A new unified theory: C-C-2W is the chemical cause of all so-called cholesterol and cholestanol lipidoses

The water-insoluble cholesterol-cholestanol-water adduct C-C-2W, chemical and physical cause of atherosclerosis and gallstones, has now been found in tendinous xanthoma as well; C-C-2W, and not cholestanol, is the initial compound deposited in hereditary CTX (cerebrotendinous xanthomatosis). From these and other findings it is theorized that what have been termed cholesterol or cholestanol lipidoses should instead be characterized as C-C-2W lipidoses. More than 1 mg of cholestanol . H2O present in the body causes crystallizaion of C-C-2W . This happens when the steroid meets cholesterol . H2O in sufficient concentration to reach a solubility product of 10(-7) mg/ml. In this light the literature can be interpreted to indicate that C-C-2W exerts negative effects on liver, intima tissue, eyes, lungs, and other parts of the body. Those effects include inflammation, cell necrosis, destruction of cell membranes, abnormal growth and, perhaps, neoplastic activity. Up to 200 g of C-C-2W in the body may be tolerated if evenly spread but not if localized in one or two areas only; e.g., the brain or the cardiovascular system. It is estimated that about 1000 g of C-C-2W, even if spread, are beyond the limit of human tolerance.     

Serum plant sterols and biliary cholesterol secretion in humans: studies with ursodeoxycholic acid

Ratios of cholestanol, campesterol, and sitosterol to cholesterol in serum are known to reflect cholesterol absorption efficiency. Here, a possible link between these ratios and biliary secretion rates of cholesterol was investigated. Biliary lipid secretion rates and serum sterols were determined in 13 patients with gallstones. Seven were treated with ursodeoxycholic acid (UDCA) (1,000 mg/d). Serum cholesterol and non-cholesterol sterols were also measured in a cross over study in 20 healthy volunteers, who received either placebo or UDCA (750 mg/d). Biliary cholesterol secretion was significantly lower, whereas the non-cholesterol sterols and their ratio to cholesterol were higher in patients with gallstones treated with UDCA. A highly significant negative linear correlation between the ratios of non-cholesterol sterols to cholesterol and biliary cholesterol secretion was observed. In volunteers, administration of UDCA for 4 weeks was followed by a significant increase in non-cholesterol sterols and their ratios. Even 4 weeks after discontinuing UDCA administration, campesterol and sitosterol were still significantly higher than pretreatment levels, which was also true for the campesterol-cholesterol ratio after 8 weeks. The results suggest that the ratios of cholestanol, campesterol, and sitosterol to cholesterol can be used as indicators of changes in biliary cholesterol secretion rates.     

Hepatic cholesterol metabolism in cholesterol gallstone disease

Hepatic cholesterol metabolism was examined in 27 Swedish patients with cholesterol gallstone disease and in 13 patients free of gallstones operated for roentgenographically suspect polyps in the gallbladder. All 40 patients underwent cholecystectomy, and a liver biopsy and gallbladder bile were obtained at surgery. The cholesterol saturation of gallbladder bile was significantly higher in patients with gallstones compared to the gallstone-free controls (131 +/- 13 vs. 75 +/- 5%, P less than 0.001). Microsomal 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity, governing cholesterol synthesis, did not differ between gallstone and gallstone-free patients (104 +/- 11 vs. and 109 +/- 22 pmol/min per mg protein, respectively). The activity of cholesterol 7 alpha-hydroxylase, catalyzing the catabolism of cholesterol to bile acids, was not significantly decreased in gallstone patients (6.2 +/- 1.1 vs. 8.0 +/- 2.0 pmol/min per mg protein). The capacity to esterify cholesterol, judged by the activity of acyl coenzyme A:cholesterol acyltransferase (ACAT), was similar in gallstone and gallstone-free patients (5.4 +/- 0.4 vs. 6.7 +/- 1.1 pmol/min per mg protein). In the presence of exogenous cholesterol, ACAT activity increased by more than fourfold in both groups. No correlation was found between the saturation of gallbladder bile and any of the mentioned enzyme activities in gallstone patients. It is concluded that distinct abnormalities in cholesterol metabolizing enzymes are not of major importance for development of gallstones in Swedish patients with cholesterol gallstone disease. The results support the contention that the etiology of cholesterol gallstones is multifactorial.     

Phospholipid peroxidation as a factor in gallstone pathogenesis

Phospholipid peroxidation markedly reduces the stability of mixed micellar systems composed of cholate, phosphatidylcholine and supersaturating levels of cholesterol. This suggests that lipid peroxidation is likely to play a significant role in the precipitation of cholesterol from gallbladder bile, thus in the pathogenesis of cholesterol gallstones. This conclusion is supported by studies of the nucleation time of cholesterol in gallbladder biles, which was significantly reduced by exposure to a stream of oxygen. This effect of phospholipid peroxidation on cholesterol solubility may occur in other biological fluids as well. In view of the increased lipid peroxidation in the elderly, it may explain the effect of age on the frequency of various diseases related to cholesterol precipitation.     

Biliary proteins: assessment of quantitative techniques and comparison in gallstone and nongallstone subjects

Although protein is the third most abundant solid in bile and is important in cholesterol crystal formation, methods for quantitating the concentration of total protein in bile have not been systematically evaluated. To establish a reliable protein assay for bile, we evaluated three protein assays (Lowry's method and the fluorescamine and Coomassie blue methods), and employed amino acid analysis as a reference technique. Large protein-to-protein variations were observed with the fluorescamine and Coomassie blue methods. Although all assays were affected by interfering substances, Lowry's method and the fluorescamine technique (after trichloroacetic acid precipitation and delipidation of bile) and the Coomassie blue method with native bile showed excellent correlations (P less than 0.0001) with those obtained by amino acid analysis. Using these reliable protein assays, we examined gallbladder bile obtained at surgery from subjects with and without gallstones. No differences in the concentrations of total biliary proteins were observed among patients with cholesterol (n = 23) or pigment (n = 7) gallstones and subjects without gallstones (n = 10). Protein values obtained by amino acid analysis also did not differ among groups. As expected, bile from patients with cholesterol gallstones was supersaturated with cholesterol while bile from nongallstone subjects and those with pigment stones was unsaturated. These results indicate that it is not possible to separate patients with and without gallstones on the basis of the total protein concentration of gallbladder bile.     

Biosynthesis of cholestanol in higher plants

To understand the early steps of C(27) brassinosteroid biosynthesis, metabolic experiments were performed with Arabidopsis thaliana and Nicotiana tabacum seedlings, and with cultured Catharanthus roseus cells. [26, 28-2H(6)]Campestanol, [26-2H(3)]cholesterol, and [26-2H(3)]cholestanol were administered to each plant, and the resulting metabolites were analyzed by gas chromatography-mass spectrometry. In all the species examined, [2H(3)]cholestanol was identified as a metabolite of [2H(6)]campestanol, and [2H(3)]cholest-4-en-3-one and [2H(3)]cholestanol were identified as metabolites of [2H(3)]cholesterol. This study revealed that cholestanol (C(27) sterol) was biosynthesized from both cholesterol (C(27) sterol) and campestanol (C(28) sterol). It was also demonstrated that cholestanol was converted to 6-oxocholestanol, and campestanol was converted to 6-oxocampestanol.     

Biosynthesis of cholestanol from bile acid intermediates in the rabbit and the rat

Biliary 7 alpha-hydroxy-4-cholesten-3-one (an intermediate in bile acid biosynthesis) may be 7 alpha-dehydroxylated in the gut and further metabolized to cholestanol (Skrede, S., and Bj?rkhem, I. (1982) J. Biol. Chem. 257, 8363-8367). We have now evaluated the quantitative importance of pathway(s) to cholestanol with 7 alpha-hydroxylated C27 steroids as intermediates. After feeding conventionally fed rabbits or rats or germ-free rats with [7 alpha-3H]cholesterol and [4-14C]cholesterol, tissue cholestanol could be isolated with about a 20% lower 3H/14C ratio than present in cholesterol. We conclude that there is a pathway to cholestanol involving 7 alpha-hydroxylated intermediates. Intestinal microorganisms are not essential for this pathway, which accounts for at most 20% of the cholestanol formed in these species. In bile fistula rats, there was also a significant conversion of intraperitoneally injected [7 beta-3H]7 alpha-hydroxycholesterol and [4-14C]7 alpha-hydroxy-4-cholesten-3-one into cholestanol. The enzymes involved in the 7 alpha-hydroxylation/dehydroxylation pathway for the biosynthesis of cholestanol are probably located in the liver. Both 7 alpha-hydroxycholesterol and 7 alpha-hydroxy-4-cholesten-3-one may be intermediates.     

Simultaneous determination of cholesterol and cholestanol in human serum by high-performance liquid chromatography using 3-(5,6-methylenedioxy-2-phthalimidyl)benzoyl azide as precolumn fluorescent labelling reagent

A fluorescent labelling reagent, 3-(5,6-methylenedioxy-2-phthalimidyl)benzoyl azide, designed for the determination of alcohols by precolumn high-performance liquid chromatography, has been applied to the simultaneous determination of cholesterol and cholestanol in human serum. The reagent reacts with cholesterol and cholestanol at 140°C for 10 min to produce the fluorescent derivatives, which can be separated on a reversed-phase column with acetonitrile—ethanol—water (60:35:7.5, v/v) as eluent. The detection limits for cholesterol and cholestanol were 45 and 50 fmol per injection (20 μl), respectively. The values of cholesterol and cholestanol in normal human sera were 135–212 mg/dl and 137–928 μg/dl, respectively.     

Effect of gallbladder hypomotility on cholesterol crystallization and growth in CCK-deficient mice

We investigated the effect of gallbladder hypomotility on cholesterol crystallization and growth during the early stage of gallstone formation in CCK knockout mice. Contrary to wild-type mice, fasting gallbladder volumes were enlarged and the response of gallbladder emptying to a high-fat meal was impaired in knockout mice on chow or the lithogenic diet. In the lithogenic state, large amounts of mucin gel and liquid crystals as well as arc-like and tubular crystals formed first, followed by rapid formation of classic parallelogram-shaped cholesterol monohydrate crystals in knockout mice. Furthermore, three patterns of crystal growth habits were observed: proportional enlargement, spiral dislocation growth, and twin crystal growth, all enlarging solid cholesterol crystals. At day 15 on the lithogenic diet, 75% of knockout mice formed gallstones. However, wild-type mice formed very little mucin gel, liquid, and solid crystals, and gallstones were not observed. We conclude that lack of CCK induces gallbladder hypomotility that prolongs the residence time of excess cholesterol in the gallbladder, leading to rapid crystallization and precipitation of solid cholesterol crystals. Moreover, during the early stage of gallstone formation, there are two pathways of liquid and polymorph anhydrous crystals evolving to monohydrate crystals and three modes for cholesterol crystal growth.     

Cholesterol gallstones in alloxan-diabetic mice

Normal and alloxan-diabetic male mice (Crj-ICR) were fed a diet containing 0.5% cholesterol for 5 and 10 weeks, and gallbladder bile was analyzed for cholesterol, phospholipids and bile acids, feces for sterols and bile acids, and plasma and liver for cholesterol, phospholipids, and triglycerides. Normal mice developed no gallstones but the diabetic mice developed cholesterol gallstones with an incidence of 70% by 5 weeks and 80% by 10 weeks after feeding of the cholesterol diet. Diabetic mice fed the ordinary diet also developed stones (23%) by 10 weeks. In the diabetic mice, the gallbladder was enlarged about threefold, and biliary lipid concentration, diet intake, and fecal excretion of sterols and bile acids increased but body weight decreased. Cholic acid and beta-muricholic acid comprised over 40% each of the total biliary bile acids in normal mice, but cholic acid increased to about 80% and beta-muricholic acid decreased to a few percent in the diabetic mice. Fecal excretion of bile acids increased after cholesterol feeding in both normal and diabetic mice, but the increased bile acid in the normal animals was beta-muricholic acid and that in the diabetic mice was deoxycholic acid. The mice that developed gallstones showed a marked increase in biliary cholesterol value and decreases in gallbladder bile and bile acid concentration, but no difference in biliary and fecal bile acid composition, bile acid synthesis, fecal sterols, or plasma and liver lipid levels. Cholesterol absorption was increased in the diabetic mice when examined by plasma 14C/3H ratio and fecal 14C-labeled sterol excretion after a single oral administration of [14C]cholesterol and a simultaneous intravenous injection of [3H]cholesterol. These data led to the conclusion that cholesterol gallstones developed in alloxan-diabetic mice fed excess cholesterol, due to the hyperphagia and the enhancement of cholesterol absorption caused by increases in the synthesis and secretion of cholic acid.     

Esterification of cholesterol and cholestanol in the whole body,tissues, and frass of Heliothis zea

The quantity of free and esterified sterols in the whole body, intestine, hemolymph, fat body, and frass of 6th-instar larvae of H. zea, fed cholesterol or cholestanol, was measured in order to determine if there was a difference in the utilization of these two molecules. The principal sterol in the tissues of the larvae was cholestanol or cholesterol, when they were fed diet containing these two molecules, respectively; there was little, if any, metabolism of dietary cholestanol to cholesterol. There was little or no difference in the amount of total sterol in the whole body, tissues, or frass of larvae fed the two different diets, indicating that the absence of a Δ⁵-bond in cholestanol does not prevent the uptake or distribution of this sterol to various tissues. However, the relative percentage of steryl ester was significantly higher in prepupae reared on a diet containing cholestanol instead of cholesterol (6–7-, 4-, 13-, 4-, and 2-fold increase, for the whole body, intestine, hemolymph, fat body, and frass, respectively). The average percentage of total sterol that was esterified in the tissues was greater in the fat body (10.8 ± 15.4 and 44.2 ± 12.3%, respectively, for larvae fed cholesterol and cholestanol) than in the hemolymph (0.5 ± 0.1 and 6.3 ± 0.8%) and intestine (1.2 ± 0.1 and 4.7 ± 1.1%). The percentage of sterol that was esterified in the frass of larvae was large (26.9 ± 3.7 and 48.2 ± 0.5%, respectively, for larvae fed cholesterol and cholestanol). Therefore, the fact that larvae of H. zea fed cholestanol, instead of cholesterol, contain this saturated molecule as their principal tissue sterol and preferentially esterify it may explain, at least in part, why their rate of growth on cholestanol is slower than on cholesterol.     

Helicobacter pylori and cholesterol gallstone formation in C57L/J mice: a prospective study

Recently, we demonstrated that cholesterol gallstone-prone C57L/J mice rarely develop gallstones unless they are infected with certain cholelithogenic enterohepatic Helicobacter species. Because the common gastric pathogen H. pylori has been identified in the hepatobiliary tree of cholesterol gallstone patients, we wanted to ascertain if H. pylori is cholelithogenic, by prospectively studying C57L infected mice fed a lithogenic diet. Weanling, Helicobacter spp.-free male C57L mice were either infected with H. pylori SS1 or sham dosed. Mice were then fed a lithogenic diet (1.0% cholesterol, 0.5% cholic acid, and 15% dairy triglycerides) for 8 wk. At 16 wk of age, mice were euthanatized, the biliary phenotype was analyzed microscopically, and tissues were analyzed histopathologically. H. pylori infection did not promote cholesterol monohydrate crystal formation (20% vs. 10%), sandy stone formation (0% for both), or true gallstone formation (20%) compared with uninfected mice fed the lithogenic diet (10%). Additionally, H. pylori failed to stimulate mucin gel accumulation in the gallbladder or alter gallbladder size compared with uninfected animals. H. pylori-infected C57L mice developed moderate to severe gastritis by 12 wk, and the lithogenic diet itself produced lesions in the forestomach, which were exacerbated by the infection. We conclude that H. pylori infection does not play any role in murine cholesterol gallstone formation. Nonetheless, the C57L mouse develops severe lesions of both the glandular and nonglandular stomach in response to H. pylori infection and the lithogenic diet, respectively.     

成石前后胆囊粘膜上皮多聚核蛋白体RNA含量变化—胆液糖蛋白和蛋白质增加的机制探讨

胆囊淤泥是胆囊结石的前身。测定胆囊粘膜上皮游离(FPR)和结合多聚核蛋白体(MBPR)的RNA含量及胆液粘液糖蛋白(MGP)的结果表明:胆囊淤泥患者胆囊粘膜合成和分泌MGP明显亢进,MBPR与相应GB中MGP的相关性比较提示此时尚有胆液淤积存在,且FPR的RNA含量也显著增高,提示细胞增殖加快。荧光胺法测定胆液蛋白质的含量发现,胆囊淤泥和胆固醇性结石患者胆囊液GB蛋白质含量显著高于色素性结石患者及“正常”对照,但蛋白质的GB浓度与HB浓度的比值显著低于胆固醇GB/HB比值,且GB中MGP与蛋白质含量呈显著正相关。说明蛋白质含量的增加与GB中过高的MGP阻止胆囊中蛋白质的清除过程有关。成石前GB中MGP和蛋白质含量的同时增高及其机制的确立,对认识及预防体内结石均有意义。     

Cholestanol metabolism in patients with cerebrotendinous xanthomatosis: absorption, turnover, and tissue deposition

To study the metabolism of cholestanol in patients with cerebrotendinous xanthomatosis (CTX), we measured the cholestanol absorption, the cholesterol and cholestanol turnover, and the tissue content of sterols in two patients. Cholestanol absorption was approximately 5.0%. The rapid exchangeable pool of cholestanol was 233 mg, and the total exchangeable pool was 752 mg. The production rate of cholestanol in pool A was 39 mg/day. [4-14C]cholestanol was detected in the xanthomas, but neither [4-14C]cholestanol nor [4-14C]cholesterol was detected in peripheral nerves biopsied at 49 and 97 days after [4-14C]cholesterol given intravenously. Of the 18 tissues analyzed at biopsy and autopsy, the cholestanol content varied from 0.09 mg/g in psoas muscle to 76 mg/g in a cerebellar xanthoma. With the assumption that the cholestanol-to-cholesterol ratio is 1.0, the relative cholestanol-to-cholesterol ratio varied from 1.0 in plasma and liver to 30.0 in the cerebellar xanthoma; cholestanol was especially high in nerve tissue. Our data indicate that CTX patients absorb cholestanol from the diet. They have a higher than normal cholestanol production rate. Cholestanol was derived from cholesterol. In CTX patients, the blood-brain barrier was intact to the passage of [4-14C]cholesterol and [4-14C]cholestanol. The deposition of large amounts of cholestanol (up to 30% of total sterols in cerebellum) in nerve tissues must have an important role in the neurological symptoms in CTX patients. In view of the intact blood-brain barrier, several other explanations for the large amounts of cholestanol in the brain were postulated.     

Increased plasma cholestanol and 5 alpha-saturated plant sterol derivatives in subjects with sitosterolemia and xanthomatosis

We have measured plasma sterol composition in 14 subjects with sitosterolemia and xanthomatosis. In addition to elevated plasma phytosterol (campesterol 16 +/- 7 mg/dl and sitosterol 35 +/- 16 mg/dl) and normal to moderately high cholesterol levels (258 +/- 96 mg/dl), concentrations of 5 alpha-saturated stanols, cholestanol, 5 alpha-campestanol, and 5 alpha-sitostanol were at least 10 times greater than controls. Diets contained plentiful quantities of cholesterol and plant sterols, but only trace amounts of cholestanol (less than 2 mg/day) and no detectable 5 alpha-campestanol and 5 alpha-sitostanol, which indicated that the 5 alpha-saturated stanols were formed endogenously. Treatment with cholestyramine reduced plasma cholesterol and phytosterol levels by 45% and 5 alpha-saturated stanols by 55%. These results indicate that abnormally high plasma concentrations of cholestanol, 5 alpha-campestanol, and 5 alpha-sitostanol are found in subjects with sitosterolemia and xanthomatosis, and that treatment with cholestyramine effectively reduced elevated plasma sterol levels.     

Identification of a phosphatidylcholine active phospholipase C in human gallbladder bile

This study describes the identification of a phospholipase C activity against phosphatidylcholine in delipidated human gallbladder bile. All biles were obtained from cholesterol gallstone patients and were negative on bacterial culture. The biliary enzyme was inhibited by EDTA and had a pH optimum of between 7-8. All of the 15 gallbladders examined contained significant phospholipase C activity (32.85 +/- 8.37 nmol/h/mg delipidated protein). The finding of a phospholipase C in gallbladder bile of patients with cholesterol gallstones may be one of the factors responsible for or related to the rapid in vitro nucleation seen in these biles.     

Comparison of the metabolism of cholesterol, cholestanol, and -sitosterol in L-cell mouse fibroblasts

The following data have been obtained from comparative studies on the metabolism of cholesterol, cholestanol, and beta-sitosterol by L-cell mouse fibroblasts. (1) When the sterols are added to the growth medium under similar conditions, cellular incorporation of cholesterol > cholestanol > beta-sitosterol; (2) only limited cellular esterification of these compounds occurs; (3) no metabolic products arising from the sterols could be detected; (4) influx of all sterols is dependent upon the concentration; and (5) exogenous cholesterol reduces mevalonate incorporation into cellular sterol to a lesser extent than acetate or glucose. The metabolism of these sterols is discussed in relation to their ability to influence de novo sterol biosynthesis.     

On the solubility of calcium deoxycholate: kinetics of precipitation and the effect of conjugated bile salts and lecithin

In view of the low solubility of calcium deoxycholate and the possible induction of cholesterol precipitation in the gallbladder by calcium insoluble salts, we find it of interest to study the precipitation of calcium deoxycholate and its dependence on other bile components. The findings of these studies were as follows: (i) Precipitation of calcium deoxycholate from mixtures of calcium chloride and monomeric deoxycholate (at concentrations below the critical micelle concentration (CMC] is very slow even at relatively high CaCl2 concentrations (more than 20 days at 50 mM CaCl2). (ii) At higher deoxycholic acid (DOC) concentrations, precipitation of micellar DOC is faster and requires much lower calcium chloride concentrations. For any given calcium concentration, the rate of precipitation is maximal at an optimal DOC concentration. In solutions containing 150 mM NaCl, the maximal rate of precipitation occurs at about 10 mM DOC, almost independent of Ca2+ concentration. At lower ionic strength (10 mM NaCl), the optimal DOC concentration is 30 mM. These observations suggest that the most important factors in determining the rate of Ca(DOC)2 precipitation are (a) the ratio between calcium ions bound to the surface of a DOC micelle, and the [DOC] (the Ca2+/DOC binding ratio) and (b) the concentration of DOC micelles. (iii) In the presence of conjugated deoxycholates, the crystallization of calcium deoxycholate is inhibited. Phosphatidylcholine has a similar, although smaller, inhibitory effect. Upon precipitation of calcium deoxycholate from a mixed micellar system containing sodium deoxycholate, phosphatidylcholine and cholesterol, the latter two components spontaneously form vesicles. The anti-nucleating effect of PC and conjugated bile salts is explained in terms of "poisoning" of the crystallization process. In view of the latter results we conclude that under normal conditions calcium deoxycholate is not likely to precipitate in the gallbladder.     

Ferroelectricity in the arterial wall: A new physical component of atherosclerosis

The appearance of two electric potentials in the aorta is explained by the blood pulse and the remote steps of atherosclerosis are elucidated from the physical point of view. When the first C-C-2W is deposited in the intimamedia, the primary cause of their retention is the stress-induced polarization of the membrane. C-C-2W possesses a permanent dipole moment which may be reversed by the field produced by the radial expansion/contraction of the arteries.The initial C-C-2W increases the polarization of the aorta walls, favoring accumulation of more oriented material on top of the first. C-C-2W is also ferroelectric and constitutes a polar continuum with the membranes. By the time fatty streaks appear, the entire aorta wall is ferroelectric and energy has been stored in the walls under the form of residual polarization. This storage gradually destroys the wall, leading to aneurism. The biological significance of the new theory is summarized in Table 2.     

Do “hot moments” become hotter under climate change? Soil nitrogen dynamics from a climate manipulation experiment in a post-harvest forest

Whole-tree forest harvest can increase soil nitrous oxide (N₂O) effluxes and leaching of nitrogen (N) from soils. These altered N dynamics are often linked to harvesting effects on microclimate, suggesting that this “hot moment” for N cycling may become hotter with climate change. We hypothesized that increases in temperature and precipitation during this post-harvest period would increase availability of soil mineral N and soil-atmosphere N₂O efflux. To test this hypothesis we implemented a climate manipulation experiment after a forest harvest, and measured soil N₂O fluxes and inorganic N accumulating on ion exchange resins. Climate treatments were: control (A, ambient), heated (H, +2.5 °C), wetted (W, +23 % precipitation), and a two-factor treatment (H+W). For all treatments, the first year after harvest had highest N₂O efflux and resin N. Wetting significantly increased cumulative soil N₂O fluxes, but only when soils were not heated too. The cumulative soil-to-atmosphere N₂O efflux from W (5.8 mg N₂O–N m^?2) was significantly higher than A (?1.9 mg N₂O–N m^?2), but H+W (~0 mg N₂O–N m^?2) was similar to A. Regardless of wetting, heating increased resin N, but only on certain dates. Cumulative resin N was on average 125 % greater in the H plots than non-heated plots. Thus, changes in temperature and precipitation each impart distinct changes to the soil N cycle. Heating increased resin N regardless of water inputs, while wetting increasing N₂O but not when combined with heating. Our results suggest that climate change may exacerbate soil N losses from whole-tree harvest in the future, but the form and quantity of N loss will depend on how the future climate changes.