Nutritional and phytochemical bounds estimation via comparative metabolomics, phylogenetics and taxonomy

Kevin Ferguson, Community Self Sufficiency, Beaverton, OR

Abstract

For cultivated and wild plant based foods, medicines, fiber, dye and other useful materials, while a great deal of chemical analysis has been known, published in journals and online in databases [1], a great deal is still unknown for many species. While nutritional, medicinal and general phytochemical analysis information per cost has improved over time, it has not kept pace with the cost for RNA-seq and, consequently, the growing databases and knowledgebases for genomes, transcriptomes, protenomes and metabolomes [5]. Having discerned metabolic pathways for biosynthesis and catabolic pathways for utilization of particular nutrients or other chemicals in one species with a largely understood genome, another organism with matching genes, metabolic pathways and similar biological environments (relative availability of nutrients, sun, water, etc.) could potentially have similar levels of respective metabolites. In this article, example metabolic pathways imply a ratio range for particular metabolites. Measured metabolite ratios of dozens of species used as food (leaves, seeds, etc.) are measured and statistical analysis shows ratios tend to fall in ranges consistent with metabololic pathway analysis. This implies that close taxonometric and/or transcriptomic relatives may be expected to have similar metabolite ratios.

Background

For cultivated and wild plant based foods, medicines, fiber, dye and other useful materials, while a great deal of nutritional and other chemical analysis has been known, published in journals and online in databases [1], a great deal is still unknown for many species. While nutritional, medicinal and general phytochemical analysis information per cost has improved over time, it has not kept pace with the cost for RNA-seq and, consequently, the growing databases and knowledgebases for genomes, transcriptomes, protenomes and metabolomes. Having discerned metabolic pathways for biosynthesis and catabolic pathways for utilization of particular nutrients or other chemicals in one species with a largely understood genome, another organism with matching genes, metabolic pathways and similar biological environments (relative availability of nutrients, sun, water, etc.) could potentially have similar levels of respective metabolites. Via RNA-seq, the expressome/transcriptome can be used to predict metabolic pathways, and via comparison with existing data for other species and/or known bounds given the metabolic and catabolic circuits, estimate levels of specific metabolites is of interest for exploring the utility of plants so far lacking direct, and often more costly, respective chemical analysis. Further, as such data becomes available and taxonometrics becomes more refined, statistical ratios of metabolites and other chemicals (such as minerals) can suggest likely bounds for a relative species. Thus, if such estimates could be sufficiently accurate, alternatives for medicines, crops, foraging, etc. might be prescreened for nutrition, medicine, toxins, allergens, etc. simply by checking existing or newly aquired (via relatively and increasingly inexpensive techniques) expressome data followed by potentially automated metabolite predictions using the above approach. Preliminary research shows that the bounds metabolite ratios as implied by metabolic pathway circuits may be rather narrow. An example aparatate derived amino acid biosynthesis follows.

Example Metabolic Pathway: Amino Acid Synthesis

The metabolic pathway for aspartate derived amino acids L-lysine, L-methionine, L-threonine and L-isoleucine has been determined for Arabidopsis thaliana, a member of the mustard family, in [4] as shown below.

The biosynthesis pathway for 3 of these 4 amino acids, namely L-threonine, L-methionine and L-lysine, is shown in [5]. Note that also via the same website as [5], the same amino acids pathway is shown for broccoli (https://pmn.plantcyc.org/BOLERACEA_OLERACEA/NEW-IMAGE?type=PATHWAY&object=PWY-724 ) and for cabbage (https://pmn.plantcyc.org/BOLERACEA_CAPITATA/new-image?object=PWY-724 ). Likewise, the same pathway is shown for hops (Humulus lupulus) and other species. One may compare still other species by clicking "change organism database" in the upper right corner of the plantcyc.org page examples given here, selecting a species, "Pathways," "Biosynthesis," "Amino Acids Biosynthesis" and finally "superpathway of L-lysine, L-threonine and L-methionine biosynthesis II". Meanwhile, a mathematical model for the biosynthesis of the same amino acids, specifically for the bacterium E. coli is given in [6]. These amino acids are among the building blocks for all protiens for plants, animals and other life forms on earth. Thus, their synthesis represents synthesis of fundamental building blocks of life, and these "nutrients" are to be found in all plant based foods and medicines, etc. except for the extreme case of highly processed foods.

**Fig.A - The Aspartate-derived amino acid pathway (from Figure 2 of [4]).**

Example Mineral Uptake Ratio: Iron and Zinc

The relationship between zinc and iron uptake has been examined in [7]): zinc interfered with translocation of iron from roots to above ground parts of Glycine max.

Procedure

Nutrition data taken from the USDA [1] for seeds and leaves for dozens of plants was used. Seeds were divided approximately by the USDA food groups into oil seeds, legumes, grains and cereals. Note that some species may be considered to be over represented since they have different varieties and/or preparation influencing database expansion. For example, in some cases data for both cooked and uncooked material is included. This has the effect of causing some peaks in histograms and reducing some variance calculations. However, there is a somewhat of a correlation between the over-representation of some foods and the varieties and species within the given family. It is beyond the scope of the current research to try to make this correlation close to 1, nor to otherwise manage the representation bias issue. This might be considered for future work.

For the selected items within each category, nutrition data was extracted for a unique ratio (a nutrient in the numerator and a nutrient in the denominator). A few exceptions for ratios used nutrient (by weigth) per 100g of the selected food item. The captions for these exceptions include "Absolute."

Data

Nutrional data was downloaded from the USDA [1]. The following lists the USDA database ID (#), and food group ID (#) along with the description included in the database. The foods were selected as identifiable whole parts of plants (with the exception of some whole flours).

Leaves:
USDA# GRP# Description
11003 1100 Amaranth leaves, raw
11004 1100 Amaranth leaves, cooked, boiled, drained, without salt
11005 1100 Arrowhead, raw
11006 1100 Arrowhead, cooked, boiled, drained, without salt
11007 1100 Artichokes, (globe or french), raw
11086 1100 Beet greens, raw
11087 1100 Beet greens, cooked, boiled, drained, without salt
11096 1100 Broccoli raab, raw
11097 1100 Broccoli raab, cooked
11098 1100 Brussels sprouts, raw
11099 1100 Brussels sprouts, cooked, boiled, drained, without salt
11100 1100 Brussels sprouts, frozen, unprepared
11101 1100 Brussels sprouts, frozen, cooked, boiled, drained, without s`+
11109 1100 Cabbage, raw
11110 1100 Cabbage, cooked, boiled, drained, without salt
11112 1100 Cabbage, red, raw
11113 1100 Cabbage, red, cooked, boiled, drained, without salt
11114 1100 Cabbage, savoy, raw
11115 1100 Cabbage, savoy, cooked, boiled, drained, without salt
11116 1100 Cabbage, chinese (pak-choi), raw
11117 1100 Cabbage, chinese (pak-choi), cooked, boiled, drained, withouo+
11119 1100 Cabbage, chinese (pe-tsai), raw
11120 1100 Cabbage, chinese (pe-tsai), cooked, boiled, drained, withoutr+
11147 1100 Chard, swiss, raw
11148 1100 Chard, swiss, cooked, boiled, drained, without salt
11152 1100 Chicory greens, raw
11161 1100 Collards, raw
11165 1100 Coriander (cilantro) leaves, raw
11203 1100 Cress, garden, raw
11207 1100 Dandelion greens, raw
11233 1100 Kale, raw
11241 1100 Kohlrabi, raw
11244 1100 Lambsquarters, raw
11250 1100 Lettuce, butterhead (includes boston and bibb types), raw
11251 1100 Lettuce, cos or romaine, raw
11252 1100 Lettuce, iceberg (includes crisphead types), raw
11253 1100 Lettuce, green leaf, raw
11254 1100 Lotus root, raw
11257 1100 Lettuce, red leaf, raw
11270 1100 Mustard greens, raw
11274 1100 Mustard spinach, (tendergreen), raw
11297 1100 Parsley, raw
11418 1100 Pumpkin leaves, raw
11427 1100 Purslane, raw
11457 1100 Spinach, raw
11503 1100 Swamp cabbage, (skunk cabbage), raw
11505 1100 Sweet potato leaves, raw
11520 1100 Taro leaves, raw
11563 1100 Tree fern, cooked, without salt
11568 1100 Turnip greens, raw
11587 1100 Vinespinach, (basella), raw
11591 1100 Watercress, raw
11613 1100 Borage, raw
11616 1100 Dock, raw
11622 1100 Kale, scotch, raw
11698 1100 Chrysanthemum leaves, raw
11700 1100 Amaranth leaves, cooked, boiled, drained, with salt
11701 1100 Arrowhead, cooked, boiled, drained, with salt
11702 1100 Artichokes, (globe or french), cooked, boiled, drained, with╖-
11703 1100 Artichokes, (globe or french), frozen, cooked, boiled, drain╣-
11749 1100 Cabbage, common (danish, domestic, and pointed types), freshµ-
11765 1100 Chard, swiss, cooked, boiled, drained, with salt
11768 1100 Collards, cooked, boiled, drained, with salt
11781 1100 Cress, garden, cooked, boiled, drained, with salt
11782 1100 Dandelion greens, cooked, boiled, drained, with salt
11789 1100 Jute, potherb, cooked, boiled, drained, with salt
11790 1100 Kale, cooked, boiled, drained, with salt
11791 1100 Kale, frozen, cooked, boiled, drained, with salt
11792 1100 Kale, scotch, cooked, boiled, drained, with salt
11793 1100 Kohlrabi, cooked, boiled, drained, with salt
11794 1100 Lambs quarters, cooked, boiled, drained, with salt
11795 1100 Leeks, (bulb and lower leaf-portion), cooked, boiled, draine.
11802 1100 New zealand spinach, cooked, boiled, drained, with salt
11984 1100 Epazote, raw
11985 1100 Fireweed, leaves, raw
11995 1100 Fiddlehead ferns, raw

Seeds, Cereal:
USDA# GRP# Description
20004 2000 Barley, hulled
20005 2000 Barley, pearled, raw
20012 2000 Bulgur, dry
20014 2000 Corn, yellow
20031 2000 Millet, raw
20036 2000 Rice, brown, long-grain, raw
20038 2000 Oats
20040 2000 Rice, brown, medium-grain, raw
20041 2000 Rice, brown, medium-grain, cooked
20045 2000 Rice, white, long-grain, regular, cooked
20052 2000 Rice, white, short-grain, raw
20053 2000 Rice, white, short-grain, cooked
20054 2000 Rice, white, glutinous, raw
20055 2000 Rice, white, glutinous, cooked
20058 2000 Rice, white, steamed, Chinese restaurant
20062 2000 Rye
20067 2000 Sorghum
20069 2000 Triticale
20070 2000 Triticale flour, whole-grain
20071 2000 Wheat, hard red spring
20072 2000 Wheat, hard red winter
20073 2000 Wheat, soft red winter
20074 2000 Wheat, hard white
20075 2000 Wheat, soft white
20076 2000 Wheat, durum
20080 2000 Wheat flour, whole-grain
20087 2000 Wheat, sprouted
20088 2000 Wild rice, raw
20089 2000 Wild rice, cooked
20090 2000 Rice flour, brown
20138 2000 Kamut, uncooked
20139 2000 Kamut, cooked
20140 2000 Spelt, uncooked
20141 2000 Spelt, cooked
20142 2000 Teff, uncooked
20143 2000 Teff, cooked
20314 2000 Corn, white
20315 2000 Corn flour, whole-grain, blue (harina de maiz morado)
20316 2000 Corn flour, whole-grain, white
20320 2000 Cornmeal, whole-grain, white
20345 2000 Rice, white, long-grain, regular, cooked, enriched, with sal¥O
20444 2000 Rice, white, long-grain, regular, raw, unenriched
20445 2000 Rice, white, long-grain, regular, cooked, unenriched, withou▐O
20446 2000 Rice, white, long-grain, parboiled, unenriched, dry
20447 2000 Rice, white, long-grain, parboiled, unenriched, cooked
20450 2000 Rice, white, medium-grain, raw, unenriched
20451 2000 Rice, white, medium-grain, cooked, unenriched
20452 2000 Rice, white, short-grain, raw, unenriched
20453 2000 Rice, white, short-grain, cooked, unenriched
20466 2000 Semolina, unenriched

Seeds, Grain:
USDA# GRP# Description
20001 2000 Amaranth, uncooked
20002 2000 Amaranth grain, cooked
20004 2000 Barley, hulled
20005 2000 Barley, pearled, raw
20006 2000 Barley, pearled, cooked
20008 2000 Buckwheat
20009 2000 Buckwheat groats, roasted, dry
20010 2000 Buckwheat groats, roasted, cooked
20011 2000 Buckwheat flour, whole-groat
20012 2000 Bulgur, dry
20013 2000 Bulgur, cooked
20014 2000 Corn, yellow
20016 2000 Corn flour, whole-grain, yellow
20020 2000 Cornmeal, whole-grain, yellow
20023 2000 Cornmeal, self-rising, bolted, plain, enriched, yellow
20024 2000 Cornmeal, self-rising, bolted, with wheat flour added, enric9N
20028 2000 Couscous, dry
20030 2000 Hominy, canned, white
20031 2000 Millet, raw
20032 2000 Millet, cooked
20033 2000 Oat bran, raw
20034 2000 Oat bran, cooked
20035 2000 Quinoa, uncooked
20036 2000 Rice, brown, long-grain, raw
20037 2000 Rice, brown, long-grain, cooked
20038 2000 Oats
20040 2000 Rice, brown, medium-grain, raw
20041 2000 Rice, brown, medium-grain, cooked
20045 2000 Rice, white, long-grain, regular, cooked
20052 2000 Rice, white, short-grain, raw
20053 2000 Rice, white, short-grain, cooked
20054 2000 Rice, white, glutinous, raw
20055 2000 Rice, white, glutinous, cooked
20056 2000 Rice, white, with pasta, dry
20057 2000 Rice, white, with pasta, cooked
20058 2000 Rice, white, steamed, Chinese restaurant
20061 2000 Rice flour, white
20062 2000 Rye
20063 2000 Rye flour, dark
20064 2000 Rye flour, medium
20065 2000 Rye flour, light
20067 2000 Sorghum
20069 2000 Triticale
20070 2000 Triticale flour, whole-grain
20071 2000 Wheat, hard red spring
20072 2000 Wheat, hard red winter
20073 2000 Wheat, soft red winter
20074 2000 Wheat, hard white
20075 2000 Wheat, soft white
20076 2000 Wheat, durum
20080 2000 Wheat flour, whole-grain
20087 2000 Wheat, sprouted
20088 2000 Wild rice, raw
20089 2000 Wild rice, cooked
20090 2000 Rice flour, brown
20129 2000 Wheat flours, bread, unenriched
20130 2000 Barley flour or meal
20138 2000 Kamut, uncooked
20139 2000 Kamut, cooked
20140 2000 Spelt, uncooked
20141 2000 Spelt, cooked
20142 2000 Teff, uncooked
20143 2000 Teff, cooked
20314 2000 Corn, white
20315 2000 Corn flour, whole-grain, blue (harina de maiz morado)
20316 2000 Corn flour, whole-grain, white
20320 2000 Cornmeal, whole-grain, white
20330 2000 Hominy, canned, yellow
20345 2000 Rice, white, long-grain, regular, cooked, enriched, with sal¥O
20444 2000 Rice, white, long-grain, regular, raw, unenriched
20445 2000 Rice, white, long-grain, regular, cooked, unenriched, withou▐O
20446 2000 Rice, white, long-grain, parboiled, unenriched, dry
20447 2000 Rice, white, long-grain, parboiled, unenriched, cooked
20450 2000 Rice, white, medium-grain, raw, unenriched
20451 2000 Rice, white, medium-grain, cooked, unenriched
20452 2000 Rice, white, short-grain, raw, unenriched
20453 2000 Rice, white, short-grain, cooked, unenriched
20466 2000 Semolina, unenriched
20481 2000 Wheat flour, white, all-purpose, unenriched
20545 2000 Rice, white, long-grain, regular, cooked, unenriched, with seP

Seeds, Legumes:
USDA# GRP# Description
16001 1600 Beans, adzuki, mature seeds, raw
16002 1600 Beans, adzuki, mature seeds, cooked, boiled, without salt
16004 1600 Beans, adzuki, yokan, mature seeds
16014 1600 Beans, black, mature seeds, raw
16015 1600 Beans, black, mature seeds, cooked, boiled, without salt
16019 1600 Beans, cranberry (roman), mature seeds, raw
16020 1600 Beans, cranberry (roman), mature seeds, cooked, boiled, with≥>
16021 1600 Beans, cranberry (roman), mature seeds, canned
16022 1600 Beans, french, mature seeds, raw
16023 1600 Beans, french, mature seeds, cooked, boiled, without salt
16024 1600 Beans, great northern, mature seeds, raw
16025 1600 Beans, great northern, mature seeds, cooked, boiled, without▄>
16026 1600 Beans, great northern, mature seeds, canned
16027 1600 Beans, kidney, all types, mature seeds, raw
16028 1600 Beans, kidney, all types, mature seeds, cooked, boiled, withÑ>
16029 1600 Beans, kidney, all types, mature seeds, canned
16030 1600 Beans, kidney, california red, mature seeds, raw
16031 1600 Beans, kidney, california red, mature seeds, cooked, boiled,ß>
16032 1600 Beans, kidney, red, mature seeds, raw
16033 1600 Beans, kidney, red, mature seeds, cooked, boiled, without sa≤>
16034 1600 Beans, kidney, red, mature seeds, canned
16035 1600 Beans, kidney, royal red, mature seeds, raw
16036 1600 Beans, kidney, royal red, mature seeds, cooked, boiled, with╤>
16037 1600 Beans, navy, mature seeds, raw
16038 1600 Beans, navy, mature seeds, cooked, boiled, without salt
16039 1600 Beans, navy, mature seeds, canned
16040 1600 Beans, pink, mature seeds, raw
16041 1600 Beans, pink, mature seeds, cooked, boiled, without salt
16042 1600 Beans, pinto, mature seeds, raw
16043 1600 Beans, pinto, mature seeds, cooked, boiled, without salt
16044 1600 Beans, pinto, mature seeds, canned
16045 1600 Beans, small white, mature seeds, raw
16046 1600 Beans, small white, mature seeds, cooked, boiled, without sa╗>
16047 1600 Beans, yellow, mature seeds, raw
16048 1600 Beans, yellow, mature seeds, cooked, boiled, without salt
16049 1600 Beans, white, mature seeds, raw
16050 1600 Beans, white, mature seeds, cooked, boiled, without salt
16051 1600 Beans, white, mature seeds, canned
16052 1600 Broadbeans (fava beans), mature seeds, raw
16053 1600 Broadbeans (fava beans), mature seeds, cooked, boiled, withoª>
16054 1600 Broadbeans (fava beans), mature seeds, canned
16056 1600 Chickpeas (garbanzo beans, bengal gram), mature seeds, raw
16057 1600 Chickpeas (garbanzo beans, bengal gram), mature seeds, cookeª>
16058 1600 Chickpeas (garbanzo beans, bengal gram), mature seeds, canne+>
16060 1600 Cowpeas, catjang, mature seeds, raw
16061 1600 Cowpeas, catjang, mature seeds, cooked, boiled, without salt+>
16062 1600 Cowpeas, common (blackeyes, crowder, southern), mature seeds+>
16063 1600 Cowpeas, common (blackeyes, crowder, southern), mature seeds+>
16064 1600 Cowpeas, common (blackeyes, crowder, southern), mature seeds->
16065 1600 Cowpeas, common (blackeyes, crowder, southern), mature seeds+>
16067 1600 Hyacinth beans, mature seeds, raw
16068 1600 Hyacinth beans, mature seeds, cooked, boiled, without salt
16069 1600 Lentils, raw
16070 1600 Lentils, mature seeds, cooked, boiled, without salt
16071 1600 Lima beans, large, mature seeds, raw
16072 1600 Lima beans, large, mature seeds, cooked, boiled, without sal+>
16073 1600 Lima beans, large, mature seeds, canned
16074 1600 Lima beans, thin seeded (baby), mature seeds, raw
16075 1600 Lima beans, thin seeded (baby), mature seeds, cooked, boiledª>
16076 1600 Lupins, mature seeds, raw
16077 1600 Lupins, mature seeds, cooked, boiled, without salt
16078 1600 Mothbeans, mature seeds, raw
16079 1600 Mothbeans, mature seeds, cooked, boiled, without salt
16080 1600 Mung beans, mature seeds, raw
16081 1600 Mung beans, mature seeds, cooked, boiled, without salt
16083 1600 Mungo beans, mature seeds, raw
16084 1600 Mungo beans, mature seeds, cooked, boiled, without salt
16085 1600 Peas, split, mature seeds, raw
16086 1600 Peas, split, mature seeds, cooked, boiled, without salt
16087 1600 Peanuts, all types, raw
16091 1600 Peanuts, spanish, raw
16093 1600 Peanuts, valencia, raw
16095 1600 Peanuts, virginia, raw
16097 1600 Peanut butter, chunk style, with salt
16100 1600 Peanut flour, low fat
16101 1600 Pigeon peas (red gram), mature seeds, raw
16102 1600 Pigeon peas (red gram), mature seeds, cooked, boiled, without>
16108 1600 Soybeans, mature seeds, raw
16109 1600 Soybeans, mature cooked, boiled, without salt
16110 1600 Soybeans, mature seeds, roasted, salted
16111 1600 Soybeans, mature seeds, dry roasted


Seeds, Oil:
USDA# GRP# Description
2001 200 Spices, allspice, ground
2002 200 Spices, anise seed
2005 200 Spices, caraway seed
2006 200 Spices, cardamom
2007 200 Spices, celery seed
2013 200 Spices, coriander seed
2014 200 Spices, cumin seed
2016 200 Spices, dill seed
2018 200 Spices, fennel seed
2024 200 Spices, mustard seed, yellow
2033 200 Spices, poppy seed
12001 1200 Seeds, breadfruit seeds, raw
12006 1200 Seeds, chia seeds, dried
12014 1200 Seeds, pumpkin and squash seed kernels, dried
12021 1200 Seeds, safflower seed kernels, dried
12023 1200 Seeds, sesame seeds, whole, dried
12024 1200 Seeds, sesame seeds, whole, roasted and toasted
12058 1200 Nuts, acorns, raw
12061 1200 Nuts, almonds
12077 1200 Nuts, beechnuts, dried
12078 1200 Nuts, brazilnuts, dried, unblanched
12084 1200 Nuts, butternuts, dried
12087 1200 Nuts, cashew nuts, raw
12093 1200 Nuts, chestnuts, chinese, raw
12097 1200 Nuts, chestnuts, european, raw, unpeeled
12098 1200 Nuts, chestnuts, european, raw, peeled
12104 1200 Nuts, coconut meat, raw
12120 1200 Nuts, hazelnuts or filberts
12127 1200 Nuts, ginkgo nuts, raw
12130 1200 Nuts, hickorynuts, dried
12131 1200 Nuts, macadamia nuts, raw
12142 1200 Nuts, pecans
12145 1200 Nuts, pilinuts-canarytree, dried
12147 1200 Nuts, pine nuts, dried
12149 1200 Nuts, pine nuts, pinyon, dried
12151 1200 Nuts, pistachio nuts, raw
12154 1200 Nuts, walnuts, black, dried
12155 1200 Nuts, walnuts, english
12158 1200 Seeds, breadfruit seeds, roasted
12160 1200 Seeds, cottonseed kernels, roasted (glandless)
12163 1200 Seeds, pumpkin and squash seeds, whole, roasted, without salå/
12174 1200 Seeds, watermelon seed kernels, dried

Results

The results are detailed in the following:

USDA food group ID, USDA nutrient ID of ratio numerator and USDA nutrient ID of ratio denominator.

the caption below each figure, including the respective names of the food group, numerator and denominator. (Excpetions are "Absolutes" that ratio absolute units per 100g of food).

figures which show ratios sorted and plotted from minimum to maximum with the plant food number (with 0 representing the index of the minimum ratio) along the horizontal axis.

statistics for the ratios of the group, including mean, max, min, stdev, etc.

histogram of ratios for the group.

Amino Acid Ratios

Perhaps not too surprisingly, the ratios of particular amino acids (by weight) to protein (by weight) varies relatively little across the food categories selected. For example, as shown in figures 23,34,43,58,69 and 77, the mean ratio of threonine to protien ranges from about 3.4 to 4.1%, with the worst standard deviation of about 0.6%. And the metabolic pathway correlated amino acid isoleucine has very similar narrow ranges (3.6 to 4.4% mean, 0.6% worst case stdev) as shown in figures 12,28,38,52,63 and 72.

Mineral Ratios

Zinc to Iron ratios off all categories have very similar statistics with peaks approximately near 0.5 as shown in figures 47, 62 and 80. Weights of other minerals were divided by the ash weight since ash is typically primarily mineral content left after water has been evaporated and organic matter has been burned off. An example is seed cereal iron to ash ratio with a mean of 0.2% and a stdev over 48 samples of .1%.