Freeze-thaw raises a handful of sensible questions. This page answers them in order, starting with the fundamentals and moving to applications.
This page was last updated on 2026-07-01 and is reviewed periodically as new material appears.
Quantification of NAD+ in biological samples typically uses liquid chromatography coupled to mass spectrometry. Enzymatic cycling assays offer higher throughput and rely on NAD+ dependent dehydrogenases to amplify signal. Both approaches require careful sample quenching because NAD+ can be rapidly consumed or converted after collection. Acidic extraction is common for NAD+, while alkaline conditions favor NADH in some protocols. Isotopically labeled internal standards help correct for losses during extraction and ionization.
Commercial NAD+ is available at research grade, often with purity specifications determined by high-performance liquid chromatography. Certificates of analysis may report water content, residual solvents, and counterion identity. Identity can be confirmed by ultraviolet absorbance near 260 nm, mass spectrometry, or enzymatic activity. Because different salt forms and hydration states exist, researchers should verify that the product matches the intended molecular form. Lot-to-lot variation in purity can affect quantitative assays and should be documented.
Beyond redox chemistry, NAD+ serves as a substrate for enzymes that cleave the molecule and transfer its ADP-ribose moiety or remove acetyl groups. Sirtuins consume NAD+ during deacetylation, poly(ADP-ribose) polymerases use it in DNA damage responses, and CD38 enzymes hydrolyze it to signaling metabolites. These consumption pathways mean that NAD+ availability can influence gene regulation, DNA repair, and calcium signaling. Cellular NAD+ concentrations decline in some tissues with age in animal models, but whether this decline is a cause or consequence of aging in humans remains an active open question.
Nicotinamide adenine dinucleotide, commonly abbreviated NAD+, is a dinucleotide coenzyme built from an adenine nucleotide and a nicotinamide nucleotide joined by a pyrophosphate linkage. Its oxidized form carries a positive charge on the nicotinamide ring, while the reduced form, NADH, carries a hydride equivalent. The molecule participates in hundreds of oxidoreductase reactions, where it accepts or donates electrons and protons. Because it can cycle between oxidized and reduced states without net consumption, NAD+ functions as a reusable electron carrier rather than a fuel molecule.
| Property | Value | Notes |
|---|---|---|
| Solubility | Freely soluble in water | Forms acidic solution; salt form may alter solubility |
| Typical storage temperature | -20 °C or lower | Desiccated and protected from light |
| Common analytical method | LC-MS | Used for biological quantification |
| UV absorbance maximum | 260 nm | Aqueous solution; pH dependent |
| Common synonym | Diphosphopyridine nucleotide | Older name abbreviated DPN |
Quality control for NAD+ materials typically combines identity, purity, and water content checks. Identity may be confirmed by ultraviolet spectrum, retention time in chromatography, or mass accuracy, while purity is assessed by HPLC peak area or quantitative nuclear magnetic resonance. Residual water and solvents can affect molar calculations and enzyme assays, so Karl Fischer titration or thermogravimetric analysis may be used. Commercial materials vary in grade and counterion form, and published methods should specify the exact salt or hydrate when reporting concentrations. Regulatory status depends on intended use, with research reagents, dietary ingredients, and clinical products treated under different frameworks.
Quantification of NAD+ in biological samples usually relies on separation techniques coupled to sensitive detection. High-performance liquid chromatography with ultraviolet detection can measure the oxidized form by its absorbance near 260 nm, while mass spectrometry provides greater specificity and can distinguish NAD+ from close analogs. Enzymatic cycling assays use coupled dehydrogenase reactions to amplify signal and estimate NAD+ concentrations in cell or tissue extracts. Because NAD+ and NADH interconvert rapidly, sample preparation must quench metabolism quickly and preserve the redox state before analysis.
NAD+ also serves as a substrate for enzymes that cleave it, including sirtuins, PARPs, and CD38. These enzymes consume NAD+ and release nicotinamide and ADP-ribose or related products. The dual roles as redox cofactor and signaling substrate connect NAD+ to DNA repair, circadian regulation, and calcium signaling. Cellular NAD+ concentrations vary by tissue, time of day, and stress exposure. How these consumption pathways interact with redox balance remains an active area of research.
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.
In aqueous solution, NAD+ is most stable under mildly acidic to neutral conditions and degrades faster at high pH or elevated temperature. The molecule can hydrolyze at the pyrophosphate bond or undergo nonenzymatic cyclization. Buffers, chelating agents, and cold temperatures slow these losses during analysis. Repeated freeze-thaw cycles are generally avoided because they can promote degradation and concentration changes. Light exposure is also controlled, though NAD+ is less photolabile than some related nucleotides.
Commercial NAD+ is supplied as a solid, often as the free acid or a salt, and purity is verified by chromatographic methods. Laboratories typically store it desiccated at minus 20 degrees Celsius or below. Working solutions are prepared fresh because even sterile aqueous solutions can lose activity over hours to days depending on pH and temperature. Documentation may include a certificate of analysis, an assay value, and a recommended retest date. Researchers should verify identity and purity when results depend on precise cofactor concentrations.
NAD+ is commonly measured by high-performance liquid chromatography with ultraviolet detection, often at 254 or 260 nm. Enzymatic cycling assays provide higher sensitivity by coupling NAD+ to a reporter reaction. Mass spectrometry can distinguish NAD+ from close analogues and confirm isotope labeling. Sample preparation usually involves rapid quenching of metabolism to prevent interconversion with NADH. Because NAD+ and NADH differ by one hydride, extraction conditions strongly affect the measured ratio.
Germany's military, the Bundeswehr (Federal Defence), is organised into the Heer (Army and special forces KSK), Marine (Navy), Luftwaffe (Air Force) and Cyber- und Informationsraum (Cyber and Information Domain Service) branches. In absolute terms, German military spending in 2025 was the fourth-highest in the world. In response to the 2022 Russian invasion of Ukraine, Chancellor Olaf Scholz announced that German military expenditure would be increased past the NATO target of 2%, along with a one-time 2022 infusion of 100 billion euros, representing almost double the 53 billion euro military budget for 2021. In 2023, military spending according to NATO criteria amounted to $73.1 billion, or 1.64% of the country's GDP, well below the NATO target of 2%. In 2026, Germany reported an estimated spending of $147.002 billion to NATO, exceeding said target at 2.69% of GDP. As of May 2024, the Bundeswehr has a strength of 180,215 active soldiers and 80,761 civilians. Reservists are available to the armed forces and participate in defence exercises and deployments abroad. Until 2011, military service was compulsory for men at age 18, but this has been officially suspended and replaced with a voluntary service. Since 2001 women may serve in all functions of service without restriction. According to the Stockholm International Peace Research Institute, Germany was the fourth-largest exporter of major arms in the world between 2021 and 2025. In peacetime, the Bundeswehr is commanded by the Minister of Defence.
=== Direct immunofluorescence testing === The presence of Immunoglobulin G, A, or M in the epidermis is normal. Detection in other locations such as intercellular and areas below the epidermis (subepidermal), as well as along the dermoepidermal junction (area that joins the epidermis and dermis), suggests paraneoplastic pemphigus.
=== Anti-atherosclerotic and anti-inflammatory effects === 1-MNA exhibits anti-atherosclerotic and anti-inflammatory properties by improving the prostacyclin- and NO-dependent secretory function of the vascular endothelium, inhibiting platelet activation, reducing inflammation within atherosclerotic plaques, and lowering systemic inflammation and TNF-α levels. The anti-inflammatory effects of 1-MNA are linked to its ability to stimulate endogenous PGI2 secretion and reduce IL-4 and TNF-α levels. These effects are mediated by endothelial mechanisms rather than a direct impact on immune cell function, ensuring that the body's immune response is not weakened.
== Overview == A chaotropic agent is a substance which disrupts the structure of, and denatures, macromolecules such as proteins and nucleic acids (e.g. DNA and RNA). Chaotropic solutes increase the entropy of the system by interfering with intermolecular interactions mediated by non-covalent forces such as hydrogen bonds, van der Waals forces, and hydrophobic effects. Macromolecular structure and function is dependent on the net effect of these forces (see protein folding), therefore it follows that an increase in chaotropic solutes in a biological system will denature macromolecules, reduce enzymatic activity and induce stress on a cell (i.e., a cell will have to synthesize stress protectants). Tertiary protein folding is dependent on hydrophobic forces from amino acids throughout the sequence of the protein. Chaotropic solutes decrease the net hydrophobic effect of hydrophobic regions because of a disordering of water molecules adjacent to the protein. This solubilises the hydrophobic region in the solution, thereby denaturing the protein. This is also directly applicable to the hydrophobic region in lipid bilayers; if a critical concentration of a chaotropic solute is reached (in the hydrophobic region of the bilayer) then membrane integrity will be compromised, and the cell will lyse. Chaotropic salts that dissociate in solution exert chaotropic effects via different mechanisms.
Gobies are dorsal finned like the lumpsuckers, yet they are not related. The Rhenanids became extinct over 200 million years before the first stingrays evolved, yet they share quite a similar appearance. Sandlance fish and chameleons have independent eye movements and focusing by use of the cornea. Acanthurids and mbuna are both aggressive, brightly colored fish that feed principally on aufwuchs, although the former is found only in marine environments, while the latter is only found in freshwater Lake Malawi. Cichlids of South America and the "sunfish" of North America are strikingly similar in morphology, ecology and behavior. The peacock bass and largemouth bass are excellent examples. The two fishes are not related, yet are very similar. Peacock bass are native of South America and is a Cichla. While largemouth bass are native to Southern USA states and is a sunfish. others will surely be described (but see the results based on DNA data). The antifreeze protein of fish in the Arctic and Antarctic, came about independently. AFGPs evolved separately in notothenioids and northern cod. In notothenioids, the AFGP gene arose from an ancestral trypsinogen-like serine protease gene. Electric fish: electric organs and electrosensory systems evolved independently in South American Gymnotiformes and African Mormyridae. Eel form are independent in the North American brook lamprey, neotropical eels, and the African spiny eel.
Sources: en.wikipedia.org
16 November – The SNP's National Executive Committee proposes cutting the number of staff at its headquarters from 26 to 16. Ruth Maguire, MSP for Cunninghame South, announces she will not seek re-election at the 2026 Scottish Parliament election because she is to undergo treatment for cancer. 17 November – At 12pm, Tesco opens its branch in Stornaway, Isle of Lewis for the first time on a Sunday, the Western Isles being the only place in the UK where the majority of shops remain closed on a Sunday. 19 November – Scottish Labour leader Anas Sarwar says his party will expand the eligibility for winter fuel payments in Scotland if they win the 2026 Scottish Parliament election. Eric Trump, the son of Donald Trump, confirms the US President-elect will visit Scotland in 2025 to open a new golf course. The Scottish Government is reported to be preparing to reduce the minimum term served by prisoners given short sentences, which could lead to the early release of between 260 and 390 prisoners in February 2025. 20 November – The Children and Young People's Commissioner Scotland launches an investigation into Police Scotland's use of force against children. 21 November – Stephen Flynn, the SNP's leader at Westminster, says he will not seek a dual mandate by standing for the Scottish Parliament while he remains an MP. Traditional Scottish singer Iona Fyfe is named as the new Rector of the University of Aberdeen, succeeding Martina Chukwuma–Ezike, and will take up the post on 1 January 2025.
When women reach menopause and the estrogen produced by the ovaries declines, fat migrates from the buttocks, hips and thighs to the waist; later fat is stored in the abdomen. Visceral fat can be caused by excess cortisol levels. At least 10 MET-hours per week of aerobic exercise leads to visceral fat reduction in those without metabolic-related disorders. Resistance training and caloric restriction also reduce visceral fat, although their effect may not be cumulative. Both exercise and hypocaloric diet cause loss of visceral fat, but exercise has a larger effect on visceral fat versus total fat. High-intensity exercise is one way to effectively reduce total abdominal fat. An energy-restricted diet combined with exercise will reduce total body fat and the ratio of visceral adipose tissue to subcutaneous adipose tissue, suggesting a preferential mobilization for visceral fat over subcutaneous fat.
=== Renal function === A 2019 systematic review published by the National Kidney Foundation investigated whether creatine supplementation had adverse effects on renal function. They identified 15 studies from 1997 to 2013 that looked at standard creatine loading and maintenance protocols of 4–20 g/day of creatine versus placebo. They utilized serum creatinine, creatinine clearance, and serum urea levels as a measure of renal damage. While in general creatine supplementation resulted in slightly elevated creatinine levels that remained within normal limits, supplementation did not induce renal damage (P value< 0.001). Special populations included in the 2019 Systematic review included type 2 diabetic patients and post-menopausal women, bodybuilders, athletes, and resistance trained populations. The study also discussed 3 case studies where there were reports that creatine affected renal function. In a joint statement between the American College of Sports Medicine, Academy of Nutrition and Dietetics, and Dietitians in Canada on performance enhancing nutrition strategies, creatine was included in their list of ergogenic aids and they do not list renal function as a concern for use. The most recent position stand on creatine from the Journal of International Society of Sports Nutrition states that creatine is safe to take in healthy populations from infants to the elderly to performance athletes. They also state that long term (5 years) use of creatine has been considered safe.
== Biosynthesis == Kynurenine gives its name to the kynurenine pathway which leads from the amino acid tryptophan to many important enzyme cofactors including niacin and nicotinamide adenine dinucleotide.
Briskets can be cooked in many ways, including baking, boiling and roasting. It is often basted during cooking. This cut of meat starts out tough because of the collagen fibers that make up the significant connective tissue. Long cooking tenderizes it as the collagen gelatinises. The fat cap, which is often left attached, helps to keep the meat from drying during prolonged cooking. Popular cooking methods in the United States include rubbing with a spice rub or marinating the meat, and then cooking slowly over indirect heat from charcoal or wood. This is a form of smoking the meat. A hardwood, such as oak, pecan, hickory or mesquite is sometimes added, alone or in combination with other hardwoods, to the main heat source. Sometimes, they make up all of the heat sources, with chefs often prizing characteristics of certain woods. The smoke from the woods and from burnt dripping juices further enhances the flavor. The finished meat is a variety of barbecue. Smoked brisket done this way is popular in Texas barbecue. Once finished, pieces of brisket can be returned to the smoker to make burnt ends. Burnt ends are most popular in Kansas City-style barbecue, where they are traditionally served open-faced on white bread. The traditional New England boiled dinner features brisket as a main-course option. In the United States, the whole boneless brisket is defined as IMPS 120.
Sources: en.wikipedia.org
Some bodybuilders inject oils or other compounds into their muscles (sometimes known as "synthol") to enhance their size or appearance. This practice can have serious health consequences and risks for humans.
== Pathophysiology == When ingested, hypoglycin A is metabolized to produce methylenecyclopropylacetic acid (MCPA). MCPA acts to inhibit the beta-oxidation of fatty acids in two ways. First, it interferes with the transport of long-chain fatty acids into the mitochondria by forming ester linkages with carnitine and coenzyme A (CoA). Also, it inhibits acyl-CoA dehydrogenases, so that only unsaturated fatty acids can be fully oxidized. Fatty acids accumulate in the liver in a microvesicular pattern that can be seen on biopsy. In the absence of fatty acid metabolism, the body becomes dependent on glucose and glycogen for energy. Octreotide can be used to reduce the secretion of insulin by the pancreas, thereby preventing severe hypoglycemia. Inhibition of beta-oxidation of fatty acids, however, also depletes nicotinamide adenine dinucleotide (NADH) and acetyl CoA—the latter an activator of pyruvate carboxylase—inhibiting gluconeogenesis. Once the liver glycogen stores are depleted, the body cannot synthesize glucose, and severe hypoglycemia results. Initial symptoms appear after about four hours, and deaths have been reported from 12 to 48 hours following consumption. Supportive care involves carefully metered IV glucose infusion and fluid/electrolyte replacement; mortality was 80% before glucose infusion was introduced in 1954. A similar outbreak of lethal hypoglycemic encephalopathy has been linked to the consumption of lychee fruit in Muzaffarpur, India.
Moreover, both the refractive index and the optical absorption are found to be dispersionless over a broad wavelength range from visible to near infrared. Finally, GO film offers flexible patterning capability by using the maskless DLW method, which reduces the manufacturing complexity and requirements. As a result, a novel ultrathin planar lens on a GO thin film has been realized recently using the DLW method. The distinct advantage of the GO flat lens is that phase modulation and amplitude modulation can be achieved simultaneously, which are attributed to the giant refractive index modulation and the variable linear optical absorption of GO during its reduction process, respectively. Due to the enhanced wavefront shaping capability, the lens thickness is pushed down to subwavelength scale (~200 nm), which is thinner than all current dielectric lenses (~ μm scale). The focusing intensities and the focal length can be controlled effectively by varying the laser powers and the lens sizes, respectively. By using an oil immersion high numerical aperture (NA) objective during DLW process, 300 nm fabrication feature size on GO film has been realized, and therefore the minimum lens size has been shrunk down to 4.6 μm in diameter, which is the smallest planar micro lens and can only be realized with metasurface by FIB. Thereafter, the focal length can be reduced to as small as 0.8 μm, which would potentially increase the numerical aperture (NA) and the focusing resolution.
15 September – The Sunday Times reports that Starmer may have broken parliamentary rules by failing to declare clothes bought for his wife, Victoria, by Labour donor Lord Waheed Alli. 16 September – Downing Street confirms that the Parliamentary Commissioner on Standards will not investigate whether Starmer broke parliamentary rules by not declaring a gift of clothes for his wife. The Welsh Government scraps plans to force parties in the Senedd to ensure 50% of their candidates are women. 18 September – Liberal Democrat leader Ed Davey unveils an expanded 33-member frontbench team following the party's gains at the general election. Former prime minister Sir John Major criticises the Sunak government's Rwanda asylum plan, describing it as "un-Conservative and un-British". Ofcom announces it will not investigate Ed Balls' interview with his wife, Home Secretary Yvette Cooper, on Good Morning Britain on 5 August despite receiving over 16,000 viewer complaints. ITV chief executive Dame Carolyn McCall defends the interview's impartiality, but says the broadcaster would not do it again. 20 September – Downing Street confirms that Keir Starmer, Angela Rayner and Rachel Reeves will no longer accept donations for clothing following controversy over gifts given to Starmer's wife. 22–25 September – The 2024 Labour Party Conference is scheduled to take place at the ACC Liverpool. 24 September – Starmer gives his keynote conference speech, setting out plans for "national renewal" but telling delegates there are no "easy answers".
Oganesson is a synthetic chemical element; it has symbol Og and atomic number 118. It was first synthesized in 2002 at the Joint Institute for Nuclear Research (JINR) in Dubna, near Moscow, Russia, by a joint team of Russian and American scientists. In December 2015, it was recognized as one of four new elements by the Joint Working Party of the international scientific bodies IUPAC and IUPAP. It was formally named on 28 November 2016. The name honors the nuclear physicist Yuri Oganessian, who played a leading role in the discovery of the heaviest elements in the periodic table. Oganesson has the highest atomic number and highest atomic mass of all known elements. On the periodic table of the elements it is a p-block element, a member of group 18, and the last member of period 7. Its only known isotope, oganesson-294, is highly radioactive, with a half-life of 0.7 ms. This half-life is too short for chemical studies. Because of relativistic effects, theoretical studies predict that it would be a solid at room temperature, and significantly reactive, unlike the other members of group 18 (the noble gases).
Sources: en.wikipedia.org
Aqueous NAD+ solutions are best kept frozen in aliquots and protected from light. Repeated freezing and thawing is avoided because it can accelerate breakdown. Dry powder stored desiccated at -20 °C or lower typically remains stable for longer periods.
Liquid chromatography-mass spectrometry provides sensitive and specific quantification in cells and tissues. Enzymatic cycling assays are also widely used for plate-based measurement. Both methods need rapid sample processing to prevent post-collection changes.
Purity refers to the proportion of the intended dinucleotide relative to related nucleotides, salts, and water. A high-purity grade supports reproducible enzymatic assays. Researchers often check purity by chromatographic and spectroscopic methods before use.
NAD+ is an oxidized dinucleotide coenzyme that carries electrons in metabolic reactions. It is also consumed by signaling enzymes, including sirtuins and PARPs. Its reduced form is NADH.