Hop Acids Research Articles

Effects of Spent Craft Brewers’ Yeast on Fermentation and Methane Production by Rumen Microorganisms

Saccharomyces cerevisiae is a key component of beer brewing and a major by-product. The leftover, spent brewers’ yeast from large breweries has been used as a protein supplement in cattle; however the possible advantages of spent yeast from smaller craft breweries, containing much higher levels of bioactive hop acids, have not been evaluated. Hops secondary metabolites from the hops (Humulus lupulus L.) used to make beer are concentrated in the yeast during brewing, and have antimicrobial activity against Gram-positive bacteria. Uncultivated suspensions of bovine rumen microorganisms produced less methane during fructose fermentation when exposed to inactivated, and freeze-dried spent craft brewers’ yeast than a bakers’ yeast control. The experiment was repeated with caprine rumen microorganisms and ground grass hay as the substrate. Likewise, in the presence of craft brewers’ yeast less methane was produced (2.7% vs. 6.9% CH4). Both experiments also revealed a decrease in acetic acid production, but not propionic acid production, when craft brewers’ yeast was included. These results indicated that spent yeast could represent a co-product for craft breweries, and a feed supplement for ruminants that has a favorable impact on methane production.

Tetrahydro-iso-alpha Acids Antagonize Estrogen Receptor Alpha Activity in MCF-7 Breast Cancer Cells.

Tetrahydro-iso-alpha acids commonly called THIAA or Tetra are modified hop acids extracted from hop (Humulus lupulus L.) which are frequently used in brewing industry mainly in order to provide beer bitterness and foam stability. Interestingly, molecular structure of tetrahydro-iso-alpha acids is close to a new type of estrogen receptor alpha (ERα) antagonists aimed at disrupting the binding of coactivators containing an LxxLL motif (NR-box). In this work we show that THIAA decreases estradiol-stimulated proliferation of MCF-7 (ERα-positive breast cancer cells). Besides, we show that it inhibits ERα transcriptional activity. Interestingly, this extract fails to compete with estradiol for ERα binding and does not significantly impact the receptor turnover rate in MCF-7 cells, suggesting that it does not act like classical antiestrogens. Hence, we demonstrate that THIAA is able to antagonize ERα estradiol-induced recruitment of the LxxLL binding motif.

Development of preparative and analytical methods of the hop bitter acid oxide fraction and chemical properties of its components.

The bitter acids in hops (Humulus lupulus L.) and beer, such as α-, β-, and iso-α-acids, are known to affect beer quality and display various physiological effects. However, these compounds readily oxidize, and the effect of the oxides on the properties of beer or their potential health benefits are not well understood. In this study, we developed a simple preparative method for the bitter acid oxide fraction derived from hops and designated the constituents as matured hop bitter acids (MHBA). HPLC-PDA-ESI/HRMS and MS(2) revealed that MHBA are primarily composed of α-acid-derived oxides, which possess a common β-tricarbonyl moiety in their structures similar to α-, β-, and iso-α-acids. We also developed a quantitative analytical method of whole MHBA by HPLC, which showed high precision and reproducibility. Using our newly developed method, the concentration of whole MHBA in several commercial beers was evaluated. Our results will promote the study of bitter acid oxides.

Antilisterial effects of different hop acids in combination with potassium acetate and potassium diacetate at 7 and 37 °C

This study was conducted to assess the effect of eight hop acid extracts (α-acid, β-acid, acid-iso, acid-tetra, potassium-iso, potassium-hexa, potassium-tetra, and potassium-rho) with/without potassium acetate/potassium diacetate salts (PAPD) on inhibition of Listeria monocytogenes in trypticase soy broth with yeast extract (TSBYE). Following overnight incubation at 37 °C, 50 ppm α-acid decreased Listeria from 6.0 to <1.0 log CFU/mL, with significant reduction to 3.6–3.8 log CFU/mL using β acid, acid-tetra, K-tetra, or K-hexa.No inhibition was seen for acid-iso, K-iso, K-rho and the control. After incubating Listeria with the five hop acids (α-acid, β-acid, acid-tetra, K-tetra, and K-hexa) at 25 ppm with/without 0.5% PAPD, Listeria population decreased from 5.7 log CFU/mL) to < 1.0 log CFU/mL in PAPD/α-acid, PAPD/β-acid, or PAPD/acid-tetra, with reduction to 1.8–4.2 log CFU/mL for the remaining except the control. When the hop acids at 25 ppm were exposed to 85 °C up to 30 min, α-acid maintained the most effective antilisteiral activity by reducing Listeria from 5.0 to <2.9 CFU/mL, regardless of heat exposure time. The minimal inhibitory concentration was 6.3 ppm for α-acid, β-acid and acid-tetra, with 12.5 ppm seen for K-tetra and K-hexa. During the incubation of hop extracts at 5 ppm/7 °C for 6 days, listeristatic effect was observed in the hop/PAPD mixtures and PAPD at 0.5 and 1%, with no listeristatic effect seen for a single addition of hop acids except β-acid. These findings indicated that the combination of hop/PAPD provided both stable and synergistic effects against Listeria growth compared to any single addition, regardless of incubation temperature and addition amount.

The development of HopGuard® as a winter treatment against Varroa destructor in colonies of Apis mellifera

The development of new veterinary medicinal products to control Varroa destructor is very important for the successful management of honey bee colonies. We tested natural plant compounds (hop acids) as active ingredients for a new veterinary drug for Varroa control. Tolerability of bees and efficacy against mites was tested in the laboratory and in the field. In laboratory tests, hop beta acids were proven to be safe for bees (5 % mortality) and effective against mites (93.3 % mortality); there is a six-fold safety margin for bees after individual dermal application compared to the lowest dosage highly toxic to mites. In a clinical study, these results were confirmed: bee colonies tolerated the treatment (<10 % mortality), while most of the mites were killed (efficacy up to 88 %). The treatment did not affect the adult bee population and winter survival of colonies.

Characterization of Hop Acids in Spent Brewer's Yeast from Craft and Multinational Sources

Spent brewer's yeast, a significant byproduct of brewing, presents a disposal issue for craft breweries. To understand value-added opportunities for its use, we measured hop acid profiles of spent yeast from three beer styles produced by a craft brewery and spent yeast from three multinational breweries using high-performance liquid chromatography with photodiode array detection/mass spectrometry. The average α-acid content of craft yeast (2,074 ± 531 μg/g) was 12-fold higher than multinational brewery samples (167 ± 89 μg/g), and the average total hop acid content of craft yeast samples (2,557 ± 622 μg/g) was fivefold higher than the multinational brewery yeast samples (487 ± 136 μg/g). Centrifuged craft yeast slurry from both light and dark ales had 7-to 10-fold, 150- to 158-fold, and >200-fold higher content of iso-α-, α-, and ß-acids, respectively, than that in the corresponding beer, demonstrating a high affinity of brewer's yeast for hop acids. Ongoing research has shown that hop acids have antioxidant, anti-inflammatory, anticancer, and antibacterial activities. The elevated content found in spent craft brewer's yeast suggests consideration for use as a high-value functional food ingredient.

Hops Βeta-Acid Extract Yields Feedlot Performance Similar to Rumensin

Hops (Humulus lupulus) have played an integral role in beer manufacturing and are widely known for their antimicrobial and preservative properties. Alpha-acids of hops are extracted and utilized to enhance beer flavor, leaving residues largely composed of β-acids. Beta-acid extracts of hops are structurally similar to ionophores and may express ionophore-like traits. Ionophores, including Rumensin (Elanco Animal Health, Greenfield, IN), are used to improve feed efficiency and to decrease the incidence of digestive disturbances in feedlot cattle. A large portion of in vitro studies show benefits from feeding hops or hop acids with results similar to ionophores; however, live animal experiments are needed to confirm these observations. The objectives of this study were to assess the effects of β-acid extracts of hops on feedlot performance in cattle fed high-concentrate diets and determine a response to varied doses of β-acid extracts of hops.

Effect of Harvest Maturity on the Chemical Composition of Cascade and Willamette Hops

Considerable expertise is required to grow high-quality hops, and brewers and hop growers alike have a common goal of obtaining the highest quality hops possible. Changes in the chemical composition of hops during plant maturation is a dynamic process requiring a comprehensive chemical and sensory analysis in order to maximize the characteristics of interest to brewers. The effect of harvest date, location, and cultivar on key chemical components of Willamette and Cascade hops was investigated for the 2010 and 2011 growing seasons. Hops were harvested at three time points (early, typical, and late) within a 3 week interval from two different farms in the Willamette Valley, Oregon. A split-plot experimental design for each cultivar was used; each farm represented a main plot and harvest years were designated as subplots. American Society of Brewing Chemists standard methods of analysis were used to measure moisture content, hop acids and their homologs, hop storage index, total essential oil content, and volatile profile by GC-FID. Additionally, difference testing, descriptive analysis, and consumer acceptance testing was conducted using beers brewed with either typical or late harvested Cascade hops from the 2010 harvest year. The response of analytes was dependent on the cultivar being examined, its location within the Willamette Valley, as well as timing of harvest. Hop acids did not change appreciably during plant maturation for the period examined, while hop oil content increased. Increases in oil quantity were strongly correlated (r > 0.90) with increases in α-pinene, β-pinene, myrcene, limonene, methyl heptanoate, and linalool concentrations. Clear sensory differences were found between beers brewed with typical and late harvested Cascade hops using triangle testing, consumer preference testing, and descriptive analysis.

Preparation of Dicyclohexylamine Alpha Acids and Dicyclohexylamine Beta Acids

The current HPLC calibration standard for measuring alpha acids and beta acids in hops and hop products is International Calibration Extract Three (ICE-3), a deoiled CO2 hop extract. Laboratories that use ICE-3 must warm and mix the calibration extract to insure that it is homogenous prior to use. Alternatively, one can prepare crystalline dicyclohexylamine salts of alpha acids (DCHA-alpha acids) and beta acids (DCHA-beta acids), which are easier to use and more stable than ICE-3.

Humulus lupulus- a story that begs to be told. A review

The hop cones of the female plant of the common hop species Humulus lupulus L. are grown almost exclusively for the brewing industry. Only the cones of the female plants are able to secrete the fine yellow resinous powder (i.e. lupulin glands). It is in these lupulin glands that the main brewing principles of hops, the resins and essential oils, are synthesized and accumulated. Hops are of interest to the brewer since they impart the typical bitter taste and aroma to beer and are responsible for the perceived hop character. In addition to the comfortable bitterness and the refreshing hoppy aroma delivered by hops, the hop acids also contribute to the overall microbial stability of beer. Another benefit of the hop resins is that they help enhance and stabilize beer foam and promote foam lacing. In an attempt to understand these contributions, the very complex nature of the chemical composition of hops is reviewed. First, a general overview of the hop chemistry and nomenclature is presented. Then, the different hop resins found in the lupulin glands of the hop cones are discussed in detail. The major hop bitter acids (α- and β-acids) and the latest findings on the absolute configuration of the cis and trans iso-α-acids are discussed. Special attention is given to the hard resins; the known δ-resin is reviewed and the e-resin is introduced. Recent data on the bittering potential and the antimicrobial properties of both hard resin fractions are disclosed. Attention is also given to the numerous essential oil constituents as well as their contributions to beer aroma. In addition to the aroma contribution of the well-known essential oil compounds, a number of recently identified sulfur compounds and their impact on beer aroma are reviewed. The hop polyphenols and their potential health benefits are also addressed. Subsequently, the importance of hops in brewing is examined and the contributions of hops to beer quality are explained. Finally, the beer and hop market of the last century, as well as the new trends in brewing, are discussed in detail. Hop research is an ever growing field of central importance to the brewing industry, even in areas that are not traditionally associated with hops and brewing. This article attempts to give a general overview of the different areas of hop research while assessing the latest advances in hop science and their impact on brewing. Copyright © 2014 The Institute of Brewing & Distilling

Mass spectrometry-based metabolomics approach to determine differential metabolites between regular and non-alcohol beers

Non-alcohol beers show taste deficiencies in relation to regular (alcohol) beers as shown by consumer evaluation. In this study, multivariate statistical analysis of data obtained by ultra-performance liquid chromatography–mass spectrometry (UPLC–MS) measurements was applied to determining differential metabolites between two regular (R1 and R2) and their related low- and non-alcohol beers (F1 and F2, respectively) from a Spanish manufacturer, as well as between F1 and F2 and two non-alcohol beers (F3 and F4) from a non-Spanish producer. Principal component analysis (PCA) of data from UPLC–MS measurements with electrospray ionization in negative mode was able to separate the six beers. Sugar content was 6-fold and 2-fold higher in F2 and F1 than in R2 and R1, respectively. Isoxanthohumol and hop acid contents decreased in F2 as compared with R2 but kept in F1 similar to R1. Results are discussed in relation to valued taste characteristics of each beer type.

Characteristics of compounds in hops using cyclic voltammetry, UV–VIS, FTIR and GC–MS analysis

The article presents the antioxidant properties of the extracts of hop EI and EII, by the electrochemical methods on a platinum electrode and comparative analysis of the composition of the extracts of hops using UV–VIS, FTIR and GC–MS methods. The hops extract EI, was obtained from the waste of the hops cone. The hops extract EII, was obtained from the hops cone itself. Hops contain a wide range of polyphenolic compounds with antioxidant properties divided in various chemical classes. Flavonoids and other polyphenolic compounds contained in hops show antioxidant capacity because of the presence of hydroxyl groups in various configurations and numbers within their molecules. The electrochemical properties and antioxidant capacity of hop samples were determined to select the most effective antioxidant. Based on the cyclic and pulse voltammograms, it was observed that hop extract EI contains polyphenols that are oxidised at a less positive potential than extract EII, i.e., it shows better antioxidant capacity. From the analysis of the UV–VIS and FTIR spectra and the GC–MS analysis, it was observed that extract EI contains less phenyl compounds than EII. In addition to flavonoids, EII contains hop acids and chlorophyll. The solutions of hop extracts show very good antioxidant capacities; therefore, they can effectively inhibit or slow negative oxidation reactions and scavenge free radicals and reactive oxygen species (ROS).

Characterization of the Formation of Branched Short-Chain Fatty Acid:CoAs for Bitter Acid Biosynthesis in Hop Glandular Trichomes

Bitter acids, known for their use as beer flavoring and for their diverse biological activities, are predominantly formed in hop (Humulus lupulus) glandular trichomes. Branched short-chain acyl-CoAs (e.g. isobutyryl-CoA, isovaleryl-CoA and 2-methylbutyryl-CoA), derived from the degradation of branched-chain amino acids (BCAAs), are essential building blocks for the biosynthesis of bitter acids in hops. However, little is known regarding what components are needed to produce and maintain the pool of branched short-chain acyl-CoAs in hop trichomes. Here, we present several lines of evidence that both CoA ligases and thioesterases are likely involved in bitter acid biosynthesis. Recombinant HlCCL2 (carboxyl CoA ligase) protein had high specific activity for isovaleric acid as a substrate (K cat /K m = 4100 s(-1) M(-1)), whereas recombinant HlCCL4 specifically utilized isobutyric acid (Kcat/K m = 1800 s(-1) M(-1)) and 2-methylbutyric acid (Kcat/K m = 6900 s(-1) M(-1)) as substrates. Both HlCCLs, like hop valerophenone synthase (HlVPS), were expressed strongly in glandular trichomes and localized to the cytoplasm. Co-expression of HlCCL2 and HlCCL4 with HlVPS in yeast led to significant production of acylphloroglucinols (the direct precursors for bitter acid biosynthesis), which further confirmed the biochemical function of these two HlCCLs in vivo. Functional identification of a thioesterase that catalyzed the reverse reaction of CCLs in mitochondria, together with the comprehensive analysis of genes involved BCAA catabolism, supported the idea that cytosolic CoA ligases are required for linking BCAA degradation and bitter acid biosynthesis in glandular trichomes. The evolution and other possible physiological roles of branched short-chain fatty acid:CoA ligases in planta are also discussed.

Kinetic Modeling of Hop Acids during Wort Boiling

Kinetic Modeling of Hop Acids during Wort Boiling

HlPT-1, a membrane-bound prenyltransferase responsible for the biosynthesis of bitter acids in hops

Female flowers of hop (Humulus lupulus L.) develop a large number of glandular trichomes called lupulin glands that contain a variety of prenylated compounds such as α- and β-acid (humulone and lupulone, respectively), as well as xanthohumol, a chalcone derivative. These prenylated compounds are biosynthesized by prenyltransferases catalyzing the transfer of dimethylallyl moiety to aromatic substances. In our previous work, we found HlPT-1 a candidate gene for such a prenyltransferase in a cDNA library constructed from lupulin-enriched flower tissues. In this study, we have characterized the enzymatic properties of HlPT-1 using a recombinant protein expressed in baculovirus-infected insect cells. HlPT-1 catalyzed the first transfer of dimethylallyl moiety to phloroglucinol derivatives, phlorisovalerophenone, phlorisobutyrophenone and phlormethylbutanophenone, leading to the formation of humulone and lupulone derivatives. HlPT-1 also recognized naringenin chalcone as a flavonoid substrate to yield xanthohumol, and this broad substrate specificity is a unique character of HlPT-1 that is not seen in other reported flavonoid prenyltransferases, all of which show strict specificity for their aromatic substrates. Moreover, unlike other aromatic substrate prenyltransferases, HlPT-1 revealed an exclusive requirement for Mg2+ as a divalent cation for its enzymatic activity and also showed exceptionally narrow optimum pH at around pH 7.0.

Chelating Properties and Hydroxyl-Scavenging Activities of Hop α- and Iso-α-Acids

Metal chelation and scavenging of highly reactive hydroxyl radicals are commonly proposed to explain the antioxidant behavior of hop acids. In the current study, an assay measuring the oxidative degradation of 2-deoxyribose (2-DR) by hydroxyl radicals was used to elucidate these mechanisms and examine the origin of the ability of hop acids to prevent oxidative damage. Hop α- and iso-α-acids were able to prevent 2-DR oxidative degradation; however, α-acids appeared to be 60% more effective than iso-α-acids. The ability of hop acids to inhibit 2-DR oxidative degradation could be inversely correlated with Fe2+ concentration, which suggests that they prevent 2-DR oxidation by chelating iron. Moreover, it was demonstrated that these hop acids were not capable of scavenging hydroxyl radicals. Further trials showed that hop α- and iso-α-acids enhanced Fe2+ autoxidation to Fe3+ when complexing it and, thus, lowered the catalytic function of the iron in the Fenton reaction system.

The Interactions of CO2, Ethanol, Hop Acids and Sweetener on Flavour Perception in a Model Beer

Beer is a complex beverage. Beer flavour is a multisensory experience in which, in addition to aroma volatiles, CO2, ethanol, bitterness (hop acids) and sweetness all contribute. To investigate the interactions between these fundamental components, a model beer system was developed using representative ingredients. Samples, selected according to a D-optimal design, were assessed by sensory profiling techniques by a trained panel. Predictive polynomial models generated from mean panel data described variations in the attributes as a function of design factors. Results show that CO2 significantly impacted on all discriminating attributes, including suppression of sweetness and modification of bitterness. A number of complex interactions with design factors showed the effects of CO2 to be dependent upon component concentration and level of carbonation. CO2 interacted with hop acids to increase carbonation and tingly perception, which increased linearly with hop acid addition but only at low levels of CO2. Ethanol was the main driver of warming perception and complexity. In agreement with other studies, ethanol enhanced sweet perception and also formed some complex interactions with hop acids and CO2 to modify various attributes, illustrating its ability to interact with both gustatory and trigeminal stimuli. Whether the mechanisms behind these interactions originate at the gustatory periphery or at higher centres in the brain is an area for further investigation. This study provides an in-depth assessment of important flavour components in beer and advances the limited data available on the effects of CO2 on sensory perception using a commonly carbonated beverage.

Effects of Ethanol, Carbonation and Hop Acids on Volatile Delivery in a Model Beer System

J. Inst. Brew. 117(1), 74–81, 2011 A model beer was created to investigate the effects of ethanol, carbonation and hop acids on volatile release (ethyl acetate, isoamyl alcohol and phenethyl alcohol) using both headspace analysis and in-nose measurement during consumption. None of the factors were found to impact on equilibrium headspace partitioning, however headspace sampling after short term decanting revealed minor and compound specific effects of each of the components. When measured in-vivo, hop acids had no significant effect on volatile delivery; however ethanol significantly increased the delivery of volatiles during consumption. This increase was sustained throughout the release profile. Carbonation was found to increase the release of ethyl acetate and isoamyl alcohol during the first release peak after swallowing, but had no significant effect on phenethyl alcohol. Furthermore, ethyl acetate was increased by carbonation in the second peak after swallowing, but this effect was not found to be persistent in subsequent peaks. These results indicate a trend between the compound’s air-water partition coefficient and the effects of carbonation in-vivo. The effects of ethanol and carbonation seemed to be independent and therefore an additive effect is possible. This study highlights the difference between data collected by headspace and in-vivo means.

What Makes Beers Different From Each Other?

W hat makes beers different from each other ? B S J Jared Rosen One might imagine that since beer has been around for thousands of years, all of its secrets and intricacies would have been unlocked by now and that there is little research left to be done. In fact, very much the opposite is true. One major overarching theme in much of this article is what causes us to perceive different varieties of beer as distinct and why we might enjoy some but not others. Before diving into the cutting edge research on one of man’s oldest beverages, let us take a moment to review how beer is actually made. Beer is more or less made from a solution of water, hops, and grain extract that has been boiled and allowed to ferment with yeast that converts the sugars from grain extract into alcohol and carbon dioxide. The hop plant is a distant relative of modern conifers and has been used in the brewing process since the middle ages. The oils (also known as hop acids) present in its cones are extracted in the boiling process and lend flavoring and a desirable level of bitterness. The grains favored by brewers are malted barley or wheat for their high content of maltose (a type of sugar). Maltose is broken down to glucose by the enzyme amylase during the boiling process. Brewer’s yeast is added in after the boiled solution of sugar and hops, known as wort, is cooled, and is then allowed to work its magic, turning glucose and other sugars into alcohol and carbon dioxide, which carbonates the beer. Over the course of thousands of years, a countless number of varieties based on this simple recipe have been created by making incremental tweaks. Different beers vary because their recipes are different, but from the consumer end all that matters is the perception of one beer versus another; thus, much research has been devoted to this end. Everything from fermentation temperature, byproducts of fermentation, and addition “About 8,000 years, probably. I like to say it’s the basis of civilization, and when people first realized they could make beer, they stopped wandering” of hops, to container type and conditions of storage, temperature of tasting, and interactions of chemical compounds that modulate taste, are parameters that brewers and researchers alike consider when trying to understand and improve this already amazing beverage. Beer has a fairly high caloric content (mainly due to alcohol and carbohydrates) so one target researchers Figure 1. Beer is one of the oldest beverages created by man. have is to and reduce the number of calories (Wang, et al., 2010). When carbohydrates are converted into alcohol, some of the complex sugars cannot be easily broken down by yeast (notably maltotriose) and remain in the beer, adding to the caloric content and leading to potentially unwanted flavors (Wang, et al., 2010). Other compounds such as acetaldehydes, diacetyls, and pentanedione can contribute undesirable flavors at high enough concentrations (Wang, et al., 2010). As a result, researchers in China have worked to genetically engineer a strain of yeast to help lower both the caloric content and some of these undesirable compounds. The yeast strain used in this study was endowed with the gene for the enzyme amylase, which helps break down some of these more complex sugars, thereby lowering the sugar content (Wang, et al., 2010). The production of acetaldehyde, which lends an unpleasant flavor, was decreased by about half by disabling one copy of the gene that codes for alcohol dehyrogenase (Wang, et al., 2010). This disrupts the metabolic pathway that results in acetaldehyde. Similar results were seen when other 1 • B erkeley S cientific J ournal • S cience of F ood • V olume 16 • I ssue 1

Bioavailability of hop-derived iso-α-acids and reduced derivatives

Iso-α-acids (IAA) and their reduced derivatives (dihydro-iso-α-acids (DHIAA) and tetrahydro-iso-α-acids (THIAA)) have been administered to Caco-2 cell monolayers (30, 60, and 120 μM) to investigate epithelial transport, in both absorptive and secretive directions. In addition, 25 mg kg(-1) IAA, DHIAA, and THIAA were applied to New Zealand white rabbits (±3-3.5 kg) in a single intravenous and oral dose. The most important pharmacokinetic parameters (C(max), t(max), half life, clearance, and AUC(0-∞)) and the absolute bioavailability were determined for each class of hop acid. The results from the in vitro Caco-2 study of IAA, DHIAA, and THIAA, showed a higher membrane permeability for IAA and THIAA, both in absorptive (P(appAB) range 1.6-5.6 × 10(-6) cm s(-1)) and secretive directions (P(appBA) range 5.7-16.3 × 10(-6) cm s(-1)), when compared to DHIAA. Factors limiting transport of DHIAA could include phase II metabolism. After oral and i.v. dosing to New Zealand white rabbits, the absolute bioavailability for IAA was determined to be 13.0%. The reduced derivatives reached higher bioavailabilities with 28.0% for DHIAA and 23.0% for THIAA. The area under curve AUC(0-∞) upon oral gavage for DHIAA and THIAA was 70.7 ± 48.4 μg h ml(-1) and 57.4 ± 9.0 μg h ml(-1), respectively, while that for IAA was 10.6 ± 5.3 μg h ml(-1). Phase I metabolism was indicated as the main factor limiting the bioavailability of IAA. Bioavailability of DHIAA is mostly influenced by phase-II metabolism as shown by enzymatic hydrolysis of plasma samples upon administration of DHIAA.