Age defying species such as the giant red sea urchin, the painted turtle, the naked mole rat and the bowhead whale may begin teaching us how to extend our own lives. All these species stand out because they exhibit negligible senescence, the ability to grow older without suffering functional declines or any age-related increase in mortality. Apparently, these species have something we lack. This something, according to a new study, is gene network stability.

A stabilized gene network promotes longer lifespan. The new data is consistent with Dr. Villeponteau’s model that keeping the optimal gene network at youth from drifting with age generates longer life. Likewise, interventions that nudge gene expression back to that of the youthful gene network reverses aging. That is the goal with all the Life Code products, particularly Stem Cell 100 and Stem Cell 100+.

The new study was undertaken by scientists from the biotech company Gero in collaboration with Robert J. Shmookler Reis, Ph.D., a professor of geriatrics at the University of Arkansas. Dr. Shmookler Reis enjoys the distinction of having extended by a factor of 10 the lifespan of the nematode Caenorhabditis elegans.

These scientists noticed that in long-lived animals, negligible senescence is accompanied by exceptionally stable gene expression. And stable gene expression, they reasoned, could be attributed to gene network repair systems. To test this idea, the scientists devised a mathematical model of a genetic network. This model not only managed to account for the age-independent mortality exhibited by long-lived animals, it also identified gene network parameters that influence longevity. These parameters include effective gene network connectivity, effective genome size, proteome turnover, and DNA repair rate.

The scientists published their findings August 28 in the journal Scientific Reports, in an article entitled, ?Stability analysis of a model gene network links aging, stress resistance, and negligible senescence.? This article argues that under a very generic set of assumptions, there exist two distinctly different classes of aging dynamics.

?If the repair rates are sufficiently high or the connectivity of the gene network is sufficiently low, then the regulatory network is very stable and mortality is time-independent in a manner similar to that observed in negligibly senescent animals,? wrote the authors. ?Should the repair systems display inadequate efficiency, a dynamic instability emerges, with exponential accumulation of genome-regulation errors, functional declines and a rapid aging process accompanied by an exponential increase in mortality.?

The authors added that the onset of instability depends on the gene-network properties only, irrespective of genotoxic stress levels. Essentially, instability can be viewed as being hard-wired in the genome of the species.

Nonetheless, the authors said that their model implies many possibilities to stabilize a regulatory network and thus extend lifespan. Some of these possibilities, in fact, have already been exploited by nature, accounting for some of the lifespan increases observed to have occurred in evolution. For example, a model parameter that the authors call the coupling rate can be adjusted by increasing or decreasing the degree of gene network connectivity.

A biological embodiment of this parameter is the degree to which the nuclear envelope reduces the effective interactions between the genes and the cellular environment. Once the nuclear envelope became available, organisms that used it enjoyed a dramatic increase in complexity and life expectancy.

Such observations have implications for potential life-extending interventions. For example, experimental reduction of the network connectivity by silencing of kinase cascades involved in regulation of transcription factors leads to a dramatic effect on the lifespan in C. elegans (up to a 10x lifespan extension by a single mutation).

The relation between stresses, stress resistance, and aging, the authors explained, can also account for the way damage to gene regulation from stresses encountered even at a very young age can persist for a very long time and influence lifespan. Such effects, the authors believe, indicate that further research into the relation between gene network stability and aging will make it possible to create entirely new therapies with potentially strong and lasting effect against age-related diseases and aging itself.

A finding reveals why the transformation process of differentiated cells into stem cells results in significant damage to the DNA. Researchers have managed to rectify this damage using a simple modification to the culture medium, which produces potentially safer stem cells for use in regenerative medicine.

Damaged tissue, such as pancreas, heart, and neuronal tissue, which is regenerated to treat cardiovascular diseases, diabetes, or neurodegenerative diseases. This is one of the ambitious scenarios to which regenerative medicine aspires and that is being announced as one of the great promises of twenty-first century biomedicine for the treatment of a long list of diseases affecting people today. The focal point is the use of stem cells, which are capable of producing different types of cells or tissue.

2006 marked a turning point in this field, when the Japanese scientist, Shinya Yamanaka, managed to generate pluripotent stem cells in the lab for the first time. These are capable of becoming any type of cell, whether insulin-producing beta cells (pancreas) or cardiomyocytes (heart), and are known as iPS cells. This cell reprogramming technique eliminated one of the great ethical dilemmas of the time: until then, pluripotent stem cells could only be obtained from embryos which, in order to achieve this, had to be destroyed.

However, as ?scar Fern?ndez-Capetillo, head of the Genomic Instability Group at the Spanish National Cancer Research Centre (CNIO), says: “the drawback of this new technology is that Yamanaka’s method damaged the stem cell genome, leading to certain safety concerns regarding these cells.” While the fact that the method damaged the DNA of iPS cells was known, the reasons were not.

According to an article published this week in Nature Communications, the team headed by Fern?ndez-Capetillo states that the damage to the genome of iPS cells lies in a very specific kind of stress that the cells are subjected to during cell reprogramming: replication stress, which occurs when the cells increase the pace of division. In addition, and based on these findings, the authors of the paper have managed to develop strategies to reduce this type of stress, resulting in pluripotent stem cells with less damage to their genome.

The results represent a significant step forward regarding the possible use of iPS cells, because after almost a decade since they were developed, there is now a more efficient way of obtaining them, with less damage to the DNA, making them potentially safer.

The CNIO Telomeres and Telomerase groups, headed by Mar?a Blasco, and the Tumoral Suppression Group, headed by Manuel Serrano have also participated in this study, together with groups from the Pasteur Institute in Paris, Toronto University and the Pompeu Fabra University in Barcelona.

Stem Cells With More Stable Genomes

The nature of the damage to the DNA observed in iPS cells has been intensely discussed for some years, due to the fact that it is linked to the rearrangement of large fragments of chromosomes which could lead to potentially dangerous mutations if used clinically.

In a paper published in Nature in 2009, the team led by Mar?a Blasco, with the collaboration of Fern?ndez-Capetillo’s group, described how the damage to the DNA had important consequences in cell reprogramming by limiting the process and making it less efficient.

Now the team headed by Fern?ndez-Capetillo has not only identified the origin of the damage, replication stress, but has managed to reduce it significantly; potentially improving the safety of induced stem cells for use in biomedicine.

To reduce damage to stem cells and thus achieve more stable genomes, the scientists have used a dual approach: genetics, increasing the production of the Chk1 protein, which repairs DNA damage due to replication stress; and chemical, based on supplementing the medium in which the cells are fed with nucleoside, the source compounds of the bricks that build DNA.

“Based on previous research performed by the group, we knew that an additional input of nucleoside reduces replication stress, probably by facilitating the successful replication of DNA as it increases the rate of cell division during the reprogramming process,” explains Sergio Ruiz, whose signature appear in first place on the paper.

The simplicity of this nucleoside-based strategy means that it can be implemented easily by laboratories around the world working with iPS cells, and thus contribute significantly to the field of regenerative biology, one of the greatest aspirations of biomedicine this century.

Journal Reference:

1.Sergio Ruiz, Andres J. Lopez-Contreras, Mathieu Gabut, Rosa M. Marion, Paula Gutierrez-Martinez, Sabela Bua, Oscar Ramirez, I?igo Olalde, Sara Rodrigo-Perez, Han Li, Tomas Marques-Bonet, Manuel Serrano, Maria A. Blasco, Nizar N. Batada, Oscar Fernandez-Capetillo. Limiting replication stress during somatic cell reprogramming reduces genomic instability in induced pluripotent stem cells. Nature Communications, 2015; 6: 8036 DOI: 10.1038/ncomms9036

One day soon, doctors may determine how physically active you are simply by imaging your brain. Physically fit people tend to have larger brain volumes and more intact white matter than their less-fit peers. Now a new study reveals that older adults who regularly engage in moderate to vigorous physical activity have more variable brain activity at rest than those who don’t. This variability is associated with better cognitive performance, researchers say.

The new findings are reported in the journal PLOS ONE.

“We looked at 100 adults between the ages of 60 and 80, and we used accelerometers to objectively measure their physical activity over a week,” said University of Illinois postdoctoral researcher Agnieszka Burzynska, who led the study with Beckman Institute for Advanced Science and Technology director Art Kramer.

The researchers also used functional MRI to observe how blood oxygen levels changed in the brain over time, reflecting each participant’s brain activity at rest. And they evaluated the microscopic integrity of each person’s white-matter fibers, which carry nerve impulses and interconnect the brain.

“We found that spontaneous brain activity showed more moment-to-moment fluctuations in the more-active adults,” said Burzynska, who now is a professor at Colorado State University. “In a previous study, we showed that in some of the same regions of the brain, those people who have higher brain variability also performed better on complex cognitive tasks, especially on intelligence tasks and memory.”

The researchers also found that, on average, older adults who were more active had better white-matter structure than their less-active peers.

“Our study, when viewed in the context of previous studies that have examined behavioral variability in cognitive tasks, suggests that more-fit older adults are more flexible, both cognitively and in terms of brain function, than their less-fit peers,” Kramer said.

The new research highlights yet another way to assess brain health in aging, Burzynska said.

“We want to know how the brain relates to the body, and how physical health influences mental and brain health in aging,” she said. “Here, instead of a structural measure, we are taking a functional measure of brain health. And we are finding that tracking changes in blood-oxygenation levels over time is useful for predicting cognitive functioning and physical health in aging.”

Sleeping in the lateral, or side position, as compared to sleeping on one’s back or stomach, may more effectively remove brain waste and prove to be an important practice to help reduce the chances of developing Alzheimer’s, Parkinson’s and other neurological diseases, according to researchers at Stony Brook University.

By using dynamic contrast magnetic resonance imaging (MRI) to image the brain’s glymphatic pathway, a complex system that clears wastes and other harmful chemical solutes from the brain, Stony Brook University researchers Hedok Lee, PhD, Helene Benveniste, MD, PhD, and colleagues, discovered that a lateral sleeping position is the best position to most efficiently remove waste from the brain. In humans and many animals the lateral sleeping position is the most common one. The buildup of brain waste chemicals may contribute to the development of Alzheimer’s disease and other neurological conditions. Their finding is published in the Journal of Neuroscience.

Dr. Benveniste, Principal Investigator and a Professor in the Departments of Anesthesiology and Radiology at Stony Brook University School of Medicine, has used dynamic contrast MRI for several years to examine the glymphatic pathway in rodent models. The method enables researchers to identify and define the glymphatic pathway, where cerebrospinal fluid (CSF) filters through the brain and exchanges with interstitial fluid (ISF) to clear waste, similar to the way the body’s lymphatic system clears waste from organs. It is during sleep that the glymphatic pathway is most efficient. Brain waste includes amyloid ? (amyloid) and tau proteins, chemicals that negatively affect brain processes if they build up.

In the paper, “The Effect of Body Posture on Brain Glymphatic Transport,” Dr. Benveniste and colleagues used a dynamic contrast MRI method along with kinetic modeling to quantify the CSF-ISF exchange rates in anesthetized rodents’ brains in three positions ? lateral (side), prone (down), and supine (up).

“The analysis showed us consistently that glymphatic transport was most efficient in the lateral position when compared to the supine or prone positions,” said Dr. Benveniste. “Because of this finding, we propose that the body posture and sleep quality should be considered when standardizing future diagnostic imaging procedures to assess CSF-ISF transport in humans and therefore the assessment of the clearance of damaging brain proteins that may contribute to or cause brain diseases.”

Dr. Benveniste and first-author Dr. Hedok Lee, Assistant Professor in the Departments of Anesthesiology and Radiology at Stony Brook developed the safe posture positions for the experiments. Their colleagues at the University of Rochester, including Lulu Xie, Rashid Deane and Maiken Nedergaard, PhD, used fluorescence microscopy and radioactive tracers to validate the MRI data and to assess the influence of body posture on the clearance of amyloid from the brains.

“It is interesting that the lateral sleep position is already the most popular in human and most animals ? even in the wild ? and it appears that we have adapted the lateral sleep position to most efficiently clear our brain of the metabolic waste products that built up while we are awake,” says Dr. Nedergaard. “The study therefore adds further support to the concept that sleep subserves a distinct biological function of sleep and that is to ‘clean up’ the mess that accumulates while we are awake. Many types of dementia are linked to sleep disturbances, including difficulties in falling asleep. It is increasing acknowledged that these sleep disturbances may accelerate memory loss in Alzheimer’s disease. Our finding brings new insight into this topic by showing it is also important what position you sleep in,” she explained.

Dr. Benveniste cautioned that while the research team speculates that the human glymphatic pathway will clear brain waste most efficiency when sleeping in the lateral position as compared to other positions, testing with MRI or other imaging methods in humans are a necessary first step.

Reference: “The Effect of Body Posture on Brain Glymphatic Transport” The Journal of Neuroscience, 5 August 2015, 35(31): 11034-11044; DOI: 10.1523/JNEUROSCI.1625-15.2015

A study from the US National Institutes of Health presents some of the most precise human data yet on whether cutting carbs or fat has the most benefits for losing body fat. In a paper published August 13 in Cell Metabolism, the researchers show how, contrary to popular claims, restricting dietary fat can lead to greater body fat loss than carb restriction, even though a low-carb diet reduces insulin and increases fat burning.

Since 2003, Kevin Hall, Ph.D. is a physicist turned metabolism researcher at the National Institute of Diabetes and Digestive and Kidney Diseases who has been using data from dozens of controlled feeding studies conducted over decades of nutrition research to build mathematical models of how different nutrients affect human metabolism and body weight.

He noticed that despite claims about carbohydrate versus fat restriction for weight loss, nobody had ever measured what would happen if carbs were selectively cut from the diet while fat remained at a baseline or vice versa. His model simulations showed that only the carb-restricted diet would lead to changes in the amount of fat burned by the body, whereas the reduced-fat diet would lead to greater overall body fat loss, but he needed the human data to back it up.

“A lot of people have very strong opinions about what matters for weight loss, and the physiological data upon which those beliefs are based are sometimes lacking,” Hall says. “I wanted to rigorously test the theory that carbohydrate restriction is particularly effective for losing body fat since this idea has been influencing many people’s decisions about their diets.”

Studying the effects of diet on weight loss is often confounded by the difficulty in measuring what people actually eat since participants may not adhere to meal plans, misjudge amounts, or are not truthful in follow-up surveys. To counter this, Hall and colleagues confined 19 consenting adults with obesity to a metabolic ward for a pair of 2-week periods, over the course of which every morsel of food eaten was closely monitored and controlled.

To keep the variables simple, the two observation periods were like two sides of a balance scale: during the first period, 30% of baseline calories were cut through carb restriction alone, while fat intake remained the same. During the second period the conditions were reversed. Each day, the researchers measured how much fat each participant ate and burned and used this information to calculate the rate of body fat loss.

At the end of the two dieting periods, the mathematical model proved to be correct. Body fat lost with dietary fat restriction was greater compared with carbohydrate restriction, even though more fat was burned with the low-carb diet. However, over prolonged periods the model predicted that the body acts to minimize body fat differences between diets that are equal in calories but varying widely in their ratio of carbohydrate to fat.

“There is one set of beliefs that says all calories are exactly equal when it comes to body fat loss and there’s another that says carbohydrate calories are particularly fattening, so cutting those should lead to more fat loss,” Hall says. “Our results showed that, actually, not all calories are created equal when it comes to body fat loss, but over the long term, it’s pretty close.”

Hall does caution against making sweeping conclusions about how to diet from this study. The study’s purpose was to explore the physiology of how equal calorie reductions of fat versus carbs affect the human body. The research is limited by its sample size; only 19 people could be enrolled due to the expense of such research and the restrictiveness of the carefully controlled protocol. However, this study clearly reaches statistical significance. In addition,, the menu that the participants followed does not emulate normal dieting and does not account for what diet would be easier to eat over extended periods.

“We are trying to do very careful studies in humans to better understand the underlying physiology that will one day be able to help generate better recommendations about day to day dieting,” Hall says. “But there is currently a gap between our understanding of the physiology and our ability to make effective diet recommendations for lasting weight loss.”

Hall recommends that for now, the best diet is the one that you can stick to. His lab will next investigate how reduced-carbohydrate and reduced-fat diets affect the brain’s reward circuitry, as well as its response to food stimuli. He hopes these results might inform why people respond differently to different diets.

Scientists from A*STAR’s Genome Institute of Singapore (GIS) have discovered metabolic rejuvenation factors in eggs. This critical finding furthers our understanding of how cellular metabolism changes during aging, and during rejuvenation after egg fertilization.

When a sperm fertilizes an egg to create a baby, two adult cells combine to form a new embryo. A similar process of combining an egg’s cytoplasm with an adult cell nucleus led to the cloning of Dolly the sheep. However, the metabolic factors underlying this fascinating process had remained unclear.

A new study from GIS suggests that old mitochondria — the oxygen-consuming metabolic engines in cells — are roadblocks to cellular rejuvenation. By tuning up a gene called Tcl1, which is highly abundant in eggs, researchers were able to suppress old mitochondria to enhance a process known as somatic reprogramming, which turn adult cells into embryonic-like stem cells.

GIS researchers found that Tcl1 does its job by suppressing mitochondrial polynucleotide phosphorylase, thereby inhibiting mitochondrial growth and metabolism.

Findings from the study were published in the scientific journal Cell Reports.

Stem cell researchers had known that egg (or oocyte) cytoplasm contains some special unknown factors that can reprogram adult cells into embryonic-like stem cells, either during egg-sperm fertilization or during artificial cloning procedures like those that created Dolly the sheep. While the Nobel Prize winner Dr. Shinya Yamanaka had invented a technology called induced pluripotent stem cell (iPSC) reprogramming to replace the ethically controversial oocyte-based reprogramming technique, oocyte-based reprogramming was still deemed superior in complete cellular reprogramming efficiency.

To address this shortfall, the GIS team led by Dr Khaw Swea-Ling, Dr Lim Bing and Dr. Ng Shyh-Chang combined oocyte factors with the iPSC reprogramming system. Their bioinformatics-driven screening efforts led to two genes: Tcl1 and its cousin Tcl1b1. After a deeper investigation, the team found that the Tcl1 genes were acting via the mitochondrial enzyme, PnPase.

“We were quite surprised, because nobody would have thought that the key to the oocyte’s reprogramming powers would be a mitochondrial enzyme. The stem cell field’s conventional wisdom suggests that it should have been some other signaling genes instead,” said corresponding author of the research, Dr Ng Shyh-Chang.

Tcl1 is a cytoplasmic protein that binds to the mitochondrial enzyme PnPase. By locking PnPase in the cytoplasm, Tcl1 prevents PnPase from entering mitochondria, thereby suppressing its ability to promote mitochondrial growth and metabolism. Thus, an increase in Tcl1 suppresses old mitochondria’s growth and metabolism in adult cells, to enhance the somatic reprogramming of adult cells into embryonic-like stem cells.

Cracking the mystery of reprogramming factors in oocytes is an important milestone. These new insights could boost efficacy of the alternative, non-oocyte-based iPSC techniques for stem cell banking, organ and tissue regeneration, as well as further our understanding of how cellular metabolism rejuvenates after egg-sperm fertilization. This could help address both the aging and the fertility problems of modern societies.

GIS Executive Director Prof Ng Huck Hui said, “This is an exciting step forward in the study of cellular aging. Although accumulated defects in mitochondrial metabolism were known to cause cellular aging, no solutions were available. Shyh-Chang’s team has uncovered a molecular pathway to solve this problem.”

The above post is reprinted from materials provided by The Agency for Science, Technology and Research (A*STAR).

Two labs in China have independently succeeded in transforming skin cells into neurons using only a cocktail of chemicals, with one group using human cells from humans and the other group using cells from mice. The two studies reinforce the idea that a purely chemical approach is a promising way to scale up cell reprogramming research that may avoid the technical challenges and safety concerns associated with the more popular method of using transcription factors. Both papers appear on August 6 in the journal Cell Stem Cell.

The importance of these studies is that they were able to change skin cells into neurons without using genetic engineering. This has already been done many times by adding new genes to cells, however that approach creates significant risks if they are used for actual human therapy since the DNA has been changed in ways that may have unknown consequences in the long run.

One of the challenges of forcing cells to change identity is that the cells you end up with may look normal but have different internal activities than their naturally forming counterparts. The two papers provide evidence that similar gene expression, action potentials, and synapse formation can be detected in transcription-factor-induced neurons as those generated from the chemical cocktails. (Both groups used mixtures of seven small molecules, but different recipes–outlined in detail in the supplemental information section of each paper–because they focused on different species.)

“We found that the conversion process induced by our chemical strategy is accompanied by the down-regulation of [skin-cell] specific genes and the increased expression of neuronal transcription factors,” said human study co-author Jian Zhao, of the Shanghai Institutes for Biological Sciences and Tongji University. “By coordinating multiple signaling pathways, these small molecules modulate neuronal transcription factor gene expression and thereby promote the neuronal cell transition.” The authors add that the direct conversion bypasses a proliferative intermediate progenitor stage, which circumvents safety issues posed by other reprogramming methods.

Zhao’s paper, co-led with cell biologist Gang Pei, also shows that the pure chemical protocol can be used to make neurons from the skins cells of Alzheimer’s patients. Most of the work using patient stem cells has been done by using transcription factors–molecules that affect which genes are expressed in a cell–to create induced pluripotent stem cells. Chemical cell reprogramming is seen as an alternative for disease modeling or even potential cell replacement therapy of neurological disorders, but the “proof-of-concept” is still emerging.

“In comparison with using transgenic reprogramming factors, the small molecules that are used in this chemical approach are cell permeable; cost-effective; and easy to synthesize, preserve, and standardize; and their effects can be reversible,” says mouse study co-author Hongkui Deng of the Peking University Stem Cell Research Center. “In addition, the use of small molecules can be fine-tuned by adjusting their concentrations and duration, and the approach bypasses the technical challenges and safety concerns of genetic manipulations, which may be promising in their future applications.”

Deng worked for four years with Zhen Chai and Yang Zhao, also of Peking University, to identify the small molecules that could create chemically induced mouse neurons. Researchers had been close for years, but a transcription factor was always necessary to complete the transformation. Through many chemical screens they identified the key ingredient, I-BET151, which works to suppress transcription in skin cells. They then found the right steps and conditions to mature the neurons post-transformation.

The authors of both papers aim to learn more about the biology behind chemically induced reprogramming and to make the protocols more efficient. While their success is promising, there are still a number of hurdles to overcome.

“We hope in the future that the chemical approaches would be more robust in inducing functional mature neurons,” Deng says. “In addition, we are attempting to generate specific neuronal subtypes and patient-specific functional neurons for translational medicine by using pure chemicals.”

Jian Zhao, of the human study, says: “It should be possible to generate different subtypes of neurons with a similar chemical approach but using slightly modified chemical cocktails.” She adds: “It also needs to be explored whether functional neurons could be induced by chemical cocktails in living organisms with neurological diseases or injury.”

References:

1.Wenxiang Hu, Binlong Qiu, Wuqiang Guan, Qinying Wang, Min Wang, Wei Li, Longfei Gao, Lu Shen, Yin Huang, Gangcai Xie, Hanzhi Zhao, Ying Jin, Beisha Tang, Yongchun Yu, Jian Zhao, Gang Pei. Direct Conversion of Normal and Alzheimer?s Disease Human Fibroblasts into Neuronal Cells by Small Molecules. Cell Stem Cell, 2015; 17 (2): 204 DOI: 10.1016/j.stem.2015.07.006

2.Xiang Li, Xiaohan Zuo, Junzhan Jing, Yantao Ma, Jiaming Wang, Defang Liu, Jialiang Zhu, Xiaomin Du, Liang Xiong, Yuanyuan Du, Jun Xu, Xiong Xiao, Jinlin Wang, Zhen Chai, Yang Zhao, Hongkui Deng. Small-Molecule-Driven Direct Reprogramming of Mouse Fibroblasts into Functional Neurons. Cell Stem Cell, 2015; 17 (2): 195 DOI: 10.1016/j.stem.2015.06.003

Lending fruits and vegetables their bright orange, red, and yellow colors, carotenoids are abundant in antioxidants, for which previous studies have associated a lower risk of premature death. A recent study assessed the potential relationships of carotenoid intake with lipid and oxidative stress markers in middle-aged men. Data analysis revealed that higher total carotenoid intake was associated with lower lipid and oxidative stress markers, and in middle-aged men higher beta-carotene intake was also associated with five of the six lower lipid stress markers.

If you decide to obtain beta-carotene from a nutritional supplement be sure and use one that is derived from carrots, algae and other natural sources rather than synthetic beta-carotene which has been associated with some negative effects in a previous study.

A total of 296 apparently healthy middle-aged men with a mean age of 50.5 years and a mean BMI of 25.8 kg/m2 were recruited to participate in the study. Dietary intake, anthropometry, blood pressure, lifestyle features, blood and urine biomarkers were assessed using validated procedures. The lipid markers included NEFA, Castelli index, and TAG:HDL ratio; oxidative stress markers included urinary 8-hydroxy-2-deoxyguanosine (8-OHdG), 8-iso-PGF2 and plasma oxidised-LDL (ox-LDL). The scientists observed a significant inverse association (P< 0?05) between NEFA concentrations and consumption of lutein plus zeaxanthin, alpha-carotene, beta-carotene and total carotenoid, while the Castelli index was negatively associated with daily intake of lycopene, beta-carotene and total carotenoids. Regarding oxidative stress biomarkers, urinary 8-OHdG and ox-LDL concentrations were also inversely associated (P< 0?05) with consumption of lycopene, lutein plus zeaxanthin, alpha-carotene, beta-carotene and total carotenoids, regardless of confounding variables. Moreover, there was a negative association of urinary 8-iso-PGF2 concentration with dietary lutein plus zeaxanthin and with the sum of all carotenoids. In conclusion, higher total daily carotenoid intake based on five investigated carotenoid types (beta-cryptoxanthin, lycopene, lutein plus zeaxanthin, alpha-carotene and beta-carotene) was associated with lower relevant lipid and oxidative stress markers in middle-aged men, with emphasis on beta-carotene that was negatively associated with five of the six lipid and oxidative stress markers evaluated in the present study.