This is a working overview of freeze-thaw, written for readers who want more than a one-paragraph summary but less than a textbook.
This page was last updated on 2026-04-24 and is reviewed periodically as new material appears.
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.
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.
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.
| Property | Value | Notes |
|---|---|---|
| Chemical name | Nicotinamide adenine dinucleotide | Oxidized form abbreviated NAD+ |
| Molecular formula | C21H27N7O14P2 | Free acid form |
| Molar mass | 663.43 g/mol | Calculated for free acid |
| CAS Registry Number | 53-84-9 | Common entry for beta-NAD+ |
| Appearance | White to off-white powder | Hygroscopic solid |
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.
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.
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.
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.
=== Intracellular receptors === Intracellular receptors exist freely in the cytoplasm, nucleus, or can be bound to organelles or membranes. For example, the presence of nuclear and mitochondrial receptors is well documented. The binding of a ligand to the intracellular receptor typically induces a response in the cell. Intracellular receptors often have a level of specificity, this allows the receptors to initiate certain responses when bound to a corresponding ligand. Intracellular receptors typically act on lipid soluble molecules. The receptors bind to a group of DNA binding proteins. Upon binding, the receptor-ligand complex translocates to the nucleus where they can alter patterns of gene expression. Steroid hormone receptors are found in the nucleus, cytosol, and also on the plasma membrane of target cells. They are generally intracellular receptors (typically cytoplasmic or nuclear) and initiate signal transduction for steroid hormones which lead to changes in gene expression over a time period of hours to days. The best studied steroid hormone receptors are members of the nuclear receptor subfamily 3 (NR3) that include receptors for estrogen (group NR3A) and 3-ketosteroids (group NR3C). In addition to nuclear receptors, several G protein-coupled receptors and ion channels act as cell surface receptors for certain steroid hormones.
List of things named after Adrien-Marie Legendre Associated Legendre polynomials Gauss–Legendre algorithm Legendre's constant Legendre's equation in number theory Legendre's functional relation for elliptic integrals Legendre's conjecture Legendre sieve Legendre symbol Legendre's theorem on spherical triangles Saccheri–Legendre theorem Least squares Least-squares spectral analysis Seconds pendulum From Summetria to Symmetry: The Making of a Revolutionary Scientific Concept
=== Standardization === Most of the dyes commonly used in microscopy are available as BSC-certified stains. This means that samples of the manufacturer's batch have been tested by an independent body, the Biological Stain Commission (BSC), and found to meet or exceed certain standards of purity, dye content and performance in staining techniques ensuring more accurately performed experiments and more reliable results. These standards are published in the commission's journal Biotechnic & Histochemistry. Many dyes are inconsistent in composition from one supplier to another. The use of BSC-certified stains eliminates a source of unexpected results. Some vendors sell stains "certified" by themselves rather than by the Biological Stain Commission. Such products may or may not be suitable for diagnostic and other applications.
== Diagnosis == Genetic testing for the m.3243A>G mutation in mitochondrial DNA is commonly used to isolate the diagnosis of MELAS syndrome from other mitochondrial disorders. This mutation is an adenine to guanine point mutation at base pair 3,243 in the mitochondrial genome. A minimum amount of mtDNA must be mutated to generate the MELAS phenotype, known as the "threshold effect". Due to mitochondrial heteroplasmy, urine and blood testing is preferable to blood alone. PCR and ARMS-PCR are commonly used, reliable, rapid, and cost-effective techniques for the diagnosis of MELAS. Magnetic-resonance imaging (MRI) is a common imaging test used to identify the presence of stroke-like lesions. These lesions are multifocal infarct-like areas of cortical edema in different stages of ischemic evolution, yet do not commonly conform to any known vascular territory, distinguishing them from a stroke. Initial lesions often occur in the occipital or parietal lobes with eventual involvement of the cerebellum, cerebral cortex, basal ganglia, and thalamus. The occipital lobe is thought to be a region prone to stroke-like lesions due to the high energy requirements of the visual cortex. Lactate levels are often elevated in serum and cerebrospinal fluid. Magnetic resonance spectroscopy (MRS) may show an elevated lactate peak in affected and even unaffected brain areas. Muscle biopsy shows ragged red fibers. However, genetic evaluation should be done first, which eliminates the need for muscle biopsy in most cases. Diagnosis may be molecular or clinical:
=== Legality === While butane-1,4-diol is not currently scheduled federally in the United States, a number of states have classified 1,4-butanediol as a controlled substance. Individuals have been prosecuted for possession of 1,4-butanediol under the Federal Analog Act as substantially similar to GHB. A federal case in New York in 2002 ruled that 1,4-butanediol could not be considered an analog of GHB under federal law, but that decision was later overturned by the Second Circuit. A jury in Federal District Court in Chicago found that 1,4-butanediol was not an analog of GHB under federal law, which was not disputed on the case's appeal to the Seventh Circuit Court of Appeals; however, this finding did not affect the outcome of the case. In the United Kingdom, 1,4-butanediol was scheduled in December 2009 (along with another GHB precursor, gamma-butyrolactone) as a Class C controlled substance. In Germany, the drug is not explicitly illegal, but might also be treated as illegal if used as a drug. It is controlled as a Schedule VI precursor in Canada.
Sources: en.wikipedia.org
== Genetics == The number of chondrocyte cells created and their maturation process can be influenced by multiple different genes and proteins. Two proteins, bone morphogenetic protein 4 (BMP-4) and fibroblast growth factor 2 (FGF2) have been seen to influence the amount of differentiation into chondrocytes. Both proteins are known to play a role in embryonic stem cell differentiation into mesodermal cells, through signaling with BMP-4 and as FGF2 acting as a stimulator. From the mesodermal germ layer, cells will continue to differentiate down into many different types of cells. On top of BMP-4 and FGF2 stimulating the mesodermal germ layer, treatment of these proteins has also been shown to amplify the number of cells that differentiate down into chondrogenic and osteogenic cells when cultured in chondrogenic and osteogenic mediums respectively. For chondrogenic cells, the treatment showed increased expression of the transcription factor Sox9, which plays a key role in chondrogenesis, the process of cartilage formation from condensed mesenchyme tissues, which then differentiate into chondrocytes. Endochondral ossification is the process by which most vertebrate axial skeletons form into hardened bones from cartilage. This process begins with a cartilage anlage where chondrocyte cells will congregate and start their maturation process. Once the chondrocytes have fully matured at the desired rate, the cartilage tissue will harden into bone. This process is similar across most vertebrates and is closely regulated due to the large importance of the skeleton in survival.
Demazin (made by Bayer Healthcare) — contains pseudoephedrine sulfate and chlorpheniramine maleate Eltor (made by Sanofi Aventis) — contains pseudoephedrine hydrochloride. Mucinex-D (made by Reckitt Benckiser) — contains 60 mg pseudoephedrine hydrochloride (also 1200 mg guaifenesin). Nexafed (made by Acura Pharmaceuticals) — contains 30 mg pseudoephedrine per tablet, formulated with Impede Meth-Deterrent technology. Nurofen Cold & Flu (made by Reckitt Benckiser) — contains 30 mg pseudoephedrine hydrochloride (also 200 mg ibuprofen). Respidina – contains 120 mg of pseudoephedrine in the form of extended release tablets. Rhinex Flash (made by Pharma Product Manufacturing, Cambodia) — contains pseudoephedrine combined with paracetamol and triprolidine. Rhinos SR (made by Dexa Medica) — contains 120 mg of pseudoephedrine hydrochloride Sinutab (made by McNeil Consumer Healthcare, a Kenvue Company) — contains 500 mg paracetamol and 30 mg pseudoephedrine hydrochloride. Sudafed Decongestant (made by McNeil Consumer Healthcare) — contains 60 mg of pseudoephedrine hydrochloride. Not to be confused with Sudafed PE, which contains phenylephrine.
A 15-hydroxyicosatetraenoate dehydrogenase metabolizes 15-hydroxyicosatetraenoic acid (i.e. 15(S)-hydroxy-5Z,8Z,11Z,13E-eicosatetraenoic acid or 15-HETE) to its 15-keto analog, 15-oxo-ETE, using NAD+ and NADH rather than NADP+ and NADPH as its co-factors. 15-Oxo-ETE appears to have a somewhat different spectrum of activities than its precursor, 15-HETE (see 15-Hydroxyicosatetraenoic acid
Marcey Lynn Waters is the Glen H. Elder Jr., Distinguished Professor of Chemistry at the University of North Carolina, Chapel Hill (UNC-CH). She is an organic chemist whose research is at the interface of chemical biology and supramolecular chemistry. Waters has received multiple awards for research, teaching, and advocating for women in science. She served as president of the American Peptide Society (APS) from 2017 to 2019. Waters graduated from the University of California, San Diego with a degree in chemistry in 1992. While an undergraduate, she worked with Prof. Charles L. Perrin studying fundamental aspects of aromaticity. Waters entered the University of Chicago for her doctoral degree in chemistry, working with Prof. William D. Wulff studying the mechanism for the Wulff-Dotz benzannulation reaction between Fischer carbene complexes. and alkynes. Walters graduated from Chicago in 1997 with a PhD in chemistry. She was an NIH postdoctoral fellow in Prof. Ronald Breslow's group from 1997 to 1999, where she worked on dinuclear metalloenzyme mimics and antiaromaticity.
Sources: en.wikipedia.org
In 1970, the focus of Freeman's research became protein crystallography and he turned his attention to the blue copper proteins (cupredoxins) and particularly the electron transport protein plastocyanin. The intensely blue colour of plastocyanin and its unusual redox properties had frustrated all attempts to synthesise a small molecule mimic. It was not until 1977 that his group finally determined the structure of plastocyanin crystallised from the poplar tree (see diagram at right); this was the first protein crystal structure determined in the Southern Hemisphere. Together with subsequent work in collaboration with Ed Solomon, this work led to understanding of the unusual geometry of the copper metal site (see diagram at left) as well as the spectroscopic and electrochemical properties characteristic of blue copper proteins. Later in his career, Freeman developed an interest in the applications of EXAFS spectroscopy to metalloprotein structure, collaborating with both James Penner-Hahn and Keith Hodgson. Working together, the Freeman and Hodgson groups were, in 1988, the first to determine a new crystal structure of a protein using the multiple wavelength anomalous dispersion (MAD) method. Throughout his career, Freeman was concerned about the influence of the tyranny of distance on the development of Australian science. For this reason, in 1972 Freeman and Alexander Boden AO, FAA, founded the Foundation for Inorganic Chemistry at the university to bring international scholars to the department to deliver a course for graduate students and give seminars for faculty.
An explanation of this relation in terms of linear elasticity theory is problematic. Linear elasticity theory predicts that stress (and hence the strain) at the tip of a sharp flaw in a linear elastic material is infinite. To avoid that problem, Griffith developed a thermodynamic approach to explain the relation that he observed. The growth of a crack, the extension of the surfaces on either side of the crack, requires an increase in the surface energy. Griffith found an expression for the constant
=== 21st century === About 89 percent of vehicular fuel needs are met by oil. Petroleum makes up 38 percent of total energy consumption in the United States, but is responsible for only 0.4 percent of electricity generation. Petroleum's worth as a portable, dense energy source powering the vast majority of vehicles and as the base of many industrial chemicals makes it one of the world's most important commodities. The top three oil-producing countries as of 2018 are the United States, Russia, and Saudi Arabia. In 2018, due in part to developments in hydraulic fracturing and horizontal drilling, the United States became the world's largest producer. About 80 percent of the world's readily accessible reserves are located in the Middle East, with 62.5 percent coming from the Arab five: Saudi Arabia, United Arab Emirates, Iraq, Qatar, and Kuwait. A large portion of the world's total oil exists as unconventional sources, such as bitumen in Athabasca oil sands and extra heavy oil in the Orinoco Belt. While significant volumes of oil are extracted from oil sands, particularly in Canada, logistical and technical hurdles remain, as oil extraction requires large amounts of heat and water, making its net energy content quite low relative to conventional crude oil. Thus, Canada's oil sands are not expected to provide more than a few million barrels per day in the foreseeable future. Many modern geopolitical conflicts are to do with petroleum.
Sources: en.wikipedia.org
NAD+ is the oxidized form, while NADH is the reduced form carrying an additional hydride equivalent. The pair participates in reversible electron transfer reactions. Their ratio helps indicate the redox state of a compartment.
NAD+ itself is not classified as a vitamin, but its precursor niacin is an essential nutrient in humans. Cells synthesize NAD+ from niacin, nicotinamide, nicotinamide riboside, or tryptophan. The intact dinucleotide is not obtained directly from typical diets in meaningful amounts.
Age-related studies often examine whether NAD+ levels decline in tissues and whether that decline affects mitochondrial function or DNA repair. Interventions using precursor molecules raise open questions about cause and effect. Current evidence does not establish that changing NAD+ levels slows human aging.
NAD+ is the oxidized form, while NADH is the reduced form carrying an added hydride. The two form a redox pair that cells use in many energy-yielding reactions.