A practical reference on freeze-thaw: what it is, how it behaves, what the literature reports, and where the honest uncertainties sit.
Reviewed 2025-09-01. Anything still debated is marked as such rather than presented as settled.
Quality control for NAD+ relies on identity, purity, and functional tests. A certificate of analysis may report high-performance liquid chromatography purity, ultraviolet spectrum, water content, and residual solvents. Because NAD+ is hygroscopic, gravimetric values can shift as material absorbs water, so purity should be interpreted alongside storage history. Mass spectrometry confirms molecular identity, while enzymatic assays show whether the material supports dehydrogenase activity. Commercial material is available as the free acid and as salts, and the counterion affects molecular weight, solubility, and how concentrations are calculated.
Laboratory measurement of NAD+ usually begins with rapid sample quenching because the molecule can change form after collection. Enzymatic cycling assays amplify signal through coupled reactions and are suited to small samples. High-performance liquid chromatography with ultraviolet detection separates NAD+ from related nucleotides. Liquid chromatography-mass spectrometry offers higher specificity and can distinguish NAD+ from close analogs. Each method has trade-offs in sensitivity, throughput, and equipment needs, so reported values depend heavily on extraction and detection choices.
Stability of NAD+ depends on form, temperature, pH, and water content. The solid is generally more stable than solutions, and it should be kept dry and cold. In solution, hydrolysis can cleave the dinucleotide, especially under alkaline conditions or at elevated temperature. Light exposure may also contribute to degradation. Buffers, chelating agents, and sterile handling can reduce losses, but no single condition preserves all preparations indefinitely. Researchers often prepare working solutions shortly before use and verify activity or purity after storage.
Stability studies show that NAD+ can hydrolyze under prolonged heat, extreme pH, or microbial contamination. Phosphate buffers near neutral pH are often used for short-term handling, though exact stability depends on concentration, temperature, and matrix. In biological samples, endogenous enzymes can rapidly degrade NAD+, making cold chain and fast processing important. Analytical reports should state extraction conditions, internal standards, and validation parameters. Without those details, comparisons across studies remain difficult and potentially misleading.
Laboratory measurement of NAD+ often begins with rapid quenching of cell or tissue samples to prevent enzymatic conversion. Acidic or alkaline extraction can precipitate proteins, but the chosen method affects recovery of oxidized and reduced forms. Enzymatic cycling assays provide high sensitivity by amplifying a NAD+-dependent reaction. High-performance liquid chromatography and mass spectrometry offer separation and structural confirmation. Each method has trade-offs in throughput, specificity, and the ability to distinguish NAD+ from close analogues.
Purified NAD+ is typically supplied as a white to off-white powder and stored desiccated at low temperature. Airtight containers limit moisture uptake, while protection from light reduces degradation of the nicotinamide ring. Aqueous stock solutions are less stable than solid material and are often aliquoted before freezing. Repeated freeze-thaw cycles can lower integrity, so working portions are kept separate. Purity is commonly checked by ultraviolet absorbance near 260 nm, high-performance liquid chromatography, or mass spectrometry.
| Property | Value | Notes |
|---|---|---|
| CAS number | 53-84-9 | Refers to the free acid form of NAD+. |
| Molecular formula | C21H27N7O14P2 | Free acid; salts include additional counterions. |
| UV absorbance maximum | 259-260 nm | Used for detection and concentration estimation. |
| Typical storage | -20 °C or below, desiccated | Protect from light and moisture; avoid repeated freeze-thaw. |
| Common analytical method | HPLC-UV or LC-MS | Enzymatic cycling is an alternative for low-abundance samples. |
Beyond redox chemistry, NAD+ is consumed as a substrate by enzymes that transfer ADP-ribose or remove acetyl groups. Sirtuins use NAD+ in deacylation reactions, poly(ADP-ribose) polymerases use it in DNA damage responses, and CD38 hydrolases convert it to signaling metabolites. Because these enzymes compete for the same pool, changes in NAD+ availability can influence multiple cellular processes. The relative contribution of each consumption route differs by cell type and condition, and precise quantitative links remain an active area of study.
Research on NAD+ spans biochemistry, aging biology, and metabolism. Studies often examine how NAD+ levels change with age, diet, exercise, or disease states, and whether precursor supplementation alters those levels. Findings in animal models do not automatically translate to humans, and measurement methods vary across studies. Questions about tissue-specific effects, long-term consequences, and causal relationships remain open. NAD+ itself is not established as a single therapeutic agent with a broad clinical role.
Nicotinamide adenine dinucleotide, abbreviated NAD+, is a dinucleotide composed of two nucleotides joined by phosphate groups. One nucleotide contains adenine; the other contains nicotinamide. The molecule exists in oxidized (NAD+) and reduced (NADH) forms, and the reversible hydride transfer between them underlies many metabolic oxidation-reduction reactions. In cells, NAD+ serves as an electron acceptor in pathways such as glycolysis, the citric acid cycle, and oxidative phosphorylation. Its concentration and redox ratio vary by compartment, tissue, and metabolic state.
NAD+ is a dinucleotide composed of two nucleotides joined by a pyrophosphate linkage. One nucleotide contains adenine, and the other contains nicotinamide. The oxidized form carries a positive charge on the nicotinamide ring and is abbreviated NAD+. It functions as a cofactor in hydride-transfer reactions, accepting electrons in catabolic pathways. In cells, it interconverts with reduced NADH, forming a redox couple central to energy metabolism. The molecule is water-soluble and does not cross cell membranes freely without specific transport or precursor pathways.
The nicotinamide ring undergoes reversible reduction at the para position, converting NAD+ to NADH. This reaction transfers a hydride equivalent, not a free hydrogen atom or electron alone. Because the redox pair has a defined reduction potential, it links oxidation of fuels to respiratory chain activity. Many dehydrogenases use NAD+ as a co-substrate and produce NADH. The ratio of NAD+ to NADH reflects metabolic state and influences flux through several pathways.
NAD+ is a dinucleotide composed of adenine, ribose, and nicotinamide moieties linked by phosphate groups. Its oxidized form carries a positive charge on the nicotinamide ring, which enables reversible hydride transfer. The molecule functions as a coenzyme in oxidoreductase reactions rather than as a dietary vitamin in its intact form. Cells maintain separate pools in cytoplasm, mitochondria, and nucleus. This compartmentalization allows distinct redox environments while preserving a shared chemical identity.
In glycolysis, NAD+ accepts electrons during the oxidation of glyceraldehyde-3-phosphate, forming NADH. The tricarboxylic acid cycle and fatty acid oxidation also generate NADH, which donates electrons to the mitochondrial electron transport chain. This flow supports ATP synthesis and helps maintain the redox balance of the cell. Other dehydrogenases use NAD+ as a cofactor for biosynthetic reductions and detoxification reactions. NADH is later reoxidized to sustain continued flux through these pathways.
Beyond electron transfer, NAD+ serves as a substrate for enzymes that cleave the molecule and transfer ADP-ribose units. Sirtuins, poly(ADP-ribose) polymerases, and CD38 consume NAD+ in regulatory reactions. These activities link NAD+ availability to DNA repair, chromatin modification, calcium signaling, and metabolic stress responses. Because consumption can exceed biosynthesis under some conditions, cellular NAD+ levels are dynamic rather than fixed. Enzyme affinity and local synthesis also influence how much NAD+ is available for signaling.
In cells, NAD+ functions primarily as an electron carrier. Dehydrogenase enzymes in glycolysis and the citric acid cycle transfer hydride from substrates to NAD+, producing NADH. NADH then delivers electrons to the mitochondrial respiratory chain, supporting ATP synthesis. In fermentation, NADH is reoxidized to NAD+ so that glycolysis can continue. The balance between NAD+ and NADH helps set metabolic flux. Beyond redox, NAD+ serves as a substrate for enzymes that cleave it, including sirtuins, poly(ADP-ribose) polymerases, and CD38. These reactions consume NAD+ and release nicotinamide and ADP-ribose products.
Biosynthesis occurs through salvage, Preiss-Handler, and de novo pathways. In mammals, the salvage pathway from nicotinamide predominates, and NAMPT is often described as rate-limiting. Nicotinamide riboside and nicotinic acid enter related routes that converge on NAD+ production. Tissue NAD+ concentrations vary widely and are maintained by a balance of synthesis and consumption. Some studies report age-related declines in certain tissues, but whether these changes cause disease or can be reversed to improve human health remains an open question.
=== Excretion === In humans, the major route of excretion of most arsenic compounds is via the urine. The biological half-life of inorganic arsenic is about 4 days, but is slightly shorter following exposure to arsenate than to arsenite. The main metabolites excreted in the urine of humans exposed to inorganic arsenic are mono- and dimethylated arsenic acids, together with some unmetabolized inorganic arsenic. The biotransformation of arsenic for excretion is primarily done through the nuclear factor erythroid 2 related factor 2 (Nrf2) pathway. Under normal conditions the Nrf2 is bound to Kelch-like ECH associated protein 1 (Keap1) in its inactive form. With the uptake of arsenic within cells and the subsequent reactions that result in the production of reactive oxygen species (ROS), the Nrf2 unbinds and becomes active. Keap1 has reactive thiol moieties that bind ROS or electrophilic arsenic species such as monomethylted arsenic (III) and induces the release of Nrf2 which then travels through the cytoplasm to the nucleus. The Nrf2 then activates antioxidant responsive element (ARE) as well as electrophilic responsive element (EpRE) both of which contribute in the increase of antioxidant proteins. Of particular note in these antioxidant proteins is heme oxygenase 1 (HO-1), NAD(P)H-quinone oxidoreductase 1 (NQO1), and γ-glutamylcysteine synthase (γGCS) which work in conjunction to reduce the oxidative species such as hydrogen peroxide to decrease the oxidative stress upon the cell.
Senate House (University of London) was built on donation of £400,000 from Rockefeller Foundation in 1926 and a foundation stone laid by King George V in 1933. It is the headquarters of the University of London since 1937. In the arts, the Rockefeller Foundation has supported the Stratford Shakespeare Festival in Ontario, Canada, and the American Shakespeare Festival in Stratford, Connecticut, Arena Stage in Washington, D.C., Karamu House in Cleveland, and Lincoln Center in New York. The foundation underwrote Spike Lee's documentary on New Orleans, When the Levees Broke. The film has been used as the basis for a curriculum on poverty, developed by the Teachers College at Columbia University for their students. The Cultural Innovation Fund is a pilot grant program that is overseen by the Lincoln Center. The grants are to be used towards art and cultural opportunities in the underserved areas of Brooklyn and the South Bronx with three overarching goals. The Rockefeller Foundation supported the art scene in Haiti in 1948 and a literacy project with UNESCO. Rusk was involved with funding the humanities and the social sciences during the Cold War period, including study of the Soviet Union. In July 2022, the Rockefeller Foundation granted $1m to the Wikimedia Foundation.
metabolic pathway A stepwise series of biochemical reactions occurring within a cell, often but not necessarily catalyzed by specific enzymes, that fulfills some activity or process related to metabolism. The reactions are linked by the sharing of reactants, products, or intermediate compounds in consecutive steps, such that the product of one reaction is used as a reactant in a subsequent reaction. Byproducts are often removed from the cell as metabolic waste. The overall pathway may be anabolic, catabolic, or amphibolic in nature. In any actively metabolizing cell, an elaborate network of interconnected metabolic pathways is required to maintain homeostasis, with degradative catabolic processes providing the energy necessary to conduct anabolic biosynthesis; for example, glycolysis, the electron transport chain, and oxidative phosphorylation provide the ATP used in fatty acid synthesis. The flux of metabolites through each pathway is regulated by the needs of the cell and the availability of substrates.
== Analytical methods == Given the large diversity of PFAS structures that exist, analytical methods for PFAS analysis generally take one of two different approaches: targeted analysis or non-targeted analysis. Targeted methods narrow focus on known PFAS of concern (e.g. PFOA, PFOS) and generally use solid-phase extraction with liquid chromatography–mass spectrometry (LC-MS) detection. For example, EPA Method 537.1 is approved for use in drinking water and can quantify 18 PFAS compounds, while EPA Method 1633A is approved for use for wastewater, surface water, groundwater, soil, biosolids, sediment, landfill leachate, and fish tissue for 40 PFAS chemicals. Regulatory limits for PFOA and PFOS set by the US EPA (4 parts-per-trillion) are limited by the capability of targeted methods to detect low-level concentrations. Non-targeted analyses often sacrifice the identification and quantification of specific PFAS compounds to better understand the amount of PFAS present as a class. For example, total organic fluorine (TOF) analysis quantifies the amount of fluoride produced when a sample is oxidized at high enough temperatures to break the carbon-fluorine bond using combustion ion chromatography. Variants of this analysis include adsorbable organic fluorine (AOF) and extractable organic fluorine (EOF), which use similar solid-phase extraction approaches as the targeted analysis, but use combustion ion chromatography (CIC) as a detector.
=== 9 May === Two people were killed by Russian shelling in Nikopol. In Russia, the governor of Belgorod Oblast claimed that eight people were injured in a Ukrainian airstrike in Belgorod city. A drone attack on Krasnodar also set a fuel depot on fire. A drone strike was also reported in an oil refinery in Salavat in the first such incident of its kind in the constituent republic of Bashkortostan. The drone used in the attack on Bashkiria was conducted by a drone light aircraft, make unknown, that travelled some 1,500 km. The SBU arrested a resident of Kharkiv on suspicion of spying for Russia. It also claimed to have arrested several individuals in Kyiv on suspicion of attempting to set off bombs on behalf of Russia. Zelenskyy implemented a number of changes in the Ukrainian defense apparatus. He appointed Brigadier General Oleksandr Trepak as commander of the Ukrainian Special Operations Forces, replacing Colonel Serhii Lupanchuk. He also dismissed Valeriy Zaluzhnyi, former commander-in-chief of Ukraine's Armed Forces, from military service "on health grounds", and reappointed Dmytro Hereha as the commander of the Ukrainian Support Forces after replacing him with Oleksandr Yakovets in March 2024. Zelenskyy also dismissed Serhii Rud as director of the State Security Administration. Two colonels were also arrested after being accused of being agents for the Russian FSB. The US Army announced that it would supply three additional HIMARS units paid for by Germany to Ukraine.
Sources: en.wikipedia.org
=== Research articles === Ifa, Demian R; Eberlin, Livia S (January 1, 2016), "Ambient Ionization Mass Spectrometry for Cancer Diagnosis and Surgical Margin Evaluation", Clinical Chemistry, 62 (1), Oxford University Press (OUP): 111–123, doi:10.1373/clinchem.2014.237172, ISSN 0009-9147, PMC 5315420, PMID 26555455 Sans, Marta; Gharpure, Kshipra; Tibshirani, Robert; Zhang, Jialing; Liang, Li; Liu, Jinsong; Young, Jonathan H.; Dood, Robert L.; Sood, Anil K.; Eberlin, Livia S. (May 31, 2017), "Metabolic Markers and Statistical Prediction of Serous Ovarian Cancer Aggressiveness by Ambient Ionization Mass Spectrometry Imaging", Cancer Research, 77 (11), American Association for Cancer Research (AACR): 2903–2913, doi:10.1158/0008-5472.can-16-3044, ISSN 0008-5472, PMC 5750373, PMID 28416487 Santagata, Sandro; Eberlin, Livia S.; Norton, Isaiah; Calligaris, David; Feldman, Daniel R.; Ide, Jennifer L.; Liu, Xiaohui; Wiley, Joshua S.; Vestal, Matthew L.; Ramkissoon, Shakti H.; Orringer, Daniel A.; Gill, Kristen K.; Dunn, Ian F.; Dias-Santagata, Dora; Ligon, Keith L.; Jolesz, Ferenc A.; Golby, Alexandra J.; Cooks, R. Graham; Agar, Nathalie Y. R. (June 30, 2014), "Intraoperative mass spectrometry mapping of an onco-metabolite to guide brain tumor surgery", Proceedings of the National Academy of Sciences, 111 (30): 11121–11126, Bibcode:2014PNAS..11111121S, doi:10.1073/pnas.1404724111, ISSN 0027-8424, PMC 4121790, PMID 24982150 Correa, Deleon N.; Santos, Jandyson M.; Eberlin, Livia S.; Eberlin, Marcos N.; Teunissen, Sebastiaan F.
In the same month, France, the United States and Russia scheduled high-level, multi-country diplomatic visits in Africa. Reporting on these trips, Associated Press quoted William Gumede of Democracy Works saying "It’s like a new Cold War is playing out in Africa, where the rival sides are trying to gain influence" used the term "new Cold War"; AP commented "some say is the most intense competition for influence [in Africa] since the Cold War". An editorial by Christian Breuer said "It is conceivable that we see the rebirth of a new era of conflict, the end of the late 20th century unipolar international security architecture under the hegemony of the United States, the end of globalisation and the beginning of a new cold war between the West and the East." In August 2022, an analysis by journalist Anshel Pfeffer in Haaretz used the term to refer to the US's "open confrontation with Russia and China", concluding that "in the new Cold War, [Israel] cannot allow itself to be neutral". In the same month, Katrina vanden Heuvel cautioned against what she perceived as a "reflexive bipartisan embrace of a new Cold War" against Russia and China among US politicians. In September 2022, Greek civil engineer and politician Anna Diamantopoulou stated that, despite unity of NATO members, "the West has lost much of its normative power", citing her "meetings with politicians from Africa, Latin America, and the Middle East". She further stated that the West will risk losing "a new cold war" unless it overcomes challenges that would give Russia and China a greater world advantage.
=== Characterization === Cox described Logan Roy as a "mysterious character in many ways. I have a lot of empathy for him. I think he's very misunderstood. I think he's a tragic figure", and compared him to Shakespeare's tragic kings, such as Henry IV. "Obviously there's Lear connotations, but it's also the father dealing with a child in his son Hal and not understanding him." David Rasche, who plays Karl Muller, suggested to Jesse Armstrong that his character became a confessor to Logan, "But I don't think Jesse wanted that. Jesse wanted Logan Roy, a man alone." Cox thinks of Logan as "quite Puritan [...] Logan's a bastard, but he's not an indecent bastard." On Logan's romantic relationships he has said "He clearly loves Marcia. There's this idea that he had this affair with Rhea, but I'm not sure that happened. Both Holly [Hunter] and I felt that hadn't actually happened, [...] The kids thinking they were having an affair was more them projecting", but also added that "he's not good at relationships at all. The female relationships in his life have been pretty disastrous". By the final season Cox noted that, Colin and Kerry, Logan's bodyguard and assistant respectively, were "the two people who matter to him most." Cox has expressed that he believes that Logan's greatest weakness was that "he loves his children desperately." In interviews with The New Yorker, Armstrong agreed with Cox that Logan loves his children, even if he had done "things which are antithetical to what most people would consider to be love."
Life Length is a biotechnology company. Founded in 2010 by Stephen J. Matlin and Dr. María Blasco Marhuenda, the company provides telomere diagnostics as well as telomerase measurement. Life Length is Spain's only federally-accredited laboratory under CLIA. It has three main facilities, with offices in Madrid and laboratories located in Tres Cantos.
Sources: en.wikipedia.org
This refrigeration method cools a contained area by melting ice, by sublimating dry ice, or evaporating liquid nitrogen. A simple example of this is a portable cooler, where ice is poured over the items that need to be refrigerated, keeping them cool and insulated from heat. Regular ice maintains freezing temperatures, but not below the freezing point, unless salt is used to cool the ice down further (as in a traditional ice-cream maker). Mixtures of other solvents with coolants like water ice, dry ice, or liquid nitrogen are also used to make cooling baths for chemistry. Dry ice can reliably bring the temperature well below the freezing point of water, to as low as 195K.
== Creation == Their lineage dates back to include more than 200 years of unconventional warfare history, with notable predecessors including the American Revolutionary War "Swamp Fox" Francis Marion, Benjamin Forsyth in the War of 1812, Frederick Funston of the Philippine–American War, the WWII OSS Jedburgh Teams, OSS Detachment 101 in Burma, the Alamo Scouts, Colt Terry of the Korean War, and Vietnam War United States Army Special Forces officer Richard J. Meadows. Some of the Office of Strategic Services were similar in terms of the mission with the original U.S. Army Special Forces function, unconventional warfare (UW), acting as cadre to train and lead guerrillas in occupied countries. The Special Forces motto, De oppresso liber (Latin: "to free the oppressed") reflects this historical mission of guerrilla warfare against an occupying power. Specifically, the three-man Jedburgh teams provided leadership to French Resistance units. The larger Office of Strategic Services "OSS" Operational Groups (OG) were more associated with Strategic Reconnaissance/Direct Action (SR/DA) missions, although they did work with resistance units. Another unit widely associated with the origins of the Army Special Forces was the First Special Service Force, a joint Canadian-American unit formed in 1942 and disbanded in 1944. Members of the First Special Service Force were retroactively awarded the Special Forces Tab upon its creation in 1983 for their part in Special Forces' history.
=== Economic return === In 2000, the Joint Economic Committee of Congress reported NIH research, which was funded at $16 billion a year in 2000, that some econometric studies had given a rate of return of 25 to 40 percent per year by reducing the economic cost of illness in the US. It found that of the 21 drugs with the highest therapeutic impact on society introduced between 1965 and 1992, public funding was "instrumental" for 15. As of 2011, NIH-supported research helped to discover 153 new FDA-approved drugs, vaccines, and new indications for drugs in the 40 years prior. One study found NIH funding aided either directly or indirectly in developing the drugs or drug targets for all of the 210 FDA-approved drugs from 2010 to 2016. In 2015, Pierre Azoulay et al. estimated $10 million invested in research generated two to three new patents.
Sources: en.wikipedia.org
Common methods include enzymatic cycling assays, HPLC with UV detection, and LC-MS. The choice depends on sample size, specificity needs, and available equipment. Rapid quenching before analysis is important because NAD+ and NADH can interconvert.
Water promotes hydrolysis, and heat accelerates degradation. Cold, dry storage slows these processes. Repeated warming and cooling can introduce moisture and condensation, so aliquoting is often used.
Yes. They may be free acid or salts, with different counterions and purity grades. The counterion changes molecular weight, so concentration calculations should account for the actual form. Certificates of analysis provide batch-specific information.
Many enzymes consume or produce NAD+ within seconds after a sample is collected. Quenching stops those reactions and helps preserve the ratio between oxidized and reduced forms. The exact quenching method depends on the tissue or cell type and the analytes of interest.