CN107209173A - 用于确定患者的独特的营养需求的方法 - Google Patents
用于确定患者的独特的营养需求的方法 Download PDFInfo
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- CN107209173A CN107209173A CN201680006639.8A CN201680006639A CN107209173A CN 107209173 A CN107209173 A CN 107209173A CN 201680006639 A CN201680006639 A CN 201680006639A CN 107209173 A CN107209173 A CN 107209173A
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Abstract
本发明涉及用于确定具有特定营养需求的患者的独特的营养需求并提供满足所述患者的独特的营养需求的组合物的方法。
Description
技术领域
本发明涉及用于确定具有特定营养需求的患者的独特的营养需求并提供满足所述患者的独特的营养需求的组合物的方法。
背景技术
多种参数或化合物限定受检者的营养状态。例如,营养物质、微量营养素和其他化合物以一定浓度存在于受检者的流体或组织中。许多疾病改变这些化合物的浓度或这些参数的值,这是因为抵抗疾病、代谢变化和/或患者的次优膳食管理而导致对这些化合物的利用率增加。结果,患有这种疾病的受检者营养不良,这是因为相关参数和化合物不再处于健康受检者的范围内并导致营养缺乏,如结构组分的不正确提供、能量供应不足、或缺乏功能组分。患有影响营养状态的疾病的受检者可受益于解决所述受检者的独特的营养需求的营养干预。因此,将需要为受检者提供包含营养物质和微量营养素的营养组合物,营养物质和微量营养素的量将重新建立等同于健康受检者的营养状态的代谢、生理和功能。
其中受检者表现出独特的营养需求的此类疾病的具体示例是炎性肠病(IBD)。
营养在IBD中的作用引起了人们的高度兴趣,特别是在小儿克罗恩氏病(CD)中,研究表明,全肠内营养(EEN)可诱导与皮质类固醇相当的轻度至中度疾病的缓解。因此,为实现安全的长期疾病管理,除护理标准(SoC)之外提供的营养干预是很有吸引力的选择。营养不良在小儿和成人IBD患者中十分常见,尤其是患有CD的那些患者,并且通常表现为会造成大体的体重减轻的蛋白质-能量缺乏,和/或维生素/矿物质缺乏。一般来说,继发于餐后腹痛和腹泻的膳食摄入不良是IBD的营养不良的最常见的原因。营养不良的程度取决于疾病的持续时间、严重程度和扩展,以及因肠切除或纤维化引起的功能丧失。据报道,IBD患者会出现脂肪和肌肉质量消耗;微量营养素缺乏也会在轻度疾病或缓解期中发生。
除了与营养不良相关的营养缺乏之外,营养也被认为是维持缓解期、特别是维持粘膜健康的有效方法。已提出,形成粘液凝胶和保护肠上皮的肠粘蛋白对于在IBD的粘膜损伤之后恢复上皮健康至关重要。维持足够的粘蛋白合成的身体能力与一些特定氨基酸的生物利用率直接相关。已知肠炎可增加肠内喂食的小型猪的胃肠道苏氨酸摄入和粘蛋白合成。因此,在如同IBD的炎症状态下,特定氨基酸可成为维持粘蛋白合成所需的条件性必需的氨基酸,从而用于营养特异性富集。
因此,需要用于识别患有一种或多种疾病或临床症状的患者的独特的膳食需求的方法,该患者的营养状态与健康受检者的营养状态不同。
发明内容
本发明的目的是提供用于确定营养状态与健康受检者的营养状态不同的患者的独特膳食需求的新方法。
本发明涉及用于通过以下方式来确定患有疾病的受检者的独特的疾病相关营养需求的体外方法:a)首先,在患有疾病的受检者的样本中确定标记物(包括营养物质、微量营养素和/或其代谢物,和/或生物标记物,或其任何组合)的状态的概况,其也被称为“营养概况”;b)其次,在来自健康受检者或来自具有不同严重程度或不同阶段的疾病的患者的样本中确定在步骤a)中所确定的相同标记物(例如,相同的营养物质、微量营养素和/或其代谢物、和/或生物标记物)的状态的类似营养概况,以及c)第三,对在步骤a)和b)中所确定的营养概况进行比较,并且从而确定患有疾病的患者的独特的营养需求。
该方法使得有可能确定患有特定疾病的患者以及患有不同严重程度或不同阶段的相同疾病的患者的营养概况和独特的营养需求。基于这些所识别的营养需求,可制备营养组合物,该营养组合物包含能够将患有疾病的患者的营养概况朝健康受检者的营养概况恢复或改善的营养物质和微量营养素。
本发明提供了包含营养物质和微量营养素的营养组合物,该营养物质和微量营养素的量能够在患有疾病的患者中重建健康受检者的营养概况或具有改善状态(即,较低的疾病严重程度)的患者的营养概况。
附图说明
图1:示出用于限定独特的营养需求的方法途径的图示。使用营养概况或营养状态来量化与特定疾病相关联的独特的营养/营养物质需求。营养概况指示总体营养状态并通过在患者和健康人群中测得的一系列营养物质和微量营养素值和/或其相关的状态标记物来进行测定。营养概况可与涉及疾病状态(复发、缓解、严重程度)的临床信息(包括临床标记物)相关。在患者组和健康组之间和/或在针对疾病活动度、严重程度或阶段定义的患者组中比较营养概况。如果在患者和健康对照之间观察到营养概况的差异,则将确定独特的营养/营养物质需求(DNR)。在图1中,“NP”是指营养概况;“Da”是指高疾病活动度或严重程度或疾病晚期阶段;“Db”是指低疾病活动度或严重程度或疾病早期疾病;“Dc”是指无疾病或健康状态;“≠”是指营养概况的差异;并且“NP(Da)”、“NP(Db)”、“NPDc”分别是指Da、Db和Dc的营养概况。
图2:以图形方式示出将DNR用于产品的营养组合物的图示,该产品的目的是适当地恢复营养水平,以便利用经临床验证的功效来缓解患者的症状、持续缓解并改善患者的生活质量。在图1中,“NP”是指营养概况;“Da”是指高疾病活动度或严重程度或疾病晚期阶段;“Db”是指低疾病活动度或严重程度或疾病早期疾病;“Dc”是指无疾病或健康状态;“≠”是指营养概况的差异;并且“NP(Da)”、“NP(Db)”、“NPDc”分别是指Da、Db和Dc的营养概况。
图3:苏氨酸和异亮氨酸代谢产物
图4:通过对苏氨酸和其他氨基酸的氧化指数的比较分析来确定IBD中的特定于苏氨酸的需求
定义
“营养状态”涉及个人或人群组(群集)的可量化的身体状态。营养状态与他们的营养品状态(营养物质的食用和利用)相关。在本发明中,使用指示所述营养状态的标记物,特别是在受检者的样本中确定的生物学标记物、生物化学标记物、生理学标记物或其他标记物来量化营养状态。
“营养概况”涉及要在样本(生物流体,例如血红细胞、血浆、血清、尿液、组织等等)中确定的营养物质和微量营养素或其相关的状态标记物的一组定量测定,并且因此需要确定对若干种、至少两种营养物质、微量营养素或相关的状态标记物的测定。
“标记物”是表示一系列或一组标记物中的一个参数/标记物的可量化的参数。所述标记物的定量指示所述标记物的状态。该参数可与诸如营养物质(例如蛋白质、氨基酸等)或微量营养素(维生素、包括矿物质等的元素)的某种化合物的量直接相关。然而,标记物也可与从样本中的营养物质和微量营养素浓度和/或状态标记物的数学组合得出的值相关。标记物也可以是功能性标记物,并且尤其涉及某些生理活性(例如,酶活性)或生理状态(氧化应激状态)。
“独特的营养需求”(DNR)是在患病受检者和健康受检者中存在差异的营养需求。例如,与健康受检者的营养概况相比,患病受检者的样本中的营养概况或营养物质的概况的定量分析可能不同。在此类情况下,所观察到的营养概况的差异可用于识别和定量特定于疾病的营养需求。
“游离形式的氨基酸”是指这样的氨基酸,其作为未与其他化合物(如其他氨基酸)结合的游离氨基酸而被包含在某种组合物中,并因此不被包含在肽或蛋白质中。
因此,“结合形式的氨基酸”是作为肽、蛋白质的一部分或与其他化合物结合的氨基酸。
“蛋白质氨基酸”是在天然产生的蛋白质中发现的那些氨基酸,其包括由平移机构用于产生蛋白质的那些氨基酸,以及在平移之后在蛋白质中被修饰的那些氨基酸。
“非蛋白质氨基酸”是在天然产生的蛋白质中没有发现的氨基酸,它们是细胞和生物体中的代谢物或结构组分。
具体实施方式
各部分标题起到阐明主题的作用,并且不应被理解为限制该主题。
本发明的概念示于图1中。
例如在一群组患者中,确定患有疾病的受检者(患病受检者,患者)的营养概况,并与一群组健康受检者(健康对照)的营养状态进行比较。营养概况包括确定营养物质、微量营养素和/或营养物质/微量营养素状态标记物的概况(至少两次测定)。在健康受检者和患有疾病的患者的样本中确定特定标记物的状态(所述标记物的定量)。患者的营养概况与健康受检者的营养概况的比较用于识别患病受检者和健康对照之间的营养物质水平和/或其相关的状态标记物的差异,该差异用于识别和定量患病人群的独特的营养需求。此外,相对于疾病的严重程度或阶段进行分层的患者人群内的营养概况的比较使得能够识别与较低疾病活动度或严重程度或阶段相关联的一组营养物质和微量营养素,并将此与患者的改善的临床症状相关联。
在下一个步骤中,可能配制将相应的独特的营养需求考虑在内的营养组合物(产品)。此外,根据疾病的严重程度或阶段进行分层的患病患者人群内的营养概况的差异也用于确定旨在将具有较高疾病活动度或严重程度的患者的营养概况恢复到具有较低疾病活动度或严重程度的患者的营养概况值的营养组合物(产品)。
例如,在已观察到相对于健康受检者中的苏氨酸和/或其一种或多种氧化代谢物的值,氨基酸苏氨酸的血液浓度降低和/或其一种或多种氧化代谢物(图3)的水平发生变化的患者中,营养组合物将包含蛋白质、肽,以便调节苏氨酸的膳食供应或游离氨基酸苏氨酸本身(图2)。所述组合物将被施用于患者,目的是使得患者能够满足其对苏氨酸的特定需求。这样便可满足患病受检者对苏氨酸的增加的需求并纠正相对于苏氨酸的最终缺陷。
一些疾病表现出不同的严重程度,伴随着不同的营养需求。在此类情况下,待确定营养状态的一群组患者将是显示出相同或相似的疾病严重程度的一群组患者。以这种方式,可能识别具有特定疾病严重程度的患者的独特的营养需求,从而提供适于该严重程度的产品。现在将更详细地描述该方法。
本发明涉及用于确定患病受检者的独特的营养需求的方法:
本发明涉及用于确定患有疾病的受检者的独特的疾病相关的营养需求的体外方法,该疾病的特征在于所述患病受检者相比于健康受检者的独特的营养需求,该方法包括以下步骤:
a.在患有疾病的患者的样本中确定标记物(包括营养物质、微量营养素和/或其代谢物和/或生物标记物)的状态的概况,该标记物的状态的概况指示所述患者的营养概况,
b.在健康受检者的样本中确定在步骤a中所确定的相同标记物的状态的概况,该标记物的状态的概况指示健康受检者的营养概况,
c.对在步骤a和b中所确定的概况进行比较,从而确定患有疾病的患者对营养物质的独特的营养需求。
可确定(定量)各种营养物质、微量营养素及其状态标记物的相关代谢物。对于每种标记物,确定其状态,即所述标记物的存在或其值。代谢相互关联的营养物质、微量营养素或相关标记物可分组,以用于进行综合分析和解读。
根据所确定的营养概况,可识别患病患者中的某些营养物质和微量营养素的缺乏。对这些缺陷的识别将允许在营养组合物中提供一组足够的营养物质或微量营养素,其水平经适当地调整以补偿这些缺陷。将这组营养物质施用于患病受检者可具有将患病受检者的营养概况恢复到健康受检者的营养概况的作用。
指示营养物质的营养状态的标记物可为直接标记物或间接标记物。直接标记物指示例如营养物质或微量营养素的量。间接标记物可从直接确定的标记物得到(例如,通过它们的组合),或者可以是营养物质或微量营养素的代谢物或分解代谢物,和/或可以是营养物质或微量营养素的生物标记物或分解代谢物;或者可与身体中的特定生理状态相关。
间接标记物可涉及确定营养物质和微量营养素的相对状态,即特定营养物质与其他营养物质的比例、特定营养物质与微量营养素的比例、特定微量营养素与特定微量营养素的比例、或上述的组合。
此外,间接标记物可涉及确定患病受检者的功能性标记物。功能性标记物是与指示受检者的健康状态的生理或生物化学参数的状态相关的标记物。例如,功能性标记物可以是测定,诸如红细胞转酮醇酶活性、红细胞谷胱甘肽还原酶活性、氧化应激状态或一氧化氮合酶活性。这些功能性标记物在健康受检者中显示出特定(参比)状态。在患有疾病的患者中,这些标记物可能具有不同的值,因此指示相对于健康受检者的不适当的营养物或微量营养素状态。为患病受检者适当地提供营养物质和微量营养素的合适的一组和合适水平的营养组合物可用于将这些功能性标记物的水平朝在健康受检者中测得的水平恢复。
样品:
对来自受检者的样本执行对营养概况的确定。
样本可以是选自全血、血浆、血清、红细胞、尿液或组织切片的样本。根据确定方法,从同一受检者获得不同的样本。也可使用来自同一受检者的样本的组合。还可设想,在第一时间点从受检者获得一个样本或一组样本,并且在第二时间点或另外的时间点获得另外的一个样本或一组样本。以这种方式,可能测定受检者的营养概况在一段时间内的变化。
根据后续分析的技术要求来对样本进行处理。
例如,生物样本可在稀释之后直接注入到电感联接的质谱仪中,以进行矿物质和微量元素分析(元素分析,包括矿物质分析)。在注入在联接至火焰离子化检测器的气液色谱系统上之前,它们也可经历蛋白质沉淀、提取、净化、衍生化等各种步骤,以用于进行脂肪酸分析,或注入在联接至质谱的高压液相色谱系统上,以进行氨基酸或水溶性维生素分析。生物样本还可被处理,以经历比色、荧光或免疫测定分析。
待确定的营养概况和标记物:
营养概况涵盖直接标记物和间接标记物。标记物可以是生物化学标记物、生物学标记物或功能性标记物、或它们的组合。这些标记物与受检者的营养状态相关,并且因此指示其相对于营养物质的营养需求。这些标记物可受到受检者所消耗的营养物质的影响。这些标记物可通过下文所述的各种方法来确定。
这些标记物的定量分析指示对营养物质的营养需求。营养物质可以是大量营养素和微量营养素。大量营养素可以是蛋白质、肽、氨基酸、脂肪、脂肪酸、碳水化合物、或胆碱。微量营养素可以是矿物质、维生素、类胡萝卜素、植物营养素。营养物质还可根据其不依赖于结构相似性的功能(功能性营养物质)而被分组。例如,抗氧化剂不依赖于其结构来提供抗氧化作用。抗氧化剂可以是维生素、矿物质、氨基酸或肽。
可确定至少10、25、50、100、250个标记物的状态。对于待确定的标记物的最大数量没有特别要求,但上限可能为25、50、100、250或1000个标记物。
蛋白质标记物:
蛋白质标记物可通过蛋白质状态的标记物或蛋白质分解代谢的标记物来进行确定,并且因此可确定对提供蛋白质或氨基酸的营养物质或者特定蛋白质和氨基酸的营养需求。蛋白质状态标记物可以选自白蛋白、前白蛋白或/和磷酸肌酸、以及它们的组合。分解代谢标记物选自氨、尿素、经修饰的氨基酸(一甲基和二甲基精氨酸)、或它们的组合。
氨基酸标记物:
氨基酸标记物可通过确定样本中氨基酸或其衍生物(包括其代谢物/分解代谢物)的数量而被确定,以作为对其状态的指示,从而可确定对提供蛋白质或氨基酸的营养物质或者特定蛋白质和氨基酸的营养需求。
氨基酸标记物选自丙氨酸、β-丙氨酸、肌氨酸、精氨酸、一甲基精氨酸、不对称二甲基精氨酸、对称二甲基精氨酸、天冬酰胺、天冬氨酸、瓜氨酸、谷氨酸、谷氨酰胺、甘氨酸、组氨酸、1-甲基组氨酸、3-甲基组氨酸、异亮氨酸、亮氨酸、赖氨酸、甲硫氨酸、鸟氨酸、苯丙氨酸、脯氨酸、丝氨酸、牛磺酸、苏氨酸、色氨酸、酪氨酸、缬氨酸、羟脯氨酸、乙醇胺、α-氨基丁酸、β-氨基异丁酸、γ-氨基丁酸、同型半胱氨酸、半胱氨酸、γ-谷氨酰-半胱氨酸、半胱氨酰-甘氨酸、同型胱氨酸、半胱氨酸、胱硫醚、甲硫氨酸亚砜、硒代甲硫氨酸、甲硫氨酸亚砜亚胺、硒代半胱氨酸、硒代胱氨酸、麦角硫因、N-甲酰基-L-甲硫氨酸、S-腺苷高半胱氨酸、S-腺苷基甲硫氨酸胺、α-酮丁酸、2-氨基丁酸、2-氨基-3-酮丁酸、α-酮基-β-甲基丁酸(或α-酮异戊酸)、α-酮异己酸或α-酮基-β-甲基戊酸。
根据一个优选的实施方案,确定苏氨酸、丝氨酸或脯氨酸的状态,优选地确定苏氨酸的状态,并且因此确定对它们的需求。
优选地,苏氨酸的状态的指示物是苏氨酸和/或苏氨酸的一种或多种分解代谢物。苏氨酸的分解代谢物选自丙酸、2-氨基丁酸、2-酮丁酸、2-氨基-3-酮丁酸、氨基丙酮、乙酰CoA、甘氨酸、或它们的组合(图3)。
优选地,异亮氨酸的状态指示物是异亮氨酸或异亮氨酸的分解代谢物。待确定的异亮氨酸分解代谢物优选地为2-酮基-3-甲基戊酸(图3)。
脂肪酸标记物:
脂肪酸标记物可通过脂肪酸的定量分析而被确定,以指示其相关状态,从而可确定对提供脂肪的营养物质、磷脂(磷脂酰胆碱、磷脂酰丝氨酸、磷脂酰乙醇胺等)或特定脂肪酸的营养需求。
脂肪酸标记物选自丁酸C4:0、己酸C6:0、辛酸C8:0、癸酸C10:0、十一烷酸C11:0、月桂酸C12:0、十三烷酸C13:0、肉豆蔻酸C14:0、十五烷酸C15:0、棕榈酸C16:0、十七烷酸C17:0、硬脂酸C18:0、花生酸C20:0、二十一烷酸C21:0、山嵛酸C22:0、二十四烷酸C24:0、肉豆蔻脑酸C14:1n-5、顺-10-十五碳烯酸C15:1n-5、棕榈油酸C16:1n-7、顺-10-十七碳烯酸C17:1n-7、C18:1n-9反式反油酸、C18:1n-9顺式油酸、顺-11-二十碳烯酸C20:1n-9、芥酸C22:1n-9、神经酸C24:1n-9、C18:2n-6反式亚油酸、C18:2n-6顺式亚麻酸、γ-亚麻酸C18:3n-6、α-亚麻酸C18:3n-3、顺-11,14-二十碳二烯酸C20:2n-6、顺-8,11,14-二十碳三烯酸C20:3n-6、顺-11,14,17-二十碳三烯酸20:3n-3、花生四烯酸C20:4n-6、顺-13,16-二十二碳二烯酸22:2n-6、顺-5,8,11,14,17-二十碳五烯酸(EPA)C20:5n-3、顺-4,7,10,13,16,19-二十二碳六烯酸(DHA)C22:6n-3或硫辛酸。
元素标记物(包括矿物质标记物):
样本中的直接元素(包括矿物质)标记物通过元素分析而被测定和/或使用其相关联的蛋白质或代谢物(例如用于确定铁状态的铁蛋白、用于确定铜状态的血浆铜蓝蛋白等)的定量而被测定。
元素标记物(包括矿物质标记物)可选自锂(Li)、硼(B)、镁(Mg)、铝(Al)、硅(Si)、磷(P)、硫(S)、钾(K)、钙(Ca)、钒(V)、铬(Cr)、锰(Mn)、铁(Fe)、钴(Co)、镍(Ni)、铜(Cu)、锌(Zn)、砷(As)、硒(Se)、溴(Br)、铷(Rb)、锶(Sr)、钼(Mo)、锡(Sn)、碘(I)、钡(Ba)、钛(Ti)、钠(Na)、氯(Cl)、氟(F)。
间接元件标记物(包括矿物质标记物)包含一个或多个元素和/或其相关联的蛋白质或代谢物的组合。
直接标记物和间接标记物可用于确定对元素(包括矿物质)的膳食需求。
维生素标记物:
维生素标记物可以是直接标记物或间接标记物。直接标记物包括对样本中的维生素和/或其代谢产物的定量。间接标记物包括一种或多种维生素和/或其代谢产物以及指示维生素的状态的功能性标记物的组合,诸如分别用于确定维生素B1(硫胺素)和维生素B2(核黄素)的状态的红细胞转酮醇酶活性、红细胞谷胱甘肽还原酶活性。
维生素标记物可用于确定对维生素的膳食需求。维生素标记物可选自水溶性维生素或脂溶性维生素。
水溶性维生素及其代谢物可选自维生素B1(硫胺素)、维生素B2(核黄素)、维生素B3(尼克酸或烟酸)、烟酰胺、甲基烟酰胺、烟尿酸、胆碱、维生素B5(泛酸)、维生素B6(吡哆醇、吡哆醛、吡哆胺)、磷酸吡哆醛、吡哆酸、维生素B8(生物素)、维生素B9(叶酸)、甲基四氢叶酸、维生素B12(氰钴胺素、甲基钴胺素)、羟基钴胺素、腺苷钴胺素。
脂溶性维生素可选自维生素A(视黄醇)、维生素K2(甲萘醌)、维生素K1(叶绿醌)、维生素E(α-生育酚、δ-生育酚)、维生素D(25-OH维生素D2、维生素D 25-OH D3、维生素D2、维生素D3、1α-25-(OH)2维生素D3)。
核苷酸标记物:
可确定核苷酸标记物,从而确定对提供核苷酸的营养物质的需求。
核苷酸可选自肌苷5'-单磷酸、腺苷5'-单磷酸、胞苷5'-单磷酸、鸟苷5'-单磷酸、肌苷5'-单磷酸、尿苷5'-单磷酸、或它们的组合。
植物营养素标记物:
可确定植物营养素标记物,从而确定对提供植物营养素的营养物质的需求。
在一个实施方案中,植物营养素可选自类胡萝卜素(例如,叶黄素、玉米黄质)、鞣花酸、黄酮类(儿茶素、表儿茶素、表没食子儿茶素)、绿原酸、白藜芦醇、芥子油苷、植物雌激素(染料木黄酮、黄豆苷元等)、或它们的组合。
肽标记物:
可确定肽标记物,从而确定对提供肽的营养物质的需求。
肽可选自还原型谷胱甘肽或氧化型谷胱甘肽。
氧化应激标记物:
可确定氧化应激标记物,从而确定对改善氧化应激状态的营养物质的需求。
在一个实施方案中,氧化应激标记物可选自4-羟基壬烯醛、丙二醛、硝基酪氨酸、羰基化蛋白质、总谷胱甘肽、还原型谷胱甘肽、氧化型谷胱甘肽、谷胱甘肽过氧化物酶活性、谷胱甘肽还原酶活性、超氧化物歧化酶活性、过氧化氢酶活性、或它们的组合。
影响氧化应激状态的营养物质可以是膳食氧化剂。膳食氧化剂可以是维生素、矿物质(例如,硒)、植物化学物质、氨基酸(例如,半胱氨酸)或肽(例如,谷胱甘肽)。这些营养物质重新建立与健康受检者相似的氧化应激水平。
氮合酶活性:
可确定氮合酶活性的标记物,从而确定对影响一氧化氮合酶活性的营养物质的需求。
在一个实施方案中,氮合酶活性的标记物可选自亚硝酸盐、硝酸盐、一甲基精氨酸、不对称二甲基精氨酸、对称二甲基精氨酸、精氨酸、瓜氨酸、鸟氨酸、精氨基琥珀酸、或它们的组合。
渗压剂标记物:
可确定渗压剂的标记物,从而确定对影响渗压剂状态的营养物质的要求。这些标记物是功能性标记物。
在一个实施方案中,渗压剂标记物可选自三甲胺N-氧化物、二甲基锍丙酸盐、三甲基甘氨酸、肌氨酸、甜菜碱、甘油磷酰胆碱、肌醇、或它们的组合。
用于定量标记物的方法:
已知用于确定样本中的上述营养状态标记物的多种方法。下面将描述如何执行所述标记物的定量的各种方法的非限制性示例。除此之外或另选地,可使用本领域已知的其他合适的方法。
本发明中的受检者的营养状态涉及可指示营养物质和微量营养素的状态的直接标记物或间接标记物的状态的概况,该标记物可以是生物化学标记物、生物学标记物、功能性标记物、或其他标记物。
直接生物化学标记物可以是特定营养物质的浓度。营养物质可以是常大量营养素(例如,蛋白质和衍生化氨基酸、碳水化合物、脂质)或微量营养素(维生素和元素(包括矿物质))。
间接生物化学标记物可以是与大量营养素、微量营养素、或它们的组合相关的营养状态指示物。
营养状态指示物可以是生物标记物,其浓度指示营养物质的状态,而不是所述营养物质本身。例如,用于铁的铁蛋白,或用于维生素D的25-羟基-维生素D同效维生素。
其他营养物质状态标记物可来自于单一营养物质或其代谢物的浓度、或营养物质及其代谢物的浓度组合(比例)、或营养物质与其他生物化学标记物(例如,营养物质/微量营养素的转运蛋白)和/或生物学标记物和/或功能性标记物(例如,特异性酶活性,如转酮醇酶活性、红细胞谷胱甘肽还原酶活性)的浓度组合。
目前有多种确定直接或间接生物化学标记物的状态的方法。在下文中,我们将讨论所述标记物的各种定量方法。然而,应当理解,相应定量方法对于本发明的方法而言并非决定性的,只要能够确定对一种特定营养物质或一组特定营养物质的营养需求即可。
氨基酸氧化指示物(IAAO)方法:
例如,对必需氨基酸的特异性需求可使用氨基酸氧化指示物(IAAO)方法进行定量(Roberts SA,Thorpe JM,Ball RO,Pencharz PB.;Am J Clin Nutr.2001Feb,73(2):276-82。(Roberts SA、Thorpe JM、Ball RO、Pencharz PB.;《美国临床营养学杂志》,2001年2月,第73卷第2期第276-282页);Elango R,Ball RO,Pencharz PB.,J Nutr.2008Feb;138(2):243-6.Review(Elango R、Ball RO、Pencharz PB.;《营养学杂志》,2008年2月,第138卷第2期第243-246页,综述))。由于N平衡的局限性,已经开发了基于稳定同位素碳氧化的方法来评估人类的必需氨基酸需求(Pencharz PB,Ball RO.Different approaches to defineindividual amino acid requirements.Annu Rev Nutr.2003;23:101-16(Pencharz PB、Ball RO,用于确定各个氨基酸需求的不同方法,《营养学年评》,2003年,第23卷第101-116页))。经验证,IAAO方法可通过最小在先适应性(minimal prior adaptation)来估计氨基酸需求(Bross R,Ball RO,Pencharz PB.,J Nutr.1998Nov;128(11):1913-9(Bross R、Ball RO、Pencharz PB.,《营养学杂志》,1998年11月,第128卷第11期第1913-1919页);Thorpe JM,Roberts SA,Ball RO,Pencharz PB.,J Nutr.1999Feb;129(2):343-8(ThorpeJM、Roberts SA、Ball RO、Pencharz PB.,《营养学杂志》,1999年2月,第129卷第2期第343-348页))。IAAO技术基于以下概念:当蛋白质合成缺乏一种必需的氨基酸时,包括所谓的指示物氨基酸(通常为L-[1-13C]苯丙氨酸)的所有其他氨基酸均因为较少使用而过量,并且因此将被氧化(Pencharz和Ball,2003年)。这主要是因为过量的氨基酸不能被存储,并且因此必须在掺入蛋白质中或发生氧化之间被分配。随着限制性氨基酸的摄入增加,指示氨基酸的氧化程度将降低,从而反映出掺入蛋白质的程度增加。一旦满足对限制性氨基酸的需求,随着测试氨基酸的摄入增加,指示氨基酸的氧化程度便将不再发生变化。指示氨基酸的氧化程度停止降低并达到平稳阶段的拐点被称为“断点”。使用双相线性回归分析所识别的断点指示对限制性(测试)氨基酸的估计平均需求。IAAO模型的一个特别优势是绝对氧化水平并不重要,关键在于在宽泛的摄入量范围内的相对氧化产生相同的断点(需求估值)。此方法是一种得到广泛认可的方法,但它具有很大的局限性。事实上,这种方法仅被应用于必需的氨基酸,在每次临床研究中仅评估一种必需的氨基酸,这对于患者是侵入性的(示踪剂的动力学研究和接受不同饮食的若干个时间点)并且非常耗时。
营养概况(或营养物质分析):
营养物质需求可使用对生物样本中的营养物质及其代谢产物的定量分析(即,被称为营养物质分析)来确定。营养物质分析使用基于各种分析技术(例如,高效液相色谱、气相色谱、质谱、分光光度法、或免疫确定法)的分析方法的组合来实现。营养物质分析包含多种营养物质和微量营养素及其代谢产物(代谢物)以及相关的蛋白质营养物质/微量营养素转运蛋白的浓度确定,或功能性生物标记物,诸如营养物质/微量营养素特异性酶活性。这种营养分析方法具有覆盖全范围的营养物质和微量营养素的优点,因此使得能够评估营养物质间的相互作用,并且由于不需要进行动力学研究,所以对于患者而言速度更快且侵入性相对较小。
关于氨基酸需求的确定,可使用对氨基酸的浓度及其特定代谢产物的同时分析来确定特定氨基酸的氧化程度。由于蛋白质氧化,对特定氨基酸的氧化程度的测定可用于推断其在蛋白质中的相对掺入,并且因此推断用于满足蛋白质合成的代谢需要的特定需求。例如,苏氨酸被氧化成2-酮丁酸、2-氨基丁酸和2-氨基-3-酮丁酸(图3)。
例如,苏氨酸及其代谢物的浓度的同时分析可用于评估苏氨酸氧化程度(参见图3左侧)。然后,将苏氨酸氧化程度与另一种必需氨基酸进行比较,作为基准,诸如异亮氨酸(参见图3右侧)。就异亮氨酸而言,使用对异亮氨酸及其氧化产物2-酮基-3-甲基-戊酸的浓度的确定来测定氧化程度(参见图2右侧)。氧化程度被定义为苏氨酸及其一种或多种代谢物的浓度组合的计算产物,诸如:
-L-苏氨酸浓度/2-酮丁酸浓度的比值
-L-苏氨酸浓度/(2-酮丁酸浓度+2-氨基丁酸浓度)的比值
-L-苏氨酸浓度/(2-酮丁酸浓度+2-氨基丁酸浓度+2-氨基-3-酮丁酸浓度)的比值
-或其任何数学组合
-或其与其他标记物或蛋白质分解代谢的产物的任何数学组合,诸如循环氨的浓度、尿素循环中的中间体(鸟氨酸、瓜氨酸、精氨酸琥珀酸、精氨酸)、对称二甲基精氨酸、和不对称二甲基精氨酸的浓度
-其与氧化应激、一氧化氮代谢(血浆/血清中的一氧化氮含量,尿液中的硝酸盐含量)的标记物的任何组合。
-其与用于监测例如IBD活性的临床标记物的任何组合,该临床标记物如:CRP、血细胞分类计数、粪便钙卫蛋白、铁状态、红细胞沉降率、蛋白质电泳、粪便中性粒细胞、维生素B12状态等。
例如,可对患病受检者和健康受检者之间的苏氨酸和异亮氨酸的氧化程度进行比较,如图4所示(此处的示例将IBD作为所关注的疾病)。从图4中可看出,只有在与健康受检者相比苏氨酸氧化程度较低的情况下,才能识别IBD对苏氨酸的营养需求增加。
因此,本发明还涉及用于确定对苏氨酸和/或异亮氨酸的营养需求的方法,该方法包括对患病受检者和健康受检者的苏氨酸和/或异亮氨酸的氧化程度进行比较。
利用超高效液相色谱串联质谱联用法来进行氨基酸定量:
氨基酸也可在从受检者获得的样本中直接测定。
样品制备
在50μL的血浆或血清中加入10μL的所标记的内标和140μL的冷甲醇(0.1%甲酸),以进行蛋白沉淀。样品然后经历涡旋混合(5min),再在4℃下以10000rpm离心10min。然后收集上清液以经历衍生化。
氨基酸衍生化
使用AccQ-Tag Ultra衍生化试剂盒氨基酸分析(AccQ-Tag UltraDerivatization Kit Amino Acid Analysis)(沃特世公司(Waters Corp.))按照以下制造商的程序来执行衍生化:将10μL标准氨基酸混合溶液或样品的上清液与70μL的AccQ-TagUltra硼酸盐缓冲液(pH=8.8)混合。通过将20μL的重构的AccQ-Tag Ultra试剂(3mg/mL的6-氨基喹啉基-N-羟基琥珀酰亚胺基氨基甲酸酯或乙腈中的AQC)加入到缓冲混合物中来执行衍生化。然后立即将样品涡旋混合,接着在55℃下温育15min。
使用超高效液相色谱串联质谱联用法(UPLC-MS/MS)来分析氨基酸
UPLC-MS/MS分析在Waters Acquity UPLC系统上执行,该系统在线联接到配备电喷雾离子化(ESI)探头的Waters Xevo TQ质谱仪。使用Waters AccQ-Tag Ultra柱(2.1mm内径×100mm,1.7μm颗粒)以及包含洗脱液A和B的二元系统来实现色谱分离。洗脱液A包含10%的AccQ-Tag Ultra洗脱液A浓缩物(沃特世公司(Waters Corp.))在90%水中的商用溶液。洗脱液B是AccQ-Tag Ultra洗脱液B(沃特世公司(Waters Corp.))的商用溶液。所用分离梯度为:0-0.54min(99.9%A),5.74min(90.9%A),7.74min(78.8%A),8.04(40.4%A),8.05-8.64(10%A),8.73-10.0(99.9%A)。自动进样器温度被设置为20℃,并且色谱柱温度设置为55℃。进样体积为2μL。使用Waters IntelliStart程序来确定每个所测得的氨基酸的锥孔电压和碰撞能量值。代表来自所有AQC加合物的碰撞诱导解离的共同的主要产物的离子m/z171用于定量各个氨基酸。使用标准氨基酸溶液至UPLC-ESI-MS/MS系统中的注入来确定氨基酸的保留时间。使用以下离子源设置:毛细管电压,2.5kV(ESI+);反溶剂气温度,600℃;反溶剂气流速,1000L/h;源温度,150℃。
分析仪设置在每个校准周期期间被确定,其中典型值如下:第一四极杆2.95(低质量数分辨率),14.35(高质量数分辨率),离子能量1:0.1;第二四极杆2.95(低质量数分辨率)和14.40(高质量数分辨率),离子能量2:0.3。使用氩气作为0.15mL.min-1的流速下的碰撞气体。利用Waters Corporation MassLynxTM软件来执行UPLC-MS/MS系统控制和数据采集。使用TargetLynxTM软件(沃特世公司(Waters Corporation))来进行数据分析。
通过气液色谱来定量脂肪酸:
脂肪酸的浓度可利用任何合适的方法如薄层色谱法或气相色谱法来确定。
在下文中,我们将举例说明气相色谱法。
样品制备
在酸性条件下执行血浆和红细胞(RBC)脂肪酸的衍生化。简而言之,将200μL的样品与螺旋盖玻璃试管中的甲醇、甲醇盐酸、己烷和内标溶液混合在一起。将试管加盖,并且在100℃下对血浆加热60min并对RBC加热90min,然后冷却至室温并且加入水以停止反应。接着将试管以1200g离心5min,并且收集上部有机相并通过气相色谱(GC)进行分析。
通过气液色谱来快速分析脂肪酸甲酯(FAME)
总FAME分析在7890Agilent气相色谱仪(美国加利福尼亚州帕洛阿尔托的安捷伦科技公司(Agilent Technologies,Palo Alto,CA,USA))上执行,该气相色谱仪配备有熔融石英BPX-70毛细管柱(10m×0.1mm内径,0.2m膜厚度;澳大利亚墨尔本的SGE公司(SGE,Melbourne,Australia))。分流进样器(35:1)和火焰离子化检测(FID)系统分别在250℃和300℃下工作。使用购自安捷伦公司(Agilent)的商业设备,烘箱的体积已减少至约5400cm3。烘箱温度程序为:在45℃下恒温保持0.5min,以100℃/min升至180℃,在该温度下恒温保持0.5min,再以9℃/min升至220℃,在此温度下恒温保持0.5min,并且然后以50℃/min升至250℃(总工作时间为7.9min)。载气(H2)流在0.7mL.min-1下保持恒定,并且FID信号的采集在50Hz下保持恒定。
对FAME的识别
使用标准FAME的混合物来确认对脂肪酸的识别。该混合物包含以下各项的甲酯:丁酸(4:0)、己酸(6:0)、辛酸(8:0)、癸酸(10:0)、十一烷酸(11:0)、月桂酸(12:0)、十三烷酸(13:0)、肉豆蔻酸(14:0)、肉豆蔻脑酸(14:1n-5)、十五烷酸(15:0)、十五碳烯酸(15:1n-5)、棕榈酸(16:0)、棕榈油酸(16:1n-7)、十七烷酸(17:0)、十七碳烯酸(17:1n-7)、硬脂酸(18:0)、反式油酸(反-18:1n-9)、油酸(18:1n-9)、反亚油酸(全反-18:2n-6)、亚油酸(18:2n-6)、花生酸(20:0)、-亚油酸(18:3n-6)、二十碳烯酸(20:1n-9)、亚麻酸(18:3n-3)、二十一烷酸(21:0)、二十碳二烯酸(20:2n-6)、山嵛酸(22:0)、二十碳三烯酸(20:3n-6)、芥酸(22:1n-9)、二十碳三烯酸(20:3n-3)、花生四烯酸(20:4n-6)、二十二碳二烯酸(22:2n-6)、二十四烷酸(24:0)、二十碳五烯酸(20:5n-3)、神经酸(24:1n-9)和二十二碳六烯酸(22:6n-3)。
含硫分子的定量:
含硫分子包含:氨基酸及其衍生物(同型半胱氨酸、半胱氨酸、γ-谷氨酰-半胱氨酸、半胱氨酰-甘氨酸、同型胱氨酸、半胱氨酸、胱硫醚、甲硫氨酸亚砜、硒代甲硫氨酸、甲硫氨酸亚砜亚胺、硒代半胱氨酸、硒代胱氨酸、麦角硫因、N-甲酰基-L-甲硫氨酸、S-腺苷高半胱氨酸、S-腺苷基甲硫氨酸胺)、肽(还原型谷胱甘肽和氧化型谷胱甘肽)和硫辛酸。
样品制备
在利用100μL的衍生化溶液进行处理之前,将血浆、血清或红细胞样品(50μL)与50μL的内标谷胱甘肽乙酯(GSHee)混合,该衍生化溶液包含10mM碘乙酸的10mM碳酸氢铵水溶液和氨树胶(0.5%v/v,pH 9.5)。将该混合物在室温下存放15分钟。停止反应,并且通过加入50μL的冷磺基水杨酸溶液(10%w/v)来使蛋白质沉淀。然后将混合物在4℃下以16000×g离心15分钟。将上清液(200μL)转移到玻璃小瓶中,并取2μL进样。
使用超高效液相色谱串联质谱联用法(UPLC-MS/MS)来分析衍生化含硫分子
UPLC-MS/MS分析在Waters Acquity UPLC系统上执行,该系统在线联接到配备电喷雾离子化(ESI)探头的Waters Xevo TQ质谱仪。使用Waters HSS T3 2.1mm+100mm,1.7μm色谱柱来实现色谱分离。使用由溶剂A(0.1%甲酸的水溶液)和B(乙腈/水20:80(v/v),含0.1%甲酸)构成的梯度以0.25mL/min的流速来执行洗脱。梯度如下:100%溶剂A 0min-2min,1%溶剂A 2min-7min,99%溶剂A 7.1min-10min。质谱仪在下列条件下工作:毛细管电压:2.5KV;源温度:150℃;反溶剂气温度:600℃;反溶剂气流速:1000L/H。对于四极杆1,低质量数分辨率和高质量数分辨率分别为2.95和14.35,其中离子能量为0.1。对于四极杆2,低质量数分辨率和高质量数分辨率分别为2.95和14.40,其中离子能量为0.3。使用氩气作为0.15mL/min的流速下的碰撞气体。使用Waters IntelliStart软件来确定每种代谢物的具有各自的优化锥孔电压和碰撞能量值的MRM通道。使用这些条件,将每个含硫分子注入到UPLC-MS/MS系统中以确定保留时间。MRM-MS方法被构建成仅监测每个MRM功能的一个传输通道。利用Waters Corporation MassLynxTM软件来执行UPLC-ESI-MS/MS系统控制和数据采集。使用TargetLynxTM软件(沃特世公司(Waters Corporation))来进行数据分析。
元素(包括矿物质)的定量:
元素(包括矿物质)可通过本领域已知的任何合适方法来进行定量。在下文中提供一个示例。
样品制备
以包含5%的1-丁醇、0.05%的EDTA、0.05%的Trition X-100和1%氢氧化铵的稀释溶液,将体积为150μL的生物流体(血浆、血清、尿液等)稀释成1:10。
使用电感耦合的等离子体三重四极杆质谱法(ICP-MS/MS)来进行元素分析
使用在低矩阵等离子体模式下工作的8800ICP-MS/MS质谱仪(日本东京的安捷伦科技公司(Agilent Technologies,Tokyo,Japan))来执行元素分析。ICP-MS系统配有集成的自动进样器,以用于直接的样品导入。将样品通过集成的蠕动泵以0.35mL min-1的流速泵送到样品导入区域中,该样品导入区域由同心雾化器和Scott双通道喷雾室组成。在等离子体中离子化后,分析物离子被传输到质谱仪中。ICP-MS系统配备有允许采用MS/MS分析模式的三重四极杆质谱分析仪。每种矿物质的定量通过外部校准来实现。为了纠正或减少等离子体波动和基质效应,采用在线稀释法以将样品与多元素内标溶液混合。为进行质量控制,在每次分析工作期间分析人类认证的参比材料(Seroorm血清微量元素L-1(SeronormTrace Elements Serum L-1),得自挪威的Sero公司(Sero,Norway))。所测定的元素列于附录1中。
使用高效液相色谱-电感耦合等离子体三重四极杆质谱联用法(UHPLC-ICP-MS/
MS)来进行元素形态分析
对于所有色谱实验,使用1290无限大双活塞UHPLC泵(日本东京的安捷伦科技公司(Agilent Technologies,Tokyo,Japan))。通过与ICP-MS联用的尺寸排阻色谱法在等度洗脱模式下用用50mM乙酸铵(pH 7.4)作为流动相来实现对与生物分子弱相关联的矿物质的分析。对于对稳定物质的分析,使用反相(RP)HPLC-ICP-MS,应用0.4mL min1的流速下的梯度洗脱。流动相A由2%的乙腈(ACN)、0.05%的TFA(三氟乙酸)组成,并且流动相B由98%的ACN、0.05%的TFA组成。典型的梯度包括在30min内乙腈从5%到80%的线性增加。对于对生物流体的分析,应用3-20μL的进样体积。通过收集与所检测到的色谱峰对应的级分来实现对矿物质的识别。最后,将所收集的级分预浓缩,脱盐并通过分子MS进行分析以用于识别生物分子。通过柱后同位素稀释分析来实现定量。这时,将同位素富集的元素溶解于2%的硝酸中,并通过蠕动泵注入,从而保持0.2mL min-1的恒定流速。
水溶性维生素:
水溶性维生素可在从受检者获得的样本中直接定量。
化学品
维生素标准品、甲酸、乙酸购自美国密苏里州圣路易斯的西格玛奥德里奇公司(Sigma-Aldrich,St.Louis,MO),Millipore水来自美国马里兰州米尔福德的沃特世公司(Waters,Milford,MA),乙腈购自美国宾夕法尼亚州拉德诺的VWR公司(VWR,Radnor,PA)。所标记的维生素标准品购自美国马萨诸塞州图克斯伯里的Cambridge IsotopeLaboratories公司(Cambridge Isotope Laboratories,Tewksbury,MA)。
样品制备
将存储于-80℃下的血浆/血清解冻并立即放在冰上。在血浆/血清中加入所标记的内标,混合并保持10min。通过将100的μL血浆/血清与100μL的10%TCA酸混合来使蛋白质沉淀。将样品在冰上提取10min,并且在4℃下以17000rpm离心10min,以供分析维生素B1、B2、B3、B6、B7及其代谢物。将100μL血清/血浆与包含乙酸和抗坏血酸的200μL的90%甲醇/水溶液混合,以用于分析维生素B9。将样品搅拌20min,离心并将使上清液在氮气流下完全干透。利用水重新悬浮样品,并使用前述的UPLC条件来进样。
使用超高效液相色谱串联质谱联用法(UPLC-MS/MS)来分析维生素B1、B2、B3、B6、
B7和B9及其代谢物
取10μL上清液注入到Waters Acquity UPLC系统中,该系统在线联接到配备电喷雾离子化(ESI)探头的Waters Xevo TQ质谱仪。使用Waters AcquityHSS T3 2.1×100mm色谱柱、0.6mL的流速、溶剂A(0.1%甲酸的水溶液)和溶剂B(0.1%甲酸的乙腈溶液)来实现色谱分离。梯度程序在9分钟内从100%A变到100%B。对维生素的检测在WatersXEVO TQS质谱仪中以设定的MRM模式执行,其中每种维生素具有2次跃迁。利用WatersCorporation MassLynxTM软件来执行UPLC-MS/MS系统控制和数据采集。使用TargetLynxTM软件(沃特世公司(Waters Corporation))来进行数据分析。
脂溶性维生素:
脂溶性维生素可在从受检者获得的样本中直接定量。
化学品
己烷、甲醇、乙醇、去离子水和乙腈均购自美国宾夕法尼亚州拉德诺的VWRinternational公司(VWR international,Radnor,PA)。维生素标准品购自美国密苏里州圣路易斯的西格玛奥德里奇公司(Sigma Aldrich,St.Louis,MO)。分析色谱柱(HSS C18,1.73×100mm)和固相萃取(SPE)板购自美国马里兰州米尔福德的沃特世公司(Waters,Milford,MA)。
样品制备
为了保护维生素免遭光降解,样品制备步骤通过在实验室中适当实现的滤光器所产生的紫外线(UV)保护下执行。使用与液-液萃取工序结合的蛋白质沉淀来提取脂溶性维生素。简而言之,将200μL丁基化羟基甲苯(BHT)的乙醇溶液太添加到200μL血清,以执行蛋白沉淀并保护样品免遭氧化。然后用2.5mL正己烷来提取样品,超声处理并离心3次。合并收集的上清液并在氮气流下干燥,以在最后在正己烷/异丙醇9:1中重构。
两种25-羟基维生素D代谢物经历不同的提取方案。简而言之,在150μL人血清中加入150μL甲醇,以进行蛋白质沉淀。收集通过离心(在4℃下以4000rpm离心15min)收集的上清液级分,并使用利用甲醇和水预处理的Oasis HLB(美国马里兰州米尔福德的沃特世公司(Waters,Milford,MA,USA))滤筒来进行SPE净化步骤。在滤筒中装入样品并用水和5%甲醇溶液洗涤。使用甲醇来进行分析的洗脱。然后干燥样品并在正己烷/异丙醇9:1中重构。
使用超高效合相色谱串联质谱联用法(UPC2-MS/MS)来分析脂溶性维生素及其代
谢物
UPC2-MS/MS分析在配备有Xevo TQS质谱仪的Waters Acquity超高效合相色谱(UPC2)系统(沃特世公司(Waters Corporation))上执行。利用HSS C18分析柱来执行色谱分离,并且该分析柱与以下泵连接:泵A(流动相,CO2)和泵B(流动相,10mM醋酸铵甲醇溶液)。在使用1.2mL的流速的14分钟的总工作时间内,所施加的梯度从2%的有机溶剂开始,直到达到40%。MS分析如下:毛细管电压:2.6kV,反溶剂气温度:500℃,锥孔气流速:150L/h,反溶剂气流速:500L/h。引用多反应监测(MRM)来执行该分析。利用Waters CorporationMassLynxTM软件来执行UPLC-MS/MS系统控制和数据采集。使用TargetLynxTM软件(沃特世公司(Waters Corporation))来进行数据分析。
例如,25-羟基维生素D同效维生素的量可用于确定维生素D的状态。
进一步的微量营养素分析:
可使用下文所述的示例性方法来执行维生素或矿物质/痕量元素状态。
维生素B12和维生素B9(叶酸)的状态可使用竞争原理而被测定。
可执行血浆/血清甲基丙二酸和总同型半胱氨酸以及全反钴胺素II的血浆浓度的定量,以用于维生素B12的状态。
可执行铁蛋白、可溶性转铁蛋白或铁调素的定量,以用于铁的状态。
可执行甲状腺素T4和T3的定量,以用于碘的状态。
可执行血浆铜蓝蛋白和铜/锌超氧化物歧化酶的定量,以用于铜的状态。
代谢物的进一步分析:
使用Cobas C111(罗氏诊断公司(Roche Diagnostics))来对血浆/血清执行进一步的分析,以用于氨和尿素定量。通过分光光度法来执行测定。氨浓度为在340nm(波长A)和629nm(波长B)下的所计算的终点降低。通过在340nm(波长A)和409nm(波长B)下的动力学降低,使用计算模式来确定尿素浓度。
组合物:
本发明的组合物包含一定量的营养物质,该营养物质在患病人中重新建立等同于健康人或健康人群组的营养概况或状态的代谢、生理和功能。
首先,确定患病人或一群组患有相同疾病或具有相同疾病严重程度的患病患者的独特的营养需求。
以这种方式,可识别未以最佳的量、而是以过低或过高的量(通常以过低的量)提供给患病患者的营养物质。
然后可将营养组合物调整为含包含一定量的营养物质,该量将使患病人的营养概况恢复到健康人的营养概况。最佳地,在食用适于患病受检者的营养组合物之后,患病受检者的营养概况将具有相同或几乎等同的营养状态。
例如,用于表现出由IAAO方法所确定的苏氨酸缺乏的患病受检者的营养组合物包含游离苏氨酸或/和与蛋白质结合的苏氨酸的形式的苏氨酸,其含量将降低或消除苏氨酸缺乏。
因此,营养组合物可为仅包括在常规饮食中表现不足的那些营养物质的补充剂。
作为另外一种选择,营养组合物可以是完全食物的形式,该完全食物提供患病人实现正常营养状态所需的所有营养物质,并且因此既包含患病受检者与健康受检者相比具有独特的营养需求的那些营养物质,也包含患病受检者与健康受检者具有相同营养需求的那些营养物质。完善食物可包含患病人与健康受试者相比具有较低需求的少量的那些营养物质。
制剂:
上述组合物可以液体或固体形式进行配制。
该组合物还可包含可由本领域的技术人员在阅读本公开之后识别的至少一种其他活性剂、载体、媒介物、赋形剂或助剂。
该组合物可呈营养组合物或药物产品的形式。营养组合物或药物产品可包含本发明的组合物。
营养组合物:
如本文所用,术语“营养组合物”包括但不限于完全营养组合物、部分或不完全营养组合物、以及特定于疾病或病症的营养组合物。完全营养组合物(即,包含所有必需的常量和微量营养素的组合物)可用作患者的唯一营养来源。患者可从此类完全营养组合物接收到100%的营养需求。部分或不完全营养组合物不包含所有必需的大量和微量营养素,并且不可用作患者唯一的营养来源。部分或不完全营养组合物可用作营养补充剂,即作为患者饮食的补充。口服补充营养组合物包含按照本发明的方法识别的患病人的与健康人相比具有较高需求的那些营养物质。
完全营养组合物通常具有热密度为0.7kcal/mL-2.0kcal/mL(2.9kJ/mL-8.4kJ/mL)的能量密度。
营养组合物可包含以下大量营养素和微量营养素:蛋白质的来源、脂质的来源、碳水化合物的来源、维生素和元素(包括矿物质)。
蛋白质来源可以是动物蛋白、乳蛋白或植物蛋白。
该营养组合物还包含一种或多种游离氨基酸。氨基酸的非限制性示例包括丙氨酸、精氨酸、天冬酰胺、天冬氨酸、瓜氨酸、半胱氨酸、谷氨酸、谷氨酰胺、甘氨酸、组氨酸、羟脯氨酸、羟丝氨酸、羟酪氨酸、羟赖氨酸、异亮氨酸、亮氨酸、赖氨酸、甲硫氨酸、苯丙氨酸、脯氨酸、丝氨酸、牛磺酸、苏氨酸、色氨酸、酪氨酸、缬氨酸。非蛋白质氨基酸的示例为瓜氨酸、HICA(α-羟基异己酸)、HIVA(α-羟基异戊酸)、HIMVA(α-羟基甲基戊酸)、或它们的组合。
游离氨基酸可以是组合物中的或与其他蛋白质来源组合的唯一蛋白质来源。每种氨基酸以总氨基酸的0.5%-25%的量存在。
脂肪来源可以是动物脂肪或植物脂肪或这两者。虽然动物脂肪具有基本上相同的热量和营养价值并且可互换使用,但是植物油因易于获得、易于制备并且饱和脂肪酸浓度较低,因此在本发明的实践中是优选的。将要使用的脂肪来源包括鱼油、卵油、藻油、玉米油、葵花油、红花油、低芥酸菜籽油、椰子油和/或大豆油、或它们的组合。
营养组合物可包含元素和矿物质,诸如硼、钙、乙酸钙、葡萄糖酸钙、氯化钙、乳酸钙、磷酸钙、硫酸钙、氯化物、铬、氯化铬、吡啶甲酸铬、铜、硫酸铜、葡萄糖酸铜、硫酸铜、氟化物、铁、羰基铁、三价铁、富马酸亚铁、正磷酸铁、铁研磨物、多糖铁、碘化物、碘、镁、碳酸镁、氢氧化镁、氧化镁、硬脂酸镁、硫酸镁、锰、钼、磷、钾、磷酸钾、碘化钾、氯化钾、乙酸钾、硒、硫、钠、多库酯钠、氯化钠、硒酸钠、钼酸钠、锌、氧化锌、硫酸锌、以及它们的混合物。矿物质化合物的非限制性示例性衍生物包括上述任何矿物质化合物的盐、碱性盐、酯和螯合物。
营养组合物还可包含维生素,这个也容易维生素B1(硫胺素、焦磷酸硫胺素、TPP、三磷酸硫胺素、TTP、盐酸硫胺素、一硝酸硫胺素)、维生素B2(核黄素、黄素单核苷酸、FMN、黄素腺嘌呤二核苷酸、FAD、乳黄素、卵黄素)、维生素B3(烟酸、尼克酸、尼克酰胺、烟酰胺、烟酰胺腺嘌呤二核苷酸、NAD、尼克酸单核苷酸、NicMN、吡啶-3-羧酸)、维生素B3-前体色氨酸、维生素B6(吡哆醇、吡哆醛、吡哆胺、盐酸吡哆醇)、泛酸(泛酸盐、泛醇)、叶酸盐(叶酸、蝶酰谷氨酸)、维生素B12(钴胺素、甲基钴胺素、脱氧腺苷钴胺素、氰钴胺素、羟基钴胺素、腺苷钴胺素)、生物素、维生素C(抗坏血酸)、维生素A(视黄醇、视黄醇乙酸酯、视黄醇棕榈酸酯、与其他长链脂肪酸的视黄酯、视黄醛、视黄酸、视黄醇酯)、维生素D(钙化醇、胆钙化醇、维生素D3、1,25-二羟基维生素D)、维生素E(α-生育酚、α-生育酚乙酸酯、α-生育酚琥珀酸酯、α-生育酚烟酸酯、α-生育酚)、维生素K(维生素K1、叶绿醌、萘醌、维生素K2、甲萘醌-7、维生素K3、甲萘醌-4、氢化甲荼锟、甲萘醌-8、甲萘醌-8H、甲萘醌-9、甲萘醌-9H、甲萘醌-10、甲萘醌-11、甲萘醌-12、甲萘醌-13)、胆碱、肌醇、6-胡萝卜素、以及它们任何组合。
完全营养组合物通常包含10en%-40en%的蛋白质、10en%-60en%的碳水化合物和20en%-80en%%的脂肪。“en%”是能量占营养组合物的总能量的百分比。
该组合物还可包含抗氧化剂、稳定剂(以固体形式提供时)或乳化剂(以液体形式提供时)。
营养组合物的形式:
在一个实施方案中,该营养组合物选自补充营养组合物、完全营养组合物、酸乳产品、发酵乳、果汁、小袋形式的干粉或谷物棒。
该营养组合物可以是也被称为用于特殊医疗用途的食物的医疗食物。医疗食物产品是专门配制的并用于疾病或医学病症的饮食管理(例如,预防或治疗疾病或不希望的医学病症)。医疗食物产品可提供临床营养,例如满足存在医学病症的患者或具有特定营养需求的其他人的特殊营养需求。
医疗食物可以是用于口服给养的保健营养组合物和/或用于肠内或胃肠外给养的营养产品的形式。就用于胃肠外给养的产品而言,其将仅包含适用于胃肠外给养的成分。适用于胃肠外给养的成分是本领域的技术人员已知的。
在一个实施方案中,医疗食物可以是营养完全的产品、饮料、膳食补充剂、膳食替代品、食物添加剂、食物产品的补充剂、待溶解的粉末、肠内营养产品、婴儿配方奶粉的形式、以及它们的组合。
在一个实施方案中,营养组合物可以是发酵乳、酸乳、新鲜奶酪、凝乳、甜食棒、早餐谷物片、早餐谷物棒、饮料、乳粉、大豆基产品、非乳发酵产品、或用于实现临床营养的营养补充剂。在一个实施方案中,组合物可以是粉末形式,特别是用于利用液体重构的粉末。在一个实施方案中,组合物可以是液体的形式,例如即饮型液体口服营养补充剂。
在一个实施方案中,营养组合物是选自片剂、胶囊剂、液体剂、咀嚼片、软凝胶、小袋、散剂、糖浆剂、液体悬浮剂、乳剂、溶液的形式、或它们的组合。在一个实施方案中,营养组合物是口服营养补充剂。作为另外一种选择,营养组合物可以是管饲物。
粘度:
如果营养组合物是液体,则其粘度低于150mPa.s,优选地低于100mPa.s,更优选地低于80mPa.s,甚至更优选地低于70mPa.s。在旋转流变仪中,使用圆锥-平板几何形状在20℃下以50 1/s的剪切速率来确定粘度。
如果组合物以经组织化处理的产品(布丁等)的形式提供以便利用勺子食用,则优选的是粘度为至少350mPa.s,优选地高于750mPa.s,更优选地介于1000和4000mPa.s之间。
治疗效用和方法:
本发明的组合物可用于治疗或预防与健康受检者营养状态不同的患病受检者的营养状态相关联的疾病或者治疗或预防所述疾病的方法。
在一个优选的实施方案中,该疾病是炎性肠病(例如,克罗恩氏病、溃疡性结肠炎、胶原性结肠炎、淋巴细胞性结肠炎、改道性结肠炎、白塞氏病、未定型结肠炎)。本发明的方法和组合物所适用的其他疾病或临床病症包括肠道易激综合症、2型糖尿病、帕金森病、阿尔茨海默病、认知衰退/损害、抑郁症、危重病症。
制备方法:
本发明提供了用于制备上述组合物的方法,该方法包括提供上述营养物质中的至少一种营养物质;并且任选地添加至少一种另外的营养物质,所述营养物质选自一种或多种氨基酸、脂肪或碳水化合物;任选地添加载体或/和水。
本领域的技术人员将理解,他们可自由地组合本文所公开的本发明的所有特征。具体地,可将针对本发明不同实施方案所描述的特征进行组合。根据附图,本发明的其他优点和特征将显而易见。
Claims (30)
1.用于确定患有疾病的受检者的独特的疾病相关的营养需求的体外方法,所述疾病的特征在于所述患病受检者相比于健康受检者的独特的营养需求,所述体外方法包括以下步骤:
a.在患有所述疾病的受检者的样本中确定标记物的状态的概况,该标记物的状态的概况指示所述受检者的营养概况,
b.在健康受检者的样本中确定在步骤a.中所确定的相同标记物的状态的概况,
c.对在步骤a.和b.中所确定的所述概况进行比较,从而确定患有所述疾病的患者对营养物质的独特的营养需求。
2.根据权利要求1或2所述的方法,其中所述疾病为炎性肠病。
3.根据前述权利要求中任一项所述的方法,其中所述营养物质选自蛋白质、氨基酸、脂肪或碳水化合物,或者其中微量营养素选自维生素或元素。
4.根据权利要求1所述的方法,其中在步骤1a)中,所述标记物的所述状态在一群组受检者中被确定,其中任选地所述群组中的所述受检者显示出相同的疾病严重程度,其中任选地所述群组中的所述受检者的疾病复发,或者其中任选地所述群组中的所述受检者的疾病缓解。
5.根据权利要求1所述的方法,其中在步骤1b)中,所述标记物的所述状态在一群组健康受检者中被确定。
6.根据前述权利要求中任一项所述的方法,其中所述样本是选自全血、血浆、血清、红细胞、尿液或组织切片的样本。
7.根据前述权利要求中任一项所述的方法,其中所述标记物的所述状态通过直接定量所述标记物或间接地定量用于指示所述第一标记物的所述状态的另外的一种或多种标记物来测定。
8.根据前述权利要求中任一项所述的方法,其中确定至少10、25、50、100、250个标记物的所述状态。
9.根据前述权利要求中任一项所述的方法,其中所述标记物是蛋白质状态和/或分解代谢的标记物,其中任选地所述蛋白质状态标记物选自白蛋白、前白蛋白或/和磷酸肌酸、以及它们的组合,并且其中任选地分解代谢的所述状态指示物选自氨、尿素、或它们的组合。
10.根据前述权利要求中任一项所述的方法,其中所述标记物是氧化应激状态的标记物,其中任选地所述氧化应激的标记物选自4-羟基壬烯醛、丙二醛、硝基酪氨酸、羰基化蛋白质、总谷胱甘肽、还原型谷胱甘肽、氧化型谷胱甘肽、谷胱甘肽过氧化物酶活性、谷胱甘肽还原酶活性、超氧化物歧化酶活性、过氧化氢酶活性、或它们的组合。
11.根据前述权利要求中任一项所述的方法,其中所述标记物是氮合酶状态的标记物,其中任选地所述氮合酶状态的所述标记物选自亚硝酸盐、硝酸盐、尿素循环的中间体(鸟氨酸、瓜氨酸、精氨酸琥珀酸、精氨酸)、一甲基精氨酸、对称二甲基精氨酸、不对称二甲基精氨酸、或它们的组合。
12.根据前述权利要求中任一项所述的方法,其中所述标记物是氨基酸状态的标记物,并且所述标记物选自氨基丙氨酸、β-丙氨酸、肌氨酸、精氨酸、一甲基精氨酸、不对称二甲基精氨酸、对称二甲基精氨酸、天冬酰胺、天冬氨酸、瓜氨酸、谷氨酸、谷氨酰胺、甘氨酸、组氨酸、1-甲基组氨酸、3-甲基组氨酸、异亮氨酸、亮氨酸、赖氨酸、甲硫氨酸、鸟氨酸、苯丙氨酸、脯氨酸、丝氨酸、牛磺酸、苏氨酸、色氨酸、酪氨酸、缬氨酸、羟脯氨酸、乙醇胺、α-氨基丁酸、β-氨基异丁酸、γ-氨基丁酸、同型半胱氨酸、半胱氨酸、γ-谷氨酰-半胱氨酸、半胱氨酰-甘氨酸、同型胱氨酸、半胱氨酸、胱硫醚、甲硫氨酸亚砜、硒代甲硫氨酸、甲硫氨酸亚砜亚胺、硒代半胱氨酸、硒代胱氨酸、麦角硫因、N-甲酰基-L-甲硫氨酸、S-腺苷高半胱氨酸、或S-腺苷基甲硫氨酸胺、α-酮丁酸、2-氨基丁酸、2-氨基-3-酮丁酸、α-酮基-β-甲基丁酸(或α-酮异戊酸)、α-酮异己酸、或α-酮基-β-甲基戊酸、或它们的组合。
13.根据权利要求12所述的方法,其中所述标记物选自苏氨酸、丝氨酸或脯氨酸,优选苏氨酸。
14.根据权利要求12所述的方法,其中所述标记物是苏氨酸标记物,其中任选地所述标记物是苏氨酸或苏氨酸的代谢物,其中任选地所述苏氨酸的所述代谢物选自丙酸、2-氨基丁酸、2-酮丁酸、2-氨基-3-酮丁酸、氨基丙酮、乙酰CoA、甘氨酸、或它们的组合。
15.根据权利要求12所述的方法,其中所述标记物是异亮氨酸标记物,其中任选地所述标记物是异亮氨酸或异亮氨酸的代谢物,其中任选地所述异亮氨酸的所述代谢物是2-酮基-3-甲基戊酸。
16.根据前述权利要求中任一项所述的方法,其中所述标记物是脂肪酸标记物,其中任选地所述脂肪酸选自丁酸C4:0、己酸C6:0、辛酸C8:0、癸酸C10:0、十一烷酸C11:0、月桂酸C12:0、十三烷酸C13:0、肉豆蔻酸C14:0、十五烷酸C15:0、棕榈酸C16:0、十七烷酸C17:0、硬脂酸C18:0、花生酸C20:0、二十一烷酸C21:0、山嵛酸C22:0、二十四烷酸C24:0、肉豆蔻脑酸C14:1n-5、顺-10-十五碳烯酸C15:1n-5、棕榈油酸C16:1n-7、顺-10-十七碳烯酸C17:1n-7、C18:1n-9反式反油酸、C18:1n-9顺式油酸、顺-11-二十碳烯酸C20:1n-9、芥酸C22:1n-9、神经酸C24:1n-9、C18:2n-6反式亚油酸、C18:2n-6顺式亚麻酸、γ-亚麻酸C18:3n-6、α-亚麻酸C18:3n-3、顺-11,14-二十碳二烯酸C20:2n-6、顺-8,11,14-二十碳三烯酸C20:3n-6、顺-11,14,17-二十碳三烯酸20:3n-3、花生四烯酸C20:4n-6、顺-13,16-二十二碳二烯酸22:2n-6、顺-5,8,11,14,17-二十碳五烯酸(EPA)C20:5n-3、顺-4,7,10,13,16,19-二十二碳六烯酸(DHA)C22:6n-3、硫辛酸、或磷脂(包括磷脂酰胆碱、磷脂酰丝氨酸和/或磷脂酰乙醇胺)。
17.根据前述权利要求中任一项所述的方法,其中标记物是元素标记物(包括矿物质标记物),其中所述元素(包括矿物质)选自锂(Li)、硼(B)、镁(Mg)、铝(Al)、硅(Si)、磷(P)、硫(S)、钾(K)、钙(Ca)、钒(V)、铬(Cr)、锰(Mn)、铁(Fe)、钴(Co)、镍(Ni)、铜(Cu)、锌(Zn)、砷(As)、硒(Se)、溴(Br)、铷(Rb)、锶(Sr)、钼(Mo)、锡(Sn)、碘(I)、钡(Ba)、钛(Ti)、钠(Na)、氯(Cl)、氟(F)。
18.根据前述权利要求中任一项所述的方法,其中所述标记物是维生素标记物,其中任选地所述维生素选自水溶性维生素或脂溶性维生素。
19.根据权利要求18所述的方法,其中所述水溶性维生素选自维生素B1(硫胺素)、维生素B2(核黄素)、维生素B3(尼克酸或烟酸)、维生素B3烟酰胺、维生素B3甲基烟酰胺、维生素B3烟尿酸、维生素B4(胆碱)、维生素B5(泛酸)、维生素B6(吡哆醇、维生素B6吡哆醛、吡哆胺)、维生素B6磷酸吡哆醛、维生素B6吡哆胺、维生素B6吡哆酸、维生素B8(生物素)、维生素B9(叶酸)、维生素B9甲基四氢叶酸、维生素B12(氰钴胺素、甲基钴胺素)、或维生素B12羟基钴胺素、腺苷钴胺素。
20.根据权利要求18所述的方法,其中所述脂溶性维生素选自维生素A(视黄醇)、维生素K2(甲萘醌)、维生素K1(叶绿醌)、维生素E(α-生育酚、-生育酚)、维生素D(25-OH维生素D2、维生素D 25-OH D3、维生素D2、维生素D3、1α-25-(OH)2维生素D3)。
21.根据前述权利要求中任一项所述的方法,其中所述标记物是核苷酸标记物,其中任选地所述核苷酸选自肌苷5'-单磷酸、腺苷5'-单磷酸、胞苷5'-单磷酸、鸟苷5'-单磷酸、肌苷5'-单磷酸、尿苷5'-单磷酸、或它们的组合。
22.根据前述权利要求中任一项所述的方法,其中所述标记物是渗压剂标记物,其中任选地所述渗压剂选自三甲胺N-氧化物、二甲基锍丙酸盐、三甲基甘氨酸、肌氨酸、甜菜碱、甘油磷酰胆碱、肌醇、或它们的组合。
23.根据前述权利要求中任一项所述的方法,其中所述标记物是植物营养素标记物,其中任选地所述植物营养素选自类胡萝卜素、鞣花酸、黄酮类、绿原酸、白藜芦醇、芥子油苷、植物雌激素、或它们的组合。
24.根据前述权利要求中任一项所述的方法,其中所述标记物是肽标记物,其中任选地所述肽选自还原型谷胱甘肽或氧化型谷胱甘肽。
25.用于制备适合施用于患有疾病的受检者的营养组合物的方法,包括:
a.利用根据权利要求1至24中任一项所述的方法来确定患有疾病的受检者的独特的营养需求,
b.制备包含一定量的营养物质的组合物,所述量能够在患有所述疾病的所述患者中使营养物质和大量营养素的营养概况恢复到在权利要求1的步骤b.中所确定的健康受检者的营养概况,或朝所述健康受检者的营养概况恢复。
26.根据权利要求25的方法制备的组合物。
27.根据权利要求26所述的组合物呈液体或粉末的形式。
28.根据权利要求26或27所述的组合物,所述组合物用于治疗特征在于所述患病受检者相对于健康受检者的独特的营养需求的疾病,其中任选地所述疾病为IBD。
29.用于根据权利要求28所述的用途的组合物,其中所述组合物包含一定量的所述至少一种营养物质,所述量满足患有所述疾病的受检者的日常需求。
30.治疗患有疾病的人类受检者的方法,包括:
a)提供根据权利要求26或27所述的组合物,
b)将所述组合物施用于患有疾病的受检者,所述疾病的特征在于所述患病受检者相对于健康受检者的独特的营养需求,其中任选地所述疾病为IBD。
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