Nuclear magnetic resonance spectroscopy of 3 beta,7 beta-dihydroxy-5-cholen-24-oic acid multi-conjugates: unusual bile acid metabolites in human urine

Complete ¹H and ¹³C resonance assignments were made for a new type of 3β,7β-dihydroxy-5-cholen-24-oic acid doubly conjugated with sulfuric acid at C-3 and N-acetylglucosamine at C-7 and its glycine- and taurine-amidated triple-conjugates by the combined use of several homonuclear and heteronuclear shift-correlated 2D NMR techniques. The effects of sulfation at C-3, N-acetylglucosaminidation at C-7, and aminoacyl amidation at C-24 on the ¹H and ¹³C chemical shifts and signal multiplicity were clarified. The shielding data serving to characterize each of the bile acid multi-conjugates are also discussed.     

Nitro as a novel zinc-binding group in the inhibition of carboxypeptidase A

2-Substituted 3-nitropropanoic acids were designed and synthesized as inhibitors against carboxypeptidase A (CPA). (R)-2-Benzyl- 3-nitropropanoic acid showed a potent inhibition against CPA (K(i)=0.15 microM). X-ray crystallography discloses that the nitro group well mimics the transition state occurred in the hydrolysis catalyzed by CPA, that is, an O,O'-bidentate coordination to the zinc ion and the two respective hydrogen bonds with Glu-270 and Arg-127. Because the nitro group is a planar species, we proposed (R)-2-benzyl-3-nitropropanoic acid as a pseudo-transition-state analog inhibitor against CPA.     

Bradykinin effects on phospholipid metabolism and its relation to arachidonic acid turnovers in neuroblastoma × glioma hybrid cells (NG 108-15)

In neuroblastoma × glioma hybrid cells (NG 108-15) labelled with [³²P]-trisodium phosphate, [³H]-inositol and [¹⁴C]-arachidonic acid, bradykinin stimulated the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP₂) while it had no effect on the release of [¹⁴C]-arachidonic acid (AA). The effect on PIP, was time- and dose-dependent with a maximal effect on [³H]-inositol- and [³²P]-labelled cells after 10–30 s of stimulation with 10⁻⁶ M bradykinin. However, the hydrolysis of [¹⁴C]-AA labelled PIP₂ was delayed compared to the effect on [³H]- and [¹⁴C]-PIP₂ and was not detectable until after 60 s of stimulation. Bradykinin stimulation resulted in an increased formation of [³H]-inositol phosphates (IP) and [³²P]- and [¹⁴P]- and [¹⁴C]-phosphatidic acid (PA) but the time course for PA formation did not allow the time-course for PIP₂ hydrolysis. A reduced labelling of [²³P]- and [¹⁴C]-phosphatidylcholine was also found in stimulated cells suggesting that PA may derive from other sources than PIP₂. In conclusion, our results indicate that bradykinin activates phospholipase C, but not phospholipase A₂, in NG 108-15 cells.     

Identification of a cDNA encoding a novel C18-Δ polyunsaturated fatty acid-specific elongating activity from the docosahexaenoic acid (DHA)-producing microalga, Isochrysis galbana

Isochrysis galbana, a marine prymnesiophyte microalga, is rich in long chain polyunsaturated fatty acids such as docosahexaenoic acid (C22:6n-3, Δ^{4,7,10,13,16,19}). We used a polymerase chain reaction-based strategy to isolate a cDNA, designated IgASE1, encoding a polyunsaturated fatty acid-elongating activity from I. galbana. The coding region of 263 amino acids predicts a protein of 30 kDa that shares only limited homology to animal and fungal proteins with elongating activity. Functional analysis of IgASE1, by expression in Saccharomyces cerevisiae, was used to determine its activity and substrate specificity. Transformed yeast cells specifically elongated the C18-Δ⁹ polyunsaturated fatty acids, linoleic acid (C18:2n-6, Δ^9,12) and -linolenic acid (C18:3n-3, Δ^9,12,15), to eicosadienoic acid (C20:2n-6, Δ^11,14) and eicosatrienoic acid (C20:3n-3, Δ^11,14,17), respectively. To our knowledge this is the first time such an elongating activity has been functionally characterised. The results also suggest that a major route for eicosapentaenoic acid (C20:5n-3, Δ^5,8,11,14,17) and docosahexaenoic acid syntheses in I. galbana may involve a Δ⁸ desaturation pathway.     

Binding of radioactively labeled carboxyatractyloside, atractyloside and bongkrekic acid to the ADP translocator of potato mitochondria

1. 1. The inhibition of the ADP-stimulated respiration of potato mitochondria by carboxyatractyloside is relieved by high concentration of ADP or by the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). Atractyloside is a much less potent inhibitor than carboxyatractyloside. The inhibition of the ADP-stimulated respiration required about 60-times more atractyloside than carboxyatractyloside.

2. 2. [³⁵S]carboxyatractyloside and [³H]bongkrekic acid bind to potato mitochondria with high affinity (K_d = 10 to 20 nM, n = 0.6–0.7 nmol per mg protein). Added ADP competes with carboxyatractyloside for binding; on the contrary ADP increases the amount of bound bongkrekic acid. [³H]atractyloside binds to potato mitochondria with a much lower affinity (K_d = 0.45 μM) than carboxyatractyloside or bongkrekic acid.

3. 3. Bound [³H]atractyloside is displaced by ADP, carboxyatractyloside and bongkrekic acid. The displacement of bound [³⁵S]carboxyatractyloside by bongkrekic acid and of bound [³H]bongkrekic acid by carboxyatractyloside is markedly increased by ADP.

4. 4. Bongkrekic acid competes with [³⁵S]carboxyatractyloside for binding. Addition of a small concentration of ADP considerably enhances the inhibitory effect of bongkrekic acid on [³⁵S]carboxyactratyloside binding.

5. 5. The adenine nucleotide content of potato mitochondria is of the order of 1 nmol per mg protein. ADP transport in potato mitochondria is inhibited by atractyloside 30- to 40-times less efficiently than by carboxyatractyloside.

不同黄腐酸用量对‘红将军’苹果产量、品质和15N-尿素去向的影响

以15年生红将军/八棱海棠为试材,运用¹⁵N同位素示踪技术,设置单施尿素(CK)及尿素配施不同用量黄腐酸处理(黄腐酸用量分别为75、150、300和450 kg·hm^-2,分别以NF₁、NF₂、NF₃和NF₄表示),研究不同黄腐酸用量对苹果植株¹⁵N-尿素吸收、利用、残留、损失及果实产量和品质的影响.结果表明: 至果实成熟期,苹果根系、一年生枝和叶片的Ndff值(植株器官从肥料中吸收分配到的¹⁵N量对该器官全氮量的贡献率)均为NF₃>NF₄>NF₂>NF₁>CK,且各处理间差异显著.植株全氮量和¹⁵N吸收量均以NF₃处理最大,其次为NF₄处理,CK处理最低.与CK处理相比,NF₁、NF₂、NF₃和NF₄处理¹⁵N利用率分别提高了14.2%、33.5%、64.2%和50.0%,而¹⁵N损失率分别降低了9.1%、18.5%、37.1%和28.7%.不同处理土壤¹⁵N残留量不同.配施黄腐酸处理0～60 cm土层¹⁵N残留量显著高于CK处理,其中以NF₃处理最多,而在60～100 cm土层显著低于CK处理.NF₃处理单株产量和纯收益较CK处理增幅最大,分别为15.8%和20.2%,其次为NF₄处理,同时,NF₃处理果实硬度、可溶性固形物含量和糖酸比均达到最高水平.通过对果实产量和氮素利用率与黄腐酸施用量进行拟合分析,得出本试验条件下适宜的黄腐酸用量为326.41～350.61 kg·hm^-2.     

不同黄腐酸用量对‘红将军’苹果产量、品质和15N-尿素去向的影响

以15年生红将军/八棱海棠为试材,运用¹⁵N同位素示踪技术,设置单施尿素(CK)及尿素配施不同用量黄腐酸处理(黄腐酸用量分别为75、150、300和450 kg·hm^-2,分别以NF₁、NF₂、NF₃和NF₄表示),研究不同黄腐酸用量对苹果植株¹⁵N-尿素吸收、利用、残留、损失及果实产量和品质的影响.结果表明: 至果实成熟期,苹果根系、一年生枝和叶片的Ndff值(植株器官从肥料中吸收分配到的¹⁵N量对该器官全氮量的贡献率)均为NF₃>NF₄>NF₂>NF₁>CK,且各处理间差异显著.植株全氮量和¹⁵N吸收量均以NF₃处理最大,其次为NF₄处理,CK处理最低.与CK处理相比,NF₁、NF₂、NF₃和NF₄处理¹⁵N利用率分别提高了14.2%、33.5%、64.2%和50.0%,而¹⁵N损失率分别降低了9.1%、18.5%、37.1%和28.7%.不同处理土壤¹⁵N残留量不同.配施黄腐酸处理0～60 cm土层¹⁵N残留量显著高于CK处理,其中以NF₃处理最多,而在60～100 cm土层显著低于CK处理.NF₃处理单株产量和纯收益较CK处理增幅最大,分别为15.8%和20.2%,其次为NF₄处理,同时,NF₃处理果实硬度、可溶性固形物含量和糖酸比均达到最高水平.通过对果实产量和氮素利用率与黄腐酸施用量进行拟合分析,得出本试验条件下适宜的黄腐酸用量为326.41～350.61 kg·hm^-2.     

Synthesis of 2,3-O-(propane-1,2-diyl)- and 2,3-O-(3-hydroxypropane-1,2-diyl)-cyclomaltoheptaose

2¹,3¹-O-(Propane-1,2-diyl)cyclomaltoheptaose has been prepared from 2-O-allylcyclomaltoheptaose by mercuration in trifluoroacetic acid, followed by reduction with sodium borohydride. 2-O-(2,3-Epoxypropyl)cyclomaltoheptaose, prepared from 2-O-allylcyclomaltoheptaose by oxidation with dimethyldioxirane, was converted into 2¹,3¹-O-(3-hydroxypropane-1,2-diyl)cyclomaltoheptaose by treatment with trifluoroacetic acid. Both derivatives containing fused 1,4-dioxane rings are mixtures of stereoisomers, in which the methyl and hydroxymethyl group, respectively, that is linked to this ring, occupies an axial or an equatorial position.     

Metabolic activation of unsaturated derivatives of valproic acid. Identification of novel glutathione adducts formed through coenzyme A-dependent and -independent processes

The ability of 2-n-propyl-4-pentenoic acid (Δ⁴-VPA) and 2-n-propyl-2(E)-pentenoic acid ([E]-Δ²-VPA), two unsaturated metabolites of valproic acid (VPA), to form reactive intermediates, deplete hepatic glutathione (GSH) and cause accumulation of liver triglycerides was investigated in the rat. With the aid of ionspray liquid chromatography-tandem mass spectrometry (LC-MS/MS), three GSH adducts were detected in the bile of Δ⁴-VPA-treated animals and were identified as 4-hydroxy-5-glutathion-S-yl-VPA-γ-lactone, 5-glutathion-S-yl-(E)-Δ³-VPA and 3-oxo-5-glutathion-S-yl-VPA. A fourth conjugate was identified tentatively as 4-glutathion-S-yl-5-hydroxy-VPA. Quantitative analysis of the corresponding N-acetylcysteine (NAC) conjugates in urine indicated that metabolism of Δ⁴-VPA via the GSH-dependent pathways accounted for approximately 20% of an acute dose (100 mg kg⁻¹ i.p.). In contrast, when rats were given an equivalent dose of (E)-Δ²-VPA, only one GSH adduct (5-glutathion-S-yl-(E)-Δ³-VPA) was detected at low concentrations in bile. In vitro experiments with rat liver mitochondria demonstrated that Δ⁴-VPA undergoes coenzyme A- and ATP-dependent metabolic activation in this organelle via the β-oxidation pathway to intermediates which bind covalently to proteins. When liver homogenates and hepatic mitochondria from rats injected with Δ⁴-VPA, (E)-Δ²-VPA or VPA were analyzed for GSH content, it was found that only Δ⁴-VPA depleted GSH pools significantly. Treatment of rats with Δ⁴-VPA and (to a lesser extent) VPA led to an accumulation of liver triglycerides, whereas (E)-Δ²-VPA had no measurable effect. It is concluded that Δ⁴-VPA undergoes metabolic activation by both microsomal cytochrome P-450-dependent and mitochondrial coenzyme A-dependent processes, and that the resulting electrophilic intermediates, which are trapped in part by GSH, may mediate the hepatotoxic effects of this compound. In contrast, (E)-Δ²-VPA is not transformed to any appreciable extent to reactive metabolites, which thus accounts for the apparent lack of hepatotoxicity of this positional isomer in the rat.     

Mechanism of reaction of nitrogen dioxide radical with hydroxycinnamic acid derivatives: A pulse radiolysis study

Nitrogen dioxide radical (NO^·₂) is known as a toxic agent produced in the metabolism of nitrates and nitrites. By the use of the pulse radiolysis technique, the mechanism of the reaction of NO^·₂ radical with hydroxycinnamic acid derivatives (HCA) was studied and the rate constants have been measured. The rate constants were found to be 7.4 × 10⁸, 7.2 × 10⁸, 8.6 × 10⁸ dm³ mol^-1s^-1 for ferulic acid, sinapic acid and caffeic acid, respectively. The reactions produce the corresponding phenoxyl radical.     

The preparation, characterisation and low temperature X-ray structure of Ru6(η-μ4-CO)2(CO)13(η6-C6Me6)

The new organometallic cluster (η²-μ₄-CO)₂(CO)₁₃(η⁶-C₆Me₆) has been prepared by the thermolysis of Ru₃(CO)₁₂ with hexamethylbenzene in octane and characterised by a single crystal X-ray diffraction study. It is isostructural with the known cluster Ru₆(η²-μ₄-CO)₂(CO)₁₃(η⁶-C₆H₃Me₃) and the metal core constitutnts the same tetrahedral Ru₄ unit with two edge-bridging Ru atoms. The mesitylene derivative has been shown to undergo rearrangement to afford the octahedral carbido cluster Ru₆C(CO)₁₄(η⁶-C₆H₃Me₃), but this conversion is not observed for the new hexamethylbenzene derivative.     

Interaction of hydroxycinnamic acid derivatives with the Cl3COO radical: a pulse radiolysis study

The electron transfer reactions between the trichloromethylperoxyl radical (Cl₃COO^·) and hydroxycinnamic acid derivatives, including chlorogenic acid, sinapic acid, caffeic acid, ferulic acid and 3,4-(methylenedioxy)cinnamic acid, have been studied by pulse radiolysis. The hydroxycinnamic acid derivatives, especially sinapic acid, are identified as good antioxidants for reduction of Cl₃COO^· via electron transfer reactions. From buildup kinetic analysis of phenoxyl radical, the rate constant for reaction of Cl₃COO^· with sinapic acid has been determined to be 8.2 × 10⁷ dm³ mol^-1 s^-1, while the rate constants of electron transfer from other hydroxycinnamic acid derivatives to Cl₃COO^· were obtained to be about 2 × 10⁷dm³ mol^-1 s^-1. The reaction of 3,4-(methylenedioxy)cinnamic acid with Cl₃COO^· was investigated as an evidence for the electron transfer mechanism.     

PATHWAYS OF INCORPORATION OF FATTY ACID INTO GLYCEROLIPIDS OF THE MURINE PERIPHERAL NERVOUS SYSTEM IN VIVO: ALTERATIONS IN THE DYSMYELINATING MUTANT TREMBLER MOUSE

In vivo glycerolipid metabolism was studied in sciatic nerves of normal and Trembler mice. The results showed that two kinetically independent pathways were implicated in the labeling of diacylglycerophospholipids from [³H]palmitate: the Kennedy pathway and a ‘direct acylation’ pathway. In normal nerves, 45% of the glycerophospholipids were labeled, with a rate constant k₃ = 3.9 × 10⁻³ min⁻¹, from phosphatidic acid and diacylglycerol intermediates, themselves formed with a rate constant of k₁ = 0.24 min⁻¹ from a free ³H-fatty acid pool, FFA₁, that represents 45% of the total injected label. The remaining 55% of the glycerophospholipids were labeled from a kinetically distinct free ³H-fatty acid pool, FFA₂, with a rate constant of k₄ = 9.8 × 10⁻², via a process that does not implicate a detectably labeled metabolic intermediate (‘direct acylation’). Glycerophospholipid labeling via the Kennedy pathway in the Trembler mouse sciatic nerves was reduced to 75% of the normal level, while labeling via the ‘direct acylation’ pathway was increased 1.4-fold. The values of the rate constants for free ³H-fatty acid utilisation (k₁ and k₄) were both increased about 2.5-fold, while that of glycerophospholipid formation from diacylglycerol (k₃) was close to normal. Copyright © 1996 Elsevier Science Ltd     

Slippage of η-allenyl/propargyl to an η structure and tautomerization of η-propargyl to η-allenyl in ligand addition and substitution reactions of platinum(II) complexes

Reactions of [(PPh₃)₂Pt(η³-CH₂CCPh)]OTf with each of PMe₃, CO and Br⁻ result in the addition of these species to the metal and a change in hapticity of the η³-CH₂CCPh to η¹-CH₂CCPh or η¹-C(Ph)=C=CH₂. Thus, PMe₃ affords [(PMe₃)₃Pt(η¹-C(Ph)=C=CH₂)]⁺, CO gives both [trans-(PPh₃)₂Pt(CO)(η¹-CH₂CCPh)]⁺ and [trans-(PPh₃)₂Pt(CO)(η¹-C(Ph)=C=CH₂)]⁺, and LiBr yields cis-(PPh₃)₂PtBr(η¹-CH₂CCPh), which undergoes isomerization to trans-(PPh₃)₂PtBr(η¹-CH₂CCPh). Substitution reactions of cis- and trans-(PPh₃)₂PtBr(η¹-CH₂CCPh) each lead to tautomerization of η¹-CH₂CCPh to η¹-C(Ph)=C=CH₂, with trans-(PPh₃)₂PtBr(η¹-CH₂CCPh) affording [(PMe₃)₃Pt(η¹-C(Ph)=C=CH₂)]⁺ at ambient temperature and the slower reacting cis isomer giving [trans-(PPh₃)(PMe₃)₂Pt(η¹-C(Ph)=C=CH₂)]⁺ at 54 °C . All new complexes were characterized by a combination of elemental analysis, FAB mas spectrometry and IR and NMR (¹H, ¹³C{¹H} and ³¹P{¹H}) spectroscopy. The structure of [(PMe₃)₃Pt(η¹-C(Ph)=C=CH₂)]BPh₄·0.5MeOH was determined by single-crystal X-ray diffraction analysis.     

[H]lysergic acid diethylamide (LSD): differential agonist and antagonist binding properties at 5-HT receptor subtypes in rat brain

[³H]Lysergic acid diethylamide (LSD) in the presence of 40 nM ketanserin labeled the 5-HT_1A receptor subtype in rat hippocampal membranes. In the presence of guanosine triphosphate (GTP), the B_max and affinity of [³H]LSD binding to the 5-HT_1A binding site were significantly decreased. [³H]LSD in the presence of 40 nM WB4101 labeled the 5-HT₂ receptor subtype in homogenates of rat frontal cortex. In contrast to the effect on [³H]LSD binding to the 5-HT_1A binding site, GTP produced no significant effect on either the B_max or the K_D of [³H]LSD binding to the 5-HT₂ binding site. Competition of 5-HT for [³H]LSD binding to the 5-HT₂ binding site was best described by a computer-derived model assuming two binding sites. In the presence of GTP, the 5-HT competition curve was shifted significantly to the right with an approx. 3-fold increase in the IC₅₀. These binding characteristics are consistent with [³H]LSD acting as an antagonist at the 5-HT₂ receptor which has multiple affinity states for agonists and is coupled to a guanine nucleotide regulatory subunit. Thus, [³H]LSD has binding characteristics consistent with it acting as an agonist at the 5-HT_1A receptor subtype but as an antagonist at the 5-HT₂ receptor subtype in rat brain.     

Multiple neurotransmitter systems influence the release of adenosine derivatives from the rabbit retina

Rabbit retinac preloaded with [³H]adenosine were superfused in vitro and the effect of neurotransmitter agonists and antagonists on the release of [³H]purines was studied. Glutamic acid, aspartic acid, kainic acid (KA), quisqualic acid (QUIS) and acid (NMDA) all stimulated the efflux of [³H] labelled and endogenous purines. Their effect was reduced in a Ca²⁺-free medium except when using a high concentration (100 μM) of KA, QUIS and NMDA. The effect of aspartic acid and of NMDA were blocked by 2-amino-7-phosphono-heptanoic acid (APH) and 2-amino-5-phosphono-valeric acid (APV). Carbachol also increased the release of adenosine-derived radioactivity and this effect was reduced by the removal of Ca²⁺ and by pretreatment with atropine. τ-Aminobutyric acid (GABA) and muscimol, induced a small increase in the release which was Ca²⁺-dependent and was blocked by bicuculline and picrotoxin. Dopamine elicited an increase in the release which was partially reduced in a Ca²⁺-free medium and was blocked by haloperidol. Glycine and 5-hydroxytryptamine (5-HT) also induced small but significant increases. The neurotransmitter antagonists had an effect of their own. Superfusion with APH and APV depressed the outflow of radioactivity whereas bicuculline, picrotoxin, strychnine and haloperidol enhanced it. The K⁺-evoked release of [³H]purines was reduced by haloperidol and by 5-HT. The observations indicate that stimulation of several important neurotransmitter receptors in the retina elicits the release of adenosine derivatives. The results with the antagonists also suggest that purines are continuously released as a result of a tonic activation of the respective membrane receptors.     

Characterization of [H]glutamate binding sites on frozen sections from rat adrenal

Some biochemical characteristics of [³H]glutamate (Glu) binding sites on frozen sections from the rat adrenal glands were studied. Adrenal frozen sections exhibited stereo-selective, saturable and temperature-dependent binding of [³H]Glu. An agonist for one of the subclasses of central Glu receptors, quisqualic acid (QA), elicited a significant inhibition of the binding, whereas neither N-methyl- -aspartic acid nor kainic acid, agonists for other subclasses of the receptors, had such a significant effect on the binding at the concentration range similar to QA. In vitro addition of sodium acetate (100 mM) resulted in a significant inhibition of [³H]Glu binding to frozen sections of the rat adrenal glands. It thus appears that there exist QA-sensitive binding sites of [³H]Glu in the rat adrenal glands which exhibit pharmacological characteristics distinctly different from those in the brain.     

Some evidence of the enzymatic conversion of bovine suppressor phosphoseryl-tRNA to selenocysteyl-tRNA

In order to clarify the mechanisms of selenocysteine incorporation into glutathione peroxidase, some evidence to show the in vitro conversion of phosphoseryl-tRNA to selenocysteyl-tRNA is reported. [³H]Phosphoseryl-tRNA was incubated in a reaction mixture composed of SeO₂, glutathione and NADPH in the presence of selenium-transferase partially purified. Analyses of amino acids on the product tRNA showed that a part (4%) of [³H]phosphoseryl-tRNA was changed to [³H]selenocysteyl-tRNA. The conversion from seryl-tRNA^su or major seryl-tRNA_IGA was not found. Selenium-transferase was essential for the conversion. [³H]Selenocysteine, liberated from the tRNA, was modified with iodoacetic acid. The product was confirmed to be carboxymethyl-selenocysteine by two-dimensional TLC. Selenocysteyl-tRNA^su should be used to synthesize glutathione peroxidase by co-translational mechanisms.     

Fractionation of gamma-emitting fission products absorbed by red kidney beans (Phaseolus vulgaris L.)

The gamma-emitting fission product nuclides ¹⁰⁶Ru, ¹²⁵Sb, ¹³⁷Cs and ¹⁴⁴Ce that accumulated in the edible pods of bean (Phaseolus vulgaris L.) plants grown in nutrient culture were subjected to chemical fractionation. The results indicated that the largest fraction of ¹⁰⁶Ru, ¹²⁵Sb and ¹⁴⁴Ce was associated with ionic forms including salts of organic acids, phosphates, carbonates and some protein-bound forms extracted with dilute mineral acids (acid fraction). The association of these radionuclides with lipids including lipophyllic pigments, free amino acids and amino sugars (ethanol fraction) was next in significance. The association of ¹³⁷Cs was, however, greater with the ethanol fraction than with the acid fraction. Considerably reduced amounts of the fission products were present in the pectates, proteins, polysaccharides and nucleic acids.     

A review of the use of isotopic and nuclear techniques in animal production

Isotopic and nuclear techniques play an important role in food and agriculture, health and industry. This paper discusses the use of these techniques and highlights potential for their use in the area of Animal Production. These techniques are discussed in two parts: (1) Isotopic methods and (2) non-isotopic nuclear methods. The isotopic techniques discussed are: stable- (¹⁵N) and radio-isotope (³⁵S or ³²P) incorporation methods for measuring microbial mass in vitro and in vivo; ¹²⁵I-labelled bovine serum albumin and ¹⁴C-labelled polyethylene glycol assays for measuring tannin in feeds; a method based on the feeding of isotope-labelled protein (¹⁵N or ¹²⁵I) complexed with tannin for ranking different tannins for their abilities to release protein for digestion in vivo; ¹⁴C-uric acid and ¹⁴C-allantoin infusion methods for development of models describing excretion of purine derivatives in urine and microbial protein supply to ruminants, which permit assessment of nutritional status of animals and determination of nutritional quality of feed resources; a ¹⁵N isotope dilution technique using ¹⁵N-leucine to distinguish feed and endogenous secretions at the ileum, for determination of true digestibility of protein-rich tree leaves and aquatic plants in pigs; progesterone radioimmunoassay (RIA) for enhancing reproductive efficiency of ruminants, and RIA based leptin and insulin like growth factor assays for assessing the nutritional status of animals; feeding of ¹⁵N enriched plant material to generate ¹⁵N-labelled excreta for research on the fate of excreta N in the environment; ¹⁵N, ¹³C and ³⁴S isotopic methods for nutrient budgeting and for following the nutrient pathways in the soil–plant–animal continuum; ³²P- or ³³P-labelled fertilizers for estimating the efficiency of P utilization in legume leaf production used for livestock feeding; double labelled water (¹⁸O and ²H labelled) method for estimating energy expenditures of grazing animals, body composition, basal metabolic rate, and milk output in cows with calves; NaH¹³CO₃/NaH¹⁴CO₃ infusion for estimation of the carbon dioxide production, which in turn is used to estimate energy expenditure in free-ranging animals; ³H- or ¹⁴C-labelled methane and ¹⁴C-labelled volatile fatty acids dilution technique for direct and indirect (using stoichiometry of carbohydrate fermentation) for determination of methane emission from livestock; ¹⁵N dilution technique requiring labeling the soil with ¹⁵N fertilizer (¹⁵N-ammonium sulphate or ¹⁵N-urea) for estimation of nitrogen fixation by leguminous trees and pastures.

The non-isotopic nuclear techniques that have been used or have the potential for use are: dual energy X-ray absorptionmetry and computer tomography for body composition determination; nuclear magnetic resonance techniques, fast atom bombardment mass spectroscopy, and mass ionisation spectroscopy for identification and structure determination of bioactive moieties of plant origin having potential for rumen manipulation or controlling internal parasites; gamma irradiation for inactivating antinutrients such as protease inhibitors, lectin, phytic acid, non-starch polysaccharides and oligosaccharides in feeds; induced mutations with gamma radiation, electron beam and fast neutrons for producing useful mutants of forage plants with improved yield, nutrient profiles and uptake.