Preparation of starch and other carbon fractions from higher plant leaves for stable carbon isotope analysis

Abstract

The measurement of the carbon isotope composition of starch and cellulose still relies on chemical isolation of these water-insoluble plant constituents and subsequent elemental analysis by isotope ratio mass spectrometry (EA/IRMS) of the purified fractions, while delta(13)C values of low-molecular-weight organic compounds are now routinely measured by gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS). Here we report a simple and reliable method for processing milligram quantities of dried plant material for the analysis of the carbon isotope composition of lipids, soluble sugars, starch and cellulose from the same sample. We evaluated three different starch preparation methods, namely (1) enzymatic hydrolysis by alpha-amylase, (2) solubilization by dimethyl sulfoxide (DMSO) followed by precipitation with ethanol, and (3) partial hydrolysis by HCl followed by precipitation of the resulting dextrins by ethanol. Starch recovery for three commercially available native starches (from potato, rice and wheat) varied from 48 to 81% for the techniques based on precipitation, whereas the enzymatic technique exhibited yields between 99 and 105%. In addition, the DMSO and HCl techniques introduced a significant (13)C fractionation of up to 1.9 per thousand, while the carbon isotope composition of native starches analyzed after enzymatic digestion did not show any significant difference from that of untreated samples. The enzymatic starch preparation method was then incorporated into a protocol for determination of delta(13)C signatures of lipids, soluble carbohydrates, starch and crude cellulose. The procedure is based on methanol/chloroform/water extraction of dried and ground leaf material. After recovery of the chloroform phase (lipid fraction), the methanol/water phase was deionized by ion exchange (soluble carbohydrate fraction) and the pellet treated with heat-stable alpha-amylase (starch fraction). The remaining insoluble material was subjected to solvolysis by diglyme (cellulose fraction). The method was shown to be applicable to foliar tissues of a variety of different plant species (spruce, erect brome, maize and soybean).

Stable isotopes and trace elements as indicators of diet and habitat use in cetaceans: predicting errors related to preservation, lipid extraction, and lipid normalization

“…DMSO also makes hydrogen bonds with water molecules, and affects cell membrane structure by increasing permeability (Gordeliy et al 1998). A direct incorporation of isotopically light C from DMSO (C 2 H 6 SO) through binding with tissue constituents, combined possibly with a loss of molecules containing the heavier isotope (see Gearing 1991), may have caused the observed δ 13 C depletion of DMSO-preserved samples, an effect also documented in previous studies (Hobson et al 1997, Todd et al 1997, Wanek et al 2001, Barrow et al 2008). An assumption underlying this hypothesis, which could not be verified given that the isotopic signature of DMSO was not measured in our study, is that DMSO was isotopically lighter in 13 C than skin.…”

“…Although the recommendation is for low-temperature cryogenic preservation (Sweeting et al 2004, Budge et al 2006, possibilities for retrospective ecological studies of marine mammals, and particularly cetaceans, commonly arise from DMSOpreserved tissue banks collected for the purpose of genetic analyses. However, biases in C and N isotope ratios or trace element concentrations associated with DMSO preservation are not fully understood (Hobson et al 1997, Todd et al 1997, Wanek et al 2001, Barrow et al 2008.…”

Compound-Specific Carbon Isotopes and Concentrations of Carbohydrates and Organic Acids as Indicators of Tree Decline in Mountain Pine

“…However, it is worth noting that needles used for the analysis from the declining mountain pine trees were green without visible damage. After collection, all needle samples were heated in a microwave for 90 s at 250 W to stop enzymatic and metabolic activities [31]. Subsequently, the samples were delivered to the laboratory at the Paul Scherrer Institute, where they were dried for 24 h at 60 °C [31] and ground with a steel ball mill to a fine powder, which was used for all further analyses.…”

“…The tubes were placed in a water bath at 85 • C for 30 min according to the protocol [19,31]. The samples were allowed to cool down for 30 min followed by centrifugation at 10,000× g for 2 min.…”

Water deficit modifies the carbon isotopic composition of lipids, soluble sugars and leaves of Copaifera langsdorffii Desf. (Fabaceae)

“…We removed 0.6 ml aliquots of the supernatant, and separated phases by adding 0.2 ml of chloroform and 0.7 ml of water. After centrifugation, the methanol/water phase (upper layer) was removed (Wanek et al 2001). The chloroform phase (lower layer) was dried in a vacuum concentrator to obtain the lipids.…”