Freeze-thaw comes up often in conversation and rarely with the context attached. Here we lay out the basics in order, then work through the practical considerations.
Updated 2026-03-10. Numbers and descriptions here follow the published literature rather than marketing material.
In glycolysis, the tricarboxylic acid cycle, and fatty acid oxidation, NAD+ is reduced to NADH at specific dehydrogenase steps. NADH then delivers electrons to the mitochondrial electron transport chain, mainly at complex I, supporting oxidative phosphorylation and ATP production. The balance between NAD+ and NADH, often expressed as a ratio, influences metabolic flux and redox homeostasis in different cellular compartments. Cytosolic and mitochondrial pools are connected but not identical, and their ratios can differ substantially because of compartment-specific enzymes and transport systems.
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.
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 |
|---|---|---|
| Common synonyms | β-NAD+, coenzyme I, DPN | DPN stands for diphosphopyridine nucleotide; older literature uses this term. |
| CAS Registry Number | 53-84-9 | Free acid form of β-nicotinamide adenine dinucleotide. |
| Molecular formula | C21H27N7O14P2 | Anhydrous free acid; molar mass 663.43 g/mol. |
| Appearance | White to off-white powder | Crystalline solid; may absorb moisture from air. |
| Solubility | Freely soluble in water | Insoluble in most nonpolar organic solvents. |
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.
Beyond electron transfer, NAD+ serves as a substrate for enzymes that cleave it and attach its ADP-ribose portion to other molecules. This group includes poly(ADP-ribose) polymerases, CD38, and sirtuins. Such reactions consume NAD+ and can influence its availability for metabolism. Cells replenish NAD+ through a salvage pathway that recycles nicotinamide and through routes starting from tryptophan or vitamin B3 forms. How these synthesis and consumption routes are coordinated across tissues remains an active area of study, and compartment-specific concentrations are difficult to measure directly.
Nicotinamide adenine dinucleotide, abbreviated NAD+, is a dinucleotide built from adenine, nicotinamide, two ribose sugars, and two phosphate groups. The oxidized form carries a positive charge on the nicotinamide ring, while the reduced form, NADH, is neutral. This pair acts as a reversible electron carrier in cells. NAD+ is present in bacteria, plants, animals, and fungi. Its structure allows it to accept and donate electrons without being consumed in the reactions it supports.
In redox reactions, NAD+ accepts a hydride ion, which consists of two electrons and one proton. The hydride adds to the nicotinamide ring at a specific carbon, converting NAD+ into NADH. Dehydrogenase enzymes use this step in glycolysis, the citric acid cycle, and fatty acid oxidation. NADH later donates electrons to the mitochondrial electron transport chain, helping to drive ATP synthesis. The balance between NAD+ and NADH reflects the metabolic state of a cell, and shifts in that balance can alter how pathways operate.
Neanderthals also consumed a variety of plants and mushrooms across their range — at Kebara Cave, Israel, over 50 species of seeds, nuts, fruits, and cereals. Neanderthals possibly employed a wide range of food preparation techniques. At Cueva del Sidrón, Spain, Neanderthals may have been roasting and smoking meat, and used certain plants—such as yarrow and camomile—for flavouring, although these plants may have instead been used for their medicinal properties. At Gorham's Cave, Gibraltar, Neanderthals may have been roasting pinecones to access pine nuts, and at Gruta da Figueira Brava, brown crabs to soften the shell before cracking them open. At Grotte du Lazaret, France, a total of twenty-three red deer, six ibexes, three aurochs, and one roe deer appear to have been hunted in a single autumn hunting season, when strong male and female deer herds would group together for rut. It is possible these Neanderthals were curing and storing all this meat before winter set in. Neanderthals at Neumark-Nord may have been rendering fat from animal bones to offset protein toxicity. Likewise, calcium measurements suggest that Neanderthals at Grotte du Bison frequently consumed bone. Neanderthals competed with several large carnivores, but also seem to have hunted them down, namely cave lions and wolves, as well as cave and brown bear both in and out of hibernation. Neanderthals and other predators may have sometimes avoided competition by pursuing different prey, namely with cave hyenas and wolves (niche differentiation).
The Zeisel determination for methoxyl group analysis indicated two methoxyl groups per molecule. John Elix and Brian Ferguson proposed a method for the total synthesis of confluentic acid in 1978. The synthesis began with the direct condensation of suitably substituted aromatic carboxylic acids and phenols, using dicyclohexylcarbodiimide. Key precursors involved were specially prepared benzoic acids, with protective measures for reactive groups. The process included steps like bromination, alkylation, and the strategic use of protecting groups for the phenol and carboxyl functionalities. The synthesis culminated in the removal of protecting groups and hydrogenolysis over palladised carbon to yield the desired depsides including confluentic acid. In 1993, G. Fegie and colleagues introduced a standardised high-performance liquid chromatographic method that enabled the separation and detection of hundreds of lichen products, confluentic acid included.
=== Australia === Dialysis is provided without cost to all patients through Medicare, with 75% of all dialysis being administered as haemodialysis to patients three times per week in a dialysis facility. The Northern Territory has the highest incidence rate per population of haemodialysis, with Indigenous Australians having higher rates of Chronic Kidney Disease and lower rates of functional kidney transplants than the broader population. The remote Central Australian town of Alice Springs, despite having a population of approximately 25000, has the largest dialysis unit in the Southern Hemisphere. Many people must move to Alice Springs from remote Indigenous communities to access health services such as haemodialysis, which results in housing shortages, overcrowding, and poor living conditions.
Aden soon became an important transit port and coaling station for trade between British India and the Far East, and Europe. The commercial and strategic importance of Aden increased considerably when the Suez Canal opened in 1869. From then and until the 1960s, the Port of Aden was to be one of the busiest ship-bunkering, duty-free shopping, and trading ports in the world. In 1937, Aden was separated from British India to become a Crown colony, a status that it retained until 1963. It consisted of the port city of Aden and its immediate surroundings (an area of 192 km2 [74 sq mi]). The Aden Settlement, and later Aden Colony, also included the outlying islands of Kamaran (de facto), Perim and Kuria Muria (see map). Prior to 1937, Aden had been governed as part of British India (originally as the Aden Settlement under the Bombay Presidency, and then as a Chief Commissioner's province). Under the Government of India Act 1935 the territory was detached from British India, and was re-organised as a separate Crown colony of the United Kingdom; this separation took effect on 1 April 1937. Through the latter years of its existence, Aden Colony was plagued by civil unrest.
== Residual topology == Residual topology is a descriptive stereochemical term to classify a number of intertwined and interlocked molecules, which cannot be disentangled in an experiment without breaking of covalent bonds, while the strict rules of mathematical topology allow such a disentanglement. Examples of such molecules are rotaxanes, catenanes with covalently linked rings (so-called pretzelanes), and open knots (pseudoknots) which are abundant in proteins. The term "residual topology" was suggested on account of a striking similarity of these compounds to the well-established topologically nontrivial species, such as catenanes and knotanes (molecular knots). The idea of residual topological isomerism introduces a handy scheme of modifying the molecular graphs and generalizes former efforts of systemization of mechanically bound and bridged molecules.
Sources: en.wikipedia.org
In 1867, the Austro-Hungarian compromise and the introduction of the dual monarchy left the Czechs and their aristocracy without the recognition of separate Bohemian state rights for which they had hoped. Bohemia remained part of the Austrian Crown Lands. In Bohemia, opposition to dualism took the form of isolated street demonstrations, resolutions from district representations, and even open air mass protest meetings, confined to the biggest cities, such as Prague. The Czech newspaper Národní listy complained that the Czechs had not yet been compensated for their wartime losses and sufferings during the Austro-Prussian War, and had just seen their historic state rights tossed aside and their land subsumed into the "other" half of the Austro-Hungarian Monarchy, commonly called "Cisleithania". The Czech hopes were revived again in 1870–1871. In an Imperial Rescript of 26 September 1870, Franz Joseph referred again to the prestige and glory of the Bohemian Crown and to his intention to hold a coronation. Under Minister-President Karl Hohenwart in 1871, the government of Cisleithania negotiated a series of fundamental articles spelling out the relationship of the Bohemian Crown to the rest of the Habsburg Monarchy. On 12 September 1871, Franz Joseph announced:
As the disease progresses, the brain undergoes a complex assortment of cellular and molecular changes, including (in addition to tauopathy) inflammation, oxidative/nitrative stress, DNA damage, epigenetic changes, excitotoxicity, endosomal/lysosomal failure, dysproteostasis, autophagy failure, lipid dysmetabolism, calcium ion (Ca2+) dyshomeostasis, post-translational protein modifications, neuronal cell cycle re-entry, mitochondrial failure, cytoskeletal disruption, glucose dysmetabolism, vascular or lymphatic impairments, and biometal dyshomeostasis. Iron dyshomeostasis is linked to disease progression in which an iron-dependent form of regulated cell death called ferroptosis could be involved. Products of lipid peroxidation are also elevated in the Alzheimer's brain compared with controls. Various inflammatory processes and cytokines also play a role in the pathology of Alzheimer's disease. Inflammation is a general marker of tissue damage in any disease, and may be either secondary to tissue damage in Alzheimer's disease or a marker of an immunological response. Cells that mediate neuroinflammation in Alzheimer's include microglia, astrocytes, oligodendrocytes, lymphocytes and myeloid cells. There is increasing evidence of a strong interaction between neurons and the immunological mechanisms in the brain. Obesity and systemic inflammation may interfere with immunological processes which promote disease progression. Microglia are especially important actors in the Alzheimer's-related inflammation.
== Career == After obtaining his PhD, Shaw spent six years working at the Centers for Disease Control and Prevention, where he was a supervisory research chemist and the chief of the radioimmunoassay laboratory. He then worked at Mercer University in Atlanta for a year as an assistant professor of pharmacy, before beginning a twelve-year stint at Smith Kline Beecham Clinical Laboratories, also in Atlanta. From 1991 until 1996, he worked at Children's Mercy Hospital in Kansas City, Missouri.
Grannygate – scandal in March 2000 over the eligibility of Shane Howarth and Brett Sinkinson representing the Wales national team Kamp Staaldraad – controversial training camp for the Springboks (South Africa's national rugby union team) before the 2003 Rugby World Cup "Bloodgate" – a scheme by the English rugby union club Harlequins to fake an injury to wing Tom Williams to allow a blood replacement to be brought on at a critical moment in their 2009 Heineken Cup quarterfinal against Leinster. The scheme, which included deliberately cutting Williams' mouth open after the match in order to cover up the fake injury, ultimately led to Quins head coach Dean Richards being banned from the sport for three years.
=== Skin and hair conditions === Flutamide has been researched and used extensively in the treatment of androgen-dependent skin and hair conditions in women including acne, seborrhea, hirsutism, and scalp hair loss, as well as in hyperandrogenism (e.g., in polycystic ovary syndrome or congenital adrenal hyperplasia), and is effective in improving the symptoms of these conditions. The dosages used are lower than those used in the treatment of prostate cancer. Although flutamide continues to be used for these indications, its use in recent years has been limited due to the risk of potentially fatal hepatotoxicity, and it is no longer recommended as a first- or second-line therapy. The related NSAA bicalutamide has also been found to be effective in the treatment of hirsutism in women and appears to have comparable effectiveness to that of flutamide, but has a far lower and only small risk of hepatotoxicity in comparison. Aside from its risk of liver toxicity and besides other nonsteroidal antiandrogens, it has been said that flutamide is likely the best typically used antiandrogen medication for the treatment of androgen-dependent symptoms in women. This is related to its high effectiveness and minimal side effects.
Sources: en.wikipedia.org
=== Food quality === Decontamination of food by ionizing radiation is a safe and efficient process for elimination of pathogenic bacteria. Ionizing radiation treatment can be applied to either raw materials or ready to eat foods, with some countries, like the United States, imposing limitations on its use. Assessed rigorously over several decades, irradiation in commercial amounts to treat food has no negative impact on the sensory qualities and nutrient content of foods.
where the initial state is the acid solution with no Ca2+ (not taking into account possible CO2 dissolution) and the final state is the solution with saturated Ca2+. For strong acid concentrations, all species have a negligible concentration in the final state with respect to Ca2+ and A− so that the neutrality equation reduces approximately to 2[Ca2+] = [A−] yielding [Ca2+] ≈ 0.5 [A−]. When the concentration decreases, [HCO−3] becomes non-negligible so that the preceding expression is no longer valid. For vanishing acid concentrations, one can recover the final pH and the solubility of CaCO3 in pure water. In the case of a weak monoacid (here we take acetic acid with pKa = 4.76) with decreasing total acid concentration [A] = [A−] + [AH], we obtain:
=== Treatment === Treatment modalities generally include both pharmacological and non-pharmacological, depending on the primary diagnosis. Additional treatment options include referral to specialist care including physical therapy and rehabilitation. Treatment recommendations differ in the acute inpatient and outpatient settings. Continuity of care and long-term follow-up is crucial in successful patient outcomes.
== History == The process is named after the Dutch physicist Frans Michel Penning who first reported it in 1927. Penning started to work at the Philips Natuurkundig Laboratorium at Eindhoven to continue the investigation of electric discharge on rare gases. Later, he started measurements on the liberation of electrons from metal surfaces by positive ions and metastable atoms, and especially on the effects related to ionization by metastable atoms.
==== Pharmacokinetics of indoline derivatives ==== Sunitinib's peak plasma level is reached 6–12 hours post-dose. The drug is 95% protein bound and has a volume of distribution of 2230 L, which indicates distribution into tissues. Sunitinib is metabolised mostly by CYP3A4 and has a half-life of 40–60 hours. Nintedanib reaches maximum plasma concentration 2–4 hours post-dose. The drug is 97,8% protein bound and is preferentially distributed in plasma. Only a minor part of the biotransformation of nintedanib is caused by CYP3A4 as the prevalent metabolic reaction for nintedanib is a hydrolytic cleavage of esterases. The half-life of nintedanib is about 10–15 hours.
Sources: en.wikipedia.org
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.
NAD+ is the oxidized form and can accept a hydride equivalent. NADH is the reduced form and donates electrons to the electron transport chain. The two forms cycle between each other during cellular respiration.
In mammals, NAD+ is synthesized mainly through salvage pathways using nicotinamide, nicotinamide riboside, or nicotinic acid. Tryptophan can also contribute through a de novo route. The salvage pathway is often considered the primary source in many tissues.
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.