A randomized, placebo-controlled trial evaluating the safety of excessive administration of kaempferol aglycone.

Kaempferol (KMP) is an important flavonoid in many fruits and vegetables. Preclinical studies on KMP have reported its pharmacological effects, including antimicrobial, antioxidant, anti-inflammatory, antitumor, antidiabetic, myocardial protective, and neuroprotective effects. Additionally, some epidemiological studies have revealed a negative association between the consumption of KMP-containing foods and the risk of developing several disorders, such as cancer and cardiovascular diseases. Thus, although a large body of literature has demonstrated the benefits of KMP supplementation, there are no reports of clinical trials evaluating the safety of KMP aglycone administration or KMP aglycone-rich food consumption. The purpose of this study was to evaluate the safety of a high dose of KMP aglycone by administrating KMP aglycone-containing supplements to healthy adults. This study had a randomized, double-blind, placebo-controlled design and a 4-week duration. Participants were randomly allocated to the KMP (n = 24) or placebo (n = 24) group. For 4 weeks, the KMP group received a capsule containing 50-mg KMP daily, a dose approximately five times higher than the estimated human dietary intake. The placebo group received a capsule containing cornstarch-based powder daily. The general toxicity parameters were evaluated by examining the characteristics of the participants, hematological and blood biochemical parameters, general urinalysis, qualitative urine tests, and adverse events. No clinical changes were observed in anthropometric and blood pressure measurements or blood and urine parameters in the KMP group compared to those in the placebo group. Furthermore, no adverse events owing to KMP aglycone administration occurred. The study results revealed that the consumption of 50-mg KMP aglycone daily for 4 weeks is safe in healthy adults.

Kaempferol increases intracellular ATP content in C2C12 myotubes under hypoxic conditions by suppressing the HIF-1α stabilization and/or by enhancing the mitochondrial complex IV activity.

Kaempferol (KMP) has numerous important biological functions, and we recently showed that it remarkably increased intracellular adenosine triphosphate (ATP) content in C2C12 myotubes under hypoxic conditions. Since intracellular ATP is generated by aerobic energy metabolism or anaerobic glycolysis, hypoxia inducible factor-1α (HIF-1α) has been shown to be associated with metabolic remodeling and causes metabolic shift from aerobic energy metabolism to anaerobic glycolysis in response to hypoxic conditions. Here, we investigate the effects of KMP under hypoxic conditions on the stabilization of HIF-1α in C2C12 myotubes and its underlying molecular mechanisms. Constitutive HIF-1α protein expression was observed in C2C12 myotubes, and its expression under hypoxic conditions was remarkably suppressed by KMP by reducing its stability; thus, resulting in an increase in ATP content. Furthermore, KMP strikingly increased the ubiquitination of HIF-1α and promoted its degradation via the ubiquitin proteasome system. Inhibition of HIF-1α by KMP resulted in the abrogation of the expression of glycolytic enzymes such as lactate dehydrogenase A and pyruvate dehydrogenase kinase isozyme 1. In addition, the metabolome profiling showed that KMP promoted oxidative energy production, while the mitochondrial complex activity assay indicated that KMP increased the activity of mitochondrial complex IV. Finally, we showed that KMP inhibited HIF-1α expression and increased intracellular ATP content in the soleus muscle of rats. Taken together, these results suggest that KMP increases intracellular ATP content under hypoxic conditions by suppressing the HIF-1α stabilization and/or by enhancing the mitochondrial complex IV activity in muscle.

Interaction of Gal-7 with HMGCS1 In Vitro May Facilitate Cholesterol Deposition in Cultured Keratinocytes.

Three-hydroxy-3-methylglutaryl coenzyme A synthase (HMGCS) 1 was identified to interact with Gal-7, a pro-apoptotic ß-galactoside‒binding protein, by yeast two-hybrid system. Their interaction was confirmed by in vitro ß-galactosidase, Biacore, and immunoprecipitation assays. A distinct interactive site of HMGCS1 was found to reside at phenylalanine 26. The expression of HMGCS1 in cultured keratinocytes was upregulated by exogenous Gal-7 and downregulated in LGALS7 small interfering RNA‒transfected cells. HMGCS1-overexpressing cells were found to induce Gal-7 expression, which suggests that Gal-7 and HMGCS1 expressions are both stimulated by positive feedback regulation. The amount of cholesterol, a final biosynthetic product of HMGCS1-involved pathway, was increased in Gal-7‒treated cells and was significantly reduced in LGALS7 small interfering RNA‒transfected cells. The increase of cholesterol level in Gal-7‒treated cells was inhibited by wild-type HMGCS1 peptide but not by phenylalanine 26‒mutated peptide, suggesting that the interaction of Gal-7/HMGCS1 is related to cellular cholesterol level. Foam cells in granulomatous tissues of the specimens from normolipidemic cutaneous xanthoma showed positive reactions with the antibodies for Gal-7 and HMGCS1 as well as for lipid markers. These results are likely to indicate that Gal-7 induction in epidermal keratinocytes causes both apoptotic cell death and HMGCS1-mediated cholesterol accumulation, which will be phagocytized by macrophages. This mechanism may explain the pathogenesis of normolipidemic cutaneous xanthoma.

Accumulation of C-reactive protein in basal keratinocytes of normal skins.

BACKGROUND: C-reactive protein (CRP) is a prototypic acute phase protein which increases dramatically in the blood during the first 48h of tissue inflammation and has been recognized as a risk factor for atherosclerosis. CRP interacts with a variety of proteins. OBJECTIVE: To know the role of accumulated CRP in the skin. METHODS: Interaction of CRP with basal keratinocytes was studied using immunohistochemical method and keratinocyte culture system. RESULTS: We found an immunohistochemical deposition of CRP on the basal keratinocyte membrane in some normal human skins (23 out of 46 skins). When added to cultured keratinocytes, heat-denatured but not native CRP was found to adhere to keratinocyte cell membrane after 1h, then internalized into cytoplasm after 24h. The heat-denatured CRP recognized at least four keratinocyte polypeptides with the molecular weights of 56, 42, 32 and 24kDa. Ligand binding assays suggested that multiple populations of receptor-ligand interactions were involved in the binding between CRP and keratinocyte. Cultured dermal microvascular endothelial cells were found to express CRP of which expression was greatly induced by interleukin-1ß (IL-1ß) treatment, suggesting that the deposited CRP in the basal keratinocytes can be derived from local dermal microvasculatures as well as from systemic circulation (serum). Treatment of cultured keratinocytes with heat-denatured CRP induced interleukin-8 (IL-8) expression, a potent leukocyte chemotactic cytokine. CRP in the medium (liquid phase) and CRP-coated dishes (solid phase) both inhibited the adhesion of keratinocytes in culture. CONCLUSION: Accumulation of CRP may regulate the skin inflammation and keratinocyte proliferation by modulating keratinocyte cytokine expression and adhesion to substrate.

Evaluation of unique elastic aggregates (elastic globes) in normal facial skin by multiphoton laser scanning tomography.

BACKGROUND: There is no reliable marker to estimate the degree of skin aging in vivo. It now has become possible to quantitatively determine the dermal characteristics of the extracellular matrix (ECM) in vivo using multiphoton laser tomography (MLT). METHODS: Fifty-seven healthy Japanese female volunteers, aged from 20 to 60 years old, were examined using multiphoton depth-resolved measurements of autofluorescence (AF) and second harmonic generation (SHG) at three sites on their right cheek. Paraffin-embedded skin specimens obtained from the faces of 12 normal individuals aged 38-68 years old were stained with Elastica van Gieson (EVG). RESULTS: We found unique elastic aggregates at a 20 µm depth from the dermo-epidermal junction (DEJ) in vivo which increased in size with aging of subjects from 20 to 60 years old. SHG fibers seemed to surround those elastic aggregates. Histological examination of specimens from normal individuals stained with EVG confirmed the occurrence of elastic aggregates with varied sizes just beneath the epidermis or hair follicles. CONCLUSIONS: The elastic aggregates are morphologically similar to previously described 'elastic globes' and can serve as a marker of the early stage of photoaging. MLT will contribute to determine age-related dermal changes using a non-invasive technique.

In vitro amyloidogenic peptides of galectin-7: possible mechanism of amyloidogenesis of primary localized cutaneous amyloidosis.

Pathogenesis of primary localized cutaneous amyloidosis (PLCA) is unclear, but pathogenic relationship to keratinocyte apoptosis has been implicated. We have previously identified galectin-7, actin, and cytokeratins as the major constituents of PLCA. Determination of the amyloidogenetic potential of these proteins by thioflavin T (ThT) method demonstrated that galectin-7 molecule incubated at pH 2.0 was capable of binding to the dye, but failed to form amyloid fibrils. When a series of galectin-7 fragments containing ß-strand peptides were prepared to compare their amyloidogenesis, Ser(31)-Gln(67) and Arg(120)-Phe(136) were aggregated to form amyloid fibrils at pH 2.0. The rates of aggregation of Ser(31)-Gln(67) and Arg(120)-Phe(136) were dose-dependent with maximal ThT levels after 3 and 48 h, respectively. Their synthetic analogs, Phe(33)-Lys(65) and Leu(121)-Arg(134), which are both putative tryptic peptides, showed comparable amyloidogenesis. The addition of sonicated fibrous form of Ser(31)-Gln(67) or Phe(33)-Lys(65) to monomeric Ser(31)-Gln(67) or Phe(33)-Lys(65) solution, respectively, resulted in an increased rate of aggregation and extension of amyloid fibrils. Amyloidogenic potentials of Ser(31)-Gln(67) and Phe(33)-Lys(65) were inhibited by actin and cytokeratin fragments, whereas those of Arg(120)-Phe(136) and Leu(121)-Arg(134) were enhanced in the presence of Gly(84)-Arg(113), a putative tryptic peptide of galectin-7. Degraded fragments of the galectin-7 molecule produced by limited trypsin digestion, formed amyloid fibrils after incubation at pH 2.0. These results suggest that the tryptic peptides of galectin-7 released at neutral pH, may lead to amyloid fibril formation of PLCA in the intracellular acidified conditions during keratinocyte apoptosis via regulation by the galectin-7 peptide as well as actin and cytokeratins.

Increase of elastic fibers in lichen sclerosus et atrophicus.

BACKGROUND: Lichen sclerosus et atrophicus (LSA) is histopathologically characterized by upper dermal hyalinization with vacuolar alteration, whereas no particular microscopic change in the mid to lower dermis has been described. The purpose of this study was to investigate any histopathologic changes involving elastic fibers in the mid to lower dermis in patients with LSA. METHODS: We surveyed 22 paraffin-embedded specimens of vulval (18 cases) and extragenital (4 cases) LSA. The sliced skin sections were stained with elastic van Gieson (EVG), and then the degree of elastic fiber change was determined. RESULTS: We found an increase in elastic fibers in the mid to lower dermis in contrast to a decrease or disappearance of elastic fibers in the superficial hyalinized dermis. The level of increase of elastic fibers varies from normal (8/22 cases) to moderate (7/22 cases) to advanced (7/22 cases) levels. CONCLUSIONS: An increase in elastic fibers in the mid to lower dermis may reflect a repairing process in response to the degraded upper dermal elastic fibers, which could be related to the pathogenesis of LSA.

Non-specific protein modifications by a phytochemical induce heat shock response for self-defense.

Accumulated evidence shows that some phytochemicals provide beneficial effects for human health. Recently, a number of mechanistic studies have revealed that direct interactions between phytochemicals and functional proteins play significant roles in exhibiting their bioactivities. However, their binding selectivities to biological molecules are considered to be lower due to their small and simple structures. In this study, we found that zerumbone, a bioactive sesquiterpene, binds to numerous proteins with little selectivity. Similar to heat-denatured proteins, zerumbone-modified proteins were recognized by heat shock protein 90, a constitutive molecular chaperone, leading to heat shock factor 1-dependent heat shock protein induction in hepa1c1c7 mouse hepatoma cells. Furthermore, oral administration of this phytochemical up-regulated heat shock protein expressions in the livers of Sprague-Dawley rats. Interestingly, pretreatment with zerumbone conferred a thermoresistant phenotype to hepa1c1c7 cells as well as to the nematode Caenorhabditis elegans. It is also important to note that several phytochemicals with higher hydrophobicity or electrophilicity, including phenethyl isothiocyanate and curcumin, markedly induced heat shock proteins, whereas most of the tested nutrients did not. These results suggest that non-specific protein modifications by xenobiotic phytochemicals cause mild proteostress, thereby inducing heat shock response and leading to potentiation of protein quality control systems. We considered these bioactivities to be xenohormesis, an adaptation mechanism against xenobiotic chemical stresses. Heat shock response by phytochemicals may be a fundamental mechanism underlying their various bioactivities.

Cytologic features of the endometrial adenocarcinoma: comparison of ThinPrep and BD SurePath preparations.

We compared the cytoarchitectural features used for the cytologic diagnosis of endometrial adenocarcinoma (EC) using ThinPrep® (TPS = ThinPrep Sample) and BD SurePath™ (SPS = SurePath Sample) preparations. In 20 patients, a direct endometrial sample using the Uterobrush was obtained. Nineteen cases of EA and one case of carcinosarcoma were studied. TPS and SPS were performed according to the manufacturer's recommendations. Moreover, after the TPS preparation, the residual material was also used to prepare an SPS sample (TP-SPS = ThinPrep-Surepath sample). The following points were investigated in both preparations: (1) number of cell clumps; SPS had a significantly higher (20.9) than TPS (1.7) and TP-SPS (10.3); (2) long axis of clumps; SPS had a significantly higher (215.4) than TPS (146.0); (3) rate of cell clumps with longer axes than 200 µm; SPS had a significantly higher (36.7) than TPS (15.2) and TP-SPS (24.2). TP-SPS showed higher values than TPS; (4) nuclear area; TPS had a significant higher (61.2) than SPS (40.8) and TP-SPS (38.6); (5) degree of overlapping nuclei; SPS (3.4) had a significantly higher number of overlapping nuclei than TPS (0.7) and TP-SPS (2.1); (6) nuclear chromatin pattern; no significant differences for the nuclear chromatin pattern were found in the three different methods. The poor performance of TPS versus SPS and TP-SPS was explained with the heavy blood contamination of the samples, and the absence of adhesive coating in the slides is used for TPS. Further investigation of technical differences in liquid-based cytology methodologies is needed.

Galectin-7 and actin are components of amyloid deposit of localized cutaneous amyloidosis.

The precursor protein of localized cutaneous amyloidosis (LCA) is believed to be cytokeratins on the basis of previous immunohistochemical studies. To identify the candidate amyloid protein biochemically, amyloid proteins were extracted with distilled water from lesional skin of LCA associated with Bowen's disease. The proteins were resolved on one- or two-dimensional polyacrylamide gel electrophoresis followed by characterization with immunoblot analysis. The proteins with multiple molecular weights of 50-67 kDa and two proteins with 25 and 35 kDa were identified as keratins, serum amyloid P component and apolipoprotein E, respectively. The unknown 14-kDa (pI = 7.0) and 42-kDa (pI = 5.4) proteins reacted with the antibody against galectin-7 and actin, respectively. The protein with the molecular weight of 14 kDa was identified as galectin-7 by MALDI-TOF mass spectrometer. Their electrophoretic mobilities were identical with normal counterparts extracted from cultured normal human keratinocytes. Galectin-7 and actin were detected by immunoblot assay in the water-soluble fractions prepared from the lesional skins of two patients with primary LCA. Immunohistochemical studies of tumor-associated (n = 9) and primary (n = 10) LCA revealed various degrees of positive immunoreactivities with the antibodies for galectin-7 and F-actin. Galectin-7 and actin, which contain considerable amount of ß-sheet structure, may be candidate amyloidogenic proteins of primary and secondary LCA.

Dermal carbonyl modification is related to the yellowish color change of photo-aged Japanese facial skin.

BACKGROUND: The photo-aged facial skin is characterized by various unique features such as dark spots, wrinkles, and sagging. Elderly people, particularly Asians, tend to show a yellowish skin color change with photo-aging. However, there has been no analytical study conducted on this unique skin color change of the aged facial skin. OBJECTIVE: The purpose of the present study is to examine whether the carbonyl modification in the dermal protein is involved in the yellowish color change that occurs in the photo-aged skin. METHODS: Normal skin samples excised from the face, abdomen and buttock of variously aged Japanese were separated into the epidermal and the dermal portions. These skin samples were histologically examined for carbonyl modification. Moreover, an in vitro constructed dermis model composed of a contracted collagen gel was treated with acrolein or 4-hydroxynonenal. All these samples were also studied colorimetrically. RESULTS: The dermal samples obtained from the photo-aged facial skin exhibited an appearance of yellowish color, whereas neither the facial epidermis nor the dermis obtained from the abdomen or buttock showed such a yellowish discoloration. The upper layer of the dermis that revealed the yellowish color showed elastosis whose elastic fibers were found to colocalize with carbonyl protein as detected by a labeled hydrazide, as well as by an immunohistochemical examination using the antibody against acrolein adduct. Experimental induction of carbonyl modification in a dermis model in vitro by a long-term treatment with acrolein or 4-hydroxynonenal was found to show the appearance of the yellowish change which was also proven by an increase in b* value of colorimetry. It was more pronounced than that induced by glycation. CONCLUSION: Our present results strongly suggest that carbonyl modification of the dermal protein is involved in the production of the yellowish color change that is noted in the photo-aged facial skin.

Collagen-derived dipeptide, proline-hydroxyproline, stimulates cell proliferation and hyaluronic acid synthesis in cultured human dermal fibroblasts.

Orally ingested collagen undergoes degradation to small di- or tripeptides, which are detected in circulating blood 2 h after ingestion. The influence of collagen-derived peptides on dermal extracellular matrix components and cell proliferation was studied using cultured human dermal fibroblasts. Of the various collagenous peptides tested here, the dipeptide proline-hydroxyproline (Pro-Hyp) enhanced cell proliferation (1.5-fold) and hyaluronic acid synthesis (3.8-fold) at a dose of 200 nmol/mL. This was concomitant with a 2.3-fold elevation of hyaluronan synthase 2 (HAS2) mRNA levels. Small interfering RNA (siRNA)-mediated knockdown of the HAS2 gene in human dermal fibroblasts inhibited Pro-Hyp-induced HAS2 mRNA transcription and cell mitotic activity. Addition of genistein or H7, a protein kinase inhibitor, abolished the Pro-Hyp-induced HAS2 mRNA stimulation. Pro-Hyp elevated phosphorylation of signal transducer and activator of transcription 3 (STAT3) within a short time period (60 min). These results suggest that Pro-Hyp stimulates both cell mitotic activity and hyaluronic acid synthesis, which is mediated by activation of HAS2 transcription.

AGE-modified collagens I and III induce keratinocyte terminal differentiation through AGE receptor CD36: epidermal-dermal interaction in acquired perforating dermatosis.

To clarify the molecular mechanism underlying the transepidermal extrusion of dermal collagen in acquired perforating dermatosis (APD) associated with diabetes mellitus and renal failure, we studied the interaction between advanced glycation end product (AGE)-modified extracellular matrix proteins and keratinocytes (KCs) in a cell culture system. The expression of involucrin (INV) and keratin 10 was significantly enhanced in normal human KCs grown on AGE-modified collagen I or III compared with cells grown on unmodified collagen I or III. Glycated collagens I and III preferentially induced the expression of AGE receptor CD36, but not of other AGE receptors. KCs induced to terminal differentiation demonstrated markedly elevated CD36 expression. Glycated collagen I- and III-induced INV expression was partially blocked by the anti-CD36 antibody (Ab). These substrates also induced epidermal matrix metalloproteinase 9 (MMP-9) expression. Lesional skin from APD patients reacted moderately or strongly with the anti-CD36 Ab as well as the anti-MMP-9 Ab in the epidermal cells surrounding the collagenous materials being eliminated. These results suggest that exposing KCs to AGE-modified interstitial collagen (types I and III) by scratching induces terminal differentiation of KCs via the AGE receptor (CD36), leading to the upward movement of KCs together with glycated collagen.

A PCR-based protocol for the generation of a recombinant West Nile virus.

Viral reverse genetics, particularly infectious cloning, is a valuable tool with applications to many areas of viral research including the generation of vaccine candidates. However, this technology is sometimes insufficient for the construction cDNA clones as the genome sequences and/or encoding proteins of some viral agents may be toxic to the host cells used for cloning. To circumvent this problem, we developed a polymerase chain reaction (PCR)-based protocol for generating a complete West Nile virus (WNV) cDNA. The fragmented cDNAs were synthesized from WNV RNA by reverse transcription-PCR, and subsequently cloned into plasmids for use as templates for WNV cDNA synthesis. The fragmented cDNAs were amplified and assembled by PCR to generate a full-length WNV cDNA. Using this cDNA as a template, WNV RNA was synthesized in vitro and transfected into mammalian cells. We also examined the generation of a mutant recombinant WNV containing a site-directed mutation within the viral genome sequence. Here, we discuss the possibility of developing a method for the generation of recombinant WNVs.

A phase I/II study of docetaxel and gemcitabine combination for chemotherapy-resistant ovarian cancer.

BACKGROUND: A phase I/II study of docetaxel (DOC) and gemcitabine (GEM) combination for treatment-resistant ovarian cancer (OC) was conducted. MATERIALS AND METHODS: Eligible patients exhibited recurrent OC within 12 months after initial treatment, or after more than 2 chemotherapy regimens. Planned dose levels (DL) were as follows: DOC 70 mg/m(2), GEM 800 mg/m(2) (DL1); DOC 70 mg/m(2), GEM 1000 mg/m(2) (DL2). DOC was administered on day 1 combined with GEM on days 1 and 8 every 3 weeks. Adverse events were assessed by NCI-CTC2.0J. Response was evaluated by RECIST or Rustin's criteria. RESULTS: The recommended dose was DL1. For all enrolled patients, the median interval from last chemotherapy was 2.5 (1-11) months and 32 patients were assessable for response. One complete response, 6 partial responses and 6 stable disease were noted. Median time to progression was 4.8 months. Toxicities were mainly hematological and manageable. CONCLUSION: This combination could be an acceptable treatment option before palliation.

Application of the TORO technique of 1H NMR to the structural analysis of cyclic peptide isomers having a slightly distorted symmetry from C2.

The extended TORO technique was applied to the structural analysis of endo-D-Tyr-gramicidin S, cyclo(-Val-Orn-Leu-D-Phe-D-Tyr-Pro-Val-Orn-Leu-D-Phe-Pro-), which has a slightly distorted symmetry from C2, by the insertion of D-Tyr and equivalent alpha-proton chemical shifts in the 1H NMR spectrum. All NMR signals of the two dominant isomers of this antibiotic with trans-trans prolines were determined by using the extended TORO technique with TOCSY and ROESY spectra. This technique is generally applicable for distinguishing overlapped signals of alpha- and amide protons from the main chains of peptides.

In utero diagnosis of an aneurysm of the vein of Galen causing hydrocephalus and heart failure.

A prenatally diagnosed aneurysm of the vein of Galen was presented in the fetus of a patient referred to our hospital at 31 weeks of gestation. Ultrasonography demonstrated polyhydramnios, cardiomegaly, dilatation of the right atrium and superior vena cava, tricuspid valve regurgitation, hydrocephalus, and a large hypoechoic mass with blood flow in the suboccipital region. Skin edema was shown thereafter. A 3262-g male was delivered by cesarean at 35 weeks of gestation. Computed tomography imaging demonstrated a large mass in the suboccipital region, after which thrombocytopenia appeared and the neonate died at 18 days of age.

Iontophoresis promotes percutaneous absorption of L-ascorbic acid in rat skin.

BACKGROUND: Percutaneous absorption of ascorbic acid is limited by its impermeability and instability. OBJECTIVE: We attempted to improve the percutaneous absorption of ascorbic acid by use of iontophoresis after topical application of ascorbic acid. METHODS: Radioactivities extracted from epidermal, dermal and blood compartments after topical application of [14C]ascorbic acid was measured in the presence or absence of iontophoresis. Autoradiography was also performed to study the histological distribution of the radioactivity of ascorbic acid. RESULTS: Iontophoresis greatly enhanced percutaneous absorption of [14C]ascorbic acid in the rat skin. Radioactive ascorbic acid in the dermis reached a maximum level at 1 h after application whereas, in the topical application method, the uptake of ascorbic acid in both epidermis and dermis was quite low. Autoradiography of skin specimens indicated that iontophoresis accelerated the absorption of ascorbic acid through both transepidermal and pilo-sebaceous routes. CONCLUSION: Iontophoretic delivery system of ascorbic acid may provide a more efficient tool for its percutaneous absorption than a simple topical application.

Study on hydration enthalpy of N-methylacetamide in water.

In order to determine the hydration enthalpy of N-methylacetamide (NMA), the enthalpy change for mixing NMA with liquid water was measured calorimetrically. NMA molecules can self-associate both in the liquid state and in aqueous solutions. The population distribution of the self-associated polymeric species in liquid NMA was evaluated by using the results of ab initio MO calculations for the monomer, dimers, and trimers of trans-NMA, while that in aqueous NMA was obtained by NMR spectroscopy. The relationship between these distributions and the mixing enthalpy was formulated on the basis of the similar energy cycle as Born-Haber type, the schematic diagram of energy cycle is given, and the hydration enthalpy of the NMA monomer was determined accurately. The enthalpy thus obtained was found to be in good agreement with that calculated by ab initio MO theory.