetes drugs, insulin, estrogens, smoking BMI, diabetes duration, and HbA1c (A1C). RESULTS: We identified 305 case patients with a recorded incident diagnosis of breast cancer. The mean +/- SD age was 67.5 +/- 10.5 years at the time of the cancer diagnosis. Long-term use of >or=40 prescriptions (>5 years) of metformin, based on 17 exposed case patients and 120 exposed control patients, was associated with an adjusted odds ratio of 0.44 (95% CI 0.24-0.82) for developing breast cancer compared with no use of metformin. Neither short-term metformin use nor use of sulfonylureas or other antidiabetes drugs was associated with a materially altered risk for breast cancer. CONCLUSIONS: A decreased risk of breast cancer was observed in female patients with type 2 diabetes using metformin on a long-term basis.

Diabetes Care. 2010 Jun;33(6):1304-8

Metformin attenuates the stimulatory effect of a high-energy diet on in vivo LLC1 carcinoma growth.

We investigated the effects of metformin on the growth of lewis lung LLC1 carcinoma in C57BL/6J mice provided with either a control diet or a high-energy diet, previously reported to lead to weight gain and systemic insulin resistance with hyperinsulinemia. Forty-eight male mice were randomized into four groups: control diet, control diet+metformin, high-energy diet, or high-energy diet+metformin. Following 8 weeks on the experimental diets, selected groups received metformin in their drinking water. Three weeks following the start of metformin treatment, mice were injected with 0.5x10(6) LLC1 cells and tumor growth was measured for 17 days. By day 17, tumors of mice on the high-energy diet were nearly twice the volume of those of mice on the control diet. This effect of diet on tumor growth was significantly attenuated by metformin, but metformin had no effect on tumor growth of the mice on the control diet. Metformin attenuated the increased insulin receptor activation associated with the high-energy diet and also led to increased phosphorylation of AMP kinase, two actions that would be expected to decrease neoplastic proliferation. These experimental results are consistent with prior hypothesis-generating epidemiological studies that suggest that metformin may reduce cancer risk and improve cancer prognosis. Finally, these results contribute to the rationale for evaluation of the anti-neoplastic activity of metformin in hyperinsulinemic cancer patients.

Endocr Relat Cancer. 2008 Sep;15(3):833-9

Metformin: new understandings, new uses.

Metformin, a biguanide, has been available in the US for the treatment of type 2 diabetes mellitus for nearly 8 years. Over this period of time, it has become the most widely prescribed antihyperglycaemic agent. Its mechanism of action involves the suppression of endogenous glucose production, primarily by the liver. Whether the drug actually has an insulin sensitising effect in peripheral tissues, such as muscle and fat, remains somewhat controversial. Nonetheless, because insulin levels decline with metformin use, it has been termed an ‘insulin sensitiser’. Metformin has also been shown to have several beneficial effects on cardiovascular risk factors and it is the only oral antihyperglycaemic agent thus far associated with decreased macrovascular outcomes in patients with diabetes. Cardiovascular disease, impaired glucose tolerance and the polycystic ovary syndrome are now recognised as complications of the insulin resistance syndrome, and there is growing interest in the management of this extraordinarily common metabolic disorder. While diet and exercise remain the cornerstone of therapy for insulin resistance, pharmacological intervention is becoming an increasingly viable option. We review the role of metformin in the treatment of patients with type 2 diabetes and describe the additional benefits it provides over and above its effect on glucose levels alone. We also discuss its potential role for a variety of insulin resistant and prediabetic states, including impaired glucose tolerance, obesity, polycystic ovary syndrome and the metabolic abnormalities associated with HIV disease.

Drugs. 2003;63(18):1879-94

Decreased mortality associated with the use of metformin compared with sulfonylurea monotherapy in type 2 diabetes.

OBJECTIVE: The aim of this study was to examine the relationship between use of metformin and sulfonylurea and mortality in new users of these agents. RESEARCH DESIGN AND METHODS: Saskatchewan Health databases were used to examine population-based mortality rates for new users of oral antidiabetic agents. Individuals with prescriptions for sulfonylurea or metformin in 1991-1996 and no use in the year prior were identified as new users. Prescription records were prospectively followed for 1-9 years; subjects with any insulin use were excluded. Causes of death were identified based on ICD-9 codes in an electronic vital statistics database. Multivariate logistic regression and survival analyses were used to assess the differences in mortality between drug cohorts, after adjusting for potential confounding variables.RESULTS: The total study sample comprised 12,272 new users of oral antidiabetic agents; the average length of follow-up was 5.1 (SD 2.2) years. In subjects with at least 1 year of drug exposure and no insulin use, mortality rates were 750/3,033 (24.7%) for those receiving sulfonylurea monotherapy, 159/1,150 (13.8%) for those receiving metformin monotherapy, and 635/4,683 (13.6%) for those receiving combination therapy over an average 5.1 (SD 2.2) years of follow-up. The adjusted odds ratio (OR) for all-cause mortality for metformin monotherapy was 0.60 (95% CI 0.49-0.74) compared with sulfonylurea monotherapy. Sulfonylurea plus metformin combination therapy was also associated with reduced all-cause mortality (OR 0.66, 95% CI 0.58-0.75). Reduced cardiovascular-related mortality rates were also observed in metformin users compared with sulfonylurea monotherapy users. CONCLUSIONS: Metformin therapy, alone or in combination with sulfonylurea, was associated with reduced all-cause and cardiovascular mortality compared with sulfonylurea monotherapy among new users of these agents.

Diabetes Care. 2002 Dec;25(12):2244-8

Metformin inhibits aromatase expression in human breast adipose stromal cells via stimulation of AMP-activated protein kinase.

AMP-activated protein kinase (AMPK) is recognized as a master regulator of energy homeostasis. In concert with the AMPK-kinase LKB1, it has been shown to provide a molecular link between obesity and postmenopausal breast cancer via its actions to inhibit aromatase expression, hence estrogen production, within the breast. The anti-diabetic drug metformin is known to increase the activity of AMPK and was therefore hypothesized to inhibit aromatase expression in primary human breast adipose stromal cells. Results demonstrate that metformin significantly decreases the forskolin/phorbol ester (FSK/PMA)-induced expression of aromatase at concentrations of 10 and 50 muM. Consistent with the hypothesized actions of metformin to increase AMPK activity, treatment with 50 muM metformin results in a significant increase in phosphorylation of AMPK at Thr172. Interestingly, metformin also causes a significant increase in LKB1 protein expression and promoter activity, thereby providing for the first time an additional mechanism by which metformin activates AMPK. Furthermore, metformin inhibits the nuclear translocation of CRTC2, a CREB-coactivator known to increase aromatase expression which is also a direct downstream target of AMPK. Overall, these results suggest that metformin would reduce the local production of estrogens within the breast thereby providing a new key therapeutic tool that could be used in the neoadjuvant and adjuvant settings and conceivably also as a preventative measure in obese women.

Breast Cancer Res Treat. 2010 Sep;123(2):591-6

Is it the time for metformin to take place in adjuvant treatment of Her-2 positive breast cancer? Teaching new tricks to old dogs.

Breast cancer is the most common malignancy diagnosed among women. According to the new molecular subclassification, basal like and Her-2 positive breast cancers have the worst outcome and these are the ones in which chemotherapy is a must as a part of adjuvant treatment. New treatment options that could be used as an adjuvant maintenance treatment are still being investigated. Insulin hormone is one of the reasons of breast cancer recurrence and death in breast cancer survivors. Targeting insulin as a therapeutic modality in breast cancer could be an option in the adjuvant treatment of breast cancer. It seems that insulin may signal to activate a cascade of proliferative and anti-apoptotic events in the cancer cell. Metformin, an oral anti-diabetic known for 50 years, may also have direct effects on cancer cells. Metformin causes Her-2 suppression via the inhibition of mTOR in breast cancer cells. Thus, we believe that the time has arrived both to target insulin reduction and to alter Her-2 oncogene based molecular pathogenetic steps in breast cancer by using metformin as an adjuvant therapy in breast cancer patients.

Med Hypotheses. 2009 Oct;73(4):606-7

Metformin and cancer: Doses, mechanisms and the dandelion and hormetic phenomena.

In the early 1970s, Professor Vladimir Dilman originally developed the idea that antidiabetic biguanides may be promising as geroprotectors and anticancer drugs (“metabolic rehabilitation”). In the early 2000s, Anisimov’s experiments revealed that chronic treatment of female transgenic HER2-/neu mice with metformin significantly reduced the incidence and size of mammary adenocarcinomas and increased the mean latency of the tumors. Epidemiological studies have confirmed that metformin, but not other anti-diabetic drugs, significantly reduces cancer incidence and improves cancer patients’ survival in type 2 diabetics. At present, pioneer work by Dilman & Anisimov at the Petrov Institute of Oncology (St. Petersburg, Russia) is rapidly evolving due to ever-growing preclinical studies using human tumor-derived cultured cancer cells and animal models. We herein critically review how the antidiabetic drug metformin is getting reset to metabolically fight cancer. Our current perception is that metformin may constitute a novel “hybrid anti-cancer pill” physically combining both the long-lasting effects of antibodies-by persistently lowering levels of blood insulin and glucose-and the immediate potency of a cancer cell-targeting molecular agent-by suppressing the pivotal AMPK/mTOR/S6K1 axis and several protein kinases at once, including tyrosine kinase receptors such as HER1 and HER2-. In this scenario, we discuss the relevance of metformin doses in pre-clinical models regarding metformin’s mechanisms of action in clinical settings. We examine recent landmark studies demonstrating metformin’s ability to specifically target the cancer-initiating stem cells from which tumor cells develop, thereby preventing cancer relapse when used in combination with cytotoxic chemotherapy (dandelion hypothesis). We present the notion that, by acting as an efficient caloric restriction mimetic, metformin enhanced intrinsic capacity of mitotically competent cells to self-maintenance and repair (hormesis) might trigger counterintuitive detrimental effects. Ongoing chemopreventive, neoadjuvant and adjuvant trials should definitely establish whether metformin’s ability to kill the “dandelion root” beneath the “cancer soil” likely exceeds metformin-related dangers of hormesis.

Cell Cycle. 2010 Mar 21;9(6)

Improved clinical outcomes associated with metformin in patients with diabetes and heart failure.

OBJECTIVE: Metformin is considered contraindicated in patients with heart failure because of concerns over lactic acidosis, despite increasing evidence of potential benefit. The aim of this study was to evaluate the association between metformin and clinical outcomes in patients with heart failure and type 2 diabetes. RESEARCH DESIGN AND METHODS: Using the Saskatchewan Health databases, 12,272 new users of oral antidiabetic agents were identified between the years 1991 and 1996. Subjects with incident heart failure (n = 1,833) were identified through administrative records based on ICD-9 code 428 and grouped according to antidiabetic therapy: metformin monotherapy (n = 208), sulfonylurea monotherapy (n = 773), or combination therapy (n = 852). Multivariate Cox proportional hazards models were used to assess differences in all-cause mortality, all-cause hospitalization, and the combination (i.e., all-cause hospitalization or mortality). RESULTS: Average age of subjects was 72 years, 57% were male, and average follow-up was 2.5 +/- 2.0 (SD) years. Compared with sulfonylurea therapy, fewer deaths occurred in subjects receiving metformin: 404 (52%) for sulfonylurea monotherapy versus 69 (33%) for metformin monotherapy (hazard ratio [HR] 0.70 [95% CI 0.54-0.91]) and 263 (31%) for combination therapy (0.61 [0.52-0.72]). A reduction in deaths or hospitalizations was also observed: 658 (85%) for sulfonylurea monotherapy versus 160 (77%) for metformin monotherapy (0.83 [0.70-0.99]) and 681 (80%) for combination therapy (0.86 [0.77-0.96]). There was no difference in time to first hospitalization between study groups. CONCLUSIONS: Metformin, alone or in combination, in subjects with heart failure and type 2 diabetes was associated with lower morbidity and mortality compared with sulfonylurea monotherapy.

Diabetes Care. 2005 Oct;28(10):2345-51

Metformin attenuates ovarian cancer cell growth in an AMP- kinase dispensable manner.

ABSTRACT Metformin, the most widely used drug for type 2 diabetes activates AMP-activated protein kinase (AMPK), which regulates cellular energy metabolism. Here, we report that ovarian cell lines VOSE, A2780, CP70, C200, OV202, OVCAR3, SKOV3ip, PE01 and PE04 predominantly express -alpha1, -beta1, -gamma1 and -gamma2 isoforms of AMPK subunits. Our studies show that metformin treatment (1) significantly inhibited proliferation of diverse chemo-responsive and -resistant ovarian cancer cell lines (A2780, CP70, C200, OV202, OVCAR3, SKVO3ip, PE01 and PE04), (2) caused cell cycle arrest accompanied by decreased cyclin D1 and increased p21 protein expression, (3) activated AMPK in various ovarian cancer cell lines as evident from increased phosphorylation of AMPKalpha and its downstream substrate; ACC (Acetyl Co-carboxylase) and enhanced beta- oxidation of fatty acid, (4) attenuated mTOR-S6RP phosphorylation, inhibited protein translational and lipid biosynthetic pathways, thus implicating metformin as a growth inhibitor of ovarian cancer cells. We also show that metformin mediated effect on AMPK is dependent on LKB1(Liver kinase B1) as it failed to activate AMPK-ACC pathway and cell cycle arrest in LKB1 null mouse embryo fibroblasts (mefs). This observation was further supported by using siRNA approach to downregulate LKB1 in ovarian cancer cells. In contrast, metformin inhibited cell proliferation in both wild type and AMPKalpha1/2 null mefs as well as in AMPK silenced ovarian cancer cells. Collectively, these results provide evidence on the role of metformin as an anti-proliferative therapeutic that can act through both AMPK dependent as well as independent pathways.

J Cell Mol Med. 2009 Oct 29

New users of metformin are at low risk of incident cancer: a cohort study among people with type 2 diabetes.

OBJECTIVE: The antidiabetic properties of metformin are mediated through its ability to activate the AMP-activated protein kinase (AMPK). Activation of AMPK can suppress tumor formation and inhibit cell growth in addition to lowering blood glucose levels. We tested the hypothesis that metformin reduces the risk of cancer in people with type 2 diabetes. RESEARCH DESIGN AND METHODS: In an observational cohort study using record-linkage databases and based in Tayside, Scotland, U.K., we identified people with type 2 diabetes who were new users of metformin in 1994-2003. We also identified a set of diabetic comparators, individually matched to the metformin users by year of diabetes diagnosis, who had never used metformin. In a survival analysis we calculated hazard ratios for diagnosis of cancer, adjusted for baseline characteristics of the two groups using Cox regression. RESULTS: Cancer was diagnosed among 7.3% of 4,085 metformin users compared with 11.6% of 4,085 comparators, with median times to cancer of 3.5 and 2.6 years, respectively (P < 0.001). The unadjusted hazard ratio (95% CI) for cancer was 0.46 (0.40-0.53). After adjusting for sex, age, BMI, A1C, deprivation, smoking, and other drug use, there was still a significantly reduced risk of cancer associated with metformin: 0.63 (0.53-0.75). CONCLUSIONS: These results suggest that metformin use may be associated with a reduced risk of cancer. A randomized trial is needed to assess whether metformin is protective in a population at high risk for cancer.

Diabetes Care. 2009 Sep;32(9):1620-5

Metformin, independent of AMPK, inhibits mTORC1 in a rag GTPase-dependent manner.

Dysfunctional mTORC1 signaling is associated with a number of human pathologies owing to its central role in controlling cell growth, proliferation, and metabolism.

Regulation of mTORC1 is achieved by the integration of multiple inputs, including those of mitogens, nutrients, and energy. It is thought that agents that increase the cellular AMP/ATP ratio, such as the antidiabetic biguanides metformin and phenformin, inhibit mTORC1 through AMPK activation of TSC1/2-dependent or -independent mechanisms. Unexpectedly, we found that biguanides inhibit mTORC1 signaling, not only in the absence of TSC1/2 but also in the absence of AMPK. Consistent with these observations, in two distinct preclinical models of cancer and diabetes, metformin acts to suppress mTORC1 signaling in an AMPK-independent manner. We found that the ability of biguanides to inhibit mTORC1 activation and signaling is, instead, dependent on the Rag GTPases.

Cell Metab. 2010 May 5;11(5):390-401

Metformin Suppresses Colorectal Aberrant Crypt Foci in a Short-term Clinical Trial.

The biguanide metformin is widely used for treating diabetes mellitus. We previously showed the chemopreventive effect of metformin in two rodent models of colorectal carcinogenesis. However, besides epidemiologic studies, little is known about the effects of metformin on human colorectal carcinogenesis. The objective of this pilot study was to evaluate the chemopreventive effect of metformin on rectal aberrant crypt foci (ACF), which are an endoscopic surrogate marker of colorectal cancer. We prospectively randomized 26 nondiabetic patients with ACF to treatment with metformin (250 mg/d, n = 12) or no treatment (control, n = 14); 23 patients were evaluable for end point analyses (9 metformin and 14 control); the two groups were similar in ACF number and other baseline clinical characteristics. Magnifying colonoscopy determined the number of rectal ACF in each patient at baseline and after 1 month in a blinded fashion (as were all laboratory end point analyses). We also examined proliferative activity in colonic epithelium (via proliferating cell nuclear antigen labeling index) and apoptotic activity (via terminal deoxynucleotidyl transferase dUTP nick-end labeling). At 1 month, the metformin group had a significant decrease in the mean number of ACF per patient (8.78 +/- 6.45 before treatment versus 5.11 +/- 4.99 at 1 month, P = 0.007), whereas the mean ACF number did not change significantly in the control group (7.23 +/- 6.65 versus 7.56 +/- 6.75, P = 0.609). The proliferating cell nuclear antigen index was significantly decreased and the apoptotic cell index remained unaltered in normal rectal epithelium in metformin patients. This first reported trial of metformin for inhibiting colorectal carcinogenesis in humans provides preliminary evidence that metformin suppresses colonic epithelial proliferation and rectal ACF formation in humans, suggesting its promise for the chemoprevention of colorectal cancer.

Cancer Prev Res (Phila Pa). 2010 Sep;3(9):1077-83

Metformin prevents tobacco carcinogen-induced lung tumorigenesis.

Activation of the mammalian target of rapamycin (mTOR) pathway is an important and early event in tobacco carcinogen-induced lung tumorigenesis, and therapies that target mTOR could be effective in the prevention or treatment of lung cancer. The biguanide metformin, which is widely prescribed for the treatment of type II diabetes, might be a good candidate for lung cancer chemoprevention because it activates AMP-activated protein kinase (AMPK), which can inhibit the mTOR pathway. To test this, A/J mice were treated with oral metformin after exposure to the tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Metformin reduced lung tumor burden by up to 53% at steady-state plasma concentrations that are achievable in humans. mTOR was inhibited in lung tumors but only modestly. To test whether intraperitoneal administration of metformin might improve mTOR inhibition, we injected mice and assessed biomarkers in liver and lung tissues. Plasma levels of metformin were significantly higher after injection than oral administration. In liver tissue, metformin activated AMPK and inhibited mTOR. In lung tissue, metformin did not activate AMPK but inhibited phosphorylation of insulin-like growth factor-I receptor/insulin receptor (IGF-1R/IR), Akt, extracellular signal-regulated kinase (ERK), and mTOR. This suggested that metformin indirectly inhibited mTOR in lung tissue by decreasing activation of insulin-like growth factor-I receptor/insulin receptor and Akt upstream of mTOR. Based on these data, we repeated the NNK-induced lung tumorigenesis study using intraperitoneal administration of metformin. Metformin decreased tumor burden by 72%, which correlated with decreased cellular proliferation and marked inhibition of mTOR in tumors. These studies show that metformin prevents tobacco carcinogen-induced lung tumorigenesis and support clinical testing of metformin as a chemopreventive agent.

Cancer Prev Res (Phila Pa). 2010 Sep;3(9):1066-76

Metformin extended release treatment of adolescent obesity: a 48-week randomized, double-blind, placebo-controlled trial with 48-week follow-up.

BACKGROUND: Metformin has been proffered as a therapy for adolescent obesity, although long-term controlled studies have not been reported. OBJECTIVE: To test the hypothesis that 48 weeks of daily metformin hydrochloride extended release (XR) therapy will reduce body mass index (BMI) in obese adolescents, as compared with placebo. DESIGN: Multicenter, randomized, double-blind, placebo-controlled clinical trial. SETTING: The 6 centers of the Glaser Pediatric Research Network from October 2003 to August 2007. PARTICIPANTS: Obese (BMI > or = 95th percentile) adolescents (aged 13-18 years) were randomly assigned to the intervention (n = 39) or placebo groups. Intervention Following a 1-month run-in period, subjects following a lifestyle intervention program were randomized 1:1 to 48 weeks’ treatment with metformin hydrochloride XR, 2,000 mg once daily, or an identical placebo. Subjects were monitored for an additional 48 weeks. Main Outcome Measure Change in BMI, adjusted for site, sex, race, ethnicity, and age and metformin vs placebo. RESULTS: After 48 weeks, mean (SE) adjusted BMI increased 0.2 (0.5) in the placebo group and decreased 0.9 (0.5) in the metformin XR group (P = .03). This difference persisted for 12 to 24 weeks after cessation of treatment. No significant effects of metformin on body composition, abdominal fat, or insulin indices were observed. CONCLUSION: Metformin XR caused a small but statistically significant decrease in BMI when added to a lifestyle intervention program.

Arch Pediatr Adolesc Med. 2010 Feb;164(2):116-23

Vitamins K2, K3 and K5 exert in vivo antitumor effects on hepatocellular carcinoma by regulating the expression of G1 phase-related cell cycle molecules.

A number of studies have shown that various vitamins K, specifically vitamin K2, possessed antitumor activity on various types of rodent- and human-derived neoplastic cell lines. However, there are only a small number of reports demonstrating in vivo antitumor effects of vitamins K. Furthermore, the mechanism of antitumor effects of vitamins K still remains to be examined. In the present study, we examined the antitumor effects of vitamins K2, K3 and K5 on PLC/PRF/5 human hepatocellular carcinoma (HCC) cells in vivo. Furthermore, to examine the mechanism of antitumor actions of these vitamins K, mRNA expression levels of various G1 phase-related cell cycle molecules were evaluated by using a real-time reverse transcription-polymerase chain reaction (RT-PCR) method. HCC-bearing animals were produced by implanting PLC/PRF/5 cells subcutaneously into athymic nude mice, and drinking water containing vitamin K2, K3 or K5 was given to the animals. Treatments with vitamins K2, K3 and K5 were shown to markedly inhibit the growth of HCC tumors. To examine the mechanism of in vivo antitumor effects of vitamins K, total RNA was extracted from HCC tumors, and the expression of G1 phase-related cell cycle molecules was quantitatively examined. Real-time RT-PCR demonstrated that the expression of the cell cycle-driving molecule, cyclin-dependent kinase 4 (Cdk4), in HCC was significantly reduced by the treatments with vitamin K2, K3 and K5. Conversely, the expression of the cell cycle-suppressing molecules, Cdk inhibitor p16INK4a and retinoblastoma, in HCC was significantly enhanced by the treatments with vitamins K2, K3 and K5. These results indicate that vitamins K2, K3 and K5 exert antitumor effects on HCC by regulating the expression of G1 phase-related cell cycle molecules. These results also indicate that vitamins K2, K3 and K5 may be useful agents for the treatment of patients with HCC.

Int J Oncol. 2005 Aug;27(2):505-11

Effect of vitamin K2 on the recurrence in patients with hepatocellular carcinoma.

BACKGROUND/AIMS: Vitamin K2 (VK2) appears to have a potent inhibitory activity for cell growth including HCC cells. We investigated whether VK2 could reduce incidence of tumor recurrence after treatment of HCC. Forty-five patients with cured or possibly cured HCC were randomly selected, assigning patients to treatment (n=21) or control group (n=24) with randomization list. METHODOLOGY: For the treatment group, forty-five mg of Glakay was given orally every day after therapy for HCC. No patients complained of adverse effects. Abdominal ultrasonography and dynamic CT were performed at 3-month intervals. Recurrence was confirmed by abdominal angiography. RESULTS: Recurrence of HCC occurred in 7 cases (33.3%) for the treatment group and 12 cases (50.0%) for the control group during mean observation periods of 19.5 and 16.5 months, respectively. Administration of VK2 was not an independent variable for the recurrence on univariate analysis. Cumulative incidence of HCC recurrence did not differ between the two groups, and the cumulative survival rate tended to be high in treatment group (p =0.054). Cox regression analysis revealed that serum albumin concentration alone was an independent factor affecting the recurrence. CONCLUSIONS: These findings suggest that VK2 does not appear to prevent recurrence of HCC after curative treatment. Our study is preliminary and large-scale trials are needed to determine whether VK2 is of benefit to decrease the recurrence of HCC.

Hepatogastroenterology. 2007 Oct-Nov;54(79):2073-7

Preventive effects of vitamin K on recurrent disease in patients with hepatocellular carcinoma arising from hepatitis C viral infection.

BACKGROUND: Despite the progression of therapeutic approaches, a high frequency of recurrence is what determines the long-term prognosis of patients with hepatocellular carcinoma (HCC). In this study, the chemopreventive effects of vitamin K2 on the recurrence and survival of patients with HCC after curative therapy were evaluated. METHODS: Sixty patients who were diagnosed to be free of HCC after radiofrequency ablation therapy or surgery were randomly assigned to either the vitamin K2 group (n = 30 patients) or the control group (n = 30 patients). All patients were positive for the hepatitis C virus (HCV) antibody and hepatitis B surface antigen positive patients were excluded from this study. Patients in the vitamin K2 group received an oral dose of menatetrenone at 45 mg per day. Disease recurrence and the survival rates were analyzed in patients with HCC. RESULTS: The cumulative recurrence-free rates in the vitamin K2 group were 92.3% at 12 months, 48.6% at 24 months and 38.8% at 36 months; and those in the control group were 71.7%, 35.9% and 9.9%, respectively (P = 0.045). The cumulative survival rates in the vitamin K2 group were 100% at 12 months, 95.0% at 24 months and 77.5% at 36 months; and those in the control group were 95.8%, 90.2% and 66.4%, respectively (P = 0.70). CONCLUSIONS: Vitamin K2 may have a suppressive effect on the recurrence of HCC and a beneficial effect on tumor recurrence. However, there was no significant difference in the survival rates. The chemopreventive effects of vitamin K2 are not sufficient. The development of a further regimen such as combination therapy is required.

J Gastroenterol Hepatol. 2007 Apr;22(4):518-22

Combination of vitamin K2 and angiotensin-converting enzyme inhibitor ameliorates cumulative recurrence of hepatocellular carcinoma.

BACKGROUND/AIMS: No chemopreventive agent has been approved against hepatocellular carcinoma (HCC) yet. Since neovascularization plays a pivotal role in HCC, an angiostatic agent is considered as one of the promising approaches. The aim of this study was to elucidate the combined effect of the clinically used vitamin K(2) (VK) and angiotensin-converting enzyme inhibitor (ACE-I) on cumulative recurrence after curative treatment on a total of 87 patients, especially in consideration of neovascularization.METHODS: VK (menatetrenone; 45 mg/day) and/or ACE-I (perindopril; 4 mg/day) were administered for 36-48 months after curative therapy for HCC. The cumulative recurrence and several indices were analyzed. RESULTS: A 48-month follow-up revealed that the combination treatment with VK and ACE-I markedly inhibited the cumulative recurrence of HCC in association with suppression of the serum level of the vascular endothelial growth factor (VEGF); a central angiogenic factor. The serum level of lectin-reactive alpha-fetoprotein was also suppressed almost in parallel with VEGF. These beneficial effects were not observed with single treatment using VK or ACE-I. CONCLUSIONS: The combination treatment of VK and ACE-I may suppress the cumulative recurrence of HCC after the curative therapy, at least partly through suppression of the VEGF-mediated neovascularization.

J Hepatol. 2009 Aug;51(2):315-21

Combined treatment of vitamin K2 and angiotensin-converting enzyme inhibitor ameliorates hepatic dysplastic nodule in a patient with liver cirrhosis.

Although it is well known that the hepatocellular carcinoma (HCC) is an ominous complication in patients with liver cirrhosis, there has been no approved drug to prevent the development of HCC to date. We previously reported that the combined treatment of vitamin K2 (VK) and angiotensin-converting enzyme inhibitor (ACE-I) significantly suppressed the experimental hepatocarcinogenesis. A 66-year-old Japanese woman with hepatitis C virus (HCV)-related liver cirrhosis developed a dysplastic nodule in the liver detected by enhanced computed tomography along with elevation of the tumor markers, namely, alpha-fetoprotein (AFP) and lectin-reactive demarcation (AFP-L3), suggesting the presence of latent HCC. After oral administration of VK and ACE-I, the serum levels of both AFP and AFP-L3 gradually decreased without any marked alteration of the serum aminotransferase activity. After one-year treatment, not only the serum levels of AFP and AFP-L3 returned to the normal ranges, but also the dysplastic nodule disappeared. Since both VK and ACE-I are widely used without serious side effects, this combined regimen may become a new strategy for chemoprevention against HCC.

World J Gastroenterol. 2007 Jun 21;13(23):3259-61

Vitamin K, an example of triage theory: is micronutrient inadequacy linked to diseases of aging?

The triage theory posits that some functions of micronutrients (the approximately 40 essential vitamins, minerals, fatty acids, and amino acids) are restricted during shortage and that functions required for short-term survival take precedence over those that are less essential. Insidious changes accumulate as a consequence of restriction, which increases the risk of diseases of aging. For 16 known vitamin K-dependent (VKD) proteins, we evaluated the relative lethality of 11 known mouse knockout mutants to categorize essentiality. Results indicate that 5 VKD proteins that are required for coagulation had critical functions (knockouts were embryonic lethal), whereas the knockouts of 5 less critical VKD proteins [osteocalcin, matrix Gla protein (Mgp), growth arrest specific protein 6, transforming growth factor beta-inducible protein (Tgfbi or betaig-h3), and periostin] survived at least through weaning. The VKD gamma-carboxylation of the 5 essential VKD proteins in the liver and the 5 nonessential proteins in nonhepatic tissues sets up a dichotomy that takes advantage of the preferential distribution of dietary vitamin K1 to the liver to preserve coagulation function when vitamin K1 is limiting. Genetic loss of less critical VKD proteins, dietary vitamin K inadequacy, human polymorphisms or mutations, and vitamin K deficiency induced by chronic anticoagulant (warfarin/coumadin) therapy are all linked to age-associated conditions: bone fragility after estrogen loss (osteocalcin) and arterial calcification linked to cardiovascular disease (Mgp). There is increased spontaneous cancer in Tgfbi mouse knockouts, and knockdown of Tgfbi causes mitotic spindle abnormalities. A triage perspective reinforces recommendations of some experts that much of the population and warfarin/coumadin patients may not receive sufficient vitamin K for optimal function of VKD proteins that are important to maintain long-term health.

Am J Clin Nutr. 2009 Oct;90(4):889-907

The effect of menatetrenone, a vitamin K2 analog, on disease recurrence and survival in patients with hepatocellular carcinoma after curative treatment: a pilot study.

BACKGROUND: The high recurrence rate of hepatocellular carcinoma (HCC) determines the long-term prognosis for patients with HCC. In the current study, the authors tested the effects of menatetrenone, a vitamin K2 analog, on recurrent HCC and survival after curative treatment. METHODS: Sixty-one patients who were diagnosed as free of HCC after surgical resection or percutaneous local ablation were assigned randomly assigned to either a menatetrenone group (n = 32 patients) or a control group (n = 29 patients). Patients in the menatetrenone group received a daily oral dose of 45 mg of menatetrenone. Disease recurrence and survival rates were analyzed in patients with HCC. RESULTS: The cumulative recurrence rates in the menatetrenone group were 12.5% at 12 months, 39.0% at 24 months, and 64.3% at 36 months; and the corresponding recurrence rates in the control group were 55.2%, 83.2%, and 91.6%, respectively (P = 0.0002). Similar results were obtained even for patients who had low baseline levels of serum des-gamma-carboxy-prothrombin. Univariate and multivariate Cox proportional hazard analyses showed that the administration of menatetrenone was the only factor related to the recurrence rate of HCC. The cumulative survival rates for the patients who received menatetrenone were 100% at 12 months, 96.6% at 24 months, and 87.0% at 36 months; and the corresponding survival rates for patients in the control group were 96.4%, 80.9%, and 64.0%, respectively (P = 0.051). CONCLUSIONS: The current study findings suggested that menatetrenone may have a suppressive effect on recurrence of HCC and a beneficial effect on survival, although a larger, placebo-controlled trial will be required to prove these effects.

Cancer. 2006 Feb 15;106(4):867-72

Effect of calcium supplements on risk of myocardial infarctionand cardiovascular events: meta-analysis.

OBJECTIVE: To investigate whether calcium supplements increase the risk of cardiovascular events. DESIGN: Patient level and trial level meta-analyses. DATA SOURCES: Medline, Embase, and Cochrane Central Register of Controlled Trials (1966-March 2010), reference lists of meta-analyses of calcium supplements, and two clinical trial registries. Initial searches were carried out in November 2007, with electronic database searches repeated in March 2010. STUDY SELECTION: Eligible studies were randomised, placebo controlled trials of calcium supplements (>or=500 mg/day), with 100 or more participants of mean age more than 40 years and study duration more than one year. The lead authors of eligible trials supplied data. Cardiovascular outcomes were obtained from self reports, hospital admissions, and death certificates. RESULTS: 15 trials were eligible for inclusion, five with patient level data (8151 participants, median follow-up 3.6 years, interquartile range 2.7-4.3 years) and 11 with trial level data (11 921 participants, mean duration 4.0 years). In the five studies contributing patient level data, 143 people allocated to calcium had a myocardial infarction compared with 111 allocated to placebo (hazard ratio 1.31, 95% confidence interval 1.02 to 1.67, P=0.035). Non-significant increases occurred in the incidence of stroke (1.20, 0.96 to 1.50, P=0.11), the composite end point of myocardial infarction, stroke, or sudden death (1.18, 1.00 to 1.39, P=0.057), and death (1.09, 0.96 to 1.23, P=0.18). The meta-analysis of trial level data showed similar results: 296 people had a myocardial infarction (166 allocated to calcium, 130 to placebo), with an increased incidence of myocardial infarction in those allocated to calcium (pooled relative risk 1.27, 95% confidence interval 1.01 to 1.59, P=0.038). CONCLUSIONS: Calcium supplements (without coadministered vitamin D) are associated with an increased risk of myocardial infarction. As calcium supplements are widely used these modest increases in risk of cardiovascular disease might translate into a large burden of disease in the population. A reassessment of the role of calcium supplements in the management of osteoporosis is warranted.

BMJ. 2010 Jul 29;341:c3691

Prospective study of calcium, potassium, and magnesium intake and risk of stroke in women.

BACKGROUND AND PURPOSE: High intakes of calcium, potassium, and magnesium have been hypothesized to reduce risks of cardiovascular disease, but only a few prospective studies have examined intakes of these cations in relation to risk of stroke. METHODS: In 1980, 85 764 women in the Nurses’ Health Study cohort, aged 34 to 59 years and free of diagnosed cardiovascular disease and cancer, completed dietary questionnaires from which we calculated intakes of calcium, potassium, and magnesium. By 1994, after 1.16 million person-years of follow-up, 690 incident strokes (129 subarachnoid hemorrhages, 74 intraparenchymal hemorrhages, 386 ischemic strokes, and 101 strokes of undetermined type) had been documented. RESULTS: Intakes of calcium, potassium, and magnesium were each inversely associated with age- and smoking-adjusted relative risks of ischemic stroke, excluding embolic infarction of nonatherogenic origin (n=347). Adjustment for other cardiovascular risk factors, including history of hypertension, attenuated these associations, particularly for magnesium intake. In a multivariate analysis, women in the highest quintile of calcium intake had an adjusted relative risk of ischemic stroke of 0.69 (95% CI, 0.50 to 0.95; P for trend=0.03) compared with those in the lowest quintile; for potassium intake the corresponding relative risk was 0.72 (95% CI, 0.51 to 1.01; P for trend=0.10). Further simultaneous adjustment for calcium and potassium intake suggested an independent association for calcium intake. The association of risk with calcium intake did not appear to be log linear; the increase in risk was limited to the lowest quintile of intake, and intakes > approximately 600 mg/d did not appear to reduce risk of stroke further. The inverse association with calcium intake was stronger for dairy than for nondairy calcium intake. Intakes of calcium, potassium, and magnesium were not related to risk of other stroke subtypes. CONCLUSIONS: Low calcium intake, and perhaps low potassium intake, may contribute to increased risk of ischemic stroke in middle-aged American women. It remains possible that women in the lowest quintile of calcium intake had unknown characteristics that made them susceptible to ischemic stroke.

Stroke. 1999 Sep;30(9):1772-9

Relation of calcium, vitamin D, and dairy food intake to ischemic heart disease mortality among postmenopausal women.

To investigate whether greater intakes of calcium, vitamin D, or milk products may protect against ischemic heart disease mortality, the authors analyzed data from a prospective cohort study of 34,486 postmenopausal Iowa women 55-69 years old and without a history of ischemic heart disease who completed a dietary questionnaire in 1986. Through 1994, 387 deaths due to ischemic heart disease were documented (International Classification of Diseases, Ninth Revision, codes 410-414, 429.2). The multivariate-adjusted relative risks for the highest versus the lowest quartiles of total calcium, vitamin D, and milk product intakes were as follows: 0.67 (95% confidence interval (CI) 0.47-0.94; p for trend = 0.09) for calcium, 1.41 (95% CI 0.93-2.15; p for trend = 0.12) for vitamin D, and 0.94 (95% CI 0.66-1.35; p for trend = 0.68) for milk products. The relative risk was 0.63 (95% CI 0.40-0.98) for high dietary calcium but no supplemental calcium intake and 0.66 (95% CI 0.36-1.23) for high supplemental calcium but low dietary calcium intake. These results suggest that a higher intake of calcium, but not of vitamin D or milk products, is associated with reduced ischemic heart disease mortality in postmenopausal women, and reduced risk may be achievable whether the higher intake of calcium is attained by diet, supplements, or both.

Am J Epidemiol. 1999 Jan 15;149(2):151-61

The influence of dietary and nondietary calcium supplementation on blood pressure: an updated metaanalysis of randomized controlled trials.

We updated our previous systematic review of the effect of supplemental calcium on blood pressure. We extended our previous searches on MEDLINE and EMBASE to May 1997 and examined citations from relevant articles. We contacted the authors of eligible trials to ensure the accuracy and completeness of data, and to identify unpublished trials. We included any study in which investigators randomized hypertensive or normotensive people to calcium supplementation or alternative therapy and measured blood pressure for at least 2 weeks. In addition to 32 trials included in the prior metaanalysis, 10 new trials contributed to this metaanalysis. Two pairs of independent reviewers abstracted data and assessed the validity of the study data according to six quality criteria. We calculated the differences in blood pressure change between the calcium supplementation and control groups and pooled the estimates with each trial weighted with the inverse of the variance using a random effects model. The predictors of blood pressure reduction that we examined included method of supplementation, baseline blood pressure, and the methodologic quality of the studies. The pooled analysis shows a reduction in systolic blood pressure of -1.44 mm Hg (95% confidence interval -2.20 to -0.68; P < .001) and in diastolic blood pressure of -0.84 mm Hg (95% confidence interval -1.44 to -0.24; P < .001). We found statistically significant heterogeneity of results across trials (P < or = .02), which persisted when we looked at the nondietary trials alone, but not when we restricted our analysis to dietary trials. Although there was a trend toward larger effects with dietary interventions, none of the possible mediators of blood pressure reduction explained differences in treatment effect. We conclude that calcium supplementation leads to a small reduction in systolic and diastolic blood pressure. The effect of supplemental calcium in the diet is at least as great as nondietary supplementation.

Am J Hypertens. 1999 Jan;12(1 Pt 1):84-92

Effects of calcium supplementation on serum lipid concentrations in normal older women: a randomized controlled trial.

PURPOSE: To determine the effect of supplementation with calcium citrate on circulating lipid concentrations in normal older women. SUBJECTS AND METHODS: As part of a study of the effects of calcium supplementation on fractures, we randomly assigned 223 postmenopausal women (mean [+/- SD] age, 72 +/- 4 years), who were not receiving therapy for hyperlipidemia or osteoporosis, to receive calcium (1 g/d, n = 111) or placebo (n = 112) for 1 year. Fasting serum lipid concentrations, including high-density lipoprotein (HDL) cholesterol and low-density lipoprotein (LDL) cholesterol, were obtained at baseline, and at 2, 6, and 12 months. RESULTS: After 12 months, HDL cholesterol levels and the HDL cholesterol to LDL cholesterol ratio had increased more in the calcium group than in the placebo group (mean between-group differences in change from baseline: for HDL cholesterol, 0.09 mmol/L (95% confidence interval [CI]: 0.02 to 0.17; P = 0.01); for HDL/LDL cholesterol ratio, 0.05 (95% CI: 0.02 to 0.08; P = 0.001). This was largely due to a 7% increase in HDL cholesterol levels in the calcium group, with a nonsignificant 6% decline in LDL cholesterol levels. There was no significant treatment effect on triglyceride level (P = 0.48). CONCLUSION: Calcium citrate supplementation causes beneficial changes in circulating lipids in postmenopausal women. This suggests that a reappraisal of the indications for calcium supplementation is necessary, and that its cost effectiveness may have been underestimated.

Am J Med. 2002 Apr 1;112(5):343-7

Effects of calcium supplementation on body weight and blood pressure in normal older women: a randomized controlled trial.

CONTEXT: Epidemiological data suggest that high calcium intakes are associated with decreased body weight and blood pressure. However, there is little evidence from randomized trials that addresses these important issues. OBJECTIVE: The objective of this study was to assess the long-term effects of calcium on body weight and blood pressure. DESIGN: This is a substudy of an ongoing, double-blind, randomized, controlled trial of calcium supplementation. End points were assessed at 30 months. SETTING: This study was performed at a university medical center. PARTICIPANTS: Normal postmenopausal women (mean age, 74 yr; mean weight, 67 kg; mean blood pressure, 134/70 mm Hg at baseline) participated in this study. INTERVENTION: Study subjects were treated with calcium (1 g/d; n = 732) and placebo (n = 739). MAIN OUTCOME MEASURES: Body weight and blood pressure were the main outcome measures. RESULTS: Weight decreased by 368 +/- 132 g (mean +/- se) with calcium treatment and by 369 +/- 134 g with placebo (P = 0.93). Fat and lean masses did not show an effect of calcium. Blood pressure showed transient reductions of 1-2 mm Hg at 6 months in the calcium group, resulting in a significant between-group difference only for systolic pressure (P = 0.048). At 30 months, the change from baseline in systolic pressure was 0.0 +/- 0.9 mm Hg in the calcium group and 2.4 +/- 0.9 mm Hg in the placebo group (P = 0.14). For diastolic pressures, the changes were -0.2 +/- 0.4 and 0.8 +/- 0.4 mm Hg, respectively (P = 0.13). In those with baseline calcium intakes less than 600 mg/d, the treatment effect was greater and did persist. CONCLUSIONS: Calcium supplementation of 1 g/d does not produce biologically significant effects on body weight, and its hypotensive effect is small and transient in most women.

J Clin Endocrinol Metab. 2005 Jul;90(7):3824-9