虚寒证的证候规律及其代谢组学的研究
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摘要
目的:
1通过文献研究,总结古今医家对虚寒证病因、证型及症状认识,分析虚寒证证本质研究情况,从而揭示虚寒证的病因、证型、症状的分布规律及证本质的研究现状;同时结合专家意见,制定具有普适性的虚寒证临床流行病学调查问卷,并为虚寒证的病因和证候规律研究提供理论和文献基础。
2通过临床流行病学调查,统计虚寒证的患病率,分析病因、证型、症状特点和规律,籍此了解虚寒证的患病情况,寻找虚寒证的主要危险因素,探讨虚寒证证型的分布情况及各证型的主要辨证指标。并为中医证候开展大样本的临床流行病学研究的技术操作方法提供一个范例。
3利用核磁共振技术,探讨虚寒证机体代谢产物的变化,阐释虚寒证的生物学本质,探索代谢组学技术在证本质研究中的应用。
方法:
1文献研究
以《中国医籍大辞典》为检索工具,对河北医科大学西校区图书馆中现有的从《内经》到清末200余本古代文献进行整理分析;以中国期刊全文数据库(河北医科大学图书馆CNKI新版检索页面)为检索库,对1996年至2006年的学术期刊中与虚寒证有关的文献进行整理,症状进行频数分析。
2虚寒证证候规律的研究
在文献研究、专家咨询的基础上,制定虚寒证调查问卷,经中医学、量表学、统计学、社会学等专家论证后,进行预调查,修改后对问卷进行信度和效度的评测,形成适合虚寒证证候学和病因学调查的具有量表性质的问卷。运用此问卷,采用随机-整群抽样方法,选择经培训合格的调查员对石家庄及其周边地区的常住居民2500人进行现场调查。回收的调查问卷经专家反复审查和诊断,对符合要求的问卷进行数据整理,运用多元统计分析(聚类分析、因子分析、主成分分析、logistic回归分析)和中医理论知识,对虚寒证的数据进行聚类挖掘和医理分析。
3代谢组学研究
选取典型虚寒证月经病患者作为研究对象,以典型虚热证月经病患者和正常健康女性为对照,于每位受试者月经期的第二天抽取空腹肘静脉血5ml,3000r/min离心20min后,取上清液2ml保存于-70℃的低温保存箱中待测。利用日本电子600兆核磁共振波谱仪对血清进行代谢产物检测。采用dalte软件( http: //qobrue. usc. es)对研究对象血清的1HNMR谱按δ0.04间隔从δ0～6分段进行数据提取,除去水峰δ4.67,对余下的各段谱峰进行积分。将峰面积的积分数据归一化之后,以文献为参比,由核磁共振图谱解析专家对每一区段进行代谢产物鉴定,按照峰的位置及其代表物质的完整性,经整理共得到24个代谢物(其中有2个为未知代谢物)和1个葡萄糖+氨基酸的区间段,将每一种代谢物的不同区间段的积分值相加得到25个总积分值。以Excel文件贮存,用于统计分析。剩下的血清采用放射免疫分析法测定血清卵泡刺激素(FSH)、黄体生成素(LH)、雌二醇(E2)、睾酮(T)、孕酮(P)的含量。
结果:
1文献研究
1.1文献研究结果表明,目前对虚寒证病因的认识与古代文献基本一致;虚寒证的辨证无统一规范,认识也尚不统一;虚寒证现有的各种辨证方法对虚寒证的临床和科研有一定的指导意义,但其多建立在临床报道和个人经验的基础上而缺少临床证候学的研究;临床和科研重视证候的本质研究而忽视证候本身内容的研究。
1.2经对现代文献中描述的虚寒证症状进行频数统计,结果显示四肢不温、畏寒、大便稀溏、倦怠四个症状对虚寒证的诊断可能有重要意义。
1.3文献研究结果提示:近十年,虚寒证本质的研究由单个指标向多个指标群的研究方向转变,引入虚寒证本质研究的新指标极少,缺乏一定的创新性。目前尚无公认的虚寒证特异性、敏感性的指标。
2虚寒证证候规律的研究
2.1根据文献研究、专家意见和预调查结果,研制了《虚寒证证候学临床流行病学调查问卷》,经临床流行病学调查和数据分析,结果表明:该问卷具有较好的信度和效度,具有一定的普适性。
2.2临床流行病学调查结果表明:虚寒证在人群中的患病率是9.946%。
2.3统计分析结果显示:虚寒证的发生与性别、年龄、学历、职业、居住地等人口学特征有关,均有统计学意义(均P<0.01);虚寒证发病的最大危险因素为房劳多产,OR=27.926,其余依次是虚寒体质、感受寒邪、饮食失宜、情志所伤、过度劳累。
2.4根据聚类分析和因子分析的结果,虚寒证的常见证候是:肾阳虚证(18.828%)、脾肾阳虚证(16.318%)、心肾阳虚证(14.644%)、脾阳虚证(10.044%)、心脾阳虚证(10.044%)、肺肾阳虚证(8.787%)、心阳虚证(6.276%)、肺阳虚证(5.021%)、肝脾阳虚证(4.184%)九个证候群。分析结果提示:虚寒证主要以两脏相兼为病多见,占56.904%,以单脏为病次之,占40.167%;其中肾阳虚证在虚寒证中所占比重较大,肝阳虚证多与其它脏相兼出现。
2.5经主成分分析和logistic逐步回归分析,得出对虚寒证各证型诊断有统计学意义的主要症状(其中主症是各证型中的主要辨证指标,按OR值由大到小的顺序排列)如下:
脾阳虚证:主症是脘腹胀满、大便稀溏、倦怠、畏寒;次症是四肢不温、食少纳呆、神疲、遇冷加重、喜暖喜按、舌淡胖。
肝脾阳虚证:主症是遇冷加重、食少纳呆、两胁胀满、恶心、眼花;次症是畏寒、头晕、四肢不温、两胁隐痛、喜热饮、喜暖喜按。
心阳虚证:主症是喜热饮、心悸、脉虚弱、怔忡;次症是畏寒、四肢不温、气短、自汗、口唇色暗、下肢浮肿、遇冷加重。
肺阳虚证:主症是易感冒、咳嗽、咳痰色白、遇劳加重;次症是畏寒、气短、四肢不温、脉迟缓无力、舌质淡白。
心脾阳虚证:主症是脉沉迟无力、心悸、大便稀溏、脘腹胀满、胸憋闷;次症是畏寒、四肢不温、喜暖喜按、头面浮肿、苔薄白。
肺肾阳虚证:主症是腰膝酸软、咳嗽、舌质淡白、易感冒、尿少;次症是四肢不温、畏寒、咳痰色白、神疲、苔白腻。
肾阳虚证:主症是尿频、腰膝酸软、面色白光白、苔白、畏寒;次症是四肢不温、遗尿、夜尿多、小便清长、舌淡胖或边有齿痕、脉细弱或迟缓无力。
心肾阳虚证:主症是心悸、尿少、腰膝酸软、遇冷加重、胸痛;次症是畏寒、四肢不温、倦怠、下肢浮肿、尿后余沥、脉细弱或沉细无力。
脾肾阳虚证:主症是大便稀溏、面色白光白、尿频、腰膝酸软、胃脘隐痛;次症是完谷不化、四肢不温、倦怠、畏寒、喜热饮、脉沉迟无力。
2.6综合研究结果与专家意见,认为畏寒、四肢不温是虚寒证特征性症状。
3虚寒证机体的代谢组学研究
3.1虚寒证组、虚热证组和正常组研究对象的血清1HNMR图谱
与正常组比较,虚寒证组和虚热证组血清1HNMR图谱在3.2～3.96ppm以及4.0ppm、1.22ppm、2.23ppm、2.41ppm、2.57ppm的峰形有明显的差异。
3.2三组血清1HNMR图谱谱峰面积积分的比较
经多元方差分析,与虚热证组、正常组比较,虚寒证代谢物中的α-葡萄糖(5.12ppm)、β-葡萄糖(3.14ppm、4.56ppm)、RegionA(3.2～3.96ppm)(葡萄糖和氨基酸)、柠檬酸(2.57ppm)、琥珀酸(2.23ppm)、不饱和脂类(5.18ppm、2.63ppm、2.34ppm、1.89ppm、1.62ppm、1.45ppm)、极低密度脂蛋白(1.16ppm)、谷氨酸(2.41ppm)、1.79ppm处未知化合物的相对积分面积明显降低,均有统计学意义;乙酰乙酸(2.11ppm)、乳酸(1.21ppm、4.0ppm)明显升高,有统计学意义(P<0.05,P<0.01;P<0.01,P<0.01)。此外,与正常组比较,虚寒证血清缬氨酸(0.92ppm)、亮氨酸(0.88ppm)、低密度脂蛋白(0.75ppm)的含量降低,有统计学意义(P<0.05,P<0.05,P<0.05),但与虚热证比较无统计学意义(P>0.05,P>0.05,P>0.05)。
与虚寒证、正常组比较,虚热证血清1HNMR图谱RegionA、赖氨酸(2.84ppm)的谱峰面积积分明显升高,有统计学意义(P<0.01,P<0.05;P<0.05,P<0.01)。
3.3虚寒证组、虚热证组和正常组血清生殖激素水平的变化
与虚热证组、正常组比较,虚寒证组患者血清中FSH、LH、E2、T的含量降低,差异均有统计学意义(P<0.01,P<0.01;P<0.01,P<0.01;P<0.01,P<0.01;P<0.01,P<0.05);与虚热证比较,虚寒证组患者血清P的含量降低,有统计学意义(P<0.01),但与正常组比较无统计学意义(P>0.05)。
与虚寒证组、正常组比较,虚热证组患者血清中LH、T、P的含量升高(P<0.01,P<0.05;P<0.01,P<0.01;P<0.01,P<0.01);与虚寒证比较,虚热证组血清中E2、FSH的含量升高,有统计学意义(P<0.01,P<0.01);但与正常组比较无统计学意义(P>0.05,P>0.05)。
3.4虚寒证的生物标志物与生殖激素的相关性分析
经多元方差分析后,将虚寒证血清中与虚热证和正常组比较均有变化的代谢物(乳酸、柠檬酸、琥珀酸、不饱和脂类、VLDL、乙酰乙酸、谷氨酸、1.79ppm处的未知化合物)分别与虚寒证机体中有变化的生殖激素FSH、LH、E2、T进行相关分析,结果乳酸与FSH、LH呈负相关(r=-0.839,P<0.05;r=-0.865,P<0.05),而与E2、T无相关性;柠檬酸与FSH、LH呈正相关(r=0.826,P<0.05;r=0.812,P<0.05),琥珀酸与FSH、LH也呈正相关(r=0.889,P<0.05;r=0.833,P<0.05),但二者与E2、T均无相关性。不饱和脂类、VLDL、乙酰乙酸、谷氨酸、1.79ppm处的未知化合物与FSH、LH、E2、T均无相关性。
结论:
本课题采用文献研究、临床流行病学调查及代谢组学的方法,对虚寒证展开了初步系统的研究,所得结论如下:
1运用文献研究的方法,探讨了虚寒证的病因和临床表现特点,从整体上把握了虚寒证的研究规律和发展方向,为下一步的研究奠定理论和文献基础。
2本课题临床流行病学调查发现虚寒证的患病率为9.946%;统计分析表明虚寒证的发生与性别、年龄、学历、职业、居住地等人口学特征有关;房劳多产是虚寒证发生的主要影响因素,其余依次是虚寒体质、感受寒邪、饮食失宜、情志所伤、过度劳累。据此可以有针对性地预防虚寒证的发生。
3虚寒证以肾阳虚证、脾肾阳虚证、心肾阳虚证、脾阳虚证、心脾阳虚证、肺肾阳虚证、心阳虚证、肺阳虚证、肝脾阳虚证九个证型为常见证型。
4经文献研究,结合专家意见以及流调结果的统计分析,确定了每种证型的主要辨证指标,畏寒、四肢不温是虚寒证的特征性症状,为虚寒证诊断标准的确立提供参考。
5通过本次临床流行病学调查,获得了中医证候的大样本临床流行病学调查的方法、质量控制及数据处理等具体经验,对中医证候学研究进行了有意义的探索。
6糖、脂肪、蛋白质代谢障碍,导致能量产生不足可能是虚寒证发生的重要原因。初步研究认为虚寒证机体代谢产物中的乳酸、柠檬酸、琥珀酸、不饱和脂类、VLDL、乙酰乙酸、谷氨酸、1.79ppm处的未知化合物可以作为虚寒证的生物标志物,提示这些生物标志物可能会作为深入研究虚寒证本质的切入点。
7虚寒证机体可能是因为糖代谢异常,脑功能受损,影响垂体、下丘脑或更高中枢的功能,使体内生殖激素分泌下降,而出现月经病。
8代谢组学分析是一种有良好发展前景的中医证候学研究方法,有助于更全面地认识机体的代谢物质组成变化,以揭示中医证候的实质。
Objective:
1 Through the literature research, we wanted to summarize the cognition of ancient and modern researcher in etiological factor, type of syndrome and symptoms of asthenia cold syndrome, analyze the essence of asthenia cold syndrome. Thereby, we hoped to reveal the research’s status quo of etiological factor, type of syndrome, symptoms and essence of asthenia cold syndrome. At the same time, we planned to combine with opinions of experts for formulating suitable clinical epidemiology investigational questionnaire of asthenia cold syndrome. And offered theoretical and literary foundation for the next study of etiological factor and symptomatic rule of asthenia cold syndrome.
2 Through the clinical epidemiology investigating, we planned to get the case rate of asthenia cold syndrome by statistics and analyze the characteristic and rule of etiological factor, type of syndrome and symptoms of asthenia cold syndrome. Accordingly, we could understand the condition of what asthenia cold syndrome had taken place and look for its main Risk factor, as well as approach the distributive condition and the main indexes of differentiation of symptoms and signs of its type of syndrome. Moreover, we wanted to offer an example for the operational method of large sample clinical epidemiology research in symptoms of traditional Chinese medicine.
3 We decided that we used the technique of nuclear magnetic resonance to approach changes of metabolin of asthenia cold syndrome and explain biological essence of asthenia cold syndrome. Then we hoped to explore the application of metabonomics’technology in the study of symptomatic essence.
Method:
1 The literature research
Using yiji dictionary of china as retrieval instrument, we retrieved all of ancient times literature from Neijing term to Qing dynasty end and to arrange and analyze them; At the same time,we used china periodical full-text data base(library CNKI retrieval page of mdition university of heibei ) to retrieve modern literature which related with asthenia cold syndrome from 1996 to 2006, then carried out frequency analysis on symptoms and arranged them.
2 The research of symptomatic rule of asthenia cold syndrome
On the literature’s research and expert’s consulation foundation, we formulated the investigational questionnaire of asthenia cold syndrome. After through the experts of Chinese traditional medicine, measuring scale, statistics, sociology demonstrasting, we beforehand investigate with investigational questionnaire. After amending, we appraised its reliability and validity by investigated again. Eventually, there formed a suitable investigational questionnaire of syndrome and etiology of asthenia cold syndrome, which possessed the qualitative of measuring scale. Used investigational questionnaire above, we adopted random cluster sampling, selected valid researcher who had been trained, then let them to survey 2500 inhabitant, who lived in shijiazhuang and its surrounding area in long-term. After these investigation questionnaires were reclaimed, they inspected and diagnosed by experts again and again. Among them, investigational questionnaires, which fit requirement, were carried out data record. Then we excavated and analyzed these data by multielement statistical analysis(cluster analysis, factor analysis, principal component analysis, logistic regression analysis) and theory of traditional Chinese medicine.
3 The metabonomics’study
We selected typical patients of asthenia cold syndrome menopathy as subjects, and selected typical patients of asthenia pyrosyndrome menopathy and health female as contrast. Every subjects took venous blood 5ml on an empty stomach at the second day of menstrual period. After centrifuging these venous blood, we took clear supernatant liquid 2ml and preserved it in - 70℃low temperature box, waiting for detection. We utilized 600MHz nuclear magnetic resonance spectrometer to detect serum metabolin composition. Then we adopted dalte software(http: //qobrue. usc. es) to get out data from subjects’serum 1HNMR spectra according toδ0.04 interval fromδ0 toδ6 segmentation. Except forδ4.669 which was water, we integrated others. After the integral of peak area had been carried out normalization, experts identified metabolin from every segment on the basis of literatures. Then according to location of peak and it representing metabolin’s integrity, we gained 24 metabolin(including 2 unkown metabolin)and 1segment of glucose and amino acids. Through added up different segment of each metabolin, we got 25 score. The scores were Stored ExceL document, waiting statistical analysis.The remain serum was used to detect FSH,LH,E2,T and P by radio immunoassay.
Result:
1 The literature research
1.1 The result of literature research shew that the cognition of etiological factor of asthenia cold syndrome mainly accord with ancient literature now; The differentiation of symptoms and signs of asthenia cold syndrome had not unified specification,and the cognition still wasn’t unification; Forthcoming various kinds differentiation of symptoms and signs methods had directional significance. But these methods mostly set up on clinical report and individual empirical foundation, but were short of research of syndromes; On the clinic and scientific research, specialists thought highly of essence of syndromes but neglected content of syndromes themself.
1.2 Through frequency statistics about symptoms of asthenia cold syndrome which had been described by modern literatures, the result shew that limbs cold, chilly, loose stool, lassitude had important significance possiblly.
1.3 The result of literature research discovered that the essential study of asthenia cold syndrome had been shifting from the study of sing index to more indexs. But new indexs that were emploied the essential study of asthenia cold syndrome were few, so this means that new ideas was lack. Nowadays, the specificity and sensitivity index of asthenia cold syndrome don’t appear.
2 The research of symptomatic rule of asthenia cold syndrome
2.1 According to the results of literature research, experts opinion and beforehand investigating, we studied and formulated the clinical epidemiological investigational questionnaire of syndromes of asthenia cold syndrome.The results of clinical epidemiological investigation and analysis of data indicated that this questionnaire had better reliability and validity. And it also had some widespread applicability.
2.2 The results of clinical epidemiological investigation signed that the case rate of asthenia cold syndrome in crowd was about 9.946%.
2.3 The statistics results shew that the occurrence of asthenia cold syndrome related with such demography characteristic as sex, age, educational background,etc. there had statistical significance(all P<0.01); Among them, fang lao and copiousness was the main risk factor, OR=27.926,which was followed by asthenia cold constitution, reception chill, improper diet, sentiment injury, excessus exertion.
2.4 According to the results of cluster analysis and factor analysis of variance, frequent type of syndromes of asthenia cold syndrome were deficiency of kidney-yang(18.828%),deficiency of spleen-yang and kidney-yang(16.318%), deficiency of heart-yang and kidney-yang(14.644%), deficiency of spleen-yang(10.041%), deficiency of heart-yang and spleen-yang(10.041%), deficiency of lung-yang and kidney-yang(8.787%), deficiency of heart-yang(6.276%),deficiency of lung-yang(5.021%), deficiency of liver-yang and spleen-yang(4.184%). analytic result also shew that main two viscus pathopoiesis together is more, occuping 56.904%; sing viscera pathopoiesis occupied 40.167%; Among them, the proportion of deficiency of kidney-yang in asthenia cold syndrome was larger. Deficiency of liver-yang appeared with other viscus together.
2.5 Through principal component analysis and logistic stepwise regression analysis, we got cardinal symptoms which had statistics significance in diagnosis of each type of syndrome of asthenia cold syndrome. They were as follows(The main symptoms arranged according to OR value ):
Deficiency of spleen-yang: Main symptoms were abdominal distention, loose stool, lassitude, chilly; Secondary symptoms were limbs cold, anorexia, mind weary, aggravation with cold, relief with warm and pressure, pale and enlarged tongue.
Deficiency of liver-yang and spleen-yang: Main symptoms were aggravation with cold, anorexia, bilateral hypochondria full, nauseated, eyes blurred; Secondary symptoms were chilly, dizzy, limbs cold, bilateral hypochondria vague pain, hot drink preference, relief with warm and pressure.
Deficiency of heart-yang: Main symptoms were hot drink preference, palmus, weakness pulse, zhengzhong; Secondary Symptoms were chilly, limbs cold, short breath, spontaneous perspiration, darkness oral lip, edema of lower limbs, aggravation with cold.
Deficiency of lung-yang: Main symptoms were catching a cold readily, cough, whiteness phlegm, aggravation with exhaustoin; Secondary symptoms were chilly, short breath, limbs cold, light body of the tongue.
Deficiency of heart-yang and spleen-yang: Main symptoms were deep and delay pulse, palmus, loose stool, abdominal distention, chest depressed;Secondary symptoms were chilly, limbs cold, relief with warm and pressure, head and face puffiness, thinness and whiteness moss.
Deficiency of lung-yang and kidney-yang: Main symptoms were acid and weak in waist and knee, cough, whiteness body of the tongue, catching a cold readily, oliguresis; Secondary symptoms were chilly, limbs cold, whiteness phlegm, mind weary, whiteness and greasiness moss.
Deficiency of kidney-yang: Main symptoms were urinary frequency, acid and weak in waist and knee, huangbai complexion, whiteness moss; Secondary symptoms were limbs cold, urinary incontinence, multi- nycturia, urine clear, pale and enlarged tongue with indentation, extenuate and weakness pulses or slow and weakness pulses.
Deficiency of heart-yang and kidney-yang: Main symptoms were palmus, oliguresis, acid and weak in waist and knee, aggravation with cold, chest pain; Secondary symptoms were chilly, limbs cold, lassitude, edema of lower limbs,droping after urine, extenuate and weakness pulses or deep and weakness pulses.
Deficiency of spleen-yang and kidney-yang:Main symptoms were loose stool, huangbai complexion, urinary frequency, acid and weak in waist and knee, gastric cavity vague pain; Secondary symptoms were wangubuhua, limbs cold,lassitude, chilly, hot drink preference,deep and delay puleses.
2.6 Synthesizeing the result of statistics and the opinion of experts, we considered chilly, limbs cold as characteristic symptoms of asthenia cold syndrome.
3 The metabonomics’research of asthenia cold syndrome
3.1 Compared with normal group, there were conspicuous changes in 3.2～3.96ppm, 4.0 ppm, 1.22 ppm, 2.23ppm,2.41 ppm, 2.57 ppmpeak forms of the serum 1HNMR spectra in asthenia cold syndrome group and asthenia pyrosyndrome group .
3.2 Through multielement ANOVA: compared with asthenia pyrosyndrome group and normal group, there were obviously low in the contents of serumα-glucose(5.12ppm),β-glucose(3.14ppm,4.56ppm), RegionA(3.2～3.96ppm)(glucose and amino acids), citrate(2.57ppm), succinate(2.23ppm), unsaturated lipid (5.18ppm, 2.63ppm, 2.34ppm, 1.89ppm, 1.62ppm, 1.45 ppm), very low density lipoprotein(1.16ppm), glutamine(2.41ppm), unkown compound(1.79ppm) of asthenia cold syndrome group. there were statistical significance; The contents of serum acetoacetate(2.11ppm), lactate(1.22ppm,4.0ppm) were obviously high, there were statistical significance, too.( P<0.05,P<0.01;P<0.01,P<0.01).In addition,compared with normal group,the contents of valine(0.92ppm), leucine(0.88ppm) and low density lipoprotein(0.75ppm) of asthenia cold syndrome cut down,there were statistical significance (P < 0.05,P < 0.05,P < 0.05),but compared with asthenia pyrosyndrome,there weren’t statistical significance(P >0.05,P >0.05,P>0.05).
Compared with asthenia cold syndrome and normal group,the integral of oppsing area of serum RegionA and lysine (2.84ppm) of asthenia pyrosyndrome obviously heightened.There were statistical significance(P<0.01,P<0.05; P<0.05,P<0.01).
3.3 The changes of genesis hormone of asthenia cold syndrome,asthenia pyrosyndrome and normal group
Compared with asthenia pyrosyndrome group and normal group, there were obviously low in the contents of serum FSH,LH,E2 and T of asthenia cold syndrome,there were statistical significance (P<0.01,P<0.01;P<0.01,P<0.01;P<0.01,P<0.01;P<0.01,P<0.05);The content of serum P of asthenia cold syndrome was lower than asthenia pyrosyndrome group,there was statistical significance(P<0.01),but compared with normal group,there wasn’t statistical significance(P>0.05).
Compared with asthenia cold syndrome and normal group, the contents of serum LH,T and P of asthenia pyrosyndrome group heightened,there were statistical significance(P < 0.01,P < 0.05;P < 0.01,P < 0.01;P < 0.01,P <0.01);The contents of serum E2 and FSH of asthenia pyrosyndrome group were higher than asthenia cold syndrome,there were statistical significance (P<0.01,P<0.01),but compared with normal group,there weren’t statistical significance(P>0.05,P>0.05).
3.4 The dependability study of between biomarkers and hormone
After analysis of variance,we carried out correlation analysis between all metabolin which had changes in comparision of asthenia pyrosyndrome group, asthenia cold syndrome and normal group and hormone which had changes in them.The results were that there were negative correlation between lactate and FSH and between lactate and LH respectively(r=-0.839,P<0.05;r=-0.865,P<0.05),but there weren’t dependablity between lactate and E2 and between lactate and T. There were positive correlation between citrate and FSH and between citrate and LH(r=0.826,P<0.05;r=0.812,P<0.05).There were also positive correlation between succinate and FSH and between succinate and LH(r=0.889,P<0.05;r=0.833,P<0.05),but the two hadn’t dependability with E2 and T. Unsaturated lipid,VLDL, acetoacetate, glutamine and 1.79ppm unkown compound hadn’t dependability with FSH,LH,E2 and T.
conclusion:
Through adopted methods of literature’s research, clinical epidemiology investigation and metabonomics, we carried out initially systematical research on asthenia cold syndrome. the conclusions were as follows:
1 Used the method of literature research, we approached etiological factor and clinical manifestation feature of asthenia cold syndrome, and held its regular pattern of research and developed direction in the whole. This established theoretical and literary foundation for the next study.
2 The clinical epidemiology investigation found that case rate of asthenia cold syndrome was about 9.946%; The results of statistical analysis shew that occurrence of asthenia cold syndrome related with such demography feature as sex, age, educational background, vocation, habitation,ect. Fang lao and copiousness was main influential factor of asthenia cold syndrome, which were followed by asthenia cold contitution, reception chill, improper diet, sentiment injury,excessive exertion. On these grounds, we could preclude the occurrence of asthenia cold syndrome directly.
3 The frequent type of syndromes in asthenia cold syndrome were deficiency of kidney-yang,deficiency of spleen-yang and kidney-yang, deficiency of heart-yang and kidney-yang, deficiency of spleen-yang, deficiency of heart-yang and spleen-yang, deficiency of lung-yang and kidney-yang, deficiency of heart-yang, deficiency of lung-yang, deficiency of liver-yang and spleen- yang.
4 Through literature research, combining opinion of experts and epidemiological investigation,we determined main indexs of differentiation of symptoms and signs of each type of syndrome. Chilly and limbs cold were characteristic symptom of asthenia cold syndrome. This offered information for establishing diagnostic criteria of asthenia cold syndrome.
5 Through clinical epidemiology investigation, we obtained methods of investigation, quality control and data processing of large sample clinical epidemiology. We carried out significant exploration on the research of syndromes of traditional Chinese medicine.
6 The dysmetabolism of suger, fat and protein resulted in energy shortage. This was main cause which led to occurrence of asthenia cold syndrome possiblly. We tentatively confirmed that lactate, citrate, succinate, unsaturated lipid, VLDL, acetoacetate, glutamine, 1.79ppm unkown compound could be thought of as biomarkers of asthenia cold syndrome.
7 The body of asthenia cold syndrome might be that glycometabolism was blocked, brain’s function was damaged.This may influence pituitary and hypothalamus or highest center and make hormone of body low, so shew menstrual disorder.
8 Metabonomics analysis was a well developed outlook method which studied syndromes of traditional Chinese medicine. It conduced more overall to understand chemical materials changes of organism, to reveal essence of syndromes of traditional Chinese medicine.
1通过文献研究,总结古今医家对虚寒证病因、证型及症状认识,分析虚寒证证本质研究情况,从而揭示虚寒证的病因、证型、症状的分布规律及证本质的研究现状;同时结合专家意见,制定具有普适性的虚寒证临床流行病学调查问卷,并为虚寒证的病因和证候规律研究提供理论和文献基础。
2通过临床流行病学调查,统计虚寒证的患病率,分析病因、证型、症状特点和规律,籍此了解虚寒证的患病情况,寻找虚寒证的主要危险因素,探讨虚寒证证型的分布情况及各证型的主要辨证指标。并为中医证候开展大样本的临床流行病学研究的技术操作方法提供一个范例。
3利用核磁共振技术,探讨虚寒证机体代谢产物的变化,阐释虚寒证的生物学本质,探索代谢组学技术在证本质研究中的应用。
方法:
1文献研究
以《中国医籍大辞典》为检索工具,对河北医科大学西校区图书馆中现有的从《内经》到清末200余本古代文献进行整理分析;以中国期刊全文数据库(河北医科大学图书馆CNKI新版检索页面)为检索库,对1996年至2006年的学术期刊中与虚寒证有关的文献进行整理,症状进行频数分析。
2虚寒证证候规律的研究
在文献研究、专家咨询的基础上,制定虚寒证调查问卷,经中医学、量表学、统计学、社会学等专家论证后,进行预调查,修改后对问卷进行信度和效度的评测,形成适合虚寒证证候学和病因学调查的具有量表性质的问卷。运用此问卷,采用随机-整群抽样方法,选择经培训合格的调查员对石家庄及其周边地区的常住居民2500人进行现场调查。回收的调查问卷经专家反复审查和诊断,对符合要求的问卷进行数据整理,运用多元统计分析(聚类分析、因子分析、主成分分析、logistic回归分析)和中医理论知识,对虚寒证的数据进行聚类挖掘和医理分析。
3代谢组学研究
选取典型虚寒证月经病患者作为研究对象,以典型虚热证月经病患者和正常健康女性为对照,于每位受试者月经期的第二天抽取空腹肘静脉血5ml,3000r/min离心20min后,取上清液2ml保存于-70℃的低温保存箱中待测。利用日本电子600兆核磁共振波谱仪对血清进行代谢产物检测。采用dalte软件( http: //qobrue. usc. es)对研究对象血清的1HNMR谱按δ0.04间隔从δ0～6分段进行数据提取,除去水峰δ4.67,对余下的各段谱峰进行积分。将峰面积的积分数据归一化之后,以文献为参比,由核磁共振图谱解析专家对每一区段进行代谢产物鉴定,按照峰的位置及其代表物质的完整性,经整理共得到24个代谢物(其中有2个为未知代谢物)和1个葡萄糖+氨基酸的区间段,将每一种代谢物的不同区间段的积分值相加得到25个总积分值。以Excel文件贮存,用于统计分析。剩下的血清采用放射免疫分析法测定血清卵泡刺激素(FSH)、黄体生成素(LH)、雌二醇(E2)、睾酮(T)、孕酮(P)的含量。
结果:
1文献研究
1.1文献研究结果表明,目前对虚寒证病因的认识与古代文献基本一致;虚寒证的辨证无统一规范,认识也尚不统一;虚寒证现有的各种辨证方法对虚寒证的临床和科研有一定的指导意义,但其多建立在临床报道和个人经验的基础上而缺少临床证候学的研究;临床和科研重视证候的本质研究而忽视证候本身内容的研究。
1.2经对现代文献中描述的虚寒证症状进行频数统计,结果显示四肢不温、畏寒、大便稀溏、倦怠四个症状对虚寒证的诊断可能有重要意义。
1.3文献研究结果提示:近十年,虚寒证本质的研究由单个指标向多个指标群的研究方向转变,引入虚寒证本质研究的新指标极少,缺乏一定的创新性。目前尚无公认的虚寒证特异性、敏感性的指标。
2虚寒证证候规律的研究
2.1根据文献研究、专家意见和预调查结果,研制了《虚寒证证候学临床流行病学调查问卷》,经临床流行病学调查和数据分析,结果表明:该问卷具有较好的信度和效度,具有一定的普适性。
2.2临床流行病学调查结果表明:虚寒证在人群中的患病率是9.946%。
2.3统计分析结果显示:虚寒证的发生与性别、年龄、学历、职业、居住地等人口学特征有关,均有统计学意义(均P<0.01);虚寒证发病的最大危险因素为房劳多产,OR=27.926,其余依次是虚寒体质、感受寒邪、饮食失宜、情志所伤、过度劳累。
2.4根据聚类分析和因子分析的结果,虚寒证的常见证候是:肾阳虚证(18.828%)、脾肾阳虚证(16.318%)、心肾阳虚证(14.644%)、脾阳虚证(10.044%)、心脾阳虚证(10.044%)、肺肾阳虚证(8.787%)、心阳虚证(6.276%)、肺阳虚证(5.021%)、肝脾阳虚证(4.184%)九个证候群。分析结果提示:虚寒证主要以两脏相兼为病多见,占56.904%,以单脏为病次之,占40.167%;其中肾阳虚证在虚寒证中所占比重较大,肝阳虚证多与其它脏相兼出现。
2.5经主成分分析和logistic逐步回归分析,得出对虚寒证各证型诊断有统计学意义的主要症状(其中主症是各证型中的主要辨证指标,按OR值由大到小的顺序排列)如下:
脾阳虚证:主症是脘腹胀满、大便稀溏、倦怠、畏寒;次症是四肢不温、食少纳呆、神疲、遇冷加重、喜暖喜按、舌淡胖。
肝脾阳虚证:主症是遇冷加重、食少纳呆、两胁胀满、恶心、眼花;次症是畏寒、头晕、四肢不温、两胁隐痛、喜热饮、喜暖喜按。
心阳虚证:主症是喜热饮、心悸、脉虚弱、怔忡;次症是畏寒、四肢不温、气短、自汗、口唇色暗、下肢浮肿、遇冷加重。
肺阳虚证:主症是易感冒、咳嗽、咳痰色白、遇劳加重;次症是畏寒、气短、四肢不温、脉迟缓无力、舌质淡白。
心脾阳虚证:主症是脉沉迟无力、心悸、大便稀溏、脘腹胀满、胸憋闷;次症是畏寒、四肢不温、喜暖喜按、头面浮肿、苔薄白。
肺肾阳虚证:主症是腰膝酸软、咳嗽、舌质淡白、易感冒、尿少;次症是四肢不温、畏寒、咳痰色白、神疲、苔白腻。
肾阳虚证:主症是尿频、腰膝酸软、面色白光白、苔白、畏寒;次症是四肢不温、遗尿、夜尿多、小便清长、舌淡胖或边有齿痕、脉细弱或迟缓无力。
心肾阳虚证:主症是心悸、尿少、腰膝酸软、遇冷加重、胸痛;次症是畏寒、四肢不温、倦怠、下肢浮肿、尿后余沥、脉细弱或沉细无力。
脾肾阳虚证:主症是大便稀溏、面色白光白、尿频、腰膝酸软、胃脘隐痛;次症是完谷不化、四肢不温、倦怠、畏寒、喜热饮、脉沉迟无力。
2.6综合研究结果与专家意见,认为畏寒、四肢不温是虚寒证特征性症状。
3虚寒证机体的代谢组学研究
3.1虚寒证组、虚热证组和正常组研究对象的血清1HNMR图谱
与正常组比较,虚寒证组和虚热证组血清1HNMR图谱在3.2～3.96ppm以及4.0ppm、1.22ppm、2.23ppm、2.41ppm、2.57ppm的峰形有明显的差异。
3.2三组血清1HNMR图谱谱峰面积积分的比较
经多元方差分析,与虚热证组、正常组比较,虚寒证代谢物中的α-葡萄糖(5.12ppm)、β-葡萄糖(3.14ppm、4.56ppm)、RegionA(3.2～3.96ppm)(葡萄糖和氨基酸)、柠檬酸(2.57ppm)、琥珀酸(2.23ppm)、不饱和脂类(5.18ppm、2.63ppm、2.34ppm、1.89ppm、1.62ppm、1.45ppm)、极低密度脂蛋白(1.16ppm)、谷氨酸(2.41ppm)、1.79ppm处未知化合物的相对积分面积明显降低,均有统计学意义;乙酰乙酸(2.11ppm)、乳酸(1.21ppm、4.0ppm)明显升高,有统计学意义(P<0.05,P<0.01;P<0.01,P<0.01)。此外,与正常组比较,虚寒证血清缬氨酸(0.92ppm)、亮氨酸(0.88ppm)、低密度脂蛋白(0.75ppm)的含量降低,有统计学意义(P<0.05,P<0.05,P<0.05),但与虚热证比较无统计学意义(P>0.05,P>0.05,P>0.05)。
与虚寒证、正常组比较,虚热证血清1HNMR图谱RegionA、赖氨酸(2.84ppm)的谱峰面积积分明显升高,有统计学意义(P<0.01,P<0.05;P<0.05,P<0.01)。
3.3虚寒证组、虚热证组和正常组血清生殖激素水平的变化
与虚热证组、正常组比较,虚寒证组患者血清中FSH、LH、E2、T的含量降低,差异均有统计学意义(P<0.01,P<0.01;P<0.01,P<0.01;P<0.01,P<0.01;P<0.01,P<0.05);与虚热证比较,虚寒证组患者血清P的含量降低,有统计学意义(P<0.01),但与正常组比较无统计学意义(P>0.05)。
与虚寒证组、正常组比较,虚热证组患者血清中LH、T、P的含量升高(P<0.01,P<0.05;P<0.01,P<0.01;P<0.01,P<0.01);与虚寒证比较,虚热证组血清中E2、FSH的含量升高,有统计学意义(P<0.01,P<0.01);但与正常组比较无统计学意义(P>0.05,P>0.05)。
3.4虚寒证的生物标志物与生殖激素的相关性分析
经多元方差分析后,将虚寒证血清中与虚热证和正常组比较均有变化的代谢物(乳酸、柠檬酸、琥珀酸、不饱和脂类、VLDL、乙酰乙酸、谷氨酸、1.79ppm处的未知化合物)分别与虚寒证机体中有变化的生殖激素FSH、LH、E2、T进行相关分析,结果乳酸与FSH、LH呈负相关(r=-0.839,P<0.05;r=-0.865,P<0.05),而与E2、T无相关性;柠檬酸与FSH、LH呈正相关(r=0.826,P<0.05;r=0.812,P<0.05),琥珀酸与FSH、LH也呈正相关(r=0.889,P<0.05;r=0.833,P<0.05),但二者与E2、T均无相关性。不饱和脂类、VLDL、乙酰乙酸、谷氨酸、1.79ppm处的未知化合物与FSH、LH、E2、T均无相关性。
结论:
本课题采用文献研究、临床流行病学调查及代谢组学的方法,对虚寒证展开了初步系统的研究,所得结论如下:
1运用文献研究的方法,探讨了虚寒证的病因和临床表现特点,从整体上把握了虚寒证的研究规律和发展方向,为下一步的研究奠定理论和文献基础。
2本课题临床流行病学调查发现虚寒证的患病率为9.946%;统计分析表明虚寒证的发生与性别、年龄、学历、职业、居住地等人口学特征有关;房劳多产是虚寒证发生的主要影响因素,其余依次是虚寒体质、感受寒邪、饮食失宜、情志所伤、过度劳累。据此可以有针对性地预防虚寒证的发生。
3虚寒证以肾阳虚证、脾肾阳虚证、心肾阳虚证、脾阳虚证、心脾阳虚证、肺肾阳虚证、心阳虚证、肺阳虚证、肝脾阳虚证九个证型为常见证型。
4经文献研究,结合专家意见以及流调结果的统计分析,确定了每种证型的主要辨证指标,畏寒、四肢不温是虚寒证的特征性症状,为虚寒证诊断标准的确立提供参考。
5通过本次临床流行病学调查,获得了中医证候的大样本临床流行病学调查的方法、质量控制及数据处理等具体经验,对中医证候学研究进行了有意义的探索。
6糖、脂肪、蛋白质代谢障碍,导致能量产生不足可能是虚寒证发生的重要原因。初步研究认为虚寒证机体代谢产物中的乳酸、柠檬酸、琥珀酸、不饱和脂类、VLDL、乙酰乙酸、谷氨酸、1.79ppm处的未知化合物可以作为虚寒证的生物标志物,提示这些生物标志物可能会作为深入研究虚寒证本质的切入点。
7虚寒证机体可能是因为糖代谢异常,脑功能受损,影响垂体、下丘脑或更高中枢的功能,使体内生殖激素分泌下降,而出现月经病。
8代谢组学分析是一种有良好发展前景的中医证候学研究方法,有助于更全面地认识机体的代谢物质组成变化,以揭示中医证候的实质。
Objective:
1 Through the literature research, we wanted to summarize the cognition of ancient and modern researcher in etiological factor, type of syndrome and symptoms of asthenia cold syndrome, analyze the essence of asthenia cold syndrome. Thereby, we hoped to reveal the research’s status quo of etiological factor, type of syndrome, symptoms and essence of asthenia cold syndrome. At the same time, we planned to combine with opinions of experts for formulating suitable clinical epidemiology investigational questionnaire of asthenia cold syndrome. And offered theoretical and literary foundation for the next study of etiological factor and symptomatic rule of asthenia cold syndrome.
2 Through the clinical epidemiology investigating, we planned to get the case rate of asthenia cold syndrome by statistics and analyze the characteristic and rule of etiological factor, type of syndrome and symptoms of asthenia cold syndrome. Accordingly, we could understand the condition of what asthenia cold syndrome had taken place and look for its main Risk factor, as well as approach the distributive condition and the main indexes of differentiation of symptoms and signs of its type of syndrome. Moreover, we wanted to offer an example for the operational method of large sample clinical epidemiology research in symptoms of traditional Chinese medicine.
3 We decided that we used the technique of nuclear magnetic resonance to approach changes of metabolin of asthenia cold syndrome and explain biological essence of asthenia cold syndrome. Then we hoped to explore the application of metabonomics’technology in the study of symptomatic essence.
Method:
1 The literature research
Using yiji dictionary of china as retrieval instrument, we retrieved all of ancient times literature from Neijing term to Qing dynasty end and to arrange and analyze them; At the same time,we used china periodical full-text data base(library CNKI retrieval page of mdition university of heibei ) to retrieve modern literature which related with asthenia cold syndrome from 1996 to 2006, then carried out frequency analysis on symptoms and arranged them.
2 The research of symptomatic rule of asthenia cold syndrome
On the literature’s research and expert’s consulation foundation, we formulated the investigational questionnaire of asthenia cold syndrome. After through the experts of Chinese traditional medicine, measuring scale, statistics, sociology demonstrasting, we beforehand investigate with investigational questionnaire. After amending, we appraised its reliability and validity by investigated again. Eventually, there formed a suitable investigational questionnaire of syndrome and etiology of asthenia cold syndrome, which possessed the qualitative of measuring scale. Used investigational questionnaire above, we adopted random cluster sampling, selected valid researcher who had been trained, then let them to survey 2500 inhabitant, who lived in shijiazhuang and its surrounding area in long-term. After these investigation questionnaires were reclaimed, they inspected and diagnosed by experts again and again. Among them, investigational questionnaires, which fit requirement, were carried out data record. Then we excavated and analyzed these data by multielement statistical analysis(cluster analysis, factor analysis, principal component analysis, logistic regression analysis) and theory of traditional Chinese medicine.
3 The metabonomics’study
We selected typical patients of asthenia cold syndrome menopathy as subjects, and selected typical patients of asthenia pyrosyndrome menopathy and health female as contrast. Every subjects took venous blood 5ml on an empty stomach at the second day of menstrual period. After centrifuging these venous blood, we took clear supernatant liquid 2ml and preserved it in - 70℃low temperature box, waiting for detection. We utilized 600MHz nuclear magnetic resonance spectrometer to detect serum metabolin composition. Then we adopted dalte software(http: //qobrue. usc. es) to get out data from subjects’serum 1HNMR spectra according toδ0.04 interval fromδ0 toδ6 segmentation. Except forδ4.669 which was water, we integrated others. After the integral of peak area had been carried out normalization, experts identified metabolin from every segment on the basis of literatures. Then according to location of peak and it representing metabolin’s integrity, we gained 24 metabolin(including 2 unkown metabolin)and 1segment of glucose and amino acids. Through added up different segment of each metabolin, we got 25 score. The scores were Stored ExceL document, waiting statistical analysis.The remain serum was used to detect FSH,LH,E2,T and P by radio immunoassay.
Result:
1 The literature research
1.1 The result of literature research shew that the cognition of etiological factor of asthenia cold syndrome mainly accord with ancient literature now; The differentiation of symptoms and signs of asthenia cold syndrome had not unified specification,and the cognition still wasn’t unification; Forthcoming various kinds differentiation of symptoms and signs methods had directional significance. But these methods mostly set up on clinical report and individual empirical foundation, but were short of research of syndromes; On the clinic and scientific research, specialists thought highly of essence of syndromes but neglected content of syndromes themself.
1.2 Through frequency statistics about symptoms of asthenia cold syndrome which had been described by modern literatures, the result shew that limbs cold, chilly, loose stool, lassitude had important significance possiblly.
1.3 The result of literature research discovered that the essential study of asthenia cold syndrome had been shifting from the study of sing index to more indexs. But new indexs that were emploied the essential study of asthenia cold syndrome were few, so this means that new ideas was lack. Nowadays, the specificity and sensitivity index of asthenia cold syndrome don’t appear.
2 The research of symptomatic rule of asthenia cold syndrome
2.1 According to the results of literature research, experts opinion and beforehand investigating, we studied and formulated the clinical epidemiological investigational questionnaire of syndromes of asthenia cold syndrome.The results of clinical epidemiological investigation and analysis of data indicated that this questionnaire had better reliability and validity. And it also had some widespread applicability.
2.2 The results of clinical epidemiological investigation signed that the case rate of asthenia cold syndrome in crowd was about 9.946%.
2.3 The statistics results shew that the occurrence of asthenia cold syndrome related with such demography characteristic as sex, age, educational background,etc. there had statistical significance(all P<0.01); Among them, fang lao and copiousness was the main risk factor, OR=27.926,which was followed by asthenia cold constitution, reception chill, improper diet, sentiment injury, excessus exertion.
2.4 According to the results of cluster analysis and factor analysis of variance, frequent type of syndromes of asthenia cold syndrome were deficiency of kidney-yang(18.828%),deficiency of spleen-yang and kidney-yang(16.318%), deficiency of heart-yang and kidney-yang(14.644%), deficiency of spleen-yang(10.041%), deficiency of heart-yang and spleen-yang(10.041%), deficiency of lung-yang and kidney-yang(8.787%), deficiency of heart-yang(6.276%),deficiency of lung-yang(5.021%), deficiency of liver-yang and spleen-yang(4.184%). analytic result also shew that main two viscus pathopoiesis together is more, occuping 56.904%; sing viscera pathopoiesis occupied 40.167%; Among them, the proportion of deficiency of kidney-yang in asthenia cold syndrome was larger. Deficiency of liver-yang appeared with other viscus together.
2.5 Through principal component analysis and logistic stepwise regression analysis, we got cardinal symptoms which had statistics significance in diagnosis of each type of syndrome of asthenia cold syndrome. They were as follows(The main symptoms arranged according to OR value ):
Deficiency of spleen-yang: Main symptoms were abdominal distention, loose stool, lassitude, chilly; Secondary symptoms were limbs cold, anorexia, mind weary, aggravation with cold, relief with warm and pressure, pale and enlarged tongue.
Deficiency of liver-yang and spleen-yang: Main symptoms were aggravation with cold, anorexia, bilateral hypochondria full, nauseated, eyes blurred; Secondary symptoms were chilly, dizzy, limbs cold, bilateral hypochondria vague pain, hot drink preference, relief with warm and pressure.
Deficiency of heart-yang: Main symptoms were hot drink preference, palmus, weakness pulse, zhengzhong; Secondary Symptoms were chilly, limbs cold, short breath, spontaneous perspiration, darkness oral lip, edema of lower limbs, aggravation with cold.
Deficiency of lung-yang: Main symptoms were catching a cold readily, cough, whiteness phlegm, aggravation with exhaustoin; Secondary symptoms were chilly, short breath, limbs cold, light body of the tongue.
Deficiency of heart-yang and spleen-yang: Main symptoms were deep and delay pulse, palmus, loose stool, abdominal distention, chest depressed;Secondary symptoms were chilly, limbs cold, relief with warm and pressure, head and face puffiness, thinness and whiteness moss.
Deficiency of lung-yang and kidney-yang: Main symptoms were acid and weak in waist and knee, cough, whiteness body of the tongue, catching a cold readily, oliguresis; Secondary symptoms were chilly, limbs cold, whiteness phlegm, mind weary, whiteness and greasiness moss.
Deficiency of kidney-yang: Main symptoms were urinary frequency, acid and weak in waist and knee, huangbai complexion, whiteness moss; Secondary symptoms were limbs cold, urinary incontinence, multi- nycturia, urine clear, pale and enlarged tongue with indentation, extenuate and weakness pulses or slow and weakness pulses.
Deficiency of heart-yang and kidney-yang: Main symptoms were palmus, oliguresis, acid and weak in waist and knee, aggravation with cold, chest pain; Secondary symptoms were chilly, limbs cold, lassitude, edema of lower limbs,droping after urine, extenuate and weakness pulses or deep and weakness pulses.
Deficiency of spleen-yang and kidney-yang:Main symptoms were loose stool, huangbai complexion, urinary frequency, acid and weak in waist and knee, gastric cavity vague pain; Secondary symptoms were wangubuhua, limbs cold,lassitude, chilly, hot drink preference,deep and delay puleses.
2.6 Synthesizeing the result of statistics and the opinion of experts, we considered chilly, limbs cold as characteristic symptoms of asthenia cold syndrome.
3 The metabonomics’research of asthenia cold syndrome
3.1 Compared with normal group, there were conspicuous changes in 3.2～3.96ppm, 4.0 ppm, 1.22 ppm, 2.23ppm,2.41 ppm, 2.57 ppmpeak forms of the serum 1HNMR spectra in asthenia cold syndrome group and asthenia pyrosyndrome group .
3.2 Through multielement ANOVA: compared with asthenia pyrosyndrome group and normal group, there were obviously low in the contents of serumα-glucose(5.12ppm),β-glucose(3.14ppm,4.56ppm), RegionA(3.2～3.96ppm)(glucose and amino acids), citrate(2.57ppm), succinate(2.23ppm), unsaturated lipid (5.18ppm, 2.63ppm, 2.34ppm, 1.89ppm, 1.62ppm, 1.45 ppm), very low density lipoprotein(1.16ppm), glutamine(2.41ppm), unkown compound(1.79ppm) of asthenia cold syndrome group. there were statistical significance; The contents of serum acetoacetate(2.11ppm), lactate(1.22ppm,4.0ppm) were obviously high, there were statistical significance, too.( P<0.05,P<0.01;P<0.01,P<0.01).In addition,compared with normal group,the contents of valine(0.92ppm), leucine(0.88ppm) and low density lipoprotein(0.75ppm) of asthenia cold syndrome cut down,there were statistical significance (P < 0.05,P < 0.05,P < 0.05),but compared with asthenia pyrosyndrome,there weren’t statistical significance(P >0.05,P >0.05,P>0.05).
Compared with asthenia cold syndrome and normal group,the integral of oppsing area of serum RegionA and lysine (2.84ppm) of asthenia pyrosyndrome obviously heightened.There were statistical significance(P<0.01,P<0.05; P<0.05,P<0.01).
3.3 The changes of genesis hormone of asthenia cold syndrome,asthenia pyrosyndrome and normal group
Compared with asthenia pyrosyndrome group and normal group, there were obviously low in the contents of serum FSH,LH,E2 and T of asthenia cold syndrome,there were statistical significance (P<0.01,P<0.01;P<0.01,P<0.01;P<0.01,P<0.01;P<0.01,P<0.05);The content of serum P of asthenia cold syndrome was lower than asthenia pyrosyndrome group,there was statistical significance(P<0.01),but compared with normal group,there wasn’t statistical significance(P>0.05).
Compared with asthenia cold syndrome and normal group, the contents of serum LH,T and P of asthenia pyrosyndrome group heightened,there were statistical significance(P < 0.01,P < 0.05;P < 0.01,P < 0.01;P < 0.01,P <0.01);The contents of serum E2 and FSH of asthenia pyrosyndrome group were higher than asthenia cold syndrome,there were statistical significance (P<0.01,P<0.01),but compared with normal group,there weren’t statistical significance(P>0.05,P>0.05).
3.4 The dependability study of between biomarkers and hormone
After analysis of variance,we carried out correlation analysis between all metabolin which had changes in comparision of asthenia pyrosyndrome group, asthenia cold syndrome and normal group and hormone which had changes in them.The results were that there were negative correlation between lactate and FSH and between lactate and LH respectively(r=-0.839,P<0.05;r=-0.865,P<0.05),but there weren’t dependablity between lactate and E2 and between lactate and T. There were positive correlation between citrate and FSH and between citrate and LH(r=0.826,P<0.05;r=0.812,P<0.05).There were also positive correlation between succinate and FSH and between succinate and LH(r=0.889,P<0.05;r=0.833,P<0.05),but the two hadn’t dependability with E2 and T. Unsaturated lipid,VLDL, acetoacetate, glutamine and 1.79ppm unkown compound hadn’t dependability with FSH,LH,E2 and T.
conclusion:
Through adopted methods of literature’s research, clinical epidemiology investigation and metabonomics, we carried out initially systematical research on asthenia cold syndrome. the conclusions were as follows:
1 Used the method of literature research, we approached etiological factor and clinical manifestation feature of asthenia cold syndrome, and held its regular pattern of research and developed direction in the whole. This established theoretical and literary foundation for the next study.
2 The clinical epidemiology investigation found that case rate of asthenia cold syndrome was about 9.946%; The results of statistical analysis shew that occurrence of asthenia cold syndrome related with such demography feature as sex, age, educational background, vocation, habitation,ect. Fang lao and copiousness was main influential factor of asthenia cold syndrome, which were followed by asthenia cold contitution, reception chill, improper diet, sentiment injury,excessive exertion. On these grounds, we could preclude the occurrence of asthenia cold syndrome directly.
3 The frequent type of syndromes in asthenia cold syndrome were deficiency of kidney-yang,deficiency of spleen-yang and kidney-yang, deficiency of heart-yang and kidney-yang, deficiency of spleen-yang, deficiency of heart-yang and spleen-yang, deficiency of lung-yang and kidney-yang, deficiency of heart-yang, deficiency of lung-yang, deficiency of liver-yang and spleen- yang.
4 Through literature research, combining opinion of experts and epidemiological investigation,we determined main indexs of differentiation of symptoms and signs of each type of syndrome. Chilly and limbs cold were characteristic symptom of asthenia cold syndrome. This offered information for establishing diagnostic criteria of asthenia cold syndrome.
5 Through clinical epidemiology investigation, we obtained methods of investigation, quality control and data processing of large sample clinical epidemiology. We carried out significant exploration on the research of syndromes of traditional Chinese medicine.
6 The dysmetabolism of suger, fat and protein resulted in energy shortage. This was main cause which led to occurrence of asthenia cold syndrome possiblly. We tentatively confirmed that lactate, citrate, succinate, unsaturated lipid, VLDL, acetoacetate, glutamine, 1.79ppm unkown compound could be thought of as biomarkers of asthenia cold syndrome.
7 The body of asthenia cold syndrome might be that glycometabolism was blocked, brain’s function was damaged.This may influence pituitary and hypothalamus or highest center and make hormone of body low, so shew menstrual disorder.
8 Metabonomics analysis was a well developed outlook method which studied syndromes of traditional Chinese medicine. It conduced more overall to understand chemical materials changes of organism, to reveal essence of syndromes of traditional Chinese medicine.
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