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 2025-11-13 and is reviewed periodically as new material appears.
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.
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.
| Property | Value | Notes |
|---|---|---|
| Molar mass | 663.43 g/mol | For the free acid form; salts have higher mass. |
| Appearance | White to off-white powder | Often hygroscopic; may clump on exposure to air. |
| Solubility | Freely soluble in water | Poorly soluble in nonpolar organic solvents. |
| Typical storage | -20 °C, desiccated | Protect from light and moisture; avoid repeated freeze-thaw. |
| Common synonyms | beta-NAD, DPN | DPN stands for diphosphopyridine nucleotide, an older name. |
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.
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.
Among her appointments are James Cleverly, who becomes Shadow Housing Secretary. Welsh Conservative member of the Senedd Laura Anne Jones joins Reform UK. 19-year-old George Finch is formally appointed as leader of Warwickshire County Council, making the Reform UK councillor the UK's youngest permanent council leader. 23 July – The Scottish Greens are forced to issue a new list of candidates for the next Holyrood election after an error was found in counting the votes. 24 July – Jeremy Corbyn and Zarah Sultana officially launch their new political party, which has the provisional name Your Party. 25 July – 220 MPs sign a letter to the prime minister urging him to recognise Palestinian statehood. Former SNP MP Mhairi Black announces that she has left the party, citing its stance over transgender rights and Palestine as her main reasons for doing so. US President Donald Trump arrives at Prestwick Airport to begin a four-day private visit to the United Kingdom during which he will meet Prime Minister Keir Starmer and First Minister of Scotland John Swinney. 29 July – Starmer announces the UK will recognise a Palestinian state in September unless Israel takes "substantive steps to end the appalling situation in Gaza". Benjamin Netanyahu, Israel's prime minister, says in response that to do so "rewards Hamas's monstrous terrorism". Ghazi Hamad of Hamas welcomes the announcement as "one of the fruits of October 7". Trump completes his four-day visit to Scotland and returns to the United States.
This referendum included a choice of different terms for a merger with Malaysia and had no option for avoiding merger altogether. On 16 September 1963, Singapore joined with Malaya, the North Borneo, and Sarawak to form the new Federation of Malaysia under the terms of the Malaysia Agreement. Under this Agreement, Singapore had a relatively high level of autonomy compared to the other states of Malaysia. Indonesia opposed the formation of Malaysia due to its own claims over Borneo and launched Konfrontasi in response to the formation of Malaysia. On 10 March 1965, a bomb planted by Indonesian saboteurs on a mezzanine floor of MacDonald House exploded, killing three people and injuring 33 others. It was the deadliest of at least 42 bomb incidents which occurred during the Konfrontasi. Two members of the Indonesian Marine Corps, Osman bin Haji Mohamed Ali and Harun bin Said, were eventually convicted and executed for the crime. The explosion caused US$250,000 (equivalent to US$2,554,118 in 2025) in damages to MacDonald House. Even after the merger, the Singaporean government and the Malaysian central government disagreed on many political and economic issues. Despite an agreement to establish a common market, Singapore continued to face restrictions when trading with the rest of Malaysia. In retaliation, Singapore did not extend to Sabah and Sarawak the full extent of the loans agreed to for economic development of the two eastern states. Talks soon broke down, and abusive speeches and writing became rife on both sides.
==== Transit time ==== The time taken for food to transit through the gastrointestinal tract varies on multiple factors, including age, ethnicity, and gender. Several techniques have been used to measure transit time, including radiography following a barium-labeled meal, breath hydrogen analysis, scintigraphic analysis following a radiolabeled meal, and simple ingestion and spotting of corn kernels. It takes 2.5 to 3 hours for 50% of the contents to leave the stomach. The rate of digestion is also dependent of the material being digested, as food composition from the same meal may leave the stomach at different rates. Total emptying of the stomach takes around 4–5 hours, and transit through the colon takes 30 to 50 hours.
Sources: en.wikipedia.org
=== Resistance === Development of bacterial resistance under therapy is a frequent occurrence and makes fosfomycin unsuitable for sustained therapy of severe infections. Mutations that inactivate the nonessential glycerophosphate transporter render bacteria resistant to fosfomycin. Still, fosfomycin can be used to treat MRSA bacteremia. Prescribing fosfomycin together with at least another active drug reduces the risk of developing bacterial resistance. Fosfomycin acts synergistically with many other antibiotics, including aminoglycosides, carbapenems, cephalosporins, daptomycin and oritavancin. Enzymes conferring resistance to fosfomycin have also been identified and are encoded both chromosomally and on plasmids. Three related fosfomycin resistance enzymes (named FosA, FosB, and FosX) are members of the glyoxalase superfamily. These enzymes function by nucleophilic attack on carbon 1 of fosfomycin, which opens the epoxide ring and renders the drug ineffective. The enzymes differ by the identity of the nucleophile used in the reaction: glutathione for FosA, bacillithiol for FosB, and water for FosX. In general, FosA and FosX enzymes are produced by Gram-negative bacteria, whereas FosB is produced by Gram-positive bacteria. FosC uses ATP and adds a phosphate group to fosfomycin, thus altering its properties and making the drug ineffective.
== History and clinical trials == In the 1970s, Jens Juul Holst and Joel Habener began research on GLP-1, initially in relation to duodenal ulcer disease. They were examining hormones secreted during eating, and testing them on pig pancreases, leading to the discovery of GLP-1's significant effects in 1988. Their work, which later contributed significantly to diabetes and obesity treatments, earned them and Daniel J. Drucker the 2020 Warren Alpert Foundation Prize. Research continued, and in 1993, Michael Nauck managed to infuse GLP-1 into people with type 2 diabetes, stimulating insulin while inhibiting glucagon and bringing blood glucose to normal levels. However, treating diabetes with GLP-1 hormones resulted in significant side effects, leading researchers financed by Novo Nordisk to start looking to develop a suitable compound for therapeutic use. In 1998, a team of researchers at Novo Nordisk led by Lotte Bjerre Knudsen developed liraglutide, a GLP-1 receptor agonist that could be used to treat diabetes. This was followed by the development of semaglutide by a team of researchers at Novo Nordisk, including Jesper Lau, Thomas Kruse, and Paw Bloch.
Another example is Girgui at Harvard University, who built a MMS based on existing underwater mass spectrometers (UMS) that can operate underwater to study the influence of microbes on the methane and hydrogen content of the ocean. He worked with a mechanical engineer to package a commercial quadrupole mass analyzer from Stanford Research Systems, a Pfeiffer HiPace80 turbopump, and a custom gas extractor into a 25 cm × 90 cm cylinder. Total cost is about $15,000. The Analytical Instrumentation Research Institute in Korea also developed a palm-portable mass spectrometer. The size and weight is reduced to 1.54 L and 1.48 kg respectively, and it used 5 W power only. The PPMS is based on four parallel disk ion traps, a small ion getter pump and a micro-computer. The PPM can perform the scan ion mass of up to m/z 300 and detect the ppm concentration of organic gases diluted in the air. The Harsh-Environment Mass Spectrometry Society is holding a biannual workshop that focuses on in-situ mass spectrometry in extreme environments, such as in the deep ocean, volcano crater, or outer space require high reliability, autonomous or remote operation, ruggedness with minimum size, weight, and power. The archives of the workshop include ~100 presentations focusing on the design and application of miniature mass spectrometers.
Sources: en.wikipedia.org
It indicates the oxidized form, which has a positive charge on the nicotinamide nitrogen. The reduced partner NADH lacks that charge and carries added electrons. The plus sign is part of the standard abbreviation, not a separate ion.
No. It also serves as a substrate for signaling and DNA-repair enzymes such as sirtuins and PARPs. Those reactions consume NAD+ and connect its availability to cellular regulation. Energy transfer remains its most abundant known role.
NAD+ is the oxidized electron acceptor, while NADH is the reduced electron carrier. They form a reversible redox pair and differ by a hydride ion. Cells maintain different ratios of the two depending on conditions and compartment.
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.